key: cord-309171-kgc7lgjp authors: dolinger, michael t.; person, hannibal; smith, rachel; jarchin, lauren; pittman, nanci; dubinsky, marla c.; lai, joanne title: pediatric crohn's disease and multisystem inflammatory syndrome in children (mis-c) and covid-19 treated with infliximab date: 2020-05-21 journal: j pediatr gastroenterol nutr doi: 10.1097/mpg.0000000000002809 sha: doc_id: 309171 cord_uid: kgc7lgjp coronavirus disease 2019 (covid-19) may lead to a severe inflammatory response referred to as a cytokine storm. we describe a case of severe covid-19 infection in a recently diagnosed pediatric crohn's disease patient successfully treated with tumor necrosis factor-alpha (tnf-α) blockade. the patient presented with five days of fever, an erythematous maculopapular facial rash, and abdominal pain without respiratory symptoms. sars-cov-2 pcr was positive. despite inpatient treatment for covid-19 and a perianal abscess, the patient acutely decompensated, with worsening fever, tachycardia, fluid-refractory hypotension, elevation of liver enzymes, and transformation of the rash into purpura extending from the face to the trunk, upper and lower extremities, including the palmar and plantar surfaces of the hands and feet. cytokine profile revealed rising levels of interleukin (il)-6, il-8, and tnf-α, higher than those described in either inflammatory bowel disease (ibd) or severe covid-19 alone. the patient was treated with infliximab for tnf-α blockade to address both moderately to severely active crohn's disease and multisystem inflammatory syndrome in children (mis-c) temporally related to covid-19. within hours of infliximab treatment, fever, tachycardia and hypotension resolved. cytokine profile improved with normalization of tnf-α, a decrease in il-6, and il-8 concentrations. this case supports a role for blockade of tnf-α in the treatment of covid-19 inflammatory cascade. the role of anti-tnf agents in patients with mis-c temporally related to covid-19 requires further investigation. coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) virus, may lead to a severe inflammatory response or cytokine storm. 1 immune dysfunction in untreated crohn's disease may augment the risk for a severe inflammatory response with covid-19. 2 we describe a pediatric patient recently diagnosed with crohn's disease who developed severe covid-19 infection successfully treated with infliximab. a 14 year old male with recently diagnosed small bowel, perianal crohn's disease presented with 5 days of fevers and abdominal pain without respiratory symptoms. physical exam was notable for tachycardia, an erythematous maculopapular facial rash, abdominal tenderness and a perianal lesion with purulent drainage. initial laboratory tests revealed a creactive protein (crp) of 79.8 mg/l (normal 0-5 mg/l), an erythrocyte sedimentation rate (esr) of 64 mm/hr (normal 0-15 mm/hr) and hypoalbuminemia of 2.9 g/dl. sars-cov-2 pcr was positive. magnetic resonance (mr) enterography revealed 28 cm of ileitis, a 2.3 cm perianal abscess and fistula. chest x-ray was negative for an acute pulmonary process. blood culture and stool pcr were negative. treatment was initiated with intravenous (iv) piperacillin/tazobactam for his perianal abscess, hydroxychloroquine and azithromycin for sars-cov-2 infection, enoxaparin for prophylaxis of venous thromboembolism, and iv fluids. repeat concentrations of il-6, il-8, and tnf-α were rising (table 1) . multidisciplinary discussion of potential therapeutic options included remdesivir, an antiviral therapy, and tocilizumab, an il-6 inhibitor, for the treatment of covid-19, iv immune globulin for the treatment of an immune-mediated vasculitis, and infliximab, an anti-tnf-α therapy. infliximab would address the management of crohn's disease and potentially, the cytokine storm attributable to mis-c associated with covid-19. given the possible dual role and rapid clinical deterioration, he received 10 milligrams/kilogram (mg/kg) of infliximab on day cytokines improved with normalization of tnf-α, a decrease in il-6, and il-8. the progression of the rash was halted after infliximab treatment on hospitalization day 8 and significantly improved by discharge (figure 1 ). crp normalized. he was given a second infusion of infliximab10 mg/kg prior to discharge, 5 days after the initial dose due to a high inflammatory burden and presence of active perianal disease. prometheus infliximab concentration prior to the second infusion was > 34 ug/ml without antibodies. follow up two weeks after discharge revealed complete resolution of clinical symptoms and normalization of all labs previously elevated. this is the first reported case of a patient with recently diagnosed crohn's disease with suspected mis-c temporally related to covid-19 treated with infliximab to co-manage both entities. despite surgical management of his active perianal disease and antibiotic therapy, the patient continued to worsen clinically. his clinical deterioration was unclear, as both ibd and mis-c temporally related to covid-19 could be implicated, with all other infectious studies negative. pro-inflammatory cytokines, tnf-α, il-1β, and il-6 are overproduced in ibd patients. 3 elevated levels of tnf-α, mean of 62.3 pg/ml, have been described in the intestinal mucosa of ibd patients. 4 serum concentration of tnf-α and il-6 (median 10.5 pg/ml and 41.5 pg/ml respectively) are also associated with severe covid-19 illness. 5 at the peak of our patient's illness, serum concentrations of tnf-α (97.8 pg/ml) and il-6 (73.4 pg/ml) were higher than is reported in either ibd or severe covid-19. inflammatory disease overlap possibly contributed to cytokine storm and mis-c. cytokine storm has been found to be a major cause of morbidity in patients with severe covid-19 infection. 1, 5 treatment targeting these pro-inflammatory cytokines, such as il-6 inhibition with tocilizumab, have shown evidence of clinical benefit in subsets of severe covid-19 patients. 6 tnf-α is also implicated in severe covid-19 cases. blockade of tnf-α is effective in the treatment of auto-inflammatory conditions in which several cytokines are elevated, suggesting anti-tnf-α therapy alone may inhibit a cytokine cascade. 7 currently, therapy targeting tnf-a has not been associated with negative outcomes in ibd patients with covid-19. 8 this case supports a role for blockade of tnf-α in the treatment of covid-19 inflammatory cascade. whether this is specific for immune dysfunction in the setting of ibd or the non-ibd covid-19 patient requires further investigation. overall, increased tnf-α levels are seen in cytokine storm due to covid-19 and in active ibd. treatment with infliximab is known to be effective for the treatment of children with crohn's disease, and as evidenced by this case, was effectual in halting a systemic inflammatory response in our patient with covid-19. the role of anti-tnf agents in patients with mis-c temporally related to covid-19 requires further investigation. purpuric rash on the dorsum of the hand and antecubital-fossa before and after treatment with infliximab on hospitalization day 8. figure 1a and 1b represent the rash on the dorsum of the hand and antecubital fossa prior to infliximab treatment on hospitalization day 8. figure 1c and 1d demonstrate the improvement in his rash on day 13 prior to discharge. the pathogenesis and treatment of the 'cytokine storm' in covid-19 international organization for the study of inflammatory bowel disease. management of patients with crohn's disease and ulcerative colitis during the covid-19 pandemic: results of an international meeting chemokine and cytokine levels in inflammatory bowel disease patients increased production of tumour necrosis factor-alpha interleukin 1-beta, and interleukin 6 by morphologically normal intestinal biopsies from patients with crohn's disease clinical and immunological features of severe and moderate coronavirus disease 2019 tocilizumab, an anti-il6 receptor antibody, to treat covid-19 related respiratory failure: a case report trials of anti-tumour necrosis factor therapy for covid-19 are urgently needed but not tnf antagonists, are associated with adverse covid-19 outcomes in patients with inflammatory bowel diseases: results from an international registry key: cord-341667-ayl71jpc authors: van reeth, kristien; nauwynck, hans; pensaert, maurice title: bronchoalveolar interferon-α, tumor necrosis factor-α, interleukin-1, and inflammation during acute influenza in pigs: a possible model for humans? date: 1998-04-17 journal: j infect dis doi: 10.1086/517398 sha: doc_id: 341667 cord_uid: ayl71jpc biologically active interferon-α, tumor necrosis factor-α (tnf-α), and interleukin-1 (il-1) were detected in bronchoalveolar lavage (bal) fluids of 3-week-old cesarian-derived colostrum-deprived pigs inoculated with h1n1 influenza virus. cytokine titers and lung virus titers were significantly higher 18–24 h after inoculation than at 48–72 h after inoculation in all 4 litters of pigs examined. all three cytokines were positively correlated with a 3to 4-fold increase in bal cell numbers (p < .036) and with a drastic neutrophil infiltration (24%–77% of bal cells vs. 0–1.5% in controls) (p < .001). in addition, cytokine production coincided with the onset of general and respiratory symptoms of influenza and with the development of a necrotizing bronchopneumonia. this study is the first demonstration of tnf-α and il-1 in bal fluids of a natural influenza virus host. it documents that pigs may be a highly valuable experimental model in human influenza virus pneumonia. biologically active interferon-a, tumor necrosis factor-a (tnf-a), and interleukin-1 (il-1) were detected in bronchoalveolar lavage (bal) fluids of 3-week-old cesarian-derived colostrum-deprived pigs inoculated with h1n1 influenza virus. cytokine titers and lung virus titers were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation in all 4 litters of pigs examined. all three cytokines were positively correlated with a 3-to 4-fold increase in bal cell numbers (p õ .036) and with a drastic neutrophil infiltration (24% -77% of bal cells vs. 0 -1.5% in controls) (p õ .001). in addition, cytokine production coincided with the onset of general and respiratory symptoms of influenza and with the development of a necrotizing bronchopneumonia. this study is the first demonstration of tnf-a and il-1 in bal fluids of a natural influenza virus host. it documents that pigs may be a highly valuable experimental model in human influenza virus pneumonia. swine influenza (si) is a highly important respiratory disease onset of fever, anorexia, tachypnea, dyspnea, and coughing. of pigs. the causative viruses are type a influenza viruses of considerable economic losses result from growth retardation h1n1 and h3n2 subtype, which are antigenically related to or weight loss. experimental viral infections of pigs have docuhuman influenza viruses. typical outbreaks involve an abrupt mented massive viral replication in lung epithelial cells, accompanied by polymorphonuclear leukocyte infiltration and epithelial degeneration [1] . however, the exact mechanisms by which si virus produces lung pathology and disease have not been studied. phragmatic lung were collected for standard histopathology, influorexia, and weight loss, particularly if produced at higher levels enza virus titrations, and fluorescent antibody stainings [7] . the [5] . evidence for a role of tnf-a and il-1 in influenza virus right lung was lavaged with 60 ml of pbs. bronchoalveolar lavage pathogenesis and disease is growing [4, 6] . (bal) cells were separated by centrifugation at 400 g and counted, the pathogenesis of human influenza has been studied aland cytospin preparations were stained with diffquik (baxter, most exclusively in volunteers and in small laboratory animals. here we studied the production of ifn-a, tnf-a, and il-1 fluid samples in 96-well microtiter plates. ifn-a activity was dein the lungs of pigs and its relation with disease or inflammation termined in a cytopathic effect reduction assay by use of mdbk cells and vesicular stomatitis virus [8] . was defined as the reciprocal of the dilution producing 50% inhibition of cytopathic effect. ifn-a specificity was demonstrated by neutralization of samples with rabbit antiserum against recombinant porcine ifn-a (gift from c. la bonnardière, jouy en josas, france). tnf-a was assayed as cytotoxic activity in pk(15) sub-experimental design, virologic examinations, and lung inflamclone 15 cells (gift from g. bertoni, bern, switzerland) in the matory parameters. four litters (18 total) of 3-week-old cesarianpresence of actinomycin d [9] . the plates were stained with crystal derived colostrum-deprived (cdcd) pigs were used. they were violet and read spectrophotometrically. the number of units of inoculated intratracheally with 10 7.0 eid 50 of influenza virus tnf-a per milliliter was defined as the reciprocal of the dilution (h1n1 a/sw/belgium/1/83), third passage in embryonated eggs. producing 50% cytotoxicity. tnf-a specificity was established control pigs were left either uninoculated or inoculated with pbs by neutralization with rabbit anti-human tnf-a (innogenetics, or with sterile allantoic fluid ( in all groups. maximum il-1 titers were 535, 245, 520, and 150 u/ml in the 4 respective groups. only in group 1 pigs was il-1 also found at 72 h after inoculation. levels of the three cytokines were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation (p õ clinical responses, influenza virus titers, bal cell numbers, percentage of neutrophils, and cytokine titers of individual pigs .016 for all three cytokines). significant correlations were noted between production of all three cytokines and bal cell num-are summarized in table 1. clinical symptoms. control pigs remained healthy. in in-bers (p õ .036 for each) and percentage of neutrophils (p õ .001 for each). cytokine production coincided with the onset fluenza virus -inoculated pigs, lethargy, shivering, anorexia, tachypnea, and labored abdominal respiration developed be-of clinical disease and development of bronchopneumonia. tween 18 and 24 h after inoculation. recovery started between 48 and 72 h after inoculation. pigs in group 1 were most discussion severely affected. influenza virus replication. control pigs tested negative for at the start of this study, it was unknown whether the lungs of 3-week-old cdcd pigs are fully capable of tnf-a and virus. virus titers and immunofluorescence scores were similar in the 4 influenza virus -inoculated groups (p ú .577). titers il-1 production. thus far, tnf-a and il-1 have only been demonstrated in the lungs of pigs 6 -8 weeks old and older, in apical and diaphragmatic lung lobes were significantly higher 18 -24 h after inoculation than at 48 -72 h after inoculation (p either conventional [11] or gnotobiotic [12] . in our study, the use of cdcd pigs was required, since we regularly detect õ .016 and .008, respectively). fluorescence was evident in all sections examined. eighteen and 24 h after inoculation, tnf-a and/or il-1 in bal fluids from conventional pigs in the absence of experimental viral infections. the age of 3 weeks bronchi/bronchioli and alveoli had, respectively, 90% and 30% of their epithelial cells fluorescing. by 48 -72 h, more of the was selected for practical reasons. because sanitary status and age may influence cytokine production, we performed a prelim-alveolar tissue became involved. lung inflammatory changes. control pigs did not have inary experiment in 3-week-old cdcd pigs. two pigs were inoculated intratracheally with 17 mg of escherichia coli o111: macroscopic or microscopic lung pathology. bal cell numbers were between 35 and 60 1 10 6 . fewer than 1.5% of cells were b4 lipopolysaccharide, a known potent tnf-a and il-1 inducer. bal fluids collected 6 and 12 h after inoculation re-neutrophils; ú95% had macrophage morphology. after influenza virus inoculation, gross lung lesions appeared vealed tnf titers of 184 and 128 u/ml and il-1 titers of 564 and 596 u/ml in the respective bioassays. consequently, 3-between 48 and 72 h after inoculation. at that time, ç85%, 18%, 18%, and 8% of lung tissue was affected in groups 1, 2, week-old cdcd pigs were found suitable for further influenza virus -cytokine studies. 3, and 4, respectively. on histopathology, bronchi/bronchioli and, to a lesser degree, alveoli showed epithelial necrosis and to our knowledge, this is the first demonstration of influenza virus -induced tnf-a and il-1 in bal fluids of a natural virus massive neutrophil infiltration at 18 -24 h after inoculation. forty-eight and 72 h after inoculation, bronchioli and alveoli host. interestingly, influenza virus was an equally effective inducer of tnf-a and il-1 as was e. coli endotoxin. tnf-a were filled with exudate containing necrotic debris and macrophages and only few neutrophils. histologic changes were most and il-1 have remarkably overlapping and synergistic effects, several of which are consistent with clinicopathologic manifes-dramatic in group 1. bal cell numbers were between 112 and 160 1 10 6 at 18 -24 h after inoculation and consisted of a tations of influenza [5] . our findings in pigs are in agreement with previous reports in mice [3, 4] and further substantiate the maximum of 56% -77% neutrophils in groups 1, 2, and 3. in / 9d43$$ap48 03-04-98 19:03:33 jinfa uc: j infect group 1 pigs, which had consistently higher values of tnf than the other groups, showed most severe disease and lesions effect of intratracheal challenge of fattening pigs previously immunised with an inactivated influenza h1n1 vaccine group 4 pigs, with barely detectable tnf levels, also had lower interferon production by leukocytes interferon-a was detected at extremely high titers in some infiltrating the lungs of mice during primary influenza virus infection. pigs. as for tnf-a and il-1, ifn-a production was tightly production of interleukin 1 and tumour necrosis factor activities in and peak ifn production coincided with the appearance of bronchoalveolar washings following infection of mice by influenza viillness. these findings support the idea that interferons contribrus peper rl, van campen h. tumor necrosis factor as a mediator of inflam-ifn-a is intrinsically pyrogenic [13] and can mediate neutromation in influenza a viral pneumonia tumour necrosis factor and interleukin 1: cytokines with vitro that ifn-a may enhance the neutrophil respiratory burst multiple overlapping biological activities kluger a in pig bal fluids, ifn could considerably add to the pyro-mj. thermal and behavioral effects of lipopolysaccharide and influenza genic and inflammatory effects of tnf-a and il-1. in interleukin-1b -deficient mice porcine respiratory coronavirus -mediated interference against influenza virus replication in the respiratory tract the other groups. most striking was the virtual lack of tnf of feeder pigs high interferon titer in newborn pig intestine and previous groups cannot be attributed to differences in viral during experimentally induced viral enteritis improved bioassay for the belonged to a genetically different line. it would be worthwhile detection of porcine tumor necrosis factor using a homologous cell line: pk(15) mur-although influenza viruses in humans primarily infect the taugh mp. inflammatory cytokine expression in swine experimentally upper respiratory tract, influenza pneumonia yearly causes high infected with actinobacillus pleuropneumoniae increased levels of tumor necrosis factor elderly. experimental research thus remains of high priority, and interleukin 1 in bronchoalveolar lavage fluids from pigs infected and commonly used animal models have some limitations ferrets, for example, upper respiratory tract infection predomi tumor necrosis factor (cachectin) is an endogenous pyrogen and induces production of innounced [15]. mice and guinea pigs, on the other hand, are terleukin 1 interferon-a enhances neutrophil respito them. also, mice show a fall in body temperature instead of ratory burst responses to stimulation with influenza a virus and fmlp. fever and, with more virulent strains, the infection is invariably lessons for human influenza from pathogenicity studies with ferrets hypothesis that tnf-a and il-1 contribute to clinicopathologic effects of influenza. first of all, both cytokines were positively we thank r. ducatelle for help with histology and d. j. shaw correlated (r ú .879) with neutrophil recruitment to the lungs, for statistics. and peak cytokine production coincided with the onset of clinical disease. furthermore, there was a clear association between individual tnf levels on the one hand and the extent of neutrophil infiltration and severity of lung pathology on the other. key: cord-335185-3qi29i6n authors: hendry, bruce m.; stafford, nina; arnold, ahran d.; sangwaiya, arvind; manglam, vijay; rosen, stuart d.; arnold, jayantha title: hypothesis: pentoxifylline is a potential cytokine modulator therapeutic in covid‐19 patients date: 2020-07-26 journal: pharmacol res perspect doi: 10.1002/prp2.631 sha: doc_id: 335185 cord_uid: 3qi29i6n we propose a new hypothesis that the established drug pentoxifylline deserves attention as a potential repurposed therapeutic for covid‐19. pentoxifylline is an immunomodulator with anti‐inflammatory properties. it is a nonselective phosphodiesterase inhibitor and through adenosine a2a receptor‐mediated pathways reduces tumor necrosis factor alpha, interleukin 1, interleukin 6, and interferon gamma and may act to reduce tissue damage during the cytokine storm host response to sars‐cov‐2 infection. this agent has been used clinically for many years and has a favorable profile of safety and tolerability. pre‐clinical data support pentoxifylline as effective in cytokine‐driven lung damage. clinical studies of pentoxifylline in radiation and cytokine‐induced lung damage in humans are positive and consistent with anti‐inflammatory efficacy. pentoxifylline is a readily available, off‐patent and inexpensive drug, suitable for large‐scale use including in resource‐limited countries. current trials of therapeutics are largely focused on the inhibition of viral processes. we advocate urgent randomized trials of pentoxifylline for covid‐19 as a complementary approach to target the host responses. covid-19 is a disease caused by the sars-cov-2 virus, characterized by an early mild to moderate viral syndrome of fever, tiredness, cough, and headache. 1 over 80% of patients have a self-limiting illness not requiring hospital admission and show clear improvement in two weeks. a minority of covid-19 patients progress through a transition phase around days 7-11 of worsening pulmonary complications. 1 these manifest as breathlessness, acute lung injury and respiratory failure, and often progress to require mechanical ventilation with subsequent high mortality. this deterioration appears to be driven by lung host responses including a cytokine storm of inflammation leading to severe tissue damage and irreversible organ failure likened to acute respiratory distress syndrome (ards). 2 patients who develop ards are at very high risk of death. the cytokine storm phase of covid-19 is associated with increased production of a range of inflammatory cytokines including interleukin-1 gamma (il gamma), interleukin-6 (il6), tumor necrosis factor alpha (tnf alpha) and interferon gamma (ifn gamma). 3 several case series have reported increased tnf alpha levels in patients with covid-19 and particularly high levels appear to be associated with a severe disease course. 3, 4 one series has described increased tnf alpha inducibility in macrophages, in the presence of sars-cov-2 virus. 4 tnf alpha, as the master regulator of cytokines, is considered key in both immune pneumonitis and acute myocardial injury witnessed in covid-19. current covid-19 therapeutic studies are mainly focused on agents designed to target viral processes or virus-host interactions, for example with remdesivir. we note that remdesivir has recently been approved by the uk government, after "numerical reduction in time to clinical improvement," but failing to meet statistical significance on the primary clinical endpoint of mortality. 5 an alternative therapeutic approach is to target the host responses that underlie the cytokine storm and associated inflammation. 3 we, and other colleagues, have called for randomized clinical trials of anti-tnf agents, such as infliximab, to treat the cytokine storm induced by sars-cov-2. 6,7 we hypothesize that pentoxifylline is an inexpensive anti-tnf immuno-modulator that will be effective in countering the cytokine storm in covid-19. pentoxifylline is a cytokine-modulating anti-inflammatory agent with many actions that might reasonably be expected to be therapeutic in the transition phase of covid-19. we first described the potential benefits of pentoxifylline as an inexpensive anti-tnf for covid-19 in april 2020. 8 pentoxifylline is a xanthine derivative drug with a wide range of actions at the cellular and molecular level. pentoxifylline has rheological actions increasing erythrocyte deformability and was originally licensed for the treatment of peripheral vascular disease on the basis of suggested improvement of microvascular and capillary blood flow. 9 more recently pentoxifylline has been determined to have extensive anti-inflammatory properties. 10 pentoxifylline inhibits 5′-nucleotidase and phosphodiesterase (pde). pde inhibition results in increased camp levels, increased protein kinase a (pka) activity and altered transcriptional regulation of pro-inflammatory genes through modulation of the nfkb/ikb pathway. 10 pentoxifylline also has beneficial therapeutic properties mediated by adenosine a2a receptor pathways in immune cells and in particular lung macrophages. pentoxifylline downregulates transcription and expression levels of tnf alpha, il1b, il6, ifn gamma, icam1, and vcam1. the 5′-nucleotidase inhibition effect of pentoxifylline reduces the production of adenosine and inosine from adenosine monophosphate (amp) and inosine monophosphate (imp), respectively. pentoxifylline appears to be able to downregulate the pathologically important pro-inflammatory adenosine receptor a2a pathway (a2ar). 11 these effects contribute to the extensive actions of pentoxifylline in reducing pro-inflammatory signals. for example, pentoxifylline reduces cytokine release from pulmonary macrophages derived from patients with sarcoidosis. 12 the lungs have the highest proportion of total resident macrophages in the human body at around 1 trillion. in rats pentoxifylline downregulates a range of inflammatory cytokines in the context of sepsis and improves lung function. 12 in animal studies pentoxifylline is effective as a therapeutic in a range of models of lung injury including radiation-induced damage, cytotoxic agent damage and aortic clamping. 13 the actions of pentoxifylline in reducing lung damage appear to be mediated by its effect on the adenosine receptor a2ar pathways. 11 the direct inhibition of 5′-nucleotidase activity by pentoxifylline likely reduces adenosine production and may therefore complement the actions on cytokine gene transcription. pentoxifylline improves glomerular damage and reduces tnf alpha in crescentic glomerulonephritis in rats. 14 pentoxifylline does not appear to alter the replication of sars-cov in mice and does not show direct antiviral activity. the anti-inflammatory properties of pentoxifylline have been confirmed in a range of clinical studies in diverse organ failure syndromes. in severe renal disease patients exhibit resistance to erythropoietin through a pro-inflammatory state characterized by increased tnf alpha, ifn gamma and il6. pentoxifylline improves erythropoietin sensitivity in these patients and this action is associated with reductions in serum tnf alpha and ifn gamma. 14 one of the largest scale clinical trials of pentoxifylline anti-inflammatory effects within the last decade, was the stopah trial. 17 examining potential benefit in acute alcoholic hepatitis, no survival benefit was demonstrated, however, no safety issues were reported in an inherently vulnerable, immunocompromised patient cohort. pentoxifylline's preferential inhibition of macrophage function in alveoli, as opposed to hepatocytes, potentially explains this outcome. 18 rainsford (2006) reviewed possible treatments for the lung complications associated with inflammatory cytokines in h5n1 "bird flu" and suggested that pentoxifylline should be considered for clinical trials in view of its pharmacology and safety profile. 19 these arguments appear equally suited to the case of covid19 . although we cannot describe the sars-cov-2 pulmonary syndrome as identical to that seen in h5n1, there are certainly parallels. tnf alpha has a pivotal role in orchestrating the production of a pro-inflammatory cytokine cascade. tnf alpha is thus considered to be a "master regulator" of pro-inflammatory cytokine production. 20 post-mortem lung biopsies in covid-19 showed interstitial edema 21 which would normally be the result of tnf induced increased capillary permeability. 22 this non cardiogenic pulmonary edema (both interstitial and intra-alveolar) is often the first stage of covid-19 acute lung injury that progresses through the cytokine storm to ards. 21 the recent description of thrombosis and endothelialitis in covid-19 23 raises the possibility that the rheological actions of pentoxifylline could be of benefit in maintaining microvascular function. pentoxifylline has over 50 years safety record data of use in humans and has an extensive evidence base for tolerability and safety. nevertheless, its safety in the context of covid-19 has not been established and this would need close monitoring in a clinical trial setting. it is available in oral form with good bioavailability, and also can be delivered by intravenous injection. the usual dose orally is 1.2 g daily in three divided doses. an inhalational formulation has been developed, originally for use in neonates. in covid-19 the cytokine storm appears to be strongly centered in lung tissue, and accordingly inhaled pentoxifylline could be an optimum method for delivery at the highest concentrations where it is most effective, with minimal systemic exposure. use of a repurposed drug for covid-19 may have multiple advantages in addition to its safety and tolerability experience. it is widely available as a generic agent, with multiple sources of supply and therefore manageable cost. there should be no patent protection issues in redirecting the agent to trials in covid-19. this should be of acute interest as limited-resource countries have now begun to amass cases of covid-19. a study of pentoxifylline in covid-19 should be feasible and ethical given the well-described adverse event profile of the drug. a randomized study in covid-19 patients presenting with, or at high risk of, pulmonary complications could be designed with pentoxifylline versus a placebo or comparator treatment, alongside standard care. pentoxifylline has been used at 400-1200 mg daily in 400 mg doses. the initial study likely should use 1200 mg daily in divided doses. if there is initial evidence of benefit with oral pentoxifylline, then study of inhaled pentoxifylline could be valuable in selected patients. in the future it will also be logical to study the combination of cytokinemodifying therapy with direct anti-viral therapeutics (such as the recently favored remdesivir). the efficacy of pentoxifylline can be assessed by randomized controlled trials with key endpoints including mortality, need for ventilatory support, time on ventilatory support, measures of oxygen exchange efficiency and time in hospital. the beneficial effects of pentoxifylline on lung injury are mediated by g protein coupled adenosine a2 receptors. 26 adenosine has multiple physiological effects through membrane-bound receptors linked to g proteins. there are four subtypes of adenosine receptors: a 1 ar, a 2a ar, a 2b ar, and a 3 ar. pentoxifylline has an antagonist effect on adenosine binding with its receptor (a2aar) and functions as an anti-inflammatory agent. 27 a1ar is predominantly found in the central nervous system and kidneys and fewer receptors are found in lungs. pentoxifylline has not been shown to have any effect on a1ar. 26 a2a adenosine receptors are mainly found in lungs and immune cells and the pentoxifylline beneficial effect is mediated by the a2aar pathway. 27 a2bar has an antagonistic effect when stimulated. however, the affinity is low and there is no evidence to support the binding of pentoxifylline to a2bar. a3ar is also unaffected by pentoxifylline. 27 activation of nlrp3 inflammasome protein by sars-cov-2 viral infection in the early stages has been put forward as a hypothesis by ratajczak and lucia. 28 the predominant effect of pentoxifylline via a2ar pathways on both lung macrophages and immune cells is anti-inflammatory, with reduced tnf alpha, interleukin-12, interleukin-6, interleukin-8 levels and increased interleukin-10 levels. the balance between the anti-cytokine a2ar mediated action of pentoxifylline and any theoretical gradient dependent passage of adenosine back into the cell to re-activate nlrp3 inflammasome protein appears to clearly favor the a2ar effect of pentoxifylline in terms of cytokine outcomes. although pentoxifylline has rheological effects which is the main indication for its long-standing use in peripheral vascular disease, no clinical benefit has been reported from controlled trials in coronary artery disease or cerebrovascular disease. although pulmonary microcirculatory abnormalities were found on post-mortem studies in covid-19, 21,23 pentoxifylline is unlikely to confer any therapeutic benefits in the pulmonary circulation because its rheological benefit has only been shown to be of clinical benefit in peripheral limb vessels. in a significant minority of covid-19 patients, coagulopathy has been found in severe covid-19 with detection of raised d-dimers. 29 however, no clinical trial has shown any benefit with anti-platelet therapy and the minor in-vitro platelet inhibition associated with pentoxifylline may not confer any therapeutic benefit related to this, in covid19. 29 if the theoretical therapeutic benefit of pentoxifylline for covid-19 based on its anti-tumor necrosis factor effect is clinically demonstrable, it may prove to be an inexpensive, and readily available, treatment strategy to target harmful cytokine excess in this disease. we advocate urgent randomized trials of pentoxifylline for patients infected with sars cov-2. bmh has received honoraria for speaking or consulting from astrazeneca, gilead sciences, otsuka, retrophin, sanofi, ucb and viiv healthcare. we would like to thank ramya arnold for her assistance with proofreading and typing up the manuscript. none of the authors have any relevant actual or potential conflicts of interest. all authors contributed equally. https://orcid.org/0000-0002-1263-6300 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan the pathogenesis and treatment of the 'cytokine storm' in covid-19 clinical features of patients infected with 2019 novel coronavirus in wuhan complex immune dysregulation in covid-19 patients with 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lung injury, oxidative stress and inflammation pentoxifylline suppresses renal tumour necrosis factor-alpha and ameliorates experimental crescentic glomerulonephritis in rats pentoxifylline in prevention of radiation-induced lung toxicity in patients with breast and lung cancer: a double-blind randomized trial the inhibition of pro-inflammatory cytokines with pentoxifylline in the cardiopulmunary bypass lung prednisolone or pentoxifylline for alcoholic hepatitis pentoxifylline inhibits tnf-alpha production from human alveolar macrophages bird flu"), inflammation and anti-inflammatory/analgesic drugs necrosis factor-α signaling in macrophages pulmonary and cardiac pathology in african american patients with covid-19: an autopsy series from new orleans influenza leaves a trail to pulmonary oedema pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 repositioning of pentoxifylline as an immunomodulator and regulator of the renin-angiotensin system in the treatment of covid-19 inhibitors of the reninangiotensin-aldosterone system and covid-19 immunomodulatory properties of pentoxifylline are mediated via adenosine-dependent pathways focusing on adenosine receptors as a potential targeted therapy in human diseases sars-cov-2 infection and overactivation of nlrp3 inflammasome as a trigger of cytokine "storm" and risk factor for damage of hematopoietic stem cells hypercoagulation and antithrombotic treatment in coronavirus 2019: a new challenge key: cord-302258-derq9b27 authors: zhang, hui; yu, jingbin; sun, hu; zhao, yunhe; wang, jitao; zhang, juan; meng, bin title: effects of ubiquitin-proteasome inhibitor on the expression levels of tnf-α and tgf-β1 in mice with viral myocarditis date: 2019-08-14 journal: exp ther med doi: 10.3892/etm.2019.7895 sha: doc_id: 302258 cord_uid: derq9b27 effects of ubiquitin-proteasome system (ups) inhibitor mg-132 on the expression levels of tumor necrosis factor-α (tnf-α) and transforming growth factor-β1 (tgf-β1) in mice with viral myocarditis were investigated to analyze the correlation of myocardial tissue score of mice between tnf-α and tgf-β1. eighty healthy male spf mice aged 6 weeks were selected and 20 mice were randomly selected as the blank group. the blank group did not receive any intervention. mortality rates of each group were recorded and compared on day 8 of modeling, and heart specimens from the remaining mice were histopathologically examined and the expression of mrna and protein of tnf-α and tgf-β1 in myocardial tissues were detected by western blot analysis. correlation between mouse myocardial histopathologic scores and expression of protein of tnf-α and tgf-β1 in myocardial tissues, as well as the expression of tnf-α and tgf-β1 in myocardial tissue in vmc mice was analyzed. the expression levels of myocardial histopathological scores, mrna and protein of tnf-α and tgf-β1 in the blank and control group were significantly lower than those in the vmc and the mg-132 group. the myocardial histopathological scores, mrna and tnf-α and tgf-β1 protein in the mg-132 group were significantly lower than those in the vmc group (p<0.05). the expression of tnf-α and tgf-β1 protein in myocardial tissues was positively correlated with the pathological score in myocardial tissue of mice (r=0.843, p<0.05; r=0.763, p<0.05), and there was a positive correlation between the expression of tnf-α and tgf-β1 protein in myocardial tissues of vmc mice (r=0.672, p<0.05). ups inhibitor mg-132, which can significantly alleviate the myocardial injury of vmc mice, reduced the expression of inflammatory factors in myocardial tissues, and improved the survival rate of mice, thus it is a potential new treatment for vmc. viral myocarditis (vmc), as one of the most common cardiac infectious diseases, is mainly caused by myocardial cells infected with the virus, causing localized or diffuse inflammation of the myocardium. the virus causes direct damage to the myocardial cells and stimulates the immune response, thus causing sustained damage to the myocardial tissues (1, 2) . coxsackie b3 virus (cvb3) is the most important virus that causes myocarditis. cvb3 can induce oxidative stress response and apoptosis in a few weeks in the pathogenesis of vmc, resulting in arrhythmia, myocardial failure and may eventually lead to sudden death, but special treatment of vmc has not been found as yet (3, 4) . ubiquitin-proteasome system (ups), as an important atp-dependent protein control system in eukaryotic cells, not only participates in physiological processes such as apoptosis, inflammatory response and intracellular signaling, but also plays an important role in maintaining cell homeostasis (5) (6) (7) . in recent years, studies have found that ups not only plays an important role in the inflammatory response of various diseases, but also plays a key role in the occurrence and development of various viral infectious diseases (8, 9) . as an aldehyde peptide proteasome inhibitor that can inhibit the activity of chyme protein, mg-132 also has a protective effect on the occurrence of inflammatory reactions in many diseases (10) . as a cytokine with a variety of biological effects produced by macrophages, tumor necrosis factor-α (tnf-α) has been shown to play an important role in the inflammatory response of vmc (11) . however, transforming growth factor-β1 (tgf-β1) is an initiating factor synthesized from the extracellular matrix of collagen fibers, and it is also one of the many factors leading to the occurrence of viral myocarditis (11) . at present, few studies have reported the role of ups in the inflammatory reaction of vmc, so we explored the effect of ubiquitin-proteasome inhibitor mg-23 on the expression levels of serum tnf-α and tgf-β1 in cvb mice, in order to understand the inflammatory mechanism of vmc. experimental animals and materials. a total of 80 healthy male spf grade mice aged 6 weeks and weighing 25.1±5.53 g were selected. all the mice were purchased from the animal experiment center of zhejiang province, with the production license of scxk (zhejiang) 2011-0166. the mice were fed in a plastic box with bedding material on a solid bottom with the temperature of 22˚c and the relative humidity between 50 and 65%, 12 h daynight rhythm was normal, and they were free to eat and drink. the study was approved by the ethics committee of central hospital of zibo (zibo, china). the cvb3 virus was purchased from cell signaling inc. at a titer of 100 tcid50 (50% tissue culture infective dose) /0.1 ml. mg-132 was purchased from calbiochem inc. at a concentration of 0.75 mg/kg. grouping and modeling. twenty mice were randomly selected as the blank group, and they were kept in routine feeding without any intervention. the remaining 60 mice were then randomly divided into the vmc, mg-132 and control group, each containing 20 mice. mice in the control group were intraperitoneally injected with 0.1 ml pbs (phosphate buffersaline) at 0.1 mmol/l, mice in the vmc and mg-132 group were intraperitoneally injected with 0.1 ml diluent of cvb3 at 100 tcid50/0.1 ml, and mice in the mg-132 group were intraperitoneally injected with 0.75 mg/kg mg-132 the day after the injection of the cvb3 virus. they were continuously injected for 7 days. mortality rates of each group were recorded and compared on day 8 of modeling, and then peripheral blood and heart samples of the remaining mice were taken for subsequent detection. pathological examination. hearts of mice were fixed with 10% formaldehyde and dehydrated routinely, then embedded with paraffin and sectioned. after sectioning, the pathological changes of mouse myocardial tissues were observed under light microscope (olympus corp.) and the pathological score of the myocardial tissues was evaluated. the judgement scores were as follows: 1 point, when the proportion of inflammatory cell infiltration and myocardial necrosis was <25%, 2 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was between 25 and 50%, 3 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was between 51 and 75%, 4 points, when the proportion of inflammatory cell infiltration and myocardial necrosis was >75%. expression levels of mrna in tnf-α and tgf-β1 detected by rt-qpcr. first, the trizol reagent (purchased from applide invitrogen, inc.) was added into the myocardial tissue of mice to extract the total rna in the blood, and the concentration and quality of total rna were detected by ultraviolet spectrophotometer (purchased from shanghai yuanxi instrument co., ltd.). total rna (2 µl) was used to compound cdna in strict accordance with the instructions of minscript reverse transcription kit (takara bio, inc.). synthetic cdna (2 µl) was used for qpcr (rt-qpcr kit was purchased from takara). total rna (2 μl) was added to a microcentrifuge tube, then 11 μl of depc h 2 o was added. a total of 12-18 μl of 10 μm oligo (dt) was added, mixed and heated at 70˚c for 10 min. after ice bath for 1 min, 2 μl 10x pcr buffer, 2 μl 25 mm mgcl 2 , 1 μl 10 mm dntp mix and 2 μl 0.1 m dtt were added, mixed and incubated for 3 min at 42˚c. a total of 1 μl of superscript ii was added, incubated at 42˚c for 30 min, and heated at 70˚c for 10 min. ice bath was followed for 5 min. the qpcr reaction system was: 2 µl reverse transcription product, 0.5 µl upstream primer, 0.5 µl downstream primer, 2x sybr green pcr master mix 10 µl, and sterilized and deionized water complemented to 20 µl. reaction conditions were: 95˚c for 2 min, 95˚c for 50 sec, 60˚c for 45 sec, extension at 72˚c for 30 sec, and for a total of 40 cycles. the expression levels of tnf-α mrna and tgf-β1 mrna were detected with glyceraldehyde-3-phosphate dehydrogenase (gapdh) as the internal reference. the primer sequences are shown in table i (the primers were synthesized and designed by shanghai gemma company), and the experiment was repeated 3 times. the result was analyzed with 2 δδ-cq method (12) . cardiac muscle tissues (100 g) of mice were taken and ripa was added lysate to extract the total protein. then the extracted protein was boiled at 100˚c for 5 min to denaturate, then separated with 10% sds-page after being cooled, transferred to the pvdf membrane, and sealed with 5% of skim milk at room temperature for 1 h. then the primary rabbit anti-mice polyclonal antibodies of tnf-α (1:500), tgf-β1 (1:500) and β-actin (1:1,000) (cat. nos. 17590-1-ap, 21898-1-ap, 14395-1-ap; proteintech group, inc.) were added at 4˚c for incubation overnight, then the secondary goat anti-rabbit polyclonal antibody (dil, 1:500; cat. no. sa00001-2; proteintech group, inc.) at room temperature for 1 h incubation, finally ecl developer was used to develop color. observation indicators. i) eight-day survival rates of mice in each group were recorded and compared. ii) the pathological score in myocardial tissues of mice in each group was table i . sequences of the primers. upstream primer downstream primer tnf-α, tumor necrosis factor-α; tgf-β1, transforming growth factor-β1; gapdh, glyceraldehyde-3-phosphate dehydrogenase. evaluated and compared. iii) the expression levels of tnf-α mrna and tgf-β1 mrna in myocardial tissues of mice in each group were compared. iv) tnf-α protein and tgf-β1 protein in myocardial tissues of mice in each group were compared. v) correlation analysis was performed between the pathological score and the protein expression levels of tnf-α and tgf-β1 in myocardial tissues of mice, and the protein expression levels of tnf-α and tgf-β1 in myocardial tissues. statistical analysis. spss 20.0 (ibm corp., armonk, ny, usa) was used for statistical analysis of the experimental data. the chi-square test was used to compare the enumeration data. kaplan-meier curve was used for survival analysis. measurement data were expressed as mean ± standard deviation. t-test was used for comparison between the two groups and univariate analysis of variance was used for multigroup comparison. lsd/t-test was used for postoperative comparison. correlation was analyzed by pearson's correlation analysis. graphpad prism 6 software (hangzhou aimeilv biotechnology co., ltd.) was used to draw the experimental images. p<0.05 was considered to indicate a statistically significant difference. comparison of survival rates of mice in each group. the survival rate of the blank group and the control group at day 8 was 100%. a total of 11 mice in the vmc group died at day 8, with a survival rate of 45%. a total of 5 mice in the mg-132 group died at day 8, with a survival rate of 75%. the 8-day survival rates of the blank group and control group were significantly higher than those of the vmc group and mg-132 group, but the 8-day survival rate of mice in the mg-132 group was significantly higher than that of the vmc group (p<0.05) (fig. 1) . histopathological scores of myocardial tissues of mice in each group. there was no inflammatory cell infiltration in myocardial tissues of mice in blank or control group. there was a large amount of inflammatory cell infiltration in myocardial tissues of mice in vmc group. the inflammatory cells in the myocardial tissues of mice in mg-132 group were significantly reduced compared with those in vmc group. the myocardial histopathological scores of mice in blank and the control group were respectively 0.33±0.04 and 0.34±0.06, the myocardial histopathological score of mice in the vmc group was 1.85±0.11, and was 1.04±0.10 in the mg-132 group. the myocardial histopathological scores of mice in the blank and the control group were significantly lower than those in vmc and mg-132 group, and myocardial histopathological score of mice in mg-132 was significantly lower than that in vmc group (p<0.05) (fig. 2) . expression levels of mrna of tnf-α and tgf-β1 in myocardial tissues of mice in each group. there was no significant figure 1 . comparison of survival rates of mice in each group. the survival rate of the blank group and the control group was 100%. the 8-day survival rates of the blank and control group were significantly higher than those of the vmc and mg-132 group, but the 8-day survival rate of mg-132 group was significantly higher than that of the vmc group, and the differences were statistically significant (p<0.05). vmc, viral myocarditis. difference in expression levels of tnf-α mrna and tgf-β1 mrna between the blank group and the control group (p>0.05), but were significantly lower than those in the vmc group and the mg-132 group. however, expression levels of tnf-α mrna and tgf-β1 mrna in myocardial tissues of mg-132 group were significantly lower than those of the vmc group (p<0.05) (table ii) . there was no significant difference in the expression levels of tnf-α protein and tgf-β1 protein between blank and control group (p>0.05), but both were significantly lower than those of vmc group and the mg-132 group. however, expression levels of tnf-α and tgf-β1 protein in myocardial tissues of mice in the mg-132 group were significantly lower than those in the vmc group (p<0.05) (table iii) . tissues of vmc mice. the expression levels of tnf-α protein and tgf-β1 protein in myocardial tissues were positively correlated with the pathological score of myocardial tissues (r= 0.843, p<0.05; r= 0.763, p<0.05), and there was a positive correlation between expression levels of tnf-α protein and tgf-β1 protein of vmc mice in myocardial tissues (r=0.672, p<0.05) (figs. 3-5 ). as a viral infectious disease, vmc currently has no specific effective treatment in clinical practice, thus, vmc is one of the most challenging diseases in the diagnosis and treatment of cardiovascular field at present (13) . besides, there are also great controversies regarding the pathogenesis of vmc, most of which support cytokine theory and immune theory (14) . as an important protein quality control system in eukaryotic cells, ups has been found to play a very important role in various viral infectious diseases in recent years. specifically, through ups, viruses can replicate and lead to oxidative stress in host cells and eventually cause cell damage (15) (16) (17) . for example, studies (18) have found that the normal ups pathway is involved in the replication of vaccinia virus. however, there are few studies on ups in vmc, and no detailed description of its mechanism has been made. in our study, the effects of ups inhibitor mg-132 intervention on myocardial cells, tnf-α and tgf-β1 in vmc mice were investigated. the results showed that the mortality of the vmc group was significantly higher than that of the blank group, control group and mg-132 group (p<0.05), suggesting vmc mice had higher mortality rates, but the mortality of vmc mice can be significantly reduced after the intervention of ups inhibitor mg-132. in addition, the expression levels of myocardial histopathological scores, mrna and protein of tnf-α and tgf-β1 in the blank group and the control group were significantly lower than those in the vmc group and the mg-132 group, and the myocardial histopathological scores, mrna and protein of tnf-α and tgf-β1 in the mg-132 group were significantly lower than those in the vmc group (p<0.05). this suggests that the intervention of ups inhibitor mg-132 can effectively improve the inflammatory infiltration in myocardial tissues of vmc mice and reduce the expression levels of inflammatory factors in myocardial tissues. ups plays an important role in inflammatory response. for example, a previous study (19) found that ups was involved in the response injury of hepatitis b coronavirus to a certain extent and could effectively alleviate the occurrence of inflammatory response. other studies (20) have shown that ups inhibitor mg-132 can inhibit the akt and erk pathways, thereby alleviating the inflammatory response and inhibiting the progression of heart failure. although these studies did not directly confirm our conclusions, they can also show that the intervention of ups inhibitor mg-132 does have a certain alleviating effect on the inflammatory response. therefore, previous studies (21) suggested that inhibition of the process of heart failure by ups inhibitors may be achieved by reducing oxidative stress. however, some studies (22) have obtained different results when different ups inhibitors were applied in the intervention of ischemic cardiomyopathy, and the reason is not clear at present. we speculate that it may be because ups inhibitors are involved in different molecular reactions, so they have different effects on inflammatory reactions or oxidative stress reactions. finally, we analyzed the correlation between the pathological score in myocardial tissues of mice and the protein expression levels of tnf-α and tgf-β1 in myocardial tissues, as well as the protein expression levels between tnf-α and tgf-β1 in myocardial tissues of vmc mice. the results showed that the expression levels of tnf-α and tgf-β1 proteins in myocardial tissues were positively correlated with the pathological score in myocardial tissues of mice, and the expression levels of tnf-α protein and tgf-β1 protein in myocardial tissues of vmc mice were also positively correlated. previous studies (23) have shown that the regulation of tgf-β can effectively stimulate the release of tnf-α in human monocytes. although it is not a study of the myocardial tissues, it also confirms our conclusion. in recent years, the role of ups in cardiovascular diseases has been gradually recognized and discovered (24) . after the establishment of vmc mouse model, we also found that the ups inhibitor mg-132 can significantly alleviate the myocardial injury of vmc mice, reduce the expression levels of inflammatory factors in myocardial tissues, and improve the survival rate of mice. ups inhibitor mg-132 may be a new treatment scheme for vmc. however, our study also has some shortcomings. we did not further explore the mechanism of action between tnf-α and tgf-β1, or describe in detail how mg-132 reduced the inflammatory response in vmc mice. this will be further explored in our subsequent experiments. hydrogen sulfide in physiology and pathogenesis of bacteria and viruses effects and related mechanism of overexpression of human thioredoxin on the inflammatory response in mice with viral myocarditis endoplasmic reticulum stress interacts with inflammation in human diseases the role of selenium in inflammation and immunity: from molecular mechanisms to therapeutic opportunities targeting the ubiquitin proteasome system in haematological malignancies the small molecules targeting ubiquitin-proteasome system for cancer therapy ubiquitin-proteasome system and hereditary cardiomyopathies nitric oxide synthases, s-nitrosylation and cardiovascular health: from molecular mechanisms to therapeutic opportunities (review) understanding selenoprotein function and regulation through the use of rodent models 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liver carcinogenesis proteasome inhibitors attenuated cholesterol-induced cardiac hypertrophy in h9c2 cells foxo proteins: cunning concepts and considerations for the cardiovascular system small molecule modulators of keap1-nrf2-are pathway as potential preventive and therapeutic agents regulation of bim in health and disease this work is licensed under a creative commons attribution-noncommercial not applicable. no funding was received. the datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request. hz, jy and hs performed rt-qpcr. yz and jw were responsible for western blot analysis. jz and bm contributed to analysis of the observation indexes. hz wrote the manuscript. all authors read and approved the final manuscript. the study was approved by the ethics committee of central hospital of zibo (zibo, china). not applicable. the authors declare that they have no competing interests. key: cord-313227-6zwkfzab authors: scala, stefania; pacelli, roberto title: fighting the host reaction to sars-cov-2 in critically ill patients: the possible contribution of off-label drugs date: 2020-05-27 journal: front immunol doi: 10.3389/fimmu.2020.01201 sha: doc_id: 313227 cord_uid: 6zwkfzab the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the etiologic agent of the 2019 coronavirus disease (covid19). the majority of infected people presents flu like symptoms and among them 15–20% develops a severe interstitial pneumonitis (ip) that may eventually evolve in acute respiratory distress syndrome (ards). ip is caused by the viral glycoprotein spike (s) binding to the angiotensin converting enzyme 2 (ace2) expressed on the surface of alveolar pneumocytes. the virus is recognized by the “pattern recognition receptors” (prr) of the immune cells that release cytokines activating more immune cells that produce a large number of pro-inflammatory cytokines, tissue factors and vasoactive peptides. affected patients might develop the “cytokine storm syndrome,” a fulminant and fatal hypercytokinaemia with multiorgan failure. in patients infected by sars-cov-2 increase in t-helper 2 (th2) cytokines (il-4 and il10) are reported in addition to the t-helper 1 (th1) cytokines (il1b, ifnγ, ip10, and mcp1) previously detected in other coronavirus infections. cytokines and other molecules involved in immune response and inflammation are conceivable therapeutic targets for ip and ards, improving symptoms and decreasing intensive care unit admissions. to this aim off label drugs may be used taking into consideration the window timing for immunosuppressive drugs in virus infected patients. some off label therapeutic options and preclinical evidence drugs are herein considered. in a report on more than 70 thousands patients of the chinese province of hubei, the majority of infected symptomatic people presented flu like symptoms (mainly fever and cough), with 15-20% of patients developing a severe interstitial pneumonitis (ip) that could evolve in acute respiratory distress syndrome (ards). the case fatality rate in the whole population resulted 2.3% (8 and 15%, for patients older than 70 and 80, respectively). in critical patients 49% of case fatality rate was registered (4) . ip is caused by the attack of the virus against the alveolar pneumocytes (aps) through the binding of the viral glycoprotein (spike, s) to the angiotensin converting enzyme 2 (ace2) expressed on the surface of the aps (5) . the virus enters in the host target cells through receptor-mediated endocytosis and quickly replicates; virus release in the extracellular space occurs through either budding or cell death. in the extracellular space the virus is recognized by the prr of immune cells (6) . this process contributes to the virus elimination through an amplification cascade in which the immune cells produce a large number of pro-inflammatory cytokines, tissue factors, and vasoactive peptides. these molecules reach the blood vessel wall causing a burst of nitric oxide, damages to the blood vessels and to the coagulation system (7) . among the most involved cells, macrophages play an important role in acute lung injury, which identify pathogen-associated molecular patterns (pamp) and trigger innate immunity (8, 9) . macrophages secrete a large number of inflammatory mediators and cytokines, such as tumor necrosis factor-alpha (tnf-α), interleukin-1beta (il-1β), interleukin-6 (il-6), inducible nitric oxide synthase (inos), and macrophage migration inhibitory factor (mif). tnf-α can directly damage cells of the pulmonary vascular endothelium, increasing capillary endothelial permeability, causing pulmonary edema, predicted by il-6 level (10). progression to acute respiratory distress syndrome (ards) is based on the acute onset of lung inflammation, determined by monocyte/macrophage polarization and function. during active infection, inflammatory monocytes/macrophages (imms), and resident macrophages undergo marked phenotypic and functional changes, from m1 proinflammatory (classically activated) to m2 inflammatory-resolving macrophages, with a dynamic continuum through discrete categories. during acute infection, monocytes/macrophages often display a phenotype of classically activated macrophages that mediate antiviral host defenses but also promote lung injury by producing nitric oxide (no), reactive oxygen species (ros), il-1, il-6, and il-8 and tnf-α. simultaneously, some macrophages may become m2 macrophages alternatively activated, exerting anti-inflammatory function and regulating wound healing by producing matrix metalloproteinases (mmps), growth factors, and anti-inflammatory cytokines, particularly tgf-β. pro-inflammatory macrophages diminish at the removal of stimulus (11) (12) (13) . evidence of a cytokine storm has been found in severe pneumonitis linked to coronavirus infection (14) . previously, in patients with sars, il1b, il6, il12, ifnγ, ip10, and mcp1 were found to be increased (15) . in patients with mers, ifnγ, tnfα, il15, and il17 were shown to participate in the severity of the pneumonitis (16) , and an elevated inflammatory innate immune response has been shown in the lower respiratory tract. although those cytokines were elevated, down-regulation of genes encoding inflammatory th1 and th2 molecules was noted (17) . interestingly, in patients infected by sars-cov-2, there is an increase in il1β, ifnγ, ip10, and mcp1, probably leading to activated t-helper-1 (th1) cell responses, and increased production of t-helper-2 (th2) immunosuppressive cytokines, such as il4 and il10 (18) . in particular, a significant increase in il2, il7, il10, g-csf, ip10, mcp1/ccl2, mip1a, and tnfα was noted in patients requiring admission to the intensive care unit (icu) compared to patients with a milder disease. as the infiltrate of monocytes, neutrophils, lymphocytes, and macrophages are the cellular actors of the inflammatory response (14) , chemokine ligands and receptors play an important role in driving immune cell migration and homing (19) . these cytokines may explain the observation of reduced levels of circulating lymphocytes. peripheral blood examinations on admission in the majority of patients with covid-19 displayed lymphopenia, elevated infection-related biomarkers (i.e., procalcitonin, erythrocyte sedimentation rate, serum ferritin, and c-reactive protein) (20) and several elevated inflammatory cytokines (i.e., tumor necrosis factor (tnf)-α, interleukin (il)-2r and il-6). patients with more severe cases had higher leukocyte and neutrophil count, lower lymphocyte count and higher neutrophil-to-lymphocyte ratio (nlr) (21) . lymphocyte subsets showed that the total number of b cells, t cells and nk cells was significantly decreased in patients with covid-19, more significantly so in severe cases. in particular, t cells (t helper, t suppressor, and tregs cells) were mostly affected by sars-cov-2. in addition, recent evidence in sars-cov infection suggests that seroconversion may also play a role in lung injury. a detrimental role of early appearance of antispike (s)-igg was demonstrated during sars-cov infection in a macaque model (22) . despite markedly reducing virus titers, anti-s-igg caused lung injury during the early stages of infection, impairing the wound-healing macrophage response and tgf-β production, while promoting pro inflammatory cytokine il-8, mcp1 production, and inflammatory macrophage accumulation (22) . interestingly, in sars patients who died in hong kong during the 2002 outbreak, the anti-spike (s) glycoprotein neutralizing antibodies appeared significantly before and reached a higher titer than in patients surviving (23) . consistently, preexisting serum antibodies, derived by exposition to influenza seasonal strains, may recognize but fail to neutralize, the new pandemic strain and were found to associate with worse clinical severity during the 2009 influenza pandemic (24, 25) . the inflammatory status together with pulmonary edema and respiratory failure define the clinical picture of the ards associated with covid-19 (26) . the most compelling emergency that the health system faces in this epidemic is the shortage of critical care units. the saturation of intensive care units (icu) precludes the rescue of patients who might be saved, increases covid-19 lethality rate and worsens the prognosis for other pathological conditions requiring icu admission. the severe ip or ards of the covid-19 requires ventilator support and can kill infected people averaging in 2 weeks from the appearance of the first symptoms (27, 28) . therapy in use for hiv and other viral disease have been empirically administered without much benefit (29) , while promising experimental antiviral drugs such as remdesivir and chloroquine, an old antimalarial drug with in vitro activity on the viral infection, are currently in clinical trials (30, 31) . in the absence of specific validated approaches, and waiting for a vaccine, a clinical empirical rational management is needed. another reasonable approach would be drugs targeting the host immune-inflammatory reaction. methylprednisolone, although somewhat controversial, seems to be overall useful in these patients (32) , while dexamethasone has been shown to be useful in patients with ards of different etiologies (32, 33) . cytokines and the other molecules involved in the immune response regulation and inflammation are conceivable targets to improve ip and ards lung injury. to this aim off label drugs may be used considering the timing for immunosuppressive drugs in virus infected patients. unfortunately, the time window is not univocally defined and data may derive from clinical studies. several therapeutic options that could be rapidly translated to clinical trials are available. some of them are listed below. tocilizumab is an anti-il6 receptor antibody (roactemra, roche) approved to treat moderate to severe rheumatoid arthritis (ra). tocilizumab has been used to counteract the side effects of immune checkpoint inhibitors and car-t therapy in cancer bearing patients (34) and, recently, to antagonize the host reaction in patients affected by ards linked to covid 19 (35) . at today covid-19 national management guidelines of chinese health authorities include the use of tocilizumab for severe pneumonia. a preliminary report on 21 critical cases of covid-19 suggests efficacy of the treatment with faster recovery and lower risk of death for treated patients, while no toxicity was associated with the reported administration schedule (one or maximum two doses) (36) . timing of administration seems to be crucial as tocilizumab may be more efficient if administered earlier than actual use (37) . anakinra is an interleukin-1 receptor antagonist (il-1ra) previously evaluated in clinical trials for ra patients. il-1beta/il-1alpha are two stimulating cytokines of monocyte-macrophage cells acting upstream of the inflammatory signaling pathway induced by inflammasome, thus anakinra should block the cytokine storm. in a small open-label study, anakinra has been tested as agent preventive of mechanic ventilation in 9 patients hospitalized for moderate-severe covid-19. amelioration of oxygen flow and blood inflammation markers was described without significant toxicity (38) . in clinically moderate and severe covid-19 patients preliminary evidence reported high levels of three cytokines, cxcl10, ccl7 and il-1, rather than il-6, (39). in chronic use anakinra could determine reaction at the site of injection and infection as the main side effects (40) . emapalumab is a fully human igg1 monoclonal antibody that binds free and receptor-bound interferon-γ. emapalumab is approved by the us fda for the treatment of haemophagocytic (hlh) (41) a rare disorder characterized by pathologic immune activation and hyperinflammation that eventually damage multiple organs. a prospective study has shown a good safety profile of emapalumab in pediatric and adolescent patients affected by hlh, with the infection susceptibility being the major threat (42) . blocking ifn γ activity could counteract the host immune hyper-reaction to sars-cov-2. mycophenolic acid has been used as immunosuppressant agent in pemphigus as a corticosteroid-sparing agent and in kidney transplant patients to avoid rejection. it inhibits inositol monophosphate dehydrogenase, that causes depletion of guanosine and deoxyguanosine nucleotide pools impairing the activity of b and t lymphocytes. the drug has also been demonstrated to inhibit mrna expression of pro-inflammatory cytokines tnf-α, il-6, and il-1β4 (43) . mycophenolic acid has been shown to have activity in vitro against zika virus replication (44) and coronavirus through a non-competitive inhibition of mers-cov papain-like protease (45). urinary infections, diarrhea, and leukopenia are the side effects more often described (46) . anti-tnfα agents used in autoimmune diseases, such as ra and ulcerative colitis, in principle, may have a role in treating severe respiratory syndrome of covid-19. infliximab is a monoclonal antibody targeting tnf alpha while etanercept is a receptor fusion protein (human igg1-fc plus soluble p75 tnf alpha extracellular domain). tnf-α is a proinflammatory cytokine produced by macrophages, lymphoid cells, endothelial cells, cardiac myocytes, adipose tissue, and brain cells such as microglia and astrocytes. its receptors are widely expressed and tnf-α plays a key role in immunological defense processes such as inducing fever, inhibiting viral replication during infections, and leading to a permanent growth arrest in cancer (47, 48) . toxicity profile includes augmented risk of infections (49) . proteasomal system regulates different cell functions, among which nuclear factor kb (nf-kb) key transcription factor for innate and adaptive immunity (50) . bortezomib inhibits proteasome and it is used in the treatment of myeloma and mantle cell lymphoma. it has been shown to have antiviral activity against herpes virus, targeting viral entry, replication, and assembly (51) . another proteasome inhibitor, vr23, possess powerful antiinflammatory activity reducing il-6 in synovial cells from ra patients, and improving lps-induced acute lung injury by decreasing neutrophil migration, tnf-α secretion, and tissue inflammation in a mice model (52) . the doselimiting toxicity of proteasome inhibitors is the peripheral neuropathy (53) a clinically relevant complication, which negatively impacts the quality of life of multiple myeloma survivors (54) . pandemic viruses decrease type i interferon (ifn) abundance (24) . in humans 17 different types of poly-adenosine 5 ′diphosphate (adp)-ribose polymerase (parp) are recognized. parps transfer adp ribose from nicotinamide adenine dinucleotide (nad+) to targeted proteins achieving a post translational modification called adp-ribosylation, generally in response to stress conditions such as dna damage, heat shock and viral attack (55) . parp11 is an adp ribosyl-transferase that inhibits interferon type i (ifn-i) antiviral activity. ifn-i is a key component of the immune response against viral pathogens that induces the expression of several genes (interferon stimulated genes -isgs) with diverse antiviral properties (56) . parp11 inhibitor, rucaparib has been shown to restore the activity of ifn-i against different viruses in a murine model (57) . there is evidence that zikv infection triggers type i ifn production by host cells, zikv is sensitive to the antiviral activity of ifn and ifn i seems crucial also in sars-cov-2 infection (58, 59) . parp inhibitors are used in subgroup of patients with breast or ovarian cancer. toxicity is mainly hematological (60) . peroxisome proliferator-activated receptor gamma (pparγagonists, rosiglitazone and pioglitazone, are drugs in clinical use for diabetes (61) . insulin resistance amplifies inflammation, associated with an increase in c-reactive protein, il-6, and tnf-α (62) and produces a pro-coagulant state with increased fibrinogen and plasminogen activator inhibitor, (pai-1) (63). pioglitazone, in clinical studies on diabetic patients, was able to reduce the plasma level of different inflammatory factors among which cpr, il-1, il-6, and tnf-α (64). thus, it is of great interest that pioglitazone can produce an anti-inflammatory effect also on lung inflammation and fibrosis (65) . considered the excellent tolerability, pparγ agonists may be tested for amelioration of virus induced lung injuries. plerixafor is a cxcr4 antagonist used for stem cell mobilization in patients undergoing autologous stem cell transplantation. cxcr4-mediated inflammatory responses is based on the efficient chemotaxis function of inflammatory cells, such as neutrophils, lymphocytes, and monocytes (66) . in murine models of acute lung insufficiency cxcr4 expression was significantly increased in macrophages sorted from bronchoalveolar lavage fluid and receptor downregulation reduced il-6 and tnf-α. administration of amd3100 significantly attenuated the influx of inflammatory cells to the airway and reduced the levels of il-4, il-5, and il-13 in an murine asthmatic model either in the lavage fluid and lung homogenate through attenuation of the th17 (67), cell population. no adverse events have been described for a single injection of plerixafor (68). fingolimod, a sphingosine-1-phosphate (s1p) receptor agonist is approved for the treatment of multiple sclerosis (ms). s1p is mainly expressed in vascular endothelial cells and lymphocytes in lung tissue. s1p1 agonists (cym-5442 and rp-002) have been reported to protect mice from death caused by severe influenza infection, attenuating cytokine production and inhibiting infiltration of innate immune cells. in a mouse model of 2009 h1n1 pandemic influenza, the s1p1 receptor agonist significantly inhibited synthesis of il-1α, il-1β, il-6, il-10, mcp-1, tnf-α, and gm-csf, and reduced deaths from lethal infections by more than 80%. in addition the combination of oseltamivir can reduce mouse mortality by 96% (69) . recently a multiple sclerosis (ms) patient in treatment with fingolimod that was diagnosed with covid-19 reported a favorable outcome (70) . as reported, the toxicity profile even for long term use, is reassuring (71) . in conclusion, while specific antiviral therapies are in rapid development (remdesivir, chloroquine, vaccine), controlling the powerful inflammatory response causing severe ip or ards is a reasonable approach. agents that are available now to improve the lung injuries due to the host reactions and reduce the lethality of the disease are badly needed, and some are already in clinical studies. drugs targeting multiple cyto/chemokines involved in sars-cov-2 ip are available for trial or for off-label use, but close attention is needed to the schedule of 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rapid, efficient mobilization of hematopoietic stem cells in sickle cell disease patients after exchange transfusion dissecting influenza virus pathogenesis uncovers a novel chemical approach to combat the infection covid-19 infection in a patient with multiple sclerosis treated with fingolimod benefitrisk profile of sphingosine-1-phosphate receptor modulators in relapsing and secondary progressive multiple sclerosis the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.copyright © 2020 scala and pacelli. this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. no use, distribution or reproduction is permitted which does not comply with these terms. key: cord-323553-bukm9m9q authors: song, woo-jin; li, qiang; ryu, min-ok; nam, aryung; an, ju-hyun; jung, yun chan; ahn, jin-ok; youn, hwa-young title: canine adipose tissue-derived mesenchymal stem cells pre-treated with tnf-alpha enhance immunomodulatory effects in inflammatory bowel disease in mice date: 2019-08-31 journal: research in veterinary science doi: 10.1016/j.rvsc.2019.06.012 sha: doc_id: 323553 cord_uid: bukm9m9q abstract canine inflammatory bowel disease (ibd) is an intractable autoimmune disorder that results in various gastrointestinal and systemic symptoms. mesenchymal stem cells (mscs), which release immunomodulatory factors such as tumor necrosis factor-α (tnf-α)-induced gene/protein 6 (tsg-6) and prostaglandin e2 (pge2), have been suggested as an alternative therapeutic option for ibd treatment in veterinary medicine. furthermore, although it is known that mscs pre-treated with pro-inflammatory cytokines show enhanced anti-inflammatory properties via the secretion of soluble factors, the underlying mechanisms of ibd remain unclear. the aim of this study was to demonstrate the therapeutic effects and corresponding mechanisms of canine adipose tissue-derived (cat)-mscs stimulated with tnf-α in mouse models of ibd. mice with dextran sulfate sodium (dss)or dinitrobenzene sulfonic acid (dnbs)-induced colitis were injected intraperitoneally with cat-mscs pre-treated with tnf-α. colitis severity was assessed and colon tissues were collected for histopathological, enzyme-linked immunosorbent assay, and flow cytometry analysis. cat-mscs stimulated with tnf-α secreted higher concentrations of immunomodulatory factors such as tsg-6 and pge2, which play a key role in inducing phenotypic alterations in macrophages. consequently, tnf-α-pre-treated cat-mscs further regulated colonic inflammatory cytokines such as interleukin (il)-1β, il-6, and il-10, and ameliorated dssor dnbs-induced colitis in mice. additionally, we demonstrated that m1 macrophages (f4/80+/inos+ cells) were decreased in colon tissues from mice treated with tnf-α-pre-treated cat-mscs, whereas m2 macrophages (f4/80+/cd206+ cells) were increased. these results may suggest a new cell-based therapeutic option for treating ibd. canine inflammatory bowel disease (ibd), which leads to gastrointestinal or systemic clinical signs, is diagnosed by ruling out the possibility of other diseases such as infection or tumor and performing histopathological assessment (cerquetella et al., 2010; craven et al., 2004) . ibd is an intractable autoimmune disease and immunosuppressive drugs are used to reduce inflammation (allenspach et al., 2006; dossin and lavoue, 2011) . however, no alternative treatments exist for dogs with ibd that do not respond to the conventional therapies. therefore, mesenchymal stem cells (mscs) that can effectively modulate inflammation might be an alternative therapeutic option (iyer and rojas, 2008) . recent studies have revealed that soluble factors released by mscs such as prostaglandin e2 (pge 2 ), hepatocyte growth factor, indoleamine 2,3-dioxygenase, and tnf-stimulated gene/protein 6 (tsg-6) contribute to immunomodulation (bassi et al., 2012; montemurro et al., 2016; teng et al., 2015) . therefore, mscs exert strong anti-inflammatory effects, although injected mscs did not migrate into inflamed tissue in a previous study (sala et al., 2015) . kang et al. and chae et al. also demonstrated that canine and feline mscs secrete soluble immunomodulatory factors (chae et al., 2017; kang et al., 2008) . in addition, our previous studies have shown that tsg-6 released from human and canine mscs ameliorates colitis in mice song et al., 2017b) . our previous study demonstrated that canine mscs pre-treated with tumor necrosis factor (tnf)-α and interferon (ifn)-γ exerted enhanced anti-inflammatory effects in vitro by releasing higher concentrations of pge 2 , an immunomodulatory factor (yang et al., 2018) . fan et al. also revealed that human mscs stimulated with interleukin (il)-1β showed enhanced efficacy in mice with colitis (fan et al., 2012) . in addition, recent studies have demonstrated that mscs pre-treated with pro-inflammatory cytokines showed enhanced secretory abilities (broekman et al., 2016; heo et al., 2011) . however, few studies have assessed the therapeutic effects of pro-inflammatory cytokine-stimulated canine mscs. therefore, in this study, we used canine adipose tissue (cat)-mscs stimulated with tnf-α, and revealed the therapeutic effects and their mechanisms in two mouse models of ibd. canine adipose tissues were obtained from healthy 4-year-old dogs using protocols approved by the institutional animal care and use committee (iacuc) of seoul national university performed in accordance with approved guidelines. the dogs were negative for canine parvovirus, canine coronavirus, and canine distemper virus infections. mscs were isolated and cultured as previously described . briefly, adipose tissue samples were washed five times in dulbecco's phosphate buffered saline (dpbs; pan-biotech, aidenbach, germany) containing 1% penicillin-streptomycin (ps; pan-biotech), and cut into small pieces in a petri dish. the samples were digested with collagenase type ia (0.1%, gibco/life technologies, carlsbad, ca, usa) for 60 min at 37°c. the samples were neutralized with dulbecco's modified eagle's medium (dmem; pan-biotech) containing 10% fetal bovine serum (fbs; pan-biotech). after centrifuging the adipose tissue mixture at 1200 ×g for 5 min, the pellet containing mscs was passed through a 70-μm cell strainer (thermo fisher scientific, rockford, il, usa) to remove undigested debris. cells were resuspended in dmem containing 10% fbs and 1% ps, seeded onto a cell culture dish at a density of 3000 cells/cm 2 , and incubated at 37°c and 5% co 2 . after 5 days, cultures were washed with dpbs to remove non-adherent cells and incubated with fresh medium. the culture medium was changed every 2-3 days until cells reached 70-80% confluence. the cells were then subcultured and seeded at a density of 10,000 cells/cm 2 in culture dishes. before experimentation, the cells were characterized using flow cytometry to evaluate the expression of several stem cell markers. cells were suspended in dpbs and monoclonal antibodies against the following proteins: cluster of differentiation (cd)29-fluorescein isothiocyanate (fitc), cd31-fitc, cd34-phycoerythrin (pe), cd73-pe (bd biosciences, san diego, ca, usa), cd45-fitc, and cd90-allophycocyanin (ebiosciences, san diego, ca, usa). cell fluorescence was analyzed with a facs aria ii system (bd biosciences). additionally, the cells' differentiation abilities were evaluated using stempro adipogenesis, osteogenesis, and chondrogenesis differentiation kits (gibco, grand island, ny, usa) according to the manufacturer's instructions. the differentiated cells were stained with oil red o, alizarin red, and alcian blue. cat-mscs at approximately 60-70% confluence were stimulated with canine recombinant tnf-α (10 ng/ml; prospec protein specialists, nj, usa) for 24 h. the cells were used for tnf-α-stimulated cat-msc groups. c57bl/6 j mice (male, 5-week-old) were purchased from nara biotech (seoul, korea) and housed under standard conditions (controlled temperature, humidity, and light cycle). all procedures involving mice were approved by the institutional animal care and use committee of seoul national university (protocol no. snu-171123-2), and the protocols were performed in accordance with approved guidelines. two different mouse models for ibd were used for this study (dextran sulfate sodium (dss)-, and dinitrobenzene sulfonic acid (dnbs)-induced colitis models), and each colitis model was made as previously described (kim et al., 2013; martín et al., 2014; morampudi et al., 2014; solomon et al., 2010) . for the first experiment, colitis was induced by 3% dss (36-50 kda; mp biomedical, solon, oh, usa) in the drinking water from day 0 to day 7, whereas mice offered normal water were used as the naive group. the following experiments were performed on day 1: cat-mscs (2 × 10 6 ) stimulated with tnf-α in 200 μl pbs; cat-mscs (2 × 10 6 ) in 200 μl pbs; or an identical volume of pbs was injected intraperitoneally into the dss-induced colitis mice. for this experiment, mice were randomly divided to the following four groups: naïve (n = 4), dss + pbs (n = 6), dss + cat-msc (n = 6), dss+ tnf-α-cat-msc (n = 6). for the second experiment, dnbs (sigma-aldrich, st. louis, mo, usa) colitis was induced by rectal administration of dnbs (5 mg/mouse in 50% ethanol) into mice. six hours after dnbs infusion, cat-mscs were administered intraperitoneally as described above. for this experiment, mice were divided into five groups: naïve (n = 4), ethanol (sham; n = 4), dnbs +pbs (n = 6), dnbs+cat-msc (n = 6), dnbs+tnf-α-cat-msc (n = 6). the mice were sacrificed on day 10 (for dss-induced colitis experiments) or day 3 (for dnbs-induced colitis experiments), and colon tissues were collected for further processing. the disease activity index (dai) was determined by a scoring system described previously . briefly, the body weight loss (grades 0-4), stool consistency (grades 0-2), rectal bleeding (grades 0-2), and general activity (grades 0-2) were monitored every 24 h. for histological analysis, colon tissue samples were fixed in 4% phosphatebuffered formaldehyde for 48-72 h, followed by embedding in paraffin, cutting into 4-μm sections, and staining with hematoxylin and eosin. ten fields per group were randomly selected and histological examinations were performed. colitis severity was calculated using the previously described scoring system. briefly, the extent of bowel wall thickening (grades 0-3), crypt damage (grades 0-3), and inflammatory cell infiltration (grades 0-2) were examined in a blind manner. tsg-6 and pge 2 in the supernatants from tnf-α-pre-treated or nontreated cat-mscs were measured using a tsg-6 elisa kit (mybiosource, san diego, ca, usa) and pge 2 elisa kit (cusabio biotech, md, usa), respectively. additionally, for in vivo experiments, total proteins were extracted from colon tissue samples using pro-prep protein extraction solution (intron biotechnology, seongnam, korea) and the concentrations of il-1β, il-6, and il-10 were measured using commercial elisa kits (all from ebiosciences) according to the manufacturer's instructions. to evaluate the mouse macrophage population, the following monoclonal antibody mixtures were used for the experiments: anti-f4/ 80-fitc and anti-inducible nitric oxide synthase (inos)-pe, or anti-f4/ 80-fitc and anti-cd206-pe (santa cruz biotechnology, santa cruz, ca, usa) were incubated with cells isolated from digested colon tissues. flow cytometry was performed using a facs aria ii system (bd biosciences) and analyzed using flowjo software (tree star, ashland, or, usa). data are shown as the mean ± standard deviation. mean values among different groups were compared by one-way analysis of variance using graphpad prism software (v.6.01; graphpad, inc., la jolla, ca, usa). p value < .05 was considered statistically significant. cells isolated from canine adipose tissue were assessed for msc characteristics. flow cytometry analysis showed that stem cell markers such as cd29, cd73, and cd90 were highly expressed in these cells. in contrast, there was no detectable expression of hematopoietic markers, including cd31, cd34, and cd45 (fig. 1a) . additionally, the cells could be differentiated into adipocytes, osteocytes, and chondrocytes (fig. 1b) . according to criteria established by international society for cellular therapy, the cells used in this study represent mscs. our previous studies demonstrated that secretory factors from canine mscs, such as tsg-6 and pge 2 , play a key role in modulating inflammation. therefore, cat-mscs were stimulated with tnf-α, a proinflammatory cytokine, to produce more immunomodulatory factors. tsg-6 and pge 2 concentrations determined from the cat-mscs pretreated with tnf-α were significantly higher than those measured from the naïve cat-mscs (fig. 1c) . next, we evaluated whether administering cat-mscs or tnf-α-stimulated cat-mscs could reduce colitis severity in mice. consistent with previous studies, administering cat-mscs resulted in a general reduction in body weight loss, dai, and colon length shortening in mice with dss-or dnbs-induced colitis ( fig. 2a -c, 3a-c). in addition, further improvement was confirmed in mice with colitis injected with tnf-α-stimulated cat-mscs ( fig. 2a-c, 3a-c) . additionally, histopathology in the inflamed colon tissue was evaluated. colons of mice treated with dss or dnbs showed severe submucosal thickening, crypt damage, and infiltration of inflammatory cells. in contrast, administering cat-mscs to mice with colitis resulted in a slight improvement, which was additionally enhanced by administering tnf-α-stimulated cat-mscs (fig. 2d, 3d) . we next explored whether cat-mscs or tnf-α-stimulated cat-mscs could improve the anti-inflammatory effects in dss-induced colitis. in the colons of mice treated with naïve cat-mscs, the production of il-1β and il-6 was significantly decreased, and production of il-10 was increased considerably compared to that in the colons of mice treated with pbs, as expected from previous studies. similar to the above results, intestinal inflammation was further improved in colons from mice treated with tnf-α-stimulated cat-mscs (fig. 4a-c) . given that the cytokines (il-1β, il-6, and il-10) modulated in the above results are mainly secreted from macrophages, we further investigated whether cat-mscs or tnf-α-stimulated cat-mscs could alter macrophage phenotypes in inflamed colon tissue. our previous study demonstrated that tsg-6 secreted from cat-mscs could increase the number of m2 macrophages in the colons of mice treated with dss. consistent with these results, our study showed that the m2 macrophage population (f4/80 + /cd206 + ) in colons of dss-induced colitis mice treated with cat-mscs was significantly increased compared with that in mice treated with pbs. in addition, the m1 macrophage population (f4/80 + /inos + ) was decreased in the cat-mscs-treated group. the phenotypic population of macrophages was further altered in the tnf-α-stimulated cat-mscs-treated group (fig. 5) . recently, a number of studies have shown that mscs can effectively ameliorate ibd in rodent models (gonzalez-rey et al., 2009; tanaka et al., 2008; wang et al., 2014) . furthermore, administering mscs as a therapeutic option for ibd in small clinical trials (both in humans and dogs) has also shown considerable promise (kim et al., 2017; perez-merino et al., 2015) . our previous studies have demonstrated that tsg-6 released from human and canine mscs plays a crucial role in immunomodulation by inducing an m2 macrophage switch song et al., 2017b) . in addition, we have shown that canine mscs stimulated with tnf-α and ifn-γ released higher concentration of pge 2 which exert anti-inflammatory effects (yang et al., 2018) . therefore, by up-regulating the secretion of theses soluble factors, mscs can enhance the immunomodulatory effects. overall, these findings highlight the efficacy of tnf-α-stimulated cat-mscs against dss-or dnbs-induced colitis in mice. in our study, we demonstrated that intraperitoneal injection of tnfα stimulated cat-mscs resulted in higher therapeutic efficacy than injecting naïve cat-mscs in two mouse models of ibd. for example, body weight loss and dai were further improved in the tnf-α-stimulated cat-mscs-treated mice by 6% and 20%, respectively, compared to the improvements in the naive cat-mscs-treated group. in addition, evaluation of colon length and histopathologic analysis highlighted the increased therapeutic effects of tnf-α-stimulated cat-mscs. in addition, concentrations of inflammatory cytokines in the inflamed colons were significantly altered in the tnf-α-cat-msc group compared with those in the cat-msc group. previous studies have revealed that human mscs stimulated with pro-inflammatory cytokines (such as tnf-α and il-1β) can improve the secretory effects of immunomodulatory soluble factors (broekman et al., 2016; heo et al., 2011) . here, we also showed that tnf-α-stimulated cat-mscs released significantly higher concentrations of tsg-6 (2.5-fold) and pge2 (3-fold), relative to naive cat-mscs. tsg-6 and pge 2 are well-known immunomodulatory factors secreted from human and canine mscs, and recent studies have demonstrated that these factors play important roles in ameliorating atopic dermatitis, rheumatoid arthritis, acute pancreatitis, and ibd kim et al., 2015; mao et al., 2017; shin et al., 2016; song et al., 2017a) . consistent with these studies, our results indicated that cat-mscs stimulated with tnf-α further reduced dss-or dnbs-induced colitis by releasing higher concentrations of tsg-6 and pge 2 . macrophages are important innate immune cells that play a key role fig. 1. (a, b) cells isolated from canine adipose tissues were characterized before their use in this study by flow cytometry analysis (a), as well as adipogenic, osteogenic, and chondrogenic differentiation analysis (b). (c) canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α released higher concentrations of immunomodulatory factors such as tsg-6 and pge2 compared to levels released by naive cat-mscs. results are shown as the mean ± standard deviation of three independent experiments. ***p < .001. in releasing inflammatory cytokines and transferring information to acquired immune cells such as t cells. it is well-established that two types of macrophages (m1 and m2) are observed in inflamed tissues (mosser and edwards, 2008; stout and suttles, 2004) and these cells play an important role in regulating inflammatory responses. melief et al. demonstrated that human mscs promote the transition of monocytes into cd206 + m2 macrophages, and consequently increase foxp3 + regulatory t cells (melief et al., 2013) . in addition, recent studies have revealed that soluble factors (such as tsg-6 and pge 2 ) released from human mscs could promote the m2 macrophage fig. 2 . canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α showed enhanced therapeutic effects on mice with dextran sodium sulfate (dss)-induced colitis. therapeutic abilities of cat-mscs were assessed by measuring body weight changes (a), disease activity index (b), colon length (c), and histopathologic analysis (d). four to six mice per group were used. results are shown as the mean ± standard deviation. *p < .05, **p < .01, ***p < .001. fig. 3 . canine adipose tissue-derived mesenchymal stem cells (cat-mscs) stimulated with tnf-α showed enhanced therapeutic effects on mice with dinitrobenzene sulfonic acid (dnbs)-induced colitis. therapeutic abilities of cat-mscs were assessed by body weight changes (a), disease activity index (b), colon length (c), and histopathologic analysis (d). four to six mice per group were used. results are shown as the mean ± standard deviation. *p < .05, **p < .01, ***p < .001. phenotype and reduce inflammation in mouse models of rheumatoid arthritis, wound healing, and ibd (shin et al., 2016; song et al., 2017b; zhang et al., 2010) . additionally, we previously demonstrated that tsg-6 released from canine mscs can induce m2 macrophage phenotypic changes in vitro and in vivo . in this study, tnf-α stimulated cat-mscs increased macrophage alteration to the m2 phenotype (f4/80 + /cd206 + ) in the colons of mice with ibd, whereas numbers of m1 macrophages (f4/80 + /inos + ) decreased in the inflamed colons. consistent with previous studies and our results, tnf-αstimulated cat-mscs reduced inflammation through altering macrophage phenotypic changes by secreting higher concentrations of tsg-6 and pge 2 . recent studies have suggested other mechanisms by which mscs might help reduce colitis severity. for example, mscs may stimulate epithelial regeneration (sémont et al., 2013; sémont et al., 2006; valcz et al., 2011) . it is well-established that mscs upregulate ki67 + cells in inflamed tissues (nakagawa et al., 2015; wu et al., 2007) . in addition, chen et al. demonstrated that mscs increase lgr5 + intestinal stem cells in colonic tissues of ibd model mice (chen et al., 2013) . another potential mechanism of msc-dependent improvement in ibd involves microbiome changes. however, in this study, tnf-α-stimulated mscs, which release higher concentrations of soluble immunomodulatory factors, showed further improved colitis in mice (soontararak et al., 2018) . therefore, we demonstrated here that the anti-inflammatory effects of mscs play an important role in their overall therapeutic effects on colitis, although mscs might reduce ibd through various mechanisms. previous studies have revealed that a high frequency of results are shown as the mean ± standard deviation. *p < .05, **p < .01, ***p < .001. intraperitoneally infused mscs aggregated with immune cells in the peritoneal cavity (sala et al., 2015; bazhanov et al., 2016) . in addition, our previous studies have shown that < 0.5% of intraperitoneally injected mscs were detected in the heart, lung, liver, spleen, kidney, brain, and colon tissues (song et al., 2017b; song et al., 2018) . based on these previous studies, it is tempting to speculate that most of intraperitoneally infused tnf-α-stimulated cat-mscs formed aggregates and ameliorated ibd at sites distant from the inflamed colon by releasing soluble factors such as tsg-6 and pge 2 . it should be acknowledged that we were not able to perform microarray screening of tnf-α-stimulated cat-mscs, although we evaluated increased tsg-6 and pge 2 from tnf-α-stimulated cat-mscs. however, it is well demonstrated that tsg-6 and pge 2 secreted from mscs could induce macrophage phenotypic alterations. therefore, our findings suggest that increased tsg-6 and pge 2 released from tnf-α stimulated cat-mscs play a key role in reducing inflammation in a mouse model of ibd. in summary, we demonstrated that tnf-α-stimulated cat-mscs further ameliorated ibd via their enhanced anti-inflammatory effects over naïve cat-mscs. additionally, we showed that cat-mscs pretreated with tnf-α could release higher levels of immunomodulatory factors such as tsg-6 and pge 2 , which contributed to induce macrophage phenotypic alterations. these results may represent a novel cellbased therapeutic option for treating autoimmune diseases such as ibd. declarations of interest: none. this study was supported by the research institute for veterinary science and bk21 plus program for creative veterinary science research. pharmacokinetics and clinical efficacy of cyclosporine treatment of dogs with steroid-refractory inflammatory bowel disease exploring the role of soluble factors associated with immune regulatory properties of mesenchymal stem cells intraperitoneally infused human mesenchymal stem cells form aggregates with mouse immune cells and attach to peritoneal organs tnf-α and il-1β-activated human mesenchymal stromal cells increase airway epithelial wound healing in vitro 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(dss) model of colitis: an overview umbilical cord-derived mesenchymal stem cell extracts reduce colitis in mice by repolarizing intestinal macrophages tsg-6 secreted by human adipose tissue-derived mesenchymal stem cells ameliorates dss-induced colitis by inducing m2 macrophage polarization in mice tsg-6 released from intraperitoneally injected canine adipose tissue-derived mesenchymal stem cells ameliorate inflammatory bowel disease by inducing m2 macrophage switch in mice mesenchymal stem cells (msc) derived from induced pluripotent stem cells (ipsc) equivalent to adipose-derived msc in promoting intestinal healing and microbiome normalization in mouse inflammatory bowel disease model functional plasticity of macrophages: reversible adaptation to changing microenvironments exogenous administration of mesenchymal stem cells ameliorates dextran sulfate sodium-induced colitis via anti-inflammatory action in damaged tissue in rats mesenchymal stem cellderived exosomes improve the microenvironment of infarcted myocardium contributing to angiogenesis and anti-inflammation the role of the bone marrow derived mesenchymal stem cells in colonic epithelial regeneration tgf-beta signaling-dependent alleviation of dextran sulfate sodium-induced colitis by mesenchymal stem cell transplantation mesenchymal stem cells enhance wound healing through differentiation and angiogenesis canine mesenchymal stem cells treated with tnf-α and ifn-γ enhance antiinflammatory effects through the cox-2/pge2 pathway human gingiva-derived mesenchymal stem cells elicit polarization of m2 macrophages and enhance cutaneous wound healing we are very grateful to the research institute for veterinary science of seoul national university, and the bk21 plus program for creative veterinary science research. key: cord-339272-trd6rkxw authors: chen, na; wu, qianchao; chi, gefu; soromou, lanan wassy; hou, jinli; deng, yanhong; feng, haihua title: prime-o-glucosylcimifugin attenuates lipopolysaccharide-induced acute lung injury in mice date: 2013-04-24 journal: int immunopharmacol doi: 10.1016/j.intimp.2013.04.014 sha: doc_id: 339272 cord_uid: trd6rkxw prime-o-glucosylcimifugin is an active chromone isolated from saposhnikovia root which has been reported to have various activities, such as anti-convulsant, anticancer, anti-inflammatory properties. the purpose of this study was to evaluate the effect of prime-o-glucosylcimifugin on acute lung injury (ali) induced by lipopolysaccharide in mice. balb/c mice received intraperitoneal injection of prime-o-glucosylcimifugin 1 h before intranasal instillation (i.n.) of lipopolysaccharide (lps). concentrations of tumor necrosis factor (tnf)-α, interleukin (il)-1β and interleukin (il)-6 in bronchoalveolar lavage fluid (balf) were measured by enzyme-linked immunosorbent assay (elisa). pulmonary histological changes were evaluated by hematoxylin–eosin, myeloperoxidase (mpo) activity in the lung tissue and lung wet/dry weight ratios were observed. furthermore, the mitogen-activated protein kinases (mapk) signaling pathway activation and the phosphorylation of iκbα protein were determined by western blot analysis. prime-o-glucosylcimifugin showed promising anti-inflammatory effect by inhibiting the activation of mapk and nf-κb signaling pathway. the acute respiratory distress syndrome (ards), a clinically important complication of severe acute lung injury (ali) in humans, highly associated with sepsis pneumonias and severe acute respiratory syndrome (sars) is a significant cause of morbidity and mortality in critically ill patients [1] [2] [3] . inflammatory stimuli from microbial pathogens, such as endotoxin (lipopolysaccharide [lps]), are well recognized for their ability to induce pulmonary inflammation, and experimental administration of lps, has been used to induce pulmonary inflammation in animal models of ali [4] [5] [6] . the development of an ali model by way of i.n. lps instillation is well suited for preliminary pharmacological studies of new drugs or other therapeutic agents because i.n. instillation of lps into mice can produce a controlled ali without causing systemic inflammation and multi-organ failure [4, 7] . lps-induced ali is considered a neutrophil-dependent ali that contributes to local recruitment and activation of neutrophils [8] ; the release of pro-inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1β, and il-6; and the formation of reactive oxygen and nitrogen species [9] [10] [11] . neutrophil recruitment in the lungs is regarded as a histological hallmark in the progression of ali [12] . several candidate therapy strategies such as fluid management, surfactants, glucocorticoids, and stem cells have been applied to treat acute lung injury and acute respiratory distress syndrome in the last decade [13, 14] . however, the mortality resulting from these conditions remains high [15] . fangfeng, the root of saposhnikovia divaricata (turcz) schischk, is widely applied for headache, febrility, vertigo and arthralgia as an important member of traditional chinese medicines (tcms). it has been proved that there are numerous pharmacologic effects of the extract from fangfeng, such as suppression of adjuvant arthritis, inhibitory effects on the peptic ulcers and analgesic, anti-convulsant, anticancer, anti-inflammatory and anticoagulant activities, etc in modern pharmacological experiments [16] . abundant compounds were isolated from it, such as chromones, conmarins, and polyacetylenes [17, 18] . prime-o-glucosylcimifugin is a major active chromone isolated from fangfeng. in recent years, it has been identified that chromones are the main active components which contribute most to its pharmacological efficacy [19] , but so far, the anti-inflammatory effects of prime-o-glucosylcimifugin on ali has not yet been studied. therefore, with a mouse model of acute lung inflammation, the present study was undertaken to examine the effect of prime-o-glucosylcimifugin on acute lung injury induced by intranasal instillation of lps in balb/c mice and investigate its possible mechanisms. prime-o-glucosylcimifugin was purchased from the national institute for the control of pharmaceutical and biological products (jilin, china). fetal bovine serum (fbs), dulbecco's modified eagle's medium (dmem), penicillin and streptomycin for cell culture were purchased from invitrogen-gibco (grand island, ny, usa). 3-(4, 5dimethylthiazol-2-y1)-2,5-diphenyltetrazolium bromide (mtt), dimethyl sulfoxide (dmso) and lipopolysaccharide (lps) (escherichia coli 055:b5) were purchased from sigma chemical co. (san diego, ca, usa). rabbit polyclonal anti-p44 erk, mouse monoclonal phosphospecific p42-p44 erk antibodies, rabbit polyclonal anti-p54 jnk, mouse monoclonal phospho-specific p46-p54 jnk antibodies, rabbit polyclonal anti-p38, mouse monoclonal phospho-specific p38 antibodies, rabbit mab iκbα and mouse mab phospho-iκbα were purchased from cell signaling technology inc. (beverly, ma). hrp-conjugated goat antirabbit and goat-mouse antibodies were provided by ge healthcare (buckinghamshire, uk). mouse tnf-α, il-6 and il-1β enzyme-linked immunosorbent assay (elisa) kits were purchased from biolegend (ca, usa). the myeloperoxidase (mpo) determination kit was purchased from jiancheng bioengineering institute of nanjing (nanjing, jiangsu province, china). all other chemicals were of reagent grade. balb/c male mice, 8 weeks old and weighing approximately 18 to 20 g, were purchased from the center of experimental animals of baiqiuen medical college of jilin university (jilin, china), and maintained at an animal facility under pathogen free conditions. the mice were fed a standard diet and water ad libitum and housed in microisolator cages under standard conditions (temperature: 24 ± 1°c, relative humidity: 40%-80%). the mice were allowed to adapt themselves to their environment for 2-3 days before experimentation. all animal experiments were performed in accordance with the national institutes of health (nih) guide for the care and use of laboratory animals and approved by the jilin university animal administration committee. the raw 264.7 mouse macrophage cell line was obtained from the china cell line bank (beijing, china). cells were cultured in complete medium (dmem supplemented with 10% heat-inactivated fbs, 3 mm glutamine and antibiotics (100 u/ml penicillin and 100 u/ml streptomycin)) at 37°c in a humidified incubator containing 5% co 2 and 95% air. cells were treated with various concentrations of prime-o-glucosylcimifugin for 1 h followed by stimulation with lps (1 mg/l). cytotoxicity studies induced by prime-o-glucosylcimifugin were evaluated by the mtt assay. raw 264.7 macrophages were seeded in 96-well plates at a density of 4 × 10 5 cells/ml in complete medium and incubated for 1 h (100 μl/well). then the cells were treated with different concentrations of prime-o-glucosylcimifugin (0-100 μg/ml, 50 μl/well) for 1 h, followed by stimulation with lps (1 mg/l, 50 μl/well) for 18 h. after 18 h, 10 μl mtt (5 g/l) was added to each well and the cells were further incubated for 4 h. the supernatant was removed and the cells were lysed with 150 μl/well dmso. the optical density was measured at 570 nm on a microplate reader. raw 264.7 cells were seeded in 24-well plates (4 × 10 5 cells/ml) and treated with 12.5, 25 or 50 μg/ml of prime-o-glucosylcimifugin for 1 h prior to stimulation of 1 mg/l lps for 24 h in a 37°c, 5% co 2 incubator. cell-free supernatants were collected and assayed. the concentrations of tnf-α, il-1β and il-6 in the supernatants of raw 264.7 cell cultures were measured by using an elisa kit, according to the manufacturer's instructions (biolegend, inc., camino santa fe, suite e, san diego, ca, usa). raw 264.7 cells (4 × 10 5 cells/ml) plated onto 6-well plates were incubated for 24 h and treated with 12.5, 25 or 50 μg/ml of prime-o-glucosylcimifugin for 1 h and then stimulated with 1 mg/l of lps for 30 min. the cells were collected and washed three times with ice-cold pbs. the cells were treated with a cell lysis buffer [50 mm tris (ph 7.6), 150 mm nacl, 5 mm edta (ph 8.0), 0.6% np-40, 1 mm na3vo4, 20 mm β-glycerophosphate, 1 mm phenylmethylsulfonyl fluoride, 2 mm p-nitrophenyl phosphate, and 1:25 complete mini protease inhibitor cocktail (boehringer, mannheim, germany)] and kept on ice for 30 min. the cell lysates were centrifuged (12,000 g at 4°c) for 5 min to obtain a cytosolic fraction. the protein concentration was determined by bca protein assay kit (beyotime, haimen, china). aliquots of the lysates were separated on 10% sodium dodecyl sulfate (sds)polyacrylamide gel electrophoresis (page) and then electroblotted onto a polyvinylidene difluoride (pvdf) membrane. the blots were blocked with 5% (w/v) non-fat dry milk for 2 h at 37°c, followed by incubation with specific primary antibody at 4°c overnight. blots were washed with tween 20/tris-buffered saline [ttbs, 20 mm tris-hcl buffer, ph 7.6, containing 137 mm nacl and 0.05% (vol/vol) tween 20] and incubated with a peroxidase-conjugated secondary antibody for 1 h. blots were again washed with ttbs and the immunoactive proteins were detected using ecl plus (thermo, usa). the mice were randomly divided into five groups: control group; lps group; lps + prime-o-glucosylcimifugin (2.5, 5 or 10 mg/kg bodyweight). prime-o-glucosylcimifugin was given intraperitoneally. one hour later, lps group and lps + prime-o-glucosylcimifugin group mice were given 50 μl lps intranasally (i.n) (200 mg/l) to induce acute lung injury. control mice were given 50 μl pbs intranasally (i.n) without lps. all the mice were alive after 7 h lps stimulation. collection of balf was performed three times through a tracheal cannula with 0.5 ml of autoclaved pbs, instilled up to a total volume of 1.3 ml. balf was centrifuged (4°c, 3000 rpm, 10 min) to pellet the cells. the concentrations of tnf-α, il-1β and il-6 in the balf were measured using sandwich enzyme-linked immunosorbent assay (elisa) kits according to the protocol recommended by the manufacturer. the cell pellets were resuspended in pbs, and the total cell number was counted using a standard hemocytometer. differences in cell numbers were examined by counting on a smear prepared by wright-giemsa staining. seven hours after intranasal instillation of lps, the mouse lungs were excised, and immediately weighed to obtain the wet weight. the dry weight was determined after heating the lungs at 80°c for 48 h. the w/d ratio was calculated by dividing the wet weight by the dry weight. mpo activity, which reflects the parenchymal infiltration of neutrophils and macrophages, was measured as described previously [20, 21] . mice were killed 7 h after lps administration under diethyl ether anesthesia. lung tissues were homogenized in 50 mm hydroxyethyl piperazine ethanesulfonic acid (hepes) (ph 8.0) containing 0.5% cetyltrimethyl ammonium bromide (ctab) and subjected to three freeze-thaw cycles. the homogenate was centrifuged at 13,000 ×g for 30 min at 4°c, and the cell-free extracts were stored at − 20°c until further use. the mpo activity was assayed using a mouse mpo elisa kit. samples were diluted in phosphate citrate buffer (ph 5.0). histopathologic examination was performed on mice that were not subjected to balf collection. the lungs were excised and fixed in 10% buffered formalin. then the tissues were dehydrated with graded alcohol, embedded in paraffin and sliced. the sections were stained with hematoxylin and eosin (h&e) and pathological changes of lung tissues were observed under a light microscope. all values are presented as mean ± sd. data were entered into a database and analyzed using spss software (spss for windows version 13.0, chicago, usa) and comparison between groups was made with one-way anova (dunnett's t-test) and student's t-test. p-values of 0.05 or less were considered statistically significant. to assess the suitable concentration of prime-o-glucosylcimifugin for the study, raw 264.7 cells were incubated with prime-oglucosylcimifugin at concentrations ranging from 12.5 to 100 mg/l in the absence or presence of lps and cell viability was measured by mtt test 18 h later. the results showed that prime-o-glucosylcimifugin at concentrations from 12.5 to 100 mg/l had no cytotoxic effect on raw 264.7 cells (fig. 1) . tnf-α, il-1β, il-6 and il-10 concentrations in the culture supernatant of raw 264.7 macrophages were measured by elisa kits (fig. 2) . raw 264.7 macrophages treated with lps alone produced significant amounts of tnf-α, il-1β, il-6 and il-10 compared to the control group. however, the production of tnf-α was slightly decreased while the levels of il-1β and il-6 were significantly inhibited in a dose-dependent manner when the cells were treated with 12.5, 25 or 50 mg/l of prime-o-glucosylcimifugin ( ⁎ p b 0.05, ⁎⁎ p b 0.01). in contrast, the concentrations of il-10 was significantly increased when the cells were treated with 50 mg/l of prime-o-glucosylcimifugin (p b 0.05 ⁎ ). in order to investigate the mechanism by which prime-oglucosylcimifugin inhibits lps-induced cytokine production, we examined the levels of lps-induced phosphorylation of erk1/2, jnk and p38 mapk in the cytoplasm by western blotting analysis using three different phosphor-special antibodies. in our study, fig. 3a showed that lps stimulation significantly increased the phosphorylation of erk1/2, jnk and p38. however, prime-o-glucosylcimifugin inhibited the phosphorylation of erk1/2, jnk and p38 in lps-induced cells. there were no changes in the expression of non-phosphorylated mapks among groups. furthermore, we examined the effect of prime-o-glucosylcimifugin on iκbα phosphorylation and degradation. the results showed that lps-induced iκbα degradation was inhibited after pretreatment with prime-o-glucosylcimifugin in a dose-dependent manner (fig. 3b ). balf was collected at 7 h after lps administration and the cytokine levels in balf were measured by elisa according to the manufacturer's instructions and as described in the materials and methods section. the levels of tnf-α, il-1β and il-6 in balf were increased dramatically compared with control group (fig. 4) . however, pretreatment with prime-o-glucosylcimifugin (2.5, 5 or 10 mg/kg) significantly down-regulated the levels of tnf-α, il-1β and il-6 in a dose-dependent manner ( ⁎ p b 0.05, ⁎⁎ p b 0.01). seven hours after lps administration, the balf was collected to evaluate the total cell counts and the number of inflammatory cells in balf, such as macrophages and neutrophils. as shown in fig. 5 , lps challenge markedly increased the number of total cells, neutrophils, and macrophages compared to the control group (p b 0.01). in addition, pretreatment with prime-o-glucosylcimifugin was found to significantly decrease the number of total cells, neutrophils and macrophages ( ⁎ p b 0.05, ⁎⁎ p b 0.01). to evaluate lps-induced changes in pulmonary vascular permeability to water, we evaluated the wet weight to dry weight ratio of the lungs. the lung w/d ratio was evidently higher at 7 h after lps administration compared with the control mice as illustrated ( ⁎⁎ p b 0.01). pretreatment of mice with prime-o-glucosylcimifugin significantly reduced the water gain (fig. 6) ( ⁎ p b 0.05, ⁎⁎ p b 0.01) . balf was collected at 7 h following lps challenge to analyze the inflammatory cytokines tnf-α (fig. 4a) , il-1β (fig. 4b ) and il-6 (fig. 4c) . the values presented are the mean ± sem (n = 6 in each group). ## p b 0.01 vs. control group, ⁎ p b 0.05, ⁎⁎ p b 0.01 vs. lps group. the mpo activity (fig. 7) was determined to assess the effects of prime-o-glucosylcimifugin on neutrophil accumulation within pulmonary tissues. lps challenge resulted in significantly increased lung mpo activity compared with the control group ( ⁎⁎ p b 0.01). however, this increase was reduced by prime-o-glucosylcimifugin ( ⁎ p b 0.05, ⁎⁎ p b 0.01). to evaluate the effect of prime-o-glucosylcimifugin on ali, we observed histological changes after prime-o-glucosylcimifugin treatment in lps-treated mice. in the lps group, the lungs were significantly damaged with inflammatory cell infiltration, alveolar wall thickening and interstitial edema. in contrast, prime-o-glucosylcimifugin (2.5, 5 or 10 mg/kg) was found to decrease these histopathological changes (fig. 8) . the results of this study indicate that prime-o-glucosylcimifugin had a promising anti-inflammatory activity. treatment with prime-o-glucosylcimifugin before lps challenge can attenuate lps-induced inflammatory responses in raw 264.7 cells and significantly protect mice against lps-induced ali. the study primarily focused on anti-inflammatory effects of prime-o-glucosylcimifugin on lps-stimulated raw 264.7 cells. lps, a glycolipid that constitutes the major portion of the outermost membrane of gram-negative bacteria, can bind to the cell membrane receptor of the monocytes/macrophages and endothelial cells, then activate the signal-transduction system, thus resulting in the synthesis and release of cytokines and inflammatory mediators [22] . excessive production of pro-inflammatory cytokines not only enhances immune responses by fighting invading pathogens, but also has deleterious effects like perturbing regular hemodynamic and metabolic balances [23] . thus, it is an important target in the treatment of inflammatory diseases to inhibit the pro-inflammatory mediator. as shown in fig. 2 , the levels of the pro-inflammatory cytokines tnf-α, il-1β and il-6 were down-regulated while the level of il-10 was up-regulated in the prime-o-glucosylcimifugin group. il-10, an anti-inflammatory, is well known to down-regulate the production of tnf-α, il-1β, il-6, il-12 and no [24] [25] [26] . a number of cytokines in combination with endotoxin can cause expression of inducible nitric oxide synthase (inos) in macrophages [27] . inducible nos is an important pharmacological target in inflammatory and mutagenesis research. it has been proved that prime-o-glucosylcimifugin had inhibitory effect on the synthesis of no induced by lps in raw 264.7 cells [28, 29] . combined with the finding of our study, it seems like prime-o-glucosylcimifugin inhibits the synthesis of no by producing il-10 in raw 264.7 cells. the anti-inflammatory actions of il-10 can interfere with the production of pro-inflammatory cytokines through the suppression of nf-κb activation by preserving the expression of iκb protein. nf-kb is a key transcriptional factor involved in regulating the expression of proinflammatory mediators, including cytokines, chemokines, and adhesion molecules, thereby playing a critical role in mediating inflammatory responses [30, 31] . under resting conditions, nf-κb is held inactive by iκb. however, nf-κb can be activated by some stimulation of various receptors including tnf receptor, toll-like receptors (tlrs) and t-cell receptor (tcr). persistent activation of nf-κb is central to the pathogenesis of many inflammatory lung disorders including chronic obstructive pulmonary, asthma, pneumonia, and acute lung injury [32] . the activation of nf-κb is implicated in the mapk signaling pathway. inhibition of mapk family pathway, such as erk, p38, and jnk, alleviates the production of pro-inflammatory cytokines [33] . therefore, we investigate the possibility that prime-o-glucosylcimifugin inhibits the production of tnf-α, il-1β, and il-6 via interfering with the activation of mapk and nf-κb. the results showed that prime-oglucosylcimifugin obviously not only inhibited nf-κb activation, but also inhibited lps-induced phosphorylation of erk1/2, jnk and p38 in raw 264.7 cells (fig. 3) . based on the above observations, our results suggest that prime-o-glucosylcimifugin had antiinflammatory ability by suppressing the expression of pro-inflammatory cytokines through blocking the activation of mapk and nf-κb pathways in vitro. acute lung injury is characterized by systemic airway inflammatory response including cytokines (e.g., tnf-α, il-6, il-8), chemokines, pro-inflammatory mediators and a variety of cells, which regulate the migration and pulmonary infiltration of neutrophils into the interstitial tissue [34] . neutrophils are an important component of the inflammatory response that characterizes acute lung injury (ali) [35] . once an inflammatory response is initiated, neutrophils are the first cells to be recruited to sites of infection or injury. although neutrophils have beneficial actions in eradicating microbial infections, excessive neutrophil activation, with resultant release of cytokines and other pro-inflammatory mediators, results in tissue injury and contributes to the development of organ dysfunction, such as ali [9] . high levels of pro-inflammatory cytokines, such as tnf-α, il-1β and il-6, perform a central role in the initiation and propagation of the inflammatory cascade in lps-induced ali [36] . cytokines, such as tnf-α, il-1β, il-6, il-8, and il-10, that are secreted by alveolar macrophages stimulate more chemotaxis and attract more neutrophils to injured lungs [37] [38] [39] . in our study, the levels of tnf-α, il-1β and il-6 in balf were lower in the prime-o-glucosylcimifugin group than in the lps group. these reductions may have contributed to the decreased neutrophil count in balf in the lps-induced ali model treated with prime-o-glucosylcimifugin. edema is a typical symptom of inflammation both in systemic inflammation and in local inflammation [40] . permeability edema which accompanies acute lung injury, severe pneumonia and acute respiratory distress syndrome (ards) is associated with alveolar fluid clearance capacity reduction, increase of capillary endothelial permeability, and alveolar epithelial barrier disruption [41] . to quantify the magnitude of pulmonary edema, we determined the w/d ratio of the lung tissue and prime-o-glucosylcimifugin was shown to inhibit this ratio. on the other hand, mpo is an enzyme located mainly in the primary granules of neutrophils, thus mpo activity in the parenchyma reflects the adhesion and margination of neutrophils in the lung [42] . in the lps-induced ali model, a large amount of pmn is recruited from peripheral blood into the lung, producing a substantial amount of mpo and reactive oxygen derivatives, and finally resulting in a cascade-like response and tissue damage [12, 43] . by contrast, administration of prime-o-glucosylcimifugin markedly suppressed lpsinduced balf neutrophilia (fig. 6 ) and mpo activity (fig. 7) , as well as ameliorated the histopathologic changes in lung tissue produced by lps challenge (fig. 8) . recent studies that have been conducted on pravastatin [44] , ceftiofur [45] , and pinocembrin [46] showed the same regulatory effects as prime-o-glucosylcimifugin, suggesting that this agent may be an important regulator of inflammatory responses. taken together, these results indicate that the protective effects of prime-oglucosylcimifugin on acute lung injury in a mouse model induced by lps may be due to its inhibition of inflammatory mediators and limitation of inflammatory response in the lung. in conclusion, the findings of this study showed that prime-oglucosylcimifugin has a promising anti-inflammatory effect and a protective effect against lps-induced ali. pretreatment with prime-o-glucosylcimifugin inhibited the release of in vitro and in vivo inflammatory responses by counteracting mapk and nf-κb activation. these findings strongly suggest that prime-o-glucosylcimifugin has a potent anti-inflammatory activity and may represent a novel strategy for the modulation of inflammatory responses. further studies are warranted to investigate the clinical usefulness of prime-oglucosylcimifugin. pathology and pathogenesis of severe acute respiratory syndrome new insights into the pathology of acute respiratory failure prevention of lps-induced acute lung injury in mice by mesenchymal stem cells overexpressing angiopoietin fas/fasl-dependent apoptosis of alveolar cells after lipopolysaccharide-induced lung injury in mice endotoxin-induced lung injury in mice: structural, functional, and biochemical responses computational identification of key biological modules and transcription factors in acute lung injury effect of surfactant on pulmonary expression of type iia pla(2) in an animal model of acute lung injury neutrophils and acute lung injury bronchoalveolar and systemic cytokine profiles in patients with ards, severe pneumonia and cardiogenicpulmonary oedema oxidant-mediated lung injury in the acute respiratory distress syndrome bronchoalveolar lavage fluids of patients with lung injury activate the transcription factor nuclear factor-kappab in an alveolar cell line contribution of neutrophils to acute lung injury stem cells in sepsis and acute lung injury therapeutic strategies for severe acute lung injury mesenchymal stem cells for acute lung injury: preclinical evidence illustrated chinese materia medica crude and prepared the constituents of ledebouriella seseloides wolff. i. structures of three new chromones studies on chinese traditional medicine fang-feng (i): structures and physiological activities of polyacetylene compounds from saposhnikoviae radix pharmacognostical studies of chinese drug fang feng identification and kinetics of leukocytes after severe ischaemia/reperfusion renal injury protective effects of sphingosine1-phosphate in murine endotoxin-induced inflammatory lung injury lps-binding proteins and receptors implication of toll-like receptor and tumor necrosis factor alpha signaling in septic shock interleukin 10 reduces the release of tumor necrosis factor and prevents lethality in experimental endotoxemia interleukin-10 attenuates the release of proinflammatory cytokines but depresses splenocyte functions in murine endotoxemia new insights into the molecular mechanism of interleukin-10-mediated immunosuppression insights into the role of nitric oxide in inflammatory arthritis inducible nitric oxide synthase inhibitor of the chinese herb i. saposhnikovia divaricate (turcz.) schischk biotransformation of prim-o-glucosylcimifugin by human intestinal flora and its inhibition on no production and dpph free radical nuclear factor-kappab activation in airway epithelium induces inflammation and hyperresponsiveness targeting mitogen-activated protein kinases for asthma different effects of farrerol on an ova-induced allergic asthma and lps-induced acute lung injury suppression of proinflammatory cytokine production by specific metabolites of lactobacillus plantarum 10hk2 via inhibiting nf-κb and p38 mapk expressions lung injury in acute pancreatitis: mechanisms, prevention, and therapy activation of ampk attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury animal models of acute lung injury acute lung injury: the role of cytokines in the elicitation of neutrophils expression of inducible nitric oxide synthase and inflammatory cytokines in alveolar macrophages of ards following sepsis activated t killer cells induce apoptosis in lung epithelial cells and the release of pro-inflammatory cytokine tnf-alpha protective effect of abamectin on acute lung injury induced by lipopolysaccharide in mice regulators of endothelial and epithelial barrier integrity and function in acute lung injury myeloperoxidase: friend and foe pulmonary neutrophil infiltration in murine sepsis: role of inducible nitric oxide synthase protective effect of pravastatin on lipopolysaccharide-induced acute lung injury during neutropenia recovery in mice ceftiofur attenuates lipopolysaccharide-induced acute lung injury in vitro and in vivo protection provided by pinocembrin against lipopolysaccharide-induced inflammatory responses this work was supported by grants from the national nature science foundation of china (no. 30972212) and the china postdoctoral science foundation (no. 20090461034) . key: cord-270414-gh9agf4x authors: fischer, y.; ritz, s.; weber, k.; sauter‐louis, c.; hartmann, k. title: randomized, placebo controlled study of the effect of propentofylline on survival time and quality of life of cats with feline infectious peritonitis date: 2011-10-12 journal: j vet intern med doi: 10.1111/j.1939-1676.2011.00806.x sha: doc_id: 270414 cord_uid: gh9agf4x background: currently there is no drug proven to effectively treat cats with feline infectious peritonitis (fip). hypothesis: propentofylline (ppf) can decrease vasculitis, and therefore prolong survival time in cats with fip, and increase their quality of life. animals: twenty‐three privately owned cats with fip. methods: placebo‐controlled double‐blind trial. fip was confirmed by histology or immunostaining of feline coronavirus (fcov) antigen in effusion or tissue macrophages or both. the cats were randomly selected for treatment with either ppf or placebo. all cats received additional treatment with glucocorticoids, antibiotics, and low molecular weight heparin according to methods. results: there was no statistically significant difference in the survival time of cats treated with ppf (8 days, 95% ci 5.4–10.6) versus placebo (7.5 days, 95% ci 4.4–9.6). the median survival time of all cats was 8 days (4–36 days). there was neither a difference in quality of life (day 7, p = .892), in the amount of effusion (day 7, p = .710), the tumor necrosis factor‐alpha (tnf‐α) concentration (day 7, p = .355), nor in any other variable investigated in this study, including a complete blood count, and a small animal biochemistry profile. conclusions and clinical importance: this study did not detect an effect of ppf on the survival time, the quality of life, or any clinical or laboratory parameter in cats with fip. therefore, ppf does not appear to be an effective treatment option in cats with a late stage of the disease fip. f eline infectious peritonitis (fip) is one of the most frequent causes of death in young cats. [1] [2] there is no proven record of cats with a confirmed diagnosis having recovered from fip. 3 thus, fip is usually lethal; no controlled study has verified the success of any treatment used to date. 1,4-6 therefore, providing objective evidence of the effectiveness of any treatment against this disease is important. several case reports can be found in the online veterinary information network (http://www.vin.com) that describe a positive effect of the methylxanthine derivative pentoxifylline (ptx) (trental a ) on the survival time in cats with fip. several veterinarians and well-known specialists in feline medicine have suggested that the use of ptx can be effective in treating cats with fip. 4, [6] [7] [8] according to those reports, ptx does not cure but is suggested to prolong the life of these cats. [4] [5] 8 in these reports it has been suggested that ptx is likely to decrease vasculitis, which is responsible for the majority of clinicopathological findings of fip. 1 the mode of action of the methlyxanthine derivatives is not fully understood, and the mechanism remains unknown. [9] [10] the ptx inhibits several cytokines, such as interleukines and tumor necrosis factor-alpha (tnf-a). 9 there are studies in rats and humans (in vivo and in vitro) describing the inhibition of cytokines by ptx, [11] [12] [13] [14] [15] and ptx and other methylxanthine derivatives seem to suppress tnf-a synthesis. 15 these proinflammatory cytokines play a major role in the pathogenesis of vasculitis. 16 therefore, it has been suggested that vasculitis may be effectively controlled with ptx because of its effect in neutralizing or suppressing these cytokines. [11] [12] [13] [14] [15] propentofylline (ppf) and ptx have mainly been trialed for use in people with peripheral vascular diseases, [17] [18] [19] [20] cerebrovascular diseases (such as alzheimer's disease, brain ischemia, or cerebrovascular insufficiency), 9, [20] [21] [22] endotoxemia, 14, 23 and ischemic heart disease. 20, 24 tnf-a also induces fibrinogen synthesis, [25] [26] [27] and is responsible for an increased production of free radicals alt alanine aminotransferase ap alkaline phosphatase ci confidence interval fcov feline coronavirus felv feline leukemia virus fip feline infectious peritonitis fipv feline infectious peritonitis virus fiv feline immundeficiency virus ifat immunofluorescent antibody technique ppf propentofylline ptx pentoxifylline rbc red blood cells spss statistical package for the social sciences tnf-a tumor necrosis factor-alpha tp total protein wbc white blood cells which cause endothelial cell damage. 28 by inhibiting the synthesis of tnf-a by activated monocytes, ptx can probably decrease fibrinogen levels, a common component of the effusion in cats with fip. 10, 19 it was previously postulated that high fibrinogen levels could be an index of tnf-a levels. this finding is supported by a close correlation between decreased fibrinogen levels and clinical improvement. 19 a study into geriatric cachexia in humans additionally showed that ptx may decrease cachexia by down-regulating proinflammatory cytokines, such as tnf-a, interleukin 1 and 6, serotonin, and interferon-c. because cats with fip are often anorectic, this was considered to be another positive effect of the methylxanthine derivatives on the well-being of cats with fip. 24, 29 ppf, another methylxanthine derivative, is licensed in several european countries (including germany) for veterinary use in dogs. it is very similar to ptx (which is not licensed in germany for veterinary use) in its chemical structure as well as in its pharmacological effects. 30-31 both ptx and ppf inhibit several cytokines, such as interleukins and tnf-a. 9 furthermore, ppf has already been applied securely and effectively to cats with feline asthma. 32 therefore, ppf instead of ptx was used in this study. the aim of this study was to evaluate the efficacy of ppf on the survival time and quality of life in cats with a confirmed diagnosis of fip in a placebocontrolled double-blind trial. the study included 23 client-owned cats. inclusion criterion to enter the study was the definitive diagnosis of fip. all cats were presented to the clinic of small animal internal medicine, lmu university of munich, germany. an informed consent of participation signed by the owners was obtained for all cats. this study fulfilled the general german guidelines for prospective studies with owners' consents and was approved by the ethics committee and the animal protection officials of the regierung von oberbayern, germany (permission no. 55.2-1-54-2531-127-09). consecutive cases of cats with confirmed fip that had owners willing to participate in the study, presented to the clinic between april 2009 and december 2010, were entered into the study. diagnosis of fip was either confirmed by detection of feline coronavirus (fcov) antigen in macrophages in the effusion using direct immunofluorescence 33 (n = 9), by histopathological examination of tissue, positive immunohistochemical staining of fcov antigen in macrophages 34 (n = 22), or by both. cats with feline immundeficiency virus (fiv) or progressive feline leukemia virus (felv) infection were not included in the study (snap felv/ fiv test b ). cats with severe clinical signs (karnofsky's score 35 <30%) or a survival time less than 72 hours after treatment initiation were retrospectively excluded (2 cats). one cat had to be excluded in retrospect because of a lack of owner compliance. seventeen of the 23 cats (74%) were european shorthair cats, 2 (9%) were british shorthair cats, and there was one (4%) of each of the following breeds: birman, persian, norwegian forest cat, and persian crossbred. the youngest cat was 13 weeks old and the oldest cat 2.8 years (mean, 0.9 years; median, 0.7 years; interquartile range, 0.42-1.25 years). fifteen (65%) cats were younger than 12 months; 20 (87%) cats were younger than 2 years. seventeen (74%) cats were male (5 neutered), and 6 (26%) female (2 neutered). the study was designed as a placebo-controlled, double-blind randomized trial. cats were randomly assigned to the ppf (n = 7) or placebo group (n = 16). the dosage of ppf was based on the dosage used of ptx to treat cats with fip in the literature and anecdotal case reports of different authors. in those reports, 10-15 mg/kg or 100 mg/cat every 12 hours was given po. 6 according to studies in humans, ppf and ptx are used at the same dosage. 36 cats in this study therefore received a median dosage of 18-25 mg/kg ppf c,d during the whole study period. alternatively, cats received the similar amount of tablets of placebo e (containing lactose, magnesium stearate, and cellulose) every 12 hours po. the ppf and the placebo pills were coded. therefore, veterinarians and owners giving the pills were blinded to identity of the treatment. the code was broken after 23 cats had been treated. all results (including survival time, karnofsky's score, blood and effusions variables, and volume of collected effusion) were obtained blinded. all cats were also treated with glucocorticoids. in case of effusion at day of presentation (n = 21), dexamethasone f (1 mg/kg) was given intraperitoneally or intrathoracically (depending on the location of effusion) every 24 hours for 6 days after thoraco-or abdominocenthesis. cats without effusion (n = 2) received dexamethasone f (1 mg/kg) sc for 6 days. after this period, all cats were treated with oral prednisolone g,h (2 mg/kg) every 24 hours until death. in addition, cats received amoxicillin/clavulanic acid i (12.5 mg/kg iv every 12 hours) for 7 days; dalteparin sodium j (75 iu/kg sc every 12 hours) for 5 days, which was gradually tapered within the next 2 days (day 6: 36 iu/kg, day 7: 18 iu/ kg); as well as fluid and nutritional treatment if necessary during the hospitalization. if the cats were not properly vaccinated, they were treated sc with one dose (4 ml) of immunglobulins k (a product containing antibodies against feline panleukopeniavirus, feline herpesvirus, and feline calicivirus). this product was given to decrease the risk of acquiring an infectious disease because of immune suppression by glucocorticoid treatment and hospitalization. glucocorticoids were given, because it is currently the only treatment thought to have a beneficial effect on cats with fip although there are no controlled studies. 3, 37 antibiotics were administered to minimize the risk of bacterial infection because paracentesis was performed daily (if effusion was present), and because of the high dosage of glucocorticoid treatment. cats also received low molecular weight heparin (dalteparid sodium) to minimize the risk of a disseminated intravascular coagulation (dic), which is often observed in cats with fip. 1,38-39 all cats were either hospitalized during the 1st 7 days after treatment initiation or had to be presented to the clinic daily. physical and ultrasound examinations were performed daily. the general condition was characterized by the karnofsky's score. the index enables judgment of quality of life and well-being in cats by means of a score of 0% (dead) to 100% (absolutely healthy and happy). 35 on day 0 (day of inclusion in the study) as well as on the control days (day 7, 14, and 28), a complete physical examination was performed, and blood was collected. a cbc was performed with an automatic analyzer (cell-dyn l ), the small animal biochemistry profile (see table 1 ) was examined using an automatic analyzer (hitachi m ). aliquots of the serum samples were preserved at -80°c for detection of tnf-a. if present, effusion was aspirated, and the amount was recorded. depending on their health status, cats were returned to their owners after day 7. the owners were asked to fill in a provided diary recording temperature, respiratory rate, weight, general condition (duration of sleeping time, eating, playing, and grooming behavior) every day, as well as any problem noticed by the owners. follow-up examinations in the clinic were scheduled on days 7, 14, and 28, including physical examination, examination of a cbc, a small animal biochemistry profile, and ultrasound to detect the presence of effusion. tnf-a was measured in the serum (on day 0, 7, 14, and 28) using an elisa. n because the elisa is only validated for cell culture supernatants, a spiking experiment using serum samples was performed. serum components can impact the accuracy of elisa results and may interfere with antibody binding or show cross-reactivity. to assess recovery of serum samples and to assess accuracy of measured values, 200 pg tnf-a were spiked into a serum sample of a healthy cat. the sample was diluted in sample diluent (pbs o + 10% fetal calf serum p ) 2-fold to yield samples containing 100, 50, and 25 pg. as a control, sample diluent was spiked and diluted accordingly. the spiked undiluted control yielded results in the expected range (89%). the spiked undiluted serum sample showed recovery of 65%, indicating inhibitors of detection in the serum. the serum at a 1 : 2 dilution showed recovery of 73% compared to 88% of the diluted con-trol. the recovery loss of 15% was considered acceptable, and interference of inhibiting components appeared to be not severe; all serum samples were therefore diluted 1 : 2 for detection of tnf-a. the elisa was performed according to the manufacturer's instructions. a 96-well microplate n was coated with capture antibody by overnight incubation. the next day, the wells were washed and samples (diluted 1 : 2) and standards n were incubated for 2 hours at room temperature. after washing, the detection antibody n was added and incubated for another 2 hours at room temperature. for detection, streptavidin-hrp n was used with tetramethylbenzidin n as a substrate solution. the reaction was stopped after 10 minutes with 0.5 m sulfuric acid. n the elisa was measured with a bio tek reader, q and the data analysis was performed using gen5 data analysis software. r all cats were randomly assigned to 2 groups, the ppf group and the placebo group. a power analysis had been performed before starting the study (using pass, 2008; http://www.ncss. com/pass). for this analysis, a clinical relevant difference in median survival time was set at 21 days, assuming that animals treated with ppf would survive at least 21 days longer than animals receiving placebo. these differences could have been detected with 18 animals per group, using a power of 80% and a significance level of 5%. however, an interim analysis on the survival time was performed after 23 cats had been treated, because most cats in the study at that time point have survived for less than 29 days, and the median survival time was not significantly different between the groups (median survival time ppf: 8.0 days; placebo: 7.5 days). therefore, it was decided to terminate the study prematurely for reasons of animal welfare, as the expected clinical relevant differences and the difference of the survival time were clearly not achievable. statistical analysis was performed using statistical software spss version 17.0 (http://www.spss.com). variables compared between both groups (ppf or placebo group) included survival time, karnofsky's score, red blood cells (rbc), hemoglobin, hematocrit, platelets, white blood cells (wbc), monocytes, lymphocytes, banded neutrophils, mature neutrophils, alanine aminotransferase (alt), alkaline phosphatase (ap), bilirubin, total protein (tp), albumin, albumin to globulin ratio, urea, creatinine, glucose, and the volume of effusion. a difference in the survival time between both groups was evaluated using a log-rank test. differences between the parameters of the 2 groups at day 0, day 7, and day 14 were investigated using a mann whitney u test. p-values <.05 were considered significant. a bonferroni correction was performed to rule out multiple test interference. a 5% significance level was assumed for all variables, and thus the p-value of .05 was divided through the number of tests performed (n = 20). therefore, a final value of p .0025 for each variable was considered significant. there was no statistically significant difference in any blood parameter or in the amount of effusion at any time point between cats treated with ppf, and those that received placebo ( table 1 ). the karnofsky's score of both groups also showed no statistically significant difference at the start of the study. cats survived between 4 and 36 days (median, 8 days). the median survival time of cats in the ppf group was 8 days (range 4-36; 95% confidence interval [ci] 5.4-10.6), and of cats in the placebo group the median survival time was 7.5 days (range 4-22; 95% ci 4.4-9.6). the difference in survival time between the 2 groups was not significantly different (p = .665) (fig 1) . twenty-two of 23 (96%) cats survived less than 29 days. these 29 days were preset as expected minimum survival time in cats receiving ppf. in a previous study, a median survival time of 8 days was detected in cats with fip. 3 in the present study, it was assumed that cats treated with ppf would live at least 21 days longer than those receiving placebo (with a median survival of 8 days), as this makes a relevant difference for the owners. 3 no statistically significant differences of any blood parameter or of effusion were apparent after the 7 and 14 day period of treatment between the ppf group and the placebo group. the karnofsky's score of both groups on the evaluated control days (day 7 and day 14) also showed no significant difference. on day 7, only 14 cats remained in the study. two of them improved 10% in the karnofsky's score, 5 cats showed no difference and the karnofsky's score of 7 cats deteriorated for at least 70%. on day 14, only 4 cats remained in the study and the karnofsky's score of all these cats had deteriorated for at least 80% compared to day 0. no statistical evaluation was performed after day 14 because only 1 cat was alive at day 28 (next control day). only in 6 cats (4 of the ppf group, 2 of the placebo group) serum samples of more than one time point were available for the comparison of the tnf-a concentration during treatment with ppf. a significant decrease was not found in any of these cats; conversely, most cats even showed increased tnf-a serum levels during the study period. in this study, there was no statistically significant difference in the survival time of cats treated with ppf versus placebo. there was also no statistically significant difference in any other variable evaluated between both groups, including the cbc and a small animal biochemistry profile (as shown in table 1 ). the median survival time (8 days) of cats with fip after definitive diagnosis in this study was nearly identical to the median survival of the study of ritz et al. 3 there are no other reports on median survival times of cats after fip was confirmed. however, in the study of ritz et al, 3 several cats survived longer than 4 weeks, which was not the case in the present study. unfortunately, the desired effect of ppf was not observed. it has been proposed that ppf may decrease the volume of effusion, by inhibiting cytokines (particularly tnf-a) and thereby reducing resulting vasculitis. there was neither a significant difference in the amount of effusion between the ppf and the placebo group, nor a decrease in tnf-a in any of the cats in which serial measurement was performed. a cure was never the ultimate goal in the use of ppf in cats with fip, because it is not an antiviral drug. however, because of the pharmacological features it was assumed to have positive effects on the well-being of the cats and the survival time. ppf was meant to inhibit the tnf-a production, 9 which is involved in the development of vasculitis. [11] [12] [13] [14] [15] the tnf-a concentration, however, did not decrease in the cats treated with ppf, but increased instead. most likely, ppf was not even able to exhibit its function within this short period of time, and the tnf-a increase mainly reflected severe progression of the disease. reasons for the lack of efficiency can be multifaceted. the most probable reason is that treatment may have been initiated too late. if signs of vasculitis were apparent, the immune-mediated process in cats with fip might have been progressed too far to be delayed by ppf. in the present study, 21 of the 23 cats already showed effusion at the day of presentation. as shown in an experimental trial, signs of fip become apparent 1-2 weeks after inoculation of the mutated feline infectious peritonitis virus (fipv). 40 as the effect of ptx is described by the manufacturer information a to be seen after 2-4 weeks after treatment initiation, and it is an assumption that the same time frame would apply to ppf, most of the cats were already dead before an effect could be reached. a further reason for lack of ppf efficiency in this study could be that the treatment intervals could have been too long. an application of ppf every 12 hours was used in this trial following the anecdotal reports describing an effect of ptx in cats. 4, 6, 32, 41 the manufacturer instruction recommends administration of ptx 3 times daily in human medicine, 20 because of a relatively short plasma half-life of 0.4-0.8 hours of the drug. a pharmacokinetic study in dogs indicated that ptx be administered every 8 hours. 42 the reasons behind the reported beneficial effects of ptx described in case reports (http://www.vin.com) are currently unknown. treatment might have been initiated earlier in these cats. alternatively, fip was not confirmed by histopathology or immunofluorescent antibody technique (ifat) in most of these cases; so, these cats could have suffered from other diseases. some of the cats might have had a "non-effusive" form of fip, which is considered to have longer survival times than in cats with effusion. 43 drug interactions between ppf and the other medications (especially the glucocorticoids) in this study are a possibility, but are not reported. in addition, in a recent study in cats with asthma, glucocorticoids and ppf were safely used in combination with no adverse interactions. the glucocorticoid dose can be reduced by addition of ppf to treatment. 32 therefore, no adverse effects were expected with the combination ppf and glucocorticoids in the present study. glucocorticoids are routinely used in cats with fip, 1,3,44-47 as it is not the virus itself that causes major damage but the cat's own immune reaction that leads to the fatal consequences. there are no evidence based studies that glucocorticoids have a positive effect in cats with fip. 37 the cats of the present study received an immunosuppressive dose of glucocorticoids (2 mg/kg). together with the stress caused by hospitalization and daily paracenthesis, glucocorticoids might be a more confounding factor. [48] [49] potentially, a lower dose of glucocorticoids, or no glucocorticoids at all, might be better for "long-term" treatment. the beneficial effects of glucocorticoids in the treatment of fip must be questioned given the median survival time is 8-9 days in the present and in the previous study, 3 both in treatment and placebo groups. 3 therefore, future treatment study protocols should include a 3rd group of cats that receive no glucocorticoids. alternatively a doubleblinded study just evaluating glucocorticoids as treatment option for fip could be performed. there was no statistically significant difference in the karnofsky's score during the treatment period between the two groups. few cats showed an increased wellbeing shortly after participating in the study. this was most likely induced by the corticosteroids and was not the effect of ppf, because this phenomenon could be observed in both groups. however, the improvement in the general condition was not long-lasting, and cats deteriorated rapidly between 4 and 21 days after treatment initiation. this study had several limitations. the 1st limitation is the unequal distribution of cats to the ppf and the placebo group. as this was a blinded, randomized trial, the distribution could not be influenced. another limitation might be the small number of cats (only 2) without initial effusion. definitive diagnosis in cats without effusion, however, is much more difficult to obtain. 33 the ppf might be more useful in cats without effusion, as it may have a chance to prevent vasculitis and therefore effusions feline infectious peritonitis feline infectious peritonitis and feline enteric coronavirus infections. part 1 effect of feline interferon-omega on the survival time and quality of life of cats with feline infectious peritonitis feline infectious peritonitis: what's new? available at: www.ancats.com.au/pdf files/feline infectious peritonitis summary diagnosis and treatment of fip in the real world 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vasculitis pharmacokinetics of pentoxifylline in dogs after oral and intravenous administration clinicopathological findings and disease staging of feline infectious peritonitis: 51 cases from 2003 to 2009 in taiwan infectious peritonitis in a cat that subsequently developed a myeloproliferative disorder beitrag zur therapie der fip effect of thromboxane synthetase inhibitor on feline infectious peritonitis in cats use of recombinant feline interferon and glucocorticoid in the treatment of feline infectious peritonitis risk factors for feline infectious peritonitis among cats in multiple-cat environments with endemic feline enteric coronavirus die diagnositik der felinen infektio¨sen peritonitis (fip): retrospektive und prospektive untersuchungen we thank the intervet deutschland gmbh, unterschleißheim, germany, for partial support of this study. key: cord-333650-4towah1t authors: malmo, jostein; moe, nina; krokstad, sidsel; ryan, liv; loevenich, simon; johnsen, ingvild b.; espevik, terje; nordbø, svein arne; døllner, henrik; anthonsen, marit w. title: cytokine profiles in human metapneumovirus infected children: identification of genes involved in the antiviral response and pathogenesis date: 2016-05-12 journal: plos one doi: 10.1371/journal.pone.0155484 sha: doc_id: 333650 cord_uid: 4towah1t human metapneumovirus (hmpv) causes severe airway infection in children that may be caused by an unfavorable immune response. the nature of the innate immune response to hmpv in naturally occurring infections in children is largely undescribed, and it is unknown if inflammasome activation is implicated in disease pathogenesis. we examined nasopharynx aspirates and blood samples from hmpv-infected children without detectable co-infections. the expression of inflammatory and antiviral genes were measured in nasal airway secretions by relative mrna quantification while blood plasma proteins were determined by a multiplex immunoassay. several genes were significantly up-regulated at mrna and protein level in the hmpv infected children. most apparent was the expression of the chemokine ip-10, the pro-inflammatory cytokine il-18 in addition to the interferon inducible gene isg54. interestingly, children experiencing more severe disease, as indicated by a severity index, had significantly more often up-regulation of the inflammasome-associated genes il-1β and nlrp3. overall, our data point to cytokines, particularly inflammasome-associated, that might be important in hmpv mediated lung disease and the antiviral response in children with severe infection. our study is the first to demonstrate that inflammasome components are associated with increased illness severity in hmpv-infected children. the negative sense single strand rna virus human metapneumovirus (hmpv) has since its discovery in 2001 emerged as a commonly detected respiratory tract pathogen involved in the npas were tested using pcr for hmpv (a1, a2a/b, b1, b2), adenovirus, human bocavirus-1, coronavirus (oc43, 229e and nl63), enterovirus, influenza a and b virus, parainfluenza virus type 1-4, parechovirus, rsv, rhinovirus, bordetella pertussis, chlamydophila pneumoniae and mycoplasma pneumoniae. nucleic acid was extracted using nuclisens easy-mag according to the manufacturer's protocol (biomerieux). all pcrs were in-house realtime assays based on taqman probes [16] . for hmpv detection, primers targeting the n-gene allowing detection of all four hmpv genotypes were used as described elsewhere [17] . genotyping was performed by sequencing an amplified f-gene pcr product [18] using a 3130 genetic analyzer (applied biosystems) and comparing sequenced data with the nucleotide blast database (www.ncbi.nlm.nih.gov/blast/). a total of 162 children tested positive for hmpv (6.1%) in npa. for the purpose of the present study, 30 children satisfying all of these criteria were recruited: an npa was sampled less than one week after onset of symptoms and within one day after admittance, either hmpv genotypes a2 or b2 were detected in npa, no viral co-infections detected in npa, and serum levels of crp <100 mg/l. clinical data were recorded prospectively or retracted from medical records. upper respiratory tract infection (urti) was diagnosed when rhinitis, pharyngitis, tonsillitis and/or otitis media (serous, simplex or purulent) was found without signs of lower respiratory tract infection (lrti). lrti was diagnosed in children with signs and symptoms of respiratory difficulty such as tachypnea, retractions and nasal flaring, signs of lower airway obstruction (wheezing, prolonged expiration, rhonchi), focal findings (crepitations) and/or a positive chest x-ray (infiltrates, atelectasis, air trapping). lrti was divided in bronchiolitis (age <2 years, tachypnea, retractions, wheezing, crepitations), obstructive bronchitis (age >2 years, signs of lower airway obstruction), asthma exacerbation with signs of lower airway obstruction, and pneumonia (cough, tachypnea, localized crepitations, x-ray infiltrates). a clinical severity score previously used to determine hmpv/rsv disease severity in children [7, 8] was calculated for each infected child as the sum of these variables 1) length of hospital stay 5 days (1 point), 2) oxygen demand (1 point), 3) ventilatory support by continuous positive airway pressure ventilation (1 point), and 4) ventilatory support by endotracheal intubation and overpressure ventilation (2 points). ten children admitted for elective surgery with no symptoms of rti in the last two weeks were randomly selected and included as controls. the clinical and virological findings are summarized in table 1 , and a detailed overview for the individual patients is presented in the s1 table. mrna quantification cdna was synthesized from rna isolated from the collected npas using the qscript kit according to the manufacturer's protocol (quanta). quantitative pcr (qpcr) was performed using perfecta sybr green reaction mix (quanta) and a steponeplus instrument (life technologies) with the temperature profile 95°c for 20 s, 40 cycles at 95°c for 3s and 60°c for 30s. fold-change in ifn-β, ifn-γ, il-28, iκbα, il-1β, il-18, nlrp3, ip-10, tnf-α, il-6 and isg54 gene expression relative to the control with highest levels of target gene mrna was calculated using the ddc t -method with gapdh as housekeeping gene. data was plotted as boxplots where the box represents 50% of the values, the horizontal line indicates the median, whiskers show the maximum and minimum values, and the closed circles indicate outliers. primer sequences used in this study are shown in the s2 table. protein quantification blood samples were collected in vacuette edta tubes (greiner bio-one) and the fractions separated according to the manufacturer's protocol. the concentrations of proteins in the plasma fraction were measured using a bio-plex multiplex system (bio-rad) and cytokine assays for ifn-β, ifn-γ, il-28a, il-1β, il-18, ip-10, tnf-α, il-6, gro-α and gro-β (bio-rad). categorical variables were analyzed with the pearson chi-square test or the 2-tailed fisher's exact test. continuous and nearly normal distributed variables were analyzed with the student's t-test or anova-test, and non-parametric variables were compared by mann-whitney u tests. a two-sided p-value less than 0.05 was considered statistically significant. normally distributed and continuous variables are presented as average±standard deviation and nonnormally distributed variables as median (range). analyses were performed using the statistical package of social science (spss, version 19.0) and sigmaplot (version 12.0). the study was approved by the regional committee for medical and health research ethics (rek, mid-norway). caregivers to all patients and controls received written information about the study. written consent was obtained at the hospital from the caregivers of all controls and the majority of the patients included in the cohort. due to practical challenges by enrolling patients 24 hours a day, 7 days a week, some patients were enrolled after discharge from the hospital. their caregivers received written information after the hospital stay and children were included if the caregivers did not resist enrollment by taking contact to the hospital within 4 weeks. this study was based on a cohort involving 2656 children hospitalized with respiratory tract infection (rti) where 162 tested positive for hmpv. from the hmpv positive children we selected samples without detectable co-infections (n = 30) as described in the methods section. further, children admitted for elective surgery without recent symptoms of rti were randomly selected and included as controls (n = 10). table 1 shows that the majority of the children were diagnosed with lower respiratory tract infection (bronchiolitis or pneumonia), and the disease severity ranged from 0-4. one half of the patients had more severe disease with a score higher than zero (s1 table) . during the period of this study, a2 (43% of the samples) and b2 (42%) were the dominant genotypes found in the cohort and therefore our focus. the length of hospitalization was average 4.1±4.2 days for a2 and 3.1±2.0 days for b2 infected children (p>0.05, student's t-test). the maximum c-reactive protein (crp) values in the patients ranged from approximately 5 to 100 mg/l with the majority below 50 mg/l, indicating that severe bacterial infections were unlikely. to determine the presence of antiviral cytokines in children infected with hmpv and controls, we initially investigated the expression of type i, ii and iii ifns. fig 1a shows that only a2 infected children had slightly elevated mrna levels of the type i ifn-β compared to the controls. as shown in fig 1b, the mrna levels of the type ii ifn-γ was not elevated in any of the patient groups relative to the controls. in correspondence with this, ifn-γ protein was not detected in the blood samples from any of the investigated children (data not shown). finally we evaluated the mrna expression of the type iii ifn il-28. fig 1c shows that both the a2 and b2 positive patients had significantly increased levels of il-28 expression. next, we wanted to investigate the possible involvement of nf-κb and inflammasome-associated genes in response to hmpv infection. fig 2 shows the mrna expression of a) iκbα, a repressor gene induced by nf-κb activation [19] , b) il-1β, c) il-18 and d) nlrp3 in hmpv infected children and controls. the expression of il-18 was significantly increased for both a2 and b2 infected children. however, for the other genes investigated their expression was not significantly increased compared to the controls. it should be noted that the standard deviations for the iκbα, il-1β and nlrp3 expression were high. this was caused by a considerable fraction of the hospitalized children having undetectable expression while others had high mrna levels relative to control subjects. in contrast, children from the control group exhibited similar levels of iκbα, il-1β and nlrp3 mrna levels. to illustrate this individual variation, differences in gene expression for individual a2 and b2 positive patients are outlined in fig 3. from this figure, it can be seen that a) iκbα, b) il-1β and c) nlrp3 were significantly upregulated in several patients. interestingly, most of the patients with upregulation of iκbα, il-1β and nlrp3 had increased expression of all three genes. to evaluate the involvement of other relevant immunomodulator genes, we measured the mrna expression of ip-10, tnf-α, il-6 and isg54. as shown in fig 4a, the expression of ip10 was increased for both the a2 and b2 infected patients. the tnf-α expression (fig 4b) was significantly higher in patients infected with b2 compared to the controls. further, the expression of il-6 ( fig 4c) was not increased at mrna level in any of the groups with hmpv infected children. finally, the expression of isg54 (fig 4d) was markedly increased for both a2 and b2 infected patients. as mentioned earlier, the expression of inflammatory genes can possibly promote pathogenic events during rti. consequently, we wanted to investigate if the gene expression profiles differed in children with a severity index of zero compared to those with more serious disease and a higher severity score. as summarized in table 2 , the mrna expression of il-1β and nlrp3 were more frequently upregulated in the children with a severity score of 1-4. finally, the concentration of several secreted cytokines in blood plasma was measured for a random subset of the patients (n = 10) and the controls (n = 10) as shown in fig 5. herein, we also included the chemokines gro-α and gro-β to evaluate implications of neutrophil recruitment to the lungs. fig 5 shows that all of the cytokines included in the analysis, except for il-28, were detected at significantly higher concentrations in patients compared to the controls. the percentage with up-regulated genes is given in the brackets. statistical significance is indicated with the p-value from a fisher's exact test comparing the two groups. further, as mentioned earlier, ifn-γ was not detected above the threshold value in the controls or patient samples. this study aimed to identify inflammatory genes associated with hmpv mediated disease. we performed an analysis of npas and blood samples collected from children hospitalized with rti. herein, we identify specific cytokines, such as ip-10, il-18, tnf-α, and isg54, that are elevated in hmpv mediated lung disease. of noteworthy interest is the up-regulation of il-1β and nlrp3 in samples from children with the most severe respiratory tract infection. this suggests that the inflammasome is involved in the innate immune response to hmpv in these patients, either as a protective mechanism or as a response to lung damage. type i and iii ifns are responsible for the induction of an antiviral state in cells immediately after infection [20] . while type i ifns are produced by many different cell types, type iii ifns seems to be mostly produced by plasmacytoid dendritic and epithelial cells where the epithelial cells are the main targets for activity [21] . due to their powerful and potentially harmful effects, the production of these cytokines is tightly regulated [22] . our results showed that the type i ifn-β mrna in npa was marginally up-regulated for the a2 infected patients, but the protein concentrations in plasma were markedly increased for both a2 and b2 positive patients. this inconsistency could be explained by the protein being produced by tissue resident cells in e.g. the lung or lymph nodes. another explanation could be a lag in the induction of protein synthesis in the epithelial cells after the elevation of mrna transcription, since it is assumed that cytokines are able to enter the bloodstream from lung tissue during inflammation. for type iii ifn il-28 we observed increased mrna expression both for a2 and b2 infected children. however, il-28 protein was not significantly elevated in blood plasma. il-28 is prominently expressed in lung epithelial cells, and suggested to prevent viral invasion through skin and mucosal surfaces [23] . hence, the lack of increased il-28 protein in blood could, similar as suggested for ifn-β, be explained by a lag in the induction of protein synthesis. the expression of il-28 has been shown to be triggered by viral infection, and it displays immunological properties similar to the type i ifn-α/β [23] . recently, it has been shown that hmpv induces expression of il-28 in mice and the cytokine was suggested to have an important protective role [24] . for rsv, which is phylogenetically and clinically closely related to hmpv, an in vitro study has shown that il-28, in contrast to ifn-α/β, is induced during infection of primary respiratory tract cells [25] . further, a study on infants hospitalized with rsv infection and bronchiolitis showed induced expression of il-28 in the airways [26] . overall, our results adds to existing data suggesting that ifn-β and il-28 are induced to some extent during hmpv infection. chemoattractants are induced during acute inflammation events to recruit effector cells of the immune system. in our study, elevated mrna expression of the chemoattractant ip-10 was observed for a2 and b2 infected patients. further, elevated levels of ip-10 protein, and also of the chemokines gro-α and gro-β were found in blood plasma. ip-10 has previously been shown to be significantly elevated seven days post infection with hmpv a2 in mice [27] . the expression of ip-10 may be induced by ifn-γ [28] , ifn-α/β [29] or tnf-α [30] secreted by different immune-cells. in our study, despite elevated ip-10, we did not measure any increased expression of the type ii interferon ifn-γ at mrna or plasma protein levels. the lack of ifn-γ detection in npas from hmpv infected children has also recently been reported elsewhere [31] . our data suggests that ifn-β and tnf-α, which was significantly detectable in plasma, might be responsible for induction of ip-10. in addition, ifn-γ-independent induction of ip-10 has been demonstrated in human peripheral blood mononuclear cells [32] . in this setting the type iii interferon il-29, which is closely related to il-28 that was shown to be upregulated herein, was correlated to ip-10 induction. of note, when infecting human epithelial cells with hmpv in vitro, we observed that il-28 mrna was induced and that il-29 showed a similar trend (data not shown), as also suggested elsewhere [24] . hence, il-28/29 could mediate ifn-γ independent ip-10 induction in hmpv infection. ifn-γ-independent ip-10 expression has also previously been reported in bal samples from patients during respiratory tract virus infection [33] . we found that mrna expression of the pro-inflammatory cytokine tnf-α was elevated only in the b2-infected children. however, the level of tnf-α protein in plasma was significantly increased both for a2 and b2 infected patients. an explanation for this could be different expression kinetics for mrna and protein. tnf-α induces nf-κb [34] and, among other functions, it is involved in the recruitment of neutrophilic cells to the lungs [35] . tnf-α production has previously been shown to be at high levels in nasopharyngeal secretions from rsor influenza-virus infected children [36] . when comparing the patients with a severity index of 0 and those with an index of 1-4, tnf-α had significantly higher levels of expression (p<0.001, student's t-test). this could indicate that the presence of tnf-α in the respiratory tract directly or indirectly affects the severity of disease in hmpv infected children. it is worth mentioning that the hmpv c t value was similar for the patients with severity index 0 and higher (s1 table) , suggesting similar extent of viral replication in these patients. we did not detect any increase in mrna levels of the pro-inflammatory cytokine il-6. on the other hand, the levels of il-6 in blood plasma were significantly increased for the hmpv infected patients. similar as for ifn-β, this observation could be explained by il-6 protein being produced by tissue-resident cells or, alternatively, downregulation of gene expression after a peaked response. a previous study comparing the expression of several inflammatory cytokines in hmpv, rsv and influenza virus, detected elevated levels of tnf-α, il-6 and il-1β protein in nasal washes from infants with rti [9] . together with our data showing increased detection of these proteins in blood from patients with rti compared to the controls, this could suggest that at least a part of the protein detected in blood might have been secreted from cells in the respiratory tract. the antiviral cytokine isg54 was expressed at the highest relative levels of any of the genes included in this study. the mrna expression was up-regulated for both hmpv a2-and b2-positive npa samples. isg54 is known to be induced by viruses downstream of pattern recognition receptors and ifns [37] . the expression of isgs is initiated by type i and type iii interferons such as ifn-β and il-28. we did indeed find that these ifns were up-regulated at mrna and/or protein level in hmpv infected children. the functional effects and clinical involvement of isg54 are largely unknown. nevertheless, isg54 has been shown to inhibit viral replication in vitro and in vivo [38] . interestingly, mice lacking isg54 have recently been shown to be subject to high mortality when infected with the mouse respiratory tract pathogen sendai virus-a negative sense, single-stranded rna paramyxovirus [39] . this suggests that isg54 has a protective function and limits pathogenesis in mice. however, in our study, we were not able to identify any differences with respect to length of hospitalization or severity index when comparing the groups of patients expressing isg54 or not (p>0.05, student's ttest), suggesting a general protective function of this cytokine in children. our study reveal that most of the hmpv-infected children exhibited low levels of mrna coding for inflammasome-associated proteins, except for il-18 which was induced in both a2 and b2 infected patients, both at mrna and protein levels. interestingly, some patients showed distinct expression profiles with elevated levels of several genes involved in the inflammasome mediated immune-response. more specific, approximately 1/4 of the patients showed increased expression of iκbα, il-1β and nlrp3. as mentioned earlier, the repressor gene iκbα expression is induced by nf-κb activation [19] . nf-κb induces mrna expression of the potent proinflammatory cytokines pro-il-1β and pro-il-18 [40] . further, increased level of nlrp3 mrna expression is the initial essential step in activating the nlrp3-inflammasome. taken together, the increased levels of nlrp3 mrna in this group of children along with enhanced il-1β and il-18 mrna expression and protein secretion could suggest that the nlrp3-inflammasome is activated to produce il-1β and il-18 in the respiratory tract of these patients. previously, it has been shown that influenza virus is able to activate the nlrp3-inflammasome in mice [14] . this was found to be critical for survival and reduction of viral titers in mice lung. further, rsv was recently shown to activate the nlrp3-inflammasome in vitro using primary human lung epithelial cells [15] . our results show that the hmpv infected children with a severity score exceeding zero more frequently exhibited elevated levels of il-1β and nlrp3 than children with severity score zero. this suggests a possible involvement of these genes in hmpv infected children with severe disease. as mentioned earlier, previous studies on rsv have shown a variation in disease severity for different genotypes, but similar studies on hmpv have been inconclusive [5] [6] [7] [8] . consequently, we wanted to see if our data suggested any differences in gene expression or disease severity for the hmpv a2 and b2 genotypes. however, we did not detect any differences between a2 and b2 with respect to expression profiles except for ifn-β and tnf-α which was significantly higher expressed in a2 and b2 positive patients, respectively, at mrna level. further, we did not detect any differences in the length of hospital stay or disease severity in the a2 and b2 infected children. our study population includes infants and up to 8 years old children. consequently there is a risk that children with both primary and secondary hmpv infections are included. these might experience different immune responses. to address this we compared children less than one year of age with older children. we found no differences in gene expression profiles (data not shown). in addition, the average age of children with severity score 1-4 was not significantly different from those with severity score zero (p = 0.28, student's t-test), indicating that the youngest children where not more disposed to severe disease compared to the older. of note, there was a difference in age distribution between hmpv-infected and control children. the controls were symptom-free and screened for the same airway pathogens as the patient samples, and no pathogens were detected. hence, we would not anticipate that the inflammatory genes are expressed above a basal level in the controls. further, to our knowledge, no studies so far have shown that basal expression is significantly different in children less than one year and older. our use of clinical samples from selected, well-classified patients is a strength of the study. for studies aimed to examine immune responses to a particular viral infection in vivo it is a challenge, especially in children, to obtain sufficient number of samples collected at similar conditions. in particular, interfering viral and bacterial co-infections are common. in our study population extensive viral analyses of each sample revealed that nearly half of the population had viral co-infections and were excluded from further analysis. in addition, bacterial infections were unlikely because most children had received one or more conjugated pneumococcal vaccinations, and only patients with maximal crp levels less than 100 mg/l were included. furthermore, we only included samples that were collected within the first week after onset of symptoms. finally, separating the samples based on the two most frequent hmpv genotypes enabled us to see if there was a genotype-specific cytokine gene expression profile or differences in disease severity. this allowed us to study the initial host immune response towards particular hmpv genotypes, which extends current literature. however, our selection criteria limited the total number of patients that were eligible for our study. consequently, it will be necessary to confirm our findings in other cohorts, in particular with respect to inflammasome-associated genes in children with severe rti. in summary, our study has identified inflammasome components that may be key players in the innate immune response against hmpv in children. the recent discovery of nlrp3 inhibitors [41] that may prevent or even treat rti-mediated inflammation, emphasizes the need to establish the role of inflammasomes in the response to rti in children. our study provide an important contribution in this regard. supporting information s1 a newly discovered human pneumovirus isolated from young children with respiratory tract disease outbreak of human metapneumovirus infection in norwegian children human metapneumovirus: lessons learned over the first decade novel human metapneumovirus sublineage correlation between respiratory syncytial virus genotype and severity of illness genetic variability of human metapneumovirus infection: evidence of a shift in viral genotype without a change in illness differences in clinical severity between genotype a and genotype b human metapneumovirus infection in children comparison of risk factors for human metapneumovirus and respiratory syncytial virus disease severity in young children differential production of inflammatory cytokines in primary infection with human metapneumovirus and with other common respiratory viruses of infancy cytokines and chemokines in respiratory secretion and severity of disease in infants with respiratory syncytial virus (rsv) infection regulation of inflammasome signaling the role of the nlrp3 inflammasome in the pathogenesis of airway disease lethal influenza virus infection in macaques is associated with early dysregulation of inflammatory related genes the nlrp3 inflammasome mediates in vivo innate immunity to influenza a virus through recognition of viral rna human respiratory syncytial virus viroporin sh: a viral recognition pathway used by the host to signal inflammasome activation coronavirus causes lower respiratory tract infections less frequently than rsv in hospitalized norwegian children real-time reverse transcriptase pcr assay for detection of human metapneumoviruses from all known genetic lineages molecular epidemiology of human metapneumovirus in ireland i kappa b: a specific inhibitor of the nf-kappa b transcription factor induction and function of type i and iii interferon in response to viral infection interferon-[lambda] in the context of viral infections: production, response and therapeutic implications posttranscriptional control of type i interferon genes by ksrp in the innate immune response against viral infection ifn-lambda (ifn-λ) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo impact and regulation of lambda interferon response in human metapneumovirus infection type-iii interferon, not type-i, is the predominant interferon induced by respiratory viruses in nasal epithelial cells interferon lambda 1-3 expression in infants hospitalized for rsv or hrv associated bronchiolitis pulmonary infection of mice with human metapneumovirus induces local cytotoxic t-cell and immunoregulatory cytokine responses similar to those seen with human respiratory syncytial virus biochemical characterization of a gamma interferon-inducible cytokine (ip-10) interferon-independent, human immunodeficiency virus type 1 gp120-mediated induction of cxcl10/ip-10 gene expression by astrocytes in vivo and in vitro essential involvement of cross-talk between ifn-γ and tnf-α in cxcl10 production in human thp-1 monocytes premature infants have impaired airway antiviral ifn[gamma] responses to human metapneumovirus compared to respiratory syncytial virus interferon lambda-1 (ifn-[lambda]1//il-29) induces elr-cxc chemokine mrna in human peripheral blood mononuclear cells, in an ifn-[gamma]-independent manner detection of respiratory viruses and the associated chemokine responses in serious acute respiratory illness tnf activates nf-κb by phosphatidylcholine-specific phospholipase c-induced "acidic" sphingomyelin breakdown role of p38 mitogen-activated protein kinase in a murine model of pulmonary inflammation severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses the broad-spectrum antiviral functions of ifit and ifitm proteins ifit1 is an antiviral protein that recognizes 5[prime]-triphosphate rna sendai virus pathogenesis in mice is prevented by ifit2 and exacerbated by interferon tlrs, nlrs and rlrs: a trinity of pathogen sensors that co-operate in innate immunity inflammasome inhibition: putting out the fire we would like to thank the research nurses at the children's clinic (st. olav's hospital) and the technicians at the department of medical microbiology (st. olav's hospital). key: cord-351387-i0zamkpd authors: witte, katrin; koch, egon; volk, hans-dieter; wolk, kerstin; sabat, robert title: the pelargonium sidoides extract eps 7630 drives the innate immune defense by activating selected map kinase pathways in human monocytes date: 2015-09-25 journal: plos one doi: 10.1371/journal.pone.0138075 sha: doc_id: 351387 cord_uid: i0zamkpd pelargonium sidoides is a medical herb and respective extracts are used very frequently for the treatment of respiratory tract infections. however, the effects of pelargonium sidoides and a special extract prepared from its roots (eps 7630) on human immune cells are not fully understood. here we demonstrate that eps 7630 induced a rapid and dose-dependent production of tnf-α, il-6, and il-10 by human blood immune cells. this eps 7630-induced cytokine profile was more pro-inflammatory in comparison with the profile induced by viral or bacterial infection-mimicking agents. the search for eps 7630 target cells revealed that t-cells did not respond to eps 7630 stimulation by production of tnf-α, il-6, or il-10. furthermore, pretreatment of t-cells with eps 7630 did not modulate their tnf-α, il-6, and il-10 secretion during subsequent activation. in contrast to lymphocytes, monocytes showed clear intracellular tnf-α staining after eps 7630 treatment. accordingly, eps 7630 predominantly provoked activation of map kinases and inhibition of p38 strongly reduced the monocyte tnf-α production. the pretreatment of blood immune cells with eps 7630 lowered their secretion of tnf-α and il-10 and caused an il-6 dominant response during second stimulation with viral or bacterial infection-mimicking agents. in summary, we demonstrate that eps 7630 activates human monocytes, induces map kinase-dependent pro-inflammatory cytokines in these cells, and specifically modulates their production capacity of mediators known to lead to an increase of acute phase protein production in the liver, neutrophil generation in the bone marrow, and the generation of adaptive th17 and th22 cells. today, plant secondary substances, especially polyphenols, get more and more into the focus of medical research. in south africa, polyphenol-rich herbal preparations made up from roots of pelargonium sidoides and pelargonium reniforme are traditionally used to treat respiratory and gastrointestinal infections, dysmenorrhea, and hepatic disorders [1] . inspired by the healing of his tuberculosis, charles henry stevens introduced this phytomedical drug to england already in 1897 [1] . more than seven decades later, a special ethanolic extract of pelargonium sidoides roots, eps 7630, was finally developed [eps 1 7630 is the active ingredient of the herbal medicinal product umckaloabo 1 (iso arzneimittel, ettlingen, germany)]. in germany, eps 7630 is approved today for the therapeutic use in patients with acute bronchitis. in addition, eps 7630 was shown to be effective in clinical trials with patients suffering from tonsillopharyngitis, rhinosinusitis, common cold or copd [2] [3] [4] [5] [6] . as an alternative to antibiotic treatments, eps 7630 has the advantage of not promoting microbial resistances [7] . the latter aspect is mainly explained by its characteristic not to interfere with the metabolism of viruses or bacteria. the main constituents of eps 7630 include coumarins (e.g., umckalin) and flavanoles (polyphenols) [8] . the latter comprise oligomeric proanthocyanidins, which are highly abundant (~40%) in eps 7630, especially oligo-and polymeric prodelphinidins. they are constructed mainly of gallocatechin and epigallocatechin components and are present with different interflavonoid bonds in pelargonium sidoides roots [9] . although the research of the last years has made substantial progress with respect to the broad antiviral and antibacterial efficacy of eps 7630, the exact mode of action of eps 7630 is not fully understood yet. in vitro, eps 7630 shows efficacy against cellular infections with influenza virus, hsv, emcv, rsv, coronavirus, parainfluenza virus, and coxsackie virus, and this appears to be mainly mediated indirectly by inhibition of virus attachment and spreading [7, [10] [11] [12] [13] . different modes of action underlying the antibacterial effects of eps 7630 have been proposed. eps 7630 inhibits the adherence of bacteria such as streptococcus pyogenes and helicobacter pylori to epithelial cells in vitro [14] [15] [16] [17] [18] . furthermore, ciliated cells isolated from the nasal epithelium enhanced their ciliary beat frequency in the presence of eps 7630, which should allow a better removal of excess mucus and bacteria [19] . regarding the infection with candida albicans, eps 7630 was shown to enhance the oxidative burst and intracellular pathogen killing by human blood phagocytes [15] . in leishmania major-infected murine macrophages, eps 7630 increased cellular nitric oxide production and mrna levels of inos and several cytokines (il-1β, il-10, il-12, il-18, tnf-α, ifn-α, ifn-γ) [13, [20] [21] [22] . however, our knowledge regarding the influence of pelargonium extract on human immune cells, in particular on their cytokine production, is still highly restricted. to gain insight into this matter we comprehensively studied the immunoregulatory effects of eps 7630 on human blood immune cells. eps 7630 is prepared from the roots of pelargonium sidoides with a drug to extract ratio of 1:8-10 using aqueous ethanol (11% w/w) as extraction solvent. dried extract of a single batch (no. psc2003/l01-11/sy06-041-a) was used to prepare a stock solution of 3 mg/ml. this solution was obtained by dissolving the powder in sterile pbs containing 10% ethanol, followed by ultrasound treatment and sterile filtration using a 0.2 μm filter unit. human peripheral blood mononuclear cells (pbmcs) were isolated from the blood of healthy donors by ficoll (biochrom) density gradient centrifugation as previously described [23, 24] . in the first setting, pbmcs were stimulated with eps 7630 (3 and 30 μg/ml), escherichia coli 0127:b8 lipopolysaccharide (tlr4 ligand; 100 ng/ml; sigma-aldrich), polyinosinic-polycytidylic acid [poly (i:c); 10 μg/ml; sigma-aldrich], a cytokine mixture of il-1β, il-2 and il-12 (10 ng/ml each; r&d systems), anti-cd3 (orthoclone; cilag) and anti-cd28 (r&d systems) monoclonal antibodies (1 μg/ml each), or were left without specific treatment (0.1% ethanol as solvent control) for 4 and 24 h, before cell culture supernatant was recovered for elisa cytokine production analysis. in another setting, pbmcs, after serum-starvation for 3.5 h, were stimulated with the same stimuli (for tcr stimulation cd3/28 coated dynabeads were used at a cell / bead ratio of 2:1) but for 10 and 30 min, and cells were recovered for western blot analysis. for eps 7630 dose-response analyses of cytokine production, isolated pbmcs were stimulated with eps 7630 at concentrations ranging from 0.1 to 10 μg/ml or were left without stimulation (0.1% ethanol control) for 48 h. in some of these concentration-response analyses, lipopolysaccharide (lps; 100 ng/ml) or poly (i:c) (10 μg/ml) was added after the first 24 h and incubation was continued for a further 24 h. to study the kinetics of cytokine production, isolated pbmcs were stimulated with 10 μg/ml eps 7630 or 0.1% ethanol (solvent control) for 4 to 72h. for intracellular tnf-α analysis, pbmcs were stimulated for 4 to 5 h with eps 7630 concentrations ranging from 1 to 30 μg/ml, with 0.1% ethanol solvent control and, if indicated, 25 ng/ml phorbol 12-myristate 13-acetate (pma)/1 μg/ml ionomycin before being prepared for flow-cytometry-based cytokine analysis. in some of these studies, pbmcs were pretreated with sp600125, pd98059, wedelolactone (all from sigma aldrich) and sb202190 (invivogen) at 10 μm each or with 0.1% dmso (solvent control, sigma aldrich) for 45 min before eps 7630 (10 and 30 μg/ml only) application. the possible influence of eps 7630 on pbmc proliferation and apoptosis was tested by stimulation of pbmcs with 0 (0.1% ethanol solvent control), 3, 10 and 30 μg/ml eps 7630 as well as with 1% dmso (positive control for apoptosis induction) for 24 h. afterwards, cells were recovered for flow cytometric analysis. cd4 + memory t-cells were purified from isolated pbmcs by negative selection using the macs system and the memory cd4 + t-cell isolation kit (miltenyi). for elisa-based cytokine production analysis, isolated t-cells were cultured in the presence or absence of eps 7630 for 48 h. for the last 24 h of culture, one part of these cells was stimulated with anti-cd3/anti-cd28-coated dynabeads (life technologies; cell/bead ratio 1:1). all cell cultures were performed using rpmi culture medium supplemented with 2 mm l-glutamin and 10% fetal bovine serum (biochrom). the study of eps 7630 effects, the collection and usage of the blood samples were approved by the clinical institutional review board of the university hospital charité berlin, and written informed consent was obtained from donors. culture supernatants were analyzed for tnf-α, il-6 and il-10 content by elisa using respective detection kits from r&d systems. in some settings, quantification of tnf-α was carried out using the immulite device and respective detection kit (dpc biermann). lysing of pbmcs, quantification of proteins in the cell lysates, sds page gel electrophoresis and blotting of the respective gel was carried out as published earlier [25, 26] . analysis of signal transduction elements was performed by incubation of blots with antibodies detecting phospho-jnk1/2, phospho-p38, phospho-erk1/2, phospho-akt, phospho-stat5 and phospho-p65 (all from cell signaling technology) and gapdh (merck millipore), followed by incubation with peroxidase-conjugated affinipure goat anti-rabbit or goat anti-mouse igg (h&l, dianova) and subsequent chemiluminescence detection using ecl reagent (lumigen). for analysis of intracellular cytokines, treated pbmcs were incubated with brefeldin a (sigma aldrich; 5 μg/ml) for the last 3 to 4 h of treatment. afterwards, cells were fixed and permeabilized using the cytofix/cytoperm kit (bd biosciences) and subsequently stained for 40 min with fluorescent antibodies detecting tnf-α (clone mab11, biolegend), cd14 (clone rmo52, beckmann coulter), and cd4 (clone sk3, bd biosciences). to assess the purity of isolated memory t-helper cells, cells were stained with fluorescent antibodies detecting the following cell surface markers as described previously [23, 24] : cd45ra (clone hi100), cd45ro (clone uchl1), cd3 (clone sk7), cd4 (clone sk3), cd56 (clone ncam16.2) (all from bd biosciences) as well as cd16 (clone 3g8), cd14 (clone rmo52), and cd19 (clone j4.119) (all from beckmann coulter). for analysis of pbmc proliferation, total cell numbers after treatment were assessed by flow cytometry. for analysis of pbmc apoptosis, treated cells were stained with fluorescent annexin v antibodies and propidium iodide using the annexin v apoptosis detection kit apc (ebioscience) according to the manufacturer's instructions. afterwards, the proportion of apoptotic cells (annexin v + cells) were evaluated. all data acquisitions and analyses were carried out using a facscalibur device and cell-quest software (bd biosciences). to test the significance of pairwise differences between the treatment groups, the wilcoxon matched-pairs signed-rank test was used (spss software, ibm). a p-value of p 0.05 was considered as indicator of significance. understanding the molecular effects of eps 7630 is key to take advantage of its medical potential and the identification of further indications. so far, data regarding its target cells and effects thereof within the human immune system are lacking. to address this issue, we first investigated whether eps 7630 influences resting human immune cells. therefore, pbmcs isolated from healthy donors were treated or not (control) with eps 7630 at two concentrations and were tested for cytokine production. as a comparison, these cells were also stimulated with toll-like receptor (tlr)3 [poly(i:c)] and tlr4 ligands (bacterial lipopolysaccharide), which mainly act on antigen-presenting cells (e.g., monocytes) and mimic viral and bacterial infection, respectively. as further positive controls a mix of cytokines (il-1β, il-2, il-12), able to activate nk-cells, as well as t-cell activating anti-cd3/anti-cd28 antibodies, were used. interestingly, cells responded to eps 7630 by secretion of tnf-α and il-6 with levels that were much higher than those induced by the mix of cytokines and tcr engagement (fig 1a) . furthermore eps 7630 slightly triggered il-10 production, and this production was clearly smaller (~3-to 5-fold) than that induced by cd3/cd28-mediated stimulation. importantly, the cytokine profile induced by eps 7630 in pbmcs was also different from that induced by tlr3 or tlr4 ligands (fig 1a) . in fact, the eps 7630-induced response was much more pro-inflammatory than that induced by the viral or bacterial infection-mimicking agents ( fig 1b) . as no relevant cytotoxicity or influence on pbmc proliferation were observed by eps 7630, the cytokine increase provoked by eps 7630 seems to be due to an induced production rate (s1 fig). a more detailed investigation revealed a concentration-dependent induction of cellular tnf-α, il-6, and il-10 secretion by eps 7630, with clear effects already seen at a concentration of 1 μg/ml (fig 1c) . additionally, a kinetic analysis of eps 7630-dependent cytokine production by pbmcs revealed, that especially tnf-α is produced very quickly (fig 1d) . afterwards, the tnf-α level continuously decline, probably as a result of binding and internalization of this cytokine by monocytes. in contrast to tnf-α, il-6 and, in particular, il-10 were produced more slowly after eps 7630 stimulation, with levels steadily increasing over time. the next study part aimed at identifying the cell type that accounts for the response observed in eps 7630-stimulated blood immune cells. since memory t helper cells are very important cytokine producers, we first isolated these cells from human pbmcs by magnet-based cell sorting (fig 2a) . surprisingly, no relevant tnf-α, il-6, or il-10 production was found by these cells stimulated with different eps 7630 concentrations (fig 2a) . in the next step, we tested whether eps 7630 might modulate the cytokine response in activated memory t helper cells. however, cd3/cd28 stimulation of these cells, pretreated with different doses of eps 7630, did not result in a relevant increase of tnf-α, il-6, or il-10 secretion (fig 2b) . moreover, flow-cytometric analysis of intracellular cytokine staining showed that no population of t helper cells was able to produce tnf-α after stimulation with eps 7630, whereas they strongly responded to pma/ionomycin activation (fig 2c and s2 fig) . these data suggest that, within pbmcs, not th-cells but other immune cells are major targets of eps 7630 action. to prove monocytes as being the cell population sensitive among pbmcs to the action of eps 7630, we analyzed intracellularly stained tnf-α in these cells after eps 7630 stimulation using flow cytometry. indeed, we observed a strong dose-dependent induction of tnf-α production in monocytes, whereby, similar to the effect on whole pbmc, this effect was already seen at 1 μg/ml (fig 3a and 3b) . to understand the action of eps 7630 at the molecular level we then investigated the signaling pathways activated by this drug. pbmcs were treated for 10 and 30 min with eps 7630 at two different concentrations (3 and 10 μg/ml) and, as comparison, with tlr3 and tlr4 ligands, the il-1β, il-2, and il-12 comprising cytokine mixture, and anti-cd3/anti-cd28 antibodies. because of the cytokine profile observed after pbmc incubation with eps 7630, we focused our analysis on map kinase, nf-κb, and pi3k pathways. respective western blot analyses revealed that eps 7630 activated the map kinases jnk1/2, p38, and erk1/2. additionally we observed a slight activation of the pi3k (phosphorylation of akt) and nf-κb (phosphorylation of p65) pathways, whereas stat5 expectedly remained unaffected (fig 3c) . in line with the different cytokine profiles induced by eps 7630 versus tlr3 and tlr4 ligands, cytokine mixture, and anti-cd3/anti-cd28 antibodies, the pattern of activated kinases by eps 7630 was unique. by pharmacological blocking using the selective inhibitors sp600125 (jnk1/ 2/3), pd98059 (mek; activator of erk1/2), sb202190 (p38α/β), and wedelolactone (iκb kinase), we observed that eps 7630-induced production of tnf-α in monocytes was strongly dependent on p38 activity and at least partly dependent on the activation of erk1/2 and nf-κb ( fig 3d) . eps 7630 is very frequently used for infection prevention, in particular in the autumn and winter. therefore, we asked whether eps 7630 pretreatment would influence the immune response in the context of viral and bacterial infections. to address this question, we pretreated pbmcs with increasing concentrations of eps 7630 (0.1-3 μg/ml) for 24 h followed by stimulation with tlr3 or tlr4 ligands (mimicking viral or bacterial infection, respectively) for another 24 h. surprisingly, we observed that eps 7630 pretreatment concentration-dependently inhibited the tlr3-and tlr4-mediated production of tnf-α and il-10 (fig 4) . in contrast, we detected eps 7630 concentration-dependent high il-6 secretion in this situation (fig 4) . these data suggest that eps 7630 pretreatment specifically modulates the cytokine production in subsequent viral and bacterial infection. the special extract of pelargonium sidoides roots, eps 7630 (umckaloabo 1 ) is therapeutically active against upper respiratory tract infections and currently approved for the application in acute bronchitis in germany. surprisingly, the large use of this preparation is not associated with a respective knowledge regarding its pharmacodynamic properties. in fact, besides the known anti-infective properties of eps 7630, directed against viruses and bacteria, little is known about its molecular effects and cellular targets. moreover, we do not understand the pathway(s), via which eps 7630 mediates its effects on cells. so far, the majority of published studies regarding the effects of pelargonium extracts concern tissue cells. only few studies involved immune cells, and most of them focused on murine macrophages [13, 15, [20] [21] [22] . especially, there are no studies that investigated the influence of eps 7630 on cytokine production by human primary immune cells. however, cytokines play an essential role in the defense against bacteria and viruses and in the intercellular communication between immune and tissue cells [27] [28] [29] [30] [31] [32] . therefore, the focus of our current study was to unravel the possible influence of eps 7630 on human immune cells. our data show that eps 7630 strongly and dose-dependently induced the production of the pro-inflammatory cytokines tnf-α and il-6 in human blood immune cells. moreover, a less prominent induction of the anti-inflammatory acting il-10 was observed. importantly, the cytokine profile induced by eps 7630 was completely different from that induced by viral or bacterial infection-mimicking agents that caused production of a more anti-inflammatory cytokine milieu. these observations suggest that eps 7630 acts as an immunostimulant and, before infection, may promote the innate immune defense and the ability of the body to quickly and efficiently eliminate potentially incoming microbes. underlying mechanisms may include in particular the activation of phagocytes by tnf-α, the induction of acute phase proteins in the liver, and the elevation of neutrophil generation in the bone marrow. within human pbmcs, monocytes but not cd4 + t cells were found to be targets of eps 7630 and to produce high amounts of tnf-α upon this treatment. this result is in line with previous data showing that pelargonium extract acted on murine myeloid cells, in which it induced the production of tnf-α and il-1β [13, [20] [21] [22] . when investigating the signaling cascades induced by eps 7630, we found a strong mapk kinase pathway activation, which included phosphorylation of jnk, erk1/2, and p38. furthermore, eps 7630 slightly provoked nf-κb and pi3k pathway activation. however, pharmacological blockade of only p38 resulted in a strongly decreased monocyte tnf-α production. the observation that this signaling pattern differed from that induced by tlr3 and tlr4 ligand, inflammatory cytokines, and cd3/cd28 engagement suggest that pelargonium extract affects monocytes via receptors different from those used by the mentioned stimuli. it has previously been reported that the majority of in vitro macrophage activation with immune stimulating botanicals is caused by lipoproteins and lipopolysaccharide derived from bacterial contamination of the herbal raw material or from endophytes colonizing these plants [33] . as described above, we observed that the eps 7630-activated pathway and cytokine pattern was very different from the response seen after stimulation of human pbmcs with bacterial lipopolysaccharide. furthermore, analysis of eps 7630 for the presence of contaminating bacterial lipopolysaccharide by a limulus amebocyte lysate (lal) assay revealed a very low content of less than 200 eu/mg, which is equivalent to about 20 ng/mg. currently, it is not known which constituents of eps 7630 are responsible for the observed effects on cytokine production. previous investigations have suggested that the antiviral activity of the extract is quantification of tnf-α, il-6, and il-10 in respective culture supernatants by elisa. mean (± sem) cytokine concentration data from 12 donors are given as percent of 0 μg/ml eps 7630 group (control). cytokine concentrations in the absence of eps 7630 in tl3l and tl4l groups were: 1267±414 and 1534±324 pg/ml (tnf-α), 1278±369 and 2135±650 pg/ml (il-6), 1011±127 and 1430±140 pg/ml (il-10), respectively. significant differences compared to 0 μg/ml eps 7630 group are indicated (* p<0.05, ** p<0.01, wilcoxon matched-pairs signed-rank test). doi:10.1371/journal.pone.0138075.g004 primarily due to its content of prodelphinidins [7] . as these complex molecules may not be systemically bioavailable, further studies are required to demonstrate whether the clinically observed therapeutic effect of eps 7630 is mediated by components different from prodelphinidins via stimulation of the immune system in the gastrointestinal tract. by our study, we also demonstrated that eps 7630 preincubation of immune cells influenced their subsequent, by viral or bacterial infection-mimicking agents induced cytokine production towards an il-6-dominated milieu, with lower tnf-α and il-10 content. this observation suggests that taking eps 7630 before infection might reduce flu-like symptoms during infection, which are associated with the action of tnf-α. furthermore, the eps 7630 caused elevated il-6 production during infection might strengthen the production of acute phase proteins, neutrophilic granulocyte generation in the bone marrow, and the establishment of adaptive th17 and th22 cells. in fact, the differentiation of th17 cells is, besides il-1β, il-23 and tgf-β, particularly dependent on the presence of il-6 [34] . furthermore, il-6 also cooperates with tnf-α in inducing the differentiation of th22 cells [32, 35] and both th17 and th22 cells play an essential role in the defense against microbes at body barriers [36, 37] . thus, by indirect induction of th17 and th22 responses, eps 7630 treatment of patients may promote the adaptive host defense against different viral, bacterial or fungal pathogens at respiratory epithelia. a historical, scientific and commercial perspective on the medicinal use of pelargonium sidoides (geraniaceae) pelargonium sidoides for acute bronchitis: a systematic review and meta-analysis treatment of acute rhinosinusitis with the preparation from pelargonium sidoides eps 7630: a randomized, double-blind, placebo-controlled trial efficacy of extract of pelargonium sidoides in children with acute non-group a beta-hemolytic streptococcus tonsillopharyngitis: a randomized, double-blind, placebo-controlled trial efficacy of a pelargonium sidoides preparation in patients with the common cold: a randomized, double blind, placebo-controlled clinical trial randomised, doubleblind, placebo-controlled trial of eps 7630 in adults with copd eps(r) 7630 (umckaloabo(r)), an extract from pelargonium sidoides roots, exerts anti-influenza virus activity in vitro and in vivo fascinating metabolic pools of pelargonium sidoides and pelargonium reniforme, traditional and phytomedicinal sources of the herbal medicine umckaloabo a detailed view on the constituents of eps 7630 the root extract of the medicinal plant pelargonium sidoides is a potent hiv-1 attachment inhibitor investigation of the influence of eps(r) 7630, a herbal drug preparation from pelargonium sidoides, on replication of a broad panel of respiratory viruses efficacy of an aqueous pelargonium sidoides extract against herpesvirus anti-infective activities of pelargonium sidoides (eps(r) 7630): effects of induced no production on leishmania major in infected macrophages and antiviral effects as assessed in a fibroblast-virus protection assay eps 7630, an extract from pelargonium sidoides roots inhibits adherence of helicobacter pylori to gastric epithelial cells extract of pelargonium sidoides (eps 7630) improves phagocytosis, oxidative burst, and intracellular killing of human peripheral blood phagocytes in vitro evaluation of an aqueous-ethanolic extract from pelargonium sidoides (eps(r) 7630) for its activity against group a-streptococci adhesion to human hep-2 epithelial cells anti-adhesive activities of flavan-3-ols and proanthocyanidins in the interaction of group a-streptococci and human epithelial cells an extract of pelargonium sidoides (eps 7630) inhibits in situ adhesion of helicobacter pylori to human stomach a new approach to pharmacological effects on ciliary beat frequency in cell cultures-exemplary measurements under pelargonium sidoides extract (eps 7630) tannins and related compounds induce nitric oxide synthase and cytokines gene expressions in leishmania major-infected macrophage-like raw 264.7 cells pharmacological profile of extracts of pelargonium sidoides and their constituents nitric oxide synthase and cytokines gene expression analyses in leishmania-infected raw 264.7 cells treated with an extract of pelargonium sidoides (eps 7630). phytomedicine cutting edge: immune cells as sources and targets of the il-10 family members? maturing dendritic cells are an important source of il-29 and il-20 that may cooperatively increase the innate immunity of keratinocytes il-22 and il-20 are key mediators of the epidermal alterations in psoriasis while il-17 and ifn-gamma are not il-22 regulates the expression of genes responsible for antimicrobial defense, cellular differentiation, and mobility in keratinocytes: a potential role in psoriasis tumor necrosis factor receptor signaling in keratinocytes triggers interleukin-24-dependent psoriasis-like skin inflammation in mice il-19 is a component of the pathogenetic il-23/il-17 cascade in psoriasis il-22 increases the innate immunity of tissues deficiency of il-22 contributes to a chronic inflammatory disease: pathogenetic mechanisms in acne inversa the th17 cytokine il-22 induces il-20 production in keratinocytes: a novel immunological cascade with potential relevance in psoriasis il-29 is produced by t(h)17 cells and mediates the cutaneous antiviral competence in psoriasis the majority of in vitro macrophage activation exhibited by extracts of some immune enhancing botanicals is due to bacterial lipoproteins and lipopolysaccharides the differentiation of human t(h)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor rorgammat production of interleukin 22 but not interleukin 17 by a subset of human skin-homing memory t cells the role of th17 cytokines in primary mucosal immunity therapeutic opportunities of the il-22-il-22r1 system the authors acknowledge brigitte ketel and beate pust for their steady excellent technical assistance. conceived and designed the experiments: rs. performed the experiments: k. witte. analyzed the data: rs k. wolk. contributed reagents/materials/analysis tools: ek. wrote the paper: k. witte rs k. wolk hdv ek. key: cord-322250-7kjakuyw authors: he, jia; yuan, renyikun; cui, xiaolan; cui, yushun; han, shan; wang, qin-qin; chen, yangling; huang, liting; yang, shilin; xu, qiongming; zhao, yonghui; gao, hongwei title: anemoside b4 protects against klebsiella pneumoniaeand influenza virus fm1-induced pneumonia via the tlr4/myd88 signaling pathway in mice date: 2020-07-02 journal: chin med doi: 10.1186/s13020-020-00350-w sha: doc_id: 322250 cord_uid: 7kjakuyw background: pneumonia refers to the inflammation of the terminal airway, alveoli and pulmonary interstitium, which can be caused by pathogenic microorganisms, physical and chemical factors, immune damage, and drugs. anemoside b4, the major ingredient of pulsatilla chinensis (bunge) regel, exhibited anti-inflammatory activity. however, the therapeutic effect of anemoside b4 on pneumonia has not been unraveled. this study aims to investigate that anemoside b4 attenuates the inflammatory responses in klebsiella pneumonia (kp)and influenza virus fm1 (fm1)-induced pneumonia mice model. methods: the network pharmacology and molecular docking assays were employed to predict the targets of anemoside b4’s treatment of pneumonia. two models (bacterial kp-infected mice and virus fm1-infected mice) were employed in our study. balb/c mice were divided into six groups: control, model group (kp-induced pneumonia or fm1-induced pneumonia), anemoside b4 (b4)-treated group (2.5, 5, 10 mg/kg), and positive drug group (ribavirin or ceftriaxone sodium injection). blood samples were collected for hematology analysis. the effects of b4 on inflammation-associated mediators were investigated by enzyme-linked immunosorbent assay (elisa) and hematoxylin and eosin staining (he) staining. proteins expression was quantified by western blotting. results: the network results indicated that many pro-inflammatory cytokines such as tumor necrosis factor α (tnf-α), interleukin-1β (il-1β), and interleukin-6 (il-6) participated in anemoside b4’s anti-inflammatory activity. the counts of neutrophil (neu) and white blood cell (wbc), the level of myeloperoxidase (mpo), and the release of pro-inflammatory cytokines tnf-α, il-1β, and il-6 increased by kp or fm1 infection, which were reversed by anemoside b4. in addition, anemoside b4 significantly suppressed the fm1-induced expression of toll-like receptor 4 (tlr4), myeloid differential protein-88 (myd88), and myeloid differentiation protein-2 (md-2), which were further validated by molecular docking data that anemoside b4 bound to bioactive sites of tlr4. therefore, anemoside b4 exhibited a significant therapeutic effect on pneumonia via the tlr4/myd88 pathway. conclusion: our findings demonstrated that anemoside b4 attenuates pneumonia via the tlr4/myd88 signaling pathway, suggesting that anemoside b4 is a promising therapeutic candidate for bacterial-infected or viral-infected pneumonia. background pneumonia refers to the inflammation of the terminal airway, alveoli and pulmonary interstitium caused by many factors, of which typical symptoms include dry cough, chest pain, fever, and dyspnea [1, 2] . pneumonia has been one of the main factors affecting people's health and even life for a long time. with the introduction of antibiotics and vaccines in the twentieth century, the survival rate of related patients has been greatly improved [3] . however, pneumonia remains the leading cause of death in developing countries, as well as in elderly, young children and chronically ill patients [4] . klebsiella pneumoniae is one of the most important opportunistic pathogens and iatrogenic pathogens, which is highly pathogenic to human beings [5] . due to the abuse of various antimicrobial agents, the multi drug resistance of klebsiella pneumoniae is widespread, which leads to great trouble for clinical treatment [6, 7] . influenza virus is the representative of orthomyxoviridae, including human influenza virus and animal influenza virus [8, 9] . influenza virus infection, whether seasonal or pandemic, often leads to pneumonia, which can cause serious health problems [10, 11] . for the treatment of viral pneumonia, apart from neuraminidase inhibitors and ribavirin, other antiviral drugs showed no significant effect on pneumonia [12] . specifically, now corona virus covid-19-induced pneumonia outbreaks world widely, leading to many deaths. as it stands now, there is not effective drugs for the treatment with pneumonia. therefore, it is urgent to find new and effective drugs for treatment with pneumonia. network pharmacology is an emerging discipline that builds and analyzes biological networks based on systems biology to reveal the role of drugs and their mechanisms [13, 14] . by constructing a "component-target-pathway" research network, we can observe how drugs act on multiple targets at the same time, thereby regulating multiple signal pathways, comprehensively reveal their drug efficacy network, and explain their mechanism of action [15] . this provides a certain guiding significance for our work. during pneumonia process, white blood cell (wbc) and neutrophil (neu) counts are always increased [16] . in addition, myeloperoxidase (mpo), an enzyme secreted by leukocytes, is present in myeloid cells, which catalyzes the formation of a variety of active oxidants during the occurrence and development of pneumonia [17] . the role of tlrs in the signaling pathway of inflammation and related diseases is highly valued [18] . when induced by external stimuli such as viruses or bacteria, the adaptor protein myeloid differentiation protein-2 (md2) directly binds and recognizes stimuli forming discrete complex, and associates non-covalently toll-like receptor 4 (tlr4) to form the final activated heterodimer that in its turn starts the intracellular signal [19] . after that, myeloid differentiation primary response 88 (myd88) is recruited to tlr4, which then activates downstream pathways and results in the pro-inflammatory cytokine release, such as tumor necrosis factor-α (tnf-α), interleukin-6 (il-6), and interleukin-1β (il-1β) [20] . this suggests that tlr4/ myd88 signaling pathway may be an effective therapeutic target for many inflammatory diseases. traditional chinese medicine has great potential in the treatment of many inflammatory and immunoregulatory diseases [21, 22] . pulsatilla chinensis (bunge) regel, a traditional chinese herb, has the functions of clearing away heat and detoxification, stopping dysentery and drying dampness, and has a good therapeutic effect on bacteria, virus infection and malignant tumors in clinic [23] . recent studies have shown that triterpenoid saponins are the main factors affecting the pharmacological activities in this herb. anemoside b4, one of the main monomer components of pulsatilla chinensis (bunge) regel, can inhibit the pathogenesis of acute kidney injury caused by cisplatin and improve renal function, which protective effects may be associated with its anti-inflammatory activities [24] . however, there are not papers involved in the therapeutic effect of b4 on pneumonia. in the present study, we used klebsiella pneumoniae-or influenza virus fm1-induced pneumonia model to investigate the antiinflammatory effects and mechanisms of anemoside b4 in vivo. klebsiella pneumoniae (bncc-102997) was purchased from beijing beina chuanglian biotechnology research institute (beijing, china). influenza virus fm1 strain was provided by absl-2 laboratory, institute of traditional chinese medicine, chinese academy of sciences (beijing, china). antibodies against myd88 (#4283), tlr4 (#14358) were obtained from cell signaling (beverly, ma, usa), and antibody against md2 (#24182) conclusion: our findings demonstrated that anemoside b4 attenuates pneumonia via the tlr4/myd88 signaling pathway, suggesting that anemoside b4 is a promising therapeutic candidate for bacterial-infected or viral-infected pneumonia. keywords: anemoside b4, pneumonia, klebsiella pneumoniae, influenza virus fm1, tlr4/myd88 was obtained from abcam (cambridge, ma, england). ribavirin (20160308) was purchased from solarbio (beijing, china). ceftriaxone sodium injection was purchased from reyoung pharmaceutical co., ltd (shandong, china). il-1β, il-6, and tnf-α elisa kits were obtained from neobioscience (shenzhen, china). mpo kit (a044-1-1) was purchased from nanjing jiancheng bioengineering institute (nanjing, china). physiological saline for injection (l219012211) was purchased from sichuan kelun pharmaceutical co., ltd. (chengdu, china). the study was approved by the ethics committee on laboratory animal management of guangxi university of chinese medicine (approval document no. syxk-gui-2019-0001). virus experiment was carried out in absl-2 laboratory, institute of traditional chinese medicine, chinese academy of sciences. all animals received humane care according to the local guide for the care and use of laboratory animals of guangxi university of chinese medicine. healthy balb/c mice (male and female, 6-8 weeks-old and weighing 18-22 g) were purchased from beijing vital river laboratory animal technology co., ltd. (beijing, china) and acclimated for 3 days (animal license #: scxk 2016-0006). all animals were housed under standard specific pathogen-free (spf) conditions and given free access to food and water with a controlled temperature (25 °c) and humidity (50%). the dried roots of pulsatilla chinensis (bunge) regel (10 kg) were powered into 100 mesh, which were further extracted by 70% ethanol to obtain liquid extract after removing the ethanol. by adding proper hot water, the extract was sequentially extracted with petroleum ether, dichloromethane, ethyl acetate, and n-butyl alcohol to get 24.2 g, 102.5 g, 34.2 g, and 685.2 g extract, respectively. the n-butyl alcohol (685.2 g) extract was further separated on a column packed with macroporous adsorption resins by gradient eluting with ethanol and water (100:0, 30:70, 60:40, 90:10) to yield four fractions: fr. 1-fr. 4. fr. 3 (180.2 g) was further separated by using medium pressure liquid chromatography (mplc) with ods column first. then sephadex lh-20 gel column eluted with meoh and semi-preparative hplc with c18 column at a flow rate of 2 ml/min eluted with meoh (60%) were employed for further purification to yield anemoside b4 (42.2 g). the purity of anemoside b4 was investigated by hplc assay. all the male mice were divided randomly into six groups (n = 20 per group): control group, model group, anemoside b4-treated group (2.5, 5, 10 mg/kg) and ceftriaxone sodium-treated group (200 mg/kg). in addition to the control group, the other five groups were administrated with klebsiella pneumoniae (1.04 × 10 9 cfu/ ml, 50 μl/per mouse) by non-invasive intratracheal (i.t.) instillation into lung of mice. mice were treated with anemoside b4 (i.v.) at the point of time 0, 3, 24, 48, 72 h or ceftriaxone sodium (i.v.) at the point of time 0 h. 72 h after klebsiella pneumoniae treatment, all experimental mice were sacrificed by dislocation (fig. 3a ). blood samples were collected for measurement of levels of wbc, neu, and pro-inflammatory cytokines. the parts of lung samples were collected and kept at − 80 °c for western blot assay. bronchoalveolar lavage fluid (balf) was also taken for pro-inflammatory cytokines. all the mice were divided randomly into six groups (n = 20 per group, half male and female): control group, model group, anemoside b4-treated group (2.5, 5, 10 mg/ kg) and ribavirin-treated group (40 mg/kg). in addition to the control group, the other five groups were administrated with influenza virus fm1 (the virus stock solution was diluted 500 times with normal saline solution) by non-invasive intratracheal (i.t.) instillation into lung of mice. each group of mice was administrated (i.v.) with anemoside b4 (2.5, 5, 10 mg/kg) or ribavirin (40 mg/kg) at the point of time 0, 24, 48, 72, and 96 h. 120 h after treatment of influenza virus fm1, all experiment mice were sacrificed by dislocation (fig. 5a ). blood samples and balf were collected for pro-inflammatory molecules. the other of lung samples were collected and kept at − 80 °c for western blot assay. systematic pharmacology software was used to predict the underlying targets and signal pathways of anemoside b4 in treating pneumonia. through searching ttd and tcmsp database, the targets of anemoside b4 were determined, implying the underlying mechanisms of b4 acting on pneumonia. blood was collected from mouse orbit. the blood sample was further processed with edta. white blood cell and neutrophil counts from blood were determined using an auto hematology analyzer (mindray, shenzhen, china). the blood sample was placed at room temperature for 2 h and then centrifuged at 3000 rpm for 20 min to collect supernatant. the bronchoalveolar lavage fluid (balf) was centrifuged at 1600 rpm for 10 min at 4 °c, and the supernatant was collected. the lung tissue was homogenized by a tissue grinder (tp-24, jieling instrument manufacturing tianjin co., ltd, tianjin, china). the samples were centrifuged for 20 min at 3000 rpm. the supernatant was immediately stored at − 80 °c. the pro-inflammatory molecules il-1β, tnf-α, and il-6 of all the supernatants were investigated by elisa kits following the manufacturer's instructions. the lung tissue samples were fixed with 4% paraformaldehyde, dehydrated by alcohol gradient, and then transparently treated with xylene, paraffin-embedded and sectioned. the sections were stained with hematoxylin and eosin (h&e), and then the pathological changes of the tissue were observed by optical microscope (uop, dsz5000x, china). lung tissues were homogenized in ripa buffer with 1% pmsf and 1% cocktail (sigma-aldrich, st. louis, mo). the lysate was centrifuged at 15,000 rpm for 25 min at 4 °c and the supernatant was harvested. the protein concentrations were examined using a bca protein kit (thermofisher, waltham, ma, usa). the denatured proteins were then separated by 10% sds-page gels and transferred to pvdf membrane (millipore, billerica, ma, usa). after blocking the pvdf membrane with 5% nonfat milk for 2 h, the pvdf membrane was incubated with primary antibodies (1:1000) at 4 °c overnight. after washed three times with tbst and incubated with secondary antibody (1:5000) for 2 h at room temperature, the protein band signals were detected with supersignal west femto maximum sensitivity substrate (pierce biotechnology) in a chemidoc mp imaging system (bio-rad, hercules, ca, usa). gapdh was used as a housekeeping protein. the molecular docking data of b4 with tlr4-md-2 (pdb code: 3fxi) was performed in ledock (http://www. lepha r.com). tlr4-md-2 structures were obtained from rcsb protein data bank (pdb code: 3fxi) [25] . data are presented as mean ± sd. all experiments were repeated at least three independent times. data were normally distributed and analyzed by one-way-anova by graph pad prism 7 software (microsoft, seattle, wa, usa). a p value < 0.05 was considered to be statistically significant. as shown in fig. 1a , the chemical structure of anemoside b4 was identified as 3-o-α-l-rhamnopyranosylthe purity of anemoside b4 was over 98%, which was determined by hplc assay (fig. 1b) . a network pharmacology-based strategy was proposed to elucidate the underlying multi-target mode of action of anemoside b4 against pneumonia (fig. 2) . this network consisted of 10 targets involved in inflammatory process [26, 27] . specifically, three targets including il-1β, il-6, and tnf-α have been reported to be related to pneumonia, which were further validated in vivo. next, we investigated the effect of anemoside b4 on pro-inflammatory cytokines in vivo. guided by the prediction results, we explored the effects of anemoside b4 on the three cytokines. the results indicated that infection of kp caused a significant increase in the secretions of tnf-α and il-6 ( fig. 3b) in mouse serum. then, the administration of b4 (from 2.5 to 10 mg/kg) reduced the production of these cytokines. he staining of lung tissue results showed that compared with the control group, kp induced obvious consolidation, alveolar damage, collapse, obvious compression, blurred border and obvious inflammatory cell infiltration; the lung tissue damage in the b4 treatment group significantly improved, alveolar collapse was significantly improved, consolidation was significantly relieved, the boundary was clearer, and compression and inflammatory changes were significantly reduced, of which the highest dose was the most obvious. in addition, significant increase was observed in the tnf-α (fig. 3d, g) , il-6 ( fig. 3e, h) , il-1β (fig. 3f ) , and mpo (fig. 3i) in the balf and lung tissue samples collected from the kp-infected pneumonia mice, which were reversed by anemoside b4. collectively, anemoside b4 significantly prevents kp-induced pneumonia. the blood counts of wbc and neu are the commonlyuse biomarkers of bacterial pneumonia clinically [16] . direct contact of the lungs with klebsiella pneumoniae can easily cause lung inflammation in mice. the blood counts of wbc (fig. 4a) and neu (fig. 4b) in the kp group were dramatically increased compared with control group. however, anemoside b4 suppressed the aberrant elevation of wbc and neu respectively, of which the effects similar to the positive drug ceftriaxone sodium. influenza virus fm1-infected pneumonia leads to an inflammatory storm, suggesting that many pro-inflammatory cytokines like tnf-α and il-6 released into blood and lung tissue [28] [29] [30] . in our study, fm1 was non-invasive intratracheally instilled into lung tissue of mice. results showed that mice infected by fm1 caused a significant rise in the secretions of tnf-α (fig. 5b, d) and il-6 ( fig. 5c, e) in both female and male mouse serum, which was suppressed by b4. thus, anemoside b4 exhibited a protective effect on fm1-induced pneumonia. to assess the protective effect of anemoside b4 on lung inflammation induced by fm1 infection, the levels of tnf-α and il-6 were examined in balf and lung tissue and he staining of lung tissue was observed. as shown in fig. 6 , there were no obvious pathological changes in lung tissues of mice in the control group; pathological changes of lung tissues in the fm1 group showed edema, structural disorder, thickened alveolar septum, alveolar cavity shrinkage, and inflammatory cell infiltration. after treatment with b4 or ribavirin, the pathological changes of lung tissue were significantly alleviated in both female and male mice (a, b) . the levels of tnf-α and il-6 ( fig. 6c-f ) in balf and lung tissue were significantly increased by fm1 infection, which was reversed by anemoside b4. our study indicated that anemoside b4 ameliorated fm1-induced lung inflammation, of which the effects were similar to the positive drug ribavirin. tlr4, a transmembrane receptor located on the surface of many cells, plays a pivotal role in inflammatory processes [31] . once stimulated, tlr4 forms a dimer and then regulates the downstream protein, thereby spawning a pathogen-specific innate immune response through releasing pro-inflammatory cytokines [32, 33] . to further study the anti-inflammatory mechanism of anemoside b4, the expression of tlr4, myd88, and md2 were analyzed in lung tissues. the results showed that fm1 activated the expression of tlr4, myd88, and md2, which was significantly suppressed by b4 (fig. 7a-c) . furthermore, molecular docking assays showed that b4 was bound to tlr4 (fig. 7d) and interacted with several amino acid sites including leu198, leu231 and his199 (fig. 7e) , which occupied the space and weakened the activation of tlr4 by fm1. taken together, anemoside b4 ameliorated fm1-induced pneumonia via the tlr4/ myd88 pathway with binding to tlr4. pneumonia is inflammation of the terminal airways, alveoli, and interstitial lungs, which can be caused by pathogenic microorganisms such as bacteria, viruses, fungi, and atypical pathogens [34] . among them, bacteria and virus are the main pathogenic factors [35] . bacterial factors including streptococcus pneumoniae, staphylococcus aureus, and klebsiella pneumoniae, etc. leads to pneumonia [36] . although antibiotics were found to effectively decrease the death induced by bacterial-induced pneumonia, yet the abuse of antibiotics leads to drug-resistant or super bacterial, which is a worldwide problem. in addition to bacterial, virus like coronavirus, influenza virus, cytomegalovirus, etc. always lead to pneumonia [37] . specifically, covid-19 virus-induced pneumonia has a higher mortality. so far, there are not effective drugs for the treatment of covid-19-pneumonia. thus, to explore an effective drug to treat with pneumonia induced by bacterial or virus is imperative and necessary. pulsatilla chinensis (bunge) regel, a traditional chinese medicine, was commonly used in the treatment of a variety of infectious diseases and malignant tumors [23] . a variety of saponins with anti-inflammatory and antitumor effects have been isolated from the root of pulsatilla chinensis (bunge) regel, among which anemoside b4 (a, b) . the level of tnf-α, il-6 (c) in female mice balf, the level of tnf-α, il-6 (d) in female mice lung tissue, the level of tnf-α, il-6 (e) in male mice balf, the level of tnf-α, il-6 (f) in male mice lung tissue were determined by elisa kits. scale bar 50 μm. value represents mean ± sd (n = 5), # p < 0.05, ## p < 0.01, ### p < 0.001 vs. control group, *p < 0.05, **p < 0.01, ***p < 0.001 vs. fm1 group is the major ingredient that quantized over 4.6% in terms of 2015 edition chinese pharmacopoeia. as it stands now, quite few studies on the pharmacological activity of anemoside b4 were found. for example, hu et al. found that anemoside b4 could alleviate intestinal dysfunction by reducing inflammatory reaction [38, 39] , which has therapeutic effects on immune system-related diseases [40, 41] . anemoside b4 can reduce the nephrotoxicity of cisplatin [42] and the renal damage caused by adenine [43] . however, there is no relevant report on the effect and mechanism of anemoside b4 on pulmonary inflammation. in this study, we investigated the effects of b4 on pneumonia. data analysis from network pharmacology shows that b4 has a potential for regulating pro-inflammatory cytokines such as il-1β, il-6, and tnf-α. among which, tnf-α is an important pro-inflammatory cytokines in the body, which plays a vital role in fig. 7 effects of b4 on the tlr4/myd88 pathway. a-c the protein expression of tlr4, myd88, and md2 were measured by western blotting. the mixed mice proteins were used in this experiment. d, e docking results of b4 with tlr4. the chemical structure of b4 is shown in pink. tlr4 is shown in other colors. value represents mean ± sd (n = 5), ### p < 0.001 vs. control group, ***p < 0.001 vs. fm1 group various physiological responses such as the synthesis and release of inflammatory mediators, neutrophil accumulation in the lungs, and complement activation [44] . and il-6, a key cytokine produced by activated t cells and fibroblasts, has a wide range of biological activities such as immunoregulation [45, 46] . studies have shown that il-6 can catalyze and amplify the inflammatory response and its expression level effectively reflects the severity of tissue cell damage, which is used as an effective indicator for clinical diagnosis of acute and chronic inflammation [47] . il-1β is an important inflammatory factor with dual sources of peripheral and central nerve, which can induce production and release of various inflammatory factors such as il-8 [48] . myeloperoxidase (mpo), a hemoglobin protein that is rich in neutrophils, can catalyze the oxidation of chloride ions to produce hypochlorous acid, kill microorganisms in phagocytic cells, and destroy various target substances [49] . it plays a key role in the body to produce and regulate inflammatory response. hematology analysis is an effective way to detect pneumonia, especially pneumonia caused by bacterial infection [50] . in most cases, a significant increase in white blood cells and neutrophils can be observed in bacterial pneumonia [51] . therefore, hematology counts and pro-inflammatory cytokines were selected as test indicators. in practical experiments, anemoside b4 exerted significant inhibitory effects on mpo, il-1β, il-6, and tnf-α. besides, anemoside b4 decreased wbc and neu cell counts in blood of the pneumonia mice. therefore, anemoside b4 has a significant protective effect on kp-or fm1-infected pneumonia in vivo. toll-like receptor 4 (tlr4), the first identified member of tlr family, is a transmembrane protein characterized by an extracellular domain containing leucine-rich repeats (lrrs) with which the md-2 molecule is associated [52] . after activated by virus, the tir domain of tlr4 interacts with the tir domain of myd88 and binds to another tir-containing adaptor protein, myd88 adaptor-like (mal), leading to an inflammatory cascade effector enzyme such as the expression of tnf-α, il-1β, and il-6 [53] [54] [55] [56] . thus, when b4 bound to tlr4, it will prevent the virus from activating tlr4, which may be an alternative strategy to treat fm1-induced viral pneumonia. previous study indicated that emodin can inhibit influenza viral-induced pneumonia via the tlr4 pathway [57] . in this study, our results indicated that b4 inhibited fm1induced expression of tlr4/myd88/md2 proteins. furthermore, our docking results indicated that b4 could directly bind to tlr4 protein. therefore, anemoside b4 suppressed the fm1 or kp-induced pneumonia via the tlr4/myd88 pathway. in summary, our study demonstrated that anemoside b4 exhibited significant protective effects on klebsiella pneumoniae-or influenza virus fm1 induced pneumonia via thetlr4/myd88 signaling pathway (fig. 8 ). community-acquired pneumonia: an overview viral pneumonia: etiologies and treatment evolving understanding of the causes of pneumonia in adults, with special attention to the role of pneumococcus pneumonia-management in the 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toll-like receptor 4 agonistic antibody promotes host defense against chronic pseudomonas aeruginosa lung infection in mice toll-like receptor 4 polymorphisms are associated with resistance to legionnaires' disease flavonoids from houttuynia cordata attenuate h1n1-induced acute lung injury in mice via inhibition of influenza virus and toll-like receptor signalling liu shen wan inhibits influenza a virus and excessive virus-induced inflammatory response via suppression of tlr4/nf-κb signaling pathway in vitro and in vivo screening of antiviral components of ma huang tang and investigation on the ephedra alkaloids efficacy on influenza virus type a emodin inhibition of influenza a virus replication and influenza viral pneumonia via the nrf2, tlr4, p38/jnk and nf-kappab pathways publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions gh designed the research. hj, ry and cx conducted the experiments. hj and gh wrote the manuscript. ry, cy, hs, wq, cy, hl, zy, ys and xq revised the manuscript. all authors read and approved the final manuscript. not applicable. the study was established according to the ethical guidelines and approved by the ethics committee on laboratory animal management of guangxi university of chinese medicine. we declare that the publisher has the author's permission to publish the relevant contribution. 1 the authors declare no competing conflict of interests. key: cord-279498-ez3yq7xi authors: suzumura, akio title: immune response in the brain: glial response and cytokine production date: 2008-12-31 journal: neuroimmune biology doi: 10.1016/s1567-7443(07)10014-4 sha: doc_id: 279498 cord_uid: ez3yq7xi abstract although the brain has been considered as an immunologically privileged site, the evidence to date suggests that this is no longer the case. cytokines such as interferon (ifn)-γ, tumor necrosis factor (tnf)-α, and interleukin (il)-3 induce class i major histocompatibility complex (mhc) antigen expression on neural cells. ifn-γ, the most potent inducer of mhc antigen, also induces class ii mhc antigen expression on microglia and astrocytes, which enable them to function as antigen-presenting cells. thus, in some pathological conditions, invading t cells can interact with neural cells to induce central nervous system (cns) damage. glial cells have also been shown to produce various cytokines and chemokines. almost all cytokines and chemokines known to occur in the immune system are also produced in the cns. in this chapter, the glial responses contributing to neuroimmune interactions are reviewed, with a focus on production and functions of cytokines in the cns. the brain has long been considered as an immunologically privileged site based on a large body of evidence: the lack of major histocompatibility complex (mhc) antigen expression on neural cells; the lack of lymphoid drainage in the central nervous system (cns); and the presence of the blood-brain barrier (bbb), which blocks the invasion of immune cells or high molecular substances including antibodies into the brain. however, as has been shown by research published in the 1980s, some cytokines or viral infections induce mhc antigen expression on both neuronal and glial cells. interferon-g (ifn-g), the most potent inducer of mhc antigen, also induces class ii mhc antigen expression on microglia and some populations of astrocytes, which enable them to function as antigen-presenting cells (apcs). in order to effectively present antigens to t cells, apcs have to express other costimulatory molecules. microglia and astrocytes have been shown to express these costimulatory molecules, and this expression is also enhanced by exposure to ifn-g. thus, if activated t cells enter the cns, either microglia or some populations of astrocytes are able to present cns antigens to expand a t-cell clone specific for a particular cns antigen. in fact, it has also been shown that activated t cells can enter the brain through an intact bbb. consequently, in certain pathological conditions, glial cells may alter their functions to actively interact with immune cells. in most cases these glial changes are mediated by cytokines. another remarkable glial response in pathological conditions is the production of cytokines. in the late 1980s, many laboratories, including ours, have demonstrated the production of cytokines by glial cells. almost all cytokines known to occur in the immune system were produced in the cns. thus, the brain should no longer be considered as an immunologically privileged site. in this chapter, i will review the glial responses in neuroimmune interactions in the cns with a focus on the production and functions of cytokines. in normal or unstimulated conditions in vivo and in vitro, neuronal and glial cells do not usually express class i or class ii mhc antigens on their surface, whereas microglia only weakly express class i mhc antigens in vitro. consequently, in the normal brain, neural cells cannot interact with their own immune cells in a specific manner. however, it has been shown that both neuronal and glial cells can be induced to express class i mhc antigens in response to lymphokines [1, 2] . as a result of this induction, the cytotoxic t cells acquire the capacity to lyse the cns cells in a mhc-restricted manner. although ifn-g is a principal factor for the induction of mhc antigen expression, tumor necrosis factor-a (tnf-a) can also induce class i mhc antigen expression on astrocytes, but not on oligodendrocytes [3] . lymphokines, especially ifn-g, also induce the expression of class ii mhc antigens on astrocytes [4] and microglia in vitro [5] . this expression is associated with the induction of mrna for class ii mhc antigens. induction of class ii mhc antigens is also observed in vivo in certain pathological conditions. in the brains of experimental allergic encephalomyelitis (eae), microglia near the infiltrating t cells are reported to be class ii mhc antigen-positive [6] [7] [8] , suggesting that a t cell-derived cytokine, most probably ifn-g, can induce class ii mhc antigen expression in vivo as well. after axotomy there are increased numbers, relative to controls, of microglia in and around facial nerve nuclei. moreover, these cells are reportedly class ii mhc antigen-positive [9] . since the bbb is not damaged in this experimental condition and since there is no definitive evidence that neural cells produce inf-g in the cns, it is unlikely that ifn-g is responsible for the induction of class ii mhc antigen expression in this model. another candidate for the induction of class ii mhc antigens in microglia is interleukin (il)-3. we have shown that il-3 induces, in a dose-dependent manner, surface expression and mrna for class ii mhc antigens in microglia, which is completely inhibited by anti-il-3 antibody [10] . although we do not detect il-3 or il-3 mrna in either microglia or astrocytes in the mouse cellular system under study, it has been reported that rat microglia produce il-3 in vitro [11] , and that il-3 mrna is detected in some populations of astrocytes and neurons by in situ hybridization [12] . therefore, it is possible that il-3 derived from degenerating neurons, reactive astrocytes, or microglia in vivo may themselves induce class ii mhc antigens on microglia in certain pathological conditions. in contrast to il-3, granulocyte-macrophage colony-stimulating factor (gm-csf) downregulates ifn-g-induced class ii mhc antigen expression in microglia. the suppression occurs in a dose-dependent manner and is neutralized by anti-gm-csf antibody [10] . as we have shown previously, gm-csf is produced by astrocytes [13] and induces the proliferation of microglia in vitro [14, 15] . it is possible that astrocytes downregulate immunoregulatory functions of microglia. however, so far, there is no evidence that gm-csf contributes to the modulation of microglia proliferation and suppression of their ia antigen expression in either physiological or pathological conditions in vivo. all the macrophage deactivating cytokines, or inhibitory cytokines, such as il-10, il-4, and transforming growth factor-b (tgf-b) downregulate the inf-g-induced class ii mhc antigen expression in microglia [16] [17] [18] . as we and other groups have shown, astrocytes and microglia produce il-10 [18] and tgf-b [19, 20] , but neither cells produce il-4, although both cell types express il-4 receptors [17, 21] . thus, microglia may downregulate their own immunoregulatory functions in an autocrine fashion, or the astrocyte may suppress the functions of microglia in a paracrine manner. it is also possible that invading t helper cells, especially t helper 2 (th2), may downregulate class ii mhc antigen expression in microglia by these inhibitory cytokines. induction of mhc antigen expression on glial cells occurs without breakdown of bbb, without invasion of immune cells. we, and another group, have shown that infection with neurotropic corona virus induces class i mhc antigen expression on oligodendrocytes and astrocytes [22, 23] and class ii mhc antigen on astrocytes [23] . these inductions permit glia to interact with invading immune cells to produce cns pathology. table 1 , and chemokines [18, 19, [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] . rat microglia reportedly produce il-3 in culture [11] . only a trace amount of il-1, but not the other cytokines, is detectable in the supernatant of unstimulated microglial culture. however, lipopolysaccharides (lps), and/or ifn-g in some cases, induce cytokine production. since microglia express receptors for most of the cytokines produced (see table 1 ), these components may function as an autocrine regulator. they also express receptors for cytokines, which are produced by other cells, but not by themselves, such as il-2 or gm-csf. thus, the latter may function as paracrine mediators (for functions of these cytokines on microglia, refer to our previous review [34] ). unstimulated microglia do not express the il-2 receptors (il-2r); however, lps treatment will induce il-2r expression on these cells. moreover, il-2 can also induce the proliferation of lps-stimulated microglia [35] . although il-2 treatment has been shown to induce the proliferation of oligodendrocytes as well [36] , we could not confirm these effects [2] . microglia also express the receptor for il-4, the cytokine produced by t cells, but not in the cns. thus, il-4 may be a paracrine mediator exerting its effects only in cases of an inflammatory process occurring in the cns, but not in the normal brain. we have shown the production of il-5 and the upregulation by ifn-g in murine microglia by means of rt-pcr for mrna expression and the bioassay to assess il-5 activity [28] . however, since we have not detected il-5 receptors on neural cells, the functions of il-5 in the cns remain to be elucidated. in contrast to murine microglia, lee et al. [37] failed to detect il-5 mrna expression in human microglia as assessed by rt-pcr, while they detected mrna for the il-5 receptor. astrocytes produce cytokines very similar to those produced by microglia (table 2) . however, microglia, rather than astrocytes, seem to be a principal source of most critical cytokines, immune response in the brain such as tnf-a, il-1, and il-12 as discussed later [26, 29] , in both pathological conditions and in culture systems. astrocytes sometimes have suppressive effects on microglia or microgliaderived cytokines. for, example, in contrast to microglia after stimulation with lps and ifn-g, astrocytes produce il-12 p40, but not p35 [29] . when the astrocyte-derived p40 forms a homodimer, it may suppress the functional heterodimer il-12 p70 produced by microglia. in addition, the production of il-12 p70 by activated microglia was inhibited by coculture with astrocytes [38] . thus, it is possible that astrocytes suppress microglial cytokine production and/or the effects of produced cytokines. the suppression of ifn-g-induced mhc class ii expression on microglia by astrocyte-derived gm-csf is another example of this type of interaction [10] . as discussed above, ifn-g activates various functions of glial cells including the induction of cytokines. the production of ifn-g was thought to be restricted to lymphoid cells. however, it has recently been shown that human fetal forebrain cells can be induced to express ifn-g mrna and produce ifn-g protein when stimulated with trypanosome lymphocyte-triggering factor (tltf) [39] . the authors claimed that astrocytes were the major producer of ifn-g in response to tltf. we have shown recently that microglia produce ifn-g in response to il-12 and/or il-18 [32] . thus, microglia may be another source of inf-g production in the cns. it has been shown that apcs such as macrophages and dendritic cells also produce ifn-g in response to il-12 [40] . candidates for nonfunctional apcs in the cns are microglia, astrocytes, and endothelial cells [4, [41] [42] [43] [44] [45] . they usually do not express class ii mhc antigen constitutively, although some populations of microglia reportedly may express class ii mhc antigens constitutively [46] . these cells induced the expression of class ii mhc molecules after treatment with certain inflammatory cytokines, especially ifn-g [4, 5, 41, 44] , in addition to expressing some of the costimulatory molecules as well [47] [48] [49] . there is published evidence that endothelial cells [50] , astrocytes [41] , and pericytes [51] can process and present protein antigens to primed cd4-positive t cells in vitro, but the specific role of these cells as apcs in vivo is still unclear. astrocytes do not usually express class ii mhc antigens in vivo, even in the presence of inflammatory cells [42] . since microglia have functional characteristics very similar to macrophages and can be induced to express class ii mhc antigens as discussed above, microglia are the most possible candidates for apcs in the cns. the expression of costimulatory molecules, such as b7, icam, lfa3, in microglia, but only a few in astrocytes, further supports this hypothesis. menendez iglesias et al. [49] detected b7-2, but not b7-1, in murine microglia only after stimulation with lps and ifn-g. satoh et al. [48] have shown that human microglia, but not astrocytes, express both b7-1 and b7-2, suggesting that microglia is a much more suitable candidate for local apcs in the cns. in fact, microglia when stimulated with ifn-g reportedly presented antigen to ovalbumin-specific or myelin basic protein (mbp)-specific t cells in vitro [44, 45] . in a carefully executed study, hickey and kimura [43] have shown that microglia function as apcs in pathological conditions in vivo. they used bone marrow chimeras of eaesusceptible and -resistant animals, and found that eae lesions developed only when the perivascular microglia were replaced with those of an eae-susceptible strain, suggesting that antigen presentation by perivascular microglia is critical for the development of eae lesions. professional apcs such as dendritic cells and macrophages produce il-12 and il-18. both cytokines have been shown to be key cytokines in the development of autoimmune processes, regulating differentiation of naïve t cells into th1. in order to exert its activity, il-12 has to form a heterodimer of p35 and p40; the homodimer of p40 suppresses the functional heterodimer. immature il-18 is cleaved by caspase-1 to become a functionally mature il-18 that induces the differentiation of th1 and the cytotoxic activity of nk and t cells. it has been reported that both microglia and astrocytes produce il-12 upon stimulation with lps [38] , while we detected functional il-12 p70 production only in microglia, but not in astrocytes, after stimulation with lps and ifn-g [30] . since soluble tnf receptors reportedly suppress il-12 production by human microglia [52] , tnf signal may also be involved in il-12 production. microglia and astrocytes also express il-18 mrna after stimulation with lps [32, 53] . lpsstimulated microglia have enough il-18 bioactivity to induce inf-g production by thymocytes and splenocytes in synergism with il-12. this suggests that microglia express caspase-1 as well. in fact, caspase-1 mrna expression is elevated in microglia in multiple sclerosis (ms) plaques [54] where il-18 is also reported to be elevated [55] . interestingly, there is a group of microglia that produce only il-12 p40, but not il-12 p35, resulting in the failure to produce functional il-12 p70 heterodimers [29] . the population did not produce il-18 even after lps stimulation (unpublished observation). therefore, microglia may have subpopulations, which regulate the differentiation of t cells in a different manner. both microglia and astrocytes produce the same cytokines, such as il-1, il-6, tnf-a, and tgf-b. however, there are several differences in the response to stimulation in these two cell types. for example, microglia produce tnf-a in response to lower doses of lps than are required for astrocytes and more rapidly than astrocytes as well. il-6 production is induced by tnf-a in astrocytes, but not in microglia [27] . similarly, gm-csf produced by astrocytes induces il-6 production in microglia, but not in astrocytes [56] . these observations indicate that microglia and astrocytes may mutually regulate their individual cytokine production. since microglia are activated in the earlier phase than are astrocytes under various pathological conditions, microglia may initiate the cascade of cytokine actions in the cns cytokine network. inhibitory signals are also included in the network (table 3) . tgf-b, produced by astrocytes and microglia, suppresses all the functions of microglia. it suppresses m-and gm-csf-induced proliferation of microglia, lps-induced activation of enzymatic activity in microglia, ifn-ginduced class ii mhc antigen expression and cytokine production by microglia. tgf-b along with il-4 and il-10 is known to be a macrophage-deactivating factor. therefore, these cytokines may function as negative regulators in the cns cytokine network by suppressing cytokine production and activation of microglia. in fact, it has been found that these inhibitory cytokines exert their influence on microglia differently. tgf-b functions as if it is a total inhibitory factor [16] . il-10 suppresses cytokine production and ifn-g-induced class ii mhc antigen expression in microglia, but does not suppress the proliferation or the activation of lysosomal enzymes in microglia [18] . il-4 also suppresses ifn-g-induced class ii mhc antigen expression in microglia [17] . however, unlike other inhibitory cytokines, il-4 induces the proliferation of microglia in either unstimulated or m-, or gm-csf-stimulated conditions. il-4 does not suppress lps-induced cytokine production, though it suppresses gm-csf-induced il-6 production by microglia [56] . we also found that il-10, but neither tgf-b nor il-4, suppressed the expression of cytokine receptors [57] . thus, it would appear that all these three inhibitory cytokines regulate the functions of microglia in a distinct manner, and that il-10 may be the most potent inhibitor for the functions of cytokines on microglia because it suppresses both cytokine production and receptor expression. several lines of evidences suggest that tnf-a plays a critical role in the pathogenesis of inflammatory demyelination, either directly or indirectly via induction of other cytokines, nitric oxide (no), or free radicals (fig. 1) . increased cerebrospinal fluid levels of tnf-a have been demonstrated in patients with ms [58] . tnf-a-positive microglia and astrocytes have been identified, especially in new active plaques. in vitro studies have demonstrated that tnf-a kills oligodendrocytes, myelin-forming cells in the cns [59, 60] , and that microglia are the principal table 3 . ", upregulate; !, no effect; #, downregulate. a il-4 upregulates il-4 receptor, but does not affect the expression of other receptors on microglia. immune response in the brain effectors for oligodendrocyte killing [61] . it has also been shown that anti-tnf-a antibody suppresses the development of eae, an animal model of ms [62, 63] . demyelination has been demonstrated to be much more severe in transgenic mice producing tnf-a in the cns [64] . in addition, tnf-a induces inflammatory cytokines or chemokines in endothelial cells and impairs the tight junctions of the bbb [65] . up until now, several substances that suppress tnf-a production have been used for the treatment of eae and ms. most of them, such as phosphodiesterase inhibitors, n-acetyl-l-cysteine, have been shown to effectively suppress the development of eae and ms [66] [67] [68] , further supporting the hypothesis that tnf-a is critical for the development of inflammatory demyelination. however, experimental demyelination could also be induced in tnf-a knockout mice, though eae was delayed in the onset and inflammatory leukocytes failed to move normally into the cns parenchyma [69] . more recently, tnf-a has been identified as a factor that promotes remyelination [70] . thus, although tnf-a is an important cytokine, it may not be the sufficient effector molecule for inflammation and demyelination. it is also possible that tnf-a may exert different effects on inflammatory demyelination, depending on whether the tnf signaling through type 1 tnf receptor (tnfr1) or tnfr2 is dominant. gliosis is a rather common pathological finding observed as a glial scar following inflammation, demyelination, ischemia, and neuronal degeneration. it consists of astrocyte proliferation, hypertrophy, and increased synthesis of glial fibrillary acidic protein (gfap), a phenotypic marker for astrocytes. evidence to date suggests critical roles for cytokines in the development of astrocytic gliosis. fontana et al. [71] first demonstrated that factors from activated lymphocytes stimulated astrocyte proliferation and designated the factor(s) as glial cell-stimulating factor (gsf). merrill et al. [36] also demonstrated increased proliferation of astrocytes after treatment with lymphokines. using enriched cultures of astrocytes and recombinant cytokines, selmaj et al. [72] showed that tnf-a is a primary factor to bring about the proliferation of rat astrocytes. however, giulian et al. [73, 74] have shown that il-1 derived from microglia [24] is the principle factor to induce astrocyte proliferation in gliosis. in contrast, yong et al. [75] claimed that the primary factor that induced the proliferation of human astrocytes was ifn-g and not il-1 or tnf-a. these differences in experimental results may be attributed to either species differences or redundancy of functions for these cytokines. alternatively, it is possible that other factors induced by either il-1, tnf-a, or ifn-g may also play a role in the proliferation of astrocytes. in view of these diverse results, it can be concluded that cytokines contribute to the pathogenesis of gliosis. however, precise identification of individual cytokine contributions to the overall process will require additional experimental inquiries. tnf-a has also been implicated as an effector for neuronal degeneration [76] [77] [78] . tnf-a exerts its cytotoxicity directly via tnfr1. alternatively, it also induces no or free radicals to form the toxic peroxinitrite. it has been shown that b-amyloid stimulates microglia to produce factors toxic to neurons. it is possible that neuronal apoptosis induced by b-amyloid is also mediated by glia-derived tnf-a [79] . combs et al. [80] concluded that the most critical factor in b-amyloid-induced, microglia-mediated neuronal apoptosis might be no, because neurotoxicity was decreased by the selective inhibitors against inducible nitric oxide synthase. apoptosis of motor neurons and dorsal root ganglion neurons by peripherin aggregates is also reportedly mediated by tnf-a [81] . tnf-a also exerts its neurotoxicity by activating astrocytes to release glutamate [82] . recently, we have shown that the most neurotoxic factor from activated microglia is glutamate [83] . tnf-a dose not exert direct neurotoxicity, but induces neurotoxicity via glutamate production by microglia. glutamate disturbs the mitochondrial respiratory chain to cause energy depletion in neurons, which results in neuronal damage toward cell death [84] . in contrast, il-1, but not tnf-a, may be involved in neurotoxicity during some variants of viral encephalitis [85] . the protein fas associated with death domain (fadd) is an adaptor protein of the tnf receptor family death pathway. a number of fadd-positive dopaminergic neurons in the substantia nigra pars compacta have been shown to be significantly decreased in patients with parkinson's disease (pd), as compared to levels in normal subjects [86] . this decrease correlated with the known selective vulnerability of nigral dopaminergic neurons in pd. on the basis of the latter, the authors concluded that the tnf-fadd pathway contributed to the susceptibility of dopaminergic neurons in pd to the effects of tnf-mediated apoptosis [86] . interestingly, cytokines described above as toxic also have protective roles for neurons against oxidative stress. tnf-a and il-1 have been shown to increase the level of manganese superoxide dismutase (mn-sod) in astrocytes, in a dose-and time-dependent manner [87] . since sod functions as protective against oxidative stress, and since the increased mn-sod activity has been demonstrated in the substantia nigra of parkinsonian patients [88] , these cytokines may function to protect degenerating neurons, via induction of sod. il-1 reportedly increases the production of nerve growth factors by astrocytes [89] . therefore, a balance between toxic and protective factors induced by cytokines may determine neuronal damage (see fig. 1 ). microglia undergo various morphological changes to become either ramified, amoeboid, or rodshaped. we have shown that all of these morphological changes could be reproduced in vitro with various cytokines [14, 15] . microglia also form a unique phenotype of multinucleated giant immune response in the brain cells (mngc), which are observed in aids encephalopathy, tuberculosis, etc. lee et al. [90] have demonstrated that treatment with il-3, il-4, ifn-g, and gm-csf induces mngc in rat microglia, while addition of il-1, il-6, or tnf-a failed to form mngc. in mouse experiments using microglia, there was no single cytokine that induced mngc in culture. however, when stimulated with il-4 or il-13 in the presence of gm-csf or m-csf, mngc formation occurred in the cultures of mouse microglia [91] . the different results between these studies may be attributable to species differences. nevertheless, the results of these studies indicate that introduction of cytokines, most probably those that are t cell-derived, can induce mngc formation without infectious agents. inducible expression of h-2 and ia antigens on brain cells expression of h-2 antigen on oligodendrocytes is induced by soluble factors from concanavalin a activated t cells tumor necrosis factor induces expression of mhc antigens on mouse astrocytes astrocytes as antigen-presenting cells. i. induction of ia antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation mhc antigen expression on bulk isolated macrophage-microglia from newborn mouse brain: induction of ia antigen expression by gamma-interferon immunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to ia positive cells with dendritic morphology an immunoelectron microscopical study of class ii major hsitocompatibility complex during chronic relapsing experimental allergic encephalomyelitis in biozzi ab/h mice microglial involvement in autoimmune inflammation of the central and peripheral nervous system expression of ia antigen on perivascular and microglial cells after sublethal and lethal motoneuron injury induction of mhc class ii antigen expression on murine microglia by interleukin-3 rat microglial interleukin-3 in situ hybridization histochemistry localization of interleukin-3 mrna in mouse brain production of granulocyte/macrophage colony stimulating factor by cultured astrocytes effects of colony stimulating factors on isolated microglia morphological transformation of microglia in vitro transforming growth factor beta suppresses activation and proliferation of microglia in vitro il-4 induces proliferation and activation of microglia but suppressed their induction of class ii major histocompatibility complex antigen expression production of interleukin-10 by mouse glial cells in culture macrophage-, and astrocyte-derived transforming growth factor beta as a mediator of central nervous system dysfunction in acquired immunodeficiency syndrome differential expression of transforming growth factor beta1,2, and 3 by glioblastoma cells, astrocytes, and microglia expression of cytokine receptors in cultured neuronal and glial cells corona virus infection induces h-2 antigen expression on oligodendrocytes and astrocytes viral particles induce ia antigen expression on astrocytes interleukin-1 of the central nervous system is produced by ameboid microglia on the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases production of tumor necrosis factor alpha by microglia and astrocytes in culture tnfa induces il-6 production by astrocytes but not by microglia production of interleukin-5 by mouse astrocytes and microglia in culture production of interleukin-12 and the expression of its receptors by murine microglia ifns are critical regulators of il-1 receptor antagonist and il-1 expression in human microglia murine microglial cells produce and respond to interleukin-18 production of interferon-g by microglia production of il-27 and il-12 family cytokines by microglia and their subpopulations cytokine network in the central nervous system and its roles in growth and differentiation of glial and neuronal cells induction of functional il-2 receptor in mouse microglia proliferation of astroglia and oligodendroglia in response to human t cell-derived factors cytokines, chemokines, and cytokine receptors in human microglia il-12 production by central nervous system microglia is inhibited by astrocytes african trypanosomes activate human fetal brain cells to proliferation and ifn-g production ifn-a production by antigen presenting cells: mechanisms emerge astrocytes present myelin basic protein to encephalitogenic t-cell lines expression of ia molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat perivascular microglial cells of the cns are bone marrow-derived and present antigen in vivo antigen presentation and tumor cytotoxicity by interferon-gamma-treated microglial cells immune regulation by brain cells in the central nervous system; microglia but not astrocytes present myelin basic protein to encephalitogenic t-cells under in vivo-mimicking conditions normal adult ramified microglia separated from other central nervous system macrophages by flow cytometric sorting the costimulatory molecule b7 is expressed in human microglia in culture and multiple sclerosis acute lesions t-cell costimulatory molecules b71 (cd80) and b7-2 (cd86) are expressed in human microglia but not in astrocytes in culture analysis of b7-1 and b7-2 costimulatory ligands in cultured mouse microglia: upregulation by interferon-gamma and lipopolysaccharide and downregulation by interleukin-10, prostaglandin e2 and cyclic amp-elevating agents antigen-specific damage to brain vascular endothelial cells mediated by encephalitogenic and nonencephalitogenic cd4 t cell lines in vitro antigen presentation by brain microvessel smooth muscle and endothelium soluble tumor necrosis factor receptor inhibits interleukin 12 production by stimulated human adult microglial cells in vitro cultures of astrocytes and microglia express interleukin 18 caspase-1 expression in multiple sclerosis plaques and cultured glial cells ccr5(+) and cxcr3(+) t cells are increased in multiple sclerosis and their ligands mip-1alpha and ip-10 are expressed in demyelinating brain lesions selective induction of interleukin-6 in mouse microglia by granulocyte-macrophage colony stimulating factor il-10 inhibits both production of cytokine and expression of cytokine receptors in microglia cytokine levels in the cerebrospinal fluid and serum of patients with multiple sclerosis tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro cytokine cytotoxicity against oligodendrocytes. apoptosis induced by lymphotoxin microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide an antibody to lymphotoxin and tumor necrosis factor prevents transfer of experimental allergic encephalomyelitis anti-tumor necrosis factor therapy abrogates autoimmune demyelination increased severity of experimental autoimmune encephalomyelitis, chronic macrophage/microglial reactivity, and demyelination in transgenic mice producing tumor necrosis factor-alpha in the central nervous system exposure of tumor necrosis factor-alpha to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin phosphodiesterase inhibitor pentoxifylline, a selective suppressor of t helper type 1-but not type 2-associated lymphokine production, prevents induction of experimental autoimmune encephalomyelitis in lewis rats oral administration of the oxidant-scavenger n-acetyl-l-cysteine inhibits acute experimental autoimmune encephalomyelitis drop in relapse rate of multiple sclerosis patients using combination therapy of three different phosphodiesterase inhibitors challenging cytokine redundancy: inflammatory cell movement and clinical course of experimental allergic encephalomyelitis are normal in lymphotoxin-deficient, but not in tumor necrosis factor-deficient, mice tnfa promotes proliferation of oligodendrocyte progenitors and remyelination glia cell stimulating factor (gsf): a new lymphokine. part 1. cellular sources and partial purification of murine gsf, role of cytoskeleton and protein synthesis in its production proliferation of astrocytes in vitro in response to cytokines. a primary role for tumor necrosis factor interleukin-1 stimulation of astroglial proliferation after brain injury interleukin-1 is an astroglial growth factor in the developing brain g-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo tumor necrosis factor alpha-induced apoptosis in human neuronal cells: protection by the antioxidant n-acetylcysteine and the genes bcl-2 and crma inhibition of p75 tumor necrosis factor receptor by antisense oligonucleotides increases hypoxic injury and beta-amyloid toxicity in human neuronal cell line neuronal death in cytokine-activated primary human brain cell culture: role of tumor necrosis factor-alpha the inflammatory response system of brain: implications for therapy of alzheimer and other neurodegenerative diseases b-amyloid stimulation of microglia and monocytes results in tnf-alpha-dependent expression of inducible nitric oxide synthase and neuronal apoptosis apoptotic death of neurons exhibiting peripherin aggregates is mediated by the proinflammatory cytokine tumor necrosis factor-a cxcr4-activated astrocyte glutamate release via tnf-a: amplification by microglia triggers neurotoxicity neuritic beading induced by activated microglia is an early feature of neuronal dysfunction toward neuronal death by inhibition of mitochondrial respiration and axonal transport tumor necrosis factor-a induces neurotoxicity via glutamate release from hemichannels of activated microglia in an autocrine manner neuronal apoptosis mediated by il-1b expression in viral encephalitis caused by a neuroadapted strain of the mumps virus (kilham strain) in hamsters fadd: a link between tnf family receptors and caspases in parkinson's disease induction of manganese superoxide dismutase by cytokines and lipopolysaccharide in cultured mouse astrocytes a selective increase in particulate superoxide dismutase activity in parkinsonian substantia nigra regulation of nerve growth factor mrna by interleukin-1 in rat hippocampal astrocytes is mediated by nfkb lymphokine induction of rat microglia multinucleated giant cell formation multinucleated giant cell formation by microglia: induction by interleukin(il)-4 and il-13 key: cord-271114-hv3gwvdi authors: allam, gamal; alsulaimani, adnan a.; alzaharani, ali k.; nasr, amre title: neonatal infections in saudi arabia: association with cytokine gene polymorphisms date: 2015-04-22 journal: cent eur j immunol doi: 10.5114/ceji.2015.50836 sha: doc_id: 271114 cord_uid: hv3gwvdi in recent years, many studies have reported potential associations between cytokine gene polymorphisms and the development, course, and outcome of sepsis, often with apparently conflicting results. the objective of this study was to investigate single nucleotide polymorphism (snp) in the interleukin (il)-1β –31 t/c, il-6 –174 g/c, tumor necrosis factor α (tnf-α) –308 g/a, and interferon γ (ifn-γ) +874 a/t genes for their possible association with susceptibility to early onset sepsis (eos) in saudi newborn infants. a total of 205 newborn infants aged 1-2 days were consecutively enrolled onto the study having met the inclusion criteria (as per the research protocol). dna was extracted from filter papers using the chelex-100 method. the cytokines snp were genotyping using taqman 5’ nuclease allelic discrimination. for cytokine measurements we used the commercially available enzyme-linked immunosorbent assay (elisa) kit. our results show that the circulating il-1β, il-6, tnf-α, and ifn-γ were significantly (p < 0.001) elevated in eos patients compared to suspected and sepsis-free control groups; and il-1β –31c, il-6 –174g, tnf-α –308g, and ifn-γ +874a alleles were associated with eos in saudi infants. in conclusion, analysis of cytokines concentrations and snp for the four tested genes can be used as a predictor of sepsis outcome in newborns. sepsis is a host condition of systemic inappropriate inflammatory response to the invasion of microorganisms [1] . although there are many advances in the development of antibiotics and there has been an explosion of knowledge about the inflammatory response, sepsis still causes one million deaths each year (42% in the first week of life) [2] . currently, it is accepted that inappropriate host inflammatory responses or inappropriate defence mechanisms play important roles in the development of sepsis [3] [4] [5] . cytokines play vital roles in the regulation of host immune response, and altered expressions of cytokines are proven to be involved in the development of sepsis [6, 7] . several risk factors for sepsis development have been identified [6] , but the cause of basic differences in susceptibility between individuals and populations remains unclear [8] . in recent years, several groups have provided accumulating evidence indicating that the genetic background of the host influences the susceptibility and prognosis of sepsis [9] [10] [11] . previous studies have suggested that the variations in the genes encoding cytokines are involved in the modulation of inflammatory responses and are responsible for inter-individual differences in the susceptibility to sepsis [5] . recent, epidemiological studies suggest that some single nucleotide polymorphisms (snp) in the genes encoding inflammatory cytokines may influence the course and outcome of sepsis [5, [12] [13] [14] . exploration of host genetic markers with prognostic value for sepsis might be helpful in managing the neonates who would be most likely to benefit from aggressive antibiotic treatment, and they put the challenges involved into perspective [15] . therefore, identification of newborns that are at high risk of developing sepsis could help us develop some effective prevention strategies. tumour necrosis factor α (tnf-α), interleukin (il)-lβ, and il-6 are proinflammatory cytokines, which have been reported to play important roles in the inflammatory response and in the pathogenesis of sepsis syndrome [16] . the study of il-1β and tnf-α, cytokines that are synthesised at the beginning of the inflammatory cascade, has given differing results. some studies have reported an increase in the concentrations of these proinflammatory cytokines in systemic circulation in septic newborns [16] [17] [18] [19] [20] , while others demonstrated similar or even lower levels in infected newborns compared to healthy newborns [21] [22] [23] [24] . such discrepancies in results among different studies could explain the different outcomes of sepsis seen in neonates [25] . therefore, the variable of clinical evolution of neonatal sepsis is related to the individual variability in cytokine production and intensity of inflammation [25, 26] , which may be due to the variation of the genetic background of the patients [27] . results of different published studies addressing the association between the snp tnf-α -308 and development of sepsis in adults and neonates are contradictory. one of the studies suggested that the tnf-α -308a allele has been associated with increased susceptibility to septic shock and mortality from septic shock in adults [28] . however, this association has not been consistently observed [29, 30] . in a study by hedberg et al. [31] the septic patients presenting the aa/ga genotypes had a mortality rate from sepsis that was three times greater than that seen among those presenting the gg genotype. moreover, a meta-analysis study has confirmed an increased risk of sepsis in carriers of the a allele for the tnf-α -308 g > a snp [10] . regarding il-1β gene polymorphisms, previous studies indicate that snps of il-1β may be associated with a poor prognosis from sepsis [32, 33] . however, other studies did not find any association between il-1β -31 polymorphisms and development of bronchopulmonary dysplasia (bpd) in preterm infants [34] . on the other hand, genetic variation within the regulatory part of the il-6 gene may affect the incidence and outcome of sepsis [35, 36] . an association of the il-6 -174 genotype with sepsis in preterm infants has been reported. harding et al. reported a higher incidence of the il-6 -174 gg genotype in infants who developed septicaemia [37] . similarly, ahrens et al. reported that the il-6 -174 gg genotype was more frequent in infants with sepsis compared to infants without infection [38] . consequently, genetic variability in the regulatory and coding regions of inflammatory cytokine genes may influence the susceptibility and/or outcome of sepsis. we reported in a previous study that a high level of c-reactive protein (crp) was associated with early onset sepsis (eos) infection in neonates living in saudi arabia, and those babies carrying the a-allele were associated with high levels of circulating crp [39] . because il-1β, il-6, and tnf-α appear before crp in eos infection [16] [17] [18] [19] , such cytokine gene polymorphisms may provide useful biomarkers for the screening of patients at risk, as well as in the identification of those most likely to benefit from specific therapeutic choices [40] . therefore, the primary aim of the current study was to investigate snps in the il-1β -31 t/c (rs1143643), il-6 -174 g/c (rs1800795), tnf-α -308 g/a (rs1800629), and ifn-γ +874 a/t (rs2430561) genes for their possible association with susceptibility to eos in saudi newborn infants. the second aim was to evaluate the association between such snps in gene promoter and the circulatory level of corresponding cytokines. a prospective cohort (cross-sectional) study was carried out over six months, from march to august 2012, in the neonatal intensive care unit (nicu), king abdel aziz specialist hospital (kaash), taif city, kingdom of saudi arabia (ksa). a total of 205 newborn infants aged 1-2 days were included in this study. patients were enrolled if they met the following inclusion criteria for participation in the study: 40 ±2 weeks gestation, an admission to the nicu at kaash for at least 24 hours, fever (≥ 38.0°c measured consecutively on two occasions at least six hours apart), and symptoms or signs of sepsis as described previously [41, 42] . the subjects were classified into three different groups according to nasr et al. [39] . group i is the control group, in which babies have no symptoms or signs of sepsis and a negative blood culture. controls babies consecutively enrolled to this study, who were admitted to the newborn nursery unit for routine check up within the first two days after birth. group ii is the suspected group of neonates, which includes any infant with signs or symptoms of sepsis, chorioamnionitis, and those born to mothers who had intrapartum temperature ≥ 38.0°c or prolonged rupture of membrane ≥ 18 hours. it is important to note that blood cultures for this group were negative. once infection was confirmed by means of a positive blood culture, the infant was put into group iii, which is the early onset sepsis (eos) group. babies were clinically examined during their stay in the nicu. once sepsis was confirmed, blood samples were taken within 12-50 hours after birth. babies suspected of having sepsis infection, before proven positive culture, were treated by clinicians (neonatologist) with double antibiotics (ampicilin and gentamicin) until the baby became symptom free or finished their course of antibiotic. full blood count was routinely taken. all investigations and treatments were provided free of charge. newborns were weighed, and those with low birth weight were assessed clinically to differentiate between intrauterine growth restriction and low birth weight using the dubowitiz scoring system. before pharmacological treatment was started, 50 μl of blood were collected on filter paper (schleicher & schuell; n° 903tm) for dna amplification and quantification of cytokines. two mls of venous blood was collected in edta vacutainer ® tubes (becton dickinson, meylan, france) for bacterial culture. dna was extracted from filter papers using chelex-100, which was stored at -80°c. 25 μl from peripheral blood or discs of the same size from filter paper were incubated overnight in 1 ml of 0.5% saponin in pbs at 4°c, and they were then washed for 15-30 minutes in 1 ml pbs at 4°c. the discs or the pellets, were boiled in 200 μl of 5% chelex-100 in water for 15 minutes, and the dna was collected in supernatants after centrifugation at 250 × g for 10 minutes [39] . the following locations were investigated in this study: il-1β -31 t/c (rs1143643), il-6 -174 g/c (rs1800795), tnf-α -308 g/a (rs1800629), and ifn-γ +874 a/t (rs2430561) genes. genotyping was performed with taqman ® 5' nuclease allelic discrimination (assay by design/demand, applied biosystems, foster city, ca) as described previously [43] [44] [45] . interleukin 1β, il-6, tnf-α, and ifn-γ levels were estimated by using a high-sensitivity sandwich elisa kit (abcam ® , cambridge, uk) according to the manufacturer's instructions. briefly, a monoclonal antibody specific for each cytokine (il-1β, il-6, tnf-α, and ifn-γ) was coated onto the wells of the microtitre plates provided. samples and standards of known concentrations were applied into the plates. after the incubation period, the biotinylated monoclonal antibodies specific for il-1β, il-6, tnf-α, and ifn-γ were added and incubated for one hour. after washing, the enzyme streptavidin-hrp that binds the biotinylated antibody was added and incubated for one hour at room temperature. a tmb substrate solution was added, and the plates were incubated in the dark for 15 minutes at room temperature. the reaction was stopped by using 2n hcl, and the absorbance was measured at 450 nm using a microplate reader (biotech, ca, usa). cytokine concentrations were determined by reference to standard curves construction. the distribution of cytokines (il-1β, il-6, tnf-α, and ifn-γ) genotype, allele frequencies and cytokine (il-1β, il-6, tnf-α, and ifn-γ) concentrations was analysed using spss version 16.0 (spss, inc., chicago, il, usa). using fisher's exact test, each snp was tested to determine if the population under investigation showed deviation from genotype frequencies expected under hardy-weinberg equilibrium (hwe) proportions [46] . tests were performed separately in the cases and controls. logistic regression analyses were performed to assess associations of genotype (independent variable). associations were quantified using odds ratios [or] with 95% confidence intervals (ci), which when they do not cross 1.00 are defined as statistically significant. the (il-1β, il-6, tnf-α, and ifn-γ) heterozygote genotypes were used as a reference in the analyses because they were the most frequent genotype in the sepsis-free controls. using the same software, we performed an overall comparison of allele frequency using a 2 × 2 test. the differences in cytokine (il-1β, il-6, tnf-α, and ifn-γ) concentrations between different study groups were analysed using kruskal-wallis test and the p-value was corrected for ties. logistic regression analyses were performed to assess the association between the cytokine (il-1β, il-6, tnf-α, and ifn-γ) serum levels in individuals with different cytokine genotypes. linkage disequilibrium (ld) analysis was performed in fstat version 2.9.3.2 to find any haplotype association within the study groups. this study received ethical clearance from the ethical committee of the college of medicine, taif university, ksa. informed consent was obtained from the neonates' parents/legal guardians, who participated in the study after we provided them with adequate information on the objectives and benefits of the project. the general characteristics of the study population are presented in table 1 . in group i, 68 individual had no symptoms for early onset sepsis (eos) with negative blood culture at a mean ± standard deviation (sd) age of 1 ± 0.12 days. group ii, suspected of eos: 68 patients had a clinical symptom of eos with negative blood culture at a mean ± sd age of 1 ± 0.12 days. group iii (eos): 69 patients had a clinical symptom with positive blood culture at a mean ± sd age of 1 ± 0. the age and sex were matched between the three groups and were not significantly different (p > 0.05, table 1 ). newborns with sepsis had significantly higher serum levels of pro-inflammatory cytokines (il-1β, il-6, tnf-α, and ifn-γ) compared to both suspected and sepsis-free control groups (overall p value < 0.001, table 1 ). as shown in table 2 , neonates with eos had significantly higher serum levels of il-1β, il-6, tnf-α, and ifn-γ compared to the suspected group (p value < 0.001, 0.001, 0.001, and 0.001, respectively). genotype frequencies for the candidate snps among the study groups with and without sepsis are shown in table 3. all polymorphisms analysed in this study were found to be in hwe (data shown in table 3 ). the il-1β cc genotype and allele were associated with eos compared to suspected patients by unadjusted analysis; for cc genotype: or = 6.22, 95% ci = (2.11-17.45), p value < 0.001; and for c allele, or = 13.43, 95% ci = (7.27-17.45), p value < 0.001 (table 4 ). the il-6 gg and cc genotypes were associated with the eos patients compared to suspected patients; for gg genotype: or = 6.83, 95% ci = (3.06-15.22), p value < 0.001; and for cc genotype: or = 4.90, 95% ci = (1.49-16.15), p value = 0.009 (table 4 ). patients carrying the g allele of the il-6 were associated with eos compared to suspected patients; or = 1.87, 95% ci = (1.11-3.20), p value = 0.002 (table 4). however, no significant association was found in eos patients carrying the c allele of il-6 compared to the suspected group. on the other hand, patients carrying the tnf-α gg genotype and g allele were significantly associated with eos compared to the suspected group; for gg genotype: or = 2.82, 95% ci = (1.34-5.97), p value = 0.007; and for the g allele: or = 2.39, 95% ci = (1. 36-4.27) , p value = 0.001 (table 4 ). the ifn-γ aa genotype and allele were associated with eos when compared to suspected patients by unadjusted analysis; for aa genotype: or = 3.27, 95% ci = (1.56-6.84), p value = 0.002; and for a allele: or = 2.74, 95% ci = (1.56-4.90), p value < 0.001 (table 4 ). in addition, we investigated if the cytokine snps were in the ld. our results indicated that cytokine snps were not in the ld (data not shown) due to the small sample size and the limited risk of losing false negative results. in order to investigate if the cytokine (il-1β, il-6, tnf-α, and ifn-γ) snps affected the circulatory level of the corresponding cytokine (il-1β, il-6, tnf-α, and ifn-γ), the association between the snps and the levels were analysed. as shown in table 5 , there was a significant association between il-1β cc genotype and the higher concentration of circulating il-1β cytokine compared to heterozygote il-1β tc genotype [or= 10.33, 95% ci = (4.71-22.63), p value < 0.001]. the il-6 gg genotypes were associated with higher level of circulating il-6 cytokine compared to individuals carrying the heterozygous il-6 gc genotype [or = 2.80, 95% ci = (1.51-5.54), p value < 0.001 (table 5) ]. individuals carrying the tnf-α aa genotype were significantly associated with lower level of circulating tnf-α cytokine compared to individuals carrying the tnf-α ga [or = 0.36, 95% ci = (0.16-0.82), p value = 0.014 (table 5)]. there was a significant association between the individuals carrying ifn-γ aa genotypes and the higher concentration of ifn-γ cytokine [or = 2.78, 95% proinflammatory cytokines play an important role in the immune response, pathogenesis of sepsis, and organ dysfunction [47] . single nucleotide polymorphisms in cytokine genes may explain, at least to some extent, the variability of the clinical course observed in sepsis and infections. the majority of previous studies related to snps in sepsis were performed on adults [36] . however, data from neonate populations with sepsis are poor, and developmental differences that affect inflammation and immune responses make it difficult to extrapolate data from adult studies to newborn populations [8, 48] . the current study focused on analyses of snps in four genes (il-1β -31 t/c, il-6 -174 g/c, tnf-α -308 g/a, and ifn-γ +874 a/t) that may play a critical role in, or are associated with, inflammatory response and sepsis severity, in order to identify possible predictive mechanisms for sepsis risk stratification. interleukin 1β is thought to be one of the key mediators in the pathogenesis of sepsis syndrome [49, 50] . data of the present study clearly showed that newborns with eos had significantly higher serum levels of il-1β compared with suspected and sepsis-free groups. this finding supports previous studies that have demonstrated an elevated level of il-1β in plasma of septic neonates when com-pared with both healthy infants and infants with clinically suspected but not confirmed sepsis [17, 18] . therefore, several studies have evaluated the role of this cytokine in the early diagnosis of neonatal sepsis [17] [18] [19] . in addition, our data clearly showed a significant association between il-1β cc genotype and higher concentration of circulating il-1β cytokine compared to heterozygote il-1β tc genotype. also, the il-1β cc genotype and allele are associated with eos as compared to suspected patients. previous data indicates that snps of il-1β may be associated with a poor prognosis from sepsis [32, 33] . however, other studies did not find any association between il-1β (c3953t) polymorphisms and outcome of sepsis [34, 51] . kang et al. [52] concluded that il-1β-31 and il-1β-511 polymorphisms are not associated with the development of bronchopulmonary dysplasia in preterm infants. this discrepancy may be explained by differences in the definition of sepsis and its severity, studied il-1β snps, and in the general characteristics of the enrolled subjects, including ethnicity. interleukin 6 plays an important role in the development, pathogenesis, and outcome of sepsis and septic shock. data from the present investigation clearly demonstrated that neonates with eos showed a highly significant table 5 . logistic regression analysis of cytokines (il-1β, il-6, tnf-α and ifn-γ) levels (pg/ml) in relation to cytokines (il-1β, il-6, tnf-α and ifn-γ) genotypes polymorphism in the combined study population serum level of il-6 compared to suspected and sepsis-free groups. this finding is in agreement with previous studies that reported that il-6 is excessively elevated in septic neonates when compared with both healthy infants and infants with clinically suspected but not confirmed sepsis [17] . many investigators have demonstrated that levels of circulating il-6 correlate with severity of sepsis and may predict outcome [35, 49, 53, 54] . interleukin 6 appears to be ideal for detecting early-onset neonatal infection with a high degree of sensitivity and specificity [18, 19, 55, 56] . since il-6 is a very early marker that appears before crp, it may be useful in the monitoring of patients with high risk of developing infection. genetic variation within the regulatory part of the il-6 gene may affect the incidence and outcome of sepsis [57] . our data showed that the il-6 gg genotypes are associated with eos when compared to suspected patients, and il-6 -174g allele is associated with eos. however, no significant association was found between il-6 -174c allele and eos. in addition, the il-6 gg genotypes are associated with higher levels of circulating il-6 cytokine compared to individuals carrying the heterozygous il-6 gc genotype. this result is in accordance with previous studies suggesting that individuals with il-6 -174c allele have significantly lower plasma concentrations of il-6 [58] , and this allele is associated with reduced il-6 plasma levels in newborns [59] . bennermo et al. [60] revealed that the il-6 -174g allele is functional in vivo with increased inflammatory response. similar results were found by studies that included caucasian infants and showed that there was a risk of neonatal sepsis associated with the snp il-6 -174 gg [37, 38] . however, contradictory results were seen by reiman et al. [61] and baier et al. [62] in studies that included very-low-birth-weight (vlbw) infants of different ethnicities; they showed that the il-6 -174c allele was associated with increased incidence of sepsis. on the other hand, other studies did not detect any association between the snp il-6 -174 and neonatal sepsis [63] [64] [65] . such contradictions in data between various studies could be attributed to the differences in study design, ethnicity, and gene-to-gene and gene-to-environmental interactions [8] . in our study, we reported that the il-6 -174g allele is associated with higher levels of circulating il-6 and eos. consequently, il-6 gene polymorphisms g-174 > c could be predictors of risk of development and/or predictors of the severity of sepsis in the saudi newborn population. tumor necrosis factor α is recognised as a primary mediator in the pathophysiology of sepsis and septic shock (reviewed in [66] ). data from the current study showed that serum levels of tnf-α were significantly higher in the eos group compared to suspected and sepsis-free control groups. this result is in accordance with previous studies that demonstrated an elevated serum level of tnf-α on day 1 of neonatal sepsis, and the role of tnf-α as an early diagnostic marker in neonatal sepsis has been evaluated [16] [17] [18] [19] . with regards to the snp in tnf-α gene promoter, the data presented here indicate that patients who are carriers of the tnf-α gg genotype and g allele are significantly associated with eos patients, compared to the suspected group. furthermore, individuals carrying the tnf-α aa genotypes are significantly associated with lower levels of circulating tnf-α cytokine, compared to individuals carrying the tnf-α ga genotype. these results are in parallel with the findings of previous studies that have demonstrated a pattern of protection that was conferred by the tnf-α -308 ga genotype against both sepsis mortality and acute respiratory distress syndrome (ards) outcome in a paediatric population [67] . similarly, in the adult population, another study has found an association between snp tnf-α -308a allele and protection against ards [68] . however, several studies have reported an association between snp tnf-α -308 g/a and sepsis outcome in the adult and paediatric population [10, 28, 29, 31, 69] , while other studies did not show any association between the snp tnf-α -308 g/a and increased risk of sepsis [50, 65, 70] . such discrepancies in results among different studies could be explained by the variations in study design, enrolled subjects, ethnicity, and gene-to-gene and gene-to-environmental interactions [8] . in addition, azevedo et al. [67] speculated that -308 g > a and -863 g > a snps in the tnf-α gene promoter seem to work in opposite directions, and could in fact reflect a different impact depending on age. data from this study clearly showed high serum ifn-γ levels in eos patients, compared to the suspected and sepsis-free control group. high levels of ifn-γ could lead to side-effects such as tachycardia, myalgia, malaise, leucopaenia, and weakness [49] . interferon γ enhances lps-induced mortality and increases levels of lps-induced circulating tnf-α [68] ; consequently, anti-ifn-γ antibodies protected against lps-and escherichia coli-induced mortality [71] . in addition, ifn-γ was shown to be a mediator of tnf-α-induced lethality [72] . therefore, ifn-γ could play an important role in sepsis development and outcome. our results demonstrated that the ifn-γ aa genotype and a allele are associated with eos when compared to suspected patients. there is a significant association between individuals carrying the ifn-γ aa genotype and the higher concentration of ifn-γ. individuals carrying the ifn-γ tt genotype are associated with lower concentrations of ifn-γ when compared to those carrying the ifn-γ at genotype. the ifn-γ + 874a allele has previously been reported to be associated with some infectious diseases [73] [74] [75] . recently, the ifn-γ + 874a allele has been shown to be associated with susceptibility to severe acute respiratory syndrome (sars). individuals with ifn-γ + 874 aa and at genotype had a 5.19-fold and 2.57-fold increased risk of developing sars, respectively [76] . the mechanism by which the ifn-γ + 874a/t allele influences the sus-ceptibility to sepsis may therefore depend on its role in the regulation of ifn-γ production [44] . in conclusion, circulating il-1β, il-6, tnf-α, and ifn-γ were elevated in eos patients; and il-1β -31c, il-6 -174g, tnf-α -308g, and ifn-γ + 874a alleles were associated with eos in saudi infants. however, due to the small sample size of this study, the cytokine snps were not in the ld. therefore, these results need to be confirmed by a large-scale multicentre prospective study and should be supported by data from other ethnic populations, with the hope that it could be used to develop an early predictor for the prognosis of sepsis in neonates. the relationship between hospital spending and mortality in patients with sepsis 4 million neonatal deaths: when? 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cholesterol-related genetic risk scores are associated with hypometabolism in alzheimer's-affected brain regions il-10, il-6 and cd14 polymorphisms and sepsis outcome in ventilated very low birth weight infants genetic polymorphisms of il-6-174 and il-10-1082 in full term neonates with late onset blood stream infections interleukin-6-174-genotype, sepsis and cerebral injury in very low birth weight infants genetic variants of tnf-[fc12]a, il-1beta, il-4 receptor [fc12]a-chain, il-6 and il-10 genes are not risk factors for sepsis in lowbirth-weight infants genetics and genomics in pediatric septic shock tumor necrosis factor (tnf) and lymphotoxin-alpha (lta) single nucleotide polymorphisms: importance in ards in septic pediatric critically ill patients the role of ifn-gamma in the pathology of experimental endotoxemia variation in the tumor necrosis factor-alpha gene promoter region may be associated with death from meningococcal disease prevalence of two tumor necrosis factor gene polymorphisms in premature infants with early onset sepsis role of interferon-gamma in experimental gram-negative sepsis evidence for ifn-gamma as a mediator of the lethality of endotoxin and tumor necrosis factor-alpha cytokine gene polymorphisms in patients infected with hepatitis b virus cytokine gene polymorphisms associated with symptomatic parvovirus b19 infection association of interferon gamma and interleukin 10 genes with tuberculosis in hong kong chinese the interferon gamma gene polymorphism +874 a/t is associated with severe acute respiratory syndrome key: cord-291076-p350i54m authors: wang, renxi; xiao, he; guo, renfeng; li, yan; shen, beifen title: the role of c5a in acute lung injury induced by highly pathogenic viral infections date: 2015-05-06 journal: emerg microbes infect doi: 10.1038/emi.2015.28 sha: doc_id: 291076 cord_uid: p350i54m the complement system, an important part of innate immunity, plays a critical role in pathogen clearance. unregulated complement activation is likely to play a crucial role in the pathogenesis of acute lung injury (ali) induced by highly pathogenic virus including influenza a viruses h5n1, h7n9, and severe acute respiratory syndrome (sars) coronavirus. in highly pathogenic virus-induced acute lung diseases, high levels of chemotactic and anaphylatoxic c5a were produced as a result of excessive complement activaiton. overproduced c5a displays powerful biological activities in activation of phagocytic cells, generation of oxidants, and inflammatory sequelae named “cytokine storm”, and so on. blockade of c5a signaling have been implicated in the treatment of ali induced by highly pathogenic virus. herein, we review the literature that links c5a and ali, and review our understanding of the mechanisms by which c5a affects ali during highly pathogenic viral infection. in particular, we discuss the potential of the blockade of c5a signaling to treat ali induced by highly pathogenic viruses. the epithelium of the lung is vulnerable to damage caused by inhaled microorganisms and other noxious particles. many studies suggested the presence of complement components at the alveolar epithelium, where inhaled airborne particles and microorganisms are deposited. [1] [2] [3] in addition, the complement system has been implicated in the development of acute lung diseases induced by highly pathogenic viruses including influenza a virus h1n1, 4 h5n1, 5 h7n9, 6 severe acute respiratory syndrome coronavirus (sars-cov), 7 middle east respiratory syndrome coronavirus (mers-cov). 8 however, the specific contributions of complement to lung diseases based on innate and adaptive immunity are just beginning to emerge. elucidating the role of complement-mediated immune regulation in these diseases will help identify new targets for therapeutic interventions. 9 complement activation leads to the formation of bioactive molecules, including the anaphylatoxins, c3a and c5a, and the lytic membrane attack complex (c5b-9). 10 the complement-activated product c5a is a strong chemoattractant and is involved in the recruitment of inflammatory cells such as neutrophils, eosinophils, monocytes, and t lymphocytes, in activation of phagocytic cells and release of granulebased enzymes and generation of oxidants. 10 c5a also displays other powerful biological activities including inducing ''cytokine storm.'' on the other hand, blockade of c5a signaling has demonstrated potential benefits in the treatment of acute lung injury (ali) induced by highly pathogenic viruses. in this article, we summarize recent developments in our understanding of the role of c5a in mediating aute lung injury induced by highly pathogenic viruses. highly pathogenic virus due to high mutation rates of viruses, every several years to decades a highly pathogenic virus emerges. especially in the recent decades, there were more than five highly pathogenic viruses such as sars coronavirus in 2002, avian influenza a/h5n1 virus in 1997, h1n1 virus in 2009, h7n9 virus in 2013, and mers coronavirus in 2012. as exemplified by coronaviruses and influenza viruses, bats and birds are natural reservoirs for providing viral genes during evolution of new virus species and viruses for interspecies transmission. 11, 12 this is the primary cause of an outbreak by jumping directly from bird to human. 13 in two months, 536 laboratory-confirmed cases and 145 deaths have been reported globally. 14 there is an h5n1 vaccine for human use, but there is currently no h7n9, sars or mers vaccine available. current vaccination strategies are still inadequate at providing protection against epidemic outbreaks. thus, it is urgent to explore the mechanism by which highly pathogenic viruses induce diseases. acute lung injury induced by highly pathogenic viral infections although highly pathogenic virus infections have the different epidemiology, there is a similar rapid progression to acute respiratory distress syndrome (ards). 15 for example, histopathological changes in the lung from patients infected with h5n1 are highly similar to those of patients with sars. 16 except for influenza a h5n1 virus, avian influenza a h7n9 virus in 2013 also caused severe pneumonia. 17 postmortem biopsy of 3 patients infected with h7n9 in 2013 showed acute diffuse alveolar damage: patient 1, who died 8 days after symptom onset, had intra-alveolar hemorrhage, whereas patients 2 and 3, who died 11 days after symptom onset, had pulmonary fibro proliferative changes. 18 patients infected with h5n1 develop rapidly progressive pneumonia, further resulting in ali or ards. 19, 20 ali may be a critical cause of death in patients with h5n1 infection. 19, 21 like h5n1 infection, h7n9 also causes serious lung pathology. in addition, sars-cov infection caused ali that may progress to life-threatening ards. mers-cov infection resulted in a more severe pneumonia than sars-cov infection. 22 respiratory distress is the most common cause of death in patients infected with highly pathogenic virus. in terms of therapy, lung protective ventilation is the cornerstone of supportive care. 23 extracorporeal membrane oxygenation is routinely used in many centers for the treatment of severe respiratory tract infections. however, due to few effective treatment options, ali is often fatal for patients infected with highly pathogenic viruses. this suggests that serious lung pathology should be of particular concern. after a microorganism infection begins, the host quickly activates the complement system to clear infected pathogens. 24 during the complement activation, the high levels of products such as c5a are commonly involved in exacerbated inflammatory reactions that can cause direct harm to the host following infections. [25] [26] [27] iav belongs to the orthomyxoviridae family with single-stranded negative-sense rna virus, 28 and has the capacity to activate the complement system. 29 in addition, the avian influenza hemagglutinins typically bind alpha 2-3 sialic acid receptors, whereas human influenza hemagglutinins bind alpha 2-6 sialic acid receptors. 30 thus, h5n1 replicates in the lower respiratory tract, then causes complement activation. 31 this suggests that upon influenza infection, the high levels of c3 and c5 including fragments c3a and c5a are produced. complement activation possibly contributes to the observed tissue damage in severe viral infection. 32 studies demonstrated that ali in h5n1-infected mice was caused by excessive complement activation such as release of c5a. 5 thus, complement activation plays a critical role in the pathogenesis of virus-induced acute lung injury. among the complement activation products, the anaphylatoxin c5a is one of the most potent inflammatory peptides. 33 increased levels of c5a were found in bronchoalveolar lavage fluid (balf) and serum from patients infected with fatally h1n1 pandemic virus. 4, 34 c5a had also been found to increase in balf of mice infected with highly pathogenic avian influenza h5n1 but not following seasonal iav infection. 35 on the other hand, balf from recovered patients with ards demonstrated significantly reduced c5a-dependent chemotactic activity. 36 thus, c5a might play a critical role in the pathogenesis of virus-induced acute lung injury. c5a-mediated inflammatory cells migrate into lung tissue compared to normal controls, sars patients had increased cellularity of balf with increased alveolar macrophages. 37 thus, mononuclear cell infiltration might have an important role in the pathogenesis of ali induced by highly pathogenic viruses like sars. anaphylatoxin c5a has been implicated in the pathogenesis of ards by mediating neutrophil attraction, aggregation, activation, and subsequent pulmonary endothelial damage. [38] [39] [40] [41] reversely, c5adependent chemotactic activity is significantly decreased in recovered patients with ards. 36 these suggest that c5a-mediated mobilization and activation of immune cells might be the central events to tissue injury caused by highly pathogenic viral infections. two chemoattractants c5a and interleukin 8 (il-8) can be synthesized by cells in the lung (e.g., macrophages, epithelial cells, endothelial cells, smooth muscle cells and neutrophils). 33 il-8 levels have also been found to correlate with neutrophil numbers and the degree of lung dysfunction. 42 c5a could strongly amplify il-8 expression from human whole blood cells induced by lipopolysaccharides and other types of toll-like receptors agonists via extracellular-signal-regulated kinases 1/2 and p38, but not c-jun n-terminal kinase. 43 the data suggest that c5a might be a critical effector molecule to mediate lymphocyte attraction by itself or indirectly by enhancing the production of il-8. altogether, c5a-mediated lymphocyte attraction plays a critical role in the pathogenesis of ali induced by highly pathogenic viruses. neutrophil extracellular traps (nets) are primarily composed of dna from neutrophils, which bind pathogens with antimicrobial proteins. nets are beneficial in antimicrobial defense and can help fight against invading pathogens. however, an excess of nets contributes to the pathology of a number of diseases including those of the lung. 44 nets are found in infection-related ali models of influenza virus. 45, 46 in vitro studies demonstrated that c5a, in association with granulocyte-macrophage colony-stimulating factor, is able to induce the release of nets. 47 c5a is also able to activate macrophages and endothelial cells and to promote vascular leakage and the release of nets. 10 thus, nets are induced by c5a during iav infection and are associated with alveolar damage in iav-induced pneumonitis. 45 the excess of net components are potent factors in lung injury. net increases the permeability of the alveolar-capillary barrier by cleaving endothelial actin cytoskeleton, e-cadherin and vecadherin. 48 the antimicrobial peptide ll-37 in net structures presents cytotoxic and proapoptotic properties towards endothelial and epithelial cells. 49 net also induces the release of proinflammatory cytokines. 48 the data suggest that c5a-mediated neutrophil extracellular traps aggravate ali in patients infected with highly pathogenic virus. c5a-mediated release of reactive oxygen species c5a is a strong chemoattractant for neutrophils and monocytes; it then activates these cells to generate oxidative burst with release of 10 a study demonstrated that ros are primary pathogenic molecules in pneumonia from mice infected with influenza virus. 50 the amount and duration of exposure of generated ros, released from respiratory, immune, and inflammatory cells, determined the extent of lung damage. 50 in lung fibroses, inflammatory cells produce a significantly greater amount of ros. critically, antioxidant treatment significantly reduces lung damage and mortality in influenza-infected mice. 51 these studies demonstrated a critical role of reactive oxygen intermediates (rois) in virus-induced epithelial damage. c5a-c5ar interaction plays a critical role in oxidative burst. 52 interception of c5a/c5ar signaling with a c5ar antagonist significantly inhibited oxidative burst in neutrophils induced with e. coli. similarly, anti-c5a blocked the oxidative burst in whole blood induced with neisseria meningitides. 53 phosphorylation of p47 phox is essential for assembly of nadph oxidase and the subsequent production of o 2 and h 2 o 2 . 10 c5a is a strong activator of mitogen-activated protein kinase (including p42/p44), which is an important kinase for p47 phox phosphorylation. except for directly affecting tissue damage, oxidant production might also be involved in signal transduction pathways. il-8 expression is enhanced by the oxidant sensitive transcription factor nuclear factor-kb 54 activated in the lungs of influenza-infected mice. 55 this means that oxygen-derived free radicals might exert much greater effects on the pathogenesis of the disease by indirectly inducing other proinflammatory mediators. thus, c5a-mediated oxygen-derived free radicals are thought to be important events in the pathogenesis of the disease. c5a-mediated release of histones histones are essential regulators of genome function in eukaryotic cells. the ns1 protein of influenza a h3n2 subtype possesses a histone-like sequence (histone mimic), and could target the human rna polymerase-associated factor 1 transcription elongation complex which has a crucial role in the antiviral response. 56 thus, the virus used ns1 histone mimic to suppress human rna polymerase-associated factor 1 transcription elongation complex-mediated antiviral response. diversely modified histone regulates gene replication, repair and transcription. after activation with influenza, h3k4me3 reduced association of interferon i (ifn-i) and ifn-iii promoters in dendritic cells (dcs) to suppress antiviral gene expression. 57 in contrast to ifns, the association of tumor necrosis factor-a (tnf-a) promoter was not disturbed. 57 histone can be excreted into cells to reduce intracellular histone to suppress antiviral gene expression. in the setting of ali both in humans and in mice, histone presence has been found in balf. 58 in addition, when polymorphonuclear leukocytes are incubated in vitro or in vivo with c5a, neutrophil extracellular histones-contained extracellular traps (nets) develop. 59 these results suggest that engagement of c5a with its receptors led to the appearance of extracellular histones in balf. extracellular histones significantly enhance inflammatory response by inducing nucleotide-binding domain and leucine-rich repeat containing family, pyrin domain containing 3 (nlrp3) inflammasome. 58 furthermore, airway instillation of histones resulted in intense lung injury and inflammation, together with fibrin clots in pulmonary veins. 60 c5a-mediated release of histones has an important contribution to the pathogenesis of ali. the process of leukocyte adhesion to endothelial cells is the first critical step in neutrophil migration into an area of inflammation. adhesion molecules on the surface of endothelial cells have an important role in inflammatory cell migration. in fact, c5a can regulate the expression of adhesion molecules. 61 c5a directly activates endothelial cells to upregulate adhesion molecules such as p-selectin. in addition, c5a and tnf-a cooperate to enhance upregulation of intercellular adhesion molecule 1 and e-selectin. 62 thus, c5a is an effective mediator in the first step in inflammatory cell migration into the lung. adhesion molecules on the surface of inflammatory cells also have an important role in inflammatory cell migration. in vitro studies demonstrated upregulation of cd1lb/cd18 expression on neutrophils induced by c5a. 10 in addition, c5a also induced the expression of b1 and b2 integrin on blood neutrophils. 63, 64 thus, enhanced adhesive interactions of neutrophils to endothelial cells promote inflammatory cell migration into inflammatory sites. the adhesion molecules effectively enhanced pro-inflammatory cytokines such as tnf-a production by pulmonary macrophages, which, in turn, promotes the inflammatory response. 62 blockade of cdllb, cd18, intercellular adhesion molecule 1, or p-selectin significantly reduced ali damage by neutrophil content of the lungs. 65 anti-c5a might protect tissue injury in various organs by limiting neutrophil sequestration through downregulating the expression of adhesion molecules. 10 these studies suggest that c5a-mediated upregulation of adhesion molecules promotes the inflammatory response. c5a-mediated adaptive immune response c5a induces innate immune cells including mast cells, neutrophils, and macrophages to release cytokines such as il-12, tnf-a and macrophage inflammatory proteins-1a. 66 il-12 is a strong activator of cd8 1 t cells, whereas tnf-a promotes transendothelial migration of t cells by up-regulating vascular adhesion molecules and induces ifn-c expression in t cells. 66 these data demonstrate that c5a indirectly induces adaptive immune response by activating innate immune cells. apart from innate immune cells, human dcs 67,68 and t cells 69 also express the c5a receptor (c5ar, cd88). thus, c5a is also a potent chemoattractant for human t cells, 69,70 b cells, 71 and dcs. 67, 68, 72, 73 in addition, during the early inflammatory stage of a pathogen infection, dcs used c5a as a homing signal to take up ag, and then were primed for helping t-cell function. 74 thus, c5a induces adaptive immune response by recruiting for dcs. cd28 and cd40l on t cells are important signaling for t-cell proliferation and differentiation induced by interaction of locallyproduced c5a with c5ar on antigen-presenting cells (apcs). accordingly, c5a could not activate cd80 2/2 cd86 2/2 and cd40 2/2 apcs to induce t cell activation. 75 the data suggest that the local interaction of c5a and c5ar on apcs is critical to cd4 1 t cell proliferation and differentiation. the binding of the c5a to the c5ar also plays an important role in cd8 1 t cell responses. 74 cd8 1 t cell activation during influenza infection requires c5a, which acts as a chemoattractant for t lymphocytes. 69, 76 thus, it is conceivable that c5a might elicit cd8 1 t cell response upon the input stimuli. accordingly, c5ar antagonist reduced the frequency and absolute numbers of flu-specific cd8 1 t cells. in patients infected with influenza a virus like h5n1, inflammatory cytokines such as il-1b, il-8, and il-6 play a major role in mediating and amplifying ali and ards by stimulating by chemotaxis c5a. 77 c5a induces innate immune cells including mast cells, neutrophils, and monocytes/macrophages to release proinflammatory cytokines such as il-12, tnf-a and macrophage inflammatory proteins-1a. 64 in addition, c5a also stimulates adaptive immune cells such as t and b cells to release cytokines such as tnf-a, il-1b, il-6, and il-8. 78, 79 many cytokines, triggered by highly pathogenic viruses like h5n1, has been called a ''cytokine storm''. 80 cytokines were rapidly induced at 24h post infection with h5n1. 81 the pro-inflammatory cytokines including il-1b and tnf-a might contribute to the severity of disease by promoting maximal lung inflammation caused by h5n1 viral infection. 82 compared to healthy volunteers, h7n9-infected patients have significantly higher levels of cytokines such as il-6, ifn-c-inducible protein 10, il-10, ifn-c, and tnf-a. 83 a dangerous cytokine storm also occurs in sars. the representative sars-cov ssrnas had powerful immunostimulatory activities in inducing pro-inflammatory cytokines tnf-a, il-6 and il-12. 84 elevated levels of some proinflammatory cytokines including moncyte chemoattractant protein-1, transforming growth factor-beta1, tnf-a, il-1, and il-6, produced by cells infected by sars-cov, might cause ali. 85 in addition, a cytokine could induce other cytokines to further enhance the proinflammatory response. take for example, elevated levels of tnf-a induced other cytokines like il-6. 86 thus, cytokine storm plays an important role in ali. anti-tnf-a (etanercept) significantly reduced the damage of ali. 87 the inhibition of macrophage migration inhibitory factor alleviated h5n1 influenza virus pneumonia in murine model by causing a significant reduction in pulmonary inflammatory cytokines il-1b, il-6 and tnf-a and ifn-c-inducible protein 10 88 a widely used antiviral agent arbidol hydrochloride efficiently inhibits both h1n1 strains and diminishes both viral replication and acute inflammation through suppression of inflammatory cytokines such as il-1b, il-6, il-12, and tnf-a. 89 these studies indicate that blockade of cytokine storm is effective in treatment of infections with highly pathogenic virus. the severe h7n9 patients were in a state of immune paralysis with general leukopenia, low antigen-presenting capacity and impaired t cell response. 90 those suffering fatal infections with h7n9 have particularly low proportions of peripheral blood t lymphocyte subgroups. 91 previous studies have demonstrated that c5a induces thymocyte apoptosis, which in turn results in decreased number of t cells in circulation and attendant immunosuppression. 10, 92 this suggests that in a striking contrast to neutrophils, thymocytes apparently receive pro-apoptotic signals from c5a. during sars-cov infection, il-6 and il-8 induced by c5a inhibits the t-cell-priming ability of dcs. 93 compared to significant up-regulation of inflammatory chemokines, the sars-cov-infected dcs showed low expression of antiviral cytokines (ifn-a, ifn-b, ifn-c, and il-12p40) . 94 these studies are in accordance with the conclusion that the n-protein of sars-cov induced ali by resulting in imbalance of pro-inflammatory and anti-inflammatory cytokines. 95 many inflammatory and anti-viral genes were differentially expressed in sars patients. plenty of pro-inflammatory cytokines such as il-1, tnf-a, and il-8 significantly increased, whereas a number of ifnstimulated genes like double-stranded rna-dependent protein kinase, interferon-induced guanylate-binding protein-1 and 2, c-x-c motif chemokine 10 decreased in the acute severe case. 96 like sars-cov, mers-cov viruses were unable to significantly stimulate the expression of antiviral cytokines (ifn-a and ifn-b) but induced comparable levels of tnf-a and il-6. 8 c5a-c5ar interaction might potentiate the mitochondrial apoptotic pathway and/or enhance the expression of proapoptotic factors, such as tnf-a, which has been linked to thymocyte apoptosis, in turn reducing the expression of antiviral cytokines. this suggests that c5a-mediated immune paralysis plays a critical role in mediating pathogenic damage in severe patients infected with highly pathogenic virus like h7n9. to evaluate the effect of c5a blockade, omci, a potent arthropodderived inhibitor of c5 activation that binds to c5 and prevents release of c5a by complement activation, was used to treat mice infected with h1n1 pandemic virus. omci significantly inhibited neutrophil and macrophage infiltration in the airways, nets formation, death of leukocytes, lung epithelial injury and overall lung damage. 4 the study suggests that targeting c5a could be a promising approach to reduce excessive inflammatory reactions associated with the severe forms of iav infections. c5ar was found to be expressed on upper (bronchial) and lower (alveolar) airway epithelial cells. an adenovirus construct (sirna) was used to silence mrna for c5ar in the lung and resulted in buildup of polymorphonuclear leukocytes, and lower levels of proinflammatory mediators in bronchoalveolar lavage fluid. 97 antagonism of c5a receptors also significantly inhibited the development of ards induced by intravenous infusion of cobra venom factor, including neutrophil migration and bronchoalveolar vascular leakage, blood pressure alterations, pro-inflammatory cytokines including tnf-a levels in bronchoalveolar lavage fluid. 98 the study indicates that c5a signaling greatly contributes to inflammation and injury in the lung and was targeted to treat highly pathogenic virus infection. in addition, interception of c5a signaling has recently shown promising beneficial effects in small animal models of ali/ards by reducing pro-inflammatory cytokines. 99 polyclonal anti-c5a antibody led to significantly reduced inflammation in lungs, alleviating ali in h5n1-infected mice. 5 the study indicates that inhibition of c5a might be an effective clinical intervention for h5n1-induced ali. however, studies in knockout mice demonstrated that c3 was required for protection from influenza infection, proper viral clearance, and associated with changes in cellular infiltration. 35 the data are in accordance with the fact that complement c5a is the leading mediator of the over-inflammatory response which induced ali, whereas the lytic membrane attack complex (c5b-9) provide a protective role in controlling viral infection. thus, we developed a neutralized humanized anti-human c5a antibody which only blocked c5a effects but did not affect the formation of c5b-9 membrane attack complex. in vitro experiments demonstrated that a novel, neutralizing, humanized anti-human c5a antibody blocked the ability of c5a to induce granulocytes to express cd11b while not affecting the ability of c5b to form the membrane attack complex. african green monkeys were inoculated with h7n9 virus and then treated intravenously with anti-human c5a antibody. anti-c5a treatment in h7n9-infected monkeys substantially attenuated ali by reducing the lung infiltration of macrophages and neutrophils, and the levels of inflammatory mediators. 6 the data suggest that humanized anti-human c5a antibody might provide a potential therapeutic reagent for h7n9-infected patients. 100 the role of c5a in the different viral infections and the effect of c5a blockade on acute lung injury were described in table 1 that the neutralized humanized anti-human c5a antibody would be a potential therapeutic option for h5n1-infected patients. 100 the complement system, a part of innate immunity, plays a critical role in host defense against pathogens. unregulated complement activation is likely to play a crucial role in the pathogenesis of lung diseases. the complement-activated product c5a displays powerful biological activities in the activation of phagocytic cells, generation of oxidants, release of histones and cytokine storm, and so on. 10 in particular, cytokine storm is believed to be responsible for many of the deaths during the 1918 influenza pandemic, 101 during the sars epidemic in 2003, 7 mers-cov in 2014, 8 and the human deaths from h1n1, 4 h5n1 102 and h7n9. 6 there is growing awareness that there are key similarities in the contribution to the cytokine storm and the manifestation of lung pathology among the chronic respiratory diseases, 103 and the cause of death such as bleeding from ebola virus. 104 c5a, as a key trigger to induce cytokine storm, could be an ideal target for many lung inflammatory diseases, and it would be important to assess the therapeutic potentials of c5a blockade in human clinical trials. we have evidence that humanized anti-c5a antibody greatly reduced lung histopathologic injury, as well as decreased lung infiltration of macrophages and neutrophils and the levels of pro-inflammatory cytokines including tnf-a in a monkey model of ali induced by h7n9 6 and herbicide, paraquat (shihui sun et al, unpublished data). thus, it is reasonable to speculate that blockade of c5a with a humanized anti-human c5a antibody would be a potential therapeutic target for highly pathogenic viral infection-induced acute lung injury. anti-c5a ab treatment also reduced lung injury and neutrophil infiltration especially on day 5 after h5n1 virus infection. also, anti-c5a ab treatment increased survival rate, with 50% mortality in the c5a ab group compared with 100% mortality in the control group on day 9 after h5n1 virus challenge. hpai h5n1 virus infected murine model h5n1 influenza virus infected mice had increased levels of c5a activation byproducts as compared to mice infected with either seasonal or pandemic 2009 h1n1 influenza viruses. h7n9-infected monkey model anti-c5a treatment in h7n9-infected monkeys substantially attenuated ali: it markedly reduced the lung histopathological injury and decreased the lung infiltration of macrophages and neutrophils. moreover, the treatment decreased the intensity of sirs; the body temperature changes were minimal and the plasma levels of inflammatory mediators were markedly reduced. the treatments also significantly decreased the virus 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influenza infection in mice role of macrophage migration inhibitory factor in influenza h5n1 virus pneumonia antiviral and anti-inflammatory activity of arbidol hydrochloride in influenza a (h1n1) virus infection emerging microbes and infections emi201528.3d 27/4/15 12:40:25 c5a in acute lung injury emerging microbes and infections severe h7n9 infection is associated with decreased antigen-presenting capacity of cd14 1 cells dynamic behavior of lymphocyte subgroups correlates with clinical outcomes in human h7n9 infection new strategies for treatment of humans with acute lung injury/acute respiratory distress syndrome severe acute respiratory syndrome (sars) coronavirus-induced lung epithelial cytokines exacerbate sars pathogenesis by modulating intrinsic functions of monocyte-derived macrophages and dendritic cells chemokine up-regulation in sarscoronavirus-infected, monocyte-derived human dendritic cells a study of pulmonary inflammatory reaction induced by nprotein of sars-cov in rat models and effects of glucocorticoids on it gene expression profiles in peripheral blood mononuclear cells of sars patients attenuation of igg immune complex-induced acute lung injury by silencing c5ar inlung epithelial cells complement inhibitors selectively attenuate injury following administration of cobra venom factor to rats protein-based therapies for acute lung injury: targeting neutrophil extracellular traps role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome preparing for the next pandemic confronting potential influenza a (h5n1) pandemic with better vaccines the cytokine network in asthma and chronic obstructive pulmonary disease clinical features and pathobiology of ebolavirus infection this study was supported by national basic research program 973 grants (2013cb530506), national nature and science fund (81471529, 81272320 and 81172800) and beijing natural science foundation (7132139, 7141007 and 7132151). key: cord-301102-jbjysyqm authors: priestnall, simon l.; mitchell, judy a.; brooks, harriet w.; brownlie, joe; erles, kerstin title: quantification of mrna encoding cytokines and chemokines and assessment of ciliary function in canine tracheal epithelium during infection with canine respiratory coronavirus (crcov) date: 2009-01-15 journal: vet immunol immunopathol doi: 10.1016/j.vetimm.2008.09.017 sha: doc_id: 301102 cord_uid: jbjysyqm one of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type i interferons (ifns). however some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. canine respiratory coronavirus (crcov) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (cird). this study aimed to quantify pro-inflammatory cytokine mrnas following infection of canine air-interface tracheal cultures with crcov. within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for crcov, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. an assay of ciliary function was used to assess potential effects of crcov on the mucociliary system. crcov was shown to reduce the mrna levels of the pro-inflammatory cytokines tnf-α and il-6 and the chemokine il-8 during the 72 h post-inoculation. the mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease. canine respiratory coronavirus (crcov) has recently been identified in the respiratory tract of dogs (erles et al., 2003) . crcov is a group 2 coronavirus and is genetically most closely related to bovine coronavirus (bcov) . the virus appears to be particularly prevalent in housed populations and within 3 weeks of entry to a kennel, dogs develop antibodies to the virus (erles et al., 2003) . serological evidence of the virus exists in three continents (decaro et al., 2007; kaneshima et al., 2006; priestnall et al., 2006 priestnall et al., , 2007 , however little is known regarding the pathogenesis of the infection and the possible role of the virus in the canine infectious respiratory disease (cird) complex. to gain further insight into the pathogenesis of crcov an infection model is required. whilst in vivo models provide the optimum research tool for viral pathogenesis studies, they are not always feasible or necessary. the ideal in vitro model for the study of host-pathogen interactions in the respiratory system would resemble, as closely as possible, the physiologic conditions in vivo. traditionally, organ cultures involving respiratory tissues have been submerged in medium and thus lacked the air-mucosal interface present in vivo. jackson et al. pioneered the development of respiratory organ cultures with an air-interface using human nasal turbinates (jackson et al., 1996) . such cultures have a number of one of the first lines of defence against viral infection is the innate immune response and the induction of antiviral type i interferons (ifns). however some viruses, including the group 2 coronaviruses, have evolved mechanisms to overcome or circumvent the host antiviral response. canine respiratory coronavirus (crcov) has previously been shown to have a widespread international presence and has been implicated in outbreaks of canine infectious respiratory disease (cird). this study aimed to quantify pro-inflammatory cytokine mrnas following infection of canine air-interface tracheal cultures with crcov. within this system, immunohistochemistry identified ciliated epithelial and goblet cells as positive for crcov, identical to naturally infected cases, thus the data obtained would be fully transferable to the situation in vivo. an assay of ciliary function was used to assess potential effects of crcov on the mucociliary system. crcov was shown to reduce the mrna levels of the pro-inflammatory cytokines tnf-a and il-6 and the chemokine il-8 during the 72 h post-inoculation. the mechanism for this is unknown, however the suppression of a key antiviral strategy during a period of physiologic and immunological stress, such as on entry to a kennel, could potentially predispose a dog to further pathogenic challenge and the development of respiratory disease. ß 2008 elsevier b.v. all rights reserved. advantages over submerged culture systems including; the presence of all cell types, intact mucociliary clearance and the interaction of the pathogen with the mucus layer and ciliated surface as in vivo. using an air-interface system for the culture of turbinate mucosa to model rhinovirus infection, jang et al. demonstrated that after 7 days of culture the mucosae did not show significant deterioration whilst pcr evidence of rhinoviral infection was recorded (jang et al., 2005) . the authors concluded that such a culture system provided a safe, reliable and physiologically relevant in vitro model for the study of the actions of viruses on the respiratory mucosa. structural damage to the respiratory epithelium and abnormal ciliary function are the visible signs of viral infection. however, during airway inflammation, cytokines present at inflammatory sites can be key components in disturbance of the epithelium and persistence of inflammatory reactions. cytokines can directly or indirectly alter the mucociliary apparatus by affecting ciliary beating or mucus secretion, predisposing to further and more serious viral or bacterial infection. the primary line of defence against viral infection requires ifn-b production in virus-infected cells, followed by establishment of antiviral functions through activation of the ifn-jak/stat signal transduction pathway (okabayashi et al., 2006) . respiratory epithelial cells also have the ability to synthesize and release pro-inflammatory cytokines, e.g. il-1b, il-6 and tnf-a and chemokines, e.g. il-8 following viral infection (adachi et al., 1997; adler et al., 1994; becker et al., 1993; brydon et al., 2005) . viral infection and pro-inflammatory cytokine production stimulate the infiltration of inflammatory cells to the respiratory epithelium. the induction of epithelial chemokines, e.g. il-8 may also be a primary event triggering increased airway reactivity in acute viral infection (kazachkov et al., 2002) . production of tnf-a or il-1 during virus infection may occur either directly or indirectly, for example as a result of cellular damage or via induction of other cytokines. tnf-a has been shown to stimulate mucus glycoprotein secretion by airway epithelial cells within 1 h of exposure (adler et al., 1994) . tnf-a, il-1b, il-6 and il-8 can also affect ciliary beat frequency indirectly through the increased synthesis of endothelin by tracheal epithelial cells (adler et al., 1994) . quantitative pcr has previously been used to demonstrate significantly increased transcription of il-6, tnf-a and a number of other pro-inflammatory and immunomodulatory cytokines/chemokines in epithelium infected with feline herpesvirus type-1 (fhv-1), suggesting a previously unknown role for fhv-1 in feline chronic nasal discharge (johnson and maggs, 2005) . this study aimed to examine the innate immune response and ciliary function of the canine tracheal epithelium following infection with crcov. an in vitro model system using canine air-interface tracheal cultures was developed for this purpose. crcov-infected cells were demonstrated using immunohistochemistry and the functional consequences of crcov infection were assessed by quantification of cytokine mrnas and measurement of ciliary function. dogs were received from a uk re-homing centre having been euthanized due to behavioural concerns and unsuitability to re-home. the whole trachea, from larynx to carina, was aseptically removed at necropsy within 2-3 h of death. the trachea was washed briefly in sterile pbs, then washed twice for 1 h at 37 8c in dulbecco's modified eagle's medium (dmem) containing 2 mm lglutamine, 50 mg gentamycin ml à1 , 100 u penicillin ml à1 , 100 mg streptomycin ml à1 and 2.5 mg amphotericin b ml à1 (sigma, poole). cultures were assembled in a similar manner to that described previously (anderton et al., 2004) . 10 mm 3 3% agarose (promega, southampton) cubes (in pbs) were placed into the centre of each well of 6-well cell culture plates (bd falcon, oxford) containing dmem with supplements (section 2.1.1). 15 mm 2 sterile filter paper squares (whatman no. 1) were placed onto the surface of the agarose. following washing, the tracheal ligament was removed and the trachea was sectioned into 10 mm 2 pieces. these pieces were positioned, epithelium uppermost, onto the agar plugs and incubated for up to 120 h at 37 8c, 5% co 2 and 96-99% rh. the viability of each trachea was assessed by measuring the ciliary function after 24 h in culture, using a latex bead clearance assay (section 2.6). tracheas with >25% of cultures failing to clear beads (score of 1) were regarded as non-viable and discarded. individual tracheal culture pieces were screened prior to culture setup for respiratory viruses (by pcr) and daily for bacterial contaminants (by culture). tracheal cultures were discarded if bacterial growth occurred within 72 h (blood agar) or 120 h (mycoplasmas). pcr assays for the detection of canine adenovirus type 2 (cav-2), canine herpesvirus (chv), canine parainfluenza virus (cpiv), crcov, canine distemper virus (cdv) and mycoplasma spp. were performed as previously described (erles et al., 2004 (erles et al., , 2003 frisk et al., 1999; kobayashi et al., 1995) on dna, extracted using the dneasy tissue kit (qiagen, crawley) according to the manufacturer's instructions, or on reverse transcribed rna (section 2.3). for each dog, tracheal culture plates were randomly assigned to a particular inoculation. tracheal cultures were inoculated after 24 h with crcov isolate 4182 (2 â 10 4 tcid 50 in 20 ml medium) , e. coli 055:b5 lps (10 mg ml à1 ) (sigma) or medium only (incl. supplements section 2.1.1). tracheal culture pieces harvested at 24 h intervals were homogenised using a ball mill (mixer mill mm 300, retsch, leeds) for 4 min at 20 hz, and rna extracted using the rneasy mini kit (qiagen) following the manufacturer's instructions for rna extraction from tissue samples. dnase digestion was performed using turbo dna-free (ambion, huntingdon) according to the manufacturer's instructions. the quantity of rna was estimated using a nanodrop-1000 spectrophotometer (nanodrop technologies, wilmington, de, usa). cdna synthesis was performed as follows: 1 mg of rna was added to 1 mg pd(n) 6 random hexamer (amersham biosciences, little chalfont) and incubated at 70 8c for 10 min. cdna was synthesised using the improm-ii reverse transcription system (promega) according to the manufacturer's instructions. a real-time quantitative rt-pcr for the detection of crcov nucleocapsid rna in tissue samples was developed and the primers are given in table 1 . a plasmid containing the crcov nucleocapsid gene target sequence was used to produce a dilution series from 1 â 10 8 to 1 â 10 1 copies ml à1 . optimisation of this qpcr is described elsewhere (mitchell et al., in press ). quantitative pcr was performed using 12.5 ml of sybr green jumpstart taq readymix (sigma) containing 2.5 mm mgcl 2 , 10 pmol ml à1 of each primer and 1 ml of cdna or plasmid in a final volume of 25 ml. amplification was performed in an opticon 2 real-time thermocycler (bio-rad, hemel hempstead) and cycling conditions are given in table 2 . cdna samples and standards were processed in triplicate and a negative control and two internal controls (previously quantified cdna samples) were included in each pcr series. 2.5. canine cytokine mrna quantification 2.5.1. production of plasmid standards for real-time quantitative rt-pcr the following cytokines were selected for mrna quantification in canine tracheal cultures; tnf-a, il-6 and il-8. partial sequences of the canine cytokine genes were amplified by pcr using previously published primers (peeters et al., 2006; peters et al., 2005) . the template cdna was obtained by reverse transcription of rna from canine macrophage-like (dh82) cells stimulated with lps. the pcr products were cloned into pgem-t easy (promega). plasmid dna was quantitated using a nanodrop-1000 spectrophotometer. the dna copy number was calculated using the following formula: copies of dna ¼ ðconcentration; mg ml à1 þâð6:023 â 10 23 þ ðdna size; bpþ â ð660 â 10 6 þ a 10-fold dilution series of each plasmid from 1 â 10 8 to 1 â 10 2 copies ml à1 was prepared and used as a template to produce a standard curve for assessment of linearity, dynamic range and primer efficiency. the pcr mixture included 12 ml of sybr green qpcr master mix reagent (finnzymes, espoo, finland), 25 pmol of each primer and 1 ml of dna in a final volume of 25 ml. the cycling conditions for each cytokine are given in table 2 . cdna samples and standards were processed in triplicate and a negative control and two internal controls (previously quantified cdna samples) were included in each pcr series. nf3 ccc tac tat tct tgg ttt 8413-8430 140 nr4 cgt ctg ttg tgt ctg tac c 8552-8534 a crcov nucleocapsid gene, nf3 and nr4 (genbank accession no. dq682406). results were analysed and quantification performed using standard curves produced by opticon monitor software v. 3.1 (bio-rad). the absolute copy number of the gene of interest was normalised to cdna concentration. cytokine mrna copies (ng cdna à1 ) were presented as the logarithm of the fold change relative to controlinoculated cultures, from the same dog, at the same time post-inoculation. this method of analysis was adopted to minimise any differences in cytokine mrna levels due to maintenance of the tracheas in vitro and natural variation in baseline levels and responses between different animals. cytokine mrna levels were also calculated relative to the crcov copy number at a given time point post-inoculation. statistical analysis of the results of canine cytokine mrna levels was performed using spss v. 16.0 (spss inc., chicago, usa). the relationship between control-and crcov-or lpsinoculated canine tracheal cultures at a given time was assessed using one-way analysis of variance (anova) and post hoc comparisons made using the bonferroni method. 2.6. latex bead clearance assay 10 ml of a suspension of 1 mm diameter latex beads (polybead polystyrene microsphere beads; polysciences europe, eppelheim, germany) was pipetted onto the surface of tracheal cultures daily. the beads covered the whole mucosa and following incubation for 30 min each tracheal culture was scored blind by visual inspection according to a scale of 0-5. zero was defined as no clearance and five was complete clearance of the whole suspension to one edge of the tracheal piece. statistical analysis of the results of latex clearance was performed using spss v. 16.0. the relationship between control-and crcov-or lps-inoculated canine tracheal cultures was assessed using 3-way repeated-measures analysis of variance (rmanova) and post hoc comparisons made using the bonferroni method. paraffin-embedded formalin-fixed crcov-inoculated tracheal culture tissue sections (4 mm) were prepared on superfrost plus microscope slides (menzel-glä ser, braunschweig, germany). slides were heated at 60 8c for 1 h, deparafinised and rehydrated. endogenous peroxidase was blocked with 3% h 2 o 2 for 10 min then washed in dh 2 o for 5 min. sections were incubated in pre-warmed (37 8c) protease xiv 0.05% (sigma) in tbs for 15 min, then rinsed in dh 2 o (2â 2 min). the shandon sequenza coverplate system (thermo fisher scientific, runcorn) was used to aid slide handling and improve tissue preservation. sections were incubated with blocking serum (2% normal goat serum [vector laboratories, peterborough] in tbs) then undiluted bovine anti-bcov polyclonal antiserum conjugated to fluorescein isothiocyanate (fitc) (vmrd inc., pullman, wa) overnight at 4 8c. sections were washed with blocking serum and incubated with biotinylated goat anti-fitc antibody (1:300 in blocking serum, vector laboratories) for 1 h at 37 8c. sections were washed with blocking serum and incubated with vectastain elite abc reagent (vectastain elite abc kit, vector laboratories) for 30 min at room temperature, followed by washing in blocking serum. colour development was carried out using the vector vip substrate kit (vector laboratories) for 8-10 min. sections were counterstained with methyl green (vector laboratories) for 4 min, dehydrated and mounted. positive cells were identified microscopically by the presence of intense purple-brown staining. the majority of cytokine primers had previously been optimised for use with real-time qpcr on canine tissue samples using taqman real-time rt-pcr assays (peeters et al., 2006; peters et al., 2005) . however for each primer set, linearity and efficiency of the pcr were assessed by analysing the correlation coefficient + slope for threshold cycle (c t ) versus log cdna concentration to ensure the accuracy of mrna quantification using a sybr green-based real-time pcr assay. for each primer set, linearity was confirmed using cdna obtained from canine tracheal cultures and the plasmid dna standard curve. high linearity was observed between c t and the log 10 dna concentration with the correlation coefficient (r) greater than 0.99 for all primer sets used. the slope of the relationship between c t and the log 10 cdna or plasmid dna concentration was used to determine the efficiency of the pcr reaction using the following equation: efficiencies for each qpcr primer set are given in table 3 . 26 sets of cdna from tracheal cultures inoculated with crcov, lps or medium-only were available for cytokine mrna and crcov quantification. levels of tnf-a mrna in control cultures (medium only) remained constant throughout the time course, however levels of il-6 and il-8 mrna increased from the time of death (à24 h) to a peak at the time of inoculation (0 h) (data not shown). levels of il-6 and il-8 mrna reduced at each subsequent 24 h of the time course. tnf-a: canine tnf-a mrna was quantified over time and the analysis was performed relative to tnf-a mrna levels in control (medium only) inoculated cultures. lpsinoculation resulted in an elevation of tnf-a mrna, relative to controls, at 24, 48 and 72 h post-inoculation, the peak being a 2.5-fold increase, at 48 h post-inoculation (fig. 1) . the tnf-a mrna levels in crcov-inoculated cultures were reduced, relative to controls, at 24, 48 and 72 h. the most marked reduction was observed at 72 h, where cytokine copies were 3.7 times lower than controls; however, at 96 h post-inoculation, tnf-a mrna copy levels were 1.5 times higher than control-inoculated cultures. il-6 and il-8: lps inoculation resulted in raised cytokine mrna copy numbers for both il-6 and il-8 and at all three times points (24, 48 and 72 h) post-inoculation (figs. 2 and 3). mrna levels of both cytokines were elevated above control levels to the highest degree at 48 h (4.9-and 6.4-fold increase for il-6 and il-8, respectively). crcov inoculation resulted in the reduction of il-6 and il-8 mrna levels at 24-72 h post-inoculation. for both cytokines, this reduction was greatest at 72 h (6.0-and 5.7-fold decrease for il-6 and il-8, respectively). il-6 and il-8 mrna levels were increased compared to controls at 96 h. there was no significance difference (p > 0.05) between measured cytokine mrna levels in control or crcovinoculated cultures at any time point. significant differences were obtained for lps inoculation at 24, 48 and 72 h (p = 0.01). the development of an accurate method for the quantification of crcov in tissue samples enabled nucleocapsid gene rna to be quantified in cdna from canine tracheal cultures inoculated with crcov. the mean crcov nucleocapsid gene copy number within tracheal cultures from different dogs at 24-96 h post-inoculation was determined (fig. 4) . in all cases, there was an overall increase in crcov nucleocapsid gene rna copies during the experiment and the peak copy number was attained at 96 h post-inoculation for each dog. however, in some dogs, there was a noticeable fall in copy number at 48 or 72 h, compared to the levels at 24 h, thus the dynamics of the infection appeared to be different for tracheal cultures from different dogs. due to this finding, cytokine mrna levels were also analysed relative to crcov copy number, to control for individual variation between the tracheas from different dogs. the quantification of canine il-6, il-8 and tnf-a mrna copies in crcov-inoculated cultures was presented as the logarithm of the fold change relative to control-inoculated cultures in the same dog at the same time point. these data were plotted against crcov nucleocapsid rna copies in the same cdna sample. the pearson correlation coefficient, r was determined for each cytokine using spss v. 16.0. there were weak positive correlations between the fold change in il-6 (r = 0.304) and il-8 (r = 0.277) mrna copies and crcov copies, however these were not considered significant (p > 0.05). a significant positive correlation (p = 0.05) was demonstrated between tnf-a mrna and crcov rna copies (r = 0.382). interestingly at low crcov rna copy number (below 2.0 logs), cytokine mrna copies appeared to be reduced relative to controls. the mean maximal clearance score, based on four individual cultures (selected arbitrarily from different areas of the trachea to reduce the effects of any potential intra-tracheal variation in ciliary function), was obtained after 30 min incubation immediately prior to inoculation (0 h) and was regarded as 100%. the mean clearance scores at each subsequent 24 h interval, up to 120 h post-mortem, were calculated as a percentage relative to the original clearance score thus giving an overall score for each condition, at each time point, per dog. assessment relative to the initial clearance was used to negate the inherent variation in ciliary function between different tracheas. a gradual reduction in latex clearance score was observed for all inoculation conditions, including the control, over 96 h (fig. 5) . after 96 h, lps-inoculated cultures retained only 15.3% of their initial clearance at 0 h and had average latex clearance scores of 30% less than the medium only controls. crcov-inoculated cultures had 43.6% of the initial clearance remaining after 96 h, compared with 45.3% for control-inoculated cultures. crcov-inoculated cultures had the greatest differential from controls at 48 h (12.6%), however by 96 h, the rate in decline of clearance score had slowed and essentially mirrored the control. ciliary clearance was found to be significantly different between control-and lps-inoculated cultures for the time period 24-96 h post-inoculation (p = 0.002). however, no significant difference (p > 0.05) was found between control-and crcov-inoculated cultures in ciliary latex clearance over the same period post-inoculation. assessment of latex clearance at 10 min intervals was used to try to detect more subtle changes in ciliary function, more specifically an altered rate of latex clearance (data not shown). ciliary clearance was found to be significantly different between control-and lpsinoculated cultures for the time period 10-60 min postinoculation (p = 0.0001). however, no significant difference (p > 0.05) was found between control-and crcovinoculated cultures, over the same period. coronaviral-antigen positive cells were detected within the epithelium of crcov-inoculated tracheal culture sections (fig. 6) . positive staining was present in the cytoplasm of ciliated columnar epithelial and goblet cells. the distribution of positive cells was relatively scant, but widespread, often occurring as small groups of positive cells. aggregates of positively stained material were observed on the luminal surface of the trachea and these were invariably located adjacent to coronaviral-antigen positive cells. less frequently, coronaviral-antigen positive cells were surrounded by areas of vacuolation within the epithelium, possibly representing a cytopathic effect of the virus. tracheas from naturally infected cird cases, where crcov was detected by rt-pcr, were also examined (fig. 6) . intra-cytoplasmic staining of epithelial cells within the trachea of naturally infected dogs was in agreement with that observed in the crcov-inoculated cultures. in vitro models, particularly those involving the respiratory system, must be physiologically relevant and closely match the in vivo environment for the accurate modelling of viral pathogenesis. many cell culture-adapted cell lines have either lost the ability to synthesize and secrete certain cytokines or else their responses to stimuli, such as viruses, are altered by growth in culture. the relevance of using isolated cell populations, removed from their normal context, for the study of the complex multicellular interactions of viruses in vivo is therefore greatly reduced. the global distribution and high seroprevalence of crcov suggest a virus that is contagious but has the ability to spread through canine populations, for the most part, clinically undetected. to appreciate further the role of crcov in canine respiratory disease, an in vitro model of the hypothesised main target tissue, the trachea, was developed. this study represents the first use of an organ culture system, incorporating an air-interface, in assessment of the functional consequences of virus replication in dogs. knowledge of the epithelial immune response is crucial for the further understanding of crcov pathogenesis. the mrna levels of pro-inflammatory cytokines and chemokines were therefore quantified following crcov inoculation. lps was chosen as a positive control as this has previously been shown to be a potent inducer of innate immunity and the corresponding cytokine profile in humans (boeuf et al., 2005) and significantly decreased ciliary activity in rat tracheal organ cultures (johnson and inzana, 1986) . the cytokines selected for investigation in this study were chosen based on their involvement in other coronavirus infections. in response to lps, mrna levels of tnf-a, il-6 and il-8 in cultures, were significantly increased from 24 h post-inoculation, relative to controls, indicating that the assay was sensitive enough to detect changes in cytokine mrnas within this system. peak levels for all three cytokine mrnas were detected at 48 h and had declined by 72 h. interestingly, crcov appeared to suppress the mrna levels of pro-inflammatory cytokines from 24 to 72 h postinoculation, however at 96 h the levels were raised for each cytokine. when correlated with actual copies of virus, it appeared that at lower copy number (as would be present during the earlier part of the time-course), there was active suppression of cytokine mrna, relative to controls. how-ever, it seemed that once the virus copies reached a threshold, of 100 copies ng cdna à1 , cytokine mrna levels were increased relative to controls, as recorded at 96 h. it is conceivable that early non-structural proteins produced by the replicating virus may act to inhibit the induction of cytokine and chemokine genes. down-regulation of ifnresponses by non-structural proteins is a strategy many viruses have evolved to remain successful pathogens (haller et al., 2006) . recently bcov has been shown not to induce a detectable pro-inflammatory response in calf intestine following inoculation (aich et al., 2007) . quantitative rt-pcr analysis of il-6 and tnf-a genes revealed that both were down-regulated following bcov infection and the authors concluded that the absence of a pro-inflammatory response may result in a more prolonged infection. sars-cov is highly sensitive to the antiviral actions of ifns, both in vitro and in vivo which may explain why the virus has been shown to actively suppress the activation of antiviral and ifn-induced effector genes via the inhibition of the crucial cytokine transcription factor, irf3 (spiegel et al., 2005; spiegel and weber, 2006) . the suppression of the immediate early interferon response, via the absence of irf3, is now thought to be a feature of all group 2 coronaviruses (versteeg et al., 2007) . the suppression of antiviral cytokines in non-immune cells by sars-cov is thought to buy time for dissemination of the virus in the host and a similar mechanism may be employed by crcov. all cultures, including controls, were subject to gradual tissue degeneration. the upregulation of pro-inflammatory cytokines would be a normal tissue response to such injury. cytokine gene expression levels in human tonsil ex vivo cultures were similar to the levels at the time of excision, except il-6 and il-8 which were markedly increased following the first 24 h of culture hypothesised to be due to initial stress of culture (bonanomi et al., 2003) . in the current model the same pattern was observed for il-6 and il-8 mrna in control cultures, whereas tnf-a mrna levels remained stable. in control-inoculated cultures increased cytokine mrna levels may have occurred as a response to maintenance in culture. the reduction in cytokine mrna in crcov-inoculated cultures, relative to controls, may have been due to crcov actively inhibiting pro-inflammatory responses through an as yet unknown mechanism. increased cytokine mrnas were observed at 96 h in crcov-inoculated cultures. the replication of crcov within the cultures may have progressed more slowly than anticipated and had the cultures been maintained for longer than 96 h, possible further increases in cytokines might have been observed. the use of an accurate method for the quantification of crcov in canine tissue samples was extremely useful in this study. whilst the assay could not differentiate between live virus and simply genetic material from non-viable particles, steps were taken to minimise this risk. the washing of tracheal pieces prior to the extraction of rna meant that only virus internalised within cells would be quantified. the assay demonstrated that the growth kinetics of crcov within this system were not linear. despite the application of a uniform virus inoculum, differences in ciliary function and the thickness and nature of the mucus layer between dogs may have resulted in variation in the physical barrier to virus receptor binding and penetration of epithelial cells. cultures from all dogs included in this study were subject to an increase in overall crcov nucleoprotein gene rna copies by 96 h postinoculation, indicating that viral replication was occurring. the differences between individual tracheal cultures were representative of the heterogeneous in vivo situation and variation between dogs was minimised by the use of control-inoculated cultures, which acted as a baseline for the responses of individual tracheas. the presence of ciliary clearance is a key strategy for the removal of pathogens from the respiratory tree and ciliary loss is a known consequence of immersion of cultures in medium. a deficient ciliary clearance mechanism in the trachea would potentiate the invasion and colonisation of more serious respiratory pathogens such as bordetella bronchiseptica. cultures were shown to retain co-ordinated ciliary beating for up to 144 h post-mortem in this system. ciliary function declined between the time of death and culture inoculation. however, following inoculation, the ciliary clearance of lps-inoculated cultures was significantly reduced with respect to both the speed and extent of clearance of latex relative to controls. whilst the effects of crcov on ciliary function were not found to be significantly different to controls, crcov was capable of limiting the ciliary function of canine tracheal epithelium at 48 h post-inoculation. between 72 and 96 h, the ciliary clearance of crcov-inoculated cultures was similar to that of controls, suggesting that any effects of crcov on the respiratory epithelium happen early in the course of infection. the crcov isolate used in the inoculation of tracheal cultures had been adapted to growth in a human rectal adenocarcinoma cell line (hrt-18 cells) and the challenge dose of virus was the maximum titre to which crcov could be cultured. the functional responses observed following virus challenge may be limited as a consequence of a loss of virulence or replicative efficiency within canine respiratory cells. ihc revealed coronavirus antigen positive intra-cytoplasmic staining of ciliated epithelial and goblet cells within canine tracheas of both crcov-inoculated cultures and from naturally infected cases of cird. whilst the number of coronaviral-antigen positive cells was greater in experimentally inoculated tissue, the same cell types and distribution were observed in naturally infected tracheal tissue. these findings were in agreement with previous work which identified coronaviral-antigen positive epithelial cells within the bronchi and major bronchioles of two cases of cird (ellis et al., 2005) . the greatest accumulations of antigen-positive material were located on the apical aspect of cells and often in association with luminal aggregations overlying the tracheal surface, possibly representing secreted antigen, retained on the surface in the periciliary mucus layer. the distribution of crcov in the canine trachea mirrors that of bcov in bovine tissues, where the primary sites of infection are the epithelial cells of the nasal cavity and trachea, leading to mild upper respiratory signs such as coughing, sneezing and rhinitis (reynolds et al., 1985) . the effects of crcov on the mucociliary and innate immune systems were observed within 24 h of inoculation. a moderate reduction in ciliary function may be due to cellular damage during viral replication. during this time mucociliary compromise and down-regulation of pro-inflammatory cytokines by crcov may present a 'window' of opportunity for the entry of other viral or bacterial pathogens, thus potentiating more serious respiratory disease. on its own, crcov may cause a transient potentially clinically silent, respiratory disease similar to the human 'common cold'. however, challenge with crcov during entry to a re-homing kennel is often accompanied by the simultaneous exposure to cpiv, chv, b. bronchiseptica and mycoplasmas, in addition to the environmental stresses that are inevitable during such an event. the simultaneous or sequential actions of these agents, primed by crcov, could lead to the development of cird and potentially outbreak situations. would like to thank sandra greaves for technical assistance. expression of cytokines on human bronchial epithelial cells induced by influenza virus a interactions between respiratory epithelial cells and cytokines: relationships to lung inflammation comparative analysis of innate immune responses following infection of newborn calves with bovine rotavirus and bovine coronavirus ciliostasis is a key early event during colonization of canine tracheal tissue by bordetella bronchiseptica interleukin-8 expression in normal nasal epithelium and its modulation by infection with respiratory syncytial virus and cytokines tumor necrosis factor, interleukin-1, and interleukin-6 cyproquant-pcr: a real time rt-pcr technique for profiling human cytokines, based on external rna standards, readily automatable for clinical use quantitative cytokine gene expression in human tonsils at 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southern italy and epidemiological relationship with canine enteric coronavirus studies on the relationship between coronaviruses from the intestinal and respiratory tracts of calves inhibition of beta interferon induction by severe acute respiratory syndrome coronavirus suggests a two-step model for activation of interferon regulatory factor 3 inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with sars coronavirus group 2 coronaviruses prevent immediate early interferon induction by protection of viral rna from host cell recognition this work was supported by a phd scholarship (s. priestnall) from the royal veterinary college. the authors key: cord-295745-iw3ftw3h authors: gershoni, jonathan m title: molecular decoys: antidotes, therapeutics and immunomodulators date: 2008-11-18 journal: curr opin biotechnol doi: 10.1016/j.copbio.2008.10.001 sha: doc_id: 295745 cord_uid: iw3ftw3h receptor–ligand interactions are fundamental to the regulation of cell physiology, enabling the communication between cells and their environment via signal transduction. receptors are also exploited by toxins and infectious agents to mediate pathogenesis. over the past 20 years, however, this bi-partite paradigm for cellular regulation, that is, receptors and their ligands, has been revised to include an unforeseen participant namely, soluble receptors or molecular decoys. decoys function as nature's modifiers of potent responses such as inflammation, stimulation of cell proliferation and triggering apoptosis. decoys not only provide the means to fine tune the regulation of these phenomena; they also serve as potential leads for the development of recombinant anti-toxins, anti-viral agents and novel therapeutics for combating cancer and inflammatory disease. one hundred years have past since ehrlich [1, 2] and langley [3, 4] proposed the existence of 'receptors' as the responsive cellular components targeted by toxins. the interplay of ligands binding to their cognate receptors as a means to regulate cell function, induce proliferation or trigger apoptosis has since become fundamental to our understanding of cell biology, immunology and neurobiology, as well as to the rational design of innovative therapeutic drugs, laying the foundations of modern pharmacology. during the past quarter of the 20th century, however, this paradigm of the receptor/ligand 'duo' has had to be revised so to incorporate a third player in this scene, soluble receptors, thus creating a triad of proteins whose balance regulates and fine tunes cellular function. soluble receptors-molecular decoys, were first proposed as novel biologics, envisioned as receptor mimetics that would function to intercept and sequester a pathogen in solution, before it would have the chance to encounter its cellular target. however, as gene cloning, expression of recombinant proteins and genomics flourished it soon became obvious that artificial decoys were in fact lagging in comparison to what turned out to be nature's basic modus operandi. for almost every membrane receptor of cytokines, growth factors and cell adhesins, soluble versions were found to be naturally produced by cells; hence 'natural decoys' that function as modifiers of the potent stimulants and regulators of inflammation and immune response. moreover, it was discovered that these same decoy receptors had been hijacked by viruses over the course of their co-evolution with their hosts. in this review both recombinant and natural molecular decoys are described. whereas sugar-based decoys [5 ,6-8] and oligonucleotide decoys [9-11,12 ,13 ] are certainly of importance, the focus here will be primarily on proteinaceous decoys, selecting illustrative examples of this class of cell regulator. out of historical justice to ehrlich and langley, anti-toxin and anti-viral decoys will be the first to be discussed, then moving on to the natural decoys and their virally hijacked versions. at the end, examples of the biotech pipeline of recombinant decoys currently in development and production are provided. the nicotinic acetylcholine receptor (nachr) is composed of five polypeptide subunits (a 2 bgd, mw 300 kda) that together form a ligand-regulated ion channel [14] . the neutrotoxins, such as d-tubocurarine and cobra toxin, are antagonists of the neurotransmitter, acetylcholine, whose binding opens the channel leading to membrane depolarization and ultimate muscle contraction. ligand overlay of protein blots [15, 16] proved to be effective for delineating a major component of the cholinergic binding site within the extracellular domain of the receptor's alpha subunit. recombinant fusion proteins expressing the ligand binding segment of the alpha subunit were found to efficiently bind alpha neurotoxins in vitro [17, 18] . the production of the recombinant cholinergic binding site, r4137, was ultimately tested in vivo and found to protect mice against lethal doses of both cobra toxin and d-tubocurarine [19] . with the in vivo proof of principle, the concept of 'decoyance' was proposed as a general application of receptor derived soluble molecular mimetics-decoys 1 , for the treatment and prevention of infectious disease. bacterial toxins are often the mediators of morbidity and death and their sequestration has been a target for decoy development. a case in point is clostridium difficile toxin a, a major cause of antibiotic associated diarrhoea and colitis [7] . the toxin is a large protein (308 kda) that binds to the trisaccharide sequence gala [1] [2] [3] galb [1] [2] [3] [4] glcnac displayed on the luminal surface of the apical plasma membrane of the intestinal epithelium. therefore, a sugarbased decoy was produced and tested by the canadian biotech company, synsorb biotech, who conjugated the trisaccharide onto an inert silicon-based support. this was then introduced orally to rats that were subsequently subjected to toxin a. the decoy-treated rats did not present the typical toxin a associated pathology of the ileal mucosa as compared with the controls. hence the decoy was able to sequester the toxin. this decoy application is particularly attractive in the incidence where antibiotic treatment is in fact detrimental; antibiotics compromise the natural flora providing clostridium with an opportunity to colonize. telovamer, a soluble, high molecular weight anionic polymer represents a 'functional decoy' able to bind and neutralize both toxin a and toxin b of clostridium difficile yet is not derived from the natural receptor for these toxins. genzyme corp has announced recently that a phase iii clinical study proved less effective than earlier phase ii results had indicated, yet further development is being pursued. another example where decoys become advantageous in light of negative effects of antibiotic treatment is hemolytic-uremic syndrome (hus) caused by shiga toxinproducing e. coli (stec) infections [8] . antibiotic therapy in such cases is contraindicated as it leads to the release of cell associated shiga toxin (stx) and induces toxin gene expression, thus leading to an increase in free toxin in the gut lumen. an elegant treatment of stec infections has been proposed by paton et al. who have constructed a recombinant probiotic e. coli strain that displays stx receptor mimics on its surface. stx binds the glycolipid receptor, globtriaosyl ceramide (gb3) that has the structure gala [1] [2] [3] [4] galb [1] [2] [3] [4] glc ceramide. the glycosyl transferase genes igtc and igte derived from neisseria were introduced into e coli r1 rendering it able to produce a chimeric lipopolysaccharide (lps) core terminating in gala [1] [2] [3] [4] galb [1] [2] [3] [4] glc. the 'probiotic bacterial decoys' were capable of preventing fatal systemic complications of stec in mice treated by oral administration of these recombinant e. coli. the threat of infectious disease took on a more sinister reality since 11 september 2001, where bio-warfare has become an ever growing concern [20] . the following two examples of anti-toxin decoys are thus especially relevant. staphylococcal enterotoxin b (seb) [21] acts as a superantigen that can indiscriminately activate a broad population of t cells triggering the massive release of inflammatory cytokines that can escalate to toxic shock syndrome and death. the mode of action of seb is to bind the vb region of the t cell receptor (tcr). despite the relatively low affinity between seb and vb (kd = 144 mm), the potency of this toxin is very high (lethal doses can be as low as nanograms per kilogram body weight). buonpane et al. [22 ] have systematically optimized the murine vb8.2 domain using serial mutagenesis and yeast-display followed by increasingly stringent screens against biotinylated seb. the highest-affinity mutant isolated had an affinity of 48 pm that is a three million fold improvement over the wild type vb8.2-seb interaction. this vb decoy proved highly effective in neutralizing the toxin, even in rabbits that had already developed early signs of toxic shock syndrome. this not only illustrates the power of decoys but emphasizes that their efficacy is apparently directly correlated with their affinity for their target. anthrax toxin certainly plays a major role in the arsenal of the bioterrorist. bacillus anthracis secretes a tripartite toxin composed of: protective antigen (pa, 83 kda) that binds macrophages; lethal factor (lf), a zinc metalloproteinase; and a ca +2 /calmodulin-dependent adenylate cyclase called edema factor (ef) [23] . pa83 binding to the integrin-like i domain of the macrophage surface protein; capillary morphogenesis protein 2 (cmg2), leads to its cleavage by furin endoprotease producing pa63. the oligomerization of pa63 into a heptamer provides the binding site for lf and ef that are then endocytosed and eventually kill the cell. various approaches have been developed to counteract anthrax toxin of which anti-pa antibodies have proven particularly effective. the concern however, is that weaponized strains of b. anthracis may be engineered to produce antigenically altered versions of pa that would escape neutralization by existing anti-pa antibodies. here the attribute of decoys is apparent as one assumes that the receptor binding surfaces of pa would not be amenable to mutagenesis and epitopic modification. scobie et al. have been able to demonstrate that soluble cmg2 can function as a potent decoy capable of protecting rats against lethal toxin challenge making this decoy one of the most effective anthrax anti-toxins known [24] . the first bona fide anti-viral decoy to be developed was soluble cd4 (scd4) for the treatment of aids. cd4 is naturally an integral membrane glycoprotein protein that traverses the plasma membrane once. its extracellular portion is composed of four immunoglobulin (ig) domains (d1-d4) where d1 binds hiv-1 gp120 [25] . the fact that cd4 is a member of the immunoglobulin superfamily has had a profound effect on its development as a decoy and on the development of decoys in general. immunoglobulins are naturally presented as membrane bound cell surface receptors (b cell receptors, bcr) yet mature into soluble serum antibodies as the result of alternative splicing. antibodies are as efficient in antigen recognition as are their cognate membrane bound bcrs. isotype switching is fundamental to antibody biology and as such, swapping v domains for other ig domains makes perfect sense. moreover, antibodies naturally are polyvalent; as in bivalent iggs, tetravalent igas and decavalent igms and therefore are avid binders of their targets. capon et al. [26] were the first to engineer an 'immunoadhesin', grafting the ligand binding ig domains d1-d2 of cd4 onto a human igg1 fc scaffold that proved to increase the serum half-life of the decoy markedly. so much so, this strategy for decoy design has been adopted by most working on soluble receptors/decoys in general [27 ] figure 1 . despite the optimism, however, it turned out that cd4based decoys simply did not have the clout required to keep hiv in check. it was quickly realized that the neutralizing potency of scd4 was preferentially greater for lab adapted strains of hiv than for the field isolates of this virus [28, 29] . the mechanism for neutralization by the decoy required induced gp120 shedding that turned out to be much more demanding for the field isolates [30] . all attempts to optimize and improve scd4 decoys [31] [32] [33] [34] [35] proved insufficient and for the moment scd4 decoys have not progressed beyond phase i/ii clinical trials. since the introduction of scd4 as an aids therapeutic, soluble receptors for various viruses (e.g. rhinovirus [36, 37] , poliovirus [38] , foot and mouth disease virus [39] , sars coronavirus [40] and hepatitis a virus [41] ) have been reported to have neutralizing activity and thus form the basis for novel therapeutics. however, to date, a commercial decoy-product effective in the prevention or treatment of viral infections has still not appeared on the market. this may be due to the fact that viruses are replicating pathogens and demand enormous efficiency for viral clearance in order for a decoy to be truly therapeutic. decoys as modifiers of regulation on the contrary, turn out to be natural components in the fine tuning of inflammation and immune responses. in 1984 ullrich et al. published the cloning of the human epidermal growth factor receptor (egfr, mw 138 kda) from a431 epidermoid carcinoma cells [42] . curiously, they also discovered that these cells contained a 2.8 kb mrna that when expressed produced a 70 kda truncated version of the egf extracellular domain. the authors concluded that this is 'particularly intriguing in view of [their] earlier observation that the v-erb-b oncogene encodes what seems to be a truncated avian receptor polypeptide corresponding to the transmembrane and cytoplasmic domains'. thus, the focus and motivation in 1984 were not neutralizing soluble receptors, but rather truncated constitutive signal transducing receptors that could explain the out-of-control cell proliferation associated with cancer. nonetheless, shortly there after reports appeared describing a diversity of naturally existing soluble receptors, most of which could be associated with one of two gene families, the tumour necrosis factor (tnf) superfamily [43 ] and the ig superfamily [44, 45 ] . it immediately became apparent that by proteolytic shedding of cell surface proteins (by metallo-proteases coined 'sheddases') or by alternative splicing, truncated versions of otherwise membrane associated receptors are readily generated [46 ] . nature produces such soluble receptors in order to crucially regulate immune responses towards cancer and infection as well as inflammation in general (figure 1 ). mantovani and his colleagues have pioneered the concept of natural decoy receptors as nature's solution for the fine tuning of its most potent defense mechanisms [47 ] . the specific example of the soluble interleukin 1 receptor type ii (il-1rii) is illustrated here as a case in point. interleukin i was one of the first cytokines to be discovered and is responsible for triggering a diversity of physiological effects such as fever, augmentation of lymphocyte responses and induction of degenerative changes in joints [48] . owing to this diversity, there was even question as to whether a single molecule could be responsible for such a variety of functions. the ultimate cloning of il-1a and il-1b in 1984, laid this debate to rest as it became clear that these cytokines function as a 'master switch' of sorts, responsible for the expression and release of numerous other cytokines (e.g. il-6 and many chemokines). availability of recombinant il1 also promptly led to the discovery of its receptors, first il-1r type 1 that is a member of the ig superfamily (contains three ig-like extracellular domains) and signal tranduces through a toll-like cytoplasmic domain. this receptor is expressed on most cells and functions in a complex with its 'associated protein'-acp. a second il1-r (type ii), expressed primarily on b cells, monocytes and polymorphonuclear cells (pmn), also exists yet is unable to signal transduce (its cytoplasmic tail is only 29aa long). colotta et al. were able to show that il-1r type ii functions as a decoy receptor and is present in both membrane associated and soluble forms [49] . furthermore, its expression is regulated by il-4. together, these illustrate the complexity and elaborate regulatory devices that exist for the control of the il-1 response. the action of il-1, mediated by its association to il-1r type i, can be moderated by sequestration of the cytokine by soluble il-1r type ii, which is itself upregulated by il-4. moreover, as il-1r type ii also binds acp, the effect of binding of decoy il-1r type ii to the complex-il-1/ il-r type i/acp causes a dominant negative shut down of signal transduction. soluble decoy receptors are not always antagonistic to their ligands as is illustrated by soluble il-6 receptor (sil-6r). this decoy binds il-6 and in doing so prolongs its half-life. furthermore, binding of il-6/sil-6r to membrane bound gp130 triggers signal transduction via a process of 'trans-signaling' [50 ] . finally, decoy action can be mediated by membrane bound receptors as well. this is particularly relevant for chemokine receptors whose decoys persist as membrane proteins that are effective in ligand binding but 'handicapped' in signal transduction. thus, 'dud' receptors serve as functional decoys to fine tune inflammation [51 ] (figure 1 ). this strategy is also employed in the regulation of programmed cell death mediated by death receptors and their decoys [52] . decoy receptors 1 and 2 are membrane bound 'dud' receptors unable to signal transduce. decoy receptor 3 (dcr3) [53] however, is a potent soluble decoy for fas ligand that tends to be overexpressed in various cancers illustrating how tipping the balance of the regulation of apoptosis can have a profound effect in cancer pathogenesis (see figure 1 ). molecular decoys: antidotes, therapeutics and immunomodulators gershoni 647 native receptors and their decoys. binding of ligands to their native receptors triggers a signal transduction cascade (for cytokines, chemokines and growth factors). often, toxins and viruses exploit existing receptors as alternative ligands and thereby elicit morbidity or gain entry into the cell. in order to prevent toxin or viral pathogenesis, or to modify the effects of the natural ligands, decoys can intercept the ligands before they reach the native receptor. three types of decoys are portrayed: 'dud' receptors are membrane associated decoys that bind the native ligands yet are unable to signal transduce. soluble receptors can be produced naturally either by alternative splicing or proteolytic cleavage. recombinant soluble receptors provide research tools and leads for the development of novel pharmaceuticals. the preferred modality for such therapeutic decoys is 'immunoadhesins', receptor binding domains grafted onto an fc scaffold. it is not surprising that the ability to intervene in immuno-regulatory processes through decoy receptors has been exploited by the large dna viruses, thus creating 'viroceptors' [54] [55] [56] [57] . the poxviruses produce a diversity of soluble cytokine receptors as well as their own versions of cytokine binding proteins. viral decoy receptors for tnf, il-1 and interferon g (ifng) provide poxviruses the means to counter act and evade the immune response, contributing directly to the virulence of the virus [58] . thus for example, the deletion of the viral ifng binding protein has no effect on viral replication in cell culture yet dampens its virulence dramatically in vivo. this point might be important in the design of safer vaccines in which viral decoy receptors can be deleted, rendering a more tolerated virus, without compromising its antigenic repertoire [59 ] . viral decoys, although derived from host receptors, have evolved to enhance their activity in immune evasion [56] . thus for example, the vaccinia viral receptor for il-1 tends to be much more specific for il-1b than its mammalian cell homologue. for the viral ifngr, which is highly stringent in the host, the opposite occurs and is broadly cross reactive as a soluble viral decoy. this decoy may actually be the result of capture of not only the receptor domain from the host but also of a helix-turnhelix (hth) derived from the tfiia host transcription factor [60] . as in the transcription factor, this hth domain allows oligomerization and is the structural element that enables the viral decoy to form tetramers and thus benefit from more avid binding. the multidomain nature of viral decoys is found in the vtnfr as well [61] . the pox tnfrs are coded by four genes named cytokine response modifiers b (crmb), crmc, crmd and crme. in crmb, in addition to its tnf binding domain, its carboxy terminus has developed the capacity to bind chemokines. thus, crmb can simultaneously sequester tnf and bind chemokines as well. chemokine binding receptors are particularly well developed in the herpes viruses [62 ] . a case in point is the human cytomegalovirus (hcmv) open reading frame us28, which is closely homologous to cc chemokine receptors and binds cc chemokines at the nanomolar range [63] . whereas us28 is effective in chemokine sequestration, its expression is not required for viral replication in cultured cells. on the contrary this membrane bound receptor may have a different role where it may enable the virus-infected cells to follow natural chemokine gradients and thus assist in the dissemination of these cells to 'preferred' tissues in the host. the viroceptors and the interplay between viruses and their hosts may actually provide new insights for the design of future therapies [64 ] . undoubtedly, the concept of exploiting the ligand binding domains of receptors as leads for therapeutic decoys is extremely attractive. however, the fact is, that for the moment, only few decoys have been fda approved as bona fide commercially viable products. nonetheless, there has been progress in this effort and the greatest promise seems to be for counter acting chronic inflammation and treating cancer. tumour necrosis factor (tnf) is a central pro-inflammatory cytokine that triggers the production of other mediators of inflammation and tissue destruction, such as il-1b and il-6 [65] . tnf is directly associated with diseases of chronic and severe inflammation such as rheumatoid arthritis (ra), crohn's disease and psoriasis and therefore has been the target for the production of specific therapeutic antagonists. etanercept (enbrel marketed by amgen and wyeth) is a dimeric immunoadhesin (see above) consisting of the extracellular ligand binding domain of the tnf receptor linked to an igg1 fc scaffold [66] . in contrast to two other antagonists based on tnf-specific monoclonal antibodies (infliximab and adalimumab), etanercept binds both tnfa and lymphotoxin (formerly tnfb) thus illustrating the advantage of a decoy over antibodies that tend to be more restrictive for their binding. etanercept also shows higher potency for ra therapy although does not seem to be as effective as the antibodies in treatment of crohn's disease ( [65] , see also [67 ] ). most certainly there are numerous off label indications that are currently being evaluated for this drug that should prove to be extremely important for the treatment of inflammatory disease and may find application in the treatment of cancer as well. indeed, cancer appears to be directly associated with chronic inflammation [68 ] . chronic inflammatory bowel disease, for example, is often a prelude to colon cancer. in the recent study of popivanova et al. [69 ] , sequestration of tnf is shown to reduce the development of colorectal cancer in a mouse model for ulcerative colitis. treatment of these mice with etanercept markedly reduces progression to colorectal cancer and extends survival dramatically. these results not only demonstrate the association of tnf mediated inflammation with colon cancer but the potential use of etanercept as an anti-cancer drug. tumour vascularization is a pivotal step in the progression to metastatic cancer [70] . so long as the tumour is not 'hooked-up' to the vasculature, oxygen and nutrients are limited, growth is restricted and the shedding of malignant cells able to 'seed' other organs is not possible. folkman was the first to propose anti-angiogenic drugs for the treatment of solid tumours [71, 72 ] . indeed, a central target for drug development is the regulator of angiogenesis, vascular endothelial growth factor, vegf [73 ] . vegf exists as a number of molecular variants and related factors such as placental growth factor, and elicits its effect by binding to its receptors vegfr1 (flt-1), vegfr2 (flk-1) and vegfr3. the first biologic to be fda approved as an anti-vegf cancer therapeutic is the monoclonal antibody bevacizumab (avastin). since then a highly potent vegf decoy, 'vegf trap' (aflibercept, being developed by regeneron pharmaceuticals and sanofi-aventis 2 ), has been produced [74, 75 ] . vegf-trap is a recombinant immunoadhesin composed of the d2 ig domain of vegfr1 linked to the d3 ig domain of vegfr2 fused to the fc of igg1 thus creating a dimeric decoy. in preclinical mouse models for non-small cell lung cancer [76] and renal cell cancer [77] vegf-trap has proven to be substantially more potent than bevacizumab. in view of these results vegf-trap is currently the subject of four phase iii clinical studies in patients with non-small cell lung cancer, pancreatic cancer, colorectal cancer and prostrate cancer [75 ] . decoy receptors are clearly a basic component in the regulatory machinery of the cell and function in the fine tuning of proliferation and death of cells and especially in immune responses. they also play a role in immune evasion of viruses. the basic concept of recombinant soluble receptors as potent therapeutics actually preceded the realization of the existence of their natural homologues. the decoy neutralization of toxins appears to be very effective and should provide genuine biologic anti-dotes. the prospects for anti-virals seem more complex as the demand for sterilizing efficiency for the moment is beyond the potency of existing decoys. markedly increasing the affinity of decoys for their cognate viruses might be necessary to make this class of therapeutic effective. for the moment the greatest success in decoys as biologic drugs is in the immunoadhesin variants of the natural cell regulators such as tnf and vegf. similar products for il-1 [78] and cytotoxic t-lymphocyte associated antigen 4 (ctla-4) [79] have also been approved, indicating that we should be seeing multiple decoys on the market in the very near future. on immunity with special reference to cell life. the croonian lecture part of a scientific master plan? paul ehrlich and the origins of his receptor concept on nerve endings and on special excitable substances in cells. the croonian lecture receptive substances': john newport langley (1852-1925) and his path to receptor theory of drug action carbohydrates as future anti-adhesion drugs for infectious diseases a comprehensive review of the role of carbohydrate 'receptors' and bacterial lectin-like molecules that mediate adhesion. the structures of the glycomoeities and their recognition are described as well as a discussion of the potential applications for anti-adhesion therapy recent progress in the understanding of the role of bacterial adhesion in the pathogenesis of urinary tract infection pothoulakis c: a receptor decoy inhibits the enterotoxic effects of clostridium difficile toxin a in rat ileum a new biological agent for treatment of shiga toxigenic escherichia coli infections and dysentery in humans overexpression of tar sequences renders cells resistant to human immunodeficiency virus replication therapeutic applications of transcription factor decoy oligonucleotides potential therapeutic applications of decoy oligonucleotides decoy oligodeoxynucleotide targeting activator protein-1 (ap-1) attenuates intestinal inflammation in murine experimental colitis a comparative analysis of ap-1 vs nfkb decoys and their application in suppressing intestinal inflammation in a mouse model marked regression of liver metastasis by combined therapy of ultrasound-mediated nf kappab-decoy transfer and transportal injection of paclitaxel, in mouse a major problem in the application of oligonucleotide decoys is the method of delivery. here the application of ultrasound is described as a means to deliver nfkb decoys in the treatment of liver disease nicotinic receptors, allosteric proteins and medicine binding of alpha-bungarotoxin to isolated alpha subunit of the acetylcholine receptor of torpedo californica: quantitative analysis with protein blots protein blotting: principles and applications mapping the main immunogenic region and toxin-binding site of the nicotinic acetylcholine receptor expression of the alpha-bungarotoxin binding site of the nicotinic acetylcholine receptor by escherichia coli transformants molecular decoys: ligand-binding recombinant proteins protect mice from curarimimetic neurotoxins molecular decoys: antidotes, therapeutics and immunomodulators gershoni 649 the evolving field of biodefence: therapeutic developments and diagnostics toxic shock syndrome and bacterial superantigens: an update neutralization of staphylococcal enterotoxin b by soluble, high-affinity receptor antagonists systematic mutagenesis leads to a marked enhancement of affinity between the vbbased decoy and its target. this illustrates the correlation between affinity and decoy efficacy anthrax toxin receptor proteins a soluble receptor decoy protects rats against anthrax lethal toxin challenge cd4: its structure, role in immune function and aids pathogenesis, and potential as a pharmacological target designing cd4 immunoadhesins for aids therapy immunoadhesins as research tools and therapeutic agents high concentrations of recombinant soluble cd4 are required to neutralize primary human immunodeficiency virus type 1 isolates two mechanisms of soluble cd4 (scd4)-mediated inhibition of human immunodeficiency virus type 1 (hiv-1) infectivity and their relation to primary hiv-1 isolates with reduced sensitivity to scd4 thermodynamic and kinetic analysis of scd4 binding to hiv-1 virions and of gp120 dissociation highly efficient neutralization of hiv with recombinant cd4-immunoglobulin molecules rationale for the use of immunotoxins in the treatment of hiv-infected humans neutralization of human immunodeficiency virus type 1 by scd4-17b, a single-chain chimeric protein, based on sequential interaction of gp120 with cd4 and coreceptor hiv-1 neutralization by chimeric cd4-cg10 polypeptides fused to human igg1 nonlinear pharmacokinetics of high-dose recombinant fusion protein cd4-igg2 (pro 542) observed in hiv-1-infected children mechanisms of receptor-mediated rhinovirus neutralization defined by two soluble forms of icam-1 rhinovirus-stabilizing activity of artificial vldl-receptor variants defines a new mechanism for virus neutralization by soluble receptors threedimensional structure of poliovirus receptor bound to poliovirus interactions of footand-mouth disease virus with soluble bovine alphavbeta3 and alphavbeta6 integrins susceptibility to sars coronavirus s protein-driven infection correlates with expression of angiotensin converting enzyme 2 and infection can be blocked by soluble receptor alteration of hepatitis a virus (hav) particles by a soluble form of hav cellular receptor 1 containing the immunoglobin-and mucin-like regions human epidermal growth factor receptor cdna sequence and aberrant expression of the amplified gene in a431 epidermoid carcinoma cells the tnf superfamily-2008 an editorial preceding a special issue on the tnf superfamily including comprehensive tables of family members co-stimulatory pathways in lymphocyte regulation: the immunoglobulin superfamily immunoglobulin superfamily cell adhesion molecules: zippers and signals a comprehensive overview of the ig family mechanisms of soluble cytokine receptor generation a systematic analysis of various mechanisms used to generate soluble receptors including 'sheddase' metallo-proteanases tuning of innate immunity and polarized responses by decoy receptors the interleukin-1 family: 10 years of discovery interleukin-1 type ii receptor: a decoy target for il-1 that is regulated by il-4 cutting edge: trans-signaling via the soluble il-6r abrogates the induction of foxp3 in naive cd4+cd25 t cells trans-signaling provides a means to enhance the effect of a decoy rather than suppress the signal as illustrated for the case of soluble il-6 tuning inflammation and immunity by chemokine sequestration: decoys and more death receptors: signaling and modulation genomic amplification of a decoy receptor for fas ligand in lung and colon cancer host-related immunomodulators encoded by poxviruses and herpesviruses viral mechanisms of immune evasion viral mimicry of cytokines, chemokines and their receptors viral hijacking of g-proteincoupled-receptor signalling networks poxvirus immunomodulatory strategies: current perspectives poxvirus-encoded gamma interferon binding protein dampens the host immune response to infection whereas the cytokine 'viroceptor' is not required for the replication of the virus, it does effect its virulence. the authors propose that modification of viruses by deletion of their viroceptors may provide for safer vaccines structure and mechanism of ifn-gamma antagonism by an orthopoxvirus ifn-gamma-binding protein a chemokine-binding domain in the tumor necrosis factor receptor from variola (smallpox) virus hcmv-encoded g-proteincoupled receptors as constitutively active modulators of cellular signaling networks modulating chemokines: more lessons from viruses virus-encoded chemokines, chemokine receptors and chemokine-binding proteins: new paradigms for future therapy an insightful analysis of the potential for novel anti-viral therapies based on viroceptors tumor necrosis factor antagonist mechanisms of action: a comprehensive review etanercept, a novel drug for the treatment of patients with severe, active rheumatoid arthritis adalimumab, etanercept and infliximab are equally effective treatments for patients with psoriatic arthritis a careful comparative analysis of anti-tnf drugs in patients suffering from psoriatic arthritis. enanercept as compared to the two mab-based drugs was found equally effective and thus it is concluded that patients should be allowed to choose the therapy best suited to them a cytokine-mediated link between innate immunity, inflammation, and cancer an excellent review linking inflammation to the development of malignant disease blocking tnf-alpha in mice reduces colorectal carcinogenesis associated with chronic colitis using a mouse model for inflammatory bowel disease and progression to colon cancer the authors illustrate the effect of etanercept. this not only demonstrates the involvement of tnf in the progression to colon cancer but also the potency of its decoy in treatment patterns and emerging mechanisms of the angiogenic switch during tumorigenesis anti-angiogenesis: new concept for therapy of solid tumors angiogenesis: an organizing principle for drug discovery? an extremely comprehensive and insightful review on the role of angiogenesis in cancer and its treatment. the article not only reviews the field in detail, it is exceptionally well illustrated and provides prof folkman's personal perspectives only months before his untimely death vascular endothelial growth factor (vegf) signaling in tumor progression a comprehensive review of a central component in the biology of tumour progression vegf-trap: a vegf blocker with potent antitumor effects a useful survey of the latest developments and clinical trials related to vegf-trap vascular endothelial growth factor trap in non small cell lung cancer vascular endothelial growth factor trap blocks tumor growth, metastasis formation, and vascular leakage in an orthotopic murine renal cell cancer model efficacy and safety of rilonacept (interleukin-1 trap) in patients with cryopyrin-associated periodic syndromes: results from two sequential placebo-controlled studies efficacy and safety of abatacept or infliximab vs placebo in attest: a phase iii, multi-centre, randomised, double-blind, placebocontrolled study in patients with rheumatoid arthritis and an inadequate response to methotrexate the author acknowledges anna roitburd-berman, natalia tarnovitski freund, yael weiss and gilad kaplan for their considerable help in putting this article together, their thoughtful reading of the drafts, their critical constructive comments and most of all, for making my lab a fun place to come to each morning-toda. jonathan m. gershoni is the incumbent of the david furman chair in immunobiology of cancer. key: cord-297857-ybqj8z1r authors: petagna, l.; antonelli, a.; ganini, c.; bellato, v.; campanelli, m.; divizia, a.; efrati, c.; franceschilli, m.; guida, a. m.; ingallinella, s.; montagnese, f.; sensi, b.; siragusa, l.; sica, g. s. title: pathophysiology of crohn’s disease inflammation and recurrence date: 2020-11-07 journal: biol direct doi: 10.1186/s13062-020-00280-5 sha: doc_id: 297857 cord_uid: ybqj8z1r chron’s disease is a chronic inflammatory intestinal disease, first described at the beginning of the last century. the disease is characterized by the alternation of periods of flares and remissions influenced by a complex pathogenesis in which inflammation plays a key role. crohn’s disease evolution is mediated by a complex alteration of the inflammatory response which is characterized by alterations of the innate immunity of the intestinal mucosa barrier together with a remodeling of the extracellular matrix through the expression of metalloproteins and increased adhesion molecules expression, such as maccam-1. this reshaped microenvironment enhances leucocytes migration in the sites of inflammation, promoting a t(h)1 response, through the production of cytokines such as il-12 and tnf-α. il-12 itself and il-23 have been targeted for the medical treatment of cd. giving the limited success of medical therapies, the treatment of the disease is invariably surgical. this review will highlight the role of inflammation in cd and describe the surgical approaches for the prevention of the almost inevitable recurrence. crohn's disease (cd) is a chronic inflammatory intestinal disease, first described as regional ileitis by crohn, ginzburg and oppenheimer in a case series presented at american medical association annual meeting in 1932 [1] . cd inflammation interests the whole intestine, being the most frequently affected part the distal ileum. patients with cd experience periods of flares and periods of remissions during their disease course. pathogenesis results from the interactions of environmental factors, immune system, susceptibility genes and host's microbiome changes, leading to disruption of the intestinal mucosa. the role of inflammatory cells in maintaining an active disease is well known and most of the therapies aim to stop the cascade of inflammatory and proinflammatory cytokines. treatment of cd is multidisciplinary: medical treatment is focused on mucosal healing and symptoms reduction; surgery maintains a key-role in treating complications such as stenosis, perforations, fistulas and abscesses. surgical recurrence is known to afflict over 80% of the operated patients [2, 3] . multiple surgical strategies have been investigated to improve outcome. introduction of laparoscopic techniques has permitted several improvements but failed in reducing recurrences; other surgical techniques are currently under evaluation in view to retard or prevent the ineluctable recurrence but the surgical cure for cd is yet to be discovered. this review is focused on cd inflammation and will discuss potential strategies to prevent recurrences, such as novel surgical approaches. crohn's disease pathogenesis is based on tissue inflammation, caused by an unrestrainable immune response against luminal bacterial antigens (fig. 1) . immune cells like cd4 t-cells, cd8 t-cells, b-cells, cd14 monocytes and natural killers, are involved in this process as they infiltrate the gut of cd patients. part of the immune-mediated susceptivity to cd resides in some innate mechanisms of defense form infectious diseases and the intestinal mucus secretion is part of those. it has been shown that variants of the muc2 gene reducing mucus production are associated to cd in a mouse model. moreover, molecules that are mediating bacterial adhesion have been correlated to the disease. this is the case of fut2, which encodes for the fucosyltransferase enzyme, responsible for the secretion of soluble forms of the abo antigens. people harboring a fut2 variants decreasing the secretion of the antigens, have an altered interaction with bacteria and are more prone to developing cd [4] . the pathogenesis is also sustained by the interaction of these cells with integrins, adhesion molecules and multiple chemokines, responsible for the production of elevated levels of inflammatory cytokines, representing the target of immune and non-immune cells and the promotion of mucosal inflammation. as such, among many adhesion molecules, some evidences on the involvement of the leucocyte maccam-1, receptor for the α4β4 integrin, seems to play a crucial role. together with leucocyte adhesion, the role of the extracellular matrix on their activation has been explored. proteins like cd44 and cd26 where shown to play a role as well as metalloproteins (mmp), being mmp1 and mmp3 abundant in the granular tissue close to cd sites of inflammation, therefore responsible for leucocytes activation [5] [6] [7] [8] . in the mucosa of cd patients, a dysregulation of various components of the immune system is invariably found. the most pronounced alteration is the hyperactivity of t cells with excessive production of cytokines, between which il-12 and ifn-γ, promoting a t h 1 lymphocytic phenotype, opposed to the t h 2 one, correlating to ulcerative colitis. moreover, tnf-α production has also been demonstrated to increase the number of cd4+ foxp3+ t reg cells, especially in the mucosa of children affected by cd [9] . the inhibition of fig. 1 immuno-mediated pathogenesis of crohn's disease. crohn's disease is a multifactorial pathology in which a major role is played by alterations at the level of immunity and inflammation. innate immunity is involved in terms of defects in the mucous barrier (mut2 and fut2 genes) while adaptive immunity relies on a t h 1 lymphocitic response and t reg cells mediated by cytokines like tnf-α, il-12, il-34 and il-23. the increased migration to the sites of inflammation is also determined by a reshaping of the extra cellular matrix through the action of metalloproteins (mmp-1 and mmp-3) and the overexpression of adhesion molecules such as maccam-1 and integrin α4β4. finally, also the host pathogen interaction between the intestinal epithelium and the microbiota has been linked to the evolution of the disease. picture created with biorender.com the effector cytokines, like tnf-α, attenuates the detrimental effects in subsets of cd patients. furthermore, the expression of the interleukins, a subgroup of cytokines implicated in the enhanced or inhibition of other cytokines in many different regulatory pathways such as maturation, growth and responsiveness of immune cells population, is to be considered anomalous in cd patients [10] . further analysis of t cell subsets has revealed the presence of t h 1 and t h 17 cells in cd, whereas the cytokines considered more involved are tnf, il12 and il23. apart from the cited cytokines, il-34 has also been associated to ibd and cd in particular. il34 expression is more pronounced in the areas of active inflammation, especially in cd, and seems to induce tnf-α and il6 expression through a erk-mediated mechanism. moreover, il-34 has been described as an inducer of ccl20 through the interaction with its receptor the m-csfr1, abundantly expressed in the inflamed colonic epithelium but not in the healthy controls. on the contrary, il-25 inversely correlates to the inflammatory state of the patients with ibd, being reduced in cd patients as opposing to healthy subject, and being reduced in the affected areas of the colon if compared to the surrounding adjacent normal tissue from the same subject. among all the possible interleukins associated to cd pathogenesis, il-12 and il-23 represent the target of still inadequate therapies because of potential side effects, such as increased risk for infection, and the blockade of specific immunological targets, capable of induction of alternative signaling or homing pathways. the latter mechanism may also partially explain the frequent lack of response to therapy with biologics such as infliximab (remicade©), a chimeric monoclonal antibody used to treat autoimmune diseases, that works by binding to tnf-α causing the reduction of il-34 expression, implicated in monocyte and macrophage differentiation, survival and function [11] [12] [13] [14] [15] . although t-cells are the main effector lymphocytes in intestinal tissue inflammation, also humoral immune system plays a crucial role. plasma cells differentiation indeed is promoted by cd4 t-cells, through a mechanism that is firmly dependent on il-2, overproduced in cd patient's gut. il-21 converts naive b-cells into b-cells expressing granzyme-b: it possesses a cytotoxic activity on the intestinal mucosa and perpetuates the epithelial damage. these proofs indicate that an altered balance between effector and counter regulatory factors is probably involved in the sustainment of the tissuedamaging immune response in cd [16] . gut microbiota also plays a recognized role in designing the inflammatory response in ibd and especially in cd. there is growing evidences that some microbial among them, anti-inflammatory drugs as mesalazine, antibiotics such as fluorochinolones and metronidazole and immunosuppressants (methotrexate). more targeted treatment options are directed towards tnf-α (infliximab, adalimumab, certolizumab) or against integrins (vedolimumab) and interleukins il-12 and il-23 (ustekinumab). picture created with biorender.com gene products can influence gene expressions in the host [17] [18] [19] . the complex network arising from this assumption is referred to as the microbial-associated molecular pattern (mamp) which is sensed by toll-like receptors on immune cells, contributing to their activation in the context of the chronic inflammation [20] . microbiome moreover represent a source of potential pathogenic inputs that can be approached through the methods used in the omics era, such as metagenomics studies, also impacting on our knowledge on geographical variations on the clinical manifestation of the disease [21] [22] [23] [24] . the inflammation is generally transmural and, on pathology examination, granulomas may be identified on biopsies, with a discontinuous distribution along the longitudinal axis. this inflammatory process often leads to irreversible tissue damage in the form of intestinal stenosis or fistulas, inflammatory masses or intra-abdominal abscesses. patients can develop one or more of these disease behavior and they often tend to evolve from inflammatory to penetrating or stricturing disease [25] . medical management (fig. 2) should be tailored based on various factors such as disease severity, subtype, behavior and location [26] . moreover, it is important to consider other factors such as age at diagnosis, extension of the lesions and extra-intestinal manifestations [27] . as matter of fact, none of the drugs used in the treatment of cd has been demonstrated to be curative or completely safe. mesalazine, which belong to the 5-asa compounds category, has been evaluated in many studies and it has never shown to definitely induce or maintain remission in cd. its benefits are related to its safety outline [28] . antibiotics are principally recommended to treat septic complications; like mesalazine, they do not show a real efficacy in the treatment of cd, except in the shortterm treatment of perianal fistula in association with anti-tnf. most frequently used agents are fluoroquinolone and metronidazole [29] . systemic corticosteroids show a fast onset of action and are indicated to induce remission. unfortunately, steroid dependency or steroid resistance can jeopardize their use that is often accompanied to a wide range of side effects like obesity, hypertension, glaucoma, cataracts and adrenal insufficiency [30] . another major part is played by immunosuppressant like thiopurines and methotrexate: thiopurines are used to maintain remission in moderate cd, usually in combination with steroids. before starting thiopurines treatment, it is mandatory to assess tpmt (thiopurine s-methyltransferase) activity, crucial for their metabolism [31] . methotrexate may be considered as an option for steroid-dependent patients. included in the side effects are hepatotoxicity and more rarely myelosuppression; they are prohibited during pregnancy because teratogenic and abortifacient [32] . anti-tnf drugs are considered the most powerful tools to treat moderate and severe form of cd, alone or in association with immunomodulators to obtain and maintain remission. the most frequently used anti-tnf are: infliximab (remicade©), a chimeric antibody that is administered intravenously; adalimumab (humira©), a fully humanized monoclonal antibody administered subcutaneously; certolizumab (cimzia©), a fab antibody fragment of humanized anti-tnf molecule [33] . more recently super-selective target monoclonal antibodies have been developed, directed against a specific pattern of inflammation. in this class there are vedolizumab (entyvio©), that targets the adhesion molecular inhibiting leukocyte migration [34] , and interleukininhibitors like ustekinumab (stelara©), a fully humanized monoclonal antibody targeting the p-40 subunit of il-12 and il-23 [35] . when cd was described for the first time, therapy was exclusively surgical. since the beginning of cd surgery experience, there was no consensus on the optimal procedure. at the mount sinai hospital in new york, dr. berg was the surgeon who operated fourteen patients presented by crohn. the "berg" operation, also known as "mount sinai" operation, implied exclusion bypass of the ileocecal region, transecting small bowel proximal to the diseased ileum, over sewing distal ileum, and anastomosing the proximal ileal end into the mid-transverse colon [36, 37] . in fact, this was a staged management, being the second planned step the resection of the diseased bowel. performing more and more cases, mount sinai surgeons noted that during the second stage of surgery, the bypassed bowel seemed to have "healed" in several cases. starting from this observation and as patients manifested clinical improvements, they decided to omit the planned second procedure. obviously, a great debate about this procedure and all early and late risks linked to it arose: blow out of the blind end, reactivation of cd in the excluded segment with abdominal pain and infections, deprivation of a large portion of the colon for water absorption. eventually, bypass procedure was abandoned due to findings of adenocarcinoma occurring in the excluded segment [19, [38] [39] [40] . surgical resection of the diseased bowel emerged as the procedure of choice for most patients with cd of the terminal ileum or with ileo-colitis, including complicated cases [41] . in tandem, advances in perioperative care, such as nutritional improvement, anesthesia and fluid and electrolyte management, guaranteed cd surgery improvements and safety. at this point physician focused on the amount of resection. in fact, it was commonly accepted practice for surgeons to resect all macroscopically involved intestine with large resection margins. consciousness of cd as a pan-enteric affection and, above all, the evidence of the inevitable recurrence and possible development of short bowel syndrome due to repetitive surgery, suggested the adoption of a conservative policy, avoiding wide resections [42] . for this reason, in order to avoid short bowel syndrome -in particular in those scenarios characterized by extensive jejunal-ileitis with fibrotic stenosing segments scattered along the diseased intestine -lee from oxford, in 198, reported another advancement in surgical management. lee was inspired by the work of indian surgeons on the management of tuberculous strictures: they observed that small bowel preservation could be achieved in patients with multiple tuberculous strictures by strictureplasty [43] . lee applied strictureplasty to the short intestinal strictures of crohn's disease and from that moment, with a great variability on techniques according to the different situations, strictureplasty assumed a fundamental role for cd surgeons [44, 45] . different strictureplasty techniques have been described. heineke-mikulicz strictureplasty consists of a longitudinal enterotomy closed in a transverse direction and it is best applied to stricture up to 7 cm in length [46] . finney strictureplasty is used for longer stenosis, up to 10-20 cm: after an antimesenteric longitudinal incision, the opened bowel segment is bent into a u shape and posterior and anterior layers are close with continuous absorbable suture [47] . michelassi strictureplasty is indicated for the treatment of multiple strictures, interesting up to 90 cm long bowel segment; in this case, a segment of diseased bowel is anastomosed to a nonaffected segment of intestine [48] . interestingly this technique has shown to induce remission in the diseased part. the mechanism is still unknown, however there seems to be a process in cd whereby obstruction is responsible for the pathogenesis of many complications [49] . this technique allows the mitigation of fecal stasis, which may play a central role in postoperative mucosal healing, modifying the microbial-mucosal interaction. possibly, the resolution of chronic obstruction may interrupt the cascade of events causing active disease [50] [51] [52] . another cornerstone in cd surgery was represented by the advent of minimally invasive surgery. feasibility and safety of laparoscopic ileo-cecal resection has been assessed and it was found not inferior in terms of outcomes when compared to open surgery [53] [54] [55] . nowadays, laparoscopy is largely accepted as the first line approach for cd, in the presence of adequate expertise [56] . laparoscopy demonstrated advantages in terms of cosmetics and postoperative recovery and assured some long-term advantages, including fewer incisional hernias, fewer adhesions and a significant impact on female fertility [57, 58] ; unfortunately, no clear differences on time to recurrence was found. from a surgical point of view cd recurrence should be considered as an inevitable consequence. the same factors that underline the pathogenesis of cd at its first stages are thought to be responsible for post-operative recurrence (por) setting, being the result of interplay of microbial, environmental, immunological and genetic variables [59] . within this contest, microbial flora role seems to be linked to the fecal stream, as demonstrated by rutgeerts et al. [60, 61] investigating the rapid recurrence of microscopic inflammation in the mucosa of excluded ileum when newly interested by fecal content. the term post-operative recurrence is used to define the appearance of new lesions after bowel resection. active surveillance for an early diagnosis is considered mandatory. rutgeerts endoscopic index is possibly the most widely used scoring system to detect recurrent lesions [62, 63] . previous studies demonstrated that the lesions are located more often in or near the area of anastomosis, usually reproducing the same initial pattern of the disease, though it has been suggested that postresection lesions should be considered new. the presence of microscopic lesion, detected during endoscopic examinations 1 year after surgery, reinforces their role as precursors of por. timing of endoscopic surveillance has been discussed taking into account available evidence; recommendation is to perform endoscopic examination after 6 months from surgery or within the first year [64, 65] . other techniques have been investigated to assess por [66] ; in particular, there is a great interest in non-invasive techniques such as ultrasonography (us). among several emerging us technique, small intestine contrast ultrasonography (sicus) resulted more sensitive in detecting small bowel lesions in cd patients [67] . sicus has been demonstrated to be comparable to ileocolonscopy, also after surgery, allowing the visualization of extra luminal lesions related to cd (bowel wall thickness, mesenteric and lymph nodes enlargement). hence, in expert hands, sicus could be considered a valid alternative for the follow-up and early diagnosis of por after surgery in cd [68] [69] [70] [71] . other recent interesting fields of investigation focus on the role of anastomosis configuration and the mesentery function in the pathogenesis of por. for what concern anastomosis, a great debate was raised regard which technique should be considered optimal, in particular between the more frequent choices: side-to-side versus end-to-end configuration and mechanic stapled versus hand sewn. in 2014 he [72] carried out a meta-analysis to compare stapled sideto-side anastomosis (sssa) and hand sewn end-to-end anastomosis (heea) in terms of postoperative early and late complications and por after ileo-colic resection for cd. the conclusion was that sssa should be preferred because of its larger luminal diameter, thus showing lower overall incidence of complications including anastomotic leak, lower recurrence and re-operation for recurrence. in 2018, feng [73] carried out a similar meta-analysis, looking specifically at the orientation of the anastomosis. feng concluded that sssa isoperistaltic is probably the optimal anastomosis because it can significantly reduce incidence of overall postoperative complications and clinical por. the underlying idea is that, with its wide lumen configuration, sssa isoperistaltic reduces recurrence by preventing early stenosis, colonic reflux and secondary ischemia. in 2018 gajendran [74] published his series, supporting the superiority of heea when compared to anti-peristaltic sssa. he also looked into the impact on quality of life and inflammatory activity. gajendran developed an experimental animal model to provide a mechanistic explanation for his clinical findings, showing that antiperistaltic orientation alters anatomy and physiology, creating an anti-peristaltic reservoir, which causes dysmotility and alteration in contractility. in particular, the animal model showed that this is due to the perpendicular surgical trans-section of the intestinal circular muscle layers: disruption of motility seems to lead to significant structural and functional changes with local stasis of enteric contents and local distension at the anastomotic site. gajendran concluded that, according to his data, the restoration of physiologic intestinal function with surgical reconstruction of the bowel as an intact tube could contribute to a better outcome in cd patients. in the same year, aaltonen's group [75] published its series regarding risk factors for anastomotic recurrence. aaltonen's et al. proposed a technical variant heea, adding an opening of the small bowel's anti-mesenteric border to ensure enough wide bowel lumen, describing this modified technique as a safe choice for ileo-colonic resection. current guidelines from the american society of colon and rectal surgeons [76] , states that anastomosis can be constructed as deemed most appropriate by the surgeon. ecco guidelines [53] more recently, seems to favor sssa, taking into account he and feng's meta-analysis [72, 73] and stressing the concept that a wider anastomosis will have a lower rate of clinical and surgical recurrence. within this context, toru kono developed a new anastomotic technique. the first kono-s anastomosis' (ksa) work was published in 2011 [77] . ksa is an antimesenteric functional end-to-end hand sewn anastomosis, configured so that the mesentery side is located in the center of the stump. both stumps are sutured to create a "supporting column" to maintain the diameter and dimension of the anastomosis, preventing distortion and stenosis associated with recurrent disease at the anastomotic side, especially on the mesenteric side, which represents a locus minoris resistentiae and so a typical recurrence location. in 2012 fichera [78] highlighted ksa innovations: the theoretical advantages of the complete exclusion of the mesentery, the initial site of cd por, with a true antimesenteric anastomosis; lower susceptibility to mechanical distortions due to the stability provided by the "supporting column"; better preservation of blood supply and innervation, achieved by dividing the mesentery close to the bowel. moreover, in 2015, katsuno published his series confirming safety, feasibility and good results and highlighted the easier endoscopic access to the ksa [79] . results from the first international multicenter study [80] , leaded by kono himself, were available in 2015 and the authors suggested resection and ksa for cd patients who are not candidates for anti-tnf therapy due to adverse effects, loss of efficacy or financial reasons. in 2018, seyfried [81] and shimada [82] published two more series and in 2020, during the ecco congress in vienna, luglio presented the results of "the supreme-cd study" [83] , the first randomized clinical trial comparing ksa and sssa in terms of endoscopic and surgical recurrence, confirming a reduction in por when ksa is performed. multicenter trials are still needed to further confirm these preliminary results. indeed, the interest on anastomotic configuration is still open and recently celentano developed a model for a v-modified side-to-side, antimesenteric, iso-peristaltic anastomosis in which a strictureplasty is added to the inlet and the outlet of the anastomosis. celentano's configuration target is the widening of the lumen of the bowel in these two critical areas, with the aim of minimizing the risk of clinical and surgical anastomotic recurrence; this is just a model ex vivo but representative of the wide interest in this field [84] . the role of the mesentery could be considered the other trending topic of the last decade. the underlying idea is that the mesentery plays a prominent role in cd pathogenesis and in recurrences. mesentery is seen as an independent organ, interposed between the intestine and the body. from its privileged location, mesentery is responsible for the conduction of local intestinal and systemic response: it is the reservoir of various cell types, in particular inflammatory ones, contained in lymph nodes and related mediators [85] . moreover, cd mesentery shows the pathognomonic phenomenon known as fat wrapping or creeping fat [86] , consisting of a peculiar form of adipose tissue hypertrophy. creeping fat is characterized by small adipocytes, increased in number with a specific gene expression profile, accompanied to immune cell infiltration, comprising regulatory m2 macrophages and t-cells [87] . guedj et al. [88] , thanks to in vitro experiments on resection specimens of ileum from patients operated for cd, proposed a mechanism in which mesenteric adipocyte, through their production of key chemokines in response to inflammatory/bacterial stimuli, orchestrate an immune response in cd-affected mesentery. between 2015 and 2016, li published two different papers regarding this topic. in the first one [89] he focused on the contribution of the mesenteric adipose tissue, measuring the fat visceral areaassimilated to the creeping fat phenomenonin ct scan performed before surgery; in this retrospective study also subcutaneous fat area and mesenteric fat index, defined as the ratio of visceral and subcutaneous fat were considered. as result, high fat visceral area was found to be and independent predictor of early clinical recurrence of cd por. in the second paper [89] , li highlighted the contribution of mesenteric nerves, vessels, lymphatics and fat mass, concluding that all these structures play a crucial role in cd pathogenesis and disease progression. he provided the basis for the copernican revolution in cd pathogenesis, querying the current dominant theory in cd, based on the unidirectional, "outside-in" axis of dysbiosis, innate immunity-adaptive immunity-mesenterybody system. emerging clinical evidence strongly suggest that the axis is bidirectional, involving also all the cited mesenteric structures, and not only endoluminal agents as already cited in the pathogenesis section. coffey and rivera [90, 91] gave other further hints to remove the veil of maya on the pathogenesis and the mesenteric role. they started from the assumption that topographic distribution of crohn's disease along the intestinal tract may have a bodily mesenteric basis in terms of tissue volume and thickness. as a proof, they noted that the largest mesenteric region is the ileocolic region, which happens to be also the commonest localization of cd. in 2018 mao [92] gave more insights on how creeping fat influences stricture formation in cd; he took into account mechanisms involved in the microenvironment at interfaces of different tissue compartments, such as creeping fat, considered as an extension of mesenteric fat beginning at the intestinal hilum, and the intestine muscularis mucosa itself. as already emphasized, in creeping fat immune and nonimmune cell types are represented and increased in number, producing mediators responsible for intestinal stricture formation. among these cells, fatmesenchymal cells seem to play a pivotal role because their interactions appear to be important in tissue remodeling in multiple organs, including the intestine. hence, creeping fat abandons the old role of innocent bystander and is acknowledged as an active participant in inflammation and immunity. mao hypothesized also that stricture formation incidence has remained unchanged because no target-therapy is available and so cells involved in this process should represent a pharmacological target. therefore, the awareness of the central mesentery's role provides a copernican revolution also in terms of clinical target. in fact, from this point of view, mesentery could be considered as an anatomical sacrarium: there are no valid pharmacotherapeutic modalities designed specifically to manipulate it. at present, the only means of targeting the mesentery are surgical, so there is the need for development of new strategies in this field. even though conservative approach to intestinal resection in cd could be considered an established dogma, in the light of the recent discovered role of mesentery, the attitude is changing. up to the present, mesentery resection has not become standard practice. this is mainly due to technical concerns regarding risk of bleeding when manipulating tissues with significant inflammation, disease-related perforation, fistula formation, adhesions, and thickened mesentery, potentially leading to complications like hemorrhage, hematomas and other injuries. overcoming these issues and starting from the previous considerations, coffey et al. [93] published a series on the inclusion of mesentery in ileocolic resection for cd. clinical findings were coherent with formulated theories: inclusion of the mesentery as part of intestinal resection is associated with reduced por, that means improved clinical outcomes, and advanced mesenteric disease resulted to be a predictor of increased risk of por. moreover de groof et al. presented a series on proctectomy in cd, demonstrated that perineal complications were more frequent after close rectal dissection than after total mesorectal excision [94] . these results suggested a pathogenic role for the mesorectal tissue in cd. in the footsteps of these assumptions, an international, multicenter, randomized controlled trial [95] is ongoing about the mesenteric excision surgery versus conservative limited resection in cd. moreover, our group is running the panacea study (pathophysiological, nodal-based approach for crohn's disease excision), a pilot yet unpublished study, based on the belief that the majority of t-cells -especially memory t-cellslies in lymph nodes [96] [97] [98] [99] . our hypothesis is that mesenteric resection, including lymph nodes, should free the organism from a great number of cells involved in intestinal inflammation. results from this study altogether with the others, will contribute to understand which of the proposed approach will be valid in reducing por. crohn's disease has been seen, in the last two decades, as a multifactorial inflammatory disease. much is known in terms of its pathogenesis from a molecular point of view from the involvement of the mucosal mucinous barrier and the role played by variants of the mut2 or the fut2 genes, which alters the barrier interaction with both pathogens and harmful substances, to the complex mechanisms involving aberrant expression of adhesion molecules. the leucocytic maccam-1 is a mediator of integrin dependent adhesion, which is part of the mechanism leading to migration of leucocytes in cd inflamed region. on the other front, the migration of inflammatory cells is also mediated by the alteration of the extracellular matrix, often mediated by mmp proteins, being mmp-1 and mmp-3 the most abundant in this clinical context. the complex milieu that is created by the interaction of the inflammatory cells with the intestinal epithelium is sustained by a complex network of cytokines and chemokines, which direct t lymphocytes towards a t h 1 response, mediated by the expression of the foxp3 transcription factor in the t reg population. among the cytokines involved in cd pathogenesis, il-34 and il-25 seem to play opposing roles, the first increased in the injured tissue, the second one diminished. other cytokines such as il-12 and il-23 have been demonstrated paying a key role in the inflammatory response and are some of the few current medical therapeutic targets for cd. anyhow, considering that a consistent part of cd therapeutic approaches remains surgical. we reviewed emerging 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student-driven research network in surgery publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. lorenzo petagna, amedeo antonelli, carlo ganini and giuseppe sica, wrote the manuscript and approved the final version. marzia franceschilli, andrea guida, sara ingallinella, bruno sensi, leandro siragusa, michela campanelli, vittoria bellato, andrea divizia, did the systematic bibliography research and contributed equally in retrieving data and analysing results. they all approved the final manuscript. cesare efrati and fabrizio montagnese, gave their clinical gastroenterology expertise, adding valuable material to the manuscript. they both critically review the whole text and approved the final version of the manuscript. the european society degenerative disease supported the publication of this review.availability of data and materials not applicable.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.author details 1 key: cord-310942-191m0e65 authors: boga, jose antonio; coto‐montes, ana; rosales‐corral, sergio a.; tan, dun‐xian; reiter, russel j. title: beneficial actions of melatonin in the management of viral infections: a new use for this “molecular handyman”? date: 2012-04-18 journal: rev med virol doi: 10.1002/rmv.1714 sha: doc_id: 310942 cord_uid: 191m0e65 melatonin (n‐acetyl‐5‐methoxytryptamine) is a multifunctional signaling molecule that has a variety of important functions. numerous clinical trials have examined the therapeutic usefulness of melatonin in different fields of medicine. clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging). the beneficial effects of melatonin can be explained by its properties as a potent antioxidant and antioxidant enzyme inducer, a regulator of apoptosis and a stimulator of immune functions. these effects support the use of melatonin in viral infections, which are often associated with inflammatory injury and increases in oxidative stress. in fact, melatonin has been used recently to treat several viral infections, which are summarized in this review. the role of melatonin in infections is also discussed herein. copyright © 2012 john wiley & sons, ltd. the methoxyindole melatonin (n-acetyl-5-methoxytryptamine) is a secretory product of the pineal gland. it was first reported as a skin lightening agent in amphibians [1, 2] . further investigations showed that another function, supported by its direct effects in regions containing high densities of melatonin receptors, such as the circadian pacemaker (the suprachiasmatic nucleus) and the pars tuberalis, is to regulate and reset circadian rhythms as well as to be involved in the measurement of day length, an environmental variable used for seasonal timing of reproduction, metabolism and behavior in species responding to photoperiodic changes [3] [4] [5] [6] [7] . in recent decades, melatonin has been reported to possess numerous additional functions and act in neural and non-neural tissues or cells that express melatonin receptors that are at lower densities than in the suprachiasmatic nucleus. thus, melatonin is involved in sleep initiation, vasomotor control, anti-excitatory actions, immunomodulation including possessing anti-inflammatory properties, antioxidant actions, and actions on energy metabolism, influences on mitochondrial electron flux, regulation of the mitochondrial permeability transition pore (mtptp), and mitochondrial protection against free radicals [8] [9] [10] [11] [12] [13] . deficiencies in melatonin production or melatonin receptor expression and decreases in melatonin levels (such as those that occur during aging) are likely to contribute to numerous dysfunctions [14] [15] [16] . in fact, several clinical trials have shown that melatonin is efficient in preventing cell damage under acute (sepsis, asphyxia in newborns) and chronic states (metabolic and neurodegenerative diseases, cancer, inflammation, aging) [17] [18] [19] [20] [21] [22] . in humans, the efficacy of melatonin as a treatment of ocular diseases, cardiovascular diseases, sleep disturbances and several other pathologies, as well as a complementary treatment in anesthesia, haemodialysis, in vitro fertilization and neonatal care, has been assessed and reported to be beneficial [23] . likewise, melatonin reduces the toxicity and increases the efficacy of a large number of drugs whose side effects are well documented [24] . the beneficial effects of melatonin are explained by its properties as a potent antioxidant, a modulator of apoptosis and a positive regulator of immune functions [25] [26] [27] [28] [29] . these actions suggest the potential to treat viral infections, which usually cause inflammatory injury and elevated oxidative stress [30, 31] . a number of reports examining the ability of melatonin to protect against viral infections have been published, as summarized in the following section. encephalomyocarditis virus (emcv) is a highly pathogenic and aggressive virus that causes encephalitis and myocarditis in rodents. administration of melatonin prevented paralysis and death of mice infected with sublethal doses of emcv [32] . melatonin also has a protective effect in mice infected with semliki forest virus (sfv), a classic encephalitis arbovirus, that invades the cns and whose replication in the mouse brain eventually leads to death. melatonin administration not only reduced the death rate but also significantly postponed the onset of the disease. furthermore, the level of virus in the blood in melatonin-treated mice was lower than in non-treated mice [33] . although attenuated west nile virus (wnv) strain wn-25 is an encephalitis virus that does not invade the brain and does not normally cause encephalitis, exposure of mice to various stressful stimuli induces wn-25 encephalitis. melatonin counteracts the immunodepressive effect of stress exposure and prevents the stress-related encephalitis and death of wn-25 infected mice [33] . venezuelan equine encephalomyelitis (vee) is an important human and equine disease caused by vee virus (veev), a mosquito-borne organism. outbreaks have occurred in northern south america from the 1920s to the 1970s with thousands of people and horses, donkeys and related species being infected. mice have been used as an animal model for this condition, because veev-infected mice show excitation and hypermotility followed by hypomotility, paralysis, coma and death. melatonin administration protects mice infected with veev by decreasing the virus load in brain and serum, reducing mortality rates, delaying the onset of the disease and deferring the time to death. furthermore, in surviving mice treated with melatonin, the veev-mediated igm antibody titres are highly elevated [34] . aleutian mink disease is a natural condition caused by persistent infection with the aleutian mink disease virus (amdv). animals in the progressive state of the disease show a marked hypergammaglobulinemia, because of high titers of non-neutralizing admv antibodies. this is thought to cause lesions in the kidney, liver, lungs and arteries. melatonin implants reduced mortality in admv-infected mink [35] . the findings in these reports document the ability of the melatonin to protect against viral infections [ table 1 ]. the potential protective mechanisms include melatonin acting as a free radical scavenger, an antioxidant enzyme inducer, a positive regulator of immune functions and an inhibitor of inflammation, as well as a regulator of programmed cell death (pcd) [ table 2 ]. free radicals are molecules formed naturally during many metabolic processes. they contain an unpaired electron in their valence orbital that makes them unstable and reactive. these reactive agents damage essential molecules in cells including lipids, proteins and dna [36, 37] . among these reactants, the superoxide anion radical (o 2 • à ), nitric oxide (no•) and especially their derivatives, the hydroxyl radical (•oh) and peroxynitrite (onoo à ), are highly biologically damaging elements produced in the host during microbial infections [38] [39] [40] [41] . phagocytes, such as neutrophils and macrophages are assumed to be the major generators of free radicals. elevated levels of o 2 • à are although ifn-g is the major cytokine inducing inos and no• overproduction in the pathogenesis of these viral infections, inos expression is downregulated by il-4, il-10 and transforming growth factor-b (tgf-b) [60] [61] [62] . ifn-g is known to be associated with type 1 helper t cell (th1) responses, and il-4 and il-10 are induced by type 2 helper t cell (th2) responses; no• biosynthesis catalyzed by inos is precisely regulated by a polarized th1-th2 balance. in other viral diseases, viral replication or viral components directly induce inos without mediation by pro-inflammatory cytokines. thus, the hiv envelope glycoprotein gp41 triggers inos expression in human astrocytes and murine cortical brain cells in culture [63, 64] . rsv directly upregulates inos in human type 2 alveolar epithelial cells (a549 cells) [65] . free radicals are produced to eliminate the pathogenic agent or to kill the virus-infected cells by a non-specific response. thus, antiviral effects of no• have been described for some dna viruses such as murine poxvirus (ectromelia virus) and herpes viruses including hsv, ebv and some rna viruses such as coxsackie virus [66] [67] [68] [69] [70] [71] . the toxic oxygen and nitrogen-based reactants, unfortunately, cannot discriminate between exogenous invading pathogens and the host cells themselves, and therefore, they also damage the host. to minimize such self-damage during the elimination of pathogens, the host employs several primitive tactics; it uses recruited phagocytes for the physical containment of pathogens in infectious foci. most bacteria, for example, can be phagocytosed and confined to septic foci, which are typically abscesses or granulomas. under these conditions, free radicals can affect bacteria rather selectively with the surrounding normal tissue remaining mostly intact. in viral infections, in contrast, free radical mediators cause non-specific oxidative/nitrosative damage in virus-infected tissue and produce oxidative stress; this occurs when the virus cannot be confined to limited areas by the non-specific host defense [56, 58, 72] . thus, no• has appreciable antiviral actions on several types of viruses including ortho-and paramyxovirus, murine vaccinia virus, coronavirus (mouse hepatitis virus), lymphocytic choriomeningitis virus, murine emcv, tickborn encephalitis virus (tbe-v) [73] [74] [75] [76] [77] [78] ; also, no• and its derivatives, especially onoo -, can be considered pathogenic in some viral infections. indeed, no• inhibition or lack of no• generation reduces the pathological consequences of viral pneumonia in mice caused by influenza virus, sev and hsv-1, hsv-1-induced encephalitis in rats, emcv-induced carditis and diabetes, and murine encephalitis induced by flavivirus (murray valley encephalitis virus, tbe-v) [55, 57, 74, [78] [79] [80] [81] [82] . a similar pathogenicity with a lack of antiviral effects has been observed for o 2 •in several experimental models of virus-induced pneumonia including those caused by influenza virus and cmv [43] [44] [45] 56, 72, 83, 84] . hcv-induced oxidative stress is emerging as a key step and a major initiator in the development and the progression of liver damage [85] . ns3, one of the non-structural proteins of hcv, was reported to induce reactive oxygen species by nadph oxidase in neutrophils [86] . high-risk human papilloma virus (hpv), which causes cervical cancer, promotes inos-dependent dna damage, leading to dysplastic changes and carcinogenesis [87] . epstein-barr virus is a herpes virus that infects the majority of the world population, generally during childhood; it has been linked to the genesis of a number of lymphoproliferative diseases and neoplasia such as the african burkitt lymphoma, nasopharyngeal carcinoma or gastric carcinoma. early stages of ebv infection generate oxidative stress either in b lymphocytes or in epithelial cells, so contributing to pathology [88] . influenza a virus causes a respiratory disease, which ranges from mild upper respiratory tract illness with or without fever to severe complications such as pneumonia. the latter disease results in respiratory failure, acute respiratory distress syndrome, multi-organ failure and even death. an abrupt increase in o 2 • à production occurs during phagocytosis, which induces injury in non-infected cells. these o 2 • à -mediated pathways contribute to a portion of the extensive tissue injury observed during severe influenza-associated complications [56] . to protect themselves against free radicalmediated damage, cells have developed an antioxidant defense that includes enzymatic and non-enzymatic mechanisms. free radical generation and a functionally efficient antioxidant defense system must be in equilibrium to avoid cellular damage caused by radicals and their derivatives. enzymes involved in the elimination of free radicals include the superoxide dismutases (sod), catalase (cat) and glutathione peroxidase (gpx). in addition to the enzymatic antioxidant system, organisms possess non-enzymatic free radical scavengers, which directly remove toxic reactants because of their electron donating ability. the best known nonenzymatic antioxidants are vitamin e (a-tocopherol), vitamin c (ascorbate), glutathione (gsh), b-carotene and, as recently described, melatonin [25] . several radical scavengers have been efficacious in ameliorating the severity of viral diseases. n-acetylcysteine, a gsh precursor, inhibits hiv in vitro [89] as did the natural thiol antioxidant, alpha-lipoic acid [90] . glutathione administration to hiv seropositive individuals by aerosol treatment can correct the glutathione deficiency [91] . the combination of several antioxidants with antiviral drugs synergistically reduces the lethal effects of influenza virus infections [92] . thus, any agent that functions as a direct radical scavenger and also stimulates antioxidative enzymes could have utility in the treatment of patients with severe complications of viral infections. melatonin is a powerful and effective •oh scavenger, which provides protection against oxidative damage of cell components. it also scavenges the peroxyl radical to a lesser degree generated during lipid peroxidation with an activity that, in some situations, is reportedly greater than that of vitamin e [22, [93] [94] [95] [96] . also, melatonin directly detoxifies the onoo à and possibly peroxynitrous acid (onooh) [97] . in vivo, melatonin stimulates several antioxidative enzymes including gpx, cat and sod, thereby potentiating its antioxidant properties [98] [99] [100] [101] . melatonin can cross anatomical barriers, including the placenta and the blood-brain barrier [102, 103] , and easily enter cells [104] . splenocytes infected with veev generated less of no•, when treated with melatonin; this finding suggests that the indoleamine protected mice infected with the veev by a mechanism involving a reduction in no• concentrations in tissue [105] . elevated production of no• and lipid peroxidation products were also found in supernatants and cellular elements of veev-infected neuroblastoma cell cultures. both no• and lipid peroxidation were decreased by melatonin treatment in a timedependent manner with an associated reduction in inos expression [106] . production of brain and serum nitrite, as well as neural lipid peroxidation products, was increased in veev-infected mice. melatonin treatment curtailed nitrite concentrations in the brain and serum of infected mice and lowered lipid peroxidation products [107] . respiratory syncytial virus is a common cause of bronchiolitis, a severe lower respiratory tract affliction that infects nearly all infants by age three worldwide. mice inoculated intranasal with rsv showed elevated oxidative stress due to rises in no• and •oh. also elevated malondialdehyde (mda) and decreases in gsh and sod activities were observed. pre-administration of melatonin in vivo resulted in marked reduction of acute lung oxidative injury induced by rsv, suppressed mda, no• and •oh generation, and restored gsh and sod levels in the lungs of rsv-infected mice [31] . rabbit hemorrhagic disease virus (rhdv) causes bleeding in the respiratory system, liver, spleen, cardiac muscle,and occasionally in the kidneys of infected rabbits with mortality over 90% in adults [108] . the activity and mrna expression of the antioxidants enzymes gpx, glutathione-s-transferase (gst) and mn-sod were significantly reduced in the liver of rhdvinfected rabbits used as a model of fulminant hepatic failure; these changes were reduced by melatonin administration in a concentrationdependent manner. melatonin treatment also caused a rise in protein expression of the nuclear factor erythroid 2 (nrf2), a transcription factor that plays a critical role by binding to the antioxidant response element in the promoter region of a number of genes encoding for antioxidant and detoxifying enzymes in several types of cells and tissues [109] . the activation of nrf2 during prevention of oxidative liver injury by melatonin in rats treated with dimethylnitrosamine has been reported [110] . during the early phase of infection and depending on the nature of the infected cells and the infecting virus, early innate defense mechanisms may be triggered to limit the extent of viral spread. the first mechanism to limit the extent of viral spread is the recognition of pathogenassociated molecular patterns (pamps), which are mostly viral nucleic acids, or their synthetic analogs produced during the viral infection, by a large repertoire of pattern recognition receptors (prrs), including toll-like receptors (tlrs), nodlike receptors (nlrs), rig-i-like receptors (rlrs) and aim2-like receptors (alrs) [111] [112] [113] [114] . such recognition initiates signaling cascades that culminate in the activation of transcription factors including nuclear factor kappa b (nf-kb), activating transcription factor 2 (atf-2), activating protein-1 (ap-1) and interferon regulatory factors 3 (irf3) and 7 (irf7). these stimulate the expression of type i ifn genes that are synthesized in most cell types and especially in plasmacytoid dendritic cells (pdc) [115] . all ifns bind to specific ubiquitously expressed cell surface receptors and induce a large number of interferon-stimulated genes (isg), whose encoded proteins mediate the antiviral effects of interferons. among these isgs, dsrna-activated protein kinase (pkr) primarily inhibits replication of rna viruses such as vesicular stomatitis virus (vsv), emcv, wnv, hcv and dna viruses including hsv-1 [116] . another group of isgs is the 20-50-oligoadenylate synthetases (oas) that requires dsrna for its activation and is a major antiviral effector against picornaviruses (e.g. emcv) and influenza a virus, as well as other rna viruses [117] . non-specific ssrna cleavage also occurs after induction of isg20, a 30-exoribonuclease, which contributes to inhibition of rna viruses such as vsv [118] . an additional, non-enzymatic mechanism of translation inhibition is pursued by the isg56/ifit family proteins, which act against hcv [119] [120] [121] . another ifn-induced protein is the human mxa, which is a key component in innate defense against orthomyxoviruses such as influenza virus as well as measles virus, vsv, hanta virus and sfv [116, 122] , the viperin (cig5), which might interfere with viral budding of enveloped viruses, such as cmv, hcv, and influenza virus [123] , and the nucleic acid-editing enzymes apobec3g and à3 f, which inhibit retroviruses [124] . a second mechanism is the triggering of effector functions of cellular components of the innate immune system, such as granulocytes, natural killer cells (nk) and natural killer t cells (nkt cells), macrophages, and dendritic cells, which are normally rapidly recruited and/or activated at the site of virus infection, causing a local inflammation [125] . during this early phase, activated nk cells release ifn-g, which is not stimulated by viral pamps but by il-12 and il-18 released by activated macrophages [126] . all of the cellular components of the innate immune system can participate in the antiviral response by killing infected cells, by producing chemokines (including eotaxin, rantes, mcp-1, il-8) that recruit inflammatory cells into the infected tissue and by producing antiviral and immunoregulatory cytokines (including tnf-a, il-1, il-3, il-4, il-5, il-6, il-12, il-18, gm-csf) that enable the adaptive immune response to recognize infected cells and perform antiviral effector functions [127] [128] [129] [130] . lymphocytes are cells of this adaptive immune system. among them, two subsets of cd4 + t cells, th1 and th2, play a key role in antiviral immunity. after being stimulated by antigen presenting cells, th1 cells produce il-2, tnf-a and ifn-g, which possess antiviral activities and regulate activation of cd8 + cytotoxic t cells, whereas th2 cells produce il-4, il-5, il-10 and il-13, which stimulate b cells to produce antibodies [131] . despite the fact that virus-specific th2 cells can be detected following primary infection by any virus, virus-specific th1 cells are usually much more abundant and reach very high numbers at the peak of the acute infection [132] . moreover, their frequencies remain elevated following resolution of the infection. melatonin is synthesized in lymphoid organs, such as the bone marrow, thymus and lymphocytes [133] [134] [135] , and there are high affinity membrane melatonin receptors as well as nuclear binding sites in circulating lymphocytes, spleen cells and thymocytes [136] [137] [138] . melatonin is known to activate both innate and adaptive immune responses leading to an increase in immune responsiveness and regulation of several immune functions [27, 28, [139] [140] [141] [142] [143] . melatonin has properties as an inflammatory regulator, because it differentially modulates pro-inflammatory enzymes, and controls the production of inflammatory mediators such as cytokines and leukotrienes. the timing of its pro-inflammatory and anti-inflammatory effects suggests that melatonin might promote early phases of inflammation, on the one hand, and contribute to its attenuation on the other hand, to avoid complications of chronic inflammation [144] . melatonin enhances the production of il-1, il-6, tnf-a and il-12 from the monocytes [145] and of il-2, ifn-g and il-6 from cultured human peripheral blood mononuclear cells [137] . it has been suggested that melatonin and ifn-g create an immunoregulatory circuit responsible for the antiviral, antiproliferative and immunomodulatory actions of . this cytokine increases serotonin and melatonin levels in lymphocytes and macrophages. the early stimulation in the production of ifn-g by melatonin suggests that earlier treatment with this indoleamine could increase the antiviral activity of ifn-g [147] . in addition to stimulating the production of several cytokines that regulate immune function, melatonin enhances immune function by directly stimulating polymorphonuclear cells, macrophages, nk cells and lymphocytes [148] . recently, considerable attention has been focused on the fact that melatonin treatment has been found to augment cd4+ t cells in lymph nodes of rats [149] . consequently, melatonin is considered an immunoenhancing agent [141, 150] . in retrovirus-infected people and mice, whereas th1 cytokine (il-2 and ifn-g) production declines, th2 cytokine (il-4, il-5, il-6, and il-10) production increases [151] [152] [153] . the excessive th2 cytokines suppress th1 cells, causing anergy of cell-mediated immunity, thus allowing the retrovirus as well as normal flora to reproduce and promote free radical generation by macrophages [154] . female c57bl/6 mice infected with the lp-bm5 mlv develop murine aids. treatment with melatonin, alone or with dehydroepiandrosterone (dhea), prevented retrovirus-induced reduction in b-cell and t-cell proliferation and in th1 cytokine secretion, as well as overproduction of th2 cytokines and tnf-a [155] . in fact, melatonin alters the balance of th1 and th2 cells mainly towards th1 responses increasing the production of th1 cytokines [156] . a link between melatonin and the immune system has been also reported in patients infected with hiv-1. although mean serum il-12 levels in hiv-1-affected individuals did not significantly differ from healthy controls, the il-12 levels of hiv-1 patients with advanced disease (cdc stage c) were significantly lower than those of patients in less advanced cdc stages b and a. taking into account that serum il-12 levels run parallel with serum melatonin concentrations as the disease advances, a relationship between immune function and melatonin has been suggested; a reduction in serum melatonin could possibly affect il-12 production thereby contributing to the progress of hiv-1 infection [157] . the protective effect of melatonin against veev by regulation of the immune system has been described by bonilla et al. [158] . the endogenous production of ifn-g, il-1b and tnf-a, but not of il-2 and il-4, is stimulated in veev-infected mice treated with melatonin [159] . nevertheless, the average mortality obtained during neutralization experiments with the corresponding anticytokine antibody suggests that although neither tnf-a nor ifn-g is essential for the protective effect of melatonin observed in murine veev infection, il-1b induced by melatonin treatment is a target cytokine to promote the immune enhanced state. this in turn causes the viral clearance or helps generate an earlier immune response against the veev infection [160] . in contrast, in the brain of veev-infected mice, melatonin stimulates the endogenous production of il-1b but reduces the concentration of tnf-a [158] . il-1b is considered one of the earliest host mediators during infectious diseases of the cns and its role in infectious processes of the brain parallels its role in the peripheral immune system [160] . although il-1b deficiency is protective against fatal sindbis virus infection [161] , mice deficient in il-1b have increased susceptibility to influenza virus [162] . in poxvirus animal models, the viral induction of this cytokine is also beneficial for the host [163] . the increase in il-1b levels detected in blood and in brain of veev-infected mice after melatonin treatment also plays a protective role, possibly by neuronal support and protection by inducing nerve growth factor secretion by astrocytes [164] . this supplies a trophic factor for many neuronal cell types in times of stress such as that produced by veev infection. the significant reduction in the concentration of brain tnf-a induced by melatonin in veevinfected mice likely diminishes the inflammatory response caused by the migration of granulocytes and macrophages to inflammatory sites within the cns [165] . these cells are recruited by colonystimulating factors produced by astrocytes stimulated by tnf-a and as a consequence of alterations in blood-brain barrier (bbb) permeability caused by the adhesive properties of astrocytes stimulated by tnf-a. tnf-a is known to induce intercellular adhesion molecules on neighboring endothelial cells [166] , alter bbb permeability and promote inflammatory cell infiltration into the cns. by reducing adhesion molecule production, which melatonin is known to do [167] , the indole would protect the brain infected with veev. respiratory syncytial virus bronchiolitis in infants is characterized by a massive infiltration of inflammatory cells into the airways. of the diverse intracellular signaling pathways, rsv is recognized by tlr3, which initiates a signaling cascade that culminates in the activation of the transcription factor nf-kb; nf-kb is a central mediator of rsv-induced airway inflammation in vivo [145, 168, 169] . rsv infection of raw264.7 macrophages time-dependently stimulates the rapid activation of tlr3 and nf-kb, as well as subsequent nf-kb dependent genes, many of which encode for pro-inflammatory cytokines and chemokines including tnf-a and il-1b. melatonin decreases tlr3-mediated downstream gene expression in rsv-infected macrophages in a dose-dependent and time-dependent manner. such inhibition of nf-kb activity, as well as of tnf-a in serum, seems to be the key event required to explain the reduction in inflammatory gene expression caused by melatonin [31, 170] . as obligate intracellular parasites, viruses are dependent on the host for each stage of replication and, therefore, constantly interface with multiple components of the host cell machinery, including cellular receptors and uptake pathways, gene expression mechanisms and the cell division apparatus. viral utilization of these systems likely causes cell stress and activates death-signaling pathways or alters expression of genes that control cell survival, evoking pcd [171, 172] . apoptosis is one type of pcd, which is dependent on cleavage of important cellular factors by effector caspases such as caspase-3 and caspase-7. two major pathways govern the activation of such effector caspases. in the intrinsic pathway, intracellular stresses sensed by the bh3-only members of the bcl-2 family promote the formation of the apoptosome by activation of caspase-9 through release of proapoptotic molecules such as cytochrome c and smac/diablo from the mitochondria. the apoptosome directly activates effector caspases. in the extrinsic pathway, occupation of death receptors such as fas and tumor necrosis factor receptor (tnf-r) by death ligands including fasl and tnfa forms a death-inducing signaling complex (disc). this results in the activation of the initiator caspase, caspase-8, which directly mediates effector caspase activation and causes cell death. the ability of melatonin to modulate apoptosis and to differentially regulate the expression of pro-apoptotic and anti-apoptotic mediators has been reported in many studies [29, [173] [174] [175] [176] [177] . rhdv infection induces liver apoptosis with increased caspase-3 expression and activity [178, 179] . these effects are attenuated by melatonin in a concentration-dependent manner. anti-apoptotic actions of melatonin on the intrinsic pathway were related to a reduced expression of bax and cytosolic cytochrome c release, increased expression of bcl-2 and bcl-xl, and inhibition of caspase-9 activity. melatonin treatment also has effects on extrinsic pathway resulting in a reduction in caspase-8 activity, tnf-r1 expression and phosphorylated janus kinase (jnk) expression, and increased expression of cellular fliceinhibitory protein (c-flip), an inhibitor of caspase-8 [179] . these findings show that inhibition of apoptotic mechanisms contributes to the beneficial effects of melatonin in rabbits with experimental infection by rhdv and supports a potential hepatoprotective role of melatonin in fulminated hepatic failure. autophagy is a type of pcd characterized by the formation of autophagosomes to remove excessive proteins and thereby maintains homeostasis within the cell. autophagy is now recognized as a component of both innate and adaptive immune responses to bacterial and viral pathogens [180] . varicella zoster virus infection provides an excellent example of autophagy in humans, because abundant autophagosomes are easily detected in the skin vesicles of both varicella and zoster [181] . autophagy is also found during viral replication of hcv [182] , rabbit calicivirus [183] and poliovirus [184] . given that melatonin modulates autophagy through redoxsensitive transcription factors [185] , the role of melatonin in such viral infections involving autophagy should be examined. beneficial effects of melatonin when combined with several drugs, such as doxorubicin, cisplatin, epirubicin, cytarabine, bleomycin, gentamicin, cyclosporin, indometacin, acetylsalicylic acid, ranitidine, omeprazole, isoniazid, iron and erythropoietin, phenobarbital, carbamazepine, haloperidol, caposide-50, morphine, cyclophosphamide and l-cysteine have been reported [24] . recently, a single blind randomized study showed a higher percent of a complete regression of symptoms of hsv-1 infection after a treatment with melatonin plus sb-73 (an extract of aspergillus sp. with antiherpetic properties) compared with the treatment with acyclovir alone [186] . effects of melatonin to increase the efficacy of other antivirals should be studied. melatonin is an endogenously produced and ubiquitously acting molecule [187] [188] [189] . because of its highly diverse actions, this indoleamine has potential to combat a wide variety of pathophysiological conditions [190] [191] [192] [193] [194] ; it has been tested in numerous clinical trials [23] with the outcomes of the treatments always being beneficial. because of its essential and basic actions on cell physiology, melatonin qualifies for the moniker "molecular handyman," as indicated in the title of this review. in relation to viral infections, melatonin also seems to be beneficial as indicated in the experimental studies summarized herein. its favorable actions against viral infections likely relate to its ability to limit the negative molecular processes normally activated when viruses invade cells. melatonin's actions include an ability to promote immune surveillance, to scavenge free radicals thereby significantly reducing the associated molecular destruction and to modulate the processes related to apoptosis. these multiple actions suggest that melatonin should be evaluated in randomized controlled trials as a preventive agent or as a treatment of viral infections particularly in older individuals where endogenous levels of melatonin have declined. it is the hope of the authors that this summary will stimulate interest in experimental examination of melatonin's antiviral actions. isolation of melatonin, a pineal factor that lightens melanocytes structure of melatonin melatonin: a coordinating signal for mammalian 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regression of herpes viral infection symptoms using melatonin and sb-73: comparison with pineal melatonin: cell biology of its synthesis and of its physiological interactions melatonin: a multitasking molecule a survey of molecular details in the human pineal gland in the light of phylogeny, structure, function and chronobiological diseases sirtuins, melatonin and circadian rhythms: building a bridge between aging and cancer melatonin, cardiolipin and mitochondrial bioenergetics in health and disease melatonin as a therapeutic tool in ophthalmology: implications for glaucoma and uveitis drug-mediated ototoxicity and tinnitus: alleviation with melatonin matrix metalloproteinases in health and disease: regulation by melatonin the authors have no competing interest. jab is a researcher of isciii/ficyt. his stay at uthscsa has been subsidized by isciii (ba 11/ 00084). key: cord-324949-sqy03dks authors: poe, francis l.; corn, joshua title: n-acetylcysteine: a potential therapeutic agent for sars-cov-2 date: 2020-05-30 journal: med hypotheses doi: 10.1016/j.mehy.2020.109862 sha: doc_id: 324949 cord_uid: sqy03dks covid-19, a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), continues to spread across the globe. predisposing factors such as age, diabetes, cardiovascular disease, and lowered immune function increase the risk of disease severity. t cell exhaustion, high viral load, and high levels of tnf-ɑ, il1β, il6, il10 have been associated with severe sars-cov-2. cytokine and antigen overstimulation are potentially responsible for poor humoral response to the virus. lower cellular redox status, which leads to pro-inflammatory states mediated by tnf-ɑ is also potentially implicated. in vivo, in vitro, and human clinical trials have demonstrated n-acetylcysteine (nac) as an effective method of improving redox status, especially when under oxidative stress. in human clinical trials, nac can be used to replenish glutathione stores and increase the proliferative response of t cells. nac has also been shown to inhibit the nlrp3 inflammasome pathway (il1β and il18) in vitro, and decrease plasma tnf-ɑ in human clinical trials. mediation of the viral load could occur through nac’s ability to increase cellular redox status via maximizing the rate limiting step of glutathione synthesis, and thereby potentially decreasing the effects of virally induced oxidative stress and cell death. we hypothesize that nac could act as a potential therapeutic agent in the treatment of covid-19 through a variety of potential mechanisms, including increasing glutathione, improving t cell response, and modulating inflammation. in this article, we present evidence to support the use of nac as a potential therapeutic agent in the treatment of covid-19. covid-19, a respiratory disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), continues to spread across the globe. sars-cov-2 is an enveloped positivesense, single stranded-rna virus in the betacoronavirus genus [1, 2] . sars-cov-2 is genomically similar to severe acute respiratory coronavirus 1 (sars-cov-1), which led to an outbreak of severe acute respiratory syndrome (sars) in the early 2000s. no cases of sars have been reported globally since 2004 [1] . similar to sars-cov-1, sars-cov-2 gains entry into cells through spike protein affinity for angiotensin converting enzyme 2 receptors (ace2) and uses host cell serine protease tmprss2 to mediate entry [3] [4] [5] . ace2 receptors are expressed in lung, heart, kidney, and intestinal tissue and primarily function physiologically in the maturation of angiotensin [6] . the pathogenicity of the sars-cov-1 stemmed from nod like receptor, pyrin domain containing 3 (nlrp3) inflammasome activation in monocytes and macrophages [7, 8] . this led to high levels of cytokines: interleukin-1β (ilβ), interleukin-18 (il18), tumor necrosis factor alpha (tnfɑ); and an immunopathological response associated with acute respiratory distress syndrome (ards), cytokine storms, organ damage, and death [7, [9] [10] [11] . although sars-cov-2 is 78% genomically similar to sars-cov-1, there are variations in the open reading frames (orfs) involved in the inflammatory monocyte and macrophage response [3, 12] . research is underway to elucidate these mechanisms further. in severe covid-19, there are increased levels of cytokines interleukin-6 (il6), interleukin-10 (il10), and tnf-ɑ [13] . in some cases, these increased cytokine levels create a "cytokine storm" and cause significant damage to lung tissue [14] . clinically, covid-19 is mild in most cases, with severe cases characterized by pneumonia and critical cases characterized by ards, sepsis, and multiple organ failure [14] . older adults, aged 65 years and older, appear to be most susceptible to covid-19 [14, 15] . the most susceptible persons to sars-cov-2 are the elderly: the typical profile of the critically ill patient is 65 years and older, presents with comorbidities, and ards [9] . these patients had a mortality rate of 67% from the time of admittance to 28 days later [9] . n-acetylcysteine (nac) is a precursor of glutathione and acts as a powerful antioxidant and free radical scavenger in the body [16] . nac has been used for paracetamol toxicity and conditions with viscous mucous secretions [17] . nac may be administered orally, intravenously, or nebulized. we hypothesize that nac could act as a potential therapeutic agent in the treatment of covid-19 through a variety of potential mechanisms, including increasing glutathione [18] , improving t cell response [19] [20] [21] , and modulating inflammation [22, 23] . in this article, we present evidence to support the use of nac as a treatment for covid-19. sars-cov-2 and the elderly sars-cov-2 is especially virulent in elderly populations. the virus gains entry primarily via ace2 receptors on alveolar type 2 epithelium [24] . hypertension, a known risk factor for developing more severe covid-19 disease, is more common in older adults [25, 26] . in adults with a history of taking angiotensin converting enzyme inhibitors and angiotensin receptor blockers to treat hypertension, there is a possible increase in ace2 receptors [27] . although the relationship has yet to be clearly defined, viral infectivity may be increased due to increased expression of ace2 secondary to treatment with acei and arbs in the elderly [28] . in sars-cov-1, the immune response was dominated by a th1 response, but in sars-cov-2 there are both th1 and th2 responses [29] . in sars-cov-1, an inflammatory monocyte and macrophage cascade activated via the nlrp3 inflammasome pathway [7, 8] . this resulted in pro-inflammatory cytokines il1β and il18, and an immunopathological response causing injury to lung tissues [7] . in sars-cov-2, there is a remarkable increase of cytokines il6, il10, and tnf-ɑ [15] , however, similar to sars-cov-1, there appears to be elevated levels of il1β, which suggests possible pro-inflammatory macrophage and monocyte activity activated by the nlrp3 inflammasome pathway [15, 30] . t cell exhaustion has been noted in severe sars-cov-2, illustrated by high levels of programmed cell death protein 1 (pd1) and low cd4+ and cd8+ counts [15] . t cell exhaustion leads to poor cd4+ and cd8+ responses, which limits viral clearance via humoral immunity [31] . the likely cause of t cell exhaustion in sars-cov-2 is hypothesized to be from excessive stimulation of t cells by viral antigen and cytokines (il6, il10, tnf-ɑ [15] . notwithstanding sars-cov-2 infection, the elderly have an increased risk of mortality due to age related proinflammatory changes [32] . one mechanism suggested is increased tnf-ɑ induced apoptosis that occurs in the elderly [33] . in severe sars-cov-2, diao et al. suggest that age related proinflammatory changes to tnf-ɑ expression may be a causative factor in t cell exhaustion [15] . additionally, elderly adults may also have decreased redox potential via lower glutathione levels [34] [35] [36] . lowered redox status of a cell increases susceptibility to oxidative stress that may lead to cell death and viral release [37] . glutathione conjugates oxidative species through phase 2 detoxification. it is a central oxidative species mediator that impacts cell cycle and apoptosis [38, 39] . l-cysteine is the rate-limiting substrate in the intracellular synthesis of glutathione [39] . nac directly affects the amino acid pool of extracellular cystine and intracellular cysteine through a series of redox reactions in the plasma [40] . nac increases extracellular cysteine, and through transport channels increases intracellular cysteine levels [18, 38, 41] . during oxidative stress, nac will increase glutathione synthesis [18, 42] . without oxidative stress, the pool of cysteine and cystine appears to primarily mediate cellular stress through thiols other than glutathione [18, 38] . individuals 60 years and older demonstrate lower plasma glutathione levels and increased oxidative stress [43] . those with diabetes have lower glutathione levels compared to control subjects [35, 43] . dietary supplementation of cysteine and glycine can increase glutathione levels and reduce oxidative stress in the elderly [36] and persons with diabetes [35] . glutathione concentrations affect the proliferative capacity of lymphocytes: low concentrations lead to less lymphocyte proliferation and high concentrations lead to more lymphocyte proliferation [44] . t cell exhaustion occurs when lymphocytes (cd4+ and cd8+) counts are low, and can be measured through program cell death protein 1 (pd1) [31] . it is commonly seen in chronic viral infections [31] . pd1 was elevated in severe covid-19 cases [15] . diao et al. referred to this state, in conjunction with significantly lowered cd4+ and cd8+ counts, as functional t cell exhaustion [15] . the investigators posited that functional t cell exhaustion is an etiological factor in severe sars-cov-2 infection. they determined the observed t cell exhaustion is associated with and likely caused by overstimulation of cytokines (il6, il10, tnf-ɑ). lymphocyte proliferation has been noted with the administration of nac. in patients with hiv, oral nac increased both whole blood glutathione levels and lymphocyte count (cd4+ and cd8+) [20, 21] . nac has also shown to decrease pd-1 levels and increase the longevity of cd8+ cells in vitro [19] . data from sars-cov-1 indicates that this coronavirus mediates the nlrp3 inflammasome pathway in infected cells by orfs 3a and 8b [7, 45, 46] . this pathway has been indicated in inflammatory cell death (necroptosis) and is likely responsible for the pathological findings found in lung biopsy of sars-1 patients [45, 47] . likewise in sars-cov-2, there is an elevated level of il1β suggesting nlrp3 activation in macrophages and monocytes [29, 30, 48] . tnf-ɑ is usually increased early in the inflammatory response [49, 50] , and can be secreted by monocytes, macrophages, and alveolar epithelium [50] . tnf-ɑ attaches to receptor sites and depending on the redox potential of the cell, may induce an apoptotic or a proliferative pathway [51] . in vitro, nac interrupts the nlrp3 inflammasome pathway in a dose dependent manner through effects on mrna expression of nlrp3 and caspase-1 activation [22] . nac lowers il1β, il18 [22] . nac has been shown to lower mucin production, and il6 and tnf-ɑ in vitro [52] . nac inhibits the downstream activities post tnf-ɑ receptor activation [52] , and while under oxidative stress nac inhibits gene expression of tnf-ɑ and il-6 [23] . there have been several clinical trials investigating the use of nac in respiratory illness in humans. intravenous nac has been used clinically for the treatment of ards. in a metaanalysis of randomized clinical trials investigating the use of nac as a treatment for ards, administration of nac resulted in a decreased length of stay in intensive care units, however, there was no change in overall short term mortality. the meta-analysis found no adverse reactions with doses similar to those used in drug-induced liver injury [53] . in both in vivo and human trials, nebulized nac may improve arterial oxygen tension [54, 55] ; and attenuate pulmonary fibrosis [56] , and ards [57] . in a randomized clinical trial, oral nac demonstrated decreases in tnf-ɑ and no adverse reactions at 1200 mg daily, however there were no changes in computed tomography scores between those treated with nac and the control group [58] . in another trial involving multiple elder care facilities, oral nac was investigated as a prophylactic and therapeutic agent for influenza. participants who were given 1200 mg daily for six months experienced fewer influenza and influenza-like episodes, decreased severity of illness, and fewer days confined to bed [59] . in this trial, nac did not alter viral seroconversion but participants taking nac were less likely to develop a symptomatic infection [59] . these findings are especially impactful given the higher-risk study population. ventilator use is common in severe cases of covid-19, with approximately 3% of all cases requiring intubation and invasive ventilation at some point during the course of illness [60] . nac has been studied as a prophylactic intervention for ventilator associated pneumonia (vap), a complication of the use of mechanical ventilation [61] . in a randomized, double-blind, placebocontrolled trial of 60 participants receiving nasogastric administration of 1200 mg nac daily, participants in the treatment group were less likely to develop vap and had a shorter duration of hospital and icu stay. additionally, the incidence of complete recovery from vap was higher in the nac group. nac has an excellent safety record in clinical trials. oral nac can cause stomatitis, nausea, vomiting, gastroesophageal reflux [17, 62] . oral nac may be used if there has been an anaphylactoid reaction to iv nac [17] . nebulized nac may cause bronchoconstriction and prolonged coughing and may worsen asthma [55, 63] . serious adverse reactions with iv nac are rare, loading dose dependent, and primarily occur in drug-induced liver injury [17, 64] . in drug-induced liver injury from paracetamol, 10 grams of nac is typically loaded in 15 minutes. this can result in upwards of 31% risk of anaphylactoid reactions ranging from pruritus, rash, angioedema, bronchospasm, and hypotension [65] . with an alternate loading schedule the overall risk of anaphylactic reaction can be lowered to 5% [65] .anaphylactoid reactions occur with iv nac and can be managed with attentive care [17] . but as noted in the ards meta-analysis no adverse reactions were recorded with a similar loading dosage used in drug-induced liver injury [53] . discussion nac has shown activity in a variety of potential therapeutic target pathways involved in the pathophysiology of sars-cov-2 infection. the pathogenic factors of sars-cov-2 that could possibly be mediated by nac are (1) t cell exhaustion, which manifests as lower counts and decreased functional capacity of cd4+ and cd8+ cells; (2) pro-inflammatory state via increase in tnf-ɑ, il1β, il18; and (3) modulation of viral activity through increased glutathione. the virus is especially virulent in the elderly population. certain physiological conditions, including diabetes, cardiovascular disease, and lowered immune function that may affect the severity of sars-cov-2 are more common in older adults. lowered redox status, common in high-risk groups including older adults and those with uncontrolled diabetes, causes alterations in the tnf-ɑ receptor activity towards a pro-inflammatory state [33, 51] . nac has been shown to replenish glutathione stores and increase the proliferative response of t cells. nac has also been shown to inhibit the nlrp3 inflammasome pathway (il1β and il18) in vitro, and decrease plasma tnf-ɑ. mediation of the viral load could occur through the ability of nac to increase cellular redox status by maximizing the rate limiting step of glutathione synthesis, and thereby decreasing the effects of virally induced oxidative stress and cell death. to test these hypotheses further, we recommend both in vitro and in vivo studies investigating nac. in vitro studies may include investigations into the action of nac on certain cell types such alveolar type 2 epithelial cells, monoctyes, or macrophages, as well changes in receptor and cytokine expression in these cell types. other studies could investigate the action of nac in human cell lines with low redox potential. human clinical trials would build on current evidence for the use of nac in other infectious respiratory illnesses. hospital-based clinical trials could administer nebulized, oral, or iv nac during various stages of illness, perhaps utilizing grouping identified by diao et al. similar to clinical trials in influenza, ards, and vap, outcome measures could include change in sars-cov-2 associated cytokines, change in lymphocyte count and activation, change in icu status, and overall mortality. while no clinical trials investigating the use of nac in covid-19, trials completed in using nac for influenza, ards, and vap have shown promising results in reducing disease severity and duration of hospital stay. currently, a protocol for using both nac and heparin has been developed by a seattle-based biotherapeutics researcher. the protocol is available for use by clinicians but at the date of this publication, data from studies using this protocol have not been published [66] . epidemiology, virology, and clinical features of severe acute respiratory syndrome -coronavirus-2 (sars-cov-2 coronavirus disease-19) severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges genomic characterisation and epidemiology of 2019 novel coronavirus: implications 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changing regimens of acetylcysteine reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial new covid-19 hope clinical trial recommendations introduced today may reduce or eliminate mechanical ventilation for coronavirus patients | biospace heather zwickey, phd hzwickey@nunm.edu james dumbauld, do pdumbauld@live.com bracey dangerfield, phd bdangerfield@nunm.edu key: cord-295523-5pv7kw6i authors: picchianti diamanti, andrea; rosado, maria manuela; pioli, claudio; sesti, giorgio; laganà, bruno title: cytokine release syndrome in covid-19 patients, a new scenario for an old concern: the fragile balance between infections and autoimmunity date: 2020-05-08 journal: int j mol sci doi: 10.3390/ijms21093330 sha: doc_id: 295523 cord_uid: 5pv7kw6i on 7 january 2020, researchers isolated and sequenced in china from patients with severe pneumonitis a novel coronavirus, then called sars-cov-2, which rapidly spread worldwide, becoming a global health emergency. typical manifestations consist of flu-like symptoms such as fever, cough, fatigue, and dyspnea. however, in about 20% of patients, the infection progresses to severe interstitial pneumonia and can induce an uncontrolled host-immune response, leading to a life-threatening condition called cytokine release syndrome (crs). crs represents an emergency scenario of a frequent challenge, which is the complex and interwoven link between infections and autoimmunity. indeed, treatment of crs involves the use of both antivirals to control the underlying infection and immunosuppressive agents to dampen the aberrant pro-inflammatory response of the host. several trials, evaluating the safety and effectiveness of immunosuppressants commonly used in rheumatic diseases, are ongoing in patients with covid-19 and crs, some of which are achieving promising results. however, such a use should follow a multidisciplinary approach, be accompanied by close monitoring, be tailored to patient’s clinical and serological features, and be initiated at the right time to reach the best results. autoimmune patients receiving immunosuppressants could be prone to sars-cov-2 infections; however, suspension of the ongoing therapy is contraindicated to avoid disease flares and a consequent increase in the infection risk. on 7 january 2020, after a cluster of cases with pneumonia of unknown origin in wuhan, hubei, china, researchers isolated a novel coronavirus, then called sars-cov-2 [1] , and covid-19, the induced disease. sars-cov-2 is believed to have zoonotic origins (probably from the huanan seafood market in wuhan) and has close genetic similarity (96%) to a bat coronavirus, suggesting it emerged from a bat-borne virus [2] . an intermediate animal reservoir such as the pangolin is also thought to be involved in its introduction to humans, but efforts to identify intermediate hosts seem to have been unsuccessful [3, 4] . since the outbreak, thanks to its contagiousness/virulence characteristics and boosted by globalization, sars-cov-2 moved around the world faster than a virus has ever done before. after three months since its appearance, it reached 1.5 × 10 6 infected patients, with 3.5 × 10 5 recovered patients and 100,000 deaths across the world [4] . due to a number of unfortunate variables, northern italy was the first country outside of asia affected by the rapid spreading of sars-cov-2, which led, at the end of march, to the highest number of deaths worldwide [4] . typical clinical manifestations of the disease generally begin after less than 5-7 days of incubation and consist of fever, cough, fatigue, and mild dyspnea. however, data from china reported that about 15-20% of patients have severe disease with interstitial pneumonia, which can progress to acute respiratory distress syndrome (ards) [5, 6] . pneumonia includes decreased oxygen saturation, with severe bilateral ground glass abnormalities, patchy consolidation, and alveolar exudates [5, 6] . in patients with ards, the virus can induce an excessive and aberrant host immune response characterized by an upregulation of pro-inflammatory cytokines, resembling the clinical and serological features of cytokine release syndrome (crs). crs is a life-threatening emergency associated with high mortality; thus, an early identification is essential. at the moment, there are no specific antiviral treatments recommended for sars-cov-2 and no vaccine is currently available. a growing awareness of sars-cov-2 infection and crs has led to exploring the use of immunomodulatory drugs as a potential treatment for the management of these patients. here, the complex relationship between infections and autoimmunity in the emergency scenario of the sars-cov-2 pandemic is discussed. we critically review the rationale for the adoption of immunosuppressive agents, commonly used in autoimmune diseases, in the treatment of sars-cov-2 infection and report current knowledge of ongoing studies. crs is a systemic inflammatory life-threatening condition typically associated with biological drug products, but also occurring during the response to some infections [7] . initially described as a reaction to the use of the anti-cd3 (okt3) monoclonal antibody (mab) [8] , this syndrome is the result of a cytokine storm. crs has been under tight evaluation since it caused the admission to the intensive care unit of six healthy individuals enrolled in the phase i trial for the anti-cd28 mab tgn1412a in 2006 [9] . crs has been also observed in patients treated with immune checkpoint inhibitors or t cell therapy (car t cells) [10] . it is noteworthy that the cytokine storm due to massive t cell stimulation is also considered a relevant mechanism to the h5n1 influenza pathogenesis [11] . crs and sepsis share several symptoms, and patients with crs are at a high risk of infections, not only for the immunosuppressive treatments, but likely also for the crs-associated immune dysregulation and tissue damages, especially at the mucosa barrier. indeed, in crs patients, infections principally involve the respiratory tract. the exacerbated reaction to infections or to biological therapy is caused by the rapid recruitment of macrophages and neutrophils, which produce pro-inflammatory cytokines and alter the fragile balance between a controlled immune response and a host-damaging reaction. damaged tissues release molecules normally not present outside the cells, including high-mobility group box 1 (hmgb1), atp, uric acid, and dna, further amplifying inflammatory responses. all these molecules are part of the so-called damage-associated molecular patterns (damps). it is noteworthy that pathogen-associated molecular patterns (pamps) and damps are recognized by the same group of innate immunity receptors, namely the pathogen recognition receptors (prrs), which include toll-like receptors (tlrs), highly expressed in neutrophils and macrophages. engagement of tlrs and other prrs leads to further activation of nf-kb and the release of cytokines (il-6, tnfα, il-1, etc.) and other mediators of inflammation. a relevant biological role has also been proposed for ferritin in crs-related conditions, as well as in autoimmune disease has been proposed. the presence of hyperferritinemia, indeed, is a well-known feature in patients with different autoimmune conditions such as ra, systemic lupus erythematosus (sle), and anti-phospholipid syndrome [12] . it has been supposed that several mechanisms involving the inhibition the h-ferritin-mediated suppression of immune cells may favor the loss of tolerance and the onset of autoimmunity [13] . ferritin can be also a pro-inflammatory signaling molecule, and hyperferritinemia has been associated with different crs-related conditions such as macrophage activation syndrome (mas) and septic shock [14] . ferritin could exert a pathogenic role in these diseases rather than being just the result of hyperinflammation. in fact, ferritin synthesis is mediated, not only by iron availability, but also by il-1, il-6, and tnf [15, 16] , which are overexpressed during crs; on the other hand, it can induce the expression of pro-inflammatory cytokines, thus becoming part of a vicious loop [17] . in particular, it has been hypothesized that during an ongoing infection, the signaling mediated by bacterial/viral cpg dna and tlr9 could activate the inflammasome, leading to il-1 and il-18 production [18] [19] [20] . through damp signaling, infection could also increase the production of hemoglobin [21] and activate macrophages, which are prominent producers of ferritin [22, 23] , amplifying the inflammatory loop [23] . moreover, a correlation between the serum levels of cd163, a marker of macrophage activation [24] , and ferritin in patients with mas has been reported [25] . different therapeutic approaches could be useful in blocking this pathway at different levels, such as plasma exchange, intravenous immunoglobulins, or mabs against il-1 and il-18 [18] . in patients affected by 2002/2003 sars-cov, immune dysregulation induced an abnormal inflammatory cytokine production by alveolar macrophages with a concomitant t cell dysfunction, involving both cd4 and cd8 t cells [26] . sars patients with a more severe disease displayed higher serum levels of pro-inflammatory cytokines (ifn-γ, il-1, il-6) and chemokines (including il-8) [27] . in crs, ifn-γ further activates immune cells, especially macrophages, which are induced to produce more inflammatory cytokines and upregulate costimulatory ligands, feeding the harmful positive loop of inflammation. it is important to remember that type i interferons (ifns) play a critical role in the normal/physiologic immune response to viruses by enhancing the toxic effects of cd8 t cells [28] , activating nk cells, and restricting viral pathogenicity to the lung microenvironment. ifnαr-deficient mice infected with the h5n1 or the 1918 influenza virus show indeed higher mortality than wild-type mice, which display systemic dissemination of the virus [29] . it is noteworthy that some studies showed that coronaviruses, in particular mers-cov, can suppress the expression of both type i and type iii ifns, evading innate immune response and contributing to its pathogenicity [30] . in in vitro models, ifn-λ showed effects against 2002/2003 sars-cov and mers-cov, suggesting a possible use to control viral infections by coronavirus. interestingly, the expression of the type iii ifn receptors is more restricted to specific cell types (neutrophils and b cells) compared with type i ifn receptors, a feature that could restrain inflammatory response to the (initial) site of infection. in an animal model, treatment with ifn-λ2/il-28a reversed the development of collagen-induced arthritis, also indicating an anti-inflammatory role in autoimmune responses [31] . however, in the contest of crs associated with covid-19, it cannot be excluded that type iii ifn receptors are upregulated also in other cell types and that these cytokines could also contribute to the complex pathogenic process [32] . in sars-cov-2 patients with a worse prognosis, il-6, il-10, and tnf-α quickly rise and reach high levels. conversely, in patients with milder symptoms, these cytokines reach lower levels, with their expression rising and declining during the illness and recovery phase, respectively [33] . the production of il-10, an anti-inflammatory cytokine, in the context of crs is often associated with the downregulation of neutrophil and monocyte function, a phenomenon termed immunoparalysis [34] . although conceptually beneficial, the persistent downregulation of hla-dr on monocytes, after sepsis, as well as after crs, leads to higher mortality rates, suggesting that the recovery from immunoparalysis is critical for patient survival [35] . as above mentioned, systemic cytokines are considered to be massively produced by macrophages in sars-cov-2 patients [36] . however, also endothelial cells play a relevant role in crs, not only as cells that are damaged by pathogens and inflammatory responses, but also as co-culprits. endothelial cells indeed produce inflammatory cytokines, including il-6, and upregulate adhesion molecules, further promoting leukocytes' recruitment and capillary leakage [37] . in line with lymphocyte recruitment into the place of infection/inflammation, two recent studies showed that in patients with severe covid-19, t cell lymphopenia is accompanied by an alteration in the distribution of circulating t cell subpopulations; patients, indeed, have increased frequencies of naive helper t cells and a reduction in memory helper t cells [38, 39] . the cytokine unbalance associated with the response to sars-cov-2 could also affect the effectiveness of the immune response both in terms of viral clearance and future immune protection. for some of the recovered patients, protective antibodies have been described. they are mainly directed towards the receptor binding domain of the spike protein, and most likely, they interfere with viral entry [40] . even if the majority of patients recover, some of them after discharge from the hospital, asymptomatic and negative rt-pcr viral rna tests remain/return positive or even relapse [41] . due to the limited number of described cases, incomplete virus clearance rather than lack of protection cannot be ruled out. noteworthy, previous studies on 2002/2003 sars-cov showed heterogeneous results on b and t cell memory [42] [43] [44] . information on the long-term quality of immune response, t and b cell immunological memory, and long-versus short-lived plasma cells towards sars-cov-2 is not available yet. altogether, these recent findings point to a major role of the host immune response, particularly of crs, as a determining co-factor in the severe life-threating form of covid-19. why some patients develop an effective immune response, which is protective and not pathogenic, and why others have a non-protective life-threatening immune response is a key question. it is likely that genetic background, which is also involved in inflammatory responses, immune-mediated diseases (including autoimmunity), and co-morbidities, may not only weaken the host, but also may share "common" pathways in inflammatory damaging responses. treatment of crs involves the use of both antiviral agents to control the underlying infection and immunosuppressants to lower the aberrant pro-inflammatory response of the host. for a better understanding, it is essential to remind about the natural course of sars-cov-2 infection. after the incubation period, indeed, the virus induces flu-like symptoms typical of mild disease; in some patients, the infection can progress to interstitial pneumonia (moderate disease) or severe pneumonia requiring oxygen therapy (severe disease), through to ards with respiratory failure [45] . this is probably the moment at which the shift from a controlled immune response to a host-damaging reaction begins to manifest clinically. then, sars-cov-2 does not directly induce tissue damage, whereas the hyperinflammatory immune activation of the host becomes the effective protagonist of the disease. the early identification of this specific moment of transition is of key importance, to allow timely immunomodulatory intervention, thus achieving a tailored approach and the best therapeutic effects ( figure 1 ). use of anti-rheumatic immunosuppressive agents in covid-19 patients, targeted to their immunomodulatory/antiviral activity and disease severity. tocilizumab, sarilumab, and anakinra have the strongest immunosuppressive effect and have been already tested in cytokine release syndrome (crs) (with tocilizumab being the only scheduled); thus, they should be administered in severe covid-19 patients at the first manifestations of hyperinflammation. baricitinib has both immunosuppressive effects (however, no data are available for crs) and antiviral activity; thus, it could be adopted in the moderate/severe form of covid-19. hydroxychloroquine (hiq) has antiviral properties and milder immunosuppressive activity than the other drugs; thus, it could be used in the moderate/severe form of to that end, several serologic markers have been proposed, such as the presence of thrombocytopenia, lymphopenia, and increased levels of d-dimer and ferritin [46] . some authors have also reported a role for il-6 and il-10 in monitoring covid-19 patients. in particular, significantly higher levels of il-6 and il-10 have been identified in severe covid-19 patients than in the milder forms, thus suggesting that these cytokines can be used to predict the transition from mild to severe infection [47] . figure 1 . use of anti-rheumatic immunosuppressive agents in covid-19 patients, targeted to their immunomodulatory/antiviral activity and disease severity. tocilizumab, sarilumab, and anakinra have the strongest immunosuppressive effect and have been already tested in cytokine release syndrome (crs) (with tocilizumab being the only scheduled); thus, they should be administered in severe covid-19 patients at the first manifestations of hyperinflammation. baricitinib has both immunosuppressive effects (however, no data are available for crs) and antiviral activity; thus, it could be adopted in the moderate/severe form of covid-19. hydroxychloroquine (hiq) has antiviral properties and milder immunosuppressive activity than the other drugs; thus, it could be used in the moderate/severe form of to that end, several serologic markers have been proposed, such as the presence of thrombocytopenia, lymphopenia, and increased levels of d-dimer and ferritin [46] . some authors have also reported a role for il-6 and il-10 in monitoring covid-19 patients. in particular, significantly higher levels of il-6 and il-10 have been identified in severe covid-19 patients than in the milder forms, thus suggesting that these cytokines can be used to predict the transition from mild to severe infection [47] . hydroxychloroquine (hiq) is an orally administered and low-cost drug widely used as a monotherapy in clinical rheumatological practice mainly to treat the mild form of rheumatoid arthritis (ra), sle, and sjogren's syndrome patients, as well as in combination therapy with conventional immunosuppressants, in more severe patients. hiq has a pleiotropic activity ranging from immunomodulatory effects, to anti-thrombotic action and antiviral properties. the immunomodulatory activity of hiq has been demonstrated in vitro to be exerted by several mechanisms. hiq interferes with lysosomal activity, impairing lysosomal and autophagosome functions and subsequently immune activation [48] . it can inhibit tlr-7 and tlr9 [49] signaling pathways and decrease the secretion of pro-inflammatory cytokines (il-6, tnf-α, il-1, ifn-γ) [50] . the anti-thrombotic mechanism is still poorly clarified; anyway, it has been reported that it can reverse platelet activation and reduce anti-phospholipid (apl) antibody titers in apl patients [51] ; it can also improve endothelial dysfunction and reduce the expression of adhesion molecules such as vcam-1 and e-selectin [52] , mechanisms that could be relevant in severe covid-19. regarding the antiviral effects, it is known that hiq is able to block the infection of different viruses, including sars-cov-1, by increasing endosomal ph and by interfering with the glycosylation of the cellular receptor [53, 54] . several in vitro studies have been conducted to explore its efficacy in blocking sars-cov-2 infection. chloroquine was found to inhibit the virus at a low micromolar concentration, with a half maximal effective concentration (ec 50 ) of 1.13 µm and a half cytotoxic concentration (cc 50 ) greater than 100 µm, which can be achievable with the standard dosing regimen [55] . in a recent study, hiq was found to have a higher antiviral effect than chloroquine, with a lower ec 50 [56] . as a consequence of these encouraging results, at least 23 clinical trials are being carried out to evaluate the efficacy and safety of chloroquine or hiq in the treatment of sars-cov-2 patients [57] . by now, the results have demonstrated efficacy in reducing the exacerbation of pneumonia, improving lung imaging findings, and promoting a virus-negative conversion without serious adverse events [58] . a guideline document promoted by the italian society of infectious and tropical disease recommends the use of chloroquine 300 mg × 2/day or hiq 200 mg x 2/day, in patients presenting with mild respiratory symptoms and comorbidities, as well as in patients with severe respiratory failure [59]. il-6 is a pleotropic cytokine with several immunological activities. it plays a role in the differentiation of mature b cells into plasma cells, and combined with tgf-β, it induces the differentiation of naive cd4 positive t cells into th17-cells and induces the production of acute-phase proteins such as crp, fibrinogen, serum amyloid a, and hepcidin [60, 61] . in bone marrow, il-6 induces the maturation of megakaryocytes into platelets and the activation of hematopoietic stem cells [60, 61] . tocilizumab and sarilumab are a humanized and human mab, respectively, recognizing the soluble and membrane-bound forms of the il-6 receptor. they are part of the first line biological therapy in patients with moderately to severely active ra and juvenile idiopathic arthritis; more recently, tocilizumab has been scheduled also for giant cell arteritis patients [61] . it is of note that il-6 in combination with tgf-β is also produced by fibroblasts and activated macrophages exerting a pro-fibrotic effect at different sites such as lungs, skin, and liver [61] . in a recent work, we proposed tocilizumab as a valid therapeutic strategy in patients with interstitial pneumonitis associated with ra, because it might contrast the pro-fibrotic effects of il-6, ameliorating both the articular and lung involvement [62] ; this observation was recently confirmed by other authors [63] . as reported above, in some patients, sars-cov-2 infection can induce an uncontrolled and aberrant host hyperimmune response that is associated with lung damage and fibrosis, leading to life-threatening multi-organ failure. serologically, an increase in the serum concentrations of il-1, il-6, il-2, il-7, il-10, tnf-α, granulocyte colony-stimulating factor, interferon-γ-inducible protein 10, monocyte chemoattractant protein 1, and macrophage inflammatory protein 1-α has been found [64] [65] [66] . furthermore, different chinese authors reported that a lower lymphocyte count and elevated crp, ferritin, d-dimer, and il-6 were poor prognostic factors in sars-cov-2 patients [67] . the efficacy of tocilizumab in resolving life-threatening crs during car t cell therapies was assessed in small patient cohorts [7] ; however, the striking positive results led to its rapid approval for the treatment of crs by the fda in 2017 followed by ema in 2018 [68, 69] . based on these data, small retrospective studies on patients affected by severe covid-19 demonstrated that tocilizumab improved ct scan ground glass lesions and oxygen saturation, normalized crp levels, and lymphocyte count in a significant percentage of patients [70, 71] . il-6 inhibitors should be initiated at the early stages of hyperinflammation, after discussion between critical care medicine and hematology/rheumatology and infection specialists; one additional dose may be considered if clinical deterioration persists (max two doses per course in severe sars-cov-2) [72, 73] . a multicenter randomized clinical trial (rct) of tocilizumab has been approved in china and is currently ongoing in patients with sars-cov-2 pneumonia and elevated il-6 levels (chictr2000029765). several clinical trials on the use of tocilizumab in patients with covid-19 are already posted on clinicaltrials.gov. these trials are enrolling different sets of the covid-19 population ranging from patients with recent onset pneumonia to life-threatening-associated crs. there is also heterogenicity among the primary outcomes, but those mainly used are the proportion of subjects with normalization of fever and oxygen saturation at 14 days, the proportion of patients requiring mechanical ventilation and intensive care unit (icu) admission, and the one-month mortality rate. the increase in lymphocyte count, decrease in crp, and amelioration of ct lung opacity are frequently reported secondary endpoints (nct04317092). another five trials have been posted on clinicaltrials.gov on the use of sarilumab in covid-19 patients, three of which have started recruitment. two studies are enrolling hospitalized covid-19 patients aiming at evaluating the safety and effectiveness of low or high dose i.v. sarilumab (nct04315298; nct04327388). the other study is recruiting patients with moderate/severe pneumonia associated with sars-cov-2, and the primary endpoint is the survival without the need for ventilator utilization at day 14 (nct04324073). in addition to il-6, also il-1 plays an important role in crs [74, 75] . il-1β and il-1α increase acute-phase signaling, homing of immune cells to the site of primary infection and epithelial cell activation, both inducing the production of many other cytokines [75, 76] . il-1β can also drive proinflammatory activity in the respiratory tract as shown by its presence in the bronchoalveolar lavage fluid of patients with lung injury [76] . anakinra is a 17 kd recombinant, non-glycosylated human il-1ra that blocks il-1α and il-1β. it was approved for treating ra, cryopyrin-associated periodic syndromes, and still's disease [77] . it has been reported to be safe and effective in the management of sepsis-associated mas, in particular those with increased liver enzymes, hypofibrinogenemia, and thrombocytopenia [78] . conversely, the other anti-il1β inhibitor, canakinumab, has not been demonstrated to be beneficial in mas [79] . as of now, four clinical trials are recruiting patients with covid-19, severe acute respiratory failure, and crs, aiming at evaluating the safety and effectiveness of anakinra alone or in combination with anti-il-6 agents (nct04330638, nct0432402, nct04357366, nct04339712). janus kinase inhibitors, also known as jak inhibitors, are a class of orally administered targeted synthetic immunosuppressants that act by inhibiting the activity of one or more of the jak family members (jak1, jak2, jak3, tyk2), thereby interfering with the jak-stat signaling pathway. several inflammatory cytokines, involved in autoimmunity diseases, by binding to their receptors, initiate a jak dependent phosphorylation cascade constituting the signaling pathway of gene transcription. hence, drugs that inhibit the activity of jak block cytokine signaling. these inhibitors have therapeutic application in the treatment of cancer, ra, psoriasis, psoriatic arthritis, and inflammatory bowel diseases [80] . by now three jak inhibitors are approved for the treatment of rheumatic conditions, tofacitinib, baricitinib, and upadacitinib. tofacitinib is a specific inhibitor of jak3 and to a lesser extent jak1 and jak2. baricitinib reversibly inhibits jak1 and jak2, with moderate activity against tyk2 and significantly less against jak3, whereas upadacitinib is a selective inhibitor of jak1 [81] . pharmacology studies, in vitro, demonstrated that all three anti-jak antibodies can inhibit jak1/2-dependent cytokines (il-6 and ifn-γ) and the jak1/tyk2-dependent cytokines (il-10 and ifn-α), whereas tofacitinib and upadacitinib are the most potent inhibitors of the jak1/3-dependent cytokines (il-2, il-4, il-15, and il-21) [81] . considering the above data, the adoption of these drugs for crs management could be useful. indeed, preclinical studies on murine models of hemophagocytic lymphohistiocytosis (hlh) and mas showed the efficacy of jak inhibition [82, 83] . jak inhibitors have also gained the attention of researchers in the scenario of sars-cov-2 infection, for their demonstrated antiviral properties. most viruses enter cells through receptor-mediated endocytosis. one of the known regulators of endocytosis is the ap2-associated protein kinase 1 (aak1); thus, aak1 inhibitors can interrupt the passage of the virus into cells and can be helpful in preventing virus infections [84] . among aak1 inhibitors, baricitinib has shown the highest affinity, being able to inhibit aak1 at the standard therapeutic dosage for ra [85] . furthermore, the use of baricitinib appears to be particularly safe also in combination with antiviral drugs considering its minimal interaction with the cyp drug-metabolizing enzymes [86] . tofacitinib showed no detectable inhibition of aak1, whereas no data are yet available on the effect of upadacitinib on aak1 [87] . currently an italian trial is recruiting patients with mild to moderate sars-cov-2 infection (nct04320277). patients will be treated with a combination of baricitinib and antiviral therapy with ritonavir. the primary endpoint of the trial is the percentage of patients requiring transfer to icu as compared with the rate of transfers observed in controls. another european trial is starting recruitment of patients with moderate to severe sars-cov-2 infection; baricitinib will be used in monotherapy compared to lopinavir/ritonavir, hiq, and il-6 inhibitor (nct04321993), all administered alone. tnf-α belongs to a large family of cytokines known as the tnf superfamily. it is produced mainly by activated macrophages, nk, t, and b cells, and exerts its action through two receptors called tnfr1 and tnfr2. after binding to its receptors, tnf-α leads to a myriad and often conflicting effects reflecting complex cross-talk mechanisms [88] . it can mediate both apoptosis and cell activation, proliferation of b cells, and enhancement of cytotoxic activity of nk cells. the nf-κb activation pathway following tnf-α binding also induces the production of pro-inflammatory cytokines such as il-4, il-6, and il-8, leading to extensive tissue damage such as vascular leakage and lung injury seen in many chronic inflammatory diseases [89, 90] . as for il-6, tnf-α is responsible for systemic inflammatory manifestations such as fever and cachexia and has been shown to be a central cytokine in the activation and maintenance of crs [75] . tnf-α is a potent antiviral cytokine that acts directly by killing the virus-infected cells prior to maximal virus replication. however, it is known that the viral spike protein of sars-cov-2 is able to induce a tnf-α-converting enzyme (tace)-dependent shedding of the ace2 ectodomain, which is coupled to tnf-α production and is crucial for the penetration of the virus into the cell [90, 91] . based on these observations, tnf-α appeared to be an attractive therapeutic target. treatment of mas patients with etanercept (a fusion protein made from the combination of two soluble human 75k tnf-r linked to an fc portion of an igg1) has already been described, but data are scarce and contradictory [92, 93] . a trial evaluating the efficacy and safety of adalimumab (human mab directed against tnf-α) in sars-cov-2 patients with severe respiratory failure and crs has recently been registered in the chinese clinical trial registry (chictr2000030089). rituximab (rtx) is a chimeric anti-cd20 mab, approved for the treatment of b cell malignancies, ra, and anca-associated vasculitis [94] [95] [96] . b cell ablation with rituximab has been observed to have efficacy in macrophage activation syndrome in patients with underlying epstein-barr virus (ebv) [97] . the proposed mechanism is the reduction of viral load via destruction of the reservoir of ebv-infected cells. unfortunately, rtx therapy has been shown to induce crs, probably caused by the rapid destruction of tumor cells and consequent changes of serum cytokine levels [98] . it seems that crs is a side effect of rtx therapy considering that it occurs mainly in patients with a very high tumor burden. thus, its use appears not to be indicated in crs secondary to sars-cov-2 infection. crs occurring during sars-cov-2 infection has a clinical and serological profile resembling that of secondary hlh. given the central role of cd8 t cells in secondary hlh, non-ablative inhibitors of t cell function are also attractive therapeutic choices [74] . cyclosporine (cys) is a cyclic undecapeptide that binds intracellularly to cyclophilin and suppresses calcium-dependent phosphatase calcineurin pathway activation. functional consequences are the block of t cell survival and activation and inhibition of il-2 production [99, 100] . it is used for the prevention of transplant rejection, as well as in different autoimmune conditions such as ra, psoriasis, and glomerulonephritis [101] . it has already been adopted as part of the standard protocol in familiar hlh patients [102] . it could also be relevant to underline that cys is able to inhibit in vitro the function of a transmembrane protein called p-glycoprotein that pumps out of the cell several drugs, including amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir, thus being crucial in the development of drug resistance to anti-retroviral therapy. cys has also been demonstrated to revert in vivo drug resistance to methotrexate in ra and psoriatic arthritis (psa) patients [103, 104] . on the basis of the anti-inflammatory activity and its ability to improve the effectiveness of antiviral therapy, low dose cys in combination with antiviral drugs might be rational in selected patients without severe renal involvement. abatacept is a dimeric fusion protein composed of the human ctla-4 extracellular domain and a human fcigg1 that binds with high-affinity cd80/cd86 molecules, thus impairing t cell activation and t-b cell cross-talk during the immune response [103, 105] . it is scheduled for use in ra and more recently psa patients. abatacept has been proposed as a valid option for interstitial lung disease associated with ra in several case series [106] and has already been proven in a few cases of mas refractory to standard intervention, demonstrating mild effectiveness and safety [107] . the main data on the use of anti-rheumatic drugs currently tested in clinical trials of covid-19 patients are resumed in table 1 . blood count (reduction in neut and plt), ast, alt, procalcitonin *, il-6 blood count, ast, alt, procalcitonin * blood count, ast, alt, procalcitonin * blood count, ast, alt, procalcitonin * * to exclude active infections from sources other than covid-19; ** sarilumab is being studied also at 11 mg/kg/i.v.; both tocilizumab and sarilumab are being tested also s.c. crs = cytokine release syndrome, plt = platelets, neut = neutrophils, alt = alanine transferase, ast= aspartate transferase, i.v. = intravenous, s.c. = subcutaneous, hiq = hydroxychloroquine, cq= chloroquine. as previously stated, sars-cov-2 infection represents an emergency scenario of an old challenge, which is the complex and interwoven link between infections and autoimmunity. this complex link has implications at the biological level in terms of individual susceptibility/resistance, as well as in the delicate balance to be reached with therapeutic options. polymorphisms in the hla locus have been shown to affect individual susceptibility with variants that confer resistance to some viral infections and predispose to autoimmune diseases and others that show more complex associations increasing the risks for both autoimmunity and infections [108] . susceptibility to several infectious diseases including hiv, hepatitis b, and influenza is associated with specific hla haplotypes. for instance, hla-a*11, hla-b*35, and hla-drb1*10 have been shown to correlate with susceptibility to influenza a (h1n1) infection. it would be important therefore to understand if specific hla loci are associated with susceptibility to sars-cov-2 or to the development of a protective immune response. while it is still early to have information on sars-cov-2 and hla, studies on 2002/2003 sars-cov did not show associations with hla-a, hla-b, and hla-drb1 allele frequencies [109] , whereas some variants of hla-drb1 seem to correlate with susceptibility to mers [110] . noteworthy, some hla-drb1 amino acid variants are associated with ra, conferring either susceptibility or resistance to this disease [108] . patients with autoimmune diseases are, indeed, at high risk of infections, due to endogenous (dysfunctional immune system) and external factors (i.e., immunosuppressants). in ra patients, the risk for infections is about double with respect to healthy individuals, and they are mainly located at the bone and joints, skin, soft tissues, and respiratory levels [111] . in ra, patient data on infection risk generally show that methotrexate (the gold standard immunosuppressants for inflammatory arthritis) and hiq are the therapies impacting the least in the increased susceptibility to infection, both being considered relatively safe [112, 113] . the risk of infections observed in ra patients treated with biologic drugs is generally reported to be higher compared with patients receiving conventional immunosuppressants [114, 115] . in a retrospective observational cohort-study, our group evaluated the role of methotrexate, corticosteroids, and tnf-α antagonists alone or in combined therapy on non-serious and serious infections in ra and spondyloarthritis (spa) patients. we identified an incidence ratio/100 patient-years of 36.3 for all infections, being 34.9 for non-serious and 1.4 for serious infections [116] . these results are similar to those reported from the corrona register on a larger ra u.s. patient population [117] . as confirmed by other authors, we also found that the combination of anti-tnf-α with corticosteroids was the most pro-infective treatment, whereas methotrexate alone was relatively safe [116] [117] [118] [119] . the corticosteroids/anti-tnf-α combination can indeed synergize in lowering tnf-α levels through different and independent mechanisms, with the consequent increase of the anti-inflammatory effect, but at the expense of a rise in the risk of infection [118, 119] . as reported by metanalyses and real-life studies, among biological agents, abatacept seems to be the safest in terms of infectious risk [120, 121] . autoimmune diseases and infections are already linked with alteration in disease activity. in autoimmune patients, indeed, infections may induce disease flare-up that may be followed by a severe clinical course, representing a frequent cause of death (20-55%) [122] . on the other hand, it is known that a higher disease activity is associated with a higher probability of developing infections [117] . in fact, high disease activity is the result of chronic inflammation against self, which can exhaust the immune resources and deviate the immune response from the danger signals delivered by pathogens [123] . conversely, infections may stimulate the immune system, thus leading to a reactivation of the underlying autoimmune disease. in view of the above, it is not surprising that, although autoimmune patients under immunosuppressive agents could be prone to sars-cov-2 infections, suspension of the ongoing conventional and biological therapy is contraindicated to avoid disease flares with a consequent increased risk of infection [124] . it could be hypothesized that the impairment of immune response caused by the ongoing therapy could be a double-edged sword. on the one hand, indeed, immunosuppression increases the risk and consequently the prevalence of sars-cov-2 infections in autoimmune patients, but on the other hand, it could decrease the risk of the aberrant hyperinflammatory response seen in patients with sars-cov-2. a registry of covid-19 in autoimmune patients has been created by the italian society of rheumatology. this registry could give useful information to clarify the above hypothesis and be of crucial help for therapeutic decision-making in this particular group of patients. whatever these results will show, one should keep in mind that co-infection can often be lethal, and thus, the use of anti-influenza and anti-pneumococcal vaccinations, already recommended in these patients [125] , assumes even more importance in this new scenario of the covid-19 pandemic. several trials evaluating the safety and effectiveness of immunosuppressants commonly used in autoimmune patients are ongoing in patients with covid-19 and crs, some of which are achieving promising results. however, such a use should follow a multidisciplinary specialist approach, be accompanied by close monitoring, be tailored to patient's clinical and serological features, and be initiated at the right time to reach the best results. it is also important to take into account that these drugs, pivotal in the treatment of many autoimmune patients, 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commons attribution (cc by) license acknowledgments: we sincerely thank raffaele d'amelio for the critical reading of the manuscript. the authors declare no conflict of interest related to the manuscript. key: cord-348391-xytmq2f2 authors: wyganowska-swiatkowska, marzena; nohawica, michal; grocholewicz, katarzyna; nowak, gerard title: influence of herbal medicines on hmgb1 release, sars-cov-2 viral attachment, acute respiratory failure, and sepsis. a literature review date: 2020-06-30 journal: int j mol sci doi: 10.3390/ijms21134639 sha: doc_id: 348391 cord_uid: xytmq2f2 by attaching to the angiotensin converting enzyme 2 (ace2) protein on lung and intestinal cells, sudden acute respiratory syndrome (sars-cov-2) can cause respiratory and homeostatic difficulties leading to sepsis. the progression from acute respiratory failure to sepsis has been correlated with the release of high-mobility group box 1 protein (hmgb1). lack of effective conventional treatment of this septic state has spiked an interest in alternative medicine. this review of herbal extracts has identified multiple candidates which can target the release of hmgb1 and potentially reduce mortality by preventing progression from respiratory distress to sepsis. some of the identified mixtures have also been shown to interfere with viral attachment. due to the wide variability in chemical superstructure of the components of assorted herbal extracts, common motifs have been identified. looking at the most active compounds in each extract it becomes evident that as a group, phenolic compounds have a broad enzyme inhibiting function. they have been shown to act against the priming of sars-cov-2 attachment proteins by host and viral enzymes, and the release of hmgb1 by host immune cells. an argument for the value in a nonspecific inhibitory action has been drawn. hopefully these findings can drive future drug development and clinical procedures. viruses are one of the oldest organisms on earth. they consist simply of a protein envelope and nucleic acids which renders them unable to replicate outside of a host [1] . nevertheless, they are extremely adaptable and can rapidly alter their structure to suit the environment. while dna viral copies are near exact, rna viruses are more often dissimilar. genetically compatible viruses can undergo recombination and create very difficult-to-treat infections [2] . by exchanging information, they can spread immunity against medical treatments, or the host immune system [3] . orthomyxoviridae, an example of a family of rna viruses which cause a disease commonly known as influenza or flu, can both rearrange compatible genes and mutate on a regular basis in order to remain invisible [4] . that is why a new flu vaccine is needed every year [5] . due to quickly developing resistance to antiviral pharmaceuticals, vaccination is currently the most effective protection against viruses. however, there is building evidence that herbal medicines, sars-cov-2 entry has been successfully inhibited in vitro by direct ace2 inhibition [5] . targeting ace2 would allow for specific intervention in the binding of sars-cov-2 [44] . direct inhibition of ace2 functioning could however have poor long-term effects on the body due to its importance in controlling blood pressure, as well as lung, kidney and cardiovascular function [45] [46] [47] . pharmacological ace inhibitors such as lisinopril are not effective in reducing ace2 expression and at only 42% homology between the two enzymes, which can be inferred from their opposing physiological function, ace inhibitors are not competitive enough at the ace-2 attachment site with the sars-cov-2 to prevent infection [48] . epidemiological studies assessing chronic conditions such as cardiovascular disease have found an association between dietary habits and prevalence [49, 50] . phenolic compounds from green tea, coffee and wine were identified as of specific preventative interest [51] [52] [53] . all phenol extracts from these foods were found to have a concentration dependent effect on inhibiting ace activity; however, no data on ace-2 was present [54] . polyphenols, including green tea phenolic compounds, have a reputation for enzymatic inhibition, and have been reported to inhibit sugar digesting enzymes as well as lipases and proteases [55] [56] [57] . this has been noted well in the human digestive tract, which poses a barrier to their further phenolic absorption, but benefits from enzymatic inhibition in various, notably low-sugar, diets [58] . this can be attributed to phenolic compound dampening saccharolytic enzyme, e.g., invertase activity by 20% [59] . crystal structure modeling of both ace-2 and sars-cov-2 s protein has identified flavonoids, a subgroup of phenolic compounds, as suitable candidates for binding and inhibiting both molecules in silico [60] . assessment has started on these compounds in vitro, and there are successes showing sars-cov-2 inhibition with phenolic compounds also effective against sars-cov [61, 62] . studies show that in order to complete the endocytic entry in to the cell, the sars-cov-2 virus needs further priming or cleavage of its s protein. this has been reported to be viable by tmprss2 [56, 63] or cathepsin l [64] proteinases, with further studies supporting that their dual inhibition completely abolishes the coronavirus family mers infectivity [65] . a broad spectrum, nontoxic protease inhibitor would be able to perform such a function. further analysis of phenolic compounds could give more pharmacological guidance in this matter. while long term proteinase and ace-2 inhibition could be detrimental to cellular function and bodily homeostasis, targeted treatment partially reducing the effectiveness of coronavirus s protein attachment to the ace2 or to the priming proteinase could have the potential to drop the sars-cov-2 viral load before a state of septic shock is reached at the peak of infection. hmgb1 might be a critical molecule to allow innate immune cells to respond to both infection and injury, and is necessary for mammalian survival as shown by a knockout murine model which displays lethal hypoglycemia [66] . serum hmgb1 levels were consistently found to be significantly higher in septic patients who did not survive than those who did [35, 67, 68] . intratracheal administration of hmgb1 has been shown to induce lung neutrophil infiltration, local production of proinflammatory cytokines (e.g., il-1, and tnf), and acute lung injury [69] . traditional medicine and hospitalization cannot offer much more against sars-cov-2 than symptomatic relief such as the use of oxygen. some hmgb1 inhibitors like anti-ifn-γ antibodies, intravenous immunoglobulin, and minocycline seem to be helpful [70] . anti-hmgb1 antibodies have also been shown to dose-dependently protect mice against lethal endotoxemia [35] , as well as endotoxin-induced acute lung injury [69, 71] while normally corticosteroids reduce inflammation, they are helpless at fighting a hmgb1 induced cytokine storm [34] . moreover, intravenous liquids containing nutrient solution and glucose may have serious side effects, such as significantly increased viral load or diabetic ketoacidosis [72] . on the other hand, insulin has a benefit of lowering hmgb1 levels [73] . interestingly, both antithrombin iii and thrombomodulin decrease hmgb1 in vitro [74] . a search of hmgb1 inhibitors shows a number of results, many of which are herbs and herbal constituents which have direct suppressive actions against this proteins activity. counter intuitively, nicotine also significantly suppresses hmgb1 in the lungs [75] . the low molecular weight fraction of an aqueous extract of the chinese herb angelica sinensis (dang gui) is protective against lethal experimental sepsis and endotoxemia in a dose dependent manner. during laboratory induced lethal endotoxemia, 90% of mice which had the extract administered daily survived, vs. 30% survival in the control group, and during laboratory induced lethal sepsis 70% of mice survived when the extract was given daily, vs. 25% control. in the sepsis model, the administration of the extract did not begin until 24 h after the onset of sepsis, at which point some of the test subjects had already succumbed. this effect was in vitro shown to be non-cytotoxic to macrophages at any of the tested concentrations [76] . hmgb1 secretion was attenuated by the extract due to reduced translocation from the nucleus to the cytoplasm of activated macrophages, showing that even late administration of the dang gui extract significantly reduces serum levels of hmgb1, and can be attributed to the rescue of mice in part due to the attenuation of systemic hmgb1 accumulation. inhibition of hmgb1 is therefore likely a key element in preventing septic shock induced death, as it is the only high mobility group protein with a cytokine-like activity which is important to starting and maintaining the septic response [77, 78] . ferulic acid and a. sinensis polysaccharide are the main components of dang gui extract, and the most biologically active. the polysaccharide has been extracted and shown to be inhibitory to viral replication in a murine leukemia virus in vivo model, also exhibiting anti-inflammatory properties [79] . ferulic acid, while anti-inflammatory via inhibition of nitric oxide (no) production, was excluded from a. sinensis mediated hmgb1 inhibition [76] , however it does show some ability to inhibit human immunodeficiency virus (hiv) proteases [80] . salvia miltiorrhiza (danshen) is also a natural remedy from the world of chinese medicine which has proven experimentally to interact with hmgb1. traditionally used to treat cardiovascular disorders, it was shown to be protective against lethal lps-induced endotoxemia and sepsis by decreasing hmgb1 levels in vivo in a murine model [81] . experiments with the danshen extract (main bioactive ingredient: danshensu) also demonstrated successful administration 24 h after the onset of sepsis. this proves that the hmgb1 inhibition by the herbal extract of danshen can be preventatively inhibitory to the septic state, and also aid to arrest and reverse it. as an in vitro enterovirus treatment, ethyl acetate extracts of danshen were shown to have the strongest antiviral effect when administered along with the virus, suggesting interference in enterovirus entry mechanisms [82] . danshen extract also possesses direct anti-viral activity as has been found to inhibit the hepatitis b reverse transcriptase [83] . just as dang gui and danshen, epigallocatechin gallate (egcg-the main catechin found in green tea extracts) is an active natural extract able to rescue mice even if it is administered after the onset of induced sepsis. camellia sinensis is a source of a polyphenolic group of compounds called catechins. the most prominent green tea catechins are egcg, epigallocatechin, and epicatechin [84] . these molecules are well known for their antitumor, antioxidative, and antimicrobial activities [85, 86] . out of the three catechins found in green tea, only egcg was found to inhibit lps and cecal ligation and puncture (clp) induced sepsis in a dose dependent manner, with up to 29% greater survival rate than control mice, even if treatment was delayed up to 24 h after the onset of sepsis. the effect was shown to coincide with egcg (10 mm) almost completely eliminating hmgb1 release and macrophage cell surface clustering [87] . green tea leaf (camellia sinensis folium) extract also reduces endotoxin-induced release of hmgb1 and is therefore proposed to possess the ability to decrease mortality from sepsis if taken regularly. complete inhibition of hmgb1 in vivo was seen under green tea extract doses as low as 10 µl/ml or 1 ml/kg, with no in vitro cytotoxicity of egcg to macrophage cultures [88] . the mechanism through which egcg interacts and modifies the kinetics of hmgb1 are still elusive. reports show that egcg can bind to lipid raft associated receptors [89] [90] [91] [92] . macrophages depend on lipid raft complexes to deliver lps and signal an inflammatory state [93] . an analysis of structural motifs of egcg could present a solution as to why it would locate to these lipid rafts, and whether its phenolic, enzyme inhibitory, activity plays a part in stopping the release of hmgb1. in addition, egcg was shown to inhibit neuraminidase activity and viral genome synthesis of influenza, resulting in less effective cellular infection and viral replication. this disruption is thought to be caused by egcg structural analogy to glycosidases which occupy cellular polysaccharides. this mimicry can inhibit haemagglutinin and neuraminidase attachment to cellular membrane polysaccharide targets and stop influenza virus invasion and release respectively [94] [95] [96] [97] . haemagglutinin proteins, while not explicitly present in sars-cov-2, are present in the coronavirus family, and are utilized in cellular entry by the human coronavirus hku1 [98] . other mechanisms have also been studied for different viral families. hepatitis b viral (hbv) entry is dependent on na+-taurocholate cotransporting polypeptide (ntcp) expressed at the membrane of human hepatocytes. a clathrin basket endocytosis, mediated by egcg, caused a drop in ntcp surface expression and reduced hbv infectivity [99] . herpes simplex virus (hsv) attachment was shown to be inhibited by the broad-spectrum activity of egcg which successfully blocks attachment to heparan sulfate or sialic acid [100] , while the speed of ebola virus infection was slowed by egcg inhibition of human cell-surface heat shock protein a5 and therefore ebola virus attachment [101, 102] ginseng, rich in ginsenoside, is another herb with potent anti-inflammatory effects. the antiseptic activity of ginsenoside rh1 (one of the main active constituents of the ginseng root extract) has been noted in hmgb1-activated human umbilical vein endothelial cells (huvecs) and mice. ginsenoside rh1 increased the survival rate in a mouse sepsis model. it also significantly reduced hmgb1 release in lps-activated huvecs. furthermore, it suppressed the production of tnf-α, il-6, activation of nf-κb and extracellular signal-regulated kinase (erk-1/2) by hmgb1. ginsenoside rh1 also inhibited hmgb1-mediated hyperpermeability and leukocyte migration in mice. in addition, treatment with ginsenoside rh1 reduced the clp-induced release of hmgb1, sepsis-related mortality and tissue injury in vivo [103] . an extract from korean red ginseng significantly protected mice in experimental sepsis by decreasing tnf, il-1, il-6, and ifn-γ production via inhibition of nf-κb activation. it is likely that korean ginseng will also reduce hmgb1 levels, because cytokines under the control of nf-kb, such as tnf and ifn-g, induce hmgb1 [104] . in fact, ginsenoside has been shown to decrease hmgb1 levels in a human uterine fibroid cell model [105] . its direct anti-viral function has also been proven against influenza strain h1n1, which was prevented from attaching to host α 2-3' sialic acid receptors by ginsenoside interaction with viral haemagglutinin when administered topically to the viral infection site intranasally [106] . the main medicinal parts of licorice are its roots and rhizomes. numerous studies have shown licorice to be antiviral against hepatitis c and hsv [107, 108] , anti-inflammatory [109, 110] , antioncogenic and antimicrobial [111] . glycyrrhizic acid (ga) and glycyrrhetinic acid (gta) are the specific chemical compounds that may be isolated from the licorice plant. it has been found that depletion of sirtuin 6 (sirt6) suppressed the number of human nasal epithelial cell cilia, and dramatically induced hmgb1 translocation from nucleus to cytoplasm in an epithelial tumor cell line. gta has been shown to have anti-inflammatory and anti-allergic activity: directly binding to hmgb1 protein extra-cellularly to inhibits its cytokine activities through a scavenger mechanism. in vitro studies using the 18-β-stereoisomer of gta to enhance sirt6 expression levels have shown inhibited translocation of hmgb1 protein from nucleus to cytoplasm and reversing its extracellular accumulation stimulated by lps. [112] ga was also found to significantly attenuate lung injury and decrease the production of inflammatory factors tnf-α, il-1 il-1β, and hmgb1, the release of which was stimulated with lps treatment. ga also induces autophagy by enhancing the number of autophagosomes, possibly helping to deal with any necrotic tissue [113] . ga can efficiently block hmgb1 directly, and reduce its devastating effects [114] . astragalus mongolicus polysaccharide (aps) pretreatment has been shown to effectively inhibit hmgb1-induced increased permeability in lung endothelial cells (ecs). signal transduction study has shown that aps inhibition of hmgb1 also affected a small guanylate rho, and its downstream effector rho kinase, in ecs, suggesting a multi layered involvement of aps [115] . rosmarinic acid (ra) extracted from perilla frutescens was shown to potently inhibit the release of hmgb1 and down-regulate hmgb1-dependent inflammatory responses in human endothelial cells. ra also inhibited hmgb1-mediated hyperpermeability and leukocyte migration in mice. furthermore, ra reduced clp-induced hmgb1 release and sepsis-related mortality [116] . ra has also proven effective in inhibiting viral replication and infection induced inflammation in a mouse model of japanese encephalitis virus mediated japanese encephalitis [117] . the ethanol extract of prunella vulgaris herb (eepv) contains polysaccharides, flavonoids, and other phenols [118, 119] . it inhibited hmgb1 release in lps-activated macrophages in a pi3k-sensitive manner and reduced serum hmgb1 level and lung hmgb1 expression in cecal ligation and clp-induced septic mice. eepv is an inducer of heme oxygenase 1, which in turn reduces hmgb1 under lps stimulus [120] . eepv also has some antiseptic and anti-inflammatory potential [121] . it has been shown to exhibit anti-hiv, as well as anti-hsv (type 1 and 2) activity [119] . the most active fraction of eepv is rich in caffeic acid, hence termed caffeic acid-rich fraction [122] . prunella vulgaris extracts have been proven to inhibit virus/cell interactions as well as host binding in hiv infection models [123] . aspalathin and nothofagin extracted from rooibos have been shown to effectively inhibit lps-induced release of hmgb1, and suppressed hmgb1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules [124] . these molecules, as part of ethanol and alkaline extracts of the rooibos plant, have also been shown to reduce influenza a viral load at a late stage of the infection in vitro [125] . vicenin-2 and scolymoside derived from honeybush can also effectively inhibit lps-induced release of hmgb1, and therefore suppress hmgb1-mediated septic responses such as hyperpermeability, the adhesion and migration of leukocytes, and the expression of cell adhesion molecules. in addition, vicenin-2 and scolymoside suppress the production of tnf-α and il-6, and activation of nf-κb and erk1/2 by hmgb1 [126] . 2.3.11. lonicera caprifolium l. intravenous treatment with lonicerae flos, the main bioactive molecule of which is the chlorogenic acid, rescued lps-intoxicated c57bl/6j mice under septic conditions and decreased the levels of cytokines such as tnf-α, il-1β, and hmgb-1 in the blood [127, 128] . extract of inulae radix in lps-activated raw264.7 cells not only inhibited nf-κb luciferase activity, phosphorylation of iκbα, and inos/no, cox-2/pge2, hmgb1 release, but also significantly suppressed expression of intracellular and vascular adhesion molecules in tnf-α activated human umbilical vein endothelial cells [129] . the main active compound in inula helenium is alantolactone, showing significant suppression of il-8, tnf-α, and il-1β release [130] . rhodiola radix is a source of salidroside. the effect and mechanism of salidroside on sepsis-induced acute lung injury is mediated by the inhibition of inflammatory responses and hmgb1 production in bacterial lps-treated macrophages and mice. salidroside can also reverse the decreased sirt1 protein expression in lps-treated macrophages and mice [131] . furthermore, salidroside was shown to alleviate the sepsis-induced lung edema, lipid peroxidation, and histopathological changes and mortality. salidroside significantly decreases the serum tnf-α, il-6, no, and hmgb1 production, pulmonary inducible no synthase and phosphorylated nf-κb-p65 protein expression, and pulmonary hmgb1 nuclear translocation in septic mice [132] . boeravinone x, isolated from abronia nana, has antiseptic effects. it was shown to inhibit lps-induced release of hmgb1, and suppressed hmgb1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. comp 1 also suppressed the production of tnf-α and il-6, and the activation of nf-κb and erk1/2 by hmgb1 [133] . cucurbitacin e (cue), a tetracyclic triterpene isolated from cucurbitaceae plants, has proven to exert anti-inflammatory and immunologically regulatory activities. recent findings highlight that cue can ameliorate human bronchial epithelial cell insult and inflammation under lps-stimulated asthmatic conditions by blocking the hmgb1-tlr4-nf-κb signaling [134] . use of the aconitum carmichaelii tuber extract, of which the majority constituent is the c19-diterpenoid improved the liver function, decreased the pathological scores, and inhibited the expression of tlr4, nf-κb, hmgb1, and caspase-3 in a model rat liver injury treatment [135, 136] . plumbagin prominently hampered hmgb1 expression and subsequently quelled inflammatory cascades, as nf-κb, tnf-α and myeloperoxidase activity. it also interrupted the reactive oxygen species-hmgb1 loop as evident by restored liver function [137] . brown algae have been recognized as a food ingredient and health food supplement in japan and korea, and phlorotannins are unique marine phenol compounds produced exclusively by brown algae. phlorotannin rich extracts of the edible brown alga ecklonia cava were investigated against the hyper-inflammatory response in lps-induced septic shock mouse model. e. cava extract significantly increased the survival rate and attenuated liver and kidney damage in mice. in addition, e. cava attenuated serum levels of no, pge2, and hmgb-1. in macrophages, treatment with e. cava extract down-regulated inos, cox-2, tnf-α, il-6, and hmgb-1. in addition, e. cava suppressed the nik/tak1/ikk/iκb/nfκb pathway. dieckol, a major compound in the extract, reduced mortality, tissue toxicity, and serum levels of the inflammatory factors in septic mice [138] . in terms of sars viral infectivity, dieckol has been shown to inhibit the sars-cov 3clpro cysteine protease, therefore inhibiting the viral ability to replicate [139] . sars-cov 3clpro shares 99.02% sequence identity with sars-cov-2 3clpro [140]. kiwifruit peel extract is rich in polyphenols, the main of which are procyanidins representing 92% w/w of the total. a kiwifruit extract inhibited the production of inflammatory molecules such as il-6, il-1β, tnf-α pro-inflammatory cytokines, hmgb1 and granzyme b serine protease by activated monocytes [141] . study has shown that pelargonidin (pel) had effectively inhibited lps-induced release of hmgb1 and suppressed hmgb1-mediated septic responses, such as hyperpermeability, adhesion and migration of leukocytes, and expression of cell adhesion molecules. furthermore, pel inhibited the hmgb1-mediated production of tnf-α and il-6, as well as nf-κb and erk1/2 activation [142] . luteolin was demonstrated to reduce the release of hmgb1 through destabilizing c-jun and suppressing hmgb1-induced aggravation of inflammatory cascade through reducing akt protein levels [143] . quercetin reduced the lung permeability changes and neutrophil and macrophage recruitment to the bronchoalveolar fluid compared to the placebo mouse model. additionally, quercetin significantly reduced cox-2, hmgb1, inos expression, and nf-κb p65 phosphorylation. these results suggest that quercetin may be a promising potential therapeutic agent against sepsis [144] . baicalein from root of scutellaria baicalensis also significantly down-regulated the expression of matrix metalloproteinase-2/9 and attenuated hmgb1 translocation from the nucleus to the cytoplasm [145] . quercetin, luteolin, and baicalein have all been found to inhibit the sars-cov 3clpro protease [146, 147] . quercetin has also been identified to target the same enzyme in mers-cov, as it has a flavonoid structure with carbohydrate attachments which favor localization to the s1 and s2 sites on the s protein [148] . resveratrol has also been shown to inhibit hmgb1. hmgb1 migrates out of the nucleus during dengue virus infection. this migration is inhibited by resveratrol treatment and is mediated by induction of sirt1 which leads to the retention of hmgb1 in the nucleus. the enhanced transcription of interferon-stimulated genes by nuclear hmgb1 also contributes to the antiviral activity of resveratrol against dengue virus [149] . in murine and human serum of septic subjects hmgb1 persists to be secreted for a long time. peak levels in cell cultures are only reached 18 h after stimulation [35] . in contrast to other cytokines such as tnf with a peak at 90 min from initial stimulus, hmgb1 generates further waves of inflammatory cytokine production through rage, and toll-like receptors 2 and 4 [150] [151] [152] [153] . hmgb1 and other inflammatory cytokines are persistently elevated in sepsis [154, 155] . all of the identified herbal extracts and flavonoids exert suppression of hmgb1 activity. this is often accompanied by a drop in nf-κb activation, and reduction of tnf-α, il-1, il-6, and ifn-γ, signifying a reduction in the inflammatory response. multiple compounds boast anti-proteinase activity. even when the extract's anti-inflammatory effect is not direct, in multiple cases there are indications that it is due to an inhibition of a surface protease, which is either an effector of the internal inflammatory changes or is in turn necessary to cause them. the anti-proteolytic activity of herbal extracts covers an anti-inflammatory, as well as anti-viral effect across a variety of different viruses. many herbal extracts contain large groups of closely related polyphenols, making it difficult to separate them, and even more difficult to attribute the effect of the extract to just one of them. however, there is a lot of similarity in the effects of all the different extracts, just as there is similarity in the chemical structure of their main constituent molecules table 1 . luteolin was demonstrated to reduce the release of hmgb1 through destabilizing c-jun and suppressing hmgb1-induced aggravation of inflammatory cascade through reducing akt protein levels [143] . quercetin reduced the lung permeability changes and neutrophil and macrophage recruitment to the bronchoalveolar fluid compared to the placebo mouse model. additionally, quercetin significantly reduced cox-2, hmgb1, inos expression, and nf-κb p65 phosphorylation. these results suggest that quercetin may be a promising potential therapeutic agent against sepsis [144] . baicalein from root of scutellaria baicalensis also significantly down-regulated the expression of matrix metalloproteinase-2/9 and attenuated hmgb1 translocation from the nucleus to the cytoplasm [145] . quercetin, luteolin, and baicalein have all been found to inhibit the sars-cov 3clpro protease [146, 147] . quercetin has also been identified to target the same enzyme in mers-cov, as it has a flavonoid structure with carbohydrate attachments which favor localization to the s1 and s2 sites on the s protein [148] . resveratrol has also been shown to inhibit hmgb1. hmgb1 migrates out of the nucleus during dengue virus infection. this migration is inhibited by resveratrol treatment and is mediated by induction of sirt1 which leads to the retention of hmgb1 in the nucleus. the enhanced transcription of interferon-stimulated genes by nuclear hmgb1 also contributes to the antiviral activity of resveratrol against dengue virus [149] . in murine and human serum of septic subjects hmgb1 persists to be secreted for a long time. peak levels in cell cultures are only reached 18 h after stimulation [35] . in contrast to other cytokines such as tnf with a peak at 90 min from initial stimulus, hmgb1 generates further waves of inflammatory cytokine production through rage, and toll-like receptors 2 and 4 [150] [151] [152] [153] . hmgb1 and other inflammatory cytokines are persistently elevated in sepsis [154, 155] . all of the identified herbal extracts and flavonoids exert suppression of hmgb1 activity. this is often accompanied by a drop in nf-κb activation, and reduction of tnf-α, il-1, il-6, and ifn-γ, signifying a reduction in the inflammatory response. multiple compounds boast anti-proteinase activity. even when the extract's anti-inflammatory effect is not direct, in multiple cases there are indications that it is due to an inhibition of a surface protease, which is either an effector of the internal inflammatory changes or is in turn necessary to cause them. the anti-proteolytic activity of herbal extracts covers an anti-inflammatory, as well as anti-viral effect across a variety of different viruses. many herbal extracts contain large groups of closely related polyphenols, making it difficult to separate them, and even more difficult to attribute the effect of the extract to just one of them. however, there is a lot of similarity in the effects of all the different extracts, just as there is similarity in the chemical structure of their main constituent molecules table 1 . attenuates serum levels of no, pge2, and hmgb-1. down-regulates macrophage levels of inos, cox-2, tnf-α, il-6, and hmgb-1. inhibits sars-cov 3cl pro . reduces hmgb1 release. inhibits nf-κb activation thus reducing tnf, il-1, il-6 and ifn-γ production. inhibits hemagglutinin. inhibits production of il-6, il-1β, tnf-α pro-inflammatory cytokines, hmgb1 and granzyme b serine protease by activated monocytes. glycyrrhizic acid decreases tnf-α, il-1β, and hmgb1 production. stimulates sirt6 expression, which leads to hmgb1 nuclear retention. blocks extracellular hmgb1. directly binds hmgb1. inhibits nf-κb, iκbα. suppresses il-8, tnf-α and il-1β, icam-1 and vcam-1 release. planch ex miq. glycyrrhizic acid decreases tnf-α, il-1β, and hmgb1 production. stimulates sirt6 expression, which leads to hmgb1 nuclear retention. blocks extracellular hmgb1. directly binds hmgb1. inhibits production of il-6, il-1β, tnf-α pro-inflammatory cytokines, hmgb1 and granzyme b serine protease by activated monocytes. glycyrrhizic acid decreases tnf-α, il-1β, and hmgb1 production. stimulates sirt6 expression, which leads to hmgb1 nuclear retention. blocks extracellular hmgb1. directly binds hmgb1. decreases tnf-α, il-1β, and hmgb1 production. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [156] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory inhibits hmgb1 expression. stimulates sirt1 expression. decreases serum tnf-α, il-6, no, hmgb1 levels and inos, nf-κb-p65 expression. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [156] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory responses characterized by excessive accumulation of various proinflammatory cytokines. a ubiquitous nuclear protein hmgb1 is released by activated macrophages/monocytes in late stages of sars-cov-2 infection, and functions as a late mediator of lethal endotoxaemia and sepsis. circulating hmgb1 levels are elevated in a delayed fashion (after 16-32 h) in endotoxaemic and septic animals. administration of recombinant hmgb1 to mice recapitulates many clinical signs of sepsis, including fever, derangement of intestinal barrier function, lung injury, and lethal multiple organ failure. anti-hmgb1 antibodies or inhibitors (e.g., ethyl pyruvate, nicotine, stearoyl lysophosphatidylcholine and chinese herbs such as angelica sinensis) protects mice against lethal endotoxaemia, and rescues them even when the first doses are given 24 h after onset of sepsis. most of the active compounds from the selected herbal medicines discussed here have aromatic ring structures, some studded with hydroxyl groups, almost all having pentose or hexose sugars, and occasionally nitro, sulphate or acetoxy functional groups as illustrated in table 1 . experimental data suggests the majority have the ability to interrupt hmgb1 release and function, while some have also been shown to directly interrupt viral attachment and release. data regarding the exact binding moieties of herbal extracts is, however, still missing. it is unclear whether these functions are possible due to their specific chemical configuration which targets only viral or host proteins responsible for attachment, invasion, replication or release of the virus particle, or weather instead their general ability to inhibit a variety of glyosidic processes. effective enzymatic inhibition, competitive or not, could be attributed to a couple of factors. these molecules are amphiphilic allowing them to effectively embed within lipid membranes and protein rafts. common among the molecules is a multi-benzene ring steroid-like adaptation which serves as the lipophilic element, while a close resemblance of motifs to human glycolipids and glycoproteins can serve as a substrate for human and viral proteinases. this mimicry could slow down a variety of inhibits hmgb1 levels. antiviral, hepatitis b virus reverse transcriptase inhibition. majority of the molecules presented above belong to the polyphenol family: rotenoids, diterpenes, phenolic acids, flavonoids, phlorotannin, triterpene saponins, stilbenes and phenylpropanoids. an exception is the sesquiterpene lactone, which has an active lactone ring with an exomethylene group, and a polysaccharide composed of simple sugars with hydroxyl groups which give the molecule polarity and an anti-inflammatory activity. images reproduced from pubchem database [156] . the pathogenesis of sepsis is attributable, at least in part, to dysregulated systemic inflammatory responses characterized by excessive accumulation of various proinflammatory cytokines. a ubiquitous nuclear protein hmgb1 is released by activated macrophages/monocytes in late stages of sars-cov-2 infection, and functions as a late mediator of lethal endotoxaemia and sepsis. circulating hmgb1 levels are elevated in a delayed fashion (after 16-32 h) in endotoxaemic and septic animals. administration of recombinant hmgb1 to mice recapitulates many clinical signs of sepsis, including fever, derangement of intestinal barrier function, lung injury, and lethal multiple organ failure. anti-hmgb1 antibodies or inhibitors (e.g., ethyl pyruvate, nicotine, stearoyl lysophosphatidylcholine and chinese herbs such as angelica sinensis) protects mice against lethal endotoxaemia, and rescues them even when the first doses are given 24 h after onset of sepsis. most of the active compounds from the selected herbal medicines discussed here have aromatic ring structures, some studded with hydroxyl groups, almost all having pentose or hexose sugars, and occasionally nitro, sulphate or acetoxy functional groups as illustrated in table 1 . experimental data suggests the majority have the ability to interrupt hmgb1 release and function, while some have also been shown to directly interrupt viral attachment and release. data regarding the exact binding moieties of herbal extracts is, however, still missing. it is unclear whether these functions are possible due to their specific chemical configuration which targets only viral or host proteins responsible for attachment, invasion, replication or release of the virus particle, or weather instead their general ability to inhibit a variety of glyosidic processes. effective enzymatic inhibition, competitive or not, could be attributed to a couple of factors. these molecules are amphiphilic allowing them to effectively embed within lipid membranes and protein rafts. common among the molecules is a multi-benzene ring steroid-like adaptation which serves as the lipophilic element, while a close resemblance of motifs to human glycolipids and glycoproteins can serve as a substrate for human and viral proteinases. this mimicry could slow down a variety of processes, hypothetically even inhibiting a virus which is adapted to deal with host specific glycoproteins and lipids, while ill prepared to digest plant analogues. generally, phenols are capable of disrupting multiple enzymatic processes, both human and viral. as the human body is still the most effective weapon against infection, it is paramount to give the immune system enough time to mount an attack. a diffuse inhibition of viral and cellular surface proteolytic processes could limit the speed of viral infection and cellular damage, giving the immune system some extra time to fight. experimental data established hmgb1 as a late mediator of lethal endotoxemia and sepsis, with a wide-over 24 h-therapeutic window, which allows for the clinical management of lethal systemic inflammatory diseases. multiple therapeutic agents from herbal medicines have been identified as candidate compounds for the task. an analysis of combinations of extracts with complimentary functions, or a pharmacological generation of an optimum molecule, could further this field. molecular studies on protease inhibitors with human-polysaccharide or viral-polysaccharide mimicking docking motifs and difficult to digest phenolic groups could prove to be the most effective remedy against a well-established viral infection. clearer proof of direct inhibition of target proteases, molecules such as hmgb1, and receptors would provide a strong basis for pharmacological development. with many animal model studies already establishing a clear link in sepsis attenuation by hmgb1 inhibition, it is now 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treatment reveals a novel role for hmgb1 in regulation of the type 1 interferon response in dengue virus infection high mobility group 1 protein (hmg-1) stimulates proinflammatory cytokine synthesis in human monocytes the receptor for advanced glycation end products (rage) is a cellular binding site for amphoterin: mediation of neurite outgrowth and co-expression of rage and amphoterin in the developing nervous system andersson, u. rage is the major receptor for the proinflammatory activity of hmgb1 in rodent macrophages involvement of toll-like receptors 2 and 4 in cellular activation by high mobility group box 1 persistent elevation of inflammatory cytokines predicts a poor outcome in ards persistent elevation of high mobility group box-1 protein (hmgb1) in patients with severe sepsis and septic shock this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-306577-gq6fss5l authors: hsueh, wei; caplan, michael s.; qu, xiao-wu; tan, xiao-di; de plaen, isabelle g.; gonzalez-crussi, f. title: neonatal necrotizing enterocolitis: clinical considerations and pathogenetic concepts date: 2002-11-11 journal: pediatr dev pathol doi: 10.1007/s10024-002-0602-z sha: doc_id: 306577 cord_uid: gq6fss5l necrotizing enterocolitis (nec), a disease affecting predominantly premature infants, is a leading cause of morbidity and mortality in neonatal intensive care units. although several predisposing factors have been identified, such as prematurity, enteral feeding, and infection, its pathogenesis remains elusive. in the past 20 years, we have established several animal models of nec in rats and found several endogenous mediators, especially platelet-activating factor (paf), which may play a pivotal role in nec. injection of paf induces intestinal necrosis, and paf antagonists prevent the bowel injury induced by bacterial endotoxin, hypoxia, or challenge with tumor necrosis factor-a (tnf) plus endotoxin in adult rats. the same is true for lesions induced by hypoxia and enteral feeding in neonatal animals. human patients with nec show high levels of paf and decreased plasma paf-acetylhydrolase, the enzyme degrading paf. the initial event in our experimental models of nec is probably polymorphonuclear leukocyte (pmn) activation and adhesion to venules in the intestine, which initiates a local inflammatory reaction involving proinflammatory mediators including tnf, complement, prostaglandins, and leukotriene c4. subsequent norepinephrine release and mesenteric vasoconstriction result in splanchnic ischemia and reperfusion. bacterial products (e.g., endotoxin) enter the intestinal tissue during local mucosal barrier breakdown, and endotoxin synergizes with paf to amplify the inflammation. reactive oxygen species produced by the activated leukocytes and by intestinal epithelial xanthine oxidase may be the final pathway for tissue injury. protective mechanisms include nitric oxide produced by the constitutive (mainly neuronal) nitric oxide synthase, and indigenous probiotics such as bifidobacteria infantis. the former maintains intestinal perfusion and the integrity of the mucosal barrier, and the latter keep virulent bacteria in check. the development of tissue injury depends on the balance between injurious and protective mechanisms. cumstances are not uniform, nec may represent a syndrome, with common findings and a variety of etiologies. intestinal necrosis, representing a latestage response, is consistent with a common pathogenesis, but disparate etiologies are possible. necrosis of the intestine can occur at any age following the sudden, complete occlusion of the blood supply to the bowel. in the newborn, thromboemboli secondary to intravascular catheters may cause bowel infarction. however, since neonatal nec cannot be traced to thromboemboli, it is considered nosologically distinct from this kind of bowel infarction. therefore, in the following discussion, nec is understood to exclude cases of bowel infarction associated with thromboembolic lesions. nec remains a leading cause of morbidity and mortality in neonatal intensive care units, with a reported incidence of approximately 10% among very low birth weight infants (ͻ 1500 g) [1] , and a mortality of 26% [2] . a disease of serious prognosis, advanced cases of nec may cause multisystem organ failure [3] . of the 2500 cases occurring annually in the united states [4, 5] , 20 -60% require surgical treatment [6] . at least 80% of patients are preterm, or have low, or very low birth weight, and the incidence of the disease is inversely proportional to the gestational age [4, 7, 8] . advances in the supportive care of premature babies, such as the use of surfactant, improved technologies for mechanical ventilation, and wider availability of skilled personnel, enable the very premature to survive, and in so doing increase the population of patients susceptible to nec. thus, it may be that despite medical advances that would potentially reduce the incidence of the disease, the incidence of nec remains unchanged over the last 20 years [1, 9] . infants of extremely low birth weight (ͻ 1000 g) and those 28 wk or less of gestational age are at greater risk of nec than infants born closer to term. the severity of the disease, risks of complications, and mortality are greater in infants of extremely low birth weight [10] . nec is uncommon in term infants, in whom it usually appears within 2 to 3 days after birth, whereas in the preterm it begins at 10 to 15 days after birth [11] . presumably, a postnatal insult is followed by the pathogenetic events that lead to the tissue devastation characteristic of nec. the initi-ating and pathogenetic factors may differ in patients of different age groups. in any case, the clinical consequences do not differ substantially in the various patient populations, including the infants of extremely low birth weight or extreme prematurity [10] . the symptoms have been staged according to widely used criteria [12, 13] . the infant manifests abdominal distension (among the most common signs of nec), vomiting, increased gastric residual, lethargy, apnea, bradycardia, or guaiac-positive stools. in stage i, there are no clear radiological signs, and these nonspecific manifestations suggest the disease but give no indication of the status of the bowel or the prognosis. in stage ii, the diagnosis is clearly established, with the appearance of pneumatosis intestinalis or free air in the portal vein. stage iii indicates more advanced disease, as manifested by shock, disseminated intravascular coagulation, acidosis, thrombocytopenia, and sometimes intestinal perforation. the predominant anatomic lesion of nec is coagulative or ischemic necrosis [14 -17] (fig. 1a-c) . the usual site is the ileocolic region. this may be because of remoteness of the ileocolic arterial branches from the main blood supply of the superior mesenteric artery, which also supplies the proximal intestine. in about half the cases, the necrosis involves both the small and large intestine; continuous or discontinuous involvement occurs in approximately equal proportions [16, 17] . the affected bowel is grossly distended, lusterless, and gray or greenish-gray, but it may be dark purple or black in the areas containing extensive hemorrhage. the soft, fragile wall may perforate when the involvement is severe and transmural. perforation tends to occur at the junction between normal and necrotic bowel, but it may appear in the midst of a devitalized region, and sometimes at more than one site. gas bubbles, which may be grossly visible in the intestinal wall, involve the entire colon more commonly in the term infant than in the premature infant [16] . ischemia in nec accounts for the necrosis, but the mechanism remains unresolved. nowicki [18] distinguished extrinsic and intrinsic mecha-neonatal necrotizing enterocolitis nisms of vascular regulation. extrinsic vascular regulation integrates the circulation of the intestine with systemic cardiovascular reflexes. an atavistic "diving reflex" (so named after the physiological changes noted in seals upon diving) [18] has been hypothesized in neonates who experience severe anoxic episodes, during which blood is diverted preferentially to the heart and the brain, to the detriment of the abdominal organs. although the diving reflex hypothesis is supported by much experimental evidence in animals [18] , it cannot satisfactorily explain all the clinical observations in nec. presumably, the reflex takes place as a result of a postulated ischemic insult during parturition [19] , whereas the manifestations of nec usually start during the 2nd wk of postnatal life. vascular reactivity in early postnatal life has been assumed to differ from that of older subjects. however, the intestinal vasculature of 2-3-day-old piglets manifests autoregulatory "escape" from sustained sympathetic stimulation, in the same manner as the intestine of older swine. experimentally, sympathoadrenergic stimulation causes transient intestinal vasoconstriction, and normal oxygen uptake is restored after 3 to 5 min [20, 21] . moreover, prospective clinical studies do not always establish an association between neonatal hypoxia or asphyxia and the development of nec: most patients with nec have no clinically apparent hypoxemia at birth [7, 18, 20] . these discrepancies by no means exclude an important participa-tion of autonomic neural influences in the development of nec. other extrinsic regulatory mechanisms, such as the participation of the renin-angiotensin axis in bowel ischemia deserve serious investigation. angiotensin receptors are densely distributed in the bowel [22] . this may explain why ischemic colitis that develops from mesenteric vasoconstriction during experimental cardiogenic shock cannot be prevented by total adrenergic blockade but is completely abolished by drugs such as captopril, which ablate the reninangiotensin axis [23] . the intrinsic vasoregulation of the intestine, defined as that "mediated by effector mechanisms produced and released within the intestine and its attendant circulation," has been studied in denervated intestinal segments and other in vivo and in vitro models [18] . a "metabolic theory" stresses homeostatic control by local tissue need for oxygen, and a "myogenic reflex theory" proposes vasoconstriction in the intestinal circulation in response to changes in venous pressure. presumably, labile, active myogenic vascular responses in the very young increase their susceptibility to intestinal ischemia [24] . other "intrinsic" vasoregulatory influences leading to intestinal ischemia include the potent agents that are considered central to a theoretical pathogenesis of nec (vide infra). the clinical situation is more complex than any hypothetical model centered upon experimental observations. as kosloske [25] observed, the chronology of clinical events is not always clear. in patients with congenital heart disease or cardiogenic shock, hemodynamic disturbances acquire a significant role in the causation of nec, but this does not gainsay the utility of clarifying the basic steps by which the disease is initiated and maintained. necrosis of the bowel can develop secondary to mesenteric thromboembolism. in neonates, thrombosis is usually an untoward effect of the placement of an umbilical artery catheter. however, in most patients with nec, no occlusion of large arteries can be identified. nec and infarction are probably different clinicopathological entities, even though both manifest coagulative necrosis. an infarct is usually single and should follow the distribution of the arterial blood supply. in contrast, nec is basically an inflammatory process, and a venule may be the initiating site of the pathophysiology. the affected areas are often multiple, are random, and are not necessarily related to the arterial supply. the early histological change of nec is coagulative necrosis, but inflammatory cells infiltrate when the disease progresses [17] . bacteria are important in nec, since the disease does not occur before the colonization of the intestine by bacteria. in a fetus whose intestinal contents are sterile, compromise of the blood supply may result in intestinal injury. in the healing process, atresia or stenosis may develop. anaerobic bacteria in the lumen of the bowel might be expected to proliferate in a segment of devitalized bowel. bacterial overgrowth in nec seems to exceed that in other diseases with ischemic bowel [17] . intestinal pneumatosis, the peculiar and characteristic finding seen in many cases of nec, is not observed in infarcts. the formation of gas bubbles within the wall of the intestine (fig. 1d) , develops largely as a result of the fermentation of intraluminal contents by bacteria, and is associated more with nec than with any other necrotizing conditions affecting the intestine. bacterial production of p-galactosidase, which reduces ph by fermentation of lactose, has been suggested to contribute to the development of nec [26] . the ability of colonizing bacteria to ferment lactose is not correlated with the production of nec [27] ; moreover, the endemic cases of nec are not consistently associated with a single infectious agent or with a particularly virulent organism that produces highly damaging toxins or that displays great entero-invasiveness or entero-aggregative ability. disparate microorganisms have been isolated from the stools of nec patients, and in some cases from both blood and stools: escherichia coli, klebsiella, enterobacter, pseudomonas, salmonella, clostridium perfringens, clostridium difficile, clostridium butyricum [28] , coagulase-negative staphylococci [29] , coronavirus, rotavirus, and enteroviruses [30] . intestinal inflammation affects about 90% of the patients with nec and is considered an appropriate host response to necrosis and proliferating bacteria [17] . inflammation tends to be less severe following sudden occlusion of the arterial circulation, as with thromboembolism, and much more conspicuous when devitalization of the bowel is gradual. according to ballance et al. [17] , the character of the inflammation in colitis of infectious origin differs from that in nec. microabscesses and crypt abscesses are common in infectious colitis, but they affect only 10% of patients with nec. moreover, extensive necrosis beyond the inflammation is a feature of nec that is generally absent in cases of infectious enterocolitis. regenerative changes in nec are usually marked by replacement of the mucosa by a cuboidal or tall epithelium displaying hyperchromatic nuclei, with mitotic activity and without mucin production. this layer covers granulation tissue or a partly reconstituted lamina propria with distorted, morphologically aberrant glands [15, 31] . regenerative changes may appear even in cases without a protracted history. ballance et al. [17] found reparative activity of recent onset in 68% of the patients, all undergoing surgery for the first time. these findings suggest that the disease may have started earlier than could be inferred from the degree of severity and/or duration of clinical symptoms. we developed a model of bowel necrosis in adult rats and mice by injecting endotoxin (lipopolysaccharide, lps) [32] , paf (platelet-activating factor, paf-acether) [33, 34] , tumor necrosis factor-␣ (tnf) [35] , or a combination of these agents. the rationale for using these agents is as follows: lps: nec is clearly associated with intestinal bacterial growth, since nec usually develops following oral feeding, and oral feeding markedly increases the growth of e. coli in the intestinal tract [36] . no single infectious agent has been isolated consistently from patients with nec. we hypothesized that resident intestinal flora such as e. coli and its toxic product, lps, would be causative agents of nec. paf: injection of lps induces endogenous production of paf [37, 38] , systemic administration of paf [39 -41] to animals mimics signs of shock, and paf antagonists prevent lps-induced shock [41, 42] . tnf: lps induces endogenous tnf production [38, 43, 44] and administration of tnf causes shock [45, 46] , whereas pretreatment of the animal with anti-tnf [46] ameliorates endotoxin shock and increases survival. paf is an endogenous phospholipid mediator produced by inflammatory cells, endothelial cells, platelets [39, 40, 47] , and bacteria of the intestinal flora, such as e. coli [48] . systemic administration of paf induces an immediate and sometimes transient hypotensive response. with large doses, the shock becomes profound, irreversible, and intestinal necrosis develops rapidly. early injury is usually detectable within 15 min. paf is probably the most potent systemically administered agent for inducing intestinal injury. in our experiments, as little as 2.5 g/kg often caused small intestinal necrosis of varying degree in the rat. since rat platelets are refractory to paf [33, 49] , the pathogenesis of necrosis cannot be due to the thromboembolic effect of paf. the necrosis, usually focal, involved the jejunum, ileum, especially the distal ileum, and/or cecum. with high doses, the entire small bowel could be affected. the necrosis began at the villus tip ( fig. 2a ) [33] , often involved more than half of the villus (fig. 2b) , and sometimes extended to the submucosa or even the serosa (fig. 2c ). although lps alone can cause hypotension and intestinal necrosis, the required dosage is often high (ͼ 5 mg/kg). lps is a potent "priming" agent for paf: a small dose of lps (0.5 mg/kg) acts synergistically with a low dose of paf (table 1 ) [33, 34, 50] . lps-induced intestinal injury is blocked by pretreatment with paf antagonists [32] , suggesting that this effect is mediated by endogenous paf. one probable reason why the small intestine, in particular the ileum, is especially sensitive to paf action, is its high content of paf receptors (paf-r). using quantitative polymerase chain reaction (pcr), we found that the ileum has the highest number of paf-r transcripts: (3.49 ϯ 0.15) ϫ 10 7 molecules/g rna [51] . the paf-r content of jejunum was only 56% of that of the ileum, and the spleen was only 30%. other organs, e.g., lung, kidney, heart, stomach, and liver, had less than 1% of that of ileum [51] . paf, even at doses below those causing bowel necrosis, almost doubled paf-r mrna in the intestine [51] . the increase was biphasic; the second peak (at 6 h) seemed dependent on endogenous paf and tnf [51] . in the small intestine, paf receptor was localized mainly in epithelial cells and eosinophils of the lamina propria [52] . paf has a short half-life in the blood, being rapidly degraded by serum acetylhydrolase into the biologically inactive lyso-paf [53] [54] [55] . paradoxically, the in vivo action of paf is prolonged. one mechanism that may account for this prolonged action is that paf induces its own production in tissues [56] . when paf antagonists were given before paf challenge, the production of paf (and paf-like phospholipids) was markedly reduced (table 2) [32, 56] . (since in these studies we assessed the biological activity, rather than chemical analysis of paf, we could not differentiate paf from paf-like phospholipids; the latter bind to paf receptor and have effects that are much like those of paf [57] ). the initial event following paf challenge is probably polymorphonuclear leukocyte (pmn) activation and pmn-endothelial adhesion. pmn-depletion markedly reduced paf-induced bowel injury [58 -60] . the major adhesion molecule involved in the paf effect is leukocyte ␤2-integrin, especially cd11b/cd18, since pretreatment with anti-cd11b or anti-cd18 antibody largely prevents pmn influx as well as paf-induced bowel injury [60] . anti-cd18 also prevents the paf-induced increase in endothelial [61] and mucosal [62] permeability. p-selectin-deficient mice and fucoidin-treated intercellular adhesion molecule-1 (icam-1) deficient mice are also protected from the adverse effects of paf [63] , suggesting a possible role of p-selectin. paradoxically, fucoidin, a potent inhibitor of selectins, shows no protective effect [62, 63] . the marked increase in pmn influx (adhered to vessels) is reflected by the increased myeloperoxidase (mpo) content in the intestine [62] . yet extravascular pmn infiltration is not found by histological examination, indicating that pmn transmigration into tissues is not required for necrosis. paf, platelet-activating factor; lps, lipopolysaccharide (bacterial endotoxin); tnf, tumor necrosis factor-␣. a all values were obtained 30 min after the injection of paf [33] . (in later studies, the dose of paf used to induce the same degree of bowel necrosis was reduced to 0.002-0.003 mg/kg, when pure c16-paf was used.) b all values were obtained 2 h after the injection of tnf [35] . c mild necrosis: involving top third of villi. d moderate necrosis: involving more than top one-third of villi, but confined to the mucosa. the final effector of paf causing cell injury is most likely reactive oxygen species (ros). one of the major endogenous sources of ros in the intestine is the xanthine dehydrogenase/xanthine oxidase system (xd/xo) [64] . xd, the precursor of xo, is constitutively and abundantly expressed in the intestinal villus epithelium [58, 65] , which catalyzes the conversion of hypoxanthine to xanthine, coupled with the reduction of nad ϩ to nadph. because xo uses molecular oxygen rather than nad ϩ as an electron receptor and thereby generates superoxide, xd to xo conversion (during ischemia) has been suggested to play the central role in intestinal reperfusion injury [64] . in normal rat intestine, the total xdϩxo content (xd/xo ratio approximately 80:20) is higher in the jejunum than in the ileum (the colon has low xdϩxo) [58] . interestingly, following paf challenge, it is the ileum that shows the most dramatic xd to xo conversion (more than twofold increase in xo) [58] . this change is rapid, detected at 15 min, and by 60 min, more than 60% of the total xdϩxo activity has converted to xo [58] . the conversion takes place mainly in the villus epithelial cells, but not in the crypt epithelium, and the major pathway is probably via activated protease [58] . how this activation is related to pmn activation and adhesion to endothelial cells, remains enigmatic. the central role of xo and ros in causing the injury is supported by pretreatment with allopurinol [58, 66] , a xanthine oxidase inhibitor, which largely prevents paf-induced bowel necrosis ( table 2 ). infusion of superoxide dismutase plus catalase also alleviates the injury [66] (table 2) . tnf has many proinflammatory actions [67] [68] [69] , such as inducing leukocyte and endothelial adhesion molecules, activating pmns and endothelial cells, and causing production of other cytokines [67] [68] [69] , including tnf itself [67] [68] [69] , eicosanoids [67, 68] , and paf [70, 71] . intravenous injection of tnf (1 mg/kg) also induces hypotension and mild intestinal injury in rats [35] . the effects of tnf and lps are synergistic: tnf (0.5 mg/kg), when combined with lps (200 mg/kg), causes profound shock and severe intestinal necrosis in rats [35] and mice [72] . paf is probably the endogenous the splanchnic bed is considered a major source of tnf production in vivo [73] . we have shown that lps (2 mg/kg) and paf (1 g/kg), at doses below those causing shock and intestinal injury, stimulate tnf gene expression and protein production in the rat's liver and small intestine, predominantly in the ileum [74] (fig. 3) . web-2086, a paf antagonist, only partially blocked lps-induced tnf mrna formation (fig. 3) , suggesting that lps induces tnf formation via both paf-dependent and paf-independent pathways. in normal intestine, tnf is constitutively expressed at very low levels within paneth cells [75] . during the acute stage of nec, tnf gene transcripts markedly increase not only within paneth cells, but also in lamina propria eosinophils, and infiltrating (but not resident) macrophages [76] . paneth cells are also rich in group iia phospholipase a 2 (pla 2 -iia) [77] , an acute phase protein, which is also upregulated by paf [78] . production of many proinflammatory cytokines, including tnf, is upregulated by transcription factors, such as nuclear factor b (nf-b) [79] . tnf activates nf-b in vitro [79, 80] , a pathway that may be involved in tnf self-activation. low doses of tnf (1 mg/kg) or paf (1 g/kg), which are below those causing shock and intestinal injury, increase the mrna of nf-b precursor, p50/p105, in the small intestine [81] . the action of paf is as potent as, but more rapid than, that of lps [81] . paf also rapidly induces nf-b nuclear translocation and activation in the intestine, mainly as p50 homodimers [82] . lps also activates nf-b, but as p50 -p65 dimer, and its effect is partly mediated via endogenous paf and tnf [83] . the role of this transcription factor is unclear, but preliminary experiments show that blocking nf-b with decoy [84] or with nf-b essential modulator (nemo) (ikk␥) binding peptide [85] attenuates paf-induced injury. a paf challenge increases gut mucosal permeability [86] . this event precedes cell necrosis, and occurs at doses below that causing necrosis [86] . paf alters the cytoskeletal structure of the intestinal epithelium and induces tyrosine phosphorylation of e-cadherin, an epithelial membrane component of the zona adherens [86] . this may be physiologic, since glucose-induced increased mucosal permeability is blocked by paf antagonists [86] . in nec, this action of paf may facilitate the entry of bacterial products, e.g., lps from the gut lumen into the tissues, triggering the inflammatory cascade. indeed, our data suggest that endogenous bacterial toxins from the intestinal lumen play a central role in paf-induced shock and bowel injury: (1) endotoxin-resistant mice are protected from paf-induced intestinal injury [87] ; (2) germ-free rats are protected from paf-induced prolonged shock and bowel injury, and the protection is lost when these animals are primed with exogenous lps [88] ; and (3) conventional rats, treated with combined antipaf induces tumor necrosis factor (tnf) mrna production in the rat liver and ileum. mrna from the liver and the small intestine was extracted 30 min after injection of a low dose (1 g/kg, iv) of paf, lps (2 mg/kg, iv), or web-2086 (1 mg/kg) plus lps. results calculated from northern blot analysis [62] . ␤-actin, a housekeeping gene, is used as the common denominator for calculation, and quantity of tnf mrna is expressed as ratio of tnf mrna/␤-actin mrna. sham-op., sham operation; lps, lipopolysaccharide (bacterial endotoxin); web, web-2086. (from hsueh et al. [132] , with permission.) biotics which markedly decrease intestinal bacteria, are protected to a large extent from the injurious effects of paf [88] (table 2) . paf probably causes intestinal injury and deleterious systemic changes via a synergistic action with endogenous bacterial toxins from intestinal bacteria [34, 89] . lps may not be the only bacterial product that synergizes with paf to produce tissue damage, since polymyxin b (which inhibits lps) alone was without protective effects [88] . paf has a prolonged in vivo action despite its short half-life in the circulation. furthermore, paf is a vasodilator in vitro [90] , but high doses cause sustained vasoconstriction of the splanchnic bed in vivo [90, 91] . these apparently paradoxical effects could be reconciled by the observations that leukotriene c 4 (ltc 4 ) [92] and norepinephrine [93] , which cause splanchnic vasoconstriction, are released after paf injection. moreover, in vivo administration of antagonists to peptide leukotrienes [34, 91] , or alpha blockers [91] , do not reverse shock, but prevent paf-induced intestinal injury ( table 2 ). the complement system, especially c5, may also participate in producing nec, since the injection of paf activates the complement system in vivo [59] , and c5 deficient mice are protected from tnf/lps-or paf-induced injury [59, 70] . nitric oxide (no) is produced endogenously by three nitric oxide synthase (nos) isoforms: the constitutive neuronal (type i) nnos, the inducible (type ii) inos, and the endothelial (type iii) enos [94] . more than 90% of the total nos in the small intestine is nnos [95] (fig. 4a,b) . although inos is constitutively present (mainly in the epithelial cells), it accounts for less than 10% of the total nos activity [95, 96] , and enos is barely detectable in the intestine. paf rapidly decreases intestinal nnos protein, mrna, and enzyme activity [95] (fig. 4c) , but has little effect on inos [96] . interestingly, the degree of injury is inversely re-lated to the nnos activity, suggesting a protective role of nnos. the protective role of no is supported by the following observation: (1) nos inhibitor l-name aggravates paf-induced necrosis [97] ; (2) inos inhibitors are protective only when there is "sufficient" nnos activity [96] ; and (3) no donors significantly reduce paf-induced bowel injury [96] . no may help to maintain the integrity of the mucosal barrier and the microvasculature, to increase blood flow, and to inhibit leukocyte adhesion [98] . several conditions that involve decreased oxygen delivery to the mesenteric circulation are associated with an increased risk of nec in human infants. these include asphyxia [99] , cyanotic congenital heart disease [100] , decreased mesenteric blood flow as reported in intrauterine growth retardation [101] , and maternal cocaine use [102] . in animal models of nec, hypoxia is associated with the development of ischemic bowel necrosis [103] but does not define the mechanism of bowel injury. we first explored the role of hypoxia in ischemic bowel necrosis using young (25-30-day-old) adult male sprague-dawley rats [104] . the animals were exposed to acute severe hypoxia by placing them in 100% nitrogen for 2 min, or to subacute moderate hypoxia by placing them in a 10% oxygen atmosphere for 15 or 30 min. plasma levels of paf were markedly elevated in the animals treated with 30 min of moderate hypoxia when compared with controls, and were also elevated in animals treated with only 2 min of severe hypoxia [104] . thirty minutes of moderate hypoxia produced mild to moderate ischemic bowel necrosis, with no evidence of necrosis in any other organs. (two minutes of severe hypoxia were not sufficient to induce bowel injury.) the bowel injury was prevented by two structurally unrelated paf antagonists, web 2086 and sri 63-441. we concluded that hypoxia results in a rapid increase in endogenous paf levels and that paf is a mediator of hypoxic intestinal injury. in addition to decreased mesenteric oxygen delivery, bacterial colonization of the gastrointestinal tract is generally held to be an important prerequisite for the development of nec [105] . the importance of bacteria can be inferred from the observations that full-blown ischemic bowel necrosis cannot be reproduced in a sterile animal model [106] , and, although bowel infarction occurs in the fetuses, typical nec has never been reported as present at birth or in a stillborn infant [15] . because hypoxia alone produced relatively mild bowel injury in our model, we hypothesized that hypoxia and bacterial endotoxin (lps) might act synergistically to produce more severe bowel injury. we treated young adult male sprague-dawley rats with either hypoxia alone (5% oxygen for 90 min), lps alone (2 mg/kg salmonella typhosa endotoxin, i.v.), or lps ϩ hypoxia (lps given at 0 min followed 90 min later by hypoxia for 90 min) [107] . both lps alone and hypoxia alone caused little or no intestinal injury, whereas combined treatment with lps and hypoxia resulted in significantly worse gross and microscopic intestinal in-jury. this injury was ameliorated by treatment with the paf antagonists, either web 2086 or sri 63-441. animals treated with lps ϩ hypoxia tended to have higher plasma paf levels than animals in the other groups, but the difference did not reach statistical significance in this study. both lps and lps ϩ hypoxia caused a significant increase in plasma tnf levels. we concluded that lps and hypoxia act synergistically to produce bowel necrosis and that paf is an important mediator in this process [107] . we explored the role of endogenous no in the pathogenesis of hypoxia-induced intestinal injury [108, 109] . inhibition of endogenous no production with l-arginine analogs significantly worsened the bowel injury produced by 90 min of 10% oxygen exposure, suggesting that endogenous no production constitutes an important defense mechanism against hypoxia-induced intestinal injury. paf levels were significantly elevated in the intestines of animals treated with hypoxia and a no synthase inhibitor, and the intestinal injury seen in these animals was prevented with the paf antagonist web 2086. in the vascular endothelium, no synthesized from l-arginine by the constitutive form of nitric oxide synthase (cnos) limits neutrophil adhesion, promotes microvascular integrity, and maintains basal vasodilator tone [110] . in a related study, inhibition of endogenous no production markedly worsened the bowel injury and intestinal neutrophil accumulation caused by paf [109] . a major challenge in understanding nec in human infants is the absence of a perfect experimental animal model. although several animal models have been used, most lack some or all of the cardinal features of the human condition. the adult rat model characterizes paf and other mediators in acute ischemic bowel necrosis, but it lacks the critical predisposing feature of prematurity. the role of paf in human nec remains speculative. in published experiments on neonatal animals, the model of barlow et al. [111] , first described in 1972, most closely resembles human nec [111] [112] [113] . in this model, newborn rat pups were removed from their mothers, exposed to maternal milk, stressed briefly with asphyxia, colonized with gram-negative enteric bacteria, and fed with artificial formula. by the 3rd day of life, most animals developed abdominal distention and discoloration, bloody stools, respiratory distress, cyanosis, hemorrhagic intestinal necrosis, and microscopic evidence of severe necrosis identical to the pathology observed in neonatal nec. maternal milk, milk leukocytes, immunoglobulin, and oral antibiotics were identified as important for the prevention of nec [114, 115] . we set out to reproduce the findings of barlow et al. and to better characterize the pathologic findings [116] . neonatal rats delivered via abdominal incision were maintained in a neonatal incubator and received the following stresses: (1) artificial formula feedings (0.1 ml every 3 h via orogastric tube, 200 cal/kg/d, advanced as tolerated); (2) asphyxia (100% n 2 for 50 s twice daily); and (3) e. coli inoculation (1 ϫ 10 9 organisms/day via orogastric tube). our data (table 3 ) confirm that asphyxia and formula feeding together are necessary to produce nec in this model. enteral bacterial inoculation was not a critical factor in our model, since more than half of the animals treated with asphyxia and formula alone developed disease compared to those treated with asphyxia, formula, and bacteria (p ϭ ns, not significant). pathologic findings were similar to human nec. grossly, the intestine was hemorrhagic, with friable, occasionally segmental lesions, but often involving most of the intestinal length. in most ani-mals, necrosis extended from villus to the submucosa (fig. 5a,b) and often transmurally (fig. 5c,d) . to evaluate the role of paf, animals stressed with asphyxia, formula feeding, and bacterial inoculation were compared with those pretreated with the paf receptor antagonists web 2170 and web 2086 [117] . web 2170 in appropriate enteral dosing (10 mg/kg q am/30 mg/kg q pm) significantly reduced the incidence of nec and death compared with controls (table 4) . a four-fold higher web 2170 dosing regimen did not alter the incidence of nec, presumably because of an agonist effect on the paf receptor at very high doses [118] . in contrast, web 2086 did not reduce the incidence of nec in stressed animals, presumably because web 2086 has a much shorter half-life than web 2170. intestinal paf concentrations were elevated (270 ϯ 80 pg/g) in animals stressed with asphyxia, formula feeding, and bacterial inoculation compared with age-matched, healthy, maternally fed controls (70 ϯ 50 pg/g, p ͻ 0.05). to further clarify the role of endogenous paf in nec, neonatal rats were treated with the paf degrading enzyme, paf-acetylhydrolase (paf-ah), as enteral supplementation in doses approximately 10-fold higher than found in human breast milk. this intervention markedly reduced the incidence of nec from 19/26 in controls to 6/26 (p ͻ 0.05) [119] . in addition, paf-ah (human, recombinant protein) was identified by immunohistochemistry throughout the intestinal tract and remained functionally active for greater than 24 h after dosing [119] . interestingly, there was no measurable human paf-ah in the circulation of animals using a sensitive monoclonal antibody/enzyme linked immunosorbent assay (elisa) technique [119] . taken together, the data support the hypothesis that endogenous paf acts as a critical mediator in this neonatal rat model of nec. experimental studies on phospholipase further support the role of paf in the neonatal rat model. phospholipase a 2 (pla 2 ) consists of a diverse family of enzymes with potent biological activity [120] . group iia pla 2 , a secretory form of pla 2 , appears to be important in the inflammatory cascade and may regulate paf production [121] . the regulation of group iia pla 2 mrna in intestine from animals stressed with asphyxia, formula feeding, and bacterial inoculation was compared with control, maternally fed animals. northern blot analysis using a cdna probe for group ii pla 2 showed an almost 3.9-fold increase in mrna in the stressed animals compared with controls (n ϭ 6 in each group; caplan et al., unpublished observations), further supporting the role of paf activation in the development of nec. additional studies were performed to understand the role of bacterial flora and long chain polyunsaturated fatty acids (pufa) on the pathophysiol-ogy of nec. since healthy breast-milk fed neonates are colonized with multiple flora including a predominance of bifidobacteria and lactobacilli, neonatal animals were treated with 10 9 bifidobacteria infantis organisms/day and evaluated for the development of nec, endotoxin translocation, mucosal permeability, and pla 2 -ii mrna expression. bifidobacteria infantis supplementation reduced the incidence of nec (7/24 vs. 19/27 control, p ͻ 0.05) but did not alter the colonization pattern of gram negative organisms [122] . bifidobacteria infantis were identified in the stool and intestinal lumen of treated animals but absent in controls. in addition, bifidobacteria infantis treatment markedly reduced pla 2 -ii gene expression in intestinal tissue (42 ϯ 29 mol/g tissue vs. 802 ϯ 320 control, p ͻ 0.01) but had no effect on mucosal permeability [122] . the results suggest that bifidobacteria infantis reduces the incidence of nec by altering paf metabolism and bacterial translocation. the role of polyunsaturated fatty acids on the inflammatory cascade, especially the omega-3 fish oil preparations, have been well recognized. since these compounds seem to reduce the incidence of nec in a human trial, we evaluated them in the neonatal rat model. animals were treated with arachidonic acid (34 mg/100 ml) and docosohexanoic acid (23 mg/100 ml) and studied for the development of nec, pla 2 gene expression, apoptosis, and endotoxin translocation. pufa supplementation did not alter the semiquantitative assessment of intestinal epithelial apoptosis, but it reduced the incidence of nec and death compared to controls, and decreased endotoxinemia at 24 and 48 h. furthermore, pufa decreased pla 2 mrna synthesis but had no effect on inos gene expression in intestinal homogenate [123] . experimental evidence strongly supports the role of paf, lps, and tnf in acute ischemic bowel necrosis and in the neonatal rat model of nec. some data from human studies suggest a similar pathophysiology in neonatal nec. local and systemic paf concentrations are elevated in neonates with nec, and feeding alone promotes paf production. we and other investigators found higher circulating plasma levels of paf and/or paf-like phospholipid in nec patients compared with agematched, illness-matched controls [124, 125] . these nec patients also had higher circulating tnf-␣ levels [124] and lower plasma acetylhydrolase activity (paf-degrading enzyme) than control babies. enteral feeding itself caused elevations of circulating paf levels in a significant percentage of preterm infants [126] , although the circulating acetylhydrolase activity was not affected by the feeding regimen. circulating paf may not adequately reflect the activity in the local environment (intestinal lumen/mucosa), but stool paf concentrations also increased with feedings [127] . fourteen days after feedings were begun, the paf levels were approximately threefold higher than prefeed-ing values (1028 ϯ 244 pg/g vs. 357 ϯ 76 pg/g, p ͻ 0.05) [126] . stool samples from seven patients with nec (stage ii or iii) had the highest levels, with a mean paf concentration eightfold higher than controls (2484 ϯ 154 pg/g) [127] . the apparent increased paf production in experimental and human nec fails to explain why nec exclusively afflicts newborn infants. several factors may predispose newborns and especially premature infants to nec, e.g., immature gastrointestinal host defense, dysfunctional mesenteric blood flow autoregulation, and low paf-degrading enzyme acetylhydrolase (paf-ah) [55, 128] . although plasma paf-ah activity is lower in nec patients than in controls [124] , paf-ah activity is low in newborns as a group, reaching normal adult values at 6 wk of life [129] . infants fed with breast milk (containing significant paf-ah activity) have a much lower risk of nec than infants fed with formula (without measurable paf-ah activity) [130] . in animal experiments, upregulation of paf-ah can prevent ischemic bowel necrosis following exogenous paf infusion [131] . these data strongly support the role of paf in neonatal nec and suggest that low neonatal paf-ah activity may in part explain the neonates' predilection of nec. we hypothesize that the initial insult in the chain of events leading to nec could be perinatal hypoxia or a mild postnatal infection, either of which results in mild mucosal damage (fig. 6 ). following formula feeding and the proliferation of the intestinal flora, bacteria may attach to the damaged intestinal epithelium because of immaturity of the "mucosal barrier," thus eliciting endogenous production of paf (and paf-like phospholipids) and tnf. the major source of paf may be epithelial cells, lamina propria cells, or endothelial cells. although gut bacteria may themselves form paf, the normal mucosal barrier probably prevents any deleterious action on the epithelium. however, in immature or mildly damaged mucosa, the close proximity of bacteria and intestinal epithelial cells may facilitate transcellular permeation of paf into the mucosa. if the acetylhydrolase is low (as in the case of premature infants), paf, which increases the intestinal epithelial permeability in vivo, may ac-cumulate locally, leading to focal mucosal "leak" and local entry of bacteria or bacterial products. paf may then synergize with lps and/or tnf, reaching the threshold necessary to trigger a cascade of inflammatory events: pmn activation and adhesion to venular endothelium, increase in vascular permeability, complement activation, nf-b translocation, induction of proinflammatory cytokines and adhesion molecules, and release of ros and inflammatory mediators (including ltc 4 , prostaglandins, and paf). eventually, vasoconstriction occurs leading to ischemia and subsequent reperfusion. activation of xanthine oxidase with massive reactive oxygen species production occurs as a consequence of ischemia and/or protease activation. the final result depends on the balance between the injurious mechanisms (inflammatory mediators, cytokines, ischemia) and the protective mechanisms (mainly nnos). an imbalance favoring the former will result in serious breakdown of the mucosal barrier and bacterial entry, thereby launching a self-perpetuating vicious cycle, leading to shock, sepsis and, sometimes, death. this work was supported by nih grants dk 34574, hd 31840, and hd00999. we thank dr. h. heuer, boehringer ingelheim, mainz, germany, for his generous gift of web 2086 and web 2170. paf-ah, paf acetylhydrolase; no, nitric oxide; c', complement; nf-b, nuclear factor-b; ltc 4 , leukotriene c 4 ; ros, reactive oxygen species; nec, necrotizing enterocolitis; pmn, polymorphonuclear leukocyte. necrotizing enterocolitis in very low birth weight infants: biodemographic and clinical correlates very low birth weight outcomes of the national institute of child health and human development neonatal network necrotizing enterocolitis-multisystem organ failure of the newborn? medical progress: necrotizing enterocolitis necrotizing enterocolitis: pathophysiology and prevention necrotizing enterocolitis: laboratory indicators of surgical disease epidemiology of necrotizing enterocolitis: a case control study a case control study of necrotizing enterocolitis occurring over 8 years in a neonatal intensive care unit necrotizing enterocolitis: a prospective multicenter investigation necrotizing 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necrotizing enterocolitis platelet-activating factor-induced ischemic bowel necrosis: the effect of platelet-activating factor acetylhydrolase necrotizing enterocolitis of the newborn: pathogenetic concepts in perspective key: cord-337414-8ndkjs1i authors: burgmaier, gruscha; schönrock, lisa m.; kuhlmann, tanja; richter‐landsberg, christiane; brück, wolfgang title: association of increased bcl‐2 expression with rescue from tumor necrosis factor‐α‐induced cell death in the oligodendrocyte cell line oln‐93 date: 2008-07-29 journal: j neurochem doi: 10.1046/j.1471-4159.2000.0752270.x sha: doc_id: 337414 cord_uid: 8ndkjs1i abstract: the present study investigated the effects of flupirtine(katadolon) on tumor necrosis factor (tnf)‐α‐mediated cell death andbcl‐2 expression in the permanent rat oligodendrocyte cell line oln‐93 (olncells). tnf‐α (500 u/ml) induced apoptosis of oln cells, which wasconfirmed by dna fragmentation using an in situ end‐labeling technique andultrastructural analysis. flupirtine significantly reduced the rate ofspontaneous cell death of oln cells already at low concentrations;tnf‐α‐mediated apoptosis was suppressed only with higher concentrationsof flupirtine (100 μm). expression of bcl‐2 protein and mrna inoln cells was detected by immunocytochemistry, western blot, and rt‐pcr.quantitative analysis of western blots revealed an ∼2.5‐fold up‐regulationof bcl‐2 protein during tnf‐α treatment. furthermore, addition of 10 or100 μm flupirtine before incubation with tnf‐α led to anapproximately threefold increase of bcl‐2 expression. exposure of oln cells toflupirtine alone moderately augmented the expression of bcl‐2 protein. ourdata demonstrate that flupirtine up‐regulates the expression of bcl‐2 proteinin oln cells; this bcl‐2 induction is associated with a reduced rate oftnf‐α‐induced cell death. multiple sclerosis (ms) is a chronic inflammatory disease of the cns leading to selective destruction of myelin sheaths and/or oligodendrocytes (lassmann, 1983) . the mechanisms behind demyelination or remyelination, however, are poorly understood, although a heterogeneous pathogenesis is suggested owing to detailed investigations of oligodendrocyte pathology in demyelinating lesions (lucchinetti et al., 1996 . in particular, it is not yet clear whether oligodendrocytes are primarily affected in the disease or whether they are destroyed together with myelin during active demyelination (itoyama et al., 1980; brück et al., 1994; ozawa et al., 1994; raine, 1994) . oligodendrocyte death is a prominent feature in ms lesions. it is, however, not yet clear whether oligodendrocytes die via apoptosis or necrosis, which are different mechanisms of cell death (selmaj et al., 1991; lucchinetti et al., 1996) . dowling et al. (1997) observed that 14 -40% of all degenerating cells in ms lesions are of oligodendroglial lineage and that most of these cells were dying by apoptosis. exposure to heat-treated cerebrospinal fluid from ms patients caused apoptotic death of astrocytes and oligodendrocytes (menard et al., 1998) . on the other hand, bonetti and raine (1997) found that oligodendrocytes express cell death-related molecules such as tumor necrosis factor (tnf) receptors but show no evidence of apoptosis. tnf is a key protein in inducing oligodendrocyte death. it mediates apoptosis of oligodendrocytes in vitro (selmaj and raine, 1988) , and its overexpression in the cns leads to demyelination and oligodendrocyte death (akassoglou et al., 1998) . a recent study revealed a strong association of tnf-␣ mrna expression and active demyelination in ms lesions (bitsch et al., 2000) . apoptosis or programmed cell death (pcd) is an active process of normal cell death during development and also occurs as a cytotoxic consequence of several stimuli, such as cytokines or irradiation (schwartz and osborne, 1993) . apoptotic cell death is accompanied by nuclear changes that include oligonucleosomal dna fragmenta-tion and chromatin condensation into a dense crescentshaped aggregate that marginates along the nuclear envelope and leads to cell shrinkage and membrane blebbing (lo et al., 1995) . several proteins are involved in the regulation of apoptosis. bcl-2 is suggested to play an important role in protecting cells from pcd. it was first described in a b cell lymphoma in which it is overexpressed; it is located in the endoplasmatic reticulum and in the inner and outer mitochondrial membranes (tsujimoto and croce, 1986; hawkins and vaux, 1994; reed, 1994) . bcl-2 seems to be involved in the apoptotic pathway by inhibiting damage of lipid membranes and cell organelles through oxygen radicals or by affecting the cell cycle (hockenbery et al., 1990) ; it was shown to be expressed by oligodendrocytes in chronic active and silent ms lesions (bonetti and raine, 1997) and to correlate positively with the survival of oligodendrocytes after a demyelinating attack (kuhlmann et al., 1999) . flupirtine is a centrally acting nonopioid analgesic that displays cytoprotective activity in cultured neurons induced to undergo apoptosis and reduces ischemic damage in rats (block et al., 1997) . flupirtine has been shown to induce and up-regulate the neuronal expression of bcl-2 protein in vitro in different experiments perovic et al., 1997) . oligodendrocytes are specifically sensitive to tnf-␣ and undergo pcd that might involve bcl-2 regulation. to elucidate whether flupirtine exerts protective effects also on glial cells via an up-regulation of bcl-2, we have used the rat oligodendroglia cell line oln-93 (oln cells) (richter-landsberg and heinrich, 1996) . these cells show characteristics of immature oligodendrocytes and provide a model system for the study of cells of oligodendroglia origin. oln-93, a permanent oligodendroglia cell line derived from primary wistar rat brain glial cultures, was used (richter-landsberg and heinrich, 1996) . these cells morphologically resemble bipolar o-2a progenitors but are differentiated into myelin basic protein-, myelin-associated glycoprotein-, and proteolipid protein-expressing oligodendrocytes. cells were kept at 37°c and 10% co 2 in dulbecco's modified eagle's medium (seromed, berlin, germany) containing 10% heat-inactivated fetal calf serum, 50 u/ml penicillin, and 50 g/ml streptomycin. culture medium was changed three times a week. for the experiments described below, oln cells were exposed to 500 u/ml recombinant human tnf-␣ (genzyme, cambridge, ma, u.s.a.) for 96 h. for cotreatment with flupirtine, oln cells were preincubated for 2 h with increasing concentrations (0, 1, 5, 10, 50, and 100 m) of flupirtine (katadolon; asta medica, frankfurt, germany). oln cells cultured in the absence of any treatment served as controls. cells (10 6 /ml) were grown on nunc (wiesbaden germany) lab-tek chamber slides in dulbecco's modified eagle's medium containing 10% heat-inactivated fetal calf serum alone or supplemented with 10 m flupirtine. cells were fixed in 4% paraformaldehyde for 20 min at room temperature and washed in phosphate-buffered saline. endogenous peroxidase activity was blocked with 3% h 2 o 2 . cells were permeabilized in 0.2% triton x-100 diluted in 5% bovine serum albumin for 20 min. rabbit anti-bcl-2 antibody (santa cruz, heidelberg, germany; diluted 1:100) was applied for 2 h at room temperature. a peroxidase-antiperoxidase technique was used and visualized with a dab-metal-enhancement kit (pierce, germany). in negative controls, the primary antibody was omitted. cellular monolayers were washed with phosphate-buffered saline and scraped off in sample buffer [1% sodium dodecyl sulfate, 10% glycerin, 1% ␤-mercaptoethanol, and 12.5% tris (0.5 m, ph 6.8) in double-distilled water] and boiled for 5 min. total protein content was determined by photometric quantification. for quantification of bcl-2 protein, 5 g of control bcl-2 protein (santa cruz) was loaded. for immunoblotting, total cellular extracts (80 g of protein per lane) were separated by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis using 12.5% polyacrylamide gels and transferred to nitrocellulose membranes according to the technique of towbin et al. (1979) . the blots were washed and incubated with rabbit anti-bcl-2 antibody (santa cruz; 1:1,500, 90 min at room temperature) followed by the secondary antibody (mouse anti-rabbit igg; dako; 1:3,000, 1 h at room temperature). after application of the peroxidase-antiperoxidase complex (dako; 1:3,000, 30 min at 37°c), the blots were washed and visualized by enhanced chemiluminescence according to the manufacturer's protocol (amersham, little chalfont, bucks, u.k.). western blots were performed from three independent experiments. the blots were scanned, and the optical density of bcl-2 bands on western blots was recorded using computer-aided software (adobe photoshop). the luminescence of bands in scanned western blots was evaluated by analyzing histograms for each band. the density of the bands is given in arbitrary units as mean values with sd. total rna extraction was performed with the rneasy total rna purification kit from qiagen (hilden, germany). one microgram of rna was reverse-transcribed using 40 units of moloney murine leukemia virus reverse transcriptase (boehringer, mannheim, germany), and the resulting cdna was amplified by pcr for 40 reaction cycles (95°c for 1 min, 58.5°c for 1 min, 72°c for 1 min). cycle dependency tests were performed to ensure capture of the pcr products in the linear range of the amplification reaction. the primers used were as follows: rat bcl-2, forward 5ј agg ggg aaa cac cag aat c 3ј, reverse 5ј tgg aag gag aag atg cca g 3ј; glyceraldehyde 3-phosphate dehydrogenase (gapdh), forward 5ј acc aca gtc cat gcc atc ac 3ј, reverse 5ј tcc acc acc ctg ttg ctg ta 3ј. the pcr mixture consisted of 4 l of cdna, 1 l of each primer (20 pmol/l), 0.2 mm deoxynucleotide triphosphates, and 2.5 u taq polymerase in a total volume of 100 l (for gapdh, 50 l), resulting in a fragment of 331 (rat bcl-2) or 451 (gapdh), respectively. the amplification product was visualized on a 1.5% agarose gel. the sequence analysis of the pcr product was performed with a purified pcr product (qiaquick pcr purification kit; qiagen). the rat bcl-2 primers for the tailing pcr cycling (95°c for 2 min; three cycles of 95°c for 20 s, 57°c for 20 s, 70°c for 30 s; 12 cycles of 95°c for 20 s, 70°c for 1 min) were as follows: forward 5ј tgt aaa acg acg gcc agt agg ggg aaa cac cag aat caa gt 3ј, reverse 5ј cag gaa aca gct atg acc tgg aag gag aag atg cca ggg gt 3ј. the sequence reaction was performed in 30 cycles (95°c for 2 min; 30 cycles of 95°c for 15 s, 57°c for 15 s, 70°c for 15 s). each lane of a 4.3% gel (licor system) was loaded with 3 l. to assess the extent of apoptosis in situ, the terminal deoxynucleotidyl transferase-mediated dutp nick end-labeling (tunel) technique was applied to oln cells with or without preincubation with flupirtine (1, 5, 10, 50, and 100 m) cultured in the presence or absence of tnf-␣ (500 u) as described in detail above. according to the manufacturer's instructions (boehringer mannheim), cells were incubated for 1 h at 37°c with the tailing mixture (containing double-distilled water, tailing buffer, 2.5 mm cocl 2 , 10 pmol of digoxigenin dna, and 25 units of terminal transferase). after washing in trisbuffered saline, oln cells were incubated for 1 h at room temperature with an alkaline phosphatase-conjugated antidigoxigenin antibody (diluted 1:250 in 10% fetal calf serum). the reaction was visualized with nitro blue tetrazolium/5bromo-4-chloro-3-indolyl phosphate. the number of oln cells with dna fragmentation was calculated from the total number of cells within the culture flask. a minimum of 100 cells was evaluated in each culture flask. oln cells were cultured in 12-well plates and exposed to tnf-␣ for 96 h. the cells were fixed in 3% glutaraldehyde (2 h, 4°c) and in 1% oso 4 (1 h, 4°c). after washing in phosphate-buffered saline, cells were dehydrated in an ascending ethanol series (10 min in 50%, 1 h in 70%, 10 min in 80%, 10 min in 96%, 15 min in 100% ethanol, and 20 min in propylene oxide) and embedded in araldite (incubation for 35 min in 1:1 and 2:1 araldite-propylene oxide solution and finally 1 h in pure araldite). thin sections were placed onto copper grids for electron microscopy and contrasted with lead citrate. electron microscopy was performed with an em10 electron microscope (zeiss). student's t test and mann-whitney u test were used for statistical analysis. immunocytochemistry showed bcl-2-positive oln cells with staining of the cytoplasm and the nuclear membrane (fig. 1a) . for quantitative determination of bcl-2 protein expression, a western blot analysis was performed. oln cells were incubated for 2 h with different concentrations of flupirtine (1, 5, 10, 50, and 100 m), with tnf-␣ (500 u/ml) alone or a combination of both as described above. cells without any treatment were used as controls. the bcl-2 control protein migrated with a molecular mass of 26 kda; each probe revealed a band of the correct size (ϳ26 kda). flupirtine alone caused a concentration-dependent induction of bcl-2 protein in oln cells (fig. 2a) . also, bcl-2 protein was induced by tnf-␣ alone and even stronger after the combined treatment with tnf-␣ and flupirtine (fig. 2b) . quantitative evaluation showed that 100 m flupirtine caused an ϳ1.6-fold increase in bcl-2 expression. an almost 2.5-fold bcl-2 induction was observed after treatment with 500 u/ml tnf-␣. the strongest induction was seen after combined treatment with flupirtine (10 and 100 m) and 500 u/ml tnf-␣, corresponding to an approximately threefold increase compared with untreated cells (fig. 3) . these data indicate that bcl-2 expression in oln cells was moderately induced by flupirtine. tnf-␣ alone led to strong bcl-2 expression, which further increased during combined treatment with these substances. the presence of bcl-2 mrna in oln cells was confirmed at the transcriptional level with the rt-pcr technique. gapdh was used as a housekeeping gene for the control. bcl-2 mrna was consistently expressed in oln cells treated with different flupirtine concentrations (1, 5, 10, 50, and 100 m) alone or in combination with tnf-␣. the expected bcl-2 pcr product (331 bp long) was present in all samples tested (fig. 4) . sequencing of the bcl-2 pcr product identified the examined cdna as the rat bcl-2 gene. the rat bcl-2 mrna consists of 1,179 bases. the present study sequenced between base 58 and 390 (331 bases). the obtained sequence was identical to the known sequence of the rattus norvegicus apart from two point mutations that were not located in the coding region. the tunel technique was used to assess the rate of apoptotic oln cells after treatment with different concentrations of flupirtine (1, 5, 10, 50, and 100 m) alone or followed by incubation with 500 u of tnf-␣ for 96 h. tunel-positive oln cells were found in each sample tested. in comparison with control and flupirtine-treated cells, however, tnf-␣ most effectively induced apoptosis. flupirtine significantly reduced the spontaneous cell death rate already at low concentrations of 1 m. tnf-␣-induced apoptosis was also prevented by flupirtine; however, higher concentrations of flupirtine were required to rescue oln cells from cell death (figs. 1b and c and 5). the nature of the cell death, apoptotic or necrotic, was further investigated by electron microscopy. ultrastructurally, the cells revealed the characteristic morphological signs of apoptosis such as chromatin condensation and the formation of apoptotic bodies. this was especially observed in oln cells stimulated with tnf-␣ (fig. 6) . the pathogenesis of demyelination and the fate of oligodendrocytes in ms are closely connected. the patterns of oligodendrocyte destruction, preservation, or proliferation are still a matter of debate. different immunological or toxic mechanisms have been suggested to be involved in oligodendrocyte destruction, including the proinflammatory cytokine tnf-␣. oln cells may be used as a model to investigate the oligodendroglia cell lineage. the present study provides evidence for an important role of the antiapoptotic protein bcl-2 in the rescue of oln cells from tnf-␣-induced apoptosis. cell death is a key event in various different cell processes. apoptosis as a physiological event has been shown to be one of the two important cell death mechanisms and is regulated by a wide range of different stimuli as well as proapoptotic and antiapoptotic genes. the fas/apo-1 (cd 95) system and the tumor suppressor gene p53 are typical members of the family of apoptosis-inducing genes, whereas bcl-2 is the prototype of an antiapoptotic protein. bcl-2 inhibits apoptosis and promotes cell survival (hockenbery et al., 1990; vaux, 1998) . the antiapoptotic activity is achieved by suppressing the apoptosis-inducing function of bax by forming heterodimers with the bax protein (oltvai et al., 1993) . it has been shown that bcl-2 up-regulation in neurons reduces apoptosis during physiological cell death (zanjani et al., 1996) . there is still considerable controversy on the role of apoptosis in the elimination of oligodendrocytes from demyelinating cns lesions. local tnf/p55tnf recep-tor signaling is capable of inducing oligodendrocyte apoptosis and demyelination in transgenic mice (akassoglou et al., 1998) . after intracerebral infection of lewis rats with jhm coronavirus, a chronic inflammatory demyelinating disease is induced, which in many aspects mimics the pathology of ms. at later stages after infection, the virus antigen was nearly completely cleared from the lesions, and oligodendrocytes were mainly destroyed by apoptosis (barac-latas et al., 1997) . similar observations were made in canine distemper virus demyelinating encephalitis (schobesberger et al., 1999) as well as in an experimental model of myelin-associated glycoprotein deficiency . in and around ms lesions, molecules belonging to the apoptotic cascade have been shown to be expressed by oligodendrocytes (bonetti and raine, 1997) . whether the expression of these molecules is associated with the presence of oligodendrocyte apoptosis, however, is still controversial (lucchinetti et al., 1996; bonetti and raine, 1997; dowling et al., 1997 dowling et al., , 1999 . the fact that oligodendrocytes can be driven into apoptosis in vitro, in contrast, is undoubted, and tnf-␣ seems to play a critical role in this process (selmaj and raine, 1988; selmaj et al., 1991) . the protooncogene p53 induces oligodendrocyte death after induction by tnf-␣ (eizenberg et al., 1996; ladiwala et al., 1999) , and tnf-␣ potentiates interferon-␥-induced cell death in oligodendrocyte progenitors (andrews et al., 1998) . the final execution phase of tnf-␣-induced oligodendrocyte death seems to be mediated by the ice/ced-3 family of caspases (hisahara et al., 1997; gu et al., 1999) . in our study, flupirtine up-regulated bcl-2 when given in high concentrations (100 m). the greatest increase in bcl-2 protein was observed when tnf-␣ was applied in combination with 10 or 100 m flupirtine. flupirtine treatment protected oligodendrocytes from tnf-␣-mediated apoptosis. however, only the 100 m concentration of flupirtine produced a highly significant reduction of oligodendrocyte apoptosis. this may be due to the fact that flupirtine does not selectively affect bcl-2 protein expression, but also modifies other proteins of the apoptosis cascade. besides up-regulation of bcl-2, flupirtine is known to decrease levels of ich-1, which is caspase 2 (osborne et al., 1997) . it is interesting that caspase 2 and 3 expression was demonstrated in oligodendrocytes undergoing apoptosis (gu et al., 1999) . a recent study identified the inhibition of caspase 3 and up-regulation of bcl-2 as mechanisms to rescue oligodendrocytes from tnf-␣-mediated apoptosis (soane et al., 1999) . thus, flupirtine may act at different levels of the apoptotic cascade and interfere with the balance of proapoptotic and antiapoptotic signals. the present study provides evidence that bcl-2 is expressed constitutively by oln cells and that the level of bcl-2 expression depends on the stimulation with different concentrations of flupirtine and/or tnf-␣. flu-pirtine increases the expression of bcl-2 and protects neurons from ␤-amyloid-induced apoptosis or prion fragment-mediated neurotoxicity perovic et al., 1997) . in the present study, flupirtine alone or in addition to tnf-␣ up-regulated bcl-2 protein and rescued oligodendrocytes from tnf-␣-induced cell death. the induction of bcl-2 expression may therefore be an interesting therapeutic target in demyelinating diseases to support oligodendrocyte survival. the question whether bcl-2 plays a role in the survival of oligodendrocytes in ms lesions was recently studied (kuhlmann et al., 1999) . as already shown by others (bonetti and raine, 1997) , bcl-2 is present in ms plaques; our study, however, found a clear association among bcl-2 expression, oligodendrocyte survival, and remyelination in ms plaques (kuhlmann et al., 1999) . bcl-2-associated mechanisms may belong to a group of different effectors that support survival of oligodendrocytes and include the activation of tyrosine kinase or cytokine receptors (casaccia-bonnefil, 2000) . new therapeutic strategies may develop from these experiments, preventing loss of oligodendrocytes from demyelinating lesions and supporting myelin regeneration. oligodendrocyte apoptosis and primary demyelination induced by local tnf/p55tnf receptor signaling in the central nervous system of transgenic mice tnf␣ potentiates ifn␥-induced cell death in oligodendrocyte progenitors patterns of oligodendrocyte pathology in coronavirus-induced subacute demyelinating encephalomyelitis in the lewis rat tumour necrosis factor alpha mrna expression fig. 6. ultrastructure of untreated and tnf-␣-treated oln cells. a: normal-appearing nucleus in untreated control cultures. b: condensed, apoptotic nucleus in a culture treated with 500 u/ml tnf-␣. magnification ϫ6,300 flupirtine reduces functional deficits and neuronal damage after global ischemia in rats multiple sclerosis: oligodendrocytes display cell death-related molecules in situ but do not undergo apoptosis oligodendrocytes in the early course of multiple sclerosis cell death in the oligodendrocyte lineage: a molecular perspective of life/death decisions in development and disease cell death and birth in multiple sclerosis brain upregulated p75 ntr neurotrophin receptor on glial cells in ms plaques plays a regulatory role in differentiation and apoptosis of central nervous system-associated cells oligodendrocyte apoptosis mediated by caspase activation analysis of the role of bcl-2 in apoptosis ice/ced-3 family executes oligodendrocyte apoptosis by tumor necrosis factor bcl-2 is an inner mitochondrial membrane protein that blocks programmed cell death immunocytochemical method to identify myelin basic protein in oligodendroglia and myelin 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pigment epithelial cells is counteracted by flupirtine patterns of oligodendroglia pathology in multiple sclerosis effect of flupirtine on bcl-2 and glutathione level in neuronal cells treated in vitro with the prion protein fragment (prp 106 -126) multiple sclerosis: immune system molecule expression in the central nervous system bcl-2 and the regulation of programmed cell death oln-93: a new permanent oligodendroglia cell line derived from primary rat brain glial cultures oligodendroglial degeneration in distemper: apoptosis or necrosis? programmed cell death, apoptosis and killer genes tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro cytokine cytotoxicity against oligodendrocytes. apoptosis induced by lymphotoxin inhibition of oligodendrocyte apoptosis by sublytic c5b-9 is associated with enhanced synthesis of bcl-2 and mediated by inhibition of caspase-3 activation electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications analysis of the structure, transcripts, and protein products of bcl-2, the gene involved in human follicular lymphoma immunopathology of apoptosis-introduction and overview increased cerebellar purkinje cell numbers in mice overexpressing a human bcl-2 transgene key: cord-354765-abayh871 authors: graham, r. s.; zachs, d. p.; cotero, v.; dagostino, c.; ntiloudi, d.; kaiser, c. r.; graf, j.; wallace, k.; coleman, t. r.; ashe, j.; pellerito, j.; tracey, k. j.; binstadt, b.; chavan, s. s.; zanos, s.; puleo, c.; peterson, e.; lim, h. h. title: calming the cytokine storm splenic ultrasound for treating inflammatory disorders and potentially covid-19 date: 2020-07-17 journal: nan doi: 10.1101/2020.07.14.20153528 sha: doc_id: 354765 cord_uid: abayh871 hyperinflammation and uncontrolled cytokine release, which can be seen in severe cases of covid-19, require therapy to reduce the innate immune response without hindering necessary adaptive immune mechanisms. here, we show results from the first in-human trials using non-invasive ultrasound stimulation of the spleen to reduce cytokine release in the context of both an acute response in healthy subjects and a chronic inflammatory condition in rheumatoid arthritis patients. splenic ultrasound results in a reduction in tnf serum levels, as well as il-1b; and il-8 transcript levels in monocytes. there is also a down regulation of pathways involved in tnf and il-6 production, and ifngammaand nfkb-regulated genes. many of these cytokines or pathways are upregulated in covid-19 patients. there is also a reduction in chemokine transcript levels and other components of the chemotactic response, suggesting that reduction of cellular migration may contribute to the therapeutic effects of ultrasound. there is no inhibition of the adaptive immune response with ultrasound treatment relating to antibody production. this is consistent with a pre-clinical animal model where enhanced antibody production was achieved with splenic ultrasound. therefore, this new splenic ultrasound approach has the potential to treat acute and chronic hyper-inflammatory diseases, as it lowers cytokine levels without disrupting the normal adaptive immune response. portable ultrasound technologies are currently being developed and translated to the clinic to treat various inflammatory disorders, with more recent efforts directed towards combatting the hyperinflammation or cytokine storm in covid-19 patients. cytokines release by cells of the innate immune system drive inflammation (1, 2). inflammatory reactions are typical in response to microbial or viral infection but can lead to health problems or life-threatening conditions if there is a persistent hyperactive innate immune response, involving cytokine toxicity and tissue damage (2, 3) . a recent and devastating example is the hyperinflammation caused by the coronavirus disease 2019 (covid-19, disease associated with sars-cov-2 viral infection). it is estimated that 17% of covid-19 cases experience a 'cytokine storm' that leads to severe or fatal respiratory disease, known as acute respiratory distress syndrome (ards) (4) (5) (6) . in addition to other acute conditions, such as sepsis and acute kidney injury (7, 8) , hyperinflammation is an issue that occurs across multiple chronic inflammatory systemic diseases, such as rheumatoid arthritis (ra) and irritable bowel syndrome (9, 10) . the current pharmacologic approaches to treating hyperinflammation are associated with multiple side effects and high costs. over the past 20 years, researchers within the field of bioelectronic medicine have investigated an unconventional approach for treating inflammation through the use of vagus nerve stimulation (11) (12) (13) . electrical stimulation of the vagus nerve activates the splenic nerve and cells within the spleen, leading to activation of the cholinergic anti-inflammatory pathway (14, 15) . this pathway depends on splenic nerve release of norepinephrine that activates acetyltransferase -expressing t lymphocytes, which in turn modulates innate immune cells to decrease systemic levels of key proinflammatory cytokines, such as il-6, il-15 and tnf (16, 17) . furthermore, electrical vagus nerve stimulation has been shown in mice and humans to provide therapeutic anti-inflammatory effects for chronic inflammatory diseases (e.g., ra and irritable bowel syndrome) and for acute inflammation (e.g., sepsis, renal ischemia, trauma/hemorrhagic shock, and acute lung injury following trauma/hemorrhagic shock; (16, (18) (19) (20) (21) . direct vagus nerve stimulation requires invasive implantation procedures. several groups have pursued non-invasive ultrasound stimulation of the spleen as a safer, non-surgical approach for modulating the cholinergic anti-inflammatory pathway. since the vagus nerve projects to the brain and multiple organs throughout the body (22) , targeting neurons or cells specifically within the spleen can reduce unintended activation or side effects. there has been a recent surge of research which demonstrates the ability to activate or modulate cells with ultrasound energy (23) (24) (25) (26) (27) (28) there are several studies in rodents showing the ability to modulate the splenic nerve or immune cells within the spleen with ultrasound, to reduce inflammation and cytokine levels (29) (30) (31) (32) (33) . one of the initial reports was in a mouse model of reperfusion injury and kidney inflammation, where ultrasound stimulation of the spleen significantly reduced kidney damage (29, 34) . recently, our research groups discovered that specific parameters of ultrasound stimulation of the spleen can drive significant anti-inflammatory effects in rodent models of both chronic inflammation (inflammatory arthritis) and acute inflammation (sepsis) (31, 32) , which was shown to be mediated through a similar cholinergic anti-inflammatory pathway accessed through vagus nerve stimulation. ultrasound is a potentially impactful clinical solution to inflammation as it can be applied non-invasively to the body with a wearable device and with energy parameters already shown to be safe for the human body based on numerous ultrasound imaging applications (35, 36) . here, we show the first in-human results of pro-inflammatory cytokine reduction with noninvasive ultrasound stimulation of the spleen. in healthy individuals, a single three-minute administration of splenic ultrasound stimulation significantly inhibits whole blood tnf production upon ex vivo exposure to endotoxin compared to sham controls. in ra patients, we observed that daily splenic ultrasound stimulation results in reduction of blood-borne transcripts encoding for pro-inflammatory markers il-1β, il-8, and nfκb, as well as suppresses pathways involved in il-6 and tnf production. ultrasound also reduces pathways involved with monocyte migration, contributing to its anti-inflammatory effect. from a safety perspective, circulating immune cell composition does not change with ultrasound treatment, and ultrasound does not inhibit the adaptive immune response in humans. our additional pre-clinical animal data further demonstrates that in addition to dampening cytokine output and circulating monocyte invasiveness, prophylactic ultrasound activation of the splenic neuroimmune pathway results in enhanced antibody response upon exposure to an inflammatory antigen. thus, activation of the splenic neuroimmune pathway may provide a low risk therapeutic approach for a broad range of health conditions, due to its pleotropic nature and physiological role in suppressing specific cytokines involved in innate immunity while enhancing the transition to and maintenance of the adaptive immune response (18, 37, 38) . together, these data suggest great potential for splenic ultrasound as a low-risk clinical therapy for a range of acute or chronic inflammatory diseases without requiring surgery or intake of artificial substances with possible side effects. considering that many of the same pro-inflammatory molecules suppressed by ultrasound are implicated as molecular drivers in covid-19 patients with severe disease, ultrasound also has potential as a non-invasive therapy to combat this pandemic. drug based suppression of these cytokines for tissue protection comes with the risk of inhibiting viral clearance or increasing susceptibility to bacterial co-infections (39, 40) . non-invasive ultrasound activation of the splenic neuroimmune pathway may provide an alternative method to combat the cytokine storm without compromising the adaptive immune response in covid-19 patients, ultimately reducing the high mortality and morbidity rates confronting this worldwide pandemic that currently has limited treatment options. to investigate how non-invasive ultrasound stimulation of the spleen affects cytokine levels directly in human subjects, healthy participants were recruited into a study designed to investigate effect of different ultrasound parameters and locations of stimulation in the spleen on various immune and metabolic responses during acute stimulation (feasibility study described at clinicaltrials.gov: nct03548116). due to the urgency of identifying potential treatment options for covid-19 patients, data pertaining specifically to ultrasound stimulation of the hilum of the spleen with the most effective intensity parameter in the study (corresponding to one cohort from the study) are presented in this paper. participants received pulsed ultrasound stimulation of the spleen for three minutes (2.2 mhz, 1 hz pulse repetition frequency, 290.4 mw/cm 2 ispta) using the ge logiq e9 ultrasound system with the c1-6 probe. an ultrasonographer first identified and targeted the hilum of the spleen, then delivered the pulsed ultrasound stimulus using a modified elastography software setting in the system. sham stimulation control subjects underwent the same procedure as the stimulated group, including transducer placement and movement of the ultrasound probe to the proper splenic location trajectory, but with no ultrasound energy being transmitted to the spleen from the transducer. since healthy subjects have low serum tnf levels, we revealed changes in inflammatory status using whole blood challenged with lipopolysaccharide (lps) ex vivo. this ex vivo cytokine production assay has been previously used to verify splenic neuroimmune activation using implanted vagus nerve stimulators and is a well-established method for non-invasively assessing activation of neuroimmune pathways (18, (41) (42) (43) . a reduction in lps-induced tnf production in blood sampled from ultrasound stimulated subjects was tested at multiple lps concentrations (fig. 1a ) and significant reduction is observed when comparing the ultrasound stimulated group to the non-stimulated controls ( fig. 1b ; p-value = 0.006 using wilcoxon unpaired rank-sum test) at the optimal lps concentration (1 ng/ml; fig.1a ). these data are consistent with earlier reports using implanted nerve stimulators or pharmaceutical activators of the splenic pathway (18, 42) . they are also consistent with the magnitude of tnf reduction using similar ultrasound stimulation parameters in a previous study activating the neuroimmune pathway in a rodent model of endotoxemia (31) . further supporting the human results, we also demonstrated that splenic ultrasound stimulation in a rodent model achieves a reduction across a number of proinflammatory cytokines beyond tnf, including il-6, ifnγ, il-1β, il-1α, and il-12 in splenic lysates (fig. s1 ), which is consistent with previous reports using implanted vagus nerve stimulators (18, 44) . tnf response after lps incubation is shown for one ultrasound stimulated subject from samples collected before (0 hours baseline) and after (2 hours post stimulation) ultrasound application. the optimal dose of lps for measuring the immunomodulatory response to ultrasound was found to be at 1 ng/ml, and this is used for comparing the tnf response between the ultrasound stimulated versus control groups. b) ultrasound stimulation of the spleen decreases whole-blood lps-induced tnf release, where blood was obtained from 18 healthy human subjects prior to and then two hours following splenic ultrasound application (stimulated: n=9, control: n=9). the data shown were taken from the 1 ng/ml lps concentration for each dose response curve for all subjects and samples. p-value was computed using the unpaired wilcoxon rank-sum test. previous animal research demonstrated that ultrasound can significantly reduce inflammation and improve clinical measures in a chronic inflammatory arthritis model (32) . expanding upon the findings in healthy human subjects in the previous section, we initiated a controlled, randomized clinical trial of splenic ultrasound treatment in ra patients (clinical study described at clinicaltrials.gov: nct03690466). participants received ultrasound stimulation to the spleen with the goal of lowering the over-active inflammatory response that occurs during ra flare-ups. up to 20 ra patients are to be recruited into the study (enrollment is ongoing). due to the urgency of identifying treatment options for this covid-19 pandemic, data from single-cell rna sequencing (scrna-seq) in peripheral blood mononuclear cells (pbmcs) that has been collected thus far in five ultrasound-treated patients have been analyzed ahead of completion of the study for inclusion into this paper. pbmcs were isolated from whole blood taken from each research participant before ultrasound treatment (day 0, pre-stimulation) and after two weeks of daily 30-minute sessions of ultrasound to the spleen (day 14, post-stimulation). from these 10 samples (subjects = 5, timepoints = 2), a total of 53,343 pbmcs were successfully sequenced from single cells (table s1 ). in silico analysis identified 13 cell-types based on gene expression patterns of standardly used marker genes (fig. s2 ). participants received stimulation with a 1 mhz transducer (1.2 w/cm 2 ; soundcare plus, roscoe medical) that was moved continuously across a 5-inch by 5-inch square, centered on the spleen, as identified by an ultrasonographer. further details on the clinical study design is presented in the materials and methods section. we aimed to identify molecular changes that occur with splenic ultrasound in humans to better understand how this ultrasound treatment can drive an anti-inflammatory response. we observed reduced monocyte expression of key pro-inflammatory cytokine genes encoding for il-1β and il-8 from all five subjects post-stimulation compared with pre-stimulation ( fig. 2a , p-values given for transcripts showing significance of p<0.05). although this result was determined by analyzing all monocytes, reduction of these two markers appears primarily in cd14+ monocytes as this is the cell type showing predominant expression of those genes (fig. 2b) . the same analysis in cd8+ t cells show a decrease in transcripts that encode for ifnγ ( fig. 2a ). the changes in transcript level of these cytokines can only be detected when comparing within the same cell type before and after treatment, within the cell that expresses the cytokine (fig. s2 ). consistent with known expression patterns of il1b and cxcl8 transcripts (encoding for il-1β and il-8), we see that these transcripts are only suppressed in the monocytes where they are primarily detected. likewise, ifng transcripts (encoding for ifnγ) are detected and found to be reduced in cd8+ t cells (fig. s2 ). in our dataset, other cytokines like tnf and il-6 did not show a significant change in monocytes; however, il-6 transcripts were detected in only 66 of the 53,343 cells. therefore, we cannot definitively conclude if there is or is not a change in transcript levels encoding il-6 with ultrasound treatment. since monocytes play a critical role in the innate immune system and the inflammatory response, we further focused our analyses on transcriptional changes that occur in circulating monocytes after ultrasound treatment. differential expression analysis of all monocytes across the five subjects, pre-and post-ultrasound stimulation, shows 938 differentially expressed genes (p-value<0.05) with 841 of those genes being down-regulated. these results reveal an overall shift in reduction of gene transcripts in monocytes with ultrasound treatment. additionally, this reduction in transcript levels did not appear as a result of sequencing differences, as we observe no obvious difference in the number of cells sequenced, the number of sequencing reads, or number of average genes detected per cell between monocytes taken from day 0 as compared to day 14 (table s1 , 'cd14 mono' and 'cd16 mono'). although these gene transcripts are defined as differentially expressed when comparing monocytes from all subjects ( fig. 2a) , we also observe that for most genes, this down-regulation is consistent across subjects ( fig. 2c ; columns represent cell-type averages for cd14+ and cd16+ cells for each subject that are similar across columns for many of the genes). showing cd14+ and cd16+ monocytes represented by orange and green dots, respectively. il-1β and cxcl8 expression on day 0 and day 14 are also mapped on these umap diagrams. c) heatmap displaying the average relative expression (for all cells in specified group) of downregulated transcripts (genes listed on the right, p-value <0.05). color denotes relative expression for post-ultrasound levels compared to (divided by) pre-ultrasound levels for each subject (represented by columns, n = 5, participant a-e shown left to right) in cd14+ cells or cd16+ cells. genes encoding for cytokine receptors, components of the jak/stat pathway, map kinases, and transcription factor (tf) are grouped and labeled on the left of the plot. these results are quite consistent given the high variability or inhomogeneity in age, sex, physical characteristics, disease properties and past treatments across subjects (participants a-e in table s2 correspond to columns 1-5 in fig. 2c , respectively). within these down-regulated genes in monocytes, we also observe that many cytokine receptors, such as those for tnf, il-6, il-17, il-13, and ifnγ were reduced with ultrasound treatment (fig. 2c) . furthermore, components of signaling pathways downstream of these cytokines are also down regulated, including components of the jak/stat pathway, map kinases, and pro-inflammatory transcription factor nfκb (fig. 2c ). together, these findings demonstrate a consistent and robust reduction in transcripts encoding key pro-inflammatory cytokines and cellular signaling molecules downstream of their receptors after 14 days of daily splenic ultrasound treatment across all five subjects. cytokines trigger multiple cellular pathways, which then contribute to systemic inflammation. our transcriptomic data reveal that many genes known to be regulated by ifnγ and nfκb are subsequently downregulated with ultrasound treatment, further promoting a cascade of suppression across multiple proinflammatory pathways with ultrasound treatment (fig. 3a) . we sought to determine if the 840 down-regulated genes in monocytes showed statistical enrichment for functional gene ontology (go)terms associated a) volcano plot showing all detected genes in monocytes, highlighting differentially expressed genes (p-value <0.05) and labeled ifnγ-regulated genes that are down regulated, as well as nfκb-regulated genes that are down regulated as characterized by kegg pathway gene lists. b) gene ontology (go) analysis using bioconductor package 'topgo', determining functional go terms significantly enriched within downregulated genes in monocytes. enriched go terms associated with cytokine production are shown, where bars represent number of significant genes in a list relative to number of genes that would be expected by chance, and with number of genes contributing to each go term represented by the shade of blue. pvalues for each term shown is from fisher exact test. with inflammation. the go system of classification assigns genes (for differentially expressed gene list) into bins based on their functional characteristics. gene sets can then be analyzed for go terms that are statistically over-represented among that set, allowing for a better understanding of which biological processes (e.g., inflammation) may be changed with treatment. out of the top statistically enriched go terms for the down-regulated genes, we observe many that contribute to pro-inflammatory cellular processes. genes suppressed by ultrasound show enrichment of go terms including "inflammatory response" (p-value = 1.50 ε-5, table s3 ), "cytokine-mediated signaling pathway," and "cellular response to cytokine stimulus" (p-values = 9.7 e-5 and 6.7e-6, fig. 3b ). we also observe enriched go terms associated with positive regulation of tnf, il-6, and il-8 among the down-regulated genes, suggesting that inducing signals for tnf or il-6 are also suppressed, despite the absences of significant change in tnf or il-6 transcript levels (fig. 3b ). these go term results show that many gene transcripts encoding for cellular inflammatory functions go down after ultrasound treatment. within circulating monocytes, we observe ultrasound-treatment associated go term enrichment of cellular components associated with il-8 functionality (fig. 3b) , as well as a robust reduction in il-8 across subjects (fig. 4a , columns represent subject cell type averages). il-8 is a chemokine (chemotactic factor), which stimulates neutrophil movement towards inflammatory sites. therefore, we further assessed if ultrasound influences other signaling molecules involved with cellular migration (e.g., to sites of inflammation). go term analysis of down-regulated genes in cd14+ monocytes show enrichment of "positive regulation of cell migration" among the top 15 significantly enriched go terms. other go terms, such as "lamellipodium assembly" (facilitates cell mobility), "ephrin receptor signaling" (promotes cell adhesion), and "actin nucleation" (involved with cell locomotion), all suggest that ultrasound treatment induces down regulation of genes involved with cell migration (fig. 4b) . furthermore, genes that more specifically contribute to "monocyte chemotaxis" are down-regulated post-ultrasound stimulation in cd14+ monocytes and many of these genes are consistently reduced across all five subjects (fig. 4a) , even with quite variable or inhomogeneous patient characteristics (table s2) . we sought to determine how ultrasound affects the circulating immune cell repertoire. in comparing the transcriptional profiles (based on principal component analysis and umap dimensional reduction) of all circulating peripheral blood mononuclear cells pre-and post-ultrasound stimulation, (table s4) . c) percent down regulation range is shown after 14 days of ultrasound stimulation for each gene (represented by dots) in each functional category described previously. we observe remarkable consistency (fig. 5a) . also, the calculated relative percent of each celltype for each subject is displayed, and based on a paired wilcoxon rank sum test for pre-and poststimulation for each cell type, we observe no significant difference in the relative proportions of percent contribution of any of the 13 cell types (fig. 5b , statistics shown in table s4 ). additionally, when comparing the number of cells sequenced for each cell type per participant there is no significant difference between timepoints (i.e. no technical bias, table s4 ). we also determined what degree of suppression occurred after ultrasound stimulation for genes involved in inflammation (genes shown in fig. 2c, 3a and 4a) . most genes show a ~5-15% reduction in transcript levels, suggesting a moderate degree of suppression, with il-8 and genes involved in cell migration showing the highest degree of suppression (>15%, fig. 5c ). these findings suggest that spleen-targeted ultrasound can potentially provide a way to blunt the elevated release of circulating cytokines and chemokines in inflammatory disorders or viral infections, without disrupting the overall immune cell repertoire. one concern in treating acute hyperinflammation is that the therapy may suppress the adaptive immune response, and the ability of the immune system to provide pathogen clearance and/or protection from co-infection with a second pathogen. therefore, we assessed whether non-invasive splenic ultrasound treatment suppresses the adaptive immune response or more specifically antibody production. our transcriptomic data do not suggest a suppression of the adaptive immune response. in fact, we observe that among genes upregulated in b cells, there is an enrichment for go terms associated with "b cell activation" (fig. 6a ). genes associated with this go term include transcripts that encode for iga, igg, and igm antibodies. interestingly, these gene transcripts are upregulated in some of the ultrasound-treated subjects (fig. 6b) , though with greater variability in results across subjects than was observed for suppression of cytokines and chemokines in monocytes. for example, we observe that for average transcript levels across b cells, transcripts encoding igg are only upregulated in 2 or 3 subjects (ighg2 and ighg4, respectively) out of the 5 subjects. regardless of the variability across subjects, these data support that ultrasound treatment does not generally inhibit b cell activation and antibody production across patients, and can even potentially enhance antibody production; thus, ultrasound is not expected to compromise the adaptive immune response. to further investigate the effects of splenic ultrasound stimulation on adaptive immune response and antibody production, as well as the variability we observed across human subjects for transcript levels that encode for iga, igg, and igm antibodies, we performed additional experiments using prophylactic ultrasound stimulation (i.e., ultrasound stimulation prior to antigen exposure) in a rodent model of endotoxemia (31) . we observe that splenic ultrasound stimulation prior to endotoxin exposure (three minutes daily, once a day for 3 days followed by lps injection on the fourth day) results in a significant increase in igm and igg antibody production 24 hours after lps exposure compared to sham and/or non-lps controls (p-values listed in fig. 6c description) . furthermore, this enhanced antibody output is dependent on the presence of the toxin/antigen, as there is no observed ultrasound effect on antibody production in the absence of lps exposure. the increase in antibody transcript levels we observed in some of the human subjects, and not in the others, may be attributed to those participants also having encountered an antigen, such as a cold or other infection that was not identified during the study. this explanation is further supported by a recent report demonstrating that brain-spleen neural pathways are capable of modulating splenic plasma cells differentiation upon presentation of novel t cell dependent antigens (45) . for naïve animals (no lps + sham), animals exposed to splenic ultrasound stimulation with no endotoxin (no lps + ultrasound), animals exposed to the endotoxin but receiving sham stimulation (lps + sham), and animals receiving both lps exposure and splenic ultrasound stimulation (lps + ultrasound). animals receiving lps + ultrasound showed significant increases in antibody production when compared to each of the other groups using an unpaired wilcoxon ranked-sum test for igm (no lps + sham, p = 0.00145; no lps + ultrasound, p = 0.00155; lps + sham, p =0.01757) and for igg (no lps + sham, p =0.02930; no lps + ultrasound, p = 0.00404; lps + sham, p =0.04298). no other comparisons using this test showed significance, p < 0.05. by presenting the first in-human data from two independent studies using different devices and protocols, we have consistently demonstrated that non-invasive ultrasound stimulation of the spleen drives anti-inflammatory effects in the context of both an acute response in healthy subjects and a chronic inflammatory condition. in ra patients after two weeks of daily 30-minute ultrasound treatment to the spleen, there was a reduction in cytokine il-1β and chemokine il-8 in circulating monocytes (fig. 2) . in healthy subjects, we also observed a significant reduction in tnf levels produced by ex vivo lps-stimulated whole blood, in individuals stimulated with 3 minutes of pulsed ultrasound (fig. 1b) . in addition to the reduction of cytokines, we observed a reduction in activation of multiple signaling pathways in circulating monocytes with ultrasound treatment. cytokines signal by binding to cell surface receptors to set off a cascade of intracellular events. important cytokines in the context of ra include tnf, il-6, and il-1β; their downstream intracellular pathways and necessary signaling molecules include cytokine receptors, components of the jak/stat pathway, map kinases and nfκb (46, 47) . many of the transcripts that encode for these critical components are downregulated with ultrasound treatment in ra patients (fig. 2b) . this finding suggests that in addition to cytokines and their immediate receptors, there is broad suppression of cellular proinflammatory pathways. down regulation of these various pro-inflammatory transcripts in ra patients occurs in the context of chronic inflammation. however, these same pathways are also involved in acute inflammatory responses (47) . genes induced by lps-stimulated human monocytes include tnf, il-1, il-6, and il-8 (48) . nfκb and associated pathways are also upregulated, and nfκb is activated via phosphorylation during lps exposure, allowing for a rapid induction of genes so the system can respond in an acute inflammatory setting. these same cellular components are downregulated with ultrasound treatment in our ra patients, suggesting that non-invasive splenic ultrasound could be used clinically to suppress acute inflammation as well. the discovery that splenic ultrasound can reduce or calm an overactive innate immune response in humans is quite timely. many of the same key inflammatory cytokines that are reduced with ultrasound stimulation are elevated in covid-19 patients. recent studies have revealed that covid-19 patients consistently produce high levels of il-1β, il-8, il-6, and tnf, which strongly suggests dysregulation in the innate immune response (34) (35) (36) (37) . more specifically, sars-cov-2 infected individuals exhibit significantly higher expression levels of il-1β and il-6 transcripts in circulating monocytes, as well as ifnγ in cd8+ t cells, shown through pbmc single-cell rna sequencing of covid-19 patients (38) . single cell rna sequencing has further shown that bronchoalveolar lavage fluid from patients with severe covid-19 disease have higher levels of il-8, il-6 and il-1β compared to patients with only moderate disease (49) . these molecules are key components of the covid-19 cytokine storm that function in acute lung injury to recruit neutrophils to alveoli, inducing lung tissue damage (39) . recently, sars-cov-2 infection in children has been associated with a novel multisystem inflammatory disease (mis-c) resembling toxic shock syndrome or atypical kawasaki disease (50) . kawasaki disease also presents with elevated transcripts levels of genes encoding for il-1, il-6, il-8, and il-17 (51) ; and along with ards, is likely to be a condition caused by the covid-19-induced cytokine storm (52) . clinical therapies that can suppress these pro-inflammatory cytokines and reduce migration of circulating immune cells to the lungs may be capable of treating patients with moderate to severe cases of covid-19 to calm the cytokine storm and prevent morbidity and mortality (fig. 7a) . ultrasound has the potential to prevent or improve severe symptoms associated with the cytokine storm in covid-19 patients. in addition to demonstrating the ability to reduce key proinflammatory cytokines and signaling pathways involved with a hyperactive innate immune response, splenic ultrasound also reduces il-8 and many components associated with cell migration, specifically monocyte migration (fig. 4) . these findings are consistent with previous animal studies showing that activation of the cholinergic anti-inflammatory pathway suppresses monocyte migration in mice (46) . taken together, one proposed mechanism of action for ultrasound treatment is that ultrasound modulates circulating immune cells initially within the spleen via activation of the cholinergic antiinflammatory pathway, which suppresses proinflammatory cytokines and signaling pathways, as well as inhibiting cellular migration, such that there is reduced inflammation at the target site (e.g., at joints in ra or alveoli for covid-19; fig. 7a ). furthermore, data in humans and preclinical animal data show an increase in antibody production with splenic ultrasound. these results demonstrate an advantageous feature of splenic ultrasound compared to pharmaceutical options for immunosuppression, such as tnf, il-6, or il-1 blocking biologics (39, 40) , in that cytokine suppression does not inhibit other functions of the adaptive immune system, such as b cell activity or antibody production. indeed, splenic ultrasound may enhance the adaptive immune system through the recently discovered brain-spleen neuroimmune pathway that enhances differentiation of antibodyproducing splenic plasma cells (sppcs) upon antigen activation (45). one limitation of the presented results in the context of covid-19 or a cytokine storm is that ultrasound stimulation of the spleen may not generate anti-inflammatory effects sufficient to suppress a severe infectious inflammatory response. the anti-inflammatory effects in this paper in response to splenic ultrasound were demonstrated acutely (single 3-minute stimulation) in healthy human subjects or to a moderate extent in ra patients. the question remains whether the antiinflammatory effects observed in healthy subjects or in patients with a chronic inflammation disorder, such as ra, will translate to patients harboring a systemic viral infection, as observed for covid-19. this question will be answered through a clinical trial that is currently being set up at the university of minnesota together with ge research to test this ultrasound approach in covid-19 patients funded by the defense advanced research projects agency (darpa; department of defense). one key asset of the upcoming covid-19 study is that ge's cart-based ultrasound system (ge logiq e10) with enhanced targeting capabilities will be used on patients. in the ongoing ra study at the university of minnesota, a commercially available ultrasound device (soundcare plus) was used on patients that did not have targeting capabilities tailored for the spleen. at the onset of that ra study, soundcare plus was one of few devices that was already fda regulated for various stimulation applications; thus, this unit was selected for the ra study. the ge logiq e10 device can be used to better target and stimulate specific locations of the spleen, which the ge team has already demonstrated with strong anti-inflammatory effects in healthy human subjects (fig. 1b) . previous animal studies have also demonstrated that stronger anti-inflammatory effects are possible with better focusing of stimulation within the spleen or longer periods on a given target (31, 32) . furthermore, sufficient activation of the cholinergic anti-inflammatory pathway has been shown to combat acute and deadly cytokine storm conditions in sepsis animal models, and the magnitude of cytokine suppression provided by ultrasound is consistent with the protection provided by implant-based or pharmaceutical methods in these studies (31, 53, 54) . in parallel with the darpa-funded study, both ge and a start-up company, secondwave systems, are developing wearable or portable ultrasound systems for neuromodulation (fig. 7b ) that can be positioned over the rib to steer and focus ultrasound energy to different locations of the spleen or other organ targets (fig. 7c) . both ge and secondwave are currently organizing clinical studies at multiples sites worldwide to assess the safety and feasibility of their therapeutic ultrasound devices. success with these initial clinical studies will propel further development and scale-up efforts for splenic ultrasound technologies to meet the large-scale demand for covid-19, as well as open up opportunities to treat future viral infections and other inflammatory disorders in combination with or in lieu of biologics. for the splenic ultrasound study involving healthy human subjects (clinicaltrials.gov: nct03548116), we provide the data from the two cohorts most relevant to the planned darpa sponsored covid study: the sham control cohort (i.e., no ultrasound, with only mechanical pressure over the spleen) and a splenic ultrasound stimulation cohort (i.e., 290.4 mw/cm 2 ispta or 1.4 mechanical index (mi) using the shear wave elastography setting on ge logiq e9) that insonified a single site (i.e., the splenic hilum; as identified by a trained ultrasonographer performing the stimulation). the trial was carried out in accordance with international conference inclusion and exclusion criteria. to be eligible to participate in the study, an individual must have met the following criteria: aged between 18 and 45 years, be without physical disability or conditions that may make them incapable of undergoing the study procedures or otherwise place them at greater harm, be without significant past medical or surgical histories that would render them at a greater risk of harm, be considered english proficient due to the study requirement to follow verbal commands during the ultrasound session, be considered active as assessed by type of activity (e.g., walking or running) and number of hours a week performing the various activities, be able to attend all study visits at approximately the same time of day, be able to comprehend the study goals and procedures, and able to provide informed consent for participation. see table s5 for subject characteristics used in the cytokine analysis shown in fig. 1 . study timeline. subjects underwent a screening visit 1 to 11 days prior to baseline visit to assess eligibility to participate in the study. eligible individuals that agreed to participate and provided written informed consent then underwent a physical and neurological examination. women of childbearing potential were asked to provide a urine sample for pregnancy testing, and approximately 21 ml of blood was drawn. on the first protocol visit (day 0), participants again underwent a physical and neurological examination, and medical history review. a baseline blood draw was then collected for cytokine and blood chemistry analysis (~35 ml). the blood draw was performed under sterile conditions using standard venipuncture techniques. individuals then underwent the ultrasound procedure based on random assignment to one of 7 groups, including the two groups reported herein (i.e., the sham control group, and a stimulated group that received 100% power stimulus applied at a single splenic target location). for ultrasound stimulus delivery, individuals were asked to lie in the right lateral recumbent position with their arms above their heads to expose the splenic region of the abdomen. after ultrasound gel was applied to the region, the ultrasound probe was placed on the participant's abdomen. this procedure was performed for both the sham control group (n=9) and ultrasound stimulated group (n=9). note that one subject from each group was excluded, in which 10 subjects were initially recruited to the study for each group (see table s5 ). stimulation paradigm. in the ultrasound stimulated cohort, the subject had the dimensions of their spleen assessed (using the b mode imaging setting on the ge logiq e9), and the probe (c1-6) was positioned over the splenic hilum. stimulation was performed using a modified elastography setting on the logiq e9 operated in two modes: an imaging/targeting mode with short ultrasound pulses (on the order of 1 microsecond) and relatively low acoustic power levels (on the order of 10 mw/cm 2 ), and a stimulation mode (from the clinical shear wave elastography setting) using longer ultrasound pulses (on the order of 1 ms) and higher acoustic power levels (290.4 mw/cm 2 ). stimulation was performed in twelve 15-second long epochs separated by 15 seconds each, and a total stimulation duration of 3 minutes. the 15 second epochs were performed during breath holds, during which an ultrasonographer was holding the probe in position above the spleen. before each stimulation period, the ultrasonographer checked the location of the probe using the imaging mode to ensure that all stimulation pulses were on target. the focal landmark used for targeting and stimulation was the hilum of the spleen in the group included in the presented analysis. for the sham control group, the movements and probe contact for the ultrasound procedure were performed by the ultrasonographer, and the total duration of the procedure remained the same as the stimulated group, except that no ultrasound power was applied (i.e., ultrasound output via the probe was turned off). a post-ultrasound blood draw was again performed, and the 2-hour blood draw time-point data is reported herein. blood analysis procedures. prior to the ex vivo cytokine test, the lps was sonicated for 30 minutes. a 10 ml blood tube (heparin; green cap) was brought into a tissue culture/sterile room and blood was transferred to a 50 ml conical tube. a blinded researcher then diluted the lps to the test concentrations in 15 pre-labeled tubes containing replicates of the different concentrations of lps used for blood exposure (dilutions were made to achieve 0, 0.1, 1, and 10 ng/ml of lps when mixed with blood). upon mixing the lps with blood, the final tubes were capped and placed on a rocker within an incubator and the samples were kept at 37 degrees celsius for 4 hours during incubation. after the incubation, the samples were removed from the incubator, centrifuged at 6000 rpm for 5 minutes, and the plasma supernatant was transferred (via pipette) for storage (frozen prior to analysis). tnf analysis was performed in triplicate from each sample using duoset elisa kit (r&d systems), as per manufacturer's instructions. this clinical study is a controlled, randomized, double-blinded trial of short-term (14 days) treatment and is listed at clinicaltrials.gov (nct03690466) for which a portion of the results are presented in this paper. the study is still ongoing, but the rnaseq data obtained thus far in five ultrasound stimulated patients is presented in this paper for its relevance in revealing the use of splenic ultrasound for potentially treating covid-19 patients. the protocol, informed consent form, recruitment materials, and all participant materials were submitted and approved by the university of minnesota's institutional review board (irb) and monitored by ctsi in accordance with its institutionally approved monitoring plan. the overall objective of the clinical trial is to demonstrate safety and efficacy of spleen ultrasound stimulation in the treatment of ra in 20 participants. for this ongoing study, 17 participants have been recruited, consented, and enrolled with all 17 patients completing the full study. the 17th participant completed the study, however the last 'in-clinic' visit was performed using clinician video conferencing due to the covid-19 pandemic, while in-person blood sample collection was still able to be performed. further details for this study can be found on clincialtrials.gov and will be presented in a future publication with complete outcome analyses. the description below only includes relevant methods and rnaseq analyses presented specifically in this paper. inclusion and exclusion criteria. to be eligible to participate in the study, an individual must have met the following criteria: 18 years of age or older, carried a diagnosis of seropositive (rheumatoid factor-or cyclic citrullinated peptide antibody-positive) rheumatoid arthritis (ra), and exhibited symptoms or signs of inadequate disease control (either modified haq score >0.3 or das-28-crp >3.2). exclusion criteria included active bacterial or viral infection, pregnancy, malignancy, or inability to provide daily self-care. the patient medical history and characteristics for each of the five subjects presented in this paper is provided in table s2 . study timeline. participants were assessed during in-clinic visits on days 0 (before treatment began), 3, 7, 10, 14, and 21. minor flexibility was allowed for scheduling clinic visits outside of holidays for special circumstances. on the first visit, the spleen was imaged by an ultrasonographer and the center of the spleen trajectory was marked on the participant's skin. participants were instructed on how to self-administer ultrasound treatment to the spleen. the procedure involved sweeping the ultrasound wand continuously across a 5-inch by 5-inch square centered on the spleen mark placed by the ultrasonographer and ensuring full contact was made with the skin during the daily 30 minute stimulation period. researchers were able to visually confirm that daily treatments were administered through daily online video sessions with the patients. these video sessions took place at about the same time each day for the 14-day treatment period. with each inclinic visit, participants received a joint examination, peripheral joint ultrasound, and blood draw, as well as completing several physical evaluations and questionnaires. full study details including analysis of all patients and assessments will be presented in a future publication. treatment. after an in-clinic training session on day 0, transcutaneous ultrasound was administered to the spleen for 30 minutes daily for 2 weeks (14 days total) via a portable device (1.2 w/cm 2 ; soundcare plus, roscoe medical) in the patient's home. the patients were randomized (1:1) to a treatment group or a control (sham) group, in which the latter received the exact same evaluations and stimulation paradigm as the treatment group, except that the device did not deliver any energy from the ultrasound transducer. ra patients and clinical assessors were blinded as to which patients received ultrasound or sham treatment. rna-sequencing and statistical analyses. 4ml of blood was collected in a green top lihep tube from each ultrasound stimulated participant (n=5) on day 0 (before the first splenic ultrasound stimulation was administered) and on day 14 (after the final treatment session). the samples were processed the same day as collection. peripheral blood mononuclear cells were isolated from whole blood using density gradient centrifugation via sepmate tubes (stemcell technologies) and following manufacturer's instructions. pbmcs were then gently frozen and stored at -80 ºc as per 10x genomics demonstrated protocols gc000039. following this protocol, once all samples had been collected and frozen, samples were thawed quickly in 20% fbs in pbs, resuspended in 10% fbs, and strained before proceeding to 10x genomics single cell protocol. samples were sequenced using umi-based approach from10x genomics, as previously described (32) and mapped to the human genome. transcriptomic analysis was performed using r statistical software, and primary filtering and normalization of each sample was done using seurat package as previously described (32, 55, 56) . samples included two timepoints from five subjects and these individual samples were merged for cell type assignment and differential expression analysis. cell types were assigned using standard marker genes (see fig. s2 ) as previously described (57) . differential expression was determined between timepoints for all cells in each cell type using a negative binomial generalized linear model. go term analysis was performed using the org.hs.eg.db and topgo packages. go term enrichment was assessed using the 'elim' algorithm with a fisher exact test cutoff of 0.01, in which the top 40 significant go terms in monocytes are shown in table s3 (58) . pre-clinical tests in rodents were performed as previously described (31) . experiments were performed under protocols approved by the institutional animal care and use committee of ge research. briefly, a vivid e9 (ge) ultrasound system was used to perform an ultrasound scan and locate the spleen. a hifu transducer and system was then positioned on the target area, and a separate ultrasound scan performed using a smaller probe (3s, ge) that was placed within the opening of the hifu transducer was used to verify alignment of the ultrasound beam with the spleen. adult sprague-dawley rats that were 8-12 weeks old (250-300 g; charles river labs) were housed at 25 degrees celsius on a 12-h light/dark cycle and acclimatized for 1 week, with handling before experiments to minimize the potential confounding measures due to stress response. water and regular rodent chow were available ad libitum. lps (0111:b4, sigma aldrich) was used to produce a significant state of inflammation in the naïve adult rodents. lps was administered to animals in the amount of 10 mg/kg, which corresponds to a ld75 dose via intraperitoneal injection. treatment. for ultrasound stimulation, animals were anesthetized with 2-4% isoflurane, and the animals were laid on a water circulating warming pad to prevent hyperthermia during the procedures. the region designated for ultrasound stimulation was shaved with a disposable razor and animal clippers prior to stimulation. ultrasound was applied to the designated area above the spleen using the probe and system described and calibrated from a previous study (31) . an ultrasound stimulus using the previously found optimal stimulation parameters (1.1 mhz, 0.5 ms pulse repetition frequency, 136.36 microsecond pulse length, 568 mw/cm 2 ispta) was then applied for 2 minutes, and lps was immediately injected post-stimulation. animals were allowed to incubate under anesthesia for one hour, and after incubation the animal was euthanized, and tissue and blood samples were collected. for experiments testing antibody production, an ultrasound stimulus was applied daily (in the morning) for three days prior to lps injection. on the fourth day, following the three consecutive treatment days, lps (10mg/kg) was injected intraperitoneally. the animals were then incubated for 60 minutes in accordance with previous studies, and tissue and blood were harvested for analysis of cytokines and antibody production. tissue collection. an incision was made starting at the base of the peritoneal cavity extending up and through to the pleural cavity. the spleen was rapidly removed and homogenized in a solution of phosphate-buffered saline, containing phosphatase (0.2-mm phenylmethylsulfonyl fluoride, 5µg/ml aprotinin, 1-mm benzamidine, 1-mm sodium orthovanadate, and 2-µm cantharidin) and protease (1-µl to 20 mg of tissue as per roche diagnostics) inhibitors. a targeted final concentration of 0.2-g tissue per ml pbs solution was applied in all samples. after collection of the whole blood, we allowed the blood to clot undisturbed at room temperature for 15-30 minutes. samples were centrifuged at 1,000-2,000x g for 10 minutes in a refrigerated centrifuge with the resulting supernatant being collected for antibody testing. samples were then stored at −80 °c until analysis. splenic tissue lysate was analyzed for cytokine concentrations using elisa kits for quantifying tnf alpha (abcam, ab100785), ifn gamma (abcam, ab239425), il-13 (abcam, ab100766), il-12 (lsbio, ls-f34357), il-10 (abcam, ab100765), il-2 (abcam, ab221834), il-1 beta (abcam, ab100768), il-1 alpha (abcam, ab113350), il-6 (abcam, ab234570) and il-4 (abcam, ab100771) as per manufacturer's instructions for tissue samples. serum was tested using elisa kits for quantifying antibody production including igg (abcam, ab189578), and igm (abcam, ab215085) as per manufacturer's instructions. figure s1 . modulation of cytokines with splenic ultrasound in rats figure s2 . cellular transcriptional signatures . s2c ), and also showing number of reads (counts) and average number of genes for each cell type. we observe that all that all cells have a relatively consistent read depth for the sequencing reaction performed. also shown are the number of genes out of the human genome that are annotated with that go term, number of genes from the list of significantly downregulated genes with ultrasound, and the number of genes that would be expected by random chance. column labeled 'classicfisher' shows p-values from the fisher's exact test and 'sig_exp' is the number of genes found to be significant in our gene list (decreased with ultrasound in monocytes) compared to the number that might be expected by chance. c d 3 d c r e m h s p h 1 s e l l g im a p 5 c a c y b p g n l y n k g 7 c c l 5 c d 8 a m s 4 a 1 c d 7 9 a m ir 1 5 5 h g n m e 1 f c g r 3 a v m o 1 c c l 2 s 1 0 0 a 9 h l a − d q a 1 g p r innate immunity and inflammation signaling in innate immunity and inflammation innate immune response to viral infection mechanisms and therapeutic relevance of neuro-immune communication acute respiratory failure in covid-19: is it "typical" ards? epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the inflammatory response in sepsis inflammation in acute kidney injury rheumatoid arthritis classification criteria: 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antioxidant stress and anti-inflammation avoiding off-target effects in electrical stimulation of the cervical vagus nerve: neuroanatomical tracing techniques to study fascicular anatomy of the vagus nerve reversible neuroinhibition by focused ultrasound is mediated by a thermal mechanism transcranial ultrasonic stimulation modulates single-neuron discharge in macaques performing an antisaccade task remote, brain region-specific control of choice behavior with ultrasonic waves effects of focused ultrasonic radiation on peripheral nerve, with observations on local heating ultrasound produces extensive brain activation via a cochlear pathway single-unit recordings reveal increased peripheral nerve conduction velocity by focused pulsed ultrasound ultrasound modulates the splenic neuroimmune axis in attenuating aki peripheral focused ultrasound stimulation (pfus): new competitor in pharmaceutical markets? noninvasive sub-organ ultrasound stimulation for targeted neuromodulation noninvasive ultrasound stimulation of the spleen to treat inflammatory arthritis therapeutic ultrasound attenuates dss-induced colitis through the cholinergic antiinflammatory pathway ultrasound prevents renal ischemia-reperfusion injury by stimulating the splenic cholinergic anti-inflammatory pathway safety of transcranial focused ultrasound stimulation: a systematic review of the state of knowledge from both human and animal studies elastography: general principles and clincial applications brain-spleen link tunes immunity vagal regulation of group 3 innate lymphoid cells and the immunoresolvent pctr1 controls infection resolution risk of infection associated with anti-tnf-alpha therapy risk of infections in rheumatoid arthritis patients treated with tocilizumab dopamine mediates vagal modulation of the immune system by electroacupuncture whole blood cytokine attenuation by cholinergic agonists ex vivo and relationship to vagus nerve activity in rheumatoid arthritis nicotine exposure alters in vivo human responses to endotoxin physiology and immunology of the cholinergic antiinflammatory pathway brain control of humoral immune responses amenable to behavioural modulation selected cytokine pathways in rheumatoid arthritis cytokines in acute and chronic inflammation lps induction of gene expression in human monocytes single-cell landscape of bronchoalveolar immune cells in patients with covid-19 acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov-2 pandemic the role of immune complexes revisited storm, typhoon, cyclone or hurricane in patients with covid-19? beware of the same storm that has a different origin therapeutic potential and limitations of cholinergic anti-inflammatory pathway in sepsis cholinergic agonists inhibit hmgb1 release and improve survival in experimental sepsis integrating single-cell transcriptomic data across different conditions, technologies, and species multiplexed droplet single-cell rna-sequencing using natural genetic variation improved scoring of functional groups from gene expression data by decorrelating go graph structure we thank all study participants. we are grateful for the assistance of individuals at the university of minnesota genomics center, especially jerry daniel and emma stanley. additionally, we would like to thank stuart sealfon and frederique ruf-zamojski for their assistance and advice processing pbmc samples for single cell sequencing. this work was supported by the united states defense advanced research projects agency (darpa) electrical prescriptions (electrx) program under the guidance of eric van gieson and gretchen table s6 . splenic ultrasound in healthy participants: subject features. characteristics and demographic data of 20 enrolled subjects for study of biological effects of ultrasound of the spleen. ten subjects received full-powered ultrasound treatment at the spleen hilum and another ten subjects were sham controls with the ultrasound device turned off. one of the subjects from the stimulated group was excluded due the selection of the wrong ultrasound imaging protocol by the clinician. one of the subjects from the control group was excluded because their baseline glucose measurement was <50 mg/dl that is considered hypoglycemic. key: cord-353887-f4yd7guj authors: tang, yujun; liu, jiajia; zhang, dingyi; xu, zhenghao; ji, jinjun; wen, chengping title: cytokine storm in covid-19: the current evidence and treatment strategies date: 2020-07-10 journal: front immunol doi: 10.3389/fimmu.2020.01708 sha: doc_id: 353887 cord_uid: f4yd7guj severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the pathogen that causes coronavirus disease 2019 (covid-19). as of 25 may 2020, the outbreak of covid-19 has caused 347,192 deaths around the world. the current evidence showed that severely ill patients tend to have a high concentration of pro-inflammatory cytokines, such as interleukin (il)-6, compared to those who are moderately ill. the high level of cytokines also indicates a poor prognosis in covid-19. besides, excessive infiltration of pro-inflammatory cells, mainly involving macrophages and t-helper 17 cells, has been found in lung tissues of patients with covid-19 by postmortem examination. recently, increasing studies indicate that the “cytokine storm” may contribute to the mortality of covid-19. here, we summarize the clinical and pathologic features of the cytokine storm in covid-19. our review shows that sars-cov-2 selectively induces a high level of il-6 and results in the exhaustion of lymphocytes. the current evidence indicates that tocilizumab, an il-6 inhibitor, is relatively effective and safe. besides, corticosteroids, programmed cell death protein (pd)-1/pd-l1 checkpoint inhibition, cytokine-adsorption devices, intravenous immunoglobulin, and antimalarial agents could be potentially useful and reliable approaches to counteract cytokine storm in covid-19 patients. in december 2019, an outbreak of a novel coronavirus-based disease was reported in wuhan, china. on 11 february 2020, the world health organization (who) named this coronavirus "severe acute respiratory syndrome coronavirus 2" (sars-cov-2) and the disease that it caused "coronavirus disease 2019" (covid19) . as of 25 may 2020, sars-cov-2 has affected over 212 countries, and about 5,529,195 cases have been confirmed around the world, of which 347,192 people have died. the reason for these deaths is suspected to be the "cytokine storm" [also called "cytokine storm syndrome" (css)]. the international classification of diseases (icd) does not include the cytokine storm or css. cron and behrens bring the current knowledge of css (1) . they define that "cytokine storm" is an activation cascade of auto-amplifying cytokine production due to unregulated host immune response to different triggers. the triggers involved infections, malignancy, rheumatic disorders, etc. another scholar described that cytokine storm is a systemic inflammatory response to infections and drugs and leads to excessive activation of immune cells and the generation of pro-inflammatory cytokines (2) . a similar entity is termed "cytokine release syndrome" (crs), which is not defined in the textbook of css (1) . crs is an acute systemic inflammatory syndrome characterized by multiple-organ dysfunction (mod). it has been reported that chimeric antigen receptor (car)-t-cell therapy could help to distinguish crs from a cytokine storm (2) . of note, the textbook described the criteria of css based on hemophagocytic lymphohistiocytosis (hlh) and secondary hlh (shlh) associated with rheumatic disorders, such as macrophage activation syndrome (mas) (1) . thus, it may be not applicable in covid-19 because the covid-19 is a contagious disease and relatively irrelevant to a genetic disorder. up to date, there is still a lack of clinical and laboratory criteria to identify the cytokine storm. in this review, we referred covid-19 associated cytokine storm as the patients who are severely ill along with a high concentration of pro-inflammatory cytokines. for patients with covid-19, the number of white blood cells, neutrophils, as well as levels of procalcitonin, c-reactive protein, and other inflammatory indices, are significantly higher in the intensive care unit (icu) cases than in non-icu cases (3, 4) . many studies showed that severely ill patients tended to have a higher concentration of pro-inflammatory cytokines, especially interleukin (il) 6, than moderately ill patients in covid-19 (5) (6) (7) (8) (9) . the result of the bronchoalveolar lavage fluid (balf) cells, which tested by transcriptome sequencing, reveals excessive chemokines releasing caused by sars-cov-2 infection, such as cxcl10 and ccl2 (10) . the high level of cytokines also indicates a poor prognosis in covid-19 (6, 11, 12) . furthermore, the pathology of postmortem examination of the lung, from who was died of covid-19, demonstrated the existence of acute respiratory distress syndrome (ards) and t-cell overactivation chen and subbarao (21) ]. method of detection (number of patients studied) cba (20) cba ( chemokines (13) . this phenomenon is due to an increase in the number of t-helper (th) 17 cells and the high cytotoxicity of the cd8 + t cells (13) . the innate and adaptive immune responses activated by sars-cov-2 infection lead to uncontrolled inflammatory responses and ultimately cause the cytokine storm (14) . the cytokine storm can lead to apoptosis of epithelial cells and endothelial cells, and vascular leakage and, finally, result in ards, other severe syndromes, and even death (15) . to lower mortality due to cytokine storm, we summarized the clinical and pathology features of the coronavirus-related cytokine storm. we explored the efficacy and safety of potential treatments and their molecular mechanism. there is still lacking sufficient evidence supporting the regulation of cytokine expression may be beneficial to the mortality of covid-19. the early-stage clinical characteristics of mers and sars are influenza-like symptoms (16) (17) (18) : pyrexia, sore throat, dry cough, myalgia, and dyspnea. those symptoms are very similar to the characteristics of early covid-19 and progress rapidly to pneumonia (3, 19, 20) . it has been found that the regulation of several cytokines is disordered in the peripheral blood of sars patients, as summarized by chen and colleagues (21) and listed in table 1 . table 1 shows an increase in levels of cytokines and chemokines and a decrease in levels of anti-inflammatory cytokines such as il-10. of note, the release of pro-inflammatory cytokines, especially interferon (ifn)-α and ifn-γ, is correlated with lethal sars (22, 23) . the cytokines with increased levels in fatal sars are il-6, il-1β, ifn, and cxcl10. these cytokines are secreted mainly by dendritic cells (dcs) and macrophages, indicating that innate immunity plays a pivotal part in lethal sars. ccr4 + ccr6 + th17 cells have many chemokine receptors and may share the same mechanism and function in cell-cell interactions in sars. cytokines secreted by dcs and macrophages induce the infiltration and recruitment of pro-inflammatory th17 cells. analyses of lungs from sars patients have revealed diffuse alveolar damage as a crucial feature. histopathological studies have shown lung consolidation and edema with pleural effusions and focal hemorrhage, all of which resemble covid-19 features (13, 24) . besides, the lungs of sars patients are infiltrated extensively with neutrophils and macrophages, which are not observed in covid-19. in peripheral blood, numbers of cd4 + and cd8 + t cells are reduced in cases of covid-19 and sars (13, 25) and are associated with death in the latter (25) . interestingly, unlike mers and sars, a high concentration of pro-inflammatory cc chemokine receptor (ccr)4 + ccr6 + th17 cells are found in covid-19 (13) . the innate and adaptive immune system takes multiple measures to respond to virus infection. mers-cov infects human epithelial cells and leads to these cells inducing significant but delayed responses by ifn, pro-inflammatory cytokines (e.g., il-1β, il-6) and chemokines (e.g., il-8) (26, 27) . sars-cov infects airway epithelial cells and results in delayed release of chemokines such as ccl3, ccl5, ccl2, and cxcl10 (28) . besides, mers-cov infects hematopoietic cells such as monocytes, macrophages, and dcs, which is not seen in those cells upon sars-cov infection (29) (30) (31) (32) . mers-cov infects the cells mentioned above to induce delayed (but increased) levels of pro-inflammatory cytokines (e.g., il-2) and chemokines (e.g., ccl2, ccl3) (27, 30) . although sars-cov is abortive in macrophages and dcs, the virus induces an increase in levels of pro-inflammatory cytokines and chemokines (31, 32) . sars-cov and sars-cov-2 infect cells using the same receptor: angiotensin-converting enzyme-2 (33) . hence, it has been postulated that both viruses can affect the same spectrum of cells. in the aspects of murine models of coronavirus, infection with sars-cov in balb/c mice has been shown to induce an increase in the number of pathogenic inflammatory monocytemacrophages (imms) (34) . through stimulation of ifnα/β receptors, the accumulating imms produce monocyte chemokines (e.g., ccl2, ccl7, ccl12) and pro-inflammatory cytokines [e.g., tumor necrosis factor (tnf), il-6, il1β], which results in further accumulation of pathogenic imms. targeting of ifn signaling, imms, or pro-inflammatory cytokines could offer protection from lethal sars-cov infection. in this way, the chemokines (produced by activated monocytes and macrophages) lead to the recruitment of neutrophils, monocytes, and t cells into the lungs (28) . after chemotaxis, activated effector t cells migrate to the lungs and destroy pneumocytes/permissive cells due to response to the virus infection (35) . the damage caused by neutrophils, monocytes, and t cells results in lung-parenchyma changes, such as diffuse alveolar damage, which leads to ards (35) . in summary, the excessive cytokines and chemokines caused by lethal coronavirus infection involve mainly antigen-presenting cells (apcs) (such as macrophages) and t cells. however, cytokines secreted by immune cells are produced to eliminate viral infection, and deficiency of such cytokines may be harmful to the body. for example, virus titers are significantly higher in toll-like receptor (tlr)3 −/− , tir-domain-containing adapterinducing interferon-β (trif) −/− , and il-6 −/− mice compared with their wild-type counterparts, and are associated with severe lung damage (36, 37) . in china, we classified the stage of covid-19 according to the guidelines (38) issued by the national health commission of the people's republic of china (nhc). according to the instructions, nhc defines severe illness of covid-19 as one of the following conditions: respiratory rate ≥30 breaths/min in the resting state; oxygen saturation ≤93%; arterial blood oxygen partial pressure (pao 2 )/fraction of inspired oxygen concentration (fio 2 ) ≤300 mmhg. critical illness as one of the following conditions: respiratory failure and requiring mechanical ventilation; shock; complication of other organ failures, and needs intensive care. the most common symptoms of covid-19 were fever, cough, shortness of breath, fatigue, and myalgia (5, 7, 39, 40) , and severe cases tend to be older with more basic diseases and suffer from dyspnea, more complications (5, 40) . in covid-19, 14% of patients progress to severe disease and 5% to critical illness (41) . a prospective study reported that the computerized tomography (ct) of the lungs of covid-19 (6) . the lung lesions increase and the scope expands as the disease progresses, and ground-glass opacity coexisted with consolidation or striated shadow. some severe patients showed diffuse lesions in both lungs. up to date, the inflammatory disorders (insufficient in chemokines) in covid-19 have been reported in many clinical studies. the covid-19 is inclined to cause a decrease of lymphocyte count and an increase of c reactive protein (crp), especially in severely ill patients (5) (6) (7) (42) (43) (44) . the major subsets of the t lymphocytes (t cell) (cd3 + cd4 + t cell and cd3 + cd8 + t cells) are reduced in the covid-19 and are significantly lower in the severe cases (5, 12, 42, 43, 45, 46) ; however, controversial results are also reported in some studies (7, 40) . the results of the other immune cells, the b cell and natural killer (nk) cell, have more inconsistency in recent researches. il-6 was observed increased in all studies, and only one study show il-10 was not elevated. about half of the studies we collected showed tnf-α was increased. only huang et al. (9) inspected the multiple types of chemokines and found that severe patients had higher levels of g-csf, gm-csf, ip-10, mcp-1, mip-1a, mip-1b, rantes, and il-8. the inflammatory disorders of covid-19 were summarized in table 2 . comparison objects il-6 il-1β il-10 tnf-α ifn-γ il-2(r) the pathologic features of covid-19 showed the lungs were infiltrated with excessive ccr6 + th17 cells and high cytotoxicity of cd8 + t cells (13) . but high cytotoxicity of cd8 + t cells does not mean they exert the normal function. the sars-cov-2 could lead to cytotoxic lymphocytes (mainly involving nk cells and cd8 + t cells) exhaustion, which is manifested as the upregulated exhaustion markers, such as nkg2. the exhaustion markers return to normal in patients who have recovered or are convalescent (47, 48) . balf cells were found extreme cytokine releases, such as ccl2, cxcl10, ccl3, and ccl4 (10). furthermore, xiong et al. (10) use the transcriptome dataset approach to discover that sars-cov-2 can activate apoptosis and p53 signaling pathway (one of the pathways responsible for the survival of the cell) in lymphocytes. these results could provide some reasons for the cause of patients' lymphopenia. another team of chen and his colleagues studied the mechanisms for lymphopenia (49) . their results demonstrate that sars-cov-2 infected the cd169 + macrophages in spleens and lymph nodes (lns), and lead to lymphoid tissue damage, such as splenic nodule atrophy and lymph follicle depletion, etc. the cd169 + macrophages express high fas and cause activation-induced cell death (aicd) through fas/fasl interactions. furthermore, sars-cov-2 selectively induced macrophages to produce il-6, not tnf-α and il-1β, to directly promotes lymphocyte necrosis. the analysis of peripheral blood mononuclear cells (pbmcs) revealed that non-structural protein (nsp) 9 and nsp10 of sars-cov-2 target nkrf (nf-κb repressor) to promote il-6/il-8 production (50) . as a consequence, it recruits neutrophils and induces uncontrollable host inflammatory response. collectively, the clinical, immunological, and pathologic features of covid-19 have something in common with sars and mers. for example, all the viruses can cause lymphopenia and influenza-like symptoms in the early stage. sars and covid-19 do not lead to the upgrade of tnf-α, but the increase of il-6 and il-10 is more prevalent in covid-19. the il-6 plays a crucial role in the pathologic of covid-19, including the chemotaxis of neutrophils and lymphocyte necrosis. importantly, covid-19 is more able to cause cytotoxic lymphocytes exhaustion. tocilizumab (tcz) is a recombinant humanized anti-human il-6 receptor monoclonal antibody, preventing il-6 binding to its receptor to exert the immunosuppression promoted by il-6. michot et al. (51) reported that 42-year-old male suffering from respiratory failure due to sars-cov-2 infection. after 4 days of tcz treatment, the crp decreased from 225 to 33 mg/l and ultimately clinically fully recovered. similarly, some case reports showed tcz is an efficacy and safety approach in covid-19, even patients with other diseases combined, such as multiple myeloma, end-stage renal disease, and sickle cell disease (52) (53) (54) . recently, a retrospective study (55) found that tcz decreased crp in all patients (n = 15) rapidly, but three of them, who are critically ill, still dead. the dead patients show continuously rising of il-6 even after the administration of tcz and methylprednisolone, indicating that repeat doses of tcz may be needed in covid-19 patients who are critically ill. another retrospective study (56) demonstrated that tcz showed a quick control of severe covid-19 manifestation, such as fever, respiratory function. all patients (n = 21, two were critically ill), have recovered and have been discharged from hospital, and no adverse event was reported during the treatment. a prospective open-label, multicenter single-arm study manifests the pilot results of the off-label application of tcz in severe patients with covid-19 (57) . the study involved 63 patients with severe covid-19, and tcz succeeded in improving respiratory and laboratory parameters, such as pa0 2 , fi0 2 , consequently, increased the likelihood of survival (the death rate of the study is 11%). it is worth mentioning that a cautionary case report by radbel et al. (58) . two patients were diagnosed with covid-19 complicated by crs and treated with tcz. unfortunately, both patients progressed to severe hlh, and one developed to viral myocarditis. all the cytokines produced by immune cells are responsible for viral clearance. suppression of cytokine release at an early stage of disease as treatment is controversial. application of synthetic disease-modifying antirheumatic drugs (dmards) and biologic dmards to downregulate cytokine expression in ra increases the risk of infection (59, 60) . the timing and the doses of the intervention still need to be inspected clearly. sars-cov-2 mainly causes a dramatic increase in il-6 and does not remarkably promote other pro-inflammatory factors, such as il-1β and ifn-γ. although treating covid-19 with tcz is an off-label use, it may be relatively appropriate and safe in coping with covid-19 associated cytokine storm basing on the current evidence. it still needs more large samples and high-quality studies to evaluate the exact efficacy and safety in covid-19. the ongoing trials of potential treatments and other treatments focus on inflammatory disorders in covid-19 are available in supplementary table 1 . glucocorticoid therapy is used widely among critically ill patients with other coronavirus infections (e.g., sars, mers). corticosteroids have been administered to icu patients infected with sars-cov-2 (3, 4, 20) . glucocorticoids exhibit pharmacologic effects at any therapeutically relevant dose through classic genomic mechanisms. some immunosuppressive effects are based on transactivation, and glucocorticoid induces gene transcription and protein synthesis of nf-κb inhibitors and lipocortin-1. through inhibition of nf-κb signaling, glucocorticoids induce inhibition of synthesis of downstream proteins such as il-1, il-6, granulocyte-macrophage colony-stimulating factor, and inducible cyclooxygenase-2 (61, 62) . glucocorticoids reduce the proliferation, activation, differentiation, and survival of t cells and macrophages (63) . glucocorticoids proffer inhibitory actions on the transcription and action of various cytokines. the th1 and macrophage-based pro-inflammatory cytokines il-1β, il-2, il-6, tnf-α, and il-17 are inhibited by glucocorticoids (63) . however, it is controversial whether corticosteroids are beneficial in the treatment of severe covid-19 patients. a comment and a meta-analysis, which mainly bases on the evidence of sars and mers (64, 65) , stated that corticosteroid would increase mortality and delayed clearance of viral in coronavirus infection diseases. thus, the corticosteroids should not be administrated for the treatment of sars-cov-2 induced lung injury or shock. newly published studies also indicate that the use of corticosteroids is not beneficial for covid-19 patients (not severe cases), and high-dose corticosteroids are associated with mortality (44, 66, 67) . most covid-19 patients discussed in these studies are not severe cases. inspecting the studies included and analyzed by the meta-analysis, only one study (68) described the numbers of patients with corticosteroids and non-corticosteroids treatment in the severe group and non-severe group. the study demonstrated the benefit of corticosteroids use in severe sars-cov infection. another comment (69) , which was written by front-line physicians from china, showed corticosteroids might have some benefit for critically ill patients with covid-19. systematic corticosteroid therapy could promote oxygen saturation and pao 2 /fio 2 . however, corticosteroids might not improve mortality in critical covid-19 patients. current evidence shows that sars-cov-2 induces an increase in a small range of cytokines. it might be overuse to administrate corticosteroids to counteract a wide range of cytokines. furthermore, sars-cov-2 causes relatively serious lymphocytopenia and lymphocytes exhaustion. glucocorticoidmediated stimulation of the "hypothalamic-pituitary-adrenal axis" might also exacerbate lymphocytopenia (70) . thus, the use of corticosteroid is a double-edged sword in covid-19. the dose, duration, and timing of corticosteroid therapy will be crucial if administrated to covid-19 patients. as stated above, lymphocytes exhaustion is one of the characteristics of covid-19, and pd-1 checkpoint-inhibitor might some help in reversing the anergy of lymphocytes. up to 4 may 2020, no study of pd-1 checkpoint-inhibitor has been reported in the treatment of covid-19. the pathway consisting of the receptor pd-1 and its ligands, pd-l1 and pd-l2, play crucial parts in the maintenance of peripheral tolerance. treatments with antibodies targeting pd-1/pd-1 ligands have elicited an increased response in different cancer types and, in tandem with antibodies targeting cytotoxic-tlymphocyte-associated antigen-4, have changed cancer therapy radically (71) . unfortunately, signaling regulated by the pd-1/pd-l pathway is also related to substantial inflammatory effects (e.g., sepsis), as this pathway plays a role in balancing protective immunity and immunopathology (72) . increased pd-l1 expression in monocytes is associated with mortality in patients with septic shock (73) . a meta-analysis of checkpoint inhibitors showed that such therapy increased the chance of survival (74) . nivolumab (anti-pd-1) and bms-936559 (anti-pd-l1) had completed phase-ib randomized studies for severe sepsis. they revealed that giving a checkpoint inhibitor did not result in unexpected safety findings or indicate a cytokine storm (75, 76) . also, cd4 + and cd8 + t cells were hyperactivated, as revealed by the high proportions of human leukocyte antigen-dr isotype and cd38, in covid-19; cd8 + t cells harbored high levels of cytotoxic granules in covid-19 patients, in which the phenotype is similar to fatal h7n9 disease (13, 77) . those results suggest that lethal covid, along with h7n9, may be related to defective activation and exhaustion of t cells, which also suggest that checkpoint-inhibitor administration may reverse this status. cytokine adsorption involves using a method, such as extracorporeal membrane oxygenation (ecmo), to filter harmful substances directly. an extracorporeal cytokine hemoadsorption device called cytosorb r (cytosorbents, monmouth, nj, usa) has been reported to capture and reduce inflammatory mediators. bruenger and colleagues reported that the plasma level of il-6 and procalcitonin decreased in one patient with severe ards after treatment with ecmo using a hemoadsorption device (78) . a 45-year-old patient with severe ards showed that venous arterial-ecmo combined with hemoadsorption therapy decreased plasma concentrations of il-6 and il-8. moreover, hemodynamic stabilization, respiratory improvement, and a decline in capillary leakage can be achieved in combination therapy (79) . two trials employing hemoadsorption therapy for infection-related cytokine storm are ongoing (nct04195126, nct03685383). a similar therapy involves dialysis. the mainly water-soluble mediators are removed from plasma, and the hemofilters can have additional adsorptive properties (80) . continuous venovenous hemofiltration and adsorption for severe septic shock are being tested in one clinical trial (nct03974386). neutralizing excessive cytokines with hemoadsorption devices might be relatively effective. the disadvantage is like corticosteroids: a wide range of cytokines would be adsorbed. thus, it would lead to the a lack of cytokines, which are at reasonable or even insufficient levels. we suggest treating the cytokine storm in covid-19 should base on the laboratory results of cytokines and chemokines. meanwhile, adjusting the parameters of the devices (e.g., treatment duration) for preventing overtreatment. ivig can elicit passive immunity, anti-inflammatory, and immunomodulatory effects that can improve treatment effects and increase survival in severe infection. an igg molecule binds to a specific target antigen through the humoral and cellular arms of the immune system. for example, igg molecule blocks the cellcell interactions mediated by cell-surface receptors (such as cd95 and cd95 ligand), neutralize the autoantibodies by anti-idiotypic antibodies, expanse the regulatory t (treg) cell populations via the blockade of immune complex binding to low-affinity fcγ receptors (fcγrs), to exert the functions of immunomodulation (81). ma and colleagues detailed a severe case of glandular fever treated with ivig (82) . levels of th1 cytokines (ifn-γ, il-12, soluble tumor necrosis factor receptor 1 (stnfr1), cxcl10, cxcl9, ccl3), and viral loads eventually recovered after the combination of prednisolone with ivig. a multicenter, doubleblind, randomized controlled trial for cases with severe influenza a (h1n1) infection demonstrated that ivig reduced the serum concentration of cytokines, viral load, and reduced mortality (83) . a meta-analysis of 17 studies (1,958 participants) found igm-enriched polyclonal and standard ig molecules decreased mortality in adults with severe sepsis or septic shock. however, a meta-analysis did not reveal a benefit in adult mortality with polyclonal ivig using high-quality trials only (84) . despite a lack of clinical evidence, the us gave emergency approval to hcq, a member of antimalarial agents, in covid-19 on 28 march (85) . a meta-analysis included the studies up to 5 april 2020 (86) and showed that four clinical trials and three observational studies are eligible for the study. unfortunately, the authors concluded that hcq has no clinical effect on patients with covid-19. however, a randomized clinical trial published on 24 april, which included the patients (n = 81) with critically ill covid-19 (such as high respiratory rate, peripheral oxygen saturation lower than 90%, shock), indicated 15.0% patients (6 of 40) have died in the low-dosage group (i.e., 450 mg twice daily on day 1 and once daily for 4 days). the critically ill death rate is over 50%, as reported by who (87) . thus, low-dosage of hcq could be beneficial for critically ill patients with covid-19. the study also indicates high dosage hcq might not be suitable for critically ill patients because of its potential safety hazards. traditional chinese medicine (tcm) has an essential role in the latest sars epidemic. several studies (88) (89) (90) (91) (92) (93) have shown that the add-on of tcm to western medicine can shorten the duration of hospitalization, alleviate symptoms, reduce mortality (including for critically ill patients), and reduce the prevalence of adverse reactions in sars. compared with a control group (western medicine only), a combination of tcm with western medicine has shown advantages in terms of symptom alleviation and preventing covid-19 (94) (95) (96) . however, the quality of the studies must be improved. the administration of tcm in a standard manner worldwide is complicated because of the different decoctions used and the matching of herbs. artemisinin can be obtained from artemisia annua, and one kind of antimalarial agents. hou and colleagues showed that extracts from artemisinin-family drugs could regulate cells from the innate and adaptive immune system, and lead to anti-inflammatory and immunomodulatory actions (97). the scope of application for artemisinin-family medicines includes infectious disease and autoimmune diseases, and artemisininfamily shows a difference in immune regulation compared with hydroxychloroquine (98-100). as stated above, ali and aki are crucial mortality factors in infectious diseases. artesunate is a derivative of artemisinin and can lessen the pathologic changes and neutrophil infiltration in the lungs of ali patients, and decrease sepsis-induced mortality (101) . by inhibiting expression of nf-κb signaling and enhancing heme oxygense-1 expression, the artesunate can lower the concentrations of tnf-α and il-6 in serum and bronchoalveolar lavage fluid. huang and colleagues discovered that dihydroartemisinin could attenuate lipopolysaccharide (lps)-induced ali through suppressing nf-κb signaling in a nuclear factor erythroid 2-related factor 2 (nrf2)-dependent fashion, thereby leading to a decrease in expression of the pro-inflammatory cytokines il-1β, tnf-α, and il-6 (102). hu and colleagues explored a new and efficacious approach for ali (103) . "artesunate liposomes" were prepared using film dispersion and then lyophilized to obtain liposomal artesunate dry powder inhalers (ladpis). after treatment with ladpis, a rapid reduction in accelerated inhalation, ali syndromes, and levels of tnf-α and il-6 has been observed in rats. besides, kidney impairment in hospitalized covid-19 patients is associated with a high risk of in-hospital death (104). cheng et al. (105) observed that dihydroartemisinin lessened glomerular injury and relieving increases in the urine albumin: creatinine ratio and serum levels of creatinine. current evidence of pathologic changes of covid-19 suggests the dysregulation of the cytokines involves mainly macrophages/monocytes. in a burn-based sepsis model balb/c mice, concentrations of adhesion molecules and neutrophil infiltration in the lungs and heart, and mortality rate are significantly increased, but those phenotypes could be reversed by artemisinin (106) . the authors discovered that artemisinin downregulates protein levels of nod-, lrr-and pyrin domaincontaining protein 3 (nlrp3) and caspase 1 in macrophages in burn-induced sepsis mice. also, a reduction in levels of the pro-inflammatory cytokines il-1β and il-18 has been observed post-therapy. nlrp3 is a sensor component expressed mainly in macrophages and which undergoes transcription by nf-κb. nlrp3 is responsible for the maturation and secretion of il-1β and il-18 (107) (108) (109) . nf-κb also increases the level of il-10 in the macrophages infected by plasmodium falciparum, and artemisinin could reduce il-10 production in animal models (110) , as well as in the clinic (111) . two studies focused on the relationship among tlr, nf-κb, nucleotide-binding oligomerization domain-containing protein (nod)2, and macrophages. tlr2 mainly locates outside the cell membrane of macrophages, dcs, and granulocytes, and recognizes bacteria (112) . tlr2 induces nf-kb activation through recruitment of tir domain containing adaptor protein (tirap) and myeloid differentiation primary response (myd)88 in macrophages and dcs. in inflammatory monocytes, tlr2 is expressed within endosomes and induces the release of type-i ifns via interferon regulatory factor 3 (irf3) and irf7 in response to viruses (113) . artesunate increases survival of mice challenged with live staphylococcus aureus/methicillin-resistant staphylococcus aureus (mrsa) compared with antibiotics alone, and its protection may be associated with reductions in tnf-α levels. artesunate reduces the expression of tlr2 mrna and nod2 mrna that upregulated by s. aureus/mrsa and also inhibits the activation of nf-κb (114) . kuang and colleagues found that the artesunate attenuated the release of tnf-α and il-6 from macrophages by inhibiting tlr4-mediated autophagic activation (115) . tlr4 also locates in the endolysosomal compartment, can recognize gram-negative bacteria and viruses (112) , shares the same pathway as the activation of nf-κb, and induces the release of type-i ifns via the tnf receptorassociated factor (traf3)-tank binding kinase 1 (tbk1)-irf3 axis (113) . however, kuang and co-workers discovered that artesunate attenuates the cytokine release by the traf6-beclin1-class iii phosphatidylinositol 3-kinase (pi3kc3) pathway. in a model of severe acute pancreatitis in rats, artesunate attenuates the release of il-1β and il-6 via the tlr4-nf-κb axis (116) . in addition, dihydroartemisinin inhibited the activation of tlr4 and irf3 in the spleen cells of systemic lupus erythematosus (sle)-prone mrl/lpr mice, which lead to a decrease in levels of ifn-α and ifn-β (117) . the mitogen-activated protein kinase (mapk) signaling pathway plays a vital part in the development, differentiation, proliferation, transformation, and apoptosis of cells (118) . the extracellular signal-regulated kinase (erk), jnk/stressactivated protein kinases (sapk), and p38 mapk are the dominant members of the mapk family. the cascades can be summarized as the erk pathway (raf-mek-erk), jnk pathway (tak1-mkk-jnk), and p38 pathway (tak1-mkk-p38). proinflammatory cytokines such as il-1 and tnf-α, ifnα, and ifnγ can induce activation of the p38 pathway, and p38 can regulate nf-κb-dependent transcription after its nuclear translocation. meanwhile, nf-κb is a crucial transcriptor for il-6, which could activate the il-6-janus kinase (jak)-signal transducer and activator of transcription (stat) pathways (119). wang and colleagues (120) found that another artemisinin derivative, sm905, suppressed generation of nitric oxide, tnf-α, il-1β, and il-6 in lps-induced macrophages. the underlying mechanism was that sm905 reduced activation of p38 and erk, and jnk suppressed iκbα degradation. furthermore, they observed that nf-κb was inhibited correspondingly in sm905-treated cells. in another lps-induced macrophage model, artemisinin has a property of prohibiting stat1 activation, and it leads to the reduction of no (an inflammatory-cascade inducer) in macrophages (121) . except for stat1, stat3, and stat5 in the splenocytes of sle-prone mrl/lpr mice could be inhibited by sm934, an artemisinin derivative (122) . artesunate therapy has been shown to improve the survival of mice infected with the herpes simplex virus. artesunate can lower levels of il-1β, il-2, il-6, ifn-γ, ccl2, ccl3, and ccl4 in these mice. these cytokines are produced primarily by apcs and th1 cells. previous studies have suggested that the artesunate can regulate th cells in virus infections. du and colleagues (123) demonstrated that the artesunate downregulated the th1 response and reduced levels of ifn-γ, tnf-α, il-12, il-18, ccl2, cxcl9, and cxcl10 in an experimental model of cerebral malaria. ra is an autoimmune disease manifested by dysfunction of various immune cells (e.g., apcs, th1, th17), which leads to a high concentration of il-1, il-6, tnf-α, and chemokines in plasma and tissues (124) . in the experimental models of ra, the proliferation of th17 cells and the production of il-17a and il-6 are inhibited by sm905 therapy and, correspondingly, the expression of retinoic acid receptor-related orphan nuclear receptor gamma t (rorγt) (a specific transcription factor for th17 cells) is also reduced (125) . fan et al. (126) demonstrated similar data and found that dc32 (an artemisinin derivative) can restore the t reg /th17 balance and reduce transcription of cxcl12 and cx3cl1. t reg can be anti-inflammatory, secrete anti-inflammatory cytokines (e.g., il-10), target th17 cells and macrophages, as well as reduce the concentration of il-1, il-6, tnf-α, and il-17 (127) . the immunosuppressive mechanisms of artemisinin on t cells include inhibiting differentiation of th17 cells by regulating the figure 1 | artemisinin-family drugs for cytokine storm in covid-19. the dysregulation of the cytokine storm involves mainly apcs. tlr2 and tlr4 locate mainly outside macrophages, dcs, and granulocytes. also, they are expressed within endosomes, play a role in recognizing bacteria and viruses. through myd88-dependent or trif-dependent pathway, tlr2 and tlr4 transmit signals for the activation of irf3 and nf-κb to induce the type i interferon and cytokines. besides, tlr2 leads to the activation of ap-1, which is responsible for the transcription of inflammatory cytokines. the cytokines target at the naïve t helper cell, to result in the naïve t helper cell to differentiate to th1 cell and th17 cell, subsequently to secrete the inflammatory cytokines and chemokines. moreover, the il-6, il-8, and il-10 secreted by monocytes and macrophages could activate cytokines receptors (i.e., il-6r, il-8r), lead to the activation of jak-stat signaling pathways and cell migration. the artemisinin-family drugs target at a variety of molecules (red and blueness nodes) in the inflammatory networks, such as nf-κb, irf3, erk (not shown in the figure), and rorγt, which inhibit the differentiation of inflammatory cells and the production of cytokines and chemokines. il-10 is an anti-inflammatory cytokine. it could be secreted by virtually all immune cells, including macrophages, dcs, nk cells, t cells, and b cells. at the moment, the high concentration of il-10 in severely ill patients with covid-19 is a mystery. on the one hand, it might play a role in antagonizing the biological function induced by il-6. on the other hand, the high concentration of il-10 might contribute to the lymphocytes exhaustion. ap-1, activating protein-1; ccl, c-c motif chemokine ligand; cxcl, c-x-c motif chemokine ligand; ikk, iκb kinase; ifn, interferon; irf3, interferon response factor 3; jak, janus kinase; jnk, jun n-terminal kinase; myd88, myeloid differentiation primary response protein 88; nf-κb, nuclear factor κ b; nlpr3, nod-, lrr-and pyrin domain-containing protein 3; mkk, mitogen-activated protein kinase kinase; smad5, smad family member 5; rorγt, retinoic acid receptor-related orphan nuclear receptor gamma t; stat, signal transducer and activator of transcription; tak1, tgfβ-activated kinase; t-bet, t-box transcription factor 21 (also known as tbx21); tlr, toll-like receptor; traf, tnf receptor-associated factor; tram, trif-related adaptor molecule; trif, tir domain-containing adaptor protein inducing interferon-β. il, interleukin. expression of rorγt and maybe also inhibition of activation of the erk pathway (ras-raf1-erk1/2) (128). in the model of ra-fibroblast-like synoviocytes (fls), artesunate decreased the production of il-6, il-8, and il-1β through preventing nf-κb translocation and iκbα degradation (129) . artemisinin-family drugs have shown efficacy and safety in treating malaria. one study reported 32 patients with severe malaria caused by plasmodium falciparum. ten patients suffered renal failure, eight had cerebral malaria, and 14 had other causes of severe malaria. after artesunate treatment, concentrations of il-6, and soluble il-6 receptor in plasma were normalized within 24 h (130). in recent years, artemisinin-family drugs have been shown to be beneficial against infection caused by the human cytomegalovirus, hepatitis-b virus, ebola virus, and human immunodeficiency virus (131) . shapira and co-workers reported the first case of the treatment of hcmv infection with artesunate (132) . germi and collaborators (133) reported that the artesunate led to an effective response in three cases with mild hcmv infection but was not efficacious in two patients with severe hcmv infection. the elevations of il-6 and il-10 are highly consistent in covid-19. il-6 targets the il-6 receptor, and the letter recruit jak, which transit cascade signal to activate signal transducer and activator of transcription 3 (stat3) (119) . some physicians suggest tofacitinib, a small molecule compound target jak1 and jak3, could be applied in the treatment of covid-19, and tofacitinib success in treating a covid-19 patient complicated with ulcerative colitis (134) . il-10, a cytokine with anti-inflammatory properties, could be secreted by virtually all immune cells, including macrophages, dcs, nk cells, t cells, and b cells (135) . we might tend to regard the high levels of il-10 as negative feedback of counteracting the increase of il-6 because il-10 can block the activity of nf-κb to downregulate the production of il-6 (135). however, an abundance of il-10 also inhibits the function and proliferation of immune cells (e.g., th1, nk cells, and cd8 t cells), which delays the clearance of viruses (135) . therefore, a mass of il-10 might be responsible for the normal levels (one study report low level) of ifn-γ (a cytokine for the clearance of viruses) and the exhaustion of lymphocytes. the il-10 inhibitor in the treatment of covid-19 also needs to be considered. even the combination of il-10 and il-6 inhibitor could be designed in future prospective studies. when using any method to regulate the dysregulation of cytokines, we might better closely monitor the laboratory index for preventing overtreatment. for example, if we use tcz to reduce the levels of il-6, we could check il-6 levels once every 2 days to keep it at a suitable concentration, which should be studied in the future. also, the dose and duration would be illuminated. the current evidence indicates that tcz, an il-6 inhibitor, is relatively effective and safe. based on the therapeutic mechanisms, we classified the remaining therapies, corticosteroids, pd-1/pd-l1 checkpoint inhibition, cytokine-adsorption devices, intravenous immunoglobulin, and antimalarial agents, as "less potential treatments." no literature of covid-19 except for corticosteroids mentions the effectiveness and safety of the less potential treatments. the benefits, dose, duration, and timing of corticosteroids still in debate, and the other less potential treatments need clinical evidence to validate. although the experimental model of infectious disease (e.g., malaria and sepsis) and autoimmune disease (e.g., ra and sle) indicates that artemisinin-family drugs could target the inflammatory networks to decrease the levels of cytokines (e.g., il-6 and tnf-α) and chemokines (e.g., il-8, cxcl10) (figure 1) . the effect and safety of antimalarial agents still need to be validated in the high-quality clinical studies and the sars-cov-2 infection disease model. a precise definition of a cytokine storm is needed urgently. mehta et al. (136) suggest that the criteria of shlh could be applied. moreover, the term needs to be placed in the icd code. the icd code would bring the standardization of disease names, the convenience of electronic medical records (emr) management, and the efficiency in information sharing. for example, the characteristic of cytokine storm would be more accessible to be collected for a retrospective study. yt: manuscript preparation and wrote the main part of the manuscript. jl: evidence collection, wrote the parts of the manuscript, and manuscript editing. dz: helped to perform the analysis with constructive discussions. zx: helped to revise the manuscript and gave many professional suggestions. jj: ideas, formulation of overarching research goals, and aims. cw: critically reviewed the manuscript, project funding, and study initiation. all authors approved the final version of the manuscript. we thank the charlesworth group (https://www.cwauthors.com. cn) for its linguistic assistance during 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use of antimalarial drugs against viral infection. microorganisms artesunate as a potent antiviral agent in a patient with late drug-resistant cytomegalovirus infection after hematopoietic stem cell transplantation success and failure of artesunate treatment in five transplant recipients with disease caused by drug-resistant cytomegalovirus case report of a sars-cov-2 infection in a patient with ulcerative colitis on tofacitinib il-10: a multifunctional cytokine in viral infections covid-19: consider cytokine storm syndromes and immunosuppression key: cord-309619-glb2y82u authors: domingo, pere; mur, isabel; pomar, virginia; corominas, héctor; casademont, jordi; de benito, natividad title: the four horsemen of a viral apocalypse: the pathogenesis of sars-cov-2 infection (covid-19) date: 2020-07-29 journal: ebiomedicine doi: 10.1016/j.ebiom.2020.102887 sha: doc_id: 309619 cord_uid: glb2y82u the pathogenesis of coronavirus disease 2019 (covid-19) may be envisaged as the dynamic interaction between four vicious feedback loops chained or happening at once. these are the viral loop, the hyperinflammatory loop, the non-canonical renin-angiotensin system (ras) axis loop, and the hypercoagulation loop. severe acute respiratory syndrome (sars)-coronavirus (cov)-2 lights the wick by infecting alveolar epithelial cells (aecs) and downregulating the angiotensin converting enzyme-2 (ace2)/angiotensin (ang-1–7)/mas1r axis. the viral feedback loop includes evading the host's innate response, uncontrolled viral replication, and turning on a hyperactive adaptative immune response. the inflammatory loop is composed of the exuberant inflammatory response feeding back until exploding in an actual cytokine storm. downregulation of the ace2/ang-(1–7)/mas1r axis leaves the lung without a critical defense mechanism and turns the scale to the inflammatory side of the ras. the coagulation loop is a hypercoagulable state caused by the interplay between inflammation and coagulation in an endless feedback loop. the result is a hyperinflammatory and hypercoagulable state producing acute immune-mediated lung injury and eventually, adult respiratory distress syndrome. in december 2019, a new epidemic disease appeared in the huanan seafood wholesale market, wuhan, hubei province, china. it was characterized by an upper respiratory tract infection rapidly evolving to bilateral pneumonia and eventually respiratory failure [1] . the etiologic agent was a new coronavirus which was named sars-cov-2, whereas the disease was called covid-19 [2] . the disease quickly expanded from its original nucleus in hubei and by march 11 , 2020 the who declared it as a pandemic. as of june 23, 2020, covid-19 has affected 188 countries around the world, with 9.131.445 confirmed cases worldwide and a death toll of 472.856 [3] . early in the course of the pandemic, clinicians and researchers realized that full-blown covid-19 evolved in at least three phases: the first phase with cough, fever, wheezing, fatigue, headache, diarrhea, and dyspnea, reminiscent of an upper tract respiratory infection. the second phase, with the rapid appearance of bilateral pneumonia, infiltrates with variable degrees of hypoxemia, and omit in the third phase in which some patients developed respiratory failure leading to death [4] . around 80% of people have sars-cov-2 infection asymptomatic or with mild to moderate illness, mostly restricted to the upper and conducting airways. the other 20% will develop symptomatic infection needing hospital admission, and 5% will require ventilatory support in the intensive care unit (icu) [5] . the clinical phases of the infection reflect the pathogenic events starting with the virus gaining access to the lungs. the clinical manifestations and pathogenic events of any infectious disease, and covid-19 in particular, should be viewed in the light of the damageresponse framework in which several factors and forces may tip the scales to the host or pathogen side [6] . therefore, sometimes the 2. the first horseman: a sneaky virus sars-cov-2 is a previously unknown b-coronavirus which shows 88% identity to the sequences of two bat-derived sars-like coronaviruses, 79 .5% identity to sars-cov, and about 50% identity to middle east respiratory syndrome (mers)-cov [2] . the genome of sars-cov-2 is a positive-sense, single-stranded rna with a size of 29.9 kb, containing at least ten open reading frames (orfs) [7] . recently, noncanonical orfs and at least 41 rna modifications with an unknown function, were identified [7] . the first orfs represent two-thirds of the viral rna. they are translated into two large polyproteins, which are later processed into 16 non-structural proteins (nsp1 to nsp16) that form the viral complex replicase-transcriptase [7] . these nsps rearrange endoplasmic reticulum into double-membrane vesicles, where viral replication and transcription take place [8, 9] . the other third of the genome encodes four main structural proteins; spike (s), envelope (e), nucleocapsid (n), and membrane (m) proteins, and several accessory proteins whose functions are currently unknown but unrelated to viral replication [10] . sars-cov-2, like sars-cov, requires the ace2 as a receptor to enter the cells [11, 12] . coronavirus s protein is a determinant of virus entry into host cells by binding the envelope spike glycoprotein to its cellular receptor ace2 [13, 14] . although it was initially thought that sars-cov achieved entry by membrane fusion, a critical proteolytic cleavage at sars-cov s protein, mediated by type ii transmembrane serine protease (tmprss2), brings about membrane fusion and viral infectivity [15] . after the virus entry, the rna genome is released into the cytoplasm and translated into two polyproteins and structural proteins [16] . the survival of sars-cov in host cells is eased by strategies to evade the immune response. the evolutionarily conserved microbial structures called pathogen-associated molecular patterns (pamps) are recognized by pattern recognition receptors (prrs) such as tolllike receptor (tlrs), retinoic acid-inducible gene-i (rig-i)-like receptors, nucleotide-binding oligomerization domain (nod)-like receptors, and c-type lectin-like receptors [17] . sars-cov induces the signal transducer and activator of transcription 1 tace tnf-a converting enzyme tbk1 tank-binding kinase 1 tlr toll-like receptor tmprss2 type ii transmembrane serine protease tnf-a tumor necrosis alpha traf3 tnf receptor-associated factor 3 xcr1 xcl1 (chemokine [c motif] ligand 1) and xcl3 (chemokine [c motif] ligand 3) receptor production of double-membrane vesicles that lack prrs and can then replicate in these vesicles [18] . furthermore, several structural and nsps encoded by sars-cov and mers-cov antagonize antiviral innate immune response. interferon (ifn) and interferon-stimulated genes (isgs) responses are counteracted by nsp1, nsp3 macrodomain, nsps-deubiquitinase, and orf3b, orf6, and orf9, thus overthrowing antiviral response [19] [20] [21] [22] [23] [24] . nsp1 inhibits ifn responses by three mechanisms, inactivation of host translational machinery, degradation of host mrnas, and inhibiting signal transducer and activator of transcription 1 (stat1) phosphorylation [25, 26] . part of the nsp3 is a papain-like protease that antagonizes ifn and cytokine production by blocking phosphorylation of ifn regulation factor 3 (irf3) and disrupting nf-kb signaling [24] . nsp7 and nsp15 are also ifn antagonists by an unknown mechanism [24] . orf3b exerts ifn antagonism through inhibition of ifnb induction by transcription factors irf3 and nf-kb, whereas orf6 antagonizes ifn by inhibiting signaling through the jak-stat pathway [24] . m and n proteins flatten ifn signaling by inhibiting tank-binding kinase 1 (tbk1)/ikb kinase e (ikke), and the negative regulation of traf3/6-tbk1-irf3/nf-kb/ ap1 signals [25, 26] . antagonism of ifn responses further promotes free virus replication resulting in increased viral pamps and damps that additionally dampen ifn signaling and stimulate prrs to induce an aberrant inflammatory response. the replicative capacity of sars-cov-2 is 3.20 folds more than that of sars cov in infected human lung tissue without significantly inducing types i, ii, and iii ifns [27] . since innate immunity is the frontline defense against sars-cov-2, a slow and poorly coordinated response may result in higher viral replication. this sequence of events, namely aecs infection, ifn signaling inhibition, and free viral replication depicts the viral vicious loop (fig. 1 ). 3. the second horseman: a gathering storm sars-cov-2 infects primarily airway and alveolar aecs, especially type ii pneumocytes, the cells that produce alveolar surfactant and are predecessors of type i pneumocytes. however, it can infect any cell expressing the receptor ace2, such as endothelial cells, pericytes, vascular smooth muscle cells, macrophages, fibroblasts, t-cells, cardiomyocytes, enterocytes, basal cell epidermal cells, and epithelial tubular distal cells [28] [29] [30] . sars-cov-2, in the face of unchecked replication because of dampened innate immunity, can replicate in high titres early after the infection [28, 31, 32] . high viral replication in aecs induces cytopathic effects, as shown by the necropsy findings of multinucleated cells (syncytia), cytoplasmic viral inclusions, and apoptosis, an ultimate cellular response to stop virus replication [33, 34] . these events are followed by the production of increased levels of proinflammatory cytokines and chemokines by aecs [35, 36] . moreover, sars-cov nucleocapsid activates interleukin-6 (il-6) expression in lung epithelial cells via cellular transportation of nuclear factor kappa b (nf-kb) [37] . massive infiltration of inflammatory cells into the lungs is, in turn, mounted by these cytokines and chemokines [32] (fig. 2) . although tissue-resident macrophages of the lungs localize to the airspace within alveoli, they do not seem to be the predominant subset in this response [38] . accumulation of inflammatory monocyte-macrophages and neutrophils in the lungs following sars-cov-2 infection promotes the additional release of cytokines and chemokines [32] (fig. 2) . besides, the sars-cov spike promotes the upregulation of il-6 and tumor necrosis alpha (tnf-a) in macrophages [39] . cytokines spill over from local inflammation to the systemic circulation. covid-19 patients have high serum levels of inflammatory cytokines, including interleukin (il)-2, il-7, il-10, granulocyte-colony stimulating factor (g-csf), interferon gamma-induced protein (ip)-10, monocyte chemoattractant protein (mcp)-1, macrophage sars-cov-2 infects primarily type ii pneumocytes through binding to the ace2 receptor. the infected and surrounding pneumocytes secret cytokine and chemokines, which attract monocyte-macrophages and neutrophils to the alveolar space, which secrete additional cytokines and chemokines. ultimately the pneumocytes suffer apoptosis/pyroptosis releasing large amounts of proinflammatory factors. endothelial cells are infected, overexpress adhesion molecules, and release chemokines and cytokines. endothelial cells undergo apoptosis, which, together with alveolar cell apoptosis, increases vascular leakage and breaks the alveolar-capillary barrier. the hyperinflammatory milieu and endothelial dysfunction activate coagulation cascades through tissue factor expression, platelet activation, and netosis all of them promoting microcirculatory thrombi formation. the break of endothelial-alveolar barrier further promotes vascular leakage resulting in interstitial and alveolar space flooding. downregulation of the ace2/ang-(1à7)/mas1r axis contributes to increasing vasoconstriction, inflammatory signals, endothelial dysfunction, vascular leakage, and prothrombotic state. sars-cov-2 = severe acute respiratory syndrome coronavirus 2; tnf-a = tumor necrosis factor alpha; il-10 = interleukin 10; mcp-1 = macrophage chemoattractant protein 1; mip-1a = macrophage inhibitory protein 1a; il-6 = interleukin 6; il-1b = interleukin 1 beta; nf-kb = nuclear factor kappa b. inflammatory protein (mip)-1a, and tnf-a. these cytokine/chemokine levels correlate with disease severity [40, 41] . covid-19 patients with severe disease have increased levels of il-6 more often than those with the mild or moderate disease [42] . although viremia is not a prominent feature in covid-19 and is usually short-lived, the degree and duration of sars-cov-2 viremia relates to the severity of disease and the serum levels of il-6 [43] . endothelial cells (ecs) are infected very early in the course of infection. because of speedy viral replication and exuberant proinflammatory cytokine/chemokine response they may suffer apoptosis [32, 33] . this apoptotic phenomenon takes place via fas/fasl or trail-dr-5-dependent mechanisms [44] . besides, inflammatory monocyte-macrophages release tnf-a which also promotes apoptosis of both lung ecs and aecs [35] . ecs and aecs apoptosis compromise lung microvascular bed and alveolar cell-capillary barrier integrity, thereby resulting in vascular leakage and alveolar edema [35] (fig. 2) . pericytes play an essential role in maintaining endothelial cell function in capillary vessels and are among the cells with the highest ace2 expression. their infection by sars-cov-2 may add to endothelial cell dysfunction leading to microcirculation disorders [29] . a striking feature of full-blown covid-19 is severe lymphopenia. cov-specific t-cells are decisive for viral clearance and limitation of additional damage to host tissues since they can dampen hyperreactive innate immune response [45, 46] . however, when exuberant inflammatory response induced by sars-cov-2 takes place, t-cell response is decreased because of tnf-a-mediated cell apoptosis, thus resulting in uncontrolled inflammatory responses [35] (fig. 1) . besides, normal t-cell activation can be suppressed by il-6, further contributing to lymphopenia [47] . in severe covid-19 patients with lymphocyte subsets examined, there is intense cd4 and particularly cd8 lymphopenia [42] , both negatively correlating with tnf-a and il-6 serum levels [41] . cd4 cells promote the production of virus-specific antibodies by activating t-dependent b cells, whereas cd8 cells are cytotoxic and can kill virus-infected cells. since cd8 cells account for about 80% of the total inflammatory lung interstitial infiltrate, highly cytotoxic cd8 lymphocytes can arbitrate immune-mediated tissue damage [34] . also, in covid-19 patients, these cells exhibit markers of functionally exhausted t-cells such as programmed cell death protein 141. there is upregulation of apoptosis, autophagy, and p53 pathways in pbmc of covid-19 patients [48] . mers-cov can induce t-cell apoptosis through activation of the intrinsic and extrinsic apoptosis pathways [49] , and sars-cov e protein can also promote t-cell apoptosis mediated by cbl-xl binding [50] . although sars-cov-2 can non-productively infect t lymphocytes, whether this infection induces t-cell apoptosis is not yet clear [51] . alternatively, pyroptosis has been suggested as a cause of lymphopenia since covid-19 patients have increased serum il1-b levels, which is the downstream indicator of cell pyroptosis [52] . in sars-cov infection, viroporin 3a triggers the activation of nod-like receptor protein 3 (nlrp3) inflammasome and the secretion of il-1-b by macrophages [53] . pyroptosis can release large amounts of proinflammatory factors [54] . whatever the cause, during the late stages of infection, depletion of t-cells may promote viral survival and, consequently, may prolong the infection. essential to control the persistent phase of sars-cov-2 infection is the appearance of humoral immunity, in which antiviral neutralizing antibodies play a significant role. however, in animal models, anti-s protein-neutralizing antibodies (anti-s-igg) may cause severe lung injury by altering inflammatory responses [55] . in sars-cov infection, the development of acute respiratory disease coincides with antiviral igg seroconversion in 80% of patients [56] and patients who died developed anti-s-neutralizing antibodies faster [57] . the presence of anti-s-igg promotes proinflammatory monocyte-macrophage lung accumulation and the production of mcp-1 and il-8. such proinflammatory cytokine release would be mediated through the binding of the virus-anti-s-igg complex to the monocytes-macrophages fcgriia receptor since its blockade reduces the production of ifn-g, tnf-a, il-1, and il-6 [55] . it would also be possible that such complexes activate the classical pathway of the complement system or induce antibody-dependent cell-mediated cytotoxicity, thus leading to cellular damage. therefore, a possible underlying mechanism would be antibody-dependent enhancement (ade) of viral infection that occurs in some patients with early, sub-optimal antibody activity that cannot completely clear the virus, leading to persistent viral replication and inflammation [58] . uncontrolled viral replication, because of a delayed innate immunity response, will cause cellular damage leading to an overexuberant and dysregulated immune kickback. this hyper reaction affecting the innate and adaptative immune responses will pave the way for immune-mediated damage of tissues and organs. this sequence of events conforms the inflammatory hurtful feedback loop (fig. 1 ). the ras plays a critical role in the control of cardiovascular and renal functions by maintaining blood pressure homeostasis and hydro-electrolyte balance [59] . initially, the ras was conceived as a linear hormonal system in which angiotensinogen synthesized in the liver is converted into the active peptide angiotensin i (ang i) through the action of renin [60] . afterward, ang i is cleaved by the ace generating ang ii [61] . two g protein-coupled receptors (gpcr) mediate the actions of ang ii, angiotensin ii receptor type 1 (at1r), and type 2 (at2r) [62] . the primary role of this canonical or classical ras pathway (ace/ang ii/at1r) is to increase the sympathetic nervous system tension, to cause vasoconstriction, increase blood pressure, and promote inflammation, fibrosis and myocardial hypertrophy [63] . the ras also possesses a non-canonical, counter-regulatory branch composed of ace2/ang-(1à7)/mas1r. the activity of the system will depend on the balance between the two branches. ace2 is the main synthesizer of ang-(1à7) by removing a single residue from ang i to generate ang-(1à9) and by cleaving a single residue from ang ii to generate ang-(1à7) [64, 65] . the functional receptor for ang-(1à7) is the gpcr mas1r [66] . the conformation of the negative or counter-regulatory axis is relevant not only because it downgrades the vasoconstrictive/ proliferative peptide ang ii to form the vasodilator heptapeptide ang-(1à7), but also because it degrades ang i to ang-(1à9), thereby limiting the availability of the substrate for ace. ang-(1à7) binds to mas1r, inducing vasodilation, inhibition of cell growth, anti-thrombotic, and anti-arrhythmogenic effects [67] . ace2 activity is controlled by a disintegrin and metalloproteinase domaincontaining protein 17 (adam-17, also called tnf-a-converting enzyme, tace). adam-17 proteolytically cleaves ace2 causing the shedding of ace2 into the interstitium, which leads to decreased ace2 activity in the tissue and elevates circulating ace2 activity [68] (fig. 3) . since blood and urine measurement of ace2 levels is feasible, they could potentially be used as a prognostic biomarker in covid-19 [69] . ras plays an essential role in the pathogenesis of inflammatory diseases in which most of the proinflammatory actions are caused by ang ii [67] . ang ii activates several cellular functions and molecular signaling pathways related to tissue injury, inflammation, and fibrosis. they involve calcium mobilization, free radical generation, activation of protein kinases and nuclear transcription factors, recruitment of inflammatory cells, adhesion of monocyte and neutrophils to endothelial and mesangial cells, upregulation of adhesion molecules and stimulation of expression, synthesis, and release of cytokines and chemokines [68] . at1r mediates most of these actions [70] . the counterregulatory ace2/ang-(1à7)/mas1r axis negatively modulates leukocyte migration, cytokine expression and release, and fibrogenesis pathways. hence, ace2 deficiency increases vascular inflammation by increasing the gene expression of vascular adhesion molecules, cytokines, chemokines, and matrix metalloproteases [71] . the loss of ace2 results in higher increases in ang ii-induced expression of inflammatory factors, enhanced vascular permeability, increased lung edema, and neutrophil accumulation [72] . the ace2/ ang-(1à7)/mas1r axis also plays an essential role in haemostasis, since it stimulates prostacyclin (pgi2) production and nitric oxide (no) release by ecs and modulates platelet activity which is less adherent having, thus anti-thrombotic activity [73] (fig. 1) . ras exhibits high activity in lung tissue, which is the leading site of ang ii synthesis. ace2 is a zinc metallopeptidase, type i integral membrane glycoprotein orientated with the n-terminal, and the catalytic site facing the extracellular space [74] . the union of ace2 with sars viral spike protein triggers enzyme internalization downregulating activity from the cell surface. once sars-cov binds to its receptor, the abundance on the cell surface, mrna expression, and the enzymatic activity of ace2 are significantly reduced [75] . proteolytic shedding of its extracellular domain is a second mechanism for downregulating ace2 at the cell surface. s protein of sars, once it binds to ace2, induces shedding by activating adam17 (tace) as do bacterial endotoxin and lipopolysaccharide (lps) [76] (fig. 3) . releasing ace2 from the cell membrane is a critical step in catalyzing substrates and implies that attenuation of ace2 activity might contribute to disease pathogenesis. the recently described induction of ace2 expression by type i ifn in human nasal epithelial cells, thus behaving as an isg, highlight an additional mechanism of ace2 downregulation by sars-cov-2 [30] . since ifn-induced isgs are crucial for host antiviral response, the absence of ace2 induction due to hampered ifn responses will further cause tissue unprotection. therefore, in covid-19, ace2 plays a pivotal role because of its multifaceted task as a facilitator of entry into aecs and its potential role in the pathogenesis of acute lung injury (ali) [75] . in the mouse model of sars, downregulation of ace2 protein expression resulted in worse pneumonia, increased ang ii levels, increased vascular permeability, enhanced lung edema, neutrophil infiltration, and further worsened lung function [72, 77] . catalytically active ace2 protein alleviated the symptoms, and active protein improved the outcome of respiratory failure [72] . in covid-19 patients, plasma concentrations of ang ii were significantly higher than in healthy individuals and ang ii levels correlated with viral load and lung injury [78] . owing to the widespread expression of ace2, covid-19 is a disseminated infection. ace2 is highly expressed in the gut and sars-cov-2 can productively infect enterocytes [28, 79] . despite ace2 the lung, and other organs, lose the protection of the non-canonical ras system as a result ace2 downregulation after sars-cov-2-induced endocytosis. consequently, the canonical ace/ang ii/at1r becomes dominant, levels of ang ii increase with the subsequent promotion of fibrosis, myocardial hypertrophy, increased ros, vasoconstriction, inflammation, and endothelial dysfunction. at1r mediates most of these actions. endocytosed sars-cov-2 spike proteins mediates adam 17-mediated proteolytic cleavage of ace2. adam-17 activity is enhanced through the activated at1r by increased levels of ang ii. tnf-a is the primary substrate of adam17. adam17 cleaves tnf-a releasing soluble tnf-a extracellularly where it has autocrine and paracrine functionalities. activation of tnf-a receptor by tnf-a also enhances adam17 activity. ace2 = angiotensin-converting enzyme 2; sars-cov-2 = severe acute respiratory syndrome coronavirus 2; ang ii = angiotensin ii; ros = reactive oxygen species; at1r = angiotensin 1 receptor; adam17 = a disintegrin and metalloproteinase domain 17; tnf-a = tumor necrosis factor alpha; tmprss2 = transmembrane protease serine 2. expression in gut being higher than in the lung, only about 10à12% of patients with covid-19 experience gastrointestinal symptoms [79] . the contribution of the digestive system to the pathogenesis of covid-19 through impairment of mucous membrane barrier and increased inflammatory cytokine production has not been determined yet. similar to mers-cov and owing to ace2 expression in the brush borders of the proximal tubules and in podocytes, kidney injury in covid-19 is characterized by diffuse proximal tubule damage with virus-like particles in tubular epithelial cells and podocytes which is indicative of direct sars-cov-2 infection [80] . these findings translate clinically into acute kidney injury and proteinuria which affect from 0.9% to 29% of covid-19 patients [80] . the consequence of impaired ace2 activity in the lung because of sars-cov-2 infection is a reduction of ang-(1à7) production. ang-(1à7) binding mas1r promotes an array of biological responses to counteract ang ii-mediated processes such as apoptosis, angiogenesis, vasoconstriction, and inflammation in the lung [81, 82] . consequently, the attenuation of ace2 catalytic function perturbs the pulmonary ras balance, resulting in enhanced inflammation and vascular permeability, leaving the lung defenceless in the face of the forthcoming raging cytokine storm. besides, infection of type ii pneumocytes will reduce the production of alveolar surfactant subsequently reducing pulmonary elasticity. moreover, the loss of type ii pneumocytes decreases restoration of type i pneumocytes which ultimately impacts on gas exchange and fibrosis [83] . the above event sequence depicts the ras vicious feedback loop (fig. 1 ). the association between covid-19 and coagulation disorders was beheld early during the pandemic when chinese physicians noticed that patients treated mainly with low-molecular-weight heparin had a decreased 28-day mortality [84] . this mortality improvement was in patients with a sepsis score higher than four or a markedly elevated d-dimer [84] . covid-19 is associated with coagulation disorders that include increases in procoagulant factors such as fibrinogen and d-dimers, both associated with poor prognosis [85, 86] . patients admitted to the icu had an increased incidence of venous thromboembolic events ranging from 25% to 36% [87] [88] [89] . moreover, standard prophylaxis for venous thromboembolism failed in 7.7% of the patients [90] . some found that most thrombotic complications were venous and primarily isolated pulmonary embolism, which suggests that it may be primary pulmonary thrombosis instead of embolic phenomena [89, 91] . in line with that, microcirculatory thrombosis is a constant finding in lung pathologic studies [33, 34] (fig. 2) . infection of ecs, together with the derangements caused by cellular infiltration and high exposure to cytokines/chemokines, eventually leads to ecs dysfunction and apoptosis [33] . all of them contribute to microvascular prothrombotic effects [92] . there is an intense interplay between haemostasis and innate immunity, called thrombo-inflammation [93] . both the intrinsic and extrinsic coagulation pathways can activate during inflammation. ecs and macrophages activate the extrinsic pathway through expression of tissue factor [94] . the intrinsic pathway can be activated by neutrophil extracellular traps (nets) released by polymorphonuclear neutrophils (pmn) in a process called netosis. nets activate ecs, platelets, and the complement system and release proteases that inactivate endogenous anticoagulants [95] . however, the role of nets in covid-19 is still a matter of discussion [96] . platelets play a dual role. first, a proinflammatory role by secreting alpha granules that recruit pmn and macrophages, which are an essential source of il-1b [97] . besides, platelets stimulate pmn to undergo netosis which in turn activates platelets, creating a feedback loop. the second role of platelets is to activate the coagulation pathway by assembling enzyme-cofactor-substrate complexes on their exposed surface [98] (fig. 1) . complement activation, which has been seen in the mouse model of sars, contributes to immune-mediated pathology [99] . activation of c3 and c5 promotes mast cell degranulation and recruitment of pmn and macrophages [100] . the prothrombotic effects of activated c3 and c5 include platelet and ecs activation, together with increasing tissue factor and von willebrand factor expression [95] . to close the loop, thrombin, and other components of the coagulation cascade can, in turn, activate c3 and c5 [95] . the primary function of thrombin is to promote clot formation by activating platelets and by converting fibrinogen into fibrin [101] . however, thrombin is a pleiotropic molecule and can increase inflammation via a proteinase-activated receptor (par), principally par-1 [101] (fig. 1) . the generation of thrombin is controlled by negative feedback loops and physiological anticoagulants such as antithrombin iii, tissue factor pathway inhibitor and the protein c system [101] . il-1b, il-6, and tnf-a promote the release of ultra-large von willebrand multimers, and the production of tissue factor and factor vii/activated factor vii, leading to increased thrombin generation while decreasing the levels of endogenous anticoagulants [101] . the ace2/ang-(1à7)/mas1r axis exerts antithrombotic effects through activation of mas1r in platelets, which then release no and pgi2 and by protecting from endothelial dysfunction [102, 103] . since this branch of the ras is not working properly in covid-19, this protective mechanism is lost ( figs. 1 and 3) . in severe covid-19, similar to other acute viral infections, a high prevalence od antiphospholipid antibodies was found, although the role of these antibodies in the prothrombotic state of sar-cov-2infected patients is still a matter of debate [104] . the progression of thrombo-inflammation may result in widespread thrombosis, which may be further enhanced by hypoxemia, hyperthermia, and hypovolemia [105] . hypoxemia triggers increased expression of hypoxia-inducible factors, which may promote additional inflammation and may activate platelets and coagulation factors. they increase tissue factor expression, increase plasminogen activating inhibitor-1, and inhibit the endogenous anticoagulant protein s [106] . in the setting of a hyperinflammatory state and endothelial injury, activation of coagulation occurs whereas the counterregulatory force ace2/ang-(1à7)/mas1r axis is inactive, leaving the field to the full expression of a hypercoagulable state. this state may clinically translate into pulmonary thrombosis, venous thromboembolism, or other thrombotic events. if these events affect microvascular lung bed, they may further promote ali and impair gas exchange. whatever the location of the thrombotic event is, it worsens the patient's prognosis. hyperinflammatory state and defective ace2/ang-(1à7)/mas1r functioning activate the fourth hurtful feedback loop. hyperinflammation induces hypercoagulation and vice versa, while ace2/ang-(1à7)/mas1r axis avoidance maximizes both (fig. 1) . the clinical spectrum of covid-19 is broad. not everyone who acquires sars-cov-2 becomes sick and the state that emerges after infection can vary among patients or within the same patient over time. consequently, it is envisaged that virus-dependent, hostdependent, and environment-dependent factors may modify the virus-host interaction explaining not only the individual susceptibility to infection but also the broad scale of damage seen in clinical disease. the initial viral titre in the airways could explain the different evolving patterns of covid-19, since this will condition the intensity of cytopathic changes, which in turn will shape the strength of immune responses [35] . sars-cov-2 replicates in high numbers very early after infection, and in turn, the magnitude of viral replication will impact on the extent of antiviral response [27] . in humans, there is a strong correlation between sars-cov and mers-cov titres and disease severity [35] . in animal models, the disease behaves differently if the virus infects airway epithelial cells or both airway and aecs (type i and type ii pneumocytes) predominantly. the viral antigen is mainly located in airway epithelial cells in mouse models permissible to infection, but which do not develop clinical disease. in contrast, in highly susceptible mice, the antigen is detected in both airways and alveolar type i and ii pneumocytes [35] . consequently, infection of aecs seems critical for both host susceptibility and the development of lung pathology. an aspect that influences sars-cov-2 infection is the state of differentiation of human airway epithelia, which, in turn, correlates positively with the expression level of ace2 in these cells [107, 108] . it is noteworthy that ace2 nasal gene expression is lower in children [109] . this fact is connected to the striking age distribution of covid-19 in which children are often spared, affecting adults with enhanced severity and mortality as age increases [5, 47] . however, the increasingly poor outcome with advancing age is influenced by the presence of common comorbidities, such as hypertension, cardiovascular disease, and diabetes, which bear a poor prognosis by themselves [47] . besides, these comorbidities relate to a decreased activity of ace2 in elderly patients, a deficit further exacerbated by sars-cov-2 infection [110] . ade is a potentially harmful, pro-inflammatory mechanism which occurs when suboptimal titres of neutralizing antibodies against sars-cov-2 are present. they are unable to control infection but instead facilitate viral entry into macrophages by a trojan horse mechanism. ade tends to happen when the time interval between coronavirus infections is long enough for antibody fall, which could be a possible mechanism for severe covid-19 in the aged [58] . females with covid-19 usually present with milder disease than males. females exhibit higher ifn and ifn regulator factor and il-10 production from pbmc, lower production of tnf-a, lower expression of tlr4 in pmn, lower numbers of nk cells, and lower pmn phagocytic activity than males [111] . oestrogens downregulate ang ii and upregulate ang-(1à7) pathways, which makes apparent gender differences in expression, activity, and tissue responsiveness of ras components [112] . besides, mas1r expression was increased in female rats but not in males after the infusion of ang ii [113] . in animal models of obesity, females appear to maintain circulating ang-(1à7) levels and are protected from hypertension and metabolic complications induced by angiotensinogen, renin, angiotensin ii, and at1r activation [114] . stimulation of counterregulatory at2r appears metabolically protective in female rodents, whilst there are inconsistent effects in males [115] . in the uk, 72% of covid-19 patients in the icu were either overweight or obese [116] , and 99% of dead north italians had obesity, hypertension, diabetes, heart disease, kidney damage, or cancer [117] . the frequent occurrence of obesity as a factor of adverse outcome is frequently shadowed by other high prevalent comorbidities in obese people making the identification of the independent role of obesity steep [118] . the association of covid-19 with obesity has been attributed to the chronic inflammatory status, the ineffective immune response or to interaction with the ras system. the immune pathways are all susceptible to genetic polymorphisms with functional consequences such as variability in cytokine expression, antigen-binding affinities, receptor ligation strength, and downstream signaling [119, 120] . high interconnection is a prominent feature of immune pathways and thus functional resultant polymorphisms may hamper the growth of an optimal immune response to covid-19. responses triggered by pamps recognition and its downstream molecules such as myeloid differentiation primary response 88 (mdy88) may be altered by tlr polymorphisms [121, 122] . hla genes present extreme allelic polymorphism. since they present viral peptides to host hla molecules to trigger an adaptative immune response, their polymorphisms may cause unevenness in antigen binding and presentation, and consequently in immune response. hla-b*46:01 has been associated to the development and increased severity of sars-cov [123] , and it has the fewest predicted binding affinity of sars-cov-2 peptides [124] . il-6 plays a central role in the hyperactive immune response in covid-19 patients. since there are functional polymorphisms in the il-6 gene that modify its protein level expression, they may affect the severity of the disease [125] . the role of ras in the pathogenesis of covid-19 is essential. single nucleotide polymorphisms and haplotypes in ace genes, such as polymorphism a/d in the ace1 gene, have been associated with circulating and tissue concentrations of ace levels and reduced expression of ace2 [126, 127] . interestingly, the prevalence of covid-19 in europe correlates inversely with ace d allele frequency [127] . a genetic variant of the ifn-induced transmembrane protein-3 gene is associated with covid-19 severity. ifn-induced transmembrane protein-3 is an immune effector protein that acts restricting membrane fusion [128] . recently, a genomewide association study in covid-19 patients with respiratory failure identified an association signal at locus 3p21.31, which includes the genes slc6a20, lztfl1, ccr9, fyco1, cxcr6 and xcr1, while there was no association signals at the hla complex [129] . lately, there has been a contention about the beneficial or detrimental role of ace inhibitors (acei) and angiotensin receptor blockers (arb) in the outcome of patients with covid-19. currently, there is no evidence to support an advantageous or harmful effect of concomitant therapy with acei or arb in covid-19 patients [130] . covid-19 is a systemic infection since it may impact any tissue or organ expressing ace2. however, the most dreadful, often lifethreatening conditions, are ali and ards. therefore, the main challenge is to avoid their development to prevent icu admission and mechanical ventilatory support. we could envisage covid-19 as a tree in which aecs viral infection and ace2 downregulation represent the roots. the tree trunk would be the hyperinflammatory and hypercoagulable state. the branches would be an end-organ disease, such as ali, myocarditis, neurological disease, liver injury, gastrointestinal involvement, and skin disease. since the chain of events triggered by sars-cov-2 infection evolves quickly, any planned intervention must come as early as possible. besides, since the pathogenesis of covid-19 involves non-viral mechanisms, any intervention planned must also address the correction or modulation of these disbalances. hence, any therapeutic intervention must be early and combine antiviral and adjuvant therapies. however, the moment of diagnosis and eventual hospital admission will mark the timeframe of interventions. to tackle the roots of the disease, potential therapeutic interventions for covid-19 should first address the viral entry into aecs. the entry of sars-cov-2 into aecs takes place after binding of the spike to the receptor ace2. specifically, the binding takes place in the receptor-binding domain of the s protein. thus, developing neutralizing monoclonal antibodies for this domain is a rational strategy to prevent the viral union and subsequent events [131] . another possible way of targeting the interaction between ace2 and s protein may be the use of soluble recombinant ace2, which may prevent the binding of the viral particle to the surface-bound, full-length ace2 [132] . in the vero-e6 monkey cell line, a soluble form of ace2 blocks sars-cov replication and reduced sars-cov-2 recovery by a factor of 1000à5000 [132, 133] . besides, since sars-cov-2 downregulates the ace2/ang-(1à7)/mas1r axis, recombinant human ace2 (rhace2) could prevent the development of ali in covid-19. rhace2 attenuated arterial hypoxemia in a piglet model of lps-induced ali [134] . in phase ii, open-label trial in humans with ards rhace2 was well tolerated, ang ii levels decreased, whereas ang-(1à7) and surfactant protein d increased [135] . however, the study was not powered to detect changes in acute physiological or clinical outcomes [135] . there is a randomized controlled trial to assess rhace2 in patients with severe covid-19 (nct 04287686). apart from ace2, sars-cov-2 entry involves tmprss2, whose inhibitor, camostat mesylate, significantly reduced lung cell line infection with sars-cov-2 [136] . endocytosis is a crucial step in sars-cov-2 infection. ap-2-associated protein kinase (aak1) regulates this process [137] . baricitinib, a janus-kinase inhibitor, has been claimed as a candidate drug for covid-19 since it inhibits aak1 [138] . arbidol inhibits viral entry by inhibiting the fusion between viral and cellular membranes [139] . however, in a small retrospective study, arbidol did not meet noninferiority versus the combination of arbidol and lopinavir/ritonavir (lpv/r) [140] . chloroquine and its safer derivative hydroxychloroquine are effective against sars-cov-2 in vitro [141] . however, recent news from the large recovery trial showed that there is no beneficial effect of hydroxychloroquine in patients hospitalised with covid-19; therefore, that arm of the study was stopped [142] . other planned large trials, such as solidarity, stopped enrolling patients to the hydroxychloroquine arm, and the national institutes of healthsponsored orchid study was also stopped [143, 144] . numerous antivirals agents are being tested in clinical trials. lpv/r could not demonstrate enough efficacy when compared with placebo [145] . the combination of ifn, lpv/r, and ribavirin showed a shorter time to negativize nasopharyngeal swabs and superiority versus lpv/ r in alleviating symptoms [146] . in two double-blind, placebo-controlled trials, remdesivir was not associated with statistically significant clinical benefits in one, whereas in the other shortened the time to recovery in hospitalized adults [147, 148] . as of now, there is no antiviral drug with proven efficacy for treating patients with covid-19. another strategy tries to modulate the exuberant inflammatory response in covid-19. the use of corticosteroids is controversial and not supported by previous experience in sars and mers [149] . however, in the recovery trial, dexamethasone reduced deaths by one third in patients receiving invasive mechanical ventilation and by one fifth in patients receiving oxygen without invasive mechanical ventilation [150] . tocilizumab, a specific il-6 receptor antagonist, is promoted to treat the hyperinflammatory state of covid-19 because of the pathogenic role il-6 plays. two observational studies have shown a clinical benefit of therapy with tocilizumab in covid-19 pneumonia with hyperinflammatory syndrome [151, 152] . anakinra, a recombinant il-1 receptor antagonist, has proven useful in a small retrospective study of covid-19 patients with ards and hyper inflammation [153] . there are additional trials in progress with tocilizumab, anakinra, and sarilumab. however, when trying to modify the cytokine response by targeting a single molecule or receptor, it should be recalled that the cytokine network is an intricate complex with a high degree of overlap, redundancy, and alternate pathways. this may explain therapy escape and eventually lack of response. therapeutic interventions for the consequences of hyperinflammatory and hypercoagulable states associated with covid-19, such as ali, ards or thromboembolic events, are beyond this review's scope. knowledge of pathophysiology is the first step to address the management of a disease appropriately. it is familiar with the mechanism that the virus uses to evade host immune defense mechanisms or those that uses to harm will permit the design of appropriate strategies to neutralize the dysfunctions or disbalances generated either by the virus or by the consequences of the infection. from the knowledge gathered, it seems that most organ damage in severe covid-19 is done through an immune-mediated mechanism, although sars-cov-2 is the necessary initiator. the spectrum of disease is comprehensive, and since not all the patients will share the same evolving pattern, the search for predictive factors to promptly identify patients more prone to evolve to life-threatening disease is of the utmost importance. in severe cases, the quick evolving pattern of the disease makes early treatment imperative, at least until reliable predictive factors become widely available. the implication of viral and host-dependent mechanisms in covid-19 pathogenesis suggests that any therapeutic strategy must combine antiviral drugs and adjuvant therapy to modulate the host's responses. all these goals will be achieved through the broad effort of basic, translational, and clinical scientists and clinicians, and will demand a high degree of commitment from patients and their families, allied professionals, and everyone engaged in the fight against covid-19. among them, politicians and health administration officers will play a unique role, since such a gigantic task will need the allocation of a vast amount of resources to overcome a health challenge to mankind like none other in recent times. while engrossed amid the pandemic, there was progress on the physiopathology of covid-19. however, gaps regarding viral, environmental, and transmissibility aspects remain-the dynamic interplay between the host and the virus and how to modify it to improve disease prognosis not being the lesser. there is a big difference in transmissibility, which is highest for sars-cov-2, among b-coronaviruses despite similar structure and functioning. asymptomatic viral shedding is the main factor. however, the role of newly described orfs and rna modifications and their functional correlations are not evident yet. although tmprss2 is involved in viral entry into the host cell, the involvement of other host proteins is still under discussion. the role of the different epithelial cells along the bronchial tree and the alveolar space needs to be ascertained. the virus's mechanisms to invade other organs beyond the lung are already poorly known. clinical disease progression is somewhat unpredictable. therefore, the identification of prognostic clinical and biological markers would optimize patients' care and resource consumption, which may be of utmost importance in pandemic times. this effort must include which role comorbidities and gender play. the definition and timing of the optimal therapeutic approach to covid-19 will represent a colossal effort, which can be accomplished only by randomized clinical trial performance. these should include concerted actions and a combination of diverse disciplines, resources, expertise, and techniques to contribute to advances in prevention, diagnosis, and therapy. this set makes up an almost flawless meaning of translational medicine defined by the european society for translational medicine (eustm) as "an interdisciplinary branch of the biomedical field supported by three main pillars: benchside, bedside, and community." for this review, our search strategy involved the review of original records, either journals or books, mainly from european and american sources, from1984 to 2020. from these sources, we hand searched reference lists of identified additional articles to retrieve additional studies. preference was given to most relevant research, but we were also keen to highlight the breadth of the topic and hence selected some publications that showcase particular areas of interest. we have searched pubmed and google scholar from database inception to may 21, 2020, for records, journals, and books for the terms "sars-cov-2 00 , "covid-19 00 , "coronavirus", "raas system", "angiotensin-converting enzyme", "angiotensin-converting enzyme 2 00 , "cytokine storm", "cytokine", "chemokine", "acute lung injury", "adult respiratory distress syndrome", "interferon", "interleukin ", "middle east respiratory syndrome", and "severe acute respiratory syndrome". references were examined in english. we declare no competing interests. the concept of the manuscript was devised by pd who also performed the overall literature searches. im, vp, hc, and jc designed the search strategy with inputs from pd and ndb. im, vp, hc, and jc carried out the literature searches and screening, and any discrepancies were discussed with pd and ndb. pd wrote the first draft of the review with inputs from all the authors. this work was partially supported by grant cov20/00070. instituto de salud carlos iii, madrid, spain. the funding source was not involved in the design of the study or in writing the report. all authors had access to the data used in the analyses, and the lead author reviewed the full report. the full study data were available to all authors. pd, ndb made the decision to submit the paper for publication. who. novel coronavirus à china genomic characterization and epidemiology of 2019 novel coronavirus: implications for virus 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adapter recruitment to tlr2 association of hla class i with severe acute respiratory syndrome coronavirus infection human leukocyte antigen susceptibility map for sars-cov-2 circulating interleukin-6 and rheumatoid arthritis: a mendelian randomization meta-analysis relationship between genetic variants of ace2 gene and circulating levels of ace2 and its metabolites the host's angiotensin-converting enzyme polymorphism may explain epidemiological findings in covid-19 infections interferon-induced transmembrane protein-3 genetic variant rs12252-c is associated with disease severity in covid-19 genomewide association study of severe covid-19 with respiratory failure use of reninàangiotensinàaldosterone system inhibitors and risk of covid-19 requiring admission to hospital: a case-population study novel antibody epitopes dominate the antigenicity of spike glycoprotein in sars-cov-2 compared to sars-cov soluble angiotensin-converting enzyme 2: a potential approach for coronavirus infection therapy? inhibition of sars-cov-2 infections in engineered human tissues using clinical-grade soluble human ace2 recombinant angiotensin-converting enzyme 2 improves pulmonary blood flow and oxygenation in lipopolysaccharide induced lung injury in piglets a pilot clinical trial of recombinant human angiotensin-converting enzyme 2 in acute respiratory distress syndrome sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ap-2-associated protein kinase 1 and cyclin g-associated kinase regulate hepatitis c virus entry and are potential drug targets baricitinib as potential treatment for 2019-ncov acute respiratory disease arbidol as a broad-spectrum antiviral: an update arbidol combined with lpv/r versus lpv/r alone against corona virus disease 2019: a retrospective cohort study remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus (2019-ncov) in vitro statement from the chief investigators of the randomised evaluation of covid-19 therapy (recovery) trial on hydroxychloroquine solidarity" clinical trial for covid-19 treatments study shows treatment does no harm, but provides no benefit a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 triple combination of interferon beta-1b, lopina-viràritonavir, and ribavirin in the treatment of patients admitted to hospital with covid-19: an open-label, randomised, phase 2 trial remdesivir in adults with severe covid-19: a randomised, double-blind, placebo-controlled, multicentre trial remdesivir for the treatment of covid-19-preliminary report clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury low-cost dexamethasone reduces death by up to one third in hospitalised patients with severe respiratory complications of covid-19 tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in pilot prospective open, single-arm multicentre study on off-label use of tocilizumab in patients with severe covid-19 interlekin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study we are indebted to jordi mancebo and m ant onia mangues for critical reading of the manuscript, and to richard pike for reviewing its writing. key: cord-306983-6w2fvtfy authors: wang, siye; le, trong quang; kurihara, naoki; chida, junji; cisse, youssouf; yano, mihiro; kido, hiroshi title: influenza virus—cytokine-protease cycle in the pathogenesis of vascular hyperpermeability in severe influenza date: 2010-10-01 journal: j infect dis doi: 10.1086/656044 sha: doc_id: 306983 cord_uid: 6w2fvtfy background. severe influenza is characterized by cytokine storm and multiorgan failure with edema. the aim of this study was to define the impact of the cytokine storm on the pathogenesis of vascular hyperpermeability in severe influenza. methods. weanling mice were infected with influenza a wsn/33(h1n1) virus. the levels of proinflammatory cytokines, tumor necrosis factor (tnf) α, interleukin (il) 6, il-1β, and trypsin were analyzed in the lung, brain, heart, and cultured human umbilical vein endothelial cells. the effects of transcriptional inhibitors on cytokine and trypsin expressions and viral replication were determined. results. influenza a virus infection resulted in significant increases in tnf-α, il-6, il-1β, viral hemagglutininprocessing protease trypsin levels, and viral replication with vascular hyperpermeability in lung and brain in the first 6 days of infection. trypsin upregulation was suppressed by transcriptional inhibition of cytokines in vivo and by anti-cytokine antibodies in endothelial cells. calcium mobilization and loss of tight junction constituent, zonula occludens-1, associated with cytokineand trypsin-induced endothelial hyperpermeability were inhibited by a protease-activated receptor-2 antagonist and a trypsin inhibitor. conclusions. the influenza virus-cytokine-protease cycle is one of the key mechanisms of vascular hyperpermeability in severe influenza. cular hyperpermeability and multiorgan failure in severe influenza remains unclear. significant increases in levels of proinflammatory cytokines such as tumor necrosis factor (tnf) a, interleukin (il) 6, and il-1b (ie, cytokine storm) affect host survival both positively and negatively [5] [6] [7] . the inflammatory response affects cell adhesion, permeability, apoptosis, and mitochondrial reactive oxygen species, potentially resulting in vascular dysfunction and multiorgan failure [8] . in addition, influenza a virus infection upregulates several cellular proteases, including ectopic trypsin [9] and matrix metalloprotease (mmp) 9 [10] . ectopic trypsin, like tryptase clara [11] , mediates the post-translational proteolytic cleavage of viral envelope hemagglutinin [12] , which is crucial for viral entry and replication [13] [14] [15] [16] [17] and subsequent tissue damage in various organs [9, 16, 17] . influenza a virus infection significantly upregulates trypsin in endothelial cells and in hippocampal neurons [9] . because trypsin efficiently converts pro-mmp-9 to active mmp-9 [18] , induction of both proteases synergistically degrades basement membrane proteins, potentially destroying tight junctions and the blood-brain barrier, followed by multiorgan failure [19, 20] . the aim of the present study was to define the pathogenic impact of cytokine storm in influenza a virus infection and the molecular mechanisms by which proinflammatory cytokines cause vascular dysfunction in animal models and in human vein endothelial cells. the results pointed to the role of the influenza virus-cytokine-protease cycle as one of the main mechanisms of vascular dysfunction in severe influenza. specified pathogen-free 3-week-old weanling c57bl/6crslc female mice were obtained from japan slc. under ketamine anesthesia, 250 or 500 plaque-forming units (pfu) of influenza a/wsn/33(h1n1) [21, 22] in 15 ml of saline or saline alone as the vehicle was instilled intranasally in mice. mice ( ) also received inhibitors against nuclear n p 10 factor-kappa b (nf-kb), such as pyrrolidine dithiocarbamate (10 mg/kg) and n-acetyl-l-cysteine (10 mg/kg) [23, 24] , and inhibitor against activator protein 1, nordihydroguaiaretic acid (2.5 mg/kg) [25] , intraperitoneally. these inhibitors were administrated once daily for 4 days immediately after viral infection (day 0). virus titers were determined in madin-darby canine kidney cells [11] . all animals were treated in accordance with the guidelines of the animal care committee of the university of tokushima. cell culture. human umbilical vein endothelial cells (lonza) were grown using the protocol supplied by the manufacturer. the cells were infected by influenza a virus wsn at a multiplicity of infection of 0.5 or treated with recombinant human il-6, tnf-a, and il-1b (10 ng/ml of each) (peprotec) in the presence or absence of antibodies against these cytokines (abcam). evaluation of vascular permeability. vascular permeability was analyzed by the evan's blue extravasation method [26] . one hour after intraperitoneal injection of 400 ml of 2% (w/ v) evan's blue dye in saline, the whole body was perfused with saline through the cardiac ventricle. the leakage of dye was detected macroscopically and by fluorescence microscope. enzyme-linked immunosorbent assay (elisa). the levels of il-6, tnf-a, and il-1b in tissue homogenates and plasma were measured using cytokine elisa kits (bd biosciences). western blotting and gelatin zymography. tissues were homogenized with 3 volumes of tris-hcl, ph 6.8, containing 2% sodium dodecyl sulphate and 0.5 m nacl, and centrifuged at 12,000 g for 30 min. human endothelial cells were lysed in radioimmune precipitation buffer (nacalai tesque) at 4њc. these extracts (30 mg protein) were electrophoresed and transferred to polyvinylidene difluoride membranes. rabbit antizonula occludens-1, anti-occludin antibodies (zymed), and anti-actin antibody (chemicon) were used. immunoreactive bands were detected using chemiluminescence (amersham bio-sciences). for gelatin zymography, the extracts (50 mg protein) were subjected to electrophoresis on 10% gelatin zymogram gels (invitrogen) as reported previously [9] . immunohistochemical staining. immunohistochemical staining was conducted as described elsewhere [9] . lung and brain sections were reacted overnight with polyclonal antibodies against human influenza a, b virus (takara) at 4њc, washed, and then reacted for 1 h at room temperature with a biotinylated second antibody. the sections were counterstained with mayer's hematoxylin. permeability assay. human endothelial cells grown to confluence on 12-well tissue culture plates with falcon cell culture inserts (1.0 mm), were exposed to the cytokines for 12 h in the presence or absence of 50 mm of aprotinin (nacalai tesque). changes in the monolayer permeability were analyzed and quantified as clearance of fluorescein isothiocyanate-dextran from the upper chamber to lower chamber as reported previously [27] . total rna was isolated from human endothelial cells using an rneasy mini kit (qiagen) and reverse transcribed using oligo primers and superscript iii rt (gibco brl) for complementary dna synthesis. the following primer pairs were used to amplify human trypsin (hprss): hprss (forward primer [f], 5'-atccaggtgagactgggagagc-aca-3', nucleotide (nt) 222-246, and reverse primer [r], 5'-gtagaccttggtgtagactccaggc-3', nt 692-716) and those of viral ns1 as reported elsewhere [28] . rt-pcr and quantification of gene expression by real time-pcr were performed using fast start sybr green master (roche diagnostics) on an abi prism 7300 system [28] . human endothelial cells were cultured on glass chamber slides until confluence. after washing twice with calcium-and magnesiumfree phosphate-buffered saline (pbsϫ), the cells were incubated with cytokines (10 ng/ml for each cytokine) for 10 h with or without pretreatment for 30 min with 20 mm protease-activated receptor (par) 2 antagonist peptide, fsy-nh 2 [29] , or 50 mm aprotinin at 37њc. the cells were also activated with 1 mg/ml trypsin or 10 mm par-2 agonist peptide [30] for 30 min. the cells were also treated for 5 h with 10 nm calcium ionophore a23187 (calbiochem) or 2 mm cacl 2 . for imaging, 10 mm fluo-3/am (invitrogen) was introduced into the cells by incubation for 30 min. the cells were then washed twice with pbsϫ and incubated with 5 mm glucose in pbsϫ at 37њc. intracellular calcium ([ca 2+ ] i ) levels were analyzed using a confocal laser scanning microscope (model cm1900; leica). statistical analysis. results are presented as mean value ‫ע‬ standard error of the mean (from 3-5 independent experiments). differences between groups were examined for statistical significance by the paired t test or 1-way analysis of variance. the wilcoxon test for comparisons of kaplan-meier survival curves was used. a p value !.05 was considered to be statistically significant. a virus wsn to study the pathogenic effects of cytokine storm on vascular dysfunction. the levels of tnf-a and il-6 in the lungs, the site of initial virus infection, were increased persistently for 6 days, and levels of il-1b peaked at days 4-6 after infection ( figure 1a ). because these cytokine responses are associated with activation of the transcription factors nf-kb and activator protein 1 [7, [31] [32] [33] , we treated mice once daily for 4 days with anti-oxidant inhibitors: pyrrolidine dithiocarbamate and n-acetyl-l-cysteine against nf-kb activation, and nordihydroguaiaretic acid against activator protein 1 activation. pyrrolidine dithiocarbamate and nordihydroguaiaretic acid significantly suppressed the upregulation of tnf-a and il-1b ( ), and n-acetyl-l-cysteine suppressed tnf-a ( p ! .001 p ! ) and il-6 ( ) at day 4 after infection ( figure 1a ). .001 p ! .01 gelatin zymography showed upregulation of ectopic trypsin in mice lung, brain, and heart during infection for 6 days ( figure 1b ). trypsin induction was inhibited by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid, probably via blockade of nf-kb and activator protein 1 binding in the promoter region of the gene (s. r. talukder, unpublished data). viral rna replication in various organs at day 4 after infection was suppressed by 11 order of magnitude by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid ( figure 1c ). suppression of viral multiplication and induction of cytokines and trypsin by treatment with pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, and nordihydroguaiaretic acid significantly improved the survival of mice at day 14 after infection (ie, the late stage of infection) ( figure 1d ). the kinetics of viral replication monitored by viral ns1 gene showed that the level of viral rna was the highest at day 4 after infection and decreased at day 6 in these organs (figure 2a ). to determine the pathogenesis of tissue injury, the viral protein accumulation in the lung and brain at day 4 after infection was analyzed by immunohistochemical staining ( figure 2b ). viral protein was detected in alveoli and terminal bronchioles in the lung and was also detected in the brain, particularly in the hippocampus, neocortex, brainstem, and brain capillaries. vascular hyperpermeability is one of the main complications of organ injury in severe influenza. vascular permeability was analyzed by infiltration of evans blue dye in the lung and brain after infection ( figure 2c and 2d) . in contrast to no dye infiltration in uninfected animals, infected mice showed a progressive increase in vascular permeability in the lung and brain at day 4 after infection. fluorescence microscopy showed leakage of dye from the blood vessels in these organs ( figure 2d ). to elucidate the mechanisms underlying vascular dysfunction in the brain, changes in the levels of tight-junction proteins, intracellular zonula occludens-1 and transmembrane occludin, and the matrix protein laminin were analyzed by western blotting. marked reductions in the expression levels of tight-junction constituents were detected at day 4 after infection, which were partly rescued by pyrrolidine dithiocarbamate, n-acetyl-l-cysteine, or nordihydroguaiaretic acid ( figure 2e ). no other tight-junction protein, claudin-5, or matrix fibronectin and type iv collagen was affected (data not shown). to clarify the linkage between upregulated cytokines and trypsin and vascular hyperpermeability after viral infection, the relationships among these findings were examined in human endothelial cells. viral infection significantly increased tnf-a and il-6 levels (2.7fold and 7.1-fold, respectively) but not il-1b levels in the culture media in a time-dependent manner over a 24-h period ( table 1) . influenza a virus infection upregulated human trypsin/ hprss gene by approximately 2-fold in the cells after infection for 6-12 h ( figure 3a ). to analyze the linkage between cytokines and trypsin in the cells, changes in the expression of hprss gene were analyzed after exposure to 10 ng/ml tnfa, il-6, and il-1b instead of viral infection for 6 h ( figure 3b ). all tested cytokines tended to upregulate hprss expression levels, especially tnf-a ( ) and il-1b ( ), although p ! .01 p ! .05 less effectively than did viral infection, and the upregulation was inhibited by simultaneous treatment of the respective neutralizing antibodies (100 ng/ml) with these cytokines (p ! for tnf-a; for il-1b). .05 p ! .01 treatment of the cells for 12 h with tnf-a, il-6, and il-1b markedly suppressed tight-junction protein zonula occludens-1 levels and occluding levels slightly, and loss of these proteins were abrogated by simultaneous treatment of the cells with 50 mm of the nonpermeable trypsin inhibitor aprotinin ( figure 4a ). furthermore, cytokine treatment disrupted the continuous and linear arrangement of zonula occludens-1 among the cells, and aprotinin inhibited the disruption ( figure 4b ). accordingly, treatment with cytokines, especially il-1b and tnf-a, tended to increase endothelial cell monolayer permeability and this effect was blocked by 50 mm of aprotinin ( ) ( figure p ! .05 4c) . these findings suggest that cytokines upregulate trypsin in vascular endothelial cells and that secreted trypsin plays an (original magnification, ϫ200) . b, immunoreactive deposits in the lung (original magnification, ϫ200). c, viral antigen (arrowheads) in epithelial cells of respiratory bronchioles and infiltrated leukocytes in alveoli (original magnification, ϫ400). d, no immunoreactive deposits in the brain before infection (original magnification, ϫ200). e, virus antigen in the cornu ammonis (ca) 1 and ca-2 and in the stratum granulosum of the dentate gyrus (dg) of the hippocampus (original magnification, ϫ200). f, virus antigen (arrowheads) in the enlarged image of ca-1 (original magnification, ϫ400). scale bars are 100 mm.c, vascular permeability in the lung and brain analyzed by evan's blue dye extravasation before (wsn-d0) and after infection at day 4 (wsn-d4). d, fluorescent micrographs of evan's blue leakage from capillaries in the brain and lung before and after infection at day 4. e, loss of tight-junction proteins, zonula occludens (zo) 1 and occludin, and laminin in the brain analyzed by western immunoblotting at day 4 after infection and its restoration by pyrrolidine dithiocarbamate (pdtc), n-acetyl-l-cysteine (nac), and nordihydroguaiaretic acid (ndga) treatments. the levels before infection are shown as control (ctr). important mechanistic role in the loss of zonula occludens-1 and increased permeability. 2+ ] i through g protein-coupled receptors, leading to cytoskeletal reorganization in the microvascular endothelium and consequent increase in permeability and tissue edema [34] . trypsin receptor par-2 is a g protein-coupled receptor activated by trypsin and tryptase and plays an important role in increasing [ca 2+ ] i [35] . to investigate the mechanisms underlying vascular hyperpermeability in severe influenza, we treated human endothelial cells with cytokines, trypsin, and par-2 agonist peptide in pbsϫ and then measured [ca 2+ ] i ( figure 5 ). marked [ca 2+ ] i mobilization was found after treatment of the cells with trypsin, par-2 agonist peptide, and cacl 2 for 10 h, whereas calcium ionophore a23187 decreased [ca 2+ ] i . treatment with tnf-a, il-1b, and il-6 also increased [ca 2+ ] i , and the mobilization was suppressed by pretreatment of the cells with par-2 antagonist, fsy-nh 2 , or aprotinin for 30 min. these results suggest that [ca 2+ ] i mobilization by proinflammatory cytokines through activation of trypsin and its receptor par-2 is one of the main mechanisms underlying increased endothelial cell permeability. the present study reports several new observations: (1) proinflammatory cytokines, tnf-a, il-1b, and il-6, when upregulated by influenza a virus infection, induce trypsin expression in various organs and human endothelial cells; (2) the upregulated trypsin induces [ca 2+ ] i mobilization via activation of the par-2, followed by loss of zonula occludens-1 and vascular hyperpermeability; (3) inhibitors of nf-kb and activator protein 1 effectively suppress the upregulation of proinflammatory cytokines and trypsin and improve the survival rates of infected mice. based on these results, we propose the influenza viruscytokine-protease cycle hypothesis as one of the mechanisms of vascular dysfunction in multiorgan failure with cytokine storm in severe influenza and influenza-associated encephalopathy ( figure 6 ). the significance of proinflammatory hypercytokinemia, or cytokine storm, in the pathogenesis of influenza a virus infigure 6 . the hypothesis of influenza virus-cytokine-protease cycle, which may affect the pathogenesis of vascular hyperpermeability and tissue destruction in severe influenza. ap-1, activator protein 1; bbb, blood-brain barrier; par-2, protease-activated receptor 2; zo-1, zonula occludens-1. fection remains unclear. the positive effects include that cytokines promote lymphocyte activation and infiltration at the sites of infection and exert direct antiviral effects. however, the negative effects of excess cytokines include the fact that the hyperinflammatory process evoked by viral infection [8, 36] may become harmful through intracellular activation of nf-kb, activator protein 1, and the janus kinase-signal transducers and activators of transcription signaling pathways [31-33, 37, 38] . the in vivo experiments presented here showed that nf-kb and activator protein 1 inhibitors markedly suppress the expression of cytokines and trypsin, viral replication, and endothelial dysfunction and result in a significant increase in the survival of infected mice. furthermore, cytokines interact with mitochondria to increase the production of reactive oxygen species, resulting in the production and activation of vasodilatory mediators, such as nitric oxide and bradykinin, and subsequent endothelial dysfunction and edema in various organs [8] (figure 6 ). the molecular mechanisms underlying tight-junction disruption in endothelial cells and vascular hyperpermeability fol-lowing the cytokine storm remain unclear. tnf-a upregulation alters the cellular redox state, reduces the expression of 4 complex i subunits by increasing mitochondrial o 2 ϫ production and depleting adenosine triphosphate (atp) synthesis, decreases oxygen consumption (resulting in mitochondrial damage) [8, 39] , and increases [ca 2+ ] i [40] . atp depletion dissociates zonula occludens-1 from the actin cytoskeleton and thereby increases junctional permeability [41] . the present results allow us to propose a new mechanism of junctional permeability regulation: upregulated trypsin by influenza a virus and/or proinflammatory cytokines induces increase in [ca 2+ ] i and loss of zonula occludens-1 in endothelial cells via par-2 signaling. in contrast to the marked upregulation of cytokines in the lungs ( figure 1a) , upregulation of cytokines in the brain was mild (data not shown), which suggests that the bloodbrain barrier destruction is the result of systemic effects of cytokines produced in the lung in severe influenza. anti-cytokine antibodies and trypsin inhibitors may effectively suppress junctional permeability. endothelial dysfunction induced by the influenza virus-cy-tokine-protease cycle in the early stage of severe influenza may also affect various circulating factors, coagulation factors, and complement systems, as well as vascular interacting cells, such as neutrophils, macrophages, and lymphocytes. multiorgan failure is the final outcome of metabolic and mitochondrial fuel disorder, immunosuppression, endocrine disorder, and tissue injury followed by endothelial dysfunction in many organs. another key pathway of acute lung injury in the highly pathogenic avian influenza virus h5n1 and acute respiratory syndrome-corona virus infection reported recently involves oxidative stress and the formation of oxidized phospholipids, which induce lung injury via toll-like receptor 4 signaling pathway [42] . in addition to these data, upregulated trypsin and proinflammatory cytokines may also affect tissue destruction and immunosuppression in the late stage of influenza a virus infection. further studies are required on the role of the influenza virus-cytokine-protease cycle in the pathogenesis of multiorgan failure, particularly in the late stage of viral infection. influenza: emergence and control influenza a and b virus infection in infants and young children during the years 1957-1976 influenza a virus associated with acute encephalopathy pcr on cerebrospinal fluid to show influenza-associated acute encephalopathy or encephalitis systemic cytokine responses in patients with influenza-associated encephalopathy induction of proinflammatory cytokines in human macrophages by influenza a (h5n1) viruses: a mechanism for the unusual severity of human disease? molecular pathogenesis of influenza a virus infection and virus-induced regulation of cytokine gene expression inflammatory cytokines in vascular dysfunction and vascular disease identification of trypsin i as a candidate for influenza a virus and sendai virus envelope glycoprotein processing protease in rat brain matrix metalloprotease-9 and tissue inhibitors of metalloproteinases i in influenza-associated encephalopathy isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelia clara cells. a possible activator of the viral fusion glycoprotein activation of influenza a viruses by trypsin treatment trypsin action on the growth of sendai virus in tissue culture cells: structural difference of sendai viruses grown in eggs and tissue culture cells identification of biological activity of paramyxovirus glycoprotein: activation of cell fusion, hemolysis and infectivity by proteolytic cleavage of an inactive precursor protein of sendai virus the molecular of influenza virus pathogenicity proteases essential for human influenza virus entry into cells and their inhibitors as potential therapeutic agents host envelope glycoprotein processing proteases are indispensable for entry into human cells by seasonal and highly pathogenic avian influenza viruses human matrix metalloproteinase-9: activation by limited trypsin treatment and generation of monoclonal antibodies specific for the activated form dystroglycan is selectively cleaved at the parenchymal basement membrane at sites of leukocyte extravasation in experimental autoimmune encephalomyelitis basement membrane and matrix metalloproteinases in monocrotaline-induced liver injury neuropathogenesis of influenza virus infection in mice persistence of viral rna in the brain of offspring to mice infected with influenza a/wsn/33 virus during pregnancy treatment with inhibitors of the nf-kb pathway improves whole body tension development in the mdx mouse the differential nf-kb modulation by s-adenosyl-l-methionine, n-acetylcysteine and quercetin on the promotion stage of chemical hepatocarcinogenesis determination of binding constant of transcription factor ap-1 and dna: application of inhibitors impaired long-chain fatty acid metabolism in mitochondria causes brain vascular invasion by a nonneurotropic epidemic influenza a virus in the newborn/suckling period: implications for influenza-associated encephalopathy human immunodeficiency virus type 1 gp120-mediated disruption of tight junction proteins by induction of proteasome-mediated degradation of zonula occludens-1 and -2 in human brain microvascular endothelial cells boost of mucosal secretory immunoglobulin a response by clarithromycin in pediatric influenza the membrane-anchored serine protease, tmprss2, activates par-2 in prostate cancer cells ligand cross-reactivity within the protease-activated receptor family active nf-kb signaling is a prerequisite for influenza virus infection differential activation of the c-jun n-terminal kinase/stress-activated protein kinase and p38 mitogenactivated protein kinase signal transduction pathways in the mouse brain upon infection with neurovirulent influenza a virus nf-kb and virus infection: who controls whom ca 2+ signaling, trp channels, and endothelial permeability protease-activated receptor 2: activation, signalling and function acute respiratory distress syndrome induced by avian influenza a (h5n1) virus in mice erk1/2 mediates tnf-a-induced matrix metalloprotease-9 expression in human vascular smooth muscle cells via the regulation of nf-kb and ap-1: involvement of the ras dependent pathway increased ap-1 and nf-kb activation and recruitment with the combination of the proinflammatory cytokines il-1b, tumor necrosis factor alpha and il-17 in rheumatoid synoviocytes tnf-induced mitochondrial damage: a link between mitochondrial complex i activity and left ventricular dysfunction acute encephalopathy associated with influenza and other viral infections molecular structure and assembly of the tight junction identification of oxidative stress and toll-like receptor 4 signaling as a key pathway of acute lung injury we are grateful to mayumi shiota for expert assistance. key: cord-346669-7n75m669 authors: wang, shixin; wei, maoti; han, yi; zhang, keju; he, li; yang, zhen; su, bing; zhang, zhilun; hu, yilan; hui, wuli title: roles of tnf-α gene polymorphisms in the occurrence and progress of sars-cov infection: a case-control study date: 2008-02-29 journal: bmc infect dis doi: 10.1186/1471-2334-8-27 sha: doc_id: 346669 cord_uid: 7n75m669 background: host genetic factors may play a role in the occurrence and progress of sars-cov infection. this study was to investigate the relationship between tumor necrosis factor (tnf)-α gene polymorphisms with the occurrence of sars-cov infection and its role in prognosis of patients with lung interstitial fibrosis and femoral head osteonecrosis. methods: the association between genetic polymorphisms of tnf-α gene and susceptibility to severe acute respiratory syndromes (sars) was conducted in a hospital-based case-control study including 75 sars patients, 41 health care workers and 92 healthy controls. relationships of tnf-α gene polymorphisms with interstitial lung fibrosis and femoral head osteonecrosis were carried out in two case-case studies in discharged sars patients. pcr sequencing based typing (pcr-sbt) method was used to determine the polymorphisms of tnf-α gene in locus of the promoter region and univariate logistic analysis was conducted in analyzing the collected data. results: compared to tt genotype, the ct genotype at the -204 locus was found associated with a protective effect on sars with or(95%ci) of 0.95(0.90–0.99). also, tt genotype, ct and cc were found associated with a risk effect on femoral head necrosis with ors(95%ci) of 5.33(1.39–20.45) and 5.67(2.74–11.71), respectively and the glucocorticoid adjusted or of ct was 5.25(95%ci 1.18–23.46) and the combined (ct and cc) genotype or was 6.0 (95%ci 1.60–22.55) at -1031 site of tnf-α gene. at the same time, the -863 ac genotype was manifested as another risk effect associated with femoral head necrosis with or(95%ci) of 6.42(1.53–26.88) and the adjusted or was 8.40(95%ci 1.76–40.02) in cured sars patients compared to cc genotype. conclusion: snps of tnf-α gene of promoter region may not associate with sars-cov infection. and these snps may not affect interstitial lung fibrosis in cured sars patients. however, the -1031ct/cc and -863 ac genotypes may be risk factors of femoral head necrosis in discharged sars patients. tnf, the gene encoding tumour necrosis factor (tnf), resides in the central part (class iii region) of the major histocompatibility complex (mhc) surrounded by a large number of other immunological genes [1] . because of the special locus of this gene, it can be deduced that this gene may associate with many diseases, and this hypothesis was confirmed by many research results [2, 3] . tnf-α is a key mediator of the inflammatory response and is critical for host defense against a wide variety of pathogenic microbes. however, the over-expression of this cytokine may lead to badness in disease recovery. the dual role of tnf, acting as an agent of both innate immunity and inflammatory pathology, poses a considerable challenge for gene regulation [2] , and this regulation mainly located on promoter region of this gene. the capacity for cytokine production in an individual has a major genetic component, and striking differences existed among individuals in terms of their ability to produce cytokines. several biallelic polymorphisms had been described within the tnfα gene, including seven in the promoter region at positions -1031t→c, -863c→a, -857c→t, -376g→a, -308g→a, -238g→a and -163g→a base pairs from the transcription start site [4, 5] . moreover, a number of studies had shown that the tnf-α promoter polymorphism had a significant effect on its transcriptional activity [6, 7] . severe acute respiratory syndrome (sars) is a newly described human infectious disease caused by a novel coronavirus-sars-cov. sars-cov infection is important because of its high infectivity and unpredictable clinical course, which is characterized by a high mortality rate [8] . till now, many researchers had reported that susceptibilities to infection sars-cov may associated with hla, mxa, oas-1 and clec4m gene polymorphisms [9] [10] [11] [12] [13] , yet these results were variable in different populations. for example, ng reported that sars-cov infection was associated with hla-b*0703 and hla-drb1*0301 in hongkong population [9] , however, lin's results showed that hla-b*4601 and hla-b*5401 were closely related to sars-cov infection [10] . chan reported that clec4m was attributed to sars-cov infection [13] , but zhi's results failed to support this conclusion. 7 these differences may be attributed to the study population used in each report, also the complex mechanism infection to sars-cov should be considered as another factor of these differences. in order to explore more host factors influencing the occurrence of sars-cov infection, we studied the polymorphisms of tnf-α gene at the promoter region, which had been ascribed to polymorphisms within the regulatory regions or signal sequences of cytokine genes [14] . after discharging from hospital, interstitial lung fibrosis was observed in sars patients. clinical data showed that the prevalence rate of this change was 21%(42/200) in cured sars patients nine months from the discharge [15] . tnf-α was one of the earliest cytokines implicated in the pathogenesis of lung fibrosis diseases and, together with il-1, has been found to over-expressed in regenerated type ii pneumocytes in human lung, thus enhanced fibroblast proliferation [16] tnf-α polymorphisms have been discovered significantly associated with increased risk of developing pulmonary fibrosis [17, 18] . given that genetic variation may potentially alter inflammation and fibrosis in the lung, the aim of this case-case control study was to examine the tnf-α polymorphisms with interstitial lung fibrosis in sars patients. in spite of interstitial lung fibrosis in cured sars patients, another sequela -femoral head necrosis was also observed in this population and the prevalence rate was 22.07%(49/221) and 23.1%(18/78) in tianjin and beijing patients respectively [15, 19] . the cause of this disease was still unknown and there were arguments about it. for example, some author considered sars-cov as the cause of femoral head necrosis, yet other authors disagreed with this view [20, 21] . previous studies showed that femoral head necrosis may caused by hormone usage [20] , yet our data failed to agree with this point. so, it need further study to explore the cause of this sequela and tnf-α polymorphisms were considered first in this report. in this paper, we aimed to study whether polymorphisms in tnf-α promoter region were associated with sars-cov infection, development, and progression of interstitial lung fibrosis and femoral head necrosis in cure sars patients. this study was reviewed and approved by ethics committees in the medical college of cpafp. the study population comprised 75 sars patients in pingjin hospital, tianjin, china, 41 health care workers of the same hospital, who had come into contact with sars patients but had not developed into sars, and 66 individuals having no contact history with sars patients. among 75 sars patients, 55 could be classified into severe and light sars according to their clinical condition history during the hospitalized period and this population also had the history of hormone therapy by reviewing the clinical treatment. anti-sars-cov antibodies of the serum samples were tested by sars elisa kits (huada diagnostics ltd, beijing, china). considered that the progression of interstitial lung fibrosis or femoral head necrosis may be affected by hormone therapy, hormone using dosage, method and lasting period were considered in this study when analyzing the associations between gene polymorphisms with disease. three kinds of hormone were used in sars patients including methylprednisolone, deltadehydrocortisone and dexamethasone. in order to simplify analyzing, deltadehydrocortisone and dexamethasone dosage were calculated into methylprednisolone using the following equation: 4 mg methylprednisolone = 5 mg deltadehydrocortisone = 0.75 mg dexamethasone. lash therapy means more than 320 mg methylprednisolone were used in a single day. cured sars patients with interstitial lung fibrosis were diagnosed by respiratory experts according to ct results following the standard proposal for therapy and diagnosis of sars patients issued by chinese ministry of health in 2004 [22] . interstitial lung fibrosis of sars patients manifested as irregular patch and strip shadow or high density strip shadow and honeycomb interstitial lung fibrosis, these changes could combine with the bronchiectasis. femoral head necrosis was diagnosed using magnetic resonance imaging (mri). an mri scan of a normal femoral head would show uniformly high signal intensity on t1and t2-weighting throughout the femoral head. agree with one of the following image could be diagnosed as femoral head necrosis in sars patients: abnormal signal with clear margin in cartilage of femoral head, or double thread image, or fracture or joint dent under cartilage, or t1wi low signal, t2wi and stir high signal of the marrow cavity edema with blur edge [22] . leucocytes were isolated within 12 h of blood collection using percoll reagent. then genomic dna was extracted using cell dna extraction kit (tiangen biotec co, beijing, china, patch number: 2004-08-13) according to the manufacturer's instructions. primers were designed according to gewaltig [23] . standard 50-μl polymerase chain reactions (pcrs) contained 5 μl(6.7 μm) forward primer 5'-gatggactcaccaggtgag-3', 5 μl(6.7 μm) reverse primer 5'-ctcatggtgtcctttccagg-3', 5 μl buffer [150 mm (nh 4 ) 2 so 4 , 500 mm tris-cl(ph = 8.8), 500 μm edta-na 2 ,15 mm mgcl 2 ,100 mm β-mercaptoethanol], 0.5 μl dna polymerase (tiangen biotec co, beijing, china), 3 μl dna template. amplification was carried out in a thermal cycler tc312 (techne, duxford cambridge, uk) with cycle parameters of 5 min at 94°c (initial dena-turation), 35 rounds of 94°c 30 s, 60°c 100 s and 72°c 150 s, and a final extension for 5 min at 72°c. the reactions were carried out in molecular bioproducts 200 μl capped tubes, as these gave optimal heat transfer in the thermal cycler. the tnf-α gene 1279 bp fragments in this paper were sequenced in double directions with forward primer 5'-gatggactcaccaggtgag-3' and reverse primer 5'-ctcatggtgtcctttccagg-3' and invitrogen company (invitrogen co, shanghai, china) using abi 373 thermal cycler carried out this job. the homozygote genotype of each snp site manifested as a single peak, yet the heterozygote with an ambiguous nucleotide position of a double color peak in the big dye chemistry pictures. according to reading the sequence graphs, the genotype was determined. the differences in values between two groups were evaluated by chi analysis for frequencies or student t test for quantitative index and binary logistic regression was done using spss 11.5 software (spss inc, chicago, illinois, usa). a total of 75 sars patients, 41 health care workers and 66 individuals were included in this study. all the populations were chinese han ethnic. the mean age was 35.0 years for sars, 35.7 for health care workers and 30.1 for individual controls (sars vs hcw, p > 0.05; sars vs individual control, p > 0.05). the proportion of male was 30.6% in sars, 26.8% in health care workers and 69.5% in individual controls (sars vs hcw, p > 0.05; sars vs individual control, p < 0.05). the sera positive rate anti-sars-cov antibody was 100.0% in sars, significantly higher than that of health care workers and individual controls (sars vs hcw, p < 0.05; sars vs individual control, p < 0.05) ( table 1) . tnf-α genotype frequencies were variable in sars, health care workers and individual controls. there were no differences of tnf-α genotype distribution at the -1031(t→c), -863(c→a), -572(a→c), -308(g→a) and according to the clinical history, symptoms of sars patients were classified into light and severe. because of the complicated clinical condition during sars outbreak, some patients' histories were incomplete and could not be classified following the severity standard [22] . the severe sars referred to those with one or more of the following: (1) dyspnea, more than 30 times per min respiratory frequencies in still condition;(2) oxygenation index less than 300 mmhg; (3) shock or multiple organ dysfunction syndrome. among all 75 patients, fifty-four were classified into light and severe. and there were no association of tnf-α polymorphisms and sars severity (table 3) . glucocorticoid using dosage, method and lasting period were not associated with interstitial lung fibrosis or femoral head necrosis in binary logistic analysis in sars patients (table 4) . and there was no difference of hormone using dosage between the interstitial lung fibrosis and non-interstitial lung fibrosis group(t = 0.72, p = 0.47) and this trend was also observed in the femoral head necrosis and non-femoral head necrosis group(t = 1.90, p = 0.064) ( table 5) . allele frequencies of tnf-α polymorphisms were listed in table 6 and there were no significant differences between interstitial lung fibrosis and non-interstitial lung fibrosis in sars patients at promoter region of tnf-α gene. allele frequencies of tnf-α gene were compared in sars patients between femoral head necrosis and non-femoral head necrosis ( four years after sars occurrence, many problems still remained unknown to us. till now, many researchers have reported that susceptibility to infection sars-cov may associate with hla, mxa, oas-1 and clec4m gene polymorphisms, yet the results are variable in different populations [9] [10] [11] [12] [13] . these differences may be attributed to the study population used in each report, also the complex mechanism infection to sars-cov should be considered as another factor of these differences. in order to explore more host factor influence the occurrence of sars-cov infection, we studied the polymorphisms of tnf-α gene at the promoter region, which have been ascribed to polymorphisms within the regulatory regions or signal sequences of cytokine genes [14] . allele distributions at -1031, -863, -857, -572, -238 and -163 were almost the same among the sars, the health care workers and individual controls, but a higher a allele frequency in sars population when compared with the control at the -308 locus(x 2 = 8.96, p = 0.003). though previous study showed that tnf-α -308 ag genotype was associated with the clearance of hepatitis b virus and the infection of helicobacter pylori caga subtype infection [24, 25] , our results failed to show the role of this locus in sars-cov infection and this conclusion agreed with that of chong wp et al [26] . we found that there was a weak protective effect of ct genotype at -204 locus of tnf-α gene against sars-cov infection. the -204 locus was a new discovered snp site of tnf-α promoter region and its role in infectious diseases might need further study. at the same time, no obvious association between the polymorphisms of tnfα promoter region with the severity of sars was observed. however, lu reported that the -238g/a polymorphism of tnf-α associated with the outcomes of hepatitis b virus infection [27] . thus, the roles of tnf-α gene in infectious diseases should be further studied. tnf-α, a key mediator of the inflammatory response, is critical for host defense against a wide variety of pathogenic microbes, but a higher concentration of this cytokine may cause severe pathology. the capacity for cytokine production in an individual has a major genetic component, and striking differences existed among individuals in terms of their ability to produce cytokines. a number of studies had shown that the tnf-α promoter polymorphism has a significant effect on transcriptional activity [6, 7] . during the process of sars-cov infection, there was a cytokine storm in patients including il-1, il-2, il-4, il-6, il-8, il-10, ifn-γ, tnf-α and tgf-β 1 [28] . the elevated levels of pro-inflammatory cytokines which may cause immuno-mediated damage to lung and other organs, resulting in acute lung injury and, subsequently, multi-organ dysfunction [29] . so, tnf-α genetic variation may potentially alter inflammation and fibrosis in the lung. after discharging from hospital, interstitial lung fibrosis was observed in cured sars patients and the prevalence rate was 21%(42/200). tnf-α was one of the earliest cytokines implicated in the pathogenesis of lung fibrosis disease and, together with il-1, has been found to over-express in regenerating type ii pneumocytes in human lung, thus enhancing fibroblast proliferation [16] .tnf-α genetic polymorphisms have been found significantly associated with increased risk of developing pulmonary fibrosis [17, 18] . during the progress of idiopathic pulmonary fibrosis, activated epithelial cells are thought to release potent fibrogenic molecules and cytokines, such as tnf-α and tgf-β 1 , which in turn foster the transformation of fibroblasts into myofibroblasts and promote their production of extracellular matrix molecules and a vicious cycle of injury and abnormal epithelial healing sets the stage for progressive fibrosis and architectural distortion of the lung parenchyma [7] . however, our data failed to show that alleles at -1031(t→c), -863(c→a), -857(c→t), -572(a→c), -308(g→a) and -238(g→a) were related to interstitial lung fibrosis when compared with non-interstitial lung fibrosis in sars patients. at the same time, -204 and -163 were homozygote genotype of tt and gg respectively in sars patients and no relationship of genotype with interstitial lung fibrosis could be calculated. this result implicated that there maybe a different mechanism of interstitial lung fibrosis of sars compared with idiopathic pulmonary fibrosis. femoral head necrosis, another sequela of discharged sars patients, prevailed with a rate of 22.07%(49/221) in tianjin [15] . however, the cause of this sequela was still unknown and there were arguments about it. for example, some author considered sars-cov as the cause of femoral head necrosis, yet other authors disagree with this view [20, 21] . previous studies showed that femoral head necrosis may caused by hormone usage, our data was far to agree with this conclusion. there was no obvious association between hormone using including hormone dosage, method and lasting period with femoral head necrosis in binary logistic analysis in sars patients. we found that the -1031 ct and cc genotypes were more frequent in sars patients with femoral head necrosis(53.7% and 6.7%, respectively) than in non-femoral head necrosis(20.0% and 0.0%, respectively). and ct and cc were related with a risk effect on femoral head necrosis with ors (95%ci) of 5.33(1.39-20.45) and 5.67(2.74-11.71), respectively when compared to tt genotype. the hormone using adjusted or of ct was 5.25(95%ci 1. 18-23.46 ) and the combined (ct and cc) genotype or was 6.0 (95%ci 1.60-22.55). also, the -863 ac genotype accounted for 43.7% of femoral head necrosis group but 10.8% of non-femoral head necrosis. compared to cc genotype, the ac genotype was manifested as another risk effect associated with femoral head necrosis with or(95%ci) of 6.42 (1.53-26.88 ) and the adjusted or was 8.40(95%ci 1.76-40.02) in cured sars patients. tnf-α can activate activity of osteoclasts and accelerate absorption of the bone and cartilage and induce occurrence of oxygen free radicals an d lipid peroxidation, which can induce ischemic necrosis of the femoral head [30] . so, the ploymophism tnf-α gene may be directly or indirectly attributed to the occurrence of femoral head necrosis in sars patients. in conclusion, there may be no association of tnf-α polymorphisms in promoter region with sars-cov infection. also, tnf-α gene polymorphisms may no affect the occurrence of interstitial lung fibrosis in cured sars patients. however, the polymorphisms may relate with femoral head necrosis. note:*:or was calculated using non-interstitial lung fibrosis as control. &: odds ratio replaced with interstitial lung fibrosis group; ‡: odds ratio replaced with non-interstitial lung fibrosis group sxw conceived of the study, and participated in its design and coordination. mtw, co-first author, carried out the molecular genetic studies and drafted the manuscript. yh, kjz and lh carried out the molecular genetic studies zy, bs and zlz participated in field investigation and samples collection of the study ylh and wlh participated in the design of the study and performed the statistical analysis all authors contributed to writing of the final manuscript all authors read and approved the final manuscript the mhc sequencing consortium: complete sequence and gene map of a human major histocompatibility complex. the mhc sequencing consortium transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes nucleotide diversity of the tnf gene region in an african village polymorphism of the 5'-flanking region of the human tumor necrosis factor (tnf)-alpha gene in japanese human leukocyte antigens class ii and tumor necrosis factor genetic polymorphisms are independent predictors of non-hodgkin lymphoma outcome the -308 tumor necrosis factor-alpha promoter polymorphism effects transcription idiopathic pulmonary fibrosis-new insights a major outbreak of severe acute respiratory syndrome in hong kong association of human-leukocyte-antigen class i (b*0703) and class ii (drb1*0301) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome association of hla class i with severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphism of oas-1 and mxa gene: a case-control study polymorphisms of interferon-inducible genes oas-1 and mxa associated with sars in the vietnamese population homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection cytokine production by normal human monocytes: inter-subject variation and relationship to an il-1 receptor antagonist (il-1ra) gene polymorphism the follow-up study on the health status of convalescent patienrs recovered from sars in tianjin co-expression of tnf alpha and il-1 beta in human acute pulmonary fibrotic diseases: an immunohistochemical analysis increased risk of fibrosing alveolitis associated with interleukin-1 receptor antagonist and tumor necrosis factor-a gene polymorphisms analysis of tumor necrosis factor-a, lymphotoxin-a, tumor necrosis factor receptor ii and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis dynamic changes of serum sars-coronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge nested case-control study of avascular necrosis of femoral head during sars patients' convalescence factors of avascular necrosis of femoral head and osteoporosis in sars patients' convalescence chinese ministry of health: proposal for therapy and diagnosis of sars association of polymorphisms of the transforming growth factor-beta1 gene with the rate of progression of hcv-induced liver fibrosis association of tnf-alpha promoter polymorphisms with the clearance of hepatitis b virus infection association between tnf-α promoter polymorphism and helicobacter pylori caga subtype infection the interferon gamma gene polymorphism +874 a/t is associated with severe acute respiratory syndrome association of -238g/a polymorphism of tumor necrosis factoralpha gene promoter region with outcomes of hepatitis b virus infection in chinese han population analysis of serum cytokines in patients with severe acute respiratory syndrome expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace2+ cells in sars patients: relation to the acute lung injury and pathogenesis of sars experimental study on gufusheng in treatment of steroid-induced ischemic necrosis of femoral head in rabbits this paper was supported by the tianjin science fund (no. 05yfszsf02900) and the science fund of medical college of cpafp(no. wy-2005-14). the author(s) declare that they have no competing interests. key: cord-351310-6p42b144 authors: bohr, adam; tsapis, nicolas; foged, camilla; andreana, ilaria; yang, mingshi; fattal, elias title: treatment of acute lung inflammation by pulmonary delivery of anti-tnf-α sirna with pamam dendrimers in a murine model date: 2020-08-13 journal: european journal of pharmaceutics and biopharmaceutics doi: 10.1016/j.ejpb.2020.08.009 sha: doc_id: 351310 cord_uid: 6p42b144 abstract to improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering rna (sirna), transfection agents are needed for efficient delivery into cells. several classes of dendrimers have been found useful as transfection agents for the delivery of sirna because their surface can readily be functionalized, and the size of the dendriplexes they form with sirna is within the range of conventional nanomedicine. in this study, commercially available generation 3 poly(amidoamine) (pamam) dendrimer was investigated for pulmonary delivery of sirna directed against tumor necrosis factor (tnf) α for the treatment of acute lung inflammation. delivery efficiency was assessed in vitro in the raw264.7 macrophage cell line activated with lipopolysaccharide (lps), and efficacy was evaluated in vivo in a murine model of lps-induced lung inflammation upon pre-treatment with tnf-α sirna. the pamam dendrimer-sirna complexes (dendriplexes) displayed strong sirna condensation and high cellular uptake in macrophages compared with non-complexed sirna. q-pcr analyses showed that the dendriplexes mediated efficient and specific tnf-α silencing in vitro, as compared to non-complexed sirna and dendriplexes with negative control sirna. also in vivo, the pamam dendriplexes induced efficacious tnf-α sirna inhibition, as compared to non-complexed sirna, upon pulmonary administration to mice with lps-induced lung inflammation. hence, these data suggest that pamam dendrimers are promising for the local delivery of tnf-α sirna in the treatment of lung inflammation via pulmonary administration. oligonucleotide-based therapeutics, including sirna, antisense oligonucleotides and mirna, are applied to intervene with the expression of specific target genes and are thereby thought to mediate more specifically disease treatment than therapeutics based on small molecules or peptides/proteins. inflammatory lung diseases are complex disorders that are usually treated with medications, which are relatively unspecific in their mode of action and are administered systemically, resulting in increased drug exposure but also undesired side effects [1, 2] . here, local sirna-based treatment may provide a much more specific and safe therapy in which certain inflammatory pathways can be targeted [3] . of the many pro-inflammatory cytokines involved in lung inflammation processes, tnfα is believed to play a central role in most inflammatory lung conditions, e.g., chronic obstructive pulmonary disease (copd), asthma, acute respiratory distress syndrome (ards) and acute lung injury (ali) [4] . more recently, it was shown to be a major target in the treatment of inflammatory flares in covid-19 infection-related ards [5] . hence, tnf-α is a promising target for sirna-based therapy against both acute and chronic lung inflammation. a sirna-based therapeutic targeting lung inflammation can be administered locally to the airways, either via inhalation or via nasal administration, where it can exert its effect directly in the inflamed tissue. local pulmonary delivery displays several therapeutic advantages, compared with systemic delivery, including (i) a quick onset of action, (ii) a reduced therapeutic dose required, and (iii) reduced side effects [6] . besides, inhalation and nasal administration represent non-invasive routes of administration, which increases patient compliance, and the fast renal clearance of sirna, observed after systemic administration, is reduced after local delivery [7] . although numerous promising therapeutic targets and oligonucleotide sequences have been identified during the past years, which have resulted in a handful of recently marketed or in advanced clinical trial products, there are still significant challenges associated with their delivery [8, 9] . generally, sirna displays poor chemical stability against nucleases and exhibits low cellular uptake and transfection [10] . hence, it is pertinent to identify efficient delivery systems that can protect the sirna from degradation, facilitate its transport across the cell membrane and mediate endosomal escape to achieve successful sirna delivery to the cytosol [11, 12] . numerous transfection agents have been identified for nucleic acid delivery, and they include, amongst others, cationic polymerand lipid-based nanocarriers, which are very efficient for cellular delivery, but they are often associated with toxicity, even at relatively low doses [13, 14] . dendrimers are synthetic globular polymers displaying a high degree of surface functionality and numerous possibilities for customizing their physicochemical properties, and they have shown great potential for pharmaceutical applications, including the delivery of nucleic acid-based therapeutics [15] [16] [17] . cationic dendrimers like the commercially available polyamidoamine (pamam) dendrimers have been shown to mediate efficient cellular uptake and transfection of sirna in vitro in multiple studies [18] . although widely used in vitro, there are only a few studies that have tested the ability of pamam dendrimers for sirna transfection in vivo [19, 20] , and to date, no studies have been performed evaluating pulmonary delivery of pamam dendrimers in vivo. in this study, we investigated generation 3 pamam dendrimers as transfection agents for pulmonary delivery of sirna targeting tnf-α and examined their efficacy and safety in a murine acute lung inflammation model. generation 3 pamam dendrimers were selected because they display very good efficiency for dendriplex formation they were prepared at different dendrimer-sirna ratios and were characterized in vitro and in vivo concerning complexation, cellular uptake, cytotoxicity, in vitro transfection efficiency and in vivo therapeutic efficacy at the rna and protein levels, respectively. tnf-α sense 5'-pgucucagccucuucucauuccugct-3', and antisense 5'-agcaggaaugagaagaggcugagacau-3', where the underlined capital letters represent 2'-o-methylribonucleotides, lower-case letters represent deoxyribonucleotides and p represents a phosphate residue [21] . a negative control sirna sequence was purchased from eurogentec (eurogentec, angers, france). the sequence of this negative control is not disclosed by the supplier. fluorescently labeled sirna with the dye tye™ 665 was provided by idt with a sequence targeting luciferase: all additional chemicals used were obtained commercially and were of analytical grade. sense 5'-pgguuccuggaacaauugcuuuuaca-3', dendriplexes were prepared in 10 mm hepes buffer at a nitrogen-to-phosphate (n/p) ratio of 5, 10, were used for upconcentrating the dendriplex suspensions by centrifugation of the samples at 13,000 g for 20 min. the particle size distribution and polydispersity index (pdi) of the dendriplexes were determined by dynamic light scattering using the photon correlation spectroscopy technique, and their zeta potential was estimated by using laser-doppler micro-electrophoresis using a zetasizer nano zs (malvern instruments, worcestershire, uk) equipped with a 633 nm laser and 173° detection optics. the complexation of dendrimers and sirna into dendriplexes was assessed using gel electrophoresis. electrophoresis was carried out applying 1% (w/v) agarose gels (promega, city, usa) containing 5 µl 2.5 mg/ml ethidium bromide solution, and samples consisting of 0.1 nmol sirna were loaded into each well of the gels. the gels were run for 20 min at 100 v in tris-borate-edta (tbe) buffer (ph 8.2). visualization of the sirna bands was performed using an mf-chemibis gel imaging system (dnr bio-imaging systems, neve yanim, israel). a murine macrophage cell line raw264.7 was purchased from the american type culture collection (atcc, molsheim, france) and cells were maintained in dulbecco's modified eagle's medium (dmem) (aldrich, st quentin fallavier, france) supplemented with 10% (v/v) fetal bovine serum (paa laboratories, pasching, austria) and 100 u/ml penicillin, 100 μg/ml streptomycin. cells were grown under a controlled atmosphere with 5% co 2 /95% o 2 at 37°c and were sub-cultured twice per week by washing and gently scraping the cells from the culture flask. cells were used between passages 5 and 12. the cellular viability of the raw264.7 cells was assessed by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay. cells were prepared in suspension, counted and seeded in 96-well plates at a density of 8 × 10 3 cells per well and left to attach overnight. the cells were subsequently incubated with dendriplexes (n/p ratio 5) at different concentrations for a period of 24 h. to each well with 100 µl culture medium, 10 µl of mtt-solution (5 mg/ml in pbs, ph 7.4) was added and the plate was incubated for 2 h at 37 °c. subsequently, the medium was replaced with 200 µl dimethylsulfoxide, and the absorbance was measured at 570 nm, using a microplate reader (fluostar optima, bmg labtech, germany). cellular uptake of sirna was assessed in raw264.7 cells using fluorescence microscopy and flow cytometry, respectively. for fluorescence microscopy, the cells were seeded in chambered cover slides µ-slide 8 well (ibidi, planegg, germany) at a density of 5 × 10 4 cells per well and cultured for 12h. the cells were then treated with fluorescently labeled sirna for 4 h (at an n/p ratio of 5 for dendriplexes and a concentration of 100 nm sirna), washed with pbs and fixed using 4% (v/v) paraformaldehyde. microscopy was performed using a zeiss lsm 510 (carl zeiss, jena, de) fluorescence microscope equipped with a 1 mw helium-neon laser and a plan-apochromat 63x objective lens (numerical aperture 1.40, oil immersion). images were captured at a 63x magnification and overlayed with differential interferential contrast (dic) images. for flow cytometry, the cells were cultured in 12-well plates at a density of 5 × 10 4 cells per well. the cells were treated with fluorescently labeled sirna for 4 h (at an n/p ratio of 5 for dendriplexes and a concentration of 100 nm sirna) and subsequently resuspended and analyzed using an accuri c6 (bd biosciences, franklin lakes, nj, usa) flow cytometer. the mean fluorescence intensity (mfi) was used to determine the relative cell uptake (the ratio between treated and non-treated samples), expressed in arbitrary units. cell transfection studies were performed as described previously [17] [22] . briefly, raw264.7 cells were seeded at a density of 1 × 10 6 cells per well in 6-well culture plates. dendriplexes prepared at an n/p ratio of 5 were transferred to culture plates to achieve a final sirna concentration of 100 nm, and they were incubated for 24 h. three hours before collection, the cells were exposed to lipopolysaccharide (lps) dispersed in pbs to obtain an lps concentration of 5 ng/ml. negative controls were only given pbs, and positive controls only received lps, but were not treated with sirna. for collection, the culture medium was removed from the wells, 1 ml ice-cold trizol (thermo fisher, villebon-sur-yvette, france) was added to each well, and the cells were scraped and homogenized by pipetting. rna extraction was performed following the stepwise instructions provided with the trizol reagent, and the total rna content of the extracts was assessed using a biomate 3 uv spectrophotometer (thermo fisher) with a traycell ultra-micro cell (hellma analytics, paris, france). additional quality control of the extracts was performed with an rna labchip, using an agilent 2100 bioanalyzer (agilent, santa clara, ca, usa). reverse transcription was performed for 1 µg rna extracts using a mix of primers [17] and an iscript cdna synthesis kit (bio-rad laboratories, (hercules, ca, usa). the cdna was diluted 1:10 (v:v) in pcr-grade water and stored at −80°c until further use. tnf-α mrna silencing was assessed by real-time reverse transcription-polymerase chain reaction (rt-pcr), essentially as previously described [22] . real-time pcr was performed using a c1000 thermal cycler instrument with a cfx96 real-time system (biorad) with the following cycling conditions: initial denaturation step at 95 °c for 5 min, followed by 35 cycles including (i) denaturation at 95 °c for 10 s, (ii) annealing at 60 °c for 10 s, and (iii) elongation at 72 °c for 10 s. the cfx manager software 3.0 (biorad) was used for crossing point (cp) analysis, and the values were normalized against the average of the two reference genes ribosomal protein, large p2 (rplp2) and glucoronidase b (gusb). the reference genes were selected based on a screening study of eight reference candidates [17] . all animal experiments were conducted following the european rules (86/609/eec and 2010/63/eu) and the principles of laboratory animal care and the national french legislation (decree no. 2013-9 and minimal stress to the animals. female swiss cd-1 outbred mice were purchased from envigo (gannat, france), and all experiments were performed using mice at the age of 6 to 8 weeks. the mice were housed in groups of four with access to water and food ad libitum and kept at a constant temperature (19-22°c) and relative humidity (45-65%). a well-known procedure for lps-induced lung inflammation [23] [24] [25] was modified using swiss cdinjection of 200 µl 20 mg/ml pentobarbital solution). subsequently, the trachea was cannulated with a catheter, and the lungs were flushed twice with 0.3 ml pbs. the bal was centrifuged at 400 g for 10 min, the supernatant was stored at -20 °c for protein quantification and tnf-α determination. mice were dosed with sirna as a prophylactic treatment before inducing lung inflammation ( figure 1 ). non-complexed sirna and dendriplexes (n/p ratio of 5) were administered via intranasal administration similar to lps administration at a sirna concentration of 1.0 mg/kg using an average volume of 30 µl sirna solution 24 h before lps challenge. the bal was extracted 4 and 72 h, respectively, after the lps challenge, and a minimum of five mice was included in each sample group. negative controls were dosed twice with pbs, and positive control mice were first dosed with pbs and subsequently treated with lps. figure 1 . schematic illustration of the in vivo experimental procedure. mice were prophylactically administered with treatments 24h before inflammation induction by lps. bal was collected either at 4 or 72h for tnf-α determination. the tnf-α protein levels were assessed by using the cytometric bead array -mouse inflammation kit (bd biosciences). the supernatants of bal samples were diluted in assay diluent from the kit, and the samples were prepared following the manufacturer´s instructions. the bal tnf-α levels were quantified using an accuri c6 flow cytometer, where 2000 roi counts were measured for each sample. all samples were prepared in duplicates and analyzed using the bd accuri c6 software. the data of the in vitro studies are reported as mean values ± sd, and the data for the in vivo studies are reported as mean values ± sem. the statistical significance was determined using either a twotailed, unpaired student's t-test or anova, with the statistical significance set at * p < 0.05, ** p < 0.01. all dendriplexes displayed an average size between 127-153 nm and a pdi between 0.19-0.27 (table 1 ). there was no clear correlation between the n/p ratio of the dendriplexes and their resulting sizes and pdis although it has previously been shown that the sizes of pamam dendriplexes decrease with an increase in n/p ratio [26] . positive zeta potential values were observed for all n/p ratios, indicating a net positive surface charge and condensation of sirna. the zeta potential increased significantly (p <0.05, anova) as a function of the n/p ratio, which can be attributed to the increase in charge ratio, and it also indicates improved sirna condensation at higher n/p ratios. gel electrophoresis studies using etbr to stain the sirna were performed to assess qualitatively the binding between sirna and dendrimer at different n/p ratios. these studies show binding between sirna and dendrimers at all n/p ratios (figure 2 ). in all cases, the major part of the sirna was retained inside the dendriplexes in the presence of etbr, as compared to free, non-complexed sirna. based on the size and binding results of the dendriplexes, subsequent experiments were performed at an n/p ratio of 5 as a compromise between the complexation and high sirna loading as shown by gel retardation assay. moreover, this ratio was selected to reduce the cytotoxicity as much as possible. cell viability studies using the mtt assay showed that the dendriplexes were well tolerated up to a sirna concentration of 800 nm (n/p ratio 5) ( figure 3a) . a similar profile was observed for noncomplexed dendrimers (not shown). the cytotoxicity measured for the dendriplexes is relatively low, as compared to other cationic transfection agents, e.g., polyethyleneimine (pei) and poly-l-lysine [27] , which indicates that pamam dendriplexes are well tolerated by raw264.7 cells. cellular uptake assessed at a subtoxic concentration of 100 nm sirna using flow cytometry showed that the uptake of non-complexed sirna was low and did not increase over time ( figure 3b ). in contrast, the cellular uptake of the dendriplexes increased gradually with time, resulting in a six-fold higher uptake transfection using tnf-α sirna and negative control sirna in macrophages activated with lps was evaluated at the mrna level using qpcr ( figure 5 ). an sirna containing 2'-o-methylated nucleotides in the antisense strand was selected to minimize the innate immune response that occurs with the administration of sirna [28] . another reason is provided by the greater stability of this chemically modified sirna [29] . non-complexed tnf-α sirna mediated little but statistically significant inhibition (26%) of the tnf-α mrna expression (p < 0.05), as compared with the negative control sirna, and 28% inhibition, as compared to the lps-challenged positive control (p < 0.05). in contrast, the pamam dendriplexes with tnf-α sirna mediated 86% inhibition of tnf-α expression, as compared to the lps-challenged positive control (p < 0.001) and 58% inhibition compared with the pamam dendriplexes with negative control sirna (p < 0.01), which represents a substantially greater inhibition compared with non-complexed sirna. hence, pamam dendriplexes display high potential for tnf-α silencing, but some unspecific silencing of pamam dendriplexes with negative control sirna was also observed. the transfection data support the observations from the cell uptake studies, indicating the improved performance of dendriplexes, as compared with non-complexed sirna. figure 5 . tnf-α mrna silencing in raw 264.7 cells by dendriplexes. samples were normalized to the lps-treated cells (control +) without sirna, and the results denote the tnf-α mrna expression level of cells treated with tnf-α sirna, relative to transfection with negative control sirna. the samples correspond to a sirna concentration of 50 nm and an n/p ratio of 5. results denote mean values ± sd (n ≥ 3). statistical significance: * p < 0.05, ** p < 0.01. to induce gene silencing in the lung, sirnas were delivered using pamam dendrimers. many studies have examined the effect of pamam dendrimers on the lung. at first, it was shown that 83% of the pamam dendrimers dosed were still remained in the lung 6.5 h post pulmonary administration to mice [30] . several studies have stressed the lung biocompatibility of pamam dendrimers and the absence of any inflammatory response in the lung [31, 32] . in several studies, it has been shown that intranasal challenge with lps results in lung inflammation characterized by an increase in the levels of tnf-α as well as other pro-inflammatory cytokines in the lungs [33] . besides, the massive recruitment of macrophages and neutrophils takes place rapidly, and the inflammatory process lasts for several days [34, 35] . chronic inflammation is usually characterized by occasional flare-ups, including copd, asthma, and cystic fibrosis, which represent the most common reason for contact between the patient and the health care system [36] . these flareups can be triggered by both external and internal stimuli, e.g., exposure to pollutants and increased stress levels. hence, the optimal treatment strategy may be a prophylactic measure taken to reduce the response to such stimuli. therefore, in this study, we investigated the therapeutic effect of a prophylactic treatment with sirna given 24 h before inducing an inflammatory response with lps. tnf-α levels were compared for mice treated with lps (control +) as well as untreated mice (control -) and were evaluated to assess the potential inflammatory response of the non-complexed sirna and the dendriplexes. dendriplexes were administered at a sirna dose of 1 mg/kg, based on previous sirna studies on pulmonary delivery, where doses ranging between 0.6-3 mg/kg were administered [37] [38] [39] . the tnf-α concentrations in the bal were measured 4 h and 72 h after exposing the mice to lps. the tnf-α levels measured after 4 h ( figure 6a macrophages. yet, based on in vivo studies it was demonstrated that dendriplexes with tnf-α sirna resulted in improved performance compared with non-complexed tnf-α sirna at early time e.g 4 h after lps challenge and lower performance compared with non-complexed tnf-α sirna 72h after lps challenge, meaning that more frequent administrations are needed in patients displaying strong lung inflammation. in this study, tnf-α was selected as a target because this proinflammatory cytokine plays a central role in lung diseases and its actions are numerous and quite diverse being linked to many lung diseases including asthma, copd, ali/ards, sarcoidosis, and interstitial pulmonary fibrosis [40] . the data show that a quick response occurs not lasting long which might be related to a very quick dissociation in the lungs. thanki et al. [7] demonstrated that although part of complexed sirna was staying in the lungs, around 50% was permeating across the air-blood barrier within 6 h and subsequently excreted via the kidneys [7] . the low stability as well might explain why the efficacy does not stand for longer times. in a previous work [17, 41] involving phosphated dendrimers, we compared two dendrimers in a lung injury model using the same anti-tnf-α sirna. it was clear from these studies that dendriplexes with the lowest kd were the more active with a long-lasting inhibition effect not only on tnf-α but other cytokines as well. this could be achieved with more frequent lung administration but would also raise the question of chronic toxicity which needs to be further evaluated. applying higher-generation pamam denderimers would have increased the stability and efficacy but again could have induced much higher toxicity. finally, two factors might explain the lower efficacy of dendriplexes at 72-hour. the first is the high stability of methylated sirna and the second is the fast elimination of dendriplexes due to the mucociliary clearance, as compared to the clearance of non-complexed molecules that can penetrate in the deep lung without undergoing this elimination process. in the current study, the performance of pamam dendrimers was investigated as a delivery system for sirna transfection, specifically for the local treatment of lung inflammation. we demonstrate a good ability to condensate sirna, high cellular internalization rate and a specific and efficient gene silencing of tnf-α in vitro in macrophages for pamam-based dendriplexes with tnf-α targeting sirna. in vivo studies in a murine acute lung inflammation model showed silencing of tnf-α for the dendriplexes although less pronounced compared to in vitro performance. the findings suggest that tnf-α targeting sirna can be used as a local treatment for overall suppression of lung inflammation and can be used as a prophylactic treatment 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anti-inflammatory effect of anti-tnf-α sirna cationic phosphorus dendrimer nanocomplexes administered intranasally in a murine acute lung injury model dendrimers for sirna delivery delivering sirna with dendrimers: in vivo applications poly (amidoamine)(pamam) dendrimer mediated delivery of drug and pdna/sirna for cancer therapy chitosan/sirna nanoparticle-mediated tnf-α knockdown in peritoneal macrophages for antiinflammatory treatment in a murine arthritis model comparison of polymeric sirna nanocarriers in a murine lps-activated macrophage cell line: gene silencing, toxicity and off-target gene expression il-17, produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: il-15 as a possible trigger local stimulation of alpha7 cholinergic receptors inhibits lps-induced tnf-alpha release in the mouse lung van der poll, the lps-induced lung inflammation in vivo 1 elucidating the molecular mechanism of pamam-sirna dendriplex self-assembly: effect of dendrimer charge density polyamidoamine dendrimers with a modified pentaerythritol core having high efficiency and low cytotoxicity as gene carriers overcoming the innate immune response to small interfering rna, hum chemical modification of sirnas to improve serum stability without loss of efficacy effect of the route of administration and pegylation of poly(amidoamine) dendrimers on their systemic and lung cellular biodistribution polyamidoamine dendrimers can improve the pulmonary absorption of insulin and calcitonin in rats pamam dendrimers as nano carriers to investigate inflammatory responses induced by pulmonary exposure of pcb metabolites in sprague-dawley rats nasal lipopolysaccharide challenge and cytokine measurement reflects innate mucosal immune responsiveness a prominent role for airway epithelial nf-κb activation in lipopolysaccharide-induced airway inflammation acute lung injury: prevention may be the best medicine living with chronic obstructive pulmonary disease: a survey of patients' knowledge and attitudes in vivo tumor targeting via nanoparticle-mediated therapeutic sirna coupled to inflammatory response in lung cancer mouse models in vivo endothelial sirna delivery using polymeric nanoparticles with low molecular weight lipid envelope-type nanoparticle incorporating a multifunctional peptide for systemic sirna delivery to the pulmonary endothelium anti-tnfα therapy in inflammatory lung diseases elucidating the role of surface chemistry on cationic phosphorus dendrimer-sirna complexation the authors would like to thank the danish council for independent research key: cord-295683-eoxxal8v authors: gong, r.; rifai, a.; dworkin, l.d. title: hepatocyte growth factor suppresses acute renal inflammation by inhibition of endothelial e-selectin date: 2006-04-01 journal: kidney int doi: 10.1038/sj.ki.5000246 sha: doc_id: 295683 cord_uid: eoxxal8v vascular endothelial activation, marked by de novo expression of e-selectin, is an early and essential event in the process of leukocyte extravasation and inflammation. evidence suggests that hepatocyte growth factor (hgf) ameliorates inflammation in animal models of renal disease, implying that hgf might inhibit specific components of the inflammatory response. this study examined the effect of hgf on endothelial e-selectin expression in acute inflammation induced by tumor necrosis factor (tnf)-α. in vitro, hgf suppressed tnf-α-induced cell surface expression of e-selectin in human umbilical vein endothelial cells (huvec) and inhibited e-selectin mediated monocytic adhesion to endothelial monolayers. hgf activated phosphatidylinositol 3-kinase (pi3k)–akt that in turn inhibited its downstream transducer glycogen synthase kinase (gsk)3. blockade of the pi3k–akt pathway with specific inhibitors abrogated hgf induced inhibitory phosphorylation of gsk3 and suppression of e-selectin. in addition, selective inhibition of gsk3 activity by lithium suppressed tnf-α-induced e-selectin expression and monocytic adhesion, reminiscent of the action of hgf. moreover, ectopic expression of an uninhibitable mutant gsk3β, in which the regulatory serine-9 is replaced by alanine, abolished hgf's suppressive effect on endothelial e-selectin. in vivo, administration of exogenous hgf reduced endothelial expression of e-selectin induced by bolus injection of tnf-α. this was associated with less sequestration of circulating fluorescence-labeled macrophages in the kidney. these findings suggest that hgf ameliorates acute renal inflammation in part by downregulating e-selectin mediated macrophage adhesion to the inflamed endothelium. inflammation, characterized by tissue infiltration by leukocytes, is a basic biological reaction and part of the innate defense to injuries induced by various pathogenic factors. 1 an inflammatory response of appropriate magnitude and timing is crucial to tissue repair and homeostasis. 1, 2 most inflammatory responses are acute and self-limiting; however, an excessive inflammatory reaction may result in critical and fatal conditions as systemic inflammatory response syndrome, severe acute respiratory syndrome, and acute renal failure. in addition, if the inflammatory response is prolonged or frequently relapsing chronic persistent inflammation develops, which may promote fibrosis and loss of organ function. 3 immunosuppressants including glucocorticoids are widely used to treat patients with excessive or chronic inflammation and reduce the likelihood of these complications, despite an increased risk of opportunistic infections. [1] [2] [3] vascular endothelial activation and dysfunction play a critical role in the inflammatory response. [4] [5] [6] [7] [8] normally, leukocytes continuously patrol the vasculature, alert for signals of inflammation. proinflammatory substances released by pathogens (e.g. lipopolysaccharide) or by damaged tissue (e.g. tumor necrosis factor (tnf)-a) upregulate the expression of adhesion molecules on the endothelium and initiate the migration of leukocytes to the inflamed area. leukocyte migration from blood to tissues involves several steps: rolling, sticking, diapedesis, and chemotaxis. 9 among these processes, rolling is the earliest and indispensable event initiating leukocyte extravasation and inflammation. rolling is mediated by the selectin family of adhesion molecules, endothelial e-selectin, platelet p-selectin, and leukocyte l-selectin. 10 both e-and p-selectins are expressed by endothelial cells; l-selectin is found only on leukocytes. p-and l-selectins are constitutively expressed whereas e-selectin is elicited by proinflammatory stimulate and is considered essential for leukocyte trafficking. 10 the importance of e-selectin in initiating inflammation is demonstrated by the potent anti-inflammatory effect of e-selectin blockade. [11] [12] [13] [14] [15] [16] hepatocyte growth factor (hgf) is a mesenchymalderived, pleiotropic multifunctional growth factor. 17 upon binding to its receptor, c-met, hgf triggers several signal transduction pathways, including phosphatidylinositol 3-kinase (pi3k)-akt, ras-mek-erk, and stat3 pathway, and modulates diverse cell processes including mitogenesis, motogenesis, morphogenesis, and antiapoptosis/survival in epithelial and endothelial cells. 17, 18 evidence suggests that hgf ameliorates both acute and chronic injury in various organs including kidney, 19 liver, 20 lung, 21 and intestine. 22 of note, inflammation is an invariable finding in both acute and chronic disease. inflammation subsides in response to hgf treatment; however, the potential beneficial effects of hgf on inflammation have been largely overlooked. recently, we reported that hgf treatment substantially attenuated inflammation in the rat remnant kidney model of chronic renal failure; 23 however, the mechanisms responsible for this action remain uncertain. in this study, we show that hgf abrogates monocyte to endothelial adhesion and ameliorates acute renal inflammation by suppressing endothelial eselectin expression. these findings suggest that the beneficial effects of hgf in both acute and chronic disease may be partially ascribed to its systemic anti-inflammatory action on the endothelium. as presented in figure 1a , tnf-a strongly stimulated eselectin expression at low doses without significantly reducing the human umbilical vein endothelial cells (hu-vec) viability. western immunoblot of whole-cell lysates showed that hgf treatment suppressed tnf-a induced total e-selectin expression in a time-dependent manner. e-selectin is found both on the cell membrane and in the intracellular reservoir in activated endothelial cells. 24 only membrane eselectin is accessible to leukocytes and functionally active in mediating the endothelial-leukocyte adhesion. to examine whether hgf treatment modulated cell surface expression of e-selectin, flow cytometry (figure 1b) and fluorescent immunocytochemistry (figure 1c -f) without cell membrane permeabilization were employed. as shown in figure 1c -f, control and hgf alone treated huvec cells are negative for e-selectin. tnf-a markedly induced e-selectin expression with a typical surface distribution pattern, and hgf pretreatment significantly decreased the surface e-selectin staining. flow cytometry analysis corroborated the immunocytochemistry findings. hgf prevented tnf-a induced surface expression of e-selectin in huvec cells. to determine whether hgf suppression of endothelial eselectin expression reduces leukocyte adhesion to endothelium, we employed the monocytic static adhesion assay. 25 few fluorescent thp-1 cells were found adherent to vehicle (figure 2d ). to quantify monocytes adherent to huvec monolayers, cells were lysed and subjected to fluorometric analysis (figure 2f ), which was in agreement with the microscopic findings. of note, addition of a specific rabbit anti-e-selectin antibody blocked monocyte adhesion, suggesting that e-selectin mediates endothelial to monocyte adhesion and that suppression of endothelial expression of e-selectin by hgf accounts for the reduction in monocytic adhesion. the pi3k-akt pathway is required for hgf suppression of eselectin after binding to its cognate receptor, c-met, hgf triggers multiple signaling pathways including the pi3k-akt pathway, ras-mek-erk pathway, and stat3 pathway. 18 hgf activated all three pathways in huvec cells, while tnf-a had only a minor effect (figure 3a ). to determine which signaling pathway mediates hgf suppression of e-selectin, we pretreated huvec with various inhibitors specific for each pathway. as shown in figure 3b , the suppressive effect of hgf on tnf-a -induced e-selectin was blocked by two different inhibitors specific for the pi3k-akt pathway, wortmannin and ly294002. in contrast, u0126, the selective inhibitor for the ras-mek-erk pathway and ppylktk-mts, the stat3 inhibitor, failed to abolish the hgf's inhibitory action ( figure 3c ). these data suggest that the pi3k-akt pathway mediates hgf's suppression of e-selectin in endothelial cells. hgf inhibits glycogen synthase kinase (gsk) 3 via pi3k-akt mediated phosphorylation in endothelial cells gsk3 is an important downstream transducer of the pi3k-akt signaling pathway. gsk3 is inactivated in response to pi3k signaling, as a result of akt-mediated phosphorylation of an n-terminal serine, serine-9 in gsk3b and ser-21 in gsk3a. 26 because phosphorylation of gsk3 at these two residues denotes gsk3 inactivation, we probed using a specific antibody against phosphorylated gsk3a (s21) and gsk3b (s9). in huvec cells, hgf treatment immediately elicited inhibitory phosphorylation of gsk3b and, to a lesser extent, gsk3a ( figure 4a ). this effect persisted for at least 90 min in the presence or absence of tnf-a, while tnf-a alone had only a minor effect. in addition, hgf-induced inhibitory phosphorylation of gsk3 was abolished by wortmannin ( figure 4b ), implying that activation of pi3k-akt pathway is required for this action. to avoid cell loss due to induction of apoptosis, the general apoptosis inhibitor boc-asp-ch 2 f was added to the culture. 27 at the optimal concentration not associated with significant apoptosis, lithium (20 mm), a selective inhibitor for gsk3, 28 induced inhibitory phosphorylation of gsk3 (figure 5a ), and attenuated basal and tnf-a-induced e-selectin in huvec (figure 5b) , reminiscent of the action of hgf. sodium, an osmolality control, had no effect. the finding that activation of pi3k-akt and subsequent inhibition of gsk3 mediates hgf suppression of e-selectin prompted us to investigate whether the pi3k-akt-gsk3 cascade regulates endothelial to monocyte adhesion. huvec monolayers were activated with tnf-a and subjected to static adhesion assay after pretreatment with hgf or different selective chemical inhibitors of the pi3k-akt-gsk3 pathway. fluorometric analysis of cell lysates demonstrated that inhibition of gsk3 by lithium attenuates tnf-a-induced endothelial to monocyte adhesion, similar to the effect of hgf. sodium, the osmolality control, had no effect. blockade of pi3k activation by wortmannin also blocked the inhibitory action of hgf on monocytic adhesion from 4 h on ( figure 6 ). gsk3 consists of two distinct isoforms, gsk3a and gsk3b. previous studies suggested that gsk3b, but not gsk3a, is essential for tnf-a or il-1b-induced inflammatory responses. 29 to further examine the role of inhibitory phosphorylation of gsk3b in hgf inhibition of e-selectin, we studied the effect of forced expression of gsk3b on eselectin in huvec cells. vectors encoding the hemagglutin (ha) tagged wild type (wt) gsk3b or uninhibitable mutant gsk3b, 30 in which the regulatory serine-9 residue was replaced by alanine (s9a-gsk3b), were transfected into huvec cells. as a control, pcdna3 was used in transfection. to evaluate the levels of expression, whole-cell lysates were analyzed by immunoblotting for ha or ha-gsk3b ( figure 7a ). immunofluorescent detection using an antibody against the ha epitope revealed that over 50% of the cells expressed the ha-tagged constructs 24 h after transfection. as shown in figure 7a -c, hgf inhibition of tnf-a induced e-selectin expression was evident in huvec cells transfected with pcdna3 or wt-gsk3b. in contrast, ectopic expression of s9a-gsk3b abolished the suppressive action of hgf on eselectin expression. collectively, these findings suggest that inhibitory phosphorylation of gsk3b at serine-9 is required for hgf inhibition of e-selectin in huvec cells. inflammation is a common finding in both acute and chronic injuries of diverse etiologies. leukocyte to endothelium adhesion mediated by the selectin family of adhesion molecules, in particular e-selectin, is an indispensable event initiating the inflammatory reaction. [6] [7] [8] [9] [10] in the present study, we found that hgf suppresses monocyte to endothelial adhesion and attenuates acute renal inflammation via inhibition of endothelial e-selectin expression. hgf exerts this action via activation of pi3k-akt-gsk3b pathway in endothelial cells. this finding complements our recent report demonstrating that hgf attenuates renal inflammation in the rat remnant kidney model of chronic renal disease. 23 hgf has also recently been reported to be anti-inflammatory in non-renal diseases. 21, [31] [32] [33] [34] in a murine model of inflammatory bowl disease (ibd), hgf gene transfection diminished inflammatory infiltrates in the intestinal epithelium. 31 similarly, arthur et al. 32, 33 reported that direct intravenous infusion of exogenous hgf to rats with ibd significantly ameliorated gross and microscopic bowl inflammation. in a murine model of airway hyperresponsiveness, airway inflammation was reduced by administration of recombinant hgf. 34 in the present study, exogenous hgf significantly attenuated tnf-a induced macrophage infiltration and acute renal inflammation in rats. the finding that hgf has systemic anti-inflammatory effects suggests that hgf may intercept common processes in the general inflammatory reaction rather than organ-specific mechanisms. leukocyte adhesion to an activated endothelium is a prerequisite for generating an inflammatory infiltrate and is found in virtually all-inflammatory diseases. 8, 9 increased expression of particular adhesion molecules is an important early marker of endothelial activation. e-selectin is of particular interest because it is only found on the inflamed endothelium in contrast to other adhesion molecules, which have a wide constitutive tissue distribution. 9 previous studies demonstrated that inhibition of endothelial activation by glucocorticoids 35 or statins 36 attenuates inflammation by decreasing endothelial expression of adhesion molecules, including e-selectin. similarly, blocking the interaction between the endothelium and leukocytes with e-selectin antibodies as well as selectin antagonists also ameliorates various inflammatory diseases. [11] [12] [13] [14] [15] [16] for instance, anti-eselectin blocking antibody or e-selectin gene disruption protected mice from ischemia-reperfusion induced acute renal failure. 12 similarly, antibody blockade of e-selectin decreased adventitial inflammation and attenuated intimal hyperplasia in rat carotid arteries after balloon injury. 13 moreover, transgenic mice producing soluble e-selectin that can competitively inhibit the binding of inflammatory cells to e-selectins on the endothelium are resistant to bleomycin induced chronic pulmonary inflammation and lung fibrosis. 14 all these studies suggest the therapeutic potential of eselectin blockade in inflammatory diseases. in our study, hgf was found to suppress endothelial e-selectin expression in cultured huvec cells and in vivo in rats injected with tnf-a. suppression of e-selectin in turn was responsible, at least in part, for hgf-induced suppression of monocyte to endothelial adhesion and inflammation in the kidney. the mechanism by which hgf regulates endothelial eselectin expression is not well studied. the pi3k-akt pathway is a major signaling cascade triggered by the binding of hgf to its cognate receptor c-met on endothelial cells. [17] [18] [19] in the present study, hgf activated pi3k-akt in endothelial cells and specific blockade of pi3k-akt overrode hgf suppression of e-selectin expression. these data are consistent with another study in a murine model of endotoxemia, 37 in which blocking the pi3k-akt pathway with wortmannin or ly294002 enhanced lps-induced e-selectin levels and exacerbated macrophage infiltration in liver and kidney. gsk3 is an important downstream substrate of the pi3k-akt signaling pathway that has been shown to regulate the inflammatory response. 29, 38 a ubiquitously expressed serinethreonine kinase, gsk3 exists in two isoforms, gsk3a and gsk3b and is a unique signal transducer in that it is constitutively active under normal conditions. gsk3 is inactivated in response to pi3k signaling, as a result of aktmediated inhibitory phosphorylation. 39, 40 we found that hgf induced inhibitory phosphorylation of both gsk3a and gsk3b in endothelial cells. selective inhibition of gsk3 by lithium also inhibited phosphorylation of gsk3 and suppressed tnf-a-induced e-selectin expression in huvec cells, reminiscent of the action of hgf. furthermore, ectopic expression of a mutant construct encoding the uninhibitable gsk3b blunted hgf-induced suppression of e-selectin, again suggesting that hgf inhibits e-selectin via inhibition of gsk3b. a regulatory role for gsk3b in e-selectin gene expression was also reported by hoeflich et al. 29 in that study, e-selectin-luciferase transcription induced by tnf-a or il-1b was reduced in gsk3b à/à cells and could be restored by the expression of exogenous gsk-3. the mechanism by which gsk3b inactivation modulates e-selectin is unclear. recent data suggest that gsk3b is an essential element for nf-kb activation. [41] [42] [43] sequence analysis reveals the presence of multiple putative kb elements in the promoter region of the e-selectin gene. 44 in addition, interaction between nf-kb and kb elements in the e-selectin promoter is required for e-selectin expression. 44, 45 therefore, it is possible that hgf inhibition of endothelial e-selectin and inflammation might be due to suppression of nf-kb via gsk3b inactivation. gsk3b has been recently suggested to be a key regulator of multiple cellular processes implicated in the pathogenesis of diabetes and chronic inflammatory diseases. 26, 38 due to its regulatory effect on gsk3b, hgf might represent a novel potential strategy for the treatment of inflammatory diseases. in summary, hgf suppresses e-selectin expression in the activated endothelium and thereby attenuates monocyte to endothelial adhesion and alleviates acute inflammation in the kidney. our findings suggest that hgf might exert its beneficial effects in various disease models at least in part through its potent anti-inflammatory action on vascular endothelium. cell culture huvec were purchased from vec technologies (rensselaer, ny, usa) and maintained in mcdb-131 complete media. huvec cells (2-8 passages) were seeded on gelatin (1.5%) coated cultures at approximately 80% confluence. after growth for 24 h in complete media, cells underwent serum starvation for 6 h in medium 199. human recombinant hgf (genentech, south san francisco, ca, usa) and human recombinant tnf-a (r&d systems, minneapolis, mn, usa) were added to the culture with fresh serum-free medium at a final concentration of 100 and 0.1 ng/ml respectively, or as otherwise indicated. in experiments with gsk3 blockade, the general apoptosis inhibitor boc-asp-ch 2 f (enzyme systems products, dublin, ca, usa) was added to the culture at the final concentration of 50 mm to reduce cell loss due to induction of apoptosis. 27 cell viability was assessed by trypan blue exclusion. human monocytes (thp-1) and ram were purchased from american type culture collection (atcc, manassas, va, usa) and were cultured in suspension respectively in rpmi supplemented with 10% fetal bovine serum and ham's f12k containing 15% fetal bovine serum. for fluorescent viable labeling, thp-1 and ram cells (1 â 10 7 ) were incubated in their respective media containing 5 mg/ ml calcein-am (molecular probes, eugene, or, usa) at 371c for 30 min. excess dye was removed by washing three times with phosphate-buffered saline. adhesion studies were performed with the thp-1 cells under static conditions. 25 briefly, huvec monolayers with equal cell numbers in 12-well plates were treated with hgf and/or tnf-a for 4 h. after addition of 1 ml fluorescence-labeled thp-1 cells (1 â 10 6 cells/ml) per well, the plates were incubated for 30 min at 371c. monolayers were gently washed three times with phosphate-buffered saline after incubation. adherent monocytes were lysed with ripa buffer 23 and fluorescent intensity was measured in a spectramax gemini em fluorescence plate reader (molecular devices, sunnyvale, ca, usa) at an excitation wavelength of 485 nm and emission at 530 nm. huvec monolayers adhered with non-labeled thp-1 cells served as negative controls. the expression vectors encoding the ha-tagged wild-type (wt-gsk3b-ha/pcdna3) and uninhibitable mutant gsk3b (s9a-gsk3b-ha/pcdna3) were kindly and respectively provided by dr jim woodgett (university of toronto, totonto, ontario, canada) 46 and dr gail vw johnson (university of alabama at birmingham, birmingham, al, usa). 30 huvec cells were transfected by electroporation using the amaxa huvec nucleofector kit (amaxa gmbh, koeln, germany). after transfection with equal amounts of expression plasmid or empty vector pcdna3 (invitrogen, carlsbad, ca, usa), huvecs were treated as indicated. flow cytometric analysis of e-selectin expression on huvec cells was performed as previously described 24 using the facs flow cytometer (becton dickinson, franklin lakes, nj, usa). primary anti-e-selectin (ctb202) mouse mab (santa cruz biotechnology, santa cruz, ca, usa) and secondary mabs were used at saturating concentrations. isotype-matched primary mab were used as negative controls. the mean fluorescence intensity in negative controls was consistently o10 fluorescence units. indirect immunofluorescence staining was performed as before. 47 briefly, cells cultured on chamber slides were fixed with 4% paraformadehyde. following donkey serum blocking for 30 min, cells were incubated with the specific primary and then secondary antibodies. finally, cells were stained with 4 0 ,6-diamidino-2phenylindole to visualize the nuclei. stained cells were mounted with vectashield mounting medium (vector laboratories, burlingame, ca, usa) and the extent of stained assessed using a fluorescence microscope. male sprague-dawley rats (harlan sprague-dawley, indianapolis, in, usa) with initial weights of 200-250 g were housed in an approved animal care facility and fed standard chow. on the day of study, rats were anesthetized, placed on a heated table to keep constant body temperature, and maintained euvolemic state as described before. 23 a polyethylene catheter was inserted in the left carotid artery as an access for drug administration. after a 45-min equilibration period, hgf (100 mg/kg wt) or an equal volume of vehicle was administrated as a bolus injection into the left carotid artery. after 30 min, a bolus injection of rat tnf-a (2 mg/kg wt) (r&d system) or vehicle was given to stimulate the systemic inflammation. to demonstrate the pro-inflammatory role of eselectin, a rabbit anti-e-selectin antibody (santa cruz biotechnology) or preimmune igg was given prior to tnf-a injection. at 4 h after tnf-a injection, fluorescent-labeled ram cells (1 â 10 4 ) resuspended in normal saline were infused via the carotid artery. rats were killed before or 30 min after ram cell infusion and various organs harvested for further investigation. one portion of the kidney was immediately frozen for cryostat sectioning. to quantify the fluorescent ram cells sequestrated in the kidney, kidney homogenates with equal amount of protein (100 mg) were subjected to fluorometric analysis in a fluorescence plate reader as described above. indirect immunofluoresent staining of e-selectin was carried out on methanol/acetone (1:1) fixed frozen cryostat sections using rabbit polyclonal anti-e-selectin antibody (santa cruz biotechnology). the alexa fluor goat anti-rabbit (molecular probes) was used as secondary antibody. as a negative control, the primary antibody was replaced by nonimmune serum from the same species; no staining occurred. frozen sections were double stained with 4 0 ,6-diamidino-2-phenylindole, counterstained with evan's blue and mounted with vectashield mounting medium. to visualize fluorescent macrophages sequestrated in the tissue, cryostat sections were directly fixed and then subjected to counterstaining. in quantitative immunofluorescence studies, all sections were stained and analyzed at the same time to exclude artifacts due to variable decay of the fluorochrome. sections were examined at â 400 magnification with a nikon microphot-fx fluorescence microscope equipped with a spot ii digital camera. captured images were analyzed with nih image (v1.62). the mean fluorescence intensity was calculated using the arbitrary fluorescence units obtained in 20 random fields per rat in three rats per group. rat kidneys were homogenized and huvec monolayers were lysed in ripa buffer. protein concentrations were determined using a bicinchoninic acid protein assay kit (sigma, st louis, mo, usa). samples with equal amounts of total protein (40-80 mg/ml) were fractionated by 7.5-10% sds-polyacrylamide gels under reducing condition and analyzed by western immunoblot as described previously. 48 the antibodies against e-selection, akt, erk2, p-stat3, stat3, gsk3, and actin were purchased from sata cruz biotechnology and those for p-akt, p-erk1/2, p-gsk3, and ha were purchased from cell signaling technology (beverly, ma, usa). for immunoblot analysis, bands were scanned and the integrated pixel density was determined using a densitometer and the nih image analysis program. all data are expressed as mean7s.d. statistical analysis of the data from multiple groups was performed by anova followed by student-newman-kuels tests. data from two groups were compared by student's t-test. the acute inflammatory reaction points of control in inflammation the human inflammatory response the endothelium in sepsis: source of and a target for inflammation mechanisms of inflammation and leukocyte activation endothelial cell activation in inflammation: lessons from mutant mouse models endothelial-dependent mechanisms in chronic inflammatory leukocyte recruitment endothelial-leukocyte adhesion molecules in human disease leukocyte-endothelial adhesion molecules the role of selectins in inflammation and disease selectins as potential targets of therapeutic intervention in inflammatory diseases protection from ischemia-reperfusion induced severe acute renal failure by blocking e-selectin e-selectin blockade decreases adventitial inflammation and attenuates intimal hyperplasia in rat carotid arteries after balloon injury role of e-selectin in bleomycin induced lung fibrosis in mice administration of an antibody to e-selectin in patients with septic shock postischemic cerebrovascular e-selectin expression mediates tissue injury in murine stroke hepatocyte growth factor: a multifunctional cytokine developmental roles of hgf/sf and its receptor, the c-met tyrosine kinase hepatocyte growth factor and the kidney hepatocyte growth factor: molecular structure and implications for a central role in liver regeneration keratinocyte and hepatocyte growth factors in the lung: roles in lung development, inflammation, and repair peptide growth factors in the intestine hepatocyte growth factor ameliorates renal interstitial inflammation in rat remnant kidney by modulating tubular expression of mcp-1 and rantes flow cytometric determination of e-selectin, vascular cell adhesion molecule-1, and intercellular cell adhesion molecule-1 in formaldehyde-fixed endothelial cell monolayers the tea flavonoid epigallocatechin-3-gallate reduces cytokine-induced vcam-1 expression and monocyte adhesion to endothelial cells the glamour and gloom of glycogen synthase kinase-3 glycogen synthase kinase 3b-mediated apoptosis of primary cortical astrocytes involves inhibition of nuclear factor kb signaling gsk3 inhibitors: development and therapeutic potential requirement for glycogen synthase kinase-3b in cell survival and nf-kb activation primed phosphorylation of tau at thr231 by glycogen synthase kinase 3b (gsk3b) plays a critical role in regulating tau's ability to bind and stabilize microtubules ameliorating effect of hepatocyte growth factor on inflammatory bowel disease in a murine model hepatocyte growth factor treatment ameliorates diarrhea and bowel inflammation in a rat model of inflammatory bowel disease hepatocyte growth factor ameliorates inflammatory bowel disease in a rat model hepatocyte growth factor attenuates airway hyperresponsiveness, inflammation, and remodeling a mechanism for the antiinflammatory effects of corticosteroids: the glucocorticoid receptor regulates leukocyte adhesion to endothelial cells and expression of endothelial-leukocyte adhesion molecule 1 and intercellular adhesion molecule 1 statins inhibit high glucose-mediated neutrophil-endothelial cell adhesion through decreasing surface expression of endothelial adhesion molecules by stimulating production of endothelial nitric oxide pi3k-akt pathway suppresses coagulation and inflammation in endotoxemic mice glycogen synthase kinase 3 (gsk-3) inhibitors as new promising drugs for diabetes, neurodegeneration, cancer, and inflammation the renaissance of gsk3 glycogen synthase kinase-3: properties, functions, and regulation glycogen synthase kinase-3b regulates nf-kb1/p105 stability a model for nf-kb regulation by gsk-3b signal transduction. a cellular rescue team three nf-kb binding sites in the human e-selectin gene required for maximal tumor necrosis factor alpha-induced expression transcriptional arrest of the human e-selectin gene glycogen synthase kinase 3b negatively regulates both dna-binding and transcriptional activities of heat shock factor 1 activation of pi3k-akt-gsk3b pathway mediates hepatocyte growth factor inhibition of rantes expression in renal tubular epithelial cells hepatocyte growth factor modulates matrix metalloproteinases and plasminogen activator/plasmin proteolytic pathways in progressive renal interstitial fibrosis this work was supported by the young investigator research fund from rhode island foundation for health (r gong), national institutes of health grant ro1-dk52314 (ld dworkin) and at001465-01a2 (a rifai). key: cord-297128-s5c9h4lm authors: hong, joung-woo; yang, ga-eun; kim, yoon bum; eom, seok hyun; lew, jae-hwan; kang, hee title: anti-inflammatory activity of cinnamon water extract in vivo and in vitro lps-induced models date: 2012-11-28 journal: bmc complement altern med doi: 10.1186/1472-6882-12-237 sha: doc_id: 297128 cord_uid: s5c9h4lm background: cinnamon bark is one of the most popular herbal ingredients in traditional oriental medicine and possesses diverse pharmacological activities including anti-bacterial, anti-viral, and anti-cancer properties. the goal of this study is to investigate the in vivo and in vitro inhibitory effect of cinnamon water extract (cwe) on lipopolysaccharide (lps)-induced tumor necrosis factor (tnf)-α and its underlying intracellular mechanisms. methods: cwe was orally administrated to mice for 6 days prior to intraperitoneal injection of lps. serum levels of tnf-α and interleukin (il)-6 were determined 1 hour after lps stimulation. peritoneal macrophages from thioglycollate-injected mice were isolated and assayed for viability, cytokine expression and signaling molecules upon lps stimulation. cwe was further fractioned according to molecular size, and the levels of total polyphenols and biological activities of each fraction were measured. results: the oral administration of cwe to mice significantly decreased the serum levels of tnf-α and il-6. cwe treatment in vitro decreased the mrna expression of tnf-α. cwe blocked the lps-induced degradation of iκbα as well as the activation of jnk, p38 and erk1/2. furthermore, size-based fractionation of cwe showed that the observed inhibitory effect of cwe in vitro occurred in the fraction containing the highest level of total polyphenols. conclusions: treatment with cwe decreased lps-induced tnf-α in serum. in vitro inhibition of tnf-α gene by cwe may occur via the modulation of iκbα degradation and jnk, p38, and erk1/2 activation. our results also indicate that the observed anti-inflammatory action of cwe may originate from the presence of polyphenols. the bark of cinnamon has been used not only as a spice and tea, but also as one of the key components of herbal remedies for the common cold, cardiovascular disease, and chronic gastrointestinal and gynecological disorders in oriental herbal medicine. accordingly, extensive studies on the pharmacological activities of the cinnamon bark have been conducted, indicating that cinnamon bark is involved in a vast range of pathological and physiological events. for instance, essential oil and water-based extracts from cinnamon have been shown to be effective against pathogenic microbes, viruses, and various types of tumor cell lines [1] [2] [3] [4] . furthermore, it has been also reported that cinnamon bark reduces the level of serum glucose through the enhancement of insulin-regulated glucose utilization in vivo [5, 6] . inflammation is a protective response for the purpose of removal of exogenous and endogenous harmful substances produced by injurious stimuli and is a part of the healing process in wounded tissues [7] . since proinflammatory cytokines such as tumor necrosis factoralpha(tnf-α), interleukin(il)-1 and il-6, lipid mediators, proteases, and oxidants produced during the typical response can cause damage to normal tissues regardless of how and where the inflammatory response is triggered, the substances involved in the inflammatory response need to be tightly regulated. if the scavenging reaction is delayed, the inflammatory response may evolve into a variety of chronic inflammatory diseases, such as atherosclerosis, rheumatoid arthritis, asthma, and neurodegenerative diseases. a vast number of molecular studies have identified several target molecules involved in inflammatory changes, and most anti-inflammatory drugs currently used suppress the biosynthesis of the inflammatory mediators mentioned earlier [8] . previous studies have indicated that the major pharmacological activities of cinnamon bark, such as its anti-bacterial, anti-inflammatory, anti-viral, and anticancer effects are derived from essential oils such as cinnamaldehyde [1, [9] [10] [11] . however, since cinnamon bark has been typically used as in the form of a water extract, where the volatile ingredients are seldom found, it is likely that the established pharmacological activities of cinnamon bark depend on a mixture comprised of a variety of water-soluble components, thereby ensuring its safety as a traditional remedy. recently, it has been found that cinnamon bark water extract (cwe) elevates glucose uptake through the promotion of insulin sensitivity and inhibits angiogenesis through blocking vascular endothelial growth factor 2 signaling [12, 13] . these results indicate that the observed pharmacological activities may have originated from polyphenolic compounds in cwe. in this study, we investigated the in vivo and in vitro effects of cwe on lipopolysaccharide (lps)-induced tnf-α and its underlying intracellular mechanisms. we also fractioned cwe according to molecular size to determine whether there exists a positive correlation between the anti-inflammatory activity of cwe and the amount of polyphenolic compounds. cinnamon bark (cinnamomi cassia p resl ) of vietnamese origin was purchased from omni herb (daegu, south korea). the plant was identified by professor choi of the department of herbology at kyung hee university. a voucher specimen sample (cc-2011) was deposited at the laboratory of herbology at kyung hee university. the plant was pulverized and soaked in one volume of water for 48 hours at room temperature, and further dissolved by sonication for 1 hour. the extract was filtered and evaporated using a freeze dryer (eyela, japan) at −70°c. the yield of cwe was about 3.62%. for size fractionation, 1.28 g of cwe was dissolved in 30 ml of distilled water and fractions were collected using 3 kda and 10kda amicon ultra centrifugal filter device (millipore, ireland). the yields of a low molecular weight (mw) fraction (below 3 kda), a middle mw fraction (between 3 kda and 10 kda), and a high mw fraction (over 10 kda) were 60%, 15% and 25% of cwe, respectively. all the final samples were dissolved in pbs and sterilized by passing through a 0.22-μm syringe filter. eight-week-old male balb/c mice were purchased from the korean branch of taconic, samtaco (osan, korea) and fed rodent chow and water ad libitum in a temperature-and humidity-controlled pathogen-free animal facility at the medical center of kyung hee university hospital. mice were maintained in accordance with the guide for the care and use of laboratory animals issued by the us national research council (1996) , and the protocol khmc-iacuc12-006 was approved by the kyung hee university medical center institutional animal care and use committee. in vivo lps injection cwe (20, 100 or 500 mg/kg of body weight) was given to mice via oral gavage for 6 days. control mice received an equal volume of normal saline during the experimental period. each group consisted of 12 mice. on day 7, lps (serotype 055:b5; sigma, st. louis, mo, usa) (1.3 mg/kg) was injected intraperitoneally 1 hour before blood sampling. blood was obtained by cardiac puncture. as a reference drug, dexamethasone (sigma) (5 mg/kg) was injected intraperitoneally 18 hours before the lps injection. blood samples were centrifuged at 800 g for 20 min. the serum samples obtained were stored at −20°c until used. for the use of in vitro culture of macrophages, normal mice were injected intraperitoneally with 2 ml of sterile thioglycollate medium (bd, france), and macrophages were collected three days later by peritoneal gavage with cold dulbecco's modified eagle's medium (dmem). the recovered peritoneal fluid was washed by centrifugation. the cells were resuspended in dmem with 10% fetal bovine serum and incubated for 3 hours at 37°c with 5% co 2 . non-adherent cells were removed. cell viability was determined using the mtt method. macrophages were seeded in 96-well plates and treated with 10, 50, 100, 200, and 400 μg/ml cwe in the presence or absence of lps for 24 hours. ten microliters of mtt solution (5 mg/ml) (sigma) was added to each well and, after 2 hours of incubation, media was aspirated and 100 μl of dimethyl sulfoxide (dmso) (sigma) was added. the optical density was read at 560 nm using a microplate reader (molecular devices, sunnyvale, ca, usa). peritoneal macrophages were seeded in 6-well plates and pre-treated with cwe for 1 hour, then stimulated with 100 ng/ml lps for 4 hours. total rna was isolated using an rneasy mini kit (qiagen, germany) and cdna was reverse-transcribed using superscript iii reverse transcriptase (invitrogen, carlsbad, ca, usa). diluted cdna was mixed with power sybr green pcr master mix (applied biosystems, foster city, ca, usa) and 2 pmol of primers for tnf-α or gapdh and. the following forward and reverse primer sequences were used: tnf-α, forward :5 0 -atg atc gcg gac gtg gaa-30 and reverse: 5 0 -agg gcc tgg agt tct gga a-30; gapdh, forward: 5 0 -ggc atg gac tgt ggt cat ga-3 0 and reverse: 5 0 -ttc acc acc atg gag aag gc-3 0 . amplification of cdna was performed in triplicate using a stepone realtime pcr system (applied biosystems). after an initial heat denaturation at 95°c for 10 min, the pcr conditions were set at 95°c for 15 s and 60°c for 1 min for 40 cycles. for each pcr, a corresponding mrna sample without rt was included as a negative control. quantification of each cdna copy number was determined according to the manufacturer's protocol. the gapdh gene was used as an endogenous control. the levels of cytokines from serum or cell supernatants were measured by enzyme-linked immunosorbent assay (elisa), according to the manufacturer's protocol (bd pharmingen, usa). peritoneal macrophages were seeded and pretreated with cwe for 1 hour and then stimulated with lps for 15 min. cells were rinsed in cold pbs and then lysed on ice in 0.1 ml of ripa buffer (50 mm tris-hcl, ph 7.5; 150 mm nacl; 1 mm edta; 20 mm naf; 0.5% np-40; and 1% triton x-100) containing phosphatase inhibitor cocktail (sigma) and protease inhibitor cocktail (roche diagnostics, mannheim, germany). after centrifugation at 13,000 g for 10 min, supernatants were collected. protein concentrations were determined using the bradford protein assay reagent (bio-rad, usa) and the samples were diluted with 6x sodium dodecyl sulfate(sds) buffer and boiled for 3 min. the samples were separated on a 10% sds-polyacrylamide gel and were transferred to polyvinylidene fluoride membranes. the membranes were blocked with 5% skim milk in tris-buffered saline with 0.1% tween 20 (tbst) for 1 hour. the membranes were incubated with iκbα, ikk, tubulin (santa cruz biotechnology, ca, usa), phospho-ikk, phospho-iκbα, phospho-jnk, jnk, phospho-erk1/2, erk1/2, phospho-p38, and p38 diluted in 5% skim milk in tbst overnight at 4°c. the blots were washed with tbst and incubated for 1 hour with anti-rabbit horseradish peroxidaseconjugated antibodies. immunoreactive bands were visualized by chemiluminescence using ecl (ge healthcare, little chalfont, buckinghamshire, uk), according to the manufacturer's instructions. total polyphenols from cwe and the size-based fractions were determined by folin-ciocalteau (fc) colorimetry as described previously [14] . gallic acid solutions were used for a calibration standard curve. twenty microliters of each fraction or total cwe in 1.58 ml of water was incubated with 100 μl of fc reagent (sigma) for 5 min at room temperature. three hundred microliters of 1.88 m sodium carbonate solution was used to quench the fc reagent-mediated reaction to form chromogens. after reading absorbance at 765 nm, the concentration of polyphenols was calculated as gallic acid equivalent per gram of extract. in vivo data are presented as mean ± sem. statistical differences among the means of multiple groups were determined by using one-way anova followed by the scheffe test. in vitro data are presented as mean ± sd. the difference between the two means was assessed using a non-paired student's t-test. calculations were carried out using spss version 12. p values of less than 0.05 were considered significant. lps is an endotoxin originating from the cell walls of gram-negative bacteria, which stimulates the expression of tnf-α and il-6 in monocytes and macrophages. these cytokines provide protective effects for the body by inducing blood clotting, leukocyte recruitment and activation of adaptive immunity, but a large quantity of such cytokines in serum results in provoking disseminated intravascular coagulation, multiple organ failure or septic shock [15] . first, we tested whether cwe treatment affects systemic inflammatory response to lps stimulation. to this end, cwe was orally administrated to mice for 6 days before intraperitoneal injection of lps and the release of serum tnf-α and il-6 was measured by elisa. based on our previous study, doses of 20, 100, and 500 mg/kg were chosen for oral administration [16] . dexamethasone was used as a reference drug to compare the suppressive activity of cwe in the presence of lps. serum levels of tnf-α were significantly reduced with the 20 and 100 mg/kg doses, while a higher dose (500 mg/kg) showed a lesser reduction than the lower dose points and was not statistical significant ( figure 1 ). this pattern was observed in our previous work in which a dose-dependent reduction in serum ifn-γ occurred only between 20-200 mg/kg of cwe [16] . in the case of il-6, a dose-dependent decrease occurred in the 20 and 100 mg/kg dosage groups although the latter group reached statistical significance. the 500 mg/kg group showed a higher level of il-6 than the control group. it seems that unidentified compounds beyond a critical level may interfere with the antiinflammatory components of cwe. next, we wanted to examine the in vitro effect of cwe in macrophages, which are the major cell source of tnf-α. we used peritoneal macrophages isolated from thioglycollate-injected mice to determine the range of cwe concentration representing no cytotoxic activity using the mtt method. concentrations of cwe up to 400 μg/ml was not cytotoxic to peritoneal macrophages treated with or without lps (figure 2 ). many herbal water extracts contain polysaccharides, which induce the secretion of tnf-α in non-stimulated macrophages in vitro [17] . we found that treatment with cwe alone ranging from 10 to 100 μg/ml stimulated the release of tnf-α into media in a concentration-dependent manner ( figure 3a ). such phenomenon must be due to the presence of water soluble polysaccharides in cwe. subsequently, we examined the effects of cwe on figure 4 in vitro effect of cwe on lps-induced iκbα degradation and map kinase activation. peritoneal macrophages were pretreated with the indicated concentration of cwe for 1 h and then stimulated with lps for 15 min. phospho-ikk, iκbα, phospho-iκbα,phospho-jnk, jnk, phospho-p38, p38, phospho-erk1/2, and erk1/2 in whole protein extracts were examined by western blot analysis. tubulin was used as an internal control. one of the five experiments is shown. lps-stimulated tnf-α secretion. there was no apparent decrease in tnf-α secretion by cwe ( figure 3a ). however, tnf-α mrna expression at 4 h after lps challenge was decreased in cells treated with 50 and 100 μg/ml of cwe ( figure 3b ). despite its inhibition of lps-induced tnf-α transcription, the unabated tnf-α levels in supernatant must have been caused by prior exposure of cells to cwe. in vitro effect of cwe on activation of iκbα, jnk, p38 and erk 1/2 upon lps stimulation in the absence of stimuli, iκbα acts as an inhibitor to block the nuclear translocation of nfκb by masking its nuclear localization signal. upon inflammatory stimulation, iκbα is subjected to phosphorylation mediated by an upstream kinase, iκbα kinase (ikk), and is subsequently released from nf-κb, followed by phosphorylation-induced proteosomal degradation [18] . we tested the effect of cwe on iκbα degradation 15 min after lps stimulation. a time course study of iκbα activity in lps-stimulated macrophages shows that phosphorylation of iκbα reaches its peak at 5 min after lps stimulation and then disappears at 15 min while a complete degradation of iκbα occurs within 15 min [19] . as expected, an almost complete loss of phospho-iκbα as well as iκbα was identified in macrophages treated with lps alone (figure 4 ). cwe at doses of 50 and 100 μg/ml inhibited iκbα degradation, although phospho-iκbα was still detected. phosphorylation of ikk was not affected. these results strongly indicate that the inhibitory effect of cwe on nf-κb signaling may occur downstream of the phosphorylation of iκbα. mitogen-activated protein (map) kinases resident in the cytoplasm of mammalian cells relay extracellular signals to the nucleus through various signal transduction pathways [20] . jnk, p38, and erk1/2, representing the family of map kinases, play a critical role in lpsinduced cytokine gene expression. we tested whether cwe can inhibit the activation of jnk, p38, and erk1/2 15 min after lps stimulation. jnk phosphorylation was reduced at all concentrations tested and phosphorylation of p38 and erk 1/2 was inhibited at 100 μg/ml of cwe, implying that different constituents of cwe may act on these pathways (figure 4 ). taken together, these results show that such inhibition occurred upstream of the map kinases. inhibitory effect of the cwe fraction containing highmolecular-weight compounds on signaling molecules in lps-stimulated macrophages since the effective oral dose of cwe is relatively low, it is reasonable to speculate that the nature of the active components might be small-molecule compounds. thus we separated cwe into a low mw fraction (below 3 kda), a middle mw fraction (between 3 kda and 10 kda), and a high mw fraction (over 10 kda). since the insulin-like and antioxidant activities of cwe originate from polyphenolic compounds, we wanted to determine the polyphenol content in these fractions [12] . as shown in table 1 , the highest concentration of total polyphenols was observed in the high mw fraction, being more than ten-fold than that of the low or middle mw fraction. next, based on the fraction yield ratio relative to the total cwe, 120 μg/ml, 30 μg/ml and 50 μg/ml of the low, middle and high mw fractions were added to macrophages and their effects on signaling molecules were examined. suppression of the degradation of iκbα and phosphorylation of jnk, p38 and erk 1/2 was detected only in cells treated with the high mw fraction ( figure 5 ). these findings indicate that the inhibitory activity of cwe in vitro is derived from the polyphenol-rich fraction. in this study, we present in vivo and in vitro evidence that cwe inhibits expression of tnf-α. in addition, lps-induced iκbα degradation and map kinase phosphorylation in macrophages was strongly inhibited by the polyphenol-rich cwe fraction. macrophages are phagocytic cells that play a critical role in clearing foreign materials, invading bacteria and cellular debris produced by tissue injuries [21] . phagocytes such as macrophages contain a variety of pattern-recognition receptors (prrs), which specifically recognize foreign organisms and modified self ligand. toll-like receptors (tlrs), complement receptor 3 and scavenger receptors are affiliated members of the prr family. among them, lps uses tlr-mediated signaling pathways such as nf-κb and map kinases to stimulate tnf-α and il-6 in macrophages. oral administration of cwe decreased serum levels of lps-induced tnf-α and il-6, but such anti-inflammatory activity was attenuated in the high dose group. in the clinical setting, cwe is used in combination with other herbal agents and thus different results could be produced. however, our experimental data imply that when used singularly the anti-inflammatory activity of cwe is subjected to dose ranges. chronic inflammatory responses found in most autoimmune diseases and metabolic diseases exhibit common characteristic processes where macrophages are initially activated and interferon (ifn)-γ-producing type-1 t helper cells subsequently stimulate macrophages to release more inflammatory cytokines. together with our previous findings that cwe prevented anti-cd3stimulated t cells from secreting ifn-γ, our current study clearly shows that cwe is able to interfere with the chronic activation of macrophages [16] . the inhibitory effect of cwe on the signaling pathways mediated by nf-κb and map kinases occurred in its polyphenol-rich high mw fraction. there is increasing evidence that polyphenols exert anti-inflammatory effects. since cwe is rich in polyphenols such as flavonoids and tannins, the anti-inflammatory effect of cwe may originate partly from polyphenolic compounds. although the high mw fraction accounts for only 25% of the yield of cwe, its polyphenol content is four times more than that of cwe. the high mw fraction may contain polyphenols conjugated with polysaccharides or tannins. procyanidins, known as condensed tannins, consist of oligomer or polymers of (epi)catechin. a higher degree of polymerized procyanidins exhibited stronger inhibition of macrophage activity [22] . therefore, it is conceivable that the polyphenol-rich high mw fraction of cwe may contain the anti-inflammatory compounds that play a major role in suppressing lpsinduced nfκb and map kinase signaling pathways. further study is required to examine whether the macromolecular polyphenols of cwe exert these antiinflammatory effects in animal models. in summary, oral treatment of cwe decreased lpsinduced tnf-α and il-6 release in serum. cwe inhibited iκbα degradation and map kinase activation in lps-stimulated macrophages in vitro. in particular, the inhibitory activity of cwe in vitro occurred in the polyphenol-rich high molecular weight fraction. mechanism of action of spanish oregano, chinese cinnamon, and savory essential oils against cell membranes and walls of escherichia coli o157:h7 and listeria monocytogenes procyanidins and butanol extract of cinnamomi cortex inhibit sars-cov infection cinnamon extract induces tumor cell death through inhibition of nfkappab and ap1 water-soluble polymeric polyphenols from cinnamon inhibit proliferation and alter cell cycle distribution patterns of hematologic tumor cell lines anti-diabetic effect of cinnamon extract on blood glucose in db/db mice cinnamon extract (traditional herb) potentiates in vivo insulin-regulated glucose utilization via enhancing insulin signaling in rats points of control in inflammation targeting innate immunity protein kinase signalling in inflammation suppression effect of cinnamomum cassia bark-derived component on nitric oxide synthase inhibitory effect of cinnamaldehyde, derived from cinnamomi cortex, on the growth of influenza a/pr/8 virus in vitro and in vivo the cinnamon-derived michael acceptor cinnamic aldehyde impairs melanoma cell proliferation, invasiveness, and tumor growth isolation and characterization of polyphenol type-a polymers from cinnamon with insulin-like biological activity novel angiogenesis inhibitory activity in cinnamon extract blocks vegfr2 kinase and downstream signaling screening of dried plant seed extracts for adiponectin production activity and tumor necrosis factor-alpha inhibitory activity on 3t3-l1 adipocytes bench to bedside: tumor necrosis factor-alpha: from inflammation to resuscitation immunomodulatory effect of water extract of cinnamon on anti-cd3-induced cytokine responses and p38, jnk, erk1/2, and stat4 activation botanical polysaccharides: macrophage immunomodulation and therapeutic potential shared principles in nf-kappab signaling distinct role of spleen tyrosine kinase in the early phosphorylation of inhibitor of kappab alpha via activation of the phosphoinositide-3-kinase and akt pathways mammalian mitogen-activated protein kinase signal transduction pathways activated by stress and inflammation the macrophage: past, present and future grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated raw 264.7 macrophages by inhibiting nfkb signaling pathway anti-inflammatory activity of cinnamon water extract in vivo and in vitro lps-induced models the authors have no conflict of interests. key: cord-280599-7ixpqd5n authors: openshaw, p j m title: what does the peripheral blood tell you in sars? date: 2004-04-01 journal: clinical & experimental immunology doi: 10.1111/j.1365-2249.2004.02448.x sha: doc_id: 280599 cord_uid: 7ixpqd5n nan (accepted for publication 18 february 2004) in the winter of 2002-3, physicians in hong kong, singapore and vietnam became alarmed by a mysterious increase in the number of patients admitted with a previously unknown illness characterized by fever, respiratory and gastrointestinal symptoms, now known as severe acute respiratory syndrome (sars). with extraordinary speed, an international cooperative effort resulted in the identification of a novel coronavirus as the cause. the clinical picture was highly suggestive of an abnormal pathological reaction to pulmonary viral infection, characterized by an over-exuberant cascade of immunological events leading to pulmonary inflammation and respiratory failure. the disease was fatal in about 10% of cases and had features reminiscent of adult respiratory distress syndrome (ards) [1] , leading to the suggestion that the pathogenesis might involve an uncontrolled release of immune mediators, a 'cytokine storm'. there was an urgent need to find out more about the disease and why it was so devastating, and taking blood and looking for cytokines in the samples was the obvious approach. the drama of attempting to discover the origins of this new disease, while at the same time trying to save the lives of affected friends and colleagues, has few modern precedents. in this and the previous issue of cei, two papers describe different approaches to measuring cytokines from blood samples from patients with sars. jones et al . [2] studied 13 patients, using elispot analysis to examine the production of seven different cytokines from unstimulated and mitogen-stimulated pbmc. most of the elispot tests showed low or subnormal results compared to 60 normal controls. however, patients with early disease, particularly those destined for poor outcome, had very high numbers of tnf and il-6 producing cells in the blood. the effects of steroid and ribavirin therapy are hard to judge and the study lacks a control group, but the authors comment that patients admitted with bacterial pneumonia showed broadly similar trends. in sars, lymphopenia is marked during the acute phase of the disease, suggesting that cells are marginated or sequestered in the lung and depleted from the periphery. this factor alone makes interpretation of peripheral cellular function during acute, evolving and transient illness very difficult. the situation can be illustrated by an analogy. in the best case, looking at cells in the peripheral blood might be similar to trying to judge a film by watching the people leaving the cinema: if they are people like you, you may well enjoy the film. asking them how they reacted (like re-stimulating lymphocytes) may give an even better view. however, looking at the people who leave while the film is still running may be highly misleading: they clearly do not resemble the crowd who stay behind to watch the film right through to the credits, let alone those who loved it so much they even hang around for the next showing. in the second paper, wong et al . [3] use a different approach to study 20 patients with sars, measuring plasma cytokines on up to 19 consecutive days of illness using a bead-based (cba) elisa assay. they found no evidence of increased tnf-a levels, but did find increases in interferon gamma in addition to a number of other cytokines and chemokines during the two weeks after onset. after steroid therapy, il-8, mcp1 and ip-10 fell rapidly. the tacit assumption underlying studies of this type is that patients with inflammatory disorders have cytokine overspill from inflamed tissues, and that measuring soluble factors in the blood will give immunopathological insights into the local disease processes. moreover, it is hoped (or even expected) that it might be possible to deduce how to treat such patients by finding a specific pattern of disordered cytokine production that could be subjected to selective immunomodulation. however, the reality faced by clinical investigators is that patient populations are heterogeneous, at various stages of the temporal evolution of disease, treated with a range of potent therapies. for example, volunteers infected with influenza have biphasic cytokine levels in the peripheral blood, with il-6 and interferon alpha dominating on day 2, followed by rises in tnf and il-8 during days three to seven [5, 6] . the site of sampling, the exact time after infection and the methods used to measure cytokines are therefore critical determinants with fundamental affects on the interpretation of such studies. these considerations often make it almost impossible to draw firm conclusions, but conclusions are almost irresistible. of clear evidence that plasma tnf levels were high [6] . however, cytokine release is often very local, as illustrated by studies of tnf production in patients with bacterial pneumonia that show tnf levels to be high in bronchial lavage fluid from the affected lung, but not in fluid from the contralateral lung or in serum [4] . perhaps this comparison is unfair, and spill-over of tnf and other cytokines into the serum does occur in some situations such as ards [7] . however, there may even be an inverse correlation between serum and locally produced cytokines, as seen in studies of tnf in the respiratory tract of children with common colds [8] . more importantly, anti-tnf therapy works well in many patients with rheumatoid arthritis or juvenile ra, but measurement of cytokines in serum and synovial fluid does not show raised levels of tnf [9] . therefore, it is clear that raised systemic (or even local) tnf levels are not required for treatment with anti-tnf therapy to work. our ability to monitor disease from measurement of parameters in the peripheral blood is thus of questionable value in determining what is happening in the site of disease or in discovering what type of therapy is likely to produce improvement. even if a particular cytokine is found to be abnormally high, we do not know whether depleting this cytokine would aid recovery, suppress symptoms or lead to uncontrolled multiplication of the pathogen causing the problem in the first place. so, what can we expect to learn by profiling cytokine production or levels in blood samples from patients with inflammatory diseases? possibly, the best we can expect is that finding some combination of cytokine levels might allow earlier diagnosis; in the case of sars, its differentiation from community acquired pneumonia, influenza or other causes of fever which require quite different approaches to patient management. this alone would be of great value, even if the relationship between those findings and the pathogenesis of disease remains uncertain. infections and the inflammatory response in acute respiratory distress syndrome prolonged disturbances of in vitro cytokine production in patients with severe acute respiratory syndrome (sars) treated with ribavirin and steroids plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome inflammatory cytokine profile in children with severe acute respiratory syndrome local and systemic cytokine responses during experimental human influenza a virus infection. relation to symptom formation and host defense compartmentalized cytokine production within the human lung in unilateral pneumonia comparison of systemic cytokine levels in patients with acute respiratory distress syndrome, severe pneumonia, and controls a comparison of cytokine responses in respiratory syncytial virus and influenza a infections in infants cytokine levels in serum and synovial fluid of patients with juvenile rheumatoid arthritis key: cord-282242-5tkhjiwl authors: gómez-laguna, j.; salguero, f.j.; barranco, i.; pallarés, f.j.; rodríguez-gómez, i.m.; bernabé, a.; carrasco, l. title: cytokine expression by macrophages in the lung of pigs infected with the porcine reproductive and respiratory syndrome virus date: 2009-08-19 journal: j comp pathol doi: 10.1016/j.jcpa.2009.07.004 sha: doc_id: 282242 cord_uid: 5tkhjiwl porcine reproductive and respiratory syndrome (prrs) is caused by a virus that predominantly replicates in alveolar macrophages. the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by the prrs virus (prrsv). expression of interleukin (il) 1α, il-6 and tumour necrosis factor (tnf)-α correlated with the severity of pulmonary pathology and the numbers of pulmonary macrophages. significant correlations were observed between prrsv infection and the expression of il-10, between the expression of il-12p40 and interferon (ifn)-γ, and between the expression of tnf-α and ifn-γ. these findings suggest that prrsv modulates the immune response by the up-regulation of il-10, which may in turn reduce expression of cytokines involved in viral clearance (e.g. ifn-α, ifn-γ, il-12p40 and tnf-α). the results also suggest that expression of ifn-γ is stimulated by il-12p40 and tnf-α, but not by ifn-α. all of these cytokines were expressed mainly by septal macrophages with weaker expression by alveolar macrophages, lymphocytes and neutrophils. there appears to be differential activation of septal and alveolar macrophages in prrsv infection, with septal macrophages being the major source of cytokines. macrophages play a significant role in the defence against pathogens by phagocytosis following recognition by surface pattern recognition receptors (prrs), by antigen presentation involving class ii molecules of the major histocompatibility complex (mhc ii) and by production of cytokines (mitchell and kumar, 2004) . cytokines may also be synthesized by several other immune or non-immune cells including lymphocytes, neutrophils and fibroblasts. the expression of cytokines following engagement of prrs by pathogen-associated molecular patterns (pamps) constitutes the main pathway involved in the activation of macrophages (zhang and mosser, 2008) . some cyto-kines may also act as inhibitors of macrophage activation. for example, interleukin (il)-12, tumour necrosis factor (tnf)-a, interferon (ifn)-a and ifn-g act as potent activators of macrophages, whereas il-10 inhibits activation of these cells (mitchell and kumar, 2004) . ifn-g and il-12 are classically involved in the subtype of immune response mediated by th1 lymphocytes, with both cytokines working in parallel (biron and sen, 2001) . the proinflammatory cytokines, including il-1a, tnf-a and il-6, are of greatest importance during the innate immune response (biron and sen, 2001) . ifn-a also participates in the innate response through antiviral activity, by inducing the differentiation of na€ ıve t cells into ifng secreting effector cells and by down-regulation of il-12 expression (biron and sen, 2001; tizard, 2008) . in contrast, il-10 is considered to be an immunosuppressive cytokine as it down-regulates the expression of several other cytokines including il-1a, tnf-a, il-6, il-10 itself, il-12 and ifn-g (biron and sen, 2001; moore et al., 2001) . porcine reproductive and respiratory syndrome (prrs) is one of the most economically significant diseases of the swine industry (neumann et al., 2005) . the syndrome is characterized by interstitial pneumonia in growing pigs and reproductive failure in gilts (rossow, 1998) . prrs is caused by a positive-strand enveloped rna virus, known as prrs virus (prrsv), which belongs to the family arteriviridae in the order nidovirales (fauquet et al., 2005) . prrsv replicates mainly in porcine alveolar macrophages and, to a lesser extent, in monocytes and dendritic cells (molitor et al., 1997; bautista and molitor, 1999) . several studies have examined the role of cytokines in the pathogenesis of prrs (van reeth and nauwynck, 2000) ; however, it is not clear how cytokines participate in macrophage activation during prrsv infection or how they regulate development of the immune response to the virus. thanawongnuwech et al. (2003) suggested that expression of ifn-g by macrophages and lymphocytes may have an inhibitory effect on the replication of prrsv. another study of a prrsv modified-live vaccine has shown that upregulation of il-10 expression is associated with a lower number of ifn-g secreting cells amongst peripheral blood mononuclear cells (pbmcs) (dı´az et al., 2006) . the role of cytokines in the interstitial pneumonia described in prrs has not yet been determined. accordingly, the aim of the present study was to characterize the production of cytokines by subpopulations of pulmonary macrophages in pigs infected by prrsv. thirty-two specific pathogen free, 5-week-old pigs from a prrsv seronegative farm were used in this study. twenty eight animals were randomly assigned to groups of four and inoculated by the intramuscular route with 1 ml of the third passage of prrsv field isolate 2982 (kindly provided by dr. e. mateu) at 10 3.0 tcid 50 . the virus was initially isolated in porcine alveolar macrophages from the serum of naturally infected piglets during a respiratory outbreak of prrs affecting a spanish farm. this field isolate has an open reading frame-5 sequence similarity of 93% with lelystad virus (genbank accession number ef429108). the inoculated animals were killed at 3, 7, 10, 14, 17, 21 and 24 days post-inoculation (dpi). another group of four pigs were sham-inoculated controls. these animals were injected intramuscularly with 1 ml of sterile rpmi 1640 medium and killed at the end of the study (24 dpi). all animals were sedated with tiletamine-zolazepam (zoletilô; virbac, barcelona, spain) followed by a lethal dose of 5% sodium thiopental (thiovetô; vet limited, leyland, lancashire). the experiment was carried out according to the guidelines of the european union (directive 86/609/eec) and was approved by the local ethical committee of centro de investigacio´n en sanidad animal (cisa-inia; valdeolmos, madrid, spain). the pigs were monitored daily for clinical signs, including rectal temperature and a clinical respiratory score, as previously described (halbur et al., 1995) . during post-mortem examination, gross lung lesions were evaluated by visual inspection and each lung lobe was scored to reflect the approximate volume or percentage of the lung tissue affected (halbur et al., 1995) . samples from the medial lobe of the right lung were fixed in 10% neutral buffered formalin and in bouin's solution, processed routinely and embedded in paraffin-wax. sections (4 mm) of formalinfixed tissue were stained with haematoxylin and eosin (he) for microscopical examination. since prrsv is most frequently detected in the apical and medial lung lobes (halbur et al., 1996) , the medial lobe was selected for immunohistochemical examination. the avidinebiotineperoxidase complex technique (abc) was used for the detection of prrsv, macrophages and cytokine proteins as described previously (hsu et al., 1981) . formalin-fixed tissue was used for detection of macrophages and tissue fixed in bouin's solution for all other immunohistochemical reactions. briefly, the sections were dewaxed and dehydrated through graded ethanol and the endogenous peroxidase activity was quenched in h 2 o 2 3% in methanol for 30 min. the sections were washed with phosphate buffered saline (pbs; ph 7.4, 0.01 m) and incubated for 30 min at room temperature with 100 ml per slide of blocking solution in a humid chamber. table 1 describes the primary antibodies and antigen retrieval methods applied. primary antibodies were incubated overnight at 4 c in a humid chamber. in each case the corresponding biotinylated secondary antibody was incubated for 30 min at room temperature. an avidineperoxidase complex (vector laboratories, burlingame, california) was applied for 1 h at room temperature. labelling was 'visualized' by application of the novaredô substrate kit (vector laboratories). sections were counterstained with mayer's haematoxylin, dehydrated and mounted. for negative controls, the primary antibody was replaced by blocking solution, normal serum and isotypematched reagents of irrelevant specificity. the number of labelled cells was determined as described previously (salguero et al., 2005) . briefly, the labelled cells were counted in 50 non-overlapping and consecutively selected high magnification fields of 0.20 mm 2 . results are expressed as the number of cells per mm 2 . immunolabelled cells were identified and counted morphologically as macrophages, lymphocytes or neutrophils. pulmonary intravascular macrophages and interstitial macrophages were grouped together and described as 'septal macrophages'. the numbers of macrophages, prrsv-infected and cytokine-expressing cells were expressed as a mean ae sd. these values were evaluated for approximate normality of distribution by the kolmo-gorovesmirnov test. differences between the means of control and inoculated animals were assessed by the kruskalewallis test followed by the manne whitney-u non-parametric test (graphpad instat 3.05, san diego, california). correlation between the presence of lung lesions and the expression of virus, macrophages and cytokines was assessed by the spearman test (graphpad instat 3.05). p < 0.05 was considered to represent a statistically significant difference. control animals did not display clinical signs or significant gross or microscopical lung lesions. although inoculated animals displayed no significant respiratory distress, they did develop dullness, weight loss and mild hyperthermia from 3 dpi. from 7 dpi until the end of the study, almost 50% of the pulmonary parenchyma of the inoculated animals was affected by interstitial pneumonia, and this was confirmed by microscopical examination of the tissue samples (figs. 1a, 2a). mac387 antibody defined macrophages in sections of lung tissue. the total number of macrophages increased in the lung of inoculated animals from 7 dpi onwards (fig. 1b) . this related primarily to an increase in the number of septal macrophages (figs. 1b and 2b). the number of alveolar macrophages decreased to 7 dpi and recovered thereafter (fig. 1b) . the number of macrophages, as determined by expression of mac387, was significantly correlated with the microscopical score of lung lesions (r ¼ 0.85; p < 0.05) ( table 2) . there was no expression of prrsv antigen in the lung of control animals. prrsv antigen was detected in the lung of infected pigs from 3 dpi until the end of the study, peaking at 7 dpi (fig. 1c) . this antigen expression was detected mainly in the cytoplasm of macrophages, and was significantly higher in alveolar macrophages than in septal macrophages (p < 0.05) (figs. 1c, 2c and 4a ). immunolabelled cells were observed not only in areas of interstitial pneumonia, but also in lung parenchyma without lesions. il-1a was observed in the cytoplasm of alveolar and septal macrophages and neutrophils, the latter appearing to be a significant source of this cytokine (fig. 1d) . expression of il-1a was always higher in inoculated animals than in controls, and had a bimodal peak at 7 and 14 dpi (p < 0.05) (fig. 1d) . the increase in il-1a at 14 dpi was attributed primarily to neutrophils (p < 0.05) (figs. 1d and 2d ). prrsv, il-1a, il-6 and tnf-a respectively. *indicates statistically significant differences (p < 0.05) between the inoculated and control group. **indicates statistically significant differences (p < 0.05) between the numbers of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. expression of il-6 and tnf-a also had a bimodal peak at 7 and 14 dpi (p < 0.05) (fig. 1e and f) . il-6 expression remained elevated until the end of the study (fig. 1e ), but the expression of tnf-a was no different to that of control animals from 21 dpi (fig. 1f ). septal macrophages were the main cell population involved in the expression of both il-6 and tnf-a (p < 0.05) (figs. 1e, f, 2e and f). alveolar macrophages and lymphocytes also expressed these cytokines, but to a lesser extent ( fig. 1e and f) . the labelling of proinflammatory cytokines was observed mainly in areas of interstitial pneumonia with moderate to severe thickening of the alveolar septa. few immunolabelled cells were observed in areas of the lung without lesions (fig. 2def) . the correlation between the lung lesion score, macrophage count and expression of proinflammatory cytokines is shown in table 2 . table 3 shows the correlation between the expression of tnf-a and ifn-g. tissue expression of ifn-a, ifn-g, il-10 and il-12p40 ifn-a was expressed in the cytoplasm of alveolar and septal macrophages and lymphocytes. septal macrophages were the main cell type involved in the expression of this cytokine, which was significantly increased at 3 dpi (p < 0.05) and decreased thereafter (figs. 3a and 4f). the number of ifn-a-expressing alveolar macrophages was also increased at 3 dpi (p < 0.05). ifn-a expression was always higher in inoculated animals than in controls (fig. 3a) . the expression of ifn-a was significantly correlated with virus expression (r ¼ 0.86; p < 0.05) ( table 3) . the kinetics of labelling for ifn-g and il-12p40 were similar throughout the study (r ¼ 0.95; p < 0.05) (table 3) , with both cytokines peaking at 7 dpi and decreasing thereafter ( fig. 3b and c) . these cytokines were expressed not only mainly by septal macrophages, but also by alveolar macrophages and lymphocytes (fig. 4c, e and g) . inoculated animals always had more ifn-g-expressing cells than controls. the expression of il-10 peaked at 7 dpi and decreased thereafter (fig. 3d ). il-10 was expressed mainly in the cytoplasm of septal macrophages ( fig. 4b and d) . the kinetics of expression of il-10 were significantly correlated with that of the virus (r ¼ 0.77; p < 0.05) ( table 3) . consecutive sections immunolabelled for prrsv antigen, ifn-g and il-10 showed co-localization of ifn-g and prrsv antigen, whereas the expression of il-10 occurred in areas without expression of ifn-g (fig. 4aec) . the number of septal macrophages expressing these cytokines was always greater than the number of labelled alveolar macrophages (fig. 3) . immunolabelling for ifn-a, ifn-g, il-12p40 and il-10 was associated with areas of mild to moderate interstitial pneumonia and was much less in areas of pulmonary parenchyma without lesions (fig. 4) . the correlations between the expression of prrsv, ifn-a, ifn-g, il-10, il-12p40 and tnf-a in the lung of prrsv-infected pigs are shown in table 3 . several reports have described changes in cytokine expression during prrsv infection, but these have not addressed local cytokine production within pulmonary lesions. the present study has characterized expression of cytokines by pulmonary macrophages in order to determine the role of these molecules in the pathogenesis of the respiratory form of prrs. the experimental infection did not lead to the animals developing respiratory symptoms, but dullness, weight loss, mild hyperthermia and lesions of the pulmonary parenchyma were observed. prrsv replication peaked at 7 dpi and was mainly localized to alveolar macrophages, which are considered as the target cell for viral replication (molitor et al., 1997; bautista and molitor, 1999) . no correlation was observed between the presence of viral antigen and the severity of the microscopical lung lesions. however, the microscopical lung lesions were significantly correlated with marked inflammatory infiltration of the septa and the number of infiltrating macrophages. moreover, the lung lesions showed significant correlation with the expression of both il-1a and il-6, but not of tnf-a, and macrophage counts were correlated with the expression of il-1a and tnf-a, but not of il-6. these observations suggest that il-1a may play a significant role in the development of interstitial pneumonia during prrs. nonetheless, when all the three proinflammatory cytokines were considered, a highly significant correlation was observed between both microscopical pulmonary lesions and macrophage counts. although prrsv replicated mainly in alveolar macrophages, proinflammatory cytokines were expressed mainly by septal macrophages, especially il-6 and tnf-a, from 14 dpi onwards. this fact indicates activation of septal macrophages, which may be induced by the synthesis of cytokines (zhang and mosser, 2008) . similar findings have been reported for other porcine viral diseases, including african swine fever, which triggers activation of interstitial macrophages expressing il-1a and tnf-a after viral replication (carrasco et al., 2002) . in the present study there was marked intra-alveolar infiltration of neutrophils expressing il-1a at 14 dpi. the earlier increase of both il-1a and tnf-a may have been responsible for the induction of this infiltration and the subsequent activation of these cells, since these cytokines are considered as neutrophil-chemoattractant and stimulant agents (van reeth and nauwynck, 2000) . furthermore, il-1a and tnfa may induce the synthesis of il-6 (van reeth and nauwynck, 2000; mitchell and kumar, 2004) ; however, in our study no correlation was observed between the expressions of these cytokines, although the maximum expression of il-6 temporally coincided with higher expression of il-1a and/or tnf-a. isolate 2982. *indicates statistically significant differences (p < 0.05) between the inoculated group and controls. **indicates statistically significant differences (p < 0.05) between the counts of alveolar and septal macrophages at a given time point. pams, alveolar macrophages. the interferons are known to play a significant role in the host immune response against viruses (van reeth and nauwynck, 2000; biron and sen, 2001) . ifn-a participates in the innate immune response and is able to induce synthesis of ifn-g (biron and sen, 2001; tizard, 2008) . in the present study, a significant correlation was observed between prrsv replication and ifn-a expression, suggesting that prrsv directly induces expression of ifn-a by macrophages. however, prrsv induces lower levels of ifn-a when compared with other porcine respiratory viral diseases, such as those caused by swine influenza virus or porcine respiratory coronavirus (van reeth and nauwynck, 2000) , which indicates that ifn-a expression may be insufficient to induce clearance of prrsv. the expression of ifn-g by macrophages and lymphocytes has been previously reported in the lung of prrsv-infected pigs (thanawongnuwech et al., 2003) . in that study, an increase in expression of ifn-g was observed at 10 dpi for infection with highly virulent strains, whereas strains of low virulence induced a higher expression at the end of the study (28 dpi). in the present study, the expression of ifn-g was undulating, showing a peak at 7 dpi, just when prrsv replication was maximal. ifn-g is known to protect macrophages in vitro against prrsv replication (bautista and molitor, 1999) ; however, that viral replication occurred throughout the period of the present study may suggest that in this experimental infection the ifn-g response was not strong enough to eliminate prrsv infection. the production of ifn-g by pulmonary macrophages is induced by the expression of other cytokines including il-12, tnf-a and ifn-a (nguyen and benveniste, 2002; mitchell and kumar, 2004; tizard, 2008) . in the present study there was good correlation between the expression of il-12p40, tnf-a and ifn-g, but poor correlation between expression of ifn-a and ifn-g. therefore, il-12p40 and tnf-a might be the most significant cytokines involved in the induction of synthesis of ifn-g in this experimental infection. royaee et al. (2004) reported correlation between virus-specific ifn-a secreting cells and virus-specific ifn-g secreting cells in pigs vaccinated with an attenuated, modified-live prrsv vaccine. high antigenic and pathogenic differences have been related to european and north american prrsv genotypes, and suggested to occur within a given genotype (halbur et al., 1995; mateu et al., 2003; stadejek et al., 2006) , which may be the cause of the discrepancies between the present study and that of royaee et al. (2004) . despite the expression of ifn-a, ifn-g, il-12p40 and tnf-a, prrsv was still replicating in the lung of infected pigs at the end of the study. il-10 is an immunomodulatory cytokine that is able to inhibit the synthesis and release of other cytokines (biron and sen, 2001; moore et al., 2001) . therefore, the expression of il-10 observed in the present study might be responsible for reduced expression of cytokines such as ifn-a, ifn-g, il-12p40 and tnf-a, that in turn may impair prolonged viral replication in the lung of infected animals. interestingly, the expression of il-10 was observed in areas of lung that showed no expression of ifn-g. moreover, the expression of il-10 was significantly correlated with prrsv replication. these results suggest that prrsv may induce the expression of il-10 and, therefore, the expression of il-10 might inhibit the expression of other cytokines, allowing a prolonged viral replication in the lung. this idea is supported by the observed immunolabelling for il-10 and ifn-g in consecutive sections of the lung and by the correlation observed between the expression of il-10 and ifn-a. in conclusion, the results of the present study indicate that activation of septal and alveolar macrophages differs throughout prrsv infection, and that the septal cells are the main source of cytokines. proinflammatory cytokines are actively expressed at the onset of the interstitial pneumonia and there is direct correlation between this expression and the infiltration of the pulmonary interstitium by macrophages. additionally, prrsv appears able to modulate the local immune response by inducing the expression of il-10 by macrophages, which may in turn reduce the levels of cytokines involved in viral clearance such as ifn-a, ifn-g, il-12p40 and tnf-a. ifn gamma inhibits porcine reproductive and respiratory syndrome virus replication in macrophages interferons and other cytokines african swine fever: expression of interleukin-1 alpha and tumour necrosis factor-alpha by pulmonary intravascular macrophages different european-type vaccines against porcine reproductive and respiratory syndrome virus have different immunological properties and confer different protection to pigs comparison of the antigen distribution of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus comparison of the pathogenicity of two us porcine reproductive and respiratory syndrome virus isolates with that of the lelystad virus use of avidinebiotineperoxidase complex (abc) in immunoperoxidase techniques: a comparison between abc and unlabeled antibody (pap) procedures genetic diversity and phylogenetic analysis of glycoprotein 5 of europeantype porcine reproductive and respiratory virus strains in spain immune diseases immunity to prrsv: double-edged sword interleukin-10 and the interleukin-10 receptor assessment of the economic impact of porcine reproductive and respiratory syndrome on swine production in the united states critical role of tumor necrosis factor-a and nf-kb in interferon-g-induced cd40 expression in microglia/macrophages porcine reproductive and respiratory syndrome deciphering the involvement of innate immune factors in the development of the host response to prrsv vaccination proinflammatory cytokines induce lymphocyte apoptosis in acute african swine fever infection porcine reproductive and respiratory syndrome virus strains of exceptional diversity in eastern europe support the definition of new genetic subtypes cell signaling: cytokines and their receptors proinflammatory cytokines and viral respiratory disease in pigs macrophage activation by endogenous danger signals the authors thank dr. e. mateu (universitat auto`noma de barcelona, spain) for providing the prrsv field isolate 2982 and dr. k. van reeth (universiteit gent, belgium) for her kind gift of f17 mab. j.g-l. was supported by a doctoral grant from the spanish ministry of education and science (ap-2004-0395). this work was supported financially by the spanish ministry of education and science project number agl2006-04146/gan. supplementary data associated with this article can be found, in the online version, at doi:10.1016/j. jcpa.2009.07.004. the authors declare no competing financial interests. key: cord-319121-et957lfl authors: mifflin, lauren; ofengeim, dimitry; yuan, junying title: receptor-interacting protein kinase 1 (ripk1) as a therapeutic target date: 2020-07-15 journal: nat rev drug discov doi: 10.1038/s41573-020-0071-y sha: doc_id: 319121 cord_uid: et957lfl receptor-interacting serine/threonine-protein kinase 1 (ripk1) is a key mediator of cell death and inflammation. the unique hydrophobic pocket in the allosteric regulatory domain of ripk1 has enabled the development of highly selective small-molecule inhibitors of its kinase activity, which have demonstrated safety in preclinical models and clinical trials. potential applications of these ripk1 inhibitors for the treatment of monogenic and polygenic autoimmune, inflammatory, neurodegenerative, ischaemic and acute conditions, such as sepsis, are emerging. this article reviews ripk1 biology and disease-associated mutations in ripk1 signalling pathways, highlighting clinical trials of ripk1 inhibitors and potential strategies to mitigate development challenges. (ripk1) is a master regulator of the cellular decision between pro-survival nf-κb signalling and death in response to a broad set of inflammatory and pro-death stimuli in human diseases 1, 2 . ripk1 kinase activation has been demonstrated in post-mortem human pathological samples of autoimmune and neurodegenerative conditions [3] [4] [5] [6] , and inhibition of ripk1 kinase activity has shown efficacy in a wide range of animal models of human diseases. as tnfr1-mediated ripk1 activation is the most comprehensively characterized paradigm, ripk1 inhibitors were originally considered to primarily offer a small-molecule alternative to anti-tnf antibody therapies for tnf-driven autoimmune conditions. however, as researchers continued to delve into the mechanisms governed by ripk1, it has become apparent that ripk1 inhibitors may offer key therapeutic options that anti-tnf therapies do not: first, ripk1 inhibitors are safe in the central nervous system (cns) as ripk1 kinase does not signal through tnfr2 which has a protective role in the cns 7 ; second, ripk1 participates in a broader set of pro-inflammatory activities than those restricted to tnf 8 ; third, ripk1 is regulated by a distinct set of signalling molecules that are genetically implicated in human autoimmune and autoinflammatory diseases, as discussed below, and thus patient stratification may be important in conducting clinical trials of ripk1 inhibitors. necrostatin-1s (nec-1s) was the first small-molecule inhibitor of ripk1 kinase to be developed and has been widely used to investigate the role of ripk1 in mechanistic studies and animal models of human diseases 1, [8] [9] [10] [11] . broad therapeutic applications of ripk1 inhibitors for the treatment of a wide range of human diseases are being investigated in clinical trials. the peripherally restricted gsk′772 is being developed for peripheral autoimmune diseases, including psoriasis, rheumatoid arthritis (ra) and ulcerative colitis 12-14 . the brain-penetrant dnl747 is in human clinical trial phase ib/iia for amyotrophic lateral sclerosis (als) 15,16 . these trials have laid the groundwork for advancing clinical applications of ripk1 inhibitors. the important role of ripk1 in driving cell death and inflammation, the established safety of inhibiting ripk1 kinase activity in humans and the ability to develop selective small-molecule kinase inhibitors of ripk1 due to the presence of a unique kinase-regulating allosteric pocket are the major factors that have contributed to ripk1's prominence as a therapeutic target. in this review, we outline the current understanding of ripk1 biology in activating cell death and nf-κb signalling, systematically review monogenic and polygenic variants of known ripk1 regulators and discuss how these mutations may contribute to disease pathology. the involvement of ripk1 in sepsis and acute ischaemic conditions is also discussed. we postulate that the improved understanding of genetic and mechanistic data may be beneficial in segmenting patients in clinical trials, particularly for neurodegenerative and inflammatory diseases. finally, we summarize the current state of ripk1 inhibitors in the clinic, including disease indications, small-molecule chemotypes, as well as ripk1 target engagement and pharmacodynamic biomarkers. distinct kinase and scaffold functions of ripk1 ripk1 is a 76-kda protein with an amino-terminal (n-terminal) kinase domain, a carboxy-terminal (c-terminal) death domain and an intermediate domain with a rhim (receptor-interacting protein homotypic interacting motif) that can bind to other nf-κb (nuclear factor κ light chain enhancer of activated b cells). a protein complex whose pathway, which can be activated in response to cytokines, free radicals, viral or bacterial antigens and other stressors, mediates the transcription of genes required for pro-survival and pro-inflammatory signalling. (tumour necrosis factor receptor 1). a type i membrane receptor that contains an intracellular death domain that, when activated, can recruit receptor-interacting serine/threonine-protein kinase 1 (ripk1) and tradd to mediate inflammatory and pro-survival functions through the nf-κb pathway, as well as cell death mediated by ripk1 kinase activity. rhim-containing proteins 2, 17 . whereas the c-terminal death domain mediates homodimerization as well as heterodimerization with other death domain-containing proteins, such as fadd, tnfr1 and fas, the n-terminal kinase domain mediates autophosphorylation in trans to promote its own activation 18, 19 . the scaffold function of ripk1 is essential for mediating pro-survival nf-κb signalling and mouse perinatal survival: ripk1 -/mice are born normally but die at postnatal day 1-3 (ref. 20 ). ripk1 deficiency reduces nf-κb-mediated transcription of pro-survival proteins, such as cellular flicelike inhibitory protein (cflip), ciap1 and a20 (ref. 21 ). the perinatal lethality of ripk1 -/mice can be rescued by inhibition of both apoptotic and regulated necrotic cell death (necroptosis) in ripk1 -/-fadd -/-ripk3 -/-, ripk1 -/-casp8 -/-ripk3 -/or ripk1 -/-fadd -/-mlkl -/mice [22] [23] [24] [25] [26] . in contrast to the phenotype of ripk1 -/mice, mice harbouring ripk1 knock-in kinase dead mutations, including d138n and k45a, or a k584r death domain mutation that blocks the dimerization-mediated activation of ripk1 are normal 19, 27, 28 . in fact, these mutant mice are resistant to various inflammatory and degenerative conditions in mouse models of disease. interestingly, both genetic and pharmacological inhibition of ripk1 kinase activity, such as treatment with nec-1s or gne684 (refs 10,29 ), offer complete resistance against the tnf-induced animal model of septic shock 30, 31, 32 . thus, the kinase activity of ripk1 promotes cell death and inflammation whereas the scaffold function of ripk1 supports postnatal survival. activation of ripk1 kinase mediated by tnfr1 signalling promotes most of the deleterious effects activated by tnf in human disease 2 . in tnf-stimulated cells, the activation of ripk1 is regulated in a transient multimeric complex associated with the intracellular domain of tnfr1, known as complex i (fig. 1) . the recruitment and activity of these proteins determines the balance between inhibited ripk1 kinase activity, where nf-κb pro-survival signalling is activated, and activated ripk1 kinase activity, which leads to inflammation and cell death. mutations that impact the recruitment to complex i or activity of these proteins leads to aberrant ripk1 kinase activity in disease, most commonly autoimmunity or inflammation (box 1). ripk1 and tradd, another death domaincontaining adaptor protein, are rapidly recruited into complex i by binding to the death domain of tnfr1. activation of ripk1 in complex i, as measured by the well-established phosphorylation of s166, can enact either apoptotic or necroptotic forms of cell death 3,9 . in complex i, the activation of ripk1 is determined by a code including complex ubiquitylation, phosphorylation and other modification events on ripk1, which include those directly organized by tradd and those mediated by proteins transcribed and translated downstream of the nf-κb pathway that are also recruited into the complex (for example, a20) 8 . this post-translational ripk1 code, which may be both cell type-specific and stimulus-specific, modulates the extent of ripk1 kinase activation, which in turn determines the mode of cell death. there is an extensive body of literature identifying cell type-specific roles for ripk1 activity using conditional mouse models that we and others have summarized previously, and so do not cover in detail in this article 1, 2 . for example, in certain cell types, such as oligodendrocytes, tnf stimulation alone may promote the activation of ripk1 and cell death 3, 4 ; in other cell types, such as fibroblasts, sustained activation of ripk1 kinase can only be achieved when tnf stimulation is combined with chemical inactivators of ripk1 repressors, such as ciap1/2 (inhibited by sm-164), tak1, tbk1 or ikks, that are both mediators of the nf-κb pathway and inhibitors of ripk1 kinase activation 18, [33] [34] [35] . ubiquitylation of ripk1 in complex i, which is organized by tradd, is essential for suppressing the aberrant activation of ripk1 kinase. tradd recruits traf2 and the e3 ubiquitin ligases ciap1 and ciap2 into complex i to mediate ripk1 k63 ubiquitylation. k63 ubiquitylation of ripk1, in turn, mediates the recruitment and activation of tak1 kinase through the polyubiquitin binding adaptors tab2 and tab3 (ref. 36 ). k63 ubiquitylation of complex i facilitates the recruitment of the lubac complex containing hoip, hoil1 and sharpin, which in turn performs m1 ubiquitylation of ripk1 and tnfr1. m1 ubiquitylation of complex i is important for the recruitment of the trimeric ikk complex through the polyubiquitin-binding adaptor subunit ikkγ/nemo 2,37 , as well as other ubiquitin binding proteins such as abin1 (a20 binding and inhibitor of nf-κb-1) and the kinase tbk1 (refs 18, 38 ). the activation of ripk1 is suppressed by direct inhibitory phosphorylation mediated by tak1, ikkα/β and τβκ1 (ref. 1 ). ciap1 may also mediate k48 ubiquitylation of ripk1 to promote its degradation by the proteasome 39 . in addition, tnfaip3, which is transcriptionally induced by the nf-κb pathway, encodes the ubiquitin editing enzyme a20, which controls the activation of ripk1 by modulating its ubiquitylation pattern 40 . tnf stimulation of fibroblasts derived from either wild-type mice or healthy patients does not lead to cell death owing to the suppression of ripk1 activation in complex i by inhibitory ubiquitylation and phosphorylation. stimulation with tnf in combination with factors that reduce the inhibition of ripk1 leads to necroptosis, ripk1-dependent apoptosis (rda) and inflammation (box 1). in experimental paradigms, ripk1 kinase activity and necroptosis can be activated by treatment with a combination of tnf, cycloheximide (chx), which blocks the activation of nf-κb-mediated transcription and translation, and zvad.fmk, which blocks the activation of caspases 10 . inhibition of caspases strongly sensitizes to ripk1 activation, as the cleavage of ripk1 by casp8 provides an important inhibitory mechanism to block the overactivation of ripk1 (refs [41] [42] [43] ). rda can be activated with a combination of tnf and the tak1 inhibitor (5z)-7-oxozeanol (known as 5z7), or tnf and sm-164, which blocks inhibitory phosphorylation and ubiquitylation of ripk1, respectively, and blocks activation of pro-survival nf-κb signalling 33 . as detailed in this review, these experimental manipulations mimic some aspects of human genetic deficiencies tnf (tumour necrosis factor). a pro-inflammatory cytokine typically secreted by macrophages/monocytes and microglia. tnf signals through two receptors: tnfr1, which mediates cell death and nf-κb signalling; and tnfr2, which mediates non-canonical nf-κb signalling. a transient complex associated with the intracellular domain of tnfr1 upon tnf stimulation that includes ripk1 and many other regulators of nf-κb activation and cell death. www.nature.com/nrd in various regulators of nf-κb activation that lead to aberrant ripk1 kinase activation and provide guidance for possible indications where ripk1 inhibitors may be efficacious (table 1) . mutations in the genes that encode ripk1 and multiple proteins that regulate ripk1 signalling can lead to immune and autoinflammatory diseases. these clinically identified mutations highlight the important role of ripk1 in regulating the innate immune response and provide mechanistic insights into the functional role of ripk1 in disease. the spectrum of immune and autoinflammatory diseases presents as a continuum between autoimmune disorders involving primarily the adaptive immune system and autoinflammatory conditions involving primarily the innate immune system, all of which can be found in diseases involving ripk1. complex i: stimulation of tnfr1 by tnf promotes the formation of an intracellular signalling complex associated with the death domain of trimerized tnfr1 that recruits two death domaincontaining proteins: adaptor protein tradd and receptor-interacting serine/ threonine-protein kinase 1 (ripk1). tradd recruits e3 ubiquitin ligases ciap1/2 and xiap to perform k63 ubiquitylation of complex i, including ripk1 k377 , which in turn recruits the lubac complex, comprised of hoip, hoil1 and sharpin. lubac mediates linear (m1) ubiquitylation of ripk1. deubiquitinase cyld and its adaptor protein spata2 modulate m1/k63 ubiquitylation of ripk1. m1 deubiquitinase otulin activates lubac. k63 ubiquitin chains on ripk1 recruit tab2/3 and tak1. m1 ubiquitin chains on ripk1 recruit the nemo-iκb kinase (ikk) complex, tbk1, a20, abin1 (a20 binding inhibitor of nf-κb-1) and optn. a20 in complex i suppresses the activation of ripk1 kinase. nf-κb activation: activation of tak1 and the ikks promote nf-κb pathway activation to mediate transcription of both proinflammatory and pro-survival genes, including a20, which modulates the ubiquitylation of ripk1 to control its activation, and cellular flice-like inhibitory protein (cflip), which modulates activation of caspase 8 (casp8). ripk1-dependent apoptosis (rda): activation of tnfr1 under a20, abin1, ciap1/2, nemo, tbk1, ikk or tak1-deficient conditions leads to ripk1 kinase activation. activated ripk1 binds to fadd and casp8 to form complex iia and promote activation of caspases and apoptosis. increased levels of a20 promote the activation of ripk1 in complex iia. necroptosis: inhibition of casp8mediated cleavage of ripk1 promotes ripk1 dimerization via the c-terminal death domain, which leads to its activation and the subsequent formation of the necrosome (complex iib) comprised of ripk1, fadd, casp8, ripk3 and mixed-lineage kinase domain-like pseudokinase (mlkl), which in turn executes necroptosis. ripk1-independent apoptosis: when the nf-κb pathway is inhibited, tnf stimulation can promote the formation of a cytosolic complex with fadd and casp8 to mediate apoptosis independent of ripk1. p, phosphate; ripk1i, ripk1 inhibitor; ub, ubiquitin. adapted from ref. 1 , springer nature limited. a form of apoptosis enacted by activated (cleaved) caspase 8 that is independent of ripk1 kinase activity. rare mutations leading to loss of function (lof) and gain of function (gof) of ripk1 have been identified in individuals with immunodeficiencies and autoinflammatory diseases (table 2) . rare biallelic lof mutations in ripk1, including missense, nonsense and frameshift mutations, have been identified in patients with combined immunodeficiency and inflammatory bowel disease (ibd) 44, 45 . whereas ripk1 -/mice die perinatally, patients with lof mutations in ripk1 exhibit paediatric onset of primary immunodeficiency characterized by an increased susceptibility to infections and early-onset ibd 20, 44, 45 . at the cellular level, the loss of ripk1 in human skin fibroblasts impairs activation of the nf-κb pathway, mapks and jun in response to tnf or poly(i:c) and increases the activation of necroptosis mediated by ripk3 and mixedlineage kinase domain-like pseudokinase (mlkl), but not apoptosis 44, 45 . the production of pro-inflammatory cytokines, including tnf, il-6 and il-10, in response to lps stimulation is severely impaired in peripheral blood mononuclear cells (pbmcs) from patients with ripk1 lof compared with control pbmcs 44 . dysregulation of cytokine production and the host defence response towards the gut microbiome likely play a key role in promoting early-onset ibd and progressive polyarthritis in these patients. casp8 inactivates ripk1 by cleaving human and mouse ripk1 after residues d324 and d325, respectively, which separates the ripk1 kinase domain from the intermediate and death domains 46 . mice with a d325a knock-in mutation that prevents cleavage by casp8 die embryonically, and can be rescued by cis-inactivation of ripk1 by d138n mutation, the loss of tnfr1, or inactivating both necroptosis and apoptosis by double knockout of ripk3 and fadd, or mlkl and fadd 31, 47, 48 . rare variants of ripk1, such as d324v and d324h, that block cleavage by casp8 have been identified in individuals with an auto somal dominant autoinflammatory disease 41, 42 . in contrast to the phenotype observed in patients with ripk1 lof mutations, patients with heterozygous noncleavable ripk1 mutations develop autoinflammatory disease characterized by recurrent fevers and lymphadenopathy. marked increases in pro-inflammatory cytokines and chemokines, such as il-6, tnf and interferon-γ (ifnγ), were found in sera from patients. impaired cleavage of ripk1 d324 variants by casp8 hypersensitized patient pbmcs to ripk1 activation, including both apoptosis and necroptosis induced by tnf, which can be blocked by the ripk1 inhibitor nec-1s (ref. 42 ). although pbmcs from patients with non-cleavable ripk1 mutations are more susceptible to inflammatory stimulation, fibroblasts from one such patient showed resistance to necroptosis and ferroptosis and reduced expression of pro-inflammatory cytokines in response to stimuli 42 . considerable changes in the gene expression patterns were found in these fibroblasts, including downregulated expression of ripk1, ripk3 and tnfr1, as well as elevated levels of the anti-oxidative glutathione and genes that offer resistance to ferroptosis. such compensatory gene expression might be necessary to overcome the deleterious effects of non-cleavable ripk1 and allow for patient survival, although these findings need to be expanded to additional patients. together, these data suggest that autoinflammatory disease caused by non-cleavable ripk1 variants may represent a canonical human ripk1 hyperactivating disease that can respond to ripk1 inhibitor treatment. dysregulation of ripk1 signalling is involved in a heter ogeneous group of monogenic immune and autoinflammatory diseases that can present with either episodic or chronic symptoms (table 2) . as many of these disease-associated genes are also involved in regulating nf-κb signalling 49 , some amount of disease patho logy can be attributed to altered nf-κb signalling. interestingly, these genes, including tnfaip3 (encoding a20), tnip1 (encoding abin1), ikbkg (encoding nemo), otulin and members of the lubac complex, are also direct regulators of ripk1 activation (table 1) . thus, exogenous triggers that lead to transient inflammation in healthy subjects may promote sustained inflammation and cell death involving different tissues and organs in individuals with aberrant ripk1 regulation. traditional treatment for autoimmune diseases has focused on managing immune hyperactivity by dampening nonspecific inflammatory responses and immune cell proliferation. however, this approach renders patients vulnerable to opportunistic infections that can be life-threatening. understanding the inflammatory box 1 | conditions that sensitize to ripk1 kinase activation multiple genetic and non-genetic conditions may promote receptor-interacting serine/threonine-protein kinase 1 (ripk1) activation with tnf signalling. • genetic loss-of-function mutations in tnfaip3 as well as changes in the levels of a20 encoded by tnfaip3, which can lead to various autoimmune and inflammatory diseases 50, [201] [202] [203] . • inactivation of apoptotic mediators, such as caspase 8 (casp8) or cellular flice-like inhibitory protein (cflip), which decreases suppression of the ripk1-mediated necroptotic pathway 233, 234 . • rare variants of ripk1, such as d324v and d324h, which block the cleavage of ripk1 by casp8 and lead to an autosomal dominant autoinflammatory disease 41, 42 . • reduction of tbk1, which directly performs inhibitory phosphorylation on ripk1 and leads to neuroinflammation and neurodegeneration in amyotrophic lateral sclerosis (als)/frontotemporal dementia 18, 118 . • reduction of lubac activity in autoimmune and inflammatory diseases by mutations in hoip, hoil1, sharpin and otulin, which leads to reduction of m1 ubiquitylation of ripk1 important for suppressing its kinase activity 65, 66, [68] [69] [70] [71] 73, 74 . • increased accumulation of ripk1 after optn loss, which promotes degeneration of oligodendrocytes and axonal demyelination and neurodegeneration in als 4,117 . • increased levels of reactive oxygen species, which result in the formation of disulfide bonds that promote ripk1 activation 135 . • ageing-related reductions in suppressors of ripk1-regulated pathways, the best characterized of which is the reduction of tak1 seen in ageing human brain samples 18, 235 . • inhibition of proteasomal or lysosomal degradation of ripk1 or other members of cell death complexes, such as ripk3 (refs 6,236,237 ). • ischaemic conditions, which lead to increased activation of ripk1 and deleterious downstream pathways by both apoptosis and necroptosis 159, 238 . www.nature.com/nrd mechanisms regulated by ripk1 may help to develop therapies that can specifically target the disease pathology in these rare diseases. furthermore, understanding the contribution of ripk1 in these rare diseases may also help to elucidate roles for ripk1 in autoimmune and inflammatory diseases that are not genetically linked to ripk1. a20 deficiency. a20, encoded by the tnfaip3 gene, is an inducible ubiquitin-editing enzyme that restricts both toll-like receptor (tlr) and tnf-induced inflammatory responses by regulating the ubiquitylation of key signalling proteins, including ripk1, traf6 and nemo 40 . mouse models with cell lineage-specific a20 deficiency phenocopy different human inflammatory diseases, suggesting an important role for a20 in restricting ripk1 activation in multiple tissues ( showed increased levels of pro-inflammatory cytokines, such as tnf, il-1β and il-6, and demonstrated clinical improvement after treatment with anti-tnf or anti-il-1β therapy. in addition, low-penetrance coding and non-coding variants in tnfaip3 have been suggested to underlie multiple autoimmune diseases, including crohn's disease, psoriasis, ra, type 1 diabetes mellitus and susceptibility to allergy and asthma [52] [53] [54] [55] . reduced a20 expression may even contribute to atopic eczema (atopic dermatitis) 56 , one of the most common inflammatory skin disorders, affecting up to 7% of adults and 25% of children globally. these studies suggest that dysregulated a20 may be a common underlying factor in the pathogenesis of inflammatory diseases. nemo deficiency syndrome. nemo, the scaffolding subunit of the ikk holocomplex comprising ikkα and ikkβ, is critical for regulating activation of the nf-κb pathway during canonical inflammatory responses 57 . nemo contains an n-terminal dimerization domain involved in binding with ikkα and ikkβ, and two distinct ubiquitin binding domains, including a uban domain that preferentially binds to m1 over k63 ubiquitin chains 58 . nemo deficiency syndrome is a complex disease caused by lof or hypomorphic mutations in the x-linked ikbkg gene, which encodes nemo, and includes clinical definitions of incontinentia pigmenti and anhidrotic ectodermal dysplasia with immunodeficiency. a case study demonstrated that anti-tnf therapy (infliximab) successfully treated inflammatory colitis in an infant with nemo deficiency 59 . nemo -/mice and epidermis-specific knockout of nemo (nemo eko ) reproduced incontinentia pigmenti phenotypes, including embryonic lethality in males and inflammatory skin lesions in heterozygous females 60, 61 . the skin inflammation of nemo eko mice can be delayed by crossing them with tnfr1 -/-62 , suggesting that ripk1 kinase may be involved in mediating inflammatory response in incontinentia pigmenti. mutant mice with specific loss of nemo in intestinal epithelial cells (iecs) (nemo iec-ko mice) show apoptosis of paneth cells and colonocytes, and microbiotadriven chronic colitis 63 . the colitis of nemo iec-ko mice was blocked by genetic or pharmacological inhibition of ripk1 kinase activity, suggesting that the pathology in this mutant line may be driven more by ripk1dependent cell death and inflammatory mechanisms than failure to activate nf-κb 30, 63 . taken together, these studies suggest that ripk1 kinase activity may play an important role in colitis and excessive inflammation in nemo syndrome. thus, ripk1 kinase inhibitors may be beneficial for the treatment of colitis and other inflammatory issues in patients deficient in nemo. however, as patients with nemo deficiency syndrome have an inability to mount a successful immune response and are hypersensitive to infection, reminiscent of patients deficient in ripk1, the inability to properly activate the nf-κb pathway may also contribute to the symptoms of nemo deficiency syndrome. otulin deficiency. otulin is the only known deubiquitylating enzyme that specifically removes m1 ubiquitin chains 64 . patients with biallelic hypomorphic mutations in otulin develop a severe form of autoinflammatory disease, known as otulipenia or otulin-related autoinflammatory syndrome (oras) 65, 66 . in otulin-deficient cells, levels of m1 ubiquitylation on multiple target proteins, such as nemo, tnfr1 and ripk1, are generally increased. as activity of the lubac activity complex is negatively regulated by m1 ubiquitylation, otulin-deficient cells have reduced lubac activity 67 . cells derived from patients with otulin deficiencies have a strong inflammatory signature including overproduction of pro-inflammatory cytokines, such as il-1β, il-6, il-18 and ifnγ, in response to lps, tnf or il-1β. these otulin-deficient cells are also sensitized to tnfinduced cell death 68 . consistent with the important role of tnf in this disease, these patients responded well to treatment with anti-tnf antibodies (infliximab) 66 . otulin -/mice and otulin c129a catalytically inactive knock-in mice die during embryonic development 65, 67 . embryonic lethality of the otulin c129a mice can be delayed by ripk1 d138n/d138n or tnfr1 -/-, whereas combined loss of ripk3 and casp8 prolongs survival to the perinatal stage, suggesting that necroptosis is activated when otulin is not functional. the lubac complex is composed of hoip, hoil1 and sharpin. patients identified with lof or hypomorphic mutations in hoip or hoil1 develop immunodeficiencies and autoinflammatory disease characterized by frequent viral and bacterial infections and multi-organ autoinflammation [69] [70] [71] . these hypomorphic mutations in hoip and biallelic biallelic lof immunodeficiency and multi-organ autoinflammation 71 ibd, inflammatory bowel disease; lof, loss of function; oras, otulin-related autoinflammatory syndrome; ripk1, receptor-interacting serine/threonine-protein kinase 1. lof mutations in hoil1 lead to a reduction in levels of the lubac complex driven by a loss of stability. lubac-deficient cells show reduced levels of ikk phosphorylation and compromised nf-κb activation. lubac is critical for suppressing ripk1 activation, and, as a consequence, enhanced ripk1 activity may be responsible for autoinflammatory symptoms in patients harbouring lubac mutations 72 . mice deficient for the gene encoding the lubac regulatory subunit sharpin, also known as chronic proliferative dermatitis mice (cpdm) (sharpin cpdm/cpdm mice), suffer from severe multi-organ inflammation, particularly in the skin with similarity to atopic dermatitis and psoriasis in humans 28, 73 . interestingly, the skin inflammation of cpdm mice can be blocked by reducing or knocking out tnf 74 , which points to a key role for this signalling node in the cpdm phenotype. inhibition of ripk1 by gne684 or ripk1-d138n protects against dermatitis in cpdm mice 28, 30, 75 . polygenic human diseases, including several autoimmune and inflammatory diseases, are associated with mutations or risk variants in multiple genes, which can include regulators of ripk1. when developing ripk1 kinase inhibitors for the treatment of these diseases, patient stratification may be important to identify those with pathogenic activation of ripk1. crohn's disease and ulcerative colitis are the two primary forms of ibd that are both characterized by intestinal inflammation and epithelial cell loss. genome-wide association studies (gwas) of crohn's disease and ulcerative colitis have identified more than 200 loci that are above the thresholds of significance 76 . however, non-genetic factors likely play a major role in conferring susceptibility as the concordance rate in monozygotic twins is only 16% in ulcerative colitis and 30-35% in crohn's disease 77 . tnfaip3, the gene encoding a20, is a recognized risk factor for both crohn's disease and ulcerative colitis. both increased and decreased expression of a20 has been associated with ibd, suggesting the importance of balancing ubiquitin editing activity in complex i. whereas mice with enterocyte-specific a20 deletion are hypersensitive to experimental colitis and tnf-induced epithelial apoptosis 78 , excessive a20 can dimerize, which sensitizes iecs to undergo ripk1-dependent apoptosis when stimulated by tnf 79 (table 3) . iecs with elevated a20 in the mucosa of patients with ibd are associated with casp3 activation. inhibition of ripk1 has been shown to ameliorate pathology in both a20-deficient and elevated conditions 79, 80 . as specific single-nucleotide polymorphisms (snps) in tnfaip3 have been found to be correlated with primary therapeutic response to anti-tnf (infliximab) therapy 81 , changes in intestinal a20 expression may be used as a biomarker for ripk1dependent pathology to stratify patients with ibd for treatment with ripk1 inhibitors. gwas have also identified atg16l1 and other autophagy-related genes as risk factors for ibd 82 . atg16l1 is a component in the macromolecular complex that lipidates lc3/atg8 to promote the formation of the canonical double-membrane autophagosome. atg16l1 t300a , a risk allele associated with ibd susceptibility, introduces a caspase cleavage site that destabilizes the atg16l1 protein product and reduces autophagy in the presence of tnf 83, 84 . atg16l1-deficient intestinal organoids show increased sensitivity to tnf-induced necroptosis, which can be effectively inhibited by nec-1s (ref. 85 ). both atg16l1 iec conditional knockout mice and a20 iec conditional knockout mice show increased severity of hypothermia induced by tnf, which can be blocked by the ripk1 inhibitor gne684 (ref. 30 ). psoriasis. gwas identified more than 60 disease susceptibility regions, including the tnfaip3, tnip1 and rela loci in psoriasis 86 . reduced levels of tnfaip3 nature reviews | drug discovery mrna and a20 protein have been found in the skin of patients with psoriasis, which can be directly correlated to disease severity 87 . furthermore, specific snps in tnfaip3 have also been found to predict the response to anti-tnf therapies 88 , suggesting the predictive value of tnfaip3 in identifying patients likely to respond to a ripk1 inhibitor treatment for psoriasis. tnip1, encoding the abin1 protein, is one of the highest scoring non-mhc genes associated with various autoimmune and autoinflammatory diseases, including psoriasis, psoriatic arthritis, systemic sclerosis and systemic lupus erythematosus (sle) across multiple gwas 54 . abin1, a ubiquitin binding protein with a uban domain that binds preferentially to linear ubiquitin chains, interacts with a20 to negatively regulate the activation of mapk and nf-κb-mediated gene transcription downstream of tnfr1 and tlrs [89] [90] [91] . abin1, which is recruited into the tnfr1 signalling complex by binding linear ubiquitin chains, in turn promotes the recruitment of a20 (ref. 38 ). abin1-deficient cells and a20-deficient cells are both sensitized to ripk1 activation and necroptosis. tnip1 -/mice die embryonically owing to liver necrosis that can be rescued by ripk3 -/or ripk1 d138n/d138n . tnip1 heterozygosity sensitizes cells to a hyperactive antiviral response mediated by an nf-κb-dependent and ripk1-independent mechanism that promotes the expression of pattern recognition receptors, such as tlr3, rig-i and mda5 (ref. 92 ). inhibition of ripk1 blocks the increased production of pro-inflammatory cytokines in tnip1 +/mice in response to viral stimuli. thus, abin1 regulates the innate immune response through the nf-κb pathway, utilizing both ripk1-dependent and independent signalling pathways. inhibition of ripk1 may reduce the exaggerated inflammatory response in patients with tnip1 mutations without affecting their host defence responses. rheumatoid arthritis (ra) is a common inflammatory autoimmune disease characterized by chronic synovial inflammation, joint destruction and systemic complications. the hla-drb1 alleles are the primary genetic risk factor associated with ra. in addition, gwas have identified ~100 non-hla loci, including tnf, associated with ra. gwas data also suggest the involvement of multiple immune cell types, including t cells, b cells, natural killer cells, dendritic cells and monocytes, as well as the pro-inflammatory cytokines produced by these cells, in mediating ra pathogenesis 93 . interestingly, snps and reduced levels of tnfaip3 have been associated with ra 94 . inhibition of ripk1 by gne684 improved pathology in a rodent model of collagen-induced arthritis 30 . is an autoimmune disease of the cns resulting in neurodegeneration that involves the loss of myelinating oligodendrocytes that normally insulate the axons of neurons. tnf is strongly linked with the aetiology of ms 95, 96 . oligodendrocytes can undergo necroptosis in the presence of tnf stimulation alone 3 . in lesions of brain samples from patients with ms or mouse models of ms, activation of ripk1, ripk3 and mlkl and formation of the necrosome were observed 3 . cflip is transcribed in response to nf-κb activation and can suppress the formation of apoptotic signalling complexes 97 . in the lesions from patients with ms, levels of cflip were found to be decreased, which may be the result of chronic neuroinflammation. decreased cflip and reduced levels of activated casp8 in patients with ms, and thus decreased casp8 inactivating cleavage of ripk1, likely explain the increased ripk1 kinase activity and necroptosis seen in ms. genetic (ripk1 d138n/d138n ) and pharmacological (nec-1s) inhibition of ripk1 kinase ameliorated disease pathology, improved animal behaviour, attenuated the production of pro-inflammatory cytokines and decreased recruitment of immune cells in two animal models of ms 3 . another inhibitor of ripk1 kinase developed by takeda also showed efficacy in an animal model of ms 98 . the contribution of peripheral immune cells and microglia to ms has been highlighted by a recent genetic association study using 47,429 ms cases and 68,374 controls 99 . this study identified 551 putative ms susceptibility genes that are highly expressed in cell types involved in adaptive immunity and innate immunity, including tnfaip3, which encodes a20. as ripk1 is highly expressed in macrophages and microglia in experimental autoimmune encephalomyelitis lesions, inhibition of ripk1 may directly reduce inflammatory responses as ripk1 activity is involved in mediating the inflammatory responses in these cells 100, 101 . thus, the beneficial effects of inhibiting ripk1 may include modulation of both inflammation and cell death in susceptible oligodendrocytes, microglia and neurons. this raises the intriguing possibility that ripk1 may act in both a cell-autonomous and a non-cell autonomous manner in ms. as the existing approved ms drugs all target adaptive immunity and do not directly address neuroinflammation that drives progression in ms, ripk1 inhibition may represent a novel therapeutic approach that targets multiple aspects of disease pathology. responses. sle is a chronic condition that affects multiple organs and tissues. multiple independent snps in the tnip1 and tnfaip3 genes are found to be associated with sle [102] [103] [104] . assembly and activation of the necroptotic machinery, including ripk1, has been implicated in netosis, a programmed suicide of neutrophils in tissues involved in mounting normal immune responses against pathogens [105] [106] [107] . netosis results in the formation of neutrophil extracellular traps (nets); this leads to the release of autoantigens from neutrophils and contributes to autoimmune pathology 108 . in several diseases in which nets drive toxicity, inhibiting ripk1 and thereby blocking other components of the necroptosis pathway has shown efficacy in disease models, including autoimmune vasculitis, venous thrombosis and sle [108] [109] [110] . more broadly, toxicity induced by neutrophil www.nature.com/nrd extravasation and net formation has been implicated in widespread diseases, ranging from autoimmune conditions to neuro degenerative diseases where an impaired blood-brain barrier results in aberrant net formation in the brain parenchyma [111] [112] [113] . the canonical and alternative complement pathways are well-characterized and essential contributors to the pathogenesis of diseases such as sle. the activation of complement in disease results in complementdependent cytotoxicity. complement can activate ripk1-ripk3-mlkl-dependent necroptosis as a contributor of complement-dependent cytotoxicity, which can be attenuated by ripk1 inhibitors 114 . furthermore, in some instances of autoimmunity where complement activation drives disease, the complement pathways can be activated by neutrophil netosis, which is also regulated by ripk1 activation 109 . taken together, these findings suggest that ripk1 may be involved in the pathogenesis of neutrophil and complement-mediated autoimmune conditions. given that ripk1 activation and necroptosis have now been genetically and mechanistically linked with human neurodegenerative diseases, there has been a keen interest in developing cns-penetrant ripk1 inhibitors to alter disease progression for diseases such as als, frontotemporal dementia (ftd), ad and other ageing-related neurodegenerative diseases 4,6,18 (fig. 2) . beyond these indications where several independent groups and clinical data have confirmed a role for pathogenic ripk1 kinase activation, limited data suggest that ripk1 activation may also play a role in the pathology of huntington disease and duchenne muscular dystrophy; however, the mechanisms underpinning this have not yet been defined 115, 116 . als is a progressive neurodegenerative disease that affects motor neurons in the brain and the spinal cord. ftd is a group of related neurodegenerative diseases that lead to the progressive degeneration of neurons in the temporal and frontal lobes of the brain, with consequences on social, emotional and language abilities. lof mutations in two genes, optn and tbk1, lead to familial als/ftd 117, 118 . loss of optn, a monogenic cause of als, resulted in activation of ripk1 and necroptosis in the spinal cords of optn -/mice, which was rescued by genetic and pharmacological inactivation of ripk1 kinase 4 . optn -/cells and the spinal cords in optn -/mice show increased levels of total ripk1 protein, due to decreased proteasomal degradation of ripk1 protein. furthermore, gain-of-function mutations have been found in p62 (encoded by sqstm1), another causal als factor that associates with optn; p62 mutants have been found to interact with ripk1 to shift the balance between necroptotic and apoptotic death 119, 120 . tbk1 haploinsufficiency is a monogenic cause of als/ftd 118, 121 . mice lacking both copies of tbk1 show profound ripk1 activation, resulting in embryonic lethality that is rescued by genetic inactivation of ripk1 (ref. 18 ). furthermore, tbk1 is responsible for directly inhibiting ripk1 by phosphorylation at t189; thus, loss of tbk1 activity increases susceptibility to ripk1 activation. age-dependent reduction of tak1 expression in human brains was shown to cooperate with heterozygous loss of tbk1 to promote late-onset als/ftd-like pathology mediated by decreased ripk1 inhibition 18, 33 . the activation of ripk1, ripk3 and mlkl has been found in post-mortem spinal cord samples from patients with als 4 . an ex vivo human co-culture system of astrocytes and motor neurons from patients harbouring a sod1 familial als mutation showed necroptotic death of motor neurons, which could be rescued with nec-1 (ref. 122 ). in addition, a sod1 g93a mouse model of als treated orally with nec-1s showed improvements in survival, motor activity and axonal pathology. taken together, these observations suggest a potential approach to genetic stratification of patients in a disease where ~90% of patients exhibit idiopathic disease without known genetic identifiers. ad is a form of progressive dementia that leads to the irreversible loss of neurons involved in cognitive and executive functions. the neuropathology of ad includes the presence of amyloid plaques and neurofibrillary tangles. chronically sustained neuroinflammation plays an important role in mediating neural dysfunction and neurodegeneration in ad. similar to what is observed in the insoluble fractions of post-mortem human cns samples from patients with ms and als 3,4 , increased activation of ripk1, ripk3 and mlkl was also found in the insoluble fractions of sporadic ad post-mortem brains 5, 6 . a recent study reported the detection of all three activated components of the necrosome machinery, pripk1, pripk3 and pmlkl, in granulovacuolar degeneration lesions in degenerating neurons in ad and preclinical stages of ad pathology 123 . interestingly, the presence of activated necrosome components correlated with neuronal loss in ad-affected brain regions, such as the hippocampal ca1 region and the frontal cortex layer iii, and tau pathology, but not aβ pathology. ripk1 may play an important role in driving neuroinflammation in ad 6 . the activation of ripk1 was shown to mediate the transcriptional induction of cst7, which encodes an endosomal/lysosomal cathepsin inhibitor named cystatin f, in microglia of mouse models of ad. cst7 is a biomarker for disease-associated microglia present in spatial proximity to aβ plaques found in both post-mortem human ad brain samples and in an ad mouse model 124 . inhibition of ripk1 suppresses the expression of a set of genes, such as ch25h, which are altered in microglia in two animal models of ad, in the sod1 g93a model of als and in ageing microglia, which enhance the ability of microglia to uptake and degrade aβ 6 . ch25h is one of the cholesterol metabolism-related genes associated with ad 125 . treatment with nec-1s has been shown to be efficacious in reducing neuro inflammation and cognitive deficits in the app/ps1 aβ-driven mouse model of ad 6 . these results suggest that ripk1 may control a neuroinflammatory pathway involved in multiple neurodegenerative diseases and in ageing. (also known as complex iib). a complex comprising activated ripk1, receptor-interacting serine/threonine-protein kinase 3 (ripk3) and mixedlineage kinase domain-like pseudokinase (mlkl) that enacts necroptosis, a regulated form of necrotic cell death. similar to the loss of oligodendrocytes seen in ms, ripk1 and necroptosis may also drive white matter loss, which has been found to be among the earliest brain pathological changes in ad, preceding the formation of neural fibrillary tangles and amyloid plaques 126 . in addition, age-related changes in brain white matterwhich include the reduction of myelinated fibres and myelin sheath degeneration co-localized with increased ubiquitin deposits -coupled with the presence of neuroinflammation may mediate the age-related cognitive decline seen in elderly individuals without ad 127 . the neuroinflammation and oligodendrocyte degeneration present in normal ageing human brains suggests the possibility of modulating ripk1 kinase to support cognitive function in ageing. in addition, ageing is associated with mitochondrial dysfunction and the subsequent production of reactive oxygen species, the latter of which has shown responsiveness to ripk1 inhibition in animal models 128, 129 . although this area requires significantly more exploration, one elucidated mechanism by which ageing sensitizes to ripk1 activation is through the reduction of tak1 expression in the cns resulting from cerebral hypoperfusion in ageing human brains 18, 130 fig. 2 | mechanisms of ripk1 in neurodegenerative diseases. activation of receptor-interacting serine/threonineprotein kinase 1 (ripk1) in neurodegenerative diseases may promote the death of neurons and oligodendrocytes cell-autonomously, and inflammation in microglia and astrocytes that acts non-cell-autonomously to promote neurodegeneration. neurons and oligodendrocytes: amyotrophic lateral sclerosis (als)/frontotemporal dementia (ftd)associated loss-of-function (lof) mutations in tbk1 and optn and ageing-related reduction in tak1 can promote ripk1-dependent apoptosis and necroptosis, which can be inhibited by ripk1 inhibitors. parkinson disease (pd)associated mutations in opa1 and drp1, als-associated mutations in sod1 and the chemical stressor mptp lead to mitochondrial dysfunction and the production of reactive oxygen species (ros). cytosolic ros can modulate cysteine residues on ripk1, which promotes activation. microglia and astrocytes: activation of ripk1 leads to the production of pro-inflammatory cytokines in microglia. activation of ripk1 in alzheimer disease (ad) leads to increased production of cst7 and ch25h, both of which are associated with the disease-associated microglia phenotype. pd/lysosomal storage disorder (lsd)-associated genes, such as gba and npc1, also result in lysosomal dysfunction, which can promote ripk1 activation, likely by promoting the accumulation of ripk1. als-associated mutations in sod1 are also known to promote mitochondrial dysfunction and ros production, which may promote glial activation of ripk1 in disease. mlkl, mixed-lineage kinase domain-like pseudokinase; p, phosphate; rda, ripk1-dependent apoptosis; ripk1i, ripk1 inhibitor. www.nature.com/nrd ripk1 at s321 may provide a biomarker for ripk1 activation in ageing brains. a non-synonymous variant in sharpin has been identified as a risk variant for late-onset ad 131 . as sharpin-deficient cpdm mice develop tnf-dependent severe dermatitis and multi-organ inflammation that can be blocked upon inhibition of ripk1 kinase 28, 30, 75 , the reduction of sharpin activity might contribute to the risk of developing late-onset ad by reducing m1 ubiquitylation of ripk1 to promote its activation. parkinson disease and lysosomal storage disorders lrrk2, the most common monogenic cause of parkinson disease (pd), which is also upregulated in sporadic disease, has been identified as a sensitizer to ripk1-dependent apoptosis 132, 133 . additionally, in vitro and in vivo models of these diseases suggest that ripk1 can be activated by mitochondrial damage or lysosomal dysfunction. induced pluripotent stem cell-derived dopaminergic neurons from patients harbouring opa1 mutations that sensitize to mitochondrial fragmentation undergo necroptosis, which can be inhibited by ripk1 inhibitors. treatment with nec-1 can also rescue in vivo loss of dopaminergic neurons in mice treated with mptp, a rodent model of pd 134 . as mitochondrial dysfunction is known to produce high levels of reactive oxygen species, it is possible that increased reactive oxygen species are directly responsible for ripk1 activation in pd 135 . consistent with ripk1 activation downstream of lysosomal inhibition seen in ad models, lysosomal dysfunction caused by niemann-pick disease, type c1 and gaucher's disease also results in ripk1-dependent activation of necroptosis 136, 137 . sepsis is a life-threatening condition caused by immune hyperreactivity to viral, bacterial and fungal infections 138 . sepsis is characterized by dysregulated production of pro-inflammatory cytokines (known as a cytokine storm), lymphopenia, coagulopathy, increased vascular permeability and eventual organ failure and death. both genetic inhibition and pharmacological inhibition of ripk1 in animal models have been shown to block tnf-induced sepsis 27, 29 . kinase inhibition of ripk1 leads to robust and highly reproducible protection against sepsis, including attenuation of hypothermia and complete rescue of lethality. these observations suggest that the kinase activity of ripk1 is critical in propagating immune hyperreactivity following infection, which may happen in septic conditions associated with severe pathogen infection. the efficacy of ripk1 inhibitors in sepsis is mechanistically attributable to the reduction of pro-inflammatory cytokines and circulating damage-associated molecular patterns, rescue of increased intestinal or vascular permeability and activation of the clotting cascade in the vascular endothelium compartment 27, 29, 139 . interestingly, the uncontrolled cytokine storm, which occurs in both patients and animal models, is thought to be driven by the interplay of inflammatory signalling and inflammatory cell death 138 . this deleterious cycle could be attenuated through ripk1 inhibition, not only through a blockade of inflammatory response but also by inhibiting necrotic cell death; the ripk1 inhibitor nec-1 has been shown to protect against lung injury in a neonatal model of sepsis induced by bacteria-driven caecal slurry 140 . patients with severe covid-19 caused by the novel sars-cov-2 infection exhibit multiple hallmarks of sepsis with increased plasma pro-inflammatory cytokines, including il-6, ifnγ, ccl2, il-10, g-csf and tnf, coagulation dysfunction and lymphopenia, which has been suggested to be a predictor for the severity of covid-19 (refs 141, 142 ). activation of ripk1 has been shown to promote the production of proinflammatory cytokines, including il-6 and tnf, as seen in patients with non-cleavable ripk1 mutations 42 . ripk1 has also been shown to promote the production of il-6, tnf, il-8 and gm-csf in mice in response to lps or poly(i:c) injection, which mimic bacterial or viral infections, respectively 92, 143 . in addition, ripk1-mediated necroptosis of hiv-infected cd4 + t cells has been implicated in the depletion of t cell populations in patients 144 , suggesting that, more broadly, lymphopenia seen in viral infections such as hiv and sars-cov-2 might be attributable to the activation of ripk1. activation of necroptosis in severe sepsis and septic shock was also supported by the elevated levels of ripk3 in the plasma of these patients 145 . taken together, these data suggest that an ripk1 inhibitor may present a novel therapeutic option to reduce the aberrant hyperinflammatory response and sepsis in the context of both viral and bacterial infections. nec-1 was shown to be protective in the middle cerebral artery occlusion mouse model of stroke 10 , which was the first example of the role of necroptosis in animal models of human diseases. subsequently, multiple in vivo studies confirmed that ripk1 kinase inhibition shows efficacy in middle cerebral artery occlusion models [146] [147] [148] [149] [150] , acute brain trauma 151 and ischaemia-reperfusion injuries in the brain, retina, heart, kidneys and liver 149, [152] [153] [154] [155] [156] . widespread ripk1 activation has been seen in ischaemia-reperfusion models of stroke, with rapid activation of necroptosis in neurons and endothelial cells, which may transition to apoptosis driven by the reduced tak1 levels as the result of cerebral hypoperfusion 130 . although there are compelling in vivo data that ripk1 inhibition is effective at rescuing pathological and behavioural attributes following acute cerebral insults, a ripk1 inhibitor has yet to be advanced into clinical trials for these conditions, which is largely due to broad challenges seen in conducting clinical trials for acute cerebral insults 157 . however, since a ripk1 inhibitor may reduce cell death under both ischaemic and haemorrhagic stroke conditions, the ability to administer a ripk1 inhibitor to stroke patients without differential diagnosis in an ambulance is an advantage that should be considered to reduce neuronal loss and preserve vascular function in the limited time window. involvement of ripk1 has also been suggested in mediating acute kidney injuries induced by cisplatin, gadolinium contrast, sepsis or ischaemia-reperfusion 156, [158] [159] [160] [161] [162] and hepatitis induced by ethanol, lps, acetaminophen or concanavalin a [163] [164] [165] [166] . interestingly, a polymorphism in the tnf promoter at position 238 (known as the tnfa allele) leading to increased levels of tnf is associated with insulin resistance and is prevalent at higher rates in patients with nonalcoholic fatty liver disease 167 . similarly, patients with nonalcoholic steatohepatitis and mice fed high-fat diets also showed increased levels of tnf, ripk3 and mlkl 168 . finally, ischaemia-reperfusion injuries are commonly seen following organ transplant. in mouse models of lung, kidney and heart transplant, inhibition of ripk1 or necroptosis in the transplanted tissue improved allograft survival [169] [170] [171] . clinical development of ripk1 inhibitors ripk1 contains a unique hydrophobic pocket that allosterically regulates the activation of its kinase activity 9, 172 (fig. 3 ). all ripk1 inhibitors described to date bind to this pocket. a phenotypic screen for small molecules that can block necroptosis led to the identification of the first specific inhibitors of ripk1, 5-(1h-indol-3-ylmethyl)-2-thiohydantoins and 5-(1h-indol-3-ylmethyl)hydantoins known as nec-1s 9,10 (table 4) . a subsequent family of benzo xazepinone ripk1 inhibitors was published by glaxo smithkline (gsk), which included gsk′481 and the clinical candidate gsk′772 (refs 173,174 ), which was followed by the identification of a dihydropyrazole chemotype of ripk1 inhibitors including gsk′963 and gsk′547 (refs 175, 176 ). published and patented compounds generated by takeda, genentech and rigel are all from the same benzoxazepinone family as the gsk′481 series [177] [178] [179] . fig. 4 . gsk′772 was the first ripk1 kinase inhibitor to enter phase ia clinical trials, and has subsequently progressed through phase ia, ib and ii clinical trials 173, 174, 180 . this peripherally restricted compound is currently being tested for peripheral autoimmune diseases. following completion of the initial phase ii clinical trials with gsk′772 in ulcerative colitis, ra and psoriasis, phase ib trials in psoriasis were reinitiated at a much higher dose (960 mg versus 60 mg daily) 181 . gsk′772 has continued to show an excellent safety profile in a phase iia clinical trial in mild to moderate plaque psoriasis, but additional data may be needed to demonstrate efficacy in this indication 182 . dnl104 (denali therapeutics) was the first brainpenetrant ripk1 inhibitor advanced into a phase ia clinical trial; although this programme was later discontinued owing to limited post-dosing liver toxicity deemed unrelated to ripk1 inhibition, this study demonstrated the safety of inhibiting ripk1 in the cns 183 . denali therapeutics subsequently initiated a phase ia clinical trial for dnl747, another brain-penetrant ripk1 inhibitor, followed by phase ib/iia trials of this compound in als and ad 184 . as of june 2020, phase ib/iia clinical trials for dnl747 are ongoing for als; however, denali announced that dnl788, a brain-penetrant back-up compound, will be entering phase ia trials in early 2021 (ref. 185 ). due to the progressive, chronic nature of ad and that >95% ripk1 kinase inhibition may be required for this disease, dnl747 was deemed to have potential dose-limiting toxicities that were not seen preclinically with dnl788, although no toxicity has been seen in the clinic to date. sanofi has partnered with denali on ripk1 inhibitors and completed a phase ia clinical trial of the peripherally restricted dnl758 for peripheral autoimmune indications, including ms 184, 186, 187 . rigel has also initiated a phase ia clinical trial targeting autoimmune diseases with r552, a benzoxazepinone presumed to be hydrophilic hydrophobic receptor-interacting serine/threonine-protein kinase 1 (ripk1) contains a unique hydrophobic pocket located between the n terminus and c terminus of the kinase domain, which allosterically regulates kinase activation. all ripk1 inhibitors discovered to date, such as necrostatin-1s (nec-1s) shown here, bind to this pocket and stabilize ripk1 in an inactive conformation (pdb: 4ith). this pocket is created owing to the outward movement of the αc-helix, resulting in the loss of an ionic pair between catalytic lys45 and glu63 of the αc-helix. the other side of the pocket is formed by the dlg motif in the inactive dlg-in conformation (catalytic asp146 facing away from the active centre) and the activation segment, which immediately follows the dlg motif. ser161 residue in the activation segment forms a critical hydrogen bond with the indole of nec-1s. gsk′772 and other benzoxazepinones also extend into the atp binding pocket, which may contribute to the increased affinity. adapted with permission from ref. 172 ad in neurodegeneration; psoriasis, ulcerative colitis and ra in autoimmune disease; and pancreatic ductal adenocarcinoma in oncology [12] [13] [14] [15] [16] 190 . phosphorylation of s166 ripk1 has been established as a biomarker of ripk1 target engagement 3, 9 . in phase ia clinical trials of dnl104, ex vivo assay-based phosphorylation of s166 ripk1 in human pbmcs was used to assess the inhibition of ripk1 kinase 183 , suggesting clini cal feasibility to determine the activation of ripk1 in blood samples for target engagement studies. in the phase ia study of gsk′772, target engagement was also measured in pbmcs ex vivo using a novel immunoassay based on a conformational change that occurred following inhibitor binding to ripk1 (ref. 180 ). production of several pro-inflammatory cytokines and chemokines regulated by ripk1 kinase activity can also be used as biomarkers of ripk1 activation 191 . of these rapidly inducible, secreted factors, gsk selected the chemokines macrophage inflammatory protein 1α (mip1α; also known as ccl3) and mip1β (also known as ccl4) as pharmacodynamic biomarkers downstream of ripk1 activation 180 . gsk′772 dose-dependently reduced mip-1α and mip1β production, which corresponded to their target engagement data. denali presented data indicating that il-1β, il-6 and mcp1 (also known as ccl2) were also ripk1 responsive, demonstrating that dnl747 dose-dependently reduced il-1β in primary human cells 192 . taken together, these studies demonstrate that ripk1 preclinical biology has provided clinically validated biomarkers of ripk1 activation in blood samples, which may also provide value as inclusion criteria to identify patients who will most benefit from ripk1 inhibitor therapy. lof or gof mutations in the gene encoding ripk1 itself, as well as in those encoding multiple regulators of the nf-κb pathway and ripk1 activation, such as tak1, nemo, a20, abin1, otulin and the lubac complex, have been identified in human inflammatory and ageing-related diseases. in addition to these monogenic diseases, dysregulation of ripk1, such as that which occurs on down-regulation or up-regulation of a20, may also be involved in mediating polygenic diseases involving ripk1. the activation of ripk1-mediated neuroinflammation may provide a common basis for the role of ripk1 in human neurodegenerative diseases. although a specific set of genetic associations between ripk1 activation and neurodegenerative diseases has been discovered, the presence of activated ripk1, ripk3 and mlkl in post-mortem human pathological samples seen in ms, als and ad demonstrates the direct relevance of this pathway even in sporadic cases. notably, the role of a20 and cflip, which are transcriptional targets of nf-κb, in suppressing the activation of ripk1 suggests that defects in activation of the nf-κb pathway, in genetic and sporadic settings, may promote the activation of ripk1 from dysregulated a20-mediated ripk1 ubiquitylation modification as well as inactivation of casp8 due to dysregulated cflip expression. phosphorylation of ripk1 s166, as well as downstream events (such as pripk3 and pmlkl) and the increased production of specific pro-inflammatory cytokines (such as ccl2, mip1α/ccl3, mip1β/ccl4, il-6 and tnf), can be used as biomarkers of ripk1 activation. however, a brain-penetrant positron-emission tomography imaging probe for ripk1 kinase activity would be particularly helpful in the development of ripk1 inhibitors for the treatment of neurodegenerative diseases. as pathways downstream of ripk1, including cell death, pro-inflammatory cytokines and nf-κb activation, can be modulated directly by disease pathogenesis, it is important to include biomarkers that specifically measure ripk1 activation when considering a clinical study. it is also important to remember the cell type specificity of ripk1 activation and the genetic context of the diseases, as sustained activation of ripk1 in specific cell types, such as fibroblasts, can only be achieved in certain settings. clinical trials of ripk1 inhibitors in patients are still in their early days, and, to date, limited chemotypes of ripk1 inhibitors with appropriate in vivo properties have been developed. development of selective, potent and safe small-molecule inhibitors of ripk1, biomarkers to reliably measure the clinical efficacy of ripk1 kinase inhibition and patient stratification remain the key challenges facing future clinical development. published online xx xx xxxx this review summarizes what is currently known about the role of ripk1 in neurological diseases regulation of rip1 kinase signalling at the crossroads of inflammation and cell death this paper provides the first evidence for the role of ripk1 in a chronic autoimmune disease with neurodegeneration -ms this paper provides the first genetic connection of ripk1 kinase and necroptosis with als necroptosis activation in alzheimer's disease ripk1 mediates a diseaseassociated microglial response in alzheimer's disease essential protective role of tumor necrosis factor receptor 2 in neurodegeneration targeting ripk1 for the treatment of human diseases identification of rip1 kinase as a specific cellular target of necrostatins this paper isolates multiple necrostatins, including nec-1, and uses necrostatins to define necroptosis as a regulated necrotic cell death mechanism that broke the traditional dogma activity and specificity of necrostatin-1, small-molecule inhibitor of rip1 kinase rip: a novel protein containing a death domain that interacts with fas/apo-1 (cd95) in yeast and causes cell death this paper demonstrates that the decline of tak1 levels in human ageing brains sensitized the activation of ripk1 to promote neuroinflammation and degeneration and the onset of als/ftd in individuals heterozygous for tbk1 death-domain dimerization-mediated activation of ripk1 controls necroptosis and ripk1-dependent apoptosis the death domain kinase rip mediates the tnf-induced nf-κb signal this paper shows that the scaffold function of ripk1 is involved in mediating nf-κb activation necroptosis in development and diseases ripk1 blocks early postnatal lethality mediated by caspase-8 and ripk3 rip1 suppresses innate immune necrotic as well as apoptotic cell death during mammalian parturition ripk1 regulates ripk3-mlkldriven systemic inflammation and emergency hematopoiesis the pseudokinase mlkl and the kinase ripk3 have distinct roles in autoimmune disease caused by loss of death-receptor-induced apoptosis ripk1 maintains epithelial homeostasis by inhibiting apoptosis and necroptosis cutting edge: ripk1 kinase inactive mice are viable and protected from tnf-induced necroptosis in vivo cutting edge: rip1 kinase activity is dispensable for normal development but is a key regulator of inflammation in sharpindeficient mice this paper demonstrates ripk1 activation in human ra and psoriasis samples by ps166 immunochemistry, efficacy of a ripk1 inhibitor in animal disease models of ibd, ra and skin inflammation, and effect of ripk1 inhibitor for pancreatic cancer metastases rip kinase-dependent necrosis drives lethal systemic inflammatory response syndrome cutting edge: ripk1 kinase inactive mice are viable and protected from tnf-induced necroptosis in vivo spata2 regulates the activation of ripk1 by modulating linear ubiquitination regulation of ripk1 activation by tak1-mediated phosphorylation dictates apoptosis and necroptosis tbk1 and ikkε prevent tnf-induced cell death by ripk1 phosphorylation serine 25 phosphorylation inhibits ripk1 kinase-dependent cell death in models of infection and inflammation tab2 and tab3 activate the nf-κb pathway through binding to polyubiquitin chains holding ripk1 on the ubiquitin leash in tnfr1 signaling abin-1 regulates ripk1 activation by linking met1 ubiquitylation with lys63 deubiquitylation in tnf-rsc ubiquitin-mediated regulation of ripk1 kinase activity independent of ikk and mk2 a20-a bipartite ubiquitin editing enzyme with immunoregulatory potential mutations that prevent caspase cleavage of ripk1 cause autoinflammatory disease a dominant autoinflammatory disease caused by non-cleavable variants of ripk1 2020) (ref. 41), this paper reports the discovery of a dominant autoinflammatory human disease caused by non-cleavable ripk1 activity of caspase-8 determines plasticity between cell death pathways biallelic ripk1 mutations in humans cause severe immunodeficiency, arthritis, and intestinal inflammation human ripk1 deficiency causes combined immunodeficiency and inflammatory bowel diseases cleavage of the death domain kinase rip by caspase-8 prompts tnf-induced apoptosis cleavage of ripk1 by caspase-8 is crucial for limiting apoptosis and necroptosis ripk1 can mediate apoptosis in addition to necroptosis during embryonic development nf-κb pathway in autoinflammatory diseases: dysregulation of protein modifications by ubiquitin defines a new category of autoinflammatory diseases loss-of-function mutations in tnfaip3 leading to a20 haploinsufficiency cause an early-onset autoinflammatory disease haploinsufficiency of a20 impairs protein-protein interactome and leads into caspase-8-dependent enhancement of nlrp3 inflammasome activation analysis of 17 autoimmune diseaseassociated variants in type 1 diabetes identifies 6q23/ tnfaip3 as a susceptibility locus sequencing of tnfaip3 and association of variants with multiple autoimmune diseases genome-wide scan reveals association of psoriasis with il-23 and nf-κb pathways farm dust and endotoxin protect against allergy through a20 induction in lung epithelial cells keratinocyte expression of a20/ tnfaip3 controls skin inflammation associated with atopic dermatitis and psoriasis regulation and function of ikk and ikk-related kinases specific recognition of linear ubiquitin chains by nemo is important for nf-κb activation infliximab therapy for inflammatory colitis in an infant with nemo deficiency nemo/ikkγ-deficient mice model incontinentia pigmenti female mice heterozygous for ikkγ/ nemo deficiencies develop a dermatopathy similar to the human x-linked disorder incontinentia pigmenti skin lesion development in a mouse model of incontinentia pigmenti is triggered by nemo deficiency in epidermal keratinocytes and requires tnf signaling nemo prevents rip kinase 1-mediated epithelial cell death and chronic intestinal inflammation by nf-κ-dependent and -independent functions otulin antagonizes lubac signaling by specifically hydrolyzing met1-linked polyubiquitin the deubiquitinase otulin is an essential negative regulator of inflammation and autoimmunity biallelic hypomorphic mutations in a linear deubiquitinase define otulipenia, an early-onset autoinflammatory disease otulin limits cell death and inflammation by deubiquitinating lubac otulin deficiency in oras causes cell type-specific lubac degradation, dysregulated tnf signalling and cell death human hoip and lubac deficiency underlies autoinflammation, immunodeficiency, amylopectinosis, and lymphangiectasia second case of hoip deficiency expands clinical features and defines inflammatory transcriptome regulated by lubac immunodeficiency, autoinflammation and amylopectinosis in humans with inherited hoil-1 and lubac deficiency cell death and inflammation -a vital but dangerous liaison a spontaneous mutation characterized by chronic proliferative dermatitis in c57bl mice linear ubiquitination prevents inflammation and regulates immune signalling rip1 kinase activity is critical for skin inflammation but not for viral propagation host-microbe interactions have shaped the genetic architecture of inflammatory bowel disease epidemiology of inflammatory bowel disease in a german twin cohort: results of a nationwide study enterocyte-specific a20 deficiency sensitizes to tumor necrosis factor-induced toxicity and experimental colitis elevated a20 promotes tnf-induced and ripk1-dependent intestinal epithelial cell death a20 prevents inflammasomedependent arthritis by inhibiting macrophage necroptosis through its znf7 ubiquitin-binding domain a20 controls intestinal homeostasis through cell-specific activities crohn disease: a current perspective on genetics, autophagy and immunity atg16l1 t300a variant decreases selective autophagy resulting in altered cytokine signaling and decreased antibacterial defense a crohn's disease variant in atg16l1 enhances its degradation by caspase 3 autophagy protein atg16l1 prevents necroptosis in the intestinal epithelium large scale meta-analysis characterizes genetic architecture for common psoriasis associated variants low tnfaip3 expression in psoriatic skin promotes disease susceptibility and severity tnfaip3 gene polymorphisms are associated with response to tnf blockade in psoriasis abin-1 is a ubiquitin sensor that restricts cell death and sustains embryonic development de-ubiquitination and ubiquitin ligase domains of a20 downregulate nf-κb signalling molecular recognition of m1-linked ubiquitin chains by native and phosphorylated uban domains abin-1 heterozygosity sensitizes to innate immune response in both ripk1-dependent and ripk1-independent manner integrative analysis of genome-wide association study and expression quantitative trait loci datasets identified various immune cell-related pathways for rheumatoid arthritis functional evaluation of tnfaip3 (a20) in rheumatoid arthritis selective modulation of tnf-tnfrs signaling: insights for multiple sclerosis treatment tnf receptor 1 genetic risk mirrors outcome of anti-tnf therapy in multiple sclerosis ref. 88), this paper provides the first insight that genetic variants in the tnfr1/ripk1 signalling pathway modulate response to anti-tnf therapy in autoimmune disease the flip side of life discovery of 7-oxo-2,4,5,7-tetrahydro-6h-pyrazolo[3,4-c]pyridine derivatives as potent, orally available, and brain-penetrating receptor interacting protein 1 (rip1) kinase inhibitors: analysis of structure-kinetic relationships international multiple sclerosis genetics consortium. multiple sclerosis genomic map implicates peripheral immune cells and microglia in susceptibility caspase blockade induces rip3-mediated programmed necrosis in toll-like receptoractivated microglia necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression a large-scale replication study identifies tnip1, prdm1, jazf1, uhrf1bp1 and il10 as risk loci for systemic lupus erythematosus association of tnfaip3 interacting protein 1, tnip1 with systemic lupus erythematosus in a japanese population: a case-control association study association of a functional variant downstream of tnfaip3 with systemic lupus erythematosus challenges in the characterization of neutrophil extracellular traps: the truth is in the details the pseudokinase mlkl activates pad4-dependent net formation in necroptotic neutrophils necroptosis and neutrophil-associated disorders a role for receptor-interacting protein kinase-1 in neutrophil extracellular trap formation in patients with systemic lupus erythematosus: a preliminary study necroptosis controls net generation and mediates complement activation, endothelial damage, and autoimmune vasculitis activated platelets induce mlkl-driven neutrophil necroptosis and release of neutrophil extracellular traps in venous thrombosis neutrophil extracellular traps (nets) in autoimmune diseases: a comprehensive review neutrophils promote alzheimer's disease-like pathology and cognitive decline via lfa-1 integrin loss of the tight junction proteins occludin and zonula occludens-1 from cerebral vascular endothelium during neutrophilinduced blood-brain barrier breakdown in vivo receptor-interacting protein kinases 1 and 3, and mixed lineage kinase domain-like protein are activated by sublytic complement and participate in complementdependent cytotoxicity necrostatin-1 ameliorates symptoms in r6/2 transgenic mouse model of huntington's disease necroptosis mediates myofibre death in dystrophin-deficient mice mutations of optineurin in amyotrophic lateral sclerosis haploinsufficiency of tbk1 causes familial als and fronto-temporal dementia sqstm1 mutations in familial and sporadic amyotrophic lateral sclerosis the autophagy machinery controls cell death switching between apoptosis and necroptosis exome sequencing in amyotrophic lateral sclerosis identifies risk genes and pathways necroptosis drives motor neuron death in models of both sporadic and familial als necrosome complex detected in granulovacuolar degeneration is associated with neuronal loss in alzheimer's disease a unique microglia type associated with restricting development of alzheimer's disease cholesterol 25-hydroxylase on chromosome 10q is a susceptibility gene for sporadic alzheimer's disease oligodendroglial cells in alzheimer's disease what's behind the decline? the role of white matter in brain aging inhibiting ripk1 limits neuroinflammation and alleviates postoperative cognitive impairments in d-galactose-induced aged mice inhibiting rip1 improves chronic stressinduced cognitive impairments in d-galactose-induced aging mice sequential activation of necroptosis and apoptosis cooperates to mediate vascular and neural pathology in stroke a rare functional variant of sharpin attenuates the inflammatory response and associates with increased risk of late-onset alzheimer's disease lrrk2 activation in idiopathic parkinson's disease regulation of a distinct activated ripk1 intermediate bridging complex i and complex ii in tnfα-mediated apoptosis pharmacological inhibition of necroptosis protects from dopaminergic neuronal cell death in parkinson's disease models rip1 autophosphorylation is promoted by mitochondrial ros and is essential for rip3 recruitment into necrosome necroptosis in niemann-pick disease, type c1: a potential therapeutic target ripk3 as a potential therapeutic target for gaucher's disease sepsis: inflammation is a necessary evil rip kinase 1-dependent endothelial necroptosis underlies systemic inflammatory response syndrome inhibition of necroptosis attenuates lung injury and improves survival in neonatal sepsis clinical features of patients infected with 2019 novel coronavirus in wuhan lymphopenia predicts disease severity of covid-19: a descriptive and predictive study ripk1 and ripk3 kinases promote cell-death-independent inflammation by toll-like receptor 4 necroptosis takes place in human immunodeficiency virus type-1 (hiv-1)-infected cd4 + t lymphocytes necroptosis regulated proteins expression is an early prognostic biomarker in patient with sepsis: a prospective observational study synergistic protective effects of humanin and necrostatin-1 on hypoxia and ischemia/ reperfusion injury necrostatin-1 prevents necroptosis in brains after ischemic stroke via inhibition of ripk1-mediated ripk3/mlkl signaling necrostatin-1 ameliorates intracerebral hemorrhage-induced brain injury in mice through inhibiting rip1/rip3 pathway necrostatin-1 improves long-term functional recovery through protecting oligodendrocyte precursor cells after transient focal cerebral ischemia in mice inhibition of necroptosis rescues sahinduced synaptic impairments in hippocampus via creb-bdnf pathway necrostatin-1 reduces histopathology and improves functional outcome after controlled cortical impact in mice role of necroptosis in autophagy signaling during hepatic ischemia and reperfusion the role of necroptosis in cardiovascular disease necroptosis, a novel form of caspase-independent cell death, contributes to neuronal damage in a retinal ischemia-reperfusion injury model phenytoin inhibits necroptosis ripk3 deficiency or catalytically inactive ripk1 provides greater benefit than mlkl deficiency in mouse models of inflammation and tissue injury challenges in acute ischemic stroke clinical trials programmed necrosis in acute kidney injury rip1 (receptor-interacting protein kinase 1) mediates necroptosis and contributes to renal ischemia/reperfusion injury the rip1-kinase inhibitor necrostatin-1 prevents osmotic nephrosis and contrast-induced aki in mice necroptosis and ferroptosis are alternative cell death pathways that operate in acute kidney failure necrostatin-1 attenuates sepsisassociated acute kidney injury by promoting autophagosome elimination in renal tubular epithelial cells absence of receptor interacting protein kinase 3 prevents ethanol-induced liver injury necrostatin-1 protects against d-galactosamine and lipopolysaccharide-induced hepatic injury by preventing tlr4 and rage signaling necrostatin-1 protects against reactive oxygen species (ros)-induced hepatotoxicity in acetaminophen-induced acute liver failure ripk1 protects from tnfα-mediated liver damage during hepatitis tumor necrosis factor α promoter polymorphisms and insulin resistance in nonalcoholic fatty liver disease necroptosis is a key pathogenic event in human and experimental murine models of non-alcoholic steatohepatitis inhibition of regulated necrosis attenuates receptor-interacting protein kinase 1-mediated ischemia-reperfusion injury after lung transplantation ripk3-mediated necroptosis promotes donor kidney inflammatory injury and reduces allograft survival ripk3-mediated necroptosis regulates cardiac allograft rejection structural basis of rip1 inhibition by necrostatins discovery of a first-in-class receptor interacting protein 1 (rip1) kinase specific clinical candidate (gsk2982772) for the treatment of inflammatory diseases dna-encoded library screening identifies benzo[b][1,4]oxazepin-4-ones as highly potent and monoselective receptor interacting protein 1 kinase inhibitors characterization of gsk′963: a structurally distinct, potent and selective inhibitor of rip1 kinase rip1 kinase drives macrophagemediated adaptive immune tolerance in pancreatic cancer discovery of 7-oxo-2,4,5, 7-tetrahydro-6h-pyrazolo[3,4-c]pyridine derivatives as potent, orally available, and brain-penetrating receptor interacting protein 1 (rip1) kinase inhibitors: analysis of structure-kinetic relationships potent and selective inhibitors of receptor-interacting protein kinase 1 that lack an aromatic back pocket group rip1 inhibitory compounds and methods for making and using the same randomized clinical study of safety, pharmacokinetics, and pharmacodynamics of ripk1 inhibitor gsk2982772 in healthy volunteers 2020) (ref. 183), this paper outlines pharmacokinetic and pharmacodynamic biomarkers used in clinical trials for ripk1 inhibitors response to inhibition of receptorinteracting protein kinase 1 (ripk1) in active plaque psoriasis: a randomized placebo-controlled study dnl104, a centrally penetrant ripk1 inhibitor, inhibits rip1 kinase phosphorylation in a randomized phase i ascending dose study in healthy volunteers denali therapeutics announces positive clinical results with its lead ripk1 inhibitor molecule and intention to initiate patient studies in multiple indications in collaboration with sanofi denali therapeutics provides broad update on its ripk1 program partnered with sanofi denali therapeutics announces that its partner sanofi has commenced dosing of dnl758 in a phase 1 study isoxazolidine derived inhibitors of receptor interacting protein kinase 1 (ripk1) rigel pharmaceuticals provides business update prior to investor & analyst call identification of a rip1 kinase inhibitor clinical candidate (gsk3145095) for the treatment of pancreatic cancer necroptosis promotes cell-autonomous activation of proinflammatory cytokine gene expression develop, defeat degeneration an association between asthma and tnf-308g/a polymorphism: meta-analysis germline mutations in the extracellular domains of the 55kda tnf receptor, tnfr1, define a family of dominantly inherited autoinflammatory syndromes traf1 signaling in human health and disease xiap deficiency is a mendelian cause of late-onset ibd xiap deficiency syndrome in humans association of peli1 polymorphisms in systemic lupus erythematosus susceptibility in a chinese population haploinsufficiency of tab2 causes congenital heart defects in humans whole-genome sequencing reveals important role for tbk1 and optn mutations in frontotemporal lobar degeneration without motor neuron disease infantile onset intractable inflammatory bowel disease due to novel heterozygous mutations in tnfaip3 (a20) multiple polymorphisms in the tnfaip3 region are independently associated with systemic lupus erythematosus novel heterozygous c243y a20/ tnfaip3 gene mutation is responsible for chronic inflammation in autosomal-dominant behcet's disease negative regulation of the nlrp3 inflammasome by a20 protects against arthritis characteristics of a20 gene polymorphisms and clinical significance in patients with rheumatoid arthritis association of tumor necrosis factor α-induced protein 3 interacting protein 1 (tnip1) gene polymorphism (rs7708392) with lupus nephritis in egyptian patients a20 deficiency in b cells enhances b-cell proliferation and results in the development of autoantibodies a20 (tnfaip3) deficiency in myeloid cells triggers erosive polyarthritis resembling rheumatoid arthritis association of two independent functional risk haplotypes in tnip1 with systemic lupus erythematosus abin1 dysfunction as a genetic basis for lupus nephritis genetic correlation between amyotrophic lateral sclerosis and schizophrenia genome-wide meta-analysis identifies novel multiple sclerosis susceptibility loci x-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired nf-κb signaling genomic rearrangement in nemo impairs nf-κb activation and is a cause of incontinentia pigmenti. the international incontinentia pigmenti (ip) consortium ref. 210), this paper identifies mutations in nemo as the cause of incontinentia pigmenti and anhidrotic ectodermal dysplasia with immunodeficiency atypical forms of incontinentia pigmenti in male individuals result from mutations of a cytosine tract in exon 10 of nemo (ikkγ) ikk-related genetic diseases: probing nf-κb functions in humans and other matters caspase-8 deficiency presenting as late-onset multi-organ lymphocytic infiltration with granulomas in two adult siblings the ubiquitin-modifying enzyme a20 restricts ubiquitination of the kinase ripk3 and protects cells from necroptosis a20 deficiency causes spontaneous neuroinflammation in mice keratinocyte-specific ablation of the nf-κb regulatory protein a20 (tnfaip3) reveals a role in the control of epidermal homeostasis a20 protects cells from tnf-induced apoptosis through linear ubiquitin-dependent and -independent mechanisms discovery of small molecule rip1 kinase inhibitors for the treatment of pathologies associated with necroptosis discovery of a highly potent, selective, and metabolically stable inhibitor of receptorinteracting protein 1 (rip1) for the treatment of systemic inflammatory response syndrome 6e11, a highly selective inhibitor of receptor-interacting protein kinase 1, protects cells against cold hypoxia-reoxygenation injury discovery of potent necroptosis inhibitors targeting ripk1 kinase activity for the treatment of inflammatory disorder and cancer metastasis structure guided design of potent and selective ponatinib-based hybrid inhibitors for ripk1 structure-activity relationship analysis of a novel necroptosis inhibitor, necrostatin-5 structure-activity relationship study of a novel necroptosis inhibitor, necrostatin-7 a novel necroptosis inhibitor-necrostatin-21 and its sar study denali therapeutics announces first-in-human dosing of its ripk1 inhibitor clinical program catalytic activity of the caspase-8-flip(l) complex inhibits ripk3-dependent necrosis ciaps block ripoptosome formation, a rip1/caspase-8 containing intracellular cell death complex differentially regulated by cflip isoforms genome-wide analysis reveals mechanisms modulating autophagy in normal brain aging and in alzheimer's disease proteasome inhibition blocks necroptosis by attenuating death complex aggregation lysosomal damage after spinal cord injury causes accumulation of ripk1 and ripk3 proteins and potentiation of necroptosis ischemic insults induce necroptotic cell death in hippocampal neurons through the up-regulation of endogenous rip3 studies in the authors' laboratories (j.y.) are supported by nih grants r21-ag059073, r01-ag047231 and rf1-ag055521. the authors thank q. zhou of zhejiang university, j. lewcock of denali therapeutics and d. vucic of genentech for helpful comments. the authors thank b. toure of nido biosciences for assistance with figures. j.y. conceived the review topic. l.m. and j.y. wrote the manuscript with support from d.o. j.y. is a consultant for denali therapeutics and sanofi. d.o. is an employee of sanofi. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-300991-ipy24zxp authors: khan, amira sayed; hichami, aziz; khan, naim akhtar title: obesity and covid-19: oro-naso-sensory perception date: 2020-07-08 journal: j clin med doi: 10.3390/jcm9072158 sha: doc_id: 300991 cord_uid: ipy24zxp through a recent upsurge of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) pandemic, the clinical assessment of most of the coronavirus disease 19 (covid-19) patients clearly presents a health condition with the loss of oro-naso-sensory (ons) perception, responsible for the detection of flavor and savor. these changes include anosmia and dysgeusia. in some cases, these clinical manifestations appear even before the general flu-like symptoms, e.g., sore throat, thoracic oppression and fever. there is no direct report available on the loss of these chemical senses in obese covid-19 patients. interestingly, obesity has been shown to be associated with low ons cues. these alterations in obese subjects are due to obesity-induced altered expression of olfacto-taste receptors. besides, obesity may further aggravate the sars-cov-2 infection, as this pathology is associated with a high degree of inflammation/immunosuppression and reduced protection against viral infections. hence, obesity represents a great risk factor for sars-cov-2 infection, as it may hide the viral-associated altered ons symptoms, thus leading to a high mortality rate in these subjects. in the month of december 2019, there was an uprising of pneumonia, marked with respiratory distress, among the residents of wuhan district, located in the north-east of china [1] . the virus responsible for this health disaster was identified as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) which belonged to the single-stranded enveloped rna viruses, and the disease was termed as coronavirus disease 2019 (covid-19) [2] . it is surprising that in the beginning of the pandemic, most of the covid-19 patients in wuhan (china) had some primary health problems, including obesity [1] . a recent cohort, conducted in 12 hospitals of the new york state on covid-19 patients, has proposed that there were 41% obese patients, admitted between march 1, 2020 and april 4, 2020 [3] . the incidence of obesity is increasing steadily in all the corners of the world, with 650 million clinically ill subjects requiring either a surgical or medical treatment [4] . the management of obesity has become a challenging task because this pathology is a favorable ground for several chronic diseases, including cardiovascular complications, type-2 diabetes mellitus, cancer, atherosclerosis, arthrosis and renal dysfunction, and respiratory tract infections (rti) in virus-affected patients [5] [6] [7] . the rti are the main physiological targets in covid-19 illness [1] . we would like to recall that during 2009 influenza pandemic, obesity was associated with reduced pulmonary immune defenses against the virus [8] . indeed, obese subjects were not only more prone to infection with the influenza (h1n1) the virus [8] . indeed, obese subjects were not only more prone to infection with the influenza (h1n1) virus, but also developed post-infection severity of illness [9] . an increase in adiposity has been shown to alter the integrity of respiratory epithelium, which might lead to dysfunctional airway fluxes [10] . due to high weight load with excessive pressure on belly and thorax, obesity will contribute to reduced pulmonary gas exchange capacities, such as forced expiratory volume (fev) and forced vital capacity (fvc) . the experiments conducted on mice have suggested that obesity is associated with high lung permeability [11] . epidemiological data confirm that there is an increased rate of pneumonia and rti in covid-19 obese patients [12] . in fact, the first report on rti in obese subjects was published by a french team wherein 47% of covid-19 patients were found to be obese with a high degree (nearly 90%) of artificial ventilation [13] . the marked inflammation leading to immunosuppression in obesity seems to favor viral infections [14] [15] [16] . sheridan et al. [17] observed that high body mass index (bmi) was associated with a high decline in influenza antibody titers and decreased cd8 + t-cell activation after 12 months postvaccination. as far as sars-cov-2 infection is concerned, tan et al. [18] assessed immunological alterations in covid-19 patients, wherein they noted an overall decline in cd4 + t-cells, cd8 + t-cells, b cells and natural killer (nk) cells. moreover, the number of immunosuppressive t-regulatory, treg (cd4 + cd25 + foxp3 + ) cells and concentrations of il-6, il-10, and c-reactive protein (crp) were upregulated in patients with severe covid-19 [18] , suggesting that sars-cov-2 infection may lead to "over-immunosuppression" in the case of obesity ( figure 1 ). the figure shows the immunosuppression in obese subjects. the adipose tissue of the obese is highly inflamed and, consequently, releases a number of cytokines, particularly il-6 and tnf-α. whose secretion is further potentiated by leptin. the lipopolysaccharide (lps)-triggered endotoxemia further aggravates inflammatory condition by inducing the release of il-6 and tnf-α from macrophages via tlr4 activation. obesity is also marked with high production of il-10, which decreases the function of dendritic cells. the prolonged inflammation will lead to immunosuppression that may favor the viral infection. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has also been shown to induce immunosuppression. once installed, sars-cov-2 will aggravate the obesity-induced lung dysfunctions. (+) and (−) show, respectively, stimulatory and inhibitory actions. since dendritic cells (dcs) are the key players in the regulation of th1/th2 dichotomy and t-cell tolerance, their importance to trigger an anti-viral response has been considered primordial [19] . o'shea et al. [20] have demonstrated that obesity impacts the functions of these cells to trigger appropriate t-cell responses. this interesting report further showed that not only the number of circulating dcs were significantly lower in obese participants than lean subjects, but also in vitro activated-dcs from obese participants expressed less cd83 (a dcs maturation marker) and also produced, in high quantities, the il-10, an immunosuppressive cytokine [21] . the il-10, in turn, has the adipose tissue of the obese is highly inflamed and, consequently, releases a number of cytokines, particularly il-6 and tnf-α. whose secretion is further potentiated by leptin. the lipopolysaccharide (lps)-triggered endotoxemia further aggravates inflammatory condition by inducing the release of il-6 and tnf-α from macrophages via tlr4 activation. obesity is also marked with high production of il-10, which decreases the function of dendritic cells. the prolonged inflammation will lead to immunosuppression that may favor the viral infection. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has also been shown to induce immunosuppression. once installed, sars-cov-2 will aggravate the obesity-induced lung dysfunctions. (+) and (−) show, respectively, stimulatory and inhibitory actions. since dendritic cells (dcs) are the key players in the regulation of th1/th2 dichotomy and t-cell tolerance, their importance to trigger an anti-viral response has been considered primordial [19] . o'shea et al. [20] have demonstrated that obesity impacts the functions of these cells to trigger appropriate t-cell responses. this interesting report further showed that not only the number of circulating dcs were significantly lower in obese participants than lean subjects, but also in vitro activated-dcs from obese participants expressed less cd83 (a dcs maturation marker) and also produced, in high quantities, the il-10, an immunosuppressive cytokine [21] . the il-10, in turn, has been shown to inhibit the ability of dcs to stimulate cd4 + t-cells and to downregulate mhc-ii, cd86 (a co-stimulatory signal protein), and antigen presentation to cd4 + t-cells [21] . obesity is also marked with high concentrations of leptin, which is also known to trigger the production of il-6 and tnf-α from adipose tissues ( figure 1 ) and to increase the risk for viral infection. indeed, tnf-α administration in mice favors the induction of an experimental autoimmune disease [22] . the adipose tissue is the main source of circulating tnf-α in obesity, as its synthesis is increased by adipocytes in obese subjects and a weight-loss results in its low concentrations [23] . in obesity, leptin further decreases the secretion of adiponectin, an anti-inflammatory adipokine. in fact, the adipose tissue of obese subjects is an inflammatory "hot spot" that is also infiltrated by macrophages [24] . besides, obesity is also marked with a change in gut microbiota that leaks the entry of lipopolysaccharide (lps) into blood circulation. the lps is directly responsible for endotoxemia, so-called, "low grade inflammation", via toll-like receptor-4 (tlr-4), by inducing the production of il-1β, tnf-α and il-6 from macrophages and, at the same time, some of the adipocytes are also differentiated into "macrophage-like" cells [25] . finally, we can state that il-6 and tnf-α are the main players of inflammation in obesity ( figure 1 ). these two cytokines, along with il-1β via the nf-kb pathway, have been proposed to be the major cause of immunosuppression [26] as they induce accumulation and activation of myeloid-derived suppressor cells (mdscs) whose expansion interrupts the maturation of macrophages, dcs and granulocytes [27] . obesity is also associated with other immunosuppressive landmarks, such as low lymphocyte subset counts and their decreased polyclonal proliferation and oxidative burst activity of monocytes, increased thymic aging, and reduced t-cell repertoire diversity, which lead to increased risk for viral infections and rti both in experimental models and clinical studies [28] . luzi and radaelli [29] have proposed that there would be high viral shedding in obese subjects, thus increasing the probabilities of spreading the viral infection. it is also noteworthy that obesity, complicated by diabetes, may further aggravate the patient's health status. indeed, bello-chavolla et al. [30] have tried to establish a link between obesity and diabetic condition in sars-cov-2 infection. these investigators concluded that obesity might increase the lethality of covid-19 in diabetic subjects. diabetes, due to the deleterious role of hyperglycemia on immune responses, represents a risk factor for covid-19 infection in obesity [31, 32] . a french nationwide study, coronado (coronavirus sars-cov-2 and diabetes outcomes), has clearly shown the deleterious role of obesity in life-threatening outcomes in a large diabetic population with covid-19 [33, 34] . a perusal of above-mentioned studies clearly demonstrates that chronic inflammation, leading to immunosuppression, may contribute to decreased protection against viral infections in obese subjects [35] . it has been recently reported that a significant number of covid-19 patients suffer from a sudden loss of their senses of smell and taste, even in clinical conditions that are not marked with common viral symptoms such as fever, dry cough or thoracic oppression [36, 37] . a large number of covid-19 patients (from 60% to 80%) from iran have complained of a complete loss of their sense of smell or taste [38] . a multicentric european study conducted on covid-19 patients demonstrated that nearly 87% of patients reported olfacto-gustatory dysfunctions [39] . a recent meta-analysis on covid-19 patients, incorporating 10 research articles from 7 countries, has reported that nearly 52% and 43% of them had, respectively, gustatory and olfactory dysfunctions [40] . in france, gautier and ravussin [41] reported that there was a sudden appearance of anosmia and/or ageusia in a small number of covid-19 patients. similarly, almost two-thirds of covid-19 patients from germany also complained of anosmia [42] . in the usa, a survey was performed in the month of april 2020 on covid-19 patients, and 37.7% of participants complained of altered smell and taste perception [43] . interestingly, the changes in gusto-olfactory perception in covid-19 patients were more prevalent in home-quarantined subjects, independently of age and gender [44] . it is important to mention that sars-cov-2 does not generate clinically significant nasal congestion or rhinorrhea as seen in general nasal infections [45] [46] [47] [48] . does sars-cov-2 infect taste buds or nasal mucosal epithelia? a recent report, conducted in mice, has demonstrated that mouse sustentacular cells, involved in the transfer of odorant messages to olfactory neurons, express angiotensin converting enzyme 2 (ace2), which is a port of entry of sars-cov-2 ( figure 2 ) [49] . j. clin. med. 2020, 9, x for peer review 4 of 12 buds or nasal mucosal epithelia? a recent report, conducted in mice, has demonstrated that mouse sustentacular cells, involved in the transfer of odorant messages to olfactory neurons, express angiotensin converting enzyme 2 (ace2), which is a port of entry of sars-cov-2 ( figure 2 ) [49] . the duration and intensity of sars-cov-2-induced inflammation will also depend on pre-existing inflammation (like in obesity) and genetic or epigenetic backgrounds of the subjects. for simplification, we do not show the structure of the tongue papillae. we show a taste bud that is the unit of lingual gustatory papillae. during viral-induced inflammation, the oro-nasal epithelia will be infiltrated by macrophages that will release the pro-inflammatory cytokines such as il-6 and tnf-α that may aggravate the epithelial integrity and lead to clinical symptoms such as loss of oro-naso-sensory (ons) functions. beside the implication of ace2, the viral-induced generalized inflammation in covid-19 patients would also affect the integrity of the olfactory epithelium. chronic rhinosinusitis has been shown to trigger alterations in the olfactory mucosa, such as goblet cell hyperplasia, squamous metaplasia, and loss of supporting cells and olfactory neurons, associated with infiltration of proinflammatory immune cells [50] . we propose that sars-cov-2 might affect the integrity or regeneration/renewal of the olfactory epithelium, impacting the physiological function of olfactory sensory neurons (figure 2 ). hence, we can cite the example of sendai virus which has been shown to impair olfaction by reducing the regeneration of the olfactory epithelium and olfactory bulb in the mouse [51] . in in vitro experiments on murine olfactory neurons infected with this virus, the number of odorant-responsive cells were decreased. by using a plausible transgenic mouse model, lane et al. [52] have demonstrated that the induction of tnf-α expression triggered inflammation in the olfactory epithelium and the reversal of tnf-α expression restored the olfactory function in these animals, demonstrating that inflammation is an important factor involved in the loss of olfactory the duration and intensity of sars-cov-2-induced inflammation will also depend on pre-existing inflammation (like in obesity) and genetic or epigenetic backgrounds of the subjects. for simplification, we do not show the structure of the tongue papillae. we show a taste bud that is the unit of lingual gustatory papillae. during viral-induced inflammation, the oro-nasal epithelia will be infiltrated by macrophages that will release the pro-inflammatory cytokines such as il-6 and tnf-α that may aggravate the epithelial integrity and lead to clinical symptoms such as loss of oro-naso-sensory (ons) functions. beside the implication of ace2, the viral-induced generalized inflammation in covid-19 patients would also affect the integrity of the olfactory epithelium. chronic rhinosinusitis has been shown to trigger alterations in the olfactory mucosa, such as goblet cell hyperplasia, squamous metaplasia, and loss of supporting cells and olfactory neurons, associated with infiltration of pro-inflammatory immune cells [50] . we propose that sars-cov-2 might affect the integrity or regeneration/renewal of the olfactory epithelium, impacting the physiological function of olfactory sensory neurons ( figure 2 ). hence, we can cite the example of sendai virus which has been shown to impair olfaction by reducing the regeneration of the olfactory epithelium and olfactory bulb in the mouse [51] . in in vitro experiments on murine olfactory neurons infected with this virus, the number of odorant-responsive cells were decreased. by using a plausible transgenic mouse model, lane et al. [52] have demonstrated that the induction of tnf-α expression triggered inflammation in the olfactory epithelium and the reversal of tnf-α expression restored the olfactory function in these animals, demonstrating that inflammation is an important factor involved in the loss of olfactory sensory neurons and olfaction sensitivity. the olfactory mucosa is very sensitive to macrophage-secreted inflammatory cytokines, such as macrophage inflammatory protein-1α (mip-1a) and monocyte chemoattractant protein-1 (mcp-1), that may influence the renewal/regeneration of nasal epithelial cells [53] . as regards taste dysfunction, ace2 was highly expressed by tongue epithelial cells, but to a lesser extent by buccal and other tissues of the mouth cavity [54] . these observations suggest that the tongue is equipped with a sars-cov-2 entry route, but we do not know whether taste papillae and taste bud cells (tbcs) express the ace2 receptor. we would like to introduce toll-like receptors (tlrs) that act as receptors for viral rna, and are abundantly expressed on taste bud cells, particularly on type ii and type iii cells [55] . the activation of tlrs by the administration of exogenous ifn-γ led to inflammation in taste bud cells and, consequently, to cell death. the autoimmune pathologies in humans or experimental rodent models have clearly demonstrated that inflammation, associated with infiltration by il-6 and ifn-γ in gustatory epithelium, impacts taste perception [56] [57] [58] . moreover, administration of exogenous ifn-γ, via stat-1 signaling, induced apoptosis of taste bud cells [59] . these observations strongly support that oral taste papillae inflammation may contribute to low oro-sensory perception of sapid molecules. beside the peripheral mechanism, different brain areas might be involved in the loss of taste and smell in covid-19. there are several reports indicating that covid-19 patients also suffer from neurological complications, such as skeletal muscle injury, delirium and acute cerebrovascular disease [47] . chigr et al. [60] have proposed that this virus might accede to the olfactory cortex either by the nasopharyngeal cavity or directly by hematogenous spread. there is no direct report on the entry of sars-cov-2 into the brainstem; however, clinical features such as vomiting, nausea and loss of appetite suggest a perturbation in the dorsal vagal complex (dvc), which belongs to the medulla oblongata, the lowest region of the brainstem that controls several physiological functions, including food intake. in the dvc, the nucleus of tractus solitaris (nts) is known to regulate food intake, not only via the vagus nerve that connects the gut, but also via chorda tympani and glossopharyngeal nerves that connect directly to the gustatory taste papillae in the tongue [61] . ralli et al. [62] have proposed that sars-cov-2 could infect the olfactory receptors in the nasal epithelium, through which it may travel to the olfactory bulb and certain brain structures, such as the medulla oblongata. this hypothesis was based on the observations in animal experiments wherein intranasal administration of sars-cov, a strain similar to sars-cov-2, could enter the brain via the olfactory nerves and spread to the thalamus and brainstem [48] . sars-cov-2, in analogy to sars-cov34 and mers-cov13 infection in transgenic mice, might attain the brainstem [63] . indeed, using the murine model of hcov infection, it was shown that sars and oc43 were able to enter the olfactory bulb via the nasal route and reach the central nervous system (cns) [64] . moreover, ct scans and mri of covid-19 patients demonstrated "bilateral inflammatory obstruction of the olfactory clefts" [65] . though we do not have experimental animal data on sars-cov-2 entry, we can state that sars-cov-2 may enter the cns, using the olfactory pathway [63] , and exert its action via ace2 that has been detected in the central nervous system [66] . the question arises whether the loss of ons perception can be considered as an early marker of sars-cov-2 infection. we should be very cautious in this regard, as the methods that have been used for the assessment of ons defects are self-reported examinations. generally, the investigators employ either a 3-armed forced choice (3-afc) test or a comparison with 6-n-propylthiouracil (prop) tasting with and without sodium chloride for oral chemosensory perception, and for the detection of olfactory thresholds, rose smell and n-butanol are employed. by using these techniques, one can be sure about the decrease (or increase) in taste detection thresholds. however, in none of the reports on covid-19 patients, such tests were employed. why do all the covid-19 subjects not complain of the loss of smell? is there any genetic or epigenetic predisposition? before going into detail, we would like to emphasize that a reduced oro-sensory perception would trigger high consumption of palatable food, thus either leading to obesity or worsening this pathology [67, 68] , though we should not ignore the implication of the food addiction component, particularly for sweet food and those rich in fat [69, 70] . the studies conducted on healthy and obese participants suggested that the latter group exhibited lower sensitivity than the former for sweet and sour taste [71] . diet-induced obesity, by maintaining mice on a high-fat diet for ten weeks, resulted in low taste bud cell number and taste-evoked calcium signaling in obese mice [72] . similar observations have been reported for bitter and salt tastes in obese subjects [73] . as regards fat taste perception, there was a decreased perception of dietary fatty acids in obese rodents and human beings [61, 74, 75] . the decrease in taste sensitivity to different taste qualities might be due to partially functional taste receptors/sensors, caused by obesity-induced downregulation [75] , genetic polymorphism [75] [76] [77] [78] or epigenetic signatures [79] . the olfaction is not only important for the detection of sense of smell, but also to appreciate the palatability of a hedonic food, as the retro-nasal detection of flavors is brought about by nasal sensory epithelial cells [80, 81] . as regards the olfactory cue, there was a significant influence of bmi on olfactory thresholds, which were increased with increasing body weight in obese subjects [82, 83] . patel et al. [84] reported that high bmi was associated with subjective olfactory dysfunction in obese patients. by employing the olfactory threshold-discrimination-identification (tdi) test, pastor et al. [85] observed that olfactory discrimination power was lesser in obese subjects than control participants. like taste modalities, the genetic polymorphism of olfactory receptor genes [85, 86] or their hypermethylation [87] , also contributes to obesity. the decreased smell perception in obesity is a multicomponent phenomenon that involves not only nasal epithelial receptor activation, but also different brain areas, such as the limbic system, thalamus and piriform cortex, as well as amygdala, which project to the orbitofrontal cortex [88] . beside the afore-mentioned factors that bring about a decrease in ons, we should not forget to mention the role of cytokine-induced (generalized or tongue-specific) inflammation in obesity. the mouse taste bud cells have been shown to produce both tnf-α and il-10 in the microenvironment of taste papillae [89, 90] . in a plausible study, kaufman et al. [74] showed that an increase in tnf-α in the tongue of obese mice was associated with a significant reduction in taste bud and taste progenitor cells in tongue papillae. moreover, tnf-α null mice were protected from obesity-induced reduced number of taste bud cells, and administration of exogenous tnf-α brought back taste buds to degeneration [91] . the adipose-specific deletion of sel1l in mice maintained on a high-fat diet resulted in reduced adiposity and showed neither an increase in tnf-α concentrations nor any sign of taste bud cell atrophy. these observations clearly indicate that tnf-α released from hyperplasic/inflamed adipose tissue in obesity may trigger a loss in gustatory taste perception. moreover, lps-induced inflammation was also found to decrease the lifespan of mature taste bud cells [92] . as regards olfactory perception, inflammation and obesity, a link between apoptosis and inflammation has been recently reported in the olfactory mucosa of obese mice fed with a moderate high-fat diet, where a significant increase in activated caspase-3 was associated with a marked loss of olfactory sensory neurons and their axonal projections, paralleled with an increased expression of iba-1, suggesting an increase in proinflammatory cells [92] . hence, if the diet-induced obese mice are re-fed a normal diet and return to normal weight, the loss in olfactory perception is also reinstated. in vitro, tnf-α has been shown to induce cell death in olfactory epithelial explants [93] . in transgenic mice, the expression of tnf-α resulted in the loss of olfactory neurons and odor perception. as regards il-6, its concentrations were found elevated in the blood of patients suffering from hyposmia [94, 95] . a perusal of above-mentions observations clearly suggests that obesity is associated with the loss of ons, and inflammation in the oro-naso epithelia plays an important role in this phenomenon. figure 2 shows that sars-cov-2 infection will install (or aggravate) an inflammatory state both in the lingual and nasal epithelia. in the lingual taste buds, the virus-induced inflammation will attenuate the gustatory perception of different taste qualities, whereas in the olfactory sensory neurons, the virus-triggered inflammation may contribute to decreased olfactory perception of odorants. it is also possible that sars-cov-2, by penetrating the olfactory bulb, may enter the brainstem and modulate ons. why do all covid-19 patients not exhibit a change in ons perception? it is possible that the alteration in oro-olfactory epithelium functions might be secondary to viral infection, which may depend on genetic (or epigenetic) and other life-style-related build-up of the patients. nonetheless, we can infer that obese subjects are at high risk for sars-cov-2 infection as they already exhibit a low ons capacity for different taste modalities. hence, the existing gustatory and olfactory sensory deficiency, due to obesity, will mask sars-cov-2-induced diminished taste and smell sensation and, thus, may aggravate the patient's health. sars-cov-2 infection may further aggravate the ons functions; mask the obesity-induced inflammation, including loss of taste and smell; and render the obese subjects more vulnerable and prone to severe pathophysiological consequences such as rti, leading to death. by now, we have observational/self-reported studies, but data regarding the duration and the time of the onset and reversal of ons symptoms in this infection are lacking. we need a complete follow-up study of these patients as a function of time on the loss of ons. as mentioned previously, we also lack the proper set-up for the detection of olfactory and taste thresholds. we still do not know whether sars-cov-2 infection alters the taste bud renewal/turn-over and taste bud physiology either upstream or downstream of the detection of sapid molecules. it is too early to predict clearly that sars-cov-2-induced changes in ons might be due to its direct or indirect deleterious effects on brain regions such as the insula, caudal orbitofrontal and anterior cingulate cortex that control the integration of both taste and smell information [96] . while we have mentioned that tongue epithelium expresses ace2 receptors [54] , we still do not know which cell type (type i, ii or iii) expresses this receptor. this information will be important to correlate the loss of a particular taste modality as type ii cells express sweet, bitter and umami receptors; type i cells express salt receptors; and type iii cells are involved in sour sensing [74, 75] . the vistas in the eating behavioral physiology with regard to sars-cov-2 infection require more detailed investigations in covid-19 patients as gustatory and olfactory receptors are also expressed in other tissues such as those in the gut, which is the main site of the release of small peptides (such as cholecystokinin and peptide-yy) that control eating behavior via the vagus nerve [97] . similarly, the olfactory bulb also expresses receptors for a number of appetite-regulating hormones and peptides such as insulin, leptin, ghrelin and orexin [98] . it is now well established that the gut microbiome of obese subjects is shifted from bacteriodetes to fermecutes, a pro-inflammatory phylum, and the effects of this change on sars-cov-2 infection susceptibility should be explored in the future. does this viral infection promote a particular microbiome in the gut and ons epithelia? a recent report has outlined that there is a significant persistent alteration in the gut microbiome in covid-19 patients [99] . can the strategies to alter the intestinal microbiota decrease the severity of sars-cov-2 infection? we think that sars-cov-2 infection is much more dangerous than what is reported now and a significant amount of clinical information remains undiscovered. this study was supported by a grant from the french national research agency (anr-11-labx-0021-lipstic). the authors 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in the absence of encephalitis in mice transgenic for human ace2 sudden and complete olfactory loss function as a possible symptom of covid-19 tissue distribution of ace2 protein, the functional receptor for sars coronavirus: a first step in understanding sars pathogenesis psychophysics of sweet and fat perception in obesity: problems, solutions and new perspectives associations between weight status and liking scores for sweet, salt and fat according to the gender in adults (the nutrinet-sante study) obesity and addiction: neurobiological overlaps fat addiction: psychological and physiological trajectory taste sensitivity, nutritional status and metabolic syndrome: implication in weight loss dietary interventions diet-induced obesity reduces the responsiveness of the peripheral taste receptor cells taste perception and implicit attitude toward sweet related to body mass index and soft drink supplementation taste of fat: a sixth taste modality? preference for dietary fat: from detection to disease cd36-and gpr120-mediated ca 2+ signaling in human taste bud cells mediates differential responses to fatty acids and is altered in obese mice genetic variation in tas1r2 (ile191val) is associated with consumption of sugars in overweight and obese individuals in 2 distinct populations genetic variation in cd36 is associated with decreased fat and sugar intake in obese childrenand adolescents taste perception and its effects on oral nutritional supplements in younger life phases taste perception, associated hormonal modulation, and nutrient intake fat sensation: fatty acid taste and olfaction sensitivity and the link with disinhibited eating behaviour variant in a common odorant-binding protein gene is associated with bitter sensitivity in people olfactory and gustatory functions and its relation to body weight higher body mass index is associated with subjective olfactory dysfunction a lower olfactory capacity is related to higher circulating concentrations of endocannabinoid 2-arachidonoylglycerol and higher body mass index in women novel common copy number variation for early onset extreme obesity on chromosome 11q11 identified by a genome-wide analysis olfactory receptor genes cooperate with protocadherin genes in human extreme obesity associations between olfactory pathway gene methylation marks, obesity features and dietary intakes central mechanisms of odour object perception interleukin-10 is produced by a specific subset of taste receptor cells and critical for maintaining structural integrity of mouse taste buds inflammation arising from obesity reduces taste bud abundance and inhibits renewal lipopolysaccharide-induced inflammation attenuates taste progenitor cell proliferation and shortens the life span of taste bud cells hyperlipidemic diet causes loss of olfactory sensory neurons, reduces olfactory discrimi-nation, and disrupts odor-reversal learning tumor necrosis factor-alpha-induced apoptosis in olfactory epithelium in vitro: possible roles of caspase 1 (ice), caspase 2 (ich-1), and caspase 3 (cpp32) velicu, i. interleukin 6 in hyposmia experience-dependent neural integration of taste and smell in the human brain olfactory receptors in non-chemosensory tissues olfaction under metabolic influences alterations in gut microbiota of patients with covid-19 during time of hospitalization key: cord-283246-dj7teo89 authors: otsuka, ryo; seino, ken-ichiro title: macrophage activation syndrome and covid-19 date: 2020-08-06 journal: inflamm regen doi: 10.1186/s41232-020-00131-w sha: doc_id: 283246 cord_uid: dj7teo89 an emerging, rapidly spreading coronavirus sars-cov-2 is causing a devastating pandemic. as we have not developed curative medicine and effective vaccine, the end of this life-threatening infectious disease is still unclear. severe covid-19 is often associated with hypercytokinemia, which is typically found in macrophage activation syndrome. sars-cov-2 infection causes this strong inflammation within the lung and propagates to respiratory and, ultimately, systemic organ malfunction. although we have not fully understood the physiological and pathological aspects of covid-19, current research progress indicates the effectiveness of anti-cytokine therapy. here, we summarize macrophage activation syndrome and its possible contribution to covid-19, and cytokine targeted attempts in severe covid-19 cases. cytokine storm is a status of the immune system in which various immune cells are extremely activated and produce large amounts of cytokines, then, in turn, exhibit systemic hyperinflammatio n [1] . it often confers multiple organ failure and a high mortality rate. various inflammatory cytokines or chemokines such as tumor necrosis factor (tnf)-α, type i and ii interferons (ifns), interleukin (il)-1, il-6, ccl2, or monocyte chemotactic protein-1 (mcp-1), as well as immunosuppressive cytokines such as il-10 or transforming growth factor-β, have been implicated. similarly, various immune cells such as t cells, b cells, dendritic cells (dcs), or macrophages are important to understand the pathophysiology of cytokine storm. among those, activation of macrophages has been particularly paid attention, as it is especially called macrophage activation syndrome (mas) [2] . mas has been suggested to be also involved in the etiology of hyperinflammatory responses in the course of treatment with chimeric antigen receptor t cell for leukemic patients. cytokine storm has been observed and discussed in various clinical conditions such as rheumatological or hematological disorders [2] . furthermore, it sometimes occurs in infectious diseases and elicits a refractory condition against intensive therapies. it is related with the induction of acute respiratory distress syndrome (ards), which is one of the severest pathological status of respiratory systems, causing pulmonary edema, decreased gas exchange, and fatal hypoxia [3] . recently, it has been suggested that cytokine storm, particularly mas, is involved in coronavirus disease 2019 (covid-19)-associated pneumonia and its exacerbation [4] . although the major body of covid-19 patients shows none to mild pulmonary symptoms, approximately 20% of patients show severe pulmonary dysfunction. among those, a certain percentage of patients undergo life-threatening, critical pneumonia, the treatment for which extracorporeal membrane oxygenation is required. the reason why only a part of severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-infected patients show such severe inflammatory condition has not been clarified. still, it is possible that the causative virus for covid-19, sars-cov-2, infect with particular types of cells such as endothelial vessels in the lung, or alveolar wall or macrophages. the infection to the cell types may induce immune responses leading to the cytokine storm, including mas. in this brief review, we discussed a possible involvement of mas in the pathophysiology of covid-19, especially in cases with severe inflammatory pneumonia. mas is a state of systemic hyperinflammation and often be observed in patients with infections, malignancy, or pediatric rheumatological diseases, such as systemic juvenile idiopathic arthritis (sjia) [2] . mas is typified by markedly upregulated expression of pro-inflammatory cytokines, which is called "cytokine storm." without any therapeutic intervention, this strong inflammation results in severe tissue injury and, ultimately, patient death. several research pieces have revealed the involvement of particular cytokines in this phenomenon, especially tnf-α, il-6, and il-1β [5, 6] . macrophages in mas state produce a high amount of these proinflammatory cytokines upon stimulation. billiau et al. reported the histopathological evidence that macrophages in the liver of patients suffering from mas were expressing tnf-α and il-6 [7] . together with il-1, tnf-α and il-6 trigger a cascade of inflammatory pathways that synergistically activate and augment inflammation [8] . thus, serum levels of these cytokines are often at a high level in mas patients [5] . inflammation is known to destruct the precise balance between coagulation and fibrinolysis. certain inflammatory cytokines such as tnfα and il-1 initiate tissue factor production from monocytes and macrophages [9] , leading to the activation of coagulation, while il-1 and il-6 increase the production of plasminogen activator inhibitor [10] . thus, the overproduction of inflammatory cytokines along with mas also promotes intravascular coagulation. standard treatment for mas includes several immunosuppressive drugs, such as steroids, calcineurin inhibitors, or anti-thymocyte globulin [5] . in spite of such broad immunosuppression, it is difficult to mitigate severe mas symptoms. therefore, previous researches have spent their efforts on the pursuit of finding a new therapeutic target. in this context, cytokines highly produced in mas patients are potential candidates, and some clinical reports provided promising results by cytokine-targeting therapy. mas occurs around 10% of sjia, a systemic inflammatory disorder of non-particular etiology characterized by arthritis and systemic features [2] . a case report on a 27-year-old female sjia patient was clinically diagnosed as mas and presented an extremely high level of tnf-α in the serum [11] . in contrast, a remarkably low level of soluble tnf receptor (tnfr) was detected. because soluble tnfr acts as an antagonist of tnf, these clinical parameters suggested overactivated tnf signaling as a cause of the hyperinflammation. although the patient was utterly unresponsive to the series of treatment including steroid pulse and cyclosporine treatment, administration of the soluble tnfr etanercept dramatically improved the symptoms. etanercept is a fusion protein of tnfr and immunoglobulin domain, which replaces the function of endogenous soluble tnfr. the case report showed complete remission of the patient and suggested a therapeutic potential of anti-tnf-α reagent. on the other hand, some researchers indicate totally opposite clinical observations that anti-tnf therapy with etanercept, in turn, triggers cytokine storm in a patient with systemic sclerosis and necrotizing fasciitis [12] . together, we need more experimental knowledge to solidify the potential of tnf-α to be a therapeutic target in mas treatment. anti-il-6 treatment with specific blocker tocilizumab has also been suggested effective in attenuating clinical symptoms of mas. shimizu et al. defined that patients with mas showed significantly lower ferritin, crp, triglycerol, fibrinogen, and aspartic aminotransferase serum levels when receiving tocilizumab treatment, indicating an alleviation of systemic inflammation [13] . however, the improvement in clinical and laboratory features of mas may compromise mas diagnosis. their analysis also showed that some cases representing laboratory mas features failed to fulfill clinical mas criteria, resulting in only 20% of possible mas patients meet the 2016 mas classification. these backgrounds underlying anti-il-6 therapy on mas may preclude the precise estimation of the treatment outcome, and the specific effect of tocilizumab on mas remains to be investigated. a case report by a research group from the university of miami miller school of medicine presented that il-1 receptor antagonist anakinra showed promising results in mas treatment [14] . il-1 is a potent stimulator of il-6 production from macrophages, and serum il-1β is often at a high level among sjia patients. the activation of the il-1 receptor signaling pathway is also suggested by gene expression profiles of peripheral blood from sjia patients with severe inflammation. other studies also indicated promising results of anakinra treatment in mas patients [15] . although its direct contribution to the onset of mas remains unclear, these observations predispose us to expect the therapeutic potential of il-1 blockade. collectively, cytokine-targeted mas therapy has been reported by various research groups. in addition to the studies mentioned above, cd28, jak1/2, and ifn-γ were also implicated as potential therapeutic targets [16] [17] [18] . particularly, when considering the cases of infection-induced mas, these specific approaches may reduce major concerns accompanied by traditional therapy with immunosuppressants, which broadly suppress immune activation. covid-19-related hyperinflammation shares its clinical features with previously reported mas symptoms. il-1β, il-2, il-6, il-7, il-17, and tnf-α were reported to be highly upregulated in patients with severe covid-19 pneumonia patients [8, 19] . particularly, plasma levels of il-2, il-7, tnf-α, g-csf, cxcl10, ccl3, and mcp1 were much upregulated in the patients treated in icu compared to those in non-icu [19] . not only the hypercytokinemia but also the increased serum levels of ferritin, crp, and d-dimer indicate the development of mas-like severe inflammation and fibrinolysis in covid-19 patients [20, 21] . despite the above clinical features shared with classical mas, some are not compatible with known mas status. hyperferritinemia is indeed a hallmark of covid-19 pneumonia. one reported data showed that the median of serum ferritin concentration was about 800 ng/ml in the wuhan patient cohort [22] ; however, it is still lower than that of mas, which often exceeds 10,000 ng/ml [23] . moreover, mas is accompanied by reduced fibrinogen and platelet count and increased d-dimer [5] , which indicates systemic disseminated intravascular coagulation. conversely, only a high d-dimer was typically found in covid-19 patients [24] . another regular feature of mas is hepatosplenomegaly which was absent in reported covid-19 patients [4, 24] . in sum, at least accumulating clinical evidence implies the coincidence of mas-like hyperinflammation with covid-19 pneumonia; however, its immunological and pathological manifestations were observed mainly in the lung, which is associated with ards. there are various etiologies under the onset of ards. clinically, ards is featured by hypoxemia, diffuse pulmonary infiltrates, pulmonary edema, and reduced lung compliance, collectively result in rapid respiratory failure [25] . in terms of plasma levels, tnf-α, il-1β, il-6, and il-8 are elevated and show a higher concentration in non-survivors than survivors of ards patients [26] . it has been reported that approximately 20% of patients developed ards during the course of covid-19 [27] , with some cases rapidly worsened and died. clinical characteristics studied in a cohort of jinyintan hospital in wuhan identified that 41.8% of covid-19 patients developed ards [20] . the patients were diagnosed as ards immediately after their admission to the hospital. of note, the study reported all of the patients' death followed ards and mechanical ventilation. one of the cell types considered to play an essential role in ards onset is macrophage residents in alveolar [28] . those alveolar macrophages would be activated upon lung infection and release pro-inflammatory cytokines such as tnf-α, il-1β, and il-6. recent genomic studies revealed that angiotensin-converting enzyme 2 (ace2) and transmembrane protease, serine 2 (tmprss2), expressed on the surface of type ii pneumocytes, would mediate sars-cov-2 entry into target cells [29, 30] . viral rna invasion into the target cells elicits the production of pro-inflammatory cytokines via the activation of nf-κb pathway [31] . besides, sars virus infection has been reported to induce pyroptosis, a state of cell death mediated by activation cascade of nlrp3 inflammasome [32, 33] . one downstream indicator of pyroptosis is il-1β; the evidence of high serum il-1β may be one indicative of pyroptosis in covid-19-related lung inflammation. pyroptosis results in releasing damageassociated molecular patterns, which stimulate neighboring macrophages to produce pro-inflammatory cytokines and chemokines. the macrophages receiving inflammation signals, in turn, recruit immune cells such as t cells into the site of inflammation. additionally, viral rna entry into macrophages also provokes macrophage activation [34] . although macrophages do not express ace2 and tmprss2, alveolar macrophages may uptake viral rna through phagocytosis and the degradation of virus-infected cells. otherwise, antibodies to sars-cov-2 virus opsonize virus particles and allow macrophages to engulf viruses via fc-receptor-mediated endocytosis. indeed, previously analyzed sars virus was detected in the cytoplasm of alveolar macrophages [35] . thus, the commencement of local inflammation induced by sars-cov-2 infection activates macrophages at that site, spreading rapidly to the entire lung, possibly due to the abundant expression of virus entry receptors, ace2 and tmprss2 [36] . accumulation of immune cells accelerates the progression of lung inflammation into ards. in severe cases, local inflammation cannot be sedated within the lung and, consequently, propagates to multiple organ failure and death (fig. 1 ). as covid-19 patients represent symptoms resembling mas, it is of our great interest to know whether anticytokine reagents used in mas are also effective in covid-19 treatment. a report of the preliminary trial by the university of science and technology of china suggests the effectiveness of tocilizumab in severe covid-19 cases [37] . they report an immediate decrease in crp and body temperature and improvement of peripheral oxygen saturation. it is noteworthy that no serious adverse effects were observed in all patients, and after tocilizumab treatment, 90% of the patients were discharged from the hospital within 3 weeks. a more recent study of 100 covid-19 patients from italy also demonstrated the rapid improvement from severe ards condition by tocilizumab treatment [38] . the therapeutic potential of blockade of il-6 signaling for covid-19 is reviewed in more detail in other review articles in this series. cavalli et al. from vita-salute san raffaele university reported the cases of covid-19-associated ards patients subjected to anakinra treatment [39] . they showed significant improvement in patients' outcomes, which was supported by 90% survival in high-dose anakinratreated patients while 56% survival in the standard treatment group at the study endpoint. the rate of ventilation-free survival was also higher, albeit not statistically significant, in anakinra-treated patients. as there are yet a small number of cases reporting the efficacy of anakinra in severe covid-19 patients, it is expected to compile additional clinical evidence. an up-to-date study indicates that the inhibition of bruton tyrosine kinase (btk) by acalabrutinib effectively improved covid-19 patient outcomes [40] . btk is involved in nlrp3 inflammasome activation cascade, which subsequently triggers il-1β production [41] . as il-1β signal inhibition by anakinra treatment showed a favorable outcome, btk targeted therapy may also be a prospective therapeutic option for covid-19. although the abovementioned clinical trials were uncontrolled ones, the effects of tocilizumab and anakinra seem promising. currently, we can find more than 40 clinical trials for surveying an efficacy of anti-il-6 receptor antibody tocilizumab and 15 trials on anakinra, either alone or combined with other drugs (https://clinicaltrials.gov). additionally, tnf, ifn-γ, and gm-csf are also targeted in other clinical trials. these trials are at least in part related with mas, because the targeted cytokines have been highly implicated with mas and macrophage functions. covid-19-related hyperinflammation is only found in part of entire patients; however, no specific treatment for this population has been reported. thus, further studies to discover an optimal treatment are urgently needed. extensive examination of mas status in covid-19 may help discover new targets. sars-cov-2 is threatening millions of lives worldwide. due to the recent rapid progress of understanding the nature of the novel spreading virus, we are getting be able to prevent its massive spread in particular areas. however, we have not yet reached the curative medicine or effective vaccine. severe cases of covid-19 are often observed with ards, representing the mas-like clinical and laboratory features. previous studies have revealed that mas symptoms can be ameliorated by anticytokine therapy. to date, anti-il-6, il-1, and tnf have shown promising outcomes in mas treatment. based on these findings, anti-il-6 or il-1 treatment was carried out in covid-19 and showed significant improvement in the patients' symptoms. the world is confronting a difficult situation. not only clinicians and researchers but also people all over the world expect the development of the best therapeutic agent to covid-19. more than 1900 clinical studies are in progress across the globe, and they may offer clues on novel treatment. review: cytokine storm syndrome: looking toward the precision medicine era the immunology of macrophage activation syndrome the role of macrophages in the pathogenesis of ali/ards the role of cytokines including interleukin-6 in covid-19 induced pneumonia and macrophage activation syndrome-like disease macrophage activation syndrome and cytokinedirected therapies therapeutic potential of interferon-γ and its antagonists in autoinflammation: lessons from murine models of systemic juvenile idiopathic arthritis and macrophage activation syndrome macrophage activation syndrome: characteristic findings on liver biopsy illustrating the key role of activated, ifn-γ-producing lymphocytes and il-6-and tnf-α-producing macrophages the trinity of covid-19: immunity, inflammation and intervention tissue factor as a link between inflammation and coagulation il-1 and il-6 induce hepatocyte plasminogen activator inhibitor-1 expression through 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neutralization of ifn-γ reverts clinical and laboratory features in a mouse model of macrophage activation syndrome clinical features of patients infected with 2019 novel coronavirus in wuhan risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease 2019 pneumonia in wuhan. china covid-19: consider cytokine storm syndromes and immunosuppression epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study the british childhood cancer survivor study: objectives, methods, population structure, response rates and initial descriptive information immune mechanisms of pulmonary intravascular coagulopathy (pic) in covid-19 pneumonia acute lung injury and the acute respiratory distress syndrome: a clinical review persistent elevation of inflammatory cytokines predicts a poor outcome in ards: plasma il-1β and il-6 levels are consistent and efficient predictors of outcome over time a trial of lopinavirritonavir in adults hospitalized with severe covid-19 diverse macrophage populations mediate acute lung inflammation and resolution sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor a pneumonia outbreak associated with a new coronavirus of probable bat origin nf-κb and virus infection: who controls whom cell pyroptosis, a potential pathogenic mechanism of 2019-ncov infection the mechanisms of nlrp3 inflammasome/pyroptosis activation and their role in parkinson's disease viral activation of macrophages through tlr-dependent and -independent pathways multiple organ infection and the pathogenesis of sars sars-cov-2 (covid-19) by the numbers effective treatment of severe covid-19 patients with tocilizumab tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study inhibition of bruton tyrosine kinase in patients with severe covid-19 bruton's tyrosine kinase is essential for nlrp3 inflammasome activation and contributes to ischaemic brain injury publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations figures and illustrations were produced using servier medical art. authors' contributions ro and ks wrote and revised the manuscript. all authors read and approved the final manuscript. availability of data and materials not applicable ethics approval and consent to participate not applicable the authors declare that they have no competing interests.received: 9 june 2020 accepted: 1 july 2020 key: cord-308433-vrkdtrfz authors: roberts, ceri a.; durham, lucy e.; fleskens, veerle; evans, hayley g.; taams, leonie s. title: tnf blockade maintains an il-10(+) phenotype in human effector cd4(+) and cd8(+) t cells date: 2017-02-15 journal: front immunol doi: 10.3389/fimmu.2017.00157 sha: doc_id: 308433 cord_uid: vrkdtrfz cd4(+) and cd8(+) effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il-10. however, the underlying cellular mechanisms that regulate expression of il-10 in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il-10 expression in human cd4(+) t cells, including il-17(+) cd4(+) t cells. here, we further characterized the regulation of il-10 expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd3-stimulated human cd4(+) t cell/monocyte cocultures led to increased percentages of il-10(+) cells in pro-inflammatory il-17(+), ifnγ(+), tnfα(+), gm-csf(+), and il-4(+) cd4(+) t cell subpopulations. conversely, exogenous tnfα strongly decreased il-10(+) cell frequencies. tnf blockade also regulated il-10 expression in cd4(+) t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il-10(+) cell frequencies in both cd4(+) and cd8(+) t cells following in vitro stimulation in a doseand time-dependent manner. blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation did not significantly regulate il-10 expression within cd4(+) or cd8(+) t cell subpopulations. we show that tnf blockade acts directly on effector cd4(+) t cells, in the absence of monocytes or cd4(+) cd25(high)cd127(low) regulatory t cells and independently of il-27, resulting in higher il-10(+) frequencies after 3 days in culture. il-10/il-10r blockade reduced the frequency of il-10-expressing cells both in the presence and absence of tnf blockade. addition of recombinant il-10 alone was insufficient to drive an increase in il-10(+) cd4(+) t cell frequencies in 3-day cd4(+) t cell/monocyte cocultures, but resulted in increased il-10 expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il-10 expression in human t cells by tnf blockade. the maintenance of an il-10(+) phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. tnf blockade maintains an il-10 + phenotype in human effector cd4 + and cd8 + t cells ceri a. roberts cd4 + and cd8 + effector t cell subpopulations can display regulatory potential characterized by expression of the prototypically anti-inflammatory cytokine il-10. however, the underlying cellular mechanisms that regulate expression of il-10 in different t cell subpopulations are not yet fully elucidated. we recently showed that tnf inhibitors (tnfi) promote il-10 expression in human cd4 + t cells, including il-17 + cd4 + t cells. here, we further characterized the regulation of il-10 expression via blockade of tnf signaling or other cytokine/co-stimulatory pathways, in human t cell subpopulations. addition of the tnfi drug adalimumab to anti-cd3-stimulated human cd4 + t cell/monocyte cocultures led to increased percentages of il-10 + cells in pro-inflammatory il-17 + , ifnγ + , tnfα + , gm-csf + , and il-4 + cd4 + t cell subpopulations. conversely, exogenous tnfα strongly decreased il-10 + cell frequencies. tnf blockade also regulated il-10 expression in cd4 + t cells upon antigenic stimulation. using time course experiments in whole peripheral blood mononuclear cell (pbmc) cultures, we show that tnf blockade maintained, rather than increased, il-10 + cell frequencies in both cd4 + and cd8 + t cells following in vitro stimulation in a dose-and time-dependent manner. blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation did not significantly regulate il-10 expression within cd4 + or cd8 + t cell subpopulations. we show that tnf blockade acts directly on effector cd4 + t cells, in the absence of monocytes or cd4 + cd25 high cd127 low regulatory t cells and independently of il-27, resulting in higher il-10 + frequencies after 3 days in culture. il-10/il-10r blockade reduced the frequency of il-10-expressing cells both in the presence and absence of tnf blockade. addition of recombinant il-10 alone was insufficient to drive an increase in il-10 + cd4 + t cell frequencies in 3-day cd4 + t cell/monocyte cocultures, but resulted in increased il-10 expression at later time points in whole pbmc cultures. together, these data provide additional insights into the regulation of il-10 expression in human t cells by tnf blockade. the maintenance of an il-10 + phenotype across a broad range of effector t cell subsets may represent an underappreciated mechanism of action underlying this widely used therapeutic strategy. keywords: tumor necrosis factor, anti-tnf, tnf inhibitors, adalimumab, interleukin-10, cd4 + t cell polarization, cd8 + t cell polarization, il-10 regulation introduction the treatment of immune-mediated inflammatory diseases has improved considerably over the last 20 years with the advent of biological therapeutics. tnfα was the first cytokine to be fully validated as a therapeutic target in ra (1) . tnfα inhibitors (tnfi) have revolutionized treatment of ra and have been used in over a million patients worldwide (2) . despite the good clinical response observed in many patients, tnfα blockade does not offer a curative treatment; approximately one-third of patients do not respond and loss of efficacy is frequently observed (3) . importantly, it is currently not possible to predict which patients will respond to tnfi therapy. in addition, in some inflammatory diseases such as sjögren's syndrome (4, 5) and multiple sclerosis (6), tnfi have not shown clinical efficacy. furthermore, and paradoxically, some patients treated with tnfi develop de novo autoimmune diseases (7) . these observations indicate that the underlying mechanisms relating to tnf blockade in humans are incompletely understood and require further exploration. the effects of tnfi are more wide-ranging than simply neutralizing the biological activity of soluble and membrane-bound tnfα (mtnfα). for example, by binding mtnfα, anti-tnf mabs can mediate cell death by complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (8) (9) (10) (11) . tnfα inhibitors have also been shown to affect downstream cytokine pathways (il-1, il-6, and il-8) (2), modulate apc function (12) , and promote regulatory t cell (treg) expansion (13) (14) (15) although opposite findings regarding the latter have been reported (16) (17) (18) (19) . recent data from our laboratory demonstrated that tnf blockade promotes il-10 expression in human cd4 + t cells (20) . it was shown both cross-sectionally and longitudinally that inflammatory arthritis patients on tnfi therapy have an increased frequency of peripheral blood (pb) il-10 + cd4 + t cells. these in vivo findings were reproduced in vitro by coculturing cd4 + t cells from healthy donors with autologous cd14 + monocytes and anti-cd3 mab, in the presence of different tnfi drugs (adalimumab, infliximab, etanercept, or certolizumab) (20) . furthermore, we showed an increase in the percentage of il-10 co-expressing il-17 + cd4 + t cells, suggesting that otherwise pro-inflammatory cells displayed anti-inflammatory potential. indeed, re-sorted tnfi-exposed il-17 + cd4 + t cells secreted increased levels of il-10, which was biologically active and could modulate markers of monocyte activation (20) . although il-17 + cd4 + t cells are recognized as an important cell population in inflammatory disease, other cd4 + t cell subsets also contribute to inflammation (21) (22) (23) (24) , as well as cd8 + t cells which can also be potent producers of proinflammatory cytokines (25) (26) (27) (28) (29) . in this study, we therefore investigated in vitro whether tnf blockade regulates il-10 expression in other pro-inflammatory cytokine-producing t cell subsets, whether blockade of other cytokines or t cell activation pathways also drives il-10 expression, and how tnf blockade may manifest its il-10-regulating effect on t cells. peripheral blood samples were obtained from healthy adult volunteers. peripheral blood mononuclear cells (pbmcs) were isolated by density gradient centrifugation using lymphoprep™ (axis-shield, oslo, norway). cd14 + monocytes and cd4 + t cells were isolated by magnetic-activated cell sorting (macs) according to the manufacturer's instructions (miltenyi biotec, bergisch-gladbach, germany), and purity was confirmed by flow cytometry. monocytes (average purity 98%) were isolated by positive selection using anti-cd14 microbeads. cd4 + t cells were isolated via negative depletion (average purity 95%), and in some experiments, cd45ro + cd4 + t cells were subsequently enriched by positive selection using cd45ro microbeads (average purity 87%). in some experiments, cd4 + t cells were sorted to very high purity (> 99%) and part of the cells depleted of cd4 + cd25 high cd127 low tregs by facs-sorting after labeling cells with cd4 percp cy5.5 (sk3), cd25 pe (m-a251), cd127 alexa fluor 488 (a019d5) mabs (all from biolegend, cambridge, uk). the study was approved by the bromley research ethics committee (06/q0705/20), and written informed consent was obtained from all participants. cells were cultured at 37°c with 5% co2 in culture medium [rpmi 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (lot# 07f7435k, south american origin)] and 1% penicillin, streptomycin, and l-glutamine (all life technologies, carlsbad, ca, usa). freshly isolated bulk cd4 + or memory (cd45ro + )-enriched t cells (0.5 × 10 6 ) and cd14 + monocytes (0.5 × 10 6 unless otherwise indicated) were cocultured with 100 ng/ml anti-cd3 mab (clone okt3, janssen-cilag ltd., high wycombe, uk). cocultures were incubated at 37°c with 5% co2 for 3 days. in some experiments, macs-isolated cd4 + t cells were cultured alone with anti-cd3/cd28 stimulation. anti-cd3 (okt3) was coated onto culture plates at 1.25 µg/ml in pbs for a minimum of 2 h at 37°c. wells were then washed three times with pbs before adding cd4 + t cells in culture medium. anti-cd28 (clone cd8.2; bd biosciences) was added to the well at a final concentration of 1 µg/ml. in some experiments, whole pbmcs were cultured with 100 ng/ml anti-cd3 mab for up to 5 days. where indicated, the following recombinant cytokines, neutralizing antibodies, or other reagents (from r&d systems unless otherwise indicated) were added at the start of the culture period: recombinant human (rh) tnfα (10 ng/ml; biosource, camarillo, ca, usa), rhil-27 (10-100 ng/ml), rhil-10 (10 ng/ml; rhil-10 used to generate data in figure s5 in supplementary material from peprotech, rocky hill, nj, usa), neutralizing/blocking antibodies to ifnγ (clone 25718, 10 µg/ml), il-17 (clone 41809, 10 µg/ml), il-1r1 (polyclonal, 2 µg/ml), il-10 (clone 23738, 10 µg/ml), il-10r (clone 8516, 10 µg/ml), and il-27 (polyclonal, 5 µg/ml). the following isotype control antibodies were used at an assay-appropriate concentration: human igg1 (abcam, cambridge, uk), mouse igg1, mouse igg2a, mouse igg2b, goat igg (all r&d systems). clinical grade biologics were purchased anti-tnf maintains il-10 + cd4 + /cd8 + t cells . antibodies to detect cd4 and/ or cd3 were added intracellularly, to allow for staining of these markers following our t cell culture conditions. stained cells were acquired using a facscantoii or lsrfortessa (bd biosciences); in most experiments, 100,000 t cell events were recorded. the gating strategies used to analyze intracellular cytokine expression in cd4 + t cells are described in figure s1 in supplementary material. all flow cytometry data were analyzed using flowjo software (version 7.6.1 or 10, tree star, inc., ashland, or, usa). il-10 was measured in cell culture supernatants using the il-10 elisa max kit (biolegend), according to manufacturer's instructions. il-27 was measured in cell culture supernatants using the duoset human il-27 elisa (ebioscience), according to manufacturer's instructions. microwell absorbance was read at 450 nm using a wallac 1420 microplate reader (perkin elmer, waltham, ma, usa). concentrations of the analytes were determined based on the standard curve included on each plate. statistical testing was performed with graphpad prism 5.0 or 6.0 (graphpad, san diego, ca, usa). data sets were tested for normality using the d' agostino and pearson omnibus normality test, followed by statistical significance testing using the appropriate tests as indicated in figure legends. data sets with n values <8 were tested non-parametrically. p values < 0.05 were considered statistically significant. tnf blockade regulates il-10 expression in human cd4 + and cd8 + t cells to investigate whether tnf blockade regulates il-10 expression in different pro-inflammatory cytokine-producing cd4 + t cells, we isolated cd4 + t cells from pb of healthy donors and cocultured the cells with cd14 + monocytes and anti-cd3 mab (100 ng/ml) in the absence or presence of the anti-tnf mab adalimumab (1 µg/ml), as previously described (20) . after 3 days, cells were restimulated with pma and ionomycin in the presence of golgistop for 3 h and stained for cytokine expression (gating strategy shown in figure s1 in supplementary material). in agreement with our previous data (20) , tnf blockade led to a significant increase in il-10 + cells within total cd4 + t cells as well as in the il-17 + cd4 + t cell subset. in addition, we found a strong increase in il-10-expressing cells within ifnγ + , tnfα + , gm-csf + , and il-4 + cd4 + t cell populations (figures 1a,b) . tnf blockade did not alter the frequencies of ifnγ + , tnfα + , or gm-csf + cd4 + t cell populations but did induce a modest increase in il-17 + cd4 + t cells, as previously shown (20) , and in most donors in il-4 + frequencies, although this did not reach statistical significance ( figures s2a, b in supplementary material). addition of an isotype control human igg1 mab did not promote il-10 expression in cd4 + t cells ( figure s3 in supplementary material). contrary to the effects of tnf blockade, addition of rhtnfα to cocultures of cd4 + cd45ro + t cells, monocytes and anti-cd3 mab led to a striking decrease in the percentage of il-10 + cells within total cd4 + t cells and within il-17 + , ifnγ + , and tnfα + cd4 + t cell subsets (gm-csf + and il-4 + cd4 + t cells were not tested) ( figure 1c ). tnfα addition did not significantly alter the frequencies of il-17 + , ifnγ + , or tnfα + cd4 + t cell subsets ( figure s2c in supplementary material). since pro-inflammatory cytokine-expressing cd8 + t cells also contribute to immune-mediated inflammatory diseases (27) (28) (29) , we investigated whether tnf blockade can regulate il-10 expression in cd8 + t cells. we adapted the culture system to stimulate whole pbmc with anti-cd3 mab (100 ng/ml) in the absence or presence of a dose range of adalimumab (0.01-10 µg/ml). after 3 days, the cultures were restimulated with pma/ionomycin in the presence of golgistop and cytokine expression was assessed within either the cd4 + or the cd8 + t cell populations. significant increases in the percentages of il-10 + cells within both cd4 + and cd8 + t cell populations were observed, including in the il-17 + and ifnγ + subpopulations (figure 2) . the regulation of il-10 expression by tnf blockade was dose responsive in both cd4 + and cd8 + t cell subsets, with significantly increased il-10 + frequencies following culture with either 1 or 5 µg/ml adalimumab ( figure 2c ). these doses reflect figure 1 | tnf blockade promotes, while tnfα impairs il-10 expression in pro-inflammatory cd4 + t cell subsets. cd4 + t cells (open symbols) or cd4 + cd45ro + t cells (filled symbols) were cocultured with autologous cd14 + monocytes and anti-cd3 mab (100 ng/ml) in the absence or presence of the anti-tnf mab adalimumab (1 µg/ml) (a,b) or rhtnfα (10 ng/ml) (c). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cd4 + t cells were gated as described in figure s1 in supplementary material. (a,b) representative dot plots (a) and cumulative data (b) showing the percentages of il-10 + cells within total cd4 + t cells (n = 35), or within il-17 + cd4 + (n = 35), ifnγ + cd4 + (n = 24), tnfα + cd4 + (n = 27), gm-csf + cd4 + (n = 12), or il-4 + cd4 + (n = 8) cells after culture in the absence or presence of anti-tnf. data were analyzed using wilcoxon matched-pairs signed rank test. (c) cumulative data showing the percentages of il-10 + cells within total cd4 + cd45ro + t cells or within il-17 + (n = 8), ifnγ + (n = 8), or tnfα + (n = 8) cd4 + cd45ro + t cells after culture in the absence or presence of rhtnfα. data were analyzed using paired t test. each connecting line represents an individual donor (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). figure 2 | tnf blockade regulates il-10 expression in cd4 + and cd8 + t cells in a dose-dependent manner. (a,b) freshly isolated peripheral blood mononuclear cells were cultured with anti-cd3 mab (100 ng/ml) in the absence (control) or presence (anti-tnf) of adalimumab (1 µg/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. cells were gated on live single cd3 + cells and il-10 expression was assessed within cd4 + (a) or cd8 + (b) t cell subsets. representative dot plots show the percentages of il-10 + cells within total cd4 + or cd8 + t cells, or within il-17 + or ifnγ + subsets after culture in the absence or presence of anti-tnf. (c) cells were cultured as described above in the presence of increasing doses of adalimumab (0-10 μg/ml). box-whisker plots represent data from n = 6 individual donors; whiskers show minimum to maximum values. data were analyzed by friedman test with comparison to control by dunn's multiple comparisons test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). the clinically effective trough levels for adalimumab in human serum following treatment (30, 31) . in agreement with data from the cd4 + t cell/monocyte cocultures, addition of rhtnfα (100 ng/ml) to anti-cd3-stimulated pbmc led to a reduction in il-10 + cell frequencies, in both cd4 + and cd8 + t cells (n = 3, data not shown). since these in vitro studies of t cell responses to tnf blockade all relied on monoclonal anti-cd3 stimulation, we tested whether tnf blockade could still elicit increased il-10 + t cell responses in the context of a more physiological antigenic stimulation. pediacel is a pentavalent vaccine consisting of purified diphtheria toxoid, purified tetanus toxoid, acellular pertussis vaccine, inactivated poliovirus, and haemophilus influenzae type b polysaccharide. revaxis is a booster vaccine containing purified diphtheria and tetanus toxoids and inactivated poliovirus. each vaccine was added to cd4 + t cell/monocyte cocultures (1:1,000 dilution) in the presence or absence of tnf blockade. after 6 days, antigen-stimulated cd4 + t cells cultured in the presence of adalimumab demonstrated elevated il-10 + frequencies as compared to cells that were not exposed to tnf blockade ( figure s4 in supplementary material). together these data demonstrate that in vitro tnf blockade provided at a physiological concentration and in a physiological setup can promote il-10 expression in cd4 + and cd8 + t cells, including in subsets expressing pro-inflammatory cytokines. tnf blockade maintains il-10 expression in cd4 + and cd8 + t cells thus far, assessment of cytokine expression following in vitro tnf blockade was carried out after 3 days of culture. time course experiments were performed to assess the kinetics of il-10 expression in cd4 + and cd8 + t cells. pbmc were cultured with anti-cd3 mab in the absence or presence of adalimumab, and the frequencies of il-10 expressing cells were analyzed 4-120 h after stimulation. the data show that early after tcr stimulation (18-24 h) the frequencies of il-10 expressing cells within cd4 + and cd8 + t cells increased rapidly irrespective of the presence of tnf blockade. however, at later time points (from 42 to 120 h), higher frequencies of il-10 + cells were sustained within cd4 + and cd8 + t cells in the presence of tnf blockade (figure 3) . these experiments suggest that tnf blockade maintains, rather than directly induces, an il-10 + phenotype in cd4 + and cd8 + t cells following tcr stimulation. blockade of tnfα, but not il-17, ifnγ, il-6r, or cd80/cd86-mediated costimulation, regulates il-10 in human cd4 + t cells our previous work demonstrated that in addition to adalimumab, other tnfα inhibitors (etanercept, infliximab, or certolizumab) as well as tnfr1/2 blocking mabs were capable of increasing frequencies of il-10-expressing il-17 + cd4 + t cells (20) . we investigated whether blockade of additional pro-inflammatory pathways could promote il-10 expression in cd4 + t cells. blockade of il-17a did not enhance the frequencies of il-10 + cells in any of the cd4 + t cell populations tested (figure 4a) . blockade of ifnγ did not affect the percentage of il-10 + cells within total cd4 + t cells, or within ifnγ or tnfα + subpopulations, but led to modestly increased frequencies of il-10 + expressing cells within the il-17 + population, although this effect was much weaker than the effect of tnf blockade in parallel cultures ( figure 4a) . addition of tocilizumab (il-6r blockade) or abatacept (ctla4-ig, which blocks cd80/cd86-mediated co-stimulation), both of which are biologic drugs routinely used in the clinic to treat rheumatoid arthritis, did not increase il-10 + frequencies in cd4 + t cells, il-17 + , ifnγ + , or tnfα + cd4 + t cell subpopulations (figure 4b) . to determine whether blockade of these pathways might regulate il-10 expression with different kinetics to tnf blockade, il-10 + frequencies were analyzed within both cd4 + and cd8 + t cells at different time points in anti-cd3-stimulated pbmc cultures exposed to these antibodies or drugs. il-10 + cd4 + and il-10 + cd8 + t cell frequencies were not regulated at any time point by blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated co-stimulation ( figure s5 in supplementary material). blockade of il-1r1 in cd4 + t cell/monocyte cocultures resulted in a significantly increased proportion of il-10 + cells within total cd4 + t cells and within il-17 + , ifnγ + , or tnfα + subpopulations ( figure 4b) . however, this effect was not replicated in either cd4 + or cd8 + t cells in whole pbmc cultures ( figure s5 in supplementary material), indicating that the capacity of il-1 blockade to regulate il-10 expression may be dependent on the in vitro culture conditions. together these data indicate that il-10 expression in cd4 + t cells and cd8 + t cells can be regulated by blocking tnfα signaling, but not by blocking ifnγ, il-17, il-6r, or cd80/cd86-mediated co-stimulation, at least in vitro. since cd14 + monocytes are major producers of tnfα, we explored whether the presence of monocytes was required for the effects of tnf blockade on regulating il-10. cd4 + t cells were sorted to a very high purity (>99%) and stimulated with platebound anti-cd3 and soluble anti-cd28 mab in the absence of monocytes or placed in coculture with monocytes and anti-cd3 mab, with or without addition of anti-tnf mab. in the cd4 + t cell only cultures, tnf blockade still brought about increased il-10 + cell frequencies within the total cd4 + population and also within il-17 + , ifnγ + , or tnfα + subpopulations, similar to the increased il-10 + frequencies observed in cd4 t cell/monocyte cocultures (figures 5a,b) . analysis of supernatants from anti-cd3/cd28-stimulated cd4 + t cells by elisa confirmed that levels of secreted il-10 were increased during culture in the presence of tnf blockade (figure 5c) . one potential interpretation of the observed increased frequencies of il-10 + cells following tnf blockade could be that there is an expansion of a pre-existing population of tregs. our previous work indicated that in three donors, macs-depletion of cd4 + cd25 + cells did not impair the anti-tnf-mediated increase in il-10 + frequencies within il-17 + cd4 + t cells and that no increase in foxp3 + cell frequencies was apparent upon tnf blockade (20) . to investigate this further, macs-isolated cd4 + t cells were facs sorted to very high purity (>99%) to yield either total cd4 + t cells or effector cd4 + cd25-cd127 + t cells (teff; depleted of cd25 high cd127 low treg). these cells were then cultured with anti-cd3/cd28 mabs in the absence of monocytes, or placed in coculture with monocytes and anti-cd3 mab, in the absence or presence of adalimumab. our data show that tnf blockade still resulted in increased frequencies of il-10-expressing cells in effector t cells in the absence of cd25 high cd127 low tregs (figure 6 ). together these data indicate that tnf blockade regulates il-10 expression directly in cd4 + effector t cells and does not require the presence of cd14 + monocytes or cd4 + cd25 high cd127 low tregs. (a) cd4 + t cells (n = 6) or cd45ro + enriched cd4 + t cells (n = 2) were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (100 ng/ml), without (control) or with anti-tnf mab (adalimumab, 1 μg/ml) or neutralizing antibodies to ifnγ or il-17 (10 μg/ml) or the appropriate isotype control antibodies (migg2a and migg2b, respectively). (b) cd45ro + enriched cd4 + t cells (n = 9) were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (100 ng/ml), without (control) or with anti-tnf mab (adalimumab, 1 μg/ml), tocilizumab (50 µg/ml), abatacept (5 µg/ml) or anti-il-1r1-blocking ab (2 μg/ml). (a,b) after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + , or tnfα + cd4 + t cell subpopulations. data were analyzed by wilcoxon matched-pairs signed rank test with comparison to the appropriate control condition (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). in support of our findings that tnf blockade can exert its effects on cd4 + t cells in the absence of monocytes, we did not find evidence for a role of the monocyte-derived anti-inflammatory mediator il-27 (32) in mediating il-10 expression in the context of tnf blockade. this was demonstrated by the findings that tnf blockade did not induce il-27 secretion, rhil-27 enhanced il-10 secretion but did not result in increased percentages of il-10 + cd4 + t cells, and il-27 blockade did not impair the increased il-10 + cd4 + t cell frequencies brought about by tnf blockade ( figure s6 in supplementary material). il-10 partly contributes to tnf blockade-mediated il-10 regulation finally, we explored whether modulation of il-10 expression in cd4 + t cells by tnf blockade was dependent on il-10 signaling itself. neutralizing antibodies against il-10 and il-10r were added to cd4 + t cell/monocyte cocultures stimulated with anti-cd3 mab, in the presence or absence of tnf blockade, and intracellular cytokine expression was assessed after 3 days. blockade of il-10 signaling in the absence of adalimumab led to significantly reduced il-10 + frequencies within total cd4 + t cells and within il-17 + , ifnγ, or tnfα + subpopulations ( figure 7a) . when adalimumab and il-10/il-10r blocking mabs were added in combination to the cocultures, a decrease in il-10 + cd4 + t cell frequencies was observed as compared to the condition treated with adalimumab alone; however, an increase was still observed as compared to il-10/il-10r blockade alone. these data suggest that pathways additional to il-10 signaling could play a role in the regulation of il-10 expression by tnf blockade. indeed, when we tested whether addition of 6) showing frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + or tnfα + cd4 + t cell subpopulations. (c) after 3 days in culture but prior to re-stimulation, supernatants were harvested from cd4 + t cell only cultures (n = 15) and analyzed by enzyme-linked immunosorbent assay for il-10 secretion. data were analyzed by wilcoxon matched-pairs signed rank test (*p < 0.05, ****p < 0.0001). roberts et al. anti-tnf maintains il-10 + cd4 + /cd8 + t cells frontiers in immunology | www.frontiersin.org february 2017 | volume 8 | article 157 exogenous il-10 (10 ng/ml) could drive il-10 expression in cd4 + t cell/monocyte cocultures, we found that this by itself was not sufficient to increase il-10 + cell frequencies in total cd4 + t cells, or within il-17 + , ifnγ + , or tnfα + subpopulations. addition of rhil-10 even decreased the percentage of il-10 + cells in total cd4 + t cells and tnfα + cd4 + t cells (figure 7b) . the biological activity of the recombinant il-10 was validated by culturing freshly isolated monocytes with 0, 10, or 100 ng/ml rhil-10 for 20 h and confirming that rhil-10 addition resulted in increased cd14 and cd163 and reduced hla-dr expression by flow cytometry, in accordance with previous studies (33, 34) (n = 2, data not shown). we next investigated the regulation of il-10 in response to rhil-10 in both cd4 + and cd8 + t cells using whole pbmc cultures assessed for il-10 expression over a 5-day time course (figure s5 in supplementary material). we found that at earlier time points (18-24 h) , rhil-10 addition reduced il-10 + cd4 + t cell frequencies (n = 6, p = 0.03, wilcoxon matched-pairs signed rank test), while at later time points (66-72 and 114-120 h), rhil-10 slightly increased il-10 + cd4 + t cell frequencies (n = 6, p = 0.03, wilcoxon matched-pairs signed rank test). il-10 + cd8 + t cell frequencies remained very low throughout, regardless of rhil-10 addition. together, these data indicate that il-10 signaling contributes in part to tnf blockade-mediated regulation of il-10 expression in cd4 + t cells, but that other factors also play a role. the effects of exogenous il-10 on regulating il-10 expression in cd4 + t cells appear to be dependent on both time and in vitro culture conditions. here, we show that t cell stimulation in the presence of tnf blockade maintains the proportion of cells expressing the antiinflammatory cytokine il-10. this phenomenon is observed in total cd4 + and cd8 + t cells as well as within a variety of pro-inflammatory cytokine-expressing (il-17 + , ifnγ + , tnfα + , gm-csf + or il-4 + ) subpopulations. tnf blockade regulates il-10 expression whether cd4 + t cells are stimulated in the presence or absence of monocytes, and when an antigenic stimulus is used in place of monoclonal anti-cd3 stimulation. we found that blockade of il-17, ifnγ, il-6r, or cd80/cd86-mediated costimulation did not regulate il-10 expression in cd4 + or cd8 + t cell subpopulations. blocking antibodies to il-1r1 resulted in increased il-10 + cd4 + t cell frequencies when added to cd4 + t cell/monocyte cocultures, but this effect was not replicated in whole pbmc cultures. one explanation could be that additional cell types are present in pbmc cultures that can produce or respond to il-1. neither cd4 + cd25 high cd127 low tregs nor il-27 was required for tnf blockade to exert its il-10-promoting effects. our data confirm and extend our previous work showing that tnf blockade promotes il-10 expression in cd4 + t cells (20) and support other findings in the literature. a small study of ra patients found that frequencies of il-10 + pbmc were increased following treatment with infliximab (14) . however, in the three patients studied, it was not confirmed which cell subset(s) expressed il-10. ebert later showed that supernatants from cultures of either monocytes (adherent pbmc) or t cells (non-adherent, cd14 − cd20 − hla-dr − pbmc), contained increased il-10 following exposure to infliximab in vitro (35) . antiga et al. demonstrated increased percentages of il-10 + cd4 + t cells in t cell blasts isolated from skin lesions of psoriasis patients, following treatment with etanercept (36), and pb il-10 + cd4 + t cells were also increased in posterior uveitis patients following treatment with a p55 tnfα receptor fusion protein (37) . earlier studies assessing mouse transgenic t cells (38) or human cd4 + t cell clones (39) also found that tnf blockade led to increased il-10 production in cell culture supernatants. in further support of our data, boks et al. showed that blockade of tnfα during in vitro priming of naïve cd4 + figure 7 | il-10 expression in cd4 + t cells is inhibited by il-10 blockade, in both the absence and presence of tnf blockade. (a) cd45ro + enriched cd4 + t cells were cocultured 1:1 with autologous monocytes in the presence of anti-cd3 (okt3,100 ng/ml), with or without anti-tnf mab (adalimumab, 1 μg/ml), with or without neutralizing antibodies to il-10 and il-10r (both 10 μg/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. box-whisker plots show frequency of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + or tnfα + subpopulations; whiskers show minimum to maximum values. data were analyzed by repeated measures anova, with comparison between selected conditions by sidak's multiple comparison test (n = 9). (b) cd45ro + enriched (filled symbols, n = 5) or bulk (open symbols, n = 1) cd4 + t cells were cocultured 1:1 with autologous monocytes and stimulated with anti-cd3 mab (100 ng/ml), with or without recombinant human il-10 (rhil-10; 10 ng/ml). after 3 days, cells were restimulated and assessed for intracellular cytokine expression as described in the methods. data show frequencies of il-10 + cells within the total cd4 + t cell population or within il-17 + , ifnγ + , or tnfα + subpopulations and were assessed by wilcoxon matched-pairs signed rank test (ns p > 0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). t cells with tolerogenic dcs favored the development of il-10 + cd4 + t cells (40) . thus, tnfi-mediated regulation of il-10 in cd4 + t cells has been indicated by a growing number of in vitro and ex vivo studies. the presence of il-10-expressing cd8 + t cells has been reported in several mouse models of infection, including acute influenza infection (41) , chronic mycobacterium tuberculosis infection (42) , and coronavirus-induced encephalitis (43) . furthermore, il-10 + cd8 + t cells have been found in patients with hiv (44) and chronic hepatitis c infection (45) . il-10 expression in cd8 + t cells can be induced by il-4, either alone (46) or in combination with il-12 (47) , or dexamethasone (48) . however, to the best of our knowledge, this is the first report showing that tnf blockade can regulate il-10 expression in cd8 + t cells. il-27 can stimulate il-10 production by various effector cd4 + t cell subsets (32, 49, 50) . our data suggest however that the capacity of tnf blockade to affect il-10 expression in cd4 + t cells is not dependent on il-27. in agreement with other work on human t cells (51, 52) , we found that addition of recombinant il-27 to anti-cd3/cd28-stimulated cd4 + t cells resulted in increased il-10 secretion, although this was not strictly dosedependent. however, in contrast to these other studies, which used naïve cd4 + t cells, our analysis of intracellular cytokine expression did not demonstrate increased il-10 + cd4 + t cell frequencies after 3 days in culture with il-27. this is possibly anti-tnf maintains il-10 + cd4 + /cd8 + t cells frontiers in immunology | www.frontiersin.org february 2017 | volume 8 | article 157 due to different kinetics of il-10 expression in a bulk cd4 + t cell population. it is of note that although in cd4 + t cell/monocyte cocultures il-10 blockade led to reduced il-10 + frequencies in both the presence and absence of tnf blockade, il-10 addition by itself was not sufficient to drive il-10 expression in cd4 + t cells. these data are in line with previous work showing that il-10 is necessary but not sufficient to enhance development of il-10-producing tr1 cells following in vitro differentiation in the presence of vitamin d3 plus dexamethasone (53) . however, when added to anti-cd3-stimulated whole pbmc cultures, rhil-10 did slightly increase il-10 + cd4 + t cell frequencies after 3 and 5 days in culture, indicating that the regulation of il-10 expression in cd4 + t cells is dependent on in vitro culture conditions. in inflammatory diseases, such as ra, the normal balance of immune regulation is disturbed and t cell-derived proinflammatory mediators can contribute to disease pathogenesis in an uncontrolled manner. this study indicates that a potentially immunoregulatory il-10 + phenotype is broadly maintained in effector t cells following exposure to tnf blockade, which may represent an underappreciated mechanism of action for this widely used therapeutic strategy. we previously provided evidence that the il-10 secreted by il-17 + cd4 + t cells following tnf blockade is biologically active (20) ; however, further work is required to investigate whether the increase in il-10 + frequencies across different t cell subpopulations is functionally relevant in the context of inflammatory disease. additional insights into the underlying molecular mechanisms via which tnf blockade maintains il-10 expression could identify potential targets for novel therapeutic strategies. author contributions cr, ld, and vf designed and performed experiments and analyzed and interpreted data. lt and he conceived the study, contributed to experimental design, and interpreted data. cr and lt wrote and revised the manuscript. all authors edited the manuscript. development of anti-tnf therapy for rheumatoid arthritis anti-tnf biologic agents: still the therapy of choice for rheumatoid arthritis direct comparison of treatment responses, remission rates, and drug adherence in patients with rheumatoid arthritis treated with adalimumab, etanercept, or infliximab: results from eight years of surveillance of clinical practice in the nationwide danish danbio registry inefficacy of infliximab in primary sjögren's syndrome: results of the randomized, controlled trial of remicade in primary sjögren's syndrome (tripss) lack of efficacy of etanercept in 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rheumatoid arthritis interleukins 27 and 6 induce stat3-mediated t cell production of interleukin 10 il-10) and viral il-10 strongly reduce antigen-specific human t cell proliferation by diminishing the antigen-presenting capacity of monocytes via downregulation of class ii major histocompatibility complex expression human monocytes express cd163, which is upregulated by il-10 and identical to p155 infliximab and the tnf-α system etanercept downregulates the th17 pathway and decreases the il-17+/il-10+ cell ratio in patients with psoriasis vulgaris anti-tnfα therapy modulates the phenotype of peripheral blood cd4+ t cells in patients with posterior segment intraocular inflammation the role of tnf alpha and related cytokines in the development and function of the autoreactive t-cell repertoire chronic exposure to tumor necrosis factor (tnf) in vitro impairs the activation of t cells through the t cell receptor/cd3 complex; reversal in vivo by anti-tnf antibodies in patients with rheumatoid arthritis inhibition of tnf receptor signaling by anti-tnfα biologicals primes naïve cd4+ t cells towards il-10+ t cells with a regulatory phenotype and function effector t cells control lung inflammation during acute influenza virus infection by producing il-10 clonal expansions of cd8+ t cells with il-10 secreting capacity occur during chronic mycobacterium tuberculosis infection highly activated cytotoxic cd8 t cells express protective il-10 at the peak of coronavirus-induced encephalitis hiv-specific il-10-positive cd8+ t cells are increased in advanced disease and are associated with decreased hiv-specific cytolysis intrahepatic virus-specific il-10-producing cd8 t cells prevent liver damage during chronic hepatitis c virus infection il-4 induces a suppressive il-10-producing cd8+ t cell population via a cdkn2a-dependent mechanism cytokine-induced il-10-secreting cd8 t cells represent a phenotypically distinct suppressor t-cell lineage glucocorticoids drive human cd8(+) t cell differentiation towards a phenotype with high il-10 and reduced il-4, il-5 and il-13 production suppression of autoimmune inflammation of the central nervous system by interleukin 10 secreted by interleukin 27-stimulated t cells a dominant function for interleukin 27 in generating interleukin 10-producing anti-inflammatory t cells il-27 is a key regulator of il-10 and il-17 production by human cd4+ t cells il-27 induces the differentiation of tr1-like cells from human naive cd4+ t cells via the phosphorylation of stat1 and stat3 vitro generation of interleukin 10-producing regulatory cd4+ t cells is key: cord-340741-bhxm4zua authors: nayak, tapas kumar; mamidi, prabhudutta; sahoo, subhransu sekhar; kumar, p. sanjai; mahish, chandan; chatterjee, sanchari; subudhi, bharat bhusan; chattopadhyay, soma; chattopadhyay, subhasis title: p38 and jnk mitogen-activated protein kinases interact with chikungunya virus non-structural protein-2 and regulate tnf induction during viral infection in macrophages date: 2019-04-12 journal: front immunol doi: 10.3389/fimmu.2019.00786 sha: doc_id: 340741 cord_uid: bhxm4zua chikungunya virus (chikv), a mosquito-borne alphavirus, is endemic in different parts of the globe. the host macrophages are identified as the major cellular reservoirs of chikv during infection and this virus triggers robust tnf production in the host macrophages, which might be a key mediator of virus induced inflammation. however, the molecular mechanism underneath tnf induction is not understood yet. accordingly, the raw264.7 cells, a mouse macrophage cell line, were infected with chikv to address the above-mentioned question. it was observed that chikv induces both p38 and jnk phosphorylation in macrophages in a time-dependent manner and p-p38 inhibitor, sb203580 is effective in reducing infection even at lower concentration as compared to the p-jnk inhibitor, sp600125. however, inhibition of p-p38 and p-jnk decreased chikv induced tnf production in the host macrophages. moreover, chikv induced macrophage derived tnf was found to facilitate tcr driven t cell activation. additionally, it was noticed that the expressions of key transcription factors involved mainly in antiviral responses (p-irf3) and tnf production (p-c-jun) were induced significantly in the chikv infected macrophages as compared to the corresponding mock cells. further, it was demonstrated that chikv mediated tnf production in the macrophages is dependent on p38 and jnk mapk pathways linking p-c-jun transcription factor. interestingly, it was found that chikv nsp2 interacts with both p-p38 and p-jnk mapks in the macrophages. this observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsp2-mapks interactions. a strong polar interaction was predicted between thr-180 (within the phosphorylation lip) of p38 and gln-273 of nsp2, whereas, no such polar interaction was predicted for the phosphorylation lip of jnk which indicates the differential roles of p-p38 and p-jnk during chikv infection in the host macrophages. in summary, for the first time it has been shown that chikv triggers robust tnf production in the host macrophages via both p-p38 and p-jnk/p-c-jun pathways and the interaction of viral protein, nsp2 with these mapks during infection. hence, this information might shed light in rationale-based drug designing strategies toward a possible control measure of chikv infection in future. chikungunya virus (chikv), a mosquito-borne alphavirus belongs to togaviridae family, is transmitted through either aedes aegypti or aedes albopictus mosquito. chikv mediated disease is one of the global challenges due to its endemics in different parts of the world (103 countries), such as tanzania (1) (2) (3) , reunion island (4-7), india (8) (9) (10) (11) (12) , italy (13, 14) , and thailand (15) (16) (17) (18) . among alphaviruses, chikv is considered as one of the most successfully evolved virus. the arboviruses including chikv have been evolving and re-emerging from centuries and their emergence and dispersion are more rapid and geographically extensive. this might be due to increase in global communication, mass immigration, vector adaptation to urbanization and land perturbation (19) . even though mortality due to chikv is very rare and restricted to children's (below 1 year), old age (above 65 years) or immune compromised patients, the pathogenesis (mainly inflammatory responses) may persist for very long periods of time both in humans and macaque model (20, 21) . currently, arboviruses raise a serious threat to the global public health, due to unavailability of effective drugs or vaccines (22, 23) . recent studies on chikv induced immune responses suggest that the host immune system is found to be both beneficiary in one hand by controlling viral infection, whereas deleterious on the other hand by promoting severe inflammatory responses (24) (25) (26) (27) (28) . studies have shown that chikv induces different inflammatory cytokines/chemokines (tnf, il-1β, il-6, ifnγ, il-8, and mcp-1) (24, (29) (30) (31) (32) (33) (34) (35) (36) (37) , which might be associated with arthritis like pathogenesis during chikv infection. in different in vivo systems (both mouse and non-human primates), predominant cellular infiltration of macrophages, monocytes, nk cells and t cells to the site of inoculation and other tissues have been observed (38, 39) . moreover, immunohistochemistry and flow cytometry based analysis of muscles and synovial biopsies revealed that macrophages are major infiltrating cells among mps (mononuclear phagocytic system) (25, 40) . blood monocytes and tissue macrophages are the major immune cells infected by chikv (21, 31, 41) . in macaque, synovial macrophages have been identified as the major host cell for longterm viral persistence (21) . this productive infection of chikv in the host macrophages might be associated with arthritis like pathogenesis despite robust immune activation (41, 42) . t cell immune responses specific to chikv is not clearly understood yet. teo th et al. have suggested that cd4 + t cells (but not cd8 + t cells) are essential for the development of chikv induced pathogenesis without affecting virus infection and dissemination in mice and this is independent of ifn-γ (43) . flow cytometry based analysis of circulating lymphocytes in chikv patients confirms that there are both cd4 + and cd8 + t cell responses during early and late phases of infection, respectively. moreover, cd95 mediated apoptosis was also detected in cd4 + t cells after 2 days of symptom appearance (44) , which might be one of the strategies to evade host immunity. purified t cells (both cd4 + and cd8 + ) from the chronic and recovered patients from 2005 to 2006 la reunion islands showed immune activation when challenged with synthetic chikv peptides and inactivated virus particles (42) . the dna vaccine based on the consensus sequences of e1/2 and capsid protein (with several modifications) of chikv resulted in robust ifn-γ and igg production suggesting that chikv induces both t and b cell specific responses (45, 46) . there are three major studied ser/thr kinases under the mitogen-activated protein kinase (mapk) family, such as p38, jnk, and erk, which are known to regulate multiple cellular pathways such as cell proliferation, activation, inflammation, cytokine and chemokine productions and different pathological conditions (47) (48) (49) (50) (51) (52) (53) . in addition, activation of mapks by different pathogens and other inflammatory diseases have been reported to induce pro-inflammatory cytokines such as tnf in the host cells (48-51, 53, 54) . the mapks have been shown to be activated by phosphorylation in specific positions (ser/tyr/thr) by several viral infections, such as coronavirus type 2, hepatitis c virus, rhinovirus and epstein-barr virus (54) (55) (56) (57) (58) . chikv is also known to induce mapks during infection in various nonimmune cells and treatment of an alkaloid berberine, reduces viral infection and joint swelling in mice (59, 60) . we have shown earlier that chikv triggers robust tnf production in the host macrophages, which might be a key mediator of virus induced inflammation (37) and macrophages are identified as the major cellular reservoirs during the late stages of chikv infection in macaques (21) . however, the precise role of mapk activation pathways in terms of chikv infection and associated robust tnf induction in macrophages (immune cell) remains largely unknown. hence, an attempt was made to understand the involvement of mapks in chikv infection and tnf induction in the host macrophages. the mouse anti-chikv-nsp2 antibody used in the current study was developed by us (61) . anti-mouse cd3 antibody, anti-tnf antibody, anti-cd69 fitc, hrp linked anti-mouse, and hrp linked anti-rabbit secondary antibodies were purchased from bd biosciences (ca, usa). anti-mouse cd28 and cd90.2 apc were procured from tonbo biosciences (ca, usa). the monoclonal antibodies for p38, p-p38, jnk, p-jnk, erk1/2, p-erk1/2, p-irf3, and p-c-jun were purchased from cell signaling technology (ma, usa). the anti-mouse alexa fluor 488 and anti-rabbit alexa fluor 647 were purchased from invitrogen (ca, usa). the rabbit polyclonal antibodies against p-p38 and p-jnk used for immunoprecipitation were purchased from santa cruz biotechnology (tx, usa). mouse igg, rabbit igg isotype control, and anti-gapdh antibody were purchased from abgenex india pvt. ltd (od, india). saponin and bovine serum albumin fraction v were purchased from sigma-aldrich (mo, usa). sb203580 (p-p38 inhibitor, sb), and sp600125 (p-jnk inhibitor, sp) were purchased from merck millipore (ma, usa). mtt assay was performed to assess the cytotoxicity of sb and sp according to the methods described before (37) . briefly, the raw264.7 cells were seeded in 96 well plates at a density of 5 × 10 3 cells per well before 18-20 h of drug treatment. then, the cells were washed in 1x pbs and incubated with different concentrations of drugs in triplicate. as both sb and sp were dissolved in the dimethyl sulfoxide (dmso), it was taken as solvent control. after 12 h, the cells were incubated with the mtt reagent to a final concentration of 10% (v/v) in rpmi media. then, the cells were placed in the incubator for upto 2 h for the formation of visible crystals. later, the media (containing mtt) were removed without disturbing the cells and 100 µl of solubilization solution was added per well followed by incubation for 15 min at room temperature (rt). the percent viable cells were calculated after taking the absorbance of the solution at 550 nm by microplate reader (bio-rad, ca, usa). raw 264.7 cell line has been well-reported to study chikv infection, replication and associated altered host immune responses (31, 37) . the raw264.7 cells were seeded in six-well cell culture plates before 18-20 h of infection with around 70% confluency. the cells were infected with the drde-06 strain of chikv with multiplicity of infection (moi) 5 as reported previously (37) . briefly, after washing the cells in 1x pbs, the virus was added over confluent monolayer for 2 h in the incubator with manual shaking at an interval of 15 min. then, the virus inoculum was washed in 1x pbs to remove unbound viruses and the cells were maintained in the complete rpmi-1640 media. the infected cells and the supernatants were collected at different time points and subjected to further processing according to the assay. sb and sp treatments were given as described before (62) . briefly, cells were pretreated with the desired concentrations of sb, sp or dmso for 2 h in serum free media (sfm). then the infection was carried out in the presence of either solvent control (dmso), sb or sp. the cells were washed thoroughly with 1x pbs after 2 h and cultured in sfm containing the drug for 3 h. then, serum was added to the cells and maintained in the incubator until harvesting (37) . viral plaque assay was performed to determine the titer of chikv as described previously (10) . in brief, after infecting the vero cells with different dilutions of cell culture supernatants (collected from chikv infected raw cells), the cells were overlaid with complete dmem containing methyl cellulose and maintained in the incubator. after the development of the visible plaques (usually 4-5 days), the cells were fixed in formaldehyde at room temperature, washed gently in tap water and stained with crystal violet. then, the numbers of plaques were counted manually under white light. flow cytometric assay was carried out as reported previously (37) . briefly, both mock and chikv infected raw264.7 cells were harvested and fixed in 4% paraformaldehyde for 10 min at rt. then, the cells were re-suspended in facs buffer and stored at 4 • c until staining. for intracellular staining (ics), the cells were permeabilized in freshly prepared 1x permeabilization buffer followed by blocking buffer (1% bsa in permeabilization buffer) for 30 min at rt. then, the cells were incubated with different primary antibodies for 30 min at rt, followed by washing with 1x permeabilization buffer twice. after that, the cells were incubated in alexa fluor r 488 and alexa fluor r 647 conjugated secondary antibodies followed by washing with 1x permeabilization buffer. the mouse igg and rabbit igg were taken as isotype control during ics. the fcr blocking reagent (miltenyi biotec, bergisch gladbach, germany) was used prior to the primary antibody incubation to prevent non-specific binding of antibodies to the fc receptors on macrophages. then, the cells were acquired by the bd facs calibur tm flow cytometer (bd biosciences, ca, usa) and analyzed by the cellquest pro software (bd biosciences, ca, usa). a total of approximately 10 × 10 3 cells were acquired per sample. sandwich elisa for cytokine analysis tnf production from the macrophage cell culture supernatants was quantified by the bd opteia tm sandwich elisa kit (bd biosciences, ca, usa) according to the manufacturer's instructions (37) . the cytokine concentrations in the test samples were calculated in comparison with the corresponding standard curve prepared by using different concentrations of the recombinant tnf in pg/ml. western blot analysis was performed to assess the levels of different protein expressions according to the protocol mentioned before (37) . in brief, both the mock and chikv infected cells were washed with ice-cold 1x pbs and the whole cell lysate (wcl) was prepared by radio immuno precipitation assay (ripa) lysis buffer. the protein concentration was quantified by the bradford reagent (sigma-aldrich, mo, usa). equal amount of protein was loaded in the 10% sds-page after mixing with 2x laemmli buffer (1:1) and blotted on to a pvdf membrane (millipore, ma, usa). then the transferred membranes were blocked with 3% bsa followed by overnight incubation with different primary antibodies. then, the membranes were thoroughly washed with tbst and incubated with the hrp conjugated secondary antibodies for 2 h at rt. after washing with tbst, the blots were subjected to chemiluminescence detection by the bio-rad gel doc with the quantity one software (bio-rad, ca, usa). for band intensity quantification, western blot images were subjected to further analysis by the quantity one 1-d analysis software while normalizing to the corresponding gapdh loading control. raw cells were infected with chikv as described above and harvested at 6 hpi. the cells were lysed with np-40 (nonidet p-40) lysis buffer (250 mm nacl, 5 mm edta, 10% glycerol, 1% np-40, 50 mm tris, ph 7.4, supplemented with protease inhibitor and phosphatase inhibitor cocktail). the resultant whole cell lysates were subjected to immunoprecipitation by immunoprecipitation kit dynabeads r protein a (thermo fisher scientific, ma, usa) according to the manufacturer's instructions. briefly, both the mock and chikv infected whole cell lysates were incubated with primary antibodies overnight on the vertical rotor at 4 • c. then, 30 µl of dynabeads r protein a was added to the cell lysate and incubated for another 4 h on the vertical rotor at 4 • c. the dynabeads r -ab-ag complexes were washed three times in lysis buffer followed by elution with elution buffer supplied in the kit. then, the eluted complexes were resuspended in 4x laemmli buffer, boiled at 90 • c for 10 min and processed further for western blot analysis as described above. the protein-protein docking was performed using the cluspro 2.0 webserver (63, 64) . this server performs three computational steps. in the first step, it does rigid-body docking using the piper. this docking program is based on the fast fourier transform (fft) correlation approach and uses pairwise interaction potential as part of its scoring function e = w1e rep + w2e attr + w3e elec + w4edars. while e rep and e attr represent the repulsive and attractive contributions, e elec denotes electrostatic energy term and edars refers to the pairwise structure-based potential (64, 65) . in the second step, 1,000 lowest energy docked structures are clustered using pairwise interface rmsd (irmsd) (64, 66) . based on the irmsd values the structure with the highest neighbors within a 9 å radius is defined as the center of the first cluster. further clustering is performed within the remaining structures to generate 30 clusters. the energy minimization is done for the structures using the van der waals terms of the charmm potential in the third step (64, 67) , following which the structures at the center of the 10 most populated clusters are taken as the output. since there was no satisfactory template available in pdb to build the homologous model of nsp2, the structure was generated earlier using the i-tasser algorithm (68) . this was used as a ligand in the study. xray crystallographic structures of jnk1 (pdb id: 3elj) and p38 (pdb id: 1a9u) were taken as receptors for the protein-protein docking. these structures were recovered from the protein data bank. the co-crystallized ligands were extracted and energy was minimized before submission of chain a of these structures as receptors. the output of docking generated four types of models using the scoring algorithms designated as balanced, electrostatic-favored, hydrophobic-favored, and van der waals+ electrostatic. amongst these, the balanced outputs were analyzed. the docking solution with largest members was taken for further visualization using the pymol software. mouse splenocytes isolation and splenic t cell purification from balb/c mice were performed as reported earlier (69) . in brief, using a 70 µm cell strainer the splenocytes were collected from mice spleens. after rbc lysis and washing with 1x pbs, cells were suspended in rpmi-1640 media supplemented with 10% fbs. according to instructions given by the manufacturer's protocol, mouse splenic t cell purification was carried out using dynabeads untouched mouse t cells kit (invitrogen, ca, usa). tcr driven t cell activation was carried out with those purified t cells (cd90.2 + ) in the presence of either chikv infected or uninfected (mock) macrophage culture supernatants (0.22 µm membrane filtered) to study the status of cd69 (a t cell activation marker) as described earlier for other infection model (70) . for tnf neutralization, anti-tnf purified antibody (bd bioscience) was incubated for 90 min with the chikv infected supernatant prior tcr stimulation. statistical analysis was performed by using the graphpad prism 5.0 software (graphpad software inc. usa). data were represented as mean ± sem. the comparison between the groups was performed by either one-or two-way anova with tukey or bonferroni post-hoc test, respectively. data presented here were representative of at least three independent experiments. p < 0.05 was considered as statistically significant difference between the groups. to determine whether any mapk (p38, jnk, and erk) is activated during chikv infection in macrophages, raw cells were infected with the virus at moi 5 and harvested at different time points (0-12 hpi). both the cells and cell culture supernatants were subjected to various downstream assays. as shown in figure 1a , the p-p38 and p-jnk expressions were increased significantly as compared to the corresponding mock cells. the p-p38 mapk expression was found to be increased around 1.5-fold as early as 3 and 6 hpi, followed by approximately 3-fold increments toward 12 hpi as compared to the corresponding mock cells. similarly, the expression of the p-jnk was found to be increased rapidly around 2-fold during early hours (3 and 6 hpi), whereas, it increased up to 3-fold with respect to the mock in later time points. the total p38 and jnk (t-p38 and t-jnk) expressions remain unaffected in both the groups. moreover, p-erk1/2 and t-erk1/2 (total-erk1/2) expressions remain unchanged throughout all the time points as compared to the corresponding mock (figures 1a,b) . this data suggests that chikv induces activation of both p38 and jnk by phosphorylation in a time-dependent manner in macrophages. since chikv induces both p38 and jnk activation in the host macrophages, next we sought to assess whether these two mapks are crucial for the viral infection and replication in the macrophages. for that, pharmaceutical inhibitors of p38 (sb203580) and jnk (sp600125) were used. first, different concentrations of both sb (0.1, 0.5 and 1.5 µm) and sp (1, 5 and 10 µm) were assessed for cytotoxicity in raw cells by mtt assay. it was observed that around 100% cells were viable in all the concentrations of sb, whereas up to 95 and 100% cells were found to be viable at 10 and 5 µm concentrations of sp, respectively (figures 2a,e) . thus, both 5 and 10 µm concentrations of sp were selected for further experiments. as sb treatment with >2 µm concentration was known to inhibit phosphorylation and activation of pkb non-specifically (71), both 0.5 and 1.5 µm concentrations of sb was used in the current study. raw cells were inoculated with chikv in the presence of sb, sp, or solvent control dmso as described above. at 12 hpi both mock and chikv infected cells were harvested and the expressions of nsp2, p-p38, and p-jnk were assessed by flow cytometry. it was observed that the percent positive cells for nsp2 were reduced from 8.19 ± 0.35 (chikv+dmso) to 2.50 ± 0.08 (chikv+sb 0.5 µm) and 1.36 ± 0.02 (chikv+sb 1.5 µm), whereas the percent positive cells for p-p38 were reduced from 8.05 ± 0.73 (chikv+dmso) to 1.31 ± 0.15 (chikv+sb 0.5 µm) and 0.46 ± 0.04 (chikv+sb 1.5 µm) (figures 2b,c) . likewise, the mfi for both the p-p38 and nsp2 were reduced at 12 hpi in the sb treated cells as compared to the dmso control ( figure 2d) . the inhibition of p-jnk by sp at 5.0 µm concentration did not affect nsp2 expression in the macrophages as compared to the dmso control (chikv+dmso; 7.63 ± 0.40, chikv+sp 5 µm; 7.21 ± 0.17, p > 0.05), despite significant reduction in the p-jnk percent positive cells (chikv+dmso; 6.08 ± 0.40, chikv+sp; 3.31 ± 0.75, p < 0.05). however, sp at the comparatively higher concentration (10 µm) did reduces nsp2 expression by around 1.5-fold (figures 2f-h) . further, plaque assay of the cell culture supernatants revealed that sb treatment reduces the number of new viral progeny release around 1.5-and 2.5-fold at 0.5 and 1.5 µm, respectively. whereas, sp at 10 µm concentration treatment reduces the number of new viral progeny release around 1.6-fold as compared to the corresponding dmso control (figure 2i) . this result indicates that the activation of both p38 and jnk mapks might be crucial for the chikv infection and replication in the host macrophages with sb being more effective comparatively in controlling infection than sp. pharmaceutical inhibitors specific to p-p38 and p-jnk reduces chikv induced tnf production in the host macrophages activation of mapks by different pathogens has been shown to induce pro-inflammatory cytokines such as tnf in the host cells (50, 54) . since, chikv triggers robust tnf production (a key mediator of inflammation) in the host macrophages (31, 37) , it was interesting to investigate whether any mapks are involved in this pathway. accordingly, macrophages were treated with either sb or sp and infected with chikv as mentioned earlier. the cell culture supernatants were subjected to sandwich elisa for the detection of tnf at early (6 hpi) and late (12 hpi) time postinfection. it was observed that both sb and sp could suppress chikv induced tnf significantly at both the time points as compared to the corresponding dmso control. at 6 hpi the tnf level for chikv+dmso was found to be 737 ± 27 pg/ml (mean ± sem), which was reduced to 466 ± 12 pg/ml (mean ± sem, p < 0.05) and 356 ± 20 pg/ml (mean ± sem, p < 0.05) in the presence of sb (1.5 µm) and sp (10.0 µm), respectively. similarly, at 12 hpi, the tnf production was 1,104 ± 29 pg/ml (mean ± sem) in the chikv+dmso sample, whereas it was reduced to 554 ± 28 pg/ml (mean ± sem, p < 0.05) for sb and 528 ± 25 pg/ml (mean ± sem, p < 0.05) for sp treatment (figure 3) . taken together, this result suggests that chikv might induce tnf via p38 as well as jnk mediated pathways in the host macrophages. tnf, one of the potent inflammatory cytokine, which can enhance tcr-dependent t cell activation (72) . we and others have previously reported that in vitro chikv infection in raw 264.7 cells leads to tnf production (31, 37) . recent studies have shown a pathogenic role of t cells during chikv infection associated to host inflammatory responses (43, 44, 46) . here we have investigated whether chikv infection induced macrophage derived tnf can facilitate mouse t cell activation associated with cell mediated immunity. for this, chikv infected culture supernatant of raw 264.7 cells were tested toward tcr driven resting t cell activation assay (69) . we have found that chikv infected macrophage culture supernatant along with tcr activation facilitated the induction of cd69 level (around 81%) as compared to uninfected culture supernatant (around 71%). interestingly, when the chikv infected macrophage culture supernatant was treated with tnf neutralizing antibody, a sharp decrease of cd69 frequency (around 63%) was observed. beside this, sb and sp treated chikv infected raw 264.7 culture supernatant along with tcr stimulation also showed downregulation of cd69 frequency in t cells (figures 4a,b) . so, the above observations may underscore that the tnf present in chikv infected culture supernatant might be able to facilitate the induction of t cell activation. often, the viral infection is associated with the activation and localization of several transcription factors (e.g., irfs, c-jun, p53), which in turn regulates host responses to viruses (73) (74) (75) (76) (77) (78) . here, the expressions of key transcription factors involved mainly in antiviral responses (p-irf3) and tnf production (pc-jun) were assessed at different hpi by western blot analysis. it was observed that both p-irf3 and p-c-jun were induced significantly in the chikv infected macrophages as compared to the corresponding mock (figures 5a,b) . this data suggest that chikv infection in the raw cell line might be associated with the elevation of key antiviral and inflammatory transcription factors. it has been reported previously that tnf is one of the key mediators for arthritis or arthritis-like diseases in humans by promoting severe inflammation. although, several other inflammatory cytokines are elevated in ra (rheumatoid arthritis), anti-tnf therapy seems to be promising for the effective treatment against it (79) . since chikv induces tnf via p38/jnk map kinase pathways and phosphorylation of cjun is reported to be associated with tnf production in other inflammatory model system (50, 80) , phosphorylation of c-jun in both mock and chikv infected macrophages was assessed by western blot analysis. surprisingly, the expression of p-cjun was reduced around 1.8-and 4.77-fold in the presence of sp at 5 and 10 µm indicating a plausible role of jnk toward cjun phosphorylation, whereas sb treatment at 1.5 µm did not affect p-c-jun expression significantly. however, both sb and sp treatment suppressed p-irf-3 expression which is induced by chikv as compared to the dmso control (figures 6a-d) . taken together, the current data depict that chikv may induce p-c-jun via jnk pathway whereas induction of p-irf-3 might be dependent on both p38 and jnk mapks. viruses are small obligatory intracellular pathogens utilizes the metabolic pathways of the host for replication. very often viruses also shut-off host translational process, which might be a strategic decision to contain antiviral responses (81, 82) . the integration of complex proteomics studies including in silico protein-protein interaction predictions keeps on unraveling the complex network of interaction with the host cell proteins. throughout the course of replication, these pathways rely heavily on the dynamic and temporarily regulated virus-host protein-protein interactions which are crucial for the virus replication, pathogenesis, and viral subversion of host defense. the identification and characterization of these interacting partners also help in the delineation of the viral protein functions precisely and might be very helpful in designing rationale drugs for an effective treatment (83) (84) (85) . the interaction of host mapk with viral protein has been shown earlier, which in turn regulates infection and replications (86) . since chikv infection modulated the phosphorylation of host p38 and jnk, their interaction with the nsp2 protein was investigated. for that, raw cells were infected with chikv and harvested at 6 hpi for further analysis. co-immunoprecipitation followed by western blot analysis showed that both the p-p38 and p-jnk proteins were pulled with the chikv-nsp2 protein in the host macrophages (figures 7a,b) . this result indicates that chikv-nsp2 interacts with both p-p38 and p-jnk and this might be playing a crucial role in the chikv infection and tnf mediated inflammatory responses. in order to unravel the amino acid residues responsible for the interaction of nsp2-mapks, protein-protein docking was carried out as mentioned above (64, 87, 88) . the balanced outputs were preferred from the docking results as this mode takes into account all possible modes of interactions. the most stable complex of nsp2-jnk1 on visualization by using the pymol software suggested the possible involvement of different residues in the interaction (supplementary table s1 ). no interaction was found between the phosphorylation lip (thr-183-x-tyr-185) of jnk1 and nsp2 (figure 8a ). this suggests a poor fit of jnk1 active site with nsp2. nonetheless, ten polar interactions were observed within 2å (figure 8b) . some of these include the interactions of met-182, arg-228, arg-189, val-196, arg-150, lys-68, and glu-346 of jnk1 with cys-217, arg-272, gln-291, gly-279, asp-280, gly-285, and lys-282 of nsp2, respectively ( figure 8b) . the most stable complex of nsp2-p38 showed a close fit of the phosphorylation lip (thr-180-x-tyr-182). in addition to that, a polar interaction was suggested between thr-180 of p38 and gln-273 of nsp2 ( figure 8c) . some of the polar interactions were also observed between lys-66, ser-329, asn-196, ser-252, ser-254, asp-177, glu-178, arg-173, lys-152, and asp-230 of p38 and asn-288, gly-285, asp-280, cys-278, asp-351, cys-257, arg-244, phe-255, arg-272, and thr-90 of nsp2, respectively ( figure 8d and supplementary table s2) . thus, these results further suggest that chikv-nsp2 interacts with p38 as well as jnk mapks during viral infection in the host macrophages. moreover, the phosphorylation lip of p38 interacts more closely with the gln-273 of chikv-nsp2, which supports the findings of ip experiments. the recent epidemics of chikungunya virus (chikv) with unprecedented magnitude and unusual clinical severity have raised a great public health concern worldwide, due to the absence of a vaccine or specific anti-chikv therapy. tnf is one of the robustly induced cytokine by chikv and in the current study, we have investigated the molecular mechanism involved in the induction of tnf in the host macrophages. our data suggested that chikv induces both p38 and jnk phosphorylation in macrophages in a time-dependent manner. moreover, p-p38 and p-jnk inhibition by sb and sp were found to reduce chikv infection. interestingly, sb mediated inhibition of chikv infection was found to be more effective even at lower concentration as compared to sp. further, inhibition of both p-p38 and p-jnk reduced chikv induced tnf in the host macrophages. moreover, chikv infected cell culture supernatant is found to facilitates t cell activation via tnf in tcr primed t cells. besides, it was observed that the expressions of key transcription factors involved mainly in antiviral responses (p-irf3) and tnf production (p-c-jun) were induced significantly in the chikv infected macrophages as compared to the corresponding mock cells. further, it was found that chikv mediated tnf production in the macrophages is dependent on p38 and jnk mapk pathways linking p-cjun transcription factor. interestingly, it was also noticed that chikv nsp2 interacts with host p-p38 and p-jnk mapks in the macrophages. this observation was supported by the in silico protein-protein docking analysis which illustrates the specific amino acids responsible for the nsp2-mapks interactions and a strong polar interaction was predicted between thr-180 (within the phosphorylation lip) of p38 and gln-273 of nsp2. however, no such polar interaction was predicted for the phosphorylation lip of jnk which indicates the differential roles of p-p38 and p-jnk during chikv infection in the host macrophages. the mapks have been shown to be activated by several viral infections (54) (55) (56) (57) . using the mouse macrophage cell line, raw264.7 cells, we report for the first time that chikv induces both p-p38 and p-jnk significantly, however, the p-erk1/2 expression remains unchanged. interestingly, the up-regulation of p-erk has been reported earlier during chikv infection in non-immune bhk cell lines (60) . another report suggested that, the nuclear localization of erk1/2 (un-phosphorylated form) in the uninfected microglia cells increases after chikv infection in astrocytes and this might be due to the release of some factor(s) from infected astrocytes in vitro (59) . in this study, it was found that inhibition of p38 signaling by sb reduces nsp2 protein expression and new viral progeny release remarkably, whereas inhibition of jnk signaling by higher concentration of sp could reduce nsp2 moderately as compared to dmso control. this result indicates that both p38 and jnk play pro-viral role in chikv infection in the host macrophages and similar observations have been reported previously in case of other viral infections (60, (89) (90) (91) . the encephalomyocarditis virus infection was suppressed in l929 cells by sb, mainly through the reduction of the viral protein synthesis (89) . whereas, in the human enterovirus 71 infection it was shown that the blockage of virus induced p-p38 leads to significant reduction in both viral protein and progeny release (90) . further investigation can be carried out on other chikv proteins and rna synthesis to understand the pro-viral role of the p-p38 in viral replication in details. mapks are known to regulate tnf production via p-c-jun in other inflammation models (50) . here, it was observed that the expression of p-c-jun is dependent on p-jnk pathway (as sp reduces p-c-jun expression in a dose dependent manner), whereas induction of p-irf3 is dependent on both mapks (p38 and jnk) during chikv infection in macrophages. therefore, it is quite possible that the p-jnk pathway induction by chikv leads to the activation of antiviral responses via p-irf3 and proinflammatory responses (tnf) via p-c-jun pathway. on the other hand, p-p38 is involved in activating both pro-viral and antiviral pathways (via induction of p-irf3) (figure 9 ). it has also been observed during this investigation that pro-inflammatory tnf production was decreased significantly during sb treatment. this might be due to the marked inhibition of chikv infection, however the possibilities of the involvement of other factors cannot be ignored. tnf may promote the activation and proliferation of t cells and thereby regulate the overall t cell mediated effector function (72) . in mouse model system, it has been demonstrated that host t cells are induced during experimental chikv infection and are associated with chikv mediated pathogenesis (43, 44, 46) . in the present study, we found that chikv infected macrophage culture supernatant may facilitate tcr driven activation of resting t cells as compared to the mock supernatant. further, the use of neutralizing anti-tnf antibody towards the regulation of the t cell activation suggests that it could be mediated via chikv induced macrophage derived tnf. additionally, presence of either sb or sp in the chikv infected macrophage supernatant also able to reduce t cell activation in vitro, indicating an effect of macrophage derived tnf on t cell activation during chikv infection. except few cases, chikv is not fatal, however, the long-term polyarthralgia, arthritis-like symptoms along with severe inflammation remain a concern for most of the chronic patients (20, 25, (92) (93) (94) (95) . tnf is one of the key mediator of arthritis or arthritis-like diseases in humans by triggering severe inflammation. despite the elevation of several other inflammatory cytokines in ra, anti-tnf therapy holds a promise for the effective treatment against it (96, 97) , which might be exploited against chikv pathogenesis in future. further, the co-immunoprecipitation analysis revealed that chikv-nsp2 interacts with both p-p38 and p-jnk upon infection in the host macrophages. this was also supported by the in silico analysis of the protein-protein interaction of chikv-nsp2 with p38 and jnk. the phosphorylation lip of p38 was found to interact with nsp2 due to the observed close fit model and a polar interaction between thr-180 of p38 and gln-273 of nsp2. residues from the n-terminus of nsp2 were also suggested to have strong (<2 å) polar interactions with the other residues around this active site. unlike this, the interaction of chikv-nsp2 showed poor fit with the phosphorylation lip of jnk and close (<2 å) polar interaction was also observed for residues from n-terminus of nsp2. these interactions might be one of the yet unknown strategies to utilize host signaling pathways through protein-protein interactions for effective viral infection (97) (98) (99) , which can be explored further in details. viral proteins are found to be phosphorylated by various kinases, which in turn regulate its functions, stability and interactions with other cellular and viral proteins (100) . however, the precise role of the viral protein phosphorylation (especially in alphavirus) has not been reported yet. in this investigation, nsp2 was found to interact with the phosphorylation lip of p38, hence, in silico analysis was carried out using ptm prediction tools, gps (group-based phosphorylation scoring method) (101, 102) and netphos 3.1, to predict the target phosphorylation sites of nsp2 in a kinase specific manner (103) . the gps is a groupbased phosphorylation algorithm, which predicts kinase-specific phosphorylation sites among different host protein kinase groups according to specific sequence pattern (101, 102) . whereas, the netphos server is based on an artificial neuronal network (ann) that allows the users to choose between generic predictions based on the given protein sequence or kinase-specific predictions (103, 104) . out of several predictions, both the softwares predicted t5, s28, and s513 sites in chikv-nsp2 with a high probability of phosphorylation by p38 (supplementary table s3 ). further, to elucidate whether positions of these amino acids in chikv-nsp2 is associated with any consensus regions of functional importance, the predicted peptides were searched in the expasy-prosite protein database. surprisingly, the peptide "fkedkayspevalne" with s513 (at the middle, red) showed a hit with alphavirus nsp2 protease domain belonging to the c9 cysteine protease family (105) . since we have shown earlier that p38 interacts strongly with chikv nsp2 (with phosphorylation lip) and the inhibition of p38 activation strongly reduces chikv infection, it might be possible that, p38 phosphorylates either nsp2 directly or through the association of other client protein(s) which in turn may modulate its function. however, further studies are required to corroborate the chikv-nsp2 phosphorylation by host kinases and its functional consequences on infection and pathogenesis. in summary, for the first time it has been shown that chikv triggers robust tnf production (a key mediator of chikv induced inflammation) in the host macrophages via both p-p38 and p-jnk/p-c-jun pathways and viral protein nsp2 interacts with both the mapks during infection. furthermore, chikv induced macrophage derived tnf was found to facilitate t cell activation in vitro. hence, this information might shed light in rationale-based drug designing for the control of the disease caused by chikv in future. an epidemic of virus disease in southern province, tanganyika territory an epidemic of virus disease in southern province, tanganyika territory, in 1952-53; an additional note on chikungunya virus isolations and serum antibodies an epidemic of virus disease in 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interest this is an open-access article distributed under the terms of the creative commons attribution license (cc by). the use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited key: cord-332071-bqvn3ceq authors: lee, jeong seok; park, seongwan; jeong, hye won; ahn, jin young; choi, seong jin; lee, hoyoung; choi, baekgyu; nam, su kyung; sa, moa; kwon, ji-soo; jeong, su jin; lee, heung kyu; park, sung ho; park, su-hyung; choi, jun yong; kim, sung-han; jung, inkyung; shin, eui-cheol title: immunophenotyping of covid-19 and influenza highlights the role of type i interferons in development of severe covid-19 date: 2020-07-10 journal: sci immunol doi: 10.1126/sciimmunol.abd1554 sha: doc_id: 332071 cord_uid: bqvn3ceq although most sars-cov-2-infected individuals experience mild coronavirus disease 2019 (covid-19), some patients suffer from severe covid-19, which is accompanied by acute respiratory distress syndrome and systemic inflammation. to identify factors driving severe progression of covid-19, we performed single-cell rna-seq using peripheral blood mononuclear cells (pbmcs) obtained from healthy donors, patients with mild or severe covid-19, and patients with severe influenza. patients with covid-19 exhibited hyper-inflammatory signatures across all types of cells among pbmcs, particularly up-regulation of the tnf/il-1β-driven inflammatory response as compared to severe influenza. in classical monocytes from patients with severe covid-19, type i ifn response co-existed with the tnf/il-1β-driven inflammation, and this was not seen in patients with milder covid-19. interestingly, we documented type i ifn-driven inflammatory features in patients with severe influenza as well. based on this, we propose that the type i ifn response plays a pivotal role in exacerbating inflammation in severe covid-19. currently, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which causes coronavirus disease 2019 , is spreading globally (1, 2) , and the world health organization (who) has declared it a pandemic. as of june 2, 2020, more than 6.1 million confirmed cases and more than 376,000 deaths have been reported worldwide (3) . sars-cov-2 infection usually results in a mild disease course with spontaneous resolution in the majority of infected individuals (4) . however, some patients, particularly elderly patients develop severe covid-19 infection that requires intensive care with mechanical ventilation (4, 5) . the mortality rate for covid-19 in wuhan, china, is estimated to be 1.4% (5) . although this rate is lower than that of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which are caused by other human pathogenic coronaviruses (6) , it is much higher than that of influenza, a common respiratory viral disease requiring hospitalization and intensive care in severe cases. in severe cases of covid-19, a hyper-inflammatory response, also called a cytokine storm, has been observed and is suspected of causing the detrimental progression of covid-19 (7) . circulating levels of pro-inflammatory cytokines, including tnf and il-6, are increased in severe cases (8) . gene expression analyses have also shown that il-1related pro-inflammatory pathways are highly up-regulated in severe cases (9) . in a murine model of sars-cov infection, a delayed, but considerable type i ifn (ifn-i) response coronavirus immunophenotyping of covid-19 and influenza highlights the role of type i interferons in development of severe covid-19 (page numbers not final at time of first release) 2 promotes the accumulation of monocytes-macrophages and the production of pro-inflammatory cytokines, resulting in lethal pneumonia with vascular leakage and impaired virusspecific t-cell responses (10) . immune dysfunction is also observed in patients with covid-19. in severe cases, the absolute number of t cells is reduced (8, 11) , and the t cells exhibit functional exhaustion with the expression of inhibitory receptors (12, 13) . however, hyper-activation of t cells as reflected in the up-regulation of cd38, hla-dr, and cytotoxic molecules was also reported in a lethal case of covid-19 (14) . immune dysfunction in patients with severe covid-19 has been attributed to pro-inflammatory cytokines (15) . in the present study, we performed single-cell rna-seq (scrna-seq) using peripheral blood mononuclear cells (pbmcs) to identify factors associated with the development of severe covid-19 infection. by comparing covid-19 and severe influenza, we report that the tnf/il-1β-driven inflammatory response was dominant in covid-19 across all types of cells among pbmcs, whereas the up-regulation of various interferon-stimulated genes (isgs) was prominent in severe influenza. when we compared the immune responses from patients with mild and severe covid-19 infections, we found that classical monocytes from severe covid-19 exhibit ifn-i-driven signatures in addition to tnf/il-1β-driven inflammation. pbmcs were collected from healthy donors (n=4), hospitalized patients with severe influenza (n=5), and patients with covid-19 of varying clinical severity, including severe, mild, and asymptomatic (n=8). pbmcs were obtained twice from three (the subject c3, c6, and c7) of the eight covid-19 patients at different time points during hospitalization. pbmc specimens from covid-19 patients were assigned to severe or mild covid-19 groups according to the national early warning score (news; mild < 5, severe ≥ 5) evaluated on the day of whole blood sampling (16) . in news scoring, respiratory rate, oxygen saturation, oxygen supplement, body temperature, systolic blood pressure, heart rate, and consciousness were evaluated (16) . severe influenza was defined when hospitalization was required irrespective of news score. patients with severe influenza were enrolled from december 2015 to april 2016, prior to the emergence of covid-19. the severe covid-19 group was characterized by significantly lower lymphocyte count and higher serum level of creactive protein than the mild covid-19 group on the day of blood sampling (fig. s1a) . multiplex real-time pcr for n, rdrp, and e genes of sars-cov-2 was performed, and there was no statistical difference in ct values for all three genes between two groups (fig. s1b ). demographic information is provided with experimental batch of scrna-seq in table s1 and clinical data in table s2 and s3. employing the 10x genomics scrna-seq platform, we analyzed a total of 59,572 cells in all patients after filtering the data with stringent high quality, yielding a mean of 6,900 umis per cell and detecting 1,900 genes per cell on average (table s4 ). the transcriptome profiles of biological replicates (pbmc specimens in the same group) were highly reproducible (fig. s1c) , ensuring the high quality of the scrna-seq data generated in this study. to examine the host immune responses in a cell type-specific manner, we subjected 59,572 cells to t-distributed stochastic neighbor embedding (tsne) based on highly variable genes using the seurat package (17) and identified 22 different clusters unbiased by patients or experimental batches of scrna-seq (fig. 1a, fig. s1d ). these clusters were assigned to 13 different cell types based on well-known marker genes and two uncategorized clusters ( fig. 1b and c, and table s5 ). in downstream analysis, we only focused on 11 different immune cell types, including igg -b cell, igg + b cell, effector memory (em)-like cd4 + t cell, non-em-like cd4 + t cell, emlike cd8 + t cell, non-em-like cd8 + t cell, natural killer (nk) cell, classical monocyte, intermediate monocyte, non-classical monocyte, and dendritic cell (dc) after excluding platelets, red blood cells (rbcs), and two uncategorized clusters. the subject c8 (asymptomatic case) was also excluded due to a lack of replicates. in hierarchical clustering, most transcriptome profiles from the same cell type tended to cluster together, followed by disease groups, suggesting that both immune cell type and disease biology, rather than technical artifacts, are the main drivers of the variable immune transcriptome (fig. s1e) . as a feature of immunological changes, we investigated the relative proportions of immune cells among pbmcs in the disease groups compared to the healthy donor group ( fig. 1d and e, and fig. s1f ). unlike the limited changes in mild covid-19, significant changes were observed in both influenza and severe covid-19 across multiple cell types among pbmcs. in severe covid-19, the proportion of classical monocytes significantly increased whereas those of dcs, non-classical monocytes, intermediate monocytes, nk cells, em-like cd8 + t cells, and em-like cd4 + t cells significantly decreased (fig. 1e) . in severe influenza, the proportion of classical monocytes significantly increased whereas those of dcs, non-emlike cd4 + t cells, em-like cd4 + t cells, igg + b cells, and igg -b cells significantly decreased. we validated the proportions of immune cell subsets from scrna-seq by flow cytometry analysis. the relative proportions of total lymphocytes, b cells, cd4 + t cells, cd8 + t cells, nk cells, and total monocytes from scrna-seq significantly correlated with those from flow cytometry analysis (fig. s1g ). first release: 10 july 2020 immunology.sciencemag.org (page numbers not final at time of first release) 3 in order to compare the effect of infection between diseases, we performed hierarchical clustering based on relative gene expression changes against the healthy donor group. unexpectedly, all types of cells among pbmcs were clustered together according to the disease groups instead of cell-types ( fig. 2a) . further investigation of the variable genes based on k-means clustering supported covid-19-specific up-or down-regulated gene expression patterns across all types of cells among pbmcs (fig. s2a) . these results indicate that, in covid-19, peripheral blood immune cells may be influenced by common inflammatory mediators regardless of cell type. despite distinct transcriptional signatures between covid-19 and influenza, severe covid-19 and influenza shared transcriptional signatures in all types of monocytes and dcs (black boxed region in fig. 2a ), possibly reflecting common mechanisms underlying the innate immune responses in severe influenza and severe covid-19. next, we sought to identify relevant biological functions in disease-specific up-or down-regulated genes in terms of the go biological pathways. first, we combined both mild and severe covid-19 as a covid-19 group and identified disease-specific changes in genes for each cell type compared to the healthy donor group using model-based analysis of single cell transcriptomics (mast) (18) . nfkb1, nfkb2, irf1, and cxcr3 were specifically up-regulated in covid-19, and cxcl10, stat1, tlr4, and genes for class ii hla and immunoproteasome subunits were specifically up-regulated in influenza (table s6) . tnf, tgfb1, il1b, and ifng were commonly up-regulated. when we directly compared covid-19 and influenza, nfkb1, nfkb2, and tnf were up-regulated in covid-19, whereas stat1, tlr4, and genes for immunoproteasome subunits were up-regulated in influenza. for each group of differentially expressed genes (degs), we identified the top 10 enriched go biological pathways and collected them to demonstrate p-value enrichment in each group of degs (fig. 2b ). both distinct and common biological functions were identified as illustrated by inflammatory response genes being highly active in both covid-19 and influenza, but genes for transcription factors, including inflammatory factors (i.e., nfkb1/2, and stat4) were up-regulated in covid-19. in contrast, a limited response in genes associated with the ifn-i and -ii signaling pathways, t-cell receptor pathways, and adaptive immune response was observed in covid-19 compared to influenza. such disease-specific gene expression patterns were exemplified at single cell resolution by gbp1 (ifn-γ-mediated signaling pathway) being specifically up-regulated in influenza, crem (positive regulation of transcription) being specifically up-regulated in covid-19, and ccl3 (inflammatory response) being commonly up-regulated ( fig. 2c and table s7) . we expanded our analysis in a cell type specific manner by conducting weighted gene correlation network analysis (wgcna) (19) for the collected genes associated with fig. 2b . we identified several modular expression patterns ( fig. 2d and table s8 ). in the covid-19 group, nfkb1/2, jun, and tnf were modularized in cd8 + t and nk cells (g6 and g7 in fig. 2d ), and il1b, nfkbid, and osm were modularized in all types of monocytes and dcs (g3 in fig. 2d ). in the influenza group, gbp1, tap1, stat1, ifitm3, oas1, irf3, and ifng were modularized in all types of t cells and nk cells (g2 in fig. 2d ), and cxcl10 and tlr4 were modularized in all types of monocytes and dcs (g5 and part of g6 in fig. 2d ). consistently, the degs between covid-19 and influenza were dominant in cd8 + t cells and all types of monocytes (fig. s2b ). to uncover disease-specific transcriptional signatures in cd8 + t cells, we performed sub-clustering analysis from emlike and non-em-like cd8 + t cell clusters using seurat (17) . each disease group-specifically enriched sub-clusters compared to the two other groups were identified in the non-emlike cd8 + t cell cluster (fig. 3a) . of the six sub-clusters from the non-em-like cd8 + t cell cluster, cluster 1 and cluster 3 were significantly enriched in the influenza and covid-19 groups, respectively ( fig. 3b and c, and s3a). clusters with the high expression of ppbp, a marker of platelets, were excluded in following analysis (e.g., cluster 6 in fig. s3a ). intriguingly, up-regulated genes in cluster 1 and cluster 3 were associated with previously defined gene sets for 'influenza a virus infection' and 'sars-cov infection', respectively ( fig. s3b ) (20) . we also found that the cluster 3-specific up-regulated genes reflect activation of immune response, including cd27, rgs1, ccl5, sell, and rgs10 ( fig. s3c and table s9 ). protein interaction network analysis of selected top 30 upregulated genes in each cluster based on string v11 (21) revealed the up-regulation of prf1, gnly, gzmb, and gzmh in cluster 1 and the up-regulation of gzmk, gzma, cxcr3, and ccl5 in cluster 3 (fig. 3d, green) . stat1, tap1, psmb9, and psme2, which are up-regulated preferentially by ifn-γ, were overexpressed only in influenza-specific cluster 1 (fig. 3d , blue). we validated these data by intracellular staining for granzyme b and pma/ionomycin-stimulated intracellular cytokine staining for ifn-γ. the percentages of granzyme b + and ifn-γ + cells among cd8 + t cells were significantly higher in the influenza group than in the covid-19 group (fig. s3d ). of the seven representative go biological pathways for the pro-inflammatory and ifn responses, pathways for responses to ifn-i and -ii were more associated with influenza-specific cluster 1, whereas pathways for the response to tnf or il-1β were more prominent in covid-19-specific cluster 3 ( we performed sub-clustering analysis from all three types of monocyte clusters to find covid-19-specific sub-clusters. however, there was no covid-19-specifically enriched subcluster ( fig. s4a and b ). next, we further focused on classical monocytes considering their crucial roles for inflammatory responses. we investigated degs between influenza and covid-19 to seek covid-19-specific transcriptional signatures in classical monocytes (fig. 4a) . interestingly, tnf and il1b, major genes in the inflammatory response, were identified as covid-19-specific and commonly up-regulated genes, respectively. to better characterize the transcriptional signatures in classical monocytes, we performed k-means clustering of up-regulated genes in at least one disease group compared to the healthy donor group. we identified five different clusters of up-regulation ( fig. 4b and table s10 ): genes in cluster 1 are commonly up-regulated in all disease groups, cluster 2 is influenza-specific, cluster 3 is associated with mild/severe covid-19, cluster 4 is associated with influenza and severe covid-19, and cluster 5 is severe covid-19specific. we examined each cluster-specific genes by gene set enrichment analysis (gsea) using cytokine-responsive gene sets originated from each cytokine-treated cells (lincs l1000 ligand perturbation analysis in enrichr) (22) . covid-19-specific cluster 3 genes were enriched by tnf/il-1βresponsive genes whereas influenza-specific cluster 2 genes were enriched by ifn-i-responsive genes in addition to tnf/il-1β-responsive genes (fig. 4c) , indicating that the ifn-i response is dominant in influenza compared to covid-19. we confirmed this result by analyzing cluster-specific genes with cytokine-responsive gene sets originated from other sources (fig. 4d ). unexpectedly, cluster 4 and 5 exhibited strong associations with ifn-i-responsive genes, in addition to tnf/il-1β-responsive genes ( fig. 4e ), indicating that severe covid-19 acquires ifn-i-responsive features in addition to tnf/il-1β-inflammatory features. next, we directly compared classical monocytes between mild and severe covid-19. when we analyzed degs, severe covid-19 was characterized by up-regulation of various isgs, including isg15, ifitm1/2/3, and isg20 (fig. 5a ). both tnf/il-1β-responsive genes and ifn-i-responsive genes were enriched in severe covid-19-specific up-regulated genes (fig. 5b ). we measured plasma concentrations of tnf, il-1β, il-6, ifn-β, ifn-γ, and il-18 in a larger cohort of covid-19 patients. among these cytokines, il-6 and il-18 were significantly increased in severe covid-19 compared to mild covid-19 whereas there was no difference in plasma concentrations of the other cytokines between the two groups ( fig. s5a) . these results indicate that cytokine-responsive gene signatures cannot be simply explained by a few cytokines because of overlapped effects of cytokines. to further investigate the characteristics of severe covid-19, we performed a trajectory analysis with monocle 2 (23) using two internally well-controlled specimens (one severe and one mild) in which both pbmc samples were collected from a single patient (the subject c7) with covid-19. trajectory analysis aligned classical monocytes along the disease severity with cluster 1 and cluster 3 corresponding to later and earlier pseudotime, respectively (fig. 5c ). representative genes in cluster 1 was enriched in the severe stage and highly associated with the both ifn-i and tnf/il-1β-associated inflammatory response (fig. 5d, fig. s5b , and table s11 ). gsea confirmed that both the ifn-i response and tnf/il-1β inflammatory response were prominent in cluster 1, but not in cluster 3 (fig. 5e ). cluster 1 exhibited a significantly higher association with a gene set from systemic lupus erythematosus, which is a representative inflammatory disease with ifn-i features, than cluster 3 (fig. 5f, left) , but was not significantly associated with a gene set from rheumatoid arthritis (fig. 5f, right) . we obtained additional evidence of the ifn-i-potentiated tnf inflammatory response in severe covid-19 by analyzing a gene module that is not responsive to ifn-i, but associated with tnf-induced tolerance to tlr stimulation. park et al. previously demonstrated that tnf tolerizes tlr-induced gene expression in monocytes, though tnf itself is an inflammatory cytokine (24) . they also showed that ifn-i induces a hyper-inflammatory response by abolishing the tolerance effects of tnf, and defined a gene module responsible for the ifn-i-potentiated tnf-nf-κb inflammatory response as 'class 1' (24) . this gene module was significantly enriched in cluster 1, but not in cluster 3 (fig. 5g ), which suggests that the ifn-i response may exacerbate hyper-inflammation by abolishing a negative feedback mechanism. finally, we validated ifn-i response and inflammatory features using bulk rna-seq data obtained using post-mortem lung tissues from patients with lethal covid-19 (25) . although the analysis was limited to only two patients without individual cell-type resolution, in genome browser, up-regulation of ifitm1, isg15, and jak3 and down-regulation of rps18 were observed commonly in post-mortem covid-19 lung tissues and classical monocytes of severe covid-19 (fig. 6a ). in the analysis with cytokine-responsive gene sets, both the ifn-i response and tnf/il-1β-inflammatory response were prominent in the lung tissues (fig. 6b) . degs in the lung tissues were significantly associated with cluster 4, which is commonly up-regulated in both influenza and severe covid-19, and cluster 5, which is specific to severe covid-19 in fig. 4b (fig. 6c) . these genes were also significantly associated with the cluster 1 identified in the trajectory analysis, but not with cluster 3 (fig. 6d ). when gene sets were defined by degs between mild and severe covid-19, the degs in post-mortem lung tissues were significantly associated with genes up-regulated specifically in severe covid-19 (fig. 6e ). severe covid-19 has been shown to be caused by a hyperinflammatory response (7) . particularly, inflammatory cytokines secreted by classical monocytes and macrophages are considered to play a crucial role in severe progression of covid-19 (26) . in the current study, we confirmed the results from previous studies by showing that the tnf/il-1β inflammatory response is dominant in covid-19 although a small number of patients were enrolled. however, we also found that severe covid-19 is accompanied by the ifn-i response in addition to the tnf/il-1β response. these results indicate that the ifn-i response might contribute to the hyper-inflammatory response by potentiating tnf/il-1β-driven inflammation in severe progression of covid-19. in the current study, we carried out scrna-seq using pbmcs instead of specimens from the site of infection, e.g., lung tissues or bronchoalveolar lavage (bal) fluids. however, hierarchical clustering based on relative changes to the healthy donor group showed that all types of cells among pbmcs were clustered together according to the disease groups as shown in fig. 2a , indicating that there is diseasespecific global impact across all types of cells among pbmcs. this finding suggests that peripheral blood immune cells are influenced by common inflammatory mediators regardless of cell type. however, we could not examine granulocytes in the current study because we used pbmcs, not whole blood samples for scrna-seq. in transcriptome studies for cytokine responses, we often analyze cytokine-responsive genes rather than cytokine genes themselves. however, we cannot exactly specify responsible cytokine(s) from the list of up-regulated genes because of overlapped effects of cytokines. for example, up-regulation of nf-κb-regulated genes can be driven by tnf, il-1β or other cytokines, and up-regulation of ifn-responsive genes can be driven by ifn-i or other interferons. in the current study, we designated the ifn-i response because many up-regulated ifn-responsive genes were typical isgs. recently, wilk et al. also performed scrna-seq using pbmcs from covid-19 patients and healthy controls (27) . similar to our study, they found ifn-i-driven inflammatory signatures in monocytes from covid-19 patients. however, they did not find substantial expression of pro-inflammatory cytokine genes such as tnf, il6, il1b, ccl3, ccl4 and cxcl2 in peripheral monocytes from covid-19 patients whereas we detected the up-regulation of tnf, il1b, ccl3, ccl4 and cxcl2 in the current study. moreover, they found a developing neutrophil population in covid-19 patients that was not detected in our study. these discrepant results might be due to different platforms for scrna-seq. wilk et al. used the seq-well platform whereas we used the 10x genomics platform that is more generally used. we also note that recent scrna-seq analyses of covid-19 sometimes lead to unrelated or contradictory conclusions to each other despite the same platform (28, 29) . although it often occurs in unsupervised analysis of highly multi-dimensional data, more caution will be required in designing scrna-seq analysis of covid-19, including definition of the severity and sampling time points. recently, blanco-melo et al. examined the transcriptional response to sars-cov-2 in in vitro infected cells, infected ferrets, and post-mortem lung samples from lethal covid-19 patients and reported that ifn-i and -iii responses are attenuated (25) . however, we noted that ifn-i signaling pathway and innate immune response genes were relatively up-regulated in post-mortem lung samples from lethal covid-19 patients compared to sars-cov-2-infected ferrets in their paper. given that sars-cov-2 induces only mild disease without severe progression in ferrets (30), we interpret that ifn-i response is up-regulated in severe covid-19 (e.g., postmortem lung samples from lethal covid-19 patients), but not in mild covid-19 (e.g., sars-cov-2-infected ferrets). indeed, severe covid-19-specific signatures discovered in our current study were significantly enriched in the publically available data of post mortem lung tissues from the blanco-melo et al.'s study although the analysis was limited to only two patients without individual cell-type resolution (fig. 6) . in a recent study, zhou et al. also found a robust ifn-i response in addition to pro-inflammatory response in bal fluid of covid-19 patients (31) . moreover, up-regulation of ifn-iresponsive genes has been demonstrated in sars-cov-2infected intestinal organoids (32) . although ifn-i has direct antiviral activity, their immunopathological role was also reported previously (33) . in particular, the detrimental role of the ifn-i response was elegantly demonstrated in a murine model of sars (10) . in sars-cov-infected balb/c mice, the ifn-i response induced the accumulation of pathogenic inflammatory monocytesmacrophages and vascular leakage, leading to death. it was proposed that a delayed, but considerable ifn-i response is critical for the development of acute respiratory distress syndrome and increased lethality during pathogenic coronavirus infection (6, 34) . currently, the management of patients with severe covid-19 relies on intensive care and mechanical ventilation without a specific treatment because the pathogenic mechanisms of severe covid19 have not yet been clearly elucidated. in the current study, we demonstrated that severe covid-19 is characterized by tnf/il-1β-inflammatory features combined with the ifn-i response. in a murine model of sars-cov infection, timing of the ifn-i response is a critical factor determining outcomes of infection (6, 10) . delayed ifn-i response contributes to pathological inflammation whereas early ifn-i response controls viral replication. therefore, we propose that anti-inflammatory strategies targeting not only inflammatory cytokines, including tnf, il-1β, and il-6, but also pathological ifn-i response needs to be investigated for the treatment of patients with severe covid-19. patients diagnosed with covid-19 were enrolled from asan medical center, severance hospital, and chungbuk national university hospital. sars-cov-2 rna was detected in patients' nasopharyngeal swab and sputum specimens by multiplex real-time reverse-transcriptase pcr using the allplex 2019-ncov assay kit (seegene, seoul, republic of korea). in this assay, n, rdrp, and e genes of sars-cov-2 were amplified, and ct values were obtained for each gene. sars-cov-2-specific antibodies were examined using the sars-cov-2 neutralization antibody detection kit (genscript, piscataway, nj) and were positive in all covid-19 patients in convalescent plasma samples or the last plasma sample in a lethal case. hospitalized patients diagnosed with influenza a virus infection by a rapid antigen test of a nasopharyngeal swab were also enrolled from asan medical center and chungbuk national university hospital from december 2015 to april 2016, prior to the emergence of covid-19. patients' clinical features, laboratory findings, and chest radiographs were collected from their electronic medical records at each hospital. this study protocol was reviewed and approved by the institutional review boards of all participating institutions. written informed consent was obtained from all patients. peripheral blood mononuclear cells (pbmcs) were isolated from peripheral venous blood via standard ficoll-paque (ge healthcare, uppsala, sweden) density gradient centrifugation, frozen in freezing media, and stored in liquid nitrogen until use. all samples showed a high viability of about 90% on average after thawing. single-cell rna-seq libraries were generated using the chromium single cell 3′ library & gel bead kit v3 (10x genomics, pleasanton, ca) following the manufacturer's instructions. briefly, thousands of cells were separated into nanoliter-scale droplets. in each droplet, cdna was generated through reverse transcription. as a result, a cell barcoding sequence and unique molecular identifier (umi) were added to each cdna molecule. libraries were constructed and sequenced as a depth of approximately 50,000 reads per cell using the nextseq 550 or novaseq 6000 platform (illumina, san diego, ca). the sequenced data were de-multiplexed using mkfastq (cellranger 10x genomics, v3.0.2) to generate fastq files. after de-multiplexing, the reads were aligned to the human reference genome (grch38; 10x cellranger reference grch38 v3.0.0), feature-barcode matrices generated using the cellranger count, and then aggregated by cellranger aggr using default parameters. the following analysis was performed using seurat r package v3.1.5 (17) . after generating the feature-barcode matrix, we discarded cells that expressed <200 genes and genes not expressed in any cells. to exclude low-quality cells from our data, we filtered out the cells that express mitochondrial genes in >15% of their total gene expression as described in previous studies (29, 35, 36) . doublets were also excluded, which were dominant in the cluster "uncategorized 1". although there was a high variability in the number of umis detected per cell, majority of cells (90.5%) were enriched in a reasonable range of the umis (1,000 -25,000), and 59% of cells with less than 1,000 umis were platelet or rbc excluded in downstream analysis. in each cell, the gene expression was normalized based on the total read count and log-transformed. to align the cells originating from different samples, 2000 highly variable genes from each sample were identified by the vst method in seurat r package v3.1.5 (17) . using the canonical correlation analysis (cca), we found anchors and aligned the samples based on the top 15 canonical correlation vectors. the aligned samples were scaled and principal component analysis (pca) conducted. finally, the cells were clustered by unsupervised clustering (0.5 resolution) and visualized by tsne using the top 15 principal components. to identify marker genes, up-regulated genes in each cluster relative to the other clusters were selected based on the wilcoxon rank sum test in seurat's implementation with >0.25 log fold change compared to the other clusters and a bonferroni-adjusted p < 0.05 (table s4) . by manual inspection, among the 22 different clusters, 20 were assigned to 11 known immune cell types, rbcs which are characterized by hba1, hba2, and hbb, and platelets. the clusters characterized by similar marker genes were manually combined as one cell type. the two remaining clusters were assigned to 'uncategorized 1' and 'uncategorized 2' because they had no distinct features of known cell types. based on the distribution of umi counts, the cluster 'uncategorized 1' was featured by relatively high umis per cell compared to other clusters and presence of higher expression of multiple cell type marker genes. the cluster 'uncategorized 2' was featured by a b celllike signatures and high expression of ribosomal protein genes, not recommended to be further analyzed according to the 10x platform guideline. in these aspects, rbcs, platelets, uncategorized 1, and uncategorized 2 were excluded in downstream analysis. to check the reproducibility of biological replicates (individuals within a same group), we calculated the spearman's rank correlation coefficient for umi counts that were merged according to each individual. the correlation coefficients of all individual pairs within the same group were visualized by a boxplot (covid-19, n=45; flu, n=10; hd, n=6). in fig. s1e , to investigate the similarity of the transcriptomes between cell types across diseases, we merged the umi counts of each cell type according to healthy donor, influenza, mild covid-19, and severe covid-19. next, the umi counts for each gene were divided by the total umi count in each cell type and multiplied by 100,000 as the normalized gene expression. based on a median expression value >0.5, we calculated the relative changes in gene expression divided by the median value for each gene. hierarchical clustering analysis was performed based on the pcc of the relative change in gene expression. in fig. 2a and fig. s2a , to compare the highly variable gene expression among mild and severe covid-19 and influenza relative to healthy donors, the normalized gene expression used in fig. s1e was divided by the values in the healthy donor group. we selected the highly variable genes in terms of the top 25% standard deviation followed by log2-transformation (pseudo-count =1). in fig. 2a , hierarchical clustering analysis was performed based on the pccs of the selected highly variable genes. for fig. s2a , to investigate the expression patterns of the selected highly variable genes (n=6,052), k-means clustering (k=50) was performed based on euclidean distance. we manually ordered the clusters and visualized them as a heat map, revealing four distinct patterns: influenza-specific (n=1,046), covid-19 specific (n=1,215), influenza/covid-19 common (n=1,483), and cell type-specific (n=2,308). to investigate the dynamic changes in cell type composition, we calculated the proportion of cell types in each individual. as a control, we calculated the relative variation in each cell type composition between all pairs of healthy donors. similarly, for each disease group, we calculated the relative variation in each cell type by dividing the fraction of the cell type in individual patient by that of individual healthy donor. after log2-transformation, we conducted statistical analysis using the relative variation in composition between the control and disease groups using a two-sided kolmogorov-smirnov test. for any two transcriptome profiles, to identify degs, we utilized the model-based analysis of single cell transcriptomics (mast) algorithm in seurat's implementation based on a bonferroni-adjusted p<0.05 and a log2 fold change > 0.25. in fig. 2b , the degs in covid-19 and influenza compared to healthy donors or covid-19 compared to influenza were identified at cell type resolution. all degs were combined according to the disease groups for further analysis. the overlapping up or down degs between covid-19 and influenza compared to healthy donors were defined as 'common up' or 'common down'. the specific degs in covid-19 or influenza were assigned as 'covid-19 up/down' or 'flu up/down', respectively. in addition, covid-19-specific up-or down-regulated genes compared to influenza were assigned as 'covid-19>flu' or 'flu>covid-19', respectively. the gene ontology analysis was performed by david. for each group of degs, the top 10 enriched go biological pathways were selected, resulting in 49 unique go biological pathways across all groups. the -log10(p-values) are shown as a heat map in fig. 2b . the weighted gene correlation network analysis (wgcna) was conducted with the genes listed in the top 10 go biological pathways of 'covid-19 up', 'flu up', and 'common up' defined in fig. 2b . the normalized gene expression values of the genes in covid-19 were divided by the values in influenza and log2-transformed (pseudo-count =1). we used default parameters with the exception of soft threshold =10 and networktype = 'signed' when we constructed a topological overlap matrix. the modular gene expression patterns were defined using cutreedynamic with a minclustersize of 5. we visualized the modular gene expression pattern as a heat map in which the cell types were ordered according to hierarchical clustering with the default parameters of hcluster in r. to find disease-specific subpopulations, each immune cell type was subjected to the subclustering analysis using seurat. briefly, the highly variable genes (n=1000) were selected based on vst and then scaled by scaledata in seurat with the vars.to.regress option to eliminate variation between individuals. the subpopulations were identified by findclusters with default parameters, except resolution (non-em-like cd8 + t cells, 0.3; classical monocytes, 0.2); the inputs were the top eight principal components (pcs) obtained from pca of the scaled expression of the highly variable genes. the subpopulations were visualized by tsne using the top eight pcs. the trajectory analysis was performed with 2000 highly variable genes in classical monocytes across mild (c7-2) and severe (c7-1) covid-19 as defined by the vst method in seurat. the following analysis was performed using monocle2. briefly, the input was created from the umi count matrix of the highly variable genes using the newcelldataset function with default parameters, except expressionfamily = 'negbinomial.size'. the size factors and dispersion of gene expression were estimated. the dimension of the normalized data was reduced based on ddrtree using reducedimension with default parameters, except scaling = false, which aligned the cells to the trajectory with three distinct clusters. to determine genes that gradually changed along the trajectory, we identified the degs using mast between clusters 1 and 3, which represent the severe stage and mild stage, respectively. the expression patterns of representative degs were visualized along the pseudotime after correction with estimated size factors and dispersion for all genes. in fig. 4b , we performed k-means clustering of degs among all pairs of mild covid-19, severe covid-19, and influenza. the log2-transformed relative gene expression of degs compared to healthy donors was subjected to k-means clustering (k=10). here, we used up-regulated degs in at least one disease group compared to the healthy donor group. we manually assigned five clusters based on gene expression patterns. the transcriptome profiles of post-mortem lung tissues from two lethal cases of covid-19 and biopsied heathy lung tissues from two donors were downloaded from a public database (gse147507). the degs were identified using deseq2 based on a bonferroni-adjusted p < 0.05 and a log2 fold change > 1. enrichment analysis using enrichr and gsea 4.0. 3 enrichr, the web-based software for gene set enrichment analysis (gsea) was used for lincs l1000 ligand perturbation analysis (22) , virus perturbation analysis, and disease perturbation analysis from the geo database. 'combined score' was calculated as a parameter of enrichment as the log(p-value) multiplied by the z-score from the fisher exact test. gsea 4.0.3 software was used to conduct the gsea when a ranked list of genes was available (fig. 5g, fig. 6c -e) (37) . results for ifn-γ-responsive genes were not presented because those were considerably overlapped with ifn-αresponsive genes, which are typical isgs. the normalized enrichment score and fdr-q value were calculated to present the degree and significance of enrichment. cryopreserved pbmcs were thawed, and dead cells were stained using the live/dead fixable cell stain kit (invitrogen, carlsbad, ca). cells were stained with fluorochrome-conjugated antibodies, including anti-cd3 (bv605; bd biosciences), anti-cd4 (bv510; bd biosciences), anti-cd8 (bv421; bd biosciences), anti-cd14 (pe-cy7; bd biosciences), anti-cd19 (alexa fluor 700; bd biosciences), and anti-cd56 (vio-bright fitc; miltenyi biotec). for staining with antigranzyme b (bd biosciences), cells were permeabilized using a foxp3 staining buffer kit (ebioscience). for intracellular cytokine staining of ifn-γ, pbmcs were stimulated with phorbol 12-myristate 13-acetate (pma, 50 ng/ml) (sigma aldrich) and ionomycin (1 μg/ml) (sigma aldrich). brefeldin a (golgiplug, bd biosciences) and monesin (golgistop, bd biosciences) were added 1 hour later. after another 5 hours of incubation, cells were harvested for staining with the live/dead fixable cell stain kit, anti-cd3, anti-cd4, and anti-cd8. following cell permeabilization, cells were further stained with anti-ifn-γ (alexa fluor 488; ebioscience). flow cytometry was performed on an lsr ii instrument using facsdiva software (bd biosciences) and the data analyzed using flowjo software (treestar, san carlos, ca). cytokines were measured in plasma samples, including ifn-β, il-18 (elisa, r&d systems, minneapolis, mn), il-1β (cytometric bead array flex kit, bd biosciences, san jose, ca), tnf, il-6, and ifn-γ (legendplex bead-based immunoassay kit, biolegend, san diego, ca). we performed the ks test to compare the distributions of two groups without assuming that the distributions follow normality. welch's t test was conducted to compare the two distributions after confirming the normality of the distributions using the shapiro-wilk normality test. a wilcoxon signed rank test was conducted to compare the differences between two groups with paired subjects. the mann-whitney test was performed to compare the means of two groups. statistical analyses were performed using prism software version 5.0 (graphpad, la jolla, ca). p<0.05 was considered significant. immunology.sciencemag.org/cgi/content/full/5/49/eabd1554/dc1 fig. s1 . clinical characteristics and assessment of the quality of scrna-seq results. fig. s2 . transcriptome features of highly variable genes. fig. s3 . characterization of disease-specific cd8+ t-cell subpopulations. fig. s4 . subpopulation analysis of classical monocytes. fig. s5 . string analysis of up-regulated genes in cluster 1 obtained from the trajectory analysis of classical monocytes. table s1 . experimental batches of scrna-seq. first release: 10 july 2020 immunology.sciencemag.org (page numbers not final at time of first release) 9 table s2 . clinical characteristics of severe influenza patients. table s3 . clinical characteristics of covid-19 patients. table s4 . the scrna-seq results. table s5 . a list of marker genes for each cluster. table s6 . a list of degs and associated biological pathways in fig. 2b . table s7 . cell types in which the gbp1, crem, and ccl3 were upregulated in fig. 2c . table s8 . a list of genes in each module obtained from wgcna in fig. 2d . table s9 . a list of up-regulated genes in non-em-like cd8+ t-cell subpopulations. table s10 . a list of genes included in each cluster defined by k-mean clustering of classical monocytes. table s11 . a list of genes up-regulated in early and late pseudotime. a new coronavirus associated 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interrogate lincs l1000 gene expression signatures single-cell mrna quantification and differential analysis with census ivashkiv, type i interferons and the cytokine tnf cooperatively reprogram the macrophage epigenome to promote inflammatory activation tenoever, i, imbalanced host response to sars-cov-2 drives development of covid-19 pathological inflammation in patients with covid-19: a key role for monocytes and macrophages a single-cell atlas of the peripheral immune response in patients with severe covid-19 single-cell landscape of bronchoalveolar immune cells in patients with covid-19 covid-19 severity correlates with airway epitheliumimmune cell interactions identified by single-cell analysis infection and rapid transmission of sars-cov-2 in ferrets heightened innate immune responses in the respiratory tract of covid-19 patients sars-cov-2 productively infects human gut enterocytes disease-promoting effects of type i interferons in viral, bacterial, and coinfections a conserved dendritic-cell regulatory program limits antitumour immunity accelerating medicines partnership rheumatoid arthritis & systemic lupus erythematosus (amp ra/sle) consortium, notch signalling drives synovial fibroblast identity and arthritis pathology pgc-1alpha-responsive genes involved in oxidative phosphorylation are coordinately downregulated in human diabetes key: cord-355847-1ru15s5a authors: convertino, irma; tuccori, marco; ferraro, sara; valdiserra, giulia; cappello, emiliano; focosi, daniele; blandizzi, corrado title: exploring pharmacological approaches for managing cytokine storm associated with pneumonia and acute respiratory distress syndrome in covid-19 patients date: 2020-06-11 journal: crit care doi: 10.1186/s13054-020-03020-3 sha: doc_id: 355847 cord_uid: 1ru15s5a sars-cov-2 complications include pneumonia and acute respiratory distress syndrome (ards), which require intensive care unit admission. these conditions have rapidly overwhelmed healthcare systems, with detrimental effects on the quality of care and increased mortality. social isolation strategies have been implemented worldwide with the aim of reducing hospital pressure. among therapeutic strategies, the use of immunomodulating drugs, to improve prognosis, seems promising. particularly, since pneumonia and ards are associated with a cytokine storm, drugs belonging to therapeutic classes as anti-il-6, anti-tnf, and jak inhibitors are currently studied. in this article, we discuss the potential advantages of the most promising pharmacological approaches. lymphocytopenia. in patients requiring icu admission, an increase in neutrophil count, d-dimer, blood urea, and creatinine levels have been detected as well as more severe lymphocytopenia. this condition is defined as a "cytokine storm" and is associated with high circulating levels of interleukins (il)-2, il-6, il-7, il-10, granulocyte colonystimulating factor (g-csf), 10 kda interferon-gammainduced protein (ip-10), monocyte chemo-attractant protein-1 (mcp-1), macrophage inflammatory protein 1α (mip-1α), and tumor necrosis factor (tnf) [4, 5] . in particular, in ards patients, a strong depletion of peripheral blood t cells, along with a decreased recruitment of lymphocytes and neutralizing antibodies and an increased production of cytokines, was detected in the lungs [6] . this network of pathogenic factors is thought to drive a severe immune-mediated interstitial pneumonia and a delayed pulmonary clearance of covid-19 [3] . current evidence supports a close relationship between cytokine storm and disease severity. indeed, icu patients displayed higher serum levels of cytokines (g-csf, ip-10, mcp-1, mip-1α, and tnf-α) than those not requiring icu. for instance, il-6 and tnf-α levels in icu patients were significantly higher when compared with non-icu ones [7] . patients with fatal outcome had higher serum concentrations of il-6 than those survived: the il-6 median levels reported by zou et al. were 11.00 pg/ml (iqr 7.50-14.40) and 6.30 pg/ml (iqr 5.00-7.90), respectively, p < 0.0001. similar findings were showed by ruan et al. 11 .4 pg/ml (iqr 8.5) in dead patients versus 6.8 pg/ml (iqr 3.61) in discharged ones [8, 9] . furthermore, a significantly close relationship between il-6 levels in critical covid-19 patients with fatal outcome (64.0 pg/ml, iqr 25.6-111.9) and rnaemia was found, in particular, the 83.3% of patients with il-6 > 100 pg/ml had positive levels of rnaemia, r 0.902. this suggests that high serum il-6 along with rnaemia could be predictors of mortality [10] . additionally, not only critically ill patients with ards have been associated with high cytokine serum levels but also non-severe patients with covid-19. indeed, il-6 median serum levels were 36.10 pg/ml (iqr 23.00-59. 20) in severe patients compared with 10.60 pg/ml (iqr 5. 13-24.18) in those with mild disease, p 0.002 [11] , and 6.69 pg/ml (iqr 4. 44-12.43) in patients with spo 2 ≥ 90% in comparison with 51.69 pg/ml (iqr 34.31-161.65) in those with spo 2 < 90%, p < 0.001, as well as the tnf-α levels (2.08 pg/ml, iqr 1.93-2.35 even in the conditions with spo 2 ≥ 90%) [12] . these data were confirmed by qin et al.; il-6 median serum levels in severe and non-severe patients were 25.2 pg/ml (iqr 9.5-54.5) and 13.3 pg/ml (iqr 3.9-41.1), respectively; and tnf-α median serum levels were 8.7 pg/ml (iqr 7.1-11.6) in severe patients and 8.4 pg/ml (iqr 6.9-10.4) in non-severe ones [6] . based on this knowledge, it has been proposed that the modulation of the above cytokines could represent an interesting approach to improve the prognosis of patients with covid-19 pulmonary complications, both pneumonia and ards. recently, the food and drug administration has allowed the emergency use of a device aiming at purifying blood of icu patients from the cytokine storm [13] . several drugs, endowed with modulating activity on cytokine pathways, including anti-il-6, anti-tnf, and janus kinase (jak) inhibitors, currently approved for the treatment of immune-mediated inflammatory diseases, have been suggested or could be yet taken into account for experimental use in covid-19 patients with ards and/or pneumonia ( fig. 1 ). tocilizumab is a humanized, immunoglobulin g1κ (igg1κ) anti-human il-6 receptor (il-6r) monoclonal antibody approved for some immune-mediated inflammatory rheumatic diseases. clinical evidence supports the view that this drug is an effective therapeutic option, with a good risk-benefit profile, in cytokine storm syndromes [14] . in china, its off label use has been tested in 21 icu ards patients with favorable results after 24-48 h in 20/21 patients [15] . moreover, a multicenter randomized clinical trial in covid-19 patients with ards, treated with tocilizumab at a dose of 4~8 mg/kg once, and an additional same dose when fever persists within 24 h after the first administration, has been approved in china [16] . the italian medicine agency has recently authorized a trial on the use of tocilizumab in covid-19 patients with ards [17] . this initiative was pushed on also by promising results published on italian newspapers. particularly, some patients treated with tocilizumab at the "pascale" cancer institute in naples showed disease improvements within 24 h and one of them did not require mechanical ventilation 2 days after starting tocilizumab [15] . another monoclonal antibody belonging to anti-il-6 drug class, siltuximab, currently approved in multicentric castleman disease with hiv-negative and human herpesvirus-8 negative, is under investigation for ards in covid-19 patients. in particular, an observational case-control study evaluating siltuximab in icu patients with ards-related covid-19 is performing at papa giovanni xxiii hospital in bergamo, italy [18] . preliminary results have shown promising outcomes as the clinical improvement in the 33% of treated icu patients [19] . in addition, a multicenter open-label randomized clinical trial is studying the benefit risk profile of siltuximab, as a single therapeutic option or in combination with anakinra, at a single dose of 11 mg/kg, in comparison with tocilizumab or anakinra, alone or in combination, in ards patients with covid-19 [20] . evidence suggested a higher binding affinity to il-6 involving siltuximab than tocilizumab but less insights are currently available on the effects of siltuximab in cytokine storm [21] . based on the results expected with tocilizumab and siltuximab, other anti-il-6 drugs, currently approved for rheumatoid arthritis, namely sarilumab and sirukumab, could be studied in ards and pneumonia patients with covid-19. notably, sarilumab has higher affinity for its target and a longer half-life than tocilizumab; thus, a sustained therapeutic effect could be achieved by administration of only one single dose [22, 23] . on march 19 th , 2020, a clinical trial evaluating the efficacy and safety of high dose and low dose of sarilumab in covid-19 patients was started [24] . subsequently, further clinical trials have followed, investigating the benefit risk profile of sarilumab in patients with covid-19related ards, at a dose of 200 mg or 400 mg, as single or repeated administration, subcutaneously or intravenously [25] [26] [27] [28] . sirukumab neutralizes il-6 specifically and directly by preventing its binding to its membrane receptor [29] , and thus, it leads to a subsequent suppression of il-6 biological actions. in a phase i trial, sirukumab showed linear pharmacokinetics with long half-life, low immunogenicity, and a good safety profile [30] . accordingly, it could represent a promising pharmacological option for counteracting the high il-6 levels occurring in ards patients. anti-tnf drugs, including infliximab, adalimumab, etanercept, golimumab, and certolizumab, could be tested also for covid-19-related ards and pneumonia. in china, a clinical trial on adalimumab in covid-19 patients was recently approved [31] . infliximab, adalimumab, and golimumab are igg 1 monoclonal antibodies, while etanercept is a fusion protein of two human tnf type 2 receptor moieties linked with the fc region of a human immunoglobulin, and certolizumab is the pegylated fab domain obtained from a humanized anti-tnf igg monoclonal antibody [32] . differences in the inhibitory mechanism were shown among these drugs, due to their different molecular binding patterns with tnf sites [33] . all anti-tnf drugs display higher binding affinity to soluble tnf than its membrane-bound form, with golimumab and etanercept showing the highest level [34] . greater binding avidity to soluble tnf was reported for etanercept than infliximab and adalimumab [35] . heterogeneity was also found in the neutralizing activity of anti-tnf drugs to soluble tnf, while that to transmembrane tnf was comparable [34] . infliximab and adalimumab displayed a greater binding activity for fcγrii and fcγriii than etanercept, but the latter was able to bind fcγri with higher affinity [36] . fcγrs play important roles in the modulation of immune responses, which rely on cytokines and vasoactive mediators [37] . in addition, a review showed that the proteins coded by the virus alter the complement system control and thus contribute to lung viral damages [3] . out of the anti-tnf drugs, the igg 1 monoclonal antibodies have a complement-dependent cytotoxicity activity [38] that could be explored in the covid-19 infection. the known differences in pharmacokinetics and pharmacodynamics among anti-tnf drugs support the need for testing these agents in covid-19-related ards and pneumonia patients without particular priorities, in order to identify the best option. other selection criteria, including the administration route, the possible positive or negative interactions resulting from combination with other drugs (i.e., hydroxychloroquine) and the costs (i.e., the use of biosimilar anti-tnf available) should be considered. anti-jak drugs (such as ruxolitinib, tofacitinib, baricitinib, oclacitinib, fedratinib, upadacitinib, and peficitinib) [39] should be considered also among the options for clinical investigations in covid-19-related ards and pneumonia patients. jaks are involved in jak/stat signaling associated with the receptors of a large variety of cytokines. in particular, stat-1 and stat-3 pathways are activated by binding of il-6 to its receptor (il-6r) [40] . tofacitinib acts as a non-selective inhibitor of all known jaks (jak1, jak2, jak3, tyk2) with moderate specificity for jak1 and jak3. baricitinib inhibits selectively jak1 and jak2 [41] . both are approved by the european medicines agency (ema); baricitinib for rheumatoid arthritis and tofacitinib for both rheumatic disorders and ulcerative colitis. ruxolitinib is a jak1/ jak2 inhibitor approved by the food and drug administration (fda) for psoriasis, myelofibrosis, and rheumatoid arthritis. upadacitinib (anti-jak1), fedratinib (anti-jak2), and oclacitinib (anti-jak1) were approved by fda for rheumatoid arthritis, myelofibrosis, and dermatitis, respectively [42] . peficitinib (anti-jak3) was approved for rheumatoid arthritis only in japan [43] . tofacitinib and upadacitinib showed potent inhibitory activities on jak1/3-dependent cytokines, both pathways being involved in lymphocyte activation. tofacitinib, baricitinib, and upadacitinib displayed also inhibitory actions on the jak2/tyk2-dependent signaling of il-3, gm-csf, and g-csf. tofacitinib was shown to act as the most potent inhibitor of g-csf (jak2/tyk2). moreover, tofacitinib, baricitinib, and upadacitinib inhibited il-6 and interferon (ifn) γ (jak1/2), as well as il-10 and ifn-α (jak1/tyk2), with tofacitinib appearing as the strongest inhibitor of il-6, ifn-γ, and il-10 signals [44] . evidence suggests that baricitinib, fedratinib, and ruxolitinib are also inhibitors of numb-associated kinases (nak), involved in viral endocytosis and replication. baricitinib showed the highest affinity for aak1 than ruxolitinib and fedratinib. thus, besides exerting putative anti-inflammatory effects in ards patients, it is expected also to reduce viral infectivity [45, 46] . of note, a clinical trial on such antiviral effect is going to start with ruxolitinib [47] , and an open-label trial is evaluating its efficacy and safety at a dose of 10 mg twice a day in covid-19 patients with ards [48] . furthermore, an expanded access program of the 5 mg ruxolitinib formulation is ongoing in severe covid-19 patients with ≥ 6 years old [49] . finally, an open label clinical trial is evaluating the benefit risk profile of baricitinib at a dose of 2 mg a day for 10 days in moderate and severe adult covid-19 patients [27] . whenever jak inhibitors could be identified as an effective pharmacological option in covid-19-related ards and pneumonia, their cost and safety issues, particularly the risk of thromboembolic events for some of them, should be taken into account [50] . several drugs 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first global approval. drugs comparison of baricitinib, upadacitinib, and tofacitinib mediated regulation of cytokine signaling in human leukocyte subpopulations covid-19: combining antiviral and anti-inflammatory treatments baricitinib as potential treatment for 2019-ncov acute respiratory disease covid-19 registered trials -and analysis -cebm treatment of sars caused by covid-19 with ruxolitinib -full text view -clinicaltrials.gov ruxolitinib managed access program (map) for patients diagnosed with severe/very severe covid-19 illness -full text view -clinicaltrials.gov restrictions in use of xeljanz while ema reviews risk of blood clots in lungs | european medicines agency publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions ic, mt, and cb contributed to the idea; ic, mt, sf, gv, ec, df, and cb to the data searching and interpretation; and ic, mt, and cb to the redaction of the manuscript and ic, mt, sf, gv, ec, df, and cb reviewed the final version. the authors read and approved the final manuscript. no funding has been received to perform this article.availability of data and materials not applicable. key: cord-301946-erzh30mt authors: kwak-kim, joanne; ota, kuniaki; sung, nayoung; huang, changsheng; alsubki, lujain; lee, sungki; han, jae won; han, aera; yang, xiuhua; saab, wael; derbala, youssef; wang, wen-juan; he, qiaohua; liao, aihua; takahashi, toshifumi; cavalcante, marcelo borges; barini, ricardo; bao, shihua; fukui, atsushi; coulam, carolyn title: covid-19 and immunomodulation treatment for women with reproductive failures date: 2020-06-12 journal: j reprod immunol doi: 10.1016/j.jri.2020.103168 sha: doc_id: 301946 cord_uid: erzh30mt covid-19 pandemic is affecting various areas of health care, including human reproduction. many women with reproductive failures, during the peri-implantation period and pregnancy, are on the immunotherapy using immune modulators and immunosuppressant due to underlying autoimmune diseases, cellular immune dysfunction, and rheumatic conditions. many questions have been raised for women with immunotherapy during the covid-19 pandemic, including infection susceptibility, how to manage women with an increased risk of and active covid-19 infection. sars-cov-2 is a novel virus, and not enough information exists. yet, we aim to review the data from previous coronavirus outbreaks and current covid-19 and provide interim guidelines for immunotherapy in women with reproductive failures. with the coronavirus disease 2019 (covid-19) pandemic, patient care has been significantly challenged not only for the covid-19 cases but for the others, including pregnant women with a history of reproductive failures (rf), such as recurrent pregnancy losses (rpl), repeated implantation failures (rif), with immune etiologies including autoimmune diseases, cellular immune dysfunction, and rheumatic conditions. these rf women with immune etiology (rfi) may have been on various immunosuppressants, immunomodulators, or anticoagulants. whether these treatment modalities increase the risk for covid-19 infection and aggravate covid-19, have been a major concern for already vulnerable pregnant women with rfi. sars-cov (severe acute respiratory syndrome coronavirus), which caused the sars outbreak in 2003, infects macrophages and t cells (perlman and dandekar 2005) and induces various cytokines, such as type i ifn, tnf-α, il-1, etc., and b cell-related antibodies (prompetchara et al. 2020) . however, it is unclear if sars-cov-2 infects the same kinds of immune effectors. sars-cov-2 has been speculated to induce the influx of neutrophils and monocytes/macrophages at the infection site, which results in hyperproduction of proinflammatory j o u r n a l p r e -p r o o f cytokines. specific t helper (th) 1 and th17 cells may be activated and contribute to exacerbating inflammatory responses. b cells and plasma cells produce sars-cov-2 specific antibodies that may neutralize viral particles (prompetchara et al. 2020) . b cell reduction was reported in the early phase of the covid-19, which in turn, affects antibody production (lin et al. 2020) , and severe lymphopenia was often manifested in severe covid-19 cases (zhu et al. 2020b) . in hospitalized severe covid-19 patients, high levels of various cytokines, including il-2, il-7, il-10, g-csf, inducible protein-10 (ip-10), monocyte chemoattractant protein-1 (mcp-1), macrophage inflammatory protein-1a (mip-1a), and tnf-α, were observed (prompetchara et al. 2020) . the primary cause of mortality in severe cases was cytokine storm, and these findings were in line with sars and middle east respiratory syndrome (mers) (price et al. 2020) the novel sars-cov-2 uses the angiotensin-converting enzyme 2 (ace2) for the cellular entry like sars-cov and mainly spreads through the respiratory tract (guo et al. 2020) . the ace2 mrna is abundantly expressed in endometrial epithelial cells in the secretory phase (vaz-silva et al. 2009 ), ovaries, and testes (honorato-sampaio et al. 2012) . renin-angiotensin system (ras) is involved in female reproductive processes, including follicular development (barreta et al. 2015) , steroid hormone production, oocyte maturation, and ovulation (reis et al. 2011). considering the reported relationship between ace2 and viral pneumonia (xu et al. 2020 ), covid-19 may attack the follicular membrane and granular cells of the ovary, affecting folliculogenesis and the quality of oocytes, and causing female infertility and pregnancy losses. additionally, it may damage endometrial epithelial cells and affect early embryo implantation, although no studies suggested that covid-19 has any specific effect on the female reproductive system. the specific effect on pregnancy has not been reported in covid-19 cases, although studies are limited. however, previous studies have confirmed that the viral pandemic, such as the sars pandemic, 2009 h1n1 influenza, or 2013 mers, was associated with an increased incidence of maternal and perinatal complications, such as spontaneous abortion, premature delivery, and intrauterine growth restriction (assiri et al. 2016) . sars-cov and mers-cov, however, did not show any vertical transmission (assiri et al. 2016) . a recent analysis of 38 pregnant women with covid-19 showed no intrauterine or transplacental transmission of sars-cov-2 from mother to fetus (schwartz 2020) . women with a history of rfi already have an increased risk of obstetrical complications. therefore, the continuation of immunotherapy is critical for these women. in this review, we aim to deliver the interim guidelines for current immunotherapy for women with rfi, concerning the covid-19 pandemic. this article was developed by the international collaboration of experts who have been working in the field of reproductive immunology. prednisone is a synthetic corticosteroid that mainly acts as an immunosuppressant. for its potent and broad-spectrum anti-inflammatory and immune-suppressive properties, prednisone has been utilized to treat autoimmune and chronic inflammatory diseases, and often the first line immunotherapeutic agent prescribed in the painful context of repeated implantation failure (rif) or rpl of immune etiologies (rhen and cidlowski 2005) . prednisone also plays an important role in mediating proper folliculogenesis, increasing production of growth factors, suppressing androgenic hormones, and suppressing nk cell activity and th1/th2 cell ratios (keay et al. 2001 ). in early reports (quenby et al. 2003) , corticosteroid therapy has been reported to be beneficial in women with consecutive miscarriages since it reduces endometrial nk cells in women with rpl (quenby et al. 2005) . however, in routine in-vitro fertilization (ivf) cycles, the cochrane review did not demonstrate the benefit of peri-implantation corticosteroid administration (boomsma et al. 2012 ). on the other hand, prednisolone, which has a relatively mild effect, was reported to improve the embryo implantation rate after ivf and protect against miscarriage when administered from embryo implantation through the early placentation phase (robertson et al. 2016) . it is reported that less than half of rif patients with immune deregulation may be prednisone responders and would benefit from its administration (ledee et al. 2018 ). low-dose prednisolone treatment was suggested to have a beneficial effect on covid-19 and have been widely used to treat covid-19 induced lung injury or septic shock (russell et al. 2020 ). however, current interim guidance from who advises against the routine use of corticosteroids for the management of suspected and confirmed covid-19 cases (world health organization 2020). currently, no data is available for women in early gestation with covid-19 infection, although previously, 57.1% of miscarriage rate was reported in women infected with sars-cov in the early pregnancy (wong et al. 2004) . therefore, pregnant women with rfi and infected with covid-19 may have an increased risk of early pregnancy loss. overall, no specific evidence exists to demonstrate whether women infected with covid-19 in the early pregnancy might get benefit from corticosteroids to prevent miscarriage. therefore, prednisone is not recommended for women with early pregnancy and covid-19 infection unless otherwise indicated. in pregnant women with rfi, low dose prednisone treatment can be continued if the covid-19 infection chance is low. if the patient has an increased risk of covid-19 infection or j o u r n a l p r e -p r o o f on high dose prednisone treatment, tapering off or decreasing the dose is recommended to reduce the susceptibility to viral infection. if women have covid-19 infection, tapering off the prednisone treatment is recommended to avoid worsening the disease, and switching into another treatment modality should be considered for the maintenance of pregnancy. women with rfi are often accompanied by thrombophilia, such as antiphospholipid syndrome and inherited thrombophilia. anticoagulants are commonly used to treat the thrombotic features of rfi during pregnancy or assisted reproductive technology (art). with the pandemic spread of sars-cov-2, many reports and guidelines concerning pregnancy are being updated, but there are only a few reports about heparin use in rfi patients. so, it is necessary to address some recommendations regarding anticoagulation treatment in rfi women with covid-19. heparin is primarily used for anticoagulation in women with rfi, and low molecular weight heparin (lmwh) is the drug of choice due to its numerous advantages over unfractionated heparin (ufh). these drugs are safe in pregnancy, and in addition to its antithrombotic effect, heparin provides a favorable environment for implantation and placental development (nelson and greer 2008) . by adhering to selectin, heparin initiates the implantation process and downregulates e-cadherin expression in the decidua, which promotes trophoblast invasion. additionally, heparin enhances the heparin-binding epidermal growth factor, which is important in preventing the apoptosis of trophoblast (nelson and greer 2008) . lmwh has been reported to have various immunological effects, including inhibition of tnf-α and il-6 in placental villous (mousavi et al. 2015 , zenerino et al. 2017 ). moreover, heparin binds to certain proteins that exhibit an antiviral effect. many different viruses, including herpes simplex virus (hsv)-1 and -2, attach to heparan sulfate on the host cell surface at the beginning of infection (trybala et al. 2000) . because of the similarity in structure between heparan sulfate and heparin, heparin interacts with viral proteins in hsv, human immunodeficiency virus (hiv), and dengue virus, which inhibits viral entry into the cells (nelson and greer 2008 (krusat and streckert 1997 , scagliarini et al. 2004 , basu et al. 2007 , pourianfar et al. 2014 , khanna et al. 2017 , gonzalez et al. 2018 , liu et al. 2018 . furthermore, replication of sars-cov nsp 15 protein was also inhibited by heparin in vitro (bhardwaj et al. 2004) . from the chinese experience of covid-19, heparin was recommended to rescue severe cases with coagulopathy induced by sars-cov-2 infection. a research group suggested the use of 40-60 mg enoxaparin/day or ufh, 10,000-15,000 iu/day may decrease the mortality of severe covid-19 patients with sepsis-induced coagulopathy (tang et al. 2020) . similarly, a review study proposed to inject lmwh 100 iu/kg, every 12 hours for 3-4 days, if the level of d-dimer becomes four times higher than the upper normal limit, unless contraindicated (lin et al. 2020 for all pregnant rfi women with thrombophilia, heparin treatment can be continued even with covid-19 infection. intravenous immunoglobulin g (ivig) is a blood product, which comprises pooled igg from the serum of thousands of donors. ivig is primarily used as a replacement treatment for immunodeficiencies but also indicated for autoimmune and inflammatory disorders. studies have reported a potential benefit of ivig when applied for patients with rfi (kwak et al. 1996) , such as increased nk cell levels and cytotoxicity, elevated th1/th2 cell ratios, and antiphospholipid antibody syndrome (ruiz et al. 1996 , kwak-kim et al. 2003 . the immunomodulatory effects of ivig are mediated through two functional domains: f(ab')2, antigen-binding fragment, and fc, crystallizable fragment. the f(ab')2 fragment plays a role in neutralizing cytokine and autoantibody, scavenging of complements, killing of target cells by antibody-dependent cytotoxicity, and blocking cell-cell interactions mediated by cell-surface receptors. the fc fragment modulates by activating and inhibiting fcγ receptor (fcrs) expression on immune cells (schwab and nimmerjahn 2013) . ivig has shown efficacy in the treatment of patients with influenza (liu et al. 2016a ) and sars (ho et al. 2004) , including sars cases with j o u r n a l p r e -p r o o f leukopenia and thrombocytopenia (wang et al. 2004) . it is speculated that massive ivig treatment (300~500mg/kg/day, 5days) may become an effective treatment to interrupt cytokine storm for severe covid-19 cases and prevent lung injury by blocking fcr in covid-19 with pneumonia (fu et al. 2020 , lin et al. 2020 (cao et al. 2020) . ivig, either conventional or created from recovered patients after covid-19 were suggested as a treatment option combined with antiviral drugs to neutralized covid-19 (jawhara 2020). ivig is a plasma protein therapy, which might be of concern for the risk of virus contamination. during the manufacturing process, viral particles are inactivated and removed by solvent-detergent, low ph incubation, nanofiltration, or others process (dichtelmller et al. 2009 , dichtelmller et al. 2011 , caballero et al. 2014 . the plasma protein therapeutics associations (ppta) has issued that the sars-cov-2 is not a concern for the safety of plasma protein therapies, including immunoglobulin, manufactured by ppta member companies (plasma protein therapeutic associations 2020). with the currently available data, it is unlikely that the use of ivig in patients with rfi will impact the chances of contracting the disease or negatively affect the clinical course in women with covid-19 infection during pregnancy. hence, ivig treatment can be continued for these women during the covid-19 pandemic. lit has been proposed for the treatment of couples with a history of rpl of unknown etiology since 1981 (taylor and faulk 1981) . however, in 2002, its effectiveness in treating of knowledge about the impact of lit on the immune response in covid-19 cases, we suggest the suspension of lit for women with rfi during covid-19 pandemic period. intralipid has been reported to be an effective treatment for women experiencing rfi who display elevated uterine nk cells (coulam and acacio 2012) . however, the level of evidence is level iii or iv because of significant heterogeneity across the studies using various methods to quantify nk cells. measurement of the number of nk cells does not address the more significant issue of the biological activity of these cells (roussev et al. 2007) . 3) continue to care for patients who are currently "in-cycle" or who require urgent stimulation and cryopreservation; 4) suspend elective surgeries and non-urgent diagnostic procedures and 5) minimize in-person interactions and increase utilization of telehealth. in view of these recommendations, fertility treatment, often combined by intralipid for reproductive problems, should be curtailed. patients with the essential fatty acid deficiency have a susceptibility to infection (fell et al. 2015) , and intralipid infusion is indicated for these patients. (roussev et al. 2007) , although intralipid infusion has been utilized for parenteral nutritional supplementation for severe covid-19 cases (caccialanza et al. 2020 ). hydroxychloroquine, a multi-purpose drug, was initially used as an antimalarial agent in 1955 (wallace 1996) . subsequently, anti-inflammatory and immunomodulatory properties of hydroxychloroquine have been reported (sciascia et al. 2016) , and it has been utilized for autoimmune diseases, such as systemic lupus erythematosus rheumatoid arthritis, antiphospholipid antibody syndrome, and rfi (sciascia et al. 2016 , ghasemnejad-berenji et al. 2018 . it exerts its role in the immune system via multiple mechanisms. one of the mechanisms is inhibiting the tolllike receptors (tlr-3, 7, and 9), which bind to nucleic acid released from trophoblast into the maternal circulation as a response of placental hypoxemia, thus inhibiting an inflammatory immune response (scharfe-nugent et al. 2012) . another mechanism is inhibiting the release of th1 cytokines, such as tnf-α and ifn-γ (weber and levitz 2000) , leading to a switch toward a th2 predominance which promotes maternal-fetal tolerance (wegmann et al. 1993 ). over the past few decades, hydroxychloroquine has been proposed to be utilized for certain viral infections since it inhibits cytokine production by t cells, including il-1, il-2, il-6, il-18, tnf-α, ifn-γ, and th17 related cytokines, reduces chemokines, such as ccl2 (mcp-1) and cxcl10 (ip-10), inhibits micro-rna expression, and decreases the synthesis of dna, rna, and proteins in thymocytes (al-bari 2015). since its function is mainly immune-modulatory, infection risk has been reported not to be increased in pregnant patients with hydroxychloroquine treatment, even though pregnancy and autoimmunity are associated with increased susceptibility and severity of infections (maddur et al. 2010 , sappenfield et al. 2013 , unless patients have autoimmune flareups or on the other types of immunosuppressant, like prednisone or cyclophosphamide (danza and ruiz-irastorza 2013) . hydroxychloroquine is a chloroquine analog. both drugs have been recommended by the control and prevention 2020, colson et al. 2020, food and drug administration 2020) . in vitro study has shown that both drugs inhibit sars-cov, sars-cov-2, and other coronaviruses, while the efficacy of hydroxychloroquine is relatively higher than chloroquine (wang et al. 2020a , yao et al. 2020 . chloroquine seems to be effective in the prevention and treatment of covid-19 pneumonia (cortegiani et al. 2020 , multicenter collaboration group of department of et al. 2020). hydroxychloroquine seems promising in attenuating the severe progression of covid-19 and suppress the cytokine storm by inhibiting the activation of t cells (devaux et al. 2020) . a recent study reported that 600mg of plaquenil daily was associated with a significant reduction of covid-19 viral load within three to six days, and its effect was reinforced by azithromycin (gautret et al. 2020 ). there are on-going studies regarding the role of hydroxychloroquine in the treatment and prevention of covid-19. in light of the available data, urgent cessation or tapering off hydroxychloroquine and chloroquine are not necessary for pregnant women with rfi, autoimmune diseases, or rheumatic conditions due to the covid-19 pandemic. if patients are not currently pregnant or planning fertility treatment in the future, it is reasonable to hold off hydroxychloroquine and resume it before the conception cycle due to the current national shortage of these drugs. in covid-19 cases, the continuation of plaquenil treatment should be further discussed with their primary care physician since the contradictory data have been reported in regards to plaquenil and covid-19. tnf-α is one of the most important mediators for acute and chronic systemic inflammatory responses. it promotes the production of other cytokines and chemokines and plays an important role in severe inflammatory conditions such as septic shock (chu 2013) . in patients with sars, plasma tnf-α levels are moderately upregulated (yoshikawa et al. 2009) , although an in vitro study has suggested that tnf-α induction may be mediated by the shedding of ace2, thus allowing cellular entry to coronavirus (haga et al. 2008) . in patients with covid-19, the levels of certain cytokines, including tnf-α, il-6, and il-10, correlate with disease severity (huang et al. 2020) . tnf-α hyperproduction in the serum of patients with covid-19 was a phenomenon that was not observed in patients with sars. therefore, it is possible that anti-tnf-α drugs, which are widely used in the clinical practice of rheumatology, may be effective in patients with covid-19 (fu et al. 2020) . a randomized controlled trial of adalimumab (humira®), a human monoclonal tnf-α antibody, on covid-19 is currently underway in china (chictr2000030089, http://www.chictr.org.cn/showprojen.aspx?proj=49889). j o u r n a l p r e -p r o o f rpl has been reported to be associated with dysregulation of th1 and th 2 immunity to trophoblasts and excessive local tnf-α production, which prevents implantation of the embryo and triggers immunological pregnancy losses (reid et al. 2001) . furthermore, tnf-α inhibitors such as humira® counteract the increased production of th1 cytokines and consequently improve the ivf and embryo transfer success rate by modulating high tnf-α levels (winger et al. 2009 ). pregnant women in the very early stages of pregnancy may benefit from the fertility-promoting effects of tnf-α inhibitor treatment. currently, no clear evidence is present if tnf-α blocker is harmful to patients with covid-19 (russell et al. 2020) . tnf-α inhibitors, as immunomodulators, it is reported that tnfα inhibitors can be continued if viral symptoms by covid-19 are mild; however, if viral symptoms worsen or high fever develops, tnf-α inhibitors should be stopped (price et al. 2020) . therefore, in pregnant women, tnf-α inhibitors may be continued. in pregnant women with covid-19, tnf-α inhibitors may be continued unless viral symptoms worsen and febrile. tacrolimus is a macrolide immunosuppressant, which is widely used in solid organ transplantation, especially the liver, kidney, and heart transplantation. it is a calcineurin inhibitor that suppresses the production of il-2 and thus, inhibits the development and proliferation of t cells (kay et al. 1989 ). it has been reported that tacrolimus regulates t cell subsets in patients with rif and shifted th1/th2 cell ratios. consequently, it promotes the development of a pregnancy and improving the pregnancy outcome of these patients (nakagawa et al.) . recently, the effectiveness of immunosuppressants, such as tacrolimus in women with rfi, has been reported (nakagawa et al. 2015 , nakagawa et al. 2017 ). recent studies have found that il-2, il-6, il-7, ip-10, g-csf, tnf-α, and il-10 were all elevated in most severe covid-19 patients (huang et al. 2020) , suggesting that cytokine storm may be associated with disease severity. furthermore, regulatory t (treg) cells were decreased in patients with severe covid-19 (chen et al. 2020) . regulating immune balance and reducing nonspecific inflammatory responses by immunosuppressants may be beneficial for covid-19 patients with severe inflammation (mehta et al. 2020) . tacrolimus has inhibitory effects on th1 immunity, which may play a critical role in patients with severe covid-19. however, currently, there is a lack of relevant reports on the use of tacrolimus in covid-19, but in severe patients, the use of related cytokine inhibitors and corticosteroid hormones has achieved some results (russell et al. 2020) . theoretically, immunosuppressants used in the post-transplant population may lead to lower immune function, and infection with the virus may cause more severe symptoms. however, an italian study showed no increased risk of severe complications in immunosuppressed patients compared to the general population (children and adults) (d'antiga 2020). it is speculated that due to the preexisting hyperactivated immune responses, the application of immunosuppressive agents may not worsen the outcomes. for patients with severe covid-19 pneumonia, tacrolimus may be an attempted method (russell et al. 2020 ). however, for mild patients, the use of immunosuppressive agents is not conducive to fight against viral infection. renal transplant recipients diagnosed with non-critical covid-19 pneumonia were successfully cured by reducing the use of immunosuppressants, including tacrolimus, mycophenolate, and prednisone and a lowdose methylprednisolone-based treatment regimen (zhu et al. 2020a ). in pregnant women with rif and tacrolimus treatment, tacrolimus can be continued with caution to avoid a viral exposure, but stopping the medication should be considered when viral symptoms present, especially with known or potential exposure for covid-19 (price et al. 2020 ). vitamin d suppresses viral replication while it has immune regulatory effects on various immune effectors (wu et al. 2019 , jakovac 2020 . it reduces the incidence of influenza infections and acute respiratory tract infections (cannell et al. 2006 , martineau et al. 2017 . previously, we reported that women with rpl have an increased prevalence of vitamin d deficiency, and vitamin d plays a major role in auto-and cellular immune responses (ota et al. 2013 , ota et al. 2015 . hence, women with rpl and vitamin d deficiency may have increased susceptibility to covid-19 infection. both sars and the current covid-19 became epidemic in wintertime when vitamin d levels were decreased since uv-b exposure for vitamin d synthesis in the skin is low (yin and wunderink 2018, zhu et al. 2020b) . the mortality rate of covid-19 was lower in countries south of latitude 35 degrees of northern hemisphere (rhodes et al. 2020) , suggesting the role of vitamin d in covid-19. since no specific treatment is present for covid-19, vitamin d supplementation as adjuvant therapy for pregnant women with rfi and covid-19 infection can be considered. at different stages of pregnancy, the immune responses and hormonal status vary, which is closely associated with the outcome of the covid-19 infection (jiao 2020) . therefore, when considering immunosuppressive and immunomodulatory treatment for pregnant women with rfi, j o u r n a l p r e -p r o o f pharmacokinetics, and pharmacodynamics of intended drugs, gestational age, and their current immune profiles should be considered not only for avoiding infection with sars-cov-2 but not to exacerbate sars-cov-2 infection. so far, low dose prednisone, ivig, tacrolimus, and heparin may have some beneficial effect on covid-19. there is no clear evidence for limiting the application of anti-tnf-α agents and intralipid in non-infected women with rfi. most immune therapy can still be used with caution on a case-by-case, and the information is 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g.; bistrian, bruce r. title: role of cytokines in aids wasting date: 1998-12-31 journal: nutrition doi: 10.1016/s0899-9007(98)00108-7 sha: doc_id: 316904 cord_uid: g7dli0a8 abstract there is now a large literature implicating cytokines in the development of wasting and cachexia commonly observed in a variety of pathophysiologic conditions. in the acquired immunodeficiency syndrome (aids), cytokines elicited by primary and secondary infections seem to exert subtle and sustained effects on behavioral, hormonal, and metabolic axes, and their combined effects on appetite and metabolism have been postulated to drive wasting. however, correlations of increased blood levels of a particular cytokine with wasting in aids have not been consistent observations, perhaps because cytokines act principally as paracrine and autocrine hormones, as well as indirectly by activating other systems. a better understanding of the mechanisms underlying the catabolic effects of cytokines is clearly needed if more efficacious strategies are to be developed for the prevention and treatment of wasting in aids. in this review we first examine the interacting factors contributing to the aids wasting syndrome. we then analyze the complex and overlapping role of cytokines in the pathophysiology of this condition, and put forward a number of hypotheses to explain some of the most important features of this syndrome. there are close interrelationships between malnutrition and infection. malnutrition enhances susceptibility to infections, exacerbates their harmful effects, and influences their outcome. infections also can produce malnutrition: the wasting syndrome and cachexia are common complications of infections, and they play an important role in the morbidity and mortality of the aids. indeed, although wasting is not universally observed in aids patients, the wasting syndrome in a human immunodeficiency virus (hiv)-seropositive individual is generally utilized to establish the diagnosis of aids 1 and is defined by a decrease in body mass greater than 10% in the absence of concomitant opportunistic infections, malignancies, and other identifiable causes of weight loss. 1 independent of the causes of wasting, aids patients who experience weight loss beyond a certain percentage of ideal body weight are at greater risk of death, thereby establishing a link between survival and the extent of body cell mass depletion. [2] [3] [4] [5] consequently, reversing wasting should improve the life expectancy and quality of life. 6 however, this has proved to be difficult and the outcome of nutritional supplementation is poor, with the tendency for weight gain to be fat and water and not lean tissue. the causes of malnutrition leading to wasting in aids are believed to be a combination of several factors, which, as shown in table i , can be classified under three main categories: 1) reduced nutrient intake, 2) malabsorption, and 3) metabolic disturbances. of the factors that may underlie the reduction in nutrient intake in hiv patients, anorexia is usually the most prominent. it is primarily the result of the cytokine responses to the infection per se, but it can also be caused or exacerbated (as an unwanted side effect) by certain medications employed in hiv patients receiving multidrug therapy for concomitant medical problems. a diminished ability to ingest nutrients can also be observed in hiv patients suffering from some central nervous system (cns) processes such as dementia or tumors. furthermore, the almost universal presence of inflammatory processes, infections, or both, involving the digestive tract of aids patients producing gingivitis, stomatitis, esophagitis, or enteritis, often impair the ability to ingest, in large part due to pain preventing intake or aggravated by food intake, as well as absorb nutrients. the net effect is that all these factors contribute to a deficit of energy and nutrient supply to meet the body's needs. in addition to reduced nutrient intake, intestinal malabsorption (due to intestinal dysfunction and inflammation) can also be an important contributory factor to malnutrition in aids cases. [7] [8] [9] [10] [11] however, a causal link between abnormal malabsorption tests and wasting is not clear. in hiv-infected children, although postnatal gain in weight and lean body mass were found to be lower than in an hiv-negative comparison group, 12 an earlier study by the same group 13 found that lactose malabsorption was not associated with higher rates of diarrhea or growth failure. this underlies the fact that carbohydrate malabsorption in hiv-infected children is not the only factor responsible for growth failure. moreover, fat malabsorption is generally more clearly related to development of malnutrition. in fact, malabsorption, particularly of fat but also certain minerals and vitamins, is common in patients with protracted diarrhea, and patients with hiv infection are prone to develop a variety of enteric infectious processes that cause diarrhea. these include those due to protozoan, viral, bacterial, and fungal pathogens (see table ii ), as well as neoplasm (lymphoma, kaposi sarcoma) and disseminated infectious processes. the role of several additional enteric pathogens (including enteroaggregative escherichia coli) in the pathogenesis of diarrhea and malabsorption has not been adequately studied and certainly deserves more attention. 14, 15 furthermore, the paucity of food in the intestine over long periods of time, in conjunction with protein-energy malnutrition, may provoke structural changes and functional intestinal atrophy. the resulting decline in digestive functions could further impair absorption of both macronutrients (fat, protein, and carbohydrates) and micronutrients (vitamins and minerals). thus, absorptive dysfunction compounds the problem of reduced nutrient intake and often aggravates anorexia through abdominal symptoms. in fact, in many if not most malabsorption syndromes, it is generally the reduction in oral intake induced by symptoms related to malabsorption (cramps, bloating, pain, and diarrhea) that is responsible for most of the energy gap between intake and total expenditure. like many pathophysiologic conditions such as sepsis and cancer, a hypermetabolic state, characterized by an increase in resting energy expenditure (ree) and disturbances in the metabolism of protein (muscle proteolysis) and fat (hypertriacyglycerolemia), has also been implicated in the aids wasting syndrome. in fact, an earlier notion that hypermetabolism is the major driving force behind wasting in aids has been particularly attractive because it provided an explanation for the excessive loss of muscle, and the difficulty of reversing the wasting syndrome (with nutritional therapy most often resulting in gain in body fat and water, with marginal or no gain in lean tissue). these metabolic abnormalities have been associated with futile cycling, such as that occurring when fatty acids are being mobilized at an accelerated rate from adipose tissue, and then reesterified into triacylglycerol for storage again in fat, 16 as well as the wasteful use of substrates underlying the conversion of glucose into fatty acids, before being stored as fat (i.e., de novo lipogenesis). 17 the contribution of these metabolic pathways to the hypermetabolic state is, however, unclear and hypertriacylglycerolemia in the hiv patients does not correlate with wasting. 18 -21 moreover, although an increase in ree has been reported in all stages of hiv infection, 22-27 the notion that hypermetabolism is the primary factor underlying wasting in aids has been challenged by more recent studies in patients with hiv indicating that the increase in ree per se is not sufficient to cause wasting. 25 first, ree is increased even in asymptomatic hiv-infected patients with normal cd4 cell counts, but such individuals can usually sustain their weight and lean body mass for prolonged periods. second, in patients who were actively losing weight or had stable weight, ree was about 10% higher than normal, but their total energy expenditure (tee) was found to be no greater than that predicted for healthy individuals. these apparent discrepancies between increased ree coexisting with normal or low tee have been reconciled with data indicating that the increased ree is largely compensated by energy saved as a result of reduced physical activity, and it is loss of appetite leading to decreased intake, coupled with malabsorption, that primarily drives wasting in aids. to what extent such reduction in physical activity is the result of lethargy and fatigue from the illness per se or that of a normal adaptive physiologic response to save energy is unknown. however, it is clear that the decrease in physical activity will alter the quality of life, which may further deteriorate because a drastic reduction in locomotor activities might lead not only to muscle atrophy and consequential functional impairments, but can also contribute to the failure to rebuild lean body mass during realimentation. furthermore, an increase in ree during active weight loss is by no means trivial, because this is counterproductive to the adaptive reduction in ree that is the normal response in order to buffer the energy deficit and hence contributes to exacerbate the negative energy balance even further. the most likely scenario in the development of the wasting in hiv-infected patients can be summarized as follows: • despite increased ree in response to hiv-infection, the patients can maintain their weight often for long periods of time, most probably through reductions in the amount of energy they expend on physical activity. this "compensation," however, may still be deleterious because it interferes with lifestyle and increases the susceptibility to muscle atrophy, and consequential functional impairment. • subsequent weight loss is primarily caused by poor appetite, malabsorption, or both, generally triggered by secondary infections. 22,25 furthermore, the persisting hypermetabolic state is counterproductive to the adaptive down-regulation of resting metabolism that normally occurs in uncomplicated starvation, thereby exacerbating the wasting process. • the rate of weight loss and the severity of wasting is likely to be dependent upon the type, severity, and outcome of the secondary infection. aids patients with malnutrition due principally to malabsorptive symptoms seem to restore body weight and lean body mass when recovering from secondary opportunistic infection or when receiving nutritional support, 28,29 similar to what has been observed in non-hiv individuals recuperating from starvation, anorexia nervosa, and other clinical conditions, although both body fat and lean tissue are being recovered, fat is being restored at a disproportionately faster rate relative to lean tissue repletion. 30 -34 however, when protein energy malnutrition is largely a reflection of systemic illness and inflammation, the recovery of lean tissue seems to be even poorer or delayed, such that body weight repletion is more as fat than body protein, similar to patients with sepsis, presumably related to inefficient protein anabolism. 33 it is against this background presentation of the interacting factors contributing to malnutrition and functional impairment in hivinfected patients-namely anorexia, malabsorption, hypermetabolism, lethargy, and impaired fat and protein metabolism-that the role of cytokines in the aids wasting syndrome is discussed in the following sections. in the 1980s, beutler and cerami 35 isolated a 17-kda protein while searching for a mediator to account for the metabolic changes (particularly hypertriacylglycerolemia) observed in animals infected with the protozoan parasite trypanosoma cruzi (chagas' disease agent). this protein was termed cachectin, as it was supposed to mediate the wasting and hypertriacylglycerolemia found in those experimental animals. 35 cachectin was subsequently found to reduce the activity of lipoprotein lipase (lpl) (potentially accounting for lower triacylglycerol turnover in vivo) and to promote lipolysis in vitro. 36 in addition, purified cachectin was able to produce anorexia, weight loss, and fever when injected into experimental animals. subsequently, when the cachectin dna sequence was isolated, it become apparent that its dna sequence was identical to that coding for tumor necrosis factor (tnf). 36 tnf had been previously isolated from the serum of animals injected with bacterial endotoxins and was able to produce necrosis of transplanted tumors in animals. 37 furthermore, administration of purified or recombinant tnf to experimental animals provokes very similar metabolic and hemodynamic changes to those observed during bacterial sepsis that could be blocked by the use of specific antisera or monoclonal antibodies. 38,39 administration of recombinant tnf to humans suggested that this cytokine could play an important role in the early activation of the hemostatic mechanism in septicemia. 40 recent experimental studies with knockout mice (for the 55-kda tnf receptor) have confirmed the importance of tnf in the pathophysiology of endotoxic shock. 41, 42 it is now well established that tnf and other cytokines (e.g., interleukins, interferons) are a group of hormonelike polypeptide mediators released by various cell types having pleiotropic actions on many cell types, and they play a regulatory role in normal and abnormal homeostasis and in host defense mechanisms (inflammatory and immune responses). cytokines share some general characteristics in their mode of action: different cytokines can have a similar effect on several target cells (redundancy); they can activate the secretion of other cytokines (producing a "cascade"); and they can also initiate their own secretion (autocrine), act in a paracrine fashion on neighboring cells, or act on distant cells as hormones. in addition to their pleiotropic actions on many body systems, they could potentially contribute to the wasting and cachexia of aids by their ability to induce anorexia, alter energy expenditure, increase muscle proteolysis and net protein breakdown, and initiate various abnormalities of lipid metabolism. a role for tnf in aids wasting syndrome was postulated in earlier studies indicating that tnf serum levels were elevated in patients with aids. 43 however, subsequent studies failed to show high-serum tnf levels in most aids patients, and no correlation appeared to exist between serum tnf levels and the magnitude of weight loss in aids patients. 44 -48 at least in some studies the choice of the methods to determine tnf activity (immunoassay versus bioassay) might account for the differences observed. pentoxifylline, a xanthine that inhibits the production of tnf by decreasing activity of the transcription factor nf-kb, has been used to highlight indirectly the possible importance of tnf in the hypertriacylglycerolemia observed in aids patients. administration of pentoxifylline was shown to reduce the triacylglycerol levels in aids patients, although this reduction was only marginally significant (p ϭ 0.06) and the study was open label (nonblinded). 49 more importantly, pentoxifylline does not alter other aspects of aids wasting, emphasizing the fact that aids wasting is not entirely tnf dependent. interleukin-1 (il-1) shares many of the characteristics of tnf and can also produce anorexia, hypertriacylglycerolemia, and stimulate hepatic fatty acid synthesis. 50,51 in addition, il-1 reduces lpl activity and produces lipolysis. 19,50 moreover, both tnf and il-1 can promote hiv-1 replication in in vitro cellular systems, which has led to the suggestion that cytokines may be important for the progression of hiv infection to aids. thus, tnf production is linked to hiv infection and the potential role of tnf in this setting is a source of this enhanced production. 52,53 naturally occurring cytokine antagonists such as the soluble form of the p55 (type i) tnf receptor (tnfsrp55) and the il-1␤ receptor antagonist (il-1ra) are produced in the body to counteract the potentially harmful effects of excessive tnf and il-1 production, respectively. 54,55 enhanced plasma levels of soluble tnf receptors have been reported to be correlated with rapid progression toward aids in hiv-1 infected patients. 56 moreover, a study suggested that enhanced tnfsrp55 and tnfsrp75 (type ii) were predictive of worsening nutritional status in hiv patients. 57 a more recent study showed that high serum levels of il-1␤, tnf, and il-8 together with an excess of the natural inhibitors il-1ra and tnfsrp55 were seen in asymptomatic hiv-1-positive african women but not in african women with aids or in hiv-negative controls. 46 this study suggests that cytokine antagonists may play a role in modulating cytokineassociated symptoms in the early phases of hiv infection. 46 alternatively, because most of the aids patients in that study were at the endstage of their disease and therefore likely to have significant protein-energy malnutrition, it might only reflect the inability or reduced ability to synthesize new proteins, including cytokines. on the other hand, another study has found no correlation between elevated soluble tnf receptor types i and ii levels and metabolic disturbances in hiv infections. 58 other studies have shown increased serum/plasma levels of il-1, tnf, il-6, and interferon-␥ in some populations of hivinfected patients. 47,48,58,59 il-6, an important mediator of the acute-phase response, reduces lpl activity in vitro and in vivo and promotes fatty acid synthesis. 60,61 in contrast to tnf and il-1, il-6 serum levels are consistently raised in aids and il-6 has been implicated in the development of cachexia in inflammatory and neoplastic processes. 47,48,59,62-64 serum levels of il-6 in hiv-infected patients are high when compared with non-infected normal subjects. 65 the levels of il-6 appear to increase according to the stage of hiv disease and appear to be higher in terminal stages of the disease. 65, 66 however, no data has been provided yet to link il-6 blood levels directly with the development of wasting and cachexia in aids patients. a major problem with studies regarding cytokines and circulating soluble receptors in the bloodstream of patients with hiv is that cytokines principally act in an autocrine and paracrine manner, thus making blood levels not necessarily relevant for a proper interpretation of their effects on tissues, organs, or body systems. moreover, cytokines are rapidly internalized by cells and they can activate the release of other substances. with respect to cytokine actions, it is therefore more adequate to think in terms of effects on tissues, organs, or systems rather than trying to simply correlate a complex clinical syndrome such as wasting with elevated circulating cytokine levels. for instance, il-6 has been more consistently found in the blood of hiv patients, and this is probably due to its longer half-life in serum as well as related to its major role in the acute-phase response as compared with il-1 and tnf, which are rapidly cleared from the bloodstream. the role of some cytokines such as tnf, il-1, il-2, il-6, and interferon-␥ in controlling food intake, energy expenditure, or both, have been underscored by many experimental studies. [67] [68] [69] [70] [71] [72] [73] these studies have demonstrated that exogenous administration of those cytokines may mimic the hypermetabolism and anorexia associated with infection. in addition, pretreatment with specific anticytokine antibodies blocked the anorectic and thermogenic effects to the exogenous administration of cytokines as well as those of cytokine-secreting tumors. furthermore, other studies utilizing techniques of intracerebroventricular microinjection have demonstrated the anorexigenic effects of several substances that can be induced by cytokines. those substances include plateletactivating factor, several chemokines/intercrines such as il-8, platelet factor-4, interferon-inducible protein-10, monocyte chemotactic protein-1/monocyte chemotactic and activating factor (mcp-1/mcaf), regulated-upon-activation of normal t cell ex-pressed and presumably secreted (rantes), as well as ␤2microglobulin, a marker for immune activation. 74 -76 these findings suggest an interaction (and perhaps redundancy or synergistic action) of several immunomodulators that are released during inflammatory and immune processes to induce anorexia. what are mechanisms (and mediators) by which anorexia and hypermetabolism are produced? several neurotransmitters, amino acids, peptides, and cytokines can potentially influence food intake during starvation and infectious processes. in addition, some neurotransmitters function during normal regulation of food behavior. prominent among them are corticotropin-releasing hormone (crh), neuropeptide y (npy), cholecystokinin (cck), norepinephrine, acetylcholine, serotonin, dopamine, glutamate, ␥-aminobutyric acid, and others. [77] [78] [79] the paraventricular nuclei (pvn) and the hypothalamus appear to be important areas for the control of compensatory feeding behavior in response to changes in energy homeostasis. 80 crh and npy appear to play a prominent role in the responses to starvation. increases of npy in the pvn area produce hunger together with activation of the hypothalamic-pituitary-adrenal (hpa) axis. 81 increases of crh produce a reduction in messenger rna (mrna) for npy together with anorexia and activation of the hpa axis, as well as increased thermogenesis, lipolysis, hyperglycemia, and inhibition of expected insulin secretion. 79, 80, 82 the paradoxical activation of the hpa axis (together with the secretion of glucocorticoids) during the secretion of both substances, crh and npy, which have opposite effects on feeding behavior, might be explained by the fact that when the hpa axis is activated, it is done so in the setting of different orchestrated responses. 83, 84 several cytokines, such as tnf, il-1, and il-6, as well as other mediators of inflammation, which were initially and collectively called "tissue corticotropin-releasing factor," can activate the hpa axis during inflammatory states. 85, 86 activation of the hpa axis by cytokines leads to the secretion of glucocorticoids, an action believed to participate in the negative feedback control of the immune response. 85, 86 during inflammatory states tnf is secreted first and promotes the cellular secretion of il-1; and the release of both cytokines leads to the secretion of il-6, which in turn acts in conjunction with glucocorticoids to elicit the production of many mediators of the acute-phase response by the liver. 62,85,87 tnf, il-1, and il-6 act in a synergistic manner, whereas glucocorticoids down-regulate the secretion of those cytokines 63,87-89 and other inflammatory mediators including nitric oxide, platelet activating factor, and prostanoids. 90 -92 tnf, il-1, and il-6 also participate in the stimulation of the hpa axis during endotoxin administration. 93 noteworthy, anti-il-6 antibodies can almost completely block the stimulation of the hpa axis by endotoxin. 93 as these cytokines do not appear to cross the blood-brain barrier in significant amounts, how can these cytokines be acting at the cns level? one explanation is that tnf, il-1, and il-6, produced peripherally, can act on crh neurons through the activation of other cells such as astrocytes or microglial cells in the brain or cells in the area postrema, which are not protected by the blood-brain barrier, to secrete cytokines or other substances. alternatively, activated endothelial cells, phagocytic cells, or activated t cells migrating through the blood-brain barrier can initiate further cellular production of cytokines to act on crh-producing neurons. to gain an insight into these mechanisms we performed a study in which transgenic mice expressing high levels of soluble tnf-r1 fusion protein (and therefore having blunted circulating tnf levels) showed reduced thermogenesis and blunted mrna expression for tnf, il-1, il-6, and crh in the brain in response to a parenteral challenge (d. arsenijevic, i. garcia, h. r. chang, and a. g. dulloo, unpublished observations). these results support the hypothesis that local production of tnf, il-1, and il-6 in the brain may mediate the increased brain production of crh to produce anorexia and hypermetabolism during endotoxin administration. furthermore, that study demonstrates that tnf is necessary for eliciting the increased production of crh and that tnf is indeed needed as an amplifier of the inflammation cascade through up-regulation of il-1 and il-6 production. this finding is in agreement with other data showing that cns administration of antibodies to neutralize il-1␤, il-6, or tnf inhibits the thermogenic and anorectic responses to peripherally injected endotoxin in the rat. 94 further systems such as the noradrenergic system may be activated during inflammatory stress. 95 the overall effect should be the enhancement of crh production, which carries the previously mentioned effects including anorexia, lipolysis, and increased thermogenesis and therefore weight loss. systemic administration of il-1 also stimulates the expression of crh mrna in the pvn together with dose-dependent activation of the hpa axis and sustained suppression of food intake. 96, 97 this effect of il-1 is partially reversed by crh antisera administration. 98 il-1 receptors have been demonstrated in hypothalamic structures. 99, 100 almost identical effects on crh release and food intake have been reported for tnf, il-6, il-2, and interferon-␥, 100 suggesting that sustained and moderate increases in the levels of those cytokines (which act synergistically) can potentially increase crh, thereby blocking the normal compensatory hypothalamic response to weight loss (i.e., increased appetite and reduced thermogenesis). and indeed, elevated levels of several cytokines, including il-1 and il-6, have been reported in the cerebrospinal fluid of aids patients. 101 thus, local production of cytokines within the cns can contribute to the wasting syndrome and cachexia observed in hiv infection and aids through chronic release of crh. another potential mechanism by which sustained production of cytokines could enhance the secretion of crh is through reduction of the sensitivity of target tissues to the effects of glucocorticoids. for instance, a decreased affinity of glucocorticoid receptors for cortisol has been described in phagocytic cells from some aids patients. 102 in those aids patients, there were elevated levels of cortisol and corticotropin associated with signs of glucocorticoid deficiency including hyponatremia and postural hypotension pointing to a glucocorticoid-resistant condition. 102 by this mechanism, hpa axis activation would be resistant to the negative feedback mechanism provided by the secretion of glucocorticoids. interestingly, il-2 in combination with il-4 has been described to produce resistance of t cells to the action of glucocorticoids by reducing the affinity of the glucocorticoid receptor for its ligand. 103 il-4 is involved in antiinflammatory mechanisms together with other cytokines, such as il-10 and il-13, and they are able to augment the expression of il-1ra. 104 -108 both insulin and glucocorticoids play an important role in the peripheral response to fasting and starvation. they also appear to play a role in the cns regulation of energy balance by regulating npy synthesis and release. 83 thus, insulin, as opposed to its peripheral anabolic effects, promotes a state of negative energy balance (through anorexia and increased energy expenditure) by reducing npy gene expression in the hypothalamus. 83 fasting (which lowers insulin levels) increases hypothalamic npy gene expression. 109 glucocorticoids have opposing effects to insulin, thus promoting a state of positive energy balance with an increase of caloric intake. 83, 110 peripherally, glucocorticoids, as well as glucagon, catecholamines, and growth hormone, may induce insulin resistance when these hormones are present in enhanced levels and in the presence of stress or infections such as in sepsis. 111 current data, however, suggest that glucagon and catecholamines are not highly elevated in aids, whereas elevated basal cortisol levels have been found frequently. 112, 113 administration of tnf to humans has been found to produce a hyperglycemic state without alterations in insulin levels, suggesting an insulin resistance-like state. 114 moreover, infusion of tnf into experimental animals produces marked insulin resistance. 115 these findings may help to partly explain the hyperglycemia and the relative insulin resistance observed in several infectious processes. however, hiv infection is characterized by high rates of insulin clearance as well as an increased sensitivity of peripheral tissues to insulin. 112 is there a role for leptin? the newly described molecule leptin and its putative receptor also appear to play a role in maintaining normal weight. leptin is the protein product of the ob gene and its circulating levels reflect energy stores in adipocytes, suggesting its role as an "adipostat." 116 -118 the leptin receptor gene is highly expressed at the hypothalamic level. 119, 120 increased leptin levels produce anorexia and increased energy expenditure, possibly by reducing the release of npy 119,121 at the hypothalamic level. thus, reduced leptin levels induce hunger and reduced thermogenesis, pointing to a role for reduced levels of leptin in the adaptive changes to fasting/starvation. on the other hand increased leptin levels may be related to resistance to obesity (anorexia and enhanced thermogenesis), and may be acting on melanocyte-stimulating hormone and the melanocortin-4 receptor, in other regions of the brain. [112] [113] [114] [115] [116] [117] [118] [119] [120] [121] [122] [123] [124] glucocorticoids and insulin have been demonstrated to up-regulate the production of leptin. [125] [126] [127] cytokines, such as tnf and il-1, and endotoxin, are able to stimulate leptin production 128 and anorexia is directly proportional to the increase of leptin pointing to a potential role of this molecule in the anorexia associated with infection. 128 however, a first study measuring leptin levels in patients with aids found that leptin levels were not increased relative to body fat in patients who were anorexic, were losing weight, or had a history of weight loss. also, leptin levels were not elevated during secondary infection, suggesting a lack of a link between increased leptin levels and anorexia in aids. 129 another study comparing the levels of leptin in hiv-infected men to age-and body-fatmatched uninfected individuals showed no differences in serum leptin levels and there was no correlation with lean body mass. 130 there was, however, correlation between leptin concentrations and percent body fat and body fat content, extending the notion that circulating leptin levels directly reflect adipose tissue mass, even in hiv-infected men with low body-fat content. 130 a significant loss of lean body mass mainly due to muscle proteolysis has long been appreciated to be characteristic of wasting and cachexia in trauma and sepsis (as well as in aids, cancer, fasting, and acidosis). well before the wealth of information that currently exists on cytokine pathophysiology, several investigators were searching for mediators, such as the so-called "muscle proteolysis factor," that could be responsible for the metabolic disturbances and wasting observed. 131 theoretically the loss of lean body mass could be due to an increase in tissue protein degradation or a decrease in tissue protein synthesis or a combination of both. accelerated protein breakdown in muscle is necessary to meet the needs of the anabolic response in liver, hemotopoietic, and wound tissue during stress and infection. on the other hand, in starvation (and in the absence of infection) there is increased appetite on refeeding, reduced energy expenditure, and relative preservation of muscle mass, but also no requirement for enhanced anabolism in those select tissues. in the wasting of aids and in the presence of cytokines there might be maintenance or declines in basal metabolic rate together with anorexia and muscle catabolism. what, therefore, would be the cause for muscle wast-ing in aids? tnf and il-1 have been shown to produce skeletal muscle catabolism in addition to their anorectic and net nitrogen loss effects, 132,133 but by different mechanisms. moreover, administration of tnf and il-1 to experimental animals have been found to produce weight loss, net nitrogen loss, skeletal muscle catabolism, and increased liver weight. 134, 135 the effects were observed independent from and additive to those resulting from semistarvation. 134, 135 recent evidence strongly suggests that activation of the ubiquitin-proteasome pathway is responsible for muscle wasting in several catabolic states. 136 the activation or the suppression of the pathway is related to the rate of ubiquitin conjugation to proteins. higher rates of ubiquitin conjugation result in enhanced muscle proteolysis. glucocorticoids promote muscle proteolysis, an action that opposes the anabolic effects of insulin, by increasing mrnas encoding ubiquitin and proteasomes subunits and therefore increasing ubiquitin-protein conjugates. 136 in the fasting state, muscle proteolysis may take place in the presence of glucocorticoids and when insulin levels are low. glucocorticoids in combination with several cytokines such as tnf, il-1, and il-6 participate in stimulation of the ubiquitinproteasome pathway producing muscle proteolysis. [137] [138] [139] in aids, all the requisite conditions for muscle proteolysis are present: increased basal levels of cortisol, 113 low circulating insulin levels, 113 and subtle release of several cytokines that may be acting synergistically. interference with tnf production by anti-tnf antibodies or by administration of pentoxifylline produces blockade of muscle proteolysis in vivo. 140, 141 pentoxifylline may be acting to block muscle proteolysis through inhibition of the proteasome-dependent activation of the transcription factor nf-kb, which is also needed for tnf production. 142 blockade of il-1 action by administering soluble il-1ra to experimental animals prevents muscle proteolysis in response to endotoxin. 143 recently, a novel glycoprotein, able to produce muscle proteolysis in vitro, as opposed to tnf and il-6, which are unable to do so and are only effective in vivo, has been described in rodents and in the urine of cachectic patients with certain cancers. 144 whether this glycoprotein also is produced in aids patients with cachexia is unknown. because cytokines such as tnf and il-1 are not able to produce muscle proteolysis in vitro, their effects on muscle proteolysis may be indirect. 143, 145, 146 several subtle endocrine alterations have been demonstrated in hiv patients that potentially may be related to cytokine production. 113 thyroid hormone, adrenal, and gonadal homeostasis could be altered during hiv infection by cytokines. the euthyroid sick syndrome can be observed with severe caloric depletion and severe illnesses, and is characterized by impaired peripheral conversion of thyroxine to t3, resulting in high normal or normal circulating levels of thyroxine and lower levels of t3. 147 in addition, enhanced rt3 levels are present due to reduced clearance, 147 whereas thyrotropin (tsh) levels appear to be within normal limits. in aids patients with anorexia and weight loss, conversion of thyroxine to t3 is decreased (euthyroid sick syndrome) as well as the levels of insulin-like growth factor-i (igf-i), whereas in stable hiv patients t3 levels are normal. 148, 149 the reduction in t3 in those patients might be the consequence of an adaptive response to caloric deprivation, as is also observed during fasting and malnourished states. maintenance of such low levels of t3 during nutritional rehabilitation may hamper the buildup of lean body mass. infusion of il-6 to patients with cancer and normal thyroid function has been shown to acutely decrease tsh and t3 and to enhance levels of rt3, but after several weeks of il-6 administration only tsh was found to be elevated. 150 similarly, infusion of tnf to normal persons produced acute de-creases of tsh and t3 and increased rt3. 151 the clinical significance of such changes in thyroid function upon cytokine administration are not completely understood but suggest that the changes observed in aids patients may be due to the effects of cytokines. in aids, increased basal levels of cortisol have been demonstrated, 113 and this might reflect the activation of the stress response. as a result of such cortisol levels, the cortisol response to provocative testing may be abnormal. cytokines can directly stimulate the release of corticotropin (acth), crh, and cortisol, 96 -100,152 which might suggest their potential role for the mild cortisol elevations in hiv patients. the demonstration of glucocorticoid resistance in some hiv patients 102 may provide an additional explanation for cortisol elevations in the serum of some aids patients. data on gonadal function in aids has been gathered mostly from male patients. there are substantial data to indicate that hiv infection is accompanied by hypogonadism. 113 hypogonadism in aids can be primary (testicular) or central in etiology, and may be due to the release of cytokines. for instance, administration of tnf to healthy men produces a rise in luteinizing hormone (lh) followed by a decrease in testosterone levels. 153 moreover, il-1 has been shown at high levels to block steroidogenesis by inhibiting the binding of lh to leydig cells. 154 a particularly noteworthy relationship is that crh, which is induced by cytokines, is also produced by leydig cells of the testes and produces autocrine effects by inhibiting testosterone biosynthesis. decreases in testosterone levels may make difficult any attempt to increase muscular mass. indeed, administration to aids patients of megestrol acetate, which stimulates appetite, also lowers testosterone levels, and results in weight gain but mainly of fat mass. 155 recent experimental data suggest that the appetite-stimulant effect of megestrol acetate involve stimulation of synthesis, transport, and release of neuropeptide y in the hypothalamus. 156 the nutritional status of an individual can be one of the major determining factors in resistance to infection. hiv infection, on the other hand, often produces malnutrition leading to wasting and cachexia. wasting syndrome in aids is multifactorial: hiv infection of gastrointestinal tissue and lymphoid tissue particularly, anorexia, inadequate nutrient intake and malabsorption, and catabolic effects on intermediary metabolism play important roles along with similar effects of repeated secondary infections. currently available preventive/therapeutic approaches for wasting in aids include baseline nutritional assessment 157 (table iii) , early diagnosis of malnutrition and maintenance of adequate nutritional intake, early diagnosis/prevention of opportunistic infections, and appetite stimulants as well as anabolic hormonal therapy. 158 -160 adequate antiretroviral therapy might be considered during early stages of hiv infection as part of the wasting preventive measures, because successful treatment of the primary infection with hiv or secondary infections is the most potent of anabolic therapies. cytokines play a complex and overlapping role in the development of wasting and cachexia in aids. they can exert behavioral, hormonal, and endocrine effects that can persist for long periods of time to produce wasting and cachexia. multipronged therapeutic approaches are therefore required to counteract their effects on different body systems. attempts have been made to modulate the release of certain cytokines such as tnf by pharmacologic means in aids patients, with the aim of arresting or reversing the wasting process. for instance, pentoxifylline has been found in pilot studies to decrease tnf serum levels as well as serum triacylglycerols in aids patients. 49, 161 however, viral load was not altered and there was no benefit in terms of weight gain. 49, 161 other pilot studies have failed to show any benefits of pentoxifylline administration to aids patients in terms of weight gain or effect on serum tnf levels. 162 similar observations have been made in cancer patients with cachexia, where pentoxifylline failed to improve anorexia or cachexia. 163 thalidomide, a drug with sedative effects that has been used for many years for the therapy of some reactive forms of leprosy (erythema nodosum leprosum), has been found to down-regulate tnf production in vitro. 164 thalidomide has shown in pilot studies to promote weight gain in hiv patients with wasting. 165, 166 thalidomide administration produced a reduction in serum tnf levels in a pilot study of hiv patients with wasting and tuberculosis. 166 however, in a recent trial in which thalidomide was effective for the therapy of aphthous ulceration of the mouth, there were increases in tnf and soluble tnf receptor type ii as well as viral load. 167 thalidomide has potent antiinflammatory properties and has been successfully used for therapy of certain types of aphthous ulcerations in aids patients as well as for the treatment of graft-versus-host disease in allogeneic bone marrow transplantations. 168, 169 an additional mechanism by which thalidomide might promote weight gain is by improving the absorption of nutrients through the gastrointestinal tract. to date, no firm evidence has been provided to indicate that pharmacologic blockade or manipulation of cytokine production is useful in the prevention or therapy of wasting and cachexia in aids. in fact, sustained blockade of cytokine production may produce harmful effects as they are needed for a proper functioning and tuning of the immune system and blockade of a particular cytokine may in turn produce blockade of other cytokines, which may have unpredictable results. for instance, administration of pentoxifylline produced an increase in mycobacterial load in macrophages from aids patients with disseminated mycobacteriumavium-intracellulare complex infection. 170 an alternative way of counteracting the effects of cytokines may include use of a cytokine with inhibitory actions on other cytokines or a drug or drugs that enhance the release of inhibitors, although these agents would be subject to the same concerns. for instance, epinephrine has been shown to increase the release of the antiinflammatory cytokine il-10, which has been previously found to inhibit tnf. 171, 172 however, it is difficult to see how this could be clinically applied, although oral or b-agonist therapy would be theoretically possible. finally, the use of antiinflammatory cytokines might be possible. potentially new approaches for prevention/therapy of wasting and cachexia of aids may include drugs that selectively can modulate the release of mediators acting at the target tissues, organs, or systems. for example, selective inhibitors or antagonists of crh that can go across the blood-brain barrier or agents that can down-regulate hypothalamic release of crh may effectively combat anorexia. also, specific inhibitors of the ubiquitinproteasome pathway might be useful to arrest muscle catabolism. however, the most effective therapy for wasting induced by infectious agents is always the successful treatment of the infection rather than the body's cytokine response to infection. ultimately the ideal nutritional solution to aids wasting will be successful antiretroviral therapy. further work in this area is greatly needed and hopefully the results of those studies will enhance our knowledge and help us to explore new therapeutic avenues for wasting and cachexia of aids. revised classification system for hiv infection and expanded surveillance case definition for aids among adolescents and adults nutritional status, gastrointestinal dysfunction, and survival in patients with aids magnitude of body-cell-mass depletion and the timing of death from wasting in aids body weight as an essential data in the management of patients with human immunodeficiency virus infection and the acquired immunodeficiency syndrome body composition studies in patients with the acquired immunodeficiency syndrome quality of life in persons with human immunodeficiency virus infection. measurement by the medical outcomes study instrument d-xylose 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hiv-induced diseases increased interleukin-6 production is associated with disease progression in hiv infection interferon-␥, more a cachectin than tumor necrosis factor cytokines, muscle proteolysis and the catabolic response to infection and inflammation the role of interleukin-6 in lipopolysaccharide-induced weight loss, hypoglycemia and fibrinogen production in vivo inhibition of central actions of cytokines on fever and thermogenesis by lipocortin-1 involves crf metabolic effects of cachectin/tumor necrosis factor are modified by site of production tumor necrosis factor and regulation of metabolism in infection role of systemic versus tissue levels tumor necrosis factor in the malnutrition (cachexia) of infection and cancer immunomodulators and feeding regulation: a humoral link between the immune and nervous systems chemokines/intercrines and central regulation of feeding modulation of feeding by ␤ 2 -microglobulin, a marker of immune activation the lateral hypothalamus: a primary site mediating excitatory amino acid-elicited eating neuropeptide regulation of appetite and weight central effects of crf on metabolism and energy balance the hypothalamus, intrinsic connections and outflow pathways to the endocrine system in relation to the control of feeding and metabolism food deprivation and ingestion induce reciprocal changes in neuropeptide y concentrations in the paraventricular nucleus hypothalamic neuropeptide y messenger ribonucleic acid levels in pre-obese and genetically obese (fa/fa) rats: potential regulation thereof by corticotropin-releasing factor feast and famine: critical role of glucocorticoids with insulin in daily energy flow hypothalamic response to starvation: implications for the study of wasting disorders the hypothalamic-pituitary-adrenal axis and immunemediated inflammation neuroendocrine-immune system interactions. the immune-hypothalamo-pituitary-adrenal axis glucocorticoid therapy for immune-mediated diseases: basic and clinical correlates glucocorticoids selectively inhibit the transcription of interleukin-1␤ gene and decrease the stability of interleukin-1␤ mrna glucocorticoid inhibition of interleukin-1 induced interleukin-6 production by human lung fibroblasts: evidence for transcriptional and post-transcriptional regulatory mechanisms the arginine-nitric oxide pathway glucocorticoids suppress group ii phospholipase a 2 production by blocking mrna synthesis and post-transcriptional expression cdna cloning and functional activity of a glucocorticoid-regulated inflammatory cyclooxygenase synergistic roles of interleukin-6, interleukin-1, and tumor necrosis factor in adrenocorticotropin response to bacterial lipopolysaccharide in vivo cytokines and thermogenesis the participation of the nervous system in the inflammatory reaction interleukin-1 stimulates corticotropin-releasing factor gene expression in rat hypothalamus interleukin-1 stimulates the secretion of of hypothalamic corticotropinreleasing factor anorexia induced by interleukin 1: involvement of corticotropin-releasing factor interleukin-1 immunoreactive inervation of the human hypothalamus immunoregulators in the nervous system human immunodeficiency virus type 1 (hiv-1) infection of the central nervous system: an evaluation of cytokines in cerebrospinal fluid cortisol resistance in acquired immunodeficiency syndrome combination of il-1 with il-4 reduces glucocorticoid receptor-binding affinity and t cell response to glucocorticoids coordinated anti-inflammatory effects of interleukin-4. interleukin-4 suppresses interleukin-1 production but up-regulates gene expression and synthesis of interleukin-1 receptor antagonist il-10 inhibits cytokine production by activated macrophages interleukin-13 is a new human lymphokine regulating inflammatory and immune responses biologic control of the tumor necrosis factor and interleukin-1 signaling cascade update on cytokines altered expression of hypothalamic neuropeptide mrnas in food-restricted and fooddeprived rats neuroendocrine control of the development of obesity: understanding gained from studies of experimental animal models insulin resistance-mechanisms, syndromes and implications evidence of endocrine involvement early in the course of human deficiency virus infection endocrine and metabolic disturbances in human immunodeficiency virus infection and the acquired immune deficiency syndrome tumor necrosis factor mimics the metabolic response to acute infection in healthy humans tumor necrosis factor impairs insulin action on peripheral glucose disposal and hepatic glucose output positional cloning of the mouse obese gene and its human homologue leptin levels in human and rodent: measurement of plasma leptin and ob rna in obese and weight-reduced subjects recombinant mouse ob protein: evidence for a peripheral signal linking adiposity and central neural networks the hypothalamic leptin receptor in humans. identification of incidental sequence polymorphisms and absence of the ob/ob mouse and fa/fa rat mutations localization of leptin receptor mrna and the long form splice variant (ob-rb) in mouse hypothalamus and adjacent brain regions by in situ hybridization activation of ␤ 3 adrenergic receptors suppresses leptin expression and mediates a leptinindependent inhibition of food intake in mice the role of neuropeptide y in the antiobesity action of the obese gene product role of the melanocortinergic neurons in feeding and the agouti obesity syndrome the alphabet of weight control transient increase in obese gene expression after food intake or insulin administration induction of ob gene expression by corticosteroids is accompanied by body weight loss and reduced food intake acute and chronic effect of insulin on leptin production in humans: studies in vivo and in vitro endotoxin and cytokines induces expression of leptin, the ob gene product, in hamsters: a role for leptin in the anorexia of infection serum leptin levels in the acquired immunodeficiency syndrome serum leptin concentrations in human immunodeficiency virus-infected men with low adiposity muscle proteolysis induced by a circulating peptide in patients with sepsis or trauma infusion of tumor necrosis factor/cachectin promotes muscle catabolism in the rat. a synergistic effect with interleukin-1 cachectic effects of recombinant human tumor necrosis factor in rats metabolic changes in rats during a continuous infusion of recombinant interleukin-1 mechanisms of host wasting induced by administration of cytokines in rats mechanisms of muscle wasting. the role of the ubiquitin-proteasome pathway evidence that tumor necrosis factor participates in the regulation of muscle proteolysis during sepsis effects of tumor necrosis factor or interleukin-1 on muscle amino acid uptake and the role of glucocorticoids interleukin-6 induces skeletal muscle protein breakdown in rats tumor necrosis factor-␣ mediates changes in tissue protein turnover in a rat cancer cachexia model pentoxifylline decreases body weight loss and muscle protein wasting characteristics of sepsis the ubiquitinproteasome pathway is required for processing of the nf-b1 precursor protein and the activation of nf-b reduced muscle protein breakdown in septic rats following treatment with interleukin-1 receptor antagonist characterization of a cancer cachectin factor the toxic effects of tumor necrosis factor in vivo and their prevention by cyclooxygenase inhibitors tumor necrosis factor can induce fever in rats without activating protein breakdown in muscle or lipolysis in adipose tissue alterations of thyroid function in patients with systemic illnesses: the euthyroid sick syndrome thyroid hormone levels in the acquired immunodeficiency syndrome (aids) or aids-related complex elevation of serum thyroxine-binding globulin (but not of cortisol-binding globulin and sex hormone-binding globulin) associated with the progression of human immunodeficiency virus infection effects of acute and chronic interleukin-6 administration on thyroid metabolism in humans tumor necrosis factor: a putative mediator of the sick euthyroid syndrome in man role of endotoxin and interleukin-1 in modulating acth, lh, and sex steroid secretion effects of tumor necrosis factor on the hypothalamic-pituitary-testicular axis in healthy men interleukin-1 inhibits leydig cell steroidogenesis in primary culture effects of megestrol acetate therapy on body composition and circulating testosterone concentrations in patients with aids megestrol acetate stimulates food and water in the rat: effects on regional hypothalamic neuropeptide y concentrations nutrition support and the human immunodeficiency virus (hiv) nutrition and hiv infection nutritional aspects of hiv infection wasting syndrome in aids: pathophysiologic mechanisms and therapeutic approaches use of pentoxifylline therapy for patients with aids-related wasting: pilot studies pentoxifylline therapy in hiv seropositive subjects with elevated tnf pentoxifylline for treatment of cancer anorexia and cachexia? a randomized, doubleblind, placebo-controlled trail thalidomide exerts its inhibitory action on tumor necrosis factor-␣ by enhancing mrna degradation effects of thalidomide on wasting syndrome in patients with aids: a randomized, double blind, placebo controlled clinical trial (abstract 536b) the effect of thalidomide on the pathogenesis of human immunodeficiency virus type 1 and m. tuberculosis infection thalidomide for the treatment of oral aphthous ulcers in patients with human immunodeficiency virus infection treatment of resistant aphthous ulceration with thalidomide in patients positive for hiv antibody thalidomide: rationale for renewed use in immunological disorders pentoxifylline aggravates impairment in tumor necrosis factor-␣ secretion and increases mycobacterial load in macrophages from aids patients with disseminated mycobacterium-avium intracellulare complex infection epinephrine inhibits tumor necrosis factor-␣ and potentiates interleukin-10 production during human endotoxemia controlling the production of interleukin-1 and tumor necrosis factor in disease for an additional perspective key: cord-023443-pvz7dll9 authors: nan title: abstracts for the scandinavian society for immunology 35th annual meeting and 20th summer school date: 2004-06-02 journal: scand j immunol doi: 10.1111/j.1365-3083.2004.01423.x sha: doc_id: 23443 cord_uid: pvz7dll9 nan the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-278339-6ddsj014 authors: gianfrancesco, milena; hyrich, kimme l; al-adely, sarah; carmona, loreto; danila, maria i; gossec, laure; izadi, zara; jacobsohn, lindsay; katz, patricia; lawson-tovey, saskia; mateus, elsa f; rush, stephanie; schmajuk, gabriela; simard, julia; strangfeld, anja; trupin, laura; wysham, katherine d; bhana, suleman; costello, wendy; grainger, rebecca; hausmann, jonathan s; liew, jean w; sirotich, emily; sufka, paul; wallace, zachary s; yazdany, jinoos; machado, pedro m; robinson, philip c title: characteristics associated with hospitalisation for covid-19 in people with rheumatic disease: data from the covid-19 global rheumatology alliance physician-reported registry date: 2020-05-29 journal: ann rheum dis doi: 10.1136/annrheumdis-2020-217871 sha: doc_id: 278339 cord_uid: 6ddsj014 objectives: covid-19 outcomes in people with rheumatic diseases remain poorly understood. the aim was to examine demographic and clinical factors associated with covid-19 hospitalisation status in people with rheumatic disease. methods: case series of individuals with rheumatic disease and covid-19 from the covid-19 global rheumatology alliance registry: 24 march 2020 to 20 april 2020. multivariable logistic regression was used to estimate ors and 95% cis of hospitalisation. age, sex, smoking status, rheumatic disease diagnosis, comorbidities and rheumatic disease medications taken immediately prior to infection were analysed. results: a total of 600 cases from 40 countries were included. nearly half of the cases were hospitalised (277, 46%) and 55 (9%) died. in multivariable-adjusted models, prednisone dose ≥10 mg/day was associated with higher odds of hospitalisation (or 2.05, 95% ci 1.06 to 3.96). use of conventional disease-modifying antirheumatic drug (dmard) alone or in combination with biologics/janus kinase inhibitors was not associated with hospitalisation (or 1.23, 95% ci 0.70 to 2.17 and or 0.74, 95% ci 0.37 to 1.46, respectively). non-steroidal anti-inflammatory drug (nsaid) use was not associated with hospitalisation status (or 0.64, 95% ci 0.39 to 1.06). tumour necrosis factor inhibitor (anti-tnf) use was associated with a reduced odds of hospitalisation (or 0.40, 95% ci 0.19 to 0.81), while no association with antimalarial use (or 0.94, 95% ci 0.57 to 1.57) was observed. conclusions: we found that glucocorticoid exposure of ≥10 mg/day is associated with a higher odds of hospitalisation and anti-tnf with a decreased odds of hospitalisation in patients with rheumatic disease. neither exposure to dmards nor nsaids were associated with increased odds of hospitalisation. epidemiology rheumatic diseases 1 therefore, people with rheumatic disease may be at higher risk for a more severe course with covid-19, including hospitalisation, complications and death. importantly, some medications used to treat rheumatic diseases, such as hydroxychloroquine and interleukin-6 (il-6) inhibitors, are being studied for the prevention and/or treatment of covid-19 and its complications including cytokine-storm. [2] [3] [4] at present, the implications of covid-19 for people living with rheumatic diseases remain poorly understood. to address this knowledge gap, a global network of rheumatologists, scientists and patients developed a physician-reported case registry of people with rheumatic diseases diagnosed with covid-19. 5 6 this report aims to (1) describe the demographic and clinical characteristics of the first 600 patients submitted to the covid-19 global rheumatology alliance (c19-gra) physician registry and (2) identify factors associated with hospitalisation for covid-19 in this population. details of the registry design have been described elsewhere. [5] [6] [7] briefly, c19-gra data regarding individuals with rheumatic diseases diagnosed with covid-19 are captured from rheumatology physicians via two parallel international data entry portals for regulatory reasons: one limited to european countries ( eular. org/ eular_ covid19_ database. cfm; hosted by the university of manchester, uk) and a second for all other sites ( rheum-covid. org/ provider-global/; hosted by the university of california, san francisco, california, usa). two patients sit on the c19-gra steering committee and they contributed to the design of the registry, the questions being asked and the analysis of the results. the c19-gra has a patient board, composed entirely of patients. these patients, and others, will be involved in disseminating the results of this analysis once published. no public were involved in the design or analysis of this project. physicians indicated whether the diagnosis of covid-19 was based on pcr, antibody, metagenomic testing, ct scan, laboratory assay or a presumptive diagnosis based on symptoms only. data elements for this analysis included physician city, state and country. countries were assigned to the six who regions ( www. who. int); the 'americas' was further divided into north and south. case information including age, sex, smoking status, rheumatic disease diagnosis, disease activity and comorbidities was collected. medications prior to covid-19 were categorised as: conventional synthetic disease-modifying antirheumatic drugs (csdmards; antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus); biologic dmards (bdmards; abatacept, belimumab, cd-20 inhibitors, il-1 inhibitors, il-6 inhibitors, il-12/il-23 inhibitors, il-17 inhibitors, tumour necrosis factor inhibitors (anti-tnf)) and targeted synthetic dmards (tsdmards) namely janus kinase (jak) inhibitors. physicians reported the approximate number of days from symptom onset to symptom resolution or to death. the primary outcome of interest was hospitalisation for covid-19. as of 20 april 2020, a total of 604 cases were entered in the registry; hospitalisation status was unknown for four cases and these were excluded from analysis. continuous variables are reported as median (iqr). categorical variables are reported as number and percentage (%). in univariable analyses, differences in demographic and rheumatic disease-specific features according to hospitalisation status were compared using χ 2 tests for categorical variables and mann-whitney u tests for continuous variables. the independent associations between demographic and disease-specific features with the odds of covid-19 hospitalisation were estimated using multivariable-adjusted logistic regression and reported as or and 95% cis; covariates included in the model were age group (<65 years vs >65 years), sex, rheumatic disease (rheumatoid arthritis (ra), systemic lupus erythematosus (sle), psoriatic arthritis (psa), axial spondyloarthritis (axspa) or other spondyloarthritis, vasculitis and other), key comorbidities (hypertension, lung disease, diabetes, cardiovascular disease and chronic renal insufficiency/end-stage renal disease), smoking status (ever vs never), physician-reported disease activity (remission, minimal/low disease activity, moderate disease activity or severe/high disease activity; or as a binary variable: remission and minimal/low disease activity vs moderate and severe/high disease activity), dmard type (no dmard, csdmard only, b/tsdmard only, csdmard and b/tsdmard combination therapy), non-steroidal anti-inflammatory drugs (nsaid) use (yes vs no) and prednisone-equivalent glucocorticoid use (0 mg/ day, 1-9 mg/day, ≥10 mg/day). categories with cell sizes <10 by hospitalisation status were collapsed to ensure sufficient power in the adjusted model. for univariable and multivariable models, patients with more than one of the following diseases recorded were classified as follows: sle>ra>psa>vascu-litis>axspa/other spondyloarthritis>other. cardiovascular disease and hypertension were collapsed as a single comorbidity in the regression model due to significant collinearity between the two variables. due to concerns regarding the possibility of confounding by indication, disease activity and prednisoneequivalent glucocorticoid use were analysed by including only one of the variables in the multivariable analysis at a time, and by including both variables in the multivariable analysis at the same time. unknown/missing data (14% smoking status, 12% nsaids, 1% glucocorticoids) were treated as a separate category in multivariable models. in exploratory analyses, the independent association between antimalarials and specific b/tsdmard therapies with hospitalisation status was estimated using multivariable logistic regression. to assess the robustness of the results, sensitivity analyses were performed. first, we repeated the above analyses after excluding patients with a 'presumptive diagnosis', meaning that the patient's physician thought he/she had symptoms consistent with the disease, but there was no evidence of the patient having: a) a confirmatory covid test; b) documentation of chest imaging showing bilateral infiltrates in keeping with covid-19 pneumonia or c) close contact with a known covid-19-positive patient. second, we limited the analyses to patients whose covid-19 outcome was known (resolved/died) or for whom at least >14 days from symptom onset (or diagnosis date if symptom onset was unknown) had elapsed, as it is unlikely that a patient would be hospitalised >2 weeks after onset. third, we excluded cases with missing/unknown values within the covariate set included in the multivariable analyses. data were considered statistically significant at p<0.05. cell counts <5 are represented by 'n<5' in tables to protect patient anonymity. all analyses were conducted in stata v. 16 .0 (statacorp). data quality was assessed by two data quality teams (one at the university of manchester, uk and the university of california, san francisco) who also confirmed there were no duplicate entries. due to the deidentified and non-interventional nature of the study, it was determined by the institutional review board that patient consent was not required. c19-gra physician registry was determined 'not human subjects research' by the uk health research authority and the university of manchester, as (14) psoriatic arthritis 74 (12) axial spondyloarthritis or other spondyloarthritis 48 (8) vasculitis 44 (7) sjögren's syndrome 28 (5) other inflammatory arthritis 21 (4) inflammatory myopathy 20 ( reported days from onset to resolution or death (n=275), median (iqr) 13 (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) n (column %) for categorical variables unless otherwise noted. percentages may not sum to 100 due to rounding. *cases could have more than one disease diagnosis. 'other' rheumatic disease category included (each n<10): undifferentiated connective tissue disease; ocular inflammation; autoinflammatory syndrome; mixed connective tissue disease; antiphospholipid antibody syndrome; calcium pyrophosphate deposition disease; systemic juvenile idiopathic arthritis; juvenile idiopathic arthritis, not systemic; igg4-related disease. †chronic obstructive pulmonary disease, asthma, interstitial lung disease or other not specified. ‡csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/tsdmard included: abatacept, belimumab, cd-20 inhibitors, il-1 inhibitors, il-6 inhibitors, il-12/il-23 inhibitors, il-17 inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmard; csdmard, conventional synthetic dmard; dmard, disease-modifying antirheumatic drug; il, interleukin; nsaid, non-steroidal anti-inflammatory drug; tnf, tumour necrosis factor. table 1 continued well as under united states federal guidelines assessed by the university of california, san francisco and patient consent was not required. we did not systematically capture how cases were identified before being entered into the registry and therefore we cannot detail this. however, we are aware of a number of large institutions that are systematically collecting all cases in their health system/district and entering them into the registry. the demographic and clinical characteristics of the first 600 cases in the c19-gra physician registry are shown in table 1. the majority of cases in the registry were from north america and europe, female and in the 50-65 age range, the countries that the cases were reported from are shown in online supplementary table 1. the most common rheumatic disease was ra (230, 38%), followed by sle (85, 14%) and psa (74, 12%). the most common comorbidities were hypertension (199, 33%), lung disease (127, 21%), diabetes (69, 12%), cardiovascular disease (63, 11%) and chronic renal insufficiency/end-stage renal disease (40, 7%). most cases were never smokers (389, 75%) and either in remission or had minimal/low disease activity (459, 80%). five patients were pregnant (1%). nearly half of the cases reported to the registry were hospitalised (277, 46%), and 9% (55) were deceased. covid-19 diagnoses were predominately made through pcr testing (437, 73%), followed by laboratory assay of unknown type (58, 10%), ct scan (42, 7%) or other (31, 5%) (individuals could be tested using more than one method). fifty-two (9%) cases had a presumptive diagnosis only (online supplementary table 2). the median number of days from covid-19 symptom onset to resolution or death was 13 (iqr: [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] . demographic and clinical characteristics stratified by sex are presented in online supplementary table 3. demographic and clinical characteristics stratified by hospitalisation status are shown in table 2. differences by age group in hospitalisation status were observed: most hospitalised patients were over age 65 (43%), compared with 16% of nonhospitalised cases (p<0.01). in unadjusted analyses, differences in hospitalisation status by disease revealed a higher percentage of people who were hospitalised had sle and vasculitis (17% and 9%, respectively) versus those who were not hospitalised (11% and 5%, respectively), while a lower proportion of patients who were hospitalised had psa and axspa or other spondyloarthritis (8% and 6%, respectively) compared with those who were epidemiology igg4-related disease. ‡csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, ciclosporin, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/ tsdmard included: abatacept, belimumab, cd-20 inhibitors, il-1 inhibitors, il-6 inhibitors, il-12/il-23 inhibitors, il-17 inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmards; csdmard, conventional synthetic dmard; dmard, diseasemodifying antirheumatic drug; il, interleukin; nsaid, non-steroidal anti-inflammatory drugs; tnf, tumour necrosis factor. not (16% and 10%, respectively). there were more comorbidities among hospitalised cases, including hypertension (45% vs 23%), lung disease (30% vs 14%), diabetes (17% vs 7%), cardiovascular disease (14% vs 7%) and chronic renal insufficiency/end-stage renal disease (12% vs 2%) (all p<0.01). there was no association between disease activity and hospitalisation status (p=0.49). nsaid use was reported less frequently among hospitalised patients than non-hospitalised patients (16% vs 25%, p=0.02), while there was a higher proportion of patients receiving high doses of glucocorticoids among those who were hospitalised than not hospitalised (16% vs 7% for doses ≥10 mg/ day, p=0.01). we found no significant difference in hospitalisation status by sex, antimalarial therapy (either monotherapy or in combination with other dmards) or reported days from symptom onset to symptom resolution or death. in a multivariable model, age over 65 years (or=2.56, 95% ci 1.62 to 4.04), hypertension/cardiovascular disease (or=1.86, 95% ci 1.23 to 2.81), lung disease (or=2.48, 95% ci 1.55 to 3.98), diabetes (or=2.61, 95% ci 1.39 to 4.88) and chronic renal insufficiency/end-stage renal disease (or=3.02, 95% ci 1.21 to 7.54) were associated with higher odds of hospitalisation (all p<0.05) (table 3) . treatment with b/tsdmard monotherapy just prior to covid-19 diagnosis was significantly associated with a lower odds of hospitalisation compared with no dmard therapy (or=0.46, 95% ci 0.22 to 0.93; p=0.03). glucocorticoid therapy at prednisone-equivalent doses ≥10 mg/ day, however, was associated with a higher odds of hospitalisation compared with no glucocorticoid therapy (or=2.05, 95% ci 1.06 to 3.96; p=0.03). neither adding disease activity to the model with glucocorticoids nor replacing glucocorticoids by disease activity changed the direction, strength or significance of the relationship between the various variables and hospitalisation status in a meaningful way (data not shown). further analyses were conducted to examine the independent association of antimalarials and specific b/tsdmards with hospitalisation. a total of 22% of cases were taking antimalarials before hospitalisation. the largest subgroup of b/tsdmard therapies was anti-tnf medications (52%). we found no significant association between antimalarial therapy and hospitalisation (or=0.94, 95% ci 0.57 to 1.57; p=0.82) after adjusting for sex, age over 65 years, rheumatic disease, smoking status, comorbidities, other csdmard monotherapy, b/tsdmard monotherapy, csdmard-b/tsdmard combination therapy (excluding antimalarials), nsaid use and glucocorticoid dose. a significant inverse association between any anti-tnf therapy and hospitalisation was found (or=0.40, 95% ci 0.19 to 0.81; p=0.01), after controlling for sex, age over 65 years, rheumatic disease, smoking, comorbidities, csdmard monotherapy, other b/tsdmard monotherapy, csdmard-b/tsdmard combination therapy (excluding anti-tnf), nsaid use and glucocorticoid dose. small numbers of non-anti-tnf b/tsdmards precluded analysing the association of these individual agents with hospitalisation (online supplementary table 4). our findings remained largely unchanged in sensitivity analyses excluding those with a presumptive diagnosis (n=52; online supplementary table 5), those with unknown outcomes (n=214; online supplementary table 6) and those with missing/unknown values (n=142; online supplementary table 7) . this manuscript describes the largest collection of covid-19 cases among patients with rheumatic diseases, with 600 cases from 40 countries. we identified factors associated with higher odds of covid-19 hospitalisation, including older age, presence of comorbidities and higher doses of prednisone (≥10 mg/ day). we did not see an association between prior nsaid use or antimalarials and hospitalisation for covid-19. we did find b/tsdmard monotherapy to be associated with a lower odds of hospitalisation, an effect that was largely driven by anti-tnf adjusted ors from models including all variables shown. *p value for multivariable logistic regression model (see 'methods' section for details). †patients with more than one disease within these five diagnoses were classified as follows: systemic lupus erythematosus>rheumatoid arthritis>psoriatic arthritis>vasculitis>axial/other spondyloarthritis>other. other rheumatic disease category included (each n<10): undifferentiated connective tissue disease; ocular inflammation; autoinflammatory syndrome; mixed connective tissue disease; antiphospholipid antibody syndrome; calcium pyrophosphate deposition disease; systemic juvenile idiopathic arthritis; juvenile idiopathic arthritis, not systemic; igg4-related disease. ‡chronic obstructive pulmonary disease, asthma, interstitial lung disease or other not specified. §csdmard medications included: antimalarials (hydroxychloroquine, chloroquine), azathioprine, cyclophosphamide, cyclosporine, leflunomide, methotrexate, mycophenolate mofetil/mycophenolic acid, sulfasalazine, tacrolimus; b/tsdmard included: abatacept, belimumab, cd-20 inhibitors, il-1 inhibitors, il-6 inhibitors, il-12/il-23 inhibitors, il-17 inhibitors, anti-tnf and janus kinase inhibitors. b/tsdmard, biologic or targeted synthetic dmards; csdmard, conventional synthetic dmard; dmard, disease-modifying antirheumatic drug; il, interleukin; nsaid, nonsteroidal anti-inflammatory drug; tnf, tumour necrosis factor. therapies. over half of the reported cases did not require hospitalisation, including many patients receiving b/tsdmards. the rate of hospitalisation was higher than in cohorts of general patients with covid-19 but this likely reflects the mechanism by which we collected the case information and should not be interpreted as the true rate of hospitalisation among patients with rheumatic disease infected with sars-cov-2. prior to this report, there had been several small case series of covid-19 in patients with rheumatic disease reported from europe. [8] [9] [10] [11] with few exceptions, 12 13 prior large descriptive studies of patients with covid-19 from china, europe and the usa have not included rheumatic disease in their baseline comorbidities. [14] [15] [16] [17] [18] [19] these studies have not allowed for further inference on the characteristics of patients with rheumatic disease and their associations with covid-19 severity. in accordance with previous studies of covid-19 in different populations, we found that patients with comorbidities such as hypertension, cardiovascular disease and diabetes had higher odds of hospitalisation. [18] [19] [20] we also found that glucocorticoid use at a prednisone-equivalent dose ≥10 mg/day was associated with an increased odds of hospitalisation, which is in agreement with prior studies showing an increased risk of infection with higher dose of glucocorticoids. 21 we did not find a significant association between antimalarial use and hospitalisation in adjusted analyses. the use of hydroxychloroquine for the treatment of covid-19, which was based on in vitro studies, has had mixed results. 2 22 studies from one group suggested a benefit on the surrogate outcome of viral clearance among hospitalised patients, but these studies either had inadequate or no comparator groups. 23 24 two randomised controlled trials of hydroxychloroquine had conflicting findings. 25 26 a phase iib randomised controlled trial comparing two doses of chloroquine among patients hospitalised with covid-19 with historical controls from wuhan detected a negative safety signal-qtc prolongation-but no clinical benefit. 27 finally, two observational studies using propensity score matching to account for confounding by indication have found no significant benefit with either hydroxychloroquine alone or combined with azithromycin on clinical outcomes including mortality 28 29 ; however, epidemiology these studies were limited by design issues and a high risk of bias due to unmeasured confounding. we also did not detect a significant association between nsaid use and hospitalisation in adjusted analyses. although no prior data in patients with covid-19 have supported a deleterious effect of nsaids on clinical outcomes, early reports cautioned against the use of nsaids suggesting harm when used during the clinical course of covid-19. 30 these observations, while anecdotal, may also relate to confounding by indication, since nsaids are also often sold over-thecounter and may not be documented in hospital records with the same accuracy as prescription medications, leading to a reporting bias. we found a lower odds of hospitalisation with b/tsdmards monotherapy in our primary multivariable analysis, which was driven largely by anti-tnf therapies. the number of cases taking other biologic drugs or jak inhibitors was small, and may have been insufficient to demonstrate other underlying effects if present. although we caution against causal inference regarding drug effects given significant potential for residual confounding in our study, we also note that there is biological plausibility for the potential benefit of biologic medications in treating covid-19, as evidenced by those with more severe disease having higher levels of cytokines, including il-6 and tnf. 31 32 the use of il-6 inhibitors is being investigated for covid-19, particularly in cases complicated by aberrant inflammatory responses or 'cytokine storm'. this is based on two initial case series of fewer than 20 patients. 33 34 anti-tnfs have also been suggested as a potential therapy in covid-19, but this has been based solely on preclinical data. 35 randomised, placebo-controlled trials are needed to clarify potential benefits or harms of biologic therapies in treating covid-19. strengths of our study include the first large analysis of patients with rheumatic diseases and covid-19. all case data were entered by rheumatology healthcare providers. the c19-gra physician registry includes cases from 40 countries suggesting that our findings are more generalisable than singlecentre or regional studies. the registry collects information on specific rheumatic disease diagnoses, which to date have not been captured in large, published case series of covid-19. 15 despite these strengths, there are important limitations to these registry data. the c19-gra registry is voluntary and does not capture all cases of covid-19 in patients with rheumatic disease. this approach to data collection places limitations on causal conclusions and temporal relationships and therefore we can only make limited inferences based on our results. there is selection bias due to several factors, including geographic location, hospitalisation status and disease severity, with the more severe cases most likely to be captured. therefore, the data cannot be used to comment on the incidence of covid-19 in this patient population or its severity. since the registry's inclusion criteria are restricted to those with rheumatic disease and covid-19, this precludes the ability to make comparisons with those who do not have rheumatic disease, or those with rheumatic disease who do not have covid-19. although physicians may be contacted for follow-up information for unresolved cases, this is a crosssectional analysis and there is the possibility that some patients may not have progressed to their maximum level of care prior to enrolment. in our dataset, 35% of cases were unresolved or had an unknown resolution status, although exclusion of these cases in sensitivity analyses did not change our conclusions. furthermore, while we have collected information on medication use prior to covid-19 diagnosis, we do not have specific data on the duration of treatment, medication dose, or additional historical treatments. at the time of this report, the c19-gra databases remain open for further case reports. with additional cases, we will be able to examine more detailed outcomes associated with specific rheumatic diseases and covid-19 treatments, as well as the outcomes of covid-19 in people with rheumatic diseases. this series of cases demonstrates that the majority of patients with rheumatic diseases captured in our registry recover from covid-19. in some cases, exposure to specific medication classes is associated with lower odds of hospitalisation; however, these findings should be interpreted with caution because of a high risk of bias. results support the guidance issued by the american college of rheumatology and the european league against rheumatism, which suggest continuing rheumatic medications in the absence of covid-19 infection or sars-cov-2 exposure. 36 37 in this series of people with rheumatic disease and covid-19, use of dmards did not increase the odds of hospitalisation. as in the general population, people with rheumatic diseases who are older and/or have comorbidities have a higher odds of covid-19-related hospitalisation. anti-tnf treatment was associated with reduced odds of hospitalisation while prednisone use ≥10 mg/day was associated with a higher odds of hospitalisation. there was no difference in antimalarials, such as hydroxychloroquine, or nsaid use between those who were or were not hospitalised. twitter pedro m machado @pedrommcmachado and philip c robinson @ philipcrobinson the risk of infections associated with rheumatoid arthritis, with its comorbidity and treatment a rush to judgment? rapid reporting and dissemination of results and its consequences regarding the use of hydroxychloroquine for covid-19 covid-19: consider cytokine storm 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efficacy of hydroxychloroquine in patients hospitalized for covid-19 infection with oxygen requirement: results of a study using routinely collected data to emulate a target trial outcomes of hydroxychloroquine usage in united states veterans hospitalized with covid-19 covid-19: european drugs agency to review safety of ibuprofen clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study effective treatment of severe covid-19 patients with tocilizumab tocilizumab treatment in covid-19: a single center experience trials of anti-tumour necrosis factor therapy for covid-19 are urgently needed college of rheumatology guidance for the management of adult patients with rheumatic disease during the covid-19 pandemic eular provisional recommendations for the management of rheumatic and musculoskeletal diseases in the context of sars-cov-2 competing interests mg reports grants from national institutes of health, niams, outside the submitted work. klh reports she has received speaker's fees from abbvie and grant income from bms, ucb and pfizer, all unrelated to this manuscript. klh is also supported by the nihr manchester biomedical research centre. sa-a has nothing to disclose. lc has not received fees or personal grants from any laboratory, but her institute works by contract for laboratories among other institutions, such as abbvie spain, eisai, gebro pharma, merck sharp & dohme españa, s.a., novartis farmaceutica, pfizer, roche farma, sanofi aventis, astellas pharma, actelion pharmaceuticals españa, grünenthal gmbh and ucb pharma. md reports no competing interests related to this work. she is supported by grants from the national institute of health, pfizer independent grants for learning and change, genentech, horizon pharma. she has performed consultant work for amgen, novartis, regeneron/sanofi unrelated to this work. lg reports personal consultant fees from abbvie, biogen, celgene, janssen, eli lilly, novartis, pfizer, sanofi-aventis, ucb and grants from eli lilly, mylan, pfizer, all unrelated to this manuscript. em reports that lpcdr received support for specific activities: grants from abbvie, novartis, janssen-cilag, eli lilly portugal, sanofi, grünenthal s.a., msd, celgene, medac, pharmakern, gafpa; grants and non-financial support from pfizer; nonfinancial support from grünenthal gmbh, outside the submitted work. gs reports no competing interests related to this work. her work is supported by grants from the national institutes of health and agency for healthcare research and quality. she leads the data analytic center for the american college of rheumatology, which is unrelated to this work. as reports grants from a consortium of 13 companies (among them abbvie, bms, celltrion, fresenius kabi, eli lilly, mylan, hexal, msd, pfizer, roche, samsung, sanofi-aventis and ucb) supporting the german rabbit register and personal fees from lectures for abbvie, msd, roche, bms, pfizer, outside the submitted work. sb reports no competing interests related to this work. he reports non-branded marketing campaigns for novartis (<us$10 000). rg reports non-financial support from pfizer australia, personal fees from pfizer australia, personal fees from cornerstones, personal fees from janssen new zealand, nonfinancial support from janssen australia, personal fees from novartis, outside the submitted work. jsh reports grants from rheumatology research foundation, grants from childhood arthritis and rheumatology research alliance (carra), personal fees from novartis, outside the submitted work. es reports non-financial support from canadian arthritis patient alliance, outside the submitted work. ps reports personal fees from american college of rheumatology/wiley publishing, outside the submitted work. jy reports personal fees from astrazeneca, personal fees from eli lilly, grants from pfizer, outside the submitted work. pm reports personal fees from abbvie, personal fees from eli lilly, personal fees from novartis, personal fees from ucb, outside the submitted work. pr reports personal fees from abbvie, nonfinancial support from bms, personal fees from eli lilly, personal fees from janssen, personal fees from pfizer, personal fees from ucb, non-financial support from roche, personal fees from novartis, outside the submitted work. zi, lj, pk, slt, sr, jfs, lt, kw, wc, jwl and zsw have nothing to disclose. patients and/or the public were involved in the design, conduct, reporting or dissemination plans of this research. refer to the 'methods' section for further details. ethics approval the c19-gra physician registry was determined 'not human subjects research' by the uk health research authority and the university of manchester, as well as under united states federal guidelines assessed by the university of california, san francisco and patient consent was not required.provenance and peer review not commissioned; externally peer reviewed. data availability statement request for access to data from the registry should be made to the data access and sharing committee of the covid-19 global rheumatology alliance.this article is made freely available for use in accordance with bmj's website terms and conditions for the duration of the covid-19 pandemic or until otherwise determined by bmj. you may use, download and print the article for any lawful, non-commercial purpose (including text and data mining) provided that all copyright notices and trade marks are retained.orcid ids laure gossec http:// orcid. org/ 0000-0002-4528-310x pedro m machado http:// orcid. org/ 0000-0002-8411-7972 philip c robinson http:// orcid. org/ 0000-0002-3156-3418 key: cord-354492-6r6qs4pp authors: messina, giovanni; polito, rita; monda, vincenzo; cipolloni, luigi; di nunno, nunzio; di mizio, giulio; murabito, paolo; carotenuto, marco; messina, antonietta; pisanelli, daniela; valenzano, anna; cibelli, giuseppe; scarinci, alessia; monda, marcellino; sessa, francesco title: functional role of dietary intervention to improve the outcome of covid-19: a hypothesis of work date: 2020-04-28 journal: int j mol sci doi: 10.3390/ijms21093104 sha: doc_id: 354492 cord_uid: 6r6qs4pp background: on the 31 december 2019, the world health organization (who) was informed of a cluster of cases of pneumonia of unknown origin detected in wuhan city, hubei province, china. the infection spread first in china and then in the rest of the world, and on the 11th of march, the who declared that covid-19 was a pandemic. taking into consideration the mortality rate of covid-19, about 5–7%, and the percentage of positive patients admitted to intensive care units being 9–11%, it should be mandatory to consider and take all necessary measures to contain the covid-19 infection. moreover, given the recent evidence in different hospitals suggesting il-6 and tnf-α inhibitor drugs as a possible therapy for covid-19, we aimed to highlight that a dietary intervention could be useful to prevent the infection and/or to ameliorate the outcomes during therapy. considering that the covid-19 infection can generate a mild or highly acute respiratory syndrome with a consequent release of pro-inflammatory cytokines, including il-6 and tnf-α, a dietary regimen modification in order to improve the levels of adiponectin could be very useful both to prevent the infection and to take care of patients, improving their outcomes. month later (on 8 january 2020), the chinese authorities declared the identification of a new type of coronavirus, informing the who a few days later that the outbreak was associated with exposure in a seafood market in wuhan city. the infection spread firstly in china and then in the rest of the world, and on the 11th of march, the who declared that covid-19 was a pandemic. coronaviruses (covs) belong to the subfamily orthocoronavirinae in the family of coronaviridae in the order nidovirales, and this subfamily includes α-coronavirus, β-coronavirus, γ-coronavirus, and delta-coronavirus [1] . coronaviruses primarily cause enzootic infections in birds and mammals and, in the last few decades, have shown to be capable of infecting humans as well [2] . in human infections with highly virulent respiratory viruses-such as avian influenza h5n1, h7n9, severe acute respiratory syndrome (sars) coronavirus, and coronavirus disease-19 (covid-19)-immunopathogenesis caused by the overproduction of pro-inflammatory cytokines may play an essential role in disease progression and mortality [3] . several recent studies have reported that covid-19 caused the destruction of the pulmonary parenchyma, including interstitial inflammation and extensive consolidation, similarly to the previously reported coronavirus infection [4, 5] . during coronavirus infection, it was observed that the lungs increased in weight, with a mild pleural effusion of clear serous fluid, named pulmonary edema, and extensive consolidation [6, 7] . in some areas, there was interstitial thickening, with mild-to-moderate fibrosis, but a disproportionately sparse infiltrate of inflammatory cells (mainly histiocytes, including multinucleated forms, and lymphocytes) [8] . a dilatation of the airspaces was observed, as was focal honeycombing fibrosis. an intra-alveolar organization of exudates was described, and the formation of granulation tissues in the small airways and airspaces was reported. these lesions were typically located in the sub-pleural region, and the cellular component mainly consisted of histiocytes, as reported in a previous paper [9] . xu et al. described in their case report the pathological findings of covid-19 associated with acute respiratory distress syndrome. at the x-ray investigation, they detected a rapid progression of bilateral pneumonia. the biopsy samples were taken from the lung; the histological examination showed bilateral diffuse alveolar damage with cellular fibromyxoid exudates [6] . considering that the mortality rate of covid-19, about 5-7% [10] , and the percentage of positive patients admitted to intensive care units being 9-11% [11] , it should be mandatory to consider and take all necessary measures intended to contain the viral infection. a recent study analyzed the data of 150 covid-19 patients, with the aim of defining the clinical predictors of mortality. the results obtained from this study suggest that covid-19 mortality might be due to virus-activated "cytokine storm syndrome", considering that the plasma levels of il-6 were higher in deceased patients compared to in discharged subjects [12] . considering that a detailed study has not been performed on the immunological response to covid-19, the only way to discuss this thematic is to refer to previous knowledge about sars-cov and mers-cov. the first response is obtained through pattern recognition receptors (prrs) including c-type lectin-like receptors, toll-like receptors (tlr), nod-like receptors (nlr), and rig-i-like receptors (rlr). moreover, several inflammatory factors are expressed such as il-6 and tnf-α; moreover, the synthesis of type i interferons (ifns) is activated, and these exert their actions against virus diffusion, accelerating macrophage phagocytosis [13] (figure 1 ). in the light of these considerations and the recent evidence in different hospitals suggesting il-6 and tnf-α inhibitor drugs as a possible therapy for covid-19, this review aims to highlight how a dietary intervention could be useful to prevent the infection and/or to ameliorate the outcome during therapy. the first laboratory report about covid-19 patients indicated several parameters that were found to be altered in blood samples; for example, d-dimer, neutrophil count, blood urea, and creatinine levels were significantly higher. in the same way, several cytokines such as il-6 and tnfα were overexpressed, indicating the immune status of the patients [14] . il-6 represents pro-inflammatory signaling produced by adipose tissue; for this reason, this endocrine cytokine could be important in regulating the host response during acute infection [15] . several papers have described the essential role of il-6 in generating a proper immune response during different kinds of viral infection in the pulmonary tract. others link this cytokine to an exacerbation of viral disease. these latter findings support the hypothesis that il-6 upregulation during viral infections may promote virus survival and the exacerbation of the clinical disease [16, 17] . indeed, il-6 has a pleiotropic function, and it is produced in response to tissue damage and infection. in particular, at the pulmonary level, innate and adaptative immune cell proliferation is strongly influenced by this cytokine. after targeting its specific receptor, il-6 starts a cascade of signaling events mainly associated with the jak/stat3 activation pathway, promoting the transcription of multiple downstream genes related to cellular signaling processes, including cytokines, receptors, adaptor proteins, and protein kinase [15] . furthermore, it has been reported that il-6 is an essential factor for the survival of mice with a viral infection. this cytokine promotes the optimal regulation of the t-cell response, inflammatory resolution, tissue remodeling promoting lung repair, cell migration, and the phagocytic activities of macrophages, as well as preventing virus-induced apoptosis in lung epithelial cells. however, experimental scientific evidence also suggests potential adverse consequences that increased levels of il-6 might have on the cellular immune response against viruses. in this context, different possible mechanisms involving this cytokine might affect viral clearance, ultimately favoring the establishment of a persistent viral state in infected hosts [18, 19] . tumor necrosis factor is a cell-signaling protein (cytokine) involved in systemic inflammation, released predominately from macrophages, but it is also released from a variety of other immune cells. it has been well described that during infection with the influenza virus, the expression of tnfα in lung epithelial cells was higher, exerting powerful anti-influenza virus activity [20] . in an animal model, it has been demonstrated that tnf-α plays a pivotal role in the development of pulmonary fibrosis. tnf-α signals via two receptors, tnf-ri and tnf-rii; the first receptor (tnf-ri) promotes the first laboratory report about covid-19 patients indicated several parameters that were found to be altered in blood samples; for example, d-dimer, neutrophil count, blood urea, and creatinine levels were significantly higher. in the same way, several cytokines such as il-6 and tnf-α were overexpressed, indicating the immune status of the patients [14] . il-6 represents pro-inflammatory signaling produced by adipose tissue; for this reason, this endocrine cytokine could be important in regulating the host response during acute infection [15] . several papers have described the essential role of il-6 in generating a proper immune response during different kinds of viral infection in the pulmonary tract. others link this cytokine to an exacerbation of viral disease. these latter findings support the hypothesis that il-6 upregulation during viral infections may promote virus survival and the exacerbation of the clinical disease [16, 17] . indeed, il-6 has a pleiotropic function, and it is produced in response to tissue damage and infection. in particular, at the pulmonary level, innate and adaptative immune cell proliferation is strongly influenced by this cytokine. after targeting its specific receptor, il-6 starts a cascade of signaling events mainly associated with the jak/stat3 activation pathway, promoting the transcription of multiple downstream genes related to cellular signaling processes, including cytokines, receptors, adaptor proteins, and protein kinase [15] . furthermore, it has been reported that il-6 is an essential factor for the survival of mice with a viral infection. this cytokine promotes the optimal regulation of the t-cell response, inflammatory resolution, tissue remodeling promoting lung repair, cell migration, and the phagocytic activities of macrophages, as well as preventing virus-induced apoptosis in lung epithelial cells. however, experimental scientific evidence also suggests potential adverse consequences that increased levels of il-6 might have on the cellular immune response against viruses. in this context, different possible mechanisms involving this cytokine might affect viral clearance, ultimately favoring the establishment of a persistent viral state in infected hosts [18, 19] . tumor necrosis factor is a cell-signaling protein (cytokine) involved in systemic inflammation, released predominately from macrophages, but it is also released from a variety of other immune cells. it has been well described that during infection with the influenza virus, the expression of tnf-α in lung epithelial cells was higher, exerting powerful anti-influenza virus activity [20] . in an animal model, it has been demonstrated that tnf-α plays a pivotal role in the development of pulmonary fibrosis. tnf-α signals via two receptors, tnf-ri and tnf-rii; the first receptor (tnf-ri) promotes intracellular signaling involving c-jun n-terminal kinase (jnk) and nuclear factor (nf)-κb, while the other receptor, tnf-rii, promotes tnf-ri-dependent cell death, without directly inducing apoptosis. although both receptors are broadly expressed, it is known that the majority of inflammatory signaling is elicited through tnf-ri [21] . in an in vitro model, it has been described that serine/threonine kinases can phosphorylate tnf-ri and its molecules, preventing tyrosine phosphorylation [22] [23] [24] . in patients with covid-19, the high serum levels of il-6 and tnf-α are negatively correlated to t cells; contrariwise, it has been demonstrated that t cell levels were restored by reducing il-6 and tnf-α concentrations [25] . these findings suggested that these cytokines could represent important targets of anti-covid-19 therapies. through the secretion of adipokines, adipose tissue participates in the regulation of several pathophysiological processes in many organs and tissues. among the adipokines, adiponectin is the most relevant. adiponectin is one of the most abundant circulating adipocytokines, accounting for 0.01% of total serum protein. adiponectin is an important regulator of cytokine responses, and this effect is isoform-specific. it is involved in a wide variety of physiological processes, including energy metabolism, inflammation, and vascular physiology. these effects are mediated by two atypical, widely expressed seven-transmembrane receptors, adipor1 and adipor2 [26] . adiponectin has beneficial effects in cardiovascular systems and blood vessels, protecting these tissues through the inhibition of pro-inflammatory and hypertrophic responses and stimulation of endothelial cell responses [27] . adiponectin circulates as three different isoforms (low molecular weight-lmw, medium molecular weight-mmw, and high molecular weight-hmw) [28] . infectious diseases are characterized by an increased production of adiponectin. several papers suggest that adiponectin may be related to disease activity and/or severity in different conditions such as rheumatoid arthritis, osteoarthritis, and systemic lupus erythematosus. since adiponectin has been found to display both pro-and anti-inflammatory activities, controversial findings have been observed regarding the role of total adiponectin in systemic autoimmune and inflammatory joint diseases. for this reason, the relative contribution of each adiponectin isoform to the inflammatory response and joint and/or tissue damage requires further study [29] . it is reported that adiponectin is regulated by transcription factors in adipose tissue, such as peroxisome proliferator-activated receptor-γ (ppar-γ) [30] . during viral infections, it has been reported that the role of the predisposition of hosts is also important, as well as their state of health and nutrition. indeed, it is well known that white adipose tissue is considered an endocrine source of biologically active substances with local and/or systemic action, called adipokines. the inappropriate secretion of adipokines seems to participate in the pathogenesis of obesity-related diseases, including endothelial dysfunction, inflammation, and atherosclerosis [31] [32] [33] . the biological function of adipokines in lung diseases seems to be mainly related to the inflammatory process. in particular, the intercorrelation between adipose tissue and the lung has become evident as the involvement of adiponectin has been demonstrated in several lung diseases such as chronic obstructive pulmonary disease (copd), emphysema, and cancer [34] . in fact, with specific regard to copd, a low-grade inflammatory state has been demonstrated [35] [36] [37] . moreover, increasing evidence suggests that adiponectin also exerts a crucial role in the vascular endothelium, maintaining vascular homeostasis and protecting against vascular dysfunctions. altogether, these findings support the anti-inflammatory role of adiponectin in copd and, in general, in other lung diseases [38] . the critical role of adiponectin in the pathophysiological conditions of the lung is also supported by the modulation of adipors with the downregulation of adipor2. it has been described that the adiponectin oligomerization state is altered in copd; moreover, the presence of adipor1 and adipor2, with a lower expression of adipor2 compared to adipor1, in lung tissue [39] has been demonstrated. the low expression of adipor2 could suggest a specific role of this receptor, mainly implicated in adiponectin's effects on inflammation and oxidative stress. mainly, it has been observed that higher levels of adiponectin are associated with a significant and specific increase in hmw adiponectin, representing the most biologically active forms. thus, hmw adiponectin increases il-6 secretion in human monocytes and human monocytic leukemia cell lines but does not suppress lipopolysaccharide (lps)-induced il-6 secretion. byn contrast, lmw adiponectin reduces lps-mediated il-6 release and also stimulates il-10 secretion [40] . furthermore, several in vitro studies have demonstrated that adiponectin in the a549 adenocarcinoma human alveolar basal epithelial cell line has an essential apoptotic effect and also reduces the production of pro-inflammatory cytokines such as tnf-α, blocking nf-κb nuclear translocation [41, 42] . indeed, adiponectin can reduce innate and adaptive immune cell proliferation and polarization, also blocking the production of pro-inflammatory cytokines such as tnf-α, il-2, and il-6, and enhancing that of anti-inflammatory cytokines such as il-10, with a decrease in the phosphorylation of ampk, p38, erk1/2, and c-jnk [43] [44] [45] [46] . data from in vitro studies on lung cells were consistent with an anti-inflammatory function of adiponectin, and adiponectin-deficient mouse models developed lung function impairments and systemic inflammation [47] . the possible role of adiponectin in inflammatory pulmonary diseases, such as asthma and chronic obstructive pulmonary disease (copd), and in critical illnesses has been the subject of recent investigations. particularly, the hmw isoform has a specific role in pulmonary diseases and critical illnesses, even if its role should be better clarified [48, 49] . an interesting study reported that systemic adiponectin concentrations in humans fall during the acute phase of lung infection: particularly, during the early phase, the pro-inflammatory state is generated by the high systemic tnf-α and il-6 concentrations, with the subsequent inhibition of adiponectin production. contrariwise, it has been described that the reduction in tnf-α and il-6 factors generates a corresponding bounce-back in systemic adiponectin concentrations [50] . although it is still unclear whether the modulation of systemic adiponectin or its signaling pathways has any therapeutic benefit in pulmonary or critical illnesses, it may serve as a novel therapeutic or preventative tool for these illnesses in the future. one obvious pharmaceutical treatment would be the exogenous administration of adiponectin by the inhalational or intravenous route. although this has been tried in mouse models [51] , the problems to be overcome prior to human administration include establishing what the biologically active molecule is and what role post-translational modifications have upon its function, and the associated difficulties in generating biologically active molecules on a large scale. considering the difficulty linked to the direct administration of adiponectin, in the last few years, other drugs have been used that indirectly improve adiponectin production. for example, a synthetic ligand of peroxisome proliferator-activated receptors can increase adiponectin mrna in adipocytes, improving the production and secretion of adiponectin [52] [53] [54] [55] . moreover, other drugs such as fibrates can increase systemic adiponectin levels by enhancing ppar-γ activity [56, 57] . another way to improve adiponectin levels is the use of angiotensin converting enzyme inhibitors [58] [59] [60] . furthermore, it is possible to stimulate adipocyte differentiation [61] and the activation of ppar [62] . finally, it has been described that calcium channel blockers [63] and a central-acting anti-hypertensive agent [64] also increase systemic adiponectin concentrations [65] . the possibility to improve the action of adiponectin through diet is intriguing; it has been described that nutritional interventions may help to regulate systemic adiponectin concentrations. in an animal model, it has been demonstrated that a diet with a high concentration of polyunsaturated fatty acids and supplemented with ω-3 can improve the plasma levels of adiponectin, increasing gene expression [66] . on the other hand, in humans, adiponectin levels are positively associated with a healthy lifestyle and the mediterranean diet, even if the mechanisms of action are not completely known [66] . finally, in light of these considerations, in covid-19 therapy, it could be very useful to combine drug therapy with a specific diet regimen. another important mediator involved in the immune response and influenced by nutrition are fatty acids, in particular, ω-3 pufas [67, 68] . in fact, during bacterial and viral infections, they are able to act on immune cells and regulate diverse inflammatory processes. ω-3 pufas are known to have anti-inflammatory properties and play an essential role in the resolution of inflammation [69] . in several lung infections, the administration of pufa can ameliorate the outcome of the patient in acute pneumonia. sharma et al. reported in their study that the dietary supplementation of ω-3 pufa can exert an overall beneficial effect against acute pneumonia through the upregulation of the host's specific and nonspecific immune defenses [70] . ω-3 polyunsaturated fatty acids (pufa, ω-3-fatty acids), the key components of fish and flaxseed oils, are increasingly consumed by the public because of their potential health benefits and can be used clinically for the treatment of metabolic, cardiac, inflammatory, and autoimmune diseases [71] . however, numerous studies have shown that these compounds are immunoregulatory and immunosuppressive and thus may increase susceptibility to infection. while reports suggest that ω-3 pufas may have beneficial effects against extracellular pathogens, few studies have been performed on systemic viral infections in mammals. jones and roper described in their study that a diet rich in ω-3 pufas did not significantly lower survival of the vaccinia virus infection, at least with short-term (~6 week) feeding in mice [71] . ω-3 pufas are metabolized into various mediators possessing anti-inflammatory properties such as resolvins and protectins. it is known that ω-3 pufas can reduce nf-κb activation by preventing nuclear p65 nf-κb translocation. furthermore, ω-3 pufas minimize the activation of erk1/2 mapk, also reducing cox-2 production. the ω-3 pufa-derived lipid mediator could markedly attenuate influenza virus replication via the rna export machinery. in addition, the treatment of protectin d1 with peramivir could completely stop mouse mortality [72] . ω-3 supplementation was previously studied in acute respiratory distress syndrome (ards). singer and shapiro suggested that the enteral administration of natural antioxidant substances could improve oxygenation and clinical outcomes in icu patients [73] . a systematic review performed in 2015 reported a positive effect only for patients suffering from ards with high mortality [74] . a more recent meta-analysis highlighted the importance of clinical trials in order to clarify the use of ω-3 fatty acids and antioxidants in patients with ards to ascertain the positive effects in order to reduce the lengths of icu stays and the numbers of days spent on ventilators [75] . although the role of ω-3 supplementation in ards should be better clarified, its pivotal role in reducing reactive oxygen species and pro-inflammatory cytokines, such as tnf-α, il-1β, il-6, and il-8 [76] , is well known. therefore, ω-3 pufas, including protectin d1, which is a novel antiviral drug, could be considered for potential interventions for covid-19. as previously described, other dietary constituents can be used to improve the patients' outcomes during lung infection, regulating the inflammatory response. among these, antioxidants play an important role in protecting lung cells against viruses and bacteria. viral infection leads to an increase in the intrapulmonary oxidative burden. in many diseases, the balance between oxidants and antioxidants (redox balance) is altered, with severe consequences [77] . the pathophysiological mechanisms by which free radicals generate various types of stress-such as oxidative, nitrative, carbonyl, inflammatory, and endoplasmic reticulum stress-lead to lung inflammation and an altered lung immune response. in this scenario, dietary antioxidants may play an important role against lung oxidative stress [77] . several studies reported the protective role of the antioxidants in lung infection and in lung inflammation [78, 79] . in particular, vitamin c, polyphenols, and flavonoids can play a protective role in lung infections, being immune modulators and inflammatory mediators. indeed, as reported by carr et al., during infection, vitamin c levels may become depleted; for this reason, vitamin c supplementation can attenuate infection. based on this evidence, these authors suggested a clinical trial with vitamin c infusion for the treatment of severe covid-19 patients [80] . among polyphenols, epigallo-catechin 3 gallate (egcg) is the most potent ingredient in green tea and exhibits antibacterial, antiviral, antioxidative, anticancer, and chemo-preventive activities. recently, numerous studies have investigated the protective effects of egcg against asthma and other lung diseases such as copd and lung pneumonia. egcg may suppress inflammation and inflammatory cell infiltration into the lungs of asthmatic mice, and may also inhibit epithelial-mesenchymal transition emt via the pi3k/akt signaling pathway through upregulating the expression of phosphatase and tensin homolog (pten), both in vivo and in vitro [81] . moreover, flavonoids can be used to attenuate lung injury in mice; it has been reported that they inhibit influenza virus and toll-like receptor signaling, blocking nf-κb translocation [82] . therefore, as summarized in table 1 , supplementation with vitamin c, flavonoids, and polyphenols can be tested in covid-19 patients, both in order to prevent viral infection and to improve patients' outcomes. table 1 . the principal antioxidants involved in lung infection and the immune-inflammatory response. red wine, oranges, red fruits and vegetables reduces inflammation and immune response, blocking nuclear nf-κb translocation polyphenols green tea, broccoli, apples vitamin c oranges, lemons, mangoes during pulmonary infections, and particularly in covid-19 patients, intracellular signaling leads to the production of pro-inflammatory cytokines, such as tnf-α and il-6, which act in concert with chemoattractants, such as cxcl1 and cxcl2, to recruit polymorphonuclear leukocytes (pmns) to the lungs, killing pathogens but generating fibrosis [83] . another important consideration during covid-19 infection is related to the modification of the secretory products of the upper and lower airways, which usually include mucin and pulmonary surfactant. during infection, mucin production is upregulated, with the function of preventing microbes from binding to and infecting epithelial cells [84] . the primary source of phospholipids (pls) in the lung is pulmonary surfactant, synthesized and released by alveolar epithelial type ii cells. the surfactant contains approximately 80-90% pls, with fatty acid chains that can be oxidized during different challenges in the lung [85] . the oxidation of these pls in the lung can occur in the setting of an increased oxidative stress situation, such as infection and inflammation [86] . the immune effects of oxidized phospholipids oxpls during infectious diseases are inevitably dictated by the balance among activation, degradation, and scavenging. it has been shown that oxpls are generated in the lung during several pulmonary infections, including influenza and avian influenza (h5n1), as well as sars coronavirus, even if the mechanisms of action are not well known [87] [88] [89] . as reported by imai et al., oxpl-induced inflammation is mediated by tlr4 and trif, driving an increase in il-6 production [89] . it is intriguing to consider that oxpl-dependent defects in phagocytosis and ros generation may lead to an increased susceptibility to respiratory infections [90] . cholesterol is the major neutral lipid in pulmonary surfactant, in which it is thought to promote the spreading, mobility, and adsorption of surfactant films [91] . as previously documented, modulating adiponectin levels can be considered an important way to reduce cytokines levels; in this way, the adverse effects related to the covid-19 infection should be attenuated. it is well described in animal models that the consumption of hyperlipidemic diets, rich in saturated fat, reduces the levels of adiponectin, while diets rich in polyunsaturated fatty acids and supplemented with ω-3 pufa increase adiponectin levels, reducing pro-inflammatory cytokines [66] . innate and adaptive immune responses are influenced not only by oxpls and cholesterol but also by the fatty acid profiles of tissues in response to pharmacological agents and diet [92] . several studies performed in animal models demonstrated how ω-3 pufa uptake into the lung tissue influences outcomes associated with infection, promoting the resolution of inflammation [93] . in another study, ω-3 pufas reduced the levels of pmns and lowered il-6 levels in lung infections [94] . these positive effects remain controversial; for example, jones and roper reported that in their experimental model, no statistically significant differences were found among the diet regimens, with and without ω-3 pufas, with respect to the susceptibility of mice to viral infection, morbidity, viral organ titers, recovery time, or mortality [71] . in conclusion, it is well known that general treatments are very important to enhance the host immune response against rna viral infection. in addition, the immune response has often been shown to be weakened by inadequate nutrition in many model systems as well as in human studies. however, the nutritional status of the host, until recently, has not been considered as a contributing factor to the emergence of viral infectious diseases. the recent reports about the pathogenesis of covid-19 suggested that one of the most important consequences of this infection is the cytokine storm syndrome [95] , which could be strictly linked with coagulopathy, generating acute pulmonary embolism caused by in-situ thrombosis [96, 97] . therefore, a great number of clinical trials are ongoing to define a useful therapy to attenuate cytokine storms [98] . for these reasons, an adequate ω-3 pufa intake may be a valid strategy against viral infection. indeed, following the recommended intake of ω-3 pufa, in the range of 0.5% and 2% of total calories (250 mg/day), may be important to protect against an excessive inflammatory response, also reducing il-6 levels. this theory found important support in a recent study that demonstrated that ω-3 pufa-derived lipid mediator protectins can suppress influenza virus replication through a mechanism that blocks the export of viral mrna. moreover, imai demonstrated that this mediator can be used in combination with the antiviral peramivir, even at late time points in infection [99] . nevertheless, the efficacy of ω-3 pufas at the clinical level is under investigation; for example, hecker et al. described a beneficial effect for a diet regimen with ω-3 pufas, describing that the pro-inflammatory cytokine levels decreased after this diet regimen [100] . the suggested positive role in the outcome and prevention of the covid-19 infection is summarized in figure 2 . innate and adaptive immune responses are influenced not only by oxpls and cholesterol but also by the fatty acid profiles of tissues in response to pharmacological agents and diet [92] . several studies performed in animal models demonstrated how ω-3 pufa uptake into the lung tissue influences outcomes associated with infection, promoting the resolution of inflammation [93] . in another study, ω-3 pufas reduced the levels of pmns and lowered il-6 levels in lung infections [94] . these positive effects remain controversial; for example, jones and roper reported that in their experimental model, no statistically significant differences were found among the diet regimens, with and without ω-3 pufas, with respect to the susceptibility of mice to viral infection, morbidity, viral organ titers, recovery time, or mortality [71] .s in conclusion, it is well known that general treatments are very important to enhance the host immune response against rna viral infection. in addition, the immune response has often been shown to be weakened by inadequate nutrition in many model systems as well as in human studies. however, the nutritional status of the host, until recently, has not been considered as a contributing factor to the emergence of viral infectious diseases. the recent reports about the pathogenesis of covid-19 suggested that one of the most important consequences of this infection is the cytokine storm syndrome [95] , which could be strictly linked with coagulopathy, generating acute pulmonary embolism caused by in-situ thrombosis [96, 97] . therefore, a great number of clinical trials are ongoing to define a useful therapy to attenuate cytokine storms [98] . for these reasons, an adequate ω-3 pufa intake may be a valid strategy against viral infection. indeed, following the recommended intake of ω-3 pufa, in the range of 0.5% and 2% of total calories (250 mg/day), may be important to protect against an excessive inflammatory response, also reducing il-6 levels. this theory found important support in a recent study that demonstrated that ω-3 pufaderived lipid mediator protectins can suppress influenza virus replication through a mechanism that blocks the export of viral mrna. moreover, imai demonstrated that this mediator can be used in combination with the antiviral peramivir, even at late time points in infection [99] . nevertheless, the efficacy of ω-3 pufas at the clinical level is under investigation; for example, hecker et al. described a beneficial effect for a diet regimen with ω-3 pufas, describing that the pro-inflammatory cytokine levels decreased after this diet regimen [100] . the suggested positive role in the outcome and prevention of the covid-19 infection is summarized in figure 2 . in addition, adiponectin plays a role in lung diseases and obesity; in the development and progression of lung disease and cancer, a pathogenic role of adiponectin was defined by both in vivo and in vitro studies. recently, immunometabolic pathomechanisms have been identified as important factors determining and modulating lung function and disease. particularly, adiponectin levels have been found to be greater in patients with copd compared with in control patients, and adiponectin-deficient mice are protected from several lung diseases [101] . moreover, it has been in addition, adiponectin plays a role in lung diseases and obesity; in the development and progression of lung disease and cancer, a pathogenic role of adiponectin was defined by both in vivo and in vitro studies. recently, immunometabolic pathomechanisms have been identified as important factors determining and modulating lung function and disease. particularly, adiponectin levels have been found to be greater in patients with copd compared with in control patients, and adiponectin-deficient mice are protected from several lung diseases [101] . moreover, it has been reported that adherence to the mediterranean diet was associated with an increase in adiponectin levels, improving cardiovascular system functionality [102] , particularly in elderly people [103] . these findings are only apparently contradictory to the first data about the mortality rate from covid-19 infections in the mediterranean area (such as in italy and spain) [104] . first of all, the data have been referred only to the tested population; moreover, it is well described that the presence of several comorbidities such as hypertension, diabetes, and cardiovascular diseases severely influenced the mortality rate reported in this area [105] . all these comorbidities can be counteracted with a correct dietary regimen. therefore, both adiponectin and ω-3 pufas appear to be attractive biomarkers for monitoring lung disease progression. finally, considering that the covid-19 infection can generate a mild or highly acute respiratory syndrome with a consequent release of pro-inflammatory cytokines, including il-6 and tnf-α, a modification of the dietary regimen in order to improve the levels of adiponectin could be very useful both to prevent the infection and to take care of the patients, improving their outcomes. given the similar pathway of action, it can be hypothesized that adiponectin and ω-3-pufa could be used as real drugs to reduce inflammation, reducing both il-6 and tnf-α levels as well as ameliorating the 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article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we wish to thank the scientific bureau of the university of catania for language support. the authors declare no conflict of interest. key: cord-023439-r04y1j22 authors: hedegaard, c. j.; bendtzen, k.; nielsen, c. h. title: the role of immune complexes consisting of myelin basic protein (mbp), anti‐mbp antibodies and complement in promoting cd4(+) t‐cell responses to mbp in health and multiple sclerosis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423k.x sha: doc_id: 23439 cord_uid: r04y1j22 multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti‐mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen‐presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp‐derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease‐associated anti‐mbp antibodies in ms patients, respectively. we have investigated the formation of mbp‐containing ic, the binding of mbp to b cells, the mbp‐elicited induction of th‐cell and b‐cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c‐inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4(+) t cells, we observed the production of tnf‐α, ifn‐γ and il‐10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il‐2, il‐4 and il‐5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp‐containing ic and thereby enhance the cytokine responses, by virtue of elevated anti‐mbp contents. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023928-9a1w174h authors: thomas, neal j.; dahmer, mary k.; quasney, michael w. title: genetic predisposition to critical illness in the pediatric intensive care unit date: 2011-12-16 journal: pediatric critical care study guide doi: 10.1007/978-0-85729-923-9_11 sha: doc_id: 23928 cord_uid: 9a1w174h much progress has been made in the past decade in the understanding of the genetic contribution to the development of human disease in general, and critical care illness specifically. with the mapping of the human genome and on-going mapping of genetic polymorphisms and haplotypes in humans, the field of critical care is now in prime position to study the impact of genetics on common illnesses that affect children who require critical care, to examine how differences of the host defense response lead to variable outcomes in outwardly appearing similar disease states, and to study how genetic differences in response to therapy will help practitioners tailor therapeutic interventions to an individual child’s genetic composition. while we are still years away from true individualized medicine, we are now closer than ever to understanding why two might children respond to the same environmental insult in vastly different ways. much progress has been made in the past decade in the understanding of the genetic contribution to the development of human disease in general, and critical care illness specifi cally. with the mapping of the human genome and on-going mapping of genetic polymorphisms and haplotypes in humans, the fi eld of critical care is now in prime position to study the impact of genetics on common illnesses that affect children who require critical care, to examine how differences of the host defense response lead to variable outcomes in outwardly appearing similar disease states, and to study how genetic differences in response to therapy will help practitioners tailor therapeutic interventions to an individual child's genetic composition. while we are still years away from true individualized medicine, we are now closer than ever to understanding why two might children respond to the same environmental insult in vastly different ways. neal j. thomas , mary k. dahmer, and michael w. quasney before being able to appreciate the advances in research that have been accomplished in relation to the genetic impact on critical illness in children in recent years, it is important to understand the basics of human genetics, and become familiar with the terminology that is utilized to discuss these remarkable advances. once the genetic basics are clear, discussion can then proceed to genetic associations that have been determined in critical illness in children. the nucleus of all cells holds chromosomes that contain deoxyribonucleic acid (dna), the genetic material that is inherited from parents. dna is responsible for determining the structure of the cell, the function and activity of the cell in response to various stimuli, and the interaction the cell has with other cells and the extracellular environment. the dna molecule consists of two chains of deoxyribonucleotides held together by complementary base pairs. the deoxyribonucleotides contain the four nucleotide bases, adenine (a), thymine (t), guanine (g), and cytosine (c) that are covalently bound together by phosphodiesterase bonds linking the 5 ¢ carbon of one deoxyribose group to the 3 ¢ carbon of the next group. the two chains of deoxyribonucleotides are linked by hydrogen bonds between the a's of one strand and the t's of the other. likewise, the g's of one strand are linked by hydrogen bonds to the c's of the complementary strand. these two complementary strands form the dna double helix ( fig. 11-1 ) , with one strand running in the 5 ¢ to 3 ¢ direction while the other strand runs in the 3 ¢ to 5 ¢ direction. the order of nucleotides bases is termed the sequence and is read in the 5 ¢ to 3 ¢ direction. the genetic information of an individual is encoded by the precise positioning and order of these base pairs. the order of nucleotide bases is termed the sequence and is read in the 5 ¢ to 3 ¢ direction. the four nucleotides of the dna double helix (from national human genome research institute's talking glossary of genetics ( http://www.genome.gov/ glossary.cfm#s )) the entire dna content of an organism is their genome . every cell of an organism contains two copies of the dna, with the exception of red blood cells which lack a nucleus and dna and sperm and egg cells which contain one copy of the dna. overall, humans have 46 chromosomes, including 22 pairs of autosomal chromosomes and one pair of sex chromosomes. each chromosome is made up of a centromere and two telomeres (ends) (fig. 11-2 ). the two arms of the chromosome are the short arm (p) and long arm (q). the parts of the genome that contain nucleotide sequences that code for proteins are the genes and it is estimated that the human genome contains about 20,000-30,000 genes. the structure of genes is very complex and highly variable. genes are made up of a variable number of exons , which contain the actual coding sequence for the proteins, and introns , which are noncoding regions which separate the exons. while the function of the introns is unclear, some disease processes have been found to be associated with certain nucleotide variations located in these intron regions ( fig. 11-3 ). genes also have regulatory regions, including promoter sequences that generally reside at the 5 ¢ end of the gene (referred to as upstream) and regions at the 3 ¢ end associated with stability of the mrna. genetic recombination , which is the re-shuffl ing of genes from generation to generation, is the basis of genetic diversity in sexually reproducing organisms. the analysis of genetic recombination is a useful method of mapping genes in the genome. genetic recombination results in an exchange of genetic material between homologous chromosome pairs. this results in segments of dna being exchanged with the other chromosome of the pair, thereby shuffl ing the genetic material. the basic principal of linkage analysis , a method used to fi nd disease causing genes, relies on the genetic recombination frequency between two loci on a single chromosome. this allows the estimation of the relative distance between them, and is crucial for the mapping of genes in the genome. recombination frequencies can be measured by genotyping individuals in a family pedigree. the closer together two loci are on a chromosome, the lower the likelihood of recombination to occur between them. if loci are very close, they are said to be linked. the reliability of genetic linkage between loci is determined using the lod score , which is an estimate of whether two loci are likely to lie near each other on a chromosome and are therefore likely to be inherited together. a lod score of 3 or more, which represents odds of 1000:1 or greater in favor of linkage, is used to indicate statistically signifi cant linkage, and therefore concludes that the two loci of interest are close. the development of genetic maps has been very useful in fi nding genes which may cause human disease. genes may be mapped to a particular location in the genome based on being inherited with respect to a marker of known map location, and with the assumption of no the entire dna content of an organism is their genome. genes are made up of a variable number of exons, which contain the actual coding sequence for the proteins, and introns, which are noncoding regions which separate the exons. nucleus telomere telomere centromere the structure of a chromosome (from national human genome research institute's talking glossary of genetics ( http://www. genome.gov/glossary.cfm#s )) genetic recombination. there are a number of polymorphic markers which may be utilized for genetic map construction, including minisatellites, microsatellites, and single nucleotide polymorphism (snps). linkage disequilibrium (ld), often referred to as allelic association, is a measure of physical association between two alleles and occurs when closely linked alleles are inherited together during many generations. no signifi cant degree of genetic recombination occurs between them, and they continue to be passed along together throughout generations. therefore, knowledge of one marker can be used to study the other. there are many potential benefi ts of identifying genes/gene variants involved in disease. these include, but are not limited to, an improved understanding of the disease etiology, insight into the mechanisms of disease pathogenesis, an ability to develop an early disease risk assessment, the potential to discover novel therapeutic drug targets, the ability to estimate the therapeutic response to specifi c pharmacologic therapies, the possibility of targeted disease prevention strategies to be utilized in high-risk populations based on genetic predisposition, and the movement from the classic symptoms-based disease defi nition towards a true molecular defi nition of complex disease processes. genetic mutations are changes that occur in the sequence of dna. mutations can be classifi ed as somatic mutations, which occur in somatic cells and are not commonly passed on to offspring, and germ-line mutations which occur in the reproductive cells and are passed on linkage disequilibrium (ld), often referred to as allelic association, is a measure of physical association between two alleles and occurs when closely linked alleles are inherited together during many generations. the structure of a gene, including the exons (coding sequence) and introns (noncoding sequence) (from national human genome research institute's talking glossary of genetics ( http://www. genome.gov/glossary.cfm#s )) to offspring. there are several different types of mutations. translocations are large-scale mutations comprised of switching of chromosomal regions between one chromosome and another chromosome. mutations can also consist of single changes in the nucleotide bases and include substitution, deletion, or insertion of nucleotides. insertions and deletions can also involve hundreds of nucleotides. mutations that occur in the coding regions can have several consequences: they can change the amino acid of the protein at a single site, they can cause a premature stop codon resulting in early termination of translation, and, consequently, lead to a truncated protein, or they may have no effect at all if the mutation leads to a nucleotide substitution that does not alter the amino acid. likewise, mutations in noncoding regulatory regions (such as promoters) may also affect the expression of the gene by altering the quantity of mrna transcribed and, hence, the level of the protein. mutations in the intron/ exon boundary region may also lead to incorrectly spliced mrnas and result in signifi cantly different proteins or differences in levels of protein products. the sequencing of the human genome has revealed that most genes are polymorphic; that is, there are small differences in the nucleotide sequences. there are estimates that the human genome may contain over 10 million of these types of variations. these differences in the nucleotide sequence are what give rise to our genetic variability; they account for inherited differences in our physical traits and the way we respond to environmental stimuli and medications. while the majority of these nucleotide variations do not cause a disease, some genetic variations may infl uence the development of certain diseases. the mutations discussed in the preceding paragraph are variations that occur in less than 1% of the population and are, thereby, rare. on the other hand, variations that occur at a frequency greater than 1% in the population are referred to as polymorphisms . if the polymorphism is a change in a single nucleotide, it is referred to as a single nucleotide polymorphism (snp) . these more common genetic variations, whether snps or small insertions or deletions of nucleotides, are the ones currently being examined in many studies for associations with susceptibility to and outcome from diseases seen in the intensive care unit setting. copy number variations (cnvs) are stretches of dna of greater than 1 kb that show differences in the expected number of copies of the dna in greater than 1% of the human population. very recently it has become clear that cnvs are also common in human genomes and contribute significantly to human genetic variation. another important concept in genetics is a locus, which refers to the location in the genome of a specifi c gene or variant. keeping in mind the above discussion of genetic variations, a locus may contain two slightly different sequences for a specifi c gene. these alternative forms of a gene are termed alleles , or variants . the alleles for a specifi c individual at a genetic locus is that person's genotype . an example is the surfactant protein b (sp-b) +1580 site. an individual's genotype at that site may either be tt, ct, or cc. individuals are heterozygous if they possess two different alleles at the locus of interest and homozygous if they possess two identical alleles at that locus. but we obviously do not contain only one genetic variation in our genome. a haplotype represents a combination of polymorphic alleles on a single chromosome delineating a pattern that is inherited together and transmitted from parent to offspring. haplotype analysis is a useful tool for analysis of disease gene discovery, as investigators may capitalize on the fact that many of the polymorphisms of interest are not transmitted independently of each other, and the presence of one gene variant can tag the presence of another polymorphism from the same chromosome. in some cases, haplotype assessment can provide a higher level of specifi city, sensitivity, and accuracy in "true" associations with disease risk or severity. by focusing on haplotypes as well as snps, researchers are now able to more accurately study genetic predisposition to various diseases of interest. with the recent report of the international hapmap consortium, and the identifi cation and cataloging of haplotypes now available, the utility of this type of study is brought into focus as an important tool to guide genetic association studies on complex human diseases. genetic polymorphisms, like the rarer mutations, may also infl uence the quantity of the mrna made if present in a regulatory region, or they may also infl uence the functional activity of the protein product. there has been an explosion of studies attempting to determine if these genetic polymorphisms may account for some of the clinical variability we as clinicians observe at the bedside in the picu. for example, can the difference in disease genetic mutations are rare changes that occur in the sequence of dna that occur in less than 1% of the population. polymorphisms are variations that occur at a frequency greater than 1% in the population; if the polymorphism is a change in a single nucleotide, it is referred to as a single nucleotide polymorphism (snp). a haplotype represents a combination of polymorphic alleles on a single chromosome delineating a pattern that is inherited together and transmitted from parent to offspring. c hapter 11 • g en etic pr edis pos ition to c r itical i lln ess severity between two children with pneumonia be associated with variations in their genes coding for one of the surfactant proteins? gene expression is the process by which the information contained within genes is used to make proteins ( fig. 11-4 ) . this occurs by a combination of two distinct processes: transcription and translation. transcription is the process by which the genetic information in dna is transcribed into messenger ribonucleic acid (mrna). mrna differs from dna in that it is singlestranded, has a modifi ed sugar backbone, and contains uracil (u) instead of t. the process of transcription involves the unwinding of the two complementary strands of dna, the enzyme rna polymerase binding to the promoter region of a gene on a single strand of dna, and synthesizing the mrna molecule by adding ribonucleotides in an order that is complementary to the dna strand. the transcribed mrna thus contains all the genetic information between the transcriptional start and stop sites on the dna including exons and introns. the non-coding intron sequences are removed by a process referred to as splicing which connects all the exons together to form the fi nal mrna product. this mrna represents the coding dna sequences for a single gene. (it should be noted that splicing variations have been identifi ed that infl uence disease processes that impact the fi nal protein product by altering the mrna sequences that are spliced together.) the mrna is then transported to the cytoplasm, and translation occurs, in which the genetic information from mrna is utilized to guide the synthesis of proteins. proteins are composed of amino acids. there are 20 different amino acids in humans, and each is encoded by a set of 3 nucleotides in the mrna. these three nucleotides are called triplets or codons . the corresponding anticodon on the transfer rna (trna) links with a codon, presenting its unique amino acid in the process of translational protein synthesis. gene expression is the process by which the information contained within genes is used to make proteins. transcription is the process by which the genetic information in dna is transcribed into messenger ribonucleic acid (mrna). translation is the process by which the genetic information from mrna is utilized to guide the synthesis of proteins. since all cells that contain a nucleus carry the full set of genetic information, it is necessary for gene expression to be selective and tightly controlled, in a way that guarantees specifi c proteins are expressed in specifi c cells under appropriate conditions. this differential expression of genes ensures that cells develop correctly, can differentiate and function as specialized cells, and can mount various responses to external stimuli. in certain disease states, expression of specifi c genes may change, thereby providing a clue as to which genes may be important in that disease process. recent advances in technology have provided a valuable tool to evaluate the expression of genes during various diseases, including sepsis. these technologies include dna microarrays, in which the basic approach is as follows: small strands of dna probes representing the genes of interest, for example, tumor necrosis factor alpha (tnf-a ), are attached to a solid substrate such as a glass slide or silicon chip (in reality, thousands of probes are applied to the same micorarray chip). mrna is then isolated from, for example, a patient without acute lung injury (ali) and one with ali. these two samples are then separately converted to complementary dna (cdna) in a manner which incorporates different fl uorophores in the two samples of cdna (e.g., red into all the cdnas from the patient with ali which would also include cdna made from the mrna coding for tnf-a , and green into all the cdnas from the patient without ali including the cdna made from the mrna coding for tnf-a ). the cdnas from the two patients are then mixed and hybridized to the chip containing the dna probes. thus, if tnf-a is up-regulated in ali and there is signifi cantly more cdna in the ali sample than the non-ali sample, then more red fl uorophore -labeled cdna would be present. when the mixture of cdnas is hybridized to the chip containing the probes, the probe for tnf-a would light up red and represent increased expression of the tnf-a gene in ali. if tnf-a is expressed at a lower concentration in the patient with ali then when the samples are mixed, more green fl uorophore-labeled cdna would be present than red fl uorophore-labeled cdna, and the probe for tnf-a on the microarray chip would light up green indicating decreased expression of tnf-a gene in ali. finally, if there is no change in the expression of the tnf-a gene, the amounts of red and green fl uorophorelabeled cdnas would be equal, and the probe for the tnf-a gene would light up yellow. in this fashion, one can identify specifi c genes that are expressed in the development of ali. several examples of the use of this technology in the critically ill patient have been published which have aided our understanding of the pathophysiology of certain icu specifi c diseases. up to this point, we have discussed the structure of dna and the process of getting from the code in the dna to protein. it is the functional aspects of these proteins that give rise to the observed traits, whether it be the color of one's eyes, the rate of metabolism of a drug, or the effi ciency with which a protein receptor on a cell surface recognizes a pathogen. the observable characteristics of an individual defi ne that individual's phenotype . this may include common physical and biochemical characteristics, but can also describe a person's disease status (such as in cystic fi brosis). phenotypes caused by mutations in a single gene may show mendelian inheritance patterns . these patterns can be autosomal dominant (where a single copy of the gene causes the phenotype), autosomal recessive (where both copies of the gene are necessary for the phenotype), or sex linked (where the mutation occurs on the x chromosome). it is crucial to note that mendelian inheritance patterns are only seen for single gene disorders. critical care diseases and syndromes, such as sepsis and acute respiratory distress syndrome (ards), are complex disorders whose genetic predisposition to the development of the disease is due to multiple genes and other factors, such as environmental exposures. the multifaceted gene-gene and gene-environment interactions make the study of these diseases extremely complex. there are many common complex disorders that display obvious familial aggregation of cases, but have no clear mendelian inheritance patterns. the most commonly studied in medicine are cancer, diabetes, hypertension, and obesity, among others. the disease aggregation may be due to complex genetic factors, the interaction of multiple genes on the development the observable characteristics of an individual defi ne that individual's phenotype. c hapter 11 • g en etic pr edis pos ition to c r itical i lln ess of the disease of interest, a host of environmental factors which place the individual at risk for the disease, or, most commonly, a combination of all of the above factors ( fig. 11-5 ). in other words, a person's genetic background may make them prone to a specifi c disease; this is called susceptibility gene variants or susceptibility genes. due to their inherited genetic make-up, the individual is at a higher inborn risk for developing the disease of interest, but only if they are exposed to the environmental stressor that is known to associate with the disease. the susceptibility gene variant will not directly lead to the disease, but will put that person at a higher risk if they are exposed to the environmental risk. for example, individuals may possess a susceptibility gene variant for the development of lung cancer, but this will only lead to the development of cancer if they are subject to a known environmental risk factor, such as smoking. alternatively, a newborn may possess one or more susceptibility gene variants for the development of bronchopulmonary dysplasia, but if they are not born prematurely and do not require mechanical ventilation in the newborn period (and therefore are not subject to the environmental stressors known to impact the development of this lung disease), they will never develop this disease despite being genetically susceptible. gene variants can also decrease the susceptibility, or increase the resistance against a disease. these are protective gene variants . to give an example, there are individuals who smoke their entire adult lives and yet never develop chronic obstructive pulmonary disease. it is likely that these individuals possess protective gene variants against the development of this disease, even in the face of a strong environmental insult. a person may possess both susceptibility and protective genetic variants for the same disease, and the mix of these variants will impact together the overall genetic risk of that person to the disease of interest. this resultant genetic risk also interacts with the environmental risk of the individual, leading to the overall risk of that person developing the disease. in critical care, it is unlikely that one gene will cause the diseases that are treated in the intensive care unit; it is more likely that multiple genes will interact with multiple environmental insults to predispose individuals to diseases processes resulting in an overall risk for an individual patient to develop a certain disease of interest. interestingly, certain populations seem to be immune to certain complex diseases. examples include the australian aborigines and inuits from greenland, in which both populations appear to be resistant to the development of type 1 diabetes. it is plausible that the disease resistance observed in these populations is due to absence of susceptibility gene variants or presence of protective gene variants in the group's gene pool. when it is determined that a complex disease has familial aggregation, it is important to take into account that families may also share environmental or social factors that predispose to the disease of interest, and therefore the entire impact may not be genetic. one example would be radon gas present in a neighborhood leading to an increased incidence of lung cancer in families living in close proximity. while it may be assumed that the genetic impact is responsible for the development of the disease, environmental exposure is the likely source. twin studies and adoption studies are utilized to attempt to determine the relative weight of genetics and environment in the development of a certain disease. genomic medicine and the concept that an individual's genetic makeup may infl uence not only the severity of and outcome from their critical illness, but also their response to the therapies, has begun to makes it's way into the intensive care unit. this section will highlight disease aggregation may be due to complex genetic factors, the interaction of multiple genes on the development of the disease of interest, a host of environmental factors which place the individual at risk for the disease, or, most commonly, a combination of all of the above factors. the complex interactions that can occur between genes that may impact a disease process (gene-gene interaction) as well as between the genes of interest and environmental factors (gene-environment interaction) studies involving analysis of gene expression and genetic association studies in patients with critical illness. while it may appear that advances have been made in the fi eld, it is important to understand that we are not yet in the age of personalized medicine and much work needs to be done. the expression of specifi c genes in many cases represents the body's specifi c and complex response to environmental stimuli, such as in the case of a severe infection, trauma, or cardiopulmonary bypass. examining gene expression may, therefore, provide a clue as to which genes are important in a specifi c critical illness. many studies have examined gene expression in critically ill patients, but most examine the expression of only a few genes. with the advent of the dna microarray technology discussed above investigators have begun to explore gene expression patterns in thousands of genes in children with septic shock using mrna isolated from whole blood representing the gene expression response in circulating white blood cells. these studies have compared gene expression from blood samples obtained within 1 day of admission to the picu with that observed in blood of healthy controls, examined longitudinal changes in gene expression in children with septic shock (days 1 and 3) and compared expression in patients with septic shock to expression in children with sepsis or systemic infl ammatory response syndrome (sirs). genes that were up-regulated, down-regulated, or unchanged between the groups were examined. the genes that were up-regulated when the septic shock group was compared to healthy controls included genes related to immunity and infl ammation as would be expected. unexpectedly the study demonstrated that many genes related to zinc biology and zinc homeostasis were down-regulated. the signifi cance of this fi nding was supported by the observation that children who did not survive septic shock had lower serum levels of zinc and the demonstration in a murine model that zinc depletion leads to increased mortality from sepsis. in addition, genes involved in t-cell receptor signaling and antigen presentation appeared decreased suggesting that septic shock may be associated with depression of the adaptive immune system. interestingly expression studies of adults with sepsis and septic shock did not identify a down-regulation of genes related to zinc biology although upregulation of genes related to immunity and infl ammation and down-regulation of genes related to the adaptive immune system was observed. the longitudinal study in children with septic shock demonstrated that in general the observed changes in up-regulated and down-regulated gene expression persisted over time. in addition, the study comparing expression in children with shock to those with sepsis or sirs indicated that while there were patterns of expression that were similar in all three groups (such as genes involved in innate immunity that were up-regulated) there were genes that were unique to the septic shock group with relation to the degree, and duration, of the response. examples include the fi nding that up-regulated genes that were involved in the il-10 signaling pathway had a greater signal which persisted for longer in the septic shock patients and down-regulation of genes for zinc biology and the adaptive immune system was greater and lasted longer than that seen in the other two groups. in addition to providing insight into the pathophysiology of sepsis and identifying potentially important proteins in early sepsis, the use of this technology may also provide physicians with a unique diagnostic tool. if the gene expression profi le of a patient who is in the early stages of sepsis is different from a patient who exhibits sirs but does not develop sepsis, then earlier therapies could be initiated before full blown sepsis is clinically evident. there is also some evidence that various subclasses of sepsis and septic shock may be able to be identifi ed using this technique. dna microarrays have also been used to investigate the expression profi le in adults with ali. mrna for pre-b-cell colony enhancing factor (pbef), a cytokine that is involved in the maturation of b-cell precursors, inhibition of neutrophil apoptosis, and perhaps regulation of endothelial cell calcium-dependent cytoskeletal arrangement was noted to be signifi cantly increased in adults with ali, a fi nding that was also consistent in both a canine and mouse model of ali. in addition to the elevated mrna levels, pbef protein in bronchoalveolar lavage fl uid was also elevated in adults with ali. it is also worth mentioning an important with the advent of the dna microarray technology, gene expression patterns in thousands of genes can lead to insight into disease pathogenesis, treatment, and outcome. c hapter 11 • g en etic pr edis pos ition to c r itical i lln ess study in a canine model of lung injury to highlight the value of gene expression arrays. the use of mechanical ventilation is invariably needed to treat patients with ali though the use of positive pressure ventilation itself may exacerbate the lung injury. gene expression arrays in a canine model of ventilator associated lung injury have identifi ed a number of genes that are regulated during ali. many of the genes can be grouped into biological processes known to important in the pathophysiology of ali; these include infl ammation (e.g., il-1b, il-6, il-1ra, mmif), coagulation (tissue factor, pai-1), and chemotaxis/cell motility (myosin light chain kinase, cell chemokine receptor 2). several other genes also appeared to be expressed including pbef, heat shock protein 70 (hsp 70), and vascular endothelial growth factor (vegf). thus, the use of expression arrays has identifi ed a number of candidate genes that may play important roles in the development of ali. as will be discussed below, genetic variations that infl uence the activity or level of the protein in several of these candidate genes have been examined in gene association studies in patients with ali. as described above, a large amount of genetic variability exists throughout our genome. whether these differences infl uence the susceptibility to or outcome from diseases in the critical care setting is an area receiving a great deal of interest. perhaps the greatest amount of focus of genetic association studies on critical illnesses is in sepsis and ali. the general approach has been to compare the frequencies of polymorphisms in specifi c candidate genes between a cohort of patients with sepsis or ali and an at-risk cohort without sepsis or ali. this section will review some of these studies. individual variability in the susceptibility to and outcome from sepsis and lung injury has long been observed in critically ill patients. why one child with pneumococcal pneumonia has little consequence of their infection and can be treated as an outpatient while another child develops refractory septic shock and respiratory failure has been attributed to a number of factors. these have included virulence of the pathogen, length of time between onset of symptoms and appropriate treatment, and comorbid conditions. while all these certainly contribute to the severity of disease, a growing body of evidence suggests that genetic variations in the individual patient may also contribute to the severity of and outcome from critical disease. these genetic polymorphisms may not be of any consequence during normal healthy periods but their importance may only become evident during a severe stressor such as an infection, trauma, cardiopulmonary bypass, or other scenarios seen in the intensive care unit (table 11 .1 ). a strong genetic infl uence on the outcome from infections was indicated by a family based study of adoptees. adoptees with a biological parent who died due to infection before the age of 50 had a relative risk of death due to infection of 5.81 (ci = 2.47-13.7); a higher relative risk than that seen when risk related to early death of a biologic parent due to cardiovascular and cerebrovascular disease (4.52; 1.32-15.4) or cancer (1.19; 0.16-8.99) was examined. thus, an individual's genetic makeup may infl uence the severity of disease in infection and sepsis. given the tens of thousands of genes in the human genome and the millions of genetic polymorphisms, on which polymorphisms and in which genes should investigators focus? one approach in choosing the candidate gene is to examine the pathways by which pathogens lead to the clinical symptoms of sepsis. the body's response to infections involves recognition of pathogen-associated products followed by an infl ammatory response that involves a large number of cellular proteins. genetic variations that lead to alterations in the amount or functional activity of any of these proteins involved in the recognition of or response to pathogen-associated products may infl uence the individual's response. examples of the infl uence of genetic variations in proteins involved in recognition of pathogens on the severity of infections include polymorphisms in the genes coding for mannose binding individual variability in the susceptibility to and outcome from critical care diseases has long been observed, and advances in genomic medicine now gives an opportunity to understand these differences. the body's response to infections involves recognition of pathogenassociated products followed by an infl ammatory response that involves a large number of cellular proteins. genetic variations that lead to alterations in the amount or functional activity of any of the proteins involved in the recognition of or response to pathogenassociated products may infl uence the individual's response. lectin (mbl) , the receptor for fc g , and toll-like receptor (tlr) 4. the heterotrimeric mbl is involved in binding bacterial surface carbohydrates and the opsonization of bacteria. a helical domain in the tertiary structure of the protein is crucial for formation of the active heterotrimer. three genetic polymorphisms in the gene coding for mbl result in amino acid changes in the helical tails of the protein and result in increased degradation and decreased serum levels of mbl. genetic association studies have demonstrated associations between variant b, c, d variants associated with decreased levels and activity and increased risk of infection fc g riia h131r r associated with decreased affi nity to igg 2 and opsonization and increased risk of infection and septic shock tlr4 asp299gly/thr399ile gly/ile associated with decreased expression, increased risk of sepsis and mortality cd-14 −159 c/t t allele associated with increased levels and susceptibility to sepsis and sepsis-related mortality in adults md-2 −1625 c/g −1625 g allele associated with higher risk of sepsis and multiple organ dysfunction score in chinese adults tnfa −308 g/a, −238 g/a, lta + 250 g/a a alleles for each polymorphism are associated with increased tnfa levels, increased mortality in sepsis and meningococcal disease, increased sepsis in adults with pneumonia, and increased mortality in bacteremia and sepsis il-6 −174 g/c g associated with increased il-6 levels in patients but c associated with increased levels in monocytes from neonates, sepsis in neonates but not adults, and severe sepsis and organ dysfunction in children il-1ra variable 86-bp repeat a2 associated with increased levels of il-1 ra and variable results of association studies examining risk of sepsis and mortality il-10 −1082 g/a, −819 c/t, −592 c/a gcc haplotype associated with increased levels and sepsis but not mortality irak-1 +1595 t/c c associated with increased nf-kb translocation and presence of shock and higher 60-day mortality in adults with sepsis hsp70a1b −179 c/t +1267 g/a −179 c/+1267a associated with decreased hspa1b and tnf a and +1267a associated with septic shock in adults with cap ace 287 bp i/d dd associated with increased serum and tissue levels and more severe meningococcal disease in children; no association with sepsis related mortality in neonates or adults pai-1 4g/5g 4g associated with increased levels and septic shock in meningococcal disease protein c −1641 a/g and −1654 c/t ac haplotype associated with increased mortality and organ dysfunction in adults with sepsis and with decreased protein c serum level; gc haplotype associated with more severe sepsis in children less than 1 year of age with meningococcemia fibrinogenbeta −854 g/a, −455 g/a, +9006 g/a gaa haplotype associated with higher levels of fi brinogen, lower 28 day mortality and less severe organ dysfunction mbl mannose-binding lectin, ig immunoglobulin, tlr toll-like receptor, rsv respiratory syncytial virus, md myeloid differentiation, tnf tumor necrosis factor, lt lymphotoxin, il-1ra interleukin 1 receptor antagonist, il-10 interleukin-10, irak-1 interleukin receptor-associated kinase 1, hsp heat shock protein, cap community acquired pneumonia, ace angiotensin converting enzyme, pai plasminogen activator inhibitor a terminology used for the various polymorphisms are the ones most commonly used in the literature and may refer to the nucleotide position, amino acid position, or name of the allele. this table is representative of polymorphisms examined in sepsis but does not include all such polymorphisms the 3 mbl genetic polymorphisms and increased susceptibility to infections, hospitalizations due to infections, number of acute respiratory infections, and risk of meningococcal infections in children, and pneumonia and sepsis in neonates. in adults these polymorphisms have been associated with recurrent respiratory infections, invasive pneumococcal infections and viral coinfections with pneumococcal pneumonia. the family of leukocyte fc g receptors is also involved in the recognition of bacteria such as streptococcus pneumoniae , haemophilus infl uenzae type b, and neisseria meningitides. fc g receptors bind the fc portion of igg bound to bacteria, thereby facilitating phagocytosis and inducing the infl ammatory response. several polymorphisms have been described in the genes coding for the various fc g receptors that alter their binding affi nity to the various subclasses of igg. two such polymorphisms have been described in the genes coding for the fc g riiib receptor and the fc g riia receptor . in the case of the fc g riiib receptor, the genetic polymorphism results in a four amino acid substitution (allotypes fc g riiib-na1 or -na2) in the receptor that alters the opsonization effi ciency. in the case of the fc g riia receptor, the genetic polymorphism results in replacing a histidine for an arginine in the extracellular domain of the receptor at amino acid position 131. the variant fc g riia receptor containing the histidine binds the fc region of igg2 with a lower affi nity and results in reduced phagocytocytosis in vitro compared with the more common fc g riia receptor containing the arginine. in association studies, a higher frequency of individuals homozygous for the na2 allotype of the fc g riiib receptor or an arginine at position 131 in the fc g riia receptor was found in patients with severe meningococcal disease or fulminant meningococcal sepsis. the fi nal examples of genetic variation in genes coding for pathogen recognition products infl uencing the severity of sepsis are the polymorphisms in the gene coding for the tlr4 receptor . this receptor is a component of a complex that includes cd-14 and myeloid differentiation (md)-2 that binds lipopolysaccharide (lps), one of the major cell wall components of gram negative bacteria. in addition, tlr4 recognizes the f protein of the respiratory syncytial virus (rsv). two genetic polymorphisms have been identifi ed in the gene coding for tlr4 that result in the change of a threonine for a glycine at amino acid position 299 and a threonine for a isoleucine at amino acid position 399. the gly299ile399 variant form of the receptor appears to be expressed on the cell surface in lower amounts and result in a lower systemic cytokine response to lps and rsv. genetic association studies have demonstrated an association between the tlr4 gly299ile399 variant and gram negative bacterial infections and septic shock as well as mortality in patients with systemic infl ammatory response syndrome. however, a number of studies have also shown confl icting results. these tlr4 variants have also been reported to be associated with susceptibility to and severity of respiratory syncytial virus infections in children. future studies with more participants will be required to determine whether variations in the tlr4 gene are involved in infection and/ or severity of disease. thus far, the focus has been on genetic variations in genes coding for proteins involved in pathogen recognition, and in each case, the variation results in an inferior host response resulting in more severe disease. currently it is thought that severe sepsis and septic shock may be the result of an imbalance in the infl ammatory response. the mechanism by which this imbalance occurs is thought to be multi-factorial. one possibility that has attracted much interest is that the host may harbor genetic variations in the regulatory regions of genes involved in the response to noxious stimuli resulting in an imbalance between pro-and anti-infl ammatory cytokines. these variations can result in an over-expression of pro-infl ammatory cytokines, such as tnf-a and interleukin (il)-6, or an under-expression of anti-infl ammatory cytokines, such as il-10. in either case, the normal infl ammatory response is dysregulated. one of the pro-infl ammatory genes in which genetic polymorphisms infl uence expression is tnf-a . as a key pro-infl ammatory cytokine, tnf-a is responsible for the activation of the infl ammatory response and by itself can produce many of the clinical manifestations of sepsis such as capillary leak, hypotension, and multiple organ dysfunction syndrome. the regulatory region of the gene coding for tnf-a has several polymorphisms that alter transcription of tnf-a , thereby infl uencing the amount of tnf-a produced. several of these polymorphisms alter nucleotides which make up the recognition sequences of some of the genetic variation of toll-like receptor 4 may be an important contributor to difference in the host response to infectious illness observed in children. transcription factors that regulate transcription. two of these polymorphisms in particular have been studied. the rarer a allele (tnf-a -308) at a location 308 base pairs upstream from the transcriptional start site results in greater transcription than the more common g allele. a second rare a allele (tnf-a -238) at a location 238 base pairs upstream from the transcriptional start site results in lower transcription than the g allele. in addition, another site located ~3,200 base pairs upstream from the transcriptional start site of the tnf-a gene and located in the gene coding for another gene, lymphotoxin (lt)-a , (also referred to as the tnfb allele, tnf-b + 252, and lt-a + 250) also appears to regulate transcription of the tnf-a gene. in genetic association studies, the frequency of the tnf-a -308 a allele has been shown to be higher in children who died from meningococcal infections and adults who died with septic shock compared with controls. genetic association studies examining the infl uence of the polymorphic lt-a + 250 site has shown a higher frequency of the a allele in adults with pneumonia presenting with the clinical symptoms of sepsis, in adults in post-operative and trauma intensive care units who develop sepsis and who have a higher mortality, and in bacteremic children who exhibit higher serum tnf-a levels and have a higher mortality. however other genetic association studies examining the tnf-a gene have reported confl icting results. recently a well designed, prospective study examining a number of polymorphisms in the gene for tnf-a (including those described above) in adults with trauma admitted to the icu demonstrated that the a allele of tnf-a -308 was associated with elevated tnf, sepsis syndrome and death in trauma patients both in their initial cohort and a replication cohort. the gene for il-6 (another pro-infl ammatory cytokine) also contains variations which multiple studies suggest are associated with the susceptibility to or outcome from sepsis. as mentioned above, the progression to severe sepsis is believed to be an imbalance in the pro-and anti-infl ammatory mediators. in addition to polymorphisms that result in increased levels of pro-infl ammatory cytokines, examples of polymorphisms that result in lower levels of anti-infl ammatory cytokines also exist. il-1 receptor antagonist (il-1ra) is one of the body's mechanisms for keeping the infl ammatory reaction in check by binding to the il-1 receptor without activating the signal transduction pathway. the gene coding for il-1ra contains a polymorphic region consisting of a variable number of 86 base-pair tandem repeats. these different il-1ra alleles have been associated with variable circulating levels of both il1-ra and il-1 b (the two genes are located close to one another on chromosome 2), and several association studies have suggested an infl uence of this variation on a variety of diseases in which infl ammation plays an important role, including the susceptibility to sepsis. il-10 is another anti-infl ammatory cytokine for which genetic polymorphisms appear to alter transcription levels. a number of studies have demonstrated an association between an increased susceptibility to sepsis and certain il-10 polymorphisms although confl icting results have also been reported. it is important to remember that the cytokines and their receptors mentioned above activate a complex signal transduction pathway composed of dozens of proteins with the end result of a well coordinated cellular response to the noxious stimulus. genetic variation in any of the proteins in the pathway may also infl uence the fi nal response. recent studies have begun to analyze components of various pathways involved in the development of sepsis. one example is il-1 receptor-associated kinase-1 (irak-1) that plays an important role in the signal transduction pathway initiated by the activation of the il-1 receptor. activation of irak-1 results in increased transcription of a variety of pro-infl ammatory genes modulated by nf-k b, a key transcription factor in the infl ammatory response. genetic variations in the gene coding for irak-1 have been shown to be associated with elevated nuclear levels of nf-k b as well as the presence of shock and a higher 60-day mortality in patients with sepsis. the association of a variant in irak-i with severity of septic shock has been independently replicated in a large multi-centered cohort of adult patients with septic shock. interestingly, this study also indicated that age might modify the relationship as this association was stronger for younger patients. many other proteins involved in these complex signaling pathways have yet to be investigated for the infl uence of genetic variations on critical illnesses. many of the genes discussed thus far have a role in infl ammation, a key component in the pathophysiology of sepsis. loss of homeostatic mechanisms regulating the coagulation/ fi brinolytic system also plays an important role in sepsis. plasminogen activator inhibitor 1 the regulatory region of the gene coding for tumor necrosis factora (tnfa ) has several polymorphisms that alter transcription of tnfa , thereby infl uencing the amount of tnfa produced. il-1 receptor antagonist (il-1ra) is a key mechanism for keeping the infl ammatory reaction in check by binding to the il-1 receptor without activating the signal transduction pathway. together with il-10, another anti-infl ammatory cytokine, genetic polymorphisms appear to alter transcription levels of these proteins. (pai-1) inhibits fi brinolysis thereby favoring the formation of microthrombi in the capillaries. the pathophysiology of multiple organ dysfunction syndrome in patients with sepsis is thought to involve, in part, intravascular fi brin deposition. thus, elevated pai-1 activity could contribute to organ failure in sepsis and elevated plasma concentrations have been observed in patients with sepsis. a genetic variation in the gene coding for pai-1 consisting of either the presence of 4 guanines or 5 guanines at a specifi c location appears to infl uence the amount of pai-1 produced. individuals homozygous for the 4g genotype (4g/4g) produce more pai-1 than individuals homozygous for the 5g genotype (5g/5g) or individuals that are heterozygous (4g/5g). association studies have demonstrated that children with meningococcal disease who were 4g/4g at this site had an increased risk of death compared to children who were 4g/5g or 5g/5g. more recent studies in both children and adults have demonstrated higher mortality in individuals homozygous for the 4g allele in a number of infectious diseases. since fi brin deposition is thought to play a role in the multiple organ system failure in patients with sepsis, genetic variations that infl uence the production of fi brin might also infl uence the severity of disease in patients with sepsis. the production of fi brinogen, the precursor to fi brin, is dependent on the transcription of fi brinogen-beta . several polymorphisms in the promoter region have been associated with higher plasma levels of fi brinogen, and higher levels of fi brinogen have been associated with improved outcomes in sepsis. association studies have demonstrated that the gaa haplotype, consisting of the genotypes at the −854, −455, and +9006 sites, is associated with higher levels of fi brinogen and with decreased mortality and less organ dysfunction. protein c has anticoagulant activity as well as anti-infl ammatory and anti-apoptotic effects suggesting that diminished activity of protein c may lead to increased fi brin deposition, infl ammation, and apoptosis. genetic polymorphisms located in the promoter region of the gene coding for protein c result in decreased levels. association studies in caucasian and han chinese adults have demonstrated increased mortality and organ dysfunction in adults with sepsis who carry the a allele at the −1641 site and the c allele at the −1654 site. this haplotype has also been reported to be associated with decreased protein c concentration. interestingly, an increased risk of more severe sepsis has been observed in children less than 1 year of age with meningococcal disease who carry the g allele at the −1641 site along with the c allele at the −1654 site. this haplotype was not associated with severe sepsis in older children suggesting a potential developmental difference in these variations on the severity of sepsis. severe lung injury in both adults and children can be precipitated by a diverse array of causes and are classifi ed as either direct injury when the insult is from the alveolar side of the alveolar/capillary membrane or indirect injury when the insult is from the capillary side. major causes of direct lung injury include pneumonia, aspiration, pulmonary contusion, and inhalation while major causes of indirect injury include sepsis, trauma without pulmonary contusion, cardiopulmonary bypass, and multiple transfusions. despite these various causes, the central pathogenesis of ali involves derangements in multiple biological processes. these include activation of infl ammation, loss of coagulation and fi brinolytic homeostasis, disruption of vascular permeability, epithelial and endothelial cell apoptosis as well as proliferation, and derangements in surfactant. some of these processes, notably infl ammation and coagulation, play key roles in the pathophysiology of sepsis as well as ali. thus, it is not surprising to fi nd that the candidate genes examined in genetic association studies for ali are in many cases the same as those examined in sepsis (table 11 .2 ). this section will review some of the genetic association studies examining the infl uence of genetic variations on the development of ali. pulmonary surfactant is synthesized by the type ii alveolar epithelial cells and is required for normal lung function. one of surfactant's primary functions is to lower the surface tension at the alveolar air-liquid interface. surfactant is composed of phospholipids and four proteins, surfactant protein (sp)-a, sp-b, sp-c, and sp-d. knockout models in mice have demonstrated that of these four proteins, only sp-b is absolutely required for post-natal genetic polymorphisms located in the promoter region of the gene coding for protein c result in decreased levels, and possibly an impact on mortality with sepsis. defi ciency in, or impaired activity of surfactant protein-b appears responsible for a number of interstitial pulmonary diseases in humans including ards. survival. defi ciency in, or impaired activity of sp-b appears responsible for a number of interstitial pulmonary diseases in humans including ards. several genetic variations exist in the genes coding for the surfactant proteins and two will be discussed here; the sp-b + 1580 t/c polymorphism and insertion/deletion polymorphism consisting of dinucleotide (ca) tandem repeats in intron 4. several studies have demonstrated an association between these polymorphisms and the need for mechanical ventilation in children (sp-b + 1580 t/c) and mechanical ventilation and ards in adults (sp-b + 1580 t/c polymorphism and insertion/ deletion of dinucleotide (ca) tandem repeats). the consequences of these variations are not fully known. the sp-b + 1580 t/c polymorphism results in an amino acid change in exon 4 in a region of the amino terminal propeptide which is thought to play a role in targeting of sp-b to lamellar bodies. the resulting amino acid change alters glycosylation of sp-b and may affect the level of sp-b by altering its processing or stability. aberrant proteolytic processing of the sp-b product encoded by the c allele is supported by a recent report demonstrating that the c allele is associated with absence of a specifi c pro-sp-b cleavage product in neonates. the intron 4 dinucleotide repeat length variation polymorphism is associated with incompletely spliced sp-b mrna. interestingly, in caucasians this length variation polymorphism in intron 4 is in linkage disequilibrium with the sp-b + 1580 t/c polymorphism; the c allele of rs1130866 is associated with the deletion variants at the intron 4 polymorphic site. further work is needed to not only defi ne the consequence of these genetic variations on surfactant function but also to further evaluate whether these genetic variations infl uence the development of ali. another study of interest involves the susceptibility to pneumonia, the leading cause of ali and ards in both children and adults. as discussed above, the 4g/4g genotype in the gene coding for pai-1 is associated with higher levels of pai-1 expression. in a large cohort of adults, those individuals with the 4g allele demonstrated a signifi cantly higher susceptibility to pneumonia. while pai-1 activity inhibits fi brinolysis leading to formation of microthrombi, it also demonstrates anti-infl ammatory activity, and in this fashion, may increase the susceptibility to infection. in patients with ali, plasma levels of pai-1 levels are elevated and protein c levels are diminished. in addition, alveolar levels of pai-1 are elevated suggesting a possible local activation of the fi brinolytic system. recent studies have demonstrated that the 4g allele of pai-1 is associated with increased mortality in patients with severe pneumonia and patients with ards. to date there are no reports indicating association of specifi c protein c variants with ali. infl ammation is one of the hallmarks of ali and as with sepsis, it is thought that one of the central components of ali is an imbalance between pro-and anti-infl ammatory cytokines in the lung. the infl uence of genetic variation in several genes involved with infl ammation on the development of ali has been examined. the tnf-a -308 a allele appears to be associated with increased mortality in adults with ards but not with the susceptibility to ards when compared with adults who were at-risk for the development of ards. the lt-a + 250 polymorphism that appears to be associated with more severe sepsis did not infl uence the severity of ards. macrophage migration inhibitory factor (mif) plays a central role in regulating the infl ammatory response by directly increasing tnf-a and il-8 and countering the antiinfl ammatory actions of glucocorticoids. mif mrna has been demonstrated in cells from bronchoalveolar lavage of patients with ali and mif concentrations in serum are elevated in patients with ali compared with controls. haplotypes composed of polymorphisms in the 3' end of the gene coding for mif are associated with the development of ali in both caucasian and african american populations. a number of studies have demonstrated association of specifi c haplotypes in another gene involved in the regulation of infl ammation, the gene for il-6, with the development of ali. whether these haplotypes are associated with elevated levels of il-6 is still unclear. association studies have also been performed examining the genetic variants discussed earlier in the promoter region of the anti-infl ammatory cytokine il-10. the −1082 gg genotype results in higher levels of il-10 and is associated with the development of ards in younger adults. furthermore, the adults with ards who carried the gg genotype at this site demonstrated lower mortality and organ failure. the genetic variants in the gene coding for mbl have also been examined for their infl uence on ali. one of the variants described previously that results in decreased serum levels of mbl, variant b, is associated with the susceptibility to ards and a greater degree of organ dysfunction and higher mortality in patients with ards. while no reports have described the tlr4 polymorphisms specifi cally in ali or ards, the gly299ile399 variant form of the receptor is associated with an increased risk of severe rsv bronchiolitis and increased risk for hospitalization for rsv in previously healthy infants suggesting a potential role for tlr4 in infl uencing the severity of lung injury. signal transduction pathways activated after stimulation of a variety of immune receptors including the tlrs and members of the family of il-1 and tnf receptors result in the upregulation of specifi c genes involved in the innate and adaptive immune responses. several transcription factors are involved in this process and genetic variation in any of these factors may infl uence the level of transcription. one such factor is nuclear factor k b (nf-k b) which under non-stimulated conditions is inhibited by the cytoplasmic inhibitor nf k bia . upon activation of cytokine-mediated signal transduction pathways, nf k bia is degraded allowing nf-k b to translocate to the nucleus. a number of polymorphisms located in the macrophage migration inhibitory factor (mif) plays a central role in regulating the infl ammatory response by directly increasing tnfa and il-8 and countering the anti-infl ammatory actions of glucocorticoids. haplotypes composed of polymorphisms in the 3 ¢ end of the gene coding for mif may be associated with the development of ali. promoter region of the gene coding for nf k bia have been described but their functional consequence is unknown. when individual nf k bia promoter polymorphisms were examined to determine if they are associated with the development of ali, none by themselves demonstrated an association. however, the haplotype of −881 g/−826 t/−297 c was found in a higher frequency in adults who developed ards especially in males with direct lung injury. another transcription factor is nf-e2 related factor 2 (nrf2) which, under nonstressed conditions, is located in the cytoplasm. under oxidative stress nrf2 translocates to the nucleus and results in transcription of several anti-oxidant enzymes. several polymorphisms within the promoter region of the gene coding for nrf2 have been identifi ed that reduce transcription. one such variant, −617 a allele, is associated with the development of ali in adult trauma patients. the role of angiotensin converting enzyme ( ace) in lung injury has recently attracted uch interest. ace is present in pulmonary endothelium and is responsible for converting ati to atii. ace levels are elevated in the bronchoalveolar lavage fl uid of adults with ards and higher levels are associated with mortality from ards. the key component is most likely atii, which has apoptotic effects on alveolar epithelial and endothelial cells in vitro . atii receptor antagonists block pneumocyte apoptosis in a model of meconium aspiration. another important component of this system is ace2, a homologue of ace expressed in human lungs, which is a negative regulator of the renin-angiotensin system as well as the probable receptor for the sars virus in humans. lung injury models using knockout mice lacking the ace2 gene have higher atii levels and exaggerated lung injury compared to wild type mice. however, the lung injury is reversed if the ace gene is inactivated in the ace2 knockout mice. this suggests that ace induces lung injury through atii and that ace2 protects against lung injury. indeed, ace inhibitors and atii antagonists appear to decrease the severity of lung injury in animal models, the risk of aspiration pneumonia in some adult populations, and reduce the 30-day mortality in adults with pneumonia. several studies have demonstrated the d/d genotype appears associated with the susceptibility to and outcome from ards. as discussed previously, expression microarrays have been invaluable in identifying other potential mediators involved in the pathophysiology of lung injury. the expression of pre-b cell colony enhancing factor (pbef) was found to be signifi cantly elevated in both animal and human studies of ali using this approach. pbef is a lesser studied cytokine involved in the maturation of b-cells, inhibition of neutrophil apoptosis, and perhaps regulation of the endothelial cell calcium-dependent cytoskeletal arrangement. two genetic polymorphisms have been identifi ed in the promoter region, −1001 t/g and −1543 c/t which appear to infl uence the development of ali. carriers of the g allele at position −1001 had a 2.75-fold increased risk of ali and the g allele remained an independent risk factor after controlling for several other variables. the t allele at position −1543 was found at a lower frequency in adults with ali. combining these two polymorphisms in a haplotype analysis demonstrated that adults with the −1001 g/ −1543 c haplotype had a higher risk of ali (7.7 fold). the consequence of these two polymorphisms remains to be elucidated though the −1543 t allele may result in reduced expression. one fi nal gene to be discussed in this section is the myosin light chain kinase (mlck) gene. three isoforms of the protein exist, and one isoform is a key component in the cytoskeletal arrangement regulating vascular permeability, angiogenesis, endothelial cell apoptosis, and leukocyte diapedesis. several polymorphisms in the gene coding for mlck have recently been identifi ed. analysis was performed not only on the infl uence of single polymorphisms on the development of ali but also a number of haplotypes using a sliding window approach. several strong associations between various single nucleotide polymorphisms as well as haplotypes and the risk of ali and sepsis were identifi ed in adults. this included one haplotype, ggt, composed of markers mylk_021, mylk_022, and mylk_011 spanning a region of 846 base pairs between the 5 ¢ untranslated region and the fi rst exon that appeared to be specifi cally associated with the risk of ali and not sepsis. the functional signifi cance of these haplotypes remains to be determined. specifi c variants in the mylk gene were also shown to be associated with trauma-induced ali, however association of specifi c genetic variants and lung injury were not observed in children or adults with pneumonia. two other areas should be mentioned in regards to the infl uence of genetic variations in the picu. the fi rst is in the area of coagulation. several examples of genetic polymorphisms in genes coding for proteins involved in coagulation and fi brinolysis were discussed above in relation to sepsis and ali. however, these and many other genetic variations that exist in other components of the coagulation cascade could also infl uence the development of thrombosis including deep venous thrombosis in critically ill patients. thrombosis of central venous catheters is a recurring problem in picus and while certain environmental factors play a role (eg, length of time catheter is in place, size of the patient and vessel), genetic polymorphisms in the patient favoring the formation of thrombi may also play a role. finally, the action of every drug that is used in the picu can potentially be infl uenced by genetic variation in the patient. whether it be inhaled b 2 -agonists, or the array of intravenous vasoactive agents, sedatives, muscle relaxants, antibiotics, steroids, etc.; all bind to protein receptors and either activate or block specifi c signal transduction pathways, many bind protein carriers or transporters, and most are metabolized by various protein enzymes. every gene coding for each of these proteins has multiple genetic variations with the potential to infl uence the levels or activities of these proteins. the area of pharmacogenomics attempts to determine the infl uence of genetic variations in genes affecting these various aspects of drug action. however, while the list of genetic polymorphisms in genes affecting drug action is growing rapidly, there are few clinical examples of the degree of infl uence that these genetic variations have on the response to drugs in the picu. for example, warfarin is the most widely used oral anticoagulant for long-term prophylaxis and treatment of thromboembolic disorders and is used in many children and adults with mechanical valves. the metabolism of and response to warfarin involves several enzymes, two of which exhibit genetic variations that dramatically alter the levels of warfarin. for one of these genes, cyp2c9, the common allele is referred to as cyp2c9*1 and is consider the wildtype while cyp2c9*2 contains a c to t nucleotide change at position 430 in exon 3 and cyp2c9*3 contains an a to c nucleotide change in exon 7. cyp2c9*2 has approximately 80% of the metabolic activity of the wild type cyp2c9*1 while cyp2c9*3 contains only 20% of the wildtype activity. by also using genetic variation in a second gene, vitamin k epoxide reductase complex subunit 1 or vkorc1, one can account for more than 50% of the observed dosing variability. current practice in the use of warfarin usually involves starting at an age and weight specifi c dose and monitoring coagulation studies. however, because of the genetic variations in these two enzymes and perhaps others, different patients take different amounts of time to achieve the appropriate therapeutic dose. knowing the specifi c genotypes of patients prior to initiating warfarin may allow for more appropriate dose selection, less time to achieve therapeutic levels, and less risk of adverse events. recently, an algorithm using the patient's genotypes at these two sites has been developed that allows for more accurate dosing in some populations. although these algorithms are being developed and tested it should be kept in mind that they do not account for drug-drug interactions. the era of the study of the genetic impact on critical illness in children is present. clinicians must be prepared to deal with the growing body of literature related to genetic infl uence on critical disease development, treatment and outcome, and be able to critically review the literature in order to determine the impact on the patients they are caring for daily. for the results of these representative genetic association studies to take the leap into clinically impacting care, they must meet certain criteria. first and foremost, the phenotype must be well defi ned; that is, the enrolling patients with ali/ards or sepsis must meet strict and well accepted criteria. second, they must be high quality studies, utilizing highly sensitive and specifi c methods for genotyping. third, the studies must use a large sample size to assure that no type i or type ii errors are made based simply upon the number of individuals genetic polymorphisms in genes coding for proteins involved in coagulation and fi brinolysis may be very important in the risk of bleeding and thrombosis in critically ill children. the area of pharmacogenomics attempts to determine the infl uence of genetic variations in genes affecting the various aspects of drug action. ace i/d but not agt (−6)a/g polymorphism is a risk factor for mortality in ards 4g4g genotype of the plasminogen activator inhibitor-1 promoter polymorphism associates with disseminated intravascular coagulation in children with systemic meningococcemia mechanisms and regulation of the gene-expression response to sepsis comparison of two polymorphisms of the interleukin-1 gene family: interleukin-1 receptor antagonist polymorphism contributes to susceptibility to severe sepsis recent advances in genetic predisposition to clinical acute lung injury novel polymorphisms in the myosin light chain kinase gene confer risk for acute lung injury macrophage migration inhibitory factor in acute lung injury: expression, biomarker, and associations polymorphisms in the mannose binding lectin-2 gene and acute respiratory distress syndrome severity of meningococcal disease in children and the angiotensin-converting enzyme insertion/ deletion polymorphism polymorphisms of human sp-a, sp-b, and sp-d genes: association of sp-b thr131ile with ards deletions within a ca-repeat-rich region of intron 4 of the human sp-b gene affect mrna splicing lipopolysaccharide hyporesponsiveness as a risk factor for intensive care unit hospitalization in infants with respiratory syncitial virus bronchiolitis fibrinogen-beta gene haplotype is associated with mortality in sepsis sepsis syndrome and death in trauma patients are associated with variation in the gene encoding for tumor necrosis factor association of tnf2, a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study variation in the tumor necrosis factor-alpha gene promoter region may be associated with death from meningococcal disease association between surfactant protein b + 1580 polymorphism and the risk of respiratory failure in adults with community-acquired pneumonia genome-level longitudinal expression of signaling pathways and gene networks in pediatric septic shock microarray analysis of regional cellular responses to local mechanical stress in acute lung injury genetic and environmental infl uences on premature death in adult adoptees the national human genome research institute's talking glossary of genetic terms irak1 functional genetic variant affects severity of septic shock genome-level expression profi les in pediatric septic shock indicate a role for altered zinc homeostasis in poor outcome pre-b-cell colony-enhancing factor as a potential novel biomarker in acute lung injury disease phenotype, preferably by a different group of investigators. finally, the impact of the genetic variant on the protein product must possess biologic plausibility as impacting the development or the outcome of the disease of study. only after all of these criteria are met should clinicians be comfortable moving to the arena of tailoring therapy based on genetic variations. 4. you are caring for two brothers with acute lung injury secondary to smoke inhalation from an apartment fi re. these two infants were apparently sleeping in the same crib when they were rescued simultaneously by fi re fi ghters. the fi rst infant was extubated within 48 h of intubation and is doing well with a minimal oxygen requirement. the second has experienced a much more severe lung insult and remains intubated on high frequency oscillatory ventilation. in attempting to understand the difference in their clinical response to the seemingly identical insult, you suspect that it may be related to a polymorphism in one of the genes that codes for surfactant protein b. in considering this possibility, which of the following is true? a. although plausible, it is unlikely to be associated with a polymorphism because polymorphisms are rare occurring in less than one percent of the population. b. it is not plausible because variances in the translation of such a complex protein require differences in an entire haplotype, and not simply a single nucleotide polymorphism. c. it is plausible because genetic polymorphisms may infl uence the quantity of mrna transcribed and/or the functional activity of the surfactant protein b. d. it is unlikely as there are no reports of associations between surfactant protein gene polymorphisms and outcomes from pulmonary disease. e. it is unlikely because dysfunctional surfactant protein b demonstrates an x-linked pattern of inheritance. key: cord-267270-r17z4d8x authors: kipar, a.; baptiste, k.; meli, m.l.; barth, a.; knietsch, m.; reinacher, m.; lutz, h. title: age-related dynamics of constitutive cytokine transcription levels of feline monocytes date: 2005-01-18 journal: exp gerontol doi: 10.1016/j.exger.2004.12.007 sha: doc_id: 267270 cord_uid: r17z4d8x monocytes/macrophages are central mediators of inflammation and immunity and therefore of major interest in the study of immunosenescence. in healthy adult cats, monocytes have been shown to constitutively transcribe proand anti-inflammatory cytokines. however, in order to characterize the effect of age, feline monocyte functions were examined for changes in cytokine transcription levels in early stages of immunosenescence. for this purpose, isolated, short-term cultured monocytes from barrier-maintained adult cats of different ages (15 mo to 10 yr) were examined for transcription of il-1β, il-6, il-10, il-12 p40 and tnf-α by real-time pcr. transcription levels of cytokines varied and were generally highest for il-1β. for il-1β, il-6 and il-12 p40, both young and old cats exhibited highest levels. the age association was significant. tnf-α appeared to be transcribed at similar levels over the examination period, whereas il-10 tended to decline with age but without any statistical significant differences. the observed age association of the constitutive transcription of some cytokines indicates a drop in monocyte activities from youth to middle age, which is then followed by a (progressive) increase with increasing age. this provides evidence that monocytes are in part responsible for the pro-inflammatory status observed with ageing. monocytes/macrophages play the central role in inflammatory responses, immunity as well as the stress response (franceschi et al., 2000) . furthermore, a chronic, agerelated, progressive stimulation of macrophages towards a pro-inflammatory status was postulated and known as 'macroph-aging' (franceschi et al., 2000) . one previous study actually showed significantly increased constitutive production of il-1b and il-6 in circulating monocytes of older female compared to young adult female human individuals, whereas no changes in the constitutive tnf-a production were observed (sadeghi et al., 1999) . on the other hand, there is evidence for decreased in vitro production of some cytokines, such as il-1b, il-6 and tnf-a, in lps-stimulated whole blood or monocytes of elderly humans (bruunsgaard et al., 1999; sadeghi et al., 1999) . in aged mice, a reduced capacity was observed of activated macrophages to produce il-1b, tnf-a and nitric oxide (wallace et al., 1995) . furthermore, aged rat monocytes/macrophages exhibit a reduced constitutive mcp-1 production (reale et al., 2003) . there is a paucity of information on the constitutive production of cytokines by monocytes/macrophages, for all species. a previous study on human monocytes demonstrated constitutive production of il-1b, il-6 and tnf-a by unstimulated human monocytes in young and old females (sadeghi et al., 1999) ; whereas another study demonstrated variable constitutive transcription of il-1b, il-6, il-10, il-12p40 and tnf-a in isolated, short term-cultured, (kipar et al., 2001) . however, with regard to monocyte-associated viral infections in the cat, certain age-related differences in susceptibility have been noted. for example, feline infectious peritonitis (fip), a coronavirus-induced, immune-mediated, fatal systemic disease, affects most frequently young animals between 6 mo and 2 yr of age (rohrbach et al., 2001) . however, aged cats develop a less intense immune response, with delayed antibody production to experimental feline immunodeficiency virus (fiv) infection compared to young animals (george et al., 1993) . in both infectious diseases, the infected monocyte is essential for viral distribution (magnani et al., 1994; kipar et al., 2004 kipar et al., , 2005 and in cases of fip, the activated monocyte also mediates the development of its pathogenetic hallmark, granulomatous phlebitis (kipar et al., 2005) . the question arises as to whether age-related constitutive changes in monocyte functions are responsible for potential differences in responses to infectious agents. thus, the purpose of this study is to evaluate constitutive transcription levels of cytokines in cats over a large age range, from 15 mo to 10 yr of age. the study was performed on 17 healthy, female or male neutered (in equal proportions) uninfected barrier-maintained cats, kept under comparable conditions at the veterinary faculties in zurich, switzerland and giessen, germany. four cats were sampled near 14 mo (13-15 mo) of age, two near 20 mo (20 and 21 mo), two near 26 mo (25-29 mo), two near 3 yr (2 yr 11 mo-3 yr 1 mo), two near 3 yr 10 mo (3 yr 9 mo-3 yr 11 mo) and five near 5 yr 1 mo (5 yr-5 yr 2 mo) during the examination period. three cats were re-sampled at an older age: one cat at 8 yr and 8 mo (previously examined at 3 yr 10 mo), two at 10 yr (previously examined at 5 yr 1 mo). 2.2. monocyte isolation, rna extraction, cdna production and real time pcr for feline (f) il-1b, fil-6, fil-10, fil-12 p40 and ftnf-a blood samples were generally taken twice from each animal, 3 weeks to 2 mo apart. peripheral blood leukocytes were isolated and short-term cultured for 15-18 h. monocytes were purified by thorough washing of plates to eliminate non-adherent cells, as previously described (kipar et al., 2001) . rna was extracted from monocytes using a commercially available kit (rneasy mini kit; qiagen, hilden, germany). contaminating genomic dna in rna extracts was digested and cdna synthesized according to published protocols (kipar et al., 2001) . realtime taqman pcr, using an automated fluorometer (abi prism 7700 sequence detection system, applied biosystems, weiterstadt, germany) was used to quantify the relative levels of feline (f) il-1b, fil-6, fil-10, fil-12 p40 and ftnf-a transcription (kipar et al., 2001) . from each cdna sample, parallel reactions were performed, in duplicates, in separate tubes for the detection of fgapdh (internal control/calibrator), fil-1b, fil-6, fil-10, fil-12 p40 and ftnf-a. amplification conditions, assay compositions, primer and probe concentrations were as previously described (kipar et al., 2001) . relative quantitation of cytokine signals was done by the comparative c t method and is reported as relative transcription or the n-fold differences, relative to the calibrator cdna (fgapdh). for each sample, differences between the target and internal control c t were calculated and served to normalize for differences in the quantity of total nucleic acid added to each reaction and in the efficiency of the rt step. the c t of the calibrator, fgapdh, was subtracted from the c t of each experimental sample to give the value dc t . for samples where a cytokine mrna signal was not observed after 45 cycles, a plausible dc t was created. this value was ascertained from the lowest c t for fgapdh (obtained from the sample with the highest expression of gapdh) subtracted from 45 (number of cycles). the resulting value was rounded to the next higher integer value. the amount of target was calculated by 2 kdc t , resulting in evaluation of the experimental samples as an n-fold difference relative to the calibrator fgapdh (kipar et al., 2001) . statistical analysis was performed, using the programme sas version 8.2 computer software proc mixed (sas institute, cary, nc, usa) a regression analysis was performed of the cytokine transcription levels versus age, both as a linear and quadratic function. a multivariable regression model was explored for all cytokines against age. final models were assessed by looking at the parameter estimates, the model deviance and the distribution of the residual values. for the identification of potential linear associations between transcription of the different cytokines and age, the pearson correlation coefficients matrix was used. all cytokines were constitutively transcribed. il-1b and tnf-a were detected in all samples. il-6 was not detected in two samples (each one from a cat aged 3 yr 1 mo and 3 yr 11 mo), il-10 was not detected in two samples (each one from a cat aged 3 yr 9 mo and 5 yr 2 mo), and il-12p40 was not detected in four samples (each one from a cat aged 3 yr 1 mo and 3 yr 11 mo and two cats aged 5 yr 2 mo). transcription levels were generally variable, both among cats of the same age (fig. 1 ) and in individual cats at different time points. il-1b was overall transcribed most intensely, followed by tnf-a, il-6 and il-10. il-12 p40 levels were generally very low (fig. 1) . for il-1b, il-6 and il-12 p40, a statistically significant association was found between the level of transcription and age. higher transcription levels were noted in younger and older cats than in those of middle age (fig. 1) . for il-1b and il-6, transcription levels were similar in the youngest and oldest cats, whereas il-12 p40 transcription was highest in the youngest cats. for il-10, an overall decline in constitutive transcription with age was indicated, but not statistically significant (fig. 1) . tnf-a levels were on average less variable throughout the examination period. a slight drop in transcription was observed in the middle-aged cats, but not statistically significant (fig. 1) . the multivariable regression analysis did not identify more suitable models to evaluate the association between cytokine transcription levels and age. results of the pearson correlation coefficients matrix are illustrated in table 1 . transcription of il-1b and il-6 appeared highly correlated, independent of age. a trend for a positive correlation between il-1b and age was found as well as a moderate negative correlation between il-12 p40 transcription and age. this report presents the results of an initial study to assess the functional state of monocytes over a wide age range in cats, by evaluating a set of both pro-inflammatory and down-regulatory cytokines (e.g. il-1b, il-6, il-10, il-12 p40 and tnf-a), in isolated, short-time cultured, unstimulated monocytes. the age range of 15 mo to 10 yr in cats used in this study covers most of adult, pre-senile life of healthy cats. this study provides evidence for a decline in constitutive transcription of some cytokines (il-1b, il-6 and il-12 p40) from early adulthood to middle age (approx. 5 yr), with their subsequent increase in older cats. the latter levels approach those seen in the youngest cats (for il-1b and il-6) or are comparatively lower (for il-12 p40). il-10 and tnf-a do not show any significant age-related changes in their constitutive transcription. nonetheless, there was a tendency for a progressive decline in il-10 transcription with age. tnf-a transcription levels, however, appear mostly stable over the entire age range, with a slight depression in the middle-aged cats. our study has two outcomes, namely to clearly emphasise the general importance of age-matched controls in studies on alterations of immune cell populations. also, this study supports the hypothesis of a general, progressively increasing 'pro-inflammatory status' of monocytes/macrophages with age (franceschi et al., 2000) . it provides evidence that monocytes are at least partly responsible for the progressive increase in blood il-6 and il-1b levels observed in older individuals (franceschi et al., 1999; bruunsgaard et al., 1999) . however, similar to a previous study on human monocytes (sadeghi et al., 1999) , the results of this study do not suggest involvement of monocytes in the induction of elevated tnf-a blood levels of older individuals, as observed in humans (bruunsgaard et al., 1999) . our study shows correlation between il-1b and il-6 production previously found in lps-stimulated human monocytes (bruunsgaard et al., 1999) , that can be explained by the stimulatory effect of il-1b on il-6 production (abbas and lichtman, 2003) . on the other hand, il-6 is known to both suppress il-1b and tnf-a production and block the effect of il-1b by inducing the production of il-1 receptor antagonist (schindler et al., 1990; jordan et al., 1995) . this might explain why there was no obvious increase in tnf-a production in the older cats. furthermore, by upregulating il-6, il-1b could actually inhibit the upregulation of tnf-a, a cytokine with far wider effects than il-1b itself (abbas and lichtman, 2003) , and thereby limit its own harmful effects in aged individuals (schindler et al., 1990; jordan et al., 1995) . the age-related decline in il-10 transcription, however, might foster il-12 p40 production due to lack of inhibition (abbas and lichtman, 2003) . our findings show that circulating monocytes might be responsible for some of the immune dysfunctions seen with increasing age, particularly 'inflamm-aging'. there is evidence for a progressive activation of monocytes with age, compatible with the previously postulated 'macrophaging' (franceschi et al., 2000) . since this progressive inflammatory status appears to occur in both ill and healthy older individuals, then it can be regarded as a characteristic of both successful and unsuccessful aging (franceschi et al., 1999; 2000) . upregulation of il-1b, il-6 and il-12 might represent adaptation of the immune system to ensure its capacity to deal with infectious agents (abbas and lichtman, 2003) . in the case of humans and non-barriermaintained animals, it might also be the consequence of the successful adaptation to stresses which occurred throughout an individual's life (franceschi et al., 2000) . previous studies on activated monocytes/macrophages from several species are equivocal. for example, aged mice have exhibited decreased il-1, tnf-a, il-6 and nitric oxide production (effros et al., 1991; wallace et al., 1995) , whereas monocytes from aged humans exhibited significantly lower il-1b, but increased il-6 and il-10 production after stimulation (sadeghi et al., 1999) . in aged mice and rats, reduced mhc class ii and monocyte chemotactic protein-1 transcription and translation, respectively, was observed (herrero et al., 2002; reale et al., 2003) . in another study higher phagocytic capacity after lps treatment, despite lower phagocytosis levels without activation was described in rat macrophages (tasat et al., 2003) . in conjunction with the present study, these findings suggest an imbalanced state of monocyte/macrophage function and thereby the innate immune response with age. they also indicate that by down-regulating certain constitutive levels of stimulatory functions such as mhc ii expression, monocytes may counteract and balance their otherwise increasingly pro-inflammatory state in aging individuals (franceschi et al., 2000) . with regard to the cat, further studies on the cytokine transcription of activated feline monocytes are needed to explain why fiv infection induces a less intense immune reaction in aged animals and why older cats are less affected by fip (george et al., 1993; rohrbach et al., 2001) . it seems plausible that despite increased or similar constitutive cytokine transcription, monocytes of older cats might exhibit an impaired reactive activation after virus infection (kipar et al., 2005) . cellular and molecular immunology impaired production of proinflammatory cytokines in response to lipopolysaccharide (lps) stimulation in elderly humans influence of age and caloric restriction on macrophage il-6 and tnf production biomarkers of immunosenescence within an evolutionary perspective: the challenge of heterogeneity and the role of antigenic load inflamm-aging. an evolutionary perspective on immunosenescence the effect of age on the course of experimental feline immunodeficiency virus infection in cats immunosenescence of macrophages: reduced mhc class ii gene expressin neutralization of endogenous il-6 suppresses induction of il-1 receptor antagonist cytokine mrna levels in isolated feline monocytes downregulated cytokine transcription in isolated monocytes of clinically healthy cats, infected with an fiv strain of low pathogenicity morphological features and development of granulomatous vasculitis in feline infectious peritonitis feline immunodeficiency virus infection of macrophages: in vitro and in vivo inhibition by dideoxycytidine-5 0 -triphosphate-loaded erythrocytes oxygen supply modulates mcp-1 release in monocytes from young and aged rats: decrease of mcp-1 transcription and translation is age-related epidemiology of feline infectious peritonitis among cats examined at veterinary medical teaching hospitals phenotypic and functional characteristics of circulating monocytes of elderly persons correlations and interactions in the production of interleukin-6 (il-6), il-1, and tumor necrosis factor (tnf) in human blood mononuclear cells: il-6 suppresses il-1 and tnf agedependent changes in reactive oxygen species and nitric oxide generation by rat alveolar macrophages decreases in macrophage mediated antitumor activity with aging the authors wish to thank mr p. fidler (zurich university), dr t. bleier, dr a. büttner, mrs h. may and mr d. schwartz (giessen university) for management of the cats. we are very grateful to dr k failing, arbeitsgruppe biomathematik und datenverarbeitung, faculty of veterinary medicine, justus-liebig-universität gießen, giessen, germany, for his contribution to the initial statistical analyses. key: cord-308008-s2t11l3h authors: limonta, daniel; torrentes‐carvalho, amanda; marinho, cíntia ferreira; de azeredo, elzinandes leal; de souza, luiz josé; motta‐castro, ana rita c.; da cunha, rivaldo venâncio; kubelka, claire fernandes; nogueira, rita maria ribeiro; de‐oliveira‐pinto, luzia maria title: apoptotic mediators in patients with severe and non‐severe dengue from brazil date: 2013-10-29 journal: j med virol doi: 10.1002/jmv.23832 sha: doc_id: 308008 cord_uid: s2t11l3h despite being the most significant arboviral disease worldwide, dengue has no antiviral treatment or reliable severity predictors. it has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. however, very little is known about the interplay between proapoptotic and antiapoptotic mediators in this disease. therefore, plasma levels of the three proapoptotic mediators fas ligand (fasl), tumor necrosis factor‐α (tnf‐α), and tnf‐related apoptosis‐inducing ligand (trail) were measured in dengue patients. patients were classified according to the world health organization classification of dengue revised in 2009. additionally, inhibitors of apoptosis protein (iaps) were determined in plasma (survivin) and peripheral blood mononuclear cells (pbmcs) lysates (ciap‐1, ciap‐2, xiap). levels of apoptotic proteins in plasma were correlated with counts of blood cells. fasl and trail levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. dengue patients with warning signs showed decreased levels of survivin compared to patients with severe dengue and controls. trail was inversely correlated with counts of lymphocyte subsets. in contrast, survivin was positively correlated with leukocyte counts. there was a trend of elevated iaps levels in pbmcs of patients with severe dengue. the results suggest a likely antiviral effect of trail in dengue. it appears that trail might be involved with apoptosis induction of lymphocytes, whereas iaps might participate in protecting leukocytes from apoptosis. further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue pathogenesis. j. med. virol. 86:1437–1447, 2014. © 2013 wiley periodicals, inc. despite being the most significant arboviral disease worldwide, dengue has no antiviral treatment or reliable severity predictors. it has been shown that apoptotic cells from blood and tissues may be involved in the complex pathogenesis of dengue. however, very little is known about the interplay between proapoptotic and antiapoptotic mediators in this disease. therefore, plasma levels of the three proapoptotic mediators fas ligand (fasl), tumor necrosis factor-a (tnf-a), and tnf-related apoptosis-inducing ligand (trail) were measured in dengue patients. patients were classified according to the world health organization classification of dengue revised in 2009. additionally, inhibitors of apoptosis protein (iaps) were determined in plasma (survivin) and peripheral blood mononuclear cells (pbmcs) lysates (ciap-1, ciap-2, xiap). levels of apoptotic proteins in plasma were correlated with counts of blood cells. fasl and trail levels were elevated in dengue patients without warning signs when compared to patients with severe dengue and controls. dengue patients with warning signs showed decreased levels of survivin compared to patients with severe dengue and controls. trail was inversely correlated with counts of lymphocyte subsets. in contrast, survivin was positively correlated with leukocyte counts. there was a trend of elevated iaps levels in pbmcs of patients with severe dengue. the results suggest a likely antiviral effect of trail in dengue. it appears that trail might be involved with apoptosis induction of lymphocytes, whereas iaps might participate in protecting leukocytes from apoptosis. further research is needed to explore the interactions between pro and antiapoptotic molecules and their implications in dengue dengue is the most prevalent mosquito-transmitted viral disease in the world and is caused by any of the four closely related, but antigenically distinct, serotypes of dengue virus (denv-1-4), of the genus flavivirus, family flaviviridae. very recently, a map of dengue risk on the basis of the global population in 2010 estimated 390 million infections worldwide, of which 96 millions manifested apparently [bhatt et al., 2013] . this estimate is more than three times higher the who predicted burden. in particular, brazil has reported the largest number of dengue cases, accounting for more than 70% in the americas. however, reliable biomarkers of dengue severity, specific antiviral therapy and a licensed antidengue vaccine are not available [world health organization, 2009; guzman et al., 2010; simmons et al., 2012] . the world health organization (who) classification of dengue, dengue fever (df), and dengue hemorrhagic fever/dengue shock syndrome (dhf/ dss) has proven to be useful for more than 30 years. still, a revised classification was proposed after a recent international multicenter study: dengue without warning signs, dengue with warning signs and severe dengue [world health organization, 2009; alexander et al., 2011] . the pathogenesis of dengue is multifactorial and not fully understood. thus far, a complex interplay among viral, immunological, and host factors has been demonstrated. this interplay includes antibodymediated immune enhancement in secondary dengue infections which increases uptake of virus-antibody complexes in monocytes and macrophages [kliks et al., 1989] . studies also have shown involvement of the storm of cytokines and other soluble mediators released by immune cells [braga et al., 2001; bozza et al., 2008] , hyperactivation of lymphocytes [rothman, 2010] , and the influence of the infecting viral serotype [balmaseda et al., 2006] . comorbidities [limonta et al., 2009 ] and genetic background [sierra et al., 2007] also are associated with the pathophysiology of this viral disease. apoptotic cell death has been proposed to play a role in dengue pathophysiology. previous studies of dengue infection have shown apoptosis in lymphocytes [mongkolsapaya et al., 2003; myint et al., 2006] , monocytes [torrentes-carvalho et al., 2009; levy et al., 2010] , and peripheral blood mononuclear cells (pbmcs) [jaiyen et al., 2009] . apoptotic cells also have been found in a variety of tissues from fatal dengue cases [huerre et al., 2001; limonta et al., 2007 limonta et al., , 2009 . caspases (cysteinyl-aspartate-cleaving proteases) are essential cellular proteins involved in apoptosis, a programmed cell death. caspase activation is inhibited by inhibitors of apoptosis proteins (iaps) [hotchkiss et al., 2009] . survivin is considered a fundamental iaps that plays a role in tumorigenesis and inflammation [altieri, 2010] . however, the role of survivin has been poorly studied in viral diseases [zhu et al., 2003; bokarewa et al., 2007] . the extrinsic or death receptor pathway is one of the two apoptotic pathways, and is mediated by transmembrane receptors. the extrinsic pathway is activated when ligands that are members of the tumor necrosis factor (tnf) superfamily tnf-a, tnf-related apoptosis-inducing ligand (trail), and fas ligand (fasl) also known as cd95l bind to the death receptors. these ligands display proapoptotic functions in membrane-bound forms or in soluble forms [locksley et al., 2001; galluzzi et al., 2012] . although tnf-a has been extensively studied in dengue [cardier et al., 2005; chen et al., 2007; levy et al., 2010] and an in vitro anti-denv action of trail has been described [warke et al., 2008b] , no data appear to be available on the interplay among circulating pro and antiapoptotic mediators in dengue patients. furthermore, to our knowledge, the revised who classification for dengue [world health organization, 2009] has not been used in studies of apoptosis in dengue cases. in the present work, apoptotic mediators were measured in plasma and pbmcs lysates from brazilian patients with dengue. for the first time, these apoptotic proteins were measured in patients with the who classification for dengue revised in 2009. the three plasma molecules evaluated (fasl, tnf-a, and trail) are fundamental apoptosis inducers. the other proteins studied, plasma survivin and pbmcs lysate proteins, are iaps. our data suggest that trail could be involved with apoptosis induction of blood lymphocytes and could also contribute to the antiviral response. conversely, iaps may participate in apoptosis protection of blood leukocytes. fasl may display a role in dengue pathophysiology, and especially in fatal cases of dengue. venous blood samples were obtained from 107 febrile dengue-suspected cases during dengue outbreaks in the southwest of brazil in 2010. seventytwo (67.3%) of the 107 cases had the diagnosis of dengue confirmed. patients were assisted at the centro de referência em dengue in campos dos goytacazes, rio de janeiro state, and at the hospital professora esterina corsini in the universidade federal do mato grosso do sul, mato grosso do sul state. pbmcs and plasma were collected at these health care facilities, then transported in liquid nitrogen to the viral immunology laboratory at the oswaldo cruz institute, and stored in liquid nitrogen until use. pbmcs and plasma samples from 10 healthy adults were collected as controls. inclusion criteria for controls were absence of fever history in the preceding three months, absence of chronic diseases, and negative anti-denv igm by elisa (panbio diagnostics, brisbane, australia). the study protocol was approved by the institutional review board of oswaldo cruz foundation (fiocruz, number 061/2008) from the ministry of health in brazil (recognized by the brazilian national ethics committee), and by the institutional review board of each brazilian hospital involved in this study. enrollment of study participants, patients under the age of 15 required parents/legal guardians consent, and control subjects was conditional on obtaining appropriate written informed consent. a case was considered positive for dengue when diagnostic assays met one or more of the following criteria: (1) igm detection in acute samples by denv-specific capture igm enzyme-linked immunosorbent assay (elisa) (panbio diagnostics), (2) denv rna detection in acute-phase serum by a serotype-specific rt-pcr assay as described elsewhere [lanciotti et al., 1992] , (3) denv isolation in acute samples by inoculation onto aedes albopictus c6/36 cells and subsequent immunoflourescent detection of viral antigens as described previously [igarashi, 1978] , and (4) detection of non-structural 1 (ns1) protein using ns1 antigen strip (bio-rad, hercules, ca) in acute serum following the manufacturer's instructions. the new who dengue classification criteria from 2009 consisting of dengue without warning signs, dengue with warning signs, and severe dengue were used for each case. dengue without warning signs comprises fever with at least two of the following: nausea, vomiting, rash, aches and pain, positive tourniquet test, and leucopenia. dengue with warning signs includes dengue patients with any of the following warning signs: abdominal pain or tenderness, persistent vomiting, clinical fluid accumulation, mucosal bleeding, lethargy or restlessness, liver enlargement >2 cm, and an increase in hematocrit concurrent with rapid decrease in platelet count. severe dengue includes at least one of the following: severe plasma leakage (leading to shock and fluid accumulation with respiratory distress), severe bleeding evaluated by clinicians, severe involvement of liver by aspartate aminotransferase (ast) or alanine transferase (alt) > 1,000 u, central nervous system with impaired consciousness, and severe involvement of the heart and other organs [world health organization, 2009 ]. upon enrollment during consultation or hospitalization, standardized forms for data collection were completed by study clinicians experienced with dengue. the forms included demographic information, vital signs, physical examinations, complete blood counts, liver enzyme values (alt, alanine transaminase; ast, aspartate transaminase), findings of ultrasounds and x-rays. about 20 ml of peripheral venous blood were collected in na-heparin anticoagulated tubes from patients and control subjects. pbmcs were separated from blood samples using the ficoll-hypaque procedure (d ¼ 1.077 g/ml; sigma-aldrich, st. louis, mo) and centrifuged at 400g for 30 min. the pbmcs layer was washed twice in rpmi 1640 medium. the viability of pbmcs was greater than 95% after trypan blue exclusion. approximately 10,000,000 pbmcs were resuspended in 1 ml of freezing solution, which consisted of 80% inactivated fbs (invitrogen, carlsbad, ca) plus 20% dmso (sigma-aldrich), and stored at à70˚c for 24 hr before transfer to liquid nitrogen. aliquots were cryopreserved for later study. lymphocyte subpopulations (cd4þ and cd8þ t cells) and monocytes (cd14þ) were enumerated by a five or six-color flow cytometry method using the following monoclonal antibodies: allophycocyanin cyanine dye (apc cy7) conjugated anti-cd4 (biolegend, san diego, ca), phycoerythrin cyanine dye (pe cy7) conjugated anti-cd8 (bd biosciences, san jose, ca), and phycoerythrin cyanine dye (pe cy5.5) conjugated anti-cd14 (southern biotech, birmingham, al). appropriate matched-isotype control antibodies were used to discriminate between positive and negative populations/subsets. subpopulations and subsets of leukocytes were determined by staining 100,000 cells of peripheral blood with appropriate combinations of monoclonal antibodies. pbmcs were suspended in pbs. the cells were then incubated on ice for 40 min in the dark with fluorochrome-conjugated monoclonal antibodies, washed twice with pbs, and fixed in 1% paraformaldehyde. fixed cells were kept in the dark until use. a minimum of 10,000 t cells' gated events were acquired using a cyan flow cytometer (dako, glostrup, denmark), and analysis was performed using flow jo v.7.6.1 software (tree star, ashland, or). plasma concentrations of the proapoptotic proteins fasl and trail, and the antiapoptotic survivin were measured by elisa (quantikine, r&d systems, minneapolis, mn) according to the manufacturer's instructions. tnf-a concentration in plasma was determined by elisa (pepro tech, inc., rocky hill, nj) following the manufacturer's instructions. analysis of cellular inhibitor of apoptosis protein 1 (ciap-1), cellular inhibitor of apoptosis protein 2 (ciap-2), and x-linked inhibitor of apoptosis protein (xiap) expression profiles was performed on lysates of pbmcs obtained from dengue patients and controls. following the manufacturer's instructions (human apoptosis array kit, proteome profiler array, r&d systems), apoptosis array data on developed xray film were quantified using quantity one image analysis software (version 4.6.3, bio-rad). a template that analyzed pixel density in each spot of the array was created and background values subtracted. a comparison of corresponding signals on different arrays was carried out to determine the relative change in iaps levels between patients and control samples. data were entered and analyzed using epidata software version 3.1 (odense, denmark). differences and comparison analysis were carried out with nonparametric mann-whitney and spearman tests using the prism software v5.0 (graphpad, san diego, ca). the kolmogorov-smirnov test was applied with p < 0.05 considered as significant. sixty-two patients with dengue and 10 control individuals were included in the apoptosis-related proteins analysis (table i ). the mean age of patients and control subjects was 40 years old. the female/ male gender distribution for patients was 30/32. patients infected with denv were recruited between the first and eleventh day after fever onset. most of the dengue infections (80.8%) were caused by denv-2. twenty-seven of the 62 patients with dengue were classified into dengue without warning signs, 20 into dengue with warning signs and 15 into severe dengue following the who dengue classification revised in 2009 [world health organization, 2009 ]. one fatal case was recorded in the severe dengue group. patients with severe dengue more commonly had bleeding, vascular leakage manifestations, and hypotension than the rest of the studied dengue patients (table i) . hematocrit values did not increase with disease severity. decreased platelet counts were significantly related to severity and all patient groups showed values below those of control subjects. values of liver enzymes (ast, alt) were increased several fold in patients with severe dengue and the increase was statistically significant (p < 0.05). the total number of leukocytes and the three cells subpopulations (cd14þ, cd4þ, and cd8þ) increased with disease severity (table ii) . however, the frequency and the absolute numbers of leukocytes did not differ significantly among the three groups of patients with dengue. in particular, cd4þ and cd8þ t lymphocyte counts were significantly higher in the control group as compared to dengue with and without warning signs groups. as shown in figure 1a , a significantly elevated level of fasl was found in patients with dengue without warning signs (362.8 ae 111.1 pg/ml) and in patients with severe dengue (298.6 ae 183.7 pg/ml) than in control subjects (248.3 ae 85.4 pg/ml). remarkably, the more elevated value of fasl was recorded in a single fatal dengue case (824 pg/ml) on the day of death. in addition, trail levels in patients with dengue without warning signs (667.7 ae 528 pg/ml) were statistically more elevated when compared to patients with severe dengue (275 ae 121.3 pg/ml) and control individuals (317.7 ae 74.6 pg/ml) (fig. 1b) . trail was the only apoptosis mediator that differentiated patients with dengue with warning signs (761.5 ae 616.2 pg/ml) and those with severe dengue. survivin levels in the plasma of dengue patients showed variability. namely, these results showed decreased survivin levels ( fig. 1c) in patients with dengue with warning signs (17.5 ae 32.8 pg/ml) compared to control individuals (175.2 ae 91 pg/ml) and patients with severe dengue (148.5 ae 157.3 pg/ml). no significant differences were found in tnf-a levels among the dengue patients studied (fig. 1d ). however, this cytokine was significantly more elevated in dengue patients without warning signs than in control subjects. cd14þ monocyte counts trail levels and platelet counts were positively associated in the dengue patients (table iii) . a negative relationship was found between trail levels and total lymphocytes. this negative correlation also occurred in both subpopulations of cd4þ and cd8þ t lymphocytes. in contrast to trail, survivin levels were positively related to peripheral leukocyte and lymphocyte counts, especially cd14þ monocyte counts. no relationship was found between survivin levels and platelet counts. fasl correlated positively with trail (r ¼ 0.343, p ¼ 0.009) (data not shown). preliminary profiling data of the iaps family members (ciap-1, ciap-2, and xiap) were evaluated in pbmcs lysates from some control subjects (n ¼ 02) and some patients with dengue (n ¼ 06). two of the patients had dengue without warning signs and four had severe dengue. a trend of increased levels in the expression of ciap-1, ciap-2, and xiap was detected in patients with severe dengue, but not in the patients with dengue without warning signs and control subjects (fig. 2) . this trend was not statistically significant. in the present observational study, levels of apoptosis-related mediators were measured in plasma and in pbmcs lysate samples from brazilian patients with severe and non-severe dengue. patients were infected with either denv-1 or denv-2. the goal was to explore the interplay between proapoptotic and antiapoptotic mechanisms in dengue disease. the findings in severe dengue patients of more hypotension, hemorrhagic and vascular leakage manifestations, decreased platelet counts and increased values of hepatic enzymes are in agreement with previous studies that used the who dengue clinical classification revised in 2009 [barniol et al., 2011; narvaez et al., 2011; de-oliveira-pinto et al., 2012] . on the other hand, in several cell lines infected by denv, the fas/fasl mechanism is involved in apoptotic-induced cell death [liew and chow, 2006; limjindaporn et al., 2007; torrentes-carvalho et al., 2009; liao et al., 2010] . modulation of the immune response by apoptosis during dengue has been reported previously in thai children who showed higher levels of plasma soluble fas in dhf than df [myint et al., 2006] . however, the present study was performed mainly in an adult population, and the measured circulating mediator was the fasl. there-fore, this is the first reported study of circulating fasl molecule in dengue. in this study, fasl values in patients with dengue without warning signs were elevated compared to those in patients with severe dengue and control subjects. however, the premortem detection of fasl in the only fatal dengue case showed the highest fasl value measured in the study. it is very likely this highest value was due to the plasma sample being obtained on the day of the death. more dengue fatalities should be studied to understand if the elevation of this proapoptotic mediator in the critical phase of dengue is related to fatal outcome and to explore the probable mechanisms of action involved. in the present study, elevated trail levels were observed in patients with dengue without warning signs compared to those with severe dengue and control subjects. in addition, trail levels discriminated patients with dengue with warning signs from patients with severe dengue. previous in vitro studies have demonstrated an increase of trail production by denv infection in a variety of human primary cells including umbilical vein endothelial cells [warke et al., 2008b] , muscle satellite cells [warke et al., 2008a] , dendritic cells, monocytes, and b cells [warke et al., 2008b; becerra et al., 2009; balas et al., 2011; wong et al., 2012] . in these human primary cells was not demonstrated an apoptotic activity of trail. conversely, antiviral action of trail was shown by recombinant trail pretreatment of monocytes and dendritic cells [warke et al., 2008b] . another study showed serum elevation of trail in venezuelan dengue patients compared to febrile patients without dengue and control subjects. however, this previous study involved only two dhf cases and did not analyze the dengue severity [becerra et al., 2009] . the findings in the present work, which used the revised who dengue classification, suggest that the antiviral action of trail that was shown previously in cell culture [warke et al., 2008b] , may also occur in vivo and hence the likely increased severity of dengue in patients lacking an elevated trail production. in a previous study [warke et al., 2008b] , trail inhibited denv in infected dendritic cells not by an apoptosis-dependent mechanism but by a mechanism linked to type i ifn signaling pathways. this finding suggests that the antiviral action of trail could be mediated by known or novel cellular proteins induced by trail. the trail hypothesis, that trail antiviral action is not direct, in dengue infection is supported by recent findings of shrestha et al. [2012] . these authors demonstrated that trail secreted by cd8þ t cells provides antiviral signals to neurons infected by west nile virus (a flavivirus closely related to denv). the effect was studied in the central nervous system of a mouse model and in primary culture of mouse cortical neurons. this work suggests that trail produced by cd8þ t cells is necessary but not sufficient to confer an antiviral effect, and that such an effect requires additional secreted cytokines and/or cytolytic activities by the cd8þ t cells. recent work by this research group [gandini et al., 2013] found that denv infection activates plasmacytoid dendritic cells, from pbmcs of healthy donors, and leads to the subsequent expression of trail in the cell membrane. these researchers also found that in dengue patients, the activation of plasmacytoid dendritic cells by membrane trail is associated with less severe dengue. this report also confirms the in vitro antiviral action of recombinant trail in monocytes infected with denv and that trail production in plasmacytoid dendritic cells is associated with ifn-a production. previous studies have shown that trail regulates megakaryocytopoiesis [zauli and secchiero, 2006 ] and promotes maturation of megakaryoblasts into platelets [melloni et al., 2005] . in addition, it has been shown in vitro that activated platelets release trail [crist et al., 2004] . however, more studies are needed to determine whether trail is related to platelet increase or trail is released by platelets during dengue infection. the inverse correlation among the levels of the proapoptotic trail and the counts of cd4þ and cd8þ t lymphocyte subpopulations supports the hypothesis that trail is related to lymphocytic cell death. a similar phenomenon has been reported in patients infected with hiv [cummins and badley, 2010] , respiratory syncytial virus [roe et al., 2004] , sars coronavirus [law et al., 2009] , measles virus [okada et al., 2000] , and in primate models of ebola virus disease [hensley et al., 2002] . therefore, apoptosis might be a contributing factor that modulates the number of lymphocytes in dengue infection, as documented previously [myint et al., 2006] . inhibitors of apoptosis protein ciap-1 (cellular inhibitor of apoptosis protein 1), ciap-2 (cellular inhibitor of apoptosis protein 2), and xiap (x-linked inhibitor of apoptosis protein) expression in peripheral blood mononuclear cell lysates from controls (solid white), dengue without warning signs patients (solid bright gray) and severe dengue patients (solid black). no statistical significance was found using the mann-whitney test for statistical analysis and by means of graphpad prism v5.0 program. horizontal lines represent the median and the vertical lines correspond to the standard deviation. trail expression is increased in primary hepatocytes and hepg2 cells infected with dengue [suksanpaisan et al., 2007] . however, the significance of trail action in hepatic cells remains unclear after the retraction of the work by matsuda et al. [2005] . in the current investigation the liver cells were not studied. however, it cannot be ruled out that expressed trail in liver cells after infection by denv is released to the circulation during the clearance of infected liver cells. in the present study, the plasma levels of tnf-a were significantly higher in dengue patients without warning signs when compared with controls. no differences were found in tnf-a levels among the three groups of dengue patients studied. although tnf-a is one of the most studied cytokines in dengue infection, the association of tnf-a with disease severity is still debated. on one hand, some studies have found an association of tnf-a with dhf [hober et al., 1993; braga et al., 2001; levy et al., 2010] . on the other hand, some studies have not found such association [laur et al., 1998; bozza et al., 2008; hoang et al., 2010; priyadarshini et al., 2010] . the discrepancies in tnf-a detection in dengue studies might be related to several factors. previous reports in dengue patients have found differences of tnf-a production in association with genetic polymorphisms of tnf-a [fernandez-mestre et al., 2004; vejbaesya et al., 2009; perez et al., 2010] . in addition, in vitro tnf-a induction can be serotype-specific [azizan et al., 2006] , and the highest plasma levels of tnf-a occur before the sixth day of dengue disease [hober et al., 1993] . the tnf-a level was not elevated in the single fatal case of dengue in this study. this finding is in agreement with previous work that did not show differences in tnf-a levels among fatal cases of dengue and survivors of df and dhf [chen et al., 2006] . the iaps participate in multiple intracellular pathways comprising cell death and survival [altieri, 2010] . for example, survivin expression is related to in vitro lymphocyte division and clonal expansion [song et al., 2005] . on the other hand, a previous study has shown an association of survivin upregulation in the circulation with the induction of adhesion molecules on the surface of blood leukocytes [mera et al., 2008] . the presence of antiapoptosis pathway proteins during dengue infection may counteract the role of proapoptosis signaling molecules. the antiapoptotic protein survivin is studied in dengue infection for the first time in the present study. this molecule showed variable levels with reduced levels in patients with dengue without warning signs as compared to control subjects and patients with severe dengue. survivin was positively correlated with counts of leukocytes, lymphocytes, and cd14þ monocytes in the dengue patients studied. although this work did not verify experimentally the exocytosis of survivin and the extracellular action of survivin on white blood cells, these correlation results suggest that survivin may be associated with proliferation of leukocytes, and specially monocytes during dengue. additionally, elevated survivin levels may provide more protection against apoptosis in these immunological blood cells from the circulation of the dengue patients studied. iaps are known for their ability to regulate caspases and cell death [silke and meier, 2013] . although preliminary, the present study showed that the expression of iaps family members in pbmcs was markedly higher in patients with severe dengue than in patients with dengue without warning signs and control subjects. this result suggests that the iaps may be involved with apoptosis inhibition in pbmcs, however, it is not clear whether this antiapoptotic effect of iaps might counteract the probable induction of apoptosis by trail in circulating lymphocytes. thus, the pro-and antiapoptotic mechanisms may be interacting but the real impact needs to be investigated further. even though the current study had novel findings of proteins related to apoptosis in dengue patients, there are still several limitations. for instance, patients were not stratified by day of infection which could explain the large standard deviation of the mean values for lymphocyte counts. measuring the numbers of lymphocytes and plasma concentrations of apoptotic proteins daily would be a more accurate way to compare groups of dengue patients. however, the higher counts of total lymphocyte subpopulations in patients with severe dengue than in patients without the severe clinical form of dengue is in agreement with previous studies [kadhiravan et al., 2010; de-oliveira-pinto et al., 2012] . in addition, the lack of data on viremia, immune status, and co-morbidities may limit the interpretation of the preliminary results of this study. the inclusion of both healthy subjects and febrile patients without dengue as the control group may have been preferable. additionally, genetic polymorphisms might affect the circulating levels of these apoptotic mediators, as previously shown in studies of trail [korner et al., 2012] , fasl [nasi et al., 2005] , and survivin [borges bdo et al., 2011] in infectious and non-infectious conditions. in conclusion, fasl may exhibit a function in the different clinical forms of dengue disease and in fatal dengue. it is hypothesized that trail has a role in inducing apoptosis of lymphocytes in dengue patients. survivin seems likely to promote survival and proliferation of blood leukocytes whereas other iaps family members may be involved in antiapoptotic pathways of pbmcs during dengue. more research should be done to characterize the complex interplay of pro and antiapoptotic mediators during the different phases of dengue disease to identify implications for dengue severity. multicentre prospective study on dengue classification in four south-east asian and three latin american countries survivin and iap proteins in cell-death mechanisms differential proinflammatory and angiogenesis-specific cytokine production in human pulmonary endothelial cells, hpmec-st1.6r infected with dengue-2 and dengue-3 virus different innate signatures induced in human monocyte-derived dendritic cells by wild-type dengue 3 virus, attenuated but reactogenic dengue 3 vaccine virus, or attenuated nonreactogenic dengue 1-4 vaccine virus strains serotype-specific differences in clinical manifestations of dengue usefulness and applicability of the revised dengue case classification by disease: multi-centre study in 18 countries gene expression profiling of dengue infected human primary cells identifies secreted mediators in vivo the global distribution and burden of dengue pathological survivin expression links viral infections with pathogenesis of erosive rheumatoid arthritis survivin -31c/g polymorphism and gastric cancer risk in a brazilian population multiplex cytokine profile from dengue patients: mip-1beta and ifn-gamma as predictive factors for severity detection of circulant tumor necrosis factor-alpha, soluble tumor necrosis factor p75 and interferon-gamma in brazilian patients with dengue fever and dengue hemorrhagic fever proinflammatory factors present in sera from patients with acute dengue infection induce activation and apoptosis of human microvascular endothelial cells: possible role of tnf-alpha in endothelial cell damage in dengue correlation of serum levels of macrophage migration inhibitory factor with disease severity and clinical outcome in dengue patients both virus and tumor necrosis factor alpha are critical for endothelium damage in a mouse model of dengue virus-induced hemorrhage expression of tnf-related apoptosis-inducing ligand (trail) in megakaryocytes and platelets mechanisms of hiv-associated lymphocyte apoptosis regulation of inflammatory chemokine receptors on blood t cells associated to the circulating versus liver chemokines in dengue fever tnf-alpha-308a allele, a possible severity risk factor of hemorrhagic manifestation in dengue fever patients molecular definitions of cell death subroutines: recommendations of the nomenclature committee on cell death dengue virus activates membrane trail relocalization and 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t-cells with disease severity in adults with dengue antibody-dependent enhancement of dengue virus growth in human monocytes as a risk factor for dengue hemorrhagic fever trail receptor i (dr4) polymorphisms c626g und a638c are associated with an increased risk for hepatocellular carcinoma (hcc) in hcv-infected patients rapid detection and typing of dengue viruses from clinical samples by using reverse transcriptase-polymerase chain reaction plasma levels of tumour necrosis factor alpha and transforming growth factor beta-1 in children with dengue 2 virus infection in french polynesia toll-like receptors, chemokine receptors and death receptor ligands responses in sars coronavirus infected human monocyte derived dendritic cells increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, c-reactive protein and apoptosis in dengue fasl/fas pathway is involved in dengue virus induced apoptosis of the vascular endothelial cells microarray and real-time rt-pcr analyses of a novel set of differentially expressed human genes in ecv304 endothelial-like cells infected with dengue virus type 2 sensitization to fas-mediated apoptosis by dengue virus capsid protein apoptosis in tissues from fatal dengue shock syndrome fatal severe dengue and cell death in sickle cell disease during the 2001-2002 havana dengue epidemic the tnf and tnf receptor superfamilies: integrating mammalian biology dengue virus-induced apoptosis in hepatic cells is partly mediated by apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand functional expression of trail and trail-r2 during human megakaryocytic development extracellular survivin up-regulates adhesion molecules on the surface of leukocytes changing their reactivity pattern original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever cellular immune activation in children with acute dengue virus infections is modulated by apoptosis evaluation of the traditional and revised who 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of dengue virus infection inhibitor of apoptosis (iap) proteinsmodulators of cell death and inflammation sustained survivin expression from ox40 costimulatory signals drives t cell clonal expansion infection of human primary hepatocytes with dengue virus serotype 2 dengue-2 infection and the induction of apoptosis in human primary monocytes tnf and lta gene, allele, and extended hla haplotype associations with severe dengue virus infection in ethnic thais efficient dengue virus (denv) infection of human muscle satellite cells upregulates type i interferon response genes and differentially modulates mhc i expression on bystander and denv-infected cells trail is a novel antiviral protein against dengue virus dengue: guidelines for diagnosis, treatment, prevention and control susceptibility and response of human blood monocyte subsets to primary dengue virus infection the role of the trail/trail receptors system in hematopoiesis and endothelial cell biology upregulation of survivin by hiv-1 vpr the authors are truly thankful to hermann g. schatzmayr, a worldwide distinguished professor from fiocruz, who inspired our investigation from the very beginning. we highly regret he could not participate in the final work because he died. we thank jaqueline bastos for the dengue diagnostics in the flavivirus laboratory at fiocruz. we also acknowledge the staff from the plínio bacelar laboratory headed by dr. carlos bacelar and prefeitura municipal de campos de goytacazes. we are grateful to the clinical and nursing staff of the participating healthcare facilities. the preparation of this manuscript was improved by the intensive course in research writing at texas a&m university (tamu). we thank the main instructor of the course, dr. barbara gastel, and her university colleagues. dr. daniel limonta received a scholarship to attend this international course. key: cord-347298-7kqrl3rv authors: hedger, m.p. title: immunology of the testis and male reproductive tract date: 2010-07-12 journal: comprehensive toxicology doi: 10.1016/b978-0-08-046884-6.01112-x sha: doc_id: 347298 cord_uid: 7kqrl3rv a large body of evidence points to the existence of a close, dynamic relationship between the immune system and the male reproductive tract, which has important implications for our understanding of both systems. the testis and the male reproductive tract provide an environment that protects the otherwise highly immunogenic spermatogenic cells and sperm from immunological attack. at the same time, secretions of the testis, including androgens, influence the development and mature functions of the immune system. activation of the immune system has negative effects on both androgen and sperm production, so that systemic or local infection and inflammation compromise male fertility. the mechanisms underlying these interactions have begun to receive the attention from reproductive biologists and immunologists that they deserve, but many crucial details remain to be uncovered. a complete picture of male reproductive tract function and its response to toxic agents is contingent upon continued exploration of these interactions and the mechanisms involved. in the decade since this chapter was first published (hedger 1997) , there has been considerable progress in the understanding of the regulation of immunity. advances such as the emergence of the innate immune system as a major theme in immunity following the discovery of the toll-like receptor (tlr) family (o 'neill and dinarello 2000) , the formalization of multiple macrophage phenotypes corresponding to the type 1 and 2 helper t (th) cell subsets (mantovani et al. 2005) , the rehabilitation of suppressor and regulatory t cells (piccirillo and thornton 2004) , and the identification of new lymphocyte subsets, including th3, th17, and natural killer t (nkt) cells (godfrey et al. 2000; macdonald 1998; romagnani 2008) , also have had considerable influence on the concept concerning the immunology of the male reproductive tract. nonetheless, while there have been advances in our understanding of the interface between the male reproductive tract and the immune system, there is still much we simply do not know. the need to understand this interface and the potential implications for reproductive toxicology continues to be just as important as ever. there is no doubt that the testis and the male germ cells in particular are susceptible to immunological damage. clinically significant testicular autoimmunity, most commonly indicated by the presence of antisperm antibodies in the serum or ejaculate, is implicated in the case of 5-12% of all male infertility patients within the developed world (baker et al. 1983; pattinson and mortimer 1987) , but the incidence is much higher in populations where access to reproductive health care is limited (ekwere 1995) . common factors suspected to contribute to testicular autoimmunity include reproductive tract infections, physical trauma, immune system dysfunction, and genetics. the release of spermatogenic antigens from the reproductive tract following vasectomy generally results in autoimmune responses, ranging in severity from sperm antibodies to orchitis, in both humans and experimental animals (alexander and anderson 1979; flickinger et al. 1990a; kojima and spencer 1983; tung and alexander 1980) . the potential for spontaneous testicular autoimmune disease in otherwise normal men remains poorly characterized, although studies in animals clearly indicate the possibility of such reactions in humans (furbeth et al. 1989; tung et al. 1981) . complicating matters is the fact that the testis is an immunologically privileged tissue, in the sense that foreign tissue grafts into the testes of experimental animals survive for prolonged periods (barker and billingham 1977; head et al. 1983a; whitmore and gittes 1978) . this dichotomy between immune susceptibility and privilege is indicative of a highly specialized interaction between the male reproductive system and the immune system, with potentially important implications for both systems. the objective of this chapter is to provide an overview of the current state of knowledge and to highlight issues for further consideration that may be of relevance to male reproductive toxicology. 11.10.2 the interface between the immune system and the reproductive tract the immune system is the body's protective arsenal against disease. it operates via the detection of potential aggressors, recognized through conserved motifs found on pathogenic organisms (innate immunity) or confrontation by completely novel (i.e., foreign) molecular structures (adaptive immunity) (vivier and malissen 2005; zinkernagel 2000) . immunity involves specialized cells possessing highly developed recognition and activation abilities (macrophages and dendritic cells) and the cells that carry out the protective responses: t and b cells in the case of adaptive immunity and nk cells and mononuclear or polymorphonuclear phagocytes (pmns) in the case of innate immunity. many of these cells play roles in both the recognition and effector arms of the immune response and in both innate and adaptive immunities (figure 1) . innate immunity provides the rapid response elements of the immune system, but is limited in its attack repertoire. adaptive immunity is much more flexible in its responses, but requires some time to become effective. once the adaptive arm has been activated toward a particular pathogen, however, it can respond rapidly in the future due to the persistence of memory lymphocytes, which form the basis of immunization (mcghee et al. 1993; schittek and rajewsky 1990) . the innate immune system operates upon recognition by specific pattern-recognition receptors of specific motifs found on bacterial, viral, fungal, and protozoan pathogens. functionally, these receptors have evolved to recognize pathogen-associated molecular patterns (pamps), such as bacterial lipopolysaccharides (lpss), lipoproteins, peptidoglycans, and viral nucleic acids (bianchi 2007; roach et al. 2005) . unlike classical ligand receptors, these receptors respond to multiple ligands possessing related, but not necessarily identical, structures. the largest and best-studied class of pattern-recognition receptors is the tlrs, which are members of a larger family of proteins that include the interleukin (il) 1 receptor (il1r), and which share a conserved cytoplasmic domain called the toll/il1r (tir) domain (akira and takeda 2004; o'neill and bowie 2007) . in humans, 10 functional tlrs (tlr1-10) have been identified (figure 2) . mammalian tlrs 11-13 are known from laboratory rodent species, but are either pseudogenes or absent in humans (roach et al. 2005) . although they are particularly associated with cells of the monocyte lineage (i.e., macrophages and dendritic cells), these receptors are also found on certain t and b cells, as well as many fibroblastic and epithelial cells, including those of the male urogenital tract (lauw et al. 2005; zhang et al. 2004) . significantly, ligands for some of the tlrs include endogenous molecules of mammalian origin (barrat et al. 2005; karikó et al. 2004; tsan and gao 2004; yasuda et al. 2005; yu et al. 2006) . tlr signaling occurs through engagement of crucial adaptor proteins, specifically myeloid differentiation primary response protein 88 (myd88) or tir domain-containing adaptor protein inducing interferon (trif), via tir domain interactions, leading to activation of tumor necrosis factor (tnf) receptorassociated factor 3 and 6 (traf3, traf6) (akira and takeda 2004; o'neill and bowie 2007; o'neill and dinarello 2000) . based on the tlr engaged, this results in nuclear translocation of the inflammatory transcription factor, nuclear factor kappa b (nfb), activation of the jun n-terminal kinase (jnk) and p38 mitogen-activated protein (map) kinases, and induction of type 1 interferon (ifn and ifn) figure 1 polarization in the immune system. the immune system is conceptually divided into innate and adaptive arms, with adaptive immunity further subdivided into type 1 (cell-mediated) and type 2 (antibody-mediated) responses. triggering of the immune response involves the induction of inflammation by various agents, which may include infections, particulates, debris from damaged cells, or noxious agents. innate immunity involves fixed pattern-recognition receptors, such as the tolllike receptors, whereas adaptive immunity depends upon highly polymorphic receptors expressed by t cells (t cell receptors) and b cells (membrane-bound and secreted immunoglobulin). in general, type 1 responses are associated with cellular immunity, autoimmune reactions, and graft rejection. type 2 responses involve antibody production by the b cells, but also play a more complex role in allergy, immunoregulation, and control of tolerance, favoring immune privilege. within these divisions, various cell types, cytokines, chemokines, bioactive lipids, and other molecules interact to integrate or mediate the responses. the direction of the adaptive immune response type is determined primarily by the activity of the antigenpresenting cells (primarily dendritic cells) under the influence of cells with immunoregulatory capacity, such as macrophages, nk cells, and nkt cells, and either type 1 or type 2 cytokines produced locally. several cell types and molecules play multiple roles within different functional divisions. abbreviations: mo, monocyte; nk, natural killer cell; pmn, polymorphonuclear cell; th; helper t cell; b, b cell; t reg , regulatory t cell; m, macrophage; nkt, natural killer t cell; ctl, cytotoxic t cell; apc, antigenpresenting cell, ros, reactive oxygen species; ifn, interferon; pge, prostaglandin e; cox, cyclooxygenase; inos, inducible nitric oxide synthase; tnf, tumor necrosis factor ; il, interleukin; il1ra, interleukin 1 receptor antagonist; tgf, transforming growth factor ; gc, glucocorticoid; mif, macrophage migration inhibitory factor. production (hertzog et al. 2003; malmgaard 2004; nakano 2004) . the pattern-recognition receptor family also includes the nucleotide binding and oligomerization domain (nod)-like receptor (nlr) family and the retinoic acid-inducible gene (rig)-like helicases (rlhs) (becker and o'neill 2007; thompson and locarnini 2007) . the nlrs detect various bacterial pamps within the cytosol. some nlrs, such as nod1 and nod2, act via nfb, while other nlrs work through induction of the cysteine protease caspase 1, which activates the proinflammatory cytokines il1 and il18 and initiates caspase-mediated proapoptotic pathways within the target cell. the rlhs, which detect the presence of viral rna in the cytosol, activate both nfb and production of type 1 ifns. activation of innate immunity leads to inflammation, characterized by increased blood and lymphatic flow within the affected tissue and recruitment of immune cells to the site through production of chemokines and expression of vascular leukocyte adhesion molecules (moser and loetscher 2001; picker and butcher 1992) . this mobilization of immune cells is accompanied by activation of complement, production of proinflammatory cytokines tlr6 tlr10 tlr11 tlr5 tlr2 tlr3 tlr9 tlr4 cd14 tlr8 tlr7 cytoplasmic cell surface cpg dna ssrna dsrna lipoproteins lps flagellin/profilin ? irf3 nfkb ap1 map kinases il1α, tnfα, il6, inos, activin a, il10, il12, cox2, ifnγ mal tram tlr2 tlr2 tlr1 apoptosis irf1,5,7 ifnα, ifnβ, tnfα myd88 traf6 traf3 traf3,6 myd88 trif figure 2 the toll-like receptors: major pathogenic ligands, adaptor proteins, signaling, and cytokine production. there are 10 active human toll-like receptors (tlr1-10). tlr11-13 are present in rodents, and tlr11 is specific to the urogenital tract. the tlrs respond to a variety of pathogen-related molecules, usually forming homo-or heterodimers during signaling: for example, tlr2 can self-associate or combine with either tlr1 or tlr6 to mediate responses to various bacterial lipoprotein classes. tlr4 forms a complex with the receptor cofactor cd14, to facilitate binding of bacterial lipopolysaccharide (lps). the tlrs can be subdivided into cell surface receptors, which largely respond to bacterial proteins, lipoproteins, and lipopolysaccharides, and intracytoplasmic receptors, which recognize single-stranded and double-stranded rna (ssrna and dsrna) and cpg-containing dna of viral or bacterial origin. most tlrs signal via the adaptor protein myd88 (myeloid differentiation primary response protein 88), except tlr3, which acts through the adaptor protein trif (tir domaincontaining adaptor protein inducing interferon ). uniquely, tlr4 can interact with either myd88 or trif, through engagement of the adaptor proteins mal (myd88 adaptor-like) or tram (trif-related adaptor molecule). downstream signaling involves the tumor necrosis factor receptor-associated factors 3 and 6 (traf3 and traf6), activation of the transcription factors nuclear factor kappab (nfb) and interferon regulatory factor 1 (irf1), or activation of the mitogenactivated protein kinases (map kinases) jun n-terminal kinase (jnk) and p38 to produce the fos/jun transcription factor activated protein 1 (ap1). these transcription factors interact to induce the expression of multiple proinflammatory genes, including interleukin (il) 1, tumor necrosis factor (tnf), inducible nitric oxide (inos), cyclooxygenase 2 (cox2), and interferon (ifn), or the type 1 interferons (ifn, ifn) and tnf. note that many details of the signaling pathways have been omitted or truncated for the sake of simplicity. and acute-phase proteins, and stimulation of phagocytosis and killing of pathogens and infected cells by macrophages, pmns, and nk cells, through production of proteases and other lytic proteins, and cytotoxic reactive oxygen species (ros) and induction of apoptotic pathways (rosenberg and gallin 2003) . moreover, activation of innate immunity is responsible for initiation of the adaptive immune response, through production of regulatory cytokines and stimulation of the antigen-presenting cell (apc) abilities of dendritic cells, macrophages, and some b cells (kelsall and rescigno 2004; spörri and reis e sousa 2005) . among the cell types that mediate the innate immune response, nk cells represent a special case. these cells are of the lymphocytic lineage, but they are capable of spontaneously attacking target cells without prior sensitization by exposure to antigen (mori et al. 2001; westermann and pabst 1992) . through a complex of specific receptors on their surface, nk cells are able to recognize and directly target transformed cells, such as virally infected or tumor cells (lanier 2001; mori et al. 2001) . in this, nk cells provide a first-line of lymphocyte defense within the context of the innate immune response, but activated nk cells also participate in adaptive immunity, both as regulators and as effector cells of the response (mocikat et al. 2003; raulet 2004) . highly specific interactions between apcs and t cells, mediated via the polymorphic proteins of the major histocompatibility complex (mhc; the human leukocyte antigen (hla) complex in humans) and antigen-specific t cell receptors (tcrs), are crucial to the adaptive immune response. the apcs process exogenous foreign proteins into short antigenic peptides, which are then incorporated into a structural groove on the external surface of the mhc protein complex during its assembly within the cell (cresswell et al. 1999; sant et al. 1999) . the tcr on the surface of a t cell that has specificity for the antigen binds to the antigen-mhc complex on the apc surface, which results in activation and proliferation of the t cell. circulating t cells express one of two coreceptors, cd4 and cd8, as part of the tcr, which direct them to recognize antigens associated with either mhc class ii or i proteins, respectively (janeway 1992) . antigens are presented to cd4 þ t cells by the so-called professional apcs that express mhc class ii antigens (i.e., dendritic cells, macrophages, and b cells) (heath and carbone 2001; unanue 1984) . the cd8 þ t cells recognize antigens presented in the context of mhc class i proteins, which are expressed by almost all cell types in the body. activation of the t cell also requires physical interaction between the apc and t cell involving costimulatory ligand-receptor pairs, particularly cd28:b7 and cd40:cd40l, as well as the production of either type 1 cytokines (ifn, il2, and il12) or type 2 cytokines (il4, il5, il10, and il13) (constant and bottomly 1997; heath and carbone 2001; moser and murphy 2000) . as a consequence, the activated t cell can have a number of different fates depending on the costimulatory molecules engaged and cytokines produced: activated cd4 þ t cells may become type 1 helper (th1) cells, which direct development of the cellular immune response involving cytotoxic cd8 þ t cells, or they may become type 2 helper (th2) cells, which promote the development of b cells into antibody-secreting plasma cells following interaction with their antigen (defranco 1987) . recently, a new th effector cell subset (th17) developmentally related to th1 cells, but producing il17, il21, and il22 has been described (ouyang et al. 2008; romagnani 2008) . these th17 cells direct a number of responses involved in protection against extracellular pathogens, including recruitment and activation of the major circulating pmn subset, the neutrophils, differentiation of b cells, and inflammatory reactions of epithelial cells. however, this cell subset has also been implicated in the development of autoimmune diseases and, along with th2 cells, in allergy (oboki et al. 2008; ouyang et al. 2008) . the absence of appropriate costimulatory molecule interactions and/or the presence of specific regulatory cytokines may also lead to t cell inactivation and deletion or generation of regulatory (i.e., suppressor) t cell subsets (gilliet and liu 2002; nossal 1994; piccirillo and thornton 2004; thompson and thomas 2002) . finally, at least some activated t and b cell clones persist as memory cells following the resolution of the immune response (dutton et al. 1998; schittek and rajewsky 1990) . the initial antigen presentation and lymphocyte activation events generally occur in the secondary immune tissues, the local lymph nodes and spleen. consequently, an effective lymphatic drainage is usually necessary for immune responses to develop in a particular tissue. however, since leukocytes recirculate through tissues, many of the important events, including antigen exposure and processing, antigen recognition by antigen-exposed lymphocytes, and lymphocyte maturation and proliferation, also occur at the reaction site itself (ascher et al. 1983; gruber 1992; pedersen and morris 1970) . moreover, while it is generally accepted that the adaptive immune system is activated by the presence of foreign antigens, there is an alternative activation model that combines elements of both the innate and adaptive immune systems. this is encapsulated by the 'danger hypothesis,' which proposes that apcs respond to substances that cause or signal damage, rather than to those that are simply foreign (matzinger 1994) . these danger signals include cd40l, the early proinflammatory cytokines il1 and tnf, ifns, and heat-shock proteins as well as substances that are normally found only inside cells (e.g., nucleotides, unmethylated cpg sequences in mammalian double-stranded dna) and hyaluron breakdown products (gallucci and matzinger 2001) . in this model, activation of the immune system occurs as a response to evidence of an extant threat, rather than toward a specific feature of the threat itself. this mechanism may contribute to the onset of certain autoimmune diseases. while an effective immune response is essential for protection of the host from a range of threats, the immune system also has its dark side. the capacity of the immune system for destructiveness is expressed in chronic inflammatory and autoimmune diseases, including those that affect the male reproductive tract (roper et al. 1998; schuppe and meinhardt 2005; schuppe et al. 2008; suominen 1995) . these examples of an immune system seemingly out of control highlight the crucial importance of effective immunoregulation. at the center of immunoregulation is the concept of tolerance, or the capacity of the immune system to ignore certain molecular patterns, and the systems that restrain the immune response once its usefulness has expired. tolerance is the ability of the immune system to discriminate self from nonself. it is fundamentally based on the removal, inactivation, or suppression of self-reactive lymphocyte subsets, so that the immune system becomes effectively incapable of recognizing and reacting to these antigens. induction of tolerance comprises both central and peripheral mechanisms. the central mechanism involves clonal deletion of t and b cell subsets that are potentially autoreactive, a process that normally occurs during gestation as part of the early maturation of the immune system (kappler et al. 1987; nossal 1994) . many details of this editing process remain incompletely understood, although the process involves broad expression by thymic medullary cells of self-antigens, including many endocrine tissue-specific antigens under the control of the transcription factor autoimmune regulator (aire) (liston et al. 2003 ). lymphocytes that recognize these self-antigens within the thymus during this process are targeted for destruction, effectively removing them from the lymphocyte repertoire. in addition, there are a number of complex mechanisms of peripheral tolerance, which involve the inactivation of antigen-specific lymphocytes in peripheral lymphoid organs by weak antigen stimulation in the absence of appropriate costimulation and the production of lymphocytes with immunoregulatory activities (gilliet and liu 2002; piccirillo and thornton 2004; thompson and thomas 2002) . peripheral immunoregulation involves several antigen-specific regulatory or suppressor t cell types, most notably the cd4 þ cd25 þ regulatory t cell (t reg ) subset. these cells selectively produce the immunosuppressive cytokines transforming growth factor (tgf) and il10, but also appear to act via direct contact with the cell being suppressed (nakamura et al. 2001; piccirillo et al. 2002) . other t cell subsets implicated in immunosuppression through their ability to produce tgf and il10 include th3 cells, tr1 (t regulatory 1) cells, and t cells, the latter being a minor t cell subset that is generally associated with epithelia (kuhnlein et al. 1994; macdonald 1998; seo et al. 2001) . the nkt cells, which are t cells with nk activity that possess unique restriction to glycolipid antigens presented by the mhc-like molecule cd1d, play a role in promoting graft survival and development of cd8 þ regulatory/suppressor t cells through production of il4 and il10 (godfrey et al. 2000; nakamura et al. 2003; sonoda et al. 2001) . the frontline nk cells are able to modulate adaptive immune responses by inducing dendritic cell death through contact-mediated lysis, but are also capable of producing ifn to stimulate the antigen-presenting activity of dendritic cells (laouar et al. 2005; raulet 2004) . it is the absence of tolerance that inhibits graft success in the very artificial condition of tissue transplantation. the immune cells of both the graft recipient and the donor tissue respond to one another, particularly with respect to polymorphic differences of the mhc proteins, leading to rejection of tissues that are not antigenically matched (gould and auchincloss 1999) . even under normal conditions, failure of preexisting tolerance may occur through a number of mechanisms, leading to autoimmune disease. somatic mutation of the antigen receptor expressed by a t or b cell may result in the creation of new self-reactive clones, subverting central tolerance (burrows et al. 2000) . autoimmunity may also occur when the inflammatory response to an infection damages or overwhelms normal mechanisms of self-tolerance, or where the infection involves organisms that express antigens that may cross-react with self-antigens (molecular mimicry) (merkler et al. 2006; regner and lambert 2001) . in addition to lymphocyte-mediated tolerance already discussed, the immune system possesses a repertoire of mechanisms that control and limit inflammatory and immune responses once they have commenced. inflammation triggers the production by the adrenal gland of corticosteroids, which inhibit the production of proinflammatory cytokines and other immune mediators, reduce the expression of leukocyte adhesion molecules, and induce lymphocyte apoptosis (buckingham et al. 1996; kapcala et al. 1995) . during inflammation, immune cells themselves produce anti-inflammatory and immunosuppressive molecules, including prostaglandins d and j and the lipoxins (herlong and scott 2006; levy et al. 2001) . moreover, activated lymphocytes have a limited lifespan, undergoing a process of activation-induced cell death following upregulation of the extrinsic apoptotic signal mediated by interaction of the fas receptor (cd95) with its ligand (fas ligand: fasl or cd95l), eventually leaving behind a relatively small number of long-lived memory cells (dutton et al. 1998; schittek and rajewsky 1990) . likewise, there are mechanisms to inhibit antibody activity and promote antibody clearance (isaacs 1990 ). classically, the term immune privilege applies to organs or tissues where survival of foreign grafts may be prolonged (barker and billingham 1977) , but more accurately, the term denotes tissues where immune responses are inhibited or suppressed (hedger 2007) . privileged tissues include the brain, eye, pregnant uterus, and the testis. initially, privilege was attributed to deficient lymphatic drainage in such tissues, or physical barriers that prevented immune cells from gaining access (barker and billingham 1977) . several privileged tissues do possess highly specialized epithelial or endothelial cell barriers that may sequester antigens away from the normal immune surveillance: the best characterized of these are the blood-brain barrier and the trophoblast in the pregnant uterus (hunt 2006; streilein 1993) . however, physical exclusion is now considered an overly simplistic explanation for immune privilege, given that lymphocyte and antibody responses to introduced antigens occur even in sites where grafts can survive for extended periods (head et al. 1983b; kaplan and streilein 1978; tafuri et al. 1995) . although barrier mechanisms and altered lymphatics may be contributory factors in some tissues, immune privilege is a consequence of tissue-specific specialized systems for controlling, subverting, or preventing local inflammatory or immune activation responses (hedger 2007) . the male reproductive tract comprises the paired testes, associated epididymis and vas deferentia, accessory glands (chiefly the prostate and seminal vesicles), and the urethra. these tissues represent a diversity of structures and, consequently, the immunology of each tissue retains distinctive features (hedger and hales 2006) . best studied in this context is the testis, which comprises two discrete tissue compartments, the seminiferous tubules and the interstitial tissue. the interstitial tissue contains the blood supply and lymphatics, as well as the innervation of the testis (fawcett et al. 1973; setchell et al. 1994) . the epithelium of the seminiferous tubules is entirely avascular. within the seminiferous epithelium, spermatogenesis is supported and maintained by the sertoli cells, which remain in physical contact with the developing germ cells at all times. during spermatogenesis, a stem cell spermatogonium sitting on the basement membrane of the seminiferous tubule becomes committed to a series of mitotic divisions, producing a cohort of spermatocytes. the spermatocytes subsequently move toward the tubule lumen, passing through tight junctions between adjacent sertoli cells, into the highly specialized environment of the adluminal compartment (wang and cheng 2007) . within this compartment, the spermatocytes divide meiotically to produce haploid spermatids and then undergo differentiation into spermatozoa, which are ultimately released into the lumen of the tubule leaving behind the majority of their cytoplasm to be digested by the sertoli cells as residual bodies. the released spermatozoa are swept by fluid secreted from the sertoli cells toward the rete testis, a collecting structure lined by a simple epithelium that is connected to the adjacent epididymis by a series of efferent ducts. sperm mature and are stored in the epididymis, until they are either released through the vas deferentia at the time of ejaculation or removed by epididymal phagocytes (barratt and cohen 1987; roussel et al. 1967) . the process of spermatogenesis is highly organized, with multiple generations of developing germ cells in each region of the epithelium maintaining distinct, progressing cellular associations or stages, which comprise the cycle of the seminiferous epithelium (14 stages in the rat, 12 stages in the mouse, 6 stages in man). these stages are arranged in sequence along the length of each seminiferous tubule, creating waves of coordinated spermatogenic development (clermont 1972) . spermatogenesis is maintained and controlled by follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells in the interstitial tissue under the influence of luteinizing hormone (lh). importantly, fsh and testosterone act upon sertoli cells and not directly on the germ cells (mclachlan et al. 1996) . these hormones determine the efficiency of the spermatogenic process largely by controlling germ cell survival, that is, the balance between differentiation and apoptosis (matthiesson et al. 2006; ruwanpura et al. 2008) . in order to maintain the cycle of the seminiferous epithelium, the developing germ cells and sertoli cells are engaged in continuous intercellular communication, although the precise nature of this communication remains very poorly understood. the testis, and spermatogenesis in particular, presents a unique challenge for the immune system. although there are reports of direct drainage of some lymphatic vessels to the thoracic duct, there is no evidence of any structural or functional deficiency in the efferent lymphatics of the testis, and allogeneic cells injected into the rat testis induce typical immune responses in the draining lymph nodes (fawcett et al. 1973; head et al. 1983b; itoh et al. 1998a; moller 1980) . nonetheless, the vast majority of spermatogenic differentiation occurs long after central tolerance is established and, while thymic expression of some testicular antigens is under the control of the aire transcription factor, it is obvious from clinical and experimental studies that germ cells express multiple autoreactive antigens and are highly immunogenic when brought into contact with the immune system (itoh et al. 1991; tung 1975) . surprisingly, this is a problem for a relatively small cohort of men only, and autoimmune orchitis or epididymitis is rarely reported in humans, except in the context of local infection (krieger 1984) . even allowing for the possibility that most cases of spontaneous autoimmune reactions against the developing germ cells are never diagnosed, since they would occur early in reproductive life, and would simply present as spermatogenic failure during infertility work-ups (schuppe and meinhardt 2005; schuppe et al. 2008) , why are such reactions not more common? it is widely believed that the blood-testis barrier plays a role in protecting testicular antigens from the immune system. however, as a result of a common misunderstanding of the nature of this vitally important structure, its actual role is usually overestimated. critically, the blood-testis barrier involves highly specialized occluding or tight junctions between adjacent sertoli cells, which separate the spermatogonial and early meiotic cells from the majority of meiotic and postmeiotic germ cells (dym and fawcett 1970; setchell et al. 1969) . the structure and functions of the barrier do not need to be discussed in detail here, as this topic is covered in much more detail in other chapters in this volume (see chapter 11.05). by preventing the passage of most molecules between adjacent sertoli cells, the bloodtestis barrier allows the creation of a highly specialized biochemical environment essential for meiotic development (cheng and mruk 2002; griswold 1988) . it also blocks access by lymphocytes, complement, and antibody (ben et al. 1986; yule et al. 1988) . by contrast, the vascular endothelium and peritubular cells surrounding the tubules play a negligible role in maintaining the blood-testis barrier in practical terms, and immune cells and proteins have relatively free access to the interstitium and basal seminiferous epithelium (mahi-brown et al. 1988; yule et al. 1988 ). thus, this barrier is quite different in properties and function from the blood-brain barrier, located on the vascular endothelium, or the trophoblastic barrier of pregnancy, which entirely segregates the developing fetal tissues from the maternal host. a reinterpretation of the blood-testis barrier as a series of cellular elements that protects the germ cells from harmful influences has been proposed more recently (bart et al. 2002) . in this model, the bloodtestis barrier consists not only of the structural elements already discussed, but also the efflux pump barrier system involving the drug transport proteins p-glycoprotein and the multidrug resistance-associated protein-1 expressed on the capillary endothelium, peritubular myoid cells, and the basal aspect of the sertoli cells (bart et al. 2004; melaine et al. 2002) . while these elements may certainly be important for protection of the testis against toxic agents, their contribution to immunoregulation is probably marginal. several lines of evidence indicate that the bloodtestis barrier cannot account for all manifestations of immune privilege in the testis. the barrier does not prevent exposure of germ cell antigens to the immune system, as antibodies and lymphocytes specific for spermatogenic antigens appear to be a normal feature of the circulating immune repertoire even under normal conditions (turek and lipshultz 1994) . spermatogenic cell autoantigens are not confined behind the sertoli cell tight junctions, and are expressed by spermatogonia and the early spermatocytes (mahi1988; yule et al. 1988 ). moreover, the barrier does not extend into the rete testis or beyond. significantly, orchitis can be passively transferred to naive mice using lymphocytes obtained from mice with active autoimmune orchitis, with the initial reaction concentrated in the interstitial tissue around the rete testis tung et al. 1987) . a similar initial pattern of development of orchitis within the rete testis region has been observed in mice actively immunized with viable germ cells (itoh et al. 1995a ). in many seasonally breeding species, annual regression of the bloodtestis barrier occurs without inducing overt inflammation or autoimmunity (pelletier 1986; tung et al. 1981) . finally, the blood-testis barrier cannot explain the enhanced survival of grafts within the interstitial tissue (i.e., outside the barrier) and does not explain survival of autoantigenic sperm in the epididymis, or remainder of the male reproductive tract. however, while the blood-testis barrier does not explain immune privilege in the testis, the breach of the tight junctions comprising the barrier remains a critical event in the development of any autoimmune destruction of the seminiferous epithelium during orchitis (mahibrown and tung 1989; mahi-brown et al. 1987) . recognition of antigen in association with mhc is essential to normal adaptive immune responses, and reduced expression of mhc class i (hla-a, hla-b, and hla-c in humans) and class ii proteins (hla-d) appears to be an important feature of immune-privileged tissues and tumors (garrido et al. 1997; haas et al. 1988; head and billingham 1985; wekerle et al. 1987) . reduction in class i and ii expression reduces the likelihood of immune-activating events involving cd4 þ helper t cells and cytotoxic cd8 þ t cells in the tissues. studies indicate a characteristic absence of expression of mhc proteins on the cells of the seminiferous epithelium under normal conditions (haas et al. 1988; head and billingham 1985; lustig et al. 1993; tung et al. 1987) , although both mhc classes are expressed in human spermatozoa (martín-villa et al. 1996) . there is evidence from studies on mouse sertoli cells in culture that mhc class ii expression can be upregulated in these cells by ifn (dal secco et al. 2008) . in contrast to the seminiferous epithelium, both mhc class i and ii proteins are expressed throughout the testicular interstitial tissue: mhc class i is expressed on most interstitial cells, including the leydig cells (haas et al. 1988; pöllänen and maddocks 1988) , while testicular macrophages and dendritic cells express mhc class ii (haas et al. 1988; head and billingham 1985; hedger and eddy 1987; mahi-brown et al. 1987; tung et al. 1987) . thus, it appears unlikely that a lack of apcs is a contributing factor in testicular immune privilege, although differences in the number and distribution of mhc class ii-expressing cells in the interstitial tissue of different species or strains may help to explain differences in susceptibility to autoimmune orchitis (flickinger et al. 1990b; kojima and spencer 1983; teuscher et al. 1987) . the fact that the testis is not isolated from the immune system, yet can tolerate both endogenous (auto-) antigens associated with spermatogenesis and exogenous (allo-or xeno-) antigens expressed by grafts, is evidence of tissue-specific inhibition of the immune response. the question then arises: if immune responses against exogenous and endogenous antigens are inhibited in the testis environment, does the testis display a reduced capacity to deal with infections and greater susceptibility to certain kinds of tumors? there is evidence that immunity may be compromised in some situations, for example, the tendency toward relapsing acute lymphocytic leukemia in the testes (hudson et al. 1985) , and several systemic viral and mycobacterial infections target the testis, including mumps, human immunodeficiency virus, tuberculosis, and the severe acute respiratory syndrome virus (krieger 1984; nistal et al. 1986; xu et al. 2006) . however, the testis does not appear to be immunodeficient. even in comparison with the rest of the male reproductive tract, orchitis due to ascending infections is relatively rare (krieger 1984; ness et al. 1997) . moreover, the extremely variable level of success of experiments by different groups using different transplant models has shown that immune privilege of the testis is both limited and conditional (dobrinski 2005; head et al. 1983a; maddocks and setchell 1988; selawry and whittington 1984; setchell et al. 1995) . just what are these necessary conditions, however, remains poorly defined. studies indicate that the effector arm of the immune response is intact within the testis, and it is the ability to recognize and react toward foreign antigens and/or mount a normal inflammatory response that is suppressed (head and billingham 1985; head et al. 1983a; mahi-brown and tung 1989; mahi-brown et al. 1987) . in summary, there appears to be a potentially unique functional interaction between the testis and the immune system, which involves control of the mechanisms normally involved in immune activation, in order to limit adaptive immune responses without drastically compromising the ability of the testis to protect itself when required. although their presence and influence is almost universally neglected, macrophages, lymphocytes, and pmns have varying degrees of access to the interstitial tissue of the testis (table 1) . they are generally excluded from the seminiferous epithelium under normal conditions. over the past 10 years, studies have started to fill in some of the details concerning these cells. macrophages are by far the most prominent immune cells in the testis. they are found in the testes of all mammalian species so far examined, but are particularly numerous in human, rat, and mouse testes (frungieri et al. 2002; hume et al. 1984; mendis-handagama et al. 1987; miller et al. 1984; vergouwen et al. 1993; wang et al. 1994) . typically, macrophages arise from myeloid precursors in the bone marrow and circulate in the blood as monocytes. they are capable of adopting a broad range of structural and functional phenotypes that are largely dictated by the tissues in which they become resident (mantovani et al. 2005; van furth 1988) . characteristically, they are robustly phagocytic and possess a potent arsenal of antimicrobial and cytotoxic activities, such as the ability to generate large amounts of ros. they produce many immunoregulatory products, including cytokines, eicosanoids (prostaglandins, thromboxanes, leukotrienes, and lipoxins), and nitric oxide, all of which are important in establishing and controlling the inflammatory process, but also have important roles in tissue remodeling, healing, and normal homeostasis. all macrophage lineages can express mhc class ii antigens and have the capacity to present to cd4 þ helper or t reg cells (unanue 1984) . thus, macrophages are the cells that link innate and adaptive immunity, and dendritic cells are a highly specialized macrophage subset that is particularly effective in this role (heath and carbone 2001) . testicular macrophages have been most extensively studied in the rat, with a few studies also in the mouse and human. studies in the rat indicate that they are heterogenous in their morphology and functions (bryniarski et al. 2004; gerdprasert et al. 2002a; itoh et al. 1995b; wang et al. 1994) . their absolute numbers are primarily under leydig cell control, but evidence suggests that fsh acting via the sertoli cell has a role in regulating their functional activity (duckett et al. 1997a,b; gaytan et al. 1994c; wang et al. 1994) . macrophages accumulate within the interstitial tissue at the time of puberty, and play critical roles in the normal development of the testis, particularly by promoting the proliferation and maturation of the leydig cell population (cohen et al. 1997; gaytan et al. 1994a,b; hardy et al. 1989; itoh et al. 1999; raburn et al. 1993; vergouwen et al. 1993) . through their ability to produce a broad array of growth factors, prostanoids, and vasoactive regulators, other developmental and homeostatic roles within the testis may be predicted as well. data concerning the immunological functions of testicular macrophages are based on a relatively small number of studies in either the rat or the mouse, and are patchy at best. indeed, there is some evidence for distinct differences even between these two closely related species. in general, testicular macrophages have intact phagocytic, cytotoxic, and antimicrobial functions (miller et al. 1984; wei et al. 1988) . rat testis macrophages express mhc class ii antigens (head and billingham 1985; hedger and eddy 1987; wang et al. 1994 ), but display a reduced capacity for lymphocyte activation in vitro . testicular macrophages in the human and mouse also express mhc class ii, although expression in the mouse appears to be more restricted (haas et al. 1988; itoh et al. 1995b; pöllänen and niemi 1987; tung et al. 1987) . a lack of expression of the essential costimulatory molecules, b7-1 (cd80) and b7-2 (cd86), has been reported in the mouse testis as well (sainio-pöllänen et al. 1996) . studies also indicate that macrophages from the rat testis have a significantly reduced ability to produce proinflammatory cytokines, such as il1 and tnf, following activation (gerdprasert et al. 2002a; hayes et al. 1996; meinhardt et al. 1996) , and stimulated mouse testicular macrophages produce the anti-inflammatory/immunosuppressive cytokines il10 and tgf 1 in vitro (bryniarski et al. 2004 (bryniarski et al. , 2005 . while the data suggest that testicular macrophages may have a predominantly anti-inflammatory and/or immunosuppressive phenotype, this has yet to be formally established. furthermore, dendritic cells, which are a highly specialized macrophage subset also found within the testicular interstitium (fijak et al. 2005; hoek et al. 1997; itoh et al. 1995b; rival et al. 2006a) , presumably play a more important role in controlling immune responses to antigens within the testis (fijak et al. 2005; rival et al. 2006a rival et al. , 2007 . in the rat testis, macrophages can be discriminated by their differential expression of two molecular markers recognized by the antibodies ed1 and ed2. antibody ed1 recognizes the lysosomal protein cd68, which is associated with antigen processing and presentation (groisman et al. 2002) , while ed2 recognizes the scavenger receptor cd163 (fabriek et al. 2005) . cd163 is involved in the clearance of hemoglobin:haptoglobin complexes and the resolution of inflammation (philippidis et al. 2004) . about 80-85% of rat testicular macrophages express this scavenger receptor and about half of these cells also lack expression of cd68 (gerdprasert et al. 2002a; meinhardt et al. 1998; wang et al. 1994 ). in the human testis, most macrophages appear to be cd68 þ , but expression of cd163 is a feature of a subset of these cells as well (frungieri et al. 2002) . the heterogeneous expression of these two markers points to the existence of multiple populations of testicular macrophages, and evidence suggests that these phenotypic differences also correspond to functional differences. in the normal and inflamed adult rat testis, cd163 þ cells lack expression of the key inflammatory mediators il1 and inducible nitric oxide synthase (inos), as measured by double-label immunohistochemistry, but these molecules are detectable in cd68 þ cells that do not express cd163 (gerdprasert et al. 2002a; o'bryan et al. 2005) . indeed, purification of cd163 þ macrophages from the rat testis using immunomagnetic beads confirms that these cells have reduced capacity to produce il1, il6, or no upon stimulation, although they retain the ability to produce prostaglandins (gerdprasert et al. 2002a; hedger, unpublished data) . this suggests that the cd163 þ macrophages have reduced proinflammatory activities, while the cd163-negative testicular macrophage population retains normal proinflammatory functions. this observation is important because macrophages that express cd163 and produce il10, but display reduced expression of cd80, cd86, inos, tnf, and il1, are polarized toward the immunoregulatory m2 subset (mantovani et al. 2005; sica et al. 2008) . the m2 macrophage subset is analogous to the th2 subset among t cells, and is associated with late-stage inflammation and its resolution, as well as tumors and other immunologically privileged tissue sites. it would appear that a significant proportion of the resident macrophages of the testis possess an immunoregulatory phenotype that is consistent with immune privilege. the functional roles of this macrophage population, as well as those of the macrophages that appear to retain normal inflammatory (i.e., m1 subset) properties, demand particular attention if we are to understand the unique immunological environment of the testis. while the functions and regulation of lymphocytes within the testis have yet to be properly characterized, by their presence alone these cells will have significant effects on testicular events. it may also be assumed that, in contrast to the largely resident population of testicular macrophages, lymphocytes circulate through the testis. this may be because they possess specificity for testicular antigens, or because they have been activated during earlier intratesticular inflammatory or infectious events (czerkinsky et al. 1999; springer 1994) . quantitative studies in the rat indicate that, at any given time, lymphocytes are approximately 10% as numerous as the macrophages in the normal testis (hedger and meinhardt 2000; hedger et al. 1998b; wang et al. 1994 ). in the rat and mouse, intratesticular lymphocytes express t cell and nk cell markers, and comprise distinct t cell, nk cell, and nkt cell subsets (tompkins et al. 1998; hedger, unpublished data) . both t cell and nk cell markers are expressed by lymphocytes in the human testis as well (hedger, unpublished data) . b cells are not usually observed in normal tissues, and this includes the testis. in the rat, many t cells possess a memory phenotype, as would be expected (hedger et al. 1998b; tompkins et al. 1998 ). reanalysis of the data for individual animals across several studies suggests that their numbers tend to be proportional to the numbers of macrophages, but gradually increase with the age of the animal (hedger et al. 1998b; hedger and meinhardt 2000; wang et al. 1994) . among the t cells of the testis, there is a strong bias toward the cd8 þ (i.e., mhc class i restricted, largely cytotoxic) subset (hedger et al. 1998b; pöllänen and niemi 1987; ritchie et al. 1984; tompkins et al. 1998; wang et al. 1994) . the prominence of nk cell subsets in the testis is also intriguing given the reduced mhc class i antigen expression in the seminiferous compartment, which would tend to make these cells more susceptible to nk cells (lanier 2001) . consistent with the anti-inflammatory/immunosuppressive phenotype of the major testicular macrophage subset, t cells and nkt cells in the rat testis constitutively produce il10 (hedger, unpublished data) . while much remains to be discovered, the existing data concerning the types and activity of testicular lymphocytes are consistent with maintaining immune privilege (i.e., the presence of immunoregulatory lymphocytes) and increased innate immunity (i.e., cytotoxic cell activity). evidence that pmns are important in testicular function comes from the fact that mast cells and eosinophils are significant elements of the interstitial tissue in many species. in the rat, mouse, dog, cat, bull, and deer, both cell types are largely absent from the testicular parenchyma, but are associated with blood vessels in the testicular capsule (anton et al. 1998) . mast cells are found throughout the interstitial tissue in equine and human testes (anton et al. 1998; nistal et al. 1984; yamanaka et al. 2000) , and both mast cells and eosinophils are present in porcine species (anton et al. 1998) . the role of these cells in vascular control and innate immunity can be predicted, but they may also play immunoregulatory roles, as mast cells do in other tissues . in humans, testicular mast cell numbers are increased in infertility (meineke et al. 2000; nagai et al. 1992; yamanaka et al. 2000) , but decline with advancing age (nistal et al. 1984) . the possibility that they accumulate due to past immune events needs to be considered, but they also appear to be actively regulated. in the adult rat testis, mast cell proliferation is under the control of the leydig cells, suggesting that these cells produce an inhibitor of mast cell activity (gaytan et al. 1990; wang et al. 1994) . neonatal estrogen treatment increases mast cell numbers in the rat testis (gaytan et al. 1989 (gaytan et al. , 1990 , and a relationship with elevated leydig cell aromatase activity is suggested by the prevalence of these cells in boar and equine testes (eisenhauer et al. 1994; raeside and renaud 1983) . neutrophils are found in the testis only under conditions of testicular inflammation or damage (gerdprasert et al. 2002a; kohno et al. 1983a; o'bryan et al. 2000b ). the majority of this chapter deals with the testis because it is the primary source of male gametes and hormones, and damage to this organ tends to have more significant consequences for fertility. the remainder of the male reproductive tract comprises the epididymis, vas deferens, accessory glands, and the urethra, which are all epithelial-lined ductal tissues of varying complexity. the immunology of these tissues appears to be quite distinct from that of the testis. while it is true that sperm spend relatively little time within most of these tissues, mature sperm reside within the epididymis for extended periods without provoking an overt immune response, and inflammation and immune cell activity can nonetheless have damaging effects in this organ. significantly, these tissues possess normal interepithelial cell junctions, but lack a completely occluding intercellular structure analogous to the blood-testis barrier (levy and robaire 1999; pelletier 2001) . secreted iga, which lacks the ability to bind complement and possesses principally antiinflammatory properties as a result, plays an important role in the male urogenital tract (clifton et al. 1992; politch et al. 2007; pudney and anderson 1995) , and the immunology of these organs tends to more closely resemble that of other elements of the mucosal immune system, such as the gastrointestinal and respiratory tracts (beagley et al. 1998; clifton et al. 1992; mestecky et al. 2005) . in the absence of the specialized mucosal-associated lymphoepithelial tissue normally found in the gastrointestinal and respiratory tracts, it is likely that the epithelial cells themselves, together with the numerous intraepithelial and stromal macrophages and lymphocytes that are normally found within these tissues, mediate local immunoregulatory mechanisms (barratt and cohen 1987; dym and romrell 1975; el-demiry et al. 1985; ritchie et al. 1984; pudney and anderson 1995; theyer et al. 1992; yeung et al. 1994) . a better understanding of the immunology of these tissues is important. inflammation, infection, and physical damage can lead to immune reactions against the sperm, which in turn may compromise fertility. moreover, many men suffer from chronic pelvic pain, generally manifesting as epididymitis or prostatitis in the absence of a detectable infection, a condition that almost certainly involves chronic inflammatory processes within the male reproductive tract. as outlined in the preceding sections, the testis is able to support immune privilege without sacrificing immune surveillance and protection. the mechanisms responsible for this immunological balancing act are still far from understood, but a number of inflammatory and immunoregulatory systems that operate within the testis have been investigated in varying degrees of detail. it is perhaps not very surprising that many of these regulatory systems also impact upon normal testis physiology, in addition to their immunological and pathological roles. given that the types and properties of leukocytes found within the testis environment are consistent with immunoregulation and enhanced innate immunity, the focus should be on which testicular elements are involved in creating this potentially unique immunological environment. most attention in this regard has been directed toward the highly versatile sertoli cell. evidence that the sertoli cell possesses specialized immunoregulatory properties comes from cotransplantation studies. sertoli cells from immature rat, murine, or porcine testes display extended survival as allografts or xenografts, and cotransplantation of sertoli cells or testis cell mixtures containing these cells confers increased survival on neural cell xenografts, and pancreatic islet allografts and xenografts (sanberg et al. 1996; selawry and cameron 1993; suarez-pinzon et al. 2000) . the ability to form a physical barrier through inter-sertoli tight junctions and the fact that sertoli cells have reduced mhc expression (haas et al. 1988; kohno et al. 1983b; sanberg et al. 1996; turek et al. 1996) undoubtedly enhance their potential to avoid t cell activation both in the intact testis and in engraftment studies. moreover, a soluble form of the nonclassical mhc class ib molecule hla-g is produced by the sertoli cells, as well as spermatocytes, spermatids, and some interstitial cells, in the rhesus monkey testis (ryan et al. 2002) . this molecule has been implicated in apoptosis of alloreactive cd8 þ cytotoxic t cells (fournel et al. 2000) . sertoli cells display lymphocytotoxic activities and produce molecules with immunosuppressive activity, such as tgf1 (de cesaris et al. 1992; suarez-pinzon et al. 2000; wyatt et al. 1988; yin et al. 2006) and fasl (bellgrau et al. 1995; yin et al. 2006 ). an active immunoregulatory role has also been suggested: murine sertoli cells treated with ifn increase expression of mhc class ii molecules and the negative costimulatory ligand b7-h1, and stimulate t reg numbers in culture (dal secco et al. 2008) . the ability of the sertoli cell to suppress other critical inflammatory events through expression of complement regulatory proteins ) and secretion of inhibitors of lymphocyte cytotoxic activities (sipione et al. 2006) is also likely to be important. finally, sertoli cells possess an enormous capacity for phagocytosis of senescent cells, cell debris, and other potentially antigenic complexes, which could otherwise activate an immune response. data concerning immunoregulatory roles of other testicular cells are less comprehensive. the evidence clearly indicates a role for leydig cells in recruiting macrophages into the testis, although testicular macrophage function may actually be determined by the sertoli cells (duckett et al. 1997a,b; gaytan et al. 1994c; wang et al. 1994) . leydig cells also produce various steroids, proteins, and other factors with immunosuppressive activity, and inhibit lymphocyte activity in vitro (born and wekerle 1982; hedger et al. 1990 ). peritubular cells, like sertoli cells, produce immunoregulatory tgf family members (konrad et al. 2000; mullaney and skinner 1993; okuma et al. 2005b) . the possibility that the germ cells themselves have a direct effect on immune cells has also been considered (hurtenbach and shearer 1982) , although it seems more likely that germ cells may influence immune responses indirectly by inducing immunoregulatory activities of the sertoli cells and other somatic cells. the relatively recent discovery of the tlrs has radically expanded our understanding of innate immunity and promises to do the same for testicular immunology. in addition to their predictable expression on testicular macrophages and dendritic cells, tlr mrna, protein, and/or ligand responses have been described in several other testicular cells. rat and mouse sertoli cells express tlr2, 3, 4, and 5 (bhushan et al. 2008; riccioli et al. 2006; starace et al. 2008; wu et al. 2008) . tlr1, 10, and 11 mrna expression has been observed in rat sertoli cells (bhushan et al. 2008) , and low levels of tlr6, 7, and 13 mrna have been detected in mouse sertoli cells (riccioli et al. 2006; wu et al. 2008 ). in the rat, germ cells also produce mrna for tlr2, 3, and 4 in a stage-specific manner, and the peritubular cells express tlr3 and 11, along with low levels of tlr2, 4, and 6 (bhushan et al. 2008) . rat leydig cells express tlr2 mrna and low levels of tlr10 (bhushan et al. 2008) . however, the cytoplasmic receptors tlr8 and 9 have yet to be observed in any nonmyeloid testicular cell type. of the other pattern-recognition receptors, nod1 mrna has been detected in rat sertoli cells, peritubular cells, and spermatogonia, while nod2 was detected in spermatogonia only (bhushan et al. 2008) . expression of tlrs and other pattern-recognition receptors in the testis is obviously important for responses toward intratesticular pathogens. infections and inflammation, both systemic and localized, have negative effects on testicular function, resulting in reduced androgen production, lowered sperm counts, and temporary loss of fertility (adamopoulos et al. 1978; baker 1998; carlsen et al. 2003) . presumably, tlrs expressed by the sertoli cells and germ cells are necessary to detect pathogens that ascend the reproductive tract and, as a consequence, lie behind the blood-testis barrier. this expression within the seminiferous epithelium also suggests a mechanism whereby inflammation can directly inhibit spermatogenesis. activation of sertoli cells by tlr ligands induces production of inflammatory mediators, such as il1, il6, and no, which have direct effects on germ cell development and leydig cell steroidogenesis (gérard et al. 1992; stéphan et al. 1995 stéphan et al. , 1997 o'bryan and hedger 2008; riccioli et al. 2006; wu et al. 2008) . moreover, while inhibition of leydig cell steroidogenesis during inflammation may involve indirect effects at the level of the pituitary, or the actions of inflammatory mediators produced by the testicular macrophages and sertoli cells, there is evidence that these cells can also respond directly to tlr ligands (bhushan et al. 2008; xiong and hales 1993a) . although the tlrs have evolved to recognize pathogen-derived molecules, such as lps (tlr4) and bacterial lipoproteins (tlr2), recent data show that they can also be activated by certain endogenous molecules. the ability of preparations of some endogenous molecules, such as heat-shock proteins, to activate tlr signaling is now attributed to contamination of these preparations by bacterial products (tsan and gao 2004) , but there is convincing evidence that mammalian mrna is a ligand for tlr3 (karikó et al. 2004 ) and mammalian cpg dna can activate tlr9 (yasuda et al. 2005) . the high mobility group box chromosomal protein 1 (hmgb1), produced by both spermatogonia and sertoli cells, is a tlr2 and 4 ligand (yu et al. 2006; zetterström et al. 2006) . expression of tlrs by the sertoli cells under normal conditions, therefore, suggests a potential role for these receptors in responding to these endogenous ligands. 11.10.4.2.2 the interleukin 1 family il1 is the best studied of all the cytokines normally expressed in the testis. it is produced in two forms, and , which are encoded by separate genes and share approximately 25% sequence homology. both forms act through the same receptor complex (il1r1), and exert a similar range of proinflammatory and physiological effects (dinarello 1996; o'neill and dinarello 2000) . most il1 signaling occurs through activation of the myd88/traf and jnk/p38 map kinase pathways, thereby regulating many proinflammatory genes through stimulation of the transcription factors nfb and activated protein 1 (ap1) (medzhitov et al. 1998) . the il1s are synthesized as precursor proteins, which are cleaved to produce 17 kda active proteins. in the case of il1, the precursor possesses low-level biological activity, but the il1 precursor protein is inactive (black et al. 1988; watanabe and kobayashi 1994) . the precursor of il1 is cleaved by the il1 converting enzyme (ice, caspase 1) during the process of secretion, whereas il1 is cleaved by the calcium-dependent membrane-associated cysteine protease (calpain) or by extracellular proteases (thornberry and molineaux 1995; watanabe and kobayashi 1994) . il1 is produced by activated monocyte/macrophages and is the main secreted form during inflammation, whereas il1 is more commonly found within the cell, where it may act as an autocrine or paracrine growth factor involved in direct cell-to-cell communication. in addition to the prototypical il1 and il1, the il1 family comprises a number of structurally related proteins, which appear to have arisen by gene duplication (dunn et al. 2001) . the closest structurally to il1/ is il18, which is processed by caspase 1 activity, but acts via a separate receptor rather than il1r1 (gracie et al. 2003) . another member of the family, which does bind to il1r1 but lacks the ability to transduce a signal, blocks il1/ action and is called il1 receptor antagonist (il1ra) (arend 1991) . production of il1 is constitutively high in the normal testis, as il1 is produced by the sertoli cells in a cyclical manner within the mature seminiferous epithelium (jonsson et al. 1999; söder et al. 1991) . its production is driven by the developing germ cells and by phagocytosis of the residual cytoplasm cast off by the spermatids at the time of their release into the tubule lumen (gérard et al. 1992; syed et al. 1995) . sertoli cell il1 production is also stimulated by lps, but not by fsh (stéphan et al. 1997) . production of il1 by the normal testis is low, but is upregulated in a subset of testicular macrophages and the leydig cells during inflammation (gérard et al. 1991; jonsson et al. 1999; o'bryan et al. 2005) . curiously, the majority of this secreted testicular il1 exists as the precursor (o'bryan et al. 2005) . the receptor il1r1 has been localized to germ cells and sertoli cells (gomez et al. 1997) . il1 stimulates dna synthesis in spermatogonia (i.e., mitosis) and early spermatocytes (i.e., meiosis), proliferation of immature sertoli cells, and a number of activities of the mature sertoli cell that support spermatogenesis, such as the production of lactate and transferrin (hakovirta et al. 1993b; hoeben et al. 1996; nehar et al. 1998; parvinen et al. 1991; pöllänen et al. 1989; söder et al. 1991) . it also regulates the ability of the sertoli cell to maintain contact with other sertoli cells and the developing germ cells, by altering the sertoli cell cytoskeleton (sarkar et al. 2008 ). in the interstitium, both il1 and il1 suppress lh-stimulated androgen production by adult leydig cells through inhibition of steroidogenic enzyme activity (hales 1992; hales 1994, 1997) . il1ra is produced by the sertoli cell, and is stimulated by fsh, il1, and lps (zeyse et al. 2000) . presumably, its role is to regulate the activity of il1/ within the testis. both il18 and its receptor have been found in the seminiferous epithelium, with il18 mrna and protein localized to spermatocytes and round spermatids (strand et al. 2005) . as is the case for il1, the majority of il18 produced within the testis is in the precursor form, indicating that the processing of these cytokines by caspase 1 in the testis is an area requiring more investigation. il18 stimulates spermatogonial dna synthesis in cultures of rat seminiferous tubules, without influencing germ cell apoptosis (strand et al. 2005) . another member of the family localized to the testis is il1f8, although its function there is entirely unknown (kumar et al. 2000) . .10.4.2.3 tumor necrosis factor and fas ligand tnf and fasl are transmembrane and secreted cytokines with typically trimeric structures and they interact with specific receptors to mediate a cell death signal (ju et al. 1995; schütze et al. 2008) . they are implicated in both immunoregulation and the control of normal spermatogenic function in the testis. tnf is a 17 kda glycosylated polypeptide and is a major early product of activated monocytes and macrophages. it binds to either of two tnf receptor subsets (tnfr1 and tnfr2) and plays a central role in initiating the inflammatory response by stimulating the production of il1 and il6 (basak and hoffmann 2008; bradley 2008) . whether tnf exerts proinflammatory or cytotoxic effects depends on the receptor subtype engaged and the expression of specific adaptor proteins within the target cell (basak and hoffmann 2008; chung et al. 2002; hsu et al. 1996; mak and yeh 2002) . consistent with its name, tnf exerts a cell death signal via tnfr1, through interaction with the tnfr-associated death domain protein (tradd) or the fas-associated death domain protein (fadd), and activation of the caspase-dependent apoptotic pathway (schütze et al. 2008) . however, interaction with tradd can also result in recruitment of other adaptor proteins leading to the binding of traf2 and activation of the nfb pathway and/or the map kinases jnk or p38 (chung et al. 2002) . moreover, tnfr2 and its related receptors, which include cd40, do not contain a death domain, and instead associate with the trafs leading to activation of cell signaling events (bradley 2008; mak and yeh 2002) . in the mouse testis, tnf is expressed by round spermatids, pachytene spermatocytes, and testicular macrophages, and mrna for tnfr1 has been located on both sertoli and leydig cells . in porcine sertoli cells, tnf receptor subunit protein expression is stimulated by fsh (mauduit et al. 1996) . there is no evidence that tnf is produced by the sertoli cell, but expression of tnf by the germ cells within the seminiferous epithelium, like that of il1 and il6, is cyclical. within the seminiferous epithelium, tnf produced by the germ cells appears to play a complex role in the control of both sertoli cell function and spermatogenesis. tnf reduces spontaneous germ cell degeneration in cultured human and rat seminiferous tubules, suggesting a germ cell survival effect presumably mediated through the sertoli cell (pentikäinen et al. 2001; suominen et al. 2004 ). conversely, tnf disrupts sertoli cell tight junction assembly by inhibiting the production of junction proteins and by regulating matrix metalloprotease and protease inhibitor activity (siu et al. 2003) . tnf also stimulates plasminogen activator inhibitor expression in rat testicular peritubular cells (le magueresse-battistoni et al. 1997) . similar to il1, tnf stimulates basal lactate production by cultured sertoli cells, but tnf generally antagonizes the actions of fsh on sertoli cell function, including the stimulation of aromatase activity and lactate production (mauduit et al. 1993; nehar et al. 1997; riera et al. 2001 ). however, delfino et al. (2003) have shown that tnf stimulates androgen receptor expression in sertoli cells via upregulation of nfb, which binds to several enhancer motifs in the androgen receptor promoter. tnf also stimulates the expression of inflammatory cytokines, chemokines, and leukocyte adhesion molecules in both sertoli cells and peritubular cells (aubry et al. 2000; de cesaris et al. 1998; stéphan et al. 1997) . in testicular pathology, tnf has been implicated as a major causative agent in the development of experimental autoimmune orchitis (eao) (yule and tung 1993) . there is a significant increase in the number of tnf-positive testicular macrophages and the number of tnfr1-positive germ cells undergoing apoptosis during eao in rats (suescun et al. 2003) . within the interstitium, tnf acts as an effective regulator of leydig cell steroidogenesis, by inhibiting lh receptor binding and steroidogenic gene expression (li et al. 1995; mauduit et al. 1991b mauduit et al. , 1998 hales 1993b, 1994) . no intratesticular cytokine has excited more controversy than the cell death signaling ligand fasl. interactions between fasl and its receptor on activated t cells are important in the regulation of the immune response (ju et al. 1995) . the death domain in the cytoplasmic region of the fas receptor recruits the fadd adaptor protein and induces t cell death via caspase-dependent apoptosis (schütze et al. 2008 ). there is evidence that fasl expression on epithelial cells in immune-privileged tissues, including sertoli cells, may be responsible for deleting activated t cells within the tissue, thereby suppressing adaptive immunity (bellgrau et al. 1995; griffith et al. 1996; saas et al. 1997; stuart et al. 1997 ). however, this hypothesis has been challenged by the observation that increased fasl expression on cells such as tumor cell lines and pancreatic islet cells does not increase protection in transplantation studies, but actually provokes a massive inflammatory reaction and rejection (allison et al. 1997; kang et al. 1997) . moreover, fasl appears to be abundantly expressed in the epithelia of several human tissues that lack any evidence for immunological privilege (xerri et al. 1997) , while absence or inhibition of fas or fasl does not automatically cause failure of immune privilege (rogers et al. 1998; suarez-pinzon et al. 2000; wahlsten et al. 2000) . it seems unlikely that fasl expression on its own is a fundamental explanation for immune privilege, although it may contribute through interaction with other immunoregulatory processes . localization of fasl and fas in the testis under normal conditions also has proven controversial, which may be attributed to differences in detection methods, limitations of some of the reagents that have been used, and the fact that these molecules are inducible (d'alessio et al. 2001; restifo 2000) . studies have shown fasl to be present in rat, mouse, porcine, and human sertoli cells and absent in most germ cells (bellgrau et al. 1995; braendstrup et al. 1999; d'abrizio et al. 2004; lee et al. 1997) , but other studies have reported that fasl expression in the rat seminiferous epithelium is confined to the germ cells (celik-ozenci et al. 2006; d'alessio et al. 2001) . the receptor fas has been found on isolated mouse sertoli cells (riccioli et al. 2000) , but in intact testes has been localized to spermatogonia and spermatocytes from the pubertal period onward (celik-ozenci et al. 2006; lee et al. 1997; lizama et al. 2007; ogi et al. 1998 ). in germ cells, fas expression is associated with cells that are undergoing apoptosis (lee et al. 1997; lizama et al. 2007) , and fas is induced by tnf and ifn in the sertoli cell (riccioli et al. 2000) . curiously, in one study using fragment cultures of human seminiferous tubules, tnf downregulated fasl expression and inhibited apoptosis of germ cells (pentikäinen et al. 2001) . less controversially, fas and fasl expression is upregulated in various models of seminiferous epithelium damage, indicating that this mechanism is important in regulating germ cell apoptosis in cases of physical and toxicological insult (lee et al. 1997; ogi et al. 1998 ). members of the il6 family of cytokines exert their actions via binding to specific receptors that associate with a common membrane signal transducer gp130, leading to the activation of the janus kinase/signal transducers and activators of transcription (jak/stat) and map kinase cascades (heinrich et al. 2003) . in addition to their functions in inflammation and the immune response, these cytokines play crucial roles in hematopoiesis, liver and neuronal regeneration, embryonic development, and fertility. members of this family that have been detected in the testis are leukemia inhibitory factor, oncostatin m, ciliary neurotropic factor, il11, and il6 itself (de miguel et al. 1996 (de miguel et al. , 1997 du et al. 1996; jenab and morris 1998; okuda et al. 1994; stéphan et al. 1997) . the principal roles of most of these cytokines in the testis appear to lie in regulating leydig cell development and the onset of spermatogenesis. a role in testicular immunology is most likely for il6, which possesses both proinflammatory and anti-inflammatory properties, and regulates the acute-phase response as well as several aspects of immune cell development and activity (kopf et al. 1994; tilg et al. 1997) . sertoli cells, leydig cells, and peritubular cells have been shown to produce il6 in vitro (cudicini et al. 1997b; okuda et al. 1994; syed et al. 1993 syed et al. , 1995 . in rat sertoli cells, il6 production is stimulated by fsh, testosterone, phagocytosis, and other inflammatory stimuli, including il1, il1, tnf, and lps (cudicini et al. 1997a,b; okuda et al. 1994 okuda et al. , 1995 stéphan et al. 1997; syed et al. 1995) . production of il6 by cultured mouse sertoli cells is inhibited by inf (riccioli et al. 2000; stéphan et al. 1997) . within the seminiferous epithelium, endogenous production of il6 follows a cyclical pattern that corresponds with the changes in the stages of the spermatogenic cycle, most probably under the influence of il1 and fsh (hakovirta et al. 1995; syed et al. 1993 syed et al. , 1995 . both the il6-specific receptor subunit (il6r) and the gp130 mrna are expressed in rat sertoli cells, and are stimulated by il1 and il6, but only the il6r subunit is stimulated by fsh (fujisawa et al. 2002) . il6 increases basal and fsh-induced transferrin and cyclic gmp secretion by the sertoli cell, and inhibits meiotic dna synthesis in preleptotene spermatocytes (boockfor and schwarz 1991; hakovirta et al. 1995; hoeben et al. 1997) . in models of eao, il6 appears to play an ameliorative or protective role within the seminiferous epithelium (li et al. 2002; rival et al. 2006b ). leydig cells are also active producers of il6 following stimulation by lh, lps, or il1 in vitro, and evidence suggests that these cells actually may be the major testicular source (boockfor et al. 1994; cudicini et al. 1997b; okuda et al. 1994 okuda et al. , 1995 . several studies have implicated il10 as a key player in testicular immunology. testicular il10 production is preferentially induced by systemic lps treatment in the adult rat testis (o'bryan et al. 2005) , and macrophages and t cells isolated from the rat testis constitutively produce il10, which may contribute to testicular immune privilege (hedger, unpublished data) . in a mouse model of eao, elevation of endogenous il10 levels by adenoviral-mediated transfection of human il10 was found to significantly reduce the incidence of immune activation, orchitis, and spermatogenic damage (watanabe et al. 2005) . although male mice deficient in il10 do not show evidence of male fertility problems, intratesticular immune responses have yet to be examined in these animals (kühn et al. 1993) . 11.10.4.2.6 nitric oxide and oxygen metabolites production of ros, particularly by macrophages, is an important component of the early response to infection (takemura and werb 1984) . significant ros include the superoxide anion (o 2 -), hydrogen peroxide (h 2 o 2 ), the hydroxyl radical (ho?), nitric oxide (no?), and the peroxynitrite anion (onoo à ). most are products of normal cellular metabolism, but their production is stimulated during inflammation through the activity of the enzymes nadph oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) and inos (forman and torres 2002; nathan 2006 ). these small molecules are cytotoxic to many microorganisms, although some play a more complex role in cell signaling processes. they also interact with crucial intracellular macromolecules, such as proteins, lipids, and dna, causing oxidative damage. although there are endogenous repair systems to correct oxidative damage, excessive production of ros contributes to the pathology of many diseases. among the ros, nitric oxide represents a special case. at high levels, no is highly cytotoxic, especially as a precursor of the peroxynitrite anion, but at low levels no acts as an intracellular and extracellular signaling molecule (bogdan 2001; schmidt and walter 1994) . it is produced by one of three related enzymes, neuronal nos (nnos or nos1), inducible nos (inos or nos2), and endothelial nos (enos or nos3), which catalyze the conversion of l-arginine to l-citrulline and no (alderton et al. 2001) . the nos enzymes are homodimeric proteins encoded by three separate genes ( table 2) . both nnos and enos are constitutively expressed enzymes whose activity is regulated through a calcium-calmodulin-mediated mechanism, whereas inos is a constitutively activated enzyme that is regulated at the transcriptional and translational levels by various inflammatory mediators, including lps, il1, and tnf (wolkow 1998) . in studies in various species, nos has been found in sertoli cells, leydig cells, peritubular cells, germ cells, testicular macrophages, and vascular endothelial cells (kim et al. 2007; lee and cheng 2004) . the nnos gene also produces a testis-specific isoform, tnnos, which has been localized to leydig cells (wang et al. 1997 ). in the normal rat seminiferous epithelium, inos is expressed by elongating spermatids and pachytene spermatocytes, particularly during the stages immediately following sperm release, with relatively lower levels of expression in sertoli cells and peritubular cells throughout the (o'bryan et al. 2000a ). in the interstitium, macrophage expression of inos appears to be largely confined to the minority of cd163-negative macrophages of the testis, and is not detectable in the majority of resident macrophages even during inflammation (o'bryan et al. 2000a; gerdprasert et al. 2002a) . testicular cell inos expression and no production are increased by inflammatory events induced by lps, testicular torsion, or testicular heating (lue et al. 2003; o'bryan et al. 2000a; shiraishi et al. 2001 ). production of no by germ cells, in particular, is implicated in the control of the formation and disassembly of the sertoli cell junctions that constitute the blood-testis barrier, as well as the junctional complexes involved in sertoli-germ cell adhesion lee et al. 2005) . moreover, pachytene and round spermatid apoptosis is significantly reduced in inos null mice, leading to an increase in daily sperm output and indicating a key role for inos in limiting germ cell survival and/or the carrying capacity of the sertoli cells (lue et al. 2003) . no inhibits leydig cell steroidogenesis directly, an action that is probably mediated through oxidative damage, and treatment with nos inhibitors counteracts the normal decrease in testosterone associated with sepsis or stress (del punta et al. 1996; sharma et al. 1998; welch et al. 1995) . similarly, production of other ros has been implicated in the loss of steroidogenic function in various inflammatory models and in leydig cell cultures (georgiou et al. 1987; quinn and payne 1984) . last but not least, no is a potent vasodilator, and plays a role in the endogenous control of testicular blood flow and formation of the interstitial fluid (lissbrant et al. 1997; o'bryan et al. 2000a) . production of no is an important mediator of germ cell death in testicular torsion (moon et al. 2005; shiraishi et al. 2001) , and may be involved in the testicular damage associated with varicocele (santoro et al. 2001; türker köksal et al. 2004) . it is evident that the major inflammatory signaling pathways mediated via nfb and the p38/jnk map kinases play important, albeit complex, intratesticular roles. notably, nfb has been implicated as an apoptosis-inducing signal for germ cells in numerous studies (lysiak et al. 2005; pentikäinen et al. 2002; rasoulpour and boekelheide 2005; starace et al. 2005) , even though it stimulates androgen receptor expression in the sertoli cell (delfino et al. 2003) . activation of p38/jnk is implicated in the stimulation of proliferation by immature sertoli cells and the regulation of blood-testis barrier dynamics, steroidogenesis, and multiple inflammatory responses, including the production of il6, inos, monocyte chemoattractant protein 1, and leukocyte adhesion molecules, in the mature sertoli cell (de cesaris et al. 1998 , 1999 ishikawa and morris 2006; ishikawa et al. 2005; petersen et al. 2005; riccioli et al. 2006) . while these pathways mediate many of the effects of inflammation on the testis, endogenous regulation of these pathways also appears to be involved in the normal functions of the seminiferous epithelium, as will be discussed later. the ifns are functionally related cytokines with antiviral activity and they comprise three main groups (, , and ), based on their structural relationships and major cellular sources (hertzog et al. 2003; malmgaard 2004) . the type i ifns (ifn and ) are produced by a variety of cell types in addition to leukocytes, and exert primarily antiviral and antiproliferative effects via the ifn receptor (ifnar). the type ii ifn is produced by nk and nkt cells, activated t cells and, under certain conditions, by macrophages and dendritic cells (schoenborn and wilson 2007; varma et al. 2002) . it acts through a separate receptor, and regulates the activity of apcs as part of the adaptive immune response in addition to its antiviral functions. hence, ifn is a type ii ifn, but a type 1 cytokine. type i ifn production is stimulated through tlr3, which detects double-stranded viral rna, and tlr4, the bacterial lps receptor (hertzog et al. 2003) . these two tlrs, which are expressed in the testis (bhushan et al. 2008; riccioli et al. 2006; starace et al. 2008; wu et al. 2008) , are able to induce the ifn transcription factor irf3, via engagement of the trif adapter protein (akira and takeda 2004; hertzog et al. 2003; o'neill and bowie 2007) . the regulation of ifn is cell specific and complex, but involves jak/stat signaling and various transcription factors, including nfb and ap1 (schoenborn and wilson 2007; varma et al. 2002) . its production is stimulated by type i ifns and by il18, among other cytokines. viral infections stimulate type 1 ifn and, somewhat more surprisingly, ifn production by sertoli cells, peritubular cells, leydig cells, and testicular macrophages, leading to typical antiviral responses in the testis (dejucq et al. 1995 (dejucq et al. , 1998a hu et al. 1998; starace et al. 2008) . a role for ifn in maintaining testicular immune privilege has been indicated by the observation that mouse sertoli cells upregulate their expression of the negative costimulatory ligand b7-h1 in response to ifn in vitro, but remain devoid of positive costimulatory molecules (dal secco et al. 2008 ). on the other hand, in vivo studies have identified ifn as a causative factor in eao in mice (itoh et al. 1998b) . moreover, ifn stimulates sertoli cell production of fas and caspase 1, and is implicated as the mediator of sertoli cell and germ cell apoptosis under various conditions (kanzaki and morris 1998; riccioli et al. 2000) . these disparate observations are indicative of a complex relationship between ifn, the local immune system, and spermatogenesis. ifn production also affects testicular steroidogenesis. normal healthy men treated with human ifn had significantly decreased serum testosterone levels in conjunction with normal serum gonadotropins, and both ifn and ifn inhibit testosterone production in primary cultures of leydig cells (orava et al. 1986 ). studies using porcine leydig cells indicate that ifn exerts its inhibitory effect on testosterone production at the level of cholesterol transport into the mitochondria, and inhibits expression of the steroidogenic acute regulatory (star) protein and the p-450 steroidogenic enzymes, cholesterol side-chain cleavage complex (p450scc) and 17-hydroxylase/c17-c20 lyase (p450c17) (orava et al. 1985 (orava et al. , 1989 . these data indicate that ifns may contribute to the decline in steroidogenic function of patients with viral infections, in particular. however, dejucq et al. (1995 dejucq et al. ( , 1998a have shown that an increase in ifn and ifn expression in sertoli and leydig cells of rats infected with sendai virus was actually associated with an increase in testosterone production. these results suggest that indirect or secondary stimulatory effects on testicular steroidogenesis may be involved, providing further evidence that ifns play a complex role in testicular function. and activin a the three tgf family members that are expressed in mammals (tgf 1 , tgf 2 , and tgf 3 ) are the archetypes of a much larger superfamily of mostly homo-and heterodimeric proteins, which includes the activins, the bone morphogenetic proteins (bmps), inhibin, and anti-müllerian hormone (lin et al. 2006) . the tgfs themselves are multifunctional growth and differentiation factors involved in many aspects of tissue remodeling and repair as well as regulation of the immune system (ashcroft 1999; chang et al. 2001; licona-limón and soldevila 2007) . nearly all cells synthesize a form of tgf and possess functional receptors for these cytokines. most tgf family ligands act via dual transmembrane serine/threonine kinase receptors, called type i and type ii, which interact upon ligand binding (cárcamo et al. 1994; ebner et al. 1993; massagué et al. 1992) . signals are transduced from the membrane to the nucleus through phosphorylation of the regulatory smad signaling proteins, with smads 2, 3, and 4 responsible for mediating tgf and activin signaling (datto et al. 1999; de caestecker et al. 1998) . all three mammalian tgf forms are differentially expressed by sertoli cells, peritubular cells, and leydig cells in the fetal and immature testis, but production declines considerably during sexual maturation gautier et al. 1994; mullaney and skinner 1993; olaso et al. 1997) . in the postpubertal testis, they have also been localized to the germ cells (caussanel et al. 1997; teerds and dorrington 1993) , and tgf receptors are found in both somatic and germ cells (caussanel et al. 1997; goddard et al. 2000; le magueresse-battistoni et al. 1995) . the tgfs appear to be involved in testicular development by controlling, among other things, apoptosis of undifferentiated spermatogonia (gonocytes) (olaso et al. 1998) , seminiferous tubule formation, and leydig cell differentiation (cupp et al. 1999; dickson et al. 2002; khan et al. 1992a; konrad et al. 2000) . in the adult testis, tgf 2 and tgf 3 regulate sertoli cell tight junction dynamics, indicating a role in regulating the permeability of the bloodtestis barrier to facilitate the passage of spermatocytes across the barrier during spermatogenesis (lui et al. 2003; xia et al. 2006) . however, the tgfs are also potent inhibitors of the activity of immune cells, especially t cells, b cells, and nk cells (ahmad et al. 1997; ashcroft 1999; cousins et al. 1991; licona-limón and soldevila 2007; wahl et al. 2006) , suggesting a prominent role in creating testicular immune privilege. accordingly, tgf 1 contributes to the lymphocyte inhibitory activity of testis extracts (pöllänen et al. 1993) and to the immunoprotective properties of sertoli cells in cotransplantation studies (suarez-pinzon et al. 2000) . activins are homodimers or heterodimers of homologous subunits, designated a -e , which are structurally related to the tgfs (chang et al. 2001; de kretser et al. 2002) . homodimers of a form activin a, which is widely expressed and has been extensively studied. relatively less is known about the other activin forms, which appear to be both less abundant and less widely distributed. activin a is a multifunctional growth factor and inflammatory regulator (phillips et al. 2001) , although activins were originally named for the ability of activin a and b, in particular, to stimulate pituitary fsh secretion (corrigan et al. 1991; ling et al. 1986 ). heterodimers of either a or b subunits with a homologous subunit, are called inhibin a and inhibin b, respectively, and act as feedback inhibitors of fsh secretion from the pituitary (robertson et al. 1986 ). unlike the activins, inhibins are not widely produced, and their chief source is the sertoli cell, the cellular target of fsh action meunier et al. 1988) . activin functions are highly regulated. like the tgfs, they act via specific type i and type ii transmembrane serine/threonine kinase receptors, linked to the smad signaling pathway (ethier and findlay 2001; vale et al. 2004) . the inhibins are competitive inhibitors of activin receptor binding, but activin homodimers and heterodimers comprising the b and c subunits also appear to act as weak competitive agonists of activin a (brown et al. 2000; makanji et al. 2008; mellor et al. 2003) . these interactions are modulated and/or facilitated by specific inhibitory coreceptor proteins, such as betaglycan and the bmp and activin membrane-bound inhibitor (bambi) (lewis et al. 2000; makanji et al. 2008; onichtchouk et al. 1999) . moreover, activin bioactivity can be effectively neutralized in the circulation by the high-affinity activin binding protein, follistatin (nakamura et al. 1991) . activin a is abundantly produced in the testis, particularly during early testicular development (barakat et al. 2008; buzzard et al. 2004) . even in the adult rat, intratesticular fluid levels of activin a are 5-10 times higher than normal circulating concentrations (o'bryan et al. 2005) . the sertoli cells appear to be the main source in the adult testis, although the a subunit is expressed in most germ cells kaipia et al. 1992) , and activin a protein is present in the resident macrophages and mast cells (okuma et al. 2005b (okuma et al. , 2006 . activin a is expressed at low levels throughout the cycle of the seminiferous epithelium, but there is a distinct peak of production immediately following spermiation, which is driven by the surge of il produced by the sertoli cell at this time (kaipia et al. 1992; okuma et al. 2006) . activin receptors are expressed by most, if not all, of the somatic and germ cells in the testis (cameron et al. 1994; de winter et al. 1992; feng et al. 1993; kaipia et al. 1993) . activin a exerts a complex regulation of germ cell, sertoli cell, and leydig cell proliferation and/or differentiation during development and in the adult (barakat et al. 2008; buzzard et al. 2003; mather et al. 1990; mauduit et al. 1991a; meehan et al. 2000; meinhardt et al. 2000) , and disorders of activin signaling are implicated in the onset of testicular cancer (dias et al. 2008) . activin a is produced by activated monocytes, macrophages, dendritic cells, bone marrow stromal cells, and some lymphocytes (erämaa et al. 1992; ogawa et al. 2006; robson et al. 2008; yamashita et al. 1992) , and plays a facilitating role in early inflammation responses (jones et al. 2007) . consistent with its homology to the tgf proteins, however, activin a also possesses a repertoire of anti-inflammatory and immunoregulatory activities, and displays characteristics of a type 2 cytokine (hedger et al. 1989; ogawa et al. 2006; wang et al. 2008; yu et al. 1998) . in various experimental systems, activin a has been found to antagonize the production and actions of il1 and il6, to inhibit critical t cell and b cell activation responses, and to induce macrophages to polarize toward the m2 phenotype (brosh et al. 1995; gribi et al. 2001; ogawa et al. 2006; russell et al. 1999) . both sertoli cells and testicular macrophages in culture respond to lps by increasing activin a production (okuma et al. 2005a ) (hedger, unpublished data) , and il1 is a very potent stimulus for activin a production by the sertoli cell (okuma et al. 2005b (okuma et al. , 2006 . the actual role of activin a in testicular inflammation remains unclear, since the high intratesticular levels of activin a are not affected by lps treatment in vivo (o'bryan et al. 2005) , but a role for activin a in modulating testicular immune responses can be predicted. androgens produced by the leydig cells have immunosuppressive properties that contribute to differences in immunity between the sexes (cutolo et al. 2004; miller and hunt 1996) . the majority of these effects are believed to be exerted at the level of the immune tissues, rather than by direct effects on circulating leukocytes, which lack classical androgen receptors (grossman et al. 1979; sasson and mayer 1981) . recently, however, it has been discovered that steroids can interact with membrane-bound g-protein-coupled receptors to trigger nongenomic responses (braun and thomas 2004; rahman and christian 2007) . studies have shown that androgens alter [ca] fluxes in lymphocytes and macrophages via such membrane-mediated interactions, affecting gene expression and function in the target cells (benten et al. 2004; walker 2003; wunderlich et al. 2002) . although studies of the role of androgens in suppressing immune responses, such as graft rejection, in the testis have been somewhat equivocal (cameron et al. 1990; selawry and whittington 1988; whitmore and gittes 1978) , the observation that immune cells can respond directly to androgens has resurrected the intriguing possibility that androgens, and possibly other testicular steroids, may directly regulate immune cell activity within the testis and adjacent draining lymph nodes. glucocorticoids play an important role in modulating both testicular immunity and steroidogenesis via a complex extratesticular regulatory loop. during inflammation, proinflammatory cytokines activate the hypothalamic-pituitary-adrenal axis leading to increased secretion of glucocorticoids, which act through the ubiquitously expressed glucocorticoid receptors (buckingham et al. 1996; sapolsky et al. 2000) . glucocorticoids suppress innate and acquired immunity at multiple levels and are an essential control in the inflammatory/immune response. they inhibit the inflammatory response primarily by repression of nfb, suppressing the production and actions of the proinflammatory cytokines, reducing adhesion molecule expression, and stimulating the production of anti-inflammatory cytokines (auphan et al. 1995; brack et al. 1997; kapcala et al. 1995) . glucocorticoids also exert inhibitory effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh 1981; hales and payne 1989; hardy et al. 2005; monder et al. 1994 ), thereby contributing to the inhibition of androgen production that accompanies local and systemic inflammatory events. prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), collectively called the prostanoids, are fundamental to many physiological processes, including inflammation and immunity. prostanoids arise from hydrolysis of membrane glycerophospholipids to release the free fatty acid arachidonic acid through the action of one of the more than 20 enzymes with phospholipase a 2 (pla 2 ) activity (kudo and murakami 2002; schaloske and dennis 2006) . arachidonic acid is in turn directed down the prostanoid synthetic pathway by the action of the cyclooxygenase (cox; also called prostaglandin-endoperoxide synthase) enzymes (figure 3) . there are two cox enzymes, which are the products of different genes: the constitutively expressed cox1 and an inflammation-induced form, cox2 (table 2 ) (smith et al. 2000; tanabe and tohnai 2002) . while conversion of arachidonic acid to the prostanoid precursor pgh 2 by cox is the rate-limiting step, production of specific prostanoids depends on the activity of the prostaglandin synthases, that is, pge synthase (pges), pgis, pgds, pgfs, and thromboxane a synthase (txas) (helliwell et al. 2004; ueno et al. 2005) . each prostanoid acts via one or more specific receptors to regulate cell growth, vascular smooth muscle constriction or relaxation, vascular permeability, and immune cell activity (bos et al. 2004; hata et al. 1990) . several prostanoids exert both proinflammatory and immunosuppressive actions through different receptor interactions, or through metabolism to other active immunoregulatory metabolites, such as 15-deoxy-pgj 2 , a ligand for the peroxisome proliferator-activated receptor (ppar) (jiang et al. 1998 ). an alternative synthetic pathway, catalyzed by the lipoxygenases, converts arachidonic acid to the related leukotrienes and lipoxins, which also possess diverse metabolic, vascular, and inflammation-regulating actions (samuelsson et al. 1987; takano et al. 1997) . most of the enzymes responsible for prostanoid production are ubiquitously expressed, and this includes the male reproductive tract (gerena et al. 2000; lazarus et al. 2002; takahashi et al. 1995; winnall et al. 2007) . in fact, the levels of e series prostaglandins in human seminal plasma are remarkably high (cooper and kelly 1975) . most testicular cells express both cox forms, and possess the capacity to produce prostaglandins in vitro (winnall et al. 2007) , although there appear to be cell type-specific and species-specific differences in the relative levels of expression (balaji et al. 2007; frungieri et al. 2006; lazarus et al. 2004 ). in the normal rat testis, cox2 is responsible for the majority of prostaglandin production (winnall et al. 2007 ), although intratesticular pge 2 levels are only marginally affected by acute inflammation (winnall et al. 2008) . this is probably due to the fact that macrophages in the rat testis express cox2 at very low levels, but are the only testis cell type to respond to an inflammatory stimulus with a significant increase in cox2 activity (winnall et al. 2007 ). these data point toward a previously unanticipated maintenance role for the socalled inducible cox2 enzyme in testicular function, as well as an anomalous inflammatory response of testicular cox2, consistent with an altered capacity of this organ to initiate and support inflammatory reactions. fertility is retained in male mice lacking either cox1 or cox2, whereas double deletions are lethal (langenbach et al. 1999; lim et al. 1997; sales and jabbour 2003) . studies on male fertility and spermatogenesis using prostaglandins, or nonsteroidal anti-inflammatory drugs such as aspirin and indomethacin, which act primarily via inhibition of cox activity, have produced conflicting conclusions (abbatiello et al. 1975; biswas et al. 1978; sanyal et al. 1980; winnall et al. 2008) . these diverse outcomes indicate the complexity of the roles of prostanoids in controlling various aspects of testicular function. prostaglandins, particularly pge 2 and pgf 2 , have been implicated in the control of leydig cell development in the immature testis, production of proinflammatory cytokines by the leydig and sertoli cells, autoregulation of steroidogenesis in the adult, and the decline in leydig cell function that occurs during aging (baker and o'shaughnessy 2001; cooke et al. 1991; haour et al. 1979; ishikawa et al. 2005; walch and morris 2002; wang et al. 2005) . production of pge 2 and pgf 2 by cox2 also mediates the effects of il1 on protein and lipid regulation by the sertoli cell involved in supporting figure 3 prostanoid and lysophosphatidylcholine synthesis and targets. synthesis of the prostanoids commences through the action of phospholipase a 2 (pla 2 ) on arachidonyl-containing phospholipids (usually a 1-acyl-2-arachidonylglycerophospholipid) to produce the free fatty acid arachidonic acid and the corresponding lysophospholipid. arachidonic acid is converted into the prostanoid precursor prostaglandin h 2 (pgh 2 ) by the action of cyclooxygenases and this is further converted into the various prostanoid subtypes, prostaglandins (pgd, pge, and pgf), prostacyclins (pgi), and thromboxanes (tx), by the action of specific prostaglandin and thromboxane synthases. synthesis of lipoxins and leukotrienes from arachidonic acid occurs via the activity of a separate lipoxygenase enzyme (not shown). the prostanoids interact with their various receptor subtypes: there are two pgd receptors (dp1-2) and four pge receptors (ep1-4). it is the cellular expression of these receptor subtypes that ultimately determines the eventual biological responses; for example, different prostanoid and receptor combinations may produce either proinflammatory or anti-inflammatory responses, or may have opposing vasodilatory and vasoconstrictive effects. when the phospholipid is a phosphatidylcholine, a lysophosphatidylcholine (lpc) is formed, which may interact with cells via g-protein-coupled receptor-mediated or direct membrane interactions, or may undergo further enzymatic modification to form other bioactive lipids. spermatogenesis and inflammation in the testis (ishikawa and morris 2006; ishikawa et al. 2005 ). in addition, pge 2 and pgf 2 produced by mature spermatozoa play a role in the acrosome reaction (breitbart et al. 1995; joyce et al. 1987) . products of the lipoxygenase pathway, such as leukotriene b 4 , are also produced by the testis, where they intervene in the regulation of leydig cell steroidogenesis by lh (papadopoulos et al. 1987; sullivan and cooke 1985) , but other functions can be expected. direct evidence for the anticipated roles of prostanoids or lipoxygenase products in testicular inflammation and immunity is lacking. initially, there was some evidence to suggest that pge 2 might be responsible for the immunosuppressive phenotype of resident testicular macrophages . this now seems unlikely, because testicular macrophages produce little pge 2 under normal conditions, and blocking cox2 activity in vivo or in vitro does not affect the production of proinflammatory cytokines in the testis in response to lps (gerdprasert et al. 2002a; winnall et al. 2007 winnall et al. , 2008 . in the rat, chronic inhibition of cox2 inhibited interstitial fluid formation in normal testes, but ameliorated the loss of fluid that usually occurs during lps-induced inflammation (winnall et al. 2008) . the latter data would seem to indicate roles for products of cox2 in the control of vascular tone in the normal and inflamed testis and this deserves further exploration. when phosphocholine (pc)-containing phospholipids are used as the substrate for pla 2 action, lysophosphatidylcholines (lpcs) are also produced (aoki et al. 2002; snyder 1990) . lpcs usually comprise a glycerophosphocholine backbone, a single long-chain fatty acid, and a free hydroxyl group (khaselev and murphy 2000) . the platelet-activating factors (pafs) are a subset of lpcs with an acetyl group in place of the hydroxyl group, which facilitates binding to the paf receptor (snyder 1990 ). the effective concentrations of these phospholipidderived molecules in biological fluids are normally tightly controlled by binding to albumin and lipoproteins (croset et al. 2000) , since some lpcs, particularly those derived from the saturated c16 and c18 fatty acids, cause cell lysis at high concentrations (weltzien 1979) . at physiological concentrations, however, lpcs induce specific responses in various cell types, including most immune cell types. nonacetylated lpcs are chemotactic toward macrophages, t cells, and nk cells, and stimulate cox2 expression by macrophages, cytokine production by t cells, and the cytotoxic activity of nk cells (légrádi et al. 2004; radu et al. 2004; whalen et al. 1999; yang et al. 2005) . in addition, lpcs induce apoptosis in t cells, indicating a role in immunosuppression as well (kabarowski et al. 2002; takeda et al. 1982) . the details of the mechanisms involved are poorly understood. while there is evidence for involvement of specific g-protein-coupled receptors (flemming et al. 2006; lin and ye 2003; radu et al. 2004; soga et al. 2005; yang et al. 2005) , lpcs may interact with g-proteins, membrane kinases, and ion channels directly, thereby bypassing the need for any specific receptor-ligand interactions. the interstitial fluid of the testis has long been known to possess potent lymphocyte-inhibiting activity in vitro (hedger et al. 1998a; pöllänen et al. 1988) . the molecules responsible for this activity have now been isolated and identified as the specific lpcs: 1-palmitoyl-sn-glycero-3-phosphocholine (16:0a-lpc), 1-oleoyl-sn-glycero-3-phosphocholine (18:1a-lpc), a 18:2a-lpc (putatively, 1-linoleoylsn-glycero-3-phosphocholine), and a 20:4a-lpc (putatively, 1-arachidonyl-sn-glycero-3-phosphocholine) (foulds et al. 2008 ). these molecules inhibit t cell proliferation in response to activation and induce apoptosis of these cells in a time-and dose-dependent manner at physiologically relevant concentrations. although paf activity has also been found in the testis (muguruma et al. 1993) , these acetylated lpcs did not appear to contribute significantly to the t cell inhibitory activity. the emergence of lpcs as regulators of critical immune events in the testis opens up new avenues of inquiry into the origins of autoimmune infertility and more general mechanisms of peripheral immunoregulation. these molecules may also have other roles in testis biology that demand consideration. 11.10.4.3.5 cell surface regulatory molecules: complement inhibitors and nonclassical mhc inhibition of complement activation is an important mechanism for reducing transplant rejection, particularly in the early phase. membrane complement inhibitors, such as cd46, cd55, cd59, and the complement receptor-related protein (crry), are expressed in several immune-privileged tissues and have been implicated in suppression of inflammation in the eye and placenta (bora et al. 1993; bulla et al. 2005) . cd46 and cd55 appear to be confined to spermatids in the adult rat testis, crry is expressed on early spermatocytes and interstitial cells, but cd59 is widely expressed throughout the seminiferous epithelium and interstitium . a role for cd59 expression by the sertoli cells in prolonging the survival of these cells in transplantation studies has been suggested . moreover, complement regulators both in seminal plasma and on the surface of the spermatozoa (cd46, cd55, and cd59) may contribute to complement evasion by the spermatozoa in the female tract . classical mhc class i molecules (hla-a, hla-b, and hla-c) are expressed on nearly all cell types, and facilitate antigen presentation by these cells and activation of cytotoxic t cells (cresswell et al. 1999; janeway 1992; sant et al. 1999) . by contrast, the nonclassical mhc class ib molecules (hla-e, -f, and -g) are much less widely expressed, and are associated with suppression of the adaptive immune response. membrane-bound and soluble variants of these mhc class ib molecules have been implicated in the apoptosis of alloreactive cytotoxic t cells, inhibition of nk cell cytolytic activity, suppression of t cell proliferation, and deviation of type 1 to type 2 responses, most notably in pregnancy (bainbridge et al. 2000; fournel et al. 2000; ishitani et al. 2006; kapasi et al. 2000; kanai et al. 2001; riteau et al. 1999) . hla-g has been found in the rhesus monkey testis (ryan et al. 2002; slukvin et al. 1999) , and hla-e is expressed on germ cells in the human testis (fiszer et al. 1997) , indicating that these molecules may contribute to immune privilege in the testis. from the evidence outlined in the previous sections, it should be evident that proinflammatory and immunoregulatory processes are involved in both normal testicular function and testicular pathophysiology. in the normal testis, roles in the maintenance of immune privilege, the control of leydig cell development, and the fine regulation of spermatogenesis are indicated (figure 4) . contributions to pathology include the development of autoimmunity within the inflammatory and immunoregulatory signaling in the control of intratesticular interactions. sertoli cells are primarily regulated by the hormones follicle-stimulating hormone (fsh) and testosterone, the latter being produced by the leydig cells on stimulation by luteinizing hormone (lh). sertoli cells and germ cells interact through production of various inflammatory mediators, including interleukin (il) 1, il6, activin a, tumor necrosis factor (tnf), and no, to facilitate and coordinate critical events throughout the cycle of the seminiferous epithelium. the development and activity of the leydig cells and the resident macrophages involve a close mutual interaction, mediated by mechanisms that remain to be completely defined. the sertoli cell and the resident macrophages, in turn, influence the activity of the antigen-presenting cells (apc; e.g., dendritic cells) and lymphocytes within the interstitial tissue to control local immune events, such as the maintenance of an immunologically privileged environment. this communication involves the immunoregulatory cytokines transforming growth factor (tgf), activin a, and il10, although other mechanisms may also be involved. male reproductive tract, disruption of steroidogenesis and spermatogenesis during systemic and local inflammatory events, and the response to vascular disturbances. 11.10.4.4.1 normal testicular function direct evidence that immune privilege is due to a specialized immune environment originally comes from studies involving the central nervous system. injection of soluble antigen into the compartments of the eye or brain produces antigen-specific suppression of cell-mediated immunity, specifically type 1 reactions (kaplan and streilein 1978; wenkel et al. 2000) . this phenomenon is called acquired immune deviation (aid), and it is possible to elicit similar inhibition of antigen-specific immune responses by prior injection of antigens into the testis (ditzian-kadanoff 1999; li et al. 1997; veräjänkorva et al. 2002) . the sertoli cell and leydig cell are almost certainly central to this process, although contributions from other testicular cells are to be expected. the mechanisms responsible presumably involve the recruitment and functional deviation of macrophages (and possibly dendritic cells) within the testis (hedger 2002) , the subsequent recruitment of immunoregulatory lymphocyte subsets (tompkins et al. 1998) , the production of immunoregulatory cytokines, such as tgf family members and il10 (o'bryan et al. 2005; suarez-pinzon et al. 2000) , local high concentrations of androgenic steroids, prostanoids, and other bioactive lipids (foulds et al. 2008; winnall et al. 2007) , complement inhibitors mizuno et al. 2006) , and testicular expression of inhibitory mhc and costimulatory molecules (dal secco et al. 2008; fiszer et al. 1997; ryan et al. 2002) . macrophages play an important role in leydig cell development and function. there is a close temporal link between the maturation of the adult leydig cell population and the increase in the number of testicular resident macrophages during puberty (hardy et al. 1989; raburn et al. 1993; vergouwen et al. 1993) . reductions in intratesticular macrophages due to specific depletion methods or in the macrophage-deficient (op/op) mouse lead to disorders of leydig cell development and mature function (cohen et al. 1997; gaytan et al. 1994a,b) , and testicular macrophage-conditioned medium stimulates leydig cell steroidogenesis under certain conditions (nes et al. 2000; yee and hutson 1985) . these supportive functions of the testicular macrophages may involve production of cytokines and other secreted mediators (khan et al. 1992b; warren et al. 1990) , as well as the distinctive intercytoplasmic specializations that develop between the two cell types (hutson 1992; miller et al. 1983 ). it has also been shown that testicular macrophages support basal steroidogenesis in the leydig cells by providing 25-hydroxycholesterol as a substrate for testosterone biosynthesis (nes et al. 2000) . endogenous regulation of inflammatory pathways within the seminiferous epithelium appears to be involved in the fine control of spermatogenesis. studies in the rat and mouse indicate that nuclear localization (i.e., activation) of the key proinflammatory transcription factor nfb, and expression of the inflammation-responsive genes, il1, tnf, il6, activin a, and inos, in the sertoli cell and the germ cells describe cyclical patterns within the seminiferous epithelium that coincide with critical events in the cycle of the seminiferous epithelium (o'bryan and hedger 2008) . most notably, release of spermatozoa from the epithelium (spermiation) in the rat testis is followed by an increase in the production of il1, il6, and activin a by the sertoli cells and of tnf and inos by the spermatocytes hakovirta et al. 1995; kaipia et al. 1992; söder et al. 1991; syed et al. 1993; o'bryan et al. 2000a; okuma et al. 2006 ). in the mouse, spermiation is accompanied by a period of elevated nuclear nfb levels in spermatocytes (delfino and walker 1998) . these responses coincide with a peak of dna synthesis by preleptotene spermatocytes and type a spermatogonia prior to meiotic and mitotic division, and reorganization of sertoli cell tight junctions to allow the meiotic cells to enter the adluminal compartment (o'bryan and hedger 2008) . given that il1, il6, and activin a are regulators of spermatogonial proliferation and meiotic progression (hakovirta et al. 1993a (hakovirta et al. , 1995 mather et al. 1990; meehan et al. 2000; meinhardt et al. 2000; parvinen et al. 1991; söder et al. 1991) , while tnf and inos/ no induce the disassembly of the intercellular tight junctions lee et al. 2005; li et al. 2006a; siu et al. 2003) , this concurrence of events is unlikely to be a coincidence. it would appear that release of the spermatozoa and/or phagocytosis of the residual cytoplasm triggers elements of the inflammatory machinery within the seminiferous epithelium. a role for tlrs or other patternrecognition receptors in this process may be postulated, and similar regulatory networks may also operate throughout the remainder of the cycle. many questions regarding the details of this functional regulation await clarification, and it must be noted that there is a curious lack of concordance between the content of nfb in the sertoli cell nucleus observed across the cycle in the mouse (delfino and walker 1998) and inflammatory cytokine production by these same cells in the rat hakovirta et al. 1995; kaipia et al. 1992; o'bryan et al. 2000a; okuma et al. 2006; söder et al. 1991; syed et al. 1993) . regardless of the questions that remain, however, key elements of the inflammatory response appear to play essential roles in the physiological regulation of the seminiferous epithelium, which are entirely separate from their roles in pathology. although it is a common assumption that male reproductive failure caused by local or systemic illness, infection, and chronic inflammatory disease is due to the negative effects of raised body temperature on spermatogenesis, there is little clinical or experimental evidence to support this belief (o'bryan et al. 2000b) . there is much more convincing evidence that the damage is due to activation of local inflammatory pathways and the activity of immune cells recruited during inflammatory events. as already outlined in the previous sections, inflammatory mediators such as il1, ros, tnf, and no, as well as glucocorticoids produced in response to inflammation, have mostly negative effects at all levels of the hypothalamic-pituitary-leydig cell axis (bambino and hsueh 1981; del punta et al. 1996; hales 1992; hales and payne 1989; hardy et al. 2005; li et al. 1995; mauduit et al. 1991b mauduit et al. , 1998 monder et al. 1994; sharma et al. 1998; welch et al. 1995; xiong and hales 1993b . the subsequent reduction in androgen production may lead to reduced spermatogenic capacity, but may also exacerbate the effects of inflammation on spermatogenesis itself. expression of tlrs on the sertoli cells is obviously important in protective responses against pathogens, but also provides a mechanism whereby pathogens can directly inhibit spermatogenesis. thus, tlr activation and the production of inflammatory mediators almost certainly alter sertoli cell functions, and this will affect the ability of these cells to support and regulate spermatogenesis, as well as activate signaling pathways that promote germ cell apoptosis (lysiak et al. 2005; pentikäinen et al. 2002; rasoulpour and boekelheide 2005; starace et al. 2005) . inflammatory events play a role in testicular damage due to vascular disturbance. spermatogenesis is an energy intensive process, and the seminiferous epithelium is not vascularized. consequently, unobstructed blood flow to the testis is essential and even minor anomalies in the testicular vasculature, such as varicocele, have cumulative negative effects on sperm production (jarow 2001) . the deleterious effects of cadmium on fertility through disruption of the testicular vasculature are well known (schrag and dixon 1985) . in experimental animals, transient torsion of the spermatic cord that renders the testis ischemic followed by reperfusion causes an increase in tnf and il1 expression, activation of the stress-related jnk/p38 map kinase signaling pathway, neutrophil recruitment and infiltration into the testis, increased oxidative stress, germ cell apoptosis, and significantly decreased serum testosterone levels (lysiak 2004; lysiak et al. 2001; rodriguez et al. 2006) . a specific role for testicular nitric oxide in mediating damage in both the testicular torsion and human varicocele models has been implicated (moon et al. 2005; santoro et al. 2001; shiraishi and naito 2007; shiraishi et al. 2001; türker köksal et al. 2004) . administration of high doses of human chorionic gonadotropin (hcg), a treatment used to correct delayed testicular descent in young boys, causes a hyperstimulation syndrome in rats comprising increased testicular blood flow and pressure, increased vascular permeability, and accumulation of interstitial fluid (hjertkvist et al. 1993; setchell and sharpe 1981; van vliet et al. 1988 ). these vascular changes are accompanied by accumulation of intravascular and interstitial neutrophils in the testis (bergh et al. 1986; widmark et al. 1987) , and spermatogenic damage (kerr and sharpe 1989a,b) . the responses to hcg can be eliminated by depletion of either the leydig cells or the neutrophils (setchell and rommerts 1985; widmark et al. 1987) , and il1 is able to replicate most of the effects (bergh and söder 1990; bergh et al. 1996) , suggesting that the response involves an inflammatory event mediated via il1 secreted by the leydig cells. thus, it appears that disruption of the seminiferous epithelium in both the ischemia/reperfusion model and hcg hyperstimulation models is directly linked to local recruitment of neutrophils. exactly how these cells exert damage on the germ cells is not yet known, but increased oxidative stress is obviously involved. the responses are quite different from those observed in lps-induced inflammation, which paradoxically causes a large reduction in interstitial fluid volume and an increase in mononuclear cells in the rat testis (o'bryan et al. 2000b; gerdprasert et al. 2002a,b) , although the germ cell damage is similar in both models. in summary, different testicular inflammation models have direct effects on spermatogenic development, in addition to causing damage to the steroiodogenic function of the leydig cells. it should also be noted that, as in most other tissues, inflammation responses within the testis do not automatically lead to autoimmune complications, but damage to various testicular functions that may be less readily apparent may lead to longer-term consequences for reproductive health (schuppe and meinhardt 2005; schuppe et al. 2008; suominen and soderstrom 1982) . 11.10.4.5 the epididymis, vas deferens, accessory glands, and urogenital tract as already noted above, the immune environment of the remainder of the male tract is quite distinct from that of the testis, or even from that of other mucosal tissues. the mechanisms whereby sperm within the male reproductive tract are protected from the immune system remain largely unknown, but an intact tract is obviously important. in humans, vasectomy leads to sperm antibody formation in approximately 70% of cases, and sperm antibodies are also associated with obstructive azoospermia and congenital absence of the epididymis and/or vas (alexander and anderson 1979; de kretser et al. 1998; hellema et al. 1979; patrizio et al. 1992) . the incidence of antibody formation in humans appears to be directly related to the distance of the lesion from the testis, implying a limited role for elements of testicular immune privilege in preventing immune reactions. there is evidence from patients and animal models that vasectomy can have deleterious effects on the testis and epididymis as well, and that at least some of these effects may have an immunological basis (aitken et al. 1999; bigazzi et al. 1976; flickinger et al. 1990a; raleigh et al. 2004) . in certain strains of rats, mice, and rabbits, vasectomy rapidly leads to orchitis and sterility, so it is not clear why this degree of damage is so rare in humans. in reality, the immunology of the male reproductive tract has yet to be investigated in depth, but two areas of recent interest are worth highlighting: the tlrs and the defensins. given the exposure to the external environment, it is not surprising that the tlrs as well as many essential tlr-related genes, such as myd88, are expressed throughout the male reproductive tract, including the epididymis, vas deferens, and accessory glands (nishimura and naito 2005; palladino et al. 2007; quintar et al. 2006; rodrigues et al. 2008) . expression is found on both epithelial cells and leukocytes in these tissues. moreover, weak expression of tlr11, originally localized to the urinary tract of experimental rodents, has been detected in the epididymis and vas deferens of the rat (lauw et al. 2005; palladino et al. 2007; zhang et al. 2004) . the importance of these molecules in acute and chronic inflammation, and the response to infection, in the male reproductive tract is an important area for future studies. moreover, changes in susceptibility to inflammation through polymorphisms in the tlrs have been associated with differences in susceptibility to the onset of cancer in the prostate (sun et al. 2005; zheng et al. 2004) . defensins are small (3-4 kda) positively charged peptides that are able to disrupt bacteria, fungi, parasites, and some enveloped viruses by forming multimeric pores in the pathogen membrane (selsted and ouellette 2005) . the -defensins are produced by most mucosal epithelial tissues, including the testis and epididymis (com et al. 2003) , and their production is stimulated via tlr activation and cytokines. a number of epididymal-specificdefensins have been identified in the mouse and the rat (li et al. 2001; jalkanen et al. 2005; yamaguchi et al. 2002; yenugu et al. 2004; zhou et al. 2004) , and a role for androgen in their regulation has been indicated (jalkanen et al. 2005; yenugu et al. 2004) . apart from their obvious role in protection against pathogens, defensins may have other functions in the reproductive tract. the novel -defensin, bin1b, which is exclusively produced and secreted by the rat caput epididymis, binds to sperm (li et al. 2001; zhou et al. 2004) , and blocking bin1b reduces sperm motility in vivo, suggesting a novel role for this molecule in sperm maturation (zhou et al. 2004) . in humans, semen is the most accessible window into the health of the male reproductive system, as it is the number and quality of sperm in the semen that provide the principal foci for male reproductive toxicity outcomes. however, semen is a complex secretion, comprising many components, including leukocytes, immunoglobulins, cytokines, prostaglandins, and various immunosuppressives. changes in the number or activity of these immunological components of the semen are also potential indicators of toxic actions within the tract. elevated numbers of leukocytes in the semen are generally considered to be an indication of infection, but leukocytes are present even in the semen of men with normal fertility barratt et al. 1991) . the origin of these cells is somewhat obscure. evidence suggests that the epididymis or vas may be a major source (anderson et al. 1991; schwartz 1990 ), but the main leukocyte subset present in most semen samples are neutrophils, which are not a normal feature of the tissues of the genital tract el-demiry et al. 1986; wolff and anderson 1988) . the impact of these cells on fertility is also poorly understood. some studies have shown a relationship between leukocytospermia and impaired sperm function (auroux 1984; aziz et al. 2004; berger et al. 1982; wolff et al. 1990) , and some data suggest that these cells might be a cause of sperm damage due to production of ros and cytokines (hill et al. 1987; tomlinson et al. 1992a ). moreover, leukocytes may attack the sperm directly, particularly in the presence of sperm autoantibodies, and may provoke or mediate hostile responses in the female tract. other studies, however, have failed to confirm a clear link between seminal leukocytes and infertility or sperm antibody formation barratt et al. 1990; tomlinson et al. 1993) , and it has even been suggested that leukocytes might play a beneficial role by removing abnormal sperm or by assisting in sperm capacitation (tomlinson et al. 1992b (tomlinson et al. , 1993 . molecules with immunomodulatory properties in seminal plasma include immunoglobulins, cytokines, and immunosuppressives. both igg and iga are the major constituents of seminal plasma, and there appears to be a negative correlation between iga concentrations and lymphosuppressive activity in seminal plasma (eggert-kruse et al. 1996; imade et al. 1997; moldoveanu et al. 2005) . these antibodies may play either protective roles in the control of immunity and infection, or destructive roles, when directed against functional elements of the sperm. proinflammatory cytokines such as il1, il2, il6, il8, il12, il17, tnf, and ifn are found in all seminal plasma samples, even those from men with apparently normal fertility (eggert-kruse et al. 2001; huleihel et al. 1999; koumantakis et al. 1998; maegawa et al. 2002; naz and evans 1998; politch et al. 2007 ). this is not particularly surprising, as these cytokines are present in most biological fluids and are modulated by the general health status of the individual. soluble il2 and il6 receptors, molecules that modulate the activity of their respective cytokines, have also been found in seminal plasma (huleihel et al. 1999) . numerous studies have shown that proinflammatory cytokines are dramatically elevated in patients with inflammation and/or leukocytospermia (eggert-kruse et al. 2001; krause et al. 2003; maegawa et al. 2002; miller et al. 2002; rajasekaran et al. 1996) . however, specific positive and negative associations with other forms of infertility have also been reported (eggert-kruse et al. 2001; gruschwitz et al. 1996; huleihel et al. 1999; loras et al. 1999; naz and evans 1998) . seminal plasma has profoundly immunosuppressive properties, as defined by the ability to inhibit various t cell and nk cell activities in vitro. this inhibitory activity has been attributed to a number of seminal factors, including prostasomes (kelly et al. 1991) , oxidized polyamines (allen and roberts 1986) , prostaglandins of the e series (skibinski et al. 1992) , nonspecific lymphocyte-suppressing proteins (maccioni et al. 2001; veselský et al. 2002) , and immunoregulatory cytokines (anderson et al. 1998; huleihel et al. 1999; loras et al. 1999; miller et al. 2002; nocera and chu 1993; rajasekaran et al. 1996; srivastava et al. 1996) . the main cytokines with immunosuppressive activity in human seminal plasma are tgf1, tgf2, il10, and activin a. immunosuppressives are believed to play a role in preventing lymphocyte responses against sperm autoantigens in the male and female reproductive tracts ochsenkühn et al. 2006 ). in the current state of understanding of the interactions between the male reproductive tract and the immune system, many questions still remain. nonetheless, it is clear that the mammalian testis in particular displays a special, if not unique, relationship with the immune system, involving distinctive cell-cell interactions and shared cytokines. recently, microarray analysis of testicular gene expression signatures in human spermatogenic failure were found to correspond with gene sets usually associated with inflammation and autoimmune disease (spiess et al. 2007 ). this relationship between reproduction and immunology impacts upon normal function, since perturbations in one system caused by toxicants will almost certainly impinge upon the other system. this extends into the characteristic responses of the testis to toxins and various pathologies (figure 5) . the complexity and potential consequences of these interactions may be appreciated by consideration of the following propositions: 1. if leukocytes play a role in maintaining normal testicular function, then drugs and toxicants that reduce macrophage and lymphocyte function or numbers may also have an indirect effect on testicular function. the effect of dichloromethylene diphosphonate on macrophages in the rat provides a good experimental example of this possibility, by demonstrating that the loss of functioning macrophages leads to a decline in androgen levels and retarded leydig cell development gaytan et al. 1994a ). inhibition of macrophage and lymphocyte functions or numbers may also compromise the immune-privileged status of the testis, potentially leading to testicular autoimmune disease. a related subset would involve chemicals that have direct action on both the immune system and testicular function, such as cyclosporin and cyclophosphamide (cavallini et al. 1990; meistrich 1984; seethalakshmi et al. 1990; wetzels 2004) , which are commonly used to suppress graft rejection response and in the treatment of some autoimmune diseases. fere with normal testicular function. the serious consequences for testicular function associated with immune activation and inflammation are illustrated by the experimental model of lps administration, which leads to leydig cell dysfunction and testicular failure (christeff et al. 1987; wallgren et al. 1993; o'bryan et al. 2000b) . any drug or toxicant that induces macrophage activation (e.g., bacterial toxins, carbon monoxide, sulfur dioxide) has the potential to induce testicular production of inflammatory cytokines, such as il1, tnf, and ifn, as well as ros, which may interfere with leydig cell function and androgen deficiency figure 5 model of toxicant actions and potential immunological consequences in the testis. toxicants may damage or alter the function of somatic cells (in particular the sertoli and leydig cells), immune cells, or germ cells, thereby inhibiting androgen production and spermatogenesis, leading to increased germ cell death. this damage may lead to an overt inflammatory event (orchitis) through activation of the proinflammatory activities of local macrophages, which may exacerbate or prolong the testicular dysfunction. these events may be followed by a full or partial recovery of normal function, but intratesticular immune cell numbers and activity may become permanently altered, with possible implications for future toxic or inflammatory episodes. in certain circumstances, which may depend upon the severity of the initial damage caused or the genetic background of the patient, the antigen-presenting activity of intratesticular dendritic cells or macrophages may also be activated. this may lead to recruitment and activation of testicular antigen-specific t cells, development of cytotoxic t cells (ctl), and b cell antibody (ig) production. this subsequent autoimmunity may have a more permanent effect on testis or sperm function (epithelial destruction or sperm antibody formation). spermatogenic development, or local immunoregulation. the danger hypothesis model of autoimmune disease may be particularly relevant in this instance (matzinger 1994) . at least one of the putative 'danger' molecules (i.e., tnf) is endogenously produced in the testis, potentially presenting a continuous challenge to the testicular immune system. 3. drugs or toxicants that induce autoimmune disease or allergy may also induce testicular autoimmune disease. there are many drugs that induce polysystemic autoimmune disease and this may also include reactions against the germ cells. an example here is polyarteritis nodosa, a necrotizing vasculitis of small arteries, which frequently involves the testis (fauci et al. 1978) . sulfonamides, penicillin, phenytoin, arsenicals, thiouracil, iodides, and thiazides are frequently suspected as causes of this condition (nusinow et al. 1985) . likewise, mutagens and other toxins that may alter t and b cell receptor repertoires to create self-reactive lymphocytes could also contribute to the onset of autoimmune disease in the male reproductive tract. 4. drugs or toxicants that affect testicular cell development or viability may also induce immune responses. there are drugs or toxicants that affect sertoli-sertoli and sertoli-germ cell junction stability, as well as agents that damage the sertoli cell or germ cells directly, resulting in massive germ cell death, which may overwhelm the normal immunoregulatory capacity of the testis. examples of this interaction are seen after testicular heating, serotonin administration, or treatment with ethane dimethane sulfonate (eds) or other testicular toxicants, which are frequently followed by transient immune and/or inflammatory events (creasy et al. 1983; hedger et al. 1995; padmanabhan and singh 1981; wang et al. 1994) . even if the epithelium recovers fully, there is the potential for sperm antibody development, leading to reduced fertility. increased exposure to estrogenic compounds is associated with activation of immune cells in the testis, most notably the macrophages, providing evidence for a mechanism whereby environmental estrogens may affect male fertility indirectly via effects on local immunity (li et al. 2006b ). 5. testicular toxicants that alter steroidogenic cell function will have a direct effect on the immune system. toxicants that damage leydig cells and reduce steroid production possibly could alter thymus function, leading to an enhanced susceptibility to autoimmune disease. balanced against this is the likelihood that other androgen-deficiency symptoms are far more likely to be manifest and treated. all these interactions, however, illustrate to one degree or another the difficulty of separating direct effects of toxic agents on the immune cells and on the testis. there is a dynamic relationship between the male reproductive tract and the immune system that should be considered when assessing the effects of various toxicants on male fertility and reproductive development. the effects of toxicants on male reproductive functions, most notably the production of androgenic steroids, will in turn impinge upon the functions of the immune system. many mechanisms underlying these interactions have begun to emerge in recent years, but a complete picture remains hidden. further studies will no doubt lead to important new concepts concerning this relationship and its implications for reproductive toxicology. proc. natl. acad. sci cellular transplantation from laboratory to clinic; halberstadt in knobil and neill's physiology of reproduction proc. natl. acad. sci proc. natl. acad. sci tight junctions sainio-pö llä nen, s.; sö der aderem, a. proc. natl. acad. sci fundamental immunology pö llä nen inflammation: basic principles and clinical correlates key: cord-259367-2e998to9 authors: jacques, alexandre; bleau, christian; turbide, claire; beauchemin, nicole; lamontagne, lucie title: macrophage interleukin-6 and tumour necrosis factor-α are induced by coronavirus fixation to toll-like receptor 2/heparan sulphate receptors but not carcinoembryonic cell adhesion antigen 1a date: 2009-09-01 journal: immunology doi: 10.1111/j.1365-2567.2008.02946.x sha: doc_id: 259367 cord_uid: 2e998to9 a rapid antiviral immune response may be related to viral interaction with the host cell leading to activation of macrophages via pattern recognition receptors (pprs) or specific viral receptors. carcinoembryonic cell adhesion antigen 1a (ceacam1a) is the specific receptor for the mouse hepatitis virus (mhv), a coronavirus known to induce acute viral hepatitis in mice. the objective of this study was to understand the mechanisms responsible for the secretion of high-pathogenic mhv3-induced inflammatory cytokines. we report that the induction of the pro-inflammatory cytokines interleukin (il)-6 and tumour necrosis factor (tnf)-α in peritoneal macrophages does not depend on ceacam1a, as demonstrated in cells isolated from ceacam1a(−/−) mice. the induction of il-6 and tnf-α production was related rather to the fixation of the spike (s) protein of mhv3 on toll-like receptor 2 (tlr2) in regions enriched in heparan sulphate and did not rely on viral replication, as demonstrated with denatured s protein and uv-inactivated virus. high levels of il-6 and tnf-α were produced in livers from infected c57bl/6 mice but not in livers from tlr2(−/−) mice. the histopathological observations were correlated with the levels of those inflammatory cytokines. depending on mouse strain, the viral fixation to heparan sulfate/tlr2 stimulated differently the p38 mitogen-activated protein kinase (mapk) and nuclear factor (nf)-κb in the induction of il-6 and tnf-α. these results suggest that tlr2 and heparan sulphate receptors can act as new viral pprs involved in inflammatory responses. the ability of innate immune mechanisms to respond to viral infection plays a major role in the survival of the host. the first step of a viral infection is the fixation of virus to its specific receptor, exposed on target cells. however, many viruses can use, as specific receptors, surface molecules located on macrophages or lymphocytes that are involved in innate defence mechanisms, thus modifying the antiviral functions of these cells. [1] [2] [3] the rapid antiviral immune response may be related to viral interactions with host cells, leading to activation of susceptible cells via pattern recognition receptors (pprs). 4 although many pprs have been identified for bacteria, few pprs are known for viruses on macrophages. tolllike receptors (tlrs) and the helicases retinoid-inducible gene (rig), melanoma differentiation-associated gene-5 (mda5) and rna-dependent protein kinase (pkr) are known to be engaged following some viral infections and to initiate the production of pro-inflammatory cytokines, chemokines and interferon a/b. 5 the occurrence of a new respiratory disease, the severe acute respiratory syndrome (sars), in which over-exuberant pulmonary inflammation is induced, has raised the possibility that some viral components may stimulate the production of inflammatory cytokines by macrophages. wang et al. demonstrated that a surface viral protein from the sars virus directly stimulated the production of tumour necrosis factor (tnf)-a and interleukin (il)-6. 6, 7 the induction of macrophage inflammatory cytokines by viral surface proteins has been occasionally observed with other viruses, such as feline infectious peritonitis virus and epstein-barr virus. 8, 9 the mechanisms triggered by coronaviruses in the induction of the inflammatory response by macrophages are not known. mouse hepatitis virus type 3 (mhv3), a coronavirus, induces a rapid acute hepatitis characterized by the death of susceptible c57bl/6 mice within 3-5 days post-infection. [10] [11] [12] histopathological studies revealed an extensive necrosis with inflammatory perivascular foci and impairment of immunosuppressive cytokines such as il-4, il-10, prostaglandin e 2 (pge 2 ) and transforming growth factor (tgf)-b. 13, 14 carcinoembryonic antigen cell adhesion molecules 1 (ceacam1), previously known as biliary glycoproteins, are members of the immunoglobulin superfamily. 15 murine ceacam1 proteins are mostly present in the liver and intestine and have been reported to act as receptors for different bacterial and viral pathogens, including mhv. 16, 17 these observations were supported by the fact that c57bl/6 mice knocked out for the ceacam1a gene are completely resistant to mhv-a59 infection. 18 ceacam1a promotes various functions, such as cellular adhesion, signalling, tumour suppression and angiogenesis. 15 activation of carcinoembryonic antigen (cea) induces the secretion of il-1a, il-6 and tnf-a by kupffer cells and then promotes the establishment of hepatic metastasis. 19 furthermore, it was demonstrated that production of il-1a and tnf-a by kupffer cells, via the activation of cea, involved tyrosine phosphorylation. 20 in addition, the signalling pathway implicated in cea-activated cells seemed to be different from the pathway involved in lipopolysaccharide (lps)-activated kupffer cells. 20 it was reported that stimulation of ceacam1a on pc12 cells by antibodies induced a rapid and transient ceacam1a tyrosine dephosphorylation, which was involved in the downstream activation of extracellular signal-related kinase (erk)-1 and erk-2 but not jun n-terminal kinase (jnk) or p38 mitogen-activated protein kinase (mapk). 21 in contrast, the replication of mhv3 in peritoneal macrophages involved the activation of the p38 and the erk-1/2 mapk in less than 30 min, suggesting that cellular activation was a result of the fixation of the virus to its receptor. 22 however, de haan et al. demonstrated that some murine coronavirus variants generated by in vitro cultures expressed an extended host range and also used heparan sulphate binding sites as an entry receptor instead of using the specific ceacam1a receptor. 23 recently, watanabe et al. have observed that another serotype, the mhv-jhm virus, can interact with heparan sulphate, but this receptor cannot allow the internalization of this virus. 24 it has been demonstrated that a few viral proteins, such as the capsid of the hepatitis b virus (hbv), the core of the hepatitis c virus (hcv), or herpes simplex virus (hsv) glycoproteins, may induce pro-inflammatory cytokines via the activation of tlr2 associated or not with membranes enriched in heparan sulphate. [25] [26] [27] [28] the recognition by tlrs of pathogen-associated molecule patterns (pamps) triggers the toll/interleukin-1 receptor-like (tir)domain-dependent association with adapters (such as myeloid differentiation primary response gene 88 (myd88) and toll-interleukin-1 receptor domain-containing adapter protein (tirap)) that recruit il-1 receptor-associated kinase (irak) and, following their phosphorylation, activates other factors involved in the activation of the transcription factor nuclear factor (nf)-jb, resulting in the expression of inflammatory cytokines such as il-1b, il-6 and tnf-a. 29 moreover, during chronic hcv infections, the serum il-6 levels correlate with the viral load and the length of the infection, 30 whereas tnf-a levels increase hepatic cellular apoptosis. 31, 32 the in vivo relevance of this observation may be related to the expression of tlrs in liver injury and the inflammatory state of chronically hcv-infected patients, 33 but the mechanisms involved have not yet been elucidated. the respective roles of ceacam1a, the coronavirus receptor, tlr2, heparan sulphate binding sites, and other unspecific viral pprs in the induction of the inflammatory responses seen in human or murine acute coronavirus infection have not been yet determined. in this study, we demonstrate that the pro-inflammatory cytokines il-6 and tnf-a, produced by mhv3-infected peritoneal macrophages, were induced by the fixation of protein s of mhv3 on tlr2 associated with regions enriched in heparan sulphate instead of cea-cam1a. furthermore, the release of il-6 and tnf-a was mostly dependent on the activation of the erk-1/2 mapk and jnk pathways and, to a lesser extent, the p38 mapk and nf-jb pathways. the pathology induced by l2-mhv3 is related to the release of intrahepatic il-6 and tnf-a via the tlr2 receptor, as demonstrated in tlr2 )/) mice and by histopathological observations. mice c57bl/6 (ceacam1a +/+ ) and sjl (ceacam1b +/+ ) mice were purchased from charles river laboratories (st-constant, qc, canada), whereas tlr2 knock-out mice (b6á129-tlr2 tm1kir /j) were purchased from jackson laboratories (bar harbor, ma). c57bl/6 ceacam1a knock-out (ceacam1a )/) ) mice were generated by dr n. beauchemin, as previously described. 34 the animals, certified as mhv3-free by the manufacturer, were housed in an environment with high efficiency particulate air (hepa)filtered air (forma scientific, marietta, oh). female mice between 8 and 12 weeks of age were used in all experiments. the study was conducted in compliance with the regulations of the animal committee of the university of quebec in montreal (uqam). the pathogenic l2-mhv3 is a cloned substrain isolated from the liver of infected dba2 mice and was produced in l2 cells as previously described. 35 the pathogenic properties of l2-mhv3 were assessed regularly. groups of four c57bl/6 and tlr2 )/) mice were infected by the intraperitoneal (i.p.) route with 1000 tcid 50 (tissue culture infective dose 50%) of l2-mhv3. mock-infected mice received a similar volume of rpmi-1640 (gibco laboratories, grand island, ny). at 96 hr post-infection (p.i.), the mice were anaesthetized by i.p. injection using ketamine hydrochloride (200 mg/kg) (vetrepharm canada inc., belleville, on, canada) and xylazine (10 mg/kg) (bayer inc., toronto, on, canada). mice were bled by section of the portal vein and aortic artery, as described by watanabe et al. 36 the liver was harvested following exsanguination as previously described. 37 briefly, the livers were pressed through a 70-lm cell strainer (falcon scientific co., montreal, qc, canada) which was then washed with 10 ml of rpmi-1640 supplemented with l-glutamine (2 mm), antibiotics (penicillin 100 u/ml and streptomycin 100 mg/ml) (gibco laboratories) and 20% fetal calf serum (fcs) (gemini bio-products, woodland, ca). the liver extracts were then deposited on 7 ml of fcs to allow debris sedimentation. the top layer was centrifuged for 10 min at 1000 g and the supernatant was collected for virus titration and cytokine quantification by enzyme-linked immunosorbent assay (elisa) after being passed through a 0á45-lm filter (sarstedt inc., montreal, qc, canada). livers obtained from mock-infected and l2-mhv3infected c57bl/6 and tlr2 )/) mice were prepared for histopathology and stained with haematoxylin-eosin. the supernatants of liver extracts from c57bl/6 and tlr2 )/) mice were used as the viral suspension. they were serially diluted in 10-fold steps using rpmi-1640 and tested on l2 cells cultured in 96-well plates. cytopathic effects, characterized by syncytia formation and cell lysis, were recorded at 72 hr p.i. and virus titers are expressed as log 10 tcid 50 . the continuous mouse fibroblast l2 cell line was grown in rpmi-1640 supplemented with 5% fcs. l2 cells were used for viral production. resident peritoneal macrophages from c57bl/6, ceacam1a )/) , sjl, and tlr2 )/) mice were obtained by peritoneal washings using rpmi-1640 supplemented with 20% fcs and 2-mercaptoethanol (gibco laboratories) and enriched by adherence to plastic. peritoneal exudate cells (10 6 ) were allowed to adhere for 2 hr and non-adherent cells were then washed away. cell viability, ranging from 90 to 100%, was assayed by a trypan blue exclusion test (sigma-aldrich, montreal, qc, canada). resident peritoneal macrophages were seeded in 24-well plates at a concentration of 10 6 cells/ml in rpmi-1640 supplemented with 20% fcs. in some experiments, agb10 (a monoclonal murine anti-ceacam1a antibody [immunoglobulin g (igg)] produced in rats and affinity-purified on a hitrap protein g column, (pharmacia, upsala, sweden) provided by dr n. beauchemin) (2 lg/10 6 cells), functional grade tlr2 monoclonal antibody (mab) (1 lg/10 6 cells; which does not inhibit completely monocyte functions) (ebioscience, san diego, ca) and heparin (400 u) (sigma-aldrich) were added for 2 hr. in other experiments, inhibitors specific for sh2 domain-containing protein (shp)-1 [sodium stibogluconate (ss), 10 lm, for 15 min] 38 (sigma-aldrich), p38 mapk (sb203580, 10 lg/ ml, for 2 hr), erk-1/2 mapk (u0126, 10 lg/ml, for 2 hr), nf-jb (sn50, 18 lm, for 2 hr) or jnk (sp600125, 25 lm, for 2 hr) (calbiochem, san diego, ca) were also added prior to infection. cells were then infected with 0á1-1á0 multiplicity of infection (moi) of infectious l2-mhv3, l2-mhv3 virus treated for 1 hr in phosphate buffer (gibco laboratories) at ph 8á0 and 37°, or l2-mhv3 treated for 1 hr with uv. cells were then incubated at 37°, under 5% co 2 , for 24 hr. supernatants were collected for cytokine quantification by elisa. determination of il-6 and tnf-a levels in liver extracts from mock-or l2-mhv3-infected c57bl/6 and tlr2 )/) mice, and in supernatants from in vitro infections, was performed using either mouse il-6 or mouse tnf-a bd opteia elisa sets (bd biosciences, mississauga, on, canada). for in vivo studies, statistical analyses were performed using an analysis of variance (anova) test. for in vitro studies, statistical analyses were performed using a student's t-test. all statistical analyses were calculated with the prism 4á03 software (graphpad software, la jolla, ca). error bars represent standard errors and a value of p 0á05 was considered significant. macrophages can produce pro-inflammatory cytokines such as il-6 and tnf-a during viral or bacterial infections. 19, 39, 40 in some murine strains, they also express ceacam1a, rendering them permissive to mhv infection. we postulated that these cells produced inflammatory cytokines when infected by mhv3 through engagement of ceacam1a, the mhv receptor. to verify this hypothesis, resident peritoneal macrophages were isolated from uninfected c57bl/6 and ceacam1a )/) mice and infected in vitro with l2-mhv3. il-6 and tnf-a released in supernatants were then quantified 24 hr p.i. by elisa. results showed that infection of c57bl/6 peritoneal macrophages with l2-mhv3 induced the production of pro-inflammatory cytokines il-6 (p < 0á001) and tnf-a (p < 0á001) ( fig. 1a,b) . surprisingly, infection of ceacam1a )/) peritoneal macrophages also induced the production of il-6 (p < 0á001) and tnf-a (p < 0á001) (fig. 1c,d) . furthermore, sjl peritoneal macrophages, which express ceacam1b, 41 induced lower levels of il-6 and tnf-a than c57bl/6 mice when infected with l2-mhv3 (data not shown). these observations suggest that ceacam1a is not triggered by mhv3 fixation to induce secretion of il-6 and tnf-a by peritoneal macrophages. to confirm that ceacam1a is not implicated in the production of mhv3-induced il-6 and tnf-a in peritoneal macrophages, these cells were isolated from c57bl/6 mice and treated or not for 2 hr with a specific anti-cea-cam1a monoclonal antibody (agb10), which is known to be a potent activator on murine dendritic cells. 42 the isolated cells were then in vitro infected with l2-mhv3 for 24 hr and released cytokines were quantified. although an increase of il-6 and tnf-a was observed in mhv3-infected peritoneal macrophages (p < 0á001 for both cytokines), binding of agb10 to ceacam1a did not trigger the secretion of il-6 and tnf-a nor inhibit the secretion of these cytokines compared with mhv3infected cells (fig. 2a,b) . preliminary results showed no difference in il-6 and tnf-a levels with 1, 2, 3 or 5 lg of agb10/10 6 cells. moreover, the isotype control mab did not induce production of il-6 and tnf-a in resident peritoneal macrophages (data not shown). however, ceacam1a may exhibit a regulatory role in association with immunoreceptor tyrosine-based inhibition motifs (itims) and shp-1 molecules. 43 to verify whether signals are induced by viral fixation to cea-cam1a leading to il-6 and tnf-a production, resident peritoneal macrophages purified from uninfected c57bl/ 6 mice were treated for 15 min with a shp-1 inhibitor (ss), and thereafter infected with l2-mhv3. although a slight increase of il-6 and tnf-a was noted in ss-treated macrophages compared with untreated cells (p < 0á001 for both cytokines), addition of the shp-1 inhibitor did not alter the secretion of il-6 and tnf-a triggered by mhv3 in macrophages (fig. 2c,d) . these results suggest that ceacam1 is not implicated in the production of the pro-inflammatory cytokines by mhv3-infected macrophages. membrane regions enriched in heparan sulphate are implicated in the secretion of il-6 and tnf-a by macrophages in the presence of mhv3 it has been reported that some isolates of coronavirus may use heparan sulphate binding sites instead of cea-cam1a as an entry receptor with the involvement of the ó 2009 blackwell publishing ltd, immunology, 128, e181-e192 s1 and s2 subunits of the spike protein of mhv. 23, 44 to determine whether that the pro-inflammatory cytokines induced by l2-mhv3 in macrophages depend on viral fixation to regions enriched in heparan sulphate, resident peritoneal macrophages from c57bl/6 and ceacam1a )/) mice were treated in vitro with heparin and infected further with l2-mhv3. the il-6 and tnf-a production was then quantified in supernatant at 24 hr p.i. production of il-6 by mhv3-infected c57bl/6 ( fig. 3a) and ceacam1a )/) (fig. 3c) peritoneal macrophages was impaired when cells from both mouse strains were treated with heparin (p < 0á001 for both mouse strains). the tnf-a levels were slightly reduced in peritoneal macro-phages from both mouse strains (p < 0á01 for c57bl/6 mice; p < 0á05 for ceacam1a )/) mice) (fig. 3b,d) . mhv3 uses tlr2 to induce the production of il-6 and tnf-a some viruses, such as hbv, hcv and herpesviruses, have been reported to promote production of the pro-inflammatory cytokines il-6, il-12 and tnf-a by their fixation to tlr2 in regions enriched in heparan sulphate. [25] [26] [27] we investigated whether tlr2 is also implicated in the establishment of an inflammatory state during the mhv infection. resident peritoneal macrophages were purified from ó 2009 blackwell publishing ltd, immunology, 128, e181-e192 e185 uninfected c57bl/6 and tlr2 )/) mice and infected further in vitro with l2-mhv3 for 24 hr, and pro-inflammatory cytokines were then quantified. results indicated that the production of il-6 and tnf-a was not induced in tlr2 )/) peritoneal macrophages infected with l2-mhv3 compared with c57bl/6 infected cells (p < 0á001 for both cytokines) (fig. 4a,b) . the addition of a functional grade tlr2 mab partially impaired il-6 (p < 0á001) and tnf-a (p < 0á05) production by resident c57bl/6 peritoneal macrophages (fig. 4c,d) . the partial effect was attributable to the intrinsic properties of the tlr2 mab, as specified by ebioscience. these results thus confirmed that tlr2 molecules are involved in the induction of a pro-inflammatory response during an mhv3 infection. to identify the intracellular signalling pathways involved in the tlr2/heparan sulphate region-dependent production of il-6 and tnf-a, peritoneal macrophages from c57bl/6 and ceacam1a )/) mice were treated with p38 (sb203580) and erk-1/2 (u0126) mapk inhibitors and infected in vitro with l2-mhv3. as shown in figures 5a,b , inhibition of both p38 and erk-1/2 mapk impaired il-6 production by mhv3-infected c57bl/6 peritoneal macrophages (p < 0á01 for the p38 inhibitor; p < 0á001 for the erk-1/2 inhibitor), whereas tnf-a production was partially impaired by the erk-1/2 inhibitor only (p < 0á001). addition of either p38 or erk-1/2 mapk inhibitors to mhv3-treated ceacam1a )/) macrophages also impaired the production of il-6 (p < 0á001 for both inhibitors) and tnf-a (p < 0á01 for both inhibitors) (fig. 5c,d) , indicating that ceacam1 may modulated the tnf-a pathway via the p38 mapk molecule. interestingly, the erk-1/2 mapk inhibitor induced a greater reduction in il-6 than the p38 mapk inhibitor in both c57bl/6 and ceacam1a )/) cells. pro-inflammatory cytokines such as il-1, il-6, il-12 or tnf-a may also be induced via the activation of nf-jb and jnk/ap-1. 45 to verify that jnk and nf-jb are involved in the production of il-6 and tnf-a from mhv3-infected peritoneal macrophages, these cells were isolated from c57bl/6 and ceacam1a )/) mice and treated with nf-jb (sn50) or jnk (sp600125) inhibitors. afterwards, the cells were infected in vitro with l2-mhv3 for 24 hr and il-6 and tnf-a levels were quantified. inhibition of nf-jb decreased levels of il-6 (p < 0á001) but not tnf-a in l2-mhv3-infected c57bl/6 peritoneal macrophages (fig. 6a,b) . however, addition of nf-jb inhibitor to mhv3-treated ceacam1a )/) cells impaired the production of both il-6 (p < 0á001) and tnf-a (p < 0á01) (fig. 6c,d) , indicating that ceacam1a may also modulate tnf-a production via the nf-jb molecule. in addition, inhibition of the jnk pathway greatly impaired the production of both il-6 and tnf-a by mhv3-infected c57bl/6 and ceacam1a )/) peritoneal macrophages (p < 0á001 for il-6 and tnf-a, in both mouse strains) (fig. 6a-d) . no cytotoxicity was observed in peritoneal macrophages from either mouse strain when they were treated with all of the above-mentioned inhibitors. moreover, despite the fact that a slight impairment in tnf-a production was noted with the p38 mapk and nf-jb inhibitors in c57bl/6 macrophages, the results obtained remained non-significant in several experiences. the mhv3 s protein, but not viral replication, induces the pro-inflammatory cytokines il-6 and tnf-a murine coronavirus spike (s) proteins can bind to cea-cam1a, the specific viral receptor, to infect susceptible cells. 16 s1 and s2 subunits may also be involved in heparan sulphate binding sites. 44 cytokine induction may depend on the fixation of viral proteins to the tlr2/heparan sulphate region at the cell surface. to test this hypothesis, peritoneal macrophages were isolated from both c57bl/6 and ceacam1a )/) mice and infected in vitro with infectious mhv3 or mhv3 treated at ph 8á0 and 37°to denature the s glycoprotein. 46 il-6 and tnf-a levels were then quantified at 24 hr p.i. production of il-6 and tnf-a was decreased in both c57bl/6 (p < 0á001 for il-6; p < 0á01 for tnf-a) and ceacam1a )/) (p < 0á001 for il-6; p < 0á01 for tnf-a) peritoneal macrophages in the presence of ph 8á0/37°-treated mhv3 compared with the levels of cytokines in the presence of infectious mhv3 (fig. 7a,b) . to determine whether intracellular viral replication may be also involved in the induction of cytokines, a similar experiment was conducted with uv-inactivated viruses. as shown in fig. 7c ,d, the inactivation of the virus with uv did not inhibit the production of il-6 and production of il-6 and tnf-a in the liver of mhv3-infected c57bl/6 and tlr2 )/) mice human viral hepatitis is strongly associated with a strong inflammatory response in the liver. 33 it has also been demonstrated that the pathogenic l2-mhv3 induces an acute hepatitis in susceptible c57bl/6 mice with inflam-matory foci in intrahepatic tissues. 13 to determine whether il-6 and tnf-a induced by fixation of s viral protein to tlr2/heparan sulphate receptors are involved in the histopathological disorders, the levels of these cytokines were quantified in total liver extracts from mhv3infected c57bl/6 and tlr2 )/) mice at 96 hr p.i. as shown in figure 8(a) , intrahepatic il-6 levels increased in mhv3-infected c57bl/6 mice (p < 0á001) compared with mock-infected mice, but not in mhv3-infected tlr2 )/) mice (p < 0á001 compared with infected c57bl/6 mice). despite the fact that intrahepatic tnf-a was induced in mhv3-infected c57bl/6 and tlr2 )/) mice (p < 0á001 for both mice), the levels remained lower in the livers from tlr2 )/) mice than in those from wild-type mice (p < 0á001 compared with infected c57bl/6 mice) (fig. 8b) . histological analysis of livers revealed an extensive necrosis with inflammatory foci only in surviving mhv3-infected c57bl/6 mice (figs 8c,d) , whereas low numbers of inflammatory foci were detected in livers from tlr2 )/) mice (fig. 8e,f) . the viral titres found in the livers of mhv3-infected c57bl/6 and tlr2 )/) mice were, respectively, 10 9á0 ± 10 0á1 and 10 7á6 ± 10 0á8 viruses/liver. thus, the levels of il-6 and tnf-a and the viral load may be related to the extensive necrosis observed in the livers of c57bl/6 mice infected with the pathogenic l2-mhv3. in this study, we have demonstrated that mhv3 protein s used tlr2 and heparan sulphate regions, instead of ceacam1a, the specific viral receptor, on the cell surface of peritoneal macrophages to promote the secretion of il-6 and tnf-a. viral fixation to tlr2/heparan sulphate triggers the activation of the p38 mapk, erk-1/2 mapk, nf-jb and jnk pathways. these results suggest a new mechanism by which surface viral proteins may induce an inflammatory response by their fixation to non-specific cell surface receptors. in addition, our results revealed a new modulating role for ceacam1a on macrophages in the secretion of tnf-a via the p38 mapk and nf-jb pathways. our results also suggest that tlr2 associated with heparan sulphate can be considered as a new viral ppr as its engagement by viral s protein initiates production of inflammatory cytokines, as already described for tlrs and helicases rig-1, mda5 and pkr. 5 we have demonstrated here that the induction of proinflammatory cytokines did not result from activation of ceacam1a, even when ceacam1a was used as a specific receptor for mhv on target cells in susceptible c57bl/6 mice. 16 in fact, ceacam1a )/) mice are completely resistant to mhv-a59 infections. 18 the irrelevance of ceacam1a engagement in the production of viralinduced inflammatory cytokine is supported by the comparable secretion of il-6 and tnf-a by macrophages from both c57bl/6 and ceacam1a )/) mice, and by experiments conducted with specific anti-ceacam1a mab and shp-1 inhibitors. pretreatment of uninfected or mhv3-infected peritoneal macrophages from c57bl/6 mice with the agb10 mab did not induce or inhibit the production of il-6 and tnf-a. furthermore, addition of an inhibitor to shp-1, implicated in the ceacam1a signalling pathway, 43 did not impair cytokine production by c57bl/6 macrophages. these results demonstrated that ceacam1a is not involved in the induction of the production of inflammatory cytokines by mhv3-infected macrophages. however, this conclusion does not totally exclude the possibility that cytokine production may be associated with intracellular pathways related to cea-cam1a, as demonstrated by a slight increase of il-6 and tnf-a in macrophages from c57bl/6 mice treated with the shp-1 inhibitor. the production of il-6 and tnf-a in sjl-derived macrophages also indicates that cea-cam1b is not also involved. however, it has been reported that incubation of dendritic cells with a specific ceacam1a mab (agb10) promotes the release of various chemokines and cytokines, such as macrophage inflammatory protein (mip)-1a, mip-2, il-6 and il-12, and increases the expression of the costimulatory molecules cd40, cd54, cd80 and cd86 on these cells. 42 ceacam1a pro-inflammatory properties may be differently regulated in different cell types. we have demonstrated that production of il-6 and tnf-a depends rather on heparan sulphate, as treatment of peritoneal macrophages from c57bl/6 and ceacam1a )/) mice with heparin impaired il-6 and tnf-a production. this observation suggests that mhv3 may use heparan sulphate or a closely related receptor to induce the production of cytokines by macrophages, rather than the ceacam1a receptor. in fact, the production of il-6 and tnf-a by mhv3-infected peritoneal macrophages is induced by viral fixation to a receptor located in regions enriched in heparan sulphate but not by viral replication, as demonstrated by experiments using viruses with denaturated s protein or uv-inactivated viruses. thus, denaturation of viral s protein by incubation at ph 8á0 and 37°inhibited the production of il-6 and tnf-a, whereas uv inactivation of mhv3, which does not alter viral fixation to the cell surface but inhibits efficient viral replication (confirmed by mhv3 immunolabelling), did not impair the production of il-6 and tnf-a in c57bl/6 or ceacam1a )/) peritoneal macrophages. therefore, these results revealed that viral replication is not essential for induction of tnf-a and il-6 and that the presence of the viral s protein is sufficient to stimulate production of cytokines by macrophages. it has been previously reported that murine coronavirus showing extended host range used heparan sulphate as an entry receptor. however, the change from a restricted ceacam1 tropism to an extended host range has been associated with only a few mutations in the s1 subunit, which are generally obtained from in vitro persistently infected cells. 44 it was also reported that treatment of susceptible cells with heparin inhibits infection with an extended-host range mhv-a59 variant. 23 the mhv3 used in this work is not a host-range variant because it was produced in vivo in the liver from susceptible mice before two or three in vitro passages into l2 cells, indicating that the ability of mhv3 to bind heparan sulphate receptors is a general property of this virus. we observed that il-6 and tnf-a production in mhv3-infected c57bl/6 peritoneal macrophages depended on erk-1/2 mapk and jnk, whereas p38 mapk and nf-jb pathways were only involved in il-6 production. it was reported that the p38 and erk-1/2 mapk pathways were activated early in mhv3-infected swiss-webster peritoneal macrophages, 22 but receptors involved in this activation were not identified. the respective roles of the p38 mapk, erk mapk and jnk pathways in mhv viral replication have not yet been determined. however, the p38 mapk and jnk pathways, but not the erk-1/2 pathway, were activated in mhv-a59-infected j774.1 cells. in addition, uv-irradiated mhv-a59 did not activate the mapks. 47 nevertheless, the downstream activation of ceacam1 seems to involve the erk-1/2 pathway in particular, whereas expression of the ceacam1 signalling pathway may be regulated through the nf-jb pathway. 21, 48, 49 however, we have demonstrated that viral fixation to the ceacam1a receptor is not directly involved in the secretion of il-6 and tnf-a by mhv3-infected peritoneal macrophages as these cytokines were also induced in cells from ceacam1a )/) mice and regulated by the mapk pathways. however, scheffrahn et al. proposed that cea-cam1a could either inhibit or stimulate the activation of the erk-1/2 mapk pathway depending on the history of the cells, i.e. according to the cell type and cell state. 50 activation of tlrs leads to the secretion of pro-inflammatory cytokines through the activation of nf-jb. indeed, inhibition of the nf-jb pathway partially inhibits il-6 and tnf-a production by macrophages isolated from ceacam1a )/) mice. furthermore, the p38 and erk-1/2 mapk pathways can be induced by peptidoglycan/ tlr2 activation in different cell types such as kupffer cells and eosinophils. 51, 52 nevertheless, no information is available on the regulation of ceacam-mediated intracellular signalling activation by tlrs. our results suggest that ceacam1a may regulate the secretion of tnf-a via the p38 mapk and nf-jb pathways. the results obtained with mhv3-infected tlr2 )/) mice suggest that tlr2 molecules are crucial in the production of il-6 and tnf-a and in determining the levels of infectious viruses during the induction of histopathological disorders. these results also suggest that viruses that possess glycoproteins and an envelope, such as coronaviruses, may induce a strong inflammatory response directly by their fixation to tlrs and membranes enriched in heparan sulphate via the s protein or by their release of free s proteins, without viral replication. the ppr role of the tlrs and the heparan sulphate receptor in the presence of infectious viruses demonstrated a new viral mechanism for amplification of the inflammatory response, thus explaining the severity of diseases induced by human or mouse coronaviruses. cytokine regulation in coronavirus infections has been poorly explored. our results indicate that mhv3 is a powerful model for such studies. we have demonstrated that the murine coronavirus mhv3 can stimulate the production of il-6 and tnf-a by activating tlr2/heparan sulphate regions at the cell surface of macrophages. in addition, these results suggest an interesting mechanism by which a virus and its viral surface proteins may regulate the immune response in addition to viral replication in macrophages. in contrast, the surface tlr4 and intracellular tlr9 are induced during a sars-coronavirus infection and correlate with the production of il-6 and ifn-c. 53 preliminary results have shown a slight impairment of il-6 and tnf-a production when ceacam1a )/) resident peritoneal macrophages infected with mhv3 were treated with a functional grade tlr4 mab (ebioscience) (data not shown). it was recently demonstrated that mhv-a59 and sars-cov produce ifn-a via the activation of intracellular tlr7 in plasmacytoid dendritic cells, thus controlling viral infection. 54 however, mhv-a59 and mhv-jhm were not able to induce interferon (ifn)-b via the activation of intracellular tlr3, indicating that the double-stranded viral rna is not accessible to cellular pprs. 55 however, the interaction between ceacam1a and other tlr molecules needs to be further investigated. the identification of these new pprs should allow coronavirus infections such as sars to be more effectively controlled or, in some cases, exploited. in other viral infections, it was reported that the interaction between hsv-1 and tlr2 contributed to lethal encephalitis, whereas human cytomegalovirus promotes the secretion of pro-inflammatory cytokines through its fixation to tlr2 and cd14. 27, 28 thus, tlrs seemed to be primordial during viral infections in the establishment of innate immunity, but a stronger inflammatory response can also aggravate the disease without an increase in viral replication. further work is in progress to elucidate the different roles of ceacam, tlrs, heparan sulphate and viral constituents in the production of inflammatory and immunosuppressive cytokines by hepatic cells infected with attenuated mhv3 variants. inhibition of natural killer cells through engagement of cd81 by the major hepatitis c virus envelope protein expression of genes for cytokines and cytokine-related functions in leukocytes infected with herpes simplex virus: comparison between resistant and susceptible mouse strains decoding the patterns of self and nonself by the innate immune system the interplay between viruses and innate immune signaling: recent insights and therapeutic opportunities up-regulation of il-6 and tnf-alpha induced by 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toll-like receptor expression in chronic hepatitis c: correlation with pro-inflammatory cytokine levels and liver injury deletion of the carcinoembryonic antigen-related cell adhesion molecule 1 (ceacam1) gene contributes to colon tumor progression in a murine model of carcinogenesis coronavirus induces il-6 and tnf-a via tlr2 heterogeneity in evolutive patterns of inbred mice infected with a cloned substrain of mouse hepatitis virus type 3 details of an isolation method for hepatic lymphocytes in mice mouse hepatitis viral infection induces an extrathymic differentiation of the specific intrahepatic alpha beta-tcrintermediate lfa-1high t-cell population sodium stibogluconate is a potent inhibitor of protein tyrosine phosphatases and augments cytokine responses in hemopoietic cell lines expression of proinflammatory cytokines by hepatic macrophages in acute classical swine fever induction of acute hepatic injury by endotoxin in mice response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus jhm carcinoembryonic antigen-related cell adhesion molecule 1 on murine dendritic cells is a potent regulator of t cell stimulation carcinoembryonic antigen (cea) inhibits nk killing via interaction with cea-related cell adhesion molecule 1 cooperative involvement of the s1 and s2 subunits of the murine coronavirus spike protein in receptor binding and extended host range nf-kappa b and rel proteins: evolutionarily conserved mediators of immune responses conformational changes in the spike glycoprotein of murine coronavirus are induced at 37 degrees c either by soluble murine ceacam1 receptors or by ph 8 murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells nuclear factor-kappa b directs carcinoembryonic antigen-related cellular adhesion molecule 1 receptor expression in neisseria gonorrhoeae-infected epithelial cells carcinoembryonic antigen-related cell adhesion molecule 1 expression and signaling in human, mouse, and rat leukocytes: evidence for replacement of the short cytoplasmic domain isoform by glycosylphosphatidylinositol-linked proteins in human leukocytes control of density-dependent, cell state-specific signal transduction by the cell adhesion molecule ceacam1, and its influence on cell cycle regulation the role of mapk in kupffer cell toll-like receptor (tlr) 2-, tlr4-, and tlr9-mediated signaling following trauma-hemorrhage intracellular signaling mechanisms regulating toll-like receptor-mediated activation of eosinophils cytokine regulation in sars coronavirus infection compared to other respiratory virus infections control of coronavirus infection through plasmacytoid-cell-derived type 1 interferon mouse hepatitis virus does not induce beta interferon synthesis and does not inhibit its induction by double-stranded rna this work was supported by a grant from nserc-canada. aj was supported by a canadian nserc studentship. key: cord-271812-ldwb05xn authors: prasad, ananda s. title: discovery of human zinc deficiency: its impact on human health and disease(1)(2)(3) date: 2013-03-01 journal: advances in nutrition doi: 10.3945/an.112.003210 sha: doc_id: 271812 cord_uid: ldwb05xn the essentiality of zinc in humans was established in 1963. during the past 50 y, tremendous advances in both clinical and basic sciences of zinc metabolism in humans have been observed. the major factor contributing to zinc deficiency is high phytate-containing cereal protein intake in the developing world, and nearly 2 billion subjects may be zinc deficient. conditioned deficiency of zinc has been observed in patients with malabsorption syndrome, liver disease, chronic renal disease, sickle cell disease, and other chronic illnesses. major clinical problems resulting from zinc deficiency in humans include growth retardation; cell-mediated immune dysfunction, and cognitive impairment. in the middle east, zinc-deficient dwarfs did not live beyond the age of 25 y, and they died because of intercurrent infections. in 1963, we knew of only 3 enzymes that required zinc for their activities, but now we know of >300 enzymes and >1000 transcription factors that are known to require zinc for their activities. zinc is a second messenger of immune cells, and intracellular free zinc in these cells participate in signaling events. zinc has been very successfully used as a therapeutic modality for the management of acute diarrhea in children, wilson’s disease, the common cold and for the prevention of blindness in patients with age-related dry type of macular degeneration and is very effective in decreasing the incidence of infection in the elderly. zinc not only modulates cell-mediated immunity but is also an antioxidant and anti-inflammatory agent. in 1869, the essentiality of zinc for the growth of aspergillus niger was observed (1) . in 1933, zinc was shown to be essential for the growth of the rats (2) . although by 1960, the essentiality of zinc for growth in various animal species was reported, it was considered improbable that zinc deficiency in humans could lead to significant clinical problems. i was trained as a clinical scientist at the university of minnesota, minneapolis, mn, and after my training, a strange set of circumstances brought me to shiraz, iran. the story of zinc began when an iranian physician presented to me at the medical center grand rounds, a 21-y-old man who looked like a 10-y-old boy and who was severely anemic. his genitalia were infantile. he had rough and dry skin, mental lethargy, hepatosplenomegaly, and geophagia. he ate only bread (whole wheat flour) and had no intake of animal protein. he consumed 0.5 kg of clay daily. he was severely iron deficient but had no blood loss. later, i discovered that this syndrome was common in the villages of shiraz, iran (3) . iron deficiency alone could not account for all the features that we observed in this case because growth retardation and testicular atrophy are not seen in iron-deficient experimental animals. an examination of the periodic table suggested to me that deficiency of another transitional element, perhaps zinc, may have also been present, which may account for growth retardation and hypogonadism. we hypothesized that a high phosphate content in the diet and geophagia may have decreased the availability of both iron and zinc, which resulted in deficiency of both elements (3) . our later studies in egypt documented conclusively that zinc deficiency occurred in humans and that zinc supplementation resulted in 12.7-15.2 cm of growth in 1 y and that genitalia became normal within 3-6 mo of zinc supplementation (4) (5) (6) . for nearly a decade, the idea that zinc deficiency occurred in humans remained very controversial. several reports, however, confirmed our observation, and in 1974, the national research council of the national academy of sciences declared zinc as an essential element for humans and established a recommended dietary allowance (rda) 4 (7) . in 1978, the fda made it mandatory to include zinc in the total parenteral nutrition fluids (8) . the details of the circumstances leading to the discovery of human zinc deficiency in the middle east have been presented in another paper. in this paper, the nutritional deficiency of zinc, which is present throughout the developing world, and a conditioned deficiency of zinc, which may complicate many diseased states, are discussed. zinc has also been recognized to have a therapeutic role in the management of certain diseases. this is also presented. chronology of zinc-related observations in humans eggleton (9) in china demonstrated in 1939 that the zinc contents of the toenails, fingernails, and skin were significantly decreased in poor chinese individuals with beriberi compared with healthy subjects, and he suggested that zinc deficiency may be a factor in beriberi. in another publication, eggleton (10) analyzed zinc and copper in 13 different organs and tissues from 26 chinese subjects of both sexes ranging in age from 156 d to 60 y who had died of miscellaneous causes. he showed that both elements were present in all the tissues examined. he also observed that whereas zinc was concentrated in all organs and tissues, copper was concentrated only in the liver, brain, kidney, and hair, in descending order. the cerebellum contained more zinc and copper than the cerebrum. by using the dithizone technique, lutz (11) analyzed zinc in various human tissues and concluded that the total zinc content of a 70-kg man was~2.2 g. this figure is remarkably close to what is accepted today (12) . absorption and excretion of zinc were first investigated by mccance and widdowson (13) . they showed that the principal route of zinc excretion was in the feces, and only a small quantity of zinc was excreted in the urine. vikbladh (14) analyzed serum zinc by the dithizone technique and reported that the normal level was 19.7 6 0.24 mmol/l, a value similar to what has been reported by modern techniques. in another paper, vikbladh (15) reported that serum zinc was decreased in many chronic diseases, including liver disease. in 1956, vallee et al. (12) reported that the serum zinc concentration was decreased in patients with cirrhosis of the liver and suggested that this represented a conditioned deficiency of zinc due to hyperzincuria. the first case of zinc deficiency in the united states was reported by caggiano et al. (16) in 1969 in a puerto rican subject with dwarfism, hypogonadism, hypogammaglobulinemia, giardiasis, strongyloidosis, and schistosomiasis. zinc supplementation resulted in improved growth and development. in 1972, hambidge et al. (17) reported the occurrence of nutritional zinc deficiency in mexican-american children from denver, co. they responded well to zinc supplementation. in 1972, halsted et al. (18) published the results of their study involving a group of 15 men who were rejected at the iranian army induction center because of "malnutrition." two women, 19 and 20 y old, were also included in their study. the unique feature was that all their subjects were 19 and 20 y old. their clinical features were similar to those reported by prasad et al. in 1961 (3) and 1963 (4) . they were studied for 6-12 mo. one group received a well-balanced diet containing adequate animal protein plus a placebo capsule. a second group was given the same diet plus a capsule of zinc sulfate containing 27 mg of elemental zinc. a third group received the diet without additional supplement for 6 mo. the 2 women lived in the house of dr. ronaghy and received the same treatment and observation program. the zinc-supplemented group gained considerably in height and showed evidence of early onset of sexual function as defined by nocturnal emission in males and menarche in females compared with those receiving only a well-balanced diet. severe deficiency of zinc acrodermatitis enteropathica. in 1973, barnes and moynahan (19) reported a 2-y-old girl with severe acrodermatitis enteropathica who was being treated with diiodohydroxyquinolone and a lactose-deficient synthetic diet but was not showing any satisfactory response to this therapy. the serum zinc concentration was significantly decreased. they, therefore, administered oral zinc sulfate to correct this deficiency. surprisingly, the skin lesions and gastrointestinal symptoms cleared after zinc supplementation. when zinc was inadvertently omitted from the child's regimen, the child suffered a relapse; however, she again completely responded to oral zinc therapy. the authors attributed zinc deficiency to the synthetic diet that the patient received. the authors, however, soon realized that zinc might have been fundamental to the pathogenesis of this rare inherited disorder and that the clinical improvement reflected correction of the zinc status in the patient. this original observation was quickly confirmed in other patients with acrodermatitis enteropathica (ae) throughout the world. the underlying pathogenesis of the zinc deficiency in these patients is due to malabsorption of zinc caused by a mutation in zip4, an intestinal zinc transporter (20) . ae is a lethal, autosomal, recessive trait that usually occurs in infants of italian, armenian, or iranian lineage 4 abbreviations used: ab, b amyloid protein; ad, alzheimer's disease; ae, acrodermatitis enteropathica; app, amyloid precursor protein; crp, c-reactive protein; dc, dendritic cells; hae, 4-hydroxyalkenal; haec, human vascular endothelial cell; hl-60, human promyelocytic leukemia cell line; icam-1, intercellular adhesion molecule 1; ifn, interferon; igf-1, insulin-like growth factor 1; mda, malondialdehyde; mnc, mononuclear cell; nf-kb, nuclear factor kb; oxldl, oxidized ldl; pha-p, phytohemagglutinin p; pma, phorbol-12 myristate 13 acetate; ra, receptor antagonist; rda, recommended daily allowance; ros, reactive oxygen species; scd, sickle cell disease; sil-1 ra, soluble interleukin-1 receptor antagonist; thp-1, human monocytic leukemia cell line; tk, deoxythymidine kinase; vcam-1, vascular cell adhesion molecule 1. (21) . the disease develops in the early months of life soon after weaning from breastfeeding. the dermatologic manifestations of severe zinc deficiency in patients with ae include bullous pustular dermatitis of the extremities and oral, anal, and genital areas around the orifices, paronychia, and alopecia. ophthalmic signs include blepharitis, conjunctivitis, photophobia, and corneal opacities. neuropsychiatric signs include irritability, emotional instability, tremors, and occasional cerebellar ataxia. weight loss, growth retardation, and male hypogonadism are also prominent clinical features. congenital malformation of fetuses and infants born of pregnant women with ae has been commonly observed (22) . ae patients have an increased susceptibility to infections. thymic hypoplasia, absence of germinal centers in lymph nodes, and plasmacytosis in the spleen are seen consistently. all t cell-mediated functional abnormalities are completely corrected with zinc supplementation. abnormal chemotoxis is also corrected with zinc therapy in ae patients. the clinical course is downhill with failure to thrive and complicated by intercurrent bacterial, fungal, viral, and other opportunistic infections. gastrointestinal disturbances are severe and include diarrhea, malabsorption, steatorrhea, and lactose intolerance. the disease, if unrecognized and untreated, is fatal. zinc supplementation, however, results in complete recovery. the ae gene has been localized to a w3.5-cm region on the 8q24 chromosome. the gene encodes a histidine-rich protein, now referred to as zip-4, which is a member of a large family of transmembrane proteins known as zinc transporters. in patients with ae, mutations in this gene have been documented (20) . total parenteral nutrition. in 1975, kay and tasman-jones (23) reported the occurrence of severe zinc deficiency in subjects receiving total parenteral nutrition for prolonged periods without zinc. okada et al. (24) and arakawa et al. (25) reported similar findings without zinc. these observations have been documented by several investigators, and, indeed, in the united states, zinc is now being routinely included in total parenteral fluids for subjects who are likely to receive such therapy for extended period. patients on total parenteral nutrition with diarrhea may lose up to 6 to 12 mg/d of zinc. the excessive loss of zinc results in severe deficiency of zinc. manifestations such as dermatologic, alopecia, neuropsychiatric, weight loss, and intercurrent infections are commonly seen. there are a negative nitrogen balance and impaired carbohydrate utilization. if zinc deficiency is not recognized and treated, the condition may become fatal (8) . zinc deficiency may also be common in very low birth weight infants because fortified human milk and preterm formula may not contain zinc; as such, physicians must be aware of this problem (26) . penicillamine therapy. a severe deficiency of zinc has also been observed in patients with wilson's disease who received penicillamine therapy as decoppering agent. this treatment may induce excessive zinc loss and cause severe deficiency of zinc (27) . in summary, the manifestations of severe zinc deficiency in humans include bullous pustular dermatitis, alopecia, diarrhea, emotional disorders, weight loss, intercurrent infections due to cell-mediated immune dysfunctions, hypogonadism in males, neurosensory disorders, and problems with healing of ulcers. severe deficiency of zinc, if untreated, may become fatal. the manifestations of a moderate deficiency of zinc include growth retardation, male hypogonadism in adolescents, rough skin, poor appetite, mental lethargy, delayed wound healing, cell-mediated immune dysfunctions, and abnormal neurosensory changes. these manifestations have been reported in subjects with nutritional deficiency of zinc as observed originally in iran and egypt (3, 4) and many subjects with conditioned deficiency of zinc. it is now apparent that a nutritional deficiency of zinc in humans is fairly prevalent throughout the world, particularly in areas where cereal proteins are primary in local diets. in turkey, geophagia is also a common problem, and the majority of the adolescents in the villages in turkey with geophagia exhibit both iron and zinc deficiencies (22) . cavdar et al. (22) observed a decreased zinc level in almost 30% of low socioeconomic status pregnant women in turkey. their diet consisted of mainly cereals. maternal zinc deficiency was associated with severe congenital malformation of the central nervous system in the fetuses, and maternal morbidity was increased. zinc and growth. growth is the first limiting effect of zinc deficiency in experimental animals (28) . zinc deficiency decreases circulating insulin-like growth factor 1 (igf-1) concentration independent of total energy intake (29) . in humans, zinc deficiency decreases circulating igf-1 concentration (30, 31) . igf-1 receptor possesses tyrosine kinase activity (28) . on activation of the receptor by igf, a cascade of phosphorylation occurs within the cell leading to regulation of cell cycle and cell division. tyrosine phosphorylation of the receptor is essential for its activation, and i hypothesize that because zinc has been shown to inhibit various protein tyrosine phosphatases (32), phosphorylation of the tyrosine kinase receptor by zinc is perhaps the most important critical step of zinc action on human growth. igf-1 activation leads to stimulation of thymidine uptake in cells (33) . in our earlier studies, we showed that in zincdeficient rats, the activity of deoxythymidine kinase (tk), an enzyme required for conversion of deoxythymidine to deoxythymidine 59-monophosphate, a precursor of thymidine triphosphate, is considerably decreased in the implanted sponge connective tissue, and this reduced activity of tk decreased dna, protein, and collagen synthesis in rats (34) . thus, it appears that zinc has multiple roles in growth. it is required for igf-1 generation, phosphorylation of igf-1 receptor, and upregulation of the activity of tk, all of which are involved in cell division and growth. although the clinical, biochemical, and diagnostic aspects of severe and moderate levels of zinc deficiency in humans are well defined, the recognition of mild deficiency of zinc remains very difficult. we therefore developed an experimental model of zinc deficiency in humans to define mild zinc deficiency. in a group of human volunteers, we induced a mild state of zinc deficiency by dietary means. adult male volunteers were hospitalized at the clinical research center of the university of michigan medical school hospital, ann arbor, mi. a semipurified diet that supplied~3.0-5.0 mg/d of zinc was used to induce zinc deficiency (35) . the volunteers were given a hospital diet containing animal protein daily for 4 wk. this diet averaged 12 mg/d of zinc, consistent with the rda. after this, they received 3.0-5.0 mg/d of zinc while consuming a soy protein-based experimental diet. this regimen was continued for 28 wk. after this, the volunteers received 2 cookies containing 27 mg of zinc supplement. this supplementation was continued for 12 wk. throughout this study, the level of all nutrients including protein, amino acids, vitamins, and minerals (both micro and macro elements) were kept constant, meeting the rda except for zinc. by this technique, we were able to induce a specific mild deficiency of zinc in human volunteers (35) (36) (37) (38) . as a result of mild deficiency of zinc, we observed a decreased serum testosterone level, oligospermia, decreased natural killer cell lytic activity, decreased il-2 activity of t helper cells, decreased serum thymulin activity, hyperammonemia, hypogeusia, decreased dark adaptation, and decreased lean body mass (36) (37) (38) . this study clearly established that even a mild deficiency of zinc in humans affects clinical, biochemical, and immunological functions adversely. zinc and immune cells. zinc is a second messenger for immune cells, and its intracellular status is directly altered by an extracellular stimulus and then intracellular zinc participates in signaling events (39) . hirano et al. (40) showed that a decrease in intracellular free zinc is critical for lpsmediated cd4+ t-cell activation by dendritic cells (dcs). lps binds to toll-like receptor 4 on dcs and initiates myd88 and trif [a domain containing adapter-inducing interferon (ifn)-b]-mediated signaling (39) . trif-mediated signaling increases the znt (a solute carrier )-5 mrna and decreases zip-6 mrna, thus resulting in a decrease in the intracellular free zinc in dcs. reduction in intracellular free zinc increases surface expression of major histocompatibility complex class ii molecules, which is important for the activation of cd4+ t cells (39, 40) . zinc affects the activity of monocytes/macrophages in several ways. zinc is involved in monocyte/macrophage development (41) (42) (43) and regulates its functions such as phagocytosis and proinflammatory cytokine production. lps stimulation of zinc-sufficient monocytes results in downregulation of inflammatory cytokines such as tnf-a, il-1b, il-6, and il-8 (37) (38) (39) . zinc inhibits the membrane phosphodiesterase, leading to elevated levels of the second messenger guanosine 39,59 cyclic monophosphate, which is followed by a subsequent suppression of the nuclear factor kb (nf-kb)-dependent mrna of tnf-a, il-1b, and other inflammatory cytokines (40) (41) (42) . additionally, zinc induces a-20, which inhibits nf-kb signaling via tnf receptorassociated factor pathways, resulting in downregulation of the mrna of inflammatory cytokines (44) (45) (46) . based on this, we propose that zinc is an important anti-inflammatory agent. zinc deficiency affects t helper subset 1 cell function adversely in humans (36) (37) (38) 43) . serum thymulin activity and generation of t helper subset 1 cell cytokine, il-2, and ifn-g were affected within 8-12 wk of institution of a zincrestricted diet (3-5 mg/d) in human volunteers, whereas plasma zinc decreased after 20 to 24 wk of the institution of the experimental diet. this suggests that t helper subset 1 cells are very sensitive to zinc restriction. t helper subset 2 cell cytokines were not affected as a result of the institution of zinc-deficient diet in humans. in the human malignant lymphoblastoid cell line of th0 phenotype cells, a human malignant lymphoblastoid cell line of the th0 phenotype, we showed that in zinc-sufficient cells, mrna levels of ifn-g, il-12 receptor b 2 , and t-bet (a transcription factor involved in t-cell differentiation) in phorbol-12 myristate 13 acetate (pma)/phytohemagglutinin-p (pha)-stimulated cells were increased compared with zinc-deficient cells (47) . although intracellular free (noncovalently bound zinc) zinc increased only slightly in pma/pha-stimulated cells, in concanavalin a-stimulated cells in zinc-sufficient medium, there was an increased sustained level of intracellular free zinc compared with the zincdeficient cells (47) . we concluded that stimulation of cells by concanvalin a via t-cell receptor, there was a release of intracellular free zinc that functioned as a signal transduction molecule for the generation of ifn-g, t-bet, and il-12 receptor b 2 mrna required for t helper subset 1 cell differentiation (47) . conditioned deficiency of zinc gi disorders. a moderate level of zinc deficiency has been observed in many gastrointestinal disorders. these include malabsorption syndrome, crohn's disease, regional ileitis, and steatorrhea. in 1968, macmahon et al. (48) were the first to report zinc deficiency in a patient who had steatorrhea. zinc deficiency in patients with malabsorption syndrome is now well recognized, and most physicians are aware of this problem. liver disease. low serum and hepatic zinc and, paradoxically, hyperzincuria were reported in patients with cirrhosis of the liver many years ago (12) . some patients with cirrhosis of the liver who had night blindness did not respond to vitamin a supplementation; however, they responded to zinc administration (21) . elevated blood ammonia levels are known to be a factor in the development of hepatic coma. it is known that zincdeficient rats have a defect in the metabolism of sulfurcontaining amino acids. zinc deficiency also affects urea synthesis and thus an abnormality related to metabolism of amino acids and ammonia may act in concert to produce hepatic coma. we reported earlier that dietary zinc restriction may lead to hyperammonemia (49) . rabbani and prasad (49) observed a decrease in hepatic ornithine transcarbamoylase activity and an increase in plasma ammonia levels in zinc-deficient rats. additionally, increased activity of the purine nucleotide enzyme adenosine monophosphate deaminase as a result of zinc deficiency has also been observed, which may also contribute to increased ammonia levels (50) . zinc therapy has been reported to be beneficial in subjects with hepatic encephalopathy (51) . more studies are needed in this important area. it is likely that some of the clinical features of cirrhosis of the liver, such as loss of body hair, testicular hypofunction, poor appetite, mental lethargy, difficulty in healing, abnormal cell-mediated immunity, and night blindness, may indeed be related to the secondary zinc-deficient state induced by hyperzincuria. renal diseases. mahajan et al. (52, 53) were the first to document that patients with chronic renal failure showed decreased concentration of zinc in plasma, leukocytes, and hair; increased plasma ammonia levels; and increased activity of plasma ribonuclease. uremic hypogeusia improved after zinc supplementation. impotence is common in uremic males and is not improved by hemodialysis. a double-blind clinical trial of zinc supplementation showed that zinc deficiency was a reversible cause of sexual dysfunction in uremia (53) . zinc deficiency in sickle cell disease. our studies have documented the occurrence of zinc deficiency in adult sickle cell disease (scd) patients (54, 55) . growth retardation, hypogonadism in males, hyperammonemia, abnormal dark adaptation, and cell-mediated immune dysfunction in scd patients have been related to a deficiency of zinc. the biochemical evidence of zinc deficiency in scd patients was decreased levels of zinc in the plasma, erythrocytes, and hair; hyperzincuria; decreased activity of certain zincdependent enzymes such as carbonic anhydrase in erythrocytes, alkaline phosphatase in the neutrophils, deoxythymidine kinase activity in newly synthesizing skin connective tissue and collagen; and hyperammonemia (54, 55) . inasmuch as zinc is known to be an inhibitor of rna, increased activity of this enzyme in plasma was considered to also be evidence of zinc deficiency. zinc supplementation in scd patients resulted in significant improvement in secondary sexual characteristics, normalization of plasma ammonia level, and correction of dark adaptation abnormality. zinc supplementation also increased zinc levels in plasma, erythrocytes, and neutrophils. the expected response to zinc supplementation in enzyme activities was also observed. increased longitudinal growth and body weight in 14-to 18-yold scd patients were observed. zinc supplementation also corrected impaired delayed-type hypersensitivity reaction and decreased natural killer cell lytic activity in scd patients (21, 54, 55) . a 3-mo placebo-controlled zinc supplementation trial (25 mg zinc as zinc acetate 3 times a day) in 36 scd patients showed that zinc-supplemented subjects had a decreased incidence of infections, increased hemoglobin and hematocrit levels, and increased plasma zinc and antioxidant power compared with the placebo group (55) . plasma nitrite and nitrate, lipid peroxidation products, dna oxidation products, and soluble vascular cell adhesion molecule 1 (vcam-1) decreased in the zinc-supplemented group compared with the placebo group. zinc-supplemented subjects showed significant decreases in lps-induced tnf-a and il-1b mrna and tnf-induced nf-kb dna binding in mononuclear cells (mncs) compared with the placebo group (55) . zinc supplementation also increased relative levels of il-2 and il-2 receptor a mrna in pha-stimulated mncs (55) . therapeutic impact of zinc zinc and infectious diseases. acute diarrhea in children. supplementation with zinc has been shown to prevent and treat diarrhea in children younger than 5 y of age, decreasing both diarrhea morbidity and mortality (56, 57) . zinc deficiency is also correlated with the risk of respiratory tract infections, but the benefit of supplementation appears to be limited to more severe episodes and in populations with a high incidence of zinc deficiency (57) . diarrhea causes the breakdown of absorptive mucosa, resulting in poor absorption of nutrients, including zinc. studies conducted earlier linked diarrheal illness to the loss of endogenous zinc (57) . children with low plasma zinc were observed to be more susceptible to diarrhea pathogens, propagating a cycle of deficiency and infection. there is extensive evidence supporting the efficacy of zinc supplementation for the prevention of childhood diarrhea (57) . in 2004, who issued a global recommendation for the daily supplementation with 20 mg zinc in children 6 mo of age and older and 10 mg of zinc in infants younger than 6 mo for 10-14 d on diarrheal onset. meta-analysis of routine supplementation for as long as 3 mo in 7 studies providing 1-2 times the rda of elemental zinc 5-7 times per week found an 18% reduction in diarrheal incidence, a 25% decrease in diarrhea prevalence, and a 33% reduction in persistent diarrhea episodes among supplemented children compared with children who received placebo (57) . a meta-analysis of 3 randomized, controlled trials providing short-course zinc supplementation with 2-4 times the daily rda for 2 wk after the onset of an episode of acute or persistent diarrhea was reported. the pooled analysis showed an 11% decrease in diarrhea incidence and a 34% decrease in diarrhea prevalence during 3-mo observation period (57) . severe infection and zinc in children. serious bacterial infections are a major cause of death in early infancy in developing countries. in 1 study, the effect of zinc as an adjunct to antibiotics in infants with probable serious bacterial infection was assessed. a randomized, double-blind, placebocontrolled zinc supplementation trial in infants 7-120 d of age with probable serious bacterial infections at 3 hospitals in new delhi, india, was conducted (58) . the patients were stratified according to whether they were underweight or had diarrhea at enrollment, and they received either 10 mg zinc or placebo orally every day in addition to standard antibiotic treatment. the primary outcome was treatment failure, which was defined as the need to change antibiotics within 7 d of randomization, the need for intensive care management, or death at any time within 21 d. significantly fewer treatment failures occurred in the zinc group (10%) compared with the placebo group (17%). ten infants receiving zinc died compared with 17 receiving placebo (58) . zinc for the treatment of the common cold. the common cold is one of the most frequently occurring diseases in the world (59, 60) . more than 20 viruses cause the common cold, and these include rhinovirus, coronavirus, adenovirus, respiratory syncytial virus, and parainfluenza virus. annually adults in the united states may develop a common cold 2-4 times in a year, and children may develop colds 6-8 times in a year. the morbidity and subsequent financial loss resulting from absenteeism from work are substantial. previously prescribed treatments have not provided a consistent relief of symptoms. eby et al. (61) in 1984 were the first to show in a doubleblind, placebo-controlled trial that zinc gluconate lozenges administered every 2 h were effective in decreasing the severity and duration of common cold. we tested the efficacy of zinc acetate lozenges in the common cold in 50 volunteers who were recruited within 24 h of symptoms of the common cold developing and conducted a randomized, doubleblind, placebo-controlled trial (59) . participants took 1 lozenge containing 12.8 mg zinc (as acetate) or placebo every 2 to 3 h while awake as soon as common cold symptoms developed. subjective symptom scores for sore throat, nasal discharge, nasal congestion, sneezing, cough, scratchy throat, hoarseness, muscle ache, fever, and headache were recorded daily for 12 d. plasma zinc and proinflammatory cytokines were assayed on day 1 and after participants were well. twenty-five in the zinc group and 23 in the placebo group completed the study. compared with the placebo group, the zinc group had shorter overall duration of cold symptoms (4.5 vs. 8.1 d, p < 0.01), cough (3.1 vs. 6.3 d, p = 0.01), and nasal discharge (4.1 vs. 5.8 d, p = 0.02), and decreased total severity scores for all symptoms (p < 0.002). in another study, we recruited 50 ambulatory volunteers within 24 h of common cold symptoms developing for randomized, double-blind, placebo-controlled trial of zinc (60) . participants took 1 lozenge containing 13.3 mg of zinc (as zinc acetate) or placebo every 2-3 h while awake. the subjective scores of clinical symptoms were recorded daily. plasma zinc, soluble il-1 receptor antagonist (ra), soluble tnf receptor 1, soluble icam-1 were assayed on days 1 and 5 (60) . compared with the placebo group, the zinc group had a shorter mean overall duration of the cold (4.0 vs 7.1 d, p = 0.001), cough (2.1 vs 5.0 d, p < 0.001), and nasal discharge (3.0 vs 4.5 d, p = 0.02). blinding of subjects was adequate, and adverse effects were comparable in the 2 groups. symptom severity scores were significantly decreased in the zinc group (p = 0.002). the mean changes between zinc and placebo groups (before vs after therapy) showed significant differences in soluble il-1-ra (p = 0.033) and soluble icam-1 level (p = 0.04). both decreased in the zinc group, and the mean changes in zinc and placebo group (before vs after therapy) showed a significant difference (p < 0.001) our results suggest that common cold viruses increase oxidative stress, which activates macrophages and monocytes and results in increased production of both the inflammatory cytokines and the anti-inflammatory product soluble il-1ra; thus, a decrease in soluble il-1ra in the zinc group suggests that zinc decreased the activation of monocytes and macrophages by decreasing oxidative stress. we previously showed that zinc functions as an antioxidant (44) . human rhinovirus type 24 "docks" with icam-1 on the surface of the somatic cells (59, 60) . thus, zinc may act as an antiviral agent by reducing icam-1 levels. we previously showed that zinc functions as a downregulator of nf-kb activity involved in the gene expression of adhesion molecules such as icam-1 (46) . we conclude that zinc acetate lozenges given within 24 h of the onset of common cold in proper doses are very effective in decreasing the duration and severity of the common cold. we propose that the beneficial effects seen in the zinc group were due to the antioxidant and anti-inflammatory effects of zinc. we also suggest that a decrease in plasma icam-1 levels due to zinc therapy may have decreased the docking of the cold viruses on the surface of somatic cells. a meta-analysis selected randomized, double-blind, placebo-controlled trials using zinc for at least 5 consecutive days to treat or at least 5 mo to prevent the common cold were included for analysis (62) . thirteen therapeutic trials (966 participants) and 2 preventive trials (394 participants) were included for analysis. studies reporting the duration and severity of cold symptoms suggest that zinc significantly reduced the overall duration and severity of common cold symptoms if the therapy was started within 24 h of the onset of the cold. zinc supplementation for prevention of the common cold showed that the incidence of the common cold, school absenteeism, and use of antibiotics were decreased (62) . in another meta-analysis, 13 placebo-controlled studies examined the therapeutic effect of zinc lozenges on common cold symptoms (63) . five of the trials used a total daily zinc dose of <75 mg and uniformly found no effect. three trials used zinc acetate in daily doses of >75 mg; the pooled results showed a 42% reduction in the duration of colds. five trials used zinc salts rather than acetate in daily doses of >75 mg; the pooled results showed a 20% reduction in the duration of common colds. in another report, the investigators included 17 randomized, controlled trials to analyze the effect of zinc on the common cold (64) . although the authors observed an increased heterogeneity due to age, zinc dose, and chemical formulation, they concluded that zinc may shorten the duration of the common cold (64) . to optimize the therapeutic effect of zinc lozenges on the common cold, one must pay attention to several issues. first, zinc therapy must begin within 24 h of the onset of cold symptoms. second, the total daily dose of elemental zinc should be >75 mg. third, the chemical formulation should be optimal so that zinc is ionized in the oral cavity at ph 7.4. zinc acetate and zinc gluconate are good salts to use; however, if citric acid, glycine, tartarate, and other binders are used, zinc is prevented from ionization. thus, it is critical that the solution chemistry of the preparation is proper. physicians and health practitioners must realize that one cannot treat common cold symptoms by swallowing zinc tablets, zinc syrup, or zinc lozenges. zinc lozenges must be used orally and allowed to dissolve slowly in the mouth, which will then allow ionic zinc to be released, absorbed, and transported to the virally infected nose. zinc therapy for wilson's disease wilson's disease is an inherited autosomal disorder of copper accumulation. the excretion of liver copper in the bile is decreased. this leads to failure of proper copper excretion in the stool and to the accumulation of copper in the liver. eventually not only the liver but also brain and other organs are damaged due to excess copper accumulation. patients typically present with liver disease, neurological disease (movement disorder), or psychiatric disturbances in the second to fourth decades of life. in many cases, the diagnosis is either missed or delayed (65, 66) . the gene for wilson's disease has been now identified. the genetic mutation leads to a defective production of a protein called atp7b (membrane-bound copper-binding atp), which is responsible for key step in biliary excretion of copper (65, 66) . the disease is recessive; thus, both copies of the atp7b gene have to be mutated to cause a failure in biliary excretion of copper and produce the disease. the gene for wilson's disease codes for a membrane-bound copper-binding atp-type protein that probably acts as a copper pump, in either the plasma membrane or the intracellular membrane. a large number of mutations in this gene causing wilson's disease have been identified. this complicates the development of an easy dna test for the diagnosis of this disease. it is important to establish an early diagnosis of wilson's disease because effective therapeutic measures may prevent accumulation of copper and serious damage to organs such as the liver and brain. ninety percent of the wilson's disease patients have low levels of ceruloplasmin, and ceruloplasmin-bound copper and nonceruloplasmin-bound copper are elevated in the plasma. measurement of the 24-h urinary copper is a good diagnostic test because this is consistently elevated in these patients (65, 66) . urinary copper, however, may be elevated in patients with obstructive liver disease also who do not have wilson's disease. a slit-lamp examination for corneal copper deposits (kayser-fleischer rings) is a very useful noninvasive diagnostic test for wilson's disease. they are positive in only 50% of the hepatic cases and are invariably present in neurologic cases of wilson's disease. the initial treatment objective is to decrease copper levels or otherwise affect copper such that new copper toxicity is avoided. it is also desirable to prevent copper from shifting from one pool to the other while decoppering is being done. initial copper control treatment may take 2-4 mo (65, 66) . several years ago, we were using 150 mg elemental zinc in 6 divided doses for the treatment of sickle cell disease (scd) patients (67, 68) . we had observed that zinc was an effective antisickling drug. we observed that at this level of zinc therapy, our treatment resulted in inducing copper deficiency in these patients (69) . this led brewer et al. (65, 66) to develop zinc as an effective anticopper drug for wilson's disease. zinc competes with copper for similar binding sites, and oral zinc decreases uptake of copper very efficiently (70) . zinc may also act by induction of intestinal cell metallothionein. metallothionein t, once induced, has a high affinity for binding copper and prevents the serosal transfer of copper into the blood. the intestinal cells turnover rapidly and take the complexed copper into the stool for final excretion. zinc not only blocks food copper but also the copper that is endogenously excreted via salivary, gastric, and other gastrointestinal juices. thus, zinc is effective in producing a chronic negative copper balance. fifty milligrams of elemental zinc (as acetate) is given orally 3 times a day for the management of wilson's disease patients. zinc is given in a fasting or postabsorptive state. the only side effect is that 10% of the subjects may have gastric discomfort. this is usually observed after the first morning dose and can be avoided if zinc is administered between breakfast and lunch or after dinner before going to bed. for maintenance therapy, zinc is the drug of choice (65, 66) . relatively speaking, zinc has no toxicity and is nonteratogenic; thus, it can be given to subjects of all ages and even the pregnant women. zinc has been approved by fda for the treatment of wilson's disease patients. age-related macular degeneration (amd) affects nearly 25% of individuals older than 65 y of age, and late-stage disease accounts for nearly 50% of legal blindness in europe and north america (71). newsome et al. (72) demonstrated that concentrations of zinc are reduced in human eyes with signs of amd and suggested that zinc deficiency may lead to oxidative stress and retinal damage. the age-related eye disease study group, supported by national eye institute/nih, conducted an 11-center doubleblind clinical trial in patients with dry-type amd (73) . a total of 3640 participants were enrolled. their ages ranged from 55 to 80 y, and the average follow-up period was 6.3 y. participants were randomly assigned to receive daily orally one of the following: 1) antioxidants (vitamin c 500 mg, vitamin e 400 iu, and b-carotene 15 mg); 2) zinc 80 mg as zinc oxide and copper 2 mg as copper oxide to prevent copper deficiency induced by zinc; 3) antioxidants plus zinc; or 4) placebo. in the group taking the antioxidant plus zinc supplements, the risk of advanced amd developing was reduced by w25% and vision loss by w19%. in the group taking zinc alone, the risk of advanced amd developing was reduced by w21% and vision loss by 11%. in the group taking the vitamins alone, the risk of advanced amd developing was decreased by 17%, and vision loss was decreased by 10%. no significant side effects were noted in subjects who received high levels of therapeutic zinc (73) . this study confirmed the antioxidant effect of zinc in humans. interestingly, only the zinc-supplemented group showed increased longevity (74) . the risk of mortality was reduced by 27% in the age-related eye disease study group studies in subjects 55-81 y of age who received only therapeutic zinc daily. at present, most ophthalmologists throughout the world are using zinc and vitamins as supplement for the treatment of the dry type of amd. the daily intake of zinc in elderly subjects in the western world including the united states is only w8-10 mg, whereas the rda is 15 mg. the elderly frequently do not eat the usual 3 meals a day and skip either breakfast or lunch. many live alone and do not cook a proper meal for themselves. our study in the detroit area showed that 35% of the well-off ambulatory elderly subjects may have a deficiency of zinc based on their plasma zinc levels. zinc deficiency and susceptibility to infections due to cellmediated immune dysfunction have been reported to occur in the elderly (75, 76) . the third nhanes (1988-1914) also found that elderly persons (older than 71 y) were at the greatest risk of inadequate zinc intake (77) . oxidative stress and increased inflammatory cytokines have been recognized as important contributing factors for several chronic diseases attributed to aging, such as atherosclerosis and related cardiovascular disorders, mutagenesis and cancer, neurodegenerative disorders, type 2 diabetes mellitus, and alzheimer's disease (ad). together, o$ 2-, h 2 o 2 , and oh radicals are reactive oxygen species (ros), and excessive generation of ros causes oxidative stress. inflammatory cytokines such as tnf-a and il-1b, generated by activated monocytes, are also known to generate greater levels of ros. chronic inflammatory processes have been implicated in high cardiovascular mortality in elderly subjects (78) . our previous studies showed that zinc supplementation in individuals 20 to 50 y of age decreased oxidative stress markers, such as malondialdehyde (mda), 4-hydroxyalkenals (haes), and 8-hydroxydeoxyguanine in the plasma; downregulated the ex vivo induction of tnf-a and il-1b mrna in mncs; and provided protection against tnf-a-induced nf-kb activation in isolated mncs (44) . we also showed previously that in the human promyelocytic leukemia cell line (hl-60), which differentiates to the monocyte and macrophage phenotype in response to pma, zinc upregulates the expression of a20 and the binding of a20 transactivating factor to dna, which results in the inhibition of nf-kb activation (44, 45) . inasmuch as zinc deficiency and susceptibility to infections due to cell-mediated immune dysfunctions have been observed in the elderly, we conducted a randomized, placebo-controlled trial of zinc supplementation in 50 healthy elderly subjects (55-87 y) of both sexes and all ethnic groups from st. patrick's senior citizen center, detroit, mi. one subject in zinc group dropped out on the second day; thus, we had complete data for 49 subjects (24 in the zinc group and 25 in the placebo group). exclusion criteria were as follows: life expectancy of <8 mo, progressive neoplastic disease, severe cardiac dysfunction, significant kidney disease, significant liver disease, and subjects who were not competent mentally. zinc supplementation consisted of 45 mg elemental zinc (as gluconate) daily for 12 mo. a comparison of the baseline data between the younger subjects (ages 18-54, n = 31) and the elderly subjects showed that the plasma zinc was lower and the percentage of cells producing il-1b and tnf-a and the generated cytokines were significantly higher in the elderly subjects (76) . vcams, vascular endothelial cell adhesion molecules, and e-selectin in the plasma also were significantly higher in the elderly. il-10 generated by t helper subset 2 cells, which is known to regulate negatively il-2 generation from t helper subset 1 cells, was significantly higher in the elderly. a similar observation with respect to il-10 generation by th2 cells in the elderly has been also reported by cakman et al. (79) . the oxidative stress markers also were significantly higher in the elderly compared with the younger adults (76) . the mean incidence of infections per subject in 12 mo was significantly lower (p < 0.01) in the zinc-supplemented group (0.29 6 0.46) than in the placebo group (1.4 6 0.95; effect size 1.46). the plasma zinc increased, and ex vivo generation of tnf-a and il-10 decreased significantly in the zinc group compared with the placebo group (76) . oxidative stress biomarkers in the plasma also decreased significantly in the zinc group compared with the placebo group (76) . in mncs isolated from zinc-deficient elderly subjects, zinc supplementation increased the ex vivo pha-induced il-2 mrna expression and plasma zinc concentration compared with the zinc-deficient subjects who received placebo. thus, our study showed that zinc supplementation (45 mg/d elemental zinc) in the elderly subjects decreased the incidence of infection by nearly 66%. after supplementation, we also observed that oxidative stress markers and the generation of inflammatory cytokines that were increased before supplementation, decreased significantly. these are highly significant effects of zinc supplementation in the elderly, and it may imply that zinc may also prove to be an excellent agent for the prevention of some of the chronic diseases. oxidative stress and increased generation of inflammatory cytokines have been implicated in the initiation and progression of many chronic diseases. these include atherosclerosis, diabetes mellitus type 2, neurodegenerative disorders, ad, and some malignancies. inasmuch as zinc is not only required for cell-mediated immunity, it is also an effective antioxidant and anti-inflammatory agent, i hypothesize that zinc will prove to be an effective therapeutic agent in the management of some of these disorders. in this section, the present rationale for the use of therapeutic zinc in the management of atherosclerosis, diabetes mellitus type 2, and ad is presented. atherosclerosis is a slowly progressive chronic inflammatory disease characterized by focal arterial lesions that ultimately block the blood vessels, which leads to angina, myocardial infarction, cerebrovascular ischemia and stroke, and even death (46, 78) . inflammation, oxidative stress, and/or endothelial dysfunction caused by known risk factors such as age, sex, smoking, hypertension, diabetes, and obesity are involved in the development and progression of atherosclerosis. our previous studies showed that zinc deficiency increases the generation of inflammatory cytokines, increases oxidative stress, and induces endothelial cell dysfunction (46) . our studies also showed that zinc deficiency may affect nearly 30%-40% of the well-off healthy ambulatory elderly subjects in the detroit area (75, 76) . in these subjects, decreased plasma zinc and increased plasma lipid peroxidation products and endothelial cell adhesion molecules compared with the healthy zinc-sufficient younger adults were observed. in a randomized, placebo-controlled zinc supplementation trial in elderly subjects, we examined the effect of supplementation on plasma c-reactive protein (crp), il-6, macrophage chemoattractant protein 1, vcam, and oxidative stress markers (46) . additionally, we examined the effect of zinc supplementation on a20, ppar-a, and nf-kb activation in haec and monocytic cell lines [hl-60 and human monocytic leukemia cell line (thp-1)]. the zincsupplemented group received 45 mg/d zinc as gluconate for 6 mo. at this level of zinc supplementation in the elderly, we have not observed any decrease in copper status (46) . the plasma zinc increased in the zinc-supplemented group, and no change was observed in the placebo group. we observed a significant decrease in lipid peroxidation product and a significant increase in antioxidant power [represented by ascorbate equivalent units (u/ml)] in the zincsupplemented group compared with the placebo group. plasma high-sensitivity crp in the zinc-supplemented group decreased significantly compared with the placebo group. plasma il-6, macrophage chemoattractant protein 1, secretory phospholipase a 2 , soluble vcam-1, and soluble e-selectin decreased after zinc supplementation, and the mean changes before and after between the 2 groups were statistically significant (46) . plasma zinc concentrations in the elderly subjects inversely correlated with the changes in plasma concentrations of high-sensitivity crp, vcam-1, macrophage chemoattractant protein 1, and mda+haes after 6 mo of supplementation (46) . zinc decreased the generation of tnf-a, il-1b, vcam-1, and mda+haes in hl-60 and thp-1 and haecs after incubation with oxidized ldl (oxldl) for 24 h compared with zinc-deficient cells. zinc increased a20 and ppar-a proteins in oxldl-stimulated thp-1 cells and haecs compared with zinc-deficient cells. the effect of zinc on nf-kb activation in thp-1 cells and haecs is shown in figures 1 and 2 . there was no significant difference in nf-kb activation by either nf-kb-driven luciferase reporter gene assay or electrophoretic mobility shift assay between the nonstimulated thp-1 cells and haecs incubated in zinc-deficient and zinc-sufficient media. however, after 24 h of oxldl stimulation, zinc-sufficient thp-1 cells and haecs showed a significant decrease in nf-kb activation compared with zinc-deficient cells. a high plasma crp concentration is a risk factor that is independent of other risk factors such as total cholesterol, ldl, age, smoking, bmi, diabetes, and hypertension (46) . crp is a widely used marker for atherosclerosis and its clinical course, complication, and prognosis (46) . in prospective studies, healthy men and women with an increased baseline level of crp were at greater risk of coronary artery disease (46) . our study showed that zinc supplementation (45 mg elemental zinc as gluconate) daily was effective in lowering plasma crp concentration. this is the first documentation to show that zinc is effective in downregulating the plasma crp level in the elderly. the increased production of ros and the activation of redox-dependent signaling cascades are involved in atherosclerosis (46, 78) . ros itself can initiate nf-kb-mediated transcriptional activation of inflammatory genes, thereby potentially acting as independent triggers of atherosclerosis. zinc deficiency increased oxidative stress and zinc supplementation decreased oxidative stress in cell culture models and humans. we confirmed in this study that zinc supplementation decreased oxidative stress in elderly subjects and human vascular endothelial and monocytic cells. thus, decreased oxidative stress by zinc may decrease the ldl oxidation and exhibit an atheroprotective effect. nf-kb is one of the major immune response transcription factors involved in the initiation and development of atherosclerosis (46, 78) . zinc plays an important role in nf-kb activation. however, the regulation of nf-kb activation by zinc is cell specific. zinc is required for nf-kb dna binding in purified or recombinant nf-kb p50 protein of the t helper cell line. additional studies also showed that zinc decreased lps-, ros-, or tnf-a-induced nf-kb activation in endothelial cells and cancer cells (46) . we reported previously (44) that compared with controls, healthy volunteers who were supplemented with zinc (45 mg/d) had a significant decrease in tnf-a and il-1b mrna and tnf-a induced nf-kb dna binding in isolated peripheral blood mononuclear cells, and zinc upregulated the expression of a-20 in hl-60 cells. in this study, we observed that zinc decreased oxldl-induced generation of tnf-a, il-1b, and vcam-1, oxidative stress markers, and activation of nf-kb and increased a20 and ppar-2 proteins in human monocytic and vascular endothelial cells. we propose that zinc inhibited nf-kb activation by inducing a20, a transactivating factor that played a role in reducing il-1b-and tnf-a-induced nf-kb activation. a20 inhibits nf-kb signaling via tnf receptor-associated factor pathways in endothelial cells (45) . our concept of the mechanism by which zinc may be beneficially regulating various pathways involved in the development of atherosclerosis is presented in figure 3 . inflammation generates ros, resulting in oxldl. oxldl activates the nf-kb inducible kinase/1kb kinase/nf-kb signaling pathway and upregulates the downstream target genes such as inflammatory cytokines, crp, adhesion molecules, inducible nitric oxide synthase, cyclooxygenase 2, fibrinogen, and tissue factor. these cytokines and molecules attract blood cells and platelets leading to coagulation, which initiates the development of atherosclerosis. we showed that zinc supplementation increased the plasma concentration of antioxidant power and decreased the plasma concentration figure 1 the effect of zinc on a20 and peroxisome proliferatoractivated receptor a (ppar-a) in the human monocytic leukemia cell line (thp-1) (a) and human aortic endothelial cells (haecs) (b and c) after oxidized ldl (oxldl) stimulation. the cells were incubated either in zinc-deficient (zn2, 1 mmol/l) or zincsufficient (zn+, 15 mmol/l) medium for 8 d (for the thp-1) and for 6 d (for haecs), followed by 24 h of stimulation with 50 mg oxldl/ml. a20 and ppar-a proteins were measured by western blot analysis. *p , 0.05 for zn2 compared with zn+ (values are sd; n = 3). gadph, glyceraldehyde 3-phosphate dehydrogenase. reproduced with permission from (46) . of inflammatory cytokines and lipid peroxidation biomarkers in elderly subjects. in view of these results, i recommend that a controlled, prospective trial of zinc supplementation in elderly subjects should be conducted to establish the atheroprotective effect of zinc. this will have an immense impact on the management of patients with coronary artery disease and patients with stroke. diabetes mellitus is one of the most common chronic diseases, and according to who, estimate nearly 300 million individuals are affected by this disorder. diabetes is one of the major causes of blindness, increased risk of cardiovascular disorder, end-stage renal disease, and nontraumatic limb amputation (80) . insulin, produced by the b cells of the pancreas, is essential for glucose clearance from the blood to muscle, fat, and liver cells. the hallmark of diabetes is a loss of control of glycemia due to lack of insulin, which may be relative or absolute depending on the type of diabetes. type 1 diabetes mellitus alone accounts for 5%-10% of all cases of diabetes. it is caused by autoimmune destruction of pancreatic b cells, resulting in virtually no production of insulin. without insulin, carbohydrate cannot be used for energy; therefore, fats become the main intracellular source of energy. this results in generation of ketone bodies leading to ketoacidosis. exogenous insulin administration is the main treatment for this situation. type 2 diabetes mellitus accounts for >90% of the cases of diabetes and is due to insulin resistance; there is no problem in insulin production initially by the b cells. eventually, however, islet b cell function also declines, leading to overt diabetes. these patients ultimately also require exogenous insulin for treatment. zinc is crucial for the pancreas and the regulation of blood glucose. insulin is stored in a crystalline form as a zinc insulin complex. hence, the zinc concentration of the pancreatic b cells is among the highest in the body. addition of zinc to insulin in vitro extended the duration of insulin action. in the 1930s, zinc ions were added in vitro to produce protamine zinc insulin to control the blood sugar in diabetic patients. in the presence of zinc ions, both insulin and proinsulin dimers aggregate into hexamers containing bound zinc (80) . znt8 mrna and protein has been shown to be almost exclusively confined to pancreatic islets and to participate in the regulation of insulin secretion (80) . znt8 is believed to be crucial for both zinc transport in the insulin granules and insulin crystallization, which could not occur unless zinc is present. zinc is a potent physiological regulator of insulin signal transduction, mainly through the inhibitory effect on protein tyrosine phosphatase 1b, the key phosphatase that dephosphorylates the insulin receptor. an adequate supply of zinc is crucial for insulin biosynthesis and storage, especially when there is hyperglycemia. in zinc-deficient states, there is a clear decrease in islet cell insulin content (80, 81) . recent studies by jansen et al. (82) showed that zinc supplementation may be a potential treatment adjunct in type 2 diabetes because zinc also promotes insulin signaling. the decrease in total body zinc in diabetic patients may be due to either hyperzincuria or decreased intestinal zinc absorption. decreased zinc in plasma, lymphocytes, granulocytes, platelets, and hyperzincuria has been observed in diabetic patients (83) . zinc deficiency contributes to diabetic complications such as increased susceptibility to infections due to immune dysfunction, increased generation of inflammatory cytokines, and increased oxidative stress. in streptozotocin-induced diabetic rats, zinc supplementation (5 mg/kg zinc sulfate) attenuated diabetes-induced renal oxidative damage and inflammation and prevented the kidney from diabetes-induced proteinuria and pathological alterations (80) . a positive correlation between hyperzincuria and glycosylated hemoglobin has been observed (80, 84) . inflammatory cytokines such as il-1b, il-6, and tnf-a play important roles in the development and complications of type 2 diabetes. insulin signaling interference by adipokines leads to insulin resistance, as demonstrated by serine phosphorylation of insulin receptor substrate by tnf-a (75, 78) . crp and il-6 were significantly increased in the diabetic group compared with the nondiabetic controls in 1 study (80, 84) . in nested case-control and prospective trials, it was shown that il-1b, il-6, tnf-a, and crp were elevated several years before the onset of type 2 diabetes (80, 84) . in our limited trial, we supplemented 9 type 2 diabetes 45 mg/d elemental zinc (as gluconate) and 7 subjects received placebo for 3 mo. the plasma zinc increased significantly and hba1 c decreased in the zinc-supplemented group (p = 0.03) plasma il-6, crp, and icam-1 showed a statistically nonsignificant decrease in the zinc group. obviously a large placebo-controlled trial is needed before any firm conclusion can be drawn. a prospective study of zinc intake and risk of type 2 diabetes was assessed among 82,297 women 33-60 y of age at baseline in 1980 and followed until 2004 in the nurses' health study (85) . during the 24 y of follow-up, they identified and confirmed 6030 cases of incident type 2 diabetes. in an age-adjusted analysis, intake of total zinc but not dietary zinc from food sources was significantly associated with a lower risk of type 2 diabetes. after adjustment for nondietary risk factors, including age, bmi, smoking, and other covariate, the highest quintiles of both total and dietary zinc intake were significantly associated with an w20% lower risk of type 2 diabetes. at the cellular blood level, zinc increases total protein phosphorylation of the insulin receptor b subunit of both preadipocytes and adipocytes. zinc inhibits tyrosine phosphatase 1b, leading to increased phosphorylation of the insulin receptor b subunit. an intracellular release of free zinc is a potent physiological regulator of insulin signal transduction through its inhibitory effect on tyrosine phosphatase 1b, the key phosphatase that dephosphorylates the insulin receptor (80) . thus, this evidence implicates an important role of zinc in diabetes management, and i recommend a clinical trial of zinc in this disease. the first description of ad was published by alois alzheimer in 1906 (86) . ad now represents the most prevalent form of dementia in modern society, accounting for 60%-80% of all dementia cases (87) . individuals with ad have a progressive cognitive decline that ultimately also erodes all higher order executive functioning. at autopsy, they exhibit a number of cardinal features within the brain. the most pronounced features are the presence of extracellular deposits known as plaques and intracellular accumulations known as neurofibrillary tangles. these 2 features are diagnostic of ad. other gross manifestations are the thinning of the cortical gray matter, the enlargement of the ventricular spaces, and generalized atrophy of the brain (87) . the potential role of zinc in dementia was first proposed by burnet (88) . since then, many reports have been published concerning the role of zinc in ad. the concentrations of zinc within the brain tend to increase from birth to adulthood and then remain stable throughout life. the serum/ plasma zinc in ad patients has been reported to be increased, unchanged, and decreased (87, 89, 90) ; thus, there appears to be a large degree of variations between different study groups. there is no difference in zinc level in frontal lobe tissue of brain between ad and controls. however, when these tissues were subfractionated, a significant decrease in zinc levels in the nuclear fraction (but not the mitochondrial or microsome fraction) of ad cases was observed (87) . decreased zinc levels were also seen in the neocortex, superior frontal and parietal gyri, medial temporal gyrus, thalamus, and hippocampus in ad patients (87) . studies showed that senile plaque cores of ad patients consist primarily of b-amyloid protein (ab) derived from a larger transmembrane protein amyloid precursor protein (app) with both zinc and copper coordinated to histidine residues located at the n-terminal end of the protein (87) . it appears that a dysregulation in various metal storage and transport proteins within the ad brain may contribute to a failure in zinc homeostasis (87) . there are several key proteins involved in the regulation of zinc in the brain. these include zinc transporting zip and znt and metallothionein. furthermore, a number of these proteins such as znt family are found not only in the neuropil but also within the plaques (87) . app is ubiquitously expressed. one function of app is to participate in the maintenance of metal ion homeostasis. an abnormal expression of app might result in dysregulation of metal ion homeostasis. recently, it was reported that app possesses iron-export ferroxidase activity that is inhibited by zinc (87) . a disturbance in zinc homeostasis may result in decreased ferroxidase activity, resulting in iron accumulation. the processing of app to generate ab peptides of varying length involves different secretases, all of which appear to interact with different metals including zinc (87) . after generation of ab, it too is capable of binding zinc. it appears that metals promote the ab aggregation pathway. another important consequence of the binding of zinc to ab is that it obscures the proteolytic cleavage site (87) , thereby inhibiting its ability to be degraded by matrix metalloproteinase. removal of the zinc with clioquinol, however, restored the sensitivity of ab degradation by matrix metalloproteinase (87) . this discussion suggests a deleterious effect of zinc binding to app and ab; however, there are several studies that suggest a potentially protective effect of zinc under certain circumstances (87) . lovell (93) reported that ab/zn ratios 1:0.1 and 1:0.01 result in a protective activity against ab toxicity. this effect is mediated in part by a modulation of na + k + atpase activity that prevents the typical calcium dyshomeostasis and cell death associated with ab toxicity (87) . garai et al. (94) suggested that, in vitro, very low concentrations of zinc may eliminate oligomeric ab, thereby limiting the toxicity mediated by soluble oligomers. it has been suggested that zinc-ab aggregates may form so as to inhibit the reduction of cu 2+ and the production of hydrogen peroxide that arises from ab-cu interaction (87) . this represents the role of zinc as an antioxidant, which results by displacing pro-oxidant cu 2+ . zinc as a therapeutic modality for ad. studies using tg576 mice, which overexpress a mutant human form of app and develop cerebral ab plaques (87) , and znt3 knockout mice have suggested that zinc may hold amyloid load in a dissociable equilibrium and provide strong support for a role of synaptic zinc in the metabolism of ab. the study also showed a reduction in cerebrovascular amyloid loads in these animals. genetic ablation of znt3 may generate a phenocopy for the cognitive and synaptic deficits present in transgenic mouse models of ad (87) . recently, a number of metal-modulatory compounds have been developed that normalize cerebral zinc ion homeostasis (87) . clioquinol treatment in aged tg 2576 mice showed a significant reduction in plaque burden parallel with a shift in brain content of ab toward more soluble species (87) . analysis of cerebral metal levels did not reveal a reduction in metals content but showed a significant increase in copper and zinc on treatment. these data obtained after clioquinol and pbt2 (a metal-modulatory compound) treatment thus showed that there is a redistribution of metals in the brain and do not chelate metals out of the system (87) . pbt2 showed profound effects in transgenic mouse models of ad (87) . it was shown that the administration of pbt2 significantly decreased both insoluble and soluble burden, decreased levels of phosphorylated tau, increased levels of synaptophysin (a surrogate marker of synapses), and rapidly improved learning and memory performances in the morris water maze (87) . this drug is currently undergoing a clinical trial for the treatment of ad patients (87) . a placebo-controlled trial of zinc supplementation was conducted for 6 mo recently in ad patients in florida (95) . the subjects received 150 mg/d of elemental zinc away from food. the new zinc product, reazin, was developed by adeona pharmaceuticals, ann arbor, mi. the product releases zinc slowly in the stomach and later in the small intestine, which maintained a sustained elevation of plasma zinc throughout the day. the sum of scores on 3 cognitive measures, alzheimer's disease assessment scale-cognitive subscale, mini-mental state examination, and clinical dementia rating scale, were assessed. the study showed that zinc had stabilized cognition, whereas the placebo group continued to deteriorate. in patients age 70 y and older, the study showed a statistically significant effect of zinc supplementation on cognitive function. recent studies showed that neuronal stress and consequent overexpression of proinflammatory proteins are the likely instigators of neuropathological changes, including both plaque and tangle formation in ad patients (96) . it is believed that microglia cells are essentially the immune cells of the brain. it was shown that neurons were activating microglia to release il-1, which in turn activated astrocytes and caused them to release s100, a soluble astrocyte cabinding protein with proinflammatory effects. thus, it appears that il-1 and s100 are associated with ad (96). il-1 was shown to upregulate the production of bapp in cord blood and brain; thus, excess inflammatory cytokine il-1 was a driving force in neurodegeneration and genesis of amyloid plaques (96) . brain with il-1 pellets had elevated levels of the mrna of a phosphorylating kinase protein mitogen-activated protein kinase 38. it was shown that il-1 was driving both the production of tau protein and its hyperphosphorylation via il-1 induction of a specific kinase: mitogen-activated protein kinase p38 (96) . il-1 may also contribute to the memory deficits in ad by decreasing the levels of neurotransmitter acetylcholine, a decrease often seen in ad patients. it was observed that the enzyme acetyl cholinesterase, which degrades acetylcholine, is upregulated by il-1 (96) . we reported in the past that zinc deficiency in humans and in cell culture models activates macrophages and monocytes by increasing oxidative stress and via upregulation of nf-kb activation generates mrna of il-1b and the cytokine protein (37, 44, 46) . i hypothesize that similar phenomenon may be taking place in zinc-deficient elderly subjects such that microglia cells are being oxidatively stressed due to zinc deficiency leading to upregulation of il-1b in brain cells. increased il-1b in turn could upregulate the generation of ab and tau proteins and hyperphosphorylation of tau protein, leading to formation of neurofibrils and damage to neuronal cells. if this hypothesis is correct, one would see an effective prevention of ad in zinc-deficient elderly patients who are supplemented with zinc (45 mg/d elemental zinc) to correct their deficiency. this hypothesis needs to be tested. if correct, zinc supplementation may prove to be a very important therapeutic adjunct of ad. chemical studies on vegetation zinc in the nutrition of the rat syndrome of iron deficiency anemia, hepatosplenomegaly, hypogonadism, dwarfism, and geophagia zinc metabolism in patients with the syndrome of iron deficiency anemia, hypogonadism and dwarfism human zinc deficiency, endocrine manifestations and response to treatment biochemical studies on dwarfism, hypogonadism, and anemia national academy of sciences guidelines for essential trace element preparation for parenteral use. a statement by an expert panel. ama department of foods and nutrition the zinc content of epidermal structures in beriberi the zinc and copper contents of the organs and tissues of chinese subjects the normal occurrence of zinc in biological materials: a review of the literature, and a study of the normal distribution of zinc in the rat, cat, and man zinc metabolism in hepatic dysfunction -serum zinc concentrations in laennec's cirrhosis and their validation by sequential analysis the absorption and excretion of zinc studies on zinc in blood studies on zinc in blood zinc deficiency in a patient with retarded growth, hypogonadism, hypogammaglobulinemia, and chronic infection low levels of zinc in hair, anorexia, poor growth and hypogeusia in children zinc deficiency in man: the shiraz experiment zinc deficiency in acrodermatitis enteropathica a novel member of a zinc transporter family is defective in acrodermatitis enteropathica biochemistry of zinc effect of nutrition on serum zinc concentration during pregnancy in turkish women zinc deficiency and intravenous feeding skin lesions during intravenous hyperalimentation: zinc deficiency zinc deficiency in two infants during parenteral alimentation for diarrhea zinc deficiency in rapidly growing preterm infants zinc deficiency following penicillamine therapy the role of zinc in growth and cell proliferation growth hormone and bone decline in somatomedin-c, insulin-like growth factor-1, with experimentally induced zinc deficiency in human subjects reduced liver insulin-like growth factor-1 gene expression in young zinc-deprived rats in associated with a decrease in liver growth hormone (gh) receptors and serum gh-binding protein picomolar concentrations of free zinc (ii) ions regulate receptor protein tyrosine phosphatase beta activity zinc deprivation of murine 3t3 cells by use of dieth-ylenetrinitrilopentaacetate impairs dna synthesis upon stimulation with insulin-like growth factor-1 (igf-i) zinc deficiency affects cell cycle and deoxythymidine kinase (tk) gene expression in hut-78 cells experimental zinc deficiency in humans decreased expression of cd73 (ecto-59-nucleotidase) in the cd8+ subset is associated with zinc deficiency in human patients changes in cytokine production and t cell subpopulations in experimentally induced zinc deficient humans dardenne m. serum thymulin in human zinc deficiency roles of zinc and zinc signaling in immunity: zinc as an intracellular signaling molecule toll-like receptor-mediated regulation of zinc homeostasis influences dentritic cell function signal transduction in monocytes: the role of zinc ions zinc in human health. amsterdam, the netherlands zinc and immune function: the biological basis of altered resistance to infection antioxidant effect of zinc in humans zinc-suppressed inflammatory cytokines by induction of a20-medated inhibition of nuclear factor-kb zinc decreases c-reactive protein, lipid peroxidation, and implication of zinc as an atheroprotective agent intracellular free zinc up-regulates ifn-g and t-bet essential for th1 differentiation in con-a stimulated hut-78 cells zinc treatment in malabsorption plasma ammonia and liver ornithine transcarbamoylase activity in zinc-deficient rats increased purine nucleotide cycle activity associated with dietary zinc deficiency zinc in human health zinc metabolism in uremia effect of oral zinc therapy on gonadal function in hemodialysis patients effect of zinc supplementation on incidence of infections and hospital admissions in sickle cell disease (scd) zinc supplementation decreased oxidative stress, incidence of infection and generation of inflammatory cytokines in sickle cell disease patients zinc supplementation in young children with acute diarrhea in india zinc in human health zinc as adjunct treatment in infants aged between 1 and 120 d with probable serious bacterial infection: a randomized, double-blind, placebo controlled trial duration of symptoms and plasma cytokine levels in patients with the common cold treated with zinc acetate duration and severity of symptoms and levels of plasma interleukin-1 receptor antagonist, soluble tumor necrosis factor receptor, and adhesion molecule in patients with common cold treated with zinc acetate reduction in duration of common cold by zinc gluconate lozenges in a double-blind study zinc for the common cold zinc lozenges may shorten the duration of colds: a systematic review zinc for the treatment of the common cold: a systematic review and meta-analysis of randomized control trials practical recommendations and new therapies for wilson's disease the uses of pharmacologic doses of zinc in the treatment of sickle cell anemia oral zinc therapy for wilson's disease hypocupremia induced by zinc therapy in adults intestinal metallothionein and the mutual antagonism between copper and zinc in the rat zinc and human health zinc content of human retinal pigment epithelium decreases with age and macular degeneration but superoxide dismutase activity increases a randomized, placebo controlled, clinical trial of high-dose supplemented with vitamins c and e, beta-carotene, for age-related macular degeneration and vision loss association of mortality with ocular disorders and an intervention of high dose antioxidants and zinc in the age-related eye disease study zinc deficiency in the elderly patients zinc supplementation decreases incidence of infections in the elderly: effect of zinc on generation of cytokines and oxidative stress zinc intake of us population findings from the third national health and nutrition survey 1988-1994 inflammation in atherosclerosis dysregulation between th1 and th2 subpopulations in the elderly zinc and diabetes the pharmacology of the insulinomimetic effect of zinc complexes disturbed zinc homeostasis in diabetic patients by in vitro and in vivo analysis of insulinomimetic activity of zinc cellular zinc in patients with diabetes mellitus tumor necrosis factor-a induced insulin resistance in 3t3-l1 adipocytes is accompanied by a loss of insulin receptor substrate-1 and glut-4 expression without a loss of insulin receptor-mediated signal transduction prospective study of zinc intake and risk of type 2 diabetes in women alois alzheimer and alzheimer's disease: a centennial perspective zinc and alzheimer's disease a possible role of zinc in the pathology of dementia serum zinc, copper, insulin and lipids in alzheimer's disease epsilon 4 apolipoprotein e allele carrier serum zinc in the progression of alzheimer's disease cerebrospinal fluid levels of transition metal in patients with alzheimer's disease zinc, copper and magnesium concentration in serum and csf of patients with neurological disorders a potential role for alterations of zinc and zinc transport proteins in the progression of alzheimer's disease selective destabilization of soluble amyloid beta oligomers by divalent metal ions copper excess, zinc deficiency, and cognition loss in alzheimer's disease what causes alzheimer's? the scientist the sole author had responsibility for all parts of the manuscript. key: cord-013803-d1sbfibq authors: abu el-asrar, ahmed m.; berghmans, nele; al-obeidan, saleh a.; gikandi, priscilla w.; opdenakker, ghislain; van damme, jo; struyf, sofie title: soluble cytokine receptor levels in aqueous humour of patients with specific autoimmune uveitic entities: scd30 is a biomarker of granulomatous uveitis date: 2019-12-05 journal: eye (lond) doi: 10.1038/s41433-019-0693-7 sha: doc_id: 13803 cord_uid: d1sbfibq purpose: soluble cytokine receptors are potential biomarkers for immune activation and have a promising potential as immunotherapeutic agents. we investigated the levels of soluble cytokine receptors in aqueous humour (ah) samples from patients with specific autoimmune uveitic entities. methods: patients with active uveitis associated with behçet’s disease (bd) (n = 13), sarcoidosis (n = 8), hla-b27-related inflammation (n = 12), vogt–koyanagi–harada (vkh) disease (n = 12) and control subjects (n = 9) were included. ah samples were analyzed with the use of multiplex assays for the proinflammatory cytokine tumour necrosis factor (tnf)-α and the soluble cytokine receptors scd30, scd163, sgp130, sil-6 receptor-α (sil-6r), stnfri and stnfrii. results: tnf-α and soluble cytokine receptor ah levels were significantly higher in uveitis patients (n = 45) compared with controls (n = 9). when nongranulomatous uveitis (bd and hla-b27-associated uveitis) was compared with granulomatous uveitis (sarcoidosis and vkh disease), the levels of scd30 and stnfri/tnf-α and stnfrii/tnf-α ratios were significantly enhanced in granulomatous uveitis. finally, when comparing the profile in the specific uveitis entities, scd30 levels were highest in patients with vkh disease. sgp130, scd163, sil-6r, stnfri and stnfrii did not differ significantly between the four different clinical uveitic subgroups. conclusions: soluble cytokine receptors are significantly upregulated in autoimmune uveitis. cd30(+) t cells might contribute to the inflammatory process in granulomatous uveitis, particularly in vkh disease. granulomatous uveitis is also characterized by significantly higher stnfrs/tnf-α ratios than nongranulomatous uveitis. patients with autoimmune uveitis present with heterogeneous clinical manifestations of intraocular inflammation and are at risk for severe visual impairment. the patients often also suffer from systemic diseases, such as behçet's disease (bd), sarcoidosis, human leucocyte antigen (hla)-b27-associated inflammation and vogt-koyanagi-harada (vkh) disease, which present with different clinical phenotypes. these specific uveitic entities presumably arise as a consequence of different underlying mechanisms, each involving distinct immune molecules [1] [2] [3] [4] . cytokines are key regulators of inflammation and are thought to play important roles in the pathophysiology of various autoimmune uveitis entities [1] [2] [3] [4] [5] . previous studies demonstrated upregulation of proinflammatory cytokines, such as tumour necrosis factor-α (tnf-α), interferon-γ (ifn-γ), interleukin (il)-6, il-15 and il-17 in the aqueous humour (ah) samples from autoimmune uveitis patients [1] [2] [3] [4] [5] . selective blockade of proinflammatory cytokines, therefore, may represent a possible way for clinical intervention. this is exemplified by the introduction and considerable clinical benefits of novel biologicals, such as anti-tnf-α agents and anti-il-6 receptor (il-6r) antibody for the treatment of autoimmune uveitis resistant to conventional treatment [6] [7] [8] . soluble cytokine receptors have been thoroughly studied in experimental and clinical studies as potential biomarkers of immune activation and/or disease activity in many clinical conditions. furthermore, soluble cytokine receptors have a promising potential as immunotherapeutic agents for several autoimmune diseases [9] . cluster of differentiation 30 (cd30), belonging to the tnf-nerve growth factor receptor superfamily, was first characterized as a cell surface antigen on hodgkin's and reed-sternberg cells [10, 11] . subsequently, cd30 was also found on activated cd45ro + memory t helper (th) cells and cd8+ t cells, but is absent on naive and resting t cells [12] . in addition, it was demonstrated that t cells isolated from peripheral blood of healthy individuals expressing cd30 produced more cytokines (ifn-γ or il-5) than cd30 negative t cells [13] . activated th cells expressing cd30 exhibited enhanced helper activity for b cell immunoglobulin production [13] . a soluble form of cd30 is released by cd30 + cells in vitro and in vivo by proteolytic cleavage. the level of scd30 was demonstrated to correlate well with surface expression of cd30 and can, hence, be applied as a marker for cd30 expression [10, 11] . cd163 is a member of the cysteine-rich scavenger receptor family class b and is expressed exclusively by the monocyte-macrophage cell lineage. as an endocytic receptor for haemoglobin-haptoglobin complexes cd163 is proposed to capture free circulating haemoglobin. cd163 may have anti-inflammatory and immunoregulatory properties, since it decreases in vitro activation and proliferation of t lymphocytes. the proteolytic cleavage of the membrane-bound cd163 by matrix metalloproteinases in response to inflammatory stimuli releases scd163. thus, scd163 may be viewed as a biomarker for macrophage activation in several inflammatory disorders [14, 15] . dysregulated expression of il-6 is implicated in the development of various chronic inflammatory autoimmune diseases [16] [17] [18] . the biological activities of il-6 are mediated by a receptor complex comprising the il-6 binding transmembrane glycoprotein il-6r-α and the transmembrane signal transducer protein gp130 that is shared by additional cytokines of the il-6 family [19] . the high-affinity complex of il-6 and il-6r interacts with two gp130 chains and activates the underlying signal transduction pathways. gp130 is broadly expressed, whereas il-6r is expressed restrictively in liver cells and certain leucocyte subtypes [19] . soluble gp130 (sgp130) isoforms are generated by alternative splicing. the soluble il-6r (sil-6r) also arises through alternative splicing, in addition to ectodomain shedding. sil-6r and intact membrane-bound il-6r bind il-6 with comparable affinity. contrary to the soluble receptors of tnf-α, which inhibit the activity of tnf-α, the il-6/sil-6r complex can via interaction with gp130 activate target cells, in the absence of the classical membrane-embedded il-6r. such cells cannot be activated by il-6 alone when sil-6r is not available. this latter process is referred to as il-6 trans-signalling, as opposed to classical signalling via surface il-6r. it is increasingly apparent that many of the activities assigned to il-6 are mediated via sil-6r [19, 20] . since sgp130 binds the complex of il-6/sil-6r, sgp130 acts as a natural inhibitor of il-6 trans-signalling in vivo, without interfering with classical signalling [19, 20] . tnf-α recognizes two distinct cell surface receptors, namely tnf-α receptor type i (tnfri) and type ii (tnfrii). in addition to membrane-bound forms, both tnfri and tnfrii also exist as soluble forms produced by proteolytic cleavage [21, 22] . the soluble receptors stnfri and stnfrii retain their ligand binding capacity and can act as natural inhibitors by sequestering tnf-α and avoiding activation of its membrane-embedded signalling receptors. as tnf-α itself is a principal inducer of stnfrs shedding and expression, determination of stnfri and stnfrii levels could provide indirect evidence of tnf-α production and reflect the activation state of the tnf-α/ tnfr system [21, 22] . we hypothesized that different immunopathogenic mechanisms are involved in each clinical subtype of autoimmune uveitis. through analysis of the soluble cytokine receptor profile in ah from patients with specific clinical entities of autoimmune uveitis, a better understanding of the underlying immune mechanisms may be obtained. in addition, it may also be of clinical importance for disease diagnosis and identification of potential targets for selective therapy. for these reasons, we analyzed the ah from patients with active uveitis associated with four systemic inflammatory diseases (sarcoidosis, vkh disease, bd and hla-b27-related inflammation) for the presence of scd30, scd163, sgp130, sil-6r, stnfri and stnfrii. furthermore, the levels of soluble receptors were correlated with the levels of tnf-α, a major proinflammatory cytokine, and with clinical disease activity. all patients included in the study were seen at the outpatient clinic of king abdulaziz university hospital and all gave informed and written consent. as controls for patients with active uveitis (n = 45; n = 13 for bd, n = 12 for hla-b27associated uveitis; n = 8 for sarcoidosis; n = 12 for vkh disease), patients who had undergone cataract extraction with no prior history of uveitis (n = 9) were included. diagnosis was made as described before following established clinical criteria, with supporting laboratory evidence as needed [4, 23, 24] . in each patient, the uveitis activity was graded according to the criteria of the standardization of the uveitis nomenclature working group grading scheme [25] . none of the patients was on topical or systemic therapy on presentation. ah sampling was done before the start of therapy. by means of limbic paracentesis 100-200 µl of ah was aspirated and processed as described [1] [2] [3] [4] . the demographic and clinical characteristics of the included patients are described in detail in a previous report in table 1 ; duplicated with permission from [4] . all procedures were performed according to the tenets of the declaration of helsinki and the protocol of this study was approved by the research center, college of medicine, king saud university. we determined the profile of tnf-α and the soluble cytokine receptor levels in the ah samples using two multiplex assays. the first multiplex contained detection reagents for the proinflammatory cytokine tnf-α (bio-plex catnr 171ak99mr2, biorad, hercules, ca, usa), the second multiplex assessed the soluble cytokine receptors scd30, scd163, sgp130, sil-6r, stnfri and stnfrii (bio-plex catnr 171al001m, biorad). the two analyses were performed according to the manufacturer's guidelines in separate plates on consecutive days on the same ah samples. in each array, 8 µl of ah was used after dilution with 56 µl of assay buffer (biorad). the data were obtained 58.2 ± 9.1 28.9 ± 4.2 [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] 25.1 ± 6.5 [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] 34.3 ± 9.3 28.3 ± 7.2 vkh vogt-koyanagi-harada when considering the whole group of patients, tnf-α and soluble cytokine receptor levels were significantly enhanced in ah of patients compared with controls ( table 2) . compared with controls, tnf-α, scd163, sgp130, sil-6r, stnfri and stnfrii levels were significantly higher in bd and hla-b-27-associated uveitis. the levels of tnf-α, scd30, scd163, sgp130, sil-6r, stnfri and stnfrii were significantly elevated in sarcoidosis. the levels of scd30, scd163, sil-6r, stnfri and stnfrii were significantly higher in vkh disease ( table 3) . next, the kruskal-wallis test was applied to compare the distribution of tnf-α and soluble cytokine receptor levels among the four disease groups. among the cytokine and soluble cytokine receptors analyzed, tnf-α and scd30 differed significantly between patients with bd, sarcoidosis, hla-b27-associated uveitis and vkh disease (p = 0.029; p = 0.001, respectively) (fig. 1a) . tnf-α levels were significantly increased in hla-b27-associated uveitis compared with vkh disease (mann-whitney test; because the biological activity of tnf-α is regulated by stnfri and stnfrii in vivo, the ratios of tnf-α to its soluble receptors were calculated for the ah samples. when considering the whole patient group, the stnfri/ tnf-α ratio was significantly higher in the ah of controls than in uveitis patients. on the other hand, the stnfrii/ tnf-α ratio was increased in uveitis patients compared with controls, but significance was not reached (table 2 ). in the four disease groups, the stnfri/tnf-α ratio was significantly higher in controls than in bd and hla-b27associated uveitis. however, the stnfri/tnf-α ratio did not differ significantly between controls and sarcoidosis or between controls and vkh disease. the stnfrii/tnf-α ratio did not differ significantly between controls and individual disease groups (table 3) . next, we evaluated the distribution of stnfri/tnf-α and stnfrii/tnf-α ratios among the four disease groups (kruskal-wallis test). no statistically significant differences were observed. when analysis was done according to subdivision of the patients into those with granulomatous uveitis (sarcoidosis and vkh disease) (n = 20) and those with nongranulomatous uveitis (bd and hla-b27-associated uveitis) (n = 25), scd30 levels and stnfri/tnf-α and stnfrii/tnf-α ratios in granulomatous uveitis were significantly higher than those in nongranulomatous uveitis. on the other hand, tnf-α levels in nongranulomatous uveitis were significantly higher than those in granulomatous uveitis. levels of scd163, sgp130, sil-6r, stnfri and stnfrii were similar in granulomatous and nongranulomatous uveitic diseases (table 4 and fig. 1b) . in the whole group of patients, the levels of tnf-α correlated significantly with the levels of stnfri (r = 0.72; p < 0.001) and stnfrii (r = 0.64; p < 0.001). significant positive correlations were found between ah levels of tnf-α (r = 0.624; p < 0.001), sil-6r (r = 0.384; p = 0.009), stnfri (r = 0.378; p = 0.01) and stnfrii (r = 0.364; p = 0.014) and clinical disease activity. on the other hand, there was a significant negative correlation between stnfri/tnf-α ratio and clinical disease activity (r = −0.337; p = 0.027). there were no significant correlations between clinical disease activity and scd30, scd163, sgp130 and the stnfrii/tnf-α ratio. in the present study of 45 patients, elevated levels of scd30 in the ah samples from patients with autoimmune uveitis might reflect a recruitment of cd30 + t cells into the ocular inflammatory microenvironment of patients with uveitis. our subgroup analysis showed that scd30 levels were significantly higher in patients with vkh disease than in patients with bd and patients with hla-b27-associated uveitis. among the four disease groups, the levels of scd30 were elevated 7.8-fold, 6.4-fold, 1.5-fold and 2.0-fold in patients with vkh disease, sarcoidosis, bd and hla-b27associated uveitis, respectively, compared with controls. in addition, scd30 levels were significantly higher in patients with granulomatous uveitis associated with sarcoidosis and vkh disease than in patients with nongranulomatous uveitis associated with bd and hla-b27-associated uveitis. our findings suggest that cd30 + t cells contribute to the inflammatory process in patients with granulomatous uveitis, particularly in vkh disease. similarly, previous studies demonstrated that cd30 + t cells are involved in the pathogenesis of several granulomatous inflammatory diseases [26] [27] [28] . recently, shinoda et al. [29] demonstrated that cd30 knockout mice showed milder symptoms in a model of experimental autoimmune encephalomyelitis and less antigen-specific th1 and th17 cells were induced. these findings suggest that cd30 expression on cd4 + t cells is implicated in the pathogenesis of autoimmune diseases of the central nervous system. previous reports demonstrated high serum levels of scd30 in both th1-and th2-dominated disorders [10, 11] . pellegrini et al. [30] reported that cd30 may be an essential costimulatory molecule and marker for an important immunoregulatory subpopulation of t cells which controls the th1/th2 balance in immune responses. increased levels of scd30 have been reported in several autoimmune diseases [31] [32] [33] [34] [35] [36] [37] . in addition, elevated scd30 serum concentrations correlated with clinical and laboratory parameters of disease activity [31, [33] [34] [35] . macrophages activated by classical pathways (m1) have proinflammatory properties. conversely, alternatively activated m2 macrophages which express cd163 have antiinflammatory, antioxidant, tissue repair, proangiogenic and fibrosing roles. therefore, scd163 may represent a marker for alternatively activated macrophages [14, 15] . the value of scd163 as a marker of macrophage activation has been confirmed in several inflammatory conditions [38] [39] [40] [41] [42] [43] [44] [45] [46] [47] . the present study is the first to demonstrate high levels of scd163 in the ocular microenvironment of patients with autoimmune uveitis, scd163 levels being elevated about 50-fold, compared with controls. these data suggest that active autoimmune uveitis may be associated with increased macrophage activation and are consistent with previous reports that macrophages are involved in the pathogenesis of autoimmune uveitis [48] . the detected rise in scd163 may be due to upregulation and/or augmented shedding of cd163. increased shedding might be correlated to elevated levels of cd163-cleaving proteinases in the ocular inflammatory microenvironment of these patients. several studies demonstrated that il-6 trans-signalling via the sil-6r is critically involved in the initiation and maintenance of several inflammatory and autoimmune diseases [19, 20] . elevated sil-6r levels have been documented in synovial fluids of patients with rheumatoid arthritis [49, 50] . in addition, elevated levels of il-6 and sil-6r correlated with the degree of joint destruction in rheumatoid arthritis [49] . in a previous study, we demonstrated upregulation of the proinflammatory cytokine il-6 in ah samples from patients with autoimmune uveitis [1] . in the present study, sil-6r levels were elevated 10.6-fold compared with controls, whereas levels of the inhibitory sgp130 were elevated only 2.6-fold. these findings suggest that the regulation of il-6 trans-signalling may be distorted in autoimmune uveitis. interestingly, selective targeting of sil-6r-mediated events may represent a novel avenue for therapeutic intervention [19, 20] . in animal models of arthritis, specific inhibition of sil-6r-mediated signalling by intra-articular injection of sgp130 effectively suppressed all parameters of disease severity [51] . similarly, in animal models of colitis, application of a gp130-fc fusion protein specifically neutralizating sil-6r suppressed colitis activity and induced apoptosis of lamina propria t cells. these results suggest that a t cell activation pathway driven by the il-6/sil-6r complex supports chronic intestinal inflammation [52] . collectively, these findings suggest that specific therapeutic targeting of sil-6r may be explored as a novel strategy for the treatment of autoimmune uveitis. indeed, neutralizing antibodies against il-6r have proven to be effective in uveitis [8] . in the present study, the levels of stnfri and stnfrii were elevated 7.8-fold and 47.1-fold, respectively, compared with controls. our results might reflect the cellular attempt to antagonize the effect of tnf-α by cleavage of the surface receptors. similarly, several studies reported increased levels of stnfri and stnfrii in inflammatory and autoimmune diseases [53] [54] [55] [56] [57] [58] [59] [60] [61] . moreover, stnfri and stnfrii levels correlated with disease activity [53, [55] [56] [57] [58] [59] . our findings also suggest the preferential shedding of stnfrii in patients with autoimmune uveitis. these results are in agreement with previous studies that demonstrated higher levels of stnfrii than stnfri levels in the synovial fluid of patients with rheumatoid arthritis [55, 56] . cellular responses to tnf-α are influenced by, amongst other factors, the extracellular concentrations of stnfri and stnfrii [21, 22] . indeed, elevated concentrations of stnfrs have been demonstrated to inhibit the activity of tnf-α both in vitro and in vivo [62] . the relative proportion of tnf-α to its soluble receptors in biological fluids has been proven to be important in a number of clinical conditions [61] [62] [63] [64] [65] . diminished ratios of stnfri/tnf-α and stnfrii/tnf-α were observed in children with meningococcal septicaemia with a fatal outcome compared with survivors [63] . increased joint destruction in polyarticular juvenile chronic arthritis may be related to the lower stnfri/tnf-α and stnfrii/tnf-α ratios [61] . considering the inhibitory effects of the soluble receptors for tnf-α, we wondered whether the ratios of stnfrs/ tnf-α would be different among the disease groups, and be differentially distributed compared with the receptor levels. indeed, the ratios were significantly higher in patients with granulomatous uveitis associated with sarcoidosis and vkh disease than in patients with acute nongranulomatous uveitis associated with bd and hla-b27-associated uveitis. these findings suggest that tnf-α would exert more destructive effects in the ocular inflammatory microenvironment of patients with acute nongranulomatous uveitis when compared with granulomatous uveitis and provides an explanation for the different clinical courses of these uveitis subgroups. this may also reflect different pathogenic mechanisms in the two disease groups. this study has several limitations regarding experimental methods and designs. firstly, our control group consisted of patients who had undergone elective cataract extraction with no prior history of uveitis. accordingly, the control patients were older than patients with uveitis. the relationship between age and human ah cytokine levels was recently investigated. increased concentration of proinflammatory and proangiogenic cytokines with age was demonstrated [66] . although the control patients might have a proinflammatory state, the statistical analysis gave clear differences in comparisons with patients with uveitis. younger controls might have yielded differences with higher statistical significance. furthermore, analysis of ah samples was performed at one-time point during the course of disease. therefore, it was not possible to evaluate the effect of the interval between onset of inflammation and ah sampling and intensity of inflammation on the levels of the studied soluble cytokine receptors. animal models are needed to explore the effect of uveitis activity and to investigate the expression of these soluble cytokine receptors at different time points. nevertheless, with the use of a multiplex assay, we analyzed ah samples from patients with several specific clinical entities of autoimmune uveitis in parallel allowing measurements of these soluble cytokine receptors simultaneously in the same samples. in conclusion, our findings suggest that cd30 + t cells might be involved in the regulation of inflammation in granulomatous uveitis, particularly vkh disease and that active autoimmune uveitis might be associated with increased macrophage activation, since we observed elevated scd163 levels. granulomatous uveitis is characterized by significantly higher stnfrs/tnf-α ratios than acute nongranulomatous uveitis. finally, specific therapeutic targeting of sil-6r-mediated signalling may represent a novel strategy for the treatment of autoimmune uveitis. to further investigate the relative importance of these soluble cytokine receptors, intervention functional studies in animal models are necessary. what was known before what this study adds • cd30 + t cells might be involved in the pathogenesis of granulomatous uveitis, particularly vogt-koyanagi-harada disease. • active autoimmune uveitis might be associated with increased macrophage activation, since we observed elevated scd163 levels. • granulomatous uveitis is characterized by significantly higher stnfrs/tnf-α ratios than acute nongranulomatous uveitis. the cytokine interleukin-6 and the chemokines ccl20 and cxcl13 are novel biomarkers of specific endogenous uveitic entities distinct cytokine and chemokine profiles in the aqueous of patients with uveitis and cystoid macular edema cytokine profiles in aqueous humor of patients with different clinical entities of endogenous uveitis differential cxc and cx3c chemokine expression profiles in aqueous humor of patients with specific endogenous uveitic entities intraocular cytokine environment in active behçet uveitis expert panel recommendations for the use of anti-tumor necrosis factor biologic agents in patients with ocular inflammatory disorders longterm clinical outcomes in patients with 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juvenile chronic arthritis tumor necrosis factor soluble receptors circulate during experimental and clinical inflammation and can protect against excessive tumor necrosis factor alpha in vitro and in vivo the j5 study group, et al. imbalance between tumour necrosis factoralpha and soluble tnf receptor concentrations in severe meningococcaemia imbalances between tumor necrosis factor-alpha and its soluble receptor forms, and interleukin-1beta and interleukin-1 receptor antagonist in bal fluid of cavitary pulmonary tuberculosis increased synovial fluid levels of interleukin-12, scd25 and stnf-rii/stnf-ri ratio delineate a cytokine pattern characteristic of immune arthropathies age-related proinflammatory and pro-angiogenic changes in human aqueous humor conflict of interest the authors declare that they have no conflict of interest.publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-275413-e2rhioty authors: rowland, raymond r.r. title: the interaction between prrsv and the late gestation pig fetus date: 2010-09-09 journal: virus res doi: 10.1016/j.virusres.2010.09.001 sha: doc_id: 275413 cord_uid: e2rhioty porcine reproductive and respiratory syndrome virus (prrsv) crosses the placenta during late gestation and productively infects the fetus. virus replication and cytokine responses were measured in tissues of fetuses recovered at 109–112 days of gestation, just prior to parturition. at the time of recovery, gross anatomical abnormalities were evident in both infected and non-infected fetuses from the infected dams. virus isolation and immunohistochemistry identified the thymus as the primary site of virus replication. steady state rt-pcr amplification of inflammatory, th1 and th2 cytokines, showed elevated ifn-γ and tnf-α mrnas in tissues from infected fetuses, which corresponded to elevated cytokine proteins in serum but not amniotic fluid. further evidence for induction of immunity was found in the hyperplastic response of lymph nodes, which included the development of germinal centers occupied cdw75+ b cells. collectively, these data support the notion that the immunocompetent fetus is capable of initiating an antiviral response, which is compartmentalized within the infected fetus. furthermore, fetal pathology may not be a direct result of virus replication in the fetus. porcine reproductive and respiratory syndrome (prrs) is caused by an enveloped positive-stranded rna virus, prrsv, belonging to the family arteriviridae cavanagh, 1997; nelsen et al., 1999; wensvoort et al., 1991) . other members of the arterivirus group include lactate dehydrogenase-elevating virus (ldv) of mice, equine arteritis virus (eav), and simian hemorrhagic fever virus (shfv; for review see plagemann, 1996) . the arteriviruses, toroviruses, roniviruses and coronaviruses form a single order, nidovirales. arteriviruses structurally resemble togaviruses, but similar to coronaviruses, replicate via a nested 3 -co-terminal set of subgenomic mrnas that possesses a common leader and a poly-a tail (reviewed in snijder and mulenberg, 1998) . the arteriviruses exhibit several important properties relevant to the study of viral pathogenesis, including cytopathic replication in macrophages, the capacity to establish and maintain an asymptomatic infec-tion, as well as cause severe and fatal disease (plagemann, 1996) . infection of adult pigs with prrsv usually produces a non-fatal disease, characterized by flu-like symptoms, a transient elevation in temperature and inappetance (reviewed in benfield et al., 1999; christianson et al., 1992) . the reproductive form of prrs occurs following the infection of late gestation pregnant gilts or sows. natural infection of the fetus with prrsv is initiated with the infection of gilts and sows at 90 days gestation. after productive replication on the maternal side, the virus crosses the placenta and productively infects the fetus. the mechanism of transplacental infection is unknown, but could be similar to the infected "trojan horse" macrophage, described for ldv (cafruny and bradley, 1996) . since the pig fetus becomes immunocompetent at about 70 days of gestation, prrsv infection occurs in an immune environment containing functional b and t cells. accordingly, virus-induced reproductive failure can present clinically as delayed returns to estrus, as well as abortions, mummified fetuses, stillborn and weak-born pigs christianson et al., 1993; collins et al., 1992; mengeling et al., 1994; rossow et al., 1999; rowland et al., 2003) . surviving neonates can exhibit the severest form of respiratory disease with mortality sometimes reaching 100% within three weeks after birth (feng et al., 2001; rossow et al., 1994; rossow, 1998) . the complex pathology following exposure to prrsv in utero represents a unique form of the disease referred to as congenital prrs (rowland et al., 2003) . the purpose of this study was to characterize the interaction between prrsv and the pig fetus by (1) identifying sites of virus replication, (2) measuring immune and inflammatory cytokines in different compartments, and (3) evaluating the response of lymph nodes. experiments involving animals were approved by the kansas state university iacu committee. pregnant sows, obtained from a closely monitored prrsv-negative herd, were challenged at 90 days gestation with a sixth passage isolate of sd-23983, a typical north american field isolate (rowland et al., 2001) . the methods for the preparation of the prrsv inoculum on marc-145 cells and infection of pigs are described in rowland et al. (2003) . virus was cultivated on marc-145 cells in mem supplemented with antibiotics (pen/step) and 7% fbs. dams, at 90 days gestation were challenged with approximately 10 5 tcid 50 of virus diluted in 5 ml of culture medium. one half of the inoculum was administered by intramuscular injection in the neck. the remaining dose was administered intranasally. mock-infected sows were challenged with medium recovered from marc-145 cells. dams were monitored for clinical signs and blood collected weekly. at between 109 and 112 days of an approximate 114 days gestation period, the dams were euthanized. the uterine horns were immediately removed and the individual fetuses with intact placenta were carefully removed and immediately necropsied. a sample of amniotic fluid was collected prior to removal. the brachial artery of each fetus was severed and blood collected using a disposable syringe and serum stored at −80 • c. maternal, accessory and fetal tissues were collected and stored in formalin for histological staining and immunohistochemistry (ihc), or storage in rnalater (ambion) for rt-pcr of cytokine mrnas. prrsv-specific antibody was measured in sera using the herdcheck ® prrs elisa (idexx) and performed by personnel at kansas state university veterinary diagnostic laboratory. serology results were reported as a sample/positive (s/p) ratio. an s/p ratio greater than 0.39 was considered positive for prrsv antibody. virus isolation (vi) in serum and tissues was performed as described in rowland et al. (2003) . briefly, serum was serially diluted in mem supplemented with pen/step antibiotics and 7% fbs and placed on 96 well plates of confluent marc-145 cells. after three days, plates were fixed in 80% acetone and stained with fitc-sdow-17 anti-nucleocapsid antibody, diluted in pbs with 5% fbs (nelson et al., 1993) . the results were reported as the log 10 of the inverse dilution of the last positive well. virus isolation from tissues was the same except that tissues were weighed and homogenized in hanks balanced salt solution and then centrifuged at 500 × g for 20 min to remove debris. the sequencing of the hypervariable region of orf5 is described in rowland et al. (1999) . total rna was prepared from serum or infected marc-145 cells using an rneasy kit (qiagen) according manufacturer's instructions. for pcr, cdna was prepared using mlv reverse transcriptase (promega) and 4msb as the primer. the sense and antisense primers for the outer amplification were 4msa, 5 -cttcgtcccttcttttcctcgtgg, and 4msb, 5 -ccgctctagagccaacgatagagtctgc, respectively. the product was re-amplified with a nested set of sense and antisense primers, 04a, 5 -accgtgtatgttaccatcacagcc and 04b, acgggaaagatgacaaaactctcc. thirty-two cycles of amplification were performed for each primer pair. the conditions for both amplifications included a 95 • c denaturing step (25 s), a 58 • c annealing step (10 s), and a 74 • c (25 s) polymerization step. the final pcr product, which contained the last 312 nucleotides of orf4, the 10 nucleotide untranslated region (utr), and the first 215 nucleotides of orf5 was sequenced directly by automated dna sequencing. pcr products were cloned into a pcr2.1 ta cloning vector (invitrogen), propagated in escherichia coli and individual plasmids sequenced using m13 forward and reverse primers. sequences were analyzed using gene jockey ii software. tissue samples for rt-pcr were immediately placed in rna-later (ambion) and stored at −80 • c. total rna was extracted from approximately 50 g of tissue using rneasy kit (qiagen) according to manufacturer's instructions. the design of cytokinespecific primers and rt-pcr procedures were performed according to reddy and wilkie (2000) . primer sequences are listed in table 1 . rna was diluted to a final volume of 50 l in nuclease free water. cdna was prepared from10 l of total rna by reverse transcription using molony murine leukemia virus reverse transcriptase (promega) and random hexamers as primers. the amplification of ␤2m mrna was used as an internal control. pcr amplification of cytokine and control cdnas consisted of 35 cycles (45 s at 94 • c, 45 s at 55 • c, and 45 s at 72 • c) and dna products electrophoresed on a 2.0% agarose gel and visualized using ethidium bromide. the identity of the dna products was confirmed by dna sequencing. tissue samples were collected and immediately placed in 10% buffered formalin. paraffin-embedded thin sections were mounted on slides, deparaffinized and stained with hematoxylin for the detection of prrsv antigen, slides were incubated for 30 min with a 1:100 dilution of mab sr-30 anti-nucleocapsid antibody (rural technologies). other antibodies included a polyclonal anti-human cd3 and b cell antibodies, anti-cdw75 and anti-cd79␣. bound antibody was detected with biotinylated goat anti-mouse or anti-rabbit ig followed by avidin-hrpo and dab chromagen (ventana medical). slides were counterstained with hematoxylin. the experiment incorporated four prrsv-infected and twomock-infected dams. all maternal serum samples were vi-negative and seronegative for prrsv prior to infection. between one and two weeks after virus challenge, all infected sows were vi-positive in serum, confirming the presence of an active infection. by the time of necropsy, the concentration of circulating virus in the dams had dipped to below detectable levels by vi. a total of 44 viable fetuses were recovered from the four infected dams (see table 2 for summary). two fetuses were dead and partially autolysed and not subjected to further study. 10 (22%) of the 44 viable fetuses were positive for prrsv by vi. since dams were vi-negative in blood at the time of necropsy, it was concluded that the presence of virus serology results, shown in parentheses, are presented as the s/p ratio. s/p ratios greater than 0.4 were considered positive for prrsv antibody. dead fetuses were partially autolysed and not tested. gross pathology key: *1, partially mummified; *2, non-viable fetus or relatively low quantity of amniotic fluid; *3, necrotic placenta; *4, merconium-and/or blood-stained amniotic fluid; *5, small, underdeveloped fetus. in the fetal circulation was the result of virus replication in the fetus and not from contamination with maternal blood. (the virus isolation technique is not subject to false positives from minute amounts of cross-contamination with viral protein or rna.) the number of infected fetuses in each litter varied from no infected fetuses (dam no. 2) to five of 14 (36%) infected fetuses for dam no. 4. fetuses that were vi-negative in serum were confirmed as prrsv-negative by vi-negative results in placenta, lung, lymph nodes and thymus (data not shown). one fetus, 1-1, was seropositive for prrsv (s/p ratio = 0.94). since there is no maternal transfer of antibody from mother to fetus (tizard, 1996) , it was concluded that this fetus generated an antibody response to prrsv in utero. 7 of the 10 infected fetuses showed some form of gross pathology, including growth retardation (two fetuses) or reduced amounts and/or merconium-stained amniotic fluid (five fetuses). ongoing virus replication in the fetus as the source of these gross pathological changes is questionable, since non-infected fetuses from infected dams exhibited similar changes. for example, fetuses from dam no. 2 (see fig. 1 ) were either merconium stained (two fetuses), non-viable, possessed reduced amniotic fluid levels (four fetuses) or small (one fetus). except for one autolysed fetus, the 23 fetuses from the two control dams showed no evidence of gross pathology (data not shown). rna viruses frequently exist as a heterogeneous population, frequently referred to as a quasispecies. the appearance or disappearance of individual viral sequences within a quasispecies population is often used as evidence to support the existence of positive or negative selection during infection (elena et al., 2000; forns et al., 1999; tsibris et al., 2009) . mutations that appear in the prrsv genome are useful as markers to identify and follow the appearance and disappearance of viruses within the population (allende et al., 2000; rowland et al., 1999) . dna sequencing of orf5 pcr products, amplified directly from the sera of infected dams, table 3 frequency of t at nucleotide position 77 of orf5. consensus nucleotide at position 77 of orf5 t:c ratio in cloned pcr products (percent) t 3/5 (60) fetus 4-09 t 6/7 (86) fetus 4-10 t 3/6 (50) fetus 4-11 c 1/4 (25) fetus 4-12 t 6/6 (100) fetus 4-13 t 5/5 (100) identified one dam, no. 4, which possessed a virus with a mutation within the hypervariable region of orf5. the change detected by sequencing the pcr product was a c to t (u for rna) nucleotide transition at position 77 that resulted in a non-conserved amino acid change from threonine to isoleucine in gp5. the t-77 mutation was not detected after sequencing the orf5 pcr products from the other dams (see table 3 ). pcr products were cloned into a ta plasmid and the individual plasmids sequenced. even though t-77 was detected in the pcr product from dam no. 4, the sequence of individual clones showed that two of the five sequences possessed a c at position 77 mutation, which indicated that viruses with the wild-type orf5 sequence were still present in the population. whole pcr and plasmid-cloned pcr products were sequenced for the five infected fetuses from dam no. 4. the frequency of the t-77 mutation ranged from 25% (fetus 4-11) to 100% (fetuses 4-12 and 4-13). these results indicate that the fetus is capable of selecting for a particular virus population, which either arises in the dam or fetus. therefore, fetal infection is a potential source of prrsv diversity. during acute infection of the postnatal pig, the largest quantity of virus and greatest number of cells supporting virus replication are found in the lung, a consequence of targeting alveolar macrophages. during the later stages of prrsv infection, secondary lymphoid organs, including tonsil and lymph nodes, become sources of virus replication (allende et al., 2000; rossow, 1998; rowland et al., 1999; . virus replication in fetal tissues was assessed using a combination of virus isolation and ihc detection of nucleocapsid antigen in formalin-fixed tissues. as summarized in table 4 , virus was isolated from all tissues from infected fetuses, including placenta, umbilical cord, heart, lung, spleen, lymph nodes and thymus. overall, the thymus contained the largest quantity of virus. nine of ten fetuses yielded measurable amounts of virus with five of the ten fetal thymuses producing titers greater than 3.0. for lung, 6 of 10 fetuses were vi-positive and only one fetal lung yielded a virus titer greater than 2.0. the recovery of virus from a tissue can represent virus in circulating blood. therefore, to determine if cells in the thymus and other tissues were a source of prrsv, tissue thin sections were stained with prrsv anti-nucleocapsid antibody. the ihc staining procedure included two sets of negative controls: tissue thin sections from non-infected fetuses and from infected fetuses stained with only secondary antibody. both controls were negative for staining (data not shown). the results in table 4 showed the largest number of positive tissues for the thymus (8 of 10 positive), followed by spleen (2 of 8 positive) and lymph node (1 of 9 positive). within the thymus, antigen-positive cells were located in both medullar and cortical regions (data not shown). prrsv antigen-positive cells were not detected in lung or tonsil. taken together, the virus titration and ihc results identify the thymus as a principal source of virus replication in the prrsvinfected fetus. in the post-natal pig, prrsv infection results in distinct pathology in the lung, including the appearance of interstitial pneumonia. representative lymph node and lung tissues from non-infected and infected fetuses are shown in fig. 2 . there was no discernable difference between lungs from infected and non-infected fetuses (compare fig. 2 panels c and d) . lymph nodes from prrsv-negative fetuses appeared largely undeveloped and devoid of well-defined germinal centers. in contrast, the lymph nodes from prrsv-infected fetuses appeared much more pronounced and enlarged. at the microscopic level, the increased lymph node volume was associated with increased numbers of cells and the formation of distinct germinal centers (see fig. 2 panels a and b) . the overall appearance is consistent with an antigen-activated lymph node. to determine the source of the increased cell volume, formalin-fixed thin sections of lymph nodes were stained with t cell-specific (cd3) and b cell-specific (cdw75 and cd79␣) antibodies. representative results for mandibular lymph nodes from infected and non-infected fetuses are shown in fig. 3 . the lymph nodes from both infected and non-infected fetuses showed exten sive areas of staining with anti-cd3, indicating the presence of t cells. lymph nodes from non-infected fetuses were negative for cwd75 staining, but possessed some regions that were positive for cd79␣. the b cell receptor for antigen (bcr) signal transduction complex is composed of a heterodimer of ig␣ and ig␤ chains, which are also known as cd79␣ and cd79␤, respectively. cd79␣ staining in the lymph node from non-infected fetuses is consistent with the presence of pro-and pre-b cells (lee et al., 2008) . the principle difference between infected and non-infected fetuses was found in an overall increase in cd79␣+ cells as well as the appearance of cdw75+ cells, which were associated with germinal centers. cdw79 is beta-galactoside alpha-2,6-sialyltransferase, which is up-regulated in activated b cells (erikstein et al., 1992) . the absence of cdw75 staining in non-infected fetuses is consistent with the overall quiescent nature of the non-stimulated fetal immune system. the up-regulation of cdw75 after prrsv infection is consistent with b cell activation and the formation of mature germinal centers in response to infection. it should be noted that infected fetuses showed different degrees of staining, a likely consequence of the different stages of fetal infection; i.e. less staining was the result of early infection. together, the overall morphology and lymphocyte marker expression results indicate that the increased volume in the lymph nodes of infected fetuses is largely the result of increased numbers of mature activated b cells, which occupy germinal centers. immune cytokines are important factors in antiviral immunity and can influence the outcome of pregnancy (arck et al., 1999; basurko et al., 2009 ). the detection of cytokine gene expression in tissues was performed using a steady state rt-pcr procedure. rt-pcr was performed on rna isolated from four fetuses randomly chosen from the two mock-infected dams and four randomly selected prrsv-infected fetuses. the tissues selected for rt-pcr amplification were lung, lymph node and placenta. lung and lymph node represent sites cytokine alterations in the post-natal pig. placenta was selected as an accessory tissue located at the fetal maternal interface. amplification of mrnas included cytokines associated with inflammatory (il-6, il-8), th1 (il-12, ifn-␥, il-2) and th2/regulatory (il-4, il-10) responses. the amplification of ␤2m mrna was included as an internal control. the determination of a cytokine response was based on the presence or absence of a pcr product. il-6, il-8, il-12 and il-10 products were detected in tissues from both control and infected fetuses. because of the qualitative nature of the pcr method, it was not possible to accurately determine quantitative differences between control and infected fetuses; and therefore, these cytokines were not subjected to further study (data not shown). il-4 mrna was not detected in any of the selected tissues for control and infected fetuses. marked differences in expression were observed for tnf-␣ and ifn-␥ mrnas. the results for ifn-␥ and tnf-␣ from lung, mandibular lymph node and placenta are presented in fig. 4 . the results for il-10 are also shown. the internal control mrna, ␤2m, was amplified from all tissues, indicating that the rna was intact. il-10 was amplified from lung and mandibular lymph nodes from two of the four control fetuses and from all infected fetuses. the presence of il-10 was not unexpected, since increased il-10 production is associated with fetal t cell responses (lin et al., 1993; rainsford and reen, 2002) . as shown in fig. 4a , ifn-␥ and tnf-␣ pcr products were not detected in lung, lymph node or placenta from the non-infected fetuses. however, ifn-␥ pcr products were obtained for lung and lymph nodes from infected fetuses. one infected fetus, 4-3, yielded a faint ifn-␥ product for placenta. for infected fetuses, tnf-␣ mrna was amplified from lung, but not lymph node or placenta (fig. 4b) . to determine if cytokine gene expression was the direct result of prrsv infection, rt-pcr for ifn-␥ and tnf-␣ was performed on the same tissues from fetuses of infected dam no. 2, which produced only prrsv vi-negative fetuses (see fig. 1 ). the analysis of mrna expression in lungs and lymph nodes from six fetuses from dam no. fig. 4 . cytokine gene expression in fetal tissues. rt-pcr for cytokine mrnas was performed on lung (l), mandibular lymph node (n) and placenta (p) for control (panel a) and infected (panel b) fetuses as described in the text using the primers in table 2 . amplification of ␤2-microglobulin mrna was used as an internal control. pcr products were electrophoresed on a 1.2% agarose gel and stained with ethidium bromide. 2 showed only the amplification of the ␤2m, but not tnf-␣ or ifn-␥ mrnas (data not shown). similar results were obtained from virusnegative fetuses located immediately adjacent to infected fetuses (data not shown). in order to confirm that tnf-␣ and ifn-␥ were produced during infection, cytokine protein levels were measured in fetal sera and amniotic fluid from infected fetuses and fetuses from mock-infected dams. as shown in fig. 5 , the concentration of tnf-␣ in serum for prrsv-infected fetuses ranged between 20 and 100 pg/ml compared to less than 30 pg/ml for control fetuses. even though the maximum quantities obtained for infected fetuses were near the lower limit of detection for the elisa test, it was clear that tnf-␣ was elevated during infection. detectable concentrations of tnf-␣ (>23 pg/ml) were found in amniotic fluid from only two control and two infected fetuses. compared to fetuses from mockinfected dams, ifn-␥ concentrations were detected in sera, with values ranging from a low of 60 to more than 500 pg/ml. a maximum level of 60 pg/ml was obtained for a single non-infected fetus. ifn-␥ was not detected in amniotic fluid from either the infected or control fetuses. the presence of ifn-␥ and tnf-␣ in serum, but not amniotic fluid, further supports the notion that the ifn-␥ and tnf-␣ responses are primarily restricted to the prrsv-infected fetus and do not extend to the accessory tissue compartments. this study characterizes the unique biology associated with the interaction between prrsv and the late gestation fetus. consistent with the body of published literature, the fetuses from the infected dams obtained in this study exhibited several anatomic pathological features typical of prrsv infection of the fetus, including lesions associated with the accessory organs, such as umbilical cord and amniotic sac (lager and halbur, 1996; mengeling et al., 1996) . one interesting observation from this study was the apparent absence of a correlation between the presence of gross abnormalities and productive fetal infection. for example, several fetuses from dam no. 2 exhibited several types of gross pathology, including death, growth retardation, or merconium/blood stained amniotic fluid. there were also examples of productively infected fetuses that showed no evidence of gross pathology (see fetuses 1-2, 3-12, 4-9, 4-11, 4-13 in fig. 1 ). the mechanism for fetal pathology remains unclear, but the results suggest that the source pathology is likely result of the infection of tissues on the maternal side and damage to maternal tissues or production of maternal factors that affect the fetus. in this study we did not observe lesions in the myometrium or placenta; however, stockhofe-zurwieden et al. (1995) reported prrs virions budding from maternal vascular endothelial cells at the maternal-fetal interface. lager and halbur (1996) reported damage to the myometrium during prrsv infection. virus-associated lesions in the myometrium are observed for horses infected with eav (coignoul and cheville, 1984) . the appar-ent discrepancy between pathology and infection helps to explain the stealthy nature of the prrsv. a significant number of apparently healthy, but infected fetuses, are likely go on to become healthy growing pigs with the capacity to shed virus. rna viruses often exist as a population of closely related sequences, frequently referred to as a quasispecies. the appearance or disappearance of individual gene sequences in the population over the course of infection is used as evidence for changes in fitness that result from selection. prrsv variants with mutations in orf5 typically appear during the serial passage of virus in culture and during infection of pigs allende et al., 2000) . the significance of mutations in orf5 as a source for increased fitness during infection is not completely understood; but is useful for following the fate of individual prrsv subpopulations over the course of a long-term infection . in this study, one dam, no. 4, showed evidence of a mixed prrsv infection as indicated by the presence of two different orf5 sequences, which were distinguished from each other by a nucleotide substitution at position 77 in orf5. the c to t change was observed in approximately 60% of the cloned plasmid products obtained from dam no. 4 (see table 2 ). however, the same frequency was not transferred to the individual fetuses, which included two fetuses that possessed only viruses with the t-77 mutation and a single fetus with a virus population dominated by c at position 77. these results suggest that fetal infection can alter the selection of prrsv variants and may represent a source of prrsv genetic diversity. to identify the targets of virus replication in the fetus, a variety of tissues were assessed for the presence of virus and virus-infected cells. vi, as opposed to more sensitive approaches, such as pcr, was selected as the means for measuring virus, because the relative insensitivity of vi avoids the possibility of false positive pcr results that might result from small amounts of contaminating maternal material. within the group of selected tissues, virus could be isolated from all tissues. however, the most consistent source and largest quantity of virus were obtained from the thymus. collectively, these data identify the thymus as a primary site of virus replication in the prrsv-infected fetus and confirm an earlier observation for prrsv by benson et al. (2002) . the specific cell population in the thymus targeted prrsv replication remains unknown. the natural predisposition of maternal immunity towards th2like responses aids in protecting the fetus by blocking cell-mediated th1-related allorejection responses (arck et al., 1999; entrican, 2002; raghupathy, 2001 ; recently reviewed in challis et al., 2009) . the negative influence of th1 cytokines on fetal development can be demonstrated experimentally in mice, which show that a single injection of il-2 or ifn-␥ into certain strains induces fetal resorption (lala, 1990; chaouat et al., 1990) . tnf-␣, when infected into mice is abortifacient (clark et al., 2004) . th1 cytokines can also have important long-term impacts. for example, newborn mice that survive maternal infection with influenza virus exhibit behav-ior similar to hyperanxiety and autism. the behavioral changes are a consequence of the altered distribution of dopamine and glutamine receptors during fetal brain development. the effect of influenza virus infection on the fetal brain can be mimicked by the administration of poly i:c, an inducer of ifn (shi et al., 2003) . furthermore, the addition of ifn-␥ to hippocampal neuron cultures alters the distribution of glutamate receptors, sufficient to affect synaptic activity between neurons (vikman et al., 2001) . virus infections during pregnancy present an interesting paradox: those cytokines that protect the fetus from viral infection, tend to inhibit fetal development or potentiate rejection of the fetus; while those cytokines that maintain and promote fetal development are associated with the inhibition of antiviral immune responses. because of the potential negative impact of th1 cytokines on fetal outcome, there is a natural predisposition for the fetus to block the induction of th1-associated cytokines. for example, the ifn-␥ response in the developing fetus can be blocked by a combination of factors, including (1) defects in the capacity of fetal dendritic cells to express mhc class ii and synthesize il-12, (2) hypermethylation of the ifn-␥ gene in fetal t cells, (3) the absence of target macrophages, and (4) the presence of an immune environment dominated by th2/regulatory cytokines (goriely et al., 2001; langrish et al., 2002; melvin et al., 1995; marodi et al., 2001; murphy et al., 2009; prescott et al., 1998; white et al., 2002,) . with this in mind, there are examples of viruses, such as epstein-barr virus (ebv), which are capable of stimulating antigen-specific ifn-␥ responses in human umbilical cord blood lymphocytes (ito et al., 1998; wilson and morgan, 2002) . these and other data provide a description of the fetus as capable of initiating a robust antiviral th1 immune response (chaouat et al., 2002; chipeta et al., 2000; murphy et al., 2009) . in this study ifn-␥ and tnf-␣ mrnas were identified in tissues from infected fetuses. rna message was not identified in tissues of fetuses from mock-infected dams or non-infected fetuses from infected dams. this indicates that altered gene expression is the direct result of fetal infection. up-regulated expression was confirmed by the presence of detectable levels of ifn-␥ and tnf-␣ proteins in serum. furthermore, the results indicate that inf-␣ and ifn-␥ cytokines response are compartmentalized within the fetus and do not extend to other compartments, such as amniotic fluid or placenta, thus lessening the probability of allorejection by the dam. from these data, it is apparent that the fetus is capable of initiating a th1-like response; however, the capacity of ifn-␥ and tnf-␣ to control prrsv infection in the fetus is not known. additional evidence for induction of virus-specific immunity was found in the development of germinal centers containing activated (cdw75+) b cells and seroconversion in at least one fetus. pro-inflammatory cytokines, such as tnf-␣ and ifn-␥ can contribute to pulmonary distress through the activation of alveolar macrophages and other cell populations. the increased quantities of ifn-␥ and tnf-␣ found in the lungs of prrsv-infected fetuses may not be sufficient to cause pulmonary damage to the fetal lung, primarily because fetal macrophages have a reduced capacity to respond to inflammatory stimuli. in addition, il-10, up-regulated in response to prrsv infection, is a potent antagonist of ifn-␥ and tnf-␣ activation of macrophages (burchett et al., 1992; marodi et al., 2001; johnsen et al., 2002; thanawongnuwech et al., 2003) . however, within days after birth, adult macrophages, including mature alveolar and intravascular macrophages emerge into an environment already enriched in ifn-␥ and tnf-␣. the outcome is the rapid recruitment, activation, and cytopathic killing of large numbers of virus-permissive macrophages (choi and chae, 2002) . this scenario as a cause of severe interstitial pnuemonia in the prrsv-infected newborn requires further investigation, but has obvious implications in the 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of ifn-gamma positive cells in porcine reproductive and respiratory syndrome virus-infected lung immunity in the fetus and newborn. in: veterinary immunology: an introduction, chapter 19 quantitative deep sequencing reveals dynamic hiv-1 escape and large population shifts during ccr5 antagonist therapy in vivo interferongamma-induced changes in synaptic activity and ampa receptor clustering in hippocampal cultures differential patterns of methylation of the ifn-gamma promoter at cpg and non-cpg sites underlie differences in ifn-gamma gene expression between human neonatal and adult cd45ro-t cells primary immune responses by cord blood cd4(+) t cells and nk cells inhibit epstein-barr virus b-cell transformation in vitro this work was partially supported by the usda national research initiative for competitive grants program grants # 97-35204-5071. key: cord-272237-gnno6elo authors: wang, ziran; hao, zhuang; yu, shifeng; huang, cong; pan, yunlu; zhao, xuezeng title: a wearable and deformable graphene-based affinity nanosensor for monitoring of cytokines in biofluids date: 2020-07-31 journal: nanomaterials (basel) doi: 10.3390/nano10081503 sha: doc_id: 272237 cord_uid: gnno6elo a wearable and deformable graphene-based field-effect transistor biosensor is presented that uses aptamer-modified graphene as the conducting channel, which is capable of the sensitive, consistent and time-resolved detection of cytokines in human biofluids. based on an ultrathin substrate, the biosensor offers a high level of mechanical durability and consistent sensing responses, while conforming to non-planar surfaces such as the human body and withstanding large deformations (e.g., bending and stretching). moreover, a nonionic surfactant is employed to minimize the nonspecific adsorption of the biosensor, hence enabling cytokine detection (tnf-α and ifn-γ, significant inflammatory cytokines, are used as representatives) in artificial tears (used as a biofluid representative). the experimental results demonstrate that the biosensor very consistently and sensitively detects tnf-α and ifn-γ, with limits of detection down to 2.75 and 2.89 pm, respectively. the biosensor, which undergoes large deformations, can thus potentially provide a consistent and sensitive detection of cytokines in the human body. human biofluids (such as saliva, tears and sweat) are attractive clinical diagnostic bio-media containing numerous cytokines (with a molecular weight lower than 70 kda) [1] [2] [3] . they can be easily collected without skin-piercing. abnormally elevated levels of cytokines in human biofluids are considered to be closely related to the attack of some severe diseases, such as coronavirus disease 2019 (covid19) , and chronic diseases [4] [5] [6] . hence, the capacity for the continuous detection of cytokine levels in human biofluids for high risk populations, daily, is of great significance for offering information on health conditions and thereby gaining valuable time, which can then be used to take effective preventative measures before the attack from such diseases [7, 8] . wearable sensors, which could be attached to the non-planar human body surface and complete on-site signal transduction, seem to be capable of performing such work. graphene, an attractive two-dimensional nanomaterial, is extremely sensitive to its surface charge distribution, and widely used as the transducer for sensors due to its outstanding electrical properties [9] [10] [11] . especially, with the aid of aptamers, the graphene field-effect transistor (gfet) can enable the sensitive, rapid and label-free detection of cytokines [12] [13] [14] . to date, efforts have been made to use gfet biosensors in wearable applications due to the high mechanical flexibility of graphene [15] [16] [17] . such sensors are fabricated on sheets of polymers, such as polydimethylsiloxane (pdms), polyester (pet) and polyethylene naphthalate (pen) [18] [19] [20] . however, these polymer sheets, whose overall thicknesses are 100 µm or more, would hardly be subject to large deformations and curvature (with radii ranging from 4 to 40 mm). as such, gfet biosensors with an extremely thin substrate that can sustain the large deformations involved in physiologically and biochemically relevant measurements on non-planar human body surfaces are still highly desirable. in this paper, we present a wearable and deformable aptameric gfet biosensor that is designed to enable the sensitive, consistent and time-resolved monitoring of cytokines in human biofluids. the biosensor is fabricated on a biocompatible and ultrathin, polymer-supporting substrate (figure 1a ). due to the employment of this substrate, whose thickness is only 2.5 µm, the biosensor, with good mechanical durability, is capable of conforming to non-planar surfaces such as the human skin or eyeball and withstanding large deformations, including bending and stretching, whilst maintaining consistent and sensitive responses. moreover, tween 80 is used to modify the graphene surface to effectively suppress nonspecific adsorption, thus enabling the biosensor to detect cytokines (tnf-α and ifn-γ, significant inflammatory cytokines, were used as representatives) in artificial tears (used as a biofluid representative). the experimental results indicate that our aptameric gfet biosensor can realize the highly sensitive detection of tnf-α and ifn-γ, with limits of detection down to 2.75 and 2.89 pm, respectively. furthermore, the time-resolved monitoring of tnf-α in artificial tears under different tensile strains with consistent sensing responses is enabled. as a result, our biosensor can be potentially used in wearable applications for monitoring an individual's health conditions and predicting the attack of chronic diseases. nanomaterials 2020, 10, x for peer review 2 of 9 enable the sensitive, rapid and label-free detection of cytokines [12] [13] [14] . to date, efforts have been made to use gfet biosensors in wearable applications due to the high mechanical flexibility of graphene [15] [16] [17] . such sensors are fabricated on sheets of polymers, such as polydimethylsiloxane (pdms), polyester (pet) and polyethylene naphthalate (pen) [18] [19] [20] . however, these polymer sheets, whose overall thicknesses are 100 μm or more, would hardly be subject to large deformations and curvature (with radii ranging from 4 to 40 mm). as such, gfet biosensors with an extremely thin substrate that can sustain the large deformations involved in physiologically and biochemically relevant measurements on non-planar human body surfaces are still highly desirable. in this paper, we present a wearable and deformable aptameric gfet biosensor that is designed to enable the sensitive, consistent and time-resolved monitoring of cytokines in human biofluids. the biosensor is fabricated on a biocompatible and ultrathin, polymer-supporting substrate (figure 1a ). due to the employment of this substrate, whose thickness is only 2.5 μm, the biosensor, with good mechanical durability, is capable of conforming to non-planar surfaces such as the human skin or eyeball and withstanding large deformations, including bending and stretching, whilst maintaining consistent and sensitive responses. moreover, tween 80 is used to modify the graphene surface to effectively suppress nonspecific adsorption, thus enabling the biosensor to detect cytokines (tnf-α and ifn-γ, significant inflammatory cytokines, were used as representatives) in artificial tears (used as a biofluid representative). the experimental results indicate that our aptameric gfet biosensor can realize the highly sensitive detection of tnf-α and ifn-γ, with limits of detection down to 2.75 and 2.89 pm, respectively. furthermore, the time-resolved monitoring of tnf-α in artificial tears under different tensile strains with consistent sensing responses is enabled. as a result, our biosensor can be potentially used in wearable applications for monitoring an individual's health conditions and predicting the attack of chronic diseases. the monolayer graphene sheet was ordered from graphenea inc. (cambridge, ma, usa). 1-pyrenebutyric acid n-hydroxysuccinimide ester (pase), ethanolamine, tween 80, human interleukin-002 (il-002), tnf-alpha and ifn-gamma were purchased from sigma-aldrich (st. louis, mo, usa). human growth hormone (gh) and human epidermal growth factor (egf) were ordered from acro biosystems (newark, de, usa). ultrathin mylar films (2.5 µm thickness of polyethylene terephthalate membranes) were ordered from chemplex industries (palm city, fl, usa). artificial tears were ordered from alcon laboratories. the aptamer specific to tnf-alpha (5 -nh 2 -tgg tgg atg gcg cag tcg gcg aca a-3 ) and ifn-gamma (5 -nh 2 -ggg gtt gtt tgt gtt ggg tgt tgt gt-3 ) was synthesized and purified by sangong biotech (shanghai, china). the gfet biosensor was fabricated following our previous nanofabrication process [13, 21, 22] . briefly, an ultrathin mylar film (2.5 µm) was placed on a glass slide as the biosensor's substrate. subsequently, drain, source and gate electrodes (4/46 nm of cr/au) were patterned onto the film using a lithography process, including e-beam evaporation and lift-off. the biosensor was then exposed to the oxygen plasma to remove the residue on the surface. a monolayer graphene sheet was transferred onto electrodes as the conducting channel, using a polymethyl methacrylate (pmma) carrier layer. after dissolving the pmma layer with acetone, the graphene was biochemically functionalized to enable the biomarker detection. the fabricated biosensor was ultra-flexible and capable of conforming to the underlying surface, such as a human wrist or eyeball (figure 1b,c) . finally, the biosensor was mounted onto a pre-stretched elastomer to obtain the necessary stretchability, which allowed the biosensor to be stretched from a 0% to 100% extension ( figure 1d ). to achieve the biochemical functionalization, the biosensor was first immersed in 10 mm 1-pyrenebutanoic acid succinimidyl ester (pase) solution for 5 h at room temperature. pase was modified on the graphene surface through π-π stacking, which was used to link the aptamer. after incubating in the 1 µm aptamer solution for 12 h, the device was washed with phosphate buffer (pbs) to remove free aptamer. ethanolamine was then used to quench the unreacted pase on the graphene by soaking in 100 mm ethanolamine solution for 1 h. finally, the biosensor was immersed in 0.05% tween 80 solution to passivate the uncoated graphene area. during the operation, a volume of 40 µl of analytes (tnf-α, ifn-γ and control proteins) at a given concentration was added to a polydimethylsiloxane (pdms) open well, which was mounted on the graphene conducting channel to hold the analyte solution. in the experiments, 20 µl of artificial tears was added to a 1 ml centrifugal tube with 980 µl of 1 × pbs. then, the mixture solution was stored at 4 • c before protein solution configurations. a microscope image of the gfet biosensor fabricated on the ultrathin film is shown in figure 2a . the surface functionalization of the graphene with pase was confirmed using raman spectra (figure 2b ). the modification of the pase split the g band, illustrating the coupling of graphene with the pyrene group on pase. furthermore, the dirac point shift ∆v dirac ,where ∆v dirac = v dirac -v dirac,0 with v dirac,0 the dirac point obtained in the solution without biomarker, before and after pase, aptamer and tween 80 functionalization was measured with the gate voltage v g increasing from −0.2 to 0.4 v at a fixed drain-source voltage v ds of 0.01 v (figure 2c,d) . ∆v dirac was observed to be monotonically increased after pase functionalization, indicating that the p-type doping of the graphene had been induced. upon the attachment of the aptamer specific to tnf-α and ifn-γ, ∆v dirac decreased by 0.04 and 0.057 v, respectively. tween 80, a chemically stable nonionic surfactant, was used to block the uncoated graphene area to suppress the nonspecific adsorption in artificial tears due to its low binding affinity to the abundant non-target molecules present in the tears [23, 24] . after the modification with tween 80, the dirac point v dirac shifted towards the direction of the negative gate voltage, illustrating that tween 80 induced the n-type doping of the graphene. thus, it was concluded that the biosensor was successfully functionalized using different aptamers. (figure 2c,d) . δvdirac was observed to be monotonically increased after pase functionalization, indicating that the p-type doping of the graphene had been induced. upon the attachment of the aptamer specific to tnf-α and ifn-γ, δvdirac decreased by 0.04 and 0.057 v, respectively. tween 80, a chemically stable nonionic surfactant, was used to block the uncoated graphene area to suppress the nonspecific adsorption in artificial tears due to its low binding affinity to the abundant non-target molecules present in the tears [23, 24] . after the modification with tween 80, the dirac point vdirac shifted towards the direction of the negative gate voltage, illustrating that tween 80 induced the n-type doping of the graphene. thus, it was concluded that the biosensor was successfully functionalized using different aptamers. the capability of the biosensor for cytokine detection was assessed using tnf-α and ifn-γ ( figure 3 )-inflammatory cytokines related to inflammation, covid-19 and cancers [25, 26] . as the tnf-α concentration increased from 0.03 to 500 nm (figure 3a) , vdirac decreased by 0.042 v, from 0.1 to 0.062 v. it could be observed that vdirac monotonically decreased with increasing tnf-α concentrations, suggesting the successful detection of tnf-α in artificial tears using the fabricated biosensor. the equilibrium dissociation constant, defined kd, was investigated to study the binding affinity between the aptamer and tnf-α (figure 3c ). the normalized δvdirac was employed to address the effect of device-to-device variations, defined as δvdirac/δvdirac,max (vdirac,max is the dirac point corresponding to the maximum tnf-α concentration tested). δvdirac/δvdirac,max was determined using the capability of the biosensor for cytokine detection was assessed using tnf-α and ifn-γ ( figure 3 )-inflammatory cytokines related to inflammation, covid-19 and cancers [25, 26] . as the tnf-α concentration increased from 0.03 to 500 nm (figure 3a) , v dirac decreased by 0.042 v, from 0.1 to 0.062 v. it could be observed that v dirac monotonically decreased with increasing tnf-α concentrations, suggesting the successful detection of tnf-α in artificial tears using the fabricated biosensor. the hill-langmuir equation [27] . based on the fitted curve, the kd was calculated to be 8.43 ± 1.67 nm. the limit of detection (lod) was estimated based on the three-sigma rule, and the sigma was obtained from the standard deviation of the experimental and fitted data. the lod was calculated to be 2.75 pm, which is many times lower than that for existing biosensors for tnf-α detection. subsequently, the biosensor was employed to test the detection of ifn-γ in artificial tears ( figure 3b,d) . as the ifn-γ concentration increased from 0.03 to 500 nm, the maximum δvdirac was 0.029 v, decreasing from 0.029 to 0 v. the sensing signal was also consistently shifted in the direction of the negative gate voltage with the increasing ifn-γ concentrations. this was expected from the electrostatic mechanism [18] . after binding with the cytokines, the aptamer together with the negatively charged cytokines was brought closer to the graphene surface, which enabled the charge redistribution in the graphene, due to electrostatic induction [12, 14] . hence, a detectable change in the drain-source current was measured. the equilibrium dissociation constant kd was estimated to be 7.36 ± 2.76 nm (figure 3d) , and the lod was calculated to be 2.89 pm. thus, the biosensor shows a consistent and sensitive response, able to detect cytokines in artificial tears with a lower lod than most other existing methods (table 1) . the equilibrium dissociation constant, defined k d , was investigated to study the binding affinity between the aptamer and tnf-α (figure 3c ). the normalized ∆v dirac was employed to address the effect of device-to-device variations, defined as ∆v dirac /∆v dirac,max (v dirac,max is the dirac point corresponding to the maximum tnf-α concentration tested). ∆v dirac /∆v dirac,max was determined using the hill-langmuir equation [27] . based on the fitted curve, the k d was calculated to be 8.43 ± 1.67 nm. the limit of detection (lod) was estimated based on the three-sigma rule, and the sigma was obtained from the standard deviation of the experimental and fitted data. the lod was calculated to be 2.75 pm, which is many times lower than that for existing biosensors for tnf-α detection. subsequently, the biosensor was employed to test the detection of ifn-γ in artificial tears (figure 3b,d) . as the ifn-γ concentration increased from 0.03 to 500 nm, the maximum ∆v dirac was 0.029 v, decreasing from 0.029 to 0 v. the sensing signal was also consistently shifted in the direction of the negative gate voltage with the increasing ifn-γ concentrations. this was expected from the electrostatic mechanism [18] . after binding with the cytokines, the aptamer together with the negatively charged cytokines was brought closer to the graphene surface, which enabled the charge redistribution in the graphene, due to electrostatic induction [12, 14] . hence, a detectable change in the drain-source current was measured. the equilibrium dissociation constant k d was estimated to be 7.36 ± 2.76 nm (figure 3d) , and the lod was calculated to be 2.89 pm. thus, the biosensor shows a consistent and sensitive response, able to detect cytokines in artificial tears with a lower lod than most other existing methods (table 1) . to investigate the specificity of the biosensor, egf and gh, two related proteins, were chosen as control proteins. the biosensor, modified with the aptamer specific to tnf-α, was firstly exposed to control proteins in artificial tears (figure 4a ). the sensing signal for tnf-α significantly increased with the adding of tnf-α concentrations. the normalized ∆v dirac /∆v dirac,max for tnf-α at 500 nm was over six times larger than that for control proteins (14.3%) at the same concentration. additionally, the dissociation constant k d for the control proteins was investigated. k d was estimated to be 1.43 × 10 10 and 1.77 × 10 9 nm for egh and gh, respectively, which is much larger than the k d for tnf-α (8.43 nm). subsequently, the biosensor modified with the aptamer specific to ifn-γ was tested using these control proteins (figure 4b) . the ∆v dirac /∆v dirac,max for ifn-γ is also six times larger than those for the control proteins (15.6%) at the same concentrations. furthermore, the k d was calculated to be 1.55 × 10 6 and 2.73 × 10 6 nm for egh and gh, respectively, which is tens of thousands of times larger than the k d for ifn-γ (7.36 nm). hence, the biosensor has a high level of specificity for the target cytokine in artificial tears. nanomaterials 2020, 10, x for peer review 6 of 9 graphene-based sensor aptamer artificial tears 2.89 pm (this work) to investigate the specificity of the biosensor, egf and gh, two related proteins, were chosen as control proteins. the biosensor, modified with the aptamer specific to tnf-α, was firstly exposed to control proteins in artificial tears (figure 4a ). the sensing signal for tnf-α significantly increased with the adding of tnf-α concentrations. the normalized δvdirac/δvdirac,max for tnf-α at 500 nm was over six times larger than that for control proteins (14.3%) at the same concentration. additionally, the dissociation constant kd for the control proteins was investigated. kd was estimated to be 1.43 × 10 10 and 1.77 × 10 9 nm for egh and gh, respectively, which is much larger than the kd for tnf-α (8.43 nm). subsequently, the biosensor modified with the aptamer specific to ifn-γ was tested using these control proteins (figure 4b) . the δvdirac/δvdirac,max for ifn-γ is also six times larger than those for the control proteins (15.6%) at the same concentrations. furthermore, the kd was calculated to be 1.55 × 10 6 and 2.73 × 10 6 nm for egh and gh, respectively, which is tens of thousands of times larger than the kd for ifn-γ (7.36 nm). hence, the biosensor has a high level of specificity for the target cytokine in artificial tears. to verify the feasibility of the biosensor for wearable applications, time-resolved measurements were employed, using the biosensor modified with an aptamer specific to tnf-α as a representative. as shown in figure 5a , the device was first exposed to different concentrations of tnf-α in artificial tears. the characterization started with the injection of the artificial tears in the pdms well, then the artificial tears spiked with various tnf-α concentrations were injected after each binding equilibrium at corresponding concentrations. the changes in the drain-source current are denoted as δids, and the maximum change is denoted as δids, max. the sensing signal was observed to increase stepwise and significantly from 0 to 0.98 with increasing tnf-α concentrations from 0 to 500 nm. moreover, it was found that equilibrium was reached after the binding of aptamer with tnf-α within 7 min at different concentrations. by contrast, the δids/δids, max-generated by related cytokines (ifn-γ and il-002)was less than 15% compared with that for tnf-α at the same concentration. to verify the feasibility of the biosensor for wearable applications, time-resolved measurements were employed, using the biosensor modified with an aptamer specific to tnf-α as a representative. as shown in figure 5a , the device was first exposed to different concentrations of tnf-α in artificial tears. the characterization started with the injection of the artificial tears in the pdms well, then the artificial tears spiked with various tnf-α concentrations were injected after each binding equilibrium at corresponding concentrations. the changes in the drain-source current are denoted as ∆i ds , and the maximum change is denoted as ∆i ds, max . the sensing signal was observed to increase stepwise and significantly from 0 to 0.98 with increasing tnf-α concentrations from 0 to 500 nm. moreover, it was found that equilibrium was reached after the binding of aptamer with tnf-α within 7 min at different concentrations. by contrast, the ∆i ds/ ∆i ds, max -generated by related cytokines (ifn-γ and il-002)-was less than 15% compared with that for tnf-α at the same concentration. nanomaterials 2020, 10, x for peer review 7 of 9 until it achieved the shape of the human eyeball (which was accompanied by the biosensor, mounted on top). the diaphragm deflection induced a large-magnitude stretching of the biosensor (100% tensile strain), serving as an example mimicking the large deformation of the wearable device for cytokine detection on the human body (figure 5d ). the sensing response to tnf-α was sensitive and consistent with the test on a flat surface (figure 5a) , and the degradation of the signal was less than 10% at the same concentration for the biosensor under two tensile strains. overall, the results demonstrate that the fabricated biosensor has promising prospects for being embedded with wearable devices for the monitoring of cytokines in body fluids. we presented a wearable gfet biosensor for the time-resolved monitoring of cytokines in artificial tears. the modification of the graphene with the aptamer enabled the highly specific detection of the target cytokine. tween 80, which occupies the uncoated area of the graphene, offers a capability of suppressing nonspecific adsorption; the biosensor hence affords a high level of sensitivity for cytokine detection in artificial tears, with a lod down to 2.75 and 2.89 pm for tnf-α and ifn-γ, respectively. in addition, the time-resolved monitoring of tnf-α in artificial tears using the biosensor under different tensile strains has been demonstrated. the sensing response for the target cytokines is consistent with that found on the flat surface. these results demonstrate that this wearable gfet biosensor is a critical step toward the general application of sensors for the monitoring of disease biomarkers in the human body. additionally, we present a potential capability for wearable applications using the biosensor mounted on a homemade diaphragm chamber for cytokine detection (figure 5b ). the diaphragm is a stretchable membrane, which can be made to swell or shrink by pumping air into the chamber to mimic the human eyeball. the biosensor was first mounted on the planar diaphragm (0% tensile strain) to test cytokine detection (figure 5c ). then, the diaphragm was inflated with compressed air until it achieved the shape of the human eyeball (which was accompanied by the biosensor, mounted on top). the diaphragm deflection induced a large-magnitude stretching of the biosensor (100% tensile strain), serving as an example mimicking the large deformation of the wearable device for cytokine detection on the human body (figure 5d ). the sensing response to tnf-α was sensitive and consistent with the test on a flat surface (figure 5a) , and the degradation of the signal was less than 10% at the same concentration for the biosensor under two tensile strains. overall, the results demonstrate that the fabricated biosensor has promising prospects for being embedded with wearable devices for the monitoring of cytokines in body fluids. we presented a wearable gfet biosensor for the time-resolved monitoring of cytokines in artificial tears. the modification of the graphene with the aptamer enabled the highly specific detection of the target cytokine. tween 80, which occupies the uncoated area of the graphene, offers a capability of suppressing nonspecific adsorption; the biosensor hence affords a high level of sensitivity for cytokine detection in artificial tears, with a lod down to 2.75 and 2.89 pm for tnf-α and ifn-γ, respectively. in addition, the time-resolved monitoring of tnf-α in artificial tears using the biosensor under different tensile strains has been demonstrated. the sensing response for the target cytokines is consistent with that found on the flat surface. these results demonstrate that this wearable gfet biosensor is a critical step toward the general application of sensors for the monitoring of disease biomarkers in the human body. simultaneous measurement of six cytokines in a single sample of human tears using microparticle-based flow cytometry: allergics vs. non-allergics measurement of cytokines in sweat patches and plasma in healthy women: validation in a controlled study acute phase protein and cytokine levels in serum and saliva: a comparison of detectable levels and correlations in a depressed and healthy adolescent sample wearable sensors for biochemical sweat analysis methylxanthine drug monitoring with wearable sweat sensors flexible electronics toward wearable sensing recent advances in cytokine detection by immunosensing ultrasensitive detection of cytokines enabled by nanoscale zno arrays high-performance flexible graphene field effect transistors with ion gel gate dielectrics the rise of graphene fully rollable transparent nanogenerators based on graphene electrodes graphene-based fully integrated portable nanosensing system for on-line detection of cytokine biomarkers in saliva an ultraflexible and stretchable aptameric graphene nanosensor for biomarker detection and monitoring modulating the linker immobilization density on aptameric graphene field effect transistors using an electric field large-area ultrathin films of reduced graphene oxide as a transparent and flexible electronic material graphene-based wireless bacteria detection on tooth enamel ultrafine graphene nanomesh with large on/off ratio for high-performance flexible biosensors measurement of cytokine biomarkers using an aptamer-based affinity graphene nanosensor on a flexible substrate toward wearable applications high-performance, flexible enzymatic glucose biosensor based on zno nanowires supported on a gold-coated polyester substrate a wearable and highly sensitive pressure sensor with ultrathin gold nanowires real-time monitoring of insulin using a graphene field-effect transistor aptameric nanosensor an integrated flexible and reusable graphene field effect transistor nanosensor for monitoring glucose rapid, label-free, electrical whole blood bioassay based on nanobiosensor systems graphene-based biosensors for detection of bacteria and their metabolic activities angiopoietin-2 sensitizes endothelial cells to tnf-α and has a crucial role in the induction of inflammation elevations of serum cancer biomarkers correlate with severity of covid-19 label-free biosensors based on aptamer-modified graphene field-effect transistors detecting multiple cell-secreted cytokines from the same aptamer-functionalized electrode detection of interferon gamma using graphene and aptamer based fet-like electrochemical biosensor the authors declare no conflict of interest. key: cord-259586-kep2dgaw authors: van reeth, kristien; van gucht, steven; pensaert, maurice title: in vivo studies on cytokine involvement during acute viral respiratory disease of swine: troublesome but rewarding date: 2002-09-10 journal: vet immunol immunopathol doi: 10.1016/s0165-2427(02)00047-8 sha: doc_id: 259586 cord_uid: kep2dgaw the early cytokines interferon-α (ifn-α), tumour necrosis factor-α (tnf-α), interleukin-1, -6 and -8 (il-1, -6, -8) are produced during the most early stage of an infection. the activities of these cytokines have been studied extensively in vitro and in rodents, but in vivo studies on the role of these cytokines in infectious diseases of food animals are few. this review concentrates on in vivo studies of cytokine involvement in infectious respiratory diseases of swine, with an emphasis on viral infections. first evidence for the role of early cytokines in pneumonia in swine came from experimental infections with mycoplasma hyopneumoniae and actinobacillus pleuropneumoniae. the role of tnf-α and il-1 in the symptoms and pathology of porcine pleuropneumonia has recently been proven by use of an adenovirus vector expressing the anti-inflammatory il-10. in the authors’ laboratory, studies were undertaken to investigate the relationship between viral respiratory disease and bioactive lung lavage levels of ifn-α, tnf-α, il-1 and il-6. out of three respiratory viruses—porcine respiratory coronavirus (prcv), porcine reproductive and respiratory syndrome virus (prrsv) and swine influenza virus (siv)—only siv induced acute respiratory disease and severe lung damage by itself. disease and lung pathology were tightly associated with the simultaneous production of ifn-α, tnf-α, il-1 and il-6. in challenge studies of siv-vaccinated pigs, levels of ifn-α, tnf-α and il-6, but not il-1 were correlated with clinical and virological protection. multifactorial respiratory disease was reproduced by combined inoculations with prcv or prrsv followed by lps from escherichia coli. in comparison with the respective single inoculations, which were subclinical, there was a true potentiation of disease and production of tnf-α, il-1 and il-6. tnf-α and il-6 were best correlated with disease. in further studies, we will use more specific strategies to dissect the role of cytokines during viral infections. interferon-a (ifn-a), tumour necrosis factor-a (tnf-a), interleukin-1-a and b (il-1-a and b), interleukin-6 (il-6) and interleukin-8 (il-8) are produced during the most early stage of an infection, before the speci®c immune response comes into play. each of these cytokines has a number of local and systemic effects, among others: upregulation of adhesion molecules on the vascular endothelium and leukocytes, activation of neutrophil and macrophage antimicrobial activities, fever, induction of acute phase proteins, catabolic effects on muscle and fat cells (for reviews see murtaugh et al., 1996; bogdan, 2000; dinarello, 2000) . tnf-a and il-1, the classical``proin¯ammatory'' cytokines, stimulate nearly all local and systemic in¯ammatory responses. il-6, which can be induced by tnf-a and il-1, is pyrogenic, induces veterinary immunology and immunopathology 87 (2002) 161±168 the release of acute phase reactants by the liver, and in turn switches off proin¯ammatory cytokine production. while tnf-a and il-1 mediate the initial adhesive reaction of neutrophils to the endothelium, il-8 appears to be essential for the directed migration of leukocytes into the infected tissue (strieter and kunkel, 1994) . ifn-a, though best known for its antiviral effects, can exert all of the systemic effects mentioned here and induces adhesion molecules in some in vitro systems. due to their diverse actions, cytokines such as ifn-a and il-6 have been classi®ed as``proin¯ammatory''bysome researchers andas``anti-in¯ammatory'' by others. the term``early cytokines'' applies to all cytokines mentioned here and will be used further. the amount of in vitro studies on cytokine actions and interactions is impressive, but there are rather few in vivo studies on cytokines in infectious disease models of food animals. still, the response of cells to cytokines can be markedly affected by the``context'', and the expression in vivo veritas is more valid for cytokine studies than for any other type of research. here we focus on in vivo research of cytokine involvement in infectious respiratory diseases of swine. this type of research encounters some particular problems. first, cytokine production during pulmonary infections is mainly restricted to the lung and diffusion of cytokines across the blood±alveolar barrier is rather limited (nelson et al., 1989) . samples of the lower respiratory tract, such as lung tissue, bronchoalveolar lavage (bal) cells or¯uid, are thus needed to study cytokine production, and these are dif®cult to obtain from living animals. for cytokine studies in particular, it is problematic to perform serial in vivo lung lavages because the procedure in itself causes lung damage with neutrophil in®ltration which can in¯uence cytokine production. also, the amount of cells and¯uid recovered in this way is rather small and not representative of the whole lung. second, the possibilities for intervention in the form of treatment with speci®c cytokine-blocking antibodies or antagonists are limited. the major problem here is the delivery of such molecules to the deeper lung over a prolonged time period. this is the reason why most studies have only demonstrated associations between lung cytokines and other parameters, without proving a true cause±effect relationship. third, respiratory infections occur with a high prevalence in swine, and the upper respiratory tract harbors commensal bacteria that can easily colonize the lungs in the presence of predisposing events. also, some of the early cytokines are induced readily by a variety of stimuli. thus, pigs of a high-health status are required for the study of cytokines at the lung level. first evidence for the involvement of early cytokines in pneumonia in swine came from experimental infections with mycoplasma and bacteria. mycoplasma hyopneumoniae is the causative agent of enzootic pneumonia, a mild chronic pneumonia with few clinical consequences if uncomplicated. characteristic peribronchiolar lymphoid hyperplasia and gross lung lesions develop by 3 and 4 weeks after experimental infection. lesions were associated with increased levels of bioactive tnf-a, il-1 and il-6 in bal¯uids (asai et al., 1993 (asai et al., , 1994 . based on this association, it is believed that these cytokines mediate lymphocyte in®ltration and activation in the pneumonic lung. porcine pleuropneumonia is caused by actinobacillus pleuropneumoniae and is characterized by a necrotizing ®brinous pneumonia with prominent neutrophil in®ltration and high morbidity and mortality. dyspnoea, increased respiratory rate, fever, depression and inappetence occur as early as 2 h after endotracheal inoculation of a. pleuropneumoniae (baarsch et al., 2000) . tnf-a, il-1, il-6 and il-8 mrna levels in the lung increased at the same time and cytokine expression was localized to the periphery of lung lesions (baarsch et al., 1995; choi et al., 1999; huang et al., 1999) . the studies by baarsch et al. (1995 baarsch et al. ( , 2000 illustrate perfectly that the results of cytokine measurements are dramatically affected by the sampling compartment and the detection method used, as well as by the time of sampling. il-6 mrna, for example, was detected by northern blotting in lung parenchyma but not in bal cells, whereas in situ hybridization (ish) on bal cells was clearly positive. similarly, tnf-a mrna in bal cells appeared to be increased in ish, but tnf-a bioactivity in bal uids was undetectable in a standard l929 cytotoxicity assay. in later experiments by the same group, however, elevated tnf-a levels were found in bal uids by elisa (morrison et al., 2000) . in summary, tnf-a as well as bioactive il-1 and il-6 have been detected in lung lavage¯uids and the role of the ®rst two cytokines has recently been proven by administration to the lung of a recombinant replication-de®cient adenovirus which expressed the anti-in¯ammatory cytokine il-10. il-10 expression reduced il-1 and tnf-a secretion, and this was associated with clinical protection and a signi®cant reduction of lung pathology (morrison et al., 2000) . in a few studies, cytokines in serum were found to be suitable indicators of ongoing bacterial infections or other disease conditions. indeed, haematogenic spread of bacteria may elicit a systemic cytokine response, and limited diffusion of cytokines from the lungs to the circulation is also possible. serum il-6, but not tnf-a or ifns, were detectable during the acute stage of infection with a. pleuropneumoniae (fossum et al., 1998) . other experimental infection studies with a. pleuropneumoniae found elevated serum tnf-a and il-1 levels (huang et al., 1999) or no cytokines at all (baarsch et al., 1995) . changes in serum tnf-a have been associated with weight loss, depression and respiratory disease in high-health boars entering conventional swine facilities (harding et al., 1997) . though the exact etiology of this socalled post-arrival disease is unde®ned, the pathogenesis seemingly involves changes in plasma or serum levels of tnf-a and acute phase reactants. at the laboratory of veterinary virology, ghent university, we are studying the signi®cance of cytokines during viral infections of the lungs of swine. we have previously demonstrated different cytokine pro-®les in bal¯uids of gnotobiotic pigs infected with porcine respiratory coronavirus (prcv), porcine reproductive and respiratory syndrome virus (prrsv) or swine in¯uenza virus (siv) (van reeth et al., 1999) . we chose to detect bioactive cytokines in these studies because bioassays have the greatest biological significance. the major ®nding was that cytokines may provide a clue for the remarkable differences in pathogenicity between the three pulmonary viruses. after intratracheal inoculation of gnotobiotic pigs with high virus doses, both prcv and prrsv were unable to induce overt respiratory signs. the infection with prcv, which replicates in alveolar epithelial cells, was asymptomatic, though gross lung consolidation consistently occurred. microscopic lung in¯ammation and airway epithelial necrosis, on the other hand, remained very mild. of the four cytokines (ifn-a, tnf-a, il-1 and il-6) examined, only ifn-a was found at high and sustained levels. prrsv, a macrophage-tropic virus, caused a transient fever, anorexia and lethargy without respiratory disease. there was massive in®ltration of alveolar septa and spaces with macrophage-like cells. lung epithelial damage, neutrophil in®ltration and gross lung consolidation, on the contrary, were minimal. only il-1 was consistently recovered from bal¯uids of prrsv-infected pigs. siv, in contrast, is a primary respiratory pathogen of swine. the disease is characterized by high fever, depression, anorexia, tachypnoea and a labored abdominal respiration. the hallmarks of the infection are a fulminant virus replication in bronchial, bronchiolar and alveolar epithelia, degeneration and desquamation of epithelial cells, neutrophil in®ltration, and the appearance of well-demarkated, dark red areas of gross pneumonia. neutrophils are the predominant cell type in bal¯uids and they are thought to be responsible for the typical breathing dif®culties and lung damage. fig. 1 illustrates the time course of pathogenetic events and the corresponding cytokine pro®le after intratracheal inoculation of gnotobiotic pigs with siv. disease, peak virus titres and neutrophil in®ltration developed as early as 18±24 h after inoculation. clinical signs and neutrophil in®ltration resolved very rapidly and virus titres had decreased signi®cantly by 72 h. excessive amounts of bioactive ifn-a, tnf-a, il-1 and il-6 were found in bal uids of these pigs. there was a tight correlation between cytokines on the one hand and disease, neutrophil in®ltration and peak virus titres on the other. as clinical signs and lung in¯ammation subsided, ifn-a and il-6 titres decreased and tnf-a and il-1 became undetectable. because of the synergistic interactions between these cytokines, the cytokinè`q uartet'' is likely to have much more potent effects than each cytokine on its own. furthermore, cytokines other than those examined here may also contribute to the pathogenesis. one candidate cytokine is il-8, the primary neutrophil chemoattractant. il-8 is one of the more dif®cult cytokines to detect by bioassay and commercial elisas for porcine il-8 have only recently become available. consequently, the secretion of il-8 in body¯uids of pigs has never been demonstrated so far and its signi®cance in diseases of swine remains to be proven. there are some striking parallels between siv and a. pleuropneumoniae with regard to clinicopathological manifestations and cytokine pro®le. with both pathogens, the clinical outcomes of an experimental infection depend on the inoculation route and dose, and cytokines are only found in clinically severe infections. in our hands, characteristic``swine¯u'' symptoms can only be reproduced by direct intratracheal inoculation of 7.0±7.5 log 10 eid 50 virus. intranasal or aerosol inoculation of a similar virus dose induced only mild clinical signs and pathology, and virus titres in the lungs were approximately 100 times lower. interestingly, the aerosol inoculation also failed to induce detectable tnf-a and il-1, while ifn-a and il-6 levels were about 100 times lower than after intratracheal inoculation. further support for a role of cytokines in in¯uenza symptoms and pneumonia comes from examinations of cytokine pro®les in siv vaccination-challenge studies (van reeth et al., 2001) . in these studies, conventional siv-negative pigs were vaccinated twice with different inactivated siv vaccines and then challenged intratracheally with virulent siv. at 24 h after the challenge, mean clinical scores, lung virus titres, neutrophil counts, and ifn-a, tnf-a and il-6 levels were clearly lower in the vaccinates than in unvaccinated challenge controls. all three cytokines were directly correlated with the viral load and with disease severity. this type of study however does not really prove the role of cytokines in disease. because replication of the challenge virus was signi®cantly reduced in the vaccinated pigs, direct pathogenic effects of the virus could probably not occur. still, our data raise questions about the role of il-1 and neutrophils, since both parameters showed little correlation with infectious virus production or disease. prcv and prrsv fail to induce multiple cytokines or respiratory disease by itself, but there are strong indications for their involvement in multifactorial respiratory disease. prrsv in particular is considered a major player in the etiology of the so-called``porcine respiratory disease complex (prdc)''. unexpectedly, most experimental dual infections with prrsv followed by bacteria were clinically mild or subclinical. in other dual infection studies, the clinical effects were extremely variable. this was also true for infections with prrsv followed by a second virus, such as prcv or siv (reviewed in . a critical evaluation shows that all of the published dual infection studies with prrsv result in additive effects and not in a true potentiation of one infection by another. in an attempt to study interactions between respiratory viruses and secondary agents in a reproducible way, we have performed subsequent inoculations of pigs with either prcv or prrsv followed by bacterial lipopolysaccharide (lps). lps is the biologically active component of endotoxin, a major constituent of the cell wall of gram-negative bacteria. the choice of lps was based on the following: 1. pigs are continually exposed to endotoxins in the respirable fraction of dust in swine con®nement units. in addition, endotoxins are released locally in the lungs during pulmonary infections with gram-negative bacteria. preparations are commercially available. combined inoculations with virus and lps therefore may avoid the variability resulting from inter-ference of a ®rst virus with replication of a second virus or bacteria. 3. lps is an extremely potent cytokine inducer and exerts most of its biological effects through cytokines. nearly all lps effects are strictly doserelated, and relatively high lps doses are required to induce cytokines and decreased lung function upon inhalation. low lps doses, on the other hand, are insuf®cient to induce cytokines and disease. it was our working hypothesis that prcv or prrsv may prime lung cells for enhanced secretion of early cytokines in response to minute amounts of lps and thereby initiate disease. in experiments with prcv, gnotobiotic pigs were inoculated intratracheally with prcv and 24 h later with lps from e. coli 0111: b4 . fig. 2 compares the effects of the combined prcv±24 h±lps inoculation with those of the respective single inoculations. all pigs were euthanatized between 3 and 72 h after lps, or at corresponding times after the single prcv inoculation. the effects of separate virus or lps inoculations were subclinical and failed to induce high and sustained cytokine levels. the combination of both agents, on the contrary, resulted in marked labored breathing, dullness and loss of appetite during the ®rst 10±12 h after lps. prior infection with prcv truly potentiated the cytokine response to lps, with 10±100 times higher titres of tnf-a, il-1 and il-6 than after the respective single inoculations. despite very high ifn-a titres in the dually inoculated pigs, similar titres may also occur after a single prcv infection (van reeth et al., 1999) . experiments with different time intervals between prcv and lps inoculations have demonstrated a distinct time frame post-viral infection during which the synergy between virus and lps is maximal. that is, typical respiratory disease was seen with 8±32 h intervals between virus and lps, but not with shorter or longer intervals. titres of tnf-a and il-6 were also critically dependent on the time interval, and those cytokines were best correlated with disease. il-1 and ifn-a, on the other hand, were not affected by the time interval and appear to play a minor if any role. the time interval experiments suggest that one virus replication cycle in the lungs must be completed at the time of lps inoculation for enhanced disease and tnf-a/il-6 production to occur. unexpectedly, there was no clear synergistic effect between prcv and lps with respect to lung pathological changes. excessive neutrophil in®ltration, oedema and haemorrhages were seen in both singly lps and prcv±lps inoculated pigs, to an almost similar degree. also, there was little correlation between these pathological features and disease or cytokines. these observations were surprising because a direct relationship between neutrophil sequestration in the lung and respiratory disorders has been demonstrated in many studies (lentsch and ward, 2001) . one attractive hypothesis is that not the in¯ammation or structural lung damage but functional lung disturbances, such as bronchoconstriction or bronchial hyperresponsiveness, contribute to respiratory disease. interestingly, tnf-a has been implicated in bronchial hyperresponsiveness in rats (kips et al., 1992) . our clinicopathological, virological and cytokine ®ndings have been remarkably similar for the combination prrsv and lps. still, prrsv and prcv differ completely from a pathogenetic point of view. unlike prcv, prrsv has a speci®c tropism for lung macrophages and the infection produces a massive and long-lasting in®ltration of the lungs with macrophagelike cells. macrophages are pre-eminent producer cells of tnf-a and il-1 upon in vitro lps exposure. because absolute numbers of macrophage-like cells increase approximately 5-fold after prrsv infection, one would assume that the lungs contain more potentially lps-responsive cells than after any other virus infection. still, tnf-a, il-1, as well as il-6 pro®les were remarkably similar to those seen in prcv±lps experiments. this raises questions about the importance of macrophages relative to other lung cells in virus±lps induced cytokine responses. the in vivo situation is probably much more complicated than any in vitro system, and several types of lung cells may be involved. in contrast with the cytokines just mentioned, ifn-a levels were some 1000 times lower than in the prcv±lps model. prrsv is a very poor inducer of ifn-a in itself (albina et al., 1998; van reeth et al., 1999) , and the subsequent lps inoculation apparently did not affect ifn-a titres. this con-®rms the idea that ifn-a is less important in the pathogenesis of virus±lps interactions. perhaps the most important difference between prcv and prrsv infection is the time frame during which the lungs are more responsive to lps. unlike prcv, prrsv persists in the lungs during at least 3±4 weeks. experimental prrsv±lps inoculations with intervals varying from 3 up to 14 days all resulted in disease and cytokine production. it seems logical therefore that prrsv will have a greater chance to interact with bacterial components in the ®eld than prcv. consequently, we will use the prrsv±lps model to test preventive or therapeutic measures and study the role of cytokines in more detail. in ongoing experiments, we are studying the effect of treatment with pentoxifylline (ptx) on the clinical response to prrsv±lps. ptx, a phosphodiesterase inhibitor, has been documen-ted to block the production of tnf-a, and may also reduce production of il-1 and il-6 (neuner et al., 1994) . like for siv, however, the true role of cytokines in prrsv±lps induced disease needs to be examined by speci®c anti-cytokine strategies. though our studies have been merely descriptive, we were the ®rst to demonstrate different cytokine patterns during different viral respiratory infections of swine and, most important, an association between cytokines and disease. the next step is to use more speci®c strategies to dissect the role of cytokines during speci®c viral infections. one important issue is that the same cytokines mediating disease may also regulate disease resistance. in fact, ifn-a has extremely potent antiviral effects, and in vitro antiviral effects have also been attributed to tnf-a and il-1. fever and neutrophils are part of the innate immune response to pathogens, and their suppression has proven deleterious in many instances. with the exception of il-8, the early cytokines are also involved in speci®c immunity by activation of t cells, b cells and macrophages. in the case of siv, virus clearance from the lungs and the development of a speci®c immune response occur within days. it is conceivable therefore that cytokines mediate both clinicopathological manifestations and the resolution of a siv infection. it remains to be seen whether selected anti-cytokine strategies can improve disease without damaging normal immune mechanisms. swine provide a unique animal model for the study of cytokines in viral respiratory diseases of humans. the pathogenesis of in¯uenza, for example, is very similar in swine and humans. moreover, the same cytokines associated with¯u symptoms in swine have been demonstrated in nasal lavage¯uids of experimentally infected human volunteers (hayden et al., 1998) . the role of cytokines in pneumonia or lower respiratory tract disease, however, cannot be studied in humans. also, the clinicopathological manifestations of in¯uenza in the mouse model differ widely from those in natural virus hosts, and pigs are an alternative and valuable animal model. interactions between respiratory viruses and endotoxins, though given very little attention, may also occur in humans. respiratory coronaviruses, for example, cause subclinical infections of the respiratory tract of humans, and the inhalation of endotoxins has similar effects in humans and swine (thorn, 2001) . finally, we can study some of the most exciting questions in pigs, such as: how do viruses induce the production of different cytokines? which cells make cytokines during different virus infections and where? how do cytokines released in the lung exert systemic effects? interferon-alpha response to swine arterivirus (poav), the porcine reproductive and respiratory syndrome virus increased levels of tumor necrosis factor and interleukin-1 in bronchoalveolar lavage¯uids from pigs infected with mycoplasma hyopneumoniae detection of interleukin-6 and prostaglandin e2 in bronchoalveolar lavage¯uids of pigs experimentally infected with mycoplasma hyopneumoniae in¯ammatory cytokine expression in swine experimentally infected with actinobacillus pleuropneumoniae pathophysiologic correlates of acute porcine pleuropneumonia the function of type i interferons in antimicrobial immunity in situ hybridization for the detection of in¯ammatory cytokines (il-1, tnf-a and il-6) in pigs naturally infected with actinobacillus pleuropneumoniae evaluation of various cytokines (il-6, ifn-a, ifn-g, tnf-a) as markers for acute bacterial infection in swineða possible role for serum interleukin-6 association of tumour necrosis factor and acute phase reactant changes with post-arrival disease in swine local and systemic cytokine responses during experimental human in¯uenza a virus infection pathogenesis of porcine actinobacillus pleuropneumonia. part ii. roles of proin¯am-matory cytokines tumor necrosis factor causes bronchial hyperresponsiveness in rats regulation of experimental lung in¯ammation interleukin-10 gene therapy-mediated amelioration of bacterial pneumonia in¯ammatory cytokines in animal health and disease compartmentalization of intraalveolar and systemic lipopolysaccharide-induced tumor necrosis factor and the pulmonary in¯ammatory response pentoxyfylline in vivo down-regulates the release of il-1 beta, il-6, il-8 and tumour necrosis factoralpha by human peripheral blood mononuclear cells acute lung injury: the role of cytokines in the elicitation of neutrophils the in¯ammatory response in humans after inhalation of bacterial endotoxin: a review proin¯ammatory cytokines and viral respiratory disease in pigs differential production of proin¯ammatory cytokines in the pig lung during different respiratory virus infections: correlations with pathogenicity a potential role for tumour necrosis factor-a in synergy between porcine respiratory coronavirus and bacterial lipopolysaccharide in the induction of respiratory disease in pigs ef®cacy of vaccination of pigs with different h1n1 swine in¯uenza viruses using a recent challenge strain and different parameters of protection the research described here was ®nancially supported by the belgian ministry of agriculture. kristien van reeth and steven van gucht are fellows of the fund for scienti®c research-flanders (fwo-vlaanderen). the authors thank lieve sys and fernand de backer for expert technical assistance. key: cord-262511-96xp1v0r authors: khabar, khalid s. a. title: rapid transit in the immune cells: the role of mrna turnover regulation date: 2007-03-30 journal: j leukoc biol doi: 10.1189/jlb.0207109 sha: doc_id: 262511 cord_uid: 96xp1v0r there have been recent, significant advances about the role of mrna turnover in controlling gene expression in immune cells. post‐transcriptional regulation of gene expression contributes to the characteristics of many of the processes underlying the immune response by ensuring early, rapid, and transient action. the emphasis of this review is on current work that deals with the regulation of mrna decay during innate immunity against microbes and t cell activation as a model of the adaptive response. in large cities, "rapid transit" is a term that refers to the efficient movement of people allowing rapid entry and exit from a transportation system. a rapid transit scheme also occurs in the immune system to allow immediate activation of the immune cells toward microbes, transient mode of action, and swift recovery. post-transcriptional regulation contributes significantly to this rapid transit by several mechanisms, including mrna stability modulation and translational control; collectively, they aim to control the expression of key gene products involved in the immune response. sequence elements, mainly in the 3ј untranslated region (3јutr), and modulation by rna-binding proteins can affect mrna stability and protein translation. many of the gene products involved in the immune system, such as cytokines, harbor destabilization sequence elements in the mrna, mostly adenylate-uridylate (au)-rich elements (ares), which comprise a heterogeneous group of sequence classes that can affect protein interactions with the mrnas and therefore influence the mrna decay characteristics [1, 2] . large-scale studies involving profiling of mrnas that are bound to rna-binding proteins indicate that many of these genes are involved in the early response to stimuli, host defense, and regulation of rna metabolism and translation [3] [4] [5] . the stabilization of cytokine mrna and other immune response gene products can occur by the activity of mrna stabilization-promoting proteins such as human antigen (hur) protein or by inactivation of rna decay-promoting proteins such as the zinc finger protein, tristetraprolin (ttp). hur is one of the most characterized rna-binding proteins; it is a mammalian homologue of embryonic, lethal, abnormal vision proteins, which was first described in drosophila. it is implicated in the stabilization of many au-rich mrnas including those that participate in immunity and inflammation such as tnf-␣, il-3, il-8, and the urokinase activator, urokinase-type plasminogen activator [6 -8] . among rna-binding proteins that promote mrna decay is ttp. it is an endotoxin-inducible, early response gene product, which destabilizes several proinflammatory cytokine mrnas including tnf-␣, gm-csf, and il-2 by binding to a specific class of ares in the 3јutr. in addition to ttp, k-homology splicing-regulatory protein (ksrp [9] ), butyrate response factor (brf1 [10] ), and certain gene products of the are rna-binding protein 1 [are/ poly(u)-binding/degradation factor 1 (auf1)] family [2] are considered negative-feedback mediators. ttp and several other rna-binding proteins appear to function by targeting au-rich mrna to the exosome, a multiprotein complex with 3ј-5ј exornase activity representing a major degradation machine in eukaryotes [11] . are-binding proteins can also function by recruitment of degradation factors other than the exosome [12, 13] . an alternative pathway of 5ј-3ј decay can also occur during degradation of au-rich mrnas in cytoplasmic rna foci-processing bodies [14] . after a brief introduction about the major signaling pathway regulating mrna turnover, the review is structured in such a way that reflects key functions of the immune cells, including recognition by innate immunity and t cell activation. signaling pathways that tend to stabilize are-mrnas are multiple and include p38 mapk, erk, c-jun n-terminal kinase, pi-3k-akt, and wnt/␤-catenin pathways [10, [15] [16] [17] . the p38 mapk pathway is one of the most common pathways that regulates mrna stability in multiple cell types. recent work extends the role of the p38 mapk pathway and its downstream target kinase, mapk-activated protein kinase 2 (mk2) [18 -22] . activation of the p38 mapk pathway drives the accumulation of ttp via the stabilization of ttp mrna and protein [18 -20] . although this remains controversial [22] , mk2-mediated phosphorylation of ttp has been reported to impair its function, possibly as a result of reduced binding affinity with the tnf-␣ are region [18, 23] . another p38 mapk downstream target with structural and functional similarity to mk2 is mk3; it is thought to cooperate with mk2 in tnf-␣ mrna stabilization [24] . ttp and other rna decaypromoting proteins, namely brf1, ksrp, and auf1, particularly when phosphorylated, are complexed with 14-3-3 proteins that influence their subcellular localization and mrna decay activity [10, 16, 25] . another player in the post-transcriptional regulation of the immune system by the p38 mapk pathway is the downstream mapk signal-integrating kinase, mnk1; this kinase is thought to phosphorylate the eukaryotic initiation factor 4e (eif4e) and heterogeneous nuclear ribonucleoprotein a1 [26, 27] . the p38 mapk pathway is central to the stabilization of many immune system mrnas such as those of cytokines during innate and adaptive immune responses. cytokines can regulate immune response via positive-and negative-feedback circuits. in general, when cytokines amplify a specific immune response, they tend to stabilize mrnas that participate in such a process. examples of those cytokines are the proinflammatory cytokines, il-1␣ and tnf-␣, which have been found to stabilize a significant number of cytokines and chemokines, including ifn-␥-inducible protein 10 (ip-10), growth-related oncogene proteins, and fractalkine (scyd1), and several genes involved in regulation of inflammatory responses such as tnf-␣-induced protein 3, nf-b␣i, and mannose-binding lectin 2 (mbl2) [28 -30] . cytokines use p38 mapk signaling and notably, its target mk2 to trigger mrna stabilization. for example, using this pathway, ifn-␥ amplifies immune responses such as increasing nk activity and up-regulating mhc expression [31] . in several inflammatory conditions, other cytokines can amplify inflammatory responses via post-transcriptional mechanisms. il-17a, in a p38 mapk-dependent manner, can increase the stability of tnf-␣-induced il-8 mrna in human airway smooth muscle, a process that is important in development of asthma [32, 33] . il-1, in addition to activated platelets, triggers stabilization of cyclooxygenase 2 (cox-2) mrna with concomitant cytoplasmic accumulation and increased binding of hur to the ares of cox-2 mrna in monocytes [33] . in general, stabilization of the mrna targets seems to occur after translocation of hur from the nucleus to the cytoplasm upon cellular activation [34] . innate immunity is the first line of defense against microbes and requires early and rapid gene expression of effector mediators to control infection effectively. recognition and effector mechanisms of innate immunity encompass post-transcriptional gene regulation, which amplifies the much-needed, early response and enforces the transient response. efficient recognition of the foreign antigens in microbes, such as bacterial surface molecules and viral dsrna, is accomplished by cells of the innate immunity, notably macrophages and dendritic cells (dc). members of the tlr family [35, 36] , which have gained considerable attention in recent years, are responsible for triggering the efficient response of the host cell to microbes. traditionally, post-transcriptional regulation in innate immunity has been studied in response to the bacterial endotoxin, lps, which binds cd14 in a complex with tlr4 on the surface of neutrophils and macrophages and initiates a cascade of signals that causes cell activation, the inflammatory response, and phagocytosis [35] . recent work gives further insights into lps-induced pathways. upon activation of tlr4 by lps, the adaptor protein myd88 is recruited to the receptor and couples with il-1 receptor-associated kinase (irak)1 and -4, causing irak activation and recruitment into a complex with tnf receptor-associated factor 6 (traf6). this pathway ultimately culminates in a cascade of signaling events including nf-b activation, mapk activation, and transcriptional activation of proinflammatory cytokines [37] . engagement of lps with tlr4 can itself lead to stabilization of its own mrna, resulting in a lps amplification response. although this has been shown in the case of vascular smooth muscle cells when exposed to lps [38] , it is likely to occur with cells of the innate immune system. tlr4 activation causes hur translocation to the cytoplasm, enhanced binding of hur to the 3јutr, and increased stability of tlr4 mrna [38] . the adaptor molecule myd88, which transduces a signal from tlr, causes stabilization of cytokine mrnas, tnf-␣, il-8, and ip-10 in macrophages when stimulated by ifn-␥ [29] . myd88 bridges ifn-␥ receptor 1 and mixed linkage kinase 3 domains, leading to p38 mapk activation [31] . lps-induced tlr4 activation can increase formyl peptide receptor 1 (fpr1) mrna stability via a pi-3k pathway in phagocytic leukocytes such as neutrophils and monocytes, leading to infiltration to sites where the invading bacteria possess the formyl peptide ligands [39, 40] . thus, tlr4 activation encompasses several post-transcriptional, positive regulatory events, which lead to amplification of lpsinduced signaling and ultimately, production of proinflammatory cytokines (fig. 1) . these cytokines and other mediators, including vascular endothelial growth factor, the solute carrier family 11 member 1, and no, can be up-regulated at the level of mrna stability in lps-stimulated, monocytic cells [30, [41] [42] [43] [44] . cytokines such as tnf-␣, il-1, il-6, and il-8 not only contribute to effector mechanisms of innate immunity but also help the activation of adaptive immunity. post-transcriptional regulation of the inflammatory response as a result of endotoxin also offers a negative-feedback control, as excessive proinflammatory cytokine production, notably, tnf-␣, can lead to deleterious, local and systemic effects including septic shock. ttp knockout mice having macrophages and neutrophils, which release higher levels of tnf-␣ and gm-csf, display diseases associated with tnf-␣ over secretion such as inflammatory arthritis [45] . some of these diseases are similar to mice with genetic knockout of the tnf-␣ are region [46, 47] . like ttp, auf1 acts as another negative-feedback mediator of the innate response to endotoxin; it has been demonstrated recently that lps challenge in auf1-knockout mice displays symptoms of severe endotoxic shock as a result of overproduc-tion of tnf-␣ and il-1␤ [48] . this is attributed to the inability to degrade the mrnas efficiently as a result of absence of auf1. although hur is an rna stabilization protein, it can have a negative modulatory function in specific situations. this effect has been shown to suppress specific, proinflammatory mrna targets such as tnf-␣ and cox-2, using in vivo models with conditional overexpression in macrophage subpopulations [49] . these three animal models of ttp, auf1, and hur offer direct in vivo evidence for the role of posttranscriptional control in innate immunity. in addition to lps, another bacterial component that is an important stimulus of innate immunity is the unmethylated cpg dna. it binds tlr9 on dc, neutrophils, monocytes, and also, in airway epithelia cells triggering a signal transduction cascade similar to those in response to lps [50 -52] . cytokines such as il-1␣ and tnf-␣ synergize with cpg dna to induce il-8 production in airway epithelia, mainly by p38 mapk activation and stability of il-8 mrna [51] . cpg dna can augment dc function and maturation using post-transcriptional mechanisms. for example, cpg dna increases mhc i mrna stability in dc by an ifn-␣/␤-dependent process [52] . hur binds to a defined, secondary rna element in the dc mrna of cd83 and increases its protein expression [53] . however, this effect does not involve mrna stabilization, probably as a result of lack of ares in cd83 3јutr [1] but most likely because of enhanced, chromosome region maintenance 1-mediated translocation of cd83 mrna to the cytoplasm by hur [53] . mature dc, which express cd83, are efficient in the stimulation of naive cd4 ϩ and cd8 ϩ t cells. so, these posttranscriptional, regulation events contribute to the efficient maturation of dc-cells, which are important apcs-and further, aid bridging innate immunity to adaptive immunity. different viruses code for different viral proteins, which can control the cell's machinery for their growth or to evade immune cells [54] . viruses can stabilize mrnas of gene products that are essential to the virus life cycle. hsv infection can lead to stabilization of the are-mrna, termed immediate early response 3 (iex-1), probably by means of its transregulatory protein icp27, which is essential in hsv early gene expression [55, 56] . the latent membrane protein 1 (lmp1) of ebv is a major player in the induction of cytokines, which in turn, support the growth and survival of the ebv-infected b cells. lmp1 activates the p38 mapk pathway and stabilizes ip-10 [57] . the kaposi sarcoma (ks) herpes virus (kshv or hsv-8), which causes angioproliferative regions in immunocompromised patients such as aids and transplant patients, promotes the production of proinflammatory cytokines. using a twohybrid system, the kshv latent protein kaposin b has been shown to interact with the c-lobe of the activation domain of mk2 and requires both of the two proline-rich, 23-amino acid direct repeats in kaposin b [58, 59] . as a result, mk2 becomes phosphorylated, and loss of destabilization of au-rich mrnas such as gm-csf and il-6 occurs [58] . il-6 is overexpressed in ks tumors and contributes to its pathogenesis [60] . replication of hepatitis c virus (hcv) in cultured liver cell lines leads to prolongation of the il-8 mrna half-life, probably by its nonstructural protein ns5a, and particularly, in the presence of tnf-␣, a potent inducer of il-8 [28] . a possible mechanism is that hcv, in a similar manner to dsrna, which is an intermediate viral product, may activate the retinoic acid-inducible gene i (rig-i) pathway, leading to il-8 mrna stabilization. rig-i along with melanoma differentiation-associated gene 5, which has been a focus of research in recent years, are rna helicases, recognizing viral dsrna and triggering ifn response [61] . two recent studies have shown that rig-i can distinguish viral, uncapped ssrna from cellular mrnas [62, 63] . these proteins and their adaptors have been explored largely for their transcriptional induction of ifn regulatory factor 3-dependent ifn and nf-b-mediated proinflammatory cytokines when compared with post-transcriptional effects. recently, it has been shown that a constitutively active rig-i protein, rig-n, stabilizes tnf-␣-induced il-8 mrna and increases reporter activity from a construct that is fused with are-bearing, il-8 3јutr [64] . il-8 is a proinflammatory cytokine, which appears to exaggerate hcv-induced hepatitis. thus, hcv-driven stabilization of il-8 mrna may explain, at least in part, the increased il-8 levels seen in the sera of hcv patients and those that are nonresponsive to ifn therapy [65] . for successful takeover of cell machinery for their own growth, viruses have the ability to shut-off cellular mrna biogenesis and induce rapid degradation of selected cellular mrnas, particularly those required for host defense. for example, the nonstructural protein, nsp1, from the severe acute respiratory syndrome coronavirus, suppresses ifn-␤ production at a post-transcriptional level by increasing its mrna decay [66] . cytomegalovirus is able to destabilize il-6 mrna with concomitant translocation of hur to the cytoplasmic compartment [67] , although this process is usually known to cause up-regulation of are and are-like-containing mrna [68] . the hsv-1 open-reading frame ul41, which encodes virion host shut-off or vhs, can act as an endornase or a component of the rnase complex, degrading cellular mrnas such iex-1 mrna [69] , although this study is in conflict with other reports that hsv-1 infection leads to iex-1 mrna stabilization [55, 56] . this discrepancy may be a result of the presence of alternative forms of transcripts with different regulatory responses owing to inclusion or exclusion of ares through alternative splicing and polyadenylation [70] . can the rna decay-promoting activity of the rna-binding proteins be harnessed as an antiviral therapeutic strategy? two recent reports show that this is a possibility [9, 71] . ksrp has been tethered to the hiv type 1 rev protein, essential in the hiv life cycle, and binds a specific response element in hiv rna. the ksrp-rev is able to bind to hiv rna and triggers its degradation, leading to a dramatic reduction in hiv repli-cation [9] . likewise, ttp can be used in the same manner, probably with an additional advantage: ttp binds directly to the au-rich hiv-1 rna and enhances multiple splicing, which leads to a reduction of hiv-1 virions [71] . activation of lymphocytes is fundamental to the immune response to antigens and requires two main signals. one signal is provided upon the engagement of tcr with the antigen presented with mhc on apcs. the other is delivered by costimulatory molecules, such as lymphocyte function-associated protein 1 (lfa-1), cd2, and cd28 on t cells, when they interact with cognate receptors on the apcs. with these two signals, the result is ap-1 (c-fos/jun) induction and commitment production of il-2, the hallmark of t cell activation and differentiation [72] . post-transcriptional control plays an important regulatory role in almost all of the events that lead to transcriptional activation (fig. 2) . a large proportion of genes is regulated by mrna stability, as revealed with the large-scale, gene-expression profiling in activated t cells [73, 74] . one of the most important signaling events in t cell activation that results in stabilization of cytokines, such as il-2, ifn-␥, tnf-␣, and gm-csf, is induced by the costimulatory cd28 [75] . this signaling pathway does not only increase the promoter activity of the cytokines [76] but also stabilizes their mrnas [75] . the increased il-2 mrna stability is thought to be controlled differently and independent of the pi-3k-dependent induction of the il-2 promoter [77, 78] . unlike il-2 transcription, cd28-induced stabilization of the il-2 mrna and possibly other cytokine mrnas does not require membrane colocalization with the tcr within cd28 in a multimolecular signaling complex, the central supramolecular activation cluster [72, 77] . the role of ttp in il-2 production during t cell activation has been studied in detail [79] . the tcr/ cd28-engaged or -immobilized, anti-cd3-induced t cells derived from ttp knockout mice have elevated il-2 levels as a result of increased stabilization of the il-2 mrna [79] . ttp is able to bind 32-base ares from the il-2 3јutr and destabilizes reporter mrna fused with a 3јutr, which contains the il-2 are [79] . like cd28, when lfa-1 on t cells binds to icam-1 on apcs, allowing strong adhesion between the two cells and promoting efficient t cell activation [80] , it stabilizes certain cytokine mrnas [81] . although it has been reported that hur translocation occurs in mouse t cells upon cd28-induced signaling, without an effect on il-2 mrna stabilization [82] , lfa-1 or cd28 engagement of human-stimulated t cells promoted hur translocation to the cytoplasm and stabilized tnf-␣ and gm-csf [81] . this discrepancy may be a result of the species source of the lymphocytes used in the two studies. ares are evolutionally conserved in mammalian, but the numbers of are pentamers may differ slightly among species, allowing differential responses [70] . a novel pathway in cytokine mrna stabilization in t cell activation and differentiation, also occurring during eosinophils activation, has been described recently involving pin1 [83, 84] . this protein isomerizes specific peptide bonds and thus, alters protein folding and activity; when it becomes activated in tcr/cd28-stimulated cd4 t cells, it associates concurrently with auf1 and hur. this results in stabilization of gm-csf mrna as a result of the loss of the mrna decay-promoting activity of auf1, which is linked to the exosome (fig. 2) . auf1 may be isomerized by pin1, allowing the stabilizing action of hur to predominate [83] . auf1 is a nucleocytoplasmic-shuttling protein and is associated with stabilization and destabilization activities; this appears to be dependent on the auf1 isoform generated from four alternative-spliced mrnas and the cell type in question. for example, when auf1 p37 and p42 isoforms are overexpressed, they bind to il-6 3јutr, facilitated by a putative rna stem-loop structure; this process promotes the stabilization of il-6 mrna [85] . ttp plays a significant role in regulating mrna stability of cytokines such as il-2 during t cell activation. thus, one can envision that for cd28-or lfa-1-induced stabilization to occur following t cell activation, ttp destabilization action must cease. this can happen by either or both of two scenarios: coordinated stabilization-destabilization kinetics and ttp phosphorylation (fig. 2) . with the coordinated kinetics model, stabilizing rna-binding proteins such as hur can occur initially following immune cell activation, allowing rapid and early response of cytokine production. this is followed by the action of mrna-destabilizing proteins such as ttp, resulting in transient cytokine production. hur translocation following tcr/cd28 engagement is seen, first within 1 h, and ttp is induced in activated t cells in a later time [86] . there is also the potential for interesting reciprocal regulation, which helps to amplify this "cycle"; hur may stabilize ttp mrna itself, thus helping to prime ttp for its destabilizing action on cytokine mrnas. ttp has also been suggested to regulate the stability of its own mrna in different cell types [20, 21] , although this has not been proved conclusively [4] ; it is also possible that this regulation extends to activated t cells. the second scenario is that ttp becomes transiently phosphorylated by one of the kinases following cd28 or lfa-1 engagement. the ultimate outcome is a transient immune response and controlled cytokine production. during restimulation of th2 cells, as opposed to priming of naïve t cells with apc, rapid dephosphorylation of eif2␣ occurs and results in derepression of translational arrest of cytokine mrnas such as il-4 mrna [87] . this process infig. 2 . enhanced mrna stabilization and translation in t cell activation. apcs, like dc, present antigens efficiently within mhc to t cells through interaction with tcr. facilitated by other costimulatory molecules, t cell activation occurs, resulting in induction of immediate early response gene products such as c-fos and c-jun. these, as ap-1 complex, with nfat activate the il-2 promoter. costimulatory molecules, such as cd28 and lfa-1 on t cells, transduce signals, which also trigger mrna stabilization and other post-transcriptional effects. for example, hur translocates from the nucleus to the cytoplasm and stabilizes are-mrnas, and pin1 isomerizes another rna-binding protein, auf1, leading to loss of its mrna decay-promoting function. these events contribute to early and rapid response of t cell activation and cytokine production. in later phase, shut-off also can be facilitated by post-transcriptional mechanisms including mrna destabilization and translational arrest. details are given in the text. pin1, peptidyl-prolyl isomerase; tia, t cell intracellular antigen. volves translation repressor proteins, tia-1, and tia-1-related protein, which operate during priming and restrict t cell function by recruiting untranslated mrnas into stress granules, discrete cytoplasmic foci triggered by eif2␣ phosphorylation [88] . also, the p38 activity, which leads to il-4 mrna stabilization, modulates differentiation of naive precursor t cells to th2 direction [89] . post-transcriptional control plays a significant part in specific pathways that regulate t cell activation and involves several types of modulators, including suppressive cytokines, mirna, and adaptors. when cytokines such as ifn-␣/␤, il-4, and il-10 dampen the immune response, they tend to destabilize mrnas involved. ifn such as ifn-␤ or -␥, when in combination with lps, induces ttp transcription and thereby reduces proinflammatory cytokines, namely, tnf-␣ and il-6 and the chemokines ccl2 (mcp-1) and ccl3 (mip-1␣) [90] . this happens in a p38 mapk-dependent manner, as revealed with ttp-knockout mouse embryo fibroblasts [90] . the ttp transcription appears to require a functional, ifn-␥-activated sequence, which recruits stat1 onto the ttp promoter. as ttp is considered an anti-inflammatory mediator and suppresses proinflammatory cytokines, it is possible that at least some of the molecules with anti-inflammatory action induce ttp or its activity. indeed, glucocorticoids can induce the transcription of ttp in the a549 lung epithelial cell line; small interfering rna to ttp prevented dexamethasone-induced reduction of tnf-␣ mrna stability and activity of reporter fused with tnf-␣ 3јutr [91] . on the contrary, this action may be tissuetype-specific, as dexamethasone may inhibit rather than induce ttp in activated macrophages [92] . il-10 is a potent, suppressive cytokine, which can inhibit macrophage action and generation of th2 lymphocytes. it is capable of inhibiting proinflammatory cytokines and th1 cytokines, such as tnf-␣, ifn-␥, il-2, il-3, and gm-csf. il-10 destabilizes tnf-␣ mrna and probably others by inhibiting the binding of hur to the ares in the 3јutr; this effect appears also to be dependent on the p38 mapk pathway [93] . another anti-inflammatory cytokine, il-4, can suppress fpr1 mrna expression via a mechanism, which acts on primary transcripts prior to maturation and depends on the constitutive instability of pre-existing mrna [94] . a possible pathway, which may involve mrna stability changes as a result of negative regulation of tcr/cd28-induced t cell activation, is the association between adaptor in lymphocytes of unknown function (alx) and membrane-associated adaptor protein (lax) adaptors. work with alx-deficient knockout mice suggested that the alx/lax association may inhibit the p38 mapk pathway and il-2 production [95] . specifically, the upstream, regulatory mapks, mkk3/6, have been found to be constitutively activated in alx-deficient mice [95] . as p38 mapk is involved in il-2 mrna stabilization, it is possible that negative regulation by alx occurs via a post-transcriptional mechanism as well. negative regulation of t cell activation and cytokine production can also occur by a mirna-processing pathway. in general, mirnas are considered post-transcriptional, negative regulatory elements, which have the ability to dampen many biological processes including innate and adaptive immunity. a role in the regulation of t cell development has been proposed recently in a few studies [96 -99] . specific deletion of the dicer-coding gene, dcr-1, in mouse t cell lineage resulted in aberrant th cell differentiation and failure to suppress ifn-␥ expression under th2-polarizing conditions [98] . although no mechanism is discussed in this study, it is possible that mirna targets the ifn-␥ mrna, which harbors an are, which may in turn be a target for mir16 by a process in which ttp promotes mrna decay. involvement of mirna in aremediated instability has been documented by demonstrating that dicer and mir16 are required in epithelial cells [100] . this ttp involvement would be an attractive option in explaining the ifn-␥ mrna dysregulation in dicer-deficient th cells. another important role of dicer in t cell development has been shown to occur in the development of regulatory t cells (treg); absence of dicer leads to a reduced number of treg and immunopathological disorders such as splenomegaly [97] . this study concludes that dicer facilitates the development of treg in the thymus and the efficient induction of the necessary forkhead transcription factor, foxp3, by tgf-␤ in naive cd4 t cells [97] . several mirnas, mir146a/b, mir132, and mir155, are lps-responsive genes in human monocytes [99, 101] . the mir155 is inducible in a human primary macrophage in response to different types of inflammatory mediators [101] , but its effects on the immune cell function remain to be elucidated. the mir146a/b bind sequences in the 3јutrs of two important adaptor molecules involved in lps signaling, traf6 and irak1, and these utrs inhibit expression of reporter constructs [99] . the role of mirna silencing machinery in not limited to negative control of the immune system as described in the above examples but itself, has a direct innate immunity function. drosha and dicer can act as intracellular, antiviral enzymes against hiv human monocytic cells, and the hiv tat can act as a suppressor of the host rna silencing [102, 103] . although many recent studies emphasize protein-protein and the adaptor interactions on signaling events, which lead to transcriptional activation, it is expected that more studies will focus on post-transcriptional mechanisms. the array of intracellular rna-protein interactions is manifested by the large number of are-mrnas and rna-binding proteins. given that many mrnas of the immune response contain ares, and multiple signaling pathways are operative such as p38 mapk and pi-3k/akt, one can imagine the vast probability of posttranscriptional interactions in the immune system. additional types of post-transcriptional mechanisms, such as alternative splicing and post-translational regulatory events, although not within the focus of this review, also contribute to the intricate and complex regulation of the immune system. as cities cope with the rapid entry of people by using rapid transit systems, post-transcriptional control of immune system helps in rapid activation of immune cells by mechanisms that involve rapid translocation of mrna stabilization proteins such as hur to effector cellular compartments and/or temporal inactivation, e.g., by phosphorylation or proteolysis of rna decay proteins such as ttp or ksrp. also, when people move out quickly from the transits at the point of exit, mechanisms in the immune system ensure a swift shut-off and recovery. this happens by the reversal of events that led to mrna stabilization, activation of decay-promoting proteins, and translational arrest (fig. 3) . these temporal and spatial events culminate in a transiently regulated immune system, which is able to cope with external and internal insults to the body without deleterious effects on the host. specific abnormal situations may arise if the transient response of the immune system becomes prolonged, such as those that occur in chronic inflammatory and autoimmune diseases. fig. 3 . rapid transit analogy with rapid activation and repression of gene expression. receptor-mediated signaling events following recognition of various types of foreign bodies or following interactions with positive and negative modulatory cytokines lead to transcriptional and post-transcriptional activities. rapid activation of immune cells and early response events such as cytokine production mimic rapid entry to transit systems, culminating in amplification of innate and adaptive responses, whereas rapid exit from the transit mimics the shut-off mechanisms in the immune cells that lead to transient response. this is orchestrated by an array of rna decay mechanisms and a vast number of instability-prone mrnas. pabp, poly(a)-binding protein; mlks, mixed lineage kinases. ared 3.0: the large and diverse au-rich transcriptome au-rich elements and associated factors: are there unifying principles? identification and functional outcome of mrnas associated with rnabinding protein tia-1 novel mrna targets for tristetraprolin (ttp) identified by global analysis of stabilized transcripts in ttp-deficient fibroblasts concurrent versus individual binding of hur and auf1 to common labile target mrnas the 3ј untranslated region of tumor necrosis factor ␣ mrna is a target of the mrna-stabilizing factor hur distinct domains of au-rich elements exert different functions in mrna destabilization and stabilization by p38 mitogenactivated protein kinase or hur stabilization of urokinase and urokinase receptor mrnas by hur is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2 tethering ksrp, a decay-promoting au-rich element-binding protein, to mrnas elicits mrna decay the are-dependent mrna-destabilizing activity of brf1 is regulated by protein kinase b rna-quality control by the exosome multiple processing body factors and the are binding protein ttp activate mrna decapping recruitment and activation of mrna decay enzymes by two are-mediated decay activation domains in the proteins ttp and brf-1 are-mrna degradation requires the 5ј-3ј decay pathway mapkap kinases-mks-two's company, three's a crowd the rna-binding protein ksrp promotes decay of ␤-catenin mrna and is inactivated by pi3k-akt signaling mitogen-activated protein kinase-dependent and -independent signaling of mrna stability of au-rich elementcontaining transcripts mitogen-activated protein kinase-activated protein kinase 2 regulates tumor necrosis factor mrna stability and translation mainly by altering tristetraprolin expression, stability, and binding to adenine/ uridine-rich element posttranslational regulation of tristetraprolin subcellular localization and protein stability by p38 mitogenactivated protein kinase and extracellular signal-regulated kinase pathways the stability of tristetraprolin mrna is regulated by mitogen-activated protein kinase p38 and by tristetraprolin itself the role of mrna turnover in the regulation of tristetraprolin expression: evidence for an extracellular signal-regulated kinase-specific, au-rich element-dependent, autoregulatory pathway structure/function analysis of tristetraprolin (ttp): p38 stress-activated protein kinase and lipopolysaccharide stimulation do not alter ttp function mk2-induced tristetraprolin:14-3-3 complexes prevent stress granule association and are-mrna decay the mitogen-activated protein kinase (mapk)-activated protein kinases mk2 and mk3 cooperate in stimulation of tumor necrosis factor biosynthesis and stabilization of p38 mapk is a p37 auf1-binding protein that facilitates auf1 transport and au-rich mrna decay posttranscriptional regulation of tnf␣ expression via eukaryotic initiation factor 4e (eif4e) phosphorylation in mouse macrophages the mnks are novel components in the control of tnf ␣ biosynthesis and phosphorylate and regulate hnrnp a1 stability of cxcl-8 and related au-rich mrnas in the context of hepatitis c virus replication in vitro toll il-1 receptors differ in their ability to promote the stabilization of adenosine and uridine-rich elements containing mrna functionally independent au-rich sequence motifs regulate kc (cxcl1) mrna myd88-mediated stabilization of interferon-␥-induced cytokine and chemokine mrna il-17a acts via p38 mapk to increase stability of tnf-␣-induced il-8 mrna in human asm expression of cox-2 in platelet-monocyte interactions occurs via combinatorial regulation involving adhesion and cytokine signaling elav protein hua (hur) can redistribute between nucleus and cytoplasm and is upregulated during serum stimulation and t cell activation the toll-like receptors: analysis by forward genetic methods pathogen recognition and innate immunity myd88 beyond toll the role of human antigen r, an rna-binding protein, in mediating the stabilization of toll-like receptor 4 mrna induced by endotoxin: a novel mechanism involved in vascular inflammation lipopolysaccharide induces formyl peptide receptor 1 gene expression in macrophages and neutrophils via transcriptional and posttranscriptional mechanisms signaling in lipopolysaccharide-induced stabilization of formyl peptide receptor 1 mrna in mouse peritoneal macrophages rna-binding protein hur is required for stabilization of slc11a1 mrna and slc11a1 protein expression differential regulation of are-mediated tnf␣ and il-1␤ mrna stability by lipopolysaccharide in raw264.7 cells induction of expression of inducible nitric oxide synthase by taxol in murine macrophage cells vegf gene expression is regulated post-transcriptionally in macrophages the tandem ccch zinc finger protein tristetraprolin and its relevance to cytokine mrna turnover and arthritis a pathogenetic role for tnf ␣ in the syndrome of cachexia, arthritis, and autoimmunity resulting from tristetraprolin impaired on/off regulation of tnf biosynthesis in mice lacking tnf au-rich elements: implications for joint and gut-associated immunopathologies endotoxic shock in auf1 knockout mice mediated by failure to degrade proinflammatory cytokine mrnas hur as a negative posttranscriptional modulator in inflammation neutrophil signaling pathways activated by bacterial dna stimulation cpg dna modulates interleukin 1␤-induced interleukin-8 expression in human bronchial epithelial (16hbe14o-) cells enhancement of dendritic cell antigen cross-presentation by cpg dna involves type i ifn and stabilization of class i mhc mrna expression of cd83 is regulated by hur via a novel cis-active coding region rna element anti-immunology: evasion of the host immune system by bacterial and viral pathogens herpes simplex virus infection stabilizes cellular iex-1 mrna herpes simplex virus icp27 is required for virus-induced stabilization of the are-containing iex-1 mrna encoded by the human ier3 gene the epstein-barr virus oncoprotein latent membrane protein 1 induces expression of the chemokine ip-10: importance of mrna half-life regulation the kaposin b protein of kshv activates the p38/mk2 pathway and stabilizes cytokine mrnas phosphorylation and function of the kaposin b direct repeats of kaposi's sarcoma-associated herpesvirus kaposi's sarcoma-associated herpesvirus immune modulation: an overview differential roles of mda5 and rig-i helicases in the recognition of rna viruses 5ј-triphosphate rna is the ligand for rig-i rig-i-mediated antiviral responses to single-stranded rna bearing 5ј-phosphates regulation of cxcl-8 (interleukin-8) induction by double-stranded rna signaling pathways during hepatitis c virus infection elevated levels of interleukin-8 in serum are associated with hepatitis c virus infection and resistance to interferon therapy severe acute respiratory syndrome coronavirus nsp1 protein suppresses host gene expression by promoting host mrna degradation posttranscriptional suppression of interleukin-6 production by human cytomegalovirus the role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy the u(l)41 protein of herpes simplex virus 1 degrades rna by endonucleolytic cleavage in absence of other cellular or viral proteins au-rich transient response transcripts in the human genome: expressed sequence tag clustering and gene discovery approach tristetraprolin inhibits hiv-1 production by binding to genomic rna adaptors as central mediators of signal transduction in immune cells patterns of coordinate down-regulation of are-containing transcripts following immune cell activation stability regulation of mrna and the control of gene expression regulation of lymphokine messenger rna stability by a surfacemediated t cell activation pathway regulation of interleukin-2 gene enhancer activity by the t cell accessory molecule cd28 engagement of cd28 outside of the immunological synapse results in up-regulation of il-2 mrna stability but not il-2 transcription cutting edge: cd28-mediated transcriptional and posttranscriptional regulation of il-2 expression are controlled through different signaling pathways tristetraprolin down-regulates il-2 gene expression through au-rich element-mediated mrna decay integrin activation under flow: a local affair lfa-1-dependent hur nuclear export and cytokine mrna stabilization in t cell activation selective cytoplasmic translocation of hur and site-specific binding to the interleukin-2 mrna are not sufficient for cd28-mediated stabilization of the mrna the peptidylprolyl isomerase pin1 regulates granulocyte-macrophage colony-stimulating factor mrna stability in t lymphocytes the peptidyl-prolyl isomerase pin1 regulates the stability of granulocyte-macrophage colony-stimulating factor mrna in activated eosinophils destabilization of interleukin-6 mrna requires a putative rna stem-loop structure, an au-rich element, and the rnabinding protein auf1 hua and tristetraprolin are induced following t cell activation and display distinct but overlapping rna binding specificities activation of the integrated stress response during t helper cell differentiation rna granules the p38 mitogen-activated protein kinase regulates effector functions of primary human cd4 t cells interferons limit inflammatory responses by induction of tristetraprolin glucocorticoids regulate tristetraprolin synthesis and posttranscriptionally regulate tumor necrosis factor ␣ inflammatory signaling inhibition of tristetraprolin expression by dexamethasone in activated macrophages il-10-induced tnf-␣ mrna destabilization is mediated via il-10 suppression of p38 map kinase activation and inhibition of hur expression il-4 inhibits expression of the formyl peptide receptor gene in mouse peritoneal macrophages negative regulation of interleukin-2 and p38 mitogen-activated protein kinase during t-cell activation by the adaptor alx ) t cell lineage choice and differentiation in the absence of the rnase iii enzyme dicer a role for dicer in immune regulation aberrant t cell differentiation in the absence of dicer nf-{}b-dependent induction of microrna mir-146, an inhibitor targeted to signaling proteins of innate immune responses involvement of mi-crorna in au-rich element-mediated mrna instability microrna-155 is induced during the macrophage inflammatory response suppression of microrna-silencing pathway by hiv-1 during virus replication human immunodeficiency virus type 1 tat protein induces an intracellular calcium increase in human monocytes that requires dhp receptors: involvement in tnf-␣ production key: cord-256838-8rzibpbl authors: eng, yi shin; lee, chien hsing; lee, wei chang; huang, ching chun; chang, jung san title: unraveling the molecular mechanism of traditional chinese medicine: formulas against acute airway viral infections as examples date: 2019-09-27 journal: molecules doi: 10.3390/molecules24193505 sha: doc_id: 256838 cord_uid: 8rzibpbl herbal medicine, including traditional chinese medicine (tcm), is widely used worldwide. herbs and tcm formulas contain numerous active molecules. basically, they are a kind of cocktail therapy. herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease interactions are complex. there is potential for both benefit and harm, so only after understanding more of their mechanisms and clinical effects can herbal medicine and tcm be helpful to users. many pharmacologic studies have been performed to unravel the molecular mechanisms; however, basic and clinical studies of good validity are still not enough to translate experimental results into clinical understanding and to provide tough evidence for better use of herbal medicines. there are still issues regarding the conflicting pharmacologic effects, pharmacokinetics, drug interactions, adverse and clinical effects of herbal medicine and tcm. understanding study validation, pharmacologic effects, drug interactions, indications and clinical effects, adverse effects and limitations, can all help clinicians in providing adequate suggestions to patients. at present, it would be better to use herbs and tcm formulas according to their traditional indications matching the disease pathophysiology and their molecular mechanisms. to unravel the molecular mechanisms and understand the benefits and harms of herbal medicine and tcm, there is still much work to be done. it is common to initiate the therapy of orthodox western medicine by fitting the pharmacologic characteristics of a drug, including pharmacokinetic and pharmacodynamic effects, to the disease pathophysiology. physicians further validate the clinical application according to evidence-based medicine (ebm); however, this is not the case for tcm. tcm developed in ancient china, and at that time, physicians managed diseases with herbs only by clinical experience, without any knowledge of disease pathophysiology, nor the pharmacologic activities of herbs, to say nothing of the molecular mechanisms of herbs. to unravel the molecular mechanism of tcm formulas, it would be better to understand how physicians prescribe them first. tcm includes herbal therapy, acupuncture, massage, and dietary therapy. in the current work, tcm will be simply defined as herbal and dietary therapies. tcm is widely popular in east asia and forms the kampo medicine in japan, as well as traditional korean medicine; importantly, traditional medicines form the mainstream of healthcare in these countries. in ancient china, several famous tcm textbooks summarized the clinical experiences of using tcm formulas against various diseases, including endemic diseases, and each formula has its indication, including specific symptoms of patients. this is quite different from using tcm formulas based on the yin-yang theory (two opposite, but complementary forces) and five elements theories (everything in the world can be classified into the natural five elements. these elements promote as well as feedback each other to keep everything in balance). kampo medicine in japan classified tcm theories into ancient formula sect and recent formula sect for prescribing tcm formulas. the physicians of the ancient formula sect (a-physicians) use the indications of formula formed before formula can have active molecules that are pharmacologically different from that ingredient or the tcm formula. obversely, the pharmacological activities of a tcm formula may differ from those of their active ingredient or active molecules of ingredients. as a consequence, unraveling the molecular mechanism of a tcm formula needs comparison of the pharmacological activities between the formula, its ingredients, and the active molecules in the ingredients. the amount of most bioactive compounds in the herbs is very low. combination of herbs to form a tcm formula can further decrease their concentrations. is it possible that herbs and tcm formulas can be effective in this low concentration of bioactive compounds? is it possible that little amount of bioactive molecules can cause interactions? in orthodox western medicine, vitamins of little amount can show their clinical effects. the molecular mechanisms are the key, instead of their amount. in the real world practice, herbs and tcm formulas are bioactive. several side effects of tcm formulas have been reported [11] [12] [13] [14] [15] [16] [17] that raise the safety issue of tcm formula. natural products and tcm formulas might not be safe; however, some a-physicians suppose that the side effects may come from the misuse of tcm formulas without fulfilling their indications, while r-physicians consider the side effects developing from the misinterpretation of the disharmony. most tcm physicians do not agree that these side effects come from tcm formulas themselves, although with the same indications or disharmony, it is common to find that some patients respond well while others do not-while some patients develop side effects, some show responses opposite to the in vitro pharmacological effects. for example, panax quinquefolius prolongs thromboplastin time, prothrombin time (pt) and thrombin time in vitro [18] . however, in combination therapy with warfarin, panax quinquefolius actually decreases seral concentration of warfarin and has shortened inr in a clinical trial [19] . with therapy using panax ginseng, one can develop thrombosis [20] , bleed [21, 22] , or remain without any particular response [23] . several factors may affect the molecular mechanisms and subsequent clinical effects of tcm formulas, including individual gene-based response, composition and amount of active molecules in tcm formulas, complex interactions, and appropriateness of use of tcm formulas. individual genetic basis is unique to metabolize tcm formulas and produces different responses. from the results of pharmacokinetic study of gan-lu-siao-du-yin, a tcm formula (submitted data), the blood concentrations of structurally-related index molecules, baicalin and baicalein, wogonin, and wogonoside, are highly variable between participants. the different patterns of blood concentrations support the unique pharmacokinetic profile based on individual genes. different concentrations of active molecules may affect the pharmacologic activities. therefore, individual gene-based metabolism could be one of the major factors affecting the molecular mechanisms and subsequent clinical effects of tcm formulas. to provide insights into action mechanisms of tcm formulas, metabolomic technologies might be helpful. a metabolomics integrative approach accepts a 'top-down' strategy to express the function of organisms through terminal symptoms of metabolic network and will gain a revolution in understanding of the holistic concept of tcm [24, 25] . such technologies have been used to investigate the biological mechanisms of different syndromes of patients by studying the functional activities of the human body from a system-wide perspective. for example, the overall biological characterization of the urine of psoriasis patients with tcm blood stasis syndrome was performed to investigate the therapeutic metabolomic mechanism of the optimized yinxieling formula [26] . in addition, metabolomics have been considered a powerful tool in diagnosis and treatment of primary dysmenorrhea by supporting information on changes of metabolites and changes in endocrinal, neural, and immune pathways [27] . the xiang-fu-si-wu formula has been demonstrated to affect some significant perturbations in sphingolipid and glycerophospholipid metabolism as well as steroid hormone biosynthesis to make the metabolic discrepancy return to the normal level [28] . tcm formulas are complex with numerous active chemical molecules in variable amounts. among these molecules with different pharmacologic activities, it is unclear which one mainly accounts for the clinical effect of a tcm formula, as the most abundant molecule might not be the most important one for a specific activity. the amount of an active molecule can be easily changed in different batches of product, or by different agriculture and collection of medicinal plants, therefore, understanding the molecular mechanisms of a tcm formula requires analysis not only of the mechanisms of the tcm formula as a whole, but those of individual active molecules and ingredient respectively as well. understanding the molecular mechanisms of an active molecule can facilitate its development into an investigational new drug (ind). meanwhile, unraveling the molecular mechanisms of a tcm formula can help to validate its traditional use and avoid its misuse and side effects. to keep a relatively constant amount of active molecules and pharmacologic activities, several things should be paid attention to, including use of right specie, use of right part of a plant, and use of a plant harvested in the right season. both use of closely related but wrong species and use of wrong part of herbs might lead to different active molecules with different pharmacologic activities, and various clinical effects and side effects. plants harvested in different seasons might contain variable amounts of active molecules thereby affecting their activities. some active molecules are secondary metabolites of plants against physical, chemical, or biological stimulants. active molecules of some plants can vary from year to year and place to place. therefore, confirmation of its authenticity is the cornerstone. in addition to this, fingerprints of the active molecules are needed to confirm the authenticity of a plant or a formula and to confirm the amount of active molecules via high-performance liquid chromatography (hplc) or liquid chromatography coupled with mass spectrometry (lcms). this is highly necessary for quality control and efficacy assessment. to have quality control of the products of tcm formulas, good manufacturing practice (gmp) procedure should be followed to avoid (a) inadequate processing that might lead to different chemical compositions of the final product; (b) inadequate storage conditions or prolonged storage that might lead to microbial contamination and early decay of the active molecules; and (c) adulteration of formulas and accidental contamination creating serious uncertainty in quality. adulteration is a plant or formula containing active pharmaceuticals or other bioactive agents for the purpose of claiming better efficacy or broader indications. accidental contamination is the plant raw materials containing heavy metals or other toxic substances from the manufacturing process due to ecological collapse. lead, mercury, and arsenic contamination in traditional chinese herbs has been reported [29] [30] [31] [32] 3.3. complex interplays between herb-drug, herb-food, herb-herb, herb-microbiome, and herb-disease in the clinical practice of orthodox medicine, the more drugs used, the more adverse drug reaction (adr) occurred [33] . this is commonly caused by drug-drug interactions. tcm formulas are mixtures of several ingredients. each ingredient has several bioactive compounds, so a tcm formula has numerous bioactive compounds. thinking of dozens or even hundreds of active molecules in a tcm formula been taken at once implies that the probability of drug interaction could be high. such interactions may be found between herb and drug, herb and food, herb and herb, herb and microbiome, and even between herb and disease. for example, in herb-drug interaction, scutellariae baicalensis is a common ingredient in tcm formulas. s. baicalensis contains baicalin, a flavonoid, as one of its major molecules. interactions between s. baicalensis and drugs are found due to baicalin affecting metabolic enzymes of drugs, displacing plasma protein binding, and regulating various transporters involved in the pharmacokinetics [34] . baicalin may inhibit the expression and hydroxylation activity of cyp3a in the liver to change the pharmacokinetics of drugs [35] . co-administration of extract of s. baicalensis, and mefenamic acid, a kind of nsaid, can potentiate its anti-inflammatory effect [36] . co-administration of baicalin and rosuvastatin, a hmg-coa reductase inhibitor commonly used to reduce serum cholesterol level, might find reduced plasma concentration of rosuvastatin in certain patients with certain genomes [37] . as for herb-food and herb-food-drug interactions, baicalin can potentiate the antioxidant activity of β-carotene, which is a terpenoid of red-orange color, abundant in plants and fruits [38] . the intakes of flavonoid-rich foods and beverages, containing baicalin and rutin, might compete with the binding site of calcium channel blockers on human serum albumin to affect their clinical effects [39, 40] . by contrast, baicalin and rutin will increase the binding affinity of curcumin on human serum albumin to change its bioavailability [41] . herbs may also interact with each other. for example, baicalin and berberine are important coexisting molecules of the combination of s. baicalensis and coptidis chinensis. berberine, but not baicalin, can increase glucose consumption. co-administration of berberine and baicalin had a synergetic effect on glucose utilization [42] . additionally, tcm herbs are commonly used as food supplements and dietary therapy. foods have been reported to modify the intestinal microbiome [43] . commensal microbiota have been thought to be involved in the development of the innate and adaptive immunity, nutrient metabolism of humans, and protection from the overgrowth of intestinal pathogens [44] . herbs can change pharmacokinetics of drugs by intestinal microbiota [34] . the intestinal microbiome is metabolically active to play an important role in the absorption of certain active molecules and change their bio-availabilities, particularly in those containing glycosidic linkages [34, 45] . therefore, change of intestinal microbiome by herbs-containing foods may affect human health care and drug therapy. as for interactions between disease and herb, more absorption of active molecules of maxing shigan decoction (mxgst), including liquiritin, glycyrrhizin, amygdalin, prunasin, ephedrine, pseudoephedrine, and methylephedrine, can be found in rsv pneumonia-infected rats vis-à-vis normal rats by reducing the clearance rates of these active molecules [46] . there are highly complex interactions between herbs and drugs, foods, herbs, microbiome, and diseases, and most of these complex interactions are not completely discovered or remain unseen, just like the submerged part of an iceberg. therefore, there are still insufficient data to completely understand the molecular mechanisms, pharmacokinetics, pharmacodynamics, and interactions of tcm formulas. acute airway infections, including acute bronchitis, viral pneumonia, and acute exacerbation of chronic obstructive pulmonary disease (copd), are commonly caused by viruses of different families, including rhinovirus, influenza, and parainfluenza virus, enteroviruses, coronavirus, adenovirus, respiratory syncytial virus, etc. [47, 48] . these viruses infect epithelia, produce inflammation, induce immune response, and cause symptoms. from the viewpoint of pathophysiology, tcm formulas used to manage airway viral infections need to have antiviral activity against such viruses listed above, and/or to induce antiviral cytokines, and/or anti-inflammatory effect, and/or to relieve symptoms commonly presented in airway infections ( figure 1 ). to simplify the molecular mechanisms and to correlate the pharmacologic activities with their clinical effects, five formulas of a-physicians will be used as examples against airway infections: ge-gen-tang (ggt; table 1 ) [49] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [50] . ggt has antiviral activities against respiratory syncytial virus (rsv) [51] and influenza virus [52] . ggt can reduce the mortality of influenza virus-infected mice [53] . among its active molecules, uralsaponins from glycyrrhiza uralensis [54] and procyanidin from cinnamomum cassia [55] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [56] . paeonol and 1,2,3,4,6-penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [57] . of its anti-inflammatory effects, ggt can suppress the interleukin-1α (il-1α) production induced by interferons (ifn) in influenza [58] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il-6, tnf-α, inos, and cox-2) [59] . ggt can stimulate il-12 and ifn-β to counteract viral infection [53] , and can also enhance the phagocytic activity of macrophages [50] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th1/th2 [60] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, ge-gen-tang (ggt; table 1 ) [49] has been reported to be effective in the treatment of common colds, chronic sinusitis, allergic rhinitis, and pneumonia. the indication to use ggt is patients with symptoms of headache, fever without sweating, and particularly stiffness of neck and shoulders. ggt can successfully reduce various symptoms of dogs infected with common cold viruses [50] . ggt has antiviral activities against respiratory syncytial virus (rsv) [51] and influenza virus [52] . ggt can reduce the mortality of influenza virus-infected mice [53] . among its active molecules, uralsaponins from glycyrrhiza uralensis [54] and procyanidin from cinnamomum cassia [55] may effectively inhibit the replication of the influenza virus. allicin in ginger (zingiber officinale) and coumarin in cinnamomum cassia might have the activity to inhibit influenza neuraminidase [56] . paeonol and 1,2,3,4,6-penta-o-galloyl-β-d-glucopyranose from paeonia lactiflora show antiviral activity against rhinovirus [57] . of its anti-inflammatory effects, ggt can suppress the interleukin-1α (il-1α) production induced by interferons (ifn) in influenza [58] . ggt was found to decrease cigarette smoking-(cs-) and lipopolysaccharide (lps)-induced elevated counts of inflammatory cells, and expression of inflammatory cytokines and proteins (il-6, tnf-α, inos, and cox-2) [59] . ggt can stimulate il-12 and ifn-β to counteract viral infection [53] , and can also enhance the phagocytic activity of macrophages [50] . among its active molecules, paeoniflorin, a major constituent of paeonia lactiflora, exerts anti-inflammatory and immunomodulatory effects by balancing the function of th1/th2 [60] . glycyrrhizin, a major constituent of glycyrrhiza uralensis, suppresses nuclear factor-kappa b (nf-κb) via the phosphoinositide 3-kinase (pi3k) pathway, inhibits the production of nitric oxides (no), prostaglandin e2 (pge2), and reactive oxygen species (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase-2 (cox-2) [61]( figure 2 ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [62] . however, this study design uses intraperitoneal injection to show the induction of no [62] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [63] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge2, il-1β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the anti-inflammatory effect [64] [65] [66] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde down-regulate no and tnf-α production to show anti-inflammatory activity [67] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [68] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [69] . 6-gingerol (6-g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il-12, but decreases il-10 and transforming growth factor-β1 (tgf-β1) levels [70] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [71] (figure 2 ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. (ros), and reduces the protein and mrna levels of inducible no synthase (inos) and cyclooxygenase-2 (cox-2) [61] (figure 2 ). meanwhile, glycyrrhiza polysaccharide, isolated from glycyrrhiza uralensis, significantly induces no production and inos transcription in peritoneal macrophages [62] . however, this study design uses intraperitoneal injection to show the induction of no [62] , instead of the traditional oral route. polysaccharides will be normally digested in the gastrointestinal tract into monosaccharide, so that polysaccharides can hardly reach intraperitoneal macrophages in the regular oral route, so this pharmacologic activity has been questioned. isoliquiritigenin, a flavonoid from glycyrrhiza uralensis, inhibits nf-κb activation to suppress inflammatory response [63] . cinnamaldehyde, from cinnamomum cassia, inhibits the secretion of pge2, il-1β and tumor necrosis factor-α (tnf-α), and the activation of nf-κb to show the antiinflammatory effect [64] [65] [66] . particularly, e-cinnamaldehyde and o-methoxy-cinnamaldehyde downregulate no and tnf-α production to show anti-inflammatory activity [67] . although it can mediate antiviral activity, tnf-α plays only a minor role in clearance of various airway viruses; rather, it is the major contributor of t-cell-mediated lung injury [68] . for enhancement of antiviral immunity, puerarin, an isoflavonoid from pueraria lobate, increased ifn-γ [69] . 6-gingerol (6-g), the main bioactive component of ginger (zingiber officinale), increases ifn-γ and il-12, but decreases il-10 and transforming growth factor-β1 (tgf-β1) levels [70] . on the contrary, gingerol was also reported to suppress t cell response and inhibit ifn-γ synthesis [71] (figure 2 ). these conflicting data may come from different study designs and raise questions about the actual pharmacological activity in humans. ma-huangtang (mht; table 2 ) [49] have been reported to be effective in the treatment of influenza, upper respiratory tract infection, acute and chronic bronchitis, and asthma [72, 73] . the indication to use mht is patients with chills, fever without sweating, headache, shortness of breath, and joint pain. clinically, mht can effectively reduce fever and flu symptoms, including myalgia, headache, arthralgia, fatigue and cough, in patients with seasonal influenza type a [74] . among its active molecules, l-ephedrine from ephedra sinica and amygdalin from prunus armeniacae, possess the antitussive effect [75] , so co-administration of ephedra sinica and prunus armeniacae shows a better antitussive effect than use singly [76] . mht clearly shows anti-inflammatory activity via suppressing the no/pge2 pathway [77] , reducing inflammatory cells infiltration and reducing pro-inflammatory cytokine, including tnf-α, il-1β, and il-6, in lung experiments [78] . in an acute bronchial asthma mice model, mht can also mitigate the pathological changes of acute asthma-like syndrome through inhibition of the toll-like receptor 9 (tlr9) pathway [79] . mht can modulate th1/th2 cytokines via decreasing il-4 & il-17 and increasing ifn-γ levels. mht can inhibit th17 cells [80] , and decreases il-4, il-5, tnf-α, cd3+, cd8+ t cell levels (th2 response), but increases il-2, ifn-γ, and cd4+ t cell levels (th1 response) to increase cd4+/cd8+ ratio [81] . among its active ingredients, ephedra sinica inhibits pge2 biosynthesis, reduces ige-mediated histamine release, reduces the mrna or protein levels of il-1β, il-6, tnf-α, cox2, and nf-κb [82] and inhibits complement activation of both classical and alternative pathways [83] . additionally, ephedra sinica can directly activate both alpha-and beta-adrenergic receptors to reduce bronchial mucosal edema and to dilate the bronchus respectively [75, 84] . to understand the molecular mechanism, several active molecules have been identified. ephedrannin a and b, from ephedra sinica, effectively suppressed the transcription of tnf-α, il-1β, and nf-κb, and the phosphorylation of p38 mitogen-activated protein (map) kinase to exert their anti-inflammatory actions on lps-stimulated macrophages [85] . glycyrrhiza uralensis and cinnamomum cassia contain active molecules mediating anti-inflammatory and immunomodulatory effects mentioned in the ggt section. for its antiviral activity, mht was initially thought to inhibit airway viruses through inducing antiviral ifn. however, herbacetin from ephedrine alkaloid-free extract of ephedra sinica might have anti-influenza activity similar to its extract containing ephedrine and pseudoephedrine [86] . the study of ephedra sinica is relative rare owing to it containing ephedrine and pseudoephedrine, which are illegal in several countries. glycyrrhiza uralensis and cinnamomum cassia also have active antiviral constituents mentioned in the ggt section ( figure 3 ). ma-xing-gan-shitang (mxgst; table 3 ) [49] , a similar formula to ma-huang-tang, is effective against influenza virus infection [87] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge2 level in the hypothalamus [88] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [89] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev1), forced vital capacity (fvc), and fev1/fvc in patients with acute exacerbation of copd [90] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [91] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and anti-pyretic effects in an lps-induced hyperthermia rat model [92] . mxgst has bronchodilation effect mediated by stimulation of β2-adrenoceptors in pigs [93] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [92] , reduce neutrophilic inflammation [93] , and in a copd rat model, can decrease il-4, il-8, and tnf-α, but increase ifn-γ [94] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure 4 ). molecules 2019, 24, x; doi: www.mdpi.com/journal/molecules ma-xing-gan-shitang (mxgst; table 3 ) [49] , a similar formula to ma-huang-tang, is effective against influenza virus infection [87] . mxgst has only one ingredient different from that of mht, i.e., using gypsum instead of cinnamomum cassia, so their pharmacologic effects and active molecules are similar, except that gypsum possesses a more powerful anti-pyretic effect by decreasing the pge2 level in the hypothalamus [88] . co-treatment with ephedra sinica and gypsum can have synergistic effects to manage fever and asthma than single use [89] . mxgst add-on therapy may improve pulmonary function indicies, such as forced expiratory volume in one second (fev1), forced vital capacity (fvc), and fev1/fvc in patients with acute exacerbation of copd [90] . additionally, at higher cumulative doses, mxgst might reduce the incidence of pneumonia and protect against admission [91] . the indication to use mxgst is patients with fever, cough with yellow and sticky sputum, chest pain, and shortness of breath. mxgst has been found to have antitussive and antipyretic effects in an lps-induced hyperthermia rat model [92] . mxgst has bronchodilation effect mediated by stimulation of β2-adrenoceptors in pigs [93] and can block acetyl-cholinergic and histaminergic receptor-induced bronchial contraction in rats [92] , reduce neutrophilic inflammation [93] , and in a copd rat model, can decrease il-4, il-8, and tnf-α, but increase ifn-γ [94] . these effects may be beneficial to manage airway viral infections with cough. the molecular mechanism of mxgst is summarized (figure 4 ). xiao-qing-longtang (xqlt; table 4 ) [49] is one of the most common prescriptions used against allergic rhinitis [95] . xqlt, at higher cumulative doses, might reduce the incidence of pneumonia and protect against admission [91] . xqlt with/without ma-xing-gan-shi-tang (mxgst) is the most frequently prescribed tcm formula for copd [96] , and is also commonly used in the treatment of patients with respiratory diseases, such as common cold, flu, bronchitis, asthma, bronchiectasis, and emphysema. the indication to use xqlt is patients with cough, watery rhinorrhea or watery sputum, but without thirst. to manage airway viral infection, xqlt is effective against human respiratory syncytial virus (rsv) infection by preventing viral attachment, internalization, syncytial formation, and by stimulating ifn-β secretion [97] , and has been proven beneficial against influenza virus in vivo through the augmentation of antiviral iga antibody [98, 99] . xqlt can reduce the airway inflammation with the decrease of eosinophils count, the ovalbumin (ova)-specific immunoglobulin e (ige) antibody, and histamine release [100] [101] [102] , can also modulate th1/th2 balance thereby reducing il-4 and restoring ifn-γ levels [101, 103] . among its active molecules, schisandrin a, a bioactive lignin of schisandra sphenanthera, inhibits the il1β-induced inflammation via suppression of mitogen-activated protein kinase (mapk) and nf-κb signal pathways [104] , and can also inhibit the nf-κb, mapk and pi3k/akt pathways, partially mediated by the activation of the nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (nrf2/ho-1) pathway to manage inflammatory and oxidative disorders caused by over-activation of macrophages [105] . schisandrin b, another bioactive lignin of schisandra sphenanthera, increases the expression of nrf2 and ho-1 and blocks the activation of nf-κb induced by lps to suppress the production of vascular cell adhesion molecule 1 (vcam-1), intercellular adhesion molecule 1 (icam-1), tnf-α, and il-8 expressions in human umbilical vein endothelial cells (huvecs) [106] . α-cubebenoate, isolated from schisandra chinensis, can block the increase of il-1β and il-6 during inflammation [107] , and inhibit lps-induced expression of inos and cox-2 [108] . oral polysaccharide from schisandra chinensis showed antitussive effect in a guinea pig model [109] . the active molecules of common ingredients, including ephedra sinica, cinnamomum cassia, paeonia lactiflora, glycyrrhiza uralensis, and zingiber officinale, have several pharmacologic activities mentioned in the above sections. most of these activities aim at inhibiting inflammation induced by airway infection, however, lectin from pinellia ternate may activate macrophages, induce neutrophil migration, cytokine release, ros overproduction and the activation of the nf-κb signaling pathway to produce pro-inflammatory activity [110] (figure 5 ). therefore, interactions, including both synergistic and antagonistic between active molecules in a tcm formula could be so complex as to affect the final effects. from more than two thousand years ago in china and japan, ye-gan-ma-huangtang (ygmht; table 5 ) [49] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev1, and asthma control test (act) score of refractory asthmatic patients [111] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev1, and fev1% predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [112] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [113] and ev71 [114] . it can regulate the serum levels of tnf-α, il-10, and il-13 to show clinical effect in management of cough and variant asthmatic symptoms in children [115] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [116] . slpcd can significantly down-regulate the mrna expression levels of th2 cytokines (il-4, il-5, il-10, and il-13) and up-regulate those of th1 cytokines (il-2 and ifn-γ) in lungs of asthmatic mice [116] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il-1β, il-6, tnf-α), and attenuating the pulmonary edema [117] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge2 production by decreasing the mrna and protein expression of inos and cox-2, respectively, as well as by suppressing nf-κb activation [118] (figure 6 ). molecules 2019, 24, x; doi: www.mdpi.com/journal/molecules from more than two thousand years ago in china and japan, ye-gan-ma-huangtang (ygmht; table 5 ) [49] has been used to manage flu-like symptoms. a meta-analysis shows that ygmht can improve the total effective rate, fev1, and asthma control test (act) score of refractory asthmatic patients [111] . when combined with salbutamol aerosol, ygmht can obviously improve their pulmonary functions, including act score, pef, fev1, and fev1% predicted value. the indication to use ygmht is patients with chill and fever, hyperinflation of lung, cough with stridor and rales associated with frothy or whitish sputum [112] . ygmht has been reported effective against enterovirus infection, including coxsackievirus [113] and ev71 [114] . it can regulate the serum levels of tnf-α, il-10, and il-13 to show clinical effect in management of cough and variant asthmatic symptoms in children [115] . additionally, a modified ygmht (also named san-long-ping-chuan-decoction; slpcd) can significantly inhibit airway inflammation, reduce inflammatory cells in bronchoalveolar lavage fluid (balf), and decrease the serum total ige levels [116] . slpcd can significantly down-regulate the mrna expression levels of th2 cytokines (il-4, il-5, il-10, and il-13) and up-regulate those of th1 cytokines (il-2 and ifn-) in lungs of asthmatic mice [116] . for this anti-inflammatory activity, aster tataricus can protect from lps-induced acute lung injury mainly through inhibiting the release of inflammatory cells (wbc, macrophage, neutrophil, lymphocyte), regulating the pro-inflammatory cytokines (il-1β, il-6, tnf-α), and attenuating the pulmonary edema [117] . among its active molecules, irigenin, a major active constituent of belamcanda chinensis, can reduce no and pge2 production by decreasing the mrna and protein expression of inos and cox-2, respectively, as well as by suppressing nf-κb activation [118] (figure 6 ). to unravel the molecular mechanism of tcm formulas, several issues need to be solved. many pharmacologic activities are obtained from in vitro and animal studies. could these activities be extrapolated into humans? there are so many active molecules with various pharmacologic activities in a tcm formula or herbs. are these molecules specific for that specific activity of a tcm formula or herbs? could they reach a minimal serum level to exert that particular pharmacologic activity in humans? active molecules may have the same activity with different potency. the most abundant one may not be the most important one for a particular activity. is it possible that there might be unidentified active molecules that actually account for a particular pharmacologic activity? several pharmacologic activities of a tcm formula cannot find a corresponding active molecule. is it possible that interactions between active molecules, e.g., synergism, but not active molecules themselves, account for a specific activity? is it possible that new active molecules are generated during preparation of the tcm formula? the genetic basis will affect the drug pharmacokinetics. is there a specific gene or single nucleotide polymorphism (snp) largely affecting the pharmacokinetic profile? does publication bias exist so that undesired pharmacologic effects are not published? is it possible that a particular gene is prone to a specific adr of a tcm formula or herbs? could a specific adr come from interactions between specific active molecules? several tcm formulas clearly show that some ingredients are not active in managing their traditional applications. could these inactive ingredients be omitted? all these questions need many studies to determine valid answers. at this moment, it would be better to use herbs and tcm formulas according to their traditional indications and the disease pathophysiology by matching their molecular mechanisms. as the world population has continued to age, the elderly have become associated with chronic diseases requiring multiple medications. increasing dissatisfaction with orthodox medicine and/or preference for alternative therapists and/or naturopathy has meant people continue to seek alternatives to maintain or improve their health, and this has spurred the growth of complementary and alternative medicine (cam) therapies. the validity of pharmacologic studies, safety issues, and validation of clinical effects of herbal medicine and tcm are limitations that should be highly considered; however, most clinicians are not familiar with herbal medicine and tcm so they do not recommend herbal therapies. more effort should be placed on unraveling the molecular mechanisms in order to solve the above issues. the reasons that limit clinicians from becoming more familiar with herbal medicine and for not recommending herbal therapies are explored below. herbal medicine and tcm form a part of complementary and alternative medicine (cam). however, evidence-based research in the field of cam therapies is still limited. there is a wrong perception that a naturally derived product is relatively safe. it is highly important to identify both usefulness and safety of cam and integrate these health approaches with orthodox medicine through rigorous scientific investigation to improve health care. it is also important for medical educators and providers to recognize the trend, the evidence, the benefits, and the risks of herbal medicine and tcm to educate clinicians for appropriate patient management and education. tcm studies published in chinese are usually not translated into english. the terminology of tcm is also difficult to translate, particularly those used by r-physicians, so medical education of herbal medicine and tcm has been neglected worldwide, except in germany and china. with their increasing use since the 1960s, understanding the molecular mechanisms, benefits, and limitations by physicians has become increasingly important to monitor their benefits and harmfulness. most of the evidence supporting clinical claims of herbs and natural products come from studies of inadequate design that do not provide tough evidence of the effects. relatively few well-designed studies support their clinical claims. sometimes, adequately powered, double-blinded, placebo-controlled clinical trials come to conclusions against previous reports, such as echinacea for upper respiratory infection [119] , or ginkgo for dementia or mild cognitive impairment [120] . additionally, the amounts of most bioactive compounds in herbs and tcm formulas are very low. is it possible that the clinical effects of herbs and tcm formulas can be effective with such a small amount of bioactive compounds? one study discussed the clinical effect of ma-huang-tang (mht) against seasonal influenza [121] . mht showed an equivalent clinical effect as neuraminidase inhibitors. however, not every tcm formula can provide such evidence. several tcm formulas, such as ma-xing-gan-shi-tang (mxgst), ge-gen-tang (ggt), and xiao-qing-long-tang (xqlt), are among the top ten most common tcm prescriptions for patients with upper respiratory tract infections (urtis) in taiwan [122] . they are commonly used for urtis, but more research is required to validate their clinical effects and mechanisms. without tough clinical evidence and clear molecular mechanisms, physicians tend to avoid herbal therapies. most clinical claims of herbal therapies are based on bench studies that do not possess external validity to support their conclusions; for example, using animal-derived cancer cell lines to study antiviral effects in humans; using cancer cell lines to study physical changes; using intraperitoneal injection to study oral medications; and using high-dose pharmacological designs to study physiological responses, etc. therefore, there is still much space for improvement of our understanding of the mechanisms of herbal medicine. herbs are pharmacologically active and can positively or negatively affect patient's health. with increasing use of herbs as dietary supplements and alternative therapy, there is an increased risk of negative impacts, such as adverse effects and interactions. herbal medicine is among the most common causes of drug-induced liver injury (dili) [123] . additionally, several adverse effects of commonly used herbs have been reviewed, including hypoglycemic or hyperglycemic effect, hypolipidemic or hyperlipidemic effect, hormonal activities, hypertensive, cardioactive [124] , and hepatotoxic effects [125] . some of them are serious even in recommended dosage, such as ephedra alkaloids (derived from ephedra sinica or ma huang) [126] [127] [128] . however, most of the molecular mechanisms causing side effects are unknown. plants containing pyrrolizidine alkaloids can lead to hepatotoxicity and venoocclusive disease, possibly by the accumulation of toxic metabolites produced via the cytochrome system [129] . some herbal treatments containing aristolochic acid (aa), including aristolochia fangchi, aristolochia debilis sieb. et zucc., aristolochia manshuriensis, aristolochia debilis, can cause aa nephropathy requiring renal replacement therapy [130, 131] ; additionally, aa is associated with urothelial cancers [132] . although most of the above issues have been identified, the unrecognized issues are just the tip of the iceberg. tcm formulas have numerous bioactive compounds to form a kind of cocktail therapy. however, the amounts of each bioactive compound in herbs and tcm formulas are very low. this may raise questions about the likelihood of their interactions with others during administration in a decoction. ginseng, a common natural product used among adults [4] , has about 30 ginsenosides as its major bioactive compounds [133] , although the actual amount of each ginsenoside is small. after oral administration, ginsenosides are metabolized and transformed by intestinal microbiota. diet can markedly influence the transformation of ginsenosides. they exert pharmacologic effects in animals, and also show various clinical effects in randomized controlled trials, including several side effects and drug interactions [133] , so small amounts of bioactive compounds do have clinical effects and side effects, which might come from the individual pharmacological activity of bioactive compounds or their synergism. the side effects may also come from individual unfavorable bioactivity or from interactions. additionally, herbal medicine and tcm therapy are commonly used in combination with orthodox medicine by patients, sometimes unknown to their doctors. the complex and unknown interactions, including herb-drug, herb-herb, herb-food, herb-microbiome, and herb-disease interactions, make use of herbal medicine more complicated and requiring frequent monitoring. the mechanisms of these interactions can be divided into molecular mimicry and pharmacologic interactions, such as pharmacodynamic and pharmacokinetic interactions. for example, with molecular mimicry, several herbs naturally has coumarin or salicylate analogue that may potentiate the bleeding risk of warfarin and salicylate, respectively. for pharmacodynamic interactions, ephedra sinica (ma huang) should not be used with sedative or anti-hypertensive agents. for pharmacokinetic interactions, st john's wort and several tcm formulas have been noted to affect the cyp450 system of liver, which may increase or decrease the effects of other drugs [134] . additionally, the herb-drug interaction may be individualized, e.g., in combination with warfarin, panax ginseng may cause thrombotic event [20] , bleeding [21, 22] , or neither [23] . currently, most of the molecular mechanisms of these identified interactions are not fully understood, to say nothing of the unrecognized interactions. quality uncertainty impacts the reproducibility of clinical efficacy and safety of commercially available natural products. variability of the quality of natural products may come from a lack of standardization of manufacturing, including misidentification of authenticity, inadequate processing, inadequate storage, adulteration of formulas, and accidental contamination [135] . most of those quality problems can be gradually solved by following the who guidelines on good agriculture and collection practices for medicinal plants [136] and botanical drug development guidance for industry of fda [137] . several health benefits of herbal medicine and tcm are claimed; for example, herbs and tcm formulas, including those discussed above, are believed to have anti-oxidative activities helpful against several diseases. this idea is based on reactive oxygen and nitrosative species (ros/rns) as metabolic byproducts that can cause damage to cellular macromolecules; thus, many diseases can be triggered by oxidative stress under high levels of ros/rns. these diseases include cancer, inflammation, and degenerative diseases. oxidative stress causes damage either with an overwhelming production of ros/rns or under insufficient levels of antioxidants or repair mechanisms, so blocking the generation of ros/rns might prevent and/or manage these diseases [138] . however, ros/rns are also signaling molecules for several physiological functions, including regulation of vascular tone, control of ventilation, and erythropoietin production, etc. actually, ros-mediated responses may protect against oxidative stress [139] . additionally, ros/rns may play a dual role in different diseases, i.e., ros/rns might contribute to, or counteract, the disease progression. it remains unclear that more dosage of antioxidants is not better and may even worsen a medical condition [138] . there are insufficient data to establish the ability of tcm to decrease ros/rns levels and establish its effects on the disease, and this affects the interpretation of any claims of benefit. to validate such claims of the benefits of herbal medicine and tcm, much work remains to be done. only when these limitations can be minimized, can the molecular mechanisms of herbs and tcm formulas be understood. consequently, clinicians can help patients by giving adequate prescriptions and suggestions to minimize the harmfulness and maximize the benefits to healthcare. herbal medicine, including tcm, is commonly used worldwide. herbal remedies contain numerous active molecules to form a kind of cocktail therapy. these active molecules may interact with each other to affect the therapeutic effects and produce side effects. understanding their pharmacological effects, interactions, side effects, clinical effects, and the underlying molecular mechanisms is very important to provide benefits, but avoid harm, to the patient. to unravel the molecular mechanisms, much work remains to be done. the abc clinical guide to herbs trends in 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virus-induced autophagy identification of suitable natural inhibitor against influenza a (h1n1) neuraminidase protein by molecular docking antiviral activity and possible mechanism of action of constituents identified in paeonia lactiflora root toward human rhinoviruses kakkon-to suppressed interleukin-la production responsive galgeun-tang attenuates cigarette smoke and lipopolysaccharide induced pulmonary inflammation via ikappabalpha/nf-kappab signaling anti-inflammatory and immunomodulatory effects of paeonia lactiflora pall. a traditional chinese herbal medicine the pharmacological activities of licorice nitrite oxide and inducible nitric oxide synthase were regulated by polysaccharides isolated from glycyrrhiza uralensis fisch isoliquiritigenin attenuates adipose tissue inflammation in vitro and adipose tissue fibrosis through inhibition of innate immune responses in mice cinnamaldehyde reduces il-1beta-induced cyclooxygenase-2 activity in rat cerebral microvascular endothelial cells cinnamaldehyde inhibits pro-inflammatory cytokines secretion from monocytes/macrophages through suppression of intracellular signaling cinnamaldehyde and 2-methoxycinnamaldehyde as nf-kappab inhibitors from cinnamomum cassia anti-inflammatory activity of cinnamon (c. zeylanicum and c. cassia) extracts-identification of e-cinnamaldehyde and o-methoxy cinnamaldehyde as the most potent bioactive compounds cellular immunity and lung injury in respiratory virus infection puerarin attenuates airway inflammation by regulation of eotaxin-3 6-gingerol as an arginase inhibitor prevents urethane-induced lung carcinogenesis by reprogramming tumor supporting m2 macrophages to m1 phenotype differential inhibition of t lymphocyte proliferation and cytokine synthesis by [6]-gingerol antipyretic effect of mao-to, a japanese herbal medicine, for treatment of type a influenza infection in children prescription pattern of chinese herbal products for adult-onset asthma in taiwan: a population-based 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pathways of complement ephedra in perspective-a current review ephedrannin a and b from roots of ephedra sinica inhibit lipopolysaccharide-induced inflammatory mediators by suppressing nuclear factor-kappab activation in raw 264.7 macrophages the pharmacological actions of ephedrine alkaloids-free ephreda herb extract and preparation for clinical application anti-influenza agents from plants and traditional chinese medicine an study on gypsum compounds and their antipyretic function and anti-inflammatory mechanisms antipyretic and anti-asthmatic activities of traditional chinese herb-pairs, ephedra and gypsum maxingshigan decoction for treating aecopd in 39 cases and nursing measures. china pharm traditional chinese medicine therapy decreases the pneumonia risk in patients with dementia antitussive, anti-pyretic and toxicological evaluation of ma-xing-gan-shi-tang in rodents the effects of ma-xing-gan-shi-tang on respiratory resistance and airway leukocyte infiltration in asthmatic guinea pigs changes in the level of cytokine in rats with chronic obstructive pulmonary disease of phlegm heat cumber lung type after treatment of maxing shigan decoction. chin the prescriptions frequencies and patterns of chinese herbal medicine for allergic rhinitis in taiwan the use of chinese herbal medicine in the treatment of chronic obstructive pulmonary disease (copd) xiao-qing-long-tang (sho-seiryu-to) inhibited cytopathic effect of human respiratory syncytial virus in cell lines of human respiratory tract in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to" to indicate a break in thought or interpretation and its mode of action in vivo anti-influenza virus activity of kampo (japanese herbal) medicine "sho-seiryu-to"-effects on aged mice, against subtypes of a viruses and b virus, and therapeutic effect proteomic analysis of anti-inflammatory effects of a kampo (japanese herbal) medicine "shoseiryuto (xiao-qing-long-tang)" on airway inflammation in a mouse model xiao-qing-long-tang attenuates allergic airway inflammation and remodeling in repetitive dermatogoides pteronyssinus challenged chronic asthmatic mice model effects of xiaoqiongtang decoction on airway inflammation and airway remodeling in patients with copd anti-allergic activity of a kampo (japanese herbal) medicine "sho-seiryu-to (xiao-qing-long-tang)" on airway inflammation in a mouse model schisandrin a inhibits the il-1beta-induced inflammation and cartilage degradation via suppression of mapk and nf-kappab signal pathways in rat chondrocytes schisandrin a suppresses lipopolysaccharide-induced inflammation and oxidative stress in raw 264.7 macrophages by suppressing the nf-kappab, mapks and pi3k/akt pathways and activating nrf2/ho-1 signaling schisandrin b inhibits lps-induced inflammatory response in human umbilical vein endothelial cells by activating nrf2 anti-septic activity of alpha-cubebenoate isolated from schisandra chinensis identification of a novel anti-inflammatory compound, alpha-cubebenoate from schisandra chinensis antitussive activity of the schisandra chinensis fruit polysaccharide (scfp-1) in guinea pigs models pinellia ternata lectin exerts a pro-inflammatory effect on macrophages by inducing the release of pro-inflammatory cytokines, the activation of the nuclear factor-kappab signaling pathway and the overproduction of reactive oxygen species meta-analysis on shegan mahuang tang for refractory asthma clinical observation on therapeutic effect of traditional chinese medicine granules made by formula of shegan mahuang decoction for patients with asthma yakammaoto inhibited human coxsackievirus b4 (cvb4)-induced airway and renal tubular injuries by preventing viral attachment, internalization, and replication yakammaoto inhibits enterovirus 71 infection by reducing viral attachment, internalization, replication, and translation effect of modified shegan mahuang decoction on cytokines in children patients with cough and variant asthma antiasthmatic effects of sanglong pingchuan decoction through inducing a balanced th1/th2 immune response network pharmacology-based investigation of protective mechanism of aster tataricus on lipopolysaccharide-induced acute lung injury inhibitory effects of irigenin from the rhizomes of belamcanda chinensis on nitric oxide and prostaglandin e(2) production in murine macrophage raw 264.7 cells echinacea for preventing and treating the common cold ginkgo biloba for preventing cognitive decline in older adults: a randomized trial a randomized, controlled trial comparing traditional herbal medicine and neuraminidase inhibitors in the treatment of seasonal influenza traditional chinese medicine treatments for upper respiratory tract infections/common colds in taiwan acg clinical guideline: the diagnosis and management of idiosyncratic drug-induced liver injury herbal medicines: a guide for health-care professionals hepatotoxicity of drug used in the treatment of gastrointestinal disorders adverse cardiovascular and central nervous system events associated with dietary supplements containing ephedra alkaloids is there an association between ephedra and heart failure? a case series efficacy and safety of ephedra and ephedrine for weight loss and athletic performance: a meta-analysis hepatotoxicity of herbal remedies rapidly progressive interstitial renal fibrosis in young women: association with slimming regimen including chinese herbs chinese herbs nephropathy: a clue to balkan endemic nephropathy? kidney int aristolochic acid as a probable human cancer hazard in herbal remedies: a review panax ginseng and panax quinquefolius: from pharmacology to toxicology progress in cytochrome p450 enzyme in toxicity of traditional chinese medicines. chin the state pharmacopoeia commission of the people's republic of china. pharmacopoeia of the people's republic of china who guidelines on good agricultural and collection practices (gacp) for medicinal plants drug development guidance for industry positive or negative actors? biomolecules free radicals in the physiological control of cell function the authors would like to thank teachers of the center for languages and culture of kaohsiung medical university for the technical support. the authors declare no conflict of interest. key: cord-307813-elom30nx authors: yip, tsz-fung; selim, aisha sami mohammed; lian, ida; lee, suki man-yan title: advancements in host-based interventions for influenza treatment date: 2018-07-10 journal: front immunol doi: 10.3389/fimmu.2018.01547 sha: doc_id: 307813 cord_uid: elom30nx influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. influenza is a major acute respiratory infection that causes mortality and morbidity worldwide. two classes of conventional antivirals, m2 ion channel blockers and neuraminidase inhibitors, are mainstays in managing influenza disease to lessen symptoms while minimizing hospitalization and death in patients with severe influenza. however, the development of viral resistance to both drug classes has become a major public health concern. vaccines are prophylaxis mainstays but are limited in efficacy due to the difficulty in matching predicted dominant viral strains to circulating strains. as such, other potential interventions are being explored. since viruses rely on host cellular functions to replicate, recent therapeutic developments focus on targeting host factors involved in virus replication. besides controlling virus replication, potential targets for drug development include controlling virus-induced host immune responses such as the recently suggested involvement of innate lymphoid cells and nadph oxidases in influenza virus pathogenesis and immune cell metabolism. in this review, we will discuss the advancements in novel host-based interventions for treating influenza disease. keywords: host factors, influenza, cytokines, metabolism, immunomodulation introduction influenza remains a source of public health concern. influenza a virus (iav) has been the cause of historical noxious pandemics, such as the spanish flu 1918 h1n1, asian flu h2n2 1957, hong kong h3n2 flu 1968, and more recently the pandemic of h1n1 2009 (swine flu). influenza also causes seasonal epidemics and outbreaks with high morbidity and mortality rates such as the 2015 h1n1 outbreak in india (1, 2) . the error-prone nature of the viral rna polymerase (rdrp) and virus' capacity for genetic re-assortment (antigenic drift and shift) result in the viral components' susceptibility to mutations, allowing the viruses to evade the immune system and increases their resistance to control strategies. currently, influenza vaccination and two classes of antiviral drugs-m2 ion channel blockers (amantadine and rimantadine) and neuraminidase (na) inhibitor (oseltamivir, zanamivir, and peramivir)-and the novel treatment option using polymerase inhibitor (favipiravir) are considered as mainstays in influenza infection treatment and control. the use of influenza vaccinations remains challenging due to antigenic drifts and shifts, with seasonal variation of new circulating species. production of vaccine is time consuming with efficacy concerns, especially in the case of pandemic. variations in vaccine efficacy caused by age should be aware, with studies suggesting that vaccineconferred protection may not be optimal in certain age groups (3) . the disadvantages of using the conventional antiviral drugs have also been a concern. significant levels of resistance to both classes of drugs have been repeatedly reported (4, 5) . high level of resistance (up to 91%) to m2 blockers has been reported in h3n2 virus strain in american isolates (6) . resistance has also been reported in h5n1 virus (7) . iav resistance to na inhibitors has also become an increasingly prevalent concern, with the recent highly fatal outbreak of influenza a(h1n1)pdm09 in india 2015 associated with oseltamivir drug resistance (8, 9) . in addition, a large cluster of influenza a(h1n1)pdm09 viruses in japan was found to have increased oseltamivir and peramivir drug resistance (5) . there is an urgent need to search for alternative targets to treat influenza virus infections, including non-viral targets such as host cellular factors; which are promising as viruses rely on the host machinery for replication. while host immune response is intended to confer a degree of protection against the infection, an impaired or exaggerated host immune response could be detrimental-iav h5n1 and h7n9 virus infection was reported to exaggerate aberrant cytokine release, resulting in a cytokine storm that caused accelerated host death (10) (11) (12) . many recent studies have focused on the investigation of targeting host factors to control virus replication as well as modulate immune response, which we have previously evaluated (13) . in this review, we will discuss the latest studies (in the past 5 years) on the investigation of novel host-based approaches with potential for influenza treatment. the replication cycle of iav can be grossly divided into four different stages: (1) entry, (2) genome nuclear import, (3) replication and protein synthesis, and (4) genome nuclear export, apical transport, assembly, and budding. as an obligate intracellular pathogen, iavs are heavily dependent on host machinery for replication and propagation. to this extent, studies employing genome-wide rna interference (rnai) to screen for host factors involved in iav replication cycle have been performed (14, 15) and an increasing number of approaches targeting these host factors to control iav replication have been investigated. entry of iav into the host cell is divided into several steps (16, 17) . first, hemagglutinin (ha) on the surface of iav binds to the terminal α-sialic acid on the host cell receptor. this induces the internalization of the viral particle by clathrin-dependent, caveolin-, and clathrin-independent endocytosis (18) . macropinocytosis was revealed as an alternative entry pathway for iav (19) , which subsequently enters the canonical endocytic pathway (20, 21) . the vesicle-containing viral particle forms an early endosome (also known as sorting endosome), which matures into a late endosome as the endocytic pathway progresses. a gradual decrease in intraluminal ph from ph 6.5 to 5.0, mediated by v-atpase proton pump (22) , takes place as the endosome matures (23, 24) . this ph drop in the endosomal lumen induces a conformational change in ha, which is activated by proteolytic cleavage to generate ha1 and ha2 from precursor molecule ha0 (25, 26) . this conformational change triggers the fusion of the viral envelope with the endosomal membrane, releasing the viral genome into the cytoplasm. acidification of the endosome causes the subsequent acidi fication of viral lumen via the iav m2 proton channel (27) , which in turn promotes the dissociation of m1 layer from both the viral envelope (24) and the viral ribonucleoprotein (vrnp) complex (28) . interestingly, a sharp decrease in ph from neutral to an acidic ph of 5.0 as utilized by acid bypass has been observed to be sub-optimal for viral replication. it is hence proposed that a gradual decrease in endosomal ph is necessary for sequential reduction in viral stiffness, dissociation of m1 from the np in the vrnp complex, destabilization of m1 layer from the viral envelope, and the eventual conformational change of the ha for the release of viral genome and proteins to the cytoplasm from late endosome (24) . proteolytic cleavage of ha0 to ha1/ha2 is an important step in iav replication. this cleavage relocates ha2, converting previously uncleaved ha0 to a metastable conformation that induces membrane fusion at acidic ph (29) . inefficient cleavage and activation of ha leads to low infectivity (30) . as identified proteins encoded by the viral genome do not possess proteolytic properties, the virus is dependent on host protease for the cleavage of ha. this provides a potential target to control iav infection. ha is commonly cleaved by trypsin-like proteases at the single arginine residue at position 329. human airway epithelium serine proteases hat and tmprss2 were identified as the host factors for cleavage at this residue (31) . aprotinin, purified from bovine lung (32) , is a protease inhibitor with a long history of clinical use as an antifibrinolytic agent in cardiac surgery (33) . its potential as an anti-iav drug has been recognized for over a decade (34) and has been shown to reduce the infectivity of a broad spectrum of iav strains (34, 35) both in vitro (26) and in vivo (36) . once withdrawn from the western drug market due to its association with mortality (33), aprotinin has been approved as a locally administered, small-particle aerosol drug for the treatment of iav infection in russia (36) . however, side-effects associated with the systemic administration of aprotinin raises the need for an alternative protease inhibitor for use in treatment of iav infections. camostat, a serine protease inhibitor, was reported to demonstrate anti-iav potential in mice dating back to 1996 (37) , but little to no research has been conducted to develop it into an anti-iav treatment. it was revisited and proven to be one of the most efficient serine protease inhibitors for the inhibition of iav replication in primary human tracheal epithelial cells in vitro when tested compounds were used at similar molarities (35) . at present, camostat is widely administered for the treatment of liver fibrosis, chronic pancreatitis, and cancer (38, 39) , making it a highly promising candidate for drug repurposing. despite the lack of association between camostat and increased mortality (as with aprotinin), reports of camostat potentially inducing acute eosinophilic pneumonia (38) warrants the need for careful consideration and further research into the repositioning of drugs from the same class. highly pathogenic iav, such as the h5 and h7 subtypes, are reported to have ha cleavage sites rich in basic residues (30) . the polybasic nature of the cleavage sites provides multiple targets for a broad spectrum of proteases, including the more ubiquitously expressed intracellular proteases such as furin (40) . this increased protease spectrum could be utilized by these viruses for the activation of ha prior to viral budding, allowing for evasion of potential inhibition by exogenously administered serine protease inhibitors. furthermore, an in vivo study utilizing mice treated with a single protease inhibitor prior to infection with h7 virus bearing a polybasic cleavage site showed poor efficacy despite good results were obtained for infection with h1n1 virus bearing single cleavage site (41) , suggesting strain specificity in using serine protease inhibitors to treat iav infections. endosomal acidification is required for the release of iav genome (in the form of a vrnp complex) into the cytoplasm (24) . research has shown that an increase in endosomal ph during the early phases of infection could inhibit iav infection in vitro (42) , bringing to light the possibility of controlling iav infection through the prevention of endosomal acidification. the v-atpase inhibitor bafilomycin a1, when used at high concentrations (10-100 nm) has been proven to inhibit iav replication through the efficient suppression of v-atpase (43, 44) . however, prominent cytotoxicity to host cells was also observed at such concentrations (44) . interestingly, lower concentrations (0.1 nm) of bafilomycin a1 lack inhibitory effects on v-atpase attenuated iav replication due to disruption of endosomal trafficking. thus, bafilomycin a1 is suggested to exert its antiviral function via distinct mechanisms at differing concentrations. diphyllin, isolated from the plant cleistanthus collinus, is a natural compound able to induce a v-atpase inhibitory effect (45) . in contrast to bafilomycin a1, diphyllin is well-tolerated in vitro without inducing obvious cytotoxic effects (46) . most notably, diphyllin is found to effectively inhibit replication of viral strains resistant to amantadine and/or oseltamivir (46) . since drug resistance to these widely administered antivirals is of major public health concern (13), diphyllin is regarded as a promising antiviral against drug-resistant iav strains. the release of iav genomic material during replication requires the fusion of the endosomal membrane with the viral envelope. since cholesterol plays a major role in controlling the fluidity of the lipid bilayer in cells, it is hence suspected to have a role in the infection cycle of iav. interferon-induced transmembrane proteins (ifitms) are proteins expressed in many vertebrates (including humans) and are found on the plasma membrane, the membranes of early and late endosomes, as well as on lysosomes (47, 48) . while humans express ifitm1, ifitm2, ifitm3, ifitm5, and ifitm10, only ifitm 1, 2, and 3 are both immune-related as well as interferon (ifn)-inducible (48) , and have been observed to restrict the replication of different viruses, including iav (49) . studies suggest that ifitms limit viral infection by reducing membrane fluidity and hence restrict the hemifusion (the mixing of lipid bilayer without the release of viral content) of viral and endosomal membranes (50) , probably via the disruption of cholesterol homeostasis of late endosomes, where viral fusion and genome release conventionally take place (51) . a recent study using rnai also demonstrated that cholesterol homeostasis can be regulated via acid phosphatase 2 (acp2)-mediated niemann-pick c2 activity and impaired the membrane fusion of iav and influenza b virus (ibv) (52) , further suggesting the importance of controlling cholesterol homeostasis in the release of viral genome to cytoplasm. on the contrary, later studies suggest that ifitm3 exerts its antiviral activity in a cholesterol-independent manner, showing that an increase in cholesterol composition of late endosomal membranes fail to inhibit viral membrane fusion (53) . in addition, studies suggested the accumulation of cholesterol level in the late endosome does not inhibit the iav genome release into cytoplasm (54, 55) . with the modulation of cholesterol levels in host endosomal membrane as a mean to inhibit iav host cell entry is still under debate, further studies are required before clear conclusions can be drawn. by comparing the mirna profiles of the iav-permissive hek 293t cells and (less permissive) hela cells, mirna-33a has been identified as a negative regulator for iav infection via the inhibition of archain 1 (arcn1, also known as δ-copi) (56). arcn1 is a subunit of the copi complex that is required for intracellular trafficking and endosome function (57) , depletion of which has been reported to inhibit iav infection (14) . despite impaired iav internalization caused by arcn1 depletion via sirna (56, 58), it was not able to recapitulate through acute inhibition of copi complex by pharmaceutical means (58) . it is hypothesized that the long-term (lasting days) perturbation on arcn1 by rnai affected the general endosomal trafficking network, a phenomena which cannot be recapitulated by acute pharmaceutical inhibition to block iav infection (58) . the potential of targeting arcn1 for iav treatment deserves further investigation, despite the favorable results from rnai studies. nuclear import of vrnp complexes from the cytoplasm following fusion of the viral and the endosomal membrane is required for replication to take place (59) . an early study suggested that vrnp complexes could be transported to the periphery of the nucleus (60), while recent studies report that vrnp complexes utilize the importin-α-importin-β1 (impα-impβ1) system for nuclear import (59, 61) and lacking of importin-α7, in an importin-α7 knockout mouse model were found to be resistant to iav infection (62) . ivermectin has long been clinically administered for the treatment of parasitosis (63) , but has recently come to attention as a potential inhibitor of impα/β (64) . ivermectin inhibition of impα/β has shown to inhibit the replication of rna viruses such as dengue virus and hiv-1 (64) . ivermectin was recently tested for the inhibition of iav in vitro, with nuclear import of vrnp complex (of both wild-type and antiviral mxa escape mutant) efficiently inhibited (65) . given ivermectin's longstanding record of clinical applications and fda-approved status, repurposing of this drug for the treatment of iav should be considered, especially while under threat of pandemic iav outbreak. following the import of the vrnp complex into the nucleus of the host cell, rdrp uses the vrna as a template to synthesize mrna or crna. synthesized crna remains in the nucleus for new vrna generation, while mrna is exported out of the nucleus for translation. viral protein products are either transported to the cell surface via golgi (in case of ha and na) or imported back into the nucleus to bind with vrna, forming new vrnp complex (59) . numerous host factors are involved in this process and hence could be possible targets for therapeutic intervention. out of the eight genome segments of iav, the m and ns segments are well known for undergoing splicing to generate at least two different mrnas per individual segment (66, 67) . cdc2-like kinase 1 (clk1) is a kinase which regulates alternative splicing of pre-mrna (68) . inhibition of clk1 by the chemical tg003 or knockdown of clk1 is shown to cause a decrease in m2 mrna generation and disrupt downstream m2 protein expression, prominently reduced iav propagation (15) . clypearin and corilagin were both found to be potent anti-iav compounds, with a higher therapeutic index than tg003 in vitro (69) . clypearin is isolated from herbs used by chinese medicine practitioners for treating respiratory tract diseases during replication, viral mrna is exported from the nucleus to cytoplasm, where protein synthesis takes place. human rna polymerase ii activity is found to be correlated with iav replication through the inhibition of nuclear export of certain viral mrnas, such as m1 mrna (70) . cyclosporine a (csa) is a fda-approved drug with immunomodulatory functions (71) that has been found to have an anti-iav effect in both cyclophilin a (cypa)-dependent and -independent manners (72) . the cypa-dependent effect was found to correlate with nuclear export of vrnp complex (see targeting nuclear export complex). the cypa-independent effect caused inhibition of host rna polymerase ii. csa is a prospective drug candidate for treatment of iav infections with a relatively high barrier for development of intrinsic drug resistance, as opposed to commonly used antivirals (73) . nuclear rna export factor 1 (nxf1) is a host factor that has been identified to be involved in the nuclear export of iav mrna. the knockdown of nxf1 in hek 293t cells revealed prominent viral mrna nuclear retention in host cell nucleus (74) . protectin d1 (pd1), an endogenously produced lipid in the respiratory tract, has been identified to have potent anti-inflammatory and antiviral effects (75) . pd1 production was notably found to be reduced in the lungs of iav-infected mice. therapeutic administration of pd1 was shown to significantly reduce iav mrna expression, lower lung viral titer, as well as improve survival of iav-infected mice. mechanistic studies revealed attenuated cytoplasmic translocation of viral mrna with such treatment. a decrease in recruitment of viral transcripts to nxf1 was observed while nuclear export of host rna remained largely unaffected, suggesting a role of pd1 in regulating nxf1 in nuclear export of viral rna. natural pd1 expression in the human airway makes this an ideal candidate for novel therapeutics in the treatment of iav infection. the eukaryotic initiation factor-4a (eif4a) family plays an important role in protein translation (76, 77) . eif4a impairment has been proven to be related to antiviral activity in a broad spectrum of rna viruses in vitro (78) , with inhibition of iav mrna translation (79) . the eif4a inhibitors, silvestrol and pateamine a were demonstrated to arrest viral protein synthesis, thus blocking viral genome replication in vitro (80) . although both silvestrol and pateamine a caused high cytotoxicity at the concentration required effective for iav inhibition, drugs targeting mrna translation for various diseases have been approved by fda or are under active development (81) . as such, inhibition of iav infections by disrupting mrna translation may well be a therapeutic approach in the future. post-translational modifications during protein maturation ensure proper function of proteins, with proteins of iav no exception. nitazoxanide, a fda-licensed drug used to treat enteritis, was found to be effective in controlling iav infection by interfering with ha n-glycosylation as well as intracellular trafficking in host cell and eventually led to a reduction in viral budding (82) . despite the mechanism of nitazoxanide being presently unknown, its ability to inhibit replication of numerous viruses [iav, respiratory syncytial virus, coronavirus, hepatitis b virus, and many others (83) ] suggests that it may act on host machinery. the drug has also been proven in vitro to inhibit the propagation of many circulating strains of human iav, including those resistant to oseltamivir or zanamivir (84) . nitazoxanide has a high barrier of resistance to iav (83) and other viral strains resistance to neuraminidase inhibitors (85), making it a very promising therapeutic target for iav treatment. the drug is currently under phase iii clinical trials (83). in the later stage of viral replication, viral rnas of iav packed with rdrp and np (known as vrnp complexes) are exported from the nucleus (59), assembled (86) , and transported to the plasma membrane [apical in polarized cells (87) ] for budding. novel targets for influenza treatment frontiers in immunology | www.frontiersin.org july 2018 | volume 9 | article 1547 exportin 1 (xpo1, also known as crm1) is well known for its function in the nuclear export of protein (88) and rna, including viral rna (89) . similar to hiv (89, 90) , iav viral rna does not directly bind to xpo1 but is instead held together by several viral proteins. the viral nuclear export protein (nep, or previously known as ns2) and the vrnp complex have been proposed as the nuclear export complex (91) . cellular xpo1 has been proven to be crucial in the nuclear export of the vrnp complex, with early studies using leptomycin b (lmb), a potent xpo1 inhibitor, revealing that in vitro inhibition of xpo1 led to nuclear retention of vrnp complex (92, 93) . however, lmb was deemed unsuitable for development as a potential drug in the phase i clinical trial due to observed cytotoxic effects (94) . verdinexor (also known as kpt-355) is a new bioavailable selective inhibitor of xpo1. it has been shown to be effective against different strains of iavs both in vitro and in vivo as prophylactic and therapeutic treatments (95, 96) . it is worth mentioning that delayed administration of verdinexor at day 4 post-infection was still deemed beneficial, with reduced viral load in vivo (96) . this suggests a prolonged therapeutic time window when compared to the mainstay antiviral drugs such as oseltamivir, where recommended administration is at the early stage of infection (within 48 h of symptom onset) (97) . currently, verdinexor has passed the phase i clinical study trials, suggesting that it does not pose severe cytotoxic effects as lmb does. in addition, a recent report demonstrated that a new drug, dp2392-e10, which binds and inhibits the function of xpo1, can suppress iav replication in vitro (98) further strengthens the concept of iav intervention by targeting xpo1. viral m1 protein is crucial in assisting the nuclear export of vrnp complex. it was commonly suggested that m1 protein links vrnp complex to viral nuclear export protein nep which interacts with xpo1 for nuclear export (59) . thus, viral m1 protein may serve as a target to inhibit nuclear export of vrnp. as previously mentioned (see inhibition of mrna export), csa inhibits iav replication via both cypa-dependent and -independent mechanisms. a recent study using a transgenic mice overexpressing cypa showed greater resistance to iav challenge (99) . in the cypa-dependent mechanism, csa enhances the binding of cypa to m1 protein (72) , increases the self-association of m1, and hinders m1 nuclear import (100) . csa also promotes the cypa-dependent degradation of viral m1 protein (72, 101) . csa seems to be a promising drug to inhibit the nuclear export of vrnp complex by inhibiting viral m1 protein stability and function. recently, cd151, a tetraspanin (defined by four transmembrane domains with conserved residues) that is expressed abundantly in lungs and interacts with integrins has been implicated in the regulation of iav replication in vitro and in vivo (102) . knockdown of cd151 in primary human nasal epithelial cells resulted in the nuclear retention of host xpo1, viral np, nep, and m1 proteins, with an increased survival rate observed in iavinfected cd151 knockout mice. co-immunoprecipitation assays suggest that cd151 interacts with viral np, m1, and nep proteins (102) ; however, the exact domains involved in interaction and the mechanism of cd151 function in nuclear export remain unclear. given that a small molecule inhibitor for cd151 is now under development (103) , more data revealing the role of cd151 in iav infection and subsequent use in targeting cd151 as anti-iav therapy is anticipated. during iav infection, raf/mek/erk signaling cascade is activated, while the inhibition of mek by u0126, probably mediated via myosin (light chain) (104), a known motor protein, impairs the nuclear export of vrnp complexes (105) . suppressing iav replication by inhibition of raf/mek/erk signaling cascade has been illustrated both in vivo (106) and in vitro (105) . the replication of ibv (107) as well as borna disease virus (108) was shown to be inhibited by u0126, suggesting the versatility of this approach in controlling infection by different viruses. despite being effective when administered locally to lungs via aerosol, u0126 has little effect when administered orally (106) . another mek inhibitor, ci-1040 (also known as pd184352) was shown to have high potency against iav in vitro (106). ci-1040 has completed phase ii clinical trials as an anti-tumor drug, with the application of ci-1040 as a potential anti-iav drug candidate recently revisited. unlike u0126, ci-1040 is orally bioavailable and oral administration of ci-1040 at 48 h post-infection protected 60% of the iav-infected mice, while the oseltamivir-treated group experienced a 100% death rate (109) . oseltamivir is known to be effective only when administered in the early stages of iav infection. this suggests the potential use of ci-1040 as an agent used in iav treatment due to its potentially longer therapeutic time window than mainstay antivirals. formyl peptide receptor 2 (fpr2) located at the host cell surface was identified as an erk stimulator (110) . antagonizing fpr2 promoted the survival of iav-infected mice (110) . furthermore, fpr2 antagonists have been described to possess antiviral activity against not only iav but also ibv infection (111) , promoting the idea that antagonizing fpr2 to suppress raf/mek/erk signaling cascade could potentially be a novel approach for the treatment of a broad spectrum of influenza viruses. after the nuclear export of the vrnp complexes, host cell's intracellular transport mechanism is required to deliver vrnp complexes to the host plasma membrane for the assembly of viral rnas and proteins at the final stage of viral replication. among the various vesicular compartments found in a cell, the rab11a + endosomes are known to recycle endocytosed membrane proteins and lipids to the plasma membrane for membrane homeostasis (112) , a property utilized by many rna viruses, including iav (87, (113) (114) (115) . iav progeny virus production was found to be significantly reduced in rab11a + knockdown human cell lines (116) . furthermore, vrnp complex plasma membrane transport perturbation was observed in rab11a knockdown cells (114, 115) ; in cells expressing deletion mutant of rab11 family interacting proteins (87) ; as well as cells treated with chemicals to interfere microtubule (114) . direct interaction of vrnp complex with rab11a has also been verified (114, 115) , demonstrating the dependence of vrna complex transport on rab11a + vesicles and the microtubule network during viral replication. since rab11a proteins do not confer any mobile properties to the vesicle, molecular motors such as kinesins are required for the active transportation of vesicles through cytoskeletons. kif13a, a kinesin-3 family member, was recently identified as a molecular motor for plasma membrane transportation of vrnp-loaded rab11a + vesicles (117) . kif13a knockdown was found to reduce progeny virus production. overexpression of a mutant form of kif13a lacking in motor capacity resulted in disruption of the plasma membrane distribution of vrnp complex during later stages of infection. this data suggest that the apical transport of viral components via rab11a or kif13a could potentially serve as therapeutic targets against iav infection. further examination is merited. tubulin acetylation and deacetylation affects microtubule stability (118) . histone deacetylase 6 (hdac6) was found to deacetylate α-tubulin, one of the subunits of microtubule (119) . a study has demonstrated that hdac6 is involved in iav replication (120) . inhibition of hdac6 by tubacin or knockdown of hdac6 gene resulted in an increase of progeny virus production with vrnp complex redistributed toward the periphery of infected cells. in addition, transportation of ha to the plasma membrane for viral budding was also found to be inhibited by hdac6. this data suggests that activation of hdac6 by its stimulant could be a potential approach to anti-iav therapy, despite hdac6 stimulants still being under development. while several studies have suggested iav transmission between cells through apical membranes (121) and intercellular connections (122) , virus budding from cell membranes remains the major route for transmission of viruses to uninfected cells. na is responsible for the cleavage of sialic acid to prevent the interaction between ha and the host cell during viral budding. besides, viral na, viral ha, m1 as well as m2, are also suggested to play an important role in the initiation of the budding process (123, 124) . in section "controlling cholesterol homeostasis, " we discussed the involvement of host cholesterol in viral membrane fusion and viral genome release to cytoplasm. recent studies have demonstrated that host cholesterol may also play an important role in viral budding. it was demonstrated that overexpression of annexin a6 (anxa6), a phospholipid binding protein, could lead to a decrease in cholesterol levels within the golgi apparatus and plasma membrane (55), ultimately causing a reduction in egression of progeny virion from infected cells (54) . this reduction could be reversed by the addition of exogenous cholesterol (55) . similar to anxa6 overexpression, addition of a hydrophobic polyamine, u18666a, could reduce cholesterol level in plasma membr ane, also inhibited viral replication (55) . since iav is assumed to bud from lipid rafts (cholesterol-rich plasma membrane domains) (123) , it was demonstrated that anxa6 overexpression or u18666a treatment could hinder progeny virus production by lowering the cholesterol content in the plasma membrane. this hypothesis was strengthened through recent studies resolving the cholesterol-binding site of viral m2 protein, suggesting that iav m2 clustering (which provides membrane curvature for scission) is mediated by cholesterol (125) . a recent report utilizing two different fda-approved cholesterol-lowering drugs, gemfibrozil and lovastatin, stated that there was reduction in stability and infectivity of progeny virus compared to that replicating within cholesterol-sufficient host cells (126) . taken together, this data suggests that controlling cellular cholesterol content would be an effective alternative with drugs available for repurposing iav treatment. further in vivo works are needed to confirm this hypothesis. the gi-type g-protein coupled receptor α2-adrenergic receptors (α2-ars) have been recently identified as a key host factor involved in iav replication (127) . apical transport of the viral protein ha is inhibited by low intracellular camp level after stimulating the α2-ar-mediated signaling. in vitro stimulation of α2-ar by its agonist clonidine inhibits iav replication. therapeutic administration of clonidine reduced pulmonary edema and improved survival rate of iav-infected mice. development of a new antiviral targeting the α2-ar-mediated signaling seems promising and deserves further investigation. although targeting host factors for viral interventions generally provides a better resistance barrier, emergence of resistance may still arise (61) . therefore, combined use of interventions targeting both virus and host factors have been recommended to reduce opportunities for viral development of resistance. one such example would be the combined administration of na inhibitor (oseltamivir) alongside an anti-host factor [such as v-atpase inhibitor diphyllin (46) , ha maturation inhibitor nitazoxanide (85) , fpr2 antagonists (111) , and xpo1 inhibitor verdinexor (96) ]. while further direct assessment for the ease of emergence of escape mutants between single and combinatory use of drugs is required, the synergistic effects of a combined, multi-drug approach observed thus far highly suggest an increased effectiveness over a single-drug approach. table 1 summarizes novel host targets regulating iav replication. compared to rnai, small molecular chemicals remain the best choice as drug candidates due to their fast acting and easy-todeliver properties. although small molecular chemicals targeting certain host factors aforementioned have yet to be developed, their rnai-identified involvement in the iav replication cycle provide leads for the development of new iav interventions. the immune system aims to protect the host from infection and clear the pathogen once an infection occurs. in addition, the complex networks formed between the host physiology and the immune system co-operatively shape the disease outcome; modulations on the networks could alleviate disease severity in iav infections. the immunological responses elicited by iav infection has been reviewed in detail (128) (129) (130) . at the initial stage of iav infection, the respiratory epithelial cells are the primary target for infection. once the infection is initiated, the recognition of infection is accomplished via the detection of pathogen-associated molecular patterns (pamps) by pattern recognition receptors (prrs) (see toll-like receptors), and lead to the expression and secretion of different cytokines and chemokines, such as il-6, il-8, tumor necrosis factor (tnf)-α, and ccl2 as well as type i and iii ifns. as sentinel cells, alveolar macrophages could also be infected, inducing cytokines and is the main source of type i ifns (128, 129) . type i ifns are known inducer for the upregulation of death receptor 5, which is the receptor for tnfrelated apoptosis-inducing ligand (trail), in lung pneumocytes (128) . il-8 and ccl2 produced by both epithelial cells and macrophages act as chemoattractants for neutrophils and monocytes, respectively. neutrophils are one of the earliest immune cells being recruited to the site of infection (131) with transmigration of neutrophils carry out by adhesion molecules, such as cd11a, cd11b, and cd18 (132) . in addition to the antiviral activity of neutrophil-released reactive oxygen species (ros), defensin and pentraxin (132) , uptaking iav by neutrophils could also help in controlling viral propagation as these cells do not support replication of iav (133) . besides controlling viral replication, neutrophils also play an important role in guiding the migration of iav-specific cd8 + t-cells in the infection site by secreting and leaving a trail of cxcl12 (134) . infiltrated monocytes will, however, differentiate into macrophages or dendritic cells (dcs). the monocytes-derived macrophages are reported to be a permissive host for iav production (135) , sustaining inflammation by producing cytokines in a magnitude larger than that of the resident alveolar macrophages. the monocyte-derived dc as well as the resident airway cd11c low b220 + plasmacytoid dc (pdc) and two types of conventional dcs (cd103 + cd11b low and cd103 − cd11b hi ) acquire the antigen of the invading pathogen through either direct infection or up-taking infected dead cells (129) . in the presence of type i ifns, dcs mature when encountering pamps from invading pathogen (129) . depending on the sub-cellular localization of the antigen, cytosolic and endosomal antigen will be loaded onto major histocompatibility complex (mhc) class i and ii molecules respectively (130) . once mature, dcs migrate from the infection site to the draining lymph nodes via the interaction of ccr7 and ccl19/ccl21 (130, 136) for antigen presentation via mhc class i and ii to naïve cd8 + and cd4 + t-cells, respectively (137) (138) (139) (140) . interestingly, monocytesderived dcs that engulfed the infected dead cells are poor antigen presenters for cd8 + t-cells and require the transfer of intact mhc class i/peptide complex to lymph node-resident cd8α + dcs which are the most efficient antigen-presenting cells to cd8 + t-cells (137) . in addition to antigen presentation, pdc are well known for their high ability in type i ifns production to limit viral propagation (141) . within the lymph node, naïve cd8 + t-cells are activated by the dcs, differentiate and clonal expand into cytotoxic t-lymphocytes (ctls) with the aid of various cytokines, including ifn-γ, il-12, type i ifns, and il2 (142, 143) , and the help from activated cd4 + t helper cells (144). differentiated ctls downregulate their lymph node homing receptor ccr7 and upregulate ccr4 and cxcr3 for the migration to the site of infection. within the site of infection, ctls control viral replication by targeting and inducing apoptosis of virus-infected cells via the secretion of perforin and granzymes as well as the ligation of death receptors on the infected cells by tnf, fas ligand, and trail. on the other hand, cd4 + t-cells are activated by the presentation of mhc class ii/ antigen complex by dcs, with co-stimulatory receptors such as cd28 expressed on the t-cells and the ligand for cd28 (cd80 and cd86) expressed on dcs playing an important role (144). activation of cd4 + t-cells lead to differentiation into different effector cells subsets, including the classical th1 and th2, and the more recently identified regulatory t cells, follicular t helper cells, th9, and th17 subsets (144). th1 cells regulate to the differentiation of ctls as mentioned whereas th2 cells contributes to the activation of b-cells through cd40l. within the pregerminal center of the lymph node, the follicular t helper cells interact with antigen-primed b-cells and promote their proliferation. antigen-primed b-cells differentiates into plasmablast and undergo antibody class-switching in the germinal center (145) . detailed functions of regulatory t cells, follicular t cells, th9, and th17 cells are discussed elsewhere (144, 145). plasmablasts enter the blood-stream, are recruited to the inflamed tissue, and terminally differentiate into plasma b cells which specialize in the production of antibody for pathogen neutralization, opsonization, and antibody-dependent cell-mediated cytotoxicity, etc. memory t-and b-cells are also developed during the maturation process, and has been discussed and reviewed elsewhere (146) (147) (148) (149) . a schematic diagram showing a summary of the immune response after iav infection has been illustrated in figure 1 . the yin and yang theory is always used to describe the importance in balancing the host immune response. in the light of this theory, the treatment strategy aims to suppress the overwhelming activation of the host immune response and in reverse to compensate any unfavorable suppression. although adaptive immune responses are important in viral clearance, the immediate innate immunity play an important role in the early control of an infection, and conversely, is a major factor for disease severity due to immunopathology. dysregulated immune responses caused by viral infections have been implicated in severe disease development (150, 151) , such as acute lung injury (ali). ali in its most severe form, known as acute respiratory distress syndrome (ards), is reported to be the most prevalent cause of mortality in iav-infected patients (152) . studies suggested that iav strains could be associated with either over-activating (human infection by avian h5n1 and h7n9) (153, 154) or suppressing (h1n1, h3n2) (155) immune response. recent history has seen the outbreak of iav pandemics of varying severity takes place at the cost of millions of lives. one such example would be the deadly spanish flu of 1918, which claimed the lives of 20-50 million of the 500 million people infected worldwide. the pathological examination of lung sections from mice infected with reconstituted 1918 iav virus revealed necrotizing bronchiolitis and severe alveolitis in tissue, with neutrophils observed as the predominant inflammatory cell type present (156) , suggesting neutrophil involvement in the pathogenesis of iav infection. the majority of immune cells in blood circulation are neutrophils; of which they are among the first innate immune cells recruited to the site of infection (131) . neutrophils characteristically control microbial infections by generating bactericidal (157) neutrophil extracellular traps (nets), consisting of granule proteins, histones, and decondensed chromatin (131) . both protective and destructive role of neutrophils in iav infections have been described. the contrasting role of neutrophils could be explained by factors such as viral strain and viral dose used in different experimental setup, etc. the protective role of neutrophils was observed when mice infected with a low, non-lethal dose of iav h3n2 strain hkx31 displayed neutrophil-mediated viral clearance via phagocytosis (132, 158) . depletion of neutrophils has found to enhance viral load in the iav-infected animals (158) . on the contrary, this protective nature is disputed due to the association of neutrophil-generated nets. extensive net formation was observed in mice infected with pr8, an iav strain highly pathogenic to mice (159) . histones and myeloperoxidase within the net induce cell death of lung epithelium and endothelium (157) , leading to the loss of integrity of the alveolarcapillary barrier, a characteristic of ali. yet, while histones have been shown to suppress iav replication in vitro (160) , in vivo study demonstrated that there was increase in lung inflammation and damage in iav-infected mice treated with histones (161) . interestingly, co-treatment of lethally infected mice with anti-histone antibody and oseltamivir resulted in an increase in animal survival when compared to infected mice groups treated solely with oseltamivir (161) . in agreement with the in vitro and in vivo data, it has been reported that net produced by cultured neutrophils from patient with h7n9 and severe h1n1 infection increased alveolar epithelial cell permeability (162) leading to ali. more importantly, plasma net level positively correlated with the disease severity index (including higher acute physiology and chronic health evaluation ii score) and multiple organ dysfunction syndrome (162) , further demonstrating the detrimental role of net in the pathogenesis of severe iav infections. studies have demonstrated the involvement of superoxide dismutase and myeloperoxidase in netosis, the formation of net (159) . the presence of anti-myeloperoxidase antibody as well as the superoxide dismutase inhibitor (detc) significantly reduced netosis. finally, tetrahydroisoquinolines (163) and a panpeptidylarginine deiminase (pad) inhibitor, named cl-amidine (164) have been suggested to inhibit netosis. despite it has been reported that during iav h1n1 infection, pad4 knockout mice displayed only slight improvement in weight loss and a slight prolonged but no end-point survival advantage was observed compared to wt mice (165) , based on the extensive findings presented above, targeting net to prevent ali in the severe case of iav infection, including the highly pathogenic avian iav, remain promising and may warrant further investigation. innate lymphoid cells are cells of lymphoid lineages that do not express antigen-specific b-or t-cell receptors (166) . similar to t-helper cells, they are classified into subsets by their ability to produce type 1 (th1), type 2 (th2), and type 3 (th17 and th22) cytokines. previous studies confirmed the involvement of ilcs of group 2 linage (ilc2) in iav infection and airway inflammation (166, 167) . on the positive side, during the recovery phase of iav infection, ilc2 expresses amphiregulin which promote airway epithelium repair (166, 168) , thus facilitating the recovery of the infected lung. on the other hand, in response to il-33 produced by macrophages, dcs, and nkt cells, ilc2 secretes il-5 and il-13 and induce airway hyper-responsiveness. recruitment of eosinophils by il-5 to the lung also mediates airway inflammation (166) . since eosinophilia is a characteristic of allergic asthma and influenza is a major cause for morbidity and mortality in asthma patients (166) , it will be of particular interest to investigate the role of ilc2 in iav infection, particularly in asthma patients. ilc1s have been initially described as immature nk cells residing in the liver and share many phenotypic similarities with nk cells (169) . it was recently appreciated that tissue-resident ilc1s other than the previously recognized nk cells are the major early source of the antiviral ifn-γ at the primary site of various viral infection, including iav (170) . interestingly, ifn-γ was found to suppress ilc2 activity and reduce il5 production which exacerbates disease severity during influenza a(h1n1) pdm09 infection (171) . this data may highlight a link between ilc1 and ilc2 and suggesting ilc1 can suppress ilc2 activity via ifn-γ production during iav infection. with ilcs finally identified, functions of these cells and their role in immune response to tumors and pathogen infections have been massively investigated in recent years. type i ifns, prostaglandin i2, corticosteroids, and testosterone have been reported to suppress ilc2 activity (172, 173) . in addition to il-33, the epithelial cytokines il-25, thymic stromal lymphopoietin, as well as the lipid mediator prostaglandin d2 were found to activate ilc2 (173) . the therapeutic potential of these ilc2 activators and suppressors is yet to be deduced. with more and more studies demonstrating the involvement of ilc in iav infection, the interplay between different ilc subtypes in iav infection would, therefore, be an interesting area to explore and modulate the ilc activity may be a future approach to combat iav infection. reactive oxygen species, generated by specialized enzymes such as nadph oxidases, are released during iav infection (174) . the nadph oxidase family consists of enzymes containing different catalytic subunit named nox1-5 and dual oxidase (duox) 1 and 2. ros have been reported to display both beneficial (limiting viral replication) and detrimental (promoting ali) effects in the course of iav infection. interestingly, the protective or destructive effect of ros is dependent on the enzyme of which the ros is generated (174) . dual oxidase1 and 2 are found to be host-protective (174, 175) . in vitro, ros generated by nuclear duox indirectly regulates the splicing of iav mrnas via the nuclear speckle-associated splicing complex (175) . in addition to altering viral mrna splicing, ros generated by doux2 has been attributed to the production of ifn-λ, an important anti-iav ifn. in response to iav infection, increased viral mrna replication was observed when duox2 was silenced in vitro (176) . increased viral replication was also observed in mice with doux silenced (175) , further depicting the protective role of doux in iav infection. unlike doux, nox2 activation could be harmful to host. iav infection was reported to induce nox2-dependent endosomal ros production (177) . ros could target the conserved cys98 on toll-like receptor (tlr) 7, and inhibit tlr7-mediated type i ifn expression during a mild iav h3n2 infection in vivo (177) . iav-infected mice treated with specific nox2 inhibitor, cholestanol-conjugated gp91ds-tat, were found to have reduction in endosomal ros production, restored tlr7 activity, and displayed a decreased viral load (177) . in addition to nox2, nox4dependent ros production has also been reported to activate mapk/erk signaling (178) , enhancing the export of vrnp complex, thus increasing viral replication (see targeting the raf/ mek/erk pathway). nox4 knockdown resulted in a reduction of viral replication in vitro (178) . targeting the different nadph oxidase isoforms, instead of scavenging ros should be considered as the therapeutic approach for iav infection, as doux-mediated ros production is beneficial (175, 176) , while nox2 and nox4 are harmful during iav infections (177, 178) . finally, ns1 (not to be confused with iav ns1 protein) has been demonstrated to be a nox inhibitor, which could inhibit the activity of nox1, nox2, and nox4. a study demonstrated that ns1 suppresses iav-induced nox2 and significantly inhibits iav virus replication (179) . besides cholestanolconjugated gp91ds-tat and ns1 aforementioned, apocynin, a phagocytic nox2 inhibitor as well as ros scavenger (180) (181) (182) , has been demonstrated to ameliorate hyper upregulation of cytokines induced by iav infection through socs1 and socs3 in vitro (154) and reduce peri-bronchial inflammation and viral titer in vivo (183) . interestingly, ebselen, another nox2 inhibitor and glutathione peroxidase mimetic, could reduce inflammatory status measured in bronchoalveolar lavage fluid (balf) of mice pre-exposed to cigarette smoke and subsequently infected with iav (184) . taken together, these reports highlight the potential use of nadph oxidases inhibitors and ros scavengers to treat iav infections. dysregulated cytokine production has been associated with the elevated mortality rate observed in severe iav infections (185, 186) . as such, the immunomodulation of cytokines are regarded as promising therapeutic tactics. recent advancements developed with this approach will be highlighted in the following section. tumor necrosis factor has two main functions during viral infection-it activates nf-κb, inducing the expression of cytokines responsible for the host immune response; and induces apoptosis through activation of a signaling cascade involving tradd, fadd, and caspase 3, 7, 8, and 10 (187) (188) (189) . tnf is known to be highly upregulated in iav-infected hosts, especially in hosts infected with highly pathogenic iav (153, 190) . however, it is both protective and counter-protective functions associated with tnf that makes it a target in the treatment of iav. the protective role of tnf is observed during infection by low pathogenic iav, where extrinsically derived tnf is responsible for attenuating tissue-damaging cd8 + t-cell response (191) . in addition to recruiting monocytic cells to the infection site, cd8 + t-cells response was observed to deteriorate lung pathology (192) and damage healthy, non-infected lung epithelial cells (193) upon iav infection. furthermore, tnf deficiency has been associated with an increased detection of il-15 and il-6 in balf (192) , which promote the survival of and proliferation of cd8 + t-cells (194, 195) and subsequent tissue damage. exacerbated lung pathology caused by the upregulation of the monocyte chemoattractant protein-1 was observed in tnf −/− mice infected with sub-lethal dose of iav (196) . in addition, decreased cd8 + t-cell contraction due to enhanced expression of the anti-apoptotic protein bcl-2 was observed in sub-lethally iav-infected tnfdeficient mice when compared to wt mice (192) . as a whole, there is substantial evidence supporting the protective role of tnf in iav infection. on the other hand, the correlation of tnf with pulmonary edema has been well-documented (197) . tnf has been observed to stimulate the expression of cxcl2 in alveolar epithelial cells in a transgenic mice model resembling extensive iav infection in lung tissue, causing alveolar damage, lung edema, and hemorrhage (198) . in addition to lung edema, tnf has also been reported to correlate with iav-associated encephalopathy (199, 200) . however, it is notable that despite iav-associated encephalopathy, direct invasion of the central nervous system is rare (201) , suggesting that iav-associated encephalopathy could instead be a result of peripheral infection. furthermore, tnf has been shown to increase the permeability of the blood-brain barrier (bbb) (202, 203) , contributing to neural damage (204) . these studies further support an anti-tnf approach as a potential therapy for severe iav infection. at present, etanercept, an anti-tnf drug administered in the treatment of rheumatoid arthritis, is the only tnf inhibitor (or even tnf directed treatment) tested for iav treatment. etanercept has been shown to protect against the in vivo lethal infection of mice with a highly virulent, mouse-adapted iav strain (205) , with observations made of an increased survival rate with decreased morbidity, expression of the proinflammatory cytokine il-6, lung injury, and edema (205) . the protective role of il-6 was demonstrated in mice challenged with sub-lethal iav infection. il-6-deficient mice displayed exacerbated pulmonary damage (206, 207) and lung injury due to an observed decline in the survival of alveolar type ii cells and alveolar epithelial cells (207) . iav suppresses the anti-apoptotic mcl-1 and bcl-xl expression, causing cell death of neutrophils which are critical in viral clearance (206) . addition of il-6 restored the expression of mcl-1 and bcl-xl in vitro and is considered as the underlying mechanism for the observed survival advantage of wt mice over il-6 knockout mice during mild iav infection. il-6 has also been shown to induce the proliferation of lung il-10 + regulatory t cells and il-27, which act to limit excessive proliferation of cd8 + t-cells and subsequent cd8 + -inflicted damage. this would hence prevent the tissue damage observed in lung immunopathology (208) . despite the apparent protective role of il-6, high levels of il-6 in serum or cerebrospinal fluid have been reported in severe neurologically complicated iav cases, with il-6 used as a marker for prognosis (199-201, 209, 210) . the role of il-6 in regulation of bbb permeability was reported (211) , with potentially detrimental neurological complications. as such, the suppression of hyper-induced il-6 as a form of therapy in severe iav infection should be considered. one such option is the anti-il6 antibodybased drug tocilizumab, which is currently administered clinically for the treatment of rheumatoid arthritis. however, study on the usage of this drug to treat hyper upregulation of il-6 due to severe iav infection has yet to be conducted. on the other hand, in a case of h1n1 virus-induced ards, the use of an extracorporeal cytokine hemoadsorption device to remove cytokines including tnf and il-6 from the bloodstream (212) has showed beneficial to the patient (213) . more research is required to confirm whether the removal or neutralization of il-6 could be a potential therapy for severe iav infections. the activation of cd8 + t-cell is crucial for viral clearance. it should, however, be tightly regulated to limit cd8 + t-cell inflicted host cell damage. il-6 mediates il-27 induction (208) . il-27 acts to suppress cd8 + t-cells and reduce morbidity through il-10 and regulatory t-cells (208) . much like other immunomodulatory approaches, the timing for applying il-27 should be carefully assessed. compared to placebo-treated iavinfected group, early administration of il-27 to iav-infected mice in fact led to poorer viral clearance, increased morbidity, and deteriorated lung histopathology, while il-27 administration during the recovery phase (5-10 days post-infection) accelerated recovery and improve lung immunopathology (214) . notably, il-27 could also suppress th17 responses and increases susceptibility to secondary s. aureus infection (215) . therefore, co-administration of antibiotics should be considered when utilizing il-27 as potential iav treatment. both type i and iii ifns have antiviral properties, with viruses counteract ifns to gain an advantage for their propagation. the iav viral protein ns1 inhibits the production of ifns by antagonizing irf-3, a key transcriptional factor for ifns. this prevents the processing of cellular pre-mrnas (including those for ifns) and directly interacts with retinoic acid-inducible gene (rig)-i receptors, which are critical in innate sensing, to suppress ifn production during infection (216, 217) . in addition to inhibiting ifn expression, the induction of socs3 inhibits ifns signaling by suppressing cytokine signaling has been documented (155) . the recognition of 5′ triphosphate on viral rna by rig-i receptor is shown to induce the expression of socs3, which in turn represses type i ifns expression (155) . due to ifns being a key contributor to antiviral immune response, an impairment of type i or iii ifn production may cause the escalation of otherwise mildly pathogenic iav infection into a life-threatening one (218) . while type i ifn has been demonstrated to inhibit iav replication in vitro (219) ; the in vivo administration of type i ifn in animal models only displayed effectiveness in a prophylactic capacity. a lowered viral titer was detected in the nasal wash of test animals. however, host susceptibility to iav infection remained unchanged (219) . notably, this protective effect is only conferred by an optimal dose of type i ifn of low to moderate amounts (10-100 units per mice daily); with higher dosages (1,000-10,000 units per mice daily) shown to increase morbidity (220) . in addition, clinical trials demonstrated that prophylac tic administration of type i ifn reduced disease severity and lowered susceptibility to iav in males and participants aged 50 or above (221) . despite relatively successful results seen in the prophylactic use of ifns, its therapeutic use is of greater clinical relevance. mice treated with type i ifn post-iav infection showed a successful reduction in lung iav titer but displayed increased morbidity and mortality in comparison to vehicle-treated mice (222) . a possible explanation for this phenomenon is the induction of excessive inflammatory response and trail-dr5-mediated epithelial cell death by type i ifn (223) , which accounts for the observed lung pathology in iav-infected animals treated with type i ifn (224) . in addition, downregulation of γδ t-cells by type i ifn has been correlated with increased susceptibility to secondary s. pneumoniae infection (225) , further arguing against the potential use of type i ifns for the treatment of iav infection. in comparison to type i ifns, the administration of type iii ifns may provide advantages in the control of iav replication (176, 222, 224) without the risk of previously reported type i ifns-mediated immunopathologic side-effects (222, 224, 226) . however, a recent study aiming to stimulate ifns signaling through the systematic administration of rig-i ligand post-iav infection demonstrated that type i, but not type iii ifns signaling is important in conferring protection during fatal iav infection in vivo (227) . though, this study did not measure the production of type i and iii ifns as well as any changes in viral load with respect to ifnar or ifnlr knockout. in addition, while human immune cells are not primary targets in iav infection, they could be susceptible to iav and become efficient host cells for virus replication. they are reported to possess a subpar response to type iii ifns (222) ; leading to the preliminary conclusion that solely using type iii ifn as treatment may not be feasible. as such, reports suggesting the use of type iii ifns over type i ifns as a front-line therapeutic agent to counter iav infections may require further investigation. the inhibition of cox-2 by selective inhibitors, nimesulide and celecoxib, was previously demonstrated to suppress the hyper upregulation of pro-inflammatory cytokines induced by highly pathogenic avian iav (228) (229) (230) . in addition, the use of zanamivir in tandem with a specific cox-2 inhibitor was shown to increase the survival rate of mice lethally infected with avian h7n9 iav, when compared to mice treated solely with zanamivir (229) . activated cox-2 regulates downstream prostaglandin production. one such example is pge2, a major type of prostaglandin recently demonstrated to play an important role during iav infection. pge2 was significantly upregulated in response to iav infection, leading to the inhibition of antiviral type i ifn production in macrophages and the subsequent increase in virus replication (231) . the use of chemicals ah6809 and gw627368x to antagonize pge2 downstream signaling molecules ep2 and ep4 respectively, was shown to induce antiviral type i ifn production. the in vivo treatment of mice lethally challenged iav with both ep2 and ep4 antagonists significantly improved the survival rate. a recent study demonstrated the ability of a modified tcm decoction to reduce peg2 production and subsequent morbidity in mice lethally challenged with iav. improved lung pathology was observed (232) . the long history of clinical tcm use supports the clinical feasibility of peg2 inhibition as an option to treat severe iav infections. pattern recognition receptors on host cells sense specific pamps present on the viral surface or generated during replication. prrs can be broadly divided into two classes by their function or location. when defined by location, prrs are classified into 3 groups-membrane-bound (tlrs and c-type lectin receptors), cytosolic (rig-i-like and nod-like receptors), and secreted (collectins and pentraxins) (233) . significant research has been conducted on prrs with regards to iav infection. tlrs and rig-i receptors have been extensively studied for their major roles in eliciting host immune responses (cytokine and ifn expression) during iav infection (234) (235) (236) . rig-i receptors have been investigated for their functional relevance to iav infection and targeting these receptors as a form of iav treatment has been extensively reviewed (237) (238) (239) . this section will cover recent research on tlrs and the targeting of different tlrs to treat iav infection. humans have been identified to express tlr1-10, while mice have been identified to express functional tlr1-9 as well as tlr11-13 (240) . most tlrs-with the exception of tlr3utilize myd88 as an adaptor protein during signal transduction. tlr3 utilizes trif as an adaptor. tlr4 is known for its ability to utilize either myd88 or trif, with the choice of adaptor dependent on its sub-cellular location (241) . different tlrs, such as tlr3, 7, and 8 (240) as well as tlr2, tlr4, and most recently tlr10 (235) , have been revealed to play a role in the orchestration of host immune responses contributing to iav pathogenesis. with tlr10 being an exception (242) (243) (244) , tlr activation largely causes the release of pro-inflammatory cytokines, with hypercytokinemia leading to ali as a major cause of mortality in severe iav infections. in addition to dysregulated cytokine release, excessive production of ros has been associated with ali development. in fact, lung injury during severe pulmonary infections, such as iav and sars, could be caused by oxidative stress (245) . iav infection activates nadph oxidase that subsequently produces oxidized papc, an endogenous phospholipid. the oxidized papc serves as an agonist for tlr4, activating a tlr4-trif-traf6-nf-κb signaling cascade to eventually trigger the release of il-6, ultimately inducing the onset of ali. in addition to oxidized papc, the induction of endogenous protein s100a9 upon intracellular prr ddx21 recognition of iav subsequently induces the activation of tlr4, further contributing to iav-induced mortality (246) . since tlr4 has been proven to be important in ali induction (and hence iav-related mortality), manipulating the stimulation and antagonism of tlr4 could potentially reduce the severity of iav infections. eritoran (e5564) is a specific tlr4 antagonist initially purposed for the treatment of sepsis, but a failed a phase iii clinical trial due to improved patient care in the placebo group prevented its eventual use in sepsis treatment (247) . in vivo administration of eritoran in mice lethally infected with iav resulted in improved clinical score, lung pathology results, and reduced viral titer. delayed administration of eritoran, at day 6 after infection beyond the recommended therapeutic time window (within 48 h after the first display of clinical symptom) for use of oseltamivir (248), also demonstrated a significant benefit to infected mice compared to non-treated group, suggesting a prolonged therapeutic time window for iav treatment when compared to mainstay antiviral drug treatment. a newer and structurally simpler specific tlr4 antagonist, fp7 (249), alongside a newly developed decoy peptide 2r9 that has been shown to disrupt tlr2, 4, 7, and 9 signaling via tirap, has been shown to protect mice from lethal iav infection (250) . these results support the potential use of tlr4 antagonism as a means to treat severe iav infection. the suppression of other tlr signaling pathways-such as blocking tlr2-mediated signaling through the use of an anti-tlr2 antibody, significantly protected against lethality when administered on day 2 and 4 post-iav infection (251) . a study also demonstrated that h5n1-infected tlr3 knockout mice had better survival than h5n1-infected wild-type mice, which is evident through the significantly faster regaining of body weight post-infection, lower viral titer in the lung, and fewer pathological changes in the lung (252) . an increasing number of tlr antagonists are now under development (253, 254) , alongside several other agents also shown to have effects on tlrs. polysaccharides isolated from r. isatidis, a traditional chinese medicinal herb used to treat iav infection, have recently been shown to inhibit pro-inflammatory cytokines such as il-6 and ccl-5 in vitro by down-regulating upstream tlr3 expression (255) . menk, an endogenous protein expressed in the adrenal medulla, was shown to both prophylactically and therapeutically increase the survival rate while reducing viral-caused lung pathology and viral titer in mice lethally challenged with iav (256) . this was determined to be caused by the downregulation of tlr7. these results suggest the potential of down-regulating tlr expression in the treatment of iav infection. the above-mentioned data suggest modulation of tlr signaling or expression as a promising approach in treating severe influenza disease and deserves immediate investigation. table 2 summarizes new immunomodulatory approaches to combat iav infections. it is well documented that patients with diabetes mellitus have a greater tendency to develop severe iav infection than healthy patients (257) . hyperglycemia increases susceptibility of the host to iav infection via viral uptake, through the promotion of v-atpase assembly (258) and immunosuppression (257) . in addition, viruses rely on host metabolism to perform essential functions during replication (259) (260) (261) (262) . these processes exert a large energy demand on the host within a very short period of time (263) ; energy of which is supplied by and is dependent on host metabolism. iav viruses have been reported to modify the metabolic state of the host. for example, increased c-myc-dependent glycolysis and glutaminolysis has been demonstrated in infected cells (264) . the changes in glucose and glutamine metabolism were reversed upon the addition of bez235, which inhibited the iav-mediated c-myc induction. administration of bez235 2 days prior to infection and up to 4 days post-infection was shown to decrease lung viral titer and improve the survival rate in iav-infected mice. small molecules such as clotrimazole and α-mangostin that target lipid metabolism have also been demonstrated to suppress iav replication in vitro (264) . in addition to being important for generating energy and biosynthesis, recent research demonstrates that cellular metabolism affects immune cell function. dysregulated immune responses observed in many diseases are associated with specific metabolic configurations. viruses, influenza inclusive (265) , were found to induce drastic alterations in metabolic levels and programs (263) . macrophages in infected hosts were observed to have marked differences in the krebs cycle, a key metabolic pathway. this is of significance due to the role of macrophages, which are immune cells critical in the pathogenesis of many inflammatory diseases (263, 265, 266) . in activated macrophages, succinate, a krebs cycle intermediate, was found to possess inflammatory signal. accumulation of succinate generates ros, leading to subsequent activation of hypoxia-inducible factor 1α and the induction of cytokines such as il-1β (267) . a recent study identified the ability of itaconate, another krebs cycle-derived metabolite, to block the production of inflammatory factors. this prevented inflammation, protecting mice from lethal levels of inflammation that can occur during infection (268) . this data suggest the critical roles of krebs cycle intermediates in regulating cytokine profiles and inflammation. metabolites generated by innate immune cells in distinct configurations could have different roles beyond that of bioenergetics, with functions in signaling regulation, transcription, and orchestrating innate immune responses. despite the lack of research conducted thus far on the application of immunometabolic approaches to influenza treatment, the prospect of manipulating immune responses by modulating immune cell metabolic state is promising. further research should focus on the identification of metabolites for modulation of immune cell function with substantial improvement of therapeutic strategies to treat iav disease. latest advancements in high-throughput technologies, e.g., meta bolomics is a useful approach to systematically investigate the changes of metabolic mechanisms during iav infections. identification of important metabolites involved during iav infection should be a new approach by modulating the host metabolism for interventions. multiple host-based intervention strategies against influenza have been developed or are under development. while approaches targeting host machinery required for virus replication seem to be promising thus far, additional research is needed to determine the effect of modulating host immune response on influenza treatment. this is increasingly important, since targeted host factors may play distinct roles in response to infection by different influenza viral strains (252) , making the management of influenza through solely targeting a single specific host factor is difficult. host-based interventions offer obvious advantages over conventional antivirals, such as a higher barrier to drug resistance (73, 83, 107) due to greater genetic stability of host factors than the mutation-prone nature of viral components. in addition, administration feasibility is a key factor to consider the usage of drugs. the mainstays of antivirals for iav infections, the na inhibitors, and m2 blockers, are recommended to be administered within 48 h of symptom onset for optimal antiviral activity. this short treatment window may not be fully fulfilled in a clinical setting. novel host-based interventions were reported to have therapeutic time windows longer than this conventional timeframe (96, 109, 214, 251) , even up to 6 days post-infection (248) , providing a clear clinical advantage over na inhibitors and m2 blockers. in addition, hypercytokinemia and ards could contribute to disease severity and mortality in instances of severe influenza infection, with virustargeting antivirals providing little to no alleviation of such complications. since host immune response is indispensable in host defense against invading pathogens, the use of immune-modulators to suppress detrimental effects while retaining beneficial protection of the host remains challenging. the timing and dosage of medication administration would be critical in determining the drug effectiveness in influenza treatment. targeting virus-induced metabolic changes to restore host normal metabolism may be a new direction to combat influenza disease. further research in the immunometabolism field, along side studies on modulating immune response to infectious disease by altering host metabolic processes; would create a new direction for future research and is expected to yield significant discoveries that may provide new therapeutic options in the treatment of iav infections. smyl conceptualized the work. il and asms drafted some review sections and tfy and smyl wrote the manuscript. influenza a(h1n1)pdm09 outbreak detected in inter-seasonal months during the surveillance of influenza-like illness in pune avian influenza a (h7n9) virus infections in humans across five epidemics in mainland china age and influenza-specific pre-vaccination antibodies strongly affect influenza vaccine responses in the icelandic population whereas disease and 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influenza highly pathogenic avian influenza a h5n1 and pandemic h1n1 virus infections have different phenotypes in toll-like receptor 3 knockout mice inhibition of toll-like receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics small-molecule inhibitors of the tlr3/dsrna complex radix isatidis polysaccharides inhibit influenza a virus and influenza a virus-induced inflammation via suppression of host tlr3 signaling in vitro novel effect of methionine enkephalin against influenza a virus infection through inhibiting tlr7-myd88-traf6-nf-kappab p65 signaling pathway influenza virus and glycemic variability in diabetes: a killer combination? front microbiol glycolytic control of vacuolar-type atpase activity: a mechanism to regulate influenza viral infection metabolism goes viral dynamics of the cellular metabolome during human cytomegalovirus infection saturated very long chain fatty acids are required for the production of infectious human cytomegalovirus progeny stealing the keys to the kitchen: viral manipulation of the host cell metabolic network viral activation of cellular metabolism targeting metabolic reprogramming by influenza infection for therapeutic intervention metabolic effects of influenza virus infection in cultured animal cells: intra-and extracellular metabolite profiling krebs cycle rewired for macrophage and dendritic cell effector functions succinate is an inflammatory signal that induces il-1beta through hif-1alpha itaconate is an anti-inflammatory metabolite that activates nrf2 via alkylation of keap1 key: cord-317628-1inxq7t5 authors: cuccarese, michael f.; earnshaw, berton a.; heiser, katie; fogelson, ben; davis, chadwick t.; mclean, peter f.; gordon, hannah b.; skelly, kathleen-rose; weathersby, fiona l.; rodic, vlad; quigley, ian k.; pastuzyn, elissa d.; mendivil, brandon m.; lazar, nathan h.; brooks, carl a.; carpenter, joseph; probst, brandon l.; jacobson, pamela; glazier, seth w.; ford, jes; jensen, james d.; campbell, nicholas d.; statnick, michael a.; low, adeline s.; thomas, kirk r.; carpenter, anne e.; hegde, sharath s.; alfa, ronald w.; victors, mason l.; haque, imran s.; chong, yolanda t.; gibson, christopher c. title: functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and covid-19 drug discovery date: 2020-08-14 journal: biorxiv doi: 10.1101/2020.08.02.233064 sha: doc_id: 317628 cord_uid: 1inxq7t5 development of accurate disease models and discovery of immune-modulating drugs is challenged by the immune system’s highly interconnected and context-dependent nature. here we apply deep-learning-driven analysis of cellular morphology to develop a scalable “phenomics” platform and demonstrate its ability to identify dose-dependent, high-dimensional relationships among and between immunomodulators, toxins, pathogens, genetic perturbations, and small and large molecules at scale. high-throughput screening on this platform demonstrates rapid identification and triage of hits for tgf-βand tnf-α-driven phenotypes. we deploy the platform to develop phenotypic models of active sars-cov-2 infection and of covid-19-associated cytokine storm, surfacing compounds with demonstrated clinical benefit and identifying several new candidates for drug repurposing. the presented library of images, deep learning features, and compound screening data from immune profiling and covid-19 screens serves as a deep resource for immune biology and cellular-model drug discovery with immediate impact on the covid-19 pandemic. acting through autocrine, paracrine, and endocrine mechanisms, endogenous immune stimuli maintain homeostasis and signal response to invasion, injury, or malignancy. immune dysregulation underlies a broad set of human diseases including inflammation 1 , autoimmune disease 2 , neuroinflammation 3 , neurodegenerative disease 4 , secondary effects of traumatic brain injury 5 , cancer 6, 7 , infection [8] [9] [10] , and cytokine storm 11, 12 . improvements in the understanding of how immune stimuli amplify or suppress the immune system, trigger new cell fate differentiation, and remodel tissue have resulted in the discovery of a wide range of successful therapeutics 13 , as demonstrated by the anti-tnf antibody adalimumab (humira), noted both for its discovery 14 and its application in rheumatic disease 15 . however, the immune system is vastly complex and dependent on cell type and context; reliably intervening in such a highly interdependent process is a formidable drug discovery challenge. with few exceptions 16 , intercellular immune signaling has been explored by studying specific factors in isolation. although cellular response to individual immune stimuli can be effectively profiled by high-dimensional molecular methods such as rnaseq 17 , proteomics 18 , and chipseq 19 , these technologies lack the speed and cost-effectiveness for systems-level analysis of large panels of immune stimuli and screening libraries across myriad inflammatory models. by contrast, image-based methods have demonstrated utility at many stages of drug discovery 20 , including compound profiling 21 , prediction of assay performance 22 , clustering by mechanism of action 23, 24 , and toxicology predictions 25 . here we present phenomics, the analysis of fluorescence microscopy images as a scalable approach to examine cellular response to a wide range of perturbations. deep-learning algorithms extract high-dimensional and dose-dependent fingerprints of cellular morphological changes, or 'phenoprints', from images to support a variety of downstream applications. these phenoprints detect subtle morphological changes far beyond human ability, and a standardized assay pipeline allows the phenoprints of millions of cellular samples to be related across time and experimental conditions. in this work, we first demonstrate the ability to use images alone to accurately quantify and relate hundreds of immune stimuli. we then show how profiles resulting from these perturbations can be employed in drug screening, particularly highlighting the utility of a relatable dataset to accurately predict the mechanism of action for unknown compounds. we used these capabilities to rapidly develop high-throughput-ready disease models for both sars-cov-2 viral infection and the resulting cytokine storm, and immediately launched large-scale drug screens that recapitulated known effective and ineffective therapies and, more importantly, identified several new potential treatments for both sars-cov-2 infection and covid-19-associated cytokine storm. in order to discern the breadth of immune response that might be captured in a single profiling assay for various applications (fig. 1) , we treated endothelial cells, fibroblasts, peripheral blood mononuclear cells (pbmc), and macrophages with diverse immune stimuli (table s1 ), crispr gene editing reagents, antibodies, and small molecules. we used a single fully automated experimental workflow in which cells are plated, treated, labeled with a panel of fluorescent stains, and imaged with high-throughput fluorescence microscopy 26 . vector representations of more than one million multi-channel fluorescence microscopy images analyzed in this manuscript were generated using a proprietary analytics workflow based on an extension of a densenet-161 neural network 27 various cell types (top left) are treated with a range of biological perturbants and treatments (bottom left), including recombinant proteins, antibodies, crispr-based genetic modifications, and small molecules. high-throughput fluorescence microscopy (middle-top) and deep learningenabled image featurization generates high-dimensional phenoprints that are used for interrogating a range of experimental questions (middle-top and middle bottom). this approach suits a suite of applications (right) with a single workflow and on a single platform. specific applications demonstrated in this paper: systems biology exploration of immune relationships, adaptation across diverse disease models, compound screening, and mechanism prediction. phenoprints are high-dimensional vector representations (embeddings) of cellular morphology derived from fluorescent images resulting from treatment with a biological perturbation. to provide landmark phenoprints across a diverse range of immune function, 446 stimuli were added to four different primary human cell types at a range of biologically relevant concentrations and compared to untreated cells and adjacent concentrations. for example, cells treated with tumor necrosis factor alpha (tnf-ɑ), the anti-pd-1 antibody nivolumab, transforming growth factor beta (tgf-β), or interferon alpha (ifn-ɑ) each displayed concentration-dependent phenotype strength measured as intra-replicate consistency as well as increasing convergence of the phenotype ( fig. 2a) . in total, 131 of 446 stimuli produced highly consistent, dose-dependent phenoprints in at least one cell type (fig. 2b) , with representation from cytokines, growth factors, chemokines, antibodies, microbial toxins, and others (fig. s2) . a subset of factors, such as innate stimulants like lps, interferons, and microbial toxins (e.g. enterotoxins) produced strong dose-dependent phenoprints in all cell types tested. by contrast, other stimuli produced phenoprints only in the expected cell type: vascular endothelial growth factor (vegf) in endothelial cells, fibroblast growth factor (fgf) family in fibroblasts, innate stimulants and interferons in macrophages and pbmcs, and tgf-β family in both macrophages and fibroblasts 28, 29 . example phenoprints for indicated immune stimuli and cells. the intra-dose cosine similarity among well replicates (n=6) for each perturbation dose, demonstrating perturbation consistency(red), and average pairwise cosine similarity between each perturbation dose (blue) and the highest dose, demonstrating phenotypic convergence. b. statistically significant phenoprints induced by immune stimuli (rectangles, colored by class as in fig. 2c ) are connected by edges to the cell type (circles) in which the phenoprint was observed. thicker edges reflect stronger interactions. c. classes of all immune stimuli in immune perturbant library. to test whether phenoprints could capture known functional relationships, we applied hierarchical clustering and confirmed appropriate grouping for factors with similar function and/or structural homology, often in a cell-type-specific manner (fig. 3a , table s2 ). for example, related factors il-4 and il-13, and all type-i ifns clustered in unique groups in any cell type in which a phenoprint was observed. chemokines (such as ccl-4 and mcp-2) and innate stimulants (such as lps and flagellin) are also readily grouped with other stimulants, but only in their appropriate cell types: macrophages and pbmcs (fig. 3a , green/purple edges). growth factors cluster in fibroblasts, consistent with their sensitivity to environmental cues for matrix remodeling and organ function, and ability to differentiate into other states such as myofibroblasts under inflammatory pressure (fig. 3a , blue edges) 30, 31 . phenoprints also captured associations between initial stimuli and overlapping secondary effects, as in the case of clusters for activators of nfkb (tnf-ɑ, il1β), interferons induced by irf3, and tlr3 ligands (poly (i:c) variants) in endothelial cells (fig. s1c ). double-stranded rna motifs such as poly (i:c) are recognized by tlr3, which signals through both nfkb and irf3 pathways 32 . fine-grained distinctions were visible in clustered phenoprints. for example, the tgf-β superfamily (tgf-β proteins, growth differentiation factors, activins, and müllerian inhibiting substance) formed a distinct cluster from other growth factor families, which included the epidermal growth factor (egf) family (egfs, tgf-ɑ, betacellulin, heparin-binding egf), platelet-derived growth factor (pdgf) family, fgf family and the insulin-like growth factor (igf) family (fig. s1d ). within these larger groupings, nearly structurally-identical tgf-β isoforms tgf-β1, tgf-β2, and tgf-β3 clustered more tightly than do other members of the tgf-β superfamily 33 . pathogen-derived toxins also revealed expected relationships. c. difficile toxins a and b produced cell-type-specific similarities with members of the human immune stimulant panel (fig. s1e ). these toxin phenoprints were similar to those of interferon proteins in macrophages, as well as to il-6 superfamily members and nfkb-mediated stimulants in huvec. this aligns with the known dual pathology of c. diff. infection, involving both inflammation through macrophage activation 34 and direct gut permeabilization effects 35 . high-dimensional morphological relationships in four cell contexts. immune stimuli are arranged based on hierarchical clustering of all factors in all cell types; only factors with a statistically strong phenoprint in at least one cell type are shown. lines between nodes are filtered for similarity and colored based on the cell type in which the association was observed. b. hierarchical clustering of phenoprints resulting from huvec treated with an annotated compound library. highlighted clusters pertain to small molecule inhibitors of agc family kinases: akt (blue), rock (orange), mek (orange)/erk (blue), and mtor inhibitors: rapalogues (blue), pi3k inhibitors (orange) and mtorc1/2 (green). c. process for reduction of high-dimensional data into two dimensions. d. projections of compound response in the context of on-and off-perturbation vectors and logistical regression for on-perturbation values by drug concentration for tnf-ɑ, il-6 (receptor chimera) ligands, and socs3 knockout in huvec (mean, n=6). contour lines depict perturbation state (orange) and target state (blue) replicates at the 50, 90, and 99th percentiles, respectively. confidence in phenotypes resulting not only from the immune perturbation but those resulting from compounds in a screening library is critical for evaluation of high-throughput screens. to this end, we generated individual compound phenoprints from an annotated bioactive library, which indeed clustered by mechanism of action ( fig. 3b ). for example, inhibitors of agc kinases akt and rock clustered within a super-group as did inhibitors of mek and erk, which is expected based on protein homology and sequential signaling, respectively. more granular sub-clusters were also observed; for example, a diverse group of inhibitors of mtor can be sub-classified into rapalogues, pi3k inhibitors and mtorc1/2 inhibitors. we next tested whether phenomics could uncover compounds that rescue the complex high-dimensional effect of various immune perturbants. we applied several disease-related phenoprints identified above and defined a corresponding perturbation vector in high-dimensional embedding space. compounds that rescued on-perturbation morphology with minimal deviation in off-perturbation morphology were selected as screen hits, as they offer a potential combination of efficacy and specificity (fig. s3a, b) . we first validated the strategy using approved anti-tnf antibodies in the context of a tnf-ɑ phenoprint (fig. 3d ). all tested antibodies rescued the phenoprint induced by tnf-ɑ (perturbed state) back towards the unperturbed phenoprint (target state), but infliximab was less potent relative to adalimumab and golimumab. it is known that infliximab, adalimumab, and golimumab all have similar affinities for tnfɑ 36 , but only adalimumab and golimumab are effective in the absence of concomitant immunosuppression 37 . although many factors can affect antibody performance, this finding suggests phenomics can differentiate subtleties of antibody efficacy beyond affinity for the ligand. next, we selected a set of clinical-stage and approved jak inhibitors to test the ability of phenomics to model compound rescue of il-6 signaling when cells are activated by the ligand or when disrupted by genetic modification. tofacitinib, baricitinib, ruxolitinib, and oclacitinib reverse the phenoprint of the il-6 receptor chimera in huvec in a dose-dependent manner. in addition to identifying compounds that block receptor-mediated signaling, we demonstrated compound-induced rescue of a phenotype resulting from knockout of an intracellular mediator. here, knockout of socs3 using crispr/cas9 leads to hyperactivation of jak signalling 38 , resulting in a phenoprint that is rescued by the same set of compounds ( fig. 3d ; comparison to inactives: fig. s3c ). well-designed phenotypic screening efforts benefit from being a more proximal model of the disease, and since they are not limited to pre-defined targets, offer the ability to uncover novel therapeutic pathways 39 . however, this carries the risk of investing time and resources into compounds that are later discovered to interact with known or disadvantageous pathways. to address this challenge, we leveraged the relatability of our nce screening and annotated compound datasets to identify novel therapeutic opportunities while rapidly deprioritizing high-risk mechanistic space, as demonstrated in a screen against the tgf-β-induced phenoprint. tgf-β, signaling through the receptor alk5, is recognized as a primary driver of fibrosis in debilitating diseases such as idiopathic pulmonary fibrosis and renal fibrosis, as well as a significant contributor to immune exclusion in the tumor microenvironment 29 . the diversity and consistency of phenoprints recapitulating known biology suggested phenomics might discover new chemical entities (nce) and predict mechanism of action. we therefore screened 90,000 diverse chemical starting points (fig. s4a ) against the tgf-β phenoprint (fig. 4a) . a novel compound of interest (rec-0104937) completely reversed the phenoprint at low micromolar concentrations. further, this same compound rescued an orthogonal functional validation assay, mitigating tgf-βinduced collagen deposition with an ec50 of 0.763 µm (fig. 4b) . we then compared the rec-0104937 phenoprint to reference phenoprints derived from a set of 6,000 diverse, well-annotated small molecules; several known alk5 inhibitors were highly similar (fig. 4c) . we experimentally validated the accuracy of these predictions in gold standard assays of alk5 activity: rec-0104937 inhibited cellular p-smad activity and cell-free biochemical alk5 activity at 0.585 µm and 0.725 µm respectively (fig. s4d, fig. 4d ) 40, 41 . because the advancement of tgf-β receptor inhibitors has been hampered by cardiac toxicity 42 , and because our research goals are to identify compounds acting against novel pathways, we rapidly deprioritized this compound in favor of others based on the primary screening data. overactive tnf-ɑ signaling is a major driver of inflammation in inflammatory and autoimmune diseases including alzheimer's disease 43 , multiple sclerosis 44 , and traumatic brain injury 45 , and significant benefit has been achieved with monoclonal antibody intervention 46 . we therefore sought to rescue the tnf-ɑ phenoprint with an intervention that not only targets a novel mechanism of action, but also benefits from advantages of small molecules over existing antibodies, such as oral availability and increased central nervous system penetration. we found 2,073 compounds that statistically altered the tnf-ɑ phenoprint at a single dose in a 90,000-compound primary screen. we tested these for dose response and selected a subset for validation. although suppression of tnf-ɑ signaling through nfkb blockade is a plausible anti-inflammatory strategy, reduction of tnf-ɑ signaling via global inhibition of nfkb leads to a challenging safety profile 47 . two molecules (rec-0150357 and rec-0082469) rescued the tnf-ɑ-induced phenoprint (fig. 4e ) and prevented secretion of il-6, a marker of tnf-ɑ stimulation (fig. 4f ) while preserving nfkb activation (fig. s4e) . by comparing the phenoprint of rec-0150357 to phenomics data from annotated compounds in prior experiments, we found strong similarity between the phenoprints of rec-0150357 and rho kinase inhibitors (fig. 4g ). this finding was corroborated by kinase profiling, revealing rock1 and rock2 inhibition at 0.097 and 0.066 µm, respectively (fig. s4f ). during intellectual property exploration, the target was further confirmed; the scaffold was previously evaluated as a rock inhibitor 48 . kinases that were inhibited to a lesser degree were cdk7 and dyrk1b (fig. s4f ). the effect of rock2 inhibition on tnf-ɑ-induced inflammation is documented 51 and similar compounds are in active development for autoimmune disease 49, 50 . therefore, we deprioritized the compound using high-dimensional primary screening data in favor of another compound, rec-0082469. the alternative scaffold also reduced tnf-ɑ-induced il-6 release ( fig. 4f ) but did not phenotypically cluster with any of the mechanistic classes annotated in our libraries, thus enabling informed prioritization of the compound for further study. in a >400 kinase biochemical screen, the compound showed no significant inhibition of any kinase at 1 µm. to investigate the potential benefit of rec-0082469 in neuroinflammation, we explored the role of the molecule to suppress microglial activation in vitro. using an immunofluorescence stain for the microglial activation marker iba-1 51 we confirmed that rec-0082469 reduced the activation of bv-2 mouse microglia ( fig. 4h ; example images fig. s4g ). given the potential of rec-0082469 to operate via a novel mechanism of action (moa) we initiated a hit optimization effort initially focused on enhancing the series potency (rec-0082469 ec50 > 1 um). we used phenomics as to assess our efforts to optimize potency while maintaining or improving efficacy against an unknown target; we succeeded in improving potency by more than ten-fold (rec-0648455 ec50 = 71 nm) and improving the on-perturbation score to 0.05 (fig. 4i ). the ongoing covid-19 pandemic presents an urgent need for quick and adaptable drug discovery in the context of a complex and poorly understood disease. we leveraged our phenomics platform to screen for approved and reference (e.g., development stage antiviral) compounds that could address two key components of covid-19 disease progression: direct effects of viral infection and the damaging effects of an unresolved inflammatory response, or cytokine storm. 1,670 and 2,913 compounds were applied to cells in the infection and cytokine storm modes, respectively, and compound rescue was evaluated with the same processing pipeline described above. many features of terminal covid-19 are the result of inflammatory pressure on endothelial cells, manifesting as barrier disruption, lymphocyte recruitment, induction of blood coagulation, and acute respiratory distress syndrome (ards) 52 . we modeled the cytokine storm associated with late-stage covid-19 in endothelial cells by applying cocktails of circulating proteins that mirror those from severe covid-19 53 patients (perturbed state) as well as healthy control patients (target state) (table s3 , fig. s5a ). we hypothesized that rescue of the perturbed state toward the target state would reveal anti-inflammatory compounds specifically relevant to the covid-19-associated cytokine storm. following identification of hit compounds, electric cell-substrate impedance sensing (ecis) was employed to confirm the activity of the aforementioned compounds in an orthogonal functional model of vascular integrity challenged with the same cytokine cocktails. presently, jak inhibitors have shown benefit in one nonrandomized trial 54 and represent one of the most common mechanisms being evaluated among hundreds of clinical trials active for covid-19. in our screen against the cytokine storm phenotype, jak inhibitors were capable of potent rescue of the severe cytokine storm phenoprint, confirming strong potential for this mechanism's efficacy in the context of a complex immune cascade (fig. 5a) . we also identified rescue by compounds in three classes of inhibitors outside of the jak/stat pathway that have been less deeply explored in the context of covid-19, including inhibitors of syk, pi3k and c-met. compounds were then applied to cells with the same inflammatory cocktail and evaluated with ecis ( fig. s5a ) to inform on benefit to vascular integrity. rescue of this orthogonal functional assay was observed for each mechanism identified in the high-dimensional assay (fig. 4g, fig. s5b -c). we next developed a model of sars-cov-2 infection to screen for repurposable compounds acting directly against viral targets or on host pathways. to define the model, we evaluated the effect of sars-cov-2 infection in multiple cell types, of which three resulted in robust phenoprints as compared to either mock infected or inactivated virus control populations: calu3 (a lung adenocarcinoma line), vero (an immortalized interferondeficient african green monkey kidney line 55 ), and primary human renal cortical epithelium (hrce) (fig. 5c, fig. s6d ). we confirmed active infection with sars-cov-2 nucleocapsid antibody staining and quantification of productive viral replication ( fig. s6a-c) . we reasoned that a primary human cell type would be most directly translatable 56 to human pathology, especially from tissues demonstrated to be directly infected by sars-cov-2 57 , and thus conducted a screen of 1660 compounds against the hrce phenotype, while testing a limited subset of those compounds in vero and calu3 cells. the majority of compounds currently under evaluation 58 in human clinical trials for covid-19 showed no or weak efficacy in the hrce model 56 . however, in these screens remdesivir and its metabolite, gs-441524, demonstrated strong efficacy and aligned with potency described in the literature (ec50 of 100 nm and 2 μm, respectively) ( fig. 5d) 59, 60 . remdesivir is a nucleoside analog that directly interferes with the viral-rna-dependent rna polymerase to inhibit viral replication and, importantly, successfully reduced recovery time for treated patients in clinical trials 61 announced after our data and analysis was publicly released. further illustrating the predictive capacity of the model, two other antivirals, lopinavir and ritonavir were not found to be efficacious and were later discontinued in clinical testing for covid-19 62 . additionally, aloxistatin (e64d), an irreversible cysteine protease inhibitor initially developed for muscular dystrophy 63 , also demonstrated suppression of the viral phenoprint in hrces (ec50 of 40 nm). recent studies have confirmed that cathepsin l, a cysteine protease, is required for sars-cov-2 entry in some cell types, and aloxistatin treatment significantly reduced entry of sars-cov-2 pseudovirions 64,65 . we then tested a subset of these antiviral compounds in additional cell types, vero and calu3, and found aloxistatin did not rescue in these models. however, another protease inhibitor, camostat mesilate, was efficacious in the calu3 model (ec50 of 260 nm), but not the vero or hrce models (fig. 5d-f, fig. s6e ). camostat inhibits tmprss2, which was recently shown to be required for sars-cov-2 entry in human airway cells 66 . similar to findings in recent clinical trials 67,68 , we found chloroquine and hydroxychloroquine to have no benefit in the hrce or calu3 models; however, they showed modest benefit in vero cells (ec50 of 730 nm and 1.65 µm, respectively) with very high offperturbation activity (fig. 5d-f) . overall, compound efficacy in human cell types was poorly recapitulated in vero cells (fig. 5d-f, fig. s6f ). taken together these findings suggest that sars-cov-2 entry protease inhibitor activity varies across cell type and species; however, remdesivir and gs-441524 show strong rescue of the viral phenoprint in all cell types tested. we identified jak inhibitors ruxolitinib and baricitinib as efficacious in both viral and cytokine storm models (fig. 5a, 5i, fig. s6g ). however, we found that high concentrations of these compounds led to increased infection in hrce cells (fig. s6h) . suppression of interferon production is a known component of sars-cov-2 infection at a low multiplicity of infection 69 . it is unclear however, what effect additional interferon suppression would have in vivo, especially at higher viral loads, warranting investigation into alternative mechanisms of cytokine storm suppression, such as pi3k or c-met inhibition. notably, bortezomib exhibited poor performance in both assay modes, is reported to impair endothelial cells in inflammatory contexts 70 , and also enhances susceptibility to viral infection 71 , particularly coronaviruses 72 . biology is massively complex and highly networked, but the tools to explore and discover novel biology and develop medicines have until recently relied on simple, univariate measurement. the genomic revolution yielded a taste of what is possible if high-dimensional biology can be scaled by massively increasing the rate of understanding of the role of thousands of genes in human biology and disease. following this advancement, several new high-dimensional approaches have been developed to add clarity to complex functional relationships and discover new therapeutics, but these are hindered from highthroughput screening application by engineering and cost bottlenecks. we present here early data from our experience using phenomics (fig. 1) as one strategy to accelerate drug discovery. we first established that diverse immune biology and pharmacology can be detected and discriminated using phenomics (fig. 2) . these data also reveal that phenomics is not simply a classification technology: deep quantification of rich, multi-parametric signal and assessment of dose response is achievable, enabling comparisons and clusterings of diverse biological perturbants alone, or in combination across diverse cell types (fig. 3) . diving more deeply on just two of the 131 immune phenoprints we uncovered that are suitable for drug screening, we explored 90,000 new chemical entity starting points in the context of tgf-β-and tnf-ɑ-induced phenoprints (fig. 4) . among hits in each context, prediction of alk5 and rock inhibition allowed us to rapidly shift resources to higher priority hits. in particular, we focused our efforts on a suppressor of the tnf-ɑ phenoprint with a high potential to potentially be active against a novel, but as of yet unknown, target. further, we drove medicinal chemistry work against this unknown target(s) using phenomics, demonstrating a 10-fold increase in potency while also increasing the magnitude of rescue. the application of phenomics can be extended to more complex disease-causing perturbations as well: the platform was rapidly adapted for the characterization and exploration of actionable therapies in the context of a novel and poorly understood disease, covid-19. within 28 days of initiating the project, we identified hits through high throughput chemical screens against covid-19 cytokine storm and sars-cov-2 infection in the relevant tissue types, without the need to develop cytokine-or virus-specific reagents and assays. we demonstrated that a handful of drugs currently in clinical trials strongly modulate the infection model (e.g. remdesivir), the cytokine storm model (pi3k inhibitors) or both (jak inhibitors), prior to their clinical trial results becoming available. conventional antiviral research relies heavily on univariate assays that measure attributes like cell death or expression level of one protein. using a single platform, we found not only conventional antivirals, but also compounds with unconventional effects on disease-associated host pathways such as inflammation. in the sars-cov-2 model remdesivir and its analog, gs-441524 demonstrated efficacy in all cell models tested. unfortunately, remdesivir is dosed via an intravenous route, typically in an inpatient setting and a time at which cytokine storm may be primarily responsible for the pathology (wherein remdesivir had no unexpected efficacy in our cytokine storm model). sars-cov-2 is able to use a variety of receptors to facilitate cell entry, with receptor specificity by cell type apparent in our data: aloxistatin (e64d), inhibiting the cathespinmediated entry pathway, and camostat, inhibiting the tmprss2-mediated pathway, each demonstrated strong response in hrce and calu3 cells respectively. nevertheless, pseudovirus entry assays 66 have shown that even in cells with both pathways active, modulating a single pathway still quantitatively reduces viral infection load. further study of the proportional activity of each pathway in relevant human tissues may be warranted. as aloxistatin is orally bioavailable, simply and inexpensively synthesized, and has a relatively strong safety profile based on chronic treatment of muscular dystrophy patients in a phase 3 trial, it deserves further study for covid-19, with the expectation that early treatment in the course of infection may be most efficacious. in our model of more advanced covid-19 symptoms driven by cytokine storm, jak inhibitors were noteworthy rescuers of the inflammatory phenoprint and moderate rescuers of the viral phenoprint at low concentrations. due to oral bioavailability and the safety profile of acute treatment they are excellent candidates for repurposing. however, we observed that jak inhibitors enhanced cellular infection of sars-cov-2 at higher concentrations, suggesting an effect on interferon signaling, a possible clinical liability that should be closely monitored during trials. supporting this finding and underlining the importance of identifying diverse options to address cytokine storm, jak inhibitors are known to increase the prevalence or severity of other viral infections including herpes zoster, jc virus, and hepatitis b [73] [74] [75] . this study also identified alternative mechanisms of action which have been much less deeply considered in the context of covid-19, such as certain syk inhibitors, c-met inhibitors and pi3k inhibitors. such molecules could be critical additions to remdesivir therapy in severe patients. this work and the recent success of dexamethasone in clinical trials for covid-19 also identified a key limitation of our current phenomics approach: when studying a cell type in isolation, phenomics surfaces compounds that act via cellautonomous mechanisms 76 . compounds that intervene in multicellular processes might be revealed by development of coculture models. taken together, our results demonstrate that systems-level modeling and drug discovery is achievable using a single phenomics platform. first, this approach simplifies and extends the ability to work across many disease models rapidly because assay development work for any new model is minimized. second, this work partially overcomes a historical limitation of phenotypic screening, predicting mechanism of action, by relating the high-dimensional phenoprint of hit compounds to those of reference molecules. finally, we show the potential of this platform in optimizing nce compounds through medicinal chemistry in a high-dimensional, target-agnostic manner. unlike other high-dimensional approaches, the relatively inexpensive nature of these image-based assays allows them to be scaled to levels of throughput comparable to more traditional lowdimensional screening modalities. in the hopes that it will be valuable to others, we have made images and embeddings from huvec treated with the immune perturbant library, and from our covid-19 primary screens (both infection and cytokine storm) available online (including raw image data, metadata, and deep learning embeddings from images) at rxrx.ai/rxrx2 and rxrx.ai/rxrx19, respectively. beyond the applications described here, the modular nature of this phenomics platform enables rapid adaptation to different libraries of immune stimulants, antibodies, or other large molecules, and incorporation of additional cellular contexts like co-culture models. in future work, comparisons of hits to phenoprints associated with knockout of each gene in the genome (achieved by arrayed whole-genome crispr knockout) may further expand our ability to predict mechanisms beyond those represented within our annotated small molecule library, bridging a key gap in phenotypic screening. critically, these data can be related over time and across disparate research programs-supporting the creation of large biological image datasets for deep-learning applications 77,78 that will accelerate drug discovery and yield functional maps of human cellular biology. human umbilical vein endothelial cells (lonza, c2519a) were cultured according to manufacturer's recommendations in egm2 (lonza, cc-3162). nhlf: normal human lung fibroblasts (lonza, cc-2512) were cultured according to manufacturer's recommendations in fgm2 (lonza: cc-3131, 4126) and used at passage four for all assays. pbmc: peripheral blood mononuclear cells (pbmcs), from healthy donors, were prepared from fresh (< 24 hours old) leukopaks (stemcell technologies inc., catalog # 70500). following 3 120 rcf washes (brake off) for platelet removal, the samples were processed by easyseptm rbc depletion reagent in accordance to manufacturer's instructions (stemcell technologies inc., catalog #18170). following isolation, pbmcs were pelleted (300 rcf) and resuspended in cryopreservation medium (cryostor® cs10 freeze media, biolifesolutions inc., part #: 210102) for long term storage. macrophage: macrophages were derived from either cryopreserved or freshly isolated pbmcs. monocytes were enriched by plastic adherence, first seeding pbmcs in serum free rpmi-1640 medium followed by 1.5 hours of incubation and washed 2x with pbs. cells were incubated for 3 d in complete medium (rpmi+ 10% heat inactivated fbs, 25 ng/ml m-csf, 10 ng/ml il-10). media was replenished after 3 d with one third of the conditioned medium and ⅔ fresh complete medium. monocyte derived macrophages (mdms) were harvested from each vessel after an additional 3 d using accutase following manufacturer's instructions (thermo -a1110501). mdms were pelleted (300 rcf) and resuspended in cryo-preservation medium. hrce: primary human renal cortical epithelial cells lonza (cc-2554) were propagated at 37°c with 5% co2 in epicm, (sciencell # 4101) supplemented with epithelial cell growth supplement (epicgs, sciencell #4152). vero: an immortalized african green monkey kidney (atcc ccl-81) were propagated at 37°c with 5% co2 in eagle's minimum essential medium (emem) supplemented with 10% fbs. calu3: human lung adenocarcinoma line (atcc htb-55) were propagated at 37°c with 5% co2 in emem supplemented with with 10% fbs. bv-2: murine microglial cells (iclc atl03001, ospedale policlinico san martino) were propagated at 37°c with 5% co2 in rpmi media + 10% heat inactivated fbs. immune stimuli (table s1 ) were solubilized in sterile phosphate buffered saline (pbs) containing 0.1% bsa (sigma cat.# a1595-50ml) to make stock solutions of .04 mg/ml in echoqualified 384w low-dead volume source plates. source plates were stored at -80°c until use. cells were seeded into 1536-well microplates (greiner, 789866) via multidrop (thermo fisher) and incubated at 37c in 5% co2 for the duration of the experiment. immune stimuli or virus were added 24 hours post-seeding (huvec, macrophage, fibroblast) or 1 h (pbmc). treatments were randomized across treatment plates with a 6-log range of immune stimuli (typically 0.001-100 ng/ml) at 6 replicates each with acoustic transfer (echo 555, labcyte) and incubated 37°c for 24 or (complete immune stimuli panel) or 48 h (for pbmc with pembrolizumab or nivolumab). active sars-cov-2 was added via multidrop 24 hours post seeding of the specified cell type. plates were stained using a modified cell painting protocol 29 . cells were treated with mitotracker deep red (thermo, m22426) for 35m, fixed in 3-5% paraformaldehyde, permeabilized with 0.25% triton x100, and stained with hoechst 33342 (thermo), alexa fluor 568 phalloidin (thermo), alexa fluor 555 wheat germ agglutinin (thermo), alexa fluor 488 concanavalin a (thermo), and syto 14 (thermo) for 35 minutes at room temperature and then washed and stored in hbss+0.02% sodium azide. live-virus experiments omitted the mitochondrial stain due to operational constraints of the biosafety level environment. one hour prior to addition of the immune stimulant or 18 hours prior to the addition of virus, cells were treated with compound via acoustic transfer (echo 555, labcyte). primary screening of new chemical entity libraries was performed at 10 or 30 µm with concentration-response confirmation spanning 100 nm to 30 µm in half-log steps. sars-cov-2 screening was completed in dose response in half-log steps between 10 nm to 3µm. after 24 h incubation (or 96 hours post viral infection), plates were imaged using image express micro confocal high-content imaging system (molecular devices) microscopes in widefield mode with 20x objectives. four sites per well were acquired with 6 channels per site. the following bandpass filters were used to visualize the channels: ff409/493/573/652, ff459/526/596, ff01-432/515/595/730-25, ff01-475/543/702, and ff01-600/37/25. kinase profiling was performed using a kinomescan tm panel of 97 or >400 kinases at eurofins-discoverx (san diego, ca). targets exhibiting > 50% inhibition were followed by kdelect ⓡ analyses for dyrk1b, cdk7, rock2 and rock1 to determine ic50's. data presented as mean and standard deviation. alt-r crispr-cas9 reagents were purchased from integrated dna technologies, inc. (idt) and prepared following the manufacturer's guidelines and protocols (alt-r crispr-cas9 crrna, alt-r crispr-cas9 tracrrna cat #1072534, alt-r s.p. cas9 nuclease v3, cat #1081059, and alt-r cas9 electroporation enhancer, cat #1075916). alt-r crispr-cas9 crrna was duplexed to alt-r crispr-cas9 tracrrna and then combined with alt-r s.p. cas9 nuclease v3, following idt guidelines, to form a functional crispr-rnp complex. this crispr-rnp complex was transfected into cells using the lonza 4d nucleofection system and standard protocols with proprietary modifications, or with a proprietary lipofectionbased process for high-throughput application. alt-r cas9 electroporation enhancer was included into the nucleofection reactions to enhance transfection efficiency following standard guidelines from idt. all images were uploaded to cloud storage and featurized by embedding them with a trained neural network using google cloud platform. this network is based on the convolutional neural network densenet-161 30 . we adapt this network in the following ways. first, we change the first convolutional layer to accept image input of size 512 x 512 x 6. like densenet-161, we use global average pooling to contract the final feature maps, which in our case are tensors of dimensions 16 x 16 x 2,208, to a vector of length 2,208. however, instead of following immediately with a classification layer, we add two fullyconnected layers of dimension 1,024 and 128, respectively, and use the 128-dimensional layer as the embedding of the image. the weights of this network were learned by adding two separate classification layers to the embedding layer, one using softmax activation and the other using arcface 78 activation, which were simultaneously optimized by training the network to recognize perturbations in the public dataset rxrx1 79 and in a proprietary dataset of immune stimuli in various cell types. due to operational constraints of the bsl-3 assay conditions, a modified assay protocol lacking one image channel was used for the live-virus experiments. to accommodate this change, we trained a separate network of the same basic architecture that used only five input channels and one fully-connected final layer of dimension 1,024. immune stimuli phenoprints were observed by calculating the mean embedding of all but one biological replicate, finding the angle between that average and the held-out replicate well, and repeating this process for every replicate to find the average cross-validated angle for that perturbation. statistical significance of these phenotypes was determined by comparing their similarity at high dose against a distribution of similarities between embeddings of images of untreated cells. we used the benjamini-hochberg multiple tests correction with a 5% false discovery rate and considered phenotypes acceptable if they had a corrected p-value<0.05 in two independent experimental batches. the similarity between a pair of immune stimuli was determined by calculating the cosine similarity between all pairs of embeddings of one immune stimulant at high concentration with the embeddings of another immune stimulant at high concentration, and testing whether the mean of this pairwisesimilarity distribution was significantly different from zero using a one-sample t-test and employing the benjamini-hochberg multiple tests correction with a 5% false discovery rate. only significant pairs are used in this paper, and the means of their pairwise-similarity distributions are the values reported in the figures. for small molecule screens, post-processing of the embedded images included normalization to remove inter-plate variance, pca to reduce the feature space, and anomaly detection to remove outliers from the control populations. the vector pointing between the barycenters of the untreated and perturbed conditions was computed, and the embedded image vectors were decomposed into the signed scalar projection (the onperturbation score) and the scalar rejection (the off-perturbation score) with respect to this vector. these scores were normalized so that the mean on-perturbation score was 0 for the untreated condition and 1 for the perturbed condition. separation of the untreated and perturbed conditions along the on-perturbation axis was assessed by z-factor. for compound moa inference, cosine similarities were computed between the embeddings of an nce compound and the set of embeddings of a compound library annotated for moa, and significantly large similarities (relative to the distribution of similarities of pairings of annotated compounds with the nce compound) were reported. for the cocktail representing severely affected patients, top concentration of the most abundant protein, cxcl-10 was selected to be 200 ng/ml based on a practical screen concentration and previously identified phenotypes for this factor. all other proteins were prepared at appropriate concentrations relative to cxcl-10. cocktails representing healthy patients and those with moderate disease severity were prepared with each concentration relative to the severe cocktail. iba1 immunofluorescence assay bv-2 microglia were thawed from liquid nitrogen and plated at 2500 cells per well in 384-well pdl/collagen-coated plates (greiner #781866). the next day, the cells were treated with compound first, followed an hour later by stimulant (recombinant murine tnf-ɑ+ifn-γ (peprotech), 200 μg/ml in 0.1% low endotoxin bsa/pbs). twenty-four hours after treatment, bv-2 microglia were fixed and stained for the microglial activation marker iba1. briefly, cells were fixed with 4% pfa for 15 min at room temperature. primary antibody solution was added to a 1:100 final dilution (iba1 antibody abcam cat #ab5076) incubated overnight at 4°c. after overnight incubation with primary antibody cells were washed with pbs, and secondary antibody solution was added (alexafluor 488 donkey anti-goat igg, 1:1000 final dilution. invitrogen cat# a11055). cells were then incubated for 1 hr at room temperature, protected from light. following secondary antibody incubation cells were washed and the plate was sealed for imaging, which was performed on an image express micro confocal high-content imaging system (molecular devices). data and error presented as mean and standard deviation. quantitative measurement of cytokines in supernatants obtained from cultured cells was performed by using a homogenous timeresolved fluorescence assay (htrf, cisbio). il-6 htrf assays were performed in accordance with the manufacturer's protocol. briefly, cells were seeded and after 24 h treated with compound over 8 concentrations and 5 replicates each. after an additional 24 h, supernatant was collected from assay plate and appropriate sample dilutions and standards were made and dispensed into barcoded labeled 384-perkinelmer proxiplates (cat# 6008280). after the recommended incubation time, the plate was read using an envision ⓡ 2105 microplate reader (perkinelmer). data and error presented as mean and standard deviation. nfkb reporter cells (tr860a-1, system biosciences) were seeded at 4000 cells per well in 384 well imaging plates (781948, greiner). compounds were added at at least 4 replicates per concentration followed by 1 ng/ml tnf-ɑ after 1 h. plates were imaged (gfp channel) once every 3 h via incucyte (sartorius). data for integrated intensity over at the 16 h time point is presented as mean and standard deviation, significance analyzed with 2-way anova. normal human lung fibroblasts (nhlf, lonza) were plated in 1536-well plates (greiner) at 0.25 x 10^6 cells/ml in fgm-2 (lonza). after 24 hours, the media was replaced with fbm media (lonza) and incubated for 24 hours. cells were treated with compounds of interest in a 11-point dose response curve at 32 replicates per concentration using an acoustic liquid handler, and incubated for 1 hour at 37°c, 5% co2. cells were then treated with 1ul of 11ng/ml tgf-β1 (r&d systems), for a final concentration of 1ng/ml. cells were incubated for 30 minutes at 37°c and 5% co2. cells were fixed with 4% pfa, blocked for 1 hour with 1% bsa/0.1% triton x-100/pbs, and then stained for psmad (cell signaling, 1:800). after an overnight incubation at 4°c, cells were stained with alexafluor 647 (thermo, 1:1000) and hoescht (thermo, 1:5000) for two hours at 25°c, washed with pbs twice, and imaged on an image express micro confocal high-content imaging system (molecular devices). images were analyzed with cellprofiler to observe nuclear translocation of psmad2. data and error presented as mean and standard deviation. normal human lung fibroblasts (nhlf, lonza) were plated in 1536-well plates (greiner) at 0.25 x 10^6 cells/ml in fgm-2 (lonza). cells were treated with compounds of interest in a 11point dose response curve at 32 replicates per concentration using acoustic transfer (echo 555, labcyte), and incubated for 1 hour at 37°c, 5% co2. cells were then treated with 1ul of 11ng/ml tgf-β1 (r&d systems), for a final concentration of 1ng/ml using multidrop (thermo fisher). cells were incubated for 96 hours at 37°c and 5% co2. cells were fixed with 4% pfa, blocked for 1 hour with 5% bsa/0.2% triton x-100/pbs, and then stained for collagen i (cell signaling, 1:500). after an overnight incubation at 4°c, cells were stained with alexafluor 750 (thermo, 1:1000), cellmask orange (thermo, 1:5000) and hoescht (thermo, 1:5000) for two hours at 25°c, washed with pbs twice, and imaged on an image express micro confocal high-content imaging system (molecular devices). images were analyzed with cellprofiler. data and error presented as mean and standard deviation electric cell-substrate impedance sensing (ecis) prior to use, 96-well ecis plates (applied biophysics, 96w20idf pet) were pre-treated with 10mm l-cysteine (sigma-aldrich, c7352-25g) and then coated with fibronectin (gibco, phe0023). human umbilical venous endothelial cells were plated in the fibronectin coated 96-well ecis plates at 55,000 cells/well in ebm-2 (lonza cc-3156) +egm-2 (lonza, cc -4176). cells were allowed to settle at room-temperature for 1 hour and then incubated for 24 hours at 37 o c, 5% co2. following incubation, the plates were placed on the ecis readers for 1 hour to establish baseline resistance. cells were then treated in a 9point dose response curve at 4 replicates per concentration using acoustic transfer (echo 555, labcyte) and returned to the incubator for 1 hour. following this, cells were treated with the cytokine storm cocktail (table s3) . resistance was measured for 24 hours following this. the assay window was defined as the time range with the greatest observable difference in membrane resistance between empty and disease control (approximately 6 hours following cocktail addition). resistance was normalized to each plate and graphed as a dose-response curve where 1 and 0 correspond to health and disease controls, respectively. after staining and imaging to establish high dimensional phenotypes, plates were rinsed once with wash buffer (1xhbss + 0.02% sodium azide) before incubating with primary antibody raised against sars-cov-2 nucleocapsid protein for 60 mins at rt (sino biological catno. 40588-t62, 1:1000 dilution). media was evacuated from wells by inverted centrifugation, and secondary antibody was added and incubated another 60 minutes at rt (thermo scientific catno. a31573, 1:2000 dilution). primary and secondary antibodies were diluted in stain base media (1xhbss, 1% bsa, 0.3% triton-x 100). plates were washed one final time using inverted centrifugation and wash buffer before imaging as described above. data presented as mean value. cell-level image segmentations and per-cell log-mean nucleocapsid staining intensities were calculated using standard image segmentation techniques (cellprofiler 80 ). these intensities were normalized by plate with respect to log-mean-intensity in the mock control cells. to adjust for optical effects that changed the background fluorescence level in infected wells, a gaussian mixture model was used to align the lowest peak in log-meanintensity across well conditions. cells were estimated to be infected if they exhibited an adjusted log-mean-intensity above the 99th percentile of intensity for the control cell population. this estimate of number of infected cells was used to compute a fraction of cells infected, which was adjusted to account for the 1% of uninfected cells expected to be above the 99% threshold. the usa-wa1/2020 strain of sars-cov-2 was propagated in vero cells. cells were grown in standard tissue culture flasks (60% confluence) and were infected at a multiplicity of infection (moi) of 0.001, in emem + 2% fbs and 50 g/ml gentamicin, incubated at 37°c with 5% co2 for 5 days. supernatants containing virus were removed from these cultures, spun down to remove cellular debris and stored at -20°c until use. viral titers were determined through standard tissue culture infectious dose 50% (tcid50) methods, where cytopathic effect (cpe) on vero 76 cells was measured by visual observation under a light microscope. to create a suitable control with inactivated virus, sars-cov-2 was irradiated with a uv lamp for 10 or 20 minutes. viral inactivation in this sample was verified using visual cpe on vero cells, where undetectable level of active virus was observed. an additional "mock" control was created using conditioned media preparations generated from uninfected vero 76 cells grown in 2% fbs in emem for five days. cellular debris were removed through centrifugation and the supernatants were frozen at -80°c until use. all experiments using sars-cov-2 were performed using biosafety level 3 (bsl-3) containment procedures at partner facilities including one at utah state university. data and error presented as mean and standard deviation. a 1536-well plate was pre-treated with compounds, at 13 concentrations (ranging from 0 to 100 ng/ml) with at least 2 replicates of each concentration, and a reaction mix containing 15 ng alk5 (thermofisher), using poly 4:1 as substrate was added to the plate. the reaction was started by the addition of 20 µm atp, the plate was mixed on a plate shaker (500 rpm for 2 min) and the reaction allowed to incubate at room temp for 60 min. the reaction was terminated by the addition of adp-glo reagent (promega v9102) (40 min incubation) and kinase detection reagent (30 min incubation). luminescence was captured using an envision xcite plate reader. data presented as mean and standard deviation. all assessments of compound diversity and similarity were performed in datawarrior (openmolecules.org). neighbors were determined to be at 83% or greater similarity as determined with the skelspheres descriptor. tables containing metrics for immune stimulant phenotypes in each cell type are provided in the supplementary materials. underlying images, metadata, and deep learning embeddings for soluble factor perturbations in huvec and all primary screens in primary cell types for covid-19 virus and cytokine storm screening have been made available at rxrx.ai. an interactive server containing drug response projections, hit scores, and structures for covid-19 screening data has been made available for custom search at covid19.rxrx.ai. the code underlying this report leverages proprietary algorithms for image processing, data standardization, outlier detection, and compound efficacy scoring. as such the code underlying this report will not be made available. instead, much of the output of these algorithms is provided in the provided supplemental tables. virus graphic in fig. 1 schematic for interpreting projection of drug response in 2-dimensional plot. contours show the 99%, 90%, and 50% distributions of on-and off-perturbation scores for the perturbation (orange) and target (blue) states. ideal rescues are compounds that rescue along the on-perturbation xaxis toward the target state with minimal increase to the off-perturbation score on the y-axis. b. potential hits are prioritized by the proximity of any dose to the target state, illustrated here with dashed lines and increasing red highlight intensity for higher-ranked dose-curve trajectories. c. projections of treatments along the on-perturbation vector: rescue of the tnf-ɑ phenoprint with clinically approved monoclonal antibodies, reversal il-6-il-6r receptor chimera, and reversal of socs3 crispr gene knockout. ec50 for high-dimensional compound rescue are indicated in parentheses representative images of hrce, calu3 and vero cells immunostained with sars-cov-2 nucleocapsid protein (pink) and modified cell paint dyes c. infection rates of each tested cell type as analyzed by nucleocapsid immunostaining. of note, hrce donors displayed significant variation in infectability and only a minority of donors exhibited infection rates high enough for screening. antibody stains were performed after the principal analysis 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across many experimental batches cellprofiler: image analysis software for identifying and quantifying cell phenotypes we declare competing interests. all authors were employees of or advisors to recursion during the course of this work. all authors have real or potential ownership interest in recursion. key: cord-252725-e3pazjdi authors: khalil, ayman; tazeddinova, diana title: the upshot of polyphenolic compounds on immunity amid covid-19 pandemic and other emerging communicable diseases: an appraisal date: 2020-10-15 journal: nat prod bioprospect doi: 10.1007/s13659-020-00271-z sha: doc_id: 252725 cord_uid: e3pazjdi polyphenols are a large family of more than 10,000 naturally occurring compounds, which exert countless pharmacological, biological and physiological benefits for human health including several chronic diseases such as cancer, diabetes, cardiovascular, and neurological diseases. their role in traditional medicine, such as the use of a wide range of remedial herbs (thyme, oregano, rosemary, sage, mint, basil), has been well and long known for treating common respiratory problems and cold infections. this review reports on the most highlighted polyphenolic compounds present in up to date literature and their specific antiviral perceptive properties that might enhance the body immunity facing covid-19, and other viral infectious diseases. in fact, several studies and clinical trials increasingly proved the role of polyphenols in controlling numerous human pathogens including sars and mers, which are quite similar to covid-19 through the enhancement of host immune response against viral infections by different biological mechanisms. thus, polyphenols ought to be considered as a potential and valuable source for designing new drugs that could be used effectively in the combat against covid‐19 and other rigorous diseases. since the first unveiling of the novel coronavirus in the city of wuhan, hubei province, china in late december 2019, exceptional and unprecedented dares presented by the rapidly rising of covid-19 pandemic health crisis are confronting people worldwide. covid-19, a single-stranded rna virus is actually complicated by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a newly identified virus alike sars-cov and mers-cov-2 that cause the severe acute respiratory syndrome and middle east respiratory syndrome respectively [1] . the main symptoms of covid-19 comprise fever, dry cough, difficulty in breathing, fatigue, mild dyspnoea, sore throat, headache, conjunctivitis and gastrointestinal problems [2] [3] [4] . initially, the pathogen strikes the respiratory tract particularly, the lower respiratory part, leading to a severe lung injury at all ages, subsequently in elderly people and those immuno-compromised individuals in particular, it may lead to a severe pneumonia associated with a systemic and strong inflammation implicated in airway damages, acute respiratory distress syndrome (ards) and subsequently, multi-organ failure and cause fatality [4, 5] . during sars-cov-2 infection, the viral immune response is divided into two main phases: incubation (non-severe stages), and massive destruction (severe stages). in the first phase, a specific adaptive immune response is vital to remove the virus and prevents the initiation of the second phase of virus infection from commencing. therefore, good general health and appropriate genetic background of the host is crucial for the development of an endogenous protective immune response at the incubation and non-severe stages that produces specific antiviral immunity. however, in the case of an impaired immune response (second phase), virus will spread and massive destruction of the affected tissues will occur, followed by an induced innate inflammation in lungs mediated by proinflammatory macrophages, granulocytes, generation of proinflammatory cytokines and chemokines such as il-6 and ip-10, macrophage inflammatory protein 1 such as mip1α, mip1β and mcp1. these inflammatory molecules attract monocytes, macrophages and t cells to the site of infection, promoting further inflammation creating overproduction of pro-inflammatory cytokines, which ultimately damage the lung structure and then the resulting cytokine storm enters the circulation system and reaches other organs, leading to a multi-organ damage [4, 6, 7] . actually, data indicated that activation of the nuclear factor (nf)-κb transcription factor (nf-κb) signaling pathway represents a major contribution to the inflammation induced post sars-cov infection and that nf-κb inhibitors are promising antiviral drugs against infections caused by the virus and potentially other pathogenic human coronaviruses [8] . in this regards, several clinical trials based mainly on anti-oxidant, anti-inflammatory, immunomodulatory drugs and other therapies are conducted to find a possible covid-19 treatment [9] . according to world health organization (who), appropriate nutrition and well-balanced diet are vital to be healthier with stronger immune systems and lower risk of chronic illnesses and infectious diseases. since, the human immune response has often been shown to be weakened by insufficient nutrition in several model systems [10] . therefore, nutrition intervention and therapy need to be considered as an integral part of the approach to patient's victim of covid-19 infection especially among the general health workers [3, 11] . in fact, several epidemiological studies have repeatedly shown an inverse association between the risk of chronic human diseases and the consumption of polyphenolic rich diet [12] . in fact, polyphenols, essential micronutrients in human diet, constitute one of the most numerous and ubiquitous group with more than 8000 polyphenolic compounds identified in the plant kingdom. in the past decades, polyphenols (phenolic acids, flavonoids, stilbenes, and lignans [13] have been intensively studied due to their countless benefits for human health particularly, their strong anti-oxidant and anti-inflammatory proprieties [14] . moreover, the newly emerging diseases such as the novel covid-19 and other infectious diseases that impose grand burden on both human health and economy worldwide, require urgent research for a novel antiviral drug preferably from a natural origin such as polyphenols [15, 16] . the polyphenols antiviral effect is due to the interactions between the phenyl rings and viral proteins and/or rna, or via their capacity to interfere with the host cell defense by regulating map kinase signaling [17, 18] . it is noteworthy that the antiviral effect of polyphenols is not significantly affected by viral mutations due to their binding capacity to the viral envelope lipids or sugar moieties [19] . these enormous features of polyphenols as a natural economical, simple and environmental friendly compounds warrant the birth of this review that highlights the current state of knowledge regarding the most important polyphenolic compounds [20] , their relative importance, their general properties in human health focusing principally on their antiviral activities, and their role in enhancing immunity in order to face the emergence of several infectious diseases such as covid-19 and its related symptoms. quercetin (3,3′,4′,5,7-pentahydroxyflavone) (fig. 1) , is a natural plant flavonoid involved in many plant processes such as pigmentation and protection against bacteria and fungi [21] . quercetin and its glycosylated (quercetin-3-o-β-dglucuronide) forms represent 60-75% of flavonoid intake [22] and it has been found ubiquitously in many fruits and vegetables including apples, berries, brassica vegetables, capers, grapes, red onions, shallots, kale, green tea, cranberries, broccoli, tomatoes, nuts, flowers, barks, leaves and honey [23, 24] . quercetin has been known for its antiviral, anti-inflammatory, anti-carcinogenic and antioxidant properties [25] as well as its exceptional ability to improve mental and physical activities and decrease infection risk [26] . in fact, quercetin is considered as a strong anti-oxidant due to its ability to inhibit lipid peroxidation and thus it protects many body organs from harmful effects of the consequential free radicals [27, 28] . in addition, the general mechanism by which quercetin exerts its antiinflammatory effects is believed to be due to its ability to restrain cox-2 and inos, nf-κb, activator protein-1 (ap-1), and mitogen-activated protein kinase (mapk) usually, associated with the inflammatory response during infections. subsequently, inhibiting the production of pro-inflammatory markers and cytokines (il-1β, il-6, ifn-γ, tnf-α, monocyte chemoattractant protein-1: mcp-1, lipoxygenase: lox) and thus enhancing the antiinflammatory cytokines like il10 [29] . moreover, a study demonstrated that both quercetin and quercitrin could inhibit lps-induced macrophage inflammation and oxidative stress by experiment and theoretical calculations [30] . quercetin blocks in vitro tnfα-mediated inflammation which prevents tnf-α from directly activating extracellular signal-related kinase (erk), c-jun nh2-terminal kinase (jnk), c-jun, and nf-κb, which are well known potent inducers of inflammatory gene expression and protein secretion. quercetin can also indirectly inhibit the inflammatory process by increasing peroxisome proliferator-activated receptor c (pparγ) which is antagonized nf-kb and ap-1 thus inhibiting the inflammatory cascades and subsequently decreases the expression of the inflammatory genes [31] . quercetin has also beneficial immuno-stimulatory effects by inducing the expression of several vital genes and the production of th-1 derived ifn-γ and down-regulating th-2 derived interleukin 4 (il-4). in fact, quercetin is seen as a universal suppressor of the accumulation and activation of immune cells, which prevents chronic inflammation [31, 32] . quercetin relaxes airway smooth muscle and potentiates β-agonist-induced relaxation via dual phosphodiesterase inhibition of purified phosphatidylinositol-specific phospholipase (cplcβ) and phosphodiesterase type 4 inhibitor (pde4) [33] . quercetin has exerted strong anti-pathogenic effects against several causal agents of upper respiratory tract infection in vitro studies [34] . quercetin inverted lung fibrosing in mice and reversed the disease progression normally caused by usual pulmonary aging markers [34] . moreover, it was found to reduce the reactive oxygenated species (ros) produced during viral infection and subsequently decrease pro-inflammatory markers such as il-8, tnf-α, il-1β and il-6 [25] and increases anti-inflammatory cytokines such as il-10 [35] , indicating that it has clear antiviral effects on several respiratory and common cold viruses through its ability to reduce virus imputation, replication and viral load in vitro, as well as lung inflammation and airways hyper-responsiveness in vivo [29] . quercetin was found to be a strong inhibitor of influenza a virus in the early stage of infection [36] and demonstrated significant inhibitory activities against dengue virus type-2 but the mechanisms of how quercetin exerts its antiviral effects are not fully understood [37] . quercetin prevented rhinovirusinduced progression of lung disease in mice with chronic obstructive pulmonary disease (copd) [38] . quercetin-3-β-galactoside, a quercetin derivate, was found to inhibit in vitro a protease essential for viral replication of sars-cov [39] and to block the enzymatic activity of mers-cov 3clpro [40] . moreover, quercetin has been reported to inhibit the proteolytic activity of sars-cov 3clpro [41] from the same family as covid-19. therefore, these properties of quercetin could render it a favorable natural compound to be exploited in the medicinal field due chiefly to its strong antioxidant properties [42] . catechins (fig. 2) [43] . catechins present in many dietary products, medicinal plants, and fruits including apples, blueberries, gooseberries, grape seeds, kiwi, strawberries, green tea, red wine, beer, cacao liquor, chocolate, cocoa. catechins particularly, those found in green tea (mainly egcg and egc) have many favorable properties for human health and were confirmed in vivo and in vitro as an anticancer, anti-obesity, antidiabetic, anti-inflammatory, anti-cardiovascular, anti-infectious, hepatoprotective, and neuroprotective [44] . catchins of green tea have been shown to decrease denaturation of tissue proteins, ros, free radical and no productions. on the contradictory, they were found to increase the production of anti-inflammatory cytokines and antioxidant enzymes [45, 46] . in fact, catchins exert a strong antiinflammatory property due to their ability to activate/deactivate inflammation-related oxidative stress-related cell signaling pathways, such as nf-κb, mapks, transcription factor nuclear factor (erythroid-derived 2)-like 2 (nrf2), signal transducer and the activator of transcription 1/3 (stat1/3). this ability of catchins to interact with those pathways might contribute to their role in inhibiting the infiltration and proliferation of immune-related cells and regulate inflammation and oxidative reactions by lowering of the production of pro-inflammatory cytokines such as il-17, chemokines, adhesion molecules, and inflammatory-related enzymes, like inos and cox-2, augmenting the production of the anti-inflammatory cytokines such as il-10, and suppress many oxidative stress-related pathways responsible for the inflammation processes [43, 47] . several studies have indicated the role of catchins in preventing infectious diseases [48] . regular consumption of green tea has been shown to decrease influenza infection and some cold symptom rates. in addition, gargling with tea catechins protects against influenza infection and common cold [49] . a study has also shown that consuming green tea supplements twice daily for 3 months resulted in a significant decrease in cold or influenza symptoms by approximately 23%. moreover, green tea consumption per day or per week was in reverse to the instance numbers of influenza a or b among school children in japan [50] . catechin compounds were also shown to exhibit high binding affinity to papain-like proteinase (plpro) which is involved in rna sars-cov-2 replication, suggesting the potential effectiveness of these compounds in the treatment of sars-cov-2 [51, 52] . egcg derivatives such as egcg-fatty acid monoesters were found to be as an innovative approach to the prevention of viral infections including emerging fatal viruses, such as ebola virus, sars virus and mers virus [53] . in fact, this action is probably due to their increased affinity for viruses and cellular membranes [54] . catechins as the major content of green tea may be a potential treatment of covid-19, caused by the new coronavirus that is nominated as severe acute respiratory syndrome-related coronavirus sars-cov-2 especially, the breath shortens and other symptoms related to this emerging virus infection. rutin (3,3′,4′,5,7-pentahydroxyflavone-3-rhamnoglucoside) (fig. 3) , is another important naturally occurring flavonoids also known as rutoside, quercetin-3-o-rutinoside, sophorin and vitamin p [55] . rutin, like other polyphenolic compounds is found in several fruits and vegetables such as apple peels, black tea, asparagus, buckwheat, onions, green tea, figs, and most citrus fruit like grapefruit, lemon, lime, apple, berries (mulberry), ash tree fruits, and cranberries. rutin was also found in several african medical plants such as araliaceae, moringaceae, leguminosae, guttiferae, euphorbiaceae, and convolvulaceae [56] . rutin has demonstrated a number of beneficial activities, including antioxidant, anti-inflammatory, cytoprotective, vasoprotective, anticarcinogenic, neuroprotective, cardioprotective, antiasthmatic, antimycobacterial, antifungal and antiviral activities [57] . in fact, rutin exerts its main activities from its ability to inhibit the nuclear factor nfκb, erk1/2 up-regulation of nrf which leads to reduced pro-inflammatory agents such as il-6, tnf-α, vcam-1, icam-1, and e-selectin [58] . a combination of rutin and chloroquine successfully discloses an antimalarial activity in white leghorn chickens infected with plasmodium (bennettinia) juxtanucleare [59] . sodium rutin sulfate, a sulfated rutin modified from the natural flavonol glycoside rutin was revealed in vitro to possess an antiretroviral effect against hiv-1 x 4 viruses iiib, hiv-1 r5 isolates ada-m and ba-l strains by blocking viral entry and virus-cell fusion through interacting with the hiv-1 envelope glycoprotein [60] . methanolic extract of capparissinaica veill, which consists mainly of rutin, was tested for its in vitro antiviral activity against avian influenza strain h5n1 using plaque inhibition assay in madin-darby canine kidney. the test showed a significant inhibition of the virus by about 73% [61] . moreover, rutin, isolated from prunus domestica was suggested as a strong inhibitor of hepatitis c virus (hcv) entry; rutin inhibits the early entry stage of hcv lifecycle by directly acting on the viral particle [62] . based on the above summarized effects of rutin, this flavonoid appears to be a potent component that could be considered in the treatment of several viral infectious diseases. resveratrol (3,5,4′-trihydroxy-trans-stilbene) (fig. 4) , a natural polyphenol non-flavonoid compound produced by several plants in response to injury or invasion by pathogens, such as bacteria and fungi [63] . resveratrol is mainly present in pigments of several fruits and vegetables, for instance rhubarb, blueberries, many red grapes, raspberries, mulberries, peanuts and also in dark chocolate and red wine. resveratrol received intensive studies due to its diverse biological activities, including, antioxidant, anti-inflammatory, antitumor, cardio protective, and antiviral activity, it also possesses other properties such as a preventive and defensive mean towards obesity, diabetes and metabolic syndrome; it can also beneficially regulate the immune system [64, 65] . resveratrol can affect several pathways, which regulate inflammation, immune response, and cellular response to a variety of stimuli [66] . in fact, resveratrol has become a household word in the context of a natural product that has the potential of promoting good health [67] . in fact, several rna virus replications have been shown to be inhibited by resveratrol including the influenza virus and rhinoviruses, which are, the main cause of common colds [68] . resveratrol could be used to prevent respiratory infections in children by a significant reduction in nasal obstruction, rhinorrhea, sneezing, cough, fever and medication usage [69] . respiratory syncytial virus (rsv) which is the main cause of severe, lower respiratory tract infections in infants was found to be improved by resveratrol intake which reduces ifn-γ levels associated with rsv-mediated airway inflammation [70] . moreover, resveratrol was found to be a potential antiviral agent in vitro against mers-cov infection [71] . moreover, stilbene derivatives to which resveratrol belongs were shown to totally inhibit the antiviral activities of sars-cov [72] . due to the numerous beneficial effects of resveratrol and its ability to interact with several cellular pathways, it could be selected as a potential candidate for treatment of covid-19 infection and its related symptoms. kaempferol also named 3,4′,5,7-tetrahydroxyflavone (fig. 5 ) is a widespread natural flavonoids found in a variety of medicinal plants, fruits, vegetables and herbs including grapes, apples, strawberries, tomatoes, broccoli, tea, spinach, beans, kale, leeks, ginkgo biloba leaves, tilia spp, equisetum spp, moringa oleifera, sophora japonica and propolis [73, 74] . kaempferol has a wide variety of pharmacological activities including antioxidant, anti-inflammatory, antimicrobial, anticancer, cardioprotective, neuroprotective, antidiabetic, anti-osteoporotic, estrogenic/antiestrogenic, anxiolytic, analgesic, and antiallergic activities [75] . in fact, many studies demonstrated the beneficial effects of dietary kaempferol in reducing the risk of chronic diseases by augmenting the body's antioxidant defense against free radicals, which reinforces body immunity [73] . kaempferol with its strong antiinflammatory, anti-oxidant properties decreased lipopolysaccharide (lps)-induced tnf-α and il-1β expression by increasing the number of activated macrophages and inhibited nf-κb translocation to the nucleus, which blocks the inflammatory cascade pathway [76] . kaempferol was established to have a high antiviral activity on both influenza viruses, h1n1 and h9n2 where kaempferol acts on the virus neuraminidase protein and its specific functional groups [77] . kaempferol is considered as an effective drug in vitro and in vivo for the potential treatment of h9n2 influenza virus-induced inflammation and lung injury. kaempferol treatment attenuated pulmonary edema, pulmonary capillary permeability, myeloperoxidase (mpo) activity, and the numbers of inflammatory cells. kaempferol reduced ros and malondialdehyde (mda). in addition, kaempferol also reduced of ros, mda, tnf-α, il-1β and il-6 production and significantly inhibited the upregulation of tolllike receptor 4 (tlr4), myeloid differentiation factor 88 (myd88) and phosphorylation of nf-κb and mapks pathways [78] . moreover, kaempferol showed an antiviral activity against influenza a replication and its spread through modulating cell-autonomous immunity through mapk signaling pathways [19] . kaempferol derivatives such as kaempferol glycosides and acylated kaempferol glucoside showed comparable antiviral activities against the 3a channel protein of sars coronavirus that may become expressed in the infected cells. therefore, inhibition of virus production and allowing the infected body to build up or strengthening its own immune system [79] . likewise, a docking study indicated that kaempferol derivatives effectively reduced the proteolytic activity of middle east respiratory syndromecoronavirus (mers-cov) by its ability to occupy the s1 and s2 sites of mers-cov 3clpro [9] . therefore, kaempferol and its derivatives might be selected as alternatives to fight covid-19 infection possibly through targeting coronavirus proteases. apigenin (4′,5,7-trihydroxyflavone) (fig. 6) , a naturally occurring flavone compound is found in large quantities in fruits, vegetables, beverages and medicinal herbs such as parsley, grapes, apples, celery, celeriac, thyme, oregano, basil, chamomile tea, beer, and wine [80] . apigenin encompasses a number of biological functions, for instance strong anti-inflammatory, antioxidant, antibacterial and antiviral activities and blood pressure reduction agent. several in vivo and in vitro studies and clinical trials suggested that apigenin is a potent therapeutic agent to overcome diseases such as rheumatoid arthritis, autoimmune disorders, parkinson's disease, alzheimer's disease, and a various type of cancers [81, 82] . in fact, apigenin stimulates different anti-inflammatory pathways, including p38/ mapk and pi3k/akt, prevents the nuclear translocation of the nf-κb, reduces cox-2 activity and strongly decreases levels of il-6, tnf-α and ifn-γ levels [80] . moreover, the influenza virus h3n2 was found to be inhibited by elsholtzia rugulosa (lamiaceae), a common chinese herb contains apigenin and luteolin among other flavonoids [83] . furthermore, apigenin has shown several antiviral activities against different viruses such as adenoviruses (adv) and hepatitis b virus in vitro, african swine fever virus (asfv), by suppressing the viral protein synthesis and reducing the asfv yield by 3 log, inhibition of viral protein synthesis through suppressing viral ires activity of picornaviruses and disrupting viral rna of enterovirus-71 (ev71) [84] . an anti-inflammatory effect of apigenin-7-glycoside in lpsstimulated acute lung injury via downregulation of oxidative enzyme expression and protein activation through inhibition of mapk phosphorylation and nf-κb pathways was also reported which may be a promising therapeutic candidate for various lung inflammatory disorders, such as lung disease and obstructive pulmonary [85] . apigenin among other flavonoids has been reported to inhibit the proteolytic activity of sars-cov 3clpro. the antiviral effect is presumed to be directly linked to suppress the activity of sars-cov 3clpro [40] and thus apigenin intake, through either a regular diet or supplements, may be beneficial for chronically infected disease such as covid19. luteolin, 3′,4′,5,7-tetrahydroxyflavone ( fig. 7) which belongs to the flavone group of flavonoids, is a natural yellow dye found widely in the plant kingdom including fruits, vegetables, and medicinal herbs such as broccoli, pepper, thyme, and celery, carrots, olive oil, peppermint, thyme, rosemary, oregano, rosemary, navel oranges, dandelion, perilla leaf, chamomile tea [86] . luteolin, like some other flavonoid compounds exerts several pharmacological activities, immuno-modulating, anti-inflammatory, anticancer, antimicrobial, antiviral, anti-oxidant, anti-allergic and neuroprotective activities [86, 87] . luteolin suppressed lps-elicited inflammatory events in mouse alveolar macrophages including cox2, the secretion of the pro-inflammatory agents, tnf-α, il-6 and inos and ros production was blocked by repressing nf-κb and ap-1 activation pathways, suggesting a possible therapeutic application of luteolin for treating lung inflammatory disorders [88] . a protection mechanism of luteolin against acute lung injury induced by lipopolysaccharide in mice was suggested by akt/nfκb inhibition pathway [89] . similar mechanism of luteion protection activity against lipopolysaccharide-induced acute lung injury and respiratory burst involved inhibition of mek/erk and pi3k/akt pathways was suggested in neutrophils [90] . moreover, luteolin inhibited epstein-barr virus (ebv) reactivation by repressing the immediate-early genes zta (zp) and rta (rp) and also inhibited sp1-luc activity in the virus promoters, suggesting luteolin is a potential dietary compound for prevention of virus infection [91] . furthermore, luteolin has been found to be a potent antifibrotic activity as it inhibits lung inflammation and suppresses of myofibroblast differentiation as well as epithelial-to-mesenchymal transition [92] . anti-asthmatic activity of luteolin was also reported in experimental mice [93] . in addition, two small molecules, tetra-o-galloyl-β-d-glucose (tgg) and luteolin, were identified, whose anti-sars-cov activities were confirmed using a wild-type sars-cov infection system. both tgg and luteolin showed at the same concentration levels to effectively inhibit the entry of hiv-luc/sars pseudo typed virus into its host, which suggests a potential clinical use of these two molecules as anti-sars drugs [41, 94] . consequently, luteolin could be selected as an alternative treatment or body immunity enhancer against covid-19 infection due to its strong anti-viral and anti-inflammatory properties during infection. genistein (4′,5,7-trihydroxyflavone) (fig. 8) , is a naturally occurring phytoestrogen found mainly in soy foods and legumes including peas, lentils or beans, fava beans, lupin, tofu, kudzu, psoralea, red clover, coffee and some medical plants such as flemingia vestita and flemingia macrophylla root, [96] . genistein possesses many therapeutic uses against numerous disorders including osteoporosis, cardiovascular diseases, a variety of tumors, diabetes, inflammation, oxidative stress and metabolic syndromes. genistein was successfully used as an immune-suppressive agent both in vitro and in vivo [97] due to its ability to inhibit the infectivity of enveloped or non-enveloped viruses, as well as single or double-stranded rna or dna viruses by different mechanisms. genistein has been shown to reduce the infectivity of a variety of viruses affecting humans and animals, including adenovirus, herpes simplex virus, human immunodeficiency virus, porcine reproductive and respiratory syndrome virus, and rotavirus [98] . an ethyl acetate extract from chongkukjang, a traditional korean fermented product prepared from soybeans, was shown as an effective therapeutic agent to treat influenza a virus infection [99] . moreover, genistein was also effective against the ionic channel of hiv, which is supposed to form a cation-permeable ion channel in the infected cell [105] . genistein may be also a useful candidate for developing a new anti-rotavirus (rv) by inhibiting rotavirus replication and upregulating aquaporin 4 (aqp4) expression via the camp/pka/creb signaling pathway [100] . a study has reported that an increasing consumption of genistein or a diet with moderate to high amounts of soy genistein is associated with a better lung function in patients with asthma [101] . the african swine fever virus (asfv) was inhibited in vitro by genistein through disrupting the viral dna replication, blocking the transcription of late viral genes as well as the synthesis of late viral proteins [102] . it has been demonstrated that pre-treatment of host cells with the kinase inhibitors (genistein and tyrphostin) leads to an inhibition of infection or transduction in cells infected with ebola virus, marburg virus, and lassa virus [103] . therefore, the strong influence of genistein as an antiviral agent against different kinds of virus infections may promote its use as a potential candidate for defeating covid-19 and related symptoms. naringenin (4′,5,7-trihydroxytlavanone) (fig. 9 ) occurs naturally in a variety of citrus fruits, bergamot, tomatoes, tomato food products, grapefruit, sour orange, cherries, cocoa and it is may be found in some herbs such as oregano and water mint. naringenin has many pharmacological effects, including antidiabetic, antiatherogenic, antidepressant, immunomodulatory, antitumor, antiinflammatory, dna protective, hypolipidaemic, antioxidant, antiasthma, antiviral, antibacterial and peroxisome proliferator-activated receptors (ppars) activator [56, 104] . naringenin blocks mapks phosphorylation by decreasing translocation and dna binding of nf-κb and ap-1, which restrain the production of proinflammatory cytokines such as il-33, tnf-α, il-1β, and il-6 [105] . it has been reported that naringenin suppressed respiratory overexpression and eosinophilic airway inflammation in asthma and thus, reduced acute neutrophilic airway inflammation by blocking the nf-κb pathway [106] . naringenin has been found to impair replication of several viruses in human cells such as dengue, zika and chikungunya viruses [107] [108] [109] and to guarantee a significant protection against lpsinduced acute lung injuries through its anti-inflammatory, antioxidant, antinitrosative and antiapoptotic effects [110, 111] . due to its strong anti-inflammatory and anti-oxidant effects, narigenin may be utilized against pneumonia companied with the spread of covid-19. gallic acid (3,4,5-trihydroxybenzoic acid) (fig. 10) , a compound found in several fruits and medicinal plants including gallnuts, grapes, tea, hops, oak bark, sumac, black raspberry, witch hazel and mostly in certain red fruits, black radish, and onions is a phenolic acid that comprises several valuable pharmaceutical properties including antioxidant, anti-inflammatory, antineoplastic, anti-cancer, gastrointestinal and cardiovascular protective, antiviral, antibiotic and antimicrobial. the strong anti-oxidant capacity of gallic acid grants it an important role in absorbing and neutralizing free radicals, with even better results than some vitamins [112, 113] . it was reported that, gallic acid has several antiviral activities such as anti-enterovirus 71 (ev71), anti-herpes simplex virus (hsv)-2 and anti-human immunodeficiency virus activity and anti-hepatitis c (hcv) [114, 115] . gallic acid of black raspberry seeds was demonstrated in vitro to have an antiviral activity against influenza a and b type viruses which suggested a promising role in targeting virus particles [116] . similarly, anti-pandemic potential role of gallic acid against influenza a (h1n1) virus was demonstrated through the down-regulation of adhesion molecules and chemokine to prevent viral attachment and inhibition of the virus mrna replication [117] . a study has reported that inhibition of human rhinoviruses (hrv) production by gallic acid is mainly due to its general role as an antioxidant and the mode of action derived from the inhibition of virus absorption [118] . moreover, gallic acid along with other related phenolic acids (caffeic acid, chlorogenic acid) was found to exert sustainable anti-viral activity against human coronavirus nl63 (hcov-nl63), one of the main circulating coronaviruses worldwide that causes respiratory tract diseases like runny nose, cough, bronchiolitis and pneumonia [119] . based on the above information, gallic acid could be proposed as an alternative treatment for covid-19 and its related symptoms. caffeic acid (3,4-dihydroxycinnamicacid) (fig. 11) is one of the most common phenolic acids, which occurs frequently in fruits, grains, medicinal herbs and dietary supplements. [120, 121] . in vitro and in vivo experiments showed that caffeic acid and its derivatives such as caffeic acid phenethyl ester have numerous physiological activities including antibacterial, antiviral, antioxidant, anti-inflammatory, anti-atherosclerotic, immunostimulatory, antidiabetic, cardioprotective, antiproliferative, hepatoprotective, anticancer, and antihepatocellular carcinoma activity [121, 122] . caffeic acid of sambucus formosana nakai ethanol extract significantly repressed the replication of human coronavirus nl63 (hcov-nl63) that causes upper respiratory tract illnesses, in a cell-type independent manner, and blocked virus attachment. the study proposed that caffeic acid could be the vital component with anti-hcov-nl63 activity [119] . in vitro caffeic acid demonstrated an antiviral activity by acting on severe fever with thrombocytopenia syndrome (sfts) virus, which causes tick-borne hemorrhagic fever in east asia, and hinders viral infection and spread by inhibiting the binding of sftsv to the cells [123] . conjugation of peramivir, an influenza inhibitor with caffeic acid has showed a better drug efficacy against influenza and was found to inhibit influenza neuraminidase [124, 125] . moreover, a study has reported antiviral activity of caffeic acid towards herpes simplex (hsv), vsv-ebola pseudotyped and vaccinia viruses and that antiviral activity increased and occurred early in the virus replication cycle with the addition of chelated inorganic ions or a metal such as iron to caffeic acid [126] . caffeic acid and its derivatives have uncountable antiviral activities that may be used against the spread of emerging covid-19 and its related symptoms. daidzein (4′,7-dihydroxyisoflavone) (fig. 12) is a naturally occurring isoflavone compound found in high concentrations in soybeans, soy products like tofu and textured vegetable protein, red clover and also in some herbs such as vitex agnus castus and black cohosh. it is also present in many other vegetables, fruits, nuts, peas, lentils, various cereals, bakery products, milk, meat and other food products [127, 128] . daidzein is classified as a phytoestrogen because it has oestrogen-like properties; with its partner genistein, daidzein compromises 90% of the intake of oestrogenic isofalvones from the diet [127, 129] . daidzein has several biological and pharmacological properties for instance antioxidant, anticancer, anti-inflammatory, neuroprotective, protective treatment of cardiovascular diseases, and autoimmune diseases [130] . soy isoflavones consumption such as genistein, daidzein, and glycitein has been associated with a reduced prevalence of chronic health disorders and regulation of the immune response, which give them an important potential role in clinical applications in immune-dysfunction [131] . daidzein markedly attenuated tnf-α-induced lung inflammation and lipopolysaccharide-induced acute lung injury through blocking of nf-κb pathway via different interaction mechanisms, which inhibited several pro-inflammatory markers such as cxcl2 in lung tissues [132] . moreover, a study has reported that daidzein exhibits an anti-fibrotic effect against bleomycin (blm) induced pulmonary fibrosis (pf) in rats [133] . furthermore, during influenza infection, reactive oxygen species (ros) and lipid peroxides are generated in several tissues such as the lung, and daidzein was reported to exert an antiviral activity through its antioxidant capacity to decease ros and lipid peroxide generations. consequently, daidzein regulates influenza virus replication via signal transduction through 5-lipoxygenase products induced by daidzein [134] . also, novel daidzein analog compounds synthesized based on structural modifications of daidzein have been demonstrated in vitro a selective antiviral agent against h1n1 tr influenza viruses [135] . due to their role in the regulation of immune responses and antioxidant activity, daidzein could be applied to enhance body immunity against emergence diseases such as covid-19 and related immunity dysfunctions. chrysin (5,7-dihydroxyflavone, also known as vitamin p) (fig. 13) chrysin exhibits many biological activities and pharmacological effects, including antioxidant, anti-inflammatory, anticancer, pro-apoptotic, antiangiogenic, antimetastatic, immunomodulatory, antiasthmatic, antibacterial and antiviral activities [136, 137] . actually, chrysin exerts its activities against various diseases by different mechanisms including the suppression of inducible nitric oxide synthase (inos) and nf-κb, inhibition of histone deacetylase and dna topoisomerases, inhibition pro-inflammatory cytokine expressions such as tnf-α and il-1β through blocking histamine release and also decreasing matrix metalloproteinase-2 (mmp-2) expression [138] . chrysin could also prevent airway inflammation by inhibiting the release of proinflammatory mediators tnf-α, il-1β, il-8, and myeloperoxidase (mop) expression via restraining erk and p38 pathway [136, 138] . chrysin has been shown to be a potent inhibitor of human immunodeficiency virus (hiv) and also has been shown to inhibit the enterovirus a71 (ev-a71) replication [139, 140] . moreover, it has been reported that chrysin inhibited the interaction between sars-cov spike (s) protein and angiotensin-converting enzyme 2 (ace2) which is the functional receptor for sars-cov [52, 141] . consequently, this huge capacity of chrysin in protecting against several diseases especially virus infections may give it a vital role in the fight against covid-19 principally, via its ability to interact with the virus receptor. ferulic acid (4-hydroxy-3-methoxycinnamicacid) (fig. 14) is a phenolic acid generally found in plant cell walls such as whole grains, spinach, parsley, grapes, rhubarb, coffee and cereal seeds, mainly wheat, oats, rye, rice barley, pineapple, maize bran, some fruits such as eggplant, orange and grapefruit, vegetables such as tomato and carrot, flax, beets, broccoli and sweet corn [142, 143] . ferulic acid is rapidly absorbed into the body and stays in the blood stream longer than any other antioxidant, even longer than vitamin c. because of these features, ferulic acid is considered to be a superior antioxidant and therefore it is widely used in health foods and nutrition [144] . in addition, ferulic acid possesses many physiological activities such as anti-inflammatory, antimicrobial, antiviral, anticancer, antiarrhythmic, antithrombotic, antidiabetic, lipid peroxidation prevention, free radical scavenger; increase no synthesis and immunostimulant properties [142] [143] [144] . sodium ferulate, the sodium salt of ferulic acid, in a combination with oxymatrine had a protective effect on lps-induced acute lung injury in mice principally by inhibiting the production of crp and tnf-α, which inhibited the myeloperoxidase (mpo) activity in lung homogenate and attenuated inflammatory cell numbers and protein concentration in the bronchoalveolar lavage fluid. similarly, sodium ferulate protects against influenza virus infection through activation of the tlr7/9-myd88-irf7 pathway, which recognizes viral nucleic acids and activates different cascades that contribute to the production of interferons (ifns) and also by inhibiting the nf-κb pathway, which resulted in blocking the influenza virus replication [145, 146] . moreover, a ferulic acid derivative had the best inhibition activity against the neuraminidase (na) influenza virus (h1n1), in vitro, due to the interactions with conserved and essential residues of na, which is comparable to those of oseltamivir and zanamivir, two available commercial na inhibitors [147] . the presented properties of ferulic acid and its derivatives sodium ferulate solely or in a combination with other compounds may warrant their use in the fight against such emergence disease similar to covid-19. hesperetin (3′,5,7-trihydroxy-4′-methoxyflavanone) ( fig. 15 ) a naturally occurring flavone is found predominantly in citrus fruits, lemons, sweet oranges, bitter orange, and grapefruit juices. in addition, it occurs in a large number of fruits and vegetables such as tomatoes and cherries, and various herbal formulations [148] . hesperetin and its metabolites display several biological activities including antioxidant, anti-inflammatory, and lipid decreasing levels, anticarcinogenic, antidiabetic, immunoregulatory, and neuroprotective [149, 150] . actually, several in vitro and in vivo studies have been reported that hesperetin, its metabolites, and its synthetic derivatives augmented the antioxidant cellular defenses such as the erk/nrf2 signaling pathway and peroxisome proliferatoractivated receptor-γ (ppar-γ), reduced inflammatory targets including nf-κb, inos, and cox-2, and other markers of chronic inflammation such as il-1β, il-6, no and tnf-α [151] [152] [153] . hesperetin has been shown a potential drug for acute lung injury (ali) induced in vivo by lipopolysaccharide by downregulated the toll-like receptor 4 (tlr4) and suppressed nf-κb activation in lung tissue [154] . hespertein plus naringenin has a protective capacity against lung fibrosis by reducing airway inflammation in murine chronic asthma model. in addition, the anti-fibrotic effects of hespertein have been indicated in the liver and kidney [155, 156] . hespertin exerted inhibitory effect on the intracellular replication of transmitted chikungunya virus (chikv) and exhibits drug-like properties which maybe a potential as a therapeutic option for chikv infection [157] . moreover, hesperetin has shown an antiviral activity against the human respiratory syncytial virus (hrsv) trough binding to m2-1 virus protein, which is an important transcriptional anti-termination factor and a potential target for viral replication inhibitor development [158] . among several compounds that exhibit an in vitro activity against sars-cov, hesperetin, was the most selective with a selectivity index of ∼300. hesperetin dose-dependently suppressed the cleavage activity of the 3c-like protease (3clpro) of sars-cov in cell-free and cell-based assays [159] . furthermore, a recent study has reported that hesperetin has the potential to inhibit angiotensin-converting enzyme 2 (ace2), the same host receptor of sars-cov and thus block infection with sars-cov-2 [160] . therefore, these studies clearly suggest that hesperetin could play a key role in the prevention and treatment of covid-19 and related pneumonia. vanillic acid (4-hydroxy-3-methoxybenzoicacid) (fig. 16) , an intermediate in the production of vanillin from ferulic acid is the main flavor component of cured vanilla beans. vanillic acid is found in the roots of angelica sinensis, a plant used in traditional chinese medicine, açaí oil, olive oil vinegar and wine [161, 162] . vanillic acid is a well-known generally regarded as a safe flavoring agent with beneficial biological activities such as anti-oxidant, anti-lipid peroxidative, anti-inflammatory, anti-apoptosis, neuroprotective/cognitive and regulation of insulin secretion [163, 164] . several studies have reported the efficiency of vanillic acid in controlling the immune response or the inflammatory ones. vanillic acid improved the activity of human lymphocyte proliferation and secretion of interferon-gamma (infγ) in human peripheral blood mononuclear cells. additional study indicated that vanillic acid has a hepatoprotective effect through its suppressive action on immune-mediated liver inflammation in concanavalin a-induced liver injury [165, 166] . vanillic acid was found to inhibit inflammatory state in mice by suppressing neutrophil recruitment and its mechanisms of action involves antioxidant effects and nfκb-related inhibition of pro-inflammatory cytokine production such as il6, cox2, il-1β and tnf-α [167, 168] . moreover, the major compounds extracted from the root of rubia cordifolia (xanthopurpurin and vanillic acid), traditionally used as a hemostatic agent inhibited effectively rotavirus multiplication by promoting virus-induced apoptosis in ma-104 cells [169] . consequently, vanillic acid may have a role in the combat against pneumonia related covid-19 infection. galangin (3,5,7-trihydroxyflavone) (fig. 17) , is a flavonoid compound found in high concentrations in alpinia officinarum hance (lesser galangal), which has been used as an herbal medicine for colds, stomachaches, and swellings, or as a food additive in asia. galangin, which has been used as a cure for many symptoms, particularly in china is also found in honey, propolis and helichrysum aureonitens [170] . several epidemiological studies in vitro and animal studies have claimed that galangin and the consumption of galangin-containing foods may affect several diseases [171] . galangin has a broad range of biological properties, including anti-oxidative and free radical scavenging, modulating enzyme activities, suppressing chemical genotoxicity, inhibitory effects on various microbes, the control of hypertension, diabetes, and chemoprevention of several cancers anti-inflammatory and anti-fibrotic activities in various disorders [172] [173] [174] . galangin attenuated in vitro and in vivo airway remodeling by inhibiting tgf-β1-mediated ros generation and mapk/akt phosphorylation in asthma; also, galangin improved ovalbumin (ova)induced airway inflammation by inhibiting nf-κb pathway, which reduced eosinophils, neutrophils, and lymphocytes infiltration and goblet cell hyperplasia. in addition, galangin reduced expression of inos, vcam-1, tnfα induced p65 nuclear translocation, expression of monocyte chemoattractant protein-1 (mcp-1), eotaxin, and c-x-c motif chemokine 10 (cxcl10) levels in lung tissue [175, 176] . in addition, galangin reduced lps-induced acute lung injury by inhibition of inflammation and oxidative stress. in fact, protective effects of galangin were associated with inhibition of nf-κb and upregulation of heme oxygenase (ho)-1. similarly, galangin exerted profound anti-asthmatic property by activating pparγ, which resulted in galangin alleviating airway inflammation in murine model of asthma. [176] [177] [178] . furthermore, at concentrations ranging from 12 to 47 µg/ ml, galangin isolated from the aerial parts of helichrysum aureonitens showed a significant antiviral activity against herpes simplex virus type 1 (hsv-1) and coxsackie b virus type 1 (coxb1) limited activity against reovirus, but no antiviral activity against adenovirus type 31 (ad31) [179] . the anti-inflammatory and anti-oxidant capacity of galangin may give it a role in the pneumonia associated with covid-19 and other emergence diseases. p-coumaric acid (4-hydroxycinnamicacid) (fig. 18) , is a phenolic compound and the most frequently occurring isomer of coumaric acid in nature. p-coumaric acid classified as a phytochemical and nutraceutical, presents in vegetables, and fruits, such as cranberry syrups, grape juices, tomatoes, apple, peanuts, carrots, garlic, potatoes, onions, beans, and cereals such as rice maize, oats and wheat, tea, beer and chocolate. p-coumaric acid and its conjugate have demonstrated several biological activities including antifungal, anti-cancer, antimicrobial, anti-viral, anti-melanogenic, antioxidant, immunomodulatory and anti-inflammatory effects, antiplatelet aggregation, anxiolytic, antipyretic, analgesic, anti-arthritis and antigenotoxicity [180] [181] [182] . in vitro and in vivo experiments showed that p-coumaric acid attenuated lipopolysaccharide-induced lung inflammation by scavenging ros production and modulating the oxidative stress under inflammatory conditions in lung injury [180] . moreover, adenostemma lavenia is a permanent medicinal herb belonging to the compositae family and is widely distributed in the tropical parts of asia mainly in taiwan. it is used to treat pulmonary congestion, pneumonia, bacterial infections of the respiratory tract, edema, and inflammation. p-coumaric acid was found to be the major constituent of this herb. in fact, adenostemma lavenia has been reported to ameliorate acute lung injury through activating ampk/nrf2/ho-1 signaling pathway and improving the anti-oxidant response [183] . p-coumaric acid among other compounds extracted from the himalayan medicinal plants traditionally used to treat bronchitis and related symptoms has demonstrated antiviral activities in vitro against different kinds of human rhinoviruses [184] . likewise, in vitro research showed that p-coumaric acid among other phenolic acid components extracted from propolis could be effective against herpes simplex virus [185] . furthermore, a recent docking study has reported that cis-p-coumaric acid found in coriander, the cis-form of p-coumaric acid, among other natural products may interfere with sars-cov-2 attachment to the host cell and can be successfully considered as anti-covid-19 agents for people with a high risk of cell stress like elders, cancer patients, and front-line medical staff. similarly, another docking study showed that p-coumaric acid, curcumin and their boronic acid derivatives are probable inhibitors of endoribonuclease nsp15 encoded by middle east respiratory syndrome coronavirus (mers-cov) [186, 187] . consequently, p-coumaric acid and its derivatives may be used as target for an alternative therapy of covid19 and its related pneumonia. eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) (fig. 19) , is a natural flavonoid isolated mainly from lyonia ovalifolia and yerba santa (eriodictyon californicum). eriodictyol is also detected in some plants of the lamiaceae family, such as peppermint (mentha piperita), oregano (origanum vulgare), and thyme (thymus vulgaris) and it is ubiquitous in fruits, vegetables, including tomatoes, mint, grapefruit, oranges, and lemons as well as in several medicinal plants such as bauhinia ungulata, arcytophyllum thymifolium, elsholtzia bodinieri, and clinopodium chinense [188, 189] . several studies have revealed that eriodictyol possesses several bioactivities, including anti-inflammation, anti-oxidation, antimicrobial, anticancer, protective effects on the neurons, kidneys, and lungs, insulin secretagogue and antidiabetic properties [190, 191] . furthermore, several studies have shown that eriodictyol exerts its anti-inflammatory and antioxidant effects through akt/nf-κb-related signaling pathways [192] . eriodictyol has immunomodulatory effects, comprising inhibition of nitric oxide (no) production by blockage of nf-κb activation and phosphorylation of p38, mapk, extracellular signal-regulated kinase 1 and 2 (erk1 and 2), jnk and cox-2 in lipopolysaccharide (lps)-induced inflammatory responses in macrophages [193] . moreover, a study demonstrated that eriodictyol could alleviate the lps-induced lung injury in mice by regulating the nrf2 pathway and inhibiting the expression of inflammatory cytokines in macrophages such as tnf-α, il-6, il-1β and mip-2, suggesting that eriodictyol may be used as a drug for the treatment of lps-induced lung injury. in addition, its anti-inflammatory and anti-oxidative capacities have drained attention to its therapeutic potential, especially, for asthma and treating colds [194, 195] . a recent study has reported that (2s)-eriodictyol 7-o-(6″-o-galloyl)-beta-d-glucopyranoside, aneriodictyol derivative, ranked as an inhibitor against sars-cov-2 3clpro receptor binding site and catalytic dyad (cys-145 and his-41) of sars-cov-2 3clpro [196] . a recent docking study has ranked eriodictyol among other compounds to be tested experimentally as an inhibitor of ace2 receptor of covid-19 [197] . these studies suggested that natural products such as eriodictyol may prove more useful candidates for covid-19 drug therapy. syringic acid (4-hydroxy-3,5-dimethoxybenzoicacid) (fig. 20) , is a phenolic acid found in many plants and dry fruits such as, dates and walnuts, olives, spices, pumpkin, grapes, acai palm, honey, red wine; cereals and other plants such as gall trees, proso millet, radish, chard and sugar apple [198, 199] . syringic acid exhibits several positive effects on human health including anti-diabetic, hepato-protective, anti-oxidant, anti-endotoxic, anti-steatotic, anti-inflammatory, anti-hypertensive, neuro-protective, anti-cancer and cardio-protection activities [198] [199] [200] . in fact, syringic acid possesses these beneficial effects on human health due to its strong anti-oxidant nature. syringic acid exerts its activities via modulation of several molecules involved in disease progression such as proteins, transcriptional factors and growth factors [200] . syringic acid identified, with other secondary metabolite compounds, in extracts of stems and leaves of bougain villeam, which it is used as a tea for easing cough, fever, sore and diarrhea besides its several beneficial activities such as antioxidant, anticancer, antibacterial, antiviral [201] . a study has reported in asthma mice model that the effect of syringic acid is prominent in the treatment of asthma by controlling the accumulation of inflammatory cells, other inflammatory markers such as il-4, il-5, il-13, and tnf-α along with enhancement of antioxidant markers, suppression of ros and controlling airway hyper-reactivity. therefore, syringic acid may be recommended for clinical trials in the treatment of asthma [202] . moreover, syringic acid has shown among other compounds, to be a potent inhibitor against h1n1 swine influenza [203] . due to its strong antioxidant capacity, synergic acid, may be used to strengthen lung and body invulnerability in front of covid-19 infections. polydatin (3,4′,5-trihydroxystilbene-3-β-d-glucoside) (fig. 21) , also named piceid is a natural resveratrol glucoside and precursor. polydatin, an active compound isolated from the root of polygonumcus pidatum sieb. et zucc, has been widely used for treatment of hyperlipemia, inflammation, infection and cancer, fever, cough, hypertension and other pharmacological benefits. polydatin is also detected in grape, peanut, hop cones, red wines, hop pellets, cocoa-containing products, chocolate products and many daily diets [204, 205] . unlike resveratrol, which passively penetrates cells, polydatin enters cells via an active mechanism using glucose carrier and it is more resistant to enzymatic oxidation than resveratrol and possesses much better solubility in water [206] . polydatin has numerous benefits which is widely reported including anti-inflammatory effect in chronic lung diseases, anti-oxidative, anti-platelet aggregative, anti-fibrosis, anti-cancer, benefits for neurological diseases and anxiolytic effects [207, 208] . a study has demonstrated that polydatin improves lipopolysaccharide (lps)-induced acute respiratory distress syndrome (ards). in addition, polydatin has mediated parkin-dependent mitophagy and protects against mitochondria-dependent apoptosis in ards [209, 210] niae (mp) infection, mp can infect both the upper and lower respiratory tracts, was found to be suppressed by polydatin treatment through inhibition of the nacht domain-, leucinerich repeat-, and pyd-containing protein 3 inflammasome (nlrp3) and the nuclear factor-κb pathway after mp infection [211] . actually, several in vivo studies have reported an important protective role of polydatin treatment via different mechanisms in ovalbumin-induced bronchial asthma as well as in lps-induced acute lung injury [212, 213] . moreover, polydatin protects the respiratory system from the damage caused by fine particulate matters (aerodynamic diameter < 2.5 μm; pm2.5) during air pollution that may cause deleterious effects such as premature death among individuals due to lung disease, lung dysfunction and asthma exacerbation, mainly by the capacity of polydatin to reduce the injury from oxidative stress and inflammation in lung tissues [214] . moreover, polydatin can attenuate hypoxic pulmonary hypertension through protein kinase c mechanisms (pkc) and it might be a promising candidate for hypoxic pulmonary treatment [215] . polydatin was found, among other phenolic compounds extracted from of polygonum cuspidatum, to inhibit influenza a virus replication in a549 cells through toll-like receptor 9-induced (tlr9) interferon beta (ifn-β) expression [216] . a recent docking study has reveal that polydatin, is a potent covid-19 main protease (mpro) inhibitor, among other natural phenolic compounds that have been demonstrated to have an anti-coronavirus activity [217] . therefore, regarding the enormous protective roles of this natural phenolic compound on the respiratory system, it is concluding that polydatin could be consecrated as a potential molecule against covid-19 infections and its related symptoms. neobavaisoflavone (7-hydroxy-3-[4-hydroxy-3-(3-methylbut-2-enyl) phenyl] chromen-4-one) (fig. 22) , is a flavonoid compound isolated mainly from psoraleacorylifolia (leguminosae) and erythrina species. psoralea corylifolia has traditionally been used in india, china and southeastern asian countries for the treatment of several diseases such as nephritis, asthma, cough, osteoporosis, hypertension and cardiovascular diseases [118, 119] . psoralea corylifolia contains, other than neobavaisoflavone, several active compounds including psoralen, isopsoralen, psoralidin, isobavachalcone, bavachin and bakuchiol [220] . moreover, psoralea corylifolia seeds have been reported to possess a high activity against the sars-cov papain-like protease (plpro) which is the main enzyme involved in sars virus replication [221] . in fact, six phenolic compounds with an antiviral activity were isolated from the ethanolic extracts of psoralea corylifolia identified as, bavachinin, neobavaisoflavone, isobavachalcone, 4′-o-methylbavachalcone, psoralidin and corylifol a. isobavachalcone and psoralidin were the greatest antiviral inhibitors of sars-cov plpro [222] . actually, neobavaisoflavone has numerous biological properties such as antibacterial, anti-fungal, anti-inflammatory, antioxidative, anti-cancer, anti-osteoporosis and anti-platelet aggregation [223, 224] . in vitro, neobavaisoflavone significantly inhibited the production of ros, reactive nitrogen species (rns) and cytokines: il-1β, il-6, il-12p40, il-12p70, and tnf-α in lps+ifn-γ-or pma-stimulated raw264.7 macrophages [223] . actually, psoralea corylifolia, which contains neobavaisoflavone and other phenolic and non-phenolic compounds maybe used as a target for a more focused research against covid-19 and related symptoms, since its extract was found to inhibit a human coronavirus strain [224] . natural compounds such as polyphenols with their countless properties seem to be a simple, safe and economical approach due to their numerous beneficial effects on human chronic diseases as well as on several bacterial and viral infectious diseases through their favorable effects on several cellular and molecular pathways, which leads to an enhanced body immunity against infection. this review summarized the most studied polyphenolic compounds, their main natural origin, their general role in human health focusing particularly on their role as an antiviral agent against respiratory tract infections and their perceptive role in the fight against covid-19, related symptoms and other emergence diseases. better design of experimental as well as human clinical studies addressing dosage and combinations of polyphenol compounds, their derivatives and other synthetized molecules are required to substantiate the benefits of such wonderful natural compounds for therapeutic and preventive purposes against infections. open access this article is licensed under a creative commons attribution 4.0 international license, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the creative commons licence, and indicate if changes were made. the images or other third party material in this article are included in the article's creative commons licence, unless indicated otherwise in a credit line to the material. if material is not included in the article's creative commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. to view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/. structure of neobavaisoflavone effective inhibition of mers-cov infection by resveratrol studies in natural products chemistry chapter 34 key: cord-272695-wmzq4lkh authors: ahmed, ahmed a.; rasheed, zafar; salem, tarek; al-dhubaibi, mohammed s.; al robaee, ahmad a.; alzolibani, abdullateef a. title: tnf-α − 308 g/a and ifn-γ + 874 a/t gene polymorphisms in saudi patients with cutaneous leishmaniasis date: 2020-05-13 journal: bmc med genet doi: 10.1186/s12881-020-01043-9 sha: doc_id: 272695 cord_uid: wmzq4lkh background: cutaneous leishmaniasis (cl) is well linked with immunogenetic factors. this study was undertaken to test the association of tnf-α − 308 and ifn-γ + 874 gene polymorphisms with the susceptibility of leishmania (l) species among cl patients in central region of saudi arabia. methods: this is a case-control study involved 169 saudi subjects with different l. species and 199 healthy controls from central region of saudi arabia. all subjects were characterized by tnf-α − 308 g/a and ifn-γ + 874 a/t gene polymorphisms using pcr. results: evaluation of genotyping and allelic frequency of tnf-α − 308 g/a in different l. species showed no significant association compared to controls (p > 0.05). except, in cases of l. tropica that showed significantly higher tnf-α − 308 a versus g allele frequency (p = 0.0004). evaluation of genotyping of ifn-γ + 874 (tt versus aa+at recessive) and allelic frequency of ifn-γ + 874 (t versus a) showed significant higher in l. major and also in total cl cases as compared to healthy controls (p < 0.05). furthermore, a strong association was observed between the susceptibility of l. major, l. tropica or total cl cases with synergistically combined high tnf-α 308/inf-γ 874 alleles. conclusions: this is the first report that shows the gene polymorphisms of tnf-α − 308 g/a and ifn-γ + 874 a/t in saudi patients with different l. species infections. data showed that the tnf-α-308 g/a gene polymorphism is not associated with the susceptibility of cl in saudi subjects. the only correlation was found in between a versus g allelic frequency in l. tropica. importantly, ifn-γ + 874 a/t polymorphism was found to be associated with the susceptibility of l. major and also with total cl subjects. moreover, data from synergistically combined high tnf-α 308/inf-γ 874 alleles strongly suggest their potential role in the susceptibility of leishmania infection. leishmania is a one of the most common forms of trypanosomiasis protozoan, which is endemic throughout the tropical and subtropical regions of the globe [1] . cutaneous leishmaniasis (cl) is the most frequent leishmania infection, known to induce dermal lesions that most likely produce permanent life-long scars. now it is well known that cl infection is primarily caused by the transmitting the parasites from the female phlebotomine sandflies bite [1] . etiology of cl infection dependents on the number of factors such as the characteristics of the parasite, type of sand-fly species, the ecological regions, current/former exposure of infection, and also on the human behavior [2, 3] . it is now well documented that the leishmania has more than 22 different species but their prevalence varies from region to region [3] . these l. species have a wide geographic distribution from south/north america to all over middle east with a significant increase of number of patients in last few years in the countries such as bolivia, brazil, peru, colombia, afghanistan, iran, syria, algeria and also in saudi arabia [2] [3] [4] [5] . furthermore, the distribution of l. species or cl also reported to have an association with the number of environmental factors including temperature and humidity. moreover, the urbanization and migration have also been documented to have an association with the global diffusion of leishmania infection [1, 4] . in saudi arabia, despite of the several important efforts taken by the government, but cl remains to be a major heath issue of the country. several studies have shown that the l. major and l. tropica are the principal reservoirs of this infection, as these species are distributed in almost all region of the country [5] . reports have also shown that the phlebotomine sandflies are commonly found in the desert regions around the farms, which assumed to be one of the responsible factors for the exposure of this parasitic infection among saudi population [5] . it is now well established that the proinflammatory cytokines play several crucial roles in onset of cl, which varies from the resistance or the host immune response to infection, especially tumor necrosis factor alpha (tnf-α) and interferon-gamma (ifn-γ) [6] . the role of tnf-α and ifn-γ was well reported in l. major as both of these cytokines are produced by t helper 1 (th1) cells, which are synergistically activates macrophages [7] . furthermore, an association of tnf-αor ifn-γ with the increase of tissue damage or ulceration was also reported in cl patients [8] . moreover, the role of th1 response against l. major infection was also demonstrated in animal model of l. major [9, 10] . out of all studied th1 cytokines, tnfα and ifn-γ were found to be critical for the initiation of preventive immunity against all studied l. species, particularly l. major [10] [11] [12] [13] . tnf-α-308g/a polymorphism was well studied in several autoimmune/inflammatory diseases, the a allele of tnfα (308g/a) was reported to have higher transcriptional activity by 6-7 folds as compared with the common g allele [14, 15] . therefore, the a allele frequency has been implicated in the pathogenesis of several infectious as well as autoimmune disorders [14, 15] . on the other hand, ifn-γ-874 a/t polymorphism was also reported to have a genetic link with several autoimmune/inflammatory disorders through + 874 t allele carriers [16, 17] . however, it is still unclear whether tnf-α − 308 g/a or ifn-γ + 874 a/t polymorphisms play a role in the leishmania infection. in view of these, we hypothesized that tnf-α-308 g/a and ifn-γ + 874 a/t gene polymorphisms may be associated with l. species infection. to test this hypothesis, dna samples were isolated from cl patients and the association between tnf-α-308 g/a or ifn-γ + 874 a/t gene polymorphisms with the l. species susceptibility was studied. the study was carried out in accordance with the code of ethics of the world medical association (declaration of helsinki as revised in tokyo 2004) for humans and the study protocol was approved by national plan for science, technology and innovation of saudi arabia (nstip/kacst # 11-med1068-09). with the institutional review board (irb) approval, the participants were recruited from the clinical units and the qassim hospitals, ksa. all patients were recruited after thorough examination based on the patients clinical appearance, microscopic and specific pcr-based observations as described previously [18, 19] . briefly, patients with compatible lesions (range table 1 . genomic dna was extracted from the lymphocytes of peripheral blood from the patients and controls by the magna pure lc instrument (roche diagnostics gmbh, roche molecular biochemical, mannheim, germany) as described previously [20] . the purity and quantity of dna was measured the by absorbance ration 260/280 as described previously [21] . the genetic variants of tnf-α 308 g/a (rs1800629), and ifn-γ 874 t/a (rs2430561) polymorphisms were amplified by the pcr based amplification refractory mutation system (arms-pcr) using the primers sequence summarized in table 2 . the arms-pcr for the tnf-α − 308 g/a gene polymorphism was performed two times for all subjects including patients and healthy human controls. one time the arms-pcr was performed by using common forward primer (5`-tctcggtttcttctccatcg-3`) and reverse primer represented for g genotype (5`-ataggt tttgaggggcatgg-3) and the second pcr was performed by the same common primer with the reverse primer that represented a genotype (5`-ataggt tttgaggggcatga-3`). detection at the correct size band with reverse primer g that means the homozygote genotype gg, whereas detection of the same size band when repeated with reverse primer a that means the homozygote genotype aa. if detected the correct band from the first run with g and also with the second run with a for the same patients, that means the genotype represents heterozygote ga. similarly, the ifn-γ + 874 a/t gene polymorphism was validated by calculation homozygote tt or gg alleles and heterozygote ta allele. the pcr amplification was carried out using primus ht dual thermal cycler pcr (mwg ag biotech, usa) in a 25 μl total volume containing about 100 ng of genomic dna template, 1 μm of each primer, 1 × go taq® green master mix (promega). the initial denaturation in pcr was carried out at 95°c for 2 min followed by 30 cycles of 95°c for 15 s, 65°c for 50 s, and 72°c for 60s. after pcr running, all amplified pcr products were electrophoresed on 2% agarose gel and fig. 1 tnf-α − 308 g/a polymorphism in cl patients. lane m indicates for dna ladder 100 bp, lanes 1 and 1'represent ga genotype for sample 1, lanes 2 and 2`represent ga for sample 2, lanes 3 and 3`represent gg genotype for sample 3, lanes 4 and 4`represent aa genotype for sample 4, lanes 5 and 5`represent gg genotype for sample 5 and lanes 6 and 6`represent gg genotype for sample 6. all tnf-α − 308 g/a genotype polymorphism detected at the same size (184 bp) by the amplification refractory mutation system pcr products were visualized under uv light using ethidium bromide stain. data were analyzed using the statistical software program spss version 17. the frequencies of studied genotypic and allelic polymorphisms among cases were compared to those of controls using fisher's exact test and odds ratio (or) with the 95% confidence interval (95% ci). a p level of < 0.05 was considered significant. the amplified pcr product for tnf-α-308 was detected at 184 base pair as shown in fig. 1 , based on these results, different species of cl, the genotypes and the alleles of the host genes polymorphism were determined and evaluated in comparison with their respective healthy controls (supplementary file (table 3 ). in contrast to l. 1.7) ]. the complete details of tnf-α 308 g/a polymorphism in different l. species and healthy human controls are summarized in table 3 . the amplified pcr product for ifn-γ + 874 were detected at 263 base pair as shown in fig. 2 , based on these results, different species of cl, the genotypes and the alleles of the host genes polymorphism were determined and evaluated in comparison with their respective healthy controls. results show in table 4 table 4 . to further re-evaluate the potential role of genotype frequencies of tnf-α 308 g/a and inf-γ + 874 a/t in the susceptibility of cl infection among the studied saudi patients, the data were analyzed by the synergistically combined genotype frequencies of tnf-α 308 g/a and inf-γ + 874 a/t in all cl patients and normal human controls. analysis from the synergistically combined tnf-α 308 g/a and inf-γ + 874 a/t genotype showed a stronger association of the high tnf-α/inf-γ genotype with the susceptibility of l. ] respectively. the complete details of the synergistically combined genotype frequencies of tnf-α-308 g/a and inf-γ 874 a/t in all studied l. species and human controls are summarized in table 5 . these results further support a potential role of tnf-α-308 g/a and inf-γ 874 a/t in the pathogenesis of cl infection among the studied saudi population. this study determined the association of the two important gene polymorphisms in saudi patients with cutaneous leishmaniasis. in one of our previous studies, we reported the prevalence of l. species among cl patients in qassim area of saudi arabia [5] . our reported data fig. 2 ifn-γ + 874 a/t polymorphism in cl patients. lane m indicate dna ladder 100 bp, lanes 1 and 1'represent aa genotype for sample 1, lanes 2 and 2`represent at genotype for sample 2, lanes 3 and 3`represent tt genotype for sample 3, lanes 4 and 4`represent at genotype for sample 4, lanes 5 and 5`represent aa genotype for sample 5 and lanes 6 and 6`represent tt genotype for sample 6. all ifn-γ +874 a/t genotypes detected at the same size (263 bp) by the amplification refractory mutation system clearly pointed out that cl patients in this region were mainly infected by l. major and l. tropica [5] . it is now well established that the parasite of leishmaniasis is endemic in all over the globe and now it becomes the major public health problem [5] . in saudi arabia, the prevalence of cl infection is on the rise and now it remains a major unsolved health problem of the country [22] . several previous studies reported an association between the susceptibility and resistance of leishmaniasis with the number of cytokines gene polymorphisms [23] [24] [25] , but none of the study did not show direct association of cytokines gene polymorphism with the cl infection nor with the any of the l. species and the controversial role of their snps with leishmaniasis remains continued [22] [23] [24] [25] . the role of th1 cytokines such as tnf-α, ifn-γ was well studied in cl patients, as their levels were found to be higher in these patients [26, 27] . in support of these, akhzari et al. found that the expression of these cytokines in cl lesion varies with the treatment response [27] . furthermore, wilhelm et al. reported that treatment of cl patients with tnf-α improves the parasitic burden and decreased the cl lesion size [28] . because of these important implications of these th1 cytokines for cl patients and frequency of occurrence of cl infection in saudi arabia, this study was designed to investigate the gene polymorphisms of tnf-α-308 g/ a and ifn-γ + 874 a/t in patients of cl with different l. species in central region of saudi arabia. analysis of tnf-α-308 g/a polymorphism on 169 saudi patients with different l. species and 199 healthy controls from the same area, showed no significant association of tnf-α-308 genotype with cl patients. these results were fully supported with the other studies of tnf-α-308 polymorphism performed on different population of cl patients [28, 29] . in contract of these results, our data also showed the cl patients infected with l. tropica, showed significantly higher tnf-α-308 a versus g allele frequency. this may be due the different genetic backgrounds of saudi populations. more specifically the frequency of tnf-α a allele in the saudi healthy controls was found in a range of 18.3%, which seem to be statically significant as compared with other [23, 29] . besides these, this study also demonstrated ifn-γ + 874 a/t polymorphism on the same cl samples obtained from qassim region of saudi arabia. our results showed that ifn-γ + 874 genotyping (tt versus aa+at recessive) and allelic frequency of ifn-γ + 874 (t versus a) showed significant higher in cl patients infected with l. major and also similar results were obtained in total cl cases as compared with their respective control humans. these are novel findings which have not been reported before. the ifn-γ + 874 tt polymorphism was reported to be associated with the transcription of ifn-γ gene in host cl, in which t allele was found to be involved in higher production of ifn-γ [17] . in addition, the resistance role of ifn-γ as well as inf-γ 874 a/t in immuno-response against leishmaniasis has also been reported in different animal models [30, 31] . all these data either directly or indirectly supported our results. furthermore, a study by kamali-sarvestani et al. also supported our results by pointing out a significantly higher frequency of t allele or tt genotype of ifn-γ + 874 a/ t polymorphism in patients infected with l. major in iranian population [29] . in contrast of these results, matos et al. reported no association between ifn-γ + 874 a/t polymorphism with the susceptibility leishmaniasis [32] . despite of these, ifn-γ + 874 a/t snp seems to be involved in the pathogenesis of leishmaniasis by influencing the amount of cytokine released in cl patients [32] . in support of these, al-bushier reported an association of ifn-γ + 874 a/t polymorphism with the susceptibility of visceral leishmaniasis [33] . to further determine the potential of tnf-α 308 g/a and inf-γ + 874 a/t snps in the susceptibility of cl infection, the analysis was performed on the synergistically combined tnf-α 308 g/a and inf-γ + 874 a/t alleles. the calculated data showed strong association between the combined tnf-α-308 g/a and ifn-γ + 874 a/t gene polymorphisms with the susceptibility of l. major, l. tropica and also with total studied cl patients. in short, the data demonstrated no association of tnf-α-308 gene polymorphism with the susceptibility of cl in saudi patients. the only association was found in between a versus g allelic frequency in l. tropica. whereas, ifn-γ + 874 a/t polymorphism was found to be associated with the susceptibility of l. major and also with the total studied cl cases. moreover, the data from the synergistically combined high tnf-α 308/inf-γ 874 alleles strongly suggested their potential role in the susceptibility of leishmania infection. who leishmaniasis control team. leishmaniasis worldwide and global estimates of its incidence world health organization: weekly epidemiological record (wer) cutaneous leishmaniasis in cuban immigrants to texas who traveled through the darién jungle, panama spatial epidemiology of cutaneous leishmaniasis in colombia: 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associated with severe acute respiratory syndrome a single nucleotide polymorphism in the first intron of the human ifn-gamma gene: absolute correlation with a polymorphic ca microsatellite marker of high ifn-gamma production interferon-gamma and interleukin-10 gene polymorphisms in pulmonary tuberculosis prevalence of leishmania species among patients with cutaneous leishmaniasis in qassim province of saudi arabia clinical manifestations and classification of old world cutaneous leishmaniasis acquired immunogenicity of dna after modification with malondialdehyde in patients with alopecia areata bisphenol a modified dna: a possible immunogenic stimulus for anti-dna autoantibodies in systemic lupus erythematosus tropical infectious diseases polymorphism in tumor necrosis factor genes associated with mucocutaneous leishmaniasis genetics of host response in leprosy lesion size correlates with leishmania antigen-stimulated tnflevels in human cutaneous leishmaniasis the role of inflammatory and anti-inflammatory cytokines in the pathogenesis of human tegumentary leishmaniasis expression of pro-inflammatory genes in lesions, spleens and blood neutrophils after burn injuries in mice treated with silver sulfodiazine rapidly fatal leishmaniasis in resistant c57bl/6 mice lacking tnf cytokine gene polymorphisms and susceptibility to cutaneous leishmaniasis in iranian patients ifn-gamma modulates the early development of th1 and th2 responses in a murine model of cutaneous leishmaniasis mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with leishmania major sp but mount a polarized t helper cell 1-type cd4+ t cell response ifng +874t/a polymorphism is not associated with american tegumentary leishmaniasis susceptibility but can influence leishmania induced ifn-gamma production impact of ifn-(+874t/a) and il-10 (−1082g/a) on the susceptibility to visceral leishmaniasis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors gratefully acknowledged the mr. casimero a. victoria for data collection.availability of data and material the raw data file was uploaded as a supplementary file.authors' contributions aa, aaz participated in study design and coordination of data collection. msad, aar, aaz were responsible for samples collection and for obtaining patients written consents. aa, ts carried out experimentation and data collection. aa, aaz and zr consulted for data interpretation and manuscript drafting. all authors have read and approved the final manuscript. this study was funded by the national science, technology and innovation plan grant from king abdulaziz city for science and technology, ksa (nstip/ kacst # 11-med1068-09). the funding body played a role in the design of the study and collection, analysis, and interpretation of data and in the manuscript drafting. the study was approved by the ethical review boards committee of the national science, technology and innovation plan, ksa (nstip # 11-med1068-09) and the study was carried out in accordance with the principles outlined in the declaration of helsinki. informed written consent was obtained for all participants. this is the first report that shows the two most important gene polymorphisms of tnf-α-308 g/a and ifn-γ + 874 a/t in saudi patients infected with different l. species. the study demonstrated no association between the host tnf-α-308 g/a gene polymorphism and cl infection in saudi patients. the only significant correlation was found in a versus g allelic frequency in cl patients infected by l. tropica. interestingly, ifn-γ-874 a/t polymorphism was found to be significantly correlated with the susceptibility of l. major and also in total leishmania subjects, suggesting its role in the production of ifn-γ, and thus enhance immune protection. furthermore, a notable association between the susceptibility of l. major, l. tropica or total studied cl patients with the synergistically combined high tnf-α 308/inf-γ 874 alleles, strongly suggested their crucial role in the onset of cl infection among the studied saudi population. supplementary information accompanies this paper at https://doi.org/10. 1186/s12881-020-01043-9.additional file 1.abbreviations cl: cutaneous leishmaniasis; l. major: leishmania major; l. tropica: leishmania tropica; l. infantum/donovani: leishmania infantum/donovani; tnf-α: tumor necrosis factor alpha; ifn-γ: interferon gamma; th1 cells: type 1 t helper cells; kacst: king abdulaziz city for science and technology; nstip: national plan for science, technology and innovation the authors declare that they have no competing interests. key: cord-344204-qq2vqzc2 authors: hariharan, apurva; hakeem, abdul rahman; radhakrishnan, subathra; reddy, mettu srinivas; rela, mohamed title: the role and therapeutic potential of nf-kappa-b pathway in severe covid-19 patients date: 2020-11-07 journal: inflammopharmacology doi: 10.1007/s10787-020-00773-9 sha: doc_id: 344204 cord_uid: qq2vqzc2 coronavirus disease 2019 (covid-19) pandemic has affected health care systems worldwide. severe presentations of covid-19 such as severe pneumonia and acute respiratory distress syndrome (ards) have been associated with the post-viral activation and release of cytokine/chemokines which leads to a “cytokine storm” causing inflammatory response and destruction, mainly affecting the lungs. covid-19 activation of transcription factor, nf-kappa b (nf-κb) in various cells such as macrophages of lung, liver, kidney, central nervous system, gastrointestinal system and cardiovascular system leads to production of il-1, il-2, il-6, il-12, tnf-α, lt-α, lt-β, gm-csf, and various chemokines. the sensitised nf-κb in elderly and in patients with metabolic syndrome makes this set of population susceptible to covid-19 and their worse complications, including higher mortality. immunomodulation at the level of nf-κb activation and inhibitors of nf-κb (iκb) degradation along with tnf-α inhibition will potentially result in a reduction in the cytokine storm and alleviate the severity of covid-19. inhibition of nf-κb pathway has a potential therapeutic role in alleviating the severe form of covid-19. on 31 december 2019, wuhan municipal health commission, china, reported a cluster of cases of pneumonia in wuhan, hubei province. a novel coronavirus was identified from these patients, and subsequently on 12 january 2020, the genetic information of the novel coronavirus (ncov) was published (wu et al. 2020a) . after full-genomic sequencing and phylogenetic analysis, it was obvious that the coronavirus that causes covid-19 is a beta-coronavirus in the same subgenus as the severe acute respiratory syndrome (sars) virus which caused the sars-cov epidemic in 2003. the structure of the receptor-binding gene region is very similar to that of the sars coronavirus; and the virus uses the same receptor, the angiotensin-1-converting enzyme 2 (ace2), for cell entry ). there has been a steady surge in the number of covid-19 cases worldwide which has crossed 40 million, with more than 1,131,000 deaths and is still on the rise (covid-19 coronavirus pandemic update worldometer info 2020). in about 14% of patients, the virus causes severe disease, including pneumonia and acute respiratory distress syndrome (ards). about 5% of patients suffer critical disease, including respiratory failure, septic shock, multi-organ failure and death (williamson et al. 2020) .these critically ill patients appear to have significantly higher levels of proinflammatory mediators and cytokines, suggestive of a "cytokine storm syndrome" (gao et al. 2019) . hyper-activation of the nuclear factor kappa-light-chainenhancer of activated b cells (nf-κb) pathway has been implicated in the pathogenesis of the severe/critical covid-19 phenotype (hirano and murakami 2020) . during previous coronavirus outbreaks, such as sars-cov and the middle east respiratory syndrome coronavirus (mers-cov) , it was reported that viral proteins such as nsp1, nsp3a, nsp7a, spike, and nucleocapsid protein all caused excessive nf-κb activation, possibly contributing to severe disease and high case-fatality rate (dediego et al. 2014; oeckinghaus and ghosh 2009; liao et al. 2005) . herein, we review current literature on the effect of sars-ncov-2 infection on nf-κb activation and discuss the potential therapeutic role of inhibitors of this pathway in the treatment of covid-19. nf-κb is a complex system of proteins present inactive in the cytoplasm along with inhibitory proteins that are known as inhibitors of nf-κb (iκbs). upon stimuli (induction), phosphorylation of iκbs by iκb kinase (ikk) leads to nuclear translocation of nf-κb, binding to their cognate dna and activates transcription of a wide variety of genes involved in host immunity, inflammation, cell proliferation and apoptosis (oeckinghaus and ghosh 2009) . the nf-κb inducers are highly variable and include bacterial lipopolysaccharides, ionizing radiation, reactive oxygen species (ros), cytokines such as tumour necrosis factor alpha (tnf-α) and interleukin 1-beta (il-1β) and viral dna and rna (zhang et al. 2017) . the activated nf-κb transcription factors promotes the gene expression of wide variety of cytokines (e.g., , chemokines (e.g., il-8, mip-1, mcp1, rantes, and eotaxin), adhesion molecules (e.g., icam, vcam, and e-selectin), acute phase proteins (e.g., serum amyloid a; saa), and inducible effector enzymes (e.g., inducible nitric oxide synthase; inos and cyclooxygenase-2; cox-2). thus nf-κb serves as a 'rapid acting' primary transcription factor which can regulate various cellular responses such as host's early innate immune response to infection, and also associated with chronic inflammatory states, viral infections, septic shock syndrome and multiorgan failure (zhang et al. 2017; li and verma 2002) . in addition, the constitutive activation of nf-κb pathways has been reported in inflammatory diseases such as multiple sclerosis and rheumatoid arthritis (liu et al. 2017 ). since the emergence of covid 19, in most severe cases an elevated level of proinflammatory factors such as, il-2, il-1, il-6, ifn-γ, mip1α, mcp1 and tnf-α have been reported (tang et al. 2020; costela-ruiz et al. 2020) . the infiltrating phagocytic cells such as monocytes and macrophages are responsible for the "cytokine storm" seen in some covid-19 patients. similarly, the inflammatory cells infiltration and diffuse pulmonary alveolar injury has been reported for patients with sars-cov and mers-cov (soy et al. 2020 ). nf-κb signal transduction pathway is considered as a prototypical proinflammatory pathway (lawrence 2009) . a study on sars-cov which was responsible for the worldwide outbreak of sars in 2003 showed that the sars-cov nucleocapsid protein (n protein) activates nf-κb in vero e6 cells in a dose dependent manner (liao et al. 2005) . in parallel, sars-cov lacking the envelope (e) gene (sars-cov-δe) showed reduced expression of proinflammatory cytokines, diminished neutrophil infiltration, reduced lung pathology which resulted in increased balb (albino, immunodeficient and inbred strain) mice survival (dediego et al. 2014; day et al. 2009 ). dediego et al. (2014 in an elegant study proved that inhibitors of nf-κb pathway increased the survival rate in both in vitro and in vivo (in mice) studies with reduced lung pathology. in vitro studies in the previous sars epidemic have shown that the spike (s) protein induces a strong cytokine response in infected mononuclear cells through the nf-κb pathway. this cytokine response was initiated through toll-like receptor (tlr) activation through a protein kinase c dependent pathway of nf-κb and could be inhibited by nf-κb blockade (dosch et al. 2009 ). thus focussing on understanding how nf-κb signalling regulates the inflammatory responses will aid in developing strategies to mitigate the "cytokine storm" and reduce the pathology of severe covid-19. in addition, identifying potential therapeutic targets associated with the nf-κb pathway will help us to manage the more severe spectrum and mortality associated with the pandemic. sars-ncov-2 is a novel virus in the coronaviridae family that has single stranded positive rna genomes. during the replication of positive stranded rna virus, production of a negative-stranded copy of the genome is a crucial step. the negative strand is used as a template for genome replication by the viral rna-dependent rna polymerase (knoops et al. 2008 ). in the course of sars-ncov-2, multiplication within the host cell results in production and accumulation of dsrna (called transcriptive intermediate) in the cellular cytoplasm. the interferon-induced dsrna-dependent protein kinase (pkr) is a threonine kinase which arrests translation within host cells in response to viral infection, an innate immune mechanism against viral replication (garcia et al. 2009; meusel et al. 2002) . in addition, upon binding to dsrna, the pkr (gets activated as a kinase enzyme), which also activates the inhibitor of iκb kinase (ikk), leading to the degradation of the inhibitors iκbα and iκbβ and the concomitant release of nf-κb (williams 1999; d'acquisto et al. 2002) . nf-κb transcription factors translocate into the nucleus where they bind to specific elements called κb-sites and initiate transcription and production of 'proinflammatory mediators' (d'acquisto et al. 2002) . this activation of nf-κb is called the "canonical pathway" as nf-κb essential modulator (nemo), a regulatory subunit of the ikk complex is involved (fig. 1) . along with the direct activation of nf-κb pathway, the pkr also mediates tnf-α (15), which activates nf-κb pathway via "noncanonical pathway". nf-κb signalling also happens because of the stimulation of a subset of the tumour necrosis factor superfamily receptors (tnfrs) through b-cell activating factor receptor (baff), lymphotoxin β or cd40. this is non-canonical pathway (without nemo) although slow, is more long lasting in terms of proinflammatory mediator production, when compared to the canonical activation ( fig. 1 ) (dorrington and fraser 2019) . one of the other major pathways through which beta coronavirus causes hyperactivation of nf-κb is via the myeloid differentiation primary response 88 (myd88) pathway through pattern-recognition receptors (pprs) ( fig. 1 ) (d'acquisto et al. 2002; birra et al. 2020) . this leads to induction of a variety of cytokines including il-6, tnf-α and chemokines (hirano and murakami 2020) . this was shown as a major factor in the higher case-fatality rates associated with sars and mers, when compared to covid-19 (wit et al. 2016) . the increase in angiotensin ii (angii) caused by reduced level of angiotensin converting enzyme ii (ace2) membrane protein which is endocytosed along with n-cov has been implicated in the progression of lung injury and propagation of severe inflammation due to dysregulation of renin-angiotensin pathway (ras) (ingraham et al. 2020) . angii acts as a proinflammatory cytokine via angiotensin-1-receptor (at1r), the latter also activates nf-κb, disintegrin and metalloprotease 17 (adam17) (devaux et al. 2020) . these three lead to production of epidermal growth factor receptor (egfr) ligands and tnf-α, which lead to a vicious cycle of further activation of nf-κb and propagation of "cytokine storm" (fig. 1 ) (gao et al. 2019; hirano and murakami 2020) . the janus kinase (jak) and signal transduction and activator of transcription factor 3 (stat3) pathway is required for full activation of the nf-κb pathway and its main stimulator is il-6. the il-6 when binds to its receptor activates jak-stat pathway allows phosphorylated stat3 to fig. 1 schematic diagram explaining the mechanism of action of nf-κb in covid-19 via various pathways leading to "cytokine storm syndrome" translocate into the nucleus which promotes ifn-γ reduction and induce cytokine release syndrome. in vascular tissues, the activated nf-κb produces il-6. thus in covid-19, both nf-κb and jak-stat pathway can be constitutively activated, which in turn can hyperactivate the il-6 amplifier response (il-6 amp), which can lead to a cascade of hyper-activation of nf-κb by stat3, leading to multiinflammatory responses (hirano and murakami 2020; battagello ds, 2020; brasier 2010) . il-6 is also considered as an important marker of cellular senescence, with rates increasing with age, all of which explains the higher mortality in elderly age group. the mapks are serine-threonine protein kinases involved in the modulation of cellular responses during stress and pathophysiological states. coronaviruses have been shown to involve all the three mapks (the jun aminoterminal kinases (jnk), p38 and extracellular signal-regulated kinases (erk1/2)) pathways for viral pathogenesis. p38 mitogen-activated protein kinases (mapks) mediated cross-talk activation of nf-κb was reported by saccani et al. (2002) . elevated levels of ang ii mediates its adverse reactions via p38 mapk pathway activation (park et al. 2007 ). p38 mapk pathway regulates the translation of cytokines tnf-α and il-1β which can activate nf-κb non-canonically (battagello et al. 2020; sun 2017) . hence it can be explained that the hyperactive p38 mapk and their cross-talk activation of nf-κb pathway might be the reason for the inflammation, thrombosis, and vasoconstriction in covid-19 (grimes and grimes 2020) the damaged human self dna at later stages of covid-19 can excessively activate sting (stimulator of interferon (ifn) genes) which leads to the production of ifn-β through activation of interferon regulatory factor 3 (irf 3). furthermore, the human sting stimulates nf-κb resulting in "cytokine storm" (jean-marie berthelot 2020). at later stages interferons cause detrimental effects through enhanced delayed innate immune response, which has been reported in sars-cov and mers-cov infections in humans (shalhoub et al. 2015; channappanavar and perlman 2017) . apart from association with the "cytokine storm", the nf-κb pathway is well known to be associated with the pathogenesis of metabolic syndrome (baker et al. 2011; urso 2020) . diabetes and obesity are attributed to a chronic inflammatory state, with high levels of glucose and free fatty acids in the body. this glucolipotoxicity, along with the inflammatory state, has a role in activation of the non-canonical nf-κb pathway. in the presence of insulin resistance the glycogen synthase kinase beta (gsk3β) get activated and blocks heat shock protein 70 which inhibits nf-κb. thus nf-κb signalling triggers a severe inflammatory response. moreover the downstream products of nf-κb signalling such as inos and nitric oxide block insulin signalling leading to worsening of the metabolic state (krause et al. 2020 ). there was a study conducted on mice which showed the association of insulin resistance with nf-κb activation via nik (nf-κb inhibitory kinase) (malle et al. 2015) . the hyperglycemic state in these patients activates tnf-α which in turn accelerates the activation of nf-κb pathway via the non-canonical pathway (hattori et al. 2000) . nf-κb is also known to be associated with atherosclerosis and vascular endothelial damage, which are predominant factors for endorgan damage in patients with metabolic syndrome. thus the overnutrition, obesity-induced excess metabolites, hyperlipidemia and hyperglycemia, are risk factors that initiate and propagate hyper-activation of the nf-κb pathway, leading to severe covid-19 and worse outcomes (catrysse and loo 2017; apicella et al. 2020; meyerovich et al. 2018) . along with diabetes and obesity, the nf-κb activation is associated with complications of hypertension as well. the ras mediated high levels of angii in the body has been noted to activate the nf-κb pathway via card11-bcl10-malt1 or cbm (caspase recruitment domain family, member 11-b-cell chronic lymphocytic leukaemia/lymphoma 10-mucosa-associated lymphoid tissue lymphoma translocation gene 1 "cbm signalosomes" which inhibit the iκb. this leads to poorly controlled hypertension and is one of the high risk factors for cardiovascular related mortality (crowley 2014) . high levels of angii have been seen in critically ill covid-19 patients, thereby implicating the role of the nf-κb pathway in the hypertensive cohort (wu et al. 2020b ). large cohort studies have shown the association with severity of covid-19 and the prevalence of comorbidities. it was reported that out of 46,248 covid-19 patients, a large subset were suffering from hypertension (17 ± 7, 95% confidence interval [ci] 14-22%), followed by diabetes (8 ± 6, 95% ci 6-11%), cardiovascular diseases (5 ± 4, 95% ci 4-7%) and respiratory diseases (2 ± 0, 95% ci 1-3%). the study showed that patients who were severely ill with covid-19, were 2.36 times more likely to have hypertension (95% ci 1.46-3.83), 3.42 times likely to have underlying cardiovascular disease (95% ci 1.88-6.22), 2.46 times more likely to have respiratory disease (95% ci 1.76-3.44), when compared to those with mild disease (dutta et al. 2020; yang et al. 2020 ). firstly, the slow viral alert signals due to immunosenescence in elderly patients esulted in greater viral replication and increased ace2 shedding. consequentially, there is increased ang ii/adam17 activity due to age-related dysregulation or alteration in ras. the ang ii binds with at1r constitutively activates nf-κb leads to vasoconstriction and inflammation and this could explain importance of age in covid-19 severity (sward 2020; muller 2020). the activated innate immunity in older patients also induces a proinflammatory profile regulated by nf-κb pathway (inflammaging) as their adaptive immunity too declined due to immunosenescence (sanghai and tranmer 2020; salminen et al. 2008) . moreover, the low levels of testosterone and estradiol (steroid hormones) in aged men and postmenopausal women, respectively fails to inhibit nf-κb thereby producing higher levels tnfα and il-6 leads to increased risk of lung damage (al-lami et al. 2020) the oxidative stress, antioxidant deprivation mechanism, increased ros and chronic low inflammation in the lungs of elderly patients might also contribute to the severity of covid-19 via nf-κb-toll-like receptor signalling pathway (delgado-roche and mesta 2020). inflammaging and immunosenescence in elderly highly contribute to the development of cytokine storm. apart from these age -related risks, cytokine storm and fatalities were also reported in young and middle-aged men might be because of genetic predisposition, virulence of viral load, epigenetic dysregulation due to life style factors and hyperactive responses by diversified immune cells. the data from epidemiological studies revealed sex-specific differences in the incidence and mortality in covid-19 patients, where the mortality rate of men is more than female. most of genes associated with immune regulation are encoded by x-chromosomes, the genetic factor, extra x chromosomes endow an active immune cells in women (al-lami et al. 2020; maleki dana et al. 2020 ). such efficient controlled innate immunity quickly recognise the virus and clear the viral load at first line of defence and thus avoiding the sustained activation of inflammatory signalling results in cytokine storm (mueller et al. 2020) . the attenuation of nf-κb pathway by sex hormone oestrogen reduce the cytokine production in female patients especially, the periovulatory dosages of oestrogen are very effective in inhibiting the cytokines il-6, il-8 and tnf-α. the activation of oestrogen receptor α by hormone replacement therapy in postmenopausal women inhibits nf-κb mediated inflammation response and cytokine production (al-lami et al. 2020). nf-κb has been implicated in extrapulmonary manifestations of covid-19, which are in addition to the systemic effects due to cytokine storm. a systematic review of 22 observational studies including 17,391 covid-19 patients reported an acute kidney injury (aki) rate of 11%, with 6.8% needing renal replacement therapy (kunutsor and laukkanen 2020) . both in vitro and in vivo studies have proposed that nf-κb activation in the renal glomerular cells via tnf-α and angii has been associated with worse renal injury and need for renal support (sanz et al. 2010 ). there are also reports of nf-κb mediated thrombotic microangiopathy within the renal microvasculature, leading to hypoxic acute tubular necrosis, leading to worse outcomes in these patients (jhaveri et al. 2020; hirsch et al. 2020) . the cardiovascular manifestations of covid-19 include myocardial injury, myocarditis, acute myocardial infarction, heart failure, dysrhythmias, and cortical venous thromboembolic events. chronic activation of nf-κb in people with known metabolic syndromes induces vascular disturbances and remodelling of cardiomyocytes, which predisposes them to higher risk of cardiac complications (hamid et al. 2011) . this when superadded with cytokine activation can lead to cardiac injury and increases mortality risk. similar to other viral infections like mers, herpes, varicella and cytomegalovirus which have the potential to activate the nf-κb pathway, covid-19 has similar neuro-pathogenesis in cases where there is thromboembolism related neuropathy and stroke (montalvan et al. 2020 ). the possible reason for this cytokine storm in covid-19 patients might be the elevated level of anti-viral inflammatory cytokines (il-10, il-6, il-2, ifn-γ and tnf-α) which were attenuated and diversified in sars and mers infections, respectively . the involvement of il-6, the noncanonical activation of nf-κb by elevated level of tnf-α create a degenerative feedback loop through protein kinase b (akt) (mozafari et al. 2020 ) and resulted in lung failure. in addition, previous reports suggested that the inflammatory cytokines tnf-α and il1β induces the granulocyte-colony stimulating factor (g-csf) via nf-κb pathway (cao et al. 2014) . these elevated levels of cytokine g-csf and gm-csf found in the cytokine storm of sars-cov 2 induce the accelerated inflammation process in covid-19 patients (zhu et al. 2020 ). immunomodulation at the level of nf-κb activation and iκb degradation along with tnf-α inhibition will potentially result in a reduction in the cytokine storm and alleviate the severity of covid-19 (fig. 2) . this has been proven in the sars setting in small animal models (dediego et al. 2014; ) . there are suggestions to repurpose currently available drugs such as cromolyn which inhibit nf-κb mediated cytokine production in the fight against covid-19 (karadesh 2020). many of the drugs currently effective in covid disease appear to have links to the nf-κb cascade of immune regulation. dexamethasone is among the two glucocorticoids (prednisolone being the other) which has the inhibitory action on the nf-κb pathway (ye et al. 2020; d'acquisto et al. 2002) *. glucocorticoids increase expression of iκb, which helps retain nf-κb within the cellular cytoplasm. they are also an immunomodulator, which reduces il-6 production and activity, which in turn reduces the cytokine feedback on nf-κb activity (auphan et al. 1995) . in the recovery (randomised evaluation of covod-19 therapy) trial, dexamethasone at a dose of 6 mg once per day for ten days, reduced deaths by one-third in ventilated patients (rate ratio 0.65) and by one-fifth in those eceiving oxygen therapy (rate ratio 0.80). the drug did not show any benefit in those getting mild to moderate disease and not requiring oxygen therapy. this supports the inference that the beneficial role of dexamethasone may be atleast partly related to the inhibition of nf-κb activation in the severe-critically ill covid-19 patients (the recovery collaborative group 2020). the role of steroids generally in covid-19 is by inhibiting the action and expression of many molecules involved in pneumonia associated inflammatory response (ledford 2020) . so it's probable that in mild and moderate disease, there isn't enough pro-inflammatory cytokines, chemokines and adhesion molecules, against which dexamethasone can increase gene transcription of anti-inflammatory cytokines and decrease pro-inflammatory mediators (patel et al. 2020) . there are also concerns in using steroids early in the course of illness due to its risk of enhancing viral replication, reducing overall innate immunity and increasing secondary bacterial infections (singh et al. 2020) . remdesivir (gs-5734) is a nucleotide analogue that perturbs viral replication by inhibiting the rna-dependent rna polymerase. by reducing dsrna related induction of nf-κb pathway, it reduces the cytokine storm and severe disease. in the adaptive covid-19 treatment trial (actt-1), where remdesivir was evaluated with the placebo, it showed quicker time to recovery for the remdesivir patients (beigel et al. 2020) . hydroxychloroquine, which was initially touted as the wonder drug and used in many regimens for the treatment of covid-19, has fallen off from popularity due to its cardiac toxicity. it reduces the levels of tnf-α, tnf-1β, igg and ifn-γ, which in turn blocks the nf-κb pathway (liang et al. 2018) . inhaled nitric oxide (no) may be useful in the fig. 2 schematic diagram showing the mechanism of action of various drugs used in covid-19 and how they inhibit the nf-κb pathway management of covid-19 ards, and there is growing evidence that it reduces the inflammatory cell-mediated lung injury by inhibiting neutrophil activation and subsequent pro-inflammatory cytokines. it also works by inhibiting the nf-κb and terminates the transcription process (matthews et al. 1996; clinicaltrials.gov. 2020a) . high dose aspirin and sulindac used in doses similar to chronic inflammatory states have potential to block nf-κb via inhibition of tnf-α and iκb kinase, and are currently being investigated in clinical trials (yamamoto et al. 1999) . camostat mesylate and nafamostat mesylate are serine protease inhibitors used to treat pancreatitis and inflammatory diseases could be used to combat nf-κb signalling pathway. these drugs are anti-inflammatory and possess anti-viral properties (catanzaro et al. 2020) . macrolide antibiotics also have immunomodulatory actions via reduction of neutrophil-related inflammation, suppression of tnf-α and il-1 reduction and mild potential for inhibition of nf-κb pathway (cheung et al. 2010; bleyzac et al. 2002) . though azithromycin was used in the treatment of covid-19 initially, both on its own and along with hydroxychloroquine, the cumulative risks of qt interval prolongation and high risk of cardiac toxicity and death have reduced the enthusiasm for this combination. nf-κb is a redox-sensitive transcription factor, which gets activated by oxidative stress (das 2020) . blocking this pathway with antioxidants such as vitamin a, vitamin c, glutathione, vitamin e, zinc and many others could have both prophylactic protection and also prevent progression of illness (nwose and bwititi 2020; bauer et al. 2020) . n-acetylcysteine has been proved to have a potent nf-κb inhibitor via downregulating the phosphorylation of iκb. apart from this, it also has an inhibitory action against tnf-α mediated activation of the nf-κb pathway (oka et al. 2000) . along with direct pathway inhibition it also has antioxidant potential which reduces the reactive oxygen species (wu et al. 2014) . it was reported to have a significant clinical improvement in critically ill covid-19 patients and is currently under assessment in a phase 4 clinical trial (assimakopoulos and marangos 2020; clinicaltrials.gov. 2020b). inhibition of tnf-α by the monoclonal antibodies such as infliximab and adalimumab, can inhibit the non-canonical activation of nf-κb pathway which has the potential to be used to alleviate symptoms in severely ill covid patients (feldmann et al. 2020; benicco et al. 2020) . it was noted that out of 536 covid-19 patients who were on regular anti-tnf-α as treatment for their existing inflammatory bowel disease (ibd), 448 (84%) patients were treated as outpatient and only 84 (15%) were hospitalised. out of this only 2% either needed intensive care admission or ventilator or had death as outcome (secure-ibd database 2020). the hormone therapy using exogenous oestrogen and testosterone also have a potential to mitigate the inflammatory response in covid 19 patients (al-lami et al. 2020) . these drugs which inhibit nf-κb and associated pathways targets individual mediators of maladaptive cytokines release rather than attenuating the whole immune response against covid-19. besides, previous reports suggests that the cytokine inhibitor therapy against tnf, il-6, il-17, il-23 and il-4 reduce undesirable inflammatory response and have no threats in viral-mediated disease progression (schett et al. 2020) . such approaches that mitigate the exaggerated immune response (by tnf and il-6) and which do not affect sars-ncov-2 clearance (by interferon type i, il-15 and ifn-γ) may exert beneficial effects against the pandemic. however, more studies are required to prove its potential effect on covid-19 patients. in summary, nf-κb pathway seems to play an important role in the natural progression of covid and conversion to a severe phenotype. nf-κb inhibition may be a possible mechanism of action of currently effective drugs against covid. more work needs to be done in identifying direct nf-κb inhibition as a therapeutic means of treating the severe form of this disease. author contributions ah: concept, critical review, data appraisal and writing the manuscript. arh: review concept, critical appraisal, writing and revising the manuscript. sr: critical appraisal and revising the manuscript. msr: review concept, critical appraisal and revising the manuscript. mr: supervision and final manuscript corrections. funding 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cord-020757-q4ivezyq authors: saikumar, pothana; kar, rekha title: apoptosis and cell death: relevance to lung date: 2010-05-21 journal: molecular pathology of lung diseases doi: 10.1007/978-0-387-72430-0_4 sha: doc_id: 20757 cord_uid: q4ivezyq in multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. the term apoptosis was first coined in 1972 by kerr et al.1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. in fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode caenorhabditis elegans.2 as time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (figure 4.1). in multicellular organisms, cell death plays an important role in development, morphogenesis, control of cell numbers, and removal of infected, mutated, or damaged cells. the term apoptosis was fi rst coined in 1972 by kerr et al. 1 to describe the morphologic features of a type of cell death that is distinct from necrosis and is today considered to represent programmed cell death. in fact, the evidence that a genetic program existed for physiologic cell death came from the developmental studies of the nematode caenorhabditis elegans. 2 as time has progressed, however, apoptotic cell death has been shown to occur in many cell types under a variety of physiologic and pathologic conditions. cells dying by apoptosis exhibit several characteristic morphologic features that include cell shrinkage, nuclear condensation, membrane blebbing, nuclear and cellular fragmentation into membrane-bound apoptotic bodies, and eventual phagocytosis of the fragmented cell (figure 4 .1). cell death is central to the normal development of multicellular organisms during embryogenesis and maintenance of tissue homeostasis in adults. 3 during development, sculpting of body parts is achieved through selective cell death, which imparts appropriate shape and creates required cavities in particular organs. in adults, cell death balances cell division as a homeostatic mechanism regulating constancy of tissue mass. deletion of injured cells because of disease, genetic defects, aging, or exposure to toxins is also achieved by apoptosis. in essence, apoptotic cell death has important biologic roles not only in development and homeostasis but also in the pathogenesis of several disease processes. dysregulation of apoptosis is found in a wide spectrum of human diseases, including cancer, autoimmune diseases, neurodegenerative diseases, ischemic diseases, viral infections, 4 and lung diseases. 5 our knowledge of cell death and the mechanisms of its regulation increased dramatically in the past two decades with the discovery nevertheless, necrosis has been shown to occur in cells having defects in apoptotic machinery or upon inhibition of apoptosis, 7 and this form of cell death is emerging as an important therapeutic tool for cancer treatment. 8 autophagy autophagy, which is also referred to as type ii programmed cell death, is characterized by sequestration of cytoplasm and organelles in double or multimembrane structures called autophagic vesicles, followed by degradation of the contents of these vesicles by the cell's own lysosomal system (see figure 4 .1). the precise role of autophagy in cell death or survival is not clearly understood. autophagy has long been regarded as a cell survival mechanism whereby cells eliminate long-lived proteins and organelles. in this regard, it is argued that autophagy may help cancer cells survive under nutrientlimiting and low-oxygen conditions and against ionizing radiation. 9,10 however, recent observations that there is there is early membrane damage with eventual loss of plasma membrane integrity and leakage of cytosol into extracellular space. despite early clumping, the nuclear chromatin undergoes lysis (karyolysis). apoptosis: cells die by type i programmed cell death (also called apoptosis); they are shrunken and develop blebs containing dense cytoplasm. membrane integrity is not lost until after cell death. nuclear chromatin undergoes striking condensation and fragmentation. the cytoplasm becomes divided to form apoptotic bodies containing organelles and/or nuclear debris. terminally, apoptotic cells and fragments are engulfed by phagocytes or surrounding cells. autophagy: cells die by type ii programmed cell death, which is characterized by the accumulation of autophagic vesicles (autophagosomes and autophagolysosomes). one feature that distinguishes apoptosis from autophagic cell death is the source of the lysosomal enzymes used for most of the dying-cell degradation. apoptotic cells use phagocytic cell lysosomes for this process, whereas cells with autophagic morphology use the endogenous lysosomal machinery of dying cells. paraptosis: cells die by type iii programmed cell death, which is characterized by extensive cytoplasmic vacuolization and swelling and clumping of mitochondria, along with absence of nuclear fragmentation, membrane blebbing, or apoptotic body formation. autoschizis: in this form of cell death, the cell membrane forms cuts or schisms that allow the cytoplasm to leak out. the cell shrinks to about one-third of its original size, and the nucleus and organelles remain surrounded by a tiny ribbon of cytoplasm. after further excisions of cytoplasm, the nuclei exhibit nucleolar segregation and chromatin decondensation followed by nuclear karyorrhexis and karyolysis. decreased autophagy during experimental carcinogenesis and heterologous disruption of an autophagy gene, beclin 1 (bcn1), in cancer cells 11, 12 suggest that breakdown of autophagic machinery may contribute to development of cancer. other interesting studies have shed some light on the relationship between autophagy and apoptosis. these investigations have shown prevention of caspase inhibitor z-vad-induced cell death in mouse l929 cells by rna interference directed against autophagy genes atg7 and bcn1 13 and protection of bax −/− , bak −/− murine embryonic fi broblasts against staurosporine-or etoposide-induced cell death by rna interference against autophagy genes atg5 and bcn1. 14 however, both of these studies were done in cells whose apoptotic pathways had been compromised. thus, it remains to be seen whether cells with intact apoptotic machinery can also die by autophagy and whether apoptotic-competent cells lacking autophagy genes will be resistant to different death stimuli. paraptosis has recently been described as a form of cell death characterized by extensive cytoplasmic vacuolation (see figure 4 .1) caused by swelling of mitochondria and endoplasmic reticulum. this form of cell death does not involve caspase activation, is not inhibited by caspase inhibitors, but is inhibited by the inhibitors of transcription and translation, actinomycin d, and cycloheximide, respectively, 15 suggesting a requirement for new protein synthesis. the tumor necrosis factor receptor family taj/troy and the insulin-like growth factor i receptor have been shown to trigger paraptosis. 16 paraptosis appears to be mediated by mitogen-activated protein kinases and inhibited by aip1/alix, a protein interacting with the calcium-binding death-related protein alg-2. 16 autoschizis autoschizis is a recently described type of cell death that differs from apoptosis and necrosis and is induced by oxidative stress. 17 in this type of death, cells lose cytoplasm by self-morsellation or self-excision (see figure 4 .1). autoschizis usually affects contiguous groups of cells both in vitro and in vivo but can also occasionally affect scattered individual cells trapped in subcapsular sinuses of lymph nodes. 18 the nuclear envelope and pores remain intact while the cytoplasm is reduced to a narrow rim surrounding the nucleus. the chromatin marginates along the nuclear membrane, and mitochondria and other organelles around the nucleus aggregate as a result of cytoskeletal damage and condensation of the cytosol. interestingly, the rough endoplasmic reticulum is preserved until the late stages of autoschizis, in which cells fragment and the nucleolus becomes condensed and breaks into smaller fragments. 19 eventually, the nuclear envelope and the remaining organelles dissipate with cell demise. genetic studies in the nematode worm c. elegans led to the characterization of apoptosis. activation of specifi c death genes during the development of this worm results in death of exactly 131 cells, leaving 959 cells intact. 2 further studies revealed that apoptosis can be divided into three successive stages: (1) commitment phase, in which death is initiated by specifi c extracellular or intracellular signals; (2) execution phase; and (3) clean-up phase, in which dead cells are removed by other cells with eventual degradation of the dead cells in the lysosomes of phagocytic cells. 20 the apoptotic machinery is conserved through evolution from worm to human. 21 in c. elegans, execution of apoptosis is mediated by ced-3 and ced-4 proteins. commitment to a death signal results in the activation of ced-3 by ced-4 binding. the ced-9 protein prevents activation of ced-3 by binding to ced-4. 22, 23 mechanisms of apoptosis caspases studies over the past decade have indicated that two distinct apoptotic pathways are followed in mammalian systems: the extrinsic or death receptor pathway and the intrinsic or mitochondrial pathway. the executioners in both intrinsic and extrinsic pathways of cell death are the caspases, 24 which are cysteine proteases with specifi city to cleave their substrates after aspartic acid residues. the central role of caspases in apoptosis is underscored by the observation that apoptosis and all classic changes associated with apoptosis can be blocked by inhibition of caspase activity. to date, 12 mammalian caspases (caspase-1 to -10, caspase-14, and mouse caspase-12) have been identifi ed. 25 caspase-13 was later found to represent a bovine homolog and caspase-11 appears to be a murine homolog of human caspases-4 and -5, respectively. caspases are normally produced as inactive zymogens containing an n-terminal prodomain followed by a large and a small subunit that constitute the catalytic core of the protease. they have been categorized into two distinct classes: initiator and effector caspases. the upstream initiator caspases contain long n-terminal prodomains and one of the two characteristic protein-protein interaction motifs: the death effector domain (ded; caspase-8 and -10) and the caspase activation and recruitment domain (caspase-1, -2, -4, -5, -9, and -12). the downstream effector caspases (caspase-3, -6, and -7) are characterized by the presence of a short prodomain. apart from the structural differences, a prominent difference between initiator and effector caspases is their basal state. both the zymogen and the activated forms of effector caspases exist as constitutive homodimers, whereas initiator caspase-9 exists predominantly as a monomer both before and after proteolytic processing. 26 initiator caspase-8 has been reported to exist in an equilibrium between monomers and homodimers. 27 although the initiator caspases are capable of autocatalytic activation, the activation of effector caspases requires formation of oligomeric complexes with their adapter proteins and often intrachain cleavage within the initiator caspase. caspases have also been divided into three categories based on substrate specifi city. 28 group i members (caspase-1, -4, and -5) have a substrate specifi city for the wehd sequence with high promiscuity; group ii members (caspase-2, -3, and -7 and ced-3) prefer the dexd sequence and have an absolute requirement for aspartate (d) at p4; and members of group iii (caspase-6, -8, and -9 and the "aspase" granzyme b) have a preference for (i/ l/v)exd sequences. several reports have suggested a role for group i members in infl ammation and that of group ii and iii members in apoptotic signaling events. the extrinsic pathway involves binding of death ligands such as tumor necrosis factor-α (tnf-α), cd95 ligand (fas ligand), and tnf-related apoptosis-inducing ligand (trail) to their cognate cell surface receptors tnfr1, cd95/fas, trail-r1, trail-r2, and the dr series of receptors, 29 resulting in the activation of initiator caspase-8 (also known as fadd-homologous ice/ced-3-like protease or flice) and subsequent activation of effector caspase-3 ( figure 4 .2). 30 the cytoplasmic domains of death receptors contain the "death domain," which plays a crucial role in transmitting the signal from the cell's surface to intracellular signaling molecules. binding of the ligands to their cognate receptors results in receptor trimerization and recruitment of adapter proteins to the cell membrane, which involves homophilic interactions between death domains of the receptors and the adapter proteins. the adapter protein for the receptors tnfr1 and dr3 is tnfr-associated death domain protein (tradd) 31 and that for fas, trail-r1, trail-r2, and dr4 is fas-associated death domain protein (fadd). 32 the receptor/ligand and fadd complex in turn recruits caspase-8 to the activated receptor, resulting in the formation of death-inducing signaling complex (disc) and subsequent activation of caspase-8 through oligomerization and self-cleavage. depending on the cell type and/or apoptotic stimulus, caspase-8 can also be activated by caspase-6. 33 activated caspase-8 then activates effector caspase-3. in some cell types, cleavage of caspase-3 by caspase-8 also requires a mitochondrial amplifi cation loop involving cleavage of proapoptotic protein bid by caspase-8 and its translocation to the mitochondrial membrane, triggering the release of apoptogenic proteins from mitochondria into cytosol (see figure 4 .2). in these cell types, overexpression of bcl-2 and bcl-xl can block cd95-induced apoptosis. 34 tumor necrosis factor-α is produced by t cells and activated macrophages in response to infection. although tnf-α-mediated signaling can be propagated through either tnfr1 or tnfr2 receptors, the majority of biologic functions are initiated by tnfr1. 35 binding of tnf-α to tnfr1 causes release of inhibitory protein silencer of death domain protein (sodd) from tnfr1, which enables recruitment of adapter protein tradd. signaling induced by activation of tnfr1 or dr3 diverges at the level of tradd. in one pathway, nuclear translocation of the transcription factor nuclear factor-κb (nf-κb) and activation of c-jun n-terminal kinase (jnk) are initiated, which results in the induction of a number of proinfl ammatory and immunomodulatory genes. 36 in another pathway, tnf-α signaling is coupled to fas signaling events through interaction of tradd with fadd. 37 the tnfr1-tradd complex can alternatively engage traf2 protein, resulting in activation of transcription factor c-jun, which is involved in survival signaling. furthermore, binding of receptor interaction protein to tnfr1 through tradd results in activation of transcription factor nf-κb, which suppresses apoptosis through transcriptional upregulation of antiapoptotic molecules such as traf1, traf2, ciap1, ciap2, and flip. the flice-associated huge protein was identifi ed to be a ced-4 homolog interacting with the ded of caspase-8 and was shown to modulate fas-mediated activation of caspase-8. 38 another class of protein, flip (flice inhibitory protein), was shown to block fasinduced and tnf-α-induced disc formation and subsequent activation of caspase-8. 39 cytotoxic t cells play a major role in vertebrate defense against viral infection. 40 they induce cell death in infected cells to prevent viral multiplication and spread of infection. 41 cytotoxic t cells can kill their targets either by activating the fas ligand/fas pathway or by injecting granzyme b, a serine protease, into target cells. cytotoxic t cells carry fas ligand on their surface but also carry granules containing the channel-forming protein perforin and granzyme b. upon recognizing the infected cells, the lymphocytes bind and secrete granules onto the surface of infected cells. perforin then assembles into transmembrane channels to allow the entry of granzyme b into the target cell. upon entry, granzyme b, which cleaves after aspartate residues in proteins ("aspase"), activates one or more of the apoptotic proteases (caspase-2, -3, -7, -8, and -10) to trigger the proteolytic death cascade (see figure 4 .2). fas ligand/fas and perforin/granzyme b systems are the main apoptotic machinery that regulates homeostasis in immune cell populations. cells can respond to various stressful stimuli and metabolic disturbances by triggering apoptosis. drugs, toxins, heat, radiation, hypoxia, and viral infections are some of the tnf-α tnfr1 complex can also elicit an antiapoptotic response by recruiting traf2, which results in nf-κbmediated upregulation of antiapoptotic genes. in cytotoxic t lymphocyte-induced death, granzyme b, which enters the cell through membrane channels formed by the protein perforin, activates caspases by cleaving them directly or indirectly. intracellular pathways: lack of survival stimuli (withdrawal of growth factor, hypoxia, genotoxic substances, etc.) is thought to generate apoptotic signals through ill-defi ned mechanisms, which lead to translocation of proapoptotic proteins such as bax to the outer mitochondrial membrane. in some cases, transcription mediated by p53 may be required to induce proteins such as bax. translocated bax undergoes conformational changes in the outer membrane to form oligomeric structures (pores) that leak cytochrome c from mitochondria into the cytosol. formation of a ternary complex of cytochrome c, the adapter protein apaf-1, and the initiator caspase-9 results in the activation of caspase-9 followed by sequential activation of effector caspase(s) such as caspase-3 and others. the action of caspases, endonucleases, and possibly other enzymes leads to cellular disintegration. for example, the endonuclease cad (caspase activated dnase) becomes activated when it is released from its inhibitor icad upon cleavage of icad by an effector caspase. antiapoptotic proteins such as bcl-2 and bcl-xl inhibit the membrane-permeabilizing effects of bax and other proapoptotic proteins. cross-talk between extra-and intracellular pathways occurs through caspase-8-mediated bid cleavage, which yields a 15 kda protein that migrates to mitochondria and releases cytochrome c, thereby setting in motion events that lead to apoptosis via caspase-9. the stimuli known to activate death pathways. cell death, however, is not necessarily inevitable after exposure to these agents, and the mechanisms determining the outcome of the injury are a topic of active interest. the current consensus appears to be that it is the intensity and the duration of the stimulus that determine the outcome. the stimulus must go beyond a threshold to commit cells to apoptosis. although the exact mechanism used by each stimulus may be unique and different, a few broad patterns can be identifi ed. for example, agents that damage dna, such as ionizing radiation and certain xenobiotics, lead to activation of p53-mediated mechanisms that commit cells to apoptosis, at least in part through transcriptional upregulation of proapoptotic proteins. 42 other stresses induce increased activity of stress-activated protein kinases, which result ultimately in apoptotic commitment. 43 these different mechanisms converge in the activation of caspases. a cascade of caspases plays the central executioner role by cleaving various mammalian cytosolic and nuclear proteins that play roles in cell division, maintenance of cytoskeletal structure, dna replication and repair, rna splicing, and other cellular processes. this proteolytic carnage produces the characteristic morphologic changes of apoptosis. once the caspase cascade is initiated, the process of cell death has crossed the point of no return. the roles of various caspases in apoptotic pathways and their relative importance for animal development have been examined in genetic studies involving knockout of different caspase genes. a caspase-1 (interleukin [il]-1b converting enzyme [ice]) knockout study suggested that ice plays an important role in infl ammation by activating cytokines such as il-1b and il-18. however, caspase-1 was not required to mediate apoptosis under normal circumstances and did not have a major role during development. 44 surprisingly, ischemic brain injury was signifi cantly reduced in caspase-1 knockout mice compared with wild-type mice, 45 suggesting that infl ammation may contribute to ischemic injury. caspase-3 deficiency leads to impaired brain development and premature death. also, functional caspase-3 is required for some typical hallmarks of apoptosis such as formation of apoptotic bodies, chromatin condensation, and dna fragmentation in many cell types. 46 lack of caspase-8 results in the death of embryos at day 11 with abnormal formation of the heart, 47 suggesting that caspase-8 is required for cell death during mammalian development. in support of this fi nding, knockout of fadd, which is required for caspase-8 activation, resulted in fetal death with signs of abdominal hemorrhage and cardiac failure. 48 moreover, caspase-8-defi cient cells did not die in response to signals from members of the tnf receptor family. 47 however, cells lacking either fadd or caspase-8, which are resistant to tnf-α-mediated or cd95-mediated death, are susceptible to chemotherapeutic drugs, serum depriva-tion, ceramide, γ-irradiation, and dexamethasone-induced killing. 48 in contrast, caspase-9 has a key role in apoptosis induced by intracellular activators, particularly those that cause dna damage. deletion of caspase-9 resulted in perinatal lethality, apoptotic failure in developing neurons, enlarged brains, and craniofacial abnormalities. 49 in caspase-9-defi cient cells, caspase-3 was not activated, suggesting that caspase-9 is upstream of caspase-3 in the apoptotic cascade. as a consequence, caspase-9-defi cient cells are resistant to dexamethasone or irradiation, whereas they retain their sensitivity to tnf-α-induced or cd95-induced death 49 because of the presence of caspase-8, the initiator caspase involved in death receptor signaling that can also activate caspase-3. overall, these observations support the idea that different death signaling pathways converge on downstream effector caspases (see figure 4 .2). indeed, caspase-3 is regarded as one of the key executioner molecules activated by apoptotic stimuli originating either at receptors for exogenous molecules or within cells through the action of drugs, toxins, or radiation. in c. elegans, biochemical and genetic studies have indicated a role for ced-4 upstream of ced-3. 50 upon receiving death commitment signals, ced-4 binds to pro-ced-3 and releases active ced-3. 50 however, when overexpressed, ced-9 can inhibit the activation of pro-ced-3 by binding to ced-4 and sequestering it away from pro-ced-3. therefore, ced-3 and ced-4 are involved in activation of apoptosis, and ced-9 inhibits apoptosis. after the discovery of caspases as ced-3 homologs, a search for activators and inhibitors analogous to ced-4 and ced-9 led to the discovery of diverse mammalian regulators of apoptosis. the plethora of these molecules and their functional diversity allowed them to be classifi ed into four broad categories: (1) adapter proteins, (2) the bcl-2 family of regulators, (3) inhibitors of apoptosis (iaps), and (4) other regulators. as stated earlier, two major pathways of apoptosis, involving either the initiator caspase-8 or the initiator caspase-9 (see figure 4 .2), have been recognized. signaling by death receptors (cd95, tnfri) occurs through a well-defi ned process of recruitment of caspase-8 to the death receptor by adapter proteins such as fadd. recruitment occurs through interactions between the death domains that are present on both receptor and adapter proteins. receptorbound fadd then recruits caspase-8 through interactions between deds common to both caspase-8 and fadd forming a disc. in the disc, caspase-8 activation occurs through oligomerization and autocatalysis. activated caspase-8 then activates downstream caspase-3, culminating in apoptosis. the inhibitory protein, flip was shown to block fas-induced and tnf-α-induced disc formation and subsequent activation of caspase-8. 39 of particular interest is cellular flip, which stimulates caspase-8 activation at physiologically relevant levels and inhibited apoptosis upon high ectopic expression. 51 cellular flip contains two deds that can compete with caspase-8 for recruitment to the disc. this limits the degree of association of caspase-8 with fadd and thus limits activation of the caspase cascade. it also forms a heterodimer with caspase-8 and caspase-10 through interactions between both the deds and the caspase-like domains of the proteins, thus activating both caspase-8 and caspase-10. 52 apoptotic protease activating factor-1 (apaf-1), a ced-4 homolog in mammalian cells, affects the activation of initiator caspase-9. 53 this factor binds to procaspase-9 in the presence of cytochrome c and 2′deoxyadenosine 5′-triphosphate (datp) or adenosine triphosphate (atp) and activates this protease, which in turn activates a downstream cascade of proteases (see figure 4 .2). 54 by and large, apaf-1 defi ciency is embryonically lethal and the embryos exhibit brain abnormalities similar to those seen in caspase-9 knockout mice. 55 these genetic fi ndings support the idea that apaf-1 is coupled to caspase-9 in the death pathway. unlike ced-4 in nematodes, apaf-1 requires the binding of atp and cytochrome c to activate procaspase-9. the multiple wd40 repeats in the c-terminal end of apaf-1 have a regulatory role in the activation of caspase-9. 56 the ced-9 homolog in mammals is the bcl-2 protein. bcl-2 was fi rst discovered in b-cell lymphoma as a protooncogene. overexpression of bcl-2 was shown to offer protection against a variety of death stimuli. 57 the bcl-2 protein family includes both proapoptotic (bcl-2, bcl-xl, bcl-w, mcl-1, nr13, and a1/bfl -1) and antiapoptotic proteins (bax, bak, bok, diva, bcl-xs, bik, bim, hrk, nip3, nix, bad, and bid). 58 these proteins are characterized by the presence of bcl-2 homology (bh) domains: bh1, bh2, bh3, and bh4 (figure 4.3) . the proapoptotic members have two subfamilies: a multidomain and a bh3-only group (see figure 4 .3). the relative ratio of pro-and antiapoptotic proteins determines the sensitivity of cells to various apoptotic stimuli. the best-studied proapoptotic members are bax and bid. exposure to various apoptotic stimuli leads to translocation of cytosolic bax from the cytosol to the mitochondrial membrane. 59 bax oligomerizes on the mitochondrial membrane along with another proapoptotic protein, bak, leading to the release of cytochrome c from the mitochondrial membrane into the cytosol. 60 other proapoptotic proteins, mainly the bh3-only proteins, are thought to aid in bax-bak oligomerization on the mitochondrial membrane. the antiapoptotic bcl-2 family members are known to block bax-bak oligomerization on the mitochondrial membrane and subsequent release of cytochrome c into the cytosol. 60, 61 after release from the mitochondria, cytochrome c is known to interact with the wd40 repeats of the adaptor protein apaf-1, resulting in the formation of the apoptosome complex. seven molecules of apaf-1, interacting through their n-terminal caspase activation and recruitment domain, form the central hub region of the symmetric wheel-like structure, the apoptosome. binding of atp/datp to apaf-1 triggers the formation of the apoptosome, which subsequently recruits procaspase-9 into the apoptosome complex, resulting in its activation 62 . activated caspase-9 then activates executioner caspases, such as caspase-3 and caspase-7, eventually leading to programmed cell death. the iaps, fi rst discovered in baculoviruses and then in insects and drosophila, inhibit activated caspases by directly binding to the active enzymes. 63 these proteins contain one or more baculovirus inhibitor of apoptosis repeat domains, which are responsible for the caspase inhibitory activity. 64 to date, eight mammalian iaps have been identifi ed. they include x-linked iap (xiap), c-iap1, c-iap2, melanoma iap (ml-iap)/livin, iaplike protein-2 (ilp-2), neuronal apoptosis-inhibitory protein (naip), bruce/apollon, and survivin. in mammals, caspase-3, -7, and -9 are inhibited by iaps. 62 there are reports suggesting aberrant expression of iaps in many cancer tissues. for example, ciap1 is overexpressed in esophageal squamous cell sarcoma 65 ; ciap2 locus is translocated in mucosa-associate lymphoid lymphoma 66 and survivin has been shown to be upregulated in many cancer cells. 67 the caspase inhibitory activity of iaps is inhibited by proteins containing an iap-binding tetrapeptide motif. 62 the founding member of this family is smac/diablo, which is released from the mitochondrial intermembrane space into the cytosol during apoptosis. in the cytosol, it interacts with several iaps and inhibits their function. the other mitochondrial protein, omi/htra2, is also known to antagonize xiap-mediated inhibition of caspase-9 at high concentrations. 68 a serine protease, omi/htra2 can proteolytically cleave and inactivate iap proteins and thus is considered to be a more potent suppressor of iaps than smac. 69 it has been reported that the heat shock proteins hsp90, hsp70, and hsp27 can inhibit caspase activation by cytochrome c either by interacting with apaf-1 or other players in the pathway. [70] [71] [72] a high-throughput screen identifi ed a compound called petcm (α-[trichloromethyl]-4-pyridineethanol) as a caspase-3 activator. further work with petcm revealed its involvement in apoptosome regulation. 73 this pathway also includes oncoprotein prothymosin-α and tumor suppressor putative hla-dr-associated proteins. these proteins were shown to promote caspase-9 activation after apoptosome formation, whereas prothymosin-α inhibited caspase-9 activation by inhibiting apoptosome formation. in an apoptotic cell, the regulatory, structural, and housekeeping proteins are the main targets of the caspases. the regulatory proteins mitogen-activated protein/extracellular signal-regulated kinase kinase-1, p21-activated kinase-2, and mst-1 are activated upon cleavage by caspases. 74 caspase-mediated protein hydrolysis inactivates other proteins, including focal adhesion kinase, phosphatidylinositol-3 kinase, akt, raf-1, iaps, and inhibitors of caspase-activated dnase (icad). caspases also convert the antiapoptotic protein bcl-2 into a proapoptotic protein such as bax upon cleavage. there are many structural protein targets of caspases, which include nuclear lamins, actin, and regulatory proteins such as spectrin, gelsolin, and fodrins. 75 degradation of nuclear dna into internucleosomal chromatin fragments is one of the hallmarks of apoptotic cell death that occurs in response to various apoptotic stimuli in a wide variety of cells. a specifi c dnase, cad (caspase-activated dnase), that cleaves chromosomal dna in a caspase-dependent manner, is synthesized with the help of icad. in proliferating cells, cad is always found to be associated with icad in the cytosol. when cells are undergoing apoptosis, caspases (particularly caspase-3) cleave icad to release cad and allow its translocation to the nucleus to cleave chromosomal dna. thus, cells that are icad defi cient or that express caspase-resistant icad mutant do not exhibit dna fragmentation during apoptosis. apoptosis plays a critical role in the postnatal lung. 76 regulated removal of infl ammatory cells by apoptosis helps in the resolution of infl ammation in the lung. 77 recent evidence also supports a role for apoptosis in the remodeling of lung tissue after acute lung injury 78 and in the pathogenesis of chronic pulmonary hypertension, 79 idiopathic pulmonary fi brosis, and chronic obstructive pulmonary disease. 80, 81 acute lung injury/acute respiratory distress syndrome acute lung injury, which clinically manifests itself as the acute respiratory distress syndrome (ards), involves disruption of the alveolar epithelium and endothelium, increased vascular permeability, and edema. two main hypotheses link the pathogenesis of ards to apoptosis, namely, the "neutrophilic hypothesis" and the "epithelial hypothesis." these two hypotheses are not mutually exclusive, and both could play important roles in the pathogenesis of ards. the neutrophilic hypothesis suggests that neutrophil apoptosis plays an important role in the resolution of infl ammation and that the inhibition of neutrophil apoptosis or the inhibition of clearance of apoptotic neutrophils is deleterious in ards. 82, 83 studies in humans showed that bronchoalveolar lavage fl uids from patients with early ards inhibit the rate at which neutrophils develop apoptosis in vitro. 84 the inhibitory effect of bronchoalveolar lavage fl uids on neutrophil apoptosis is mediated by granulocyte/macrophage colony-stimulating factor, and possibly by il-8 and il-2. 85,86 a membrane surface molecule, cd44, has been shown to play an important role in the clearance of apoptotic cells in vivo and in vitro. 87 in a model of bleomycin-induced lung injury, cd44-defi cient mice failed to clear apoptotic neutrophils, which was associated with worsened infl ammation and increased mortality. 87 activation of phagocytic cells inhibits production of proinfl ammatory cytokines, including il-1β, il-8, il-10, granulocyte/ macrophage colony-stimulating factor, and tnf-α and increases release of anti-infl ammatory mediators such as transforming growth factor-β, prostaglandin e 2 , and platelet-activating factor. 88, 89 the net effects of these changes could favor resolution of infl ammation. the epithelial hypothesis suggests that the apoptotic death of alveolar epithelial cells, in response to soluble mediators such as fas ligand, contributes to the prominent alveolar epithelial injury characteristic of ards. several lines of evidence suggest a role for the fas/fas ligand system in epithelial cell apoptosis. 90 fas is expressed on alveolar and airway epithelial cells, 91, 92 and its expression increases in response to infl ammatory mediators such as lipopolysaccharide. fas-mediated lung cell apoptosis is modulated by surfactant protein a, which inhibits apoptosis in vivo. 93 chronic obstructive pulmonary disease chronic obstructive pulmonary disease, caused primarily by smoking, generally refers to chronic bronchitis and emphysema. several factors, including protease/antiprotease imbalance, oxidative stress, cigarette smokederived toxins, and infl ammation mediated by neutrophils, macrophages, and cd8 + t cells, have been shown to contribute to the disease process. furthermore, matrix metalloproteinase 94 and vascular endothelial growth factor receptor inhibition, 95, 96 but not fas/fas ligand, have been shown to play role in the development of emphysema. asthma allergic asthma is characterized by intermittent or persistent bronchoconstriction and has been linked to airway remodeling and chronic infl ammation, with increased numbers of eosinophils, cd4 + t cells, and mast cells. although at present a role for apoptosis in asthma is not confi rmed, studies ex vivo have shown reduced apoptosis of circulating peripheral cd4 + t cells and eosinophils in asthma, which might contribute to infl ammation. corticosteroids used to reduce infl ammation in asthma have been shown to induce eosinophil apoptosis. 97 pulmonary fi brosis is characterized by epithelial damage, fi broblast proliferation, and deposition of collagen. although the mechanism of alveolar epithelial cell apoptosis in pulmonary fi brosis is not known, several reports have suggested fas pathway, 98 angiotensin pathway, 99 activated t cell-derived perforin, 100 il-13 stimulation, 101 and transforming growth factor-β1 activation 102 to play critical roles. because insuffi cient apoptosis is often associated with tumorigenesis, modulation of apoptotic and antiapoptotic targets seems to be an attractive approach to cancer therapy. lung cancers can be divided into small cell lung cancers (sclcs) and non-small cell lung cancers (nsclcs). 103 the sclcs are relatively more sensitive to anticancer drugs and irradiation than are the nsclcs, 104 but the molecular basis for this difference is not clearly known. evaluation of apoptosis-associated substances has shown that caspase-8, fas, and fas ligand are often downregulated in sclcs but not in nsclcs. 105 an investigation of the basis for these differences revealed that there were no differences in the levels of bax and bcl-xl, but the expression of bcl-2 was found to be signifi cantly higher in sclc than in nsclc cell lines. the observation that in some cases bcl-2 can be converted into a proapoptotic bax-like death molecule may offer an explanation for the paradoxic expression of bcl-2 in sclc. 106 the lack of expression of procaspase-1, -4, -8, and -10 107 reported in sclc suggests that these caspases probably do not contribute to spontaneous apoptosis in these cells. apoptosis regulators apaf-1 and procaspase-3 are overexpressed and are functional in nsclc cell lines. in both types of lung cancer, apoptotic stimuli result in cytochrome c release and activation of caspase-9 and caspase-3, but only sclc cell lines showed a relocalization of caspase-3 into the nucleus 108 ; this suggests that the resistance of nsclc cell lines is probably due to defective relocalization of caspase-3. the expression of caspase-9 and caspase-7 in nsclcs was found to be similar to normal lung tissue. 109 however, these cell lines express the apoptosis inhibitor and splice variant of caspase-9 casp9b. in vitro, chemotherapy-resistant nsclc cell lines exhibit decreased caspase-9 and caspase-3 expression, 110 which suggests an inhibition of apoptosis induction via apoptosome formation in nsclc. additionally, both nsclc and sclc cells express high and almost equal levels of survivin. 107 the resistant nsclc cells showed higher expression of c-iap2, and the radiosensitive sclc cells exhibited increased expression of xiap. 111 these results suggest no correlation between the level of expression of the iaps and the difference in the radiosensitivity between nsclc and sclc cells. cell death has become an area of intense interest and investigation in science and medicine because of the recognition that cell death, in general, and apoptosis, in par-ticular, are important features of many biologic processes. involvement of many genes in the death process suggests that cell death is a complex phenomenon with many redundant mechanisms to ensure defi nitiveness. the realization that defective cell death plays a central role in the pathogenesis of diseases has stimulated work on therapies targeted to these processes, and this work will undoubtedly continue in the future. apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics genetic control of programmed cell death in the nematode c. elegans programmed cell death in animal development apoptosis: defi nition, mechanisms, and relevance to disease apoptosis as a therapeutic target for the treatment of lung disease four deaths and a funeral: from caspases to alternative mechanisms dual signaling of the fas receptor: initiation of both apoptotic and necrotic cell death pathways alkylating dna damage stimulates a regulated form of necrotic cell death a novel response of cancer cells to radiation involves autophagy and formation of acidic vesicles autophagy: in sickness and in health tissue protein turnover during liver carcinogenesis reduced autophagic activity in primary rat hepatocellular carcinoma and ascites hepatoma cells regulation of an atg7-beclin 1 program of autophagic cell death by caspase-8 role of bcl-2 family proteins in a non-apoptotic programmed cell death dependent on autophagy genes an alternative, nonapoptotic form of programmed cell death paraptosis: mediation by map kinases and inhibition by aip-1/alix autoschizis: a novel cell death inhibition of the development of metastases by dietary vitamin c:k3 combination autoschizis: a new form of cell death for human ovarian carcinoma cells following ascorbate/menadione treatment. nuclear and dna degradation the molecular biology of apoptosis evolutionary conservation of a genetic pathway of programmed cell death interaction between the c. elegans cell-death regulators ced-9 and ced-4 interaction and regulation of the caenorhabditis elegans death protease ced-3 by ced-4 and ced-9 caspases: enemies within vital functions for lethal caspases mechanism of xiapmediated inhibition of caspase-9 insights into the regulatory mechanism for caspase-8 activation a combinatorial approach defi nes specifi cities of members of the caspase family and granzyme b. functional relationships established for key mediators of apoptosis signalling by cd95 and tnf receptors: not only life and death apoptosis control by death and decoy receptors the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation fadd, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis caspase-6 is the direct activator of caspase-8 in the cytochrome c-induced apoptosis pathway: absolute requirement for removal of caspase-6 prodomain two cd95 (apo-1/fas) signaling pathways induction of cell death by tumour necrosis factor (tnf) receptor 2, cd40 and cd30: a role for tnf-r1 activation by endogenous membrane-anchored tnf tumor necrosis factor (tnf) receptor 1 signaling downstream of tnf receptor-associated factor 2. nuclear factor kappab (nfkappab)-inducing kinase requirement for activation of activating protein 1 and nfkappab but not of c-jun nterminal kinase/stress-activated protein kinase involvement of mach, a novel mort1/fadd-interacting protease, in fas/apo-1-and tnf receptor-induced cell death the ced-4-homologous protein flash is involved in fas-mediated activation of caspase-8 during apoptosis viral fliceinhibitory proteins (flips) prevent apoptosis induced by death receptors memory and distribution of virus-specifi c cytotoxic t lymphocytes (ctls) and ctl precursors after rotavirus infection fasdependent cd4 + cytotoxic t-cell-mediated pathogenesis during virus infection transcriptional regulation during p21waf1/cip1-induced apoptosis in human ovarian cancer cells activation of c-jun nh2-terminal kinase/stress-activated protein kinase (jnk/ sapk) is critical for hypoxia-induced apoptosis of human malignant melanoma characterization of mice defi cient in interleukin-1 beta converting enzyme reduced ischemic brain injury in interleukin-1 beta converting enzyme-defi cient mice caspase-3 is required for dna fragmentation and morphological changes associated with apoptosis targeted disruption of the mouse caspase 8 gene ablates cell death induction by the tnf receptors, fas/apo1, and dr3 and is lethal prenatally fadd: essential for embryo development and signaling from some, but not all, inducers of apoptosis reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9 the ins and outs of programmed cell death during c. elegans development c-flip(l) is a dual function regulator for caspase-8 activation and cd95-mediated apoptosis the fl ip side of flip apaf-1, a human protein homologous to c. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3 an apaf-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9 apaf1 (ced-4 homolog) regulates programmed cell death in mammalian development autoactivation of procaspase-9 by apaf-1-mediated oligomerization bcl-2 inhibits death of central neural cells induced by multiple agents bcl-2 family proteins role of hypoxia-induced bax translocation and cytochrome c release in reoxygenation injury association of bax and bak homo-oligomers in mitochondria. bax requirement for bak reorganization and cytochrome c release bcl-2 prevents bax oligomerization in the mitochondrial outer membrane mechanisms of caspase activation and inhibition during apoptosis diablo promotes apoptosis by removing miha/xiap from processed caspase 9 iap family proteins-suppressors of apoptosis identifi cation of ciap1 as a candidate target gene within an amplicon at 11q22 in esophageal squamous cell carcinomas the apoptosis inhibitor gene api2 and a novel 18q gene, mlt, are recurrently rearranged in the t(11;18)(q21;q21) associated with mucosa-associated lymphoid tissue lymphomas a novel anti-apoptosis gene, survivin, expressed in cancer and lymphoma a serine protease, htra2, is released from the mitochondria and interacts with xiap, inducing cell death omi/htra2 catalytic cleavage of inhibitor of apoptosis (iap) irreversibly inactivates iaps and facilitates caspase activity in apoptosis hsp27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3 heat-shock protein 70 inhibits apoptosis by preventing recruitment of procaspase-9 to the apaf-1 apoptosome negative regulation of cytochrome c-mediated oligomerization of apaf-1 and activation of procaspase-9 by heat shock protein 90 distinctive roles of phap proteins and prothymosin-alpha in a death regulatory pathway caspase-dependent cleavage of signaling proteins during apoptosis. a turn-off mechanism for anti-apoptotic signals caspasemediated proteolysis during apoptosis: insights from apoptotic neutrophils programmed cell death contributes to postnatal lung development granulocyte apoptosis and its role in the resolution and control of lung infl ammation apoptosis is a major pathway responsible for the resolution of type ii pneumocytes in acute lung injury mechanisms of structural remodeling in chronic pulmonary hypertension induction of apoptosis and pulmonary fi brosis in mice in response to ligation of fas antigen essential roles of the fas-fas ligand pathway in the development of pulmonary fi brosis granulocyte apoptosis and the control of infl ammation macrophage engulfment of apoptotic neutrophils contributes to the resolution of acute pulmonary infl ammation in vivo modulation of neutrophil apoptosis by granulocyte colony-stimulating factor and granulocyte/macrophage colony-stimulating factor during the course of acute respiratory distress syndrome g-csf and il-8 but not gm-csf correlate with severity of pulmonary neutrophilia in acute respiratory distress syndrome interleukin-2 involvement in early acute respiratory distress syndrome: relationship with polymorphonuclear neutrophil apoptosis and patient survival resolution of lung infl ammation by cd44 macrophages that have ingested apoptotic cells in vitro inhibit proinfl ammatory cytokine production through autocrine/paracrine mechanisms involving tgf-beta, pge2, and paf phosphatidylserinedependent ingestion of apoptotic cells promotes tgf-beta1 secretion and the resolution of infl ammation recombinant human fas ligand induces alveolar epithelial cell apoptosis and lung injury in rabbits fas expression in pulmonary alveolar type ii cells expression of fas (cd95) and fasl (cd95l) in human airway epithelium natural protection from apoptosis by surfactant protein a in type ii pneumocytes upregulation of gelatinases a and b, collagenases 1 and 2, and increased parenchymal cell death in copd inhibition of vegf receptors causes lung cell apoptosis and emphysema oxidative stress and apoptosis interact and cause emphysema due to vascular endothelial growth factor receptor blockade glucocorticoid-induced apoptosis in human eosinophils: mechanisms of action increased circulating levels of soluble fas ligand are correlated with disease activity in patients with fi brosing lung diseases bleomycin-induced apoptosis of alveolar epithelial cells requires angiotensin synthesis de novo the perforin mediated apoptotic pathway in lung injury and fi brosis interleukin-13 induces tissue fi brosis by selectively stimulating and activating transforming growth factor beta(1) early growth response gene 1-mediated apoptosis is essential for transforming growth factor beta1-induced pulmonary fi brosis united states lung carcinoma incidence trends: declining for most histologic types among males, increasing among females progress in understanding the molecular pathogenesis of human lung cancer loss of expression of death-inducing signaling complex (disc) components in lung cancer cell lines and the infl uence of myc amplifi cation conversion of bcl-2 to a bax-like death effector by caspases differences in expression of pro-caspases in small cell and non-small cell lung carcinoma defective caspase-3 relocalization in non-small cell lung carcinoma increased expression of apaf-1 and procaspase-3 and the functionality of intrinsic apoptosis apparatus in non-small cell lung carcinoma rescue of death receptor and mitochondrial apoptosis signaling in resistant human nsclc in vivo expression of inhibitor of apoptosis proteins in small-and non-small-cell lung carcinoma cells key: cord-023411-iszb5qlk authors: astrinidou‐vakaloudi, a.; xytsas, s.; diamanti, i.; . ioannidis, h; pangidis, p. title: presence of helicobacter pylori antibodies in haemodialysis patients date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423p.x sha: doc_id: 23411 cord_uid: iszb5qlk aim: renal dysfunction may influence the colonization of gastric mucosa by urea‐splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end‐stage renal disease (esrd), receiving long‐term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(+) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-275730-650sjwyy authors: gogoi, himanshu; mansouri, samira; jin, lei title: the age of cyclic dinucleotide vaccine adjuvants date: 2020-08-13 journal: vaccines (basel) doi: 10.3390/vaccines8030453 sha: doc_id: 275730 cord_uid: 650sjwyy as prophylactic vaccine adjuvants for infectious diseases, cyclic dinucleotides (cdns) induce safe, potent, long-lasting humoral and cellular memory responses in the systemic and mucosal compartments. as therapeutic cancer vaccine adjuvants, cdns induce potent anti-tumor immunity, including cytotoxic t cells and nk cells activation that achieve durable regression in multiple mouse models of tumors. clinical trials are ongoing to fulfill the promise of cdns (clinicaltrials.gov: nct02675439, nct03010176, nct03172936, and nct03937141). however, in october 2018, the first clinical data with merck’s cdn mk-1454 showed zero activity as a monotherapy in patients with solid tumors or lymphomas (nct03010176). lately, the clinical trial from aduro’s cdn adu-s100 monotherapy was also disappointing (nct03172936). the emerging hurdle in cdn vaccine development calls for a timely re-evaluation of our understanding on cdn vaccine adjuvants. here, we review the status of cdn vaccine adjuvant research, including their superior adjuvant activities, in vivo mode of action, and confounding factors that affect their efficacy in humans. lastly, we discuss the strategies to overcome the hurdle and advance promising cdn adjuvants in humans. vaccines, by no doubt, are one of the most crowning achievements of medical science. the success of edward jenner's smallpox vaccine and louis pasteur's anthrax and rabies vaccines demonstrated the protection from the disease without transferring the disease itself. these pioneer works lay the foundation for immunology as edward jenner is considered the father of immunology. vaccine development has come a long way since then and significantly reduced disease burden worldwide. today, a new era of recombinant and subunit vaccines with improved reactogenicity and safety profile has emerged. however, these new generations of vaccines require the addition of an adjuvant to enhance their vaccine property. vaccine adjuvants determine the magnitude and quality of vaccine responses while maintaining a safety profile. since its discovery by alexander thomas glenny in the 1930s, alum is the choice of an adjuvant in commercially available vaccines [1] . as a vaccine adjuvant, alum enhances antibody production but is limited to the th2 response, which is not adequate for the protection from intracellular pathogens that require th1 and cd8 + cytotoxic t cell responses. despite intensive research on new vaccine adjuvants, very few adjuvants have been licensed for commercial use today (table 1) . furthermore, along with the new vaccine adjuvants such as mf59, as03, and as04, the development of new safe, potent vaccine adjuvants that generates long-lasting and balanced vaccine responses for intracellular and extracellular pathogens is a high priority for public health. cancer-related death is the second leading cause of death. the current regimen of cancer therapies are effective in extending the patients' life but generally fail to prevent relapse and death. immunotherapy, by activating the immune responses and generating memory cytotoxic cd8 + t cells, can potentially prevent tumor metastasis, regression, and cure cancer. cancer development is accompanied by an immunosuppressive microenvironment and t cell dysfunction and exhaustion [24] [25] [26] . methods to break tumor tolerance and activate anti-tumor immunity are highly desirable. cdns have a potent anti-tumor activity. demaria et al. showed that intratumoral injection of cgamp enhanced the anti-tumor cd8 + t cell response inhibiting the growth of injected tumors in mice models of melanoma and colon cancer [27] . intratumoral injection of cgamp in b16f10 lung metastasis induced a systemic cd8 + t cell response to restrict the growth of distant tumors [28] . nicolai et al. showed that the cda activated natural killer (nk) cells-dependent and cd8 + t cell-independent mechanism for tumor rejection originated from different tissue types [29] . cda induced tumor regression even in rag2 -/mice but was strongly depleted in rag2 -/-il2rg -/mice lacking nk cells, b, and t cells [29] . moreover, suppressing the nk cell activity by nk1.1 antibody resulted in rapid tumor growth of mc-38-b2m-/-(colorectal), b16-f10-b2m-/-(melanoma), ct26-b2m-/-(colorectal), c1498-b2m-/-(leukemic), and rma-b2m-/-(lymphoma) tumour models [29] . the study was clinically relevant as human tumors such as hodgkin's lymphoma often exhibit a partial loss of mhc i thereby unable to activate a ctl response. activating nk cell-mediated anti-tumor immunity then becomes essential. a major hurdle in cancer immunotherapy is the "cold" tumors, which lack inherent immunogenicity reflecting in the inability of checkpoint inhibitors to attenuate tumor growth. a study by francica et al. showed that the cdn mediated tumor necrosis factor (tnf)-α production by innate immune cells are responsible for acute tumor clearance, and blocking tnf-α inhibits tumor necrosis and clearance against "cold" tumors [30] . lastly, cdns effectively induce dc cross-presentation to the mhc class i molecules to produce a ctl response [18] . a potent antigen-specific cd8 + memory t cell response by in vivo targeting of the dec-205 receptor in dc, thereby provided an effective approach to target viral infections [18] . combinatorial methods such as intramuscular immunization of cda with alum have shown significant enhancement in antigen-specific cd4 + and cd8 + t cell responses while maintaining a safety profile [31] . the mucosal surface is a natural point of entry for many respiratory pathogens such as influenza, streptococcus pneumoniae, mycobacterium tuberculosis, staphylococcus aureus, b. anthracis, coronavirus, rotavirus, etc. moreover, immunization at one mucosal surface, particularly intranasal vaccination in mice, monkeys, and humans has generated not only a local iga response, but also enhanced the iga response in salivary glands, upper and lower respiratory tracts, and even in distant genital tracts, and the small and large intestines [32] [33] [34] [35] [36] [37] . furthermore, rectal immunization of mice with a non-living peptide-based vaccine effectively induced a systemic ctl response [38] . despite its role in providing a frontline of defense, immunization via the mucosal route has not been significantly explored, which is reflected by the limited number of available mucosal vaccines against influenza (flumist), salmonella typhi (vivotif), rotavirus (rotateq, rotatrix), vibrio cholerae (dukoral, shanchol), and adeno virus [39] . a major obstacle in the development of a mucosal vaccine is the availability of a potent mucosal adjuvant, which not only generates a local mucosal iga response but also enhances mucosal th and ctl responses along with a systemic reaction. the most common mucosal immunostimulatory molecule used for vaccine studies are the bacterial enterotoxins, cholera toxin, and e. coli heat-labile toxin. mechanistically, these toxins form a pentameric subunit, which binds to the gangliosides (preferentially gm1), thereby facilitating the uptake. recent studies, however, raised safety concerns. studies found that the enterotoxins are accumulated in the olfactory epithelium nerves and the olfactory bulb that induce inflammatory responses in meninges, the olfactory nerve, and glomerular layers of the olfactory bulb promoting neuronal damage [40] [41] [42] . cdns have received enormous interest as potential mucosal adjuvants. in 2007, ebensen et al. first demonstrated cdns as a mucosal adjuvant [9] . they showed that cdg/β-gal i.n. immunized mice not only mounted a systemic igg response but also generated an enhanced β-gal specific iga response after 42 days in lung balf and vaginal lavage [9] . intranasal immunization of cdg/β-gal also enhanced the serum igg2a and igg1 production by 640-and 320-fold as compared to only β-gal immunized mice [9] . the systemic cellular response was also enhanced by cdg/β-gal immunization, as was observed with the production of interferon (ifn)-γ (2000-fold), il-5, and il-2 in the ex vivo recall assay compared to only β-gal immunized mice [9] . cdns also induce mucosal th17 [7, 19] . the mucosal adjuvant potential of cdns was further strengthened when madhun et al. showed that i.n. administration of a plant-based h5n1 influenza antigen induced high frequencies of multifunctional th1 cells [8] . two doses of the cdg adjuvanted vaccine were able to mount a very high mucosal iga response and protected against antigenically drifted h5n1 viruses [8] . in comparison, the same vaccine when it immunized i.m. did not generate a th1 response, which is critical for viral infections [8] . the safety profile of the vaccine adjuvant is crucial while administration avoids unwanted side effects. histopathology studies and cytokine profiling suggested that the cdg adjuvanted vaccine also induced the production of anti-inflammatory cytokine-like il-10, thereby maintaining a balanced pro-and anti-inflammatory cytokine profile [43] . cdg also induced the production of il-22, thereby facilitating lung epithelium repair along with effectively clearing s. pneumoniae from the organs [43] . thus, cdns have shown promising and safe mucosal vaccine activities in animal models. distinct from the cdn-sting-type i ifns-immunity paradigm, andrew mellor's group described a cdn-sting-type i ifns-ido1-tolerance signaling that mediates potent t-reg responses and suppresses inflammation [44] . they first showed that dna nanoparticles (dnp, containing pei and plasmid dna lacking tlr9 ligands) activate sting-dependent ido-1 production that induces tolerance [44] . later, they showed that the cdg treatment delayed the experimental autoimmune encephalitis (eae) onset and reduced disease severity [45] . they identified a new cdg-sting/type i ifns/ido1/tregs pathway that suppresses cns-specific autoimmunity [45] . recently, the same group showed that the cdn-sting-ido-1 activity in the tumor microenvironment (tme) promoted the growth of low antigenicity lewis lung carcinoma (llc) [46] . lastly, they showed that treating pre-diabetic nod mice with cgamp delayed the type i diabetes (t1d) onset [47] that depends on type i ifns. thus, cdn may be used as immune-modulatory drugs as well. ifnβ (avonex ® , biogen, ma, usa; rebif ® , merck, darmstadt, germany) has been used to treat multiple sclerosis for over 20 years. however, the in vivo mechanism of ifnβ-induced tolerance and targeted cell population remain unknown [48] . cdn-induced ifnβ may have a similar tolerance genetic effect in vivo, particularly if cdns are targeted to the tolerogenic dcs in vivo. noteworthy, mellor's group found that while the cgamp treatment delayed the t1d onset in nonobese diabetic (nod) mice, the cdg treatment did not [47] . a further analysis showed that cdg, unlike cgamp, did not generate type i ifns in nod mice, though nod mice still produced tnf in response to cdg [47] . lastly, they found that the nod mice contain an n210d sting polymorphism that may explain differential type i ifns responses by cgamp and cdg [47] . previous studies had similar findings on the selectivity of cdns in activating sting variants in mice. the r231a [49] or r231h [50] change in the mouse sting rendered the variants selectively to lose the response to cdg but not cgamp. notably, both human and mouse sting genes have the n210 residue while sting genes from the rat, pig, cat, dog, ferret, and hamster have the d210 residue. cdg may lose its type i ifns-dependent anti-tumor and tolerance-inducing function in these model organisms. cdns carry negative charges that may prevent them from crossing the plasma membrane to activate sting in the cytosol. indeed, in vitro cell activation by cdns requires transfection reagents or membrane permeabilization to deliver cdns into the cytosol. the only cdns that directly activate cells in culture without the need for membrane permeabilization is the synthetic cdn rprpss-cda [51] . in vivo, i.n., i.p., s.c. administration of cdns can all induce vaccine responses in mice without the need for transfection reagents [8, 9, 15, 31, 52] . these cdns are likely taken up by dcs and phagocytes directly by pinocytosis or phagocytosis [43] . with the advent of nanotechnology, nanoparticle, and microparticle mediated delivery vehicle such as liposomes, emulsions, virus-like particles, biodegradable polymers, immune-stimulating complexes (iscom) have received tremendous attention. these new delivery methods provide a sustained release of the cargo from the matrix and improve bioavailability and dosing frequency [53, 54] . thus, nanoparticle or microparticle encapsulated cdns have been fabricated and studied as an alternative delivery approach of cdns in vivo [10, [55] [56] [57] (table 2) . table 2 . cyclic dinucleotide as vaccine adjuvants. rprp-ss-cdg/ml-rprp-ss-cgamp in addvax [squalene based oil-in-water nano emulsion] (s.c.) or in pbs (i.n.) [11] th1, th17 responses; protection against mtb infection cgamp in pulmonary surfactant mimicking liposome (i.n.) [10] cd8 + t cell response; 100% protection against a 10ld 50 virulent strain of ca09 h1n1 cdg in peg lipid nanoparticles (s.c.) [58] tfh and gc b cell induction cgamp in ultra-ph sensitive nanoparticle ex vivo human pbmc [59] il-6, il-8, g-csf, tnf-a, type i ifns, mip-1α production from hiv infected pbmc, and inhibited hiv i replication cdg in pbs (i.n., i.m. s.c., i.p.) [4, 5, 8, 9, 13, 15, 43] systemic and mucosal humoral and cellular immune responses that protect from bacterial infections cda with alum (i.m.) or in pbs (s.c. or i.n.) [7, 18, 31] balanced igg1 and igg2a responses; th1/th2/th17 responses table 2 . cont. (stingel) synthetic ml-rprp-ss-cda in multidomain peptide hydrogel (s.c.) [60] slow-release of cdn and highly effective in maintaining tumor clearance and rejecting tumor growth and provide durable anti-tumor immunity cdg in ysk05 liposome (s.c.) [61] activation of antigen-specific ctl activity and restrict murine tumor growth completely adu-s100 (synthetic ml-rprp-ss-cda) in combination with anti-pd1 or anti-ctla4 antibody (intratumoral) [62] low-dosage promotes local sting activation primarily in monocytic cell lineages whereas a high-dose resulted in cellular activation at distal sites a combination of adu-s100 and α-cpi provide durable immunity even against cold tumors synthetic rprp-ss-cdg and ml-rprp-cda in pbae nanoparticles and anti-pd1 antibody (intratumoral) [56] enhanced shelf-life stability for up to nine months. a 10-fold lower dose of pbae/cdn nanoparticles with α-pd1 antibody resulted in significantly reduced tumor growth and reduction as compared to soluble cdn cgamp in acetalated dextran mps (i.p., i.m., i.v., and intratumoral) [57] tumor reduction at 100-1000 fold lower dose as compared to soluble cgamp the synergistic effect of nk cells and cd8 + t cells resulted in early anti-tumor efficacy in a triple-negative breast cancer model cgamp in ph-responsive polymersomes (intratumoral) [63] recruitment of active neutrophil and neutrophil chemokine cxcl1 in tme, the polarization of m2 macrophage towards inflammatory profile; enhanced cdn accumulation in lymph nodes and inhibited contralateral tumor growth. increased cd8 + and cd4 + t cells with enhanced cd8 + /cd4 + t cell ratio in tme cgamp in cationic liposomes (variable peg levels) (i.v.) [64] durable anti-tumor immunity against a b16f10 orthotopic skin melanoma model the anti-tumor activity of cgamp in liposome was obtained at 10-100-fold lower concentration than soluble cgamp cdg in cationic silica nps (intratumoral) [65] a single intratumoral dose showed 37.5% protection and 100% protection upon tumor re-challenge intratumoral treatment with cdg/np in ova expressing b16-f10 melanoma cell line induced ifnγ and tnfα producing ova-specific cd8 + t cells in pbmc adu-s100 (synthetic ml-rprp-ss-cda) in saline (intratumoral) [29, 30] nk cell-mediated and cd8 + t cell dispensable tumor regression and clearance in local and distal tumor sites. increased cytokines and chemokines and innate immune cells (macrophages, neutrophils) in the tme and tumor dlns leading to complete tumor clearance cgamp in phosphatidylserine liposome/radiotherapy (inhalation) [66] enhanced cytosolic release of cgamp, efficient sting signaling, and type i ifn production in apcs resulting in a pro-inflammatory tme and suppressed tregs production in tme. liposome/cgamp and radiation therapy-induced complete regression of lung metastasis and inhibited metastasis in both the irradiated and non-irradiated lung stingvax soluble cda/synthetic rr-s2-cda/synthetic ml-rr-s2-cda (s.c.) [17] enhanced ifn-γ + tumor-infiltrating cd8 + t cells. stingvax in combination with synthetic cda-induced type i ifn, mhc i, cd80, cd83, cd86 in monocytes and dc obtained from donor pbmcs synthetic dmxaa (i.p., i.v., intratumoral) [16, 67, 68] tumor-specific cd8 + t cell, and long term protection in a c1498.siy acute leukemia model anti-tumor immunity in distal tumor sites macrophage repolarization in non-small cell lung cancer model compared to the soluble cgamp [55] . however, the antigen-specific antibody levels of cgamp immunized vs. cgamp/lh hydrogel immunized mice were comparable [55] . moreover, in 2015, hanson m.c. et al. showed that cdg encapsulated in a pegylated liposome enhanced its retention in the lymph node by 15-fold, which otherwise disseminated into the blood following s.c. immunization [58] . incorporation of hiv gp41 antigen membrane-proximal external protein (mper) with a cdg encapsulated liposome enhanced 5.3-fold antigen-specific tfh cell formation as compared to cdg or liposome only immunized mice [58] . there were improved germinal center b cell production and 11-fold increased antigen-specific igg titers [58] . lastly, the humoral responses were more durable than vaccines administered with the tlr agonist mpla [58] . microparticles have the advantage of biocompatibility, tunable biodegradability, ease of synthesis, and stability at elevated temperatures. acetylated-dextran encapsulating cgamp microparticles enhanced type i ifns responses by nearly 1000-fold in vitro and 50-fold in vivo [69] . cgamp microparticles (i.m.) increased antigen-specific antibody titers by~100-fold, enhanced th1 responses, and expanded germinal center b cells [69] . the encapsulated cgamp with hemagglutinin antigen achieved nearly complete protective immunity against a lethal challenge of influenza strain a/puerto rico/8/1934 h1n1 (pr8) seven months post-immunization [69] . the dose of cdn adjuvants (200 ng) used was 100-fold lower than previous reports [69] . lastly, the encapsulated cgamp elicited no observable toxicity in animals [69] . most recently, wang j. et al. designed a biomimetic pulmonary surfactant encapsulating the cgamp (ps-gamp) perth h3n2 vaccine [10] . the ps-gamp adjuvanted flu vaccine (i.n.) provided heterotypic cross-protection against the michigan15 h1n1 strain 30 days post-immunization in ferrets [10] . these biomimetic liposomes activated sting pathways in both alveolar macrophages and alveolar epithelial cells and enhanced the recruitment of cd11b + dc and the differentiation of cd8 + t cell and humoral response [10] . it generated heterotypic protection against h3n2, h5n1, h7n9 viruses as early as two days after a single dose of immunization and promoted the formation of a durable cd8 + trm in mice [10] . schulz et al. demonstrated that administering the cgamp/dextran microparticle (10 µg cgamp) via i.t. or i.v., i.m., or s.c. routes significantly decreased the b16f10 myeloma tumor size in mice [57] . a 100 ng of cgamp/dextran microparticle (i.t.) was sufficient to reduce the tumor size as compared to the soluble cgamp, which had to be immunized at a 10× higher concentration to obtain the same effect [57] . liposomes composed of a phospholipid bilayer can easily penetrate the cell membrane. miyabe et al. synthesized a ph-sensitive synthetic lipid ysk05 for cytosolic delivery of cdg as a result of the high fusogenic property of the lipid vesicle at acidic ph [61] . in a mouse model infected with eg7-ova cells, s.c. immunization of liposome/cdg (300 ng) completely inhibited tumor growth while the soluble cdg could not restrict tumor growth [61] . a similar study using cationic pegylated liposome and cgamp was evaluated against metastatic lung tumors in the b16f10 melanoma mode [64] . intravenous injection of liposome/cgamp in tumor-bearing mice led to over 200-fold increase of lung ifnβ [64] . liposome/cgamp treated mice showed a significantly reduced median tumor nodule length in the lung when compared to pbs-treated controls [64] . in comparison, free cgamp treated mice failed to reduce the tumor length [64] . furthermore, liposome/cgamp formulation generated tumor-specific memory and provided nearly total protection against re-challenge even after 60 days of treatment [64] . separately, in a b16.f10 melanoma model, cgamp encapsulating peg-dbp ph-responsive nanoparticles enhanced sting signaling in the tumor microenvironment (tme) [63] . a single intratumoral administration of polymersome encapsulated cgamp increased neutrophil infiltration, reduced macrophages immunosuppressive capacity, and increased dc activation in the tme [63] . in contract, mixing cgamp with the pre-formulated vesicle did not have any immunomodulatory effect [63] . the polymersome encapsulated cgamp resulted in an 11-fold decrease in tumor growth [63] . the intravenous administration of polymersome encapsulated cgamp reduced the doubling time (dt) by 50% [63] . combinatorial administration of the formulation with an immune checkpoint blockade further reduced the dt by 40%. importantly, the mice that received combinatorial therapy exhibited no evidence of tumor burden even after 55 days of treatment [63] . finally, intratumoral administration of the polymersome encapsulated cgamp did not cause any organ cytotoxicity [63] . intratumoral injection of immunostimulants is effective in the local site. however, it often fails to generate a systemic response and is ineffective on distant tumor sites. using a phosphatidylserine coated liposome loaded with cgamp (np-cgamp) in combination with radiotherapy (ir), liu and y. et al. detected significantly elevated lung ifnβ, and tnfα, ifnγ, il-6, il-12p40 in a lung metastasis mouse mode [66] . the combinatorial therapy inhibited metastases in both the ir-and non-ir-treated lungs and caused complete regression of lung metastases in some mice [66] . this combinatorial therapy also promoted an anti-tumor memory response [66] . in summary, cdns can be administered directly via i.n., s.c., or encapsulated in nano/microparticles via i.m., s.c., i.n., i.t., or i.v. routes. encapsulated cdns lower the dose (>10-fold less) needed to induce effective vaccine responses in vivo. furthermore, encapsulated cdns offer the advantage of long term storage upon lyophilization, which is desirable in developing countries [70, 71] . however, nanoparticles are often related to unwanted cytotoxicities such as cell necrosis and apoptosis [72] that may cause safety concerns in prophylactic vaccines for the general public. nevertheless, for cancer immunotherapy, the acute toxicity may be further harnessed for tumor disintegration following local treatment [73] . [74] . however, irf3 -/mice responded to cdg immunization [74] . irf3 mediates sting activated type i ifns production. the results, thus, suggested that the in vivo cdn adjuvanticity may be mediated by a sting-dependent, but irf3-type i ifns independent pathway (figure 1 ). in 2014, blaauboer s. et al. showed that cdg mucosal adjuvanticity depends on tnfr1, but not type i ifns signaling [52] . in 2019, mansour s. et al. showed that tnfr2 signaling is critical for cdg mucosal adjuvanticity in vivo [77] . tnfr2 is activated only by membrane tnf, suggesting that both secreted and membrane tnf are required for cdg adjuvant responses. type i ifns induction is the hallmark function of sting. thus, the discovery that type i ifns are dispensable for cdn-induced antibody and memory th induction was unexpected. nevertheless, several subsequent studies largely substantiated this conclusion [11, 23, 30, 58] . in the cytoplasm, cdns bind to sting dimers located on the endoplasmic reticulum (er) membrane, which undergoes conformational changes and activation. the sting (stimulator of interferon genes) activation recruits kinases tank binding kinase 1 (tbk 1) or iκb kinase (iκκε). tbk 1 phosphorylates interferon regulatory factor 3 (irf3), which dimerizes and translocates to the nucleus to activate type i ifns. iκκε phosphorylates nuclear factor-κb (nf-κb) inhibitor iκbα leading to dissociation of iκbα from nf-κb and translocation of the later to the nucleus to activate pro-inflammatory cytokines such as tnf-α. mcwhirter s. m. et al. demonstrated that i.p. immunization with cdg/(human serum albumin) hsa failed to generate an immune response in ifr3 -/-/irf7 -/-mice [74] . however, irf3 -/-mice responded to cdg immunization [74] . irf3 mediates sting activated type i ifns production. the results, thus, suggested that the in vivo cdn adjuvanticity may be mediated by a sting-dependent, but irf3-type i ifns independent pathway (figure 1 ). in 2014, blaauboer s. et al. showed that cdg mucosal adjuvanticity depends on tnfr1, but not type i ifns signaling [52] . in 2019, mansour s. et al. showed that tnfr2 signaling is critical for cdg mucosal adjuvanticity in vivo [77] . tnfr2 is activated only by membrane tnf, suggesting that both secreted and membrane tnf are required for cdg adjuvant responses. type i ifns induction is the hallmark function of sting. thus, the discovery that type i ifns are dispensable for cdn-induced antibody and memory th induction was unexpected. nevertheless, several subsequent studies largely substantiated this conclusion [11, 23, 30, 58] . mechanistically, blaauboer s. et al. showed that cdg simultaneously activates parallel irf3-type i ifns and rela-tnf signaling in bmdm and bone marrow dendritic cells (bmdcs) [52] . in 2019, mansouri s. et al. demonstrated that intranasal administration of cdg differentially activates rela in lung conventional dendritic cell 1 (cdc1), relb in lung conventional dendritic cell 2 (cdc2), and both rela and relb in lung monocyte-derived dcs (modcs) [77] . relb fl/fl cd11c cre mice are deficient in cdginduced memory th responses but retained cdg-induced antibody production [77] . the rela fl/fl cd11c cre mice lost antibody and memory th cell induction in lung mucosa but maintained antibody production and memory th induction in the systemic compartment [78] . these results suggested that cdns activate rela and relb in vivo to generate tnf, both soluble tnf and membrane tnf, that promote antibody production and memory th cells induction in vivo (figure 1 ). in the cytoplasm, cdns bind to sting dimers located on the endoplasmic reticulum (er) membrane, which undergoes conformational changes and activation. the sting (stimulator of interferon genes) activation recruits kinases tank binding kinase 1 (tbk 1) or iκb kinase (iκκε). tbk 1 phosphorylates interferon regulatory factor 3 (irf3), which dimerizes and translocates to the nucleus to activate type i ifns. iκκε phosphorylates nuclear factor-κb (nf-κb) inhibitor iκbα leading to dissociation of iκbα from nf-κb and translocation of the later to the nucleus to activate pro-inflammatory cytokines such as tnf-α. mechanistically, blaauboer s. et al. showed that cdg simultaneously activates parallel irf3-type i ifns and rela-tnf signaling in bmdm and bone marrow dendritic cells (bmdcs) [52] . in 2019, mansouri s. et al. demonstrated that intranasal administration of cdg differentially activates rela in lung conventional dendritic cell 1 (cdc1), relb in lung conventional dendritic cell 2 (cdc2), and both rela and relb in lung monocyte-derived dcs (modcs) [77] . relb fl/fl cd11c cre mice are deficient in cdg-induced memory th responses but retained cdg-induced antibody production [77] . the rela fl/fl cd11c cre mice lost antibody and memory th cell induction in lung mucosa but maintained antibody production and memory th induction in the systemic compartment [78] . these results suggested that cdns activate rela and relb in vivo to generate tnf, both soluble tnf and membrane tnf, that promote antibody production and memory th cells induction in vivo (figure 1 ). though type i ifns are dispensable for cdn-induced antibody production, cdn-induced anti-tumor immunity was dependent on type i ifns produced in the tumor microenvironment [27, [79] [80] [81] [82] [83] . bmdcs from wt and ifnar -/showed a decreased cross-presentation of the siinfekl epitope in response to cdg/ovalbumion (ova) [83] . intranasal cda immunization had an impaired cd8 + t cell function in ifnar -/mice [83] . dcs bridge innate and adaptive immune responses and direct vaccine responses. in 2015, blaauboer s. et al. showed that sting fl/fl cd11c cre mice, which lack the cdn receptor sting in cd11c + cells, including dcs, are defective in cdg-induced humoral and cellular vaccine responses [43] . thus, cdns are recognized by sting in dcs to generate adjuvant responses. dcs are a functionally heterogeneous population. blaauboer s. et al. showed that intranasal administration of cdg activates cdc1, cdc2, and modcs in the lung [43] . interestingly, mansouri s. et al. showed that the activation of modcs by cdg is indirect in vivo [77] . for the reason that is still not clear, lung modcs did not take up intranasally administered cdg [77] . instead, modcs are activated by tnf from cdg-stimulated cdc2 [77] . in 2019, mansouri s. et al. demonstrated that cdg vaccine adjuvanticity is mediated by cdc2 [77] . cdc1, though, are activated by cdg in vivo, and are largely dispensable for the humoral and memory th2/th17 induction [77] . modcs, on the other hand, are critical for generating lung mucosal iga, tfh cells, and lung memory th cells [77, 78] . in 2020, mansouri s. et al. showed that lung modcs differentiated into bcl6 + and bcl6mature modcs by secreted and transmembrane tnf from cdc2, respectively in vivo [78] . the bcl6 + and bcl6mature modcs then drive the induction of lung cd4 + memory t cells and lung tfh cells, respectively, to promote lung mucosal vaccine responses [78] (figure 2 ). bmdcs from wt and ifnar showed a decreased cross-presentation of the siinfekl epitope in response to cdg/ovalbumion (ova) [83] . intranasal cda immunization had an impaired cd8 + t cell function in ifnar -/-mice [83] . dcs bridge innate and adaptive immune responses and direct vaccine responses. in 2015, blaauboer s. et al. showed that sting fl/fl cd11c cre mice, which lack the cdn receptor sting in cd11c + cells, including dcs, are defective in cdg-induced humoral and cellular vaccine responses [43] . thus, cdns are recognized by sting in dcs to generate adjuvant responses. dcs are a functionally heterogeneous population. blaauboer s. et al. showed that intranasal administration of cdg activates cdc1, cdc2, and modcs in the lung [43] . interestingly, mansouri s. et al. showed that the activation of modcs by cdg is indirect in vivo [77] . for the reason that is still not clear, lung modcs did not take up intranasally administered cdg [77] . instead, modcs are activated by tnf from cdg-stimulated cdc2 [77] . in 2019, mansouri s. et al. demonstrated that cdg vaccine adjuvanticity is mediated by cdc2 [77] . cdc1, though, are activated by cdg in vivo, and are largely dispensable for the humoral and memory th2/th17 induction [77] . modcs, on the other hand, are critical for generating lung mucosal iga, tfh cells, and lung memory th cells [77, 78] . in 2020, mansouri s. et al. showed that lung modcs differentiated into bcl6 + and bcl6 -mature modcs by secreted and transmembrane tnf from cdc2, respectively in vivo [78] . the bcl6+ and bcl6-mature modcs then drive the induction of lung cd4 + memory t cells and lung tfh cells, respectively, to promote lung mucosal vaccine responses [78] (figure 2 ). cellular mechanism of cdn adjuvanticity by lung dcs. intranasal immunization of cdn promotes its uptake by functionally distinct lung dc subsets: cdc1, tnfr2 + cdc2, and tnfr2 -cdc2. upon the cdn uptake, cdc1 and tnfr2 + cdc2 mature and migrate towards the lung draining lymph node where they direct naïve t cells towards th1, th2, and th17 effector cells. the tnfr2 -cdc2 population, on the cdn uptake, is activated but does not migrate. instead, the tnfr2 -cdc2 produces transmembrane tnf, which engages tnfr2 on monocyte-derived dcs (modcs) to trigger lung modcs activation. activated lung modcs induce tfh, gc formation, and iga production in the lung. figure 2 . cellular mechanism of cdn adjuvanticity by lung dcs. intranasal immunization of cdn promotes its uptake by functionally distinct lung dc subsets: cdc1, tnfr2 + cdc2, and tnfr2 -cdc2. upon the cdn uptake, cdc1 and tnfr2 + cdc2 mature and migrate towards the lung draining lymph node where they direct naïve t cells towards th1, th2, and th17 effector cells. the tnfr2 -cdc2 population, on the cdn uptake, is activated but does not migrate. instead, the tnfr2 -cdc2 produces transmembrane tnf, which engages tnfr2 on monocyte-derived dcs (modcs) to trigger lung modcs activation. activated lung modcs induce tfh, gc formation, and iga production in the lung. the in vivo cellular mechanism of cdns may differ depending on their delivery methods and composition during immunization. however, activating dcs is likely a common mechanism for cdns to induce vaccine responses. for example, to enhance memory th responses, the cdn composition needs to activate cdc2. to enhance lung mucosal iga and lung memory th responses, modcs activation is a must. lastly, to generate cytotoxic cd8 + t cells responses, the cdn composition needs to activate cdc1 that specializes in antigen cross-presentation. the anti-tumor activity of sting depends on type i ifns production. however, the cellular source of type i ifns remains debatable. tumor-infiltrating dendritic cells (dcs) were identified as the major cellular source of ifnβ at the tumor site [84] . in addition to dcs, tumor cells themselves are induced to contribute to the production of ifn-β [84] . similarly, sting was essential for radiation-induced adaptive immune responses, which relied on type i ifn signaling on dcs [80] . other studies identified endothelial cells as the type i ifns producer. upon intratumoral cgamp injection in the mouse melanoma model, type i ifns were produced by endothelial cells, not dcs [27] . similarly, endothelial cells but not dcs were the principal source of spontaneously induced type i ifns in growing tumors [27] . similar results were obtained using an ex vivo model of cultured human melanoma explants [27] , and endothelial sting expression was correlated with enhanced t-cell infiltration and prolonged survival in human colon and breast cancer [79] . it was suggested that endothelial cells initiate spontaneous and therapeutic anti-tumor immunity of cdn [27, 79] . cancer-associated fibroblasts (cafs) and cancer infiltrating macrophages also produce type i ifns [85] . following transcytosis of cytoplasm (likely containing cgamp) from cancer cells into cafs, the sting pathway was activated, and ifnβ1 was produced in cafs. ifnβ then drives interferon-stimulated transcriptional programs in both cancer cells and cafs [85] . intratumoral administration of cgamp recruited cd45 + cd11b mid ly6c + cell subset to mouse tumor microenvironment of 4t1 breast cancer, squamous cell carcinomas, ct26 colon cancer, or b16f10 melanoma tissue [28] . the infiltrating cells express f4/80, mhc class ii, and secreted ifnβ, as well as tnfα, t cell-recruiting chemokines, cxcl10, and cxcl1 [28] . lastly, though local activation of tumor-specific cd8 + effector t cells is responsible for durable anti-tumor immunity by cdns via type i ifns production [62] , cdns also activate nk cells to kill cd8 + t cell-resistant tumors [29, 86] . cgamp administration triggered sting activation and ifnβ production in myeloid cells and b cells but not nk cells [86] . type i ifns then activate the nk cell to kill tumor cells [29, 86] . in all, sting in non-hematopoietic cells and hematopoietic cells are needed for the maximal therapeutic efficacy of cdns [79] (figure 3 ). in vivo cdn-induced, type i ifns-mediated anti-tumor immunity. the cdn immunization in the tumor microenvironment (tme) activates the sting pathway in dcs, endothelial cells, fibroblasts, and myeloid cells, leading to the production of type i ifns. type i ifns act as a stimulus for dc to induce cd8 + t cell cross-priming. type i ifns also activate natural killer (nk) cells leading to nk cell-mediated cytotoxicity and tumor cell killing. the tolerogenic responses induced by dnps and cdns depend on sting and ifn-αβ receptor genes, but not ifnγ receptor genes [45] . cd11b + dcs were identified as the only cells to produce ifnβ [44] . the cd11b + dcs consist of cdc2 and modcs. it is unknown (i) which dcs subset is responsible for the cdn tolerogenic function; (ii) the unique ifnβ signaling that induces t-regs production. in vivo cdn-induced, type i ifns-mediated anti-tumor immunity. the cdn immunization in the tumor microenvironment (tme) activates the sting pathway in dcs, endothelial cells, fibroblasts, and myeloid cells, leading to the production of type i ifns. type i ifns act as a stimulus for dc to induce cd8 + t cell cross-priming. type i ifns also activate natural killer (nk) cells leading to nk cell-mediated cytotoxicity and tumor cell killing. the tolerogenic responses induced by dnps and cdns depend on sting and ifn-αβ receptor genes, but not ifnγ receptor genes [45] . cd11b + dcs were identified as the only cells to produce ifnβ [44] . the cd11b + dcs consist of cdc2 and modcs. it is unknown (i) which dcs subset is responsible for the cdn tolerogenic function; (ii) the unique ifnβ signaling that induces t-regs production. murine studies have consistently shown promising results for cdn adjuvants in infectious diseases and cancer therapeutics. however, cdn human clinical trials have failed to yield the desired effect. merck's synthetic cdn mk-1454 (nct03010176) administered intratumorally failed to generate any response against metastatic solid tumors patients (head and neck squamous cell carcinoma, triple-negative breast cancer, and anaplastic thyroid carcinoma). aduro biotech's synthetic cdn, adu-s100, also had disappointing clinical responses (weak response in only 5% of the treated patients) (nct02675439). two confounding factors may explain the ineffectiveness of cdns in humans. the cdn adjuvanticity depends on sting in vivo [52] . unlike murine sting, the human sting gene is highly heterogeneous and shows population stratification [51, [87] [88] [89] . human sting genes have five alleles that have a population frequency above 1%. they are r232 (wt), h232, haq (r71h-g230a-r293q), aq (g230a-r293q), and q293. the haq-sting is extremely popular in east asians but very rare in africans [88] . in contrast, aq and q293 are only found in africans [51] . in a recent clinical trial, sebastian m. et al. showed that haq carriers had low anti-pps antibodies production in response to pneumovax ® 23 immunization (clinicaltrials.gov: nct02471014) [90] . the result is in line with the previous data from a haq knock-in mouse mode [51] . lastly, kennedy r. b. et al. demonstrated that pbmc from h232/h232 individuals had an impaired sting-mediated innate immunity (~90% decrease of ifnα induction) to poxviruses [91] . a large human population carries these sting alleles [51] . for example, the haq/haq, haq/wt, haq/h232, and h232/h232 humans account for~65% of east asians and~30% of caucasians [51, 92] . thus, large human populations carry sting alleles that impact cdn vaccine responses. the median age of patients in one cdn clinical trial was 61 (clinicaltrials. gov: nct02675439). how age affects cdn efficacy was not well-addressed. in early 2019, wannemuehler and m et al. showed that cdn induced serum antibody production in 20-month-old female balb/c mice [93] . they did not examine memory t cells responses or distinguish high-and low-affinity antibodies production [93] . they immunized mice (s.c.) with 20 µg cdn [93] , a rather high dose of cdn for mice. in late december 2019, compans and r et al. showed that cgamp (5 µg) adjuvanted with 1 µg of hemagglutinin (ha) (i.d.) did not induce antibodies or protective immunity in the 19-month-old female balb/c mice [94] . one hundred percent aged mice immunized (i.d.) with cgamp/ha died subsequently from the influenza infection [94] . in comparison, 75% of aged mice immunized with quil-a, a saponin adjuvant, with ha antigen immunization (i.d.) survived from the influenza infection [94] . in 2020, gogoi h et al. reported that cdg (5 µg, i.n.)-induced high-affinity, durable antibodies, and th1/th17 responses were severely reduced in one-year-old (~equivalent age of 42.5-year-old in humans) and two-year-old (~equivalent age of 70-year-old in humans) c57bl/6j mice [95] . aging decreases the response to vaccination [96] [97] [98] [99] . thus, similar to most vaccines, cdn vaccine adjuvanticity is also negatively impacted by age. cdn adjuvants induce balanced, durable humoral, cellular mucosal immune responses, and potent anti-tumor immunity that is highly desirable for vaccine protection from a broad spectrum of pathogens and cancers. sting-targeting cdns, thus, will continue to garner attention from the community. to advance cdns as a human vaccine and cancer adjuvants, more rigorous research to understand their mode of action in vivo are needed. for example, examine the therapeutic efficacy of cdns in vivo using mice that have the equivalent age of human cancer patients and develop cdn compositions that may enhance their adjuvant activities in aged mice. accumulating evidence in humans [90, 91, 100, 101] and mouse models [50, 51, 102] indicated that the haq and h232 alleles are impaired in the sting function. cdn-adjuvanted vaccines aim to protect the general public from the pandemic and endemic. cdn derivatives that activate haq and h232 sting in vivo are highly desirable. notably, the nod mice, which contain an n210d of sting [47] , and the r231h mouse [50] react to cgamp but not cdg for type i ifns production. thus, it is possible to design a cdn that activates h232 or even haq sting in vivo. alternatively, he y. et al. recently developed a cgamp-sting∆tm tetramer that showed promising anti-tumor activity in vivo and activates type i ifns in the absence of sting gene [103] . meanwhile, for cdns that already entered clinical trials, it is worth adopting the personalized medicine concept and analyzing the data based on sting genotypes or designing the study based on the patients' sting genotypes and age. the majority of the human population (r232/r232) will get the immediate benefit of the superior and unparalleled vaccine adjuvant and anti-tumor activity of cdns. towards an understanding of the adjuvant action of aluminium systems analysis of human vaccine adjuvants correlates of adjuvanticity: a review on adjuvants in licensed vaccines c-di-gmp is an effective immunomodulator and vaccine adjuvant against pneumococcal infection 3',5'-cyclic diguanylic acid elicits mucosal immunity against bacterial infection evidence for cyclic diguanylate as a vaccine adjuvant with novel immunostimulatory activities bis-(3',5')-cyclic dimeric adenosine monophosphate: strong th1/th2/th17 promoting mucosal adjuvant intranasal c-di-gmp-adjuvanted plant-derived h5 influenza vaccine induces multifunctional th1 cd4 + cells and strong mucosal and systemic antibody responses in mice the bacterial second messenger cyclic digmp exhibits potent adjuvant properties pulmonary surfactant-biomimetic nanoparticles potentiate heterosubtypic influenza immunity sting-activating adjuvants elicit a th17 immune response and protect against mycobacterium tuberculosis infection sublingual targeting of sting with 3'3'-cgamp promotes systemic and mucosal immunity against anthrax toxins cyclic di-gmp stimulates protective innate immunity in bacterial pneumonia c-di-gmp protects against intranasal acinetobacter baumannii infection in mice by chemokine induction and enhanced neutrophil recruitment c-di-gmp as a vaccine adjuvant enhances protection against systemic methicillin-resistant staphylococcus aureus (mrsa) infection. vaccine direct activation of sting in the tumor microenvironment leads to potent and systemic tumor regression and immunity sting agonist formulated cancer vaccines can cure established tumors resistant to pd-1 blockade the sting activator c-di-amp exerts superior adjuvant properties than the formulation poly(i:c)/cpg after subcutaneous vaccination with soluble protein antigen or dec-205-mediated antigen targeting to dendritic cells the member of the cyclic di-nucleotide family bis-(3', 5')-cyclic dimeric inosine monophosphate exerts potent activity as mucosal adjuvant cholera toxin and its b subunit promote dendritic cell vaccination with different influences on th1 and th2 development as04, an aluminum salt-and tlr4 agonist-based adjuvant system, induces a transient localized innate immune response leading to enhanced adaptive immunity the potential of 3',5'-cyclic diguanylic acid (c-di-gmp) as an effective vaccine adjuvant cyclic dinucleotide-adjuvanted dengue virus nonstructural protein 1 induces protective antibody and t cell responses t-cell exhaustion in the tumor microenvironment t cell dysfunction and exhaustion in cancer sting activation of tumor endothelial cells initiates spontaneous and therapeutic antitumor immunity intratumoral administration of cgamp transiently accumulates potent macrophages for anti-tumor immunity at a mouse tumor site nk cells mediate clearance of cd8(+) t cell-resistant tumors in response to sting agonists tnfalpha and radioresistant stromal cells are essential for therapeutic efficacy of cyclic dinucleotide sting agonists in nonimmunogenic tumors the combination vaccine adjuvant system alum/c-di-amp results in quantitative and qualitative enhanced immune responses post immunization comparison of the oral, rectal, and vaginal immunization routes for induction of antibodies in rectal and genital tract secretions of women differential induction of mucosal and systemic antibody responses in women after nasal, rectal, or vaginal immunization: influence of the menstrual cycle intranasal immunization is superior to vaginal, gastric, or rectal immunization for the induction of systemic and mucosal anti-hiv antibody responses nasal immunization of nonhuman primates with simian immunodeficiency virus p55gag and cholera toxin adjuvant induces th1/th2 help for virus-specific immune responses in reproductive tissues antibody responses in the lower respiratory tract and male urogenital tract in humans after nasal and oral vaccination with cholera toxin b subunit a comparative evaluation of nasal and parenteral vaccine adjuvants to elicit systemic and mucosal hiv-1 peptide-specific humoral immune responses in cynomolgus macaques mucosal immunization with hiv-1 peptide vaccine induces mucosal and systemic cytotoxic t lymphocytes and protective immunity in mice against intrarectal recombinant hiv-vaccinia challenge recent progress in mucosal vaccine development: potential and limitations cutting edge: the mucosal adjuvant cholera toxin redirects vaccine proteins into olfactory tissues use of the inactivated intranasal influenza vaccine and the risk of bell's palsy in switzerland nasal vaccination, escherichia coli enterotoxin, and bell's palsy the mucosal adjuvant cyclic di-gmp enhances antigen uptake and selectively activates pinocytosis-efficient cells in vivo cutting edge: dna sensing via the sting adaptor in myeloid dendritic cells induces potent tolerogenic responses activation of the sting adaptor attenuates experimental autoimmune encephalitis sting promotes the growth of tumors characterized by low antigenicity via ido activation stimulator of interferon genes agonists attenuate type i diabetes progression in nod mice immunomodulatory activity of interferon-beta sting is a direct innate immune sensor of cyclic di-gmp selective loss of responsiveness to exogenous but not endogenous cyclic-dinucleotides in mice expressing sting-r231h. front the common r71h-g230a-r293q human tmem173 is a null allele mpys/sting-mediated tnf-alpha, not type i ifn, is essential for the mucosal adjuvant activity of (3'-5')-cyclic-di-guanosine-monophosphate in vivo positively charged nanoparticles for improving the oral bioavailability of cyclosporin-a solid lipid nanoparticles (slns) to improve oral bioavailability of poorly soluble drugs submicron-sized hydrogels incorporating cyclic dinucleotides for selective delivery and elevated cytokine release in macrophages biodegradable sting agonist nanoparticles for enhanced cancer immunotherapy a microparticle platform for sting-targeted immunotherapy enhances natural killer cell-and cd8(+) t cell-mediated anti-tumor immunity nanoparticulate sting agonists are potent lymph node-targeted vaccine adjuvants innate immune activation by cgmp-amp nanoparticles leads to potent and long-acting antiretroviral response against hiv-1 stingel: controlled release of a cyclic dinucleotide for enhanced cancer immunotherapy a new adjuvant delivery system 'cyclic di-gmp/ysk05 liposome' for cancer immunotherapy magnitude of therapeutic sting activation determines cd8(+) t cell-mediated anti-tumor immunity endosomolytic polymersomes increase the activity of cyclic dinucleotide sting agonists to enhance cancer immunotherapy liposomal delivery enhances immune activation by sting agonists for cancer immunotherapy induction of necrotic cell death and activation of sting in the tumor microenvironment via cationic silica nanoparticles leading to enhanced antitumor immunity an inhalable nanoparticulate sting agonist synergizes with radiotherapy to confer long-term control of lung metastases sting pathway activation stimulates potent immunity against acute myeloid leukemia dmxaa causes tumor site-specific vascular disruption in murine non-small cell lung cancer, and like the endogenous non-canonical cyclic dinucleotide sting agonist, 2'3'-cgamp, induces m2 macrophage repolarization a robust microparticle platform for a sting-targeted adjuvant that enhances both humoral and cellular immunity during vaccination achieving long-term stability of lipid nanoparticles: examining the effect of ph, temperature, and lyophilization short-and long-term stability study of lyophilized solid lipid nanoparticles for gene therapy safety of nanoparticles in medicine zinc oxide nanoparticles: a promising nanomaterial for biomedical applications a host type i interferon response is induced by cytosolic sensing of the bacterial second messenger cyclic-di-gmp the n-ethyl-n-nitrosourea-induced goldenticket mouse mutant reveals an essential function of sting in the in vivo interferon response to listeria monocytogenes and cyclic dinucleotides mpys is required for ifn response factor 3 activation and type i ifn production in the response of cultured phagocytes to bacterial second messengers cyclic-di-amp and cyclic-di-gmp immature lung tnfr2(-) conventional dc 2 subpopulation activates modcs to promote cyclic di-gmp mucosal adjuvant responses in vivo mucosal vaccine adjuvant cyclic di-gmp differentiates lung modcs into bcl6 + and bcl6 − mature modcs to induce lung memory cd4 + t h cells and lung t fh cells respectively sting activation reprograms tumor vasculatures and synergizes with vegfr2 blockade sting-dependent cytosolic dna sensing promotes radiation-induced type i interferon-dependent antitumor immunity in immunogenic tumors the host sting pathway at the interface of cancer and immunity sting pathway agonism as a cancer therapeutic type i ifn and not tnf, is essential for cyclic di-nucleotide-elicited ctl by a cytosolic cross-presentation pathway growing tumors induce a local sting dependent type i ifn response in dendritic cells sting and irf3 in stromal fibroblasts enable sensing of genomic stress in cancer cells to undermine oncolytic viral therapy tumor-derived cgamp triggers a sting-mediated interferon response in non-tumor cells to activate the nk cell response identification and characterization of a loss-of-function human mpys variant tmem173 variants and potential importance to human biology and disease response to comment on "the common r71h-g230a-r293q human tmem173 is a null allele obesity and sting1 genotype associate with 23-valent pneumococcal vaccination efficacy polymorphisms in sting affect human innate immune responses to poxviruses single nucleotide polymorphisms of human sting can affect innate immune response to cyclic dinucleotides sting pathway stimulation results in a differentially activated innate immune phenotype associated with low nitric oxide and enhanced antibody titers in young and aged mice combination of sting pathway agonist with saponin is an effective adjuvant in immunosenescent mice new modc-targeting tnf fusion proteins enhance cyclic di-gmp vaccine adjuvanticity in middle-aged and aged mice immunosenescence and vaccine failure in the elderly: strategies for improving response age-related defects in cd4 t cell cognate helper function lead to reductions in humoral responses cd4 + t cells and immunosenescence-a mini-review aging of the cd4 t cell compartment the common haq sting variant impairs cgas-dependent antibacterial responses and is associated with susceptibility to legionnaires' disease in humans multiple homozygous variants in the sting-encoding tmem173 gene in hiv long-term nonprogressors sting differentially regulates experimental gvhd mediated by cd8 versus cd4 t cell subsets self-assembled cgamp-stingdeltatm signaling complex as a bioinspired platform for cgamp delivery this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-023425-3sjsogvq authors: røntved, c. m.; dernfalk, j.; ingvartsen, k. l. title: do high and low tumour necrosis factor‐α responders exist in dairy cows? date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423v.x sha: doc_id: 23425 cord_uid: 3sjsogvq a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor‐α (tnf‐α) ex vivo. initially, a time‐ and dose‐dependent study was carried out to find the optimal stimulation conditions for the tnf‐α response. the tnf‐α response peaked between 3 and 4 h at 38.5 °c. a dose in the range of 5–10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf‐α response. thirty‐eight danish–holstein dairy cows were investigated for their tnf‐α responsiveness ex vivo in the periparturient period. heparin‐stabilized blood samples were collected seven times over a period of 4 months (weeks −3, −1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf‐α responsiveness occurred over time. moreover, the mean tnf‐α responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf‐α response, whereas others had high a tnf‐α response. we are currently investigating whether high and low tnf‐α responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf‐α responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023388-btbf6wkg authors: hoffmann, h. j.; nielsen, l. p.; blumberga, g.; dahl, r. title: decrease in fine t‐cell subset ratio mt2/mt1 during steroid reduction of asthmatic patients date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ah.x sha: doc_id: 23388 cord_uid: btbf6wkg combining inhaled long‐acting β‐2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t‐cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4(+)cd45ra(–)cd62l(+)cd11adim) and mt1 (cd4(+)cd45ra(–)cd62l(–)cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750–1000 µg daily or equivalent, were randomized to receive, double‐blinded, either seretide(®)[salmeterol and fluticasone propionate (sfc, n = 16)] 50 µg/500 µg bd or fp 500 µg bd (n = 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 µg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p = 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p = 0049 from first to final visit). in patients receiving laba + ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-336510-qzm9wgde authors: ellermann-eriksen, svend title: macrophages and cytokines in the early defence against herpes simplex virus date: 2005-08-03 journal: virol j doi: 10.1186/1743-422x-2-59 sha: doc_id: 336510 cord_uid: qzm9wgde herpes simplex virus (hsv) type 1 and 2 are old viruses, with a history of evolution shared with humans. thus, it is generally well-adapted viruses, infecting many of us without doing much harm, and with the capacity to hide in our neurons for life. in rare situations, however, the primary infection becomes generalized or involves the brain. normally, the primary hsv infection is asymptomatic, and a crucial element in the early restriction of virus replication and thus avoidance of symptoms from the infection is the concerted action of different arms of the innate immune response. an early and light struggle inhibiting some hsv replication will spare the host from the real war against huge amounts of virus later in infection. as far as such a war will jeopardize the life of the host, it will be in both interests, including the virus, to settle the conflict amicably. some important weapons of the unspecific defence and the early strikes and beginning battle during the first days of a hsv infection are discussed in this review. generally, macrophages are orchestrating a multitude of anti-herpetic actions during the first hours of the attack. in a first wave of responses, cytokines, primarily type i interferons (ifn) and tumour necrosis factor are produced and exert a direct antiviral effect and activate the macrophages themselves. in the next wave, interleukin (il)-12 together with the above and other cytokines induce production of ifn-γ in mainly nk cells. many positive feed-back mechanisms and synergistic interactions intensify these systems and give rise to heavy antiviral weapons such as reactive oxygen species and nitric oxide. this results in the generation of an alliance against the viral enemy. however, these heavy weapons have to be controlled to avoid too much harm to the host. by il-4 and others, these reactions are hampered, but they are still allowed in foci of hsv replication, thus focusing the activity to only relevant sites. so, no hero does it alone. rather, an alliance of cytokines, macrophages and other cells seems to play a central role. implications of this for future treatment modalities are shortly considered. virus-host interactions are crucial for the outcome of infections. several strategies have been utilized by viruses to overcome the host defence. for the virus to be successful, these evasive strategies have to be balanced with the pathology induced and the possibilities of transmission to new susceptible individuals. the mammalian host utilizes ubiquitous and redundant antiviral defence mechanisms. in different viral infections, different parts of the host defence seem to be crucial. however, the redundancy ensures that other systems are ready to take over, if one of them fails. the final outcome of a viral infection depends on a delicate regulation and timing of these antiviral effector mechanisms in response to the invading virus. a viral infection of an individual thus involves a conflict between the virus and the host, which could conceptually be viewed upon as a human controversy escalating to invasion and armed struggle. to understand the resulting course of events it is important to know each party of the conflict and to conduct an analysis of the powerful weapons held by each of the combatants. the present review analyzes the early non-specific events in the conflict upon herpes simplex virus (hsv) infection. initially, each participant of the conflict, the infecting hsv and the non-specific antiviral weapons of the host, are described. subsequently, the early events of the conflict, the armament, early strikes and the opening battle between hsv and the host are discussed. insight into the early non-specific defence mechanisms are important for our understanding of the conflict and may indicate how to intervene during serious systemic infections. herpesviruses are ubiquitous viruses generally infecting humans early in life. the majority of humans has had a primary infection with one or more herpesviruses and harbour these viruses in a latent state for the rest of their lives. the initial infection is most often asymptomatic, but can be symptomatic depending on the herpesvirus in question and the age and immune status of the host. the viruses are phylogenetically old and humans and herpesviruses have evolved together [1] . this co-evolution has created viruses which are well adapted to the human host and environment. thus, herpesviruses are capable of coping with the human immune defence in a balanced manner generally without serious threads to the life of the host. infection with a foreign herpesvirus, normally hosted by another species, does not always hold this balance, and the pathology is unpredictable. this is seen when humans are infected with the simian b virus, which often shows serious clinical outcome [2] . the human herpes simplex viruses were initially identified by lowenstein, who passed it onto rabbits in 1919, and found it to be sensitive to alcohol and higher temperatures [3] . the viruses were classified into two serologically different types by schneweiss in 1962 [4] , and these are now known to belong to the subfamily of alphaherpes-virinae together with varicella-zoster virus. these alphaherpesviruses all show neurotropic latency, and mucosal or skin lesions are frequently seen as a result of viral reactivation from sensory nerves. the two types of herpes simplex virus confer the genera simplexvirus 1 and -2, which were formally designated by the international committee on taxonomy of viruses as human herpesvirus (hhv) 1 and 2 [5] . herpes simplex virus (hsv) type 1 and type 2 are very closely related, showing a homology at the dna level of 83% in protein coding regions and less in noncoding regions [6] . the genetic map of the two herpes simplex viruses is colinear [6] , and the genomes are of approximately the same size, hsv-1 of 152 kbp [7] and hsv-2 of 155 kbp, and code for corresponding genes [6] . the minor sequence variations give different cleavage sites for restriction endonucleases, which has been used intensively as an important epidemiological tool [8] [9] [10] . as all other herpesviruses the herpes simplex viruses are enveloped, icosahedral dna viruses with a capsid of approximately 100 nm ( fig. 1 ) [1] . the envelope holds at least 10 different glycoproteins protruding from the outer side (gb, gc, gd, ge, gg, gh, gi, gk, gl, and gm). the glycoproteins are primarily responsible for attachment to cellular receptors and fusion of membranes (especially gb and gd) [11] [12] [13] [14] . in addition, there are two unglycosylated proteins in the viral envelope. the glycoproteins of the envelope have several immunoregulatory effects besides their primary more mechanical functions in viral attachment and entry [15] [16] [17] [18] [19] . in the space between the envelope and the capsid, the complete viral particles posses an almost amorphous structure which was termed the tegument by roizman and furlong [20] . the tegument consists of several viral proteins involved in the initial phases of viral infection and replication such as transport of the viral dna out of the capsid [21] , early shutoff of cellular protein synthesis (vhs) [22] , and initiation of transcription of viral genes (α-trans-inducing factors) [23] . besides the tegument seen in complete viral particles, tegument-like structures are seen in enveloped particles lacking a capsid and dna, the so called light particles [24, 25] . the capsid is composed of a complex icosahedral structure of 162 capsomeres, each with a central channel running from the outside to the interior of the capsid. inside the capsid the double stranded linear dna is packed as a spool with the ends in close proximity [21, 26, 27] . the genome consists of a long (l) and a short (s) segment which are covalently linked [28] , and contains a high density of genetic information with about 94 open reading frames (orf) and encodes approximately 84 polypeptides [7, 29] , of which only 37 are required for replication of the virus in cultured cells [30, 31] . the viral genes are expressed in a cascade in groups classified as immediate early (ie, α), early (β), early late (γ 1 ) and late (γ 2 ) genes, each with a certain characteristic group of promoters regulating the sequential expression [29, 32] . generally, the α-gene products are transcription inducers, the β-gene products are viral enzymes such as the thymidine kinase and the viral dna polymerase, and the products of γgenes are the structural proteins of the viral particle [33] . the viral transcriptional chain is closed by some of the tegument proteins (e.g. vp16/vmw65) which are γ-gene products with structural properties in the tegument of the viral particle and besides this harbour transcriptioninducing capacity upon α-gene promoters crucial in the induction of the next replication cycle of the virus [32, 34] . the hsv infection is initiated by adsorption of the viral particle via gb or gc to a cellular receptor, which is a heparan sulphate chain on cellular proteoglycans [35] . thus hsv adsorption can be inhibited by heparin and soluble heparan sulfates [36, 37] . this initial binding, in which gc is important but dispensable, is of greater significance for hsv-1 than for hsv-2, a divergence which could have implications for the different pathogenic patterns of the two strains [38, 39] . furthermore, trapping of hsv to heparan sulfate motives in the tissues, e.g. basal laminas, may be of importance for containment of the infection at a specific site [40] . binding to the heparan sulfate-containing cellular receptors, which are in size with the hsv particle itself, is reversible, and serves to concentrate the viral particle in near proximity to the cell ( fig. 1 ) [35, 39, 41] . a crucial step is then conducted by gd binding to an entry receptor, of which three classes has been described [42] . these include herpes virus entry mediator (hvem), later designated as herpes virus entry protein a (hvea), which is a member of the tumour necrosis factor receptor family, nectin-1 (hvec) and nectin-2 (hveb), both members of the immunoglobulin superfamily, and heparan sulfate sites modified by 3-o-sulfotransferases [43] [44] [45] [46] . the differential use of these receptors is of importance for hsv entry of different cell types and infection of polarized cells [47] [48] [49] [50] [51] , exemplified by nectin-1, which is of importance in infection of the vaginal mucosa [52] . upon binding to one of these entry receptors, conformational changes in gd lead to interaction with gb or gh-gl dimer, which results in membrane fusion by a mechanism not known in detail ( fig. 1) [41, 53] . the membrane fusion can take place both with the plasma membrane on the surface of the target cell and with an endosomal membrane after intraluminal ph-reduction, as it is seen for some other enveloped viruses [50,54-56]. following these initial steps of infection several immunomodulatory cellular events are induced, but the potential importance of signalling through receptors involved in adsorption and membrane fusion is only scarcely analysed [57] . the receptor molecule hvem is by its normal ligand capable of inducing activation of nuclear factor κb (nf-κb) and activation of t cells. by interaction with hsv-gd these receptor responses are inhibited. thus, the hsv interaction with at least one of its receptors has multiple potentials for modulation of the host response to the infection [58, 59] . upon fusion, the hsv nucleocapsid is transported by microtubules to a nuclear membrane pore where the viral dna is released into the nucleus [60,61]. both viral tegument products and cellular kinases are responsible for the initiation of α-gene transcription [62] . in these initial events the determination of whether it will lead to a lytic infection cycle or a latent infection seems to be directed largely by the infected cell type in question [63, 64] . a key event in this seems to be early induction of latency-associated transcripts (lats) with sequences antisense to the infected cell protein null (icp0) and icp4 [65] [66] [67] . in the hsv composition and entry figure 1 hsv composition and entry. electron micrograph of negatively stained hsv particle with indications of major structural elements. important mediators of adsorption to cells (1), receptor binding (2) and fusion of membranes (3) during the process of infection are drawn stylistically. initial phase of the lytic replication cycle, the ie-gene products, besides being transcription factors for the next wave of viral proteins, intimately regulate cellular functions in favour of viral replication and immune evasion [33, 68] . of these, the icp0, a promiscuous transactivator without much dna-binding capacity, forces the cell to a pre-dividing state optimal for viral protein synthesis [69, 70] . furthermore, icp0 is active in inhibiting immune mechanisms such as interferon production and antiviral effects of interferons [71-73] and induces degradation of cellular proteins, involving the proteasome [74,75]. very early in infection, the first transcriptional activity is seen just inside the nuclear membrane at the site were the viral dna enters the nucleus [76] . the produced icp0 colocalizes with the promyelocytic leukaemia (pml) nuclear bodies and initiates degradation of these, an event which seems to be important for productive replication of the virus [77, 78] . icp4 binds to parental viral dna which is juxta-localized to the pml bodies, and later, when the bodies are degraded, replication compartments are formed, in which also icp27 can be found [76, 79, 80] . icp27 affects the posttranscriptional polyadenylation and splicing of rna, and it is thus an element of the delayed host protein shutoff [81] . immune evasion is additionally induced by the ie protein icp47 which binds to transporter associated with antigen processing, tap1/tap2 and blocks the presentation of viral peptides by the major histocompatibility complex (mhc)-i [82]. the hsv progeny is formed in the nucleus of the infected cell, where the viral dna is packed into preformed capsids. these are assembled with the tegument proteins and bud through the inner nuclear membrane to the perinuclear space [11] . the route of virus from here to the external side of the cell is controversial. apparently two routes of viral egress are possible [11] . one way is by continuous passage through vesicles and the golgi apparatus, where the membrane proteins are modified. the other route is by fusion of the newly acquired envelope with the outer nuclear membrane or the membrane of a vesicle, generating naked nucleocapsids in the cytoplasm. from here a new budding event should take place, for instance into the golgi apparatus. the progeny virus thus acquires the envelope from other membranes, than the inner nuclear lamella, as it is indicated by analysis of membrane lipids [83] . increasing evidence is pointing at this latter possibility of de-envelopment and re-envelopment as the dominating route of hsv egress [84-86]. the progress of hsv infection in tissues is influenced by the capacity of hsv to infect adjacent cells directly through cell junctions. the virus is thus avoiding exposure to extracellular substances such as antibodies and complement. the glycoproteins ge and gi are crucial for this kind of polarised transmission which primarily takes place in epithelial infections [47, 87] . as it is the case at the molecular level, the two herpes simplex viruses show similarities in their clinical appearance, both giving rise to primary infections of mucosal membranes and showing latency in sensory nerve ganglia [1] . the primary infections with hsv are often asymptomatic, especially at young age, but in a minority of cases vesicular or ulcerative lesions are seen. although hsv-1 and -2 can give rise to indistinguishable clinical infections, there are differences in the anatomical distribution of these infections, as described in 1967 by dowdle et al. [88] . hsv-1 is predominantly giving rise to infections above the waist, and hsv-2 to infections below the waist. this pattern is, however, not as straightforward as primarily described. in the last decades changes in both prevalence and distribution of hsv infections have been seen. the overall prevalence of hsv infection is very different in different countries and ethnic and social populations [89] [90] [91] . a decline in hsv prevalence has been observed in the western countries, probably because of improved socioeconomic conditions [92] [93] [94] . in parallel to the decline in prevalence, the aetiology of herpes genitalis has changed in several countries, presumably because of altered human habits and conditions of life [92] . in some areas of the world the proportion of genital infections caused by hsv-1 is still low (4-20 %) the aetiology of a genital infection is not insignificant, in that the frequency of recurrence is higher in hsv type 2infected individuals than in those infected with type 1 [89, 95] . the frequency of primary and recurrent infections with both hsv-1 and -2 has been reported to be higher among women than men [97, 103, 105] . overall, these epidemiological changes could have implications for the risk of neonatal infection from vaginal delivery, in that more women are seronegative at delivery and thus a higher number have the risk of caching a primary hsv infection. on the other hand, less hsv is circulating, reducing the risk of those who are susceptible. clinical appearance and pathogenesis as described above, primary infection with hsv is most often asymptomatic, especially in younger children [106] . however, some individuals experience a symptomatic primary infection with vesicular herpetic gingivostomatitis or in adolescence more often a pharyngitis [107] . as it is the case with orofacial infections, a primary genital hsv infection can be both asymptomatic and symptomatic with ulcerative lesions and with or without generalized symptoms such as fever, headache etc. [108, 109] . rarely, the infection disseminates to one or several organs giving rise to infections such as necrotising hepatitis, meningitis, encephalitis or to disseminated intravascular coagulopathy [110] [111] [112] [113] . such a clinical course, although uncommon, is most often seen in immunosuppressed patients e.g. transplant patients, neonates or pregnant women [114] [115] [116] . in pregnancy, primary infection with hsv without previous seroconversion at the time of delivery seems to be the main risk factor for infection of the newborn [109, 117] . genital hsv reactivations at labour only seem to posses a minor risk for neonatal infection of the baby [117, 118] , but in spite of this, approximately 70% of neonates infected are born by asymptomatic women [63] . the amount of virus in vaginal secretions during reactivations is much lower than the amount of virus in primary infections, and in reactivated cases maternal antibodies furthermore seems to be protective for the neonate [117, [119] [120] [121] [122] . when transmitted, the course of hsv infection in the newborn varies. in the pre-acyclovir era about one third of cases were mucocutaneus infections only involving the skin, mouth and eyes, one third were infections of the central nervous system (cns) with or without mucocutaneus involvement, and the last third were disseminated infections involving multiple organs, including the liver, lungs, adrenals, and often the cns [119] . of these, neonates with a generalized infection had a one-year mortality of approximately 60%, those with cns-infections had intermediary mortality, and nearly no mortality was seen in the group of patients with only mucocutaneus involvement [119] . in infected with multi-organ involvement the deaths are often set off by infection of liver or lungs or by coagulopathy. sequelae, such as mental or neurological disabilities are seen in some of those with cns involvement [123] . now a day, after initiation of high-dose acyclovir treatment, the mortality and sequela rates have dropped [124] . the clinical pattern of neonatal hsv infections has changed in that less of the mucocutaneus infections disseminate to generalized infections when treated [123] . even with high-dose acyclovir, improvements in treatment protocols are still needed, because the mortality is still as high as 30% in disseminated infections. reduction in the time from debut of symptoms to initiation of therapy is vital and passive immunotherapy with hsv-specific antibodies could posses a potential as adjuvant to the antiviral treatment [123, 125, 126] . other adjuvant treatment modalities are still needed in both neonatal infections and in generalized infections at later ages. the pathology of hsv infections is mainly caused by a direct cytopathic effect of the virus, resulting in cellular lysis and focal necrosis of the infected area [119, 127, 128] . in tissues capable of regeneration, this is not devastating, provided that the lesions do not totally destroy the organ or result in functional disability during the infection. in the brain, however, the capacity for regeneration is small, and larger necroses induced by viral infection will result in life-long sequelae [119, 123] . a delicate balance exists between the direct hsv-induced pathology and the immunopathology induced by immune reactions to the virus and the toxic and functional side effects of these reactions [129] . immunopathogenesis seems to be the main aspect of hsv stromal keratitis, which often leads to blindness [130, 131] . the scarification from this infection has even been attributed to autoimmunity by molecular mimicry [132] . weak immune response to the virus leads to severe infections because of massive viral replication and dissemination. an immense immune reaction, especially with high amounts of virus to trigger a response, can bring about increased symptoms of infection, local symptoms such as high intra-cerebral pressure or pulmonary complications, as well as generalized or septic symptoms [129, [133] [134] [135] [136] . it is thus clear that early control of hsv replication in the initial phases of infection is crucial for the host. early containment or at least inhibition of viral replication can prevent dissemination of the infection, and the early nonspecific immune reactions thus have the potential to inhibit development of a symptomatic infection. obviously the host will benefit from an attenuated or asymptomatic course of infection, but hsv -with the potential of subsequent reactivation from a latent site -could also benefit from such a course of infection, in that the host will survive and the activity of the host in society will not be hampered by symptoms from infection. thus, the hsv has excellent chances to reach new susceptible hosts which bring the virus and the host in a situation of mutual benefit [33] . with phagocytic activity. in 1892 metchnikoff named them macrophages (large eaters) in contrast to microphages (the polymorphonuclear leukocytes) [137] , and in 1924 aschoff defined the reticuloendothelial system by the criteria of uptake of vital dye [138] . the macrophages are now more precisely defined as an important member of the mononuclear phagocyte system, defined in 1969 by van furth and colleagues [139] . in the tissues they constitute a dynamic pool of cells with many functional capabilities, among which the capacity of phagocytosis, microbial killing, motility, and adherence to surfaces are classic [139] . the macrophages originate from the bone marrow, where proliferating promonocytes give rise to monocytes which enter the blood stream [140] . after a mean circulation time of approximately 11/2 day, the blood monocytes migrate to the tissues [140] . in the tissues the monocytes differentiate into macrophages with characteristics determined by the environment of the tissue in question [141] . the tissue macrophages in the major organs are represented by kupffer cells in the liver, alveolar and interstitial lung macrophages, spleenic and sinusoidal lymph node macrophages, microglia in the brain, osteoclasts in bone, and langerhans cells of the skin. thus, macrophages are strategically situated all over the body taking care of debris from the organism itself and foreign material, among others invading microorganisms, including viruses [142, 143] . macrophages in different organs have different characteristics and functional capabilities and can not totally substitute one another in studies on macrophages [141, [144] [145] [146] [147] . likewise, macrophages from different species can possess differences in their functional capability, e.g. the capacity for nitric oxide (no) production [148, 149] . macrophages in tissues are, as described above, in part originating directly from monocytes, but they are also in part originating from local proliferation. this local proliferation in the tissues is performed by newly recruited monocytes, and in the steady state situation they only constitute a small fraction of the mononuclear phagocytes present [150] . of the monocytes produced in the bone marrow of mice and passing through the blood, approximately half are targeting the liver, 15 % are going to the lungs, 25 % to the spleen and 7 % to the peritoneal cavity [150] [151] [152] . in the lungs, 70% of tissue macrophages in the steady-state originate from monocyte influx and 30% from local proliferation [153] . this proportion might vary between different tissues, as the lifespan of tissue macrophages in different organs also varies from around 6 days in mouse spleen to approximately one month for alveolar macrophages [151, 152] . in the skin, langerhans cells are a very stable and long-lived population of cells staying there for at least 18 month in the steady-state situation. however, in inflammation the langerhans cells are within 2 weeks replaced and supplemented by circulating mononuclear cells [154] . when an inflammatory process is initiated, the dynamics of monocytes and macrophages are changed. monocytes and other white blood cells are produced and recruited from the bone marrow, and the white blood cell count in the circulation is increased. the monocytes are mainly passing through the blood to become tissue macrophages, and the number of macrophages in the inflamed tissue can be increased by more than ten times [155] . in inflamed tissue the local proliferation of macrophages does not seem to increase, although the number of newly recruited cells is high, indicating that the differentiation of monocytes in the tissues is accelerated [155] . the differentiation of monocytes and activation of macrophages have been a focus of interest for many years because of the observation that macrophage activation is crucial in the defence against many intracellular pathogens [156] [157] [158] [159] . it became clear relatively early that lymphocytes and soluble factors secreted by these (lymphokines) are important in activation of macrophages for killing of intracellular bacteria, e.g. listeria [160] . in the killing of bacteria, interferon (ifn)-γ was shown to be an important stimulator of macrophage activation [161] . as mechanisms in performance of the killing simple toxic substances of reactive oxygen species (ros) and nitric oxide were identified and seem to conduct their action in synergy [162] [163] [164] . the toxic substances are chemically simple, but their production and regulation in macrophages are very complex and still a matter of intense studies [149] . the state of the activated macrophage has changed conceptually from being viewed as one specific condition of the cell towards a more dynamic picture, provoked by the fact that macrophages activated by different means show different phenotypical characteristics [163, 165] . the activated macrophage is now viewed as a cell with floating characteristics of many functional capacities regulated by a multitude of stimulating substances, such as the cytokine environment, hormones, and pathogenic and foreign substances [147, 166] . among variables, controlling macrophage activity in infected individuals, are the genetic constitutions of the host. the genetic background has been shown to be of importance for the regulation of both basic proliferation and function of macrophages and for the more specific antimicrobial responses [167, 168] . soluble mediators of lymphocyte activities were described as early as 1953, but the first lymphokines/cytokines found and characterized were the type i interferons. soon after, many other soluble mediators of lymphocyte and monocyte/macrophage activities were found [169] [170] [171] . the term lymphokine was introduced by dumonde et al. in 1969, to describe lymphocyte derived factors, and the term monokine was used as a description of factors coming from the mononuclear phagocyte system, both acting on many cells, primarily leukocytes [172] . because of a broader view on origin and function of these factors, the term cytokine is now more often used. each cytokine was originally named according to biological activity in a functional assay, which often gave several different names to one cytokine, and thus confusion at the molecular level. to straighten this out, a numerical nomenclature of interleukins (between leukocytes) was introduced in 1979 [173] . this numbering system has clarified the field, but since it has no mnemonic functional anchorage it has drawn critique since then [174] [175] [176] . the cytokines are generally smaller proteins, some composed of two subunits, utilizing specific receptors on target cells for induction of their functional effects. they are structurally related in three families, with the prototypes being il-1, il-2 and il-17 [176] . functionally, cytokines are highly potent regulatory proteins acting in a paracrine or autocrine manner at picomolar concentrations [177] . the cytokine receptors are also structurally clustered in families, and functionally utilize a battery of overlapping kinases and nuclear binding proteins in their signalling pathway and thus have overlapping functions [178] . the final functional capacity of the effector cell thus reflects the cytokine environment experienced by the cell [177] . thus the cytokines comprise a network of factors inducing or inhibiting each others secretion and function in different cells, giving rise to a constantly floating landscape of a large array of functional capacities [177] . in the early hours of a viral infection, the cytokines produced by cells infected or coming into contact with viral products are vital in conduction of the innate immune response to the infection [168, 179] . the interferons (ifns) were described and named in 1957 by isaacs and lindenmann [170] , who characterized the substances involved in the previously described interference of one virus with the replication of another unrelated virus, and the interfering activity of inactivated influenza virus with the subsequent infection of chorioallantoic membranes [180] [181] [182] . the ifns were the first cytokines described in detail, and thus provided the fundamental basis for the understanding of the cytokine concept [183] . the ifns are divided into three major groups. the two original groups of ifns are designated type i and type ii, type i being the so called non-immune ifn, and type ii the immune ifn. type ii (ifn-γ) is produced in high amounts as part of a specific immune reaction, whereas the type i ifns can be produced by many cell types in response to, in immunological terms, non-spe-cific stimulation. the many functions of ifns and the growing understanding of signalling and regulation indicate that ifn analogues may play a major role in the next generation of new antiviral compounds [171] . the type i ifns are a diverse group of cytokines, consisting of ifn-α, ifn-β, ifn-ε, ifn-κ, ifn-ω, ifn-δ, ifn-τ, and ifn-ξ/limitin [171, 184] . the first five of these are expressed in humans, and their relative production depends on the stimulus and the cell type in question. the ifn-α family consists of multiple species and some of these in different allelic forms in both humans and mice. in humans 13 ifn-α genes and one pseudogene and in mice 14 ifn-α genes and 3 pseudogenes have been identified, clustering on chromosome 4 in mouse and chromosome 9 in man [185] . the functional importance of such a diversity is largely unknown. the subtypes differ in potency and have previously been shown to vary in their profile of activities [186, 187] , but new studies show correlation between antiproliferative and antiviral effects of various ifn-α species [185] . thus, it seems that the importance of the diversity could come from varying expression patterns of the different ifn-α species. most of the α ifns are n-glycosylated, but glycosylation does not correlate with activity of the molecule, but rather with in vivo stability, and recombinant ifns are shown to have activity comparable with that of the naturally produced molecules [185, 188] . only one ifn-β species exists, coded by a gene situated in the ifn type i cluster on chromosome 4 in mouse and chromosome 9 in man, as described above [185] . the natural ifn-α and -β have a molecular weight of 19 -26 kda and most species retain stability at ph 2 [189] . all type i ifns bind to one common receptor composed of two subunits, ifn-α-receptor(r)1 and ifn-αr2. the ifnα/β receptor (ifnar) signal through the jak/stat-pathway by phosphorylation of the janus kinase (jak)1, tyrosine kinase (tyk)2, signal transducer and activator of transcription (stat)1 and stat2, and induces genes with an ifn-stimulated response element (isre) in their promoter [171, 190] . generally the type i ifns exhibit a huge range of biological effects, such as antiviral and antiproliferative effects, stimulation of immune cells such as t cells, natural killer (nk) cells, monocytes, macrophages, and dendritic cells, increased expression of mhc-i, activation of pro-apoptotic genes and inhibition of anti-apoptotic mechanisms, modulation of cellular differentiation, and inhibition of angiogenesis [171] . the newly discovered ifn-ξ/limitin also interacts with the ifn-α/β receptor, and is regarded as a type i ifn [184, 191] . antiviral activity of ifn-ξ has been shown against many viruses including hsv, and it exhibits both immunomodulatory and anti-tumour effects, but the lymphosuppressive activity is less than that of ifn-α [184, 192] . a human homolog of ifn-ξ could thus have interesting potential in the therapy of tumours and viral infections. the type ii ifn is represented by only one member, the ifn-γ [193] . structurally, ifn-γ is distinct from the type i ifns, and it signals through a different receptor. for many years ifn-γ was thought only to be expressed by t cells. later the large granular lymphocytes (nk cells) were recognised as important producers by the fact that ia-antigen (mhc-ii) expression on mouse macrophages could be induced by listeria monocytogenes infection in scid mice lacking t cells [194] [195] [196] . in recent years it has, however, been clear that other cell types, originally thought not to be producers of ifn-γ, are in fact capable of ifn-γ expression. so now macrophages, b cells, nkt cells and professional antigen-presenting cells are also recognized as ifnγ producers in certain situations [197] [198] [199] [200] [201] [202] . induction and production of ifn-γ in antigen-presenting cells and nk cells seem to be vital in the early non-specific response to infections and of importance in the linkage to the adaptive specific responses coming up later [202] [203] [204] . the induction of ifn-γ production in non-t cells (e.g. nk cells) is conducted by cytokines, especially il-12 in synergy with other proinflammatory cytokines, largely produced by mononuclear phagocytes [205, 206] . ifn-γ exerts its effects through a distinct class ii cytokine receptor, the ifn-γ receptor (ifngr), composed of two subunits, ifn-γr1 and ifn-γr2. upon binding of a homodimer of ifn-γ to the receptor complex, jak2 autophosphorylates and then transphosphorylates jak1. activated jak1 in turn phosphorylates ifn-γr1, which allows binding of the stat1 homodimer to the receptor and subsequent phosphorylation of stat1 [204] . the ifngr and stat1 are preformed as hetero-and homo-dimers, and upon receptor binding, the ifn-γ-ifn-γr1-stat1 complex seems to be internalized and translocated to the nucleus, where the activated stat1 homodimer binds to dna at gas elements and induces the first wave of responses [204, [207] [208] [209] [210] [211] . many of these initial ifn-γ induced products are transcription factors participating in further regulation of the many ifn-induced cellular response. among these products are the ifn regulatory factors (irfs) which stimulate or inhibit transcription of genes possessing an isre in the promoter region [204, 212] . for many years the key mediator of macrophage activation during antigen-induced processes was recognised as macrophage activating factor (maf) [213] . only later, the crucial importance of these effects was attributed to ifn-γ [214, 215] . ifn-γ has antiviral activity, but the most important effects of ifn-γ seem to be activation of macro-phages, antigen-presenting cells, and nk cells and inhibition of t-helper type 2 (th2) cells, resulting in a th1driven cell-mediated response to infection [204] . experiments in knock out (ko) mice with deficient ifn-γ, ifngr, or stat1 expression have shown that this system is of major importance, but not vital, in the host response to viral infections [216] [217] [218] [219] . besides the two traditional groups of ifns, a new group of ifn-like cytokines has been described in various species and named il-28a (ifn-λ2), il-28b (ifn-λ3), and il-29 (ifn-λ1) [171, 220] . these cytokines are antiviral proteins interacting with a distinct heterodimeric class ii cytokine receptor composed of ifn-λr1 and il-10r2, but sharing with the type i ifns some intracellular signalling pathways through the isre [221] . thus, they have a largely similar antiviral effect as the type i ifns [220] . tumour necrosis factor (tnf, former designated tnf-α) and lymphotoxin (lt; former tnf-β) were for many years also known as cachectin from their involvement in cachexia of cancer patients [222] . tnf is a prototype and the second member of the tnf ligand superfamily (tnfsf2), now encompassing over 40 known signalling molecules, among which the ltα, ltβ, and light (ltlike, exhibits inducible expression, and competes with hsv glycoprotein d for hvem, a receptor expressed by t lymphocytes) are some of the more prominent ligands [58, 223] . each member is the ligand of one or two distinct receptors of the tnf receptor family sharing a high degree of homology. the current nomenclature of these ligands and receptors has now been gathered on the internet [224] . tnf is a type ii transmembrane glycoprotein coded from the human chromosome 6 and from chromosome 17 in mice [223] . it is synthesized as a 26 kda transmembrane pro-tnf, primarily located in the membranes of the golgi apparatus [225] . the pro-tnf is cleaved by a metalloprotease releasing the 17 kda extracellular portion of the molecule [222, 226] . production and release of tnf from the cell is regulated at both the transcriptional and translational level and by post translational modification as described above [227] . during hsv infection both preand post-transcriptional regulatory mechanisms are involved in tnf production [228] . tnf is produced by many cell types of immune origin, primarily mononuclear phagocytes, neutrophils, nk cells and t cells, and has diverse effects on different cells [222] . both membrane bound and soluble tnf interact as homotrimers with two different receptors, the p55 tnfr1 (tnfrsf1a) and the p75 tnfr2 (tnfrsf1b) [222] . as most other receptors of this family, tnfr1 holds a death domain important in the pro-apoptotic pathway. tnfr1 is expressed virtually on every cell type except erythrocytes, whereas tnfr2 is mostly expressed on endothelial and bone marrow derived cells [227] . the tnfr2 activates nf-κb (p50, p65/rela, and p52/relb) by ubiquitin-mediated degradation of inhibitor-κb (iκb) after phosphorylation by an iκb kinase (ikk). besides inducing apoptosis, tnfr1 also activates nf-κb (p50/ p65) [229, 230] . furthermore, the activator protein 1 (ap-1) is activated by mitogen-activated protein kinases (mapks) and together with nf-κb primarily acts in the proinflammatory pathways. thus, signalling from the tnf receptor family induces a delicate balance between life and death (apoptosis) of the cell. both of the tnf receptors can by proteolytic cleavage be converted to soluble receptors with the capacity to compete with their signalling ancestors, but also act to stabilize the trimeric tnf and thus maintain its activity [227, 231] . the tnf superfamily seems to have evolved with the adaptive immune system in vertebrates and is crucial for the embryonic development of lymphoid tissue [223] . furthermore, tnf is, as a proinflammatory cytokine, involved in activation of many immune cells and is thus an important factor of both the early non-specific and the specific immune response [232] . the importance of the tnf superfamily in antiviral defence is illustrated by the fact that different viruses have developed mechanisms for interference with nearly every step of activity of this system [227, 229] . il-12 is the prime member of a small group of heterodimeric cytokines, all with the capacity to induce production of ifn-γ in a variety of cells. il-12 was first described as an nk cell stimulatory factor (nksf) and identified as a heterodimeric molecule composed of a p40 and a p35 subunit, which are covalently linked [233] . the p35 subunit has homologies to il-6, and p40 is homologous to the extracellular domain of the haematopoietin receptor family, particularly the il-6rα chain [234] . the two il-12 subunits are coded from different chromosomes, i.e. the human chromosomes 3 and 5 and the mouse chromosomes 6 and 11, respectively [235] . these genes are regulated separately, and coordinated induction in the same cell is required for secretion of the biologically active il-12p70 heterodimer [236] . il-12 is produced by monocytes, macrophages, dendritic cells, neutrophils and b cells [235, 237] . in the initial response of spleen cells in mice injected in vivo with extracts of toxoplasma gondii or with lipopolysaccharide (lps), the cellular source was found to be dendritic cells, but cultured macrophages have by themselves also been shown to produce il-12p40 upon hsv-2 infection [238, 239] . such differences could depend on variations in the signalling mechanisms involved, which is also illustrated by the observation that the production in dendritic cells and macrophages has dif-ferent kinetics. this difference could be brought about by differences in the requirement for co-stimulation with ifn-γ [240] . a collaborative action of dendritic cells and macrophages could be important, as indicated for il-12 induction by influenza virus and other inducers [241] . the receptor for il-12 is found on nk cells, t cells and dendritic cells and consists of two subunits (β1 and β2), which signal by the β2 subunit through the jak/stat pathway, primarily by activated stat4 [235] . the primary effect of il-12 is induction of ifn-γ production in nk cells and t cells, and il-12 activates the cytotoxic potential of these cells. the ifn-γ locus in nk cells is constitutively demethylated and is thus ready for transcription of the gene, which is in contrast to that of t cells, [242] . macrophages and nk cells are then stimulated by ifn-γ, resulting in activation for enhanced antimicrobial capacity [243, 244] . il-12 and ifn-γ in conjunction are the main responsible factors for activation of a th1-driven adaptive cellular immune response, important for the long-term control of intracellular pathogens [235] . il-12 stimulates proliferation of naïve t cells, and in conjunction with ifnγ inhibits th2 cell differentiation and the production of th2 cytokines (e.g. il-4, il-5, and il-13) [235] . thus il-12 holds a key position in induction and control of the th1 response. the il-12-induced ifn-γ production is synergistically enhanced by other cytokines such as tnf and il-1 [240] , and ifn-γ production can even be induced in macrophages by co-stimulation with il-18 [197, 245, 246 ], a cytokine which by itself does not possess major ifn-γinducing capacity [240] . a positive feed-back loop is initiated by the il-12-induced production of ifn-γ, in that ifn-γ is an important primer of il-12 production, thus accelerating the system [247]. furthermore, t cells enhance il-12 production through signals of the proinflammatory tnf family [240] . in virus-infected macrophages a similar autocrine feed-back loop involving il-12, il-18, ifn-α/β, and ifn-γ could be speculated [248] . this potentially harmful situation, with accelerating ifnγ production, regulated in a positive feed-back loop by il-12, is inhibited by cytokines possessing anti-inflammatory properties. among these il-10 holds a crucial position as an inhibitor of il-12 production, an effect which is also conducted by transforming growth factor-β (tgf-β) [249] [250] [251] .the th2 cytokines of the other side of the adaptive response, il-4 and il-13, inhibit il-12 induction in the early phases of stimulation, but later they can be potent inducers of il-12 production, although they still inhibit many of the ifn-γ-induced activities [212, 252, 253] . phagocytosis of apoptotic cells by macrophages inhibits production of il-12, a regulatory mechanism which seems to be important in restriction of the damages induced by uncontrolled defence mechanisms [254] . injection of high doses of il-12 to virus-infected mice is toxic, and leads to death with the pathology of tnf-related toxic shock, an effect which was explained by increased sensitivity to the toxic effects of tnf, and found to be dependent on the genetic constitution of the host [255, 256] . the small il-12 cytokine family also includes two other heterodimeric cytokines, il-23 and il-27, and a homodimer of il-12p40. the latter is found in vivo in mice and functions as an antagonist of il-12, but it is debated whether it exists in humans [257, 258] . il-23 is composed of the il-12p40 and a p19 subunit and likewise binds to a receptor with one of the il-12 receptor subunits (il-12rβ1) and a distinct il-23r subunit [240, 259] . the production and function of il-23 is quite similar to that of il-12, but il-23 has a unique capacity to induce proliferation of memory t cells [235] , and it has been found in nervous ganglia of hsv-infected mice on day 3 of infection [260] . il-23 drives il-17 production of nk cells, which mobilizes neutrophils and promotes production of the proinflammatory cytokines il-1, il-6, and tnf [261] . il-27 is the newest recognized member of the family, constructed of two distinct subunits (ebi3 and p28), but still with functional capacities alike those of il-12 [262] . the functional implications of these later discovered members of the il-12 family is not yet clear, but it seems as if they are contributors to the overall effects of the il-12 family and fine-tune the system [235, [263] [264] [265] [266] . the induction of ifn-γ and activation of nk cells is not only mastered by members of the il-12 cytokine family. other cytokines, like il-15, are also implicated in development, function, and activation of these cells [267, 268] . generally, the il-12 cytokine family has shown itself of importance in early defence against several viral infections, and as a vital inducer and regulator of the adaptive immune response against viruses and other intracellular pathogens [219, 256, 261, 269] . upon an accelerating pro-inflammatory response induced by initial viral replication the organism has to embank the ifn-γ-activated potentially harmful actions of macrophages and nk cells. important mediators of this embankment are il-4 and il-13, which as described above repress the induction of il-12, and thus put a brake on the positive feed-back loop of ifn-γ production [249, 252] . furthermore, il-4 suppresses the production of other proinflammatory cytokines such as tnf and il-1 [270] . most importantly, il-4 and il-13 are potent inhibitors of the efferent arm of the pro-inflammatory system, and thus inhibit production of reactive oxygen species and nitric oxide. the production of these two potentially harmful effector mechanisms of activated macrophages is hampered by inhibition of production of the responsible enzymes in these reactions, the nadph oxidase and the inducible nitric oxide synthase (inos) [271] [272] [273] . the primary producer cells of il-4 and il-13 are the th2 cells, but these cytokines are also produced by basophils and mast cells [274] [275] [276] . the receptors for il-4 and il-13 are expressed on most cells and are composed as dimers of four different chains. il-4 is the ligand of two receptors: a high-affinity heterodimer of il-4rα and the il-2r common γ-chain and another heterodimeric receptor composed of il-4rα and il-13rα1. il-13 binds to three complexes: a high-affinity heterodimer of il-13rα and il-4rα and two homodimers composed of either il-13rα1 or il-13rα2, which are both coded from genes on the human x-chromosome [276] . the immunomodulatory signalling is conducted through the jak/stat-pathway utilizing jak1, jak3 and stat6. phosphorylated and homodimerized stat6 binds to stat binding elements (sbe), which includes gas, and either trans-activates or inhibits transcription of the adjacent genes [212] . the functions of il-4 and il-13 are nearly overlapping with only discreet discrepancies [276, 277] . il-4 was discovered in 1982 on the basis of another important effect of the cytokine, namely the ability to induce proliferation of b cells, and it was from this effect in the early years called b cell growth factor [278] . as this, some other effects of il-4 are stimulating, in that it furthermore activates other th2-like effects such as b cell class-switching and expression of mannose receptor and fc receptor for ige on macrophages [276] . despite the anti-inflammatory profile il-4 has in vivo been shown to confer some resistance to hsv infection [279, 280] . il-4 is thus not only an inhibiting cytokine but essentially an immunomodulatory cytokine with regulatory effects on macrophages as well. the early innate defence mechanisms have for many years been regarded as important for the course of many viral infections, including infections with hsv [281] . the control of viral replication and dissemination during the first days of an hsv infection seems to be vital for the final outcome. if the viral replication is not halted by natural defence mechanisms during induction and maturation of the antigen-specific immune response, the adaptive immune system can be overwhelmed by massive viral infection at the dawn of activity of the specific reactions. the mechanisms of the anti-herpetic natural defence have been analysed extensively. it became relatively early clear that antiviral activity of macrophages [281] and nk cells [282] and early activity of the ifn-system [283] were important mediators of innate resistance to hsv. the relative contribution of each of these players in the early defence has been much debated, and as more interactions and molecular mechanisms are now elucidated, it seems clear that all of these players each hold a crucial position in an integrated antiviral natural defence system. an important model used in the study of resistance mechanisms in defence against generalized infection with hsv is a mouse model, where mice infected intra-peritoneally or intra-venously experience a generalized infection with hsv replication in most organs, including the liver, spleen, and eventually the brain [284] . the dissemination of infection to the brain and the severity of infection of the peripheral organs depend in part on the age of the mice, as is the case in humans, where neonates have difficulties in controlling a hsv infection [281, [285] [286] [287] . the course of infection in mice also depends on the type of hsv in question. furthermore, in 1975 lopez described a differential susceptibility of inbred mice to generalized infection with hsv, and this genetic difference in sensitivity has since been used for analysis of resistance factors of importance for the anti-herpetic defence [288] . in generalized infections, the genetics of the relative resistance to hsv-2 was shown to segregate with the x-chromosome [289] . this pattern of resistance to the generalized infection was for both hsv-1 and -2 attributed to a genetically determined difference in the capacity for ifn-α/β production [179, 290, 291] , and it was shown that the x-linked pattern of resistance segregated with the hsv-2-induced production of ifn-α/β in macrophages during the first hours of infection [168] . furthermore, macrophages from female mice respond to hsv with higher ifn-α/β production than macrophages from male mice [168] . this observation is in line with female mice being more resistant to hsv infection in vivo [291] . early production of ifn-α/β has been correlated to resistance of hsv infections in several other studies. treatment of mice with antibodies to ifn-α/β increases and accelerates mortality of a generalized hsv-1 infection and with higher doses of virus, mice are dying already after three to four days, a period where antigen-specific mechanisms are still in the induction and proliferation phase [292] . furthermore, mice treated with mercuric chloride showed higher titres of hsv-2 in the first days of infection, an effect which could be correlated to impaired production of ifn-α/β [293, 294] . in studies on peripheral hsv infections, such as cutaneous or corneal infections, ifn-α/β has been shown to be produced locally and to restrict the local replication of hsv and infection of nervous ganglia cells of the area, an effect which has also been correlated to the genetic constitution of the host [295] [296] [297] [298] . the genetic background for the x-linked trait of hsv resistance and ifn-α/β production of macrophages remains unravelled. induction of ifn-α/β upon hsv infection seems to be governed by different mechanisms in different cells [299] . ifn-α/β can be induced early by both infectious and uv-inactivated hsv in various cells, with the infectious virus being the more potent inducer in mouse peritoneal macrophages, whereas the uv-inactivated virus showed most potency in human peripheral blood mononuclear cells (pbmc) [168, [299] [300] [301] [302] . production of ifn-α/β was induced by gd of hsv-1 in pbmc, but not in murine macrophages [17, 303, 304] . in pbmcderived dendritic cells, however, the cellular mannose receptor was shown to be involved [299, 305] . furthermore, different toll-like receptors (tlrs) have been shown to react with hsv [306] . tlrs are transmembrane pattern recognition receptors (prrs) that detect redundant microbial molecular motives and induce antiviral and proinflammatory cytokines in response to alerting signals. in dendritic cells, tlr9-signalling, induced by the gc-rich hsv genome, has been shown to govern the induction of ifn-α/β, but tlr9-ko mice are still capable of controlling hsv infections in vivo [307] [308] [309] . however, in mouse macrophages the tlrs do not seem to be crucial for ifn-α/β induction upon hsv infection [304] . this is in agreement with the observation that the majority of ifn-α/β produced by spleen cells and dendritic cells and the total production from bone marrow macrophages was independent of tlr9 or myd88, which is necessary for signalling by most tlrs [308] . in this study, heat inactivated virus was shown still to induce ifn-α/β in cells utilizing tlr9. as resident peritoneal macrophages do not produce ifn-α/β in response to even high doses of heat inactivated hsv, this gives an additional indication of independency from tlr9 of ifn-α/β production in macrophages [300] . moreover, efficient induction of ifn-α/β by hsv in macrophages required dsrna-activated protein kinase (pkr) activity and infectivity of the virus [304] . this is in agreement with the observation that dsrna, which is produced by most viruses during replication, induces ifn through pkr, and not through tlr3, which also binds dsrna [310] . furthermore, another mechanism of ifn induction by dsrna through a rna helicase has been proposed [311] . the different induction patterns in different cells types, and the fact that ifn-α/β seems largely to be induced by other mechanisms than tlrs, explain the fact that knocking out tlr-signalling by myd88 did not influence the in vivo infection with hsv in mice [309] . other tlrs have also been shown to mediate signals in hsv infections. in hsv encephalitis in tlr2-ko mice, viral replication seemed unchanged or slightly increased during the first 4 days of infection, and the production of il-6 and monocyte chemoattractant protein 1 were impaired, but interestingly pathological changes and mortality were reduced [134] . in relation to the x-linked resistance pattern of hsv infection and ifn production upon hsv infection, it is interesting that some of the tlrs are coded from the xchromosome [312] . these are the tlr7 and tlr8, which are triggered by guanosine-or uridine-rich ssrna in the endosomal compartment of cells [313, 314] . there are, however, no indications that this pathway is implicated in ifn induction in cells during hsv infection, but the question has still not been directly addressed. regulation of the ifn-α/β gene induction is in part governed by activation of the transcription factors irf-3 and -7, which are induced by ifn-α/β itself, resulting in a positive feed back loop, an effect which has been known for years without knowledge of the signalling mechanisms [315] [316] [317] . thus, one possible explanation for the genetic differences in hsv-induced ifn-α/β production could be an elevated physiological level of this ifn self-stimulating system [318] [319] [320] [321] . an analysis of the levels of irf-3 and -7 in normal macrophages from these mice could be of interest. analysis of the levels of the ifn-induced enzyme 2'-5'-oligoadenylate synthetase (oas) in uninfected cells showed low but slightly higher levels in cells from relatively resistant mice [322] . with lps, cells from the relatively resistant (c57bl/6) mice show an early induction pattern of ifn-α/β, peaking within 2 hours, whereas cells from the susceptible balb/c mice demonstrate a delayed response, peaking 7 hours after induction [323] . among other transcription factors involved in induction of the various ifn-α/β genes are the heterodimeric nf-κb family, which is activated by tlrs, il-1r, and tnfr [324] . during a hsv infection nf-κb is activated and translocated to the nucleus [325] . many regulatory mechanisms of nf-κb activation exist, one of them exerted through tnf, which is produced by macrophages very early during hsv infection ( fig. 2) [293, 300, 325, 326] . thus, the responsible mechanisms might be exerted by other regulatory signals, influencing the magnitude of the hsv-triggered ifn-α/β induction pathway, and perhaps not by this pathway in itself [327] . a number of x-linked immunodeficiencies have been described, one of them being the wiskott-aldrich syndrome with defects in a protein expressed in haematopoietic cells, facilitating reorganization of the actin cytoskeleton, and thus influencing the mobility of immune cells and chemotaxis of macrophages. patients with this x-linked immunodeficiency show aggravated herpetic infections, and cells from some patients seem to produce lower amounts of ifn in response to hsv [328] [329] [330] . cells from patients with another x-linked immunodeficiency with mutations in the cd40-ligand, a member of the tnf family, showed decreased ifn-α/β production when infected with hsv-1, but these patients apparently show a normal response to viral infections [331, 332] . this supports the notion above that other regulatory signals might be involved. the overall effects of the ifn-α/β system, besides the production as described above, are determined by the sensitivity of cells to the secreted ifn-α/β. the effector mechanisms of ifn-α/β on hsv replication are not fully elucidated. several ifn-α/β-activated systems are involved, including the dsrna-activated pkr, which phosphorylates, and thereby inhibits, the elongation initiation factor (eif)-2α, resulting in inhibition of translation [333] . another important mediator of the antiviral activity is the oas system, which activates 2'-5'oligoadenylate-dependent rnase l with the capacity to degrade single-stranded rna [333] . lately, the pml bodies have been described as crucial for the anti-hsv effect of ifn-α/ β [334] . in mice exhibiting a relatively hsv-resistant phenotype, the direct antiviral effect of ifn-α/β in embryonic cells was found to be approximately three-fold higher than in cells from susceptible mice [322] . data from another study showed comparable results on ifn-α/β sensitivity concerning the replication of encephalomyocarditis virus (emcv) in cells from the same mouse strains [335] . this phenomenon was inherited as a co-dominant autosomal trait without any apparent influence of x-linked genes [322, 336] . further studies in mouse fibroblasts have revealed that tnf intensify the antiviral effect of ifn-α/β and, thus, the in vivo situation seems more complicated ( fig. 2) [300, 337] . in the original publication on genetics of hsv susceptibility in inbred mice, lopez reported fibroblasts from the different mice to replicate hsv equally, and the same was found in the cells showing differential sensitivity to ifn-α/β [288, 322] . in line with these results, the ifn-activated oas, an inhibitor of hsv replication, was induced to a higher degree in cells from the resistant mice upon ifn-α/β treatment [322, 333, 338] . furthermore, the level of stimulated and unstimulated oas was generally found to vary between different inbred mouse strains [339] . thus, the genetic difference in antiviral action of type i ifns seems to affect the replication of several different viruses and to correlate with resistance to hsv. the viral host protein synthesis shutoff, exerted by the hsv vhs-protein of the tegument, has major effects on the cytokine production of infected cells and reduces the effect of ifn-α/β on hsv replication [340] . furthermore, the tegument proteins have been shown to induce cellular inhibitors of the jak/stat pathway, resulting in inhibition of both ifn signalling and production [341, 342] . the ie protein icp0 inhibits activation of irf-3 and thereby also restricts ifn-induced pathways [71-73], and icp0, icp4 and icp27 induce late shutoff of protein synthesis with decreased mrna stability and thus reduced cytokine production [81,343]. as outlined, it thus seems hsv has evolved several mechanisms to evade the consequences of the ifn-α/β system, which underline the importance of these cytokines in the antiviral defence. during hsv infection macrophages are activated and possess an increased antiviral potential [281, 344] . classically, the macrophage antiviral activity has been described as intrinsic or extrinsic [345] . resting macrophages possess a high degree of intrinsic activity against hsv, generally being non-permissive to viral replication. the macrophages are thus a blind end for the hsv infection, and they can in that way protect other cells from infection, for example as a barrier lining the liver sinusoids [344] . the extrinsic antiviral activity refers to the ability of macrophages to inactivate virus outside the macrophage itself or to inhibit viral replication in other cells [346] . the intrinsic antiviral activity depends among other factors on macrophage differentiation and has been correlated to ifn activity, either physiological levels of "spontaneous" preinfection-synthesized or rapidly acting autocrine ifn-α/β [344] . in that respect, macrophages from mice of the resistant phenotype showed higher intrinsic activity by being less permissive to hsv replication [281, 347] . one potential antiviral mechanism of macrophages may be the production of ros. these were originally assigned to bacterial killing, but the effect of ros has also been correlated to antiviral functions, although they might not be of major importance [348] . the ros are mainly produced by nadph-oxidases (nox), which are membrane-bound multi-component enzymes primarily situated in the phagolysosome [349] . activation of the nahpd-oxidase, by phosphorylation and fusion of the enzyme subunits, primarily results in production of superoxide anion (o 2 -), which by superoxide dismutase can be converted to hydrogen peroxide (h 2 o 2 ). the h 2 o 2 in turn is then by fe 2+ (fenton reaction) or by fe 3+ and o 2 -(haber-weiss reaction) converted to hydroxyl radical (·oh), hydroxyl anion (oh -) and singlet oxygen ( 1 o 2 ), or by the myeloperoxidase to hypochlorous acid (hocl) [349, 350] . small amounts of ros are also produced by the mitochondria and may be of importance as signalling molecules from tnf [351, 352] . during hsv infection in vivo, macrophages are activated and achieve an increased capacity to react with a respiratory burst of ros when appropriately triggered, i.e. by phorbol esters (fig. 2) [353] . this macrophage activation is induced early in response to hsv infection, reaching a plateau within the first 12 hours of i.p. infection [353] . in vitro, macrophages were shown to be the cell type responding with an oxidative burst, and this capacity peaked after only 8 hours of infection with hsv [353] . this hsv-induced capacity for an increased respiratory burst was shown to be governed by autocrine ifn-α/β as a sine qua non phenomenon [300, 353] . nevertheless, tnf was also found to influence the macrophage activation. by itself, tnf reduced the macrophage capacity for a respiratory burst, but in combination with ifn-α/β it synergistically enhanced the ifn-induced activation [293, 300] . interestingly, a secreted portion of the hsv-gg acts as a phagocyte chemoattractant and induces production of ros by signalling through the receptor activated by the phorbol esters [354] . the hsv-induced activation of macrophages in vivo is influenced by the genetic constitution of the host, with the most pronounced activation of macrophages originating from resistant mice, as expected on the basis of the genetics of ifn-α/β production in response to the infection. furthermore, the genetics of the efferent part of the ifn-α/β-mediated hsv-induced activation of macrophages, displayed a co-dominant autosomal trait, as was the case with the antiviral effect of ifn-α/β in fibroblasts [336] . thus, the genetically-determined sensitivity to ifnα/β seems to be expressed in different cell-types. the influence of tnf on the genetics of this phenomenon has not been addressed. in contrast to these observations, the genetics concerning the antiproliferative effect of ifn-α/β in bone marrow cells seems to be reversed [335, 355] . this might, however, be linked, in that ros are shown to activate various signalling molecules, mediate apoptosis, and exhibit antiproliferative effects depending on the dose and time of exposure [352] . little is known on the potential antiviral effect of ros. by examining peroxidized lipids, which is an oxidative product from ros in tissues, it has been documented that these are produced during the acute hsv infection in vivo, and speculations on antiviral mechanisms have focused on induction of apoptosis [348, 356, 357] . hsv triggers apoptosis of infected cells by several pathways, and the importance of this phenomenon is indicated by the fact that the virus has evolved mechanisms to counteract each of these pathways [358] . macrophages generally suppress apoptosis in hsv infections, as seen by increased apoptosis in macrophage-depleted mice [359] . several studies on the mechanisms involved in the early battle against hsv, performed in in vivo animal models, have pointed to ifn-α/β as a crucial player. in adoptive transfer experiments, the effect of adult mouse spleen cells on the initial phase of a generalized hsv infection in suckling mice was conducted by ifn-α/β [360] . furthermore, administration of a hematopoetic growth factor to neonatal mice increases the number of dendritic cells, b cells and nk cells, and confers resistance in a cutaneous model of hsv infection. the effect in this model could also largely be attributed to the actions of ifn-α/β, with some additional contributions by ifn-γ [361, 362] . in ko-mice ifn-α/β was able to control the initial phase of a generalized hsv infection without contributions from nk, t-or b cells, but these latter players were necessary for survival and long term control of the infection [363] . the importance of an early, local ifn-response in models including in vivo progression and evaluation of final outcome of infection is more unclear, in that many other viral and host factors are of importance in these more complicated models with several stages of infection and involvement of different organs. such models are, however, more close to the normal human hsv infection, starting at an epithelial surface, but to expect that one resistance factor in such a complicated system will come out clear as the responsible factor for the outcome downstream the sequence of events, is too simplistic. nevertheless, induced expression of ifn-α/β in the eye by plasmid dna or an adenovirus vector was shown to inhibit early local replication of hsv and the concomitant spread of virus to the brain and death from encephalitis [333, 364] , and in ifn-α/βr ko-mice hsv replicated to much higher titres than in normal mice [297] . this tells us that the innate and adaptive immune systems exhibit much redundancy, and that ifn-α/β is of vital importance in local inhibition of hsv replication. the multitude of antiviral mechanisms, be it innate or adaptive, have varying effects and importance in the different phases of infection, such as initial local infection, dissemination to other organs, establishment of latency and reactivation, and conclusions can not be drawn from one situation to another. the reactions discussed above, involving production of ifn-α/β and tnf, take place within the first 6 to 12 hours of a hsv infection, and thus are reactions, which can execute an effect within the first replication cycle of the virus. a little later, other cytokines such as il-12, il-18 and ifn-γ are produced and give rise to other weapons in the battle against the virus. they will, in turn, within the next replication cycle execute their actions, with potential harmful consequences for either parts of the conflict. a few hours after the type i ifn and tnf response, macrophages react upon hsv infection with production of il-12, which is seen from 8 to 12 hours after infection and on [238, 365] . the same was found with other viruses 12 to 24 hours after infection [366] . in these and other studies, the producers of il-12p40 during viral infection seem to be inflammatory cells, including macrophages, and not the infected stromal cells [365, 367] . the il-12 induction during hsv infection requires infectious virus, and it was shown to be regulated at the transcriptional level [238] , as it is also the case when it is induced by lps [247, 367] . the dependence on infectivity is, however, in conflict with results from in vivo production of il-12p40 and ifn-γ in draining lymph nodes from sites injected with uv-inactivated hsv [302] . high doses of uv-inactivated virus were used, and some minimal transcription of viral genes could have taken place, although the virus was not replication competent. transcription of the il-12p40 gene in macrophages requires de novo protein synthesis during the inducing hsv infection, which could explain the relatively late appearance of il-12 production [238, 367] . the κb-sequence of the il-12p40 promoter binds nf-κb in hsv-infected cells, and the production of il-12p40 was found to be repressed by an inhibitor of nf-κb activation [238] . both these observations indicate that signalling through nf-κb is of significance in hsv-induced il-12 production. in human macrophages, tnf has been shown to inhibit il-12p40 production, but not p35 production, by a mechanism not involving nf-κb [368] . furthermore, il-12 has in a mouse model been shown to stimulate tnf expression [255] , indicating that tnf can participate in a negative feed-back loop in the regulation of the il-12 system [369] . likewise, ifn-α/β has been shown to inhibit il-12 production in both humans and mice [370] [371] [372] . the implication of such inhibition by ifn-α/β and tnf, which are secreted very early in hsv infections, well before the production of il-12, has so far not been elucidated. as described earlier, the il-12p40 induction is influenced by ifn-γ in a positive feed back loop. ifn-γ could activate il-12 transcription through binding of irf-1, -2, and -8 to an isre site in the promoter-region of il-12 [373, 374] . upon hsv infection, ifn-γ is produced as part of the nonspecific response to the virus. a marked synergism between hsv and ifn-γ in il-12 induction has been demonstrated [238] , indicating that the il-12 / ifn-γ autoaccelerating system is of importance during hsv infections. the ifn-γ-inducing activity of the produced il-12 is pronounced in mouse peritoneal cells after 24 hours of infection with hsv [238] . in a study by kirchner et al. ifn-γ was detected as early as on day 3 of in vivo hsv infection, and the ifn-γ production was correlated to the genetics of hsv resistance [375] . during hsv infection, the production of ifn-γ is mainly induced as a concerted action of several factors and not by il-12 alone. ifn-α/β by itself was shown to be a weak inducer of ifn-γ production by nk cells, but in synergy with il-12 the production of ifnγ was markedly enhanced [376] . in elicited peritoneal macrophages, hsv induced efficient ifn-γ production through cooperation of il-12, ifn-α/β and il-18 [377] . in such a proinflammatory environment even other cells than nk and t cells, e.g. macrophages, might produce lower levels of ifn-γ [200, 202, 245] . il-12 signals through stat4, but stat4 translocation to the nucleus of nk cells has also been seen after ifn-α/β stimulation [206, 378] . likewise, ifn-α/β induces stat4 phosphorylation in t cells [379] , indicating that il-12 and ifn-α/β at this point act through a shared signalling pathway. furthermore, the synergistic action of il-12 and il-18 in ifn-γ production by macrophages was shown to be dependent on stat4 [197] . in addition to these factors, tnf and il-1 have also been shown to act in synergy with il-12 in ifn-γ induction [206, 380, 381] and vice versa, ifn-γ has been shown to synergize with hsv in induction of tnf production [325] . this further emphasizes the concept of positive feed-back mechanisms in the regulation of early ifn-γ production. the important direct effect of ifn-α/β on hsv replication was found to be enhanced synergistically by ifn-γ in both cell culture and in vivo in mice [363, [382] [383] [384] . this is, however, in conflict with an early study, which could not reveal any synergism between ifn-α/β and ifn-γ on the replication of hsv in human blood mononuclear cells [385] . synergistic action of the two types of ifn is further supported by the observation of synergism between ifn-γ and tnf on hsv replication in corneal cells, and the fact that this was exerted through production of ifn-β [386] [387] [388] . the effect was, however, greatly dependent on the cell type examined, which could explain the above-mentioned inconsistency. synergism between tnf and ifn-γ in inhibition of hsv replication has now been shown to be mediated by activation of a tryptophan-depleting enzyme [389] . thus, relatively small amounts of early ifn-γ produced by nk cells in response to il-12, ifn-α/β, tnf, and il-18 could in collaboration with the already present ifn-α/β and tnf have important local effect on hsv replication in permissive cells ( fig. 3 ). this conclusion is further supported by observations in ko mice, indicating that collaborated action of ifn-α/β and ifn-γ is of importance in control of subcutaneous hsv infections [362] . in vivo studies on hsv infections in immunodeficient, ko, and antibody-treated mice have shown that the il-12, second early wave of response figure 3 second early wave of response. regulatory pathways controlling production and action of ifn-γ during early hsv infection. when infected with hsv macrophages (mφ) produce several cytokines, including il-12, which stimulate production of ifn-γ, primarily in nk cells. ifn-γ then induces no production in macrophages and stimulate the direct antiviral activity of ifn-α/β in other cells. stimulatory pathways are indicated by green arrows (→), and inhibitory pathways are drawn in red. -23 / ifn-γ system is able to control the infection, affecting both the survival rate and the hsv titres early in infection [390, 391] . the effect of il-12 in hsv infections seems to be conducted in synergy with il-18 [390] , as it has also been shown for vaccinia virus [392] . in hsv corneal infections in ko mice, il-12 was shown to participate in the immune pathogenesis [393] , but in another study utilizing il-12 encoding plasmid dna, corneal expression of il-12 reduced the angiogenesis, and thus the pathology of the infection [394] . however, both studies agreed that il-12 does not affect the local titres of hsv in the eye. after a thermal injury, wide-spread hsv infections are an important risk, and treatment of injured mice with il-12 combined with soluble il-4r results in augmentation of the ifn-γ production and decreased viral replication and mortality [395] . in mice infected with murine cytomegalovirus (mcmv) production of il-12-induced ifn-γ by nk cells has been demonstrated in vivo, and the system was further shown to lower the viral titres [396, 397] . the il-12 / ifn-γ system seems, however, not to be of importance in all viral infections, in that the latter study could not detect any production of early il-12 or ifn-γ in a model of infection with the arenavirus lymphocytic choriomeningitis virus. analyses of the il-12, -23 / ifn-γ system in humans with genetic defects and in ko-mice reveal more redundancy in man than in mouse and indicate that the system is of more importance in dna-than in rna-virus infections [219] . the producers of early ifn-γ, the nk and nkt cells, and the cytokine il-15 and the transcription factor t-bet, which are both crucial for the differentiation and function of these cells, have all been shown to be decisive for the early control of hsv infection in vivo [268, [398] [399] [400] . although nk cells but not ifn-γ was shown to be decisive for survival from ocular infections [401] , such an effect of ifn-γ has been seen by others [402] . furthermore, a review of genetic functional nk cell defects found nk cells and their innate ifn-γ production to be of central importance in herpesvirus infections [403] . overall, it can be concluded that the il-12 / ifn-γ system is active in hsv infections and possesses an important antiviral potential, capable of controlling viral replication during the early phases of infection. in macrophages exposed to ifn-γ, the enzyme inducible nitric oxide synthase is induced, which eventually results in production of no from molecular oxygen and a guanidino nitrogen by conversion of l-arginine to l-citrulline [404] . upon hsv infection, the inos gene is induced, as shown by detection of inos-mrna in infected mouse peritoneal cells and corneal neutrophils [405, 406] . the production of no in hsv-infected cultures of resting mouse peritoneal cells, which comprise a mixed population of macrophages, lymphocytes, nk cells etc., is dependent on the virus being infectious [405] . this is in line with the requirement of infectious hsv for il-12 production and thus for production of ifn-γ as described previously [238] . no could itself be involved in a positive feed-back, in that signalling of il-12 utilizing tyk2 requires the activity of no [407] . when exogenous ifn-γ is added to virus-infected cells, a marked synergism is seen. this synergistic effect of hsv on the ifn-γ-induced no production in macrophages was shown to be mediated by autocrine secretion of tnf [325, 405] . in line with this, mice with a targeted disruption of the tnf gene showed impaired resistance to hsv and increased viral replication within the first days of infection [408] , and antibodies to tnf and an inhibitor of no production impaired early control of hsv infection in peripheral nervous tissue [409] . the induction of inos and the following production of no in response to ifn-γ and hsv is a relatively slow reaction, coming up after about 18 hours of infection [405] . in in vivo vaginal hsv infections inos mrna could be detected after 24 hours of infection [410] . thus, the production of this relatively toxic substance is part of the second wave of innate defence mechanisms. the retarded production of no and the requirement for two or more signals for induction of inos are logic considering the in hsv-infected macrophages exposed to ifn-γ, inos is induced synergistically though tnf-induced nf-κb activation and translocation to the nucleus, as shown by binding of a heterodimeric complex of p55/p65 and a homodimer of p55 to the κb-site of the inos promoter during infection [325] . the crucial position of nf-κb in the induction of inos and production of no is also indicated by experiments showing that antibodies to tnf inhibit activation of nf-κb and production of no in hsvinfected cells and abolish the synergism between the virus and ifn-γ, an observation which was also seen with inhibitors of nf-κb activation [325] . further analysis of the signalling mechanism has revealed that the synergism upon hsv infection is influenced by physical interaction of irf-1 and the nf-κb subunit p65 and controlled by the isresite and the distal κb-site of the inos promoter ( fig. 5 ) [411] . a further support for this notion comes from the observation that the dna-binding capacity of nf-κb and the nuclear translocation of irf-1 have similar kinetics upon hsv infection [411] and the fact that irf-1 is essential for inos induction [412] . induction of other genes such as ifn-β and vascular cell adhesion molecule 1 also involve physical interaction of irf-1 and nf-κb [413] , and both irf-1 and irf-2 have in other cells types been shown to form complexes with nf-κb [414, 415] . another potential mechanism in the synergistic induction of inos could involve complex formation of ifn consensus sequence-binding protein (icsbp or irf-8) and irf-1, which is also important for high-output no production but has still not been studied in hsv infections [416] . thus, high-output no production from activated macrophages is controlled by a "double-lock" signalling mechanism restricting the production of this antiviral toxic substance to sites of active viral replication, and sparing uninfected tissue from the detrimental effects ( fig. 4 ). the antiviral effects of no have been documented in several viral infections, although there clearly exist viruses and conditions where no does not exhibit major antiviral properties. no is thus not a magic bullet against virus infections [417] . in hsv infections, no has been shown to confer a substantial part of the antiviral activity induced by ifn-γ in a macrophage cell line and to participate in the extrinsic anti-hsv effect of macrophages [418] [419] [420] [421] . an exogenously added donor of no has in several cell lines been shown to reduce the replication of hsv [422] . in vivo, analysis of mice treated with an inhibitor of no production showed higher titres of hsv in the lungs but increased survival rates due to reduced inflammation [135] . recently, a study using another inhibitor of no production has confirmed the anti-herpetic effect of no during a hsv respiratory infection, but in this study mice with inhibited no production showed increased inflammatory responses, symptoms of infection, and mortality ifn-γ il-4 (at high ifn-γ / stat1) [423] . replication of hsv during vaginal infection was increased in the presence of an inhibitor of no production, and this enhanced viral replication was most prominent during the first 24 hours of infection [410] . in inos-ko mice, the herpes virus mcmv replicates to higher titres in various organs and in macrophages, and this results in impaired survival of the animals [424] . weanling mice with a targeted disruption of the inos gene showed increased hsv replication, but apparently without differences in hsv titres during the first days of infection [425] , and in adult ko mice, we could not detect any significant effect of no during the early days of a generalised hsv infection (ellermann-eriksen, unpublished results). probably, these in vivo results are due to redundancy of the antiviral system. [426] . the final effects of no on hsv infections therefore appear to be balanced between antiviral versus toxic effects, and the final outcome seems to depend on the timing, infectious dose, and tissues involved. thus no production in the early phases of hsv infection is one of the effector mechanisms of the innate immune response inhibiting hsv replication, but when overproduced, no might itself result in pathology, as discussed in the following section. as outlined above, positive feed-back mechanisms exist at the afferent side of the early cytokine response, involving especially the production of ifn-γ, il-12, ifn-α/β and tnf, and synergisms at the efferent side, resulting in highoutput no production. as a result of coordinated induction of the inos gene by several transcription factors, activated by especially ifn-γ and tnf, a potent early antiviral system is activated. however, no causes damage to dna, proteins and lipids in cells and tissues and could thus be deleterious for the host [427] [428] [429] [430] . a study in ko-mice indicates that no can be responsible for inflammation and life-threatening symptoms to hsv infection of the lungs [135] . this effect of no on pulmonary symptoms is also observed in influenza virus infections [431] , although no inhibits replication of both influenza virus and severe acute respiratory syndrome coronavirus [432, 433] . consequently, when this system is activated, it has to be controlled and eventually closed down, as it would otherwise induce unnecessary harm to the host. such negative regulations of the inos gene induction in ifn-γ activated macrophages is conducted by il-4 and il-13 [272, 273, 434] . furthermore, tgf-β can exhibit downregulation of no production through several post-transcriptional regulatory mechanisms, but the contribution of these pathways have not been analysed in hsv infections [435, 436] . il-4 production during hsv infection has in vaginal and cns infections been demonstrated on day 2 of infection and to increase for the next days [437, 438] . in peritoneal cells from mice infected i.p. pro-duction of il-4 could be detected at day 5 of infection [439] . at low ifn-γ concentrations, il-4 has been shown to inhibit inos induction through stat6 competition with stat1 binding to the gas element of the irf-1 promoter region. this results in reduced expression of the transcription factor irf-1, which is crucial for induction of inos [440] . generally, stat6 was shown to be a key factor in il-4-and il-13-induced inhibition of inos gene transcription induced by ifn-γ (fig. 5 ) [441] . at higher ifn-γ concentrations, activated stat6 is no longer able to compete with the high amounts of activated stat1 dimer [440] . however, in this situation il-4 is still able to inhibit the production of no from ifn-γ-stimulated macrophages [272, 273, 434] . in the presence of high levels of ifn-γ, il-4 is not able to alter the induction of irf-1, but the production of irf-2 is increased [434] . the human promoter region of irf-2 contains a sbe, and the induction of irf-2 could thus potentially be mediated by stat6 binding to this element [442] . this is in agreement with the fact that irf-2 is known to compete with the binding of irf-1 to isre sites and to antagonize the transactivating activity of irf-1 in the regulation of other ifninduced genes [443] [444] [445] . inhibition of inos expression by high concentrations of irf-2 relative to irf-1 has thus been proposed as a controlling mechanism in situations with high levels of ifn-γ ( fig. 5 ) [434] . furthermore, another mechanism could evolve from the observation that il-4 signalling can result in disruption of the complex formation of icsbp and irf-1 and thereby inhibit inos induction [416] . other mediators of il-4-induced repression of inos induction might exist, in that another dnabinding transcriptional repressor competing with irf-1 has been described [446] . in ifn-γ activated macrophages the il-4-and il-13induced inhibition of inos induction can thus be overruled by hsv infection, leading to a sustained no production ( fig. 4) [439, 447] . this effect of hsv infection is mediated through tnf production and nf-κb activation [439, 447] . however, pre-treatment with il-4 has in a theiler's murine encephalomyelitis virus model showed inhibition of nf-κb activation [448] . in thioglycollateinduced peritoneal cells, lps and tnf could only overcome the inhibiting effect of il-4 in situations, where il-4 was added simultaneously or after the stimulators [272] , a sequence of events which, however, is in agreement with the sequence of cytokine production in hsv infections. when activated, the nf-κb p65 physically interacts with irf-1 and trans-activate inos transcription in hsvinfected and tnf-treated cells [411, 449] . it is thus tempting to speculate that the nf-κb-irf-1 complex has higher affinity for the combined dna-binding site and thus is able to obstruct the binding of irf-2 to the isre site of the inos promoter and in that way turn the competition towards transcriptional activity ( fig. 5 ) [411] . this will block the inhibiting effect of il-4 in foci of hsv replication and open up for no production at sites where the antiviral effect is of more importance than the potential toxicity. in treatment of hsv infections, we have for many years had a very powerful tool in the antiherpetic drug acyclovir and related compounds. but there are still therapeutic problems in the group of patients with generalized or cns infections, and therefore it is tempting and timely to hypothesize on possible future treatment strategies. as described, it is clear that relatively discrete but early actions of the non-specific defence systems are crucial for the long term outcome of the infection. the same holds for the antiviral therapy, and early presumptive therapy and rapid diagnostics could thus potentially improve the final outcome. in the seeking for improved antiviral treatment, adjuvant therapy with anti-hsv antibodies could potentially accelerate the clearance of viral particles, and block viremic dissemination in patients, who are still seronegative at the time of treatment. immunomodulatory treatment modalities imitating the early non-specific antiviral defence, working as described in this review, could be considered. the key players exhibiting the least toxicity by themselves could be used, taking advantage of potential synergy with other cytokines in the foci of hsv infection. in the future, molecules with affinity for various receptors are expected to be produced, and when we know the signalling mechanisms in detail and all the potential interactions, molecular signalling could be addressed directly by pharmaceuticals. in consequence of the crucial position of the type i ifns in innate response to hsv, future analogues of ifn-α/β seem obvious as candidates for adjuvant treatment of severe hsv infections. this could be supplemented with il-12, which would give the highest ifn-γ production in foci of hsv infection because of other cytokines such as ifn-α/β, tnf and il-18 being present there. with focussed production of ifn-γ at sites of active viral replication and treatment with ifn type i analogues the focal antiviral activity could be increased markedly, without too much activity in areas without infection. to hamper systemic consequences of the enhanced proinflammatory reactions, such pro-inflammatory treatment could perhaps benefit from concomitant treatment with il-4 or other stat6-activating therapeutics in the future. this would further focus the activity to sites of active hsv replication. in situations with massive viral replication in nearly all organs, high-dose aciclovir should perhaps only be supplemented with anti-inflammatory medications and inhibitors of tnf, since many of these individuals risk to die from septic reactions. the author(s) declare that they have no competing interests. the family herpesviridae: a brief 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herpes simplex virus 1 interaction with toll-like receptor 2 contributes to lethal encephalitis suppression of herpes simplex virus type 1 (hsv-1)-induced pneumonia in mice by inhibition of inducible nitric oxide synthase (inos, nos2) evaluation of systemic inflammatory responses in neonates with herpes simplex virus infection lecons sur la pathologie comparée de l'inflammation das reticulo-endotheliale system the mononuclear phagocyte system: a new classification of macrophages, monocytes, and their precursor cells the origin and kinetics of mononuclear phagocytes functional heterogeneity in liver and lung macrophages a quantitative study of the kinetics of blood clearance of p32-labelled escherichia coli and staphylococci by the reticuloendothelial system blood clearance of p32-labeled vesicular stomatitis and newcastle disease viruses by the reticuloendothelial system in mice murine kupffer cells. mononuclear phagocytes deficient in the generation of reactive oxygen intermediates 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in mouse typhoid suppression of macrophages antimicrobial activity by a tumor cell product infections in patients with inherited defects in phagocytic function studies on the mechanisms of macrophage activation. i. destruction of intracellular leishmania enriettii in macrophages activated by cocultivation with stimulated lymphocytes macrophage activation and differentiation tumor necrosis factor and granulocyte macrophage-colony stimulating factor stimulate human macrophages to restrict growth of virulent mycobacterium avium and to kill avirulent m. avium: killing effector mechanism depends on the generation of reactive nitrogen intermediates hydrogen peroxide from mouse peritoneal macrophages nadph oxidase, nramp1 and nitric oxide synthase 2 in the host antimicrobial response in vitro studies on normal, stimulated and immunologically activated mouse macrophages. iii. intracellular multiplication of listeria monocytogenes the chemokine system in diverse forms of macrophage activation and polarization differences in the response of inbred mouse strains to the factor increasing monocytopoiesis x-linkage of the early in vitro alpha/beta interferon response of mouse peritoneal macrophages to herpes simplex virus type the effect of tuberculin on sensitized and normal leucocytes virus interference. i. the interferon interferons, interferon-like cytokines, and their receptors lymphokines": non-antibody mediators of cellular immunity generated by lymphocyte activation revised nomenclature for antigen-nonspecific t-cell proliferation and helper factors international lymphokine nomenclature committee international union of immunological societies. nomenclature committee working group on lymphokines interleukin is as interleukin does the cytokine network cytokines and cytokine receptors the role of interferon in the resistance of c57bl/6 mice to various doses of herpes simplex virus type 1 a protective action of neurotropic against viscerotropic yellow fever virus in macacus rhesus interference of inactive virus with the propagation of virus of influenza henle w: interference phenomena between animal viruses; a review interferons and their actions interferon-zeta/limitin: novel type i interferon that displays a narrow range of biological activity characterization of the murine alpha interferon gene family antiproliferative and antiviral activities of human leukocyte interferons a species of human alpha interferon that lacks the ability to boost human natural killer activity structural and functional differences between glycosylated and non-glycosylated forms of human interferon-beta (ifn-beta) human cytokines -handbook for basic and clinical research 1st edition signal transduction by type i interferons limitin: an interferon-like cytokine that preferentially influences b-lymphocyte precursors antiviral activity of limitin against encephalomyocarditis virus, herpes simplex virus, and mouse hepatitis virus: diverse requirements by limitin and alpha interferon for interferon regulatory factor 1 immune specific induction of interferon production in cultures of human blood lymphocytes regulation of macrophage ia expression in mice with severe combined immunodeficiency: induction of ia expression by a t cellindependent mechanism a t cell-independent mechanism of macrophage activation by interferon-gamma regulation of interferon-gamma gene expression the production of ifn-gamma by il-12/il-18-activated macrophages requires stat4 signaling and is inhibited by il-4 interleukin 18 together with interleukin 12 inhibits ige production by induction of interferon-gamma production from activated b cells il-12 up-regulates il-18 receptor expression on t cells, th1 cells, and b cells: synergism with il-18 for ifn-gamma production ifn-gamma expression in macrophages and its possible biological significance cutting edge: cross-talk between cells of the innate immune system: nkt cells rapidly activate nk cells ifn-gamma production by 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interferon-gamma as the lymphokine that activates human macrophage oxidative metabolism and antimicrobial activity monoclonal antibodies to murine gamma-interferon which differentially modulate macrophage activation and antiviral activity characterization of herpes simplex virus type-1 infection and herpetic stromal keratitis development in ifn-gamma knockout mice targeted disruption of the mouse stat1 gene results in compromised innate immunity to viral disease role of interferon-gamma in immunity to herpes simplex virus the role of il-12, il-23 and ifn-gamma in immunity to viruses il-28, il-29 and their class ii cytokine receptor il-28r ifn-lambdas mediate antiviral protection through a distinct class ii cytokine receptor complex tumor necrosis factors the tnf superfamily the new tnf nomenclature scheme 2005 localization and post-golgi trafficking of tumor necrosis factoralpha in macrophages metalloprotease-disintegrins: links to cell adhesion and cleavage of tnf alpha and notch tumor necrosis factor (tnf)-alpha and tnf receptors in viral pathogenesis expression of tnf-alpha by herpes simplex virus-infected macrophages is regulated by a dual mechanism: transcriptional regulation by nf-kappa b and activating transcription factor 2/jun and translational regulation through the au-rich region of the 3' untranslated region viruses and the tnf-related cytokines, an evolving battle nf-kappab and the innate immune response soluble tumor necrosis factor (tnf) receptors conserve tnf bioactivity in meningitis patient spinal fluid biological functions of tumor necrosis factor cytokines and their receptors identification and purification of natural killer cell stimulatory factor (nksf), a cytokine with multiple biologic effects on human lymphocytes the il-12 family of heterodimeric cytokines: new players in the regulation of t cell responses the biology of il-12: coordinating innate and adaptive immune responses coexpression of two distinct genes is required to generate secreted bioactive cytotoxic lymphocyte maturation factor production of natural killer cell stimulatory factor (interleukin 12) by peripheral blood mononuclear cells ellermann-eriksen s: herpes simplex virus type 2 induces secretion of il-12 by macrophages through a mechanism involving nf-kappab in vivo microbial stimulation induces rapid cd40 ligand-independent production of interleukin 12 by dendritic cells and their redistribution to t cell areas interleukin-12 and the regulation of innate resistance and adaptive immunity the reciprocal interaction of nk cells with plasmacytoid or novel p19 protein engages il-12p40 to form a cytokine, il-23, with biological activities similar as well as distinct from il-12 herpes simplex virus type 1 infection induces upregulation of interleukin-23 (p19) mrna expression in trigeminal ganglia of balb/c mice il-12 and il-23: master regulators of innate and adaptive immunity il-27, a heterodimeric cytokine composed of ebi3 and p28 protein, induces proliferation of naive cd4(+) t cells mice lacking il-12 develop polarized th1 cells during viral infection effects of il-12 and il-23 on antigen-presenting cells at the interface between innate and adaptive immunity il-12: the role of p40 versus p75 novel il-12 family members shed light on the orchestration of th1 responses critical role for il-15 in innate immunity nk and nkt cell-independent contribution of interleukin-15 to innate protection against mucosal viral infection il-23 induces stronger sustained ctl and th1 immune responses than il-12 in hepatitis c virus envelope protein 2 dna immunization interleukin-4 suppresses inflammatory cytokine gene transcription in porcine macrophages interleukin-4 suppresses the expression of macrophage nadph oxidase heavy chain subunit (gp91-phox) mechanism of suppression of nitric oxide synthase expression by interleukin-4 in primary mouse macrophages effect of il-4 and il-13 on ifn-gamma-induced production of nitric oxide in mouse macrophages infected with herpes simplex virus type 2 heterogeneity of helper/inducer t lymphocytes. i. lymphokine production and lymphokine responsiveness mouse splenic and bone marrow cell populations that express high-affinity fc epsilon receptors and produce interleukin 4 are highly enriched in basophils alternative activation of macrophages differential responses of human monocytes and macrophages to il-4 and il-13 identification of a t cell-derived b cell growth factor distinct from interleukin 2 recombinant herpes simplex virus type 1 expressing murine interleukin-4 is less virulent than wild-type virus in mice the effect of uv-b irradiation on primary and secondary hsv-1 infections in interleukin-4 knockout mice role of macrophages in natural resistance to virus infections resistance to herpes simplex virus -type 1 (hsv-1) immunobiology of infection with herpes simplex virus role of macrophages in hepatitis induced by herpes simplex virus type 1 and 2 in mice re-evaluating natural resistance to herpes simplex virus type 1 macrophages and age-dependent resistance to hepatitis induced by herpes simplex virus type 2 in mice experimental infection of inbred mice with herpes simplex virus. iii. comparison between newborn and adult c57bl/6 mice genetics of natural resistance to herpesvirus infections in mice genetics of macrophage-controled resistance to hepatitis induced by herpes simplex virus type 2 in mice an xlinked locus influences the amount of circulating interferon induced in the mouse by herpes simplex virus type 1 x-linked resistance of mice to high doses of herpes simplex virus type 2 correlates with early interferon production role of interferon in the pathogenesis of virus diseases in mice as demonstrated by the use of anti-interferon serum effect of mercuric chloride on macrophage-mediated resistance mechanisms against infection with herpes simplex virus type 2 influence of mercuric chloride on resistance to generalized infection with herpes simplex virus type 2 in mice innate and acquired immunity to herpes simplex virus type 1 ectopic expression of dna encoding ifn-alpha 1 in the cornea protects mice from herpes simplex virus type 1-induced encephalitis virgin hw: interferons regulate the phenotype of wild-type and mutant herpes simplex viruses in vivo neural infection in mice after cutaneous inoculation with hsv-1 is under complex host genetic control multiple mechanisms for hsv-1 induction of interferon alpha production by peripheral blood mononuclear cells autocrine secretion of interferon-alpha/ beta and tumour necrosis factor-alpha synergistically activates mouse macrophages after infection with herpes simplex virus type 2 herpes simplex virus type 1-induced interferon production and activation of natural killer cells in mice transient ifngamma synthesis in the lymph node draining a dermal site loaded with uv-irradiated herpes simplex virus type 1: an nk-and cd3-dependent process regulated by il-12 but not by ifn-alpha/beta inhibition of herpes simplex virus type 1-induced interferon synthesis by monoclonal antibodies against viral glycoprotein d and by lysosomotropic drugs viral activation of macrophages through tlr-dependent and -independent pathways the mannose receptor mediates induction of ifn-alpha in peripheral blood dendritic cells by enveloped rna and dna viruses reading the viral signature by tolllike receptors and other pattern recognition receptors toll-like receptor 9-mediated recognition of herpes simplex virus-2 by plasmacytoid dendritic cells herpes simplex virus type-1 induces ifn-alpha production via toll-like receptor 9-dependent and -independent pathways herpes simplex virus type 1 activates murine natural interferon-producing cells through toll-like receptor 9 viral infection switches non-plasmacytoid dendritic cells into high interferon producers the rna helicase rig-i has an essential function in double-stranded rna-induced innate antiviral responses three novel mammalian toll-like receptors: gene structure, expression, and evolution species-specific recognition of single-stranded rna via toll-like receptor 7 and 8 toll-like receptors in innate immunity positive feedback regulation of type i ifn genes by the ifninducible transcription factor irf-7 regulation of ifn regulatory factor-7 and ifn-alpha production by enveloped virus and lipopolysaccharide in human plasmacytoid dendritic cells the effect of priming with interferon on interferon production by two lines of l cells studies on expression of spontaneous and induced interferons in mouse peritoneal macrophages by means of monoclonal antibodies to mouse interferons injection of mice with antibody to mouse interferon alpha/beta decreases the level of 2'-5' oligoadenylate synthetase in peritoneal macrophages specific interferon genes are expressed in individual cells in the peritoneum and bone marrow of normal mice inhibition by interleukin-4 of constitutive beta interferon synthesis in mouse macrophages genetically determined difference in the antiviral action of alpha/beta interferon in cells from mice resistant or susceptible to herpes simplex virus type 2 genetic control of the innate immune response signaling to nf-kappab nf-kappab activation is responsible for the synergistic effect of herpes simplex virus type 2 infection on interferon-gamma-induced nitric oxide production in macrophages virus-cell interactions regulating induction of tumor necrosis factor alpha production in macrophages infected with herpes simplex virus tumor necrosis factor acts synergistically with autocrine interferon-beta and increases interferon-beta mrna levels in human fibroblasts is wiskott-aldrich syndrome a cell trafficking disorder? the wiskott-aldrich syndrome natural killer cell function and interferon generation in patients with primary immunodeficiencies functional defects of dendritic cells in patients with cd40 deficiency cd40 and dendritic cell function distinctive roles for 2',5'-oligoadenylate synthetases and double-stranded rna-dependent protein kinase r in the in vivo antiviral effect of an adenoviral vector expressing murine ifn-beta promyelocytic leukemia protein mediates interferon-based anti-herpes simplex virus 1 effects mouse genes influence antiviral action of interferon in vivo differential sensitivity of macrophages from herpes simplex virus-resistant and -susceptible mice to respiratory burst priming by interferon-alpha/beta antiviral activity of tumour necrosis factor. synergism with interferons and induction of oligo-2',5'-adenylate synthetase differential effect of murine alpha/beta interferon transgenes on antagonization of herpes simplex virus type 1 replication continuous production of interferon in normal mice: effect of anti-interferon globulin, sex, age, strain and environment on the levels of 2-5a synthetase and p67k kinase the role of the ul41 gene of herpes simplex virus type 1 in evasion of non-specific host defence mechanisms during primary infection induction of suppressor of cytokine signaling-3 by herpes simplex virus type 1 contributes to inhibition of the interferon signaling pathway herpes simplex virus 1 gene products occlude the interferon signaling pathway at multiple sites suppression of proinflammatory cytokine expression by herpes simplex virus type 1 the interferon-macrophage alliance macrophages and other nonspecific defences: role of modulating resistance against herpes simplex virus macrophage extrinsic antiviral activity during herpes simplex virus infection role of interferon in persistent infection of macrophages with herpes simplex virus possible role of reactive oxygen species in antiviral defense oxidative killing of microbes by neutrophils reactive oxygen and reactive nitrogen intermediates in innate and specific immunity reactive oxygen intermediates in tnf signaling molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells herpes simplex virus type 2 primes mouse macrophages for an early and genetically determined respiratory burst mediated by interferon-alpha/beta a proinflammatory peptide from herpes simplex virus type 2 glycoprotein g affects neutrophil, monocyte, and nk cell functions strain dependence of the antiproliferative action of interferon on murine erythroid precursors herpes simplex virus type 1 latency in the murine nervous system is associated with oxidative damage to neurons the antiviral activity of tumour necrosis factor on herpes simplex virus type 1: role for a butylated hydroxyanisole sensitive factor herpes simplex virus 1 induces and blocks apoptosis at multiple steps during infection and protects cells from exogenous inducers in a cell-type-dependent manner a role for apoptosis induced by acute herpes simplex virus infection in mice the role of natural killer cells and interferon in resistance to acute infection of mice with herpes simplex virus type 1 treatment of neonatal mice with flt3 ligand leads to changes in dendritic cell subpopulations associated with enhanced il-12 and ifn-alpha production flt3 ligandtreated neonatal mice have increased innate immunity against intracellular pathogens and efficiently control virus infections interplay between alpha/beta and gamma interferons with b, t, and natural killer cells in the defense against herpes simplex virus type 1 type i interferons and herpes simplex virus infection: a naked dna approach as a therapeutic option herpes simplex virus type 1-mediated up-regulation of il-12 (p40) mrna expression. implications in immunopathogenesis and protection interleukin-12 gene expression after viral infection in the mouse the il-12 response to herpes simplex virus is mainly a paracrine response of reactive inflammatory cells inhibition of il-12 production in human monocyte-derived macrophages by tnf pure e: inhibition of interferon gamma induced interleukin 12 production: a potential mechanism for the anti-inflammatory activities of tumor necrosis factor interferon-alpha/beta inhibition of interleukin 12 and interferon-gamma production in vitro and endogenously during viral infection type i interferons and il-12: convergence and cross-regulation among mediators of cellular immunity interferon alpha/beta and interleukin 12 responses to viral infections: pathways regulating dendritic cell cytokine expression in vivo il-12 is dysregulated in macrophages from irf-1 and irf-2 knockout mice irf-8/icsbp and irf-1 cooperatively stimulate mouse il-12 promoter activity in macrophages in vitro production of immune interferon by spleen cells of mice immunized with herpes simplex virus type i interferons enhance production of ifn-gamma by nk cells interferon (ifn)-alpha/beta, interleukin (il)-12 and il-18 coordinately induce production of ifn-gamma during infection with herpes simplex virus type differential secretion of tnf-alpha and ifn-gamma by human peripheral bloodderived nk subsets and association with functional maturation the role of stat4 in species-specific regulation of th cell development by type i ifns interleukin 12 and tumor necrosis factor alpha are costimulators of interferon gamma production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist il-1 alpha and tnf-alpha are required for il-12-induced development of th1 cells producing high levels of ifn-gamma in balb/c but not c57bl/ 6 mice inhibition of replication of herpes simplex virus in mouse macrophages by interferon alpha/beta interferon and gamma interferon synergize to inhibit the replication of herpes simplex virus type 1 mathematical analysis demonstrates that interferons-beta and -gamma interact in a multiplicative manner to disrupt herpes simplex virus replication effect of interferon on replication of herpes simplex virus types 1 and 2 in human macrophages mechanism of inhibition of hsv-1 replication by tumor necrosis factor and interferon gamma synergistic anti-hsv effect of tumor necrosis factor alpha and interferon gamma in human corneal fibroblasts is associated with interferon beta induction synergistic anti-herpes effect of tnf-alpha and ifn-gamma in human corneal epithelial cells compared with that in corneal fibroblasts daubener w: inhibition of human herpes simplex virus type 2 by interferon gamma and tumor necrosis factor alpha is mediated by indoleamine 2,3-dioxygenase interleukin-12 (il-12) and il-18 are important in innate defense against genital herpes simplex virus type 2 infection in mice but are not required for the development of acquired gamma interferon-mediated protective immunity interleukin-12-and gamma interferon-dependent innate immunity are essential and sufficient for long-term survival of passively immunized mice infected with herpes simplex virus type 1 il-12 and il-18 act in synergy to clear vaccinia virus infection: involvement of innate and adaptive components of the immune system reduced severity of hsv-1-induced corneal scarring in il-12-deficient mice il-12 suppresses the expression of ocular immunoinflammatory lesions by effects on angiogenesis therapeutic protective effects of il-12 combined with soluble il-4 receptor against established infections of herpes simplex virus type 1 in thermally injured mice requirement for natural killer cell-produced interferon gamma in defense against murine cytomegalovirus infection and enhancement of this defense pathway by interleukin 12 administration an absolute and restricted requirement for il-12 in natural killer cell ifn-gamma production and antiviral defense. studies of natural killer and t cell responses in contrasting viral infections interleukin-15 and natural killer and nkt cells play a critical role in innate protection against genital herpes simplex virus type 2 infection in the absence of t cells, natural killer cells protect from mortality due to hsv-1 encephalitis protective immunity to genital herpes simpex virus type 2 infection is mediated by t-bet the role of natural killer cells in protection of mice against death and corneal scarring following ocular hsv-1 infection kinetics of cytokine production in the cornea and trigeminal ganglion of c57bl/6 mice after corneal hsv-1 infection human natural killer cell deficiencies and susceptibility to infection nitric oxide as a secretory product of mammalian cells herpes simplex virus type 2 synergizes with interferon-gamma in the induction of nitric oxide production in mouse macrophages through autocrine secretion of tumour necrosis factor-alpha production of key molecules by ocular neutrophils early after herpetic infection of the cornea requirement for type 2 no synthase for il-12 signaling in innate immunity absence of tumour necrosis factor facilitates primary and recurrent herpes simplex virus-1 infections macrophage control of herpes simplex virus type 1 replication in the peripheral nervous system nitric oxide and hsv vaginal infection in balb/c mice interferon (ifn)-gamma and herpes simplex virus/tumor necrosis factor-alpha synergistically induce nitric oxide synthase 2 in macrophages through cooperative action of nuclear factor-kappa b and ifn regulatory factor-1 requirement for transcription factor irf-1 in no synthase induction in macrophages endothelial interferon regulatory factor 1 cooperates with nf-kappa b as a transcriptional activator of vascular cell adhesion molecule 1 nf kappa b and interferon regulatory factor 1 physically interact and synergistically induce major histocompatibility class i gene expression interferon regulatory factor-2 physically interacts with nf-kappa b in vitro and inhibits nf-kappa b induction of major histocompatibility class i and beta 2-microglobulin gene expression in transfected human neuroblastoma cells complex formation of the interferon (ifn) consensus sequence-binding protein with irf-1 is essential for murine macrophage ifn-gamma-induced inos gene expression does nitric oxide play a critical role in viral infections? inhibition of viral replication by interferon-gammainduced nitric oxide synthase interferon-gamma induced type i nitric oxide synthase activity inhibits viral replication in neurons inhibition of viral replication by nitric oxide and its reversal by ferrous sulfate and tricarboxylic acid cycle metabolites nitric oxide and macrophage antiviral extrinsic activity evidence for antiviral effect of nitric oxide. inhibition of herpes simplex virus type 1 replication early inhibition of nitric oxide production increases hsv-1 intranasal infection role of nitric oxide synthase type 2 in acute infection with murine cytomegalovirus mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced th1 cell responses phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase epr characterization of molecular targets for no in mammalian cells and organelles dna strand breakage, activation of poly (adp-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite nitric oxide regulation of superoxide and peroxynitrite-dependent lipid peroxidation. formation of novel nitrogen-containing oxidized lipid derivatives altered immune responses in mice lacking inducible nitric oxide synthase rapid interferon gamma-dependent clearance of influenza a virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice osterhaus ad: inhibition of influenza virus replication by nitric oxide nitric oxide inhibits the replication cycle of severe acute respiratory syndrome coronavirus interleukin-4-mediated inhibition of nitric oxide production in interferon-gamma-treated and virus-infected macrophages mechanisms of suppression of macrophage nitric oxide release by transforming growth factor beta spontaneously increased production of nitric oxide and aberrant expression of the inducible nitric oxide synthase in vivo in the transforming growth factor beta 1 null mouse effect of the deletion of us2 and us3 from herpes simplex virus type 2 on immune responses in the murine vagina following intravaginal infection small amounts of exogenous il-4 increase the severity of encephalitis induced in mice by the intranasal infection of herpes simplex virus type 1 herpes simplex virus type 2 infection of macrophages impairs il-4-mediated inhibition of no production through tnfalpha-induced activation of nf-kappab il-4-induced stat6 suppresses ifngamma-stimulated stat1-dependent transcription in mouse macrophages impaired il-13-mediated functions of macrophages in stat6-deficient mice structure and regulation of the human interferon regulatory factor 1 (irf-1) and irf-2 genes: implications for a gene network in the interferon system structurally similar but functionally distinct factors, irf-1 and irf-2, bind to the same regulatory elements of ifn and ifn-inducible genes absence of the type i ifn system in ec cells: transcriptional activator (irf-1) and repressor (irf-2) genes are developmentally regulated recognition dna sequences of interferon regulatory factor 1 (irf-1) and irf-2, regulators of cell growth and the interferon system b lymphocyte-induced maturation protein (blimp)-1, ifn regulatory factor (irf)-1, and irf-2 can bind to the same regulatory sites inhibition of no production in macrophages by il-13 is counteracted by herpes simplex virus infection through tumor necrosis factor-alpha-induced activation of nk-kappa b interleukin-4 and interleukin-10 modulate nuclear factor kappab activity and nitric oxide synthase-2 expression in theiler's virus-infected brain astrocytes interaction of interferon regulatory factor-1 and nuclear factor kap-pab during activation of inducible nitric oxide synthase transcription i wish to thank all members of the group for highly constructive discussions and especially søren c. mogensen for critical review of the manuscript. for excellent technical help with the electron microscopy i thank ruth nielsen. furthermore, i wish to thank the department of clinical microbiology, aarhus university hospital, skejby and department of medical microbiology and immunology, university of aarhus for their hospitality and support. key: cord-023391-bq5w3jk9 authors: utermöhlen, o.; karow, u.; baschuk, n.; herz, j.; loegters, t. t.; krönke, m. title: delayed elimination of the lcm virus from acid sphingomyelinase‐deficient mice due to reduced expansion of virus‐specific cd8(+) t lymphocytes date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423l.x sha: doc_id: 23391 cord_uid: bq5w3jk9 the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn‐γ generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase(–/–) mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8(+) t cells. the secretion of ifn‐γ in response to contact with target cells as well as the cytolytic activity of virus‐specific cd8(+) t cells was severely impaired. additionally, both phases of the lcm virus‐specific dth response, mediated by cd8(+) and cd4(+) t cells consecutively, were diminished in asmase(–/–) mice. however, the secondary memory response of virus‐specific ctl was not altered, and the virus was effectively controlled for at least 3 months by asmase(–/–) mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus‐specific cd8(+) t cells during the acute infection of mice with the lcm virus. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023429-x52gbklw authors: ruseva, m.; gajdeva, m.; takahashi, k.; ezekowitz, a.; thiel, s.; jensenius, j. c. title: mannan‐binding lectin inhibits humoural responses date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423an.x sha: doc_id: 23429 cord_uid: x52gbklw chronic hepatitis b virus (hbv) infection affects about 200–400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n‐linked glycosylation side and is recognized by both mbl‐a and mbl‐c in a ca‐dependent manner. hbsag–mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl‐a and mbl‐c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag‐specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10‐fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein‐ag‐mbl‐rich complexes inhibit b‐cell responsiveness via putative mbl receptors. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023935-o2ffxgnn authors: lorts, angela; cornell, timothy t.; shanley, thomas p. title: sepsis date: 2011-12-16 journal: pediatric critical care study guide doi: 10.1007/978-0-85729-923-9_27 sha: doc_id: 23935 cord_uid: o2ffxgnn the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and influenced by multiple host and non-host factors including: the developmental stage, the presence of co-morbidities, pathogen-related factors, and genetic influences on both the host inflammatory response as well as the response to pharmacologic agents, all combining to have a profound influence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of inflammation. the health care provider faced with the management of a child with septic shock relies on a comprehensive understanding of the numerous disciplines embodied in the practice of pediatric critical care medicine. the child with septic shock may have simultaneous derangements in the function of virtually every system of the body including: cardiovascular, respiratory, immune, renal, coagulation, hepatic, metabolic and neurologic. the degree to which physiologic alterations are manifest in a given patient is variable and infl uenced by multiple host and non-host factors including: the developmental stage, the presence of comorbidities, pathogen-related factors, and genetic infl uences on both the host infl ammatory angela lorts , timothy t. cornell, and thomas p. shanley response as well as the response to pharmacologic agents, all combining to have a profound infl uence on outcome. the clinician must possess a systematic and multifaceted approach to these critically ill patients. the goal of this chapter is to provide a comprehensive description of the epidemiology, biology and pathophysiology (at both the cellular and organ level) of sepsis, as well as outlining the current principles of managing septic shock. it will be apparent that optimal management requires a strong working knowledge of cardiovascular physiology, infectious diseases, multiple organ interactions, immunity, coagulation, pharmacology, and the molecular biology of infl ammation. before reviewing the epidemiology of pediatric sepsis, it must be appreciated that the conclusions of prevalence studies have been obscured in the past by several factors including a lack of a reliable case defi nition. it has only been in the 1990s that consensus defi nitions for sepsis and septic shock were achieved. it was hoped that the development of standard defi nitions would not only enable accurate characterization of the epidemiology of sepsis, but also serve to stratify patients early in the course of sepsis for the purpose of clinical studies aimed at testing novel therapies. the most widely used defi nition of pediatric sepsis/septic shock is based on the 1992 american college of chest physicians/society of critical care medicine (accp/sccm) consensus conference, with adaptations for the pediatric population. the following four defi nitions resulted from these discussions: sirs , sepsis , septic shock , and severe sepsis . although there is overlap between some of these terms (particularly between septic shock and severe sepsis), each is intended to defi ne a particular patient population. longstanding clinical observations have identifi ed the presence of tachycardia, tachypnea, hyperthermia and leukocytosis as signs of infection, though these responses may also be present in the absence of any apparent infectious source. as a result, this physiologic response was defi ned as the systemic infl ammatory response syndrome (sirs). sirs defi nes a state of infl ammation/immune activation in a child and is based on the presence of at least two of the four criteria listed in table 27 -1 . thus, patients with diverse clinical conditions such as sepsis, pancreatitis, burns, or severe trauma can meet criteria for sirs. it has been argued that the sirs defi nition is non-specifi c and that too broad a range of patients are ultimately classifi ed as having sirs. nevertheless, the criteria have been widely used in both prescriptive and interventional studies to enhance the "capture" of all patients at risk for the subsequent development of severe sepsis or septic shock. sirs i s a state of infl ammatory/ immune activation and is based on the presence of at least two of the four following clinical criteria: temperature >38°c or <36°c, heart rate >90th percentile for age, respiratory rate >90th percentile for age, or hyperventilation to paco 2 < 32 mm hg. the defi nition attempts to "capture" all patients at risk for the subsequent development of severe sepsis or septic shock. sepsis is defi ned as a sirs response which is secondary to an infection, either documented by microbiology cultures or other clinical evidence of infection. severe sepsis is defi ned by sepsis criteria plus evidence of insuffi cient end organ perfusion (table 27-1 ) . finally, septic shock is defi ned by sepsis criteria plus hypotension (two distinct measurements <3rd percentile for age) after the administration of at least 20 ml/kg of crystalloid or colloid, in addition to the criteria listed for severe sepsis (table 27-1 ) . these criteria have been used extensively for conducting clinical investigations and have proven to be of value despite criticism for lack of both sensitivity and specifi city. the latest consensus conference was convened in 2007 to further refi ne the diagnostic criteria and therapeutic recommendations, with specifi c considerations for the pediatric population. published in 2008, the surviving sepsis campaign aims to improve the outcome in sepsis worldwide. the refi nement of pediatric-specifi c criteria for septic shock is also intended to aid future clinical trials and epidemiologic investigations in pediatric sepsis. the few published pediatric-specifi c studies illustrate the importance of sepsis in this age range. proulx analyzed the incidence and outcome of sirs, sepsis, severe sepsis, and septic shock in a single institution. over 1,000 admissions were analyzed over a 1-year period. sirs was present in 82% of patients, while 23% had sepsis, 4% had severe sepsis, and 2% had septic shock. the overall mortality for this population was 6% with a majority of deaths occurring in patients with multiple organ dysfunction syndrome (mods). an epidemiologic study using discharge international classifi cation of disease, 9th revision (icd-9) codes reviewed hospital records from seven large states representing nearly one-quarter of the united states population. while the criteria used for inpatient coding at discharge are not identical to accp/sccm consensus conference criteria, the study estimated an incidence of 42,371 cases of severe sepsis in individuals less than 20 years of age (0.6 cases/1,000 population). the highest incidence was in neonates (5.2 cases/1,000 population), compared to children ages 5-14 who had an incidence of 0.2 cases/1,000 population. the overall mortality rate was 10.3% (4,364 deaths nationally) consistent with the frequent observation that the mortality rate remains lower than comparable adult data. the study estimated an annual national health care cost of $1.7 billion associated with severe sepsis in children. a follow-up study with the same methodology appeared to show a 13% increase in the absolute number of cases of severe sepsis from 1995 and 1999 with the majority of this increase accounted for by severe sepsis in children less than 1 year of age. the mortality rate had decreased to 9.0% during this time period. collectively, these data illustrate that sepsis is a major health problem on the basis of incidence, mortality, and health care costs. there remains a need for further, well-designed epidemiologic studies of pediatric sepsis. future studies will enhance our understanding of not only epidemiology, but also the impact of new diagnostic and therapeutic approaches resulting from improved design of interventional trials specifi c to the pediatric population. sepsis is a systemic disease and can impact the functioning of all organ systems. the most common clinical manifestations of sepsis include: fever or hypothermia, tachypnea, tachycardia, leukocytosis or leukopenia, thrombocytopenia, and change in mental status. one of the earliest signs of infection is fever which results from the pyrogenic effect of cytokines, particularly interleukin (il)-1 b and tumor necrosis factor (tnf)-a . presentation with hypothermia can also occur, but is more common in infants. one traditional classifi cation of shock states divides this clinical state into three broad categories: hypovolemic, cardiogenic and distributive shock. the shock associated with 555 c hapter 27 • s eps is sepsis is unique in that all three forms are likely to be present. hypovolemic shock results from capillary leak, increased insensible losses, and decreased effective blood volume secondary to venodilation. cardiogenic shock is related to direct myocardial depression, the cause(s) of which remains the focus of investigation. finally, distributive shock is often apparent as brisk capillary refi ll, widened pulse pressure and bounding peripheral pulses and is caused by abnormally decreased systemic vascular resistance from pathologic vasodilation. the particular pattern of these hemodynamic physiologic perturbations manifested by any individual patient can be variable. some children have increased cardiac output with diminished systemic vascular resistance characteristic of distributive shock or the so-called "warm" shock state. in stark contrast to adults, in which this hemodynamic profi le (increased cardiac output/decreased systemic vascular resistance) is most common, children more frequently present with depressed cardiac output and elevated systemic vascular resistance. these patients appear cool with diminished pulses and poor capillary refi ll that is characteristic of the "cold" shock state. while important to recognize that patients may transition from one state to another, the presence of hypotension is often a late and particularly ominous sign that requires prompt intervention as its presence is associated with increased mortality. patients with sepsis often present with alterations in their respiratory system, notably tachypnea that refl ects a compensatory respiratory alkalosis aimed at neutralizing a metabolic acidosis related to hypoperfusion and anaerobic metabolism. chest x-ray fi ndings can reveal a small heart in the presence of hypovolemia with few vascular markings. alternatively, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation in some children with sepsis can result in pulmonary edema. rapid progression to acute respiratory failure from ards is not uncommon. all organ systems and ultimately cellular functions are affected by poor perfusion and decreased oxygen delivery related to depressed cardiac and respiratory function. in addition, there may be direct injurious effects of bacterial toxins and circulating cytokines such as triggering of programmed cell death or apoptosis. the neurologic state of a child with sepsis is frequently altered and can range from agitation or irritability to frank obtundation. this depressed mental status can be present even in the absence of meningitis as a manifestation of cerebral hypoperfusion. skin manifestations are not uncommon and can include petechiae and purpura that are ominous signs of disseminated intravascular coagulation (dic) and purpura fulminans secondary to meningococcemia. diffuse erythema secondary to toxic shock syndromes can be present. there is also an increasing appreciation of sepsis-induced microvascular angiopathy contributing to distal skin and organ ischemia. an initial thorough and detailed physical exam provides both important clues to the diagnostic possibilities of pediatric septic shock and the underlying hemodynamic profi le. however, serial exams are imperative to follow pathophysiologic changes and to gauge the impact of therapeutic interventions in reversing the manifestations of shock. data from both clinical and basic science studies have supported the hypothesis that pathogens and/or their products initiate a host immune response that triggers widespread infl ammation causing tissue injury and organ dysfunction. potential initiating pathogens include gram-negative and gram-positive bacteria, viruses, fungi and protozoa. in some cases, overwhelming spread of pathogens (e.g. bacteremia) with release of toxins (e.g. endo-or exotoxins) may directly injure the host resulting in organ dysfunction. higher order organisms have evolved an immune system to eradicate pathogens which has evolved to include two systems: the innate or natural immune system and the acquired or adaptive immune system. the innate immune system is responsible for the highly conserved function of recognizing pathogens and mounting an effector response. it includes a series of molecules located on the cell surface termed pattern-recognition receptors (prr) which are capable of recognizing a broad array of conserved structures on a variety of children with sepsis may have hemodynamic characteristics that transcend traditional classifi cation. they often have elements of hypovolemia, cardiac dysfunction and abnormal vascular tone. in the septic child, the combination of capillary leak, decreased myocardial function and the result of fl uid resuscitation may result in rapid progression to acute respiratory failure. pathogens (so-called pathogen-associated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide (lps) on gram-negative bacteria, lipoteichoic acid on gram-positive bacteria, mannans on yeast, double-stranded rna of rna viruses and unmethylated, cpg dna from bacteria. the effector responses that are regulated by the innate immune system (e.g. phagocytes, complement) are activated immediately upon infection and are designed to rapidly inhibit the replication of microorganisms. these cell surface pattern-recognition receptors (prr) are expressed on most antigen presenting cells of the innate immune system and represent diverse families of proteins. one group of prrs, the toll-like receptors (tlr), has been identifi ed as perhaps the most critical pathogen recognition receptor family in the context of sepsis biology. other families of prr include the c-type coll agenous lectins (collectins) that bind to a variety of carbohydrate moieties on cells, bacteria and viruses. most members of this family share structural homology to the complement protein c1q and can functionally substitute for c1q in activating the complement cascade. another family of prr possesses leucine-rich regions critical for protein-protein interactions that are necessary for immune recognition. examples of these leucine rich receptors include cd14, a receptor on the cell surface of macrophages that binds to lps and the macrophage scavenger receptor that binds to bacterial cell walls. unbound circulating prrs exist and include pentraxins, such as c-reactive protein, an acute phase reactant synthesized by the liver and lipopolysaccharide-binding protein (lbp) which binds to lps to optimize its binding to the cd14/toll-like receptor cellular complex. another key component of innate immunity is the complement system. the complement system is a complex cascade of proteins that possesses a broad array of anti-pathogen activities including: opsonization (c3), neutrophil chemotaxis (c5a), perforating cytotoxicity (c6-9, mac complex) and the ability to bind to and directly lyse viruses (c1). an in depth discussion of the role of complement in the response to infection is beyond the scope of this chapter, but has been recently summarized. in summary, the host possesses a ubiquitous and diverse set of pathogen recognition receptors which function to protect the host from infectious challenges, but at the expense of triggering powerful effector responses. paramount to effector responses of the innate immune system is a proinfl ammatory action of numerous cytokines and chemokines. these biologically active proteins are critical to the activation and recruitment of cellular components of the adaptive immune system. while necessary for pathogen clearance, this acute, proinfl ammatory immune response must also ultimately subside in order to reestablish homeostasis and avoid cellular and tissue damage. a key pathophysiologic feature of sepsis is that this immune response often appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response seen frequently in response to infection can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). lps recognition: recent epidemiologic surveys of the causative agents of sepsis have indicated an increase in the incidence of gram-positive organisms such that there is a roughly equivalent prevalence between these and gram-negative organisms. historically, sepsis research has focused on the role of gram-negative bacteria in evoking a pathologic response. the structure of endotoxin shows three domains: an outer polysaccharide hydrophilic chain which determines the o-antigenicity, an acidic core region, and a lipid-rich region. gramnegative organisms possess endotoxins with variable repeats of mono-and heteropolysaccharides with complex side chain structure to provide a basis for distinct antigenicity. the o-region is linked via an acidic core to the lipid a region that is highly conserved and responsible for much of the toxicity attributed to intact lps. a series of seminal observations have determined the molecular mechanisms by which the classic pamp, lps, initiates a proinfl ammatory response. first, a strain of lps-resistant mice, the c3h/hej strain, was identifi ed and its resistance was found to be attributed to a single genetic mutation. second, it was shown that the lethal effects of endotoxin could be conferred by transfer of hematopoietic cells. endotoxin tolerant mice could be rendered the cells of the innate immune system contain cell surface molecules termed patternrecognition receptors (prr). these receptors are capable of recognizing a broad array of conserved structures on a variety of pathogens (so-called pathogenassociated molecular patterns, or pamp's). examples of pamp's include: lipopolysaccharide, lipoteichoic acid, viral rna and bacterial dna. toll-like receptors (tlr) are pathogen recognition receptors that have a critical role in sepsis. tlr4 is active in recognition of lps on gram-negative bacteria whereas tlr2 is active in the recognition of lipotechoic acid on gram-positive bacteria. a hallmark of sepsis is an immune response that appears to become unregulated resulting in an overwhelming proinfl ammatory response and host autodestruction. this characteristic systemic infl ammatory response is seen frequently in response to infection, but can also be observed in association with non-infectious triggers (e.g. trauma, burns, pancreatitis, cardiopulmonary bypass). c hapter 27 • s eps is lps-sensitive after reconstitution with hematopoietic cells derived from the monocyte/macrophage lineage from an lps-sensitive strain. third, stimulation of monocyte-derived cells with endotoxin resulted in production of several cytokines and chemokines critical to the systemic infl ammatory response. among these, tnf and il-1 were shown to be critical initiators of the septic response and could in fact mimic the endotoxin response. finally, the elucidation of the lps receptor assisted the identifi cation of those signal transduction pathways by which endotoxin triggers infl ammatory gene expression. lps receptor: membrane bound cd-14 was shown to be required for lps signaling. however, it lacked a transmembrane extension required for cytoplasmic signaling indicating the presence of additional components of the receptor complex. investigators working with drosophila had identifi ed a gene, toll, which was responsible for dorsoventral polarization in embryonic development. when toll was functionally mutated, it was demonstrated to play a key role in host defense against aspergillus fumigatus . homology between the toll-like receptors and the mammalian il-1 family of receptors was discovered and provided additional evidence that this family was crucial to the human innate immune response. finally, it was determined that the c3h/hej mouse strain which is hyporesponsive to lps possessed a mutation in toll-like receptor 4 (tlr4), providing further evidence that this receptor was necessary for lps signaling. tlr4 is one of ten mammalian toll-like receptors that have been cloned to date, each being activated by a specifi c set of ligands. since these discoveries, other members of the lps-receptor complex have been elucidated and include both md-2 and myd88. it is also known that circulating lpsbinding protein (lbp) facilitates lps binding to the cell surface receptor complex. together these components are able to "sense" lps at the cell surface and transmit this signal via a series of complex pathways. similarly, the products of gram-positive organisms, notably the cell wall component lipotechoic acid, activate cell activation through the related toll-like receptor 2 (tlr2). after engagement of cell surface receptors (e.g. tlr2 and tlr4), several important signal transduction pathways are activated that elicit a number of transcriptional factors responsible for infl ammatory gene expression. among these, the nuclear factor-k b (nf-k b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. in the case of nf-k b, stimulation of the lps receptor causes phosphorylation of the inhibitor of k b kinases (i k k) which in turn phosphorylates the intracellular inhibitor of nf-k b, i kappa b (i k b). upon phosphorylation, i k b undergoes poly-ubiquination followed by proteosomal degradation. the removal of i k b effectively unmasks a nuclear translocation sequence on nf-k b enabling it to proceed into the nucleus to bind to nf-k b consensus sequences present on the promoter regions of many infl ammatory genes: cytokines including tnf, chemokines including il-8, adhesion molecules including e-selectin and others such as inos (see fig. 27 -1 ). the role of nf-k b in sepsis is supported by studies demonstrating that survivors and non-survivors of sepsis are distinguishable on the basis of nf-k b binding activity in peripheral blood mononuclear cells. in addition, in sepsis-induced ards, increased activation of nf-k b in macrophages obtained by bal is found in ards patients when compared to icu controls. to a similar degree, the mapk signaling pathways are important in mediating the septic response. three mapk pathways exist: p38 protein kinase, extracellular-regulated protein kinase (erk), and c-jun-terminal kinase (jnk). evidence exists for the role of each of these signaling pathways in sepsis. tnf production by neutrophils and macrophages is dependent on p38 activation. lps stimulation of monocytes activates jnk with downstream activation of activating protein-1 (ap-1) and subsequent il-1 b production. lps induction of tnf is in part dependent on erk pathway activation. together, these two pathways, nf-k b and mapk's, appear to be critical to the propagation of signals from the cell surface to the nucleus where expression of infl ammatory gene products occurs. as such, these pathways remain valid targets for future strategies in modulating the septic response. nuclear factork b (nfk b ) and the mitogen activated protein kinase (mapk) pathways play a prominent role in regulating the expression of a number of infl ammatory gene products key to propagating the sepsis response. while numerous proteins have been shown to play a role in the septic response, a full review of each protein's function is beyond the scope of this study guide. instead, we aim to highlight some known principle mediators in this cascade. evidence that tnf mediates the septic response stems from numerous observations: it is produced by hematopoietic cells, its expression is temporally related to the development of septic shock, recombinant tnf induces experimental septic shock in animals, and passive immunization against tnf attenuates endotoxin-mediated responses. tnf possesses numerous functions in infl ammation such as driving adhesion molecule and chemokine expression to facilitate leukocyte-endothelial cell adhesion; upregulating tissue factor and inhibition of protein c to create a pathologic procoagulant state in the vasculature; and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. in human studies, levels of tnf have been shown to correlate with mortality, with the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. the name, il-1, is now used to describe the family of proteins including two agonists (il-1 a and il-1 b ) and one antagonist, the il-1 receptor antagonist protein (il-1ra). il-1 b which is secreted, mediates much of the systemic effects attributed to il-1 release in sepsis. synthesized as a propeptide, il-1 b requires proteolytic cleavage by the il-1 converting enzyme (ice) to become bioactive. il-1 b utilizes the 80-kda type i receptor which is tnf possesses numerous functions in infl ammation such as inducing adhesion molecules and chemokines that facilitate leukocyte-endothelial cell adhesion and inducing nitric oxide synthase (inos) which mediates pathologic vasodilation. tnf also upregulates tissue factor and inhibits protein c to create a pathologic procoagulant state in the vasculature. clinically, levels of tnf correlate with mortality, the development of shock and purpura fulminans and with the development of sepsis-induced ards and shock. p p iκ κ κ κk associated with a number of adapter proteins (e.g. myd88,tnf receptor-associated factor 6 (traf6) and interleukin-1 receptor-associated kinase (irak)) to propagate signals through both the nf-k b and ap-1 pathways. il-1 b infusion elicits fever, hypotension and leukocytic infi ltration to the lungs. in a manner similar to tnf, il-1 stimulates monocyte activation and phagocytosis, increases adhesion molecule expression, and increases tissue factor expression while inhibiting thrombomodulin secretion, thus creating a procoagulant state. when detected in the circulation of septic patients, il-1 levels also correlate with mortality. of note, the il-1ra is a circulating inhibitor of il-1 b that binds to the il-1 receptor without initiating a signal. the expression of il-1ra has been shown to follow peak expression of il-1. it is speculated that il-1ra is an endogenous regulator of il-1 effects. however, in clinical trials, il-1ra infusion failed to improve mortality in sepsis. furthering our molecular understanding of sepsis-induced organ dysfunction was the identifi cation of the "leukocyte-endothelial cell adhesion cascade". this cascade is characterized by cytokine activation of the selectin family of adhesion molecules (e.g. e-selectin) on the endothelium which initiate a process of neutrophil "rolling" via interaction with sialyated moieties constitutively present on circulating neutrophils. activation of the "rolling" neutrophil results in both increased expression and activation of the integrins which in turn bind to intercellular adhesion molecule (icam)-1 that is upregulated on the endothelial cell surface by tnf and il-1 b . this integrin-icam-1 interaction mediates fi rm adhesion of the neutrophil to the endothelial cell surface. finally, in response to various chemotactic cytokines or chemokines, neutrophils migrate to the site of infl ammation. release of both oxygen-and nitrogen-based radical species and proteases by the neutrophils may ultimately contribute to cellular injury and organ dysfunction. nitric oxide (no) is responsible for endothelium-derived relaxation of blood vessels. three isoforms of nitric oxide synthase are responsible for production of no: type i, a neuronal isoform (nnos); type ii, an inducible isoform (inos) and type iii, a constitutive, endothelial isoform (enos). tnf and il-1 b are capable of inducing inos and increased levels of circulating stable byproducts of no are found in both septic adults and children who simultaneously display low systemic vascular tone. this supports the hypothesis that no plays a principal role in septic shock via pathologic vasodilation. it has also been suggested that tnf and il-1 b may be the so-called "myocardial depressant factors" by increasing circulating no through induction of inos ; however, it is not clear that no is the exclusive mediator of these effects. in light of the evidence supporting the role of no in septic shock, clinical trials employing no synthesis inhibitors in septic shock were initiated. though early clinical reports and small studies reported that nos inhibitors could signifi cantly improve blood pressure, this was at the expense of decreasing cardiac output secondary to increased afterload. as a not uncommon hemodynamic profi le in pediatric septic shock is decreased cardiac output and elevated systemic vascular resistance, it is not cleat that nos inhibitors will have a therapeutic role in pediatric (or adult) sepsis in the future. studies employing agents directed against the early mediators of the septic response have been mostly ineffectual. this has led to the hypothesis that additional molecules with delayed kinetics of expression may infl uence the outcome in sepsis. as an example, it was observed that lps-challenged mice often die long after peak expressions of tnf and il-1 b suggesting that late-acting proteins may contribute to endotoxin-induced mortality. investigators searching for late expressed proteins identifi ed a member of the high mobility group (hmg)-1 non-histone chromosomal protein family in conditioned media 16 h after lps-stimulation of macrophages. this protein renamed hmgb1 is a known ligand for the receptor for advanced glycation end products (rage). the rage receptor is expressed on monocytes and vascular smooth muscle. binding by hmgb1 activates both the nf-k b and mapk pathways. increased expression of hmgb1 was found in endotoxemic mice and in critically ill patients with surgical sepsis where increased levels correlated with non-survival. in animal models, the blockade of hmgb1 can inhibit the infl ammation associated with endotoxemia, cecal ligation and puncture and lps-triggered acute lung injury. identifi cation of hmgb1 and similar "late" mediators may provide a broader therapeutic window for successful immune modulating therapy in sepsis. regulatory processes and mediators exist for the purpose of modulation and eventual resolution of infl ammation and the septic response. an absence in the decline of proinfl ammatory mediators such as tnf and il-6 over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous cytokine antagonists including soluble tnf receptors, the il-1ra and additional anti-infl ammatory cytokines, such as il-10 and transforming growth factor-β (tgf-b ). il-10 has multiple anti-infl ammatory properties including inhibition of cytokine production from activated monocytes. il-10 inhibits expression of those cytokines known to contribute to sepsis, as well as important chemokines, including il-8. in addition, il-10 increases expression of other anti-infl ammatory molecules such as il-1ra and soluble tnf receptors. exogenous administration of il-10 in various experimental models has been used in an attempt to decrease infl ammatory cytokines and diminish organ injury. human studies showed that patients who did not survive ards had lower levels of il-10 in their bal fl uid compared to survivors. furthermore, the inability to increase il-10 in response to meningococcal infection was associated with increased mortality. thus, il-10 and additional regulatory cytokines (e.g. tgf-b , il-13) possess a number of anti-infl ammatory properties and are important contributors to the endogenous regulation of the acute septic response. dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. a common hematologic alteration in sepsis is the development of disseminated intravascular coagulation (dic) which is an acquired state of activation of coagulation and intravascular fi brin formation resulting in vascular thrombosis. in addition to proinfl ammatory cytokines, tissue factor (tf) activation also plays a prominent role in activating the coagulation cascade, initiating fi brin formation and contributing to the development of dic. concurrent with enhanced production of fi brin, there is decreased fi brinolysis related to increased plasminogen activator inhibitor type 1 (pai-1), as well as dysfunction and/or depletion of antithrombin iii, protein c, protein s and tissue factor pathway inhibitor (tfpi). at iii which inhibits thrombin by forming thrombin-antithrombin (tat) complexes is decreased in sepsis related to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. protein c is also noted to be depleted among patients with sepsis and septic shock. regulation of activation of protein c to activated protein c (apc) in the coagulation cascade is mediated in a complex manner and will not be discussed here. apc, upon dissociation from its receptor, binds to its co-factor, protein s, to subsequently inactivate factors va or viiia, thus playing a key role in inhibiting coagulation. it is both antithrombotic and profibrinolytic. apc also possesses anti-infl ammatory activity. in models of endotoxemia, apc infusion decreases cytokine production and attenuates neutrophil activation. these antiinfl ammatory effects appear to be independent of apc's anticoagulant effect. following an absence in the decline of proinfl ammatory mediators such as tnf and il-6 over the course of sepsis is an associated risk factor for mortality. monocyte activation results not only in production of proinfl ammatory cytokines, but also expression of a number of endogenous anti-infl ammatory cytokines including soluble tnf receptors, the il-1ra and additional anti-infl ammatory cytokines, such as il-10 and tgfb . important anti-infl ammatory molecules include il-10, il-1ra and soluble tnf receptors. il-10 inhibits expression of proinfl ammatory cytokines known to contribute to sepsis, as well as important chemokines, including il-8. in addition, il-10 increases expression of other anti-infl ammatory molecules such as il-1ra and soluble tnf receptors. at iii inhibits thrombin by forming thrombin-antithrombin (tat) complexes. at iii is decreased in sepsis due to degradation by elastases from activated neutrophils, dilution secondary to volume resuscitation, and impaired hepatic synthesis. despite a correlation between low at iii levels and mortality in patients with sepsis, replacement trials of at iii have failed to show a signifi cant effect on improving mortality. these encouraging pre-clinical studies, clinical trials examining the effect of apc on mortality from sepsis were commenced culminating in the protein c worldwide evaluation in severe sepsis (prowess) trial. in this study, apc was associated with a statistically signifi cant reduction in 28-day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome ( fig. 27-2 ). it is not uncommon to observe that patients exposed to seemingly identical pathogen insults display strikingly different pathophysiology and outcomes. it is believed that genetic differences among hosts are at least in part responsible for this variability in sepsis responses. as mentioned previously, the insensitivity to lps in the c3h/hej mouse line was mediated by a mutation in the coding sequence for tlr4. similar fi ndings of an attenuated response to pathogen stimulation have now been reported in patients with mutations in both the tlr4 and tlr2 gene. the polymorphism in tlr2 appears to confer an increased predisposition to severe gram-positive bacterial infections. more recently, a polymorphism within the cd14 promoter gene (c to t transition at base pair -159) was identifi ed with a particular genotype over-represented among septic shock patients compared to healthy controls. among the septic patients, the presence of this genotype also was associated with a signifi cantly higher mortality (71% versus 48%). these studies support the concept that genetic alterations in those genes known to participate in the septic response affect the host immune response and likelihood of survival. for a more complete review of the numerous examples of genetic alterations in key infl ammatory genes, the reader is directed to the suggested readings. the administration of activated protein c was associated with a statistically signifi cant reduction in 28-day mortality in septic adults. however, a recent pediatric study was stopped after an interim analysis showed that apc administration was highly unlikely to show improvement in outcome. the coagulation cascade in sepsis (reprinted with permission from bernard 2001 ) as the cellular response to sepsis has become better understood, the approach to treatment of sepsis has become broader. the treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. as reviewed above, sepsis is an immunologically complex response to an invasive pathogen necessitating tight physiologic regulation in order to eradicate the organism while maintaining cellular and organ homeostasis. in cases where the immunologic and infl ammatory responses continue to escalate, numerous pathways are altered and may ultimately prove amenable to immune modulating therapy, but this approach has been unsuccessful to date. the initial priority in the treatment of the septic child is respiratory and cardiovascular stabilization. the primary goals of therapy in those initial hours following clinical presentation are to maintain oxygenation and ventilation, achieve normal perfusion and blood pressure, and re-establish appropriate urine output for age. children with sepsis may have altered mental status which, if profound, raises concern about the ability to protect the airway. tachypnea associated with a primary or compensatory respiratory alkalosis is commonly present. the combination of increased lung vascular permeability and aggressive fl uid resuscitation to restore intravascular volume and maintain blood pressure may contribute to the subsequent development of pulmonary edema. in children with lung edema, the related changes in lung compliance and loss of functional residual capacity can dramatically increase the work of breathing ultimately necessitating tracheal intubation and mechanical ventilatory support. arterial blood gas analysis may show hypoxemia and metabolic acidosis; however, the decision to provide mechanical ventilatory support should not be based solely on laboratory fi ndings. the presence of increased work of breathing, hypoventilation or obtundation are all indications for instituting mechanical ventilatory support which holds additional benefi t in decreasing the overall oxygen consumption, especially when combined with sedation and paralysis. it should be stated, however, that children with warm shock can commonly be managed without endotracheal intubation so long as they are not obtunded or fl uid overloaded. disorientation or lethargy with intact responsiveness does not require placement of an artifi cial airway as many institutions manage these patients without intubation. the work of breathing associated with hyperventilation in the absence of pulmonary edema is not clinically signifi cant. furthermore, there is no evidence that decreasing work of breathing in the presence of distributive shock will result in redistribution of nutrient fl ow to vital organs, the very nature of distributive shock. however, it is more common for infants to present with cardiac dysfunction and pulmonary edema or seriously altered mental status requiring endotracheal intubation and mechanical ventilation. correction of intravascular volume depletion should be made prior to the institution of positive pressure ventilation. the decrease in venous return after the initiation of positive pressure ventilation may lead to further hemodynamic compromise in the child with intravascular volume depletion. caution should also be taken in choosing sedative agents for intubation, using agents that have the least impact on tenuous hemodynamics (e.g. ketamine). controversy exists over the adrenal suppressive effect of a single dose of etomidate when used for intubation in septic children and adults. the pediatric critical care clinician should be aware of the concern for adrenal suppression following a single dose of etomidate used for the intubation of children with septic shock and the published guidelines which do not recommend its use in this setting. following intubation, attention must be paid to matching the mechanically provided minute ventilation to that which was present during spontaneous respiratory effort so that respiratory compensation of acidemia is preserved. if it is deemed that positive pressure ventilation is not needed, supplemental oxygen should be provided to maintain normal oxygen saturations. treatment of sepsis involves four important components: initial resuscitation, elimination of pathogen, maintenance of oxygen delivery, and carefully directed regulation of the infl ammatory response. with regard to fl uid status, septic children have decreased effective intravascular volume related to a number of causes. poor oral intake of fl uid for a period of time prior to clinical presentation is common. increased vascular permeability leads to intravascular volume loss due to extravasation of fl uid from the vascular space, so-called "third spacing". finally, the no-mediated vasodilation reviewed above increases vascular capacitance thereby decreasing the effective circulating volume. thus, when sepsis is suspected, it is imperative to expeditiously achieve vascular access and initiate fl uid resuscitation with 20 ml/kg of isotonic fl uid as quickly as possible. while debate continues as to the most effective fl uid for resuscitation, no pediatric literature exists to support colloid over crystalloid, the latter of which was recently demonstrated to be equally effective in a large adult icu trial. there is some support for using colloid fl uid in patients with a narrow pulse pressure; however, this practice is not supported by any large, well-designed clinical studies. while following the clinical exam for signs of intravascular volume overload (new onset of rales, increased work of breathing, development of a gallop, or hepatomegaly), fl uid should be administered quickly with the goal of monitoring heart rate response, urine output, capillary refi ll time and level of consciousness. initial fl uid resuscitation of the child with septic shock commonly requires a volume of up to or greater than 60 ml/kg in the fi rst hour and one retrospective study demonstrated an increased survival in children given fl uid volumes of 40 ml/kg or more within the fi rst hour. the accm clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock recommend that the child with septic shock be repeatedly examined for the development of "rales, gallop rhythm, hepatomegaly, and increased work of breathing" during volume loading, and that in the absence of such fi ndings, volumes up to 200 ml/kg can be administered in the fi rst hour. these guidelines further state that "the rate of fl uid administration should be reduced substantially when there are clinical signs of adequate cardiac fi lling without hemodynamic improvement." despite on-going fl uid resuscitation, hypotension and inadequate organ perfusion may persist requiring the initiation of inotropes and/or vasopressors. in children, vasoactive medicines should only be given in addition to fl uid resuscitation. however, consensus guidelines recommend that vasoactive infusions may be necessary in some cases to sustain perfusion pressure even when hypovolemia is not yet resolved. dopamine is the most common fi rst choice agent selected for hemodynamic support in those patients with fl uid-refractory shock. dopamine provides inotropic support at lower concentrations; however, it is often necessary to increase it to higher doses that provide vasopressor activity (up to 20 m g/kg/min) to maintain adequate tissue perfusion. the decision of which agent to add in the setting of dopamine-refractory shock should be based on the underlying cause of cardiovascular compromise. for example, if hemodynamic instability is related to low cardiac output from direct cardio-depressant effects then increased inotropy from dobutamine or low-dose epinephrine may be indicated. if hypotension persists secondary to decreased vascular tone, then agents such as epinephrine and norepinephrine dosed in the alpha agonist range should be considered. finally, in children who demonstrate low cardiac output and/or increased afterload from vasoconstriction (i.e. increased systemic vascular resistance), agents with primarily inotropic or vasodilator function including milrinone, dobutamine or short-acting nitrovasodilators can been considered in the fl uid-resuscitated, normotensive child. as in evaluating the adequacy of fl uid resuscitation, similar clinical parameters should be followed in titrating vasoactive medications. appropriate endpoints include: capillary refi ll time less than 2 s, normal peripheral pulses, warm extremities, urine output greater than 0.5 ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava (svc) oxygen saturation greater than 70%. invasive monitoring can further assist the clinician with goal directed endpoints. although frequent beside examination remains integral to the care of the child in septic shock, an additional task in the initial resuscitation phase is placement of appropriate and necessary vascular access and monitors. central venous access is a necessity for the child with fl uid refractory shock to provide for delivery of vasoactive medicines and large volumes of fl uid. these catheters can be useful for following the central venous pressure (cvp) during fl uid adequate volume resuscitation of the child with septic shock commonly requires a volume of up to or greater than 60 ml/kg in the fi rst hour. appropriate endpoints of sepsis resuscitation include: capillary refi ll time less than 2 s, normal peripheral pulses, warm extremities, urine output greater than 0.5 ml/kg/h, improved mentation, resolving acidemia, decreasing serum lactate and when available, superior vena cava oxygen saturation greater than 70%. administration. finally, when the tip is located in the superior vena cava, blood sampling can provide an approximate measure of the mixed venous oxygen saturation which has been validated as a critical target in adult shock resuscitation. the decision regarding the access site for a central venous catheter is dictated by a number of mitigating factors such as the experience level of the operator and the presence of coagulopathy. femoral catheters, in the absence of abdominal pathology, can be used to estimate cvp with good correlation. the cvp measured in the abdominal inferior vena cava must be assessed carefully as a low cvp can be a reliable indicator of hypovolemia, however, a normal or high cvp in the presence of abdominal distention does not automatically exclude the presence of hypovolemia. multiple adult studies have demonstrated that even an accurate intra-thoracic cvp may be a poor approximation of left ventricular end diastolic pressure and volume. the cvp can be elevated despite the presence of hypovolemia if pulmonary hypertension, right ventricular dysfunction with poor diastolic compliance, tricuspid regurgitation, cardiac tamponade or an intracardiac left-to-right shunt exists. even though precise determination of the true mixed venous saturation requires the presence of a pulmonary artery catheter, the approximations derived from the svc saturation have proven a useful target in septic adults. in contrast, because of differences in oxygen extraction between the upper extremities, abdomen, and lower extremities, venous oxygen saturations from a low lying femoral line do not accurately correlate with those measured in the pulmonary artery. consensus guidelines recommend therapeutic endpoints of superior vena cava oxygen saturation >70% or mixed venous (pulmonary artery) oxygen saturation >65%. placement of an intra-arterial catheter provides continuous monitoring of systemic blood pressure, pulse pressure and hemodynamic variation with respiration, as well as a means for drawing arterial blood gases, lactate levels and additional laboratory studies. the arterial blood also provides the most accurate measure of arterial oxygen content and can be used to both assess the function of the lungs and to maximize oxygen delivery. in the ventilated patient, variation in the amplitude of the arterial waveform has been found to correlate closely with intravascular volume status (see also chapter 3 ). systolic pressure variation (spv), also referred to as "reverse pulsus paradoxus," is the variation in beat-to-beat amplitude of the arterial pulse during positive pressure ventilation. a single positive pressure breath normally affects the arterial pressure in a biphasic manner. the initial response to a positive pressure breath is to "squeeze" pulmonary vascular blood into the left atrium (the opposite, "pooling" of blood, occurs with negative pressure inspiration) leading to a rise in systolic pressure. in addition, positive intrathoracic pressure reduces the afterload on the left ventricle by virtue of the pressure gradient from the thorax outward further augmenting this early rise in arterial pressure. these two effects produce an upward movement of the systolic blood pressure coincident with the positive pressure breath, referred to as the d up component of spv. following this d up, a fall in systolic pressure occurs a few beats later as the decreased venous return (preload) to the right ventricle that occurred during positive pressure inspiration is now evident as decreased preload to the left ventricle after a few cardiac cycles. the transient reduction in right ventricular volume and output leads to a smaller left ventricular stroke volume and a brief reduction in arterial pressure that occurs later in the ventilator cycle ( d down). an exaggerated spv (>10 mm hg) has been seen early in the setting of hypovolemia. this is due to a greater d down component. several studies have shown that an increase in the spv occurs prior to a fall in arterial pressure and may a better predictor of hypovolemia than a low pulmonary capillary wedge pressure (pcwp) (<10 mm hg). an increase in the spv due to a greater d down component can also occur due to high airway pressures causing decreased venous return. recently, pulse pressure variation (ppv) has also been found to be a sensitive indicator of preload, and more importantly, fl uid responsiveness. ppv is defi ned as the maximal pulse pressure (systolic minus diastolic blood pressure) less the minimum pulse pressure divided by the average of these two pressures. the use of systolic pressure variation and pulse pressure variation is limited to those patients on mechanical ventilation. these measurements should occur when there is no spontaneous breathing. in the presence of a consistent delivered tidal volume, systolic pressure variation can be used to track the adequacy of intravascular volume over time (fig. 27-3 ) . the decision to use a pulmonary catheter (pac) with the goals of optimizing left ventricular preload, monitoring cardiac index and measuring oxygen delivery remains controversial. caveats to interpretation of pac data include the presence of an intracardiac shunt and an abnormally functioning mitral valve or other obstructed left heart lesion as either shunting, regurgitation, or inaccurate pressure determinations will alter cardiac index and/or the pulmonary capillary wedge pressure measurements. as previous data in adults have shown no benefi t of pac use, a recent consensus statement regarding their use stated that the role of pac remains unclear. studies of children with septic shock have shown that the information obtained from a pac aided in identifying hemodynamic profi les different from those presumed by care givers and has directly infl uenced care decisions. it was concluded by a recent consensus panel to be of potential benefi t in improving the management of pediatric patients. pulmonary artery catheter placement should be considered for pediatric patients who remain in shock after resuscitation and initiation of the usual vasoactive agents in whom the fl uid status and cardiac function remains unclear. in this setting, therapeutic endpoints are a cardiac index of >3.3 and <6.0 l/min/m 2 and systemic and pulmonary vascular resistances within the normal range. early identifi cation of a possible offending pathogen and aggressive source control represent a crucial component of septic shock therapy. prompt initiation of appropriate antimicrobial therapy against the causative pathogen has been shown to be one of the most important predictors of outcome. in a 1980's study of over 1,100 adults, providing appropriate systolic pressure variation (spv) and pulse pressure variation (ppv) can accurately predict which patients will be responsive to further fl uid resuscitation (gunn and pinsky 2001 ) . antimicrobial coverage at least 1 day prior to identifi cation of the organism was associated with improved survival. the pathogen itself has prognostic signifi cance. fungal infections, while accounting for only a minority of sepsis cases, carry the lowest survival rate, followed by gram-positive and gram-negative bacteria. survival rate has been reported to be the highest in patients in whom no pathogen was identifi ed. because of the importance of appropriate antimicrobial therapy, the decision of which agents to empirically start must balance potential side effects versus maximizing coverage. in this respect, it is important to be familiar not only with the most common causative pathogens, but also the local icu nosocomial risks and pathogen resistance patterns. initially, broad antibiotic coverage is initiated. neonates are most frequently placed on ampicillin and an aminoglycoside (e.g. gentamicin) or a third generation cephalosporin such as cefotaxime. in infants and children over the age of 4-6 weeks, the decision to start vancomycin empirically should be considered in light of the increasing antibiotic resistance of streptococcus pneumoniae and rising incidence of community acquired methicillin resistant staphylococcus aureus (mrsa). in addition, a 3rd or 4th generation cephalosporin (e.g. ceftriaxone) should be used. suspicion of a gram-negative infection or nosocomial infection requires additional coverage, usually in the form of an aminoglycoside, for the possibility of pseudmonas species and other resistant gram-negative organisms. because of its broad coverage, including many anaerobic species, and low renal toxicity, piperacillin/tazobactam is empirically administered with increasing frequency. the antiviral agent, acyclovir, should be administered if there is suspicion of a herpes virus infection. in immunocompetent children, the decision to start empiric antifungal therapy remains controversial. in the child who is not improving over the initial days of empiric coverage or in whom there is a higher risk for fungal infection (e.g. presence of indwelling devices, immunosuppression or other signifi cant co-morbidities), antifungal coverage may be indicated. the development of agents equally as effective as amphotericin, but with substantially reduced nephrotoxicity such as fl uconazole and caspofungin, may ultimately sway the risk/benefi t analysis towards more aggressive, earlier initiation of empiric antifungal coverage in select, high risk populations. the ability to narrow the spectrum of treatment once the causative organism has been identifi ed will reduce the number of potential side effects and curtail the development of pathogen resistance related to imprudent use of broad spectrum antibiotics. the current mainstay of supportive care in sepsis remains the maintenance of adequate oxygen delivery in the face of myocardial depression, capillary leak, acidosis, and massive cytokine release. while some early adult studies have suggested improved outcomes when achieving supra-normal levels of oxygen delivery, this approach in pediatric sepsis remains unproven. this is likely due to the fact that septic patients may have a perturbed ability to extract oxygen in addition to suboptimal oxygen delivery. clinically, this impairment in cellular oxygen uptake may be refl ected by an inappropriately high central venous oxygen saturation (s cv o 2 ) in the face of a progressive and therapy refractory acidosis. optimizing appropriate oxygen delivery remains a clinical goal and incorporates the need for maximizing oxygen carrying capacity. while there is no recommended hemoglobin level for children, the most recent nih consensus conference suggested a hemoglobin concentration of 10 g/dl for adults with cardiopulmonary compromise as part of a protocol toward achieving the therapeutic goal of s cv o 2 >70%, with improved outcomes demonstrated when this goal was achieved during initial resuscitation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload defi ned earlier in this chapter. similar clinical parameters can be assessed in response to blood transfusion. perhaps the best assessments are determinations that provide indirect evidence of the balance between oxygen delivery and consumption such as lactate levels and mixed venous oxygen saturation. in the context of fl uid loading with blood transfusion, empiric administration of diuretics to eliminate extra fl uid should be avoided until hemodynamic stability has been achieved or if the child exhibits signs of intravascular volume overload. finally, the nutritional status of the septic child must be addressed. patients with sepsis often have poor nutrition prior to admission to the picu and often may not be fed in the fi rst few days of illness. this state combined with the increased metabolic rate associated with sepsis place the septic patient at risk for protein calorie malnutrition. intestinal hypoperfusion in combination with absence of local enterocyte nutrition can cause mucosal barrier dysfunction and may contribute to translocation of bacteria and endotoxin from the intestine into the blood stream. while the use of enteral feeding in critical illness has been shown to improve survival and decrease hospital stay, its use must be balanced with the risk of stressing intestinal function in the face of poor splanchnic perfusion, especially in the child requiring the use of vasopressors such as epinephrine and norepinephrine. regardless of which mode of nutrition is chosen, the goal of achieving nitrogen balance is important for allowing recovery and return to physiologic homeostasis. in the absence of enteral feedings, protection from stress-related gastrointestinal ulcer formation is advised. because poor outcome in sepsis has been attributed to a dysregulated proinfl ammatory state, anti-infl ammatory agents, such as corticosteroids, have long been proposed as a potential therapeutic strategy. anecdotally, it has been observed that some patients treated with antibiotics appear to acutely worsen in a time frame consistent with the onset of antibiotic activity. this observation has been attributed to massive release of bacterial products following the lysis of high numbers of bacteria. to this end, investigators had shown that animals treated with anti-infl ammatory drugs prior to receiving antibiotics demonstrated a less severe response to bacterial lysis. despite encouraging preclinical studies, two subsequent large adult trials using high dose steroids early in sepsis showed no improvement in mortality. more recently, studies using lower doses of steroids over a longer period of time have suggested a possible benefi t including a reduced time to cessation of vasopressor therapy. these more recent observations have stimulated a resurgence in the use of corticosteroids in sepsis. adrenal insuffi ciency is frequently unrecognized in children with septic shock and a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. therefore, identifying "at risk" patients with a corticotropin stimulation test and treating this group may improve outcome in sepsis. a study in which adult septic shock patients who were classifi ed as "nonresponders" based on corticotropin stimulation results were treated with hydrocortisone and fl udrocortisone for 7 days and had a signifi cantly reduced risk of death. however, this initial observation was not observed in larger follow-up studies. thus, the use of hydrocortisone and the application of a corticotropin stimulation test in septic patients remains highly controversial. in pediatrics, it is currently recommended that any child with fl uid-and catecholamine-refractory shock (inadequate response to two or more vasoactive agents), has a known history of adrenal insuffi ciency, or has previously received exogenous steroids should be considered for steroid replacement with hydrocortisone (usual dose between 50 and 100 mg/m 2 /day divided every 6 h). because the host response to sepsis is mediated by circulating infl ammatory molecules, it has been hypothesized that extracorporeal removal of these mediators via hemofi ltration or exchange transfusion may affect outcome. case reports suggest that arterial oxygenation and hemodynamics can be improved with use of hemofi ltration during sepsis and multiple organ failure. however, there exist many mitigating factors in evaluating the pediatric experience and the effi cacy of hemofi ltration remains unproven. challenges with instituting extracorporeal hemofi ltration include diffi culty with vascular access in smaller children, potential fl uid and electrolyte imbalance, hypothermia, anticoagulation requirements and acutely compromised hemodynamics during initiation. in addition, it is not known whether benefi cial proteins such as albumin, immunoglobulins, clotting factors and counter-regulatory cytokines are removed during this process. while experience shows that hemofi ltration can be safely performed in children with sepsis, it remains unclear if it will improve outcome. part of the infl ammatory response involves cytokines that cause widespread activation of the coagulation cascade with suppression of fi brinolysis as reviewed above. it is encouraging adrenal insuffi ciency is frequently unrecognized in children with septic shock. a low basal circulating cortisol level, especially in association with an abnormal corticotropin stimulation test, has been associated with higher mortality rates. that administration of activated protein c in adults with septic shock was associated with a signifi cant decrease in 28-day mortality. though activated protein c should not be routinely used in pediatric sepsis, indications for its use may be determined from further trials in pediatric sepsis. in the meantime, many other potential immune modulating therapeutic agents have been identifi ed and are currently under investigation. unfortunately, many of the antiinfl ammatory agents tried to date (anti-il-1, anti-bradykinin, anti-endotoxin, anti-tnf-a , soluble tnf receptor and anti-platelet activating factor) have not shown any benefi t in large, randomized clinical trials. it is hoped that improvements in study design which include thoughtful stratifi cation of patients, timely identifi cation of the presence or absence of a pathogen and consideration of genetic factors that infl uence outcome will eventually assist in discovering and targeting pharmacologic agents that ultimately improve the outcome of the pediatric patient with septic shock. vascular bed-specifi c hemostasis: role of endothelium in sepsis pathogenesis corticosteroids for severe sepsis and septic shock: a systematic review and meta-analysis immunological therapy of sepsis: experimental therapies effi cacy and safety of recombinant human activated protein c for severe sepsis signal transduction during innate and adaptive immunity role of nfkappab in the mortality of sepsis new insights into its pathogenesis and treatment the accp-sccm consensus conference on sepsis and organ failure stress doses of hydrocortisone reverse hyperdynamic septic shock: a prospective, randomized, double-blind, single-center study clinical practice parameters for hemodynamic support of pediatric and neonatal septic shock: 2007 update from the american college of critical care medicine toll-like receptors: molecular mechanisms of the mammalian immune response prognostic values of tumor necrosis factor/cachectin, interleukin-1, interferon-alpha, and interferon-gamma in the serum of patients with septic shock. swiss-dutch j5 immunoglobulin study group hemodynamic support in fl uid-refractory pediatric septic shock surviving sepsis campaign guidelines for management of severe sepsis and septic shock surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock organization and regulation of mitogenactivated protein kinase signaling pathways lps induction of gene expression in human monocytes implications of arterial pressure variation in patients in the intensive care unit signal transduction by the c-jun n-terminal kinase (jnk) -from infl ammation to development the i kappa b kinase (ikk) and nf-kappa b: key elements of proinfl ammatory signalling use of corticosteroid therapy in patients with sepsis and septic shock: an evidence-based review myocardial dysfunction in septic shock: part ii. role of cytokines and nitric oxide rationale for restoration of physiological anticoagulant pathways in patients with sepsis and disseminated intravascular coagulation genomic polymorphisms in sepsis the contrasting effects of dopamine and norepinephrine on systemic and splanchnic oxygen utilization in hyperdynamic sepsis early enteral nutrition in acutely ill patients: a systematic review source control in the management of severe sepsis and septic shock: an evidence-based review role of the coagulation system in the local and systemic infl ammatory response new rationale for glucocorticoid treatment in septic shock anti-infl ammatory cytokines pediatric considerations epidemiology of sepsis and multiple organ dysfunction syndrome in children pulmonary artery catheter consensus conference: consensus statement drotrecogin alfa continuous plasmafi ltration in sepsis syndrome. plasmafi ltration in sepsis study group early goal-directed therapy in the treatment of severe sepsis and septic shock innate immune mechanisms triggering lung injury effects of nitric oxide in septic shock signal transduction pathways in acute lung injury: nf-k b and ap-1 sepsis remains one of the most pressing clinical challenges for the pediatric intensivist. it is apparent that while a great deal is now understood about the biological and molecular mechanisms involved in sepsis, this knowledge has not yet had a dramatic impact on improving outcome. at present, therapeutic modalities for sepsis remain largely supportive and founded on the fundamental physiologic principle of providing adequate oxygen delivery. with this approach, mortality in pediatric sepsis improved modestly over the past decades. however, the fact that over 4,000 children per year continue to die in association with severe sepsis argues that further advances be made. realization of the goal of improving survival requires investigators committed to achieving further mechanistic insights into the physiologic, molecular, and genetic biology of sepsis, in concert with large pediatric-specifi c interventional trials. a 5 year old female is admitted to the pediatric intensive care unit with septic shock. she is well oxygenated on a 40% oxygen face mask. she has already received 60 ml/kg of 0.9% normal saline and has been started on a dopamine infusion at a rate of 5 mcg/kg/min. in monitoring her response to these interventions, which of the following should not be used as a therapeutic endpoint to monitor her progress? a. capillary refi ll time b. echocardiographically measured ejection fraction c. mental status d. serum bicarbonate level e. urine output ³ 1 ml/kg/h 10. a 12 year old male with acute lymphocytic leukemia is admitted to the pediatric intensive care unit with vancomycin resistant enterococcus bacteremia. his vital signs reveal a temperature of 39.6°c, a heart rate of 145 bpm, a respiratory rate of 20 breaths/min, and a blood pressure of 108/35 mm hg. he is lethargic, but arousable. his pulses are bounding and his capillary refi ll is brisk. an arterial blood gas reveals a ph 7.31, a paco 2 33 mm hg, a pao 2 65 mm hg, an oxygen saturation of 93%, and a base defi cit of (−10). the oxygen saturation of venous blood sampled from the superior vena cava is 88%. a. the young man is bacteremic, but not in shock as evidenced by his bounding pulses, brisk refi ll, and normal systolic blood pressure. b. the young man is in shock with inadequate oxygen extraction at the tissue level evidenced by the elevated superior vena cava saturation. c. the young man has a primary metabolic acidosis, but has a normal oxygen extraction as oxygen saturation in the superior vena cava is normally higher than elsewhere in the body. d. the young man has a primary metabolic acidosis, but is not in shock, evidenced by his high superior vena cava saturation. e. the young man has a primary respiratory alkalosis that would benefi t from supplemental oxygen therapy.11. there is suffi cient data to justify the use of which of the following adjuvant therapies in pediatric sepsis? a. the administration of activated protein c to a child in septic shock without thrombocytopenia or coagulopathy b. the administration of anti-tnf-a monoclonal antibodies to a septic patient to decrease the proinfl ammatory response c. the administration of stress dose hydrocortisone to a septic patient whose serum cortisol level fails to increase sufficiently in response to a corticotropin stimulation test d. the early initiation of high volume, continuous veno-venous hemofi ltration to remove proinfl ammatory cytokines e. the transfusion of packed red blood cells to maintain a hemoglobin ³ 12 g/dl in order to provide supranormal oxygen delivery 12. it has become clear that dysregulation of the coagulation cascade occurs in sepsis as refl ected by activation of procoagulant pathways, consumption of clotting factors, alterations in fi brinolysis, and reduced anticoagulant activity. which of the following components of coagulation is increased during sepsis? a. antithrombin iii (at iii) b. plasminogen activator inhibitor type 1 (pai-1) c. protein c d. protein s e. tissue factor pathway inhibitor (tfpi) key: cord-023415-hhvmsn5b authors: karlsson, h.; larsson, p.; wold, a. e.; rudin, a. title: pattern of cytokine responses to gram‐positive and gram‐negative commensal bacteria is profoundly changed when monocytes differentiate into dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423at.x sha: doc_id: 23415 cord_uid: hhvmsn5b the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigen‐presenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte‐derived dcs were stimulated with uv‐inactivated gram‐positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram‐negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il‐12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il‐12p70, tnf, il‐6 and il‐10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram‐positive strains correlated with a lower surface expression of toll‐like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram‐negative bacteria. ifn‐γ increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram‐negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram‐positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023410-eblcf902 authors: kollgaard, t. m.; reker, s.; petersen, s. l.; masmas, t. n.; vindelov, l. l.; straten, p. t. title: clonally expanded cd8(+) t cells in allogeneic bone marrow transplantation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bm.x sha: doc_id: 23410 cord_uid: eblcf902 allogeneic bone marrow transplantation (bmt) is a potentially curative therapy for patients with haematologic malignancies. several lines of evidence demonstrate that donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t‐cell‐depleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft‐versus‐host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft‐versus‐tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t‐cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8(+) t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t‐cell receptor clonotype mapping based on rt‐pcr and denaturing gradient gel electrophoresis (dgge). using this gel‐based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8(+) t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-103625-p55ew8w7 authors: ramana, chilakamarti v. title: regulation of early growth response-1 (egr-1) gene expression by stat1-independent type i interferon signaling and respiratory viruses date: 2020-08-14 journal: biorxiv doi: 10.1101/2020.08.14.244897 sha: doc_id: 103625 cord_uid: p55ew8w7 respiratory virus infection is one of the leading causes of death in the world. activation of the jak-stat pathway by interferon-alpha/beta (ifn-α/β) in lung epithelial cells is critical for innate immunity to respiratory viruses. genetic and biochemical studies have shown that transcriptional regulation by ifn-α/β required the formation of interferon-stimulated gene factor-3 (isgf-3) complex consisting of stat1, stat2, and irf9 transcription factors. furthermore, ifn α/β receptor activates multiple signal transduction pathways in parallel to the jak-stat pathway and induces several transcription factors at mrna levels resulting in the secondary and tertiary rounds of transcription. transcriptional factor profiling in the transcriptome and rna analysis revealed that early growth response-1 (egr-1) was rapidly induced by ifn-α/β and toll-like receptor (tlr) ligands in multiple cell types. studies in mutant cell lines lacking components of the isgf-3 complex revealed that ifn-β induction of egr-1 was independent of stat1, stat2, or irf9. activation of the mek/erk-1/2 pathway was implicated in the rapid induction of egr-1 by ifn-β in serum-starved mouse lung epithelial cells. interrogation of multiple microarray datasets revealed that respiratory viruses including coronaviruses regulated egr-1 expression in human lung cell lines. furthermore, egr-1 inducible genes including transcription factors, mediators of cell growth, and chemokines were differentially regulated in the human lung cell lines after coronavirus infection, and in the lung biopsies of covid-19 patients. rapid induction by interferons, tlr ligands, and respiratory viruses suggests a critical role for egr-1 in antiviral response and inflammation with potential implications for human health and disease. interferons (ifns) are pleiotropic cytokines that play a central role in innate and adaptive immunity (1, 2) . there are 3 major types of interferons. the type i interferons consists of ifn-alpha/beta (ifn-α/β ). type ii interferon represented by interferon-gamma (ifn-γ), and type iii interferons represented by interferon lambda (ifn-λ ) . the biological effects of ifn are mediated mainly by the rapid and dramatic changes in gene expression (3)). type i ifn signaling involves the binding of ifn-α/β to its receptor (ifnar) and activation of receptor-associated janus protein tyrosine kinases jak1 and tyk2 and the phosphorylation of stat1 and stat2 to form a heterodimer. this heterodimer associate with the interferon responsive factor 9 (irf9) to form the interferon-stimulated gene factor-3 (isgf-3) complex on interferon-stimulated response elements (isre) in the promoters of type i ifn responsive genes to regulate transcription (1, 2, 4) . stat1 homodimer or heterodimer of stat1/stat2 can also bind to gamma activated sequence (gas) in the promoter and regulate transcription of some type i ifn responsive genes (1, 4) . in addition to this classical canonical jak-stat pathway, non-canonical pathways involving stat2, irf9, and unphosphorylated isgf-3 components in the regulation of gene expression have been described (5) . furthermore, several signal transduction pathways are activated by ifn receptors in parallel to jak-stat including extracellular signal-regulated kinases (erk-1/2) and phosphoinositide 3'-kinase/akt pathways (6, 7) . the first wave of interferon signaling is followed by induction of transcription factors such as interferon regulatory factors (irfs) that sustain the secondary and tertiary transcriptional responses. tlr recognition of pathogens by immune cells results in the production of multiple cytokines such as ifn-α/β , tumor necrosis factor-α (tnf-α ), and interleukin-β (il-1 β ) in innate immunity (8) . activation of multiple signal transduction pathways and cross-talk between the pathways enables fine-tuning of gene expression in innate immunity (9) . cross-talk between tnf-α and ifn signaling pathways regulate inflammatory gene expression to influence the immune responses (10, 11 ) . clinical significance of the balance between immune modulation and inflammation in signal transduction pathways has been demonstrated in a variety of autoimmune diseases (12, 13) . early growth response 1 (human egr1/ mouse egr1; referred in this manuscript as egr-1) belongs to a family of immediate-early response genes that contain a conserved zinc finger dna-binding domain and binds to a gc-rich sequence in the promoters of target genes (14) . a variety of signals, including serum, growth factors, cytokines, and hormones stimulate egr-1 expression (15, 16) . egr-1 has been shown to play an important role by regulating inflammatory gene expression in a variety of lung diseases and in mouse lung injury models including asthma, emphysema, airway inflammation, and pulmonary fibrosis (17) (18) (19) . ischemia-mediated activation of egr-1 triggers the expression of pivotal regulators of inflammation including the chemokine, adhesion receptor, and pro-coagulant gene expression (20) . egr-1 stimulates chemokine production in interleukin-13 mediated airway inflammation, and remodeling in the lung (21) . high levels of expression of egr-1 and egr-1 inducible genes were reported in atherosclerosis, an inflammatory disease (22) . egr-1 may have a potential role in liver injury and in acute pancreatitis (23) (24) (25) . lipopolysaccharide (lps) induction of egr-1 was mediated by the activation of erk-1/2 pathway and serum response elements (26) . in this study, transcription factor profiling in interferon-mediated gene expression data sets and rt-pcr revealed that egr-1 was rapidly induced by ifn-α/β and tlr ligands in multiple cell types. studies in mouse and human fibroblast mutant cell lines revealed that egr-1 induction by type i interferons was independent of 4 transcription factors stat1, stat2 or irf9 . furthermore, the regulation of egr-1 by ifn-β was mediated by the activation of the erk-1/2 pathway in serum-starved mouse lung epithelial cells. respiratory pathogens including coronaviruses (sars-cov-1 and 2) and influenza viruses regulated the expression of egr-1 in human lung cell lines and in lung biopsies and peripheral blood cells of covid-19 patients, these studies suggest that the regulation of egr-1 may play an important role in the antiviral response and inflammatory disease. gene expression in response to interferon, tnf-α. and tlr agonist treatment in human peripheral blood mononuclear cells (pbmc), human hepatoma cells (huh-7), mouse bone marrow-derived macrophages (bmdm) were reported previously (27) (28) (29) . supplementary data was downloaded from the journal publisher websites and geo datasets were analyzed with the geor2r method (ncbi). cluster analysis was performed using gene expression software tools at www.heatmapper.ca. gene expression datasets representing human lung cell lines infected with respiratory viruses and from covid-19 patients were reported previously (30, 31) . gene expression resources from immgen rna seq skyline were used ( http://rstats.immgen.org/skyline_covid-19/skyline.html ). outliers of expression were not included in the analysis. mouse lung epithelial (mle-kd) and macrophage (raw264.7) cell lines were used (11, 32) . human fibrosarcoma cell line (2ftgh) and mutant cell lines lacking stat1 (u3a), stat2 (u6a), and irf9 (u2a) were described previously (33) . wild -type and stat1-knockout mouse embryo fibroblast (mef) cell lines were used (34) . cells were 5 maintained in dmem supplemented with 10% fbs and 1% penicillin and streptomycin. cells were plated at 60-70% density and maintained in full medium for a day. cells were incubated in serum-free medium for another 24 hours. cells were treated with tnf-α (20 ng/ml) or ifn-β were performed using ambion retroscript (austin, tx), according to the manufacturer's protocol. primer sequences for egr-1, irf1, and gapdh were obtained from the molecular reagents section of mouse genome informatics (mgi). human egr-1 and β− actin primer sequences were previously described (22) . pcr products were resolved on a 1% agarose gel containing ethidium bromide and visualized with u.v. light and images were captured on a digital system. image files were processed with imagej (nih) software. specific gene expression was normalized to gapdh or β -actin and fold changes in the treated samples were calculated with respect to controls. mle-kd cell extracts were prepared and proteins were separated by electrophoresis using 8%-10% sds-page gels. proteins in the gel were electrophoretically transferred to polyvinylidene difluoride membranes (bio-rad, ca) and subjected to immunoblotting with the antibodies for phosphorylated erk-1/2 (thr202/tyr 204) or total erk-1/2 from cell signaling technology (beverly, ma). blots were visualized by enhanced chemiluminescence western detection system (pierce, il). (figure 2a and 2b). these results were consistent with previous studies in human fibroblasts (15) . tnf-α induction of egr-1 was much higher than ifn α/β in 2ftgh cells ( figure 2b ). interestingly, tnf-α but not ifn-α induced egr-1 mrna in hela cells suggesting differential pathway regulation (15) . furthermore, ifn-λ induction of egr-1 was higher than with ifn-α/β in huh7 cells ( figure 2c ) irf9 (u2a) demonstrated that ifn-β induction of egr-1 was independent of isgf-3 components ( figure 4b ). tnf-α and tlr ligands activate several mitogen-activated protein kinase (mapk) pathways such as extracellular signal-regulated kinase (erk), jun n-terminal kinase (jnk) and p38 (37) . activation of the erk-1/2 pathway leading to the phosphorylation of transcription factors such as elk1 and srf1 was implicated in the rapid induction of egr-1 in response to multiple stimuli (26, 38) . (42) . detailed promoter analysis revealed that multiple distal and proximal cis-elements were involved in egr-1 induction (43). the generation of an inflammatory response is a complex process involving multiple cytokines acting in parallel and in concert in innate and adaptive immunity (36, 44) . influenza virus-infected dendritic cells and macrophages, which reside in close proximity to lung epithelium, can produce significant amounts of tnf-α and type i ifn in response to virus infection (45) . ifn-α / β is involved in signaling cross-talk with tnf −α that enhance or dampen the severity of inflammatory response (9, 10) . interaction between interferon-α/β and tnf-α signaling was reported in autoimmune diseases. (12, 13) . a select list of genes was induced by both tnf-α and interferon-a /β in pbmc and bmdm gene expression datasets ( figure 6 ). tnf-α induction of these genes was much higher than ifn α/β in the mouse bmdm cells. interestingly, egr-1 was induced by both cytokines in pbmc and bmdm. these genes are involved in transcriptional regulation and integrating signal transduction pathways that are likely to play an important role in the cytokine storm and shift to hyperinflammatory gene expression in response to coronavirus infections (30,65). egr-1 is a zinc finger transcription factor that binds to a gc-rich sequence in the promoter regions of target genes. a shortlist of egr-1 regulated genes was generated from the truust transcription factor database and published studies in different cell types and under different stimuli (20) (21) (22) 46) . egr-1 regulates genes involved in cell growth (egr2, atf3, pdgfrb, cdkna1, ccnd1, tnfsf10, and chemokines involved in inflammation such as (cxcl10, cxcl2, ccl2, ccl3). it is important to note that many of the egr-1 target genes were also known targets of nf-kb, ap1, and stat1 in cytokine signaling (46) . furthermore, egr-1 interacts with several other transcription factors such as ets1, ets2, elk1 that are common in cytokine responsive genes (46) . previous studies have shown that transcription factors of innate and adaptive immunity show functional connectivity involving shared interacting protein partners and target genes. (47) . cluster analysis revealed that egr-1 induction was correlated with the expression of egr-1 target genes by ifn-α/β in pbmc and in bmdm cells ( figure 7 ). severe acute respiratory syndrome coronaviruses (sars-cov) and influenza viruses are major respiratory pathogens in humans with seasonal epidemics and potential pandemic threats (48, 49) . gene expression profiling studies of human lung epithelial cell lines such as calu-3, a549, and nhbe1 in response to respiratory viruses were reported recently (30, 31) . interrogation of infection also induced egr-1 mrna in a549 cells, 24 hours post-infection ( figure 12c ). these studies suggest that egr-1 expression is a common host response to many respiratory viruses gene expression profiling studies of a limited number of human lung biopsies and pbmc of healthy controls and covid-19 patients were reported recently (30, 31) . interrogation of the gene expression data in lung biopsies revealed that egr-1 expression levels were significantly lower in covid-19 patients compared with healthy controls. expression of egr-1 target genes related to cell growth such as ccnd1, egr2, and pdgfrb were also down-regulated ( figure 13 and data not shown). in contrast, expression of egr-1 target genes involved in inflammatory responses such as cxcl10, ccl2, ccl3, ccl4, and tnfsf10 were dramatically enhanced in covid-19 patients compared with healthy controls (figure 14 and data not shown). interestingly, stat1 expression levels were significantly increased in covid-19 patients compared with healthy controls (figure 14 ). there are several limiting factors to interpreting the data. the lung is a complex tissue consisting of more than thirty cell types with differential contributions with respect to cell mass and gene expression. for example, type i and type ii cells in mouse lungs constitute the major and minor cell types and express distinct cell markers (35) . without information on the expression levels in distinct cell types, it is difficult to correlate egr-1 expression with target genes in the whole lung tissue. in support of this view, in a mouse model of cd8 + t cell -mediated lung injury, chemokine expression was dependent on egr-1 activation in alveolar type ii cells (63) . another intriguing possibility is that enhanced stat1 expression compensates for decreased egr-1 expression in some cell types in the lung. it is important to consider that in addition to changes in mrna levels, the phosphorylation status of stat1 and egr-1 may play an important role in the transcriptional regulation of chemokine target genes. it is likely that differential expression in distinct cell types may account for the disparity of egr-1 has emerged as a key regulator of cell growth, reproduction, and response to tissue injury (16, (61) (62) (63) . egr-1 was rapidly induced by interferons and pro-inflammatory cytokines such as tnf-α, and il-1 β (15, 63, 64) . recent studies have demonstrated dramatic changes in type i interferon, tnf-α, and il-1 β production by immune cells and cytokine-mediated lung inflammation in covid-19 patients (30, 57, 65, 66) . however, the role of egr-1 in innate immunity and antiviral response to respiratory viruses in general and sars-cov-2, in particular, remains to be investigated. transcriptional factor profiling in the transcriptome revealed that egr-1was induced by ifn-α/β , tlr ligands, and tnf-α in human and mouse cells. studies in mutant cell lines lacking stat1, stat2, and irf9 revealed that ifn-α/β induction of egr-1 was independent of isgf-3 components and mediated by the activation of erk-1/2 pathway. furthermore, respiratory viruses such as sars-cov-2 induced egr-1 and its target genes in several lung epithelial cell lines and in covid-19 patients. activation of egr-1 by ifn-α/β and tnf-α and cross-talk between the pathways modulates signal transduction and inflammatory response in innate immunity. how cells respond to interferons the jak-stat pathway at 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severe covid-19 key: cord-016523-pznw2ciu authors: fouqué, amélie; legembre, patrick title: the cd95/cd95l signaling pathway: a role in carcinogenesis date: 2014-08-29 journal: cancer immunology doi: 10.1007/978-3-662-44006-3_9 sha: doc_id: 16523 cord_uid: pznw2ciu apoptosis is a fundamental process contributing to tissue homeostasis, immune response, and development. cd95, also called fas, is a member of the tumor necrosis factor receptor (tnf-r) superfamily. its ligand, cd95l, was initially detected at the plasma membrane of activated t lymphocytes and natural killer (nk) cells where it contributes to the elimination of transformed and infected cells. given its implication in immune homeostasis and immune surveillance combined with the fact that various lineages of malignant cells exhibit loss-of-function mutations, cd95 was initially classified as a tumor suppressor gene. nonetheless, in different pathophysiological contexts, this receptor is able to transmit non-apoptotic signals and promote inflammation and carcinogenesis. although the different non-apoptotic signaling pathways (nf-κb, mapk, and pi3k) triggered by cd95 are known, the initial molecular events leading to these signals, the mechanisms by which the receptor switches from an apoptotic function to an inflammatory role, and, more importantly, the biological functions of these signals remain elusive. and the loss of its extracellular membrane integrity, leading to the release of different apoptogenic factors in the cytosol. among them, cytochrome c associates with the caspase-9/ apaf1 complex to form the apoptosome and trigger apoptosis [ 6 ] . these two signaling pathways share common features, and both require the aggregation of initiator caspases as their preliminary events. during interactions with respective ligands, members of the death receptor superfamily recruit adaptor proteins such as fas-associating protein with a death domain (fadd) [ 7 , 8 ] or tumor necrosis factor (tnf) receptor 1-associated death domain protein (tradd) [ 9 ] , resulting in the aggregation and activation of the initiator caspase-8 and caspase-10 to form the death-inducing signaling complex (disc) [ 10 ] . in a similar manner, release of cytochrome c and atp by mitochondria promotes the formation of the apoptosome with the cytosolic apaf-1, thereby aggregating and activating the initiator caspase-9, which in turn cleaves caspase-3 [ 11 ] . it should be kept in mind that death receptors cd95 [ 12 ] , tnfr1 [ 13 ] , dr4 [ 14 ] , dr5 [ 15 ] , and dr6 [ 16 ] have been cloned based on their ability to elicit apoptosis. although studies have revealed the ability of fas/cd95, dr4, and dr5 in triggering non-apoptotic signaling pathways even immediately after their cloning [ 17 , 18 ] , most, if not all, studies have been focused on characterizing the molecular events leading to cell death. accordingly, several agonistic molecules were developed in order to kill cancer cells, neglecting the impact of non-apoptotic signals in pathophysiological contexts. more recent data changed this vision by evaluating the biological role of death receptor-mediated non-apoptotic signaling pathways in chronic infl ammatory disorders and carcinogenesis. in this chapter, apoptotic signaling pathways induced by death receptors are discussed. moreover, recent evidences pointing to the nonapoptotic signals transmitted by the same receptors are brought up, which may imply their tremendous impact on tumor progression and the design of therapeutic tools. death receptors tnfr1, fas, dr3, dr4, dr5, and dr6 belong to the tumor necrosis factor receptor (tnf-r) superfamily. these type i transmembrane proteins share common features, such as extracellular amino-terminal cysteine-rich domains (crds) [ 19 , 20 ] , which contribute to ligand specifi city [ 21 ] , and preassociation of the receptor at the plasma membrane [ 22 -24 ] and a conserved 80 amino acid sequence located in their cytoplasmic tail called death domain (dd), which is necessary for disc formation and initiation of the apoptotic signal [ 25 , 26 ] . tnf-α exerts its effects by binding to two receptors, tnfr1 and tnfr2 [ 20 ] . recently, progranulin was identifi ed as a ligand of tnfr with a higher affi nity than tnf-α. progranulin antagonizes tnf-α signaling and plays a critical role in the pathogenesis of infl ammatory arthritis in mice [ 27 ] . tnfr1, a 55 kda protein expressed in almost all cell types, presents a dd in its intracellular region; whereas tnfr2, a 75 kda protein, is mainly detected in oligodendrocytes, astrocytes, t cells, myocytes, thymocytes, endothelial cells, and human mesenchymal stem cells [ 28 ] . uncertainty remains on the tnfr2 signaling pathway, which has been previously reviewed [ 28 ] . the crd1 of cd95, tnfr1, and tnfr2 is involved in homotypic interactions, leading to pre-association of the receptor as a homotrimer in the absence of ligand [ 23 , 24 , 29 ] . this domain has been designated as the pre-ligand binding assembly domain (plad) [ 29 ] . receptors of the tnfr superfamily do not possess any enzymatic activity on their own and rely on the recruitment of adaptor proteins for signaling. among these adaptor proteins, tradd or fadd are instrumental in the implementation of cell death processes [ 7 -10 ] . tnf is synthesized as a 26 kda transmembrane type ii protein (m-tnf) of 233 amino acids [ 30 ] which can be cleaved by the metalloprotease tace [ 31 , 32 ] to release the 17 kda soluble form of the cytokine (cl-tnf). in contrast to cl-tnf, which only activates tnfr1, m-tnf can bind and activate both tnfr1 and tnfr2 [ 33 ] . activation of tnfr1 leads to the induction of cellular processes ranging from cell death (apoptosis or necroptosis) to cell proliferation, migration, and differentiation; the implementation of such different cellular responses refl ects the formation of different molecular complexes after receptor activation [ 28 ] . binding of tnf to tnfr1 causes the formation of two consecutive complexes. while the plasma membrane complex (complex i) elicits a non-apoptotic signaling pathway, a second, internalized, complex (complex ii or disc) triggers cell death [ 2 ] . in the presence of tnf, the adaptor protein tradd interacts with tnfr1 and recruits other proteins involved in the signaling of the receptor, such as traf2, ciap1, ciap2, and rip1, to form complex i. at the plasma membrane, this complex activates the nf-κb signaling pathway, which in turn promotes the transcription of antiapoptotic genes such as ciap-1, ciap-2, and c-flip [ 34 ] . the linear ubiquitin chain assembly complex (lubac) is also recruited to complex i via ciap-generated ubiquitin chains [ 35 ] . hoil-1, hoip, and sharpin constitute the lubac complex. hoil-1 and hoip add a linear ubiquitin chain by catalyzing the head-to-tail ligation of ubiquitin [ 36 ] to rip1 and nemo (ikkγ) in complex i [ 37 ] , thereby activating nf-κb. tnf-induced caspase activation is mediated by a second, intracellular complex ii, which is formed when complex i dissociates from the receptor, along with fadd and caspase-8 recruitment [ 2 ] . nf-κb activation leads to c-flip overexpression, preventing formation of complex ii. contrariwise, when nf-κb activation is blocked, c-flip, whose protein halflife is short [ 38 ] , is absent, and cells experience death [ 2 ] . rip1 is deubiquitinated by enzymes such as cezanne [ 39 ] and cyld [ 40 ] and the complex composed of tradd and rip1 moves to the cytosol to form complex ii. fadd is recruited to tradd by dd-dd interaction and binds caspase-8 [ 2 ] . noteworthy, when caspase-8 activity is inhibited or its expression is extinguished, disc is unable to trigger the apoptotic signaling pathway, but tnfr1 or cd95 stimulation leads to the activation of another cell death signal, namely, necroptosis [ 41 , 42 ] . to prevent the induction of the necroptotic signal, caspase-8 cleaves and inactivates rip1 and rip3 [ 43 ] . the fi ne-tuned control of necroptosis by members of the apoptotic signaling pathway in the organism has been elegantly confi rmed by experiments showing that the embryonic lethality of mice harboring the single ko of caspase-8 or fadd is rescued by an additional ko of the rip3 gene [ 44 -46 ] . many studies on tnf demonstrated its pivotal role in fueling infl ammation, a multistep process that promotes autoimmunity (e.g., rheumatoid arthritis, ankylosing spondylitis, crohn's disease, psoriasis, and refractory asthma) and cancer. many tnf inhibitors, such as neutralizing monoclonal antibodies (mabs) (e.g., infl iximab, adalimumab, and golimumab), have been developed to treat these chronic infl ammatory disorders, demonstrating that altering ligand/receptor interactions with neutralizing mabs is an invaluable opportunity to treat certain chronic infl ammatory disorders. other tnf-α antagonists, such as etanercept, a tnfr2-immunoglobulin fc fusion protein, can improve the clinical course of rheumatoid arthritis [ 47 ] . while fi ndings accumulate to decipher the molecular mechanisms involved in the induction of apoptotic and non-apoptotic signaling pathways by tnfr1 and to elucidate how the receptor can switch from one signal to the other, the mechanistic links involved in the implementation of non-apoptotic signaling pathways by cd95 remain elusive. however, recent fi ndings have revealed its proinfl ammatory effects [ 48 -54 ] . in 1989, identifi cation of the mab apo-1 by peter krammer et al. revealed the existence of a 52 kda protein whose aggregation was able to transmit an apoptotic signal in cancer cells [ 55 ] . this receptor was identifi ed in 1991 by nagata and colleagues and called fas (cd95 or apo-1) [ 12 ] . its ligand, fasl, was cloned in 1993 by the same group and was found to be mainly expressed at the surface of activated t lymphocytes [ 56 ] and natural killer (nk) cells [ 57 ] ; however, its expression was also detected in different tissues in which the presence of acute or chronic infl ammation is undesirable including the eyes [ 58 ] and testes [ 59 ] . in addition, two mouse models, in which either the level of cd95 expression was downregulated [due to an insertion of a retrotransposon in intron 2 of the receptor gene, these mice are called lymphoproliferation (lpr) [ 60 -62 ] the cd95 gene ( apt -1 ) consists of nine exons, with exon 6 encoding the transmembrane domain [ 66 ] (fig. 9.1 ). cd95 can be resolved under denaturing conditions between 40 and 50 kda by sds-page. the receptor is a type i transmembrane protein harboring three crds. similar to the tnf receptor [ 29 ] , cd95 is preassociated at the plasma membrane as a homotrimer, and this quaternary structure is mandatory for transmission of the apoptotic signals in the presence of cd95l [ 23 , 24 ] . homotrimerization of cd95 occurs mainly through homotypic interactions of the cd95-crd1 [ 22 -24 ] . binding of cd95l or agonistic anti-cd95 mabs to cd95 alters both the conformation and the extent to which the receptor is multimerized at the plasma membrane. the intracellular region of cd95 encompasses an 80 amino acid stretch designated as the dd ( fig. 9 .1 ), which consists of six antiparallel α-helices [ 67 ] . upon addition of cd95l, cd95 undergoes conformational modifi cation of its dd, which induces a shift of helix 6 and fusion with helix 5, promoting both oligomerization of the receptor and recruitment of the adaptor protein fadd [ 68 ] . a consequence of the opening of the globular structure of cd95 is that the receptor becomes connected through this bridge, which increases the magnitude of its homo-aggregation. this long helix allows the stabilization of the complex by recruiting fadd. overall, the cd95-dd:fadd-dd crystal structure provides some insights into the formation of the large cd95 clusters observed using imaging or biochemical methods in cells stimulated with cd95l. in addition, it also confi rms that alteration in the cd95 conformation plays an instrumental role during signal induction [ 68 ] however, this elongated c-terminal α-helix favoring the cis -dimerization of cd95-dd was challenged by driscoll et al. who did not observe the fusion of the last two helices at a more neutral ph (ph 6.2), compared to the acidic condition (ph 4) used in the initial study to resolve the cd95-dd:fadd-dd structure [ 68 ] . consequently, at ph 6.2, association of cd95 with fadd predominantly consisted of a 5:5 complex, which occurred via a polymerization mechanism involving three types of asymmetric interactions but without major alteration of the dd globular structure [ 69 , 70 ] . it is likely that the low ph condition used in the study performed by scott et al. altered cd95 conformation and resulted in the formation of nonphysiological cd95:fadd oligomers [ 68 ] . nonetheless, it cannot be excluded that a local decrease in the intracellular ph affects the initial steps of the cd95 signaling pathway in vivo , through promoting the opening of the cd95-dd and eventually contributing to the formation of a complex eliciting a sequence of events different from the one occurring at physiologic ph. once docked on cd95-dd, fadd selfassociates [ 71 ] and binds procaspases-8 and procaspases-10, which are auto-processed and released in the cytosol as active caspases, which cleave many substrates leading to the execution of the apoptotic program and cell death. the complex cd95/fadd/caspase-8/ caspase-10 is called disc ( fig. 9 .2 ) [ 10 ] . due to the importance of disc formation in the fate of cells, it is not surprising that numerous cellular and viral proteins were reported to hamper the formation of this structure, such as flip [ 72 , 73 ] and ped/pea-15 [ 74 ] , which interfere with the recruitment of caspase-8/caspase-10 ( fig. 9 .2 ). fig. 9 .2 type i/ii cells. binding of transmembrane cd95l to cd95 leads to disc formation. disc consists of fadd and procaspase-8. c-flip and pea-15 bind to fadd and prevent caspase-8 recruitment. at the disc level, aggregation of procaspase-8 promotes its autocleavage and activation. cleaved caspase-8 is then released in the cytosol where it promotes the cascade of caspase activation leading to apoptosis. type i cells are characterized by an effi cient disc formation, which releases suffi cient caspase-8 to directly activate caspase-3. by contrast, type ii cells present a weak disc formation, and the low amount of released caspase-8 activates the mitochondrion-dependent apoptotic pathway to amplify death signal following the discovery of cd95 and the fi rst steps of its signaling pathway, peter and colleagues described that cells can be divided in two groups with regard to the kinetics through which they respond to cd95-mediated apoptotic signals, the magnitude of disc formation and the role played by the mitochondrion in this pathway [ 75 ] . disc formation occurs rapidly and effi ciently in type i cells releasing a large amount of activated caspase-8 in the cytosol, while type ii cells have diffi culty forming this complex, and the amount of active caspase-8 is insuffi cient to directly activate the effector caspase-3 and caspase-7 [ 75 ] . nonetheless, type ii cells experience cell death upon cd95 engagement and are even more sensitive to the cd95mediated apoptotic signal compared to type i cells [ 75 -77 ] . this discrepancy can be partly explained by the fact that the low amount of activated caspase-8 in type ii cells is suffi cient to cleave bid, a bh3-only protein, which constitutes the molecular link between caspase-8 activation and the apoptotic activity of mitochondria. indeed, after cleavage by caspase-8, truncated bid (tbid) translocates to mitochondria, where it triggers the release of proapoptotic factors ( fig. 9 .2 ) [ 78 , 79 ] . although cd95 stimulation activates the mitochondrion-dependent apoptotic signal in type i and type ii cells, it seems that only type ii cells are addicted to this signal as they display a higher amount of the caspase-3 inhibitor xiap compared to type i cells [ 80 ] . among the inhibitor of apoptosis protein (iap) family, xiap, c-iap1, and c-iap2 inhibit caspase-3, caspase-7 [ 81 , 82 ] , and procaspase-9 [ 83 ] activity by direct binding, thereby preventing access to substrates. furthermore, xiap can function as an e3 ligase whose activity is involved in the ubiquitination of active caspase-3 and its subsequent degradation through the proteasome [ 84 ] . to detach xiap from caspase-3 and restore the apoptotic signal, cells require the release of smac/diablo (second mitochondria-derived activator of caspase/direct iap-binding protein with low pi) by the mitochondrion [ 85 , 86 ] , explaining why type ii cells are more addicted to this organelle compared to type i cells ( fig. 9.2 ). to summarize, disc formation and iap amount are two cellular markers allowing a clear discrimination between type i and type ii cells. even though iap overexpression can account for the mitochondrion dependency observed in type ii cells, it remains unclear why disc formation is hampered in type ii cells and/or enhanced in their type i counterparts. recently, high activity of the lipid kinase phosphoinositide 3-kinase (pi3k) or downregulation of its neutralizing phosphatase, phosphatase and tensin homologue on chromosome 10 (pten), was found in type ii cells, while this signal is blocked in type i cell lines [ 87 , 88 ] . the pi3k signaling pathway was reported to prevent the aggregation of cd95 [ 89 ] , probably by retaining the receptor outside of lipid rafts [ 87 , 90 ] . pea-15, also known as ped, is a protein containing a death effector domain (ded) that has been shown to inhibit the cd95 and tnfr1 apoptotic signals ( fig. 9. 2 ) [ 74 ] . activation of pi3k and its downstream effector, serine-threonine kinase akt, leads to phosphorylation of pea-15 at serine 116 [ 87 , 90 ] ; this posttranslational modifi cation promotes its interaction with fadd, ultimately inhibiting disc formation [ 91 , 92 ] . notably, the existence of type i and type ii cells is not only an in vitro observation, but has been identifi ed physiologically in human body. cd95-mediated apoptotic signal cannot be altered in thymocytes or activated t cells expressing a bcl-2 transgene, conferring to their type i nature [ 93 ] , whereas hepatocytes expressing the same transgene resist cd95-induced apoptosis and thus behave as type ii cells [ 94 , 95 ] . germinal mutations in apt -1 have been reported in patients developing a syndrome termed autoimmune lymphoproliferative syndrome type ia (alps, also called canale-smith syndrome) [ 96 -98 ] . alps patients show chronic lymphadenopathy and splenomegaly, expanded populations of double-negative α/β τ lymphocytes (cd3 + cd4 − cd8 − ), and often develop autoimmunity [ 96 , 97 , 99 , 100 ] . in agreement with the notion that cd95 behaves as a tumor suppressor, alps patients display an increased risk of hodgkin and non-hodgkin lymphoma [ 101 ] . predominance of post-germinal center (gc) lymphomas in patients exhibiting either germ line or somatic cd95 mutations can be explained by the fact that, inside germinal centers of the secondary lymphoid follicles, the cd95 signal plays a pivotal role in the deletion of self-reactive maturating b lymphocytes [ 102 ] , in addition to the fact that apt1 belongs to a set of rare genes (i.e., pim1, c-myc, pax5, rhoh/ttf, and bcl-6) subject to somatic hypermutation [ 103 , 104 ] , which may affect biological function. in addition to post-gc lymphomas, signifi cant amounts of mutations in the cd95 gene were found in tumors of various histological origins (reviewed in [ 54 ] ). extensive analysis of cd95 mutations and their distribution in apt -1 reveals that, with some exceptions, most are gathered in exons 8 and 9 encoding the cd95 intracellular region ( fig. 9. 3 ) [ 105 ] . remarkably, most of these mutations are heterozygous, mainly localized in cd95-dd, and lead to inhibition of the cd95-mediated apoptotic signal. indeed, in agreement with the notion that cd95 is expressed at the plasma membrane as a pre-associated homotrimer [ 23 , 24 ] , formation of heterocomplexes containing wild-type and mutated cd95 prevents fadd recruitment and abrogates the ignition of the apoptotic signal in a dominant manner. extensive analysis and positioning of various cd95 mutations described in the literature seem to highlight mutation "hot spots" in the cd95 sequence ( fig. 9.3 ). among these hot spots, (fig. 9.3 ) . strikingly, the pivotal role played by these amino acids in stabilization or formation of intra-and inter-bridges between cd95 and fadd may explain these hot spots. for instance, both r234 and d244 contribute to the homotypic aggregation of the receptor and fadd recruitment [ 67 ] . nevertheless, the observation of death domain hot spots is in contradiction with the study of scott and colleagues demonstrating that the region of the cd95-dd interacting with the fadd-dd extends over a disperse surface through weak binding affi nity [ 68 ] . most alps type ia patients affected by malignancies do not undergo loss of heterozygosity (loh), which formed the hypothesize that preservation of a wild-type allele may contribute to carcinogenesis [ 106 , 107 ] . in the same line, it was demonstrated that expression of a unique mutated cd95 allele blocks the induction of apoptotic signals, while it fails to prevent nonapoptotic signals such as nf-κb and mapk [ 106 , 107 ] , whose induction promotes invasiveness in tumor cells [ 105 , 108 ] . in addition, mutations found in the intracellular cd95-dd exhibit a higher penetrance of alps phenotype features in mutation-bearing relatives compared to extracellular mutations. these results suggest that unlike dd mutations, cd95 mutations localized outside the dd somehow prevent the apoptotic signal but may fail to promote non-apoptotic pathways, which may contribute to disease aggressiveness. in addition to cd95 downregulation or expression of the mutated allele of the receptor, the plasma membrane distribution of cd95 represents an additional pathway for tumor cells to develop resistance to cd95l-expressing immune cells. indeed, the plasma membrane is a heterogeneous lipid bilayer comprising compacted or liquid-ordered domains, called microdomains, lipid rafts, or detergent-resistant microdomains (drms). these domains are described as fl oating in a more fl uid or liquid-disordered 2-d lipid bilayer and are enriched in ceramides [ 109 ] . it has been elegantly shown that while cd95 is mostly excluded from lipid rafts in activated t lymphocytes, tcr-dependent reactivation of these cells leads to rapid distribution of the death receptor into lipid rafts [ 110 ] . this cd95 compartmentalization contributes to reducing the apoptotic threshold leading to the clonotypic elimination of activated t lymphocytes through activation of the cd95-mediated apoptotic signal [ 110 ] . similarly, the reorganization of cd95 into drms can occur independent from ligand upon addition of certain chemotherapeutic drugs (e.g., rituximab [ 111 ] , resveratrol [ 112 , 113 ] , edelfosine [ 87 , 114 , 115 ] , aplidin [ 116 ] , perifosine [ 115 ] , cisplatin [ 117 ] ). the molecular cascades that underlie this process remain elusive. nevertheless, a growing body of evidence leads us to postulate that alteration of intracellular signaling pathway(s), such as the aforementioned pi3k signal [ 87 , 90 ] , may change biophysical properties of the plasma membrane, such as membrane fl uidity, which in turn may facilitate cd95 clustering into large lipid raft-enriched platforms, favoring disc formation and induction of the apoptotic program [ 118 ] . accumulation of cd95 mutations is not the only mechanism by which malignant cells inhibit the extrinsic signaling pathway. posttranslational modifi cations in the intracellular tail of cd95, such as reversible oxidation or covalent attachment of a palmitic acid, were reported to alter the plasma membrane distribution of cd95 and thereby its subsequent signaling pathway. for instance, s-glutathionylation of mouse cd95 at cysteine 294 promotes clustering of cd95 and its distribution into lipid rafts [ 119 ] . this amino acid is conserved in the human cd95 sequence and corresponds to cysteine 304 (or c288 when subtraction of the 16 amino acid signal peptide is taken into consideration [ 12 , 120 ] ). interestingly, janssen-heininger and colleagues emphasize that death receptor glutathionylation occurs downstream of caspase-8 and caspase-3 activation whose catalytic activity damages the thiol transferase glutaredoxin 1 (grx1), an enzyme implicated in the denitrosylation of proteins [ 119 ] . the consequence of grx1 inactivation is the accumulation of glutathionylated cd95, which clusters into lipid rafts, sensitizing cells to the cd95-mediated apoptotic signal. based on these fi ndings, caspase-8 activation occurs prior to aggregation of cd95 and redistribution into lipid rafts, both of which are requisite to form the disc and subsequently activate larger amounts of caspase-8. in agreement with these observations, activation of caspase-8 was reported to occur in a two-step process. first, an immediate and small amount of activated caspase-8 (<1 %) is generated when cd95l interacts with cd95 that orchestrates acid sphingomyelinase (asm) activation, ceramide production, and cd95 clustering, which in turn promote disc formation and the outburst of caspase-8 processing essential to mount the apoptotic signal [ 121 ] . s-glutathionylation consists in a bond between a reactive cys-thiol and reduced glutathione (gsh), a tripeptide consisting of glycine, cysteine, and glutamate; its attachment to the protein will alter its structure and function in a manner similar to the addition of a phosphate [ 122 ] . s-glutathionylation is not the only posttranslational modifi cation of cd95 on a cysteine. s-nitrosylation of cysteine 199 (corresponding to c183 after subtraction of signal peptide sequence) and 304 (c288) in colon and breast tumor cells also promotes the redistribution of cd95 into drms, the formation of the disc, and the transmission of the apoptotic signal [ 123 ] . two reports have brought into light that covalent coupling of a 16-carbon fatty acid (palmitic acid) to cysteine 199 (c183) elicits the redistribution of cd95 into drms, the formation of sds-stable cd95 microaggregates resistant to denaturing and reducing treatments, and internalization of the receptor [ 124 , 125 ] . although their order remains to be fi ne-tuned, these molecular steps play a critical role in the implementation of apoptotic signals. of note, similar to s-nitrosylation, both the aforementioned s-glutathionylation at c304 (c288) and palmitoylation at c199 (c183) promote the partition of cd95 into lipid rafts and enhance the subsequent apoptotic signal. further investigation is required to address whether these posttranslational modifi cations are redundant and occur simultaneously in dying cells or are elicited in a cell-specifi c and/or in a microenvironmentspecifi c manner. understanding the molecular mechanisms controlling these posttranslational modifi cations would be of great interest in order to identify the mechanism by which tumor cells block them, leading to their resistance to the extrinsic signaling pathway. using a powerful magnetic method to isolate receptor-containing endocytic vesicles, it has been shown that cd95 promptly associates with endosomal and lysosomal markers when incubated with an agonistic anti-cd95 mab [ 126 ] . in addition, expression of a cd95 mutant in which the dd-located tyrosine 291 (y275) is changed to phenylalanine does not seem to alter the capacity to bind fadd but compromises cd95lmediated cd95 internalization occurring through an ap-2/clathrin-driven endocytic pathway [ 126 ] . more strikingly, expression of the internalization-defective cd95 mutant y291f abrogates the transmission of apoptotic signals, but fails to alter the non-apoptotic signaling pathways (i.e., nfkb and erk), and even promotes them ( fig. 9.3 ) . these fi ndings provide insight into the presence of a region in the dd, interacting with ap2 and promoting a clathrin-dependent endocytic pathway in a fadd-independent manner. regarding the role of palmitoylation in receptor internalization, the interplay between lipid alteration and the ap2/clathrin-driven internalization of cd95 remains to be elucidated. it has been recently demonstrated that cd95 engagement evokes a rapid and transient ca 2+ signaling, which stimulates the recruitment of protein kinase c-β2 (pkc-β2) from the cytosol to the disc [ 127 ] . this kinase transiently brakes disc formation, providing a checkpoint before the irreversible commitment to cell death [ 128 ] . these fi ndings raised the following questions: what are the ca 2+ -dependent molecular mechanisms transiently inhibiting disc formation, and do tumor cells use this signal to escape the immune response and/or resist chemotherapy? in 1998, inhibition of caspase activity was shown to sensitize fi broblastic l929 cell line to tnfmediated necrotic cell death [ 42 ] . with respect to cd95 signal, tschopp et al. showed that fadd and rip1 participate in the implementation of a non-apoptotic signaling pathway, which leads to a necrotic morphology without chromatin condensation and with loss of plasma membrane integrity [ 41 ] . of note, bid cleavage was not observed in this necrotic signal. while fadd plays a crucial role in both apoptotic and necrotic pathways, rip1 recruitment to cd95 occurs independently of this adaptor protein. indeed, yeast two-hybrid experiments showed that rip1 can bind directly to the cd95 dd, while this interaction is lost when a bait corresponding to mutated cd95-dd (replacement of val 238 to asn) is used [ 129 ] . in addition, rip3 (ripk3, a member of the rip kinase family) is an indispensable factor for the induction of the necrotic signaling pathway [ 78 -80 ] . a growing body of evidence supports the existence of necroptosis (programmed necrosis). in addition, identifi cation of necrostatin, a chemical inhibitor of necroptosis [ 130 ] , which specifi cally inhibits rip1 kinase activity [ 131 ] , has accelerated the pace of discovery in this fi eld of cell death. interplays exist between apoptosis and necroptosis; for instance, caspase-8, a potent inhibitor of necroptosis for both cd95 and tnfr1 [ 132 ] , plays a critical role in necroptosis by its ability to process and inactivate rip1 and rip3 [ 133 , 134 ] . at least for tnf signaling, the necrotic signal relies on the activity of cyld, a deubiquitinat-ing enzyme that is also cleaved and inactivated by caspase-8 [ 135 ] . overall, these fi ndings suggest that the apoptotic machinery controls the necrotic one. this concept has been recently established in vivo by double-ko experiments [ 44 -46 , 136 ] . the ko of fadd or caspase-8 is deleterious in mice mainly by the fact that these two apoptotic factors are benefi cial in inhibiting a rip1-/rip3-dependent necrotic signal; thus, their loss unleashes the necroptotic program and leads to embryonic lethality. yet, most studies on necroptosis have focused on the tnf signaling pathway, whereas the mechanism by which cd95 can elicit this cell death pathway, and how the switch in this receptor occurs between non-apoptotic, apoptotic, and necroptotic signals remains unclear. importantly, the impact of each cell death on antigen presentation, and on the effi ciency of immune response after elimination of infected or transformed cells, remains unclear. oncogenic cytokine? the transmembrane cd95l (cd178/fasl) is present at the surface of activated lymphocytes [ 64 ] and nk cells [ 137 ] where it orchestrates the elimination of transformed and infected cells. in addition, cd95l is expressed on the surface of neurons [ 138 ] , corneal epithelia and endothelia [ 58 , 139 ] , and sertoli cells [ 59 ] to prevent the infi ltration of immune cells and thus to prohibit the spread of infl ammation in these sensitive organs (i.e., brain, eyes, and testis, respectively), commonly called "immune-privileged" sites. the description of physiological immune privilege was followed by tumor-mediated immune privilege, since two groups reported that the ectopic expression of cd95l by malignant cells participated in the elimination of infi ltrating t lymphocytes and thus could play a role in the establishment of a tumor site whose access was denied to immune cells [ 140 , 141 ] . however, these observations are controversial since ectopic expression of cd95l in allogenic transplant of β-islets [ 142 , 143 ] and in tumor cell lines [ 144 ] led to a more rapid elimination of these cells than control cells, due to increased infi ltration of neutrophils and macrophages endowed with antitumor activity. among the weapons at the disposal of immune cells, transmembrane cd95l contributes to the elimination of pre-tumor cells. therefore, pretumor cells that escape the immunosurveillance will be shaped to develop resistance to cd95, a process termed immunoediting [ 145 ] . in other words, imprinting of the immune system on pretumor cells will select malignant cells with increased resistance towards the cd95l-induced signal. as previously mentioned, these alterations of the cd95 signal not only block the cd95-mediated apoptotic signal but also promote the transmission of non-apoptotic signals by cd95l, which may play a critical role in carcinogenesis [ 106 -108 , 146 ] . in agreement with this hypothesis, a complete loss of cd95 expression is rarely observed in malignant cells [ 147 ] . accumulating evidence indicates that the apoptotic ligand cd95l behaves as a chemoattractant for neutrophils, macrophages [ 50 , 143 , 144 ] , t lymphocytes [ 53 ] , and malignant cells in which the cd95-mediated apoptotic signal is nonproductive [ 108 , 148 ] . nonetheless, the biological role of cd95l has to be clarifi ed due to the fact that pathophysiologically the ligand is present in at least two forms with different stoichiometries. indeed, cd95l is a transmembrane cytokine whose ectodomain can be cleaved by metalloproteases such as mmp3 [ 149 ] , mmp7 [ 150 ] , mmp9 [ 151 ] , and adam-10 (a disintegrin and metalloproteinase 10) [ 152 , 153 ] and released as a soluble ligand in the bloodstream. based on the data demonstrating that a hexameric cd95l represents the minimal level of selfassociation required to signal apoptosis [ 154 ] and that cleavage by metalloproteases releases an homotrimeric ligand [ 154 , 155 ] , this soluble ligand has long been considered as an inert ligand competing with its membrane-bound counterpart for cd95 binding, thus acting as an antagonist of the death signal [ 155 , 156 ] . it has been recently demonstrated that this metalloprotease-cleaved cd95l (cl-cd95l) actively participates in the aggravation of infl ammation and autoimmunity in patients affected by systemic lupus erythematosus (sle) by inducing the non-apoptotic nf-κb and pi3k [ 51 , 53 ] signaling pathways ( fig. 9.4 ) . unlike transmembrane cd95l, induction of the pi3k signaling pathway by its metalloprotease-cleaved counterpart occurs through the formation of a complex devoid of fadd and caspase-8 which recruits the src kinase c-yes instead [ 53 , 148 ] ; this unconventional receptosome was designated motilityinducing signaling complex (misc) [ 53 , 157 ] ( fig. 9.4 ). even though experiments by the authors did not detect any trace of caspase-8 in the misc, this enzyme has been shown to participate in cell migration. the protease activity of caspase-8 can be abolished by its phosphorylation at tyrosine 380 by src kinase [ 158 ] . this posttranslational modifi cation was observed in cells stimulated with egf and in colon cancer cells exhibiting constitutive activation of src; from a molecular standpoint, this modifi cation does not alter caspase homodimerization or recruitment in disc [ 158 ] . moreover, the egfrdriven phosphorylation of caspase-8 at y380 turns out to be a potent inducer of the pi3k signaling pathway by recruiting the pi3k adaptor p85 alpha subunit [ 159 ] . ultimately, caspase-8 phosphorylation triggers cell migration. nonetheless, it is noteworthy that cd95-induced migration and invasion do not appear to require an intact dd (reviewed in [ 160 ] ), suggesting that either the caspase-8-dependent mode of cell migration occurs as an alternative signal for death receptors or that it only participates in non-death receptor-induced cell motility. it would be interesting to address this question in the future. to date, it can only be surmise that phosphorylation of caspase-8 at y380 upon egfr stimulation may prime certain cancer cells to become unresponsive to the apoptotic signal triggered by cytotoxic cd95l and meanwhile promote cell migration, an essential event in the course of cancer cell metastasis ( fig. 9.4 ) . it is noteworthy that in a similar manner, a decrease in the plasma membrane level of cd95 or expression of a mutated cd95 allele, as observed in alps patients and malignant cells, inhibits the implementation of the apoptotic signal but does not affect the transmission of non-apoptotic signals, such as nf-κb, mapk, and pi3k [ 106 , 107 , 147 ] , suggesting that these signals may stem from a different domain than cd95-dd or rely on different thresholds to be elicited. in summary, although the cd95/cd95l interaction can eliminate malignant cells by implementation of the disc or can promote carcinogenesis by sustaining infl ammation and/or by inducing metastatic dissemination [ 50 , 51 , 53 , 108 , 147 , 148 , 161 ] , the molecular mechanisms underlying the switch between these different signaling pathways remain enigmatic. an important question to be addressed is how the magnitude of cd95 aggregation controls the formation of "death"vs . "motility"-iscs. addressing these questions will lead to the development of new therapeutic agents with the ability to contain the spread of infl ammation or impede carcinogenesis at least in pathologies involving increased soluble cd95l such as cancers (e.g., pancreatic cancer [ 162 ] , large granular lymphocytic leukemia, breast cancer [ 157 ] , and nk cell lymphoma [ 163 ] ) or autoimmune disorders (e.g., rheumatoid arthritis and osteoarthritis [ 164 ] , graft-versus-host-disease (gvdh) [ 165 , 166 ] or sle [ 53 , 167 ] ). altogether, these studies support the notion that the death function of cd95 may correspond to its "day job," while the receptor may act as "a night killer" by fueling infl ammation in certain pathophysiological contexts. strikingly, while the soluble form of cd95l generated by mmp7 (cleavage site inside the 113 elr 115 sequence, fig. 9 .5 ) induces apoptosis [ 150 ] , its counterpart processed between serine 126 and leucine 127 does not [ 51 , 53 , 155 ] . to explain this discrepancy, one may speculate that the different quaternary structures of the naturally processed cd95l underlie the in the presence of cl-cd95l, cd95 triggers misc formation. this complex is devoid of fadd and caspase-8, but, instead, recruits the src kinase c-yes that implements the pi3k signaling pathway. cd95 engagement is also capable of nf-κb and mapk activations through a yet unknown mechanism. right panel : it was reported that procaspase-8 can be phosphorylated by the tyrosine kinase src upon egfr stimulation. this posttranslational modifi cation not only blocks the catalytic activity of caspase-8 but also promotes the recruitment of the p85 subunit of pi3k. we surmise that this caspase-8 phosphorylation may favor the non-apoptotic signals induced by cd95 implementation of a "death"vs . "non-death"inducing signaling complexes and downstream signals. in agreement with this notion, soluble cd95l bathed in the bronchoalveolar lavage (bals) of patients suffering from acute respiratory distress syndrome (ards) undergoes oxidation at methionines 224 and 225 ( fig. 9 .5 ), which enhances the aggregation level of the soluble ligand followed by its cytotoxic activity [ 168 ] . the same authors observed that the stalk region of cd95l, corresponding to amino acids 103-136 and encompassing the metalloprotease cleavage sites (fig. 9 .5 ), participates in the multimerization of cd95l, which accounts for the damage of the lung epithelium in ards [ 168 ] . of note, in ards bals, additional oxidation occurs at methionine 121 ( fig. 9 .5 ), which in turn prevents the processing of cd95l by mmp7, and explains why this cytotoxic ligand keeps its stalk region [ 168 ] . nonetheless, preservation of this region in soluble cd95l raises the question that whether an unidentifi ed mmp7independent cleavage site exists in the juxtamembrane region of cd95l, near the plasma membrane, or the ligand detected in ards patients corresponds to the full-length cd95l embedded in exosomes [ 169 , 170 ] . indeed, this peculiar exosome-bound cd95l can be expressed by human prostate cancer cells (i.e., lncap), and evokes apoptosis in activated t lymphocytes [ 171 ] . overall, these fi ndings emphasize that it will be of great interest in the future to fi nely characterize the quaternary structure of the naturally processed cd95l from the sera of patients affected by cancers or chronic/acute infl ammatory disorders, to better understand the molecular mechanisms implemented by this ligand and thus predict its subsequent biological functions. apoptosis is a fundamental process contributing to tissue homeostasis, immune response, and development. cd95, also called fas, is a member of the tumor necrosis factor receptor (tnf-r) superfamily. its ligand, cd95l, was initially detected at the plasma membrane of activated t lymphocytes and natural killer (nk) cells where it contributes to the elimination of transformed and infected cells. given its implication in immune homeostasis and immune surveillance combined with the fact that various lineages of malignant cells exhibit loss-of-function mutations, cd95 was initially classifi ed as a tumor suppressor gene. nonetheless, in different pathophysiological contexts, this receptor is able to transmit non-apoptotic signals and promote infl ammation and carcinogenesis. although the different non-apoptotic signaling pathways (nf-κb, mapk, and pi3k) triggered by cd95 are known, the initial molecular events leading to these signals, the mechanisms by which the receptor switches from an apoptotic function to an infl ammatory role, and, more importantly, the biological functions of these signals remain elusive. apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics induction of tnf receptor i-mediated apoptosis via two sequential signaling complexes apoptosis in alzheimer's disease -an update apoptosis in parkinson's disease: signals for neuronal degradation human ice/ced-3 protease nomenclature the biochemistry of apoptosis a novel protein that interacts with the death domain of fas/apo1 contains a sequence motif related to the death domain fadd, a novel death domaincontaining protein, interacts with the death domain of fas and initiates apoptosis the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation cytotoxicity-dependent apo-1 (fas/cd95)-associated proteins form a deathinducing signaling complex (disc) with the receptor cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade the polypeptide encoded by the cdna for human cell surface antigen fas can mediate apoptosis molecular cloning and expression of the human 55 kd tumor necrosis factor receptor the receptor for the cytotoxic ligand trail trail-r2: a novel apoptosismediating receptor for trail identifi cation and functional characterization of dr6, a novel death domain-containing tnf receptor fas transduces activation signals in normal human t lymphocytes divergent signalling via apo-1/fas and the tnf receptor, two homologous molecules involved in physiological cell death the tnf receptor superfamily of cellular and viral proteins: activation, costimulation, and death the tnf and tnf receptor superfamilies: integrating mammalian biology the molecular architecture of the tnf superfamily precise mapping of the cd95 preligand assembly domain identifi cation and characterization of a ligand-independent oligomerization domain in the extracellular region of the cd95 death receptor fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations a novel protein domain required for apoptosis. mutational analysis of human fas antigen a novel domain within the 55 kd tnf receptor signals cell death the growth factor progranulin binds to tnf receptors and is therapeutic against infl ammatory arthritis in mice signal transduction by tumor necrosis factor receptors a domain in tnf receptors that mediates ligand-independent receptor assembly and signaling human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha the transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kda tumor necrosis factor receptor nf-kappab antiapoptosis: induction of traf1 and traf2 and c-iap1 and c-iap2 to suppress caspase-8 activation recruitment of the linear ubiquitin chain assembly complex stabilizes the tnf-r1 signaling complex and is required for tnf-mediated gene induction a ubiquitin ligase complex assembles linear polyubiquitin chains linear ubiquitination prevents infl ammation and regulates immune signalling rapid turnover of c-flipshort is determined by its unique c-terminal tail nf-kappab suppression by the deubiquitinating enzyme cezanne: a novel negative feedback loop in pro-infl ammatory signaling ripk-dependent necrosis and its regulation by caspases: a mystery in fi ve acts fas triggers an alternative, caspase-8-independent cell death pathway using the kinase rip as effector molecule inhibition of caspases increases the sensitivity of l929 cells to necrosis mediated by tumor necrosis factor phosphorylation-driven assembly of the rip1-rip3 complex regulates programmed necrosis and virus-induced infl ammation rip3 mediates the embryonic lethality of caspase-8-defi cient mice catalytic activity of the caspase-8-flip(l) complex inhibits ripk3-dependent necrosis fadd prevents rip3-mediated epithelial cell necrosis and chronic intestinal infl ammation lasker clinical medical research award tnf defi ned as a therapeutic target for rheumatoid arthritis and other autoimmune diseases fas engagement induces neurite growth through erk activation and p35 upregulation fas engagement accelerates liver regeneration after partial hepatectomy cd95-ligand on peripheral myeloid cells activates syk kinase to trigger their recruitment to the infl ammatory site membrane-bound fas ligand only is essential for fas-induced apoptosis a novel juxtamembrane domain in tumor necrosis factor receptor superfamily molecules activates rac1 and controls neurite growth the naturally processed cd95l elicits a c-yes/calcium/pi3k-driven cell migration pathway cd95-mediated cell signaling in cancer: mutations and post-translational modulations monoclonal antibody-mediated tumor regression by induction of apoptosis molecular cloning and expression of the fas ligand, a novel member of the tumor necrosis factor family involvement of fas ligand and fasmediated pathway in the cytotoxicity of human natural killer cells fas ligand-induced apoptosis as a mechanism of immune privilege a role for cd95 ligand in preventing graft rejection lymphoproliferation disorder in mice explained by defects in fas antigen that mediates apoptosis aberrant transcription caused by the insertion of an early transposable element in an intron of the fas antigen gene of lpr mice the defect in fas mrna expression in mrl/lpr mice is associated with insertion of the retrotransposon, etn autoimmunity in mice bearing lprcg: a novel mutant gene generalized lymphoproliferative disease in mice, caused by a point mutation in the fas ligand the many roles of fas receptor signaling in the immune system structure of the human apo-1 gene nmr structure and mutagenesis of the fas (apo-1/cd95) death domain the fas-fadd death domain complex structure unravels signalling by receptor clustering solution nmr investigation of the cd95/fadd homotypic death domain complex suggests lack of engagement of the cd95 c terminus the fas-fadd death domain complex structure reveals the basis of disc assembly and disease mutations homotypic fadd interactions through a conserved rxdll motif are required for death receptor-induced apoptosis inhibition of death receptor signals by cellular flip viral flice-inhibitory proteins (flips) prevent apoptosis induced by death receptors ped/pea-15: an anti-apoptotic molecule that regulates fas/tnfr1-induced apoptosis apo-1/fas) signaling pathways two cd95 tumor classes with different sensitivities to antitumor drugs cd95 engagement mediates actin-independent and -dependent apoptotic signals signal transduction mediated by bid, a pro-death bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways bid-defi cient mice are resistant to fas-induced hepatocellular apoptosis xiap discriminates between type i and type ii fas-induced apoptosis the c-iap-1 and c-iap-2 proteins are direct inhibitors of specifi c caspases x-linked iap is a direct inhibitor of cell-death proteases iaps block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases ubiquitinprotein ligase activity of x-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in fas-induced cell death smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition bcl-2 and bcl-xl inhibit cd95-mediated apoptosis by preventing mitochondrial release of smac/diablo and subsequent inactivation of x-linked inhibitor-of-apoptosis protein localization of fas/cd95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway pten loss promotes mitochondrially dependent type ii fas-induced apoptosis via pea-15 phosphatidylinositol 3′-kinase blocks cd95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of cd95 actin-independent exclusion of cd95 by pi3k/akt signalling: implications for apoptosis phosphorylation of pea-15 switches its binding specifi city from erk/mapk to fadd protein kinase b/akt binds and phosphorylates ped/pea-15, stabilizing its antiapoptotic action bcl-2 and fas/apo-1 regulate distinct pathways to lymphocyte apoptosis bcl-2 protects from lethal hepatic apoptosis induced by an anti-fas antibody in mice a bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-fas antibody injection fas gene mutations in the canale-smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity dominant interfering fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome mutations in fas associated with human lymphoproliferative syndrome and autoimmunity chronic lymphadenopathy simulating malignant lymphoma lymphoproliferative syndrome with autoimmunity: a possible genetic basis for dominant expression of the clinical manifestations the development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline fas mutations and defective lymphocyte apoptosis flice-inhibitory protein is a key regulator of germinal center b cell apoptosis primary diffuse large b-cell lymphomas of the central nervous system are targeted by aberrant somatic hypermutation the origin of cd95-gene mutations in b-cell lymphoma does cd95 have tumor promoting activities? the relevance of nf-kappab for cd95 signaling in tumor cells induction of apoptosis and activation of nf-kappab by cd95 require different signalling thresholds cd95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells cd95 signaling via ceramide-rich membrane rafts ligand-independent redistribution of fas (cd95) into lipid rafts mediates clonotypic t cell death fas receptor clustering and involvement of the death receptor pathway in rituximabmediated apoptosis with concomitant sensitization of lymphoma b cells to fas-induced apoptosis resveratrol-induced apoptosis is associated with fas redistribution in the rafts and the formation of a death-inducing signaling complex in colon cancer cells redistribution of cd95, dr4 and dr5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells intracellular triggering of fas aggregation and recruitment of apoptotic molecules into fas-enriched rafts in selective tumor cell apoptosis edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy cisplatin-induced cd95 redistribution into membrane lipid rafts of ht29 human colon cancer cells redistribution of cd95 into the lipid rafts to treat cancer cells? redox amplifi cation of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and s-glutathionylation of fas purifi cation and molecular cloning of the apo-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. sequence identity with the fas antigen ceramide-mediated clustering is required for cd95-disc formation s-glutathionylation uncouples enos and regulates its cellular and vascular function s-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells palmitoylation is required for effi cient fas cell death signaling palmitoylation of cd95 facilitates formation of sds-stable receptor aggregates that initiate apoptosis signaling the role of receptor internalization in cd95 signaling cd95 triggers orai1-mediated localized ca2+ entry, regulates recruitment of protein kinase c (pkc) beta2, and prevents deathinducing signaling complex formation the cd95 signaling pathway: to not die and fl rip: a novel protein containing a death domain that interacts with fas/apo-1 (cd95) in yeast and causes cell death chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury identifi cation of rip1 kinase as a specifi c cellular target of necrostatins the roles of fadd in extrinsic apoptosis and necroptosis cleavage of the death domain kinase rip by caspase-8 prompts tnf-induced apoptosis cleavage of rip3 inactivates its caspaseindependent apoptosis pathway by removal of kinase domain caspase 8 inhibits programmed necrosis by processing cyld programmed cell death: apoptosis meets necrosis fas involvement in cytotoxicity mediated by human nk cells fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? cd95 ligand (fasl)-induced apoptosis is necessary for corneal allograft survival melanoma cell expression of fas(apo-1/cd95) ligand: implications for tumor immune escape the fas counterattack: fasmediated t cell killing by colon cancer cells expressing fas ligand transgenic expression of cd95 ligand on islet beta cells induces a granulocytic infi ltration but does not confer immune privilege upon islet allografts fas ligand expression in islets of langerhans does not confer immune privilege and instead targets them for rapid destruction regulation of the proinfl ammatory effects of fas ligand (cd95l) cancer immunosurveillance, immunoediting and infl ammation: independent or interdependent processes? dominant-negative fas mutation is reversed by down-expression of c-flip cd95 promotes tumour growth yes and pi3k bind cd95 to signal invasion of glioblastoma stromelysin-1 (mmp-3) in synovial fl uid of patients with rheumatoid arthritis has potential to cleave membrane bound fas ligand identifi cation of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human fas ligand matrix metalloproteinase-9 regulates tnf-alpha and fasl expression in neuronal, glial cells and its absence extends life in a transgenic mouse model of amyotrophic lateral sclerosis the fas ligand intracellular domain is released by adam10 and sppl2a cleavage in t-cells adam10 regulates fasl cell surface expression and modulates fasl-induced cytotoxicity and activation-induced cell death two adjacent trimeric fas ligands are required for fas signaling and formation of a deathinducing signaling complex conversion of membrane-bound fas(cd95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity membrane fas ligand kills human peripheral blood t lymphocytes, and soluble fas ligand blocks the killing cd95l cell surface cleavage triggers a pro-metastatic signaling pathway in triple negative breast cancer src kinase phosphorylates caspase-8 on tyr380: a novel mechanism of apoptosis suppression caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility how cd95 stimulates invasion fas and nf-kappab signalling modulate dependence of lung cancers on mutant egfr production and pro-apoptotic activity of soluble cd95 ligand in pancreatic carcinoma fas ligand in human serum soluble fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis levels of soluble fasl and fasl gene expression during the development of graft-versushost disease in dlt-treated patients increased soluble fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease serum levels of soluble fas ligand in patients with silicosis the biological activity of fasl in human and mouse lungs is determined by the structure of its stalk region diacylglycerol kinase alpha regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of fas ligand-containing exosomes in t lymphocytes modulation of the immune response using dendritic cell-derived exosomes tumor exosomes expressing fas ligand mediate cd8+ t-cell apoptosis key: cord-023445-c4tqioz1 authors: lauridsen, c.; jensen, s. k. title: supplementation of vitamin c to weaner diets increases igm concentration and improves the biological activity of vitamin e in alveolar macrophages date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423u.x sha: doc_id: 23445 cord_uid: c4tqioz1 vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay‐c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr‐(α‐tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023950-nv0pbbu2 authors: schnyder, bruno; schnyder-candrian, silvia title: dual role of th17 cytokines, il-17a,f, and il-22 in allergic asthma date: 2012-07-19 journal: il-17, il-22 and their producing cells: role in inflammation and autoimmunity doi: 10.1007/978-3-0348-0522-3_10 sha: doc_id: 23950 cord_uid: nv0pbbu2 the proinflammatory role of t helper (th) 17 cells and therefore of its cytokines, il-17 (il-17a), il-17f, and il-22, in autoimmune disorders has been favored, although there is evidence that not only il-17a but also il-17f and il-22 have a dual role as negative regulators. here we review the concept of the dual function of il-17a, il-17f, and il-22 in the light of recent strategies to use neutralization of these cytokines as potential alternative to neutralizing tnf and il-1 treatments in chronic inflammatory disorders. expectedly, in allergic lung inflammation, neutralization of il-17a inhibited neutrophil recruitment. however, this il-17a antibody treatment concomitantly increased eosinophil recruitment by neutralizing il-17a’s dual role as negative regulator. il-17a negatively regulated dendritic cell function and activation of t helper cell (th)2 cytokine production. furthermore, il-17a inhibited th2-characteristic chemokine and adhesion molecule expression. on a mechanistic level, il-17a acted on iκb-β by preventing degradation and in turn leading to reduced nf-κb activation or il-17a inhibited transcription factor irf-1. therefore, anti-il-17a therapy, although presenting a promising lead in chronic inflammatory disorders, bears a potential risk of exacerbating allergic asthma. il-17a and il-17f homodimers and il-17f/il-17a heterodimer transduce their signals through the receptor composed of il-17ra and il-17rc [2, 3] . il-17a and il-17f are produced by the memory t cells termed th17, a t helper cell lineage distinct from th1 and th2 cells, which is negatively regulated by interferon-g and il-4 [4, 5] . unchecked activation of th17 cells by il-23 is linked to chronic inflammation in experimental autoimmune encephalomyelitis (eae) and type ii collagen-induced arthritis, two heretofore prototypical "th1" disease models [6, 7] . the il-23-th17 cell axis has been implicated to contribute to the allergic th2 response [8] ; for review, see [9] . il-23 as well as the co-expressed th17 cells cytokines il-17a and il-22 [10] is found in the lung of allergic patients [11] [12] [13] and in lung homogenates of ovalbumin (ova)-sensitized and challenged mice [8, 14] . in vitro production of il-17a and il-22 was triggered with il-23 and even further enhanced in the presence of ova, in cultures of mediastinal lymph node (mln) cells isolated from antigen ova-sensitized and challenged mice [8, 14] . therefore, the role of il-17a and il-22 was addressed in the allergic response. il-17a was indeed required during antigen sensitization to develop a th2 response in allergic asthma, as shown in il-17r-deficient mice [8] . neutralization of il-17a, however in this model, augmented the allergic response, while recombinant il-17a administration reduced pulmonary eosinophil recruitment and bronchial hyperreactivity. recombinant il-17a reduced eosinophil-chemokine eotaxin (ccl11) and thymus-and activation-regulated chemokine (tarc/ccl17) in lungs in vivo, and antigen uptake by dendritic cells and il-5 and il-13 production in regional lymph nodes were also reduced by recombinant il-17a [8] . these findings demonstrated a novel negative regulatory role of il-17a. a beneficial role of il-17a has been confirmed in the model of chronic fungal-induced asthma. in this model, the protective role of tlr6 was dependent on il-23 and the production of il-17a. tlr6 deficient mice showed reduced il-23 and il-17a expression and an exacerbated th2 response, which was normalized by addition of recombinant il-23 which recovered the il-17a production [15] . furthermore, murdoch and lloyd provide evidence in the model of acute allergen-induced response that the gdt cell-dependent normalization of lung function and resolution of inflammation was dependent on the production of il-17 [16] . therefore, endogenous il-17a has a dual role. while it is essential during antigen sensitization to establish allergic th2 response, in sensitized mice il-17a attenuates the th2 response. a new role as negative regulator of antigen-driven th2 response was ascribed for il-17f. il-17f dampened antigen activation of dcs resulting in a reduced th2 response and reduced inflammation [14] . these findings may provide a possible explanation why yang and colleagues found an increased allergic response in il-17f-deficient mice [17] . il-22, similar to il-17a [7, [18] [19] [20] , has been found in diseased tissues from patients with different chronic inflammatory diseases, involving infiltrating activated t cells, such as rheumatoid arthritis, psoriasis, inflammatory bowel disease, and copd [21] [22] [23] [24] [25] . il-22 is expressed by il-9-activated mast cell and by th17 cells initiated by tgf-b in the context of il-6 and other proinflammatory cytokines [10] . il-22 is increased in lung homogenates of ova-sensitized and challenged mice [14] . neutralization of endogenous il-22 in ova-sensitized mice increased the eosinophilic response [14] , whereas administration of recombinant il-22 attenuated the acute allergic response [26, 27] . these data provide evidence that il-17a,f and il-22 besides their inflammatory role have a negative regulatory function in allergic lung inflammation. it has in the past abundantly been described that full acquisition of pathogenic function in experimental autoimmune encephalomyelitis (eae) by effector th17 cells is mediated by il-23. however, as shown most recently, stimulation of the myelin-reactive t cells with tgfb plus il-6, instead of il-23, completely abrogated their pathogenic function despite upregulation of il-17a production [28] . these regulatory th17 cells failed to upregulate the proinflammatory chemokines crucial for central nervous system inflammation. in contrast, the regulatory th17 cells produced il-10, which had potent anti-inflammatory activities. this study by cua's group [28] did not show whether il-17a directly conveyed negative regulation of inflammation, which was rather due to coexpressed il-10 in eae. several experimental approaches listed below have indeed demonstrated direct inhibitory functions of recombinant and endogenously produced il-17a in vitro and in vivo. furthermore, an il-17a-induced expression of the anti-inflammatory il-10 has been demonstrated in macrophages, yet il-17a has moderate effects on monocytes and macrophages [29] . first, expression of recombinant murine il-17a in vaccinia virus increased viral virulence significantly in mice [30] , suggesting that il-17a negatively regulated the antiviral host defense. second, administration of recombinant il-17a ameliorated and negatively regulated the late phase of experimental autoimmune neuritis (ean), a model of peripheral nerve demyelination [31] . third, in vitro studies provided possible mechanisms of how il-17a acts as a suppressor. il-17a inhibited the chemokines rantes (ccl5), fractalkine (cx3cl1), and ctack (ccl27) [32] [33] [34] and the mononuclear leukocyte adhesion molecule vcam-1 in tnf-activated mesenchymal cells [35] . fractalkine, ctack, rantes, and vcam-1 are involved in inflammatory responses of both th1 and th2 types. rantes and vcam-1 are essential in the recruitment of mononuclear cells, and vcam-1 is involved in the formation of germinal centers (present in autoimmunity). therefore, existence of the novel negative regulatory role of il-17a needs to be revisited in multiple inflammatory and immune disorders. revisiting the role of il-17a in multiple immune responses would help address the question as to whether il-17a acts like a regulatory t cell (treg) cytokine such as tgfb or il-10, which reduces allergic pulmonary challenges as well as vast t cell responses [36] [37] [38] . indeed, in initial experiments il-17a was described as a treg cytokine in cell cultures, inhibiting vast t cell responses [36, 39] . furthermore, treg cells and tgfb promote under proinflammatory conditions the development of th17 cells and production of il-17a [40] . therefore, il-17a as a downmodulator of the dendritic cells and th2 response provides evidence for a novel feedback mechanism by which treg cells may control a th2 response in the effector phase of allergic asthma. protective role of il-22 has also been ascribed in other models than allergic lung inflammation. il-22 provided protection to hepatocytes during acute liver inflammation [41] and protected against cona-or tetrachloride-induced liver injury [42] . second, delivery of il-22-ig fusion gene ameliorated experimental autoimmune myocarditis (eam) in rats [43] . third, local il-22 gene delivery led to rapid amelioration of intestinal inflammation in a mouse model of ulcerative colitis, and conversely, inhibition of il-22 activity by local overexpression of its antagonist, il-22 binding protein, prevented recovery and goblet cell restitution in acute colitis (dss) [44] . therefore, even though il-22 is an upregulator of proinflammatory gene expression and has proinflammatory function, there is growing evidence that il-22 has, similar to il-17a, a protective role in inflammatory diseases such as colitis, eae, and allergic lung inflammation. il-17a production is induced by tgfb in a proinflammatory milieu, including presence of il-6, il-1, or tnf, and its production is sustained by il-23. in contrast, il-17a production is inhibited by il-4, ifng, il-25 (il-17e), or il-27. in lungs, il-17a has been shown to originate from antigen-specific th17 cells as well as from an inkt subpopulation, which is nk1.1 negative [45] . in an allergic lung response, il-17a production was induced by il-23 and controlled by il-4 receptor signaling [8] . in mice lacking il-4 responsiveness, il-17a was overproduced correlating with reduced effector functions of allergic asthma. the inhibition of the th2 response was indeed ascribed to endogenous il-17a, as assessed using il-17a neutralizing antibody treatment in vivo. in the absence of il-4 responsiveness, not only il-17a but also il-22 production was significantly increased upon allergen challenge, and similar to il-17a, inhibition of endogenous il-22 by neutralizing antibodies increased the allergic response, indicating that both il-17a and il-22 contribute to the inhibition of the th2 response. this increase in il-17a and il-22 in the absence of il-4 responsiveness was not due to differences in il-23 concentrations since pulmonary ova-induced il-23 concentrations are equal in il-4ra ko and wt mice [8] . therefore, the il-4 signals affect directly il-22 and il-17 production and do not act on il-23 production [8] . however, the molecular mechanism of il-22 expression, being either il-23 dependent or independent, in allergic lung inflammation needs to be investigated in future experiments. neutrophil involvement has been ascribed to both infectious disease and allergic inflammation [46] [47] [48] . in allergy, neutrophil recruitment is in part due to endogenous il-17 [8, 49] , but not due to il-22, since neutralization of il-22 did not diminish neutrophil but increase recruitment in the allergic response. this is in line with a previous report, which demonstrated that il-22 alone and in synergy with il-10 decreased il-8 production by human alveolar epithelial cell lines [50] . therefore, a novel mechanism of how il-4 promotes a th2 response was proposed, by suppression of the novel suppressor molecules il-17a and il-22. this added a novel function of il-4 to the list of its proallergic effects, including differentiation of th2 lymphocytes, inhibition of t lymphocyte apoptosis, induction of ige production, promotion of eosinophil transmigration into the lungs, mucus hypersecretion, and bronchoconstriction [8, [51] [52] [53] . on a mechanistic level, il-17a elicits dual effects and reportedly promotes expression of proinflammatory (hemopoietic, cxc-chemokines, acute phase) factors [54, 55] , whereas it inhibits the production of mononuclear cell recruiting molecules like tnf-induced vcam-1 and cc-chemokine rantes [35] . this dual effect of il-17a in human cell cultures predicted a reduced mechanism of mononuclear cell recruitment in vivo. cc-type chemokines rantes (ccl5), tarc (ccl17) and eotaxin (ccl11) were induced by antigen ova in vivo. tarc primarily attracts ccr4-positive th2 cells. il-17a reduced tarc production, which correlated with reduced lymphocyte counts and th2-derived il-5 concentrations in lung tissues. the expression of the major eosinophil attractant, eotaxin, was also reduced by il-17a and accompanied by reduced eosinophil infiltration in the airways. indeed, reductions or absence of these cc chemokines reportedly ablates allergic asthma [56] [57] [58] . therefore, diminished cell attraction seems to be the pivotal mechanism of how il-17a attenuates the allergic inflammation. further il-17a effects like acute phase il-6 and prostaglandin (pg)e2 elevations may also have corroborated to reduce locally the allergic inflammation in the lungs. il-6 elevations inhibited aeroallergen-induced th2 inflammation [59, 60] . pge2 elevations reduced pulmonary allergy specifically via the e3 receptor [61] . therefore, while il-17a may upregulate negative regulators il-6 and pge2, it has a direct inhibitory effect on the local production of th2 cytokines il-4, il-13, and il-5 in the lung and regional lymph nodes [8] . mechanistically, il-17a inhibits dendritic cell activation and antigen uptake, which leads to reduced activation of t cells and reduced il-4, il-13, and il-5 production, resulting in reduced allergic response. however, the inhibition of the th2 response by il-17a represented a reduction rather than a complete blockade. intact anti-allergen ige concentrations in the circulation may explain why il-17a did not completely block but rather reduced pulmonary allergy and asthma. elevated ige concentrations reportedly correlate with and contribute to allergic reactions [62] , although it is not sufficient for the development of allergy. therefore, il-17a acts as negative regulator of established th2 response locally in lungs. in the experimental models of allergic lung inflammation, il-22 dampened the hallmarks of an allergic th2 response in vivo, by inhibition of dcs and their expression of co-stimulatory molecules upon antigen treatment [14] . il-22 attenuates further the allergic response by inhibiting the induction of tarc (ccl17), il-13 and il-25 as shown in vivo [26, 27] . in vitro, il-22 prevented tnf-a/il-13-induced tarc and il-13 production in murine clara cells [26] and il-25 production in the lung epithelial cell line mle-15 induced by il-1b or lps [27] . therefore, these data corroborate that il-22 not only has a negative regulatory function in experimental models of autoimmunity, and inflamed liver and colon but also in established th2 response in the lung. intranasal administration of recombinant il-17a reduced eosinophil recruitment and a th2 response, while neutrophil recruitment was not induced when applied locally at low doses of 2.5 mg/kg il-17a to allergen-treated mice [8] . these findings were supported by the following cell culture data [35] . il-17a inhibited tnf-induced chemokine rantes expression in human synovial fibroblasts and mouse lung fibroblasts. this inhibitory activity of il-17a was sixfold more potent than its stimulatory activity on tnf-a-induced il-6 or il-8 secretion (ic50 ¼ 0.2 ng/ml vs. ed50 ¼ 1.2 ng/ml), measured in the same cells. furthermore, neutralization of the human il-17a receptor (il-17r) by antibodies competitively reversed the il-17a-induced il-6 upregulation. however, anti-il-17r antibody only partially neutralized the inhibitions of rantes production by il-17a. yet, il-17r was essential for the rantes inhibition, as assessed in il-17r-deficient cells. therefore, inhibitory and stimulatory functions of il-17a involve receptor il-17r but show distinct dose responses and in turn different sensitivities to an il-17r antagonizing antibody. these findings suggest a higher efficiency of the inhibitory over the stimulatory il-17a functions and may explain why a net negative regulatory effect of il-17a manifests in chronic inflammation in vivo where il-17a production is low. il-17a interferes at tnf-activated nf-kb signaling in human synoviocytes [35] . this inhibition is immediate, within 20 min, and proposes a direct effect of il-17a rather than via expression of secondary mediators. the reduced degradation of specifically ikb-b, but not ikb-a, provides a late inflammatory phase control mechanism by il-17a for the following reason. it has been shown that inhibitor ikb-a is of importance for the transient inactivation of nf-kb, whereas ikb-b as part of a multimeric complex is involved in the persistent inactivation of nf-kb [63, 64] . the fact that ikb-b but not ikb-a is affected by il-17a further supports the possibility that il-17a is implicated in the regulation of the chronic phase of inflammation and immunity. however, interferences of il-17a on tnf-induced nf-kb activity virtually depend on the cell type and promoter targeted by tnf. for example, unlike the synoviocytes described above [35] , tnf-induced nf-kb binding was only moderately and not statistically significantly reduced by il-17a in colonic myofibroblasts [32] . in those cells, it was proposed that il-17a interfered at the tnf-induced rantes production mainly through inhibition of irf-1. this in turn prevented the cooperation of irf-1 with the nf-kb activity. furthermore, in macrophages it has been shown that il-17a inhibited tnf expression transiently, the effect of il-17a being biphasic with an early decrease of tnf release (at less than 30 min) and a marked stimulation later on (by 6 h) [29] . after 60 min and later, il-17a also inhibited camp production and the transcription factor activities of creb, ap-1, as well as nf-kb in the macrophages [29] . il-22, on the molecular and cellular level, acts by activating stat3. in vitro stimulation of epithelial cell with il-22 resulted in the phosphorylation of stat3, and diminished tnf/il-13 induced tarc and il-13 production [26] . beneficial effect of il-22 through stat3 activation has previously been published. so il-22 enhanced hepatocyte survival, by enhancing expression of transcription factor stat3, bcl-2, and bcl-xl in inflamed liver and colon [41, 42] . stat3 activation in intestinal epithelial cells and il-22 was linked to mucosal wound healing [65] . il-22 activated stat3 and induced il-10 by colon epithelial cells [66] . prevention of dc activation and antigen (ova) sensitization induced by transfer of ex vivo ova-loaded dc has previously been ascribed to the prototype immune regulator il-10 [25] and newly for il-22 [14] . whether the negative regulatory effects of il-22 in allergic lung inflammation are due to il-22 or indirectly induced needs further investigation. inflammation but are still distinct the th17-derived il-22 and il-17a negatively regulated allergy and dc functions. its expressions were further induced by allergic stimulation together with il-23 and were controlled by il-4ra signals . while neutralizing il-22 or il-17 antibodies augmented the allergic response, il-22, il-17a, and il-17f reduced the response by inhibiting dc functions. therefore, the data demonstrate that il-22 and il-17 are novel endogenous negative regulators of allergy, and il-22, il-17, or th17 cells may represent an interesting therapeutic target in lung allergy. il-17a and il-22 are both co-expressed in th17 cells [10] , yet they are induced through independent pathways. recent studies showed that il-22 but not il-17a and il-17f production strongly depends on aryl hydrocarbon receptor signals [67] . furthermore, il-22 production is induced in absence of il-6, while il-17a production depends on the presence of il-6 [22] . additional il-22 production is ascribed to il-9 activated mast cells [68] . we found that il-17a, but not il-22 production, was markedly dependent on the intracellular signaling of the il-1 receptor and its adaptor myd88 pathway in lymph node cell cultures originating from ova-treated mice [14] . a review by eyerich and colleagues provides an overview on overlaps and differences between il-17 and il-22 [69] . together this suggests collaborative and nonredundant pathways leading to th17 cytokines and allergy. 8 anti-il-17 and anti-il-22 therapies: a risk or advantage? the pathological role of il-17a, il-17f, and il-22 in autoimmune disorders has convincingly been documented and hence favored so far, although there is emerging evidence that il-17a, il-17f, and il-22 also have a beneficial role as negative modulator in antigen-specific immune processes and allergic asthma [8, 14, [26] [27] [28] 31] . increased il-17a concentrations in allergic asthma, chronic bronchitis, chronic obstructive pulmonary disease (copd), cystic fibrosis, and acute respiratory distress syndrome (ards) (for review, see [11] ), but also rheumatoid arthritis (ra), were linked to the pathology of the diseases [20] . il-17a neutralization inhibits experimental murine arthritis [20] and is a potential alternative therapy to tnf neutralization in rheumatoid arthritis. the novel negative regulatory function of il-17a indicates, however, that such a therapy bears the potential risk of exacerbating allergic asthma. therefore, an anti-il-17a treatment in chronic inflammatory disorders seems very promising, inhibiting neutrophil recruitment in inflamed lungs and joints, while the exacerbating th2 response in experimental allergic response by anti-il-17a antibodies may exclude respective groups at risk from such a therapeutic anti-il-17a treatment. because of the proinflammatory function of il-17a and il-22, neutralizing therapies targeting either il-17 or il-22 are considered in allergic asthma [70, 71] . however, inhibiting il-17a or il-22 may bear the potential risk of opportunistic infections. il-22 together with tnf-a was found to be important to keep the epidermal integrity during infection with candida albicans [72] . mutations in stat3 in animal models and humans confer a defect in il-17 function, resulting in increased susceptibility to respiratory infections with bacteria and fungi [70, 73, 74] . neutralization of il-22 resulted in exacerbation of bacterial infections, suggesting a protective role in mucosal/epithelial host defense [75] . and il-22 was protective during the development of lung fibrosis induced by chronic exposure to bacillus subtilis [76] . still, because of the novel function of il-22 and il-17 as endogenous negative regulators of allergy, il-22, il-17, or th17 cells may represent an interesting therapeutic target in lung allergy. interleukin-17 cutting edge: interleukin 17 signals through a heteromeric receptor complex the human il-17f/il-17a heterodimeric cytokine signals through the il-17ra/il-17rc receptor complex interleukin 17-producing cd4+ effector t cells develop via a lineage distinct from the t helper type 1 and 2 lineages a distinct lineage of cd4 t cells regulates tissue inflammation by producing interleukin 17 divergent pro-and anti inflammatory roles for il-23 and il-12 in joint autoimmune inflammation interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain interleukin-17 is a negative regulator of established allergic asthma role of il-23 and th17 cells in airway inflammation in asthma interleukin (il)-22 and il-17 are coexpressed by th17 cells and cooperatively enhance expression of antimicrobial peptides neutrophilic airway inflammation and il-17 activation of peripheral th17 lymphocytes in patients with asthma th17 immunity in patients with allergic asthma interleukin-22 is a negative regulator of the allergic response the protective role of tlr6 in a mouse model of asthma is mediated by il-23 and il-17a resolution of allergic airway inflammation and airway hyperreactivity is mediated by il-17-producing {gamma}{delta}t cells regulation of inflammatory responses by il-17f therapeutic efficacy of il-17 neutralization in murine experimental autoimmune encephalomyelitis requirement of il-17 receptor signaling in radiation-resistant cells in the joint for full progression of destructive synovitis blocking of interleukin-17 during reactivation of experimental arthritis prevents joint inflammation and bone erosion by decreasing rankl and interleukin-1 il-22 increases the innate immunity of tissues interleukin-22, a t(h)17 cytokine, mediates il-23-induced dermal inflammation and acanthosis interleukin-22, a member of the il-10 subfamily, induces inflammatory responses in colonic subepithelial myofibroblasts expression of interleukin-22 in rheumatoid arthritis: potential role as a proinflammatory cytokine il-10-treated dendritic cells il-10-treated dendritic cells decrease airway hyperresponsiveness and airway inflammation in mice il-22 is produced by innate lymphoid cells and limits inflammation in allergic airway disease il-22 attenuates il-25 production by lung epithelial cells and inhibits antigen-induced eosinophilic airway inflammation tgf-beta and il-6 drive the production of il-17 and il-10 by t cells and restrain t (h)-17 cell-mediated pathology il-17 stimulates the production and expression of proinflammatory cytokines, il-beta and tnf-alpha, by human macrophages interleukin 17 modulates the immune response to vaccinia virus infection enhancement of acute phase and inhibition of chronic phase of experimental autoimmune neuritis in lewis rats by intranasal administration of recombinant mouse interleukin 17: potential immunoregulatory role il-17 selectively down-regulates tnf-alpha-induced rantes gene expression in human colonic subepithelial myofibroblasts constitutive and inflammatory mediator-regulated fractalkine expression in human ocular tissues and cultured cells il-17 suppresses tnfalpha-induced ccl27 production through induction of cox-2 in human keratinocytes il-17 reduces tnf-induced rantes and vcam-1 expression functional assay for human cd4 + cd25+ treg cells reveals an age-dependent loss of suppressive activity suppression of airway eosinophilia by killed mycobacterium vaccae-induced allergen-specific regulatory t-cells depletion of murine cd4+ t lymphocytes prevents antigen-induced airway hyperreactivity and pulmonary eosinophilia specificity of regulatory cd4 + cd25+ t cells for self-t cell receptor determinants tgfbeta in the context of an inflammatory cytokine milieu supports de novo differentiation of il-17-producing t cells interleukin-22 but not interleukin-17 provides protection to hepatocytes during acute liver inflammation hydrodynamic gene delivery of interleukin-22 protects the mouse liver from concanavalin a-, carbon tetrachloride-, and fas ligand-induced injury via activation of stat3 hydrodynamicbased delivery of an interleukin-22-ig fusion gene ameliorates experimental autoimmune myocarditis in rats il-22 ameliorates intestinal inflammation in a mouse model of ulcerative colitis identification of an il-17-producing nk1.1(neg) inkt cell population involved in airway neutrophilia dual effects of p38 mapk on tnf-dependent bronchoconstriction and tnf-independent neutrophil recruitment in lipopolysaccharide-induced acute respiratory distress syndrome chemokines and their receptors as potential targets for the treatment of asthma neutrophils and immunity: challenges and opportunities interleukin-17 orchestrates the granulocyte influx into airways after allergen inhalation in a mouse model of allergic asthma interleukin-22: a potential immunomodulatory molecule in the lung requirement for il-13 independently of il-4 in experimental asthma anti-interleukin-4 therapy immunologic basis of antigen-induced airway hyperresponsiveness t-cell-derived interleukin-17 regulates the level and stability of cyclooxygenase-2 (cox-2) mrna through restricted activation of the p38 mitogen-activated protein kinase cascade: role of distal sequences in the 3 0 -untranslated region of cox-2 mrna stimulus-specific induction of a novel nuclear factor-kappab regulator, ikappab-zeta, via toll/interleukin-1 receptor is mediated by mrna stabilization eosinophil recruitment to the lung in a murine model of allergic inflammation. the role of t cells, chemokines and adhesion receptors inhibition of airway inflammation by aminoterminally modified rantes/cc chemokine ligand 5 analogues is not mediated through ccr3 intervention of thymus and activation-regulated chemokine attenuates the development of allergic airway inflammation and hyperresponsiveness in mice endogenous and exogenous il-6 inhibit aeroallergen-induced th2 inflammation enhanced airway inflammation and decreased subepithelial fibrosis in interleukin 6-deficient mice following chronic exposure to aerosolized antigen suppression of allergic inflammation by the prostaglandin e receptor subtype ep3 anti-ige efficacy in murine asthma models is dependent on the method of allergen sensitization i kappab-beta regulates the persistent response in a biphasic activation of nf-kappa b ikappabbeta, but not ikappabalpha, functions as a classical cytoplasmic inhibitor of nf-kappab dimers by masking both nf-kappab nuclear localization sequences in resting cells stat3 links il-22signaling in intestinal epithelial cells to mucosal wound healing interleukin-22 activates stat3 and induces il-10 by colon epithelial cells the aryl hydrocarbon receptor links t(h)17-cell-mediated autoimmunity to environmental toxins cloning and characterization of il-10-related t cell-derived inducible factor (il-tif), a novel cytokine structurally related to il-10 and inducible by il-9 il-17 and il-22: siblings, not twins interleukin-17 regulation: an attractive therapeutic approach for asthma update in the mechanisms of allergen-specific immunotherapy il-22 and tnf-a represent a key cytokine combination for epidermal integrity during infection with candida albicans the role of interleukin-17 in the helicobacter pylori induced infection and immunity deficiency of th17 cells in hyper ige syndrome due to mutations in stat3 il-22 mediates mucosal host defense against gram-negative bacterial pneumonia ) gd t cells protect against lung fibrosis via il-22 key: cord-016168-3hyb9stq authors: nan title: pathogenesis of fever date: 2009 journal: clinical manual of fever in children doi: 10.1007/978-3-540-78598-9_3 sha: doc_id: 16168 cord_uid: 3hyb9stq #x203a; although infection is the most common cause of fever, fever is also a common finding in hypersensitivity reaction, autoimmune diseases, and malignancy. › febrile response is mediated by endogenous pyrogens (cytokines) in response to invading exogenous pyrogens, primarily microorganisms or their direct products (toxins). › these endogenous pyrogens act on thermosensitive neurons in the hypothalamus, which ultimately upgrade the set point via prostaglandins. › the body reacts by increasing the heat production and decreasing the heat loss until the body temperature reaches this elevated set point. › fever, in contrast to hyperthermia, will not climb up relentlessly because of an effective central control of the hypothalamic center. › cytokines play a pivotal role in the immune response by activation of the b cells and t lymphocytes. the production of fever simultaneously with lymphocyte activation constitutes the clearest and strongest evidence in favor of the protective role of fever. › the protective processes of the immune response are optimal at high temperature (around 39.5°c). › not all effects resulting from fever generation benefit the host; some are harmful and even lethal. this occurs mainly by overproduction of the cytokines or imbalance between cytokines and their inhibitors, such as severe and fulminate infections and septic shock. research in fever has been centered on the hypothesis that fever results from physiological processes that are set in motion by an external stimulus. egyptian scholars recognized that local inflammation was responsible for fever. in 1868, billroth (1829-1894) attempted to confirm this ancient observation by injecting pus into animals, thereby producing a febrile response. in 1943, menkin carried out similar experiments and isolated a product termed "pyrexin" [1] . beeson in 1948 isolated a fever-inducing substance from a leukocyte, leukocyte pyrogens, which later became known as endogenous pyrogen (ep). interleukin-1 (il-1) was first identified as a cytokine by gery and waksman and proved to be identical with ep [2] . • fever (pyrexia) is a regulated body temperature above the normal range occurring as a result of il-1-mediated elevation of the hypothalamic set point. once fever is established, body temperature is regulated, as in health, by a net balance between heat production and loss. • hyperthermia is unregulated elevated body temperature above the normal range due to imbalance between heat production and loss. interleukins are not involved and therefore the hypothalamic set point is normal. • a pyrogen is a substance (infectious organisms or their product toxins, or cytokines) that provokes fever. • exogenous pyrogens are substances that originate outside the body and that are capable of inducing interleukins. • endogenous pyrogens are substances that originate inside the body and that are capable of inducing fever by acting on the hypothalamic thermoregulatory center. il-1, tumour necrosis factor (tnf) and interferon (inf) are endogenous pyrogens. • cytokines are proteins produced throughout the body, mainly by monocytes, macrophages, and t cells to regulate the immune responses within the body and control inflammatory and haematopoietic processes and may induce fever. as they enter the circulation and act on distant organs, they are considered as hormones. cytokines are proinflammatory cytokines, anti-inflammatory cytokines, interleukins, or lymphokines. • pro-inflammatory cytokines (il-1, il-6, tnf-α, inf-γ, granulocyte-macrophage colony-stimulating factor, gm-csf) are responsible for initiating an effective defense against exogenous organisms. their overproduction may be harmful by causing shock, multiple organ failure, and death. • anti-inflammatory cytokines (il-1 receptor antagonist, il-4, il-10) antagonize proinflammatory cytokines. their overproduction may also be harmful by suppressing the immune function. • interleukins are cytokines acting specifically as mediators between leukocytes, and hence their name. if their amino acid sequence is known, they are assigned an interleukin number. if their sequence is not known, then they are named according to the biological property. il-1 and il-6 play a major part in the pathogenesis of fever. • lymphokines are cytokines that are secreted by lymphocytes (il-2, il-3, il-4, il-5, il-6, il-9, il-10, il-13, il-14, tnf-β). • acute-phase response is the term used for haematological, endocrinological, and metabolic changes that follow (within hours or days) the onset of fever in response to local damage to a tissue. these changes are induced by several cytokines and are beneficial to the host. during the response, various acute-phase proteins, notably c-reactive protein (crp) and serum amyloid a, are synthesized by liver and released into circulation in large amounts. crp plays a role in complement activation, opsonization (engulfing and destroying microbes by phagocytes), and increasing platelet aggregation. exogenous pyrogens (exps) initiate fever, usually within 2 h of exposure, by interacting with macrophages or monocytes, leading to il-1 induction. other mechanisms to initiate fever include the following: some endotoxins, produced by bacteria, act directly on the hypothalamus to alter the set point. il-1 is not involved. radiation of the hypothalamus, ddt (dichloro-diphenyl trichloroethane) poisoning, and scorpion venom may also induce fever by a direct effect on hypothalamus. • exps may activate lymphocytes to secrete lymphokines, particularly inf-y, which in turn stimulate macrophages and monocytes to produce il-1. • some bacteria produce exotoxins that stimulate macrophages and monocytes to release il-1. this mechanism operates in scarlet fever and toxic shock syndrome. in toxic shock syndrome, the shock is due to the toxin. diseases involving exotoxins produced by gram-positive bacilli are less fever inducing than those produced by pyrogenic gram-positive cocci. • borrelia spirochetes (the cause of relapsing fever) do not contain endotoxin, and the attachment of these bacteria to the mononuclear cells induces the production of il-1. other bacteria, such as pneumococci, have no endotoxin or other pyrogens, and the mechanisms responsible for fever are presumably immunological. • gram-negative bacteria. the pyrogenicity of gram-negative bacteria (e.g., escherichia coli, salmonella) is due to a heat-stable factor, endotoxin. the active components are lipid and carbohydrate (lipopolysaccharide, lps) elements of the outer membrane of these bacteria. endotoxin causes a dose-related progressive increase in temperature. in severe cases, it causes vasodilatation, capillary leakage, and hypotension. infection with gram-negative endotoxin (e.g., septicemia) does not elicit fever in many situations: • neonates, young infants, children with fulminating infection with septic shock (complicates septicemia in 20% of cases) and with malnutrition may present with normal temperature or hypothermia. • septicemia presenting with hypothermia is a well-known clinical entity possibly due to inhibition of il-6 and il-1 by il-10 [3] . • gram-positive bacteria. the main pyrogen of staphylococci is peptidglycan of the cell wall. endotoxin is more active per unit weight than peptidoglycan, which may explain the comparatively worse prognosis associated with gram-negative infection. • viruses. it is well known in clinical practice that viruses cause fever. mechanisms by which viruses may produce fever include direct invasion of macrophages, immunological reaction to viral components involving antibody formation, induction by inf, and necrosis of cells by viruses. • fungi. live or killed fungal products are exogenous pyrogens that induce fever. the induction of fever mainly occurs when the fungi are in the bloodstream. children with neoplastic diseases who develop fever associated with neutropenia are at high risk for developing invasive fungal infection. • phagocytosis is largely responsible for fever in blood transfusion reactions (once an infection is excluded) and immune hemolytic anaemia. • antigen-antibody complexes. an exogenous antigen may react with circulating, sensitized antibodies to form a complex that induces il-1 production (immune fever). examples of immunologically mediated fever include systemic lupus erythematosis and adverse drug reactions. fever associated with penicillin hypersensitivity results from interaction of antigen-antibody complexes with leukocytes, which release il-1. • other non-microbial pyrogens include some hormones, drugs, and intracranial lesions such as bleeding and thrombosis. • steroids. these are endogenous antipyretics, which suppress fever development through its inhibitory effects on il-1 and tnf-α production. certain steroids, however, are pyrogenic in humans. the most known steroid is etiocholanolone, an androgenic metabolite that may induce the release of il-1. this steroid produces fever only when injected intramuscularly (not intravenously), hence fever may result from il-1 released by subcutaneous tissue at the injection site. this steroid is thought to be responsible for fever in a few patients with adrenogenital syndrome and fevers of unknown origin. mononuclear cells are leukocytes (3-8% of the leukocytes) and are largely responsible for the production of il-1 and fever induction. polymorphonuclear granulocytes are no longer thought to be responsible for il-1 production because fever may occur in their absence, for example agranulocytosis. the mononuclear cells are either circulating monocytes in the peripheral blood or tissue macrophages (histocytes) scattered in organs such as lung (alveolar macrophages), lymphnodes, placenta, peritoneal cavity, and the subcutaneous tissue. the origin of both monocytes and macrophages is the granulocytes-monocyte colonyforming unit (gm-cfu) in the bone marrow. monocytes enter the circulation either to remain there for a few days as circulating monocytes or to migrate to the tissue where they undergo functional and morphological transformation into macrophages, when their life span is several months. these cells play an important role in: • host defense, including engulfing and destroying the microbe (phagocytosis), recognition of antigen, and presenting it to attached lymphocytes and • activation of t lymphocytes and tumor cell destruction. situations associated with reduced function of the monocyte-macrophage system (mms) include newborn infants, corticosteroid and other immunosuppressive therapy, systemic lupus erythematous, wiskot-aldrich syndrome (immune deficiency involving b and t cells, eczema, and thrombocytopenia), and chronic granulomatous disease. the two major monocyte-macrophage products (cytokines) are il-1 and tnf. il-1 is stored in an inactive form in the cytoplasm of secreting cells and is enzymatically converted to an active form before it is released across the cell membrane into the circulation. it affects distant organs and therefore acts as a hormone. the kidney is the principal site for its removal. il-1 consists of three structurally related polypeptides, two agonists (il-1 α and il-1β), and an antagonist (il-1 receptor antagonist = il-1ra) that inhibits the activities of the two agonists. the relative amount of il-1 and il-1ra in a disease influences whether the inflammation remains active or suppressed. il-1 is produced by: • macrophages as the main source of il-1 production. • hepatic kupffer cells, keratinocytes, pancreatic langerhans's cells. • astrocytes in the brain tissue, which may contribute to the immunological responses within the cns and the fever secondary to cns bleeding. • cells from certain malignant tumors (e.g., hodgkin's disease, acute leukemia and renal carcinoma). this explains the frequent association of fever in these conditions in the absence of infection. and • stimulating the liver to synthesize certain acute-phase proteins, such as fibrinogen, haptoglobin, ceruloplasmin, and crp. at the same time, the synthesis of albumin and transferrin decreases. characteristically, there is a decreased concentration of iron and zinc and an increased concentration of copper. the low iron is the result of reduced intestinal assimilation of iron and increased liver storage of iron. these changes contribute to host defense by depriving invading microorganisms of essential nutrients, such as iron and zinc (the process is referred to as nutritional immunity). • appetite suppression, which results in a significant reduction of food intake, seen commonly in febrile illness [4] . this process occurs within the brain. il-1 receptor antagonist attenuates the appetite-reducing effects by il-1. • stimulating osteoclast differentiation and activation, resulting in bone loss. • production of factor s, a peptide identical to il-1 with sleep promotion effect leading to slow-wave sleep. it is produced in astrocytes of the brain. the factor may explain the observation of increased sleep in febrile illnesses. • stimulating the production of collagenase and prostaglandin e2 (pge2), which play a major role in the pathogenic progression of various arthritides, particularly rheumatoid arthritis. • stimulating the pituitary-adrenal axis, causing increasing production of glucocorticoid hormones. • increasing protein breakdown, leading to myalgia, which commonly accompanies fever. this proteolysis, mediated by pge2, is blocked by the pge2 inhibitor indomethacin. amino acids released during proteolysis can be metabolized within the muscle as a direct source of energy and are reused for the synthesis of new proteins. other amino acids may become substrates for gluconeogensis. in all these circumstances the activity of il-1 is enhanced at elevated temperatures. antagonism of il-1 (il-1ra) has therapeutic effects in many diseases, including: • various forms of hereditary periodic syndromes and neonatal-onset multisystem inflammatory disease (chap. 1). • hiv replication. il-1ra has suppressive effects on the virus. • bone erosions and loss, occurring, for example, in rheumatoid arthritis. there is little or no il-1 in healthy humans at rest. a long list of diseases, including meningitis, septicemia, crohn's disease, rheumatoid arthritis, neonatal hypoxic-ischaemic encephalopathy, and acute organ rejection are associated with increased levels of il-1 and poor outcome for the patients. malnutrition (kwashiorkor and marasmus), on the other hand, is associated with a significant impairment of macrophage function and il-1 production. tnf, discovered in 1968, is a cytokine produced by monocytes and microphages (tnf-α), lymphocytes (tnf-β), natural killer cells, kupffer cells, and astrocytes of the brain in response to invasive or injurious stimuli. like il-1, tnf is regarded as an endogenous pyrogen because its acts on the hypothalamus to induce fever. unlike il-1, tnf has no direct effect on stem-cell and lymphocyte activation. tnf in small quantities has diverse beneficial biological effects, including: • sharing many biological properties with il-1, for example, enhancing host defense against infection, promoting normal tissue remolding, including wound healing, and enhancing chemotaxis of macrophages and neutrophils as well as increasing their phagocytic and cytotoxic activity. • being the earliest and most important mediator of inflammation. • stimulating il-1 production. • having a direct effect against certain tumor cells (e.g., by damaging the nuclear dna and producing free radicals). its use against human cancers, however, has been associated with poor outcome, mainly due to its systemic side effects. when large amounts are released in tissue, however, tnf may lead to. • lethal tissue injury and shock (septic or toxic shock). • wasting (tnf is identical to cachectin), by inhibiting the activity of lipoprotein lipase, and producing negative nitrogen balance and glucose release, often associated with chronic infection and some tumors. high serum levels of tnf correlate with the activity and prognosis of many infectious diseases, including bacterial meningitis, leishmaniasis, human immunodeficiency virus (hiv) infection, malaria, and intestinal inflammatory diseases. increased tnf production in kawasaki disease may play a role in the immune activation and damage to vascular endothelial cells occurring in the disease. il-6 is the third most studied cytokines. it is: • a pro-inflammatory cytokine that is secreted by macrophages and t lymphocytes to stimulate immune response • synergistic with il-1 and tnf-α, including induction of fever (il-6 responds earlier than il-1) and acute phase response. it parallels the duration of fever. il-6 is an early marker (within 3-4 h of endotoxin stimulation) • • increased in many diseases, for example, sepsis, autoimmune diseases and juvenile idiopathic arthritis, kawasaki disease, and epidural fever [5] anti-il-6 receptor is available to treat il-6-related immune-inflammatory diseases. other cytokines with their main effects are shown in table 3 .1. the antigen-specific cells of the immune system are lymphocytes, of which there are two main types: • b cells are responsible for antibody production, whereas; • t cells regulate antibody synthesis and mediate cytotoxic function as well as inflammatory response of delayed-type hypersensitivity. t-cells are either: -th1 cells, which produce inf-γ, il-2, and tnf-β and promote cell-mediated immunity and phagocytic activity, or -th2 cells that produce il-4, il-5, il-6, il-9, and il-10. these promote antibody production and play a crucial role in allergic responses (immediate-type hypersensitivity). il-1 has an essential role in the activation of lymphocytes. the t lymphocyte recognizes antigen only after the antigens are processed and presented to them by macrophages; only then do t lymphocytes become active. interferon is known for its ability to 'interfere' (hence the name) with viral replication in infected cells. there are three molecules, tnf-α, β, and γ, differing in biological activity and amino-acid sequences. il-2 is probably the second most important lymphokine (after inf) that is released by activated t lymphocytes in response to il-1 stimulation. it has an essential effect on the growth and function of t cells, natural killer cells, and b cells. cases of severe congenital combined immunodeficiency due to a specific defect in the production of il-2 have been reported. its effects are: • antitumor cytotoxicity (e.g., against neuroblastoma, melanoma) as a result of proliferation and activation of activated cytotoxic t lymphocytes and those that include malaise, fever, anorexia, and myalgia of the four hematopoietic-colony stimulating factors (erythropoietin, granulocyte-colony stimulating factor (g-csf), macrophage-colony stimulating factor (m-csf), and granulocyte-macrophage-colony stimulating factor (gm-csf) ), the latter (gm-csf) appears to have the most potential clinical benefits . [6] it is a proinflammatory cytokine, which is produced mainly by lymphocytes, although monocytes, macrophages, and mast cells are also capable of producing it. gm-csf's principal function and potential therapeutic uses are the following: the administration of gm-csf may be associated with the development of fever, which is blocked by nonsteroidal anti-inflammatory drugs such as ibuprofen. thermoregulation requires intact peripheral mechanisms that balance heat production and loss, and a functioning hypothalamic thermoregulatory center regulating these mechanisms. heat production occurs by various mechanisms: • at rest, as many organs such as brain, muscles, viscera, liver, heart, thyroid, pancreas, and adrenal glands contribute to heat production at the cellular level involving adenosine triphosphate (atp). in the newborn infant, brown fat localized mainly in the neck and scapular area produces heat through nonshivering thermogenesis. this tissue is highly vascularized and contains a large quantity of mitochondria. fatty acid oxidation in these mitochondria can increase heat production to twofold in response to cold. • older children and adults conserve heat by vasoconstriction and generate heat by shivering in response to cold. blood flow, regulated by the cns, plays a vital role in distributing heat throughout the body. in a warm environment or when core temperature is elevated, the hypothalamic thermoregulatory center activates efferent fibers of the automatic nervous system to produce vasodilatation. the increased blood flow to the skin causes heat loss from the core through the skin surface to the surroundings in the form of sweating. in colder environments or with decreased core temperature, reduced skin blood flow promotes retention of body heat. pathological uncontrollable increase of heat production occurs in malignant hyperthermia (chap. 2) in response to a rise in body temperature, heat is lost from the body via the four physical modalities of radiation, evaporation, convection, and conduction. failure of heat loss has been incriminated as the cause of infantile heat stroke, which carries a high mortality rate. heat loss occurs through the following mechanisms: • in general, 60% of the total heat is lost by radiation, which is the transfer of heat from the skin surface to the external surroundings by mean of electromagnetic waves. • about one-quarter is lost by evaporation from the skin and lungs, which occurs as water is converted from liquid to gas: 243 kj (58 kcal) is lost for every 100 ml of water. • convection (12% of the heat loss) is the transfer of heat through the movement of air or fluid surrounding the skin surface. • conduction (3% of the heat loss) is the heat transfer between two objects in direct contact and at different temperatures. this is the primary mode of heat loss from the core to the surface. a child in the lying position with a large contact surface has a higher heat loss through conduction than in the standing position. simultaneously, the hypothalamus stimulates vasodilatation to increase insensible loss (for every 1°c elevation of body temperature, there is a 10% insensible loss) and activates the sweat glands to increase perspiration production. physical factors obviously affect the ability to respond to temperature changes. the greater heat loss in the newborn infant is mainly due to a greater surface area compared to that of an older child. failure of heat loss occurs in anhidrotic ectodermal dysplasia and during anticholinergic drug overdose. fever generation includes the following stages: • the specific area of the il-1 action is the pre-optic and anterior hypothalamus, which contains clusters of thermosensitive neurons localized within the rostral wall of the third ventricle. the site is called organum vasculosum laminae terminalis (ovlt), which has emerged as an interface between circulation and brain. the firing rate of these thermosensitive neurons changes according to the temperature of the area's blood supply and the input from the skin and muscular thermoreceptors. warm-sensitive neurons have firing rates that increase with warming and decrease with cooling, whereas the firing rates of cold-sensitive neurons increase with cooling or decrease with warming. • il-1 enters the perivascular space of the ovlt through the fenestrated capillary wall to stimulate cells to produce pge2, which diffuses into the adjacent pre-optic/hypothalamic region to cause fever. the view that the ovlt is the major port of entry for pyrogenic cytokines has recently been challenged [7] . in the endothelial and perivascular cells of the blood-brain barrier (bbb), pyrogenic cytokines are switched to pge2. these cells probably represent a structure termed circumventricular organ system (cvos), which consists of small clusters of neurons and is adjacent to the bbb. this structure serves as a communication channel between blood and neurons of the hypothalamus. when circulating pyrogenic cytokines are detected by the cvos, pge2 is induced [8] . • the ultimate result of these complex mechanisms is an upward shift of the thermostatic set point to a febrile level that signals efferent nerves, especially sympathetic fibers innervating peripheral blood vessels, to initiate heat conservation (vasoconstriction) and heat production (shivering). this is aided by behavioral means aimed also to increase body temperature, such as seeking a warmer environment or covering up with a blanket. the resulting temperature increase continues until body temperature approximates to the temperature of the elevated set point. the cations na + and ca 2+ as well as cyclic adenosine monophosphate (camp) may also contribute to the alteration of body temperature, although their exact role is not yet clear. the raised set point is reset back to normal if the concentration of il-1 falls or if antipyretics are administered, which blocks prostaglandin synthesis. recently, the peptide angiotensin 11 has been shown to lower body temperature at the final step of fever [9] . prostaglandin e2 has been found to exert a negative feedback on the release of il-1, thereby tending to terminate the mechanisms that initially induced the fever. in addition, arginine vasopressin (avp) acts within the cns to reduce pyrogen-induced fevers. the normalization of temperature is initiated by vasodilatation and sweating through increased skin blood flow controlled by sympathetic fibers. the generation of fever involves the following steps: • numerous substances from outside the body, exogenous pyrogens, initiate the fever cycle. endotoxin of gram-negative bacteria, with their pyrogenic component lipopolysaccaride, is the most potent exp. fever is also a common finding in children without obvious evidence of infection, for example, hypersensitivity reaction, autoimmune diseases, and malignancy. the exps stimulate monocytes, fixed-tissue macrophages, and reticuloendothelial cells to produce and release identical substances, now collectively termed il-1, which has multiple biological functions essential for the immune response. • il-1 acts on the hypothalamic thermoregulatory center through mediators, of which pge2 is the most important, to raise the thermostatic set point. il-1 thereby acts as an endogenous pyrogen. the hypothalamic thermoregulatory center accomplishes heat production by inducing shivering and heat conservation through vasoconstriction. at an established degree, fever is regulated (even at a temperature of over 41.0°c) and heat production approximates loss, as in health, though at a higher level of the set point. therefore fever does not climb up relentlessly. • in addition to the function as an endogenous pyrogen, il-1 activates t lymphocytes to produce various factors, such as inf and il-2, which are vital for immune response. the production of fever simultaneously with lymphocyte activation constitutes the clearest and strongest evidence in favor of the role of fever. chemical basis of fever potentiation of the lymphocyte response to mitogens: cellular source pf potentiating mediators involvement of the accurate nucleus of the hypothalamus in interleukin-1 induced anorexia elevated maternal and serum interleukin-6 levels are associated with epidural fever promising stratagems for reducing the burden of neonatal sepsis fever and hypothermia in systemic inflammation: recent discoveries and revisions the neurobiology of the human febrile responses angiotensin ii: its effects on fever and hypothermia in systemic inflammation key: cord-023387-tyeh14wz authors: hvas, c. l.; kelsen, j.; agnholt, j.; höllsberg, p.; dahlerup, j. f. title: probiotic bacteria induce regulatory cytokine production via dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423au.x sha: doc_id: 23387 cord_uid: tyeh14wz probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001;18:213–25), and dendritic cells were matured from their peripheral blood mononuclear cells. t‐cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf‐α, ifn‐γ, il‐10 and gm‐csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf‐α and il‐10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn‐γ and tnf‐α were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25(+)), in part mediated by dendritic cells. future studies will address whether this shift to a cd25(+) phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-269986-jdcw59r2 authors: regan, andrew d.; cohen, rebecca d.; whittaker, gary r. title: activation of p38 mapk by feline infectious peritonitis virus regulates pro-inflammatory cytokine production in primary blood-derived feline mononuclear cells date: 2009-02-05 journal: virology doi: 10.1016/j.virol.2008.11.006 sha: doc_id: 269986 cord_uid: jdcw59r2 feline infectious peritonitis (fip) is an invariably fatal disease of cats caused by systemic infection with a feline coronavirus (fcov) termed feline infectious peritonitis virus (fipv). the lethal pathology associated with fip (granulomatous inflammation and t-cell lymphopenia) is thought to be mediated by aberrant modulation of the immune system due to infection of cells such as monocytes and macrophages. overproduction of pro-inflammatory cytokines occurs in cats with fip, and has been suggested to play a significant role in the disease process. however, the mechanism underlying this process remains unknown. here we show that infection of primary blood-derived feline mononuclear cells by fipv wsu 79-1146 and fipv-df2 leads to rapid activation of the p38 mapk pathway and that this activation regulates production of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-alpha) and interleukin-1 beta (il-1 beta). fipv-induced p38 mapk activation and pro-inflammatory cytokine production was inhibited by the pyridinyl imidazole inhibitors sb 203580 and sc 409 in a dose-dependent manner. fipv-induced p38 mapk activation was observed in primary feline blood-derived mononuclear cells individually purified from multiple spf cats, as was the inhibition of tnf-alpha production by pyridinyl imidazole inhibitors. coronaviruses are a diverse family of enveloped positive-stranded rna viruses that infect a wide range of species including humans. coronaviruses are divided into three groups in which group 1 and 2 infect mammals and group 3 infects birds (perlman et al., 2008) . feline coronaviruses (fcovs) belongs to group 1 and are classified as either serotype i or ii depending on the sequence of their spike (s) protein (rottier, 1999) . in addition, each serotype is divided into two biotypes designated as either feline enteric coronavirus (fecv) or feline infectious peritonitis virus (fipv) based on their pathological outcome in cats (vennema et al., 1998) . fecv is ubiquitous amongst felines and causes mild to often unapparent enteritis, while fipv leads to a lethal systemic infection marked by severe granulomatous inflammation (pedersen et al., 1984a (pedersen et al., , 1984b weiss and scott, 1981) . the mechanism underlying this drastic difference in disease between the two biotypes remains elusive, namely because fecv and fipv isolates from the same serotype are virtually indistinguishable on the genetic and antigenic level. however it has been shown that the two biotypes possess markedly different abilities to infect cells of the immune system, with fipv isolates possessing an extended tropism that allows for the infection of macrophages and monocytes (stoddart and scott, 1989) . recent studies have suggested that this alteration in tropism may be due to mutations in the s protein that affect protein cleavage and fusion activation during entry (regan et al., 2008; rottier et al., 2005) . viral pathogens that infect immune cells (e.g. human immunodeficiency virus (hiv) and dengue virus) are known to induce aberrant cytokine production, a process which is proposed to play a role in the pathological outcome of their respective diseases (fantuzzi et al., 2003; kedzierska and crowe, 2002; leong et al., 2007) . studies of cats with fip have shown that cytokine expressions are altered as compared to healthy animals (dean et al., 2003; kiss et al., 2004) . specifically it has been noted that expression of the pro-inflammatory cytokine tumor necrosis factor alpha (tnf-alpha), interleukin-1 beta (il-1 beta) and interleukin-6 (il-6) are significantly increased in cats with fip, and are likely produced by infected macrophages and monocytes (kiss et al., 2004; takano et al., 2007a takano et al., , 2007b . it has been shown that tnf-alpha is able to induce feline t-cell apoptosis, making it the most likely causative agent of t-cell lymphopenia in fipvinfected cats (dean et al., 2003; takano et al., 2007a) . in addition tnfalpha has been shown to increase expression of the fcov receptor aminopeptidase n (apn) causing target cells to be more susceptible to viral infection and further exacerbate the disease (takano et al., 2007b) . however despite their critical role in the pathological outcome of fip, the mechanism regulating fipv-induced upregulation of pro-inflammatory cytokines remains undescribed. mitogen-activated protein kinases (mapks) are a family of proteins that serve as components of signaling pathways within cells in order to process and respond to extracellular stimuli (raman et al., 2007) . typically, receptors on the cell surface initiate signaling cascades, which lead to phosphorylation and translocation of mapks to the nucleus where they regulate transcriptional activators (whitmarsh, 2007) . in recent years, it has become clear that mapks also regulate processes outside of the nucleus such as mrna translation and cytoskeletal remodeling (frevel et al., 2003; huang et al., 2004) . three major mapk pathways have been identified which are conserved in all eukaryotic cells ranging from yeast to mammals. these pathways are designated as extracellular signal-regulated kinases 1 and 2 (erk1/2), c-jun n-terminal kinases (jnk1) and p38 mapk (pearson et al., 2001) . in general the erk pathway is activated by proliferative stimuli, while the jnk and p38 mapk pathways are activated by extracellular stresses such as ultraviolet light, heat and osmotic shock (pearson et al., 2001) . p38 mapk was originally identified as the target of pyridinyl imidazole compounds that were shown to inhibit the production of il-1 and tnf-alpha in lipopolysaccharide (lps)-stimulated human monocytes (lee et al., 1994) . subsequent studies have shown that the p38 mapk pathway is responsible for the phosphorylation of a large group of transcriptional and translational response elements which directly regulate the expression of a wide variety of proinflammatory cytokines (kumar et al., 2003) . due to its involvement in cytokine regulation, we reasoned that the p38 mapk pathway might play a role in the increased production of pro-inflammatory cytokines observed in cats with fip. in this study we examined the activation of the p38 mapk pathway in response to infection by fipv in primary feline blood-derived mononuclear cells. we also investigated the role of p38 mapk in tnf-alpha, il-1 beta and il-6 production, and the effect of p38 mapk inhibitors on these processes. the p38 mapk pathway has been shown to be activated by multiple viral pathogens during infection (adamson et al., 2000; banerjee et al., 2002; dumitru et al., 2006; erhardt et al., 2002; holloway and coulson, 2006; zachos et al., 1999) . to determine whether the p38 mapk pathway is activated during the infectious lifecycle of fipv, primary feline blood-derived mononuclear (pfbm) cells were inoculated with either fipv-1146 or fipv-df2 at an moi of 100. untreated cells and infected cells ranging from 15 min to 12 h p.i. were lysed and analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). untreated cells showed a minimal level of p38 mapk phosphorylation, however addition of either virus isolate caused rapid phosphorylation of p38 mapk (n600% increase) within 15 min p.i. (figs. 1a and c) . p38 mapk underwent dephosphorylation by 60 min and then showed a second phase of phosphorylation later in infection between 6 and 12 h p.i., which was less pronounced (fig. 1a) . to determine whether viral replication was required for fipv-induced p38 mapk activation, uv-inactivated virus was added to pfbm cells and analyzed by western blot as described above (fig. 1b) . uv-treated fipv also induced p38 mapk phosphorylation, however the activation was not biphasic, and instead remain sustained throughout the 12 h time-course (fig. 1b) . membranes were re-probed with anti-p38 mapk (n-20) pab to show that an equal amount of p38 mapk was present in each sample (figs. 1a and b) . to further confirm that the p38 mapk pathway is activated by fipv, pfbm cells were inoculated with fipv-1146 at an moi of 100 before fixing the cells for immunofluorescent microscopy. p38 mapk again showed a rapid phosphorylation by 15 min p.i. while the total amount of p38 mapk remained unchanged (fig. 2) . in addition the p38 mapk in infected cells showed increased nuclear localization as compared to untreated cells, a phenomenon highly associated with the regulation of transcriptional activators (fig. 2) . these data indicate that the p38 mapk pathway is activated during infection of pfbm cells by fipv, and that viral replication is dispensable for this activation to occur. the p38 mapk pathway was first discovered by investigating the target of pyridinyl imidazole compounds which blocked lps-induced cytokine induction in human monocytes (lee et al., 1994) . to test the effect of pyridinyl imidazole inhibitors on fipv-induced p38 mapk phosphorylation, pfbm cells were treated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. 15 min p.i. cells were lysed and analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). cells which were pretreated with dmso alone showed rapid fipv-induced phosphorylation of p38 mapk, however those which were pretreated with either sb 203580 or sc 409 showed no activation as compared to uninfected cells (fig. 3) . these data demonstrate that fipv-induced p38 mapk activation is blocked by pyridinyl imidazole inhibitors. activation of the p38 mapk pathway has been shown to be required for replication of some viruses including the murine coronavirus mouse hepatitis virus (mhv) (banerjee et al., 2002) . to investigate whether activation of the p38 mapk pathway is required for replication of fipv, pfbm cells were treated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 or fipv-df2. 12 h p.i. cells were fixed and stained for with the anti-fipv n protein mab (17b7.1). as shown in fig. 4 , pretreatment with p38 mapk inhibitors has no significant effect on fipv replication in pfbm cells. activation of p38 mapk by viral pathogens has been shown to induce the production of pro-inflammatory cytokines such as tnfalpha, il-1 beta and il-6 (banerjee et al., 2002; griego et al., 2000; lee et al., 2005a lee et al., , 2005b sloan and jerome, 2007; wang et al., 2004; yurochko and huang, 1999) . to investigate whether tnfalpha production in fipv-infected pfbm cells is regulated by p38 mapk activation, pfbm cells were treated with 10 μm of either sb 203580, sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. 24 h p.i. supernatants were collected, concentrated and analyzed by western blot with the anti-tnf-alpha (n-19) pab. cells which were pretreated with dmso alone showed significant production of tnf-alpha, however those which were pretreated with either sb 203580 or sc 409 showed no detectable production of tnf-alpha as compared to uninfected cells (fig. 5a ). to quantify the production of tnf-alpha, infections were performed as described above, except at 24 h p.i. supernatants were collected and analyzed by anti-tnf-alpha capture elisa. infected cells which were pretreated with dmso alone showed significant production of tnf-alpha (n750 pg/ml) however pretreatment with 10 μm sb 203580 and 10 μm sc 409 resulted in a 8-fold and 4-fold reduction in tnf-alpha production respectively (fig. 5b) . uninfected cells produced no tnf-alpha, or were below the detection level of the assay (data not shown). overall these data indicate that production of the pro-inflammatory cytokine tnf-alpha in fipvinfected pfbm cells is regulated by activation of the p38 mapk pathway. to show that the reduction of fipv-induced tnf-alpha production by sb 203580 and sc 409 was specific to the pyridinyl imidazole inhibitors, pfbm cells were treated with either sb 203580 or sc 409 at a range of concentrations (10 μm, 1 μm or 0.1 μm) or 0.1% dmso for 2 h before inoculating with fipv-1146 or fipv-df2 at an moi of 100. as seen in our previous data, pfbm cells which were pretreated with dmso alone produced significant amounts of tnfalpha however pretreatment with sb 203580 and sc 409 resulted in a significant reduction in tnf-alpha production in a dose-dependent manner (fig. 6) . it is known that individual animals can vary in their reaction to infection by fipv (kiss et al., 2004) . to determine whether or not fipvinduced p38 mapk activation was specific to a single animal, pfbm cells were individually prepared from six additional spf cats (07pgp2, 07pgv4, 07pgv5, 07fgr2, 07fgv6, 07fjm5). pfbm cells individually purified from each animal were inoculated with fipv-1146 at an moi of 100. untreated cells and infected cells (15 min p.i.) were lysed and . 24 h p.i. supernatants were collected and tnfalpha production was quantified by anti-tnf-alpha capture elisa (b). tnf-alpha produced from untreated cells was below the detection limit of the assay (b10 pg/ml). fig. 6 . fipv-induced tnf-alpha production is inhibited in a dose-dependent manner by sb 203580 and sc 409. pfbm cells were pretreated with either sb 203580 or sc 409 at a range of concentrations (10 μm, 1 μm or 0.1 μm) or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and tnf-alpha production was quantified by anti-tnf-alpha capture elisa. tnf-alpha from untreated cells was below the detection limit of the assay (b10 pg/ml). analyzed by western blot with the anti-phospho-p38 mapk mab (3d7). consistent with our previous data, untreated cells showed a minimal level of p38 mapk phosphorylation while addition of fipv caused a rapid phosphorylation of p38 mapk in pfbm cells from all six cats (fig. 7) . membranes were re-probed with anti-p38 mapk (n-20) pab to show that an equal amount of p38 mapk was present in each sample (fig. 7) . in addition, the regulation of tnf-alpha production by p38 mapk was analyzed in pfbm cells from all six spf cats. pfbm cells from each animal were treated with 10 μm sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and analyzed by anti-tnf-alpha capture elisa. while the baseline level of tnf-alpha production differed slightly amongst all of the cats tested, treatment with the p38 inhibitor sc 409 resulted in the same trend observed in our previous experiments: a significant reduction in tnf-alpha levels (between 3fold to 8-fold) (fig. 8) . these data taken together suggest that fipvinduced activation of the p38 mapk pathway in pfbm cells represents a common mechanism by which this virus promotes tnf-alpha production in cats. cats with fip have also been reported to show increased levels of the pro-inflammatory cytokines il-1 beta and il-6. to investigate whether il-1 beta and il-6 production in fipv-infected pfbm cells is regulated by p38 mapk activation, pfbm cells were treated with 10 μm of either sb 203580, sc 409 or 0.1% dmso for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and analyzed by anti-il-1 beta and anti-il-6 capture elisa. infected cells which were pretreated with dmso alone showed significant production of il-1 beta (n200 pg/ml) however pretreatment with 10 μm sb 203580 and 10 μm sc 409 resulted in a 7-fold and 4-fold reduction in il-1 beta production respectively (fig. 9) . neither infected nor uninfected pfbm cells produced significant levels of il-6 ( fig. 9) . overall, these data indicate that both tnf-alpha and il-1 beta production in fipv-infected pfbm cells is regulated by p38 mapk activation, a situation that does not apply to il-6. modulation of signaling pathways by viruses is becoming recognized as a key pathogenic determinant in viral diseases mediated by aberrant host immunological responses. in the case of fip, cytokine production is markedly altered between animals with disease as compared to healthy animals, with overproduction of the proinflammatory cytokine tnf-alpha in particular being indicative of a poor outcome (kiss et al., 2004) . feline tnf-alpha causes apoptosis in feline t-cells (implicating it as the causative agent of t-cell lymphopenia), and upregulates the fipv receptor apn making target cells more susceptible to infection in vitro (dean et al., 2003; kiss et al., 2004; takano et al., 2007a takano et al., , 2007b . it has been shown previously that fipv-infected monocytes upregulate the expression of tnf-alpha, however the mechanism regulating this process remains undescribed. in this study we show that infection by fipv causes a rapid activation the p38 mapk pathway in pfbm cells, and that this process directly regulates production of the pro-inflammatory cytokines tnf-alpha and il-1 beta. as shown in fig. 1 , fipv-induced p38 mapk activation in pfbm cells occurs in a biphasic temporal pattern which mimics that observed with other viral pathogens that activate mapk pathways during infection such as influenza virus (pleschka et al., 2001) . at present we are unable to define the mechanism by which fipv particles are able to activate the p38 mapk pathway, however the rapid nature of the initial activation suggests that it occurs early during entry; likely due to interactions between the s protein and its receptor. this model is further supported by the observation that uv-inactivated virus also induce rapid activation of the p38 mapk pathway. this activation is markedly different than that reported in mhv infected cells, where activation did not occur until 6-12 h p.i., and uv-treated viral particles did not induce phosphorylation of p38 mapk (banerjee et al., 2002) . it notable that the fipv receptor apn localizes to lipid rafts (navarrete santos et al., 2000; nomura et al., 2004) which are known to be a signaling portal for the p38 mapk pathway (calzolari et al., 2006; head et al., 2006; olsson and sundler, 2006; sugawara et al., 2007; wang et al., 2006; zeidan et al., 2008) . in fact it has recently been shown that rhinovirus activates the p38 mapk pathway through the actions of lipid rafts and rhoa (dumitru et al., 2006) . further investigation will be necessary to determine the role of apn and lipid rafts in the initial phase of fipv-induced p38 mapk activation and tnf-alpha/il-1 beta production. interestingly, uv-inactivated fipv induced prolonged p38 mapk activation, rather than the biphasic activation induced by untreated viral particles. this suggests that fipv may activate p38 mapk during entry, but then suppresses p38 mapk during the early phase of replication. the second phase of fipv-induced p38 mapk activation induced by untreated viral particles (6 h p.i.) may be caused by the production of pro-inflammatory cytokines. it has been shown that tnf-alpha can itself activate the p38 mapk through signaling associated with the cytoplasmic domain of its receptors tnf receptor 1 (tnfr1)-associated death domain protein (tradd) and tnf receptor-associated factor 2 (traf2) (carpentier et al., 1998; hsu et al., 1995 hsu et al., , 1996 . therefore tnf-alpha produced during the initial phase of fipv-induced p38 mapk activation, may be the cause of the latter phase of activation. pretreatment with the pyridinyl imidazole inhibitors sb 203580 and sc 409 blocked production of tnf-alpha and il-1 beta suggesting that p38 mapk directly regulates production of the cytokines in fipvinfected pfbm cells. the upregulation of il-6 production was not fig. 8 . fipv-induced tnf-alpha production by pfbm cells from six spf cats is inhibited by sc 409. pfbm cells were individually prepared from three male (07pgp2, 07pgv4, 07pgv5) and three female (07fgr2, 07fgv6, 07fjm5) spf cats. cells from each animal were inoculated with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and tnf-alpha production was quantified by anti-tnf-alpha capture elisa. tnf-alpha from untreated cells was below the detection limit of the assay (b 10 pg/ml). observed in fipv-infected pfbm cells, suggesting that another cell type may be responsible for its production in cats with fip. at this time the mechanism by which p38 mapk regulates pro-inflammatory cytokine production in fipv-infected pfbm cells is unknown, however regulation of cytokines by mapks in analogous systems occurs by affecting either transcriptional regulation, translational regulation, or both (kumar et al., 2003) . for example the recently emerged severe acute respiratory syndrome coronavirus (sars-cov) is also known to infiltrate immune cells such as monocytes and macrophages and activate the p38 mapk pathway (belyavsky et al., 1998; franks et al., 2003; gu et al., 2005; nicholls et al., 2003) . sars-cov infection causes a p38 mapk-dependent phosphorylation of downstream transcriptional regulators such as activating transcription factor 1 (atf-1) and signal transducer and activator of transcription 3 (stat-3), as well as translational regulators such as mapk activate protein kinase 2 (mapkapk2) and the eukaryotic initiation factor 4e (eif4e) (mizutani, 2007; mizutani et al., 2004a mizutani et al., , 2004b . as seen in fig. 2 it appears that fipv causes increased p38 mapk nuclear localization suggesting that the activation of transcription factors likely play a role in proinflammatory production in pfbm cells, however this also does not exclude a role for translational regulation. future studies examining the role of downstream transcriptional and translational regulators in fipv-infected pfbm cells should clarify the mechanism regulating this process. another aspect complicating the treatment of fip is the diverse reactions to infection displayed by cats with the disease (kiss et al., 2004) . our results suggest that activation of the p38 mapk pathway and its regulation of tnf-alpha production is common to pfbm cells of all cats, however further sampling of animals throughout different geographic regions will be required to confirm this conclusion. pyridinyl imidazole compounds have been shown to be efficacious therapeutic agents for blocking the mediators of chronic inflammatory diseases such as rheumatoid arthritis (kumar et al., 2003) . in fact, several p38 mapk inhibitors have shown promise in animal models of inflammatory diseases and some have even reached human clinical trials (kumar et al., 2003) . our results show a clear activation of p38 mapk by fipv during infection, and that this activation is responsible for pro-inflammatory cytokine production which is a key contributor to the pathological changes observed in cats with fip. this raises this possibility that p38 mapk inhibitors, alone or in conjunction with other therapies, may possess therapeutic benefits in the treatment of cats with fip. primary feline blood-derived mononuclear (pfbm) cells were individually purified from four male spf cats (animal id# 07pjo7, 07pgp2, 07pgv4, 07pgv5) and three female spf cats (animal id# 07fgr2, 07fgv6, 07fjm5) (liberty research, waverly, ny) using a standard ficoll-paque gradient (ge healthcare) as specified by the manufacturer. cells were seeded in 24-well plates with tissue culturetreated glass coverslips and allowed to attach overnight. after washing, cells were incubated in the presence of 5% co 2 at 37°c in rpmi-1640 media ph7.4 supplemented with 10% fetal bovine serum (fbs), 2 mm glutamine, 100 u/ml penicillin and 10 μg/ml streptomycin. the purity of pfbm preparations were routinely checked by immunofluorescence microscopy using the marker dh59b (veterinary medical research & development inc., pullman, wa). crandell-reese feline kidney cells were obtained from the american type culture collection (atcc) and cultured and maintained according to atcc guidelines. fipv wsu 79-1146 (fipv-1146) was obtained from the atcc. fipv-df2 was provided by dr. ed dubovi (animal health diagnostic center, new york state college of veterinary medicine, cornell university). both viruses were grown by inoculating crfk cells at a moi of 0.01 and collecting supernatant after cpe was observed in 80% of cells which typically occurred between 48 and 72 h. supernatant was fig. 9 . production of il-l beta and il-6 by fipv-infected pfbm cells. pfbm cells were pretreated with 10 μm of either sb 203580 or sc 409 (or 0.1% dmso as a control) for 2 h before inoculating with fipv-1146 at an moi of 100. 24 h p.i. supernatants were collected and il-1 beta and il-6 production was quantified by anti-il-1 beta or anti-il-6 capture elisa. clarified by a low speed centrifugation step (1250× g for 10 min) and viral particles were then pelleted by centrifugation at 28,000 rpm in a sw28 rotor (sorvall) for 60 min. pellets were resuspended in phosphate-buffered saline (pbs). virus titers were determined by plaque assays on crfk cells using standard techniques. for uvinactivation, a thin layer of viral suspension was exposed to uv light (30 w) at a distance of 10 cm for 5 min. inactivation was verified by performing infection assays in crfk and pfbm cells as described. the anti-phospho-p38 mapk (thr180/tyr182) (3d7) rabbit monoclonal antibody (mab) was obtained from cell signaling technologies (danvers, ma). the anti-p38 mapk (n-20) goat polyclonal antibody (pab) and anti-tnf-alpha (n-19) goat pab were obtained from santa cruz biotechnology (santa cruz, ca). the anti-fipv nucleocapsid (n) protein mab (17b7.1) was provided by dr. ed dubovi (animal health diagnostic center, new york state college of veterinary medicine, cornell university). anti-cd127a mab dh59b was obtained from veterinary medical research and development, inc. (pullman, wa). the feline tnf-alpha elisa kit (tnf-alpha/tnfsf1a), feline il-1 beta elisa kit (il-1 beta/il-1f2), feline il-6 elisa kit, and associated antibodies and detection reagents were obtained from r&d systems (minneapolis, mn). the p38 mapk inhibitors 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1h-imidazole (sb 203580) and 4-(3-(4-chlorophenyl)-5-(1-methylpiperidin-4-yl)-1h-pyrazol-4-yl) pyrimidine (sc 409) were obtained from calbiochem (san diego, ca). pfbm cells were incubated in low-serum media (1% fbs) for 12 h before inoculation with the specified virus at an moi of 100, or pretreatment with the specified inhibitor for 2 h followed by infection. for p38 mapk activation experiments, cells were lysed at the specified time-points in lysis buffer (1% triton x-100, 50 mm tris-hcl, 150 mm nacl, 1 mm edta, 1 mm dtt, 50 mm beta-glycerophosphate, 100 mm sodium vanadate, ph 7.4) supplemented with 1× complete protease inhibitor cocktail (roche). lysates were clarified by centrifugation at 13,000 rpm in a table-top centrifuge at 4°c for 15 min before freezing at −80°c for later analysis. for immunofluorescence assays, cells were fixed at the specified time-points with 3% paraformaldehyde. for analysis of cytokine production, supernatant was collected 24 h postinoculation (p.i.) before freezing at −80°c for later analysis. fixed cells were labeled with the specified antibodies as described previously (chu et al., 2006) . cells were viewed on a nikon eclipse e600 fluorescence microscope, and images were captured with a sensicam em camera and analyzed with iplab software. sds sample buffer was added to lysates and the reaction was heated at 95°c for 10 min before separation using a 4-20% sds-page gel at 200 v for 2 h. gels were electroblotted to pvdf membrane at 200 a for 2 h, blocked with 5% bovine serum albumin and probed with the specified antibody at 4°c for 12 h. membranes were developed using either anti-rabbit antibody (southern biotech, birmingham al) or anti-goat antibody (santa cruz biotechnology, santa cruz ca) linked to horseradish peroxidase and ecl substrate (pierce, rockford il) and images captured using a fujifilm las-3000 ccd camera. for western blot analysis of tnf-alpha production, supernatants were concentrated 50× using icon 9 kda molecular weight cut-off spin columns (pierce, rockford il) and analyzed by western blot as described above. western blot densitometry analysis of signal intensity was performed using imagej software. for quantification of cytokine production, supernatants were processed with the specified elisa kits (r&d systems) using standard capture elisa techniques as specified by the manufacturer. epstein-barr virus immediate-early proteins bzlf1 and brlf1 activate the atf2 transcription factor by increasing the levels of phosphorylated p38 and c-jun n-terminal kinases murine coronavirus replication-induced p38 mitogen-activated protein kinase activation promotes interleukin-6 production and virus replication in cultured cells coronavirus mhv-3-induced apoptosis in macrophages tfr2 localizes in lipid raft domains and is released in exosomes to activate signal transduction along the mapk pathway traf2 plays a dual role in nf-kappab-dependent gene activation by mediating the tnfinduced activation of p38 mapk and ikappab kinase pathways the avian coronavirus infectious bronchitis virus undergoes direct low-ph-dependent fusion activation during entry into host cells in vivo cytokine response to experimental feline infectious peritonitis virus infection rhinoviral infections activate p38map-kinases via membrane rafts and rhoa hepatitis c virus core protein induces cell proliferation and activates erk, jnk, and p38 map kinases together with the map kinase phosphatase mkp-1 in a hepg2 tet-off cell line monocyte/macrophage-derived cc chemokines and their modulation by hiv-1 and cytokines: a complex network of interactions influencing viral replication and aids pathogenesis lung pathology of severe acute respiratory syndrome (sars): a study of 8 autopsy cases from singapore p38 mitogen-activated protein kinase-dependent and -independent signaling of mrna stability of au-rich element-containing transcripts role of p38 mitogen-activated protein kinase in rhinovirus-induced cytokine production by bronchial epithelial cells multiple organ infection and the pathogenesis of sars microtubules and actin microfilaments regulate lipid raft/caveolae localization of adenylyl cyclase signaling components rotavirus activates jnk and p38 signaling pathways in intestinal cells, leading to ap-1-driven transcriptional responses and enhanced virus replication the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation tradd-traf2 and tradd-fadd interactions define two distinct tnf receptor 1 signal transduction pathways map kinases and cell migration the role of monocytes and macrophages in the pathogenesis of hiv-1 infection disease outcome and cytokine responses in cats immunized with an avirulent feline infectious peritonitis virus (fipv)-ucd1 and challenge-exposed with virulent fipv-ucd8 p38 map kinases: key signalling molecules as therapeutic targets for inflammatory diseases a protein kinase involved in the regulation of inflammatory cytokine biosynthesis hiv-1 gp120-induced tnf-{alpha} production by primary human macrophages is mediated by phosphatidylinositol-3 (pi-3) kinase and mitogen-activated protein (map) kinase pathways p38 mitogenactivated protein kinase-dependent hyperinduction of tumor necrosis factor alpha expression in response to avian influenza virus h5n1 the pathology of dengue hemorrhagic fever signal transduction in sars-cov-infected cells tyrosine dephosphorylation of stat3 in sars coronavirus-infected vero e6 cells phosphorylation of p38 mapk and its downstream targets in sars coronavirus-infected cells aminopeptidase n/cd13 is associated with raft membrane microdomains in monocytes lung pathology of fatal severe acute respiratory syndrome human coronavirus 229e binds to cd13 in rafts and enters the cell through caveolae the role of lipid rafts in lps-induced signaling in a macrophage cell line mitogen-activated protein (map) kinase pathways: regulation and physiological functions pathogenic differences between various feline coronavirus isolates pathogenicity studies of feline coronavirus isolates 79-1146 and 79-1683 influenza virus propagation is impaired by inhibition of the raf/mek/erk signalling cascade differential regulation and properties of mapks differential role for low ph and cathepsin-mediated cleavage of the viral spike protein during entry of serotype ii feline coronaviruses the molecular dynamics of feline coronaviruses acquisition of macrophage tropism during the pathogenesis of feline infectious peritonitis is determined by mutations in the feline coronavirus spike protein herpes simplex virus remodels t-cell receptor signaling, resulting in p38-dependent selective synthesis of interleukin-10 intrinsic resistance of feline peritoneal macrophages to coronavirus infection correlates with in vivo virulence the lipid raft proteins flotillins/reggies interact with galphaq and are involved in gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase a "possible" involvement of tnf-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis tnf-alpha, produced by feline infectious peritonitis virus (fipv)-infected macrophages, upregulates expression of type ii fipv receptor feline aminopeptidase n in feline macrophages feline infectious peritonitis viruses arise by mutation from endemic feline enteric coronaviruses sustained activation of p38 mitogen-activated protein kinase and c-jun n-terminal kinase pathways by hepatitis b virus x protein mediates apoptosis via induction of fas/fasl and tumor necrosis factor (tnf) receptor 1/tnf-alpha expression hsp70 enhances macrophage phagocytosis by interaction with lipid raft-associated tlr-7 and upregulating p38 mapk and pi3k pathways pathogenesis of feline infectious peritonitis: pathologic changes and immunofluorescence regulation of gene transcription by mitogen-activated protein kinase signaling pathways human cytomegalovirus binding to human monocytes induces immunoregulatory gene expression herpes simplex virus type 1 infection stimulates p38/c-jun n-terminal mitogen-activated protein kinase pathways and activates transcription factor ap-1 leptin-induced cardiomyocyte hypertrophy involves selective caveolae and rhoa/rock-dependent p38 mapk translocation to nuclei we thank marc antoniak for helpful advice and discussions during the course of this work, and ed dubovi for kind provision of reagents. we also thank a damon ferguson for technical assistance. adr was supported grant t32ai007618 (training in molecular virology and pathogenesis) from the national institutes of health. work in the author's lab was supported by the winn feline foundation and the george sydney and phyllis redmond miller trust. key: cord-023430-5zuewjv2 authors: nilkaeo, a.; bhuvanath, s. title: interleukin‐18 inhibition of oral carcinoma cell proliferation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bg.x sha: doc_id: 23430 cord_uid: 5zuewjv2 interleukin‐18 (il‐18), a pro‐inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn‐γ production and stimulation of nk‐cell‐cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il‐18 effect on an oral carcinoma (kb) cell line. with rt‐pcr technique, kb‐cell line was found to express il‐18 receptors (il‐18rα and il‐18rβ), indicating that this oral carcinoma line is a target for il‐18 study. we showed that recombinant human il‐18 inhibited kb‐cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il‐18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il‐18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death‐controlling genes (bcl‐2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb‐cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il‐18, as this cytokine is an important regulator of anticancer mechanisms. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-034340-3ksfpaf7 authors: nan title: proceedings of the 26th european paediatric rheumatology congress: part 2: virtual. 23 26 september 2020 date: 2020-10-28 journal: pediatr rheumatol online j doi: 10.1186/s12969-020-00470-5 sha: doc_id: 34340 cord_uid: 3ksfpaf7 nan introduction: juvenile idiopathic arthritis (jia) represents the most common pediatric chronic rheumatic disease. children with jia present an increased risk of infections, due to the immune-regulatory effects of disease modifying antirheumatic drugs (dmards); many of these infections are vaccine-preventable. nevertheless, suboptimal vaccinations rates are reported in children with jia. objectives: to evaluate vaccination coverage in a population of children with jia and to describe the prevalence of the adverse events following immunization (aefis) in our cohort. methods: a single-centre retrospective study was conducted by reviewing medical records of all jia patients, diagnosed according to ilar criteria, admitted to the pediatric rheumatology unit of university of naples federico ii from january to december 2019. parents were asked to provide the vaccinations records in form of the vaccination booklet. the occurrence of aefis was explored by telephone interviews. introduction: intrarticular corticosteroid injections (iaci) are widely used in the management of patients with juvenile idiopathic arthritis (jia). general anesthesia can be avoided in case of a small number of joints to inject or in older children. however, pain and anxiety may reduce the patient compliance to iaci, and may compromise the accuracy of the procedure. in order to overcame such problems, the use of appropriate methods of pain and anxiety control is advisable. objectives: to assess the effectiveness and satisfaction of patients undergoing iaci with the use of topical numbing agent or under minimal sedation. methods: patients with jia who underwent an iaci of up to 3 joints were recruited. depending on age and number of joints to treat, a group of patients (group a) were injected with the application 30 minutes prior the procedure of a topical numbing agent (prilocaine+lidocaine) to the skin over the injection site. another group of patients (group b) were treated under minimal sedation (ketorolac/tramadol or morphine + midazolam). the physician was asked to record the degree of motion and pain of the patient during the procedure and the patient (or parents for patients aged less than 4 years) was asked to report the degree of pain and satisfaction on a visual analogue scale (vas) from 0 to 10. results: twenty-seven patients were enrolled for a total of 30 procedures, 17 and 13 of them in group a and b, respectively. the median age at the procedure was 10 years for group a and 11 years for group b. for group a median pain scores for patients, parents and physicians were 2, 2 and 1.5, respectively. in patients of group b who underwent the iaci under ketorolac/tramadol the median pain scores for patients, parents and physicians were 3, 5.25 and 2.5, whereas in patients treated with morphine median pain scores were 6, 6 and 2, respectively. overall, we found that pain as reported by the patient/parent were higher with increase in the number of sites injected (and, consequently, duration of procedure) and age of patient. amount of motion during procedures was overall negligible. the majority of patients/parents was satisfied for the procedures. only 2 patients treated with midazolam had psychomotor agitation during the iaci. conclusion: iaci in a small number of sites without the use of general anesthesia is well tolerated by patients. the level of pain perceived from patients is irrespective of the power of the painkiller used, but seems to correlate with the duration of the procedures. it is possible that, in the paediatric age, the psychoemotional component seems to be decisive, with a progressive loss of tolerance with the increase in the number of injected joints. for gc-ms analysis of the steroid hormone metabolites age and sexmatched healthy controls were matched to each patient. patients were excluded if they were treated with corticosteroids in the preceding 3 months. results: of the 35 metabolites measured, 23 were significantly lower in jia patients before the etanercept treatment compared to the healthy control group. one day after the injection only 5 metabolites were still significantly lower in the jia patients and all the other 30 metabolites normalized and were similar to the control group. urine metabolite ratios reflecting cyp21 and 11β-hsd2 enzymatic activity indicate that these two enzyme activities were lower in jia patients. the slowest recoveries noted were for metabolites of dheas and 17 oh pregnenolone. conclusion: prior to etanercept treatment almost all urine adrenal metabolites were significantly lower mainly due to the active inflammatory process. immediately after the treatment many metabolites raised to normal values as in the control group. the two adrenal enzymes that were found to be affected in jir are cyp21 and 11β-hsd2. blocking tn alpha immediately restore adrenal function in jia. introduction: patients with juvenile idiopathic arthritis (jia) receive adalimumab treatment. adalimumab is a monoclonal antibody that blocks tnf-α and is structurally and functionally similar to human igg 1 . nevertheless, there are reports of the development of anti-drug antibodies. the production of these antibodies may be associated with treatment failures (a decrease in the effectiveness of therapy or drug inefficiency that developed over time) and hypersensitivity reactions. to our knowledge, there is currently limited information on the availability of adalimumab antibodies (aaa) in patients with jia. objectives: to evaluate the prevalence rate and the clinical significance of aaa in patients with jia on adalimumab treatment. methods: 26 patients with jia were examined, 17 of whom had the oligoarticular form of the disease, 7 of them with uveitis, and 9 patients had the polyarticular form of the disease, 3 of them with uveitis. among them, there were 13 (50%) girls and 13 (50%) boys. the mean age was 11.0 ± 3.4 years; the mean disease duration was 4.1 ± 2.2 years. patients received adalimumab (at least 1 year before the study) with concomitant administration of methotrexate (mtx) or adalimumab only -13 children who did not receive mtx for at least 3 months prior to the study as a result of either adverse events of mtx administration (5 patients) or permanent drug remission (8 patients). before starting adalimumab therapy, all participants were treated with mtx. the mean duration of adalimumab treatment for these patients was 1.8 ± 1.0 years. the serum aaa level of antibodies was determined using the enzyme immunoassay (eia) method. this method determines both free and bound antibodies to adalimumab at reference values less than 10 au/ml. a and was used every 2 weeks for 3 months. the values were presented as mean ± standard deviation. data processing and analysis were carried out using pearson's chi-squared test and spearman's correlation test. results: 8 (31%) of the 26 patients enrolled in the study had aaapositive results. the mean aaa level in positive patients was 40.8 ± 20.1 au/ml. further disease relapses tended to occur significantly more often in aaa-positive patients than in aaa-negative ones (χ2 = 5.46, p = 0.019). thus, 5 of 8 (62.5%) aaa-positive children had at least 1 exacerbation of the disease within 3 months, compared with 3 of 18 (16.7%) in aaa-negative ones. 7 out of 8 (87.5%) aaapositives did not take mtx for at least 3 months compared to 6 out of 18 (33.3%) in aaa-negative ones. thus, aaas are found to be significantly more frequent without concomitant administration of mtx in the treatment of jia (χ2 = 6.5, p = 0.01). there were no observed adverse events or side effects during adalimumab therapy. no significant correlation was found between the presence of aaa and sex, introduction: advances on molecular medicine, illumination of the cytokine network and the immune pathways shed light on the etiopathogenesis for a better understanding of juvenile idiopathic arthritis (jia). however, the fact that the course of the disease differs individually strongly suggests the effect of external factors. objectives: the current study was undertaken to evaluate sociodemographic and sociocultural features, parent behavior, the gestation and breastfeeding period, nutritional status of early childhood in our patients with jia, and to determine their relationship with disease activity, damage index, remission time, and relapse rate. methods: the study was conducted with a face-to-face questionnaire method with the parents of 171 patients with jia and 183 healthy children. the medical patient records were reviewed. juvenile arthritis disease activity score (jadas) 27, wallace clinical inactive disease criteria, juvenile arthritis damage index (jadi), and relapse rates were used to assess the general medical condition of each patient. results: the median age of jia patients (n = 171) was 13 (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20) , with a female ratio of 59,1%. age at disease onset was 7 (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) years. the first remission time was 5(1-17) months. the patients were evaluated according to disease subtypes and treatment modalities. there was no difference in the duration of breastfeeding according to the distribution of the subtypes (p = 0,97). when the breastfed and formula-fed patients were compared, there was a marginally significant difference in terms of first remission time (p = 0,05), whereas there was a significant difference in relapse rate in patients who introduced to cow milk early (<12 months) (p = 0,019). the early risk factors and their relationship with the disease are presented in table 1 . both breastfeeding durations and maternal literacy levels showed a significant difference in terms of relapse rates (p = 0,01; p=0,03, respectively). there was no significant difference in breastfeeding durations and gestational risks between the patients and the healthy group (p = 0,1; p= 0,65), respectively. however, the smoking rate among family members was significantly higher in the patient group (p = 0,03). conclusion: in patients with juvenile idiopathic arthritis, breastfeeding rate and duration did not differ when compared to healthy controls. however, breastfeeding duration, cow's milk commence age, and maternal literacy appeared to be relevant to the relapse rates. going to preschool both influence the remission time and relapse rate. these findings suggest a role for parental attitude and nutritional status during early childhood in the course of jia. none declared introduction: immunogenicity and low trough concentrations have been associated with adalimumab treatment failure in several studies of paediatric inflammatory diseases, indicating the possible value of therapeutic drug monitoring (tdm). adalimumab efficacy may be improved by changing dose or treatment intervals based on drug concentrations. however, lack of standardization, assay heterogeneity, and paucity of research hinder the implementation of tdm in clinical practice. objectives: to assess the relationship of trough concentrations, immunogenicity and adalimumab response in paediatric patients with jia. methods: monocentric cohort study of patients ≤18 years with jia treated with adalimumab due to active arthritis. clinical data and plasma samples were collected during routine follow-up. adalimumab trough concentrations were measured by liquid chromatographytandem mass spectrometry (lc-ms/ms). anti-adalimumab antibodies were measured in samples with trough concentrations <5mg/l. disease activity was evaluated using the clinical juvenile arthritis disease activity score with 71 joint count (cjadas71). response to adalimumab was defined as at least 50% reduction of disease activity within 3 months of therapy followed by clinical inactive disease or minimal disease activity after 6 months. the latter was defined as cjadas71 ≤1.5 and ≤2.5, for oligoarthritis and polyarthritis, respectively, or an active joint count equal to zero when cjadas71 was unavailable. results: 36 adalimumab trough samples were available from 35 jia patients. although there was no significant difference in median adalimumab dose, trough concentrations were significantly lower in patients with secondary failure compared to primary failure or an adequate adalimumab response (p-values <0.01). in addition, there were 11 samples with trough concentrations <5mg/l, 9 in the group with secondary failure and 2 in the group with adequate adalimumab response (table 1) . conclusion: adalimumab trough concentrations were significantly lower in jia patients with secondary failure compared to primary failure or an adequate response to adalimumab. anti-adalimumab antibodies were present in 8 out of 11 samples with trough concentrations <5mg/l. adalimumab trough concentration measurements may identify jia patients that would benefit from increased doses or shorter treatment intervals. in addition, jia patients with primary failure and adequate adalimumab trough concentrations may respond better to biologic agents with other therapeutic targets. although biologic agents have improved disease outcome of patients with jia, concentration measurements using reliable and cost-effective methods, such as lc-ms/ms, could further improve efficacy of biologic agents and guide treat-to-target strategies. introduction: most studies of physical fitness (pf) in juvenile idiopathic arthritis (jia) have shown decreased levels of maximal oxygen consumption (vo 2 max) compared to healthy peers. in jia, boys have higher pf-levels than girls and younger children have higher levels than adolescents, congruently with data of healthy peers. previously, we have shown that more than half of jia-patients had below normative average of vo 2 max. however, monitoring physical activity (pa) using accelerometry, 68% of boys and 39% of girls with jia fulfilled the recommendations of who of ≥1 hour of pa per day, which was comparable to normative values (61%/39%). moreover, patients reporting engagement more than 7 hours per week in club-sports exceeded accelerometry values of healthy peers. objectives: to explore the association between pf and specific sport habits in 10 to 16-year-old jia-patients, related to gender, bmi, disease activity, and pain, and comparing the most fit quartile (q4) of patients (respectively boys and girls) to the least fit quartile (q1). methods: sixty patients with jia performed an indirect ergometertest of vo 2 max (watt max test) and answered the physical activity and sport questionnaire (pasq). objective pa-monitoring for one week was conducted using the gt1m accelerometer. cut-offs for moderate-high and high intensity pa were set to >1000 and >2500 counts per minute, respectively. disease activity was assessed with the jadas-27, current pain and worst pain last week were measured on visual analogue scales (vas) and in a one-week pain diary using the faces pain scale-revised (fps-r). results: girls with jia (n=36) had lower mean pf than the boys (n= 24) (36.5±8.2/43.4±6.73 ml/kg/min), being below normative values, respectively. in both genders the most fit boys and girls (q4; 49.3-57/ 40.9-54) had average to well-above normative average pf. the least fit boys (q1; 33.5-37.4) all had pf-levels well-below normative average. in girls q1-levels (18.7-30.9) were well-below to below normative average. we found significant differences between most fit (q4) and least fit (q1) patients regarding patient´s global wellbeing (p=0.040) and pain diary (p=0.026). these differences were not significant when separating genders, though differences were more pronounced in girls. the least fit girls (q1) had significantly higher disease activity (jadas-27) than the most fit girls (q4)(p=0.019). the most fit boys and girls (q4) engaged equally in high intensity sports (soccer: 3/24; 2/36, handball: 0/24; 2/36, gymnastics: 2/24; 4/ 36, rowing: 1/24; 0/36). however, more boys than girls played soccer (11/24; 3/36), whereas more girls preferred sports of lower intensity (riding: 8/36; 0/24). eight of 11 boys in soccer and 2 boys in gymnastics or rowing had below average to well-above normative average of pf (q3+q4: 41.6-57). three girls in gymnastics, 2 girls in soccer, and 2 girls in handball were in q4 (40.9-54) with levels of average to well-above average pf. the third girl in soccer was in q2 (31-36.3) with levels of well-below to below normative average. none of the riding girls were in q4 and only 1 was in q3 (36.3-40.8) (below to average normative pf). comparing accelerometer-monitored values of pa-intensity in girls with low (q1) and high (q4) pf, pavalues of q1 were significantly lower than in q4. the same tendency was observed in boys, but not to significance. conclusion: our results are in accordance with most other studies of pf in jia, adding to the knowledge of gender-specific differences in pf and type and behavior in sport activities. it emphasizes the need of regular pf-testing and guidance in high intensity pa and sport in order to improve pf and avoid the risks of inactivity and lifestyle diseases in jia. pg/ml), which also showed the highest the frequency of detection of its increase. it was absent in sjia (7.52±4.74 pg/ml). the highest values of il-17r (1849836.4±176751 pg/ml) were in the middle age group. the data obtained suggest the compensatory value of increasing il-17r and the simultaneous initiation of inflammatory and anti-inflammatory processes during exacerbation of jia. assessment of the ratios of stimulating and inhibiting cytokines showed in patients with uveitis, the ratio of ifn-γ/il-1β (4379.29 ±476.83) was higher than with other jia (from 60.84 ± 14.92 in ojia to 105.20± 66.01 in pjia) and ifn-γ/il-17r (4474.01 ±3899.19 versus from 20.14± 11.48 in ojia to 934.55±931.37 in sjia). an increase of il-1β/il-17r ratio was characteristic only for sjia (34.12±26.17). all of these ratios increased with disease activity (r=0.22-0.37) & they did not reflect the unpleasant course of the disease. methods: in this multicenter, case-control study, 113 fecal samples were collected from 91 children with jia, with 72 of these samples collected from untreated children (67 of whom were treatment-naïve children). samples from 28 children with jia were collected during treatment with mtx as single treatment and samples from 13 children during treatment with etn. of those 13 children, four were treated with etn as single treatment and nine had a combination of etn and mtx. we compared 28 children on single treatment with mtx with 57 untreated children (52 treatment-naïve), and 13 children on treatment with etn (nine in combination with mtx) with 62 untreated children (57 treatment-naïve). we also did pairwise comparisons of samples taken before any medication was given (n = 22) and samples taken during ongoing treatment with mtx (n = 14) or etn (n = 8, four in combination with mtx). the microbiota was characterized by sequencing amplicons from the v3 and v4 regions of the 16s rrna gene. alpha diversity of the fecal samples was measured using the chao-1 index and the shannon diversity index. to compare these indices between treated children and untreated children, we used a logistic regression model with age at sampling as a covariate. for pairwise analyses, we used the wilcoxon signed-rank test. to analyze the community composition of the microbiota, principal coordinate analysis (pcoa) plots based on bray-curtis distances were generated for visual comparisons, and analysis of similarity (anosim) was used to test for differences. analyses for relative abundances of taxa were performed at three taxonomic levels (phyla, families, and genera), and logistic regression with age as a covariate was used for calculations of differences between treated and untreated children, while the wilcoxon signedrank test was used for pairwise comparisons. significance was set to p < 0.05 and corrections for multiple comparisons were made using the benjamini-hochberg method. results: analyses showed no significant differences in α-diversity between children treated with mtx or etn and untreated children, and pairwise comparisons of samples before and during treatment with mtx or etn also showed no differences. pcoa plots for children treated with mtx or etn, in comparison with untreated children, did not show any clustering. anosim showed no significant differences between treated and untreated children. pcoa plots were also made for the pairwise comparisons of children sampled before and during treatment, and according to that analysis the community compositions of microbiota did not change in any uniform way during treatment with either mtx or etn. furthermor, analyses of relative abundances of specific taxa did not reveal any significant results in any of the comparisons, after adjustment for multiple analyses. conclusion: treatment with mtx or etn did not alter the composition of fecal microbiota in children with jia. introduction: juvenile idiopathic arthritis (jia) is the most common rheumatic disease in childhood and an important cause of shortterm and long-term disability if patients are not treated appropriately. by definition, jia clinically presents with peripheral joint inflammation of unknown origin, persisting for at least six consecutive weeks and starting before the age of 16 years. the predominant subtypes, i.e. oligoarticular (oligo) and polyarticular (poly) jia, have long been assumed autoimmune diseases caused by dysregulation of the adaptive immune system, with a central role for autoreactive t cells belonging to the th1 and th17 lineages and autoantigens that may include aggrecan, fibrillin, matrix metalloproteinase (mmp)-3 and heat shock proteins. nevertheless, the original t cell-centered hypothesis has been challenged since it does not cover nor completely explain the full spectrum of immune-pathological phenomena observed in patients. lien.desomer@uzleuven.b objectives: emerging evidence suggests a potentially important role for neutrophils in jia pathogenesis. here, we investigated extensively the phenotypical features of neutrophils present in the peripheral blood and inflamed joints of jia patients. methods: synovial fluids and parallel blood samples from patients with oligo-or polyjia and blood samples from healthy children were collected. multicolor flow cytometry panels allowed for in-depth phenotypical analysis of neutrophils, focusing on the surface expression of adhesion molecules, activation and maturation markers, chemoattractant-and toll-like receptors. multiplex technology was exploited to quantify pro-and anti-inflammatory cytokines in plasma and synovial fluids. results: the vast majority of synovial fluid neutrophils displayed a strongly activated, hypersegmented phenotype with decreased lselectin (cd62l) expression and increased numbers of nuclear lobes, upregulation of adhesion molecules cd66b, cd11b and cd15 and downregulation of chemokine receptors cxcr1/2. an elevated percentage of cxcr4-expressing aged neutrophils was detected in synovial fluids from patients. strikingly, significant percentages of synovial fluid neutrophils showed a profound upregulation of atypical neutrophil markers, including cxcr3, icam-1 and hla-dr. conclusion: our data indicate that neutrophils present in inflamed joints of jia patients are strongly activated cells with elevated proinflammatory and antigen presenting potential. this detailed molecular analysis supports the notion that a complex intertwining between these innate immune cells and adaptive immune events drives jia. none declared the main factors, associated with incomplete vaccination againts measels, parotitis, rubella and diphtheria in 170 juvenile idiopathic arthritis patients n. lyubimova 1 , i. objectives: the aim of our study was to evaluate the rate and the main factors of incomplete vaccination against measels,parotitis, rubella (mmr) and diphtheria in jia patients. methods: in the present study were included data 170 jia(55 boys and 115 girls)aged from 2 to 17 years,who received scheduled vaccination before the age of 2 years and before jia onset against measles,parotitis,diphtheria and rubella.incomplete vaccination means the reduced number of vaccine to age.in all patients the ig g anti-vaccine antibodies levels were detected with elisa.jia categories were:oligoarthritis -73,polyarthritis -61,systemic-16 and enthesitisrelated arthritis-20.data presented with median and 25%>75% results: incomplete vaccination against mmr was in 50 (42%)diphtheria in 85 (50%) of the jia patients. the main differences in the studied groups are in the table.there were no differences in sex,jia categories and treatment, except biologics compare to complete and complete vaccination in all vaccines.no differences in antimeasels(p=0.18),antiparotitis (p=0.1) and anti-rubella(p=0.17)vaccination between complete and incomplete vaccination group.number of patients with protective level of anti-vaccine antibodies was similar, except parotitis(70% vs 84.2%, p=0.035).the anti-diphtheria antibodies igg level was lower in the patients with incomplete vaccination group (0.07 iu/ml [95%ci:0.03; 0.22] vs 0.2 [95%ci:0.06; 0.4], p=0.001)as well as number of patients with protective level(34% vs 54%, p=0.002). in the multiple regression model only jia onset age(p=0.00001)and methotrexate treatment duration(p=0.003) were predictors of incomplete vaccination against mmr and methotrexate treatment duration(p=0.005) and biologic treatment(p=0.05) for diphtheria incomplete vaccination.incomplete mmr vaccination influence on the maintenance of the protective anti-parotitis level(p=0.036)in regression model.in correlation analysis the number of vaccination influences on anti-diphtheria antibodies level(p=0.017)and number of patients with protective level of anti-diphtheria antibodies(p=0.017). the main predictors in logistic regression for incomplete vaccination for mmr were:onset age<6 years(or=7. 8 conclusion: younger onset of jia age, longer duration of jia and methotrexate treatment, biologics and more than 1 biologics are the main predictors of incomplete vaccination againt mmr and diphtheria. introduction: the prevalence of autoimmune thyroid disorders (aitd) has been reported to be higher in patients with juvenile idiopathic arthritis (jia) in comparison to the general population. nevertheless, there is a lack of studies investigating risk factors for aitd development in children with jia. objectives: to investigate the co-occurrence of jia and autoimmune thyroiditis in southern italy and to identify potential predisposing factors to anti-thyroid antibodies (ata) positivity in a jia population. methods: a single-centre retrospective study was conducted. all jia patients admitted to the pediatric rheumatology unit of the university of naples federico ii, from january 2001 to december 2019, tested for ata at least once and with a minimum of 6-months follow-up, were included. for each patient, demographic, clinical and laboratory data were extracted from clinical charts. differences between patients affected by jia with or without ata were analyzed. results: three hundred thirty jia patients (247 females; median age 12.5 years, iqr 9.1-16.1) were included in study. median age at jia onset was 4 years (iqr: 2.2-7.8). twenty-three patients [7% (95% ci 4.5-10. 3)] presented ata positivity. twenty-one of them (91.3%) were females. anti-thyroperoxidase was positive in 18/23 patients (78.2%) while 12 patients presented anti-thyroglobulin positivity (52.1%). both antibodies were present in 8/23 (34.8%). 19 patients showed the typical ultrasound findings of autoimmune thyroiditis, resulting in a prevalence of hashimoto's thyroiditis of 5.7% (95% ci 3.5-8.8) in our cohort. three female patients developed subclinical hypothyroidism, whereas one male patient presented subclinical hyperthyroidism. the remaining 19 patients were euthyroid. no statistically significant difference was observed in regard to age of jia onset, follow-up duration and jia subtype between the patients with or without ata. the proportion of females was marginally significantly higher (p=0.059) in the group with ata positivity compared to children without thyroid antibodies (91.3% vs 73.6%, respectively). 56.5% of patients with ata showed ana positivity compared to 37.5% of patients without ata (p=0.07). family history for aitd was significantly higher in children with thyroid antibodies positivity (p= 0.01). anti-tnf-alpha inhibitors were administered in only 3 children (13%) with thyroid antibodies before their detection compared to 35 .5% of patients without thyroid antibodies (p=0.028). multivariate regression analysis showed that patients with a family history for aitd were about four times more likely to develop ata (or 3.75, 95% ci 1.401-10.017, p=0.008) and confirmed that ata positivity is less likely to occur in patients undergone anti-tnf-alpha therapy (or 0.127, 95% ci 0.031-0.518, p=0.004). conclusion: a high prevalence of ata positivity and hashimoto's thyroiditis in patients with jia was found in our wide southern italian cohort. as expected, a positive family history of aitd was found to be associated with a higher risk to ata development during the follow-up. this finding supports the usefulness of an active screening for aitd in jia children, in particular in patients with relatives affected by thyroid disorders. notably, patients treated with tnf-alpha inhibitors resulted less likely to develop thyroid antibodies. further studies are needed to investigate the effect of anti-tnf-alpha therapy on thyroid autoimmunity in jia. introduction: the knee is considered by far the joint most frequently affected at jia onset. nonetheless, jia onset may present with unusual musculoskeletal involvement, eventually leading to a delay in the diagnosis and treatment. objectives: to identify the type and number of musculoskeletal sites affected at jia onset in consecutive patients seen at the study center in an 8 years period. methods: records of patients with new diagnosis of jia from june 2012 to may 2020 available information in the medical history and standardized joint assessment at diagnosis, were retrospectively reviewed. systemic jia subtype according to ilar classification criteria were excluded. demographic and clinical features, including the type and number of joints at disease onset and diagnosis, were registered. data were analyzed through descriptive statistics. results: of a total of 333 caucasian patients included in the study (75.7% females), 241 patients (72.4%) had oligoarthritis, 79 (23.7%) rf-negative polyarthritis, 7 (2.1%) rf-positive polyarthritis, 1 (0.3%) psoriatic arthritis, 5 (1.5%) enthesitis-related arthritis (era). antinuclear antibody (ana) were positive in 188 patients (56.5%). the median age at onset was 4.8 years (iqr 2.3-9.3). at diagnosis 103 (30.9%) patients had only 1 active joint, 143 (43.0%) had 2-4 active joints, 87 (26.1%) had ≥ 5. as expected the knee, the tibiotalar and the wrist were the most frequently affected joints (77.2%, 41.1%, 21.0%, respectively); cervical spine was involved only in patients with polyarthritis (n=13). notably, of 103 patients with monoarthritis at diagnosis 98 presented with large joints involvement, among which n=2 isolated elbow and n=2 isolated wrist, and 5 with small joints involvement (table 1) . no sufficient data were available regarding the involvement of tendons and bursae, since the standard joint assessment form did not include them. nonetheless, additional 4 patients, not included in the sample analysis, had isolated tenosynovitis involvement at diagnosis (n=1 both-sided ulnar extensor tendons; n=2 isolated tenosynovitis of the flexor digiti proprius; n=1 tenosynovitis of 2 flexors digiti proprii). conclusion: our study confirms the knee, the tibiotalar and the wrist as the most frequently affected joints at jia diagnosis. on the other hand, musculoskeletal sites, such as small joints of hands and feet, the hip and the shoulder, usually involved in polyarticular jia, can be the site of disease presentation in oligo-and also mono-articular jia. further, jia may present with isolated tendon involvement. our results foster not to delay jia diagnosis in persistent synovitis occurring in infrequent joints and to include musculoskeletal sites, other than joints, in the standard articular evaluation. this could be realized by merging clinical and imaging (i.e. ultrasound) musculoskeletal examinations in the same assessment. none declared introduction: treatment response in jia is currently viewed as a binary outcome: response or non-response. however, jia is a heterogeneous disease and it is likely that different, identifiable subgroups of children and young people (cyp) may demonstrate different patterns of disease following treatment. identifying these response subgroups can assist the tailoring of stratified treatment approaches in jia. objectives: to identify subgroups of cyp defined by different trajectories of juvenile arthritis disease activity score (jadas) components following methotrexate (mtx) initiation for jia. methods: mtx-naïve cyp with jia were selected if enrolled prior to january 2018 in the bspar etanercept cohort register or the biologics for children with rheumatic diseases study at point of starting mtx. jadas components (active joint count, physician's global assessment (pga, 0-10cm), parental global evaluation (pge, 0-10cm) and standardised esr (0-10) were calculated based on data collected in the year following mtx initiation. multivariate group-based trajectory models were used to explore mtx response clusters across the different jadas components, which were log1p transformed for analysis. optimal models were selected based on a combination of model fit (bic, relative entropy, average posterior probabilities), parsimony and clinical plausibility. clinical and demographic characteristics and achievement of acr pedi 30/90 by six months were compared across identified groups. results: of 658 cyp selected, the majority were female (68%) and of white ethnicity (86%), with rf-negative jia the most common disease category (35%). six subgroups of cyp were identified with differing patterns of disease activity following mtx initiation. two groups improved across all jadas components: fast improvers (11%), and slow improvers (16%). persistent pga (8%), and persistent pge (13%) groups maintained one persistent disease feature but otherwise improved. one group relapsed (7%) and a final group had persistent disease overall (44%). there were no differences in active joint counts at mtx initiation between subgroups and all ilar categories were represented across each subgroup. however, cyp in persistent disease and slow improver groups had higher chaq, pga and pge scores at mtx initiation. those with persistent disease were also older at mtx initiation. the majority of cyp fulfilled acr pedi 30 response (>60% across every group). acr pedi 90 achievement was low at 6 months for slow improvers (30%) and high in the relapse group (68%). between 41% and 73% achieved acr pedi 90 response in groups with persistent disease in one jadas component. we identify different patterns of disease activity within cyp initiating mtx, suggesting a simple responder/non-responder analysis at a set point may be over-simplistic. commonly used response measures did not adequately describe these heterogeneous response patterns. understanding both clinical factors associated with, and biological mechanisms underpinning, these subgroups would aid stratified medicine in jia. introduction: despite modern treatment and improved disease control, pain is the most common complaint in juvenile idiopathic arthritis (jia). knowledge about pain thresholds and pain sensitivity among young adults with jia is sparse. objectives: to study pressure pain thresholds (ppts) in young adults with jia, 16 years after disease onset compared with controls. methods: consecutive newly diagnosed children with jia and a disease onset between 1997-2004 from central norway, were included in this prospective population-based long-term follow-up study. children with onset 1997-2000 were part of the nordic jia cohort 1,2 . age-and sex-matched controls were drawn from the national population register of norway. inactive disease and remission were defined according to wallace 3, 4 . at the follow-up between 2015-17, data from a clinical examination and blood tests were included in addition to an investigator-blinded quantification of ppts. a digital algometer was used to manually apply pressure three times with an even rate at the upper and lower limb. ppts from jia and controls, and from subgroups of jia defined by disease status, were compared with multilevel models in stata. results: among the 96 participants with jia, 71% were female, median age was 22.7 (iqr 18.7-26.2) years, median disease duration was 16.1 (iqr 14.2-17.1) years, 47% had an oligoarticular disease (persistent or extended), and 45% were in remission off medication. in the control group, 71% were female and median age was 23.5 (iqr 20.2-26.7) years. results from the multilevel regression model showed significantly lower ppts among participants with jia compared to controls (table 1 ). in the jia group, participants with inactive disease had lower ppts than both jia in remission off medication and jia with active disease ( table 1 ). the results were consistent for both upper and lower limb. conclusion: in this long-term follow-up study of young adults with jia, we found significantly lower ppts compared to controls. this may indicate that young adults with jia have altered pain sensitivity possibly due to accumulated earlier pain experiences. introduction: juvenile idiopathic arthritis (jia) represents the most common chronic rheumatic disease in childhood. non-steroidal antiinflammatory drugs (nsaids) and intra-articular steroids are the first line treatment for jia. systemic steroids, disease modifying antirheumatic drugs (dmards) and biologic drugs are used in children with severe disease. it is not possible at onset of disease to predict when a child can suspend pharmacological treatment, so children affected from jia have to continue pharmacological treatment for several months or years. anecdotal reports showed that rarely jia could present renal involvement due to uncontrolled inflammation or to long exposure to drugs. objectives: because no cohort studies investigating renal injury in children with jia are available, we designed this kind of study in our population. methods: we retrospectively evaluated 110 patients suffering from jia. jia diagnosis was made according to ilar criteria, treatment was assigned with acr recommendations. for each patient we recorded the type and the duration of pharmacological treatment and the presence of renal injury. renal injury was defined by the presence of hypertension (systolic and/or diastolic blood pressure >95 th percentile for age, sex and height), proteinuria (persistentconfirmation within 3 months-urinary protein/creatinine ratio>0.5 mg/mg for children <2 years old and >0. 2 introduction: juvenile idiopathic arthritis (jia) is a pediatric rheumatic disease with partially unknown etiology and pathogenesis. neutrophils are the most common immune cell present in synovial fluid from inflamed joints in oligoarticular jia, but the role of neutrophils in the immunopathogenesis of oligoarticular jia has not been investigated. objectives: to characterize neutrophil phenotypes and effector functions in the circulation and in the inflamed joint during active arthritis in children with oligoarticular jia. methods: paired samples of blood and synovial fluid from 17 children with oligoarticular jia were investigated regarding surface markers, phagocytic ability and oxidative burst. healthy blood neutrophils exposed to cell-free jia synovial fluid and healthy oral cavity neutrophils were studied as controls for synovial fluid exposure and transmigration respectively. results: synovial neutrophils had a shifted phenotype, characterized by high surface levels of neutrophil activation markers cd11b and cd66b, and mannose receptor cd206 and decreased levels of adhesion molecule cd62l compared to circulating neutrophils. in comparison to oral cavity neutrophils, synovial neutrophils had higher levels of cd11b and a different overall phenotype, suggesting that the phenotype shift in synovial compared to circulating neutrophils is not dependent on transmigration alone. jia synovial fluid in itself induced activation of healthy blood neutrophils, measured as increased cd11b levels. synovial fluid neutrophils had impaired ability to phagocytose opsonized e. coli and to produce oxygen radicals upon neutrophil activation with phorbol-myristateacetate (pma) compared to circulating neutrophils. the impaired effector functions in synovial neutrophils was not dependent on the synovial fluid alone, as addition of cell-free synovial fluid to blood neutrophils did not alter phagocytosis and oxidative burst. conclusion: jia synovial fluid neutrophils are activated, have a "polarized" phenotype and impaired effector functions compared to neutrophils in the circulation. this study will help bridge the current knowledge-gap regarding the role of neutrophils in the immunopathogenesis in oligoarticular jia. methods: a case report is described. data was extracted from the medical chart of the patient and a literature review was undertaken. results: a 7-year-old girl was transferred to our tertiary center after being admitted for prolonged intermittent fevers, abdominal pain, fatigue and polyarthralgias. on examination, there was symmetrical proximal muscle weakness, a vasculitic lower limb rash, facial erythema with eyelid edema (fig. 1 ) and oral mucositis. initial laboratory exams revealed pancytopenia, high muscle enzymes, increased erythrocyte sedimentation rate with moderately elevated reactive c-protein, and hypocomplementemia. she also had non-nephrotic proteinuria, without hematuria.further investigations showed a positive direct antiglobulin test, antinuclear antibodies, antidouble-stranded dna, anti-mi 2 and anti-ku. serositis (pericardial and pleural effusions, ascitis) and hepatosplenomegaly were present. lower limb mri documented diffuse muscle edema. the diagnosis of an overlap syndrome of jsle and iim was established. while being treated for concomitant bacteremia, the patient became ill-appearing, with persistent fevers, worsened cytopenias, low fibrinogen and high ferritin and triglycerides, and a macrophage activation syndrome (mas) diagnosis was assumed. the patient received antibiotics and intravenous immunoglobulin, followed by methylprednisolone pulses, iv cyclosporine (cyc), hydroxychloroquine and supportive therapy with progressive improvement. due to hypertension (possibly related to cyc) and persistent proteinuria a renal biopsy was performed showing class iv lupus nephritis. after achieving clinical stability, cyc was switched to mycophenolate mofetil as an induction treatment, which is ongoing. conclusion: imm with sle os is uncommon, and has seldom been described in children. in addition to fulfilling sle criteria, our patient had clinical, laboratory and imagiologic evidence of imm. the presence of myositis specific antibodies (especially anti-mi 2) further supports the diagnosis of an os rather than an atypical presentation of a lupus myopathy. juvenile dermatomyositis appears to be the imm subtype -it is associated with anti-mi 2, and mild heliotrope and eyelid edema are compatible. facial rash sparing the nasolabial folds is more suggestive of sle. mas is a rare but life-threatening condition that should be suspected in rheumatologic conditions and might be triggered by infections or disease flares. its identification may be particularly challenging at presentation, especially in sle where cytopenias are common. the reported prevalence in adult sle ranges from 0.9% to 4.6%; disease-specific criteria have been proposed. mas has occasionally been described in iim. in a patient with a predisposing condition, persistent fevers and illappearance must always prompt a mas workup, since early diagnosis and treatment are paramount. due to an early referral to a pediatric rheumatology center, the patient received a prompt diagnosis and treatment, which probably improved her prognosis. results: four of the five patients were female (80%) and all aged between 6 and 10 years. four of them had calcinosis at the time of diagnosis, although they may have had symptoms for 12 to 18 months prior to diagnosis. skin involvement was severe requiring multiple systemic and topical therapeutic agents in four out of the five patients -significantly more affected than the muscles. one patient had amyopathic subtype with normal childhood myositis assessment score (cmas) throughout. none of them had cardiac involvement. all had weakly positive anti-nuclear antibodies (ana); but were negative for myositis antibodies except the patient with most severe skin involvement and calcinosis (patient 2), who was positive for anti-tif1gamma antibodies. two of the three patients with calcinosis at onset had cyclophosphamide as the second line agent (chosen due to calcinosis) following systemic corticosteroids with complete resolution of the lesions after six cycles at 500mg/m2. one patient responded to infliximab, which failed to work after 20 months, following which cyclophosphamide was tried with good response. the other two patients were given cyclophosphamide after they failed to respond to rituximab, which did work for muscle disease. one patient had recurrent episodes of calcinosis needing surgical curettage despite initial response to cyclophosphamide and later ivig. introduction: systemic juvenile idiopathic arthritis (sjia) is a unique form of childhood arthritis. according to current understanding sjia is primarily driven by innate immune mechanisms at disease onset, but can progress towards chronic destructive arthritis, which can involve t cellular immunity. for yet incompletely understood reasons, sjia can be complicated by macrophage activation syndrome (mas), a severe hyperinflammatory condition characterized by a catastrophic cytokine storm resulting in multiple organ failure and high mortality. objectives: the sjia/mas working party (wp) aims to promote knowledge and international multidisciplinary collaboration among experts in the field of mas and sjia and to foster translational research in order to improve the care and outcome of these patients methods: currently 60 pres members participate to the mas/sjia wp. the wp arranges an annual meeting during the pres congress, open to all members activities. the mas/sjia wp core team frequently report about ongoing activities by email. results: several studies are currently ongoing. a project aimed to establish and validate a risk score for mas in sjia patients using routine laboratory parameters of disease activity and severity has already completed the construction phase. recently, building of a validation cohort comprising data form 182 patients from 10 paediatric rheumatologic centers has been accomplished and is awaiting analysis (claudia bracaglia). a second project focused on mas patients with systemic thrombotic microangiopathy (tma) has just completed the collection of 27 patients with mas and tma from 18 centers in 9 countries and results will soon be published (francesca minoia). furthermore, the mas/sjia wp participated in the data collection phase of a project on the development of new criteria for primary hlh (jan-inge henter and annacarin horne introduction: hemophagocytic lymphohistiocytosis (hlh) is an immunological disorder characterized by clinical signs and symptoms of severe uncontrolled inflammation, due to massive release of inflammatory cytokines. a delay in diagnosis is common, and is one of the factors that determine the poor outcome. hlh is classified into primary (phlh) and secondary (shlh). it is important to differentiate between the two as management differs. objectives: to describe the clinical and laboratory profile of hlh in infancy. methods: the electronic case files of children (age<1 year) diagnosed with hlh at the aims, kochi, kerala, between january 2012 and december 2019, was retrospectively reviewed and described. results: eight infants, with age range 1.5 months to 7 months, were clinically diagnosed with hlh. all were immunised and had normal development for age. none had a family history suggestive of hlh. third degree consanguinity was present in parents of patient no.5 and second degree for patient no.7. duration of symptoms before presentation ranged from 2 days to 68 days. duration of follow up with us ranged from 12 days to 192 days, for those who expired. all, eight of them, had fever, anemia, thrombocytopenia, hyperferritinemia, transaminitis, raised ldh and crp. lymphadenopathy was present only in patient no.4. before starting specific treatment patient no. 7 had pseudomonas sepsis, patient no.5 had roseomonas gilardii infection; patient no.3 and 4 were igm cmv positive but their pcr was negative. both of them had received prior blood transfusion. before making a definitive diagnosis of hlh patients were treated for puo, sepsis ? cause and acute liver failure. there was a delay in diagnosis for all patients except patient no.7. all of them were treated with hlh 2004 protocol with modification according to clinical status of the patient. later, broad spectrum antibiotics, antifungals and antivirals were used for all. anakinra was tried for patient no.5. five patients (phlh) succumbed to sepsis and mods and three (one phlh and two shlh) are continuing follow up. hsct was not done in any of them. other clinical features are shown in table 1 . conclusion: making a timely diagnosis of hlh is difficult. differentiating phlh from shlh is very important as the management differs. genetic testing should be done for all infants with hlh. negative genetic study doesn't rule out phlh. the only curative treatment for phlh is hsct. shlh infants, once their primary condition is treated, can have normal survival. hyperbilirubinemia, splenomegaly, neutropenia, hepatomegaly, tissue hemophagocytes and hypertriglyceridemia were more common in phlh. health, kolkata diagnosed as having mas, admitted between july 2008 and april 2020 was tabulated and retrospectively analyzed . objectives: to evaluate the clinical features, laboratory findings and outcomes in pediatric mas, assess the response to different pharmacological therapies, and finally to identify possible factors associated with an unfavourable outcome. methods: the data of patients diagnosed with mas over the study period was analyzed for the clinical and laboratory features, treatment details, response to therapy and outcome. results: 35 patients were diagnosed as having mas. primary illness was sjia in 29 (82%), sle in 5 (14%) and kawasaki disease (kd) in 1(4%). all had fever with varying degrees of multi systemic involvement. hyperferritinemia was universally present. in the absence of anakinra in india, pulse methylprednisolone with cyclosporine was used for treating the majority.10 patients (28.5%) expired. patients on biologics and steroids can present with a silent mas which may be difficult to diagnose. conclusion: mas is a near fatal complication with protean manifestations and multi organ dysfunction. hyperferritinemia is characteristic, higher values being associated with increased mortality. patients resistant to steroids and cyclosporine had a poor prognosis. early recognition with aggresive management forms the backbone of a successful outcome as reflected by improved prognosis over successive years. late presentations with multiorgan dysfunction are associated with the poorest outcomes. methods: case report's description results: a two-year-old boy presented with one month history of fever associated with limping gait, cervical lymphadenopathy and skin rash. laboratory tests showed elevation of inflammatory markers and ferritin. by exclusion criteria, sjia was diagnosed and steroid therapy started. after a soft tissue bacterial infection, fever relapsed and laboratory tests were consistent with mas (day 1): hb 8.5 g/dl, plt 44000/mm3; fdp 1522 ug/l, crp 100 mg/l, ferritin 2200 ug/l. high doses intravenous metilprednisolone and oral cyclosporin a (csa) were started. on day 2 he presented a systemic capillary leak syndrome and acute myocarditis. he was admitted into the pediatric intensive care unit (picu) where intravenous immunoglobulin and subcutaneous anakinra (ana) were added. on day 4, due to an introduction: sjögren's syndrome is a systemic autoimmune disease characterized by dry syndrome and lymphocytic infiltration of the exocrine and extraglandular glands. pulmonary involvement in primary sjögren's syndrome occurs in 9-20% of patients, with very heterogeneous manifestations, and occasionally as an initial mani-festation¹. diffuse interstitial lung involvement is one of the most characteristic pulmonary manifestations and the most frequent subtypes in lung biopsy are interstitial lymphocytic pneumonia and nonspecific interstitial pneumonia². objectives: 14-year-old girl presented to our hospital because of bilateral interstitial involvement with ground glass areas in lower lobes of both lungs on thorax and abdominal ct scan after for kidney stones follow-up. the patient had grade 1 mmrc dyspnoea and dry cough but denied having symptoms of arthralgia or arthritis, photosensitivity, oral and genital ulcers, raynaud's phenomenon or episodes of dry mucosa. she had no history of autoimmune disease nor family antecedents of any autoimmune disease. a physical examination disclosed no finger clubbing or swollen superficial lymph nodes but indicated crackles on pulmonary auscultation. laboratory work showed elevated acute phase reactants, positive rheumatoid factor, positive antinuclear antibodies (1/ 40), positive cytoplasmic antineutrophil antibodies (1/320) and igg and iga hypergammaglobulinemia. an examination for autoantibodies were negative for anti-ss-a, anti-ss-b, anti jo-1, anticentromere and anti-scl-70 antibodies. iontophoresis with pilocarpine and 6-minute walk test was also normal. pulmonary function tests demonstrated a mild restrictive impairment and a reduced percent diffusion capacity for carbon monoxide of 55%. fibreoptic bronchoscopy showed acute inflammation in bronchial mucosa. flow cytometry of bronchoalveolar lavage and cytology showed lymphocytosis with a 15% of cd4 and 85% of cd8 lymphocytes in bronchoalveolar lavage fluid. finally, a transbronchial lung biopsy lead to a definitive diagnosis, showing mixed interstitial inflammation and lymphocytic follicular hyperplasia with formation of germinal centers, suggestive of a lymphoid interstitial pneumonia of unreleased autoimmune etiology. throughout time, the patient reported progression of her symptoms with increasing dyspnoea, persistent dry cough, xerostomia and arthralgia. schirmer and rose bengal dye test were negative, and a salivary gland biopsy showed interstitial plasmacytosis and no igg4 plasma cells expression which suggested sjogren's disease. a high resolution computerized axial tomography was requested, suggesting organizing pneumonia in the context of sjogren's disease. methods: several studies indicate that lung involvement in sjögren is more frequent in advanced stages of the disease and rarely as an initial manifestation. sjögren's syndrome in paediatric age is rare and the subtype of secondary sjogren's is the most common. the course is longer, and the symptoms are more heterogeneous than in adulthood 5 . the diagnosis in children is delayed, because children less frequently report dryness and frequently present with extraglandular clinical features suggestive of other autoimmune diseases. a systematic review on primary sjögren's syndrome in male and paediatric population reported a 2.4% of pulmonary involvement in paediatric patients. 6 pulmonary involvement is associated with an increase in the mortality of patients with sjögren's, therefore, it is essential to periodically monitor patients with respiratory symptoms, making an early diagnosis and treatment of the disease. results: -conclusion: we present a case of a patient with childhood sjögren's disease with atypical onset of disease with lung involvement. introduction: sarcoidosis is a multi-system disorder. little is known about its pathogenesis. in children, the early onset sarcoidosis phenotype including blau syndrome is more often seen. 1, 2 the diagnosis of sarcoidosis is confirmed by demonstrating a typical non-caseating granuloma on a biopsy specimen. other granulomatous diseases should be excluded, in particular mycobacterial infections, crohn's disease and immunodeficiencies. the clinical presentation may vary depending on the organs involved and the age of the patient. 3, 4 objectives: we are reporting the case of a boy with a presentation of bone sarcoidosis at a young age. this is a rare phenotype in children. methods: clinical details were retrospectively collated using routine clinical records. confirmation of diagnosis was confirmed with bone biopsy. results: a 5 year old non-identical twin boy of ghanaian descent born in the uk had a slowly growing, painless frontal bone mass which started to develop from 7 months of age. he was developmentally normal, with no history of fever, rashes or joint pains. examination findings revealed frontal bossing while the remainder of the musculoskeletal examination was normal. there was no evidence of rashes, hepatosplenomegaly and ocular examination was normal. the patient was initially referred for neurosurgical review with suspected fibrous dysplasia, after an initial mri scan of the head revealed abnormal marrow signal and expansion of the frontal bone, with no soft tissue swelling. however, the ct scan of the calvarium was not suggestive of fibrous dysplasia. consequently, bone biopsy was performed demonstrating inflammation with granuloma formation. he was referred to infectious diseases and rheumatology. there was no travel history and no tb contact. quantiferon tb was negative. infectious work-up was negative especially for mycobacterial infections. rheumatology work-up identified on skeletal survey another bone location: a well-defined lytic lesion in the right distal fibula that was biopsied. infection cultures and pcr were negative. histopathology identified fibrous tissue and poorly formed granulomas. laboratory investigations revealed a mild microcytic anaemia with iron deficiency and eosinophilia. he had normal serum calcium and vitamin d and his esr was 25 mm/hr. ana, anca and rheumatoid factor were negative, and complement c3 and c4 were normal. his serum angiotensin converting enzyme (ace) level was raised at 125 nmol/ ml/min (normal <40 nmol/ml/min). investigations revealed mild renal impairment with normal urinary tests including normal calcium, protein and tubular proteins. ultrasound of the kidneys was normal. chest x-ray was normal. lung function was performed and was normal. dlco couldn't be performed due to low lung volume. vascular and inflammation genetic panel identified a variant in the nemo gene. functional studies excluded nemo deficiency and patient did not display any of the clinical features. however, a pattern of dysregulated t cells response was identified. he was treated with oral steroids and methotrexate. the oral steroids were successfully weaned off. he has been successfully treated with methotrexate 10mg s/c to initially stabilise disease with no bone growth, and had no significant side effects. repeat mri 2 years later showed increased burden of disease with other newly affected sites however, including the right femoral diaphysis and signal changes in the left tibial metaphysis. based on the mri and increasing musculoskeletal pain, decision was made to escalate to anti-tnf (adalimumab) with good clinical response. conclusion: bone sarcoidoisis is rare in children but this should be considered in the differential diagnoses when granulomatous inflammation is identified on histopathology. response to steroids and methotrexate is usually good but some patients will need escalation to anti-tnf. the most worrisome non-rheumatic condition causing persistent night pain in children which closely mimics arthritis is malignancy 1, 4 . it is vital to pick up subtle clues at an early stage especially in absence of hematological manifestations , organomegaly and lymphadenopathy. to reveal early clinical clues in pediatric patients with predominant musculoskeletal (msk) night pains who were initially diagnosed as suffering from some form of chronic arthritis but ultimately turned out to be affected by malignancy. methods i gathered a data of five pediatric patients fulfilling above mentioned criteria who were seen at dev children's hospital between january 2019 and march 2020. it included demographics, clinical presentation and laboratory results. all above cases reemphasize the need for an extremely detailed history pertaining to characteristics of pain & pattern recognition in pediatric rheumatology. prolonged fever , persistent msk night pain, persistent limp, upper limb and hip joint involvement which is unlikely for jia at onset are proven to be the earliest subtle clues which should not be missed. 1 other constitutional symptoms, respiratory, cardiovascular, ophthalmologic or osteoarticular involvement were absent. growth was unaffected. auditory tests were normal. systemic antibiotic treatment and local steroids were ineffective. laboratory findings were unremarkable, with only mild elevation of esr (28mm/1 st hr). ana and anca were absent in repeat meausrements (3 months intervals). cardiovascular disease was excluded. abdominal us was normal. on the basis of relapsing bilateral auricular chondritis and confirmatory histological findings revealing inflamed cartilage from the pinna of the ear with chondrocyte degeneration, perichondrial infiltrates of lymphocytes, plasma and polymorphonuclear cells and replacement of cartilage with fibrous tissue perivascular infiltrates of polymorphonuclear cells and lymphocytes, relapsing polychondritis was diagnosed. one month nsaids trial, pending histology results was ineffective. methotrexate sc and steroids 1mg/kg/d gradually tapered over a 3-month period were given with significant improvement of auricular inflammation and normalization of markers of inflammation. auricular chondritis worsened after steroid withdrawal and adalimumab was added to treatment with significant improvement of auricular inflammation in 2 months. in the following 8 months auricular chondritis relapsed during uris with mild elevation of esr (25mm 1st hr) and crp (13 mg/l). after 15 months of treatment, in an effort to prolong the intervals of adalimumab administration, bilateral auricular chondritis relapsed. after 24 months of mtx and 21 months of adalimumab administration inflammation was put in complete remission. the following year no flares or involvement of other systems were observed, under methotrexate and adalimumab treatment. conclusion: in this patient isolated auricular relapsing polychondritis was unresponsive to nsaids. steroids and methotrexate greatly improved inflammation but did not induce complete remission. complete remission was achieved by addition of adalimumab to methotrexate treatment, which also allowed for steroids discontinuation. none declared first ever single center study revealing spectrum of rheumatic diseases in 114 children from an indian state of gujarat d. b. pandya, on behalf of dr mehul mitra, pankaj buch, sonal shah, there is very limited information and awareness about pediatric rheumatic and immunodeficiency diseases amongst primary physicians 1, 2, 3 in gujarat and to make this matter even worse, we are not having a single exclusive pediatric rheumatology and immunology centre for a population of around 60 million. to guesstimate a status of children with rheumatic and immunodeficiency diseases in gujarat and spectrum of these diseases at dev children's hospital. methods i gathered a retrospective data of 174 patients who attended dev children's hospital between january 2019 and january 2020. out of these, 114 children with confirmed diagnosis of inflammatory rheumatic diseases and suspected primary immunodeficiencies were included. patients with non-inflammatory musculoskeletal(msk) pains and non-rheumatic diseases causing msk pains were excluded. my collected data included referral details, demographics, clinical presentation, laboratory results and diagnosis. majority of the cases were referred by pediatricians, orthopedicians, hemato-oncologist and general physicians. main reasons for referral were joint involvement , undiagnosed fever , multisystem disease and elevated inflammatory markers. many physicians had put a diagnosis like rheumatoid/rheumatic arthritis, autoimmune disease or connective tissue disease. almost 80% of patients had been evaluated with rf, aso titer, ana and joint imaging irrespective of clinical pattern by their primary physicians before referral. fever , msk involvement, extreme fatigue, constitutional symptoms, skin and mucosal involvement were prominent complaints noted by me. family history of rheumatic, primary immunodeficiency (pid) or consanguinity was found in 1/3 of patients. anemia of chronic disease, elevated esr and thrombocytosis were almost universal laboratory findings in our cohort. rheumatic diseases in children are not anymore rare but due to lack of expertise and awareness , these children are not getting diagnosed. many cases were advised unnecessary rheumatological investigations even before referral. results: a 10-year-old female patient was referred to the rheumatology clinic at our hospital with a previous history of fever of 39°c (102.2ºc), loss of appetite, and acute polyarthritis of wrist, knees, and ankles. at that time, laboratory exams revealed a hemoglobin of 11.1 g/dl, c reactive protein 78.6 mg/l, and antistreptolysin o titers of 400 ui/ml (normal range <200ui/ml. clinical symptoms were relieved only after using nsaids. after 6 months, the patient returned to our hospital with a 7-month history of weight loss and claudication related to pain and daily morning stiffness (15 minutes) on her right ankle. new laboratory findings demonstrated positive antinuclear antibodies 1:320, negative rheumatoid factor, and alpha-1-acid glycoprotein of 171 mg/dl (normal range: 44-113mg/dl). clinical signs suggestive of chronic arthritis with exuberant swelling of the ankles were observed on physical examination (figure a). she was screened for tuberculosis (tb) and had a positive (18mm) tuberculin skin test (figure b). chest ct revealed infiltrative soft tissue mass in the posterior mediastinum, with homogeneous contrast enhancement (figure c). magnetic resonance imaging of both ankles was performed and demonstrated bilateral and symmetrical tibiotalar arthritis and prominent tenosynovitis of extensors, flexors, and fibularis tendons (figure d). right ankle synovial biopsy revealed no granulomas and joint fluid culture was negative for mycobacterium tuberculosis, confirming reactive arthritis (poncet's) and tenosynovitis, that may follow mycobacterial infection with no infective agent in the joints. conclusion: to our knowledge, there is no report of poncet's disease associated with inflammatory tenosynovitis, showing the particularity of this case. the patient's symptoms resolved after two months of anti-tb therapy. introduction: cacp is characterized by congenital or early-onset camptodactyly (usually bilateral); non-inflammatory arthropathy (more frequently in the wrists, knees, ankles, elbows, and hips); coxa vara (reduction of the angle between the neck and shaft of the femur); and non-inflammatory pericardial effusion (a late manifestation, less frequently reported). recognizing the radiological aspects of this syndrome and differentiating it from jia is crucial since cacp has no effective treatment and jia is usually treated with nsaids and methotrexate (2, 3) . objectives: to report a rare case of cacp syndrome mimicking jia. methods: case report and literature review. results: a 5-year-old male patient presented with arthropathy characterized by painless progressive swelling and restricted movement of the hands, hips, knees, and ankles since the first year of life. he had a family history of camptodactyly from his paternal grandfather. on physical examination, symmetric camptodactyly of the hands and feet was observed (a). he had no history of rash or weight loss and inflammatory markers were unremarkable. the echocardiogram was normal. the pelvic radiograph showed a widening of the joint space and bilateral coxa vara. magnetic resonance imaging (mri) of the hips (b) and knees (c) was performed and depicted large joint effusions (arrows, b and c) with normal synovial thickness and mild synovial enhancement in all joints, without bone marrow edema-like signal. a synovial biopsy of the knee was performed and revealed mild synovial hyperplasia without inflammatory cells. the patient was diagnosed with camptodactyly-arthropathy-coxa vara-pericarditis syndrome (cacp -omim 208250), a recently described genetic disorder with no gender predominance identified to date (1). conclusion: an important differential diagnosis of cacp is juvenile idiopathic arthritis (jia), a painful inflammatory chronic arthritis that can cause not only joint effusions due to synovial inflammation, but arthritis was the most frequent extraglandular manifestation. renal tubular acidosis represented the typical expression of renal involvement (19 cases). neuromyelitis optica and aseptic meningoencephalitis (6 and 9 cases, respectively) were the most typical neurologic manifestations. two cases of interstitial lung disease and one of pulmonary hypertension were reported. almost all patients had autoantibodies, mostly ana (200/224 patients) and anti-ssa/ro (170/208 patients). the schirmer test was performed in less than half of the patients, of whom 62% tested positive. a positive result of minor salivary biopsy was reported in 129/140 cases with available data. juvenile idiopathic arthritis was the most frequently associated disease, followed by systemic lupus erythematosus (16 and 8 cases, respectively). no significant differences between patients with or without parotitis were found except that patients with parotitis showed increased levels of crp more frequently than those without it (p= 0.00). patients with anti-ssa/ro had more frequently a positive schirmer test (p= 0.04). the presence of rf was significantly associated with dry mouth (p= 0.00), arthritis (p= 0.00), and rash (p= 0.04). a positive minor salivary biopsy was more common in children with dry eyes than in those without this clinical feature (p= 0.02). arthritis was more frequent in patients with other diseases than in those with primary ss (p= 0.00). we further investigated ss features according to the age groups (≤ 6 years, 7-11 years, ≥ 12 years). parotid involvement was inversely proportional to the age and occurred more frequently in younger patients (79% of those ≤ 6 years; p= 0.03). interestingly, the rate of anti-ssa/ro positivity increased with age (97% of those ≥ 12 years; p= 0.00). conclusion: even though parotitis was the most frequently reported feature, a wide range of clinical manifestations in children with ss has been reported so far. a better knowledge of css features will help to pave the way for the development of css specific diagnostic criteria. none declared introduction: pachydermodactyly (pdd) is a rare benign fibromatosis, characterized by progressive painless swelling of soft tissue of proximal interphalangeal (pip) joints without inflammation signs. generally pdd affects pip joints of the fingers, rarely of the thumb. the involvement is typically symmetrical, in few cases unilateral. it usually occurs more frequently in young males. etiology is unknown, but it arises from mechanical stimulation of periarticular skin (i.e repetitive rubbing, interlacing, and cracking of fingers). pdd has to be considered in the differential diagnosis of arthritis (i.e. juvenile idiopathic arthritis, jia) and many syndromes (i.e. progressive pseudorheumatoid dysplasia). prognosis is good with cessation of mechanical stimulation 1 the recurrent paroxysmal appearance of inflammatory lumps (local erythematous tender swellings, which partially respond to antiinflammatory agents), accompanied by elevated inflammatory markers during flares, suggest that fop may be an autoinflammatory disease. the episodic formation of bone, often following a trivial injury, suggests that innate immune-related triggers induce tissue transformation through the bmp pathway. moreover, interleukin-1β (il-1β), a well-known mediator of the innate immune system, has been linked to ho and mineralization in mesenchymal stem cell cultures derived from human bone marrow. we hypothesized that treating fop patients with anti-il-1 agents could help ameliorate the progression of this devastating disease. we report our experience treating two fop patients with anakinra and canakinumab. objectives: to decrease the frequency of fop paroxysms, and/or limit the symptoms and extent of residual lesions, by using anti-il-1 agents. methods: patients' data and blood il-1 levels were analyzed to characterize the efficacy of anti-il-1 treatments in ameliorating the natural progression of fop. results: a 13.5 year old boy and a 5 year old girl were diagnosed with fop, both clinically and genetically (the typical r206h mutation was found). various treatments, including high-dose corticosteroids, pamidronate infusions, celecoxib, monteleukast and sirolimus, did not change the course of the disease. both patients are receiving canakinumab (the male patient was initially treated with anakinra). the male patient has been treated for over 2 years. flare rate was markedly reduced from one new lump every 8 days to approximately one every 25 days ( figure 1 ). the lumps involved in almost all of these flares are the same: at the left scapular base and within the sternocleidomastoid muscle. the female patient has been treated for a year, and has not experienced any ho flares during canakinumab treatment. temporarily withholding canakinumab in both patients, led to serious flares 8 weeks after the last dose. notably, while undetectable levels of il-1β (<0.125 pg/ml) were found in the three plasma samples obtained from the male patient during treatment with anakinra or canakinumab, high levels (up to 21.52 pg/ml, about 90-fold higher compared to average levels measured in healthy controls) were found in his plasma samples collected during the flare ( figure 2 ). in contrast, il-18 and il-6 plasma levels, measured before, during and after withholding treatment, were comparable or slightly higher than those observed in healthy controls ( figure 3a , b). conclusion: we report here, for the first time, that anti-il-1 agents were found efficacious in treating two fop patients. we also found markedly increased il-1β levels during flares, which normalized following the treatment. we suggest a role for il-1β in the pathogenesis of this disease. although it is too soon to conclude whether fop may be included under the umbrella of auto-inflammatory syndromes, anti-il-1 agents can be effective in ameliorating the natural progression of fop. introduction: musculoskeletal symptoms are one of the common reasons for applying to rheumatology departments in general practice 1 . although inflammatory causes are generally considered in the foreground, it is known that non-inflammatory causes including genetic diseases may also be responsible. the absence of signs of inflammation (morning stiffness, redness, tenderness) and normal inflammatory markers in laboratory findings may support nonrheumatologic diseases 2 . objectives: to present genetic disorders that can mimic rheumatologic symptoms and to answer when genetic diseases should be considered in the differential diagnosis in patients presenting with rheumatological complaints. methods: we retrospectively evaluated 60 patients who applied to hacettepe university pediatric rheumatology department with musculoskeletal compliants between january 2015 and december 2019 and had been consulted to genetics departmant. the rate and degree of consanguinity, clinical diagnosis, indication for consultation, accompanying musculoskeletal and other findings had been recorded. the diagnosis of genetic diseases were based on physical examination, radiological evaluations and genetic analysis. results: a total of 60 patients, 19 boys (31.6%), with a mean age 12.46 ± 1.41 years were included in the study. the rate of consanguinity was 25.0%. the most frequent referral to the genetic department was the presence of skeletal anomalies (n:12) such as camptodactyly, clinodactyly, and bone shortness accompanying joint findings. other causes include short stature (n:4), joint deformity (n:5), joint hyperlaxicity (n:10), dysmorphic findings such as atypic facial appearance (n:9), accompanying diseases that may be part of a syndrome (n:11), genetic diagnosis suspicion according to the results of radiological examination (n:4) and joint findings without clinical and laboratory signs of inflammation (n:5). distribution of joint involvement in 20 patients with genetic disease were hands, knees, and hips respectively. in the laboratory evaluation of patients presenting with joint swelling and arthralgia, acute phase reactants (erythrocyte sedimentation rate and c-reactive protein concentrations) were within normal reference values. one third of the patients (33.3%) had a final diagnosis of a genetic disease. the diagnoses of these patients were as follows; cacp (camptodactyly, arthropathy, coxa vara deformity and pericarditis) syndrome (n:3), trichorhinophalangeal syndrome (n:1), progressive pseudoromatoid dysplasia (n:2), lig4 syndrome (n:1), 3m syndrome (n:1), h syndrome (n:1), spencd (spondyloenchondrodysplasia, n:3), and nonspecific connective tissue disease (n:8). conclusion: genetic syndromes with musculoskeletal findings are often unrecognized and misdiagnosed as rheumatologic diseases leading to unnecessary procedures and treatments. summarizing the genetic diagnosis spectrum that can be detected in these patients will increase the awareness of physicians. results: according to the results of observation, the disease was more common in the age group of 7-11 years (65%), to a lesser extent among children in the group of 12-15 years (35%), less often in the group of 3-7 years (5%). when examining infectious agents, zoonotic infection was detected in 41% (listeria monozytogenes, yersinia enterocolitica). clinical course of nodular erythema in this group was characterized by an expressed activity of the inflammatory process with multiple elements in the lower and upper extremities, joint syndrome, increased esr to 45± 3.8 mm per hour, crp 28± 2.5 mg\l. the disease was preceded by an episode of acute infection with an increase in body temperature, intoxication, in some cases with short-term intestinal syndrome, pharyngitis. the rashes were persistent and recurrent, with a slow regression of laboratory activity. streptococcal etiology of nodular erythema was detected in 37% of cases. there was an increase in esr to 25±3.8 mm per hour, crp 15± 2.7 mg/l, a significant increase in antistreptolysin on average 480± 34% iu / ml. with an increase in individual cases to 870 iu/ml. in 13% of cases, erythema nodosum developed after an intestinal infection. among the pathogens were identified sh. disenteria, e. coli, yersinia enterocolitica, enterovirus. the disease was characterized by moderate activity, a good response to etiological therapy and a short course of nsaids . an interesting fact was the development of nodular erythema in 4% of cases caused by the epstein-barr virus in groups of children from 3 to 7 years and 7-9 years. they had clinic picture with normothermia, no symptoms of intoxication, periodically occurring elements of nodular erythema on the shins, no blood changes. therapy aimed at eliminating the virus gave a positive result and did not require specific anti-rheumatic therapy. in 5% of cases, the etiology of nodular erythema was not defined. the clinical course of nodular erythema in children depends on the infectious agent that was the trigger of the pathological process. the higher activity and duration of the disease is caused by zoonotic infection, which requires more active antiinflammatory therapy with corticosteroids, which may be associated with the activation of autoimmunity. this group of children was taken for further observation as a group at risk of developing systemic connective tissue disease. changes in the etiological structure of nodular erythema and treatment tactics require further study. introduction: sjögren syndrome (ss) is a chronic autoimmune disorder characterized by inflammation of the lacrimal and salivary glands leading to oral and ocular dryness. childhood ss is rare and poorly defined and underdiagnosed owing to the lack of childspecific diagnostic or classification criteria. objectives: the purpose of this study is to describe 12 cases with pediatric ss in order to better clarify the characteristics of the disease in the pediatric age. methods: we retrospectively reviewed medical records of patients (pts) with pediatric ss referring to three italian pediatric rheumatology centers. due to lack of childhood validated ss-specific criteria, physician diagnosis was the only inclusion criteria. results: we collected data on 12 pts (9 females). the mean age of disease onset is 10.0 yrs (median 10.2, range 4-17). the mean age of diagnosis is 11.83 (median 11.45, range 6-18). the follow up period varied from 0.1 to 9.3 yrs (mean 3.95, median 5.0). the most common manifestations were articular involvement (mainly with arthralgia) (9/12 pts) and parotid/salivary glands swelling (8/12 pts). xerostomia and xerophthalmia were found in 6/12 pts and in 4/12 respectively. vaginal dryness was reported only by one pt. fever and fatigue occurred in 3/12 and 7/12 pts respectively. we also recorded 3 cases of circulating immune complexes manifestations in 3 pts, purpura (n=2) and glomerulonephritis (n=1). we observed an endocrine involvement in 3 pts (1 metabolic syndrome, 2 autoimmune thyroiditis). abdominal pain was found in 4/12 pts. all pts were positive for autoantibodies (positivity for ana or anti-ssa or anti-ssb or fr) at presentation. rf test results were available in 8 pts, all positive. positive ana (titer>1/320) and anti-ssa were present in 10/12 pts and in 9/12 respectively. hypergammaglobulinemia (range 1,6-8.04 g/dl) was found in 8/11 pts (1 na). abnormal schirmer test was observed in the half of cases (6/12). minor salivary gland biopsy was performed in 10 pts resulting in histological evidence of focal lymphocytic sialadenitis in 9/10. sonographic evaluation of salivary glands was abnormal in all of the patients (10/10). with regard to treatment, 6/12 pts received corticosteroids and eight were also treated with one or more dmards such a hydroxychloroquine (n=8), methotrexate (n=3), azathioprine (n=1), leflunomide (n=1). biological therapy was used in 3 patients for systemic involvement: 1 received belimumab and then rituximab, while the other patients received rituximab. conclusion: xerostomia and keratoconjunctivitis sicca were not common in our series while recurrent parotid swellings were more frequent than what reported in adults. pediatric recurrent parotitis should increase the suspicion for sjögren syndrome. current diagnostic criteria for ss do not include parotitis and therefore, the incidence of ss may be under-recognized in childhood. the disease is not always benign and patients with severe course may need second line treatment including immunosuppressant and biologics. introduction: improving our understanding of pediatric rheumatological (pr) patient population is crucial for pediatric rheumatologists to know rheumatic disease epidemiology and to raise awareness leading to early detection. we didn't find studies of pr disorders presenting in the first year of life. objectives: the aim of this study is to assess the prevalence of pr disorders with onset in the first year of life. methods: we retrospectively studied patients observed in our pediatric rheumatology unit between january 1 st of 1987 and december 31 st of 2019. we defined acute (<2 weeks), subacute (≥2 and <6 weeks) and chronic (≥6 weeks). results: a total of 3751 patients were observed in 32 years. diseases' onset occurred in the first decade of life in 2290 patients (61%) and in the first year of life in 158 (4,2%). among the latest group, chronic inflammation was the most frequent group of diagnosis (30%), followed by recurrent inflammation (23%), acute inflammation (11%), infection (9%), infiltrative/ degenerative disorders (8%) and subacute inflammation (3%). the remaining patients (16%) were diagnosed with other disorders classified as miscellaneous. among chronic inflammation group, 14 patients were diagnosed with juvenile idiopathic arthritis (4 systemic); 14 had neonatal lupus and one patient had polyarteritis nodosa. among recurrent inflammation group, 13 patients were later diagnosed with pfapa (periodic fever, aphthous stomatitis, pharyngitis and adenitis), 8 were diagnosed with behçet disease and 6 had an autoinflammatory disorder. acute vasculitis was diagnosed in 13 patients (9 kawasaki disease and 4 acute hemorrhagic edema of infancy). among infectious diseases group, there were two cases of congenital syphilis with arthritis and two cases of osteomyelitis secondary to bcg vaccination. conclusion: rheumatological diseases presenting in the first year of life are not exceptional. although many patients didn't have a definitive diagnosis at the beginning of the symptoms, many of them were later diagnosed with rheumatic disorders, mostly chronic inflammation (30%), which requires early diagnosis, specific treatment and long-term follow-up. rheumatic diseases must be considered as differential diagnosis in the first year of life in order to avoid delayed intervention and long term disabilities and sequelae. (1), on the other side measles-induced mas has rarely been reported (2). objectives: we present the case of a child known to have sjia in remission, who presented a measles primary infection and a secondary kd complicated by mas. methods: a 5 years old girl, not fully vaccinated and known to have sjia in remission under methotrexate, presented for frequent high grade fever of 3 days duration associated with flat flash red spots on the face and trunk as well as the palms and soles. a koplik's spot was identified. conjunctivitis and coryza were also present. initial viral serology, including measles, returned negative. fever persisted and on day 7, edema of both hands and feet appeared with bilateral cervical adenopathy, erythematous tonsils, gingivitis, cracked lips and hepatomegaly was noted. all cultures were negative and chest x-ray was normal. inflammatory markers rose up. viral serology was repeated and measles igm came back positive. cardiac ultrasound ruled out coronary aneurism and the ophthalmic exam showed no uveitis. kd criteria were met and 2g/kg of intravenous immunoglobulins (ivig) were administered. after 48 hours of clinical improvement, fever reappeared and the patient returned to be ill looking although the rash regressed. we noted high ferritine(2016 ng/ml) together with low c3, decrease in platelets(170 x10 3 /ml) and elevation of hepatic enzymes, ldh and cpk, without increase in the inflammatory biomarkers. mas was suspected and a bone marrow aspirate showed the presence of mild macrophage hemophagocytosis. antibodies for lupus and auto-immune myositis were all negative. steroids were given, fever disappeared, and spectacular clinical and biological improvements were objected. 2 weeks later, desquamation of all extremities was noted. sars-cov-2 was not investigated because historically this case presented 1 year earlier than the pandemic. results: we hereby report, for the first time, kd and mas triggered by measles infection in a child with sjia in remission. the exact mechanism involved in kd-induced mas and measles-induced mas has not yet been defined but a defective immune response is suspected (3). conclusion: significant similarities and overlap between measles, kd, sjia and mas make an early diagnosis very challenging (1)(3). the recent covid19 pandemic emphasizes how a viral illness can be responsible of kd and sometimes degenerating in mas. we report this clinical case as an example of a systemic inflammatory syndrome (sis) taking place after a viral infection to measles. in the era of covid19 pandemic and secondary sis in children, an additional challenge is present in regions lacking measles vaccine coverage. none declared the musculoskeletal manifestations of scurvy: a diagnostic challenge for the rheumatologist p2 was a 5-year-old boy, with autism spectrum disorder, malnutrition and severe food selectivity, admitted to our unit for refusal to bear weight and bruises in lower limbs. the auxological evaluation showed a strongly dystrophic aspect. coagulation profile and main organ function markers were normal. at nutritional biochemical parameters evaluation, iron and vitamin c deficiencies were detected (vitamin c: 2 μmol/l). oral vitamin c therapy was started, with prompt clinical response. p3 was a 7-year-old boy with autism spectrum disorder, admitted to our unit for lameness and difficulty in walking for a month. at clinical examination, a mottled skin at lower limbs was noted. joint examination was normal. auxological parameters and main blood tests were adequate for age. given the presence of food selectivity, he underwent serum vitamin c dosage (11 μmol/l); hence he started oral vitamin c therapy, with rapid clinical improvement. p4 was a 2 years old boy who was referred for coxalgia and fever. at clinical examination, pale skin, gingival hyperemia, and pain in mobilization of the left hip were present. microcytic anemia was detected, but main organ and inflammatory markers were normal. no evidence of infection was present. x-ray of femur and knee showed morpho-structural alteration of the distal metaphysis bilaterally. a low intake of fruit and vegetables was reported; hence, dosage of vitamin c was performed, resulting reduced (2.5 μmol/l). he started vitamin c oral therapy with clinical response. p5 was a 13-year-old girl with behavioral disorder and intellectual disability, admitted for fever and right knee swelling which appeared two days after a right leg burning. c-reactive protein and esr were elevated and ultrasound exam confirmed intra-articular knee effusion. suspecting a septic arthritis, antibiotic therapy was started with laboratory normalization and partial clinical improvement. considering the persistence of knee swelling after nine days of intravenous antibiotic therapy, the presence of gingival hyperemia and history of food selectivity, vitamin c dosage was practiced (12 μmol/l). oral vitamin c was administered with complete clinical resolution. conclusion: although scurvy is considered a disease of the past, it still occurs nowadays. food selectivity associated to autism is a major risk factor for vitamin c deficiency in childhood. rheumatologists should take into account the diagnosis of scurvy in the diagnostic approach of musculoskeletal disorders in children, especially when development disorders are present. 15.4%), juvenile dermatomyositis (n=1), sarcoidosis (n=1), granulomatous polyangiitis (gpa) (n=1), sting-associated vasculopathy with onset in infancy (savi) (n=1), and oligoarticular jia (n=1). respiratory symptoms were present in 6 (46.2%) patients at the time of primary diagnosis. in other patients, the time period between the diagnosis of the rheumatic disease and the onset of the respiratory symptoms ranged from 1 to 12 years. cough, the most common symptom, was present in 10 (76.9%) patients. six patients manifested with cough and sputum. six (46.2%) patients had shortness of breath and one patient had hemoptysis. on the physical examination of one patient, rales and clubbing were detected. high resonance computerized tomography (hrct) was performed in all patients. hrct findings were as follows; lymphadenopathy in 8 patients (61.5%), ground glass appearance in 10 patients (76.9%), consolidation in one patient, pleural effusion in one patient, pulmonary nodule in 4 patients (30.8%), fibrosis in one patient, cystic lesions in 3 patients (23.1%), septal thickening in 5 patients (38.5%), bronchiectasis in one patient, and reverse halo sign in one patient. in echocardiographic examination, only one patient had pulmonary hypertension. three patients underwent open lung biopsy, and diagnosis was made with pathological examination of the lung tissue. of these three patients, two (15.4%) had lymphocytic interstitial pneumonia (lip), and one patient had chronic inflammation and focal fibrosis. infectious lung disease was not detected in any patient. ten patients (76.9%) had interstitial lung disease associated with rheumatic disease, one patient had pulmonary hemorrhage, one patient had pulmonary involvement of gpa, one patient had pulmonary involvement of sarcoidosis. there was no statistically significant difference between the first and last spirometry and dlco values during the follow-up period. mortality was 7.5% (1/ 13) in this cohort. active disease was significantly associated with abnormal tc, hdl, and tg levels (p=0.04*), (p=0.03*) and (p=0.04*) respectively. multivariate analysis of the factors affecting abnormal cholesterol level revealed that sle is a significant predictor of abnormal cholesterol level . presence of jsle increase risk of abnormal cholesterol 9 times more than cases without jsle. the overall percent predicted was 80%. active disease is a significant risk factor for abnormal tg with increased risk of abnormal tg by 3.2 among cases with active disease than cases with inactive disease. the overall percent predicted was 75.6%. conclusion: children with rheumatic diseases showed significant lipid profile abnormalities. abnormal tc, hdl and tg are significantly associated with active disease. presence of jsle increase risk of abnormal cholesterol. active disease is a significant risk factor for abnormal tg. therefore, lipid levels should be monitored regularly and managed in patients with paediatric rheumatic diseases to minimize the longterm risk of cvd. methods: non-experimental, cross-sectional and descriptive study. a confidential survey was conducted online, aimed at residents and attendings who deal with musculoskeletal pain. were addressed with the definitions of arthralgia, arthritis, myalgia, allodynia and hyperesthesia (between five to seven options) with only one correct answer. correct definitions: arthralgia (pain localized to the joint or periarticular structures, as a only manifestation); arthritis (criterion one or criterion two: 1 -joint swelling or intra-articular effusion / 2 -limitation of joint mobilization associated with at least one of the following: a) pain b) tenderness c) swelling d) heat); myialgia (pain with muscular origin or referred to muscle, regardless of its etiology); allodynia (pain resulting from usually non-painful stimulus); hyperesthesia (coexistence of allodynia plus hyperalgesia -exaggerated responses to tactile and thermal nociceptive and nonnociceptive stimuli the association of pure red cell aplasia (prca) with thymoma led to the discovery of the autoimmune mechanisms involved in the pathogenesis of this rare disease. till date many adult case reports have revealed a strong link between prca and autoimmune diseases, endocrine disorders, rheumatic diseases, autoinflammation and immune dysregulation. [1] [2] [3] [4] [5] objectives to stimulate a search for the genetic and immunological roots for a 2.5 years old girl with syndromic face, pure red cell aplasia, type 1 diabetes and polyarthritis. methods this is a story of a 2.5 years old girl with pure red cell aplasia, type 1 diabetes and polyarthritis. she was normal till 7 months of age. at the age of 8 months, she was diagnosed with type 1 diabetes. she was evaluated by her paediatrician in view of generalized hypotonia, deformed pinna, low set ears, midfacial hypoplasia, blue sclera, umbilical hernia and retracted eyelids. she had multiple episodes of seizures during next few months. to me, she was presented with one year history of polyarthritis with severe pallor requiring frequent blood transfusions. family history was inconclusive. musculoskeletal examination showed polyarthritis involving right knee, bilateral ankles, fingers and toes. further examination revealed haemolytic facies and hepatosplenomegaly. i was not able to make out any facial dysmorphism mentioned earlier by her paediatrician. results: table 1 conclusion early age of onset, pure red cell aplasia, autoimmune and endocrine manifestations with some doubtful facial dysmorphism inspired me to suspect some known or unknown immune dysregulation syndrome in this child. genetic analysis would be the best possible option in this scenario if financial condition permits. introduction: galactosialidosis (gs) is a rare inherited lysosomal storage disorder (lsd) which is characterized by a defect in the lysosomal glycoprotein catabolism. here we report, for the first time, a case of a child affected by gs who presented with recurrent episodes of extensive joint inflammation in both knees. knowledge on gs related inflammatory joint pathology is lacking, which hampers evaluation of possible mechanisms that could give an explanation for the significant arthritic joint abnormalities as observed in our patient. objectives: the aim of this study is to describe the clinical presentation as well as the laboratory, radiologic and microscopic features of this extremely rare presentation of gs. furthermore, we conduct a literature review on lsd's complicated by arthritis in order to evaluate potential mechanisms that could explain the extensive inflammatory joint swelling observed in our patient. methods: in this study we present a 12-year-old turkish boy who was diagnosed with gs (late infantile form) at 17 months of age. from the age of 8 years, the boy presented with episodes of inflammatory joint pathology of the knee. informed consent was obtained. alongside the case report, a literature review using medline was conducted. an extensive list of known lsd's was combined with the terms: "arthritis", "joint inflammation", "synovitis" and "synovial inflammation". cases in which joint inflammation was based on a probable cause other than the underlying lsd were excluded. results: in the present case, owing to comprehensive examinations (i.e. laboratory tests, imaging and microscopic examination) multiple possible causes for the recurrent inflammatory joint pathology could be rejected (i.e. no signs of infectious arthritis, reactive arthritis, osteoarthritis, arthritis secondary to a malignancy or crystal induced arthritis). a diagnosis which could explain the clinical picture is the jia subtype: ana negative oligo-articular jia. however, microscopic examination showed numerous foamy macrophages with extensive vacuolization in the synovial tissue of the inflamed joint, which is not associated with jia. given the evidence of storage products within the macrophages of the inflamed synovial tissue and no conclusive diagnosis, gs itself should be considered as the primary cause for the recurrent arthritis. an in-depth literature review using medline for data on inflammatory joint pathology in lsd's showed that 7 lsd subtypes (i.e. fabry disease, farber lipogranulomatosis, gaucher disease type 1, mucopolysaccharidosis ix, a-mannosidosis, fucosidosis and cystinosis) could present with disease related arthritis. multiple potential arthritic mechanisms secondary to storage product accumulation in lsd's have been described, such as: dysregulation of innate immunity and increased upregulation of numerous pro-inflammatory proteins. conclusion: given the evidence of storage products within macrophages of the inflamed synovial tissue and the absence of other etiological clues, our hypothesis is that gs itself is the primary cause for the inflammatory joint pathology in our patient. although, gs cannot be linked directly to joint inflammation, lysosomal defects have been associated to pro-inflammatory effects that possibly could result in arthritic disease. future identification of other patients with gs is required to support the hypothesis of an arthritic clinical phenotype of gs and to assess underlying pathophysiology. introduction: joint pain (jp) is a relatively common complaint among children and adolescents. a painful joint in children for many years continues to maintain the status of the most common symptom of juvenile arthritis. however this symptom should not always be interpreted as a manifestation of rheumatic diseases. objectives: the aim of current review is to debate of the structure in children with the chief complaint of jp. methods: we retrospectively analysed our series of 600 children which attending outpatient department with complaint about pain lasting longer than two months in one or more joints. the clinical, instrumental and laboratory pictures were collected. special attention was paid to certain aspect of medical complaints, a complete and accurate history and physical examination. different categories as possible etiologies of jp in children were systematize and detailed. results: all children were divided into several groups based on their anatomical and physiological characteristics of osteoarticular system: the first group consisted of 240 children under 6-7 years old, the second group -220 children 7-12 years old, the third group -140 children over 12 years old. research suggests that more preschool children were experience bilateral lower extremity pain by "post-walk genesis" due to natural hypermobility, immaturity of sensory innervation of the joints and imbalance of the leg muscles (e.g. growing pains). the second most common cause of jp was associated with intra -or postinfectious factor (viral, streptococcal and chronic focal of infection). the frequency of juvenile arthritis and other rheumatic diseases in children of this age group did not exceed 10%. special attention was paid to fever, chills, malaise, nightpain and constitutional symptoms with changes in blood lab tests to exclude osteomyelitis (inc specific cause), malignancies manifestation and other bone tumors (less 5%). the most common causes of joint pain of school-age children were hypermobility syndrome and enthesopathy (primary, secondary). secondary enthesopathy were result of changes in nutrition, rapid growth and excessive exercise. also enthesopathy were manifestation of endocrine, gastrointestinal or infectious diseases. the proportion of children with the onset of chronic inflammatory arthropathy also did not exceed 10%. hypermobility child's syndrome was characterized by harmless pain (inc low back pain), linked to physical activity (less morning stiffness). over the past decade, we've seen a gradual increase in the number of children (95% were girls) with knee pain by diagnosed patellofemoral and mediopatellar plica syndromes, patellar tendinitis or idiopathic cause. in most cases children was complicated by syndrome of increased anxiety. the share of true chronic inflammatory arthropathies, including spondylitis, in children of this age group did not exceed 10%. fibromyalgia were diagnosed less 5%. introduction: a significant part of patients in rheumatologist's practice is children and teenagers with complaints of pain. the further volume of examination and the choice of treatment course depends on the capability of the rheumatologist to define the inflammatory and non-inflammatory genesis of pain. that makes the problem of differential diagnosis very important. objectives: to conduct a comparative analysis of patients with a principal pain complaint to determine if there are significant differences in the groups with the inflammatory and noninflammatory pain genesis. methods: the retrospective study included children who consulted a rheumatologist in the outpatient clinic in the period 2018-2020 without preliminary selection (n = 176). of them there were selected children with principal pain complaint (n = 120). according to the diagnosis, the children were divided into 2 groups: those who have inflammatory genesis of pain (a, n =59) and those with noninflammatory genesis of pain (b, n =61). the group a included children with such diagnoses as: reactive, poststreptococcal and juvenile idiopathic arthritides. the group b included children with arthralgia, chronic pain syndrome, orthopedic pathology, fibromyalgia. results: 1. groups a and b differ in the average age of the first complaints onset (t-criterion for equality of means) with a high degree of statistical significance (group a = 7,4 years; group b = 9,3 years; p = 0.019). which means that in group a more often than in group b first complaints appear in the age between 1 to 10 while in group b more often than in group a it happens in the age between 11 to 16. 2. there was a statistically significant difference in the means between groups a and b in time between the onset of first complaints and the first visit to a rheumatologist (p = 0.03) also in favor of this conclusion speaks the fact that in group a the number of visits to a rheumatologist in the same year when the first complaints appear is almost 2 times higher than in group b. 56% of cases in group a consulted the rheumatologist the same year when the first complaints appeared in comparison to group b where only 31% of patients did the same. below is the table with distribution of cases by the number of years between the first complaints onset and the first visit to a rheumatologist in both groups: conclusion: in children with arthritides, the first pain complaints appear at an earlier age (an average of 7.4), and in group b (an average of 9.3). patients with arthritis more often visit a rheumatologist earlier (within 1 year after the first complaints) than those with non-inflammatory genesis of pain complaints. the most common cause of recurrent musculoskeletal pain is growing pain (gp) in children. differential from rheumatic diseases could be challenging in some cases since there are no diagnostic criteria for gp. objectives: to analyze gp characteristics in a large cohort of patients in comparison with other non-inflammatory and inflammatory diseases causing limb pain, and to simplify the gp's diagnosis process by using machine learning (ml) techniques. methods: this is a multicenter cross-sectional study. introduction: it is a well-known fact that the period of intensive growth in children is associated with the processes of active bone mass accumulation and coincides with them in time. one of the most distinctive indicators of an increase in the disease incidence among children for the recent decade (+105,3%) can be found in the skeletal disorders resulting from disrupted calcium metabolism and vitamin d deficit. the latter is widespread in ukraine as it is observed in 92% of schoolers. objectives: establish the specifics of the structural and functional status of the bone tissue in children during the growth spurt, taking account of the degree of vitamin d3 sufficiency. methods: the examination covered 147 children aged 9-17 who were divided into three groups depending on the presence of the growth spurt (gs) and its intensity: group 1 -35 children who had become 8-12 cm taller for the year in question; group 2 -32 children who had become taller by 12 cm or more, group 3 -80 children who had experienced no growth spurt. inclusion criteria were the following: no chronic somatic or endocrine pathologies, no musculoskeletal disorders or mineral homeostasis disruptions; physical exertion corresponding to their age; the children had not been taking any complexes of vitamins and minerals, including vitamin d 3 for 6 months before the examination. conclusion: children aged 9-17 showed deficiency of vitamin d 3 reaching 100% which had no correlation with the presence or intensity of the growth spurt. in children who experienced growth spurt, a reduced bmd proved more frequent and correlated with the spurt intensity, however, it did not depend on sufficiency of vitamin d 3. therefore, during the growth spurt, disrupted mineralization of the bone tissue was influenced not only by the vitamin d deficit but also by the correlation between the bone tissue mineralization rate and intensity of growth in the children. methods: a self-reported 25 question online survey on qol of patients with sjia and aosd was developed by the non-profit organizations, the autoinflammatory alliance, kaisz/vaisz, enca and sjia foundation in english and translated to dutch. respondents were recruited by convenience sampling through online social media posts. data on flares, triggers, family history, and correlation of symptoms with labs were collected in addition to detailed information on qol during and in-between flares. results: between 2017 and 2019, there were 109 responses; 54 were from parents of children with sjia, 18 from adults with sjia, and 37 from adults with aosd. interestingly, adults (whether diagnosed with sjia or aosd) were more likely to report pain, fatigue, joint swelling or arthritis, nausea & vomiting, and diarrhea during flares than children. adults were also more likely to describe flares >one month. 80% of patients reported being "greatly" or "severely" limited during flares. between flares, 20% reported being "greatly" or "severely" limited while 59% were "somewhat" limited. 80% felt their condition affected their studies, job, and career, including 66% of children with sjia, 100% of adults with sjia, and 92% with aosd. respondents were asked open-ended questions regarding their experience with disease flares and impact on their lives, and specifically how sjia and aosd affected work, career and schooling. responses regarding the disease experience were classified into 7 theme areas: 1) experience with disease onset and process of diagnosis; 2) health care access, quality, and drug safety concerns; 3) physical impact of the disease including pain and chronic fatigue; 4) social impact of the disease; 5) mental health and emotional impact of the disease; 6) impact on work, career, and employment; and 7) broad impact on life and lifestyle. responses regarding effect on work, career, and schooling were categorized into 3 theme areas: 1) physical impact negatively influencing school/work productivity; 2) lost work and wages, including unemployment and needing disability benefits, and parents missing work to care for the child; and 3) the socialemotional impact as well as negative effects on mental health. about half of patients regardless of age reported the name sjia did not represent well the disease, specifically that it did not emphasize the systemic symptoms, and that the disease gets confused with other types of arthritis. adult patients with sjia did not like to have juvenile in the name. conclusion: children and adults with sjia and aosd report high levels of qol limitation and effect on school, work, and career, both during and between flares. our qualitative data emphasizes the importance of multidimensional evaluation of disease with ongoing input from the patients, which will provide a foundation for asking more relevant research questions to foster better care and improve qol. results: in total 111 participants were included in the study : 62 juvenile idiopathic arthritis (jia) patients, and 49 their parents. the mean age of the patients was 13.7 ± 2.3 years. significant differences were found between patients and healthy children in such hrqol survey categories like "autonomy" and "financial resources" (p < 0.05). although quality of life in children's with juvenile idiopathic arthritis was lower than in healthy children in hrqol survey category "self perception" (p < 0.05). after analyzing data no significantly differences were found between patients and parents' assessment scores in hrqol survey categories (p > 0.05). conclusion: juvenile idiopathic arthritis has a moderate negative influence on hrqol survey categories "self perception", "autonomy" and "financial resources" (p < 0.05) according kidscreen-52 queationnaire. introduction: juvenile dermatomyositis (jdm) is often first identified by parents and carers as the red facial rash develops. the rash can progress and lead to young people being misdiagnosed with eczema, scarlet fever or psoriasis. however, over time the obvious signs of jdm can become "invisible" as treatment calms the rashes and masks the outward signs of jdm, until a flare occurs, when the rashes can be a marker for disease activity or progression. as part of a larger study, children around the united kingdom were asked to discuss their views on whether they wanted people to be able to see their jdm. objectives: to understand the implications for children and young people from having a disease that has visible and invisible phases and whether they want others to see their jdm, or not. methods: children and young people around the united kingdom who were already consented and enrolled into the uk juvenile dermatomyositis cohort and biomarker study were asked to complete a bespoke questionnaire. there was a mix of open and closed questions, and it was administered in paper format to all children and young people between the ages of 8 and 19 years of age for self-completion, either on the paper forms, or via a secure web-based software system. the questionnaires were administered at the end of 2018. numeric data were described and qualitative data were analysed using standard content analysis. results: 246 questionnaire packs were sent out, with 127 (52%) being returned. of these 4 could not be used due to practical reasons, such as only demographic data being completed, which left a sample of 123. 98 (80%) of the 122 who responded said other people could not see their jdm, with only 11 (9%) saying it was visible and 13 (11%) saying they did not know if others can see it. 1 did not respond to the question as said their jdm has gone away. they were then asked whether it was a good or bad thing for others to be able to see their jdm or for others not to be able to see it. 41 young people left comments as to why it was a good thing, 36 left comments as to why it was a bad thing and 14 left comments to why they said don't know, table 1 presents the top ranking response for the three multichoice answers. conclusion: this study has highlighted the disparity between young people wanting others to see their jdm so that they gain more understanding and empathy from those around them, but equally, wanting their jdm to be invisible, so that they feel the same as their peers. whilst many paediatric rheumatic conditions are in fact invisible, our data illustrate that jdm often gives children and young people a taste of both visible and invisible phases of disease activity. as one young person said "it's not good, nor badit's good that it's invisible sometimes so i can blend in without the disabled stereotype. however, sometimes it needs to be seen so i can be understood and not challenged". disclosure of interest: none declared multidimensional assessment report (j-fimar) which includes comprehensive patient self-report questionnaire and numerical rating scales to measure pain, fatigue, headache, sleep quality, physical function, psychological state, health-related quality of life, satisfaction with illness course. the j-fimar has been devised according to the outcome measure in rheumatology (omeract) guidelines. discriminant ability of the multidimensional tool was evaluated by testing it in a control group including healthy controls and patients affected by active juvenile idiopathic arthritis (jia). the psychosocial consequences of chronic pain were evaluated by using the children depression index (cdi) and the multidimensional anxiety scale for children (masc). the objective sleep quality was measured by overnight polysomnography. results: table 1 shows characteristics and the most represented somatic symptoms in our cohort of jfs patients at the study enter. polysomnography was performed in 21 patients with sleep disturbance; 8/21 (38.1%) showed an electroencephalographic pattern of alpha wave intrusion in slow wave sleep (sws). the presence of objective sleep disorders was significantly correlated to cdi score rs -0,775 (p≤0,0001) and masc 0,61 (p=0,005). from november 2016 to april 2020 j-fimar was completed by 43 jfs patients (f 35 (81.4%), median age 14.7 years [7.1-17.6], median disease duration 1.9 years [0.1-7.8]) in 125 visits. all patients filled out the questionnaire in a short time (<15 minutes) and considered it simple and easy to understand. jfs patients showed significantly higher score for pain, fatigue, poor physical function and measure of psychological distress than healthy controls and jia patients (p<0.05 for each item). conclusion: jfs patients presented significantly higher pain experience, functional disability, and impaired quality of life than patients with active jia. a relevant percentage of jfs patients experience sleep disturbances, which were correlated with mood and anxiety disorders. our multidimensional tool was feasible and able to quantify global jfs severity. this multidimensional tool, by measuring the main domains affected by the disease, could be promising to individualize treatment strategy and to test its efficacy. disclosure of interest: none declared introduction: fatigue is a subjective state of overwhelming, sustained exhaustion and decreased physical and mental capacity, which is not relieved by rest. fatigue is the most common complaint in children and teens with an autoinflammatory disease, besides the disease related flares. the purpose for this study was to show that fatigue is a serious issue for children and young people with autoinflammatory diseases. we hypothesized that age, gender and/ or the type of autoinflammatory disease could have differing effects on the fatigue experience. objectives: we aimed to investigate fatigue in children and young people (cyp) with autoinflammatory disease, including how this affected them on a daily basis. methods: cyp with autoinflammatory diseases were invited to complete an online survey, providing details about their fatigue and how it affected them. the survey was developed by the non-profit organizations autoinflammatory alliance and kaisz/vaisz, in english. respondents were recruited by convenience sampling through online social media posts. data on age, gender and disease were collected in addition to information on their experience of fatigue on school and social interaction. a total of 114 cyp (age range 7-18 years) with an autoinflammatory disease responded to the survey (52% female). results: the majority of respondents (81%) reported experiencing both mental and physical fatigue. respondents were asked how much their fatigue affected them, on a scale of 0 to 10; overall, the mean fatigue score was 6.6. however, young people aged 15 or over reported a significantly higher impact than those aged 11-14 years (mean 7.5, p=0.012). different autoinflammatory diseases were surveyed: crmo 25%, caps 20%, pfapa 12% also unclassified said (usaid) with 23%. in the open-response portion of the survey, 81% of respondents reported that fatigue was physical, as well as mental, in their experience. most (89%) reported that someone had doubted their fatigue in the past; 29% had found their teachers had doubted them, 26% had friends who doubted them, and 24% reported that they felt their doctors had doubted them. children and young people also felt a number of activities made their fatigue worse (table 1) ." conclusion: cyp with autoinflammatory diseases experience physical and mental fatigue. health professionals and teachers should listen to patients reporting fatigue, validate their experience, and help find ways to support them. identifying resources to help the patients with fatigue, and referrals to therapy and mental health resources as needed may help the patients to better cope and manage their chronic disease. further studies will include patient engagement in designing questionnaires about all aspects of life and autoinflammatory disease will help our understanding of these complex conditions and how they affect patients. introduction: scleromyositis is the most common overlap syndrome but is rarely observed in childhood. this disorder involves two different autoimmune diseases: systemic scleroderma (ssc) and polymyositis (pm). objectives: to describe the clinical course of a ssc/pm syndrome in a young girl. methods: case report results: an 11-year-old female was admitted to the neurological unit of our hospital for creatine phosphokinase (cpk) increase and hypertransaminasemia associated to sporadic episodes of right calf pain. familiarity for muscular dystrophy was reported in the maternal branch. muscle tone and trophism were preserved at initial neurological evaluation. laboratory investigation confirmed increased muscle enzyme levels, including cpk (x70) (ck-mm 94.5%, ck-mb 5.5%), aldolase (x7), cardiac troponin (x10) and myoglobin (x10). suspecting a primary muscle disease, she underwent a total body stir-mri which showed a diffuse edema of gluteus medius bilaterally and a muscle biopsy revealing a marked muscle damage with dystrophic aspects and normality of the tested proteins. a genetic extended panel for congenital myopathies resulted negative. after 4 months, a new clinical examination showed the occurrence of general skin induration, sclerodactyly and tightening of the face skin. appearance of dysphagia was also reported, and muscle enzyme increase persisted. in suspicion of an ssc/pm overlap syndrome, she was referred to our unit. nailfold capillaroscopy showed capillary dilatation and branching, megacapillaries and diffuse microhemorrhages. reduction of esophageal contractions amplitude and hypotensive lower esophageal sphincter pressure were observed at esophageal manometry test. high-resolution ct of lungs and pulmonary function testing were normal. skin biopsy showed sclerodermiform findings. immunological studies revealed a positivity of antinuclear antibody (1:320) and anti-ku. anti-pm-scl resulted negative. an oral corticosteroid therapy (prednisone, 1.5 mg/kg/day) was started in association with subcutaneous methotrexate (15 mg/m 2 /week) and intravenous immunoglobulins (ivig) (2gr/kg every two weeks). improvement of skin manifestation, joint mobility, as well as normalization of serum cpk levels were observed. over 3 months, prednisone and ivig were slowly discontinued up to the ongoing dosage of 0.9/mg/day and 2 gr/kg every 4 weeks, respectively. mtx is still ongoing at the same dosage. conclusion: the diagnosis of overlap connective tissue disease syndromes may be challenging in pediatric age. different symptoms may be prevalent at different stages throughout the course of the disease. in our patient, a localized myositis preceded the ss onset by about four months. even though the use of high dose of corticosteroids is associated to a higher incidence of renal crisis in patients with css, a combined therapy with high doses of oral steroids, ivig and mtx was safe and effective in skin, muscle and joint symptoms in our patient. results: a total of 336 images were obtained from 118 healthy children included in the study and 708 capillary measurements were made. capillary density was significantly higher in group 4 than in groups 1 and 2. arterial width was significantly lower in group 1 as compared to group 3 and 4, and in group 2 as compared to group 4. apical loop width and capillary distance were significantly lower in group 1 compared to group 2 and 3 and 4. there was no significant difference between the age groups in terms of capillary length and venous width. there was no difference between the groups in terms of capillary morphology. in total 336 image evaluations, capillary tortuosity was <50% in 67.8%, and > 50% in 4.2%, and capillary crossing were <50% in 52.5% and> 50% in 3.4%. while the enlarged capillary was 4.7% and the avascular area was 4.2%, capillary branching, capillary meandering, microhemorrhage, and giant capillary were not detected in any case. there was a good level of agreement between the researchers, as 20 cases with 120 capillaries were evaluated with a good level of agreement (table 1) . conclusion: this is the first study to evaluate capillary morphology in healthy turkish children. this study also adds that some special forms such as enlarged capillary and avascular area, which is always named as pathological in adult age, can be seen in healthy children. these data will be guiding in capillaroscopic studies in various patient groups, particularly in children with collagen vascular diseases. methods: 308 patients with jia were tested for hla-b27. they were divided into 2 groups: 1) hla-b27 positive and 2) hla-b27 negative. results: 100 patients (32,5%) were hla-b27 positive and all of them are fulfilled the eular criteria of entesitis-related arthritis (era). the group 2 consists of 208 patients (67.5%). there's no statistical difference between both groups in active joint count, ana-positivity and uveitis frequency, the rate of use methotrexate and time before biologics. no difference in axial cervical spine 12 (12.0%) vs 21 (10.1%) (p=0.613) and sacroiliac joints 18 (18.0%) vs 23/207 (11.1%) (p=0.097) involvement was observed. hla b27(+) patients often received pulse therapy with methylprednisolone due to increased inflammatory activity and severe arthritis (22% vs 11.1%, p=0.011). other parameters are listed in table1. conclusion: patients with hla-b27 positivity were characterized by male predominance, more often hip involvement, higher laboratory activity and the need for more frequent use biologics. the rate of axial involvement wasn't different in hla-b27 positive and negative patients, that needs further study and creating more accurate classification criteria for jsa. disclosure of interest: none declared introduction: although gut is increasingly recognized as origin and/or target of inflammation in adult onset spondyloarthritis (spa), the incidence of gut involvement in juvenile spa (jspa) patients is still largely unknown, mostly due to the lack of reliable non-invasive tests. objectives: we performed a cross-sectional study of fecal calprotectin (fcal), a surrogate marker of gut inflammation, in patients with jspa, other forms of juvenile idiopathic arthritis (jia) and noninflammatory (ni) conditions. methods: fcal was measured by commercially available assay in stool samples of enthesitis related (era), psoriatic (psa) and patients with other jia subtypes (oligo-and poly-articular) who fulfilled ilar criteria, as well as in children with ni causes of musculoskeletal pain (ni-msd), regardless of the gastrointestinal (gi) symptoms (table 1 ). fcal was compared among different groups of patients and correlated with demographic data, clinical characteristics, treatment modalities and disease activity measured by jspada. the values were also dichotomized to <50 mg/kg, 50-200 mg/kg, and >200 mg/kg, which was regarded as normal, slightly increased and increased, respectively. ileocolonoscopy was performed in one patient. our study has shown that fcal levels are significantly higher in era patients compared to other jia (p=0.03) and/or ni-msd (p=0.03) patients. moreover, almost a third of patients with era had levels of fcal above the range regarded as normal, which adds to the number of evidences for a gut inflammation in this particular type of jia. besides, the fcal levels were higher in those with axial involvement, which further suppots the association of gut and axial inflammation in children with era. although endoscopy remains a gold standard for the diagnosis of gut inflammation, fcal can help to select children with era who might benefit from this invasive procedure, regardless of the gi symptoms, as shown in one patient with the highest fcal concentration in our study. moreover, fcal levels seems not to be influenced by disease characteristic and/or concomitant therapy intake. therefore, fcal should be a part of diagnostic workup in children with any type of jia, but most importantly in those with era. evaluate potential predictor variables of magnetic resonance imaging (mri) sij remission methods: retrospective review of prospectively collected data. we included patients with era (according to ilar criteria) continuously treated with anti-tnf agents for ≥ 12 months who had at least two mris of the sacroiliac joints performed before starting anti-tnf therapy (baseline) and during the follow up ( > 12 months after anti-tnf treatment). si joints were examined using t1-weighted images, t2 fastsuppressed and short-tau inversión recovery. the sparcc-sis was scored by two pediatric radiologists. sparcc-sis assessed the presence, depth and intensity of bone marrow edema (bme) on consecutive six slices in the iliac and sacrum bones . scoring is composed by: bme (0-48), bm intensity (0-12), bm depth (0-12 introduction: several paediatric patients manifest conditions commonly misdiagnosed as spider bites, which however, can include other arthropods bites; bacterial, viral, and mycotic infections; vasculitis; dermatological diseases; miscellaneous conditions as drug reactions, chemical injuries. objectives: in italy, spiders which are likely to be associated with severe toxin mediated tissue damage are uncommon, especially in urban zones. however, a minor trauma may be a precipitating factor for pyoderma gangrenosum particularly over the legs, in association with inflammatory bowel disease, haematologic diseases and juvenile idiopathic arthritis (jia). methods: we describe a 11-years old boy with pyoderma gangrenosum complicated spider bite in association with systemic jia (sjia). the patient was in clinical remission after the start of the sjia, occurred two months before, still treated with tapering doses of steroids and canakinumab, with the normalization of inflammatory parameters (crp, esr, saa, ferritin) and clinical manifestations. only a mild arthritis of the knee persisted and for this reason he was still treated with steroids. furthermore, he developed hyperglycemia, requiring insulin treatment. the first dermatological manifestation which he referred was a red dot of the leg skin. in a few days, the erythema enlarged, involving an area of 7 x 7 cm, with oedema, pain, and blisters, evolving in a necrotic lesion, with purulent exudate, surrounded by a haemorrhagic zone. results: haematological controls revealed neutrophilic leucocytosis, increased crp and procalcitonin. he started treatment with intra venous administration of teicoplanin plus ceftriaxone, with no resolution of the clinical manifestations and the reduction of leukocytosis, crp, procalcitonin. a culture swab was performed and was positive for pseudomonas aeruginosa, confirmed by pcr on the culture. he started ciprofloxacin and surgical curettage of the lesion, with the resolution of the lesion and the normalization of biochemical parameters. conclusion: the aspect of the lesion and its evolution were evocative of a spider bite suggested by anamnestic records, complicated by a pyoderma gangrenosum secondary to pseudomonas aeruginosa. the underlying disease, the immune suppressive treatment, with steroids and biological drugs, the hyperglycaemic pattern of the patient allowed the severe evolution of the spider bite. children in treatment with immune suppressive and/or biologic drugs are at high risk of infections. skin lesions, as arthropods bites, can be a facility for superinfection, with possible haematological and systemic diffusion. the strict application of the ilar 1 requirement for the presence of documented arthritis for the diagnosis of sjia, early in the disease course, may result in unnecessary delays in initiating appropriate treatment. in preliminary printo 2 classification criteria for sjia, this mandatory requirement of documented arthritis has been modified. to measure performance of preliminary printo classification criteria for sjia in our indian cohort. methods i gathered a data of seven sjia patients who attended dev children's hospital between jan 2019 and jan 2020. my data included demographics,clinical presentation, laboratory parameters and outcome of these patients. all these patients were diagnosed at an early stage by clinical judgement irrespective of fulfilment of ilar criteria. i applied preliminary printo classification criteria for all. average age of selected children (4 girls and 3 boys) was 5.1 years. conclusion a preliminary printo classification criteria for sjia has been validated in our cohort. there are many raised inflammatory markers in most of these patients other than wbc count. these markers should be considered to be added in supportive laboratory criteria to be more specific towards the diagnosis. it is important to add pid in exclusion list especially in a case of sjia with mas at onset. 3 trial registration identifying number leningrad's regional children' two patient stopped treatment within 6 months from therapy start: due to primary inefficiency (1) and allergic reaction (1). five (5/11) patients were-co-administered with cdmards, other 5with oral gc, and 6 subjects had been previously exposed to other biologic drugs. whole 5 patients stopped therapy due to secondary inefficiency: 2 patients were switched on toc, other children were switched on eta (1) introduction: systemic juvenile idiopathic arthritis (sjia) is a rare, complex auto-inflammatory disease with significant morbidity including fever, rash, serositis and articular problems. with the availability of interleukin-1 (il-1) and il-6 inhibitor treatment, morbidity has significantly reduced and the outcome for sjia patients has improved. however, differences in access to care and differences in treatment strategies between countries in and outside of europe remain a concern. objectives: the single hub and access point for paediatric rheumatology in europe (share) consortium aimed to develop best practices for paediatric rheumatic diseases in order to decrease differences in care between european countries. here, we present the final results of the literature review and a series of consensus meetings on defining overarching, diagnostic and therapeutic recommendations for diagnosis and treatment of sjia. methods: the share methodology has been previously published, including the use of the eular standardized operating procedure for developing best practice recommendations. as per these guidelines, a methodologist provided supervision during the process and consensus meetings. conclusion: hscore seems to perform slightly better than ms-score for the diagnosis of mas in our cohort. early inhibition of il-1 is discussed to play an important role in the disease course of sjia 1 . assuming that pretreatment with other dmards leads to a later start of therapy with canakinumab, this analysis evaluates the effectiveness of canakinumab as first-line vs. second-line dmard. to evaluate the effectiveness of canakinumab as first used biological dmard in sjia compared to canakinumab in sjia-patients pretreated with other dmards. methods sjia-patients documented in the german biologic registry for pediatric rheumatology (biker), who were exposed to canakinumab, were identified. for the first-line (fl) group dmard naïve patients were selected, prior treatment with corticosteroids and/or nsaids was allowed. patients receiving any dmard prior to canakinumab entered the second-line (sl) group. both groups were compared in a retrospective intention-to-treat-analysis. effectiveness canakinumab treatment showed good effectiveness in sjia both as first-and second-line dmard. after 6 months the use of canakinumab as first-line dmard is associated with higher response rates compared to second-line use. our data support the hypothesis that early treatment with canakinumab is associated with good therapeutical response and a positive effect on the disease course of sjia. results: a total of 11 children (9 girls) with sle were identified. median age of symptom onset and diagnosis was 14 years(range 8-17 years) and 11 years respectively. the presenting manifestations were fever(5), oral ulcers(3), alopecia(3), malar rash(4), photosensitivity(5), renal involvement (5), seizures(1) and gastrointestinal complaints (1) apart from some unusual manifestations of isolated peripheral arthritis(1), isolated bilateral pleural effusion(1), macrophage activation syndrome(2). laboratory investigations: hemogram revealed anemia in 8 children and thrombocytopenia in 5. urine examination showed nephrotic range proteinuria in 1 child and subnephrotic proteinuria in 2. microscopic hematuria was noted in 2 pateints. renal function tests were deranged in 2 cases. ana, anti dsdna positivity and hypocomplimentemia were present in all. renal biopsy was done in 4 patients, 2 had class iv, one class iii and one had class v lupus nephritis. all patients were initiated on hydroxychloroquine and photoprotection. children with renal involvement were given pulse methylprednisolone followed by tapering doses of oral prednisolone and intravenous, monthly cyclophosphamide. azathioprine was used as maintenance therapy in all. subcutaneous weekly methotrexate was used in 2 patients. one child (mas) died during disease course. disease continues to be in remission in rest. conclusion: we found a significant female preponderance in our study group. renal involvement was the commonest presentation. some unusual presentations were also seen. early recognition of sle is critical for timely initiation of appropriate treatment. this is the first report of a cohort of pediatric sle from this part of india. introduction: autoantibodies in ahai may be igg/igm/iga. ahai can be divided into primary or secondary (e.g. sle, lymphoproliferative diseases, infections, medications). it is also classified based on the temperature at which the antibody reacts to erythrocytes, and can be warm (igg or iga) or cold (igm or c3). in warm ahai, the antibodies react at temperatures ≥37ºc, not activating the complement system and not undergoing agglutination in vitro. in cold ahai, antibodies react at temperatures below 37ºc, activating the complement system with in vitro agglutination.mixed aiha (warm and cold) is rare and occurs in <10% of aiha cases and can occur at any age, but is extremely rare in children. the prevalence of the mixed form is less than 1/1,000,000 patients with ahai. objectives: to report a rare case of mixed ahai and idiopathic intracranial hypertension(iih) in a 15-years old female patient with a previous diagnosis of sle and aps. methods: case report and literature review. results: a 15-years old female adolescent previously diagnosed with sle/aps since 2017 was in remission on hydroxychloroquine(400mg);azathioprine(150mg);aspirin(100mg);vitamind3(1.000iu);calcium(1g), and sunscreen. in april 2020 , she had a relapse presenting with fatigue, myositis, headache, hypocomplementemia, and severe autoimmune hemolytic anemia (hb of 4g/dl) (sledai-2k=18 points). mixed ahai was diagnosed base on a direct/indirect coombs test 4/4+;directantiglobulintesting showing anti-iga(weak),anti-igm(3+/4+),anti-igg(3+/4+),anti-c3c(weak),anti-c3d (3+/4+);igg1/3subclasses with a reaction of 1:100(2+/4+);an eleven cell antibody panel positive revealing a cold and warm antibody, and adsorption technique revealing a cold and warm autoantibody. chest ct showed bibasilar subsegmental atelectasis, head ct/mri was normal and lp showed a high opening pressure of 45cmh2o with a normal cell count. after the procedure, the patient reported improvement in the pain and was diagnosed with iih. the patient was screened for secondary causes for ahai (table 1) due to the unusual mixed type pattern and serology was positive for chlamydia trachomatis (igm) and mycoplasma pneumoniae (indeterminate-igm/positive-igg) suggesting a recent infectious trigger causing reactivation of the underlying disease with a probable cross-reactivity. the patient treated with 10-days of clarithromycin. before the infectious screening came back negative, ahai was treated with a single dose of ivig(1g/kg) and then, with 3-days of methylprednisolone(1g/day). azathioprine was replaced by mycophenolate mofetil. due to headache recurrence, acetazolamide(500mg/day) was started, and the patient referred no pain. the patient was discharged with a resolution of the symptoms. objectives: to our knowledge, the association of gbs and bbe has been described in adults only. methods: we here describe a child presenting at sle disease-onset with an overlap of peripheral (gbs) and central (bbe) nervous system manifestations, highlighting the possible association between these two entities in children. results: an 11-year-old healthy girl presented with acute ataxia, ophtalmoparesis and altered level of consciousness, rapidly followed by areflexia, facial paresis, swallowing difficulties, sensory deficits, paresis in all four limbs and respiratory insufficiency. these symptoms were accompanied by pleuro-pericardial serositis, proteinuria and hypertension. immunological investigations revealed the presence of positive ana and ds-dna antibodies. the renal biopsy showed a stage iii lupus nephritis. hence, the clinical, laboratory findings and biopsy report led to the diagnosis of psle. brain and spine mri did not show any abnormalities; diffuse slowing compatible with nonspecific encephalopathy was seen on eeg. nerve conduction studies (ncs) confirmed the clinical suspicion of acute polyradiculoneuropathy with proximal interruption of motor nerve conduction, compatible with guillain-barré-like syndrome. csf analysis (performed twice) remained normal. the patient was treated with glucocorticoids, intravenous immunoglobulins, cyclophosphamide as well as plasmapheresis. the neurological and physical symptoms improved gradually with complete neurological recovery four months after onset. conclusion: overlapping forms of bbe/gbs have never been described in association to sle in children. our patient's presentation and evolution fulfilled the criteria for such an overlap, occurring at psle onset. although sle and bbe/gbs are rare entities, our case suggests that there may be a common underlying immune background. this association should be recognized early for rapid and appropriate treatment initiation. infantile antiphospholipid antibody syndrome: acquired and de novo apl appearance in four infants t. giani 1 , g. ferrara 2 , a. mauro 3 , r. cimaz 4 introduction: antiphospholipid syndrome (aps) is a rare condition in the neonatal age. in most cases it is considered a passively acquired autoimmune disease, due to a transplacental passage of maternal antiphospholipid antibodies (apl). exceedingly unusual is the de novo production of apl in newborns and infants. objectives: to describe four infants who developed an early brain stroke with increased and persistent levels of apl, even after six months of life. methods: we reviewed the clinical charts of four such infants, followed from diagnosis up to two years after the disappearance of apl. conclusion: common characteristics of these four children are the development of brain stroke and the increased and persistent apl levels even after six months of life. this opens the window on a gray zone related to the origin of these antibodies (maternal or neonatal) and on their role in the pathogenesis of the infantile brain stroke. patients had over 20% of their monitoring completed but only 2 had over 80%. aspects of monitoring that were more time intensive or were required less regularly were most frequently overlooked. there was a statistically significant increase in the percentage of completed monitoring in those patients for whom the lupus checklist was used compared to patients where a checklist was not used (p=0.00). conclusion: there is significant room for improvement in the monitoring of these patients with jsle in the rheumatology clinic. this audit illustrates that more diligent use of the lupus checklist and an overall improvement in sustained use of the checklist will help to improve monitoring of these patients. evidence suggests that checklists are underutilised in medicine and wider implementation of this simple tool could improve patient outcomes. 3, 4, 5 interventions such as in person or electronic reminders, or audits with feedback to physicians could improve usage over time. the application of the lupus checklist or a similar document in other paediatric clinics is important for comprehensive monitoring of a condition as complex as jsle and has the potential to prevent ongoing damage and medication toxicity in this high-risk population. juvenile onset and this cluster have may more severe kidney, neuropsychiatric or hematological involvement. objectives: the aim of this study was to assess the clinical and laboratory characteristics, disease activity, and treatment response of patients with juvenile sle (jsle). methods: this is a retrospective study involving patients between 1 july 2016 and 1 january 2020. the data of patients diagnosed with jsle and followed up for a minimum of 6 months, were collected. the sledai-2k scores at initiation and at the follow-up (1st, 3rd, 6th, and 12th months of treatment) were examined. the sledai-2k score was considered to be ≤4, for disease remission status. results: a total of 49 children were included in to the study. the female/male ratio was 4.4/1 and the median age of the patients at the diagnosis was 13 (iqr: 11.1-15.2) years. the median follow-up of patients was 19 (iqr: 12-25) month. four of the patients were diagnosed with monogenic sle. two siblings were diagnosed with c3 deficiency and two were diagnosed with familial chilblain lupus. the most common clinical findings were found musculoskeletal complaints (69.4%), malar rash (51%), oral ulcers (38.8%), and fever (30.6%), respectively in over all the group. the frequency of involvement of the system and organs was as follows; mucocutaneous 77.6%, musculoskeletal 69.4%, renal 44.9%, hematological 34.7%, serous membranes 16.3%, neuropsychiatric 12.2%, respectively. all patients had anti-nuclear antibody positivity, while 46.9% had anti-ds dna, 14.3% had anti-sm and 8.2% had antiphospholipid antibody positivity. while all patients received hydroxychloroquine treatment, 22.4% of the patients were received were mycophenolate mofetil, 22.4% were azathioprine, 14.3% cyclophosphamide, 12.2% methotrexate and 10.2% were rituximab. the median sledai-2k score was 14 (iqr: 10-18.5) at admission, besides it was found to 6 (iqr: 4-12), 4 (iqr: 2-6), 2 (iqr: 0-6) in the 1st, 6th and 12th months of treatment, respectively. while 98% of the patients had active disease at admission, 67.3% at 1 months, 32.7% at 6 months and 22.4% at 12 months still had active disease (sledai-2k >4). patients with initially high sledai-2k scores had significantly lower remission rates in the first month (p=0.003). it was observed that patients with high sledai-2k scores in admission were more resistant to conventional immunosuppressive treatments and the use of rituximab was more frequent in these patients. at least one major organ (renal, hematological, neurological) were affected in 57% of patients. the remission rate of these patients at 6 months was found significantly decreased compared to the others (p <0.005). renal biopsy was performed in 21 patients (42.9%). 12 of them had type 4 lupus nephritis (ln), 5 had type 2, 2 had type 3, and 1 had type 5. it was observed that patients with renal involvement were the group that reached remission latest. conclusion: the presence of high initial sledai-2k scores and the major organ involvement have poor predictive value to achieve inactive disease. a two year old girl of consanguineous parents presented to hospital at 13 months of age with fever and erythematous macular rash on her cheeks which spread to her nose, chin, and ears. the rash started a month prior, and progressed over her entire body. a skin swab grew staphylococcus aureus but the rash didn't respond to topical antibiotics. review of systems was unremarkable except for longstanding oral thrush and diaper rash. birth and family history were unremarkable. on exam she had a diffuse, erythematous, morbilliform eruption over her face and body. she had facial swelling, orbital edema and vasculitic oral ulcers. she had leukopenia mainly neutropenia, low hemoglobin, with normal platelets. her liver enzymes and erythrocyte sedimentation rate (esr) were high while c-reactive protein, immunoglobulins, c3 and c4 were normal. cultures were negative, however she was positive for adenovirus, mycoplasma and ebv (ebv load was 6000 iu/ml ). autoimmune hepatitis work up was negative. the direct coombs test, antinuclear antibodies (1:640), ro, rnp and smd were positive. ch50 came low as well as c1q level of 4 mg/dl (normal range 12-22 mg/dl). lymphocyte subsets showed reduced cd4 and nk cells. bone marrow aspiration showed active marrow. skin biopsy showed chronic non-specific inflammation (immunofluorescence and electron microscopy were not available). echocardiogram showed dilatation of the left coronaries. she was treated with intravenous immunoglobulin (ivig) for kawasaki disease with no improvement. therefore pulse steroid 30mg/kg followed by 2 mg/kg was initiated. her rash, facial swelling and abnormal blood counts improved dramatically. whole exome sequence showed homozygous variant c.469g>t p.g157c at the c1qa gene. while tapering steroids she flared so subcutaneous methotrexate was started. unfortunately, she continued to have rash, leukopenia and high liver enzymes, so treatment was switched to mycophenolate mofetil and hydroxychloroquine. however she did not improve and started to have recurrent bacterial and viral infections that included cellulitis, gastroenteritis and upper respiratory tract infection. we started her on regular ivig, which helped with infections and allowed for weaning of steroids. however she developed alopecia and lower limb spasticity with delayed walking. mri brain and spine was normal. upon reanalysis of the wes, two other homozygous mutations at kif1c and apg7 were identified and associated with spastic paraplegia, but reported as variants of unknown significant. fresh frozen plasma (ffp) transfusions were started, initially weekly, then every two weeks and subsequently every four weeks. the rash disappeared, leukopenia and esr improved and we were able to discontinue steroids conclusion: early-onset sle with a severe course of disease raises the possibility of a genetic etiology. we are reporting, for the first time, a rare missense mutation g>t in exon 3 of the c1qa gene that resulted in an amino acid substitution that is pathogenic. interestingly, she had other mutations associated with neurological manifestation that never reported together before and altered her phenotype. she has responded well to ffp as has been reported in a few case reports results: a total of 148 psle under the age of 13 years were included, 30% (n = 44) were males. the overall mean age at diagnosis was 7.6 ± 3.5 years and median disease duration was 9.5 (5-13) years. huv was diagnosed in 34.5% (n = 51) of psle cohort. psle with uv were more likely to be males (57% vs 15%; p < 0.001), diagnosed at a younger age (5.9 vs 8.5 years; p < 0.001), have a family history of sle (53% vs 36%; p = 0.044) and have conjunctivitis more frequently (32% vs 5.3%; p < 0.001) than psle without uv. psle with uv were also less likely to have cns involvement (7.6% vs 20%; p = 0.045) and hematological manifestations such as leukopenia (9.4% vs 24%; p = 0.028) and thrombocytopenia (5.7% vs 18%; p = 0.045). in addition, psle with uv were more likely to be associated with low c3 complement count (94% vs 66%; p < 0.001) and positive cytoplasmic anca (11% vs 0%; p = 0.022).however, the psle with uv cohort were less likely to be associated with ana (65% vs 83%; p = 0.016), dsdna (56% vs 72%; p = 0.042) and perinuclear anti-neutrophil cytoplasmic antibodies (33% vs 55%; p = 0.047). conclusion: we report a high occurrence of huv in psle cohort (34.5%) associated with unique demographic, clinical features and laboratory features. the debate regarding whether huv is a rare subset or unusual type of sle, or is a separate entity altogether, continues. however, the overlap in clinical, laboratory and genetic mutation supports the notion that huv and sle fall into the same spectrum of autoimmune disease with similar disease pathogenesis. however, further studies are needed to reach clear conclusions regarding the relationship between huv and sle. introduction: the last decade has brought a lot to the approaches to the diagnosis and treatment of juvenile arthritis. in russia, the actualization of the problem of diagnosis and treatment of jia required the development of federal standards, which provide the most detailed algorithms for medical care, both at the stage of inpatient and outpatient care. in the regions of the russian federation, the effective use of these documents required a whole range of additional educated activities, both with students of medical universities, as well as with the medical and nursing community, in addition, a set of work was carried out to create a regional regulatory framework. in the total biological therapy pool, 67% of patients receive tnf-alpha inhibitors, antibodies to il-6 receive 27% of patients, antibodies to il-1 -6,25%. it is worth noting that when using biological agents in 60% of cases, the criterion of an inactive disease was achieved by 4-5 months, which was characterized by the absence of acute inflammatory symptoms, normalization of esr and crp. monitoring of patients with jia receiving biological agents required the conduct of a number of educational activities for medical personnel, the creation of an additional methodological base. for further training of young specialists at the regional medical university, a program of an additional educational course in pediatric rheumatology was developed and introduced. a regional patient organization was established and also required a set of information activities by the medical community. conclusion: in the saratov region of the russian federation, about 20% of patients with jia receive biological therapy, which corresponds to the average indicators according to the literature. in the structure of the biological drugs used, the group of tnf-alpha inhibitors is preserved -67%. the introduction of modern methods of treatment using biological agents in jia has significantly increased the effectiveness of treatment, but it required the organization of additional information support for medical personnel. disclosure of interest: none declared introduction: immunogenicity and development of anti-drug antibodies have been associated with treatment failure and adverse events during biologic treatment. anti-drug antibodies (adas) have been reported in 21% of juvenile idiopathic arthritis patients treated with adalimumab. however, their role in reducing adalimumab efficacy is still debated due to conflicting results. no study has been directed toward identification of neutralizing adas in paediatric rheumatic disorders. objectives: aim of our study was to detect adas, along with their clinical relevance, using a new theranostic peptide-base assay in a cohort of children with inflammatory chronic diseases on adalimumab treatment. methods: six candidate adalimumab derived peptide antigens (hc-cdr1, hc cdr2, hc cdr3, lc cdr1, lc cdr 2, lc cdr3) have been developed and optimized to be tested. their performance has been compared with commercial elisa kit and a spr-based optical assay (biacore®). assays have been performed in sera of a cohort of children receiving adalimumab due to an inflammatory chronic disease. mean age, disease duration, concomitant treatment with methotrexate (mtx), ana positivity, disease activity parameters and scores at the time of ada determination have been recorded. chisquare, and fisher exact test were used to compare data. pearson's and spearman's correlation tests were used to determine correlation coefficients for entered variables. results: eighteen (14 f, median age 12.6, range 3.8-16, yrs) patients were enrolled: 16 affected by juvenile idiopathic arthritis, 7 of whom complicated by jia -associated chronic uveitis, and 2 patients affected by chronic idiopathic uveitis. peptide assay revealed adas in 8 children, biacore in 6, commercial elisa in 5. of note, we found total concordance among the 3 tests just in 2 patients. no significant correlation has been proven among the 3 ada determinations. biacore and elisa determination showed significant concordance (r s : 0.72, p<0.006). the presence of hc cdr3 and lc cdr 3 resulted significantly correlated with disease activity (r s : 0.57, p<0.05), and, inversely, with disease remission on treatment (r s = -0.523, p<0.05). no patient experienced severe adverse events and no correlation with adas has been revealed conclusion: in chronic rheumatic disorders, novel reliable methods are urgently required to guide clinical decision and support decisions about switching within or between drugs in refractory children. the 3 different methods, since based on different antigenic probes, detect different antibody populations. the present peptide-based assays might contribute to identify neutralizing adas in patients treated with adalimumab. further validation in larger cohort is required. introduction: non-bacterial multifocal osteomyelitis (nbo) is a rare polygenic autoinflammatory disease, which is difficult to diagnose and treat. because of combination of bone lesions with arthritis and/ or axial skeleton damage in most cases the diagnosis of juvenile idiopathic arthritis (jia) or juvenile ankylosing spondylitis (jas) may be establish as a concurrent diagnosis, so this allows to legal use of biologics (ba) for the treatment. objectives: to analyze the single center experience of clinical and laboratory features of multifocal nbo in patients (pts) who were treated by ba for the last 8 years. methods: the study involved a retrospective cohort of multifocal nbo pts treated by different ba in our clinic from 2013 to 2020. all of them underwent standard rheumatological examination. in order to examine all localizations of the bone damage, a scintigraphy and/ or "whole body" mri scan was performed. results: among the whole group of pts with nbo (n=40) we identified 13 pts treated by ba (tnf-inhibitors only). the majority were girls (n=9, 69 %). age at disease onset was 10.2 years in average (me 10.2 range 1.3-16.5). for legal reason of ba administration, we classified our patients according to rheumatological features as jia or jas. 7 pts had jia (5 girls), 6 pts had jas (4 girls). among 13 pts 9 had oligoarthritis (69%), 4 had polyarthritis of low limbs (hip, knee, ankle). axial involvement was represented by active erosive sacroiliitis with deep bone marrow edema on mri scan in 9 pts (69%), active spondylitis of several bodies in thoracic spinein 2; erosive arthritis with partial ankyloses of facet joints of neck in 3 pts, multiple syndesmophytes in 1 girl. we found that definite axial lesions in nbo developed in very young children (in 2 y.old at minimum), much earlier than in "idiopathic" jas. hla b27 was presented in 5 pts (39%), 5 pts had ana in high titer (all of those hla b27-negative). the pts had bone lesions in different parts of skeleton: vertebral bodies -5 pts, clavicle -1, sternum, ribs -1, extremities bones, metaphysic mostly (tibial, fibular -7 pts), sacroiliac region -4 pts. extraskeletal manifestations were observed in 3 pts, one in each condition -uveitis, psoriasis pustulosus, acnae conglobate. in a girl with very severe course of disease, not responded to any therapy nbo was combined with familial mediterranean fever. high level of laboratory activity were detected before biologics in 10 pts (77%): esr acceleration up to 60 mm/h, increase of crp up to 80 mg/l. treatment included nsaids (all), methotrexate (7 pts), sulfasalazine (6 pts, but it was withdrawn in all pts), bisphosphonates (1 pt), prednisolone (3 pts). because of high activity of nbo with appearance of new bone lesions and persistent arthritis tnf inhibitors were administrated: etanercept in 10 pts, adalimumab -4 (2 as first line, 2second line), golimumab -1. at the start of ba the average age was 13.7 years (range 7.2-17.9); mean disease duration was 3,4 years (range 0.3-8.1). there were 2 cases of withdrawals. due to inefficacy etanercept was switched to adalimumab. disease activity decreasing was reached in the most of the patients (12 from 13). among them 2 pts developed the whole remission with resolving of active arthritis and bone marrow edema spots. skin lesions (psoriasis pustulosis and acnae conglobate) were significantly improved. there were no adverse events during the tnf therapy. conclusion: our experience of the therapy with tnf inhibitors in patients with high nbo activity has shown that this is a good and safe therapeutic option that is expected to prevent progression and bone destruction. . ae were reported for 71.7% of patients, most within 24 to 48 hours after the first or second injection: flu-like symptoms (57.5%), hypocalcaemia (37.5%) and hypophosphatemia (20%). underweight patients (body mass index < 18.5 kg/m²) accounted for 50% of hypocalcaemia. the frequency of all the ae not significantly decreased with the reduction of the first dose. only one serious hyponatremia occurred corresponding to a patient with renal failure before treatment. conclusion: our results were similar to those previously published: bisphosphonates are safe for osteoporosis in children. in the literature, sae are very rare in children, being limited to anecdotal osteopetrosis in cases of higher doses and long-term treatment, and delayed bone healing. anecdotal osteonecrosis of the jaw in adults has never been described in children. the use of bisphosphonates beforehand requires dietary measures (vitamin d and calcium supplementation). furthers systematic collection on efficacy and safety parameters for each bisphosphonates drug should confirm these data. introduction: the use of biosimilars in rheumatology has increased significantly over the last 5 years and has resulted in considerable cost savings. objectives: to assess the effectiveness and tolerability of the adalimumab biosimilar abp 501 in patients with jia. methods: a database of patients prescribed adalimumab in our service has been screened to identify patients with jia, who switched from the originator to the biosimilar. only patients who had a clinical review since they had started the biosimilar were included. a paired-samples t-test was conducted to compare the number of active joints at the clinic appointment before and after the initiation of the biosimilar treatment. the frequency and type of side effects, the clinical response and the number of patients who switched back to the originator have been collected. results: sixty-one patients who switched to the biosimilar abp 501 between february 2019 and february 2020 were included. they were comprised of 30 enthesitis-related arthritis (era), 13 polyarthritis, 9 oligoarthritis, 6 psoriatic and 3 systemic jia patients. their baseline characteristics and outcomes are summarised in table. the mean duration of follow-up after the switch to biosimilar was 10 months (range 2-23). eleven patients (18%) reported side effects; the most common side effect (n=7, 63.6%) was injection site reactions and the remaining 4 consisted of anaphylaxis, druginduced lupus, dizziness and bone pain, respectively. seven patients (11.5%) reverted to the adalimumab originator, 4 as a result of side effects, 3 because of ineffectiveness and one patient for both reasons. in addition, 3 patients were changed to a different biologic, one patient due to allergy to both the originator and biosimilar and the other two patients had active disease on the originator and biosimilar adalimumab. two patients stopped the biosimilar and remained off any biologic, in the first case this was due to a side effect and in the second case it was patient's choice. on the whole, 78.7% of patients had remained on abp 501 at their last visit. there was no significant difference in the active joint count before the biosimilar was started (mean 0.55+/-1.11) and after the switch (mean 0.6+/-1.59), (p= 0.855). introduction: golimumab (gol) is approved for polyarticular juvenile idiopathic arthritis (pjia) in patients of ≥2years but long-term safety data are limited. objectives: prospective monitoring of long-term safety and effectiveness of gol in routine care using the biker-registry. methods: baseline demographics, clinical characteristics, disease activity and safety parameters were compared to a contemporary 1:2 matched control cohort using alternative tnf inhibitors or methotrexate without exposure to a biologic. efficacy outcomes were jadas10, joint counts and functional status. safety assessments were based on adverse events (ae) reports. results: in this ongoing study, 65 pts initiating gol were matched to 130 with alternative tnfi and 65 biologic-naïve pts. pts starting gol had a longer disease duration (p<0.0001) and use of gol was significantly more often second line (84.6% vs 22.3%, p< 0.0001) and thus disease activity was lower at baseline. pts in the gol cohort used less corticosteroids, otherwise patients were comparable with pts treated with other tnfi (table 1 ). in gol treated ps a marked clinical response was noted at 6 months and beyond, indicating the effectiveness of gol in the treatment of pjia. a significant decrease of the mean jadas 10 11.3 to 5.3 (p= 0.0008) after 6 months of treatment was observed, as well as jia acr 30/50/70/90 response rates of 61/59/42/29%. jadas remission and minimal disease activity was observed in 27% and 53.7% after 6 months and in 39% and 54% after 12 months of treatment. rates of ae, sae and infectious ae were comparable in the gol cohort (87.5/100py, 3.4/100py and 11.1/100py), the alternative tnfi cohort (92.3/100py, 2.9/100py and 9.7/100py) and the mtx only cohort (121.2/100py, 2.1/100py and 18.5/100py). sae reported in the gol cohort were flares of uveitis and of jia (each 1) and fibromyalgia syndrome (1) . sae reported in the alternative tnf cohort was two serious infections (both influenza), one knee ligament injury, one flare of arthritis and one hyperventilation . no case of pregnancy, malignancy or death was reported. conclusion: golimumab seems an effective in treatment of pjia. tolerability was acceptable and comparable to alternative tnfi or mtx. recruitment to the project is ongoing. disclosure of interest none declared introduction: methotrexate (mtx) is one of the most commonly used disease-modifying anti-rheumatic drug in rheumatology practice. it has some side effects that can impair quality of life. the most common of them is associated with the gastrointestinal tract. objectives: the aim of the study is to evaluate and compare the frequency of methotrexate intolerance in adult and pediatric patients. methods: patients with rheumatologic diseases followed in hacettepe university pediatric rheumatology and rheumatology departments who used oral or parenteral methotrexate for at least 3 months were included in the study. methotrexate intolerance was assessed using 'methotrexate intolerance severity score (miss) questionnaire. the miss questionnaire consisted of 5 parts: abdominal pain, nausea, vomiting, fatigue and behavioral symptoms. the patients scored the severity of each symptom separately; 0: no symptoms, 1: mild symptoms, 2: moderate symptoms, 3: severe symptoms. a total score of 6 or more was defined as mtx intolerance. visual analogue scale (vas) ranging from 0 cm to 10 cm was performed to each patient concurrently with the miss questionnaire. in the pediatric patient group, miss questionnaire and vas assessment were applied to both patients and families. results: a total of 100 patients, 50 of whom were children, enrolled in the study. the mean age for children and adults were 11.78 (± 3.4) and 52.9 (± 11.8) respectively. the most frequent diagnosis of patients was juvenile idiopathic arthritis (78.0%) in children and rheumatoid arthritis in adults (68.0%). the mean mtx dose in adults and pediatric group was 12.5 (±3) mg vs 14.5 (± 3.6) mg (p: 0.004). the prevalence of mtx intolerance in children and adults were 66.0% (n:33) and 14.0% (n:7) respectively. the mean miss score in the pediatric group was higher compared with the adults (12.4±9.4 vs 1.84±4.5, p<0.001). similarly, the mean vas scores were higher in pediatric group (1.2±2.4 vs 4.2±3.2 (p<0.001)). there was a strong correlation between miss and vas scores between family and child evaluations (p <0.01, r = 0.95 / p <0.01, r = 0.94). abdominal pain, nausea, vomiting and behavioral symptoms were observed more frequently in children compared to adults. results: 3(4%) out of 73 patients were diagnosed with psoriasis denovo. one patient was treated with ada (a girl with undifferentiated arthritis who had positive hla-b 27, anf and family history of psoriasis -her grandmother had psoriasis), 2 patients were treated with eta (both female, one patient had undifferentiated arthritis, the other had enthesitis-related arthritis; both patients had positive hla -b 27 and anf negative). 2 patients achieved significant improvement after changing tnfalpha inhibitor (1-ada, 1-eta), 1 patient (was treated with eta) had significant improvement after discontinuation of biological therapy. conclusion: this single-center observational study demonstrates the possibility of developing psoriasis de-novo in patients with jia receiving tnf-alpha inhibitors. although more extensive research is needed, our data suggest that discontinuing the tnf-alpha inhibitor or switching to another tnfalpha inhibitor in patients with psoriasis de-novo should be considered as a treatment strategy in such cases. objectives: long-term surveillance of patients newly initiating toc treatment for at least 5 years compared to a cohort of patients newly initiating a comparator biologic using the biker-registry. methods: baseline demographics, clinical characteristics and disease activity, efficacy and safety parameters were compared. efficacy outcomes were jadas10, joint counts and functional status safety was assessed by adverse events (ae) reports. results: 161 patients with 161 matched controls have been recruited. patients starting on toc were older at treatment start (12.1 vs. 10.1 years (y); p<0.0001) and had a longer disease duration (p< 0.0001). toc was significantly more often a second line biologic (p< 0.0001). baseline jadas10 (17+/-10 vs 15+/-6), chaq-di (0.63+/-0.63 vs 0.65+/-0,64), esr 18+/-15 mm/h vs. 21+/-21 mm/h and active joint counts (7+/-7 vs. 6+/-5) were comparable. upon toc a substantial response with a significant reduction in jadas 10 from 16.8 to 3.4 (p<0.0001) after 12 months of treatment was observed. there were no significant differences between patients from the toc cohort and their matched controls in the jia acr 30/50/70/90 criteria, jadas 10, jadas remission and minimal disease activity was reached by comparable numbers (toc 37% and 58%; control cohort 37% and 60%). the total number of ae was comparable (toc cohort n=201 ae; (77/ 100py); control cohort n=207; (65/100py; rr 1.2; 95%ci 0.99-1.4). more serious ae (sae) were reported with toc. serious infections were documented at lower frequency with toc. uveitis events were documented at significantly higher frequency with tnf inhibitors most likely due to a selection bias (table 1) . sae with toc were depression (n=3) in 2 with suicidal intent, exacerbation of jia (n=2), septic arthritis, gastrointestinal infection, abdominal pain, colitis, paronychia and fracture. sae in the control cohort were depression, osteomyelitis, gastrointestinal infection and disease flare. no significant differences regarding cytopenias and elevated transaminases were observed. no gastrointestinal perforation, no vascular events and no deaths occurred. conclusion: toc was effective and comparable to treatment with alternative biologics. tolerability was acceptable. as toc was given as a second-line biologic in the vast majority of patients comparisons between the 2 cohorts have to be interpreted carefully. observation is ongoing. conclusion: in this retrospective cohort study in pediatric patients on rtx-treatment, we found undetectable low drug levels in adapositive patients, indicative for their neutralizing capacity. consequently, the lack of b-cel depletion leads to reduced treatment efficacy. patients with sle seem more susceptible to develop ada. if ada are detected, continuation of treatment seems non-effective and changing medication is advised. certainly when considering that, in this study, anaphylactic reactions only occurred in ada-positive patients. none declared objectives: the aim of this study was to evaluate retrospectively the long-term efficacy and safety of adalimumab in patients with jiaassociated uveitis. methods: we have retrospectively analysed nineteen jia patiens data with associated uveitis from our centre registry between 2010 and 2020, treated with adalimumab after failure of treatment with corticosteroids and metotrexate. demografic data and blood samples were collected at different time points while uveitis activity was evaluated by slit-lamp biomicroscopy. adverse events were recorded. results: registry records provided 10 years follow up of 19 jia patients data with associated uveitis. eleven patients were females (57.90 %) diagnosed as oligo/extended oligoarticular jia while eight (42.10 %) were males diagnosed as enthesitis related arthritis (era). before adalimumab was prescribed, all patients were previously treated with metotrexate during 3.5 years in avarage dose of 10 mg/ m 2 weekly. the mean uveitis duration, before adalimumab administration was 9 months. ten years long follow up period have showed that there were no new relapsis of uveitis while patients were receiving adalimumab and metotrexate. all of our patients were able to gradually tapper and stop treatment with topical steroids two months after adalimumab commencing. seven patients were able to stop biological treatment after 4.3 years of adalimumab usage. uveitis relapsed three monts after the adalimumab discontinuation only in one patient. two patient were lost to follow up during the transitional period. no serious adverse events were recorded. conclusion: during the long term follow up period adalimumab have shown good efficacy and safety profile in jia patients with active inflammatory ocular disease. introduction: post-streptococcal syndrome is a systemic immunemediated complication of beta-haemolytic streptococci infection, mostly seen as post-streptococcal arthritis, rheumatic fever or glomerulonephritis. uveitis is an uncommon manifestation of this syndrome. objectives: case report methods: case report results: a previously healthy 7-year-old female was admitted at the emergency department with prolonged fever, arthritis and red eye. she had a 4-month history of febrile episodes every two weeks, with axillary temperature ranging from 37,8 to 39ºc. migratory arthralgia affecting both knees and tibiotarsal joints showed up two months after the fever onset and worsened in the previous week, with refusal to walk. non-painful bilateral red eye for several weeks was mentioned. other symptoms were absent. recent infections were denied and family history was irrelevant. physical examination revealed lower limb muscular atrophy, knees pain and impaired function and bilateral tibiotarsal arthritis with inability to walk. ophthalmological observation showed a bilateral non-granulomatous anterior uveitis. sequential laboratory work up revealed a maximum eritrocitary sedimentation rate of 135 mm/h, maximum c-reactive protein of 5,3 mg/dl, microcytic hypochromic anemia, positive antistreptolysin o titer (asot) (initial result of 1250 that increased to 2500 in 4 weeks and later decreased to 500) and negative anti-nuclear antibodies. cardiac involvement was excluded. the diagnosis of rheumatic fever with concomitant poststreptococcal uveitis was assumed and the patient was treated with oral and topical ophthalmic corticosteroids with prompt clinical resolution of fever, acute polyarthritis and uveitis. no relapse occurred in a 5-year follow-up. conclusion: juvenile idiopathic arthritis (jia) is the most common cause of uveitis in childhood. although our patient clinical course could initially raise the possibility of systemic jia (sjia), the criteria that define this entity weren't all present and clinical and laboratory findings were more supportive of rheumatic fever. besides, uveitis occurs exceptionally in sjia, which turned this diagnosis even less reasonable. in our rheumatology unit, among 563 patients diagnosed with jia in 32 years, 89 had uveitis. however, in the group of 51 patients with sjia only one had ocular involvement, a boy with isolated vitritis. post-streptococcal uveitis (psu) typically presents as bilateral, non-granulomatous anterior uveitis, as described in this case. as streptococcal infection is very common among children and many patients may experience subclinical infection. psu should be considered in all patients with uveitis along with positive asot and negative routine investigations for other causes. although psu has been described in literature, to the best of our knowledge, this is the first reported case of concomitant rheumatic fever and psu. . ada was first tapered to every 3 weeks by 76% of the responders and then to every 4 weeks by 49% before discontinuing. fewer respondents used or tapered ifx, toc or aba. around 65% tapered the interval and 20% tapered the dose and interval for aba, 26% for toc and 37% ifx there were differences in the duration of tapering prior to discontinuation of specific medications. for ada it was 6 months in 62%, 12 months in 36% ,and 24 months in 10%. for ifx it was 6 months in 27%, 12 months in 45%, and 24 months in 33%. for toc it was 40% after 4 weeks, 87% after 6 weeks and 53% after 24 weeks. for aba i.v. it was 30% after 8 weeks, and 90% after 12 weeks. if combination therapy was used, 36% tapered the bdmard first, 62% csdmard first, and 12% both simultaneously. conclusion: this is the first survey to describe "real world" medication tapering and discontinuation practices of pediatric rheumatologists and ophthalmologists globally. most physicians start to taper medication after 24 months of remission on medication and discontinue after the 6 to 12 months of tapering. we would like to thank all the participating colleagues, who took time to fill out our surve introduction: jia-associated uveitis (jia-u) occurs in 10-20% of children with juvenile idiopathic arthritis (jia) and typically asymptomatic, and sight-threatening complications occur in 50% of children, (i.e. cataracts, vision loss). frequent ophthalmic examinations are important for early diagnosis and monitoring of uveitis activity. even after uveitis is controlled, risk of disease exacerbation still exists. therefore, frequent ophthalmic screening and monitoring is important for detection and management of jia-associated uveitis (jia-u). s100 proteins, cytokines, and chemokines detected in aqueous humor of patients with uveitis are also detected in tears. biomarker discovery using tears is promising since collection is noninvasive, feasible, well-tolerated, and close to the target organ. objectives: we aim to determine if s100 proteins, cytokines, and chemokines levels differ in tears of children with jia and jia-u and in children with jia-u by uveitis activity. methods: tears were collected using schirmer strips from children ≥5 years old with oligo-or polyarticular rf negative jia with (jia-u) and without uveitis (jia-no-u), and in children with jia-u at time of active and inactive eye disease. activity was defined by standardization of uveitis nomenclature (sun) criteria. active uveitis was anterior chamber inflammation grade ≥0.5+ cells. s100a8, a9, and a12 were measured by elisa, and il-18, il-8, ip-10, mcp-1, rant es, and sicam-1 by luminex assays. biomarker levels were compared in children with 1) jia-no-u (n=8) to active jia-u (n=8), and 2) jia-u (n=8) at time of active and inactive uveitis. results: children with jia-no-u and jia-u were matched by jia subtype and arthritis activity. they had primarily oligoarticular jia (63%), active arthritis (25%), and were on systemic medication (75%). at time of active uveitis, 75% had grade 0.5+, and 25% had 1+ and mean interval between time of active and inactive disease was 11 months. we found that levels of biomarkers in tears of children with jia-no-u compared to active jia-u were similar. although not statistically significant, levels of s100a12 (mean difference 12,190 pg/ml [95% ci -4847 to 29,227], p= 0.14) and sicam-1 (5329 pg/ml [95% ci -5372 to 16,031], p=0.28) were higher when uveitis was active compared to inactive. conclusion: our results suggest that s100a12 and sicam-1 are potential biomarkers of uveitis activity in jia-u, but not uveitis diagnosis. thus, neutrophils may play a role in the pathogenesis of anterior uveitis which has been reported in an animal model of acute anterior uveitis. identifying biomarkers using tears provides a noninvasive and objective method of monitoring uveitis. limitations are our heterogeneous cohort that varied by arthritis severity and immunosuppression, and minimally active uveitis. we were underpowered to detect statistically significant differences and continue to collect tears prospectively in children with jia-u with goal of n=28. despite low uveitis activity, we were still able to detect differences. further studies in larger and diverse cohorts are necessary to assess the role of s100a12 and sicam-1 in jia-u. objectives: to report an extremely rare presentation of gpa in a 12 year old with acute digital ischemia. a 12 year old boy, with a background of poorly controlled type 1 diabetes and hypothyroidism, initially presented to hospital unwell with diabetic ketoacidosis. treatment was initiated promptly with good response. furthermore, he was found to have weight loss, productive cough and hearing loss over the past 3 months. he was haemodynamically stable, but very pale and cachectic. he had reduced air entry and crackles on the right. there was hypertonia and clonus in his lower limbs. blood tests showed microcytic hypochromic anaemia (hb 82g/l), normal white cell count, thrombocytosis and raised inflammatory markers (crp 138mg/l and esr 68 mm/hr). his chest x-ray showed enlargement of the right hilum with consolidation/ atelectasis extending into the middle and lower lobes. mri scans of head and spine were normal apart from fluid opacification in the paranasal sinuses. he was screened for infections including tuberculosis and started on intravenous antibiotics. on day 13, he developed painful bluish discolouration of his left hand, particularly his thumb, index and middle fingers. his left radial and brachial pulses weren't palpable. a heparin infusion was started. a doppler scan showed occlusion of radial and ulnar arteries proximal to the wrist with no clear thrombus. he had a ct thoracic aorta with contrast which showed proximal left radial artery occlusion and distal ulnar artery occlusion with no evidence of proximal embolic source or vasculitis. it showed multiple perihilar masses (lymph nodes) in the right lung and peripheral parenchymal masses in both lungs, suggestive of atypical infection or connective tissue disease. blood tests still showed raised inflammatory markers(crp 107mg/l, esr 86 mm/hr and platelets 658 10 9 /l). an autoantibody screen showed positive anca with strongly positive anti pr3(>100 u/ml); other autoantibodies, including ana, ds dna and anti-phospholipid antibodies, were negative. he developed further ischaemia with bluish, painful discoloration of his right foot, especially right great toe, with a weakly palpable dorsalis pedis pulse. doppler scan revealed occlusion/narrowing of the posterior tibial artery 6cm proximal to the ankle. following vascular team advice, he was started on ilioprost infusion to aid reperfusion of the extremities involved, with good results. based on clinical and lab features of systemic inflammation, evidence of upper airway involvement(bilateral conductive hearing loss and sinusitis on mri scan), parenchymal lesions on ct chest and strong pr3 positivity, a diagnosis of gpa was made. results: our patient responded well to therapy including multiple pulses of high dose methylprednisolone and cyclophosphamide, with improvement of all organs involved and no further digital ischemia. conclusion: although gpa is very rare in children, it is associated with high morbidity and mortality. many studies show that the spectrum of paediatric gpa is not vastly different from adults, except for higher gender bias towards female, more constitutional and musculoskeletal symptoms and higher risk of subglottic stenosis. although there are a handful of case reports of digital ischaemia in adults with gpa, to our knowledge this is the first case report of acute digital ischaemia in paediatric gpa. early diagnosis and prompt treatment with a multidisciplinary team approach is paramount for good outcome. introduction: adenosine deaminase-2 deficiency (dada2) is a monogenic vasculitis syndrome whose presentation ranges from recurrent fevers and livedo reticularis to systemic vasculitis, hematologic and immunologic abnormalities, and early-onset stroke. it is characterized by biallelic loss-of-function mutations in the encoding gene of ada2 protein and low levels of ada2 enzymatic activity in the peripheral blood. the genotype and phenotype features of dada2 has a wide spectrum. treatment with anti-tnf inhibitors is effective in controlling vascular inflammation and reducing strokes. objectives: to describe two sisters with different presentations of dada2 and a deletion mutation on exon 7 of the ada2 gene. methods: medical data was used to describe the clinical manifestations of two siblings. parental informed consent was obtained. results: patient 1: a 10-year-old female had presented with fever, rash, arthralgia, hepatosplenomegaly, and coombs positive autoimmune hemolytic anemia (aiha) at the age of 7 years. she had been followed with a suspected diagnosis of systemic lupus erythematosus (sle) and steroids, azathioprine, mycophenolate mofetil had been used. her ana and complement levels were normal. because of unmet classification criteria of sle, genetic testing had been done, and no mutation found in the ada2 gene. cranial mr and mr angiography was normal. she was referred to our clinic after 2.5 years of the first manifestation. physical examination revealed raynaud phenomenon on both hands and feet, livedo reticularis, arthritis, and splenomegaly. laboratory tests indicated an increase in acute phase reactants, cd19, cd20, and switched memory b cell lymphopenia, and hypogammaglobulinemia. because of prolonged fevers, a thorax ct was obtained and aneurisms of the renal artery were seen. abdominal ct angiography indicated multiple aneurysms of both renal, intercostal, and hepatic arteries. repeated genetic analysis of the ada2 gene showed a homozygous deletion mutation on exon 7. she has been followed on anti-tnf and iv immunoglobulin without severe symptoms for a year. patient 2: the older sister had been followed with a diagnosis of familial mediterranean fever with e148q heterozygous mutation because of recurrent fever, abdominal pain, erysipelas-like erythema, elevated acute phase reactants, and splenomegaly. she did not have any other cutaneous or systemic findings. because of parental consanguinity, the ada2 gene was analyzed and a homozygous deletion mutation on exon 7 was found. she has been followed without any symptoms after anti-tnf treatment. throat swab was negative. abdomen ultrasound showed bowel wall thickening, testis ultrasound was normal. hsp diagnosis was confirmed. methylprednisolone iv was administered for three days, then oral prednisone was started. purpuric lesions, abdominal pain persisted, so we decided to add mmf (600mg/ m 2 /day) and prednisone was tapered in a month. results: thanks to mmf vasculitis lesions and abdominal symptoms disappeared in few days. mmf was continued for a month, tapered in 6 months. there was no evidence of relapse in a 6 months follow up. conclusion: these cases suggest that mmf may be useful to induce and maintain remission of recurrent hsp with gastrointestinal involvement. multicenter clinical trials with long-term follow up to confirm the efficacy of mmf in the treatment of hsp with gastrointestinal involvement are needed. introduction: henoch-schönlein purpura (hsp), the most common childhood vasculitis. cholecystitis is extremely rare in patients with hsp. this is the first case of a libyan child presenting with hsp complicated by calculus cholecystitis hsp nephritis. objectives: our aim is to present an unusual case of gall bladder involvement in an 8-year-old libyan female affected by hsp. methods: a case reports study results: : we report an unusual case of gall bladder involvement in an 8-year-old libyan female with hsp. she was referred to a rheumatology clinic due to hsp with chronic calculus cholecystitis and distended small bowel with fluid-like fecal material with no evidence of intussusception on an abdominal ultrasound. the patient had a one-month history of abdominal pain, purpuric lesion on lower limbs and swelling in both feet. she was admitted 3 times to another hospital before being referred to the rheumatology clinic. an abdominal sonography revealed a distended small bowel with fluid-like fecal material with no evidence of intussusception and chronic calculus cholecystitis; they treated her with urosdoxycholic acid tab at 250mg per day and ibuprofen syrup. then referred to our rheumatology clinic. after 40 days, she showed a purpuric rash over her lower extremities, mainly over her thighs and buttocks, microscopic hematuria, no arthritis, no fever, no abdominal pain; her blood pressure was normal at90\55mmhg, and she had normal laboratory tests (cbc, wbc 7.7, hgb 10.8, platelets 356 esr 20ml\hour, crp 1mg\dl was negative, c3 was 150mg\dl within normal range 90-180mg\dl, c4 was 35.4 mg\dl within normal range 10-40, anca, ana, as well antidsdna ab yielded negative, antistreptolysin-o (aso) titer was 250 todd , lft included total bilirubin , direct , indirect gpt,got, u�, creatinine ) except urine routine showed mild microscopic hematuria rbc 100 hpf , protein was nil ) urine for pro-tein disclosure of interest none declared p161 correlation of serum neopterin levels with disease activity and moneta 1 1 division of rheumatology, irccs, ospedale pediatrico bambino gesù, roma; 2 university of genova a multinational study of thrombotic microangiopathy in macrophage syndrome clinical impact of a targeted next-generation sequencing gene panel for autoinflammation and vasculitis laboratory biomarkers to facilitate differential diagnosis between measles and kawasaki disease in a pediatric emergency room: a retrospective study a rare case of measles-associated hemophagocytic lymphohistiocytosis in an infant. cureus children's interstitial and diffuse lung disease respiratory complications of the rheumatological diseases in childhood on behalf of dr nishant dharsandiya and dr j.p. keshrani paediatric rheumatology & immunology, dev children's hospital arthritis care res (hoboken) disclosure of interest: none declared development and initial validation of the ms score for diagnosis of macrophage activation syndrome in systemic juvenile idiopathic arthritis development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome double-blind, placebo-controlled study of anakinra in pediatric and adult patients with still's disease l. schanberg 1 , p. nigrovic 2 duke children's hospital & health center, durham; 2 boston children's hospital, boston; 3 children's mercy kansas city, kansas city; 4 university of alabama at birmingham, birmingham; 5 nationwide children's hospital, columbus; 6 university of iowa hospitals and clinics children and adolescents with sle: not just little adults severe disease presentation and poor outcomes among pediatric systemic lupus erythematosus patients in south africa the checklist manifesto: how to get things right clinical review: checklists -translating evidence into practice the who surgical safety checklist: a review pehlivanoğlu 3 p283 gastrointestinal henoch-schönlein purpura treated with mycophenolate mofetil: description of two case reports venous vessel wall thickness in lower extremity is increased in male patients with behcet's disease increased vein wall thickness in behçet disease polyarteritis nodosa: a contemporary overview eular / printo / pres criteria for henoch -schönlein purpura , childhood polyarteritis nodosa , childhood wegener granulomatosis and childhood takayasu arteritis : ankara six patients were treated with canakinumab and 2 patients with anakinra. conclusion: it is known that excessive production of il-1β can cause inflammatory bone loss and abnormality. vitamin d deficiency and osteopenia/ osteoporosis may cause additional musculoskeletal problems besides arthritis and joint destruction in caps. we think that ca metabolism and bone mineral density measurements should be a part of routine controls in patients with caps. disclosure of interest none declared ab007 clinical and genetic features of patients with periodic syndrome associated with mutation of the tumor necrosis factor receptor gene and juvenile arthritis having mutations in tnfrsf1a gene m active arthritis in 8/9, it was poly in 2, oligo in 6. when assessing the clinical symptoms and laboratory activity of patients with jia, it was revealed that in the onset of the disease, systemic manifestations were observed in 8/12: fever in 8/12, rash in 4/12, hepatosplenomegaly in 5/ 12, pneumonitis in 2/12, carditis in 1/12 and lymphadenopathy in 5/12. high laboratory activity was recorded in 11/12. active arthritis in 10/12, it was polys in 4, oligo in 6. in all 100% of patients, the nucleotide variants of the tnfrsf1a gene were identified in the study. 9/21 of patients were diagnosed with traps. the most frequent heterozygous variant of tnfrsf1a gene with nucleotide substitution of c.362g>a was found in 7/9 of patients, in 1/9 of patients it was found homozygous variant with nucleotide substitution of c.362g>a, in 1/9 of children it was found heterozygous variant with deletion of c.337_339del. all of these variants are pathogenic. 12/21 of patients were diagnosed with ja: juvenile arthritis with a systemic onset was in 7/12, paucarticular arthritis was in 2/12, in 1/12 it was poly rf-and in 1/12 it was psoriatic arthritis. it is worth noting to note that in 6/12 a heterozygous version of the tnfrsf1a gene was detected with a nucleotide substitution c.362g> a, however, considering the absence of clinical manifestations of autoinflammatory disease and active articular syndrome in these patients, children were diagnosed with ja. in addition dvoryakovskaya: none declared, a. mamutova speaker bureau of: novartis, k. isayeva: none declared, r. denisova speaker bureau of: novartis covid-19 and relapsing kawasaki disease: a case report during the pandemia m. c. maggio ab009 introduction: the pandemia of covid-19 remains a global health alarm with high incidence of lethality, especially in older age groups who suffer from underlying medical conditions. however, children are less likely to manifest severe conditions. objectives: covid-19 was correlated to a higher incidence and a suspected increased risk of kawasaki disease (kd) in children anamnestic records revealed a previous kd, without coronary artery lesions (cal), 1 year before. results: he was treated with antibiotics, intravenous infusion of immunoglobulins (ivig) (2 gr/kg), acetylsalicylic acid (asa) (50 mg/kg in 4 doses/day) and reached defervescence into 2 days. echocardiography excluded cal. the nasopharyngeal swab for sars-cov-2 was doubt. the second throat swab done the day after ivig infusion, was negative; however, the third nasopharyngeal swab for sars-cov-2, done 4 days after ivig infusion, was positive. chest x-ray showed a significant lung interstitial thickening. il-6 levels were < 6.25 pg/ml (n.v. < 6.25 pg/ml). he continued treatment with antibiotics, asa (5 mg/kg/day), with the progressive resolution of the clinical symptoms and of the normalization of laboratory findings. conclusion: the peculiar outcome of the patient is the correlation of covid-19 with kd, recently reported as associated. kd is considered as a multifactorial autoinflammatory disease, induced by a cytokine hypersecretion with a systemic vasculitis. covid-19 is considered a cytokine storm syndrome, with a severe systemic vasculitis. sars-cov-2 infection could be the trigger that could lead to hyperinflammation of kd. the ivig infusion could explain the transient negative swab for sars-cov-2, with the successive positive relieve lasting 7 days, and the normal levels of il-6, detected after ivig infusion. relapsing kd is rare (1.7-3.5%); in our patient this event could be triggered by the documented sars-cov-2 infection. disclosure of interest none declared disclosure of interest none declared ab012 spectrum of systemic inflammatory syndrome in children during covid 19 pandemic in india d. b. pandya, on behalf of dr haresh dobariya pediatric rheumatology & immunology, dev children's hospital ab025 rituximab for treatment of resistant paediatric mctd v. paisal, s. compeyrot-lacassagne paediatric rheumatology the diagnosis and classification of of mixed connective tissue disease mixed connective tissue disease in children -case series the value of rituximab treatment in primary sjögren's syndrome juvenile idiopathic arthritis a multifaceted approach is essential for robust rehabilitation m methods: in a retrospective study 92 children (89% girls) aged median (iqr) 4,2 (1,6 -7,6) years with oligoarticular onset jia without extra-articular manifestations (oligo-ja) who did not received dmards were monitored. all children were met ilar criteria. ttriamcinolone acetonide (ta) was administered intra-articular at a dose of 20-40 mg with an injection interval of 3-6-12 months which was depended on the activity of the disease. the maximum allowable number of consecutive isolated intra-articular injections (is-iai) was 3-4. a total of 218 active joints were injected with ta: knees -156 injections, ankles -62 injections. all children were divided into two groups: active / inactive arthritis based on the effectiveness of local corticosteroid treatment. the average follow-up was 48 and physicians' assessment of jia disease activity efficacy is-iai of ta was no associated significantly with number of active joint of onset oligo-ja, cjadas10, serum level of crp mg/ml, esr mm/h, il6 pg/ml and tnf-α pg/ml, titer of anf. the mean inflamed synovial fluid of il6 levels 2208 abstracts from conferences and relevant studies were added. rcts were included if (i) patients were aged ≤ 20 years, (ii) patients had a previous defined pediatric rheumatic diagnosis and (iii) rct met predefined outcomes. studies were excluded in case of (i) observational or single arm study or (ii) sample size ≤ 5 patients. study design, location, duration, treatment, population, sample size, age criteria, gender, concomitant treatments and primary outcome was extracted. results: out of 550 screened references, 62 references reporting 35 unique rcts in pird. all 35 rcts reported efficacy while 34/35 rcts provided safety outcomes and 15/35 rcts provided pk data. ten of 17 reviewed bdmards are approved for pirds by the food and drug administration (fda). of these, seven had ≤ 2 rcts. the most common intervention was tnf inhibitors (63%) treatment with intravenous immunoglobulin (ivig) significantly reduces the risk of caas. however, up to 20% of cases are ivig resistant with a higher risk of cardiovascular complications. currently several second-line treatments are available for refractory kd. nonetheless, the existing literature is still unable to identify which treatment is the most effective. recent studies suggest that a il-1 receptor antagonist (anakinra) may be an effective therapy in refractory kd. objectives: we report the case of a 3 year-old boy diagnosed with kd refractory to conventional treatment, who developed giant caas successfully treated with subcutaneous (sc) anakinra. methods: case report. results: a 3 year-old boy was referred to our pediatric rheumatology unit 18 days after the onset of a typical refractory kd. he had been previously treated at a local hospital with two doses of ivig (2 g/kg), infused respectively 8 and 11 days after the onset of the fever. afterwards, given the persisting fever, doses of pulse intravenous (iv) methylprednisolone (mpdn 30 mg/kg/day) have been used for 3 days followed by oral prednisone (2 mg/kg/day). treatment with acetylsalicylic acid (60 mg/kg/day q8h) was also started. following a transient defervescence the day after the first iv pulse mpdn, fever relapsed and the echocardiography showed caas of left main coronary artery (lmca), left anterior descending (lad) and right coronary artery (rca) rationale and study design for a phase i/iia trial of anakinra in children with kawasaki disease and early coronary artery abnormalities (the anakid trial) the use of interleukin 1 receptor antagonist (anakinra) in kawasaki disease: a retrospective cases series there are few reports of acute kidney injury (aki) in kd, defined as serum creatinine level elevation to more than 1.5 times of baseline level. objectives: to describe the case of kawasaki disease complicated by aki methods: a 5-year-old female was admitted to our rheumatology unit with persistent fever (6 days), widespread polymorphous exanthema, change in lips and in oral mucosa. family history was unremarkable. she had no chronic underlying disease nor history of previous hospitalization. at admission, she appeared stable. body temperature was 38.9°c, o2 saturation was 96% in ambient area, blood pressure was 118/75 mm hg, heart rate was 90 bpm, respiratory rate was 21 breaths per minute. on examination she presented widespread polymorphous exanthema, changes in lips and in oral mucosa, cervical lymphadenopathy and bilateral conjunctival injection. results: exams revealed: white blood cells 11980/μl, hb 10.4 g/dl, platelets 389.000/μl, albumin 2.5 g/dl, serum sodium 126 meq/l, serum chloride 90 meq/l. transaminases were in normal range. creatinine was 1.5 mg/dl disclosure of interest none declared ab041 paediatric extra-pulmonary large vessel arteritis, a forme fruste of pediatric behcet's disease? we presented two siblings from a consanguineous marriage with different clinical presentations of dada2. further, we emphasize that genetic testing should be repeated in the presence of clinical suspicion. introduction: there are several scoring systems developed in japan that are clinically used to stratify high risk kd patients and thus identify the ones that may benefit from early adjunctive therapy. there are increasing reports from all over the world on poor performance of these scores in other ethnic populations. objectives: the aim of our study was to evaluate the kobayashi, egami, sano and kawamura scores in our population which is homogenous caucasian. methods: hospital database was retrospectively searched for code m30.3 of the international classification of diseases, 10th revision, clinical modification code: mucocutaneous lymph node syndrome [kawasaki] , over the period from january 2006 to december 2019. all patients who were seen in this period for the first time for complete or incomplete kawasaki disease, as defined by the american heart association, were included. we applied ivig resistance prediction scores (kobayashi, sano, egami and kawamura scores) to our cohort. only patients who received 2g/ kg ivig within the first 10 days of the disease were included in this analysis. the scores of prediction models were calculated for each patient and patients were assigned to high-or low-risk group accordingly. results: during the study period a total of 169 children were diagnosed with kd (61.5 % males, median age 3.28 years). all of them were caucasian except one child who was biracial (caucasian and african american). among them, 158 children were hospitalized in the acute phase of the disease and 11 children were seen in the subacute phase of the disease. 151 children were followed-up for at least one year to evaluate persistent coronary artery aneurysms (caa), which were observed in 8 (5.3 %) patients. among them, 2 were not treated with ivig and 2 received ivig after 10 days of illness. 125 patients were treated with ivig within first 10 days of illness and were included in the calculation of ivig resistance prediction scores. 24 (19.2 %) were ivig resistant. sensitivity of kobayashi, sano, egami and kawamura scores were 0.53, 0.47, 0.61and 0.58, respectively. specificity of those scores were 0.77, 0.87, 0.75 and0.58, respectively. we found no difference in demographic or clinical characteristics between ivig resistant and ivig responsive patients. patients with ivig resistance had significantly higher alt (p = 0.025), neutrophil-to-lymphocyte ratio (p = 0.036) and lower serum sodium (p = 0.009). conclusion: by applying the japanese scores to our population, we were able to identify most of the low-risk, but missed many of the high-risk patients. our results are consistent with caucasi n based population studies available to date. introduction: varicella zoster virus (vzv) related arterial ischemic stroke (ais) has been described in literature in pediatric age. however, the long-term course of post-vzv vasculopathy need to be inquired: clear information about prevalence of recurrence and severity of clinical outcome are lacking, even if a favorable evolution was initially described, and therapeutic protocols are not currently standardized. objectives: we aimed to describe the clinical, laboratory and neuroradiologic features of children affected by ais due to post-vzv referred to our institute and to present our experience in their therapeutic management. methods: we selected 22 pediatric patients (6 females) with ais and a cns confirmed vzv reactivation and/or with a vzv history in the previous 12 months. other causes of pediatric stroke (systemic disease, cardiac disease, trauma, major thrombophilia) were excluded. clinical, neuroimaging, laboratory and treatment data were reviewed, focusing on pediatric score outcome measure (psom) and executive functions final outcome. results: average age of ais onset, vzv primary infection and interval between infection and ais were: 4 years 10mo (range: 1 year and 8 mo-9 years and 11 months), 4 years and 5 months (range 8 months-9.4 years), and 7 months (range 10days-34 months), respectively. the ais involved the nucleo-capsular region in 18 cases, the cerebral cortex in 9 cases, the thalamus in 4 cases, and the pons in 3 subjects. seventeen patients had inflammatory focal cerebral arteriopathy (ifca). virological confirmation (vzv-dna or anti-vzv igg in the cerebrospinal fluid) was obtained in 11 patients. three patients were treated with trombectomy and one with rtpa. thirteen patients were treated with antiviral agents associated with steroids in 8 cases, with different administration schedules. only in one case steroid treatment was given without association with antiviral agents. one patient received a short course of steroid and antiviral treatment at the time of the stroke and then a more prolonged course after six months at the time of the virological diagnosis. prophylactic antiaggregants were administered to all patients. mean follow-up was 2 years and 5 months (range 6 mo -10 years) ; ifca was persistent in 12 cases and transient in 5 subjects. four patients presented a recurrence of post vzv arteriopathy, two of them presenting new stroke events. twelve patients presented a variable motor deficit at last follow up. the mean psom score of the cohort at the last visit was 1 (range 0-2). executive functions were evaluated at last follow up in twelve patients, showing no deficit in seven patients, a mild deficit in two patients and a severe deficit in the last three. conclusion: albeit a favourable evolution was initially described, our experience suggests that vzv-related ais may result in persistent fca and significant neurological impairment in the majority of cases. therapeutic approach, particularly involving steroid administration, still need to be validated. introduction: iga vasculitis/ henoch schönlein purpura (igav/hsp) is the most common vasculitis of childhood and renal involvement is the most serious long-term complication. a better understanding of the pathophysiology of the progression to kidney disease is required for better treatment to be achieved and current biomarkers of ig a vasculitis with nephritis (igavn) lack the predictive value. objectives: in this study, an untargeted metabolomics approach was performed to reveal the underlying molecular mechanism of disease pathogenesis and to find potential biomarkers of plasma samples from patients with igav and igavn.methods: igav was diagnosed according to the ankara criteria in 2008 (1). forty-five patients, including 39 active igav patients (h), 6 igavn (n), and 6 age-and gender-matched healthy controls (c), were enrolled in the study. plasma samples from subjects were collected on the same day of igav(hsp) diagnosis and before steroid or other immunosuppressive treatment initiated. this study has utilized liquid chromatography-mass spectrometry (lc-ms/ q-tof) to investigate the alterations in plasma metabolomic profiles. three separate pools, health controls, active igav , and igavn were created. peak picking, grouping, and comparison parts were performed (metabolite profiling) via xcms (https://xcmsonline.scripps.edu/) software. results: totally 2618 peaks were detected for group h, n and c. among them 355 peaks were found to be statistically significant and reliable (p< 0.05) and 155 of these peaks were found to be changed (fold change >1.5) between the groups c and h. on the other hand, 66 peaks were found to be changed (fold change >1.5) between the groups h and n. the number of the peaks on the intersection of the peaks found to be changed between the groups (c and h) and (h and n) was 39. based on putative identification results, 15 peaks were matched with 11 metabolites. we found an up-regulated level of dhap(18:0), prostaglandin d2/i2, 5methyltetrahydrofolic acid, porphobilinogen and n-acetyl-4-o-acetylneuraminic acid/n-acetyl-7-o-acetylneuraminic acid, 5-aminopentanamide /5-aminopentanoic acid, glycocholic acid, saccharopine, n2-succinyl-l-ornithine, gamma tocopherol, and galactosylsphingosine /glucosylsphingosine in igav patients. in conclusion, we have identified a number of metabolites that may be associated with the pathogenesis of igav. we also suggest that dhap (18:0), prostaglandin d2/i2, porphobilinogen, 5-methyltetrahydrofolic acid and n-acetyl-4-oacetylneuraminic acid/n-acetyl-7-o-acetylneuraminic acid may serve as biomarkers for predicting kidney disease since they were increased only in the patients who developed renal involvement at follow-up. children were divided into four groups: those with jia who didn't receive mtx yet (group 1); those who received mtx less than one gram during whole treatment (group 2); those who received mtx from 1 to 3 grams (group 3); children, received more than 3 grams of mtx (group 4). the autoimmune inflammatory process in jia can cause formation of pathological changes in the liver, even before the start of treatment. it is confirmed by a statistically significant correlation of bfgf level in 1st group with liver steatosis according to ultrasound examination (r = 0.8) and the level of c-reactive protein (r = 0.7). this indicates a close relationship between the intensity of the inflammatory process and collagen synthesis activation, which can further provoke liver fibrosis. alterative processes in the liver associated with autoimmune inflammation, as evidenced by the presence of a positive correlation between the level of alt and bfgf (r = 0.5). upon reaching mtx dose 1 gram and 3 grams, it is possible that compensatory processes in the liver are triggered, as evidenced by the negative correlation between the content of bfgf and hgf (r = -0.6).conclusion: the use of modern markers with routine laboratory and instrumental studies is appropriate for the timely determination of the risks of developing irreversible pathological changes in the liver during jia treatment with mtx. objectives: the aim of our study is to evaluate the efficacy of (iag) injections in hip in children with (jia) and to assess the factors predicting the improvement of this management. methods: this is a retrospective study, between 2006 and 2009, including patients with jia diagnosed according to the ilar criteria. the socio-demographic data were collected as well as the parameters of the disease. the activity was evaluated by jadas. the functional impact was assessed by the lequesne score. the treatments taken have been specified as well as the infiltrations received. the improvement after infiltration was assessed by jadas and lequesne score.results: fourteen patients were included, with mean age 17.21 +6.8 . the mean age at the onset of symptoms was 11 +0.5 [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] . subtypes of jia according to the ilar were: enthesitis-related arthritis in 7 cases, seropositive polyarticular jia in 2 cases, seronegative polyarticular jia in 2 cases, oligoarticular jia in 2 cases and juvenile psoriatic arthritis in one case. all the patients had hip arthritis, inaugural in 90% of the cases. of these, 92.8% had a flexion deformity and lower limb inequality. the average lequesne index was 8.5 +4.6. the treatments taken were methotrexate in 57.14% of the cases, sulfasalazine in 14.28% of the cases, and the combination of the two in 21.4% of the cases. eleven patients underwent hip infiltration, and three of them required more than one. eighty one percent improved thereafter. the number of infiltrations was not statistically associated with the lequesne index (p = 0.069). improvement after infiltration was negatively associated with the prior existence of an inequality of the lower limbs (p = 0.04). the existence of a flexion deformity was not associated with good results after infiltration (p = 0.476, r=-0,624). ten patients (90%) among those who had an infiltration did not have to resort to surgery. conclusion: iag injection is an adjunct therapy in aji with hip involvement offering a good results and delay surgery in the majority of cases. the presence of lower limb inequality is associated with less improvement of iag. conclusion: synovial rice bodies are rarely described in juvenile idiopathic arthritis, even less at disease onset. their presence has not been associated to a worse disease prognosis or joint outcome but awareness of the existence of this particular form of intraarticular loose bodies may encourage the clinician to use lower gauge needle during arthrocentesis procedure; this can prevent arthroscopy, as occurred in our case 1. arthroscopy may be necessary in some cases to achieve full drainage of the joint. in our series the duration of arthritis correlated with the size of rice bodies and the number and agressiveness of procedures needed to evacuate them. objectives: we described a case of liver involvement in sle presenting with emphasis on the differential diagnosis with autoimmune hepatitis. methods: case report study results: : an 8-year-old female patient was referred to the rheumatology clinic with complaints icteric sclera for 10 months anorexia, malaise, pain in the both knees, ankles joints and both wrists accompanied by swelling, and remarkable motion limitations. laboratory revealed t bilirubin 4.9 mainly direct 3.9 with elevated liver enzymes got 401, gpt 189, alkp 520, high glutamyl transpeptidase 56u\l her wbc 7.4 hgb 10, ptl 317, except very high esr 105ml\hr, crp was positive 190mg\dl, viral screen (hcv, hbsag, hiv) was normal, serology tests ana was positive with high titer 1280, anti ds-dna ab was positive 320, anti-sm was negative, anti lkm1 antibodies negative, anti smooth muscle ab negative soluble liver antigen were negative, antimitohondrial ab( m1,m2,m3). ultrasound abdomen revealed mild enlarged spleen, abnormal diffused increased liver echogenicity with early stage of liver cirrhosis treated her by fresh frozen plasma 5 times, vit k 10mg once\ day then was referred to rheumatology clinic regarding her serology tests & developed arthritis of her joints suspected psle! she was performed liver biopsy showed lesions necrotic inflammatory portal and lobular severe in eosinophilic polynuclear with cirrhosis evoking a syndrome of overlap associating a primary biliary cirrhosis and an autoimmune hepatitis. laboratory data revealed liver dysfunction and liver biopsy suggesting autoimmune hepatitis, and she underwent treatment for hepatitis (prednisolone with azathioprine), urosdoxycholic acid with fat-soluble vitamins k, d&a, e. however, with the elimination of jaundice and decreased hepatic enzyme levels, the prednisolone dose was tapered within 2 months and stopped before they were referred to rheumatology clinic. on her review of systems, she has malar rash, generalized fatigability. on physical examination, we found malar rash, levidoreticularis of her skin, swelling and limitation of movement in the knees, ankles, wrists joints. there was hepatosplenomegaly. laboratory data revealed liver treatment for hepatitis, ana still high titer 1:1280, antids dan positive with titer 307 iu\ml, antisma was negative .wbc 4.5, hgb 11.8, plt 268, esr 68ml\hr, her ultrasound abdomen: revealed slightly heterogeneous liver with coarse echotexture without focal lesion with liver span 14 cm.these paraclinical results together with the clinical findings strongly suggested systemic lupus erythematosus (sle) as the definitive diagnosis. indeed, in this case, aih was associated with sle, prednisolone orally for 2 months, after that dose was tapered and continued, rapid clinical improvement in arthritis, malaise, and general condition. azathioprine was continued. in addition, daily hydroxycholoquine sulfate overlapping of sle and aih should be suspected when aih patients present with a malar or other skin rash. the prompt diagnosis and adjustment of further treatment plans can improve disease outcomes and prevent liver disease progression. introduction: juvenile systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by multi-visceral involvement with an unpredictable prognosis. the diagnosis is usually made in young women aged between 20 to 40years, however, it can affect people at any age and it is classified as a juvenile illness when it starts before the age of 16. objectives: we are reporting the epidemical, clinical, therapeutical and evolutional characteristics of a series done in the pediatric pole in setif with 13 girls and 1 boy. methods: the average age of onset is 13 years. the average time limits of the diagnosis is 7 months. the clinical features is done with cutaneous, articular manifestations and fever respectively in 100% 71% and 57% of the cases ,followed by kidney damage in 42% of the cases , the cardiac, pulmonary and ophthalmological participations are reported with low percentage. haematological involvement was detected in 85% of the patients and the inflammatory syndrome was almost constant. a positive titer of anti-nuclear antibodies and anti-dna is objectified, as well as a reduction in the complement rate. antibodies anti gp 2 and anti cardiolopine are positive in 57% of cases. kidney damage was diagnosed in 42% of the cases , and only one case of overlap syndrome with dermatomyosits was reported. concerning the neurological form it was present in only one addolecent girl ,and only one case of familial lupus.results: the diagnosis is based on the classification of the american college of rheumatology (acr) 1982 revised on 1997 and the new criteria slicc"systemic lupus international collaborating clinics" . the clinical characteristics of our series relies on global data of literature with the predominance of cutaneous and articular involvement. with however some specific characteristics which are individualized by a more advanced age of onset, 13 years on average in our study versus 10 years and 12 years, the rarity of familial forms (1 case), a lower percentage of kidney damage (42% versus 63% and 80%).the therapeutic management was based on corticosteroid therapy and hydroxychloroquine in the majority of cases, the use of immunosuppressants has been reserved for severe forms. conclusion: lupus is an autoimmune disease with protean clinical manifestations, the prognosis of which is dominated by renal, neurological and thrombotic disorders. cortisonic treatments and immunosuppressants have significantly improved the prognosis for life . trial registration identifying number: lupus is an autoimmune disease with protean clinical manifestations, the prognosis of which is dominated by renal, neurological and thrombotic disorders. cortisonic treatments and immunosuppressants have significantly improved the prognosis for life . onset of inactive and active oligo-ja were not significantly differ. the analysis revealed a correlation between a short phase of beneficial effect after is-iai of ta and risk of activity disease (with an inactive phase of arthritis less than 3 months, the risk activity was or = 2.09, p <0.001; with an inactive phase less than 2 months -or = 8.9, p < 0.001). rtx was administered to patients who had received high-dose cs with 2-3 dmards; in all cases combined pulse therapy cs № 2-10 was preliminarily used. rtx 500 mg № 2 was applied after 6mo-2y from the debut of the disease. in all 5 cases, its use led to clinical improvement after 1-5 mo with normalization of laboratory activity indicators, in 4 cases a decrease in the level of b cells to 0-0.56 in μl was noted (3 with agammaglobulinemia). after 2 months 3 patients had severe infectious complications, 2 of them ended fatally. 2 another patients had a second stroke. the 1st patient survived, had a kidney allotransplantation, there is no disease activity. the 2nd patient, in connection with the development of the demyelinating process of cns, attempted to continue therapy using golimumab with ivig. it led to an increase in the infectious syndrome, therefore, we decided to refrain from continuing with itnf as well. the patient died after 2 years from the administration of rtx due to the progression of neurological disorders. 2 cases with auto-inflammatory syndromes were: chronic infantile neurologic cutaneous and articular syndrome received tcz; it was unsuccessful (hyperthermia and rash persisted, eye lesions progressed, there were no increase in height), later switched to anakinra. family mediterranean fever, received adalimumab (ada). the 1-year-course of ada leaded to the disappearance of articular and abdominal syndrome while maintaining persistent increased levels of esr and crp and periodic fever. the use of tcz in 2 patients with ssd was more successful. the first patient received it subcutaneously for 1 year, cs&dmards (3 were used) had already been canceled, lung and kidney lesions were contained, blood pressure normalized, escsg-ai decreased from 7 to 1, mrss decreased from 18 to 14. in the second case, the patient received tcz for 6 months i/v, decrease of escsg-ai 6.5 to 1, mrss 33 to 21 were noted, the dose of cs was halved, he also continued treatment with cyclophosphamide. we introduce a 13-year-old girl patient who has been admitted to our clinic with suspicion of an erythema nodosum. she had painful subcutaneous nodules for 4 weeks, especially on the lower extremities and her face. macroscopically, central necrotizing skin rashes could be seen. she had frank arthritis of both knee and ankle joints. the comprehensive serological diagnosis (including hepatitis serology and anti-streptolysin titer) were normal except for a slight increase in crp 0,9 mg/dl and esr 36 mm/h. the patient also complained of abdominal pain and bloody stools. calprotectin was 3613 μg/g. a gastro-coloscopy revealed a small mariske and a minimal inflammation of the ileocecal valve, without signs of vasculitis or chronic bowel disease. a skin biopsy revealed leukocytoclastic vasculitis of the small arteries. angiography of the intestinal arteries was rejected by the family. initially we started a treatment with methylprednisolone pulses followed by oral prednisolone. the patient showed a very good response with quick resolution of the skin symptoms and abdominal pain. the medication could be quickly tapered and discontinued at full remission after one month results: pan is classified as a cutaneous pan (cpan) when there are exclusive skin manifestations, besides arthralgia or arthritis. a systemic pan must be diagnosed with the involvement of internal organs. however, cutaneous pan may evolve into systemic pan. in our patient, the skin and joints were primarily affected. if the existing gastrointestinal complaints are part of a systemic pan or chronic bowel disease could not be cleared yet, due to refusal of further investigations. conclusion: cpan must be considered as a suspected diagnosis in patients with necrotizing skin nodules. as transition of the cutaneous into the systemic form cannot be predicted regular monitoring is mandatory. introduction: prevalence of behcet's disease in children is not known, but is probably very low. extra-pulmonary large vessel arteritis in these cases is even rarer as a presenting manifestation. objectives: to report two cases of paediatric extrapulmonary large vessel arteritis with a 'behcet like disease'. methods: we present case reports of two cases who presented to paediatric rheumatology opd to our department. ms. f, a 16 year old girl was referred to us with history of short duration of fever, generalized lymphadenopathy, neutrophilic leucocytosis, thrombocytosis, hyperglobulinemia and high inflammatory markers. on detailed history and examination she was found to have a healed palatal ulcer and her maternal aunt was found to have a history of recurrent oral ulcer, genital ulcer and enthesitis. patient's montoux test was positive but the gene expert for mtb was negative. md-ct showed a circumferential thickening of aorta, subclavian and bilateral renal artery with stenosis at origin of both renal arteries indicating a vasculitis. few necrotic nodes were also noted in lungs. lymph node biopsy suggested a reactive hyperplasia. tissue typing showed presence of hla b 44, b 51. she improved clinically with oral prednisolone and mycophenolate mofetil and had no recurrence till her recent follow up visit. second case, master fkn an 11 year old child was referred to us with a background of 2 week history of fever, non migratory arthritis, raised inflammatory markers and a symptomatic severe aortic regurgitation with pandiastolic flow reversal on 2d echo. his evaluation showed negative montoux, normal igg4 levels and hla b35 b 51 on tissue typing. his aortic wall thickness resolved with 1 mg/kg oral prednisolone and mycofenolate mofetil. results: both these cases have features similar to behcet's disease. these cases do not fulfil isg, icbd 2014 or icbd criteria for pediatric behcet's disease. however, the aortitis and other clinical features responded well to the treatment in both cases.conclusion: paediatric case with extra-pulmonary large vessel arteritis that do not meet criteria for behcet's disease but have specific clinical or laboratory features do respond well to immunosuppression. therefore, after ruling out other causes of the large vessel vasculitis, a possibility of form fruste of behcet's disease should be under consideration. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. these abstracts have been published as part of pediatric rheumatology, volume 18, supplement 2, 2020: proceedings of the 26th european paediatric rheumatology congress (pres 2020). the full contents of the supplement are available at https://ped-rheum. biomedcentral.com/articles/supplements/volume-18-supplement-2. please note that this is part 2 of 2. key: cord-021266-afs9eb40 authors: el gendy, fady m.; el-mekkawy, muhammad said; el-naidany, sherin sobhy; el-torgoman, shimaa tarek title: the role of tumor necrosis factor alpha −308 g>a promoter polymorphism in pediatric community acquired pneumonia date: 2020-02-10 journal: nan doi: 10.1186/s43054-020-0019-1 sha: doc_id: 21266 cord_uid: afs9eb40 background: tumor necrosis factor alpha (tnf-α) −308 g>a promoter polymorphism might be associated with excessive production of the proinflammatory cytokine tnf-α, modulating host response to pulmonary infections. our objective was to evaluate the association of tnf-α gene −308 g>a polymorphism with susceptibility to, and severity of, community-acquired pneumonia (cap). results: this was a cross-sectional study including 45 egyptian children hospitalized for cap in addition to 45 healthy children who served as a control group. pneumonia severity was assessed on admission by the world health organization (who) guidelines; pediatric respiratory severity score (press) score; predisposition, infection, response and organ failure (pirom) score; and respiratory index of severity in children (risc) score. genotyping of tnf-α polymorphism was performed to all individuals by polymerase chain reaction and restriction fragment length polymorphism (pcr-rflp). patients were monitored till hospital discharge. frequency of ag genotype was lower among patients compared with control [odds ratio (or) and 95% confidence interval (ci) = 0.13 (0.03–0.63); p = 0.012]. prevalence of genotypes aa+ag was lower among patients compared with controls [or and 95% ci = 0.34 (0.12–0.99); p = 0,048]. the “a” allele prevalence was higher among controls, but no significant association was found with cap [or and 95% ci = 0.58 (0.25–1.35); p = 0.21]. when press score was used to classify patients into “severe pneumonia” and “non-severe pneumonia,” no significant association of any of the alleles or genotypes with cap severity was found. conclusion: tnf-α −308 g>a polymorphism confers protection from pediatric cap but is not associated with indicators of cap severity. larger studies are needed to confirm these findings in pediatric patients from different ethnicities. pediatric community-acquired pneumonia (cap) is defined as the presence of signs and symptoms of pneumonia in a previously healthy child due to an infection which has been acquired outside hospital [1] . despite marked decrease in childhood mortality and pneumoniaspecific mortality, cap remains the major cause of mortality in younger children globally, causing about 900,000 child deaths in 2013 [1] . in response to cap, an inflammatory reaction is produced locally in the lung which consists of both pro-inflammatory and anti-inflammatory cytokines, including interleukins (il), granulocyte colony-stimulating factor (g-csf), granulocytemacrophage colony-stimulating factor (gm-csf), and tumor necrosis factor alpha (tnf-α) [2] . tnf-α is potent pleiotropic pro-inflammatory cytokine produced mainly by activated macrophages, lymphocytes, and endothelial cells [3] . human tnf-α is a 17-kda protein that exists in a soluble form or a membrane-bound form. tnf-α gene is located within the major histocompatibility complex (mhc) on the short arm of chromosome 6 [4] . historically, the name "tumor necrosis factor" referred to a "factor" induced by bacterial infections that leads to tumor regression [5] . it was later discovered that tnf-α has a wide range of biological effects on host defense against pathogenic agents. it can induce cell survival, proliferation, and differentiation. it can also cause both apoptosis and necrosis under certain conditions [6] . tnf-α function is double-faceted. on the one hand, local production of tnf-α is beneficial in the acute situation since it increases expression of adhesion molecules on the vascular endothelial cells that help immune cells to migrate to sites of infection [4] . moreover, tnf-α activates phagocytes to engulf infectious agents. on the other hand, systemic or prolonged elevation of tnf-α level may be harmful. high level of circulating tnf-α is associated with toxic shock induced by endotoxins [7] . tnf-α injection into experimental animals causes a syndrome similar to septic shock [8] and infusion of tnf-α into humans results in systemic inflammatory response syndrome [9] . tnf-α and il-1 act synergistically to induce a shock-like state characterized by increased vascular permeability, pulmonary edema, and hemorrhage [10] . in addition, tnf-α was found to play a major role in the pathogenesis of inflammatory diseases like rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, behçet's disease, and inflammatory bowel diseases [11] . a large number of polymorphisms of tnf-α gene promoter have been described which are thought to affect production of tnf-α. one of these single-nucleotide polymorphism (snp) occur at nucleotide number 308 before the transcription start site where nucleotide "g" is changed to "a" (−308 g>a). this snp could potentially affect tnf-α synthesis at the transcriptional level. the "a" allele was suggested to be associated with higher tnf-α levels which may alter the course of immune response [12] . although fairly well evaluated in adult cap patients, previous pediatric literature evaluating the role of tnfα −308 g>a polymorphism in cap is scarce. the aim of the present study was to test the hypothesis that tnf-α −308 g>a polymorphism is a risk factor for susceptibility to cap and for having a severe disease course. this was a cross-sectional study conducted on 90 egyptian children from september 2018 to april 2019. the local ethical committee approved the study protocol. the study population included a group of 45 children with a diagnosis of cap who were consecutively enrolled from the ward and the pediatric intensive care unit (picu) of a university hospital after obtaining a written informed consent from the parents. another group of 45 age-and sex-matched healthy children served as a control group. children from the age of 2 months to 5 years hospitalized with a diagnosis of cap were eligible for inclusion in the study after obtaining an informed parental consent. we specifically chose this age range since it is the one included in the world health organization (who) classification of pneumonia severity which was the main classification used in the present study for the purpose of evaluating the relation of tnf-α polymorphism to cap severity. the exclusion criteria were (1) children with known immunodeficiency or taking immunosuppressive drugs, (2) suspected tuberculosis, (3) chronic respiratory disorders, (4) coexistence of another infection with cap, e.g., gastroenteritis, (5) persistent asthma, (6) severe malnutrition, and (7) a pre-existent cardiac disease. in addition, children were excluded from the control group if the parents gave a history of previous cap episode. cap was diagnosed in the presence of signs and symptoms of acute lower respiratory infection that has been acquired outside the hospital and that was confirmed by demonstration of an infiltrate on chest x-ray [13] . patients were evaluated through full history, general examination, and local examination of the chest to detect signs of lower respiratory tract infection and those indicating respiratory distress, e.g., chest indrawing. moreover, pneumonia severity was assessed by some clinical scoring systems, namely the pediatric respiratory severity score (press) score [14] ; predisposition, infection, response and organ failure (pirom) score [15] ; respiratory index of severity in children (risc) score, and the who classification [16] . laboratory investigations included complete blood count (cbc), c-reactive protein (crp), blood gas analysis, serum electrolytes, and renal function tests. blood culture was performed for patients needing picu admission. in addition, analysis of tnf-α −308 g>a promoter polymorphism was performed to all patients and controls by polymerase chain reaction followed by restriction fragment length polymorphism (pcr-rflp) as previously described [17] :a 2-ml venous blood sample was drawn from all study participants and stored in edta tubes. genomic dna was extracted from peripheral blood using a using thermo scientific gene jet genomic dna purification kit (lithuania). dna was eluted and stored at − 20°c for further pcr procedure. a 116 bp dna fragment was amplified, using the forward primer 5′-agg caa tag gtt ttg agg gcc at-3′ and the reverse primer 5′-aca ctc ccc atc ctc cct gct-3′. pcr amplification was performed by applied bio systems 2720 thermal cycler (singapore). the pcr reaction involved the following steps: an initial denaturation of 95°c for 2 min then 35 cycles of 95°c for 30 s; 60°c for 15 s; 74°c for 15 s; and a final extension step at 74°c for 10 min. the amplified pcr product was cleaved by ncoi restriction enzyme (10 u/μl; thermo fisher scientific; waltham, ma, usa) where1 μl of ncoi was added to 10 μl pcr reaction mixture, 18 μl nuclease free water, and 2 μl 10x buffer tango. the mixture was gently mixed and spun for a few seconds then incubated at 37°c for 16 h then subjected to agarose gel (3%) electrophoresis and visualized by ethidium bromide staining and uv transillumination. a 100-bp dna ladder (biolabsinc, new england) was used. the presence of a single band at 116 bp indicated aa homozygous genotype. the presence of two bands at 96 and 20 bp indicated gg homozygous genotype, and finding of three bands at 116, 96, and 20 bp indicated ag heterozygous genotype. we used three genetic models, namely "genotype/codominant," "recessive," and "dominant" models to test the association of −308 tnf-α polymorphism with cap. in the genotype/co-dominant model, the frequencies of each of the different genotypes (gg vs aa vs ag) among patients and controls were compared. in the recessive model, the frequency of aa genotype was compared versus that of ag+gg among patients and controls. in the dominant model, the frequency of aa+ag genotypes was compared versus gg. qualitative data was expressed as number (%) and analyzed using a chi-square test. normally distributed continuous variables were expressed as the mean ± standard deviation while continuous variables with non-normal distribution were presented as the median (minimummaximum). normally distributed continuous variables were compared using a t test while non-normally distributed continuous variables were compared using the mann-whitney u test (for two variables) or the kruskal-wallis test (for more than two variables). logistic regression analysis was used to test the association of different alleles and genotypes with cap. this yielded odds ratio (or) and 95% confidence interval (95% ci). if the odds ratio is > 1, this means positive association (the variable increases the risk of occurrence of an event). if the odds ratio < 1, this indicates a negative association (the variable decreases the risk of occurrence of an event). all statistical analyses were performed using ibm spss software version 23.0 (spss, inc., chicago, il, usa). a p value < 0.05 was considered statistically significant. forty-five patients and 45 controls were enrolled. their basic characteristics are shown in table 1 . 37.8% of patients had previous one or more episodes of pneumonia. sixty percent had lobar consolidation; the remaining had patchy or interstitial infiltrate. the median pirom, press, and risc scores were 0, 3, and 2 respectively. only three children (6.6%) had severe pneumonia under the who classification. of note, none of our patient cohort died. the frequencies of different alleles and genotypes were compared among patients and controls under the recessive, dominant, and genotype models ( table 2) . under the genotype model, the ag genotype frequency was significantly lower among patients compared with control. the odds ratio was< 1 [or and 95% ci = 0.13 (0.03-0.63); p = 0.012], implying that the association was negative, that is, ag genotype decreases the risk of acquiring cap. under the dominant model, the frequency of the pooled genotypes aa+ag was significantly lower among patients compared with controls. the or and 95% ci were 0.34 (0.12-0.99) and p = 0.048, implying that the presence of either aa or ag significantly reduced susceptibility to cap. under the recessive model, no significant difference was found in the prevalence of aa genotype compared with gg+ag genotypes [or = 2.09 (0.36-12.07); p = 0.4] the frequency of "a" allele was lower among patients but no significant association was found with cap [or and 95% ci = 0.58 (0.25-1.35); p = 0.21] the relation of genotypes and alleles to cap severity when press score was used to classify patients into "severe pneumonia" and "non-severe pneumonia," no significant association of any of the alleles or genotypes with cap severity was found under the dominant, recessive, or genotype models ( table 3) . the median press, risc, and pirom scores tended to be lower among patients with aa genotype, but the difference was not significant (table 4 ). scientists have been increasingly aware that the invading pathogens are not entirely to blame for the severity of infectious processes since the excessive inflammation produced by the host plays a significant role. in the present study, we evaluated the role of one polymorphism in the promoter of tnf-α gene. our main finding was that the frequency of ag genotype and that of the aa+ag genotypes were significantly lower among cap patients compared with controls and logistic regression analysis indicated a significant negative association of genotypes with cap. the prevalence of "a" allele was also lower among patients, but the difference was not statistically significant. together, these findings could be construed as implying that tnf-α g>a promoter polymorphism confers protection from cap development. surprisingly, our findings came against the hypothesis that more tnf production by the "a" allele increases susceptibility to cap. however, it is also reasonable to suggest a counter hypothesis: more tnf-α production ensures more complete pathogen eradication. this could explain the finding reported by a study of patients with influenza pandemic (h1n1) wherein the frequencies of "a" allele and ga genotype were significantly lower while those of the g allele and gg genotype were significantly higher among patients compared with controls [18] . similarly, another study reported that the patients with influenza-related pneumonia were more frequently homozygous for the g allele of the tnf 308 g/a polymorphism compared with controls [19] . notwithstanding, one should be cautious while drawing firm conclusions from our current research due to the small sample size. moreover, the inflammatory [12] . consequently, the level of tnf-α is likely the product of several of these polymorphisms and not just −308 g>a [20] , necessitating a thorough evaluation of all polymorphisms in future larger studies. it should be pointed out that, to the best of our knowledge, our current study is the first to evaluate the role of −308 g>a polymorphism in pediatric cap patients, but a relevant study of 120 very low birth weight mechanically ventilated infants found that the incidence of nosocomial pneumonia was not significantly different between infants with gg genotype and those having aa/ag [21] . in another study of 69 adult patients with pneumococcal disease (61 with cap, 5 with meningitis, and 3 having both), no significant difference in the distribution of tnf-α was found between patients and controls [22] . likewise, a large multicenter study found no significant difference in the distribution of alleles or genotypes of −308 g>a polymorphism between adult cap patients and controls [23] . similarly, a meta-analysis of 12 studies (the great majority were adult studies) concluded that −308 g>a polymorphism (aa+ag vs gg) was not associated with cap or hospital-acquired pneumonia (hap) risk, but when subgroup analysis was performed, the polymorphism was found to be associated with pneumonia in asians but not in caucasians [24] . it seems that the latter multicenter study and metaanalysis somewhat discouraged researchers from pursuing further research on the issue. however, it should be noted that the situation in pediatric patients is far from clear and the influence of ethnicity needs a more thorough evaluation. in addition to the effect of tnf-α polymorphism on cap susceptibility, we evaluated a potential influence of the polymorphism on cap severity. we found no significant association of alleles or genotypes with any indicators of cap severity, including clinical severity scores, but we were not able to assess the influence of tnf-α polymorphism on mortality since none of our patients died. it is likely that the small sample size could be responsible for our failure to demonstrate an association of tnf-α with any indicators of cap severity. however, previous studies generally point to lack of association of this polymorphism with cap outcome. consistent with our findings, genotyping of 77 children with respiratory syncytial virus infection revealed no association of tnf-α −308 g>a polymorphism with any of the clinical outcomes, including severity scores of lower respiratory illness, oxygen saturation, lengths of oxygen supplementation, intensive care unit (icu) and hospital stays, and the presence or absence of pneumonia and otitis media [25] . however, another study of 277 chinese adult patients with severe pneumonia-induced sepsis found that tnf-α "a" allele increased the risk of septic shock (or = 4.28). moreover, the combined ga+aa increased the risk of septic shock even after adjustment for confounding factors (or = 2.96) however, no association of alleles or genotypes was found with mortality [26] . in a multicenter study, no significant association of allele or genotype of −308 g>a polymorphism with adult cap severity and outcome was noted [23] . similarly, a meta-analysis concluded that genotype (aa+ag) was not associated with a higher risk of mortality from pneumonia compared with gg, but carriers of "a" allele had higher risk of having severe pneumonia [24] . the unexpected lack of association of tnf-α −308 g>a polymorphism with cap outcome might be explained by the presence of additional factors which cooperate together to determine the level of inflammatory response which, in turn, is presumed to influence the final prognosis. these factors include the following: first, variations in the levels of pro-inflammatory cytokines other than tnf-α; second, variation in the level of tnfα from the influence of other promoter polymorphisms; third, variations in the responses of different individuals to the same tnf-α level due to polymorphisms in tnfα receptor genes or in the genes encoding signal transduction pathway molecules; fourth, the balance between pro-inflammatory and anti-inflammatory genes; fifth, the inflammatory response to cap in children might be somewhat different from that in adults; and sixth, the pattern of cytokine response might vary according to the pathogen type since different organisms interact with different pattern recognition receptors (prr), with different signaling pathways [27] . so, it should be born in mind that if the excessive inflammatory response is associated with severe cap, it is not tnf-α alone which determines the level of this inflammation, and some patients may demonstrate excessive tnf-α gene expression but its effects become overwhelmed by the other factors. undoubtedly, a large association study or a meta-analysis of a greater number of studies is needed for more clarification of the issue. the main limitation of the present study is the small sample size. another limitation is the lack of measurement of serum tnf-α level to evaluate its relation to different alleles and genotypes. in addition, patients with severe pneumonia were a minority among our patient cohort and it is possible that our findings could have been different if more patients had severe pneumonia. these limitations need to be avoided in future studies. in conclusion, tnf-α −308 g>a polymorphism appears to confer protection from pediatric cap among egyptian children but it is not associated with indicators of cap severity. larger studies in different populations are needed to confirm the role of this polymorphism in pediatric cap. table 4 the effect of genotype on pneumonia severity scores global and national burden of diseases and injuries among children and adolescents between 1990 and 2013: findings from the global burden of disease 2013 study cytokine concentrations in plasma from children with severe and non-severe community acquired pneumonia the transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kda tumor necrosis factor receptor tumour necrosis factor-alpha (tnf-alpha): the good, the bad and potentially very effective the treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases tumor necrosis factors: gene structure and biological activities shock and tissue injury induced by recombinant human cachectin role of inflammatory mediators in the pathophysiology of acute respiratory distress syndrome tumour necrosis factor in man: clinical and biological observations interleukin 1 induces a shock-like state in rabbits. synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition tnf-mediated inflammatory disease tumor necrosis factor alpha -308 gene locus promoter polymorphism: an analysis of association with health and disease british thoracic society guidelines for the management of community acquired pneumonia in children pediatric respiratory severity score (press) for respiratory tract infections in children application of a prognostic scale to estimate the mortality of children hospitalized with community acquired pneumonia development of the respiratory index of severity in children (risc) score among young children with respiratory infections in south africa tnf -308g > a promoter polymorphism (rs1800629) and outcome from critical illness plasma cytokine levels and cytokine gene polymorphisms in mexican patients during the influenza pandemic a(h1n1) pdm09 tnf-α, il-10, and enos gene polymorphisms in patients with influenza a/h1n1 complicated by pneumonia genetic polymorphisms within tumor necrosis factor gene promoter region: a role for susceptibility to ventilator-associated pneumonia tumor necrosis factor [alpha]-308 polymorphism associated with increased sepsis mortality in ventilated very low birth weight infants pneumococcal septic shock is associated with the interleukin-10-1082 gene promoterpolymorphism genetic variability in the severity and outcome of communityacquired pneumonia associations between tnf-α polymorphisms and pneumonia: a meta-analysis cytokine gene polymorphisms moderate illness severity in infants with respiratory syncytial virus infection association of tumor necrosis factor α -308g/a and interleukin-6 -174g/c gene polymorphismwith pneumonia-induced sepsis using cluster analysis of cytokines to identify patterns of inflammation in hospitalized patientswith community-acquired pneumonia: a pilot study none authors' contributions fme designed the study and provided a critical revision of the manuscript. mse analyzed and interpreted the patient data and wrote the manuscript. sse performed the genetic molecular testing. ste recruited the patients and collected the clinical data. all authors read and approved the final manuscript." availability of data and materials not applicable the study protocol was approved by menoufia faculty of medicine committee for medical research ethics. this committee does not grant reference numbers for the approved researches. a written informed consent was obtained from the parents. the authors declare that they have no competing interests. key: cord-023433-d1b7qvhs authors: siassi, m.; hohenberger, w.; croner, r. title: expression of human collectins in colorectal carcinoma date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bo.x sha: doc_id: 23433 cord_uid: d1b7qvhs introduction: the human collectins, mannan‐binding lectin (mbl), surfactant protein‐a (sp‐a) and surfactant‐protein‐d (sp‐d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy‐kit). gene expression profiles were analysed using gene‐chips (affymetrix, hg‐u133). we analysed the data for the expression of mbl, its associated serine proteases mannan‐binding lectin‐associated serine protease 1/2 (masp 1/2), sp‐a and sp‐d. the signal intensity of the genes of interest was compared using the mann–whitney u‐test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp‐a and masp‐2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this study. only sp‐a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-001039-qocuprwb authors: hayasaka, daisuke; shirai, kenji; aoki, kotaro; nagata, noriyo; simantini, dash sima; kitaura, kazutaka; takamatsu, yuki; gould, ernest; suzuki, ryuji; morita, kouichi title: tnf-α acts as an immunoregulator in the mouse brain by reducing the incidence of severe disease following japanese encephalitis virus infection date: 2013-08-05 journal: plos one doi: 10.1371/journal.pone.0071643 sha: doc_id: 1039 cord_uid: qocuprwb japanese encephalitis virus (jev) causes acute central nervous system (cns) disease in humans, in whom the clinical symptoms vary from febrile illness to meningitis and encephalitis. however, the mechanism of severe encephalitis has not been fully elucidated. in this study, using a mouse model, we investigated the pathogenetic mechanisms that correlate with fatal jev infection. following extraneural infection with the jaoars982 strain of jev, infected mice exhibited clinical signs ranging from mild to fatal outcome. comparison of the pathogenetic response between severe and mild cases of jaoars982-infected mice revealed increased levels of tnf-α in the brains of severe cases. however, unexpectedly, the mortality rate of tnf-α ko mice was significantly increased compared with that of wt mice, indicating that tnf-α plays a protective role against fatal infection. interestingly, there were no significant differences of viral load in the cns between wt and tnf-α ko mice. however, exaggerated inflammatory responses were observed in the cns of tnf-α ko mice. although these observations were also obtained in il-10 ko mice, the mortality and enhanced inflammatory responses were more pronounced in tnf-α ko mice. our findings therefore provide the first evidence that tnf-α has an immunoregulatory effect on pro-inflammatory cytokines in the cns during jev infection and consequently protects the animals from fatal disease. thus, we propose that the increased level of tnf-α in severe cases was the result of severe disease, and secondly that immunopathological effects contribute to severe neuronal degeneration resulting in fatal disease. in future, further elucidation of the immunoregulatory mechanism of tnf-α will be an important priority to enable the development of effective treatment strategies for japanese encephalitis. japanese encephalitis virus (jev), which belongs to the genus flavivirus in the family flaviviridae, is a causative agent of acute central nervous system (cns) disease in humans and domestic animals [1] . pigs and birds are amplifiers or reservoir hosts of jev in the environment, providing a source of virus for blood feeding culex spp. mosquitoes [1] which may subsequently feed on and infect humans. japanese encephalitis (je) is a major public health issue in asia and the asia-pacific region [1, 2] . annually, 30,000-50,000 cases and 10,000-15,000 deaths are reported and more than 50% of survivors may suffer from neurological disability [2] . human infections are largely subclinical with a rate varying from 1:25 to 1:1000 [3, 4] . the clinical symptoms vary from mild to severe disease including a non-specific febrile illness, meningitis, encephalitis and meningoencephalitis, the latter being observed in the most severe cases [4] [5] [6] . . following an incubation period of 6-16 days, patients may develop fever, headache, vomiting, rigor and nausea [1, 4] . subsequently, encephalitic cases develop neurological symptoms including seizure, tremor, photophobia, decreased sensorium, generalized and localized paresis, movement disorder [4, 6] . signs of meningeal irritation such as neck stiffness may be present. these clinical features are not unique to je, thus, laboratory diagnosis is required to distinguish it from other neurological disorders. the variety of disease symptoms and the prognosis in je cases appears to be dependent on complex interactions between viral and host factors [4] . in particular, host factors appear to be important determinants of disease severity and a number of specific proinflammatory cytokines and chemokines are observed in severe je cases [7] . for example, it has been demonstrated that increased levels of tnf-α in cerebrospinal fluid (csf) and serum correlated with cases of severe disease [8] . however, how these biological cytokines and chemokines contribute to the severe disease has not been fully elucidated. therefore, understanding the mechanism of the specific host response in severe cases is an important priority to develop a specific treatment for the infectious disease. the laboratory mouse model is commonly employed to study the cns pathology induced by encephalitic flaviviruses [9] [10] [11] . in common with human cases, mice develop relatively similar neurological dysfunction and the pathologic changes in infected mouse brains are similar to those observed in human cases [6, 9] . although extraneural infection frequently does not result in detectable viremia or virus burden in mice, it is believed that initial virus replication occurs in dendritic cells (dcs) such as langerhans cells at the site of infection, and the infected dcs migrate to draining lymph nodes [6] . virus then invades the cns and hosts develop cns disease, although the mechanism by which the blood-brain-barrier is crossed is not completely understood [12] [13] [14] [15] [16] [17] [18] . cns pathology is the consequence of viral infection of the affected cells and the resulting inflammatory responses in the cns. flavivirus variants may induce different degrees of pathology, however, the host immune response is likely to be a more critical determinant of clinical outcome [19] . inflammatory responses mainly contribute to virus clearance and recovery from fatal disease. for example, cd8 + t cells are reported to have an important function in controlling virus infection [20] [21] [22] [23] [24] [25] , although one report showed only a subsidiary contribution of cd8 + t cells in jev infection [26] . on the other hand, in recent studies it was suggested that immunopathological mechanisms may contribute to the severity of outcome following some encephalitic flavivirus infections [19, [27] [28] [29] [30] . for example, it was reported that cd8 + t cell function enhances pathogenicity during wnv and mvev infections [29, 30] . furthermore, in tick-borne encephalitis virus (tbev)-infected mice the inflammatory response was reported to contribute significantly to the fatal outcome [28] . microglia are the resident macrophages in the brain and are activated in response to a number of different pathological states [31] . following jev infection, activated microglia play a significant role in the development of pathology by producing pro-inflammatory cytokines such as il-1, il-6 and tnf-α [32, 33] . although these pro-inflammatory cytokines have dual roles, acting both as protectors and degenerators of neurons [31] , tnf-α is believed to be one of the key factors that mediate immunopathology in the cns during encephalitic flavivirus infection. for example, it was suggested that tnf-α directly mediates neuronal apoptosis by the engagement of tnf receptor 1 (tnfr1), the tnfr-associated death domain (tradd) and neuronal death contributes to glial activation and subsequent neuroinflammation [31, 34, 35] . it was also shown that tnf-α and il-1β mediate rantes gene expression for the recruitment of immune cells and glutamate released by jev-infected microglia, involves tnf-α signaling and contributes to neuronal death [36, 37] . on the other hand, other studies have shown that tnf-α has a protective role against wnv infections and restricts wnv pathogenesis by promoting trafficking of mononuclear leukocytes into the cns [38, 39] . furthermore, neuronal tnf-α expression during wnv encephalitis may be an adaptive response to diminish cxcl10-induced death [40] . at this stage of our knowledge, therefore, the precise role of the tnf-α response in encephalitic flaviviral pathogenesis remains to be clarified. immunomodulatory cytokines also affect disease outcome of encephalitic flavivirus infection. il-10 is reported to have an effect on immunoregulation [41] . it was suggested that il-10 mediates protection from acute encephalitis and plays a central role in determining the clinical outcome of jev infection [42] . insufficient anti-inflammatory cytokine production of il-4 and il-10 in the brain is associated with increased tissue pathology [43] . il-10 displays a neuroprotective function during jev infection and regulates deleterious effects of proinflammatory cytokines [44] . furthermore, an experiment using il-10 ko mice showed that il-10 signaling plays a negative role in immunity against wnv infection and blockade of il-10 signaling helps to control viral infection [45] . thus, the precise role of the il-10 response following encephalitic flavivirus infection also remains to be resolved. in general, evaluation of virus pathogenicity and virulence in mouse models utilizes either the subcutaneous or intradermal route of infection. this is considered to be a reproducible model of natural human infections following the bite of an infected mosquito or tick and in the past, death was used as an index of pathogenesis [46] . however, it is known that peripheral infections with some strains of encephalitic flaviviruses do not exhibit normal dose response curves based on mortality. although this was first reported in the 1940's [47] , the reason for these apparent discrepancies were not fully understood. we previously showed that the oshima strain of tbev caused dose independent mortality and the fatality rate did not increase more than 50% with increasing virus challenge doses from 10 2 to 10 6 plaque forming unit (pfu) [48] . in our study of tbev, we suggested that the variation of fatal outcome in individual mice appeared to be due to variation in individual host responses [48] . the purpose of this study was to investigate the host factors that influence disease severity following jev infection in a mouse model. in particular, we focused on the variation of disease outcome in individual mice following extraneural infection with jev. we first compared the pathogenicity of two jev strains, which cause either dose-dependent or doseindependent mortality responses. we next compared severe or mild cases of mice infected with jev exhibiting doseindependent mortality and investigated the specific host responses such as tnf-α and il-10 expression in the cns. we also examined the roles of the specific cytokines observed in severe cases using appropriate knockout (ko) mice. the animal experiments were performed in accordance with the recommendations in the fundamental guidelines for proper conduct of animal experiment and related activities in academic research institutions under the jurisdiction of the ministry of education, culture, sports, science and technology. the experimental protocols were approved by the animal care and use committee of the nagasaki university (approval number: 091130-2-7/0912080807-7). the jath160 strain of jev was kindly provided by tomohiko takasaki, national institute of infectious disease, japan. stocks of jev jaoars982 and jath160 viruses were obtained from cell culture medium of baby hamster kidney (bhk) cells infected with viruses previously prepared in suckling mouse brains [49] . the bhk cells were maintained in eagle's minimal essential medium (emem; nissui pharmaceutical co.) containing 8% fetal calf serum (fcs) and antibiotics. c57bl/6j (b6) mice were purchased from japan slc corporation. b6 background il-10-/-mice were purchased from jackson laboratory, usa [50] . tnf -/-mice were kindly provided by yoichiro iwakura, research institute for biomedical sciences, tokyo university of science [51] . these mice were mated in the facility of nagasaki university. five to six week old mice were subcutaneously inoculated with a range of 10 0 -10 6 pfu of jev diluted in emem containing 2% fcs. mock infected mice were inoculated with emem from the supernatant medium of bhk cells. mice were weighed daily and observed for clinical signs including behavioral symptoms and signs of paralysis. thirteen days post infection (pi), dying and recovering mice were distinguished by the degree of weight ratio, and namely mice exhibiting more than 25% or less than 10% weight loss were recognized as dying or recovering mice, respectively. following subcutaneous inoculation with 10 4 pfu of jev, mice were euthanized and blood, spleen, brain and spinal cord were collected following perfusion with cold phosphate-buffered saline (pbs). brains were dissected to provide four separate fractions, ie the brain cortex, thalamus, cerebellum and brainstem. until they were used, these tissues were stored at -80° c. each tissue was homogenized in ten volumes of pbs containing 10% fcs and diluted with emem with 2% fcs. virus titers were determined by plaque forming assays using bhk cells and were expressed as pfu/g tissue [48] . mouse brains and spleens were collected after perfusion with cold pbs. freshly isolated brains and spleens were immediately immersed in rnalater (ambion). total rna was extracted using rneasy lipid tissue mini kit (qiagen) according to the manufacturer's instructions. the expression levels of cytokines were measured by real time-pcr as demonstrated previously [52] . the copy numbers were calculated as a ratio of the copy numbers of internal control glyceraldehyde-3-phosphate dehydrogenate. mice inoculated with jev were anesthetized and perfused with 10% phosphate-buffered formalin. fixed tissues were routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin. immunohistochemical detection of the jev antigens was performed as described previously [53] . rabbit polyclonal antibody against e protein was used to detect jev antigens. brains were collected from four mice in each group of dying and recovering mice at 13 days pi. the brains were homogenized and passaged in bhk cells for 2 days. viral rna was extracted from the supernatant medium of the bhk cells using qiaquick pcr purification kit (qiagen) according to manufacturer's protocol. reverse transcription was performed by using superscript iii reverse transcriptase (invitrogen) and random hexamers. pcr was performed to cover the whole genome sequence using takara ex taq dna polymerase (takara bio inc.). the cycle sequencing reaction was performed by using bigdye terminator v 3.1 cycle sequencing kit (life technologies) and the dna sequence was determined with applied biosystems 3730 dna analyzer (life technologies). serum samples were collected from infected mice. the levels in the serum were measured by using competitive enzyme immunoassay and sandwich enzyme-linked immunosorbent assay kits for corticosterone (assaypro), tnfα and il-10, il-12 (endogen) according to the manufacturer's instructions. recovery of leukocytes was performed by applying previously described methods [22, 54] . briefly, after perfusion with cold pbs, brains and thymus were removed and placed on ice in rpmi containing 5% fcs (nissui pharmaceutical co.). brains were strained and homogenized gently with a 70 µm cell strainer (bd biosciences). after washing with rpmi, the cell suspension was layered onto a 70% and 30% percoll gradient (ge healthcare bio-sciences ab) and centrifuged at 800 × g for 45 min at 23° c. the leukocytes were collected from between the 70% and 30% interface. thymocytes and splenocytes were also recovered from these mice. cells were strained with a 70 µm cell strainer (bd biosciences) and lysed with rbc lysis buffer (sigma-aldrich). after washing, cells were resuspended in rpmi medium. isolated cells were counted and kept on ice until the staining procedure. brain leukocytes were washed and blocked with rat anti-mouse cd16/32 (fc receptor) (beckman coulter) in facs buffer (pbs containing 0.1% bsa and 0.1% sodium azide). cells were stained with a mixture of different fluorescentlabeled antibodies directed at surface phenotypic markers, cd45-fitc, f4/80-pe, nk1.1-percp-cy5.5, cd4-pe-cy7, cd8-apc, cd19-alexa fluor 700, cd3e-efluor 450 (beckman coulter) and then fixed with 4% paraformaldehyde overnight. the stained cells were analyzed by galios™ flow cytometer (beckman coulter). leukocytes were recognized by characteristic size (forward scatter), granularity (side scatter) and cd45 expression. thymocytes were recognized by their characteristic size and cd4 + cd8 + double positive cells were recognized by the expression of cd4 + and cd8 + . kruskal-wallis test, and mann whitney test were used for statistical analysis to assess the significant differences of viral loads, expression levels of cytokines, and numbers of leukocytes. gehan-breslow-wilcoxon test was performed to assess the survival curves of jev-infected mice groups. p value <0.05 was considered statistically significant. in this study, we used inbred b6 mice to minimize the influence of the genetic background of individuals. subcutaneous infection with jaoars982 did not lead to a normal dose dependent curve of mortality ( figure 1a ). the mortality rate was not significantly increased when challenge doses ranged from 10 2 to 10 6 pfu per mouse, although the infectivity in the mice increased sequentially ( figure 1a ). on the other hand, jath160 infection exhibited a dose dependent mortality curve and the infectivity in the mice was consistent with the mortality ( figure 1a ). these observations indicate that individual jaoars982-infected mice exhibit a variable prognosis independent of virus challenge dose, whereas all jath160-infected mice died. because a virus challenge dose of 10 4 pfu of either jaoars982 or jath160 induced 100% infectivity ( figure 1a ), this dose was used for all further investigations to compare the pathogenesis. jaoars982-infected mice did not start to die until 13 days pi and the mean survival time (mst) was 15.5 ± 2.56 days ( figure 1b ). mice that died exhibited generalized clinical signs involving slowness in movement, ataxia, piloerection and anorexia. continuous weight loss was observed in mice that died, whereas survivors regained weight from 13 to 15 days pi onwards ( figure 1c ). on the other hand, jath160-infected mice started to die at 9 days pi and all mice had died by 15 days pi ( figure 1b ) following continuous weight loss ( figure 1c ). mst was 12.8 ± 0.89 days and was significantly shorter than that of jaoars982-infected mice (mann whitney test, p=0.0173). thus, we hypothesized that the cause of fatal disease was different between jaoars982and jath160-infected mice. infectious virus was detectable in the brain cortex and thalamus at 5 days pi in both jaoars982 and jath160infected mice without significant difference in titer ( figure 1d ). however, at 9 days pi viral loads of jath160-infected mice were significantly higher than those of jaoars982-infected mice in every region of the brain cortex, thalamus, brainstem, cerebellum and spinal cord ( figure 1d ). it is important to note that viral load in the brain cortex was higher than in other regions of the cns in both jaoars982 and jath160-infected mice ( figure 1d ), indicating that the brain cortex is the main target region for jev infection. thus, we next examined the cytokine levels in the brain cortex to compare the immune responses. the levels of tnf-α, ifn-γ, il-2 and il-10, but not il-4 and il-5 were significantly higher in jath160infected mice than in jaoars982-infected mice ( figure 1e , figure s1a ). the cytokine levels of tnf-α, ifn-γ, il-2 and il-10 were very low or undetectable in mock-infected mice and in infected mice at 5 days pi ( figure 1e ). corresponding to the viral loads, histopathological examination showed that a large number of neurons displayed jev antigens and severe cuffing was observed in the brain cortex of jath160-infected mice at 9 day pi ( figure 1f ). jaoars982-infected mice also exhibited jev antigen-positive neurons and cuffing, but at lower levels than those observed in jath160-infected mice ( figure 1f ). mock-infected mice showed no jev antigen-positive neurons or inflammatory reactions ( figure 1f ). these results confirm that during the early phase of infection, jath160-infected mice developed severe encephalitis with extensive neuronal infection which contrasts with the less extensive neuronal infection induced in jaoars982-infected mice. infectious virus was either not detectable or very limited in spleens (data not shown). interestingly, the levels of tnf-α and il-2 in spleens were up-regulated in jaoars982-infected mice at 5 and 9 days pi, however, they were not elevated in jath160-infected mice ( figure s1b ). the levels of ifn-γ, il-4, il-5 and il-10 were not significantly different between jaoars982 and jath160-infected mice ( figure s1b ). these observations suggest that i) inflammatory responses in peripheral organs were different from those in the cns, and ii) jath160 infection induced no significant expression of tnf-α in the spleen. in view of the observation that individual mice displayed different disease progress when infected with jaoars982 under identical conditions, we attempted to identify specific factors relating to disease severity outcome. initially, we mice were recorded for 21 days and no mice died after 21 days. infectivity was determined by anti-jev igg antibody seroconversion for more than 1:1000 of igg elisa titer. (b and c) b6 mice were subcutaneously infected with 10 4 pfu of jaoars982 (n=30) and jath160 (n=15). survival curves p: gehan-breslow-wilcoxon test. (c) the averages ratio of weight change of living mice at the time points compared with those of day 0 following subcutaneous infections with 10 4 pfu of jaoars982 (n=30) and jath160 (n=15). error bars represent the standard deviations. (d) viral loads in distinct regions of the cns following subcutaneous infections with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=15) and jath160 (day 5: n=5, day 9: n=8). p: mann whitney test. (e) mrna levels of tnf-α, ifnγ, il-2 and il-10 quantified by real-time pcr in the brain cortex of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (f) histopathological features of brain cortex in b6 mice infected with 10 4 pfu of jaoars982 and jath160 at 9 days pi. jev antigens were detected using e-protein specific jev antibody (insets). each experiment represents six and four mice infected with jaoars982 and jath160, respectively. jaoars982infected mice showed slight inflammatory infiltration in meninges. in brain cortex, a few degenerated cells were presented (arrow) and were virus antigen-positive cells. in jath160-infected mice, severe inflammatory reactions were seen in meninges and perivascular area (asterisks). many virus antigen-positive cells were detected in degenerated neuronal cells of the cortex (arrows). doi: 10.1371/journal.pone.0071643.g001 attempted to discriminate severe and mild disease groups during the observation period by following the progression of weight change of individual mice. we discriminated dying and recovering mice based on whether they showed less than 0.75 or more than 0.90 of the weight ratio at 13 days pi ( figure 1c ). it was difficult to predict if mice would survive or die between 0.75 and 0.9 of the designated weight ratio, because within this window both dead mice and survivors were recorded. having established this defining parameter between severe and mild disease groups, we then attempted to examine specific factors relating to disease severity. we initially considered the possibility that the divergence of disease severity might be due to the selection of quasispecies variants from the jaoars982 virus population in the cns. accordingly, we compared the virus sequences recovered from the brains of dying and recovering mice (figure 2a) . however, no specific virus sequence differences were detected in viruses from either the severe or mild disease severity groups (dsg) (figure 2a) . furthermore, recovered viruses from either severe or mild dsg exhibited similar mortality patterns to those of the parent jaoars982 virus ( figure 2b) . noticeably, the viruses recovered from severe dsg mice did not induce 100% lethal infection in subsequent mouse virulence tests ( figure 2b ). these results indicate that quasispecies variants of jaoars982 did not contribute to the divergence of disease progression observed in all experiments with this virus; thus, other factors such as host response seem most likely to be the determinants of disease severity. we next compared the viral loads in the cns between severe and mild dsg in jaoars982-infected mice at 13 days pi. viral loads in brain cortex, thalamus and brainstem but not cerebellum and spinal cord were significantly higher in severe dsg mice than in mild dsg ( figure 3a ). however, in the brain cortex, the variance of virus titer in the mild dsg mice ranged from the minimal detection limit to 10 8 pfu/g of tissue, whereas all mice exhibited more than 10 6 pfu/g of tissue in the severe dsg ( figure 3a ). these results imply that 45.8% (11/24) of mice in the mild dsg produced high viral loads similar to those in the severe dsg. thus, it is likely that high viral infection alone is not a critical determinant of severe disease and additional factors contribute to the fatal encephalitis. therefore, to compare the specific immune responses in severe cases, we further subdivided the mice into three subgroups, severe group (sg), mild group with high viral load (>10 6 pfu/g of tissue) (mhg) or low viral load (<10 6 pfu/g of tissue) (mlg), and compared their cytokine levels in the brain cortex ( figure 3b ). all groups exhibited increased levels of inflammatory cytokines of tnf-α, il-10, ifn-γ and il-2, but not il-4 and il-5 in the brain cortex compared with the uninfected group (ug) ( figure 3b) . interestingly, the level of tnf-α in the sg was significantly increased when compared with those in the mhg and mlg ( figure 3b ). the level of il-10 in the sg was also significantly higher than in the mlg ( figure 3b ). although the difference was not significant, the level tended to be higher than that recorded in the mhg ( figure 3b ). on the other hand, ifn-γ did not show significant differences between the three groups, and il-2 levels in the sg were lower than in the mhg ( figure 3b ). histopathological examination revealed inflammatory infiltration with mononuclear cells in the brain cortex of both severe and mild cases of jaoars982-infected mice ( figure 4a to c). in severe cases, jev antigens were detected in neurons, and degenerated neurons were observed in a wide area of the brain cortex and medulla ( figure 4a ). on the other hand, in mild cases, there was variation of pathological features in some jaoars982-infected mice. other mild cases showed neuronal infections similar to those observed in severe cases but there was little neuronal degeneration in the brain cortex ( figure 4b ). other mice exhibited very limited evidence of neuronal infection and neuronal degeneration ( figure 4c ). mock-infected mice showed none of these pathological changes ( figure 4d ). in summary, both neuronal infection and cns pathology were associated with severe disease outcome. in particular, increased levels of tnf-α and il-10 in the brains appeared to be associated with severe disease, although it was not clear whether the increased levels were the cause or result of severe disease. interestingly, the levels of tnf-α in the spleens of sg and ug mice were similar and relatively low, whereas the levels of both mhg and mlg mice were significantly higher ( figure s2a ). il-10 levels in sg, mhg and mlg mice were high compared with those in ug mice. no significant differences were observed between the sg, mhg and mlg ( figure s2a ). on the other hand, the ifn-γ level in sg mice was lower than those recorded in the ug, mhg and mlg ( figure s2a ). there were no significant differences of il-2, il-4 and il-5 levels between ug, sg, mhg and mlg mice ( figure s2a ). some mice in the sg showed high levels of tnf-α in the serum, although no significant difference was observed when compared with other groups ( figure s2b ). il-10 in the serum of sg mice was significantly increased compared with ug and mhg mice ( figure s2b ). corticosterone levels in the serum were also significantly increased in sg mice compared with other groups ( figure s2b ). corticosterone, a major glucocorticoid hormone, is a strong immunosuppressant and is elevated under stress response conditions such as those preceding death [55, 56] . furthermore, severe cases resulting from infection with jaoars982 exhibited a significant reduction of cd4 + and cd8 + doubly-positive cells in the thymus ( figure s2c ). thymic depletion and body weight loss are the main features of the systemic stress response [55, 56] . these observations therefore suggest that sg mice exhibited a severe systemic stress response accompanied by immune suppression. thus, the roles of inflammatory cytokines appeared to be different in peripheral and cns tissues. to investigate in more detail, the role of tnf-α and il-10 during jev infection, we infected tnf-α ko and il-10 ko b6 comparison of viral genome sequences (nucleotide and corresponding amino acid -in parentheses) between recovered viruses from brains of severe (s14, s15, s18 and s19) and mild (m4, m10, m13 and m24) cases of jaoars982-infected b6 mice. (b) weight changes of b6 mice infected with 10 4 pfu of recovered viruses from severe (s14, s15, s18 and s19) and mild (m17 replaced to m4, m10, m13 and m24) cases. five mice in each group were inoculated subcutaneously and observed for 21 days. closed and open symbols identify mice that died or survived, respectively, during observation period. mice with jaoars982, and observed the disease courses compared with those of infected fully immunocompetent b6 mice. unexpectedly, the mortality rates of tnf-α ko and il-10 ko mice were increased compared with those of wt mice (77.3%, 43.2% and 26.7%, respectively) ( figure 5a ). msts of fatal cases in tnf-α ko and il-10 ko mice (12.6 ± 1.05 and 11.5 ± 0.80 days) were significantly shorter than those of wt mice (15.5 ± 2.14 days) (p=0.0087 and p=0.0039, respectively). consequently, these observations indicate that tnf-α and il-10 protect significant proportions of mice from fatal infection by pathogenic jaoars982 virus. importantly, tnf-α had a particularly pronounced protective effect. following inoculation with jaoars982 virus, there were no significant differences of infectious viral loads in the brain cortex between wt, il-10 ko and tnf-α ko at 5, 9 and 11 days pi ( figure 5b ). however within individual mice in each mouse group the range of viral infectivity varied from the lowest detection limit to 10 9 pfu/g of tissue at 9 and 11 days pi ( figure 5b) . it therefore appears that the increased mortality in il-10 ko and tnf-α ko mouse was not simply due to the increased viral loads in the brains, but other factors must also have contributed to the fatal disease in these ko mice. it was difficult to distinguish between dying and recovering mice on the basis of their clinical signs at 9 to 11 days pi. however, high viral loads in the brain cortex appeared to be necessary for fatal outcome. thus, we compared the inflammatory responses in the brain cortex of mice that contained high viral loads with more than 10 6 pfu per g of tissue ( figure 5b) . surprisingly, tnf-α ko mice exhibited significantly enhanced levels of ifn-γ, il-1β, il-2, il-4, il-5 and il-6 in the brain when compared with the wt and il-10 ko mice at 9 and 11 days pi ( figure 5c and figure s3a ). furthermore, at 5 days pi, the levels of il-4 and il-5 were higher in tnf-α ko ( figure s3b ). il-10 ko mice also exhibited the increased levels of ifnγ, il-1β, il-2, il-4 and il-6 compared with those of wt mice at 9 days pi, although the differences were smaller than those between tnf-α ko and wt mice ( figure 5c ). uninfected mice showed some significant differences of cytokine levels between the three groups, but the levels were very low compared with infected mice and were not significant ( figure s3c) . furthermore, the levels of perforin, granzyme b and fasl at 9 days pi, and granzyme a at 11 days pi were significantly increased in tnf-α ko mice compared with those of wt mice ( figure 5d and figure s3d ), whereas il-10 ko mice exhibited the increased level of perforin at 9 days pi ( figure 5d ). these cytokines are associated with immune-mediated neurodegeneration. these findings suggest that immunopathological effects in the cns contribute to the accelerated mortality in tnf-α ko mice infected with jaoars982. thus, il-10 and in particular tnf-α mediate immunomodulatory effects against such inflammatory responses. histopathological examination of tnf-α ko mice revealed severe neuronal loss in extensive areas of brain cortex when compared with wt mice (figure 6a and b) . however, the proportion of infiltrated cells involving leukocytes (cd45), t cells (cd3, cd4 or cd8), b cells (cd19), nk cells (nk1.1) and macrophages (f4/80) did not appear to differ significantly between tnf-α ko and wt mice ( figure 6c ). these observations suggest that the increased levels of cytokines in tnf-α ko mice were due to qualitative differences of their expression in inflammatory cells, rather than quantitative increases of infiltrating cytokine producing cells. in the spleens of mock-infected mice, there were no significant differences of ifn-γ levels between wt, il-10 and tnf-α ko mice ( figure s4a ). however, following jaoars982 infection the levels of ifn-γ in tnf-α ko mice were significantly increased compared with those of wt mice at 5 and 9 days pi ( figure s4b and c) . on the other hand, il-2 and il-4 levels in tnf-α ko mice were significantly higher than those of wt and il-10 ko mice during mock infection ( figure s4a ) and following jaoars982 infections ( figure s4b to d) . also, the level of il-5 in tnf-α ko was decreased compared with wt and il-10 ko mice at 5 days pi ( figure s4b) . these observations suggest that the patterns of cytokine levels observed in spleens were different from those of the brain and therefore that peripheral responses are unlikely to contribute to the increased disease severity in tnf-α ko mice. although the high virulence of jath160 is probably attributable to viral factors, we attempted to assess whether or not tnf-α might also contribute significantly to the pathogenicity observed following infection with jath160 virus. accordingly, mice were inoculated with jath160 virus at a challenge dose of 10 4 pfu per mouse, all wt, il-10 ko and tnf-α ko mouse groups died. however, tnf-α ko mice presented with significantly shorter survival times than b6 wt mice (9.57±1.19 days and 12.8±0.89 days, respectively, mann whitney test: p=0.0002) ( figure 7a ). it is important to note that viral loads in the brains were not significantly different for either wt, tnf-α ko or il-10 ko mice at 5 and 7 days pi ( figure 7b ). tnf-α ko mice exhibited significantly increased levels of ifn-γ and il-5 in the brains compared with wt and/or il-10 ko mice at 5 and 7 days pi following jath160 inoculation ( figure 7c and d) . however, levels of il-2 and il-4 in the brains were not increased when wt and tnf-α ko groups at 5 and or 7 days pi were compared ( figure 7c ). moreover, levels of perforin, granzyme a, granzyme b and fasl were not increased in tnf-α ko when compared with wt mice at 7 days pi. however, histopathological data showed that tnf-α ko mice presented with severe acute necrotic changes in the brain cortex compared which was not the case for wt and il-10 ko mice at 9 days pi ( figure 7d ). in the spleens, similar to the jaoars982 infection, the levels of ifn-γ, il-2 and il-4 in il-10 ko and tnf-α ko mice were significantly increased compared with those of wt mice at 7 days pi following jath160 infection, whereas the level of il-5 was decreased in tnf-α ko ( figure s5b) . these results suggest that the shorter survival time of jath160-infected tnf-α ko mice when compared with wt mice may be partially attributable to an immunopathological effect, whereas direct neuronal infection is likely to be the main cause of neurodegeneration in jath160-infected mice. this study provides the first evidence that tnf-α has an immunoregulatory effect on pro-inflammatory cytokines in the cns during jev infection and this results in protection from fatal disease due to infection with this virus. following jaoars982 virus infection, tnf-α ko mice exhibited significantly increased mortality rates when compared with wt mice. although it has been suggested that tnf-/-mice show developmental defects of the humoral immune system 10 4 pfu of jath160 at 9 days pi. jev antigens were detected using e-protein specific jev antibody (insets). each experiment represents four, five and six mice of wt, tnf-α ko and il-10 ko mice, respectively. the b6 wt and il-10 ko mice showed severe inflammatory reactions in the brain cortex. on other hand, the tnf-a ko mice exhibited acute necrotic changes with slight inflammatory reactions in the brain cortex. including a lack of primary b cell follicles [38, 57, 58] , tnf-α ko mice that we used in this study did not show significant depletion in the anti-jev igm response (data not shown) or in cytokine expression ( figure s4 ). in addition, no significant increases of viral propagation were observed in the peripheral and cns tissues. interestingly, there were no significant differences of viral load in the cns between wt and tnf-α ko mice. however, high inflammatory responses were observed in the cns of tnf-α ko mice. in particular, perforin, granzyme a, granzyme b and fasl, which are known to be associated with immune-mediated neurodegeneration, were significantly increased in the brains of tnf-α ko mice when compared with those of wt mice. these observations suggest that immunopathological effects contribute to the severe neuronal degeneration and fatal disease in tnf-α ko mice. il-10 ko mice also exhibited increased mortality and upregulated levels of inflammatory cytokines in the cns compared with wt mice and in common with tnf-α ko mice, there were no significant differences in viral loads. however, it is important to note that the levels of inflammatory cytokines of tnf-α ko mice and the resultant mortality were dramatically higher than those observed in il-10 ko mice. il-10 has an immunoregulatory function on various cells in the innate immune system including cytotoxic and helper t cells, nk cells and b cells [59] . il-10 signaling has a negative role in immunity against wnv infection [45] . it is also known that tnf-α is a critical regulatory cytokine exerting homeostatic and pathological effects in the csf [60] . therefore, our data imply that tnf-α mediates greater efficacy of immunoregulatory function during jev infection. in preparatory studies of jev infection, we attempted to inject tnf-α intravenously or intracerebrally after jev inoculation to examine whether or not this improved the disease outcome. however, there was no significant improvement in the condition of the mice or protection from death. administration of anti-mouse tnf-α antibody also showed no improvement of disease outcome. although we cannot totally exclude the possibility that failure of tnf-α administration to improve disease outcome, may have been the result of the technical design of the experiments, different responses of tnf-α in the cns when compared with peripheral tissues may partly explain our observations. therefore, further investigation of the immunoregulatory mechanism of tnf-α in vivo and in vitro will be required to understand the basis of the immunopathological effects observed during jev infection. in this study, we focused on the variation of disease severity in mice following jaoars982 infection to detect specific responses that may be associated with severe disease. thus, we discriminated severe and mild cases of mice by their weight ratio at 13 days pi. using this simple and effective approach, we had previously shown that specific immune responses were detected in severe disease cases when compared with mild cases following tbev infection [48] . loss of appetite probably caused the weight loss due to decreased food and water intake. however, undernourishment did not appear to be the simple cause of death, because our preliminary data showed that an infusion of glucose solution to compensate for weight loss did not prevent fatal disease. we first considered whether or not viral quasispecies could account for the diversity of disease outcome. our results did not support this possibility. we also identified specific immune responses including tnf-α and il-10 up-regulation in the brains of severe cases when compared with mild cases. in human cases, an increased level of tnf-α in the csf appears to correlate with je disease severity [8] . therefore, this jevinfected mouse model appears to be a reproducible model of severe je disease in human cases. furthermore, from the results of increased fatality in tnf-α ko mice, we propose that increased levels of tnf-α in the brains of severe cases in wt mice were probably produced in response to the disease severity, to alleviate the pathological impact of the encephalitis. it has been reported that immunopathological effects do contribute to flavivirus encephalitis [27] . cytolytc leukocytes such as cd8 + t cells induce cytopathology during some encephalitic flavivirus infections [28] [29] [30] and these leukocytes kill virus-infected cells using two distinct mechanisms viz., fas and granular exocytosis which involve perforin, granzyme a and b [61] [62] [63] [64] . in tnf-α ko mice, we showed that the increased levels of inflammatory cytokines including fas and the granular exocytosis correlated with severe encephalitis and fatal outcome. however, in wt mice, the apparent immunopathological features were not observed in dying mice 13 days pi. although it was difficult to identify dying and surviving mice before 13 days pi by their clinical signs, jaoars982-infected mice showed varying levels of fas and granular exocytosis in the brains at 11 days pi and some of them exhibited similar or higher levels compared with the tnfα ko mice ( figure s3d ). thus, fatal cases may exhibit severe encephalitis caused by immunopathological responses during the early phase of infection and thereafter severe clinical signs may appear in some mice. jaoars982-infected mice exhibited a variety of immune responses and different prognoses in individual mice. however, it was not clear how the immune response differentiated between dying and recovering mice. in order to explain these variable immune responses, we previously showed that specific t cell receptor (tcr) repertoires were present in dying mice during tbev infection [65] . furthermore, we also showed that specific tcr repertoires were detected in the dying mice compared with the recovering mice following jaoars982 infection (shirai, et al., unpublished results). these data raise the possibility that there may be a variety of specific t cell clones effecting either protective or pathogenetic functions in dying and recovering mice. dose independent mortality induced by encephalitic flaviviruses has been recognized but has been an unresolved problem since the 1940's [27, 47] . recently, it was suggested that induction of more vigorous innate immune responses might control early virus dissemination following increasing infectious challenge doses of virus [6, 26, 66] . we have also recently discovered that interferon alpha receptor knockout induces dose-dependent mortality following extraneural infection with jaoars982 (hayasaka, et al., unpublished results) . in addition, we previously reported that late death following tbev infection appears to be a key feature of dose independent mortality within the encephalitic flaviviruses [48] . in the current study, jaoars982-infected mice also displayed increased times to death and the variation of acquired immune responses which either showed protective or pathological effects, appeared to be correlated with severe disease. therefore, we propose that in addition to innate immune response, subsequent acquired immune responses, which varied contingently in individuals, appeared to be a determining factor associated with dose-independent mortality. interestingly, jath160-infected mice did not show increased levels of tnf-α in the spleen at 5 and 9 days pi. however, it is uncertain if the low level of tnf-α in the spleen directly contributed to the subsequent cns infection and the neuropathogenesis during jath160 infection. it is important to note that there were 17 amino acid differences in the genomic sequences of jath160 and jaoars982. therefore, it will be important to determine whether or not specific amino acid substitutions can influence tnf-α expression and thus contribute to the pathogenesis of the lethal process during jath160 infection. in conclusion, jath160-infected mice developed severe encephalitis and all mice died due to severe infections of the cns (figure 8 ). on the other hand, jaoars982-infected mice exhibited varying degrees of encephalitis and different prognoses (figure 8 ). we therefore propose that fatal outcome is attributable both to immunopathological changes in addition to high levels of cns infection. at this stage we cannot define the critical factors involved in the immunopathogenetic process ( figure 8 ). furthermore, up-regulation of tnf-α and il-10 in the brain appear to be important determinants of the pathogenetic response ( figure 8 ). clearly, further elucidation of the contribution of immunopathology and the suppressive impact of tnf-α, are important priorities to enable the development of effective treatment strategies for je. figure s1 . cytokine levels of spleen in b6 mice infected with jaoars982 and jath160. (a) mrna levels of il-4 and il-5 quantified by real-time pcr in the brain cortex of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (b) mrna levels of tnf-α, ifnγ, il-2, il-10, il-4 and il-5 quantified by real-time pcr in the spleen of b6 mice infected with 10 4 pfu of jaoars982 (day 5: n=5, day 9: n=12), jath160 (day 5: n=5, day 9: n=5) and mock (n=8). p: mann whitney test. (tif) figure s2 . cytokine levels of spleen in severe and mild cases of jaoars982-infected mice. (a) mrna levels of tnf-α, il-10, ifnγ, il-2, il-4 and il-5 quantified by real-time pcr in the brain cortex of jaoars982-infected b6 mice at 13 days pi. uninfected group: u (n=8), severe group: s (n=8), mild group with high viral load of >10 6 pfu/g of brain tissue: mh (n=11), mild group with low viral load of <10 6 pfu/g of brain tissue: ml (n=13). p: kruskal-wallis test, p: mann whitney test. (b) the levels of il-10, tnf-α and corticosterone measured by enzyme-linked immunosorbent assay in the plasma of jaoars982-infected b6 mice at 13 days pi uninfected group (u group, n=6), severe group (s group, n=6), mild group with high viral load of >10 6 pfu/g of brain tissue (mh group, n=7), mild group with low viral load of <10 6 pfu/g of brain tissue (ml group, n=6). p: kruskal-wallis test, p: mann whitney test. (c) cd4 and cd8 expressions of thymocytes from mock, mild and severe cases of jaoars982-infected b6 mice at 13 days pi. each experiment represents four and fifteen mice of severe and mild cases, respectively. (tif) flaviviruses. in: dm knipepm howleyde griffinra lambse straus. fields virology past, present, and future of japanese encephalitis natural japanese encephalitis virus infection among humans in west and east japan shows the need to continue a vaccination program overview: japanese encephalitis new initiatives for the control of japanese encephalitis by vaccination: minutes of a who/cvi meeting immunobiology of japanese encephalitis virus proinflammatory cytokines and chemokines in humans with japanese encephalitis correlation of tumor necrosis factor levels in the serum and cerebrospinal fluid with clinical outcome in japanese encephalitis patients flavivirus encephalitis: pathological aspects of mouse and other animal models a preliminary neuropathological study of japanese encephalitis in humans and a mouse model comparative study of mouse brains infected with japanese encephalitis virus by intracerebral or intraperitoneal inoculation west nile virus encephalitis: sequential histopathological and immunological events in a murine model of infection pathogenesis of neurotropic arbovirus infections flaviviruses. in: dm knipepm howleyde griffinra lambma martin. fields virology interleukin-1beta but not tumor necrosis factor is involved in west nile virus-induced langerhans cell migration from the skin in c57bl/6 mice tick-borne encephalitis tickborne flaviviruses: dissecting host immune responses and virus countermeasures pathogenesis of west nile virus infection: a balance between virulence, innate and adaptive immunity, and viral evasion encephalitis caused by flaviviruses neuronal cxcl10 directs cd8+ t-cell recruitment and control of west nile virus encephalitis a temporal role of type i interferon signaling in cd8+ t cell maturation during acute west nile virus infection role of cd8+ t cells in control of west nile virus infection cd8+ t cells use trail to restrict west nile virus pathogenesis by controlling infection in neurons cd40-cd40 ligand interactions promote trafficking of cd8+ t cells into the brain and protection against west nile virus encephalitis cxcr3 mediates region-specific antiviral t cell trafficking within the central nervous system during west nile virus encephalitis pivotal role of antibody and subsidiary contribution of cd8+ t cells to recovery from infection in a murine model of japanese encephalitis immunopathology of flavivirus infections cd8+ tcells mediate immunopathology in tick-borne encephalitis cd8+ t cells mediate recovery and immunopathology in west nile virus encephalitis lack of both fas ligand and perforin protects from flavivirusmediated encephalitis in mice role of pro-inflammatory cytokines released from microglia in neurodegenerative diseases proinflammatory mediators released by activated microglia induces neuronal death in japanese encephalitis the involvement of microglial cells in japanese encephalitis infections tumor necrosis factor receptor-associated death domain mediated neuronal death contributes to the glial activation and subsequent neuroinflammation in japanese encephalitis tumor necrosis factor receptor-1-induced neuronal death by tradd contributes to the pathogenesis of japanese encephalitis tnf-alpha and il-1beta mediate japanese encephalitis virus-induced rantes gene expression in astrocytes glutamate released by japanese encephalitis virus-infected microglia involves tnf-alpha signaling and contributes to neuronal death tumor necrosis factor alpha protects against lethal west nile virus infection by promoting trafficking of mononuclear leukocytes into the central nervous system the immune adaptor molecule sarm modulates tumor necrosis factor alpha production and microglia activation in the brainstem and restricts west nile virus pathogenesis tnf-alphadependent regulation of cxcr3 expression modulates neuronal survival during west nile virus encephalitis interleukin-10 and related cytokines and receptors immunomodulatory cytokines determine the outcome of japanese encephalitis virus infection in mice an insufficient antiinflammatory cytokine response in mouse brain is associated with increased tissue pathology and viral load during japanese encephalitis virus infection kinetics of cytokine profile during intraperitoneal inoculation of japanese encephalitis virus in balb/c mice model il-10 signaling blockade controls murine west nile virus infection steps of the tick-borne encephalitis virus replication cycle that affect neuropathogenesis influence of age on the susceptibility of mice to infection with certain neurotropic viruses mortality following peripheral infection with tick-borne encephalitis virus results from a combination of central nervous system pathology, systemic inflammatory and stress responses distribution and characterization of tick-borne encephalitis viruses from siberia and far-eastern asia interleukin-10-deficient mice develop chronic enterocolitis failure of germinal center formation and impairment of response to endotoxin in tumor necrosis factor alpha-deficient mice accumulation of t-cells with selected t-cell receptors in the brains of japanese encephalitis virus-infected mice participation of both host and virus factors in induction of severe acute respiratory syndrome (sars) in f344 rats infected with sars coronavirus pathogeneses of respiratory infections with virulent and attenuated vaccinia viruses virus infection as a stressor: influenza virus elevates plasma concentrations of corticosterone, and brain concentrations of mhpg and tryptophan the thymus is a common target organ in infectious diseases immune and inflammatory responses in tnf alpha-deficient mice: a critical requirement for tnf alpha in the formation of primary b cell follicles, follicular dendritic cell networks and germinal centers, and in the maturation of the humoral immune response peyer's patch organogenesis is intact yet formation of b lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kda receptor interleukin-10 and the interleukin-10 receptor tumor necrosis factor-alpha and the roles it plays in homeostatic and degenerative processes within the central nervous system fas and perforin pathways as major mechanisms of t cell-mediated cytotoxicity two distinct pathways of specific killing revealed by perforin mutant cytotoxic t lymphocytes molecular mechanisms of lymphocyte-mediated cytotoxicity and their role in immunological protection and pathogenesis in vivo granzymes are the essential downstream effector molecules for the control of primary virus infections by cytolytic leukocytes tcell clones expressing different t-cell receptors accumulate in the brains of dying and surviving mice after peripheral infection with far eastern strain of tick-borne encephalitis virus chimeric live, attenuated vaccine against japanese encephalitis (chimerivax-je): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated japanese encephalitis antigen we thank tomohiko takasaki key: cord-276564-o21ncldx authors: miller, r.; wentzel, a.r.; richards, g. title: covid-19: nad(+) deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on sirt1 activity date: 2020-06-29 journal: med hypotheses doi: 10.1016/j.mehy.2020.110044 sha: doc_id: 276564 cord_uid: o21ncldx the sars-cov-2 hyperinflammatory response is associated with high mortality. this hypothesis suggests that a deficiency of nicotinamide adenine dinucleotide (nad(+)) may be the primary factor related to the sars-cov-2 disease spectrum and the risk for mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad(+) levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these groups have also been observed to have high mortality following infection with covid-19. further consumption of nad(+) in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. this provides a unifying hypothesis as to why these groups are at high risk of mortality and suggests that nutritional support with nad(+) and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. covid-19: nad + deficiency may predispose the aged, obese and type2 diabetics to mortality through its effect on sirt1 activity we hypothesize that reduced nicotinamide adenine dinucleotide (nad + ) levels with consequent deficient activity of the nad + dependent molecule sirt1, which modulates cytokine production, may be the factor that predisposes the aged, obese, type 2 diabetics and other vulnerable groups to an increased mortality. the sars-cov-2 hyperinflammatory response is associated with high mortality. this hypothesis suggests that a deficiency of nicotinamide adenine dinucleotide (nad + ) may be the primary factor related to the sars-cov-2 disease spectrum and the risk for mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad + levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these groups have also been observed to have high mortality following infection with covid-19. further consumption of nad + in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. this provides a unifying hypothesis as to why these groups are at high risk of mortality and suggests that nutritional support with nad + and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. background covid-19 may be asymptomatic or manifest in 3 clinical phases, an initial upper respiratory tract infection, with a few patients thereafter progressing to a pneumonic phase, and an even smaller number to the hyperinflammatory phase which may be lethal. (1) the aim of any therapy would be to intervene at an early stage, either prophylactically or therapeutically, to prevent progression of the disease to a point where mechanical ventilation (mv) is required, or significant organ dysfunction occurs. (2) risk factors for a poor outcome include older age, comorbidity (in particular diabetes, hypertension and cardiac disease), non-asthmatic respiratory disease, obesity, immunosuppression and male sex. (2, 3) the independent associations of advancing age, male sex, chronic respiratory conditions (though not well controlled asthma), chronic cardiac and chronic neurological disease with in-hospital mortality, are in line with other international reports. (4) it is difficult however to determine why these conditions specifically are linked to mortality. docherty et al. report that severe sars-cov-2 infections are rare in those under 18 years of age, comprising only 1.4% of those admitted to hospital. only 0.8 % of those in this study were under 5 years of age, and this "j" shaped age distribution was starkly different from the "u" shaped distribution seen in seasonal influenza. (5) it has not been clear from observational studies however, why sars-cov-2 mostly spared children, but it has been speculated that it is due to differential expression of the ace2 receptor in the developing lung. (6) similarly, pregnancy has not been reported to be associated with mortality, in contrast to the situation with influenza. (4, 7) while the general concept of an excessive or uncontrolled release of pro-inflammatory cytokines is well known, an actual definition of what a hyperinflammatory response or "cytokine storm" is, is lacking. furthermore, there is a poor understanding of the molecular events that precipitate this response and the contribution it makes to pathogenesis. it is also not known what therapeutic strategies might be used to prevent this catastrophic progression of the disease or lessen its severity once initiated. (8) in this phase, there is an unbalanced and exacerbated inflammatory response with the release of inter alia, tumor necrosis factor (tnf-α), interleukin 1β (il-1β), interleukin 6 (il-6), as pro-inflammatory mediators together with interleukin 10 (il-10) and interferon β as anti-inflammatory mediators. the complex interactions between tnf-α, the interleukins, chemokines and interferons in sars-cov-2 are currently poorly understood; however, they are associated with and related to a significant viraemia. (9, 10) the fact that most international studies have indicated that certain risk factors were common suggested that a similar systemic abnormality might be present in those at high risk, predisposing to severe illness or mortality. in this context, the nicotinamide adenine dinucleotide (nad + )-and zincdependent molecule, the silent information regulator 1 (sirt1) represents a potential common thread in the aetiology of the hyperinflammatory response and increased mortality. sars-cov-2 targets and binds to the angiotensin-converting enzyme 2 receptor (ace2r), a membrane-associated aminopeptidase also expressed in the vascular endothelium, renal and cardiovascular tissue, and small intestine, testis, and respiratory epithelia. (11) the ace2r acts as the host cell receptor for the virus which binds via the spike protein on the viral capsid. (12) this stimulates clathrin-dependent endocytosis of both the ace2r and virus, events that are essential for infectivity. this process induces adam 17 activity which reduces expression of ace2 on the cell surface. (13) nicotinamide adenine dinucleotide (nad + ) nad + is a cofactor found in every cell of the body, and it is involved in multiple metabolic pathways. it is a fundamental housekeeping molecule that catalyses electron transfer in metabolic reductionoxidation reactions, functioning as an electron shuttle in the production of adenosine triphosphate (atp). increased age is a strong predictor of sars-cov-2-associated in-hospital mortality after adjusting for comorbidity. (6) older patients have also been identified as having the lowest levels of nad + , (14) while, conversely, those with the lowest risk, infants and children have the highest levels. the decline in nad + levels with ageing is mainly dependent on cd38, a 45kda transmembrane molecule, encoded on chromosome 4. in leukocytes, it acts as a receptor in adhesion and signalling pathways. (15) cd38 expression is increased with insulin resistance, and may exacerbate the age-dependent decline of nad + .(9) nad + and nadp profoundly affect age-influencing factors such as oxidative stress and mitochondrial activity, and nad + -dependent sirtuins also mediate the ageing process. (10) as humans, age, antioxidant defence mechanisms such as glutathione production are also depleted and the associated increase in reactive oxidative species (ros) causes all cells (16) to enter a state of pseudohypoxia in which the ratio of nad + /nadp declines. (17) (18) (19) oxidative stress also activates the nad + -dependent enzyme, poly adp ribose polymerase 1 (parp1). (20) hyperactivity of parp1 results in depletion of cellular nad + pools, leading to atp deficiency, energy loss, and subsequent cell death. these processes have the potential to enhance the pro-inflammatory cascade. nad + deficiency impairs sirt1 function (21) and its successful activation. whereas extreme niacin deficiency is associated with the development of pellagra, more subtle decreases occur in diabetes, ageing and hypertension with resultant attenuation of responsiveness to inflammatory stimuli. (22) silent information regulator 1 (sirt1) sirtuins are an ancient family of seven nad + -dependent deacylase and mono-adp-ribosyl transferase signalling proteins that are intrinsically involved in metabolic regulation and cellular homeostasis. of particular interest is sirt1, which downregulates adam 17 (a disintegrin and metalloproteinase domain 17), also called tnf-α converting enzyme (tace), by increasing expression of timp3 the gene that encodes for tissue metalloproteinase inhibitor 3. (23) in so doing it decreases levels of tnf-α, il-1b and il-6. an increase in tnf-α causes sirt1 to down-regulate adam 17, thereby controlling tnf-α formation in a negative feedback loop that secondarily influences il-1b and il-6 production, which are dependent on tnf-α. (23) sirt1 is known to play a crucial role in obesity-associated metabolic diseases, cancer, ageing, cellular senescence, cardiac ageing and stress, prion-mediated neurodegeneration, inflammatory signalling in response to environmental stress, embryonal development of the heart, brain, spinal cord and dorsal root ganglia, and placental cell survival. (24) in its inactive or open state, it contains a zn ++ module and an nad +binding site. (25) whereas certain conditions such as ulcerative colitis, crohn's disease, short bowel syndrome, renal failure, alcoholism, and inadequate meat intake are specifically associated with zinc deficiency there is evidence that zinc intake among older adults might be marginal. an analysis of the third national health and nutrition examination survey (nhanes iii), 1988-1994 data found that 35-45% of adults aged 60 years or older had a zinc intake below estimated average requirements. (26) when nad + binds to the sirt1 molecule in the presence of the zn ++binding module, it undergoes a structural change, enveloping the nad + molecule and causing it to be "closed" or activated. (25) the presence of both the zn ++ and the nad + moieties are imperative for its function. sirt1 downregulates adam17 and cytokine production adam17 is a proteinase encoding gene. tnf-α and the cytokine receptor for il-6 must be proteolytically cleaved in order to be systemically active, and adam17 provides this function. if adam17 expression is not downregulated by sirt1, tnf-α and il-6 are released, resulting in an uncontrolled hyperinflammatory response as may occur with covid-19. (23, 27-30) sirt 1, by inhibition of adam17 and thereby tnf-α and il-6, performs an anti-inflammatory function. (31) (32) (33) (34) (35) (36) (37) (38) if oxidative stress is severe, increased adam17 attempts to ameliorate tissue injury by converting active iron (fe2 + ) to its inert form (fe3 + ) which is stored in hepatocytes and macrophages and as ferritin by means of the fenton reaction (fe2 + +h 2 o 2 →fe 3+ + ho • + oh − ), (fe 3+ + h 2 o 2 →fe 2+ + ho 2 • + h + ). this also potentially transforms haemoglobin to methaemoglobin, reducing its capacity to bind to oxygen. (39) covid-19 replication and sirt1 sirt1 not only controls and modifies the inflammatory response, but along with the sirtuin family (sirt1-7) is also a primary defence against dna and rna viral pathogens. (40) in some respiratory infections and cardiovascular conditions, sirt1 promotes autophagy (the destruction of damaged or redundant cellular components occurring in vacuoles within the cell), and in so doing inhibits apoptosis and provides protection against hypoxic stress. [37] [38] [39] [40] upregulation of sirt1 directly decreases viral replication and inhibits the activation of adam17, thereby decreasing tnf-α, il-1b and il-6. conversely depletion of sirt1 allows for increased viral replication with little or no inhibition of adam17 activity, causing uncontrolled increases in tnf-α, il-6 and il-1b. whereas an increase in tnf-α would usually increase sirt1 activity to downregulate adam17, in the presence of a deficiency of nad + or zn ++ , this would not occur due to insufficient activation of sirt1, causing an unchecked increase in tnf-α. in both obesity and type 2 diabetes mellitus, intracellular nad + levels are decreased in multiple tissues, including adipose tissue, skeletal muscle, liver and the hypothalamus. (41) furthermore, both conditions are characterised by low-grade inflammation associated with activation of both il6 and tnf-α. (42, 43) obesity or type 2 diabetes mellitus would increase the risk for cytokine storm due to an inability to activate sirt1. sirt1 maintains vascular endothelial function, preventing or reducing the potential for the metabolic syndrome, ischaemia-reperfusion injury and inflammation in obesity. with increasing age however, nad + levels and sirtuin activity decline and this is exacerbated by obesity and sedentary lifestyles (22) . sirt1 is an effective inhibitor of oxidative stress in vascular endothelial cells (ec) (44) via various signalling pathways. (45) the endothelial glycocalyx (eg) is a web of membrane-bound glycoproteins on the luminal side of endothelial cells, associated with various glycosaminoglycans that cover the vascular endothelium. (46) the eg separates cellular blood components from the endothelium and maintains osmotic tension of the intravascular compartment. (44, 45) conditions causing damage to, and shedding or fragmentation of the eg, (as seen in sarscov-2 under severe oxidative stress induced by the hyperinflammatory response), exposes the endothelium, allowing adhesion, clumping and activation of platelets with degranulation and release of vasoactive substances. the eg has anticoagulant properties as it is a binding site for mediators such as heparin cofactor ii, antithrombin, thrombomodulin and tissue factor pathway inhibitor (tfpi). heparin cofactor ii and dermatan sulphate inhibit thrombin, and antithrombin activity is enhanced when bound to heparan sulphate. conversely, exposure of the endothelial cell surface protein, thrombomodulin, which contains a cofactor for thrombin, chondroitin sulphate, promotes coagulation via activation of tissue factor (46) as seen in sar-cov-2. the eg is already compromised in systemic inflammatory states, such as diabetes, hyperglycaemia, surgery, trauma and sepsis. (46) under conditions of more severe oxidative stress, as in the hyperinflammatory response, widespread damage may lead to its destruction, with the occurrence of capillary leak and oedema formation, accelerated inflammation, platelet aggregation, hypercoaguability and a loss of vascular responsiveness. (47) inflammatory mediators that are implicated in this process are tnf-α, bradykinin, c-reactive protein and mast cell tryptase. given the above, it is possible that activation of sirt1 may be a crucial factor in the prevention of the hyperinflammatory response and may be necessary for a successful defence against viral attack. vulnerable patient groups would potentially be less likely or unable to ensure sufficient activation of sirt1 due to low nad + levels or associated nutritional deficiencies including zn ++ , and as such contribute to an inability to control viral replication and reduce the uncontrolled expression of pro-inflammatory cytokines. the sars-cov-2 hyperinflammatory response is associated with high mortality. a deficiency of nad + , in the context of an elevated cd38, may be the primary factor related to the sars-cov-2 disease spectrum and the risk of mortality, as subclinical nutritional deficiencies may be unmasked by any significant increase in oxidative stress. nad + levels decline with age and are also reduced in conditions associated with oxidative stress as occurs with hypertension, diabetes and obesity. these same groups have also been observed to have high mortality following infection with covid-19. further consumption of nad + in a pre-existent depleted state is more likely to cause progression to the hyperinflammatory stage of the disease through its limiting effects on the production of sirt1. given that activation of sirt1 is dependent on the availability of nad + and zinc and that high levels of oxidative stress deplete nad + , thereby decreasing sirt1 activity, nutritional support with nad + precursors and sirt1 activators, could minimise disease severity if administered prophylactically and or therapeutically. the significance of this hypothesis, if proven, has far-reaching consequences in the management of covid-19 especially in third world countries, where resources and finances are limited. robert miller is managing director 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in sepsis robert miller is managing director of battle brew tactical nutrition key: cord-023414-xxw5kptr authors: chistensen, h. r.; frøkiær, h. title: characterization of a large panel of lactic acid bacteria derived from the human gut for their capacity to polarize dendritic cell date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ap.x sha: doc_id: 23414 cord_uid: xxw5kptr dendritic cells (dcs) are the principal stimulators of naïve t helper (th) cells and play a pivotal regulatory role in the th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut‐derived lactobacillus and bifidobacterium spp. was screened for dc‐polarizing capacity by exposing bone marrow‐derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc‐surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin‐12 (il‐12) and tumour necrosis factor (tnf)‐α, while the differences for il‐10 and il‐6 were less pronounced. bifidobacteria tended to be weak il‐12 and tnf‐α inducers, while both strong and weak il‐12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il‐12 induction inhibited il‐12 and tnf‐α production induced by an otherwise strong cytokine‐inducing strain of lactobacillus casei, while il‐10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il‐12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei‐induced upregulation of cd86 was reduced in the presence of a weak il‐12‐inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg‐driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-256837-100ir651 authors: smith, steven b.; dampier, william; tozeren, aydin; brown, james r.; magid-slav, michal title: identification of common biological pathways and drug targets across multiple respiratory viruses based on human host gene expression analysis date: 2012-03-14 journal: plos one doi: 10.1371/journal.pone.0033174 sha: doc_id: 256837 cord_uid: 100ir651 background: pandemic and seasonal respiratory viruses are a major global health concern. given the genetic diversity of respiratory viruses and the emergence of drug resistant strains, the targeted disruption of human host-virus interactions is a potential therapeutic strategy for treating multi-viral infections. the availability of large-scale genomic datasets focused on host-pathogen interactions can be used to discover novel drug targets as well as potential opportunities for drug repositioning. methods/results: in this study, we performed a large-scale analysis of microarray datasets involving host response to infections by influenza a virus, respiratory syncytial virus, rhinovirus, sars-coronavirus, metapneumonia virus, coxsackievirus and cytomegalovirus. common genes and pathways were found through a rigorous, iterative analysis pipeline where relevant host mrna expression datasets were identified, analyzed for quality and gene differential expression, then mapped to pathways for enrichment analysis. possible repurposed drugs targets were found through database and literature searches. a total of 67 common biological pathways were identified among the seven different respiratory viruses analyzed, representing fifteen laboratories, nine different cell types, and seven different array platforms. a large overlap in the general immune response was observed among the top twenty of these 67 pathways, adding validation to our analysis strategy. of the top five pathways, we found 53 differentially expressed genes affected by at least five of the seven viruses. we suggest five new therapeutic indications for existing small molecules or biological agents targeting proteins encoded by the genes f3, il1b, tnf, casp1 and mmp9. pathway enrichment analysis also identified a potential novel host response, the parkin-ubiquitin proteasomal system (parkin-ups) pathway, which is known to be involved in the progression of neurodegenerative parkinson's disease. conclusions: our study suggests that multiple and diverse respiratory viruses invoke several common host response pathways. further analysis of these pathways suggests potential opportunities for therapeutic intervention. respiratory viruses account for seasonal colds, bronchiolitis, acute otitis, sinusitis, croup, community-acquired pneumonia, and exacerbation of both chronic obstructive pulmonary disease and asthma [1] . the prevalence of pandemic orthomyxoviridae influenza a virus (flu) from april 2009 to 2010 was estimated to be approximately 60 million cases, 270,000 hospitalizations, and 12,000 deaths [2] . paramyxoviridae respiratory syncytial virus (rsv) infection results in nearly two million children requiring medical care with about 57,000 children younger than five years hospitalized annually [3] . in one survey, rsv was the most prevalent pathogen in children under five years with an acute respiratory infection, followed by adenoviridae adenovirus (adeno), and picornaviridae human rhinovirus (hrv) [4] . while initially effective, pathogen gene targeted treatments exert evolutionary selection on the infectious species often leading to the emergence of drug resistant strains. as a result, there are increasing clinical reports of resistance against many drugs that directly act on viral proteins or their dna [5, 6] . in particular, resistance to different classes of antiviral drugs is becoming more clinically prevalent in respiratory virus infections as seen with rsv and flu treated with the antiviral drugs palivizumab [7] , and oseltamivir [8] , respectively. pathogens elucidate two broad types of biochemical responses in the host. first is the activation of the host immune system. while the immune response is critical in combating pathogen infections, its over-activation often exacerbates tissue damage initiated by viral invasion [9, 10] . the second response is the up-regulation of host genes, such as protein biosynthetic pathways, that are crucial for sustaining pathogen invasion, replication and evasion [11] . interestingly, genetically distinct respiratory viruses often modulate common host proteins and biological pathways during infection [1] . for example, many respiratory viruses trigger similar general airway inflammatory responses such as the expression of cytokines interleukin-6 (hugo gene name il6), interleukin-8 (il8) and interleukin-11 (il11), and granulocyte macrophage-colony stimulating factor (csf2). these inflammatory responses in turn initiate iga production, b cell differentiation and t cell stimulation [12] [13] [14] [15] [16] . as a consequence, diagnosis for specific viral infections is difficult since diverse respiratory viruses cause similar, often indistinguishable patient symptoms [1] . however, because distinct respiratory viruses converge on similar immune responses, opportunities also exist for targeting host proteins and pathways which will potentially affect multiple viral pathogens [17] . moreover, human targets might be less susceptible to the evolution of drug resistance due to constraints on the virus to find alternative host pathways for its proliferation. individuals may experience a co-infection or sequential infections of multiple viruses or bacteria which can complicate both disease diagnosis and drug prescription decisions. furthermore, patients infected by multiple pathogens may have further complications due to drug-drug interactions, cumulative drug toxicities and immune system suppression, as observed during hiv and mycobacterium tuberculosis co-infections [18, 19] . indeed, a study in children under five years showed pervasive clinical occurrences of co-infections involving combinations of rsv, hrv, paramyxoviridae parainfluenza virus, flu, coronaviridae sars-coronavirus (coron), paramyxoviridae metapneumonia virus (mpneu), parvoviridae human bocavirus and adeno [4] . therefore, in addition to minimizing drug resistance, there is a need for new therapeutic approaches to safely and effectively treat co-infections by multiple viral and/or bacterial pathogens, particularly where strain-specific diagnostics or treatments are unavailable. the development of new antiviral therapeutics requires a greater understanding of the global host response when challenged by different types of viruses. such knowledge may lead to the identification of novel human genome targets that are shared across multiple viral infections as well as opportunities for repositioning existing drugs for the treatment of infectious diseases [20] . several recent studies have generated multiple mrna microarray gene expression datasets derived from experiments involving the infection of human cell-lines or animal models with one or more of the major respiratory viruses [21] [22] [23] . through a systematic analysis of these respiratory virus-human host gene expression datasets, we determined common sets of genes and pathways involved in host responses to viral infections. among the most significant pathways, we identified several potential new opportunities for repurposing existing drugs for the treatment of respiratory viral infections. we performed a large-scale analysis of published mrna microarray datasets from studies involving a wide range of respiratory viruses in human host infection models. we focused on human mrna array datasets in order to avoid complications inherent in cross-species comparisons. in order to ensure consistency in experimental conditions and reduce biases due to noisy or poor quality datasets, we instituted an iterative process of database querying, data filtering, and common pathway analysis across all published human mrna datasets for twelve relevant respiratory viruses. these viruses initially included the double stranded dna viruses herpesviridae human cytomegalovirus (cmv) and adeno; the positive sense single stranded rna viruses coron, picornaviridae coxsackievirus (cox), hrv, picornaviridae echovirus (echo), and picornaviridae enterovirus (entero); and the negative sense single stranded rna viruses flu, mpenu, rsv, bunyaviridae hantavirus (hant) and sin nombre virus (snv). this list was later narrowed to include only the subset listed in table 1 based on filtering processes outlined in the materials and methods and shown in figure 1 . a total of seven different respiratory viruses were analyzed, represented by fifteen unique gene expression omnibus (geo) datasets (indicated by geo series or gse accession numbers), nine different human cell types, and seven different array platforms for a total of 28 unique comparisons. note that one dataset (gse17156) contained two different viruses (flu and rsv) that were analyzed. after querying the geo database and prescreening for obvious non-candidate datasets such as those not associated with human array platforms, there were at least 23 datasets associated with at least one of the twelve respiratory viruses. however, after considering all conditions for geo dataset candidacy, at least four of these datasets were excluded. in one case, an adeno dataset (gse1291 [pmid unpublished]) had less than three samples per treatment group, as did a cox (gse712 [pmid unpublished]) and a cmv (gse19345 [24] ) dataset. as another example, a cmv dataset (gse675 [25] ) lacked a healthy/control treatment group. additionally, at least four datasets had some comparison groups that did not fit the filters for inclusion. for instance, an hrv (gse13396) dataset's original study design was to observe differences in hrv infectivity between asthmatic and non-asthmatic patients. the asthmatic comparison group data were eliminated from the analysis because of potential difficulties in distinguishing between host inflammatory responses due to viral infections from those associated with chronic asthma. similarly, a combined flu, hrv and rsv dataset (gse17156) contained two main patient groups. one group was classified as developing symptoms after exposure to a single virus under study, while the other group did not develop any symptoms after exposure. only the group that developed symptoms for each of the three viruses was considered for further analysis and the asymptomatic group was omitted. in total, 19 geo datasets, representing 42 unique comparisons (different time points and/or virus strains) were analyzed for quality because they met the four requirements for dataset candidacy. no single dataset exhibited overall poor quality control (qc), and therefore, all 19 datasets representing 42 comparison groups were analyzed for differential expression. however, qc analysis across all candidate datasets revealed two outliers in gse17156 (samples gsm429252 and gsm429279), two in gse11348 (samples gsm286647 and gsm286733), and one outlier each in dataset gse24132 [26] (sample gsm594166), gse1739 (sample gsm30367), and gse19580 (sample gsm487986) for a total of seven samples removed from five different datasets. an illustration of the kernel density and principle component analysis (pca) plots generated during the qc analysis is shown in figure 2 for gse17156's rsv treatment (median of 141 hours post infection) and rsv control (baseline) groups. additional qc analysis results including median of absolute deviation (mad) score plots and pair-wise correlation maps are shown in figure s1 . initially, all samples except gsm429279 showed acceptable kernel density (figure 2a) , pca (figure 2c ), mad score ( figure s1a ) and pair-wise correlation ( figure s1c ) plots. the sample gsm429279 was removed because: a) it did not conform to the kernel density of the other samples; b) it fell outside of the hotelling t2 alpha threshold of 0.05 (represented by the superimposed elliptical on the pca plot), and; c) it was an outlier in both the mad score and pair-wise correlation plots. a second qc round was performed, which resulted in a further non-conforming sample, gsm429252, being discarded. subsequent qc analysis generated acceptable results in kernel density (figure 2b ), pca (figure 2d ), mad score ( figure s1b ), and pair-wise correlation ( figure s1d ), thus this dataset passed our criteria for inclusion in the analysis. all datasets exhibiting acceptable quality were analyzed for probe differential expression. an example volcano plot is shown in figure s2 for rsv treatment group at peak symptoms versus control group (data originating from gse17156). cutoff levels of 1.5-fold increase or decrease in probe expression levels, respectively, and p-values ,0.05 were used throughout (represented by red lines in figure s2 ). all comparison groups had at least some differentially expressed probes, although the number varied greatly indicating potential falsely discovered probes (for example, a comparison group within gse18816 had 111 differentially expressed probes while a comparison group within gse11408 had 2533 differentially expressed probes). however, the conservative pathway enrichment approach we employed tends to attenuate falsely discovered genes. there were three comparison groups that did not meet the least square mean (lsm) threshold requirement and were excluded from the differentially expressed probe list: two of the for each comparison group, the differentially expressed probes were mapped to their corresponding genes, and then a p-value was assigned for each pathway map using the software genego (accessed june 2011). next, the comparison group's significant pathway lists were combined to find the union of all significant pathways (that is, the combined pathway list where all treatment groups have at least one significant pathway). a total of 459 out of the approximately 650 pathway maps available in metabase were determined to be significant. comparison groups having ,5% significant pathways of the total significant pathways (that is, comparison groups containing less than 23 significant pathways) lead to the exclusion of eleven comparison groups from the union list. excluded groups were: hrv at 8 hours (eliminating one comparison group from gse11348), hrv at 72 hours (eliminating one comparison group from gse17156), both strains of flu at 1 hour and 3 hours each and another strain at 6 hours (eliminating three comparison groups from gse18816), rsv at 24 hours (eliminating all comparison groups from gse24132), cmv at 24 and 72 hours (eliminating all comparison groups from gse24434 [27] ), and flu at 8 hours (eliminating all comparison groups from gse24533 [28] ). at the end of the final step in our filtering process, a total of 15 datasets, or 28 comparison groups remained (tables 1, s1 and s2). there were 67 enriched pathways in which all seven respiratory viruses were represented by at least one comparison group (table s3 ). the list is ranked first by the viral frequency, followed by the sum of the normalized viral expression (nve) for each pathway. also shown are the differentially expressed as well as the total number of network objects across all 28 comparisons. the top 20 enriched pathways are listed in table 2 along with the percentage and names of the differentially expressed genes with a viral frequency of at least five in each pathway. of these, the top five pathways were chosen for further analysis and mapping. these pathways are epidermal growth factor receptor (egfr) signaling, cd40 signaling, interferon-gamma (ifng) signaling, histamine receptor h1 (hrh1) signaling, and interleukin-17 (il17) signaling (figures s5. s6, s7, s8, s9; table s4 ). additionally, the parkin-ubiquitin proteasomal system (parkin-ups) pathway was chosen for further analysis because it has not been previously associated with the innate immunity and might be an interesting new mechanism of host response to respiratory viral infection ( figure 3 ). the nves for differentially expressed genes with frequencies of at least six viruses are shown in table 3 along with their associated pathways. the list is ranked by the greatest viral frequency, and then by number of pathways in which the gene is differentially expressed. the nve values for all genes, along with associated pathways, ranked by the greatest viral frequency, followed by the number of pathways in which the gene is differentially expressed are in table s5 . we ensured that the nve was not bias toward any particular comparison group, and indeed no single dataset contributed to the overall nve for any single virus (table s2) . hierarchical clustering on the quantile normalized fold change values for all genes having expression values in at least 26 out of 28 comparisons (at least 90% comparisons) and significant in at least seven comparisons ( figure s3 ) as well as for genes with nve of at least six viruses ( figure s4 ) did not reveal any dominant clustering by gse or virus type. the most consistently up-regulated genes (up-regulated in at least six viruses and down-regulated no more than one virus) are: nuclear factor of kappa light polypeptide gene enhancer in b-cells inhibitor alpha (nfkbia), tumor necrosis factor alpha-induced protein 3 (tnfaip3), chemokine c-c motif ligand 2 (ccl2), interferon regulatory factor 1 (irf1), prostaglandin-endoperoxide synthase 2 (ptgs2), chemokine c-c motif ligand 20 (ccl20), dual specificity phosphatase 1 (dusp1), eukaryotic translation initiation factor 2-alpha kinase 2 (ei-f2ak2), tnf receptor superfamily member 6 (fas), suppressor of cytokine signaling 1 (socs1), tnf receptor-associated factor 1 (traf1), and ubiquitin-conjugating enzyme e2l 6 (ube2l6). there were no consistently down-regulated mrnas (downregulated in at least six viruses and up-regulated in no more than one virus). we sought drug repurposing candidate targets from the top five enriched pathways and the parkin-ups pathway by searching the drugbank database, version 3.0 (http://www.drugbank.ca/ accessed august 2011) [29] [30] [31] , for drugs targeting any of the 67 differentially expressed genes with a viral frequency of at least five (table s6 ). of these, thirteen genes, or almost 20% of the original 67 genes, were associated with at least one approved small molecule or protein therapy. there genes were: prostaglandinendoperoxide synthase 2 (ptgs2), tnf, matrix metallopeptidase 9 (mmp9), jun proto-oncogene (jun), interleukin 1 beta (il1b), ccl2, cd86, coagulation factor iii (f3), phosphoinositide-3kinase regulatory subunit 1 (pik3r1), intercellular adhesion molecule 1 (icam1), nuclear factor of kappa light polypeptide gene enhancer in b-cells 2 (nfkb2), caspase 1 (casp1), and tubulin beta 3 (tubb3). a selection of these genes, along with other characteristics to evaluate their potential as drug targets such as involvement in immune response [29] [30] [31] , jackson laboratory knock-in/knock-out mouse (jax) phenotype [32] , approved or marketed small molecule drug or protein therapy, and current indications for that drug, are listed in table 4 . note that the current indication may not be for the gene target listed. mimosine (gene target: ccl2) and glucosamine (gene targets: nfkb2 and mmp9) did not have a current indication, while the interactions of natalizumab (gene target: icam1) and gallium nitrate (gene target: ilb1) with their gene targets were unclear. additionally, therapies associated with ptgs2 are cyclooxygenase (cox-2) inhibitors which have known side-effect issues thus were not explored further. therefore, nfkb2, icam1 and ptgs2 were excluded from table 4 , leaving ten genes for potential drug repurposing. the potential cases for drug repurposing are discussed more in-depth for four targets; f3, il1b, tnf and casp1. our study used a systematic process to minimize potential technical noise that could have arisen from our comparative analysis of fifteen unique datasets from nine different cell types, and seven different array platforms. these measures included candidate dataset filtering followed by qc, differential gene expression and pathway enrichment analyses. a total of 14 out of 42, about one third of the total comparisons, were removed as a result of this filtering process, which is indicative of our conservative analysis approach. we had previously used largescale and merged-sam analyses in integrating large-scale microarray datasets involving cancer tissues from multiple laboratories [33, 34] . however, the small sample size datasets used in the present study required a more rigorous methodology to identify data outliers. to our knowledge the qc analysis performed with each geo dataset is unique to this study. although no dataset was completely disregarded after qc analysis, some samples were clear outliers, thus potentially skewing the data. kauffmann and huber have demonstrated improvements in signal-to-noise ratios after performing post normalization qc analysis to remove array outliers within an experiment [35] . those authors used ma-plot and boxplots of the log-ratios to determine outliers instead of mad scores, pca and pair-wise correlations employed in this study. fundamentally, the concept of data improvement after outlier removal applies regardless of the qc analysis approach. signaling 26 jun, myc, nfkbia, stat1, fos, jak2, hbegf, dusp1, dusp4, ptk2, gsk3b, mmp9, nfkb2, pik3r1, prkca, sos2, tgfa cd40 signaling 31 il8, jun, nfkbia, tnfaip3, ccl2, fas, il6, irf1, jak2, ptgs2, traf1, ccnd2, cd86, icam1, lyn, map2k3, map3k14, mapk14, nfkb2, pik3r1, tp53, traf5 ifng signaling 24 myc, stat1, cdkn1a, eif2ak2, irf1, jak2, socs1, stat2, camk2g, cebpb, icam1, mapk14, pik3r1, prkca, ptpn11 hrh1 signaling 25 il8, jun, nfkbia, fos, il6, tnf, csf2, f3, gnaq, gnb4, gng12, icam1 despite the diverse nature of the microarray data analyzed here, we found a large overlap between comparison groups in significant pathways, especially the immune system. of the top twenty enriched pathways, eighteen are associated with immune response ( table 2) . for example, egfr signaling is known to be activated during infection by respiratory viruses flu [36] and entero [37, 38] . cd40 signaling is associated with coron [39] , rsv [40] , and the general immune response [41] . interferon gamma (ifng) signaling is initiated by flu [42] and rsv [43] , while interleukin 1 signaling is stimulated by flu [42] . as components of the general immune response, interferon and interleukin pathways are activated by infectious agents such as hepatitis c virus (hcv), hiv and tuberculosis as well as chronic diseases like crohn's disease, diabetes, and metastatic melanoma [44, 45] . the overall relationships between the transitory host immunity response launched by pathogenic infections versus that seen in chronic autoimmune and neurodegenerative diseases are complex and an intense area of investigation [46] . in addition, there are considerations about subtle shifts in gene function roles in different cell tissue types amongst the various diseases. thus, we are cautious about any linkages between pathways involved in infections and those of chronic diseases as implied by our analysis without further validation studies. parkin (park2) is an e3-ubiqutin ligase associated with the progression of the neurodegenerative disorder parkinson's disease. [47] . as a central hub protein in the parkin-ups pathway, park2 ubiquinates proteins encoded by septin 5 (sept5) [48] , tubulin alpha and beta [49] , and the glycosylated form of synuclein, alpha (snca) [50] for degradation by the 26s proteasome. park2 also ubiquinates synuclein, alpha interacting protein (sncaip) for regulation of snca [51] , interacts with stip1 homology and u-box containing protein 1 e3 ubiquitin protein ligase (stub1) to enhance ubiquitination of g proteincoupled receptor 37 (gpr37), [52] (which associates with f-box and wd repeat domain containing 7 (fbxw7)), and cullin 1 (cul1) to ubiquitinate cyclin e [53] . park2 is deactivated by protolytic cleavage by casp1 and caspase 8 (casp 8) [54] and can be activated by either heat shock protein 70kd (hspa4) or stub1 [52] . the parkin-ups pathway is not commonly associated with general immune response to viral infection. however, other ubiquitylation proteins, such as isg15, are known to play roles in host defense [55, 56] . associations between influenza infection and neuroinflammation in early onset autosomal recessive parkinson's disease have been recently suggested [57] [58] [59] . at least one factor in the progression of parkinson's disease is the formation of neuotoxic lewy bodies due to increases in snca. increases in snca are believed to be the result of loss-of-function mutations in park2 which cause disruptions in the protein's localization and solubility [60] [61] [62] . polymorphisms in the gene park2 have also been associated with susceptibility to infectious diseases such as leprosy, typhoid fever and paratyphoid fever, although the exact mechanism is still unclear [63, 64] . jang et al. observed activation of snca in mouse nervous tissue long after pathogenic h5n1 flu infection where the increased levels of snca mirror those found in parkinson's disease [57] . similarly, recent findings from rohn and catlin indicate flu as a potential causative factor for parkinson's disease [58] . indeed, links between flu and other neurodegenerative diseases have been suggested, and these include seizures, transverse myelitis, expressive aphasia, syncope, encephalitis, neuromyelitis optica, and central nervous system disease in general [65] [66] [67] . park2 itself has a low signal at the mrna level which might be due to its significant regulation by post-translation processes [52, 54] . further studies are needed to determine the mechanism our analysis suggests several potential repurposing opportunities for launched drugs against host-viral targets (table 4 ). this assumption is based on the occurrence of genes that are differentially expressed in infection models for at least five of the seven respiratory viruses, have involvement in a number of relevant pathways related to host immune response, and encode for known drug targets. the drugs associated with this gene list do not have current indications as anti-viral therapies, although pranlukast and clenbuterol are prescribed for relief of lung disorders such as bronchospasm after allergic reactions and asthma bronchoconstriction during asthma attacks, respectively. also, minocycline, sometimes called minocin, is a broad-spectrum tetracycline antibiotic as well as a caspase 1 (casp1) inhibitor while chloroquine is a well-known anti-malaria drug [29] [30] [31] . in fact, eight of the ten drug repurposing gene targets are involved in activation of the innate immune response, while the remaining two have some evidence of virus modulation. potential drug repurposing opportunities for f3, il1b, tnf, and mmp9, as well as the parkin-ups pathway gene product casp1, are discussed below. coagulation factor iii (f3). f3 normally binds to the native cofactor vii or viia to induce the blood coagulation cascade. treatment with recombinant coagulation factor viia promotes blood coagulation in hemophiliacs [29] [30] [31] . esmon et al. [68] suggest that coagulation could be used therapeutically to modulate inflammation responses and vice versa, but also caution about the danger of increased incidence of thrombosis. the consistent upregulation of f3 across five viruses suggests that the immunecoagulation axis is already initiated and supplemental f3 activation may cause thrombosis complications. further study is needed to develop therapeutics that could balance between innate immune response triggered by coagulation factor viia therapy and stabilization of the antithrombotic state. interleukin 1 beta (il1b). il1b is a cytokine involved in inflammatory response, cell proliferation, differentiation, and apoptosis. il1b is specifically cleaved into its active form by the protease casp1 after which it activates the nlrp3 inflammasome [29] [30] [31] 69] . indeed, il1b is consistently up regulated across cmv, flu, hrv, mpenu and rsv which likely correlates with inflammasome activation. however, overexpression of il1b causes multiple inflammatory disorders [69] . antagonists or neutralizers of il1b, such as canakinumab, could potentially reduce inflammation damage associated with viral infection. tumor necrosis factor (tnf). tnf has a wide range of biological functions including modulation of immune response to pathogen assault. mouse tnf knock-out phenotypes include abnormal immune system physiology, increased susceptibility to viral infection, and both increased and decreased susceptibility to bacterial infection [29] [30] [31] . in our study, tnf is mostly up regulated in infections by cmv, coron, cox, and flu but directionally ambiguous for mpneu and not expressed under rsv. while total disruption of tnf function would be deleterious to the host, there are instances where partial tnf inhibition provides a clinical benefit in patients with viral complications [70, 71] . pranlukast is a cysteinyl leukotriene receptor-1 antagonist that reduces bronchospasm caused by an allergic reaction, usually with asthmatic individuals. this drug inhibits tnf-alpha by blocking macrophage cysteinyl leukotriene 1 (cysltc4, d4) receptors [72] or suppression of nf-kappa b activation [73] . pranlukast has been recently shown to be beneficial not only in cases of respiratory syncytial virus postbronchiolitis, but also in a wide variety of other diseases with strong inflammatory complications such as cystic fibrosis, cancer, atherosclerosis, eosinophils cystitis, otitis media, capsular contracture, and eosinophilic gastrointestinal disorders [71] . amrinone is a type 3 pyridine phosphodiesterase inhibitor used in the treatment of congestive heart failure and is an inhibitor of tnf [74] . phosphodiesterase inhibitors have been shown to alter immune response [75] [76] [77] [78] and, in one case, specifically through tnf [79] . amrinone is known to modulate pro-and antiinflammatory factors in endotoxin-stimulated cells [80] . type 4 phosphodiesterase inhibitors have been used to treat rsv-induced airway hyper-responsiveness and lung eosinophilia [81] . therefore, indirect evidence suggests that armirone may be beneficial in respiratory viral infection situations by inhibiting tnf via type 4 phosphodiesterase, although this has yet to be seen in clinical studies. matrix metallopeptidase 9 (mmp9). mmp9 encodes a matrix metallopeptidase that degrades type iv and v collagens, and is implicated in arthritis and metastasis [29] [30] [31] . we can only speculate on the role mmp9 plays in infection. our analysis finds the gene to be up-regulated for three viruses while down-regulated for two different viruses. in other studies, mmp9 has been observed to be up-regulated after exposure to double stranded rna and is important to airway injury [82] , specifically by rsv [83] . mmp9 expression is induced by il1b [84] which, as mentioned above, is an activator of the nlrp3 inflammasome [85] . mmp9 inhibitors such as marimastat, minocycline or captopril, could be beneficial assuming that the protein is coopted by the infecting virus for tissue remodeling. blocking mmp9 may also reduce inflammatory damage by down-regulating the inflammasome. caspase 1 (casp1). in the case of the parkin-ups pathway, inhibiting tubulin-beta formation may reduce viral proliferation given that flu utilize acetylated tubulin for protein trafficking [86] and increases in neuronal class iii tubb occur after cox infection [87] . a casp1 inhibitor such as minocycline could be used to increase park2 ubiquitinase activity, in turn decreasing the tuba or tubb availability. as mentioned above, casp1 is a component of the nlrp3 inflammasome, activating the precursor to il1b [69] . therefore, a casp1 inhibitor would have an antagonist relationship with il1b, hence the inflammasome. further, casp1 inhibitors would be agonists for park2, thereby reducing accumulation of snca. in this regard, casp1 inhibitors may not only prevent unnecessary nlrp3 inflammasome activation via ilb1, but may also reduce accumulation of neurotoxic lewy bodies through activation of park2. however, caspases are not specific to the parkin-ups pathway and inhibition in this regard may result in toxicity or other complications [88] . additionally, mouse jax phenotypes for casp1 show both increased and decreased susceptibility to bacterial infection, as well as decreased inflammatory response. while casp1 inhibition may prove beneficial in terms of increasing inflammatory responses, it is ambiguous in terms of benefit for bacterial infections. in our analysis, the expression of casp1 and tubb3 is also somewhat variable across virus types. therefore, more study is needed specifically on the role of caspase and tubulin in host response to respiratory virus infection. modulation of any human host pathway for the treatment of viral infections has potential drawbacks with respect to toxicity and other side-effects. for example, although interferon is widely used to help combat viral pathogens, the treatment is known to cause an array of side-effects related to toxicity including confusion, lethargy, impaired mental status, numbness, tingling, fevers, chills, headaches, anorexia and sepsis [89, 90] . another caveat is that some proteins are beneficial if up-regulated during initial viral infection but have detrimental effects if over-activated for prolonged periods. thus determining the desired mechanism and direction of therapeutic intervention requires careful study. although targeting host-pathogen interactions is a challenging therapeutic approach, there are considerable upside benefits with respect to overcoming pathogen-mediated drug resistance and the capability of treating multiple, co-infecting pathogens. our study suggests several potential human-host proteins that could be targets of future therapeutics as well as some possible drug candidates for further investigations of repurposing against respiratory virus infections. the national center for biotechnology information's geo database (http://www.ncbi.nlm.nih.gov/geo/ (accessed between january and july 2011) was searched for human mrna datasets for twelve respiratory viruses. (figure 1 ) reduced the number of viruses with suitable datasets to seven species (table 1) . all analyzed geo datasets contain at least one ''treatment group'' and ''control group''. ''treatment'' was the experimental variable under study, usually a virus type, strain, or time point. ''group'' was a collection of individual ''samples'', or replicates, each of which originates from their own microarray chip. ''comparison group'' was the treatment group compared to a control group. a particular dataset may have more than one comparison group. all criteria for dataset inclusion in the final analysis were chosen prior to the analysis. dataset candidacy filtering consisted of four criteria: 1) the dataset must contain at least 3 samples per treatment or control group because a sample size any less would mean a loss in statistical power for subsequent analysis; 2) the microarray platform must be supported by either affymetrix, agilent or illumina due to probe mapping abilities of the software used in subsequent analysis; 3) each gene expression profile had to be derived from human cells and probed using a human-based genome microarray platform and not other species; and 4) the dataset must contain at least one wild-type infection treatment group (i.e., unmodified virus strain or infectivity mechanism) and at least one healthy control group (i.e., no genetic or media modifications such as gene knock outs or inhibitors, respectively). prior to quality control (qc) analysis, we pre-screened and preprocessed each dataset. normalized raw data and the study design table were imported from the geo databases (the data was assumed to be normalized by robust multi-array average, but in some cases the published study used an alternative normalization method). where appropriate, the intensity values were log 2 transformation. various experimental parameters such as time point, virus strain and number of replicates were extracted from the study design tables. samples irrelevant to the main study design were marked for segregation or exclusion from our downstream analysis, but not excluded from quality assessment. these were classified as ''failing to meet treatment specification'' at the candidate filtering step. studies that had a large number of missing intensity values (over 10%) were annotated and flagged. the qc analysis assessed each sample in the dataset for kernel density, pca, mad, and pair-wise pearson correlation such that: 1) the kernel density was normally distributed; 2) after pca values were within the hotelling t2 alpha level threshold of 0.05 [91] [92] [93] ; 3) mad score scores were in the range of +3 to 23 with no outliers [94] ; and 4) inner-treatment group pair wise correlations for samples derived from a single cell were $0.97 or $0.90 if taken from individual donors [94] . figures were created using array studio software, version 4.1. (omicsoft corporation, research triangle park, nc, usa [95] ). during subsequent analysis, each comparison group was treated separately, regardless of dataset origination, in order to gain a wider, less bias view of representative genes and pathways. once a comparison group passed the qc analysis filters, lsm values were calculated for each probe using array studio in order to reduce the number of false positives due to low probe intensity values. probes within each of the filtered datasets were tested for biological and statistical relevance using the array studio implementation of fold change and statistical models, respectively. specifically, to determine a probe's fold change expression when compared to control, the geometric mean of each probe's log 2 transformed intensity value within a treatment was generated, and then normalized to the corresponding control group's geometric mean. the treatment versus control data were fitted to a general linear model, and associated p-values for each probe were calculated using a modified t-test [96] . thus, to be considered differentially expressed, each probe within a comparison group must have a p-value ,0.05 after general linear model test and a fold change in either direction of 1.5. to visualize a comparison group's significance and fold change, volcano plots were generated using array studio of a probe's 2log(p-value) versus its transformed fold change (fc) value according to the following piece-wise function: the differentially expressed probes were mapped to their corresponding genes using metacore/metabase (genego), a software/database package that creates biological pathways and networks from gene lists (database accessed june 2011) [97, 98] . if more than one probe mapped to a gene, the probe with the highest magnitude fold change value was used for that gene. thus, the mapped differentially expressed probe list became the differentially expressed gene list for each comparison group. the differentially expressed gene lists from each comparison group were analyzed for enriched pathways using genego. a pvalue for each of the 658 pathway maps in the metabase were generated for each comparison group using a hypergeometric test [99] . in order for a pathway to be considered enriched, each comparison group must contain pathways that have a p-value ,0.01 and occur in .5% of the total studies. the enriched pathway list was ranked by its viral frequency, which is defined by the number of viruses represented by at least one comparison group, and then by the sum of normalized viral expression or nve for each enriched pathway. the nve for each pathway was calculated using the number of comparisons containing significant pathways within a virus type relative to the number of comparisons within that virus type. for example, if one out of four flu comparisons for pathway a were significant, the nve for flu would be 1/4. ranking the pathways in this fashion resulted in a clearer determination of pathways shared across multiple viruses, irrespective of time, strain type, or number of comparison groups. after examining the ranked pathway list described above, the top five significant pathways and an additional pathway representing a unique mechanism were further analyzed. with each map, the proteins were labeled according to the number of viruses in which the transcript was differentially expressed thus yielding the viral frequency for that protein. in cases where a protein complex was made up of subunits, the greatest magnitude fold change value for any subunit was chosen to represent the entire complex. genego was used for the visualization of this pathway map. similar to the pathway nve, the nve for each gene within these six chosen pathways was calculated using the number of comparisons containing either up or down regulated genes for each protein within a virus type relative to the number of comparisons within that virus type. for example, if two out of three rsv comparisons for gene x were up-regulated, gene x's nve for rsv would be 2/3. we performed complete linkage and correlation distance hierarchical clustering using arraystudio on quantile normalized fold change values to determine the separation qualities of the analyzed data [100] . clustering was performed on genes that had expression values for at least 90% of the total number of comparisons. we used the matlab function 'knnimpute' to impute missing fold change values using k-nearest neighbors estimation (matlab version 7.11 (r2010b), mathworks, cambridge ma, 2010) [101, 102] . approved or marketed small molecule and protein therapeutics for each of the differentially expressed proteins modulated by 5 or more respiratory viruses were obtained from the drugbank database, version 3.0 (http://www.drugbank.ca/ accessed august 2011) [29] [30] [31] . we only considered those drugs that were launched products with experimental and clinical evidence of direct interaction with gene product in question. the therapy's interaction with the target and approved indication were identified using a combination of drugbank, the drug manufacturer's information page, and the national center for biotechnology information's pubchem (http://pubchem.ncbi.nlm.nih.gov/ accessed september 2011) [103] and gene (http://www.ncbi.nlm. nih.gov/gene/ accessed september 2011) databases. supplemental evidence of mechanism of action was obtained from immune or infection-related jackson laboratory knock-in/knock-out mouse (jax) phenotype (http://www.jax.org/ accessed september 2011) [32] . (table 3 ). the horizontal axis contains each of the 28 different comparisons labeled by virus, gse and time point. the vertical axis shows the clustering of 27 genes from the top five and parkin-ups pathways that have an nve of at least 6 and have an expression value in at least 26 comparisons. for genes present in more than one of the five pathways, the number of participating pathways is indicated by the count of ''*'' before the gene name. color scheme is as described for figure s3 . (tif) figure s5 epidermal growth factor receptor signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/ mc_legend.pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s6 cd40 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend.pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s7 interferon-gamma signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s8 histamine receptor h1 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. (tif) figure s9 interleukin-17 signaling pathway with viral frequency. viral frequencies superimposed for each of most frequently differentially expressed proteins, where red circles are differential expression of genes by 7 viruses, orange circles are differential expression of genes by at least 6 viruses, and blue circles are differential expression of genes by 5 viruses. see metacore website at http://www.genego.com/pdf/mc_legend. pdf for figure legend and table s4 for pathway map gene products' corresponding hugo gene names. 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viral infections in humans systemslevel comparison of host-responses elicited by avian h5n1 and seasonal h1n1 influenza viruses in primary human macrophages gene expression profiles during in vivo human rhinovirus infection: insights into the host response rhinovirus-induced modulation of gene expression in bronchial epithelial cells from subjects with asthma identification of human metapneumovirus-induced gene networks in airway epithelial cells by microarray analysis differential recognition of tlr-dependent microbial ligands in human bronchial epithelial cells identification of gene biomarkers for respiratory syncytial virus infection in a bronchial epithelial cell line we thank vinod kumar for his assistance in computational analysis. key: cord-023407-s85g7g0x authors: huang, y.‐m.; liu, x.; steffensen, k.; sanna, a.; arru, g.; sominanda, a.; sotgiu, s.; rosati, g.; gustafsson, j.‐å.; link, h. title: anti‐inflammatory liver x receptors and related molecules in multiple sclerosis patients from sardinia and sweden date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423d.x sha: doc_id: 23407 cord_uid: s85g7g0x the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing‐remitting ms from sassari, sardinia and stockholm, sweden. sex‐ and age‐matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real‐time pcr. lxr‐α was lower (p < 0.05) in ms (mean ± sem: 3.1 ± 0.2; n = 37) compared to hc (3.6 ± 0.1; n = 37). lxr‐α was lower in ms from stockholm (2.6 ± 0.2; n = 22) compared to corresponding hc (3.4 ± 0.1; n = 22; p < 0.01) and compared to ms (3.8 ± 0.2; n = 15; p < 0.001) and hc (4 ± 0.2; n = 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr‐β (−4.1 ± 0.4) compared to corresponding hc (−2.9 ± 0.3). ms from stockholm was associated with higher abca‐1 (6.1 ± 0.4 versus 5.0 ± 0.3; p < 0.05) and higher estrogen receptor‐β‐cx (2.4 ± 0.4 versus 0.8 ± 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ± 0.2) compared to ms from sassari (1.4 ± 0.3; p < 0.01), ms (1.3 ± 0.4; p < 0.01) and hc from stockholm (1.2 ± 0.3; p < 0.01). ms from sassari had lower cyclooxygenase‐1 compared to corresponding hc (5.1 ± 0.4 versus 6.6 ± 0.3; p < 0.01) and lower prostaglandin‐e (−0.03 ± 0.5) compared to the hc (1.4 ± 0.5; p < 0.05) and ms (2.7 ± 0.4; p < 0.05) and hc from stockholm (1.9 ± 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-005664-n4xv247l authors: plötz, frans b.; vreugdenhil, harriet a.; slutsky, arthur s.; zijlstra, jitske; heijnen, cobi j.; van vught, hans title: mechanical ventilation alters the immune response in children without lung pathology date: 2002-01-15 journal: intensive care med doi: 10.1007/s00134-002-1216-7 sha: doc_id: 5664 cord_uid: n4xv247l objective: this study was undertaken to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in infants without preexisting lung pathology. design and setting: prospective observational study in pediatric intensive care unit in a university hospital. patients: twelve infants who were subjected to an uncomplicated diagnostic cardiac catheterization procedure were studied. all subjects were ventilated with a volume control mode, 0.3 fio(2), 4 cmh(2)o peep, and 10 ml/kg tidal volume. volatile (servoflurane) anesthetics were given. measurements and results: tracheal aspirates and blood samples were obtained before and after 2 h of mechanical ventilation. in tracheal aspirates and in supernatants of stimulated whole-blood cultures cytokine concentrations were measured. in the tracheal aspirates the immune balance was characterized by a proinflammatory response pattern, with a significant increase in tnf-α and il-6 concentrations; concentrations of anti-inflammatory mediators remained very low. the functional capacity of peripheral blood leukocytes to produce inf-γ, tnf-α, and il-6 in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of anti-inflammatory mediators. the th1 immune response by peripheral blood leukocytes was decreased. the observed change in th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. blood leukocytes to produce inf-γ, tnf-α, and il-6 in vitro was significantly decreased. this was accompanied by a significant decrease in the killing activity of natural killer cells. conclusions: two hours of servoflurane and mechanical ventilation using a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs the immune balance favors a proinflammatory response pattern without detectable concentrations of antiinflammatory mediators. the th1 immune response by peripheral blood leukocytes was decreased. the observed change in th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. it has become clear that alterations in the immune balance may prevent an appropriate and effective response to various stimuli [1, 2] . cd4 + t-cells can be divided functionally into th1 and th2 cells based on their cytokine profiles [3] . th1 cells secrete interferon (ifn) γ while th2 cells secrete interleukin (il) 4, il-5, il-10, and il-13. macrophages secrete proinflammatory and anti-inflammatory cytokines such as il-1β, tumor necro-sis factor (tnf) α, il-12, and il-10. for example, an alteration in the th1/th2 balance, resulting in a th2 dominance, is thought to contribute to enhanced pulmonary disease in respiratory syncytial virus bronchiolitis [4] . on the other hand, new evidence indicates that a disturbance of the balance between proinflammatory mediators and anti-inflammatory mediators may initiate or amplify the inflammatory response in patients with the acute respiratory distress syndrome (ards) [1, 5] . for example, the ratio of il-1β to il-1 receptor antagonist is markedly elevated in patients with ards, favoring the unopposed proinflammatory activity of il-1β. the observation that low intrapulmonary concentrations of il-10 and il-1 receptor antagonist at the onset of ards are associated with a poor outcome suggests that a lack of inhibitory cytokines is correlated with a poor prognosis. it has also been suggested that mechanical ventilation produces alterations in the immune balance [6] . experimental studies have demonstrated that mechanical ventilation results in an inflammatory reaction in the lungs and that the degree of inflammation depends on the ventilatory strategy and mode [7, 8, 9, 10, 11] . this inflammatory reaction may not be limited to the lungs but may initiate or propagate multiple system organ failure [12, 13, 14] . a possible explanation for the spillover of inflammatory mediators as a result of mechanical ventilation is loss of compartmentalization [15] . the important concept of compartmentalization refers to the fact that the inflammatory response remains compartmentalized in the area of the body were it is produced [16, 17] . haitsma et al. [18] have shown in rats that injurious ventilatory strategies, although not conclusive, disturb the compartmentalization of the early cytokine response in both the lung and the systemic circulation [15] . infants who undergo cardiac catheterization may have multiple risk factors that may affect the inflammatory milieu in their lungs, including mechanical ventilation, exposure to anesthetic agents, and the stress of the procedure. the present study was designed to examine the hypothesis that mechanical ventilation in association with anesthesia would alter the cytokine profile in the lungs, and/or systemic circulation, of patients without preexisting lung pathology. the study included 12 children (median age 3.5 years, range 1-11) who were undergoing a diagnostic cardiac catheterization procedure. the children had a history of a congenital heart disease, some of whom had been (partially) corrected: atrial-ventricular septal defect, transposition of the great arteries [2] , aortic valve insufficiency, ventricular septal defect [2] , tetralogy of fallot, coarctation of aorta, tricuspid atresia, pulmonary atresia [2] , double outlet right ventricle. patients with a history of allergic or respiratory diseases, known chromosomal or immunological disorders, and patients recently hospitalized or mechanically ventilated were excluded. all subjects were intubated and ventilated with a volume control mode and a fractional inspiratory oxygen of 0.3, a maximum peak inspiratory pressure of 19.1±2.0 cm h 2 o, a mean positive end-expiratory pressure (peep) of 3.8±1.0 cm h 2 o, and a mean tidal volume of 9.95±0.95 ml/kg (measured body weight). the end-tidal co 2 was maintained between 35-45 mmhg. if peep, inspiratory oxygen concentration, or tidal volume needed to be adjusted to maintain an adequate oxygenation or to maintain normocapnia, the patient was excluded from the study. heart rate and blood pressure of the individual patients remained constant during the procedure. all patients received servoflurane (3.75%) anesthetic during the procedure. the study was approved by the medical ethics committee, and parents gave informed consent. tracheal aspirates and blood samples were obtained immediately after intubation, before the start of mechanical ventilation, and after 2 h of mechanical ventilation. tracheal aspirates were obtained as previously described [19] . the suction catheter was rinsed with 0.5 ml sterile normal saline and added to the suction trap. the aspirate was placed immediately on ice. thereafter 10% dithiothreitol (10%; 100 µl per 1 ml aspirate) was added, and the samples were centrifuged at 1500 rpm for 5 min. supernatants were stored at -80°c until analysis. blood samples were drawn from a venous catheter. heparinized blood was diluted 1:10 in rpmi-1640 medium (roswell park memorial institute life technologies, grand island, n.y., usa), and whole-blood cultures were set up. the whole-blood culture stimulated with lipopolysaccharide (lps) is a suitable ex vivo method to study monocyte cytokine production under conditions in which many of the physiologically relevant cellular interactions remain intact [20, 21] . to induce lymphocyte cytokine production (il-4, ifn-γ) anti-cd2,1 and anti-cd2,2 (1:12000) plus anti-cd28 (1:3000) monoclonal antibodies (clb, amsterdam, the netherlands) were added, and cultures were incubated for 72 h at 37°c in 5% co 2 in air. all cultures were performed in quadruplicate. to induce the production of monocyte il-6, il-8, tnf-α, lps (difco laboratories, detroit, mich., usa) (1 ng/ml) was added to the diluted blood samples and cultures were incubated for 24 h at 37°c in 5% co 2 in air. to induce monocyte il-10 production lps (1 ng/ml) was added, and cultures were incubated for 48 h at 37°c in 5% co 2 in air. to induce monocyte il-12 production lps (100 ng/ml) and ifn-γ (20 ng/ml) were added, and cultures were incubated for 24 h at 37°c in 5% co 2 . addition of ifn-γ results in a more optimal il-12 response in the presence of lps. cytokine assays tnf-α, il-4, il-6, il-8, il-10, il-12, and ifn-γ were measured via enzyme-linked immunosorbent assay (clb). the detection limit was 4-6 pg/ml for tnf-α, 0.6 pg/ml for il-4, 1 pg/ml for il-6, 4-8 pg/ml for il-8, 3-5 pg/ml for il-10, 3 pg/ml for il-12, and 4-6 pg/ml for ifn-γ. when cytokines were not detectable, the minimum detectable level was used in the calculations. the composition of peripheral leukocytes was determined by analyzing the forward-sideward scatter. for lymphocyte subset analysis, whole blood was incubated with conjugated monoclonal antibodies under saturating conditions specific for cd3, cd4, cd8, and cd16/56 (simultest, becton and dickinson, mountain view, calif., usa). subsequently, red blood cells were lysed and samples were analyzed with a flow cytometer (facs-star+, becton and dickinson). the difference between negative and positive fluorescence was determined by measuring unstained cells and cells stained with an irrelevant isotype control body. natural killer cell activity natural killer cell (nk) cell activity was analyzed by determining the capacity of peripheral blood cells to kill 51 cr-labeled k562 target cells as described previously [22] . cortisol was measured by a chemiluminescence immunoassay performed on the fully automated advia centaur immunoanalyzer (bayer, leverkusen, germany). all values were expressed as mean ±sd and were analyzed by the nonparametric wilcoxon signed-rank test. differences were considered significant at the level of p<0.05. the concentrations of tnf-α in the supernatant of the tracheal aspirates increased significantly 2 h after me-chanical ventilation (p=0.01; fig. 1 ). there was a trend towards an increase in il-6 levels (p=0.05; fig. 1 ). il-8 concentrations showed high interindividual variation both before and after mechanical ventilation. the concentrations of the anti-inflammatory cytokines il-10 and ifn-γ remained unchanged just above the detection level (fig. 1 ). the capacity of lymphocytes to produce cytokines was determined in whole-blood cultures stimulated with anti-cd2/cd28 [20, 21] . after 2 h of mechanical ventilation a significant decrease in ifn-γ production was observed in the cultured supernatants (p=0.01), but no significant changes in il-4 concentrations were observed (fig. 2) . the capacity of monocytes to produce cytokines was determined in whole-blood cultures stimulated with lps. after 2 h of mechanical ventilation there was a decrease in the production of proinflammatory cytokines il-6 (p<0.05) and tnf-α by peripheral blood monocytes (p<0.05; fig. 2 ). il-8 concentrations showed high interindividual variation before and after mechanical ventilation. the amount of il-10 and il-12 produced by monocytes was unaltered in all patients as a result of 2 h of mechanical ventilation (fig. 2) . we observed significant changes in the cellular composition of the whole-blood samples. there was a increase in the percentage of granulocytes (p<0.05) and a decrease in the percentage of lymphocytes (p<0.05; table 1 ). the percentage of cd3 and cd4 increased slightly but significantly (p<0.05). the percentage of cd16/56 tended to decrease (table 1) . as a result of 2 h of mechanical ventilation the killing capacity of nk cells to lyse k562 target cells decreased significantly (p<0.01). the mean percentage of activity decreased from 35.1±5.1 to 22.3±4.3. this remarkable decrease in killing capacity of nk cells cannot be explained by a decrease in the total numbers of nk cells (table 1) . the major finding of the present study is that exposing infants with normal lung function to 2 h of volatile anesthetics, mechanical ventilation, and cardiac catheterization is associated with remarkable changes in immune responses. we observed a proinflammatory response in the lungs with a significant increase in tnf-α, while antiinflammatory cytokine concentrations in tracheal aspirates remained virtually unchanged, just above the detection level. in addition, the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular ifn-γ, tnf-α, and il-6. this was accompanied by a significant decrease in the activity of nk cells. this indicates that this procedure is associated with a change in the th1/th2 balance with a decreased th1 immune response. a major question from our study is which aspect of the total procedure consisting of exposure to volatile anesthetics, ventilation, and catheterization is responsible for the observed changes in the inflammatory response of our patients. a recent review article summarized the effect of anesthetic agents on the immune response and concluded that there is little evidence to support the concept of clinically relevant immune modulation by anesthetics during major surgery [23] . no clinical study has examined the effect of servoflurane on the immune response in infants and young children. experimental studies have shown that during mechanical ventilation of uninjured lungs several volatile anesthetics may augment gene expression of proinflammatory cytokines in rat alveolar macrophages [24] . however, servoflurane was not associated with a significant increase in gene expression of proinflammatory cytokines or with concentrations of tnf-α in the lavage fluid of these rats over that with mechanical ventilation alone [24] . kotani et al. [25] demonstrated in mechanically ventilated adult patients that intravenous propofol or volatile isoflurane produced a similar increase in gene expression of all proinflammatory cytokines on alveolar macrophages. this is remarkable since the route of administration of these anesthetics are completely different. one would have expected that by directly acting on alveolar macrophages the volatile anesthetic -isoflurane -would induce faster and probably more pronounced gene expression. it therefore remains questionable whether these clinical observed effects are all attributable to general anesthesia. any effect of anesthesia is likely to be overwhelmed by the neuroendocrine stress response during major sur-gery [23] . however, in our study the response of the hypothalamo-pituitary-adrenal axis to the catheterization procedure is probably negligible. serum cortisol levels measured before and after mechanical ventilation were similar. other factors such as hemorrhage, hypotension, ischemia/reperfusion, and blood transfusion, which may affect immune competence during major surgery, were negligible in our study. thus the catheterization procedure can therefore not considered to be major surgery. we are therefore left with the possibility that the changes in the immune response in our study were the result of mechanical ventilation, although we are aware that definitive conclusions cannot be made. several experimental studies have reported that injurious ventilatory strategies increase tnf-α mrna expression and lung lavage levels of tnf-α protein [7, 9, 11] . in these studies tidal volumes were very high (40 ml/kg), and/or there was preexisting lung injury. pretreatment with intratracheal instillation of anti-tnf-α antibodies improved oxygenation, reduced infiltration of leukocytes, and ameliorated pathological findings [26] . the results of the experimental studies clearly demonstrated that tnf-α plays a pivotal role in initiating an inflammatory cascade induced by mechanical ventilation. the lung macrophage may be the critical mechanosensor cell capable of producing tnf-α in response to stretching mechanical forces [27], although there is evidence that the pulmonary epithelium may also be a key player in this regard [28] . it is remarkable, however, that such a significant proinflammatory response was observed with the ventilatory strategy we adopted. our patients had normal lungs, and a tidal volume of 10 ml/kg should not cause overdistention, since the patients would not have the marked heterogeneities in pulmonary compliance that exist in patients with ards [29, 30] . this is supported by the observation that peak inspiratory pressures remained low (19.1±2.0 cmh 2 o) throughout the 2-h period. to our knowledge, only one other study has examined the effect of mechanical ventilation on release of cytokines in patients with normal lung function [31] . wrigge et al. [31] observed that after 1 h of mechanical ventilation plasma levels of pro-and anti-inflammatory mediators remained low and did not differ from baseline. unfortunately, the local production of cytokines in the lung was not measured. the observed effects in our study may be explained by a two-hit hypothesis in which any one factor by itself does not induce an effect, but a combination of factors act synergistically to cause the changes in immune response, i.e., mechanical ventilation and volatile anesthetics. it remains speculative what causes the onset of the peripheral immune response. one of the mechanisms could be that tnf-α produced locally in the lung causes leukocyte redistribution from the systemic circulation into the alveolar space [9, 11] . mechanical ventilation may recruit t cell subsets with distinctive properties with respect to homing and trafficking into inflamed sites [32] . we observed that the functional capacity of peripheral blood leukocytes to produce proinflammatory cytokines in vitro was significantly decreased, in particular inf-γ, tnf-α, and il-6. ifn-γ is associated with a th1 response, which is considered to be beneficial in terms of an appropriate and effective response to various stimuli, including trauma, infection, and perhaps mechanical ventilation [2] . ifn-γ is also important in stimulating the cytolytic activity of nk cells and cd8 + cytotoxic t lymphocytes. the decrease in ifn-γ production was also accompanied by a significant decrease in the killing activity of nk cells. the altered th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. in conclusion, 2 h of servoflurane and mechanical ventilation with a tidal volume of 10 ml/kg is associated with remarkable changes in the immune response in infants without preexisting lung pathology undergoing cardiac catheterization. in the lungs a proinflammatory response pattern dominates without detectable concentrations of anti-inflammatory mediators. we observed a decrease in the th1 immune response by peripheral blood leukocytes. the altered th1/th2 balance in favor of th2 cytokine activity may be a systemic adaptation to the proinflammatory milieu in the lung. further studies possibly using different anesthetic agents, different operative procedures, and different ventilatory strategies are needed to establish the mechanisms and clinical relevance of our findings. cytokines and the acute respiratory distress syndrome (ards): a question of balance dominance of t-helper 2-type cytokines after severe injury human th1 and th2 subsets: doubt no more anti-il-4 treatment at immunization modulates cytokine expression, reduces illness, and increases cytotoxic t lymphocyte activity in mice challenged with respiratory syncytial virus the acute respiratory distress syndrome ventilator-induced lung inflammation: is it always harmful? injurious ventilatory strategies increase cytokines and c-fos m-rna expression in an isolated rat lung model role of high-frequency ventilation in surfactant-depleted lung injury as measured by granulocytes inflammatory chemical mediators during conventional ventilation and during high frequency oscillatory ventilation ventilator pattern influences neutrophil influx and activation in atelectasis-prone rabbit lung intraalveolar expression of tumor necrosis factor-alpha gene during conventional and high-frequency ventilation multiple system organ failure: is mechanical ventilation a contributing factor? effect of mechanical ventilation on inflammatory mediators in patients with acute respiratory distress syndrome: a randomized clinical trial mechanical ventilation as a mediator of multisystem organ failure in acute respiratory distress syndrome compartmentalized lung cytokine release in response to intravascular and alveolar endotoxin challenge loss of compartmentalization of alveolar tumor necrosis factor after lung injury ventilator-induced lung injury measurement of interleukin 10 in bronchoalveolar lavage from preterm ventilated infants a convenient whole blood culture system for studying the regulation of tumor necrosis factor release by bacterial lipopolysaccharide monocyte il-10 production during respiratory syncytial virus bronchiolitis is associated with recurrent wheezing in a one year follow-up study the authors thank the pediatric cardiologists and cardioanesthetists for their technical assistance. key: cord-004919-d7tilk8v authors: baker, rahaf; liew, jean w.; simonson, paul d.; soma, lori a.; starkebaum, gordon title: macrophage activation syndrome in a patient with axial spondyloarthritis on adalimumab date: 2018-12-07 journal: clin rheumatol doi: 10.1007/s10067-018-4387-5 sha: doc_id: 4919 cord_uid: d7tilk8v macrophage activation syndrome (mas) is a rare and potentially fatal condition characterized by excessive activation and uncontrolled proliferation of t lymphocytes and macrophages, leading to overwhelming systemic inflammation and cytokine release. mas has been reported with viral infections, autoimmune disorders, malignancies, and medications. we describe a case of a patient with axial spondyloarthritis (axspa) treated with adalimumab, who presented with mas. to the editor: macrophage activation syndrome (mas) is a rare and potentially fatal condition characterized by excessive activation and uncontrolled proliferation of t lymphocytes and macrophages, leading to overwhelming systemic inflammation and cytokine release. mas has been reported with viral infections, autoimmune disorders, malignancies, and medications. we describe a case of a patient with axial spondyloarthritis (axspa) treated with adalimumab, who presented with mas. a 34-year-old middle eastern man with long-standing hla-b27-positive axspa on adalimumab presented with several days of persistent fevers. axspa had been diagnosed on the basis of a history of recurrent iritis, patellar tendon enthesitis, and inflammatory lower back pain. the back pain, although initially responsive, became refractory to non-steroidal antiinflammatory medications and a sacroiliac joint steroid injection. adalimumab was started in april 2018, and for the next rahaf baker and jean w. liew contributed equally to this work. key points • mas is a rare condition of systemic inflammation with high mortality; having a strong index of suspicion can lower mortality in these patients. • medications, including tnf inhibitors, should be considered as possible triggers for mas. • consider high-dose steroids and anakinra, either alone or in combination, for the treatment of mas. if a medication is suspected, remove the offending agent. 2.5 months, he had dramatic improvement in his inflammatory back pain, and no extra-articular manifestations. in june 2018, he presented to the hospital and endorsed 4 days of high fevers with mild lower chest discomfort and cough. he described unremitting fevers that occurred multiple times daily, which were associated with significant weakness and rigors. the review of systems was otherwise negative. he had no other pertinent past medical history or medications. his family history was non-contributory. his social history was notable for moving to the usa from saudi arabia 10 years ago with recent, frequent travel around the usa and mexico. he had traveled to las vegas for a work conference a few days prior to his presentation. on admission, his temperature was 40.2°c, blood pressure was 129/68 mm/hg, and pulse was 60 beats per minute. he was ill-appearing and his physical exam was otherwise only notable for splenomegaly with tenderness on palpation. his initial labs revealed lymphopenia (absolute lymphocyte count 650 cells/μl), mild anemia (hemoglobin 12 g/dl), thrombocytopenia (132,000 cells/μl), and transaminitis (ast 53 u/l, alt 76 u/l). erythrocyte sedimentation rate (esr) was 42 mm/h and c-reactive protein (crp) was 202 mg/dl. an extensive infectious disease workup was undertaken, with all tests reported as negative (table 1) . computed tomography (ct) of the chest, abdomen, and pelvis showed mild splenomegaly with residual thymic tissue and no lymphadenopathy. after a week of hospitalization, the patient remained febrile despite treatment with antipyretics. he developed a transient, faint, erythematous maculopapular rash over his trunk. further labs demonstrated elevated ferritin (2312 mg/dl), fibrinogen (871 mg/dl), and triglycerides (289 mg/dl). crp peaked at 400 mg/dl. a diagnosis of mas was considered. on the seventh day of hospitalization, he was started on anakinra 100 mg twice daily without adequate fever control; thus, oral prednisone 60 mg daily was added. he quickly defervesced with improvement of his condition, labs, and inflammatory markers (fig. 1) . a bone marrow biopsy, which was obtained on hospital day 7, showed erythroid hyperplasia and hemophagocytic histiocytes without evidence of malignancy (fig. 2) . soluble interleukin-2 receptor levels, however, were low. after an 11-day hospitalization, he was discharged on prednisone 60 mg daily and anakinra 100 mg once daily. adalimumab was completely discontinued. upon outpatient follow-up, he had continued symptomatic improvement with normalization of the esr, crp, ferritin, and fibrinogen. he was tapered off prednisone over 2 months but his inflammatory back pain returned. mas is a potentially fatal syndrome that can present in patients with inflammatory conditions and is considered to be similar to hemophagocytic lymphohistiocytosis (hlh) [1, 2] . the finding of abundant activated hemophagocytic macrophages, or histiocytes, has led to their classification as a histiocytic disorder [3] . the underlying pathogenesis of mas is poorly understood, but it is proposed to be triggered by an inciting event such as infection, malignancy, autoimmune conditions, or drugs, that leads to immune dysregulation [4] . the resultant overwhelming cytokine storm drives phagocytosis of blood cell precursors and the infiltration of macrophages into tissues, causing multiorgan dysfunction. currently, there are no diagnostic criteria for mas in adults, and the proposed classification criteria rely on our understanding from the pediatric population [1] . the findings of high fevers, cytopenias, hyperferritinemia, and hypertriglyceridemia should alert the clinician to the possibility of mas. although significantly elevated ferritin is specific for hlh in pediatrics, it is not as specific to hlh or mas in the adult population [5] . evidence of hemophagocytic cells on bone marrow biopsy is not required for diagnosis and is not always found on presentation in mas. the most consistent diagnosis for our patient was mas, as he had high persistent fevers without an infectious cause, lymphopenia and anemia, marked hyperferritinemia, hypertriglyceridemia, splenomegaly, and the presence of hemophagocytes on bone marrow biopsy. the diagnosis of adult-onset still's disease (aosd) was excluded due to his axspa [6] . the leading explanations for his mas were adalimumab, an undetected viral infection, or his underlying axspa. infection, particularly viral, is the most common reported cause of mas cited in the literature [2] . our patient had extensive infectious workup that was negative; however, his mas may have been triggered by an undetected virus. in rheumatologic conditions, mas has been associated with underlying activity of systemic juvenile idiopathic arthritis and aosd, and in rare cases of systemic lupus erythematosus [1] . spa is rarely associated with mas; only three were found in our review of the literature ( table 2 ). of these three cases, only one occurred in the absence of tnf inhibitor use or clear source of infection [7] . our patient had well-controlled spa that was stable prior to presentation; therefore, we felt that spa was a less likely trigger for his mas. recently, mas has been reported more frequently in association with tnf inhibitor use, although it is unclear whether this is due to heightened clinician awareness or increasing incidence. on our literature review, we found 10 reports of mas associated with etanercept, infliximab, or adalimumab (table 3) . of the two cases that described the timing of mas relative to tnf inhibitor administration, one was in a patient with ra 2 months after discontinuation of etanercept, and one was in aosd 2 months after initiation of adalimumab [12, 14] . four of the cases reported had associated infections, including visceral leishmaniasis, disseminated histoplasmosis, liver abscess, or primary ebv, which were felt to be the primary trigger for mas, though the tnf inhibitor was implicated as a contributing risk factor for the infection [8, 13, 15] . it is plausible that adalimumab triggered mas in our patient as he presented at 2.5 months after initiation of adalimumab, which is consistent with the timeline from other case reports, and he had no obvious infection. the pathogenesis of mas secondary to tnf inhibitors is unknown. these medications have been successful in treating some refractory cases of mas, but their use may also rarely induce or aggravate autoimmune diseases, or trigger mas in the setting of infection [1] . it has been proposed that tnf inhibitor blockade of macrophage activity is coupled with a compensatory immune system activation and rebound cytokine response [16] . this leads to an overall immune system dysregulation and may trigger hemophagocytosis. the explanation for this paradoxical effect on immune response will require further research. as for the treatment of mas, we note that in mas associated with tnf inhibitors only, six of the eight patients had clinical and laboratory improvement after treatment with highdose corticosteroids. two patients died despite receiving highdose corticosteroids; however, these patients presented with severe or late systemic disease that was previously not well controlled on prednisone [9, 12] . in the cases of mas with an associated infection, three of the four patients received highdose corticosteroids in addition to treatment of underlying infection, and all four patients responded well and survived [8, 13, 15] . in the three reported cases of mas in spa, all three patients received high-dose prednisone and clinically stabilized [7, 8] . dual treatment with high-dose steroids and anakinra, an il-1 receptor antagonist, induced remission in our patient. both agents have been shown to rapidly induce remission and ameliorate the cytokine storm in refractory and severe cases of mas [4] . anakinra was trialed first in our patient given his prolonged and high fevers, but given the antibiotics (amoxicillin, clavulonic acid, erythromycin), iv immunoglobulin (2 g/kg), and 7 methylprednisolone boluses followed by prednisone healing of abscess *the tnf inhibitor was a likely risk factor for the infection, which likely triggered the onset of mas ebv, epstein barr virus; pcr, polymerase chain reaction duration of symptoms and the likely presence of other cytokines besides il-1, it alone was not sufficient in inducing remission. in conclusion, it is important to recognize mas as a possible life-threatening complication of autoimmune and inflammatory diseases. currently, the diagnosis of mas relies heavily on the pediatric diagnostic criteria, and the treatment on case reports and case series in adults. it is important to increase awareness of this rare disease among clinicians in order to prevent mortality and improve outcomes of these patients. disclosures none. publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. classification criteria for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis how i treat hemophagocytic lymphohistocytosis in the adult patient revised classification of histiocytoses and neoplasms of the macrophage-dendritic cell lineages macrophage activation syndrome in the era of biologic therapy marked hyperferritinemia does not predict for hlh in the adult population mechanisms, biomarkers and targets for adult-onset still's disease ankylosing spondylitis presenting with macrophage activation syndrome syndrome d'activation macrophagique sous biothérapies: deux cas macrophage activation syndrome induced by etanercept in a patient with systemic sclerosis macrophage activation syndrome associated with etanercept in a child with systemic onset juvenile idiopathic arthritis macrophage activation syndrome following initiation of etanercept in a child with systemic onset juvenile rheumatoid arthritis macrophage activation syndrome after etanercept treatment visceral leishmaniasis and macrophagic activation syndrome in a patient with rheumatoid arthritis under treatment with adalimumab possible macrophage activation syndrome following initiation of adalimumab in a patient with adult-onset still's disease a rare trigger for macrophage activation syndrome syndrome d'activation macrophagique apres traitement par infliximab pour maladie de crohn fistulisee key: cord-017470-sjk7a34u authors: arlati, sergio title: pathophysiology of acute illness and injury date: 2018-06-14 journal: operative techniques and recent advances in acute care and emergency surgery doi: 10.1007/978-3-319-95114-0_2 sha: doc_id: 17470 cord_uid: sjk7a34u the pathophysiology of acute illness and injury recognizes three main effectors: infection, trauma, and ischemia-reperfusion injury. each of them can act by itself or in combination with the other two in developing a systemic inflammatory reaction syndrome (sirs) that is a generalized reaction to the morbid event. the time course of sirs is variable and influenced by the number and severity of subsequent insults (e.g., reparative surgery, acquired hospital infections). it occurs simultaneously with a complex of counter-regulatory mechanisms (compensatory anti-inflammatory response syndrome, cars) that limit the aggressive effects of sirs. in adjunct, a progressive dysfunction of the acquired (lymphocytes) immune system develops with increased risk for immunoparalysis and associated infectious complications. both humoral and cellular effectors participate to the development of sirs and cars. the most important humoral mediators are pro-inflammatory (il-1β, il-6, il-8, il-12) and anti-inflammatory (il-4, il-10) cytokines and chemokines, complement, leukotrienes, and paf. effector cells include neutrophils, monocytes, macrophages, lymphocytes, and endothelial cells. the endothelium is a key factor for production of remote organ damage as it exerts potent chemo-attracting effects on inflammatory cells, allows for leukocyte trafficking into tissues and organs, and promotes further inflammation by cytokines release. moreover, the loss of vasoregulatory properties and the increased permeability contribute to the development of hypotension and tissue edema. finally, the disseminated activation of the coagulation cascade causes the widespread deposition of microthrombi with resulting maldistribution of capillary blood flow and ultimately hypoxic cellular damage. this mechanism together with increased vascular permeability and vasodilation is responsible for the development of the multiple organ dysfunction syndrome (mods). • the inflammatory reaction is a highly adaptive, integrated response with a global protective effect against microbial pathogens or tissue damage. it provides for the elimination of pathogens, removal of cellular debris, and promotes tissue repair and healing. • multiple trauma or severe infection causes a widespread inflammatory reaction that causes diffuse endothelial activation (chemotaxis), damage (permeability edema), and vasodilation (hypotension-shock). • inflammation and coagulation are strictly coupled. as a general rule hyperinflammation means hypercoagulability. coagulation has beneficial effects when inflammation is localized, but it becomes catastrophic when disseminated intravascular coagulation ensues. • humoral inflammatory mediators include cytokines, complement, thrombin, acute phase proteins, kinins, and paf. endothelial cells, monocytes, antigenpresenting cells (macrophages and dendritic cells), and neutrophils are the main effector cells. • neutrophils are responsible for tissue damage. their widespread activation accounts for the noxious effects to innocent tissues with multiple organ damage. • a counter-inflammatory response mounts immediately after the hyperinflammatory reaction. such response is proportionate to inflammation along the whole course of disease. • an immediate decrease of the acquired (lymphocytic) immune response occurs in parallel with inflammation leading to dysfunction and progressive immunoparalysis. • apoptosis of the cells of the immune system is the main responsible of immunoparalysis. this exposes to increased risks for opportunistic infections and sepsis/septic shock. (compensatory anti-inflammatory response syndrome, cars) [1] . although cars opposes to the systemic inflammatory response syndrome (sirs) [2] , this is a double-edged sword because the risk of septic complications is increased. if unresolved, sirs and cars become the underlying players of a catabolic syndrome that leads to mods and ultimately death [3] . in the past sirs was viewed as an exaggerated response to inflammatory stimuli, but the latest experimental and observational data indicate that it is a rather predictable side effect of especially severe morbid events. in practice, sirs and cars result from the growing sophistication of icu care that keeps patients alive during the early (acute) phase of traumatic and septic diseases. the protracted survival of formerly rapid lethal conditions makes now appreciable their natural evolution. recent acquisitions also suggest that sirs and cars develop simultaneously rather than in sequence as previously believed. as a result a mixed antagonist response syndrome (mars) was coined to reflect the balance between sirs and cars [4] (fig. 2.1 ). however, the phenotypic predominance of the hyperinflammatory state is the rule in early sepsis, hypoxia, or trauma as influenced by antigenic load, microbial virulence, host genetic factors, age, nutritional status, and comorbidities [5] . in the past cars was believed to develop after sirs exhaustion by repeated noxious stimuli (second hit theory) [5, 6] . according to this theory, recurrent morbid insults augment the inflammatory response by repeated stimulation of the inflammatory cascade. in this sense, sirs would no longer depend by the initial insult but rather by the intensity and frequency of subsequent hits. however, the continuous challenge of the inflammatory system mounts an antiinflammatory response that ultimately becomes predominant. so cars does not develop simultaneously with sirs, but only a minimal overlap would exist between the two phe-nomena. this pathophysiological view is derived from the observation that cars prevails in the later stages of disease when the increased susceptibility to infections is associated with a weakened pro-inflammatory response (sirs exhaustion). this concept translated into the linear transition from acute (early) sirs to chronic (late) cars with possible alternating recurrence of the two phases (mars), (fig. 2.2) . however, this view is no longer accepted as the historical belief of the "cytokine storm" after a catastrophic acute illness (e.g., meningococcal sepsis) giving the spectacular inflammatory reaction is not the rule [7] . instead, the most common picture is by far a patient over 65 years of age with sepsis or recovering from multiple trauma/surgery and evidence of immunosuppression without the typical exaggerated acute phase inflammatory response [7] . in the past "cytokine storm" was synonymous of sirs that is hyperinflammation defined by excessive release of classical proinflammatory cytokines including il1, il6, il8, and tnfα. however, this concept is too narrow as it was quickly noted that "cytokine storm" is not the typical occurrence in late (chronic) sepsis or even in acutely septic patients with a weakened immune system [8, 9] . similarly, it seems incorrect to define cars on the basis of elevated release of antiinflammatory cytokines in the blood. the current concept is rather that the magnitude of cytokine release depends on the premorbid immune-inflammatory status of the patient [10] . otherwise stated the healthier the patient, the stronger will be the release of cytokines after stimulus. as a corollary, the more protracted is the disease, the more faded will be the inflammatory response over time (e.g., recurrent sepsis in postsurgical or trauma patient). however, an acute inflammatory response although typical of the acute phase may occur at any time of the disease profile if the host is sufficiently immunologically responsive. this view holds for both the pro-inflammatory and anti-inflammatory cytokines, so it is incorrect to define the patient's inflammatory status on the basis of his/her cytokine profile [10] . therefore, the mixed cytokine response pattern better represents the patient's inflammatory status leading to the paradigm "sepsis: always in mars" [11] . thus a hyperinflammatory status at the onset of sepsis or multiple trauma reflects the ability of the host to release a great amount of proinflammatory and anti-inflammatory mediators. such ability is destined to fade over time with progression to a late (chronic) inflammatory status. in recent years, the immunocompetent cells have emerged as a new relevant player for the appearance of immunosuppression or immunoparalysis that often characterizes the host's response during the more chronic disease stages. at present, the process of immunosuppression (decreased t-cell proliferation and production of il2, decreased monocyte and macrophage function) is believed to occur in parallel with the hyperinflammatory status of early sepsis of traumatic or surgical origin [12, 13] . animal studies indicate that in non-survivors, the immune cell suppression progresses indefinitely up to anergy from the very beginning to the more chronic stages in a timeindependent manner [10] . as a result, the phenotype of immunosuppression does not often correspond to the cytokine pattern of peripheral blood. prior to early deaths, cellular immunosuppression develops rapidly together with high pro-inflammatory and antiinflammatory cytokine release (mars-like) [14] . conversely, the chronic state is preceded by a progressive (subacute or chronic) impairment of immune cells function with robust pre-lethal signs of anergy and a deteriorating but mars-like cytokine profile (simultaneous presence of both pro-inflammatory and anti-inflammatory mediators in the blood) [10] . the spread of the inflammatory process starting from a single organ or tissue is by far the most frequent event in the pathophysiology of acute diseases. the inflammatory response is a highly coordinated process, which has evolved to limit the spread of noxious stimuli, eliminates pathogens and necrotic cellular debris, and promotes the healing of damaged tissues. it is subjected to multiple activations and control mechanisms and whose efficiency is largely dependent on genetic predisposition, age, and neurovegetative and hormonal milieu derived from the stress response. finally, inflammation and immunity are tightly related in a complex network of multiple interconnections and reverberating loops. however, extremely intense or repeated stimulations may disturb their tuned response so that the inflammatory mediators spill over the anatomical barriers and multiple organs dysfunction syndrome ensues. cardinal inflammation phenomena are local vasodilation, increased endothelial permeability, and chemotactic cells activation from the natural (granulocytes and monocytes) and acquired immune system (lymphocytes). the vascular mechanisms that lead to the four cardinal signs of inflammation are resumed in fig. 2. 3. neutrophils and monocytes are activated to infiltrate the site of infection with subsequent phagocytosis and lysis of bacterial products or cellular debris. in the meanwhile, the activated coagulation system seals the site of inflammation and provides a meshwork of fibrin that helps in the reparation process. therefore, increased membrane permeability, tnfα, il-1β, il-6 il-8, il-12, ifγ cytokine profile il-4, il-10,tgf-β sirs cars mars fig. 2. 2 temporal profile of pro-inflammatory and anti-inflammatory cytokines according to the hypothesis of sequential development of sirs and cars. mars is a transitional state in-between them capillary vasodilation, chemotaxis, and phagocytosis are defensive mechanisms that act in concert to ensure the maximum of protection against any threat or danger. a multiplicity of cell types (e.g., endothelium, monocytes, platelets) as well as humoral factors (complement, leukotrienes, kinins) acts following a synergistic and often redundant logic to activate, propagate, and maintain the inflammation so that the host defense is guaranteed. nevertheless, the uncontrolled diffusion of inflammatory mediators causes hypotension and tissue edema by generalized vasodilation and increased endothelial permeability. furthermore, the diffuse deposition of microthrombi by disseminated intravascular coagulation worsens the oxygen supply to tissues this, in turn, contributing to ischemic cells damage, further inflammation (ischemia/reperfusion injury), and mods. the local action of the inflammatory system is similar to a well-refined military strategy. after an enemy attack (e.g., trauma) that engages the local garrison (resident macrophages and glial cells), the combat zone is rapidly enclosed and sealed by reinforcement troops (chemotactic activation of pmns and intravascular coagulation). thereafter, the soldiers get into the battlefield (increased membrane permeability and leukocytes migration) and shoot at the enemy destroying him (phagocytosis, proteases, and toxic oxygen products). however, after a massive attack, the hurried and often disorganized mobilization of the reserve troops makes difficult or even impossible to implement an effective defense. the recruitment of soldiers is often chaotic and uncoordinated (widespread chemotactic activation), the effective concentration of troops is impossible (generalized increase of endothelial permeability), and military patrols often shoot at innocent people (organ damage). thus, pneumococcal pneumonia can transform into severe sepsis or septic shock if a generalized inflammatory reaction develops by either cellular (neutrophils, monocytes, macrophages, endothelium) or humoral effectors (complement, contact phase proteins, leukotrienes, cytokines, chemokines) resulting into increased capillary permeability (tissue edema), vasodilation (hypotension), and coagulation activation (ischemic organ damage). generally, the pathophysiological mechanisms that lead to a systemic inflammatory reaction are infections, trauma, and ischemiareperfusion damage. each of them can act by itself or in combination with the other two. for example, multiple trauma causes the activation of immune-inflammatory mechanisms by itself (tissue necrosis), or as a consequence of ischemia-reperfusion damage (e.g., gut ischemia by posttraumatic mesenteric hematoma or post-ischemic muscular tissue reperfusion after hemorrhagic shock). apart from the abovementioned mechanisms, the uncontrolled activation of the immune system (autoimmune diseases), massive cytokines production (metastatic cancer, leukemia, or lymphoma), and the unrestrained activation of serum proteases (acute pancreatitis) are less frequent causes of generalized inflammatory activation. the widespread activation of the inflammatory system (systemic inflammatory reaction syndrome, sirs) originates from the site of trauma, infection, or hypoxic cell damage (ischemia/reperfusion). infection, traumatic or hypoxic injury, causes the release of a heterogeneous pattern of endogenous and exogenous molecules that trigger the innate immune system as chemoattractants and activators of antigen-presenting cells. infection from bacterial, viral, and fungal agents releases signaling substances that are recognized by the innate immune system due to their characteristic molecular pattern (pathogen-associated molecular patterns, pamps) [15] . conversely, traumatic or hypoxic cell injury releases the so-called damage-associated molecular patterns (damps) [16, 17] which represent the correlate of pamp for danger signals of endogenous origin. pamps and damps are grouped into the larger family of "alarmins" in assignment to the term danger signals [15] . otherwise stated pamps and damps constitute a physiologic signaling system that alerts the body to the presence of foreign invaders or noxious stimuli. "alarmins" activate specific receptors of the superfamily of the toll-like receptors (tlrs) [18, 19] , expressed on endothelial and innate immune cells like macrophages, dendritic cells (antigenpresenting cells, apcs), monocytes, and pmns [20] . apcs act as an intermediate between innate and acquired immune system. their main function is to process antigen material and to present it to effector t cells of the immune system. tlr receptors recognize a variety of peptides that are important signaling molecules for activation and production of a multiplicity of inflammatory mediators. in addition, damps are potent activators of the complement system [21] [22] [23] whose anaphylatoxins attract monocytes and pmns on the endothelium. the high mobility group box protein (hmgb) is one of the most studied "alarmin." hmbg is a protein molecule derived from the nucleus of damaged cells [15, 24] . it is released by activated myeloid cells (e.g., neutrophils) [20] , macrophages, dendritic cells [25] [26] [27] , or necrotic cells [26] and acts as a chemoattractant for monocytes, macrophages, dendritic cells, neutrophils, and ϒδ cells [3] . it also participates to the secretion of proinflammatory cytokines [28] and mediates the monocyteendothelial interaction by increasing vascular leakage [24, 29] . fragments of dna and histones are other well-known potent "alarmins." they originate from damaged tissues and microbial digestion by resident tissue macrophages or activated neutrophils. peptides and mitochondrial dna are vigorous alert molecules probably because of their vestigial origin from intracellular bacteria [3] . in addition to "alarmins," the release of phospholipids by damaged cell membranes (cellular hypoxia and trauma) or exogenous lipopolysaccharides (e.g., lps-endotoxin) and polymers (lipoteichoic acid and peptidoglycans) alert the innate immune system by activating the complement and the contact phase proteins system (fxii, kallikrein, and kininogen). phospholipids activate the phospholipases a 2 and c [30] with the production of arachidonic acid metabolites as leukotriene b 4 , prostaglandin e 2 , and thromboxane a 2 [31] . the activation pathway of phospholipase a 2 and c is detailed in fig. 2. 4. in addition, mast cells release histamine and bradykinin with resulting vasodilation and increased capillary permeability and edema [32] . just 20-30 min after trauma or microbial invasion, the innate immune cells become activated, fig. 2 .5. the first line of the innate immune defense is represented by pmns, mostly neutrophils, and monocytes. pmns are chemoattracted by locally produced cytokines (e.g., tnf-α), leukotrienes, platelet-activating factor (paf), and complement fragments (c5a) [33] [34] [35] [36] . these inflammatory mediators also activate pmns to express adhesion surface molecules for appropriate ligands on the activated endothelium [37] . this receptor-ligand interaction allows for adhesion of leukocytes to the capillary wall. thereafter pmns migrate through the endothelial barrier into the tissues by opportune signaling receptors on the inner surface of the endothelium. in the meanwhile, resident macrophages secrete cytokines (tnfα and il-1) and chemokines (il-8) which help the immune and inflammatory cells in selfregulating and crosstalking each other. cytokines and chemokines are pleiotropic molecules with a great variety of effects that act in a paracrine and autocrine fashion. among the most important cytokines secreted in the hyper-acute phase, tnf-α and il-1β act within 1-2 h after trauma or sepsis, while il-6, il-10, and the chemokine il-8 are subacute mediators [38] . among the most relevant properties of cytokines, there is the ability to activate monocytes (innate immune system) and lymphocytes (acquired immune system) [39] . under the appropriate cytokine pattern, the circulating monocytes differentiate into resident macrophages, while lymphocytes mature into different cell lines having immune-stimulatory (th-1), immune-depressant (th-2), and immune-modulatory (treg) action [40] . pro-inflammatory cytokines trigger both the recruitment and the phagocytic activity of leukocytes [41] . they also stimulate the release of proteases and increase the production of free oxygen radicals from activated neutrophils [41] . 1. one of the best characterized cytokines is tnf which can produce sirs when injected experimentally in high doses [39] . its actions include the stimulation of many cell types and survival or apoptosis [42] , cytokine secretion (il-6, il-8, ifϒ, il-10), activation of the arachidonic acid pathways (thromboxane a2 and prostaglandin e2) and nitric oxide, induction of fever and production of selectins, and paf (see below). tnf secretion is stimulated by many physiological stresses (hemorrhage, hypoxemia) as well as ischemia-reperfusion, endotoxin, and complement system. macrophages and monocytes are the main tnf producers, although activated t cells are also implicated. tumor necrosis factor acts via its receptors tnfr1 and tnfr2 with effects that depend on the specific receptor binding and environmental factors influence. binding of tnf-α to tnfr1 leads to transcription of pro-inflammatory genes via the nf-kb transcription factor. however, tnfr1 possesses a death domain at the cytoplasmic tail so that its stimulation also induces apoptosis via the caspase enzymes cascade (see below). although these two effects seem contradictory, they might represent an adaptive mechanism that has evolved to protect cells against excessive stimulation (see below: kidney dysfunction). in contrast to the almost ubiquitous tnf1r, tnfr2 is expressed on immune and endothelial cells only and becomes activated by membrane-bound tnf-α [42, 43] . binding to tnfr2 leads to activation and proliferation of neutrophils and many immune cells as nk cells, b cells, and peripheral t cells [44] . 2. interleukin-1-beta is the other well-studied cytokine that acts synergistically with tnf-α. its actions include fever induction, cytokine and chemokine secretion [45] , t cell and macrophage stimulation and various inflammatory mediators (including adhesion molecules), acute phase proteins, and no synthesis. it also induces the release of neutrophils from the bone marrow and stimulates the expression of proteases and metalloproteases by tissues. il-1β is secreted by macrophages, monocytes, and endothelial cells. stimuli for il-1β include tnf, complement, endotoxin, hemorrhage, and ischemia. the effects of il-1β are mediated by the type il-1 receptor (il-1ri) which belongs to the il-1 receptor superfamily. besides il-1β, another molecule that binds il-1ri is the so-called soluble il-1 receptor antagonist (il1-ra) suggesting a controlling role in il-1-mediated immune response. 3. the third important cytokine in the early inflammatory response is il-6. its secretion is induced by tnf and il-1 [46] . although it does not induce sirs when injected into experimental animals, il-6 has vasodilatory properties as it induces the production of nitric oxide by the endothelial cells. il-6 is a subacute cytokine with marked regulatory properties on immune cell growth and differentiation. it inhibits apoptosis of neutrophils and stimulates the hepatic acute phase protein synthesis but also exhibits an anti-inflammatory action [47] by inducing the release of soluble tnfr and il-1ra [48] . it seems, therefore, to play a dual role in the inflammatory response by acting both as a pro-inflammatory and anti-inflammatory mediator [49, 50] . clinical studies demonstrated increased plasma levels in non-survivor septic patients so that il-6 has been proposed as a prognostic marker [51] [52] [53] . 4. the chemokine il-8 is the most powerful chemoattractant for pmns and monocytes [54] . chemokines control the traffic of the immune cells, which are the main effectors of the immune system. their binding to g-protein-coupled receptors leads to dose-dependent effects including chemotaxis and activation of immune cells. after stimulation by il-1, tnf-α, complement, microbial products (e.g., lps), hypoxia, and reperfusion, il-8 increases neutrophils degranulation, adherence, and chemotaxis. interestingly, chemokines act as chemoattractants in lower doses and potent activators of immune cells function in higher doses. recently a group of so-called silent chemokine receptors has been described suggesting a role as decoy or scavenger receptors. the duffy antigen receptors for chemokines (darc), first described as blood group antigen, have been shown to bind il-8 and other chemokines thus suggesting a regulatory action against excessive leukocyte activation in the systemic circulation [39] . the cellular source and the main effects of pro-inflammatory cytokines are resumed in table 2 .1. apart from cytokines, other important mediators of the hyper-acute phase are the arachidonic acid metabolites (leukotrienes, prostaglandins, and thromboxane). they are responsible for the activation of pmns and endothelium and platelet aggregation. these substances are generated by activation of the phospholipases a2 and c (pla 2 and plc) which follows the entry of ca 2+ ions into damaged cells. pla 2 also induces the release of paf from the endothelium. paf is an important inflammatory mediator with marked effects on macrophages, endothelial cells, and platelets aggregation. the inflammatory response is promptly propagated and amplified by important circulating mediators as the complement system, the contact phase proteins (kallikrein-kinins), and the coagulation cascade. 1. the complement cascade is a series of enzymatic cleavage reactions that are activated via the classical route by antigen-antibody complexes (igm and igg) or alternatively by bacterial degradation products (e.g., lps, endotoxin) [32, 55, 56] . the latter pathway does not require prior immunization and thus represents an immediate defense mechanism against microbes. conversely, the "natural antibodies" activate the complement by forming antigen-antibody complexes with different molecular species from exogenous (microbial) or endogenous (necrotic cells) origin [57] [58] [59] . "natural antibodies" are physiologically circulating antibodies that arise in serum independently from prior host immunization [60] . they are mainly produced by a subpopulation of cd5 + b cells and are poly-reactive with different antigens as peptides, phospholipids, and polysaccharides [60] [61] [62] . in musculoskeletal trauma and ischemia-reperfusion injury, the intense antigenic stimulation leads to binding of natural igm antibodies and development of post-traumatic "innate autoimmunity" [63] . complement activation leads to the formation of opsonins, anaphylatoxins, and the membrane attack complex, a multiprotein complex responsible for the increased capillary permeability [32] . anaphylatoxins and opsonins are involved in important inflammatory processes such as opsonization, chemotaxis, and neutrophils degranulation. complement also promotes the synthesis of acute phase proteins by the liver and stimulates the degranulation of mast cells with histamine release (vasodilation) [32, 64] . in practice, the activated complement is involved in all the most relevant inflammatory processes as immune cells attraction to the site of trauma or infection, microbial phagocytosis and lysis, antigens opsonization, platelets activation, and histamine release. the complement cascade is, therefore, a fundamental component of the innate host defense as it can be rapidly activated in a non-specific manner after injury or infection. it is phylogenetically ancient due to its basic role in the host response to bacterial pathogens. however, the systemic activation of the complement leads to severely deleterious effects such as shock and tissue edema due to massive vasodilation and increased membrane permeability. 2. coagulation factor xii and kallikrein, together with kininogen, represent the contact phase system. these proteins activate each other by forming an integrated, highly amplified system for activation of the coagulation cascade. kallikrein also stimulates the formation of the vasodilator bradykinin from kininogen. kinins are potent vasodilators and vigorous effectors of increased vascular permeability [65] . a close interconnection exists between hemostasis and the inflammatory system. for example, the activated platelets aggregate with circulating leukocytes to stimulate the immune cells system with production of endothelial damage [66] . platelets also release pro-inflammatory mediators that excite the immune system [67] thus creating a vicious circle that produces more and more sirs. the activated coagulation cascade after trauma or infection is primarily intended to stop the loss of blood and to seal the lesion. thereafter, phagocytic and immune cells eliminate necrotic cells and invading microbes. however, the uncontrolled activation of the coagulation cascade causes the widespread deposition of microthrombi, this in turn causing hypoxic cellular damage and increased risk for hemorrhagic complications due to consumption of platelets and coagulation factors [68] [69] [70] [71] . disseminated intravascular coagulation (dic) is a pathophysiological mechanism that frequently occurs after multiple trauma [68, 70, 71] and severe infections [68, 70] . dic is responsible for diffuse microthrombi deposition, capillary blockade with heterogeneous microcirculatory perfusion, tissue ischemia, and production of hypoxic cellular damage [68, 72] . 3. acute phase proteins are synthesized in the liver by induction of the locally (kupffer cells) produced cytokines (tnf-α, il1-β, and il6). this process is amplified by circulating cytokines that spillover from injured or infected tissue (overflow theory). acute phase proteins include c-reactive protein (crp), α-1-antitrypsin, α-2macroglobulin, ceruloplasmin, lipopolysaccharide-binding protein (lpb), fibrinogen, and prothrombin [73] . these proteins have several inflammatory and anti-inflammatory properties. for example, crp increases the surface expression of tissue factor (tf) [74] , a procoagulant membranebound 4.5 kd protein on pmns, monocytes, and tissue macrophages (see below). conversely, α-1-antitrypsin neutralizes leukocyte proteases and free oxygen radicals, while α-2-macroglobulin and ceruloplasmin are inhibitory cofactors for the synthesis of cytokines [74] . finally, high concentrations of lpb suppress lps activity [75] . at this step of the inflammatory process, the injured or infected tissue is invaded by activated pmns, mainly neutrophils that release proteases (elastase), exert their phagocytic and digestive properties (phagolysosomes), and produce free oxygen radicals (e.g., hydrogen peroxide) [76] and reactive no species (e.g., peroxynitrite) [77] . both neutrophils and macrophages kill bacteria. the first step involves phagocytosis of opsonized microbes. opsonization means that bacteria are covered with host proteins (e.g., antibodies and complement fragments) and appropriately recognized by cell surface receptors such as tlr and receptors for the fc portion of the immunoglobulins. the phagocytosed bacteria are typically inserted into a vacuole, the phagosome, which fuses with primary (azurophilic) or secondary granules to form phagolysosomes. azurophilic granules contain antimicrobial proteases such as elastase and bactericidal/permeability increasing proteins, while secondary granules contain antiseptic peptides as lysozyme, lactoferrin, and metalloproteases. fusion of granules with phagosomes creates a hostile environment that kills the pathogen ( fig. 2.6 ). at the same time, the resident macrophages and circulating monocytes begin to phagocyte cellular and microbial opsonized bacteria bind to complement receptors (crs) and fc receptors (fcrs) on the surface of phagocytic cells. thereafter the bacterium is taken into the cytoplasm and included into a phagosome. the fusion of phagosome with specific and azurophilic granules gives a phagolysosome that destroys the bacterium by toxic and enzymatic digestion debris. these cells produce cytokines and express membrane surface antigens that originate from the digestion process (see fig. 2 such molecules activate the endothelium to express adhesion molecules for further neutrophils chemoattraction. in the meanwhile, macrophages interact with t helper cells (cd4 + ) that secrete interferon ϒ (ifϒ) and other cytokines for phagocytic cells activation. finally, their interaction with b lymphocytes produces antimicrobial antibodies. this is a highly coordinated process, able to eliminate foreign bodies or cellular debris so ensuring the most effective protection against any insult of whatever origin. besides the plethora of pro-inflammatory mediators, many anti-inflammatory substances play a counter-regulatory role in the development of cars. il-4, il-10, il-13, and transforming-growth-factor β (tgf-β) are among the most important anti-inflammatory and immunosuppressant mediators. the main actions of anti-inflammatory cytokines are shown in table 2 .2. besides these anti-inflammatory cytokines, several immune effector cells develop their actions immediately after the onset of the acute inflammatory response. the nature of their response is largely determined by the expressed membrane antigenic pattern as it determines their subsequent cellular interactions and biochemical reactions. from the native, multipotent cd4 + t helper 0 cell (th0), two main cell lines differentiate depending on cytokine environment, either into th1 helper cell by il-12 or th2 helper cell by il-4 [78] . thereafter il-12 is produced, and opportune co-stimulatory molecules (cd80/86) are expressed together with mhcii antigens that bind to corresponding t-cell ligands. this binding results into t-cell activation and proliferation. the production of interferon γ stimulates the macrophages to kill intracellular bacteria [79] . th1 helper cells are therefore involved in the classical inflammatory response. overactivation of th1-type cells produces type 4 delayed hypersensitivity. 2. conversely, th2-type lymphocytes show marked antiinflammatory properties by producing anti-inflammatory cytokines as il-4, il-5, il-10, and il-13 [80] . overactivation causes type 1 ige-mediated allergy and hypersensitivity. other important players of the cell-mediated immune response in trauma and sepsis are the regulatory t (tregtype) cells and t17-type cells. 3. treg-type cells formerly known as suppressor t cells are modulator and deactivator of the immune response. they are immunosuppressive and generally downregulate the activation and proliferation of effector t cells (t cells that responds to a stimulus including helper and killer lymphocytes). treg-type cells produce il-10 and tgfβ a potent anti-inflammatory cytokine. elevated levels of circulating treg cells have been observed in the blood of immune-paralyzed infected patients [81, 82] . 4. the th17-type cells are a subset of t helper cells so named because of production of the cytokine il-17. they exert protective effects on the gastrointestinal mucosal barrier function [83] . there is general agreement that th17-type lymphocytes are protective against extracellular bacterial and fungal infections [84, 85] . the decrease of th17 cells population contributes to immunoparalysis [86] . it is widely accepted that the development of a successful immune response requires the balance between th1 and th2 helper cells. in the early stage of sepsis and trauma, a phenotypic th1-type cells profile predominates with ifϒ production which activates the bactericidal action of macrophages and induces b cell production of opsonizing and complement-fixing antibodies. conversely, later (chronic) stages are characterized by a shift from the th1-type profile into a predominantly th2-type response leading to immunoparalysis, reduced antigen recognition and cellular anergy (see below) [87] . other more complex anti-inflammatory actions include the decreased production of monocytederived cytokines by reduced transcriptional factors (nf-kb) for the encoding genes [88] . also, the reduced expression of membrane surface receptor cd14 on monocytes (lps-receptor) blunts their pro-inflammatory activity. also the expression of mhc (major histocompatibility complex) class ii molecules hla-dr (human leukocyte antigen) is downregulated [89] with interference in the elimination of infected cells and maturation of several immune cell lines. these anti-inflammatory effects play an important compensatory role in the development of the counter antiinflammatory response syndrome (cars). ideally cars is the physiological response to the risks of tissue damage by uncontrolled inflammation. the main sirs and cars events are summarized in fig. 2 .8. the highly integrated, redundant, and coordinated immune-inflammatory system makes easy its comparison with a great classical orchestra. as a classical orchestra, there are players (neutrophils), soloists (monocytes and macrophages), and the musical score (humoral mediators). this score may be harmoniously classical (locally coordinated inflammatory response) or dissonantly dodecaphonic (sirs). but who is the conductor? this fundamental role is covered by the endothelium. the acquisition that the endothelium orchestrates a complex, often redundant network of the immune-inflammatory players is quite recent. the endothelium is the interface between coagulation and inflammation in sepsis and trauma. endothelial cells line the vessels in every organ by forming a barrier with organ-specific properties. so the vascular permeability is virtually null in capillaries of the brain and almost complete in the kidney with intermediate levels in the liver, muscle, and lung. the main properties of the endothelium are: 1. lining the vascular system thus separating the blood from the cells and interstitium 2. regulating the vascular tone with a net vasodilatory effect after trauma, hypoxic damage, or sepsis, the injured endothelium becomes prothrombotic and anti-fibrinolytic ( fig. 2.9 ). the endothelium is involved in all the three major pathogenic pathways of septic and traumatic coagulopathy: tissue factor-mediated thrombin generation, dysfunctional anticoagulation, and fibrinolysis impairment [90] . moreover, its activation by inflammatory stimulus leads to platelet and leukocyte adhesion and inhibition of vasodilation. endothelial injury can be documented microscopically by a visible change of cell shape or damage and defects of endothelial lining. indirect evidence of the damaged endothelium is the finding of elevated soluble markers of cell injury notably thrombomodulin, intercellular adhesion molecule, and e-selectin and von willebrand factor [91, 92] . after endothelial damage, inflammatory fluids and cells can shift from the blood into the interstitial space. endothelial injury is sustained over time so that its property loss is long-standing [93, 94] . experimental animal and human studies demonstrated that after injury, the full recovery of endothelial lining occurs within 21 days [93] . the denudation of the vessel wall exposes tissue factor (tf), the principal activator of the extrinsic coagulation pathway with the risk of intravascular coagulation [93, 94] . normally the outer endothelium expresses various membrane-bound molecules with anticoagulants properties among which are cell surface heparin. antithrombin iii (atiii) further inhibits thrombin activity by preventing its binding with receptor (trr). also tissue factor (tf) is not expressed on the endothelium, while its main physiological inhibitor, tissue factor pathway inhibitor (tfpi), is normally released by the intact endothelium. finally the tis-sue plasminogen activator (tpa) catalyzes the zymogen plasminogen into plasmin, while tissue plasminogen activator inhibitor type-1 (tpai-1) is reduced. after hypoxia, injury, or sepsis, the damaged endothelium expresses large amounts of tf, while tfpi release is inhibited. also thrombomodulin receptor is internalized thus reducing apc formation, while decreased atiii synthesis and degradation by serine protease increases the activity of thrombin. finally tpai-1 synthesis and release are increased thus reducing the formation of plasmin like molecules [95] and thrombin-binding protein thrombomodulin (tm). heparin-like molecules are supported by glycosaminoglycans which cover the endothelial surface [90] . they act as cofactors for the antithrombinmediated inhibition of thrombin and activate factor x (stuart-power factor) [95] . tm is the major responsible for thrombin inactivation as when bound to thrombin, forms a potent protein c (pc) activator complex that equips the endothelium with anticoagulant properties. however, the inflammatory or septic stimuli decrease the anticoagulant properties of the endothelial cells by loss or internalization of tm [96] , while the contemporaneous stimulation of the thrombin receptor increases the inflammatory pathways. finally, the action of neutrophil proteases also contributes to reduced tm expression. moreover, the release of tissue plasminogen activator (tpa) is decreased, while its main physiological inhibitor, the plasminogen activator inhibitor-1 (pai-1), increases so that the profibrinolytic properties of the endothelium are diminished. tf is increasingly expressed on both monocyte and macrophage membranes as well as on other cell types, while the function of its main physiological inhibitor, the tissue factor pathway inhibitor (tfpi), is decreased by reduced synthesis of glycosaminoglycans on endothelial surface [97] . in practice, any single procoagulant or profibrinolytic has its physiological inhibitor. thus, tf is inhibited by tfpi [98] , the coagulation system is counteracted by the activated protein c system [99] , and tissue plasminogen activator (tpa) is coupled with tissue plasminogen activator inhibitor (tpai) [100] . the imbalance between these systems results in a net procoagulant state with increased fibrin deposition, microvascular thrombotic occlusion, and risk for tissue ischemia [101] . direct endothelial damage as occurs after trauma or ischemia-reperfusion injury contributes to activation of coagulation by decreased production of endothelial-derived substances as pgi and no. the anti-adhesive properties of such molecules reduce leukocytes and platelets aggregation and counteract the procoagulant effectors. localized coagulation has protective effects in discrete traumatic injuries, but its widespread activation is deleterious to the host with risk for multiple organ dysfunction and death [102, 103] . endothelial activation refers to the increased expression of adhesion molecules on endothelium surface. complementary molecules are also expressed on the outer membrane of neutrophil and monocyte cells [104] . the surface expression, modulation, and activity of these molecules are highly regulated by locally produced cytokines (endothelial and monocyte cells) as the chemokine il8 or paf, il1-β, and tnfα. the first step of the adhesion process consists of a "rolling" of leukocytes on the endothelial surface. this process involves selectins which are molecules expressed on leukocytes (l-selectin), endothelial cells (e-selectin), and platelets (p-selectin). selectins act as receptors that permit a loose binding with the endothelial surface thus allowing for leukocyte rolling in proximity of opportune signaling molecules expressed on the endothelium. the second step involves the linkage of integrins with receptors of the immunoglobulins superfamily expressed on the endothelium surface. integrins are a group of three heterodimer proteins expressed on the outer surface of activated leukocytes and collectively termed the cd11/cd18 complex. adhesion molecules include icams, e-selectins, pecam (platelet endothelial cell adhesion molecules), and vecam (vascular endothelial cell adhesion molecules). the final step consists of migration of activated leukocytes to the borders of endothelial cells and interaction with the endothelial-expressed adhesion molecules icam, pecam, e-selectin, and vecam [105] . adhesion can occur without intervention of adhesion molecules in the lung and liver. it has been suggested that actincontaining stress fibers increase at the leukocyte periphery, this, in turn, causing decreased pmns deformability and sequestration into the pulmonary and hepatic capillary beds [106] . the main effects of endothelial cells activation are summarized in fig. 2. 10. thrombin is the main effector of increased endothelial permeability thus reaffirming the bidirectional interplay between the coagulation and the inflammatory cascade ( fig. 2.11) . thrombin can cleave a set of protease-activated receptors (par1-4) expressed on the endothelium [90, 107] that induces rho-dependent cytoskeletal derangement in endothelial cells [108, 109] . par1 receptor activates rho kinase which inhibits the dephosphorylation of myosin light chains (mlc) [110] . phosphorylated mlc causes the actin-myosin interaction at the cell-to-cell contacts with contraction and rounding of endothelial cells and increased of vascular permeability. this facilitates the passage of protein molecules and leukocytes from the blood into the interstitial space (fig. 2.12) . furthermore, the formation of gaps in the endothelial barrier exposes the proteins of the external coagulation pathway to the abundant tf amount expressed by the basement membrane and vessel adventitia. clot formation is thus initiated, while collagen fibers from the extracellular matrix prompt vwf to polymerize and platelets to adhere. endothelial dysfunction refers to decreased no release and reduced expression or synthesis of the constitutive no synthase enzyme (enos). abnormality of endothelial relaxation properties lasts for many days after the acute insult (endothe-lial stunning) [111] . the impairment of no and pgi release by endothelial dysfunction have a profound impact upon cellular oxygenation with direct effects on tissues and organs well-being (fig. 2.10 ). according to the concept of intrinsic metabolic regulation, oxygen supply and demand are constantly matched. local vascular relaxation is therefore in dynamic equilibrium with the nervous-mediated vasoconstriction so that capillary blood flow is finely tuned to peripheral requirements. the endothelium plays a crucial role as a sensor of the local blood flow because of functional and structural coupling with the smooth muscle of arterioles and arteries. sensing is realized by depolarization/hyperpolarization of the endothelial cells, while communication involves electronic spread via endothelium/muscle cell-cell gap junctions [112] . therefore, the hyperpolarization of the capillary endo-thelium induces the upstream vasodilation of the feeding arterioles and arteries [113, 114] . when inflammation impairs this finely tuned cell-cell communication, a "malignant intravascular inflammation" ensues [115] . the abnormal vascular reactivity, increased fibrin deposition, and cells adhesion may well account for heterogeneity of intra-organ perfusion and impaired oxygen delivery. the relationship between organ perfusion heterogeneity, impaired organ oxygen supply, and development of metabolic acidosis has been demonstrated in an animal model of endotoxic shock [116] . in an in vivo model of septic shock, a 36% reduction of perfused capillary density with increased perfusion heterogeneity and mean intercapillaries distance contributed to functional shunting [117] with impaired oxygen extraction after endotoxin challenge or fecal peritonitis [118, 119] . the other fundamental player of the systemic immuneinflammatory activation after trauma and ischemiareperfusion or sepsis is the monocyte/macrophage cell. monocytes are the circulating form of resident macrophages and dendritic cells. resident macrophages can be found in the liver (kupffer cells), lung (alveolar macrophages), lymphoid tissue (spleen and lymph nodes), and kidney (intra-glomerular mesangial macrophages). all these cells are very active in the synthesis of cytokines and removal of particulate antigens. in addition, they play a central role in antigen presentation to the innate th1-type cells. apcs are fundamental for the eradication of pathogens, foreign antigens, or autologous cellular debris. their key functions include the recognition, uptake, and killing of invading organisms. tissue macrophages are initially activated after tissue injury by ischemic necrosis of mesenchymal cells in an il1-α-dependent process. after the necrotic insult il1-α is released from the dying cells with subsequent binding to the il-1 macrophage surface receptors. il1-α activates the assembly of inflammasome within the macrophage cytosol. inflammasomes are multiprotein cytoplasmic complex that cooperate with the toll-like receptors to respond to various insults by processing cytokines and promoting the inflammatory response. in the presence of pathogenic stimuli (e.g., alarmins), the inflammasome opens up so triggering the conversion of immature pro-inflammatory cytokines (e.g., il1-β) into mature forms and activating proapoptotic enzymes (e.g., caspases). thereafter, the extracellular il1-β secretion and upregulation of il1-β-induced chemokines, together with the increased expression of adhesion molecules, and cytokines secretion on the nearby endothelium [120, 121] allow for recruitment of circulating neutrophils and monocyte. no synthesis is activated with resulting vasodilation, opening of endothelial gaps, and loosening of endothelial barrier properties [122] (fig. 2.8 ). monocytes and macrophages are also pivotal cells for the interaction between myeloid and endothelial cells. after the expression of chemoattractant molecules (il8, monocyte chemoattractant protein-1 mcp-1, or macrophage inhibitory protein-1 mip-1), the endothelium attracts monocytes to the inflammatory focus where they migrate into the tissue and become macrophages. finally, the activated monocytes express large amounts of tf to propagate the procoagulant activity. after major uncomplicated trauma, the ability of apcs to express mhcii surface antigens is reduced [122] and returns to normal within a week [123] . after severe injuries, the apcs show a continuously decreased function. this deactivation leads to reduced expression of mhcii surface antigens and decreased ability to secrete cytokines which result into the increased susceptibility to infections [124] . many research findings have focused on the existence of premature programmed cell death (apoptosis) after injury, ischemia-reperfusion, and sepsis [125, 126] . although apoptosis is an adaptive mechanism in several tissue and organs, namely, the lymphoid tissue and gut, its role seems deleterious in acute inflammatory states. several studies suggest that the programmed cell death contributes to the derangement of cellular homeostasis in parenchymal (lung endothelial cells, kidney tubular cells, and skeletal muscle cells) and lymphoid organs with increased risk for sepsis and development of multiple organ dysfunction [87] . accelerated apoptosis induces lymphocytopenia [127] and decreased monocyte survival as reflected by loss of monocyte cd14 expression (co-receptor for the detection of bacterial lipopolysaccharide-lps) [82] . autoactivation of cytosolic and mitochondrial caspases are the two major pathways involved in apoptosis [128] (fig. 2.13) . briefly, the extrinsic pathways activate caspases via binding to members of the tnf-receptor superfamily, while the mitochondrial induced pathway requires the emission of cytochrome c, otherwise essential for mitochondrial survival, into the cytosol with subsequent caspases activation [129] . caspases are a family of proteolytic enzymes synthesized as inactive zymogens and activated by appropriate stimuli to express a death effector domain [130] . apoptosis is a series of coordinated processes [131] that lead to dna fragmentation, chromatin condensation, and blebs formation in the plasma membranes. most important this controlled form of cellular degradation carries out with minimal effects on surrounding tissues. therefore, apoptosis in a crucial physiological process during fetal development and throughout life as it maintains the normal development and regulation of cellular proliferation. for example, during organ development apoptosis eliminates those cells that are no longer necessary. it has been estimated that without apoptosis, about 2 tons of bone marrow and lymphoid tissue would accumulate in the body [132] ! conversely accelerated apoptosis of lymphocytes is as detrimental as delayed apoptosis in neutrophils. neutrophil cells are constitutively apoptotic as this ensures a tight conr e c e p to r the two main pathways of apoptosis are extrinsic and intrinsic pathway. each requires specific triggering signals and activates its own initiator caspase which in turn activate the executioner caspase-3. the execution pathway includes cell shrinkage, chromatin condensation, cytoplasmic blebbing, and formation of apoptotic bodies. finally phagocytosis of apoptotic bodies is performed by adjacent parenchymal cells or macrophages trol of the inflammatory response, but upon delayed apoptotic stimuli (cytokines), neutrophils become persistently activated and contributes to extensive organ damage by continuous release of toxic products (protease enzymes and reactive oxygen species) [133] . apoptosis causes a profound depression of immune functions with immunoparalysis and depletion of several immune cells including helper (cd4 + ) and suppressor (cd8 + ) t cells, b lymphocytes, and apcs (antigen presenting cells) [134] . postmortem studies have confirmed immune cells apoptosis in all age groups [134, 135] . it can be speculated that apoptosis of gut-associated lymphoid tissue (galt) [136] may be involved in bacterial translocation (see below). several findings support the current view of immunoparalysis as a dominant feature of patients surviving the early hyperinflammatory phase. although focal regions of inflammation ischemia and necrosis undoubtedly occur and contribute to the development of multiple organ dysfunction, death is the consequence of failure to control the primary infection or the development of new hospital-acquired infections often with opportunistic pathogens. this does not necessarily mean that infection is the main responsible for the patient's death and in fact many postmortem results are inconclusive. however, the severe decrease of innate immune function and the widespread hibernation of nonimmune cell type (cellular hibernation response) [137, 138] make apoptosis a primary mechanism for multiple organ dysfunction and ultimately death. interestingly cancer and sepsis show similar immune defects [139] , and increased survival after improving host immunity in cancer patients is encouraging to the field of sepsis. the most frequent manifestations of immunoparalysis include profound and persistent lymphopenia, loss of delayed type 4 hypersensitivity reaction [140] , reactivation of latent viruses infections (herpes simplex virus and cytomegalovirus) [141, 142] , and infection by low-virulent pathogens (e.g., stenotrophomonas spp., candida spp., acinetobacter spp.) [143, 144] . decreased pro-inflammatory cytokines production including ifϒ, tnf, and il-2, increased il-10 and other anti-inflammatory cytokines, decreased monocyte/macrophage and dendritic cells function with increased expression of apoptotic surface markers, and decreased cell survival are the hallmarks of immunoparalysis. dendritic cells and monocytes play a pivotal role in the development of decreased immune function. their reduced survival (apoptosis susceptibility) and function abnormalities (reduced hla-dr expression and increased production of il-10) induces t helper cell anergy and treg cell proliferation. the reduced capacity of monocytes to release pro-inflammatory cytokines (e.g., tnf, il-1, il-6, il-12), whereas the release of anti-inflammatory mediators (e.g., il-10, il-1ra) is not impaired or even enhanced [145] suggest for cellular reprogramming toward the anti-inflammatory pathway [146] . monocyte dysfunction is known as endotoxin tolerance that is reduced cellular response to an endotoxin challenge. the main consequence of endotoxin tolerance is the increased susceptibility to nosocomial infections and death. decreased immune t-cell function and t-cell exhaustion are the other leading causes of immunoparalysis. 1. the high antigenic load and the elevated pro-and antiinflammatory cytokine profile of the early phase of trauma and sepsis are ideal for the development t-cell exhaustion. phenotypic features of exhausted t cells are a decreased production of pro-inflammatory cytokines, mainly ifϒ and tnf, decreased cellular proliferation and cytotoxic function by excessive expression of pd-1 inducible co-stimulatory molecule and programmed cell death ligand pdl-1, and decreased expression of the il-7 receptor [148] . il-7 is a multipotential cytokine that acts to reverse immunosuppression by multiple mechanisms. it has shown to induce the two-to threefold increase of both naïve (th0-type) and cd4/cd8 cells in cancer [149] and hiv patients [150] so reversing their major pathological abnormality, i.e., profound lymphopenia. other effects include blockade of apoptosis [151] , reversal of t-cell exhaustion [152] , increased ifϒ [152] production leading to macrophage activation, and increased adhesion molecules expression on activated t lymphocytes [153] . the decrease of th1-type, th2-type [86, [154] [155] [156] , and th17-type cells [86, 157] with maintenance of the number of treg cells leads to the relative increase of regulatory functions and downregulation of effector t-cell response [86, 157] . the reduction of th17-type cells contributes to the increased susceptibility to fungal infection due to their important role in protecting against extracellular bacterial and fungal invasion [158] . the relative increase of treg cells is deleterious as being is associated with decreased effector t-cell proliferation and function. their increased resistance to apoptosis with respect to the other t-cell type is probably responsible for their increased proportion [159, 160] . treg cells can also suppress innate immune cells thus inhibiting myeloid-derived cell function [161] . therefore, increased treg cells impair the immune function by acting both on innate and adaptive immunity. neutrophils, nk cells, and ϒδ t cells are other effectors whose function is decreased or impaired during immunoparalysis. 2. briefly circulating neutrophils are markedly increased during the hyperinflammatory phase due to delayed apoptosis and release of immature cellular forms [162, 163] . loss of chemotactic activity is their most frequently encountered dysfunction [164, 165] . a subset of neutrophils with suppressive properties similar to the myeloid-derived suppressor cells (mdscs) contributes to the development of immunoparalysis by production of large amounts of il-10 [166] and interference in proliferation and function of effector t cells [167] . mdscs are a heterogeneous group of myeloid cells that expand during chronic inflammatory states and cancer as a result of altered hematopoiesis. mdscs possess strong immunosuppressive properties of both humoral and cellular type [168] . 3. nk cells are markedly reduced both in peripheral blood and tissues where they are most abundant [169] . impaired ifϒ by nk cells increases the host susceptibility to viral infections and reactivation of latent viruses, notably herpes simplex and cytomegalovirus [141, 142] . 4. ϒδ t cells are a subset of lymphocytes that possess functions common to the innate and adaptive immune systems. they are particularly abundant in the intestinal mucosa with innate-like defense mechanism (ifϒ and il-17 cytokines production). intestinal ϒδ t cells are a first-line defense against infections [170] , and their decreased number exposes to the risk of microbial invasion of the blood and peritoneal cavity. the main effects of chronic inflammation upon innate and acquired immune cells are shown in figs. 2.14 and 2.15. multiple organ dysfunction syndrome (mods) is defined as "the presence of altered organ function in an acutely ill patient such that homeostasis cannot be maintained without intervention" [2] . the mechanisms implicated in the development of mods include: anti-inflammatory cytokines secretion cytotoxic function cytokines secretion. ag-specific antibodies production. cd8 + t-cell b cell t reg cell lymphocytopenia results from massive loss of t helper and t suppressor lymphocytes. t regulatory cells are more resistant to apoptosis so that an immunosuppressive phenotype results. surviving t cells shift from a proinflammatory th-1 cell-type to an anti-inflammatory th-2 cell-type profile 3. immune system dysregulation 4. mitochondrial dysfunction although the above mechanisms can interact in a variety of ways and combinations, immune system dysregulation and mitochondrial dysfunction seem to play a prevalent role. as detailed above, the immune system dysregulation is the imbalance between pro-inflammatory and anti-inflammatory mechanisms. cars is a coordinated anti-inflammatory response to severe inflammatory stimuli that allows for the maintenance of immune system homeostasis. the "purpose" of cars is to limit the noxious effects of sirs while not interfering with pathogen elimination or healing of the injured tissue. however, cars may be dangerous when its effects lead to a condition known as "immunoparalysis" with an increased risk of nosocomial sepsis and septic shock. mitochondrial dysfunction with its hypoxic cellular implications is also important in the pathogenesis of mods. pmns production of reactive oxygen species (superoxide and peroxynitrite) and inflammatory cytokines induces oxidative stress that leads to uncoupling of oxidative phosphorylation. the derangement in cellular energy metabolism is known as cytophathic hypoxia, a functional concept that points out the imbalance between adequate oxygen delivery and poor oxygen utilization at the mitochondrial level. when cytophathic hypoxia develops, the result is cellular dysfunction and death. although "bad" from a clinical point of view, cytophathic hypoxia may be a cellular adaptive response [171] as it can be viewed as a cellular hibernation-like state. however, if this phenomenon occurs for too long, irreversible cellular damage may result [172] . bacterial translocation is another proposed mechanism for sustained inflammation and development of multiple organ dysfunctions. although the "sustained-hit model" [173] predicts that the persistent stimulation of natural and acquired immune system by noxious stimuli is responsible for the maintenance of the inflammatory status, recent acquisitions implicate the apoptotic loss of δϒ t cells in the intestinal mucosa [170] . there is good evidence that ischemia-reperfusion injury is the main mechanism for loss of gut barrier function [174] . as oxygenation of the villi is dependent on a counter-current mechanism such that o 2 tension at the tip of the villi is lower than that of arterial blood, any decrease in splanchnic blood flow may be associated with bacterial translocation. at this point endotoxin, bacteria, or gut ischemia can stimulate the gut-associated lymphoid tissue to generate an immune-inflammatory response that affects distant organs. otherwise stated the gut becomes a pro-inflammatory organ, releasing cytokines and other inflammatory mediators which give origin to sepsis and mods. according to the point of view of the intensive care medicine, the whole body is constituted by seven physiologically interdependent organ systems: nervous, respiratory, cardiovascular, hepatic biliary, renal, digestive, and coagulation system. although many of them can be involved, the respiratory and cardiovascular systems are most frequently damaged with acute lung injury (ali)/acute respiratory distress syndrome (ards) and sepsis-induced hypotension being the commonest clinical presentations. it is well established that the number or dysfunctional organs affects the prognosis. thus the probability to die with only one single dysfunctional organ is fairly less than half with respect to two or more organ dysfunction [175] . moreover, a multicenter study established that the mortality increases almost linearly from 21.2 to 76.2% when organ dysfunction increases from 1 to ≥4 organ systems [176] . mortality is also influenced by comorbidities notably chronic renal failure, diabetes, and cancer [176, 177] . cumulative comorbidities also increase the risk for the development of mods with the highest mortality rate [178] . by converse, survivors suffer from a persistent chronic illness that may last for months or even years. this illness is characterized by prolonged icu stay, slow recovery from organs dysfunction, recurrent infections, progressive or permanent cognitive deficit, and loss of overall sense of well-being and function [179, 180] . so the duration of healthy life expectancy is reduced although the patient's overall life span is intact. the common pathophysiological denominator of mods is the loss of cell membrane barrier function. this finding is strikingly evident from the autopsies of patients who die as a result of multiple organs dysfunction syndrome. despite the clinical evidence of acute myocardial dysfunction, progressive cholestatic jaundice, continuing impairment of renal function, and similar dysfunction of other organ systems, the histopathological findings are remarkably normal or limited to mild tissue edema. therefore, the preserved organ morphology is strikingly in contrast with the impairment of organ function. the loss of cellular and tissue (endothelial) barrier function seems the cornerstone for this sort of hibernation that prevents the multiplicity of organ expression, variability, and communication. the commonest clinical presentations of single organ system dysfunctions are shown below. recent acquisitions indicate that the brain is often involved in sepsis and mods as it is one target of sirs either by direct activation of resident pro-inflammatory cells (neuroglia) or by mediators generated elsewhere (complement, cytokines). although the cns is considered an immuneprotected organ due to the blood-brain barrier, the abundance of glial cells with their signaling receptors makes the brain one of the preferred target for organ system dysfunction. therefore, the local inflammatory reaction that follows traumatic or hypoxic (e.g., post-cardiac arrest) brain damage can spread outside the cns thus exacerbating sirs. conversely, the circulating cytokines and other inflammatory mediators may increase the permeability of the blood-brain barrier thus causing brain damage [181] . cytokines activate monocytes to transform into glial cells (equivalent to resident macrophages in extra-cerebral tissues) so that inflammation continues in the nervous tissue [182] . circulating cytokines also can stimulate the afferent fibers of the vagus nerve that activate the central nervous system. after stimulation of vagus nerve endings, cerebral endothelial cells are activated resulting in the breakdown of the blood-brain barrier [183, 184] . the activation of cerebral endothelium also induces microvascular dysfunction, loss of cerebral vascular tone (autoregulation), and coagulopathy (microthrombi deposition) [185] . receptors for the commonest cytokines have been found in the hippocampus, one of the most involved regions for mnemonic elaboration and neuroplasticity [181] . chronic inflammation of the hippocampus can lead to irreversible cognitive decline, especially in the elderly. histopathologic lesions of the brain in sepsis include cerebral edema, infarct and ischemic lesions, and microabscesses [185] . sepsisassociated encephalopathy has been reported in 23% of people in the icu [186] . however, the true incidence is difficult to estimate because of lack of clear definition and subjective nature of its assessment. moreover, sedative drugs may blunt the symptoms thus making the diagnosis difficult. symptoms include changes in consciousness, awareness, cognition, and behavior. symptoms seem correlated with the global severity of illness as assessed by the acute physiology and chronic health evaluation ii score (apache ii score). it is by far the commonest organ system involved. ali and ards are the two clinical manifestations of respiratory dysfunction. ali is defined as the presence of bilateral pulmonary infiltrates on chest radiograph and arterial hypoxemia (pao 2 /fio 2 < 300). if the pao 2 /fio 2 drops below 200, the condition is classified as ards [187] . ards can originate from two distinct pathways: 1. direct pulmonary damage as occurs after trauma, infection, aspiration, or inhalation injury 2. indirect damage as occurs with sepsis, pancreatitis, extrapulmonary trauma and burns, blood transfusions, and fat embolism [188, 189] respiratory dysfunction span from extremely severe forms requiring immediate tracheal intubation, mechanical ventilation, and paralysis to milder forms with mild hypoxia and tachypnea. the lung is the most frequent organ involved in sepsis and trauma probably because it is the single organ with the largest vascular bed and it harbors one of the largest populations of resident macrophages. ards is characterized by neutrophil infiltration, alveolar-capillary barrier disruption, pulmonary vascular leakage, and edema. in most severe forms the alveoli contain eosinophilic protein-rich exudate with presence of hyaline membrane [190] . electron microscopic examination reveals extensive damage to type i alveolar pneumocytes with cellular swelling and blebbing of the plasma membranes [190] . pulmonary edema and absorption atelectasis decrease the pulmonary volume with the occurrence of refractory hypoxia by increased pulmonary shunt. moreover, the widespread deposition of microthrombi and hypoxic pulmonary vasoconstriction impose a pressure overload upon the right ventricle with myocardial dysfunction and failure. the initial clinical presentation is often subtle with tachypnea, dyspnea, dry cough, and agitation. hypoxia with hypocapnia is the most prominent laboratory feature. the magnitude of hypoxia is often unexplained by the mild or absent roentgenographic signs. ct scan reveals diffuse but nonuniform ground-glass consolidations often with a non-conformal gravitational distribution [191] . in more severe forms, multiple patchy areas of lung consolidation coalesce in massive airspace consolidation [191] . characteristically the distribution is uniform and affects all lung zones extending to the extreme periphery of the lung fields. hypoxia and interstitial/alveolar edema stimulate the carotid body chemoreceptors and intraparenchymal lung mechanoreceptors to hyperventilate with decreased arterial carbon dioxide tension. in the latest phases, normocapnia ensues mainly as result of increased respiratory work due to alveolar collapse, decreased pulmonary volume, and respiratory compliance. the mechanical disadvantage of decreased pulmonary compliance causes the abnormal increase of trans-pulmonary pressure (the pressure across the alveoli) that is the driving force for pulmonary expansion. the shear stress upon the delicate alveolar and interstitial structure causes further inflammation as a vicious cycle ensue with endothelial activation and worsening of pulmonary function that influences each other. mortality from ards ranges from 15 to 80% [189, 192] with average values of about 30% according to most recent ventilatory strategies [193] . it is one of the commonest organ dysfunction of septic and/ or traumatic origin. some studies report an incidence of up to 66% [194, 195] with a mortality rate as high as 70% [195, 196] . the cardiovascular dysfunction has long been recognized as a consequence of the generalized inflammatory reaction [197] . its most prominent clinical features are (1) myocardial dysfunction with biventricular dilation and reduced ejection fraction [198] and (2) arterial hypotension despite fluid volume infusion and decreased response to vasoactive drugs. it generally accepted that the decreased cardiac function is the product of circulating myocardial depressant factors (endotoxin, il1β, tnfα, il6, paf, and others) and mitochondrial damage [199] [200] [201] . some experimental evidence indicates that endotoxin acts by inducing an inflammatory response through heart tissue macrophage, mast cells, and infiltrating blood leukocytes. the clinical picture includes the following: arterial hypotension, mental impairment, warm and dry skin, oliguria, elevated arterial blood lactates, and higher than normal mixed or central venous saturation. the hemodynamic pattern shows a high cardiac output and decreased peripheral vascular resistance [202, 203] . the picture of low cardiac output state (pale and wet skin, oliguria, and hypotension) is rarely seen and indicates a more severe form of cardiac depression [202, 203] . when examined in detail, the cardiac function appears depressed as, despite the high output state, the ejected volume is lower than normal (reduced stroke volume), the ventricle is dilated (increased diastolic volume), and its contractile state is impaired with reduced ejection fraction (increased end-systolic volume). these changes are often masked by the decreased peripheral vascular resistance. the reduced afterload allows for the maintenance of a high output state as the dysfunctional ventricle pump blood against a dilated arterial system. when the ventricular dysfunction is severe, the clinical picture resembles that of cardiogenic shock with low cardiac output, high arterial lactates, and intense peripheral vasoconstriction. several studies have shown that cardiac dysfunction is biventricular in origin and that is potentially reversible by 7-10 days [204] . the other essential feature of cardiovascular dysfunction is arterial hypotension by decreased peripheral arterial resistance. because a major fraction of peripheral vascular resistance is located in peripheral arteries, this segment is designated as the resistance vessels. resistance vessels control the blood flow to organs, and it is under the control of either intrinsic (e.g., metabolic) or extrinsic (e.g., neural and humoral) factors. intrinsic regulation plays a fundamental role in pressure-flow autoregulation (ability of an organ to keep constant the blood flow despite fluctuations in arterial pressure), reactive hyperemia (ability to increase the blood flow after relief of a vascular obstruction), and functional hyperemia (ability to cope the organ blood flow in accordance with local metabolic needs) [205, 206] . in sepsis, intrinsic regulation is compromised in all the vascular beds with the exception of the cerebral vasculature [207, 208] . this can explain the mismatch between whole oxygen supply and metabolic demands of septic shock. the increased cardiac output does not cope with the cellular energy requirements so that hypoxia and increased acid production develop. by converse, the extrinsic regulation of resistance vessels is responsible for maintenance of arterial pressure and blood flow to the whole organs. during sepsis and after trauma or ischemia-reperfusion injury, a marked hypo-responsiveness to vasopressor agents is a frequent finding with development of arterial hypotension and tissue hypoperfusion. it is generally accepted that the decreased responsiveness to vasoconstrictor stimuli is consequent to increased production of endothelial nitric oxide (no) by the induced nitric oxide synthase (inos) [209] . in normal arterioles, a small amount of no is produced by endothelial nos (enos) as maintenance of cardiovascular homeostasis [209] . however, cytokines and endotoxins induce inos to produce large quantities of no [209, 210] . although inos induction can be viewed as an adaptive response to noxious stimuli, the resulting hypotension by massive no production is deleterious for organs and tissues. also, there is some evidence that vascular smooth muscle can be another relevant source of no production. in smooth muscle, neural nos (nnos) is upregulated thus contributing to no generation in septic animals [211] . another factor implicated in arteriolar hypo-responsiveness may be the complex of morphological alterations of the endothelial lining of the arterioles. experimental studies show that the endotoxin challenge results into endothelium disruption with cell swelling, pseudopod formation, and frank denudation of the endothelial lining [212, 213] . finally, the loss of capillary to arteriolar signaling may induce the hypo-responsiveness of arterioles to vasopressor stimuli. in normal conditions, capillaries may regulate the arteriolar tone by the upstream spread of electronic signaling along the endothelium. in sepsis, the cell-to-cell communication is impaired so that the modulation of blood flow delivery to capillaries is severely compromised [214] . taken all together the above mechanisms may lead to the inappropriate redistribution of blood flow to tissues and organs [215, 216] . because the arterial control of blood flow is responsible for the convective flux of oxygen to capillaries, the alteration of arterial tone may cause regional hypoxia [215] and ultimately multiple organ dysfunction. treatment principles of hyperdynamic shock are briefly outlined in chap. 3. the coagulation system is designed: 1. to stop active blood loss 2. to seal the site of lesion, thus allowing for pathogens destruction and removal of debris 3. to initiate the healing process the most severe form of coagulation disorder is the disseminated intravascular coagulation (dic). this pathophysio-logic mechanism results from the imbalance between coagulation and fibrinolysis with upregulation of the procoagulant pathway, downregulation of anticoagulant pathway (namely, atiii, protein c, and s system), and decreased fibrinolysis. coagulation is regulated by three main anticoagulant mechanisms: antithrombin, the protein c system, and the tissue factor pathway inhibitor (tfpi). the loss of physiologic anticoagulation in sepsis results from the action of several humoral (il1-β, tnf-α, and il6, c-reactive protein) and cellular (vascular endothelial cells, monocytes, macrophages, and platelet) pro-inflammatory mediators. furthermore, monocytes and macrophages are the main source of tissue factor (tf) a potent thrombin generator via the vii (extrinsic) pathway. the widespread fibrin deposition causes the formation of microthrombi with diffuse microcirculatory flow disturbance. tf is a membrane-bound protein that possesses the most important procoagulant activity [217] . after its expression on the cellular membranes, it forms complexes with the circulating factor vii of the coagulation cascade (extrinsic pathway) thus activating the formation of thrombin [218] . tf is abundant in the subcutaneous tissue and virtually in all blood-tissue barrier [219] . as seen above, tf is also inducible on monocyte and macrophage surfaces. in meningococcal septicemia, large quantities of tf have been localized in microparticles (mps) shed from platelets and leukocyte aggregates [220] . after trauma or infection, large amount of tf can be expressed by the denuded endothelium, damaged subcutaneous, and muscular tissues with priming of the coagulation cascade. similarly, sepsis can induce disseminated intravascular coagulation by cytokines production and monocyte adhesion to the activated endothelium with further tf expression. after thrombin formation, the von willebrand factor (vwf) is released from the weibel-palade bodies of endothelial cells. vwf binds to platelet glycoprotein iib to stabilize the adhesion of platelets and entrapped white blood cells aggregates to the injured endothelium [221] . normally vwf multimers are cleaved by the protein complex adamts13, and this process is stimulated under conditions of shear stress. sepsis is associated with decreased circulating adamts13 levels resulting in increased levels of ultra-large vwf multimers [221] . after adhesion to the endothelium (white thrombus formation), platelets enhances the inflammatory and coagulation cascade by expression of tf on activated monocytes and endothelial cells [222] . clinical conditions associated with overt dic are summarized in table 2 .3. the role of dic in the pathogenesis of mods is widely supported by experimental and postmortem findings. firstly, the demonstration of intravascular thrombi at autopsy appears related to the clinical dysfunction of the organ [223] . secondly, experimental studies demonstrated the deposition of intravascular and extravascular fibrin in the kidneys, lung, liver, and brain [223] . finally, clinical studies demonstrated the prognostic importance of dic as being an independent predictor of death [224] . the clinical diagnosis of dic relies upon the simultaneous presence of: 1. consumption of coagulation factors (prolonged clotting times) 2. consumption of coagulation inhibitors (e.g., decreased atiii activity) 3. platelets consumption or count of less than 100,000/mm 3 4. presence of fibrin degradation products (increased d-dimer) 5. presence of a condition known to be associated with dic in the past decade, clinical simple scoring systems for dic were introduced (table 2. they have individual strengths (jaam has the highest sensitivity, while isth is the most specific) and collective weaknesses (poor diagnostic sensitivity for late-onset dic) [228] . fibrinogen levels are not sensitive indicators of dic as fibrinogen synthesis is increased during sirs as occurs in the acute phase protein reaction. finally, the diagnosis of dic can be difficult in the presence of hepatic dysfunction or portal vein thrombosis as many of diagnostic clues of dic are present in these pathologic conditions. coagulation dysfunction affects up to 30% of patients with sepsis [229] , while thrombocytopenia (<10 5 platelets/mm 3 ) occurs in up to 60% with an inverse relationship between platelet count and severity of disease [230, 231] . acute kidney injury (aki) is defined as an absolute increase in serum creatinine of 1.5-fold from baseline or a urine output <0.5 ml/kg/h for 6 h [232] . overall aki frequency was reported over 20% in a large cohort study of 325,395 patients admitted to icu with odds of death increasing in parallel with increased severity of acute kidney injury [233] . crude mortality has been found ranging between 28 and 82% [234, 235] . the severity of aki is classified using the risk, injury, failure, loss and end-stage renal disease (rifle) criteria listed in table 2 .5, [236] . two main pathways are responsible for aki: one is characterized by acute tubular necrosis, renal hypoperfusion, and ischemia-despite common belief, this form accounts for only 22% of sepsis-induced aki [237] -and the second form is specific to mods, and its predominant mechanism is apoptosis induced by inflammatory cytokines [238] . this pathogenic mechanism is more adherent to the hyperdynamic circulatory state that follows post-traumatic sirs and sepsis [239] . experimental studies suggest that the decrease in glomerular filtration rate in aki is the result of a disproportionate vasodilation of the efferent than the afferent glomerular arterioles [240] . in normal condition, the vascular tone of afferent and efferent vessels is regulated by humoral intra-glomerular factors (mainly angiotensin, thromboxane a2, and pgi2). the interplay between vasoconstrictor (angiotensin and thromboxane a2) and vasodilator (pgi2) factors causes the tone of efferent vessels to increase with respect to the afferent arterioles. this allows for the glomerular capillary pressure to ensure normal filtration. during sirs or sepsis, the dilation of the efferent arterioles decreases the glomerular pressure and hence the filtration rate [241] . this pathophysiologic mechanism can explain the positive effects of norepinephrine or vasopressin infusion in patients with sirs and oliguria. despite the common belief, norepinephrine and vasopressin have been reported to increase the urinary output in patients with hyperdynamic shock. their vasoconstrictor effect is probably helpful in restoring the tone of the efferent arteriole with resulting increase of capillary perfusion pressure. in addition to glomerular hemodynamic changes, circulating or locally (mesangial cells) produced tnfα induces tubular cell apoptosis with imbalance in water and electrolytes homeostasis [242] . aki is an important form of organ dysfunction because of its association with high mortality rates. in a multinational, multicenter study, the prevalence of aki has been estimated to be 5-6% of people with only 40% of them discharged alive [243] . septic shock is the most common cause of aki with a prevalence of 65% [244] . trauma patients who developed aki showed 29% of mortality rate as compared with 9% of the overall mortality rate [245] . the gut has long been recognized as the "motor" of mods due to its capability of amplifying the sirs response during shock and gut hypoperfusion. the incidence of gastrointestinal dysfunction is difficult to estimate due to the subjective nature of diagnosis and the lack of a clear diagnostic definition. symptoms and signs of intestinal dysfunction include hyporexia, anorexia, inability to tolerate enteral feeding, decreased intestinal motility, and diarrhea often of hemorrhagic type [246, 247] . after a catastrophic hypoperfusion of the gut as occurs with cardiac arrest or severe multiple jmhw japanese ministry of health and welfare [225] , isth international society of thrombosis and haemostasis [226] , jaam japanese association of acute medicine [227] a if hematologic malignancy, 0 pts for bleeding and platelets and add 3 pts to the total score loss of renal function >4 weeks end-stage renal disease (e) loss of renal function >3 months modified from [236] trauma, the normal intestinal flora is destroyed and substituted by an increasing number of pathogens [248] . this phenomenon is detrimental because the normal flora protects against the colonization of microbial pathogens thus preventing bacterial translocation (see above) [249] . however, the change of normal flora together with reduced intestinal motility and disruption of normal gut mucosal barrier increase the risk for bacterial translocation and development of mods. the loss of mucosal barrier function is the result of endotoxin, inflammatory cytokines, and decreased production of tight junction proteins [250] . hepatic dysfunction is schematically divided into two clinical forms: hypoxic hepatitis (hh) and jaundice-induced cholestasis (jic). hh can result from absolute (e.g., cardiogenic) or relative decrease in cardiac output with reduced oxygen supply to the liver. during septic shock, cardiac output and oxygen delivery are increased albeit not enough to cope with the high oxygen requirement of the liver and the inability of cells to extract oxygen [45] . moreover, the hepatic arterial buffer response and other vascular mechanisms of defense against portal blood flow reduction are impaired [251] . hh is also the product of oxidative stress after ischemia/reperfusion injury and the action of endotoxin and other inflammatory mediators which lead to activation of kupffer cells and activation and recruitment of leukocytes. the diagnosis of hh requires: 1. a clinical setting of impaired oxygen delivery (usually due to cardiac and/or respiratory failure) 2. a greater than 20-fold increase in serum aminotransferase activity 3. the exclusion of other causes of liver cell necrosis [252, 253] another landmark of hh is the acute drop of prothrombin levels which gives origin to a hemorrhagic syndrome [254] . these acute biochemical changes, especially those reflecting acute liver cells necrosis, decrease rapidly within 2 or 3 days and normalize within approximately 2 weeks. the delayed elevation of serum bilirubin is often seen but without apparent jaundice. jaundice-induced cholestasis is a more subtle form of hepatic jaundice usually associated with severe infections [255, 256] . the increase of bilirubin is a late event in these septic patients [257] , and histological studies reveal the intrahepatic origin of the cholestasis [258] . the reduction of bile salt excretion in the gut is responsible for the atrophy of the mucosal gut barrier [259] with loss of integrity and increased risk of bacterial translocation. moreover, the gut is deprived by the bacteriostatic action of bile salts with proliferation of pathogens and increased production of endotoxin [260, 261] . this creates a vicious circle that perpetuates the infection and hence further cholestatic liver damage. the production and transport of bile salts are a complex, highly coordinated, and energy-dependent mechanism. during sepsis many of these mechanisms are impaired because of a lack of energy due to hypoxia or hypoperfusion or both [262] . the major limiting factor for the bile flow at the canalicular level is the atpdependent bile salt export pump that is severely impaired in the septic liver [262] . the severity of hyperbilirubinemia appears directly related to the severity of impairment in the various steps of bile formation. no specific therapies can be recommended for acute liver dysfunction. the most recommended measures are merely supportive including the normalization of perfusion pressure and restoration of oxygen delivery [263] , the maintenance of normal oxygen tension, and serum glucose levels. the early use of enteral nutrition is also helpful in counteracting the atrophy of gut mucosal barrier and reducing the intestinal pathogens [264] . a high-impact trauma victim was admitted to ed with head trauma and severe hemorrhagic shock by ruptured spleen and multiple bone fractures. the patient was unconscious; his blood pressure was 85/55 mmhg with 135 bpm pulse rate. metabolic acidosis (ph 7.27, paco2 32 mmhg) and hyperlactacidemia (7.5 mmol/l) were also present. splenectomy and external fixation of bone fractures were quickly performed. during surgery, the blood pressure remained at about 100/60 with an average 120 bpm pulse rate. despite several liters of crystalloids and transfusion of blood products (4 prc and 4 ffp), an infusion of norepinephrine up to 0.15 μg/kg/min was started. at icu admission, the patient had stable vital signs (ap 115/50 mmhg) a mild tachicardia (hr 115 bpm), and compensated metabolic acidosis (ph 7.37, paco2 30 mmhg) with elevated arterial blood lactates (7 mmol/l). after 12 h from icu admission, the resuscitation phase appeared complete. at 24 h, fever of 38.8 °c, leukocytosis 21,000 cells/mm 3 , respiratory alkalosis, and tachycardia prompted the execution of a total body ct scan. a. sepsis b. sirs c. intra-abdominal abscess d. fever of cns origin a. il-4 and il-10 b. il-1 and tnf c. il-6 and il-7 d. the ct scan excluded intra-abdominal abscesses and revealed large bilateral psoas muscle and pelvic hematomas. microbiological cultures from urine, bronchial tube drainage fluids were negative. within 5 days fever and tachycardia disappeared so that the definitive fixation of bone fractures was scheduled for the following week. at day 10, a new appearance of fever and leukocytosis occurred. a. infection b. sirs c. drugs-induced fever d. fever of unknown origin the chest roentgenogram showed a right perihilar infiltrate and purulent bronchial secretions. broad spectrum antibiotics were started, but 24 h thereafter the patient develops hypotension, oliguria, metabolic acidosis, altered mental status, and decreased platelet count. a a multi-sensitive klebsiella pneumoniae was isolated from blood cultures, and antimicrobial therapy allows for rapid control of the septic source and resolution of the clinical picture. the patient was discharged from intensive care after 15 days and returned at home within 60 days from the hospital admission. please see chap. 58 for the correct answer. sir isaac newton, sepsis, sirs and cars of critical care medicine consensus conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis the systemic immune response to trauma: an overview of pathophysiology and treatment sepsis chronically in mars: systemic cytokine responses are always mixed regardless of the outcome, magnitude, or phase of sepsis the pathophysiology and treatment of sepsis sepsis syndromes: understanding the role of innate and acquired immunity immunosuppression in sepsis: a novel understanding of the disorder and new therapeutic approach inflammatory cytokine response in patients with septic shock secondary to generalized peritonitis cd11c expressions on monocytes surface and cytokine production in patients with sepsis, severe sepsis and septic shock sepsis: multiple abnormalities, heterogeneous responses, and evolving understanding sepsis: always in mars selective defects on t lymphocytes function in patients with lethal intraabdominal infection lipopolysaccharideinduced tumor necrosis factor alpha production and not monocyte human leukocyte antigen-dr expression is correlated with survival in septic trauma patients immunodepression in sepsis and sirs assessed by ex-vivo cytokine production is not a generalized phenomenon: a review pamps and alarmins: all we need to know about danger how tissue injury alarms the immune system and causes a systemic inflammatory response circulating mitochondrial dampss cause inflammatory response to injury bacterial endotoxin stimulates macrophages to release hmgb1 partly through cd14-and tnf-dependent mechanisms the impact of endogenous triggers on traumaassociated inflammation trauma alarmins as activators of damage-induced inflammation early complementopathy after multiple injuries in humans molecular mechanisms of inflammatory and tissue injury after major trauma-is complement the "bad guy the role of complement in trauma and fracture healing alarmin(s) news about danger: workshop on innate danger signals and hmgb1 proinflammatory cytokines (tumor necrosis factor and interleukin 1) stimulate release of high mobility group protein-1 by pituicytes release of chromatin protein hmgb1 by necrotic cells triggers inflammation the nuclear protein hmgb1 is secreted by monocytes via a non-classical, vesicle-mediated secretory pathway high mobility group 1 protein (hmg-1) stimulates proinflammatory cytokine synthesis in human monocytes regulation of monocyte migration by amphoterin (hmgb1) inflammatory mediators in relation to the development of multiple organ failure in patients after severe blunt trauma the complement system in trauma-related and ischemic tissue damage: a brief review interleukin-6 potentiates neutrophil priming with plateletactivating factor trauma primes cells trauma: the role of the innate immune system neutrophil priming and activation in the pathogenesis of postinjury multiple organ failure neutrophil recruitment under shear flow: it's all about endothelial rings and gaps inflammatory serum markers in patients with multiple trauma. can they predict outcome systemic inflammation after trauma differentiation of effector c4 t cell populations postinjury neutrophil priming and activation: an early vulnerable window cytokines in patients with polytrauma tnf receptor type 2 mediates thymocyte proliferation independently of tnf receptor type 1 function and regulation of tumor necrosis factor type 2 interleukin-1 beta cytokine regulation of interleukin 6 production by human endothelial cells anti-inflammatory cytokines interleukin-6 (il-6) as an anti-inflammatory cytokine. induction of circulating il-1 receptor antagonist and soluble tumor necrosis factor receptor p55 inflammatory cytokines and cell response in surgery cox g il6 is an anti-inflammatory cytokine required for controlling local or systemic acute inflammatory responses procalcitonin as a diagnostic marker and il-6 as a prognostic marker for sepsis interleukin-6 levels act as a diagnostic marker for infection and a prognostic marker in patients with organ dysfunction in the intensive care unit interleukin 6 is a useful marker for early prediction of the severity of acute pancreatitis chemokine signaling in inflammation complement activation in injured patients occurs immediately and is dependent on the severity of the trauma the role of the complement system in traumatic brain injury femur fracture induces site-specific changes in t-cells immunity reperfusion injury of ischemic skeletal muscle is mediated by natural antibody and complement innate autoimmunity natural antibodies, autoantibodies and complement activation in tissue injury role of biological modifiers regulating the immune response after trauma the role of neuroinflammation in traumatic brain injury evidence for a role of kallikrein-kinin system in patients with shock after blunt trauma molecular and functional interactions among monocytes, platelets, and endothelial cells and their relevance for cardiovascular diseases platelets: bridging hemostasis, inflammation and immunity coagulation abnormalities in acute lung injury and sepsis priming for enhanced alveolar fibrin deposition after hemorrhagic shock: role for tumor necrosis factor new treatment strategies for disseminated intravascular coagulation based on current understanding of the pathophysiology systemic inflammation and disseminated intravascular coagulation in early stage of ali and ards: role of neutrophil and endothelial activation extracellular proteolysis in brain injury and inflammation: role for plasminogen activators and matrix metalloproteinases the acute phase response function of c-reactive protein high concentrations of lipopolysaccharide-binding protein in serum of patients with severe sepsis or septic shock inhibit the lipopolysaccharide response in human monocytes neutrophil-mediated tissue injury and its modulation role of nitric oxide in inflammation the expanding universe of t-cell subsets: th1 and th2 and more signalling events involved in interferon-gamma-inducible macrophage nitric oxide generation th1 and th2 subsets: paradigms lost? marked elevation of human circulating cd4 + cd25 + regulatory t cells in sepsis induced immunoparalysis human cd4 + cd25 + regulatory t lymphocytes inhibit lipopolysaccharide-induced monocyte survival through a fas/fas ligand-dependent mechanism immunity to fungal infections th17 cytokines and the gut mucosal barrier giammarellos-bourboulis ejg. deficient candida-specific t-helper 17 response during sepsis longitudinal study of cytokine and immune transcription factor mrna expression in septic shock immunoparalysis after multiple trauma il-10-induced micro-rna-187 negatively regulates tnf-alpha, il-6 and il-12p40 production in tlr4-stimulated monocytes persisting low monocyte human leukocyte antigen-dr expression predicts mortality in septic shock inflammation endothelium and coagulation in sepsis plasma levels of the three endothelialspecific proteins von willebrand factor, tissue factor pathway inhibitor, and thrombomodulin do not predict the development of acute respiratory distress syndrome elevated circulating e-selectin, intercellular adhesion molecule 1, and von willebrand factor in patients with severe infection a single endotoxin injection in the rabbit causes prolonged blood vessel dysfunction and a procoagulant state vascular endothelial cell dysfunction in septic shock anticoagulant properties of the vascular endothelium initiation and regulation of tissue factor-dependent blood coagulation reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor the tissue factor-factor viia complex: procoagulant activity, regulation, and multitasking protein c anticoagulant pathway and its role in controlling microvascular thrombosis and inflammation plasminogen activator inhibitor 1: physiological and pathophysiological roles organ distribution of fibrin in disseminated intravascular coagulation recombinant e. coli-derived tissue factor pathway inhibitor reduces coagulopathic and lethal effects in the baboon gram-negative model of septic shock delayed treatment with recombinant human tissue factor pathway inhibitor improves survival in rabbit gram-negative peritonitis traffic signals on endothelium for lymphocyte recirculation and leukocyte emigration mechanisms of endothelial cell injury in acute inflammation leukocyte trafficking in alveoli and airway passage endothelial injury in sepsis mechanistic coupling of protease signaling and initiation of coagulation by tissue factor activation of endothelial cell protease activated receptor 1 by the protein c pathway regulation of myosin phosphatase by rho and rho-associated kinase (rho-kinase) stunning" following a brief exposure to endotoxin: a mechanism to link infection and infarction? incidence of myoendothelial gap junctions in the proximal and distal mesenteric arteries of the rat is suggestive of a role in endothelium-derived hyperpolarizing factormediated response endothelial cell pathway for conduction of hyperpolarization and vasodilation along hamster feed artery microvascular recruitment in hamster striated muscle: role for conducted vasodilation the splanchnic circulation: no longer a silent partner pathological supply dependence of systemic and intestinal o2 uptake and endotoxemia microvascular perfusion is impaired in a rat model of normotensive sepsis heterogeneity of gut capillary transit times and impaired gut oxygen extraction in endotoxemic pigs effect of a maldistribution of microvascular blood flow on capillary o2 extraction in sepsis cutting edge: critical role of mesothelial cells in necrosis-induced inflammation through the recognition of il-1 alpha released from dying cells interleukin-1 in the pathogenesis and treatment of inflammatory diseases interleukin-10 down regulates mhc class ii alphabeta peptide complexes at the plasma membrane of monocytes by affecting arrival and recycling immediate il-10 expression following major orthopaedic trauma. relationship to anti-inflammatory response and subsequent development of sepsis the significance and challenges of monocyte impairment: for the patient and the surgeon depletion of dendritic cells, but not macrophages, in patients with sepsis leukocyte apoptosis and its significance in sepsis and septic shock apoptosis and surgical trauma: dysregulated expression of death and survival factors on peripheral lymphocytes cell death: critical control points how cells die: apoptosis pathways human caspases: activation, specificity, and regulation cell death during sepsis: integration of disintegration in the inflammatory response to overwhelming infection the sirens' song dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome apoptotic cell death in patients with sepsis, shock and multiple organ dysfunction prolonged lymphopenia, lymphoid depletion and hypoprolactinemia in children with nosocomial sepsis and multiple organ failure rapid onset of intestinal epithelial and lymphocyte apoptotic cell death in patient with trauma and shock mechanisms of organ dysfunction in critical illness: report from a round table conference held in brussels mechanisms of sepsis-induced organ dysfunction sepsis-induced immunosuppression: from cellular dysfunctions to immunotherapy delayed hypersensitivity: indicator of acquired failure of host defenses in sepsis and trauma herpes simplex virus lung infection in patients undergoing prolonged mechanical ventilation cytomegalovirus reactivation in critically immunocompetent patients the late phase of sepsis is characterized by an increased microbiological burden and death rate predictors of 30-day mortality and hospital costs in patients with ventilator-associated pneumonia attributed to potentially antibiotic-resistant gram-negative bacteria bench to bedside review: endotoxin tolerance as a model of leukocyte reprogramming in sepsis endotoxin tolerance: new mechanisms, molecules and clinical significance lipopolysaccharide structure-function relationship in activation versus reprogramming of mouse peritoneal macrophages immunosuppression in patients who die of sepsis and multiple organ failure interleukin-7 and immune reconstitution in cancer patients: a new paradigm for dramatically increasing overall survival effects of recombinant human interleukin-7 on t-cell recovery and thymic output in hiv-infected patients receiving antiretroviral therapy: result of a phase i/iia randomized, placebo-controlled, multicenter study overexpression of bcl-2 in transgenic mice decreases apoptosis and improves survival in sepsis il-7 restores lymphocyte function in septic patients il-7 promotes t cell viability, trafficking, and functionality and improves survival in sepsis lymphocyte subset responses to trauma and sepsis cd4+ t-lymphocyte alterations in trauma patients early circulating lymphocyte apoptosis in human septic shock is associated with poor outcome early assessment of leukocyte alterations at diagnosis of septic shock icu-acquired immunosuppression and the risk for secondary fungal infections regulatory t cell populations in sepsis and trauma increased proportion of cd4(+) cd25(+)foxp3(+) regulatory t cells during the early-stage sepsis in icu patients innate immune functions of immature neutrophils in patients with sepsis and severe systemic inflammatory response syndrome neutrophil paralysis in sepsis the function of neutrophils in sepsis expression and function of the chemokine receptor cxcr1 and cxcr2 in sepsis neutrophils are significant producers of il-10 during sepsis a subset of neutrophils in human systemic inflammation inhibits t cell responses through mac-1 cd11+/gr-1+ myeloid suppressor cells cause t cell dysfunction after traumatic stress adib-conquy m. captain study group. toll-like receptors expression and interferon-gamma production by nk cells in human sepsis association of ϒδ tcells with disease severity and mortality in septic patients multiorgan failure is an adaptive, endocrine-mediated, metabolic response to overwhelming systemic inflammation the multiple organ dysfunction syndrome evolving concepts in the pathogenesis of postinjury multiple organ failure current status of bacterial translocation as a cause of surgical sepsis the impact of acute organ dysfunction on patients' mortality with severe sepsis epidemiology of severe sepsis in the united states: analysis of incidence, outcome and associated costs of care the epidemiology of sepsis in patients with malignancy the role of infections and comorbidity. factors that influence disparities in sepsis the lingering consequences of sepsis: the hidden public health disaster? hmgb1 mediates cognitive impairment in sepsis survivors cytokines and the nervous system ii: actions and mechanisms of action resolving postoperative neuroinflammation and cognitive decline understanding brain dysfunction in sepsis sepsis-associated encephalopathy: review of neuropsychiatric manifestations and cognitive outcome the neuropathology of septic shock impact of encephalopathy on mortality in the sepsis syndrome. the veterans administration systemic sepsis cooperative study groups acute respiratory distress syndrome: the berlin definition pulmonary and extrapulmonary forms of acute respiratory distress syndrome epidemiology of ards and ali pulmonary edema (including respiratory distress syndrome) chapter 14: pulmonary edema is the mortality higher in the pulmonary vs the extrapulmonary ards? a meta-analysis ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome the epidemiology of acute organ system dysfunction from severe sepsis outside the intensive care unit epidemiology of severe sepsis in japanese intensive care units: a prospective multicenter study cub-réa study group. incidence and impact of organ dysfunctions associated with sepsis myocardial dysfunction in septic shock: part 1. clinical manifestation of cardiovascular dysfunction profound but reversible myocardial depression in patients with septic shock prevention of endotoxin-induced sarcoplasmic reticulum calcium leak improves mitochondrial and myocardial dysfunction tumor necrosis factor-alpha decreases sarcoplasmic reticulum ca2+-atpase expressions via the promoter methylation in cardiomyocytes endotoxin and cytokines induce direct cardiodepressive effects in mammalian cardiomyocytes via induction of nitric oxide synthase patterns of septic shock in man: a detailed study of 56 patients bacteremia due to gram-negative bacilli other than the salmonella: a clinical and therapeutic study right ventricular dysfunction and dilatation, similar to left ventricular changes, characterize the cardiac depression of septic shock in humans microcirculation of the intestinal mucosa tissue perfusion and organ function: ischemia/reperfusion injury sepsis-induced vasoparalysis does not involve the cerebral vasculature: indirect evidence from autoregulation and carbon dioxide reactivity studies cerebral hemodynamics vascular reactivity and metabolism during canine endotoxin shock control of vascular reactivity inducible nitric oxide synthase and vascular reactivity in rat thoracic aorta: effect of aminoguanidine endotoxin induced skeletal muscle contractile dysfunction: contribution of nitric oxide synthases leukocyte activation does not mediate myocardial leukocyte retention during endotoxemia in rabbits endothelial structural integrity is maintained during endotoxic shock in an interleukin-1 type 1 receptor knockout mouse capillary and arteriolar responses to local vasodilators are impaired in a rat model of sepsis vascular reactivity and tissue oxygenation cardiac output and redistribution of organ blood flow in hypermetabolic sepsis the role of the tissue factor pathway in initiation of coagulation structural biology of tissue factor, the initiator of thrombogenesis in vivo cell biology of tissue factor, the principal initiator of blood coagulation cellular origin and procoagulant properties of microparticles in meningococcal sepsis inflammation-associated adamts13 deficiency promotes formation of ultra-large von willebrand factor tissue factor expression by monocytes: regulation and pathophysiological roles microvascular thrombosis and multiple organ dysfunction syndrome treatment effects of drotrecogin alfa (activated) in patients with severe sepsis with or without overt disseminated intravascular coagulation criteria for diagnosis of dic based on the analysis of clinical and laboratory findings in 345 dic patients collected by the research committee on dic in japan towards definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation a multicenter, prospective validation of disseminated intravascular coagulation diagnostic criteria for critically ill patients: comparing current criteria prospective evaluation of three different diagnostic criteria for disseminated intravascular coagulation the hematologic system as a marker of organ dysfunction in sepsis incidence, risk factors, and outcome of severe sepsis and septic shock in adults: a multicenter prospective study in intensive care unit. french icu group for severe sepsis coagulation system and platelets are fully activated in uncomplicated sepsis epidemiology of sepsis acute kidney injury incidence and outcomes of acute kidney injury in intensive care units: a veterans administration study incidence classification and outcomes of acute kidney injury developing a consensus classification system for acute renal failure acute dialysis quality initiative workgroup: acute renal failuredefinition, outcome measures, animal models, fluid therapy and information technology needs the histopathology of septic acute kidney injury: a systematic review glucocorticoids potently block tumor necrosis factor-alpha and lipopolysaccharide-induced apoptotic cell death in bovine glomerular endothelial cells upstream of caspase 3 activation renal blood flow distribution in experimental septic acute renal failure pathophysiology of septic acute kidney injury: what do we really know? angiotensin ii in experimental hyperdynamic sepsis production of tumor necrosis factor by rat mesangial cells in response to bacterial lipopolysaccharide beginning and ending supportive therapy for the kidney (best kidney). acute renal failure in critically ill patients: a multinational multicenter study cooperative antimicrobial therapy of sepstic shock (catss) database research group. acute kidney injury in septic shock: clinical outcomes and impact of duration of hypotension prior to initiation of antimicrobial therapy incidence and outcome of early acute kidney injury in critically ill trauma patients altered gastrointestinal motility in critically ill patients: current understanding of pathophysiology, clinical impact, and diagnostic approach gastrointestinal dysmotility: clinical consequences and management of the critically ill patient dramatic changes of the gut flora immediately after severe and sudden insults gut flora in health and disease epithelial barrier leak in gastrointestinal disease and multiorgan failure mechanism and role of intrinsic regulation of hepatic arterial blood flow: hepatic arterial buffer response hypoxic hepatitis: underlying conditions and risk factors for mortality in cirtically ill patients hypoxic hepatitis: clinical and hemodynamic studies in 142 consecutive cases hypoxic hepatitis hepatic dysfunction during bacterial sepsis liver function in septic shock the use of maximum sofa score to quantify organ dysfunction/failure in intensive care. results of a prospective, multicenter study. working group on sepsis relate problems of the esicm sepsis and cholestasis pathophysiology of increased intestinal permeability in obstructive jaundice the value of bile replacement during external biliary drainage: an analysis of intestinal permeability, integrity, and microflora effect of internal biliary drainage on plasma levels of endotoxin, cytokines, and c-reactive protein in patients with obstructive jaundice sepsis and cholestasis surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock guidelines for the provision and assessment of nutrition support therapy in the adult critically ill patient: society of critical care medicine (sccm) and american society for parenteral and enteral nutrition key: cord-023441-q83y12sk authors: draborg, h.; roggen, e. l.; soni, n. k.; patkar, s.; friis, e. p.; lyngstrand, s. t.; christensen, l. l. h.; batori, v.; danielsen, s.; ernst, s. title: recominant expression and immunological characterization of house dust mite allergen der p 1 date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ag.x sha: doc_id: 23441 cord_uid: q83y12sk the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige‐mediated allergic reactions, while maintaining its ability to trigger proper th‐cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro‐der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro‐enzyme to a fungal signal peptide. the n‐glycosylation site of der p1 was mutated resulting in a deglycosylated pro‐enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active‐site‐titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro‐der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige‐binding assays and t‐cell proliferation assays. by in silico epitope mapping of a modelled 3‐dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-006610-me8rhkcg authors: nör, jacques e.; polverini, peter j. title: role of endothelial cell survival and death signals in angiogenesis date: 1999 journal: angiogenesis doi: 10.1023/a:1009053411094 sha: doc_id: 6610 cord_uid: me8rhkcg angiogenesis, the process of new microvessel development, is encountered in a select number of physiological processes and is central to the pathogenesis of a wide variety of diseases. there is now convincing evidence that regulated patterns of endothelial cell survival and death, a process known as apoptosis, play a central role in the periodic remodeling of the vasculature, and in the timely evolution and regression of angiogenic responses. in this review we discuss the current evidence suggesting a role for inducers and inhibitors of angiogenesis as well as other mediators that modify endothelial cells functions in the survival and death of endothelial cells. we also discuss how dysregulation of apoptosis can lead to aberrant angiogenesis as demonstrated in the pathogenesis of retinopathy of prematurity and cancer. angiogenesis and apoptosis are biological processes that are indispensable for normal organ and tissue homeostasis. programmed cell death, also known as apoptosis, maintains normal tissue and organ homeostasis by removing super¯uous, damaged or senescent cells. mounting evidence suggests that apoptosis is also involved in the homeostasis of the vascular system. recent studies suggest that endothelial cell apoptosis is necessary for repair of damaged blood vessels and for sprouting and branching of capillaries during angiogenesis. events that govern the survival and death of endothelial cells have emerged as major factors that contribute to angiogenic responses during embryonic development, in the maintenance of organ and tissue homeostasis in adult organisms, and in pathological conditions such as tumor development. in this review we will brie¯y summarize the literature on apoptosis and angiogenesis, and examine more closely the current knowledge about how the death machinery contributes to vascular remodeling and angiogenesis. lastly, we will show how disruption of the apoptotic program can contribute to the unrelenting and persistent angiogenesis that is the hallmark of angiogenesis dependent diseases. programmed cell death is necessary for the development and survival of multicellular organisms. every second, hundreds of thousands of cells are generated in the human body and a similar number of them die [1] . the overwhelming majority of these cells die by apoptosis, a morphologically stereotyped series of cellular events that result in the deletion of cells without in¯ammation [2, 3] . the process of programmed cell death was ®rst described in 1842 in the neuronal system of the developing toad embryo [4] . however, it was only in 1972 that the term apoptosis was proposed to de®ne this genetically regulated form of cell death [5] . programmed cell death involves expression of genes and protein synthesis, and is distinguished from necrosis where cell injury and or in¯ammation results in membrane damage and cell lysis. apoptosis is a key component of a number of physiological processes. it accounts for the death of cells necessary for tissue remodeling, for the cell loss that accompanies atrophy of adult tissues following endocrine stimuli, and the removal of cells no longer needed at the late stages of wound healing [1, 2, 6] . apoptosis is the mechanism by which autoreactive lymphocytes are eliminated during development or after termination of immune responses [7] . it also eliminates excessive neutrophils that are produced continuously in the bone marrow [8, 9] . apoptosis also maintains organ and tissue homeostasis by eliminating cells that are potentially destructive. disruption of the function of a cell caused by viral infection generates a signal that induces apoptosis of infected cells in order to eliminate the virus from the organism [10] . perhaps one of the best examples of how apoptosis guards against intrusion by potentially harmful cell populations is the protective role that this process plays in the prevention of cancer. apoptosis is an ecient mechanism designed to recognize and eliminate cells that have acquired genetic lesions that are potentially lethal to the host [7] . in this context, cell death is a physiologically protective event. in summary, apoptosis is a mechanism that regulates the reshaping of tissues and organs, insures the survival of the ®ttest cells and their optimal adaptation to the environment, and eliminates infected, damaged, or cells at risk for neoplastic conversion [1, 7, 11, 12] . at the cellular level, apoptosis is characterized by a predictable, well-choreographed series of morphological and biochemical events. cells undergoing apoptosis shrink in volume, detach their neighbors, lose their microvilli, and detach from one another [2] . the outer membrane bulges (blebs), the chromatin condenses into dense granular caps, and the nuclear membrane eventually breaks down. at this stage, surface convolution takes place and the cell is disassembled into a series of condensed membrane-bound, apoptotic bodies that are phagocytosed by macrophages or by other adjacent cells [2] . the events that initiate the apoptotic program are tightly regulated by an intricate system of controls and checkpoints that are designed to minimize accidental or inappropriate activation of the death cascade. although it is well recognized that apoptosis plays a central role in numerous in physiological and pathological processes, the genetic and biochemical signals that comprise the cell death machinery are still remain to be de®ned. we will begin this review with an overview of what is currently known about the molecular regulation of apoptosis and the intracellular and extracellular signals that initiate this cascade. each cell possesses its own death machinery, which can be activated on demand. our understanding of the molecular events that control apoptosis emerged from studies in the nematode caenorhabditis elegans [13, 14] . during development of c. elegans, 1090 cells are generated of which 131 undergo apoptosis [15] . genetic studies in this worm led to the identi®cation and ordering of key components of the cell death machinery. the apoptotic program is a highly conserved mechanism that is mediated by regulators, adaptors, and executioners (eectors) of apoptosis [1, 14] . figure 1 shows a comparison of intracellular pathways that control apoptosis in nematodes and mammals. in c. elegans, upstream signals promote the binding of egl-1 (regulator) protein to ced-9 (regulator) that, in its turn, releases ced-4 (adapter). unbound ced-4 induces proteolytic cleavage of ced-3 (executioner) and a series of downstream events culminating in cell death [16±19] . in humans, the bcl-2 family of proteins is composed of at least 16 members that are critical regulators of apoptotic pathways and function to either inhibit or to promote cell death [20±22] . these proteins are located within the intracellular membranes of mitochondria, the nucleus, and endoplasmic reticulum and are believed to form ion-channels/pores when these proteins form homo and/or heterodimers [22, 23] . the pro-apoptotic proteins bad and bid (human homologues of the nematode egl-1) regulate the activity of the antiapoptotic bcl-2 and bcl-x l (homologues of ced-9). bad and bid lack membrane anchoring domains and move from cytosol to the surface of membranous organelles, where they can bind to other members of the bcl-2 family [24] . for example, the dimerization of bad with bcl-2 mediates downstream events that result in apoptosis [22] . therefore, this step of the apoptotic pathway is dynamically controlled by the`regulated' translocation of mobile proteins such as bid and bad, and their binding to anchored proteins such as bcl-2 and bcl-x l . none of the known antiapoptotic bcl-2 family members (regulators) interacts directly with caspases figure 1 . mechanisms that control cell survival and death in the nematode c. elegans and in mammals. the current understanding of genetic pathways of apoptosis is described for c. elegans (top) and mammals (bottom). both, the nematode genes and their mammal homologues, are functionally characterized as regulators, adaptors, or executioners of apoptosis. symbols: inhibition (a), activation (!). modi®ed from vaux and korsmeyer [1] . (executioners) [9, 10] . the function of adaptors (also called caspase activators) is necessary to transduce proapoptotic signals from regulators to the caspases [25] . for example, apaf-1 is a human homologue of ced-4 [26] that oligomerizes and activates procaspase-9 when it is not bound to antiapoptotic proteins of the bcl-2 family [27, 28] . therefore, antiapoptotic members of the bcl-2 family may inhibit caspa1se activation by preventing oligomerization of the adapter protein apaf-1 the key executioners of apoptosis are caspases, a family of cysteine proteases that are normally present in the cell as proenzymes that require proteolytic cleavage to become activated [25, 31] . based on their sites of action in the apoptotic pathway, these proteases were divided into upstream caspases (initiator caspases) and downstream caspases (eector caspases) [31] . in mammals, apaf-1 is dissociated from pro-survival bcl-2 family members and activated by datp and cytosolic cytochrome c, a protein that is released from the mitochondria during apoptosis [26, 31, 32] . upon activation, apaf-1 undergoes a conformational change, which enhances its ability to interact with caspase-9 (an initiator caspase) through its caspase recruitment domain (card) [26, 29, 31, 33, 34] . upon this interaction, caspase-9 is processed by autocatalysis and subsequently activates downstream eector caspases such as caspase-3 [25, 33, 35] . when the downstream caspases are activated, they cleave icad (inhibitor of caspase activated dnase), allowing cad (caspase activated dnase) to enter the nucleus and degrade chromosomal dna [36±38] . a number of cytokines and extracellular proteins initiate apoptosis by signaling through speci®c cell membrane receptors [39] . the best characterized death receptors are cd95 (also known as fas) and tnfr1 (also termed p55), that belong to the tumor necrosis factor (tnf) receptor gene superfamily [25, 31, 39] . they have cysteine-rich extracellular domains and a cytoplasmic sequence termed the`death domain' (dd) [40, 41] . binding of the cd95 ligand or tnf to their respective receptors activates fadd (adapter or caspase activator) that engages procaspase-8 molecules in a death-inducing signaling complex (disc) [42] . the aggregation of several procaspase-8 molecules mediates their auto-processing and activation [43, 44] . active caspase-8 signals through caspase-3 [45] and triggers downstream death-inducing events similar to the ones described above. peptides that speci®cally inhibit caspase activity (e.g. zvad-fmk, zdevd-fmk) have been shown to prevent dna fragmentation and enhance cell survival in vitro and in vivo [46±49]. the use of caspase inhibitors for treatment of diseases that are caused by excessive apoptosis is already being evaluated in clinical trials. however, there are still several unanswered questions regarding their ecacy and safety for therapeutic use [50] . therapeutic strategies designed to target the recently discovered caspase-independent pathways of apoptotic death may eventually prove to be a more reliable and safer alternative to the use of caspase inhibitors [51±53]. during the early stages of embryogenesis a primary vascular plexus develops from endothelial cell precursors or angioblasts by a process termed vasculogenesis [54, 55] . subsequently, these cells proliferate and organize into primitive blood vessels establishing the initial vascular network. vascular endothelial growth factor (vegf) and its receptors vegfr-1, vegfr-2 are important molecules in the initial phase of vascular development. knockout mice lacking any one of these genes die before day 10.5 of embryonic development by absence or delayed endothelial cell dierentiation and failure of vasculogenesis [56, 57] . after the primary vascular plexus is formed, endothelial cells start to proliferate and form new capillaries [55] . the emerging vascular plexus is rapidly remodeled to resemble a mature system by a process termed angiogenesis. this term was ®rst used in 1935 to describe the formation of new blood vessels from pre-existing capillaries in the placenta [58] . recent studies have demonstrated the presence of endothelial cell precursors in peripheral blood as well as the ability of these cells to target sites of active angiogenesis [59] . these ®ndings raise the possibility that vasculogenesis and angiogenesis may not be as distinct from one another as once thought. despite their low turnover (measured in years) in adult tissues, endothelial cells still retain the capacity to divide and form new blood vessels in response to speci®c stimuli [60] . capillary blood vessels consist of endothelial cells and pericytes that are programmed to form a complete capillary network when stimulated by mediators of angiogenesis [61] . the process of angiogenesis starts with proteolytic degradation of interstitial tissue and the basement membrane of the parent vessel. this is followed by a series of well orchestrated events in which endothelial cells migrate towards the angiogenic stimulus and divide [62] . endothelial cells elongate and align to form a sprout, and the lumen is formed by a curvature inside each endothelial cell [63] . individual sprouts elongate and eventually join with each other forming loops through which blood begins to¯ow. pericytes originating from migrating and dedierentiation of arterial smooth muscle cells, position themselves along the original sprouts and reposition themselves in the basement membrane that invest newly formed capillaries [64, 65] . the acquisition of a coating with pericytes has been recently described as the end of the`plasticity window' in which the vascular architecture is ®ne tuned according to the availability of oxygen [66] . a tightly controlled balance between cell proliferation and cell death dictates tissue integrity and tissue homeostasis. traditionally, regression of neovascular responses has been associated with inhibition of endothelial cell proliferation, migration, and adhesion [67] . only recently has the role of endothelial cell survival and death signals in sustaining and disrupting neovascularization been recognized. it is now well established that key regulators of angiogenesis function, at least in part, by modulating the survival of endothelial cells during the processes of vessel repair and angiogenesis. for the purposes of discussion we have divided the regulators of angiogenesis in two broad groups according to their role in endothelial cell survival and death: inducers of endothelial cell apoptosis and enhancers of endothelial cell survival (table 1) . many but not all of these mediators we will describe below have been shown to function as either antiangiogenic or proangiogenic factors. when viewed in this context perhaps all mediators of angiogenesis will eventually be found to function as either inducers of endothelial cell death or survival factors. tsp1 is the ®rst member of a family of multifunctional extracellular matrix (ecm) glycoproteins [68±70]. it is intimately associated with matrix surrounding blood vessels where it is able to interact with a variety of growth factors, matrix molecules and cations [71±76]. tsp1 has been implicated in several steps in the angiogenic response [70, 77±79] . it potently inhibits endothelial cell proliferation [80±83], migration [82, 84] and induces vascular disassembly. endothelial cell sprouting is enhanced by exposure to anti-tsp1 antibodies [76, 85] , and endothelial cells transduced with antisense tsp1 [82] exhibit an enhanced ability to form sprout-like structure on arti®cial matrices in vitro. the potent anti-angiogenic activity of tsp1 has been localized to two domains within the central stock region of the molecule: the procollagen homology region and the properdin-like type i repeats [72, 86±88] . the antiangiogenic eect of tsp1 involves signalling through the endothelial cell membrane receptor cd36 [89] . recent data suggest that the antiangiogenic eect of tsp1 is mediated in part by its ability to induce endothelial cell apoptosis. tsp1 as well as peptide fragments derived from its antiangiogenic domains have been shown to induce apoptosis of human umbilical vein endothelial cells (huvecs) in vitro [90] . angiostatin a 38 kda internal fragment of plasminogen that contains the ®rst four disul®de-linked kringle structures was puri®ed from plasma of mice bearing a lewis lung carcinoma, sequenced and named angiostatin [91±93]. it was characterized as a speci®c inhibitor of endothelial cell proliferation in vitro and suppressor of tumor growth and metastatic dissemination in vivo [91, 92, 94] . angiostatin can be generated by the hydrolysis of circulating plasminogen by a macrophage-derived metalloelastase [95] , matrilysin and gelatinase b [96] , or stromelysin [97] . the antiangiogenic function of angiostatin is believed to be mediated, at least in part, by its ability to induce apoptotic death of endothelial cells [98, 99] . treatment of endothelial cells with angiostatin resulted in decreased cell numbers without signi®cant eects on dna synthesis, suggesting that the decreased proliferation rates observed in cells exposed to angiostatin were due to enhanced endothelial cell apoptosis [98, 99] . the domains responsible for angiostatin pro-apoptotic function appear to reside in kringle domains 1, 2 and 3 [99] . tumor necrosis factor (tnf) is a multifunctional cytokine secreted by activated macrophages [100±102] and t cells [103] . tnf is an important mediator of in¯ammatory processes and immune responses, where it was shown to induce tumor regression by hemorrhagic necrosis [101, 104] . tnf's function in tumor serum albumin accutin extracellular atp alkyllyso-phospholipid et16-ome 2-methoxyestradiol regression has been attributed to its cytotoxic eect on endothelial cells that results in disruption of the tumor capillary bed [105] . several studies have shown that binding of tnf to endothelial cells results in their apoptotic death [105±108]. the ability of tnf to induce endothelial cell apoptosis is enhanced by concomitant exposure to inhibitors of protein synthesis [107±109]. this suggested that tnf might also protect endothelial cells from apoptosis by utilizing a pathway(s) that is dependent on protein synthesis [110] . in fact, tnf was shown to induce expression of the antiapoptotic protein a1 (a bcl-2 homologue) and where overexpression of a1 was shown to inhibit apoptosis when endothelial cells were exposed to tnf and actinomycin d [110] . the recent ®nding that overexpression of bcl-2 further supports this tnf associated protective mechanism. bcl-x l also protects endothelial cells from tnf-mediated apoptosis after sensitization with cyclohexamide [111] . tnf seems to mediate a multitude of intracellular signal transduction pathways that are triggered upon its binding to endothelial cell membrane receptors. these include activation of: (a) nf-jb, which mediates expression of il-8, vegf, and e-selectin in endothelial cells [112±115]; (b) protein kinase c, which leads to the upregulation of a1 expression and subsequent enhancement of cell survival [110] ; (c) sp1, which mediates expression of vegfr-2 [116] ; and (d) ceramides and c-jun, which result in endothelial cell death [110, 117] . despite extensive research, the eects of tnf in endothelial cells are still unclear. while some reports demonstrate that tnf induces endothelial cell apoptosis, other investigations have shown that tnf is angiogenic [115, 118, 119] . there are a number of possible explanations for these con¯icting results. (a) tnf's ability to induce angiogenesis in vivo may be due to the synthesis of angiogenic factors such as il-8, vegf or bfgf or macrophages [115, 119] . (b) responses mediated by tnf are dose-dependent. while tnf stimulates angiogenesis at low concentrations [118] , it inhibits it in higher concentrations [120] . this suggests that the induction of endothelial cell death or survival depends on the expression levels of tnf that accumulate extracellularly, which in turn determines whether it transmits a death signal or survival signal to endothelial cells. transforming growth factor-b (tgf-b) tgf-b has been shown to induce apoptosis of huvec in vitro by downregulating bcl-2 expression [121] . choi and ballerman have demonstrated that tgf-b induces apoptosis of renal glomerular endothelial cells and capillary sprouting in vitro [122] . the authors found that endothelial cell sprouting is inhibited in dominant negative mutants if tgf-b type ii receptors are blocked. they concluded that tgf-b-induced endothelial cell apoptosis is necessary for capillary morphogenesis [122] . escherichia coli endotoxins (lps) cause acute pulmonary endothelial cell injury in vivo [123] and endothelial cell apoptosis in vitro [124] . recently, a study has associated lps with the pathogenesis of endotoxic shock syndrome that is characterized by generalized in¯ammation, circulatory collapse and death [125] . co-injection of lps with its putative eector (tnf-a) induces endothelial cell apoptosis that is associated with enhanced expression of the pro-apoptotic lipid ceramide [125] . the ability of lps to induce apoptosis of cultured sheep pulmonary artery endothelial cells was attenuated by collagen [124] and by overexpression of the heat shock protein-70 (hsp-70) [126] . vitamin c and e have also been shown to prevent lps-mediated apoptosis in huvec [127] . the protective eect of these vitamins was associated with enhanced expression of the antiapoptotic protein bcl-2 and decreased expression of the pro-apoptotic protein bax [127] . several other inducers of endothelial cell apoptosis have been described in the literature. interferon-c induces apoptosis of normal endothelial cells through activation of the protein kinase c pathway [128] . amyloid b-peptide, a molecule involved in neuronal degeneration observed in the brain of patients with alzheimer's disease, has been implicated in the induction of endothelial cell apoptosis [129] . it was suggested that amyloid b-peptide-induced endothelial cell apoptosis might be directly involved with the vascular damage frequently observed in these patients [129] . cholesterol oxides are molecules involved in the initiation and progression of atherosclerosis [130] . physiological concentrations of cholesterol oxides induce apoptosis of endothelial cells and cause damage to the vessel wall [131] . this observation might explain, at least in part, the role of cholesterol oxides in the etiology of vascular injury in vivo [132] . accutin, a rgd containing small peptide from the disintegrin family, was recently puri®ed from the viper venom of agkistrodon acutus [133] . in vitro, accutin induces apoptosis of endothelial cells by inhibiting their adhesion to ®brinogen, ®bronectin, or vitronectin [133] . furthermore, accutin is anti-angiogenic when evaluated in the chick chorioallantoic membrane (cam) assay. it was suggested that the mechanisms responsible for this anti-angiogenic eect involve selective blockade of the endothelial cell integrin a v b 3 and consequent induction of apoptosis [133] . endothelial cell injury is a component of the`increased pulmonary edema' that manifests in patients with acute respiratory distress syndrome (ards) [134] . dawicki and collaborators showed that extracellular atp and adenosine induce apoptosis of pulmonary artery endothelial cells [135] . they speculate that atp released from activated platelets and cells undergoing cytolysis in ards patients causes apoptosis of lung endothelium, and thereby exacerbates pulmonary injury [135] . pharmacological agents can also induce endothelial cell apoptosis. alkyllyso-phospholipid et16-ome, a putative anti-tumor drug, induces apoptosis when added to the culture medium of human umbilical vein endothelial cells [136] . moreover, 2-methoxyestradiol, an endogenous estrogen metabolite of the oral contraceptive 17-ethylestradiol, induces apoptosis of bovine pulmonary artery endothelial cells in vitro and inhibits angiogenesis in vivo [137] . lastly, an important role for accessory cells and changes in blood¯ow in endothelial cell apoptosis and capillary regression has been proposed in a series of studies by richard lang and colleagues [138±142] . during regression of the pupillary membrane, a transient capillary network found in the anterior chamber of the developing rodent eye, macrophages initiate apoptosis of endothelial cells which leads to capillary regression. this selective ablation of endothelial cells results in vessel obstruction and a block in plasma¯ow within the clogged capillary segment. endothelial cells then die in a`synchronous' manner because they are deprived of essential survival factors present in plasma. recently this group has determined that the major survival factor in plasma that is responsible for maintaining the integrity of this embryonic organ is vegf. the signi®cance of vegf as a survival factor will be discussed in greater detail below. in 1983, vascular permeability factor (vpf) was iden-ti®ed in tumors and characterized as an endothelial factor that enhances the permeability of microvessels to circulating molecules [138] . in 1989, two independent groups reported the cloning and sequencing of an endothelial cell speci®c mitogen with heparin binding properties that was called vascular endothelial growth factor (vegf) [143±145]. further sequencing and characterization led to the conclusion that vpf and vegf shared the same biological functions and were encoded by the same gene. alternative splicing of vegf mrna originates ®ve human vegf isoforms, of which vegf 165 is the predominant molecular species [147, 148] . vegf has been extensively characterized as a potent permeability factor [150] , endothelial cell speci®c mitogen and chemoattractant [143, 151] an angiogenic factor [148, 149] , and a mediator of adhesion of natural killer cells to tumor endothelium by inducing expression of vcam-1 and icam-1 [152] . more recently, the role of vegf as an enhancer of endothelial cell survival has been investigated. vegf was shown to protect endothelial cells from apoptosis induced by tnf by inducing upregulation of b3 integrin and ®bronectin [153] . it was proposed that the sustained endothelial cell survival observed in cells exposed to vegf was mediated by their enhanced adhesion to matrix [153, 154] . at approximately the same time, another group of investigators determined that vegf enhanced the survival of microvascular endothelial cells cultured in hydrophobic polystyrene [155] . interestingly, the mechanism suggested for vegf-induced endothelial survival in this experimental design was dependent on vitronectin and a 5 b 5 integrin, not a 5 b 3 . the ability to enhance endothelial cell survival was speci®c to vegf, since other angiogenic factors such as bfgf were tested and did not exhibit the same eect [155] . the mechanisms underlying vegf's survival function are starting to be unveiled. vegf was shown to upregulate expression of the antiapoptotic protein bcl-2 and its homologue a1 in endothelial cells in vitro [156, 157] . overexpression of bcl-2 was sucient to enhance endothelial cell survival and protect against apoptosis induced by growth factor deprivation [157, 158] . vegf-mediated bcl-2 upregulation in endothelial cells unequivocally potentiates angiogenic responses in vitro and in vivo [157] . vegf's survival signal was shown to be mediated by the flk-1/kdr receptor and engagement of the phosphatidylinositol 3 0 -kinase/akt transduction pathway [159] . another group has characterized vegf's survival function for endothelial cells cultured in collagen as dependent on the activation of the mitogen activated protein kinase [mapk], rather than the akt/pkb, signaling pathway [160] . one of the most potent stimuli for vegf secretion and angiogenesis is oxygen deprivation. hypoxia is associated with a variety of responses at the cellular and tissue level. reduced levels of oxygen induce glycolysis to enhance energy production. it also enhances erythropoietin synthesis to increase the oxygen carrying capacity of the blood, and vegf secretion which enhances tissue oxygenation by increasing vessel permeability and promoting neovascularization. an important component of the transcriptional response to hypoxia is the transcription factor hif-1. this hypoxia-inducible transcription factor functions by controlling the expression of a series of target genes that regulate tissue oxygenation in a number of physiological and pathological settings. recent work from several laboratories has established a key role for hif-1 in the hypoxic response during embryonic development and angiogenesis [161±163]. for example teratocarcinomas derived from embryonic stem cells null for hif-1a exhibited a dramatic reduction in growth and vascularization [161±163] . this ®nding correlated with a reduced capacity of these cells to release vegf. furthermore it was shown that hif-1a null mutant embryos exhibited a complete lack of cephalic vascularization, reduced numbers of somites and abnormal neural fold formation. carmeliet and collaborators have reported that embryonic stem cells null for hif-1a showed reduced hypoxia-induced expression of vegf. this was associated with a reduction in the formation of large, mature vessels and impaired vascular function [163] . the consequences of aberrant expression of hifa have been revealed in several recent reports that examined the molecular basis of unregulated angiogenesis in the hereditary cancer syndrome, the von hippel-lindau (vhl) disease [164±166] . individuals aected by this disorder develop hemangioblastomas in the retina, cerebellum and spine as well as the adrenals and kidney. interestingly, individuals with this disease exhibit a molecular and biochemical phenotype reminiscent of oxygen deprivation. it was recently reported [164] that the vhl gene binds to the transcription factors hif-1a and hif-2a where it targets them for destruction. cells lacking the vhl gene cannot degrade these two transcription factors. this in turn drives excessive vegf synthesis and angiogenesis. although a number of questions remain, these recent observations suggest that vegf may utilize a number of dierent molecular pathways to enhance the survival of endothelial cells. the fgf family consists of nine structurally related polypeptides. bfgf was originally puri®ed from the bovine pituitary gland [167] , sequenced, and characterized as an angiogenic factor [168] . araki and colleagues in 1990 [169] were the ®rst to implicate fgf as a survival factor for endothelial cells. enhanced activity of protein kinase c was associated with the ability of bfgf to protect endothelial cells against apoptosis induced by growth factor deprivation [170] or ionizing radiation in vitro and in vivo [170±172]. in contrast, tyrosine phosphorylation, but not protein kinase c activation, was shown to mediate bfgf's protective eect [173] . the role of bfgf in endothelial cell survival was further characterized by the ®nding that its removal from culture medium of murine aortic endothelial cells was sucient to activate the pro-apoptotic cysteine protease interleukin-1b-converting enzyme (ice) and mediate dna fragmentation [174] . more, recently, the anti-apoptotic function of bfgf was associated with its ability to enhance expression of bcl-2 [175] . endothelial cells rapidly undergo apoptosis when their interactions with the extracellular matrix are inhibited, in a process called`anoikis' [154] . integrins were identi®ed as key transducers of extracellular matrix signals that were required for maintaining cell survival [154] . there is increasing evidence that integrins also play a critical role in the regulation of angiogenesis by modulating endothelial cell survival [176±180] . the ®nding that a v b 3 is preferentially expressed in newly formed microvessels and that monoclonal antibodies to a v b 3 induce endothelial cell apoptosis restricted to these vessels was immediately considered a major breakthrough as a potential anti-angiogenic tumor therapy [177±180] . the authors hypothesized that if a v b 3 ligation is prevented; the endothelial cells will no longer receive necessary survival signals from the ecm and undergo apoptosis by default. the signaling pathways that mediate endothelial cell survival upon activation of a v b 3 have been extensively studied. ligation of endothelial cell a v b 3 during angiogenesis promotes a speci®c signal that leads to inhibition of p53 expression and its inducible partner p21 waf1 (mediator of cell cycle arrest), and suppression of the pro-apoptotic bax pathway [181] . the activation of the transcription factor nf-jb was shown to be dependent on the gtp-binding protein ras and the tyrosine kinase src, and is necessary for a v b 3 -mediated endothelial cell survival [182] . another study has identi®ed the activation of the phosphatidylcholine-speci®c phospholipase c and production of diacylglycerol (dag) as essential components of integrin mediated survival signals in endothelial cells [183] . exposure of human endothelial cells to gamma interferon (ifn-gamma) was shown to inhibit the a v b 3 -mediated survival pathway and to induce endothelial cell apoptosis in vitro [184] . treatment of patients with ifn-gamma also resulted in enhanced endothelial cell apoptosis in metastatic melanoma [184] . inactivation of the integrin pathway during endothelial cell apoptosis may be due to cleavage of its cytoplasmic domain. calpain-mediated proteolysis of the b 3 cytoplasmic domain was observed during apoptosis of human umbilical vein endothelial cells, and its inhibition with sodium orthovanadate (a phosphatase inhibitor) rescued these cells from apoptosis [180] . nitric oxide is a multifunctional molecule that is synthesized by endothelial cells in low doses through the activity of the enzyme nitric oxide synthase (enos) [185, 186] . inhibition of no was associated with enhanced endothelial cell apoptosis in con¯uent cultures of bovine aortic endothelial cells [187] . the authors suggested that no has an important role in maintaining vascular homeostasis and architecture [186] . enhanced endothelial cell apoptosis has been associated with the pathogenesis of atherosclerosis [188, 189] . the incidence of coronary disease in post-menopausal women increases concomitantly with a decrease in the synthesis of estrogens, and the administration of this hormone is being considered one of the most eective anti-atherogenic therapies available for women [190] . estradiol, a key estrogen metabolite, has been now characterized as a potent anti-apoptotic mediator for endothelial cells. estradiol protected endothelial cells against apoptosis induced by tnf-a [192] . it was also shown that estradiol's anti-apoptotic function was mediated by increased tyrosine phosphorylation of a focal adhesion kinase that stabilized focal adhesion contacts [189] . adrenomedullin, a potent vasorelaxant/hypotensive peptide, was shown to suppress serum deprivationinduced apoptosis of rat endothelial cells via a camp-independent mechanism [192] . human umbilical vein and microvascular endothelial cell apoptosis were inhibited by physiological concentrations of serum albumin [193] . the authors suggested that the removal of excessive blood vessels in remodeling tissues might be mediated by a reduced supply of serum albumin to the endothelial cells. lastly, the inhibitor of apoptosis protein family (iaps) has received considerable attention over the past 5 years [194] . initially discovered in baculoviruses they appear to be highly conserved across several species including humans [195, 196] . the iaps appear to suppress apoptosis through direct caspase inhibition (primarily caspase 3 and 7) and by modulation of the transcription factor nf-jb [197] . although a role for the iaps in endothelial survival and angiogenesis has yet to be established, given the wide spread distribution of these proteins, it would not be entirely surprising to ®nd that they play a role in endothelial cell survival and angiogenesis. in response to angiogenic stimuli, endothelial cells undergo a series of tightly controlled events that result in sprouting and development of a capillary network and the remodeling of established vessels. when newly formed blood vessels are no longer necessary, they undergo regression. figure 2 depicts the process of programmed cell in both angiogenesis and angiosuppression. traditionally, the initiation of angiogenesis has been described as involving degradation of basement membrane followed by proliferation and migration of endothelial cells to form a capillary loop. in 1995, choy and ballermann [122] raised the intriguing possibility that capillary morphogenesis depended on the selective apoptosis of endothelial cells mediated by transforming growth factor-b1 (tgf-b1). this homodymeric polypeptide is strongly expressed in sites of tissue morphogenesis [198] . it has been shown to induce angiogenesis in vivo [199] and endothelial cell sprouting in vitro [200] . during the process of capillary morphogenesis, tgf-b1 mediates secretion of plasminogen activator that cleaves the proenzyme plasminogen and activates plasmin, which in turn degrades ecm proteins [122] . this results in detachment and apoptosis of endothelial cells from selective areas of the developing capillary, and allows for the initiation of a new capillary loop [122, 201] . targeted apoptosis of endothelial cells seem to have an important physiological role in allowing for the communication between the newly formed capillary and their`parent' venules. the role of endothelial cell proliferation and migration in the pathogenesis of angiogenesis-dependent diseases and the mediators responsible for aberrant angiogenic responses have been extensively characterized. more recently researchers have turned their attention to characterizing the impact of endothelial survival and death signals in pathological angiogenesis. the accumulated evidence to date suggests that disruption in the cell death machinery is central to the pathogenesis of a number of angiogenesis-dependent diseases (figure 3 ). two excellent examples of this phenomenon are retinopathy of prematurity, that is caused by excessive endothelial cell apoptosis in its initial stages [202] ; and (b) cancer, that is associated with an unrelenting angiogenic response [203] . these two diseases were selected for discussion here because there is a considerable body of literature about their pathogenesis. however, it is anticipated that in time new data will emerge linking changes in the survival pro®le of endothelial cells to other angiogenesis dependent diseases. with increasing survival of premature infants weighing less than 1000 g, the incidence of retinopathy of prematurity-induced blindness has also increased steadily [204] . the pathogenesis of retinopathy of prematurity has been directly associated to the fact that premature infants are placed in oxygen chambers and exposed to a hyperoxic environment to provide enough oxygen for their immature lungs [205] . the exposure of the immature retina to hyperoxia results in excessive apoptosis of endothelial cells [67, 206, 208] . when the lungs of the infant mature, the transition to room air causes excessive retinal neovascularization that is mediated by the relative ischemia in the retina resulting from the initial figure 3 . the balance expression of endothelial survival and death signals characterizes physiological angiogenesis. (a) during the initiation phase of a physiological angiogenic response there is an increase in the level of proangiogenic factors coincidental with a reduction in the level of angiogenesis inhibitors. this temporary shift to the angiogenic phenotype is accompanied by a transient upregulation in survival signals that is induced by proangiogenic factors. also, the number of endothelial cells undergoing apoptosis is reduced due to the diminished level of angiogenesis inhibitors and/or increased resistance of endothelial cells to the pro-apoptotic eects of angiogenesis inhibitors. once the metabolic demands of the tissue have been met, i.e., during the reparative phase of tissue injury, the level of proangiogenic mediators in the tissue decreases while the level of angiogenesis inhibitors begin to rise. this results in increased endothelial cell apoptosis, and the rapid regression of neovessels. (b) in angiogenesis dependent diseases the angiogenic phenotype is prolonged resulting in a protracted angiogenic response. the reduction in the level of angiogenic inhibitors along with a relative or absolute increase in the level of proangiogenic factors results in prolonged upregulation of endothelial cell survival signals and a marked decrease in endothelial cells undergoing apoptosis. we propose that prolonged upregulation of survival signals and/or a reducing in death signals contributes to sustained neovascularization that characterizes angiogenesis dependent diseases. disruption of blood supply [206, 208±210] . the resulting aberrant neovascularization is the cause of the severe ocular disease observed frequently in these infants, including blindness [211] . the pathogenesis of retinopathy of prematurity has been extensively studied at the molecular level, and vegf is believed to play a major role in this process. it is known that vegf is very responsive to subtle changes in oxygen tension. for example, its expression levels are increased in hypoxic areas and result in local induction of neovascularization that re-establishes normal oxygen supply [145, 212, 213] . recent reports demonstrated that vegf expression is downregulated soon after the level of oxygen rises in the retina [206, 207, 210, 212] , and precedes blood vessel regression caused by endothelial cell apoptosis [193] . when the child starts breathing room air, the retina becomes hypoxic because most blood vessels have been previously disrupted. in attempt to re-establish normal oxygen tension to the area, vegf is upregulated above physiological levels and causes excessive neovascularization that is responsible for the ocular pathology [206, 207, 210, 212] . this knowledge provides a better rationale for administrating exogenous vegf in infants prior to exposing them to the hyperoxic environment of oxygen chambers in an attempt to prevent endothelial cell apoptosis and maintain the retinal vasculature [66, 206, 210] . this clinical setting underlines the important function of vegf as an angiogenic mediator and endothelial cell survival factor. it also demonstrates that this growth factor provides a signal that is necessary to sustain endothelial cell survival in vivo and that its premature downregulation results in the disruption of established as well as newly formed microvessels. judah folkman and colleagues ®rst proposed the hypothesis that`solid tumors are angiogenesis-dependent' in 1971 [214] . since then the validity of this statement has been widely con®rmed [214] . the observation that tumors grow as`cylinders' and enter into dormancy unless they acquire a new blood supply strengthened the concept that tumors were angiogenesis dependent [215, 216] . a recent study con®rmed this hypothesis by demonstrating the existence of an inverse relation between spontaneous apoptosis of tumor cells and intratumoral microvessel density [217] . the intense competition for limited oxygen and nutrient supplies, as well as for physical space inside the mass of a solid tumor, generates a hostile microenvironment for normal and neoplastic cells. however, it is clear that tumor cells have developed mechanisms for enhancing their survival and enabeling them to grow in nutrient deprived environments. a question arises with regards to normal endothelial cells that populate tumor-associated blood vessels. how do they survive in this hostile environment and sustain tumor neovascularization? tumor-associated endothelial cells receive a continuous input of survival signals from the ecm and depend on these signals to remain viable and functional. these conclusions are supported by the ®ndings obtained in two elegant experiments from eli keshet's laboratory. vegf expression was`shut down' with an inducible vegf expression system (`tet-o ') in xenografted glioma tumors in nude mice. the authors observed that upon vegf withdrawal endothelial cells became apoptotic, tumor neovascularization decreased, and extensive tumor necrosis took place [202] . in a second model system, these investigators demonstrated that castration of scid mice bearing an androgendependent tumor resulted in decreased intratumoral expression of vegf and tumor regression [218] . the observation that endothelial cells began to undergo apoptosis before neoplastic cells con®rmed the hypothesis that tumor-associated endothelial cells require speci®c survival signals mediated by vegf to remain viable. these ®ndings reported above suggest that tumor regression mediated by withdrawal of vegf is not due to inhibition of endothelial cell proliferation. the turnover of tumor-associated endothelial cells is thought to occur over several days or weeks. however, in this study the tumors started to regress after 24 h. therefore, the lack of endothelial cell proliferation and migration cannot be the only mechanism responsible for the vascular regression observed in these tumors. an alternative hypothesis is that vegf is required for maintaining endothelial cells survival and to sustain tumor angiogenesis. when this positive`survival' signal is eliminated endothelial cells become more responsive to inhibitors of angiogenesis leading to endothelial cell apoptosis, vessel disassembly, and tumor regression ( figure 4 ). angiogenesis is absolutely necessary for embryonic morphogenesis and for maintaining tissue and organ homeostasis in adult organisms. disruption of this biological process has been unequivocally associated with several diseases that are now described as being angiogenesis-dependent. traditionally, angiogenic mediators have been categorized as promoting endothelial cell proliferation, migration, and adhesion. there is now compelling evidence that angiogenesis is modulated by a tightly controlled series of cellular and biochemical events that determine whether endothelial cells survive or die. the events that govern the survival and death of endothelial cells signi®cantly in¯uence the stability and duration of an angiogenic response. after all, angiogenesis would be of little bene®t to developing organisms or adult tissues undergoing repair if endothelial cells did not survive and eventually die in a predetermined manner. similarly, in pathological settings such as in neoplasia, tumor cells would be unable to withstand the onslaught of nutrient deprivation and physiological signals designed to cause their demise if endothelial cells were not able to organize into a microvascular network. thus as we look to develop novel strategies designed to retard pathological angiogenic responses or enhance physiological angiogenesis, we will have to consider factors that in¯uence endothelial cell survival as an integral component of any therapeutic strategy. . this diagram depicts how aberrant expression of endothelial survival and death signals might contribute to the unrelenting angiogenesis that is a hallmark of tumor development. in (a) vascular remodeling is a feature of normal microvascular homeostasis. in this setting the levels of inducers and inhibitors of angiogenesis are in balance. the proportion of long-lived endothelial cells populating established vessels is balanced by those endothelial cells undergoing periodic, selective apoptosis. with the emergence of neoplastic cell populations (b) the level of inducers begin to rise in association with a reduction in the level of inhibitors of angiogenesis. this is re¯ected in a greater number of longlived endothelial cells and fewer endothelial cells undergoing apoptosis. lastly in (c) as tumor neovascularization is accelerated coincidental with an increase in the tumor mass, the level of stimulators of angiogenesis that enhance endothelial cell survival greatly exceed the level of inhibitors and thus death signals. as a consequence death signals are minimized and long-lived endothelial cells accumulate in even greater numbers. we predict this would result in a more sustained and stable vascular supply to the tumor and contribute to tumor growth and progression. cell death in development apoptosis and the regulation of cell numbers in normal and neoplastic tissues: an overview apoptotic pathways: the roads to ruin untersuchungen uè ber die entwicklungsgeschichte der geburtsshelferkroete (alytes obstertricians) apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics the role of apoptosis in wound healing apoptosis in the pathogenesis and treatment of disease analysis of hematopoiesis in max 41 transgenic mice that exhibit sustained elevations of blood granulocytes and monocytes cell suicide for beginners conversion of lytic to persistent alphavirus infection by the bcl-2 cellular oncogene the origin of programmed cell death mechanisms and genes of cellular suicide mechanisms and functions of cell death genetics of programmed cell death in c. elegans past, present and future post-embryonic cell lineages of the nematode, caenorhabditis elegans nunä ez g. interaction and regulation of subcellular localization of ced-4 by ced-9 interaction of ced-4 with ced-3 and ced-9 ± a molecular framework for cell death interaction between the c. elegans cell death regulators ced-9 and ced-4 nunä ez g. caenorhabditis elegans egl-1 disrupts the interaction of ced-9 with ced-4 and promotes ced-3 activation bcl-2 and the regulation of programmed cell death bcl-2 family: regulators of cell death bcl-2 family proteins double identity for proteins of the bcl-2 family bid: a novel bh3 domainonly death agonist caspases: enemies within apaf-1, a human protein homologous to c. elegans ced-4, participates in cytochrome c-dependent activation of caspase-3 caspase-9, bcl-xl, and apaf-1 form a ternary complex bcl-x l interacts with apaf-1 and inhibits apaf-1-dependent caspase-9 activation nunä ez g. wd-40 repeat region regulates apaf-1 self-association and procaspase-9 activation autoactivation of procaspase-9 by apaf-1-mediated oligomerization caspases: the proteases of the apoptotic pathway mitochondria and apoptosis cytochrome c and datpdependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade regulation of cell death protease caspase-9 by phosphorylation multiple species of cpp32 and mch2 are the major active caspases present in apoptotic cells a caspase-activated dnase that degrades dna during apoptosis, and its inhibitor icad cleavage of cad inhibitor in cad activation and dna degradation during apoptosis molecular cloning and characterization of human caspase-activated dnase death receptors: signaling and modulation the tnf receptor superfamily of cellular and viral proteins: activation, costimulation, and death a novel domain within the 55 kd tnf receptor signals cell death flice is activated by association with the cd95 death-inducing signaling complex (disc) autoproteolytic activation of pro-caspases by oligomerization synthetic activation of caspases: arti®cial death switches pro-caspase-3 is a major physiologic target of caspase-8 flice, a novel fadd-homologous ice/ced-3-like protease, is recruited to the cd95 (fas/apo-1) death±inducing signaling complex bcl-x(l) can inhibit apoptosis in cells that have undergone fas-induced protease activation a caspase inhibitor fully protects rats against lethal normothermic liver ischemia by inhibition of liver apoptosis caspase inhibition reduces apoptosis and increases survival of nigral transplants caspases: key mediators of apoptosis the bcl-2 protein family: arbiters of cell survival defects in the ubiquitin pathway induce caspase-independent apoptosis blocked by bcl-2 enforced dimerization of bax results in its translocation, mitochondrial dysfunction and apoptosis mechanisms of angiogenesis heterozygous embryonic lethality induced by targeted inactivation of the vegf gene role of thē t-1 tyrosine kinase in regulating the assembly of vascular endothelium isolation of putative progenitor endothelial cells for angiogenesis progress in applied microcirculation increased capillary endothelial cell protease activity in response to angiogenic stimuli in vitro angiogenesis in vitro rat aortic smooth muscle cells become perycites during angiogenesis in vitro what is the role of endothelial cells in angiogenesis? a plasticity window for blood vessel remodelling is de®ned by pericyte coverage of the preformed endothelial network and is regulated by pdgf-b and vegf the pathophysiology of angiogenesis the structural and functional properties of thrombospondin the structure of endothelial cell thrombospondin. characterization of the heparin-binding domains diversity of function is inherited in matricellular proteins: an appraisal of thrombospondin-1 a tumor suppressordependent inhibitor of angiogenesis is immunologically and functionally indistinguishable from a fragment of thrombospondin peptides derived from two separate domains of the matrix protein thrombospondin-1 have anti-angiogenic activity thrombospondin 1 synthesis and function in wound repair unique distribution of the extracellular matrix component thrombospondin in the developing mouse embryo thrombospondin-1, an inhibitor of angiogenesis, is regulated by progesterone in the human endometrium thrombospondin exerts an antiangiogenic eect on cord formation by endothelial cells in vitro the functions of thrombospondin and its involvement in physiology and pathophysiology thrombospondin-1 speci®c inhibition of endothelial cell proliferation by thrombospondin platelet thrombospondin modulates endothelial cell adhesion, motility, and growth: a potential angiogenesis regulatory factor downregulation of endothelial cell thrombospondin 1 enhances in vitro angiogenesis endothelial cell mitogenesis induced by lpa: inhibition by thrombospondin-1 and thrombospondin-2 recombinant truncated thrombospondin-1 monomer modulates endothelial cell plasminogen activator inhibitor 1 accumulation and proliferation in vitro evidence that tenascin and thrombospondin-1 modulate sprouting of endothelial cells modulation of endothelial cell proliferation, adhesion, and motility by recombinant heparinbinding domain and synthetic peptides from the type i repeats of thrombospondin expression and analysis of cooh-terminal deletions of the human thrombospondin molecule thrombospondin-1 suppresses tumorigenesis and angiogenesis in serum-and anchorageindependent nih 3t3 cells cd36 mediates the in vitro inhibitory eects of thrombospondin-1 on endothelial cells thrombospondin 1 and type i repeat peptides of thrombospondin 1 speci®cally induce apoptosis of endothelial cells angiostatin: a circulating endothelial cell inhibitor that suppresses angiogenesis and tumor growth angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a lewis lung carcinoma angiostatin: an endogenous inhibitor of angiogenesis and of tumor growth kringle domains of human angiostatin. characterization of the anti-proliferative activity on endothelial cells macrophage-derived metalloelastase is responsible for the generation of angiostatin in lewis lung carcinoma angiostatin-converting enzyme activities of human matrilysin (mmp-7) and gelatinase b/type iv collagenase (mmp-9) generation of an angiostatin-like fragment from plasminogen by stromelysin-1 (mmp-3) angiostatin induces endothelial cell apoptosis and activation of focal adhesion kinase independently of the integrin-binding motif rgd multiple forms of angiostatin induce apoptosis in endothelial cells tumour-necrosis factor from the rabbit. ii. production by monocytes tumor necrosis factor, other cytokines and disease macrophages induce apoptosis in normal cells in vivo simultaneous production of tumor necrosis factor-alpha and lymphotoxin by normal t cells after induction with il-2 and anti-t3 tumor necrosis factor: an updated review of its biology tumor necrosis factor induces apoptosis (programmed cell death) in normal endothelial cells in vitro alveolar epithelial cells regulate the induction of endothelial cell apoptosis induction of endothelial cell apoptosis by tnf alpha: modulation by inhibitors of protein synthesis activation and injury of endothelial cells by cytokines inhibition of tumor necrosis factor signal transduction in endothelial cells by dimethylaminopurine endothelial cell death induced by tumor necrosis factor-alpha is inhibited by the bcl-2 family member, a1 bcl-2 and bcl-xl serve an anti-in¯ammatory function in endothelial cells through inhibition of nf-kappab nf-kappa b and i kappa b alpha: an inducible regulatory system in endothelial activation transcriptional regulation of endothelial cell adhesion molecules: nf-kappa b and cytokine-inducible enhancers h 2 o 2 and tumor necrosis factor-alpha induce dierential binding of the redox-responsive transcription factors ap-1 and nf-kappab to the interleukin-8 promoter in endothelial and epithelial cells involvement of interleukin-8, vascular endothelial growth factor, and basic ®broblast growth factor in tumor necrosis factor alpha-dependent angiogenesis tumor necrosis factoralpha regulates expression of vascular endothelial growth factor receptor-2 and of its co-receptor neuropilin-1 in human vascular endothelial cells programmed cell death induced by ceramide macrophageinduced angiogenesis is mediated by tumour necrosis factoralpha tumor necrosis factor type alpha, a potent inhibitor of endothelial cell growth in vitro, is angiogenic in vivo induction of vascular endothelial growth factor by tumor necrosis factor alpha in human glioma cells. possible roles of sp-1 transforming growth factor beta 1 induces apoptotic cell death in cultured human umbilical vein endothelial cells with down-regulated expression of bcl-2 inhibition of capillary morphogenesis and associated apoptosis by dominant negative mutant transforming growth factor-b receptors endotoxin and lung injury collagen is a survival factor against lps-induced apoptosis in cultured sheep pulmonary artery endothelial cells lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation the heat-shock response attenuates lipopolysaccharide-mediated apoptosis in cultured sheep pulmonary artery endothelial cells vitamin c and e prevent lipopolysaccharide-induced apoptosis in human endothelial cells by modulation of bcl-2 and bax the dierential response to interferon gamma by normal and transformed endothelial cells amyloid beta-peptide induces cell monolayer albumin permeability, impairs glucose transport, and induces apoptosis in vascular endothelial cells angiotoxicity and arteriosclerosis due to contaminants of usp-grade cholesterol induction of apoptosis in endothelial cells treated with cholesterol oxides evidence for apoptosis in advanced human atheroma. colocalization with interleukin-1 beta-converting enzyme accutin, a new disintegrin, inhibits angiogenesis in vitro and in vivo by acting as an integrin a v b 3 antagonist and inducing apoptosis role of mesenchymal cell death in lung remodeling after injury extracellular atp and adenosine cause apoptosis of pulmonary artery endothelial cells programmed cell death in response to alkyllysophospholipids in endothelial cells 2-methoxyestradiol, an endogenous estrogen metabolite, induces apoptosis in endothelial cells and inhibits angiogenesis: possible role for stress-activated protein kinase signaling pathway and fas expression macrophages are required for cell death and tissue remodeling in the developing mouse eye apoptosis during macrophage-dependent tissue remodelling a relationship between¯ow and apoptosis during programmed capillary regression is revealed by vital analysis vegf deprivationinduced apoptosis is a component of programmed capillary regression tumor cells secrete a vascular permeability factor that promotes accumulation of ascites¯uid pituitary follicular cells secrete a novel heparin-binding growth factor speci®c for vascular endothelial cells isolation and characterization of a newly identi®ed endothelial cell mitogen produced by att-20 cells isolation and characterization of a vascular endothelial cell mitogen produced by pituitary-derived folliculo stellate cells vascular endothelial growth factor vascular endothelial growth factor (vegf) and its receptors vascular endothelial growth factor, platelet-derived growth factor, and insulin-like growth factor-1 promote rat aortic angiogenesis in vitro vascular endothelial growth factor is a secreted angiogenic mitogen tumors: wounds that do not heal. similarities between tumor stroma generation and wound healing dierential endothelial migration and proliferation to basic ®broblast growth factor and vascular endothelial growth factor during angiogenesis, vascular endothelial growth factor and basic ®broblast growth factor regulate natural killer cell adhesion to tumor endothelium vascular endothelial growth factor inhibits endothelial cell apoptosis induced by tumor necrosis factor-alpha: balance between growth and death signals the extracellular matrix as a cell survival factor vascular permeability factor/vascular endothelial growth factor inhibits anchorage-disruption-induced apoptosis in microvessel endothelial cells by inducing scaold formation vascular endothelial growth factor induces expression of the antiapoptotic proteins bcl-2 and a1 in vascular endothelial cells vascular endothelial growth factor (vegf)-mediated angiogenesis is associated with enhanced endothelial cell survival and induction of bcl-2 expression bcl-2 gene prevents apoptosis of basic ®broblast growth factor-deprived murine aortic endothelial cells vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3 0 -kinase/akt signal transduction pathway. requirement for flk-1/kdr activation distinct signal transduction pathways are utilized during the tube formation and survival phases of in vitro angiogenesis hypoxia-inducible factor-1 modulates gene expression in solid tumors and in¯uences both angiogenesis and tumor growth hif-1a is required for solid tumor formation and embryonic vascularization role of hif-a in hypoxia-mediated apoptosis, cell proliferation, and tumor angiogenesis the tumor suppressor protein vhl targets hypoxia-inducible factors for oxygen-dependent proteolysis structure of the vhl-elonginc-elonginb complex: implications for vhl tumor suppressor function rbx1, a conponent of the vhl tumor suppressor complex and scf ubiquitin ligase primary structure of bovine pituitary basic ®broblast growth factor (fgf) and comparison with the amino-terminal sequence of bovine brain acidic fgf nucleotide sequence of a bovine clone encoding the angiogenic protein, basic ®broblast growth factor apoptosis of vascular endothelial cells by ®broblast growth factor deprivation role of protein kinase c in the inhibition by ®broblast growth factor of apoptosis in serum-depleted endothelial cells basic ®broblast growth factor protects endothelial cells against radiation-induced programmed cell death in vitro and in vivo radiation-induced apoptosis in microvascular endothelial cells angiogenic stimuli are essential for survival of vascular endothelial cells in threedimensional collagen lattice involvement of interleukin-1 beta-converting enzyme in apoptosis of bfgf-deprived murine aortic endothelial cells fibroblast growth factor-2 inhibits endothelial cell apoptosis by bcl-2-dependent and independent mechanisms role of integrins in angiogenesis integrin alpha v beta 3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels requirement of vascular integrin alpha v beta 3 for angiogenesis integrins, angiogenesis and vascular cell survival cleavage of the cytoplasmic domain of the integrin beta3 subunit during endothelial cell apoptosis suppression of p53 activity and p21waf1/cip1 expression by vascular cell integrin a v b 3 during angiogenesis nf-kappab mediates alphavbeta3 integrin-induced endothelial cell survival relationships between phosphatidylcholine-speci®c phospholipase c and integrins in cell-substratum adhesion and apoptosis in vascular endothelial cells evidence for the involvement of endothelial cell integrin alphavbeta3 in the disruption of the tumor vasculature induced by tnf and ifn-gamma endothelial cytosolic proteins bind to the 3 0 untranslated region of endothelial nitric oxide synthase mrna: regulation by tumor necrosis factor alpha role of nitric oxide in the control of apoptosis in the microvasculature role of nitric oxide in autocrine control of growth and apoptosis of endothelial cells natural course of the impairment of endothelium-dependent relaxations after balloon endothelium removal in porcine coronary arteries. possible dysfunction of a pertussis toxin-sensitive g protein 17b-estradiol inhibits apoptosis of endothelial cells estrogen and coronary heart disease in women estrogenreceptor-mediated inhibition of human endothelial cell apoptosis. estradiol as a survival factor adrenomedullin as an autocrine/paracrine apoptosis survival factor for rat endothelial cells serum albumin is a speci®c inhibitor of apoptosis in human endothelial cells iap family of proteins-suppressors of apoptoisis an apoptosis inhibiting baculovirus gene with a zinc ®nger-like motif an apoptosis inhibiting gene from a nuclear polyhedrosis virus encoding a polypeptide with cys/his sequence motifs iaps block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases enhanced expression of tgf-beta and c-fos mrnas in the growth plates of developing human long bones transforming growth factor type beta: rapid induction of ®brosis and angiogenesis in vivo and stimulation of collagen formation in vitro phenotypic modulation of endothelial cells by transforming growth factor-beta depends upon the composition and organization of the extracellular matrix disruption of epithelial cell±matrix interactions induces apoptosis conditional switching of vascular endothelial growth factor (vegf) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by vegf withdrawal angiogenesis in cancer, vascular, rheumatoid and other disease retinopathy of prematurityinduced blindness: birth weight-speci®c survival and the new epidemic vascular endothelial growth factor acts as a survival factor for newly formed retinal vessels and has implications for retinopathy of prematurity roles of vascular endothelial growth factor and astrocyte degeneration in the genesis of retinopathy of prematurity oxygen-induced retinopathy in the rat: relationship of retinal nonperfusion to subsequent neovascularization hypoxic regulation of vascular endothelial growth factor in retinal cells regulation of vascular endothelial growth factor by oxygen in a model of retinopathy of prematurity ocular sequelae in extremely premature infants at 5 years of age retinal vascular endothelial growth factor (vegf) mrna expression is altered in relation to neovascularization in oxygen induced retinopathy identi®cation of a human vpf/vegf 3 0 untranslated region mediating hypoxia-induced mrna stability tumor angiogenesis: therapeutic implications the relation between cell proliferation and the vascular system in a transplanted mouse mammary tumour angiogenic factors spontaneous apoptosis is inversely related to intratumoral microvessel density in gastric carcinoma endothelial cell death, angiogenesis, and microvascular function after castration in an androgen-dependent tumor: role of vascular endothelial growth factor key: cord-023372-ft8cp9op authors: rahman, q. k.; wikman, m.; vasconcelos, n.‐m.; berzins, k.; ståhl, s.; fernández, c. title: the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th‐1 type of response date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aw.x sha: doc_id: 23372 cord_uid: ft8cp9op finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less‐conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200‐specific antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th‐1 antibody response. in contrast, no priming effect was observed for ex vivo ifn‐γ production but stimulation with the hsp‐chimeric fusion protein induced a stronger secretion of ifn‐γin vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023417-by18aczt authors: vilhelmsson, m.; ekman, g. j.; zargari, a.; scheynius, a. title: the malassezia sympodialis allergen mala s 11 with sequence similarity to manganese superoxide dismutase induces maturation and production of inflammatory cytokines in human dendritic cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ae.x sha: doc_id: 23417 cord_uid: by18aczt the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t‐cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen‐presenting dendritic cells. monocyte‐derived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il‐6 (200‐fold), tnf‐α (100‐fold) and il‐8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross‐reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023375-x4p187u7 authors: alitalo, a.; meri, t.; lankinen, h.; cheng, z.‐z.; jokiranta, s.; seppälä, i.; lahdenne, p.; brooks, c.; hefty, p. s.; akins, d. r.; meri, s. title: lysine‐dependent binding of ospe to the c‐terminus of factor h mediates complement resistance in borrelia burgdorferi date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aj.x sha: doc_id: 23375 cord_uid: x4p187u7 serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid‐encoded, surface‐exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h‐binding proteins of approximately 27–35 kda has been described. the ospe‐related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe‐related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h‐binding regions of ospe‐related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c‐terminal regions of both human and mouse factor h (scrs 18–20) specifically bind to ospe‐related lipoproteins. we also found fhr‐1, whose c‐terminal scrs 3–5 are homologous to scrs 18–20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i‐v) in ospe that could directly interact with factor h. deleting the c‐terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c‐termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c‐terminal‐binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h‐binding regions were mutated to alanines, we observed that lysines in the factor h‐binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe‐related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c‐termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h‐binding proteins may account for their susceptibility to serum lysis. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023403-jzdrvfvr authors: ahlfors, e.; sveinhaug, m. m.; nango, g.; johansen, c.; lyberg, t. title: proliferation of cells in the oral mucosa, the ear skin and the regional lymph nodes in mice sensitized and elicited with a hapten date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ac.x sha: doc_id: 23403 cord_uid: jzdrvfvr during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti‐brdu antibody and developed using abc‐kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4–24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten‐exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-006770-m5wqk6rh authors: rook, graham a. w.; taverne, janice; playfair, john h. l. title: evaluation of tnf as antiviral, antibacterial and antiparasitic agent date: 1991 journal: biotherapy doi: 10.1007/bf02172089 sha: doc_id: 6770 cord_uid: m5wqk6rh nan the toxic effects associated with release of tnf during acute infections have been well publicised. they are very striking, but make little "biological sense" in evolutionary terms. however it is now becoming clear that tnf plays an important role in resistance to infection, and the toxic effects may represent protective mechanisms which have got out of control. in this paper we first review the evidence for this protective role and the mechanisms which are likely to be involved, and then consider whether it may be possible to exploit the beneficial effects without concomitant toxicity. protective effects in vivo deduced from effects of neutralizing antibody to tnf a lethal infection with listeria monocytogenes causes detectable levels of tnf to appear in the serum of mice, whereas a sublethal infection does not [1] . nevertheless tnf plays a role in these sublethally infected animals, because neutralising antibody raised against murine tnf will exacerbate the disease [1] [2] [3] and 1 mg of rabbit antibody to murine tnf given 2 hours before infection with 0.1 ld50 of l. monocytogenes resulted in 100% mortality by 7 days. on the other hand such antibody had little effect if given on day 3 or later, suggesting that the protective role of tnf is mostly during the early phase of the infection. similarly neutralising anti-tnf caused enhanced proliferation of m. boris (bcg) in mice if given early, before the classical t cell-dependent granulomata have formed [4] . this effect of antibody to tnf is not confined to experiments with facultative intracellular bacteria, since infections with plasmodium vinckei [5] and leishmania major [6] were also exacerbated. direct evidence for the protective role of tnf in vivo has been obtained by the injection of recombinant tnf. this has been found to limit infection with leishmania major [6] , plasmodium spp. [7, 8] , trypanosoma cruzi [9] , toxoplasma gondii [9] and mycobacterium avium [10] , and to accelerate clearance of legionella pneumophila [11] . it will also protect mice from streptococcus pneumoniae and from klebsiella [ 12] . moreover, it was observed that c3h/hej mice, which produce little cytokine in response to the lps of gram negative bacteria, were 1000-fold more susceptible to a lethal infection with escherichia coli than the congenic, lps-sensitive c3h/hen mice. the c3h/hej mice could be protected from > 20 ld50's by pretreatment with a combination of il-1 and tnf [13] . therefore, in this model of gram negative infection the tnf itself seems to be protective. conversely, tnf can be an essential component of a lethal pathway which accompanies gram negative septicaemia, and neutralisation of tnf, rather than administration of yet more of the cytokine, was protective in a baboon model [14] . such apparent discrepancies indicate that the timing and dose of tnf may be critical. there is evidence for this in other models. thus tnf can protect mice from lymphocytic choriomeningitis virus (lcm) if it is given before severe inflammation has developed in the brain, but causes accelerated death if given after this has occurred [ 15] . the timing of the administration of tnf to mice infected with trypanosoma musculi is also critical, though the reverse of the situations described above. in the case early treatment seems to result in increased growth of the parasite, while if given late, after the parasitaemia has stabilised, the tnf can enhance clearance [ 16] . in order to make sense of these apparently conflicting observations, it is necessary to consider the source of tnf during infection, and the mechanisms by which tnf may exert its protective effects. tnf plays a protective role during infection with such a wide variety of organisms, that it is not surprising to find a similarly wide distribution of microbial components able to induce its production. the lipopolysaccharides (lps) of the gram negative organisms are the most studied, but the somewhat analogous lipoteichoic acids (lta) of gram positive cocci [17] and phosphatidyl inositol mannosides (lipoarabinomannan or lam) of the mycobacteria [18] are equally potent. other bacterial components which appear to have this property include the toxic shock syndrome toxin of staphylococci (tsst-1) [19] , a streptococcal cell wall preparation [20] , and muramyl dipeptide (mdp), a synthetic analogue of part of the ubiquitous bacterial cell wall peptidogylcan appears to prime for enhanced release of tnf by other bacterial components [21] . perhaps all micro-organisms trigger release of tnf, and in addition to the organisms already discussed above, legionella [11], listeria [1] , malaria parasites [22] , and candida [23] all have this property though the active components have not been identified. in the case of malaria, both the blood-stage parasite and released antigens are active, and there is some evidence that the latter may be predominantly glycolipid in nature and act as t-independent antigens [24] . it remains possible that some organisms do not have the ability to trigger release of tnf. however there have been sporadic reports of lymphokines able to cause release of tnf by pathways which do not appear to involve triggering of appropriately activated cells by a microbial component. if this is so, tnf could be involved in protection against organisms which lack a tnf "trigger". similarly it is possible that membraneassociated tnf is involved in local events which do not require release of free tnf. macrophages and monocytes infected with hiv spontaneously release tnf [25, 26] . moreover, the free virus is able to trigger tnf release from uninfected monocytes by cross-linking membrane cd4 [27] . the importance of these observations is discussed later. many workers have investigated the possibility that tnf is directly toxic for microorganisms. most of the results have been negative, and remain unpublished. however tnf has been reported to be toxic for trypanosoma musculi in vitro [ 16] . tnf can indirectly cause killing of organisms by activating phagocytes. there is strong evidence that tnf can prime neutrophils, so that they subsequently give an exaggerated burst of superoxide or h202 production when exposued to stimuli such as zymosan [28] , phorbol myristate acetate (pma) or f-met-leu-phe [29] . the increased oxidative response to zymosan [28] , and to opsonised amoebae [30] was attributed to increased expression of cr3 [28] which may also be responsible for the increase in adhesion of tnf-exposed neutrophils to endothelial cells [31] . these priming effects are rapid. increased adhesion to endothelial cells [31] is apparent within 5 minutes of exposure to tnf, and priming for enhanced superoxide production is apparent within 20 mins. longer exposure to tnf (20 mins to several hours) increased phagocytosis of latex, and adcc [32] , and in some reports, leads directly to superoxide or h202 release without any further stimulus [28, 33] . however some release of lactate dehydrogenase (ldh) is seen at this time [28] , implying cell death, so the interpretation is not clear. perhaps the neutrophils are triggered to release tnf while phagocytosing their dying colleagues. nevertheless this may not be an in vitro artefact, and such events could presumably occur in vivo. neutrophils exposed to tnf for several hours in this way are able to disrupt endothelial cell monolayers in vitro [33] . whatever the details of the activation pro-cess, it is clearly rapid, and can result in enhanced clearance of candida [34] , and legionella [ 11] . there is a report that platelets can also be activated by tnf. if platelets are incubated overnight with schistosomulae in the presence of tnf, the percentage killing is increased, though tnf is not itself toxic to the larvae [35] . exposure to tnf will cause inhibition of multiplication of trypanosoma cruzi in murine peritoneal macrophages. the tnf can be added after infection has taken place [36] . other workers found that the effect was not seen using lps-insensitive c3h/hej macrophages, and that it could be blocked by adding catalase [37] . they suggested that the inhibition was dependent on release of h202 from the tnf-activated cells by contaminating lps. tnf did not inhibit growth of t. cruzi in human fibroblasts [36] , perhaps because these cells do not make h202. tnf had no effect on the intracellular growth of toxoplasma gondii [36] . nevertheless the same group detected a protective effect of tnf during infection with t. gondii in vivo [9] , suggesting that direct activation of macrophages is likely to be only one of several protective pathways enhanced by tnf. bermudez and young report that tnf will cause kill of aids-derived strains of mycobacterium avium by human monocytederived macrophages, and by murine peritoneal macrophages in vitro [38] , while gamma interferon caused increased growth of the organisms in both human and murine cells. these findings are in conflict with much previously published data. gamma interferon induced total stasis of m. tuberculosis [39] and of m. avium strains not derived from patients with aids (unpublished observations) in murine peritoneal cells. on the other hand tnf had no effect at all on growth of m. tuberculosis in these cell types (rook et al., unpublished) though a small effect is seen using bone-marrow-derived murine macrophages (s. kaufmann, personal communication). therefore, either the aidsderived strains are quite different, or technical problems are significant. as outlined earlier, tnf does exert some protective effect against m. avium in vivo [ 10] , but for reasons to be explained below, this does not constitute evidence that its mode of action is by direct activation of the macrophages. as in the case of toxoplasma [9] , there is more than one mechanism at work. tnf is able to activate nk cells [40] , and it has been suggested that in synergy with unidentified microbial components, tnf causes release of gamma interferon from these cells [3] . this could be an important pathway providing the source of gamma interferon in scid mice [3] , and a simple rationale for the role of tnf in the early t cell-independent phase of response to listeria [1, 2] , and bcg [4] in vivo. moreover it provides a possible explanation for the protective effect of tnf against m. avium [10] and toxoplasma [9] in mice. this pathway "makes sense" from an evolutionary point of view, because it means that ifn-gamma is available early after infection, before the t cell response has developed. it will clearly be essential to check whether it is involved in the experiments in mice where tnf or anti-tnf administered early after infection, are found to modify the disease [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . it should be noted that if this is how tnf protects mice against m. avium, it is irrelevant to human infections with mycobacteria, since all authors agree that ifn-gamma has no effect on the growth of mycobacteria in human macrophages [39] . tnf increases the killing of schistosomulae by eosinophils [41] but a study of direct influences of tnf on degranulation, enzyme release, and oxidative metabolism of eosinophils revealed minimal effects so the mechanism of the tnf-enhanced cytotoxicity is not known [42] . basophils and mast cells tnf does not appear to activate these cell types [43] . other sections of this issue deal in detail with the immunomodulatory and pro-inflammatory effects of tnf, and the relationships between tnf and the production of other cytokines and mediators. clearly these topics are all relevant to its role in protection against infection, but they are not reviewed again here. only certain important experiments directly involving infection are outlined. exposure of hep-2 cells (derived from a human carcinoma of the larynx) to tnf renders them resistant to invasion by salmonella typhimurium [44] and inhibits intracellular growth of chlamydia trachomatis [45] . the latter effect is partly reversed by neutralising antibodies to ifn-beta, and also by addition of tryptophan. the authors concluded that the tnf induced secretion of ifn-beta, which in synergy with the tnf augmented tryptophan-depleting enzyme activity. tnf did not inhibit growth of t. cruzi in human fibroblasts [36] . the ability of tnf to influence cells not belonging to the immune system is particularly relevant in the context of viral infections. several cell lines have been shown to be protected from viral cytopathic effect by tnf, and virus yield and viral protein synthesis were reduced. cell lines in which these effects were observed include hep-2, human embryonic lung fibroblasts (hel & wi-38), and mouse embryonic fibroblasts (mef) [46] , and human lung (a549) and renal (7860) carcinomas [47] . susceptibility to the tnf-mediated effect was not related to the transformed phenotype, or to inherent sensitivity to the cytotoxic effect of tnf [46] . tnf will protect from both rna (emcv and vsv) and dna viruses (adenovirus-2, hsv-2) and acts synergistically with gamma interferon [47] . in at least one cell line the protection is mediated indirectly via induction of ifn-beta [48] though this is probably not so in every case, and several pathways may exist. tnf inhibits activation of human b cells by epstein-barr virus in the presence of macrophages. this seems to be due to production by the macrophages of an unidentified soluble factor [49] , and might represent a useful role for tnf in malaria, which can act as a cofactor with ebv in the genesis of b cell (burkitt's) lymphoma. thus the a547 human lung carcinoma is rendered sensitive to the cytotoxic effect of tnf by infection with adenovirus or vsv [47] , and similar observations have been made with herpes virus [50] and hiv [51] . it seems likely that this mechanism can be either protective or severely disabling depending on how many cells in any one vital organ are infected, and so killed, at the time when tnf is released or administered. this could also partly explain the detrimental effect of late administration of tnf to mice infected with lcm virus [15] . it is possible that the ability of tnf to kill some transformed or virus-infected cells is 171 part of a broader property enabling it to destroy cells which are functionally disturbed. we have found recently that if l929 fibroblasts are infected with m. tuberculosis (which they take up in large numbers) they become sensitive to killing by very low levels of tnf in the absence of emetine or actinomycin d. the intracellular mycobacteria do not cause any obvious alterations in protein synthesis, (assessed by 35s-methionine labelling, followed by analysis of the sds-page-separated proteins in a beta scanner), but when such cells are exposed to a normally non-toxic level of tnf, protein synthesis ceases promptly (filley, rook et al., in preparation) . this phenomenon is also seen using a normally tnf-resistant subclone of the l929 line supplied by dr. n. matthews. since neutralising antibody to tnf exacerbates infection with listeria or m. boris (bcg) if given early, before granulomata have formed [1, 2, 4] , it was suggested that induction of granuloma formation might be a function of tnf. in fact these experiments could equally be explained by the tnf/nk cell/ifn-gamma pathway described earlier. nevertheless it has been shown that tnf conjugated to sepharose beads can cause granuloma formation in mouse lungs [52] . induction of tolerance to tnf treatment of rats with a single low intravenous dose of tnf protected them from a potentially lethal dose given 24 hours later. similarly it protected against the lethal effects of lps, or caecal ligation and puncture [53] . this suggests that tnf could conceivably be used prophylactically in patients in whom septicaemic episodes were anticipated. the mode of action may be tachyphylaxis. on the other hand tnf is known to lead secondarily to production of il-6 [54] , and this cytokine induces a pattern of acute phase response involving protease inhibitors, caeruloplasmin and haptoglobin, which may have an antiinflammatory effect [55] . endogeneously raised levels of tnf correlating with bad clinical outcome in vivo in numerous clinical situations high serum levels of tnf correlate with a poor clinical outcome. these include septicaemia [56] , and the adult respiratory distress syndrome (ards) [57] . similarly in malaria tnf has been implicated in severe disease [58] , and in such complications as anaemia [59] , and abortion [60] , and in cerebral malaria in a mouse model [61] . this has given rise to suggestions for the treatment of gram-negative shock, severe malaria, etc., with regimes aimed at reducing tnf levels, either directly by antibodies or inhibitors, or indirectly by vaccine-induced immunity against the triggering molecules [24] . in patients with aids high levels of tnf correlate with encephalopathy [62] . although tnf is reported to kill hiv-infected cells [51] the consequences of the interaction of these cells with tnf may not be protective. several authors agree that tnf increases virus yield from hiv-infected cells in vitro [51, [63] [64] [65] [66] , and the mechanism is partly understood [66] . therefore administration of tnf to individuals infected with hiv may be contraindicated, and there is a report that tnf caused rising levels of circulating hiv antigen in patients undergoing a trial of tnf therapy for kaposi's sarcoma [67] . since hiv also induces tnf production [25] [26] [27] , this positive feedback may constitute a significant part of the pathogenesis of the disease. it is also interesting that tuberculosis is an early complication of hiv, (unlike m. avium infection which occurs late when t cells are depleted), and seems to lead to fur-ther aggravation of the hiv infection. perhaps this is attributable to synergistic induction of tnf by hiv [25] [26] [27] and by mycobacterial lipoarabinomannan [ 18] leading to further activation of the virus. detrimental consequences of administered tnf administration of tnf can induce cachexia, anaemia, inflammation and haemorrhagic necrosis [68] . however, the systemic toxicity of tnf is greatly increased in the presence of il-1 or lps [69] . this may be one of the obstacles which prevents its use in the treatment of infection, since these substances are likely to be present in relevant patients. moreover microbial products, and certain types of inflammatory response "prepare" tissue sites so that they become exquisitely sensitive to tnf, and liable to undergo haemorrhagic necrosis in its presence [70] [71] [72] . this "preparation" of an inflamed site probably involves changes in the properties of endothelial cells (reviewed in [73] ), and can be brought about both by t cell-independent (lps, [70] ) and t cell-dependent (low doses of soluble mycobacterial antigen [72] ) responses to microbial products. this phenomenon probably explains the shwartzman reaction (discussed in [72] ) in which a skin site prepared by an injection of lps undergoes necrosis if a further dose of lps is given intravenously 24 hours later. this tendency for sites of microbial inflammation to undergo necrosis in the presence of tnf is largely a vascular phenomenon. however, it could be further aggravated if the infection involved resulted in the cells of the relevant organ becoming themselves sensitive to the cytotoxic effects of tnf as described earlier for virus-infected [47, 50, 51] and tuberculosis-infected cells. this has been postulated as the basis of fulminant hepatitis [74] and a combination of necrotising vasculitis and death of infected cells could help to explain the very rapidly fatal effect of tnf administered late to mice with lcm virus [ 15] . should we conclude that the treatment of infectious disease with tnf will be impossible, in spite of the conclusive evidence that this cytokine is an essential part of the normal response to infection? the situation is not quite that bleak. obviously it will be essential to avoid activating hiv, or triggering shock, shwartzman reactions, or fulminant haemorrhagic necrosis of infected organs or foci. on the other hand several recent studies revealed that we know very little about the circumstances under which tnf is or is not toxic. rodents can be desensitised to the toxic effects of tnf [13, 75] but we do not yet know whether such desensitisation also eliminates all the many protective effects of the cytokine. still more remarkable is the observation that monophosphoryl lipid a (a non-toxic derivative of lps) will induce serum levels of tnf in mice infected with listeria monocytogenes which are comparable to the levels induced by lps itself, and yet whereas the result is fatal in the lps-treated animals, it is not in those receiving the monophosphoryl compound [76] . again this implies that the toxicity of tnf involves other agonists, or can be blocked by regulatory substances, but we do not know which, or whether the protective effects would be similarly decreased. there is clearly much scope for further experiment. production of tumor necrosis factor during murine listeriosis endogeneous tumour necrosis factor (cachectin) is essential to host resistance against listeria monocytogenes infection tumor necrosis factor is involved in the t cell-independent pathway of macrophage activation in scid mice the inducing role of tumor necrosis factor in the ment of bactericidal granulomas during bcg infection application of anti-tnf to plasmodium vinckei-infected mice is followed by an increase of parasitaemia tumour necrosis factor plays a protective role in experimental murine cutaneous leismaniasis recombinant tumour necrosis factor inhibits malaria parasite /n vivo but not in vitro inhibition of murine malaria (plasmodium chabaudi) in vivo by recombinant interferon-gamma or tumor necrosis factor, and its enhancement by butylated hydroxyanisole effect of recombinant tumour necrosis factor on acute infection in mice with toxoplasma gondii or trypanosoma cruzi treatment of experimental disseminated mycobacterium avium complex infection in mice with recombinant il-2 and tumor necrosis factor protective effects of tumor necrosis factor in experimental legioneua pneumophila infections of mice via activation of pmn function effects of tnf in bacterial infections pretreatment with recombinant murine turnout necrosis factor alpha/cachectin and murine interleukin 1 alpha protects mice from lethal bacterial infection anti-cachectin/tnf monoclonal antibodies prevent septic shock during lethal bacteraemia tumour necrosis factor inhibits the development of viral meningitis or induces rapid death depending on the severity of inflammation at the time of administration effect of tumour necrosis factor on growth of trypanosoma musculi in vivo and in vitro antitumour effects of streptococcal lipoteichoic acids on meth a fibrosarcoma lipoarabinomannan from mycobacterium tuberculosis induces the production of turnout necrosis factor from human and murine macrophages toxic shock syndrome toxin 1 as an inducer of human tumor necrosis factors and gamma interferon release of tumour necrosis factor (tnf) into mouse peritoneal fluids by ok432, a streptococcal preparation production of tumour necrosis factor in nude mice by muramyl peptides associated with bacterial vaccines soluble malarial antigens are toxic and induce the production of tumour necrosis factor in vivo tumor necrosis factor induction by candida albicans from human natural killer cells and monocytes the malaria vaccine: anti-parasite or anti-disease? purified blood monocytes from hiv 1-infected patients produce high levels of tnf alpha and il-1 groopman je. production of tumor necrosis factor alpha and interteukin 1 beta by monocytic cells infected with human immunodeficiency virus interleukin-1 and tumor necrosis factor alpha can be induced from mononuclear phagocytes by human immunodeficiency virus type 1 binding to the cd4 receptor stimulation of neurophils by tumour necrosis factor biochemical mechanisms in the priming of neutrophils by tumour necrosis factor tumor necrosis factor alpha potentiates neutrophil antimicrobial and candida albicans and associated increases in oxygen radical production and lysosomal enzyme release stimulation of the adherence of neutrophils to the umbilical vein endothelium by human recombinant tumour necrosis factor activation of human polymorphonuclear functions by interferon gamma and tumor necrosis factors receptor binding and activation of polymorphonuclear neutrophils by tumour necrosis factor alpha growth inhibition of candida albicans by human polymorphonuclear neutrophils: activation by interferon gamma and tumor necrosis factor recombinant tumour necrosis factors mediate platelet cytotoxicity to schistosoma mansoni larvae activity of recombinant tumour necrosis factor on toxoplasma gondii and trypanosoma cruzi recombinant tumour necrosis factor enhances macrophage destruction of trypanosoma cruzi in the presence of bacterial endotoxin tumour necrosis factor, alone or in combination with il-2, but not ifn-gamma, is associated with macrophage killing of mycobacterium avium complex activation of macrophages to inhibit proliferation of mycobacterium tuberculosis: comparison of the effects of recombinant gamma-interferon on human monocytes and murine peritoneal macrophages in vivo and in vitro induction of natural killer cells by cloned human tumour necrosis factor tumour necrosis factor enhances eosinophil toxicity of schistosoma mansoni larvae modulation of human peripheral blood eosinophit function by tumour necrosis factor alpha comparative effect of recombinant il-1, -2, -3, -4, and -6, ifn-gamma, granulocyte-macrophage-colony-stimulating factor, tumor necrosis factor-alpha, and histaminereleasing factors on the secretion of histamine from basophils in vitro treatment of hep-2 cells with human tumour necrosis factor alpha and human interferons reduces invasiveness of salmonella typhimuriuml reversion of the antichlamydial effect of tumour necrosis factor by tryptophan and antibodies to beta interferon antiviral effect of recombinant tumour necrosis factor in vitro tumour necrosis factors alpha and beta inhibit virus replication and synergise with interferons the in vitro antiviral activity of tumor necrosis factor (tnf) in wish cells is mediated by ifn-beta induction tumour necrosis factor selectively inhibits activation of human b cells by epstein-barr virus human tumour necrosis factor alpha kills herpesvirus-infected, but not normal cells cytocidal effect of tumor necrosis factor on cells chronically infected with human immunodeficiency virus (hiv): enhancement of hiv replication the role of monokines in granuloma formation in mice: the ability of interleukin-1 and tumour necrosis factor alpha to induce lung granulomas prevention and treatment of endotoxin and sepsis lethality with recombinant human tumor necrosis factor circulating interleukin 6 during an infusion of tumour necrosis factor and interferon gamma hepatocyte-stimulating factor iii shares structural and functional identity with leukemiainhibitory factor association between tumour necrosis factor in serum and fatal outcome in patients with meningococcal disease tnf in bronchopulmonary secretions of patients with adult respiratory distress syndrome elevated tumor necrosis factor alpha and interleukin-6 serum levels as markers for complicated plasmodium facciparum malaria tumour necrosis factor may contribute to the anaemia of malaria by causing dyserythropoiesis and erythrophagocytosis tumor necrosis factor in malariainduced abortion tumor necrosis factor (cachectin) as an essential mediator in murine cerebral malaria elevated serum levels of tumor necrosis factor are associated with progressive encephalopathy in children with acquired immunodeficiency syndrome tumor necrosis factor alpha induces expression of human immunodeficiency virus in a chronically infected t-cell clone augmentation of human immunodeficiency virus type i gene expression by tumor necrosis factor alpha tumor necrosis factor enhances replication of human immunodeficiency virus (hiv) in vitro tumor necrosis factor alpha activates human immunodeflciency virus type 1 through induction of nuclear factor binding to the nf-kappa b sites in the long terminal repeat intravenous recombinant tumor necrosis factor in the treatment of aids-related kaposi's sarcoma cachectin/tumor necrosis factor induces cachexia, anaemia, and inflammation interleukin-i potentiates the lethal effect of tumour necrosis factor alpha/cachectin in mice synergy between tumour necrosis factor and bacterial products causes haemorrhagic necrosis and lethal shock in normal mice the role of gamma-interferon, vitamin d3 metabolites and tumour necrosis factor in the pathogenesis of tuberculosis the role of cytokines in the immunopathology of tuberculosis and the regulation of agalactosyl igg tnf as an activator of vascular endothelium does tumor necrosis factor play a role in the pathogenesis of fulminant hepatitis? med hypotheses sensitisation and desensitisation to lethal effects of tumour necrosis factor and il-1 induction of tumour necrosis factor, ifngamma, and acute lethality in mice by toxic and non-toxic forms of lipid a key: cord-007858-1ijxilpb authors: xu, g.l.; yao, l.; rao, s.y.; gong, z.n.; zhang, s.q.; yu, s.q. title: attenuation of acute lung injury in mice by oxymatrine is associated with inhibition of phosphorylated p38 mitogen-activated protein kinase date: 2005-04-08 journal: j ethnopharmacol doi: 10.1016/j.jep.2005.01.026 sha: doc_id: 7858 cord_uid: 1ijxilpb oxymatrine is one of the alkaloids extracted from chinese herb sophora japonica (sophora flavescens ait.) with activities of anti-inflammation, inhibiting immune reaction, antivirus, protecting hepatocytes and antihepatic fibrosis. however, the effect of oxymatrine on acute lung injury (ali) has not been known yet. in this study, the effect of oxymatrine on ali was investigated using an oleic acid-induced ali mouse model. morphological findings showed that the oleic acid group demonstrated a marked lung injury represented by prominent atelectasis, intraalveolar and interstitial patchy hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the existence of inflammatory cells in alveolar spaces. while in the oxymatrine/dexamethasone group, these changes were less severe and in the vicinity of the control group. furthermore, pretreatment with oxymatrine significantly alleviated oleic acid-induced lung injury accompanied by reduction of lung index and wet-to-dry weight ratio, decreases in serum tnf-α level and inhibition of phosphorylated p38 mapk. these findings suggest that oxymatrine has a beneficial effect on acute lung injury induced by oleic acid in mice and may inhibit the production of proinflammatory cytokine, tnf-α, by means of the inhibition of p38 mapk. acute lung injury (ali) is characterized by an intense pulmonary inflammatory response, with neutrophil accumulation, interstitial edema, disruption of epithelial integrity and leakage of protein into the alveolar space (john et al., 2001) . acute respiratory distress syndrome (ards) is characterized by increased capillary endothelial permeability that leads to segmental accumulation of high protein content in interstitial edema. fibrin and platelet plugs occlude the microvasculature of the lung and neutrophil sequestration and activation in the interstitium leads to further segmental alveolar damage and flooding. these processes result in decreased pulmonary compliance, compromised gas exchange and extensive intra-pulmonary shunting with concomitant ventilation-perfusion mismatching. at present, ards is often classified as a continuum of injury ranging from the milder form 'acute lung injury (ali)' to the more severe form 'ards' (christopher et al., 1999) . ali/ards is associated with the development of multiple organ dysfunction syndrome (mods), which plays an important role in the death of patients with sepsis, pneumonia, aspiration of gastric contents, trauma, multiple transfusions and ischemia reperfusion. severe acute respiratory syndrome (sars) is a novel global infectious disease induced by a virus from the family coronaviridae. clinical investigation shows that pathological changes in sars patients are similar to those of acute lung injury, as revealed by alveolar cell collapse, severe exudation, acute inflammatory reaction and hyaline membrane formation (zhong, 2003; du et al., 2004) . currently, no corrective therapy is available for the 0378 it is generally accepted that the development of mods follows the gradual route of ali-ards-mods. during this pathway, the lung behaves as a central organ and is the first target subject to injury. because pulmonary dysfunction may lead to severe hypoxemia and further other multiple organ dysfunction or failure, the treatment of the above mentioned diseases should concentrate on the development of ali and take measures as soon as possible . the research and development of anti-sars drugs should also comply with this law. potential new therapeutic strategies for sars have been shown to include a wide search for drugs, which is conducive to reduction of lung injury and management of symptoms (zhong, 2003; du et al., 2004) . as a traditional chinese medicine, sophora japonica has been used for treatment of many diseases for thousands of years. matrine-like alkaloids have been found to be the chief active components of chinese herb sophora japonica including matrine, oxymatrine, sophocarpine, sophoramine, sophoridine et al. the content of oxymatrine in the composite of sophora japonica is up to 2.8% (qi et al., 1996) . basic and clinical researches have shown that oxymatrine has the following pharmacological effects: anti-inflammation, inhibiting immune reaction, antivirus, protecting hepatocytes and antihepatic fibrosis (liu et al., 1994 (liu et al., , 2003 liu and chiou, 1996; chen et al., 2001; dong et al., 2002; xiang et al. 2002; yang et al., 2002) . chinese bureau of science and technology had announced during the outbreak of sars that the composite sophora japonica injection has distinct effects in the treatment of sars (zhong, 2003) . considering the pharmacological effects of oxymatrine, we speculate that oxymatrine may play a central role in the composite injection although the effect of oxymatrine on sars has not been reported to date. in order to test this hypothesis and because the pulmonary pathological changes in sars are similar to those of acute lung injury, the effect of oxymatrine on acute lung injury was investigated using an ali mouse model in this study. we also studied whether the p38 mapk intracellular signal pathway was involved in the development of ali and discussed whether oxymatrine could become a therapeutic candidate drug for ali, ards and sars. oxymatrine was obtained from yixin pharmaceutical co. ltd. (zhejiang, china) with hplc purity >98%. oleic acid was purchased from linfeng chemical co. ltd. (shanghai, china). albumin was obtained from sigma. rabbit polyclonal antibodies for phosphorylated and nonphosphorylated p38 mapk were provided by cell signaling technology. tween 20 and glycine were purchased from amresco co. polyvinylidene difluoride (pvdf) transfer membranes for western blotting were obtained from roche molecular biochemicals (quebec, canada). protein assay dye reagent was purchased from jiancheng bioengineering co. (nanjing, china) . hrp conjugate goat anti-rabbit igg and pipa lysis buffer were purchased from shengnengbocai biotech inc. (shanghai, china) . mouse tnf-␣ elisa assay kit was obtained from jingmei biotech co. (guangdong, china). all other reagents were of the highest grade available commercially. male kunming strain mice weighing 18-24 g were obtained from laboratory animal center, school of medicine, southeast university (nanjing 210009, china). the mice were housed in climate-controlled quarters (24-18 • c at 50% humidity) with a 12 h light/dark cycle and free access to food and water. all experiments were conducted according to the "guide for the care and use of laboratory animals", published by the national institutes of health (nih publication 86-23, revised 1986). mice were randomly assigned into six groups. the oleic acid group received 0.3 ml/kg, i.v. of oleic acid (mixed with 0.1% bovine serum albuman). the control group received 0.3 ml/kg, i.v. of saline. the three oleic acid + oxymatrine groups were treated with oxymatrine for three consecutive days before oleic acid injection (oxymatrine; 12.5, 25 and 50 mg/kg, i.p.) (xiang et al., 2002) . the dexamethasone group received intraperitoneal injection of dexamethasone at dose of 2.0 mg/kg 2 h before oleic acid injection. the oleic acid and control groups were treated with an equivalent volume of saline instead of oxymatrine. mice were sacrificed 6 h after oleic acid injection. immediately after each animal had been sacrificed, the thorax was opened and the lung was removed en bloc by observers unaware of the nature of the experiment. the left lower lobe was excised and fixed in 10% buffered formalin. the lungs were embedded in paraffin, and the sections stained with hematoxylin and eosin were examined by light microscopy for evidence of lung injury, as described in the following: alveolar congestion, hemorrhage, edema, infiltration/aggregation of neutrophils in the airspace or vessel wall, thickness of the alveolar wall and hyaline membrane formation. electron microscopy of lungs was carried out on samples fixed in phosphate buffer (ph 7.4) containing 2.5% glutaraldehyde and post-fixed in osmium tetraoxide. transmission electron micrographs were produced with a hitachi h-7000 electron microscope. the wet weight of the whole lung was weighed on an automatic electric balance and the lung index was calculated according to the following formula: lung index (%) = the wet weight of the whole lung/body weight × 100%. subsequently, the right lung was excised and weighed to obtain the 'wet' weight. the lung was then dried in an oven at 80 • c for 7 days to obtain the 'dry' weight. to assess tissue edema, the ratio of wet weight to dry weight (w/d ratio) was calculated. the left lung was cut into pieces and subjected to histological examination and electron microscopy observation. blood samples were harvested from the eyes before mice were killed. the blood was allowed to clot for approximately 1 h at room temperature and then centrifuged at 3500 × g for 5 min to obtain the serum. the serum was stored at −20 • c until assayed. the tnf-␣ was quantitated according to the instructions available in elisa kits. the levels in mice samples were calculated from a standard curve generated from recombinant mice tnf-␣. the detection range of this assay for tnf-␣ is 26-2000 ng/l. samples with a concentration exceeding the limits of the standard curve were repeated after dilution. three mice in each group were adopted 30 min after oleic acid injection. the lungs were frozen in liquid nitrogen for measurements immediately after they were removed. before the assay, the specimens were cleared of fat and debris. all specimens had a wet weight of 100 mg. phenylmethylsulfonylfluoride (pmsf; 1 mmol) was added just before use. the samples were homogenized in 500 l of pipa lysis buffer using a microhomogenizer on ice. all debris and nuclei were removed by centrifugation at 9000 × g at 4 • c for 10 min. the supernatant obtained was used as the whole cell lysate. protein concentrations were determined with bovine serum albumin as the reference standard using protein assay dye reagent. an 80 g of proteins were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (sds-page). the membranes were first incubated with 5% nonfat milk in tris-buffered saline (tbs). after washing three times in 0.1% tween 20-tbs (tbst), the membranes were incubated with p38 mapk and phosphorylated p38 mapk antibodies separately (1:1000) in tbst over night at 4 • c, and then washed three times in tbst. they were incubated with horseradish peroxidaselinked goat anti-rabbit igg (1:1000) for 1 h at room temperature and detected with the tmb substrate for horseradish peroxidase. data from experiments are expressed as mean ± s.e.m. and statistically analyzed using the student's unpaired t-test. a value of p < 0.05 was considered significant. light microscopic findings in the lung at 6 h after oleic acid injection demonstrated a marked lung injury resembling those seen in lung of patients with ali/ards, represented by prominent atelectasis, intraalveolar and interstitial patchy hemorrhage, edema, thickened alveolar septum, formation of hyaline membranes and the existence of inflammatory cells in alveolar spaces (fig. 1a) , which were not observed in the control group (fig. 1b) . in oleic acid + oxymatrine groups, these changes were less severe than in the oleic acid group and were in the vicinity of the control group (fig. 1c ). in the oleic acid + dexamethasone group, these changes were not pronounced and were close to the high dose oxymatrinetreated group (fig. 1d) . as shown in fig. 2 , electron microscopic findings in the lung after oleic acid injection included epithelial cell swelling (endoplasmic reticulum dilation and mitochondria swelling), the existence of blood cells and inflammatory cells in alveolar spaces (fig. 2b-d) , which were not observed in the control group ( fig. 2a) . these changes were improved or not evident in oleic acid + oxymatrine/dexamethasone groups ( fig. 2e and f). values of the lung index and wet-to-dry lung weight ratio (w/d) in various groups of experimental animals were shown in fig. 3 . oleic acid injection resulted in an increase in the lung index and w/d ratio of the lung, as compared to the control (p < 0.01, p < 0.01). both were attenuated by 25 mg/kg (p < 0.05, p < 0.01) and 50 mg/kg (p < 0.01, p < 0.01) oxymatrine treatment. in contrast, 12.5 mg/kg oxymatrine did not diminish the above two values significantly although they were lower than the oleic acid group. the lowest level was found in animals receiving dexamethasone treatment. as tnf-␣ plays a pivotal role in mediating oleic acidinduced ali, we also assessed the regulation of tnf-␣ production by oxymatrine (fig. 4) . the serum tnf-␣ level in the oleic acid group was significantly higher than that in the control group (p < 0.01). treatment with oxymatrine inhibited the increase in a dose dependent manner and the level at the highest dose reverted to the control level. serum level of tnf-␣ in the dexamethasone group was evidently lower than that in the oleic acid group and similar to the control group. this suggested that tnf-␣ increases induced by oleic acid could be suppressed by oxymatrine. as shown in fig. 5 , the level of p38 phosphorylation in the oleic acid group was significantly higher than that in the control group. pretreatment with oxymatrine inhibited phosphorylation of p38 in a dose dependent manner. p38 phosphorylation in the highest oxymatrine group was similar to that of the control group. in contrast, the total p38 protein remained unchanged. xiang and colleagues used oxyimatrine to evaluate effect of oxymatrine on fulminant hepatitis and hepatocyte apoptosis in mouse models and oxymatrine was administered at dose of 50 mg/kg intraperitoneally (i.d. × 3 days) (xiang et al., 2002) . initially, we administered oxymatrine at the dose of 50 mg/kg intraperitoneally twice a day for three consecutive days, resulting in a protective effect that could be easily monitored by macroscopic and/or microscopic observation. then we used, oxymatrine at doses of 25, 12.5 and 6 mg/kg to investigate the relationship between dose and effect. results showed that no protective effects were observed at 12.5 mg/kg or below. so 50, 25 and 12.5 mg/kg were ultimately selected as the test dosage. our results showed that a series of pathological changes were observed under light and electron microscopy after an intravenous administration of oleic acid in mice, which mimicked the pathological changes of clinical ali/ards satisfactorily. furthermore, the lung index and wet/dry weight ratio were greater in ali mice than in control group. these findings are in agreement with other reports (kenji et al., 2001) , demonstrating that the oleic acid-induced ali mouse model is reproducible and our ali model is successful. the underlying mechanism of ali induced by oleic acid is associated with cytokines releases such as tnf-␣, which stimulates monocytes to produce il-1.as the core of the cytokine-network, tnf-␣ and il-1 play important roles not only in the production of other inflammatory cytokines, but also in the migration and adherence of neutrophils to endothelial cells (yoshimi et al., 2000) , which together with endothelial cell injury by cytokines result in the production of superoxide radicals and subsequently injure alveolar epithelial cells. all these cause ultimate pulmonary dysfunction. in the present study, our data revealed that serum tnf-␣ level was higher in oleic acid group than that in control group. oxymatrine evidently decreased serum tnf-␣ level, lung index as well as wet-to-dry ratio and reduced pulmonary injury induced by oleic acid. these findings not only corroborate the direct relationship between tnf-␣ and ali but also suggest that oxymatrine have a protective effect on oleic acid-induced ali. fig. 2 . ultrastructure of the lung tissue in mice without or with oleic acid injection (8000×). representative photomicrographs showing: epithelial cell swelling (endoplasmic reticulum dilation and mitochondria swelling) (b), the existence of red blood cells (c) and inflammatory cells (d) in alveolar spaces were observed in oleic acid group. these changes were not observed in the control group (a) and improved or not evident in oleic acid + oxymatrine/dexamethasone groups (e, f): a, normal epithelial cell; b, swollen epithelial cell; c, red blood cell in alveolar spaces; d, inflammatory cell in alveolar spaces. fig. 3 . effect of oxymatrine on lung index and wet-to-dry weight ratio (w/d) in mice with lung injury induced by oleic acid. the mice were given oxymatrine (12.5, 25 and 50 mg/kg, i.p.) for three consecutive days and dexamethasone (2 mg/kg, i.p.) 2 h before injection of oleic acid. the control and oleic acid groups received normal saline. the mice were then sacrificed 6 h after oleic acid administration and lung index, wet-to-dry weight ratio (w/d) were calculated. mean ± s.e.m., n = 6; ** p < 0.01 when compared with control; # p < 0.05 and ## p < 0.01 when compared with oleic acid group. fig. 4 . effect of oxymatrine on serum tnf-␣ level in mice with lung injury induced by oleic acid. the mice were given oxymatrine (12.5, 25 and 50 mg/kg, i.p.) for three consecutive days and dexamethasone (2 mg/kg, i.p.) 2 h before injection of oleic acid. the control and oleic acid groups received normal saline. the mice were then sacrificed 6 h after oleic acid administration and serum tnf-␣ level were determined by elisa assay. mean ± s.e.m., n = 6; ** p < 0.01 when compared with control; ## p < 0.01 when compared with oleic acid group. bands of p38 map kinase were identified in the five groups, and mean density levels in the five groups were almost the same. phosphorylated p38 map kinase was augmented in the oleic acid group compared with the control group, whereas expression of phosphorylated p38 map kinase was markedly attenuated in the oxymatrine group compared with the oleic acid group. it has been reported that oxymatrine concentration is higher in lung and heart than in other organs. this signifies the anti-ali effect of oxymatrine has a pharmcokinetic basis (wang and wang, 2000) . additionally, dexamethasone as positive control exerted a significant preventive effect on oleic acid-induced ali injury. this might be explained by its potent anti-inflammation effect described as follows: diminution of alveolocapillary permeability; reduction of alveolar epithelial response to pathogen; stability of cell and lysosome membrane; enhancement of surfactant release; prevention of microthrombogenesis and blockade of neutrophil activation (wang et al., 2001; su et al., 2004) . under the stimuli of different ali/ards pathogens, a wide and complicated signal transduction process occurs in many different cells. although the detailed mechanism is still unknown, the p38 mitogen-activated protein kinase (mapk) has been paid special attention. p38 mapk is a cytokinesuppressive anti-inflammatory drug target first discovered by lee et al. (1994) . most reports demonstrate that p38 mapk is responsible for regulating inflammatory responses (nick et al., 2000 (nick et al., , 2002 fang et al., 2002) . on the other hand, arcaroli et al. (2001) reported that p38 did not have a central role in the development of ali after either hemorrhage or endotoxemia. thus, the role of p38 mapk in the development of ali is still uncertained. as a result, p38 mapk has not been proposed as a valid target for clinical management of ali/ards. in order to examine the role of p38 mapk in ali, we then analyzed the expression of p38 mapk and evaluated the effects of oxymatrine. the expression of p38 map kinase was shown using western blotting analysis. results showed that phosphorylated p38 map kinase was augmented in the oleic acid group compared with the control group, whereas expression of phosphorylated p38 map kinase was attenuated in the oxymatrine group compared with the oleic acid group in a concentration-dependent manner. this suggested that the administration of oxymatrine before ali markedly attenuate the activation of p38 map kinase during lung injury. it has been reported that tnf-␣ is augmented via activation of the p38 map kinase-related intracellular signal pathway (johnson et al., 1989; lee et al., 1994) . oxymatrine markedly attenuated the phosphorylation of p38 map kinase and disturbed the mechanism for the production of tnf-␣, thus resulting in the lower serum tnf-␣ level. from these findings, we conclude that oxymatrine ameliorates ali by attenuating the production of proinflammatory cytokines, and that this attenuation is associated with suppression of p38 map kinase activation. in addition, our results confirm that p38 map kinase does play an important role in the development of oleic acid-induced ali. based on our results and previous reports, we expect that p38 map kinase may become a promising target for clinical management of ali, although it needs to be supported by further animal experiments and clinical data. reports have shown that there are at least three types of map kinase: extracellular signal-regulated protein kinase (erk1/2; p42/p44); c-jun n-terminal protein kinase (jnk); p38 map kinase (davis, 1993; kyriakis et al., 1994; bogoyevitch et al., 1995; han et al., 1995; raingeaud et al., 1995; xia et al., 1995) . the nf-b pathway is also reported to be involved in the process of ali. together with the three types of mapk, nf-b may create a network to regulate inflammatory responses in ali. in this study, we only studied the effect of oxymatrine on p38 mapk. further study is needed to elucidate whether oxymatrine has modulative effects on other pathways. in china, pure oxymatrine injection has been available in hospital for treatment of hepatitis and tumor for many years. however, it has not been used for ali/ards/sars in clinic. since our results indicate that oxymatrine prevents mice from oleic acid-induced ali, we hope that oxymatrine can be used to treat ali/ards/sars although further research should be carried out on more animal experiments before clinical trials. in this paper, we found oxymatrine had a beneficial effect on ali in mice for the first time. 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key: cord-023390-5hcgdlmt authors: bhuvanath, s.; nilkaeo, a. title: inflammatory cytokine modulation of cancer cell proliferation date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bi.x sha: doc_id: 23390 cord_uid: 5hcgdlmt inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell‐mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il‐1( and il‐18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf‐7), oral carcinoma cell line (kb), colon cancer cell line (caco‐2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-001293-dfaxj3bv authors: cavaillon, jean-marc; eisen, damon; annane, djilalli title: is boosting the immune system in sepsis appropriate? date: 2014-03-24 journal: crit care doi: 10.1186/cc13787 sha: doc_id: 1293 cord_uid: dfaxj3bv a relative immunosuppression is observed in patients after sepsis, trauma, burns, or any severe insults. it is currently proposed that selected patients will benefit from treatment aimed at boosting their immune systems. however, the host immune response needs to be considered in context with pathogen-type, timing, and mainly tissue specificity. indeed, the immune status of leukocytes is not universally decreased and their activated status in tissues contributes to organ failure. accordingly, any new immune-stimulatory therapeutic intervention should take into consideration potentially deleterious effects in some situations. refinements of supportive care of patients with severe sepsis have decreased their overall mortality, but no adjuvant drug therapy has emerged despite strenuous efforts in the field. twenty years have passed since the first patients with sepsis were included in clinical trials based on the understanding that tnf orchestrates the inflammatory response and should be the target for therapeutic intervention. in response to the failure of therapies aiming to target either the up-stream microbial activators or the effector molecules of the inflammatory cascade, a new concept has emerged of boosting the immune system to counter immunosuppression that develops in patients who survive the initial, hyperinflammatory period of sepsis [1] . inflammation is a highly sophisticated and complex response that fundamentally 'aims' to protect the host. in this review, we argue against the promulgation of what we believe is a misleading perception of sepsis inducing secondary immunosuppression. the possible negative consequences of immune system-boosting therapy are paradoxical properties have also been reported for il-10, the prototypic anti-inflammatory cytokine. its proinflammatory activity been established in human volunteers receiving endotoxin injection [6] . our own in vitro studies showed that adherence of human monocytes modulated the effect of il-10 on expression of 16 genes, including 'suppressor of cytokine stimulation' (socs) molecules, in the opposite direction as compared with non-adherent cells [7] . these observations illustrate the statement by moore and colleagues that 'il-10 can effect very different outcomes depending on timing, dose, and location of expression. in some scenarios, the expected immuno-suppressive activities are observed, while in others, il-10 enhances immune or inflammatory responses' [8] . among other cytokines classified as antiinflammatory, transforming growth factor-beta (tgfβ) may behave as pro-inflammatory mediator as tgfβtransgenic mice are more sensitive to lps-induced shock [9] and some of its inflammatory activities reflect its capacity to favor the differentiation of t helper (th) 17 and production of the pro-inflammatory il-17. the classification of non-cytokine inflammatory mediators also relies on an overly simplistic division between proand anti-inflammatory properties. this is well illustrated by prostaglandin e 2 (pge 2 ), a key mediator of infectious immunopathology. on one hand, pge 2 induces fever, increases vascular permeability, increases vasodilatation, and causes pain while also inhibiting production of tnf, increasing production of il-6, inhibiting 5-lipoxygenase and leukotriene a4 generation, and inducing 15lipoxygenase and the generation of the lipoxins involved in inflammation resolution. on the other hand, pge 2 has inhibitory properties on macrophages, neutrophils, th1 lymphocytes, natural killer (nk) cells, and cytotoxic lymphocytes but activates mast cells, th2, th17, and regulatory t lymphocytes (t reg ) [10] . this panoply of pge 2 -stimulated events amply demonstrates the inability to simply characterize the activities of this and the other molecules mentioned as pro-or anti-inflammatory. in an early anti-tnf monoclonal antibody intervention study, a significant improvement in day 3 survival was observed between the antibody-treated group and the placebo group [11] . although this was not a prespecified primary outcome, it is interesting to see that the treatment targeting tnf consisting of a single early injection was beneficial within a short period of time after sepsis onset, reinforcing the idea that tnf plays a key deleterious role in the early events of sepsis. once anti-tnf treatments were better targeted to the sickest patients by adding biological inclusion parameters (plasma il-6 level), survival was significantly improved on day 28 [12] . synergistic effects between immune modulators are a key characteristic of their effect. this explains how a non-lethal dose of one cytokine can lead to mortality when injected with a non-lethal dose of another cytokine. similarly, it may explain how the removal of some inflammatory mediators by coupled plasma filtrationadsorption was protective in an endotoxin-shock model while levels of circulating bio-active tnf were unaffected [13] . clear demonstrations of cytokine-mediated tissue damage exist. nevertheless, because of their ambiguous role mentioned above, identification of their precise role during sepsis has led to controversy. in animal models of sepsis, the role of tnf may vary depending upon the type of infection [14] . many model parameters influence conclusions of the relative role of the different mediators studied. identical cytokines have been found to be protective or deleterious depending upon the model. this has been the case for ifnγ [15] and granulocytemacrophage colony-stimulating factor (gm-csf) [16] among others (for example, il-17, il-33, 'tnf-related apoptosis-inducing ligand' (trail), and tgfβ). host-protective innate immune responses and consequent inflammation are inextricably linked and overlapping. consequently, the same cellular actors are key elements defending the host against infection while simultaneously contributing to deleterious events. for example, neutrophil extracellular traps that catch and kill bacteria and fungi are associated with the release of elements such as histones and mitochondria that behave like damage-associated molecular patterns perpetuating the inflammatory process. beneficial or deleterious roles of the same leukocyte subset have been reported depending upon the experimental model. for example, a peritonitis model using nude mice (lacking t cells) suggested that t lymphocytes contribute to protective immune responses [17] . by contrast, in an escherichia coli sepsis murine model, t lymphocytes markedly contributed to severity [18] . similarly, t reg improved survival in polymicrobial sepsis [19] whereas, in another report, reduced t reg activity led to improved survival [20] . the 'half angel/half devil' role of nk cells during severe infection is also described. nk cells contribute to systemic inflammation during polymicrobial sepsis but play a critical protective role in host defense against staphylococcus aureus lung infection (as reviewed in [21] ). although apoptosis of dendritic cells (dcs) is particularly increased during sepsis, they are protective in murine polymicrobial sepsis [22] . transcriptomic analysis of dcs in trauma patients shows a large number of upregulated inflammatory genes, suggesting their contribution to systemic inflammation and organ failure [23] . apoptosis of lymphocytes, dcs, and nk cells is a hallmark of sepsis. hotchkiss and colleagues [24] provided key experiments demonstrating that lymphocyte apoptosis was deleterious and its prevention highly protective. in addition to the depletion of apoptotic lymphocytes that contribute to the alteration of the immune status, apoptotic t cells themselves can further produce an immunosuppressive milieu following their release of tgfβ [25] . in contrast, the apoptosis of neutrophils is reduced. interestingly, injection of apoptotic neutrophils in lps-challenged mice with or undergoing cecal ligature puncture improved outcomes [26] . this may be due to the capacity of apoptotic neutrophils to limit the production of il-1 and tnf by lps-activated monocytes and to favor the production of il-10 and tgfβ [27] . favoring neutrophil apoptosis while differentially preventing that of lymphocytes and dcs would represent a considerable interventional challenge! in sepsis, apoptosis does not only affect immune cells. apoptosis of epithelial cells, endothelial cells, neurons, and cardiac myocytes is reported with crucial effects of loss of altered barrier function ( figure 1 ): in the lungs -acute lung injury and adult respiratory response syndrome [28] ; in the kidneys -acute kidney injury [29] . enhanced translocation of bacteria and bacterial products occurs consequent on intestinal epithelial cell apoptosis [30] , contributing to the concept of the gut as the motor of multiple organ failure (mof). sepsis-induced cardiac myocyte apoptosis produces altered contractility and cardiac dysfunction [31] . apoptosis of endothelial cells [32] induces vascular leakage. finally, microglial and neuronal apoptosis may follow autonomic failure that precedes shock and mof [33] . in addition to epithelial apoptosis, tight junction alterations enhance organ dysfunction. it has been demonstrated that nitric oxide favors disruption of epithelial cell tight junctions in numerous organs, including liver, gut, and lung. leukotrienes favor protein extravasation as shown in the kidney of septic mice [34] . still, cytokines remain the main orchestrators of these tissue injuries. in a model of acute kidney injury, it was nicely demonstrated that inflammatory cytokines, including tnf and il-17, cause small intestine and liver injury [35] . among others, il-17a is critical for generation of intestinal ischemia/reperfusion injury and subsequent liver and kidney injury [36] . all together, the altered functions of epithelial cells, endothelial cells, neurons, and cardiac myocytes contribute to mof that may influence outcomes in sepsis more than altered immune status. the concomitant occurrence of inflammation, anti-infectious response, and altered immune status in sepsis when roger bone coined the concepts of systemic inflammatory response syndrome (sirs) and compensatory anti-inflammatory response syndrome (cars), he conceived that one or the other would be predominating figure 1 during sepsis, many types of cells (but not neutrophils) display enhanced apoptosis, leading to various deleterious consequences. aki, acute kidney injury; ali/ards, acute lung injury/acute respiratory distress syndrome; nk, natural killer. [37] . however, we contend that cars should be considered an adapted compartmentalized response with the aim of silencing some acute pro-inflammatory genes and maintaining the expression of certain genes involved in the anti-infectious process. despite our views [38] , authors still propose a two-wave concept with sirs appearing before cars, although they admit that 'rigorous examination of previous studies provides evidence that both proinflammatory and opposing anti-inflammatory response(s) occur concomitantly in sepsis' [1] . tamayo and colleagues [39] studied a large panel of circulating cytokines in patients with sirs or sepsis, concluding that both pro-and anti-inflammatory mediators play roles from the very beginning of this life-threatening condition. similarly, meta-analysis of 12 transcriptomic studies including 784 individuals led to the conclusion that 'the arbitrary distinction of separating sepsis into proinflammatory and anti-inflammatory phases is not supported by gene-expression data' [40] . immune status has been studied frequently by measuring tnf or other inflammatory cytokine production by circulating monocytes in response to lps [41] . we studied patients undergoing abdominal aortic surgery, showing that reduced expression of human leukocyte antigen (hla)-dr on cd14 high monocytes occurs during surgery [42] . similarly, hla-dr expression was already reduced on monocytes taken very soon after severe trauma at accident scenes [43] . altered tnf production capacity of circulating cells in response to tlr2 or tlr4 agonists is also observed very soon after injurious insults, such as on admission of patients after cardiac arrest [44] . even if soon after the initial insult the intensity of the inflammatory response reaches its peak, there is a persistent inflammation associated with altered immune status in surviving patients [45] . the more severe the insult, the more profound is the alteration and the more chance the patients have to develop adverse clinical outcome. the immune status of leukocytes during sepsis and sirs varies depending on the compartment in which they reside terms such as 'immunoparalysis, immunosuppression, and anergy' are far too extreme to describe the immune status of circulating leukocytes in patients with sepsis or sirs. altered immune status of circulating leukocytes is not globally present. indeed, some functions like phagocytosis remain unaltered [46] , and ex vivo cytokine production in response to heat-killed s. aureus (hksa) remains unchanged in patients with sepsis [47] compared with healthy controls. this is in full agreement with the observation that lps primes hksa-induced tnf production in macrophage cell lines instead of leading to cross-tolerance [48] . while the concept of endotoxin tolerance is considered to partially mimic the alteration of immune status in sepsis, it is worth mentioning that cross-tolerance between microbial agonists is not invariant. for example, candida albicans and fungal cell wall β-glucan also prime lps-induced proinflammatory cytokine production [49] . these observations led us to propose the concept of leukocyte reprogramming [50] to explain the fact that tolerised macrophages retain anti-infectious properties. in addition, in tissues, there are numerous examples to illustrate the hyper-activity of these cells. for example, in mice with polymicrobial sepsis alone or as a 'second hit' after traumatic hemorrhage, it was nicely demonstrated by chaudry's group [51] that the ex vivo production of tnf or il-6 after lps activation was significantly reduced among peripheral blood mononuclear cells and splenic macrophages but that it was enhanced in alveolar and kupffer cells. similarly, in a murine model of trauma, the cytokine productive capacity of kupffer cells and alveolar macrophages was enhanced [52] . indeed, macrophage functions differ depending on the compartment from which they derive. we established [53] that the specific cytokine and cellular microenvironment within the lung was responsible for this particular resistance of alveolar macrophages to endotoxin tolerance, which can also be observed in human alveolar macrophages [54] . similarly, in kidneys, in response to a second challenge with lps, the expression of tnf and inducible nitric oxide synthase was further enhanced [55] . this may explain why unilateral nephrectomy could be protective in a murine peritonitis model and after lps injection [56] . most importantly, despite the fashionable concept of m1/m2 macrophages, the response of macrophages to il-4 and ifnγ is in fact completely different depending upon their origin [57] . as a consequence of this great heterogeneity of immune cells within different compartments, each tissue behaves independently, contributing to the global inflammatory response with a specific pattern, as illustrated by differential cytokine expression in liver, lungs, heart, brain, muscle, kidney, intestine, and spleen [58] . another example of the different behavior of leukocytes in various compartments is the frequent occurrence of hemophagocytosis (>60%) directly observed in the bone marrow of the critically ill [59] . this phenomenon is associated with extreme production of inflammatory cytokines. accordingly, it has been proposed that when hemophagocytosis is diagnosed in critical care patients, aggressive immunosuppressive therapy be undertaken without delay [59] . differences between cells harvested from different compartments after sepsis have also been reported for spleen and peritoneal myeloid dcs [60] . the major differences between compartments are further illustrated by the fact that gene deficiency may differentially affect outcomes of infection. for example, il-10 deficiency protects against francisella tularensis pulmonary infection but aggravates cutaneous infection [61] . similarly, we showed that scavenger receptor-a (sr-a), 'macrophage associated receptor with a collagenous base' (marco), cd36, or tlr2 deficiency protect mice against peritoneal s. aureus infection while these deficiencies aggravated pneumonia [62] . interestingly, when streptococcus pneumoniae was the pathogen used to colonize the murine nasopharynx, marco ko mice (but not sr-a ko mice) had significantly impaired clearance of pneumococcal colonization [63] . furthermore, inflammatory foci cells may not behave similarly to cells from other healthy compartments. for example, it was shown that neutrophils derived from sputum of patients with chronic bronchitis or cystic fibrosis are insensitive to inhibitory effects of il-10 in contrast to circulating neutrophils [64] . the concomitant presence of inflammation within tissues and altered immune status within the hematopoietic compartment is short-lived in murine models rendering them inappropriate to study patients with concomitant sepsis and cars [65] . in addition, mice are highly resistant to bacteria like s. aureus and their serum contains factors that limit inflammatory response intensity as compared with human serum [66] . a most provocative report comparing transcriptomic patterns of circulating cells from trauma patients, human endotoxemia-model participants, and murine-model equivalents revealed total absence of correlation [67] . when most therapeutic approaches have been validated in preclinical studies performed with murine models, one understands why those were not the most appropriate ones. the scientific community needs to reconsider models used to validate therapeutic approaches. if murine responses do not resemble human processes, maybe other species, like the pig, should be preferred. porcine monocytes and lps-activated macrophages are closer to their human counterparts than murine cells [68] . of course, murine models remain valuable to further decipher the mechanisms of sepsis. the best example is the two-hit model, which demonstrated that the nature of the first hit and its severity, the nature of the infection, and the route of infection may influence the outcome in a completely opposite direction [69] . are patients with sepsis dying of immune failure -dissecting the arguments used to describe compensatory immunosuppression occurring after sepsis? the clinical observations used to argue that immunosuppression occurs in sepsis patients surviving the initial inflammatory cascade [1] are in essence that patients develop nosocomial infections due to opportunistic pathogens, including reactivated chronic viral infections, and that patients who die after sepsis have unresolved foci of infection. these underpinning observations require further consideration. representing bacteria such as enterococcus faecium, stentrophomonas maltophilia, and pseudomonas aeruginosa along with candida as 'opportunistic pathogens' overstates the role of sepsis-induced immune dysregulation as the primary cause of nosocomial infection in these patients. these multiply-instrumented, high-intensity care, bed-bound, vulnerable patients often have breaches in their integument and mucous membranes (airways, surgical sites, indwelling catheters) and perturbed microbiomes from antibiotic treatments. overgrowth of antibioticresistant microorganisms and barrier defects predispose them to secondary infections, even without overt defects in their immune defenses [70] . these are all organisms of normal virulence that cause nosocomial infections in sepsis patients because of the selection pressure of potent antibiotics and the presence of biofilm affected/colonized intravascular and urinary catheters. additionally, reactivation of herpes simplex virus (hsv) and cytomegalovirus (cmv) may have some clinical relevance in critically ill patients. cmv-emia is quite common in patients with sepsis (30% in some studies) and is at least associated with worse outcome in icu patients in recent meta-analyses. whether cmv could cause immune compromise itself, be a reflection of immune compromise, or simply be an indicator of poor outcome in patients with sepsis remains unclear [71] . reactivation of oro-labial hsv is extremely common in sepsis, and hsv can frequently be detected in respiratory secretions. however, only one study has reliably investigated lower respiratory tract infection in critically ill, immunocompetent patients, showing that 21% of patients with ventilator-associated pneumonia (vap) had bronchopneumonitis due to hsv [72] . in 55% of these patients, the vap appeared to be due to hsv alone. however, acyclovir treatment had no impact on the outcome in patients with hsv bronchopneumonitis [72] . of greater relevance to predisposition to nosocomial infection in sepsis patients remaining in icus for prolonged periods are physical breaches in innate immune system barriers. intravascular catheters, endotracheal tubes with consequently increased dead space, and increased gastric ph due to peptic ulcer prophylaxis regimens are all, along with broad-spectrum antibiotics, potent promoters of nosocomial infection. post-mortems (pms) identifying unresolved infection foci are not reliable proof that patients are dying of sepsis. pneumonia is frequently present in patients in whom supportive care is withdrawn due to failure to thrive. where pneumonia has been found more frequently at pm than was appreciated ante-mortem, the extent of pulmonary involvement was not quantified [73] . in this series, there was clear agreement by clinical and pm assessment that mof was the commonest cause of death [73] . these data call into question the relevance of unresolved, pm infection in patients dying in the icu as a direct indicator of immunosuppression following as a direct consequence of previous sepsis. if the patients die with infectious foci and altered immune status, it does not mean they die because of them. because of the monocyte deactivation in sepsis, it was proposed to restore it with the use of either ifnγ or gm-csf, two cytokines that counteract endotoxin tolerance. the first attempt was successfully performed in nine septic patients who received subcutaneous ifnγ that restored ex vivo cytokine production and hla-dr expression by monocytes [74] . the authors claimed that overall mortality was lower in the treated group compared with historical controls. in mechanically ventilated trauma patients, ifnγ was aerosolized. however, in a previous phase iii study in burn patients, ifnγ had failed to protect patients from infection or decrease mortality [75] . we must recall that ifnγ injection increases mortality in animal models of polymicrobial infection [15] . all together, these data have limited the routine use of ifnγ in icu patients, although a dutch clinical trial is ongoing. gm-csf has been demonstrated to be able to restore some immune status parameters. however, a metaanalysis concluded that gm-csf did not significantly reduce in-hospital mortality, although it significantly increased infection recovery [76] . although no adverse effects were reported, it is worth recalling a case report of a patient who developed a fatal adult respiratory distress syndrome after gm-csf treatment [77] . in animal models, gm-csf favors lps-induced lung inflammation, amplifying lps-induced bronchoconstriction [78] . gm-csf favors production of tnf and il-1. in a recent study, it was confirmed that gm-csf synergizes with lps, promoting il-1β secretion [79] . lethal injection of lps in gm-csf receptor ko mice led to far lower mortality among these mice as compared to wild type mice. given all the efforts made by some authors to convince the scientific community of the use of gm-csf, it is challenging to read the conclusion of this present paper given that gm-csf has been previously underestimated as a target for therapeutic intervention in many bacterial infections and inflammatory disorders associated with the production of il-1β. il-7 is another cytokine that is promoted for the treatment of sepsis and that is supported by murine and human ex vivo tissue data [1, 80] . one can conjecture that systemic treatment with il-7 may act in undesired places, as illustrated by the following: il-7 worsens graft-versus-host-induced tissue inflammation [81] ; favors inflammation in colitis [82] , contributes to arthritis severity [83] ; upregulates chemokines, ifnγ, macrophage recruitment, and lung inflammation [84] ; and, finally, increases production of inflammatory cytokines by monocytes and t cells [85] . many other cytokines (for example, il-2, il-12, il-15, and tnf) can boost the immune system and are reported to be beneficial in murine sepsis models. however, one wonders whether systemic treatment with any immunostimulating cytokine may act on tissue leukocytes boosting the inflammatory process while boosting immune status as well. in this perspective, the attempt to treat peripheral mononuclear cells of sepsis patients ex vivo with il-2 before re-injecting them is an interesting approach that prevents the delivery of this cytokine to the bloodstream, allowing it to act strictly on the desired cells [86] . in this study, the mortality was 8% in the extracorporeally treated group of patients (n = 121) but was 21% in the patients receiving standard treatment (n = 52). rather than repeating the mistakes of past experimental treatments for sepsis in which therapies were developed after successful preclinical models that may be far from mimicking human disease, it would be ideal to proceed in the future with new treatments in which extensive human data are available prior to embarking on expensive licensure studies. furthermore, identifying currently licensed drugs with tolerable safety profiles as potential sepsis agents leap-frogs costly drug development and early-phase human studies. in animal models, extant licensed drugs, such as chloroquine [87] and androstenenediol [51] , have successfully restored immune status. most interestingly, in the latter case, the treatment protected mice against polymicrobial sepsis and boosted altered ex vivo cytokine production observed with peripheral blood cells and spleen macrophages, dampening production observed with alveolar macrophages and kupffer cells. a similar compartmentalized adapted specificity was reported with estradiol [88] . other approaches involve pro-resolving lipid mediators [89] , although it is uncertain whether they may also adversely boost immune status. the recently recognized aspirin-triggered lipoxins, anti-inflammatory mediators of inflammation resolution, make aspirin a possible inexpensive agent for both prevention and treatment of sepsis. considerable observational cohort data show improvements in mortality in patients with sepsis pretreated with aspirin [90] . this approach is being prospectively studied as part of an aspirin primary prevention trial. could other immunomodulatory approaches be considered with less putative dangerous consequences on inflamed tissues. this may be the case of thymosin-α1. indeed, a very promising study demonstrated its efficiency to improve clinical outcome in patients with severe sepsis [91] , after a preliminary investigation had demonstrated a better performance with respect to organ failure scores in thymosin-α1-treated patients with sepsis arising from intra-abdominal infection due to carbapenem-resistant bacteria [92] . however, one must call for caution since thymosin-α1 can also favor the production of inflammatory cytokines and nitric oxide and further increases the percentage of t reg cells [93, 94] . still, very little is known of its effect on leukocytes present in different compartments. the cell surface molecules containing in their intracytoplasmic domain an immunoreceptor tyrosine-based inhibition motif -such as programmed death-1 (pd-1), b and t lymphocyte attenuator (btla), and cytotoxic t-lymphocyte antigen 4 (ctla-4) -could also be interesting targets for new therapeutic approaches. the expression of pd-1 on t cells and its ligand (pd-l1) on monocytes is upregulated in critically ill [95] or septic shock [96] patients. increased expressions were associated with increased occurrence of secondary nosocomial infections and mortality after septic shock [97] . not only are pd-1-deficient mice markedly protected from the lethality of sepsis, accompanied by a decreased bacterial burden and suppressed inflammatory cytokine response [98] , but also blockade of pd-1 or pd-l1 improves survival in a murine model of sepsis, reverses immune dysfunction, inhibits lymphocyte apoptosis, and attenuates organ dysfunction [99] [100] [101] . the relevance of these observations in human settings is still needed. ctla-4 is a high-avidity receptor for cd80 and cd86. enhanced ctla-4 expression was demonstrated more frequently in patients with sepsis than in non-infected critically ill patients or control subjects [102] , and blocking ctla-4 improved survival in bacterial and fungal experimental sepsis [103, 104] . however, the use of such an approach seems tricky since, in animal models at high dose, anti-ctla-4 could worsen survival [103] , and the use of abatacept (a soluble ctla-4 dimerized with an fc fragment of immunoglobulin) led to increased survival in invasive pneumococcal infection [105] . similarly, btla expression is enhanced in patients with sirs or sepsis [106] and, in a murine model of sepsis, btla-deficient mice displayed an enhanced resistance [107] . in contrast, these mice displayed enhanced susceptibility to endotoxin-induced shock [108] . accordingly, the exact role of btla needs to be further deciphered before strategies targeting btla could be proposed to treat patients with sepsis. new therapeutic approaches to treat sepsis should take into consideration that the immune status of leukocytes in the peripheral blood might be quite different from those present in inflamed tissues. we believe that a systemic approach to immune stimulation is not appropriate if immune cells are boosted generally, independent of their location. an ideal drug would limit the overzealous inflammatory process that leads to organ failure and favor homeostatic responsiveness of leukocytes ( figure 2 ). this is the challenge we have to address if we wish to avoid further decades of disillusionment. figure 2 new therapeutic interventions should address both the events in the tissues that lead to organ failure and the altered immune status of leukocytes restricted to some specific compartments. cars: compensatory anti-inflammatory response syndrome; cmv: cytomegalovirus; ctla-4: cytotoxic t-lymphocyte antigen 4; dc: dendritic cell; gm-csf: granulocyte-macrophage colony-stimulating factor; hksa: heat-killed staphylococcus aureus hsv: herpes simplex virus; ifnγ: interferon-gamma lps: lipopolysaccharide; marco: macrophage-associated receptor with a collagenous base; mof: multiple organ failure; nk: natural killer pd-1: programmed death-1; pd-l1: programmed death-1 ligand prostaglandin e 2 ; pm: post-mortem; sirs: systemic inflammatory response syndrome; sr-a: scavenger receptor-a; tgfβ: transforming growth factor-beta th: t helper; tlr: toll-like receptor; tnf: tumor necrosis factor; t reg : regulatory t lymphocyte; vap: ventilator-associated pneumonia immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach paradoxical anti-inflammatory actions of tnf-alpha: inhibition of il-12 and il-23 via tnf receptor 1 in macrophages and dendritic cells tnf suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis interleukin 1 receptor signaling regulates duba expression and facilitates toll-like receptor 9-driven antiinflammatory cytokine production systemic interferon-gamma suppresses the development of endotoxin-induced uveitis in mice proinflammatory effects of il-10 during human endotoxemia adib-conquy m: adherence modifies the regulation of gene expression induced by interleukin-10 a: interleukin-10 and the interleukin-10 receptor hepatic 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experimental sepsis by inhibiting lymphocyte apoptosis and reversing monocyte dysfunction pd-l1 blockade attenuated sepsis-induced liver injury in a mouse cecal ligation and puncture model decreased response to recall antigens is associated with depressed costimulatory receptor expression in septic critically ill patients dose-dependent effect of anti-ctla-4 on survival in sepsis blockade of the negative co-stimulatory molecules pd-1 and ctla-4 improves survival in primary and secondary fungal sepsis inhibition of t cells provides protection against early invasive pneumococcal disease b and t lymphocyte attenuator expression on cd4+ t-cells associates with sepsis and subsequent infections in icu patients btla expression contributes to septic morbidity and mortality by inducing innate inflammatory cell dysfunction b and t lymphocyte attenuator inhibits lps-induced endotoxic shock by suppressing toll-like receptor 4 signaling in innate immune cells cite this article as: cavaillon et al.: is boosting the immune system in sepsis appropriate? critical care key: cord-002079-jne14jqf authors: macparland, sonya a.; ma, xue-zhong; chen, limin; khattar, ramzi; cherepanov, vera; selzner, markus; feld, jordan j.; selzner, nazia; mcgilvray, ian d. title: lipopolysaccharide and tumor necrosis factor alpha inhibit interferon signaling in hepatocytes by increasing ubiquitin-like protease 18 (usp18) expression date: 2016-05-27 journal: j virol doi: 10.1128/jvi.02557-15 sha: doc_id: 2079 cord_uid: jne14jqf inflammation may be maladaptive to the control of viral infection when it impairs interferon (ifn) responses, enhancing viral replication and spread. dysregulated immunity as a result of inappropriate innate inflammatory responses is a hallmark of chronic viral infections such as, hepatitis b virus and hepatitis c virus (hcv). previous studies from our laboratory have shown that expression of an ifn-stimulated gene (isg), ubiquitin-like protease (usp)18 is upregulated in chronic hcv infection, leading to impaired hepatocyte responses to ifn-α. we examined the ability of inflammatory stimuli, including tumor necrosis factor alpha (tnf-α), lipopolysaccharide (lps), interleukin-6 (il-6) and il-10 to upregulate hepatocyte usp18 expression and blunt the ifn-α response. human hepatoma cells and primary murine hepatocytes were treated with tnf-α/lps/il-6/il-10 and usp18, phosphorylated (p)-stat1 and myxovirus (influenza virus) resistance 1 (mx1) expression was determined. treatment of huh7.5 cells and primary murine hepatocytes with lps and tnf-α, but not il-6 or il-10, led to upregulated usp18 expression and induced an ifn-α refractory state, which was reversed by usp18 knockdown. liver inflammation was induced in vivo using a murine model of hepatic ischemia/reperfusion injury. hepatic ischemia/reperfusion injury led to an induction of usp18 expression in liver tissue and promotion of lymphocytic choriomeningitis replication. these data demonstrate that certain inflammatory stimuli (tnf-α and lps) but not others (il-6 and il-10) target usp18 expression and thus inhibit ifn signaling. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with usp18 representing a potential target for intervention in various inflammatory states. importance inflammation may prevent the control of viral infection when it impairs the innate immune response, enhancing viral replication and spread. blunted immunity as a result of inappropriate innate inflammatory responses is a common characteristic of chronic viral infections. previous studies have shown that expression of certain interferon-stimulated genes is upregulated in chronic hcv infection, leading to impaired hepatocyte responses. in this study, we show that multiple inflammatory stimuli can modulate interferon stimulated gene expression and thus inhibit hepatocyte interferon signaling via usp18 induction. these findings represent a new paradigm for how inflammation alters hepatic innate immune responses, with the induction of usp18 representing a potential target for intervention in various inflammatory states. i nterferon (ifn) is a key endogenous mediator of viral clearance by the innate immune response. one excellent example of this is hepatitis c virus (hcv) infection of the liver (1) . interferon signaling drives the expression of multiple interferon-stimulated genes (isgs), which mediate viral clearance. isgs, however, can also be induced by other factors, including inflammatory stimuli such as tumor necrosis factor alpha (tnf-␣) (2) . hcv infection, which induces chronic inflammation of the liver, is associated with high serum tnf-␣ (3, 4) . interestingly, anti-tnf-␣ treatment has been shown to lead to an improved virologic response to ifn-␣/ribavirin antiviral therapy for hcv (5) , while other data show safety of combined treatment but no effect on hcv viral loads of hcv treatment-naive individuals (6) . the purpose of the present study is to define an important causal link between inflammation and the host hepatic innate immune response, with broad viral relevance. our recent work in the host innate immune response to chronic hcv infection suggested an association between hepatocytes and the liver resident immune cells that drive liver inflam-mation (7) (8) (9) . there is a dichotomous response to chronic hcv infection in the liver, and this response predicts who will and who will not respond to exogenous ifn-␣ treatment. in patients that do not respond to therapy with ifn/ribavirin, hepatocytes have strong preactivation of the ifn-response, with high expression of a subset of isgs (7) (8) (9) . in patients exhibiting a sustained virologic response on therapy with ifn/ribavirin, tissue macrophages show a high expression of isgs (7, 9) . the expression of isgs in macrophages or hepatocytes is more predictive of treatment outcome than il28b polymorphisms (7) ; this finding suggests a link between hepatic inflammatory cell activation and viral clearance. liver kupffer cell inflammatory responses are induced by and play a role in the control and clearance of multiple viral infections of the liver (10, 11) . although the mechanisms underlying these patterns are undoubtedly complex, the association between liver macrophages and hepatocytes raises the question of how the hepatocytes react to the cytokines produced by the macrophages (and other inflammatory cells). if isg expression in hepatocytes reflects a response to inflammatory stimuli, then how the liver responds to a viral infection will be modulated by the inflammatory milieu. in hcv infection, we have found that the isg15/usp18 pathway is an important regulatory pathway. both isg15 and usp18 are isgs that are strongly upregulated in the livers of ifn treatment-resistant patients (7) (8) (9) . isg15 is a ubiquitin-like protein that is strongly upregulated by ifn and conjugates to multiple cellular proteins (12, 13) . usp18 is an isg15-specific protease that is also upregulated by ifn-␣ (12, 13) . both isg15 and usp18 have been suggested to blunt type 1 ifn signaling (12, (14) (15) (16) . in addition to its ability to remove isg15 from its conjugated proteins, usp18 has been shown to bind to the type 1 ifn receptor and blunt ifn signaling (15) . upregulation of usp18 may represent a negative-feedback loop counteracting the effects of type 1 ifn. furthermore, knockdown of usp18 increases both isg induction and anti-hcv activity of ifn-␣ (14) , and data have shown that ifn-␣ treatment given to mice in vivo increases hepatic usp18 and blunts the effect of a subsequent dose of ifn-␣ (12) . the mechanisms controlling usp18 expression in the liver are poorly understood but will have relevance to understanding innate immune mechanisms in chronic viral infection. although the majority of work on usp18 has centered on its upregulation by type 1 ifn, interferon stimulated genes have multiple upregulatory stimuli other than ifn-␣ (2, 17) . for example, inflammatory stimuli, e.g., endotoxin and lipopolysaccharide (lps), have been shown to upregulate usp18 in peritoneal exudate macrophages (18) . if similar stimuli lead to increased usp18 expression in hepatocytes, then this pathway could represent a novel link between inflammatory and innate immune responses in the liver. the present study is based on the hypothesis that liver inflammation will directly impact hepatocyte expression of usp18 and therefore will impact ifn signaling. this link will have relevance to multiple diseases, since inflammatory stimuli such as tnf-␣ and lps have been shown to play roles in many liver diseases, including viral hepatitis (19) (20) (21) . the link may also help to explain our observations in chronic hcv infection, since chronic hcv infection is characterized by increased serum tnf-␣ (3, 4), increased hepatocyte usp18, and impaired ifn responsiveness (8) . quite apart from hcv, the importance of the link between hepatic inflammation and innate immune response lies in the downstream effects of the impairment of innate immunity. we speculate that by blocking ifn-␣ signaling, usp18 expression may lead to an enhanced susceptibility to infection with interferon-sensitive viruses and enhanced viral proliferation. support for this notion is found in a mouse study in which expression of usp18 in macrophages led to lower ifn responsiveness, leading to locally restricted replication of vsv (22) . in the present study, treatment of hepatic cells with lps and tnf-␣, but not il-6 or il-10, led to upregulated usp18 expression in hepatocytes. the enhanced usp18 expression was associated with decreased ifn-␣-stimulated expression of p-stat1 and isgs, a phenomenon reversed by usp18 knockdown. as an in vivo correlate of our in vitro findings, experimentally induced hepatic ischemia/reperfusion injury induced usp18 mrna expression in liver and enhanced lymphocytic choriomeningitis (lcmv) replication, an effect not seen in usp18 ϫ/ϫ mice. these data demonstrate that certain inflammatory stimuli (tnf-␣ and lps), as well as ischemic injury, but not other cytokines (il-6 and il-10) can lead to enhanced hepatocyte usp18 expression and thereby inhibit ifn signaling. these findings lend new knowledge to our understanding of how inflammation can modulate hepatic innate immune responses, with usp18 representing a potential target for intervention to reverse any proviral effect of inflammation. phorylated-stat1 (p-stat-1; cell signaling technology, boston, ma), rabbit polyclonal anti-stat-1 (santa cruz biotechnology), goat polyclonal anti-ube1 antibody (santa cruz biotechnology), or mouse monoclonal anti-actin (sigma) antibodies, followed by anti-mouse or anti-rabbit igg conjugated to horseradish peroxidase (calbiochem, billerica, ma). an enhanced chemiluminescence detection kit (amersham pharmacia biotech, uppsala, sweden) was used to determine the levels of protein expression. flow cytometric quantification of usp18 expression in huh7.5 cells. usp18 expression on huh7.5 cells treated with lps or tnf was quantified by using flow cytometry as previously described (28) . briefly, cells were fixed, permeabilized, and stained with mouse anti-human usp18 polyclonal antibody (abnova) or a relevant isotype-matched control antibody. staining was detected with a secondary goat anti-mouse igg labeled with fluorescein isothiocyanate (fitc; santa cruz biotechnology). the cells were acquired using a facscalibur cytometer (bd biosciences, san jose, ca). a minimum of 10,000 events were collected. the resulting data were analyzed using flowjo software (tree star, inc.). the experiments were repeated four times. sirna targeting murine usp18 and ube1l. usp18 small interfering rna (sirna) is a pool of three target-specific 19-to 25-nucleotide (nt) sirnas designed to knock down usp18 gene expression that was obtained from santa cruz biotechnology. the ube1l sirna used was a pool of three to five target-specific 19-to 25-nt sirnas designed to knockdown mouse ube1l gene expression and was obtained from santa cruz. usp18 and ube1l sirna was transfected into 5 ϫ 10 5 primary murine hepatocytes according to the manufacturer's instructions using the santa cruz sirna reagent system sc-45064 (santa cruz) and as previously described (14) . inhibition of inflammatory signaling in primary mouse hepatocytes. to inhibit lps-or tnf-␣-stimulated nf-b activation, primary mouse hepatocytes were incubated with the following inhibitors for 30 min prior to 6 h of stimulation with lps or tnf-␣: p6062, a protein kinase a inhibitor (29); nsc33994, a jak2 inhibitor (30); p3115, a protein kinase c inhibitor (31); pd098059, a mitogen-activated protein kinase kinase inhibitor (32); silibinin, an inhibitor of ikk␣ (33) ; and wortmannin, a phosphatidylinositide 3-kinase inhibitor (34) . the concentrations, sources, and main targets of these inhibitors are described in table 1 . after lps or tnf-␣ stimulation, hepatocytes were harvested and usp18 and il-1␤ (a surrogate of nf-b activation) mrna expression was evaluated as described below. rna isolation and quantitative real-time pcr analysis. huh7.5 cells and primary murine hepatocytes were treated with ifn-␣ (100 u/ml), tnf-␣ (20 ng/ml), lps (100 ng/ml), il-6 (100 ng/ml), or il-10 (10 ng/ ml) over a 24-h time course, and usp18 expression was determined by quantitative pcr (qpcr) as previously described (8, 9) . briefly, total rna was prepared from cells using the trizol reagent (invitrogen) according to the manufacturer's instructions. the reverse transcription reactions of the extracted rna were performed with a first-strand cdna synthesis kit (amersham pharmacia biotech) according to the manufacturer's directions. first, 1 g of extracted rna was added in a total volume of 15 l of combined cdna reaction reagents with random hexamer oligonucleotides as the first-strand primer in a 1.5-ml reaction tube. samples were heated to 65°c for 10 min, chilled on ice for 5 min, and incubated at 37°c for 1 h, followed by a 10-min incubation at 80°c. the specific primers for all of the detected genes for the pcrs were based on genbank-published sequences: mus musculus ubiquitin-specific peptidase 18 (usp18; nm_011909) forward primer (5=-tacagcagagagcagcagga) and reverse primer (5=-cacatgtcggagcttgctaa); mus musculus myxovirus (influenza virus) resistance 1 (mx1; nr_003520) forward primer (5=-tctgaggagagccagacaat-3=) and reverse primer (5=-actctggtccccaatgacag); mouse hypoxanthine guanine phosphoribosyltransferase (hprt; nm_013556) forward primer (5=-tcagt caacgggggacataaa) and reverse primer (5=-ggggctgtac tgcttaaccag); mus musculus il-1␤ (nm_008361) forward primer (5=-gaaatgccaccttttgacagtg) and reverse primer (5=-tgg atgctctcatcaggacag); homo sapiens ifn-␣2b (ifna2b; ay255838) forward primer (5=-gcttgggatgagaccctccta) and reverse primer (5=-cccaccccctgtatcacac); homo sapiens ifn-␥ (ifng; nm_000416) forward primer (5=-tcggtaactgacttgaatg tcca) and reverse primer (5=-tcgcttccctgttttagctgc); homo sapiens actin, beta-like 2 (actbl2; nm_001017992) forward primer (5=-gtctgccttggtagtggataatg) and reverse primer (5=-tcgagg acgccctatcatgg); homo sapiens ubiquitin specific peptidase 18 (usp18; nm_017414) forward primer (5=-aggagaagcgtccctt tcca) and reverse primer (5=-tggtccttaatcaggttccagag); and homo sapiens myxovirus (influenza virus) resistance-1 (mx1; nm_001144925) forward primer (5=-ggtggtccccagtaatgtgg) and reverse primer (5=-cgtcaagattccgatggtcct). quantitative real-time pcr was performed on an abi prism 7900ht machine (applied biosystems, foster city, ca) with sybr green realtime pcr master mix (applied biosystems) according to the directions provided by the manufacturer. all of the rna samples and controls were assayed in duplicate. real-time pcr conditions were as follows: 10 min at 95°c, followed by 45 cycles of 15 s at 95°c and 15 s at 60°c monitor fluorescence in sybr channel during a 60°c annealing/extension step. the results were analyzed by using applied biosystems sds2.2 software (applied biosystems). experimental hepatic ischemia/reperfusion injury model. hepatic ischemia/reperfusion injury (hiri) was simulated as before (35) . partial (70%) hepatic ischemia was induced for 60 min in mice, after which the surgical clamps were removed. control (sham) animals underwent anesthesia and laparotomy alone. animals were euthanized 6 or 24 h after ischemia/reperfusion, the affected liver segments were removed, and target gene expression was determined by qpcr in whole liver tissue. lcmv strain we was propagated in l929 cells (atcc ccl-1) as previously described (36) . in additional experiments, after 60 min hiri or sham laparotomy, mice were infected with 2 ϫ 10 6 pfu of lcmv we by intravenous tail vein injection. the ischemic liver lobes were harvested at day 7 postinfection. snap-frozen liver tissue samples were homogenized in ␣-mem (multicell, usa) supplemented with 5% fbs, l-glutamine, and 100 u of penicillin/ml plus 100 g of streptomycin/ml using the tissuelyser lt system (qiagen, netherlands). viral titers were examined on mc57 cells (atcc crl-2295) using a focus-forming assay as previously described (37) . statistical analysis. all data were analyzed using prism version 5.0 software (graphpad software, san diego, ca). statistically significant differences in fig. 1e were calculated by using a two-tailed student t test (prism software). the impact of hiri on lcmv replication (see fig. 6a and b) was evaluated by one-way analysis of variance with the tukey's post hoc test. usp18 is induced in hepatocytes by lps and tnf-␣ but not by il-6 and il-10. we have previously shown that usp18 can modulate the type 1 ifn response (14) . we sought to determine whether inflammatory stimuli could increase usp18 expression in hepatocytes. liver tissue inflammation is mediated by both proand anti-inflammatory stimuli, acting through diverse pathways. we therefore compared the effects of three proinflammatory stimuli (tnf-␣, lps, and il-6) and one anti-inflammatory stimulus (il-10), all four signaling through independent receptors and signaling cascades (38, 39) . in a 24-h time course experiment, usp18 mrna expression was measured after stimulation with lps (100 ng/ml), tnf-␣ (20 ng/ml), il-6 (100 ng/ml), or il-10 (10 ng/ml). usp18 mrna expression was induced by tnf-␣ and lps but not by il-6 or il-10 ( fig. 1a) . meanwhile, neither tnf-␣ nor lps treatments had a marked effect on mx1 mrna expression (fig. 1b) , indicating that the effects we observe are not due to a generalized upregulation of isgs or to type 1 ifn signaling. tnf-␣ and lps treatment also led to augmented usp18 protein expression by western blotting (fig. 1c ) and by intracellular staining (fig. 1d to f). treatment of huh7.5 cells with tnf-␣ or lps induced expression of usp18 in 74.1% ϯ 8.9% and 70.5% ϯ 5.2%, respectively, compared to untreated controls in which usp18 expression was 31.0% ϯ 4.7% (fig. 1fi) . usp18 induction was also demonstrated with a significant shift in the mean fluorescence intensity of usp18 staining after tnf-␣ or lps simulations (fig. 1fii ). in these assays, the degree of protein expression did not correlate completely with mrna expression, a finding that is consistent with many genes in the liver (40) in that the degree of mrna induction was higher than the observed usp18 protein expression. however, the functional effects we observed in terms of ifn-␣ responses were robust. tnf-␣ and lps block ifn-␣ signaling in huh7.5 hepatoma cells. to determine whether inflammatory stimuli such as tnf-␣ and lps can interfere with type 1 ifn signaling, we sought to determine whether pretreatment of hepatoma cells with tnf-␣ or lps blocked ifn-␣ signaling. huh7.5 cells were treated with tnf-␣ (20 ng/ml) or lps (100 ng/ml) for 24 h. the cells were subsequently treated with ifn-␣ for 6 h. we chose 6 h to measure mx1 expression based on previous descriptions of ifn-stimulated mx1 induction in the literature (41) and based on our observations with primary mouse hepatocytes. control cells were left untreated, followed by a 6-h ifn-␣ treatment at 24 h. after the 6-h ifn-␣ treatment, the cells were harvested, and mx1 expression was measured by qpcr as a measure of ifn-inducible gene expression. as expected, a 6-h exposure to ifn-␣ strongly induced mx1 expression, whereas a 24-h exposure to tnf-␣ and lps did not (fig. 2, columns 2, 3, and 5 ). both tnf-␣ and lps pretreatment inhibited the effect of 6 h of exposure to ifn-␣ (fig. 2, columns 2, 4, and 6 ), indicating that ifn-␣ signaling was impaired in the presence of these inflammatory stimuli. having shown that certain inflammatory stimuli both increase hepatocyte usp18 and blunt ifn-␣ signaling, we next sought to determine whether the blunting of ifn-␣ signaling is mediated via increased usp18. we addressed this question by selective knockdown of usp18 mrna. thus, huh7.5 cells transfected with usp18 sirna or control sirna were treated with lps (100 ng/ml), tnf-␣ (20 ng/ml), and/or ifn-␣ (100 iu/ml) for 2 h or pretreated with lps (100 ng/ml) or tnf-␣ (20 ng/ml) for 24 h and then either left untreated or treated with ifn-␣ (100 iu/ml) for 2 h (a 2-h time point was selected for optimal expression of pstat1 in huh7.5 cells). the expression of pstat1, stat1, and isg15 conjugates (as a marker of usp18 knockdown), usp18, and actin was detected by western blotting. as seen in fig. 3a , usp18 expression was largely knocked down in cells transfected with usp18 sirna. ifn-␣-induced phosphorylation of stat-1 was inhibited by pretreatment (for 24 h) with tnf-␣ or lps (fig. 3a , sirna control lanes f and h versus lane d), but this effect is reversed by knockdown of usp18 (fig. 3a , sirna usp18, lanes f and h versus lane d). of note, usp18 knockdown did not increase pstat1 in response to ifn-␣ before 24 h; these findings are consistent with previous observations (12) . we next wanted to confirm whether our findings from hepatoma cells would hold true in primary mouse hepatocytes. with this in mind, primary murine hepatocytes from usp18 ϩ/ϩ mice were isolated, transfected with usp18 sirna or control sirna, and then exposed to ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h. after a washing step, the cells were treated with ifn-␣ for an additional 6 h. mx1 isg mrna expression was measured by qpcr (normalized to expression of the hprt housekeeping gene) as an index of downstream ifn-␣ effect. ifn-␣, lps, and tnf-␣ treatment blocked the effect of the final dose of ifn-␣ (fig. 3b, columns 2, 3, 5, and 7) . however, knockdown of usp18 reversed this effect and augmented the effect of ifn-␣ (fig. 3b, columns 9, 10, 12, and 14) . the data from this experiment are consistent with those obtained with human huh7.5 cells (data not shown). thus, the ability of tnf-␣ and lps to block ifn signaling is seen in both primary and immortalized hepatocytes. as noted earlier, usp18 has dual roles: it both strips isg15 from its target proteins and impairs type 1 ifn signaling independent of its protease activity (15) . to determine whether the usp18-dependent blunting of hepatocyte ifn signaling was due to its ability to strip isg15 from its conjugates, we blocked the process of isgylation by knockdown of the isg15 e1 enzyme, ube1l. murine hepatocyte ube1l was knocked down via transfection with sirna specific for ube1l. usp18 ϩ/ϩ murine hepatocytes transfected with ube1l sirna or control sirna were stimulated with ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h and then treated with an additional dose of ifn-␣ for 6 h. knockdown was measured in the hepatocytes transfected with ube1l sirna by western blotting probing for ube1l expression and isg15 conjugate formation (fig. 4a) . next, to examine the effect of this knockdown on isg induction, wild-type murine hepatocytes transfected with ube1l sirna or control sirna were stimulated with tnf-␣ (20 ng/ml) and lps (100 ng/ml) for 24 h and then restimulated with ifn-␣ (100 iu/ ml) for 6 h, at which time mx1 isg mrna expression was measured by qpcr. as seen in fig. 4a , ube1l knockdown was achieved, resulting in a decrease in isg15 conjugates. however, as seen in fig. 4b , we observed that neither the presence nor the relative absence of isg15 conjugation impacted the ability of tnf-␣ and lps to block ifn-␣ signaling since there was no change in isg expression after 6 h of ifn-␣ treatment when usp18 ϩ/ϩ hepatocytes transfected with anti-ube1l sirna were compared to hepatocytes transfected with irrelevant sirna (fig. 4b , sirna control, columns 2, 5, and 7, and ube1l sirna, columns 9, 12, and 14). these results are consistent with there being no role for isgylation (and, thus, for the ability of usp18 to strip isg15 from its protein conjugates) in the ability of tnf-␣ and lps to block type 1 ifn signaling in hepatocytes, and this finding is in agreement with previous data showing that usp18 blocks ifn signaling independent of its enzymatic activity (15) . we next sought to determine whether tnf-␣ and lps could induce usp18 expression in hepatocytes via the induction of ifn-␣. as observed in fig. 3a , stat1 phosphorylation shows distinct activation profiles with ifn-␣ stimulation compared to tnf-␣ or lps stimulation (fig. 3a , sirna control and sirna usp18, lanes b and c compared to lane d). although these data strongly suggest that the effect of lps and tnf-␣ stimulation are a result of direct induction of isgs and not secondary to the induction of ifn-␣, we wanted to confirm whether lps and tnf-␣ could induce type 1 or type 2 ifn (ifn-␣ or ifn-␥) in primary murine hepatocyte. thus, primary murine hepatocytes were treated with ifn-␣ (100u/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 6, 12,18, or 24 h; they were then lysed and assessed for ifn-␣, ifn-␥, and usp18 expression by qpcr. we observed that neither ifn-␣, lps, nor tnf-␣ induce much, if any, hepatocyte expression of ifn-␣ or ifn-␥, although the same doses induce strong expression of usp18 (fig. 5) . thus, the induction of isgs by lps and tnf-␣ is very unlikely to reflect the induction of hepatocyte type 1 or type 2 ifn, which suggests that the observed usp18 induction is not due to type 1 or type 2 ifn secretion and autocrine stimulation of the ifn-␣ receptor. experimental hepatic ischemia/reperfusion induces usp18 expression and enhances lcmv replication. we then assessed whether tissue-wide hepatic inflammatory stress increases liver usp18 expression, as an in vivo confirmation of our in vitro findings. this was approached by inducing partial (70%) hepatic ischemia/reperfusion injury (hiri) for 60 min in mice, euthanizing the animals at 6 or 24 h (time points relevant to our in vitro time course) and measuring induction of usp18 by qpcr. we chose hiri as a very well-characterized inflammatory stress, known to be driven both by tnf-␣ and lps (35) . as seen in fig. 6a , hiri alone induced usp18 mrna expression in whole livers by ͼ10fold that of untreated animals. thus, in vivo liver inflammation leads to increased usp18 expression at the organ level. after determining that hiri induces usp18 expression, we next wanted to examine the impact of hiri on viral control. thus, we induced 70% hiri for 60 min prior to lcmv we infection of usp18 ϩ/ϩ and usp18 ϫ/ϫ mice. as seen in fig. 6b , hiri led to a significant increase in lcmv viral titers in usp18 ϩ/ϩ mice, an effect that was not observed in usp18 ϫ/ϫ mice. having shown that lps/ tnf-␣ stimulation increases hepatocyte usp18 expression, we then sought to determine whether we could pharmacologically inhibit the induction of usp18 by lps and tnf-␣. we focused on small-molecule agents that have been linked to the nf-b signaling pathway, because of the central role of this pathway in inflammatory activation in response to a large number of inflammatory mediators, including lps and tnf-␣ (42) . we were particularly interested in inhibitors, such as silibinin, that in addition have been linked to clinical suppression of hepatic viral production (in the case of silibinin and hcv) (43, 44) . thus, we preincubated primary mouse hepatocytes for 30 min with various inhibitors of lps and tnf-␣ signaling and measured their impact on usp18 induction, while simultaneously measuring expression of proinflammatory cytokine il-1␤ mrna, since nf-b is also known to promote il-1␤ transcription (45, 46) . as seen in fig. 7a and as table 1 , tnf-␣ induced expression of usp18 was potently downregulated by all inhibitors tested, and this inhibition coincided with impaired il-1␤ mrna expression (fig. 7b) . meanwhile, only two inhibitors potently (ͼ80%) inhibited usp18 expression as well as il-1␤ mrna expression in response to lps stimulation; nsc33994 (an inhibitor of jak2) and silibinin (an inhibitor of ikk␣) (fig. 7a and b and table 1 ). these data suggest that the induction of usp18 by tnf-␣ and lps, and pos-sibly other inflammatory stimuli, is promoted by nf-〉 signaling and that hepatocyte usp18 expression in particular-compared to il-1␤-may be an attractive target for pharmacologic manipulation in the setting of liver inflammation. in this study we examined the role of various inflammatory stimuli in the induction of usp18 and the downstream establishment inflammatory stimuli, we examined the induction of stat-1 phosphorylation in huh7.5 cells with or without usp18 knockdown (a) and the expression of isg mrna (mx1) in primary mouse hepatocytes and the ability of usp18 knockdown to restore isg induction after lps and tnf-␣ stimulation (b). (a) huh7.5 cells were transfected with anti-usp18 sirna or control irrelevant sirna. huh7.5 cells were then pretreated with lps (100 ng/ml), tnf-␣ (20 ng/ml), or ifn-␣ (100 u/ml) or left untreated for 24 h and then exposed to ifn-␣ or left untreated for an additional 2 h. as controls, huh7.5 cells were treated with lps, tnf-␣, and ifn-␣ for 2 h only. the expression of usp18, pstat1, stat1, and isg15 conjugates and actin proteins was measured by western blotting. (b) primary murine hepatocytes from usp18 ϩ/ϩ mice were isolated, transfected with anti-usp18 sirna or control irrelevant sirna, and pretreated with ifn-␣ (100 iu/ml), lps (100 ng/ml), or tnf-␣ (20 ng/ml) for 24 h (ifn-␣ pre, lps pre, or tnf-␣ pre, respectively). after being washed, the cells were cultured in the presence or absence of ifn-␣ for 6 h (ifn-␣ final). mx1 isg mrna expression was measured by qpcr and normalized to the expression of the hprt housekeeping gene. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. of an ifn-␣ refractory state. we used multiple methods to show that certain inflammatory stimuli, including lps and tnf-␣ are able to induce the expression of usp18, which results in downstream downregulation of ifn-␣-induced isg expression. the role of usp18 in the ifn-␣ refractory state has been previously demonstrated by human and mouse usp18 knockdown studies (12, (14) (15) (16) . other inflammatory stimuli, including il-10 and il-6, did not induce usp18 or impair expression of isgs, including usp18. in vivo, hepatic inflammatory stress (ischemia/reperfusion injury) led to increased hepatic usp18 gene expression that was associated with poor control of lcmv infection. thus, the hepatic inflammatory milieu, contributed to by individual inflammatory cytokines and stimuli, modulates usp18 expression. these results demonstrate one mechanism by which liver inflammation directly impacts the hepatocellular innate immune response. usp18 is known to be induced by multiple inflammatory stimuli in macrophages (18) and lymphocytes (47) . our findings in hepatocytes are consistent with work done by other groups in immune cells. for example, lps treatment of a murine macrophage cell line upregulates usp18 in an irf3-dependent manner (18) . the cytokine specificity of our results is also consistent with finding that il-6 alone is not able to induce usp18 in murine t cells (47) . only when t cells were treated with il-6 and another proinflammatory cytokine, such as transforming growth factor ␤, il-1␤, and il-23, was usp18 expression induced (47) . these data point out that usp18 can be induced by inflammatory stimuli in multiple cell types in the absence of ifn. usp18 is therefore well positioned to act as the mediator of "cross talk" between innate immunity and inflammatory responses. in the present study, we show that increased usp18 expression following exposure of hepatocytes by inflammatory stimuli blunts the ifn response. the binding of usp18 with the ifn-␣ receptor has been shown to inhibit the interaction of stat-1 with the ifn-␣ receptor and thus block downstream ifn signaling (12, 15) . our data are consistent with this mechanism, since the blunting of hepatocyte ifn signaling after exposure to inflammatory stimuli is independent of usp18-mediated removal of isg15 from its target proteins. the degree of tnf-␣ and lps-induced ifn-␣ refractoriness was not changed by ube1l knockdown, although isgylation was considerably reduced. however, other mechanisms may also be at play. recent work has demonstrated that usp18 has the ability to deubiquitinate the transforming growth factor-activated kinase 1 (tak1) complexes required for nf-b activation in t cells and that overexpression of usp18 leads to decreased nuclear activation and impaired formation of tak1 complexes (47) . usp18-mediated nf-b inhibition may be of importance not only for t cell adaptive immunity but also for liver inflammation. the role of tak1 in innate and adaptive immunity has been previously demonstrated (48) , and studies have also shown that tak1 deletion interferes with hepatocyte homeostasis and leads to hepatic injury (49) . thus, increased hepatocyte usp18 in response to inflammatory stimuli may constitute a negative-feedback cycle with relevance to multiple aspects of the liver's response to infection. our in vivo experiments demonstrated that inflammation resulting from ischemia/reperfusion injury induces usp18 expression, which coincides with diminished lcmv control in mouse livers. the general finding that liver inflammation promotes lcmv production is in agreement with work showing that polymicrobial sepsis, characterized by high tnf-␣ and inflammation, leads to increased susceptibility to lcmv infection (50) . in our present study, hepatic ischemia/reperfusion injury did not enhance lcmv production in usp18 knockout mice. these data are intended as proof-of-concept but do suggest that the role of usp18 in hepatic viral infection and inflammation deserves further investigation. our in vitro and in vivo findings suggest a new model for how inflammation alters hepatic innate immune responses. in this model, liver inflammation leads to increased hepatocyte usp18, which in turn makes the liver more susceptible to infections targeting the hepatocyte, such as hcv. this mechanism may help to explain the clinical observation that hcv infection of a transplanted, hcv-naive liver graft (that has gone through an ischemia/reperfusion cycle) is more aggressive than the original infection and that the severity of the reinfection correlates with the severity of the ischemia/reperfusion injury suffered during the transplant (51, 52) . however, we have also found that usp18 is necessary but not sufficient on its own to induce an ifn-␣ refractory state (53) . with this in mind, we hypothesize that the innate immune response and its ability to control ifn-sensitive viruses will depend on cumulative effect of multiple intrahepatic signals, including the induction of usp18. if these results are to be translated into a clinical setting, then one approach is to target usp18 induction pharmacologically. using multiple inhibitors of tnf-␣/lps signaling, we found that two inhibitors, silibinin (an inhibitor of ikk␣) and nsc33994 (a jak2 inhibitor) possess the ability to inhibit tnf-␣ and lps-induced usp18 expression and proinflammatory effects. these findings raise the possibility of pharmacologically targeting usp18 expression during inflammatory events to prevent the establishment of an ifn-␣ refractory state. previously, inhibition of jak2 signaling led to a protection of mouse livers from ischemic insult (54) . as well, silibinin has been shown to reduce of hcv liver graft reinfection (43) and enhance hcv clearance in ifn-␣ nonresponders (55) via multiple mechanisms, including by direct inhibition of hcv ns5b rna polymerase (44) . usp18 modulation may be one mechanism by which silibinin exerts its anti-hcv effects. the present study focuses on a relatively small number of inflammatory stimuli; while tnf-␣ and lps are important to a large number of liver diseases, they are far from being the only drivers of the hepatic inflammatory response and tissue levels of usp18 are likely influenced by other stimuli as well. for example, we previously demonstrated that increased usp18 expression in the liver is and enhances lcmv replication. to examine the in vivo effect of an inflammatory stimulus on usp18 expression and viral replication, a hepatic ischemia/reperfusion model was used. (a) partial (70%) hepatic ischemia was induced for 60 min, after which the portal vascular clamp was removed. control animals (sham) underwent anesthesia and a laparotomy alone. animals were euthanized 6 and 24 h after ischemia/reperfusion (i/r), and the affected liver segments were removed. usp18 mrna expression was determined by qpcr in whole liver tissue and normalized to hypoxanthine-guanine phosphoribosyltransferase (hprt) expression. data are expressed as means ϯ the sem with n ϭ 3 to 4 mice/group. a p value of ͻ0.05 was considered significant. (b) after 60 min of hiri or sham laparotomy, mice were infected with 2 ϫ 10 6 pfu of lcmv we. liver lobes were harvested at day 7 postinfection. viral titers were examined on mc57 cells using a focus-forming assay. data are expressed as means ϯ the sem with n ϭ 3 to 10 mice/group. a p value of ͻ0.05 was considered significant. inhibition of nf-b activation impairs lps-and tnf-␣-stimulated usp18 induction. to investigate the link between lps/tnf-␣ stimulation and usp18 induction, inhibitors of nf-b activation were added to primary mouse hepatocytes for 30 min prior to stimulation with 20 ng of tnf-␣/ml or 100 ng of lps/ml for 6 h (the inhibitor names, targets, and concentrations used are given in table 1 ). inhibitor-treated cells were also left unstimulated as a control. as a control for usp18 induction, hepatocytes were stimulated with 100 u of ifn-␣/ml for 6 h. usp18 (a) and il-1␤ (b) mrna expression levels were evaluated by pcr and normalized to the hprt housekeeping gene expression prior to determining the fold increase versus mock-treated samples. the data were generated from pooled triplicate experiments analyzed in duplicate. error bars represent the sem for duplicate pcrs. predictive of patients with chronic hcv infection who will not respond to ifn-based anti-hcv therapy (8) . although we found that tnf-␣ hepatic mrna expression was increased in treatment nonresponders, it was relatively more increased in treatment responders (9) . we suspect that whereas increased tnf-␣ does contribute to usp18 expression, there are other stimuli, for example, lps and perhaps the recently described interferon-lambda 4 (56) , that also modulate hepatic usp18 expression. we propose that the ultimate usp18 expression is due to the liver's coordinate response to multiple stimuli. the finding that hepatic usp18 expression is modulated by inflammatory stimuli is a new paradigm for the interaction of the liver inflammatory microenvironment and viral infection. taken together, these data may suggest that usp18 represents a good target for intervention in numerous inflammatory states and in the clinical setting of hcv-related liver transplantation. hiri-induced upregulation of usp18 could lead to worsened outcomes posttransplant, including a quicker, more aggressive reinfection of the new organ with higher hcv rna titers. we suggest that the finding that ifn responses and usp18 expression are tightly linked to the hepatic inflammatory response helps to explain the finding that tnf-␣ antibody treatment improves treatment outcomes of chronic hcv with treatment combinations, including ifn-␣ (5). mechanism of action of interferon and ribavirin in treatment of hepatitis c interferon, mx, and viral countermeasures hepatitis c virus infection induces inflammatory cytokines and chemokines mediated by the cross talk between hepatocytes and stellate cells tumor necrosis factor alpha gene expression and the response to interferon in chronic hepatitis c etanercept as an adjuvant to interferon and ribavirin in treatment-naive patients with chronic hepatitis c virus infection: a phase 2 randomized, double-blind, placebocontrolled study safety of anti-tumor necrosis factor-alpha therapy in patients with rheumatoid arthritis and chronic hepatitis c virus infection hepatic celltype specific gene expression better predicts hcv treatment outcome than il28b genotype hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis c viral infection cell-type-specific gene expression signature in liver underlies response to interferon therapy in chronic hepatitis c infection tissue macrophages suppress viral replication and prevent severe immunopathology in an interferon-idependent manner in mice the role of kupffer cells in hepatitis b and hepatitis c virus infections alpha interferon induces long-lasting refractoriness of jak-stat signaling in the mouse liver through induction of usp18/ubp43 the isg15/usp18 ubiquitin-like pathway (isgylation system) in hepatitis c virus infection and resistance to interferon therapy silencing of usp18 potentiates the antiviral activity of interferon against hepatitis c virus infection ubp43 is a novel regulator of interferon signaling independent of its isg15 isopeptidase activity usp18-based negative feedback control is induced by type i and type iii interferons and specifically inactivates interferon alpha response gene induction pathways mediated by distinct irfs during viral infection lipopolysaccharide activates the expression of isg15-specific protease ubp43 via interferon regulatory factor 3 tumor necrosis factor-alpha in liver ischemia/reperfusion injury the role of intestinal endotoxin in liver injury: a long and evolving history viral and host factors induce macrophage activation and loss of toll-like receptor tolerance in chronic hcv infection enforced viral replication activates adaptive immunity and is essential for the control of a cytopathic virus complete replication of hepatitis c virus in cell culture tnf-alpha-induced sphingosine 1-phosphate inhibits apoptosis through a phosphatidylinositol 3-kinase/ akt pathway in human hepatocytes oncostatin m is a potent inducer of hepcidin, the iron regulatory hormone lipopolysaccharide, immune activation, and liver abnormalities in hiv/hepatitis b virus (hbv)-coinfected individuals receiving hbv-active combination antiretroviral therapy protein interferon-stimulated gene 15 conjugation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis hepatitis c virus persisting after clinically apparent sustained virological response to antiviral therapy retains infectivity in vitro protein-kinase inhibitor-(6-22)-amide peptide analogs with standard and nonstandard amino-acid substitutions for phenylalanine-10: inhibition of campdependent protein-kinase bone marrow-derived myofibroblasts promote colon tumorigenesis through the il-6/jak2/stat3 pathway nonmuscle myosin is regulated during smooth muscle contraction pd-098059 is a specific inhibitor of the activation of mitogen-activated protein-kinase kinase in-vitro and in-vivo silibinin inhibits constitutive and tnf alpha-induced activation of nf-b and sensitizes human prostate carcinoma du145 cells to tnf alpha-induced apoptosis wortmannin is a potent phosphatidylinositol 3-kinase inhibitor: the role of phosphatidylinositol 3,4,5-trisphosphate in neutrophil responses protective strategies against ischemic injury of the liver interleukin-10 determines viral clearance or persistence in vivo quantification of lymphocytic choriomeningitis virus with an immunological focus assay in 24-well or 96-well plates dubbing down innate immunity malignant pirates of the immune system a comparison of selected mrna and protein abundances in human liver kinetic differences in the induction of interferon stimulated genes by interferon-alpha and interleukin 28b are altered by infection with hepatitis c virus nf-b in the liver-linking injury, fibrosis, and hepatocellular carcinoma successful prevention of hepatitis c virus (hcv) liver graft reinfection by silibinin mono-therapy differential in vitro effects of intravenous versus oral formulations of silibinin on the hcv life cycle and inflammation dexamethasone inhibits il-1␤ gene expression in lps-stimulated raw 264.7 cells by blocking nf-b/rel and ap-1 activation nf-b regulates il-1␤ transcription through a consensus nf-b binding site and a nonconsensus cre-like site usp18 inhibits nf-b and nfat activation during th17 differentiation by deubiquitinating the tak1-tab1 complex essential function for the kinase tak1 in innate and adaptive immune responses disruption of tak1 in hepatocytes causes hepatic injury, inflammation, fibrosis, and carcinogenesis polymicrobial sepsis increases susceptibility to chronic viral infection and exacerbates cd8 ϩ t cell exhaustion recipient age affects long-term outcome and hepatitis c recurrence in old donor livers following transplantation prolonged rewarming time during allograft implantation predisposes to recurrent hepatitis c infection after liver transplantation the ubiquitin specific protease usp18 is necessary but not sufficient for a hepatocyte ifn refractory state: variable roles in type i and type iii ifn responsiveness blockade of janus kinase-2 signaling ameliorates mouse liver damage due to ischemia and reperfusion silibinin is a potent antiviral agent in patients with chronic hepatitis c not responding to pegylated interferon/ribavirin therapy a variant upstream of ifnl3 (il28b) creating a new interferon gene ifnl4 is associated with impaired clearance of hepatitis c virus we thank dong er zhang for the gift of the usp18 ϫ/ϫ mice. s.a.m. thanks the casl/cihr hepatology fellowship program and the national cihr research training program in hepatitis c for financial support. key: cord-023419-lnmc6vv5 authors: steinhauer, c.; wingren, c.; borrebaeck, c. a. k. title: high‐throughput proteomics on antibody‐based microarrays: the importance of probe and surface design date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ax.x sha: doc_id: 23419 cord_uid: lnmc6vv5 in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive high‐throughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single‐chain fv antibody library, genetically constructed around one framework, the ncoder‐library, containing 2 × 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on‐chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in‐house‐designed substrate, macroporous silicon coated with nitrocellulose (map3‐nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double‐(his)6‐tag, we increased the binding efficiency of sinfab‐molecules to ni2(+)‐coated solid supports, thereby allowing nonpurified probes to be directly applied. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023438-g0k0vvdc authors: krog, j.; jepsen, c. f.; tønnesen, e.; parner, e.; hokland, m. title: the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423aa.x sha: doc_id: 23438 cord_uid: g0k0vvdc materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr‐release assay using k562 as nk‐sensitive target cells. the pbmcs were characterized, using 4‐colour flow cytometry, with special emphasis on the nk‐cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-002326-3qb1ym4w authors: yang, runkuan; zhu, shengtao; tonnessen, tor inge title: ethyl pyruvate is a novel anti-inflammatory agent to treat multiple inflammatory organ injuries date: 2016-12-03 journal: j inflamm (lond) doi: 10.1186/s12950-016-0144-1 sha: doc_id: 2326 cord_uid: 3qb1ym4w ethyl pyruvate (ep) is a simple derivative of pyruvic acid, which is an important endogenous metabolite that can scavenge reactive oxygen species (ros). treatment with ep is able to ameliorate systemic inflammation and multiple organ dysfunctions in multiple animal models, such as acute pancreatitis, alcoholic liver injury, acute respiratory distress syndrome (ards), acute viral myocarditis, acute kidney injury and sepsis. recent studies have demonstrated that prolonged treatment with ep can ameliorate experimental ulcerative colitis and slow multiple tumor growth. it has become evident that ep has pharmacological anti-inflammatory effect to inhibit multiple early inflammatory cytokines and the late inflammatory cytokine hmgb1 release, and the anti-tumor activity is likely associated with its anti-inflammatory effect. ep has been tested in human volunteers and in a clinical trial of patients undergoing cardiac surgery in usa and shown to be safe at clinical relevant doses, even though ep fails to improve outcome of the heart surgery, ep is still a promising agent to treat patients with multiple inflammatory organ injuries and the other clinical trials are on the way. this review focuses on how ep is able to ameliorate multiple organ injuries and summarize recently published ep investigations. [figure: see text] pyruvate is the final product of glycolysis and the starting substrate for the tricarboxylic acid (tca) cycle, and this important metabolic intermediate is also an effective scavenger of hydrogen peroxide and other ros [1, 2] . pharmacological administration of pyruvate is able to improve organ function in animal models of oxidantmediated cellular injury [1, 2] ; however, pyruvate is unstable in aqueous solutions and this certainly limits its therapeutic potential. ep, a simple derivative of pyruvic acid, is also an ros scavenger, but exerts pharmacological effects, such as the anti-inflammatory effects, which are quite distinct from those exerted by pyruvate anion [1, 2] . treatment with ep has been shown to improve survival and/or ameliorate multiple organ dysfunctions in a wide variety of preclinical models of critical illnesses [1, 2] . up to date, about 340 ep related papers have been published; about 100 new papers published after 2010 have not been summarized and reviewed. this review focuses on how ep is able to ameliorate inflammatory injures of multiple vital organs and summarizes new findings from recently published ep investigations. acute pancreatitis (ap) is a relatively common disease, its severe form is potentially fatal and sap is associated with high mortality, ranging from 15-40% [3] [4] [5] [6] [7] [8] . the inflammatory cytokines play a crucial role in the pathogenesis of sap [3, 8, 9] ; furthermore, the damaged pancreatic acinar cells and the activated inflammatory cells produce a large amount of oxygen radicals in ap, and these ros molecules can damage the lipid membranes of pancreatic acinar cells, they can also injure the capillary endothelium in the circulation to accelerate the progress of sap [7] . currently, therapeutic efforts are limited to supportive measures, because no effective specific treatment exists. the effect of ep on acute pancreatitis ep has been repeatedly reported to ameliorate sap in different animal models [4, 6, 10, 11] . ep treatment [ep source in this review was all from sigma-aldrich unless otherwise noted. ep dissolved in commercially available ringer's lactate solution (rls). commercially available rls was used as the control solution. ep (40 mg/kg) was intraperitoneally injected every 6 h for 48 h)] significantly ameliorates pancreatic injury and necrosis [4, 6] ; ep therapy also markedly reduces pancreatic expression of tnf-α, il-6, hmgb1 and nf-kb dna binding [4, 6] ; treatment with ep reduces the number of inflammatory cell infiltration and decreases the pancreatic level of lipid peroxidation, which is a parameter of ros [4] . because early inflammatory cytokines (such as tnf-α and il-6), late inflammatory mediator hmgb1 and ros play a significant role in the pathogenesis of sap [4, 6, 10, 11] , and ep can reduce the levels of these inflammatory cytokines and scavenge ros. therefore, ep may attenuate pancreatic injury during sap. the effect of ep on severe acute pancreatitis related multiple organ injuries about 20-30% of all acute pancreatitis patients develop sap in clinical practice, and the mortality rate in sap is 20-30% [3] . sap starts as a local inflammation of pancreatic tissue that induces the development of multiple extrapancreatic organs dysfunction [6, 8] . during sap, the concentrations of both early (tnf-α, il-6) and late inflammatory cytokines are significantly increased [6, [10] [11] [12] [13] , these cytokines play a significant role in the pathogenesis of sap [6, 12] . the late inflammatory cytokine hmgb1 is particularly important because extracellular hmgb1 can aggravate the pancreatic inflammatory process [14] and hmgb1 can also contribute to multiple distant organ injuries in the following experimental models as well: hmgb1 contributes to liver injury in ischemia-reperfusion [15] . exogenous hmgb1 injection is able to induce liver injury in normal mice [16] . hmgb1 impairs hepatocyte regeneration during acetaminophen hepatotoxicity and blockade of hmgb1 improves hepatocyte regeneration in acetaminophen overdose-induced fatal liver injury [17] . anti-hmgb1 treatment protects against apap hepatotoxicity in rats [18] . hmgb1 also contributes to renal ischemia reperfusion injury [19] , sepsis-induced kidney injury [20] and severe acute pancreatitis related kidney injury [21] . hmgb1 is also found to significantly contribute to hemorrhagic shock related acute lung injury (ali) [22] ; hypoxia-induced ali [23] and severe acute pancreatitis related ali [24] . hmgb1 is also an important factor that not only significantly contributes to gut mucosal injury [16] , but also mediates gut bacterial translocation (bt) [25] , and blockade of hmgb1 can even prevent gut bt [25] . gut mucosal injury and intestinal bt in sap is particularly important because the intestine is the biggest reservoir of bacteria in the body and leakage of bacteria or microbial products, notably lps, from the lumen of the gut into the systemic circulation, which drives the development of systemic inflammation and multiple organ dysfunction syndrome (mods) in experimental models [26] . sap frequently induces gut barrier dysfunction [6, 9, 27, 28] . the small intestine becomes damaged by intestinal ischemia-reperfusion during sap [29, 30] , and the failure of gut barrier is associated with bt [29] , which is evident in sap [6, 27, 28, 31] . sap patients have significantly increased serum lps [32] , 68.8% of the sap patients have bacteraemia and these bacteria are highly likely gut-derived opportunistic pathogens [27] . furthermore, bt and infected pancreatic necrosis in acute necrotizing pancreatitis derive from small bowel rather than from the colon [28] . bt and/or gut derived lps play a critical role in the development of systemic inflammatory response syndrome (sirs) and mods during sap [29, 33] , because bt not only contributes to pancreatic infection [28, 33] , but also induces/triggers sirs/sepsis in critical illness [33, 34] . the profound sirs/sepsis can lead to mods and mortality in sap [9, 29, 31, 33] , this is one of the underlying mechanisms that ap frequently affects extrapancreatic organs [6, 25] , and the incidence of mods in sap is not available but certainly higher than the 20-30% of mortality rate in sap [3] . systemic inflammation with multi-organ dysfunction is the cause of death in a murine ligation-induced sap, and sirs and mods can lead to the preponderance of mortality (75%) in this lethal sap model [9] while bile duct ligation does not have mortality, even though obstructive jaundice is prone to sepsis [9] . therefore, the gut is thought to act as the starter of sirs [29] and hmgb1 may be an important factor that links gut barrier dysfunction and mods during sap. ep (40 mg/kg was intraperitoneally injected every 6 h for 48 h) can ameliorate sap related multiple organ injuries [6, 10, 11] at least partly because ep not only reduces the inflammation in these organs [6, 10, 11] , but also inhibits nearly 90% of the hepatic tissue hmgb1 to release [12] and other related cells to release hmgb1 [10, 12] , thereby decreases the circulating hmgb1 level in sap [10, 14] . thus, hmgb1 is an important factor that not only directly contributes to multiple organ injuries, but also mediates gut bt to trigger/induce systemic inflammation/sepsis, the latter can lead to mods. currently, the circulating hmgb1 level is thought to be reliable to predict the severity of sap [10, 14] , this is likely because hmgb1 is linked to multiple organ injuries during sap [10, 12, 14] , the liver is an important source of circulating hmgb1 [12] , and the circulating hmgb1 level could reflect the severity of liver injury, which is one of the important parameters for diagnosing sap [6, 12] . sap associated bt was reduced by 90% following ep treatment [6] breaking the link between bt and mods. therefore, ep could be a better treatment option against sap related multiple organ injuries in experimental models [6, 10, 11] . the liver is the largest organ in the body, hepatocytes account for 70-80% of the hepatic cytoplasmic mass and non-parenchymal cells make up 20-30% of the hepatic cytoplasmic mass [35] . kupffer cells (kcs) are the most abundant mononuclear phagocytes in the body and a predominant source of inflammatory cytokines released into the systemic circulation [8] . the amount of cytokines released from the liver represents about 50% of the total cytokine content in the body [36] , suggesting that the liver is the major contributor of the circulating cytokines. alcoholic hepatitis is associated with considerable morbidity; 46% of patients with the severe alcoholic hepatitis die within 30 days of the onset [37] . general measures for treatment include abstinence from alcohol and supportive care. alcoholic hepatitis is reversible if the patient stops drinking, it usually takes several months to resolve, however, abstinence from alcohol is difficult, and the treatment is still challenging. in an experimental murine model, alcohol induces fatty change and piecemeal necrosis; alcohol administration also induces significantly increased hepatic lipid peroxidation, nf-kb activation, tnf-α mrna expression; furthermore, alcohol administration induces a large amount of gut bt, which can serve as the "second hit" to contribute to the alcoholic liver injury. all of these alcohol-induced effects are ameliorated by treatment with ep (40 mg/kg was intraperitoneally injected every 6 h for 48 h) instead of ringers lactate solution, suggesting that ep ameliorates hepatic inflammatory response and hepatic lipid peroxidation, and resultantly decreases hepatocellular injury duo to acute alcoholic intoxication [37] . obstructive jaundice and cholangitis are common diseases that are prone to sepsis that can lead to mortality [38] . in an experimental murine model of common bile duct ligation model [38] , obstructive jaundice induces evident hepatocellular necrosis and significantly increased circulating alt and total bilirubin levels. obstructive jaundice also induces increased hepatic lipid peroxidation and increased hepatic expression of transcripts for tnf-α, il-6, and inos. furthermore, bile duct ligation also induces a large amount of gut bt, which can serve as the "second hit" to contribute to the liver injury. all of these changes can be significantly attenuated by delayed treatment with ep (40 mg/kg was intraperitoneally injected every 8 h for 72 h) instead of rls, suggesting that ep ameliorates hepatic inflammation, lipid peroxidation and necrosis in obstructive jaundice [38] . in addition, ep treatment increases nf-kb dna binding, which often modulates inflammation when hepatic necrosis is not evident [37] but modulates regeneration when hepatic necrosis is evident [38] , this concept has been proved in acetaminophen hepatotoxicity in which massive hepatocyte necrosis is a predominant feature [39, 40] . therefore, it is likely that ep enhances nf-kb dna binding to improve hepatocyte regeneration in obstructive jaundice. ep ameliorates acute liver injury secondary to severe acute pancreatitis sap frequently affects the liver and the inflamed liver play a significant role in the pathogenesis of sap [6, 12, 13] . in a lethal experimental sap murine model, sap induces significantly increased hepatic lipid peroxidation, nf-kb activation, hepatic expression of transcripts for tnf-α, il-6, inos and cox-2 [12] . sap also induces focal hepatocyte necrosis and a large number of inflammatory cell infiltration [6, 12, 13] , all of these changes can be significantly decreased by delayed treatment with ep instead of ringers lactate solution [12] . in particular, sap can induce the loss of nearly 85% of hepatic tissue hmgb1 tested by western blot using whole hepatic tissue, and this effect can be reversed by ep treatment (40 mg/kg intraperitoneally injected every 6 h for 48 h) [12] , suggesting that the liver is an important resource of the circulating hmgb1, and ep is a potent hmgb1 inhibitor [12] . in another sap rat model, sap induces significantly increased hepatic lipid peroxidation, hepatic nf-kb dna binding and hepatic expression of transcripts for tnf-α, il-1 and hmgb1 [13] , all of these changes can be significantly reduced by ep treatment (40 mg/kg intraperitoneally injected every 6 h for 48 h) [13] . fatty liver is common world widely, and its treatment is problematic. in a high-fat induced rat model, the fatty liver induces increased serum alt and increased hepatic tnf-α level; these changes can be significantly decreased by ep intake (supplemented in 0.3% drinking water for 6 weeks) but ep does not affect the development of a fatty liver [41] , suggesting that ep can protect against inflammation induced liver cell damage but ep cannot prevent the development of fatty liver in animal experiment. hepatic ischemia-reperfusion (i/r) is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma and hemorrhagic shock [42] . hepatic i/r induces significantly increased hepatic expression of tnf-α, il-6, hmgb1 and nf-kb activation, and hepatic i/r also induces markedly increased hepatic expression of bcl-2, bax, beclin-1 and lc3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, all of these changes are significantly reduced by ep treatment (1 h before the ischemia procedure, a single dose of ep was intraperitoneally injected to animals in the 20 mg/kg group, the 40 mg/kg group and the 80 mg/kg group, liver tissue samples were obtained 4 h, 6 h and 16 h after i/r), suggesting that ep ameliorates hepatic i/r injury via its antiinflammatory and its anti-apoptosis effect. drug-induced acute fatal liver injury is the leading cause of acute liver failure (alf) in the developed countries [43] [44] [45] [46] , and alf has a high mortality and the treatment is still quite challenging [43] . concanavalin a induces autoimmune hepatitis with significantly increased hepatic expression of tnf-α, il-6, il-1, hmgb1 and nf-kb activation, ep treatment (1 h before the con a injection, a single dose of ep was intraperitoneally injected to the animals in the 40 mg/kg ep group and the 80 mg/kg ep group; liver tissue samples were obtained 3 h, 6 h and 24 h after con a injection) reduces all of these changes and resultantly ameliorates concanavalin induced autoimmune hepatitis [44] , which can be fatal. d-galactosamine induces acute fatal liver injury with significantly increased serum tnf-α, hmgb1, ifngamma and endotoxin, all of these changes can be significantly decreased by ep treatment (a single dose of 40 mg/kg ep was intraperitoneally injected 2 h after alf induction, samples were taken 22 h after ep injection), therefore, ep can ameliorate acute fatal liver injury induced by d-galactosammine [45] . acute-on-chronic liver failure (aclf) rats have significantly increased serum endotoxin, tnf-α, hmgb1, ifn-gamma and il-18, ep administration (40 mg/kg was intraperitoneally injected at 3 h, 6 h, 12 h, 24 h after the induction of aclf, and samples were taken 48 h after the induction of aclf) decreases all of these changes to protect against aclf in rats [46] . acetaminophen is the leading cause of drug induced alf, and ep treatment (40 mg/kg was intraperitoneally injected every 8 h for 24 h) can reduce liver injury at early phase by its potent anti-inflammatory effect [43] . diabetes can lead to an increased oxidative stress that significantly contributes to diabetes-induced liver injury [47] . diabetes induces significantly increased total antioxidant status and hepatic peroxidation. ep therapy (50 mg/kg was intraperitoneally injected twice a day for 14 days) (instead of ringer solution) significantly decreases all of these changes and resultantly ameliorates diabetes-induced liver injury in a streptozocin induced diabetic rat model [47] . the effect of ep on acute lung injuries ep on sap related acute lung injury acute lung injury (ali), also addressed as mild acute respiratory distress syndrome (mards), is a significant health problem associated with high mortalities [8, 48] ; mards is also a severe complication and a major feature of mods to sap [8] . in patients with ap, up to 20% of all deaths occur prior to admission to hospitals, and mards is the predominant cause of death in these cases [48] . in sap, the mards is the main contributing factor to early deaths, i.e. within the first week after admission [49] . the inflammatory mediators and the profound sirs are thought to play a key role in the development of mards [3, 50, 51] because the significantly increased serum inflammatory mediators activate alveolar macrophages to release chemotactic mediators that play an important role in recruiting neutrophils, which work together with the elevated circulating pro-inflammatory mediators to severely damage alveolar epithelium and microvascular endothelium, and resultantly causes the increased permeability of the alveolar-capillary barrier and pulmonary edema. this theory is supported by the following evidence in which the alveolar-capillary barrier is severely injured and the pulmonary permeability is significantly increased in an experimental acute necrotizing pancreatitis [6] . the antiinflammatory agent ep (40 mg/kg intraperitoneally injected every 6 h for 48 h) markedly decreases the lung permeability and alleviates pulmonary edema at least partly by reducing pulmonary inflammation and neutrophils infiltration in a couple of experimental sap models with acute lung injury [6, 10, 52, 53] . increased local and systemic levels of il-6 are associated with inflammatory process, including neutrophil infiltration of the alveolar space, resulting in lung injury [54] . ep treatment reduces the il-6-induced release of il-8 and decreases il-6induced neutrophil adhesion to the lung epithelial cells [54] , and this anti-inflammatory effect is via akt and nf-kb p65 pathway [55] . therefore, ep reduces secretory and adhesive potential of lung epithelial cells under inflammatory conditions [54, 55] . the effect of ep on endotoxin-induced acute lung injury lps intravenous injection induces significantly increased plasma tnf-α, il-6; lps administration also induces significantly increased expression of ho-1 and inos in lung tissue. in addition, lps also induces lung edema and infiltration of neutrophils, all of these changes can be reduced by ep treatment (intravenously infused for 4 h into the animals in the following 3 different ep concentration groups: 20, 40 and 60 mg/kg, and lung tissue samples were harvested 6 h after ep administration) [56] . in a murine model, lps intratracheal administration significantly increases the release of tnf-α, il-6, il-1 and hmgb1 into bronchoalveolar lavage, all of these changes can be markedly decreased by ep treatment (100 mg/kg was intraperitoneally injected at 0 h, 12 h, 24 h and 48 h after the induction of ali), and early administration of ep can improve survival [57] . prolonged exposure to hyperoxia results in ali, accompanied by significantly increased levels of proinflammatory cytokines and a large number of leukocyte infiltration in the lungs [58] . hmgb1 plays a critical role in mediating hyperoxia induced ali through the recruitment of leukocytes into the lung [58] , a single dose of ep treatment (40 mg/kg intraperitoneal injection prior to hyperoxia exposure, and lung tissue samples were taken 24 h after hyperoxia exposure) attenuates the hyperoxia induced ali probably by inhibiting hmgb1 secretion from hyperoxic macrophages [58] , suggesting that ep may treat oxidative inflammatory lung injury in patients receiving hyperoxia through mechanical ventilation. in cultured macrophages, lps stimulates the macrophages to release tnf-α, il-6, and hmgb1, and these changes can be effectively prevented by ep treatment (cells were incubated with 25 mm ep for 48 h) [59] . lps stimulates macrophages to release hmgb1 and up-regulates inos expression, ep treatment (cells were incubated with 25 mm ep for 24 h) can reverse these effects by inducing heme oxygenase-1 (ho-1) via a p38 makp-and nrf2dependent pathway [60] . hmgb1 is a ubiquitous nuclear protein that can be actively secreted by immunocompetent cells, including monocytes, macrophages and neutrophils, and this highly conservative nuclear protein is an important late inflammatory mediator in sepsis [61] . hmgb1 can also be passively released by dying cells or necrotic tissue [25] . hmgb1 plays an important role in modulating inflammatory cascade in activated macrophages: hmgb1 stimulates macrophages to release tnf-α and il-6, while hmgb1neutralizing antibody can block tnf-α release [62, 63] and knocking-out hmgb1 receptor can reverse il-6 release [63] . in macrophages cultures, lps stimulates macrophages to release high concentrations of early inflammatory cytokines such as tnf-α, il-6 and il-1 and the late mediator hmgb1 [61] [62] [63] , and ep treatment reduces these changes by specifically inhibiting the activation of p38 mitogen activated protein kinase and nf-kb, two signalling pathways that are critical for cytokines release [61] . sepsis is a serious clinical syndrome, which is mediated by an early (such as tnf-α and il-1) and late (such as hmgb1) pro-inflammatory cytokine response to infection [61] . delayed treatment with ep (40 mg/kg intraperitoneally injected 24 h, 30 h, 48 h, and 54 h after cecal puncture, the experiment finished 120 h after cecal puncture) significantly increases survival and markedly reduces circulating levels of hmgb1 in mice with sepsis [61] . ep increases survival and decreases serum hmgb1 by up-regulation of ho-1 level in septic animals [60] . in an established septic animal model, sepsis induces significantly increased plasma tnf-α, il-6 and il-1; sepsis also increases hepatic lactate, lactate/pyruvate levels and down-regulates hepatic atp and energy charge levels; all of these effects can be reversed in the septic mice treated with a single dose of ep (40 mg/kg intraperitoneal injection, and the liver samples were taken 6 h after ep injection), suggesting that ep protects against sepsis by regulating energy metabolism and inhibiting systemic inflammation [64] . in addition, ep improves sepsis outcome by reducing mitochondrial swelling and dysfunction in experimental sepsis [65] . septic patients have significantly increased serum hmgb1 levels, which can induce endothelial cell hyperpermeability via bax and bcl-2 regulated apoptosis, ep can reverse these detrimental effects to prevent endothelial cell injury in experimental sepsis [66] . furthermore, ep can effectively reduce vascular endothelial inflammation and this effect at least partly depends on the attenuation of endoplasmic reticulum stress [67] . acute kidney injury (aki) is a common serious complication of sap and sepsis. endotoxin and ros play an important role in the pathogenesis of aki and sap. sepsis is a common cause of acute renal failure (arf), and the incidence of sepsis increases markedly after age of 50 [68] . sepsis induces significantly increased plasma tnf-α, creatinine and causes tubular damage and multiple organ injury, sepsis also induces increased mrna for tnf-α, tissue factor, pai-1, and urokinase-like plasminogen activator; all of these changes can be significantly decreased by ep treatment (a single dose of 40 mg/kg was intraperitoneally injected 12 h after cecal puncture and experiment finished 24 h after cecal puncture), therefore, ep attenuates sepsis-induced arf in an animal model [68] . diabetic nephropathy is a common complication [69] . diabetic rats have increased laminin, type iv collagen and fibronectin deposition in the glomeruli and these animals also have albuminuria and increased nadphdependent ros generation; all of these changes can be significantly decreased by ep treatment (40 mg/kg intraperitoneally injected every other day for 12 weeks) [69] , suggesting that ep might be a novel therapy to treat diabetic nephropathy. cisplatin-induced nephrotoxicity is common in clinical practice. cisplatin induces significantly increased perfusion pressure, serum urea, creatinine, total oxidant status and tissue lipid peroxidation, all of these changes can be significantly decreased by ep therapy (50 mg/kg was intraperitoneally injected once a day for 5 days) [70] , suggesting that ep has a protective effect against cisplatin nephrotoxicity. inflammation plays important roles in the pathogenesis of coxsackievirus b3 (cvb3)-induced acute viral myocarditis (avmc) [71] . cvb3 virus induces increased levels of hmgb1, tnf-α, il-1, il-17 in the heart and serum of the avmc mice, and all of these changes can be significantly decreased by ep treatment (40 mg/kg/ day and 80 mg/kg/day intraperitoneally injected on day 5, day 6 and day 7 post infection), and this protective effect is associated with inhibition of hmgb1/rage/nf-kb pathway [71] . hmgb1 is a late inflammatory cytokine that triggers and amplifies the inflammation cascade following ischemia/reperfusion (i/r), and ep can inhibit hmgb1 release in i/r models [72] . i/r procedure induces increased levels of hmgb1, tnf-α, il-6, these changes can be significantly reduced by ep treatment (a single dose of ep with 40 mg/kg concentration was intravenously injected prior to the 48 h reperfusion) instead of pbs, the accumulation of hmgb1 is deleterious to the heart following myocardial i/r [72] . in another rat cardiac i/r model, ep treatment significantly preserves cardiac function, enhances tissue atp levels, attenuates myocardial oxidative injury and reduces apoptosis following myocardial ischemia [73] . in another regional heart i/r rat model, ep therapy (a single dose of 40 mg/kg was intraperitoneally injected 1 h prior to the 24.5 h i/r procedure) inhibits i/r-induced nf-kb translocation and neutrophil activation in myocardium, ep also decreases the serum levels of inflammatory cytokines, and resultantly ep improves cardiac function and reduces infarct size after regional i/r injury [74] . in another prolonged rat myocardial ischemia model, ep therapy enhances myocardial atp levels, attenuates myocardial oxidative injury, and resultantly decreases infarct size and preserves cardiac function [75] . the effect of ep on acute brain injury ep attenuates traumatic brain injury in a rat model of unilateral cortical contusion injury (cci), ep treatment (40 mg/kg was intraperitoneally injected 1 h, 12 h and 24 h after brain injury, brain samples were harvested 72 h after brain injury) significantly decreases the number of dead/dying cells in the ipsilateral hippocampus and improves recovery of beamwalking, neurological scores after injury, suggesting that ep treatment after cci is neuroprotective and improves neurobehavioral recovery [76] . traumatic brain injury (tbi) can cause brain cell death/dying, and the/dead/ dying cells can release nuclear protein hmgb1 that can activate inflammatory pathways, therefore, the hmgb1 inhibitor ep (75 mg/kg was intraperitoneally injected at 5 min, 1 h, 6 h after brain injury, and brain samples were harvested 24 h after brain injury) significantly decreases the expressions of hmgb1, tlr4, nf-kb dna binding and inflammatory mediators, such as, tnf-α, il-1 and il-6. ep treatment also ameliorates beam walking performance, brain edema and cortical apoptotic cell death, suggesting that the protective effects of ep maybe mediated by the reduction of hmgb1/tlr4/nf-kb-mediated inflammatory response in the injured rat brain [77] . many tbi survivors sustain neurological disability and cognitive impairment due to the lack of defined therapy to reduce tbi-induced long-term brain damage, ep (40 mg/kg was intraperitoneally injected at 15 min, 12 h, 24 h, 36 h, 48 h, 60 h after brain injury, and brain samples were taken 28 days after brain injury) confers longterm neuroprotection against tbi, possibly via breaking the vicious cycle among matrix metalloproteinase-9mediated blood-brain barrier disruption, neuroinflammation and long-lasting brain damage [78] . experimental tbi is known to produce an acute increase in cerebral glucose utilization, followed rapidly by a generalized cerebral metabolic depression. early administration of ep (40 mg/kg was intraperitoneally injected at 0 h, 1 h, 3 h, 6 h after brain injury, and brain samples were harvested 24 after brain injury) enhances cerebral glucose metabolism and neuronal survival, therefore, ep therapy is able to attenuate cerebral metabolic depression and neuronal loss after traumatic brain injury [79] . intracerebral haemorrhage (ich) is a devastating disease with no specific treatment. increasing evidence indicates that inflammatory response plays an important role in ich-induced brain damage [80, 81] . in a murine model of ich, ep treatment (3 concentrations of ep at 10 mg/kg, 50 mg/kg and 100 mg/kg were intraperitoneally injected to animals in 3 separate groups at 1 h, 6 h, 12 h after the induction of ich, and brain samples were harvested 72 h after the induction of ich) reduces brain edema and improves neurological function after ich. ep also protects neurons from haemoglobin-induced cell death in vitro and neuronal cell degeneration in ich mice. ep exerts anti-inflammatory effects via inhibiting microglia activation, nf-kb activation and decreasing tnf-α, il-1 production. these results indicate that ep protects ich induced brain damage via anti-cell death and antiinflammatory actions [80] . in another rat ich model, ep treatment (40 mg/kg was intraperitoneally injected at 1 h, 6 h and 12 h after the induction of ich, and brain samples were harvested 72 h after the induction of ich) significantly reduces inflammatory cell infiltration and expression of il-1, matrix metalloproteinase-9 in the perihematoma after ich. ep therapy also shows less brain edema, less haemorrhage and greater neurobehavioral function. the results suggest that ep ameliorates inflammatory damage after ich via hmgb1-rage signalling pathway [81] . subarachnoid hemorrhage (sah) is also a devastating disease with no specific treatment [82] . in a rat model of sah, ep treatment (a single dose of ep at 100 mg/kg concentration was intraperitoneally injected 1 h after the induction of sah, and brain samples were harvested 24 h after the induction of sah) inhibits microglia activation and reduces the expression of inflammatory cytokines tnf-α and il-1; ep therapy also inhibits apoptosis and prevents the disruption of tight junction proteins to stabilize the bbb [82] . in a rat cerebral ischemia model, ep administration (intraperitoneally injected at the doses of 1, 4, 20 and 40 mg/kg at 4 h and 24 h after the brain ischemia injury, and the size of infarct was assessed after 2 days of reperfusion) significantly reduces infarct volume and also suppresses the infarct volume related motor impairment, neurological deficits, microglial activation and inflammatory cytokine expression. furthermore, the neuroprotective effect is still evident even when the ep treatment is given as late as 24 h after the cerebral ischemia induction, suggesting that ep can protect against cerebral ischemia injury with a wide therapeutic window [83] . neonatal hypoxic-ischemic (hi) brain injury causes severe brain damage in newborns. following hi injury, rapidly accumulating oxidants injure neurons and interrupt ongoing developmental processes [84] . ep therapy (a single dose of ep at 25 mg/kg was intraperitoneally injected 30 min after hi brain injury, and brain samples were harvested at 3 h, 6 h, 12 h, 24 h,48 h,72 h, 7 days and 4 weeks after hi brain injury) and the insulin-like growth factor-1 (igf-1) treatment protect the neonatal rats brain against hi injury and improve neurological performance and these effects are additive [84] . inflammatory bowel disease is characterized by overproduction of inflammatory mediators and reactive oxygen that induce intestinal damage and chronic inflammation. inflammatory bowel disease is common but the treatment is still challenging [85, 86] . in a rat tnbs-induced colitis model, ep treatment (20 mg/kg, 40 mg/kg and 100 mg/kg were orally administered to 3 separate groups once a day for 7 days) significantly recovers the mucosal cytoarchitecture by reducing neutrophil infiltration and decreasing the levels of multiple inflammatory mediators (il-1, il-17, il-6, il-23, inos) [86] . ep therapy (40 mg/ kg was intraperitoneally injected once a day for 7 days) also ameliorates experimental colitis in mice by inhibiting the hmgb1-th17 and th1/tcl responses [85] . as inflammation is linked to cancer growth, the antiinflammatory agent ep is expected to have anti-tumor activity, and ep administration (40 mg/kg and 80 mg/kg intraperitoneally injected once a day for 9 days) significantly inhibits hepatic tumor growth [87] . the low-cost ep (40 mg/kg intraperitoneally injected twice a day for 3 weeks) elicits a potent immune-based antitumor response through inhibition of indoleamine 2, 3-dioxygenase (ido), a key tolerogenic enzyme for many human tumors [88] . ep treatment (40 mg/kg intraperitoneally injected once a day for 2 weeks) inhibits tumor angiogenesis by inhibition of the nf-kb signalling pathway [89] . hmgb1 and rage are significantly expressed in gastric adenocarcinoma, and ep treatment (40 mg/kg and 80 mg/kg intraperitoneally injected once a day for 2 weeks) inhibits gastric cancer growth via regulation of the hmgb1-rage and akt pathways [90] . ep administration (cultured cancer cells were treated with ep at 10 mm and 20 mm for up to 120 h) also inhibits growth and invasion of gallbladder cancer cells via down-regulation of the hmgb1-rage axis [91] . hepatocellular carcinoma (hcc) develops in response to chronic hepatic injury. although p53 is usually regarded as a tumor suppressor, its constant activation can promote pro-tumorigenic inflammation, at least in part, via inducing hmgb1 release. ep administration (40 mg/kg was intraperitoneally injected once every other day for 7 weeks) prevents tumorigenesis in rat livers by restoring p53, and ep treatment does not affect p53-mediated hepatic apoptosis [92] . furthermore, ep treatment (cultured cancer cells were treated with 10 mm, 20 mm ep up to 72 h) induces apoptosis and cell-cycle arrest in g phase in hepatocellular carcinoma cells [93] . in addition, ep treatment (cultured cancer cells were treated with 10 mm, 20 mm, 40 mm ep up to 120 h) defangs some malignancy-associated properties of prostate cancer cells including proliferation, invasion and anchorage-independent growth [94] . taken together, ep may have a potential as a new multi-functional compound for cancer therapy. ep does not inhibit nuclear translocation of nf-kb family members but attenuates nf-kb dna binding in an experimental colitis model [95] , more specifically, ep inhibits nf-kb activation by alkylating a critical cysteine residue (cys 38 ) in the p65 subunit of the nf-kb heterodimer, and alkylation of cys 38 interferes with dna-binding by the transcription factor [1, 2] . ep also interacts with nf-kb subunits, rel a and p50 to inhibit their functions at multiple points, for example, ep is able to inhibit the nuclear association of rel a after tnf-α treatment [96] . at least some of the antiinflammatory effects ep are related to its ability to scavenge ros, since ep is an anti-oxidant [1, 2, 12] , and oxidative stress is able to activate nf-kb-dependent gene transcription [1, 2, 12] . ep not only prevents nuclear-to-cytoplasmic translocation of hmgb1 [95] , but also inhibits cytoplasmic hmgb1 to be released extracellularly [12] . moreover, ep inhibits hmgb1 release from primary microglial cells via direct intracellular ca (2+) chelation [97] , and ep also regulates inflammation and exerts a neuroprotective effect via dual functions, chelating intracellular zn (2+) and promoting nad replenishment [98] . ep administration on translational/clinical practice ep therapy has been confirmed to be effective and safe in multiple sap animal models and multiple liver injury models, it would be reasonable to focus on sap and alcoholic hepatitis clinical trials next step. intravenous infusion is used to administer ep (from critical therapeutics inc, lexington, ma, usa) to healthy volunteers and high-risk patients undergoing coronary artery bypass graft and/or cardiac valvular surgery with cardiopulmonary bypass. ep (90 mg/kg) was administered intravenously starting after the induction of general anesthesia followed by 5 more doses of 90 mg/kg administered every 6 h. ep treatment did not reduce major complications within 14 or 28 days of surgery. ep solution has a ph of less than 7, so ep administration may contribute to acidosis, however, no significant safety concerns were discovered during the clinical trial because the adverse event profile for patients receiving ep (n = 49) was similar to that of patients receiving placebo (n = 53). ep administration has been proved to be safe in a large number of experimental animals, no severe side effect has been reported. ep is a novel anti-inflammatory agent and ros scavenger, this safe and low-cost compound is able to treat multiple inflammatory organ injuries and systemic inflammation in experimental animal models. ep also has a potential to inhibit multiple tumor growth, and this anti-tumor activity may be associated with its anti-inflammatory effect. the biochemical basis for the anti-inflammatory and cytoprotective actions of ethyl pyruvate and related compounds central role of neutrophil in the pathogenesis of severe acute pancreatitis protective effect of ethyl pyruvate on pancreas injury in rats with severe acute pancreatitis early jejunal feeding initiation and clinical outcomes in patients with severe acute pancreatitis ethyl pyruvate ameliorates distant organ injury in a murine model of acute necrotizing pancreatitis role of free radicals in the development of severe acute pancreatitis acute lung injury in acute pancreatitis-awaiting the big leap systemic inflammation with multiorgan dysfunction is the cause of death in murine ligation-induced acute pancreatitis delayed ethyl pyruvate therapy attenuates experimental severe acute pancreatitis via reduced serum high mobility group box 1 levels in rats ethyl pyruvate improves survival and ameliorates distant organ injury in rats with severe acute pancreatitis ethyl pyruvate ameliorates liver injury secondary to severe acute pancreatitis therapeutic treatment with ethyl pyruvate attenuates the severity of liver injury in rats with severe acute pancreatitis high-mobility group box 1 protein and its role in severe acute pancreatitis the nuclear factor hmgb1 mediates hepatic injury after murine liver ischemia-reperfusion hmgb1 b box increases the permeability of caco-2 enterocytic monolayers and impairs intestinal barrier function in mice high mobility group b1 impairs hepatocyte regeneration in acetaminophen hepatotoxicity diet restriction inhibits apoptosis and hmgb1 oxidation and promotes inflammatory cell recruitment during acetaminophen hepatotoxicity glycyrrhizic acid ameliorates hmgb1-mediated cell death and inflammation after renal ischemia reperfusion injury glutamine administration ameliorates sepsis-induced kidney injury by downregulating the highmobility group box protein-1-mediated pathway in mice blokade of high mobility group box-1 protein attenuates experimental severe acute pancreatitis induction of acute lung inflammation in mice with hemorrhagic shock and reperfusion: role of hmgb1 ethyl pyruvate inhibits hypoxic pulmonary vasoconstriction and attenuates pulmonary artery cytokine expressions downregulation of hmgb1 protects against the development of acute lung injury after severe acute pancreatitis neutralization of hmgb1 is associated with bacterial translocation during acetaminophen hepatotoxicity leaky gut hypothesis: a historical perspective bacteraemia in patients with acute pancreatitis as revealed by 16 s ribosomal rna gene-based technique bacterial translocation and infected pancreatic necrosis in acute necrotizing pancreatitis derives from small bowel rather than from colon role of the gut barrier in acute pancreatitis the crosstalk between gut inflammation and gastrointestinal disorders during acute pancreatitis gut origin sepsis, macrophage function, and oxygen extraction associated with acute pancreatitis in the rat effect of traditional chinese medicine on intestinal mucosal permeability in early phase of severe acute pancreatitis bacterial translocation in acute pancreatitis mesenteric lymph: the bridge to future management of critical illness physiology and pathophysiology of liver inflammation, damage and repair kupffer cell blockade reduces hepatic and systemic cytokine levels and lung injury in hemorrhagic pancreatitis in rats ethyl pyruvate ameliorates acute alcoholic-induced liver injury and inflammation in mice ethyl pyruvate reduces liver injury in a murine model of extrahepatic cholestasis ringer's lactate improves liver recovery in a murine model of acetaminophen toxicity drug hepatotoxicity: environmental factors the effect of ethyl pyruvate supplement on rat fatty liver induced by a high-fat diet ethyl pyruvate ameliorates hepatic ischemiareperfusion injury by inhibiting intrinsic pathway of apoptosis and autophagy ethyl pyruvate reduces liver injury at early phase but impairs regeneration at late phase in acetaminophen overdose ethyl pyruvate pretreatment attenuates concanavalin a-induced autoimmune hepatitis in mice ethyl pyruvate prevents inflammatory factors release and decreases intestinal permeability in rats with d-galactosamine-induced acute liver failure ethyl pyruvate protects against experimental acute-on-chronic liver failure in rats protective effect of ethyl pyruvate on liver injury in diabetic strptozotocin-induced rats fatal acute pancreatitis occurring outside of the hospital: clinical and social characteristics pancreatitis-associated acute lung injury: new insights lung injury in acute pancreatitis: mechanism, prevention, and the therapy acute pancreatitis-associated lung injury: pathophysiological mechanisms and potential future therapies ethyl pyruvate reduces lung injury matrix metalloproteinases and cytokines and improves survival in experimental model of severe acute pancreatitis ethyl pyruvate significantly inhibits tumor necrosis factor-α interleukin-1 and high mobility group box 1 releasing and attenuates sodium taurocholate-induced severe acute pancreatitis associated acute lung injury pre-or post-treatment with ethanol and ethyl pyruvate results in distinct anti-inflammatory response of human lung epithelial cells triggered by interleukin-6 ethanol, ethyl and sodium pyruvate decrease the inflammatory response of human lung epithelial cells via akt and nf-kb in vitro but have low impact on hepatocellular cells ethyl pyruvate reduces acute lung injury via regulation of inos and ho-1 expression in endotoxemic rats ethyl pyruvate reduces mortality in an endotoxin-induced severe acute lung injury mouse model inhibition of extracellular hmgb1 attenuates hyperoxia-induced inflammatory acute lung injury ethyl pyruvate inhibited hmgb1 expression induced by lps in macrophages ethyl pyruvate induces heme oxygenase-1 through p38 mitogen-activated protein kinase activation by depletion of glutathione in raw 264.7 cells and improves survival in septic animals ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation hmgb1 modulates inflammatory responses in lps-activated macrophages a critical cysteine is required for hmgb1 binding to toll-like receptor 4 and activation of macrophage cytokine release ethyl pyruvate protects against sepsis by regulating energy metabolism ethyl pyruvate reduces hepatic mitochondrial swelling and dysfunction in a rat model of sepsis expression of hmgb1 in septic serum induces vascular endothelial hyperpermeability endoplasmic reticulum stress mediates the anti-inflammatory effect of ethyl pyruvate in endothelial cells ethyl pyruvate decreases sepsis-induced acute renal failure and multiple organ damage in aged mice ethyl pyruvate ameliorates albuminuria and glomerular injury in the animal model of diabetic nephropathy protective effects of ethyl pyruvate in cisplatin-induced nephrotoxicity ethyl pyruvate attenuated coxackievirus b3-induced acute viral myocarditis by suppressing of hmgb1/rage/nf-kb pathway role of high-mobility group box 1 in myocardial ischemia/reperfusion injury and the effect of ethyl pyruvate effects of ethyl pyruvate on cardiac function recovery and apoptosis reduction after global cold ischemia and reperfusion ethyl pyruvate has anti-inflammatory and delayed myocardial protective effects after regional ischemia/reperfusion injury ethyl pyruvate preserves cardiac function and attenuates oxidative injury after prolonged myocardial ischemia beneficial effects of sodium or ethyl pyruvate after traumatic brain injury in the rat beneficial effects of ethyl pyruvate through inhibiting high-mobility group box 1 expression and tlr4/nfkb pathway after traumatic brain injury in the rat ethyl pyruvate protects against blood-brain barrier damage and improves long-term neurological outcomes in a rat model of traumatic brain injury pyruvate treatment attenuates cerebral metabolic depression and neuronal loss after traumatic experimental brain injury ethyl pyruvate ameliorates intracerebral hemorrhage-induced brain injury through anti-cell death and anti-inflammatory mechanisms blockade of high mobility group box-1 signaling via the receptor for advanced glycation end-products ameliorates inflammatory damage after acute intracerebral hemorrhage ethyl pyruvate alleviates early brain injury following subarachnoid hemorrhage in rats inhibition of the cerebral ischemic injury by ethyl pyruvate with a wide therapeutic window combination treatment with ethyl pyruvate and igf-i exerts neuroprotective effects against brain injury in a rat model of neonatal hypoxic-ischemic encephalopathy ethyl pyruvate ameliorates experimental colitis in mice by inhibiting the hmgb1-th17 and th1/tc1 responses intestinal antiinflammatory activity of calcium pyruvate in the tnbs model of rat colitis: comparison with ethyl pyruvate ethyl pyruvate administration inhibits hepatic tumor growth immunotherapeutic suppression of indoleamine 2, 3-dioxygenase and tumor growth with ethyl pyruvate ethyl pyruvate, an anti-inflammatory agent, inhibits tumor angiogenesis through inhibition of nf-kb signaling pathway inhibition effects of ethyl pyruvate administration on human gastric cancer growth via regulation of hmgb1-rage and akt pathways in vitro and in vivo ethyl pyruvate administration suppresses growth and invasion of gallbladder cancer cells via downregulation of hmgb1-rage axis p53 promotes inflammation-associated hepatocarcinogenesis by inducing hmgb1 release ethyl pyruvate inhibits proliferation and induces apoptosis of hepatocellular carcinoma via regulation of hmgb1-rage and akt pathways exploring glyoxalase 1 expression in prostate cancer tissues: targeting the enzyme by ethyl pyruvate defangs some malignancy-associated properties ethyl pyruvate decreases hmgb1 release and ameliorates murine colitis interaction of ethyl pyruvate in vitro with nf-kb subunits, rel a and p50 ethyl pyruvate inhibits hmgb1 phosphorylation and release by chelating calcium neuroprotective effect of ethyl pyruvate against zn (2+) toxicity via nad replenishment and direct zn αα2 +) chelation not applicable. this investigation was partly supported by sigrid juselius funding in finland and south-eastern norway regional health authority, grant number 2013121. data sharing not applicable to this article as no datasets were generated or analyzed during the current study. author contributions rky designed and drafted the manuscript. stz carried out literature search and drafted the manuscript. tit drafted and revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. not applicable.ethics approval and consent to participate not applicable.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord-023394-ptfjxpo6 authors: isa, a.; norbeck, o.; pöhlmann, c.; tolfvenstam, t. title: mapping of the ex vivo cellular immune response against the complete human parvovirus b19 genome during acute infection date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423n.x sha: doc_id: 23394 cord_uid: ptfjxpo6 background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self‐limiting disease, but also capable of causing both significant pathology and long‐term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifnγ enzyme‐linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850–1850 sfc/million pbmcs, roughly corresponding to 0.3–0.6% b19‐specific cd8(+) cells circulating in peripheral blood at 10–80 weeks post‐infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow‐up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post‐infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-002209-xs6qigg4 authors: kıray, hülya; lindsay, susan l.; hosseinzadeh, sara; barnett, susan c. title: the multifaceted role of astrocytes in regulating myelination date: 2016-09-17 journal: exp neurol doi: 10.1016/j.expneurol.2016.03.009 sha: doc_id: 2209 cord_uid: xs6qigg4 astrocytes are the major glial cell of the central nervous system (cns), providing both metabolic and physical support to other neural cells. after injury, astrocytes become reactive and express a continuum of phenotypes which may be supportive or inhibitory to cns repair. this review will focus on the ability of astrocytes to influence myelination in the context of specific secreted factors, cytokines and other neural cell targets within the cns. in particular, we focus on how astrocytes provide energy and cholesterol to neurons, influence synaptogenesis, affect oligodendrocyte biology and instigate cross-talk between the many cellular components of the cns. astrocytes were long considered secondary to neurons in central nervous system (cns) function, and erroneously dismissed as "brain glue" (glia is the greek term for glue). research over the past two decades, however, has shown astrocytic roles extending to a range of brain functions far beyond basic physical and metabolic neuronal support (sofroniew and vinters, 2010) . astrocytic regulation of myelination was first hypothesised by műller in 1904, who claimed that the demyelinating disease, multiple sclerosis (ms), was rooted in astrocytic dysfunction (müller, 1904; williams et al., 2007) . evidence has since continued to grow supporting the premise that astrocytes could be important in regulating myelination (sofroniew and vinters, 2010; williams et al., 2007; barnett and linington, 2013; moore et al., 2011) . glial fibrillary acidic protein (gfap) has been used extensively in the study of astrocytes. increased gfap expression has been associated with astrocyte reactivity in cns lesions and is a pathological hallmark of disease and/or injury. fig. 1 illustrates astrocytes immunolabelled with gfap and nestin, another marker thought to label reactive astrocytes (kamphuis et al., 2012) . in experimental allergic encephalomyelitis (eae), a widely used animal model of ms, where demyelination is induced by myelin antigens, administered together with adjuvant that contains bacterial components (traugott and lebon, 1988; tsukada et al., 1991; villarroya et al., 1996) , gfap expression was seen on more numerous and much larger astrocytic processes in chronic lesions compared to normal appearing white matter (webster et al., 1985; eng et al., 1971) . thus, the degree of gfap immunoreactivity appears to reflect the level of reactive astrogliosis. this was reviewed in detail by sofroniew and vinters (2010) , who described a continuum of phenotypic changes, that range from mild to severe, the latter resulting in glial scar formation (sofroniew and vinters, 2010; nash et al., 2011a) . attempts have also been made to define the astrocyte phenotype in more detail along this continuum (liberto et al., 2004) . it has been suggested that mild astrogliosis is associated with astrocyte "activation" and severe astrogliosis is associated with "reactivity", with the former promoting recovery of cns function after injury and the latter walling off the injured area and preventing repair (liberto et al., 2004) . although activated astrocytes have been associated with less detrimental effects on the cns and reactive astrocytes as more damaging, it is clear that these properties are not all or nothing and reactive astrocytes can fig. 1 . expression of astrocyte reactivity markers. rat neurosphere-derived astrocytes cultured on pll-coated glass coverslips express the reactivity markers gfap (green) and nestin (red). astrocytic effects on re/myelination can be classified into 4 main groups. they contribute to re/myelination by: 1) providing an energy source (lactate) and cholesterol for neurons. glucose taken up by endothelial cells lining the blood brain barrier is later transferred to astrocytes which transform it to glycogen, which can then be used to produce lactate. 2) playing a role in synaptic signal transmission by regulating the fluid, ph/ion (e.g. potassium, k + ), glio/neurotransmitter homeostasis and contributing to synapse modulation through secreted molecules, such as thrombospondins (thbss) . 3) affecting the survival, proliferation and maturation of oligodendrocytes by secreting growth factors, some of which are regulated by iron homeostasis provided by astrocytes. chemokines may also influence oligodendrocyte membrane ensheathment of axons. 4) altering reactivity status through their release of chemokines/cytokines, which in turn affects the cross-talk between all neural cells including microglia. also be beneficial to cns repair. interestingly, in studies using gfap null mice, it was seen that animals had abnormal myelination, nonmyelinated axons in the optic nerve with an age related reduction in myelin thickness (liedtke et al., 1996) . non-conservative mutations in the gfap gene have also been linked to the white matter brain disorder, alexander disease (brenner et al., 2001; li et al., 2002) . therefore, the evidence for direct or indirect astrocytic roles on re/myelination has been established both by in vitro and in vivo studies. considering the limitations in the current treatments for demyelinating cns diseases and injuries, it is crucial to identify other approaches to regulate myelination in search of novel strategies for repair. astrocytes have been shown to promote myelination through their supportive roles on neuron survival and maintenance of neuronal activity, and their direct action on proliferation, differentiation and migration of oligodendrocytes (fig. 2) . this review will focus on the interaction of astrocytes with neural cells to synergistically promote myelination. it is apparent that astrocytes can affect myelination under a range of normal and pathological conditions, but it is important to understand how this is regulated. many molecules can trigger or even regulate astrogliosis, including large polypeptide growth factors and cytokines (john et al., 2003; moore et al., 2011) , mediators of innate immunity such as lipopolysaccharide (lps) and other toll-like receptor ligands (farina et al., 2007) , neurotransmitters (bekar et al., 2008) , purines, reactive oxygen species, and molecules related to hypoxia and glucose deprivation (swanson et al., 2004) . for the purpose of this review we will focus on cytokines and chemokines. although these compounds are primarily considered in the context of chemotaxis in immune cells, here we will highlight their roles on astrocyte activation and reactivity. these molecules can be produced in an autocrine or paracrine fashion by various cell types in the cns including neurons, oligodendrocyte lineage cells, microglia, pericytes and endothelial cells. not only do these factors influence astrocyte phenotype but they also can affect a range of neural and immune cell types. 2.2. astrocyte activation (mild astrogliosis): a pro-reparative phenotype? astrocytes can be activated directly or indirectly. for example, in response to injury, microglia become activated and release the cytokine interleukin 1β (il-1β, herx et al., 2000) , which is an early injury signal (auron, 1998) . the delay of astrocyte activation in mice lacking il-1β, as well as in mice lacking il-1 type 1 receptor suggests that microglial activation is necessary for astrocyte activation (herx et al., 2000) . it has also been suggested that ciliary neurotrophic factor (cntf; a member of the il-6 family of cytokines)treated astrocytes in vitro had a phenotype that was more supportive of cns repair and thus are, by definition, activated (albrecht et al., 2002; . under cntf treatment, astrocytes upregulate expression of classical reactivity markers such as gfap, vimentin, and clusterin, show cellular and nuclear hypertrophy, and increase their proliferation rate (winter et al., 1995; levison et al., 1996; 1998; hudgins and levison, 1998) . there is a growing body of evidence for the promotion of neuronal survival by cytokine-activated astrocytes, potentially through secretion of various neurotrophic growth factors in the vicinity of neurons including nerve fig. 3 . simplified schematic of the effects of cytokine-activated astrocytes on re/myelination. astrocytes can be influenced by various cytokines to change their reactivity status to one that falls within the continuum of phenotypes, namely more activated or reactive; both of which will secrete factors that can modulate myelination in a positive or negative way. astrocytes with more severe reactivity present increased gfap expression, proliferation rate, and cellular hypertrophy with a more stellate morphology. the milder "activated" astrocytes can secrete a range of factors including; neurotrophic factors, growth factors, and cytokines that will stimulate re/myelination by promoting neuronal survival, neurite outgrowth, neurogenesis, and/ or oligodendrocyte precursor cell (opc) survival, proliferation, and/or maturation. conversely astrocytes that tend to have a more severe "reactive" phenotype, possibly induced by proinflammatory cytokines/cns tissue damage, may secrete cytokines and chemokines that lead to myelin and oligodendrocyte damage in vitro, suppress remyelination, delay disease recovery in experimental autoimmune encephalomyelitis (eae), and suppress myelination in myelinating embryonic rat mixed spinal cord cultures. however, these reactive scar forming astrocytes can also protect cns tissue by preventing immune cells from invading and exerting a pro-inflammatory response and have been shown to even ameliorate eae. growth factor (ngf), brain-derived neurotrophic factor (bdnf), activity dependent neurotrophic factor (adnf), hepatocyte growth factor (hgf), leukaemia inhibitory factor (lif), fibroblast growth factor-2 (fgf-2) and cntf (schwartz and nishiyama, 1994; rudge et al., 1995; uchida et al., 1998; dreyfus et al., 1999; messersmith et al., 2000; albrecht et al., 2002; fig. 3 ). moreover, cultured spinal cord astrocytes, treated with cntf, support the survival of a significantly greater number of ventral spinal motor neurons and promote neurite outgrowth better than unstimulated astrocytes (albrecht et al., 2002) . other researchers have shown that cytokine-activated astrocytes can promote neurogenesis, possibly by stimulating the differentiation of neural stem cells (nscs) residing in the subventricular zone and the dentate gyrus in adult animals (liberto et al., 2004) . because these multipotent nscs can migrate beyond their sites of origin and can later differentiate into oligodendrocytes, neurons and microglia, they have the potential to enhance recovery from cns injury and disease. importantly, activated astrocytes have also shown positive effects on myelination. our own investigations have shown that cntf activated astrocytes can promote the percentage of myelinated fibres in cns rat cultures (nash et al., 2011b) . further evidence of this has been shown in mice infected with the a-59 strain of the mouse hepatitis virus (mhv-a59), an animal model for ms (jordan et al., 1989 , messersmith et al., 2000 . these animals have been shown to secrete increased levels of cntf during the remyelination phase and cntf mrna is induced in the remyelinating regions in cells exhibiting astrocytic features (albrecht et al., 2003) . it is suggested that the increase in il-1β levels at early stages of cns pathology stimulates the induction of cntf mrna and protein in astrocytes (stöckli et al., 1991; guthrie et al., 1997; dallner et al., 2002; liberto et al., 2004) , a phenomenon which appears to be important for remyelination (herx et al., 2000) . this could be due to fgf-2 signalling as cntf treatment elevates astrocytic levels of fgf-2 mrna significantly, whereas, il-1 β shows no effect (albrecht et al., 2003) . since fgf-2 can enhance opc proliferation (albrecht et al., 2003) , it may produce more opcs for subsequent myelination (redwine et al., 1997; messersmith et al., 2000) . moreover, if the gp130 receptor, the ubiquitous signal transducer for cntf and all il-6 family members, is genetically removed from astrocytes, astrocyte survival was poor, there was a reduction in the development of astrogliosis, and larger areas of demyelination formed with a greater proinflammatory t cell response (haroon et al., 2011) . therefore, cntf seems to be an important cytokine involved in astrocyte reactivity and myelination. interestingly, il-1β can also stimulate the astrocytic production of another il-6 family cytokine, lif, (aloisi et al., 1994) , which has been shown to promote survival and differentiation of oligodendrocytes (khan and de vellis, 1994; mayer et al., 1994; bugga et al., 1998) . lif also decreases disease severity when exogenously administered in both chronic and relapsing-remitting eae mice (aloisi et al., 1994; butzkueven et al., 2002; ishibashi et al., 2006) . positive effects of lif on the survival and maturation of oligodendrocytes also provides evidence for the positive roles of lif on myelination (khan and de vellis, 1994; mayer et al., 1994; bugga et al., 1998) . however, other pro-inflammatory cytokines such as tumour necrosis factoralpha (tnf-α) and interferon-gamma (ifn-γ) have also been shown to potentiate reactive astrogliosis (yong et al., 1991 john et al., 2003 as discussed below. in contrast to their positive effects on myelination, astrocytes can also have a more detrimental effect on cns repair via the secretion of chemokines/cytokines when in a more severe, reactive state (fig. 3) . one such cytokine is tnf-α, which has been shown to induce myelin and oligodendrocyte damage in vitro (selmaj and raine, 1988) . tnf-α mrna expression in ms plaques positively correlates with the extent of demyelination and has been shown to be present in microglia/macrophages and to a smaller percentage of astrocytes (bitsch et al., 2000) . on the other hand, studies have shown tnf-α expression is mainly associated with gfap positive fibrous astrocytes in chronic active ms lesions at the lesion edge (hofman et al., 1989) as well as foamy macrophages and endothelial cells (selmaj et al., 1991) . however, the fact that astrocytes appear as the major or minor tnf-α expressing cell types in ms lesions might be because astrocytes internalize the protein in a receptor-mediated manner (aránguez et al., 1995; kuhlmann et al., 2006) rather than producing it themselves as suggested by hofman et al. (1989) and bitsch et al. (2000) . moreover, it is possible that astrocytes require a longer period of time to become reactive upon injury and only produce tnf-α first at the lesion edge of acute ms plaques and later both at the lesion edge and in the lesion centre of chronic active plaques (selmaj et al., 1991) . in situ hybridisation for tnf-α mrna has been detected in gfap-positive astrocytes in mice suffering from pneumococcal meningitis (izadpanah et al., 2014) which also suggests that astrocytes can indeed produce tnf-α in cns pathologies. since tnf-α effects the maturation of oligodendrocytes (cammer and zhang, 1999) , remyelination failure in the cns lesions could be because tnf-α prevents the in situ differentiation of oligodendrocytes. interestingly, direct cell contact between pre-oligodendrocytes (preols) and astrocytes has been shown to be a prerequisite for tnf to induce apoptosis in preols of rodent mixed glial cultures (kim et al., 2011) . nevertheless, it is possible that tnf-α increases production of pdgf in demyelinated spinal cord lesions of mhv-a59-injected mice (redwine and armstrong, 1998; frost et al., 2003) as suggested by the increase of pdgf-β transcription and pdgf-αβ protein levels in embryonic human astrocytes upon tnf-α treatment (silberstein et al., 1996) . therefore, astrocytes might have a positive role on remyelination both by producing tnf-α and by secreting pdgf upon stimulation with tnf-α. pdgf could in turn support the survival and enhance the proliferation of opcs in demyelinating lesions (woodruff et al., 2004; vana et al., 2007) . consequently, it is yet difficult to conclude whether reactive astrocytes associated with increased tnf-α levels in cns lesions are predominantly stimulatory or inhibitory to remyelination. ifn-γ, another cytokine found in ms plaques, has been reported to not only activate astrocytes, but is also expressed by reactive astrocytes and by immune cells that astrocytes have stimulated (pulver et al., 1987; traugott and lebon, 1988; miljkovic et al., 2007; hashioka et al., 2009; ionescu et al., 2011) . similar to tnf-α, ifn-γ has been shown to suppress remyelination and to delay disease recovery in transgenic eae mice, where ifn-γ expression by astrocytes was stimulated temporally in the recovery stage (lin et al., 2006) . astrocyte-directed expression of ifn-γ in transgenic mice has also resulted in regional hypomyelination and selective disruption of brain histogenesis, which led to ataxia and shorter life span (laferla et al., 2000) . furthermore, knocking down ifn-γ receptor expression in astrocytes three days before immunization suppressed eae and demyelination by inhibiting inflammatory cell infiltration (ding et al., 2015) . these animals presented lower mean clinical scores even when the receptor silencing was initiated after disease onset or at disease peak (ding et al., 2015) . despite the abovementioned evidence suggesting inhibitory roles for reactive astrocytes on myelination, hindinger et al. (2012) have proposed that ifn-γ signalling in astrocytes is indispensable for the alleviation of eae since levels of demyelination and axonal loss are increased during acute eae in mice with an astrocytic expression of a dominant negative allele for ifn-γ receptor. nevertheless, their approach blocked ifn-γ signalling in astrocytes without decreasing the expression of ifn-γ receptor, which would lower the levels of ifn-γ available for immunoregulatory cells; whereas, ding et al. (2015) have knocked down the expression of the receptor itself. therefore, blocking or lowering ifn-γ signalling in astrocytes with a carefully planned strategy might provide new disease-modifying treatments that will limit demyelination. reactive astrocytes also secrete c-x-c motif chemokine 10 (cxcl10, ransohoff et al., 1993) , particularly around active ms lesions (omari et al., 2005; carter et al., 2007) . cxcl10 mrna expression increases significantly during peak disease and decreases during the recovery phases in animal models of ms (godiska et al., 1995; glabinski et al., 1997; fife et al., 2001) . a direct effect of cxcl10 on the inhibition of myelination was shown in dissociated rat spinal cord cells plated on astrocytes treated with cxcl10 and its neutralizing antibody. in these experiments cxcl10 identified by microarray analysis, was upregulated in an astrocyte phenotype that was inhibitory to cns myelination in vitro. specifically, cxcl10 appeared to inhibit oligodendrocyte process extension (nash et al., 2011b) . consequently, cytokines stand out as an important family of molecules to activate astrocytes and to initiate different forms of astrocyte reactivity that could be either beneficial or inhibitory for the cns milieu in terms of re/myelination. it should be noted that the secretion of such pro-inflammatory cytokines that can contribute to the lack of remyelination within ms plaques is not restricted to reactive astrocytes since other glial and inflammatory cell types will also secrete them. moreover, the up regulation of such cytokines by reactive astrocytes can also be protective for cns injury. for example, the astrocytic scar can restrict leukocyte migration from within areas of damaged tissue into the otherwise healthy non damaged cns tissue in close proximity to the scar protecting it from immune mediated damage (faulkner et al., 2004; okada et al., 2006; herrmann et al., 2008; voskuhl et al., 2009 ). a vital supportive role played by astrocytes following injury is the provision of an energy source, which is important if axons are to be myelinated. this energy is metabolized from glucose which enters the brain via the endothelial cells lining the blood brain barrier (bbb), which are in close contact with astrocytes. unlike endothelial cells, astrocytes biochemically transform glucose into glycogen, the principal source of stored energy in all cell types (pellegri et al., 1996; pfeiffer-guglielmi et al., 2003) . in addition, it has been suggested that astrocytes under low glucose concentrations can degrade stored glycogen into lactate which in turn increases extracellular lactate levels to provide energy for nearby axons when deprivation occurs after injury (tekkök et al., 2005) . the lactate derived from astroglial glycogen via glycolysis is transferred directly to the axon at the node of ranvier (hirrlinger and nave, 2014) . the importance of lactate during demyelination is only just emerging. whether astrocytes maintain the energy levels of only axons as previously thought, or if this extends to oligodendrocytes as well, is an interesting concept. oligodendrocytes are known to consume lactate at higher levels than other cns cells, therefore making them an important user of any lactate production. furthermore, promotion of myelination via oligodendrocytes has been shown when endogenous lactate is applied (rinholm and bergersen, 2012) therefore at least some astrocytic lactate production may be targeted to myelinating oligodendrocytes (sánchez-abarca et al., 2001; rinholm et al., 2011) . however, energy regulation in the cns is more complex as recent evidence has shown that oligodendrocytes in turn can transfer glycolysis products such as lactate to axons via monocarboxylic acid transporters (mct1, mct2), which reside in internodal myelin and the axonal compartment (fünfschilling et al., 2012) . cholesterol is essential to every cell in the body as it is an important component of cellular membranes. in the cns it is vital for normal brain development, is a precursor to many signalling molecules such as steroid hormones, and importantly, is a major structural component of myelin sheaths (siegel et al., 1999) . the bbb prevents the transport of either hepatic or dietary cholesterol, meaning that cholesterol must be derived by de novo synthesis within the cns (orth and bellosta, 2012) . astrocytes are proposed to be one of the primary cellular sources of cholesterol (pfrieger and ungerer, 2011) and mediate its secretion by their expression of several apolipoproteins, molecules that bind cholesterol (boyles et al., 1985; lin et al., 1986; xu et al., 2006; kurumada et al., 2007) . there is sufficient evidence to suggest that there is horizontal transfer of cholesterol (boyles et al., 1985; lin et al., 1986; xu et al., 2006; kurumada et al., 2007) with both astrocytes and oligodendrocytes producing cholesterol to maintain myelin sheath formation and neurons. this transfer between cells is critically relevant to neurodegenerative diseases, since the availability of cholesterol is thought to be an essential rate limiting factor to myelin production (may et al., 2004; liu et al., 2010) . therefore, in addition to their roles in providing energy to neurons, astrocytes also emerge as important cholesterol-suppliers in the cns, which is vital for myelination. a further function of astrocytes is the removal of excitotoxic molecules from the extracellular space, thus supporting neuronal survival. they can actively remove excitotoxic glutamate and convert it to glutamine by increasing their levels of glutamate transporters and glutamine synthetase (faden et al., 1989; eng et al., 1997; krum et al., 2002) , thereby preventing neuronal cell death during brain pathology. astrocytes can also release gliotransmitters such as glutamate, purine, gaba and d-serine into the synaptic cleft upon excitation by changes in neuronal synaptic activity and can thereby regulate neuronal excitability (parpura et al., 1994; bezzi et al., 1998; mothet et al., 2000; coco et al., 2003; halassa et al., 2007) . neurotransmitter released from the neuronal synapse can reach adjacent astrocytes, stimulating increases in intracellular ca 2+ concentrations, which then leads to the secretion of gliotransmitters (porter and mccarthy, 1997) . these regulatory molecules then can feedback to presynaptic nerve terminals to modulate synaptic neurotransmission (araque et al., 1998) . these observations have even given rise to the currently accepted 'tripartite synapse' hypothesis, where astrocytes form an active, integral regulatory component of the synapse (araque et al., 1999; halassa et al., 2007) . recently, electron microscopy has also shown microglia interacting with neuronal synapses (tremblay et al., 2010) and playing a role in synapse maturation, synaptic remodelling, and synaptic activity (ji et al., 2013) . evidence has shown that microglia secrete immune factors that play an important role in synaptic connections and illustrates the complexity of cross-talk between neural cells and the immune system. these researchers have suggested a change in name from tripartite synapse to the quad-partite synapse (schafer et al., 2013; wu et al., 2015) . in addition to playing a role in synaptic signal transmission, astrocytes can also modulate synapses. it has been demonstrated that astrocytes secrete molecules such as thrombospondins that might be required for the formation, function and pruning of developing synapses (ullian et al., 2001; christopherson et al., 2005) . both presynaptic and postsynaptic activity in purified rat retinal ganglion cultures have been enhanced in the presence of astrocytes; and immunohistochemistry of rat brain cryosections from various developmental stages have shown that glial generation precedes the appearance of synapses (ullian et al., 2001) . astrocytes might also be functional in synaptic remodelling and pruning in healthy or diseased adult cns (barres, 2008) . because synaptic signal transmission can trigger astrocytes to secrete the cytokine lif, which in turn increases the number of myelinated axons in dorsal root ganglion cultures co-cultured with oligodendrocytes (ishibashi et al., 2006) , the support and maintenance of healthy signal transmission appears important for the regulation of myelination. furthermore, astrocytes may contribute to synaptic transmission by supporting maintenance of the synaptic interstitial fluid by regulating ion, ph and transmitter homeostasis. astrocyte processes contain transporters for potassium uptake and aquaporin 4 water channels (nielsen et al., 1997; rash et al., 1998; amiry-moghaddam et al., 2003; solenov et al., 2004) , which maintain the transmitter homeostasis of the synaptic interstitial fluid (steinhäuser et al., 1994; gundersen et al., 1996; mennerick et al., 1996; bergles and jahr, 1997; fujita et al., 1999) . connexin channels and connexin proteins are important candidates which play a role in the regulation of neuronal activity and survival. for example, astroglial connexins decrease neuronal excitability by removing extracellular potassium and glutamate; while also providing metabolic supply to neurons (wallraff et al., 2006; rouach et al., 2008; froger et al. 2010; pannasch et al., 2011) . on the other hand, the elevation of connexin expression at lesion sites in cns pathologies might also be associated with other, possibly protective, roles (nagy et al., 1996; koulakoff et al., 2008; kuchibhotla et al., 2009; mei et al., 2010; karpuk et al., 2011) . studies using connexin knockout animals allude to the importance of connexins in promoting communication between astrocytes and between astrocytes and oligodendrocytes on myelin integrity. myelin damage has been observed in cx47, cx43/cx30 and cx30/cx47 single/double knockout mice (menichella et al., 2003; odermatt et al., 2003; lutz et al., 2009; tress et al., 2012) . interestingly, the level of oligodendrocyte gap junction connexins cx47 and cx32 were reduced both within and around lesions during early stages of inflammatory demyelination in eae mice, (traugott and lebon, 1988; tsukada et al., 1991; villarroya et al., 1996; meeuwsen et al., 2003) . these mice also presented decreased expression of cx43, the major astrocytic partner of cx47, when spinal cord sections were analysed immunohistochemically (markoullis et al., 2012) . cx43 expression was increased at late eae stages, where remyelination was observed, leading to the suggestion that astrocytic protein cx43 might play an important role in recovery from neuroinflammation (markoullis et al., 2012) . in the 1980s advancements were made on the ability to grow purified cultures of glial cells, with raff and colleagues in particular developing techniques to purify opcs from the optic nerve, a tissue devoid of neuronal cell bodies (raff et al., 1983) . with the development of these culture techniques it was shown that astrocytes play important roles in opc differentiation (noble and murray, 1984; noble et al., 1988; raff et al., 1988; noble et al., 1989) and in the rate of oligodendrocyte axonal ensheathment (watkins et al., 2008) . further in vitro studies, where conditioned medium collected from primary astrocyte monolayers was incubated with other neural cells, showed enhanced neuronal survival, proliferation of opcs, and protection of oligodendrocytes from stress (noble and murray, 1984; yoshida et al., 1995; yamamuro et al., 2003; zhu et al., 2006; arai and lo, 2010) . it is likely that astrocytes support opc survival and proliferation by providing soluble factors such as platelet derived growth factor (pdgf) and fgf-2 (bögler et al., 1990; ferrara et al., 1988; pringle et al., 1989) . astrocytes are important providers of secreted growth factors, for both neuronal and glial proliferation and survival. for example, cntf, although shown to be important in the activation of astrocytes, is constitutively expressed by white matter astrocytes, and is a key player in opc survival and maturation in vitro and in vivo as discussed earlier (stöckli et al., 1991; dallner et al., 2002; stankoff et al., 2002; cao et al., 2010) . cntf has also been reported to enhance the migration of subventricular zone-derived progenitors (vernerey et al., 2013) , protect oligodendrocytes from apoptosis, and decrease myelin destruction in demyelinating pathological conditions (linker et al., 2002) . in studies when cntf was injected subcutaneously at the remyelination phase of cuprizoneinduced demyelination, an increase was seen in myelin oligodendrocyte glycoprotein (mog) expression in the cerebral cortex (salehi et al., 2013) . moreover, intraperitoneal injections of cntf and intravenously transplanted mesenchymal stem cells that overexpress cntf resulted in a reduced loss of neurons and disease severity, and increased neuronal functional recovery in eae mice (kuhlmann et al., 2006; lu et al., 2009) . however, in these experiments it is difficult to see if the effect is on the activation status of the astrocytes as discussed above, or the direct effect on the opc. astrocytes have also been shown to exhibit a crucial role in opc remyelination via their iron exporter ferroportin (fpn) in mice, where focal demyelination was induced by the injection of lysophosphatidylcholine (lpc) into their spinal cords (schulz et al., 2012) . in these astrocyte-specific fpn ko mice, fewer remyelinating axons and a reduction in opc proliferation were observed following lpc-induced demyelination compared to control animals. this could either be due to direct effects on opcs through limited iron supply or indirect effects via iron-deficient microglia, which expressed significantly lower levels of tnf-α and il-1β when stimulated by lps compared to control microglia (schulz et al., 2012) . because the expression of fgf-2 and insulin-like growth factor-1 (igf-1) was significantly upregulated by il-1β and tnf-α, respectively, and the expression of transforming growth factor beta (tgf-β) was stimulated by il-1β in purified astrocyte cultures, it has been suggested that iron-deprivation in the milieu would lower the expression of il-1β and tnf-α in microglia and thus lead to reduced growth factor expression in astrocytes, which would in turn render opc proliferation and possibly differentiation (schulz et al., 2012) . as discussed throughout this review astrocytes can play important roles not just in myelination during development, but also in remyelination in adult tissue after cns injury. their reparative roles might be related to their level of reactivity, so it is important to identify markers that can define these different astrocyte phenotypes although at present these are not easy to define. one approach is to use microarrays. as described, nash et al. (2011b) identified cxcl10 as inhibitory to myelination, but others using a lower-scale cdna array that contained probes for cytokines, chemokines, growth factors and their receptors have identified other pro-inflammatory cytokines including tnf-α, il-1β, or ifn-γ (meeuwsen et al., 2003) . another large array was carried out on gfp-astrocytes purified at various time points after the onset of two models of disease, namely ischemic stroke (middle cerebral artery occlusion) and neuroinflammation induced by lps injection (zamanian et al., 2012) . the resulting data suggested that astrocytes could present different mrna expression profiles depending on the insult despite the presence of reactive gliosis in both types of cns damage. however, although there was upregulation of a core set of genes (lipocalin 2 and serpina3n), it was clear that changes in the astrocyte after injury are highly heterogeneous, and that changes in astrocyte activity may depend on the injury type. hopefully, more specific markers of "good" or "bad" astrocytes for cns repair will be identified and allow more specific identification of how these astrocytes influence repair. there is abundant evidence to suggest that astrocytes contribute to re/myelination mainly by: 1) providing the right conditions for neurons to myelinate by i) supplying neurons with energy and cholesterol, ii) removing excitotoxic molecules from the extracellular environment, and iii) regulating the fluid, ion, ph, and neuro/gliotransmitter homeostasis. 2) playing a role in the survival, proliferation, maturation and function of oligodendrocytes and the migration of opcs into the lesioned areas in the cns. 3) influencing microglia. the manner by which astrocytes affect myelination can often be seen to correlate with its level of reactivity. astrocyte reactivity can be induced by the milieu of cytokines present after injury, which can be beneficial or inhibitory in re/myelination depending on the context and severity of the 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activity and oligodendrocyte pathology cooperation between two growth factors promotes extended self-renewal and inhibits differentiation of oligodendrocyte-type-2 astrocyte (o-2 a) progenitor cells apolipoprotein e associated with astrocytic glia of the central nervous system and with nonmyelinating glia of the peripheral nervous system mutations in gfap, encoding glial fibrillary acidic protein, are associated with alexander disease analysis of neuronal and glial phenotypes in brains of mice deficient in leukemia inhibitory factor lif receptor signaling limits immune-mediated demyelination by enhancing oligodendrocyte survival maturation of oligodendrocytes is more sensitive to tnf alpha than is survival of precursors and immature oligodendrocytes transplantation of ciliary neurotrophic factor-expressing adult oligodendrocyte precursor cells promotes remyelination and functional recovery after spinal cord injury induction of the genes for cxcl9 and cxcl10 is dependent on ifn-gamma but shows differential cellular expression in experimental autoimmune encephalomyelitis and by astrocytes and microglia in vitro thrombospondins are astrocytesecreted proteins that promote cns synaptogenesis storage and release of atp from astrocytes in culture cntf and cntf receptor alpha are constitutively expressed by astrocytes in the mouse brain silencing ifn-γ binding/signaling in astrocytes versus microglia leads to opposite effects on central nervous system autoimmunity expression of neurotrophins in the adult spinal cord in vivo an acidic protein isolated from fibrous astrocytes expression of glutamate uptake transporters after dibutyryl cyclic amp differentiation and traumatic injury in cultured astrocytes the role of excitatory amino acids and nmda receptors in traumatic brain injury astrocytes are active players in cerebral innate immunity reactive astrocytes protect tissue and preserve function after spinal cord injury bovine brain astrocytes express basic fibroblast growth factor, a neurotropic and angiogenic mitogen cxcl10 (ifn-gamma-inducible protein-10) control of encephalitogenic cd4+ t cell accumulation in the central nervous system during experimental autoimmune encephalomyelitis inhibition of cytokine-induced connexin43 hemichannel activity in astrocytes is neuroprotective pdgf and fgf2 regulate oligodendrocyte progenitor responses to demyelination differential expressions of glycine transporter 1 and three glutamate transporter mrna in the hippocampus of gerbils with transient forebrain ischemia glycolytic oligodendrocytes maintain myelin and long-term axonal integrity synchronous synthesis of alpha-and beta-chemokines by cells of diverse lineage in the central nervous system of mice with relapses of chronic experimental autoimmune encephalomyelitis chemokine expression in murine experimental allergic encephalomyelitis selective excitatory amino acid uptake in glutamatergic nerve terminals and in glia in the rat striatum: quantitative electron microscopic immunocytochemistry of exogenous (d)-aspartate and endogenous glutamate and gaba astroglial ciliary neurotrophic factor mrna expression is increased in fields of axonal sprouting in deafferented hippocampus the tripartite synapse: roles for gliotransmission in health and disease gp130-dependent astrocytic survival is critical for the control of autoimmune central nervous system inflammation interferon-gamma-dependent cytotoxic activation of human astrocytes and astrocytoma cells stat3 is a critical regulator of astrogliosis and scar formation after spinal cord injury central nervous system-initiated inflammation and neurotrophism in trauma: il-1 beta is required for the production of ciliary neurotrophic factor ifn-γ signaling to astrocytes protects from autoimmune mediated neurological disability adapting brain metabolism to myelination and long-range signal transduction tumor necrosis factor identified in multiple sclerosis brain ciliary neurotrophic factor stimulates astroglial hypertrophy in vivo and in vitro cultured adult porcine astrocytes and microglia express functional interferon-γ receptors and exhibit toxicity towards sh-sy5y cells astrocytes promote myelination in response to electrical impulses brain parenchymal tnf-α and il-1β induction in experimental pneumococcal meningitis microglia: an active player in the regulation of synaptic activity cytokines: powerful regulators of glial cell activation expression of viral and myelin gene transcripts in a murine cns demyelinating disease caused by a coronavirus gfap isoforms in adult mouse brain with a focus on neurogenic astrocytes and reactive astrogliosis in mouse models of alzheimer disease neuroinflammation leads to region-dependent alterations in astrocyte gap junction communication and hemichannel activity regulation of an oligodendrocyte progenitor cell line by the interleukin-6 family of cytokines astrocytes promote tnf-mediated toxicity to oligodendrocyte precursors neurons control the expression of connexin 30 and connexin 43 in mouse cortical astrocytes changes in astroglial glt-1 expression after neural transplantation or stab wounds synchronous hyperactivity and intercellular calcium waves in astrocytes in alzheimer mice continued administration of ciliary neurotrophic factor protects mice from inflammatory pathology in experimental autoimmune encephalomyelitis stage-specific association of apolipoprotein a-i and e in developing mouse retina regional hypomyelination and dysplasia in transgenic mice with astrocyte-directed expression of interferongamma acute exposure to cntf in vivo induces multiple components of reactive gliosis ciliary neurotrophic factor stimulates nuclear hypertrophy and increases the gfap content of cultured astrocytes gfap mutations in alexander disease pro-regenerative properties of cytokine-activated astrocytes gfap is necessary for the integrity of cns white matter architecture and long-term maintenance of myelination immunoreactive apolipoprotein e is a widely distributed cellular protein. immunohistochemical localization of apolipoprotein e in baboon tissues interferon-gamma inhibits central nervous system remyelination through a process modulated by endoplasmic reticulum stress cntf is a major protective factor in demyelinating cns disease: a neurotrophic cytokine as modulator in neuroinflammation cholesterol involvement in the pathogenesis of neurodegenerative diseases overexpression of cntf in mesenchymal stem cells reduces demyelination and induces clinical recovery in experimental autoimmune encephalomyelitis mice deletion of astrocyte connexins 43 and 30 leads to a dysmyelinating phenotype and hippocampal ca1 vacuolation disruption of oligodendrocyte gap junctions in experimental autoimmune encephalomyelitis neuronal lrp1 functionally associates with postsynaptic proteins and is required for normal motor function in mice ciliary neurotrophic factor and leukemia inhibitory factor promote the generation, maturation and survival of oligodendrocytes in vitro cytokine, chemokine and growth factor gene profiling of cultured human astrocytes after exposure to proinflammatory stimuli astroglial connexin immunoreactivity is specifically altered at β-amyloid plaques in β-amyloid precursor protein/presenilin1 mice connexins are critical for normal myelination in the cns components of glial responses to exogenous and synaptic glutamate in rat hippocampal microcultures fibroblast growth factor 2 (fgf2) and fgf receptor expression in an experimental demyelinating disease with extensive remyelination astrocytes stimulate interleukin-17 and interferon-gamma production in vitro how factors secreted from astrocytes impact myelin repair d-serine is an endogenous ligand for the glycine site of the n-methyl-d-aspartate receptor pathologische anatomie und pathogenese elevated connexin43 immunoreactivity at sites of amyloid plaques in alzheimer's disease astrocyte phenotypes and their relationship to myelination functional duality of astrocytes in myelination specialized membrane domains for water transport in glial cells: highresolution immunogold cytochemistry of aquaporin-4 in rat brain purified astrocytes promote the in vitro division of a bipotential glial progenitor cell platelet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type-2 astrocyte progenitor cell the complex relationship between cell division and the control of differentiation in oligodendrocyte-type-2 astrocyte progenitor cells isolated from perinatal and adult rat optic nerves connexin 47 (cx47)-deficient mice with enhanced green fluorescent protein reporter gene reveal predominant oligodendrocytic expression of cx47 and display vacuolized myelin in the cns conditional ablation of stat3 or socs3 discloses a dual role for reactive astrocytes after spinal cord injury cxc chemokine receptors on human oligodendrocytes: implications for multiple sclerosis cholesterol: its regulation and role in central nervous system disorders astroglial networks scale synaptic activity and plasticity glutamatemediated astrocyte-neuron signalling cloning, localization and induction of mouse brain glycogen synthase immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies cholesterol metabolism in neurons and astrocytes astrocytic neurotransmitter receptors in situ and in vivo pdgf a chain homodimers drive proliferation of bipotential (o-2a) glial progenitor cells in the developing rat optic nerve cultured human fetal astrocytes can be induced by interferon-gamma to express hla-dr a glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium platelet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture astrocyte expression of mrna encoding cytokines ip-10 and je/mcp-1 in experimental autoimmune encephalomyelitis direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord in vivo proliferation of oligodendrocyte progenitors expressing pdgfalphar during early remyelination in situ expression of fibroblast growth factor receptors by oligodendrocyte progenitors and oligodendrocytes in adult mouse central nervous system neuroscience: the wrap that feeds neurons regulation of oligodendrocyte development and myelination by glucose and lactate astroglial metabolic networks sustain hippocampal synaptic transmission changes in neurotrophic factor expression and receptor activation following exposure of hippocampal neuron/ astrocyte cocultures to kainic acid ciliary neurotrophic factor role in myelin oligodendrocyte glycoprotein expression in cuprizone-induced multiple sclerosis mice oligodendrocytes use lactate as a source of energy and as a precursor of lipids the "quad-partite" synapse: microgliasynapse interactions in the developing and mature cns iron efflux from astrocytes plays a role in remyelination neurotrophic factor gene expression in astrocytes during development and following injury tumor necrosis factor mediates myelin damage in organotypic cultures of nervous tissue identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions basic neurochemistry: molecular, cellular and medical aspects cytokine-regulated expression of platelet-derived growth factor gene and protein in cultured human astrocytes astrocytes: biology and pathology sevenfold-reduced osmotic water permeability in primary astrocyte cultures from aqp-4-deficient mice, measured by a fluorescence quenching method ciliary neurotrophic factor (cntf) enhances myelin formation: a novel role for cntf and cntf-related molecules properties of gaba and glutamate responses in identified glial cells of the mouse hippocampal slice regional distribution, developmental changes, and cellular localization of cntf-mrna and protein in the rat brain transfer of glycogen-derived lactate from astrocytes to axons via specific monocarboxylate transporters supports mouse optic nerve activity interferon-gamma and ia antigen are present on astrocytes in active chronic multiple sclerosis lesions microglial interactions with synapses are modulated by visual experience panglial gap junctional communication is essential for maintenance of myelin in the cns tumor necrosis factor and interleukin-1 in the csf and sera of patients with multiple sclerosis histological investigation of spinal cord lesions in the spinal hyperostotic mouse (twy/twy): morphological changes in anterior horn cells and immunoreactivity to neurotropic factors control of synapse number by glia plateletderived growth factor promotes repair of chronically demyelinated white matter ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain baumann n. myelin-induced experimental allergic encephalomyelitis in lewis rats: tumor necrosis factor alpha levels in serum and cerebrospinal fluid immunohistochemical expression in glial cells and macrophages of optic nerve and spinal cord reactive astrocytes form scar-like perivascular barriers to leukocytes during adoptive immune inflammation of the cns the impact of astrocytic gap junctional coupling on potassium buffering in the hippocampus distinct stages of myelination regulated by gamma-secretase and astrocytes in a rapidly myelinating cns coculture system immunocytochemical study of myelinassociated glycoprotein (mag), basic protein (bp), and glial fibrillary acidic protein (gfap) in chronic relapsing experimental allergic encephalomyelitis (eae) astrocytes-friends or foes in multiple sclerosis? a role for ciliary neurotrophic factor as an inducer of reactive gliosis, the glial response to central nervous system injury platelet-derived growth factor regulates oligodendrocyte progenitor numbers in adult cns and their response following cns demyelination microglia: dynamic mediators of synapse development and plasticity profile and regulation of apolipoprotein e (apoe) expression in the cns in mice with targeting of green fluorescent protein gene to the apoe locus possible involvement of astrocytes in neuroprotection by the cognitive enhancer t-588 gamma-interferon promotes proliferation of adult human astrocytes in vitro and reactive gliosis in the adult mouse brain in vivo neurotrophic effects of conditioned media of astrocytes isolated from different brain regions on hippocampal and cortical neurons genomic analysis of reactive astrogliosis astrocyte-conditioned medium protecting hippocampal neurons in primary cultures against corticosterone-induced damages via pi3-k/akt signal pathway this work was funded by the wellcome trust (hk, wt083434), medical research council (sl, mr/j004731/1) and medical research scotland (sh, phd-718-2013). key: cord-023374-87ob1exq authors: sukhija, s.; gupta, v. k.; shah, a.; thiel, s.; sarma, p. u.; madan, t. title: levels, complement activity and polymorphisms of mannan‐binding lectin in patients of bronchial asthma with allergic rhinitis date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ai.x sha: doc_id: 23374 cord_uid: 87ob1exq activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan‐binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl‐induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age‐matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl‐induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p = 0.0024, or = 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with ‘a’ allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that ‘a’ allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-022631-s4n24xij authors: jonsson, m. v.; brun, j. g.; skarstein, k.; jonsson, r. title: germinal centres in primary sjögren's syndrome indicate a certain clinical immunological phenotype date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423h.x sha: doc_id: 22631 cord_uid: s4n24xij ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin‐stained paraffin‐embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc‐like lesions were detected in 33/130 (25%) of the pss patients. seventy‐two pss patients lacking these structures (gc‐) were randomly selected for comparison. focus score was significantly increased in the gc(+) patients compared to the gc(–) patients (p = 0.035). in the gc(+) group, 54.5% of the patients presented with anti‐ro/ssa compared to 43.7% in the gc(–) group. anti‐la/ssb was detected in 31.3% of the gc(+) patients compared to 25.7% of the gc(–) patients. sixty‐one percentage of gc(+) patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gc(–) patients (p = 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc(–)). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-022353-q2k2krnm authors: w. quimby, fred; h. luong, richard title: clinical chemistry of the laboratory mouse date: 2007-09-02 journal: the mouse in biomedical research doi: 10.1016/b978-012369454-6/50060-1 sha: doc_id: 22353 cord_uid: q2k2krnm the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events such as, the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events is partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. the power of these models to elucidate the genetic basis of disease cannot be overemphasized. this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and knockout mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. it has been more than 20 years since publication of the first edition of the mouse in biomedical research, and since that time new emphasis has been placed on the mouse as a model for the pathophysiology and treatment of human diseases. during this time, the frontier of clinical chemistry in the mouse has advanced and expanded because of two major events: the increasing reliance on mice in biomedical research, and increasing availability of practical yet sophisticated techniques and instrumentations that have allowed for the detection of a wider variety of biomarkers of disease. the progression of these two events has been partially driven by the increasing regulatory demands related to safety/toxicity assessment of novel drug development. abbreviations used in this chapter are summarized in appendix 6-1. for the last quarter century, mice have been the most frequently used mammal in biomedical research, rising from 61% of all mammals used in 1983 to 71% in 1993 (national center for research resources 1997 . the nature of their use has changed dramatically during this time. during the first half of the 20th century, a great deal of emphasis was placed on the development of inbred strains. the availability of inbred strains has led to major breakthroughs in cancer, biology, and immunology (quimby 2002) . by 1970 there were approximately 250 inbred strains available. in addition, outbred stocks continue to be utilized in a wide variety of studies but particularly in the fields of toxicology and pharmacology. during the second half of the 20th century, investigators developed congenic lines and recombinant inbred strains, each having an impact on elucidating the role of individual genes and assigning new traits to linkage groups respectively (paigen 2003a ). however, beginning in the early 1980s scientists began to genetically engineer mice by either adding a new gene (transgenic) or deleting a normal mouse gene (knockout, ko) (paigen 2003b) . the power of these models to elucidate the genetic basis of disease cannot be overemphasized. over the past 15 years, publications citing transgenic and ko mice have increased exponentially and at the time of this writing the mutant mouse resource of the jackson laboratory listed more than 10,000 such unique lines. during this same period the results of both the human and mouse genome projects were published (international human genome sequencing consortium 2001; mouse genome sequencing consortium 2002) . this provided complete nucleotide sequences for each genome allowing investigators to quickly develop the equivalent murine model for many of the inherited human diseases. transgenic and ko mice have helped clarify disease pathogenesis in virtually every area of medicine and often elucidated biochemical pathways, previously unknown, which are now subject to testing and quantification. as a result, clinical chemistry in the mouse has grown from evaluation of 15-20 analytes found in plasma (or urine), to hundreds of biomarkers that can monitor disease status at the cellular level. due to the massive amount of new information characterizing each of the standard strains and mutant lines of mice, a mouse phenome database has been developed to manage data and provide researchers with the ability to explore raw phenotypic data (including clinical chemistry) and summary analyses. the resource allows investigators to select the appropriate murine model for physiology testing, drug discovery, toxicology studies, mutagenesis, modeling human disease, quantitative trait loci (qtl) analyses, identification of new genes, and unraveling the influence of environment on genotype (bogue and grubb 2004) . by far, the most important new technology in the rapidly growing and changing field of clinical chemistry is the discipline of proteomics. proteomics combines the disciplines of molecular biology, biochemistry, and genetics to the analysis of the structure, function, and interactions of proteins produced by the genes of a particular cell, tissue, or organism. the field of proteomics has grown mainly due to the development of new instruments that are based on sophisticated techniques, yet amenable to practical implementation. for example, the development of surface-enhanced laser desorption/ionization (seldi) platform time-of-flight (tof) mass spectroscopy (ms) allowed petricoin et al. (2002a) to identify five peptides in the sera of women with ovarian cancer that were not found in women without ovarian cancer, even though the structure and function of some of these peptides were not actually known. building on these findings, zhang et al. (2004) combined the seldi-tof ms with protein separation procedures to develop a multianalyte immunoassay for rapidly screening potential cancer patients. this process of identifying proteins (biomarkers) that are predictive for a disease is known as plasma protein profiling. similar technology has been used to develop plasma protein profiles for neoplastic conditions in humans, such as prostate cancer petricoin et al. 2002b; qu et al. 2002) and bladder cancer (adam et al. 2001; vlahou et al. 2001) , as well as a variety of non-neoplastic conditions, such as ischemic versus hemorrhagic stroke , severe acute respiratory syndrome (kang et al. 2005 ), alzheimer's disease (carrette et al. 2003) , and creutzfeldt-jakob disease (sanchez et al. 2004) . plasma protein profiling has also begun in mouse models. xiang et al. (2004) used a combination of two-dimensional gel electrophoresis (2d-ge) and matrix-assisted laser desorption/ionization (maldi) tof ms to quantify serum protein profiles of c57bl/6 mice harboring the lewis carcinoma with and without treatment using acetazolamide. they found upregulation of many peptides associated with tumor growth and metastasis, and many of these same peptides were modified during treatment. two specific targets of acetazolamide antitumor activity were subsequently identified as histone h2b and croci (a ubc-like peptide). park et al. (2004) used similar technology to determine plasma protein profiles for high (c57bl/6) and low (c3h) atherosclerosis prone strains of mice on normal or atherogenic diets. they identified 30 proteins in which the levels had changed after eating an atherogenic diet. of these, 14 were differentially changed in c57bl/6 mice and an additional 16 were changed in both strains. in addition, 28 proteins were differentially expressed between the two strains regardless of diets. four of these proteins were upregulated in c57bl/6 and 11 were upregulated in c3h. nine of those protein markers were definitively identified and their roles in the pathogenesis of atherosclerosis discussed in the "atherosclerosis" section. plasma protein profiling in mice has the potential to become an important discovery tool for translational research, particularly in identifying cancer-associated plasma biomarkers in humans. for example, juan et al. (2004) also used 2d-ge and maldi tof ms to identify plasma biomarkers in balb/c-nude mice harboring human xenotransplanted tumors. in mice bearing a human stomach cancer cell line, serum amyloid a (saa) was elevated. the authors then went back to screen the sera of human stomach cancer patients and were able to demonstrate that, when compared to controls, humans with stomach cancer also had significantly elevated levels of saa. all of these examples illustrate the power of proteomics. proteomic pattern diagnostics offers a means to look at molecular diagnostic information in human or mouse serum, without preconceived assumptions about the existence or identity of the biomarkers. this allows for the development of sensitive and specific peptide assays for identified biomarkers of disease. practical applications of proteomics have been facilitated by the creation of instrumentation that can analyze multiple analytes from small sample volumes with fast through-put (such as multiplex technology, which are discussed in the "multiplex technology" section). in an attempt to manage all the experimental data arising from the fields of proteomics, metabolomics and transcriptomics (gene array), the chemical effects in biological systems (cebs) knowledge base was developed (xirasagar et al. 2004 ). this is a useful, searchable database that integrates experimental findings from all three disciplines, by biosource identification (animal, test article, genotype, and investigator). by making the cross correlation from data arising from three different streams it is hoped that combination groups of predictive markers for disease and toxicity will emerge. the cost of bringing a new drug to market is estimated at $0.8-1.7 billion (food and drug administration 2004), of which a large proportion is spent on safety/toxicity assessments during the preclinical phase of the development of a drug. although expensive, safety/toxicity regulations are necessary, as illustrated by the fact that a new drug entering phase i clinical trials has only an 8% chance of reaching the marketplace due to toxicity issues. the ability to decrease the costs of drug development due to safety/toxicity issues is becoming increasingly reliant on the use of the laboratory mouse as a model for human disease and the identification of sensitive biomarkers for toxicity and disease. the underlying basis of using laboratory mice as models for human disease was demonstrated by everett and harrison (1983) , who showed that the predictive reliability of mice for quantitative toxicity of five chemotherapeutic drugs in humans was at least as good, if not better, that the predictive reliability demonstrative by dogs and monkeys. more recently, newell et al. (2004) presented data on predictive performance of rats and mice to demonstrate qualitative human toxicity when given 39 novel cancer chemotherapeutic agents. they claim that nonrodent species are unnecessary for identification of a safe phase 1 starting dose for human trials. furthermore, the two rodent species (rats and mice) used gave similar quantitative and qualitative results. the arrival of genetically engineered mice has further enhanced the relevance of using mice in research, especially in terms of basic mechanistic research and applied screening for genotoxicity and carcinogenicity. transgenic mice carrying a human gene and expressing the protein are said to be "humanized." these animals are invaluable in drug assessment especially when the drug interacts only with the human protein (bolon 2004) . zambrowicz and sands (2003a) showed that the ko phenotype of mice correlated well with the molecular targets of the 100 best selling drugs available to the u.s. market. zambrowicz et al. (2003b) compared the physiology of ko mice, where the deleted gene was known to produce a novel target for each of the 24 new drugs in the developmental pipeline of the 10 largest pharmaceutical companies, and found that 85% of these targets demonstrated a sound biologic rationale for the selected disease. transgenic and ko mice have been used successfully to screen new drug candidates for safety and to elucidate basic mechanisms of toxicity. a large number of drug metabolizing enzyme (dme) ko lines have been employed in safety screening. dmes may be involved in the safe metabolism of a drug or they may generate toxic intermediates. removal of the dme gene may result in the animal being more sensitive to the test article, and it may provide protection against toxic effects of the drug. for instance, ko mice lacking the nqo-1 gene have increased menadione toxicity, and mice lacking the cyp2e1 gene are resistant to acetaminophen hepatotoxicity (henderson and wolf 2003) . murine models are also available for evaluation of chemical mutagenicity (big blue mouse; stratagene, la jolla, ca and muta mouse; covance research products, denver, pa). these marker genes are present in mice of different genetic backgrounds (bolon 2004) . ko lines have been created with the increased sensitivity to chemically induced carcinogenesis. mice carrying a single p53 allele, or over expressing ha-ras, or having a complete deletion of the xpa (xeroderma pigmentosum) gene, have all been used for screening xenobiotics in vivo (bolon 2004) . these same transgenic and ko models are useful in screening environmental chemicals for toxicities (jacobson-kram et al. 2004) . it is clear that the mouse will continue to be essential for discovery and in evaluating the safety of new drugs. equally relevant is the development of clinical chemistry assays needed to study the associated metabolic events in mice (especially in transgenic and ko lines) and assess serum/plasma biomarkers. these biomarkers should either be quantitative measures of the biologic effects (which provide informative links between mechanism of action and clinical effectiveness) or surrogate markers (which are quantitative measures that predict effectiveness). in this arena, the field of proteomics is likely to make a dramatic impact on clinical chemistry. although we hope all readers of this chapter will benefit from this section on assays and instruments, the primary purpose of this chapter is to briefly introduce the reader to areas where methods in clinical chemistry are changing and provide sources of information for services, reagents (including test kits), and instrumentation, specifically for testing biomarkers (including traditional analytes) in mouse serum, plasma, or urine. those seeking a detailed description of clinical chemistry methods and instruments should refer to the fundamentals of clinical chemistry (burtis and ashwood 2001) . some dramatic changes have occurred over the past two decades that have revolutionized the discipline of clinical chemistry. historically, most analytes were measured by a colorimetric end point assay (based on the binding of an analyte to another molecule creating a new substance that absorbs light of a specific wavelength). some of these analytes and the newer biomarkers are now measured by enzymatic tests that have higher specificities and sensitivities. another change has been the substitution of electrochemical assays, such as ion-selective electrodes, for flame photometry used in the quantitation of sodium and potassium. perhaps the change that has had the largest impact on clinical chemistry is the development of monoclonal antibodies and their use as reagents in immunoassays (to be discussed later). many commercial companies now offer services that measure analytes and biomarkers in the blood of mice using a combination of colorimetric-, enzymatic-, electrochemical-, and immunologic-based methods, and many of the instruments used are capable of running multiple assay types simultaneously. the use of laboratories that offer validated assays specifically for mouse blood is important to ensure accurate and precise results. a summary of a few laboratories that provide validated murine assays is presented in table 6 -1. the increasing availability of mouse-specific reagents has resulted in many new assay techniques that provide a high degree of sensitivity and specificity. growth in the number of reagents capable of quantitating analytes in mouse serum is illustrated in table 6 -2. techniques employing nonisotopic labels for detection such as enzyme cascade, fluorescence, chemiluminescence, and electrochemiluminescence, are rapidly replacing older radioimmunoassay (ria) techniques. enzyme immunoassays, such as enzyme-linked immunosorbent assay (elisa), enzyme-multiplier immunoassay technique (emit), and cloned enzyme donor immunoassay (cedia), provide quantitative results based on photometric methods. enzyme immunoassay is popular because it generates compounds that can be quantitated photometrically. typical enzymatic labels include ~-galactosidase, horseradish peroxidase, alkaline phosphatase, and glucose-6-dehydrogenase. in addition these elisa test kits are compact, easy to use, and quantitated with inexpensive instruments. these kits (usually in a well format) are available for quantitation of a wide range of mouse serum and plasma biomarkers (table 6-3) . fluoroimmunoassay (fia), which utilizes a fluorescent molecule as an indicator label, was previously subject to problems associated with background fluorescence. today this problem has been largely resolved by using chelates of lanthanide as a label. modifications of fia have eliminated the need to separate free from bound label (homogenous assay). chemiluminescent and electrochemilumiscent immunoassays are similar to enzyme immunoassays, except that quantitation of results is based on the emission of light after a chemical label is exposed to an oxidation reaction (chemiluminescence) or to an electrochemical reaction (electrochemiluminescence). the number of biomarkers that can be quantified by commercially available test kits based on immunoassays is likely to grow at unparalleled rates as a result of proteomics research where mice are the favored model. mulitplex immunoassay is a unique technology that combines four distinct components in a manner that allows for simultaneous analysis of up to 90 different analytes from 50 ktl of serum/plasma. the core component is an inexpensive, consumable, 5.6 ~tm diameter polystyrene microsphere that are encoded into 100 different fluorescent color sets using two fluorophores (red and infrared) at multiple concentrations. the second component is a biologic assay (combined with an mmp-3 (matrix metalloproteinases) pro-mmp-9 mmp-9 tissue inhibitor of metalloprotease 1 (timp-1) orange fluorescent reporter molecule) that is built onto the surface of the microspheres. a diverse range of biologic assays can be built onto the microsphere surface, including immunoassays, nucleic acid assays, enzymatic reaction assays, or receptor-ligand analysis assays. the third component is a flow cytometer that focuses the microspheres into a single file in front of a two interrogating lasers, which allow for high throughout. one laser is a red diode emitting at 635 nm, which illuminates each microsphere. the resulting red and infrared fluorescence provides classification information for that particular microsphere set. the other laser is a green yag diode emitting at 532 nm that excites the orange fluorescent reporter molecules of the surface of the microsphere, providing a quantitative signal for that particular biologic assay. the last component is digital signal processing data acquisition hardware that provides the speed necessary to read the microspheres at up to 5000 per second. because multiplex technology can analyze a wide range of biomarkers simultaneously, dynamic reference results (plasma profiles) can be developed based on the changing concentrations of the biomarkers during the course of a disease. the known and potential applications of developing plasma profiles of diseases are powerful. plasma profiles can be used to screen and identify diseases (especially during the early development of a disease) and characterize the efficacy of drugs targeted against these diseases. multiplex technology can help elucidate new biomarkers of disease. furthermore, plasma profiles could also potentially be developed to monitor the blood concentrations of drugs as well. investigators seeking additional information on availability of reagents and test kits should refer to "clinical laboratory reference" (nelson 2006 ) and the linscott's directory of immunologic and biologic reagents (linscott 2005) . sections of these two references provide the names and addresses of sources. the catalogs of individual companies may be fruitful, but many reagents for use in human blood and have not been validated for use in other species (including the mouse). each of the various instruments mentioned in this section may be found in the 8th annual analyzers buyer's guide (advance for administrator of the laboratory 2003). this guide lists all manufacturers and gives detailed information on each instrument including technology platforms, methods used, through-put capability, purchase price, maintenance costs, reagent package cost, as well as a variety of options available. contact information, including web site, is available for each manufacturer. additional information regarding clinical chemistry on animal blood is available on the web site of the division of animal clinical chemistry at the american association of clinical chemistry (www.aacc.org/divisions/animal). with a mean body size of 35 g, adult mice have approximately 2.4 ml of blood volume, allowing 75 ~tl to be removed weekly without consequence to health and welfare (loeb 1997; loeb and quimby 1999) . four blood collection sites for acquiring 75 ~tl of blood repetitively from adult mice include the orbital plexus, the tail vein, the jugular vein, and cardiac puncture (quimby 1999b) . general anesthesia is recommended for orbital plexus and cardiac puncture, although terrilrobb et al. (1996) claim that topical application of proparacaine hydrochloride is an acceptable procedure for collecting orbital plexus blood. the jugular vein is punctured using a lancet directed at the rear of the jaw, exposing the jugular vein and its tributaries and the submandibular and facial veins (golde et al. 2005) . collection of blood from any of these veins at this site works well, and the animal does not require anesthesia or a restraint device. the blood should be collected with a small centrifuge tube or capillary tube. nerenberg and zedler (1975) describe a vacuum apparatus used to collect larger volumes of blood from the tail vein. lewis et al. (1976) found that heparinization of mice before tail bleeding increases the yield. prewarming the mouse under a lamp or through immersion in warm water facilitates tail bleeding. the use of heparinized microhematocrit tubes, during tail or orbital plexus bleeding can maximize the plasma volume with minimal hemolysis (quimby 1999b) . hem and smith (1998) recommend a saphenous vein prick method over the orbital plexus method for collecting repeated small volumes from conscious mice. macleod and shapiro (1988) used indwelling fight atrial catheters for repetitive bleeding of conscious, unstressed mice. disadvantages of specific procedures have been described. sakaki et al. (1961) reported sympathetic nervous system stimulation associated with tail bleeding and the mixing of venous and arterial blood with the orbital plexus method. patrick et al. (1983) found that, compared to jugular vein collection, cardiac puncture was associated with higher plasma glucose concentrations and lower creatine kinase (ck) activity. plasma glucose concentrations are higher in blood collected from the orbital sinus compared to the tail vein. differences associated with the method of anesthesia are also important. halothane, methoxyflurane, isoflurane, and pentobarbital sodium have been widely used and are considered safe. however, caution should be exercised in the interpretation of certain chemistry values. higher plasma glucose levels are reported with tail vein sampling in mice anesthetized with pentobarbital or methoxyflurane compared with conscious mice (chuang and luo 1997) . plasma glucose levels are higher after collection from the orbital plexus if pentobarbital or proparacaine hydrochloride is administered. methoxyflurane has no glucose elevating effect on samples collected from the orbital plexus. cunliffe-beamer (1983) claims that carbon dioxide narcosis provides sedation and analgesic for 1-2 minutes and is an appropriate anesthetic for orbital plexus bleeding. when greater volumes of blood are required, four collection sites can be used that require anesthesia and subsequent euthanasia of the mouse. these sites include the jugular vein (ambrus et al. 1951) , the abdominal aorta (lushbough and moline 1961) , the brachial artery (young and chambers 1973) , and the heart (cubitt and barrett 1978; mitruka and rawnsley 1977) . blood collection from a newborn mouse can be accomplished by decapitation. injection of two units of heparin subcutaneously several minutes before decapitation may allow collection of up to 40 ~tl of blood (20 ~tl of plasma). although collection of urine is possible in mice, it is impractical due to anatomic and physiologic restrictions. the total urinary bladder volume is less than 0.5 ml, and total urinary output per day is less than 2 ml (jung et al. 2003) , meaning that mice frequently micturate. this restricts the amount urine that can be collected at any one time point and, therefore, will also restrict the number of analytes that can be assessed from any one urine sample. to be able to collect enough urine for assessment from a single live mouse, mice can be placed in a plastic cage without absorbent bedding material and urine then collected from the bottom of the cage after 4-6 h. however, a disadvantage of this technique is that the urine is exposed to the contamination by feces and other environmental contaminants and organisms. pooled urine samples collected from a group of live mice using the same technique can increase the total amount of urine collected but will not allow specific results to be related back to specific individuals from that group. mice removed quickly from their cage and held over a piece of parafilm will frequently void and the parafilm helps prevent the urine from spreading. urine can also be collected directly from the urinary bladder from mice after euthanasia. this technique allows for a sterile sampling, but the volume that can be collected is restricted by total urinary bladder volume (0.5 ml). often, the volume is much less because mice typically void urine from the urinary bladder at the time of death. reference ranges refer to the range of an analyte or biomarker in a population that has not been selected for the presence of disease or abnormality. reference ranges are usually generated from a large number of individuals from a population, so reference ranges for the same analyte or biomarker can vary between two different populations. table 6-4 lists the reference ranges for selected analytes in two inbred strains and an outbred stock. in some situations, reference ranges that have been published or generated by laboratories cannot be relied on to accurately some variables known to adversely affect the host include environmental factors, pathogens, and shipment. in addition, nutrition, time of sample collection, and storage techniques may all contribute to variability. age has an effect on analytes in the mouse. in an early study, barrett et al. (1975) found that serum calcium levels in 4-monthold c3h/fg and a/fg inbred mice were significant higher than 7-month-old mice of the same strains. more recent and more comprehensive analyses completed by loeb et al. (1996) reinforces the effect that age has on clinical chemistry parameters in five inbred strains and two f1 hybrids, including serum protein. the effect sex has on chemistry parameters is most demonstrable with sex hormones, including follicle-stimulating hormone (fsh), luteinizing hormone (lh), and progesterone. table 6 -3 illustrates these differences between male and female mice, and between female mice in estrus and out of estrus. strain-associated changes appearing in healthy animals have been documented for complement components (goldman and goldman 1976) , cholesterol (dunnington et al. 1981; meade and gore 1982) , testosterone (ivanyi et al. 1972) , cortisol-binding protein (goldman et al. 1977) , and serum protein (borovkov and svirdov 1975; loeb 1997) . cyclic biorhythms, whether circadian or ultradian, influence blood levels of various analytes in mice. blood levels of adrenocorticotropic hormone (acth), corticosterone, growth hormone (gh), and lh may peak one or more times daily, and thus special attention must be given to both the time of collection and the order of sampling individuals between groups if between-group comparisons are to be made (loeb 1997) . the degree of hydration, exposure to noise, degree of confinement, and environmental temperatures has all been shown to affect serum chemistry analytes (quimby 1999b) . diet is known to influence the blood levels of many analytes. perhaps the best studied is the effect of atherogenic diets on serum cholesterol. similarly, significant differences in both serum cholesterol and urea nitrogen are seen in mice maindetermine the presence or absence of disease or abnormality, tained on a semipurified (ain-76) diet. the mouse is unique these situations include evaluating analytes and biomarkers in j among mammals because murine muscles do not contain transgenic and ko mouse populations (especially in experiments in which population sizes are small) and evaluating certain analytes and biomarkers (such as those assessing immune function). instead, baseline data compiled from controls (the population of wild-type mice from which the transgenic and ko mice were derived from) should be used. the usefulness of compiled baseline data depends on controlling a large number of variables known to influence chemistry determinations. several studies have demonstrated significant differences in selected serum analyte concentrations with age difference in the same strain, between sexes in the same strain, and among strains (everett and harrison 1983) . carnosine or anserine. carnosine serves as a source of histidine when histidine is restricted. unlike other mammals, mice on histidine-free diets show signs of histidine deficiency. fasting may also affect the levels of certain analytes (quimby 1999b) . the presence or absence (axenic mice) of intestinal microbial flora is associated with dramatic changes in immunoglobulin (ig) levels and may be associated with changes in other analytes as well. for example, axenic mice have significantly lower levels of iga compared to conventional mice (moreau et al. 1982) . the presence of pathogens may be associated with dramatic changes in various analytes, even with subclinical infection. for instance, infection with lactic dehydrogenase-elevating virus (ldv) is associated with major elevations in serum lactic dehydrogenase, isocitric dehydrogenase, malic dehydrogenase, aspartate aminotransferase, and glutathione reductase activities (quimby 1999b ). asummarized from loeb and quimby (1999) . alterations in clinicopathologic parameters can be attributed to stress in mice. landi et al. (1982) found that plasma corticosterone concentrations in mice tested within 48 h after arrival (by plane or truck transport) were significantly higher than tested after 48 h post arrival. mice sensitized on arrival with sheep red cells as an antigen had significantly lower antibody titers, fewer plaque-forming cells, and a decreased delayed type of hypersensitivity reaction when compared to normal mice allowed to acclimate to the facility for 48 h before being sensitized. elevated serum corticosterone levels can arise from excessive handling of mice prior to blood collection. the effect of sample storage at various temperatures and for varying periods has been described. falk et al. (1981) evaluated the effect of storage time (after freezing) on 20 serum analytes in six laboratory species. they found that in the mouse, only creatine kinase activity changed significantly with storage up to 28 days. hemolysis is a common problem during blood collection in mice. hemolyzed samples are associated with changes in various enzymes, such as increased ck activity and decreased lipase activity. to minimize this, everett and harrison (1983) recommend heparinized plasma and careful selection of collection sites for routine chemical determinations. the effect of lipemia, various anticoagulants, and pharmacologic agents on clinical chemistry values has been reviewed for domestic animals (meyer and harvey 1998) . using several methods. regardless of the distribution of data, it is generally useful to describe the limits that include 95% of the test results in a disease-free population. for values exhibiting a gaussian distribution, parametric methods (such as mean and standard deviation) are appropriate. for gaussian distributed data, this is the range that includes two standard deviations above and below the mean. certain murine analytes have non-gaussian distributions and must be evaluated using nonparametric methods. a variety of methods are available, including the percentile method and logarithm-transformed data analyzed with parametric methods. the method of percentile estimates is more vulnerable to bias due to extreme values (outliers) than is the log-transformed parametric method. boyd (1985) asserts that a sample size of at least 120 is required to give 90% confidence intervals using the percentile method, whereas a sample size of 50 may give reliable ranges if parametric analyses are used. neither statistical method just described will replace raw data in certain situations, such as when assessing analytes for immune function, nor when using data derived from wild-type mice as comparison baseline data for transgenic and ko mice. the issue of quality control and test validity has been thoroughly discussed for common domestic animals and is applicable to chemistry determinations in mice (meyer and harvey 1998) . quality assurance in clinical chemistry determinations is important to ensure that consistently accurate and precise results are achieved. everett and harrison (1983) stressed the importance of a clinical pathology quality assurance program that includes regular assays of pooled and commercially prepared pre-assayed sera. in addition, they encourage participation in a subscription quality assurance program, such as with a veterinary laboratory association. the within-day and day-to-day coefficient of variation should be known for each analyte measured. due to the tremendous number of variables known to influence clinical chemistry values in mice, it is often prudent to test adequate numbers of control specimens along with the experimental samples. this technique is often impractical when tests are being conducted strictly for diagnostic purposes, and in those situations, compiled values that are controlled for as many variables as possible may be sufficient. the values that define a reference range for a particular analyte or biomarker in normal or healthy mice may be described this section is intended to serve as a review of specific tests for routine analytes. additionally, a more comprehensive discussion is presented for analytes and biomarkers involved in two areas of translational research of intense interest (obesity/diabetes and atherosclerosis) and novel biomarkers of disease (such as immune function tests). only analytes and biomarkers for which there are currently available commercial tests for mice serum, plasma, and/or urine are discussed; certain analytes and biomarkers for which commercially available tests are not available for the mouse (such as calcitonin) are not covered. for more complete information for the routine analytes, please refer to loeb and quimby (1999) . glucose is the main source of energy in mice. blood glucose concentrations depend on the rates of entry and the removal rate from the blood. the rate of entry is dependent on intestinal absorption of dietary sources of glucose, the breakdown of body glycogen stores (glycogenolysis), and synthesis from gluconeogenic metabolites (gluconeogenesis). the removal rate is mainly dependent on insulin, which is released from [3-cells of the pancreatic islets. insulin promotes cellular uptake of blood glucose (mainly in muscle, liver, and fat) by stimulating the translocation of glucose transporters, glut-1 to glut-7 to the cell membrane. when removed from circulation, blood glucose may either be utilized (to maintain cell function) or converted to fat and glycogen in liver and muscle as an energy store. however, the effect of insulin is modulated by other hormones (such as glucagon, corticosterone, gh, epinephrine, somatostatin, and amylin) that ultimately result in the tight control the levels of blood glucose, depending on tissue demands for energy. glucagon is released from t~-cells of the pancreatic islets in response to low circulating glucose, stimulating the liver to increase circulating glucose through glycogenolysis and gluconeogenesis. corticosterone and gh antagonize the action of insulin. epinephrine suppresses insulin release and stimulates glucagon release and glycogenolysis. somatostatin suppresses both insulin and glucagon secretion, whereas amylin increases blood glucose, blood insulin, and insulin resistance (burtis and ashwood 2001; kaneko 1999) . glucose determinations are generally conducted on fresh serum. however, plasma glucose determination is acceptable if delay of greater than 30 minutes before separation of erythrocytes is anticipated. in this case, fluoride should be used as the anticoagulant because it inhibits glycolysis by erythrocytes. blood glucose in mice is measured using the hexokinase or glucose oxidase. these tests may be performed as an analytical method or by using glucose oxidase coated test strips (for urine glucose), or small portable analyzers (seidelmann et al. 2005) . assessment of long-term average blood glucose levels in mice is also available by rias measuring glycosylated hemoglobin and glycosylated serum proteins (collectively known as fructosamines) (gould et al. 1986 ). the mouse phenome database lists serum glucose levels of 41 strains with blood collected after a 4-h fast on 7-9 week old males and females on a standard laboratory diet. for all mice, the overall mean was 179 + 30.9 mg/dl (naggert et al. 2003) , but blood glucose levels varied with age, sex, and strain. females of a strain tended to have lower levels than males, with lp/j mice showing the lowest values (f = 125 + 22.0 and m = 146 _+ 18.8 mg/dl) and c57b1/10j mice showing the highest values (f = 230 _+ 25.5; m = 263 _+ 57.3 mg/dl). in mice serum glucose levels decrease between the 3rd and 12th month of age and in c57bl/6 and balb/c strains the glucose level rises again after 24 months (loeb 1997) . serum or plasma glucose levels may also vary depending on the site of collection and anesthetic used (quimby 1999b) . patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with higher blood glucose. differences associated with the method of anesthesia are also important. higher plasma glucose levels are reported after tail vein sampling under either pentobarbital or methoxyflurane compared to sampling conscious mice (chuang and luo 1997) . plasma glucose levels were higher after orbital plexus sampling of mice if pentobarbital or proparacaine hydrochloride was provided. methoxyflurane had no glucose elevating effect on samples collected from the orbital plexus; however, in conscious animals, plasma glucose levels were higher in blood collected by retro-orbital sinus compared to tail bleeding. hyperglycemia (increased blood glucose levels) in the mouse can be due to increased peripheral resistance of tissues to insulin (such as with exogenous corticosteroid administration, increased endogenous corticosterone release, glucagons administration, and gh administration), and diabetes (see discussion later). hypoglycemia (decreased blood glucose levels) can be due to excessive circulating insulin (such as with excessive insulin administration and transgenic mice with [3-cell tumors of the pancreatic islets), reduced glycogen stores (such as with advanced liver disease), excessive glucose use (such as with pregnancy, septicemia, and neoplasia), and reduced glucose intake (such as with starvation and diseases of malabsorption). diabetes in mice is defined as persistent blood glucose levels of greater than 300 mg/dl in fasting mice. this level also corresponds to the renal threshold for glucose urine excretion in mice, so urine glucose assessment is useful in this regard for mice. nonobese diabetic mice (a model of type 1 diabetes) have nonfasting plasma glucose levels in young prediabetic mice between 130-180 mg/dl, which rises to >300 mg/dl between 10-30 weeks of age (leiter 1997) . genetically modified mice have been identified for virtually every ligand and receptor regulating glucose metabolism and, depending on the gene mutation, may exhibit altered levels of plasma glucose. for example, adenosine monophosphate-activated protein kinase (ampk) is a critical enzyme in energy metabolism (including cellular glucose uptake and fatty acid oxidation in muscle, and fatty acid synthesis and gluconeogenesis in hepatocytes). using ko mice, shaw et al. (2005) demonstrated that kinase lkb 1 is an important activator of ampk in the liver under energy-stress conditions, and that ko mice deficient in kinase lkb1 were persistently hyperglycemic. the authors also demonstrated that kinase lkb 1 is the target of the type 2 diabetic therapeutic drug, metformin. glucose tolerance tests (gtt) have been performed in mice. one-, 3-, and 4-h tests have been conducted. in the 1-h test, glucose concentrations are evaluated before and 1 h following the administration of 2 mg/g glucose administered intraperitoneally (ip) (oldstone et al. 1984) . the 3-h test compares pre-injection serum to serum collected at 15, 30, 60, and 120 minutes following administration of 3 mg/g glucose given ip (hotamisligil et al. 1996) . a sensitive 4-h gtt has also been described in which mice are given 10 ml/kg of 10% glucose orally (gates et al. 1972) . the hypoglycemic response to insulin in mice has also been described (hotamisligil et al. 1996) . kaku et al. (1988) measured glucose, glycosylated hemoglobin and insulin levels in six inbred strains undergoing gtt (either in fed or fasted mice). to estimate the number of genes involved in phenotypic differences in glucose tolerance, the least glucose tolerant strain (c57bl/6) was bred to the most tolerant strain (c3h/hej) and f1 hybrids and backcross animals tested. the authors concluded that glucose tolerance in six commonly used inbred strains is a polygenic trait. thrifty genes have been hypothesized to give early human hunter-gatherers a survival advantage by providing an economic mechanism to store energy during periods of famine (zimmet and thomas 2003) . because the most efficient mechanism for energy storage is through promotion of adipose tissue, it seems reasonable that at least some of these "thrifty" genes may function in this manner. another hypothesis holds that during periods of famine it is essential to conserve glucose for use by the brain and that the mechanism responsible involves insulin resistance in peripheral tissues (neel 1962) . should both hypotheses be true it is easy to envision an association between obesity and type 2 diabetes, especially where sedentary lifestyle and unrestricted access to food occur together (lazar 2005) . one candidate thrifty gene encodes the hormone leptin. leptin is produced by adipose tissue, and its absence leads to obesity and insulin resistance. during times of high adipose storage blood levels of leptin are high and it promotes energy metabolism and inhibits food intake. the opposite occurs during starvation. but leptin is only one of a number of adipokines, secreted by adipose tissue, which aid in the regulating appetite and metabolism. in fact the location of the adipose tissue, the size of average adipocytes, and adipocyte metabolism of glucose and corticosteroids each modify the endocrine function of adipose tissue (lazar 2005) . among the proteins secreted by adipose tissue are adiponectin, adipsin, resistin, visfatin, tumor necrosis factor-a (tnf-a), interleukin-6 (il-6), macrophage chemoattractant protein-1 (mcp-1), plasminogen activator inhibitor-1 (pai-1), angiotensinogen, saa, and a-acid glycoprotein. like adipocyte-derived free fatty acids, which have been shown to contribute to insulin resistance in liver and muscle, most of these proteins are capable of modulating glucose metabolism and insulin action. adiponectin and visfatin work synergistically with insulin to enhance glucose uptake by muscle and block glucose synthesis by the liver (hug and lodish 2005) . blood levels of visfatin increase in obesity and the cytokine can bind to and stimulate the insulin receptor (fukuhara et al. 2005) . blood levels of adiponectin negatively correlate with body mass and are lower in obese humans and mice, suggesting that reduced mrna expression of the adiponectin gene may be involved with obesity (masaki et al. 2004) . increases in adiponectin downregulate the hepatic expression of tnf-a. it mediates its antidiabetogenic effects via receptors on peripheral tissues, especially liver. tnf-a, resistin, and il-6 each induce resistance to insulin. however tnf-a also suppresses expression of adipocyte specific fred w. quimby and richard h. luong t genes; resistin maintains glucose during fasting; and il-6 production increases in the obese. the cytokines tnf-~ and il-6 are proinflammatory, are also produced by monocytes, and act on the liver to produce acute phase reactants. they also induce suppressor of cytokine signaling-3 (socs-3), an intracellular signaling molecule that impairs neuronal signaling by leptin and insulin, and thus causes resistance to the central actions of both hormones (schwartz and porte 2005) . resistin mediates its effects principally by decreasing the expression of gluconeogenic enzymes in the liver (banerjee et al. 2004 ). certain cytokines, increased endoplasmic reticulum stress, chronic hyperglycemia, chronic hyperlipidemia, and oxidative stress may all induce apoptosis of insulin producing ~l-cells in the islets of the pancreas (rhodes 2005) . insulin receptor substrate-2 (irs-2) is a key molecule promoting ~-cell growth and survival. these molecules act immediately downstream from surface receptors for insulin and insulin-like growth factor-1 and inhibition of irs-2 has been shown to lead to insulin resistance. inhibition may result from accumulation of reactive oxygen species in ~-cells chronically exposed to increased glucose metabolism or from chronic exposure to elevated fatty acid (which through production of long chain acyl-coa active protein kinase c-isoforms degrade irs-2). leptin has been shown to modulate interleukin-l~ (il-i~), a potent inducer of apoptosis. tnf-t~ and il-6 induce ~-cell apoptosis by activating the transcription factor nuclear factor k:i] (nf-~:[3). centrally the actions of both insulin and leptin take place in the mediobasal hypothalamus, where the neurons exert potent effects on food intake and energy expenditure. here neurons co-express neuropeptide y (npy) and agouti-related peptide (agrp), which stimulate food intake and reduce energy expenditure. leptin and insulin inhibit these neurons. under conditions of reduced leptin and insulin signaling, npy increases, inducing hyperphagia, weight gain, insulin resistance, and glucose intolerance. the anabolic effects of agrp arise from its antagonism of the melanocortin receptors mc3r and mc4r, which serve to limit food intake. blockage of mc3r and mc4r leads to weight gain and insulin resistance. precursor proopiomelanocortin (pomc) and pomc neurons in the arcuate nucleus are stimulated by leptin and insulin and the resultant production of melanocortin and its binding to mc3r and mc4r inhibits food intake and promotes weight loss (schwartz and porte 2005) . however, in the absence of leptin, as seen in lep ~176 mice, neurons of the arcuate nucleus of the hypothalamus are permanently disrupted and treatment in adulthood cannot reverse this defect (bouret et al. 2004) . orexin a (hypocretin-1) and orexin b (hypocretin-2) are neuropeptides produced in the lateral hypothalamus by neurons with axonal projections to many sites including those that control feeding behavior and sleep/wakefulness (taylor and samson 2003) . orexin ko mice develop hypophagia with obesity and insulin-resistant diabetes (hara et al. 2001 ). ghrelin, a peptide made predominantly by the stomach, is also known to act centrally and affect food intake and increase secretion of gh (ghigo et al. 2004; korbonits et al. 2004 ). in the periphery leptin has been shown to specifically repress rna levels and enzymatic activity of hepatic stearoyl-coa desaturase-1 (sld-1), which catalyzes the biosynthesis of monounsaturated fatty acids. this effect was found to be an important metabolic action of leptin (cohen et al. 2002) . leptin resistance, a common feature of obesity in mice and humans, has also been shown to result, in part, from the shedding of membrane-bound hepatic leptin receptors into the plasma, where soluble receptors modulate circulating leptin levels and possibly its biologic activity (cohen et al. 2005) . thus the connections between factors regulating obesity and insulin resistance (diabetes) are complex and occur both centrally and peripherally. further investigations in mice will involve quantifying glucose, adipokines, insulin, leptin, soluble leptin receptor, and sld-1. for further discussion of mouse models of obesity and diabetes please refer to chapter 19 in this text. metabolism and food intake insulin, leptin, amylin (also called islet amyloid polypeptide, iapp), glucagon, ghrelin, obestatin, orexin a, orexin b, gh, and corticosterone have been measured in mice, and ria and elisa kits are commercially available (see table 6 -3). in addition, these hormones may be measured as part of a multiplex panel (see "multiplex technology" section). leptin values in c57bl/6, 129, and fvb/n strains range from 1-3 ng/ml, insulin values range from 2.2-5.2 ~t iu/ml, corticosterone levels range from 5-40 ~tg/dl, and gh ranges from 1-90 ~tg/ml. epinephrine is measured by ria, and the range for mice is 0-200 pg/dl (depaolo and masoro 1989). adipokines secreted entirely by adipose tissue include adiponectin, adipsin, and resistin. adiponectin and resistin have been measured in mouse serum and commercial elisa test kits are available for this species. a polyclonal antibody that cross-reacts with a conserved sequence of mouse adipsin has been created (searfoss et al. 2003) . tnf-ct, il-6, and visfatin are adipokines that are synthesized also by macrophages and lymphocytes; as such they provide a common link between regulation of obesity, resistance to insulin and inflammation (lazar 2005) . both tnf-t~ and il-6 may be measured by commercially available elisa kits (see the "cytokines and chemokines" section). a method has also been published for measurement of visfatin (fukuhara et al. 2005 ). reagents for quantifying the soluble leptin receptor and sdl-1 are not commercially available at this time, although methods for measurement of these analytes in mouse serum have been published (cohen et al. 2002; cohen et al. 2005 ). the four main types of lipids in plasma are free cholesterol, esterified cholesterol, triglycerides, and phospholipids. lipids are derived from the diet (mainly from long-chain fatty acids), although endogenous recycling (mainly in the liver) occurs. plasma lipids have poor water solubility; thus, they require water-soluble protein molecules for their transport in plasma. the complex of plasma lipids and proteins are known as apoproteins (also known as apolipoproteins), which contain a core of nonpolar lipids surrounded by a surface layer of phospholipids, free cholesterol, and apoproteins. apoproteins are classified according to physical and chemical parameters, and include (in order of increasing density) chylomicrons, very low-density lipoproteins (vldl), intermediate density lipoproteins (idl), low-density lipoproteins (ldl), and high-density lipoprotein (hdl). particle density is proportionally related to the amounts of phospholipid, protein, and triglycerides they contain; increasing particle density corresponds with increasing proportions of phospholipid and protein, and decreasing density corresponds with decreasing proportions of triglyceride. chylomicrons and vldl are often referred to as triglyceride-rich lipoproteins. apoproteins on the surface of the particles serve as ligands for receptors, cofactors of enzyme interaction and structural components (wagner et al. 1999 ). table 6 -5 lists the mouse apoproteins and describes their known function. the largest lipoprotein particle is the chylomicron, which transports dietary lipids in the form of triglycerides from the intestines. chylomicrons contain apoproteins a and b48 and are absorbed into the lymphatics from the intestine, eventually entering the blood. they deliver fatty acids to the tissues with assistance from lipoprotein lipase (lpl) and release glycerol into the blood. as chylomicrons lose triglycerides they become smaller and are called remnants. these remnants acquire apoprotein e from plasma hdl and are rapidly cleared by the liver by the apoprotein e receptor or chylomicron remnant receptor. vldl is the second most prevalent lipoprotein particle in normal mouse blood (after hdl) and it transports triglyceride from liver to extrahepatic tissues. vldl is synthesized in the liver with apoprotein b 100, c, and e attached. lpl aids in the release of fatty acids (from triglycerides) to peripheral tissues. ldl, which is present in very low concentrations in normal mice, carries cholesterol to extrahepatic tissues. ldl contains apoprotein b 100 and is the primary source of cholesterol deposited in the intima of arteries in mice. apoprotein b 100 binds to the ldl receptor (ldlr). hdl is synthesized in the liver and contains large amounts of free cholesterol and apoproteins a, c, and e. during metabolism hdl free cholesterol is esterified by the action of lecithin:cholesterol acyltransferase (lcat). the exchange of cholesterol ester for triglycerides (from vldl) results in a less dense hdl subfraction that is completely removed from the circulation by the liver. exchange of esterified cholesterol for triglycerides is mediated by cholesteryl ester transfer protein (cetp) in humans; however, this activity is absent in mice. phospholipid transfer from vldl to hdl is mediated by phospholipids transfer protein (pltp). high levels of soluble hepatic lipase are found in mouse plasma (lusis, 2000; wagner et al. 1999) . hdl is thought to transport cholesterol from peripheral tissue to liver by a process known as reverse cholesterol transport (rct). this process is believed to be facilitated by the adenosine triphosphate (atp)binding cassette transporter a1 (abca1), which transports phospholipids and cholesterol to the acceptors apoprotein a-1 and apoprotein e (aiello et al. 2002) . hdl also serves as a reserve of apoprotein c and apoprotein e necessary for vldl metabolism. table 6 -6 lists the apolipoprotein receptors found in mice and describes their ligands and functions. atherosclerosis is a major factor in heart disease, stroke, and peripheral vascular disease in humans, and as such, is the principal cause of death in western countries. the etiology is complex, involving both genetic and environmental components. although there are some examples of single gene defects in humans that lead to atherosclerosis, these conditions are rare and do not explain disease prevalence. atherosclerosis is characterized by the formation of plaques in the intima of large and medium size arteries, usually in locations where blood flow is disturbed. the plaques contain a variety of cells including endothelial cells, monocytes or macrophages, smooth muscle cells, and lymphocytes, which secrete products that modulate progression of plaque formation. in addition plaques contain a complex mix of collagen, proteoglycans, occasional cartilaginous tissue with calcification and lipoprotein (primarily ldl). disease risk correlates directly with elevated circulating levels of ldl cholesterol and risk is indirectly correlated to levels of hdl. thus, although ldl promotes atherosclerosis, hdl protects against it (national cholesterol education program 1993). the combination of high ldl and low hdl is termed dyslipidemia and is found in human patients with insulin resistance syndrome. mice do not develop spontaneous atherosclerosis, because they have low circulating levels of ldl and lack cetp activity. however, genetically engineered mice that over-or under-express genes involved in lipid metabolism have contributed greatly to our understanding of lipoprotein metabolism (marschang and herz 2003) . in particular, transgenic mice have been developed with plasma lipid profiles that are similar to those of humans with atherosclerosis (breslow 1994 (breslow , 1996 . in humans and transgenic mice with elevated ldl (or low hdl), ldl passively diffuses across the endothelium, especially in areas where endothelial cell morphology has been altered by the sheer forces generated by blood turbulence. once ldl is within the intima there is an interaction between ldlapoprotein b and matrix proteoglycans resulting in ldl trapping. ldl undergoes modifications associated with oxidation, lipolysis, proteolysis, and aggregation that contribute to inflammation and uptake of ldl by tissue macrophages. lipoxygenases (los) insert molecular oxygen into polyenoic fatty acids producing hydroperoxyeicosatetraenoic acid (hete). endothelial cells release hete into the vessel wall, where it initiates oxidation of ldl. further oxidation of ldl is aided by myeloperoxidase and sphingomyelinase. mice lacking los have diminished atherosclerosis. macrophages and monocytes recognize oxidized ldl via their surface scavenger receptors and become loaded with cholesterol ester, forming a fatty streak appearing along the vessel wall. hdl, on the other hand, removes excess cholesterol from peripheral tissue and inhibits lipoprotein oxidation. hdl carries paraoxonase, which degrades oxidized phospholipids (lusis 2000) and protects against subsequent oxidative damage. oxidized ldl stimulates endothelial cells to express adhesion molecules (such as icam, vla-4, and vcam) and monocyte colony-stimulating factor (m-csf) on their luminal surface, which attracts additional monocytes and lymphocytes. oxidized ldl also inhibits nitric oxide production. infiltrating thymic-derived lymphocytes (t cells) and macrophages initiate migration of smooth muscle cells from the media to the intima, where they synthesize matrix components. mcp-1 is also released by macrophages, inducing monocyte differentiation, migration, and scavenger receptor expression. the accumulation of excess free cholesterol can be inhibited by activation of acyl coa:cholesterol acyltransferase (acat)mediated cholesterol esterification and cellular cholesterol effiux. one mechanism responsible for cholesterol effiux is secretion of apoprotein e by macrophages that promotes effiux via hdl. if this fails, as in the case of cholesterol-laden macrophages in plaques, apoptosis of the macrophages ensues. loading of the endoplasmic reticulum with free cholesterol activates er resident protein kinase and the unfolded protein response (upr) that initiates apoptosis through activation of caspase-12 (feng et al. 2003a) . as macrophages undergo apoptosis they create a necrotic core in the plaque, promoting extracellular cholesterol cleft formation. smooth muscle cells create a fibrous cap over the plaque near the luminal surface. the lesion advances as t cells, activated by cd40-cd40l ligation, release cytokines such as interferon-3, (ifn-y) and tnf-ct. these substances activate matrix degrading proteases and adhesion molecules, promoting additional inflammation. as the inflammation progresses the fibrous cap becomes compromised, leading to physical rupture and the generation of a thrombogenic surface. oxidized ldl increases the production of tissue factor that, on the thrombogenic surface, initiates the coagulation cascade and thrombus formation. each of these details have been elucidated using genetically modified mice (auerbach et al. 1992; choudhury et al. 2004; feng et al. 2003b; lusis 2000; reardon and getz 2001; tailleux et al. 2003; trigatti et al. 2004) . for a further discussion of these models readers are urged to read chapter 16 in this book. for measurements of plasma lipids, serum is the preferred sample. serum can be stored at 4~ for 5-7 days without adverse effect on measurements. a. serum cholesterol and triglycerides serum cholesterol in mice can be measured using the enzymatic oxidation method of roschlay (meade and gore 1982) , the lipid research clinic program protocol (morrisett et al. 1982) , or the abell technique (mitruka and rawnsley 1977) . the most common enzymatic method employs cholesterol ester hydrolase (to convert cholesterol ester to cholesterol), cholesterol oxidase (which oxidizes cholesterol to hydrogen peroxide and cholest-4-en-3-one) and peroxidase (which catalyzes a reaction involving hydrogen peroxide, phenol and 4-aminoantipyrine to form the dye quinonelmine). all of these components of this test are combined in a single reagent mix. the dye absorbance is measured at 500 nm (choudhury et al. 2004) . triglyceride (tg) levels, which are mainly reflective of the tg content of chylomicrons and vldl in the mouse, are quantified using a variety of enzymatic methods. the most popular method combines lipoprotein lipase, glycerol kinase, and glycerol-phosphate oxidase with peroxidase and 4-aminoantipyrine (usually in two reagents). in this method, serum tgs are converted to glycerol by lpl via hydrolysis. next the glycerol is phosphorylated in an atp-dependent reaction catalyzed by glycerol kinase. glycerol-phosphate is catalyzed to dihydroxyacetone and hydrogen peroxide by glycerophosphate oxidase and peroxidase catalyzes the final reaction in which peroxide and 4-aminoantipyrine are converted to a stable dye and absorbance can be read at 500 nm (tsimikas et al. 2000) . reagents for both total cholesterol and total tgs may be used in an automated analyzer or purchased directly from a chemical company with instructions for manual laboratory analysis. total serum cholesterol and tg levels vary by strain, gender, age, length of fast, and diet. jiao et al. (1990) studied total plasma cholesterol (tpc) and tg levels of various inbred strains fed a standard diet. after 18-20 h of fasting, tpc levels ranged from 55 mg/dl (akr/j) to 128 mg/dl (nzb/b 1nj), and tg levels ranged from 13 mg/dl (c57bl/6) to 67 mg/dl c3h/hej; each much lower than seen in normal humans. albers and piagen (1999) quantified total cholesterol levels in 15 strains give a standard laboratory diet and fasted for 4 h before blood collection. levels (in 6-to 8-week-old mice) varied greatly between strains with c57blks/j females having the lowest level (39 mg/dl) and nzb/b 1nj males having the highest levels (127 mg/dl). for a given strain, males always had higher levels than females. trends in total hdl levels in the same strains paralleled total cholesterol levels. triglyceride levels also varied greatly among strains but did not parallel cholesterol levels. among the strains tested, c57bl/6j mice had the lowest tg levels (71-80 mg/dl) and c3h/hesnj the highest (f, 162 mg/dl; m, 231 mg/dl). differences between genders of the same strain were less pronounced, compared to cholesterol and males generally had higher levels than females. a transient cause of plasma lipid elevation (hyperlipidemia) in the mouse is related to recent feeding (postprandial hyperlipidemia), especially on a high-fat diet. causes of persistent hyperlipidemia include diabetes, sustained feeding of a high-fat diet, and nephrotic syndrome. b. hdl, ldl, idl, and vldl levels of individual apolipoproteins can be measured by density gradient ultracentrifugation combined with either electrophoretic, immunologic, chemical, or morphologic analyses. although published reference ranges are not available, the distribution and characterization of murine apoproteins has been reported (camus et al. 1983 ). similar methods were used to evaluate plasma apoproteins in mice consuming atherogenic diets (moltisett et al. 1982) . more recently the profile of murine plasma lipoprotein cholesterol has been determined by fast protein liquid chromatography with on-line post-column analysis of superose 6 gel-filtration eluates (sehayek et al. 2003; strauss et al. 2001) . in contrast to humans, mice normally have low levels of ldl in their plasma (15 mg/dl), with the major plasma lipoprotein being hdl. the low ldl concentration is due to editing of 70% of apoprotein b mrna transcripts in mouse liver leading to apoprotein b48 containing particles that are cleared much faster than ldl in humans (lusis 2000) . in contrast, human apoprotein b mrna transcript editing only occurs in the intestine. a much simpler methodology has been developed to measure hdl and non-hdl lipoproteins in mice, based on precipitation and enzymatic analysis. when phosphotungstate and magnesium salt is added to serum (or plasma), all lipoprotein, except hdl, is precipitated and can be removed. the remaining solution is then tested for cholesterol as described previously. the non-hdl-cholesterol component is calculated by subtracting hdl-cholesterol from total cholesterol in the nonprecipitated sample (sehayek et al. 2003) . some strains develop elevated cholesterol levels associated with increases in non-hdl levels after consuming high fat diets (breslow 1994) . c. mouse apoproteins commercial antibodies against mouse apoprotein a1 and apoprotein a2 are available and elisas have been described for the measurement of these apoproteins in plasma and in hdl fractions of plasma (dansky et al. 1999) . plasma apoprotein a1 levels can also be measured by multiplex analysis. an assay for mouse apoprotein j (apoj or clusterin) has also been described (navab et al. 1997) . apoj, which is a ubiquitous glycoprotein postulated to have multiple functions, is associated with hdl and is the amyloid-associated protein associated with amyloid plaque formation in alzheimer's disease in humans. d. other analytes associated with lipid metabolism and atherosclerosis in mice elisa kits are commercially available for the quantitation of many mouse coagulation proteins including: fibrinogen, factor vii, d-dimer, tissue factor, and von willebrand's factor antigen. elisa kits are also available to quantify many murine products of arachidonic acid metabolism including hete. reagents, elisa kits, and multiplex assays are available for murine inflammatory cytokines and chemokines (see the "cytokines and chemokines" section), as well as monocyte colony stimulating factor. dansky et al. (1999) have described assays for the quantitation of murine aryl esterase and paraoxonase. table 6 -7 lists the mouse enzymes involved in lipid metabolism with references that describe their measurement. the innate and adaptive immune responses have been extensively studied (see volume 4 of this series) and therefore we will not attempt to describe all the features of mouse immunity in detail here. readers are directed to volume 4, molecular and cellular immunology of the mouse, for a general overview, and the 15 chapters that follow for a more detailed description. this section describes the growing number of quantifiable soluble serum proteins and lipids associated with immunity and inflammation in the mouse (see table 6 -3). furthermore, in addition to immunodeficiency, autoimmunity, and allergy, investigations of atherosclerosis, obesity, diabetes, cancer, as well as infectious diseases, each have immunologic and inflammatory components. 1. immunoglobulins (ig) as in humans, mouse immunoglobulins (ig) are molecules composed of four polypeptide chains; two of lower molecular weight called light (l) chains, and two with higher molecular weight called heavy (h) chains. disulfide bonds link one l chain to one h chain, and the two h chains to each other. immunoglobulin h chains are composed of four to five domains, including an n-terminal variable region domain and four constant-region domains. in addition, structural differences in the constant-region domains of the heavy chain are used to classify the five different classes of immunoglobulin, igm, igg, iga, ige, and igd. l chains are composed of only two domains and structural differences in these domains are used to classify l chains as either kappa or lambda type. in the mouse, 95% of serum ig has kappa l chains. any individual antibody secreting b cell (or plasma cell) will make a single ig molecule composed of two identical h chains and two identical l chains. mice make four subtypes of igg: igg1, igg2a, igg2b, and igg3. certain strains, c57bl/6, c57b1/10, sjl, and nod, do not make igg2a but rather make a novel igg2c. the igg subtypes in mice are not exact homologues of human subtypes (mestas and hughes 2004) . prenatal (transplacental) transfer of maternal ig as well as postnatal transfer across the intestinal epithelium is limited to the igg2a, 2b, and 3 subclasses of immunoglobulin and is mediated by neonatal fc receptors (fcrn) located in the placenta or on the intestinal brush border of the proximal small intestine (bankert and mazzaferro 1999) . mouse igg2b fixes complement by the classical pathway and igg 1 and igg2a fix complement by the alternative pathway. ige and igg1 are homocytotropic antibodies capable of binding to receptors on mast cells and basophils and mediate immediate hypersensitivity reactions. although igg and ige circulate in the mouse as monomers, igm circulates as a pentamer and iga circulates as a polymeric molecule. normally very little igd can be detected in serum. serum levels of igm, iga, igg, and ige are influenced by the rate of synthesis and rate of catabolism. like humans, the rate of igg catabolism in mice is directly proportional to the serum concentration of the subclass. the average half-life of murine igg is 4.5 days. the catabolic rate of iga is independent of serum concentration. mice synthesize from 50-130 mg/kg/day of total ig, although this is dependent on strain and level of antigenic stimulation. certain strains have a propensity to develop specific t-helper (th, cd4+) cell subclasses in response to antigenic stimulation, and these strains are referred to as having principally a th1 or th2-1ike phenotype. typically cd4+ lymphocytes modulate immune responses by the cytokines they secrete. those secreting il-1, ifn-y, and lymphotoxin are generally referred to as th1 (and are favored responses for immunity against viruses and intracellular pathogens) and those secreting il-4, il-5, il-10, and il-13 are referred to as th2 (and enhance humoral immunity while suppressing cell mediated immunity). when confronted with the same antigen, balb/c mice exhibit a th2-dominant response, and c57bl/6 mice exhibit a thl-dominant response. il-4 participates in immunoglobulin (antibody) class switching (see volume 4, chapter 5). consequently, th2 strains are the models of choice for investigations of allergic inflammation because they produce higher concentrations of il-4 induced immunoglobulin classes (ige and igg1). the cba/n strain is deficient in its ability to produce igm and igg3. immunoglobulin levels are also greatly reduced in germ-free mice, offspring of mice on zinc-deficient diets, and mice on protein-deficient diets (quimby 1999b) . quantifying the various classes and subclasses of murine ig can be done using various immunoassays including: radial immunodiffusion, ria, or enzyme immunoassay. both ria and enzyme immunoassay have the higher degrees of sensitivity that are needed to accurately quantitate levels of ige and igd in murine serum. enzyme immunoassay has become the favored assay to avoid isotope handling and disposal. the reported concentrations of normal balb/c mice are: igg1 (6.5 mg/ml), igg2a (4.2 mg/ml), igg2b (1.2 mg/ml), igg3 (0.1-0.2 mg/ml), iga (0.7 mg/ml), igm (1.0 mg/ml), and ige and igd are both less than 0.01 mg/ml (bankert and mazzaferro 1999) . in addition, there are many commercial sources of enzyme-linked antibodies that supply reagents (and instructions) for developing assays to measure antigen-specific antibody concentrations by subclass of antibody. the complement system is composed of 40 or more chemically and immunologically distinct proteins capable of interacting with antibodies, certain bacterial products, and cell membranes. a brief summary of this system is described later. please refer to two recent publications (quimby 1999a; turnberg and botto 2003) for more details about the structural and functional aspects of each protein of the complement system. the role of the complement system in mouse immunity is described in volume 4 (overview) of this series. the sequential activation of individual complement proteins from inactive to active substances is a dynamic event called the complement cascade. the ability of the first component of complement, c1, to bind specific sites on the heavy chain of mouse igg2b and activate a sequence of reactions leading to production of a molecular unit capable of lysing a target cell membrane has established the complement system as the primary mediator of antibody-antigen reactions. recent findings suggest that the pentraxins, c-reactive protein (crp), serum amyloid protein (sap), and pentraxin 3 (ptx3) can bind to complement component clq and activate the classical pathway. this may be an important mechanism for removal of apoptotic cells that might otherwise predispose to autoimmune disease (nauta et al. 2003) . each protein of the complement system is normally present in the circulation as an inactive molecule. although the complement cascade may be activated by any of four separate pathways, the central event for each is activation of c3 to c3b yielding a small c3 cz chain fragment. the two c3 convertases are c4b2a (for the classical, lectin, or pentraxin pathways) and c3bbb for the alternative pathway. each c3 convertase cleaves c3 and adds the c3b fragment to the convertase complex forming c5 convertase. cleavage of c5 leads to the membrane attack complex (mac) common to all activation pathways (goldsby et al. 2003) . assemblage of the mac on the surface of a target cell leads to the formation of a large channel enabling ions and other small molecules to diffuse out leading to cell death. the activated components of complement also participate in chemotaxis, phagocytosis, cell adhesion, and b-cell differentiation. there are many notable differences between mice and humans regarding expression of complement components and regulation of cascade activation. mice have both an active and inactive form of circulating c4 and the genes (both on chromosome 17) are designated ss and slp, respectively. many inbred strains do not synthesize active c5 (e.g., dba/2 and a/j strain) due to a post-translational defect and therefore they cannot generate a mac. some strains are deficient in the production of c8 (e.g., dba/2j strain). complement component 6 exists as two allelic forms in mice with 90 and 100 kda molecular weights. similarly there are several notable differences in the regulators of complement activation. most of these regulators are found in the regulator of complement activation (rca) locus on murine chromosome 1 and, as in humans, restrict assembly and stability of convertase enzymes. in mice, decay acceleration factor (daf) is encoded by two genes. however, only the product of daf-1 is widely dispersed on all tissues. this membrane-anchored protein inhibits c3 cleavage by accelerating decay of c3 convertases. in mice it is the major regulator of c3 in the skin (not kidney) and is a ligand for cd97 which, on cross-linking, leads to lymphocyte activation. cd59 is a membrane-anchored inhibitor of c5b-9 formation (mac) and prevents c9 from binding the c5b-8 complex. deficiencies of cd59 in humans lead to hemolytic anemia but not in mice. membrane cofactor protein (mcp) is a major cofactor for factor i in humans causing cleavage of c3b and c4b deposited on self-tissue. in mouse mcp is only expressed in the testis. complement receptors 1 and 2 are encoded by separategenes in humans but are produced by alternative splicing of a single gene in mouse. mice have a complement receptor 1 (cr-1) related gene/protein y (crry) not found in humans, which is a membrane-anchored c3 inhibitor. it is the major regulator of c3 in mouse kidney and has some of the functions as mcp and cr1 in humans. crry has daf activity and serves as a cofactor for factor i (which enzymatically cleaves c3b). the mouse has been widely used in studies on the biosynthesis and molecular biology of individual components of complement. the genes encoding 39 components, subcomponents, receptors, and inhibitors have been identified in mice. the complement components may be quantified by assays designed to measure the functional properties of these proteins or their antigenic properties. tests designed to measure antigenic properties of complement are generally simpler, less subject to error, and less expensive; however, they have the disadvantage of measuring both active and inactive forms of their proteins and therefore may not correlate well with functionally active protein. functional assays measure the ability of the entire classical or alternative pathway, or individual components of pathways to lyse (hemolyze) antibody-coated (sensitized) or noncoated (for the alternative pathway) red cells in suspension or in agarose gel. these assays are precise and sensitive (quimby 1999a) . antigenic assays include radial immunodiffusion, electroimmunodiffusion (rocket electrophoresis), automated immunoprecipitation, crossed immunoelectrophoresis, and more recently elisa (quimby 1999a) . elisa kits are commercially available for quantifying murine c lq, c3, c4, c3a (desarg), and c3a. in addition antibodies are commercially available for these as well as murine c4a, c4d, c5, and c6. complement components c2, c5, c6, c7, and factor b may be quantified using functional assays (quimby 1999a) . membrane-bound complement regulators, crry, cd59, and daf, can all be detected using previously published antibodies (lin et al. 2001 (lin et al. , 2002 . the principle mediator of the lectin activation pathway is mannose-binding lectin (mbl). after binding to mannose residues on the surface of microorganisms, two mbl-associated serine proteases (masps), masp-1 and masp-2, bind to mbl. this complex causes cleavage and activation of c4 and c2 (masp1 and 2 mimic the activities of c14 and cls). commercially available elisa kits are available for murine mbl-a and mbl-c quantitation. other activators of complement include members of the pentraxin family. in mice this may include crp or sap, although the concentration of crp in normal mice is very low. immunologic reagents are available to quantify both pentraxins in mice (gentry 1999; quimby 1999b ). circulating immune complexes (cic) are multimolecular substances composed of antigen, antibody, and activated complement components. in mice, igm, igg1, igg2a, and igg2b have complement activation regions. although cic may form following exposure to circulating foreign antigens, such as those associated with microorganisms, they are also common manifestations of spontaneous autoimmune disease such as that seen in (nzb x nzw)f1, mrl/lpr, bxsb, and krn strains. cic are cleared from the circulation by both cr-1, cr-2, and fcr. tissue deposition of immune complexes may lead to vasculitis. cic have been quantified in mice by capitalizing on their binding to various complement receptors, by precipitation of clq with polyethylene glycol, or by immunoassay. commercially available elisa kits are commonly employed today (quimby 1999b ). more than 100 inbred strains or mutant lines spontaneously develop autoimmune disease or are susceptible to autoimmune disease induction. the details of many of these lines may be found in volume 4, chapters l l and 12 in this series. autoimmune diseases in mice include: thyroiditis, rheumatoid arthritis, sj6gren's syndrome (ss), hemolytic anemia, lupus erythematosus, type 1 diabetes mellitus, experimental allergic encephalitis, oophoritis, orchitis, gastritis, ulcerative colitis, and polyendocrine disease (boyton and altmann 2002; ravirajan and isenberg 2002; sakaguchi 2000) . antibodies directed to self-antigens in the mouse have been quantified using a wide range of methods from immunofluorescence to elisa. table 6 -3 lists the commercially available elisa kits used to quantify murine autoantibodies. those antigenic targets associated with systemic lupus erythematosus include: cardiolipin, double-stranded deoxyribonucleic acid (dsdna), single-stranded deoxyribonucleic acid (ssdna), histone, [~-2 glycoprotein, proliferating cell nuclear antigen (pcna), neutrophil cytoplasmic antibody (canca), ribosomal p, and smith. antigenic targets for arthritis include: rheumatoid factor (rf), collagen type 1, collagen type 2, and ssdna. antibodies against insulin and glutamic acid decarboxylase are seen in type 1 (juvenile) diabetes. mixed connective tissue diseases are characterized by antibodies to ribonuclearprotein (rnp). antibodies to myeloperoxidase (mpo) may be observed in vasculitis. mice with ss develop autoantibodies to ss antigens a and b (ssa and ssb, respectively). panels containing 14 autoantigens are available as multiplex assays for quantifying murine autoantibodies. a. interleukins (il) (ils are cytokines that are secreted by leukocytes and act on other leukocytes. interleukins have been classified based on the secretory cell type (i.e., monokines vs. lymphokines), and they have been classified based on whether they are primarily involved in innate (il-1, il-6, il-12, tnf-t~, ifn-t~), or adaptive (il-2, il-4, il-5, il-10) immunity. commercially available elisa kits are available to quantify most murine interleukins, as demonstrated in table 6 -3. i. interleukin-1 (il-1) il-1 is a name for two proteins, il-lt~ and il-1 [3, that are encoded by separate genes. along with il-1 receptor antagonist, il-18, il-6, and tnf-t~, these proteins modulate acute inflammation. the effects of il-1 are pleotrophic and involve bone remodeling, insulin secretion, appetite regulation, fever induction, neuronal development, and many others. both il-1 o~ and il-1 [3 are secreted as 269-271 amino acid (aa) pro-cytokines that are enzymatically cleaved into bioactive 17-kda segments. unlike il-1 [3, the intact procytokine of il-1 ct is also bioactive, both within the cytoplasm and on the cell surface, where it is anchored to the cell membrane via a mannose glycosylation residue that attaches to the membrane-associated lectin. there is 78% sequence identify between mouse and human il-i~ genes and 58% identity between mouse and human il-1 t~ genes. a third gene encodes il-1 receptor antagonist, a soluble 25-kda molecule with 19% sequence homology to il-lt~ and 26% homology with il-113. mouse il-lra is 75% homologous with human il-lra. there are two il-1 receptors, types i and ii, but only il-1ri is capable of signal transduction. il-lra inhibits the action of il-lt~ and il-1b by binding il-1ri. a 60-kda form of il-1ri has also been described that is soluble and preferentially binds il-lra. il-1rii has no signal transducing associated protein and serves to modulate levels of il-1 t~, il-i[3, and il-lra by binding them on the cell surface. it can also occur as a soluble receptor. with the recent finding of six new members in the il-1 ligand family (in humans), a revised nomenclature for both il-1 ligands fred w. quimby and richard h. luong and il-1/il-18 receptor families has been developed (sims 2002) . further details may be found in volume 4 (overview and chapter 8) of this series. ii. interleukin-2 (il-2) il-2 is a lymphokine secreted by activated t-helper cells. it acts in an autocrine fashion to induce the expression of il-2 receptor on t cells, resulting in t-cell proliferation (cogoli-greuter et al. 2004) . il-2 also acts in a paracrine fashion modulating the activities of b cells, natural killer (nk) cells, and lymphocyte activated killer (lak) cells. il-2 is a glycoprotein of 133 amino acids (in humans) with 63% homology between mouse and human. the il-2 receptor (il-2r) is a multisubunit cellular receptor belonging to the class 1 cytokine receptor family (hematopoietin receptor family). the il-2r has a-, [~-, and y-chains. [3-and y-chains interact to transduce the il-2r signal and the y-chain is shared with receptors for il-4, il-7, il-9, and il-15. iii igg1 in mice and is responsible for the downstream events leading to differentiation and activation of th2 cells. il-4 also induces expression of adhesion molecules like vcam, th2 cytokines such as il-5, il-6, and il-9 and chemokines like eotaxin-1 and-2. il-4 primes mast cells and basophils leading to enhanced activation during allergic challenge (mueller et al. 2002) . homology between mouse and human molecules is low (25%) and each is species-specific in its biologic activity. it induces the growth of b-1 progenitors and igm production by b-1 cells. il-5 induces class switch, favoring production of iga, igg1, and ige. on eosinophils, il-5 induces iga and igg receptors and stimulates leukotriene (lt), c4, and paf secretion, in addition to inducing eosinophil growth and maturation. the receptors for il-5 consist of a ligand binding tx-subunit and a non-ligand binding (common) signal transducing ~-subunit that is shared by receptors for il-3 and granulocyte-monocyte colony-stimulating factor (gm-csf) (sato and miyajima 1994) . vi. interleukin-6 (il-6) il-6 is secreted by a wide variety of cells including t cells, b cells, monocytes, fibroblasts, hepatocytes, keratinocytes, astrocytes, and endothelial cells. it has broad pleiotropic effects on host defense, acute phase responses, immune responses, and hematopoiesis. il-6 is classified as an inflammatory cytokine and based on a helical cytokine structure and subunit makeup, il-6 is the prototypic member of a family of molecules that includes leukemia inhibitory factor (lif), oncostatin m (osm), ciliary neurotrophic factor (cntf), cardiotrophin (ct-1), and il-11. mouse il-6 is 25 kda and contains four cysteines and contains o-glycosylation sites and shares 40% homology with the human molecule (van snick 1990). the il-6 receptor has two subunits, a nonsignal transducing subunit binding with low affinity (~-subunit), and a signal transducing subunit (~-subunit) that does not bind il-6 by itself but participates in high-affinity binding. the soluble il-6r~ chain binds il-6 and the complex induces expression of mcp-1, which attracts monocytes into areas of inflammation (kaplanski et al. 2003) . vii. interleukin-7 (il-7) il-7, previously called lymphopoietin-1, is expressed by stromal cells, especially in the bone marrow and thymus, where it promotes thymopoiesis of t cells and the differentiation of pro-b cells into pre-b cells (aspinall et al. 2004; goldsby et al. 2003) . mouse il-7 has 65% amino acid sequence homology with human il-7 and both proteins exhibit cross-species activity. viii. interleukin-8 (il-8) il-8 is not expressed in the mouse; however, another protein, kc, is secreted and has many properties of the human chemokine, gro, which is known to bind the il-8 receptor (see the "cytokines and chemokines" section). ix. inrelclevlcln-io (il-io) il-10 is the prototypic member of the il-10 cytokine family comprising ill0, il-19, il-20, il-22, il-24 (fisp), and il-26. il-10 is a 178 amino acid protein with an 18 amino acid signaling sequence. both mouse and human il-10s have two intrachain disulfide bonds and form non-disulfide linked homodimers. mouse and human il-10 are 72% homologous. il-10 is a th2 cytokine, which inhibits ifn-y and gm-csf production by th1 cells. additionally it induces cd8 + t-cell chemotaxis, inhibits t-cell apoptosis, participates in iga class switch in b cells, induces histamine release from mast cells, and promotes tnf-tx and gm-csf production by nk cells. il-10 inhibits secretion of the neutrophil chemokines mip-lt~ and mip-i~ and blocks production of il-1~ and tnf-ct by neutrophils. it is immunosuppressive to dendritic cells and induces the differentiation of a subset of regulatory cd4 + t cells (trl) (grouz and cottrez 2003; morel et al. 1997 ). x. il-11, also known as adipogenesis inhibitory factor (agif), is a pleiotropic cytokine with effects that overlap that of il-6. il-11 is a member of the il-6 cytokine family and as such has a four-helix bundle fold motif. it binds to the multimeric il-11 receptor that shares the promiscuous gpl30 signaling ~-subunit with other receptors in this family. the il-11r ~ chain is unique and binds il-11 but does not have a cytoplasmic domain; instead binding leads to homodimerization of the ~-chain that activates the janus kinases. il-11 stimulates proliferation and differentiation of monocytes and megakaryocytes causing thrombopoiesis. it also activates osteoclasts and enhances bone resorption, decreases new bone formation, and stimulates chondrocyte and synoviocyte production. il-11 protects small intestinal epithelial cells from chemotherapy and radiation injury and ameliorates inflammatory bowel disease (schwertschlag et al. 1999) . il-11 inhibits adipogenesis, regulates neuronal differentiation, and regulates t-cell function (enhances th2 and inhibits th1 cytokine production). overexpression of il-11 in the lung causes airway remodeling, fibrosis, and mononuclear nodules analogous to the clinical picture in chronic asthma. il-11 is also required for the uterine decidualization response (robb et al. 2002; zheng et al. 2001) . xi. il-12, also known as natural killer cell stimulatory factor (nksf), is a heterodimeric cytokine composed of a 40-kda (p40) subunit and a 35 kda (p35) subunit. the p40 subunit is shared by il-23, a cytokine with similar activities. macrophage, monocytes, and dendritic cells produce il-12 after activation of toll-like receptors (tlr) on these cells by bacterial ligands. il-12 induces production of ifn-7by th1 and nk cells and intact il-12 skews the balance between th subsets in favor of th1 cells. il-12 binds to the il-12 receptor that is composed of two subunits, [31 and ~2, on the surface of nk and th1 cells. il-12p40 interacts with il-12r[31, and il-12p35 binds il-12r[32. negative feedback regulation of il-12 production involves down regulation of tlr signaling by phosphoinositide 3-kinases (piks). thus il-12 is centrally involved at the interface of innate and adaptive immunity (fukao and koyasu 2003" ottenhoff et al. 2002) . xit, il-13, along with il-4 and il-5, is a member of the type-2 cytokine family and as such is involved in inflammation, mucus production, tissue remodeling, and fibrosis. this single-chain protein shares 58% amino acid sequence homology with human il-13. il-13 is produced by activated t cells, mast cells, and nk cells and promotes th2 responses including synthesis of ige. signaling is mediated by the type-2 il-4 receptor which consists of il-4r~ and il-13rc~i chains. another il-13 binding protein, il-13r~2, strongly inhibits the activity of il-13 (goldsby et al. 2003; mentink-kane and wynn, 2004) . xiii. il-17, also known as cytotoxic t lymphocyte-associated antigen-8 (ctla-8), is produced by t cells and is pleiotropic in activity. il-17 is a 158 amino acid residue polypeptide with a 21 amino acid signal sequence and a mature polypeptide of 137 amino acids. it is a disulfide-linked homodimer. based on the presence of spatially conserved cysteine residues in the il-17 family of proteins, there are six family members in humans and mice, il-17, il-17b, il-17c, il-17d, il-17e, and il-17f (aggarwal and gurney 2002) . like il-17 itself, several of the family members appear to modulate immune function. produced by mouse cd4 ⧠t cells, il-17 induces il-6, mcp-1, prostaglandin-e2 (pge2) and granulocyte colony-stimulating factor (g-csf) by fibroblasts, keratinocytes, epithelial cells, and endothelial cells. it induces icam-1 surface expression, proliferation of t cells, and the differentiation of cd34 + marrow progenitors into neutrophils (fossiez et al. 1998 ). the ubiquitously distributed receptor is a type 1 transmembrane glycoprotein of 830 amino acids in length. xiv. il-18 is a 24-kda, nonglycosylated polypeptide that lacks a classical signaling sequence. its structure resembles il-1 and the propeptide undergoes proteolytic cleavage by interleukin-1 [3-converting enzyme (ice) or another caspase to produce an 18-kda bioactive molecule. there is 64% sequence homology between mouse and human il-18. il-18 induces the production of ifn-7 by t cells and nk cells and the expression of fas ligand (fasl) on a variety of all types. il-18 activates nf-~:[3 and the induction of various chemokines. il-18 plays an important role in the early antibacterial host response (weijer et al. 2003) . xv. and il-20 induces keratinocyte differentiation and proliferation. il-21 is a four-helix-bundle cytokine similar in structure to il-15 and sharing sequence homology with il-2 and il-4. murine il-21 is 57% homologous to human il-21. the il-21 receptor utilizes the common 7-chain. il-21 is produced by the th2 cells. the actions of il-21 are pleiotropic and seen on b cells, t cells, nk cells, and dendritic cells. il-21 induces apoptosis in resting and activated b cells, an effect counteracted by activation of cd40. it also upregulates production of igg1 and inhibits ige, in fact it inhibits many il-4 activities. il-21 also inhibits dendritic cell differentiation. il-21 enhances the activity of activated nk cells and mediates the proliferation and expansion of t-cell subsets. il-22 induces acute phase reactants by hepatocytes and reduces il-4 production by th2 (mehta et al. 2004) . recombinant antigens and antibodies are commercially available for murine il-20, il-21, and il-22. b. the transforming growth factor-~ superfamily (tgf-~sf) of cytoi~nes members of tgf-~ family share 25-40% sequence homology with tfg-~i and a monomeric structure that consists of two antiparallel pairs of [3-strands forming a flat curved surface, a separate long cz-helix, and a disulfide rich core with a characteristic cysteine knot. most tgf-~sf members are disulfide-linked homodimers; however, three members lack the seventh conserved cysteine residue and are not covalent homodimers. members of the tgf-[3sf include tgf-[31, tgf-132, tgf-133, activins, inhibins, bone morphogenic proteins (bmp), growth differentiation factors (gdf), glial-derived neutrophic factors (gdnf), and mtillerian inhibiting substance (mis). tgf-[31 is stimulatory for cells of mesenchymal origin and inhibitory for cells of epithelial or neuroectodermal origin. in the murine immune system tgf-[31 is the mediator of immune suppression via cd4+cd25+tr cells and, at least in the case of suppressing cd8 + effector t cells, involves tgf-[3 receptor ii on these cells (powrie 2004; von boehmer 2005) . in addition tgf-[3 is known to inhibit b-cell proliferation and it promotes isotype switch to iga. oral tolerance to th2 responses (against food allergins) is mediated by tgf-~i1 (mucida et al. 2005 ). tgf-[3 has a wide range of effects on cell growth differentiation and malignant transformation (letterio 2005) . murine tgf-~i 1 may be quantified in serum or plasma using commercially available elisa kits. antibodies are also available which specifically bind murine bmp, activin a, activin c, and gdf-1,-3,-5,-8, and-9, although they are not recommended for elisa development. c. the tumor necrosis factor superfamily (tnfsf) tnf-related ligands share many features but high amino acid sequence homology is not one of them. with the exception of nerve growth factor and tnf-~, all ligands are type ii tramsmembrane proteins (extracellular c-terminus) that contain a short cytoplasmic segment and a long extracellular region. tnf-~ is fully secreted and has a nonfunctional transmembrane segment. tnfsf members form trimeric structures and their monomers are composed of ~-strands oriented into a twosheet structure. receptors for the tnfsf ligands also belong to a superfamily, tnfrsf (gruss and dower 1995) , and are characterized as type i transmembrane proteins (with their amino termini outside of the cell), with extracellular cysteine-rich structural motifs. tnfrsf members exist both as membrane and soluble forms. commercially available elisa kits or matched antibody for development of assays are available to quantify the murine receptors and ligands of the tnfsf discussed in this section. i. tumor necrosis factor-ix (tnf-ix) tnf-tx is expressed as a 26-kda membrane glycoprotein and the soluble glycoprotein is generated by proteolytic cleavage via tnf-ct converting enzyme (tace). the 17-kda homotrimer cleavage product circulates. mouse tnf-tx has 79% sequence homology with human. tnf-tx is expressed widely on tissues throughout the body (goetz et al. 2004) . tnf-tx is a strong mediator of inflammation and immune function, or regulates on growth and differentiation, and is cytotoxic for many transformed cells. ii iii. cd40l cd40l (tnfsf5, cd154) is a 39-kda, type ii, transmembrane glycoprotein that can be proteolytically cleaved to 15-to 18-kda soluble forms with full biologic activity. it forms natural trimeric structures and the mouse cd40l shares 73% sequence identity with human cd40l. cells expressing cd40l include b cells, cd4 + and cd8 ⧠t cells, monocytes, nk cells, and y t cells (toubi and shoenfeld 2004) . on binding cd40, the complex initiates signals important for cell proliferation or apoptosis. cross-linkage between t and b cells allows cd40 to transduce the tyrosine kinases lyn and syk, and activate phospholipase c, ip3, and dag. when combined with other cytokines, ligation of b-cell cd40 provides the second signal allowing differentiation of b cells to plasma cells (goldsby et al. 2003) . iv. cd30l cd30l (tnf5f8, cd153) is a 40-kda glycoprotein with 72% sequence homology between murine and human molecules. cd30l is expressed on monocytes, macrophages, b cells, activated t cells, neutrophils, megakaryocytes, resting cd2 ⧠t cells, erythroid precursors, and eosinophils. ligation to cd30 can induce either proliferation or apoptosis. v. fas ligand (fasl) fasl (also known as tnfsf6), is a 40-kda glycoprotein that, after cleavage, forms a 70-kda homotrimer that is active only in membrane form in the mouse. polymorphisms in fasl also exist and a single amino acid substitution in position 273 (phe to leu) results in the generalized lymphoproliferative disease (gld) mutation. fasl is expressed on cells of the adaptive and innate immune systems, as well as cells of the lung and intestine. there is 77% sequence homology between murine fasl and human fasl (lynch et al. 1994) . ligation of fas by fasl on mature t cells leads to activation of the caspase cascade and apoptosis. this is a major homeostatic mechanism regulating the size of the t-cell pool and for eliminating t cells that repeatedly encounter self-antigens (goldsby et al. 2003) . vi. tnf-relateo activation-induced cytokines (trance) trance, also called rank ligand and osteoprotegerin ligand (opgl), is an osteoclast differentiation factor. mouse and human share 85% sequence homology, and trance is expressed on t cells and t-cell rich organs such as thymus and lymph nodes. vii. tnf-receeror superfamily (tnfrsf) tnfrsf members mediate the cellular effects of tnfsf members. tnfri and tnfrii bind tnf-~ and it appears tnf-~ complexes with lt-[3 and the complex binds to tnfri or lt-[~ receptor. cd40 is associated with b-cell proliferation but is expressed on many cells throughout the body. mouse cd40 shares 62% sequence homology with human cd40; however, the mouse molecule has a 28 amino acid extension of its cytoplasmic tail. cd30 has 480 amino acid residues and a 90 amino acid deletion in the extracellular region compared to human. it is expressed on cd4 + and cd5 + t cells and ligation results in production of il-5. murine fas lacks 8 amino acid residues found in human and shares only 50% sequence homology. soluble forms result from alternative gene splicing and circulate as dimers or trimers. fas is expressed by cd34 stem cells, fibroblasts, nk cells, keratinocytes, hepatocytes, b and t cells (and their precursors), and eosinophils. osteoprotegerin (opg) inhibits the action of osteoclasts and is a secreted member of the tnfrse although it has no transmembrane segment and circulates as a disulfide-linked homodimer. murine troy, also named toxicity and jnk inducer (taj) and tnfrsf19, shares 92% homology with human in its extracellular domains. d. interferons interferons are a group of related but distinct proteins that share more than 95% amino acid sequence homology. members of the type i interferon family share a common cell surface receptor composed of two subunits. commercially available elisa kits may be used to quantify murine io interferon-~ (ifn-~) ifn-~ is induced in a wide variety of cells, including monocytes and macrophages, in response to viral infection. one known inducer is double stranded ribonucleic acid (dsrna). induce resistance to viral replication by binding the ifn-~/~ receptor, which activates the jak-stat pathway, inducing several genes. one of those genes is ribonuclease, which degrades viral rna. binding of ifn-~ to nk cells enhances their lytic activity for virally infected cells. ifn-~ is secreted by leukocytes. iii inreturelcon-'t(ifn-y) ifn-y is secreted by th1 cells, nk cells, and cytotoxic t cells (tc), which activates macrophages to secrete tnf-ct, express class ii major histocompatibility complex (mhc) molecules, and produce antimicrobial activities. ifn-y secretion by th1 also induces antibody-class switch to igg2a, which supports phagocytosis and complement fixation. ifn-y promotes differentiation of tc from cd8 + precursors that will be involved in the effector response to viral infections and intracellular pathogens. ifn-y also inhibits the expansion of th2 cells. ifn-y secretion is induced by successful stimulation of t cells by antigen presenting cells. e. chemo~s chemokines, along with adhesion molecules, are the principle controllers of leukocyte migration and as such directly affect leukocyte retention and relocation during hematopoiesis and at sites of immune defense and inflammatory disease (moser et al. 2004) . chemokine-induced signaling is via g-protein coupled cell surface receptors. although most chemokines are secreted proteins, two chemokines, cxcl16 and cx3cl 1, are membrane bound. two primary subfamilies are recognized based on the arrangement of two nh2-terminal cysteine residues that are either located adjacent to each other (cc) or are separated by a single amino acid (cxc). two minor subfamilies include chemokines with a single cysteine resides (xcl1, xcl2) and a chemokine with three amino acids separating the cysteine residues (cx3cl1). functionally conserved regions of the n-terminus of each member mediate receptor binding and extracellular matrix fixation (or binding cell surface glycosaminoglycans). many chemokines are designated with a name given at the time of their identification; all have also been assigned a name based on their structural motif (cc, cxc, xc, cx3c) followed by l for ligand. receptors are heterotrimers and their activated g-protein subunits stimulate phospholipase c[3, piks, and c-src tyrosine kinases (moser et al. 2004) . receptors are designated by the type of chemokine they bind (i.e., cxc, cc, xc, or cx3c), followed by r. mice lack the cxcr1 family of chemokines and receptors found in humans. table 6 -8 lists the murine chemokine receptors and the known ligands. table 6 -3 lists the murine chemokines for which there are either commercial test kits or matched antibodies for kit development. two main functional groups define chemokines. inflammatory chemokines recruit effector leukocytes to sites of infection, inflammation, and repair. homeostatic chemokines control the navigation of leukocytes during hematopoiesis in the bone marrow and thymus, control homing of cells to spleen, lymph nodes, and peyer's patches during the adaptive immune response and control immune surveillance in peripheral tissues. some chemokines participate in both inflammation and homeostasis and are called dual-function chemokines. many dual function chemokines are highly selective for the recruitment of t cells. other chemokines have ill-defined functions regarding homeostasis and inflammation but participate in other vital activities such as the role of pf4 (cxcl4) in thrombosis and the role of cxcl 10 in gut epithelial cell turnover (cliffe et al. 2005) . most inflammatory chemokines are thought to be induced and the variety of stimuli that induce their expression is broad. by contrast most homeostatic chemokines are thought to be constitutively expressed. an exception is the inducing effect of lymphotoxin and tnf-~ on b-cell attracting chemokine-1 (bca-i, cxcl13), ccl19, and secondary lymphoid tissue chemokine (slc, ccl21), which also participate in inflammation. leukocytes also release several kinds of proteases that degrade chemokines at their n-terminus, resulting in the loss of receptor binding, antagonist generation, or enhancing their biologic function. leukocyte cd26, dipeptidyl peptidase iv is known to act on cxcl9, cxcl10, cxcl11, and cxcl12. murine sulphostin inhibits the action of cd26 and stimulates g-csf and granulopoiesis (abe et al. 2005) . matrix metalloproteases (mmps) are enzymes that degrade extracellular matrix proteins, including stromal cell derived factor-1 (sdf-1, cxcl12) and mcp-1. table 6 -3 lists the murine mmps that can be quantified by commercially available elisa kits. in addition to mmps, cathepsins have been shown to modify chemokines. table 6 -9 lists the murine chemokines and the proteases that degrade them and stimuli that induce them. certain chemokines may antagonize the activity of other chemokines; for instance, three agonists for the receptor cxcr3 are also antagonists for receptor ccr3 (which is agonized by eotaxin [ccl11]). because cxcr3 and ccr3 are differentially expressed on th1 and th2 cells, these chemokines [monokine induced by ifn-y (mig/cxcl9), ifninducible protein-10 (ip-10/cxcl10), and ifn-inducible t-cell chemoattractant (i-tac/cxcl11)] modulate the th subpopulation allowed to enter tissue sites favoring th1 immune polarization (moser et al. 2004) . table 6 -10 summarizes some known effects of murine chemokines. because chemokines and their receptors are known to modulate many inflammatory diseases including the autoimmune diseases, they have become target for new therapeutics. the chemokine antagonist, met-rantes, has been shown to be an effective inhibitor of allergic airway disease in mice. likewise, deficiencies of chemokines and their receptors in mice have modified disease progression in atherosclerosis, autoimmunity, and also prolong allograft survival (mackay 2001 ). in addition, many viral genomes are known to encode structural genes for chemokine antagonists, which appears to be a principal mechanism used by many viruses to evade the host immune system. these present another target for drug intervention for viral infections. finally, pepducins, derived from the intracellular loops of cxcr1, cxcr2, and cxcr4, specifically inhibit receptor g-protein signaling in mice and prevent fatal sepsis and disseminated intravascular coagulation (kaneider et al. 2005 ). certain growth factors (see table 6 -3) and cytokines activate phospholipases after binding their cell surface receptors. these phospholipases act on membrane phospholipids to release arachidonic acid, a precursor for several eicosanoids. arachidonic acid is metabolized by any one of three biochemical pathways: the cyclooxygenase (cox) pathway, which forms pgs and thromboxane, the lo pathway, which forms hetes and leukotrienes (lts), and the cytochrome p-450 monooxygenase pathway, which forms epoxides and hetes. the cox enzymes, cox-1 and cox-2, catalyze the first step in the synthesis of pgs by converting arachidonic acid to prostaglandin h 2 (pgh2). pgh 2 is chemically unstable and is the precursor for enzymatic and nonenzymatic production of pgd 2, pge 2, and pgfza. thromboxane synthetase converts pgh 2 to thromboxane a 2 (txa2) that is quickly converted to thromboxane b 2 (txb2). in vascular tissue, pgh 2 is converted to pgi 2 or prostacyclin by prostacyclin synthetase (natarajan and nadler 2004; reimers 1999) . although cox-1 is constitutively expressed in most tissues, cox-2 is induced by bacterial lipopolysaccharides, il-i~, il-ll~, and tnf-~ (see the "cytokines and chemokines" section). in the circulation, pge 2 and pgi 2 cause vasodilation, whereas pgfza and txb 2 are potent vasoconstrictors. in the kidney, pge1, pge2, pgd2, pgg2, pgi2, and pgh2 produce vasodilation, increased renal blood flow, and urinary excretion of sodium. renal production is increased by pgd2, pge2, and pgi2. pgg2, pgh2, and txa 2 modulate platelet aggregation and, following platelet adhesion, they release catecholamines, serotonin, and adenosine diphosphate, which enhance platelet aggregation. pgi 2 is a potent inhibitor of platelet aggregation. pge1 increases, whereas pge2 decreases, the ability of red cells to pass through capillaries. pge2 and pgfza inhibit the activities of t and b cells and the production of ils and chemokines, which attract monocytes. pgd 2 is a potent inducer of chemotaxis for th 2 cells and plays a major role in allergic airway disease. through the varied activities of vasodilation, vascular permeability, and leukocyte migration, the pgs are potent modulators of inflammation. in addition pge1 inhibits mackay 2001 rossi et al. 1999 niess et al. 2005 mackay 2001 huang and xiang 2004 yamaguchi et al. 2005 insulin secretion and release after glucose challenge (see the "glucose and carbohydrate metabolism" section), and it inhibits the lipolytic effects of glucagons, adrenocorticotropic hormone, and epinephrine (see the lipid metabolism section) and inhibits the secretion of corticosterone, prolactin (prl), gh, thyroid stimulating hormone (tsh), and lh. in fact, pgs have a multitude of effects associated with female reproduction (reimers 1999 table 6 under the action of 5-lo, arachidonic acid is converted to 5-hydroxyperoxyeicosatetraenoic acid (5-hpete) and then leukotriene a4 (lta4), which is unstable. lta4 is metabolized to ltb4 by lta4 hydrolase or to cysteinyl leukotrienes (ltc4, ltd4h, and ltc45) by ltc4 synthase. 5-lo expression is limited to neutrophils, eosinophils, monocytes, and mast cells; however, lta4 hydrolase is expressed in erythrocytes, t cells, platelets, airway and intestinal epithelial cells, fibroblasts, heart, kidney, and adrenal cortex. because the latter cells and tissues do not express 5-lo, lta4 must be delivered to them via myeloid cells by a process known as transcellular biosynthesis (maclouf 1993) . receptors for ltb4 (blt1 and blt2) have been identified and are either widely expressed (blt2) or are confined to myeloid cells, t cells, and lung cells (blt1). in addition to attracting myeloid cells to sites of inflammation, ltb4 is a potent inducer of th1 and th2 t effector cell chemotaxis (as potent as cxcl12 in the mouse) and cd8+t-effector cell chemotaxis (as potent as rantes). like pgd2, ltb4 plays a major role in allergic airway disease (luster and tager 2004) . additional information on murine prostanoids and their receptors is available in recent reviews (jala and haribabu 2004; kobayashi and narumiya 2002) . all the arachidonic acid metabolites discussed in this section may be quantified using commercially available elisa kits (see table 6 -3). e. enzymes ap is an inducible enzyme in which serum activity is increased due to increased synthesis. the exact physiologic function of ap is not known but is thought to transport metabolites across cell membranes. quantification of serum ap is based on a reaction between ap and a suitable phosphorylated substrate that is susceptible to ap activity. as in other species, there are two major forms of ap in mice: intestinal ap (lap) and tissue nonspecific ap (tnap). unlike other domestic animal species, iap activity contributes to serum ap activity, in addition to tnap. lap is located along the brush-border of the enterocytes. intestinal ap activity was shown to vary four-fold between two strains of swiss mice (nayudu and moog 1967) . this difference in activity was under polygenic control and influenced by a strain-specific factor in milk. tnap is found in various tissues, and in each location, post-translation modifications may result in a different isoenzyme. hoshi et al. (1997) used immunohistochemistry to localize these isoenzymes in mice to the following locations: bone tissue (specifically the entire cell surface of preosteoblasts and the basolateral cell membrane of osteoblasts), cartilage (mostly in chondrocytes of the proliferative and hypertrophic zones), the incisors (particularly the cells of the stratum intermedium, the subodontoblastic layer, the proximal portion of secretory ameloblasts, and the basolateral portion of odontoblasts), kidneys (on the brush borders of proximal renal tubules in kidney), liver (on cell membrane of the biliary canaliculi), and the placenta (on trophoblasts). serum ap activity can vary due to the type of assay used, age, sex, and strain. different ap assays vary in the type of substrate used, the ph of the reaction, and the incubation temperature of the reaction, all of which can affect quantitation of ap activity. for example, the hausamen technique can detect renal and intestinal ap activity, but not hepatic ap activity (hausamen et al. 1967 ). loeb et al. (1996 ) and frith et al. (1980 demonstrated that serum ap activity decrease significantly after 3 months of age in balb/c and c57bl/6 mice of both sexes but increase again in very old (36 month old) mice. the high levels of serum ap seen in juveniles is related to increases in the bone ap isoenzymes, which is associated with osteoblastic activity due to rapid growth. picketing and picketing (1984) showed that serum ap activity is lower in males than in females. quantification of serum ap measures the total ap activity from all sources, and therefore elevations in ap activity can be a nonspecific determinant of tissue dysfunction, depending on the tissue (and therefore ap isoenzyme) involved. for example, changes in the serum activity of ap due to liver disease only occur if cholestasis is concurrently involved. mice lacking the gene that modifies tnap into the bone isoenzyme suffer from skeletal hypomineralization (anderson et al. 2004 ). alt is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. alt (also known as glutamic pyruvic transaminase [gpt]) reversibly catalyzes the conversion of alanine to pyruvate. quantification of serum alt is based on a reaction between alt and a suitable substrate (such as alanine). in mice, alt is found in the highest concentrations in the liver, although activity has also been demonstrated in intestine, kidney, heart, muscle, and brain (clampitt and hart 1978) . despite its widespread tissue distribution, alt is mostly used as an analyte to assess hepatocellular damage (masaki et al. 2005; taieb et al. 2005 ). an 11,000% increase in serum alt activity has been reported following infection with mouse hepatitis virus, and significant increases follow infection by helicobacter hepaticus (mccathey et al. 1997 ). ast is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ast (also known as glutamic oxaloacetate transaminase [got]) reversibly catalyzes the conversion of aspartate to oxaloacetate. quantification of serum ast is based on a reaction between ast and a suitable substrate (such as aspartate). in mice, ast is found in a variety of tissues, including liver, skeletal muscle, cardiac muscle, erythrocytes, blood vessels, brain, intestine, kidney, lung, testes (papadimitriou and van duijn 1970) . the highest specific activity of ast is found in mouse cardiac muscle and lowest in skeletal muscle (herzfeld and knox 1971) . in the liver, ast is found mainly in periportal hepatocytes based on histochemical studies (papadimitriou and van duijn 1970) . activity in lung, kidney, intestine, and skeletal muscle is very low when measured by the technique of bergmeyer and bernt (1974) . loeb et al. (1996) demonstrate significant age-associated increases in serum ast activity in two inbred and two f1 hybrid strains. although widely distributed, alt is mainly used to assess hepatocellular damage, cardiac muscle damage (naraoka et al. 2005; ray et al. 2005) , and testicular injury (santos et al. 2005) . alt activity has been used as an indicator of hepatic injury of mice infected with mouse hepatitis virus (fassati et al. 1969 ). ldh is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. ldh reversibly catalyzes the conversion of pyruvate to lactate. quantification of serum ldh is based on a reaction between ldh and a suitable substrate (such as pyruvate or lactate). as in other species, ldh in the mouse is a tetrameric enzyme consisting of either a or b subunits. there are five isoenzymes of ldh (based on differences subunit a and b composition), and these are ldh-1 (b4), ldh-2 (a1b3), ldh-3 (a2b2), ldh-4 (a3b1), and ldh-5 (a4). specific isoenzyme distribution depends on differential expression of either the a and b subunits (quimby 1999b) . for instance, all embryonic murine tissues contain ldh-5 because the a subunit is only expressed during early fetal development. as the embryo matures, subunit expression can involve both a or b subunits in different tissues, meaning that by birth and sexual maturity, each tissue contain a characteristic ldh subunit profile. in adult mice, the heart and erythrocytes contain ldh-1 and ldh-2, whereas most other tissues have ldh-3. skeletal muscle and hepatocytes fail to express subunit b and therefore are composed predominantly of ldh-5. serum ldh activity can vary due to age and sex. ldh levels have been shown to be higher in males versus females of the balb/c strain (frith et al. 1980) . serum ldh levels increase with age in balb/c and c57bl/6 mice of both sexes (frith et al. 1980) . serum ldh can also be elevated falsely by hemolysis. quantification of serum ldh measures the total ldh activity from all sources. however, damage to a particular tissue will result in increased activity of that isoenzyme in serum. in general, the highest activity of ldh in the mouse is in skeletal muscle, with decreasing activity in the heart, liver, kidney, and intestine. serum ldh-5 activity rises within 72 h after inoculating mice with the mouse hepatitis virus (fassati et al. 1969) . mice infected with the ldh virus (ldv) exhibit increased serum concentrations of ldh, isocitric dehydrogenase, malic dehydrogenase, phosphohexase isomerase, and ast (notkins 1965) . along with ast and ck, ldh is considered an excellent marker for cardiac injury (naraoka et al. 2005; ray et al. 2005) . otc is a mitochondrial enzyme in which serum activity is increased due to leakage across damaged mitochondrial membranes. otc is found primarily in the liver of mice, and there increases in serum activity reflect severe injury to hepatocytes. abnormal otc activity has been described in mice having the sparse-fur (spf/y) mutation. they serve as a model for the most common inborn error of urea synthesis in humans. assays for mouse otc have been developed to monitor activity levels following gene transfer or liver transplantation (batshaw et al. 1999; ye et al. 2001 ). ck is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. ck reversibly catalyzes the phosphorylation of adenosine diphosphate (adp) to ate using creatine phosphate as the donor for the phosphate group. quantification of serum ck is based on a reaction between ck and a suitable substrate (such as creatine phosphate). as in other species, cytoplasmic ck is a dimeric enzyme consisting of either m or b subunits. there are three isoenzymes of ck (based on differences subunit m and b composition), and these are ck-1 (bb), ck-2 (mb), and ck-3 (mm). brain contains ck-1, skeletal muscle contains ck-3 (mm), and cardiac muscle contains ck-1, ck-3, and ck-2 (mb) (quimby 1999b ). in the mouse, the greatest ck activity is found in skeletal muscle, with much less activity found in the heart and brain. mitochondrial ck (ck-mt) is found in mitochondria of many tissues. serum ck activity is affected by age, sex, and method of collection and anesthesia. patrick et al. (1983) found that compared to jugular vein collection, cardiac puncture was associated with lower ck activity. levels of serum ck activity have been reported for balc/cann mice and c57bl/10 mice of varying ages and sex (sub et al. 1994) . serum ck activity is a useful and specific marker enzyme of muscle injury, because ck in central nervous tissue does not cross the blood-brain barrier. sub et al (1994) compared normal c57bl/10 mice with heterozygous male and homozygous female mice carrying the mdx (mutant dystrophin) allele, and found that homozygous females have 12-to 15-fold increases and heterozygous males (mdx/y) have 30 fold increases in plasma ck compared to wild-type mice. plasma ck levels correlated with skeletal muscle necrosis in these dystrophic mice. mice have been engineered that lack cytoplasmic ck and ck-mt . mitochondria from heart or skeletal muscle from double kos had higher adp concentrations compared to wild-type animals, suggesting the higher concentrations contribute to the control of the reduced cytosolic atp free energy potentials seen in double kos. aldolase is a cytosolic enzyme that can alter its distribution between soluble and particulate forms, according to the metabolic status of the tissue. in adult mice, nine aldolase isoenzymes are known to occur in tissues with significant activities in the muscle, brain, liver, kidney, and spleen. in the mouse liver aldolase, together with fructokinase and triokinase, metabolize fructose (hagopian et al. 2005) . everett and harrison (1983) report no apparent advantages in mice in the measurement of aldolase over other enzymes known to have specific liver or muscle activity. sdh is a cytoplasmic and mitochondrial enzyme in which serum activity is increased due to leakage across damaged cytoplasmic and mitochondrial membranes. sdh (also known as iditol dehydrogenase [idh]) reversibly catalyzes the conversion of fructose to sorbitol. quantification of serum sdh is based on a reaction between sdh and a suitable substrate (such as fructose). sdh is located primarily liver, kidney, and seminal vesicles. the activity of sdh is usually low in the serum and rises during hepatic injury. however, the labile nature of sdh during handling makes is less suitable overall as a indicator of hepatic dysfunction compared to over liver-specific enzymes (such as ast). mice with the gene for sdh knocked out have been used to study the role of sorbitol accumulation in diabetic albuminuria (ii et al. 2004 ). amylase is a cytoplasmic enzyme in which serum activity is increased due to leakage across damaged cytoplasmic membranes. amylase hydrolyzes complex carbohydrates to form maltose and glucose in the presence of free calcium ions. quantification of serum amylase is based on a reaction between amylase and a suitable substrate (such as starch). similar to humans and pigs (but not dogs, cats, cattle, and horses), expression of amylase in mice is related to two distinct but closely linked loci (meisler et al. 1983) . salivary amylase is the gene product of amy-1 (salivary), and appears to be a single enzyme. pancreatic amylase is the gene product of amy-2, and based on electrophoretic studies in inbred mice, there appear to four isoenzyme classes: a1, a2, b 1, and b2. similar to other domestic animal species, pancreatic amylase is filtered through the glomerulus, but unlike other domestic species, pancreatic amylase is not resorbed by renal tubular epithelial cells and is excreted rapidly in the urine. therefore normal serum amylase activity in mice consists mainly of salivary amylase (mackenzie and messer 1976) . despite this, elevations in serum amylase activity is usually considered a reliable marker for pancreatitis in mice (nathan et al. 2005) . ross et al. (1974) reported two-to three-fold increases in serum amylase activity in mice infected with coxsackievirus of salivary and pancreatic trophism. alterations in the activity of specific pancreatic isoenzymes have been shown in streptozotocin-induced diabetes in mice (quimby 1999b) . there are two pancreatic lipase isoenzymes in mice. serum lipase activity has been used to monitor cerulean-induced acute pancreatitis in mice (cuzzocrea et al. 2004) . recently a new colipase-dependent lipase has been described in suckling mice (d'agostino and lowe 2004) . the enzyme 5'-nucleotidase was measured in the serum of normal mice using a simple one-step kinetic method (dooley and racich 1980) . a reference range of 10.9 + 4.5 (sd) u/1 has been recorded in 100 mice, and it is thought but not proven to be a good indicator of hepatic injury (clampitt and hart 1978) . glutamate dehydrogenase (gdh) has been measured in the tissues and serum of mice. gdh is known to play a key role in insulin secretion (carobbio et al 2004) . the activity of gdh is fivefold greater in the liver than in the kidney and brain, and the authors speculated that its measurement in serum would be a sensitive indicator of hepatic cell injury. serum gdh activity is also elevated in mice on caloric restriction (hagopian et al. 2003 ). corticosterone is the major glucocorticoid secreted by the adrenal cortex of mice. it functions as a regulator of carbohydrate, protein, and fat metabolism and modifies the host response to stress. the male mouse has a well-defined diurnal concentration pattern, with a maximum concentration of 9 ktg/dl at the start of the dark cycle and a minimum concentration of 5 btg/dl shortly before the end of the dark cycle (ottenweller et al. 1979) . in contrast, female mice have a minimum concentration of 13.5 ktg/dl at the beginning of the dark cycle and a maximum of 40 ~tg/dl well into the dark period (scheving et al. 1983) . the length of the dark cycle was different in each study. it may be measured using radioimmunoassay, elisa, or fluorometric assay in mouse serum or plasma. corticosterone circulates in both free and protein bound forms. in the mouse, greater than 99% circulates bound to cortisol-binding globulin (cbg) and albumin. the diurnal variation in corticosterone levels is paralleled by the diurnal pattern of cbg. urinary and salivary corticosterone is derived only from the free plasma fraction. corticosterone synthesis and secretion may be influenced by many drugs (woodman 1997) . measurements of corticosterone in unrestrained mice using indwelling catheters have elucidated the necessity of eliminating stress for accurate interpretation of data (macleod and shapiro 1988) . both handling and crowding laboratory mice can cause elevations in corticosterone (balcombe et al. 2004; fullwood et al. 1998) . c57bl/6 ob/ob mice have elevated corticosterone levels that increase markedly after 40 weeks of age, preceding elevations in serum glucose (garthwaite et al. 1980) . lean c57bl/6 controls had lower serum concentrations of corticosterone that declined between 5 and 20 weeks of age. food restriction (to 60% of ad libitum) profoundly affects the diurnal increase in plasma corticosterone in mice. at 20:00 hours (8 p.m.), the daily maximum in this study, food restricted balb/c mice had 300% more corticosterone compared to controls. these mice also had significantly reduced thymic weight and inflammation in response to the injection of carrageenan subcutaneously. the authors report that the magnitude of carrageenan-induced inflammation fluctuates with a diurnal trough, which coincides with peak corticosterone levels (klebanov et al. 1995) . corticosterone is synthesized in response to acth, which is made in the anterior pituitary. acth release is episodic (not at fixed intervals) and does not involve steroid feedback. acth concentration peaks in the early evening in mice and can be reversed by reversing the light cycle. acth is highly conserved with the mouse acth differing from human by only two amino acid residues. ria may be used to measure acth in the mouse (daily levels vary from 2.6-5.4 ng/ml) and to demonstrate rhythmic cycling. samples must be collected at 5-to 30-minute intervals. commercial elisa kits for acth are also available (quimby 1999b) ko mice incapable of synthesizing corticotrophin releasing hormone or acth may require corticosterone supplementation in drinking water and are particularly susceptible to hypoglycemia during fasting. fetuses of homozygous ko crh null mothers must be supplemented from gestational day 12 to weaning with 30 mg/ml corticosterone in drinking water (mugila et al. 1995) . oxytocin gene ko mice respond to a psychogenic stressor with more anxiety-related behavior and more corticosterone production (amico et al. 2004 ). lh promotes follicular development, increases estradiol secretion in the preovulatory follicle, causes follicular rupture, converts the preovulatory follicle into a corpus luteum, increases progesterone production by the corpus luteum, and, in the male, increases production of testosterone from leydig cells (woodman 1997) . lh is a glycoprotein composed of t~-and ~-chains. the amino acid sequence of the t~-chain is identical to that of follicle-stimulating hormone (fsh); however, the ~l-chain is specific and confers the receptor binding properties on the hormone. a homologous ria for rat lh using antisera to rat lh and purified rat lh has been employed to measure lh in mice (quimby 1999b) . lh and folliclestimulating hormone (fsh) are secreted by the same anterior pituitary cell in response to stimulation by gonadotrophinreleasing hormone (gnrh), which is synthesized in the hypothalamus. gnrh is identical in mouse and humans, and its differential stimulatory effect on gonadotropes, producing fsh or lh is controlled through gnrh pulse frequency. pulsatile secretion of lh leads to great variations in blood concentration. in female mice, there is a 10-to 25-fold increase in basal lh levels in the afternoon of proestrus. in a study comparing young cycling c57bl/6 female mice to old females (40% of which not cycling), flurkey et al. (1982) found lower lh levels in the older group and a more rapid rise in lh among younger cycling mice. these authors found that reproductive failure in male mice correlated with the loss of episodic release of lh. female homozygous ko mice lacking the gene for the immediate early transcription factor ngfi-a were infertile secondary to lh-[3 deficiency. although ovaries from these mice had a similar number of follicles, they lacked corpora lutea (lee et al. 1996) . basal levels of serum progesterone were lower in ngfi-a females (6.3 + 2.4 ng/ml) compared to wild type (11.3 +_ 3.6 ng/ml), whereas serum estradiol levels were similar in deficient (50.7 + 36.9 pg/ml) and wild-type (52.0 _+ 34.3 pg/ml) mice. decreased amounts of mrna encoding lh-[3, but not fsh-[3, were observed in ngfi-a-deficient mice; no changes were observed in mrna levels for prl or gnrh receptor between deficient and wild-type mice. an ovariectomy normally removes gonadal feedback inhibition, allowing for increased amounts of lh-~ and fsh-[3 in the pituitary; however, ovariectomy of ngfi-a deficient female mice lead to an increase in fsh-~ only. homozygous ngfi-a deficient males did make lower amounts of lh-~ compared to wild-type males, but they made significantly more lh-[3 than did ngfi-a-deficient females. the levels of lh in males appeared to be sufficient for fertility, and they had normal serum testosterone levels. these results suggest that ngfi-a acts synergistically with transcription factor sf-1 to regulate the promoter for lh-[3 gene expression. the simultaneous activation of both sf-1 and ngfi-a by gnrh appears to confer the specificity of lh-~ synthesis. female transgenic mice overexpressing lh are anovulatory, develop granulosa cell tumors, and undergo precocious puberty (mann et al. 2003) . excellent reviews detailing the use of transgenic and ko mice in elucidating the secretion and function of lh have been published (bums and matzuk 2002; huhtaniemi et al. 2002; wells and murphy 2003) . fsh stimulates the growth and maturation of ovarian follicles in the female and promotes the latter stages of spermatogenesis in males. it acts principally on the sertoli cells of the seminiferous tubules of the testis and induces the secretion of androgen-binding protein and inhibin. in females, fsh and lh have distinct secretory patterns that are synchronized to the estrous cycle; mouse estrous cycles have a 4-to 5-day periodicity. in addition to estrous cycle dependent rhythms, both lh and fsh have ultradian pulses and show circadian periodicity, with highest levels occurring during the dark period of the light cycle. testosterone, estrogen, and progesterone all provide feedback regulation of lh and fsh secretion (woodman 1997) . because rat and mice share considerable sequence homology for the fsh-~ chain, the rat ria kit from national institute of diabetes and digestive and kidney diseases (niddk) can be used to measure fsh in the mouse (dalterio et al. 1981) . a mouse specific ria is commercially available. in cycling female mice, the plasma levels of fsh increase from 80 to 120 ng/ml during proestrus and 250-300 ng/ml during estrus. although bronson and desjardins (1977) found age associated decreases in serum lh and testosterone in male cbf 1 mice experiencing decreased sperm production, no similar decreases were observed in fsh concentration. no significant differences in the twofold increased fsh levels were seen among young versus old males following castration. this appears to differ from results obtained from young versus old rats (finch et al. 1977) . the regulation and function of fsh has been studied in transgenic and ko mice (cooke and saunders 2002; wells and murphy 2003) . testosterone promotes spermatogenesis and provides feedback regulation of gonadotrophin synthesis. through its action on the epididymis, proteins necessary for spermatozoal maturation are synthesized. dihydrotestosterone (dht) promotes the growth and differentiation of accessory sex organs (depaolo and masoro 1989) . in addition to their effects on reproductive organs, testosterone and dht have physiologic functions on the central nervous system, cardiac tissue, and the liver. in the mouse, testosterone stimulates the growth of kidneys and synthesis of erythropoietin (woodman 1997) . lh stimulates synthesis of testosterone from cholesterol by the interstitial leydig cells of the testis. in target tissues, the enzyme 5r converts testosterone to dht. in the fetal testes, leydig cells first become identifiable during the rise of testosterone. in most mammalian species, leydig cells disappear when testosterone levels fall during gestation. however, the mouse is an exception and leydig cells do not undergo regression postnatally (aoki 1970) . testosterone and dht circulate in both free and protein bound forms. ninety-eight percent of total testosterone is bound to protein, mainly albumin. mice, unlike humans, dogs, monkeys, and cats, do not have circulating sex hormone-binding globulin. in mice, a pulse release pattern may be seen within their diurnal rhythm of testosterone. testosterone can be measured in mice using ria or elisa. plasma testosterone was not found to change during the average lifespan of c57bl/6 and dba/2j mice (finch et al. 1977) . this is in contrast to cbf1 mice, wistar and long-evans rats, and humans, which experience greater than a 30% decrease in testosterone at midlife (bronson and desjardins 1977) . in contrast to testosterone levels in older mice, castration-induced elevation of lh was impaired in 28-month-old c57bl/6j mice compared to 12-month-old mice. androgen receptor ko mice have been described (cooke and saunders 2002) . 5. estradiol (e2) the granulosa cells of the mature graafian follicle are the main source of 17[3-estradiol (e2) in mammals. fsh stimulates the activation of aromatase in follicles, which is responsible for the conversion of androgens to e2. e 2 is also synthesized by the testis, adrenal, liver, and skin, although in much lower amounts than by the ovary. e2 provides negative feedback control over lh secretion, and it stimulates prl secretion in mice. e 2 promotes the growth and development of the female reproductive tract, external genitalia, and the ductal system of the mammary glands. in addition, e 2 sensitizes the follicle to fsh. e 2 is measured in mice using ria and elisa. high circulating levels of e 2 precede the preovulatory surge in lh. ryan and schwartz (1980) reported basal levels of e 2 in mice of 1 to 5 pg/ml. holinka et al. (1978) studied circulating levels of progesterone and estradiol in young and old c57bl/6 female mice during gestation. they found that the old multiparous females have delayed and reduced preparturitional rise is plasma e 2 compared to young females. because preparturitional e 2 is thought to regulate uterine progesterone levels, the decline in e 2 seen in older females coupled with elevated progesterone may delay the onset of labor and lead to prolonged gestation. when sex steroid hormones such as e 2 bind their receptor, they induce a conformational change that allows the complex to bind dna response elements on nuclear target genes and associate with coactivators and transcription factors to form an active transcriptional complex. this complex is responsible for initiating the transcription of the target gene. one coactivator that associates with the active transcriptional complex involving sex hormones is steroid receptor coactivator-1 (src-1). to better understand its physiologic role during sex hormone-receptor binding, ko mice that were deficient in src-1 were created (xu et al. 1998 ). deficient male mice had a 34% reduction in testosterone-stimulated prostate growth and small testes compared to wild-type mice. deficient females had a half-normal uterine growth response to exogenous e 2 and only a partial ductal growth response (in the mammary gland) following exogenous e 2 and progesterone. serum concentrations of e 2 and testosterone were elevated 1.2 to 1.5 times wild-type levels, indicating an abnormality in the endocrine feedback control system. although src-1 was shown to clearly be necessary for optimal sex hormone-induced cellular activation, the lesion created in this ko mouse was not nearly as profound as that seen in mice with disrupted e 2 receptors (korach et al. 1996) . mice with the genes for the e 2 receptors and aromatase knocked out have been described (simpson et al. 2005 synthesized by the corpus luteum before implantation and by both corpus luteum and placenta following implantation, progesterone is necessary for preparing the uterus for implantation and maintaining pregnancy. in addition, a small amount of progesterone is synthesized by the adrenal gland. activation of the corpus luteum to secrete progesterone requires both lh and prl. progesterone provides negative feedback control over lh. in mice, progesterone is measured using ria or elisa. in cycling mice, the levels of progesterone increased from 5 to 35 ng/ml on proestrus and returned back to baseline on estrus. flurkey et al. (1982) compared the plasma levels of lh, progesterone, and prl in cycling young (10-week) and old (8month) female c57bl/6j mice. they showed age-related deficits in the preovulatory levels of all three hormones. the lh elevations during the ascending and descending portions of the preovulatory surge were smaller; the slower rises in lh during the ascending phase of the surge correlated with decreased progesterone in older females. there was no correlation between lh or progesterone level and length of estrus cycle. holinka et al. (1978) observed changes in plasma progesterone levels in young and old female mice during gestation. older mothers had a much slower decline in circulating progesterone than younger mothers between gestational days 17-23. because a major decrease in plasma progesterone is thought to be essential for the onset of uterine contractions and parturition in mice, the authors hypothesized that the attenuated decline in older mothers may account for their prolonged gestation. surgimoto et al. (1997) engineered mice lacking the pgfzareceptor (fp) and found that deficient female mice had normal estrous cycles, ovulation, fertilization, and implantation but did not undergo parturition. these pregnant females were characterized by a decline in preparturition progesterone levels due to delayed luteolysis and failure to develop labor. close examination revealed that near term there was no expression of the oxytocin receptor in the uterus of fp-deficient mice. when the ovaries of fp-deficient pregnant females were removed on day 19 of pregnancy, progesterone levels fell, uterine oxytocin receptors were expressed, and pups were born alive within 24 h. this study demonstrated that the role of pgf2~ is to initiate luteolysis, resulting in an immediate decline in progesterone levels. the subsequent induction of oxytocin receptors and their activation by bound oxytocin initiate labor. it is feasible that the age-associated prolongation of gestation in old female mice may involve a defect in pgfz,~-induced luteolysis. this mechanism for the induction of labor also explains the well-known observation that aspirin-like drugs (that inhibit cox metabolism) may delay parturition in women. prl, a 23-kda single-chain polypeptide, is principally made by the pituitary gland but may be also be found in brain, thymus, spleen, lymph nodes, mammary gland, myometrium, sweat gland, lacrimal gland, and bone marrow. more than 300 functions have been attributed to prl, involving reproduction and lactation, growth and development, endocrinology and metabolism, brain and behavior, immunomodulation, and electrolyte balance. it mediates effects by endocrine, paracrine, and autocrine mechanisms. in mice, prl is of major importance for maintenance of the structure, life span, and function of the corpora lutea and the development and maintenance of the mammary gland and lactation. however, prl-receptor ko males have no major defect in fertility. prl plays a role in anxiety-related behaviors, bone development, and abdominal fat deposition in both sexes (kelly et al. 2001) . its role in immune function is more controversial, although it appears to modulate immunity during times of stress (dorshkind and horseman 2000) . prl mediates its effects by binding the prl receptor (prlr). mice have two major forms of prlr that are created via alternative splicing of a single gene. the prlrs are members of the class i cytokine receptor superfamily. although most functions of prl are mediated by the unmodified 23-kda peptide, post-translational modification allows variants of prl to bind its receptor eliciting transcription of genes necessary for tissue specific changes (harris et al. 2004) . prl secretion is under inhibitory control by dopamine and among its secretagogues are thyrotropin-releasing hormone (trh), vasoactive intestinal peptide, gastrin, serotonin, ~-endorphin, oxytocin, angiotensin ii, gnrh, and arginine vasopressin. prl in mice is quantified using ria using the nih rp-1 reference standard. male mice measured during the light phase have <1 ng/ml whereas the range during the dark phase was 10-20 ng/ml (depaolo and masoro 989). oxytocin, a 21-kda nonapeptide, is synthesized as a prohormone in neurons whose bodies are located in the supraoptic and paraventricular nuclei at the base of the hypothalamus. once synthesized, the prohormone is packaged into neurosecretory granules and transported down the axons of these neurons transport synthesized oxytocin to the pars nervosa of the pituitary gland. during transport, the molecule is cleaved to yield the 10-kda carrier proteins, neurophysin i, and the l l-kda oxytocin. the primary stimulus for oxytocin release is mechanical stimulation of the mammary gland and distention of the reproductive tract (reimers 1999) . there is a single oxytocin receptor (otr). investigations using ko mice have shown that oxytocin is required for milk let down by the mammary gland as well as for postpartum alveolar proliferation. in males, oxytocin is required for normal spermiation and sperm transfer. in both genders, oxytocin helps control normal blood pressure and salt intake. kos display reduced aggression and a striking deficit in the ability to recognize a previously encountered mouse (young and gainer 2003) . oxytocin may be quantified in mice using commercially available elisa kits or by species-specific ria. values in one recent publication showed plasma oxytocin at 1.5 _+ 0.6 pg/ml in male c57bl/6 mice ). avp is synthesized and released by the same neurons previously described for oxytocin, although the two secretory granules rarely colocalize in the same neuron. the carrier protein for avp is neurophysin ii. avp is released from neuron secretory granules by electrical signals from osmoreceptors measuring the osmolarity of extracellular fluid. action potentials, generated in the receptors, cause calcium influx into axon terminals and exocytosis of ave avp is transported in the blood to the kidneys, where it binds receptors in the distal segment of the nephron and collecting ducts leading to increased resorption of water (reimers 1999) . there are three avp receptors, v l ar, vlbr, and v2r; and these are all g-protein coupled receptors. nonsense mutations of the avp gene (as seen in the brattleboro rat) or kd mice develop neurohypophysial (central) diabetes insipidus. mice with the v2r knocked out develop nephrogenic diabetes insipidus characterized by polyuria, polydipsia, and failure to concentrate urine. when the v1 ar gene is knocked out, mice develop an enhanced proliferation of splenic b cells and enhanced igg1 and igg2b production to thymicdependent antigens. when v lbr is knocked out the mice display a marked reduction in social aggression and a deficit in social recognition (young and gainer 2003) . avp can be measured by ria or elisa and the reported values in normal male c57bl/6 mice are 5.1 + 1.5 pg/ml in plasma ). tsh is a 28-kda glycoprotein made by the anterior pituitary gland on stimulation by thyrotropin-releasing hormone (trh). trh is made in the hypothalamus. tsh is secreted in a pulsatile manner, similar to acth. tsh secretion is regulated by triiodothyronine (t3), which acts on the hypothalamus to inhibit secretion of trh and acts on the pituitary gland to inhibit tsh synthesis (reimers 1999) . tsh plays a role in stimulating the thyroid gland to produce thyroxine and it influences the outcome of t-cell development in thymus and intestine. in addition to the thyroid gland, tsh receptors are found on many different populations of hematopoietic cells in bone marrow, subsets of dendritic cells, monocytes, and lymphocytes in the spleen and lymph nodes (klein 2003; scofield et al. 2005) . ko mice have been created in which genes for trh (wells and murphy 2003; yamada et al. 2003) and tsh receptors (biesiada et al. 1996) have been deleted. in addition, spontaneous mutations in the pitl gene lead to deficiencies of gh, prl, and tsh (yeap and leedman 1999) . tsh is measured by ria and reference ranges established around a 3.1 u/mg reference standard were 1-90 ng/ml (depaolo and masoro 1989). 11. thyroxine (t4) and triiodothyronine (t3) t 4 is synthesized entirely in the thyroid gland, whereas t 3 is produced primarily by conversion of t 4 to t 3 in extrathyroidal tissues. in the thyroid, thyroglobulin is synthesized and stored, and later hydrolyzed to form t 4. the tyrosine residues, held in peptide linkage within thyroglobulin, are first iodinated and later iodotyrosines are chemically coupled to form hormonally active t 4 and t 3. production of t 4 depends on adequate supplies of iodine. both t 3 and t 4 circulate in the blood primarily bound to albumin and m-globulin; however, mouse transthyretin does not bind t 4. approximately 85% of t3, the active thyroid hormone, in blood is made through the monodeiodination of the outer ring of t 4 in a variety of tissues. certain drugs, food deprivation, and illness can all effect monodeiodinase activity (reimers 1999) . t3 actions are mediated via two t3 receptors, tr~x and tr[3, which act as hormone-inducible transcription factors and belong to a large superfamily of nuclear hormone receptors including the steroid hormone, vitamin d, retinoic acid, and peroxisomal proliferator receptors (yen 2003) . in mouse and human tr~ and tr[3 encode nine mrna isoforms through alternative splicing; four isoforms tr~i, trc~2, tr[31, and tri]2, are expressed as proteins. trc~ ~ ko mice, which lack all products from the tr~ locus, are fertile and have normal basal thyroid status but have increased sensitivity to thyroid hormones in the pituitary and liver following provocative testing with increasing doses of t3. tr~x -/-have a disruption of the first coding exon in the tr~x locus, which prevents transcription of tr~i and try2 mrnas but not tra~i or tra~2. these pups die shortly after weaning unless supplemented with t3 for the first 2.5 months of life, after which they develop normal thyroxine levels. however, both genders are infertile. try2 -/-overexpresses tr~i and are hypothyroid but have inappropriately normal tsh levels. they exhibit some signs of hyperthyroidism, such as increased heart rate, weight loss, and elevated body temperature. tr~ -/-lack all tr[3 isoforms and display resistance to thyroid hormones demonstrating the key role of tr~ in set-point control of the pituitary-thyroid feedback axis. tr[32 -/-ko mice have resistance to thyroid hormones and elevated t3, t4, and tsh. they have defective tsh suppression by t3. several double kos (both tr~ and tri]) have been developed with profound resistance to t3. these targeted mutants have helped to elucidate the full function of thyroid hormones involving: bone formation and mineralization, abnormal development of skeletal muscle, disrupted development of the cones in the retina, abnormalities in the auditory system, cochlear and vestibular structures, delayed small intestine development, impaired thermogenesis, and altered development of the central nervous system and immune system (o'shea and williams 2002). both total t 4 and t3 can be measured by ria in mice, and reference ranges vary greatly by strain and age. although t3 is the active hormone, its serum levels are so low that it is a less reliable indicator of thyroid status. total t 4 ranges in swiss webster and icr mice are 5.5 _+ 0.7 and 4.7 + 0.3 jag/ml, respectively. total t3 levels range from 65-85 ng/100 ml in both sw and icr stocks (depaolo and masoro 1989). many factors, including autoantibodies, are known to interfere with thyroid hormone assays (despres and grant 1998; reimers 1999) . 12. parathyroid hormone (pth) pth is synthesized by the chief cells of the parathyroid gland and secreted as an 84 amino acid peptide. circulating levels of ionized calcium induce synthesis of pth. low calcium stimulates pth synthesis, and high calcium inhibits secretion. the primary function of pth is to control calcium concentrations in extracellular fluid and prevent hypocalcemia by increasing calcium resorption from bone, glomerular filtrate, and intestines (reimers 1999) . pth mediates its effect via its g-protein coupled receptor, pth1r. interestingly a second protein, pth related protein (pthrp), which is secreted by a variety of tissues and acts by autocrine and paracrine signaling, uses the same pth1r. pthrp modulates a wide range of physiologic and developmental responses (goltzman and white 2000) . mice with targeted mutations of the pth, pthrp, and pth1r genes have demonstrated the critical role of these proteins in regulating both the switch between proliferation and differentiation of chondrocytes and their replacement by bone cells (schipani and provost 2003) . serum levels of pth may be quantified in mice using a commercially available elisa kit, which can also be used to quantify the protein in rats. 13. other hormones elisa kits are commercially available to quantify many additional hormones in the serum of mice, including insulin, leptin, gh, epinephrine, orexin a, orexin b, adiponectin, adipsin, and resistin. each of these hormones have been briefly discussed in the "glucose and carbohydrate metabolism" section. the liver has many complex functions including protein metabolism, carbohydrate metabolism, and lipid metabolism. the liver is involved with the synthesis of many plasma proteins (including albumin), the conversion of ammonia to urea, and the production of glucose from glucogenic amino acids). the liver is implicated with the regulation of blood glucose levels (via glycogenolysis and gluconeogenesis, as discussed in the "glucose and carbohydrate metabolism" section), the removal of glucose from the blood via glut-1 and glut-2 membrane transporters, and storage in the form of glycogen. the liver is also involved in cholesterol and triglyceride synthesis, the formation of lipoproteins (discussed in the "lipid metabolism" section), and the synthesis of carbohydrates from fats. there are also other more specific functions that the liver facilitates, including the synthesis of heme, the synthesis of many coenzymes, detoxification/biotransformation of exogenous and endogenous substances via the cytochrome p-450 microsomal enzymes, and the synthesis of bile. the role of the liver in these functions for various animals has been reviewed (tennant 1999; woodman 1988 ). the elevation of plasma or serum enzymes usually confined to the cytosol or mitochondria of hepatocytes is helpful in elucidating liver injury. many factors influence the duration of elevated serum enzyme levels including molecular size, intracellular location, rate of plasma clearance, rate of enzyme inactivation, and hepatic production. hepatic necrosis is associated with elevations of alt, ast, sd, and otc in mice, and extrahepatic cholestasis is associated with elevated ap (tennant 1999) . please refer to the "enzymes" section for a description of each enzyme. bilirubin is a pigment that is produced by the degradation of the hemoglobin by cells of the mononuclear phagocyte system. there are two main types of bilirubin. unconjugated bilirubin is a non-water soluble molecule that is transported in blood bound to albumin. hepatocytes uptake unconjugated bilirubin, where it undergoes glucuronidation to produce the water-soluble form, conjugated bilirubin. conjugated bilirubin is then excreted via the hepatobiliary system and excreted in bile. quantification of serum bilirubin levels is based on the van den bergh or diazo reaction. diazo reacts directly with conjugated bilirubin, and with unconjugated bilirubin only after addition of alcohol to the reaction. therefore, measuring serum bilirubin involves the following steps. first, the level of conjugated bilirubin is measured. second, alcohol is added to the reactants, which allows quantification of total bilirubin levels. finally, the level of unconjugated bilirubin is determined by subtracting the conjugated measurement from the total measurement. the excretory capacity of the liver may be assessed by measuring serum bilirubin. elevated levels of unconjugated bilirubin are usually observed in situations of increased erythrocyte breakdown, such as in hemolytic diseases. unconjugated hyperbilirubinemia is also seen in disease in which hepatic uptake, conjugation, and secretion of bilirubin are diminished. increased levels of conjugated bilirubin are usually associated with intrahepatic cholestasis or extrahepatic bile duct obstruction. conjugated bilirubin in blood is normally filtered by the glomerulus in small amounts. conjugated hyperbilirubinemia may result in bilirubinuria (tennant 1999) . bilirubin clearance from blood and the role of the constitutive androstane receptor in this process has been studied in normal and transgenic mice (huang et al. 2003; . studies documenting the bilirubin-metabolism/detoxifying enzymes, their regulatory nuclear receptors, and lipid transporters in mice have been reported (wagner et al. 2005) . bile acids are cholesterol breakdown products that are secreted by hepatocytes into the hepatobiliary system, and ultimately into the intestinal tract. bile acids aid digestion by emulsifying dietary lipid aggregates, and solubilizing and transporting lipids in an aqueous environment (in particular fat-soluble vitamins). in the liver, bile acids also play a role regulating the secretion of apolipoprotein b (elzinga et al. 2003) . extensive enterohepatic recirculation results in almost 99% return of bile acids secreted by the liver from the intestinal tract. absorption occurs mainly in the ileum under the influence of the apical na ⧠bile acid transporter on epithelial cells (kida et al. 2003) . some absorption also takes place in the large intestine. absorbed conjugated bile acids (from ileum) pass unaltered to the portal circulation where they are removed by na+-taurocholate cotransporting polypeptide located on the basolateral hepatocyte membrane (jung et al 2004) . there are two types of bile acids, cholic acid and chenodeoxycholic acid. in mice, they are conjugated with taurine before secretion by the liver. the liver-specific enzymes, bile acid coa ligase and bile acid-coa:amino acid n-acyltransferase, are responsible for conjugation (inoue et al. 2004 ). stedman et al. (2004) described normal serum bile acid levels in normal and transgenic mice. elevated serum bile acid levels in mice is usually due to decreased bile acid recycling by the liver, and mainly includes diseases associated with decreased hepatic functional mass and cholestasis (tennant 1999) . hoda and green (2003) measured increased levels of serum bile acids after bile duct ligation. albumin is a nonglycosylated protein and is the most abundant protein in plasma. it serves as the most important determinant of plasma oncotic pressure, and it is a major transport protein for both endogenous metabolites and xenobiotics. it is made exclusively by the liver. serum albumin may be measured by radial immunodiffusion, dye binding reactions (using bromcresol green or bromcresol purple), electrophoresis, and elisa. serum albumin levels decrease with age in many inbred strains (quimby 1999b) . hypoalbuminemia is usually reflective of decreased hepatic synthesis (due to hepatic disease, inflammation, and malabsorptive/maldigestive diseases), increased loss due to hemorrhage or via the intestinal tract (protein-losing enteropathies) and kidneys (protein-losing nephropathies) (tennant 1999) . most of the proteins associated with coagulation are synthesized by the liver (except factor viii), and measurement of fibrinogen or prothrombin time has served as a marker for decreased synthesis due to hepatic injury (tennant 1999) . prothrombin time will also be increased due to consumption of clotting components and in vitamin k deficiency. fibrinogen is also an acute phase reactant, and plasma elevations are seen during inflammation (tennant 1999) . fibrinogen may be quantified in mouse plasma by elisa. ceruloplasmin is copper-containing acute phase reactant and iron transport protein made exclusively in hepatocytes of mice. its serum levels may be decreased during hepatic injury and increased in response to inflammatory stimuli (min et al. 1991; shim and harris 2003) . many of the complement components are also made in the mouse liver, especially c2, c3, c4, and factor b. decreased circulating levels may be seen due to hepatic injury or due to consumption during activation. please refer to the "complement" section for further details on complement function and measurement. two dyes, sulfobromophthalein and indocyanine green, have been used to assess hepatocellular or bile tract function. following an intravenous bolus, these dyes are rapidly cleared from the plasma by hepatocytes and excreted into the bile. both dyes have been used to assess liver function in mice (hurwitz et al. 1994; huang and vore 2001) . the kidney plays a complex role in maintaining homeostasis in the body and is involved in such functions as water and electrolyte balance, nutrient conservation, maintenance of blood ph, and removal of the end products of nitrogen metabolism (such as urea, creatinine, and allantoin). in addition, the kidney produces and responds to a variety of hormones. however, assessing renal function is complicated by the large functional reserve of the kidneys. for instance, increases in urea nitrogen do not occur until 70-75% of renal mass has become functionally compromised. additionally, assessment of analytes in urine is difficult due to the small size of mice (as discussed in the "sampling" section). urea is produced in the liver as a waste product of protein catabolism (specifically a breakdown product of ammonia). urea is a small molecule that freely diffuses across cell membranes, and therefore the urea concentration is the same in blood, serum, and plasma. traditionally, urea concentrations is measured in terms of urea nitrogen (the amount of nitrogen contained within urea). determination of urea nitrogen is usually made from serum, but whole blood can be used (hence the term blood urea nitrogen [bun] ). quantification of urea nitrogen is based on spectrophotometry assays, measuring the amount of urea that is hydrolyzed by urease. frith et al. (1980) investigated urea nitrogen levels in balb/c and c57bl/6 mice. urea nitrogen levels decreased after 3 months of age but increased again after 12 months in both sexes. in balb/c mice, levels were higher in males than females, whereas in c57bl/6 mice, females had significantly higher levels than males. elevated urea nitrogen (azotemia) can be caused by prerenal, renal, and postrenal conditions. prerenal causes include increased protein catabolism (such as with inflammation, starvation, and high-protein diets). renal causes usually are associated with conditions that compromise more than 70-75% of functional renal mass and include conditions such as renal amyloidosis, glomerular immune complex disease, and polycystic disease. postrenal include any cause that results in obstruction of the lower urinary system. decreased urea nitrogen is found with disease associated with hepatic insufficiency and low-protein diets. creatinine is a degradation product of creatine and creatine phosphate and represents an end-product of muscle metabolism. quantification of creatinine is based on spectrophotometry assays. baseline serum levels are directly related to muscular conditioning and total muscular mass, which varies between individuals. pathologically elevated serum creatinine levels are caused by the same prerenal, renal, and postrenal causes that elevate urea nitrogen in serum. therefore, quantification of serum creatinine offers no interpretative advantage over urea nitrogen (everett and harrison 1983) . proteinuria is a common finding in normal mice, and includes rodent-specific proteins such as uromucoid, small quantities of c~-and [3-globulins, and a family of prealbumins known as major urinary protein (mup). the mup has three electrophoretic variants, designated as mup-1, mup-2, and mup-3, that are under both genetic and hormonal control. a regulatory locus designated mup-1 with codominantly expressed alleles a and b (located on chromosome 4) controls the urinary levels of the three variants. the mup is synthesized in the liver, secreted into blood, and excreted into urine. males are have higher levels of proteinuria than are females, with levels of 5 mg/ml, and age-related increases are seen in mice of both sexes. increases in other urinary proteins have been associated with a variety of renal diseases in mice. the concentration of urine (amount of solutes dissolved in urine) can be measured by urine specific gravity (usg) or osmolality. urine concentration in healthy mice is very high. watts (1975) determined the usg of healthy cba mice, which ranged from 1.060 to 1.080. the author also found the usg increased from 20 to 80 days of age. the urine concentrating ability of transgenic mice expressing human sickle cell hemoglobin (kos for mouse hb) was assessed by ryan et al. (1997) . they found that sickled cells caused vascular, tubular, and glomerular changes, as well as corresponding hyposthenuria (4-h water deprived osmolality of affected mice was 807 + 285 mosm compared to 1541 + 360 mosm in controls). i. electrolytes 1. sodium, potassium, chloride, and phosphorus sodium and potassium levels are easily measured in murine serum using flame photometry (lithium reference) or ion-specific electrodes. serum sodium levels are slightly higher in mice than in most other mammalian species, with reported values of 174 + 23 (sd) meq/1 (finch and foster 1973) , 147 + 15 (sd) meq/l (everett and harrison 1983) , and 155-161 meq/1 (loeb et al. 1996) . no differences in serum sodium were seen during aging, between sexes, or among strains. serum chloride has been measured using mercuric thiocyanate and the chloridimetric or ion-specific electrode techniques, and inorganic phosphorus in mice has been measured using the phosphomolybdate technique. serum inorganic phosphorus levels decreased as mice aged between 1 and 12 months. this change was documented in balb/c and c57bl/6 strains, as well as in outbred mice (loeb et al. 1996) . total serum calcium reflects both ionized (active calcium) and protein-bound calcium (mainly bound to albumin). ionized calcium is biologically active in bone formation, neuromuscular activity, cellular biochemical processes, and blood coagulation. decreased serum albumin levels are also expected to decrease total calcium levels, by decreasing the amount of protein-bound calcium. however, hypoalbuminemia does not result in clinical signs of hypocalcemia. in mice, total serum calcium has been measured using the sodium alizarin sulfonate technique or atomic absorption spectrometry. two reports, using different techniques, list similar reference ranges of 9 + 1 (sd) mg/dl, whereas a third report lists reference ranges of 5.6 + 0.4 (sd) mg/dl for male and 7.4 + 0.50 (sd) mg/dl for female albino mice. the latter values reported for male mice were significantly lower than those for six inbred strains of mice in two different age-groups using the same techniques (quimby 1999b) . likewise, no differences associated with sex were reported by everett and harrison (1983) , who also examined random bred albino mice. however, bonella et al. (1968) demonstrated significantly higher levels in female swiss albino mice. serum calcium levels appear to decline between 3 and 12 months of age in the balb/c and c57bl/6 strains as well as in outbred mice . frith et al. (1980) found that female c57bl/6 mice had lower calcium levels than males. bonella et al. (1968) demonstrated significantly elevated calcium levels when blood was collected by orbital puncture versus cardiac puncture. hypercalcemia in mice can results as a response to increased levels of pth (such as with hyperparathyroidism) or pthrp (such as with certain neoplasms like multiple myeloma). in mice, the response of parathyroid chief cells to hypercalcemia appears different from other species. grone et al. (1992) studied this response in nude mice with pthrp secreting tumor cells or infusions of pthrp, and found prominent membranous whorls and increased cytoplasmic area of chief cells in mice with hypercalcemia (17.0 + 3.1 mg/dl), which marked these cells as distinctly different from parathyroid chief cells of other species. hypocalcemia can be due to hypoalbuminemia (as previously discussed), renal disease, pancreatitis, and hypomagnesemia. alcock and shils (1974) found that mice fed magnesium-deficient diets developed hypocalcemia, as did mice receiving intramuscular injections of heparin. total serum protein has been evaluated in mice using either the lowry or biuret methods, although the lowry method is preferred for analysis of small volume samples. hypoproteinemia is seen in hepatic injury (also see the "liver function tests" section) or during protein-losing enteropathies or nephropathies. hyperproteinemia is seen during dehydration. age-related changes have been described in a number of strains, such as increased total serum protein in older balb/c and b6 mice and lower levels in dba/2 mice (loeb et al. 1996) . each of the major classes of serum protein ((xl-, (x2-, ~-, and y-globulins) in mice can be distinguished by electrophoresis. changes in serum levels have been associated with a wide variety of diseases in mice. for instance, hypergammaglobulinemia was found in scid mice injected with human cag multiple myeloma cells . the acute phase reactants ceruloplasmin and fibrinogen have been discussed in the "serum proteins" section and crp and sap in the "complement" section. saa is an acute phase reactant synthesized by the liver and may be induced by the inflammatory cytokines il-1 or il-6 (see the "cytokines and chemokines" section). normally saa is quickly cleaved to a small molecular weight product, but during chronic infection or failure in the degradation pathway, tissue deposition of amyloid may occur. differences in serum concentrations have been described in various mouse strains and during disease. commercial ria and elisa kits are available for its quantitation in mice (quimby 1999b) . alpha-1-fetoprotein (afp) is encoded by the afp gene which is nearly identical in its organization to the mouse albumin gene, albl. it is likely afp arose due to gene duplication. afp is present in fetal serum but the synthesis declines shortly after birth. serum levels are regulated by two loci, afr-1 and afr-2, which are not linked to afp. the action of these genes may be modified by tgf-~ and p53 (wilkinson et al. 2005) . levels in mouse serum have been quantified by immunoelectrophoresis (zizkovsky 1975 ) and both polyclonal antibodies and recombinant dna-derived mouse afp are available for assay development (boismenu et al. 1997) and are elevated in mice with certain neoplasms and during hepatic regeneration (jin et al. 2005; quimby 1999b ). sulphostin, a novel inhibitor of dipeptidyl peptidases iv (dppiv) that stimulates hematopoiesis in mice proteomic approaches to biomarker discovery in prostate and bladder cancers 8th annual analyzers buyer's guide il-17: prototype member of an emerging cytokine family increased atherosclerosis in hypeflipidemic mice with inactivation of abca-1 in microphages diet-induced changes in plasma phospholipids transfer protein activity, lipids, and lipoproteins. mpd: 801, 802, 828. mouse phenome database web site comparison of magnesium deficiency in the rat and mouse apoc-1 and apoc-iii as potential plasmatic markers to distinguish between ischemic and hemorrhagic stroke comparison of methods for obtaining blood from mice anxiety and stress responses in female oxytocin deficient mice impaired calcification around matrix vesicles of growth plate and bone in alkaline phosphatase-deficient mice hormonal control of leydig cell differentiation interleukin-7 an interleukin for rejuvenating the immune system a spectrophotometric microtiter-based assay for the detection of hydroperoxy derivatives of linoleic acid mast cell lineage development and phenotypic regulation laboratory routines cause animal stress regulation of fasting blood glucose by resistin biochemistry of immunoglobulins variations in serum calcium between strains of inbred mice correction of ureagenesis after gene transfer in an animal model and after liver transplantation in humans with ornithine transcarbamylase deficiency biology of the congenitally hypothyroid hyt/hyt mouse the chemokine stromal cell-derived factor-1 regulates the migration of sensory neutron progenitors methods of enzymatic analysis the mouse phenome project purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in escherichia coli genetically engineered animals in drug discovery and development: a maturing resource for toxicologic research effects of heparin on serum calcium in mice albumin content in blood of inbred mice strains lentiviral shrna silencing of murine bone marrow cell ccr2 leads to persistent knockdown of ccr2 function in vivo trophic action of leptin on hypothalamic neurons that regulate feeding the interpretation of serum biochemistry test results in domestic animals transgenic models of autoimmune disease lipoprotein metabolism and atherosclerosis susceptibility in transgenic mic mouse models of atherosclerosis reproductive failure in aged cbf male mice: interrelationships between pituitary gonadotropic hormones, testicular function, and mating success stromal cell derived factor 1/cxcl12 selectively counteracts inhibitory effects of myelosuppressive chemokines on hematopoieteic progenitor cell proliferation in vitro minireview: genetic models for the study of gonadotropin actions recruitment of cxcr3 and ccr5 t-cells and production of interferon-gamma inducible chemokines in rejecting human arteries tietz fundamentals of clinical chemistry distribution and characterization of the serum lipoproteins and apolipoproteins in the mouse, mus musculus disparate 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dehydrogenase, and aspartate aminotransferase and their isoenzymes as indicators of the development of experimental hepatitis in mic the endoplasmic reticulum is the site of cholesterol-induced cytotoxicity in macrophages niemann-pick c heterozygosity confers resistance to lesional necrosis and macrophage apoptosis in murine atherosclerosis hematologic and serum electrolyte values of the c57bl/6j male mouse in maturity and senescence hormone production by the pituitary and testes of male c57bl/6j mice during aging age effects of luteinizing hormone, progesterone, and prolactin in proestrus and acyclic c57bl/6j mice innovation~stagnation: challenge and opportunity on the critical path to new medical products interleukin-17 hematologic and clinical chemistry findings in control balb/c and c57bl/6 mice pi3k and negative regulation of tlr signaling visfatin: a protein secreted by visceral fat that mimics the effects of insulin floor space needs for laboratory mice: c57bl/6males in solid-bottom cages with bedding a longitudinal hormonal profile of the genetically obese mouse return to normal of blood-glucose, plasma insulin, and weight gain in new zealand obese mice after implantation of islets of langerhans acute phase proteins ghrelin: more than a new frontier in neuroendocrinology tumor necrosis factors a rapid, simple, and humane method for submandibular bleeding of mice using a lancet palatal cytosol cortisol-binding protein associated with cleft palate susceptibility and h-2 genotype relationship in the functional levels of early components of complement to the h-2 complex of mice developmental and tissue-specific regulation of parathyroid hormone (pth)/pth-related peptide receptor gene expression ccl1-ccr8 interactions: an axis mediating the recruitment of t-cells and langerhans-type dendritic cells to sites of atopic skin inflammation measurement of glycosylated haemoglobins and glycosylated plasma proteins in animal models with diabetes or inappropriate 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luteinizing hormone reception function loperamide effects on hepatobiliary function, intestinal transit, and analgesia in mice redox state-dependent and sorbitol accumulationindependent diabetic albuminaria in mice with transgene-derived human aldose reductase and sorbitol dehydrogenase deficiency hepatocyte nuclear factor 4 alpha is a central regulator of bile acid conjugation genetic association between h-2 gene and testosterone metabolism in mice use of transgenic mice in carcinogenicity hazard assessment leukotrienes and atherosclerosis: new roles for old mediators endothelial lipase and hdl metabolism targeted mutation of plasma phospholipids transfer protein gene markedly reduces high density lipoprotein levels genetic heterogeneity of lipoproteins in inbred strains of mice: analysis by gel permeation chromatography afp gene expression after acute diethylnitrosamine intoxication is not afr2 regulated identification of tumor-associated plasma biomarkers using proteomic techniques: 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between the development of the intestinal iga immune system and the establishment of microbial flora in the digestive tract of young holoxenic mice split activity of interleukin-10 on antigen capture and antigen presentation by human dendritic cells: definition of a maturative step genetic susceptibility and resistance to diet-induced atherosclerosis and hyperlipoproteinemia chemokines; multiple levels of leukocyte migration control initial sequencing and comparative analysis of the mouse genome oral tolerance in the absence of naturally occurring tregs structure, binding, and antagonists in the il-4/il-13 receptor system corticotropinreleasing hormone deficiency reveals major fetal, but not adult, glucocorticoid need diet effects on bone mineral density and content, body composition, and plasma glucose, leptin and insulin levels. mpd: 143. mouse phenome database web site evaluation of h-fabp as a marker of ongoing myocardial damage using hgh transgenic mice lipid inflammatory 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chemistry values of the n:nih(s) mice and parameter variations due to sampling techniques use of proteomic patterns in serum to identify ovarian cancer lessons from kitty hawk: from feasibility to routine clinical use for the field of proteomic pattern diagnostics alkaline phosphatase activity of the mouse immune regulation in the intestine boosted decision tree analysis of surface enhanced laser desorption/ionization mass spectral serum profiles discriminates prostate cancer from non-cancer patients animal models in biomedical research complement the mouse transgenic models of tolerance and autoimmunity: with special reference to systemic lupus erythematosus short-term and long-term in vivo exposure to an ephedra-and caffeine-containing metabolic nutrition system does not induce cardiotoxicity in b6c3f1 mice model models of atherosclerosis hormones type 2 diabetes: a matter of beta-cell life and death leukemia inhibitory factor and interleukin-ll: cytokines with key roles in implantation virus induced pancreatic disease; alterations in concentrations of glucose and amylase in blood lungkine, a novel cxc chemokine, specifically expressed by lung bronchoepithelial cells changes in serum hormone levels associated with male-induced ovulation in group housed adult female mice ko transgenic mouse model of sickle cell disease animal models of autoimmunity and their relevance to human diseases hematological comparison of the mouse blood taken from the eye and tail cystatin c as a potential cerebrospinal fluid marker for the diagnosis of creutzfeldt-jakob disease efficacy of 2, 3-dimercapto-l-propanesulfonic acid (dmps) and diphenyl diselenide on cadmium induced testicular damage in mice multimeric cytokine receptors: common versus specific functions bilateral lesions in the suprachiasmatic nuclei affect circadian rhythms and h-thymidine incorporation into deoxyribonucleic acid in mouse intestinal tract, mitotic index of corneal epithelium, and serum corticosterone pthrp, pth, and the pth/pthrp receptor in endochondral bone development diabetes, obesity, and the brain hematopoietic, immunomodulatory and epithelial effects of interleukin-11 intestinal tsh production is localized in crypt enterocytes and in villus 'hotblocks' and is coupled to il-7 production: evidence for involvement of tsh during acute enteric virus infection adipsin, a biomarker of gastrointestinal toxicity mediated by a functional gamma-secretase inhibitor loci controlling plasma non-hdl and hdl cholesterol levels in a c57bl/6j x casa/rk intercross quantitative trait loci mapping of genetic modifiers of metabolic syndrome and atherosclerosis in low-density lipoprotein receptor-deficient mice the kinase lkb 1 mediates glucose homeostasis in liver and therapeutic effects of metformin genetic defects in copper metabolism estrogen: the good, the bad, and the unexpected il-1 and il-18 receptors, and their extended family b-cell-and monocyte-activating chemokine (bmac), a novel 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and in vivo the protein profile of acetazolamidetreated sera in mice bearing lewis neoplasm cebs object modes for systems biology data, sysbio-om partal hormone resistance in mice with disruption of the steroid receptor coactivator-1 (src-1) gene platelet factor 4 gene transfection into tumor cells inhibits angrogenesis tumor growth and metastasis mice lacking the thyrotropin-releasing hormone gene: what do they tell us? differences in the human and mouse amino-terminal leader peptides of ornithine transcarbamylase affect mitochondrial import and efficacy of adenoviral vectors combined pituitary hormone deficiency: lessons from the murine models molecular basis of resistance to thyroid hormone a mouse bleeding technique yielding consistent volume with minimal hemolysis transgenesis and the study of expression, cellular targeting, and function of oxytocin, vasopressin and their receptors kos model the 100 best-selling drugs: will they model the next 100? predicting drug efficacy: kos model pipeline drugs of the pharmaceutical industry three biomarkers identified from serum proteomic analysis for the detection of early stage ovarian cancer cxc chemokine ligand 129 stromal cell-derived factor 1 alpha and cxcr4-dependent migration of ctls toward melanoma cells in organotypic culture il-ii: insights in asthma from overexpression transgenic modeling genotype, obesity, and cardiovascular disease: has technical and social advance outstripped evolution? specific fetal serum proteins of 13 mammalian species t3 t4 tace taj tc tg tgf-i3 tgf-~isf th tlr tnap tnf tnfrsf tnfsf tof tpc trance trh tsh tx upr usg vldl serum amyloid a serum amyloid protein standard deviation stromal cell-derived factor-1 sorbitol dehydrogenase surface-enhanced laser desorption/ionization secondary lymphoid tissue chemokine stearoyl-coa desaturase-1 suppressor of cytokine signaling-3 steroid receptor coactivator-1 sj6gren's syndrome single-strand deoxyribonucleic acid triiodothyronine thyroxine tumor necrosis factor-a converting enzyme toxicity and jnk inducer cytotoxic t-cells triglyceride transforming growth factor-j3 transforming growth factor-13 superfamily t-helper toll-like receptors tissue nonspecific alkaline phosphatase tumor necrosis factor tumor necrosis factor receptor superfamily tumor necrosis factor superfamily time-of-flight total plasma cholesterol tumor necrosis factor-related activation-induced cytokines thyrotropin-releasing hormone thyroid-stimulating hormone thromboxane unfolded protein response urine specific gravity very low-density lipoprotein key: cord-003013-h8txbd3p authors: kim, sena; joe, yeonsoo; surh, young-joon; chung, hun taeg title: differential regulation of toll-like receptor-mediated cytokine production by unfolded protein response date: 2018-04-24 journal: oxid med cell longev doi: 10.1155/2018/9827312 sha: doc_id: 3013 cord_uid: h8txbd3p the ability of the host immune response is largely mediated by the proinflammatory cytokine production. physiological and pathological conditions of endoplasmic reticulum (er) trigger unfolded protein response and contribute to the development or pathology of inflammatory diseases. under er stress, unfolded protein response (upr) signaling pathways participate in upregulating inflammatory cytokine production via nf-kappab, mapk, and gsk-3β. moreover, it has been suggested that er stress crosstalks with toll-like receptor (tlr) signaling pathway to promote the production of proinflammatory cytokines. in addition, tlr stimulation can lead to upr activation to promote inflammation. in this review, we will cover how proinflammatory cytokine production by upr signaling can be induced or amplified in the presence or absence of tlr activation. the endoplasmic reticulum (er) is a crucial site involved in maintaining cellular functions, such as synthesis, modification, releases and translocation of proteins, biosynthesis of steroids, cholesterol and other lipids, metabolism of carbohydrates, and storage of calcium [1] [2] [3] . numerous physiological and pathological conditions including imbalance in the er folding capacity, accumulation of misfolded proteins, altered cellular metabolism, hypoxia, oxidative stress, infection, disruption of er calcium ion balance, or n-linked glycosylation, can trigger the er stress and initiate the unfolded protein response (upr) to restore er homeostasis and ensure cell survival [2, 4] . however, if the er stress cannot be resolved, the upr will initiate er stress-associated programmed cell death to protect the organism by removing the stressed cells. when er stress occurred in a cell, three individual er-resident transmembrane branches of upr begin with the dissociation from er chaperon bip/grp78, followed by homodimerization and autophosphorylation of protein kinase rna-like endoplasmic reticulum kinase (perk) and inositol-requiring enzyme 1α (ire1α) to activate the cytoplasmic kinase domains [5, 6] . in contrast, activating transcription factor 6 (atf6) is translocated to the golgi apparatus and then activated via proteolytic cleavage [7] . activated perk phosphorylates eif2α, which transiently attenuates global mrna translation, therefore reducing protein flux into the er. interestingly, certain mrnas contain small orfs in their 5′utr such as activating transcription factor 4 (atf4) can escape inhibition of translation. as a sustained translational inhibition is not compatible with cell survival, atf4 induces gadd34, a regulatory subunit of protein phosphatase (pp1) acting as regulator of phosphorylation of eif2α, to restore mrna translation. atf4 also induces the expression of the transcription factor ddit3/chop, which is involved in er stress-mediated apoptosis. ire1α has functions of endoribonuclease and serine-threonine kinase. an endoribonuclease activity of ire1α is a specific splicing of the xbp1 mrna, which allow the translation of the spliced xbp1 (xbp1s) transcriptional factor. xbp1s has a major role in the induction of a wide variety of chaperones or proteins involved in proteinrefolding, er-associated degradation system, lipid metabolism, and proinflammatory responses [8] . after dissociation from bip, atf6 is transported via coat protein copii-covered vesicles to the golgi compartment, where it undergoes intramembrane proteolysis by the golgi enzyme site 1 protease (s1p) and s2p to produce active n-terminal fragment that translocate to the nucleus. active n-terminal fragment patf6-n directly induces the expressions of er capacity and folding-related genes (such as grp78, grp94, gadd153, and xbp1) ( figure 1 ). toll-like receptor (tlrs) can recognize pathogenassociated molecular patterns (pamps) and dangerassociated molecular patterns (damps) and induce tlrmediated intracellular signaling cascades to eliminate the pathogens through the production of proinflammatory cytokines including tnf-α, il-6, il-1β, and il-8, but its uncontrolled activation can damage the host [9] . sustained proinflammatory cytokine productions often contribute to the development of many inflammatory and autoimmune diseases. upon ligands binding to tlrs, tlrs recruit adaptor proteins, myeloid differentiation primary response 88 (myd88), and/or tir domain-containing adapterinducing interferon-β (trif) and transduce signals through interleukin-1 receptor-associated kinase (irak), tnf receptor-associated factor (traf), tgf-β-activated kinase 1 (tak1), and receptor-interacting protein 1 (rip1). in general, the myd88-dependent response mediates the induction of proinflammatory cytokine, whereas the trif-dependent response mediates the induction of type 1 ifn response. traf3 interacts with both myd88 and trif, but it differentially regulates mapk signaling pathway and type i ifn signaling pathway, respectively [10] . tlr2 and 4 activation elicit traf3 ubiquitination which is the key to selective proinflammatory cytokine production through mapk activation or type i ifn response through interferon regulatory factor 3 (irf3). traf3 ubiquitination by traf6 and ubiquitin ligase ciap1/2 resulted in mapk activation and induction of proinflammatory cytokines in a myd88-dependent manner [11] . blockade of traf3 ubiquitination inhibits proinflammatory cytokine production through suppression of mapk activation without anti-inflammatory cytokine production, il-10, and type i ifn responses. thus, myd88-dependent signaling cascades by tlr stimulation result in the activation of nf-kappab and mapk, which are the central mediators of cytokine production ( figure 1 ). er stress has been shown to regulate proinflammatory cytokine production, which are mediated by tlr signaling cascade components such as nf-kappab, mapk, and gsk-3β. studies have demonstrated that various metabolic syndromes are associated with chronic metabolic inflammation and impairment of er function and established a link between inflammatory responses through the tlr signaling and er stress response [12] [13] [14] [15] . in recent studies on inflammatory diseases due to er stress, inflammation was observed in models of tunicamycin-induced acute liver failure and various cancers [16, 17] . these reports may indicate the link between er stress and inflammation, but the regulation of tlr-mediated proinflammatory cytokine production by upr signaling is not completely understood. ozcan et al. found a mechanism of er stress-induced inflammation in white adipose tissue in high-fat diet-induced obese mice [18] . according to a study, it was provided that ire1α aggravates inflammation and the phenotypes of obesity through the switching of m1-m2 macrophage polarization in a cell-autonomous manner [19, 20] . in addition, er stress shares tlr-mediated signaling components with pro-and anti-inflammatory cytokine productions, leading to the activation of nf-kappab and mapks. the xbp1 deletion or chemical chaperone treatment in macrophages alleviates proinflammatory cytokine production by lps. in this review, we discuss er stress and tlr-mediated signaling pathways for the regulation of proinflammatory cytokine production that linked er stress to inflammation. upr is an important modulator in the induction of inflammatory cytokines, and upr activation was shown to be sensitive to inflammation [21] . it has been well established that nf-kappab activation is required for the induction of proinflammatory cytokines and has been linked to upr [8, [22] [23] [24] . the perk-induced inhibition of translation results in decreased translation of iκb, which is a negative regulator of the nf-kappab, therefore leading to greater activation and translocation of nf-kappab transcription factor to the nucleus [25, 26] . a kinase activity of ire1α directly triggers iκb phosphorylation in a traf2-dependent manner, which results in the activation of nf-kappab [27] . atf6 also activates nf-kappab via phosphorylation of the akt [28] . these results indicate that upr is sufficient to induce the proinflammatory mediator production such as il-6, il-1β, tnf-α, and il-8 through nf-kappab activation. mapks (jnk, p38, and erk) are also important inflammatory signaling molecules that induce inflammatory cytokines in response to er stress [29, 30] . chen et al. showed that hiv protease inhibitors (pis) induce proinflammatory cytokines, tnf-α, and il-6 through er stress-mediated erk activation, and these effects are diminished in chop knockout macrophages [31] . thus, chop is responsible for hiv pi-induced erk activation and proinflammatory cytokine production. the ire1α can activate jnk in a traf2dependent manner, leading to the increased expression of proinflammatory cytokines through activator protein 1 (ap1) [32] . mijosek et al. demonstrated that er stress induces phosphorylation of erk, p38, and jnk through mainly perk and atf6 pathways in human primary bronchial epithelial cells resulting in increased expressions of il-6 and il-8 [33] . mapk signaling pathways are involved in regulating the expression of inflammatory cytokines and er stress-induced inflammatory cytokine productions that are also dependent upon the mapk activation ( figure 1 ). tlr in proinflammatory cytokine production for the eradication of invading microbial pathogens. fritz et al. demonstrated that tlr4 and nod1 and nod2 agonists lead to promote the production of proinflammatory cytokine in human monocytes and dendritic cells [34] . roles of tlrs and nod1/nod2 on innate immune response have been proposed, but the nod1/nod2 signaling during er stress-induced inflammation is not clear. under er stress condition, ire1α branch in er transmembrane is oligomerized and autophosphorylated. activated ire1α binds traf2 (tnf receptor-associated factor 2) to induce proinflammatory cytokine production via nf-kappab signaling [35] . yan and liu showed that chemical er stress inducers, thapsigargin and dithiothreitol, induced proinflammatory cytokine il-6 in a nod1/2-dependent manner. in addition, brucella abortus strain rb51 induces il-6 production through traf2-, nod1/2-, and rip2-dependent signaling pathway and could be abolished by er stress inhibitor, tauroursodeoxycholic acid (tudca), or an ire1α kinase inhibitor. recently, yan and liu demonstrated that leucine-rich repeat kinase 2-(lrrk2-) dependent nod1 activation by er stress has a new positive regulator of rip2 in inducing inflammatory cytokine in macrophages [36] . therefore, these studies suggest that nod1/2 activation with tlr is a new mechanism for er stress-induced proinflammatory cytokines. inflammasome regulates il-1β and il-18 expression and maturation [37] . studies have revealed that upr-induced nf-kappab activation and ros generation are responsible for il-1β and il-18 secretions [38] . kim and il-1β production in rb51-infected or er stress inducertreated bmdm, but not lps + atp-treated bmdm [41] . brucella abortus strain rb51 induces immune signaling through the induction of er stress. notably, er stressinduced mitochondrial damage was required for the noncanonical nlrp3 inflammasome activation, which was mediated by ire1α, caspase-2, bid, and mitochondrial content release (mitochondrial-derived damage associated molecular patterns, mtdmap) in an asc-independent manner. overall, er stress regulates il-1β and il-18 production during chemical stress or microbial infection through canonical and noncanonical inflammasome activation. numerous studies have demonstrated that gsk-3β is a key regulator of inflammatory cytokine production in er stress and tlr signaling [42, 43] . gsk-3β was found to strongly promote the production of proinflammatory cytokines by tlr-induced myd88-dependent and myd88-independent pathways, and the inhibition of gsk-3β was shown to protect the host from several inflammatory diseases such as colitis, arthritis, and sepsis-induced organ failure [43] . studies by martin et al. reported that stimulation of monocytes or peripheral blood mononuclear cells with tlr2, tlr4, tlr5, or tlp9 agonists induces substantial increases in il-10 production while suppressing the release of proinflammatory cytokines after gsk-3β inhibition [42] . in addition, lps-induced gsk-3β activation induces nf-kappab activation and inhibits c/ebpβ, creb, and ap-1 transcriptional activities, diminishing the production of the antiinflammatory cytokine, il-10. moreover, the inhibition of gsk-3β suppresses stat3 activity resulting in reduced levels of il-6 in lps-treated mice and lps-cultured primary glial cells [44, 45] . er stress was also associated with increased activity of gsk-3β through the reduction of serine phosphorylation and promotion tyrosine phosphorylation by akt inhibition and ire1α activation, respectively [44, 46, 47] . the pi3k-akt pathway has been shown to negatively regulate proinflammatory cytokine production through gsk-3β inactivation [44] . guha and mackman showed that the inhibition of pi3k-akt pathway enhances lps-induced tnf-α production through mapks (erk, p38, and jnk) and nuclear translocation of nf-kappab which induces the transactivation of p65 through gsk-3β activation [48] . likewise, the inhibition of gsk-3β with licl reduces lps-induced tnfα in pbmcs and thp-1 monocytic cells, suggesting that gsk-3β positively regulates proinflammatory cytokine, tnf-α, through the reduction of transactivation of p65 without nuclear translocation of nf-kappab. therefore, the activation of gsk-3β by er stress-mediated pi3k-akt inhibition enhances the nf-kappab-dependent inflammatory cytokine production. recently, kim et al. demonstrated that gsk-3β has a role in inducing inflammatory cytokine during er stress [49] . the authors showed that the production of the proinflammatory cytokines il-1β and il-6 is triggered by the er stress inducers thapsigargin and tunicamycin or lps in a gsk-3β-dependent manner. however, tnf-α is regulated by ire1α-mediated xbp1 splicing, independently of gsk-3β ( figure 2 ). these findings provide evidence for previously uncharacterized functions for gsk-3β on er stress in the regulation of the immune response. recent studies have shown that er stress influences toll-like receptor-mediated intracellular signaling cascades involved in the activation of innate and adaptive immune responses [21, 50, 51] . mahadevan et al. demonstrated that tlr4 ko bmdm treated with er stress inducers shows decreased production of proinflammatory cytokines (il-6, il-23p19, and tnf-α) compared with wt bmdm [52] . on the other hand, tlr2 ko bmdm exposed to er stress shows no decreased production of proinflammatory cytokine and increases the production of mip-1α, mip-1β, and mcp-1, suggesting that tlr2 may normally function as a negative regulator in response to er stress-induced proinflammatory cytokine responses. on the other hand, shimasaki et al. showed that er stress enhances tlr2-dependent proinflammatory cytokine production (tnf-α, il-8, and il-6) through atf4mediated tlr2 upregulation [53] . these data suggest that er stress-related proteins affect proinflammatory cytokine production through modulating tlr signaling. toll-like receptor-mediated activation of intracellular signaling pathways results in increased production of proinflammatory cytokines including tnf-α, il-6, and il-1β. similarly, the activation of tlr ligands affects upr signaling pathways. microbial infection and tlr ligand treatment lead to the induction of upr-related protein activation and gene expression, suggesting that upr appears to closely interact with host immune response [54] [55] [56] . it was demonstrated that tlr2 and tlr4 activate ire1α-xbp1 axis without perk phosphorylation and atf6 activation and promote the production of proinflammatory cytokines [57] . in addition, xbp1-deficient macrophages reduce il-6 production in response to tlr agonists or infection with pathogen [8] . these evidences suggest that tlr-mediated ire1α activation is intimately linked with inflammatory signaling pathways. several reports have shown that the concomitant treatment of tlr ligands and er stress-inducing chemicals synergize the production of proinflammatory cytokines (ilβ and il-6) [33, 57] . this amplified cytokine production can be regulated at the level of both the transcription and translation. according to some reports, this synergism depends on the p38, erk, and gsk-3β activations. mijosek et al. demonstrated that er stress-mediated p38 and erk activation is able to boost the production of inflammatory cytokines such as il-6 and il-8 in tlr-(tlr4, tlr3, and tlr5) stimulated airway epithelial cells. this synergic effect is mainly mediated by the activation of p38 and erk via perk and up-regulated p38 expression via atf6 [33] . on the other hand, pharmacological activation of xbp1 or overexpressing xbp1s with the er stress inducers synergistically augments tlr-mediated il-6 and tnf-α productions. in addition, kim et al. showed that er stress inducer significantly augments lps-induced proinflammatory cytokines (i.e., tnf-α, il-1β, and il-6) and gsk-3β activity in raw264.7 macrophages and bmdm [49] . rao et al. demonstrated that kupffer cells isolated from 4-pba-treated ischemic liver or atf6-downregulated kupffer cells from ischemic liver produce significantly less tnf-α and il-6 after stimulation with lps [58] . thus, atf6 activation in ischemic liver induces enhancement of proinflammatory cytokine production of macrophage in response to tlr4. thus, the er stress and tlr activation synergize the production of proinflammatory cytokines. some reports have shown that immune-enhancing drugs can boost the immune response to protect septic patients with the later immunosuppressive stage. given that er stress can restore cytokine production under endotoxin tolerance, it may be helpful to use er stress induction to increase the cytokine production in the immune-depressed state. thus, it is possible that er stress under endotoxin tolerance condition might restore the immune capability to defend the host from infection. indeed, er stress inducers enhance clearance of bacteria through recovery of immune response under endotoxin tolerance condition (unpublished data). thus, er stress figure 2 : interactions of downstream pathways of ire1α for the transcriptional regulation of il-1β and tnf-α in response to er stress. er stress-induced ire1α activation differentially regulates il-1β and tnf-α through gsk-3β activation and xbp1 splicing, respectively. ire1αmediated gsk-3β activation induces transcription of il-1β but inhibits xbp1 splicing. thus, sb216763, a gsk-3β inhibitor, selectively inhibits il-1β gene expression and increases tnf-α production in response to er stress. in contrast, ire1α-mediated xbp1 activation results in the transcription of tnf-α. stf083010, ire1α rnase inhibitor, suppresses tnf-α production without affecting il-1β production. in addition, activation of xbp1 by er stress inducers synergistically augments lps-mediated tnf-α production. likewise, gsk-β activation by er stress inducer augments lps-mediated il-1β. the inflammatory response due to er stress is frequently observed in the development of nonmalignant immunological disorders, such as rheumatoid arthritis and neurodegenerative diseases [12] . evidences have shown that er stress enhances tlr-induced intracellular cascades to produce proinflammatory cytokines (table 1) . however, more research is needed to understand the role of er stress in host immune responses and to exploit this knowledge to design new drugs for patients with various inflammatory and metabolic diseases. overall, this review emphasizes that the er stress-induced inflammatory cytokine productions are shared with tlr-mediated signaling pathways and taking advantage of er stress may be used as therapeutic option to prevent inflammatory diseases and protect secondary infection in septic patients through recovery of immune responses. the authors declare that there are no conflicts of interest regarding the publication of this article. chapter five -endoplasmic reticulum and the unfolded protein response: dynamics and metabolic integration endoplasmic reticulum stress and unfolded protein response in infection by intracellular parasites the role of er stress in lipid metabolism and lipotoxicity misfolded proteins, endoplasmic reticulum stress and neurodegeneration the unfolded protein response, degradation from the endoplasmic reticulum, and cancer endoplasmic reticulum stress, unfolded protein response, and cancer cell fate activation of mammalian unfolded protein response is compatible with the quality control system operating in the endoplasmic reticulum the interplay between endoplasmic reticulum stress and inflammation inhibition of toll-like receptor signaling as a promising therapy for inflammatory diseases: a journey from molecular to nano therapeutics different modes of ubiquitination of the adaptor traf3 selectively activate the expression of type i interferons and proinflammatory cytokines essential cytoplasmic translocation of a cytokine receptor-assembled signaling complex endoplasmic reticulum stress and the inflammatory basis of metabolic disease endoplasmic reticulum stress and inflammation in obesity and diabetes xbp1 links er stress to intestinal inflammation and confers genetic risk for human inflammatory bowel disease s-nitrosylation links obesityassociated inflammation to endoplasmic reticulum dysfunction endoplasmic reticulum stress-activated glycogen synthase kinase 3β aggravates liver inflammation and hepatotoxicity in mice with acute liver failure liver inflammation and metabolic signaling in apc min/+ mice: the role of cachexia progression chemical chaperones reduce er stress and restore glucose homeostasis in a mouse model of type 2 diabetes the metabolic er stress sensor ire1α suppresses alternative activation of macrophages and impairs energy expenditure in obesity er stress promotes inflammation through re-wired macrophages in obesity emerging functions of the unfolded protein response in immunity er stress-induced inflammation: does it aid or impede disease progression a molecular web: endoplasmic reticulum stress, inflammation, and oxidative stress from endoplasmicreticulum stress to the inflammatory response endoplasmic reticulum stress pathway perk-eif2α confers radioresistance in oropharyngeal carcinoma by activating nf-κb er stress activates nf-κb by integrating functions of basal ikk activity, ire1 and perk a new paradigm: innate immune sensing of viruses via the unfolded protein response activation of the akt-nf-κb pathway by subtilase cytotoxin through the atf6 branch of the unfolded protein response er stress depresses nf-κb activation in mesangial cells through preferential induction of c/ebpβ the unfolded protein response in immunity and inflammation hiv protease inhibitor lopinavir-induced tnf-α and il-6 expression is coupled to the unfolded protein response and erk signaling pathways in macrophages coronavirus infection, er stress, apoptosis and innate immunity endoplasmic reticulum stress is a danger signal promoting innate inflammatory responses in bronchial epithelial cells synergistic stimulation of human monocytes and dendritic cells by toll-like receptor 4 and nod1-and nod2-activating agonists nod1 and nod2 signalling links er stress with inflammation lrrk2 enhances nod1/2-mediated inflammatory cytokine production by promoting rip2 phosphorylation il-1β processing in host defense: beyond the inflammasomes dysregulated il-1β secretion in autoinflammatory diseases: a matter of stress? endoplasmic reticulum stress is sufficient for the induction of il-1β production via activation of the nf-κb and inflammasome pathways endoplasmic reticulum stress activates the inflammasome via nlrp3-and caspase-2-driven mitochondrial damage allosteric inhibition of the ire1α rnase preserves cell viability and function during endoplasmic reticulum stress toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3 glycogen synthase kinase 3β in toll-like receptor signaling glycogen synthase kinase 3: a point of convergence for the host inflammatory response ifn-γ suppresses il-10 production and synergizes with tlr2 by regulating gsk3 and creb/ ap-1 proteins central role of glycogen synthase kinase-3β in endoplasmic reticulum stress-induced caspase-3 activation endoplasmic reticulum stress-induced apoptosis is partly mediated by reduced insulin signaling through phosphatidylinositol 3-kinase/akt and increased glycogen synthase kinase-3β in mouse insulinoma cells the phosphatidylinositol 3-kinase-akt pathway limits lipopolysaccharide activation of signaling pathways and expression of inflammatory mediators in human monocytic cells endoplasmic reticulum stress-induced ire1α activation mediates cross-talk of gsk-3β and xbp-1 to regulate inflammatory cytokine production the molecular chaperone grp78 contributes to toll-like receptor 3-mediated innate immune response to hepatitis c virus in hepatocytes cellular stress response and innate immune signaling: integrating pathways in host defense and inflammation transmission of endoplasmic reticulum stress and pro-inflammation from tumor cells to myeloid cells endoplasmic reticulum stress increases the expression and function of toll-like receptor-2 in epithelial cells bacteria, the endoplasmic reticulum and the unfolded protein response: friends or foes? diverse roles of endoplasmic reticulum stress sensors in bacterial infection initiation of innate immune responses by surveillance of homeostasis perturbations tlr activation of the transcription factor xbp1 regulates innate immune responses in macrophages atf6 mediates a proinflammatory synergy between er stress and tlr activation in the pathogenesis of liver ischemia-reperfusion injury key: cord-005983-2ascbu62 authors: eigler, a.; loher, f.; endres, s. title: suppression der synthese des tumornekrosefaktors date: 2001 journal: internist (berl) doi: 10.1007/s001080050721 sha: doc_id: 5983 cord_uid: 2ascbu62 klinische studien aus den vergangenen 5 jahren belegen die zentrale rolle des tumornekrosefaktors (tnf) im pathophysiologischen geschehen der rheumatoiden arthritis und des morbus crohn. die vorliegende arbeit gibt einen überblick über die regulation der synthese und die vielfältigen wirkungen dieses zytokins. so wurden erhöhte tnf-konzentrationen bei verschiedenen infektiösen und entzündlichen erkrankungen sowie in verbindung mit speziellen therapien nachgewiesen. darauf aufbauend werden experimentelle therapeutische strategien zur hemmung der tnf-bildung dargestellt. klinische studien aus den vergangenen 5 jahren belegen die zentrale rolle des tumornekrosefaktors (tnf) im pathophysiologischen geschehen der rheumatoiden arthritis und des morbus crohn.die vorliegende arbeit gibt einen überblick über die regulation der synthese und die vielfältigen wirkungen dieses zytokins.so wurden erhöhte tnf-konzentrationen bei verschiedenen infektiösen und entzündlichen erkrankungen sowie in verbindung mit speziellen therapien nachgewiesen.darauf aufbauend werden experimentelle therapeutische strategien zur hemmung der tnf-bildung dargestellt. tumornekrosefaktor · rheumatoide arthritis · morbus crohn · zytokine · signaltransduktion der tumornekrosefaktor-α (tnf-α) nimmt eine dominierende rolle im so genannten zytokinnetzwerk und damit in der pathogenese zahlreicher infektiöser und entzündlicher erkrankungen ein. tnf wurde nach seiner fähigkeit benannt, bei versuchstieren in transplantierten tumoren eine hämorrhagische nekrose zu verursachen [1] . zehn jahre nach beschreibung dieser eigenschaft wurde tnf-α 1985 aufgereinigt und sequenziert [2] und das gen von verschiedenen arbeitsgruppen parallel kloniert [3, 4, 5] . seither sind zahlreiche biologische eigenschaften dieses zytokins identifiziert worden, zusätzlich zur lyse von tumorzellen und zur induktion von kachexie, die ursprünglich zur erkennung von tnf geführt hatten [6] . schon im jahre 1893 war von dem new yorker chirurgen coley beobachtet worden, dass schwere infektionen in manchen fällen zur regression eines malignen tumors führten [7, 8] [9] . die tnf-wirkungen werden durch quervernetzung membrangebundener rezeptormoleküle vermittelt (tnf-rezeptor i, tnfri, p55; tnf-rezeptor ii, tnfrii, p75; [10] ). in der tnfri-knockout-maus wurde eine resistenz gegenüber dem endotoxinschock demonstriert, andererseits war eine infektion mit listeria monocytogenes letal [11] . die extrazellulären anteile beider tnf-rezeptoren liegen auch als lösliche form im serum vor, können weiterhin tnf binden und damit die akuten wirkungen des tnf abschwächen [12, 13] . unter bestimmten bedingungen konservieren diese natürlichen tnf-inhibitoren jedoch auch tnf [14, 15, 16] . nach bindung an die membranständigen rezeptoren übt tnf eine reihe unterschiedlicher wirkungen aus (abb. 1). die signaltransduktion distal des tnf-rezeptors wird unter anderem von phospholipasen c [17] und sphin-gomyelinasen vermittelt, die ceramide aus sphingomyelin freisetzen und ceramidabhängige proteinkinasen aktivieren [18] . nachdem die gabe von anti-tnf-antikörpern in tierexperimenten des septischen schocks positive ergebnisse erbracht hatten, wurden hohe erwartungen an eine therapie des septischen schocks beim menschen geweckt. diese wurden jedoch nicht erfüllt. dagegen wurden bei chronisch entzündlichen erkrankungen -der rheumatoiden arthritis und dem morbus crohn -eindrucksvolle erfolge mit anti-tnf-antikörpern und mit tnf-rezeptorfusionsproteinen erzielt. auf diese studienergebnisse gehen 2 weitere arbeiten in diesem heft näher ein. die empfindlichste methode, um zirkulierendes tnf nachzuweisen, basiert auf der extrem hohen bindungsaffinität und -spezifität des p55-tnf-rezeptors. bei gesunden probanden konnte mit dieser methode kein tnf im plasma nachgewiesen werden [19] . die nachweisgrenze dieses assays ist 200 attomolar (10 -18 mol/l), das entspricht 120.000 tnf-trimeren oder 10 femtogramm in 1 ml plasma. im gegensatz dazu werden bei akuten erkrankungen wie dem septischen schock tnf-konzentrationen im nanomolaren bereich (10 -9 mol/l) nachgewiesen. die synthese des tnf wird in monozyten und makrophagen durch unterschiedliche exogene substanzen wie z. b. endotoxin und β-glukane sowie auch durch endogene mediatoren wie il-1 induziert. hohe konzentrationen von tnf im plasma konnten bei zahlreichen infektiösen und entzündlichen erkrankungen nachgewiesen werden (tabelle 1). hohe konzentrationen von tnf wurden auch in therapieassoziierten syndromen nachgewiesen wie der gabe von interleukin-2 (il-2) zur therapie von soliden tumoren und nach der applikation von anti-cd3-antikörpern zur therapie der akuten abstoßungsreaktion nach organtransplantation. die hemmung der bildung [20] oder der aktivität [21] von tnf während der gabe von anti-cd3-antikörpern schwächt deren nebenwirkungen ab. ein nutzen durch tnf-antagonismus erscheint somit grundsätzlich bei den in der tabelle 1 aufgeführten erkrankungen möglich. [22] . thalidomid verkürzt die halbwertszeit der tnf-rna [23] . antisense-oligonukleotide führen zu einer spezifischen hemmung der tnf-translation [24] . dabei müssen jedoch spezielle experimentelle bedingungen geschaffen werden, da oligonukleotide sonst auch zu einer induktion der tnf-synthese führen können [25] . in den folgenden abschnitten wird näher auf strategien zur hemmung der tnf-synthese eingegangen, die im tierexperiment oder schon beim menschen in klinischen studien untersucht worden sind. im jahre 1988 konnten kunkel et al. [26] erstmals zeigen, dass substanzen, die zyklisches adenosinmonophosphat erhöhen, die tnf-synthese in murinen makrophagen hemmen. diese stoffe umfassen phosphodiesteraseinhibitoren, die den abbau von camp verhindern, und prostaglandine, die die adenylylzyklase über g-proteine aktivieren und auf diese weise zu einer verstärkten camp-bildung führen. darüber werden die campabhängigen proteinkinasen a aktiviert, was in einer phosphorylierung von zielproteinen wie etwa camp-responsiveelements (cre)-bindungsproteinen mündet. diese transkriptionsfaktoren binden an spezifische sequenzen in der promotorregion bestimmter gene. eine entsprechende cre-spezifische sequenz wurde in der 5'-flankierenden region des tnf-gens beschrieben [27] . die expression des tnf ist weiterhin abhängig von der aktivierung des transkriptionsfaktors nf-κb (nuclear factor-κb). mehrere nf-κb-bindende regionen wurden in der promotorregion des tnf-gens identifiziert [28] . nf-κb ist physiologischer weise an seinen spezifischen inhibitor iκb im zytosol gebunden. im falle einer aktivierung dissoziiert dieser komplex, und nf-κb gelangt in den zellkern. die aktivierung von nf-κb kann durch antioxidantien gehemmt werden [29] . eine hemmung der interaktion des nf-κb mit seinem motiv wurde für pentoxifyllin [30] berichtet, einem unspezifischen phosphodiesteraseinhibitor. unter den klinisch eingesetzten phosphodiesterasehemmern ist pento-xifyllin die substanz, die am eingehendsten bezüglich der tnf-hemmenden wirkungen untersucht wurde. so wurden patienten, die anti-cd3-monoklonale-antikörper zur behandlung einer akuten transplantatabstoßungsreaktion erhielten, zur hemmung der nebenwirkung durch tnf mit pentoxifyllin behandelt [20, 31] . als interessante alternative zum einsatz des unspezifischen phosphodiesterasehemmstoffes pentoxifyllin, erscheint der spezifische typ-iv-phosphodiesteraseinhibitor rolipram, der 500-mal wirksamer die tnf-synthese hemmt [32] . die typ-iv-phosphodiesterase überwiegt in monozyten [33] und bildet daher einen guten angriffspunkt für die hemmung camp-abhängiger funktionen dieser zellen. außerdem konnte für die kombination von rolipram mit prostaglandinen (prostaglandin e2 und prostazyklinanaloga) eine synergistische wirkung in bezug auf die camp-erhöhende [34] und die tnf-hemmende wirkung [35] gezeigt werden. diese synergie könnte relevant für den einsatz von rolipram bei lokalen entzündungsprozessen mit hohen interstitiellen konzentrationen an prostaglandin e2 sein. die entzündungshemmende wirkung von camp-erhöhenden substanzen wird jedoch nicht nur über eine hemmung der tnf-synthese vermittelt. so wird z. b. auch das antiinflammatorische zytokin il-10 durch camp-erhöhung verstärkt exprimiert [36, 37] . mehrere tierexperimentelle arbeiten belegen die wirksamkeit einer spezifischen hemmung der phosphodiesterase in vivo. in einem rattenmodell der experimentellen autoimmunenzephalo[38] . die therapeutische wirkung wurde in einem primatenmodell (marmoset-affen) der autoimmunenzephalomyelitis bestätigt [39] . weiterhin konnte durch die therapie mit rolipram die suppression der tnf-synthese und ein überlebensvorteil in einem rattenmodell des ards gezeigt werden [40] . in einem mausmodell der kollageninduzierten arthritis konnte rolipram eine hemmung der tnf-synthese und einen therapieerfolg erzielen [41] . die autoren konnten die wirksamkeit von rolipram in einem maus-kolitis-modell nachweisen [42] . rolipram wurde in den frühen 1980er jahren synthetisiert [43] , als antidepressivum in klinischen studien untersucht, aber nicht bis zur marktreife gebracht. rolipram wurde nie beim menschen mit der absicht untersucht, die tnf-synthese zu hemmen. derzeit werden von mehreren pharmazeutischen unternehmen spezifische typ-iv-phosphodiesterasehemmstoffe entwickelt oder bereits in klinischen studien eingesetzt. überwiegend werden im rahmen dieser studien patienten mit asthma bronchiale untersucht. erstmals 1991 wurde gezeigt, dass ein teil der antiinflammtorischen wirkung von thalidomid (früher contergan ® ) über eine hemmung der tnf-synthese (abb. 3) vermittelt wird [44] . thalidomid wurde bei mehreren erkrankungen eingesetzt, die mit erhöhten tnf-konzentrationen einhergehen. so liegen arbeiten zur therapie der chronischen graft-versus-host-erkrankung,der chronischen polyarthritis, der sarkoidose und des hiv-assoziierten wasting-syndroms vor (übersicht in [45] ). zwei kürzlich erschienene offene klinische studien zur gabe von thalidomid bei patienten mit steroidabhängigem m. crohn beschrieben ein klinisches ansprechen schon nach 4 wochen in 58% und 55% der behandelten patienten. in einem kritischen editorial wurden diese ergebnisse vorsichtig positiv bewertet und ein möglicher einsatz bei patienten gesehen, die infliximab nicht vertragen [45] . eindringlich wurde jedoch aufgrund der bekannten teratogenität von thalidomid vor einem unkritischen einsatz gewarnt. die gabe von thalidomid darf nur unter strengsten kontrollmaßnahmen erfolgen und auch unter dieser vorgabe erst nach vorliegen von positiven ergebnissen von randomisierten, kontrollierten klinischen studien. interleukin-10 wurde ursprünglich als ein produkt einer bestimmten untergruppe von t-helferzellen (th2-zellen) identifiziert, das die proliferation, entwicklung und funktion einer anderen t-helferzellgruppe (th1-zellen) hemmt [46] . il-10 wird von verschiedenen zellen wie b-zellen, b-zell-lymphomen, monozyten, keratinozyten und mastzellen gebildet. entsprechend des ursprünglichen namens -zytokinsynthese-inhibierender faktor -hemmt il-10 die produktion verschiedener proinflammatorischer zytokine, insbesondere tnf und il-1 in monozyten und makrophagen. ein protektiver effekt für il-10 konnte in einem mausmodell der letalen endotoxinämie gezeigt werden.auch in einem kolitismodell der maus wurde ein therapieerfolg mit il-10 erreicht [47] . in gesunden probanden hemmt il-10 die ex-vivo-synthese proinflammatorischer zytokine. nach endotoxingabe bei freiwilligen probanden wurde eine verminderte tnf-konzentration nach ilob und welche der hier aufgezeigten strategien zur hemmung des tnf klinische relevanz erlangen werden, ist derzeit noch nicht abzusehen. insbesondere die meist nur mündlichen berichte von gehäuftem auftreten von lymphomen und karzinomen unter therapie mit anti-tnf-antikörpern dämpfen die euphorie, die diese innovative therapieform ausgelöst hat. für tumornekrosefaktor (tnf) ist durch therapiestudien bei menschen eine notwendige mediatorrolle bei m. crohn und bei chronischer polyarthritis belegt. der therapieerfolg von blockierenden antikörpern und rezeptorfusionsproteinen unterstreicht die bedeutung der suche nach weiteren therapeutischen angriffspunkten an diesem zytokin. eine vielzahl körpereigener mediatoren und exogener wirkstoffe wurden identifiziert, die die synthese von tnf in therapeutischen dosierungen hemmen. dass diese synthesehemmung auch einen günstigen einfluss auf den erkrankungsverlauf nimmt, wurde im vergangenen jahr in pilotstudien zur gabe von thalidomid bei m. crohn gezeigt. aus der großen gruppe der tnf-synthesehemmenden substanzen sind spezifische phosphodiesteraseinhibitoren (vom typ iv) in der klinischen entwicklung am weitesten fortgeschritten. sie werden derzeit in phase-iii-studien für patienten mit asthma bronchiale geprüft. falls sie zur zulassung kommen, wird eine indikationsausweitung auf andere tnf-vermittelte erkrankungen untersucht werden. an endotoxin-induced serum factor that causes necrosis of tumors human tumor necrosis factor.production, purification, and characterization human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin cloning and expression in escherichia coli of the gene for human tumour necrosis factor molecular cloning of the complementary dna for human tumor necrosis factor cachectin and tumor necrosis factor as two sides of the same biological coin the treatment of malignant tumors by repeated inoculations of erysipelas: with a report of ten original cases late results of the treatment of inoperable sarcoma by the mixed toxins of erysipelas and bacillus prodigiosus structure of tumour necrosis factor the tumor necrosis factor ligand and receptor families mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock, yet succumb to l.monocytogenes infection a tumor necrosis factor-binding protein purified to homogeneity from human urine protects cells from tumor necrosis factor toxicity two tumor necrosis factor-binding proteins purified from human urine.evidence for immunological cross-reactivity with cell surface tumor necrosis factor receptors stabilization of the bioactivity of tumor necrosis factor by its soluble receptors soluble tumor necrosis factor (tnf) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both tnf carriers and tnf antagonists cytokine-binding proteins: stimulating antagonists tumor necrosis factor induces rapid production of 1'2'diacylglycerol by a phosphatidylcholine-specific phospholipase c functional dichotomy of neutral and acidic sphingomyelinases in tumor necrosis factor signaling receptor-mediated label-transfer assay (relay) -a novel method for the detection of plasma tumor necrosis factor at attomolar concentrations pentoxifylline suppresses okt3-induced tumor necrosis factor-α formation in renal transplant recipients evaluation of recombinant human soluble dimeric tumor necrosis factor receptor for prevention of okt3-associated acut clinical syndrome dexamethasone and pentoxifylline inhibit endotoxininduced cachectin/tumor necrosis factor synthesis at separate points in the signaling pathway thalidomide exerts its inhibitory action on tumor necrosis factorα by enhancing messenger rna degradation specific suppression of human tumor necrosis factor-α synthesis by antisense oligodeoxynucleotides oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin prostaglandin e 2 regulates macrophage-derived tumor necrosis factor gene expression signal transduction and gene regulation: the nuclear response to camp function and activation of nf-κb in the immune system nuclear factor kappab -an oxidative stressresponsive transcription factor of eukaryotic cells (a review) cooperative inhibition of nf-kappa b and tat-induced superactivation of human immunodeficiency virus type 1 long terminal repeat pentoxifylline reduces the first-dose reactions following okt3 the specific type iv phosphodiesterase inhibitor rolipram suppresses tumor necrosis factor-α production by human mononuclear cells phosphodiesterase inhibitors: new opportunities for the treatment of asthma enhanced tumor necrosis factor suppression and cyclic adenosine monophosphate accumulation by combination of phosphodiesterase inhibitors and prostanoids cicaprost and the specific type iv phosphodiesterase inhibitor rolipram synergize in suppression of tumor necrosis factor-α synthesis up-regulation of monocytic il-10 by tumor necrosis factor-a and camp elevating drugs anti-inflammatory activities of camp-elevating agents: enhancement of interleukin-10 synthesis and concurrent suppression of tumor necrosis factor production the antidepressant rolipram suppresses cytokine production and prevents autoimmune encephalomyelitis prevention of autoimmune demyelination in non-human primates by a camp-specific phosphodiesterase inhibitor effects of rolipram on responses to acute and chronic antigen exposure in monkeys suppression of tnf-alpha expression, inhibition of th1 activity, and amelioration of collagen-induced arthritis by rolipram specific type iv phosphodiesterase inhibitor rolipram mitigates experimental colitis in mice potential antidepressant activity of rolipram and other selective cyclic adenosine 3' ,5'-monophosphate phosphodiesterase inhibitors thalidomide selectively inhibits tumor necrosis factor alpha production by stimulated human monocytes new life in a sleeper: thalidomide and crohn's disease two types of mouse t helper cell.iv.th2 clones secrete a factor that inhibits cytokine production by th1 clones role of interleukin-10 in a murine model of dextran sulfate sodium-induced colitis key: cord-000324-to4g9he9 authors: spentzas, thomas; shapley, rebekah kh; aguirre, carlos acuna; meals, elizabeth; lazar, lauren; rayburn, mark s; walker, brett s; english, b keith title: ketamine inhibits tumor necrosis factor secretion by raw264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant staphylococcus aureus date: 2011-01-25 journal: bmc immunol doi: 10.1186/1471-2172-12-11 sha: doc_id: 324 cord_uid: to4g9he9 background: infections caused by community-associated strains of methicillin-resistant staphylococcus aureus (ca-mrsa) are associated with a marked and prolonged host inflammatory response. in a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage tnf response to antibiotic-exposed ca-mrsa bacteria via its antagonism of n-methyl-d-aspartate (nmda) receptors. raw264.7 cells were stimulated for 18 hrs with 10(5 )to 10(7 )cfu/ml inocula of either of two prototypical ca-mrsa isolates, usa300 strain lac and usa400 strain mw2, in the presence of either vancomycin or daptomycin. one hour before bacterial stimulation, ketamine was added with or without mk-801 (dizocilpine, a chemically unrelated non-competitive nmda receptor antagonist), apv (d-2-amino-5-phosphono-valerate, a competitive nmda receptor antagonist), nmda, or combinations of these agents. supernatants were collected and assayed for tnf concentration by elisa. results: raw264.7 cells exposed to either lac or mw2 in the presence of daptomycin secreted less tnf than in the presence of vancomycin. the addition of ketamine inhibited macrophage tnf secretion after stimulation with either of the ca-mrsa isolates (lac, mw2) in the presence of either antibiotic. the nmda inhibitors, mk-801 and apv, also suppressed macrophage tnf secretion after stimulation with either of the antibiotic-exposed ca-mrsa isolates, and the effect was not additive or synergistic with ketamine. the addition of nmda substrate augmented tnf secretion in response to the ca-mrsa bacteria, and the addition of apv suppressed the effect of nmda in a dose-dependent fashion. conclusions: ketamine inhibits tnf secretion by mrsa-stimulated raw264.7 macrophages and the mechanism likely involves nmda receptor antagonism. these findings may have therapeutic significance in mrsa sepsis. infections caused by community-associated strains of methicillin-resistant staphylococcus aureus (ca-mrsa) present a major public health problem because of recent increases in the incidence of these infections [1, 2] . in a 2007 report, the centers for disease control concluded that staphylococcus aureus is now the most important cause of serious and fatal infection in the united states [3] . the prototypical usa400 strain, mw2, (cdc nomenclature for this strain of mrsa) was first isolated in 1999 from a midwest child with fatal ca-mrsa pneumonia [4] . in 2003, the prototypical usa300 ca-mrsa strain, lac, was isolated from los angeles county patients with skin and soft tissue infections, severe pneumonia and sepsis. recently, concerns about ca-mrsa infections were heightened after reports of severe invasive staphylococcal infections in some patients infected with the novel 2009 h1n1 influenza a virus [5, 6] . ca-mrsa isolates express many virulence factors [7, 8] , including several cytolysins: α-toxin, γ-toxin, panton-valentine leukocidin (pvl), phenol-soluble modulins (psms), δ-toxin and, unlike traditional hospital-associated (ha-mrsa) isolates, may express superantigens such as tsst-1 [9] . these bacterial components can stimulate massive cytokine release and lead to septic shock, acute respiratory distress syndrome (ards) and death. it is likely that strategies designed to modulate the excessive and prolonged host inflammatory response could improve the outcome of fulminant mrsa infections. monocytes and macrophages play important roles in host defense against staphylococci and other pyogenic bacteria [10] , but excessive systemic or local production of inflammatory mediators by macrophages could be deleterious in patients with severe staphylococcal infections. we previously reported that raw264.7 murine macrophages exposed to any of a series of six pediatric clinical isolates of s. aureus (two ca-mrsa, two ha-mrsa, and two methicillin-susceptible strains) in the presence of daptomycin (vs. vancomycin) secreted less tnf and accumulated less inducible nitric oxide synthase (inos) protein [11] . vancomycin is a cell-wall active antibiotic that triggers bacterial lysis; it is the antibiotic most commonly used to treat severe mrsa infections in children [12] . daptomycin is a novel antibiotic that is rapidly bactericidal against staphylococci but does not appear to cause rapid bacterial lysis; the mechanism of its action is not certain but it is reported to trigger depolarization of the bacterial membranes and inhibition of both dna and rna synthesis [13, 14] . the rapid lysis of staphylococci, streptococci and other pyogenic bacteria exposed to cell-wall active antibiotics such as beta-lactams and vancomycin results in exaggerated release of bacterial products and an augmented and potentially harmful host inflammatory response [15, 16] . therefore, optimal treatment of sepsis and other severe bacterial infections might include the use of antibiotics and/or other medications that blunt the host inflammatory response and dampen the cytokine cascade [16] . ketamine is one of the recommended anesthetics in pediatric septic shock [17] [18] [19] , which is frequently caused by staphylococci [12, 20] . the reasoning for ketamine's use in staphylococcal septic shock is its blood pressure supporting effect. it increases cardiac output and blood pressure, possibly via a catecholamine release mechanism [17, 21] . some data suggest that ketamine has anti-inflammatory effects [22] [23] [24] [25] . for example, it has been reported that ketamine suppresses macrophage tnf secretion in response to gram-negative bacterial lps in vivo and in vitro [22, 23, 25] . there is also one report that ketamine suppresses tnf production by human whole blood in vitro after exposure to staphylococcal enterotoxin b [24] . the mechanisms responsible for the anti-inflammatory effects of ketamine are not known [22] [23] [24] [25] .the present study examined the hypothesis that ketamine could suppress macrophage tnf production in response to whole bacteria, in this case clinical isolates of methicillin-resistant staphylococcus aureus (mrsa). given the important role of tnf in sepsis [26] [27] [28] [29] , and the importance of staphylococcal sepsis in children, such suppression could have a therapeutic impact. although membrane-bound toll-like receptors (tlr2 and tlr4) are essential for lipopolysaccharide (lps)induced tnf production [30] , this is not the case for staphylococcus aureus. because s. aureus is able to "attack" or form pores in macrophages, tnf secretion occurs even in the absence of tlr 2 and tlr4 sensors (possibly via nod1 and nod2, intracytoplasmic sensors of peptidoglycan-derived muropeptides) [31] . therefore, another mechanism independent of toll-like receptors must exist for ketamine's anti-inflammatory action, at least in staphylococcal infections. we also tested the effects of two chemically unrelated nmda receptor antagonists, the anti-convulsant mk-801 (dizocilpine) [32, 33] , a non-competitive inhibitor of nmda receptors, and apv (d-2-amino-5-phosphonovalerate), a competitive nmda receptor antagonist [34, 35] , as well as the nmda substrate itself, on macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria. for these studies, we utilized two well-characterized clinical isolates: lac (los angeles county), representative of the usa300 group of organisms and closely related to the dominant ca-mrsa clone associated with soft tissue infections and serious invasive disease in the memphis area [1] , and mw2, a clinical isolate from a midwestern child with fatal ca-mrsa sepsis [4] , representative of the usa400 group of organisms that constitute the other main lineage of ca-mrsa isolates in the united states. bacteria were grown to late logarithmic phase at 37°c in tryptic soy broth (becton dickinson and co., sparks, md) and washed three times in endotoxin-free phosphate-buffered saline. concentrations were determined by colony counts. a range of concentrations of bacteria (10 5 -10 7 cfu/ml) was studied, based upon our previously published data with other ca-mrsa strains [11] and our preliminary experiments using lac and mw2 (data not shown). minimum inhibitory concentrations (mics) for these strains were determined by the microbiology laboratory at le bonheur children's hospital using the e-test method: both strains were fully susceptible to vancomycin and daptomycin (lac: mic vancomycin 1.0 μg/ml; daptomycin 0.75 μg/ml; mw2: mic vancomycin < 0.5 μg/ml; daptomycin 0.75 μg/ml). cell culture raw264.7 murine macrophage-like cells were purchased from the atcc and cultured in dulbecco's modified eagle's medium (mediatech inc., herndon, va) supplemented with 10% fetal bovine serum (hyclone, logan, ut) and 2 mm glutamine (gibco, carlsbad, ca). experiments were done in 24-well tissue culture plates (becton dickinson, lincoln park, nj) with 1 × 10 6 cells per well. either vancomycin or daptomycin was added to the cell cultures immediately before the addition of live staphylococci (10 5 -10 7 cfu/ml). cells were then incubated for 18 hours. daptomycin was obtained from cubist pharmaceuticals (lexington, ma). vancomycin was purchased via the department of pharmacy at le bonheur children's hospital (lbch) from hospira (lake forest, il). clinically achievable concentrations of each of the antibiotics, as previously tested in our laboratory [11] , were used (20 μg/ml). these experiments were repeated in parallel in the presence of ketamine (100 μm) and/or mk-801 (dizocilpine, 150 μm), apv (d-2-amino-5-phosphonovalerate, 300 μm ("low") or 3 mm ("high"), or nmda (30 μm). the modulation of mrsa-stimulated macrophage tnf production by ketamine was subsequently examined also at a range of concentrations of 10 μμ, 50 μμ, 100 μμ and 150 μμ. the selected concentration (100 μm) is based on the achievable anesthetic concentrations [36] [37] [38] [39] and on the pre-existing literature related to ketamine's tnf suppressive effect on murine macrophage models when stimulated by lps [23] [24] [25] 27] . the concentrations for the other factors were selected from the available literature, mk-801 [40] [41] [42] , apv and nmda [32] have previously been studied in cell culture models and have been shown to not cause cytotoxicity at the tested concentrations. ketamine and/or mk-801 or apv or nmda were added to the macrophage cultures one hour prior to bacterial challenge. the source of ketamine was ketalar ® , a racemic mixture (1:1) of optically active isomers (r and l) of this drug, purchased from the lbch pharmacy. emphasis in the experiment was placed on correlation with the clinical situation; thus racemic ketamine, the most commonly clinically used product, was selected. dizocilpine (mk-801), apv and nmda were purchased from sigma chemical co. (st. louis, mo). after incubation, cell-free supernatants were collected and assayed for tnf concentrations by using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer (ebioscience, san diego, ca). tnf is a key cytokine produced by macrophages during mrsa stimulation. in our preliminary studies, we also measured secretion of other cytokines and found that il-1, il-6, and no secretion were strongly correlated with tnf secretion in response to these bacteria (r 2 = 0.84, 0.87 and 0.93, respectively). we focused on tnf secretion for these studies. the tested concentrations of vancomycin, daptomycin, ketamine, mk-801, apv, and nmda had no effect on the viability of the raw264.7 cells, as determined by visual inspection of the monolayer, low power microscopic inspection of the monolayer and exclusion of 0.2% trypan blue dye. for the single comparison experiments (ketamine or mk 801 or apv), tnf secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times (a total of at least nine samples). the four preliminary runs and all the exposures (total of 16) where the inocula were different from 10 5 to10 7 cfus/ml at the verifying colony count were excluded from the final analysis. experiments with different exposure times (6, 10, 14, 24 hours) were conducted to determine whether the inhibition increased over time. in the multiple comparison experiments (ketamine and mk 801 synergistic action), tnf was measured from at least four well replicates. all experiments were performed separately for lac and for mw2 mrsa strains. there is an intrinsic experimental variation of absolute values of tnf production (up to 25%) because of cell culture and macrophage growth characteristics. the design was composed of factorial multiple measurements and the results were analyzed according to a mixed linear model, (glimmix) sas 9.2 (sas institute, cary, nc) and r 2.9.1 and ggplot2 software. we set pre-planned (a priori) contrasts, i.e., we set all our comparisons in advance of multiple setting experiments. significant differences were presumed at a probability value of p < 0.05. the results were graphed using error bars with 95% confidence intervals. differences in the means were estimated either with asymptotic techniques for normally distributed data or bootstrapping techniques for non-normally distributed data. ca-mrsa strains mw2 and lac stimulated less tnf secretion by raw264.7 murine macrophages in the presence of daptomycin than in the presence of vancomycin as previously observed with two usa300 ca-mrsa strains isolated from memphis children with invasive staphylococcal infections [11] , macrophages exposed to either of the two prototypical ca-mrsa strains studied (the usa300 strain, lac, or the usa400 strain, mw2) secreted significantly less tnf in the presence of daptomycin as compared with vancomycin (more than 50% reduction in each strain; figure 1 ). macrophage tnf secretion in response to mw2 was 34,535 ± 1,536 pg/ ml in the presence of vancomycin and 15,377 ± 1,267 pg/ml in the presence of daptomycin, a reduction of 55%, significant at p < 0.05. similarly, macrophage tnf secretion in response to lac in the presence of vancomycin was 33,345 ± 1,535 pg/ml, and 14,432 ± 1,536 pg/ml in the presence of daptomycin, a reduction of 57%, significant at p < 0.05. we previously reported similar findings in six s. aureus clinical isolates (including two pediatric ca-mrsa isolates of the usa300 group), suggesting that this effect of daptomycin is conserved in many different s. aureus isolates. the addition of ketamine (100 μμ) to macrophage cell cultures inhibited tnf secretion in response to vancomycin-or daptomycin-exposed ca-mrsa isolates ( figure 2) . the effect was similar on both strains, lac and mw2, in the presence of vancomycin (upper panel) or daptomycin (lower panel). in the initial experiments we analyzed the effect of one hour pre-incubation with ketamine on the macrophage response to vancomycin-exposed ca-mrsa bacteria (mw2 and lac). in response to vancomycinexposed mw2, pre-incubation with ketamine reduced macrophage tnf secretion by approximately 29% (p < 0.05), i.e., from 33,085 ± 867 pg/ml to 23,347 ± 862 pg/ml. pre-incubation with ketamine led to a similar reduction (25%; p < 0.05) in macrophage tnf secretion response after stimulation with vancomycinexposed lac (from 28,365 ± 735 pg/ml to 21,432 ± 736 pg/ml). we next studied the effect of ketamine pre-incubation on macrophage tnf secretion after stimulation with daptomycin-exposed mw2 or lac. once again, the addition of ketamine resulted in significant inhibition of macrophage tnf secretion in response to mw2 (23,185 ± 1,267 pg/ml to 17,354 ± 853 pg/ml, a reduction of approximately 25%; p < 0.05) or lac (approximately 18% reduction, p < 0.05; figure 2 ). adding ketamine after the mrsa inocula did not alter the response. figure 1 the ca-mrsa isolates lac (usa300) and mw2 (usa400) stimulated less tnf secretion by raw264.7 murine macrophages when exposed to daptomycin (dap) than when exposed to vancomycin (van). lac or mw2 were added to raw264.7 cells at a final concentration of 10 5 to 10 7 cfu/ml (retrospective confirmation) in the presence of either vancomycin or daptomycin at 20 μg/ml. cells were incubated for 18 hours; supernatants were collected and analyzed for tnf content by an enzyme-linked immunosorbent assay (elisa). results are depicted as means with 95% confidence intervals shown as "error bars" (see methods). the "*" indicates significance at p < 0.05. control represents the mean tnf macrophage production by macrophages not stimulated with bacteria. mrsa mw2 and lac exposed to ketamine one hour prior to stimulation, ketamine (100 μm) was added to the indicated wells. cells were then incubated for 18 hours; supernatants were collected and analyzed for tnf content by elisa. results are depicted as means with 95% confidence intervals shown as "error bars" (see methods). the "*" indicates significance at p < 0.05. control represents the mean tnf macrophage production by macrophages not stimulated with bacteria. the nmda inhibitor mk-801 (dizocilpine) inhibited macrophage tnf secretion after stimulation with antibiotic-exposed ca-mrsa strains pre-incubation of raw264.7 cells for one hour with the nmda receptor antagonist, mk-801 (150 μμ), also inhibited tnf secretion by these cells after stimulation with antibiotic-exposed ca-mrsa strains (mw2 or lac, figure 3 ). in response to stimulation with mw2 in the presence of vancomycin, pre-incubation with mk-801 significantly inhibited tnf secretion by these cells, i.e., from 32,407 ± 1,188 pg/ml to 23,337 ± 1,272 pg/ml (approximately 28% reduction; p < 0.05, figure 3 , upper panel). mk-801 also inhibited macrophage tnf secretion in response to vancomycin-exposed lac, causing a 34% reduction (figure 3, upper panel) . pre-incubation with mk-801 also significantly inhibited macrophage tnf secretion in response to daptomycin-treated mw2 or lac (figure 3 , lower panel). in response to stimulation with mw2 in the presence of daptomycin, pre-incubation with mk-801 inhibited tnf secretion by these cells by approximately 26% (from 22,305 ± 648 pg/ml to 16,437 ± 642 pg/ml, p < 0.05). mk-801 inhibited macrophage tnf secretion in response to daptomycin-exposed lac by approximately 33% (from 22,164 ± 864 pg/ml to 14,647 ± 832 pg/ml, p < 0.05). pre-incubation of raw264.7 cells with combinations of mk-801 and ketamine did not affect the magnitude of inhibition of macrophage tnf secretion observed in the presence of ketamine (or mk-801) alone. figure 4 depicts results for macrophages stimulated with vancomycin-or daptomycin-exposed mw2; responses to antibiotic-exposed lac were similar (data not shown). one hour prior to stimulation, either ketamine at 100 μm, mk-801 at 150 μμ, or both were added to the indicated wells. cells were then incubated for 18 hours; supernatants were collected and analyzed for tnf content by elisa. lane 1 (control) represents the mean tnf production by macrophages not stimulated with bacteria. the mean includes wells exposed to ketamine, mk-801, both ketamine and mk-801, and neither. in the absence of bacteria, tnf secretion was minimal and was not affected by ketamine and/or mk-801. nmda augments macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria: both ketamine and a competitive nmda receptor antagonist, apv, block this effect we further examined the role of nmda receptors in modulating the macrophage tnf response to the ca-mrsa bacteria by studying the effects of a competitive nmda receptor antagonist, apv, and the effects of the nmda substrate itself ( figure 5 ). we found that apv (at either 300 μm or 3 mm) also inhibited macrophage tnf secretion in response to vancomycin-exposed mw2 (p < 0.05, figure 5 ). the magnitude of the inhibition was comparable to that observed with either ketamine or mk-801 (and, as in the case of mk-801, was not additive or synergistic with ketamine). furthermore, the addition of the nmda substrate (30 μm) resulted in a marked augmentation of the macrophage tnf response to the antibiotic-treated ca-mrsa bacteria (p < 0.05), and this effect was blocked by ketamine and by the competitive nmda receptor antagonist, apv ( figure 5 ). we next studied the effects of a range of concentrations of ketamine and found that inhibition of macrophage tnf secretion in response to vancomycinexposed lac or mw2 was consistently observed at concentrations of ketamine at the lowest concentration tested (10 μm) and was greater at concentrations of 50 -150 μm ( figure 6 ). we also examined the kinetics of inhibition of macrophage tnf secretion by incubating raw264.7 cells for 6, 10, 14, 18 and 24 hours after exposure to ketamine at a concentration of 100 μm 1 hour prior to stimulation with vancomycin-exposed lac or mw2. we found that the magnitude of suppression of tnf secretion was similar at all times studied (figure 7 ). we found that exposure of murine macrophages to ketamine inhibited tnf secretion by 18-34% after stimulation with ca-mrsa bacteria in the presence of antibiotics. the magnitude of the effect was comparable in response to both mw2 (usa400) and lac (usa300) bacteria and was similar in the presence of either vancomycin (a lytic antibiotic associated with a greater tnf response to the bacteria) or daptomycin (a non-lytic antibiotic associated with a blunted tnf response to the bacteria). our data suggest that ketamine administration to macrophages stimulated by ca-mrsa is associated with blunting of the tnf response to these virulent pathogens, and suggest that these findings may have therapeutic significance in mrsa sepsis. furthermore, these data confirm and extend our previous observations that ca-mrsa bacteria exposed to daptomycin (versus vancomycin) trigger less tnf secretion by macrophages. the potentially beneficial antiinflammatory effects of daptomycin and ketamine were additive (figures 2, 3) . bacteria were added at a final concentration of 10 5 to10 7 cfu/ml (retrospective confirmation) in the presence of vancomycin at 20 μg/ml. one hour prior to stimulation, apv ("low" concentration of 300 μm or "high" concentration of 3 mm), ketamine (100 μm), or nmda (30 μm) were added, alone or in combination, as indicated. cells were then incubated for 18 hours; supernatants were collected and analyzed for tnf content by elisa. the control lane represents the mean tnf macrophage production by macrophages not stimulated with bacteria. the mean includes wells exposed to apv, ketamine, or nmda alone or in combination. in the absence of bacteria, tnf secretion was minimal and was not affected by apv, ketamine, or nmda. lanes 0-9 depict mean tnf secretion by macrophages exposed to vancomycin-treated mw2 alone (lane 0) or in the presence of the indicated concentrations of apv, ketamine, and/or nmda (lanes 1-9). tnf secretion was reduced by approximately 30-40% when macrophages were pre-incubated with apv, ketamine, or apv + ketamine (lanes [1] [2] [3] [4] [5] . the magnitude of inhibition by ketamine and high-dose apv was similar and there were no additive or synergistic effect observed with combinations of ketamine and apv. addition of nmda (30 μμ) led to a substantial increase in the amount of tnf secreted in response to the mw2 strain (lane 9), and this augmented response was blocked by both apv and ketamine. the "*" on "0.control" and "8.nmda+lo_apv" bars indicates significance at p < 0.05. the "**" on "0.control_mw2" and "9.nmda" bars indicates differences between the pretreated wells, and that tnf production after mrsa stimulation with nmda substrate (9.nmda) is significantly higher than that at the baseline mrsa stimulation (0.control_mw2) at p < 0.05. an improved understanding of the pathogenesis of sepsis and other life-threatening infections caused by ca-mrsa bacteria could expedite the development of novel strategies for the diagnosis, treatment, and/or prevention of these serious infections. ca-mrsa infections often are associated with severe and prolonged host inflammatory responses [43] [44] [45] [46] . prompt antibiotic treatment of these and other serious bacterial infections is indicated, but paradoxically has the potential to trigger excessive release of bacterial products and the subsequent augmentation of the host inflammatory response [15, 16] . macrophages are important sources of many of the proinflammatory cytokines (including il-1β, il-6, il-8, il-12, and tnf) secreted in response to staphylococci and other gram-positive bacteria [15, 16, 41] . although the cytokine cascade is essential for normal host defense, excessive or inappropriate inflammation can be harmful. therefore we need an improved understanding of these interactions in order to develop better adjunctive therapies for patients with severe bacterial infections. in a previous study, we found that exposure of either of two ca-mrsa strains isolated from memphis children (or any of four other s. aureus isolates from children with invasive staphylococcal infections) to daptomycin (compared with vancomycin) led to a less pronounced macrophage inflammatory response, characterized by diminished secretion of tnf and reduced accumulation of the inducible nitric oxide synthase (inos) [11] . in this study, we found that this differential effect of daptomycin (versus vancomycin) was also observed when macrophages were stimulated with either of the two prototypical ca-mrsa strains most widely studied today: the usa400 isolate, mw2, and the usa300 isolate, lac. importantly, ketamine pre-incubation inhibited macrophage tnf secretion in response to both ca-mrsa strains in the presence of daptomycin as well as in the presence of vancomycin, and the greatest suppression of tnf secretion was noted in the presence of both daptomycin and ketamine. the mechanism(s) responsible for the anti-inflammatory properties of ketamine are not known, but its neurological and psychotropic actions are believed primarily to be mediated by antagonism of nmda receptors [21, 47] . glutamate is the brain's primary excitatory neurotransmitter. nmda receptors are found in many cell bacteria were added at a final concentration of 10 5 to10 7 cfu/ml (retrospective confirmation) in the presence of vancomycin at 20 μg/ml. the ketamine concentration was 100 μm. results are depicted as percentile reduction with 95% confidence intervals, i.e., the percent of tnf reduction at the specific exposure time that occurs in comparison to inoculation without ketamine at the same time. the "*" indicates statistically significant difference at p < 0.05. types, including blood lymphocytes, lung macrophages, and multiple hematopoietic precursors in bone marrow cells [40, 42, 47, 48] . both ketamine and the chemically unrelated anticonvulsant dizocilpine (mk-801) are noncompetitive antagonists of the nmda receptor, one of the three known glutamate receptors [32, 33, 47] . apv is a competitive inhibitor of the classical nmda receptor and acts on the nr2 component of the receptor (30, 33) . we found that mk-801 and apv also inhibited macrophage tnf secretion in response to antibiotictreated mw2 or lac cells. the magnitude of the inhibition by mk-801 (approximately 30%) and apv (25-35%) was comparable to that observed with ketamine (18-34%), and combinations of mk-801 and ketamine or of apv and ketamine did not exhibit additive or synergistic inhibition of tnf secretion. furthermore, adding nmda led to augmented macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria, and the nmda receptor antagonist, apv, blocked this effect. the suppression of tnf induced by ketamine was observed across a range of concentrations and throughout the incubation period. our study has its limitations. to translate the present findings, we are currently working on a clinical model to assess the clinical significance of ketamine's anti-inflammatory effects in patients with bacterial sepsis. although studies of the effect of ketamine on macrophage responses to purified bacterial components such as gram-negative lipopolysaccharide (lps) or gram-positive lipoteichoic acid (lta) are instructive [23, 24, 49] , we argue that characterization of the macrophage responses to whole organisms is more likely to provide clinical insights. indeed, the pioneering experiments of carswell and old that identified tnf used whole bacteria as stimuli in macrophage sepsis simulation settings [49] , and we have previously demonstrated that macrophage responses to live, antibiotic-treated staphylococci serve as a powerful model system. furthermore, the model examines the effect of ketamine only in the presence of antibiotics (either vancomycin or daptomycin). in practice, this is a common clinical scenario. our data suggest that clinically achievable concentrations of both ketamine and daptomycin could potentially inhibit the excessive macrophage inflammatory response that is observed in patients with severe staphylococcal infections. in the battle of sepsis everything counts. adjunctive therapies of sepsis are greatly needed. studies in animal models and clinical trials will be required to determine whether the anti-inflammatory effects of ketamine and/or other agents that block nmda receptors could be beneficial in the treatment of severe staphylococcal infections. emergence of community-associated methicillin-resistant staphylococcus aureus at a memphis, tennessee children's hospital methicillin-resistant s. aureus infections among patients in the emergency department invasive methicillin-resistant staphylococcus aureus infections in the united states prevention: four pediatric deaths from community-acquired methicillin-resistant staphylococcus aureus-minnesota and north dakota emergence of communityacquired 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determination of ketamine in plasma and its application to human samples ketamine infusions: pharmacokinetics and clinical effects kicman at: detection of ketamine and its metabolites in urine by ultra high pressure liquid chromatography-tandem mass spectrometry pharmacokinetics of ketamine in man expression of a functional n-methyl-daspartate-type glutamate receptor by bone marrow megakaryocytes new in vitro model of traumatic neuronal injury: evaluation of secondary injury and glutamate receptor-mediated neurotoxicity glutamate acting on nmda receptors stimulates neurite outgrowth from cerebellar granule cells panton-valentine leukocidin is not a virulence determinant in murine models of community-associated methicillin-resistant staphylococcus aureus disease toxic shock syndrome-associated staphylococcal and streptococcal pyrogenic toxins are potent inducers of tumor necrosis factor production association between staphylococcus aureus strains carrying gene for panton-valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients is panton-valentine leukocidin the major virulence determinant in community-associated methicillin-resistant staphylococcus aureus disease? glutamate modulation of human lymphocyte growth: in vitro studies systemic administration of mk-801 protects against ischemia-induced hippocampal neurodegeneration in the gerbil an endotoxin-induced serum factor that causes necrosis of tumors ketamine inhibits tumor necrosis factor secretion by raw264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant staphylococcus aureus english's laboratory is supported by cubist pharmaceuticals (the makers of daptomycin), but cubist did not fund this project. the authors would like to thank andrea patters for her editorial assistance, thomas t. spentzas for his help with the graphics and dr sunny anand for reviewing the manuscript. the authors declare that they have no competing interests. key: cord-003686-1pfk4qve authors: kaneko, naoe; kurata, mie; yamamoto, toshihiro; morikawa, shinnosuke; masumoto, junya title: the role of interleukin-1 in general pathology date: 2019-06-06 journal: inflamm regen doi: 10.1186/s41232-019-0101-5 sha: doc_id: 3686 cord_uid: 1pfk4qve interleukin-1, an inflammatory cytokine, is considered to have diverse physiological functions and pathological significances and play an important role in health and disease. in this decade, interleukin-1 family members have been expanding and evidence is accumulating that highlights the importance of interleukin-1 in linking innate immunity with a broad spectrum of diseases beyond inflammatory diseases. in this review, we look back on the definition of “inflammation” in traditional general pathology and discuss new insights into interleukin-1 in view of its history and the molecular bases of diseases, as well as current progress in therapeutics. in terms of general pathology, inflammation is one of the adaptive responses to various injuries including physical, chemical, and biological factors. the roman encyclopedist a. cornelius celsus described four cardinal signs of inflammation in one concise sentence: "now the signs of an inflammation are four: redness (rubour) and swelling (tumour), with heat (calour) and pain (dolour)" [1] . a century and a half later, galen added a fifth sign: "disturbance of function" (funcio laesa) [2] . the classical signs of inflammation are considered to be related to cells and tissues responding to pathological cell injury caused by internal stimuli, including damage-associated products and metabolites, and external stimuli, including bacteria and viruses [3] [4] [5] [6] . the host bears the receptors that facilitate recognition of these damage-associated molecular patterns (damps) and/ or pathogen-associated molecular patterns (pamps) that are not host-derived. these receptors are termed pattern recognition receptors (prrs) [7] . prrs directly or indirectly detect infection and/or noxious chemicals, resulting in inflammation that is coupled with the induction of immune responses and a tissue reparative component [8] . the signal transduction triggered by these prrs leads to the acute inflammatory mediator expressions that regulate the elimination of pathogens and infected cells [9, 10] . there are several known prrs: toll-like receptors (tlrs), rig-i-like receptors (rlrs), nod-like receptors (nlrs), and c-type lectin receptors (clrs). the majority of nod-like receptors such as nlrp1, nlrp3, nlrc4, nlrp6, and nlrp12 can interact with apoptosis-associated speck-like protein containing a caspase-recruitment domain (asc) and caspase-1, and the resulting complex is a sensor of cell injury called "inflammasome", an interleukin (il)-1β-processing platform that plays a crucial role in il-1β maturation and secretion from cells. other pyrin-domain (pyd)-containing proteins such as aim2, ifi-16, and pyrin are also known to form inflammasomes. among them, nlrp3 inflammasomes monitor membrane integrity and pore-forming toxins, crystals, and many other noxious stimuli and are involved in il-1β processing and maturation [11] [12] [13] [14] . it is now widely accepted that an inflammatory response is the extreme end of a spectrum that ranges from a homeostatic state of inflammation to a stress response and finally inflammation [8, 15] . the homeostatic state of inflammation, which is not inflammation from the perspective of general pathology, was suggested to be maintained by prrs expressed in stromal and/or immune cells, detecting endogenous ligands in parenchymal cells and/or pathogens, leading to chronic inflammatory responses ranging from the basal homeostatic state to disease-causing inflammation [15, 16] . in addition to several forms of inflammation including classical inflammation, homeostatic inflammation, a distinct nomenclature for low-grade inflammation, such as para-inflammation (an adaptive response against stress or malfunction) and meta-inflammation (metabolically triggered inflammation), has been proposed [17] [18] [19] . as discussed above, there are various factors involved in forms of inflammation; in particular, since il-1 is a downstream cytokine of the sensor of cell injury, the inflammasome, it is important for regulating inflammation and tissue damage beyond inflammation [20] . il-1 is a master regulator of inflammation via controlling a variety of innate immune processes [21] . from a historical point of view, il-1 has a wide range of biological functions, which include acting as a leukocytic pyrogen, a mediator of fever and a leukocytic endogenous mediator, and an inducer of several components of the acute-phase response and lymphocyte-activating factor (laf) [22, 23] . laf was later shown to be a macrophage-derived immune mediator acting on t-and b-lymphocytes and was designated as il-1 in the second international lymphokine workshop held in switzerland in 1979 [24, 25] . in addition, serum blocking factors in breast cancer patients identified by the leukocyte adherence inhibition test were reported. the serum adherence-promoting factors were regulated by il-1 [26] [27] [28] . to date, the tumor microenvironment has been characterized by dominant immunosuppression, being infiltrated by tumor immunosuppressive myeloid-derived suppressor cells (mdscs), regulatory t cells (tregs), and tumor-associated macrophages (tams) [29, 30] . il-1 is capable of inducing the recruitment of tams and mdscs, which promote tumor development in breast cancer [31] . currently, human sequence algorithm technologies suggest that the il-1 family comprises a total of 11 members with similar or distinct biological effects [32, 33] . il-1α, il-1β, il-1ra, il-18, il-33, il-36α, il-36β, il-36γ, il-36ra il-37, and il-38 have been identified and characterized (table 1 ) [32] . among them, il-1α, il-1β, il-18, il-33, and il-36 are receptor-agonistic, and il-1ra, il-36ra, and il-38 are receptor-antagonistic. il-37 is the only anti-inflammatory cytokine. although the function of each il-1 family member is now being investigated, il-1 is the most characterized among these members. there are two individual forms of il-1, il-1α and il-1β, isolated from two distinct cdnas, but they are indistinguishable in terms of their biological functions [34] . although the homology between il-1α and il-1β is not high (27%) in terms of amino acid sequences, il-1α and il-1β are structurally similar and show the same functions by sharing a common receptor, il-1 type 1 receptor (il-1r1), and both have the same central β-barrel along with adjoining loops [35, 36] . the difference between il-1α and il-1β is an n-terminal extension of 14 residues beyond the n-terminus of il-1α and il-1β [37] . the molecular weight of each precursor is approximately 31 kda, and il-1α and il-1β are processed by specific proteases to mature forms. the n-terminal domain of il-1α contains a nuclear localization sequence (nls) and shows transcription activity [38] . il-1α is produced as a 271-amino acid (aa) precursor protein. for transcription of the il-1α gene, transcription factor specificity protein 1 (sp1) activates the il-1α promoter activity in the 5′-upstream gc box (− 60 to − 45 bp) [39] and nf-κb, which is also activated by il-1α itself, and stimulates the consensus promoter region (− 103 to − 70 bp) to induce its own gene expression and production in an autocrine manner [40] . the precursor of il-1α translocates into the nucleus to bind to chromatin and also exists in a membrane-anchored form. upon stress responses, il-1α is processed by ca 2+ -dependent protease calpain or other proteases, such as cytotoxic t-lymphocytes (ctl)/natural killer (nk)-granzyme-b, mast cell chymase, or neutrophil elastase to the c-terminal 159 aa as mature il-1α [41] . the il-1α processing separates nls from its precursor, which is not linked to secretion or cell death [21] ; however, il-1α is a key danger signal that induces inflammation on release from necrotic cells [42] . the il-1α precursor triggers il-1r1 on resident macrophages in necrotic tissues, producing il-1β as well as chemokines as post-necrotic inflammation [43] . il-1β is produced as a 269-aa precursor protein and processed by caspase-1, which is also known as il-1β-converting enzyme (ice), activated in inflammasomes, to the c-terminal 153 aa as mature il-1β [11, 12, 34, 44] . the il-1β precursor is also processed by other serine proteases [45] . neutrophils derived from caspase-1-deficient mice release mature il-1β processed by elastase in response to lipopolysaccharide (lps) stimulation [46] . the neutrophil proteases, such as elastase, chymases, granzyme a, cathepsin g, and proteinase-3, cleave the il-1β precursor into a secreted, biologically active form [47] [48] [49] . these alternatively cleaved forms of functional il-1β were detected in synovial fluid of a patient with inflammatory polyarthritis and gout [50] . occasionally, massive neutrophil infiltration appeared in excess-inflammation-damaged tissues and organs, such as in septic shock or systemic inflammatory response syndrome. thus, the nlrp3 inflammasome-related inflammation induced by a variety of factors described above may be a target of anti-il-1 therapy [51] . currently, a two-step model of the initiation of nlrp3 inflammasome activation is suggested. the first step mediates transcriptional and post-translational priming of nlrp3 (step1), and the second step is activation of inflammasomes (step 2). step 1 is the first synthesis of a biologically inactive il-1β precursor by nf-κb binding to the consensus binding site (− 296 to − 286 bp) to transcribe the il-1β gene. step 2 is processing into mature, biologically active il-1β by caspase-1 activated by a cytosolic activation platform called inflammasome [52, 53] . the inflammasome is a large protein complex, which consists of prrs, such as nlrs, aim2, rig-i or pyrin, an adaptor protein asc, and caspase-1. among them, the nlrp3 inflammasome is a prototype inflammasome, which has been reported to be activated by a wide range of pamps and damps [54, 55] . various nlrp3-activating pamps have been reported, i.e., bacteria-derived rna or dna, pore-forming toxins, lethal toxins, flagellin/rod proteins, muramyl dipeptide (mdp), m2 protein, virus-derived rna or dna, fungus-derived β-glucans, hypha mannan, zymosan, and protozoon-derived hemozoin [56] . nlrp3-activating damps have also been reported, i.e., self-derived glucose, β-amyloid, hyaluronan, atp, cholesterol crystals, monosodium urate (msu) crystals, calcium pyrophosphate dihydrate (cppd) crystals, environment-derived alum, asbestos, silica, alloy particles, uv radiation, and skin irritants [56] ; however, their diverse physiological and chemical signals leading to the direct activation of nlrp3 have not been fully elucidated. instead, efflux of potassium has been identified as the common activator of most known nlrp3 step 2 signals [57, 58] . the nlrp3 activation by potassium efflux suggested to lead nlrp3-nek7 interaction to drive inflammasome activation [59] [60] [61] . the mechanism underlying the secretion of il-1β has been suggested to overlap with il-1α secretion [41] . also, mitochondrial and phagosomal reactive oxygen species (ros) have been proposed to activate the nlrp3 inflammasome. alternatively, non-canonical pathways of nlrp3-inflammasome activation associated with proinflammatory caspases, caspase-4, caspase-5, and caspase-11 have been proposed. in this process, lps is recognized by the caspase-recruitment domain (card) of respective caspases, leading to activation [62] [63] [64] [65] . caspase-8 or proteases in neutrophils are also processed and activate il-1β. several prrs, such as nlrp1, nlrp3, nlrc4, pyrin, and aim2, convert the assembly of the adaptor molecule asc into a high-molecular-weight complex, called the pyroptosome [66] . then, the caspase-1 precursor is recruited to the pyroptosome to also form helical structures, which enable its proximity-induced proteolytical auto-activation. with caspase-1 precursor maturation into the active p102/p202 heterotetramer, it cleaves the il-1β precursor, leading to pyroptotic cell death. this cell death is mediated by the caspase-1-dependent cleavage of gasdermin-d (gsdmd) [67] [68] [69] . in turn, the mature n-terminal fragment of gsdmd translocates to the inner leaflet of the plasma membrane to form round and pore-like structures of approximately 15 nm in diameter [70] [71] [72] [73] . tissue distributions of interleukin-1 il-1α and il-1β are expressed in a wide range of tissues and a variety of cells, especially in macrophages in lymphoid organs including the thymus, spleen, lymph nodes, peyer's patches, and bone marrow. in non-lymphoid organs, il-1α and il-1β are expressed in tissue macrophages in the lung, digestive tract, and liver. they are also expressed in cellular subepithelial endometrial tissue of the uterus, in the glomeruli, in outer cortical areas of the kidney, and in various specific cell types, including neutrophils, keratinocytes, epithelial and endothelial cells, lymphocytes, smooth muscle cells, and fibroblasts [74, 75] . there are two cell surface il-1 receptors, il-1r1 and il-1 type 2 receptor (il-1r2), a decoy receptor. il-1 binds to il-1r1, which requires the formation of a heterodimer with the il-1 type 3 receptor (il-1r3) (also known as il-1racp) accompanied by adaptor il-1 receptor-associated kinase (irak) and myeloid differentiation primary response protein 88 (myd88) [76] . il-1r1 initiates inflammatory responses when binding to the ligands il-1α and il-1β and has been reported to be expressed by tlymphocytes, fibroblasts, epithelial cells, and endothelial cells. il-1r2, which does not initiate signal transduction, is expressed in a variety of hematopoietic cells, especially in b-lymphocytes, mononuclear phagocytes, polymorphonuclear leukocytes, and bone marrow cells. notably, expression levels of il-1r1 and il-1r2 are different among the cell types; for example, neutrophils predominantly express il-1r2. as a result, much higher concentrations of il-1β are required to activate neutrophils, whereas low concentrations of il-1β are sufficient to activate endothelial cells. the il-1r1-mediated signaling pathways also differ according to the cell types [77, 78] . il-1r3 is a co-receptor for il-1r1, responsible for signaling after binding ligands il-1α and il-1β, and has been reported to be ubiquitously expressed by all cells responsive to il-1. il-1r3b is a brain-specific isoform of il-1r3 generated by alternative splicing, and it has been reported to be expressed in the brain, cerebellum, and spinal cord [79] . activated il-1 is incapable of functioning until recognized by cell surface receptors. the complex contains a motif of gtpase activity and activates gtpase-activating protein and protein kinases [80] [81] [82] . in contrast, il-1r2 is thought to reduce the biological response to il-1. the proximity of the two cytoplasmic domains of il-1r1 and il-1r3 is thought to initiate signal transduction by the hydrolysis of gtp. this is followed by c-jun n-terminal kinase (jnk) and p38 map kinase [83] . irak and tumor necrosis factor (tnf) receptor-associated factor (traf) 6 activate nf-κb, as well as p38, jnks, extracellular signal-regulated kinases (erks), and mitogen-activated protein kinases (mapks) [84] . the nf-κb activation pathway is dependent on the iκ-b kinase (ikk) complex, composed of ikkα, ikkβ, and nf-κb essential modulator (nemo), via associations with tak1, tak2, traf2, and traf6 in the il-1r1-signaling pathway [85] . these signals play important roles in both acute and chronic inflammation in various diseases [86] . single nucleotide mutation of the cias1 gene results in nlrp3 mutation, which induces constituted inflammasome activation causing cryopyrin-associated periodic syndrome (caps). this is termed autoinflammatory disease, including familial cold autoinflammatory syndrome (fcas), muckle-wells syndrome (mws), and neonatal-onset multisystem inflammatory disease (nomid)/chronic infantile neurologic, cutaneous, and arthritis (cinca) syndrome, which leads to greater il-1β secretion without any damps or pamps [87] [88] [89] [90] [91] [92] . the most common autoinflammatory disease is familial mediterranean fever (fmf). fmf is autosomal recessive; however, mutations in the causative mefv gene, encoding mutated pyrin, leads to active pyrin inflammasome [93] . inflammatory diseases like those above, characterized by the enhanced secretion of il-1β, include a group of autoinflammatory diseases such as nlrp12 autoinflammatory syndrome; hyperimmunoglobulinemia d and periodic fever syndrome (hids)/mevalonate kinase deficiency (mkd); pyogenic arthritis, pyoderma gangrenosum, and acne (papa) syndrome; pyoderma gangrenosum, acne, and suppurative hidradenitis (pash) syndrome; pyogenic arthritis, acne, pyoderma gangrenosum, and suppurative hidradenitis (papash); majeed syndrome; and tnf-receptor-1-associated syndrome (traps) [93] [94] [95] [96] [97] [98] [99] [100] . on deficiency of the il-1-receptor antagonist (dira), excess il-1β induces other proinflammatory cytokines and chemokines [101] . excess stress responses disrupt body homeostasis under physiological conditions and lead to excess cytokine production. nlrp3 inflammasomes have also been reported to be involved in low-grade subclinical inflammation induced by chronic exposure to high levels of free fatty acids and glucose, leading to increased apoptosis and impaired insulin secretion of β-cells in obese type 2 diabetes mellitus (t2d) patients [102] [103] [104] . indeed, islet amyloid polypeptide (iapp) oligomers activated nlrp3 inflammasomes to induce significant il-1β production by infiltrating macrophages in an in vivo study [105, 106] . higher concentrations of glucose activate nf-κb and il-1 precursors in cells [102] . minimally oxidized low-density lipoproteins stimulate tlr4, which triggers il-1β expression [104, 105] , and accumulations of islet amyloid polypeptides are deposited and mediate nlrp3 inflammasome activation in islet macrophages [107] . another oligomer of amyloid, amyloid β, can induce il-1β via nlrp3 inflammasomes in a process involving the phagocytosis of amyloid β in glial cells in patients with alzheimer's disease (ad) and subsequent lysosomal damage and release of cathepsin b [108] . ros are considered to be involved in the activation of nlrp3 inflammasomes, and it was suggested that direct interaction between amyloidogenic peptide and nlrp3 could initiate nlrp3 inflammasome formation in a cell-free system [109, 110] . both il-1α and il-1β gene polymorphisms have been reported to be associated with central obesity and metabolic syndrome in a population with coronary heart disease in an epidemiologic study [111] . thus, these diseases are il-1dependent cytokinopathies (interleukinoneopathies). besides the above diseases, numerous inflammatory diseases related to excess il-1 signaling have also been identified [112] [113] [114] . for example, high il-1β levels in humans and mice result in increased th17-dominant immunopathology, and il-1β expression was limited to macrophages and neutrophils, which account for a large proportion of the cd45α cells in the cervix upon chlamydia muridarum infection [115] . consequently, il-1β promotes the differentiation of monocytes into conventional dendritic cells (dcs) and m1-like macrophages and supports the proliferation of activated b-lymphocytes and their differentiation into plasma cells [116] [117] [118] . il-1 in combination with il-2 promoted not only the expansion of nk cells but also cd4+ cd8+ t-lymphocytes [119] . il-1β generated by activated antigen-presenting cells (apcs) induced type 1 immune responses, which produced ctl and led to the polarization of cd4+ t -lymphocytes towards t-helper cell type 1 (th1) [120, 121] . il-1β plays a role in resolving acute inflammation resulting in the initiation of adaptive anti-tumor responses; however, chronic inflammatory conditions increase the risk of developing cancer [122] . in human breast cancer, higher expression of il-1β is associated with tumor invasiveness and aggressive tumor biology [123] . expression of il-1α, il-1β, and their receptors in human breast cancer tissues results in the activation of a population of cells and subsequently contributes to angiogenesis, tumor proliferation, and tumor invasion in the microenvironment [124] . in a spontaneous mmtv-pymt mouse mammary gland tumor model, mature il-1β levels in primary mammary tumors and metastasis sites were significantly elevated, being associated with inflammasome activation and the infiltration of myeloid cells in tumor microenvironments. in this model, cd11b + gr1 + and cd11b + gr1 − myeloid cell populations were also significantly increased in both tumor tissues [31] . il-1β generated in a tissue with a tumor microenvironment dominated by tams promotes tumor growth and metastasis in breast cancer [122, 125] . il-1, by promoting mdscs and sustaining the immunosuppressive activity of tams, contributes to the suppression of effective adaptive anti-tumor immune responses [126] . actually, the sphingolipid sphingosine-1-phosphate (s1p) on tams promotes lymphangiogenesis and lung metastasis via nlrp3/il-1β in mouse breast cancer model [127] . for example, obesity induces an increase in tumor-infiltrating mdscs with activated nlrc4 inflammasome, leading to il-1β production, which drives tumor progression through adipocyte-mediated vascular endothelial growth factor (vegf) a expression and angiogenesis [128] . a recent report showed that il-1β orchestrates tumor-promoting inflammation in patients with high-risk her2-negative breast cancer who would benefit from il-1-blocking therapeutics with anakinra (described later on). the report indicates that while anakinra downregulates gene expressions for il-1β, il-1r1, il-1r2, and il-1r3, increased gene expressions of nk cells and ctls are observed [129] . although il-1 has been well-characterized, il-18 and other il-1 family members have been less comprehensively investigated. il-18 can be processed by caspase-1 and proteinase-3 as well as il-1β, to be activated [130] [131] [132] . considering the pathogenesis of il-1-related diseases, il-18 could be involved [133] . il-18 was originally identified as interferon (ifn)-γ-inducing factor [134] . il-18 is the most structurally related to il-1β. il-18 is synthesized as a 24-kda inactivated precursor and is cleaved by caspase-1 to a biologically active 17-kda mature form [131, 132] . although il-1β is biologically active within the pg/ml range, il-18 requires 10-20 ng/ml and sometimes higher levels for in vitro activation [135, 136] . since the il-18 precursor is expressed ubiquitously in tissues [137] , il-18 signaling is thought to be regulated concentration-dependently. mature il-18 forms a signaling complex with the il-18 receptor alpha chain (il-18rα) with low affinity. if the cell expresses an il-18 receptor β chain (il-18rβ), a high affinity complex is formed like the il-1r accessory chain il-1r3. the complex of the heterodimer recruits myd88 through the toll-il-1 receptor (tir), four iraks, and traf-6, leading to the degradation of i-κb and activation of nf-κb, as that for il-1 signaling [83] . il-18 is involved in regulation of the th1 response by modulating the production of ifn-γ. for example, in synergy with either il-12 or il-15, which upregulates the expression of the il-18rβ co-receptor, il-18 induces the production of ifn-γ by t cells [138] . il-18 induces ifn-γ production by nk cells, and nk cells express ccr7 and produce high levels of ifn-γ [139] . the combination of il-18 and il-12 induced high levels of ifn-γ upon hypoglycemia, intestinal inflammation, and inanition [140] . some human autoimmune diseases are associated with the elevated production of ifn-γ and il-18. autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis (ra), type-1 diabetes mellitus, crohn's disease and psoriasis, and graft versus host disease are thought to be mediated by il-18 [141] . so far, several anti-il-18 therapies have been reported. an anti-il-18, multicenter, randomized, single-blind, placebo-controlled, parallel-group, phase iia trial for the treatment of t2d was reported whereby anti-il-18 monoclonal antibody, gsk1070806, was well-tolerated; however, the anti-il-18 therapy did not lead to any improvements in glucose control [142] . interleukin-18 binding protein (il-18bp) was purified from urine by chromatography on il-18 beads that abolished il-18 induction of ifn-γ, il-8, and activation of nf-κb in vitro [143] . the il-18 inhibition using il-18bp significantly decreased mdscs in the tumor microenvironment in a preclinical osteosarcoma mouse model [144] . il-18bp (tadekinig α®) was successful in the treatment of still's disease and nlrc4-mutated autoinflammatory macrophage activation syndrome (mas), for which anti-il-1 treatment had failed [145, 146] . several inhibitors of il-1 signaling have been clinically approved (fig. 1) . one is a recombinant human intrinsic il-1 receptor antagonist (il-1ra), anakinra [147] . anakinra is the pharmaceutical name of a recombinant form of intrinsic human il-1ra, a 17.2-kda protein consisting of 153 amino acid residues. il-1ra was first reported in 1985 as a bioactive il-1 inhibitor of 22-25 kda in the supernatants of human monocyte culture, and it was independently identified as an il-1 inhibitor from the urine of febrile patients [148, 149] . anakinra was the first biological drug of a selective il-1r1 antagonist to receive approval from the us food and drug administration (fda). since anakinra is an il-1 receptor antagonist, it can prevent the activity of both il-1α and il-1β by competitively blocking their binding to il-1r1 and il-1r2. anakinra has been applied for a wide range of diseases including autoinflammatory diseases, non-cancer inflammatory diseases, and malignancies [150] . to date, no serious adverse effect of anakinra has been reported [151] . another is rilonacept (ril on' a sept), a soluble decoy receptor (fig. 1) . rilonacept is a recombinant fusion protein consisting of the extracellular portion of human il-1r1 and il-1r3 fused with the fc portion of human igg1 [152] [153] [154] . rilonacept binds to both il-1α and il-1β with high affinity and inhibits the activity of both with a long-term inhibitory effect. rilonacept was first approved by the fda for the treatment of caps in 2008. subcutaneous injection with a loading dose and a weekly injection of half the loading dose was administered [154] . there are no known severe adverse effects of rilonacept due to il-1 signaling inhibition. these drugs could modulate the immune response. the most common side effects (> 10% of treated patients) are inflammation of the upper respiratory tract or sinuses, headache, and redness at the injection site [154] . the third is canakinumab (fig. 1) . canakinumab, a specific human monoclonal igg1 antibody targeting il-1β, is intravenously or subcutaneously infused to neutralize the bioactivity of human il-1β [155, 156] . canakinumab does not react with il-1α or il-1r1. therefore, canakinumab is a more specific inhibitor of il-1β, expected to have no effect on il-1α-dependent host defense [154] . early clinical trials established the administration of canakinumab every 2 weeks as safe and effective against several inflammatory diseases [155, 156] . there are several agents currently undergoing clinical trials. il-1α production is a very early step in the sterile inflammatory response at the center of the malignant phenotype that drives angiogenesis, tumor stromal remodeling, tumor invasiveness, metastasis, and cachexia [150, [157] [158] [159] . thus, il-1α may be a particularly important target for the treatment of cancer. a neutralizing true human igg1κ a b d c fig. 1 interleukin-1 receptors and inhibitors of il-1 signaling. a il-1r1 interacts with both il-1α and il-1β and promotes signal transduction, together with its co-receptor il-1r3 (il-1racp). il-1ra is a protein that binds to il-1r1 but not il-1r3, and it is as an inhibitor of il-1 signaling. il-1r2 is a decoy receptor because it lacks a cytoplasmic segment. b anakinra is a recombinant form of intrinsic human il-1ra. it works as an antagonist of il-1r1, and it is able to inhibit both il-1α and il-1β. c rilonacept is a recombinant fusion protein including the extracellular protein of human il-1r1 and il-1r3 fused with the fc portion of human igg1. it binds to both il-1α and il-1β with high affinity and has a long-term inhibitory effect. d canakinumab and mabp1 are monoclonal antibodies against il-1β and il-1α, respectively. they bind to and neutralize their targets specifically monoclonal antibody specific for human il-1α, mabp1, has been developed, and it was well-tolerated with no dose-limiting toxicities or immunogenicity [160, 161] (fig. 1) . mabp1 treatment for patients with advanced colorectal cancer in a randomized, double-blind, placebocontrolled, phase 3 study revealed that mabp1 improved clinical performance in patients with advanced colorectal cancer [161] . mabp1 is a promising treatment for patients with hidradenitis suppurativa not eligible for the anti-tnf-α antibody adalimumab [162] . gevokizumab is an anti-il-1β monoclonal antibody, igg2, which improved glucose control and β-cell function in a diet-induced-obesity mouse model [163] and in the presence of il-1β-driven inflammatory diseases [164] . ly2189102 is a humanized monoclonal antibody (igg4) that binds to il-1β to neutralize its activity. its affinity is comparatively high (2.8 pmol/l). previous clinical studies evaluated not only its safety and pharmacokinetics but also its effects on ra (nct00380744). weekly treatment of t2d patients with ly2189103 for 3 months resulted in modest reductions in glycated hemoglobin and blood glucose [165] . population pharmacokinetics (pk) of ly2189102 were characterized using data from 79 t2d subjects (study h9c-mc-bbdk) who received 13 weekly subcutaneous doses of ly2189102 (0.6, 18, and 180 mg) and 96 ra subjects (study h9c-mc-bbde) who received five weekly intravenous (iv) doses (0.02-2.5 mg/kg) [166] . no additional study has been reported. amg 108 is a fully human, igg2 monoclonal antibody that binds to human il-1r1, inhibiting the activity of il-1α and il-1β [167] . patients with osteoarthritis received placebo or amg 108 subcutaneously (sc, 75 or 300 mg) or intravenously (iv, 100 or 300 mg) once every 4 weeks for 12 weeks or received placebo or 300 mg amg 108 sc, once every 4 weeks for 12 weeks; however, there was non-significant but numerically greater improvement in pain compared with the placebo group based on womac pain scores [168] . amg108 is now termed medi-8968 which has been studied in not only osteoarthritis, but also chronic obstructive pulmonary disease. in all cases, the benefit is limited [168, 169] . ebi-005 is a protein chimera of il-1β and il-1 receptor antagonists (il-1ra or anakinra). ebi-005 binds to il-1r1 and inhibits il-1 signaling and has been studied for the treatment of ocular surface inflammatory diseases [170] . since il-1β is known to be processed and activated by caspase-1, caspase-1 could be an indirect target for il-1β signaling. to examine this, the highly selective caspase-1 inhibitor vx-765 was applied to a rat model of myocardial infarction (mi) and mouse model of ad [171, 172] . the recombinant human il-1-receptor antagonist anakinra is markedly effective against caps such as mws, fcas, and nomid/cinca. weekly rilonacept treatment markedly improved the clinical symptoms of caps and normalized the levels of saa in those at risk of developing amyloidosis [90, 153, 173, 174] . in several case reports of patients with fmf, anti-il-1 treatment with anakinra, canakinumab, or rilonacept in colchicine-resistant patients was highly effective [175] [176] [177] [178] . it was also reported that there was a rapid and lasting response of pyoderma gangrenosum to targeted treatment with anakinra in a patient with papa syndrome [179] . anakinra and canakinumab therapies were also reported to be effective in patients with mkd/hids [180] . in the case of traps, although tnf-α is considered to be mainly involved in clinical manifestations, marked improvement following il-1β blockade occurred [112, 181] . an open-label, phase ii study was reported whereby 19 patients with active recurrent or chronic traps (19/20, 95%; 95% ci 75.1% to 99.9%) achieved the primary efficacy endpoint. canakinumab treatment for traps rapidly improved the median time to clinical remission to 4 days (95% ci 3 to 8 days) [182] . skin findings also promptly improved upon anakinra treatment in a patient with dira [183] . monotherapy involving canakinumab for the treatment of fmf has been reported [184] . a nationwide report on il-1 treatment of patients with fmf revealed that 172 fmf patients (83 [48%] female; mean age, 36.2 years [range, ) were included; the mean age at onset was 12.6 years (range, 1-48), and the mean colchicine dose was 1.7 mg/day (range, 0.5-4.0). anakinra was administered to 151 patients, and canakinumab was administered to 21 patients. anti-il-1 treatment was used in 84% of colchicine-resistant patients and 12% of amyloidosis patients. during the mean of 19.6 months of treatment (range, 6-98), the attack frequency per year was significantly decreased (from 16.8 to 2.4; p < 0.001), and symptoms of 42.1% of colchicine-resistant patients with fmf were ameliorated. in this study, the complete remission rate was 40% and inefficacy rate was 8% in patients treated with anakinra, whereas the complete remission rate was 65% and inefficacy rate was 6% for patients treated with canakinumab [185] . although the response rates were not significant (p = 0.144 and χ2 = 3.872606) in the study above [185] , in our opinion, long-acting canakinumab may be more efficacious than anakinra, considering the necessity of daily subcutaneous anakinra injection because of its short half-time clearance of less than 12 h [185] . there are suspected etiologies of autoinflammatory disorders, but all lack a known genetic basis. in patients with adult-onset still's disease (aosd), anakinra monotherapy is significantly effective and has become the standard therapy, especially in prednisone-resistant patients. commercially available anti-il-1 agents (anakinra/kineret®, canakinumab/ ilaris®, or rilonacept/arcalyst®) for patients with treatmentresistant aosd are effective. canakinumab and anakinra were also effective for patients with schnitzler syndrome, an adult-onset autoinflammatory disease characterized by focal urticaria and systemic inflammation including fever with bone and muscle pain, in the first placebo-controlled study, and several clinical trials are currently ongoing [186] [187] [188] [189] . il-1 blockade therapy using anakinra is successful in patients with psoriatic arthritis, ankylosing spondylitis, and ra. on the other hand, its efficacy and safety are insufficient, precluding use for patients with systemic lupus erythematosus or sjögren syndrome, and il-1β inhibition using canakinumab had no effect on the decline in β-cell function after diabetes onset in patients with type 1 diabetes mellitus resulting from autoimmune-mediated β-cell loss [190] [191] [192] [193] [194] . as for ra, the enhanced release of other proinflammatory cytokines such as tnf-α and il-6 plays important roles in the inflamed synovium of ra patients [195] . in patients for whom tnf-α blockers are contraindicated, anakinra is effective in controlling the disease activity of ra and has been licensed for treatment at a dose of 100 mg/day by subcutaneous injection every day [196, 197] . compared with anakinra, tnf inhibitors, such as the anti-tnf-α monoclonal antibody infliximab, or etanercept that fuse the tnf receptor to the end of the igg1 antibody, dominate the field of biologics for ra because of the sense of well-being experienced by patients within hours of treatment [198] . tocilizumab, a humanized anti-il-6 receptor (il-6r) monoclonal antibody, has also been shown to improve the symptoms of patients with ra [199] . however, those agents are associated with the risk of reactivating bacterial pathogens such as tuberculosis (tb) and malignancies [197] . notably, no cases of tb reactivation were reported in 7964 ra patients after anakinra treatment, whereas 8 cases of tb reactivation were reported in 10,281 ra patients after tocilizumab treatment, and 7 and 10 cases of tb reactivation were reported in 2626 and 3167 ra patients after tnf-inhibitor treatment with golimumab and certolizumab pegol, respectively. this suggests the low risk of tb reactivation in ra patients treated with anti-il-1 therapy [197] . anakinra is safe and may be associated with a dose-related survival advantage in patients with septic shock syndrome complicated by acute respiratory distress syndrome, disseminated intravascular coagulation, and renal dysfunction, and subsequent secondary hemophagocytic lymphohistiocytosis (hlh), or macrophage-activating syndrome (mas) [200, 201] . for sepsis with mas, anakinra treatment led to significant improvements in hepatobiliary dysfunction and disseminated intravascular coagulation in patients (65.4% anakinra vs. 35.3% placebo) and the 28-day survival rate, with the hazard ratio for death being 0.28 (0.11-0.71; p = 0.0071) for the treatment group on cox regression. the data included 763 adults in the original study cohort, randomized to receive either anakinra or placebo [202] . for metabolic syndromes il-1 inhibition by anakinra, rilonacept, or canakinumab is efficacious for gout patients [203] . il-1 plays a role in the progression of atherosclerosis as well [204] . in patients with a history of mi, canakinumab significantly reduced the high-sensitive serum crp concentration from the baseline, as compared with a placebo, without affecting the ldl-cholesterol concentration. a 150-mg dose of canakinumab resulted in a significantly reduced risk of recurrent cardiovascular events compared with a placebo [205] . the inhibition of il-1 with anakinra improved glycemia and the pancreatic β-cell function and reduced systemic inflammation [205] . although il-1β inhibition with canakinumab had similar effects on major cardiovascular events among those with and without diabetes, treatment over a median period of 3.7 years did not reduce incident diabetes [206] . the blockade of il-1 with anakinra improved glycemia and the β-cell secretory function and reduced markers of systemic inflammation [104] . anakinra also prevented transthyretin extracellular deposition in the sciatic nerve in a familial amyloidotic polyneuropathy mouse model [207] . during ischemic disease, such as mi or cerebral infarction, or tissue injury, cell death by necrosis takes place and the il-1α precursor is released in sterile inflammation [208] . when there is no time for the synthesis of il-1α, il-1α is ready to function as soon as it leaves the dying cell in the first few hours following tissue ischemia or injury [209] . in fact, animal studies showed that the inhibition of il-1 is effective in limiting atherosclerosis and cardiovascular events and improving the symptoms of acute mi and ischemic stroke [210, 211] . two pilot studies of il-1 inhibition in st-segment elevation mi revealed a reduced acute inflammatory response and favorable overall performance at the 3-month follow-up [212] . il-1β is thought to play an important role in cancer invasiveness, progression, and metastases via inflammation in the tumor microenvironment. a further analysis in the canakinumab anti-inflammatory thrombosis outcomes study (cantos), a randomized trial of the role of inhibition of il-1β in atherosclerosis, revealed that anti-inflammatory therapy with canakinumab targeting the il-1β innate immunity pathway could significantly reduce lung cancer mortality [213] . moreover, treatment of patients with metastatic breast cancer-related with anakinra eliminates a systemic transcriptional signature of il-1-associated inflammation in blood cells. the inflammatory signature in primary breast cancers identifies a subset of patients that could potentially benefit from il-1β-targeted therapies [129] . safety profiles of both anakinra and canakinumab were reported [214] . in this study, several clinical and therapeutic data on patients treated with either anakinra or canakinumab were retrospectively analyzed. four hundred and seventy-five patients participated; anakinra and canakinumab were prescribed in 421 and 105 treatment courses, respectively. eighty-nine adverse events were recorded; 13 (14.61%) were classified as serious adverse events during a mean follow-up of 24.39 ± 27.04 months. [214] . in addition, anakinra is applied to metastatic cancer. a trial involving metastatic colorectal cancer patients showed significantly increased survival when anakinra was added to standard chemotherapy for colorectal cancer and patients with her2-negative breast cancer [129, 215] . the il-1 blockade will reduce il1-driven inflammation and immunosuppression that may contribute to the tumor metastatic table 2 effective anti-il-1 therapy for inflammatory diseases autoinflammatory diseases: cryopyrin-associated periodic syndrome (caps) [87, 88] familial mediterranean fever (fmf) [95] pyogenic arthritis, pyoderma gangrenosum and acne syndrome (papa) [96] nlrp12 autoinflammatory syndrome [97] tumor necrosis factor receptor-1-associated syndrome (traps) [100] hyperimmunoglobulinemia d and periodic fever syndrome (hids)/ mevalonate kinase deficiency (mkd) [180] deficiency of the interleukin-1-receptor antagonist (dira) [183] autoimmune diseases: psoriatic arthritis [191] ankylosing spondylitis [192] rheumatoid arthritis (ra) [196] metabolic syndrome: gout [203] atherosclerosis [204] type 2 diabetes mellitus (t2d) [204] amyloidosis [207] neurodegenerative disease: alzheimer's disease (ad) [111] infections and inflammatory responses: septic shock syndrome [199] acute respiratory distress syndrome (ards) [199] disseminated intravascular coagulation (dic) [199] hemophagocytic lymphohistiocytosis (hlh) [200] macrophage-activating syndrome (mas) [200] ischemic diseases: myocardial infarction (mi) [209] stroke [209] malignant rumor: breast cancer [129] microenvironment [216] . the timeline of therapeutics is summarized in fig. 2 . we described il-1 as an important cytokine for not only inflammation related to cell injury but also homeostasis of cells, tissues, and organs in view of the general pathology. in addition, we also described recent expanding il-1 signal-targeting for the treatment of diseases. once the balance of il-1 signaling is disrupted, it may markedly contribute to the pathogenesis of not only inflammatory disease, but also malignancies. il-1-targeted biologics have been expanding, as there are no known serious adverse effects such as lymphoproliferative disorder or virus reactivation like tnf or il-6-targeting therapies. therefore, il-1 is expected to become an attractive molecular target to treat a wide range of diseases such as autoinflammatory diseases, autoimmune diseases, infectious disease, metabolic syndromes, ischemic diseases, and malignant tumors [217, 218] (table 2) . availability of data and materials not applicable. the manuscript was written by nk and jm, and all authors read and approved the final manuscript. ethics approval and consent to participate not applicable. not applicable. disturbance of function (functio laesa): the legendary fifth cardinal sign of inflammation, added by galen to the four cardinal signs of celsus robbins & cotran pathologic basis of disease inflammation and metabolic disorders endoplasmic reticulum stress and the inflammatory basis of metabolic disease inflammatory mechanisms in obesity approaching the asymptote? evolution and revolution in immunology stress, inflammation, and defense of homeostasis pattern recognition receptors and inflammation editorial overview: special section: effects of endogenous immune stimulants: from a defence system against infection to a homeostatic mechanism linking metabolism with inflammation the pyrin-card protein asc is an activating adaptor for caspase-1. srinivasula sm the inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proil-β nucleotide-binding oligomerization domain-like receptors and inflammasomes in the pathogenesis of nonmicrobial inflammation and diseases regulation of inflammasome signaling homeostatic inflammation, an emerging concept homeostatic inflammation in innate immunity inflammation, metaflammation and immunometabolic disorders origin and physiological roles of inflammation it's time to redefine inflammation il-1 family members in the pathogenesis and treatment of metabolic disease: focus on adipose tissue inflammation and insulin resistance immunological and inflammatory functions of the interleukin-1 family potentiation of the t-lymphocyte response to mitogens. i. the responding cell macrophage sensitivity to endotoxin: genetic control by a single codominant gene biochemical and biological characterization of lymphocyteactivating factor (laf) produced by the murine macrophage cell line, p388d interleukin 1: the first interleukin detection of anti-tumour cell mediated immunity and serum blocking factors in cancer patients by the leucocyte adherence inhibition test cell-mediated immunity and specific serum factors in human cancer: the leukocyte adherence inhibition test leukocyte adherence inhibition by soluble tumor antigens in breast cancer patients the inflammatory microenvironment in tumor progression: the role of tumor-associated macrophages blockage of the nlrp3 inflammasome by mcc950 improves anti-tumor immune responses in head and neck squamous cell carcinoma targeting inflammasome/il-1 pathways for cancer immunotherapy il-1 family nomenclature the interleukin-1 family: back to the future partial purification of human lymphocyte-activating factor (laf) by ultrafiltration and electrophoretic techniques crystallographic refinement of interleukin 1β at 2.0 a resolution tructure of interleukin 1 at 2.7-a resolution structure and function of interleukin-1, based on crystallographic and modeling studies the precursor form of il-1α is an intracrine proinflammatory activator of transcription the interaction with sp1 and reduction in the activity of histone deacetylase 1 are critical for the constitutive gene expression of il-1 alpha in human melanoma cells molecular analysis of constitutive il-1alpha gene expression in human melanoma cells: autocrine stimulation through nf-κb activation by endogenous il-1α inflammasome activators induce interleukin-1α secretion via distinct pathways with differential requirement for the protease function of caspase-1 identification of a key pathway required for the sterile inflammatory response triggered by dying cells the interleukin-1α precursor is biologically active and is likely a key alarmin in the il-1 family of cytokines cloning, sequence and expression of two distinct human interleukin-1 complementary dnas inflammasome-independent regulation of il-1-family cytokines treating inflammation by blocking interleukin-1 in a broad spectrum of diseases expression and alternative processing of il-18 in human neutrophils rapid and specific conversion of precursor interleukin 1β (il-1β) to an active il-1 species by human mast cell chymase multiple biological activities of human recombinant interleukin 1 granzyme a is an interleukin 1 beta-converting enzyme processing of precursor interleukin 1β and inflammatory disease nf-κb regulates il-1β transcription through a consensus nf-κb binding site and a nonconsensus cre-like site mechanisms of interleukin-1β release activation and regulation of the inflammasomes mechanisms and functions of inflammasomes the inflammasome nlrs in immunity, inflammation, and associated diseases activation of the nalp3 inflammasome is triggered by low intracellular potassium concentration k + efflux is the common trigger of nlrp3 inflammasome activation by bacterial toxins and particulate matter a genome-wide crispr (clustered regularly interspaced short palindromic repeats) screen identifies nek7 as an essential component of nlrp3 inflammasome activation nek7 is an essential mediator of nlrp3 activation downstream of potassium efflux nlrp3 activation and mitosis are mutually exclusive events coordinated by nek7, a new inflammasome component noncanonical inflammasome activation by intracellular lps independent of tlr4 cytoplasmic lps activates caspase-11: implications in tlr4-independent endotoxic shock inflammatory caspases are innate immune receptors for intracellular lps caspase-4 mediates non-canonical activation of the nlrp3 inflammasome in human myeloid cells bax/bak-induced apoptosis results in caspase-8-dependent il-1β maturation in macrophages cleavage of gsdmd by inflammatory caspases determines pyroptotic cell death caspase-11 cleaves gasdermin d for non-canonical inflammasome signalling gasdermin d is an executor of pyroptosis and required for interleukin-1β secretion gsdmd membrane pore formation constitutes the mechanism of pyroptotic cell death gsdmd p30 elicited by caspase-11 during pyroptosis forms pores in membranes poreforming activity and structural autoinhibition of the gasdermin family inflammasomeactivated gasdermin d causes pyroptosis by forming membrane pores detection of il-1 alpha and il-1 beta gene expression by in situ hybridization. tissue localization of il-1 mrna in the normal c57bl/6 mouse the interleukin-1 family: 10 years of discovery mass spectrometric analysis of the endogenous type i interleukin-1 (il-1) receptor signaling complex formed after il-1 binding identifies il-1racp, myd88, and irak-4 as the stable components mouse neutrophils express the decoy type 2 interleukin-1 receptor (il-1r2) constitutively and in acute inflammatory conditions il-1 receptor type 2 suppresses collagen-induced arthritis by inhibiting il-1 signal on macrophages the interleukin-1 receptor family interleukin-1-induced activation of a protein kinase co-precipitating with the type i interleukin-1 receptor in t cells nf-κb activation by interleukin-1 (il-1) requires an il-1 receptor-associated protein kinase activity evidence from sequence information that the interleukin-1 receptor is a transmembrane gtpase interleukin-1 (il-1) pathway the interleukin-1 receptor/toll-like receptor superfamily: signal transduction during inflammation and host defense targeting of tak1 in inflammatory disorders and cancer tnf and map kinase signalling pathways mutation of a new gene encoding a putative pyrin-like protein causes familial cold autoinflammatory syndrome and muckle-wells syndrome de novo cias1 mutations, cytokine activation, and evidence for genetic heterogeneity in patients with neonatal-onset multisystem inflammatory disease (nomid): a new member of the expanding family of pyrinassociated autoinflammatory diseases chronic infantile neurological cutaneous and articular syndrome is caused by mutations in cias1, a gene highly expressed in polymorphonuclear cells and chondrocytes neonatal-onset multisystem inflammatory disease responsive to interleukin-1beta inhibition pattern of interleukin-1beta secretion in response to lipopolysaccharide and atp before and after interleukin-1 blockade in patients with cias1 mutations genetic and molecular basis of inflammasomemediated disease innate immune sensing of bacterial modifications of rho gtpases by the pyrin inflammasome papa, pash and papash syndromes: pathophysiology, presentation and treatment the b30.2 domain of pyrin, the familial mediterranean fever protein, interacts directly with caspase-1 to modulate il-1β production pyrin binds the pstpip1/cd2bp1 protein, defining familial mediterranean fever and papa syndrome as disorders in the same pathway clinical presentation and pathogenesis of cold-induced autoinflammatory disease in a family with recurrence of an nlrp12 mutation lack of isoprenoid products raises ex vivo interleukin-1 secretion in hyperimmunoglobulinemia d and periodic fever syndrome clinical, genetic, and therapeutic diversity in 2 patients with severe mevalonate kinase deficiency dramatic improvement following interleukin 1beta blockade in tumor necrosis factor receptor-1-associated syndrome (traps) resistant to anti-tnf-α therapy an autoinflammatory disease with deficiency of the interleukin-1-receptor antagonist glucose-induced beta cell production of il-1beta contributes to glucotoxicity in human pancreatic islets thioredoxin-interacting protein links oxidative stress to inflammasome activation free fatty acids induce a proinflammatory response in islets via the abundantly expressed interleukin-1 receptor i activation of the nlrp3 inflammasome by islet amyloid polypeptide provides a mechanism for enhanced il-1β in type 2 diabetes iapp boosts islet macrophage il-1 in type 2 diabetes resident macrophages mediate islet amyloid polypeptide-induced islet il-1β production and β-cell dysfunction the nalp3 inflammasome is involved in the innate immune response to amyloid-β iapp/amylin deposition, which is correlated with expressions of asc and il-1β in β-cells of langerhans' islets, directly initiates nlrp3 inflammasome activation amyloid β directly interacts with nlrp3 to initiate inflammasome activation: identification of an intrinsic nlrp3 ligand in a cell-free system association of interleukin-1 gene polymorphisms with central obesity and metabolic syndrome in a coronary heart disease population pattern of interleukin-1β secretion in response to lipopolysaccharide and atp before and after interleukin-1 blockade in patients with cias1 mutations autoinflammation: translating mechanism to therapy central delivery of iodine-125-labeled cetuximab, etanercept and anakinra after perispinal injection in rats: possible implications for treating alzheimer's disease critical role for interleukin-1β (il-1β) during chlamydia muridarum genital infection and bacterial replication-independent secretion of il-1beta in mouse macrophages negative regulation of cytokine signaling influences inflammation interleukin-1β triggers the differentiation of macrophages with enhanced capacity to present mycobacterial antigen to t cells the role of interleukin 1 in human b cell activation: inhibition of b cell proliferation and the generation of immunoglobulin-secreting cells by an antibody against human leukocytic pyrogen ril 2-induced proliferation of human circulating nk cells and t lymphocytes: synergistic effects of il 1 and il 2 il-1β strikingly enhances antigen-driven cd4 and cd8 t-cell responses the nlrp3 inflammasome in kidney disease and autoimmunity cancer-related inflammation expression of interleukin-1β in human breast carcinoma the interleukin-1 family of cytokines and receptors in human breast cancer: implications for tumor progression cancer: an infernal triangle il-1 and il-1 regulatory pathways in cancer progression and therapy s1pr1 on tumor-associated macrophages promotes lymphangiogenesis and metastasis via nlrp3/il-1β obesity-associated nlrc4 inflammasome activation drives breast cancer progression il1 receptor antagonist controls transcriptional signature of inflammation in patients with metastatic breast cancer neutrophil proteinase 3-mediated induction of bioactive il-18 secretion by human oral epithelial cells activation of interferon-gamma inducing factor mediated by interleukin-1beta converting enzyme caspase-1 processes ifn-gamma-inducing factor and regulates lps-induced ifn-gamma production interleukin-18 and il-18 binding protein endotoxin-induced serum factor that stimulates gamma interferon production a novel role for interleukin-18 in adhesion molecule induction through nf-κb and phosphatidylinositol (pi) 3-kinase-dependent signal transduction pathways differences in signaling pathways by il-1beta and il-18 gene expression, synthesis, and secretion of interleukin 18 and interleukin 1beta are differentially regulated in human blood mononuclear cells and mouse spleen cells interferon-gammainducing factor enhances t helper 1 cytokine production by stimulated human t cells: synergism with interleukin-12 for interferon-gamma production m-csf induces the expression of a membrane-bound form of il-18 in a subset of human monocytes differentiating in vitro toward macrophages ifn-gammadependent and -independent mechanisms in adverse effects caused by concomitant administration of il-18 and il-12 natural killer cells in human autoimmune diseases a study to investigate the efficacy and safety of an anti-interleukin-18 monoclonal antibody in the treatment of type 2 diabetes mellitus interleukin-18 binding protein: a novel modulator of the th1 cytokine response inhibition of il-18-mediated myeloid derived suppressor cell accumulation enhances anti-pd1 efficacy against osteosarcoma cancer prolonged treatment with tadekinig alfa in adult-onset still's disease life-threatening nlrc4-associated hyperinflammation successfully treated with il-18 inhibition interleukin 1 receptor antagonist. a new member of the interleukin 1 family effects of immune complexes on production by human monocytes of interleukin 1 or an interleukin 1 inhibitor identification of a specific interleukin 1 inhibitor in the urine of febrile patients an expanding role for interleukin-1 blockade from gout to cancer a pilot study to evaluate the safety and efficacy of the longacting interleukin-1 inhibitor rilonacept (interleukin-1 trap) in patients with familial cold autoinflammatory syndrome efficacy and safety of rilonacept (interleukin-1 trap) in patients with cryopyrinassociated periodic syndromes: results from two sequential placebocontrolled studies church ld, mcdermott mf. canakinumab, a fully-human mab against il-1 for the potential treatment of inflammatory disorders antiinflammatory therapy in clinical care: the cantos trial and beyond il-1α secreted by colon cancer cells enhances angiogenesis: the relationship between il-1α release and tumor cells' potential for liver metastasis interleukin-1α secreted by pancreatic cancer cells promotes angiogenesis and its therapeutic implications il-1 is required for tumor invasiveness and angiogenesis mabp1, a first-in-class true human antibody targeting interleukin-1α in refractory cancers: an open-label, phase 1 dose-escalation and expansion study mabp1 as a novel antibody treatment for advanced colorectal cancer: a randomised, double-blind, placebo-controlled, phase 3 study mabp1 targeting il-1α for moderate to severe hidradenitis suppurativa not eligible for adalimumab: a randomized study xoma 052, an anti-il-1β monoclonal antibody, improves glucose control and β-cell function in the diet-induced obesity mouse model il-1β inhibition in cardiovascular complications associated to diabetes mellitus double-blind, randomized study evaluating the glycemic and anti-inflammatory effects of subcutaneous ly2189102, a neutralizing il-1β antibody, in patients with type 2 diabetes population pharmacokinetic modeling of ly2189102 after multiple intravenous and subcutaneous administrations a phase 2 randomized, double-blind study of amg 108, a fully human monoclonal antibody to il-1r, in patients with rheumatoid arthritis a randomized, double-blind study of amg 108 (a fully human monoclonal antibody to il-1r1) in patients with osteoarthritis of the knee a randomised, placebo-controlled trial of anti-interleukin-1 receptor 1 monoclonal antibody medi8968 in chronic obstructive pulmonary disease preclinical development of ebi-005: an il-1 receptor-1 inhibitor for the topical ocular treatment of ocular surface inflammatory diseases the highly selective caspase-1 inhibitor vx-765 provides additive protection against myocardial infarction in rat hearts when combined with a platelet inhibitor caspase-1 inhibition alleviates cognitive impairment and neuropathology in an alzheimer's disease mouse model interleukin-1-receptor antagonist in the muckle-wells syndrome prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1 receptor antagonist the efficacy of anakinra in an adolescent with colchicine-resistant familial mediterranean fever anti-il-1 treatment for secondary amyloidosis in an adolescent with fmf and behçet's disease rilonacept for colchicine-resistant or -intolerant familial mediterranean fever: a randomized trial canakinumab for the treatment of autoinflammatory recurrent fever syndromes targeted treatment of pyoderma gangrenosum in papa (pyogenic arthritis, pyoderma gangrenosum and acne) syndrome with the recombinant human interleukin-1 receptor antagonist anakinra efficacy of interleukin-1-targeting drugs in mevalonate kinase deficiency beneficial response to interleukin 1 receptor antagonist in traps canakinumab treatment for patients with active recurrent or chronic tnf receptor-associated periodic syndrome (traps): an open-label, phase ii study deficiency of interleukin-1 receptor antagonist responsive to anakinra canakinumab as monotherapy for treatment of familial mediterranean fever -first report in central and eastern europe region nationwide experience with off-label use of interleukin-1 targeting treatment in familial mediterranean fever patients efficacy and safety of canakinumab in patients with still's disease: exposure-response analysis of pooled systemic juvenile idiopathic arthritis data by age groups rapid responses to anakinra in patients with refractory adult-onset still's disease adult onset still's disease-the evidence that antiinterleukin-1 treatment is effective and well-tolerated (a comprehensive literature review) efficacy and safety of canakinumab in schnitzler syndrome: a multicenter randomized placebo-controlled study anakinra for rheumatoid arthritis: a systematic review an open-label pilot study of the efficacy and safety of anakinra in patients with psoriatic arthritis refractory to or intolerant of methotrexate open label trial of anakinra in active ankylosing spondylitis over 24 weeks a systematic review of the off-label use of biological therapies in systemic autoimmune diseases interleukin-1 antagonism in type 1 diabetes of recent onset: two multicentre, randomised, double-blind, placebo-controlled trials revolutionary change in rheumatoid arthritis management with biological therapy interleukin-1 in the pathogenesis and treatment of inflammatory diseases tuberculosis risk in patients treated with nonanti-tumor necrosis factor-α (tnf-α) targeted biologics and recently licensed tnf-α inhibitors: data from clinical trials and national registries blockade of tnf-α rapidly inhibits pain responses in the central nervous system study of active controlled monotherapy used for rheumatoid arthritis, an il-6 inhibitor (samurai): evidence of clinical and radiographic benefit from an x ray reader-blinded randomised controlled trial of tocilizumab il-1ra sepsis syndrome study group. initial evaluation of human recombinant interleukin-1 receptor antagonist in the treatment of sepsis syndrome: a randomized, open-label, placebo-controlled multicenter trial therapeutic role of anakinra, an interleukin-1 receptor antagonist, in the management of secondary hemophagocytic lymphohistiocytosis/sepsis/multiple organ dysfunction/macrophage activating syndrome in critically ill children interleukin-1 receptor blockade is associated with reduced mortality in sepsis patients with features of macrophage activation syndrome: reanalysis of a prior phase iii trial anti-interleukin-1 therapy in the management of gout interleukin-1β inhibition and the prevention of recurrent cardiovascular events: rationale and design of the canakinumab anti-inflammatory thrombosis outcomes study (cantos) cantos trial group. antiinflammatory therapy with canakinumab for atherosclerotic disease anti-inflammatory therapy with canakinumab for the prevention and management of diabetes interleukin-1 signaling pathway as a therapeutic target in transthyretin amyloidosis interleukin-1-receptor antagonist in type 2 diabetes mellitus the sterile inflammatory response differential release of chromatin-bound il-1 discriminates between necrotic and apoptotic cell death by the ability to induce sterile inflammation blocking interleukin-1 as a novel therapeutic strategy for secondary prevention of cardiovascular events comparative safety of interleukin-1 blockade with anakinra in patients with st-segment elevation acute myocardial infarction (from the vcu-art and vcu-art2 pilot studies) effect of interleukin-1β inhibition with canakinumab on incident lung cancer in patients with atherosclerosis: exploratory results from a randomised, double-blind, placebo-controlled trial working group" of systemic autoinflammatory diseases of sir (italian society of rheumatology). safety profile of the interleukin-1 inhibitors anakinra and canakinumab in real-life clinical practice: a nationwide multicenter retrospective observational study fluorouracil and bevacizumab plus anakinra for patients with metastatic colorectal cancer refractory to standard therapies (irafu): a single-arm phase 2 study interleukin-1 beta-a friend or foe in malignancies? blocking il-1: interleukin 1 receptor antagonist in vivo and in vitro treating inflammation by blocking interleukin-1 in humans all authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord-005872-w1x1i0im authors: volk, t.; kox, w.j. title: endothelium function in sepsis date: 2000 journal: inflamm res doi: 10.1007/s000110050579 sha: doc_id: 5872 cord_uid: w1x1i0im endothelial cells can be the prime target for an infection and infected endothelial cells may serve as an initiating system for a systemic response as these cells are able to secrete many mediators known to be of paramount importance. endothelial cell functions in turn are regulated by these circulating mediators. cellular interactions with leukocytes revealed protective and destructive functions. single cell and animal studies indicate that endothelial permeability is increased and apart from clinical obvious edema formation in septic patients, the endothelial component remains unknown. endothelial coagulation activation has been shown in vitro, however human data supporting an endothelial procoagulatory state are lacking. defects in endothelium dependent vasoregulation in animal models are well known and again human studies are largely missing.¶an imbalanced production of reactive oxygen species including nitric oxide has been found to be involved in all endothelial functions and may provide a common link which at present can be supported only in animal studies. sepsis is considered the leading cause of death in noncoronary intensive care units. it has been defined as a systemic inflammatory reaction to an infection. among many cellular disturbances endothelial cells play a major role in the pathogenesis of this disease as these cells are critically involved in maintaining a delicate balance between vasoconstriction and vasodilation, blood cell adherence and nonadherence, anticoagulation and procoagulation, permeability and thightness. all these functions are believed to be imbalanced and impairment is believed to precede clinically recognizable alterations (e.g. bleeding, edema, organ dysfunction and shock) but it is by no means clear whether these changes are the cause or consequence of endothelial dysfunction in sepsis. endothelial functions have largely been studied in vitro from the first successful attempts of culturing isolated human endothelial cells dating back to the early 70ties. endothelial cells from and within different organs or different species behave differently and tend to change properties the longer they are kept in culture. to overcome isolation variabilities several endothelial cell lines are currently available which have retained at least some features. however, experiments from single cell cultures are flawed in many ways and results from these may never be of any relevance to medical practice. it is inherent to any cell culturing that data obtained from these experiments often are restricted to cell type, time of culture and general working conditions. in vitro stressed endothelial cells almost uniquely tend to activate a functional program leading to a proinflammatory, procoagulatory and hyperpermeable phenotype. in this review we want to summarize investigations of disturbed endothelial functions including infection, mediator and cellular interactions, permeability, coagulation and vasoactivc properties from molecular findings to patient care. staphylococcus aureus is the most prevalent bacterial pathogen isolated from patients with blood stream infection in north america [1] . s. aureus has been reported to directly infect human umbilical vein endothelial cells (huvec) thereby inducing secretion of cytokines and functional upregulation of adhesion molecules [2] . internalized s. aureus may lead to apoptosis in huvec [3] or persistence as small colony variants [4] . group a streptococci can enter huvec [5] , a process which may render these cells particularly sen-sitive to otherwise subtoxic concentrations of hydrogen peroxide [6] . group b streptococci (gbs) are the most common cause of neonatal sepsis and pneumonia. gbs-induced endothelial cell injury can be confirmed by histological findings at autopsy, in animal studies and in vitro [7] . gbs invasion and subsequent damage of endothelial cells may be inhibited by cytochalasin d in huvec implicating that cytoskeletal interactions are important for toxicity. however, invasion of brain microvascular endothelial cells by gbs may be dose dependently cytotoxic due to beta-hemolysin production [8] . streptococcus pneumoniae may enter activated endothelial cells using paf-receptors [9] . this receptor engagement may also serve as a sorting signal for endothelial transcytosis [10] . infection and activation of endothelial cells by listeria monocytogenes is believed to be a critical component of the pathogenesis of this disease and includes ceramide generation, transcription factor activation and increases in adhesion molecule expression on huvec [11] . listeria have been described to enter huvec either directly via internalin b or by a cell-to-cell spread from infected monocytes [12] . gram negative bacteria have lipopolysaccarides (lps) within their cell wall. cellular binding of lps usually is accomplished by cd14. endothelial cells lack cd14 receptors and lps effects on endothelial cells generally require the presence of cd14 in the serum. lps effects from gram negative live bacteria (b. fragilis, e. cloacae, h. influenzae, k pneumoniae) on endothelial cells have been demonstrated by transcription factor activation and subsequent surface expression of e-selectin and tissue factor which was not seen from viable or heat-killed gram-positive bacteria (s. aureus, e. faecalis, s. pneumoniae) [13] . neisseria meningitidis adherence on endothelial cells has been reported to be influenced by pilus protein c expression and cd66 on endothelial surfaces [14, 15] and may cause tissue factor expression due to the presence of lps within the cell wall. haemophilus influenzae generally is not toxic to endothelial cells except three clones of biogroup aegyptius causing brazilian purpuric fever [16] . toxicity has been reported to be independent of endotoxin, phagocytosis and replication since irradiation, cycloheximide, cytochalasin d and methylamine have no effect on the ability of the bacterium to invade and cause a cytotoxic response. piliated pseudomonas aeruginosa adheres to and enters human endothelial cells leading to progressive damage [17] or may persist by lysis of endosomal membranes [18] . escherichia coli may invade human brain microvascular endothelial cells involving specialized proteins [19, 20] . endothelial infection by chlamydia pneumoniae also activates endothelial cells to produce cytokines and adhesion molecules and become procoagulant [21] [22] [23] . bartonella quintana, the cause of trench fever transmitted by the body louse, has recently been implicated in culture-negative endocarditis and bacteraemia amongst homeless people [24] . infection and damage of endothelial cells by b. quintana has been demonstrated in vitro and in vivo [25] . interaction of bartonella henselae with endothelial cells may result in bacterial aggregation on the cell surface and the subsequent internalisation of the bacterial aggregate by a unique struc-ture, the invasome [26] . rickettsia rickettsii, an obligate intracellular gram-negative bacterium which causes rocky mountain spotted fever, inhibits endothelial apoptosis allowing to remain inside the host endothelium [27] but is also known to cause a proinflammatory endothelial phenotype. rickettsia conorii causes mediterranean spotted fever which causes endothelial infection with secretion of cytokines, increases in adhesion molecules and induction of surface tissue factor [28, 29] . attachment of borrelia burgdorferi, the agent inducing lyme disease, to endothelial cells may be accomplished by cd14, alpha(v)beta3 and alpha5beta1 integrins or different classes of proteoglycans [30] [31] [32] . b. burgdorferi activates the transcription of chemokine and adhesion in molecule gene expression in endothelial cells [33] . plosmodium infected erythrocytes may bind to endothelial cells via p-selectin, cd36, intercellular adhesion molecule 1 (icam-1) or platelet endothelial cell adhesion molecule 1 (pecam-1) on the endothelial surface [34] [35] [36] . phagocytosed live candida albicans stimulates cytokine secretion and inducible cyclooxygenase expression in endothelial cells [37, 38] . some viral diseases are known to primarily infect endothelial cells and alter their function. dengue virus infection of huvec leads to production of chemoattractant proteins rantes and il-8 [39] , herpes and measles virus infection of brain microvascular endothelial cells increases lymphocyte adhesion by increasing icam-1 [40] and measles virus and cytomegalovirus increases tissue factor expression on huvec [41, 42] . hemorrhagic fever caused by hantaviruses may be accomplished by integrin mediated endothelial infection [43] . on the other hand, ebola virus infects endothelial cells with a transmembrane glycoprotein and inhibits inflammatory responses [44] . exotoxins are known to directly activate endothelial cells. platelet activating factor (paf), no˙and pgi 2 secretion from huvec has been demonstrated by e. coli hemolysin via inositolphosphate/diacylglycerol formation and by s. aureus alpha-toxin via transmembrane ca 2+ entry [45] . there is increasing evidence that hemolytic uremic syndrome results from the systemic action of verocytotoxin producing e. coli on vascular endothelial cells [46] . alpha toxin from clostridium perfringens is a phospholipase c and has been reported to induce adhesion molecule expression and secretion of chemokines from endothelial cells [47] . brain capillary endothelial cells have been reported to express mbec1, a protein that may serve as the c. perfringens enterotoxin receptor [48, 49] . the activity of small gtpase rho has been shown to be altered by c. difficile toxin b [50] and pasteurella mulrocida toxin [51] , which leads to alterations of endothelial permeability. p. aeruginosa exotoxin a may directly injure endothelial cells by a motif shared by many toxins [52] . listeriolysin and phosphatidylinositol-specific phospholipase c secreted from l. monocytogenes has been shown to induce phosphatidyinositol metabolism and diacylglycerol formation in the absence of bacterial uptake by the endothelial cells [53] . some bacterial exotoxins have been used for years as pharmacological tools like pertussis toxin for studying g-protein dependent endothelial cell functions. lipoteichoic acid and peptidoglycan from cell wall components of gram positive bacteria have been shown to induce sepsis in animal models. while lipoteichoic acid has been reported to directly activate endothelial cells [54] , peptidoglycan seems monocyte dependent [55] . most in vitro experiments, however, were performed with different sources of lps as a surrogate activator modelling gram negative bacterial infection. endothelial stimulation using lps in relevant concentrations are usually performed in the presence of serum containing soluble cd14-receptor because endothelial cells generally lack cd14. alternatively, proinflammatory cytokines including tumor necrosis factor alpha (tnf-a), interleukins (il-1, il-4) and interferon gamma (ifng) acting on endothelial cells have extensively been investigated and shown to alter endothelial in vitro functions [56, 57] . the inflammatory response in endothelial cells has been linked to an alteration in reactive oxygen production including superoxide (o 2 -˙) , hydrogen peroxide (h 2 o 2 ), nitric oxide (no˙), hydroxyl radicals (oh˙) and secondary reaction products thereof. o 2 -˙i s believed to be present in unstressed conditions in less than nanomolar quantities within cells. mitochondrial and cytoplasmatic superoxide dismutase readily reacts with o 2 -˙f orming h 2 o 2 which has been measured in micromolar concentrations in human blood. sources of endothelial o 2 -˙p roduction apart from mitochondrial leakage may include metabolism of cytp450 or other metabolic byproducts and production by a nadh oxidase system or by nitric oxide synthase in the absence of l-arginine. some bacterial pathogens are known to be able to produce h 2 o 2 by themselves, however interaction with the endothelium may greatly enhance cellular alterations. streptococcal hemolysin, streptolysin s, is capable of interacting with h 2 o 2 to injure vascular endothelial cells [6] . iron bound to the p. aeruginosa siderophore, pyochelin, augments oxidantmediated endothelial cell injury by modification of transferrin to form iron complexes capable of catalyzing the formation of oh˙from o 2 -˙a nd h 2 o 2 [58] . r. rickettsii infection of the endothelial cell line ea.hy 926 and huvec has been demonstrated to cause glutathione depletion, a major intracellular antioxidant, and reduced glutathione peroxidase activity leading to increased amounts of intracellular peroxide [59] . an increase in reactive oxygen species production has been shown in endothelial cells after incubation with lps, il-1, tnf-a and ifn-g [60] [61] [62] [63] [64] [65] . tnf-a has many times been reported to increase, e.g., adhesion molecule expression inhibitable by various antioxidants [62, [66] [67] [68] . nitric oxide, like o 2 -˙, is chemically not very reactive at all. endothelial production has been established by either constitutive no-synthase (nos) iii in a ca 2+ and phosphorylation dependent manner or by inducible nos ii. direct biochemical actions of no˙include metalcomplex containing proteins, other radical species, oxygen and oxygen derivatives leading to oxidation, nitrosation and nitration. relevant concentrations in vivo have been reported to range from nm to 100 mm. no˙directly reacts with oxyhemoglobin (fe 2 -o 2 ) leading to methemoglobin (fe 3+ ) and nitrate (no 3 -). no˙reacts with o 2 forming nitrite (no 2 -) via intermediate no 2 and n 2 o 3 . mainly n 2 o 3 is participating in n-or s-nitrosylations leading to nitrosamines or nitrosothioles, the latter may serve as a circulating source or as a transporter across cell membranes. reduced glutathione has a high affinity to n 2 o 3 and may also be important in toxicity related to no˙autoxidation. toxicity related cellular targets of no˙may include inhibition of cytochrome c oxidase, inhibition of catalase or dna damage. on the other hand, no˙has been reported to inhibit iron catalysed fenton reaction and to inhibit lipid peroxidation. iron nitrosyl formation in heme containing proteins are the best characterized reactions for no˙in biology. this type of interaction includes very sensitive stimulation of guanylate cyclase and inhibition of cytochrome c oxidase. e. coli hemolysin and s. aureus alpha toxin induce no˙formation in cultured porcine pulmonary endothelial cells [69] . transformed mouse endothelial cells stimulated by the combination of ifn-g and tnf-a killed intracellular r.conorii by a mechanism that required the synthesis of no˙ [70] . ifn-g, tnf-a and il-1 stimulated murine endothelial cells have been shown to kill schistosoma mansoni through the production of nitric oxide [71] . both no˙and o 2 -˙p roduction may have a limited influence on endothelial viability under resting conditions. cultured bovine and porcine aortic endothelial cells showed decreased no˙production after 1 h of lps incubation which was related to decreases in capacitative ca 2+ signals [72] . posttranscriptional destabilization of nos iii mrna may have accounted for this early decrease in no˙production [73] . nos ii, which is believed to produce larger amounts of no˙is also known to be regulated by many proinflammatory stimuli with significant cell type variablilities. at sites of endothelial involvement in an inflammatory process both vascular non-endothelial and non-resident cells are known to produce no˙. as the amount and physiological consequences of no˙produced from noss at the microcirculatory level is not known, it may well serve also to reduce inflammatory processes as recently implicated from a coculture experiment [74] . that an increase in reactive oxygen species is present in human sepsis has been documented by almost any study trying to quantify secondary reaction products by several methods, however the cellular sources remain speculative ( table 2 ). in 1990 beckman et al. showed that the presence of both no˙and o 2 -˙p roduced peroxynitrite (onoo -) which may decompose to produce ho˙like molecules and thereby kill endothelial cells [75] . at ph 7 its lifetime is in the order of a second. half of the onoo formed is rapidly equilibrated to peroxynitrous acid (onooh) and breaks down to no 3 11 sepsis immunohistochemical endothelial nitrotyrosine≠ [176] 3 septic shock no 2 -/no 3 -, plasmatic nitrotyrosine≠ [177] 3 septic lung injury immunohistochemical endothelial nitrotyrosine≠ antioxidant capacity was defined as the ability of plasma to inhibit * ferryl myoglobin production by hydrogen peroxide addition to metmyoglobin or ** oxo-iron induced damage to deoxyribose, phospholipids and dna. tbars, thiobarbituric acid reactive substances; xod, xanthine oxidase. models and therefore question the view that this molecule solely is detrimental. toxicity induced by an interaction between hydrogen peroxide and nitric oxide has led to conflicting results. rat lung microvascular endothelial cells and porcine pulmonary artery endothelial cells exposed to h 2 o 2 have been reported to be protected by no˙donors [76, 77] , whereas toxicity in bovine aortic endothelial cells [78] and in rat liver microvascular endothelial cells [79] was increased in the presence of no˙donors. no˙in the presence of h 2 o 2 may also produce oh˙like molecules independent of the presence of iron [80] . neutrophils may add to the complexity of toxic reactions. myeloperoxidase from activated neutrophils, which produces hocl and oh˙molecules in the presence of o 2 -˙, has recently been demonstrated to convert no 2 into no 2 , thereby damaging endothelial cells [81] . high doses of reactive oxygen species (including no˙) have been shown to cause apoptosis of endothelial cells, whereas low doses were protective [82] . in mice disseminated endothelial apoptosis has been suggested to be responsible for organ failure and shock induced by endotoxin or tnf-a [83] . both lps or tnf-a usually do not cause endothelial cell death unless protein synthesis is blocked probably due to simultaneous increases in antiapoptotic protein synthesis [84] . however, postmortal investigation in humans deceased from or with sepsis did not confirm these results [85] . most of the genes activated in vitro during the endothelial stress response are controlled by at least two transcription factor families: activator protein 1 (ap-1) and nuclear factor kappa b (nfkb). nfkb has gained wide interest as a target in inflammatory diseases as it seems to be invariably upregulated [86] . a variety of agents including cytokines and reactive oxygen stress cause ikb to dissociate from the complex after phosphorylation by ikb-kinase complex. h 2 o 2 has been reported to activate transcription factor nfkb in porcine aortic endothelial cells [87] whereas huvec were unresponsive [88] . no˙has in most cases been shown to inhibit transcription factor nfkb and its influence on transcription factor ap-1 is unclear. inhibition of nfk-b as a therapeutic means has been suggested. however, as this transcription factor is also involved in protective gene regulation, its inhibition can make cells sensitive to e.g. tnf-a [89] . data on activated intracellular signalling pathways in sepsis patients are scarce but include nfkb within mononuclear cells [90] . usually any blood cell is kept off endothelial surfaces. this is believed to be accomplished by net electrical charge, biomechanical characteristics of flowing blood and the secretion of no˙. if blood cells touch the endothelium a ca 2+ -signal is induced [91] , but it is unclear at present whether this has any functional consequence. it is tempting to speculate that ca 2+signals may then in a context sensitive manner augment proadhesive processes or antiadhesive endothelial properties. activated endothelial cells are potent producers of cytokines like il-1, a major proinflammatory cytokine, and chemotactic peptides including il-8 for neutrophils, macrophage inflammatory protein-1 alpha (mip-1a), monocyte chemoattractant proteins 1-4 and rantes (regulated on activa-tion, normal t cell expressed and secreted) for monocytes, t-lymphocytes and dendritic cells, growth related protein (gro) and gamma-interferon-inducible protein (ip-10) for activated t-lymphocytes, epithelial neutrophil activating peptide 78 (ena-78), vascular monocyte adhesion-associated protein (vmap-1) and endothelial monocyteactivating polypeptide ii (emap-ii) for monocytes [92] . many adhesion molecules are expressed on the surface of endothelial cells in a highly complex yet regulated manner. p-selectin, e-selectin, icam-1, vascular cell adhesion molecule 1 (vcam-1), pecam-1 are well known for their stimulus-, cell-, time-and organ specific dependence of expression and their importance in regulation of leukocyteendothelial interactions. moreover, apart from being passive adhesion molecules, all of the above mentioned molecules have been shown to signal inside endothelial cells upon receptor engagement. shed receptors from endothelial surfaces may also serve as endogeneous antiadhesive molecules demonstrating even more the dynamic and complex nature of these processes. soluble forms of adhesion molecules have been shown to be present in high amounts in human sepsis (table 3) , however whether this is beneficial or detrimental is not known and the assumption that these parameters may serve as practical indexes remains to be established. particularly ifn-g has been repeatedly shown to increase endothelial hla-dr expression rendering them capable of mhc class ii restricted interactions with cd4 + t-cells. costimulatory cd80 (b7-1) and cd86 (b7-2) are usually not present on endothelial surfaces leading to the conventional view that endothelial cells are semiprofessional antigen presenting cells. however, under certain conditions both costimulatory molecules and cd40 can be upregulated on endothelial surfaces indicating excessive antigen presentation [93] . migration of lymphocytes into inflamed mouse tis-vol. 49, 2000 endothelial function in sepsis 189 sues has been reported to depend on psgl-1 and esl-1 binding endothelial p-selectin and e-selectin, respectively [94] . this phenotype was found to be restricted to th1 lymphocytes, but human sepsis seems to be predominated by a th2 type [95] . both tnf-a and ifn-g are able to promote transmigration of leukocytes, whereas coapplication of both cytokines inhibit this process [96] . t-cells migrating through the endothelial barrier in a pecam-1-dependent manner are subject to inhibitory signals which may limit their activation in tissues [97] . tnf bound to monocytes has been shown to inhibit endothelial apoptosis, whereas this process is promoted in the presence of lymphocytes [98] . unperturbed endothelial cells express fas-ligand which has been implicated as a means of signalling apoptosis to constitutively fas receptor (cd95) bearing cells, whereas tnf-a treated endothelial cells decrease fas ligand expression thereby allowing leukocyte survival during extravasation [99] . how the endothelium might interact specifically with lymphocytes or monocytes in septic patients can only be speculated on. neutrophils interacting with endothelial cells have repeatedly been shown to cause toxicity due to the release of enzymes and reactive oxygen species. if uncontrolled, these cells are believed to mediate significant tissue damage. however whether uncontrolled activation or rather deactivation is present in human sepsis is a matter of debate. endothelial cells have been reported to inhibit some neutrophil functions [100] . transmigrated neutrophils may actively participate in the endothelial resealing process by the secretion of adenosin precursors [101] . however, massive leukocyte extravasation as one would expect from most animal studies has never been shown in septic patients [102] . endothelium is known to regulate transvascular fluid flux, flux of nutrients, mediators and cells by either paracellular or transcellular vacuolar channel related pathways. lateral junction proteins including the vascular endothelial cadherin-complex, platelet-endothelial cell adhesion molecule-1, occludin, zona occludens-1, recently described junctional adhesion protein, cd151/platelet endothelial tetraspan antigen and cd81/target of antiproliferative antigen are known to participate in this process. paracellular permeability is achieved by either an active contraction or the controlled release of an intrinsic tone mediated in most cases by the action of myosin light chain kinase (mlck) acting on non muscle myosin. generally an increase in the concentration of camp keeps cultured endothelial monolayers tight and cgmp has been reported to assist this function in human aortic and foreskin vessels [103] . endothelial retraction may be initiated by increases in intracellular ca 2+ concentration, but elevation of ca 2+ in the presence of maintained camp-kinase dependent phosphorylation is not edemagenic. these antagonistic effects of ca 2+ and camp in endothelial permeability regulation have recently been reviewed by moore et al. [104] . many reports documented increases in endothelial permeability involving exotoxins like s. aureus alpha-toxin and p. aeruginosa cytotoxin [105, 106] or endotoxins [107] . for example, p. multocida toxin has been shown to activate rho/rho kinase, which inactivates mlc phosphatase. the resulting increase in mlc phosphorylation caused endothelial cell retraction and a rise in endothelial permeability [51] . lps induced increases in paracellular permeability by caspase activated cleavage of adherens junction proteins [108] . counteracting lipid peroxidation during lps activation may inhibit increases in permeability [109] . tnf-a stimulated endothelial cadherin complex is disrupted in a proteasome dependent manner [110] . the resulting increase in permeability has been reported to lower camp and activate phosphediesterase ii and iv [111] . h 2 o 2 induced hyperpermeability of porcine pulmonary endothelial cells has been reported to be effectively reduced by cgmp elevating drugs including phosphodiesterase ii inhibition or no˙donators [112] . the electroneutral na-k-cl cotransport system is thought to function in the maintenance of a selective permeability. il-1, tnf-a and lps upregulate the expression of a bumetanide-sensitive na-k-cl cotransporter subtype in huvec and in murine lung and kidney endothelial cells [113] . septic rats show different increases in albumin flux accross several endothelial beds [114] . increases in venular permeability have been shown to be preventable by the antioxidants n-acetyl-cystein or tirilazad mesylate in e. coli infused rats [115, 116] . il-10, an antiinflammatory cytokine not produced by endothelial cells, was shown to participate as an inhibitor of endothelial permeability induced by lps in mice [117] . clinically, increases in endothelial permeability may be obvious in many septic patients, but only recently venous congestion plethysmography showed a selectively elevated filtration capacity as a measure of endothelial dysfunction in septic patients [118] . which of the many pathways of increased permeability might be turned on and whether it persists remains unknown. in principle endothelial cells are believed to be anticoagulatory by virtue of their surface expression of glycosaminoglycan-antithrombin iii complex, thrombomodulin, heparin releasable tissue factor pathway inhibitor and production of adenosine by ecto-adpases, their secretion of protein s, prostacyclin and no˙. no˙production was shown to participate in heparan sulfate preservation in porcine aortic endothelial cells [119] and may be responsible for prostacyclin secretion by activating cyclooxygenase-1 under resting conditions [120] . endothelial release of plasminogen activator (t-pa) and plasminogen activator inhibitor 1 (pai-1) may determine the fibrinolytic potential of plasma. endothelial cells specifically bind coagulation factors xii, iia, ix, viia and xa. xa was shown to bind to endothelial effector cell protease receptor-1 and thereby cause release of no˙, il-6, il-8, mcp-1 and functional upregulation of icam-1, e-selectin and vcam-1 [121] . when endothelium is perturbed by physical or chemical factors transformation to a prothrombotic surface is invariably seen in in vitro models. thrombomodulin surface expression on huvec can easily be downregulated by lps, il-1 and tnf-a and upregulated by increasing camp [122] . a tnf-a induced decrease in surface thrombomodulin has been suggested via activation of phosphodiesterase ii and iv thereby decreasing camp in baec [111] . vascular endothelial growth factor may counteract il-1, tgf-b and lps induced suppression of both thrombomodulin surface antigen and mrna [123] . adenosin nucleotides are released from damaged as well as lps stimulated and shear stressed huvec [124] . endothelial cells have atp diphosphohydrolase (cd 39) on their surface to degrade atp via adp and amp to adenosine. adenosine is known to have antiaggregatory properties due to stimulation of prostacyelin and no˙production. however, activating endothelial cells with tnf-a has been reported to cause loss of atp diphosphohydrolase activity, which was preventable in the presence of antioxidants [125] . tissue factor (tf) expression on endothelial cells by bacteria, lps, il-1 and tnf-a is well known. tissue factor pathway inhibitor (tfpi), a serine protease inhibitor of xa and xa/viia/tf complex on endothelial surfaces, which immediately blocks tissue factor activation, has been shown to be decreased under proinflammatory conditions. another counterbalancing mechanism includes shear stress in tnf-a stimulated huvec [126] . however, an anticipated increase in endothelial tissue factor expression has not convincingly been demonstrated in animal or human sepsis [127, 128] . fibrinolytic systems on endothelial surfaces are also believed to be altered in sepsis. increases in pai-1 has been reported after stimulation with il-1 or lps [129, 130] . however soluble pai-1 in septic patients was not found to be different from nonseptic patients [131] . once thrombin formation has occured, its cleavage of endothelial proteinase activated receptor (par) in turn may lead to secretion of il-8 and il-6 [132] , upregulation of icam-1 and vcam-1 [133] , relaxation via no˙production or to vasoconstriction by an as yet unidentified factor [134] . these data may demonstrate an interconnection between coagulation activation, inflammation and vasoregulation mediated by the endothelium. coagulation activation is clearly present in septic patients, however endothelial participation in this process is unclear. potent procoagulatorv sources may well include bacterial surfaces per se [135] or monocytes [128] . up to the late 60ies the endothelium was viewed as a passive organ, which at best was able to remove vasoactive hormones in the lung. in 1976-1978 sir john vane's group (noble laureate 1982) reported that endothelial cells can synthesize i series prostaglandins (like prostacyclin) and thereby relax arteries and inhibit platelet aggregation. however, whether pgi 2 regulates basal vascular tone is unclear. after a new technician used an unintended vessel preparation robert furchgott realized after a series of contradicting results that acetylcholine (ach) was no longer able to relax precontracted arteries when endothelium was removed and termed the nonprostanoid mediator an endothelium derived relaxing factor (edrf). in 1986 superoxide was shown to participate in vasoregulation. the presence of superoxide dismutase prolonged the action of edrf whereas addition of o 2 -˙i nactivated edrf. even direct effects of several reactive oxygen species have been suggested to be relevant in cerebral or coronary circulation. collectively robert furchgott, louis ignarro and ferrid murad received the noble laureate in 1998 for demonstrating that no˙is a major edrf. endothelial cells produce more relaxing factors which pharmacologically can be separated from nitric oxide action and these were termed endothelium derived hyperpolarizing factors (edhfs). these factors may particularly be important in coronary and gastrointestinal vessels. epoxyeicosatrienoic acids, anandamide, the endogenous ligand of cannabinoid receptors or simply the release of k + may constitute edhfs. conceptually shear may in larger vessels primarily determine production of no˙, whereas cyclic strain determines physiological edhf release. soon after the discovery of edrf endothelin-1 (et-1), a vasoconstricting peptide produced from endothelial cells was isolated. low doses of et-1 can induce no˙release and subsequent relaxation via et breceptors on endothelial cells. endothelin secretion is thought to occur abluminally leading to et a -receptor activation on smooth muscle cells and subsequent vasoconstriction. many other factors clearly contribute to endothelial control of vasoregulation by e.g. transcellular production of vasoconstricting prostanoids like thromboxane a 2 or prostaglandin h 2 or the enzymatic conversion of angiotensin i to angiotensin ii by angiotensin converting enzyme. a hallmark of sepsis is the heterogeneous pattern of vasoconstriction and vasodilatation in different organs, culminating in a fall in total peripheral vascular resistance concomitant with regional maldistribution of blood flow. vasoactive substances produced by the endothelium under experimental septic conditions are known to be altered by factors such as no˙, pgi 2 , angiotensin converting enzyme (ace) activity, endothelin and adrenomedullin. endothelium dependent vasoregulation has largely been studied in animal models (table 4 ). in 1985 endothelium dependent vasoregulatory failure was seen as a defect in reactive hyperemia related vasodilator release [136] and decreased dilatation of arterioles induced by ach [137] . in a rat model of cecal ligation and puncture decreased vasoconstriction was found after administration of norepinephrine in septic animals which was largely reversible by removal of the endothelium [138] . parker et al. [139] showed in explanted coronary arteries and aortas of guinea pigs treated with lps intraperitoneally for 16 h that endothelium dependent relaxation induced by acetylcholine and adp was depressed, whereas relaxation induced by substance p or receptor independent relaxations by ca 2+ -ionophore a23187 was unaffected. edrf release and bioactivity from explanted aortas of these animals was decreased after adp or ach stimulation, whereas a23187 induced edrf release was unaltered [140] . this group also demonstrated reduced adp and ach responses after 4 h of lps endotoxemia in guinea pigs and that adp may produce constricting thromboxane in septic animals [141] . in contrast, coronary arteries of rabbits treated for 5 weeks with low doses of lps stimulation with ach but not adp showed increased relaxation of explanted vessels [142] . wang et al. [143] isolated subepicardial arterioles from rats treated 48 h intraperitoneally with feces containing life e. coli and showed in a pressurized no-flow chamber that relaxation by alpha 2 agonist clonidine and adp was reduced in an endothelium dependent manner, which could be inhibited by the nos inhibitor lnma. also, in this model mesenteric arter-vol. 49, 2000 endothelial function in sepsis iolar relaxation after adp and clonidine was decreased, whereas in skeletal muscle these agonists caused vasoconstriction [144] . pulmonary arteries of rats treated with lps showed depressed endothelin-1 induced contractions which were even augmented in endothelium denuded vessels. the authors [145] concluded that a vasoconstrictor eicosanoid is produced in lps treated animals by pulmonary endothelium upon et-1 stimulation. swine infused with live p. aeruginosa showed no alteration in endothelium dependent bradykinin and endothelium independent nitroprusside relaxation, whereas ach induced relaxation was found to be reduced in explanted peripheral arteries [146] . chaudry's group investigated endothelium dependent relaxation in rats treated with cecal ligation and puncture. a time dependent alteration was demonstrated with increased ach induced vasorelaxation early after challenge whereas depressed vasodilatation after 5-20 h was found in explanted aortas with no alteration in nitroglycerine induced relaxation [147] . the decreased endothelium dependent response to ach was also found in superior mesenteric arteries and small intestinal arteries [148] . in the same model this group demonstrated a reduction of immunodetectable nos iii in explanted aortas [149] . porcine coronary arterioles incubated for 20 h with e. coli lps (100 mg/ml) decreased bradykinin induced edhf secretion [150] . carotid and coronary arteries from rabbits also showed decreases in edhf-release in an ex-vivo assay after treatment with lps, tnf-a and il-1 [151] . collectively these data indicate that the majority of sepsis models consistently show a disruption of receptor coupled relaxation mechanism leading to an intraendothelial signalling deficit. to quantify endothelial function in humans several methods are available. endothelium dependent relaxation after pharmacological stimulation or flow dependent relaxation after vessel obstruction are frequently quantified by high resolution ultrasound techniques [152] , alternatively flow and size can be determined angiographically. however, definite functional measurements in human sepsis are scarce. endothelium dependent relaxation has been investigated in isolated superficial hand veins of healthy volunteers after lps exposure. reduced vasorelaxation by bradykinin and arachidonic acid in noradrenalin precontracted vessels were noted which persisted for more than 2 days [153] . reactive hyperemia is believed to mainly test endothelial no˙production upon shear stress if vessel diameters and flow is monitored. implications from indirect measurements support an endothelial dysfunction in septic patients [154] [155] [156] . however, data on pharmacological stimulation of endothelium dependent relaxation in humans are currently not available. 192 t. volk +/-ec: presence or absence of endothelium; ne: norepinephrine; ach: acetylcholine; adp, adenosine diphosphate; sp, substance p; a23187, ca 2 + -ionophore; cox, cycloogygenase; snp, sodium nitroprusside; ntg, nitroglycerine; et, endothelin; bk, bradykinin; edhf, endothelium derived hyperpolarizing factor; pres., preserved. table 4 . endothelium dependent relaxation is impaired in animal sepsis models. a normal response to infection or other insults is a self limiting process that through temporal expression of regulators and effector molecules causes resolution. the failure to resolve the causative infection may lead to sepsis. cellular and animal models of sepsis using bacteria, endotoxins, exotoxins, cytokines or some peptides all consistently produce endothelial impairment which is usually regarded as dysfunctional. blocking the majority of pathways used by these inducing agents has often lead to the inhibition of such endothelial alterations. many aspects of these induced alterations can be expected to reveal exciting new pathways and complex interactions at various molecular and cellular level. the past has taught us that inhibitors of presumably activated pathways consistently failed to improve survival in septic patients. this has stimulated many researchers to reconcile the results of experimental and clinical models in sepsis. compared to activating pathways, considerably less is known about how an inflammatory response is endogenously counterregulated. cytokines induce a whole host of signal inhibiting proteins and endogenous counterregulating systems are just beginning to be elucidated. activation of endogenous counterregulatory systems may become the predominant feature of the so-called compensatory antiinflammatory response syndrome. endothelial responses to endogenously present antiinflammatory mediators have hardly been investigated in sepsis models. almost all endothelial cell studies in sepsis indicated that an imbalance in reactive oxygen species production is associated with the above described dysfunctions. animal studies in which endothelial function could be improved pharmacologically also consistently indicate that reversal of imbalanced reactive oxygen production may be a common link (table 4 ). it seems that at times an adequate production of nitric oxide is lacking whereas superoxide and/or derivatives are overproduced. however, as endothelial functional measurements in septic humans become available, we will hopefully get a clearer picture of what might happen in our patients. [199] clp, cecal ligation and puncture; lpo, lipid peroxidation; ros, reactive oxygen species. table 5 . treatment of endothelial vasoregulatory dysfunction in animal sepsis models. bacterial pathogens isolated from patients wit bloodstream infection: frequencies of occurrence and antimicrobial susceptibility patterns from the sentry antimicrobial surveillance program (united states and canada, 1997) staphylococcus aureus infections internalization of staphylococcus aureus by endothelial cells induces apoptosis staphylococcus aureus small colony variants are induced by the endothelial cell intracellular milieu genetic inactivation of the extracellular cysteine protease enhances in vitro internalization of group a streptococci by human epithelial and endothelial cells interaction of viable group a streptococci and hydrogen peroxide in killing of vascular endothelial cells group b streptococci invade endothelial cells: type iii capsular polysaccharide attenuates invasion invasion of brain microvascular endothelial cells by group b streptococci streptococcus pneumoniae anchor to activated human cells by the receptor for platelet-activating factor pneumococcal trafficking across the blood-brain barrier. molecular analysis of a novel bidirectional pathway two distinct phospholipases c of listeria monocytogenes induce ceramide generation, nuclear factor-kappa acivation, and e-selectin expression in human endothelial cells internalin b is essential for adhesion and mediates the invasion of listeria monocytogenes into human endothelial cells activation of human endothelial cells by viable or heat-killed gram-negative bacteria requires soluble cd14 interaction of neisseria maningitidis with the components of the blood-brain barrier correlates with an increased expression of pilc the ndomain of the human cd66a adhesion molecule is a target for opa proteins of neisseria meningitidis and neisseria gonorrhoeae human microvascular endothelial tissue culture cell model for studying pathogenesis of brazilian purpuric fever pseudomonas aeruginosa selective adherence to and entry into human endothelial cells endothelial function in sepsis 193 cincomitant endosome-phagosome fusion and lysis of endosomal membranes account for pseudomonas aeruginosa survival in human endothelial cells endothelial cell glcnac beta 1-4glcnac epitopes for outer membrane protein a enhance traversal of escherichia coli across the blood-brain barrier escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10 characterization of a strain of chlamydia pneumoniae isolated from a coronary atheroma by analysis of the omp1 gene and biological activity in human endothelial cells chlamydia species infect human vascular endothelial cells and induce procoagulant activity signal transduction pathways activated in endothelial cells following infection with chlamydia pneumoniae bartonella (rochalimaea) quintana endocarditis in three homeless men bartonella quintana invades and multiplies within endothelial cells in vitro and in vivo and forms intracellular blebs interaction of bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome nf-kappa b-dependent inhibition of apoptosis is essential for host cellsurvival during rickettsia rickettsii infection rickettsia conorii infection enhances vascular cell adhesion molecule-1-and intercellular adhesion molecule-1-dependent mononuclear cell adherence to endothelial cells il-6 and il-8 production from cultured human endothelial cells stimulated by infect on with rickettsia conorii via a cell-associated il-1 alpha-dependent pathway the role of cd14 in signaling mediated by outer membrane lipoproteins of borrelia burgdorferi integrins alpha(v)beta3 and alpha5beta1 mediate attachment of lyme disease spirochetes to human cells different classes of proteoglycans contribute to the attachment of borrelia burgdorferi to cultured endothelial and brain cells borrelia burgdorferi upregulates the adhesion molecules e-selectin, p-selectin, icam-1 and vcam-1 on mouse endothelioma cells in vitro characterization of plasmodium falciparum-infected erythrocyte and p-selectin interaction under flow conditions intercellular adhesion molecule-1 and cd36 synergize to mediate adherence of plasmodium falciparum-infected erythrocytes to cultured human microvascular endothelial cells pecam-1/cd31, an endothelial receptor for binding plasmodium falciparum-infected erythrocytes candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells secreted aspartyl proteinases and interactions of candida albicans with human endothelial cells dengue virus infection of human endothelial cells leads to chemokine production, complement activation, and apoptosis adhesion molecule expression and lymphocyte adhesion to cerebral endothelium: effects of measles virus and herpes simplex 1 virus measles virus induction of human endothelial cell tissue factor procoagulant activity in vitro effects of viral activation of the vessel wall on inflammation and thrombosis cellular entry of hantaviruses which cause hemorrhagic fever with renal syndrome is mediated by beta3 integrins ebola virus inhibits induction of genes by double-stranded rna in endothelial cells human endothelial cell activation and mediator release in response to the bacterial exotoxins escherichia coli hemolysin and staphylococcal alpha-toxin infection by verocytotoxin-producing escherichia coli alpha toxin from clostridium perfringens induces proinflammatory changes in endothelial cells brain capillary endothelial cells express mbec1, a protein that is related to the clostridium perfringens enterotoxin receptors phospholipase c and perfringolysin o from clostridium perfringens upregulate endothelial cellleukocyte adherence molecule 1 and intercellular leukocyte adherence molecule 1 expression and induce interleukin-8 synthesis in cultured human umbilical vein endothelial cells glucosylation of small gtp-binding rho proteins disrupts endothelial barrier function pasteurella multocida toxin increases endothelial permeability via rho kinase and myosin light chain phosphatase evidence for a structural motif in toxins and interleukin-2 that may be responsible for binding to endothelial cells and initiating vascular leak syndrome the listerial exotoxins listeriolysin and phosphatidylinositol-specific phospholipase c synergize to elicit endothelial cell phosphoinositide metabolism lipoteichoic acid-induced neutrophil adhesion via e-selectin to human umbilical vein endothelial cells (huvecs) endothelial and epithelial cells do not respond to complexes of peptidoglycan with soluble cd14 but are activated indirectly by peptidoglycan-induced tumor necrosis factor-alpha and interleukin-1 from monocytes cytokines and endothelial cell biology cytokine regulation of endothelial cell function: from molecular level to he bedside pseudomonas siderophore pyochelin enhances neutrophil-mediated endothelial cell injury superoxide dismutase-dependent, catalase-sensitive peroxides in human endothelial cells infected by rickettsia rickettsii superoxide release from interleukin-1b-stimulated human vascular cells: in situ electrochemical measuremeut lipopolysaccharide enhances oxidative modification of low density lipoprotein by copper ions, endothelial and smooth muscle cells e-selectin expression in human endothelial cells by tnf-alpha-induced oxidant generation and nf-kappab activation superoxide responses of endothelial cells to c5a and tnf-alpha: divergent signal transduction pathways lactosylceramide mediates tumor necrosis factor-alpha induced intercellular adhesion molecule-1 (icam-1) expression and the adhesion of neutrophil in human umbilical vein endothelial cells effect of antioxidants on lipopolysaccharide-stimulated induction of mangano superoxide dismutase mrna in bovine pulmonary artery endothelial cells ambient but not incremental oxidant generation effects intercellular adhesion molecule 1 induction by tumour necrosis factor alpha in endothelium icam-1 and vcam-1 expression induced by tnf-alpha are inhibited by a glutathione peroxidase mimic glutathione peroxidase mimics prevent tnfalpha-and neutrophilinduced endothelial alterations pore-forming bacterial toxins potently induce release of nitric oxide in porcine endothelial cells cytokine-induced, nitric oxide-dependent, intracellular antirickettsial activity of mouse endothelial cells endothelial cells are activated by cytokine treatment to kill an intravascular parasite, schistosoma mansoni, through the production of nitric oxide escherichia coli endotoxin inhibits agonistmediated cytosolic ca2 + mobilization and nitric oxide biosynthesis in cultured endothelial cells expressional control of the "constitutive" isoforms of nitric oxide synthase (nos i and nos iii) inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide nitric oxide donor prevents hydrogen peroxide-mediated endothelial cell injury nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells protective effects of tetrahydrobiopterin against nitric oxide-induced endothelial cell death endothelial damage induced by nitric oxide: synergism with reactive oxygen species hydroxyl radical formation resulting from the interaction of nitric oxide and hydrogen peroxide halliwell b van d v. formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils nitric oxide inhibits lipopolysaccharide-induced apoptosis in pulmonary artery endothelial cells lipopolysaccharide induces disseminated endothelial apoptosis requiring ceramide generation lipopolysaccharide induces the antiapoptotic molecules, a1 and a20, in microvascular endothelial cells apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction nuclear factor-kappab: a pivotal transcription factor in chronic inflammatory diseases oxidant-sensitive and phosphorylation-dependent activation of nf-kappa b and ap-1 in endothelial cells endothelial activation by hydrogen peroxide. selective increases of intercellular adhesion molecule-1 and major histocompatibility complex class i adenovirus-mediated expression of a dominant negative mutant of p65/rela inhibits proinflammatory gene expression in endothelial cells without sensitzing to apoptosis role of nfkappab in the mortality of sepsis endothelial function in sepsis initial contact and subsequent adhesion of human neutrophils or monocytes to human aortic endothelial cells releases an endothelial intracellular calcium store 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plethysmography (vcp) endothelial-derived nitric oxide preserves anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells does elevated nitric oxide production enhance the release of prostacyclin from shear stressed aortic endothelial cells? hypotension and inflammatory cytokine gene expression triggered by factor xa-nitric oxide signaling up-regulation of thrombomodulin in human umbilical vein endothelial cells in vitro thrombomodulin-dependent anticoagulant activity is regulated by vascular endothelial growth factor increased release of atp from endothelial cells during acute inflammation loss of atp diphosphohydrolase activity with endothelial cell activation fluid shear stress attenuates tumor necrosis factoralpha-induced tissue factor expression in cultured human endothelial cells cell biology of tissue factor, the principal initiator of blood coagulation on behalf of the subcommittee on tissue factor pathway inhibitor (tfpi) of the scientific and standardization committee of the isth effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells induction of plasminogen activator inhibitor type 1 and type 1 collagen expression in rat cardiac microvascular endothelial cells by interleukin-1 and its dependence on oxygencentered free radicals plasminogen: an important hemostatic parameter in septic patients potential mechanisms for a proinflammatory vascular cytokine response to coagulation activation thrombin-activated human endothelial cells support monocyte adhesion in vitro following expression intercellular adhesion molecule-1 cd54) and vascular cell adhesion molecule-1 (vcam-1; cd106) dual endothelium-dependent vascular activities of proteinase-activated receptor-2-activating peptides: evidence for receptor heterogeneity activation of the contact-phase system on bacterial surfaces -a clue to serious complications in infectious diseases reactive hyperemic 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with septic shock evidence for in vivo peroxynitrite production in human acute lung injury elevated von willebrand factor antigen is an early plasma predictor of acute lung injury in nonpulmonary sepsis syndrome increased plasma levels of soluble thrombomodulin in patients with sepsis and organ failure endothelial cell activity varies in patients at risk for the adult respiratory distress syndrome soluble e-selectin levels in sepsis and critical illness. correlation with infection and hemodynamic dysfunction die zirkulierenden adhäsionsmoleküle sicam-1 und sb-selectin bei patienten mit sepsis elevated circulating e-selectin, intercellular adhesion moleculc 1, and von willebrand factor in patients with severe infection increased circulating thrombomodulin in children with septic shock systemic endothelial activation occurs in both mild and severe malaria. correlating dermal microvascular endothelial cell phenotype and soluble cell adhesion molecules with disease severity increased plasma von willebrand factor in the systemic inflammatory response syndrome is derived from generalized endothelial cell activation blood levels of endothelin-1 and thrombomodulin in patients with disseminated intravascular coagulation and sepsis plasma levels of endothelial cell protein c receptor are elevated in patients with sepsis and systemic lupus or erythematosus: lack of correlation with thrombomodulin suggests involvement of different pathological processes demonstration of rickettsia conorii-induced endothelial injury in vivo by measuring circulating endothelial cells, thrombomodulin, and von willebrand factor in patients with mediterranean spotted fever influence of angiotensin-converting enzyme inhibitor enalaprilat on endothelial-derived substances in the critically ill mesenteric and skeletal muscle microvascular responsiveness in subacute sepsis influence of group b streptococci on piglet pulmonary artery response to bradykinin effects of antisense oligonucleotide to inos on hemodynamic and vascular changes induced by lps pentoxifylline maintains vascular endothelial cell function during hyperdynamic and hypodynamic sepsis a novel nonanticoagulant heparin prevents vascular endothelial cell dysfunction during hyperdynamic sepsis effect of endotoxin-enhanced hepatic reperfusion injury on endonthelium-dependent relaxation in rat aorta splanchnic vascular endothelial dysfunction in rat endotoxemia: role of superoxide radicals endothelial dysfunction in a rat model of endotoxic shock. importance of the activation of poly (adp-ribose) synthetase by peroxynitrite endothelial cell dysfunction in septic shock key: cord-009326-dvhkk405 authors: lee, jae min; choi, sun sil; park, mi hyeon; jang, hyunduk; lee, yo han; khim, keon woo; oh, sei ryang; park, jiyoung; ryu, hyung won; choi, jang hyun title: broussonetia papyrifera root bark extract exhibits anti-inflammatory effects on adipose tissue and improves insulin sensitivity potentially via ampk activation date: 2020-03-14 journal: nutrients doi: 10.3390/nu12030773 sha: doc_id: 9326 cord_uid: dvhkk405 the chronic low-grade inflammation in adipose tissue plays a causal role in obesity-induced insulin resistance and its associated pathophysiological consequences. in this study, we investigated the effects of extracts of broussonetia papyrifera root bark (pre) and its bioactive components on inflammation and insulin sensitivity. pre inhibited tnf-α-induced nf-κb transcriptional activity in the nf-κb luciferase assay and pro-inflammatory genes’ expression by blocking phosphorylation of iκb and nf-κb in 3t3-l1 adipocytes, which were mediated by activating ampk. ten-week-high fat diet (hfd)-fed c57bl6 male mice treated with pre had improved glucose intolerance and decreased inflammation in adipose tissue, as indicated by reductions in nf-κb phosphorylation and pro-inflammatory genes’ expression. furthermore, pre activated amp-activated protein kinase (ampk) and reduced lipogenic genes’ expression in both adipose tissue and liver. finally, we identified broussoflavonol b (bf) and kazinol j (kj) as bioactive constituents to suppress pro-inflammatory responses via activating ampk in 3t3-l1 adipocytes. taken together, these results indicate the therapeutic potential of pre, especially bf or kj, in metabolic diseases such as obesity and type 2 diabetes. inflammation is a protective response against infection, tissue stress, and injury in any tissue and defends and restores physiological functions. however, dysregulated inflammatory processes result in chronic inflammation, which is increasingly seen as a major driver of numerous diseases such as obesity and type 2 diabetes [1] . obese adipose tissue produces inflammatory cytokines, including tumor necrosis factor (tnf)-α, monocyte chemokine protein (mcp)-1, and interleukin (il)-6 [1] . subsequently, the elevated inflammatory stimuli induce the activation of the inhibitor of κb (iκb) kinase (ikk)/nf-κb and c-jun n-terminal kinase (jnk) pathways, which negatively regulate insulin action in not only adipose tissue, but also other peripheral tissues, such as liver [2] . thus, the accumulation of pro-inflammatory responses in adipose tissue may be one of the causal factors for insulin resistance. a previous study has demonstrated that pro-inflammatory gene expression is elevated in adipose tissue in the early onset of obesity, but in other tissues, such as liver and skeletal muscle, there is no differences in the expression of inflammatory gene expressions [3] . thus, adipose tissues appear to act as priming tissues that respond to a high-fat diet (hfd) and initiate inflammation in obesity. therefore, understanding the inflammatory responses in adipose tissues of obese individuals is of clinical importance. it has been well demonstrated that amp-activated protein kinase (ampk) is a master regulator for energy sensing, which responds to control energy homeostasis. ampk can be activated by various conditions. starvation, hypoxia, exercise, and oxidative damages are the main cellular stresses for activating ampk [4] . there are two well-known upstream kinases: liver kinase b1 (lkb1) and ca 2+ /calmodulin-dependent protein kinase kinase (camkk) can activate ampk via phosphorylation. several reports clearly demonstrated that one of the major roles of ampk is regulating metabolic requirement. for example, ampk stimulates energy production pathways through fatty acid oxidation, mitochondrial biogenesis, and glucose catabolism. on the other hand, it inhibits energy-consuming pathways, including fatty acids' synthesis and amino acids' biogenesis [5] . thus, dysfunctions of ampk or downstream signaling pathways could result in metabolic diseases, such as obesity and type 2 diabetes [4] . interestingly, it has been reported that ampk could suppress the nf-κb transcriptional activity [6] . the activation of ampk by aicar (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) can inhibit colitis [7] , autoimmune encephalomyelitis [8] , and inflammation after lung injury [9] . in contrast, disrupting ampk-mediated signaling in hematopoietic-derived cells induced the infiltration of adipose tissue macrophages (atms) and hepatic steatosis [10] . in addition, pro-inflammatory responses inhibited the activation of ampk in adipose tissue and induced the expression of pro-inflammatory genes in vivo [11] . it has been reported that the infiltration of atms is significantly increased in ampkα1 −/− mice, and these mice showed increased expression of pro-inflammatory genes, such as il-6 or tnf-α, in adipose tissue [12] . together, these observations that ampk can suppress inflammation have a significant impact on obese-mediated inflammation in adipose tissue. paper mulberry (broussonetia papyrifera) is a deciduous tree that is distributed throughout asia, and its barks, roots, and fruits are used in traditional chinese medicine. it has been shown that broussonetia papyrifera has anti-tyrosinase and antioxidant activity [13, 14] and anti-inflammatory activities in cells [15] . constituents of the roots of this plant, broussochalcone a, kazinol a, and kazinol i, have been reported as inhibitors of lipopolysaccharide-induced nitric oxide (no) production by suppressing nf-κb activation in macrophages [16, 17] . moreover, kazinol b, a b. papyrifera-derived prenylated flavan, has been shown to inhibit no production [16] . interestingly, kazinol b enhances glucose uptake via akt (a serine/threonine kinase) and ampk activation in adipocytes [17] . collectively, these reports suggest that b. papyrifera might ameliorate inflammation, but to what degree it elicits systemic insulin sensitivity, and by what mechanism, remains unclear. in the present study, we aimed to demonstrate that roots of b. papyrifera improve pre-established insulin resistance and identify major bioactive compounds that modulate obese-associated inflammation in adipose tissue. the root bark of b. papyrifera was sampled at mugo-ri, gonyang-myeon, sacheon-si, gyeongsangnam-do, south korea, in june 23, 2015 (by dr. jin-hyub paik). the collected raw materials were deposited in the korea research institute of bioscience and biotechnology (kribb) and the international biological material center (ibmrc) (kribb 0059119) [18] . of the collected roots, only barks were used for obtaining a better yield. the target compounds were isolated from dried root bark of b. papyrifera as previously described [18] . briefly, the total b. papyrifera root bark extracts (tpre, yield 10.05%) were separated by spot-ii mplc (medium-pressure liquid chromatography) (gilson, middleton, wi, usa) using reversed-phase silica gel (ymc-pack ods-aq hg, 20 × 250 mm, 10 µm, kyoto, japan) eluted with meoh-h 2 o to give tpre nos. 1-8 ( figure s1a and figure 1b) . broussoflavonol b and kazinol j were found to be components of tpre no. 6 (pre) based on the uplc-pda-qtof-ms chromatograms ( figure s1c ). among these fractions, tpre no. 6 (pre) was subjected to a gx-271 (automated liquid handler) semipreparative hplc system (gilson, middleton, wi) using a reversed-phase column (ymc-pack ods-aq-hg, 10µm) and eluted with the meoh-h 2 o gradient system (55% → 100% meoh, 70 min) by 25 repeated injections of the samples (2 g/ml methanol dilutions) to yield four fractions (pre nos. 6-1~6-4). a further preparation.-hplc procedure was repeated several times using each condition (see below). the fraction 6-3-5 (345.1 mg) enriched with broussoflavonol b was further isolated by prep.-hplc (gilson, middleton, ma, usa). broussoflavonol b (44.9 mg) and broussonol d (29.4 mg) were isolated using a ymc-pack pro c8 column. the fraction 6-3-7 enriched with kazinol j was further isolated by prep.-hplc (plc 2020) with a gradient system of meoh-h 2 o, and (-)-(2s)-kazinol i (7.6 mg), kazinol j (48.7 mg), broussoflavonol c (215.7 mg), and broussonol g (40.6 mg) were obtained. the isolated compounds were purified as described previously, and purity the was more than 95.0%, as determined by ultra-performance liquid chromatography [18] . two compounds were characterized using spectroscopic data, including 1 h, 13 c nmr, and hrms, in comparison with previously published data [16, 19] . an acquity uplc™ system (waters corporation, milford, ma, usa) equipped with a binary solvent delivery manager and a photodiode array (pda) was used for uplc (ultra-performance liquid chromatography) analysis. hrms analysis was performed using an ultra-performance liquid chromatography quadrupole time of flight mass spectrometry (uplc-qtof-ms) equipped with an electrospray ionization (esi) interface (waters q-tof premiertm, waters corporation, milford, ma, usa). the nmr analysis was carried out using a fourier transform (ft)-nmr spectrometer (jeol ecz500r, jeol ltd., akishima, tokyo, japan) for 1d spectra ( 1 h nmr and 13 c nmr). the overall processes are described in figure s1d . all extracts and single compounds for the experiments were prepared by dissolving in dimethyl sulfoxide (dmso, sigma, st. louis, mo, usa). 3t3-l1, raw264.7, and hek293 cells were obtained from the american tissue culture collection (atcc, manassas, va, usa) and cultured in dulbecco's modified eagle's medium (dmem, life technologies, ny, usa) with 10% bovine calf serum (invitrogen, gaithersburg, ca, usa) and 10% fetal bovine serum (atlas, co, usa), respectively. adipocyte differentiation was induced by treating cells with dmem containing 10% fbs, 0.5 mm isobutylmethylxanthine (ibmx), 1 µm dexamethasone, and 850 nm insulin. after 48 h, the medium was replaced every other day with dmem containing 10% fbs and 850 nm insulin. all chemicals for cell culture were obtained from sigma-aldrich (st. louis, mo, usa) unless otherwise indicated. after 6-7 days from initiation of differentiation ( figure s2a ), we treated compounds as indicated concentrations and time. after treating fully differentiated adipocytes with pre, bf, or kj for 24h, cell morphology was monitored by an inverted microscope (zeiss, oberkochen, germany) ( figure s2b ). for oil red o staining, 3t3l1 preadipocytes or differentiated 3t3l1 adipocytes treated with/without pre, bf, or kj as indicated concentrations were stained with the oil red o staining kit according to the manufacturer´s recommendations (biovision, inc., milpitas, ca, usa). hek-293 cells were transfected with nf-κb-responsive luciferase reporter (promega, san luis obispo, wi, usa) and prl-renilla using lipofectamine 2000 (invitrogen, gaithersburg, ca, usa). following an overnight transfection, the cells were treated with pre, bf, or kj for 24 h, followed by treatment with tnf-α (10 ng/ml) for 6 h. dmso was used as the vehicle. the cells were harvested, and reporter gene assays were carried out using the dual-luciferase kit (promega, san luis obispo, wi, usa). luciferase activity was normalized to renilla activity. raw 264 macrophages were seeded in 96 well plates (2.0 × 10 5 cells/ml) and treated with pre, br or kj as the indicated concentration with/without lipopolysaccharide (lps, sigma, st. louis, mo, usa) for 24 h. for no production, the amount of no was calculated by measured nitrate in media using griess reagent according to the manufacturer´s recommendations (sigma, st. louis, mo, usa). for cell viability, cells were determined using the mtt solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), sigma, st. louis, mo, usa) at 0.5 mg/ml. the purple formazan crystals were dissolved in dmso, and the absorbance was recorded on a microplate reader at a wavelength of 570 nm. all animal experiments were performed according to the procedures approved by ulsan national institute of science and technology's institutional animal care and use committee (unistiacuc-19-04). seven-week-old male c57bl/6j mice (dbl, samsung, korea) were fed a high fat diet (60% kcal fat, d12492, research diets inc., new brunswick, nj, usa) for 10 weeks the mice were housed (n = 4/cage) and granted free access to food and water. food and water were changed once a week. for glucose tolerance tests (gtt), mice were intraperitoneally (i.p.) injected daily with 40 mg/kg of pre or vehicle (saline containing 5% dmso and 5% tween 80 (sigma, st. louis, mo, usa) for 7 days and fasted for 16 h (6 p.m. to 10 a.m.) prior to i.p. injection of d-glucose (2 g/kg body weight). pre solution prepared at 4 mg/ml in saline containing 5% dmso and 5% tween 80 was injected in an amount of 10 µl/g of body weight. fasting insulin was determined using the ultrasensitive mouse insulin elisa kit (crystal chem., eik grove village, il, usa). once mice were sacrificed, isolated adipose tissue and liver were weighed and immediately frozen in liquid nitrogen and then used for western blot analysis and gene expression analysis. liver sections were embedded in paraffin and stained with hematoxylin and eosin (h&e) to visualize hepatocytes and lipid droplets in the tissues. sections were analyzed by an inverted microscope (zeiss, oberkochen, germany). each sample (cells or tissues) was lysed with ripa lysis buffer containing protease and phosphatase inhibitor (sigma-aldrich, st. louis, mo, usa). an equal amount of protein was separated on sds-page and transferred onto nitrocellulose membranes (ge healthcare, chigago, il, usa). the membranes were blocked in a 5% bovine serum albumin (bsa) blocking buffer and incubated with specific primary antibodies for phospho-nf-κb, nf-κb, phospho-iκb, iκb, phospho-ampk, ampk, phospho-acc, and acc (cell signal technology, danver, ma, usa) at 4 • c overnight. the signals were detected using an ecl detection kit (ge healthcare, chigago, il, usa), followed by incubation with horseradish peroxidase-conjugated secondary antibodies (thermofisher, gaithersburg, ca, usa). we quantified the band intensity by using the imagej program (nih, bethesda, md, usa). total rna was isolated from cells or tissues using trizol reagents (invitrogen, city, ca, usa). the rna was reverse-transcribed using the abi reverse transcription kit. quantitative pcr reactions were performed with sybr green fluorescent dye using an abi9300 pcr machine. relative mrna expression was determined by the ∆∆-ct method normalized to tata-binding protein (tbp) levels. data were presented as the means +s.e.m. statistical significance was estimated by an unpaired t-test for comparisons between two conditions. a one-way anova was used for comparisons between more than two conditions. dunnett's post hoc test was used for multiple comparisons. all statistics were performed with graphpad prism 7.0 software (graphpad, san diego, ca, usa). to investigate the effects of pre on inflammation, we first tested nf-κb transcriptional activity of tpre because nf-κb is an essential regulator of pro-inflammatory response ( figure s1 ). as shown in figure 1a , tpre suppressed nf-κb transcriptional activity induced by tnf-α, and it was dose-dependent. to further evaluate the effect of tpre for inhibiting nf-κb activity, we partitioned tpre using medium-pressure liquid chromatography into eight sub-fractions ( figure s1 ), which were then used to assay nf-κb transcriptional activity. of the eight sub-fractions, four fractions significantly inhibited nf-κb transcriptional activity induced by tnf-α. sub-fraction 6 (pre) had the most potent activity for inhibiting nf-κb activation and dose-dependently repressed nf-κb transcriptional activity ( figure 1b ,c). 5-aminoimidazole-4-carboxamide ribonucleotide (aicar), an amp analog, was used as the positive control for ampk activation. nutrients 2020, 12, 773 5 of 12 data were presented as the means +s.e.m. statistical significance was estimated by an unpaired t-test for comparisons between two conditions. a one-way anova was used for comparisons between more than two conditions. dunnett's post hoc test was used for multiple comparisons. all statistics were performed with graphpad prism 7.0 software (graphpad, san diego, ca, usa). to investigate the effects of pre on inflammation, we first tested nf-κb transcriptional activity of tpre because nf-κb is an essential regulator of pro-inflammatory response ( figure s1 ). as shown in figure 1a , tpre suppressed nf-κb transcriptional activity induced by tnf-α, and it was dosedependent. to further evaluate the effect of tpre for inhibiting nf-κb activity, we partitioned tpre using medium-pressure liquid chromatography into eight sub-fractions ( figure s1 ), which were then used to assay nf-κb transcriptional activity. of the eight sub-fractions, four fractions significantly inhibited nf-κb transcriptional activity induced by tnf-α. sub-fraction 6 (pre) had the most potent activity for inhibiting nf-κb activation and dose-dependently repressed nf-κb transcriptional activity ( figure 1b and figure 1c ). 5-aminoimidazole-4-carboxamide ribonucleotide (aicar), an amp analog, was used as the positive control for ampk activation. next, we further tested the effect of pre on inflammatory responses. differentiated adipocytes were treated with tnf-α, and we examined the effects of pre on the expression of pro-inflammatory genes. as shown in figure 2a , pre repressed the tnf-α-mediated pro-inflammatory gene, and this effect was dose-dependent. furthermore, pre blocked lipopolysaccharide (lps)-induced proinflammatory response in raw264.7 cells, as previously reported ( figure s3 ) [18] . ampk is a wellknown, important inflammatory suppressor, and ampk signaling critically regulates inflammation in many cell types [6] . therefore, to further investigate the molecular mechanism involved in preassociated repression of the nf-κb signaling pathway, we first tested whether pre activated ampk. in 3t3-l1 adipocytes, pre enhanced the phosphorylation of ampk, while pre did not affect ampk protein level. this phosphorylation was blocked by compound c, a specific inhibitor of ampk ( figure 2b) . similarly, the phosphorylation of acetyl-coa carboxylase (acc), a substrate of ampk, was enhanced by pre treatment, and its phosphorylation was blocked by compound c treatment. next, we further tested the effect of pre on inflammatory responses. differentiated adipocytes were treated with tnf-α, and we examined the effects of pre on the expression of pro-inflammatory genes. as shown in figure 2a , pre repressed the tnf-α-mediated pro-inflammatory gene, and this effect was dose-dependent. furthermore, pre blocked lipopolysaccharide (lps)-induced pro-inflammatory response in raw264.7 cells, as previously reported ( figure s3 ) [18] . ampk is a well-known, important inflammatory suppressor, and ampk signaling critically regulates inflammation in many cell types [6] . therefore, to further investigate the molecular mechanism involved in pre-associated repression of the nf-κb signaling pathway, we first tested whether pre activated ampk. in 3t3-l1 adipocytes, pre enhanced the phosphorylation of ampk, while pre did not affect ampk protein level. this phosphorylation was blocked by compound c, a specific inhibitor of ampk ( figure 2b) . similarly, the phosphorylation of acetyl-coa carboxylase (acc), a substrate of ampk, was enhanced by pre treatment, and its phosphorylation was blocked by compound c treatment. pre treatment decreased tnf-α-induced phosphorylation of iκb and nf-κb in adipocytes ( figure 2c ). in addition, pretreatment with aicar specifically inhibited tnf-α-mediated pro-inflammatory signaling. pretreatment with compound c blocked pre-induced suppression of phosphorylation of iκb and nf-κb in 3t3-l1 adipocytes ( figure 2c ). together, these results strongly suggest that pre suppresses tnf-α-mediated pro-inflammatory gene expression by activating ampk. nutrients 2020, 12, 773 6 of 12 pre treatment decreased tnf-α-induced phosphorylation of iκb and nf-κb in adipocytes ( figure 2c ). in addition, pretreatment with aicar specifically inhibited tnf-α-mediated pro-inflammatory signaling. pretreatment with compound c blocked pre-induced suppression of phosphorylation of iκb and nf-κb in 3t3-l1 adipocytes ( figure 2c ). together, these results strongly suggest that pre suppresses tnf-α-mediated pro-inflammatory gene expression by activating ampk. next, we determined whether pre exhibited anti-diabetic activities in vivo. ten-week-hfd-fed c57bl/6 mice were used for a glucose tolerance test (gtt). pre and glucose were administrated intraperitoneally, because the components of pre have been reported to inhibit alpha-glucosidase to exclude the possibility that oral glucose and pre administration suppress glucose absorption from the gut [20] . the pre-treated group showed increased glucose tolerance compared to that in control mice ( figure 3a ). plasma glucose levels in pre-treated mice, as determined by the area under the curve (auc), were significantly suppressed compared to those of control mice. furthermore, the fasting insulin level showed a tendency to decrease ( figure 3c ), suggesting that pre ameliorates obesity-induced glucose intolerance. body weight ( figure 3b ), adipose tissue weight ( figure s4a ), and liver weight ( figure s4b) were not changed by treatment with pre. after 3t3-l1 adipocytes were pre-incubation with/without compound c for 1 h, cells were treated with pre or aicar for an additional 2 h. the expression of phospho-ampk, ampk, phospho-acc, and acc was analyzed by western blotting. (c) 3t3-l1 adipocytes were pretreated with the indicated concentration of pre for 24 h and aicar for 2 h before 20 ng/ml tnf-α treatment for 30 min. compound c (comp. c) was pretreated for 1h before pre treatment. the expression of phospho-iκb, iκb, phospho-nf-κb, nf-κb, and tubulin was analyzed by western blotting. next, we determined whether pre exhibited anti-diabetic activities in vivo. ten-week-hfd-fed c57bl/6 mice were used for a glucose tolerance test (gtt). pre and glucose were administrated intraperitoneally, because the components of pre have been reported to inhibit alpha-glucosidase to exclude the possibility that oral glucose and pre administration suppress glucose absorption from the gut [20] . the pre-treated group showed increased glucose tolerance compared to that in control mice ( figure 3a ). plasma glucose levels in pre-treated mice, as determined by the area under the curve (auc), were significantly suppressed compared to those of control mice. furthermore, the fasting insulin level showed a tendency to decrease ( figure 3c ), suggesting that pre ameliorates obesity-induced glucose intolerance. body weight ( figure 3b ), adipose tissue weight ( figure s4a ), and liver weight ( figure s4b our initial results showed that pre activated ampk and suppressed pro-inflammatory mediators in vitro. therefore, we tested whether pre activated ampk and nf-κb in obese adipose tissue in vivo. upon treatment with pre, nf-κb phosphorylation was significantly decreased, whereas ampk phosphorylation was increased in epididymal white adipose tissue (ewat) ( figure 4a ). next, we assessed the effects of pre on inflammation in adipose tissue. as shown in figure 4b , the expression of pro-inflammatory genes (il-1β and inducible nitric oxide synthase (inos)) was significantly reduced in ewat after treatment with pre. however, marker genes of macrophages (f4/80, cd11b, and cd68) or marker genes of the m2 macrophage, including arginase-1 (arg-1), mannose receptor c-type 1 (mrc-1), macrophage galactose binding lectin (mgl), and ym-1 (chitinaselike 3) were not changed ( figure s5 ). interestingly, pre-treated hfd-fed mice showed significantly decreased expression of lipogenic genes (fatty acid synthase (fasn), sterol regulatory element-binding protein 1 (srebp-1c), and acc-1) in adipose tissue ( figure 4c ). these results indicated that pre had anti-inflammatory and anti-lipogenic activities in adipose tissue. our initial results showed that pre activated ampk and suppressed pro-inflammatory mediators in vitro. therefore, we tested whether pre activated ampk and nf-κb in obese adipose tissue in vivo. upon treatment with pre, nf-κb phosphorylation was significantly decreased, whereas ampk phosphorylation was increased in epididymal white adipose tissue (ewat) ( figure 4a ). next, we assessed the effects of pre on inflammation in adipose tissue. as shown in figure 4b , the expression of pro-inflammatory genes (il-1β and inducible nitric oxide synthase (inos)) was significantly reduced in ewat after treatment with pre. however, marker genes of macrophages (f4/80, cd11b, and cd68) or marker genes of the m2 macrophage, including arginase-1 (arg-1), mannose receptor c-type 1 (mrc-1), macrophage galactose binding lectin (mgl), and ym-1 (chitinase-like 3) were not changed ( figure s5 ). interestingly, pre-treated hfd-fed mice showed significantly decreased expression of lipogenic genes (fatty acid synthase (fasn), sterol regulatory element-binding protein 1 (srebp-1c), and acc-1) in adipose tissue ( figure 4c ). these results indicated that pre had anti-inflammatory and anti-lipogenic activities in adipose tissue. our initial results showed that pre activated ampk and suppressed pro-inflammatory mediators in vitro. therefore, we tested whether pre activated ampk and nf-κb in obese adipose tissue in vivo. upon treatment with pre, nf-κb phosphorylation was significantly decreased, whereas ampk phosphorylation was increased in epididymal white adipose tissue (ewat) ( figure 4a ). next, we assessed the effects of pre on inflammation in adipose tissue. as shown in figure 4b , the expression of pro-inflammatory genes (il-1β and inducible nitric oxide synthase (inos)) was significantly reduced in ewat after treatment with pre. however, marker genes of macrophages (f4/80, cd11b, and cd68) or marker genes of the m2 macrophage, including arginase-1 (arg-1), mannose receptor c-type 1 (mrc-1), macrophage galactose binding lectin (mgl), and ym-1 (chitinaselike 3) were not changed ( figure s5 ). interestingly, pre-treated hfd-fed mice showed significantly decreased expression of lipogenic genes (fatty acid synthase (fasn), sterol regulatory element-binding protein 1 (srebp-1c) , and acc-1) in adipose tissue ( figure 4c ). these results indicated that pre had anti-inflammatory and anti-lipogenic activities in adipose tissue. next, we examined whether pre prevented hepatic steatosis, which is increased by obesity. histological observations revealed that pre significantly suppressed hepatic steatosis in obese mice induced by hfd ( figure 5a and figure s6 ). because ampk plays crucial roles in suppressing hepatic steatosis [4] and pre activated ampk in adipose tissue, we examined ampk signaling in liver. as shown in figure 5b , treatment with pre increased ampk phosphorylation in liver. furthermore, pre significantly decreased lipogenic gene expression (srebp-1c and stearoyl-coa desaturase-1 (scd-1)) ( figure 5c ). in addition, pre increased the expression of acyl-coa synthetase long-chain (acsl), very-long-chain acyl-coa dehydrogenase (vlcad), and short-chain acyl-coa dehydrogenase (scad), which are involved in fatty acid oxidation ( figure 5d ). taken together, these results suggested that pre activated ampk, which improved fatty liver. genes (b) and lipogenic genes (c) were analyzed by quantitative real-time pcr. data are shown as the mean±s.e.m. (n = 5) *p < 0.05; **p < 0.001; ***p < 0.0001 vs. hfd-fed vehicle group. next, we examined whether pre prevented hepatic steatosis, which is increased by obesity. histological observations revealed that pre significantly suppressed hepatic steatosis in obese mice induced by hfd ( figure 5a and figure s6 ). because ampk plays crucial roles in suppressing hepatic steatosis [4] and pre activated ampk in adipose tissue, we examined ampk signaling in liver. as shown in figure. 5b, treatment with pre increased ampk phosphorylation in liver. furthermore, pre significantly decreased lipogenic gene expression (srebp-1c and stearoyl-coa desaturase-1 (scd-1)) ( figure. 5c ). in addition, pre increased the expression of acyl-coa synthetase long-chain (acsl), very-long-chain acyl-coa dehydrogenase (vlcad), and short-chain acyl-coa dehydrogenase (scad), which are involved in fatty acid oxidation (figure. 5d ). taken together, these results suggested that pre activated ampk, which improved fatty liver. to identify the bioactive compounds in pre that activate ampk, we isolated 20 compounds via methanolic extraction ( figure s1 ). among them, we found that broussoflavonol b (bf) and kazinol j (kj) dramatically increased ampk phosphorylation in 3t3-l1 adipocytes ( figure 6a ). both bf and kj increased ampk and acc phosphorylation, and compound c significantly blocked them ( figure 6b ). furthermore, bf and kj significantly suppressed tnf-α-induced nf-κb transcriptional activity ( figure 6c ). consistent with nf-κb activity, treatment with bf and kj downregulated tnf-αstimulated pro-inflammatory gene expression (il-6, mcp-1, and inos-only in the bf-treated group) in adipocytes ( figure 6d ). in addition, both bf and kj decreased iκb degradation and nf-κb phosphorylation, and compound c significantly blocked them ( figure 6e ). they also suppressed lps-mediated no production in raw264.7 ( figure s7 ). together, these results strongly indicated that bf and kj were bioactive compounds of pre and could block an inflammatory response through blocking the nf-κb signaling pathway via ampk activation. to identify the bioactive compounds in pre that activate ampk, we isolated 20 compounds via methanolic extraction ( figure s1 ). among them, we found that broussoflavonol b (bf) and kazinol j (kj) dramatically increased ampk phosphorylation in 3t3-l1 adipocytes ( figure 6a ). both bf and kj increased ampk and acc phosphorylation, and compound c significantly blocked them ( figure 6b ). furthermore, bf and kj significantly suppressed tnf-α-induced nf-κb transcriptional activity ( figure 6c ). consistent with nf-κb activity, treatment with bf and kj downregulated tnf-α-stimulated pro-inflammatory gene expression (il-6, mcp-1, and inos-only in the bf-treated group) in adipocytes ( figure 6d ). in addition, both bf and kj decreased iκb degradation and nf-κb phosphorylation, and compound c significantly blocked them ( figure 6e ). they also suppressed lps-mediated no production in raw264.7 ( figure s7 ). together, these results strongly indicated that bf and kj were bioactive compounds of pre and could block an inflammatory response through blocking the nf-κb signaling pathway via ampk activation. (b) 3t3-l1 adipocytes were pre-incubated with/without compound c (10 μm) for 1 h, and then, cells were treated with bf or kj for an additional 2 h. the expression of phospho-ampk, ampk, phospho-acc, and acc was analyzed by western blotting. (c) hek-293 cells were transfected with the nf-κb-responsive luciferase reporter and prl-renilla. the cells were treated with bf and kj as the indicated concentration for 24 h, followed by treatment with tnf-α (10 ng/ml) for an additional 6 h. the cells were harvested, and luciferase activity was measured. data are shown as the mean ± s.e.m (n = 3) *p < 0.05; **p < 0.001; ***p < 0.0001 vs. the tnf-α-only-treated group. (d) after 3t3-l1 adipocytes were pretreated with/without compound c, cells were incubated with/without bf or kj for 24 h and stimulated with tnf-α (20 ng/ml) for 30 min. the expressions of phospho-iκb, iκb, phospho-nf-κb, nf-κb, and tubulin were analyzed by western blotting. (e) 3t3-l1 adipocytes were pre-incubated with the indicated concentration of bf or kj for 24 h, followed by treatment with 20 ng/ml tnf-α for 5 h. total rna was isolated, and the mrna expression level of each gene was analyzed by quantitative real-time pcr. data are shown as the mean ± s.e.m (n = 3) *p < 0.05; **p < 0.001; ***p < 0.0001 vs. the tnf-αonly-treated group. obesity is very closely related to chronic and low-grade inflammation. many reports have suggested that insulin resistance accompanies chronic inflammation and abnormal mediator secretion in obese adipose tissue [21] , indicating that reduction of tissue inflammation could be an indispensable target for improving obesity-related metabolic syndromes. it has been reported that the cells were treated with bf and kj as the indicated concentration for 24 h, followed by treatment with tnf-α (10 ng/ml) for an additional 6 h. the cells were harvested, and luciferase activity was measured. data are shown as the mean ± s.e.m. (n = 3) * p < 0.05; ** p < 0.001; *** p < 0.0001 vs. the tnf-α-only-treated group. (d) after 3t3-l1 adipocytes were pretreated with/without compound c, cells were incubated with/without bf or kj for 24 h and stimulated with tnf-α (20 ng/ml) for 30 min. the expressions of phospho-iκb, iκb, phospho-nf-κb, nf-κb, and tubulin were analyzed by western blotting. (e) 3t3-l1 adipocytes were pre-incubated with the indicated concentration of bf or kj for 24 h, followed by treatment with 20 ng/ml tnf-α for 5 h. total rna was isolated, and the mrna expression level of each gene was analyzed by quantitative real-time pcr. data are shown as the mean ± s.e.m. (n = 3) * p < 0.05; ** p < 0.001; *** p < 0.0001 vs. the tnf-α-only-treated group. obesity is very closely related to chronic and low-grade inflammation. many reports have suggested that insulin resistance accompanies chronic inflammation and abnormal mediator secretion in obese adipose tissue [21] , indicating that reduction of tissue inflammation could be an indispensable target for improving obesity-related metabolic syndromes. it has been reported that roots of b. papyrifera have been used as a suppressant for edema in traditional chinese medicine [22] . the core phytochemicals in the roots of b. papyrifera are flavonols, flavans, and chalcones [23] . there is emerging evidence that these core phytochemicals have anti-inflammatory activities. [15, 16, 24] . in the present study, we focused on the anti-inflammatory effects of pre in adipose tissue and investigated whether it could ameliorate systemic insulin resistance in obese mice. pre potently inhibited tnf-α-induced inflammatory responses by suppressing nf-κb activation in adipocytes. this effect improved not only glucose tolerance, but also hepatic steatosis in hfd-induced obese mice. using the fda guideline to calculate human equivalent doses (hed), the hed of this effective dose of pre (40 mg/kg) was 195 mg/day in humans. this dose was lower than that of metformin (850 mg~2550mg/day) even though pre was a crude extract. furthermore, bf and kj, the isolated phytochemicals from pre, were the bioactive compounds that suppressed inflammatory responses in 3t3-l1 adipocytes through blocking nf-κb signaling. of significance, these effects were partially dependent on ampk activation in adipocytes ( figure s8) . ampk has been shown to have strong anti-inflammatory activity via inhibiting inflammatory responses in various in vivo models [6] . various studies have shown that ampk inhibits pro-inflammatory signaling in many tissues and cells, especially adipocytes and macrophages, which are the main cell types of adipose tissue [25, 26] . in addition, ampk ameliorates insulin resistance in obesity [10, 11, 25, 26] . it has been reported that ampk activators specifically suppressed the expression of pro-inflammatory genes and the activation of nf-κb-mediated signaling [27, 28] . the molecular mechanism for nf-κb activation is: (1) the degradation of iκb in a ubiquitin-dependent manner is triggered through its phosphorylation by ikk; (2) iκb degradation results in nuclear translocation of nf-κb [29] . based on these results, we proposed the roles of pre and its constituents to inhibit nf-κb transcriptional activity through the blockade of phosphorylation-mediated iκb degradation and nf-κb phosphorylation; this effect was reversed by inhibition of ampk. thus, pre-mediated nf-κb inactivation would likely be accompanied by inhibition of the tnf-α-stimulated ikk/iκb/nf-κb signaling pathway. in addition to the identified signaling pathways, it remains to be elucidated whether other molecular mechanisms for the ampk-mediated anti-inflammatory effect of pre exist. ampk has several phosphorylation targets [30] , but there are some reports suggesting that ampk could inhibit nf-κb activity and its signaling indirectly via sirtuin 1 (sirt1) [11] , the forkhead box o (foxo) family [30] , and peroxisome proliferator-activated receptor γ co-activator (pgc-1α) [31] , which are known as downstream mediators of ampk. these mediators could inactivate the p65 subunit of nf-κb, and the expression of pro-inflammatory genes is subsequently repressed. thus, further studies related to the underlying mechanism of how pre and its active components regulate ampk activation and its resultant anti-inflammatory effect are needed. obesity-associated adipocyte dysfunction by chronic inflammation in adipose tissue contributes to developing hepatic steatosis: (1) excessive free fatty acids from adipose tissue, which stimulate inflammation, could be delivered to liver; thus, triglycerides could be accumulated in liver; (2) adipose tissue secretes pro-inflammatory cytokines including tnf-α, which exacerbate inflammation in liver and induce hepatic steatosis [1] . here, we demonstrated that pre did not alter ewat mass and adiposity, by it ameliorated adipose tissue inflammation indicated by significant decreases in the pro-inflammatory signaling and pro-inflammatory genes' expression. furthermore, pre enhanced ampk activation, which regulates gene expression related to fatty acid oxidation and lipogenesis. thus, improved adipose tissue inflammation and ampk activation by pre could ameliorate hepatic steatosis. we demonstrated that pre and its active components had potential therapeutic effects on ameliorating inflammation in both adipose tissue and liver. furthermore, pre could reduce hepatic steatosis and improve glucose homeostasis. in addition, we proposed that the activation of ampk by pre and its active components could be the underlying molecular mechanism by which they have anti-inflammatory effects. taken together, these finding demonstrated the beneficial effects of b. papyrifera root and its phytochemicals and indicated their potential as candidates for targeting ampk for the treatment of obesity and/or type 2 diabetes. the following are available online at http://www.mdpi.com/2072-6643/12/3/773/s1: figure s1 : scheme of extract and isolation, figure s2 : adipogenic induction of 3t3l1 preadipocytes into adipocytes, figure s3 : the effect of pre on adipose tissue and liver weight, figure s3 : the effects of pre on lps-induced pro-inflammatory gene expression in raw264.7 cells, figure s4 : the effect of pre on adipose tissue and liver weight, figure s5 : the effect of pre on macrophage infiltration and polarization in hfd-fed obese mice, figure s6 : the effect of pre in liver steatosis, figure s7 : the effect of bf and kj on lps-induced no production and cell viability in raw264.7 cells, figure s8 : pre improves insulin sensitivity via suppressing inflammation by activation of ampk in adipose tissue. chronic inflammation in fat plays a crucial role in the development of obesity-related insulin resistance inflammation and insulin resistance inflammation is necessary for long-term but not short-term high-fat diet-induced insulin resistance ampk as a therapeutic target for treating metabolic diseases. trends endocrino ampk: mechanisms of cellular energy sensing and restoration of metabolic balance exploiting the anti-inflammatory effects of amp-activated protein kinase activation ampk agonist downregulates innate and adaptive immune responses in tnbs-induced murine acute and relapsing colitis singh, i. 5-aminoimidazole-4-carboxamide ribonucleoside: a novel immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis ampk agonists ameliorate sodium and fluid transport and inflammation in cystic fibrosis airway epithelial cells hematopoietic ampk β1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity macrophage alpha1 amp-activated protein kinase (alpha1ampk) antagonizes fatty acid-induced inflammation through sirt1 amp-activated protein kinase α1 protects against diet-induced insulin resistance and obesity antityrosinase and antioxidant effects of ent-kaurane diterpenes from leaves of broussonetia papyrifera antioxidant lignans from the fruits of broussonetia papyrifera comparison with various parts of broussonetia papyrifera as to the antinociceptive and anti-inflammatory activities in rodents inhibition of nitric oxide production on lps-activated macrophages by kazinol b from broussonetia kazinoki kazinol b from broussonetia kazinoki improves insulin sensitivity via akt and ampk activation in 3t3-l1 adipocytes anti-inflammatory flavonoids from root bark of broussonetia papyrifera in lps-stimulated raw264.7 cells evaluation of polyphenols from broussonetia papyrifera as coronavirus protease inhibitors polyphenols from broussonetia papyrifera displaying potent alpha-glucosidase inhibition inflammation, stress, and diabetes five new diprenylated flavonols from the leaves of broussonetia kazinoki the genus broussonetia: a review of its phytochemistry and pharmacology prenylated polyphenols from broussonetia kazinoki as inhibitors of nitric oxide production role of amp-activated protein kinase in adipose tissue metabolism and inflammation adenosine 5-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype luteolin reduces obesity-associated insulin resistance in mice by activating ampkα1 signalling in adipose tissue macrophages berberine suppresses proinflammatory responses through ampk activation in macrophages nf -κb, inflammation, immunity and cancer: coming of age amp-activated protein kinase and its downstream transcriptional pathways tnf-α reduces pgc-1α expression through nf-κb and p38 mapk leading to increased glucose oxidation in a human cardiac cell model the authors declare no conflict of interest. key: cord-013183-t25gecuw authors: beloumi, dhekra; blasco, agustín; muelas, raquel; santacreu, maría antonia; garcía, maría de la luz; argente, maría-josé title: inflammatory correlated response in two lines of rabbit selected divergently for litter size environmental variability date: 2020-09-01 journal: animals (basel) doi: 10.3390/ani10091540 sha: doc_id: 13183 cord_uid: t25gecuw simple summary: animal welfare is a priority objective for the livestock industry. litter size environmental variability has been related to environmental sensitivity. a divergent selection experiment for environmental variance of litter size variance was carried out successfully in rabbits over thirteen generations. the low line showed a lower inflammatory response and susceptibility to infectious disorders than the high line. in conclusion, the decrease of environmental sensitivity seems to increase the adaptation of the animal to the environment, and thus, its welfare. abstract: a divergent selection experiment for environmental variance of litter size variance was carried out in rabbits over thirteen generations. the aim of this study was to evaluate the inflammatory response in the two lines of the experiment, in order to analyse the effect of selection on susceptibility to diseases after challenging to stressful situations, such as 24 h after the first delivery. a total of 78 females were used in this study, 39 from each line. the line selected for litter size heterogeneity (the high line) showed lower white blood leukocyte count (wbc; −0.87 × 10(3)/µl), lower percentage of basophils (−0.11%), higher concentration of tnf-α (+13.8 pg/ml), and greater concentration of crp (+38.1 µg/ml) than the line selected for litter size homogeneity (the low line). the high line had also higher concentrations of bilirubin, cholesterol, gamma-glutamyl transferase (ggt) and alkaline phosphatase (alp) compared to the low line (difference between lines were +0.08 µmol/l, +0.14 µmol/l, +0.35 u/l and +2.4 u/l, respectively). the high line showed higher inflammatory response than the low line, in accordance with a larger susceptibility to infectious disorders. in conclusion, the line selected to increase litter size environmental variability seems to have poor capacity coping with environmental stressors. therefore, selection for litter size environmental variability can be a useful way to improve animal welfare. in the last few decades, animal welfare has become a priority objective for farmers and the livestock industry [1] . animal welfare is defined as the capacity of animals to cope with their environment [2] . susceptibility to stress and diseases are closely related to this adaptation [3] , playing an important role for the immune system in this process [4] . inflammation is the immune system's response that is triggered in response to microbial invasion or tissue damage in order to maintain the body's homeostasis [5] . inflammation is a complex process which involves a high number of molecules [6] . the cytokines interleukin-6 (il-6) and tumour necrosis factor alpha (tnf-α), together with the acute-phase protein c-reactive protein (crp), are mainly used as inflammatory biomarkers. levels of these biomarkers help to detect the presence of inflammation and severity of disease [7] [8] [9] [10] . the liver has an important role in the synthesis of acute-phase proteins, such as crp, in response to cytokines such as il-6, tnf-α and il-1β [11] . moreover, liver metabolism has been related to the inflammatory process [12] . in prolific species such as pigs, rabbits and mice, environmental variability in body weight and in litter size has been related to immune response and resistance to diseases (see review by lung [13] ). a divergent selection experiment for environmental variance of litter size at birth was performed in rabbits. after ten generations of selection, the lines showed a remarkable divergent response (2.7 kits 2 in the low line vs. 4.4 kits 2 in the high one) [14] . in a previous study, argente [15] found lower basal levels of cortisol and crp in females selected for litter size homogeneity (the low line) than those selected for litter size heterogeneity (the high line). after, vaccination against viral haemorrhagic disease and myxomatosis, the low line showed a better immune response than the high one [15] . this would indicate better ability to cope with environmental stressors, such as infections, in the homogenous line. the objective of this study was to evaluate the inflammatory response in the two lines of the divergent selection experiment for litter size environmental variance, in order to analyse the effect of selection on susceptibility to diseases after stressful situations, such as 24 h after the first delivery. for this purpose, additional inflammatory and biochemical markers to the ones studied by argente [15] were measured in the thirteenth generation of the experiment. all experimental procedures were approved by the miguel hernández university of elche research ethics committee, according to council directives 98/58/ec and 2010/63/eu (reference number 2017/vsc/pea/00212). a divergent selection experiment on environmental variability of litter size was carried out over thirteen generations. selection was based on the phenotypic variance of litter size of each doe, after correcting litter size for both year-season and parity-lactation status (first parity, and lactating or not at mating in other parities) [14] . a total of seventy-eight primiparous female rabbits from the thirteenth generation, 39 from the line selected for litter size heterogeneity (the high line) and 39 from the line selected for litter size homogeneity (the low line), were used in this experiment. all the animals were reared in the farm of the miguel hernández university of elche (spain). the rabbits were fed a standard commercial diet (17% crude protein, 16% fibre, 3.5% fat, nutricun elite gra ® , de heus nutrición animal, la coruña, spain). food and water were provided ad libitum. does were housed in individual cages (37.5 cm × 33 cm × 90 cm) under a constant photoperiod of 16 h continuous light: 8 h continuous darkness, and with controlled ventilation. the experiment took place from march to july. table 1 shows the distributions of does and their liveweight per month. the temperature ranged from 15.2 • c to 32.1 • c. reproduction was organized in discrete generations. all does were mated at the same age, i.e., at 18 weeks of age. following the blood-sampling procedure described in [16] , two blood samples of 3 ml were drawn from the central artery of each doe's ear 24 h after the first delivery at twenty-two weeks of age. delivery is a stressful event to does which may have an influence on haematological and biochemical parameters. the first blood sample was collected into a tube with tripotassium ethylenediaminetetraacetic acid (k3-edta). this sample was divided into two aliquots. one aliquot was used for haematology, and the other one was centrifuged (at 4000 rpm for 15 min) in order to determine concentrations of c-reactive protein (crp), interleukin 6 (il-6), tumour necrosis factor alpha (tnf-α), and cortisol. the plasma samples obtained by centrifugation were stored at −80 • c until further analysis. the second blood sample was collected into a lithium heparin tube. after centrifugation, the concentrations of bilirubin, cholesterol, alkaline phosphatase (alp), gamma glutamyl transpeptidase (ggt), albumin (alb), bile acid (ba), and blood urea nitrogen (bun) were assessed. haematological parameters such as white blood leukocyte count (wbc) and the percentage of lymphocytes, neutrophils, monocytes, basophils and eosinophils were done by the haematology analyser abacus junior vet (diatron, austria). c-reactive protein (crp) concentration was quantified using a commercially available elisa kit for rabbits (catalogue number 2210-5; life diagnostics inc., west chester, pa, usa). interleukine 6 (il-6) concentration was analysed using a commercially available elisa kit for rabbits (catalogue number csb e06903rb; cusabio, houston, tx, usa). the concentration of tumour necrosis factor alpha (tnf-α) was quantified using a commercially available elisa kit for rabbits (catalogue number el rb0011; elabscience, houston, tx, usa). cortisol plasma concentration was performed using an available elisa kit for rabbits (catalogue number csb e06956rb; cusabio, houston, tx, usa). plasma concentrations of bilirubin, cholesterol, alkaline phosphatase, gamma glutamyl transferase, albumin, bile acid, and blood urea nitrogen were evaluated using the vetscan ® mammalian liver profile rotor from abaxis company. data were analysed using the following model: where ms i is the month of blood sampling effect with five levels, l is the line effect with two levels (high and low line), b is the regression coefficient, x ijk is the covariate weight and e ijk is the residual term. residuals were assumed to be independently normally distributed with the same variance. a bayesian analysis was used, with bounded flat priors for all unknown parameters. marginal posterior distributions were estimated for all unknowns using gibbs sampling. marginal posterior distributions of the differences between lines were computed with the program rabbit, developed by the institute for animal science and technology (valencia, spain). monte carlo markov chains of 60,000 iterations, with a burn-in period of 10,000, and only one out of every 10 samples was saved for inferences. convergence was tested using the z criterion of geweke, and monte carlo sampling errors were computed using time-series procedures. bayesian statistic gives a new approach to the description of the uncertainly against classical statistics. for example, we can provide the difference between lines (d h-l ) and the precision of our estimation, finding the shortest interval with 95% probability of containing the true value, that can be asymmetric around the estimation (this is called the highest posterior density interval at 95% probability). notice that in the bayesian context, there is nothing like 'significance' [17] , but we can calculate the actual probability of the difference between the high and low line |d h-l | being higher than zero; this is much more informative than p-values and significance (see blasco [17] for details). we consider that there is enough evidence for the high and the low lines being different when the probability of this difference in absolute value |d h-l | is more than 90%. however, this is not a significance test, but a way to help the discussion, since we have all actual probabilities of the differences between lines and the reader can consider other probability as being relevant enough to differentiate both lines. table 2 shows the features of the estimated marginal posterior distributions of the differences between lines (d h-l ) for the haematological parameters. the high line had lower white blood leukocyte count (wbc; −0.87 × 10 3 /µl, p = 0.95) and lower percentage of basophils than the low line (−0.11%, p = 0.93). we did not observe differences between lines for the percentages of lymphocytes, neutrophils, monocytes, and eosinophils. according to table 3 , the high line showed higher concentration of tnf-α (+13.8 pg/ml, p = 0.90) and greater concentrations of crp in comparison with the low line (+38.1 µg/ml, p = 1.00). concentrations of interleukin 6 (il-6) and cortisol were similar in both lines (p = 0.55 and p = 0.60, respectively). features of the marginal posterior distributions of the differences between the high and the low lines for biochemical parameters are given in table 4 . the concentrations of bilirubin and ggt were higher in the high line than the low line (p = 0.88 and p = 0.89, respectively). cholesterol and alp levels were also higher in the high line than in the low one (+0.14 µmol/l and +2.4 u/l, p > 0.90). there is some evidence of differences between lines in ba (p = 0.84). no differences between the high and low lines were found for alb and bun (p = 0.61 and p = 0.54). haematological parameters provide valuable information on the health status of the animal. in the present study, we evaluated the haematological profile in the two lines at 24 h after the first delivery. we found that there was no difference between lines, except for wbc and the percentage of basophils. the wbc values obtained were within the normal range for rabbits (2.71 − 12.23 × 10 9 /l), reported by [18] . total leucocyte counts are involved in the immunity reaction and defence of the organism [19, 20] . susceptible rabbits to acute infections may have a decrease in wbc count [21, 22] . on the other hand, basophils are essential for linking innate and adaptive immunity [23] . therefore, a higher basal concentration of wbc and basophils in the low line would be related to better disease resistance and good immunity. lymphocytes and neutrophils are important for the immune system. neutrophils provide the first line defence against infection in innate immune response [24] . lymphocytes are involved in humoral and cell-mediated immunity response [25] . in a previous study, argente [15] found a higher basal concentration of neutrophils and lower concentration of lymphocytes in the low line. however, no differences between lines were found in this study. we would like to note that sample collection was carried out at different environmental stress conditions, i.e., at first mating in the previous study and at 24 h after the first delivery in the current study, and that could affect the values of immunological parameters. cortisol concentration was used as a biochemical indicator of stress and pain [26] . in the present study, the cortisol concentrations in the high and the low lines were higher than the normal range of cortisol concentration (2.6-3.8 µg/dl) for rabbits reported by [27] . this may be due to the moment of recording. indeed, the samples were collected 24 h after parturition. it is known that parturition is considered one of the most stressful and painful events for the dam [28] . such a stressful event could increase the cortisol concentration up to several hours postpartum [29] . this finding implies that both lines were under similar level of stress. it is well known that a high level of stress leads to dysregulation of the immune system, [30] increasing predisposition to disease [31] . inflammation is a biological response of the immune system that can be triggered by variety of factors, including pathogens and damaged cells [5] . in order to evaluate the inflammatory process in both divergent lines, i.e., their susceptibility to diseases, we assessed the plasma levels of two cytokines, interleukin-6 and tumour necrosis factor alpha, and c-reactive protein. interleukin-6 (il-6) is a pro-inflammatory cytokine partly produced by the combined action of il-1β and tnf-α and has an effect on inflammation and the immune response [32] . tnf-α is a cytokine with pro-inflammatory activities produced by macrophages and have an important role in the innate defence mechanism [33] . a higher susceptibility to diseases is related to a higher concentration of tfn-α [34] . both divergent lines were exposed to the same environment. however, the high line showed higher concentration of tnf-α than the low line. a high level of tfn-α agrees with higher inflammatory response in the high line, and therefore a higher susceptibility to diseases in this line. c-reactive protein (crp) is an acute-phase protein and an important etiological factor in inflammation [35] . its production is mainly hepatic, by hepatocytes as a response to stimulation with il-6, tnf-α and il-1β (reviewed by stoner et al. [11] ). thus, crp level in blood is considered an inflammatory biomarker [36] . female rabbits of the high line showed greater crp concentration. a higher basal crp concentration in this line would confirm a higher sensitivity to disease, to the presence of chronic inflammation, and to a lesser tolerance to usual microorganisms in the farm microenvironment [6, 37, 38] . we note that selection for litter size environmental variability increased the difference in cpr between lines from eighth generation (5.6 µg/ml [15] to thirteenth generation (38.1 µg/ml), in agreement with a correlated response to selection for litter size environmental variability on the animal's susceptibility to diseases. infection and inflammation seem to have consequences on liver metabolism in order to reduce this inflammation [39] . we assessed the biochemical indicator relationship between liver health and inflammation in both lines. delivery is a stressful event to does which could affect to biochemical parameters concentrations. however, the levels of bilirubin, cholesterol, alp, gct, ba, alb, and bun in our lines were within the wide range of values reported in rabbits by [18, 27] (3.4-8.5 µmol/l, 0.3-3.0 µmol/l, 9.1-94.6 iu/l, 2.5-14.5 iu/l, 0.7-19.6 µmol/l, 25-50 g/l, and 6.14-8.38, respectively). when we compared both lines, the concentrations of bilirubin and cholesterol were higher in the high line. according to fan et al. [40] , the increase in the concentration of cholesterol in rodents is a response to the production of inflammatory cytokines (mainly tnf-α and il-1). bilirubin is an endogenous antioxidant which promotes lipid peroxidation prevention [41] . inoguchiet et al. [42] reported that bilirubin plays a protective role against chronic inflammation. together, it seems that greater basal concentrations of cholesterol and bilirubin are related to higher sensitivity to inflammation and susceptibility to diseases in the high line. gamma glutamyl transferase (ggt) is an inflammation regulator which increases first in the case of a hepatic disorder [43] . females from the high line showed higher ggt concentration than those from the low line; this would suggest a liver dysfunction and high susceptibility to diseases [44] . the high line has a higher alkaline phosphatase (alp) concentration than the low line. alp is a good indicator of liver diseases and general health [45] . an increased concentration of alp is an indication of liver dysfunction and a disruption in the inflammatory system [46] . these results agree with the greater susceptibility to diseases of this line. recently a genome-wide association study was performed on our lines, identifying several genes with functionality in the immune system and stress [47] . this finding corroborates the decisive role of the immune system in the environmental variation of litter size. our study shows the high line having higher inflammatory response under stressful situations such as 24 h after delivery, and consequently this line displays a greater susceptibility to diseases and stress. therefore, selection for litter size environmental variability can be a useful way to improve animal welfare. robustesse et canalisation: vision de généticiens welfare assessment and relevant ethical decisions: key concepts bien-être animal: context, definition, évaluation inflammation as "common soil" of the multifactorial diseases inflammation: the common pathway of stress-related rethinking il-6 and crp: why they are more than inflammatory biomarkers, and why it matters sex differences in the association between stressor-evoked interleukin-6 reactivity and c-reactive protein correlation of 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diabetes: a new aspect on old molecule reference values for heamatological and biochemical parameters in saanen goats breeding in afyonkarahisar province gamma-glutamyl transferase: a predictive biomarker of cellular antioxidant in adequacy and disease risk serum activity of alanine amino transferase (alt) as an indicator of health and disease van den ingh, t.s. hepatocyte derived micro rnas as sensitive serum biomarkers of hepatocellular injury in labrador retrievers identification of functional mutations associated with environmental variance of litter size in rabbits this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license animals 2020, 10, 1540 key: cord-002119-kl431ev6 authors: garcia, elisa; aguilar-cevallos, jorge; silva-garcia, raul; ibarra, antonio title: cytokine and growth factor activation in vivo and in vitro after spinal cord injury date: 2016-06-23 journal: mediators inflamm doi: 10.1155/2016/9476020 sha: doc_id: 2119 cord_uid: kl431ev6 spinal cord injury results in a life-disrupting series of deleterious interconnected mechanisms encompassed by the primary and secondary injury. these events are mediated by the upregulation of genes with roles in inflammation, transcription, and signaling proteins. in particular, cytokines and growth factors are signaling proteins that have important roles in the pathophysiology of sci. the balance between the proinflammatory and anti-inflammatory effects of these molecules plays a critical role in the progression and outcome of the lesion. the excessive inflammatory th1 and th17 phenotypes observed after sci tilt the scale towards a proinflammatory environment, which exacerbates the deleterious mechanisms present after the injury. these mechanisms include the disruption of the spinal cord blood barrier, edema and ion imbalance, in particular intracellular calcium and sodium concentrations, glutamate excitotoxicity, free radicals, and the inflammatory response contributing to the neurodegenerative process which is characterized by demyelination and apoptosis of neuronal tissue. traumatic spinal cord injury (sci) is a complex, lifedisrupting medical condition due to the detrimental effects on social, familiar, and personal life, which include in the majority of cases permanent paralysis due to the low regenerative capacity of the central nervous system (cns). sci triggers a series of interconnected mechanisms that can be divided into the primary and secondary injury. the direct and immediate physical disruption of neurons, glial cells, and blood vessels makes up the primary injury. in turn, the secondary injury consists of a cascade of autodestructive cellular and molecular mechanisms that exacerbate the primary injury and lead to an enlargement of the initial area of trauma [1] [2] [3] [4] . several mechanisms take part in this latter phase of the injury, including vascular disruption, increased blood-spinal cord barrier permeability, ionic dysregulation, edema, excessive intracellular calcium concentration, glutamate excitotoxicity, lipid peroxidation, an autoreactive inflammatory reaction, and apoptosis [5] . ultimately, the sum of these processes causes cell death, demyelination, and axonal degeneration at the epicenter of injury and the surrounding regions. these cellular and molecular changes that occur early after sci alter gene expression profiles, which is characterized by a significant upregulation of genes with roles in transcription, inflammation, and signaling proteins [6] . evidence suggests that the consequent inflammation mediated by cytokines, growth factors, and related molecules plays a role in both the damage and repair of injured neural tissue [7] [8] [9] . the critical balance between these processes plays a major participation in the progression and outcome of a neurodegenerative process [10] . cytokines encompass a large family of small signaling proteins involved in intercellular communication that are normally associated with the immune response and its 2 mediators of inflammation modulation but have pleiotropic effects in the physiology of health and disease including cellular growth, survival, and differentiation. these molecules, which can be classified as peptides, proteins, or glycoproteins, are secreted by numerous cells and can be grouped into a proinflammatory or antiinflammatory category on the basis of the final balance of their effects [10] . subsequently, growth factors are proteins synthesized by a wide variety of cells that stimulate cellular survival, chemotaxis, proliferation, and differentiation [11, 12] . the aim of this review is to expose the role of cytokines and growth factors within the pathogenesis of sci, since the study of these molecules could bring to light novel potential therapeutic targets that could reduce the degenerative processes that occur after sci. cord injury barrier. the blood-cns vascular barriers consist of complexes of adherence junction proteins and tight junctions, astrocyte endfeet, perivascular microglia, pericytes, and continuous capillary endothelial cells embedded in the basement membrane that separate and protect the cns from metabolites and neurotoxic substances present in the systemic circulation [13] [14] [15] . this infrastructure allows the blood brain barrier (bbb) and blood spinal cord barrier (bscb) to regulate the transport of molecules, the interaction between the cns and the immune system, and helps maintaining homeostasis in the brain and spinal cord. one of the earliest events ensuing traumatic sci is the disruption of the bscb by a mechanical force that destroys neural tissue and tears neuronal and endothelial cell membranes [5] . the resulting inflammatory response disturbs the microenvironment of the spinal cord, alters vascular permeability, facilitates the entry of peripheral immune cells, and exposes the adjacent noninjured tissue to potentially noxious molecules [16, 17] . these molecules include early inflammatory cytokines such as interleukin 1 (il-1 ) and tumor necrosis factor (tnf ); in addition, they might include nitric oxide (no • ), reactive oxygen species (ros), elastase, and matrix metalloproteinase-9 (mmp-9) [17] . the importance of the bscb is evidenced by the positive correlation between increased barrier disruption and improved motor locomotion 14 days after sci [18] [19] [20] . an additional consequence of such disruption is a series of regulatory changes in the transport systems for selective cytokines that may induce regenerative or destructive effects. in particular, there is an upregulation of the transport system of tnf after sci that remains saturable despite bscb disruption. the increase of tnf takes place before other cytokines in sci and is mediated by the receptor-based transport composed by tnfr1 (p55) and tnfr2 (p75) [21] . tnf has a role in inflammation, myelin destruction, apoptotic neuronal cell death, and astrocyte toxicity. nevertheless, this cytokine is also capable of stimulating neurite outgrowth, secretion of growth factors, and tissue remodeling [21] . it has been suggested that tnf has a dual role: deleterious in the acute phase, but beneficial in the chronic phase after sci [22] . similarly, leukemia inhibitory factor (lif) utilizes a transport system mediated by lifr (gp190), which is upregulated by barrier disruption, but remains saturable despite this event [21, 23] . lif is involved in the activation of microglia/macrophages and in the proinflammatory response in sci [24] . contrastingly, lif has been shown to prevent oligodendrocyte apoptosis in mice with sci after overhemisection, notably contralateral to the spinal cord lesion, through the induction of the jak/stat and akt signaling pathways as well as by potentiating the expression of the antiapoptotic molecule, ciap2. reduced oligodendrocyte apoptosis after sci with lif administration resulted in a substantial decrease in demyelination shown by the preservation of lamellated myelin surrounding viable axons and deposition of the degraded myelin basic protein. the data suggest that lif signals survival in oligodendrocytes after sci, prevents the secondary wave of demyelination, and thereby reduces inhibitory myelin deposits and enhance locomotor recovery [25] . imbalance. immediately after contusive sci, the rupture of the blood-cns barrier causes water to accumulate in the extracellular compartment and results in the production of neural tissue edema [26, 27] . this is a process that may aggravate the initial injury and result in paraplegia or even death [13] . the subsequent increment in vascular permeability and the formation of edema could also be in part mediated by the vascular endothelial growth factor (vegf) and proto-oncogene tyrosine-protein kinase (src/c-src) which exists downstream of vegf [28] . it is worth noting that administration of vegf has resulted in an increase in permeability of the bscb from the acute to chronic phase, which is interesting since it is regarded to be a component involved in angiogenesis, neurogenesis, and locomotor recovery [29] . as the secondary injury progresses, this fluid accumulation in the cns becomes characterized by ionic imbalance, which consists of an increase in the intracellular concentration of na + and ca 2+ , in conjunction with an elevated extracellular concentration of k + and mg + [30] [31] [32] . consequently, the na + and ca 2+ ions attract water molecules into the cell and cause edema. the resulting fluid accumulation then propels the compression of adjacent tissues and the development of ischemia, which leads to more autodestructive phenomena such as free-radical production, lipid peroxidation, and inflammation. it is important to note that the edema that occurs after contusive sci is directly related to the initial trauma and motor dysfunction experienced by the affected individual [27, 33] . astrocytes are the principal regulators of water transport in the cns, where they are additionally linked to the maintenance of ion homeostasis, spatial buffering of extracellular potassium, calcium signal transduction, adult neurogenesis, and neurotransmitter uptake and release [34] [35] [36] . a molecule expressed in astrocyte endfeet, astrocyte processes, and the basolateral membrane of ependymal cells is aquaporin 4 (aqp4), the predominant water channel in the cns [36] . recent studies indicate that aqp4 regulates the beforementioned astrocytic functions [36] . moreover, the absence of aqp4 has been shown to reduce proinflammatory cytokines in astrocytes such as tnf and interleukin-6 (il-6) after cns injury [37] . it is important to mention that the role of aqp4 in the resolution of edema is still under debate [37] . nevertheless, evidence demonstrates that aqp4 has an essential role in the formation and distribution of edema and that it is intrinsically involved in the development of the inflammatory process after an insult to the cns [37] . on the other hand, neurons regulate synaptic transmission and neural plasticity by the activation of membrane receptors and channels in adjacent neurons. released neurotransmitters can bind to inhibitory (gaba)ergic receptors or excitatory glutamate receptors such as amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (ampa), n-methyl-daspartate (nmda), kainate, and metabotropic receptors [38] . in the locomotor networks of the spinal cord, ca 2+ activated, apamin-sensitive k + channels (sk) control the firing of constituent neurons and regulate the locomotor rhythm. voltagegated ca 2+ channels (vgccs), such as n-type ca 2+ channels, are considered the main activators of sk channels [39] , which during early development play a role in neurite outgrowth and functional neuromuscular synapse organization [40] . nmda receptors, besides controlling evoked neurotransmitter release, also play a role in the activation of sk channels in dendrites [39, 40] . sk channels have been found to regulate hippocampal synaptic plasticity, learning, and memory, particularly sk2 channels [41] . synaptic transmission involves ca 2+ and employs calmodulin (cam) dependent kinases (camkiiv), protein kinase c, protein kinase a, ip3 kinase, ca 2+ -dependent phosphatase calcineurin b, cyclic amp phosphodiesterase, adenylyl cyclase, lca 2+ -dependent neuronal nitric oxide synthase (nos), and calpains, which are ca 2+ activated proteases [42, 43] . in the first few minutes following sci, oxidative stress, lipid peroxidation, and membranous deposition of protein aggregates take place. these processes impair ca 2+ pumps and cell membrane channels, including those present in the endoplasmic reticulum. this downregulation is evidenced by an increased concentration of cytosolic ca 2+ from extracellular pools and intracellular ca 2+ storages [44] . in normal conditions, the energy-dependent ca 2+ buffering system within axons removes the excess ca 2+ . however, when adenosine triphosphate (atp) is depleted by the excessive energy demands of demyelination, this normal ca 2+ buffering fails and the level of intracellular ca 2+ rises until it becomes toxic [44] . the result is the chaotic activation of processes such as proliferation, differentiation, apoptosis, and gene transcription in cells [45] . in addition to the before-mentioned channels, axons also have a high concentration of voltage-gated na + channels spread along the length of their bodies. thus, when axonal demyelination occurs, there is a dramatic increase in na + influx into the cell during the action potential propagation. the elimination of such an excess concentration of intracellular na + can come at a steep metabolic expense in a similar fashion to ca 2+ removal, since the na + /k + atpase maintains the na + electrochemical gradient by atp consumption [46, 47] . when atp levels fall below a certain threshold, there is a concomitant increase in the intra-axonal concentration of na + and ca 2+ . consequently, glutamate is released, and the na + /ca 2+ exchanger, which normally pumped out 1 ca 2+ in exchange for 3 na + , is reversed [46, 47] . it is also important to mention that the subsequent release of atp after the lesion increases in peritraumatic areas for 6 or more hours [48] . this excessive release of atp by the traumatized tissue after sci is followed by the activation of high affinity purinergic p2x receptors. it is important to note that the p2x7 receptors may also contribute to the excessive influx of ca 2+ since they are upregulated in response to the atp release induced by sci. this might explain why spinal cord neurons respond to atp with excessive firing, followed by irreversible increases in ca 2+ that end up in cell death [49, 50] . furthermore, p2x7rs have been associated with cells of the immune system that mediate cytotoxic cell death (because of changes in transmembrane ion fluxes, swelling, and vacuolation) and those that mediate inflammatory responses, including proinflammatory mediators such as il-1 and tnf [49, 50] . glutamate receptors are involved in the excitatory neurotransmission of the mammalian cns, where they participate in various changes in the efficacy of synaptic transmission, and induce excitotoxic damage in a variety of acute and chronic neurological disorders [51, 52] . the process of excitotoxicity refers to the excessive receptor activation by this excitatory amino acid that results in neuronal death [53] . just 15 min after sci, glutamate levels at the epicenter and surrounding regions become six times higher than physiological levels due to the overstimulation of ionotropic receptors and the massive increase of intracellular ca 2+ and na + . this glutamate influx provokes overexcitation and endotoxicity by the secondary increase of intracellular ca 2+ and the activation ca 2+ dependent signaling pathways as previously mentioned [54] [55] [56] . moreover, the augmented expressions of genes related to neurotransmitter receptors (nmda, ampa, ach, gaba, glur, and kainate) increase demyelination and oligodendrocyte destruction [57, 58] . an important mechanism for the reduction of excessive extracellular glutamate is the activity of glutamate transporters such as glial glutamate transporter 1 (glt-1) and glutamate aspartate transporter (glast), which are primarily expressed by astrocytes [59] . unfortunately, the excitotoxicity induced by the extracellular glutamate concentration is enhanced by the reduced uptake by astrocytes and the microglia release tnf , il-1 , and ros that exacerbated the neural damage [60] . tnf and il-1 have been shown to cause oligodendrocyte death when the latter are placed in coculture with both astrocytes and microglia. both cytokines inhibit glutamate transporters in astrocytes and thus expose oligodendrocytes to an excessive glutamate concentration. it is important to note that antagonists of ampa/kainate glutamate receptors such as nbqx (2,3-dioxo-6-nitro-7-sulfamoilbenzo(f)quinoxalina) and cnqx (6-cyano-7-nitroquinoxaline-2,3-dione) blocked il-1 toxicity towards oligodendrocytes [61] . tnf causes excitotoxicity through a series of interconnected, deleterious mechanisms. first, microglia release this cytokine in the inflammatory response, which induces additional release of tnf . in turn, it causes the release of glutamate that acts on metabotropic receptors of microglia and stimulates more tnf release. subsequently, astrocytes are stimulated to release glutamate, which is not effectively transported back into the soma. lastly, the rise in the excitatory/inhibitory ratio causes the excessive ca 2+ entry and excitotoxic neuronal death previously described. the consequent neuronal death caused by the excessive glutamate concentrations further stimulates microglia to remain in an active state, which includes the production and release of tnf in a vicious cycle [53] . tnf potentiates cytotoxicity by glutamate through an increased localization of glutamate receptors such as ampa and nmda while decreasing inhibitory gaba receptors on neurons [62] , which explains why nbqx blocked tnf toxicity to oligodendrocytes [61] . in the destruction of neurons, nerve fibers, glial cells, and blood vessels at the site of injury, where approximately 30% of neurofilament constitutive proteins are degraded in 1 h, and 70% are lost within 4 h after the injury [63] . proteins such as cathepsin b, y, and s, members of the cysteine lysosomal proteases and papain superfamily, have been linked to neurofilament destruction. this link results from the fact that cathepsin b can degrade myelin basic protein, cathepsin y can produce a bradykinin, and cathepsin s can degenerate extracellular molecules through inflammatory mediators. in particular, only cathepsin s is able to retain its activity after prolonged incubation at neutral ph, more than 24 h [64, 65] . the expression of this protease is restricted to cells of the mononuclear phagocytic system such as microglia and macrophages [64] . a basement membrane heparan sulfate proteoglycan (hspg), perlecan, which was found to promote mitogenesis and angiogenesis, can be degraded by cathepsin s in vitro. hspgs have roles in adhesion, protease binding sites, and growth factor regulation as is the case for basic fibroblast growth factor (bfgf) [66] . furthermore, cathepsin s degrades laminin, fibronectin, collagens, and elastin at acidic or neutral ph [65] . it is known that tnf , interferon-(ifn ), il-1 , and granulocyte macrophage colony stimulating factor (gmcsf) stimulate the release of active cathepsin s into an environment with a neutral ph [65] . subsequently, a change in lipid metabolism and the homeostasis of lipid mediators is an alternate route by which genes are thought to modulate the susceptibility of nervous tissue to trauma. interestingly, altered protein cleavage, one of the main driving forces of protein aggregation in neurodegenerative disorders, can be further enhanced by trauma occurring in the presence of specific lipid-binding proteins, important molecules in charge of the distribution of lipids and the transport of cholesterol among cells in the cns. apolipoprotein e (apoe) is one particular example of this phenomenon, since a reduction in its availability causes a reduction in the recovery after neurotrauma or an ischemic insult. apoe fragments are produced by traumainduced proteolytic cleavage, which, in turn, might disrupt the cytoskeleton by the phosphorylation of tau and the promotion of neurofibrillary tangles. at the same time, apoe4 increases the inflammatory effect of neurotrauma by a significant increase of il-6, tnf , and no • in the injured tissue [67, 68] . radicals. microvascular disruption, ionic imbalance, increased intracellular calcium levels, glutamate excitotoxicity, mitochondrial dysfunction, arachidonic acid breakdown, and the activation of inos contribute to the formation of free radicals (fr) [69] . fr are reactive molecules produced by the metabolism of the cell that possess an unpaired electron, which easily reacts with biomolecules by oxidizing them [70] . a fr is made up of sulphur (s), nitrogen (n), chloride (cl), or carbon (c). these elements associate with oxygen and form other fr such as no • . metals such as fe, mn, co, ni, and cu can also be considered fr since they have unpaired electrons [71, 72] . many of these molecules are either reactive oxygen species (ros) such as delta and sigma oxygen ( the mechanical reduction of the superoxide anion mediated by nad(p)h oxidases causes the anion to react with no and form a neurotoxic compound known as peroxynitrite [73] . at physiologic ph, peroxynitrite first reacts with proteins and phospholipids and then breaks down into other cytotoxic products such as no • , nitrogen dioxide (no 2 ), and oh − radicals. hall and braughler demonstrated the occurrence of early posttraumatic lipid peroxidation (lp) as early as 5 min after injury. lp is a mechanism that disrupts the normal structure and function of the lipid bilayers that surround the cell and membrane-bound organelles. when peroxynitrite or other fr takes an electron off a polyunsaturated lipid, it generates a lipid radical (l • ) that can further interact with molecular oxygen and yield a lipid peroxyl radical (loo • ). then, if the resulting lipid peroxyl radical loo • is not reduced by antioxidants, lp associated with sci induces early damage to the spinal microvascular endothelium (within 2-3 h). as a direct consequence of this damage, there are crater formation, platelet adherence, leucocyte presence, and the formation of microemboli, events that are concurrent with the reduced blood flow to the white matter of the spinal cord. the damage to the myelin sheath unhinged a demyelination process that is the particularity of a neurodegenerative process [74] . the cns is particularly sensitive to lp because of its high content of peroxidation-susceptible lipids (arachidonic, linoleic, and docosahexaenoic acid) and the primarily radical-mediated oxidative protein damage. considering the timeframe of the injury, the oxidative damage to dna and lipids, in addition to protein nitration, is observed within the first week after injury [75] [76] [77] [78] [79] [80] [81] . one of the degradation products of peroxynitrite, no • , alters the mitochondrial electron transport chain and induces the production of fr. these molecules have direct deleterious effect on enzymes with iron-sulfur clusters in their catalytic core, such as ubiquinone succinate [82] . after sci, the concentration of no • increases 3 to 5 times more than baseline levels and reaches its peak at 12 h. meanwhile, there is an increased production of inducible nitric oxide synthase (inos) and peroxynitrite [83] . the resulting elevated no • concentration induces cell damage and lipid peroxidation, increases vascular permeability, and causes edema [84] . hence, due to its involvement in the previous processes, no • participates in the development of the excessive glutamate and calcium concentrations that induce excitotoxicity [85] . it is known that no • is produced by different synthases. however, only inos is capable of producing a high concentration of no • for a prolonged period of time [86] . collectively, astrocytes, neutrophils, monocytes, and microglia induce the expression of inos at the presence of proinflammatory stimuli such as lipopolysaccharide (lps), ultraviolet radiation (uv), and tnf , il-6, il-1, and ifn [87] . in some studies, the expressions of inos and its protein activity were found 3 h, 4 h, 24 h, and 72 h after sci [83, 88, 89] . the inflammatory response is a characteristic phenomenon of innate immunity that does not require a previous exposition to the agent but does increase substantially with subsequent expositions as the response becomes specific and direct. cellular immunity consists of specialized cells that can recognize, endocyte, and eliminate different types of microorganisms or noxious substances. on the other hand, the humoral response is composed by soluble macromolecules that circulate in the blood and extracellular liquid that acts upon the pathogen [90, 91] . sci presents different patterns of gene expression depending on the cell type and activation phase [92] . numerous studies have suggested that the inflammatory response in sci is beneficial, because it can eliminate tissue debris and induce the release of various neurotrophic factors [17, 93, 94] . nevertheless, this inflammatory response tends to go out of control when it exacerbates autoreactive mechanisms that cause neural destruction. traumatic sci triggers inflammatory reactions in which various types of cells, cytokines, and neuroprotective/neuroregenerative molecules are involved [95] . 2.6.1. cells of the inflammatory response. immediately after the rupture of the blood-spinal cord barrier, the consequent inflammatory response involves the participation of chemical mediators, and resident (astrocytes and microglia) and peripheral (macrophages, lymphocytes) immune cells [96, 97] . additionally, oligodendrocytes, neurons, and endothelial cells participate in the cellular response after sci [98] , in which microglia and endothelial cells function as antigenpresenting cells (apc) [96] . throughout the inflammatory response, the infiltration of immune cells is the principal contributor to neural degeneration [95] . these cells are guided to the lesion site from the periphery by the effect of chemokines and cytokines that are mainly released by microglia, astrocytes, and peripheral macrophages, which make up the principal resource of these molecules in the lesion site [5, [99] [100] [101] [102] . the released cytokines include il-1 , il-1 , il-6, tnf , gm-csf, and lif [60] . the neurons of human patients expressed these molecules, 30 min after sci, and microglia, 5 h after the lesion; however, the expression decreased by the 2nd day [103] . similar results were obtained in mice and rats since the expression by local neurons was found at 1 h, and at 6 h by microglia, which decreased to baseline on day 1 after sci [104] . the expression of tnf and il-1 by microglia and astrocytes was identified 5-15 min after the lesion. the peak expression was at 1 h for tnf and 12 h for il-1 [104] . after sci, two waves of cellular infiltration have been characterized. the first wave consists of polymorphonuclear leukocytes (pmn) that predominate throughout the first hours following the lesion. they are activated by il-1, interleukin-2 (il-2), and il-6 in particular [105] and might be mainly recruited by chemokine (c-c motif) ligand 2 (ccl2), chemokine (c-x-c motif) ligand 1 (cxcl1), and chemokine (c-x-c motif) ligand 2 (cxcl2, also called macrophage inflammatory protein 2-alpha (mip2-alpha)) [106] . these cells become apparent in the walls of veins and venules adjacent to the lesion in the first 3-4 h and can be observed inside the tissue 8-24 h after the lesion. it has been found that these cells represent 90% of the infiltrating cells 12 h after the injury [107] . the inflammatory response is evidenced by the increased quantity of leukocytes in the cerebrospinal fluid, the infiltration of pmn in the lesion site, the increment in the leukotriene levels (ltb4 in particular), and the activity of myeloperoxidase. in addition, a significant increase in the expression of intercellular adhesion molecule 1 (icam-1) can be identified, which favors the infiltration of neutrophils from the first 3 to 12 h after the lesion [108] . the second wave of infiltration is characterized by the presence of monocytes and macrophages, which can be observed around 3-4 days after sci [106] . various chemokines are known to mediate macrophage infiltration such as ccl2, cxcl1, and cxcl2 [106] . this demonstrates how important the recruitment of macrophages is after an injury to the cns [109, 110] . activated microglia become evident in the first day after sci [108] ; moreover, there is a peak in the proliferation and recruitment of microglia from day 3 to day 7 [111, 112] . the overexpression of lif has been found to cause a dramatic increase in the proliferation of microglia/macrophages and astrocyte activation [24] . the pathological proliferation of macrophages and microglia might contribute to the subsequent exacerbation of the initial damage [113, 114] , even though macrophages have an important role in the clearing of denatured dendrites [115] . microglia at the injury site rapidly express the alarmin il-1 , while infiltrating neutrophils and macrophages produce il-1 which plays a role in the infiltrating mechanism of these cells. interestingly, the expression of il-1 mediates the suppression of the survival factor tox3 (tox high mobility group box family member 3) in oligodendrocytes, which in the absence of such cytokine would provide protection of this cell population, and functional recovery after sci [7] . diverse studies have reported that the recruitment of leukocytes to the injured spinal cord is a physiological response that is associated with the production of cytokines and protein kinases that are involved in the repair of the site of lesion. neutrophils, for example, are the first cells to be recruited with the objective of clearing the lesion site from possible pathogens and cellular debris through phagocytosis. however, the activation of these cells also induces the release of a significant amount of neurotoxins such as ros, rns, chemokines, and a variety of enzymes that promote tissular destruction [105, 116, 117] . the taoka report provides evidence demonstrating that after sci the maximum peak of neutrophil migration perfectly correlates with the extent of the damage and the motor alterations observed after the lesion [105] . the infiltration of monocytes and macrophages after sci has for its objective the removal of cellular debris and the stimulation of new blood vessel and parenchymal cell formation. the infiltration of these cells regulates the activation and proliferation of t lymphocytes since they play the role of apc [117] . microglia are pluripotent cells capable of developing different phenotypes proportional to the severity of the lesion, which determines the intensity of the inflammatory response, the quantity of recruited cells, and the magnitude of the immunological response. this can be explained by the interaction between microglia and t lymphocytes, since it induces an antigen specificity that regulates the immune response and the subsequent phases [118] . microglial cells are distributed throughout the cns, where they serve as a pathological sensor that is activated in response to noxious stimuli such as physical trauma or vascular obstruction [119] . activated microglia migrate to the site of injury, proliferate, and transform from the resting phenotype (ramified cells) to amoeboid phagocytic cells [120] . in fact, after sci, activated microglia can be seen at the epicenter of the lesion initially at 12 h [60] . in addition, there is an upregulation of surface receptors such as cr3 (complement receptor type 3) and mhc ii (major histocompatibility complex) whose implications are covered in several papers of our group. these activated microglia can then release a series of cytokines, chemokines, and enzymes, which are proportional to the activating stimulus as mentioned previously. the series encompass il-1 , il-6, tnf , tgf-1 (transformation growth factor-1), m-csf [121] , inos, ngf (neural growth factor), nt-3 (neurotrophin-3), and bndf (brain neuronal derived factor) [122, 123] . interestingly, monocyte derived microglia and macrophages might be able to induce regeneration by the secretion of neurotrophic factors, particularly tgf-1 [17] . activated microglia and macrophages have been implicated in the secondary pathology that accompanies traumatic or autoimmune injuries to the brain and spinal cord [124] . the associated implications usually refer to the activation of these cells towards an inflammatory m1 phenotype; however, these cells can also be activated towards an m2 macrophage phenotype that responds to il-4 and il-13. this contrasting phenotype is characterized by the production of several extracellular matrix proteins that may promote tissue remodeling, repair, neurotrophic support, and axonal regeneration [125] [126] [127] [128] . taking into account the excessive release of glutamate and the feedback of the inflammatory response after sci, it is no surprise that microglia acquire a reactive phenotype that expresses very low quantities of the mhc ii molecules and is not capable of maintaining an adequate interaction with t lymphocytes [118, 129] . the m1 phenotype is characterized by the excessive release of no • , il-1, il-6, and tnf , which leads to toxicity. in these conditions, astrocytes and postsynaptic neurons show signs of damage, evidenced by the expression of ros, which can induce apoptosis [121] . nevertheless, activated microglia remove the cellular debris after the lesion and are capable of promoting revascularization in the site of injury, which facilitates the release of trophic factors and nutrients for the survival and proliferation of infiltrating cells in the lesion site [118] . furthermore, microglia are capable of expressing glutamate transporters, which apparently help buffer the excessive concentrations of glutamate, and consequently protect cells from toxicity [129, 130] . it is important to take the before-mentioned into account since activated microglia and peripheral macrophages make up the majority of inflammatory cells present in the site of lesion, especially since these cells are morphologically different and respond to different modulatory signals [131] in an early response of the innate immunity to the lesions of the cns, which have diverse etiologies such as ischemia and neurotrauma [132, 133] . therefore, given that in the brain and the spinal cord there is a considerable heterogeneity of macrophages, the relative contribution of one population of cells in the local inflammatory reaction can dictate whether a cascade of events initiates as a regenerative or a destructive process. this all depends on the macrophage phenotype activated, particularly microglia [117] . in regard to t-lymphocyte infiltration in humans, it might be detected months after the injury, and b-lymphocytes are not usually found [134] . on the other hand, mice models present both cell types 7 days after sci, with a peak at 42 days [135] . lymphocytes are the cells that modulate the intensity of the inflammatory response. traditionally, their participation after sci has been linked with the damage to neural tissue since they are capable of producing proinflammatory cytokines such as inf and il-1 . on one hand, inf is linked directly to neuronal destruction since it induces the expression of other proinflammatory cytokines (tnf , il-6, il-12, and il-1 ), and proinflammatory molecules such as ros and inos, since it participates in the induction of transcription factors including nf (nuclear factor kappa beta) and ap-1 (activator protein-1) [118, 136, 137] . in addition, inf is known to participate in the induction of a cytolytic response by tcd8+ (cd, cluster of differentiation) since it is the principal inductor of mhci through the phosphorylation of stat1 (signal transducers and activators of transcription-1) [138] . moreover, the chemoattractant, c-x-c motif chemokine 10 (cxcl10) recruits cd4 th1 cells via the cxcr3a receptor [95] . after the induction of protective autoreactivity, which is a strategy based on the modulation of the immune response by neural derived peptides, diverse studies have reported that the presence of t lymphocytes with a th2 phenotype in the lesion site favors functional recovery [139] [140] [141] . this is due to their ability to synthesize ngf, bdnf, and diverse neurotrophins (nt3, nt4, and nt5) [142, 143] . in a classical th1 activation pattern, however, the inflammatory response after sci can be responsible for the necrosis of the lesion site and the surrounding area [144] [145] [146] . to further explore this phenomenon the cytokine expression was analyzed in comparison to sham animals and the dominant phenotype was found to be th1 and th17 predominantly according to expectations [147, 148] . this is due to its important role in the generation of free radicals, cytokines, and chemokines that exacerbate the damage to the neural tissue for weeks or even months. it is important to point out that this noxious effect occurs when the immune response is not modulated, since it is correlated with excitotoxicity, lipid peroxidation, and the development of an autoreactive response towards neural constituents [17, 93, 139, 149, 150] . contrastingly, this response can either increase the damage to the neural tissue, promote protection [150] [151] [152] [153] , or even promote restoration of the injured tissue [126, 127, 144] as a function of neuromodulation. the autoreactivity observed after sci is characterized by the specific immune response, with lymphocytes being the only cells capable of specifically recognizing the antigens, and initiating the adaptive immune response. despite the existence of mechanisms by which these autoreactive t cells are eliminated or inactivated, these are not sufficient, and consequently they can be found in practically every healthy individual. thus, autoreactivity can be part of a normal immune response that can find its origin in several infectious and inflammatory diseases. however, when this mechanism is excessive, the result is an autoimmune disease [154] [155] [156] . there are multiple diseases that are considered to be autoimmune or to have an autoimmune component. multiple sclerosis (ms) is one of such diseases. it is an inflammatory, demyelinating disease, in which an autoimmune response to mbp [157] has been reported. interestingly, after sci, an autoreactive phenomenon similar to the pathophysiology of ms can be observed. consequently, it is well-known that the lymphocyte role after sci is fundamental, because these cells are responsible for the generation of autoimmunity in individuals with genetic susceptibility [89, 158, 159] . these events can become chronic if the proinflammatory environment is not regulated. if not regulated, the response would involve the participation of other immune cells, other signaling pathways, and other patterns of gene expression. the persistent influx of immune cells from the systemic circulation as neutrophils, macrophages, lymphocytes, basophils, and eosinophils is correlated with additional elevation of proinflammatory cytokine levels and neural tissue destruction that would unavoidably make tissue recovery more difficult [108, 160, 161 ]. cytokines comprise a large family of small signaling proteins that affect nearly every biological process including embryonic development, disease, nonspecific infection response, cognitive functions, aging, cellular growth, survival, and differentiation [10, 162] . these "cytokines," which can be classified as peptides, proteins, or glycoproteins, encompass interferons, interleukins, the chemokine family, the tumor necrosis factor family, adipokines, and mesenchymal growth factors [10, 163] . these molecules are produced by one cell and go on to act on another cell in order to bring a change in the function of the target cell. the difference with hormones is that cytokines are products of most cells while not being of a particular tissue or cell. the majority of cytokines function by binding to specific cell surface receptors; this action triggers intracellular signaling and activates transcription factors such as ap-1 and nf [162] . interestingly, the diverse properties of a single cytokine can be explained by the following mechanisms: the first mechanism involves the presence of the receptor of a certain cytokine in one particular type of cell (e.g., il-33 receptor on mast cells) [164] . the second mechanism is explained by the presence of the receptor to a specific cytokine on most cells (e.g., activation of nf by il-1, or tnf induction of cox-2). the third mechanism encompasses the ability of cytokines to induce or function as coactivators (e.g., il-18 induces ifn when il-12 is present, but when it is not, il-18 induces fas ligand) [165] . despite the fact that cytokines are studied in every discipline of biology, the effects of these molecules are mostly studied in the realm of inflammation, immunology, cancer, and atherosclerosis [162] . in these areas, cytokines can be grouped into a proinflammatory or anti-inflammatory category on the basis of the resulting balance of their added effects [10] . in the cns, cytokines have homeostatic physiologic and neuromodulatory functions. surprisingly, they also have the capability of contributing to neuronal damage and destruction when their concentration exceeds a certain threshold. one of the reasons as to why they cause such damage and destruction lies in the uncontrolled inflammatory response observed after sci, which emphasizes the reason behind the augmented study of these molecules in inflammation-related research. the upregulation of these cytokines, as well as the consequent cellular infiltration they cause, plays a crucial role in the determination of the extent of the secondary tissue damage and neural degeneration observed after the injury [95, 166, 167] . therefore, taking into account that the production and release of proinflammatory cytokines and chemokines (table 1) is the first inflammatory event that develops after sci, the importance of these molecules becomes clear [166, 167] . in regard to the realm of inflammatory cytokines, there is a clear diversity in their functions. for starters, certain molecules are capable of inducing vascular permeability and cellular fluid loss, which include components of the complement cascade (c3a and c5a), which in turn cause the release of histamine, prostaglandins, and leukotrienes from resident mast cells. specific inflammatory cytokines such as tnf , il-1, and il-6 are synthetized by various cells in the cns and are known as mediators of the peripheral immune response 8 [118, 192] il-4 (i) high levels 24 h ai, concentrations remain during 7 days and decrease 3 days ai (i) neuroinflammatory regulation in various pathological conditions (ii) confers regenerative properties to macrophages (iii) controls free radical production in peripheral macrophages and microglia [166, [193] [194] [195] [196] [197] [198] [199] il-13 (i) detected 1 day ai (i) macrophage activation onto m2 phenotype [166, 199] ip-10/cxcl10 (i) expressed locally 30 [200, [205] [206] [207] mediators of inflammation 9 [200, [205] [206] [207] mcp1/ccl2 (i) detected from 1 h ai with pl at 24 h and remains low up to 24 days ai (i) macrophage and pmn infiltration mediator [106, 184, 200, 205, 206] min: minutes; ai: after injury; pl: peak levels. mediators of inflammation [168] [169] [170] . on one hand, tnf immediately recruits neutrophils to the site of the lesion by the induction of adhesion molecules such as icam-1 and vcam-1 (vascular cell adhesion molecule-1), as it stimulates the release of il-8, which is a chemotactic factor for neutrophils. furthermore, tnf alters the permeability of endothelial cells and damages the blood-spinal cord barrier. moreover, this cytokine is able to exert cytotoxic activity towards oligodendrocytes and contributes to demyelination. in addition, tnf also stimulates the proliferation and hypertrophy of astrocytes, hereby promoting the formation of the fibroglial scar, which acts as a barrier to a possible regeneration of the cns as a biological measure of last resort in response to an uncontrolled chronic inflammation [168] [169] [170] . on the other hand, studies have shown that a direct injection of il-1 into the spinal cord leads to enhanced vascular permeability and lymphocyte recruitment. subsequently, il-6 has been found to promote the activation and infiltration of macrophages and microglia [161, 171] . in fact, it is known that il-6 is a major player in chemokine infiltration, because it has the ability to interact with other cytokines and neurotrophic factors [172, 173] . interestingly, several studies have revealed that the continuous inhibition of il-6 is detrimental to functional recovery because it also participates in axonal regeneration and gliosis, in line with the role of tnf in chronic inflammation [174, 175] . thus, it is important to take into account that the mediation of the early inflammatory tissue damage may actually worsen the functional outcome [176] . this leads to a conflict, since the role of inflammation after sci appears to be contradictory when the beforementioned and following points are taken into account [177] . on one hand, proinflammatory cytokines, il-1 and il-6, are beneficial at low concentrations due to their induction of neurotrophin expression and the mediation of leukocyte activation/recruitment to the injury site by the induction of adhesion molecules in the cell surface such as icam-1, p-selectin, and e-selectin [172, 173] . on the other hand, at higher concentrations, these inflammatory cytokines activate transcription factors such as nf , ap1, and atf, factors that stimulate the expression of neurotoxic genes, including cox-2, inos, and proinflammatory proteases in different target cells [88, 178, 179] . pan found that the mrnas of cytokines such as tnf , il-1 , il-1 , and il-6 could be detected 15 min after injury. from these cytokines, il-1 and il-1 continually reached peak levels until the 6 h but were not present from the 12 to 24 h after sci. in addition, by 4 h after contusive sci, significantly increased mrna levels of il-1a and il-6 were clearly detected by qrt-pcr [180, 181] . digging further into the time frame of expression, western blot studies found that the mature form of il-1 is expressed by the 2 h. this evidence suggests that the inflammatory cytokine is released very quickly after tissue damage. the expression of these genes was identified 1 h after contusive rat sci by cdna microarrays [57] . the procedure was then repeated in spinal cord injury patients, and the same results were observed [103] . moreover, hayashi found that after sci the mrnas of cytokines such as tnf and il-1 were upregulated in as little as 1-3 h after the lesion [148, 182, 183] . on another note, tnf mrna peaked quickly 60 min after the injury and fell slightly by the 120 min. tnf mrna remained elevated by day 1 after sci, returned to a low level by day 3, and was not detected by day 5 [184] . il-6 mrna increased slowly, reached peak levels by 6-12 h, and fell by 24 h [180] . it is important to note that the levels of these mrnas were nearly undetectable in sham-injured animals. another study found that, between 12 h and 72 h after sci, the gene expression of proinflammatory cytokines such as il-1, il-3, il-6, and their receptors was strongly upregulated [6] . tnf and il-1 induce both il-1 and tnf mrnas. consequently, the downregulation of the signaling of il-1 and tnf reduces the induction of il-1 mrna [163] . this suggests that the activity of these cytokines contributes to their own mrna regulation [163, 180] . from the 3 h and up to 24 h, tnf , il-1 , il-6, and lif were found to be strongly upregulated in and around the contused area. these cytokines were produced at the same time range. it is worth noting that another wave of expression was observed for tnf and il-1 at 14 days, which correlates with an increased blood-spinal cord barrier function [104] . in particular, the overexpression of lif has been found to cause a dramatic increase in the proliferation of microglia/macrophages and astrocytic activation [24] . tnf is released significantly faster than other proinflammatory cytokines, because this is stored in a preformed state on the cell surface and in the granules of mast cells. it is not a surprise that role of this cytokine is similar to that of il-1 given the facts stated above [185] . it is important to note that tnf is the principal promoter of wallerian degeneration since it activates resident schwann cells in the peripheral nervous system and facilitates macrophage recruitment into the injury site [186] . in addition, these macrophages release proteases, fr, and cytokines [187] . similar to the facts stated above, the extracellular expression of tnf [187] in the surrounding white matter was detected 3 h posterior to contusion sci, with a peak that took place from day 1 to day 3 [166] . thus far, the time frames of expression have been described. the following information regards the receptors of such molecular products. from the two subtypes of tnf receptor that exist, each subtype has a different distribution and presence that depends on the particular cell type. for instance, tnf-r1 is expressed constitutively on most cell types, whereas the expression of tnf-r2 in astrocytes requires induction by tnf , il-1 , and ifn [188] . a large amount of evidence indicates that tnf-r1 augments neuronal death and tnf-r2 promotes neuroprotection [189] . what has been observed in the lesion concludes that the expression of tnf-r1 and tnf-r2 is increased within 15 min after traumatic sci in adult rats and reaches its peak at 4 h for tnf-r2 and 8 h for tnf-r1. the expression of both receptor subtypes then goes on to decline after day 1 and day 3, respectively [190] . it is important to note that these receptors are initially found on the epicenter of the lesion site. posteriorly, they spread radially towards distant areas during their peak expression and later become confined to the lesion area. these receptors are expressed by several cells, which include neurons, oligodendrocytes, and astrocytes [189, 190] . these cells might work individually or synergistically to mediate the biological activity of tnf , which makes an interesting research topic, given that these receptors are known to be involved in antiapoptotic activities through the tnf-r/nf signal transduction pathway [191] . on a last note, tnf participation in the expression of inos in microglial cells [137] causes an exacerbated neural destruction as a direct consequence of the induction of the nf pathway, which can then contribute to the expression of ifn . ifn within the nervous system is classically associated with the inflammatory response after injury as mentioned in the previous paragraph [213] . this molecule is believed to be normally involved as one component of the physiological response to tissue damage and trauma. cd4+ and cd8+ t cells together with natural killer (nk) cells are the major sources of ifn . nevertheless, evidence shows that this cytokine is also produced within the nervous system by neurons and glial cells in the absence of infiltrating immune cells [214] . in various animal models, ifn promotes macrophage signaling, production of proinflammatory cytokines and chemokines, recruitment of macrophages to the cns, and the activation of cns resident and infiltrating apc populations. moreover, ifn is also the most potent inductor of mhci, and it is upregulated in the cns after injury [215] . in low concentrations, ifn may participate in the homeostasis of the synaptic circuitry [216, 217] . as previously mentioned, ifn is involved in the upregulation of mhc i, which has been shown to play an important role in the synaptic plasticity process following axotomy. furthermore, ifn has been shown to regulate phosphorylation and nuclear translocation of signal transducer and activator of transcription 1 (stat1) and to influence neuronal excitability by the expression of the peripheral nerve-type sodium channel gene pn1 [192] . it is important to note that several studies found that ifn and il-17 had the highest levels of gene expression, since this indicates that the phenotype found after sci is predominantly th1 and th17 and the ifn release could be detected from 1 h to 12 weeks, depending on microenvironment [147, 148] . interleukin-17 (il-17) is primarily produced by th17 cells and has an important role in inflammation and autoimmune disease [201] . a key regulator in its production is il-6. nevertheless, tgf-and interleukin-21 (il-21) are also capable of stimulating il-17 production. similarly, interleukin-23 (il-23) is also able to promote il-17 production just as interleukin-22 (il-22) does. in one study, serum levels of il-6, il-21, and il-23 were increased in large quantities 1 h after sci, had a peak at 24 h, and had a positive correlation with increased il-17 [202] . signal transducer and activator of transcription 3 (stat3) and rar-related orphan receptor gamma (ror ) are two transcription factors capable of mediating il-17 production and th17 differentiation. as a result, a closed circuit is formed, in which il-17, the stat3 signaling pathway, and il-17 related cytokines promote neuroinflammation as they costimulate one another. il-17 expression and production was detected from 1 h to 72 h after contusion injury [202] . stat3 is a primary transcription factor of the downstream signaling of il-6 [203] . the phosphorylation of stat3 in this pathway induces a proinflammatory gene expression that correlates with il-17 quantities in spinal cord neurons and astrocytes. interestingly, through this very same pathway, anti-inflammatory th2 cells can be suppressed by il-6 inhibition of foxp3 expression in a stat3 dependant manner. it is important to recognize that stat3 was found higher in the sci rat group, whose expression peaked at 24 h [202] . it is worth noting that il-17 antagonistic therapy in rheumatoid arthritis (ra) suggests that the inhibition of the pathological role of il-17 may be a promising therapeutic approach in humans [204] . chemokines are functionally related cytokines that induce specific actions in the immune system. they are released in response to an infection, inflammation, or trauma [184] . chemokines are grouped into two families: the family (cxc), which participates in the recruitment of polymorphonuclear cells, and the family (c-c), which provides the priming signal for macrophages, lymphocytes, eosinophils, and basophils. the family includes gamma-interferon inducible protein (ip-10/cxcl10), platelet factor 4, il-1, and melanoma growth stimulatory activity (mgsa/gro/kc) [218] . in particular, the chemokine cxcl10 has been shown to inhibit angiogenesis, growth, and chemotaxis of endothelial cells via the cxcr3b receptor. consequently, the neutralization of cxcl10 promotes angiogenesis through the expression of eight genes related to angiogenesis and vasculature remodeling after sci [95] . an important member of the family is the monocyte chemoattractant protein (mcp-1/ccl2). it is detected in astrocytes and perivascular mononuclear cells in experimental allergic encephalomyelitis (eae). mcp-1 levels are related to the parallel development of clinical disease and macrophage infiltration [205, 206] . the same case applies to macrophage inflammatory protein 1 alpha (mip-1 /ccl4) and macrophage inflammatory protein 1 beta (mip-1 ) [219] . their expression has been shown predominantly in myeloid and lymphoid cells [207] , where an increased expression of mip-1, mip-2 (cxcl2/3), and mcp-1 after sci plays a role in the inflammatory process, since these molecules recruit circulating leukocytes to the injury site [220] . mcp-1 mrna was present in the normal spinal cord, was increased 1 h after sci, peaked at 24 h, and returned to a low level by day 14. mcp-1 is expressed by astrocytes that surround white matter. in addition, mip-1 mrna was present in the normal spinal cord, where it increased at 1 h after sci, peaked from 3 to 6 h, decreased by day 1, remained unchanged until day 7, and returned to a low level by day 14. mip-1 expression in astrocytes was observed from day 3 to day 6 following injury. additionally, the expression of this molecule was found at the contusion site and in rostral and caudal sections to this location. by day 5 after injury, the expression of mip-1 returned to baseline levels. moreover, ip-10 mrna presented low levels in the normal spinal cord, increased its levels at 1 h, peaked at 6 h, and remained high up to day 5 after sci. it decreased to baseline levels by day 14 [184] . another study found the chemokines, mcp-1, mip-1, mip-1 , mip-2, and ip-10, to be expressed locally at 30 min with a peak at 6 h after sci. it is worth noting then that chemokines remain present 24 d after injury-at lower levels-in contrast with the rest of the cytokines [200] . molecules of the inflammatory response. the changes in gene expression that contribute to the secondary injury are characterized by protracted neuronal loss and neurological dysfunction. therefore, the predominant downregulation of these factors might play a role in cell survival and may lead to the development of novel interventions that promote recovery [181, 221, 222] . in order to develop a viable therapy, it is essential to identify the specific molecular pathways that become altered as a function of time after sci [223] . for instance, activated macrophages and microglia after cns injury produce various neurotrophic factors and molecules that enhance regeneration [93, 224] . however, this response highly depends on the temporal sequence that proceeds the injury [108] . this consequently indicates that there is a proper and timely regulation of inflammatory reactions that can take place and be of paramount importance to the design of therapeutic strategies involving cytokines, growth factors, or neurotrophins [98, 116] . (1) cytokines. a particular cytokine involved in this beneficial aspect of the inflammatory response is il-4. this cytokine exerts an anti-inflammatory effect after cns damage [193] [194] [195] . for instance, endogenous il-4 has been shown to participate in the regulation of neuroinflammation in various pathologic conditions [196] [197] [198] . this anti-inflammatory cytokine and its receptor subunit il-4 have a role in spinal cord trauma. this is illustrated by the high level expression of il-4 24 h after contusive sci in rats, whose elevated concentration persisted for 7 days but was decreased 3 days after sci. interestingly, on day 1 after sci, an increased expression of il-13 was observed. this is noteworthy since this interleukin shares the same receptor with il-4 for signal transduction [166, 199] . moreover, the cytokine expression of the contused spinal cord was not significantly affected by il-4 attenuation for the proinflammatory cytokine levels of il-1, il-6, and tnf . in fact, the opposite effect was observed, since the event correlated with a marked increase in the extent of macrophage quantity 7 days after sci, which was preceded by an increase in the level of mcp-1 [166] . these results suggest that the expression of il-4 regulates the extent of macrophage activation in the acute phase of the injury [166] . in addition, il-4 has been shown to exert a neuroprotective effect against microglia-mediated neuronal toxicity by the regulation of fr formation [194] . on similar lines, macrophages stimulated with il-4 are reported to be less neurotoxic and to have an increased regenerative capability. this evidence makes il-4 injections a possible therapeutic application [166] . il-10 and tgf have been reported to act as neuroprotective molecules in a manner similar to il-4 [225] . for instance, it has been shown that an intrathecal infusion of tgf is able to enhance axonal growth after spinal contusion through the epidermal growth factor receptor (egfr) that is primarily upregulated by astrocytes surrounding the lesion. here, tgf stimulates proliferation, migration, and transformation to an axon phenotype supportive of growth [226] . on the other hand, a potential treatment for certain aspects of the secondary injury such as inflammation, excitotoxic damage, and neuronal apoptosis is the administration of il-10 since its anti-inflammatory effects involve the downregulation of il-1 , il-2, il-6, tnf , ifn , matrix metalloproteinase-9, nitric oxide synthase, myeloperoxidase, and ros [227] . in addition, proapoptotic factors such as cytochrome c, bax, and caspase 3 are downregulated by the effects of il-10. other effects of this cytokine include the upregulation of antiapoptotic factors such as b-cell lymphoma 2 (bcl-2). furthermore, il-10 provides trophic support to neurons by its receptor, in addition to increased tissue sparing, neuroprotection, and functional recovery. in the nervous system, il-10 receptor expression has been found in microglia, astrocytes, and oligodendrocytes acting as antagonist for the production of proinflammatory cytokines [225, 227] . in the first moments after sci, the elevated synthesis and release of proinflammatory mediators plays a role in the secondary degeneration [103] . this might be a therapeutic opportunity. for instance, an antagonist of proinflammatory cytokines such as il-1 receptor antagonist has demonstrated a neuroprotective effect after global ischemia, excitotoxicity, and traumatic brain injury in rodents [228] . (2) growth factors. after mechanical trauma, astrocytes and neurons release fibroblast growth factor (fgf) which is thought to counteract excitotoxic or ischemic damage by the activation of antiapoptotic signals in stressed neurons [229] . acidic fibroblast growth factor (afgf) is a potent mitogenic and chemotactic agent for vascular endothelial cells, dermal fibroblasts, and epidermal keratinocytes. moreover, it has a role in the regeneration process since it contributes to angiogenesis. in the normal uninjured spinal cord, afgf mrna was found to be present in low levels. after sci (table 2) , however, the factor increased in the 1 h, stayed at that level, peaked from day 5 to day 7, and remained high from day 14 to day 21 [209] . many therapeutic strategies seek to induce a higher expression of neurotrophic factors. a particular strategy that has shown significant results is the combination of peripheral nerve grafts with afgf after transection sci in rats. this strategy induced higher il-4, il-10, and il-13 levels in the graft areas of rat spinal cords. moreover, this strategy has been shown to regulate th2 cytokine production, m2 response, and neurotrophic factor production, where the latter can indirectly regulate the inflammatory response and neural destruction [211] . it is worth noting that the use of afgf with fibrin glue in combination with surgical neurolysis for nonacute sci has been proven feasible and safe in clinical trials which have shown significant improvements in asia motor and sensory scale scores and impairment scales, neurological levels, and functional independence measures, 24 months after treatment [230] . bdnf detected 6 h ai, increases up to 6 weeks, and decreases 12 weeks ai (i) m2 macrophage phenotype induction (ii) production of several extracellular matrix proteins for tissue remodeling and repair, neurotrophic support, and axonal regeneration (iii) increases growth, angiogenic, and axonal guidance factors [125, 150, 209, 210] afgf detected 1 h ai, pl 5-7 days, and remains elevated up to 14-21 days ai (i) potent mitogenic and chemotactic agent for vascular endothelial cells, dermal fibroblasts, and epidermal keratinocytes (ii) promotes th2 cytokine and neurotrophic factor production and m2 response [209, 211] bfgf detected 1 h ai, pl at day 3, remains elevated 5-7 days, and returns to low levels 14-21 days ai (i) angiogenesis [209] tgf-detected at 24 h ai (i) immunosuppressant (ii) tissue stabilization and structural preservation (iii) neural regeneration and repair [212] min: minutes; ai: after injury; pl: peak levels. after the injury, the expression of basic fibroblast growth factor (bfgf), another growth factor involved in angiogenesis, was found in astrocytes localized at the site of contusion and in the surrounding white matter. unlike afgf, bfgf mrna was not detected in the uninjured spinal cord. it was only detected 1 h after sci, in increased quantities at 6 h, and at its peak 3 days after sci. afterwards, it remained high from day 5 to day 7, only to return to a low level by days 14 to 21 [209] . before going further, it is important to note that growth factors such as tgf-may act as immunosuppressants. moving on, 24 h after sci, genes related to growth and differentiation became present. these included tgf-, nerve growth factor (vgf), platelet derived growth factor (pdgf-), galanin, and neuropeptide y. these genes have been suggested as aids in tissue stabilization, structural preservation, repair, and regeneration after sci. for instance, increased pdgf and vgf levels after sci may prevent the death of axotomized neurons and a decrease in their energy metabolism [212] . subsequently, the increased abundance of galanin and neuropeptide-y transcripts may produce an antinociceptive effect in the injured spinal cord [231] . moreover, it is known that cannabinoid receptor 1 (cb1) is colocalized with the neuropeptide cck. in this relationship, the neuropeptide acts as an endogenous opioid antagonist [232] . therefore, the downregulation of cb1 and the expression of the cck precursor might help explain why there is a relative resistance of neuropathic pain to the analgesic action of morphine in sci patients [233] . similar results have been found in several transcripts, and the previously mentioned genes have shown an increased abundance in comparison to sham animals [57, 223, 234] . (3) neurotrophins. neurotrophins constitute a family of molecules that has assumed a central role in studies dealing with recovery after sci [235] . four members of this family are involved in neuron survival and the regeneration process after sci: ngf, brain derived neurotrophic factor (bdnf), neurotrophin-3 (nt-3), and nt-4/5. neurotrophins emit signals when they bind to low and high affinity receptors in the membrane of their target cells. for instance, the low affinity p75 receptor binds all neurotrophins [208] . another signaling method used by neurotrophins is carried out by three high affinity tyrosine kinase receptors, collectively known as trk receptors. trka, trkb, and trkc compose the trk family of tyrosine-protein kinases. these three receptors mediate the biological properties of the ngf family of neurotrophins. trka is the particular receptor for ngf, while trkb serves as a receptor for both bdnf and nt-4. lastly, trkc is the primary receptor for nt-3. however, this particular neurotrophin can activate trka and trkb receptors when present in high concentrations [236] . through semiquantitative rt-pcr in a spinal cord contusion model, it was found that the expression of neurotrophin family members and their receptors was significantly diminished 6 h after the lesion. yet, in contrast to this pattern of trk receptor expression, p75ntr showed a significant upregulation after contusive sci [237] . interestingly, an increase in bndf was observed up to 6 weeks after compression sci with a decrease 12 weeks afterwards [210] . similarly, an increased expression of growth, angiogenic, and axonal guidance factors, as well as extracellular matrix molecules, can be observed in the chronic phase (days to years) following sci [150, 209] . the series of interconnected deleterious mechanisms of the secondary injury is orchestrated by the expression of specific genes, in particular those of signaling proteins such as cytokines, chemokines, and growth factors. the 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inappropriate inflammation and immunosuppression are components of the response to surgery, trauma, injury, and infection in some individuals and these can lead, progressively, to sepsis and septic shock. the hyperinflammation is characterized by the production of inflammatory cytokines, arachidonic acid-derived eicosanoids, and other inflammatory mediators, while the immunosuppression is characterized by impairment of antigen presentation and of t helper cell type-1 responses. long-chain n−3 fa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly (by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism) and indirectly (by altering the expression of inflammatory genes through effects on transcription factor activation). thus, long-chain n−3 fa are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. as such, an emerging application of n−3 fa is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. parenteral or enteral nutrition including n−3 fa appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients. the systemic inflammatory response syndrome is the name given to the uncontrolled inflammatory response to insult or injury involving excessive production of inflammatory cytokines such as tumor necrosis factor (tnf)-α, interleukin (il)-1β, il-6, and il-8 (1, 2) . sepsis has been defined as "the systemic inflammatory response syndrome that occurs during infection" (1) . sepsis is the leading cause of death in critically ill patients in western countries. using records from 1995 for state hospitals in the united states it was estimated that there were more than 750,000 cases of sepsis with a 28.6% mortal-ity rate (215,000 deaths) and a total cost of almost us$17 billion (3) . death from septic shock is the result of multiple organ failures and represents the extreme end of a continuum of events of increasing severity and decreasing likelihood of survival (4,5; fig. 1 ). the systemic inflammatory response syndrome, sepsis, and septic shock may together be termed as "septic syndromes." the involvement of inflammatory cytokines in septic syndromes has been long recognized and vervloet et al. (6) wrote "these mediators [i.e., inflammatory cytokines] are largely, if not completely, responsible for the clinical signs and symptoms of the septic response to bacterial infection." in support of this idea, patients with sepsis were found to have markedly elevated circulating concentrations of tnf-α, tnf receptor 1, il-1β, il-1 receptor antagonist (il-1ra), and il-6, and those patients with the highest concentrations were more likely to die (6) (7) (8) (9) . in addition, circulating white cells from septic patients exhibited high levels of activated nuclear factor kappa b (nfκb), a transcription factor that promotes the expression of numerous genes associated with inflammation, and again levels of activated nfκb were higher in those patients who went on to die (9) . animal studies also support a role for inflammatory cytokines in the septic response. these studies have often used bacterial endotoxin (also called lipopolysaccharide) as a surrogate for infection, although endotoxin is a fragment of the gram-negative bacterial cell wall and not a viable organism. mice injected with endotoxin exhibit high circulating concentrations of tnf-α, il-1β, il-6, and il-8, and survival of these animals can be improved by administering anti-cytokine antibodies (10, 11) , cytokine receptor antagonists (12) , or anti-inflammatory cytokines such as il10 (13) , or by knocking out the tnf-α receptor (14) . despite this evidence, it is important to note that some studies report that many septic patients do not show detectable or elevated circulating concentrations of tnf-α or il-1β (15) (16) (17) (18) . furthermore, it appears that inflammatory cytokines do play a beneficial role in sepsis. for example, in some animal models, blocking tnf-α increases mortality (19) (20) (21) , while a tnf-α antagonist increased mortality in a clinical trial (22) . thus, the situation regarding the pathological role of inflammatory cytokines in sepsis is unclear; it may be that a little is beneficial but that excess is harmful and that complete blocking negates the beneficial effects. another consideration is that there may be large between-individual differences in the generation of inflammatory cytokines, in the sensitivity to the harmful effects of these cytokines, and in the effects of blocking these cytokines. thus, there may be significant variation in the susceptibility of individuals to exhibit the systemic inflammatory response syndrome and to progress toward septic shock. this may partly relate to the extent and site of the initial injury, partly to the nature and site of the infection, if any, and partly to aspects of the patient's well-being prior to receiving the injury (e.g., nutritional state). it is now recognized that genetics may also play a role. in fact there are likely to be genetic variations in many aspects of the septic response to infection and injury. these most likely relate to adaptations of various population groups to withstand infection and injury in different ecological settings. in the context of this article, genetic variations in the propensity to produce inflammatory cytokines are of relevance. it is now recognized that there are single base variations in genes or in their promoter regions called single nucleotide polymorphisms or snps (pronounced "snips"). snps have been described for tnf-α, tnf-β, il-1β, il-6, il-10, tnf receptors, il-1 receptors, il-1ra, and for many other genes involved in the septic response (23) . these snps are of functional significance since they partly determine the extent of expression of the gene once it is activated (23) . thus tnf-α production by monocytes in response to endotoxin is higher in individuals who have a g rather than an a at -308 in the tnf-α gene promoter region (24) . intriguingly, tnf-α production is also affected by a polymorphism in the tnf-β gene: tnf-α production by monocytes in response to endotoxin was higher if there was an a at +252 in the tnf-β gene than if there was a g (25) . genotypes affecting tnf-α production appear to be of relevance with respect to sepsis mortality. for example, possession of a g at -308 in the tnf-α gene was found in 39% of patients with septic shock compared with 18% of controls, and among patients with septic shock this polymorphism was significantly more common among patients who died (52% vs. 24% among survivors) (26) . in controlling for age, it was identified that, for the same clinical score, patients with a g at -308 of the tnf-α gene had a 3.7-fold higher risk of death than those without a g (26) . in another study, patients with sepsis who were homozygous for a at +252 in the tnfβ gene displayed significantly higher plasma tnf-α concentrations than heterozygotes or homozygotes for g, and they showed 88% mortality compared with 37% for heterozygotes and 25% for g homozygotes (27) . in a more recent study, postoperative patients who were homozygous for a at +252 in the tnf-β gene had a 1.5-fold higher risk of developing severe complications than heterozygotes (28) . furthermore, among the patients who developed sepsis, those who were homozygous for a at +252 in the tnf-β gene were more likely to die (71 vs. 20% for heterozygotes and 0% for homozygotes for g) (28) . these findings raise the possibility of being able to identify patients at high risk of complications and mortality on the basis of genetic polymorphisms. although there has been much focus on the potential detrimental role of inflammatory cytokines in sepsis, other mediators including arachidonic acid-derived eicosanoids, reactive oxygen species, nitric oxide, and adhesion molecules are involved in the pathological processes that accompany critical illness. prostaglandin (pg) e 2 is implicated in sepsis, burns, and critical illness (29, 30) , while leukotriene (lt) b 4 and oxidants released by neutrophils are involved in acute respiratory distress syndrome [see kollef and schuster (31) ]. in addition to hyperinflammation, patients with sepsis also display immunosuppression (32) (33) (34) . there are reports that septic patients have high circulating concentrations of the antiinflammatory cytokine il-10 and that these are strongly correlated with mortality (35, 36) . note that this is contrary to the predicted effect of il-10 since this cytokine down-regulates tnf-α production and its early administration is protective in murine endotoxemia (37) (38) (39) . however, the apparently harmful effect of il-10 may relate to the timing of its production. lymphocytes from patients with burns or trauma produce low levels of the t helper (th) 1-type cytokines [e.g., interferon (ifn)-γ] associated with host defense against bacteria and viruses but high levels of the th2-and treg-type cytokines (il-4, il-10) associated with inhibition of host defense against bacteria and viruses (33, 35) . there also appears to be decreased monocyte expression of human leukocyte antigens (hla) (40) (41) (42) (43) , the proteins involved in antigen presentation to t cells, and this is associated with impaired ability of monocytes to stimulate t cells (43) . interestingly, il-10 downregulates both th1-type cytokine production and hla expression (44, 45) , and this might be the origin of the harmful effect of this cytokine in septic patients. recent studies have revealed impaired proliferative or secretory functions of t cells from patients with sepsis, trauma, or burns (46, 47) . the traditional view is that the immunosuppressed phase of septic syndromes lags behind the hyperinflammatory phase (fig. 2) ; that is, initially sepsis is characterized by increased generation of inflammatory mediators (the systemic inflammatory response syndrome), but as it persists there is a shift toward an anti-inflammatory, immunosuppressed state sometimes called the compensatory anti-inflammatory response syndrome. however, some recent studies challenge this and suggest that the hyperinflammatory and immunosuppressed states coexist. some authors report that immunosuppression is present at the onset of sepsis (46, 48, 49) , rather than being a later compensatory response. for example, tschaikowsky et al. (49) identified that significantly decreased monocyte expression of hla-dr was evident at the onset of severe sepsis in postsurgical patients; in survivors there was some recovery of expression but in nonsurvivors there was a further decrease or even a permanent suppression of hla-dr expression. these authors identified that the timing of the peak of the systemic inflammatory reaction (identified as the time of maximum c-reactive protein concentration) coincided with the timing of the lowest monocyte expression of hla-dr. from this they concluded that decreases in monocyte hla-dr expression occur simultaneously with "signs of hyperinflammation" and as early as the onset of severe sepsis (49) . thus, it appears that immune cells and cytokines have both detrimental and protective roles in patients as they move through the stages of sepsis. however, the traditional view that hyperinflammation precedes immunosuppression, as shown in figure 2 , may be a simplification of the real situation, and this increases the challenge to finding interventions that might benefit high-risk patients. human immune and inflammatory cells are rich in polyunsaturated fa (pufa), especially arachidonic acid (20:4n-6) [see calder (50) ]. classically the influence of pufa on immunity and inflammation has been viewed as relating to their influence on eicosanoid generation (51) (52) (53) (54) . arachidonic acid is the principal substrate for cyclooxygenase (cox) and lipoxygenase (lox) enzymes giving rise to 2-series pg and thromboxanes (tx) or 5-hydroxyeicosatetraenoic acids (hete) and 4-series lt, respectively. these mediators have cell-and stimulus-specific sources and frequently have opposing effects (table 1 ). for example, pge 2 is produced mainly by monocytes, macrophages, and, to a lesser extent, neutrophils and inhibits the production of tnf-α and il-1β [see miles et al. (55) and references therein; 56,57] and il-12 (57, 58) , while ltb 4 is produced mainly by neutrophils, other granulocytes, and, to a lesser extent, monocytes and macrophages and increases the production of tnf-α and il-1β [see rola-pleszczynski et al. (59) and references therein]. thus, the overall physiological (or pathophysiological) outcome will depend upon the cells present, the nature of the stimulus, the timing of eicosanoid generation, the concentrations of different eicosanoids generated, and the sensitivity of target cells and tissues to the eicosanoids generated. recent studies have demonstrated that pge 2 induces cox-2 in fibroblasts cells and so upregulates its own production (60), induces production of il-6 by macrophages (60) it is frequently considered that the effects of arachidonic acid are solely related to its role as an eicosanoid precursor. cell culture studies have shown that arachidonic acid activates nfκb in a monocytic cell line (67) , and induces tnf-α, il-1α and il-1β in osteoblasts (68), il-6 in macrophages (60) and osteoblasts (69) , and cox-2 in fibroblasts (60) , and it appears that these effects are exerted directly by arachidonic acid rather than by an eicosanoid metabolite. what is evident from these studies is that arachidonic acid may be able to regulate inflammatory mediator production in its own right and, if so, that it has effects that are sometimes the opposite of those of pge 2 , for example, with respect to tnf-α production. a series of cell culture-based studies with human endothelial cells has suggested that another n-6 fa, linoleic acid (18:2n-6), may also play a role in inflammation through activation of nfκb and increased production of tnf-α, il-6, and other inflammatory mediators (70) (71) (72) (73) (74) (75) (76) . increased consumption of long-chain n-3 pufa, usually as components of fish oil, by humans results in increased amounts of epa (20:5n-3) and dha (22:6n-3) in cells involved in immunity and inflammation [see calder (50) ]. the incorporation of these fa from the diet into immune/inflammatory cells of humans is near-maximal within a few weeks (77, 78) and occurs in a dose-dependent manner (79) . incorporation of epa and dha into human cells is partly at the expense of arachidonic acid [see calder (50) ], and the functional significance of this is that it decreases the amount of arachidonic acid available as a substrate for eicosanoid synthesis. thus, fish oil supplementation of the human diet has been shown to result in decreased production of pge 2 (80-83), txb 2 (82), ltb 4 and 5-hete (84, 85) , and lte 4 (86) by inflammatory cells. however, the mechanism of the effect of long-chain n-3 fa on eicosanoid generation extends beyond simply decreasing the amount of arachidonic acid substrate. for example, epa competitively inhibits metabolism of arachidonic acid by cox (87-89) and 5-lox (87, 90) . in vitro studies also report that dha can inhibit cox activity (91, 92) but not that of 5-lox (90, 92) . interestingly, however, both epa and dha suppressed cytokine-induction of cox-2 and 5-lox gene expression in cultured bovine chondrocytes and in human osteoarthritic cartilage explants (93, 94) . by inhibiting cox and lox activities and by suppressing the up-regulation of the genes for these enzymes in response to inflammatory stimuli, long-chain n-3 fa act to oppose generation of eicosanoids from arachidonic acid. the final element of the effects of longchain n-3 fa on eicosanoid production is the ability of epa to act as a substrate for cox and lox enzymes, so giving rise to a different family of eicosanoids: the 3-series pg and tx, the 5series lt, and the hydroxyeicosapentaenoic acids (hepe). epa, which appears to be a good substrate for 5-lox (86, 90) , is also a substrate for cox enzymes (95, 96) . thus, fish oil supplementation of the human diet has been shown to result in increased production of ltb 5 , lte 5 , and 5-hepe by inflammatory cells (84) (85) (86) , although generation of pge 3 has been more difficult to demonstrate (97) . the functional significance of this is that the mediators formed from epa are believed to be less potent than those formed from arachidonic acid. for example, ltb 5 is 10-to 100-fold less potent as a neutrophil chemotactic agent than ltb 4 (98, 99) . recent studies have compared the effects of pge 2 and pge 3 on production of cytokines by cell lines and by human cells. bagga et al. (60) reported that pge3 was a less potent inducer of cox-2 gene expression in fibroblasts and of il-6 production by macrophages. pge 2 and pge 3 had equivalent inhibitory effects upon production of tnf-α (55,56) and il-1β (55) by human mononuclear cells stimulated with endotoxin and upon production of ifn-γ production by mononuclear cells stimulated with mitogen (56, 66) . however, il-2 production appeared to be less sensitive to pge 3 than pge 2 (66) . studies using the isolated, perfused rabbit lung have identified contrasting effects of arachidonic acid-and epa-derived eicosanoids. infusion of escherichia coli hemolysin caused hypertension mediated by txb 2 and increased vascular leakage mediated by 4-series lt (100) . inclusion of arachidonic acid in the perfusate increased txb 2 and 4-series lt generation, arterial pressure, and vascular leakage (100,101). in contrast, inclusion of epa in the perfusate decreased txb 2 and 4-series lt generation, decreased arterial pressure and vascular leakage, and increased generation of txb 3 and 5-series lt (100) . perfusion with fish oil attenuated the hypertension induced by calcium ionophore (102) . compared with soybean oil infusion, fish oil decreased the concentration of ltc 4 by 50% and increased the concentration of ltc 5 from barely detectable to very similar to that of ltc 4 (102) . in addition to long-chain n-3 fa modulating the generation of eicosanoids from arachidonic acid and to epa acting as substrate for the generation of alternative eicosanoids, recent studies have identified a novel group of mediators, termed e-series resolvins, formed from epa by cox-2 that appear to exert anti-inflammatory actions (103) (104) (105) . in addition, dha-derived mediators termed d-series resolvins, docosatrienes, and neuroprotectins also produced by cox-2 have been identified, and these too appear to be anti-inflammatory (106) (107) (108) . this is an exciting new area of n-3 fa and inflammatory mediators, and the implications for a variety of conditions may be of great importance. cell culture studies investigating the direct effects of arachidonic acid on inflammatory mediator production have also investigated effects of long-chain n-3 fa. epa did not activate nfκb in a monocytic cell line (67), while epa and dha inhibited endotoxin-stimulated production of il-6 and il-8 by cultured human endothelial cells (109, 110) . more recent studies showed that epa did not induce tnf-α, il-1β, or il-1α (68) or il-6 (69) in osteoblasts, and even countered the upregulating effect of arachidonic acid (68) ; that epa and dha could totally abolish cytokine-induced up-regulation of tnf-α, il-1α, and il-1β in cultured bovine chondrocytes and in human osteoarthritic cartilage explants (93, 94) ; and that epa or fish oil inhibited endotoxin-induced tnf-α production by monocytes (111) (112) (113) (114) . epa was also less potent than arachidonic acid in inducing cox-2 expression by fibroblasts and il-6 expression by macrophages (60) . epa prevented nfκb activation by tnf-α in cultured pancreatic cells, an effect that involved decreased degradation of the in-hibitory subunit of nfκb (iκb), perhaps through decreased phosphorylation (115) . similarly, epa or fish oil decreased endotoxin-induced activation of nfκb in human monocytes (111, 113, 114) , and this was associated with decreased iκb phosphorylation (113, 114) , perhaps due to decreased activation of mitogen-activated protein kinases (116) . these observations suggest direct effects of long-chain n-3 fa on inflammatory gene expression via inhibition of activation of the transcription factor nfκb. animal feeding studies with fish oil support the observations made in cell culture with respect to the effects of long-chain n-3 fa on nfκb activation and inflammatory cytokine production. compared with feeding corn oil, fish oil lowered nfκb activation in endotoxin-activated murine spleen lymphocytes (117) . feeding fish oil to mice decreased ex vivo production of tnf-α, il-1β, and il-6 by endotoxin-stimulated macrophages and decreased circulating tnf-α, il-1β, and il-6 concentrations in mice injected with endotoxin [sadeghi et al. (118) and references therein]. several studies in humans involving supplementation of the diet with fish oil have demonstrated decreased production of tnf-α, il-1β, and il-6 by endotoxin-stimulated monocytes or mononuclear cells (a mixture of lymphocytes and monocytes) (80) (81) (82) 119) . the study of caughey et al. (82) reported a significant inverse correlation between the epa content of mononuclear cells and the ability of those cells to produce tnf-α and il-1β in response to endotoxin. recent studies have confirmed the ability of dietary fish oil to decrease production of tnf-α (120) and il-6 (120,121) by human mononuclear cells. furthermore, these studies provide for the first time information on the dose-response relationship between dietary intake of long-chain n-3 fa and production of these cytokines. it should be noted that there are also several studies that fail to show effects of dietary long-chain n-3 fa on production of inflammatory cytokines in humans [see calder (50) for references]. it is not clear what the reason for this is, but the dose of n-3 fa used and other technical factors are likely to be contributing factors. one other factor that has recently been identified is polymorphisms in genes affecting cytokine production (122) . it was found that the effect of dietary fish oil on cytokine production by human mononuclear cells was dependent on the nature of the -308 tnf-α and the +252 tnf-β polymorphisms. this study raises the possibility of being able to identify those who are more likely and those who are less likely to experience specific anti-inflammatory effects of fish oil. thus, examination of fa composition and of eicosanoid profiles, cell and tissue culture work, and animal and human feeding studies have revealed a range of anti-inflammatory actions of long-chain n-3 fa ( table 2 ). these may be of benefit in sepsis, particularly during the "early" hyperinflammatory phase. the benefits of fish oil in animal models of experimental endotoxemia have been clearly demonstrated. for example, dietary fish oil or fish oil infused intravenously significantly enhanced survival of guinea pigs to intraperitoneal endotoxin compared with safflower oil (123, 124) . dietary fish oil resulted in a decreased concentration of circulating postendotoxin eicosanoids (pge 2 , txb 2 , 6-keto-pgf 1α ) in rats and in decreased eicosanoid generation by alveolar macrophages (125, 126) . furthermore, compared with dietary safflower oil, fish oil resulted in lower circulating tnf-α, il-1β, and il-6 concentrations following endotoxin administration to mice (118) . dietary fish oil also appears to decrease sensitivity to inflammatory cytokines (127, 128) . fish oil decreased endotoxininduced metabolic perturbations in guinea pigs and rats (129, 130) and improved heart and lung function and decreased lung edema in endotoxic rats (126, (131) (132) (133) and pigs (134) (135) (136) . in addition to effects on production of inflammatory eicosanoids and inflammatory cytokines, long-chain n-3 fa decreased generation of arachidonic acid-derived partial replacement of arachidonic acid in cell membrane phospholipids eicosanoids (many with inflammatory actions) inhibition of arachidonic acid metabolism by phospholipase a 2 , cox, and 5-lox decreased induction of cox-2, 5-lox, and 5-lox-activating protein however, it is the effects of lower amounts of long-chain n-3 fa that are of relevance to the patient setting. several studies in humans, typically providing long-chain n-3 fa as fish oil, and investigating aspects of cell-mediated immunity have been performed. phagocytic uptake of escherichia coli appears unaffected by dietary long-chain n-3 fa in humans (138) (139) (140) (141) . one study reported that fish oil decreased expression of hla-dp, -dq, and -dr on human monocytes (142), suggesting impaired ability to present antigen, but there have been no studies attempting to confirm this finding. meydani et al. (81) reported that fish oil providing 2.4 g epa plus dha per day decreased t-lymphocyte proliferation in older but not younger women. however, that study also reported increased oxidative stress in the older subjects (143) , and it may be that the effect of n-3 fa was due to excessive lipid peroxidation. several other studies report no effect of various doses of longchain n-3 fa on lymphocyte proliferation (78, 121, 140) , although there are studies reporting a decrease (144, 145) . one recent study reported that long-chain n-3 fa caused a dosedependent increase in proliferation of t cells (83) . it is noteworthy that the fish oil used was given in combination with an antioxidant mix. this might be important in terms of preventing excessive lipid peroxidation and so in determining the overall effect of n-3 fa. the study by meydani et al. (81) also reported decreased production of il-2 in the older women, but this effect has not been confirmed by others in either older (145) , young (121, 141) , or mixed-age (78, 140) subjects. a recent study reported a dose-dependent increase in ifn-γ production following n-3 fa supplementation as fish oil (83) . that antioxidants were given in combination with fish oil may have been important in generating this finding. thus, the effects of long-chain n-3 fa on aspects of cellmediated immunity are rather unclear, although recent human studies suggest that adverse immune effects are not exerted at modest doses (see previous discussion for references) and that enhanced t-cell responses (proliferation and ifn-γ production) may occur at modest doses so long as antioxidants are also given (83) . in terms of sepsis, the true test of immunocompetence occurs when live pathogens are administered. this is a different situation from using endotoxin that is not living and that therefore does not require a robust cell-mediated immune response to eliminate it. as indicated previously, it is clear that long-chain n-3 fa protect against the deleterious effects of endotoxin. however, the situation regarding live pathogens is much less clear. this is because animal studies, frequently using high intakes of n-3 fa, report opposing findings. infusion of fish oil into rats also receiving low-dose endotoxin decreased the number of viable bacteria in mesenteric lymph nodes and liver (146) . fish oil did not decrease bacterial translocation across the gut, and so the authors concluded that fish oil must have improved bacterial killing. compared with linoleic acid-rich vegetable oils, fish oil fed to rats before exposure to live bacteria (147, 148) resulted in increased survival, which was associated with decreased production of pge 2 . more recently, infusion of fish oil after induction of sepsis by cecal ligation and puncture decreased mortality (and pge 2 production) compared with vegetable oil (149) . intragastric administration of fish oil into chow-fed rats before cecal ligation and puncture improved survival compared with saline or vegetable oil infusion (150) . compared with vegetable oil feeding to mice, fish oil feeding increased survival to an intramuscular injection of klebsiella pneumoniae (151) . the findings from these studies (146) (147) (148) (149) (150) (151) contrast with those reporting that fish oil feeding decreases the survival of mice to oral salmonella typhimurium (152) and to intraperitoneal listeria monocytogenes (153) , of guinea pigs to mycobacterium tuberculosis (154) , and of neonatal rabbits to staphylococcus aureus (155) . thus, animal studies do not provide a clear picture of the effect of high-dose fish oil on ability to survive an infectious challenge. there are few human studies that address exposure to long-chain n-3 fa and infection; most intervention studies performed to date have been too small and of too short duration to monitor infection as an outcome. however, it is worth noting that an epidemic of measles in greenland triggered by its introduction to a naive population by an infected danish sailor showed the same characteristics as previous epidemics in other naive populations (156) . this suggests that the very n-3 fa-rich diet of the greenland inuits did not worsen their response to the virus and this could indicate that these fa do not increase infectious susceptibility in humans. surgery is typically accompanied by an inflammatory response that may be exaggerated in some patients, especially if the surgery is major. if the patient is exposed to pathogenic organisms and is unable to cope with these, then sepsis may develop. artificial nutrition is frequently used post-surgery and this may involve parenteral (i.e., intravenous) infusions, especially where the gastrointestinal tract is not fully functional (e.g., post-abdominal surgery). lipids are included in parenteral nutrition to provide an alternative source of calories to glucose and the lipid source used most frequently has been soybean oil, which is rich in the n-6 fa linoleic acid, although it also contains a proportion of α-linolenic acid (18:3n-3). a meta-analysis of total parenteral nutrition suggested that inclusion of lipids might be detrimental (p = 0.09 for lipids vs. no lipids) (157) , at least in very ill patients. it is not clear why this is, although a number of in vitro experiments have shown that soybean oil-based lipid emulsions can exert immunosuppressive effects [see calder et al. (158) for references], which would clearly be detrimental in patients at risk of infection and sepsis. clinical trials provide conflicting evidence, some showing some immunosuppressive effects (159, 160) and others not (161) (162) (163) , at least in some patient groups. the concern about potential harm, the view of sepsis as a hyperinflammatory state followed by an immunosuppressed state (fig. 2) , and the idea that n-6 fa might be "proinflammatory and immunosuppressive" has led to the development of alternative lipid emulsions for parenteral applications. emulsions using a mix of medium-chain triglycerides and soybean oil or based upon olive oil instead of soybean oil have been developed, but these will not be discussed here. however, of relevance to the present discussion is the development of emulsions that include fish oil as a partial replacement for soybean oil. several such emulsions have been tested in surgical patients. intravenous infusion of a lipid emulsion containing fish oil for 5 d into patients who had undergone major abdominal surgery resulted in much higher ltc 5 production by blood leukocytes stimulated ex vivo at 6 d postoperation (164) . in another study, patients who had undergone abdominal surgery received soybean oil or a mix of medium-chain triglycerides, soybean oil, and fish oil (50:40:10, by vol) for 5 d post surgery (165) . leukocytes from these patients produced more ltb 5 and ltb 5 isomers at postoperative days 6 and 8. patients who had undergone major gastrointestinal surgery received a medium-chain triglyceride/soybean oil mix (50:50, vol/vol) or a mix of medium-chain triglycerides, soybean oil, and fish oil (50:30:20, by vol) for 5 d postsurgery (166) . patients receiving fish oil got 3 (days 1 and 2) and 6 g (days 3, 4, and 5) of long-chain n-3 fa per day. neutrophils from these patients produced less ltb 4 and more ltb 5 at postoperative days 6 and 10. plasma tnf-α concentrations were lower in the fish oil group at day 6, while plasma il-6 concentrations were lower at day 10. the study did not report clinical outcomes. a more recent study infused a fish oil-rich formula on the day before abdominal surgery and on days 1 to 5 following abdominal surgery (167) . on days 4 and 5 the patients also received standard total parenteral nutrition that included 50 g of fat/d (n = 12; n = 11 in the control group). tnf-α production by endotoxin-stimulated whole blood tended to be lower at postoperative day 5 in the fish oil group, but this was not significant. serum il-6 concentrations were significantly lower at days 0, 1, and 3 in the fish oil group. monocyte expression of hla-dr was preserved in the fish oil group but declined at postsurgery days 3 and 5 in the control group. no differences in infection rates or mortality were observed. however, postoperative stay in intensive care tended to be shorter in the fish oil group (4.1 vs. 9.1 d) as did total hospital stay (17.8 vs. 23.5 days), although neither of these was a significant effect. postoperative stay on medical wards was significantly shorter in the fish oil group. another recent study compared the effects of lipid-free total parenteral nutrition or parenteral nutrition including 10% soybean oil or 8.3% soybean oil plus 1.7% fish oil for 5 d after large bowel surgery (168) . there were no differences between the groups with respect to the numbers of circulating lymphocytes, b cells, cd4 + cells, cd8 + cells, or natural killer cells before surgery or at days 3 and 6 postsurgery, although these were affected by surgery itself. there were no differences between groups with respect to t-lymphocyte proliferation, but il-2 production was increased in the fish oil group and the postsurgery decline in ifn-γ production was prevented by fish oil. these studies indicate that inclusion of fish oil in parenteral nutrition regimens for gastrointestinal surgical patients modulates generation of inflammatory eicosanoids (164) (165) (166) and cytokines (166, 167) and may help to counter the surgery-induced declines in antigen-presenting cell activity (167) and t cell cytokine production (168) . importantly, these studies do not reveal deleterious immunologic effects of fish oil infusion in these patients. furthermore, the only one of these fairly small studies to have examined hard end points like length of hospital stay suggests some clinical benefit from fish oil infusion in these patients (167) . however, larger studies are required to evaluate the effects of this approach on complication rates, hospital stay, and mortality rate. a very recent report from a larger cohort of patients receiving parenteral nutrition postsurgery does indicate benefit of inclusion of fish oil in the regimen (169) . patients received fish oil postoperatively (n = 86) or controls received a 50:50 medium-chain triglyceride-soybean oil mix (n = 110). there were no differences between the two groups with respect to the proportions of patients who died or developed wound infections or with respect to length of hospital stay. however, the proportion of patients who were readmitted to intensive care (5%) was significantly lower in the fish oil than in the control group (17%). a group of patients also received the fish oil-containing emulsion for 2 d preoperatively (n = 53). here there were a number of very significant benefits. this group showed a significantly decreased need for mechanical ventilation (17 vs. 31% in the control group), a significantly shorter length of hospital stay (22 vs. 29 d) , significantly less need for readmission to intensive care (5 vs. 17%), and a significantly lower mortality rate (3 vs. 15%) (169) . this study demonstrates a benefit from the inclusion of long-chain-3 fa in parenteral nutrition regimens used in abdominal surgery patients. however, it also demonstrates a much greater benefit if the fa are additionally provided before surgery, which, of course, is only possible in elective surgery. the greater benefit of preoperative infusion of longchain n-3 fa may relate to better incorporation of the fa into leukocytes and other tissues. enteral nutrition is an alternative form of artificial nutrition. it describes provision of nutrients directly into the gastrointestinal tract via a tube and is sometimes referred to as "tube feeding." enteral nutrition is used in patients with a functional gastrointestinal tract and is considered preferable to parenteral nutrition. the influence of enteral feeds including long-chain n-3 fa in their composition has been examined in surgical patients, generally in those who have undergone surgery to remove cancerous regions of the intestine. these studies have frequently used an enteral formula named impact ® (novartis, basel, switzerland), which contains arginine, long-chain n-3 fa, and nucleotides, each of which is lacking from control formulas. thus, any effects observed cannot be ascribed to a particular component of impact. the effect of impact on immunoinflammatory outcomes in surgical patients has been widely examined. daly et al. (170) reported that impact results in time-dependent incorporation of epa into mononuclear cells and that this is associated with a timedependent decrease in pge 2 production. studies have reported that impact increases phagocytosis by monocytes but not by neutrophils (171, 172) , increases t-cell proliferation (173) and cell-mediated immunity (172, 174) , and decreases circulating concentrations of il-6 (172, 175) . several of these studies report significantly improved clinical outcomes related to lower infection rate (170, 172, 173, 175) and decreased length of hospital stay (170, 172, 175) . studies of impact and similar enteral formulas investigating clinical outcomes in postsurgical patients have been subject to meta-analyses (176) (177) (178) , which conclude that this approach to enteral nutrition significantly decreases infectious complications and length of hospital stay in elective surgery patients. it is possible that the modulation of inflammation and the improvements in immune function reported in these patients receiving impact contribute to the improved clinical outcomes. however, it is not possible to ascribe these benefits to long-chain n-3 fa. critically ill patients frequently require artificial support, depending upon the extent of organ damage or failure, and this will include nutritional support. the influence of enteral feeds including long-chain n-3 fa has been examined in critically ill patients; again, many of these studies have involved impact. a study in intensive care unit patients (a mix of trauma, sepsis, and major surgery patients) reported that impact resulted in higher t-cell proliferation at days 3 and 7 (179), while a study of severe trauma patients reported greater hla-dr expression at day 7 (180) . these studies did not report improvements in clinical outcomes. studies of impact and similar enteral formulas investigating clinical outcomes in trauma and critically ill patients have been subject to metaanalysis (176) (177) (178) . the most recent of these concluded that this approach to enteral nutrition decreases length of hospital stay but has no effect on infectious complications or mortality in critically ill patients (178) . another trial performed in patients with moderate and severe acute respiratory distress syndrome used an enteral preparation that differed mainly in lipid source from the control (181) . the control group of patients (n = 72) received a formula in which the lipid source was 97% corn oil plus 3% soy lecithin. the experimental group (n = 70) received a lipid source that was 32% canola oil, 25% medium-chain triglycerides, 20% borage oil, 20% fish oil, and 3% soy lecithin. the experimental formula also contained more vitamin c and vitamin e than the control and it contained β-carotene, taurine, and carnitine, which the control formula did not. patients receiving the experimental formula got about 7 g of epa, 3 g of dha, 6 g of γ-linolenic acid, 1.1 g of vitamin c, 400 iu of vitamin e, and 6.6 mg of β-carotene per day for 6 d. by 4 d the numbers of total leukocytes and of neutrophils in the alve-olar fluid declined significantly in the experimental group and were lower than in the control group. arterial oxygenation and gas exchange were improved in the experimental group. these patients had a significantly decreased requirement for supplemental oxygen, decreased time on ventilation support (11.0 vs. 16.3 d), and a shorter length of stay in intensive care (12.8 vs. 17.5 d) . total length of hospital stay tended to be shorter in the experimental group (29.6 vs. 34.6 d). significantly fewer patients in the experimental group developed new organ failure (8 vs. 28%). the mortality rate was 12% in the experimental group and 19% in the control group, but this difference was not statistically significant. more recently, new data from this study have become available (182) . patients receiving the experimental formula had significantly lower concentrations of il-8 in their alveolar fluid and tended to have lower concentrations of ltb 4 and tnf-α. it is possible that the lower concentrations of ltb 4 and il-8, both of which are potent leukocyte chemoattractants, may have been responsible for the lower neutrophil infiltration reported in the experimental group, and indeed neutrophil counts were significantly associated with these concentrations (182) . this study establishes that the experimental treatment decreases production of inflammatory mediators and infiltration of inflammatory leukocytes and that this can result in significant clinical improvement in extremely ill patients. because of the many differences in composition between the experimental and control formulas used it is not possible to ascribe the effects and benefits to any particular nutrient. however, the effects on ltb 4 , il-8 and tnf-α concentrations are consistent with effects of long-chain n-3 fa reported elsewhere. recently, data from studies using parenteral nutrition with fish oil in sepsis patients have become available (183, 184) . patients received a standard soybean oil-based emulsion or an emulsion containing fish oil for 5 (178) or 10 (177) d. blood leukocyte counts and serum c-reactive protein concentration tended to be lower, and production of ltb 5 by stimulated neutrophils was significantly higher in patients receiving long-chain n-3 fa (177). production of tnf-α, il-1β, il-6, il-8, and il-10 by endotoxin-stimulated mononuclear cells did not increase during infusion of the fish oil-containing emulsion whereas production of the four proinflammatory cytokines was markedly elevated during the first 2 d of soybean oil infusion (178) . these studies establish that infusion of long-chain n-3 fa into patients with sepsis can modulate inflammatory mediator production and related inflammatory processes. however, the impact of this on hard clinical outcomes in these patients is not yet clear. in summary, long-chain n-3 pufa from fish oil decrease the production of inflammatory cytokines and eicosanoids. they act both directly, by replacing arachidonic acid as an eicosanoid substrate and by inhibiting arachidonic acid metabolism, and indirectly, by altering the expression of inflammatory genes through effects on transcription factor activation. thus, long-chain n-3 pufa are potentially useful anti-inflammatory agents and may be of benefit in patients at risk of developing sepsis. an emerging application of n-3 pufa is in surgical or critically ill patients where they may be added to parenteral or enteral formulas. parenteral or enteral nutrition including n-3 pufa appears to preserve immune function better than standard formulas and appears to partly prevent some aspects of the inflammatory response. studies to date are suggestive of clinical benefits from these approaches, especially in postsurgical patients. definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis strategies for the treatment of 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with eicosapentaenoic acid and docosahexaenoic acid on in vitro neutrophil and monocyte leukotriene generation and neutrophil function dietary ω-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils n-3 fatty acids and cysteinyl-leukotriene formation in humans in vitro, ex vivo and in vivo manipulation of platelet aggregation by prostaglandins and their fatty acid precursors: pharmacological basis for a therapeutic approach interaction between peroxidase and cyclooxygenase activities in prostaglandin-endoperoxide synthase eicosapentaenoic acid inhibits prostaglandin d2 generation by inhibiting cyclo-oxygenase-2 in cultured human mast cells effects of exogenous arachidonic, eicosapentaenoic, and docosahexaenoic acids on the generation of 5-lipoxygenase pathway products by ionophore-activated human neutrophils effect of docosahexaenoic acid (dha) on arachidonic acid metabolism and platelet function docosahexaenoic acid is a strong inhibitor of 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docosahexaenoic acid in urine brain, human blood and glial cells: autocoids in anti-inflammation novel docosanoids inhibit brain ischemia-reperfusion-mediated leukocyte infiltration and pro-inflammatory gene expression neuroprotectin d1: a docosahexaenoic acid-derived docosatriene protects human retinal pigment epithelial cells from oxidative stress the omega-3 fatty acid docosahexaenoate reduces cytokine-induced expression of proatherogenic and proinflammatory proteins in human endothelial cells docosahexaenoic and eicosapentaenoic acids inhibit in vitro human endothelial cell production of interleukin-6 fish oil decreases macrophage tumor necrosis factor gene transcription by altering the nfκb activity modulation of lipopolysaccharide-stimulated macrophage tumor necrosis factor-α production by ω-3 fatty acid is associated with differential cyclooxygenase-2 protein expression and is independent of interleukin-10 nf-κb inhibition by ω-3 fatty acids modulates lps-stimulated macrophage tnf-α transcription eicosapentaenoic acid prevents lps-induced tnf-α expression by preventing nf-κb activation the anti-catabolic effects of n-3 fatty acids fish oil modulates macrophage p44/42 mitogen-activated protein kinase activity induced by lipopolysaccharide fish oil suppressed cytokines and nuclear factor kappab induced by murine aids virus infection dietary lipids modify the cytokine response to bacterial lipopolysaccharide in mice n-3 pufa supplementation, monocyte pca expression and interleukin-6 production inhibition of tumour necrosis factor-α and interleukin-6 production by mononuclear cells following dietary fish-oil supplementation in healthy men and response to antioxidant co-supplementation comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell function in healthy human subjects the ability of fish oil to suppress tumor necrosis factor-α production by peripheral blood mononuclear cells in healthy men is associated with 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composition and function during porcine endotoxemia dietary fish oil and fish and borage oil suppress intrapulmonary proinflammatory eicosanoids biosynthesis and attenuate pulmonary neutrophil accumulation in endotoxic rats effects of eicosapentaenoic and gamma-linolenic acid on lung permeability and alveolar macrophage eicosanoid synthesis in endotoxic rats effects of a fish oil diet on pig's cardiopulmonary response to bacteremia effects of endotoxin on pigs prefed omega-3 vs omega-6 fatty acids-enriched diets select dietary fatty acids attenuate cardiopulmonary dysfunction during acute lung injury in pigs fatty acids and lymphocyte functions the effect of highly purified eicosapentaenoic and docosahexaenoic acids on monocyte phagocytosis in man influence of dietary supplementation with long-chain n-3 or n-6 polyunsaturated fatty acids on blood inflammatory cell populations and functions and on plasma soluble adhesion molecules in healthy adults lack of effect of foods enriched with plant-or marine-derived n-3 fatty acids on human immune function the influence of different combinations of γ-linolenic acid, stearidonic acid and epa on immune function in healthy young male subjects fish oil supplementation inhibits the expression of major histocompatibility complex class ii molecules and adhesion molecules on human monocytes effect of long-term fish oil supplementation on vitamin e status and lipid peroxidation in women dietary supplementation with omega 3 polyunsaturated fatty acids decreases mononuclear cell proliferation and interleukin 1 beta content but not monokine secretion in healthy and insulin dependent diabetic individuals dietary supplementation with γ-linolenic acid or fish oil decreases t lymphocyte proliferation in healthy older humans fish oil-supplemented parenteral diets normalize splanchnic blood flow and improve killing of translocated bacteria in a low-dose endotoxin rat model dietary omega-3 fatty acids decrease mortality and kupffer cell 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and multiple organ failure in patients after severe trauma and the enteral nutrition in ards study group (1999) effect of enteral feeding with eicosapentaenoic acid, γ-linolenic acid, and antioxidants in patients with acute respiratory distress syndrome enteral nutrition with eicosapentaenoic acid, gamma-linolenic acid, and antioxidants reduces alveolar inflammatory mediators and protein influx in patients with acute respiratory distress syndrome ) ω-3 vs, ω-6 lipid emulsions exert differential influence on neutrophils in septic shock patients: impact on plasma fatty acids and lipid mediator generation parenteral nutrition with fish oil modulates cytokine response in patients with sepsis key: cord-007621-rapinodd authors: vidovic, maria; sparacio, shaun m.; elovitz, michal; benveniste, etty n. title: induction and regulation of class ii major histocompatibility complex mrna expression in astrocytes by interferon-γ and tumor necrosis factor-α date: 2002-11-13 journal: j neuroimmunol doi: 10.1016/0165-5728(90)90103-t sha: doc_id: 7621 cord_uid: rapinodd astrocytes can function as antigen-presenting cells (apc) upon expression of class ii major histocompatibility complex (mhc) antigens, which are induced by interferon-γ (ifn-γ). previous data from this laboratory had shown that the cytokine tumor necrosis factor-α (tnf-α) enhances ifn-γ-mediated class ii antigen expression on astrocytes. we have now investigated the effect of ifn-γ and tnf-α on class ii mhc mrna expression in astrocytes using northern blot analysis. astrocytes do not constitutively express mrna for class ii mhc. kinetic analysis of class ii mhc mrna expression in ifn-γ-treated cells demonstrated an 8 h time lag, which was followed by an increase over the next 16 h. optimal expression of class ii mrna was detected after a 24 h incubation with ifn-γ. this level of expression was further enhanced by the simultaneous addition of ifn-γ and tnf-α to the astrocytes, while tnf-α alone had no effect on class ii mrna expression. tnf-α does not act by increasing the stability of ifn-γ-induced class ii mrna, indicating its action is not at that specific level of post-transcriptional control. furthermore, astrocyte class ii mrna expression was inhibited when cycloheximide (chx) was added together with ifn-γ or ifn-γ/tnf-α, and when chx was added up to 4 h after treatment with ifn-γ or ifn-γ/tnf-α. these results indicate that astrocyte class ii mrna expression is mediated by newly synthesized proteins induced by ifn-γ and/or ifn-γ/tnf-α. the expression of class ii antigens on astrocytes, and cytokine modulation of their expression, may be important in the initiation and perpetuation of intracerebral immune responses. the non-neuronal cells of the central nervous system (cns) are made up of the macroglia (astrocytes, oligodendrocytes and ependymal cells) and the microglia. collectively, these glial cells perform a variety of active roles during development of the brain (rakic, 1971; silver and sapiro, 1981) and subsequently in the maintenance of normal cns physiology (hertz, 1981; janzer and raft, 1987) . recent work has suggested that glial cells such as astrocytes and microglia may be involved in immunological events occurring in the brain. the astrocyte can be stimulated to secrete a number of immunoregulatory molecules, including interleukin-1 (il-i) (fontana et al., 1982) , interleukin-3 (il-3) (frei et al., 1985) , interleukin-6 (il-6) (frei et al., 1989; benveniste eta[., 1990) , prostaglandins (fontana et al., 1982) , leukotriene 134 (llartung et al., 1988) , tumor necrosis factor-c~ (tnf-co (robbins et al., 1987; lieberman et al., 1989; chung and benveniste, 199(i) and ifn-c~/,8 (tedeschi et al., 1986) . the microglia can also be stimulated to secrete 1l-1 (giulian et al., 1986) , il-6 (frei et al., 1989) and tnf-a (frei et al., 1987) , thus providing the cns with numerous endogenous sources of cytokines necessary, for immunological response. more importantly, the astrocyte and microglia can function as antigen-presenting cells (apc) in the cns (fierz et al., 1985; frei et al., 1987) . these cells are able to internalize, process, express and present antigen to encephalitogenic t cells (fontana et al., 1984) . however, such a function is only possible upon expression of class i1 major histocompatibility complex (mhc) molecules. indeed, astrocytes can be induced to express class ii antigens both in the cns and in vitro, following exposure to interferon-7 (ifn-t) (wong et al.. 1984 : fierz et al., 1985 or virus (massa et al., 1986) . mhc-encoded class 11 molecules are heterodimerit glycoproteins which have a central role in the regulation of immune responses (benacerraf, 1981) . the expression of class ii antigens is primarily restricted to b cells, monocytes/macrophages and dendritic cells (hammerling et al., 1975) , although certain non-lymphoid cells can be induced to express class ii upon exposure to ifnq,, and function as apc. these include pancreatic beta cells (markmann et al.. 1988) , keratinocytes (gaspari et al., 1988) , brain endothelial cells (mc-carron et al., 1985) , and most pertinent to this study, astrocytes (fontana et al., 1984) . abnormal control in the level of expression of class i1 genes, and aberrant expression in cells normally class ii negative have been implicated in autoimmune phenomena. because of the importance of class ii mhc antigens, many studies have been directed toward understanding the regulatory mechanisms involved in class ii mhc gene expression. it is generally accepted that induction of class ii gene expression by ifn-y occurs at the transcriptional level (basta et al., 1987; blanar et al., 1988 : fertsch-ruggio et al., 1988 : rosa and fel-ious, 1988 : amaldi et al., 1989 , and that transacting factors interacting with cis-acting dna regulatory elements are involved in the transcriptional regulation of class ii mhc expression (accolla et al., 1985 " salter et al., 1985 sherman et al., 1987 sherman et al., , 1989 blanar et al., 1988 , ama[di et al., 1989 celada et al., 1989) . these trans-acting regulatory factors have been postulated to function positively or negatively, and to be expressed ubiquitously, or in a tissue-or stage-specific manner. although ifnq, is considered the primary inducer of class ii antigens, there is evidence for other cytokines contributing to class i1 expression. we have previously shown that tnf-~ enhances ifnq,-induced class ii antigen expression on astrocytes, and that this is a synergistic interaction as tnf-a alone has no effect on class i1 expression (benveniste et al., 1989) . the present study was undertaken to extend these previous findings, and to examine, at the molecular level, the effect of 1fn-y and tnf-a on astrocyte class ii gene expression. we report that astrocytes express class ii mrna 8 h after treatment with ifn-2¢ or 1fn-t/tni'-~,, indicating a long lag period between exposure to the cytokines and initiation of class ii gene expression. tnf-~ does not act to stabilize ifnq,-induced class ii mrna, suggesting it may act at other levels of post-transcriptional control or at the transcriptional level. furthermore, the expression of class ii mhc mrna was completely inhibited by cycloheximide (chx), suggesting a role for newly synthesized proteins in astrocyte class ii mhc expression. as astrocytes can be stimulated to secrete tnf-a (robbins et al., 1987; lieberman et al., 1989; chung and benveniste, 1990) , and express high affinity tnf-a receptors (benveniste et al., 1989) , tnf-a can act in an autocrine fashion to enhance class ii gene expression in astrocytes. by modulating class ii gene expression and thereby stimulating the apc function of astrocytes, ifn-y and tnf-a in concert may play a pivotal role in the regulation of intracerebral immune responses. rat recombinant ifn-y (specific activity: 4 x 106 u/mg) was purchased from amgen biologicais (thousand oaks, ca, u.s.a.), and human recombinant tnf-a (specific activity: 5.6 x 107 u/mg) was the generous gift of genentech (south san francisco, ca, u.s.a.). monoclonal antibody to glial fibrillary acidic protein (gfap) was obtained from boeringher mannheim (indianapolis, in, u.s.a.), and monoclonal antibody to rat class i1 mhc antigens (clone ox-6) was from accurate corporation (westbury, ny, u.s.a.). second antibody was affinity-purified goat anti-mouse lg conjugated to fluorescein-isothiocyanate (fitc) from southern biotechnology (birmingham, al, u.s.a.). cycloheximide and actinomycin-d were purchased from sigma chemical company (st. louis, mo, u.s.a.) . primary glial cell cultures were established from neonatal rat cerebra by a modification of the mccarthy and de vellis technique (1980) as previously described (benveniste and merrill, 1986) . meninges were removed from rat brains prior to glial cell dissociation and culture. culture medium (cm) was duibecco's modified essential medium (dmem), high glucose formula supplemented with glucose to a final concentration of 6 g/l, 2 mm glutamine, 0.1 mm non-essential amino acid mixture, 0.1% gentamicin, and 10% fetal bovine serum (hyclone, logan, ut, u.s.a.). after 10 days in primary culture, oligodendrocytes were separated from the glial cultures by mechanical dislodging, and the astrocytes were obtained by trypsinization (0.25% trypsin/0.02% edta) and replated at a density of 6-10 x 10 6 cells/100 mm 2 tissue culture plate and allowed to adhere for at least 24 h. the cells were counted using trypan blue; cell viability was 99-100%. the astrocytes were monitored for purity by immunofluorescence, and by non-specific esterase staining for contaminating microglia as previously described (benveniste and merrill, 1986) . the primary astrocytes were plated (5.0 x 104) on 12 mm glass coverslips, incubated in culture medium for 2 days, washed twice with phosphate-buffered saline (pbs), and fixed for 10 s in cold acetone. the cells were then stained for gfap, an intracellular antigen unique to astrocytes (bignami et al., 1972) , using a monoclonal antibody to gfap (1:4) for 30 min at room temperature, followed by a 30 min incubation with goat anti-mouse ig/fitc (1:20). the coverslips were then mounted in 30% glycerol, and visualized by fluorescent microscopy. astrocyte cultures were routinely > 97% positive for gfap, and less than 2% of the cells were microglia based on their positive staining for non-specific esterase. total cellular rna was isolated from confluent monolayers of astrocytes that were incubated for various intervals (0-48 h) without or with ifn-y and/or tnf-a. in some experiments, the protein synthesis inhibitor, chx (5 #g/ml) or the rna synthesis inhibitor, actinomycin d (5 #g/ml), were added to the cytokine-treated astrocytes for 0-24 h. rna isolation followed the procedure of chomczynski (1987) . the cells were collected, washed 2 times with cold pbs, and pelleted. rna was extracted with guanidinium isothiocyanate and phenol, and precipitated with ethanol. samples (15 ~g) of total cellular rna were denatured with formaldehyde for 15 rain at 55°c, and rna was size fractionated by electrophoresis through a 1.0% agarose gel" containing ethidium bromide for visualization of 28 s and 18 s ribosomal rna bands. the visualization of rna bands was useful for assessing the integrity of the rna and for varifying the amount of rna loaded. the rna was then transferred to nitrocellulose paper in 20 x standard saline citrate (ssc) (3 m naci and 0.3 m sodium citrate) at 4°c. after the transfer, the nitrocellulose paper was air-dried and the rna cross-linked in a uv stratalinker oven. prehybridization was performed at 42°(7 in a solution containing 50% (v/v) formamide, 5 x ssc, 1× denhardt's solution, 50 p,g/ml of denatured salmon sperm dna, and 0.1% sodium dodecyl sulfate (sds) for 8-24 h. hybridization was carried out at 420( ` for 48 h in prehybridization solution containing 10% dextran sulfate, 0.05 mm na phosphate buffer and denatured ?2p-labeled murine class i1 e-e~ cdna probe (2 x 1w' cpm/ml). the blots were then washed in 2 x ssc (twice for 20 min) at room temperature, followed by 1 x ssc containing 0.1% sds (twice for 30 min) at 42°c and finally in 0.1 x ssc for 30 rain at 42°c. the blots were dried between whatman filter paper and exposed to kodak x-omat ar film plus intensifying screens at -70°c. the autoradiographs were quantitated by scanning densitometry with a bio-rad model 620 video densitometer. filters were stripped to remove bound class 11 mhc probe, and rehybridized with a second control probe, cyclophilin. a edna probe (peacll) specific for mouse class ii e-a (mathis et al., 1983) was the generous gift of dr. jerold woodward, university of kentucky. the 1.08 kb ecori insert was isolated, and labeled with [et-s2p]deoxyctp using an amersham nick translation kit according to the manufacturer's instructions. a specific activity of 0.5-1 x 1() ~ cpm/~g dna was routinely attained. a edna probe for rat cyclophilin (plb15) (danielson et al., 1988) was the generous gift of dr. jim douglass, the oregon health sciences university. primary rat astrocytes were resuspended in dmem containing 10% fetal bovine serum (fbs), and plated at 4-5 x 10 ~ cells/well into 6-well (35 mm) plates (costar, cambridge, ma, u.s.a.). the plates were incubated overnight to allow recovery of the cells from trypsinization and to assure adherence of the astrocytes. after 24 h the original medium was aspirated off and fresh serum-free medium (1 ml) was added to the wells. triplicate wells of primary rat astrocytes were treated with 100 u/ml of recombinant rat ifn-y and/or 50 ng/ml of recombinant human tnf-a for various incubation periods (0 3 days). at each time point, the cells were trypsinized and stained for class it antigens, as previously described (benveniste et al., 1989) . briefly. astrocytes were incubated with 30 ~1 of ox-6 monoclonal antibody for 60 rain in the cold. washed 3 times with pbs containing 0.5% fbs and 0.02% azide (pbs-fbs-azide), and then incubated with 30 /tl of goat anti-mouse ig-fit(" (1:20) for another 30 rain in the cold. after washing 3 times with pbs-fbs-azide, the cells were fixed in a final volume of 100 ~1 of 1% paraformaldehydc and analyzed on the facstar (becton-dickinson, mountain view, ca, u.s.a.) for class ii antigen expression. negative controls were incubated with 30 /tl of pbs-fbs-azide in place of first antibody, or with an irrelevant monoclonal antibody of the same isotype. the gate window of forward-angle light scatter lay between channels 10 and 255: the gate window for log of green fifc fluorescence lay between channels 0 and 255. ten thousand cells were analyzed for each sample. the level of class i1 mhc mrna was examined in astrocytes following treatment for various times with ifn-y, tnf-a or a combination of the two cytokines. to determine the steady-state level of mrna for class ii, northern blot analysis was performed using a edna probe for murine class ii genes (e-a), with total rna isolated from cultured astrocytes. as seen in fig. 1 , a 1.3 kb class ii mhc mrna transcript was present in ifn-~, treated astrocytes (lanes 2 and 4) and absent in untreated cells (lanes 1 and 3) . class it m}tc mrna expression was more pronounced when the cells were cultured with ifn-y in serum-free medium (sfm) (fig. 1, lane 4) as opposed to serum-containing medium (fig. 1, lane 2) , thus, all the subsequent experiments were conin primary rat astrocytes. northern blot of rna from astrocytes that were incubated in serum containing media (lanes 1 and 2) or serum-free medium (sfm) (lanes 3 and 4) without (lanes 1 and 3) or with if'n-), (100 u/ml) (lanes 2 and 4) for 24 h. total rna was extracted and size fractionated by gel electrophoresis. hybridization was performed with a cdna probe (e-a) specific for a murine class 1i mhc gene. the blot was then exposed at -70°c for 24 h to kodak x-omat ar film plus two intensifying screens, kb, kilobases. ducted in sfm. optimal expression of class ii mrna was detected when cells were stimulated with 100-250 u/rnl of ifn-'r (data not shown). some variability in the concentration of ifn-t required for induction of class ii mrna was noted, and this variability was dependent on the lot of ifn-7 used. therefore, it was necessary to do a dose-response study for each lot of ifn-t used. for this study, 100 u/ml of ifn-t was sufficient for maximal expression of class ii mrna. the optimal time required for class ii mrna expression following treatment of astrocytes with ifn-t is illustrated in fig. 2 . astrocytes were incubated in sfm without or with ifn-~, for 12, 24 or 48 h prior to harvesting. a low level of class ii mhc mrna was detected at 12 h following treatment with ifn-~,, with maximal expression detected after a 24 h incubation with ifn-t. there was a 2.7-fold increase in class ii mhc mrna expression from 12 to 24 h, and a slight reduction at 48 h. we have previously shown that the level of class ii protein expression, based on fluorescenceactivated cell sorting (facs) analysis, was enhanced when the cells were treated with both ifn-t and tnf-a (benveniste et al., 1989) . similarly, in this present study, the incubation of as-2kb fig. 2 . kinetic analysis of ifn-y treatment on astrocyte class ii mhc mrna expression. astrocytes were cultured in sfm without (lanes 1, 3, and 5) or with ifn-'r (lanes 2, 4, and 6) for 12 h (lanes 1 and 2) , 24 h (lanes 3 and 4) or 48 h (lanes 5 and 6). total rna was extracted and analyzed for class 11 mrna by northern blot hybridization method. the blot was exposed to kodak x-omat ar film plus two intensifying ~reens at -70°c for24 h. trocytes with both ifn-y and tnf-a resulted in an enhanced expression of class ii mrna compared to ifn-7 alone (fig. 3) . optimal enhancement of class ii mrna was demonstrated using tnf-a at 50 ng/ml (fig. 3, lane 4) , which correlates with the concentration of tnf-a used for synergistic induction of class ii mhc protein (benveniste et al., 1989) . a 2.2-fold increase in class ii mrna expression in the presence of 50 ng/ml of tnf-a was detected, compared to ifn-y alone. as expected, tnf-a alone did not induce mrna for class ii antigens (data not shown). class ii mhc mrna expression induced by ifn-y/tnf-a was also enhanced when experi-2kb 1 2 3 4 5 , for 24 h. rna was isolated for analysis by northern blot hybridization method. the blot was probed with labeled e-a cdna, and exposed at -70°c for 24 h to kodak x-omat ar film plus two intensifying screens. lg4 ments were performed in sfm (data not shown), indicating that a serum component(s) has a slight inhibitory effect on class i1 mrna expression. in other cell types, a lag phase of approximately 6-8 h precedes the appearance of class ii mrna induced by ifn-7 (basta et al,, 1988; blanar et al., 1988, rosa and fellous, 1988; amaldi et al., 1989) . we performed a more indepth analysis of the kinetics of induction of class ii mrna by ifn-7 and ifn-7/tnf-a in astrocytes. analysis of mrna was performed at different times after induction with ifn-y (fig. 4 , lanes 2, 4, 6, and 8) and ifn-y/tnf-a (fig. 4, lanes 3 , 5, 7, and 9). no class ii mrna was detected until 8 h following treatment with ifn-7, with maximal exvression detected 24 h after exposure to ifn-y. similarly, class 11 mrna was not detected until 8 h in astrocytes that were stimulated with ifn-7/tnf-a; however, the intensity of the rna signal was increased in the presence of both cytokines, as expected. thus, there was an 8 h time lag before class ii mrna was detected in astrocytes. at early time points (8 and 12 h), mrna doublets are seen which ultimately merge into a diffuse, 6 and 7) . and 24 h (lanes 8 and 9). astrocytes in sfm alone (lane 1). the blot was exposed to kodak x-omat ar film plus two intensifying screens at -70 o c for 4 days. more intense 1.3 kb band at 24 h. this may be due to multiple transcription initiation sites described for the e-a gene (mathis et al., 1983) . in addition, a larger mrna species of 2.4 kb is seen at 24 h. the significance of this band is unknown at this time. results for the induction of class ii mhc antigen expression and mrna accumulation are summarized in fig. 5 . tnf-a increases ifn-v-induced class ii expression by increasing levels of mrna for the class ii molecule. however, it is not known whether tnf-a acts by increasing transcription or by stabilizing the mrna. experiments were conducted to assess class ii mrna stability in the presence of ifn-7 or ifn-y/tnf-a. class ii mrna was induced in astrocytes with either ifn-"/or ifn--//tnf-a for 24 h, then actinomycin d (a transcription inhibitor) was added for various times (1, 2, 4, 8, 16, and 24 h). total cellular rna was isolated and analyzed by northern blotting. preliminary results indicated that a decrease in class ii mrna was not detected until 8 h of actinomycin d treatment (data not shown). subsequent experiments were performed utilizing rna extracted after 8, 16 and 24 h of actinomycin d treatment. as shown in fig. 6 actinomycin d treatment, tnf-a did not appreciably affect the stability of e-a mrna compared to the stability of ifn-y-induced e-a mrna. in fact, it appears that tnf-a contributes to an accelerated destabilization of class ii mrna. the approximate half-life of e-a mrna in the presence of ifn-v/tnf-a was 19 h, compared to greater than 24 h in the presence of ifn-v alone. these same blots were reprobed for cyclophilin mrna to demonstrate that the integrity and quantity of rna loaded in each lane was similar (data not shown). these data indicate that tnf-a does not act by mrna stabilization to enhance if/q-v-induced class ii expression. the 8 h delay in class ii mrna expression after ifn-v or ifn-v/tnf-a stimulation of as-195 trocytes suggests that signal transmission initiated by these cytokines involves a number of intermediary steps, possibly the expression of newly synthesized gene products. to test this, we examined whether protein synthesis was required for induction of class 1i mrna by ifn-v and ifny/tnf-a. chx, an inhibitor of protein synthesis, was added to astrocytes at a concentration (5 /.tg/rnl) that inhibited protein synthesis by more than 92%, while still maintaining cell viability (data not shown). astrocytes were cultured for 24 h in the presence of ifn-y, ifn-y/tnf-a, chx alone, ifn-y plus chx, ifn-y/tnf-a plus chx, rna extracted, and then analyzed. fig. 7 demonstrates the effect of chx on the induction of class ii mrna by ifn-v and tnf-a. no mrna for class ii was detected in cells treated with chx alone, ifn-y plus chx, or ifny/tnf-a plus chx (fig. 7, lanes 4, 5, and 6 ). inhibition of protein synthesis completely abolished the induction of class ii mrna by ifn-y and ifn-y/tnf-ct. however, there was no inhibition of cyclophilin mrna expression (fig. 7b) , and no alteration in the pattern of ethidium bromide staining of rna in all the samples treated with chx alone or chx plus the cytokines (fig. 7c) , indicating that chx did not cause a generalized inhibition of mrna expression in astrocytes. cyclophilin was used as a control for these experiments as rna levels do not change upon treatment with ifn-v or ifn-v/tnf-a. that newly synthesized protein(s) is required for the induction of the class ii mhc gene in astrocytes treated with ifn-y or ifn-y/tnf-a was suggested by results in fig. 7 . the duration of protein synthesis required to allow expression of the class ii mhc gene in astrocytes was examined in cells that were pretreated with ifn-y or ifn-7/tnf-a for different lengths of time prior to the addition of chx. class ii mhc mrna was measured 24 h after the treatments were started. as shown in fig. 8a , when chx was added simultaneously with ifn-y/tnf-a or 1-2 h after ifn-y/tnf-a treatment, there was no detectable expression of class ii mhc mrna. however, when astrocytes were incubated with ifnhowever, in samples that were treated for 12 h with ifn-y/tnf-c~ before ctlx was added, there was still a 25% reduction in the expression of class ii mhc signal compared to the positive control of 1fn-y/tnf-a alone (fig. 8a, lane 2) , suggesting that continuous synthesis of protein is required for optimal expression of the class 11 mhc gene. chx treatment had no effect on the expression of cyclophilin rna (fig. 8b) . similar results were seen when astrocytes were incubated with ifn-t and chx, except that the expression of class ii mhc mrna was detected only after cells were incubated with ifn-7 for 8 h prior to the addition for 24 h. total rna was extracted, northern blot hybridization performed and the blot was exposed to kodak x-omat ar film plus two intensifying screens at -70°c for 3 days. autoradiograph of class ii mrna (a). autoradiograph for cyclophilin mrna was obtained by stripping class i1 probe and rehybridizing with a second probe to detect cyclophilin mrna expression (b). photograph of the original gel stained with ethidium bronude to show that there was no alteration in the quantity or the quality of rna in all the samples treated with chx (c). ~q 7/tnf-a for 4 h prior to addition of chx, and class ii rna measured 24 h later, a low level of class ii rna was detected. the increase in the level of class i1 mhc mrna detected parallels the increase in the amount of time the cells were treated with ifn-t/tnf-a before the addition of chx, i.e., the longer the treatment with ifn-7/tnf-a before the addition of chx, the stronger the mrna signal. these results, therefore, suggest that protein synthesis, initiated within 4 h of the cells encountering ifn-t/tnf-a, is critical for subsequent class ii mhc mrna expression. the blot was exposed at -70 ° c for 3 days to kodak x-omat ar film plus two intensifying screens (a). autoradiograph for cyclophilin mrna obtained by stripping class ii probe and rehybridizing with a second probe to detect cyclophilin mrna (b). photograph of the original gel stained with ethidium bromide (c). of chx (data not shown), indicating that 8 h of protein synthesis was critical for ifn-~-induced class ii mrna expression. in this study we have shown that primary neonatal rat astrocytes, upon stimulation with ifn-~,, express mrna transcripts for class ii mhc genes, and that tnf-a enhances the expression of ifn-~,-induced class ii mrna. these results support previous findings that ifn-~, and tnf-a synergize in the induction of class ii mhc protein expression in rat astrocytes (benveniste et al., 1989) . kinetic analysis demonstrated that class ii mrna was first detected after 8 h of treatment with ifn-y, followed by an increase in mrna expression over the next 16 h. when astrocytes were treated with ifn-~, and tnf-a simultaneously, the kinetics of class ii mrna expression did not change; however, the overall amount of steady-state class ii mrna was increased. optimal expression of class ii mrna was detected 24 h after incubation with ifn-3, alone or ifn-~,/tnf-a. although the predominant forms of gene regulation occur at the transcriptional level, a number of control mechanisms can act on rna once its transcription has been initiated. post-transcriptional regulatory mechanisms include (1) changes in mrna stability, (2) alternative rna splicing, (3) poly a addition, and (4) control of translational initiation. in our experiments, tnf-a did not increase the stability of ifn-3,-induced class ii mrna, indicating that tnf-a did not act at that level of post-transcriptional control. preliminary results from our laboratory suggest that the increase in class ii mrna occurs primarily by an increase in transcription of the e-a gene since nuclear run-on assays detected no transcription of the class ii genes without induction by ifn-y, and enhanced transcription in the presence of ifn-~, plus tnf-a. further experimentation is necessary to determine conclusively if tnf-a acts solely at the transcriptional level, or whether both transcriptional and post-transcriptional events result in increased class ii mhc mrna and protein. 197 the time required for the appearance of class ii mhc mrna following treatment with ifn-~, or ifn-~,/tnf-a (8 h) suggests that cytokine signal transmission is complex and may involve a number of intermediary steps. we examined whether protein synthesis was required for ifn-), or ifn-"t/tnf-a-induced expression of astrocyte class ii genes. the expression of class ii mrna was completely inhibited when chx was included with ifn-~, and ifn-'t/tnf-~ treatment, indicating that newly synthesized protein is required for astrocyte class ii mhc gene expression. a minimum of 4 h of active protein synthesis was required for subsequent ifn-t/tnf-a-induced class ii mrna expression, while 8 h was required for subsequent ifn-), expression. however, in experiments where chx was added 12 h after treatment with ifn-), or ifn-),/tnf-~, there was still a 45% and 25% reduction, respectively, in the expression of class ii mrna compared to astrocytes incubated with the cytokines alone. this indicates that the synthesis of novel proteins is required continuously for optimal class ii gene expression in astrocytes. other studies have shown that protein synthesis was required for up to 12 h after ifn-~, was added to murine p388d cells to detect an increase in the level of i-aa (boettger et al., 1988) , while in peritoneal mouse macrophages, a 30% decrease in i-at~ mrna levels was observed even when chx was added after 12 h of ifn-'rtreatment (fertsch et al., 1987) . in contrast, celada et al. (1989) demonstrated that protein synthesis was only required for 30 rain after murine macrophages were treated with ifn-), for an increase in i-aft mrna to be detected. thus, different cell types have varying requirements for active protein synthesis to express class ii mrna in response to ifn-),. other reports on ifn-t-induced expression of class i1 genes have indicated that protein synthesis is not required. induction of dra mrna in the human glioblastoma cell line u373-mg (basta et al., 1988) , dermal fibroblasts (collins et al., 1986) and i-aa in murine wehi-3 cells (woodward et al., 1989) , occurs in the absence of protein synthesis. this suggests that the expression of class ii mrna in these cells is mediated by pre-existing trans-acting factors that are triggered by ifn-'t (woodward et ai., 1989) . it is also important to note that primary astrocytes (this study) and glioblastoma cells (basta et al., 1988) differ in their requirements for protein synthesis for class 11 expression, illustrating fundamental differences between normal astrocytes and transformed glial cells. tnf-a may be an important enhancer of class i1 expression in the cns as it can function in an autocrine fashion on the astrocyte. in addition to responding to tnf-a and expressing specific high affinity receptors for this factor (benveniste et al., 1989) , astrocytes can also secrete tnf-a (robbins et al., 1987; sawada et al., 1989; chung and benveniste, 1990) . more importantly, ifn-t primes the astrocyte to produce tnf-a (chung and benveniste, 1990) , thus ifn-t can influence both tnf-a and class ii gene expression in the astrocyte. although class ii expression on astrocytes has been conclusively demonstrated in vitro, in vivo studies have generated conflicting results. direct injection of ifn-t into the brains of mice induced class ii antigens on astrocytes, indicating that astrocytes have the potential to express these antigens in vivo (wong et al., 1984) . many laboratories have examined whether astrocytes express class i1 antigens in a variety of immune-mediated disease states to better understand the possible role of the astrocyte as a local apc. traugott et al. (1985) demonstrated class ii expression on astrocytes in active chronic multiple sclerosis (ms) lesions, and then confirmed these studies by performing double-staining for both class ii and gfap (traugott and lebon, 1988) . a study by hofman et al. (1986) also identified class ll-posirive astrocytes in ms brain by double-staining. rodriguez et al. (1987) have studied class i1 expression on glial cells in an animal model of cns demyelination induced by theiler's virus. in susceptible strains of mice (bio.s and bio.asr2), the majority of class ii-positive glial cells had morphological characteristics of astrocytes, while uninfected mice or resistant strains (bio.s, (9r)) were class ii negative. in sjl mice with acute or chronic relapsing experimental allergic encephalomyelitis (eae), an animal model for ms, some class ii-positive ceils were identified as astrocytes (sakai et al., 1986) . however, other studies investigating the eae model in lewis rats failed to detect class ii-positive astrocytes in the brain (hickey et al., 1985 : matsumoto et al., 1986 vass et al., 1986) . thesc conflicting results may bc due solely to technical problems involved with antigen fixation and staining methodologies, or may indicate that the ability of astrocytes to function as apc in vivo may only bc relevant in certain diseases or specific stages of disease. another possibility may be the loss of class ii-positive astrocytes by class ii mhc-restricted t cell-mediated cytotoxicity as shown by sun and wekerle (1986) . the disease eae appears to be strain-specific as brown-norway rats and balb/c or c57bl/6 mice are resistant, whereas lewis rats and sjl mice are susceptible (linthicum and frelinger, 1982) . recent studies have demonstrated that astrocytes derived from susceptible strains express much higher levels of class ii antigen upon treatment with either ifn-~, or virus compared to astrocytes prepared from eae-resistant strains (massa et al., 1987a, b) . this hyperinduction of class ii in eae-susceptible animals was astrocyte specific as both peritoneal macrophages and microglial cells of susceptible and resistant strains showed identical patterns for class ii induction. this differential expression of class ii on astrocytes in response to ifn-t compared to microglia suggests that regulation of class i1 expression on astrocytes may correlate with antigen-presenting capacity and ultimately, disease development in the cns. we have begun, at the molecular level, to dissect the regulatory mechanisms utilized by primary rat astrocytes for class i1 mhc gene expression. future studies will focus on the regulation of gene expression at the transcriptional level, and ifn-t/tnf-a-induced trans-acting regulatory factors required for class ii gene expression. reactivation by a trans-acting factor of human mhc-ia gene expression in interspecies hybrids between an ia-negative human b cell variant and an la-positive mouse b cell lymphoma induction of hla class ii genes by ifn-y is transcriptional and requires a trans-acting protein identification of an interferon-y response region 5' of the human histocompatibility leukocyte antigen dret chain gene which is active in human glioblastoma multiform lines detailed delineation of an interferon-y-responsive element important in human hla-dra gene expression in a glioblastoma multiform line role of mhc gene products in immune regulation stimulation of oligodendroglial proliferation and maturation by interleukin-2 tumor necrosis factor-a enhances interferon-y mediated class i! antigen expression on astrocytes induction and regulation of interleukin-6 gene expression in rat astrocytes localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence a (1988) transcriptional activation of hla-dra by interferon y requires a trans-acting protein cycloheximide, an inhibitor of protein synthesis, prevents y-interferon-induced expression of class ii mrna in a macrophage cell line lnterferon-y activates multiple pathways to regulate the expression of the genes for major histocompatibility class ii i-a#, tumor necrosis factor and complement component c3 in mouse macrophages single step method of rna isolation by acid guanidinium thiocyanate-phenolchloroform extraction tumor necrosis fac-199 tor-alpha production by astrocytes: induction by lipopolysaccharide, interferon-gamma and interleukin-1 recombinant human tumor necrosis factor increases mrna levels and surface expression of hla-a,b antigens in vascular endothelial cells and dermal fibroblasts in vitro plbl5: a cdna clone of the rat mrna encoding cyclophilin induction of macrophage la antigen expression by rlfn-y and down-regulation by ifn-a/fl and dexamethasone are mediated by changes in steady-state levels of la mrna induction of macrophage la antigen expression by rlfngamma and down regulation by ifn-alpha/beta and dexamethasone are regulated transcriptionally astrocytes as antigen presenting cells, t. induction of la antigen expression on astrocytes by t cells via immune interferon and its effect on antigen presentation production of prostaglandin e and an interleukin-l-like factor by cultured astrocytes and c6 glioma cells astrocytes present myelin basic protein to encephalitogenic t-cell lines astrocytes of the brain synthesize interleukin-3-1ike factors antigen presentation and tumor cytotoxicity by interferon-y-treated microglial cells on the cellular source and function of interleukin-6 produced in the central nervous system in viral diseases class ii mhc-bearing keratinocytes induce antigen-specific unresponsiveness in hapten-specific th1 clones interleukin-1 of the central nervous system is produced by ameboid microglia tissue distribution of la antigens: la on spermatozoa, macrophages and epidermal cells primary rat astroglial cultures can generate leukotriene b 4 functional interactions between astrocytes and neurons expression of la molecules by astrocytes during acute experimental allergic encephalomyelitis in the lewis rat immunoregulatory molecules and il-2 receptors identified in multiple sclerosis brain astrocytes induce bloodbrain barrier properties in endothelial cells production of tumor necrosis factor and other cytokines by astrocytes stimulated with lipopolysaccharide or a neurotropic virus acute autoimmune encephalomyelitis in mice. i1. susceptibility is controlled by the combination of h-2 and histamine sensitization genes antigen presenting function of class !i mhc expressing pancreatic beta cells viral particles induce la antigen expression on astrocytes inducibility of la antigen on astrocyte by murine coronavirus jhm is rat strain dependent hypersensitivity of la antigen on astrocytes correlates with strain-specific susceptibility to experimental autoimmune encephalomyelitis the murine e-a immune response gene lmmunohistochemical analysis of the rat central nervous system during experimental allergic encephalomyelitis, with special reference to la-positive cells with dendritic morphology presentation of myelin basic protein by murine cerebral vascular endothelial cells preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissues neuron-glial relationship during granule cell migration in developing cerebellar cortex. a golgi and electromicroscopic study in maccacus rhesus production of cytotoxic factor for oligodendroeytcs by stimulated astrocytes immune response gene products (la antigens) on glial and endothelial cells in virus-induced demvelination regulation of hla-dr gene by ifn-~,. transcriptional and post-transcriptional control ) la expression in chronic relapsing experimental allergic encephalomyelitis induced by long-term cultured t cell lines in mice evidence for two trans-acting genes regulating hla class ii antigen expression pr(xluction of tumor necrosis factor-alpha by microgila and astrocytes in culture upstream dna sequences required for tissue-specific expression of the hla-dra gene class ii box consensus sequences in the hla-dra gene: transcriptional function and interaction with nuclear proteins axonal guidance during development of the optic nerve: the role of pigmented epithelia and other intrinsic factors ) la-restricted encephalitogenic t lymphocytes mediating eae lyse autoantigen-presenting astrocytes astrocytes produce interferon that enhances the expression of h-2 antigens on a subpopulation of brain cells interferon-'), and la antigen are present on astrocytes in active chronic multiple sclerosis lesions on the presence of la-positive endothelial cells and astrocytes in multiple sclerosis lesions and its relevance to antigen presentation the distribution of la antigen in the lesions of rat acute experimental allergic encephalomyelitis inducible expression of h-2 and la antigens on brain cells mhc class ii transcription in different mouse cell types: differential requirement for protein synthesis between b cells and macrophages this work was funded in part by grants rg 1954-a-2 and rg 2205-a-3 from the national multiple sclerosis society (e.n.b) and grant bns-8708233 from the national science foundation (e.n.b). we acknowledge the support of the university of alabama at birmingham flow cytometry core facility (am 20614).we thank mr. keith berry for facs analysis, and ii yup chung, j. gavin norris and john r. bethea for helpful discussions. key: cord-017640-i8h48ny6 authors: fouqué, amélie; legembre, patrick title: the cd95/cd95l signaling pathway: a role in carcinogenesis date: 2020-01-03 journal: cancer immunology doi: 10.1007/978-3-030-30845-2_11 sha: doc_id: 17640 cord_uid: i8h48ny6 apoptosis is a fundamental process contributing to tissue homeostasis, immune response, and development. cd95, also called fas, is a member of the tumor necrosis factor receptor (tnfr) superfamily. its ligand, cd95l, was initially detected at the plasma membrane of activated t-lymphocytes and natural killer (nk) cells where it contributes to the elimination of transformed and infected cells. given its implication in immune homeostasis and immune surveillance combined with the fact that various lineages of malignant cells exhibit loss-of-function mutations, cd95 was initially classified as a tumor suppressor gene. nonetheless, in different pathophysiological contexts, this receptor is able to transmit non-apoptotic signals and promote inflammation and carcinogenesis. although the different non-apoptotic signaling pathways (nf-κb, mapk, and pi3k) triggered by cd95 are known, the initial molecular events leading to these signals, the mechanisms by which the receptor switches from an apoptotic function to an inflammatory role, and, more importantly, the biological functions of these signals remain elusive. inhibition of this cellular process is observed in different pathologies, such as cancer and autoimmunity, while amplification of the apoptotic signal was reported in neurodegenerative disorders including alzheimer's and parkinson's diseases [3, 4] , as well as infection by human immunodeficiency virus (hiv). the origin of the apoptotic signal has been used to distinguish two main signaling pathways. the intrinsic pathway stems from accumulation of dna damage, deregulation of mitochondrial function, or virus infection and induces the release of proapoptotic factors from the mitochondria, whereas extrinsic signals are transmitted by the binding of apoptotic ligands to death receptors present at the cell surface. interconnections exist between these two signaling pathways: both leading to the activation of a family of cysteine proteases specific for aspartic acid residues, called caspases [5] . the apoptotic role of mitochondria is associated with reduction in its transmembrane potential and the loss of its extracellular membrane integrity, leading to the release of different apoptogenic factors in the cytosol. among them, cytochrome c associates with the caspase-9/apaf-1 complex to form the apoptosome and trigger apoptosis [6] . these two signaling pathways share common features, and both require the aggregation of initiator caspases as their preliminary events. during interactions with respective ligands, members of the death receptor superfamily recruit adaptor proteins such as fas-associated protein with a death domain (fadd) [7, 8] or tumor necrosis factor (tnf) receptor 1-associated death domain protein (tradd) [9] , resulting in the aggregation and activation of the initiators caspase-8 and caspase-10 to form the death-inducing signaling complex (disc) [10] . in a similar manner, release of cytochrome c and atp by mitochondria promotes the formation of the apoptosome with the cytosolic apaf-1, thereby aggregating and activating the initiator caspase-9, which in turn cleaves caspase-3 [11] . it should be kept in mind that death receptors cd95 [12] , tnfr1 [13] , dr4 [14] , dr5 [15] , and dr6 [16] have been cloned based on their ability to elicit apoptosis. although studies have revealed the ability of fas/cd95, dr4, and dr5 in triggering non-apoptotic signaling pathways even immediately after their cloning [17, 18] , most, if not all, studies have been focused on characterizing the molecular events leading to cell death. accordingly, several agonistic molecules were developed in order to kill cancer cells, neglecting the impact of non-apoptotic signals in pathophysiological contexts. more recent data changed this vision by evaluating the biological role of death receptor-mediated non-apoptotic signaling pathways in chronic inflammatory disorders and carcinogenesis. in this chapter, apoptotic signaling pathways induced by death receptors are discussed. moreover, recent evidences pointing to the non-apoptotic signals transmitted by the same receptors are brought up, which may imply their tremendous impact on tumor progression and the design of therapeutic tools. death receptors tnfr1, fas, dr3, dr4, dr5, and dr6 belong to the tumor necrosis factor receptor (tnfr) superfamily. these type i transmembrane proteins share common features, such as extracellular amino-terminal cysteine-rich domains (crds) [19, 20] , which contribute to ligand specificity [21] , and pre-association of the receptor at the plasma membrane [22] [23] [24] and a conserved 80-amino acid sequence located in their cytoplasmic tail called death domain (dd), which is necessary for disc formation and initiation of the apoptotic signal [25, 26] . tnf-α exerts its effects by binding to two receptors, tnfr1 and tnfr2 [20] . recently, progranulin was identified as a ligand of tnfr with a higher affinity than tnf-α. progranulin antagonizes tnf-α signaling and plays a critical role in the pathogenesis of inflammatory arthritis in mice [27] . tnfr1, a 55 kda protein expressed in almost all cell types, presents a dd in its intracellular region, whereas tnfr2, a 75 kda protein, is mainly detected in oligodendrocytes, astrocytes, t-cells, myocytes, thymocytes, endothelial cells, and human mesenchymal stem cells [28] . uncertainty remains on the tnfr2 signaling pathway, which has been previously reviewed [28] . the crd1 of cd95, tnfr1, and tnfr2 is involved in homotypic interactions, leading to pre-association of the receptor as a homotrimer in the absence of ligand [23, 24, 29] . this domain has been designated as the pre-ligand binding assembly domain (plad) [29] . receptors of the tnfr superfamily do not possess any enzymatic activity on their own and rely on the recruitment of adaptor proteins for signaling. among these adaptor proteins, tradd or fadd is instrumental in the implementation of cell death processes [7] [8] [9] [10] . tnf is synthesized as a 26 kda transmembrane type ii protein (m-tnf) of 233 amino acids [30] which can be cleaved by the metalloprotease tace [31, 32] to release the 17 kda soluble form of the cytokine (cl-tnf). in contrast to cl-tnf, which only activates tnfr1, m-tnf can bind and activate both tnfr1 and tnfr2 [33] . activation of tnfr1 leads to the induction of cellular processes ranging from cell death (apoptosis or necroptosis) to cell proliferation, migration, and differentiation; the implementation of such different cellular responses reflects the formation of different molecular complexes after receptor activation [28] . binding of tnf to tnfr1 causes the formation of two consecutive complexes. while the plasma membrane complex (complex i) elicits a non-apoptotic signaling pathway, a second, internalized complex (complex ii or disc) triggers cell death [2] . in the presence of tnf, the adaptor protein tradd interacts with tnfr1 and recruits other proteins involved in the signaling of the receptor, such as traf2, ciap1, ciap2, and rip1, to form complex i. at the plasma membrane, this complex activates the nf-κb signaling pathway, which in turn promotes the transcription of antiapoptotic genes such as ciap1, ciap2, and c-flip [34] . the linear ubiquitin chain assembly complex (lubac) is also recruited to complex i via ciap-generated ubiquitin chains [35] . hoil-1, hoip, and sharpin constitute the lubac complex. hoil-1 and hoip add a linear ubiquitin chain by catalyzing the head-to-tail ligation of ubiquitin [36] to rip1 and nemo (ikk-γ) in complex i [37] , thereby activating nf-κb. tnf-induced caspase activation is mediated by a second, intracellular complex ii, which is formed when complex i dissociates from the receptor, along with fadd and caspase-8 recruitment [2] . nf-κb activation leads to c-flip overexpression, preventing formation of complex ii. contrariwise, when nf-κb activation is blocked, c-flip, whose protein half-life is short [38] , is absent, and cells experience death [2] . rip1 is deubiquitinated by enzymes such as cezanne [39] and cyld [40] , and the complex composed of tradd and rip1 moves to the cytosol to form complex ii. fadd is recruited to tradd by dd-dd interaction and binds caspase-8 [2] . noteworthy, when caspase-8 activity is inhibited or its expression is extinguished, disc is unable to trigger the apoptotic signaling pathway; but tnfr1 or cd95 stimulation leads to the activation of another cell death signal, namely, necroptosis [41, 42] . to prevent the induction of the necroptotic signal, caspase-8 cleaves and inactivates rip1 and rip3 [43] . the fine-tuned control of necroptosis by members of the apoptotic signaling pathway in the organism has been elegantly confirmed by experiments showing that the embryonic lethality of mice harboring the single ko of caspase-8 or fadd is rescued by an additional ko of the rip3 gene [44] [45] [46] . many studies on tnf demonstrated its pivotal role in fueling inflammation, a multistep process that promotes autoimmunity (e.g., rheumatoid arthritis, ankylosing spondylitis, crohn's disease, psoriasis, and refractory asthma) and cancer. many tnf inhibitors, such as neutralizing monoclonal antibodies (mabs) (e.g., infliximab, adalimumab, and golimumab), have been developed to treat these chronic inflammatory disorders, demonstrating that altering ligand/receptor interactions with neutralizing mabs is an invaluable opportunity to treat certain chronic inflammatory disorders. other tnf-α antagonists, such as etanercept, a tnfr2-immunoglobulin fc fusion protein, can improve the clinical course of rheumatoid arthritis [47] . while findings accumulate to decipher the molecular mechanisms involved in the induction of apoptotic and non-apoptotic signaling pathways by tnfr1 and to elucidate how the receptor can switch from one signal to the other, the mechanistic links involved in the implementation of non-apoptotic signaling pathways by cd95 remain elusive. however, recent findings have revealed its proinflammatory effects [48] [49] [50] [51] [52] [53] [54] . in 1989, identification of the mab apo-1 by peter krammer et al. revealed the existence of a 52 kda protein whose aggregation was able to transmit an apoptotic signal in cancer cells [55] . this receptor was identified in 1991 by nagata and colleagues and called fas (cd95 or apo-1) [12] . its ligand, fasl, was cloned in 1993 by the same group and was found to be mainly expressed at the surface of activated t-lymphocytes [56] and natural killer (nk) cells [57] ; however, its expression was also detected in different tissues in which the presence of acute or chronic inflammation is undesirable including the eyes [58] and testes [59] . in addition, two mouse models, in which either the level of cd95 expression was downregulated (due to an insertion of a retrotransposon in intron 2 of the receptor gene, these mice are called lymphoproliferation (lpr) [60] [61] [62] ) or the cd95l affinity for cd95 was reduced (due to the germ line mutation f273l in cd95l, called generalized lymphoproliferative disease (gld), which decreases cd95l binding to cd95 [63, 64] ), have provided some insight into the pivotal role played by this interaction in immunosurveillance and immune tolerance [65] . the cd95 gene (apt-1) consists of nine exons, with exon 6 encoding the transmembrane domain [66] (fig. 11.1 ). cd95 can be resolved under denaturing conditions between 40 and 50 kda by sds-page. the receptor is a type i transmembrane protein harboring three crds. similar to the tnf receptor [29] , cd95 is pre-associated at the plasma membrane as a homotrimer, and this quaternary structure is mandatory for transmission of the apoptotic signals in the presence of cd95l [23, 24] . homotrimerization of cd95 occurs mainly through homotypic interactions of the cd95-crd1 [22] [23] [24] . binding of cd95l or agonistic anti-cd95 mabs to cd95 alters both the conformation and the extent to which the receptor is multimerized at the plasma membrane. the intracellular region of cd95 encompasses an 80-amino acid stretch designated as the dd ( fig. 11 .1), which consists of six antiparallel α-helices [67] . upon addition of cd95l, cd95 undergoes conformational modification of its dd, which induces a shift of helix 6 and fusion with helix 5, promoting both oligomerization of the receptor and recruitment of the adaptor protein fadd [68] . a consequence of the opening of the globular structure of cd95 is that the receptor becomes connected through this bridge, which increases the magnitude of its homo-aggregation. this long helix allows the stabilization of the complex by recruiting fadd. overall, the cd95-dd/fadd-dd crystal structure provides some insights into the formation of the large cd95 clusters observed using imaging or biochemical methods in cells stimulated with cd95l. in addition, it also confirms that alteration in the cd95 conformation plays an instrumental role during signal induction [68] . however, this elongated c-terminal α-helix favoring the cis-dimerization of cd95-dd was challenged by driscoll et al. who did not observe the fusion of the last two helices at a more neutral ph (ph 6.2), compared to the acidic condition (ph 4) used in the initial study to resolve the cd95-dd/fadd-dd structure [68] . consequently, at ph 6.2, association of cd95 with fadd predominantly consisted of a 5:5 complex, which occurred via a polymerization mechanism involving three types of asymmetric interactions but without major alteration of the dd globular structure [69, 70] . it is likely that the low ph condition used in the study performed by scott et al. altered cd95 conformation and resulted in the formation of nonphysiological cd95/fadd oligomers [68] . nonetheless, it cannot be excluded that a local decrease in the intracellular ph affects the initial steps of the cd95 signaling pathway in vivo, through promoting the opening of the cd95-dd and eventually contributing to the formation of a complex eliciting a sequence of events different from the one occurring at physiologic ph. once docked on cd95-dd, fadd selfassociates [71] and binds procaspase-8 and procaspase-10, which are auto-processed and released in the cytosol as active caspases, which cleave many substrates leading to the execution of the apoptotic program and cell death. the complex cd95/fadd/caspase-8/ caspase-10 is called disc (fig. 11 the fate of cells, it is not surprising that numerous cellular and viral proteins were reported to hamper the formation of this structure, such as flip [72, 73] and ped/pea-15 [74] , which interfere with the recruitment of caspase-8/ caspase-10 ( fig. 11 .2). following the discovery of cd95 and the first steps of its signaling pathway, peter and colleagues described that cells can be divided in two groups with regard to the kinetics through which they respond to cd95-mediated apoptotic signals, the magnitude of disc formation, and the role played by the mitochondrion in this pathway [75] . disc formation occurs rapidly and efficiently in type i cells releasing a large amount of activated caspase-8 in the cytosol, while type ii cells have difficulty forming this complex, and the amount of active caspase-8 is insufficient to directly activate the effectors caspase-3 and caspase-7 [75] . nonetheless, type ii cells experience cell death upon cd95 engagement and are even more sensitive to the cd95-mediated apoptotic signal compared to type i cells [75] [76] [77] . this discrepancy can be partly explained by the fact that the low amount of activated caspase-8 in type ii cells is sufficient to cleave bid, a bh3-only protein, which constitutes the molecular link between caspase-8 activation and the apoptotic activity of mitochondria. indeed, after cleavage by caspase-8, truncated bid (tbid) translocates to mitochondria, where it triggers the release of proapoptotic factors ( fig. 11 .2) [78, 79] . although cd95 stimulation activates the mitochondrion-dependent apoptotic signal in type i and type ii cells, it seems that only type ii cells are addicted to this signal as they display a higher amount of the caspase-3 inhibitor xiap compared to type i cells [80] . among the inhibitor of apoptosis protein (iap) family, xiap, ciap1, and ciap2 inhibit caspase-3, caspase-7 [81, 82] , and procaspase-9 [83] activity by direct binding, thereby preventing access to substrates. furthermore, xiap can function as an e3 ligase whose activity is involved in the ubiquitination of active caspase-3 and its subsequent degradation through the proteasome [84] . to detach xiap from caspase-3 and restore the apoptotic signal, cells require the release of smac/diablo (second mitochondria-derived activator of caspase/ direct iap-binding protein with low pi) by the mitochondrion [85, 86] , explaining why type ii cells are more addicted to this organelle compared to type i cells (fig. 11 .2). to summarize, disc formation and iap amount are two cellular markers allowing a clear discrimination between type i and type ii cells. even though iap overexpression can account for the mitochondrion dependency observed in type ii cells, it remains unclear why disc formation is hampered in type ii cells and/or enhanced in their type i counterparts. recently, high activity of the lipid kinase phosphoinositide 3-kinase (pi3k) or downregulation of its neutralizing phosphatase, phosphatase and tensin homologue on chromosome 10 (pten), was found in type ii cells, while this signal is blocked in type i cell lines [87, 88] . the pi3k signaling pathway was reported to prevent the aggregation of cd95 [89] , probably by retaining the receptor outside of lipid rafts [87, 90] . pea-15, also known as ped, is a protein containing a death effector domain (ded) that has been shown to inhibit the cd95 and tnfr1 apoptotic signals (fig. 11 .2) [74] . activation of pi3k and its downstream effector, serine-threonine kinase akt, leads to phosphorylation of pea-15 at serine 116 [87, 90] ; this posttranslational modification promotes its interaction with fadd, ultimately inhibiting disc formation [91, 92] . notably, the existence of type i and type ii cells is not only an in vitro observation, but has been identified physiologically in the human body. cd95-mediated apoptotic signal cannot be altered in thymocytes or activated t-cells expressing a bcl-2 transgene, conferring to their type i nature [93] , whereas hepatocytes expressing the same transgene resist cd95-induced apoptosis and thus behave as type ii cells [94, 95] . mutations? germinal mutations in apt-1 have been reported in patients developing a syndrome termed autoimmune lymphoproliferative syndrome type ia (alps, also called canale-smith syndrome) [96] [97] [98] . alps patients show chronic lymphadenopathy and splenomegaly, expanded populations of double-negative α/β-τ-lymphocytes (cd3 + cd4 − cd8 − ), and often develop autoimmunity [96, 97, 99, 100] . in agreement with the notion that cd95 behaves as a tumor suppressor, alps patients display an increased risk of hodgkin and non-hodgkin lymphoma [101] . predominance of post-germinal center (gc) lymphomas in patients exhibiting either germ line or somatic cd95 mutations can be explained by the fact that, inside germinal centers of the secondary lymphoid follicles, the cd95 signal plays a pivotal role in the deletion of self-reactive maturating b-lymphocytes [102] , in addition to the fact that apt-1 belongs to a set of rare genes (i.e., pim1, c-myc, pax5, rhoh/ttf, and bcl-6) subject to somatic hypermutation [103, 104] , which may affect biological function. in addition to post-gc lymphomas, significant amounts of mutations in the cd95 gene were found in tumors of various histological origins (reviewed in [54] ). extensive analysis of cd95 mutations and their distribution in apt-1 reveals that, with some exceptions, most are gathered in exons 8 and 9 encoding the cd95 intracellular region (fig. 11. 3) [105] . remarkably, most of these mutations are heterozygous, mainly localized in cd95-dd, and lead to inhibition of the cd95-mediated apoptotic signal. indeed, in agreement with the notion that cd95 is expressed at the plasma membrane as a pre-associated homotrimer [23, 24] , formation of heterocomplexes containing wild-type and mutated cd95 prevents fadd recruitment and abrogates the ignition of the apoptotic signal in a dominant manner. extensive analysis and positioning of various cd95 mutations described in the literature seem to highlight mutation "hot spots" in the cd95 sequence (fig. 11.3 ). among these hot spots, arginine 234, aspartic acid 244, and valine 251 account for a significant amount of the documented cd95 mutations. indeed, among the 189 mutations annotated in the 335 amino acids of cd95, 30 (~16%) are localized on these three amino acids (fig. 11.3) . strikingly, the pivotal role played by these amino acids in stabilization or formation of intra-and interbridges between cd95 and fadd may explain these hot spots. for instance, both r234 and d244 contribute to the homotypic aggregation of the receptor and fadd recruitment [67] . nevertheless, the observation of death domain hot spots is in contradiction with the study of scott and colleagues demonstrating that the region of the cd95-dd interacting with the fadd-dd extends over a disperse surface through weak binding affinity [68] . most alps type ia patients affected by malignancies do not undergo loss of heterozygosity (loh), which formed the hypothesis that preservation of a wild-type allele may contribute to carcinogenesis [106, 107] . in the same line, it was demonstrated that expression of a unique mutated cd95 allele blocks the induction of apoptotic signals, while it fails to prevent nonapoptotic signals such as nf-κb and mapk [106, 107] , whose induction promotes invasiveness in tumor cells [105, 108] . in addition, mutations found in the intracellular cd95-dd exhibit a higher penetrance of alps phenotype features in mutation-bearing relatives compared to extracellular mutations. these results suggest that unlike dd mutations, cd95 mutations localized outside the dd somehow prevent the apoptotic signal but may fail to promote non-apoptotic pathways, which may contribute to disease aggressiveness. in addition to cd95 downregulation or expression of the mutated allele of the receptor, the plasma membrane distribution of cd95 represents an additional pathway for tumor cells to develop resistance to cd95l-expressing immune cells. indeed, the plasma membrane is a heterogeneous lipid bilayer comprising compacted or liquid-ordered domains, called microdomains, lipid rafts, or detergent-resistant microdomains (drms). these domains are described as floating in a more fluid or liquid-disordered 2d lipid bilayer and are enriched in ceramides [109] . it has been elegantly shown that while cd95 is mostly excluded from lipid rafts in activated t-lymphocytes, tcr-dependent reactivation of these cells leads to rapid distribution of the death receptor into lipid rafts [110] . this cd95 compartmentalization contributes to reducing the apoptotic threshold leading to the clonotypic elimination of activated t-lymphocytes through activation of the cd95-mediated apoptotic signal [110] . similarly, the reorganization of cd95 into drms can occur independent from ligand upon addition of certain chemotherapeutic drugs (e.g., rituximab [111] , resveratrol [112, 113] , edelfosine [87, 114, 115] , aplidin [116] , perifosine [115] , cisplatin [117] ). the molecular cascades that underlie this process remain elusive. nevertheless, a growing body of evidence leads us to postulate that alteration of intracellular signaling pathway(s), such as the aforementioned pi3k signal [87, 90] , may change biophysical properties of the plasma membrane, such as membrane fluidity, which in turn may facilitate cd95 clustering into large lipid raft-enriched platforms, favoring disc formation and induction of the apoptotic program [118] . accumulation of cd95 mutations is not the only mechanism by which malignant cells inhibit the extrinsic signaling pathway. posttranslational modifications in the intracellular tail of cd95, such as reversible oxidation or covalent attachment of a palmitic acid, were reported to alter the plasma membrane distribution of cd95 and thereby its subsequent signaling pathway. for instance, s-glutathionylation of mouse cd95 at cysteine 294 promotes clustering of cd95 and its distribution into lipid rafts [119] . this amino acid is conserved in the human cd95 sequence and corresponds to cysteine 304 (or c288 when subtraction of the 16-amino acid signal peptide is taken into consideration [12, 120] ). interestingly, janssen-heininger and colleagues emphasize that death receptor glutathionylation occurs downstream of caspase-8 and caspase-3 activation whose catalytic activity damages the thioltransferase glutaredoxin 1 (grx1), an enzyme implicated in the denitrosylation of proteins [119] . the consequence of grx1 inactivation is the accumulation of glutathionylated cd95, which clusters into lipid rafts, sensitizing cells to the cd95-mediated apoptotic signal. based on these findings, caspase-8 activation occurs prior to aggregation of cd95 and redistribution into lipid rafts, both of which are requisite to form the disc and subsequently activate larger amounts of caspase-8. in agreement with these observations, activation of caspase-8 was reported to occur in a two-step process. that is, an immediate and small amount of activated caspase-8 (<1%) is generated when cd95l interacts with cd95 that orchestrates acid sphingomyelinase (asm) activation, ceramide production, and cd95 clustering, which in turn promote disc formation and the outburst of caspase-8 processing essential to mount the apoptotic signal [121] . s-glutathionylation consists in a bond between a reactive cys-thiol and reduced glutathione (gsh), a tripeptide consisting of glycine, cysteine, and glutamate; its attachment to the protein will alter its structure and function in a manner similar to the addition of a phosphate [122] . s-glutathionylation is not the only posttranslational modification of cd95 on a cysteine. s-nitrosylation of cysteine 199 (corresponding to c183 after subtraction of signal peptide sequence) and 304 (c288) in colon and breast tumor cells also promotes the redistribution of cd95 into drms, the formation of the disc, and the transmission of the apoptotic signal [123] . two reports have brought into light that covalent coupling of a 16-carbon fatty acid (palmitic acid) to cysteine 199 (c183) elicits the redistribution of cd95 into drms, the formation of sds-stable cd95 microaggregates resistant to denaturing and reducing treatments, and the internalization of the receptor [124, 125] . although their order remains to be fine-tuned, these molecular steps play a critical role in the implementation of apoptotic signals. of note, similar to s-nitrosylation, both the aforementioned s-glutathionylation at c304 (c288) and palmitoylation at c199 (c183) promote the partition of cd95 into lipid rafts and enhance the subsequent apoptotic signal. further investigation is required to address whether these posttranslational modifications are redundant and occur simultaneously in dying cells or are elicited in a cell-specific and/or in a microenvironmentspecific manner. understanding the molecular mechanisms controlling these posttranslational modifications would be of great interest in order to identify the mechanism by which tumor cells block them, leading to their resistance to the extrinsic signaling pathway. using a powerful magnetic method to isolate receptor-containing endocytic vesicles, it has been shown that cd95 promptly associates with endosomal and lysosomal markers when incubated with an agonistic anti-cd95 mab [126] . in addition, expression of a cd95 mutant in which the dd-located tyrosine 291 (y275) is changed to phenylalanine does not seem to alter the capacity to bind fadd but compromises cd95l-mediated cd95 internalization occurring through an ap2/clathrin-driven endocytic pathway [126] . more strikingly, expression of the internalization-defective cd95 mutant y291f abrogates the transmission of apoptotic signals, but fails to alter the non-apoptotic signaling pathways (i.e., nf-κb and erk), and even promotes them (fig. 11.3) . these findings provide insight into the presence of a region in the dd, interacting with ap2 and promoting a clathrin-dependent endocytic pathway in a fadd-independent manner. regarding the role of palmitoylation in receptor internalization, the interplay between lipid alteration and the ap2/clathrin-driven internalization of cd95 remains to be elucidated. it has been recently demonstrated that cd95 engagement evokes a rapid and transient ca 2+ signaling, which stimulates the recruitment of protein kinase c-β2 (pkc-β2) from the cytosol to the disc [127] . this kinase transiently brakes disc formation, providing a checkpoint before the irreversible commitment to cell death [128] . these findings raised the following questions: what are the ca 2+ -dependent molecular mechanisms transiently inhibiting disc formation, and do tumor cells use this signal to escape the immune response and/or resist chemotherapy? in 1998, inhibition of caspase activity was shown to sensitize fibroblastic l929 cell line to tnfmediated necrotic cell death [42] . with respect to cd95 signal, tschopp et al. showed that fadd and rip1 participate in the implementation of a non-apoptotic signaling pathway, which leads to a necrotic morphology without chromatin condensation and with loss of plasma membrane integrity [41] . of note, bid cleavage was not observed in this necrotic signal. while fadd plays a crucial role in both apoptotic and necrotic pathways, rip1 recruitment to cd95 occurs independently of this adaptor protein. indeed, yeast two-hybrid experiments showed that rip1 can bind directly to the cd95-dd, while this interaction is lost when a bait corresponding to mutated cd95-dd (replacement of val 238 to asn) is used [129] . in addition, rip3 (ripk3, a member of the rip kinase family) is an indispensable factor for the induction of the necrotic signaling pathway [78] [79] [80] . a growing body of evidence supports the existence of necroptosis (programmed necrosis). in addition, identification of necrostatin, a chemical inhibitor of necroptosis [130] , which specifically inhibits rip1 kinase activity [131] , has accelerated the pace of discovery in this field of cell death. interplays exist between apoptosis and necroptosis; for instance, caspase-8, a potent inhibitor of necroptosis for both cd95 and tnfr1 [132] , plays a critical role in necroptosis by its ability to process and inactivate rip1 and rip3 [133, 134] . at least for tnf signaling, the necrotic signal relies on the activity of cyld, a deubiquitinating enzyme that is also cleaved and inactivated by caspase-8 [135] . overall, these findings suggest that the apoptotic machinery controls the necrotic one. this concept has been recently established in vivo by double-ko experiments [44] [45] [46] 136] . the ko of fadd or caspase-8 is deleterious in mice mainly by the fact that these two apoptotic factors are beneficial in inhibiting a rip1-/rip3-dependent necrotic signal; thus, their loss unleashes the necroptotic program and leads to embryonic lethality. yet, most studies on necroptosis have focused on the tnf signaling pathway, whereas the mechanism by which cd95 can elicit this cell death pathway, and how the switch in this receptor occurs between non-apoptotic, apoptotic, and necroptotic signals remains unclear. importantly, the impact of each cell death on antigen presentation, and on the efficiency of immune response after elimination of infected or transformed cells, remains unclear. oncogenic cytokine? the transmembrane cd95l (cd178/fasl) is present at the surface of activated lymphocytes [64] and nk cells [137] where it orchestrates the elimination of transformed and infected cells. in addition, cd95l is expressed on the surface of neurons [138] , corneal epithelia and endothelia [58, 139] , and sertoli cells [59] to prevent the infiltration of immune cells and thus to prohibit the spread of inflammation in these sensitive organs (i.e., brain, eyes, and testis, respectively), commonly called "immune-privileged" sites. the description of physiological immune privilege was followed by tumor-mediated immune privilege, since two groups reported that the ectopic expression of cd95l by malignant cells participated in the elimination of infiltrating t-lymphocytes and thus could play a role in the establishment of a tumor site whose access was denied to immune cells [140, 141] . however, these observations are controversial since ectopic expression of cd95l in allogenic transplant of β-islets [142, 143] and in tumor cell lines [144] led to a more rapid elimination of these cells than control cells, due to increased infiltration of neutrophils and macrophages endowed with antitumor activity. among the weapons at the disposal of immune cells, transmembrane cd95l contributes to the elimination of pre-tumor cells. therefore, pretumor cells that escape the immunosurveillance will be shaped to develop resistance to cd95, a process termed immunoediting [145] . in other words, imprinting of the immune system on pre-tumor cells will select malignant cells with increased resistance toward the cd95l-induced signal. as previously mentioned, these alterations of the cd95 signal not only block the cd95mediated apoptotic signal but also promote the transmission of non-apoptotic signals by cd95l, which may play a critical role in carcinogenesis [106] [107] [108] 146] . in agreement with this hypothesis, a complete loss of cd95 expression is rarely observed in malignant cells [147] . accumulating evidence indicates that the apoptotic ligand cd95l behaves as a chemoattractant for neutrophils, macrophages [50, 143, 144] , t-lymphocytes [53] , and malignant cells in which the cd95-mediated apoptotic signal is nonproductive [108, 148] . nonetheless, the biological role of cd95l has to be clarified due to the fact that pathophysiologically the ligand is present in at least two forms with different stoichiometries. indeed, cd95l is a transmembrane cytokine whose ectodomain can be cleaved by metalloproteases such as mmp3 [149] , mmp7 [150] , mmp9 [151] , and adam-10 (a disintegrin and metalloproteinase 10) [152, 153] and released as a soluble ligand in the bloodstream. based on the data demonstrating that a hexameric cd95l represents the minimal level of selfassociation required to signal apoptosis [154] and that cleavage by metalloproteases releases an homotrimeric ligand [154, 155] , this soluble ligand has long been considered as an inert ligand competing with its membrane-bound counterpart for cd95 binding, thus acting as an antagonist of the death signal [155, 156] . it has been recently demonstrated that this metalloprotease-cleaved cd95l (cl-cd95l) actively participates in the aggravation of inflammation and autoimmunity in patients affected by systemic lupus erythematosus (sle) by inducing the non-apoptotic nf-κb and pi3k [51, 53] signaling pathways (fig. 11.4) . unlike transmembrane cd95l, induction of the pi3k signaling pathway by its metalloproteasecleaved counterpart occurs through the formation of a complex devoid of fadd and caspase-8 which recruits the src kinase c-yes instead [53, 148] ; this unconventional receptosome was designated motility-inducing signaling complex (misc) [53, 157] (fig. 11.4 ). even though experiments by the authors did not detect any trace of caspase-8 in the misc, this enzyme has been shown to participate in cell migration. the protease activity of caspase-8 can be abolished by its phosphorylation at tyrosine 380 by src kinase [158] . this posttranslational modification was observed in cells stimulated with egf and in colon cancer cells exhibiting constitutive activation of src; from a molecular standpoint, this modification does not alter caspase homodimerization or recruitment in disc [158] . moreover, the egfr-driven phosphorylation of caspase-8 at y380 turns out to be a potent inducer of the pi3k signaling pathway by recruiting the pi3k adaptor p85 alpha subunit [159] . ultimately, caspase-8 phosphorylation triggers cell migration. nonetheless, it is noteworthy that cd95-induced migration and invasion do not appear to require an intact dd (reviewed in [160] ), suggesting that either the caspase-8-dependent mode of cell migration occurs as an alternative signal for death receptors or that it only participates in non-death receptor-induced cell motility. it would be interesting to address this question in the future. to date, it can only be surmised that phosphorylation of caspase-8 at y380 upon egfr stimulation may prime certain cancer cells to become unresponsive to the apoptotic signal triggered by cytotoxic cd95l and meanwhile promote cell migration, an essential event in the course of cancer cell metastasis (fig. 11.4) . it is noteworthy that in a similar manner, a decrease in the plasma membrane level of cd95 or expression of a mutated cd95 allele, as observed in alps patients and malignant cells, inhibits the implementation of the apoptotic signal but does not affect the transmission of non-apoptotic signals, such as nf-κb, mapk, and pi3k [106, 107, 147] , suggesting that these signals may stem from a different domain than cd95-dd or rely on different thresholds to be elicited. in summary, although the cd95/ cd95l interaction can eliminate malignant cells by implementation of the disc or can pro-mote carcinogenesis by sustaining inflammation and/or by inducing metastatic dissemination [50, 51, 53, 108, 147, 148, 161] , the molecular mechanisms underlying the switch between these different signaling pathways remain enigmatic. an important question to be addressed is how the magnitude of cd95 aggregation controls the formation of "death"-vs. "motility"-iscs. addressing these questions will lead to the development of new therapeutic agents with the ability to contain the spread of inflammation or impede carcinogenesis at least in pathologies involving increased soluble cd95l such as cancers (e.g., pancreatic cancer [162] , large granular lymphocytic leukemia, breast cancer [157] , and nk cell lymphoma [163] ) or autoimmune disorders (e.g., rheumatoid arthritis and osteoarthritis [164] , graft-versus-host-disease (gvhd) [165, 166] , or sle [53, 167] ). altogether, these studies support the notion that the death function of cd95 may correspond to its "day job," while the receptor may act as "a night killer" in the presence of cl-cd95l, cd95 triggers misc formation. this complex is devoid of fadd and caspase-8, but, instead, recruits the src kinase c-yes that implements the pi3k signaling pathway. cd95 engagement is also capable of nf-κb and mapk activations through a yet unknown mechanism. right panel: it was reported that procaspase-8 can be phosphorylated by the tyrosine kinase src upon egfr stimulation. this posttranslational modification not only blocks the catalytic activity of caspase-8 but also promotes the recruitment of the p85 subunit of pi3k. we surmise that this caspase-8 phosphorylation may favor the nonapoptotic signals induced by cd95 by fueling inflammation in certain pathophysiological contexts. strikingly, while the soluble form of cd95l generated by mmp7 (cleavage site inside the 113 elr 115 sequence, fig. 11 .5) induces apoptosis [150] , its counterpart processed between serine 126 and leucine 127 does not [51, 53, 155] . to explain this discrepancy, one may speculate that the different quaternary structures of the naturally processed cd95l underlie the implementation of "death"-vs. "non-death"-inducing signaling complexes and downstream signals. in agreement with this notion, soluble cd95l bathed in the bronchoalveolar lavage (bals) of patients suffering from acute respiratory distress syndrome (ards) undergoes oxidation at methionines 224 and 225 ( fig. 11 .5), which enhances the aggregation level of the soluble ligand followed by its cytotoxic activity [168] . the same authors observed that the stalk region of cd95l, corresponding to amino acids 103-136 and encompassing the metalloprotease cleavage sites (fig. 11 .5), participates in the multimerization of cd95l, which accounts for the damage of the lung epithelium in ards [168] . of note, in ards bals, additional oxidation occurs at methionine 121 ( fig. 11 .5), which in turn prevents the processing of cd95l by mmp7, and explains why this cytotoxic ligand keeps its stalk region [168] . nonetheless, preservation of this region in soluble cd95l raises the question that whether an unidentified mmp7-independent cleavage site exists in the juxtamembrane region of cd95l, near the plasma membrane, or the ligand detected in ards patients corresponds to the full-length cd95l embedded in exosomes [169, 170] . indeed, this peculiar exosome-bound cd95l can be expressed by human prostate cancer cells (i.e., lncap) and evokes apoptosis in activated t-lymphocytes [171] . overall, these findings emphasize that it will be of great interest in the future to finely characterize the quaternary structure of the naturally processed cd95l from the sera of patients affected by cancers or chronic/acute inflammatory disorders, to better understand the molecular mechanisms implemented by this ligand and thus predict its subsequent biological functions. apoptosis is a fundamental process contributing to tissue homeostasis, immune response, and development. cd95, also called fas, is a member of the tumor necrosis factor receptor (tnfr) superfamily. its ligand, cd95l, was initially detected at the plasma membrane of activated t-lymphocytes and natural killer (nk) cells where it contributes to the elimination of transformed and infected cells. given its implication in immune homeostasis and immune surveillance combined with the fact that various lineages of malignant cells exhibit lossof-function mutations, cd95 was initially classified as a tumor suppressor gene. nonetheless, in different pathophysiological contexts, this receptor is able to transmit non-apoptotic signals and promote inflammation and carcinogenesis. although the different non-apoptotic signaling pathways (nf-κb, mapk, and pi3k) triggered by cd95 are known, the initial molecular events leading to these signals, the mechanisms by which the receptor switches from an apoptotic function to an inflammatory role, and, more importantly, the biological functions of these signals remain elusive. apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics induction of tnf receptor i-mediated apoptosis via two sequential signaling complexes apoptosis in alzheimer's diseasean update apoptosis in parkinson's disease: signals for neuronal degradation human ice/ced-3 protease nomenclature the biochemistry of apoptosis a novel protein that interacts with the death domain of fas/apo1 contains a sequence motif related to the death domain fadd, a novel death domain-containing protein, interacts with the death domain of fas and initiates apoptosis the tnf receptor 1-associated protein tradd signals cell death and nf-kappa b activation cytotoxicity-dependent apo-1 (fas/cd95)-associated proteins form a deathinducing signaling complex (disc) with the receptor cytochrome c and datp-dependent formation of apaf-1/caspase-9 complex initiates an apoptotic protease cascade the polypeptide encoded by the cdna for human cell surface antigen fas can mediate apoptosis molecular cloning and expression of the human 55 kd tumor necrosis factor receptor the receptor for the cytotoxic ligand trail trail-r2: a novel apoptosismediating receptor for trail identification and functional characterization of dr6, a novel death domain-containing tnf receptor fas transduces activation signals in normal human t lymphocytes divergent signalling via apo-1/fas and the tnf receptor, two homologous molecules involved in physiological cell death the tnf receptor superfamily of cellular and viral proteins: activation, costimulation, and death the tnf and tnf receptor superfamilies: integrating mammalian biology the molecular architecture of the tnf superfamily precise mapping of the cd95 pre-ligand assembly domain identification and characterization of a ligand-independent oligomerization domain in the extracellular region of the cd95 death receptor fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations a novel protein domain required for apoptosis. mutational analysis of human fas antigen a novel domain within the 55 kd tnf receptor signals cell death the growth factor progranulin binds to tnf receptors and is therapeutic against inflammatory arthritis in mice signal transduction by tumor necrosis factor receptors a domain in tnf receptors that mediates ligand-independent receptor assembly and signaling human tumour necrosis factor: precursor structure, expression and homology to lymphotoxin a metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells cloning of a disintegrin metalloproteinase that processes precursor tumour-necrosis factor-alpha the transmembrane form of tumor necrosis factor is the prime activating ligand of the 80 kda tumor necrosis factor receptor nf-kappab antiapoptosis: induction of traf1 and traf2 and c-iap1 and c-iap2 to suppress caspase-8 activation recruitment of the linear ubiquitin chain assembly complex stabilizes the tnf-r1 signaling complex and is required for tnf-mediated gene induction a ubiquitin ligase complex assembles linear polyubiquitin chains linear ubiquitination prevents inflammation and regulates immune signalling rapid turnover of c-flipshort is determined by its unique c-terminal tail nf-kappab suppression by the deubiquitinating enzyme cezanne: a novel negative feedback loop in pro-inflammatory signaling ripk-dependent necrosis and its regulation by caspases: a mystery in five acts fas triggers an alternative, caspase-8-independent cell death pathway using the kinase rip as effector molecule inhibition of caspases increases the sensitivity of l929 cells to necrosis mediated by tumor necrosis factor phosphorylation-driven assembly of the rip1-rip3 complex regulates programmed necrosis and virus-induced inflammation rip3 mediates the embryonic lethality of caspase-8-deficient mice catalytic activity of the caspase-8-flip(l) complex inhibits ripk3-dependent necrosis fadd prevents rip3-mediated epithelial cell necrosis and chronic intestinal inflammation lasker clinical medical research award tnf defined as a therapeutic target for rheumatoid arthritis and other autoimmune diseases fas engagement induces neurite growth through erk activation and p35 upregulation fas engagement accelerates liver regeneration after partial hepatectomy cd95-ligand on peripheral myeloid cells activates syk kinase to trigger their recruitment to the inflammatory site membrane-bound fas ligand only is essential for fas-induced apoptosis a novel juxtamembrane domain in tumor necrosis factor receptor superfamily molecules activates rac1 and controls neurite growth the naturally processed cd95l elicits a c-yes/calcium/pi3k-driven cell migration pathway cd95-mediated cell signaling in cancer: mutations and post-translational modulations monoclonal antibody-mediated tumor regression by induction of apoptosis molecular cloning and expression of the fas ligand, a novel member of the tumor necrosis factor family involvement of fas ligand and fasmediated pathway in the cytotoxicity of human natural killer cells fas ligand-induced apoptosis as a mechanism of immune privilege a role for cd95 ligand in preventing graft rejection lymphoproliferation disorder in mice explained by defects in fas antigen that mediates apoptosis aberrant transcription caused by the insertion of an early transposable element in an intron of the fas antigen gene of lpr mice the defect in fas mrna expression in mrl/lpr mice is associated with insertion of the retrotransposon, etn autoimmunity in mice bearing lprcg: a novel mutant gene generalized lymphoproliferative disease in mice, caused by a point mutation in the fas ligand the many roles of fas receptor signaling in the immune system structure of the human apo-1 gene nmr structure and mutagenesis of the fas (apo-1/cd95) death domain the fas-fadd death domain complex structure unravels signalling by receptor clustering solution nmr investigation of the cd95/fadd homotypic death domain complex suggests lack of engagement of the cd95 c terminus the fas-fadd death domain complex structure reveals the basis of disc assembly and disease mutations homotypic fadd interactions through a conserved rxdll motif are required for death receptor-induced apoptosis inhibition of death receptor signals by cellular flip viral flice-inhibitory proteins (flips) prevent apoptosis induced by death receptors ped/pea-15: an anti-apoptotic molecule that regulates fas/tnfr1-induced apoptosis two cd95 (apo-1/fas) signaling pathways two cd95 tumor classes with different sensitivities to antitumor drugs cd95 engagement mediates actin-independent and -dependent apoptotic signals signal transduction mediated by bid, a pro-death bcl-2 family proteins, connects the death receptor and mitochondria apoptosis pathways bid-deficient mice are resistant to fas-induced hepatocellular apoptosis xiap discriminates between type i and type ii fas-induced apoptosis the c-iap-1 and c-iap-2 proteins are direct inhibitors of specific caspases x-linked iap is a direct inhibitor of cell-death proteases iaps block apoptotic events induced by caspase-8 and cytochrome c by direct inhibition of distinct caspases ubiquitinprotein ligase activity of x-linked inhibitor of apoptosis protein promotes proteasomal degradation of caspase-3 and enhances its anti-apoptotic effect in fas-induced cell death smac, a mitochondrial protein that promotes cytochrome c-dependent caspase activation by eliminating iap inhibition bcl-2 and bcl-xl inhibit cd95-mediated apoptosis by preventing mitochondrial release of smac/diablo and subsequent inactivation of x-linked inhibitor-of-apoptosis protein localization of fas/cd95 into the lipid rafts on down-modulation of the phosphatidylinositol 3-kinase signaling pathway pten loss promotes mitochondrially dependent type ii fas-induced apoptosis via pea-15 phosphatidylinositol 3′-kinase blocks cd95 aggregation and caspase-8 cleavage at the death-inducing signaling complex by modulating lateral diffusion of cd95 actin-independent exclusion of cd95 by pi3k/akt signalling: implications for apoptosis phosphorylation of pea-15 switches its binding specificity from erk/mapk to fadd protein kinase b/akt binds and phosphorylates ped/pea-15, stabilizing its antiapoptotic action bcl-2 and fas/apo-1 regulate distinct pathways to lymphocyte apoptosis bcl-2 protects from lethal hepatic apoptosis induced by an anti-fas antibody in mice a bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-fas antibody injection fas gene mutations in the canale-smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity dominant interfering fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome mutations in fas associated with human lymphoproliferative syndrome and autoimmunity chronic lymphadenopathy simulating malignant lymphoma lymphoproliferative syndrome with autoimmunity: a possible genetic basis for dominant expression of the clinical manifestations the development of lymphomas in families with autoimmune lymphoproliferative syndrome with germline fas mutations and defective lymphocyte apoptosis flice-inhibitory protein is a key regulator of germinal center b cell apoptosis primary diffuse large b-cell lymphomas of the central nervous system are targeted by aberrant somatic hypermutation the origin of cd95-gene mutations in b-cell lymphoma does cd95 have tumor promoting activities? the relevance of nf-kappab for cd95 signaling in tumor cells induction of apoptosis and activation of nf-kappab by cd95 require different signalling thresholds cd95 ligand induces motility and invasiveness of apoptosis-resistant tumor cells cd95 signaling via ceramide-rich membrane rafts ligand-independent redistribution of fas (cd95) into lipid rafts mediates clonotypic t cell death fas receptor clustering and involvement of the death receptor pathway in rituximabmediated apoptosis with concomitant sensitization of lymphoma b cells to fas-induced apoptosis resveratrol-induced apoptosis is associated with fas redistribution in the rafts and the formation of a death-inducing signaling complex in colon cancer cells redistribution of cd95, dr4 and dr5 in rafts accounts for the synergistic toxicity of resveratrol and death receptor ligands in colon carcinoma cells intracellular triggering of fas aggregation and recruitment of apoptotic molecules into fas-enriched rafts in selective tumor cell apoptosis edelfosine and perifosine induce selective apoptosis in multiple myeloma by recruitment of death receptors and downstream signaling molecules into lipid rafts cytoskeleton-mediated death receptor and ligand concentration in lipid rafts forms apoptosis-promoting clusters in cancer chemotherapy cisplatin-induced cd95 redistribution into membrane lipid rafts of ht29 human colon cancer cells redistribution of cd95 into the lipid rafts to treat cancer cells? redox amplification of apoptosis by caspase-dependent cleavage of glutaredoxin 1 and s-glutathionylation of fas purification and molecular cloning of the apo-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. sequence identity with the fas antigen ceramide-mediated clustering is required for cd95-disc formation s-glutathionylation uncouples enos and regulates its cellular and vascular function s-nitrosylation of the death receptor fas promotes fas ligand-mediated apoptosis in cancer cells palmitoylation is required for efficient fas cell death signaling palmitoylation of cd95 facilitates formation of sds-stable receptor aggregates that initiate apoptosis signaling the role of receptor internalization in cd95 signaling cd95 triggers orai1-mediated localized ca2+ entry, regulates recruitment of protein kinase c (pkc) beta2, and prevents death-inducing signaling complex formation the cd95 signaling pathway: to not die and fly rip: a novel protein containing a death domain that interacts with fas/apo-1 (cd95) in yeast and causes cell death chemical inhibitor of nonapoptotic cell death with therapeutic potential for ischemic brain injury identification of rip1 kinase as a specific cellular target of necrostatins the roles of fadd in extrinsic apoptosis and necroptosis cleavage of the death domain kinase rip by caspase-8 prompts tnf-induced apoptosis cleavage of rip3 inactivates its caspase-independent apoptosis pathway by removal of kinase domain caspase 8 inhibits programmed necrosis by processing cyld programmed cell death: apoptosis meets necrosis fas involvement in cytotoxicity mediated by human nk cells fas ligand expression by astrocytoma in vivo: maintaining immune privilege in the brain? cd95 ligand (fasl)-induced apoptosis is necessary for corneal allograft survival melanoma cell expression of fas(apo-1/cd95) ligand: implications for tumor immune escape the fas counterattack: fasmediated t cell killing by colon cancer cells expressing fas ligand transgenic expression of cd95 ligand on islet beta cells induces a granulocytic infiltration but does not confer immune privilege upon islet allografts fas ligand expression in islets of langerhans does not confer immune privilege and instead targets them for rapid destruction regulation of the proinflammatory effects of fas ligand (cd95l) cancer immunosurveillance, immunoediting and inflammation: independent or interdependent processes? dominant-negative fas mutation is reversed by down-expression of c-flip cd95 promotes tumour growth yes and pi3k bind cd95 to signal invasion of glioblastoma stromelysin-1 (mmp-3) in synovial fluid of patients with rheumatoid arthritis has potential to cleave membrane bound fas ligand identification of novel matrix metalloproteinase-7 (matrilysin) cleavage sites in murine and human fas ligand matrix metalloproteinase-9 regulates tnf-alpha and fasl expression in neuronal, glial cells and its absence extends life in a transgenic mouse model of amyotrophic lateral sclerosis the fas ligand intracellular domain is released by adam10 and sppl2a cleavage in t-cells adam10 regulates fasl cell surface expression and modulates fasl-induced cytotoxicity and activation-induced cell death two adjacent trimeric fas ligands are required for fas signaling and formation of a death-inducing signaling complex conversion of membranebound fas(cd95) ligand to its soluble form is associated with downregulation of its proapoptotic activity and loss of liver toxicity membrane fas ligand kills human peripheral blood t lymphocytes, and soluble fas ligand blocks the killing cd95l cell surface cleavage triggers a pro-metastatic signaling pathway in triple negative breast cancer src kinase phosphorylates caspase-8 on tyr380: a novel mechanism of apoptosis suppression caspase-8 interacts with the p85 subunit of phosphatidylinositol 3-kinase to regulate cell adhesion and motility how cd95 stimulates invasion fas and nf-kappab signalling modulate dependence of lung cancers on mutant egfr production and pro-apoptotic activity of soluble cd95 ligand in pancreatic carcinoma fas ligand in human serum soluble fas ligand in the joints of patients with rheumatoid arthritis and osteoarthritis levels of soluble fasl and fasl gene expression during the development of graft-versushost disease in dlt-treated patients increased soluble fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease serum levels of soluble fas ligand in patients with silicosis the biological activity of fasl in human and mouse lungs is determined by the structure of its stalk region diacylglycerol kinase alpha regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of fas ligand-containing exosomes in t lymphocytes modulation of the immune response using dendritic cell-derived exosomes tumor exosomes expressing fas ligand mediate cd8+ t-cell apoptosis key: cord-023389-ilrp8vb7 authors: wefer, j.; harris, r. a.; lobell, a. title: protective dna vaccination against mog(91‐108)‐induced experimental autoimmune encephalomyelitis involves induction of ifnβ date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423j.x sha: doc_id: 23389 cord_uid: ilrp8vb7 dna vaccine coding for the encephalitogenic peptide mog(91‐108) protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t‐cell immunoglobulin‐ and mucin‐domain‐containing molecule (tim‐3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen‐specific lymphocyte population upregulating ifnγ upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigen‐specific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen‐specific upregulation of ifnβ upon recall stimulation and (4) reduced il‐12rβ2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnβ. we are currently investigating the cellular mechanisms behind this ifnβ‐mediated protection. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-018764-02l423mk authors: clark, ian a.; griffiths, michael j. title: the molecular basis of paediatric malarial disease date: 2007 journal: pediatric infectious diseases revisited doi: 10.1007/978-3-7643-8099-1_9 sha: doc_id: 18764 cord_uid: 02l423mk severe falciparum malaria is an acute systemic disease that can affect multiple organs, including those in which few parasites are found. the acute disease bears many similarities both clinically and, potentially, mechanistically, to the systemic diseases caused by bacteria, rickettsia, and viruses. traditionally the morbidity and mortality associated with severe malarial disease has been explained in terms of mechanical obstruction to vascular flow by adherence to endothelium (termed sequestration) of erythrocytes containing mature-stage parasites. however, over the past few decades an alternative ‘cytokine theory of disease’ has also evolved, where malarial pathology is explained in terms of a balance between the proand anti-inflammatory cytokines. the final common pathway for this pro-inflammatory imbalance is believed to be a limitation in the supply and mitochondrial utilisation of energy to cells. different patterns of ensuing energy depletion (both temporal and spatial) throughout the cells in the body present as different clinical syndromes. this chapter draws attention to the over-arching position that inflammatory cytokines are beginning to occupy in the pathogenesis of acute malaria and other acute infections. the influence of inflammatory cytokines on cellular function offers a molecular framework to explain the multiple clinical syndromes that are observed during acute malarial illness, and provides a fresh avenue of investigation for adjunct therapies to ameliorate the malarial disease process. although many species of malarial parasite exist, only plasmodium falciparum, vivax, ovale, and malariae are classically associated with human infection. the former two species are most frequently associated with malarial disease in humans, with severe malarial disease almost exclusively associated with p. falciparum infection. falciparum malaria is responsible for considerable morbidity (300-500 million annual clinical cases) and death across the globe, with a particular burden of mortality among children in sub-saharan africa. infection with p. vivax is rarely fatal, but is associated with considerable morbidity outside the african continent. it should also be recalled that malaria causes social and economic disruption on a uniquely large scale [1] . severe adult malaria is a clinical syndrome originally classified using 10 defining and 5 supportive (often overlapping) clinical features unified by the presence of asexual malarial parasites in the peripheral blood smear [2] . based on observations of children in coastal kenya, paediatric severe malaria has similarly been distilled into three main (again often overlapping) clinical syndromes, anaemia, respiratory distress (an indicator of an underlying metabolic acidosis) and impairment of consciousness [3] . these clinical syndromes are discussed below. in the review mentioned above [3] , the authors' judicious use of the term impaired consciousness, rather than cerebral malaria (cm), promoted the useful concept that the neurological features (and in-turn the underlying mechanisms) associated with severe malaria are not necessarily unique to malarial disease. indeed, over 60 years ago, it was noted that the clinical features of malaria can resemble those exhibited in patients with fulminant bacterial or viral infections [4] . severe malaria has been intensively studied, and there appears to be a complex interplay between host infection and disease. this is highlighted by the different clinical manifestations of severe malaria exhibited by children and adults. these differences are undoubtedly, in part, a function of patient age. however, age is just one of a series of interacting factors, e.g. geographical region, level of malaria transmission, degree of previous malaria exposure, length of illness prior to treatment and host immunity that may influence the clinical presentation of severe malaria. this variation in clinical presentation has been mirrored by a similar multitude of proposals regarding the functional mechanisms underlying pathogenesis of severe malaria. one concept of pathogenesis consistently articulated has been the 'mechanical theory'. historically, this theory was developed from two fundamental differences between p. falciparum and p. vivax infection. firstly, erythrocytes parasitised with p. vivax do not sequester. secondly, death following p. vivax infection is rare. consequently, pathogenesis is believed to be due to obstruction of micro-vascular flow by erythrocytes containing mature-stage falciparum parasites adhering to the endothelium (termed sequestration). more recently the 'cytokine theory of disease' has also gained credence. this theory can be applied to disease following both falciparum and vivax infection. the lower mortality associated with p. vivax being explained by a relatively milder degree of pro-inflammatory imbalance during the host's response to p. vivax infection. the main theme of this chapter is to examine the increased understanding of the functions of inflammatory cytokines gained over the past 15 years, and explore how these insights are changing attitudes in malarial disease research. we also discuss how two theories (mechanical and cytokine) can, as proposed first in a recognisable form at least 65 years ago [5] , be complementary. the majority of the clinical cases of malaria occur in sub-saharan africa. nevertheless, malaria also accounts for considerable morbidity and mortality in other continents particularly south east asia [6] . in malaria-endemic regions (e.g. sub-saharan africa), where the resident population have continuous exposure to malarial parasites, most of the severe cases are seen in children [7] . in hypoendemic regions (e.g. south east asia), where parasite exposure is more intermittent, cases of severe malaria are also common in adults (tab. 1). clinical features associated with malaria mortality vary between children and adults, but acidosis and coma are associated with malarial mortality in both populations [7, 8] . acute renal failure (arf) and pulmonary oedema, a marker for adult respiratory distress syndrome (ards), are almost exclusively reported among adults [9, 10] , whereas mortality associated with hypoglycaemia is frequently reported among children [11] . why malarial disease displays such age-related differences in pathophysiology is unclear. however, these differences are not exclusive to malaria. ards, which is more frequently observed as a complication of trauma in adults compared with children [12] , is believed to reflect an exaggerated pro-inflammatory response within the lung [9] . a possible lead for future studies on these age-related differences in malaria is suggested by a report of peritoneal macrophages collected from healthy adults producing much less interleukin (il)-10 (an anti-inflammatory cytokine), but the same levels of pro-inflammatory cytokine, than those from healthy children, giving adults a much higher pro-inflammatory status [13, 14] . the mechanism of malarial arf pathogenesis is postulated to be multifactorial, involving mechanical, haemodynamic, and immunological factors [15] . the observation that arf is more frequently observed as a complication of trauma in adults than children [12] suggests that age-related variations in cytokine response may again influence pathogenesis. hypoglycaemia is regarded as a more frequent complication of sepsis in paediatric populations compared with adults [16] . hypoglycaemia in children may, in part, be associated with a higher basal metabolic rate, and lower glycolytic [17] and gluconeogenic substrate reserves compared to adults [18] . however, these substrates are not always limiting during acute paediatric malaria, suggesting functional impairments of glucose metabolism may also occur [19] . such functional impairments may, in part, be influenced by increases in inflammatory cytokines as the infection progresses. once the malarial parasite was identified as the cause of disease, it quickly became apparent that illness and death were linked with parasite invasion into bloodstream and subsequent parasite growth within (and release from) the erythrocytes. by the start of the 20th century, two major theories, capillary blockage and toxicity of the parasites themselves, had been proposed to explain morbidity and mortality. thus, the study of malarial disease is not a settled story requiring regular updates, but one containing, from its beginning, an unresolved tension. vascular occlusion and malarial toxin (nowadays vascular occlusion and inflammatory cytokines) have been alternative approaches to understanding malarial disease as a whole, as well as the coma, for over a century, and the two have often been discussed side by side [5, 20, 21] . the presence of hyperlactataemia, hypoglycaemia, and metabolic acidosis, all three consistent with a patient being forced to rely on anaerobic glycolysis for energy production, have provided a consensus that hypoxia is central to disease pathogenesis in falciparum malaria. as sum-marised below, the modern literature offers two main theories for cellular hypoxia during infection; insufficient oxygen delivery to cells and impaired oxygen utilization within the cells. both mechanisms may be governed by the host inflammatory cytokine response to infection. this chapter focuses on how an increased understanding of the molecular functions of cytokines during disease demonstrates a closer alignment between the pathogenesis of falciparum infection and other systemic infectious diseases. one hundred and twenty years ago, golgi (of the golgi apparatus [22] ), noted onset of malarial fever and illness at a predictable short interval after the regular shower of new parasites were released from bursting red cells. the nature of the putative toxin so released was much discussed in the first decade of the 20th century [23] . it was assumed to be directly toxic, in the manner of tetanus toxin. the proposal that malarial products were not harmful in themselves, but only through causing the infected host to harm itself through generating toxic amounts of molecules (pro-inflammatory cytokines) that, in lower concentrations, inhibit growth of malarial parasites did not arise until 1981 [24] . indeed, acceptance of the broad applicability of this concept to infectious disease in general is now sufficient for its evolution to be a subject for research [25] . tumour necrosis factor (tnf) is regarded as a major player, malaria being the first disease in which it was proposed to cause systemic illness and pathology [24] . multiple tnf promoter polymorphisms have since been independently associated with severe malaria across several geographical populations [26] . a longitudinal study in burkino faso has also demonstrated several tnf promoter polymorphisms associated with the regulation of host-parasite density [27] . the tnf concept has since begun to dominate the sepsis literature [28] , and the virulence of different strains of influenza, a disease that is a standard clinical misdiagnosis for imported malaria, has recently been expressed in terms of their capacity to induce tnf [29] . the critical role of tnf in both malaria and influenza pathogenesis is consistent with the clinical similarities between the diseases. indeed, tnf infusions in tumour patients produce side effects mimicking both diseases [30] , as discussed below. although tnf is the prototype pro-inflammatory cytokine linked with severe malaria, other cytokines (and mediators) including interferon (ifn)[31] , its corresponding receptors ifn-receptor-1 [32] and ifn-receptor-1 [33] , il-1 [34] , il-4 [35] and il-10 [36] have all be identified through genetic association analysis to be linked with their potential regulation of malarial disease severity. all the above cytokines typically act as homeostatic agents, but can cause pathology if produced excessively. when this happens they also induce a late-onset, but long-acting cytokine termed the high mobility group box 1 (hmgb1) protein, which prolongs and amplifies inflammation [37, 38] . this molecule, normally in the cellular nucleus and previously known only for several physiological functions, now shows great promise as a therapeutic target in sepsis, in that countering it after the onset of illness protects well in experimental sepsis [39, 40] . it accumulates, in proportion to degree of illness, in serum from african children infected with falciparum malaria [41] . once neutralising anti-tnf antibodies became available for human use, they were tested for efficacy against malarial disease. unfortunately, a central tenet of the cytokine concept of infectious disease (that the proinflammatory cytokines that cause disease are the same mediators that, in lower concentrations, are responsible for the innate immunity that controls parasite growth) was not taken into consideration. tnf has been shown to inhibit a mouse malarial parasite in vivo [42] , and p. falciparum in vitro, provided white cells to generate the next down-stream mediator, possibly nitric oxide (no) [43] , were present [44] . this is consistent with findings in human subjects [45] . thus, it is not surprising that anti-tnf antibody, by removing inhibitory pressure from the pathogen, can enhance the disease in falciparum malaria [46] , as shown 5 years earlier in human sepsis [47] . cytokines as a disease mechanism extends beyond malaria as noted above, the idea that excessive production of inflammatory cytokines underlies the pathology of illness is used widely, from malaria across a range of conditions, infectious or otherwise. as reviewed recently [48] , this now includes the illnesses caused by rickettsias, protozoa other than malaria, and viruses. increased circulating levels of these cytokines have been detected in the serum very soon after onset of illness in virtually all those infectious diseases in which they have been sought. some cytokine increased, and consequences are shown in table 2 . when rtnf was under trial in volunteers as an anti-tumour agent [49, 50] nearly 20 years ago, virtually all of the symptoms and signs they share were reproduced as side effects. this includes headache, fever and rigours, nausea and vomiting, diarrhoea, anorexia, myalgia, thrombocytopaenia, immunosuppression, and central nervous system manifestations, all of which have been shown to be caused by a mechanism involving inflammatory cytokines. the rate, timing and intensity of cytokine release vary in different disease states, and provide them with somewhat individual clinical pictures, but the fundamentals remain. nevertheless, the clinical patterns generated are remarkably close, in that, at least in some populations, clinical features cannot predict a diagnosis of malaria from other causes of fever [51] . mature erythrocytic forms of p. falciparum are not seen in peripheral blood smears, and cause the erythrocytes they inhabit to adhere to the walls of venules and capillaries. from this observation arose the widely held view that much of the pathology following malarial infection is explained through parasite sequestration causing impairment of microvascular flow. sequestration certainly occurs, since the life cycle dictates this. however, whether the temporal and anatomical patterns of sequestration are the same in both individuals with fatal disease and in parasite tolerant individuals has not been ascertained. consequently, whether sequestration is the principal instigator of local pathology, or whether sequestration is an associated feature of all malarial infections with local pathology determined by other factors in the host response to the infection, e.g. a local imbalance of inflammatory mediators, has not been fully elucidated. erythrocyte cyto-adherence (irrespective of whether this adhesive process is directly or indirectly due to parasite sequestration) has repeatedly been shown to be mediated through a series of host-derived ligands. cd36 and thrombospondin were the first described endothelial receptors that bound infected red blood cells (rbcs) [52, 53] , with most studied wild parasite isolates demonstrating adhesion to cd36 [54] . more recently, it has been shown that p. falciparum also interacts with other host adhesion receptors, i.e. intercellular adhesion molecule-1 (icam-1 cd54), vascular cell adhesion molecule-1 (vcam-1 cd106) and e-selectin [55, 56] . certain adhesive phenotypes, such as rosetting (the spontaneous tethering of infected and non-infected rbcs) and clumping (tethering of infected rbcs through platelets) have been preferentially associated with severe malarial disease [57, 58] . cd36 is involved in both mechanisms of adhesion, and a non-sense mutation in the gene encoding for cd36 has also been associated with protection from severe malaria [59] . polymorphisms in the gene encoding icam-1 have also been associated with susceptibility to severe disease [27] . furthermore, icam-1, together with vcam and e-selectin, are up-regulated by tnf, with circulating levels of these ligands shown to be increased in severe malaria compared to uncomplicated infection [60] . sequestration during falciparum malaria appears to be concentrated in the brain and placenta. there is some evidence to suggest that the propensity of inflammatory cytokines to up-regulate cell adhesion molecules, secondary to local variation in the density of thrombomodulin, is potentially higher in the microvasculature of the brain and placenta compared to other tissues. as reviewed [61] , tnf and il-1 increase tissue factor expression on endothelial cells, thereby initiating pathways that generate thrombin [62] . when thrombin binds to thrombomodulin on the endothelial cell surface, protein c is activated, which in turn can lead to further downstream activation of the coagulation cascade. therefore vasculature with lowest thrombomodulin densities on the endothelial cell surface (brain least, placenta next least, and other organs more [63] ) will have more unbound thrombin available for its other functions on activated endothelium. these other functions include up-regulation of adhesion molecules such as selectins, icam-1, vcam-1 [64] and monocyte chemotactic protein-1 (mcp-1) [65] . therefore, up-regulation of adhesion molecules within the cerebral vessels may occur as a local endothelial response to systemic inflammation and may not necessarily be precipitated by parasite sequestration. anaemia is another obvious way in which too little oxygen reaches cells, and thus their mitochondria [66] . as recently reviewed [67] , critical illness associated with an inflammatory response invariably causes multifactorial anaemia. obviously a high parasite load in malaria indicates that the infected rbcs will soon burst when the next generation of erythrocytic forms escapes, but anaemia does not correlate with parasitaemia, and sometimes is extreme when very few parasites are, or have been, present. the severe anaemia in transgenic mice expressing human tnf [68] incriminates the inflammatory response itself, so anaemia and mitochondrial dysfunction (see mitochondrial dysfunction section below), both consequences of systemic inflammation, can be expected to coexist, and both contribute to total energy depletion. the lifespan of an rbc is, in part, limited by how long it can remain flexible enough to squeeze through fenestrations in specialised vessels in the red pulp of the spleen, and thus avoid phagocytosis by adjacent macrophages. normally this loss is balanced by erythropoiesis, and haematocrit remains normal. if rbcs develop a premature loss of deformability they are removed from the circulation earlier. this loss of deformability happens to both infected and non-infected red cells in malaria, whether caused by p. vivax or p. falciparum. under physiological conditions, erythrocytes (and other cells) control the passive influx of osmotic active solutes (especially na + ) via an active, energy-dependent elimination of these solutes using na + /k + -atpase. this prevents intracellular accumulation of osmotically active solutes, preventing a subsequent influx of water, cell swelling and loss of cell integrity. during human [69] and monkey [70] malaria infection, intracellular na + accumulates within erythrocytes (both parasitised and non-parasitised) implying that this na + /k + pump is impaired during the disease process. parallel changes in the ionic content of erythrocytes have been documented in a sepsis model of infection [71] . similarly, reduction in erythrocyte deformability was shown to be associated with increased no, an inhibitor of this membrane pump [72] , in another sepsis model [73] . since inhibition of the na + /k + pump in vitro correlates with both reduced red cell deformability and decreased red cell filterability [74] , any factor that inhibits the na + /k + pump could potentially worsen anaemia. identification of inducible no synthase (inos) activity, as one factor influencing red cell deformability, suggests that a pro-inflammatory milieu [75] may again govern the reduction in red cell deformability observed during malaria infection. originally observed in uraemic patients, poor red cell deformability was recognised in a small pilot study of malaria patients in 1985 [76] . it was reported soon afterwards in sepsis [77, 78] , and subsequently studied in falciparum malaria with a view to understanding both circulatory obstruction [79] and anaemia [80] . it seems clear that a short life (poor deformability), and a slow replacement rate (dyserythropoiesis, below) can combine to cause severe anaemia in various diseases, particularly in chronic infections such as malaria. when red cells have a shortened lifespan, e.g. secondary to reduced erythrocyte deformability, replacement by new recruits is vital to avoid anaemia. unfortunately, the same inflammatory cytokines that shorten lifespan also retard replacement. some years ago researchers began to stress the contribution of bone marrow dyserythropoiesis to the anaemia of falciparum malaria [81, 82] . a group in oxford [83] , seeking an explanation for this dyserythropoiesis through an electron microscopy study of bone marrow, observed sequestration of parasitised red cells and argued that this caused the bone marrow dysfunction in falciparum malaria by restricting blood flow and thus inducing hypoxic changes. this idea proved inadequate, however, when this same group subsequently reported dyserythropoiesis and erythrophagocytosis in vivax malaria, in which parasitised red cells do not sequester [84] . some time ago an undefined product in macrophage supernatants [85] , later identified as tnf [86] , was found to inhibit the growth and differentiation of erythroid progenitor cells. when rtnf became available, the dyserythropoiesis and erythrophagocytosis seen in terminal plasmodium vinckei-infected mice was reproduced by giving a single injection early in the course of the infection [87] . phagocytosis of erythroblasts in bone marrow, a phenomenon also reported by wickramasinghe et al [83, 84] in human malaria, also occurred. decreased erythropoiesis was subsequently reported in mice receiving continuous tnf infusions via implanted osmotic pumps, and mice expressing high levels of human tnf have been shown to become markedly anaemic during malaria infections [68] , even though parasite numbers, and therefore red cell loss post-schizogony, are considerably reduced. the past decade has seen an expansion of this line of enquiry into human malaria, and also the number of cytokines, both pro-inflammatory and anti-inflammatory [88, 89] in absolute amounts and ratios [90, 91] , that have been investigated in this context. investigations have been extended to include other pro-inflammatory cytokines, such as il-12 [92] and fasl [93] , and examined the role in anaemia of the persistence of cytokine production during malaria infection [94] . another inflammatory cytokine, macrophage inhibitory factor (mif) that is increased in malaria, and induced by tnf, has been shown to cause dyserythropoiesis in in vitro studies on bone marrow cells [95, 96] . thus, inflammatory cytokines generated during malaria are a major determinant of the degree to which anaemia influences the amount of oxygen that reaches tissues in malaria. mitochondria are vital to energy (atp) generation through cellular respiration. cellular respiration requires oxygen and pyruvate, as well as multiple cofactors and active transport molecules. within the matrix of the mitochondrion organelle, pyruvate is catabolised via the krebs cycle and oxidative phosphorylation (involving nadh and fadh2) to generate atp. when this series of reactions are 100% efficient (unlikely in vivo), 1 molecule of glucose generates 2 molecules of pyruvate, which are further catabolised to water and carbon dioxide with the concomitant generation of 36 molecules of atp. in comparison, during anaerobic glucose catabolism, pyruvate is converted to lactate with the concomitant generation of 2 molecules of atp, a process that also facilitates regeneration of nadh and fadh2. evidence is accumulating that inflammatory cytokines, as released in malaria, sepsis, and viral diseases, induce mitochondrial dysfunction and dysregulate cellular respiration, resulting in the incomplete catabolism of pyruvate. the process, termed 'cytopathic hypoxia' [97] , mimics cellular hypoxia, in that it results in the incomplete catabolism of pyruvate and accumulation of lactate. awareness of this mechanism began with oxygen tension being shown to be increased in septic rats [98] and patients [99] . a cytokine model of mitochondrial dysfunction has since been developed in which impairment of cellular respiration occurs following induction of sepsis (or exposure to pro-inflammatory cytokines), despite sufficient oxygen supply [97, 100, 101] . more recently, impairment of enzyme activity associated with the mitochondrial complexes has been demonstrated in muscle biopsies retrieved from rodent models of sepsis [102] and septic patients [103, 104] . the observation that the inflammatory cytokines implicated in mitochondrial shutdown are prominent in both sepsis and malaria [105, 106] supports such organelle dysfunction being equally plausible in malaria. researchers are also becoming aware that, beyond energy production, mitochondria also play a vital role in cell homeostasis through generation and detoxification of reactive oxygen species [107] . the accelerated oxidative damage that accompanies sepsis could be both a cause and a consequence of cytokine-induced mitochondrial dysfunction. interestingly, the ultrastructural damage reported to accompany mitochondrial dysfunction in sepsis [102] reflects maegraith's observations in monkey malaria [108] [109] [110] decades ago. metabolic acidosis, often associated with hyperlactataemia, has been described in african children with severe falciparum malaria [111, 112] . it is not unique to this disease, being seen in viral, rickettsial and bacterial infections [113] as well as acute gastroenteritis, where its prevalence is higher than in malaria [114] . the terms hyperlactataemia and lactic acidosis are often mistakenly used interchangeably in the malaria literature. as often reviewed in the basic literature [115] [116] [117] [118] , protons (h + , the basis of acidosis) are not formed when atp and lactate are generated during glycolysis, but on the subsequent hydrolysis of atp in tissues. every time a molecule of atp undergoes hydrolysis, a proton is released. if this occurs under aero-bic conditions, these protons are consumed within atp regeneration from adp, and ph remains normal, i.e. acidosis does not occur. in contrast, if the mitochondria are not functioning adequately, whether through insufficient oxygen supply or an inability to use it, atp regenerates under anaerobic condition, and the protons are not consumed. hence, once the buffering capacity of the body is exceeded, acidosis occurs. in short, metabolic acidosis requires the ratio of glycolytic (i.e. anaerobic) atp hydrolysis to mitochondrial (i.e. aerobic) atp hydrolysis to reach a point at which the buffering systems can no longer cope. pathological changes in the buffering system can be a major determinant of when this occurs. high lactate levels have traditionally been seen not only as a marker for poor oxygen delivery in disease states, but also a consequence of it, and the cause of the acidosis. for some time hyperlactataemia has been regarded as a functionally relevant marker for a poor prognosis in both sepsis [119] and malaria [66, 112, 120] . although the sepsis world now discusses several origins for the lactate increase, including inflammation-induced mitochondrial dysfunction [97] , in falciparum malaria it is still generally attributed to a reduced oxygen supply, mostly through microvascular occlusion by sequestered parasitised erythrocytes [121] . other mechanisms are known to contribute to acidosis in malaria, independent of lactate production, e.g. acute renal failure [8] . impaired hepatic clearance [8, 112] , production by parasites, and, in some areas, thiamine deficiency [122] are also argued to contribute to lactate accumulation independent of impaired cellular respiration. thus, as described below, although acidosis and hyperlactataemia can be associated, they are independent cellular mechanisms. lactate anion has complex roles in biology. hyperlactataemia may be associated with acidosis, a normal ph, or alkalosis [123] . a recent editorial in critical care medicine [124] has lucidly summarised the key points of the mechanism of metabolic acidosis in sepsis, a condition that shares systemic inflammation and a range of its consequences with severe malaria (tab. 2). these authors argue against lactate as the cause of the acidosis associated with hypoxia. instead, they note the evidence that during hypoxia, be it from limited oxygen supply or utilisation, the unconsumed protons that cause acidosis arise from the hydrolysis of non-mitochondrial atp. since these reactions are independent of lactate levels, it is difficult to see how therapeutically reducing levels of this anion, as has been proposed [125] , could increase survival rate in falciparum malaria any more than in sepsis [126] . indeed, in theory it could harm comatose patients, since there is evidence that lactate helps brain tissue survive hypoxic and hypoglycaemic episodes [127] [128] [129] , and the lactate shuttle is proving to be how astrocytes protect neurons from metabolic stress [130] . even when considerable lactate is generated in acute inflammatory states, other, unidentified, anions contribute much more than it does to the strong ion difference that, through influencing the body's buffering capacity, influences acidosis in sepsis [131, 132] and falciparum malaria [114, 133] . thus, lactate accumulation can only partially account for the high anion gap observed during the metabolic acidosis associated with severe malaria. in summary, lactate is an imprecise but useful marker for metabolic acidosis in malaria. in turn, acidosis is an imprecise but useful marker of impaired cellular respiration. whether impaired cellular respiration arises from (a) poor supply of oxygen to mitochondria (through vaso-occlusion, low circulating volume, anaemia or cardiac insufficiency) or impaired mitochondrial function (in response to severe systemic inflammation) the outcome is essentially the same. the resulting high anion gap metabolic acidosis is strongly predictive of death in severe malaria. greater understanding of the multiple factors influencing the metabolic acidosis could provide further insight into the underlying pathophysiological process and may provide additional therapeutic options. when glycolysis is enhanced for any period glycogen stores are soon depleted, and gluconeogenesis supervenes. however, its substrate supplies are limiting [134] , and the hypoglycaemia often reported in severe malaria [135] and sepsis [19, 136] occurs. hypoglycaemia is therefore a secondary cause of harm in these diseases, and is an inevitable consequence of exuberant, mostly anaerobic, glycolysis. cm is a clinical syndrome characterised by coma (inability to localise a painful stimulus) at least 1 h after termination of a seizure or correction of hypoglycaemia, detection of asexual forms of p. falciparum malarial parasites on peripheral blood smears, and exclusion of other causes of encephalopathy [137] . a relatively consistent feature of acute cm in children is raised intracranial pressure (icp). studies in african children have demonstrated a raised cerebrospinal fluid (csf) opening pressure during lumbar puncture in 80% of cm children [138] , raised icp during intracranial pressure monitoring (23/23 icp > 10 mmhg) [139] and papilloedema (a late sign of raised icp) in 44% of cm patients who died [140] . where computer tomography has been performed, there was evidence of diffuse brain swelling in 40% of patients [139] . the cause of the raised icp is likely to be multi-factorial and has been postulated to involve both vasogenic and cytotoxic patterns of cerebral oedema. vasogenic oedema is characterised by accumulation of interstitial fluid within the brain secondary to increased permeability of the blood-brain barrier (bbb). it has been demonstrated in bacterial cerebral infections, but evidence of significant disruption of the bbb is not conclusive in cm [141] . others have proposed that icam-1 binding by infected erythrocytes may generate a cascade of intracellular signalling events that disrupt the cytoskeletal-cell junction structure and cause focal disruption to the bbb [142] . adult post-mortem analysis has shown cerebrovascular endothelial cell activation (increased icam-1 endothelial staining, reduction in cell junction staining, and disruption of junction proteins), particularly in vessels containing infected erythrocytes [143] . however, disruption of intercellular junctions is not associated with significant leakage of plasma proteins (fibrinogen, igg, or c5b-9) into perivascular areas or csf [143] . in thai adults, transfer of radioactively labelled albumin into csf was not raised during unconsciousness compared with convalescence [144] . similarly, the albumin index (ratio of concentrations of albumin in csf to those in blood) was not altered significantly in vietnamese adults [145] or significantly different between malawian children with cm who died and those who survived [143] . cytotoxic oedema is increasingly being recognised as an important mechanism of cerebral oedema in traumatic brain injury [146] . as previously discussed, this type of cell swelling involves disturbance of the "pumpleak equilibrium" maintained, under physiological conditions via active elimination of osmotically active solutes through the energy-dependent na + /k + -atpase. thus, cytotoxic oedema can occur secondary to an imbalance in supply and demand of energy within the cells. several mechanisms, such as sustained increase in neuronal activation, impaired substrate delivery (structural and functional) and impaired mitochondrial utilisation of available substrates, including oxygen, may coexist to generate this imbalance. all these mechanisms could contribute to atp depletion and na + /k + atpase failure, leading to cytotoxic oedema in cm. cm is clearly associated with increased neuronal activity. a recent review identified that 80% of african children with cm have a history of seizures, with prolonged and recurrent seizures associated with a poor outcome [147] . impaired vascular flow during acute cm may limit substrate delivery within the brain and contribute to energy imbalance. in the past, a common premise was that parasite sequestration precipitated cerebral vaso-occlusive/ischaemic (i.e. stroke-like) events that manifested clinically as cm. however, cm demonstrates several features that are atypical for stroke. in children, focal neurological signs do not tend to accompany coma, although a sub-set of patients do exhibit hemiparesis or focal brainstem deficits during the agonal period [148] . the incidence of residual neurological deficits following recovery from coma is relatively low (11% [147] ) when compared to childhood stroke (93% had residual neurological deficit [149] ). where computer tomography has been performed in children, diffuse brain swelling was observed [150] rather than focal lesions more typical of stroke. although retinal haemorrhages have been observed in 46% of malawian children with cm (and in 63% of patients who died), these lesions were also seen in 30% of children with sma in the same study [140] . consequently, although associated with cm, retinal haemorrhages do not confirm that focal cerebral vaso-occlusive/ischaemic events underlie cm. similarly, histological examination of 32 fatal cm cases of african children at autopsy demonstrated that one third had little or no evidence of local vascular change in the brain, as indicated by sequestered parasites, monocyte clusters, micro-haemorrhages, local vascular inos [151] or haemoxygenase -1 (ho-1) [152] staining. accepting that cm may occur without ischaemia does not exclude temporary or less severe reductions in vessel flow occurring during acute cm (associated or independent of parasite sequestration) that may contribute to impaired substrate delivery and lead to energy imbalance. as previously discussed, energy imbalance may also be impaired due to the uncoupling action of inflammatory cytokines on mitochondrial atp production. in gambian and ghanaian children, concentrations of tnf and its receptor were higher in those with cm than in those with mild or uncomplicated malaria [153, 154] . polymorphisms in the tnf promoter region have also been associated with increased risk of cm and death [155] or neurological sequelae [156] . cytokines may also up-regulate inos in brain endothelial cells, increasing production of no, which could then diffuse into brain tissue and disrupt neuronal (and/or mitochondrial) function [157, 158] . in the brain, mitochondrial function may also be influenced by neuronal excitotoxins. within the simplified model of dissociated neuronal culture, mitochondria appear to play a critical role in neuronal homeostasis during excitotoxin exposure. mitochondria are not only involved with maintaining atp production but also calcium homeostasis, and generation and detoxification of reactive oxygen species [107] . excitotoxin production may also be influenced by cytokine release. tnf administration has been shown to alter brain metabolism of tryptophan to produce more kynurinine [159, 160] . thus, as part of a general inflammatory reaction, increased excitotoxin generation during acute malaria may contribute to cellular energy imbalance. elevated levels of neuronal excitotoxins (quinolinic and picolinic acid) in the csf have been associated with a fatal outcome in malawian children with cm [161] . similarly, a graded increment of quinolinic acid concentration in csf was observed across patient outcome groups of increasing severity in african children [162] . although a subset of the malawaian autopsy patients [163] demonstrated negligible histological change in their brains, they did demonstrate inflammation, as indicated by inos, mif [151] and ho-1 [152] , staining in other tissues. these systemic changes were shared with the comatose sepsis cases in the study, and therefore are consistent with the premise that coma may in part be secondary to a host inflammatory response to systemic infection. below are further examples of systemic responses to infection that present with diffuse cerebral syndromes, including coma. in the past, the term cm has been restricted to falciparum malaria, and patients with p. vivax infection exhibiting symptoms of severe malaria, including coma, have been dismissed as undiagnosed falciparum co-infections. however, the use of more sensitive diagnostic techniques makes such dismissal less tenable. two such studies report adults exhibiting severe malaria with p. vivax (but not p. falciparum) infection detectable on pcr and serological and testing [142, 143] . the patients exhibited multiple organ failure including cerebral symptoms, renal failure, circulatory collapse, severe anaemia, haemoglobinuria, abnormal bleeding, acute respiratory distress syndrome, and jaundice. vivax malaria has been associated with a strong systemic inflammatory response [164] , but this was not investigated in the above studies. sepsis-associated encephalopathy (sae) syndrome has multiple features that resemble cm. it is characterised by a diffuse disturbance of cerebral function (typically impairment of consciousness) that occurs in the context of systemic response to infection without direct neuroinvasion (i.e. meningitis, macroscopic cerebritis and brain abscesses are excluded). sae is associated with generalised slow waves on the electroencephalogram (eeg), with the depth of coma linked with mortality. mild sae cases often recover completely, while survivors of severe sae may have persistent neurological deficit [165] ). in line with adult cm, the severity of encephalopathy parallels the severity of systemic organ failure [141] . inflammatory cytokines have been demonstrated to be higher in the serum than in the csf, suggesting that sepsis encephalopathy is a consequence of the systemic inflammatory response to infection [141] . an animal model in which prior administration of a neutralising antibody to tnf prevented the sepsis encephalopathy of pancreatitis [166] is consistent with this. further postulated reversible mechanisms of pathogenesis include changes in regional cerebral blood flow, neurotransmitter imbalance, mitochondrial dysfunction, bbb impairment and oxidative stress [167] . severe influenza infection can present with encephalopathy, yet as in malaria, the pathogen is not neuroinvasive [168] . seizures and coma occur after high fever [169] , commonly accompanied by thrombocytopaenia [169] , with metabolic acidosis and hyperlactataemia in severe cases (t. ichiyama, personal communication). similar to adult malaria, neurological sequelae occur concurrently with multiple organ failure [170] . tnf, il-6, stnfri, and soluble e-selectin are increased in serum and csf [171, 172] , and serum nitrite/nitrate levels are increased [173] . detailed examination of brain has revealed apoptosis of neurons and glial cell, histological evidence of active caspase-3 and caspase-cleaved parp, cerebral oedema, and bbb impairment [174] . these parallel changes are set out in table 3 . it is clear, therefore, that the presence of sequestering parasitised red cells is not necessary to generate these changes, which are also demonstrable in the falciparum malaria encephalopathy. notably, high levels of inflammatory cytokine are present in each disease. seizures are a very common component of acute malaria illness in children. a recent review documented that 80% of african children had a history of seizures, with 60% exhibiting seizures during hospital admission [175] . the molecular basis of the seizures is unclear. multiple mechanisms have been postulated, including fever, hypoxia and/or cytokine stimulation leading to an imbalance of neurotransmitters and excitotoxins or neuronal damage [11, 148] . recently, lang and co-workers [176] demonstrated that falciparum parasitaemia is associated with the generation of specific antibodies for voltage-gated calcium channels directed against neurones. higher antibody concentrations were detectable in sera from patients exhibiting cm or malaria with seizures than uncomplicated malaria, suggesting that these antibodies may influence seizure propensity. only the erythrocytic form of malaria is associated with disease, so valuable information about which african children are likely to have more, or less, severe malaria has inevitably been obtained from examining the inborn rbc abnormalities that endemic malaria has selected across the tropics. the coinciding geographic distributions of malaria transmission and the thalassaemias prompted haldane to put forward the 'malaria hypothesis', which proposed that common erythrocyte abnormalities are selected because of the fitness advantage they confer against malaria [177] . sickle cell haemoglobin (hbs) has also been repeatedly shown to be associated with malaria resistance, with heterozygotes for the hbs trait demonstrating 10% of the population at risk for severe malaria in certain populations [178] . other haemoglobinopathies (e.g. hbc [179, 180] and hbe [181] ) and deficiencies in rbc enzymes (e.g. glucose-6-phosphate dehydrogenase deficiency [182] ) have also been linked with protection against severe malaria. the mechanisms of protection afforded by haemoglobinopathies are likely to be multi-factorial. studies have demonstrated evidence to support several independent mechanisms including: reduced parasite invasion of rbcs and diminished intraerythrocytic growth of parasites in patients with the hbs trait [183] , enhanced phagocytosis of parasite-infected erythrocytes (ies) [184] and enhanced immune responses against ies [185] . recent in vitro studies observed that hbc modifies the quantity and distribution of the variant antigen p. falciparum erythrocyte membrane protein 1(pfemp1) on the ie surface. pfemp1 has been implicated in numerous ie adhesive interactions. in the latter study the authors demonstrated that hbc reduces the level of ie adhesion to endothelial monolayers, in addition to ie rosetting (the adhesion of ies to uninfected erythrocytes) and ie agglutination by sera. these findings provide the prospect that hbc pro-tects against severe malaria by mitigating the obstruction and inflammation caused by the pfemp1-mediated adherence of ies [186] . however, sequestration is believed to enhance parasite survival by enabling ies to avoid splenic clearance, so any reduction of sequestration by hbc can be expected to limit parasite fitness. multiple epidemiology studies (e.g. [179, 187, 188] ) have failed to identify any significant impact of hbc on the frequency or density of parasitaemia in naturally exposed populations. consequently, the influence of the changes in ie surface conformation needs to be confirmed and further examined in vivo [189] . a recent study re-confirmed that african children with -thalassaemia trait are significantly less likely to be hospitalised with severe malaria, particularly with coma or severe anaemia (hb < 5 g/100 ml). it is intriguing that the -thalassaemia patients did not demonstrate a lower incidence of uncomplicated malaria nor any reduction in peripheral parasite density [190] . thalassaemia has also been associated with increased incidence of clinical vivax and falciparum malaria during early life [191] . the findings raise speculation that the trait may indirectly afford enhanced immunity through increased non-lethal exposure to malarial parasites. such a mechanism is appealing, since it would be equally plausible across a range of haemoglobinopathies, including hbc. variations in erythrocyte membrane proteins also have a profound influence on malaria susceptibility. most notably the absence of duffy antigen protein confers absolute protection to p. vivax infection. more recently, the duffy antigen has also been associated with a protection against falciparum malaria [192] . enzymes involved with iron handling may also have a critical influence on malaria morbidity. a recent study from the gambia demonstrated that children in an endemic malaria area possessing the haptoglobin 2,2, isotype had a significantly increased risk of anaemia [193] . however, a lack of parallel alterations in other haematinic indices leaves the mechanism of this process unclear. malarial protection within individuals exhibiting multiple rbc abnormalities appears even more complex. a recent study observed that the concurrent presence of sickle cell and -thalassaemia trait among african children had a negative influence on the risk of malaria infection [194] . the results warn geneticists that gene epistasis may have a profound influence on overall malarial susceptibility. in tropical countries many hospital deaths from falciparum malaria happen before anti-malarial drugs have had time to kill the parasites. two approaches could help rectify this -addressing public-health problems resulting in delayed presentation, and identifying the physiological processes and molecular pathways that lead to these early deaths, with a view to developing evidence-based adjunct therapies. therapies being explored in sepsis, and based on disease pathogenesis data common to sepsis and malaria, may prove to be transferable from either of these diseases to the other. as noted above, circulating levels of a late-appearing inflammatory cytokine, hmgb1, are increased in falciparum malaria [41] as well as in sepsis. results from animal models on the role of hmgb1, although untested in humans, have inspired enthusiasm for inhibition of this molecule as a potential intervention for human sepsis. for instance, anti-hmgb1 antibodies provided dose-dependent protection [37] and reduced mortality [195] against experimental sepsis in mice. late administration of ethyl pyruvate, which inhibits hmgb1 release from macrophages, also conferred protection against endotoxaemia in mice [196] . treatments directed towards critical downstream consequences of malaria infection and inflammation, such as those intended to limit acidosis, are also a focus of investigation. one current approach is to identify which acute malaria patients most benefit from early volume expansion [197] . controlling lactic acidosis via sodium dichloroacetate (dca), an inhibitor of pyruvate dehydrogenate kinase (maintaining pyruvate dehydrogenase in its active form), is also being examined. dca reduced lactate levels in acute malaria patients [198] , although the study was unable to determine whether treatment improved outcome. an earlier large sepsis study also demonstrated that dca reduced lactate, but again with no improvement in outcome [126] . as outlined in the section 'is hyperlactataemia a cause or marker of the acidosis of malaria?', some researchers argue, in view of the strong ion difference contributing to acidosis and the postulated mitochondrial dysfunction during acute malaria infection, that lactate reduction per se may have limited impact on prognosis. other adjunct therapies are also being examined. improving rbc deformability provides one potential therapeutic approach. in vitro studies with n-acetylcysteine (nac), reported to scavenge free radicals, showed improvement in red cell deformability through in vitro studies [199] . unfortunately, an initial in vivo trial of nac in malaria patients had no effect on mortality [200] . blocking endothelial activation is also a focus of research, with initial in vitro studies providing some encouraging results [201] . in conclusion, continuing to identify the host responses to malaria infection that lead to disease is providing insights into novel molecular mechanisms. this information is beginning to guide the design of much needed additional therapies against this disease. there is little doubt that poor oxygen supply through vascular occlusion or anaemia could contribute to the body relying on excessive glycolysis to generate energy, resulting in hyperlactataemia, hypoglycaemia, and metabolic acidosis, and altered consciousness. however, inflammatory cytokines control these changes, as well as inhibit the capacity of mitochondria to use oxygen. thus, as described throughout this review, inflammatory cytokines are likely to have various pivotal roles across the multiple pathological processes involved in malarial disease (fig. 1 ). indicators of life-threatening malaria in african children the pathophysiologic and prognostic significance of acidosis in severe adult malaria malaria and the lung indicators of mortality in african adults with malaria severe falciparum malaria in children: current understanding of pathophysiology and supportive treatment the injured child is resistant to multiple organ failure: a different inflammatory response? endotoxin induces an exaggerated interleukin-10 response in peritoneal macrophages of children compared with adults il-1 beta induces an exaggerated pro-and anti-inflammatory response in peritoneal macrophages of children compared with adults current knowledge in falciparum malaria-induced acute renal failure pediatric considerations hypoglycemia in infancy and childhood glucose production and gluconeogenesis in adults with uncomplicated falciparum malaria hypoglycaemia in african children with severe malaria severe and complicated falciparum malaria in melanesian adults in papua new guinea cerebral malaria: the sequestration hypothesis the cytokine theory of human cerebral malaria golgi c (1886) sull infezione malarica the prevention of malaria possible importance of macrophage-derived mediators in acute malaria evolutionary causes and consequences of immunopathology how malaria has affected the human genome and what human genetics can teach us about malaria a high frequency african coding polymorphism in the n-terminal domain of icam-1 predisposing to cerebral malaria in kenya induction of cachectin (tumor necrosis factor) synthesis by influenza virus: deficient production by endotoxin-resistant (c3h/hej) macrophages induction of proinflammatory 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and malaria in adults from southeast asia lactate homeostasis and lactic acidosis lactic acidosis in sepsis: another commentary dichloroacetate for lactic acidosis in severe malaria: a pharmacokinetic and pharmacodynamic assessment a controlled clinical trial of dichloroacetate for treatment of lactic acidosis in adults protection by lactate of cerebral function during hypoglycaemia brain lactate, not glucose, fuels the recovery of synaptic function from hypoxia upon reoxygenation: an in vitro study the effect of intravenous lactate on cerebral function during hypoglycaemia lactate as a pivotal element in neuron-glia metabolic cooperation unaccounted for anion in metabolic acidosis during severe sepsis in humans unmeasured anions identified by the fencl-stewart method predict mortality better than base excess, anion gap, and lactate in patients in the pediatric intensive care unit unidentified acids of strong prognostic significance in severe malaria glucose homeostasis in children 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receptor in influenza virus-associated encephalopathy analysis of cytokine levels and nf-[kappa]b activation in peripheral blood mononuclear cells in influenza virus-associated encephalopathy cytochrome c and tumor necrosis factor-alpha values in serum and cerebrospinal fluid of patients with influenza-associated encephalopathy high concentration of serum nitrite/ nitrate obtained from patients with influenza-associated encephalopathy apoptosis and microglial activation in influenza encephalopathy pathogenesis, clinical features, and neurological outcome of cerebral malaria antibodies to voltage-gated calcium channels in children with falciparum malaria the rate of mutation of human genes common west african hla antigens are associated with protection from severe malaria hemoglobin c associated with protection from severe malaria in the dogon of mali, a west african population with a low prevalence of hemoglobin s haemoglobin c protects against clinical plasmodium falciparum malaria 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and other diseases in children living on the coast of kenya high incidence of malaria in alpha-thalassaemic children high prevalence of human antibodies to recombinant duffy binding-like alpha domains of the plasmodium falciparum-infected erythrocyte membrane protein 1 in semi-immune adults compared to that in non-immune children seasonal childhood anaemia in west africa is associated with the haptoglobin 2-2 genotype negative epistasis between the malaria-protective effects of alpha(+)-thalassemia and the sickle cell trait reversing established sepsis with antagonists of endogenous high-mobility group box 1 ethyl pyruvate prevents lethality in mice with established lethal sepsis and systemic inflammation randomized trial of volume expansion with albumin or saline in children with severe malaria: preliminary evidence of albumin benefit pharmacokinetics and pharmacodynamics of dichloroacetate in children with lactic acidosis due to severe malaria oxidative stress and rheology in severe malaria a pilot study of n-acetylcysteine as adjunctive therapy for severe malaria inhibition of endothelial activation: a new way to treat cerebral malaria key: cord-017309-pt27efu1 authors: gupta, g. s. title: selectins and associated adhesion proteins in inflammatory disorders date: 2012-03-20 journal: animal lectins: form, function and clinical applications doi: 10.1007/978-3-7091-1065-2_44 sha: doc_id: 17309 cord_uid: pt27efu1 inflammation is defined as the normal response of living tissue to injury or infection. it is important to emphasize two components of this definition. first, that inflammation is a normal response and, as such, is expected to occur when tissue is damaged. infact, if injured tissue does not exhibit signs of inflammation this would be considered abnormal and wounds and infections would never heal without inflammation. secondly, inflammation occurs in living tissue, hence there is need for an adequate blood supply to the tissues in order to exhibit an inflammatory response. the inflammatory response may be triggered by mechanical injury, chemical toxins, and invasion by microorganisms, and hypersensitivity reactions. three major events occur during the inflammatory response: the blood supply to the affected area is increased substantially, capillary permeability is increased, and leucocytes migrate from the capillary vessels into the surrounding interstitial spaces to the site of inflammation or injury. the inflammatory response represents a complex biological and biochemical process involving cells of the immune system and a plethora of biological mediators. cell-to-cell communication molecules such as cytokines play an extremely important role in mediating the process of inflammation. inflammation and platelet activation are critical phenomena in the setting of acute coronary syndromes. an extensive exposition of this complex phenomenon is beyond the scope of this article (rankin 2004). play fairly broad roles in the generation of immune responses. the three selectins act in concert with other cell adhesion molecules e.g., intracellular adhesion molecule (icam-1), vascular cell adhesion molecule-1 (vcam-1), and leukocyte integrins to effect adhesive interactions of leukocytes, platelets, and endothelial cells. the structure and functions of selectins, which belong to c-type lectins family, have been reviewed in chaps. 26, 27, and 28. the selectin family of lectins consists of three closely related cell-surface molecules with differential expression by leukocytes (l-selectin), platelets (p-selectin), and vascular endothelium (e-and p-selectin). structural identity of a selectins resides in its unique domain composition (chap. 26). e-, p-, and l-selectin are >60 % identical in their nh2 terminus of 120 amino acids, which represent the lectin domain (chaps. 26, 27, and 28) . the ligands (counter structures) of selectins are sialylated and fucosylated carbohydrate molecules which, in most cases, decorate mucin-like glycoprotein membrane receptors. their common structure consists of an n-terminal ca 2+ -dependent lectin-type domain, an epidermal growth factor (egf)-like domain, multiple short consensus repeat (scr) domains similar to those found in complement regulatory proteins, a transmembrane region, and a short cytoplasmic c-terminal domain. together this arrangement results in an elongated structure which projects from the cell surface, ideal for initiating interactions with circulating leucocytes. the lectin domain forms the main ligand binding site, interacting with a carbohydrate determinant typified by fucosylated, sialylated, and usually sulphated glycans such as sialyl lewis x (s-le x ). the egf domain may also play a role in ligand recognition. the short consensus repeat (scr) domains (two for l-selectin, six for e-selectin, and nine for p-selectin) probably act as spacer elements, ensuring optimum positioning of the lectin and egf domains for ligand interaction. the egf repeats have comparable sequence similarity. each complement regulatory-like module is 60 amino acids in length and contains six cysteinyl residues capable of disulfide bond formation. this feature distinguishes the selectin modules from those found in complement binding proteins, such as complement receptors 1 and 2, which contain four cysteines (chap. 26). the selectins cell-surface receptors play a key role in the initial adhesive interaction between leukocytes and endothelial cells at sites of inflammation. selectins (p, e and l) and their ligands (mainly p-selectin ligand) are involved in the rolling and tethering of leukocytes on the vascular wall. activation of endothelial cells (ec) with different stimuli induces the expression of e-and p-selectins, and other adhesion molecules (icam-1, vcam-1), involved in their interaction with circulating cells. lymphocytes home to peripheral lymph nodes (plns) via high endothelial venules (hevs) in the subcortex and incrementally larger collecting venules in the medulla. hevs express ligands for l-selectin, which mediates lymphocyte rolling (horstman et al. 2004) . for structure and functions of selectins, the readers are advised to consult chaps 26-28. in this chapter we will emphasize mainly on the role of selectins in inflammatory disorders including cancer. atherothrombosis, defined as atherosclerotic plaque disruption with superimposed thrombosis, is the leading cause of mortality in the western world. atherosclerosis is a diffuse process that starts early in childhood and progresses asymptomatically through adult life. later in life, it is clinically manifested as coronary artery disease (cad), stroke, transient ischaemic attack (tia), and peripheral arterial disease. from the clinical point of view, we should envision this disease as a single pathologic entity that affects different vascular territories. a suggestive analogy is that tia and intermittent claudication are the unstable angina of the brain and lower limbs, respectively; and stroke and gangrene are the myocardial infarction. circulating platelets display reversible interactions with atherosclerotic lesions. atherosclerotic arterial disease is associated with an increased share of platelets unable to express p-selectin and an increased fraction of platelets that microaggregate in citrate anticoagulant. these platelet alterations are not completely explained by either focal arterial injury or abnormal rheology associated with arterial stenosis but appear to be an effect of the atherosclerotic process (mcbane et al. 2004 ). the pathogenesis of arterial thrombotic disease involves multiple genetic and environmental factors related to atherosclerosis and thrombosis. venous thrombosis is a world wide health problem in the general population. injury to the endothelium leads to dysfunction. the causes of injury include lipids, immune complexes, microorganisms, smoking, hypertension, aging, diabetes mellitus and trauma. the selectins are thought to be largely responsible for the initial attachment and rolling of leukocytes on stimulated vascular endothelium. platelet activation is an important process in the pathogenesis of atherothrombosis. platelet adhesion, activation, and aggregation at the sites of vascular endothelial disruption caused by atherosclerosis are key events in arterial thrombus formation. platelet tethering and adhesion to the arterial wall, particularly under high shear forces, are achieved through multiple high-affinity interactions between platelet membrane receptors (integrins) and ligands within the exposed subendothelium, most notably collagen and von willebrand factor (vwf) . platelet adhesion to collagen occurs both indirectly, via binding of the platelet glycoprotein (gp) ib-v-ix receptor to circulating vwf, which binds to exposed collagen, and directly, via interaction with platelet receptors gp vi and gp ia/iib. platelet activation, initiated by exposed collagen and locally generated soluble platelet agonists (primarily thrombin, adp, and thromboxane a2), provides the stimulus for the release of platelet-derived growth factors, adhesion molecules and coagulation factors, activation of adjacent platelets, and conformational changes in the platelet a(iib)b3 integrin (gp iib/iiia receptor). platelet aggregation, mediated primarily by interaction between the activated platelet gp iib/iiia receptor and its ligands, fibrinogen and vwf, results in the formation of a plateletrich thrombus (steinhubl and moliterno 2005) . p-selectin expression in platelets is elevated in disorders associated with arterial thrombosis such as coronary artery disease, acute myocardial infarction, stroke, and peripheral artery disease. during thrombosis, p-selectin is expressed on the surface of activated endothelial cells and platelets. p-selectin mediates rolling of platelets and leukocytes on activated endothelial cells as well as interactions of platelets with leukocytes. platelet p-selectin interacts with psgl-1 on leukocytes to form platelet-leukocyte aggregates. furthermore, this interaction of p-selectin with psgl-1 induces the upregulation of tissue factor, several cytokines in leukocytes and the production of procoagulant microparticles, thereby contributing to a prothrombotic state. p-selectin is also involved in platelet-platelet interactions, i. e. platelet aggregation which is a major factor in arterial thrombosis. p-selectin interacts with platelet sulfatides, thereby stabilizing initial platelet aggregates formed by gpiib/iiia-fibrinogen bridges. inhibtion of the p-selectin-sulfatide interaction leads to a reversal of platelet aggregation. thus, p-selectin plays a significant role in platelet aggregation and platelet-leukocyte interactions, both important mechanisms in the development of arterial thrombosis. following activation, p-selectin is rapidly translocated to the cell surface (merten and thiagarajan 2004; wang et al. 2005 ). platelet activation occurs in peripheral blood of patients with rheumatic mitral stenosis (ms). the plasma levels of soluble p-selectin are elevated in permanent atrial fibrillation (af) patients; the plasma levels of soluble p-selectin in the left atrium do not significantly differ from those in the right atrium, femoral vein, or femoral artery. the venous plasma levels of sp-selectin in patients with moderate-to-severe ms are significantly higher than those in healthy volunteers or patients with lone af. in addition, in patients with ms, there was no difference in the plasma levels of sp-selectin between the left and right atrial blood and between peripheral and atrial blood. moreover, there was no change in spselectin levels as a result of percutaneous transluminal mitral valvuloplasty (ptmv) (chen et al. 2004) . lip et al. (2005) studied the relations of plasma vwf (an index of endothelial damage and dysfunction) and sp-selectin levels in relation to the presence and onset of clinical congestive heart failure (chf) and degree of left ventricular dysfunction in patients taking part in spaf (stroke prevention in af). while plasma vwf was higher among patients with af and chf, plasma p-selectin concentrations were not affected by presence, onset, or severity of heart failure. atherosclerosis is a complex chronic inflammatory disease of the arterial wall. though the inflammatory nature of atherosclerosis has been established, the initial events that trigger this response in the arterial intima remain obscure. studies reveal a significant rate of genomic alterations in human atheromas. the accumulation of genomic rearrangements in vascular endothelium and smooth muscle cells are important for disease development. it is well accepted that the induction of ec adhesion molecules is a critical component in acute inflammatory responses as well as allogeneic interactions in vascularized allografts and, possibly, atherogenesis. inflammation and genetics are both prominent mechanisms in the pathogenesis of atherosclerosis and arterial thrombosis. accordingly, population studies have explored the association of ischaemic heart disease with gene polymorphisms of the inflammatory molecules: tumor necrosis factors (tnf) a and b, transforming growth factors (tgf) b1 and 2, p and e selectins, and platelet endothelial cell adhesion molecule (pecam) 1. the partly conflicting data provide some evidence that alterations in the genetics of the inflammatory system may modify the risk of ischaemic heart disease. (ros) as an initial event in recent years, reactive oxygen species (ros) are considered as initial event in causing atherosclerosis. ros are a family of molecules including molecular oxygen and its derivatives produced in all aerobic cells. excessive production of ros, outstripping endogenous antioxidant defense mechanisms, has been implicated in processes in which they oxidize biological macromolecules, such as dna, protein, carbohydrates, and lipids. many ros possess unpaired electrons and thus are free radicals. these include molecules such as superoxide anion (o 2 à ), hydroxyl radical (ho • ), nitric oxide (no • ), and lipid radicals. other reactive oxygen species, such as hydrogen peroxide (h2o2), peroxynitrite (onoo à ), and hypochlorous acid (hocl), are not free radicals per se but have oxidizing effects that contribute to oxidant stress. the cellular production of one ros may lead to the production of several others via radical chain reactions. for example, reactions between radicals and polyunsaturated fatty acids within cell membrane may result in a fatty acid peroxyl radical (r-coo • ) that can attack adjacent fatty acid side chains and initiate production of other lipid radicals. lipid radicals produced in this chain reaction accumulate in the cell membrane and may have a myriad of effects on cellular function, including leakage of the plasmolemma and dysfunction of membrane-bound receptors. of note, end products of lipid peroxidation, including unsaturated aldehydes and other metabolites, have cytotoxic and mutagenic properties. a decline in no bioavailability may be caused by decreased expression of the endothelial cell no synthase (enos), a lack of substrate or cofactors for enos (fig. 44.1 and 44.2) . in mammalian cells, potential enzymatic sources of ros include the mitochondrial respiration, arachidonic acid pathway enzymes lipoxygenase and cyclooxygenase, cytochrome p450s, xanthine oxidase, nadh/nadph oxidases, no synthase, peroxidases, and other hemoproteins. although many of these sources could potentially produce ros that inactivate no • , 3 sources have been studied extensively in cardiovascular system. these include xanthine oxidase, nadh/nadph oxidase, and no synthase (cai and harrison 2000; hamilton et al. 2004; vijya lakshmi et al. 2009 ). during initial step in atherosclerosis, there is rapid targeting of monocytes to the sites of inflammation and endothelial injury; the adhesion of leukocytes to activated endothelial cells is mediated by icam-1. the induction of ec adhesion molecules is a critical component in acute inflammatory responses as well as allogeneic interactions in vascularized allografts and, possibly, atherogenesis. the "inflammatory triad" of il-1, tnf, and lps are potent stimulators of the ec activation and adhesion molecules e-selectin or elam-1 (or also known as cd62e), icam-1 and vcam-1. pecam-1 plays also a key role in the transendothelial migration of circulating leukocytes (diapedesis) during vascular inflammation. icam-1 and vcam-1 are inflammatory predicators of adverse prognosis in patients with acute coronary syndromes (acs) (postadzhiyan et al. 2008) (fig. 44.2) . levels of p-selectin are increased in the blood of patients with familial hypercholesterolemia (fh) in spite of long-term intensive extracorporeal ldl-elimination, documenting the activity of atherosclerosis. low levels of p-selectin and mcp-1 after hypolidemic procedure can be used as a marker showing the effectivity of the extracorporeal ldl-cholesterol elimination (blaha et al. 2004 ). in an extended study, the levels of expression of tissue factor, icam-1, p-and e-selectin, and pai-1 were found low, whereas those of endothelial protein c receptor and vcam-1 were high (merlini et al. 2004 ). polymorphisms in the e-selectin gene are associated with accelerated atherosclerosis in young (age <40 years) patients, further suggesting a role of inflammation in atherosclerosis. a further change in endothelial physiology is an increase in the surface expression of e-selectin, which regulate adhesive interactions between certain blood cells and endothelium. intravascular fibrinolysis induced by tissuetype plasminogen activator or urokinase may contribute to the initiation of atherosclerosis by inducing p-selectin and platelet activating factor as well as to plaque rupture, either directly or indirectly, by activating metalloproteinases. as e-selectin is only expressed on activated endothelium, it provides an opportunity to study pathophysiological aspects of this cell in cardiovascular and other disease. however, seselectin can be found in the plasma, which has potential role in the pathogenesis of cardiovascular disease as raised levels have been found in hypertension, diabetes and hyperlipidemia, although its association in established atherosclerosis disease and its value as a prognostic factor is more controversial (holvoet and collen 1997) . polymorphisms for three genes, p-selectin, l-selectin, and e-selectin (genes p-sel, l-sel, and e-sel, respectively) showed that the selectin cluster is linked to markers at chromosome 1q23 (vora et al. 1994 ). significant genomic alterations were found on 1q22-q25 in sel-l gene. the message indicated somatic dna rearrangements, on loci associated to leukocyte adhesion, vascular smooth muscle cells growth, differentiation and migration, to atherosclerosis development as an inflammatory condition (arvanitis et al. 2005) . wenzel et al. (1999) and yoshida et al. (2003) described an adenine to cytosine (a/c) substitution for cdna position 561 resulting in an amino acid exchange from serine to arginine at position 128 (s/r or ser 128 arg) was detected in the epidermal growth factor (egf) domain. a higher mutation frequency was observed in patients aged 50 years or less with proven severe atherosclerosis as well as in patients aged 40 years or less. if ser 128 arg substitution had an effect on the adhesion of blood cells to the endothelium, the polymorphism could be of interest with respect to association studies in a number of pathological conditions, such as cardiovascular diseases. the ser 128 arg polymorphism is associated with a higher risk for early severe atherosclerosis. yoshida et al. (2003) suggested that the eselectin ser 128 arg polymorphism could functionally alter leukocyte-endothelial interactions as well as biochemical and biological consequences, which may account for the pathogenesis of myocardial infarction . leu 554 phe e-selectin mutations in hypertension and cad wenzel et al. (1996 wenzel et al. ( , 1999 detected 17 mutations, five of which resulted in an amino acid substitution. in e-selectin, exchange at ser 128 arg in egf domain and leu 554 phe in membrane domain, and a dna mutation from guanine to thymine (position 98) presented different allele frequencies in young patients with severe atherosclerosis, compared with an unselected population. the bi-allelic a/c polymorphism in the e-selectin gene may be implicated in the clinical expression of erythema nodosum (en) secondary to sarcoidosis (amoli et al. 2004 ). however, the e-selectin polymorphism may be associated with severity of atherosclerotic disease, but it is unknown if it is actually a risk factor for atherosclerosis (ghilardi et al. 2004) . a strong relationship was confirmed between 561a > c and 98g > t polymorphisms of e-selectin gene and susceptibility to cad by zak et al. (2008) . a body mass index (bmi)specific effect of leu 554 phe polymorphism of e-selectin gene on blood pressure has been reported by marteau et al. (2004) who strengthened the view that e-selectin is implicated in hypertension (marteau et al. 2004) . serum levels of e-and p-selectin in patients with essential hypertension (eh) are significantly higher than in controls, where as differences in serum levels of soluble l-selectin, vcam-1, or icam-1 between the patients with eh and the controls were not different (sanada et al. 2005 ). gene p-selectin thr 715 pro (a/c) polymorphism genetic analyses of p-selectin in the progression of atherosclerosis have provided conflicting results regarding the role of variation within the p-selectin gene and risk for heart disease. miller et al. (2004b) suggested that the thr 715 pro c allele was rare in blacks (0.8 %) and intermediate in south asians (3.0 %) compared to whites (11.2 %). sp-selectin levels were significantly lower in the individuals with the ac or cc compared to the aa genotype in both whites and south asians. thus, in whites and south asians the c allele of the thr 715 pro p-selectin polymorphism is associated with (hamilton et al. 2004) . excessive production of ros has been implicated in processes in which they oxidize biological macromolecules, such as dna, protein, carbohydrates, and lipids. many ros possess unpaired electrons and thus are free radicals. these include molecules such as superoxide anion (o 2 à ), hydroxyl racial (ho • ), nitric oxide (no • ), and lipid radicals. the cellular production of one ros may lead to the production of several others via radical chain reactions. a decline in no bioavailability may be caused by decreased expression of the endothelial cell no synthase (enos), a lack of substrate or cofactors required for enos action. low-density lipoprotein (ldl) is oxidized to oxidized form of ldl (ox-ldl) and initiates the atherosclerotic process in the vessel wall (see fig. 44 .2). abbreviations: o2à, superoxide; no, nitric oxide; onooà, peroxynitrite; h2o2, hydrogen peroxide; ohà, hydroxyl radical; sod, superoxide dismutase; gsh, reduced glutathione; gssg, oxidised glutathione; vsm, vascular smooth muscle lower sp-selectin levels (miller et al. 2004b ). the p-selectin thr 715 pro polymorphism is not associated with incident chd or ischemic stroke in either whites or african-americans (volcik et al. 2006 ). in cad, inflammatory biomarkers have been extensively investigated; more evidence exists for c-reactive protein (crp; chap. 8). fatty acid (fa) composition in serum has been associated with crp and e-selectin but not with other inflammatory markers (petersson et al. 2009 ). studies suggest that, besides crp, other inflammatory biomarkers such as cytokines, s-cd40 ligand, serum amyloid a (saa), selectins (e-selectin, p-selectin), icam-1, vcam-1, and several others may have a potential role for the prediction of risk for developing cad and may correlate with severity of cad (eikemo et al. 2004; fang et al. 2004; potapov et al. 2005; zakynthinos and pappa 2009) . the combination of natriuretic peptide (bnp) and e-selectin offers increased predictive value. plasma levels of adhesive molecules are correlated in patients with stable ihd. cigarette smoke condensate (csc)-induced surface expression of icam-1, e-selectin, and vcam-1 in huvec. fang et al. (2005) reported a significant decrease in c allele frequency of pecam-1 gene and showed that leu 125 val polymorphism of pecam-1 gene and elevated soluble pecam-1 were related to severe coronary artery stenosis in cad patients. sp-selectin is associated with myocardial damage: platelets are known to be activated during myocardial infarction (mi). though, the levels of sp-selectin, se-selectin and specam-1 did not differ significantly in the pathogenesis of atherosclerosis, sp-selectin was substantially increased in patients with acute myocardial infarction (ami). yip et al. (2006) tested the hypothesis that platelet activity shown by cd62p is enhanced and predictive of both the extent of myocardial damage and 30-day clinical outcome in patients with st-se ami undergoing primary coronary stenting. xu et al. (2006) suggested that activated-platelets play an important role in the process of myocardial ischemiareperfusion injury, and platelet-derived p-selectin is a critical mediator. p-selectin expression, along with cd40 ligand and tissue factor is significantly increased in infarcted rabbits with respect to controls. clopidogrel administration reduced p-selectin expression and cd40 ligand (molero et al. 2005) . ox-ldl also augments expression of monocyte chemoattractant protein 1 (mcp-1) and macrophage colony stimulating factor (m-csf). mcp-1 mediates the attraction of monocytes and leukocytes and their diapedesis through the endothelium into the intima. m-csf plays an important role in the transformation of monocytes to macrophage foam cells. macrophages express scavenger receptors, which internalize oxldl in their transformation into foam cells. migration of smooth muscle cells (smcs) from the intima into the media is another early event initiating a sequence that leads to formation of a fibrous atheroma hyperhomocysteinemia and selecins in mi: hyperhomocysteinemia is regarded as an independent risk factor for vascular diseases, and homocysteine is supposed to contribute to oxidative stress and endothelial damage. hyperhomocysteinemia is significantly associated with mi in comparison with controls with an odd ratio of 6.26 (khare et al. 2005) . folic acid corrected and reduced hyperhomocysteinemia in a large majority of the cases. although the levels of sp-selectin, se-selectin and specam-1 decrease after folic acid therapy, it was only se-selectin which was significantly reduced. apart from their lipid-lowering capacity, statins also exert anti-inflammatory and antioxidant effects. dna polymorphism in mi: some polymorphisms may increase the risk of mi within specific ethnic groups or in certain populations. p-selectin expression is increased in atherosclerotic plaques, and high plasma levels of this molecule have been observed in patients with unstable angina. dna polymorphisms in p-selectin gene may be a possible candidate for mi. the p-selectin gene is situated on chromosome 1q21-q24, spans >50 kb and contains 17 exons. four polymorphisms (ser 290 asn, asn 562 asp, leu 599 val and thr 715 pro) predicted a change in the amino acid sequence of p-selectin. in patients with mi from four regions of france and northern ireland (the ectim study) the p-selectin polymorphisms provided a heterozygosity of 91 %. the polymorphisms were tightly associated with one another and displayed patterns of linkage disequilibrium suggesting the existence of highly conserved ancestral haplotypes. study illustrates the complexity of the relationship between gene variability and disease and the necessity to explore in detail the polymorphisms of candidate genes (herrmann et al. 1998; tregouet et al. 2002) . the e-selectin gene arg128, 98 t, and phe554 alleles and pecam1 leu1 25 val and ser 563 asn polymorphisms may increase the risk of atherosclerosis, but not necessarily the risk of mi. this association seems to be more pronounced in younger patients and may be especially important in patients with a low risk for developing atherosclerosis. reports indicated that screening for cd14-260 c/t genotypes is unlikely to be a useful tool for risk assessment and it remains unclear whether cd14 polymorphisms significantly increase the risk of mi. the a 252 g polymorphism of lymphotoxin-a (lta) gene, a member of the tnf family, is strongly related with the onset of ami (auer et al. 2003) . quantitative real-time rt-pcr confirmed that lta increased the expressions of e-selectin and vcam1 both in huvec and hcaec, suggesting the roles of lta in the development of atherosclerosis. aminian et al. (2007) determined the possible role of gly 241 arg and lys 469 glu polymorphisms in development of cad and acute or chronic mi. although the frequency of gly-arg and arg-arg genotypes were higher in the control group compared to the chd patients, no strong corelation was found between gly 241 arg and lys 469 glu polymorphisms and occurrence of chd and mi in population from iran. in ischemic event in patients with atherosclerotic ischemic stroke, though the platelet aggregability was decreased after day 3 compared to that at day 1 of stroke onset, platelet cd63 and p-selectin/cd62p expression remained high even 90 days after the events. this suggested that platelet hyperactivation in atherosclerotic ischemic stroke might be sustained for a considerable period (cha et al. 2004; nadar et al. 2004b; yip et al. 2006 ). blood levels of icam-1 and cd62p expression in different typing of patients with ischemic stroke are different. evidences suggest that mps (meridian-phlegm stagnancy) group of patients is the key pathogenic factor of ischemic stroke. mucosal tolerance to e-selectin after booster tolerization can relieve cerebral ischemia-reperfusion injury and induce ischemia tolerance in rats. the mechanisms may involve decreased frequencies of cd8 + t cells, heightened mrna expression of il-10 and lowered mrna expression of e-selectin in the ischemic hemisphere (yun et al. 2008) . selakovic et al. (2009) defined changes of soluble cams in cerebrospinal fluid and plasma in the patients with the acute brain infarction, in which significant increase in the level of soluble adhesion molecules occurs within the first seven days. studies show that hypoxia/reoxygenation stimulates icam-1 and apoptosis (antonova et al. 2009 ). cerebral arteriovenous malformations (avms) showed significant upregulation of e-selectin, vcam-1 and icam-1 (storer et al. 2008; chan and sukhatme 2008; tuttolomondo et al. 2009 ). li et al. (2008) showed that icam-1 lys 469 glu polymorphism was involved in the causation of ischemic stroke, especially in female but not in male (rodrigues et al. 2008) . two allelic variants were related to ischemic stroke. multivariable regression analysis after adjustment for vascular risk factors demonstrated that alleles arg of ser 128 arg and phe of leu 554 phe polymorphisms are independent risk factors for ischemic stroke. the combination of two minor alleles of e-selectin genes appeared to be the strongest susceptibility factor for ischemic stroke (haidari et al. 2009 ). sarecka-hujar et al. (2010) could not confirm the relationship between the 98 g > t polymorphism of the e-selectin gene and childhood ischemic stroke. the g allele of the e-selectin 98 g > t polymorphism was more frequently transmitted to the children after stroke compared to the t allele. there is a need for further studies in these areas. the association between blood pressure and different adhesion molecules appeared to be present in women younger than 50 years, who were likely to be pre-menopausal (miller et al. 2004a) . serum levels of e-and p-selectin in patients with essential hypertension (eh) are significantly higher than in the controls (sanada et al. 2005) . after adjustment for age, only se-selectin concentrations were significantly associated with blood pressure. higher levels of plasma sp-selectin were confirmed in hypertensive patients alone with vegf (nadar et al. 2004a) . it is stated that decrease in blood pressure may reduce the rate of progression of atherosclerosis by affecting the expression of e-and p-selectin in the endothelium, the platelets, or both. in vitro studies indicate that complement activation regulates the expression of p-selectin on endothelial cells. this suggests that in disorders such as ischemia/reperfusion injury, in which both complement and p-selectin have been shown to play a role, complement activation is a primary event and the effects of p-selectin are secondary. in mouse kidney model of i/r injury, results indicated that complement and p-selectin-mediated pathways of renal reperfusion injury are mutually independent (farrar et al. 2004 ). induction of circulating polymorphonuclear neutrophils (pmns) might contribute to the superior outcome following stenting and early intervention compared to conventional balloon angioplasty (ptca). a substantial increase in se-selectin levels early after ptca and stent implantation may predict development of restenosis (heider et al. 2006; kilickap et al. 2004) . after reperfusion of myocardial vessels, p-selectin expressed on majority of vessels (77 %) though the expression decreased during subsequent remaining duration of reperfusion (chukwuemeka et al. 2005) . in rats, the mrna expression for several genes was associated with inflammation after transient middle cerebral artery occlusion (mcao). gene expression increased in the injured hemisphere for il-1b, il-6 and icam-1. tnf-a mrna was upregulated in the injured versus uninjured hemisphere, while e-selectin mrna showed a significant increase from 6 to 24 h after mcao (berti et al. 2002) . both p-selectin and lfa-1 may be important targets to control pathologic inflammation in i/r-induced tissue injury in the colon (riaz et al. 2002) . the study in intestinal ischemia and reperfusion injury (ir/i) using murine models demonstrated the importance of p-selectin in warm and cold ir/i. the blockade of p-selectin using rpsgl1-lg or the absence of p-selectin ko mice confers a survival advantage and reduction in tissue injury. the mechanism appears to be independent of neutrophil infiltration (carmody et al. 2004 ). enterocyte apoptosis is increased following intestinal i/r injury. hyperoxia following intestinal i/r in rat increased e-selectin expression in the jejunum and ileum and a concomitant increase in neutrophil recruitment in the ileum, accompanied by increased cell apoptosis (braun et al. 2004; sukhotnik et al. 2008) . germ cell-specific apoptosis that occurs after i/r of murine testis is dependent on neutrophil recruitment to the testis and is dependent on e-selectin. blockage of e-selectin may be a strategy to treat postischaemic testis (celebi and paul 2008) . allergic inflammation is characterized by recruitment of specific leukocyte subpopulations from blood into tissue and requires a series of cell adhesion-molecule-mediated interactions between postcapillary vascular endothelium and the leukocyte cell surface. three major groups are involved: selectins, integrins, and the immunoglobulin gene superfamily. p-and e-selectin mediate initial leukocyte adhesion, whereas beta 2-integrin/icam-1 and vla-4/ vcam-1 pathways mediate leukocyte arrest and transendothelial migration. because vla-4 expression is restricted to eosinophils and lymphocytes, vcam-1 has been implicated in selective eosinophil recruitment characterizing allergic inflammation. however, additional factors such as profile of cytokine release are likely to operate since tissue eosinophilia has been observed in the absence of vcam-1 expression (smith et al. 1993a ). e-selectin is highly expressed on vascular endothelium in atopic dermatitis and psoriasis, and in patients with measles. the cutaneous lymphocyte-associated antigen (cla), which is expressed on peripheral skin-homing helper memory t cells in healthy persons, is at least partly the sialyl 6-sulfo le x determinant (ohmori et al. 2006 ) and a ligand for selectins. the differential polyadenylation of e-selectin transcripts may provide the molecular basis for the observed chronic expression of e-selectin in human dermal disorders. in atopic dermatitis, patients express icam-1 and icam-3, e-selectin and l-selectin (60 %) in the dermis, without expression of e-and l-selectins in the epidermis. a high expression of adhesion molecules in the skin lesions of atopic dermatitis patients may play an important role in the pathogenesis of atopic dermatitis (lugovic et al. 2006) . the blood markers for atopic dermatitis, including soluble forms of eselectin, vcam-1 and icam-1 were reduced after treatment with cetiridine (izu and tokura 2005) . the extracts from dust mites, dermatophagoides farinae, d. pteronyssinus and euroglyphus maynei with and without endotoxin (lps) stimulated endothelial cells to express icam-1, vcam-1, and e-selectin and to secrete il-6, il-8, mcp-1, and gm-csf. serum levels of se-selectin are higher in children with measles than in children with atopic dermatitis, atopic asthma and healthy controls. but it was not correlated with measles. there was no correlation between se-selectin and tnf-a level (park et al. 2008) . pollinosis from parietaria judaica is one of the main causes of allergy in the mediterranean area. the treatment of endothelial cells with pollen extract causes an increase of e-selectin and vcam-1 protein levels as well as an increase of il-8 production. the stimulation of cell adhesion molecules was paralleled by an increase of adhesion of polymorphonuclear cells (pmns) to hmvec-l monolayer (taverna et al. 2008 ). allergic rhinitis is an inflammatory disease of the nasal mucosa, caused by an ige-mediated reaction after exposure to the allergen. persistent inflammation is induced by the presence of an inflammatory cell infiltrate, together with icam-1 expression in the epithelial cells of the mucosa exposed to the allergen to which they are sensitized, in the absence of clinical symptoms (montoro et al. 2007 ). nasal polyposis is a chronic non-infectious inflammatory disease of the nasal and paranasal cavity mucosa. eosinophil migration from blood stream to nasal polyps involves different molecules such as icam-1, vcam-1, and l-, p-and eselectins. patients with nasal polyposis exhibit a higher expression of vcam-1, e-selectin, and l-selectin compared to healthy controls . staphylococcal enterotoxin a (sea) and staphylococcal enterotoxin b (seb) infection increased icam-1 expression and cytokine secretion (wang et al. 2007 ). excessive leukocyte accumulation is involved in the pathogenesis of the sepsis-induced acute lung injury. studies suggest that p-selectin has a substantial role in the pathogenesis of the lung injury induced by lps (ohnishi et al. 1999 ). in bleomycin-induced fibrosis in mice, the l-selectin and/or icam-1 deficiency inhibited skin and lung fibrosis with decreased th2 and th17 cytokines and increased th1 cytokines. in contrast, p-selectin deficiency, e-selectin deficiency with or without p-selectin blockade, or psgl-1 deficiency augmented the fibrosis in parallel with increased th2 and th17 cytokines and decreased th1 cytokines. yoshizaki et al. (2010) suggest that l-selectin and icam-1 regulate th2 and th17 cell accumulation in skin and the lung, leading to the development of fibrosis, and that p-selectin, e-selectin, and psgl-1 regulate th1 cell infiltration, resulting in the inhibition of fibrosis (yoshizaki et al. 2010) . adult respiratory distress syndrome (ards) appears to develop as the acute lung injury in the course of many severe diseases, as the result of damage of alveolar-capillary barrier. clinical observations suggest that analysis of e-, p-selectin and icam-1 concentrations in the serum of patients with ards may be helpful in monitoring the course and treatment of the disease (skiba-choińska and rogowski 1996). in the pathogenesis of paracoccidioidomycosis, gonzalez et al. (2005) suggest that during early stages, up-regulation of icam-1, vcam-1, cd18 and mac-1 expression may participate in the inflammatory process. the house dust mite (hdm) is the common indoor allergen associated with bronchial asthma. icam-1, vcam-1, and e-selectin are newly synthesized prior to spontaneous asthma attacks, and their expression may play a key role in eosinophil infiltration into the airway (ohkawara et al. 1995) . crude extract of d. farinae induces icam-1 expression in eol-1 cells through signaling pathways involving both nf-kb and jnk ). kirchberger et al. (2006) demonstrated that signaling via icam-1 induces adhesiveness of mononuclear phagocytes, which critically involves pecam-1 and is mediated via lfa-1/icam-3. the most common acute infection in humans, human rhinovirus (hrv) is a leading cause of exacerbations of asthma and chronic obstruction pulmonary disease. icam-1 is a critical target-docking molecule on epithelial cells for 90 % hrv serotypes. icam-1 regulates not only viral entry and replication but also signaling pathways that lead to inflammatory mediator production (lau et al. 2008; lee et al. 2008 ). the sicam-1 but not se-selectin from patients with asthma is significantly higher than healthy controls. although serum levels of sicam-1 are higher in asthmatics, it may be necessary to establish individual baseline values for serial estimation to evaluate their clinical relevance (bijanzadeh et al. 2009 ). the serum levels of sicam-1 were significantly higher in obese nonasthmatic and obese asthmatic children versus control and lean asthmatic children . p-selectin is an important controller of the inflammation by mediating selective eosinophil cell influx to the lung. it can be used as a sensitive marker in mild asthma (sjosward et al. 2004 ). adhesion molecule expression and interactions are involved in initiation and propagation of autoimmune diseases including rheumatoid arthritis (ra), systemic lupus erythematosus, sj€ ogren's syndrome, autoimmune thyroid disease, multiple sclerosis, systemic sclerosis (ssc) and diabetes mellitus. increased adhesion molecule expression and avidity changes occurring with cellular activation are the principal methods regulating leukocyte adhesion. although differences between specific autoimmune diseases exist, key interactions facilitating the development of autoimmune inflammation appear to include l-selectin/p-selectin/e-selectin, lfa-1/ icam-1, very late antigen-4 (vla-4)/vcam-1, and a4b7/ madcam or vcam-1 adhesion. a vast array of adhesive interactions occurs between immunocompetent cells, endothelium, extracellular matrix, and target tissues during the evolution of an autoimmune disease. dermatitis herpetiformis (dh) and bullous pemphigoid (bp), the autoimmune diseases, are characterized by destruction of the basement membrane zone (bmz) and anchoring fibres by autoantibodies and infiltration. skin biopsies from patients with dh, with bp, and from healthy subjects showed the expression of e and l selectins mainly in the skin leukocytes in all samples where as b1, b3 integrins was detected mainly in basal keratinocytes. integrins and selectins seem to play an important role in the destruction of bmz in dh and bp (erkiert-polguj et al. 2009 ). p-selectin levels were significantly higher than normal in ra and ssc, but not in sle. in contrast, mean l-selectin levels were significantly higher than normal in sle, but not in ra or ssc. where as soluble il-2 receptors in patients with active ra, ssc and sle were almost double the normal level, showing a strong positive correlation only between l-selectin and sil-2r, and only in patients with sle. these findings indicated a distinct pattern of immune cell activation in chronic diseases that share an over-activation of t-lymphocytes (sfikakis and mavrikakis 1999). adhesion molecules have been implicated in the development and progression of cardiovascular disease, particularly in people with diabetes. diabetes mellitus type 1 (type 1 diabetes or t1dm, also called insulin-dependent diabetes mellitus-iddm, or, formerly, juvenile diabetes) is a form of diabetes mellitus that results from autoimmune destruction of insulin-producing b cells of the pancreas. the chronic hyperglycemic state in t1dm patients produces an aggression to vascular endothelium leading to a premature development of atherosclerosis. in both boys and girls, seselectin is an early marker of endothelial dysfunction and a probable risk marker of atherosclerosis in children with t1dm (carrizo et al. 2008) . the levels of c-reactive protein, e-selectin, and cytokines in association with severity index were significantly increased in t1dm and type 1 diabetic patients with microvascular complications (t1dm-mv patients) compared with control subjects (devaraj et al. 2007 ). nerve microvasculitis and ischemic injury appear to be the primary and important pathogenic alterations in lumbosacral radiculoplexus neuropathy (lrpn) of patients with diabetes mellitus (dlrpn) and without diabetes mellitus (lrpn). the up-regulation of inflammatory mediators target different cells at different disease stages and that these mediators may be sequentially involved in an immune-mediated inflammatory process that is shared by both dlrpn and lrpn (kawamura et al. 2008) . adhesion molecules are upregulated in endothelial cells of the placental bed in pregnancies complicated by t1dm in association with increased adherence of peripheral blood monocytes. the increase in monocyte adhesion to decidual endothelial cells from diabetic pregnancies was associated with increased endothelial cell expression of icam-1, but not vcam-1. icam-1 expression in normal decidual endothelial cells was stimulated by pro-atherogenic and proinflammatory stimuli (xie et al. 2008; telejko et al. 2009 ). type 2 diabetes: in contrast to t1dm, type 2 diabetes mellitus (t2dm) results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency (formerly referred to as non-insulin-dependent diabetes mellitus, niddm for short). endothelial dysfunction in type 2 diabetic patients is associated with inflammation, increased levels of circulating soluble adhesion molecules (vcam-1 and e-selectin), and inducing production of ros, and urinary albumin excretion (potenza et al. 2009 ). diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment (riad et al. 2008; west et al. 2008) . serum levels of cams in diabetic patients: abnormal levels some of serum icam-1, vicam-1, e-selectin, pselectin, l-selectin have been detected in t2dm. high-fat load and glucose alone produce an increase of nitrotyrosine, icam-1, vcam-1, and e-selectin plasma levels in normal and diabetic subjects. a decrease in neutrophil surface cd62l expression and significantly higher concentrations of sicam-1, svcam-1, se-selectin, vwf, hscrp, il-6 and fibrinogen in patients with diabetic microangiopathy in comparison with diabetic group without microangiopathic complications and healthy controls suggested that: (1) diabetic microangiopathy is accompanied by increase in cd11b expression and decrease in cd62l (l-selectin) expression on peripheral blood neutrophils; (2) neutrophil activation and intensified adhesion; (3) the development of diabetic microangiopathy is accompanied by an increase in soluble adhesion molecules and inflammatory markers concentrations in the blood (lim et al. 2004; mastej and adamiec; 2008) . levels of e-selectin positively correlated with high triglyceride levels in type 2 diabetic subjects with silent ischemia (adamikova et al. 2008; okapcova and gabor 2004; rubio-guerra et al. 2008) . though, baseline plasma levels of vascular markers (hscrp, sicam-1, svcam-1, e-selectin and p-selectin) were significantly elevated, they did not improve after aerobic exercise. the se-selectin and vwf are elevated in chronic heart failure patients with dm but not in those without dm. high seselectin levels may be associated with ischaemic events in patients with dm (kistorp et al. 2008) . diabetic nephropathy (dn) and diabetic heart: diabetic nephropathy (dn) is the leading cause of end stage renal disease (esrd) (malatino et al. 2007) . although the pathogenesis of dn is multifactorial, local inflammatory stress may result from both the metabolic and hemodynamic derangements observed in dn. the current evidence supporting the role of inflammation in the early phases of clinical and experimental dn has been reviewed (fornoni et al. 2008) . inflammatory markers such as il-18 and tnf-a are increased in the serum of patients with diabetes and dn. this occurs at an early stage of disease, and correlates with the degree of albuminuria. the pharmacologic interventions for dn by angiotensin converting enzyme inhibitors, angiotensin receptor blockers and aldosterone antagonists may have anti-inflammatory effects, which are independent of their hemodynamic effect. diabetic retinopathy: the association between soluble adhesion molecules levels and retinopathy in type 2 diabetic patients has been clarified. se-selectin levels are elevated in diabetic patients compared to control subjects, with no significant difference in sicam-1 and svcam-1 levels. the progression of retinopathy was not associated with an increase in soluble adhesion molecules. however, nowak et al. (2008) observed that serum levels of sicam-1 and selam-1 were significantly elevated and the concentration svcam-1 was elevated but not significantly in diabetic patients. increase in sicam-1 and svcam-1 levels, as well as their correlation with high vitreous il-6 and tnfa concentrations in patients with diabetic retinopathy seems to confirm the inflammatory-immune nature of this process. significantly increased tnf-a concentration in the vitreous body was related to the rise of vcam-1 (adamiec-mroczek and oficjalska-młyńczak 2008; leal et al. 2008; khalfaoui et al. 2008) . intravitreal injection of corticosteroid has been used to treat diabetic macular edema. plasma levels of vwf and sp-selectin (but not se-selectin) are significantly higher among rheumatoid disease (rd) patients compared to controls. levels of vwf progressively rise with increasing cardiovascular risk (bhatia et al. 2009 ). serum levels of icam-1, icam-3, vcam-1, l-selectin, and e-selectin have been detemined in children with a variety of pediatric rheumatic diseases. a trend toward higher levels of se-selectin was found in vasculitis vs other diagnoses. the sicam-1 was higher in patients with active vs inactive disease across all diagnoses. report suggests that (1) elevated e-selectin levels in vasculitis likely reflect the high degree of endothelial activation and possibly overt vascular damage in those conditions. (2) the correlation of sl-selectin with c4 in sle may indicate that downregulation of shedding of cell surface l-selectin is involved in continued adherence of leukocytes to endothelium, possibly causing further damage and immune complex deposition in this condition. (3) the trend toward inverse correlation between se-selectin and vwf:ag in diabetes mellitus is interesting. (4) levels of sicam-i may be a useful marker of active vs quiescent disease in general in the pediatric rheumatic diseases, although lack of correlation with disease activity indices indicates that it is too insensitive to smaller differences in disease activity to be recommended for routine clinical use (bloom et al. 2002) . considerable evidence indicates that patients with rheumatoid arthritis (ra) are at greater risk of developing atherosclerosis and cardiovascular disease. atherosclerotic cardiovascular mortality is increased in ra patients. the markers proposed for assessing ra activity include rheumatoid factor, anti-citrullinated protein/peptide antibodies, igm anti-igg advanced glycation end products, markers of bone/ cartilage metabolism, mannose-binding lectin, e-selectin, il-6, and leptin. various studies have investigated the correlation between some of these markers and other variables that might indicate disease activity, e.g., inflammatory activity tests and disease activity scores. however, there is as yet insufficient evidence that any of these markers, in isolation or in combination, are useful in the assessment of ra activity. many numerous endothelial cells become positive for eselectin and e-selectin mrna in ra synovial membranes and the e-selectin expression appeared to correlate with inflammatory activity. p-selectin deficiency in mice resulted in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. mice deficient either in e-selectin or in e-selectin and p-selectin (e/p-selectin mutant) also exhibit accelerated development of arthritis compared with wild type mice in cia model. the strong vascular expression of e-selectin indicates an activation of endothelial cells in the recruitment of cells associated with the chronic inflammation of ra (foster et al. 2009; da mota et al. 2009 ). e-selectin and icam-1 are upregulated on the synovial endothelium, while vcam-1 plays an important role in synovial lining layer cells and within the synovial stroma. the expression of cams may be blocked by mabs and modified by nonsteroidal anti-inflammatory drugs and disease-modifying antirheumatic drugs. (cobankara et al. 2004) . serum soluble adhesion molecules concentrations are down-regulated following anti-tnf-a antibody therapy combined with methotrexate (mtx) (klimiuk et al. 2004 (klimiuk et al. , 2007 lev€ alampi et al. 2007; bosello et al. 2008) . in comparison with osteoarthritis (oa), patients with early ra are characterized by high serum concentrations of sicam-1, svcam-1, and se-selectin (yildirim et al. 2005) , while ldl-cholesterol was decreased in all ra patients (pemberton et al. 2009 ). p-selectin deficiency in mice results in accelerated onset of joint inflammation in the murine collagen-immunized arthritis model. mice deficient either in e-selectin or in e-selectin and p-selectin (e/pselectin mutant) also exhibit accelerated development of arthritis compared with wild type mice, suggesting that these adhesion molecules perform overlapping functions in regulating joint disease. ruth et al. (2005) suggested that eselectin and p-selectin expression can significantly influence cytokine and chemokine production in joint tissue, and that these adhesion molecules play important regulatory roles in the development of ra in e/p-selectin mutant mice (singh et al. 2008) . in ra patients, p-selectin expression, pmc and scd40l levels were increased when compared with controls. the increase in markers of active platelets, p-selectin and scd40l, and platelet-monocyte levels might be associated with the increased cardiovascular mortality in ra. psoriatic arthritis (psa) is associated with the development of endothelial dysfunction and increased atherosclerotic complications. endothelial activation might have a role in the pathogenesis of both psoriasis and psa. among parameters of platelet activation, only pmc might play a role in the pathogenesis of psa (pamuk et al. 2008 (pamuk et al. , 2009 systemic lupus erythematosus (sle): elevated serum concentrations of et-1, s-thrombomodulin (tm), and seselectin reflect persisting endothelial cell activation in sle, and point to an important role of et-1 in the pathogenesis of internal organ involvement (kuryliszyn-moskal et al. 2008 ). the s-e-selectin, tm and s-vcam-1 are significantly elevated in lupus nephritis (ln) with renal vascular lesions (vls) than in ln without vls. a positive correlation was found between tm and serum creatinine in patients with vascular lesions. therefore, serum tm and s-vcam-1 can be biomarkers of vls in ln patients (yao et al. 2008 , rho et al. 2008 ). autoimmune thyroiditis: autoimmune thyroiditis is multifactorial in etiology with genetic and environmental factors contributions. patients with untreated graves' disease (gd) show high serum level of se-selectin, which correlated with the activity of the disease. the expression of icam-1 and vcam-1 was increased in ec from patients from graves' disease (gd) and hashimoto's thyroiditis (ht). results suggest that both the lfa-1/icam-1, icam-3 and vla-4/vcam-1 pathways could play a relevant role in autoimmune thyroid disorders (marazuela et al. 1994 ). in patients with gd, the 721 g-a polymorphism was associated with an earlier age of gd onset (before age 40) and that the 1405a-g polymorphism could predispose to graves ophthalmopathy. it was concluded that g241r and k469e amino acid substitutions in the icam1 molecule could influence the intensity/duration of the autoimmunity process and the infiltration of orbital tissues (kretowski et al. 2003) . chen et al. (2008) suggested that common sele variants may be associated with susceptibility to gd in chinese population, though the limitation of sample size and multiple test problems exists (chen et al. 2008 ). sjogren's syndrome: cams are involved in the lymphoid cell infiltration of the salivary and lacrimal glands in sjogren's syndrome (ss) patients. biopsies from ss patients showed a marked expression of vcam-1 and icam-1 in the venules surrounded by infiltrated cd4 + cd45ro + t cells. e-selectin was expressed on vascular endothelium with weak intensity (saito et al. 1993) . pisella et al. (2000) reported that a significant increase of hla-dr and icam1 expression by epithelial cells was consistently found in patients with keratoconjunctivitis sicca (sjogren syndrome). these markers were well correlated with each other and correlated inversely with tear break-up time and tear production. cytokine-mediated up-regulation of vcam-1 and icam-1 that facilitates the recruitment of vla-4 and lfa-1 expressing t cells might contribute to lymphoid cell infiltration in the salivary and lacrimal glands in ss cams mediate the extravasation of leukocytes and their accumulation in inflamed intestinal mucosa. eosinophilic inflammation is a common feature of numerous eosinophilassociated gastrointestinal (egid) diseases. increased intestinal expression of e-selectin has been associated with multiple organ failure and an adverse outcome. vcam-1 is not altered in in mucosa of patients with inflammatory bowel disease (ibd) regardless of the activity of the inflammatory process. in contrast, e-selectin was not detected in normal colonic mucosa or in colonic mucosa of patients with ibd. however, high levels of e-selectin were consistently found on endothelial surfaces in association with active inflammation in affected areas of colonic mucosa in patients with either ulcerative colitis or crohn's colitis. in addition, e-selectin appeared to be present within neutrophils which had migrated into crypt abscesses in affected mucosa. thus e-selectin may play an important role in facilitating leukocyte migration into sites of active ibd involvement (koizumi et al. 1992) icam-1 was expressed to a greater degree in ulcerative colitis (uc) specimens. serum icam-1 levels in uc patients showed lower levels than those in the control group and were found to vary according to degree of clinical severity (ogawa et al. 2008) . characterization of integrin expression on colonic eosinophils revealed that colonic cc chemokine receptor 3 + eosinophils express icam-1 counter-receptor integrins al, am, and b2. it appears that b2-integrin/icam-1-dependent pathways are integral to eosinophil recruitment in colon during gi inflammation associated with colonic injury (forbes et al. 2006) . mccafferty et al. (1999) examined the role of p-selectin in intestinal inflammation in p-selectin deficient mice alone or in combination with either icam-1 or e-selectin and suggested that anti-adhesion therapy might play only a limited, beneficial role and often a detrimental role in intestinal inflammation. the se-selectin levels of crohn's disease patients with active disease are higher than those with remission of the disease. l-selectin does not change in patients with active disease compared to those with remission. thus, determination of se-selectin in children with crohn's disease is of significance in estimation of inflammation activity (adamska et al. 2007 ). khazen et al. (2009) investigated mutations in cam genes in tunisian patients, implicated in determining susceptibility to ulcerative colitis (uc) and crohn's disease (cd). a significant increase in allele frequencies of 206 l of l-selectin and the associated genotype f/l was observed in patients with uc and cd compared with controls; the l206 allele and f/l206 genotype frequencies were significantly increased in uc patients with left-sided type; whereas, the f/l206 genotype was significant in cd patients with ileocolonic location. no significant differences in allele or genotype frequencies were observed for icam-1 k469e, e-selectin, and pecam-1 polymorphisms between uc patients, cd patients, and controls. khazen et al. (2009) suggest an association of inflammatory bowel disease with allele l206 of l-selectin gene, whereas genotype l/f was associated with a subgroup of uc (left-sided type) and cd patients with more extensive location of disease and stricturing behavior. however, vischer et al. (2008) did not reveal any difference in mrna and protein expression levels for any construct or a major impact of missense variants on icam-1 biological function. pulse-chase experiments showed that two variants, k469e and arg478 to trp (r478w), had a prolonged half-life compared with wildtype icam1, whereas two other variants, g241r and pro352 to leu (p352l), had a decreased half-life, implying differences in protein degradation. celiac disease: celiac disease is a chronic intestinal inflammatory disease that develops in genetically susceptible individuals after gluten ingestion. the icam-1 gene, located in the celiac disease linkage region 19p13, encodes icam-1 involved in inflammatory processes. increased levels of icam-1 were observed in intestinal biopsies and in sera of celiac disease patients. in addition, an association between the icam1 polymorphism g241r and celiac disease patients has been described in a french population.though, in spanish population results discard the importance of icam1 g241r in celiac disease (dema et al. 2008 ). behçet syndrome: behçet's disease/syndrome (bd/bs) is a multisystemic inflammatory disorder of which oral aphthous ulceration is a major feature. cd3 and gd t-cell expression and other adhesion molecules including vcam-1 and icam-1 were upregulated, whereas cd40 showed little change in bd. the changes in cell-cell and cellextracellular matrix interactions may affect cell homeostasis and participate in the formation of oral ulcers in bd (kose et al. 2008 ). however, demirkesen et al. (2008) found no significant differences between the bs and control groups in regard to e-selectin, p-selectin, vcam-1, pncam-1 except for icam-1. systemic sclerosis or systemic scleroderma: systemic sclerosis or systemic scleroderma is a systemic autoimmune disease or systemic connective tissue disease that is a subtype of scleroderma. severe fibrosis and increased expression of profibrotic cytokines are important hallmarks in the gastric wall of patients with systemic sclerosis (ssc; scleroderma). the cd4 + /cd8 + t cell ratio is significantly increased in ssc specimens. t cells strongly express the activation markers vla-4, lfa-1, and icam-1. endothelial cells showed corresponding surface activation with strong expression of vcam-1 and icam-1. these results provide the evidence that endothelial/lymphocyte activation leading to prominent cd4 + t cell infiltration may play a key pathogenetic role within the gastric wall of patients with ssc (manetti et al. 2008) . in patients with ssc with and without pulmonary arterial hypertension (pah), serum sicam-1, svcam-1, sp-selectin and specam-1 levels were higher than in healthy donors (hd) at baseline and fell to normal values after 12 months of bosentan therapy. endothelial activation occurs in ssc, and that changes in the t cell/endothelium interplay take place in ssc-associated pah. bosentan seems to be able to hamper these changes and restore t cell functions in these patients (iannone et al. 2008 ). soluble adhesion molecules play a significant role in hepatitis. biliary atresia (ba) is a congenital or acquired liver disease and one of the principle forms of chronic rejection of a transplanted liver allograft. in the congenital form, the common bile duct between the liver and the small intestine is blocked or absent. the acquired type most often occurs in the setting of autoimmune disease, and is one of the principle forms of chronic rejection of a transplanted liver allograft. the serum se-selectin of ba patients was higher than that of controls. subgroup analysis showed that there was an increase in se-selectin levels of ba patients with jaundice compared to those without jaundice. also, se-selectin was positively correlated with serum alanine transferase (alt), a marker for liver injury, but not with serum gamma glutamyl transpeptidase (ggt) (vejchapipat et al. 2008) . cholangitis without a modifier-from greek chol-, bile + ang-, vessel + itis-, inflammation) is an infection of the bile duct (cholangitis). in secondary cholangitis, icam-1 expression is increased along with de novo vcam-1 and e-selectin appearance on the endothelium of microvessels in chronic exacerbated cholangitis (gulubova et al. 2008) . primary biliary cirrhosis (pbc) is an autoimmune disease of the liver marked by the slow progressive destruction of the small bile ducts (bile canaliculi) within the liver. patients with pbc, primary sclerosing cholangitis and chronic active hepatitis (autoimmune) show significant increase in sicam-1 compared with normal healthy subjects. significant elevation in sicam-1 is also detected in patients with inactive alcoholic cirrhosis, suggesting that impaired liver may, in part, account for the increased serum level in patients with autoimmune liver disease. in contrast, se-selectin did not differ significantly from healthy controls. although, peripheral blood mononuclear cells (pbmc) may be a source of sicam-1, thomson et al. (1994) suggested that pbmc may not be a significant source of sicam-1 in this disease. the differential expression of cams in the liver is consistent with the suggestion of selectins involvement in neutrophil rolling in the vasculature and icam-1 in transendothelial migration and adherence to parenchymal cells (essani et al. 1995) . wu et al. (2009) investigated the relationships between the polymorphisms of e-selectin gene and plasma seselectin levels in relation to disease progression in a hepatitis b virus (hbv)-infected chinese han population. the frequency of c allele (ac or cc) of the a 561 c polymorphism was significantly increased in patients with liver cirrhosis (lc) compared to normal population. there was no difference in allele distribution of the g 98 t polymorphism. the a 561 c polymorphism of e-selectin gene may be associated with disease progression in patients with chronic hbv infection and control the expression of plasma soluble levels, while the g 98 t polymorphism may be related to fibrotic severity in chinese population (wu et al. 2009 ). mice with targeted deletion of the p-selectin gene developed unpolarized type 1/type 2 cytokine responses and severely aggravated liver pathology following infection pathogen schistosoma mansoni. liver fibrosis increased 6 fold, despite simultaneous induction of ifn-g and increase in inflammation in absence of p-selectin. this suggested a critical role of p-selectin in the progression of chronic liver disease caused by schistosome parasites (wynn et al. 2004 ). axonal degeneration was confirmed as the major pathological feature of critical illness polyneuropathy (cip). expression of e-selectin was significantly increased in endothelium of epineurial and endoneurial vessels, suggesting endothelial cell activation (fenzi et al. 2003) . increasing evidence indicates that inflammatory responses are implicated in the pathogenesis of cerebral vasospasm after aneurismal subarachnoid hemorrhage (sah). murine sah model provided the evidence of effective prevention of sah-induced vasospasm by a mab implied the possible role of e selectin in the pathogenesis of vasospasm after sah (lin et al. 2005) . neuroinflammation is present in the substantia nigra (sn) of patients of parkinson disease (pd). a large number of icam-1-positive reactive astrocytes have been observed in the sn of patients with neuropathologically confirmed pd, including three of familial origin. the icam-1-positive reactive astrocytes were mainly concentrated around residual neurons in areas of heavy neuronal loss and extracellular melanin accumulation (miklossy et al. 2006 ). the svcam-1 plasma levels were higher in late onset alzheimer's disease (load) and vascular dementia (vd) compared with controls. among patients (load, vd, and not-dementia (cdnd), se-selectin levels were higher in individuals with most severe cerebrovascular disease on ct scan. increased svcam-1 plasma levels in load and vd suggest the existence of endothelial dysfunction in both types of dementia. results support the possible role of e-selectin in the pathogenesis of cerebrovascular disease (zuliani et al. 2008 ). upregulation of icam-1, lfa-1, mac-1 and subsequent leukocyte infiltration appears to be significant events of pancreatic and pulmonary injuries in acute pancreatitis (ap) . proinflammatory cytokines and oxidative stress seem to be involved in the development of local and particularly systemic complications in ap patients. acute pancreatitis patients show vcam-1 and p-selectin concentrations significantly lower and l-selectin concentrations significantly higher than the healthy subjects. only e-selectin was significantly higher in severe than in mild disease (pezzilli et al. 2008) . kleinhans et al. (2009) showed that the endothelial cell expression of pecam-1, vcam, e-selectin, and p-selectin was upregulated in severe porcine pancreatitis. in acute pancreatitis, plasma levels of se-selectin and soluble thrombomodulin (stm) serve as endothelial markers; the former is an endothelial activation marker, while the latter is an endothelial injury marker (chooklin 2009; ida et al. 2009 ). in patients affected by microscopic polyangiitis (mpa) and associated with myeloperoxidase (mpo)-anti-neutrophil cytoplasmic antibodies (anca), higher sicam-1 and seselectin levels during active phase and their slower decline during the treatment period, could be a prognostic risk factor for chronic renal failure development (di lorenzo et al. 2004; musial et al. 2005 ). an increased level of se-selectin in patients susceptible to restenosis supports a role for white blood cell/endothelial interaction in restenosis after angioplasty (sainani and maru 2005) . the impairment of vascular endothelial function was obvious in uremic patients with maintaining hemodialysis (mhd). the changes of icam-1 and e-selectin could be accepted as biochemical criterions of vascular endothelial injury . diuresis, serum creatinine, urea, and enzyme elimination are pathological among patients with acute renal failure (arf). higher elimination rates of sicam-1 and higher values of se-selectin compared to patients without arf indicated additional parameters for early signs of kidney damage (dehne et al. 2008) . both circulating and urinary tnf-a levels are increased in inflammatory chronic renal diseases. tnf-a appeared to play a crucial role in the immunopathogenesis of nephritis by the induction of chemokine, icam-1 and vcam-1 expression via the activation of the intracellular mapk signaling pathway, which may contribute to macrophage and lymphocyte infiltration li et al. 2008) . snps in selectin genes and iga nephropathy: although intensive efforts have been made to elucidate the genetic basis of ig a nephropathy (igan), genetic factors associated with the pathogenesis of this disease are not well understood. a case-control study, based on linkage disequilibrium among snps in selectin gene cluster on chromosome 1q24-25 revealed two snps in the e-selectin gene (sele8 and sele13) and six snps in the l-selectin gene (sell1, sell4, sell5, sell6, sell10, and sell11), that were significantly associated with igan in japanese patients. sele8 and sell10 caused amino acid substitutions from his to tyr and from pro to ser for his-to-tyr substitutions; and sell1 could affect promoter activity of the l-selectin gene. the tgt haplotype at these three loci was associated significantly with igan. these snps in selectin genes may be useful for screening populations susceptible to the igan phenotype. (takei et al. 2002) transplant rejection: soluble adhesion molecules are not valuable markers for stable kidney graft (stx) rejection reaction. however, patients with chronic renal failure showed increased levels of adhesion molecules, which could reflect an impaired elimination (alcalde et al. 1995) . the expression levels of icam-1 and vcam-1 show positive correlation with the severity of graft rejection and can provide evidence for early diagnosis and prevention of cr. chronic allograft failure (caf) is the major cause for late graft loss in renal transplantation. icam-1 polymorphisms may represent a predetermined genetic risk factor for caf. this was substantiated by the polymorphism in exon 4 at the mac-1 binding site and in exon 6 at fifth ig-like domain (mclaren et al. 1999) . khazen et al. (2007) found no evidence for an association of any polymorphism with acute rejection in e-and l-selectin. during kidney reperfusion, eselectin, icam-1, and vcam-1 concentrations correlated positively with hypoxanthine concentrations during reperfusion, whereas concentrations of icam-1 correlated negatively with xanthine concentrations, indicating metabolic changes in renal tissue (domanski et al. 2009 ). serum cam levels have been analyzed in many organ diseases, including diseases of nervous system, endocrine disorders and others. immune dysfunction has been proposed as a mechanism for pathophysiology of autisticspectrum disorders. levels of sp-selectin and sl-selectin were significantly lower in patients than in controls. furthermore, sp-selectin levels were negatively correlated with impaired social development during early childhood (iwata et al. 2008) . in multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (eae), inflammatory cells migrate across the endothelial blood-brain barrier (bbb) and gain access to the cns. the role of e-and p-selectin in this process has been controversial. d€ oring et al. (2007) suggest that absence of e-and pselectin did neither influence the activation of myelinspecific t cells nor the composition of the cellular infiltrates in the cns during eae. thus, e-and p-selectin are not required for leukocyte recruitment across bbb and the development of eae in c57bl/6 and in sjl mice (d€ oring et al. 2007) . no significant differences in allelic or genotypic frequency in all the snps (rs6133, rs4987310 and rs5368 substitutions) tested were found in the italian population (fenoglio et al. 2009 ). the pathophysiology of cluster headache (ch) is supposed to involve the lower posterior part of the hypothalamus, the trigeminal nerve, autonomic nerves and vessels in the orbital/retro-orbital region. remahl et al. (2008) compared serum levels of sicam-1, svcam-1 and se-selectin in patients with episodic ch and in patients with biopsypositive giant cell arteritis (gca), a vasculitic disorder of large and medium-sized arteries. within the ch group, sicam-1, svcam-1 and se-selectin showed an increasing trend in remission compared with active cluster headache period, but se-selectin only was significant. remahl et al. (2008) suggest that cluster headache is not a vasculitic disorder of medium-sized arteries, but ch patients may have an immune response that reacts differently from that of healthy volunteers. adhesion molecules have a role in many vasculitic disorders. compared to controls, takayasu's arteritis (ta) patients had elevated levels of se-selectin, svcam-1, and sicam-1. compared to controls, patients with inactive ta also had elevated levels of se-selectin, svcam-1, and sicam-1. there was no difference between active ta and controls. the se-selectin had a trend towards increased levels in inactive versus active ta, but there was no difference in svcam-1 and sicam-1 levels between the groups. patients with inactive ta had elevated levels of se-selectin, svcam-1, and sicam-1 that might indicate persistent vasculopathy in clinically inactive disease (tripathy et al. 2008 ). serum levels of svcam-1, sicam-1, stm, p-selectin, eselectin and crp levels as inflammation markers are increased in patients of b-thalassemia intermedia and not influenced by treatment (kanavaki et al. 2009 ). pseudoxanthoma elasticum (pxe) is a hereditary disorder predominantly affecting the skin, retina and vascular system. p-selectin concentrations were increased in male and female pxe patients and levels correlated with the abcc6 gene status of the patients. patients harboring two mutant abcc6 alleles had 1.5-fold increased p-selectin concentrations in comparison to patients with at least one wild-type allele. eand l-selectin levels were within normal range and the allelic frequencies did not differ between from controls. elevated p-selectin levels in pxe patients are potentially due to oxidative stress and elevated protease activity in pxe (g€ otting et al. 2008) . fabry disease, an x-linked systemic vasculopathy, is caused by a deficiency of a-galactosidase a resulting in globotriaosylceramide (gb 3 ) storage in cells. accumulation of gb 3 in the vascular endothelium of fabry disease is associated with increased production of reactive oxygen species (ros) and increased expression of cams. increased gb 3 induces expression of icam-1, vcam-1, and e-selectin. reduction of endogenous gb 3 by treatment of the cells with an inhibitor of glycosphingolipid synthase or a-galactosidase a led to decreased expression of adhesion molecules. this study indicates that excess intracellular gb 3 induces oxidative stress and up-regulates the expression of cams in vascular endothelial cells (shen et al. 2008 ). recent reports have expanded the concept that inflammation is a critical component of tumor progression. many cancers arise from sites of infection, chronic irritation and inflammation. it is now becoming clear that the tumor microenvironment, which is largely orchestrated by inflammatory cells, is an indispensable participant in the neoplastic process, fostering proliferation, survival and migration. in addition, tumor cells have co-opted some of the signaling molecules of the innate immune system, such as selectins, chemokines and their receptors for invasion, migration and metastasis. these insights are fostering new anti-inflammatory therapeutic approaches to cancer development the complexity of the tumor microenvironment has been revealed in the past decade. the cams in the process of inflammation are responsible for recruiting leukocytes onto the vascular endothelium before extravasation to the injured tissues. some circulating cancer cells have been shown to extravasate to a secondary site using a process similar to inflammatory cells. the most studied ligands for cams expressed on cancer cells, s-lewis a and s-lewis x antigens, are shown to be involved in adhesion to endothelial cells by binding to e-selectin. this process, shared by inflammatory cells and cancer cells, may partially explain the link between inflammation and tumorigenesis. the adhesion of colon cancer cells to e-selectin can be directly affected by changes in the expression level of sialosyl le a antigen. the specific lack of expression of sialosyl le a carbohydrate structure on the surface of colon cancer cells completely abolished their adhesion to e-selectin. it is proposed that glycoproteins as well as gangliosides carrying sialosyl le a structures, when properly exposed and present in high density on surface of cancer cells, can effectively support the adhesion of cancer cells to e-selectin (klopocki et al. 1998; kobayashi et al. 2007 ). in addition to endogenous ligands for l-, p-, and e-selectins (chap. 26, 27, and 28), several proteins are found in cancer cell lines or solid tumors that act as ligands for e, l, and p selectins. selectin ligands present in cancers are: (1) glycodelin a (gda) is primarily produced in endometrial and decidual tissue and secreted to amniotic fluid. gda is expressed in ovarian cancer where it can act as an inhibitor of lymphocyte activation and/or adhesion (jeschke et al. 2009 ); (2) the cysteine-rich fibroblast growth factor receptor (fgf-r) represents the main e-selectin ligand (esl-1) on granulocytes. hepatic stellate cells (hsc) are pericytes of liver sinusoidal endothelial cells, which are involved in the repair of liver tissue injury and angiogenesis of liver metastases. hsc express fgf-r together with fuct7 and exhibit a functional e-selectin binding activity on their cell surface (antoine et al. 2009 ). (3) although bcell precursor acute lymphoblastic leukemia (bcp-all) cell lines do not express the ligand psgl-1, a major proportion of carbohydrate selectin ligand was carried by another sialomucin, cd43, in nall-1 cells. cd43 plays an important role in extravascular infiltration of nall-1 cells and the degree of tissue engraftment of bcp-all cells may be controlled by manipulating cd43 expression (nonomura et al. 2008 ). (4) thomas et al. (2009a) identified podocalyxin-like protein (pclp) as an alternative selectin ligand. pclp on ls174t colon carcinoma cells possesses e-/l-, but not p-, selectin binding activity. pclp functions as an alternative acceptor for selectin-binding glycans. the finding that pclp is an e-/l-selectin ligand on carcinoma cells offers a unifying perspective on the apparent enhanced metastatic potential associated with tumor cell pclp overexpression and the role of selectins in metastasis (thomas et al. 2009b ). (5) e-selectin has been shown to play a pivotal role in mediating cell-cell interactions between breast cancer cells and endothelial monolayers during tumor cell metastasis. the counterreceptor for e-selectin was found as cd44v4. however, cd44 variant (cd44v) isoforms was functional p-, but not e-/l-selectin ligands on colon carcinoma cells. furthermore, a~180-kda sialofucosylated glycoprotein(s) mediated selectin binding in cd44-knockdown cells. this glycoprotein was identified as carcinoembryonic antigen (cea). cea serves as an auxiliary l-selectin ligand, which stabilizes l-selectin-dependent cell rolling against fluid shear (thomas et al. 2009b ). zen et al. (2008) identified a~170 kda human cd44 variant 4 (cd44v4) as e-selectin ligand, which has a high affinity for e-selectin via sle x moieties. angiogenesis plays an important role in a variety of pathophysiologic processes, including tumor growth and rheumatoid arthritis. studies on capillary morphogenesis and angiogenesis in vitro have suggested a role for e-selectin in the process of differentiation into tube-like structures. soluble e-selectin is a potent mediator of human dermal microvascular endothelial cell (hmvec) chemotaxis, which is predominantly mediated through the src and the phosphatidylinositiol 3-kinase (pi3k) pathways (kumar et al. 2003) . gastrin-17 (g17) has marked proangiogenic effects in vivo on experimental gliomas and in vitro on huvecs and transiently decreased the expression of e-selectin, but not p-selectin, whereas il-8 increased the expression of e-selectin. specific antisense oligonucleotides against e-and p-selectin decreased huvec tubulogenesis processes in vitro. this showed that gastrin has marked proangiogenic effects in vivo on experimental gliomas and in vitro on huvecs. this effect depends in part on the level of e-selectin activation, but not on il-8 expression/release by huvecs (lefranc et al. 2004 ). adhesion molecules are thought to have a role in the host defense against carcinogenesis. significantly increased p-selectin, s-vcam-i and s-icam-i levels were observed in patients with bladder cancer, and s-vcam-i levels correlated with tumor stage (coskun et al. 2006) . selectins mediate attachment of leukocytes to activated endothelium as well as the adhesion reaction of tumor cells during malignancy (borsig 2007) . in a breast tumor xenograft model, the effect of combined tnf-a and ifn-g therapy involved the selective destruction of the tumor vasculature and death of tumor cells. concomitant with these changes rt-pcr analysis revealed the increase of stromal mrna levels for a series of stromal cytokines, cytokine receptors including tnf-a, sicam-1, vcam-1, p-selectin, which could be implicated in the observed events (de kossodo et al. 1995) . squamous cell carcinomas: in order to evaluate the risk of postoperative haematogenic recurrence of esophageal squamous cell carcinoma (scc) patients, shimada et al. (2003) examined the preoperative serum levels of seselectin and pathological status of the patients. the patients with a high serum soluble e-selectin concomitant with expression of s-lewis antigens had a significant risk of postoperative haematogenic recurrence. sccs of suninduced skin cancers are particularly numerous in patients on t cell immunosuppression. blood vessels in sccs did not express e-selectin, and tumors contained few cutaneous lymphocyte antigen (cla + ) t cells, the cell type thought to provide cutaneous immunosur-veillance. clark et al. (2008) found that sccs evade the immune response at least in part by down-regulating e-selectin and recruiting t reg cells. cutaneous t-cell lymphoma (ctcl): the ctcl is characterized by accumulation of malignant cd4 + t cells in the skin. in malignant t cells from sezary syndrome (ss), a leukemic variant of ctcl, in dermal microvessels in mouse skin, hoeller et al. (2009) found that ss cells rolled along dermal venules in a p-selectin-and e-selectin-dependent manner at ratios similar to cd4 + memory t cells from normal donors. chemokine ccl17/tarc was sufficient to induce the arrest of ss cells in the microvasculature. together, experiments suggested molecular adhesion cascade operant in ss cell homing to the skin in vivo. patients with ctcl showed increased levels of sicam-1 and sicam-3 when compared with healthy individuals and patients with inflammatory dermatosis. the se-selectin and svcam-1 levels were not affected (lópez-lerma and estrach 2009). hodgkin's disease: increased sicam-1 and se-selectin have been observed in hodgkin's disease/lymphoma (hd/ hl) patients at diagnosis and svcam-1 at diagnosis correlated with both sicam-1 and se-selectin levels. chemotherapy resulted in a significant decrease of sicam-1 and se-selectin (syrigos et al. 2004 ). serum sicam-1 level increases at advanced stages of untreated multiple myeloma (mm) patients, but did not differ significantly from controls. a positive correlation of il-6 appeared with sicam-1 and se-selectin (uchihara et al. 2006) . epstein-barr virus (ebv)-positive nk/t cells showed affinity to vascular components. ebv-positive nk lymphoma cells express icam-1 and vcam-1 at much higher levels than those in ebv-negative t cell lines. furthermore, nk lymphoma cell lines exhibited increased adhesion to cultured endothelial cells stimulated with tnf-a or il-1b. the up-regulated expression of vcam-1 on cytokine-stimulated endothelial cells can be important to initiate the vascular lesions (kanno et al. 2008) . non-small cell lung cancer: serum levels of icam-1 increased in advanced stage non-small cell lung cancer (nsclc) patients, whereas se-selectin levels were not significantly different from healthy controls reports suggest that higher serum icam-1 can be useful for diagnosis while e-selectin levels have prognostic significance and could be a potential prognostic factor in nsclc patients (dowlati et al. 2008; guney et al. 2008 ). the cyfra 21-1 and se-selectin showed good performance in detecting lung cancer from normal groups. however, cyfra 21-1 was superior to se-selectin in discriminating lung cancer from benign lung diseases (swellam et al. 2008 ). maspin, a serine protease inhibitor belonging to serpin family, is known as a tumor-suppressor protein and also exhibits an inhibitor effect on angiogenesis. positive correlations were found for maspin positivity and lymph node metastases; eselectin positivity and lymph node metastases, and p-selectin positivity and lymph node metastases and lymphovascular invasion. correlations do exist between maspin, e-and p-selectin expressions with each other and with tumor stage. inactive cytoplasmic maspin cannot act as a tumor suppressor. expression of e-and p-selectins in tumor cells facilitates the occurrence of metastases, lymphovascular invasion, and perithyroidal soft tissue invasion. further studies are needed to reveal detailed interactions between maspin, e-selectin, and p-selectin expression (bal et al. 2008) . primary hyperparathyroidism: patients with primary hyperparathyroidism (phpt) have impaired vasodilation. based on small number of patients, a study suggested that classic cardiovascular risk factors seem to be the main determinants for the high plasma levels of se-selectin and vwf in phpt. together with unaltered thrombomodulin and se-selectin levels, the vwf decrease in plasma after parathyroidectomy reflects a specific mechanism of its endothelial calcium-and/or pth-stimulated secretion in some phpt patients without risk factors (fallo et al. 2006 ). colorectal cancer: plasma level of sp-selectin, seselectin and icam-1 were significantly higher in colorectal cancer (crc) patients. the highest levels of se-selectin and icam-1 were observed in patients with liver metastasis. there was no correlation between sp-selectin and seselectin, but a significant correlation was seen between seselectin and icam-1 in all patients. plasma concentration of e-selectin and icam-1 may indicate tumor progression and liver metastasis (dymicka-piekarska and kemona 2009; sato et al. 2010) . the interaction between re-selectin and crc cells alters the gene expression profile of cancer cells. a dna microarry analysis indicated that e-selectin-mediated alterations were significantly more pronounced in metastatic crc variants sw620 and km12sm than in the corresponding non-metastatic local sw480 and km12c variants. the number of genes altered by e-selectin in metastatic variants was 10-fold higher than the number of genes altered in the corresponding local variants. analysis indicated that e-selectin down regulated (at least by 1.6folds) the expression of seven genes in a similar fashion, in both metastatic cells. confocal microscopy indicated that eselectin down-regulated the cellular expression of hmgb1 protein and enhanced the release of hmgb1 into the culture medium. the released hmgb1 in turn, activated endothelial cells to express e-selectin (aychek et al. 2008) . the entrapment of malignant cells within the hepatic sinusoids and their interactions with resident nonparenchymal cells are considered very important for the whole metastatic sequence. in the sinusoids, cell connection and signaling is mediated by multiple cell adhesion molecules, such as the selectins. the three members of the selectin family, e-, p-and l-selectin, in conjunction with sialylated lewis ligands and cd44 variants, regulate colorectal cell communication and adhesion with platelets, leucocytes, sinusoidal endothelial cells and stellate cells. therefore, trials have already commenced aiming to exploit selectins and their ligands in the treatment of benign and malignant diseases. multiple pharmacological agents have been developed that are being tested for potential therapeutic applications (schnaar et al. 2008; paschos et al. 2010; zigler et al. 2010 ). the degree of selectin ligand expression by cancer cells is well correlated with metastasis and poor prognosis for cancer patients. initial adhesion events of cancer cells facilitated by selectins result in activation of integrins, release of chemokines and are possibly associated with the formation of permissive metastatic microenvironment. while eselectin is one of the initiating adhesion events during metastasis, it is becoming apparent that p-selectin and lselectin-mediated interactions significantly contribute to this process as well (gout et al. 2008; l€ aubli and borsig 2010 ). extravasation of cancer cells is a pivotal step in the formation of hematogenous metastasis. extravasation is initiated by the loose adhesion of cancer cells to endothelial cells via an interaction between endothelial selectins and selectin ligands expressed by the tumor cells. metastatic spreading is a dreadful complication of neoplastic diseases that is responsible for most deaths due to cancer. it consists in the formation of secondary neoplasms from cancer cells that have detached from the primary site. leukocytes and tumor cells use e-selectin binding ligands to attach to activated endothelial cells expressing e-selectin during inflammation or metastasis. the formation of these secondary sites is not random and several clinical observations indicate that the metastatic colonization exhibits organ selectivity. this organ tropism relies mostly on the complementary adhesive interactions between cancer cells and their microenvironment. e-selectin and slewis antigens might play important role in breast tumor, lymph node and liver metastasis. high levels of se-selectin have been reported in melanoma and some epithelial tumors, especially in colorectal carcinoma. but se-selectin may not be used as a predictive marker of metastasis in colorectal carcinoma, though high levels of se-selectin may support diagnosis of liver metastasis (uner et al. 2004; eichbaum et al. 2004) . it appeared that serum levels of se-selectin are associated with the clinical course of liver metastases from breast cancer. eichbaum et al. (2004) observed a possible trend for certain unfavorable prognostic parameters (e.g., young women, low-graded tumors, human epidermal growth factor receptor 2 over-expression) that could be related to higher serum levels of se-selectin. role of e-selectin in diapedesis of cancer cells: diapedesis is a vital part of tumor metastasis, whereby tumor cells attach to and cross the endothelium to enter the circulation. e-selectin was found to regulate initial attachment and rolling of colon cancer cells and also the subsequent diapedesis through the endothelium. evidence indicates that eselectin-dependent paracellular extravasation is independent of icam and vcam and that it requires the activation of extracellular signal-regulated kinase (erk) mitogenactivated protein kinase downstream of e-selectin. studies establish the role of e-selectin in diapedesis of circulating cancer cells woodward 2008) . polymorphisms within e-selectin gene, especially the s 128 r polymorphism, may increase the risk of metastases by facilitating adhesion of tumor cells to endothelium. blood dna from patients treated for stage ii or iii colorectal cancer (crc) and from healthy controls was assessed for three polymorphisms within e-selectin gene (s 128 r, g 98 t and l 554 f) and one within the p-selectin gene (v 640 l). the s 128 r polymorphism was detected in 22.3 % patients and was correlated with g 98 t polymorphism. in multivariate analysis, the s 128 r polymorphism was associated with shorter event-free survival (efs) and overall survival (os) in whole population, in patients with stage ii crc, and in patients with stage iii crc. l 554 f and v 640 l polymorphisms had no prognostic value. the s 128 r polymorphism is a constitutional factor associated with a higher risk of relapse and death in patients treated for crc and its detection may permit better selection of patients suitable for adjuvant therapy, especially among those with stage ii disease (hebbar et al. 2009 ). metastasis is thought to involve the formation of tumor-platelet-leukocyte emboli and their interactions with the endothelium of distant organs. a link between these observations shows that p-selectin, which normally binds leukocyte ligands, can promote tumor growth and facilitate the metastatic seeding of a mucinproducing carcinoma. p-selectin-deficient (p-sele à/à ) mice showed three potential pathophysiological mechanisms: (1) intravenously injected tumor cells home to the lungs of psele à/à mice at a lower rate; (2) p-sele à/à mouse platelets fail to adhere to tumor cell-surface mucins; and (3) tumor cells lodged in lung vasculature after intravenous injection often are decorated with platelet clumps, and these are markedly diminished in p-selectin à/à animals (kim et al. 1998 ). however, the surgical procedure did not totally eliminate the factors responsible for platelet activation and did not normalize platelet activation (dymicka-piekarska et al. 2005; hanley et al. 2006 ). role of sialyl-lewis antigens: during inflammation, eand p-selectins appear on activated endothelial cells to interact with leukocytes through sialyl-lewis x (sle x ) and sialyl-lewis a (sle a ). these selectins can also interact with tumor cells in a sialyl-lewis-dependent manner and hence, they are thought to play a key role in metastasis. diverting the biosynthesis of sialyl-lewis antigens toward nonadhesive structures is an attractive gene therapy for preventing the hematogenous metastatic spread of cancers. the transduced a1,2-fucosyltransferase-1 (fut1) efficiently fucosylated the p-selectin ligand psgl-1 without altering p-selectin binding . the metastasis of cancer cells and leukocyte extravasation into inflamed tissues share common features. carbohydrate antigen sle a (ca19-9) is the most frequently applied serum tumor marker for diagnosis of cancers in the digestive organs. the normal counterpart of the determinant, namely disialyl-le a is predominantly expressed in non-malignant epithelial cells of the digestive organs. the disialyl-le a determinant carries one extra sialic residue attached through a 2 ! 6 linkage to glcnac moiety compared to cancerassociated sle a , which carries only one 2 ! 3 linked sialic acid residue (monosialyl lewis a) (fig. 44.3) . disialyl-le a in normal epithelial cells serves as a ligand for immunosuppressive receptors such as sialic acid binding ig-like lectins (siglec-7 and -9) expressed on resident monocytes/ macrophages and maintains immunological homeostasis of mucosal membranes in digestive organs. epigenetic silencing of a gene for a 2 ! 6 sialyl-transferase in the early stages of carcinogenesis results in impairment of 2 ! 6 sialylation, leading to incomplete synthesis and accumulation of sle a , which lacks the 2 ! 6 linked sialic acid residue, in cancer cells. simultaneous determination of serum levels of sle a and disialyl-le a , and calculation of the sle a / disialyl-le a ratio provides information useful for excluding a false-positive serum diagnosis. during cancer progression in locally advanced cancers, tumor hypoxia induces transcription of several glyco genes involved in sle a synthesis. expression of the determinant, consequently, is further accelerated in more malignant hypoxia-resistant cancer cell clones, which become predominant clones in advanced stage cancers and frequently develop hematogenous metastasis. sle a , as well as its positional isomer sle a , serves as a ligand for vascular e-selectin and facilitates hematogenous metastasis through mediating adhesion of circulating cancer cells to vascular endothelium. patients having both strong sle a expression on cancer cells and enhanced e-selectin expression on vascular beds are at a greater risk of developing distant hematogenous metastasis (kannagi 2007) . in a human-mouse model, the selectin ligand s-le a is involved in in vivo extravasation of colorectal carcinoma (crc) cells. highly metastatic crc cells expressing high levels of s-le a extravasate more efficiently than non-metastatic crc cells expressing low levels of s-le a . down-regulating the expression of s-le a in crc cells by genetic manipulations, significantly reduced crc extravasation. the arrest and adhesion of crc cells, and possibly of other types of cancer cells as well, to endothelium depend on the expression of the selectin ligand sle a by the tumor cells (ben-david et al. 2008) . 3'-sulfo-le a is known to be the potent ligand of e-selectin which is important in cell adhesion and migration. the serum 3'-sulfo-le a can provide important information in patients with primary gastric cancer, which might be useful as a predictive marker especially for the detection of tumor metastasis (zheng et al. 2009 ). specialized carbohydrates modified with sle x antigens on leukocyte membranes are ligands for selectin adhesion molecules on activated vascular endothelial cells at inflammatory sites. the sle x expression of invasive micropapillary carcinoma was higher than that of invasive ductal carcinoma, which was also associated with lymph node metastasis. e-selectin combined with sle x might play an important role in lymph node metastasis in invasive micropapillary carcinoma. the expression pattern of sle x in invasive micropapillary carcinoma suggested that the reversal of cell polarity of invasive micropapillary carcinoma might be as an important factor for the morphogenesis and possibly the pathogenesis, especially their higher rates of lymph node metastasis (wei et al. 2010) . the activity of core 2 b1,6 n-acetylglucosaminyltransferase (c2gnt1) in leukocytes greatly increases their ability to bind to endothelial selectins. c2gnt1 is essential for the synthesis of core 2-branched o-linked carbohydrates terminated with sle x (c2-o-sle x ). e-selectin and its ligand-sle x are closely correlated with the metastasis of hepatocellular carcinoma. c2-o-sle x is a potentially useful early predictor of metastasis (zhang et al. 2002) . the expression profiles of c2-o-sle x in the malignant progression and metastasis of colorectal adenocarcinomas is upregulated in colorectal adenocarcinomas and metastatic liver tumors (st hill et al. 2009 ). endothelial p-selectin as a target of heparin action: metastasis can be effectively inhibited by the anticoagulant heparin in different tumor models. at the cellular level, many of the antimetastatic effects of heparin in vivo are due to its action on p-selectin-mediated binding. ludwig et al. (2007) addressed the potential contribution of endothelial p-selectin expression to adhesive events between the microvasculature and melanoma cells in vivo. heparin not only inhibits p-selectin-mediated melanoma cell rolling but also attenuates melanoma metastasis formation in vivo, supporting the concept that endothelial p-selectin expression may represent an additional target of heparin in experimental melanoma lung metastasis (ludwig et al. 2007) . the low molecular weight heparin (lmwh) significantly improved colonic inflammation in rats with trinitrobenzene sulphonic acid (tnbs) induced colitis. the effect is possibly related to inhibition of proinflammatory cytokine il-8, but not involved platelet surface p-selectin expression (xia et al. 2004 ). the survival benefits in patients with cancer treated with lmwh may result from a lmwhmediated effect on the immune system or on the cross-talk between platelets and tumor cells. however, survival observed with lmwh in patients with cancer apparently cannot be explained by a lmwh effect on these circulating markers (di nisio et al. 2005) . nonetheless, in vivo antimetastatic effects of heparins reflect their action on pselectin-mediated binding. therefore, these commonly used anticoagulants widely differ in their potential to interfere with p-selectin mediated cell binding. importantly, the superior inhibitory capacity on p-selectin function of unfractionated heparin and lmwh nadroparin as opposed to lmwh enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. hence, p-selectin inhibition constitutes a valuable feature to identify anticoagulants that are suitable for anticancer therapy (ludwig et al. 2006) . stevenson et al. (2005) studied metastasis inhibition by clinically relevant levels of various heparins and investigated the structural basis for selectin inhibition differences. five clinically approved heparins were evaluated for inhibition of p-selectin and l-selectin binding to carcinoma cells and showed differing abilities to inhibit selectins, likely explained by size distribution. it should be possible to size fractionate heparins and inhibit selectins at concentrations that do not have a large effect on coagulation. gao et al. (2005) prepared periodate-oxidized, borohydride-reduced heparin (ro-heparin) and tested its anticoagulant and anti-inflammatory activities. compared with heparin, ro-heparin had greatly reduced anticoagulant activity. intravenous administration of this compound led to reduction in the peritoneal infiltration of neutrophils in a fig. 44.3 structures of three carbohydrate determinants, disialyl lewis x (le x ), sialyl lewis a and lewis a (le a ). in panel, note that the only difference between the three determinants is the linkage of sialic acid residues. the a(2 ! 6) sialic acid residue in disialyl le a is synthesized by a sialyltransferase st6galnacvi, which shows a significant decrease in its mrna level upon malignant transformation mouse acute inflammation model. in vitro studies showed that the effect of ro-heparin on inflammatory responses was mainly due to inhibiting the interaction of p-selectin with its ligands. these results indicate that ro-heparin may be a safer treatment for inflammation than heparin, especially when selectin is targeted. to clarify the mechanism of heparin antimetastatic activity, several biological effects are being investigated. cancer progression and metastasis are associated with enhanced expression of heparanase, which is inhibited efficiently by heparin. heparin is also a potent inhibitor of selectinmediated interactions. p-and l-selectin were shown to contribute to the early stages of metastasis, which is associated with platelet-tumor cell thrombi formation. low anticoagulant heparin preparations still inhibited metastasis efficiently indicating that anticoagulation is not a necessary component for heparin attenuation of metastasis. modified heparins characterized for heparanase inhibitory activity are also potential inhibitors of selectins. selectin inhibition is a clear component of heparin inhibition of metastasis. the contribution of selectin or heparanase inhibition by heparin can provide evidence about its antimetastatic activity (borsig 2007) . one of the mechanisms by which heparin inhibits metastasis is by blocking the p-selectin-based interaction of platelets with tumor cell. the sulfate groups at c6/ n and especially c6, but not c2 and c3, of heparin play a critical role in p-selectin recognition and that 2-o,3-odesulfated heparin can block p-selectin-mediated a375 human melanoma cell adhesion. thus chemical modification of heparin, especially 2-o,3-o-desulfation, may result in a therapeutic agent that is anti-metastatic because it blocks unwanted p-selectin-dependent adhesion but that lacks dose-limiting anticoagulant effects . heparin-induced thrombocytopenia: the pathophysiology of heparin-induced thrombocytopenia (hit) is a complex process which involves platelets, vascular endothelium, and leukocytes. the activation products from these sites also contribute to the activation of coagulation and to the fibrinolytic deficit. many of the markers of hemostatic activation processes have been found to be at increased levels during acute phases of the hit syndromes. since the pathophysiology of hit involves the activation of platelets, endothelium, and leukocytes, it is expected that activation products related to these hemostatic systems, including soluble selectins, will also be increased in circulating blood. these alterations may provide an index of the pathophysiologic process. fareed et al. (1999) reviewed on the circulating levels of p-, e-, and l-selectins in hit patients and their modulation after therapeutic intervention. with the availability of recombinant hirudin, it is now possible to provide alternate anticoagulants to hit patients. however, fareed et al. (1999) suggest that the immunoactivation of platelets and other cells may require additional adjunct therapeutic approaches. activated protein c (apc) is the major physiological anticoagulant with concomitant anti-inflammatory properties. turunen et al. (2005) suggest that apc has an antiinflammatory role in i/r injury in clinical renal transplantation (turunen et al. 2005) . bimosiamose prolongs survival of kidney allografts. binding of the p-, l-, and e-selectins to sle x retards circulating leukocytes, thereby facilitating their attachment to the blood vessels of allografts. selectin inhibitor bimosiamose (bimo) inhibits the rejection process of kidney allografts in a rat model in association with reduced intragraft expression of p-selectin glycoprotein ligand-1, cx (3)cl1, ccl19, ccl20, and ccl2. thus, bimo blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines (langer et al. 2004 ). brain death (bd), a significant antigen-independent process, the donor-related injury up-regulates variety of inflammatory mediators in peripheral organs. one of the immediate responses is the expression of selectins by endothelial cells of the transplanted tissues, which in turn trigger a cascade of nonspecific events that may enhance host alloresponses. using a rat model in which donor bd accentuates subsequent renal allograft injury, gasser et al. (2005) tested the effects of therapy with rpsgl-ig alone, or in combination with sirolimus (srl) and cyclosporin a. it was found that in contrast to the effects of standard doses of srl or cyclosporine, rpsgl-ig decreased inflammation in the early posttransplant period such that lower doses of maintenance immunosuppression were sufficient to maintain long-term graft function. intestinal transplantation (itx) is severely limited by ischemia-reperfusion (i/r) injury. t lymphocyte is an important regulatory cell in this inflammatory process (farmer et al. 2005a) . rpsgl-ig treatment leads to marked improvement in the outcome. the mechanism of action seems to involve the blockade of neutrophil and lymphocyte infiltration that leads to a decreased inflammatory response possibly driven by th2 cytokines (farmer et al. 2005b) . it was suggested that liver transplantation and liver resection, together with portal clamping time, might be a potential stimulus for platelet activation. becker et al. (2004) indicated that neither liver transplantation nor liver resection influences gpiib/iiia and p-selectin expression on circulating platelets (becker et al. 2004 ). endothelial activation contributes significantly to the systemic inflammatory response to bacteraemia. release of soluble endothelial markers into the circulation has been demonstrated together with elevated plasma levels of cams and has been reported in bacteraemic patients. it has been proposed that the infection of endothelial cells with staphylococcus aureus, streptococcus sanguis, or staphylococcus epidermidis induces surface expression of icam-1 and vcam-1 and monocyte adhesion. in general, leukocyte/endothelial cell interactions such as capture, rolling, and firm adhesion should be viewed as a series of overlapping synergistic interactions among adhesion molecules resulting in an adhesion cascade. these cascades thereby direct leukocyte migration, which is essential for the generation of effective inflammatory responses and the development of rapid immune responses (golias et al. 2007) . helicobacter pylori is a common bacterial pathogen that infects world's population up to 50 %. carbohydrate components on h. pylori (sequences related to le x or le a antigens) are responsible for the persistent inflammation through interactions with leukocyte-endothelial adhesion molecules of the host. h. pylori isolates from patients with chronic gastritis, duodenal ulcer and gastric cancer interact with e-and l-selectins (galustian et al. 2003) . expression of e-selectin was specifically upregulated in h. pylori-induced gastritis but not in gastritis induced by acetylsalicylic acid or pouchitis. the upregulated e-selectin expression was localized to the gastric mucosa rather than being a systemic response to the infection (svensson et al. 2009 ). although mice with mutations in individual selectins showed no spontaneous disease and had a mild or negligible deficiencies of inflammatory responses, bullard et al. (1996) , in contrast, found that mice with null mutations in both endothelial selectins (p and e) develop a phenotype of leukocyte adhesion deficiency characterized by mucocutaneous infections in response to intraperitoneal s. pneumoniae peritonitis. these mice provide strong evidence for the functional importance of selectins in vivo (bullard et al. 1996) . anthrax lethal toxin (lt), a key virulence factor of bacillus anthracis, enhanced vcam-1 expression on primary human endothelial cells suggesting a causative link between dysregulated adhesion molecule expression and the poor immune response and vasculitis associated with anthrax. results suggest that lt can differentially modulate nf-kb target genes and highlight the importance of vcam-1 enhancement (warfel and d'agnillo 2008) . vascular endothelium stimulation in vitro that lead to the upregulation of cams is known for the pathogenic spirochaetes, including rlic10365 of leptospira interrogans. the recombinant proteins of l. interrogans in e. coli as a host were capable to promote the upregulation of icam-1 and e-selectin on monolayers of huvecs. in addition, pathogenic and non-pathogenic leptospira are both capable to stimulate endothelium e-selectin and icam-1, but the pathogenic l. interrogans serovar copenhageni strain promoted a higher activation than the non-pathogenic l. biflexa serovar patoc (atzingen et al. 2009; gómez et al. 2008) . chlamydia pneumoniae has been associated with cardiovascular disease and atherosclerosis. to determine the ability of c. pneumoniae to elicit inflammation, h€ ogdahl et al. (2008) infected human coronary artery endothelial cells (hcaec) with c. pneumoniae. secretion of il-8, mcp-1, and icam-1 was significantly increased after c. pneumoniae infection of hcaec in comparison with uninfected controls, where as release of eselectin or mmp-1did not change. this suggested that c. pneumoniae initiates and propagates vascular inflammation in ways that contribute to coronary artery disease (h€ ogdahl et al. 2008) . the sicam-1, svcam-1, and se-selectin are present in gingival crevicular fluid (gcf) and changes in their levels may be a sensitive indicator to differentiate healthy sites from those with periodontitis (hannigan et al. 2004; tamai et al. 2007) . porphyromonas gingivalis is a gram-negative bacterium that is an important etiologic agent of human adult periodontitis. e. coli lps and isoforms of p. gingivalis lps were potent in stimulating the expression of inflammatory markers, with e. coli lps being more potent . dna samples from blood of periodontitis patients genotyped for e-selectin ser 128 arg and l-selectin phe 206 leu revealed a significant difference in the ser 128 arg polymorphism of e-selectin, but not in l-selectin, between periodontal patients and controls; the 128arg allele was present more frequently in patients. houshmand et al. (2009) suggested that ser 128 arg polymorphism of e-selectin might contribute to the susceptibility of iranian individuals to periodontitis. cams in subjects with hiv disease: swingler et al. (2003) suggested that while both soluble cd23 and icam1 promote resting cell hiv1 infection, productive infection of cycling cells requires soluble icam1. swingler et al. (2003) noted that these results may explain in part the existence of a resting t-cell reservoir infected with hiv-1. subjects with hiv disease have multiple risk factors for cardiovascular disease, including elevated levels of icam-1 and vcam-1. many of the variables associated with icam-1 and vcam-1 levels can be related to their impact on inflammation (melendez et al. 2008 ). the lfa-1, icam-1, and icam-3 are enriched at virological synapse (vs). the cognate adhesion molecule interactions at vs are important for hiv-1 spread between t cells (jolly et al. 2007 ). zuccarello et al. (2002) described a distinct form of familial chronic mucocutaneous candidiasis characterized by earlyonset infections by different species of candida, restricted to the nails of the hands and feet and associated with low serum concentration of icam-1. phan and filler (2009) measured the effects of c. albicans on the endothelial cell production of e-selectin and tnf-a in vitro. during invasive pulmonary aspergillosis, a. fumigatus hyphae invade the abluminal endothelial cell surface, whereas they invade the luminal endothelial cell surface during haematogenous dissemination. infection with hyphae stimulates endothelial cells to synthesize e-selectin, vcam-1, il-8, and tnf-a in vitro. in neutropenic mice infected with wild-type a. fumigatus, increased pulmonary expression of e-selectin and tnf-a occurred only when neutropenia had resolved. in nonneutropenic mice immunosuppressed with corticosteroids, a. fumigatus stimulated earlier pulmonary expression of eselectin and vcam-1, while expression of icam-1 and tnf-a was suppressed. in both mouse models, expression of e-selectin was associated with high pulmonary fungal burden, angioinvasion, and neutrophil adherence to endothelial cells (chiang et al. 2008; kamai et al. 2009 ). 44.9.3.1 falciparum malaria significant differences are observed between falciparum malaria patients and the healthy people in term of levels of both se-selectin and thrombomodulin (tm). the levels of both se-selectin and tm correlated positively with temperature, levels of ifn-g and levels of tnf-a; and negatively with hemoglobin levels. trends of positive correlations were observed between sp-selectin or vwf and temperature (matondo et al. 2008) . evidence from autopsy and in vitro binding studies suggests that adhesion of erythrocytes infected with plasmodium falciparum to the human host icam-1 receptor is important in the pathogenesis of severe malaria. fernandez-reyes et al. (1997) identified a mutation (k29m) in the icam1 gene, which they designated 'icam1 kilifi,' that was associated with susceptibility to cerebral malaria with relative risks of 2.23 and 1.39 for homozygotes and heterozygotes, respectively. the available epidemiological, population genetic and functional evidence link icam-1(kilifi) to severe malaria susceptibility (fry et al. 2008; cojean et al. 2008) . increased serum concentrations of soluble sicam-1, cd54 and of soluble e-, but not soluble p-and l-selectins were detected in malagasy patients living in hyperendemic focus of schistosoma mansoni. serum levels of icam-1 were significantly correlated with the disease severity (esterre et al. 1998) . studies in several models of inflammation have underscored the importance of p-and e-selectins in the migration of t cells to inflamed tissues. cd4 + t cells recruited to the cutaneous compartment during infection with leishmania major express p-and e-selectin ligands. results suggest that by blocking p-and e-selectins, the immune pathology associated with cutaneous leishmaniasis might be ameliorated without compromising immunity to infection (zaph and scott 2003) . invasive amebiasis offers a new model that poses an inadequate immune response leading to a continuous and prolonged activation of endothelial cells (ecs) by amebas, amebic molecules and cytokines, leading to necrosis. hyperactivated endothelial cells continuously express icam-1 and e-selectin, pro-coagulant molecules (tissue factor, vwf, and the plasminogen activator inhibitor), resulting in ever greater inflammation and thrombosis (campos-rodríguez et al. 2009) sepsis is a multifactorial, and often fatal, disorder typically characterized by widespread inflammation and immune activation with resultant endothelial activation. though bacterial sepsis is most common, sepsis occurs with fungal, parastic and mycobacterial organisms. during bacterial sepsis in vivo, in wild-type mice and mice with e-, p-, or e-/ p-selectin deficiencies, a phenotypic abnormality in e-selectin-deficient mice suggested that e-and p-selectin are important in the host defense against s. pneumoniae infection (munoz et al. 1997) . p-selectin is an important mediator of eosinophil recruitment to the cornea from limbal vessels to the corneal stroma, suggesting that p-selectin interactions may be potential targets for immunotherapy in eosinophil-mediated ocular inflammation (kaifi et al. 2000) . staphylococcus aureus is one of the most significant pathogens in human sepsis and endocarditis. peptidoglycan induced surface expression of ec inflammation markers icam-1 and vcam-1, which supported the adhesion of monocytes to these ecs (mattsson et al. 2008) . teoh et al. (2008) assigned adiponectin as a modulator of survival and endothelial inflammation in experimental sepsis and a potential mechanistic link between adiposity and increased sepsis. newborn infants with clinical diagnosis of sepsis demonstrated significantly higher plasma se-selectin levels in infected infants. infants with gram-negative sepsis had higher se-selectin levels than did those with gram-positive sepsis. c-reactive protein was the best test for diagnosis of neonatal sepsis (zaki and el-sayed 2009). hofer et al. (2008) compared two different models of sepsis lps-induced endotoxemia and cecal ligation perforation (clp) bacteremia in rats with respect to changes in endothelial expression of cams as a marker for capillary breakdown of the blood brain barrier. increased icam-1 expression might be an early factor involved in these pathogenic events. although the role of pecam-1 could not be determined, it was possible to show its expression on cerebral endothelium in all groups (hofer et al. 2008) . in mouse models of sepsis, shapiro et al. (2009) demonstrated increased circulating levels of se-selectin, sicam-1, svcam-1 and sp-selectin at 24 h, while clp was associated with increased levels of se-selectin alone. in real-time pcr, mrna levels for p-selectin, icam-1 and pai-1 were increased in skin from endotoxemic mice. in clp, mrna levels for p-selectin, icam-1, e-selectin and pai-1 were elevated, while vcam-1 expression was reduced in skin. most, but not all of these changes correlated with alterations in immunohistochemical staining (shapiro et al. 2009 ). the field of selectin inhibition has matured significantly in recent years in the ability to inhibit selectin/ligand interactions with drug-like molecules and to demonstrate disease modification in human trials. a comprehensive review of new developments in the field of selectin inhibition through discussion of patents/patent applications from 2003 to august 2009 has been reported by bedard and kaila (2010) treatment of human endothelial cells with cytokines such as il-1, tnf-a or ifn-g induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. interfering with either leukocyte adhesion or upregulation of adhesion protein is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. the induction of e-selectin, vcam-1, and icam-1 genes requires the transcription factor nf-kb. pharmaceutical agents, which prevent the induced expression of one or more of cell adhesion molecules on endothelium, might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. e-selectin expression is induced on the endothelial cell surface of vessels in response to inflammatory stimuli but is absent in the normal vessels. thus, e-selectin is an attractive molecular target, and high affinity ligands for e-selectin could be powerful tools for the delivery of therapeutics and/or imaging agents to inflamed vessels. zimmerman and blanco (2008) reviewed the structure and regulation of lfa-1 and different classes of inhibitors that interfere lfa-1/icam-1 interactions. alicaforsen (isis 2302), an antisense to icam-1, designed to inhibit icam-1 expression did not reveal significant effect in crohn's disease. however, topical enemas for ulcerative colitis demonstrated some effect in secondary outcomes, and initial studies in pouchitis are promising (philpott and miner 2008) . icam-1 antibody (uv3) was highly effective at slowing the growth of tumors and/or prolonging survival in scid mice xenografted with human multiple myeloma, lymphoma, melanoma and other cell lines (brooks et al. 2008) . a structurally diverse collection of small molecule inhibitors has been characterized and developed either to bind the idas site of a l i-domain or to the midas of the b 2 i-like domain. cams play important roles in a critical step of tumor metastasis and arrest of tumor cells onto the venous or capillary bed of the target organ. in this process, il-1b induces nuclear translocation of nf-kb in huve cells, followed by induction of cell surface expression of e-selectin, icam-1, and vam-1, and subsequent adhesion of those cancer cells expressing sialyl le x antigen, which is a ligand to e-selectin. the adhesion of tumor cells to il-1b-treated huve cells can be inhibited by anti-nf-kb reagents such as n-acetyl l-cysteine, aspirin, or pentoxifylline. these observations indicate the involvement of nf-kb in cancer metastasis and the feasibility of using anti-nf-kb reagents in preventing metastasis (tozawa et al. 1995) . incubation of huvec with n,n,n-trimethylsphingosine (tms) resulted in a dose-dependent inhibition of il-1b-induced e-selectin expression. sphingosine or n,n-dimethylsphingosine had no effects on the expression. this inhibitory effect of tms on il-1b-dependent endothelial cell activation may partly explain the known anti-inflammatory or anti-metastatic effect of tms in vivo (masamune et al. 1995) . cimetidine inhibits the expression of e-selectin on vascular endothelial cells in gastric-and colorectal cancer patients, treated for chemotherapy (kawase et al. 2005) . since the expression of e-selectin and mac-1 is regulated either directly or indirectly by nf-kb, studies provide in vivo evidence that tepoxalin is a potent inhibitor of nf-kb mediated events in animal models and this novel molecular mechanism clearly defines it as a new class of anti-inflammatory compounds. e-selectin transcription requires binding of transcription factors, nf-kb, atf-2, and hmg-i(y). huve cells treated with tnf-a showed e-selectin surface expression, which peaked at 4 h and then declined. however, atf-2 binding was unchanged after stimulation with tnf-a. the termination of e-selectin expression is controlled at the level of transcription, with loss of protein-dna interactions at only one of three nf-kb-binding sites in the e-selectin promoter (boyle et al. 1999 ). e-selectin is synthesized following x-ray exposure to doses as low as 0.5 gy. x-ray-induced expression of eselectin and icam-1 has been proposed to contribute to radiation injury in normal tissues. e-selectin expression does not require cytokine synthesis, but involves nf-kb activation (hallahan et al. 1995) . nfkb inhibition using nfkb inhibitors abrogates x-ray induced inflammatory mediators (hallahan et al. 1998) . andrographolide, the principal component of medicinal plant andrographis paniculata, has been shown to inhibit nf-kb activity and may attenuate allergic asthma via inhibition of the nf-kb signaling pathway. andrographolide attenuated ovainduced lung tissue eosinophilia and airway mucus production, mrna expression of e-selectin, and inducible nos in lung tissues. findings implicate a potential therapeutic value of andrographolide in the treatment of asthma jiang et al. 2007 ). transforming growth factor (tgf-b) has been shown to decrease the adhesiveness of endothelial cells for neutrophils, lymphocytes, and tumor cells. tgf-b inhibits the basal e-selectin expression and tnf-stimulated expression. while tgf-b had no effect on the expression of vcam-1 and icam-1, the effect was additive with il-4 in inhibiting the expression of e-selectin. thus, perivascular tgf-b appears to act as an inhibitor of inflammatory responses involving neutrophils and a subset of lymphocytes (gamble et al. 1993) . ifn-g down-regulates the induction by a viral mimetic, polyinosinic-polycytidylic acid [poly-(i:c)], of e-selectin. the inhibitory effect of ifn-g on poly(i:c)induced e-selectin was specific for dsrna. results indicated the role for ifn-g in the regulation of e-selectin gene expression in response to dsrna by a transcriptional mechanism independent of nf-kb, as well as by a minor decrease in message stability (faruqi and dicorleto 1997) . retinoic acid inhibits the expression of vcam-1 but not e-selectin: several genes are regulated by tocopherols which can be categorized, based on their function. genes that are related to inflammation, cell adhesion and platelet aggregation include e-selectin, icam-1, and others (azzi et al. 2004) . retinoic acid and synthetic derivatives are known to exert anti-inflammatory effects in cutaneous diseases. pretreatment with all-trans-retinoic acid (t-ra) specifically prevented tnfa-induced vcam-1 expression, but not icam-1 and e-selectin induction (gille et al. 1997) . the tnfa-mediated activation of the human vcam-1 promoter was also inhibited after t-ra treatment, while the icam-1 promoter activation was unaffected, indicating that the selective inhibition of cam expression is regulated in part at the level of gene transcription. furthermore, the transcriptional inhibition by t-ra appears to be mediated by its effects upon the activation of nf-kb-dependent complex formation. the specific inhibition of cytokine-mediated vcam-1 gene expression in vitro provides a potential basis by which retinoids exert their biological effects at sites of inflammation in vivo (gille et al. 1997) . radiationinduced expression of e-selectin was also blocked by t-ra, whereas 9-cis retinoic acid was ineffective. application of statins and t-ra might have clinical impact in protecting against e-selectin-promoted metastasis, which might arise as an unwanted side effect from radiation treatment (holler et al. 2009; nubel et al. 2004 ). methylation of e-selectin promoter gene represses nf-kb transactivation: the e-selectin promoter in cultured endothelial cells is under-methylated in comparison with non-expressing hela cells. thus, methylation is likely to play a role in blocking e-selectin expression in nonendothelial cells (smith et al. 1993) . in intestine, muc2 is the main mucin carrying s-le x , which interacts with eselectin. this interaction may contribute to the extravasation of tumor cells and thus to the metastases. in several colorectal carcinoma cell lines the methylation of the 5'-flanking region of muc2 correlated with the suppression of the muc2 gene. the increase in muc2 expression after the inhibition of the methylation with 5-aza-2' deoxycytidine strongly supports the notion that the suppression of muc2 gene is related to the methylation of the promoter (riede et al. 1998 ). the advances in the development of adhesion molecule blocking agents, as well as an insight into the potential of these molecules in cardiovascular therapy have been reviewed from time to time (lutters et al. 2004 ). prophylactic dosing of a recombinant p-selectin ligand decreases venous thrombosis in a dose-dependent fashion in both feline and nonhuman primate animal models. additionally, treatment of 2-day iliac thrombi with a recombinant protein, p-selectin inhibitor, significantly improves vein reopening in nonhuman primates (register 2009 ). it is interesting to note that p-selectin inhibition decreases thrombosis without adverse anticoagulation. myers et al. (2005) evaluated an orally bioavailable inhibitor of p-selectin (psi-697), which decreased thrombosis. since, p-selectin is expressed on the surface of activated endothelial cells and platelets during thrombosis, targeting the plasminogen activator (pa) to pselectin would enhance local thrombolysis and reduce bleeding risk. a urokinase (upa)/anti-p-selectin antibody (husz51) fusion protein is known to increase fibrinolysis in a hamster pulmonary embolism (dong et al. 2004) aspirin reduces risks of myocardial infarction, stroke and cardiovascular death (serebruany et al. 2004 ). the impact of cyclooxygenase (cox)-2 antagonist treatment on acute coronary risk is controversial. prolonged cox-2 inhibition attenuates crp and il-6, does not modify p-selectin and mmp-9, and has no deleterious effect on endothelial function in stable patients with a history of recurrent acute coronary events and raised c-reactive protein (crp) (bogaty et al. 2004) . statins used in the control of hypercholesterolemia exert a protective effect on the endothelium reflected by a reduced level of circulating adhesion molecules. statins exert a beneficial effects on endothelial function and atherosclerotic plaque, modulating oxidative stress and inflammation, with subsequent, well documented, primary and secondary prevention of cad. following statin treatment, sp-selectin, and icam-1 and highly sensitive crp decreased compared to baseline levels. other proteins (svcam-1, se-selectin and platelet ecam-1) did not show significant changes. in contrast to crp, the reduction of spselectin concentrations correlated directly with the lowering of total cholesterol and inversely with the progression of cad (marschang et al. 2006) while diclofenac is capable of inhibiting the expression of e-selectin, icam-1 and vcam-1, the sjc13 is selective in inhibiting the expression of e-selectin and vcam-1, but not icam-1 in endothelial cells. nonsteroidal anti-inflammatory agents, such as sodium salicylate and aspirin, inhibit nf-kbdependent gene activation. salicylate blocked the tnf-ainduced increase in mrna levels of adhesion molecules and gave a dose-dependent inhibition of tnf-a-induced surface expression of vcam-1 and icam-1 with higher doses required to inhibit e-selectin expression. ibuprofen appeared a potent inhibitor of il-1a and tnf-a-induced surface expression of vcam-1 and a less potent inhibitor of icam-1. indomethacin, a nonsalicylate cyclooxygenase inhibitor, had no effect on surface expression of adhesion molecules, suggesting that the effects were not due to inhibition of cyclooxygenase (pierce et al. 1996) . methimazole, used in treating autoimmune diseases, may also diminish pathological inflammation by suppressing e-selectin expression. the phenyl methimazole can also reduce cytokineinduced e-selectin expression and consequent leukocyte adhesion. compound 10, which dramatically inhibits tnfa-induced vcam-1 mrna and protein expression in human aortic endothelial cells, has a modest inhibitory effect on tnf-a induced e-selectin expression and has no effect on icam-1 expression (dagia et al. 2004) . a thieno(2,3-d)pyrimidine, a-155918 inhibits the tnfainduced expression of e-selectin, icam-1, or vcam-1 on hevcs (stewart et al. 2001) . co-treatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced icam-1, vcam-1, and e-selectin expression on human endothelial cells. one of the potent flavones, apigenin, exhibited a dose-and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level (gerritsen et al. 1996) . enalapril and losartan but not placebo induced a small but stable decrease of cardiovascular icam-1 and vcam-1, while e-selectin and leukocyte expression of icam-1 remained unchanged. the lowering of plasma adhesion molecules may indicate an antiatherogenic effect of angiotensin ii blockade in hypercholesterolemia (graninger et al. 2004 ). targeting interaction of selectins and appropriate carbohydrate ligand is a promising approach to treat chronic inflammation. b-1,3-glucan sulfate (ps3) has inhibitory activity toward l and p-selectins under static conditions (alban et al. 2009 ). access to synthetic carbohydrates is an urgent need for the development of carbohydrate-based drugs, vaccines, adjuvants as well as novel drug delivery systems. besides traditional synthesis in solution, synthetic carbohydrates have been generated by chemoenzymatic methods as well as automated solid-phase synthesis. synthetic oligosaccharides have proven to be useful for identifying ligands of carbohydrate-binding proteins such as c-type lectins and siglecs using glycan arrays. furthermore, glyconanoparticles and glycodendrimers have been used for specific targeting of lectins of the immune system such as selectins, dc-sign, and cd22 (lepenies et al. 2010) . compounds that target both heparanase and selectins offer a promising approach for cancer therapy. borsig et al. (2011) reported semisynthetic sulfated tri mannose c-clinked dimers (stmcs) which are endowed with heparanase and selectin inhibitory activity. stmc hexasaccharide is an effective inhibitor of p-selectin in vivo. p-selectin-specific stmc attenuated metastasis in animal models, indicating that inhibition of tumor cell interaction with the vascular endothelium is critical for cancer dissemination. the small size, the stability of the c-c bond, and the chemically defined structure of stmcs make them superior to heparin derivatives and signify stmcs as valuable candidates for further evaluation. steroids down-regulate the expression of cams in endothelial cells stimulated by lps in vitro. low-dose hydrocortisone is a new treatment of patients with septic shock, a state that is characterized by an endothelial injury. treatment with glucocorticoids differently affected the pattern of evolution of scams, with se-selectin being decreased and sicam-1 being increased. expression of sp-selectin and svcam-1 was not affected (leone et al. 2004) . methotrexate (mtx) markedly reduces the expression of vascular e-selectin. a positive correlation between disease severity and the frequency of cutaneous lymphocyte-associated antigen (cla)-positive t cells in the blood of untreated patients with psoriasis has been observed. it is suggested that mtx decreases the expression of cla and e-selectin and that this may be a major mechanism for the therapeutic effect of mtx on psoriatic skin lesions (sigmundsdottir et al. 2004) . assessment of selected adhesion molecule and proinflammatory cytokine levels in the vitreous body of patients with type 2 diabetes-role of the inflammatory-immune process in the pathogenesis of proliferative diabetic retinopathy levels of adhesion molecules bear a relationship to triglyceride levels in type 2 diabetic subjects with proven silent ischemia value of e-selectin and l-selectin determination in children and youth with inflammatory bowel disease ps3, a semisynthetic b-1,3-glucan sulfate, diminishes contact hypersensitivity responses through inhibition of l-and p-selectin functions circulating adhesion molecules during kidney allograft rejection icam-1 polymorphisms (g241r, k469e), in coronary artery disease and myocardial infarction e-selectin polymorphism in erythema nodosum secondary to sarcoidosis expression of e-selectin ligand-1 (cfr/esl-1) on hepatic stellate cells: implications for leukocyte extravasation and liver metastasis activation and damage of endothelial cells upon hypoxia/reoxygenation. effect of extracellular ph genomic rearrangements on vcam1, sele, apeg1and aif1 loci in atherosclerosis lp95, a novel leptospiral protein that binds extracellular matrix components and activates e-selectin on endothelial cells genetic polymorphisms in cytokine and adhesion molecule genes in coronary artery disease e-selectin regulates gene expression in metastatic colorectal carcinoma cells and enhances hmgb1 release regulation of gene expression by a-tocopherol e-selectin, and p-selectin expressions in papillary thyroid carcinomas and their correlation with prognostic parameters a novel antiinflammatory role for andrographolide in asthma via inhibition of the nuclear factor-kb pathway platelet p-selectin and gpiib/iiia expression after liver transplantation and resection selectin inhibitors: a patent review the involvement of the sle-a selectin ligand in the extravasation of human colorectal carcinoma cells quantitative real-time rt-pcr analysis of inflammatory gene expression associated with ischemia-reperfusion brain injury plasma indices of endothelial and platelet activation in rheumatoid disease: relationship to cardiovascular co-morbidity soluble intercellular adhesion molecule-1 and e-selectin in patients with asthma exacerbation selectins and monocyte chemotactic peptide as the markers of atherosclerosis activity soluble adhesion molecules in pediatric rheumatic diseases impact of prolonged cyclooxygenase-2 inhibition on inflammatory markers and endothelial function in patients with ischemic heart disease and raised creactive protein: a randomized placebo-controlled study antimetastatic activities of modified heparins: selectin inhibition by heparin attenuates metastasis sulfated hexasaccharides attenuate metastasis by inhibition of p-selectin and heparanase tnf-a blockade induces a reversible but transient effect on endothelial dysfunction in patients with long-standing severe rheumatoid arthritis transcriptional arrest of the human e-selectin gene expression of e-selectin and its transcripts during intestinal ischemia-reperfusion injury in pigs the antitumor activity of an anti-cd54 antibody in scid mice xenografted with human breast, prostate, non-small cell lung, and pancreatic tumor cell lines infectious susceptibility and severe deficiency of leukocyte rolling and recruitment in e-selectin and p-selectin double mutant mice endothelial dysfunction in cardiovascular diseases: the role of oxidant stress invasive amebiasis: a microcirculatory disorder? p-selectin knockout mice have improved outcomes with both warm ischemia and small bowel transplantation soluble e-selectin in children and adolescents with type 1 diabetes blocking e-selectin inhibits ischaemiareperfusion-induced neutrophil recruitment to the murine testis increased platelet cd63 and pselectin expression persist in atherosclerotic ischemic stroke receptor tyrosine kinase epha2 mediates thrombin-induced upregulation of icam-1 in endothelial cells in vitro increased plasma levels of soluble p-selectin in rheumatic mitral stenosis the common variants of e-selectin gene in graves' disease aspergillus fumigatus stimulates leukocyte adhesion molecules and cytokine production by endothelial cells in vitro and during invasive pulmonary disease pathogenic aspects of pulmonary complications in acute pancreatitis patients effect of rosiglitazone treatment on circulating vascular and inflammatory markers in insulin-resistant subjects changes in pselectin expression on cardiac microvessels in blood-perfused rat hearts subjected to ischemia-reperfusion human squamous cell carcinomas evade the immune response by down-regulation of vascular e-selectin and recruitment of regulatory t cells successful treatment of rheumatoid arthritis is associated with a reduction in serum seselectin and thrombomodulin level cytoadherence characteristics to endothelial receptors icam-1 and cd36 of plasmodium falciparum populations from severe and uncomplicated malaria cases p-selectin or intercellular adhesion molecule (icam)-1 deficiency substantially protects against atherosclerosis in apolipoprotein e-deficient mice circulating e-selectin and tumor necrosis factor-alpha in extraarticular involvement and joint disease activity in rheumatoid arthritis protein biochip array of adhesion molecule expression in peripheral blood of patients with nasal polyposis serum p-selectin, soluble vascular cell adhesion molecule-i (s-vcam-i) and soluble intercellular adhesion molecule-i (s-icam-i) levels in bladder carcinoma patients with different stages autoantibodies and other serological markers in rheumatoid arthritis: predictors of disease activity? phenyl methimazole inhibits tnf-a-induced vcam-1 expression in an ifn regulatory factor-1-dependent manner and reduces monocytic cell adhesion to endothelial cells changes in endogenous cytokines, adhesion molecules and platelets during cytokine-induced tumor necrosis evaluation of se-selectin and sicam-1 as parameters for renal function icam1 r241 is not associated with celiac disease in the spanish population comparative study of adhesion molecule expression in nodular lesions of behçet syndrome and other forms of panniculitis evidence of increased inflammation and microcirculatory abnormalities in patients with type 1 diabetes and their role in microvascular complications circulating levels of soluble adhesion molecules in patients with anca-associated vasculitis plasma cytokine and p-selectin levels in advanced malignancy: prognostic value and impact of low-molecular weight heparin administration circulating adhesion molecules and purine nucleotides during kidney allograft reperfusion p-selectin-targeting of the fibrin selective thrombolytic desmodus rotundus salivary plasminogen activator alpha1 e-and p-selectin are not required for the development of experimental autoimmune encephalomyelitis in c57bl/6 and sjl mice cell adhesion molecules, vascular endothelial growth factor, and basic fibroblast growth factor in patients with non-small cell lung cancer treated with chemotherapy with or without bevacizumab-an eastern cooperative oncology group study does colorectal cancer clinical advancement affect adhesion molecules (sp-selectin, se-selectin and icam-1) concentration? soluble p-selectin concentration in patients with colorectal cancer serum levels of soluble e-selectin are associated with the clinical course of metastatic disease in patients with liver metastases from breast cancer markers for endothelial activation during open heart surgery cell adhesion molecules-update expression of selected adhesion molecules in dermatitis herpetiformis and bullous pemphigoid differential induction of mrna for icam-1 and selectins in hepatocytes, kupffer cells and endothelial cells during endotoxemia serum concentrations of sicam-1, se-, sp-and sl-selectins in patients with schistosoma mansoni infection and association with disease severity biochemical markers of endothelial activation in primary hyperparathyroidism markers of low-grade inflammation and soluble cell adhesion molecules in chinese patients with coronary artery disease association of leu125val polymorphism of platelet endothelial cell adhesion molecule-1 (pecam-1) gene & soluble level of pecam-1 with coronary artery disease in asian indians selectins in the hit syndrome: pathophysiologic role and therapeutic modulation disruption of pselectin signaling modulates cell trafficking and results in improved outcomes after mouse warm intestinal ischemia and reperfusion injury cd62 blockade with p-selectin glycoprotein ligand-immunoglobulin fusion protein reduces ischemia-reperfusion injury after rat intestinal transplantation independent pathways of p-selectin and complement-mediated renal ischemia/reperfusion injury ifn-g inhibits double-stranded rnainduced e-selectin expression in human endothelial cells candidate gene analysis of selectin cluster in patients with multiple sclerosis enhanced expression of e-selectin on the vascular endothelium of peripheral nerve in critically ill patients with neuromuscular disorders a high frequency african coding polymorphism in the n-terminal domain of icam-1 predisposing to cerebral malaria in kenya icam-1-dependent pathways regulate colonic eosinophilic inflammation role of inflammation in diabetic nephropathy circulating endothelial cells and rheumatoid arthritis: relationship with plasma markers of endothelial damage/dysfunction variation in the icam1 gene is not associated with severe malaria phenotypes interactions of the gastrotropic bacterium helicobacter pylori with the leukocyteendothelium adhesion molecules, the selectins-a preliminary report transforming growth factor-beta inhibits e-selectin expression on human endothelial cells p-selectin-mediated acute inflammation can be blocked by chemically modified heparin, ro-heparin selectin blockade plus therapy with low-dose sirolimus and cyclosporin a prevent brain death-induced renal allograft dysfunction microvascular endothelial cells from e-selectin-deficient mice form tubes in vitro ser128arg gene polymorphism for e-selectin and severity of atherosclerotic arterial disease retinoic acid inhibits the regulated expression of vascular cell adhesion molecule-1 by cultured dermal microvascular endothelial cells leukocyte and endothelial cell adhesion molecules in inflammation focusing on inflammatory heart disease putative outer membrane proteins of leptospira interrogans stimulate human umbilical vein endothelial cells (huvecs) and express during infection expression of adhesion molecules in lungs of mice infected with paracoccidioides brasiliensis conidia circulating p-,l-and e-selectins in pseudoxanthoma elasticum patients selectins and selectin ligands in extravasation of cancer cells and organ selectivity of metastasis angiotensin receptor blockade decreases markers of vascular inflammation implication of adhesion molecules in inflammation of the common bile duct in patients with secondary cholangitis due to biliary obstruction serum levels of intercellular adhesion molecule icam-1 and e-selectin in advanced stage non-small cell lung cancer e-selectin genetic variation as a susceptibility factor for ischemic stroke e-selectin gene induction by ionizing radiation is independent of cytokine induction nuclear factor kappab dominant negative genetic constructs inhibit x-ray induction of cell adhesion molecules in the vascular endothelium strategies to reduce oxidative stress in cardiovascular disease variant isoforms of cd44 are p-and l-selectin ligands on colon carcinoma cells soluble cell adhesion molecules in gingival crevicular fluid in periodontal health and disease e-selectin gene s128r polymorphism is associated with poor prognosis in patients with stage ii or iii colorectal cancer role of adhesion molecules in the induction of restenosis after angioplasty in the lower limb the p-selectin gene is highly polymorphic: reduced frequency of the pro715 allele carriers in patients with myocardial infarction tumor necrosis factor-alpha upregulates the expression of ccl2 and adhesion molecules of human proximal tubular epithelial cells through mapk signaling pathways in vivo imaging of cutaneous t-cell lymphoma migration to the skin injury of the blood brain barrier and up-regulation of icam-1 in polymicrobial sepsis expression of chemokines and adhesion molecules in human coronary artery endothelial cells infected with chlamydia (chlamydophila) pneumoniae pravastatin limits radiation-induced vascular dysfunction in the skin thrombosis and atherosclerosis endothelial microparticles as markers of endothelial dysfunction e-selectin and lselectin polymorphisms in patients with periodontitis endothelial activation and systemic inflammation in obese asthmatic children bosentan regulates the expression of adhesion molecules on circulating t cells and serum soluble adhesion molecules in systemic sclerosisassociated pulmonary arterial hypertension significance of endothelial molecular markers in the evaluation of the severity of acute pancreatitis serum levels of p-selectin in men with high-functioning autism the various effects of four h1-antagonists on serum substance p levels in patients with atopic dermatitis immunohistochemistry, glycosylation and immunosuppression of glycodelin in human ovarian cancer andrographolide inhibits the adhesion of gastric cancer cells to endothelial cells by blocking eselectin expression adhesion molecule interactions facilitate human immunodeficiency virus type 1-induced virological synapse formation between t cells impaired eosinophil recruitment to the cornea in p-selectin-deficient mice in onchocerca volvulus keratitis (river blindness) polarized response of endothelial cells to invasion by aspergillus fumigatus soluble endothelial adhesion molecules and inflammation markers in patients with b-thalassemia intermedia carbohydrate antigen sialyl lewis a-its pathophysiological significance and induction mechanism in cancer progression adhesion of epstein-barr virus-positive natural killer cell lines to cultured endothelial cells stimulated with inflammatory cytokines inflammatory mediators in diabetic and non-diabetic lumbosacral radiculoplexus neuropathy increase in e-selectin expression in umbilical vein endothelial cells by anticancer drugs and inhibition by cimetidine adhesion molecules (icam-1 and vcam-1) and diabetic retinopathy in type 2 diabetes evaluation of markers of endothelial damage in cases of young myocardial infarction adhesion molecule polymorphisms in acute renal allograft rejection polymorphism in icam-1, pecam-1, e-selectin, and l-selectin genes in tunisian patients with inflammatory bowel disease increase in soluble eselectin level after ptca and stent implantation: a potential marker of restenosis p-selectin deficiency attenuates tumor growth and metastasis engagement of icam-1 by major group rhinoviruses activates the lfa-1/icam-3 cell adhesion pathway in mononuclear phagocytes biomarkers of endothelial dysfunction are elevated and related to prognosis in chronic heart failure patients with diabetes but not in those without diabetes the role of vascular adhesion molecules pecam-1 (cd 31), vcam-1 (cd 106), e-selectin (cd62e) and p-selectin (cd62p) in severe porcine pancreatitis reduction of soluble adhesion molecules (sicam-1, svcam-1, and se-selectin) and vascular endothelial growth factor levels in serum of rheumatoid arthritis patients following multiple intravenous infusions of infliximab soluble cell adhesion molecules (sicam-1, svcam-1, and se-selectin) in patients with early rheumatoid arthritis role of sialosyl lewis(a) in adhesion of colon cancer cells-the antisense rna approach endothelial cell adhesion molecules and cancer progression expression of vascular adhesion molecules in inflammatory bowel disease expression of cytokeratins, adhesion and activation molecules in oral ulcers of behçet's disease intercellular adhesion molecule 1 gene polymorphisms in graves' disease src and phosphatidylinositol 3-kinase mediate soluble e-selectin-induced angiogenesis clinical significance of selected endothelial activation markers in patients with systemic lupus erythematosus house dust mite induces expression of intercellular adhesion molecule-1 in eol-1 human eosinophilic leukemic cells selectin inhibitor bimosiamose prolongs survival of kidney allografts by reduction in intragraft production of cytokines and chemokines syk associates with clathrin and mediates phosphatidylinositol 3-kinase activation during human rhinovirus internalization selectins promote tumor metastasis m€ uller cells do not influence leukocyte adhesion to retinal endothelial cells contribution of soluble intercellular adhesion molecule-1 to the migration of vascular smooth muscle cells characterization of gastrin-induced proangiogenic effects in vivo in orthotopic u373 experimental human glioblastomas and in vitro in human umbilical vein endothelial cells cell adhesion molecules as a marker reflecting the reduction of endothelial activation induced by glucocorticoids applications of synthetic carbohydrates to chemical biology effects of infliximab on cytokines, myeloperoxidase, and soluble adhesion molecules in patients with juvenile idiopathic arthritis association between the ser128arg variant of the e-selectin and risk of coronary artery disease in the central china intercellular adhesion molecule-1 gene k469e polymorphism and ischemic stroke: a case-control study in a chinese population soluble cd40 ligand, soluble p-selectin, interleukin-6, and tissue factor in diabetes mellitus: relationships to cardiovascular disease and risk factor intervention monoclonal antibody against e selectin attenuates subarachnoid hemorrhage-induced cerebral vasospasm effects of congestive heart failure on plasma von willebrand factor and soluble p-selectin concentrations in patients with non-valvar atrial fibrillation p. gingivalis and e. coli lipopolysaccharides exhibit different systemic but similar local induction of inflammatory markers a distinct profile of serum levels of soluble intercellular adhesion molecule-1 and intercellular adhesion molecule-3 in mycosis fungoides and sézary syndrome the ability of different forms of heparins to suppress p-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo p-selectin: a common therapeutic target for cardiovascular disorders, inflammation and tumour metastasis the role of adhesion molecules in atopic dermatitis blocking endothelial adhesion molecules: a potential therapeutic strategy to combat atherogenesis circulating e-selectin as a risk marker in patients with end-stage renal disease endothelial/lymphocyte activation leads to prominent cd4+ t cell infiltration in the gastric mucosa of patients with systemic sclerosis adhesion molecules from the lfa-1/icam-1,3 and vla-4/vcam-1 pathways on t lymphocytes and vascular endothelium in graves' and hashimoto's thyroid glands reduction of soluble p-selectin by statins is inversely correlated with the progression of coronary artery disease the leu554phe polymorphism in the e-selectin gene is associated with blood pressure in overweight people n,n,ntrimethylsphingosine inhibits interleukin-1 beta-induced nf-kb activation and consequent e-selectin expression in human umbilical vein endothelial cells neutrophil surface expression of cd11b and cd62l in diabetic microangiopathy transgene expression of a(1,2)-fucosyltransferase-i (fut1) in tumor cells selectively inhibits sialyl-lewis x expression and binding to e-selectin without affecting synthesis of sialyl-lewis a or binding to p-selectin markers of vascular endothelial cell damage and p. falciparum malaria: association between levels of both se-selectin and thrombomodulin, and cytokines, hemoglobin and clinical presentation staphylococcal peptidoglycan initiates an inflammatory response and procoagulant activity in human vascular endothelial cells: a comparison with highly purified lipoteichoic acid and tsst-1 the impact of peripheral arterial disease on circulating platelets intestinal inflammation in adhesion molecule-deficient mice: an assessment of p-selectin alone and in combination with icam-1 or e-selectin adhesion molecule polymorphisms in chronic renal allograft failure endothelial adhesion molecules are associated with inflammation in subjects with hiv disease evaluation of microbiologic and hematologic parameters and e-selectin as early predictors for outcome of neonatal sepsis expression of endothelial protein c receptor and thrombomodulin in human coronary atherosclerotic plaques p-selectin in arterial thrombosis role of icam-1 in persisting inflammation in parkinson disease and mptp monkeys cellular adhesion molecules and blood pressure: interaction with sex in a multi-ethnic population association between the thr715pro p-selectin gene polymorphism and soluble p-selectin levels in a multiethnic population in south london effect of clopidogrel on the expression of inflammatory markers in rabbit ischemic coronary artery allergic rhinitis: continuous or on demand antihistamine therapy? host defense against systemic infection with streptococcus pneumoniae is impaired in e-, p-, and e-/p-selectin-deficient mice serum vcam-1, icam-1, and l-selectin levels in children and young adults with chronic renal failure decreased venous thrombosis with an oral inhibitor of p selectin plasma and platelet-derived vascular endothelial growth factor and angiopoietin-1 in hypertension: effects of antihypertensive therapy platelet morphology, soluble p selectin and platelet p-selectin in acute ischaemic stroke. the west birmingham stroke project cd43, but not pselectin glycoprotein ligand-1, functions as an e-selectin counterreceptor in human pre-b-cell leukemia nall-1 blood serum levels of vascular cell adhesion molecule (svcam-1), intercellular adhesion molecule (sicam-1) and endothelial leucocyte adhesion molecule-1 (elam-1) in diabetic retinopathy ionizing radiationinduced e-selectin gene expression and tumor cell adhesion is inhibited by lovastatin and all-trans retinoic acid clinical significance of intercellular adhesion molecule-1 in ulcerative colitis in situ expression of the cell adhesion molecules in bronchial tissues from asthmatics with air flow limitation: in vivo evidence of vcam-1/vla-4 interaction in selective eosinophil infiltration identification of cutaneous lymphocyte-associated antigen as sialyl 6-sulfo lewis x, a selectin ligand expressed on a subset of skin-homing helper memory t cells protective effect of anti-pselectin monoclonal antibody in lipopolysaccharide-induced lung hemorrhage the levels of soluble adhesion molecules in diabetic and nondiabetic patients with combined hyperlipoproteinemia and the effect of ciprofibrate therapy increased platelet activation markers in rheumatoid arthritis: are they related with subclinical atherosclerosis? elevated plateletmonocyte complexes in patients with psoriatic arthritis elevated serum soluble eselectin levels in korean children with measles the engagement of selectins and their ligands in colorectal cancer liver metastases biomarkers of oxidant stress, insulin sensitivity and endothelial activation in rheumatoid arthritis: a cross-sectional study of their association with accelerated atherosclerosis relationships between serum fatty acid composition and multiple markers of inflammation and endothelial function in an elderly population serum adhesion molecules in acute pancreatitis: time course and early assessment of disease severity endothelial cell stimulation by candida albicans antisense inhibition of icam-1 expression as therapy provides insight into basic inflammatory pathways through early experiences in ibd salicylates inhibit i kb-a phosphorylation, endothelial-leukocyte adhesion molecule expression, and neutrophil transmigration flow cytometric analysis of conjunctival epithelium in ocular rosacea and keratoconjunctivitis sicca circulating soluble adhesion molecules icam-1 and vcam-1 and their association with clinical outcome, troponin t and creactive protein in patients with acute coronary syndromes natriuretic peptides and e-selectin as predictors of acute deterioration in patients with inotrope-dependent heart failure endothelial dysfunction in diabetes: from mechanisms to therapeutic targets biological mediators of acute inflammation primate models in women's health: inflammation and atherogenesis in female cynomolgus macaques (macaca fascicularis) comparison of soluble icam-1, vcam-1 and e-selectin levels in patients with episodic cluster headache and giant cell arteritis novel cardiovascular risk factors in premature coronary atherosclerosis associated with systemic lupus erythematosus enhancement of endothelial nitric oxide synthase production reverses vascular dysfunction and inflammation in the hindlimbs of a rat model of diabetes fundamental and distinct roles of p-selectin and lfa-1 in ischemia/reperfusion-induced leukocyte-endothelium interactions in the mouse colon increased methylation of promotor region suppresses expression of muc2 gene in colon carcinoma cells hydralazine reduces leukocyte migration through different mechanisms in spontaneously hypertensive and normotensive rats the effect of trandolapril and its fixed-dose combination with verapamil on circulating adhesion molecules levels in hypertensive patients with type 2 diabetes accelerated development of arthritis in mice lacking endothelial selectins the endothelial leukocyte adhesion molecule. role in coronary artery disease expression of cell adhesion molecules in the salivary and lacrimal glands of sjogren's syndrome elevation of serum soluble e-and p-selectin in patients with hypertension is reversed by benidipine, a long-acting calcium channel blocker association analysis of the e-selectin 98 g > t polymorphism and the risk of childhood ischemic stroke and e-selectin as markers of hematogenous metastases and as predictors of prognosis in colorectal cancer carcinoembryonic antigen and cd44 variant isoforms cooperate to mediate colon carcinoma cell adhesion to e-and l-selectin in shear flow temporal patterns of soluble adhesion molecules in cerebrospinal fluid and plasma in patients with the acute brain infraction lack of uniform platelet activation in patients after ischemic stroke and choice of antiplatelet therapy adhesion and lymphocyte costimulatory molecules in systemic rheumatic diseases skin biopsies demonstrate site-specific endothelial activation in mouse models of sepsis globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in fabry disease endothelial cells high serum soluble e-selectin levels are associated with postoperative haematogenic recurrence in esophageal squamous cell carcinoma patients methotrexate markedly reduces the expression of vascular e-selectin, cutaneous lymphocyte-associated antigen and the numbers of mononuclear leucocytes in psoriatic skin synoviocyte stimulation by the lfa-1-intercellular adhesion molecule-2-ezrin-akt pathway in rheumatoid arthritis decreased serum levels of p-selectin and eosinophil cationic protein in patients with mild asthma after inhaled salbutamol adhesion molecules and their role in pathogenesis of ards adhesion molecules in allergic inflammation dna-methylation of the e-selectin promoter represses nf-kb transactivation the high affinity selectin glycan ligand c2-o-slex and mrna transcripts of the core 2 b-1,6-n acetylglucosaminyl-transferase (c2gnt1) gene are highly expressed in human colorectal adenocarcinomas the role of the platelet in the pathogenesis of atherothrombosis differential metastasis inhibition by clinically relevant levels of heparins-correlation with selectin inhibition, not antithrombotic activity discovery of inhibitors of cell adhesion molecule expression in human endothelial cells. 1. selective inhibition of icam-1 and e-selectin expression inflammatory molecule expression in cerebral arteriovenous malformations effect of 100 % oxygen on e-selectin expression, recruitment of neutrophils and enterocyte apoptosis following intestinal ischemia-reperfusion in a rat expression and significance of icam-1 and its counter receptors lfa-1 and mac-1 in experimental acute pancreatitis of rats selective upregulation of endothelial e-selectin in response to helicobacter pylori-induced gastritis soluble cytokeratin-19 and e-selectin biomarkers: their relevance for lung cancer detection when tested independently or in combinations hiv-1 nef intersects the macrophage cd40l signaling pathway to promote resting-cell infection prognostic significance of soluble adhesion molecules in hodgkin's disease association between singlenucleotide polymorphisms in selectin genes and immunoglobulin a nephropathy possible requirement of intercellular adhesion molecule-1 for invasion of gingival epithelial cells by treponema medium effects of parietaria judaica pollen extract on human microvascular endothelial cells circulating asymmetric dimethylarginine, endothelin-1 and cell adhesion molecules in women with gestational diabetes adiponectin deficiency promotes endothelial activation and profoundly exacerbates sepsis-related mortality podocalyxin-like protein is an e-/l-selectin ligand on colon carcinoma cells: comparative biochemical properties of selectin ligands in host and tumor cells different roles of galectin-9 isoforms in modulating e-selectin expression and adhesion function in lovo colon carcinoma cells circulating intercellular adhesion molecule-1 (icam-1) in autoimmune liver disease and evidence for the production of icam-1 by cytokine-stimulated human hepatocytes effects of antinuclear factor kb reagents in blocking adhesion of human cancer cells to vascular endothelial cells specific haplotypes of the p-selectin gene are associated with myocardial infarction mechanisms by which eselectin regulates diapedesis of colon cancer cells under flow conditions soluble endothelial cell adhesion molecules and their relationship to disease activity in takayasu's arteritis activated protein c reduces graft neutrophil activation in clinical renal transplantation immuno-inflammatory and thrombotic/fibrinolytic variables associated with acute ischemic stroke diagnosis transactivation of the icam-1 gene by cd30 in hodgkin's lymphoma serum levels of soluble e-selectin in colorectal cancer elevated serum soluble e-selectin is associated with poor outcome and correlated with serum alt in biliary atresia oxidative stress in cardiovascular disease molecular investigation of the functional relevance of missense variants of icam-1 p-selectin thr715pro polymorphism predicts p-selectin levels but not risk of incident coronary heart disease or ischemic stroke in a cohort of 14595 participants: the atherosclerosis risk in communities study polymorphisms and linkage analysis for icam-1 and the selectin gene cluster platelet, not endothelial, p-selectin is required for neointimal formation after vascular injury staphylococcal enterotoxins a and b enhance rhinovirus replication in a549 cells anthrax lethal toxin enhances tnf-induced endothelial vcam-1 expression via an ifn regulatory factor-1-dependent mechanism selectively desulfated heparin inhibits p-selectin-mediated adhesion of human melanoma cells e-selectin and sialyl lewis x expression is associated with lymph node metastasis of invasive micropapillary carcinoma of the breast dna polymorphisms in adhesion molecule genes-a new risk factor for early atherosclerosis functional characterization of atherosclerosis-associated ser128arg and leu554phe e-selectin mutations l-arginine prevents metabolic effects of high glucose in diabetic mice crossing the endothelium: e-selectin regulates tumor cell migration under flow conditions polymorphisms and plasma soluble levels of e-selectin in patients with chronic hepatitis b virus infection p-selectin suppresses hepatic inflammation and fibrosis in mice by regulating interferon-g and the il-13 decoy receptor effects of low molecular weight heparin on platelet surface p-selectin expression and serum interleukin-8 production in rats with trinitrobenzene sulphonic acid-induced colitis intercellular adhesion molecule-1 (icam-1) expression is necessary for monocyte adhesion to the placental bed endothelium and is increased in type 1 diabetic human pregnancy activated platelets contribute importantly to myocardial reperfusion injury circulating thrombomodulin and vascular cell adhesion molecule-1 and renal vascular lesion in patients with lupus nephritis serum e-selectin and erythrocyte membrane na + k + atpase levels in patients with rheumatoid arthritis platelet activity is a biomarker of cardiac necrosis and predictive of untoward clinical outcomes in patients with acute myocardial infarction undergoing primary coronary stenting e-selectin polymorphism associated with myocardial infarction causes enhanced leukocyteendothelial interactions under flow conditions cell adhesion molecules regulate fibrotic process via th1/th2/th17 cell balance in a bleomycin-induced scleroderma model mucosal tolerance to e-selectin provides protection against cerebral ischemiareperfusion injury in rats synergistic effects between 561a > c and 98 g > t polymorphisms of e-selectin gene and hypercholesterolemia in determining the susceptibility to coronary artery disease inflammatory biomarkers in coronary artery disease th1 cell-mediated resistance to cutaneous infection with leishmania major is independent of p-and eselectins cd44v4 is a major e-selectin ligand that mediates breast cancer cell transendothelial migration e-selectin and its ligand-slex in the metastasis of hepatocellular carcinoma serum 3'-sulfo-le a indication of gastric cancer metastasis cell adhesion: implication in tumor progression inhibitors targeting the lfa-1/ icam-1 cell-adhesion interaction: design and mechanism of action familial chronic nail candidiasis with icam-1 deficiency: a new form of chronic mucocutaneous candidiasis markers of endothelial dysfunction in older subjects with late onset alzheimer's disease or vascular dementia key: cord-007613-g4s0v8ra authors: rimstad, espen; reubel, gerhard h.; dean, gregg a.; higgins, joanne; pedersen, niels c. title: cloning, expression and characterization of biologically active feline tumour necrosis factor-α date: 2000-03-10 journal: vet immunol immunopathol doi: 10.1016/0165-2427(94)05345-s sha: doc_id: 7613 cord_uid: g4s0v8ra we report the cloning, expression and characterization of biologically active feline tumour necrosis factor-α (ftnf-α). messenger rna was extracted from feline peritoneal macrophage cultures and used to synthesize cdna for polymerase chain reaction (pcr) amplification. the pcr products were cloned into the plasmid vector pcrii and sequenced, showing 99.3% homology with a published ftnf-α gene sequence. subcloning into the vector pgex-2t and subsequent expression resulted in a 43 kda fusion protein of ftnf-α and glutathione s-transferase (gst). thrombin cleavage of the fusion protein yielded a 17 kda protein. this protein cross-reacted with a monoclonal anti-human tnf-α antibody in western blotting, but not with a polyclonal anti-murine tnf-α serum. recombinant ftnf-α (rftnf-α) and rftnf-α-gst had a cd(50) of 15 ng ml(−1) and 230 ng ml(−1), respectively, in the l929 cytotoxicity assay. cats given rftnf-α-gst intravenously manifested the typical biological effects of tnf-α, including fever, depression, and piloerection. the rftnf-α-gst upregulated il-2 receptor and mhc-ii antigen expression on peripheral blood mononuclear cells stimulated in vitro, but had no effect on tnf-α receptor and mhc-i antigen expression. tumour necrosis factor-alpha (tnf-a ) is a cytokine with multifunctional activity. although its original activity was recognised against tumour cells (carswell et al., 1975) , it is now known to play an important role in immune and inflammatory responses as well as in the pathogenesis of many human and animal diseases (reviewed by jhattelh, 199 1). tnf-a may also play a crucial role in the pathogenesis of human aids (matsuyama et al., 199 1) . tnf-c~ stimulates human immunodeficiency virus (hiv) replication in both established lymphoid and primary t cell cultures (suzuki et al., 1989) . this enhanced replication is mediated through tnf-a inducible nuclear factors like nfkb and the kb enhancer elements of the hiv ltr (osborn et al., 1989) . induction of hiv gene expression is regulated by interactions of dna binding proteins with specific gene sequences (folks et al., 1989; osborn et al., 1989; poli et al., 1990) . the levels of tnf-(w are increased in patients with aids and may upregulate virus replication in an autocrine fashion (poli et al., 1990 ). in addition tnf-a, may play an important role in some clinical manifestations of hiv infection; dramatic improvement in aphthous stomatitis and esophagitis is seen in aids patients treated with a tnf-a inhibitor (thalidomide) (nicolau and west, 1990) . tnf-a! is an important reagent, therefore, for studies of hiv pathogenesis. the nucleotide sequences for human, mouse, sheep, pig, rabbit and cat tnf-a, has previously been reported (pennica et al., 1985; shirai et al., 1985; ito et al., 1986; drews et al., 1990; mcgraw et al., 1990; green and sargan, 1991) . both human and murine recombinant tnf-a proteins have been expressed in different systems, and used in several studies (pennica et al., 1985; shirai et al., 1985) . however, recombinant feline tnf-a (rftnf-cu) protein has not been expressed. the aim of this study was to clone the cdna of feline tnf-cu and to express it in e~/zerichia coli in a biologically active form. our goal is to use rftnf-a to study immunodeficiency virus pathogenesis using the feline immunodeficiency virus (fiv) infection model. fiv infection has been shown to be a valid animal model for hiv studies because similar changes in tnf-a! expression have also been observed in fiv infected cats (lawrence et al., 1992; lehmann et al., 1992; pedersen, 1993) . adult specific pathogen free (spf) cats were obtained from the breeding colony of the feline retrovirus research laboratory, university of california, davis. animals were housed in quarters provided by the animal research service, university of california, davis. peritoneal macrophages were obtained by peritoneal saline lavage from two specific pathogen free cats as previously described (stoddart and scott, 1988; brunner and pedersen, 1989) . cats were inoculated intraperitoneally with 0.75 ml of human diphtheria/pertussis/tetanus vaccine, and the peritoneal cavities lavaged 4 days later. cells were pelleted by centrifugation and resuspended in rpm1 medium with 10% fetal bovine serum (fbs ) and cultured for 8 h. adherent cells were determined to be virtually 100% macrophages by non-specific alpha-naphthyl esterase staining (stoddart and scott, 1988 ) . the macrophage cultures were then stimulated with 100 ng ml-' of e. coli lipopolysaccharide (lps ) (sigma, st. louis, mo) for 2 h, and frozen at -70°c until further use. the 2 h duration of lps stimulation was based upon the kinetics of tnf-a, production after lps stimulation in macrophages from other animal species (green and sargan, 1991 ). messenger rna was extracted both from 4 x 1 o6 unstimulated and 4 x 1 o6 lps stimulated macrophages using an mrna extraction kit (micro-fast trac, invitrogen, san diego, ca); 0.08 pg mrna from each source was used for cdna synthesis. synthesis of cdna was performed at 42°c for 2 x 60 min using oligo-dt primers (cdna-cycle kit, invitrogen). the cdna was then used in a polymerase chain reaction (pcr) with a 100 ~1 reaction mixture consisting of 10 mm tris-hcl (ph 8.3), 50 mm kcl, 1.5 mm mgcl,, 0.0 1% gelatine, 200 pm of each dntp, 30 pmol of each primer, and 2.5 u of pfu dna polymerase. three primers, making up two pairs p 1 /p3 and p2/p3, were constructed from a previously published sequence of feline tnf-a (mcgraw et al., 1990) : p 1, gggatccatgagcactgaaagcatgatccg; p2, ggggatcccagaa-cactcagatcatcttctc; p3, ggctgcagaattcacagggcaat-gatcccaaagta. the primers had restriction sites for bamhi, psti and ecorl in the 5'-ends to make directionally cloning of the pcr products possible. the forward primer p 1 was located at the start of the ff nf-a gene and the forward primer p2 at the assumed start of the coding sequences for the mature tnf-cr protein, while the backward primer p3 was located at the 3'-end of the coding sequences of the gene. the mixtures were overlaid with 30 ~1 mineral oil and heated at 94°c for 5 min and then cycled 35 times at 94°c for 1 min, 55 "c for 1 min, 72°c for 1 min with a final extension at 72°c for 7 min. the pcr products were subjected to electrophoresis on a 1.7% agarose gel using 3 v cm-' for 2 h in 0.5x tbe-buffer ( 1 xtbe = 0.09 m tris-borate, 2 mm edta ) and then stained with ethidium bromide. to assure that stimulated macrophage mrna was the actual source for cdna, cdna derived by reverse transcription of mrna from unstimulated macrophage cultures, as well as feline genomic dna, were extracted and used as targets in separate and parallel pcrs. the amplified fragments generated by both the p 1 /p3 and p2/p3 primers were separately cloned into the plasmid vector pcri1 (ta-cloning kit, invitrogen), and the nucleotide sequences were determined by conventional dideoxy sequencing of both strands. the cloned fragment from the amplification with p2/p3 was digested out with bamhi/ecori, and subcloned directly into the expression vector pgex-2t (pharmacia, uppsala, sweden). the pgex-2t vector has been used previously to express fiv-pl7 and -p24 proteins (reid et al., 199 1) . this expression vector contains an open reading frame encoding glutathione s-transferase (gst), followed by unique restriction endonuclease sites for bamhi, smal and ecorz, followed in turn by termination codons in all three frames. a thrombin cleavage site is constructed into the vector between gst and the protein to be expressed (chang, 1985 ) . the resulting plasmid, designated pgex-ft nf, was used to transform the e. coli strain xll-blue. a 100 ml overnight terrific-broth (t-broth) culture of e. coli containing pgex-ff nf was diluted 1: 10 in t-broth and incubated for 1 h at 37' c. all bacterial cultures contained 50 ,ug ml-' ampicillin and 12.5 ,ug ml-' tetracycline. expression of the recombinant protein was induced by adding isopropyl-p-d-thiogalactopyranoside (iptg) to a concentration of 0.3 mm. the culture was further incubated at 37°c for 5 h and then centrifuged for 10 min at 5oooxg. the supematant was discarded. the bacterial pellet was resuspended in 10 ml ice-cold phosphate-buffered saline (pbs), and sonicated twice for 30 s; samples were kept on ice. triton-x-100 was added to a concentration of 1%, the solution was centrifuged for 5 min at 10 000 xg, and the supernatant collected. one milliliter of a 50% slurry of glutathione-agarose beads was added to the supernatant and gently mixed for 3 min at room temperature, followed by three times washing with pbs. the fusion protein was eluted by adding 1 ml of 50 mm tris-cl (ph 8.0) with 10 mm reduced glutathione. the elution was repeated three times. the purity of the rftnf-cu, the efficiency of the elutions, and its relative molecular size were estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page). the part of the rfi'nf-cr-gst that bound to the glutathione-agarose beads, was washed with thrombin cleavage buffer (50 mm tris-cl, ph 7.5, 150 mm nacl, 2.5 mm cacl,) and incubated with 1% thrombin for 1 h at room temperature (chang, 1985) . the cleaved protein products were analyzed on sds-page. the concentrations of the purified recombinant proteins were analysed as described earlier (bradford, 1976) . peripheral blood mononuclear cells ( pmbcs) from four different normal donor cats were purified on ficoll-hypaque density gradients, and resuspended in growth medium (rpmi, 10% fbs, 1 ,ug ml-' concanavalin a (cona) ) to a concentration of 1 o6 cells ml-'. tenfold increasing concentrations (0, 1, 10, 100 and 1000 ng ml-' ) of rftnf-a-gst or rfi'nf-a were added to quintriplicate wells and the cells maintained in culture for 48 h before being analyzed for cell surface receptor expression. control wells contained growth medium without cona and no tnf-a. pmbcs from each culture were pelleted by low speed centrifugation, washed twice with pbs containing 2% fbs and 0.1% sodium azide (pbs/fbs/nan, ). the pelleted cells were then resuspended in one of the following reagents and incubated for 15 min: ( 1) 15 ~1 of tissue culture supernatant from mouse hybridoma cell line 9f23 containing antibodies against the alpha subunit of the feline interleukin-2 receptor (il-2r) (kindly provided by dr. k. ohno, tokyo, japan) (ohno et al., 1992) ; (2) 10 ~1 of tissue culture supematant from mouse hybridoma cell line 42.382, which contains antibodies to feline mhc class ii antigen (rideout et al., 1992 ) ; ( 3 ) 15 ~1 of tissue culture supematant from mouse hybridoma w6/32 which contains antibodies against feline mhc class i antigen (pollack et al., 1988) ; (4) 10 pg of rftnf-a. control cultures were left untreated. following incubation, cells were pelleted, and washed twice with pbs/ fbs/nan3. the tnf-a treated cells were incubated for an additional 15 min with 25 ~1 of mouse monoclonal antibody to human tnf-a receptor (biosource international, camarillo, ca), washed twice with pbs/fbs/nan,, and pelleted. cell pellets were then resuspended in 25 ~1 of a 1:25 dilution of goat f(ab' )2 anti-mouse igg-fitc (caltag laboratories, san francisco, ca) and 10 ~1 propidium iodide ( 100 mg ml-' ) were added to each tube and the samples incubated at 37°c for 15 min. samples were then washed twice with pbs/fbs/nan, buffer and 10 000 cells were analyzed immediately by flow immunocytometry using a 488 nm argon laser, standard filter configuration for two color analysis and consort 30 software (facscan, becton-dickinson, san jose, ca). data were analyzed with lysys software. events were gated on forward and log side scatter light characteristics and dead cells were eliminated from analysis based on propidium iodide staining. live cells were evaluated using log green fluorescence and analysis regions were set such that less than 2% of control cells were in the positive analysis region. western blot analysis of rffnf-cr and rftnf-a-gst were performed as described earlier (lutz et al., 1980) . polyclonal rabbit anti-murine tnf-a (genzyme, cambridge, ma) and monoclonal anti-human tnf-a (biosource international, camarillo, ca) were used. two adult new zealand white rabbits were immunised subcutaneously with 100 pg of rftnf-a-gst at weeks 0 and 2, and with the same amount of rffnf-(y on weeks 4, 6, 14, and 20. the first dose of antigen was in freund's complete adjuvant, while subsequent doses were in freund's incomplete adjuvant. major antibody activity was demonstrated against both gst and tnf-a component by western blotting. serial dilutions of recombinant proteins were tested for cytotoxic activity using the murine tibroblast cell line l929 as described (flick and gifford, 1984) . briefly, l929 cells were seeded into flat bottomed 96-well microtiter plates and incubated overnight in eagle's minimum essential medium (mem) supplemented with 5% fbs. medium was then replaced with mem containing 5 ,ug ml-' actinomycin d (sigma, st. louis, mo), samples were tested in quadruplicates of 100 ~1 and incubated for 16 h at 37°c and 5% co*. the cell supernatant was removed thereafter and the monolayer stained with crystal violet for 10 min. the absorbance of washed stained cell monolayers was measured at a wavelength of 595 nm using an automatic plate reader (biorad, hercules, ca). medium and recombinant feline immunodeliciency virus p24-gst (fiv-rp24-gst) were used as a negative control and recombinant mouse tnf-cr (rmtnf-ar, genzyme ) as a positive control. the concentration of ffnf-c~ resulting in 50% of the absorbance of the controls was considered the 50% cytotoxic dose ( cdso). recombinant ff nf-a ( 500 ng ml-' ) was incubated with different dilutions of both preimmune and immune rabbit anti-rltnf-a serum for 30 min at room temperature. the ffnf-cu antiserum mixtures were then tested with l929 cells as described above. the first study involved three groups of adult spf cats, each consisting of two animals. each group was injected i.v. with 25 or 50 pg kg-' of rftnf-cu-gst or 50 pg kg-' of gst dissolved in pbs. the cats were observed for clinical symptoms for a period of 48 h and rectal temperature was measured every 20 min the first 3 h. in a second study, two adult cats were each inoculated intravenously with 50 pg kg-' of rftnf-a-gst dissolved in pbs. clinical symptoms and rectal temperatures were measured 0,2,6, 12,24 and 48 h following treatment. a 700 bp dna fragment was amplified using primer pair p 1 /p3 and cdna from lps stimulated macrophages as target (fig. 1, lane 1) . no pcr product was amplified from cdna produced from mrna of unstimulated macrophages (fig. 1, lane 2 ) . three fragments of 1.7 kb, 400 bp and 250 bp were amplified from genomic dna (fig. 1, lane 3) . primer pair p2/p3 amplified a 500 bp dna fragment from cdna of stimulated macrophages (fig. 1, lane 4) . no pcr products were amplified from cdna derived from the mrna of unstimulated macrophages (fig. 1, lane 5 ) , while an 850 bp fragment was amplified from genomic feline dna (fig. 1, lane 6 ) . the primers pl /p3 of lps-stimulated, unstimulated feline macrophages, and genomic dna as targets, respectively. lanes 4, 5 and 6: rt-pcr using primers p2/p3 of lps-stimulated. unstimulated feline macrophages, and genomic dna as targets, respectively. sizes of the 1.7 kb and 850 bp amplified fragments from genomic feline dna corresponded to the distance between the pi-p3 and p2-p3 primers in the genomit ftnf-cu nucleotide sequence, respectively. similarly the 700 bp and 500 bp bands amplified from cdna from lps-stimulated macrophages corresponded to the estimated respective sizes of mrna for the pre-and mature-proteins of tnfo!. sequence analysis of the p 1 /p3 amplified product from cdna from stimulated macrophages showed a 99.3% homology to previously reported genomic dna sequence of ftnf-a gene, and 98.7% homology on the amino acid level. there were 9 1% and 8 1% sequence homologies between the mrna of ttnf-cu and the respective human and murine tnf-(y genes. the homologies between the deduced amino acid sequence of the mature part of ffnf-a, i.e. between primer pair p2/p3, to those of human and murine tnf-a, were 92% and 78%, respectively. a single band with a molecular size of 43 kda was observed in sds-page gels of the purified fusion protein rftnf-gst expressed by pgex-ffnf (fig. 2, lane 1) . thrombin cleaved the fusion protein into two fragments one with a size of 17 kda, which corresponds to the size of tnf-(u in other species (marmenout et al., 1985; pennica, et al., 1985) (fig. 2, lane 2) , the other was retained on the glutathione agarose beads after thrombin cleavage had thus a size of 26 kda, which corresponded to the size of gst (smith et al., 1986) . the amount of purified rftnf-gst produced was about 4 mg 1-l bacterial culture medium. however, the amount of rftnf-a after thrombin cleavage was only about one-tenth of this. no cross-reactivity was observed between polyclonal rabbit anti-murine tnf-(y antibodies and rffnf-a in western blot. as predicted from sequence homologies and previous studies (lehmann, et al., 1992) monoclonal anti-human tnfcr antibodies reacted specifically to both rffnf-gst and rff nf-(r (fig. 3 ) . l929 mouse tibroblast cells were susceptible to the toxic effects of both rffnfa-gst and rttnf-a (fig. 4) . at concentration below 125 ng ml-' the rffnf-a was significantly more toxic than a similar concentration of rftnf-lu-gst. the cds,, for the l929 cells was estimated to be 230 ng ml-' and 15 ng ml-' for rftnf-cy-gst and the rffnf-a, respectively. the cytotoxic effect of purified rffnf-a on l929 cells was completely neutralised by a 1: 10 dilution, and partially neutralised by a 1: 100 dilution, of rabbit anti rttnf-a! serum. preimmune serum had no inhibitory effect on the cytotoxicity. cats given rff nf-cu-gst, became clinically ill between 15 min and 10 h after treatment. a fever peaked at 4-5 h after treatment and disappeared after 10 h and was the most prominent syndrome (fig. 5) nously. an increase in the rectal temperature was apparent within 2 h in cats treated with rttnf-cu-gst but not with gst alone. depression, immobility caused by malaise, protrusion of the nictitating membranes, piloerection (especially along the dorsum of neck and back and on the tail) and hemoconcentration. clinical signs peaked at 4 h following treatment and had largely disappeared by 10 h. the clinical signs and their severity were similar in cats given either 25 and 50 pg kg-' of the fusion protein, except for one cat that was treated with 50 pg kg-' of rft nf-a-gst and developed moderately severe hypovolemic shock and signs of cerebellar hypoxemia (disorientation, loss of balance ). these signs appeared 4 h following treatment and lasted for about 1 h before spontaneously resolving. no clinical signs of illness were seen in two control cats that were given only the gst portion of the fusion protein (fig. 5 ) , although some variation was evident between the four individual donor cats, rftnf-a induced a dose related increase of ig2r and mhc class ii antigen expression on the cell surface of in vitro stimulated feline pmbcs (fig. 6 ) . at the highest rftnf-cr concentration, this increase was 13-23% for il-2r and 7-30% for mhc class ii antigen expression. recombinant ftnf-a had no stimulatory effect in vitro on mhc class i or tnf-cu receptor expression. biologically active recombinant feline tnf-a was expressed in e. coli. the entire itnf-a gene was cloned from cdna by pcr since the feline genomic tnf-a gene is interrupted by three introns, making it difficult to clone a functional tnf-a gene directly from genomic dna. although genomic dna was not used in the experiment the pcr primers were constructed from a tnf-a sequence that was itself derived from genomic dna . the cdna encoding the functional tnf-a gene was derived from mrna by reverse transcription. the mrna was extracted from peritoneal macrophages that were induced to produce high levels of tnf-a (and specific mrna) by e. coli lps stimulation. this procedure allowed for the ultimate construction of a plasmid containing only the relevant portions of the tnf-(r gene in a continuous linear configuration. the protein expression system used the pgex-2t vector. the gst fusion protein encoded by the plasmid pgex-itnf had a molecular size of 43 kda of which rftnf was making up 17 kda. this is in about the same size range as human and murine tnf-a (marmenout et al., 1985) ) and correlates well with the estimated molecular size ( 17.9 kda) of the 157 amino acid long mature feline tnf-cw. the reduction in the yield of rffnf-(y after the fusion protein had been cleaved with thrombin could have been caused by the lower solubility of the cleaved protein compared with the fusion protein. a possible obstructive effect of the agarose beads on the efficacy of thrombin cleavage could not be ruled out. analysis of the nucleotide sequence of the cloned mrna encoding for the preprotein of feline tnf-cy showed a homology of 99.3% with the coding sequences of a previously sequenced feline tnf-a gene ) and 98.7% homology at the amino acid level (98.8% homology for the mature part). the small nucleotide sequence divergence (0.7%) between these two ftnf-a genes reflects either limited genetic variations between individual cats and/or small errors in reverse transcription/pcr. if the differences were due to errors, however, the errors were not sufficient to alter the biological activity of the protein. the fusion protein and rftnf-a, both appeared to be biologically active. both proteins killed tnf-a-sensitive l929 mouse cells; rftnf-cr-gst had a toxic effect on these cells comparable to rftnf-cx at concentrations above 125 ng ml-', but at levels below 125 ng ml-' rff nf-cu was significantly more toxic than rfinf-(r-gst. this indicated some interference by the gst moiety on the rftnf-cu portion of the molecule. polyclonal rabbit anti-human tnf-a prevented the toxic effect of both rftnf-a-gst and rffnf-a on l929 cells, again demonstrating that both the cleaved and fused fi'nf-a! proteins were biologically and antigenitally intact. the rftnf-cr-gst was also biologically active when injected intravenously into cats. the onset and the character of clinical signs resembled those observed for tnf-(i! in other species (creagan et al., 1988) . since i.v. administration of recombinant gst did not induce any clinical signs, it can be deduced that the in vivo effects were caused by the rff nf-a portion of the fusion protein and not by the gst. as expected, it was shown that rftnf-a-gst upregulated the expression of ig2r and mhc class ii antigens in normal cultures of feline pmbcs. the induction of ig2r and mhc class ii antigen mrna by tnf-a involves the activation of transcriptional factors (maniatis et al., 1987) . tnf-a activates nfkb proteins that can induce expression of genes possessing kb-like enhancer elements in their regulatory regions (lowenthal et al., 1989b) . included in this group are the genes encoding ig2r (lowenthal et al., 1989a) , mhc class ii antigen (pessara and koch, 1990) , and human, feline and simian immunodeliciency viruses (folks et al., 1989; osbom et al., 1989; dewhurst et al., 1990; poli et al., 1990; sparger et al., 1992) . feline tnf-a possessed considerable antigenic cross-reactivity with human, but not with murine tnf-a! in western blotting. the degree of antigenic crossreactivity corresponded with the deduced amino acid sequence of the tnf-cz coding regions of the three species; ffnf-cu showed a genetic homology of 92% and 78% with human and murine tnf-a, respectively. in conclusion, our results demonstrate that rff nf-cz could prove to be a useful reagent for the study and treatment of feline disease. studies of the cytokinevirus interactions in the fiv infection of cats could be useful for human aids research. a rapid and sensitive method for the quantification of microgram quantities of proteins utilising the principle of protein dye binding infection of peritoneal macrophages in vitro and in vivo with feline immunodeficiency virus an endotoxininduced serum factor that causes necrosis of tumours thrombin specificity. requirement for a polar amino acid adjacent to the thrombin cleavage site of a polypeptide substrate a phase i clinical trial of recombinant human tumour necrosis factor sequence analysis and acute pathogenicity of molecularly cloned sivsmm_rbj14 gene sequence of porcine tumour necrosis factor alpha comparison of in vitro cell cytotoxic assays for tumour necrosis factor tumor necrosis factor a induces expression of human immunodeticiency virus in a chronically infected t-cell clone sequence of the cdna encoding ovine tumour necrosis factoralpha: problems with cloning by inverse pcr molecular cloning of the gene encoding rabbit tumour necrosis factor biologic activities and mechanisms of action of tumour necrosis factor-a/cachectin decreased mitogen responsiveness and elevated tumour necrosis factor production in cats shortly after feline immunodeficiency virus infection tumor necrosis factor a levels in cats experimentally infected with feline immunodeticiency virus: effects of immunization and feline leukemia virus infection tumor necrosis factor-alpha activation of the il2 receptor-alpha gene involves the induction of kappa b-specific dna binding proteins tumor necrosis factor a induces proteins that bind specifically to kb-like enhancer elements and regulate interleukin 2 receptor a-chain gene expression in primary human t-lymphocytes humoral immune reactivity to feline leukemia virus and associated antigens in cats naturally infected with feline leukemia virus regulation of inducible and tissue-specific gene expression molecular cloning and expression of human tumour necrosis factor and comparison with mouse tumour necrosis factor i99 1. cytokines and hiv infection: is aids a tumour necrosis factor disease ? gene sequence of feline tumour necrosis factor alpha thalidomide: treatment of severe recurrent aphthous stomatitis in patients with aids production of a monoclonal antibody that defines the alpha subunit of the feline il-2 receptor tumor necrosis factor alpha and interleukin i stimulate the human immunodeficiency virus enhancers by activation of the nuclear factor kb the feline immunodeficiency virus cloning and expression in e. co/i of the cdna for murine tumour necrosis factor tumor necrosis factor a regulates expression of the major histocompatibility complex class ii-associated invariant chain by binding of an nf-kb-like factor to apromotor element tumor necrosis factor a functions in an autocrine manner in the induction of human immunodeficient virus expression the detection ofconven-e. rimstad et al. / veterinary immunology and immunopathology 45 (i 99s) 297-310 tional class i and class ii i-e homologue major histocompatibility complex molecules on feline cells immunodiagnosis of feline immunodeficiency virus infection using recombinant viral p 17 and ~24 persistent upregulation of mhc class ii antigen expression on t lymphocytes from cats experimentally infected with feline immunodeliciency virus cloning and expression in escherichia coli of the gene for human tumour necrosis factor m, 26000 antigen of schistosoma japonicum recognized by resistant wehi 129/j mice is a parasite glutathione s-transferase regulation of gene expression directed by the long terminal repeat of the feline immunodeficiency virus isolation and identification of feline peritoneal macrophages for in vitro studies of coronavirus-macrophage interactions augmentation of in vitro hiv replication in peripheral blood mononuclear cells of aids and arc patients by tumour necrosis factor this work was supported by public health service grants ai-25802-03 and ca-50179-01. key: cord-023392-axd0901z authors: hansen, t. k.; tarnow, l.; thiel, s.; steffensen, r.; parving, h.‐h.; flyvbjerg, a. title: association between mannose‐binding lectin and vascular complications in type 1 diabetes date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423i.x sha: doc_id: 23392 cord_uid: axd0901z complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose‐binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02–2.27), p = 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 µg/l (iqr 753–4867 µg/l) versus 1491 µg/l (iqr 577–2944), p = 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 µg/l (iqr 636–5231 µg/l) versus 1741 µg/l (iqr 656–3149 µg/l), p = 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-013366-sbdtpsz6 authors: ramírez-pérez, sergio; hernández-palma, luis alexis; oregon-romero, edith; anaya-macías, brian uriel; garcía-arellano, samuel; gonzález-estevez, guillermo; muñoz-valle, josé francisco title: downregulation of inflammatory cytokine release from il-1β and lps-stimulated pbmc orchestrated by st2825, a myd88 dimerisation inhibitor date: 2020-09-21 journal: molecules doi: 10.3390/molecules25184322 sha: doc_id: 13366 cord_uid: sbdtpsz6 the inflammatory process implicates homeostasis disruption and increased production of inflammatory mediators. myeloid differentiation primary response 88 (myd88) is an essential protein recruited after lipopolysaccharide (lps) and interleukin (il)-1β stimulation, a process that converges in nuclear factor kappa b (nf-κb) activation, as well as a transcription of several genes of both proand anti-inflammatory cytokines. the inhibition of myd88 has shown efficacy by decrease inflammatory response, and has demonstrated potential application as a therapeutic target in chronic diseases. in this study, we investigate the effect of myd88 dimerisation inhibitor st2825 on cytokine production from rhil-1β and lps-stimulated peripheral blood mononuclear cells (pbmc) from healthy blood donors (hbd). st2825 significantly downregulates the production of ifn-γ, il-6, il-12, il-2, il-15, il-7, vegf, il-1ra, il-4, il-5, il-13 and il-9 (p < 0.05) in lps-stimulated pbmc. moreover, st2825 had a relatively low impact on il-1β signalling pathway inhibition, showing that only a few specific cytokines, such as ifn-γ and il-1ra, are inhibited in rhil-1β-stimulated pbmc (p < 0.01). in conclusion, myd88 dimerisation inhibitor st2825 showed high efficacy by inhibiting proand anti-inflammatory cytokine production in lps-stimulated pbmc. moreover, although rhil-1β induced a sustained cytokine production (p < 0.05), st2825 did not show a significant effect in the secretion of neither pronor anti-inflammatory cytokines in rhil-1β-stimulated pbmc. myeloid differentiation primary response 88 (myd88) represents an important molecule associated with activation of several signalling pathways, which are implicated in the inflammatory immune response [1] . interleukin (il)-1β and toll-like receptors (tlr) signalling pathways are one of the most strongly studied mechanisms in which myd88 plays a primary role [2] [3] [4] [5] . the il-1β activity implicates binding between this cytokine and the il-1r type i (il-1ri), this binding triggers the interaction of toll-il-1-receptor (tir) domains followed by myd88 recruitment and downstream signalling cascades, which converge in the activation of several transcription factors and pro-inflammatory cytokine production [2, 6] . regarding inflammation-associated tlr activation, molecules 2020, 25 , 4322 2 of 14 tlr4 has been the best-studied molecule in both innate and adaptive immunity [7, 8] . tlr4 signalling pathway can be activated myd88-dependent or independent manner [7] ; nevertheless, tlr4-dependent lps-regulated signalling involves the recruitment and homodimerisation of myd88 and leads to pathways of intracellular signal transduction which converge in the production of inflammatory mediators implicated in the regulation of the inflammatory process [8, 9] . overactivated myd88-dependent lps and il-1β inflammatory signalling pathways have displayed high expression of pro-inflammatory mediators not only in healthy blood donors (hbd), but also in patients with chronic, systemic and autoimmune diseases [10] [11] [12] [13] [14] [15] . due to this fact, the identification of new molecules that regulate these signalling pathways has taken high relevance [5, 11, [16] [17] [18] . the chemical molecule st2825 acts as an inhibitor of myd88 dimerisation and its activity has been demonstrated through the inhibition of tlr9-dependent cpg-regulated signalling, and inhibition of il-12, il-1β, il-6 and tumor necrosis factor alpha (tnf-α) expression in lps-stimulated raw 264.7 cells [19] [20] [21] [22] . however, the effect of the st2825 molecule on cytokine production mediated by il-1β and lps-stimulated peripheral blood mononuclear cells (pbmc) has yet been clarified. in the present study, pbmc obtained from hbd were stimulated with both rhil-1β and lps to identify pro-and anti-inflammatory cytokine profiles, as well as, the inhibitory activity of st2825 molecule on the cytokine secretion. our results indicate that inhibition of myd88 dimerisation mediated by st2825 molecule causes a decrease secretion of both pro-and anti-inflammatory cytokines in supernatants of lps-stimulated pbmc; however, st2825 showed high impact neither pro-nor anti-inflammatory cytokine secretion in rhil-1β-stimulated pbmc. to determine the specific concentration of st2825 in which the secretion of cytokines was inhibited, curves of different concentrations of st2825 were performed ( figure 1 ). tnf-α quantification was taken as a positive control, and the concentration of this cytokine was determined in supernatants of lps-stimulated pbmc after 24 h ( figure 1a ). the concentration of tnf-α in supernatants of pbmc without stimuli was 36.62 pg/ml; whereas, a high concentration of tnf-α in lps-stimulated pbmc was identified (906.7 pg/ml). regarding st2825 stimuli three different concentrations were taken 10, 30 and 50 µm and tnf-α levels were determined, the medium values were 234.6 pg/ml (p = not significant [n.s.]), 28.33 pg/ml (p < 0.05), and 15.60 pg/ml (p < 0.01), respectively. similarly, the concentration of tnf-α was determined in supernatants of rhil-1β-stimulated pbmc ( figure 1b ). tnf-α levels from rhil-1β-stimulated pbmc were 51.78 pg/ml, for rhil-1β plus 10 µm of st2825 were 8.24 pg/ml (p = n.s.), and after add 30 and 50 µm of st2528 to rhil-1β-stimulated pbmc, the tnf-α levels were 0 pg/ml for both (p < 0.05) ( figure 1b ). lps has been implicated in the production of pro-inflammatory cytokines through tlr4 activation. our results indicate that lps is a potent inductor of several pro-inflammatory cytokines in pbmc. statistically significant differences were found between pbmc treated with rpmi alone and lps (p < 0.01). in addition, st2825 molecule was used as a negative regulator of tlr4-dependent lps-regulated signalling pathway. st2825 in lps-stimulated pbmc decreased secretion of interferon gamma (ifn-γ) (p < 0.001), il-6 (p < 0.05), il-12 (p < 0.05), il-2 (p < 0.05), vascular endothelial growth factor (vegf) (p < 0.05), il-15 (p < 0.05) and il-7 (p < 0.01) ( figure 2 ; table s1 ). since our study included males and females; in order to identify differential effects on cytokine production release a statistical analysis by gender was performed. however, our results showed non-statistically significant differences between males and females (data not shown). as a control for the effect of st2825 alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st2825. for ifnγ, tnfα, il-1ra and il-2, statistically significant differences were found (p < 0.05). the levels of these cytokines in the presence of st2825 were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st2825 than in the basal response of untreated pbmc was not observed (table s2) . molecules 2020, 25, x 3 of 14 as a control for the effect of st2825 alone, a statistical analysis was performed by comparing the production of inflammatory cytokines studied in the presence or absence of st2825. for ifnγ, tnfα, il-1ra and il-2, statistically significant differences were found (p < 0.05). the levels of these cytokines in the presence of st2825 were significantly lower than in pbmc treated with rpmi alone; furthermore, a higher cytokine secretion as an effect of st2825 than in the basal response of untreated pbmc was not observed (table s2) . the concentration of anti-inflammatory cytokines was determined in supernatants of lpsstimulated pbmc. interestingly and contrary to expectations, after 24 h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il-1ra, il-4, il-5, il-13, il-10 and il-9 (p < 0.01). additionally, st2825 inhibited the secretion of il-1ra (p < 0.001), il-4 (p < 0.05), il-5 (p < 0.05), il-13 (p < 0.01) and il-9 (p < 0.001), but not il-10 (354.7 pg/ml, p = n.s.) ( figure 3 ; table s1 ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st2825 alone, have a similar response to unstimulated cells (table s2 ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st2825 can inhibit the observed response; this is a relevant finding that has not been reported till date. (b) the soluble levels of tnf-α in the supernatant of rhil-1β-stimulated pbmc at 10 ng/ml and rhil-1β (10 ng/ml) plus different concentrations of st2825 (10, 30 and 50 µm) were determined. significant inhibition was identified at 30 µm (p < 0.05) and 50 µm (p < 0.01) of st2825 for lps; while for rhil-1β significant inhibition was identified at 30 µm (p < 0.05) and 50 µm (p < 0.05) of st2825. data provided in medians and interquartile ranges (n = 4), φ kruskal-wallis test was performed, and dunn's test obtained statistically significant differences. the concentration of anti-inflammatory cytokines was determined in supernatants of lps-stimulated pbmc. interestingly and contrary to expectations, after 24 h of stimulation with lps in pbmc, we observed anti-inflammatory cytokine production of il-1ra, il-4, il-5, il-13, il-10 and il-9 (p < 0.01). additionally, st2825 inhibited the secretion of il-1ra (p < 0.001), il-4 (p < 0.05), il-5 (p < 0.05), il-13 (p < 0.01) and il-9 (p < 0.001), but not il-10 (354.7 pg/ml, p = n.s.) ( figure 3 ; table s1 ). moreover, it was observed that anti-inflammatory cytokine secretion from pbmc stimulated with st2825 alone, have a similar response to unstimulated cells (table s2 ). based on these results, pbmc stimulated with lps can secrete pro-and anti-inflammatory cytokines and st2825 can inhibit the observed response; this is a relevant finding that has not been reported till date. the role of il-1β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil-1β-stimulated pbmc (table 1 ). in an exciting way and as previously reported, il-1β represents an important cytokine capable of inducing th17-related cytokine profile. in this study, we observed high production of these cytokines: however, only il-17a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after 24 h of rhil-1β stimulation (p < 0.05). furthermore, the concentration of cytokines, such as il-12 (p < 0.01), vegf (p < 0.05) and il-15 (p < 0.05), were found higher after rhil-1β stimulation. on the other hand, only il-4 and il-9 significantly increased after rhil-1β stimulation (p < 0.01). regarding the st2825 effect on rhil-1β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il-1β signalling pathway inhibition (table 1) . st2825 molecule only inhibited the secretion of ifn-γ (p < 0.01) and il-1ra molecules 2020, 25, 4322 4 of 14 (p < 0.001). the present study shows that the specific inhibition of critical components in the il-1 signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd88-independent mechanisms could regulate the production of cytokines in pbmc. the role of il-1β has been widely described in several chronic conditions, as well as in several cell types. our results showed high secretion of pro-inflammatory cytokines in rhil-1β-stimulated pbmc (table 1 ). in an exciting way and as previously reported, il-1β represents an important cytokine capable of inducing th17-related cytokine profile. in this study, we observed high production of these cytokines: however, only il-17a, granulocyte-monocyte colony-stimulating factor (gm-csf) and granulocyte colony-stimulating factor (g-csf) increased significantly after 24 h of rhil-1β stimulation (p < 0.05). furthermore, the concentration of cytokines, such as il-12 (p < 0.01), vegf (p < 0.05) and il-15 (p < 0.05), were found higher after rhil-1β stimulation. on the other hand, only il-4 and il-9 significantly increased after rhil-1β stimulation (p < 0.01). regarding the st2825 effect on rhil-1β-stimulated pbmc and contrary to our expectations; our results showed that this molecule had a relatively low impact on the il-1β signalling pathway inhibition (table 1) . st2825 molecule only inhibited the secretion of ifn-γ (p < 0.01) and il-1ra (p < 0.001). the present study shows that the specific inhibition of critical components in the il-1 signalling pathway is not enough to avoid the secretion of inflammatory mediators, the above suggests that various myd88-independent mechanisms could regulate the production of cytokines in pbmc. the role of inflammatory key mediators, such as il-1β and lps, in activating the innate immune response and subsequently leading to inflammation has been widely described in several studies [2, 3] . il-1β and lps-signalling pathways trigger cascades of intracellular activation mediated by myd88 recruitment, which converges in the activation of transcription factors, such as nf-κb and its translocation to the nucleus [2, 3, 23, 24] . the expression of receptors associated with these pro-inflammatory mediators can be given in a variety of cells of the immune system, within the main ones are professional antigen-presenting cells, treg cells and effector t cells [24] [25] [26] [27] . our results show that after 24 h, pro-inflammatory cytokine levels of il-1β, tnf-α, ifn-γ, il-6, il-12, il-17a, g-csf, gm-csf, il-2, vegf, il-15 and il-7 significantly increased in lps-stimulated pbmc. recent studies performed in vitro have described that in both pbmc and thp-1 cells can be possible to carry out a differentiation towards m1 macrophages by stimulation with lps, ifn-γ or gm-csf; m1 macrophages have been characterised by the expression of pro-inflammatory cytokines, such as il-6, il-12, tnf-α and il-1β [13, 28, 29] . regarding cytokines, such as il-6 and tnf-α, previous studies performed in lps-stimulated pbmc from hbd reported increased levels of these cytokines in comparison with those levels found in unstimulated pbmc [13, 30] . an important cytokine involved in the innate immune response mediated by nk and nkt cells is il-15, which increased after lps stimulation; concerning this result, one study reported that lps-stimulated monocytes could produce il-15 [9] . concerning the expression of gm-csf and vegf, previous studies conducted in lps-stimulated pbmc after 24 h showed high levels of both cytokines, as we observed in this study [15] . an earlier report indicated that gm-csf expression increased in lps-stimulated pbmc; nonetheless, its expression remains low compared with other pro-inflammatory cytokines, such as tnf-α or il-6, which could suggest that this cytokine is produced only for a specific population of monocytes [13] . the activation of several transcription factors after lps stimulation has been demonstrated in a large number of studies. rothfuchs et al. reported that ifn-γ mrna expression was strongly diminished in tlr4 -/macrophages compared with the wild type phenotype; moreover, tlr4 activation causes myd88-dependent ifn-α production, which results in an autocrine effect that regulates ifn-γ mrna expression by stat1 activation [31] . on the other hand, increased expression of ror-γt and the phosphorylated form of nf-κb1 were found after lps stimulation, and this effect was associated with the differentiation of th17 cells and high expression of il-17a [32] . peyssonnaux et al. reported that hif-1α was highly expressed in lps-stimulated macrophages; interestingly, hif-1α/rorγt/p300 complex can be linked to the promoter region of il17a and can also promote its transcription [9, 33, 34] . vegf is a cytokine produced in response to the hypoxia process and subsequently to hif-1α activation which, as mentioned above, hif-1α is a transcription factor induced in response to lps [15, 33] . transcription factor activation followed by high production of pro-inflammatory cytokines in a tlr4-lps-dependent pathway has been widely described. however, posttranscriptional mechanisms implicated in tlr4 activation have also recently been described. for example, arid5a protein is a crucial factor for the production of il-6; this protein binds to the 3'-utr region of the il-6 mrna and leads to its stabilisation and subsequently, to its efficient expression in vivo [35] . moreover, nyati et al. described an alternative lps signalling pathway that is independent on p38 activation, but dependent on the mitogen-activated protein kinase (mapk) phosphatase 1 (mkp-1) [35] . this phosphatase mkp-1 can induce the translocation from the nucleus to the cytoplasm of au-rich element rna-binding protein 1 (auf-1), and auf-1 acts by stabilising the mrna of il-6, tnf-α and il-10 [35] . according to the present study, we observed that the production of anti-inflammatory cytokines significantly increases in lps-stimulated pbmc. several studies have reported that tlr4-dependent lps-regulated signalling pathway causes a predominantly pro-inflammatory response in pbmc. however, some interesting studies have reported that the expression of irf4 could also be expressed after lps/ifn-γ or il-4 stimulation; irf4 is a key transcription factor involved in the differentiation of m2 macrophages [28, 36, 37] . however, a significant limitation in our study is that the expression of cytokines was determined after 24 h of stimulation. in this regard, several reports indicate that pro-inflammatory cytokines have an early maximum expression peak within the first eight hours of lps stimulation (il-12, il-1β, il-6, tnf-α and il-15) and expression of cytokines, such as il-10 and il-1ra, exhibit a maximum expression peak at 20 and 48 h, respectively [9] . this behaviour on the cytokine profile could also be explained by a process known as "tlr tolerance", which is characterised by reducing expression of pro-inflammatory cytokines and high expression of m2 activation markers after sustained exposure to tlr ligands [38] . additionally, a specific type of m2 polarisation in human mononuclear cells after induction of lps tolerance has been reported [38] , and after lps tolerance recovering, macrophages can express both m1 and m2 polarisation states [39] . stimulation with rhil-1β triggered the production of several pro-inflammatory cytokines. interestingly, as previously described in other studies, this cytokine induces the release of th17-related cytokine profile; il-17a, gm-csf and g-csf were significantly expressed in our research [40] [41] [42] . nevertheless, a new t helper cell subset characterised by high production of gm-csf has been currently described [43] . this cell subset was identified as gm-csf producing cd4 + t cell (th-gm-csf), and its differentiation depends on il-1β signalling pathway, and subsequent irak1 and nf-κb activation [43] . furthermore, th-gm-csf cells are able to produce high concentrations of pro-inflammatory cytokines, such as il-12, il-6 and tnf-α [43] . our results also showed increased expression of il-12 and vegf; regarding il-12 production, langlet et al. reported that this cytokine could be expressed in an il-1β-dependent activation [44] . moreover, previous reports have shown that vegf expression requires activation of hif-1α, and activation of hif-1α occurs after il-1β stimulation [45] [46] [47] . concerning the results of the inhibition of myd88 dimerisation and according to expectations, st2825 molecule significantly inhibits the pro-inflammatory and anti-inflammatory cytokine production mediated by the activation of lps-tlr4 pathway. about these results, long et al. reported a significant decrease in cytokine expression of il-12, il-1β, il-6 and tnf-α from raw264.7 cells treated with lps plus st2825 [22] . several studies have reported that st2825 causes decreased recruitment and activation of specific molecules and transcription factors involved in the activation of tlr4 and the lps-dependent immune response activation. the main inhibited molecules, due to the activity of st2825, are irak1, irak4, traf6, p-ikk, p-ikbα, p-nf-κb and hif-1α [48, 49] . st2825 has also been proposed as a novel drug targeting in diseases like lymphoma, leukaemia, human hepatocellular carcinoma, and traumatic brain injury [50] [51] [52] . however, its role as a possible inhibitor in the production of cytokines produced after stimulation with lps remains undetermined. the decrease recruitment of molecules implicated in the myddosome formation may explain the inhibition of pro-and anti-inflammatory cytokines as a consequence of tlr4-dependent lps-regulated signalling. at the same time, signalling pathway activation after il-1-il-1ri-il-1racp complex formation has been widely described [3, 6, 23, 53, 54] . this process implicates recruitment and homodimerisation of myd88, as well as intracellular signalling cascades that converge in the transcription of genes of pro-inflammatory mediators [3, 6, 23, 53, 54] . specific myd88 dimerisation inhibition has been tested in many studies where the role of this molecule in an il-1β-dependent activation pathway was evident [21, 55, 56] . another study performed in healthy human articular chondrocytes reported decreased map kinase (mapk) activation after myd88 dimerisation inhibition and il-1β stimulation [57] . an interesting study conducted by wang et al. (2019) previously reported that st2825 decreases the expression of several molecules involved in the myddosome formation, such as phosphorylated btk and iκb, along with the decreased secretion of il-10 and ifn-β from b-cell lymphoma cell lines [58] . in our study, il-10 release decreases on both il-1β and lps-stimulated pbmc treated with st2825; nonetheless, the effect was not statistically significant. in relation to the ifn response, a decreased secretion of ifn-γ on both il-1β and lps-stimulated pbmc was observed our study as an effect of st2825. however, our results did not show a substantial inhibitory effect on cytokine production from pbmc treated with il-1β plus st2825. in regard to this result, loiarro et al. (2007) previously described an inhibitory effect observed on nf-κb activity after stimulation with il-1β stimulation (30 ng/ml) plus st2825 (30 µm) on hek293t cells [19] . nevertheless, although nf-κb activity decreases, st2825 did not deplete nf-κb activation, which could provide signalling activation enough to produce some of the inflammatory cytokines observed in our study [19] . indeed, since this st2825 chemical compound affects only the association of tir domains of myd88 and the disruption of this tir domain interaction inhibits the recruitment of irak4. by extension, irak1 and the subsequent signalling cascade, dimerisation inhibition of myd88 might affect only one specific signalling pathway [19] . these results might suggest that alternative il-1β signalling pathways independent of myd88 homodimerisation and recruitment in pbmc could be active as well. this approach arises from previous studies in which new receptors associated with the recognition of il-1β have been observed. il-1racpb is a unique receptor expressed on neurons, and its expression implicates activation of certain signalling pathways, such as p38 mapk, but not nf-κb; it has even been observed that this alternative signalling pathway is independent on myd88, irak1 and traf6 [59, 60] . moreover, heinz et al. reported a new molecule known as unc5cl; this protein contains death domains (dd) similarly to those found in myd88 [61] . unc5cl is considered as a pro-inflammatory signalling inducer and involves recruitment of irak1, irak4, traf6 and converges in nf-κb and jnk activation in a myd88-independent manner [61] . currently, there are not reports in which these new molecules have been reported in pbmc; however, the possibility to perform future studies that elucidate this observation remains open. moreover, in spite of the fact that st2825 did not show a substantial inhibitory effect on downregulation of inflammatory cytokine from il-1β-stimulated pbmc, it might be necessary to consider the use of other il-1 inhibitors, such as il-37 and il-38 [62, 63] , which could be useful to compare a differential response orchestrated by st2825 on il-1β-stimulated pbmc and the inhibitory effect of both il-37 and il-38. in this regard, conti et al. mentioned that il-37 suppresses the innate and acquired immune response and inhibits the inflammation through its binding with il-18 receptor-α chain (il-18rα) [64] . additionally, il-37 can activate the mtor signalling pathway and increase the adenosine monophosphate (amp) kinase [64] ; therefore, il-37 and il-38 might represent cytokines of great interest in experimental models where the suppressive effect of several inflammatory mediators is required. furthermore, a weakness of this study was not to measure cell viability after stimulation with st2825. differences (that were not statistically significant) have been previously reported for st2825 in other studies at 10, 20 or 30 µm on cell viability [19, 20, 58] . our study used pbmc, reduction on specific pbmc subpopulations might contribute to the behaviour observed on these inflammatory cytokines. therefore, considering this limitation will be important in future studies to analyse the observed response of this molecule on cell viability and the percentage of apoptosis by the effect of st2825. the understanding of inflammatory mechanisms regarding activation and effector function on physiological processes in the first instance can provide us with an overview of the behaviour of specific factors involved in the regulation and maintenance of the inflammatory process and how it can lead to the development of chronic inflammation. this study provides information about the inhibitory activity of st2825 on cytokine production (figure 4) , possibly through the tlr4 signalling pathway regulation in lps-stimulated pbmc. moreover, a relatively low impact on il-1β signalling pathway inhibition orchestrated by st2825 in pbmc was observed ( figure 4) ; possibly due to the various mechanisms of activation that remain unexplored on this signalling pathway, which could be cell subset-dependent. nevertheless, future studies focused on the identification of specific down-stream factors implicated in both il-1β and lps signalling pathway activation to elucidate how the regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st2825 chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. regulation of cytokine production takes place in distinct pbmc subpopulations will be required. therefore, our results not only provide valuable evidence about the potential use of the st2825 chemical molecule to inhibit the inflammatory cytokine release in pbmc from hbd, but also leave open the possibility to study the effect of this molecule in chronic diseases, such as rheumatic and autoimmune diseases, in which the inflammatory process plays a critical role. ten hdb over 18 years of age were included in the study, five females and five males. a blood sample (30 ml) was collected from each participant to obtain pbmc and perform cell culture experiments. the presence of infections and body mass index ≥ 25 kg/m 2 were taken as exclusion criteria. all subjects included in the present study signed the informed consent letter before their inclusion, and the present study was performed following the declaration of helsinki amendments. the pbmc were isolated from a blood sample following the density gradient separation method using histopaque ® -1077 (sigma aldrich, st. louis, mo, usa; ρ 1.076-1.078 g/ml). the obtained pbmc were washed and resuspended in rpmi-1640 medium supplemented with penicillin (50 u/ml, sigma aldrich, st. louis, mo, usa) and streptomycin (50 µg/ml, sigma aldrich, st. louis, mo, usa) at a concentration of 1% for both. the trypan blue test analysed the cell viability, and the total of separated cells per ml was quantified directly in a neubauer chamber. the pbmc were placed in 24-well plates after the density adjustment at 1 × 10 6 cells/ml (final volume of 1000 µl). cells were cultured in rpmi-1640 medium without serum and supplemented with antibiotic and antifungal. untreated cells were taken as the control group for each experiment. the effect of lps (30 ng/ml) in the secretion of tnf-α was studied in samples from four hbd to demonstrate the positive stimulation of the cells. the concentration of st2825 necessary to inhibit the rhil-1β and lps response in pbmc was determined at 0, 10, 30 and 50 µm. subsequently, pbmc culture was performed with lps (30 ng/ml), lps (30 ng/ml) plus st2825 (30 µm) or without stimulation (control group). similarly, pbmc were stimulated with rhil-1β (10 ng/ml), rhil-1β (10 ng/ml) plus st2825 (30 µm), st2825 (30 µm) alone or without stimulation (control group). st2825 molecule was added to the corresponding well 15 min before the stimuli with rhil-1β or lps. each experiment was done with two biological replicates and was incubated for 24 h at 37 • c in a humidified 5% co 2 atmosphere. once the incubation time had elapsed, supernatants were collected and stored at −80 • c for the subsequent quantification of pro-inflammatory and anti-inflammatory cytokine secretion. the concentration of il-1β, tnf-α, ifn-γ, il-6, il-12, il-17a, m-csf, gm-csf, il-2, vegf, il-15, il-7, il-1ra, il-4, il-5, il-13, il-10 and il-9 and were quantified from culture supernatants by multiplex immunoassay method using the bio-plex pro tm human cytokine 27-plex assay (bio-rad laboratories, inc., hercules, ca, usa) according to the manufacturer's instructions. luminex magpix ® (luminex corporation, austin, tx, usa) instrument was used for xmap assays. all data were shown as medians and interquartile ranges. the differences between the non-parametric quantitative variables were analysed by u of mann-whitney test to compare two groups and both the kruskal-wallis test and the dunn's post hoc test for multiple comparisons. data analysis was performed using graphpad prism v5.0 software, and a p-value < 0.05 was considered statistically significant. myd88: a central player in innate immune signaling understanding early tlr signaling through the myddosome the il-1 family of cytokines and receptors in rheumatic diseases signaling in innate immunity and inflammation future potential therapeutic targets for ra the interleukin-1 receptor family toll-like receptors: critical proteins linking innate and acquired immunity toll-like receptors in immunity and inflammatory diseases: past, present, and future lps-induced cytokine production in human monocytes and macrophages lipopolysaccharide and il-1β coordinate a synergy on cytokine production by upregulating myd88 expression in human gingival fibroblasts the il-1β signalling pathway and its role in regulating pro-inflammatory and pro-labour mediators in human primary myometrial cells effect of pro-inflammatory/anti-inflammatory agents on cytokine secretion by peripheral blood mononuclear cells in rheumatoid arthritis and systemic lupus erythematosus elispot analysis of lps-stimulated leukocytes: human granulocytes selectively secrete il-8, mip-1beta and tnf-alpha increased production of il-6 and il-17 in lipopolysaccharide-stimulated peripheral mononuclears from patients with rheumatoid arthritis analysis of cytokines and chemokines produced by whole blood, peripheral mononuclear and polymorphonuclear cells discovery of small molecule inhibitors of myd88-dependent signaling pathways using a computational screen the role of intermediary domain of myd88 in cell activation and therapeutic inhibition of tlrs toll-like receptors as therapeutic targets for autoimmune connective tissue diseases advance: inhibition of myd88 dimerisation and recruitment of irak1 and irak4 by a novel peptidomimetic compound pharmacological inhibition of tlr9 activation blocks autoantibody production in human b cells from sle patients targeting the toll-like receptor/interleukin 1 receptor pathway in human diseases: rational design of myd88 inhibitors polygonatum sibiricum polysaccharides play anti-cancer effect through tlr4-mapk/nf-κb signaling pathways interleukin-1 (il-1) pathway the regulatory roles of toll-like receptor 4 in secretions of type 1/type 2 relative cytokines by splenocytes and dendritic cells exposed to clonorchis sinensis excretory/secretory products expression of interleukin 1 receptors on human peripheral t cells the interleukin-1 family: back to the future optimised flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types i and ii thp-1 and human peripheral blood mononuclear cell-derived macrophages differ in their capacity to polarise in vitro m2 macrophages and their role in rheumatic diseases peripheral blood mononuclear cells from neovascular age-related macular degeneration patients produce higher levels of chemokines ccl2 (mcp-1) and cxcl8 (il-8) intracellular bacterial infection-induced ifn-gamma is critically but not solely dependent on toll-like receptor 4-myeloid differentiation factor 88-ifn-alpha beta-stat1 signaling lipopolysaccharide directly stimulates th17 differentiation in vitro modulating phosphorylation of relb and nf-κb1 cutting edge: essential role of hypoxia inducible factor-1alpha in development of lipopolysaccharide-induced sepsis e3 ubiquitin ligases siah1/2 regulate hypoxia-inducible factor-1 (hif-1)-mediated th17 cell differentiation tlr4-induced nf-κb and mapk signaling regulate the il-6 mrna stabilising protein arid5a interleukin-4 induced interferon regulatory factor (irf) 4 participates in the regulation of alternative macrophage priming negative regulation of toll-like-receptor signaling by irf-4 endotoxin tolerance represents a distinctive state of alternative polarisation (m2) in human mononuclear cells identification of a unique hybrid macrophage-polarisation state following recovery from lipopolysaccharide tolerance interleukin-1 and il-23 induce innate il-17 production from gammadelta t cells, amplifying th17 responses and autoimmunity pivotal role of dermal il-17-producing γδ t cells in skin inflammation critical regulation of early th17 cell differentiation by interleukin-1 signaling interleukin-1β-induced irak1 ubiquitination is required for th-gm-csf cell differentiation in t cell-mediated inflammation pkc-alpha controls myd88-dependent tlr/il-1r signaling and cytokine production in mouse and human dendritic cells interleukin 1 induces hypoxia-inducible factor 1 in human gingival and synovial fibroblasts il-1beta-mediated up-regulation of hif-1alpha via an nfkappab/cox-2 pathway identifies hif-1 as a critical link between inflammation and oncogenesis regulation of hypoxia-inducible factor-1α (hif-1α) expression by interleukin-1β (il-1 β), insulin-like growth factors i (igf-i) and ii (igf-ii) in human osteoarthritic chondrocytes dioscin reduces lipopolysaccharide-induced inflammatory liver injury via regulating tlr4/myd88 signal pathway tlr4 promotes the expression of hif-1α by triggering reactive oxygen species in cervical cancer cells in vitro-implications for therapeutic intervention effect of st2825 on the proliferation and apoptosis of human hepatocellular carcinoma cells inhibition of myeloid differentiation factor 88(myd88) by st2825 provides neuroprotection after experimental traumatic brain injury in mice myd88 inhibitor st2825 suppresses the growth of lymphoma and leukaemia cells overview of the interleukin-1 family of ligands and receptors the interleukin (il)-1 cytokine family-balance between agonists and antagonists in inflammatory diseases interactive sites in the myd88 toll/interleukin (il) 1 receptor domain responsible for coupling to the il1beta signaling pathway peptide-mediated interference of tir domain dimerisation in myd88 inhibits interleukin-1-dependent activation of nf-{kappa}b irak1 and traf6 knockdown in human chondrocytes inhibits interleukin-1-induced matrix metalloproteinase-13 gene expression and promoter activity by impairing map kinase activation disrupting myddosome assembly in diffuse large b-cell lymphoma cells using the myd88 dimerisation inhibitor st2825 neuron-specific effects of interleukin-1β are mediated by a novel isoform of the il-1 receptor accessory protein a central nervous system-restricted isoform of the interleukin-1 receptor accessory protein modulates neuronal responses to interleukin-1 the death domain-containing protein unc5cl is a novel myd88-independent activator of the pro-inflammatory irak signaling cascade interleukin-1 family cytokines and mast cells: activation and inhibition car-t cell therapy causes inflammation by il-1 which activates inflammatory cytokine mast cells: anti-inflammatory role of il-37 induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies this article is an open access article distributed under the terms and conditions of the creative commons attribution the authors declare no conflict of interest.molecules 2020, 25, 4322 key: cord-023402-8qfmo6rq authors: reinholdt, j.; baxendale, h.; ekström, n.; kayhty, h.; poulsen, k.; kilian, m. title: pneumococcal iga1 protease activity interferes with opsonophagocytosis of streptococcus pneumoniae mediated by serotype‐specific human monoclonal iga1 antibodies date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423t.x sha: doc_id: 23402 cord_uid: 8qfmo6rq bacteria‐specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody‐treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as fabα (monovalent) deprived of fcα which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody‐coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero‐steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type‐4 polysaccharide‐induced phagocytosis of iga1 protease‐deficient type‐4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type‐4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type‐4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease‐producing target bacteria was almost exclusively in the form of fabα. conversely, iga1 from protease‐deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody‐mediated opsonophagocytosis. besides, in these experiments, iga‐mediated opsonophagocytosis was independent of complement. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023373-6wh1kb3p authors: melchjorsen, j.; bowie, a. g.; matikainen, s.; paludan, s. r. title: differential requirements for toll‐like receptor signalling for induction of chemokine expression by herpes simplex virus and sendai virus date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423r.x sha: doc_id: 23373 cord_uid: 6wh1kb3p toll‐like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress‐associated molecules. tlr–ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus‐induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus‐encoded inhibitor of tlr‐signalling a52r or dominant‐negative myd88 totally inhibited hsv‐induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv‐induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr‐dependent and ‐independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023431-zjyrhlxn authors: sigmundsdóttir, h.; johnston, a.; gudjónsson, j. e.; valdimarsson, h. title: differential effects of interleukin‐12 and interleukin‐10 on superantigen‐induced expression of cutaneous lymphocyte‐associated antigen and αeβ7 integrin (cd103) by cd8(+) t cells date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423ab.x sha: doc_id: 23431 cord_uid: zjyrhlxn the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin‐10 (il‐10), il‐12 and transforming growth factor (tgf)‐β are important regulators of chronic inflammatory disease, where cutaneous lymphocyte‐associated antigen (cla) and αe integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue‐specific homing but may help to retain t cells within epithelial layers. we have previously shown that il‐12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8(+) but not cd4(+) t cells. while il‐12 increased superantigen‐stimulated expression of cla, this cytokine strongly inhibited the cd103 expression, and a combination of il‐12 and tgf‐β completely abrogated the induced cd103 expression. conversely, il‐10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1‐mediated inflammatory responses, il‐10 may also inhibit the migration of cd8(+) t cells into the skin while il‐12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il‐10 and il‐12, and the balance between these cytokines could influence the t‐cell migration of inflammatory cells into epithelial tissues. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-023421-1d1gf7az authors: sønder, s. u. s.; hedegaard, c. j.; bendtzen, k. title: monitoring patients treated with type 1 interferons: antiviral versus mxa induction assays date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423bb.x sha: doc_id: 23421 cord_uid: 1d1gf7az interferon‐α/β (ifn‐α/β) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large‐scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large‐scale screening in specialized laboratories. the read‐out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn‐α or ‐β, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-016960-xhzvp35g authors: berencsi, györgy; szomor, katalin n. title: fetal and neonatal illnesses caused or influenced by maternal transplacental igg and/or therapeutic antibodies applied during pregnancy date: 2012-03-08 journal: maternal fetal transmission of human viruses and their influence on tumorigenesis doi: 10.1007/978-94-007-4216-1_9 sha: doc_id: 16960 cord_uid: xhzvp35g the human fetus is protected by the mother’s antibodies. at the end of the pregnancy, the concentration of maternal antibodies is higher in the cord blood, than in the maternal circulation. simultaneously, the immune system of the fetus begins to work and from the second trimester, fetal igm is produced by the fetal immune system specific to microorganisms and antigens passing the maternal-fetal barrier. the same time the fetal immune system has to cope and develop tolerance and t(reg) cells to the maternal microchimeric cells, latent virus-carrier maternal cells and microorganisms transported through the maternal-fetal barrier. the maternal phenotypic inheritance may hide risks for the newborn, too. antibody mediated enhancement results in dengue shock syndrome in the first 8 month of age of the baby. a series of pathologic maternal antibodies may elicit neonatal illnesses upon birth usually recovering during the first months of the life of the offspring. certain antibodies, however, may impair the fetal or neonatal tissues or organs resulting prolonged recovery or initiating prolonged pathological processes of the children. the importance of maternal anti-idiotypic antibodies are believed to prime the fetal immune system with epitopes of etiologic agents infected the mother during her whole life before pregnancy and delivery. the chemotherapeutical and biological substances used for the therapy of the mother will be transcytosed into the fetal body during the last two trimesters of pregnancy. the long series of the therapeutic monoclonal antibodies and conjugates has not been tested systematically yet. the available data are summarised in this chapter. the innate immunity plays an important role in fetal defence. the concentration of interferon is relative high in the placenta. this is probably one reason, why the therapeutic interferon treatment of the mother does not impair the fetal development. at term, the amount of maternal igg antibody is higher in the neonate than in the mother. this pattern holds when the igg antibody is an anti-tnfa medication. a specific fc receptor neonate (fcrn) facilitates transfer of the igg antibodies across the syncytiotrophoblast into the fetal circulation (kane and acquah 2009 ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to the mother. by discontinuing this drug 8-10 weeks prior to delivery, the baby will likely be born with no or minimal serum levels, thus avoiding immunosuppression in a young infant. though there are some suggestive data that certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules (clowse 2010) . in early human placenta the exchange tissue area (villi) is formed around the entire surface of the conceptus. this placental shape is called diffuse placenta. by the third month of pregnancy, only the villi near the initial site of implantation have persisted, leading to the formation of the disc-shaped placenta. although chorioallantoic placenta in humans begins functioning already by the end of the fourth week of pregnancy, this process is completed with the formation of diskshaped placenta (sadler 2004) . during the first trimester, the human fetus is surrounded by two fluid cavities, i.e., the inner amniotic cavity and the outer extra-embryonic coelomic cavity. the chorioallantoic placenta is only formed of fetal vessels' endothelium and trophoblastic layer, bathed directly in the maternal blood (van der aa et al. 1998 ). for 8 out of 15 monoclonal therapeutic compounds, for which toxicity studies (in a broad sense) were performed, no significant maternal, fetal, or neonatal toxicity was observed. for the remaining seven products, the most common adverse effects on reproduction and development were reduced fetal weight, increased abortion rates, and reduction in fertility, indicating the general toxicity of these compounds (pentsuk and van der laan 2009 ). twenty-four (59%) of the 41 children had one or more congenital anomalies that are part of vacterl association, but only one child (with maternal etanercept administration) was diagnosed with vacterl association i.e. v: vertebral defects, a: anal atresia or imperforate anus, c: cardiac abnormalities [atrial septal defect, ventricular septal defect, and tetralogy of fallot], t: tracheoesophageal fistula or tracheal atresia/stenosis, e: esophageal atresia, r: radial and or renal abnormalities, and pre-axial l: limb abnormalities (carter et al. 2006 (carter et al. , 2009 ). fc-fcgr interactions play a critical role in the biological function of antibody and are likely to be instrumental in preventing or modulating lentiviral infections. antibody responses that depend on fc-fcgr interactions may help widen the spectrum and increase the potency of vaccine-induced antibodies (forthal and moog 2009) . maternofetal transmission of non-neutralising dengue virus specific antibodies result in the development of hemorrhagic fever of the newborns even in the case of the first infection with a heterotypic virus. the mechanism is the formation of non-neutralised neonatal virus and heterotypic maternal igg complexes, which will be taken up by endothelial and hematopoetic cells by (virus-abfc)-fcgr pinocytosis. the risk disappears after the 8th month of age of the children, when the maternal igg disappeared from the circulation of the children (libraty et al. 2009 ). this phenomenon has been observed in the case of other flaviviruses and enteroviruses, too (ferenczi et al. 2008; wang et al. 2010b ). dengue serotype cross-reactive cytotoxic lymphocytes (ctl) clones showing high avidity for antigen produce higher levels of inflammatory cytokines than serotype-specific clones. high avidity cross-reactive memory ctl may produce inflammatory cytokines during the course of secondary infection, contributing to the pathogenesis of vascular leak. these cells appear to be subsequently deleted leaving a more serotype-specific memory ctl pool. antibody-dependent enhancement ade is neither a sufficient nor an absolutely necessary precondition for the development of severe shock syndrome or hemorrhagic disease. ctl response to a viral infection can be modulated by the infection history of an individual in a manner likely to contribute to disease severity l€ uhn et al. 2007 ). respiratory syncytial virus (rsv) enhancer activity of transplacental maternal antibodies have been observed first by osiowy et al. (1994) . rsv infection is also enhanced by non-neutralising antibodies (johnson and graham 2004) . vaccination using inactivated virus particles were caused immune enhancement in the case of rsv in contrast to other, for example influenza viruses (openshaw et al. 2001; ye et al. 2004) . cytotoxic t-cells, cytokines and interferon induction by toll-like receptors may potentiate this enhancement (mobbs et al. 2002; boukhvalova et al. 2006; tregoning et al. 2008; lee et al. 2008a, b; nguyen et al. 2010; ubol and halstead 2010) . tumour necrosis factor b1 block of cell-replication enhances rsv-replication (gibbs et al. 2009 ). the evidence of the enhancer effect of non-neutralizing antibodies to rsv can be prevented by the treatment of children at increased risk with neutralizing monoclonal antibodies directed against the fusion (f) protein of rsv (motavizumab and/or palivizumab). this monoclonal antibodies were not found to cause harmful effects in connection with the preventive treatment of neonates and infants at risk (martin-mateos 2007; nieri et al. 2009; weisman 2009; groothuis et al. 2011 ). anti-rsv-f protein specific antibodies can prevent also the rsv-s. pneumoniae enhancement of respiratory infections (hament et al. 2005) . the blocking of influenza specific anti-neuraminidase antibodies using antiidiotypes indirectly enhanced the hemagglutination-inhibition titers of antisera (dowdle et al. 1972) . influenza h1 specific antisera were found to enhance virus replication in a macrophage-like cell line p388d1, when p388d1 cells, previously had been treated with neuraminidase to remove the viral receptors (ochiai et al. 1990 ). maternal antibodies systemic lupus erythemadosus (sle), is characterised by antibodies towards dsdna and ro52 (e3 ligase regulating tlr signalling) which, are present several years before the onset of disease and the neonatal disease is caused by some of these transplacental antibodies (watson et al. 1984) . systemic lupus erythematosus (sle) is the most common autoimmune disease affecting women of reproductive age and is associated with poor maternal and fetal outcomes. cd4(+)cd25(+) t reg cells are a subset of t lymphocytes with potent immunosuppressive activity that play crucial roles in controlling immunological self tolerance. evidence suggests that they are augmented in pregnancy, especially in the first trimester, suggesting an important role in early placental development. the literature describing t reg cells in sle is conflicting, but sle is associated with reduced numbers and functionally defective t reg cells, which may predispose pregnant women with the disease to pregnancy complications. this article discusses the role of t reg cells in sle and pregnancy, and how these cells may contribute to poor pregnancy outcome in sleaffected women (blois et al. 2007; clark et al. 2005) . sle was induced by interferon a administration, and by a specific stimuli i.e. sunshine exposure and smoking. the hla-dr3 haplotype was also found to be a risk factor (klareskog et al. 2010) . alcohol consumption was shown to be protective in these illnesses. vaccinations in adult age was found to be innocuous concerning the risk of ra according to the results of a case-control study when common vaccinations 5 years before onset of ra had been followed up. the effects of environment, however, are unknown for the neonatal heart block in p200 and ro52 positive women, but the active immunisation does not increase the risk of it (bengtsson et al. 2010a, b) . in sle and sj€ ogren's disease pregnants with high antibody titers against the p200 epitope of ro52 are those who almost exclusively carry the risk that their fetuses will develop neonatal heart block between gestational weeks 20-24. monitoring during these period the anti-ro52 antibodies, steroid treatment or preventive in utero pacemaker treatment may reduce the risk (wahren-herlenius 2010). foetal genes, maternal age and infectious agents may contribute to the risk of congenital heart block. especially inherited high level interferon production was also shown to be a risk factor for sle (niewold et al. 2007) . heparin treatment and intravenous gamma globulin (ivig) treatment and specific anti-idiotypes reduce the risk of the disease (clark et al. 2010) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. the id:anti-id ratio was significantly higher in mothers whose offspring developed neonatal lupus compared to mothers who gave birth to a healthy child (p < 0.0001). removal of anti-id antibodies substantially increased the reactivity against la(349-364) in sera from five of seven mothers tested. ivig from batches administered to mothers who gave birth to a healthy child had an id:anti-id activity ratio of <1, in contrast to that given to mothers who gave birth to a child with neonatal lupus (brucato et al. 2010 (brucato et al. , 2011 routsias et al. 2011) . the main pathomechanism of the development of neonatal lupus erythematosus is the transcytosis of maternal antibodies, and probably microchimeric cells. the pathogenesis of the maternal disease is extremely complex as summarised recently by perl (2010) and perl et al. (2010) . mitochondrial hyperpolarization underlies mitochondrial dysfunction, depletion of atp, oxidative stress, abnormal activation, and death signal processing in lupus t cells. nitric oxide production, expression of endogenous retroviral and repetitive elements such as hres-1, (the long interspersed nuclear elements 1), trex1, interferon alpha (ifn-alpha), toll-like receptors 7 and 9 (tlr-7/9), high-mobility group b1 protein, extracellular signal-regulated kinase, dna methyl transferase 1, histone deacetylase, spleen tyrosine kinase, proteasome function, lysosome function, endosome recycling, actin cytoskeleton formation, the nuclear factor kappa b pathway, and activation of cytotoxic t cells were shown to be components of the pathogenesis (varghese et al. 2011) . the hres-1 human endogenous retrovirus (erv) encodes a 28k nuclear autoantigen and a 24-kd small gtpase, termed hres-1/rab4. hres-1/p28 is a target of cross-reactive antiviral antibodies, whereas hres-1/rab4 regulates the surface expression of cd4 via endosome recycling. hres-1/rab4 is overexpressed in lupus t cells where it correlates with increased recycling of cd4 and cd3 and contributes to downregulation of cd3/tcrs via lysosomal degradation. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulates interferon and anti-dna antibody production. erv proteins may trigger lupus through structural and functional molecular mimicry, whereas the accumulation of erv-derived nucleic acids stimulate interferon and anti-dna antibody production in sle (perl 2010; perl et al. 2010) . these complex of pathogenetic factors many of them influenced by pregnancy results the clinical disease of the newborns (ruiz-irastorza and khamashta 2011). neonatal lupus erythematosus (nle) is an inflammatory disorder of neonates characterized by transient cutaneous lesions and/or congenital heart block. the cutaneous lesions usually heal with minimal scarring within 5-10 months, but may be delayed for many months in occasional cases. the maternal antibodies disappear from the circulation of the newborns within 8-10 months, the long-lasting clinical symptoms indicate, that irreversible events, or impairment of fetal cells occur during the fetal life. photosensitivity is recognized as a component of this syndrome. u1ribonucleoprotein (u1-rnp) specific antibodies can be detected in the circulation of the newborns. skin and cardiac manifestations coexist in only 10% of patients. hepatic, hematological and, less commonly, pulmonary, neurological and gastrointestinal abnormalities may also be present (watson et al. 1984; perez et al. 2011) . neonatal lupus is a model of passively acquired autoimmunity in which a mother-, who may have systemic lupus erythematosus (sle) or sj€ ogren's syndrome (ss) or may be entirely asymptomatic-synthesizes antibodies to ssa/ro and/or ssb/ la ribonucleoproteins that enter the fetal circulation via trophoblast fcrn receptors and presumably cause tissue injury (lee 1990 ) as mentioned above. congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with ro/ssa autoantibodies. during pregnancy, the antibodies are transported across the placenta and affect the fetus. it has been previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the ro52 component of the ro/ssa antigen correlate with the development of congenital heart block. the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the 200-239 aa stretch (ottosson et al. 2005 ). smith (sm) antigen, which is highly specific for sle is composed of at least nine different polypeptides with molecular weights ranging from 9 to 29.5 kda, b (b1, 28 kda), b 0 (b2, 29 kda), n (b3, 29.5 kda), d1 (16 kda), d2 (16.5 kda), d3 (18 kda), e (12 kda), f (11 kda), and g (9 kda). sle patients develop antibodies against the sm complexes of the small nuclear rnas u1 to u6 in the mother and recently these were also detected in neonatal le patient (ortiz-santamaria et al. 2010) . in neonatal lupus, mothers with high anti-idiotypic antibody activity against anti-la autoantibodies are at lower risk of giving birth to an unhealthy child, as compared with mothers without anti-idiotypic antibodies. usually anti-idiotypic antibodies may confer protection from the harmful effect of autoantibodies in certain autoimmune diseases (tzioufas and routsias 2010) . ivig enhanced the anti-id antibody response in pregnant women with anti-la/ssb antibodies. a high id:anti-id ratio in both the ivig preparation and the maternal serum may explain the absence of an effect of ivig in preventing recurrent neonatal lupus in some cases (routsias et al. 2011) . the children of the mothers suffering from sle and sj€ ogren's syndromes are at risk also for other rheumatic/autoimmune diseases without carrying antibodies reactive with ssa/ro or ssb/la antigens. among the siblings without neonatal lupus developed later juvenile rheumatoid arthritis, hashimoto thyroiditis, psoriasis and iritis, diabetes mellitus, congenital hypothyroidism and nephrotic syndrome (martin et al. 2002; winter and schatz 2003) . experimental systemic lupus erythemetosus could be induced in mice using immunisation with anti-idiotype antibodies (ab2) specific to the anti-dna-specific monoclonal antibodies (mendlovic et al. 1989) . identical twins of mothers suffering from sle had also transient neonatal bullous skin disease (nakajima et al. 2011). autoimmunity has been defined as a normal physiological state with control mechanisms that prevent autoimmunity from progressing to overt pathology. the onset of a subset of rheumatoid arthritis (ra) begins with appearance of citrullinated protein antigen (cpa) positivity when tolerance to certain citrullinated proteins/peptides is broken supported by certain hla-dr haplotypes (hla-dr04 and hla-drb1 in smokers). local expression of peptidylarginine deiminases and occasional elevation of inflammatory cytokines can be measured (klareskog et al. 2010) . methotrexate was significantly more effective than placebo in preventing progression to this disease state in anti-cpa-positive patients with undifferentiated arthritis (ua) whereas no difference between methotrexate and placebo was seen in the acpa-negative group of patients with undifferentiated arthritis (ua; arthritis without arthralgia). the risk of ra was lower in this group without treatment (ehrenstein et al. 2004; van dongen et al. 2007 ). the treatments using therapiesinfliximab (a tumour necrosis factor (tnf-a) blocker) and abatacept (t cell costimulation inhibitor; anti-cd80 and anti-cd86) did not result in significant improvement of the acpa patients with ua (saleem et al. 2008; emery et al. 2010) . the analysis of myeloid-related proteins in serum is an excellent tool for the diagnosis of systemic onset of juvenile idiopathic arthritis (jia), allowing early differentiation between patients with systemic-onset of jia and those with other inflammatory diseases. mrp-8/mrp-14 and il-1b represent a novel positive feedback mechanism activating phagocytes via two major signaling pathways of innate immunity (tlr4) during the pathogenesis of systemic-onset of jia (frosch et al. 2009 ). the rheumatoid arthritis was found to be improved during pregnancy probably due to galactosylation of igg molecules (f€ orger and østensen 2010). women with ra can acquire the susceptibility allele through microchimeric cells. very high amounts (0.9% of total pbmcs) of drb1*04 microchimerism in patients with ra were observed. similarly, drb1*01 microchimeric dna was observed in significantly higher quantities in women with ra compared with healthy control subjects. in contrast, there was no difference between women with ra and control subjects when microchimerism for non-ra-associated alleles (hla-dqb1*02 and drb1*15/16) was analyzed (rak et al. 2009 ). by analogy to graft-versus-host disease (gvhd), pioneer studies on microchimerism in women with scleroderma proposed direct and indirect recognition mechanisms as underlying reactions of microchimeric cells (nelson 2002) . in gvhd, donor cells invade the recipient, which is not what is observed with microchimerism in patients with ra. however, the presence of drb1*01 microchimerism (0.03%) and drb1*04 microchimerism (0.9%) is not negligible, with frequencies among total host pbmcs similar to the frequencies of antigen-specific cd4+ t cells, which thus are sufficient to impact the host immune reactions. the haplotypes of the patients influence the progression of the rheumatoid arthritis, too (liu et al. 2007 ). autoimmune neonatal bullous skin disease caused by placental transfer of maternal igg autoantibodies is rare. it has been reported in neonates born to mothers with pemphigus vulgaris, pemphigus foliaceus, and gestational pemphigoid. vertically acquired congenital autoimmune blistering disorders appear to be self-limited and resolve with supportive therapy, concomitant with the presumed clearance of maternal autoantibodies from the neonate's circulation (abrams et al. 2011 ). antiphospholipid antibody syndrome (apas) is regarded as the most frequently acquired risk factor for thrombophilia. thrombophilia is the tendency to thrombosis. the antiphospholipid antibody syndrome (apas) is a disorder of recurrent vascular thrombosis, pregnancy loss and thrombocytopenia, associated with persistently raised levels of anti-phospholipid antibodies (apa). the apa are phospholipids (part of a cell's membrane) recognized by the body as foreign and antibodies are produced against them. maternal autoimmune diseases significantly reduce the pregnancy outcome of the women. the most frequent illnesses were antiphospholipid syndrome, antiphospholipid syndrome associated with a rheumatic disease (aps/rd), other rd patients, isolated autoantibodies (autoabs) in the absence of a definite autoimmune disease (aabs) and reactive arthritis or spondyloarthropathies. of these patients, 50.6% had previous pregnancy complications with an anamnestic livebirth rate of 43.4%. in these patients, 10.4% of pregnancies resulted in preterm delivery and 10.9% newborns had low weight at delivery. aps/rd patients had the worse outcome: 17.6% resulted in miscarriage, 14.3% resulted in growth restriction and 50% resulted in preterm delivery. this result was mainly due to patients with aps/systemic lupus erythematosus (sle) that had the lowest gestational age at delivery (30.8 ae 3.56 weeks) and the lowest newborn weight (canti et al.2011) . antiphospholipid syndrome was suggested to be the result of antibodies, directed against cardiolipin as a result of antigenic mimicry. the suspected antigen is beta-2-glycoprotein-i (b2gpi) (sherer et al. 2007) . clinically significant are lupus anticoagulant, anticardiolipin antibodies and anti-b 2 glycoprotein-i (anti-b2 gp-i) antibody. neonates born to mothers with primary aps are at risk of prematurity, being small for gestational age, and having thrombocytopenia (chou et al. 2009 ). antiphospholipid antibodies (apl) can impair the physiologic development of a fetus during pregnancy not only by causing thrombosis of the placental vessels, but also by directly binding throphoblast cells and modifying their functions (tincani et al. 2009 ). transplacentally transferred antiphospholipid antibodies act as a risk factor, but are not usually a sufficient condition for thrombosis and other thrombophilic risk factors should be systematically evaluated. long-term studies of children born to antiphospholipid-antibody-positive mothers provided the evidence of possible neurodevelopmental changes in these children and regular neuropsychological assessments are recommended. antiphospholipid-antibody-related thromboses in children are frequently associated with multiple antiphospholipid antibody positivity and concomitant presence of inherited prothrombotic disorders can be also detected in addition to nonthrombotic manifestations, particularly hematological, skin and neurological manifestations (avcin 2008) . treatment of pregnant women with apas results in marked improvement in the live birth rate (4.6-85.7%). however, complications like preeclampsia and intrauterine growth restriction (iugr) occur even after treatment, requiring strict monitoring and timely delivery. aberrant concentrations of fetuin a and heat shock protein might have also role in the preeclamptic inflammation (molvarec et al. 2009a, b; dadhwal et al. 2011 ). foetal/neonatal disease is due to transplacental thyrotrophin receptor stimulating antibodies (trab). it's extremely important recognising and treating graves' disease in mothers as soon as possible, because a thyrotoxic state may have adverse effects on the outcome of pregnancy and both on the foetus and newborn. neonatal grave's disease tends to resolve spontaneously within 3-12 weeks as maternal thyroid stimulating immunoglobulins are cleared from the circulation but subsequent development may be impaired by perceptual motor difficulties. hashimoto's thyroiditis is a very common autoimmune thyroid disease. in presence of maternal hashimoto's thyroiditis, there are usually no consequences on foetal thyroid, even if antitpo and antitg antibodies can be found in the newborn due to transplacental passage. however there are some reports describing foetal and neonatal hyperthyroidism in the affected mothers' offspring (radetti et al. 2002; hemminki et al. 2010) . a number of autoimmune diseases; especially autoimmune thyroid diseases, erythema nodosum and sarcoidosis parity might somehow be involved in maternal disease development (jørgensen et al. 2011) . maternal thyroid status assessment and treatment improves fetal outcomes and neuropsychological developmental of the newborn (staii et al. 2010 ). juvenile myasthenia gravis is associated with antibodies to the acetylcholine receptor (achr) in most patients. thymoma is rare, but often malignant in children. the frequency of juvenile myasthenia gravis with antibodies to the musclespecific kinase (musk) varies markedly in different countries. neonatal myasthenia gravis associated with musk antibodies is often a severe and protracted albeit transient disease (béhin et al. 2008; evoli 2010) . transient neonatal myasthenia gravis (mg) is a human model of passively transferring the disease. although all newborn babies of myasthenic mothers have anti-achr antibodies at birth (morel et al. 1988; tzartos et al. 1990) , only a small percentage of them (10-15%) express the myasthenic syndrome (namba et al. 1970) . the myasthenic symptoms usually appear a few hours after birth and their average duration is about 3 weeks. neonatal myasthenia gravis is transiently transferred from the mothers to the newborn. nicotinic acetylcholine receptor (achr) antibodies result in loss of achrs and also directly block the function of the remaining achr molecules, thereby causing a defect in neuromuscular transmission. the majority, though not all, of both myasthenic and non-myasthenic infants were found to have a repertoire of anti-achr specificities very similar to their mothers. no significant differences were observed between sera from the two groups of mothers. adequate treatment in mothers can reduce both frequency and severity of neonatal disease. neonatal disease will recover following ivig treatment (béhin et al. 2008; o'carroll et al. 2009 ). the absence of neonatal myasthenia gravis might be caused by the antigenic differences between the fetal and adult enzymes similar to those detected in rats (hall et al. 1985; hesselmans et al. 1993 ). the human fetal acetylcholine receptor (achr) is present until 33 weeks gestation, when the fetal (g) subunit is replaced by the adult (e) subunit. the term "fetal acetylcholine receptor inactivation syndrome" has been proposed for the illness, when other developmental disorders were also caused by the maternal antibodies (oskoui et al. 2008 ). neonatal guillain-barré syndrome (gbs) was observed to occur 7-12 days postpartum in children born to mothers with gbs. serum from mother and infant depressed quantal content by approximately 90% and reduced the amplitude of postsynaptic currents by 30-40% in mouse, newborn and juvenile rats. the antibody nature of the blockade could be confirmed by showing that monovalent fab fragments were similarly effective as purified immunoglobulin (ig) g. both cellular and humoral immune mechanisms are operative in guillain-barré syndrome (gbs). transplacentally transferred blocking antibodies may be specifically directed at epitopes of the mature but not the fetal neuromuscular junction (luijckx et al. 1997; buchwald et al. 1998 buchwald et al. , 1999 . guillain-barré syndrome and sydenheim's chorea are diseases, which were shown to be associated with the immune response after microbial infections. the occurrence of guillain-barré syndrome noted in infants whose mother had harmless autoimmune antibodies during pregnancy (buchwald et al. 1999; sladky 2004) . the syndrome's occurrence within families is also of interest. the mmp9 c(-1562)t and tnfa c(-863)a snp were associated with severe weakness and poor outcome, indicating that these snps may be one of the factors predisposing to a severe form of gbs (geleijns et al. 2007 ). the associated features of er22/23ek carriers consist of favorable metabolic and body compositional conditions. in contrast, the n363s polymorphism was reported to be associated with an enhanced sensitivity to glycocorticoids. haplotypes carrying the minor allele of the bcli polymorphism of the glycocorticoid receptor gene was related to the phenotype and outcome of gbs explaining the family dependence of the syndrome and other neonatal disorders (dekker et al. 2009 ). mutations in about a dozen of genes have been linked to the development of permanent neonatal diabetes mellitus (pndm). the most frequent causes of pndm are heterozygous mutations in the kcnj11, ins and abcc8 genes. although pndm is a rare phenomenon (one case in about 200,000 live births), this discovery has had a large impact on clinical practice as most carriers of kcnj11 and abcc8 gene mutations have been switched from insulin to oral sulphonylureas with an improvement in glycemic control (aguilar-bryan and bryan 2008; rubio-cabezas et al. 2011). the majority of transient neonatal diabetes mellitus (tndm) cases have an abnormality in chromosome 6q24. half of the ndm cases are transient (tndm) and the other most frequent causes of ndm are missense mutations in the pancreatic b-cell k atp channel genes kcnj11, ins and abcc8 (chr11), and in the preproinsulin gene, ndm has been linked to numerous other genetic causes including point mutations in gck (chr7), glis3 (chr9), eif2ak3 (chr2), pdx1 (chr13), ptf1a (chr10), slc2a2 (chr3), hnf1b(chr17) or foxp3 (chrx) (aguilar-bryan and bryan 2008; bonnefond et al. 2010) . genetic mutations were identified in~75% of non-consanguinous probands with pndm/mdi, using sequential screening of kcnj11, ins and abcc8 genes in infants diagnosed within the first 6 months of age. this percentage decreased to 12% in those with diabetes diagnosed between 7 and 12 months. patients belonging to the latter group may either carry mutations in genes different from those commonly found in pndm/mdi or have developed an early-onset form of autoimmune diabetes. islet-cell antibodies (ica), glutamic acid decarboxylase autoantibodies (gada), tyrosine phosphatase-related proteins-islet antigen 2 autoantibodies (ia-2a), insulin autoantibodies (iaa), zinc transporter 8 autoantibodies (znt8a) were found in the sera of the children, suggesting autoimmune origin of their disease (russo et al. 2011 ). biliary atresia (ba) is a devastating disease of infants, invariably leading to cirrhosis, end-stage liver disease, and death if untreated. it has been shown using microarray technique, that the t-cell regulatory gene rras seems to be a key factor in the development of the disease (zhao et al. 2011) . a recent review reported that ba may involve a primary perinatal hepatobiliary reoviral or rotaviral infection and a secondary autoimmune-mediated bile duct injury (mack 2007 ). the maternal virus infections followed by maternofoetal microchimerism seems to be a very impressive explanation of the etiology (muraji et al. 2008) . in a mouse model, oral vaccination before mating with rotateq and rotarix prevented most rhesus rotavirus-induced ba (turowski et al. 2010 ). biliary atresia is probably the endresult of different aetiological factors, among which viruses and other agents may cross the placenta. otherwise it cannot be understand, why only one of the twins obtain the disease (morris et al. 1977) . the anti-idiotypes transcytosed by different efficiency into the circulation of the twins, might be an additional explanation for the asymmetric disease. maternal symptomless paraproteinemia was also found to be transmitted to the fetus. the paraprotein was detected after birth for 3 months in the serum of the child and caused prolonged immunosuppression without later consequences (littlewood et al. 1970; littlewood and payne 1977) . transient neonatal acquired von willebrand syndrome (avws) has been observed peripartum. its clinical management is analogous to monoclonal gammopathy of undetermined significance (mgus) of the mother since it is the consequence of transplacental transfer of maternal igg antibodies (simone et al. 1968; nageswara rao et al. 2009 ). immune trombocytopenic purpura associated with pregnancy. fetal and neonatal alloimmune thromocytopenia (fmait) results from transplacental transfer of maternal antibodies that develop in response to alloimmunization against paternal human platelet antigens (hpas) expressed on fetal platelets. this thrombocyte loss could be prevented using modified igg molecules (ghevaert et al. 2008) . at present seven biallelic human platelet antigen (hpa) systems have been determined and can be typed using genomic dna. platelet genotyping is a valuable tool in confirming platelet antigen specificities of alloantibodies detected in patients' sera to complement the clinical history in the diagnosis of alloimmune platelet disorders such as fetal and neonatal alloimmune thrombocytopenia (fnait). prenatal platelet typing of the fetus in suspected cases of fnait became also available (curtis 2008) . half of the infants were borne with low platelet counts and 6 of 16 required replacement therapy upon birth (ozkan et al. 2010; gasim 2011) . the maternal, transplacental igg binding to the fetal platelets was suggested to prevent their recirculation by fcgr binding to and phagocytosis by macrophages. the pathogenesis of immune trompcytopenic purpura and that associated to myelodysplasia and leukemia were shown to be different (psaila and bussel 2008; psaila et al. 2011 ). neonatal endarteritis has been diagnosed in the newborns of mothers suffering from endarteritis nodosa. fatal myocardial infarction in a neonate due to coronary arteries is compared with two lethal cases of mucocutaneous lymph node syndrome and/or infantile periarteritis nodosa (mlns/ipn). cutaneous polyarteritis was transmitted to the newborn, too (kitzmiller 1978; krapf et al. 1981; stone et al. 1993) . during the last decade no publication could be found on neonatal endarteritis. probably the pathogenesis of this disease has been reevaluated in the light of the molecular diagnostic findings. hereditary autoinflammatory syndromes are caused by monogenic defects of innate immunity and are classified as primary immunodeficiencies. these syndromes are characterized by recurrent or persistent systemic inflammatory symptoms and must be distinguished from infectious diseases, autoimmune diseases, and other primary immunodeficiencies. the sited review describes the epidemiological, clinical and laboratory features, prognosis, and treatment of the main autoinflammatory syndromes, namely: familial mediterranean fever; tnf receptor associated periodic syndrome; the cryopyrinopathies; mevalonate kinase deficiency; pediatric granulomatous arthritis; pyogenic arthritis, pyoderma gangrenosum and acne syndrome; majeed syndrome; and deficiency of interleukin 1 receptor antagonist. the cryopyrinopathies discussed include neonatal-onset multisystem inflammatory disease (also known as chronic infantile neurologic, cutaneous and articular syndrome), muckle-wells syndrome, and familial cold autoinflammatory syndrome (jesus et al. 2010) . primary immunodeficiencies (pids) were analyzed to gain insight into the physiopathology of sle. some pids have been consistently associated with sle or lupus-like manifestations: (a) homozygous deficiencies of the early components of the classical complement pathway in the following decreasing order: in c1q, 93% of affected patients developed sle; in c4, 75%; inc1r/s, 57%; and in c2, up to 25%; (b) female carriers of x-linked chronic granulomatous disease allele; and (c) iga deficiency, present in around 5% of juvenile sle. mutations of the complement system (c1-inhibitor; c1q, c1r, c1s, c2, c3, c4, c5, c6, c7, c8 and c9 deficiencies were shown to facilitate the development of sle in addition to the facilitation of bacterial infections (c1 and c4, encapsulated bacteria; c5 to c9 neisseria). other autoimmune diseases, i.e. polyendocrinopathy candidiasis ectodermal dystrophy (apeced), immune dysregulation polyendocrinopathy enteropathy x-linked (ipex), and autoimmune lymphoproliferative syndrome (alps), suggesting that mechanisms considered as critical players for induction and maintenance of tolerance to autoantigens, such as (1) aire-mediated thymic negative selection of lymphocytes, (2) foxp3 + regulatory t cell mediated peripheral tolerance, and (3) deletion of auto-reactive lymphocytes by fas-mediated apoptosis, were not found to be associated with sle physiopathology (blois et al. 2007; carneiro-sampaio et al. 2008 ). behçet syndrome (bs) is a multisystem chronic inflammatory disorder, which is characterized by relapsing oral and genital ulceration and iridocyclitis. while being of unknown etiology, vasculitic changes of possible autoimmune origin are common to all involved organs, and thrombotic complications, which may adversely affect gestation, are frequently seen was shown to possess genetic etiology on the basis of the comparison of its incidence among monozygotic and dizygotic twins (masatlioglu et al. 2010 ). pregnancy does not have a deleterious effect on the course of bd and may possibly ameliorate its course. however, it seems that bd may adversely affect pregnancy. the miscarriage rate was higher, and the pregnancy complications and cesarean section rates were significantly elevated (jadaon et al. 2005) . overall, parity was associated with an 11% increased risk of female predominant autoimmune diseases. pregnancies resulting in liveborn children therefore seem to contribute only little to the general female predominance in autoimmune diseases. crohn's disease and ulcerative colitis; in idiopathic inflammatory bowel diseases (ibd), including crohn's disease and ulcerative colitis, there is pathogenic build-up of cd4 + t cells at sites of inflammation, mediated in part by il-6, which provides (as described earlier) an anti-apoptotic signal to t cells through the induction of bcl-2 and bclxl and promotes th17 lineage differentiation. il-17 is upregulated in patients with both types of ibd. il-6 levels are elevated markedly in the serum of patients with ibd, decrease with treatment of inflammation and are predictive of ibd relapse. microbial pattern-recognition receptors, such as the tlrs and nod2 (nucleotide oligomerization domain 2), which activate nf-kb, have been implicated in these conditions. neutralisation with an anti-il-6r antibody largely prevented the colitis in mice (arad et al. 2010) . most women and men with ulcerative colitis (uc) and crohn's disease (cd) can expect a healthy child with neither preterm birth nor low birthweight. no neonatal forms have been described (ludvigsson and ludvigsson 2002; van assche et al. 2005) . later crohn's disease was shown to impair the fetal outcome in the case of the affected mothers (naganuma et al. 2011 ). colitis-associated-cancer (cac); tgf-b suppresses the formation of cancer by inhibiting il-6 trans-signaling and human samples of colon cancer have low-levels of il-6r, as do samples of inflamed colon. adenomatous polyposis coli (apc) gene of mice with apc mutation develop cancer, genetic deletion of the tlr-adaptor protein myd88 decreased the number of cancers markedly. because myd88 activation in turn activates nf-kb, it is not surprising that il-6 production was decreased greatly in mice deficient in myd88 and supports prior data showing that il-6 is one of the effector signals in the tlr-nf-kb activation pathway (becker et al. 2003) . tgf-b was found to suppresses tumor progression in colon cancer by inhibition of il-6 trans-signaling. huntington disease was found to be of congenital origin, and the diagnosis can be obtained before implantation (hdcrg 1993; peciña et al. 2010) . the causal mutation is the expansion of a cag trinucleotide repeat tract in exon 1 of a large gene on chromosome 4 that results in the extension of a polyglutamine tract at the n-terminus of the encoded, ubiquitously expressed protein called huntingtin. from the maternal blood the dna from the fetal-maternal transport can be also applied for the prenatal diagnosis (bustamante-aragones et al. 2008). hypoparathyroidism with autoimmunity due to the 22q11.2 deletion syndrome. the majority of patients acquired autoimmune antibodies, but without antiparathyroid antibodies (lima et al. 2011 ). wegener granulomatosis (wg) is systemic disease of unknown etiology characterized by necrotizing granulomatous inflammation, tissue necrosis, and variable degrees of vasculitis in small and medium-sized blood vessels. the classic clinical pattern is a triad involving the upper airways, lungs and kidneys. ninety percent of patients present with symptoms involving the upper and/or lower airways, and 80% will eventually develop renal disease (mubashir et al. 2006) . pregnancy in patients with wg requires preconceptional planning, careful clinical management, and vigorous treatment of active disease. the management is individualized and the pregnancy outcome is variable. antenatal management and therapeutic options are important (koukoura et al. 2008) . no neonatal disease have been described, and ivig treatment could be successfully applied in the steroid resistant patients (bellisai et al. 2004 ). kawasaki disease (kd) is an illness mostly of infants and young children, the majority being less than 4 years old. the peak age of onset of illness is 6-11 months. it is unusual in very young infants. from 1970 to 1995, only six instances of kd occurred in infants 30 days of age or younger in japan, and infants 90 days of age or younger accounted for only 1.67% of all patients. the youngest infant of kd to date (a neonate 2 week old) was reported by stanley and grimwood. the youngest reported from india is a 46-day-old infant (thapa et al. 2007 ). therapeutic monoclonal antibodies (mabs) are most commonly of the igg1 subclass, which is transported most efficiently to the fetus. in all animal species used for testing developmental toxicity, fetal exposure to igg is very low during organogenesis, but this increases during the latter half of gestation such that the neonate is born with an igg1 concentration similar to the mother. the therapeutic monoclonal antibodies might cause developmental and reproductive toxicity (dart) requiring testing of antibody-based therapeutics (pentsuk and van der laan 2009). guillain-barré syndrome during pregnancy can be also treated with ivig and plasmapheresis. (niklasson et al. 1998; goyal et al. 2004; bahadur et al. 2009; modi et al. 2010; ohlsson and lacy 2010) . polyclonal antisera, administered for passive immunization, are mixtures of different proteins, sharing binding activity against the antigen (ag) determinants of the same immunogen preparation. immunoadhesins (or immunoglobulin fusion proteins) are antibody-like molecules resulting from the fusion of a constant region (e.g., fc portion) of an immunoglobulin and the ligand-binding region of a receptor or an adhesive molecule. antibody fragments such as fab, scfv, diabodies, and minibodies are molecules devoid of part or whole of the fc portion. therefore, they have faster clearance and better tissue/tumor penetration than whole immunoglobulins and they will perform better than whole iggs in conditions where a short half-life is desirable, such as in radio-imaging and/or radio-therapy. they have no adcc and cdc triggering activity. the smallest proteins retaining antigen binding are a single variable domain antibody (nieri et al. 2009 ). ivig prevention improves the risk of heart block of sle patients (friedman et al. 2010) . several members of the idiotype-anti-idiotype network and antibody dimers including antigen-antibody complexes were found in the ivig preparations (luijten et al. 1988; osterhaus et al. 1989; clark et al. 2010 ). 105ad7 anti-idiotype monoclonal antibody can mimic the cd55 antigen. the molecular basis of 105ad7 mimicry has been identified with three cdr regions of 105ad7 showing similarity to three regions of cd55. these regions have been analysed for potential t-cell epitopes, and sequences that are predicted to bind to hla/a1,3,24 and to hla/dr1,3,7 have been identified within the cdrh3 region of 105ad7. 105ad7 can stimulate cd4 and cd8 responses in colorectal cancer patients with the appropriate haplotype. only a few patients produce a sustained memory response (durrant et al. 2000; reinartz et al. 2003) . most of these vaccines for the lymphoma therapy use the tumor b cell idiotype (the unique variable region of the surface immunoglobulin) as a tumor-specific antigen. in spite of several problems anti-idiotype vaccines prospect towards integration of this strategy in the therapeutic armamentarium for lymphoma (houot and levy 2009) . clinical studies have been published using different preparations and summarised at the end of table 9 .1. abagovomab (aca125) acts as an antigen mimic of the carbohydrate ovarian cancer antigen 125 (wagner et al. 1997) . the frequency of peripheral t reg s was increased during abagovomab therapy in a high percentage of patients. despite higher t reg counts compared with baseline levels, the suppressive capacity of t reg s was reduced in a subset of patients. the data further indicate that the ability of t cells to proliferate in response to ca-125 in vitro could be associated with diminished t reg activity. importantly, ca-125-specific immunity could not be enhanced by in vitro t reg depletion, as ca-125 induced cd25 + foxp3 + t reg s with suppressive capacity. abatacept (orencia) human igg fc domain 1 ctla-4; anti-cd80 and anti-cd86. abatacept (abt, orencia, bristol-myers squibb ltd) rheumatoid arthritis tumor necrosis factor inhibitor in phase 3 clinical trial, but it was found effective only in very early phase of the disease (malottki et al. 2011; emery et al. 2010) . prophylactic withdrawal of drugs before pregnancy is mandatory (østensen et al. 2008) . at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (østensen and f€ orger 2011) . abciximab (reopro) igg1k-fab, chimera -integrina2b3 (platelet-gp)haemostasis/trombosis (nieri et al. 2009 ). using immunohistochemistry, reopro was only detected attached to maternal and fetal platelets, and to the trophoblastic surface of the placental villi (miller et al. 2003a, b) . the effect of abciximab to icam-1 was excluded, but an effect to monocytes could not be excluded (voisard et al. 2006) . adalimumab (ada, humira; abbott) human recombinant, igg1k, anti-tnfa, as, psa, cd, pp, jia (van schouwenburg et al. 2010) tnf-alpha blocker therapy (adalimumab). the therapy of pregnants showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (østensen et al. 2008) . expression of hla-g on pmbcs is up-regulated in ankylosing spondylitis (as), correlates with acute phase reactants and decreases after tnf-alpha blocker therapy (chen et al. 2010) . anti-tnfa is used for the treatment of infectious bowel diseases even during pregnancy, which might influence the development of the fetal immune system (arsenescu et al. 2011) . anti adalimumab anti-idiotype antibodies (bartelds et al. 2007 malottki et al. 2011; nieri et al. 2009 ). (2007) ? teplizumab anti-cd3 t.1 diabetes herold et al. (2002 herold et al. ( , 2005 ? integrin a2b3 direct exposure to anti-tnf treatment during pregnancy was not related to a higher incidence of adverse pregnancy outcomes than infectious bowell diseases overall (schnitzler et al. 2011 ). the anti-tnf agent was started prior to conception and continued until there was evidence of fetal cardiac activity. in women treated with a combination of anti-tnf therapy, anticoagulation therapy, and ivig, the live birth rates (71%) were greater than those in women treated with anticoagulation therapy alone (19%) or in those receiving a combination of anticoagulation therapy and ivig (54%). fetal outcomes, including gestational age and birth weight, were similar across the groups, and no congenital anomalies were reported after anti-tnf agent exposure (winger and reed 2008; vinet et al. 2009 ). rheumatoid arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriasis, psoriatic arthritis, crohn's disease (østensen and f€ orger 2011) . all users requested at least one time repeatedly the treatment in norway (mahic et al. 2011) . long term treatment did not resulted in reactivation of chronic hepatitis b virus infection (mori 2011) . the treatment caused the improvement in two bone formation markers -b-alkaline phosphatase and osteocalcin (veerappan et al. 2011) . alemtuzumab (mabcampath, genzyme) humanized, igg1k, anti-cd52 anti-ca125 immunoglobulin directed against cd52 antigen expressed on t-and b lymphocytes, monocytes, macrophages, nk cells, and a subpopulation of granulocytes, but not on hematologic precursors. induction of cdc or adcc on an fcreceptor g-binding mechanism. pancreas transplant recipients on alemtuzumab maintenance therapy suffered frequently from red cell aplasia, and autoimmune hemolytic anaemia. (elimelakh et al. 2007 ) cord-blood-hematopoetic-stem-cell expansion and increase the availability of cord-blood units for transplantation (lim et al. 2008) . in contrast to ivig and rituximab the compound may be an effective therapy for complex immunohematologic disorders complicating hematopoietic stem cell transplantation. the paper emphasizes the importance of t-cells in transplant associated immune cytopenias (chao et al. 2008 ). b-ccl (nieri et al. 2009 ). alicaforsen is a human monoclonal antibody a1b2-integrin, also known as leukocyte function antigen (lfa)-1, and its ligand, intercellular adhesion molecule-1 (icam-1), is important for the recruitment of leukocytes to inflammatory sites (bosani et al. 2009 ). anti-cd34 and anti cd105 intratumoral microvessel density (imvd) monoclonal antibodies. anti-cd105 was more effective against non-small cell lung cancer than anti-cd34 (tanaka et al. 2001) . anti-cd137 mab (bms-663513, bristol-myers squibb) 4-1bb (cdw 137), a member of tumor necrosis factor receptor (tnfr) superfamily stimulating t-cells, nk-cells and dcs . anti-cd137 mab enhances rituximabdependent cytotoxicity against the lymphoma cells (lee et al. 2005; kohrt et al. 2011) . anti-leu-2b or anti-leu-2c, igg2a, marker of t-cell subpopulation (clement et al. 1984; champlin et al. 1990) . apomab (genentech) igg1, against extracellular domain dr5/tumor necrosis factor related (trail) receptor 2 apoptosis inducing ligand (nieri et al. 2009 ). basiliximab (simulect) chimeric, igg1k, anti-cd25, il2r antagonist (aktas et al. 2011 ) 10% reduced fetal body weight (pentsuk and van der laan 2009) . bavituximab (peregrine pharmaceuticals, inc., tustin, ca), anti-phosphatidylserine bavituximab combined with radiotherapy holds promise as a vascular targeting and immune enhancement strategy for the treatment of human glioblastoma (he et al. 2009 ). hcv therapy (immunostimulant) (dammacco et al. 2010; quer et al. 2010) . belatacept (ctla4-ig) is a new recombinant molecule that interferes with the signal of t lymphocyte activation and prevents acute rejection after renal transplantation. hla-g acts as a naturally tolerogenic molecule in humans. patients treated with ctla4-ig displayed significantly higher soluble hla-g (shla-g) plasma concentrations than patients treated with calcineurin inhibitors or healthy donors (bahri et al. 2009 ). ctla4-ig-treated dc acted as tolerogenic apc through shla-g secretion as they suppressed t cell alloproliferation, which could be restored by using a neutralizing anti-hla-g ab (bahri et al. 2009 ). the use of anti tumour necrosis factor mabs are not recommended (partlett and roussou 2011) . cytotoxic t-lymphocyte-associated protein 4 (ctla-4) is a negative regulator of t cell activation and may modulate peripheral self-tolerance (kaufman et al. 1999) . bevacizumab (avastin) humanized, igg1k, anti-vascular endothelial growth factor-a (vegf-a) used for the treatment of non-small cell lung cancer, (nsclc) colorectal cancer (cc); (nieri et al. 2009 ). efd rabbit: dose-dependant decrease in maternal bodyweight, increase in fetal malformations and late resorptions; (pentsuk and van der laan 2009 ). intravitreal bevacizumab therapy during pregnancy for off-label ocular indications can result in significant visual improvement without adverse fetal events related to treatment (tarantola et al. 2010) . intravitreal 1.25 mg bevacizumab may reach the systemic circulation in plasma concentrations of 100 ng/ml (csáky and do 2009) . two pregnants have lost their babies within 10 days following intravitreal injections of bevacizumab (petrou et al. 2009 ). placenta gf levels are elevated in the plasma of colorectal and rectal carcinoma patients receiving bevacizumab (xu and jain 2007) . prevention of angiogenesis by msc in pancreatic cancer (beckermann et al. 2008) . blinatumomab (epcam, antigen -epithelial cell-adhesion molecule, -present on 85% of cancer cells) and mt-103 and mt110 present on all non-hodgin lymphoma cells (armstrong and eck 2003; nieri et al. 2009 ). cd3/cd19 bispecific, single chain recombinant antibody topp et al. 2011) . catomaxomab (removab) bifunctional recombinant anti epidermal cell adhesion molecule epcam and anti-cd3. it is inducing adcc in ovarian and stomach cancer. epcam is the ligand for human leukocyte immune-globulin like receptor (lair-1) (armstrong and eck 2003; nieri et al. 2009; bokemeyer 2010; r€ ussel et al. 2011) . ceavac (mimicking carcinoembryonic antigen) colon cc (foon et al. 1999 ). certolizumab (cimzia®; ucb) pegylated humanized antibody fab' fragment of tnf-a monoclonal antibody; ra, cd; (østensen and f€ orger 2011) . certolizumab does not cross the placenta as easily as the igg derived drugs due to the pegylation of the molecules, thus reducing the harmful consequences to the fetus (clowse 2010) . cetuximab (erbitux) chimeric, igg1k, anti-epidermal growth factor receptor-1 (her-1), apoptosis, cdcc colorectal cancer (cc); squamous cell cancer of head and neck (scchn) weight loss and reduced food consumption in high-dose group dose-dependent increase in abortion rates (not known, if could be associated with treatment) weight loss and reduced food consumption in high-dose group (powell et al. 2008; pentsuk and van der laan 2009; rech and vonderheide 2009) . chaglycd3 (cd3-specific) a humanized antibody, an aglycosylated human igg1 antibody directed against cd3 was shown to reduce the insulin-requirement of the patients. residual beta-cell function was better maintained with chaglycd3 than with placebo (keymeulen et al. 2005) . cixutumumab (img-12 or cix) targeting insulin-like growth factor receptor (igf-ir) treatment for multiple cancers. human igg1, blocks interaction between igf-ir and its ligands, igf-i and -ii, and induces internalization and degradation of igf-ir. its combination of cetuximab (mab against egfr) inhibited the growth of pancreatic cancer and promoted its regression. an antiangiogenic mechanism was associated with cix treatment. reviewed recently (quatrale et al. 2011) . hyperglycemia is a regular side effect, but the fetal consequences during pregnancy have not been evaluated yet (mckian and haluska 2009) . eyelash trichomegaly in adults (bouché et al. 2005; garrido et al. 2007) . cnto 328 chimeric monoclonal antibody with high affinity for human il-6 myeloma multiplex sensitivity to glycocorticoid (voorhees et al. 2009 ). ct-011 (curetech) humanized anti-pd-1 igg1 mab that binds to mouse and human pd-1, programmed death receptor 1. pd-1 but not ctla-4 blockage abrogates the protective effect of regulatory t cells in a pregnancy murine model. mkrtichyan et al. 2011; stagg et al. 2011; wafula et al. 2009 ). dacetuzumab (sgn-40; seattle genetics) humanized; and hcd122 fully human (novartis/xoma); cd40, tumour necrosis factor receptor; b-cells, dcs, macrophages lymphomas . daclizumab (zenapax) humanized, igg1k, anti-a-chain of cd25, il-2r antagonists (elimelakh et al. 2007; aktas et al. 2011 ) transplant rejection. eculizumab (soliris) humanized, igg2/4j, anti-human complement c5; paroxismal nocturnal haemoglobinuria (pnh); (thomas et al. 1996; nieri et al. 2009; danilov et al. 2010 ). there was no evidence of complement blockade from cord blood samples taken at delivery. eculizumab appears safe to use in this setting and is likely to prevent many of the complications usually observed (kelly et al. 2010) . edrecolomab (panorex) igg2a, epcam antigen cdc-adcc cancer (nieri et al. 2009 ). efalizumab (raptiva) humanized, igg1k, anti-integrin-cd11a -psoriasis (nieri et al. 2009 ). progressive multifocal leukoencephalopthy was found to be a rare, but lethal disease associated with long term efalizumab therapy (kothary et al. 2011 ). epratuzumab, a humanized igg1 unconjugated anti-cd22 antibody effective against non-hodgkin lymphoma and follicular lymphoma (leonard et al. 2008; watanabe 2011) . consequences of the application during pregnancy has not been reported. etanercept (etn, enbrel, wyeth pharmaceuticals) human igg fc domain 1 tnfr2/p75; anti-tnfa as, psa, pp, jia; 81 ng/ml cord blood. 21 ng/ml 1 week postpartum 2 ng/ml; 3 weeks postpartum; undetectable 12 weeks postpartum (clowse 2010) . reduction of post-partum microchimerism (rak et al. 2009 ). complications of the therapy may be acute anterior uveitis (etanercept), psoriasis (infliximab > etanercept) and infectious bowel disease (ibd) (etanercept > infliximab) which were observed in association with the treatment using tnfantagonists. the paradoxical consequences, however, affected less than 5% of the treated patients (fouache et al. 2009 ). eternacept, which is a recombinant human p75 soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued. the recombinant receptor proved to be useful for the treatment of rheumatoid arthritis (malottki et al. 2011 ), but its use in pregnants has not been approved (østensen et al. 2008) . etanercept treatment (25 mg â 2/week) has been stopped 6 weeks before pregnancy. the treatment had to be reinitiated from the 20th week of pregnancy and no fetal complication was observed (umeda et al. 2010). the etanercept treatment was initiated 7 weeks before pregnancy (25 mg/sq-m 2 â weekly). the cord blood contained 81 ng/ml etanercept in contrast to the maternal serum (3,849-2,849 ng/ml). no detectable etanercept was found in the newborn's blood 12th week after delivery, although the breast milk contained 3.5 ng/ml etanercept (murashima et al. 2009 ). all patients requested at least one times the repetition of the treatment according to a publication from norway (mahic et al. 2011) . fontolizumab used for the preventive treatment of newborns at risk for respiratory syncytial virus infection (rsv) resulted a significant decrease in c-reactive protein levels suggested a beneficial biological effect. (nieri et al. 2009; reinisch et al. 2010) . galiximab, a human-primate chimeric anti-cd80 antibody: galiximab is a human-primate chimeric anti-cd80 antibody with excellent tolerability and singleagent effectiveness for recurrent follicular lymphoma (fl), resistant to other therapeutical means (watanabe 2011) . gemtuzumab (mylotarg k ) igg4k; humanised; cd33-monocyte, myeloid cell drug targeting (nieri et al. 2009 ). golimumab (simponi®; centocor ortho biotech) human monoclonal igg1 antibody ra, as, psa (østensen and f€ orger 2011) . hcd122 (novartis/xoma) cd40-specific fully human igg1 mab with antagonistic activity that mediates adcc and blocks cd40l-induced survival and proliferation of normal and malignant b cells (chatenoud and bluestone 2007) . ibritumomab tiuxetan (zevalin) murine, igg1k, anti-cd20; radiol (yttrium 90) imc-c225 (nieri et al. 2009 ). infliximab (ifx, remicade, schering-plough ltd) chimeric, igg1k, anti-tumor necrosis factor alpha (tnfa) rheumatoid arthritis (ra) and crohn's disease (cd) (saleem et al. 2008; van schouwenburg et al. 2010) . fatal case of disseminated mycobacterial infection has been reported in an infant who received bcg vaccine at 3 months of age. the mother had been treated with infliximab throughout her pregnancy. vaccination with live bacteria and viruses should be postponed in infants exposed to infliximab in utero, until serum levels are undetectable which may require more than 6 months (djokanovic et al. 2011) . the fetal concentration of infliximab was found to be higher than that of the mother. this might be a risk for the postnatal development of the immune system (zelinkova et al. 2011 ). due to the high rate of igg transfer near term, babies have been found to have similar blood levels of infliximab to their mothers (clowse 2010) . all reported pregnancy outcomes under treatment with infliximab showed no increase in miscarriage, prematurity or structural malformations in neonates compared with non-exposed pregnancies (østensen et al. 2008) . only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide. the potency of this agent in moderate-to-severe ulcerative colitis (uc) and cd has been one of the most important advances in the care of inflammatory bowel disease (ibd) in the past decade (d'haens and daperno 2006) . anti-infliximab anti-idiotypes. no association was found between the patients' allotypes and the presence or concentration of anti-infliximab antibodies ) reduction in fertility (not known, whether related with male or female animals) (pentsuk and van der laan 2009). vacterl association? acute graft versus host disease (couriel et al. 2009) were described, but it proved to be useful for the treatment of rheumatoid arthritis (malottki et al. 2011) . inotuzumab ozogamicin (cmc-544), the calicheamicin-conjugated anti-cd22 monoclonal antibody and rituximab combination were used for the treatment of ankylosing spondilitis, psoriatic arthritis and ulcerative colitis; (nieri et al. 2009; smith et al. 2010 ) the concentration in the blood of the newborn was 39.5 mg/ml 6 week post-partum and slowly declined over 6 months (clowse 2010) . ipilimumab (fcgriib binding) overcoming tcla-4-mediated immunosuppression, increasing anticancer immune-response (melanoma malignum; mdx-010; bristol-myers squibb/medarex); ctla-4 (cd152) t cells; t reg cells; colon and prostatic cancer; (nieri et al. 2009; houot et al. 2011) . iratumumab (sgn-30 and mdx-060) cd30-specific igg used for the treatment of hodgkin's lymphoma. myelosuppression, fatigue, elevated liver enzymes were documented during therapy (klimm et al. 2005) . lfb-r593, a fully human anti-rhesus d (rhd) antibody, for the prevention of feto-maternal allo-immunization in rhd-women, as a substitute for human polyclonal anti-rhd immunoglobulins (urbain et al. 2009 ). lfb-r603, a monoclonal antibody directed against cd20, for the treatment of b cell malignancies. antibody-dependent cellular cytotoxicity (adcc) activity and enhanced affinity to fcgriii (cd16), both correlated to a glycosylation pattern characterized by a low fucose content (urbain et al. 2009 ). mapatumumab; trail receptor activation (death receptor 4) mediator of apoptosis in cancer cells (nieri et al. 2009 ). matuzumab (humanised anti-egfr monoclonal antibody; reviewed by seiden et al. 2007 ; and recently by quatrale et al. 2011) . melimmune: anti-idiotype antibody that mimic the high molecular weight chondroitin sulfate proteoglycan antigen of melanoma cells (pride et al. 1998; murray et al. 2004; ward et al. 2011) . mitumomab (bec2, imclone systems) bec-2 anti-idiotype (giaccone et al. 2005; bottomley et al. 2008) bec2 is an anti-idiotypic antibody that mimics gd3, a ganglioside that is expressed on the surface of tumor cells and is of neuroectodermal origin. ganglioside gd3 can be used as a vaccine against small cell lung cancer (sclc) (nieri et al. 2009 ). mln-02 anti-a4b7 integrin antibody of igg1 type, humanised (reviewed by bosani et al. 2009 ) approved for the treatment of crohn's disease. motavizumab (humanised mouse monoclonal antibody). motavizumab targets a highly conserved epitope in the a antigenic site of the rsv fusion (f) protein, which is important in the invasion of rsv from cell to cell. motavizumab, which differs from palivizumab by just 13 amino acids, has exhibited a 70-fold enhancement in binding to the rsv f protein compared with the first-generation mab, with an 11-fold faster association rate and sixfold slower disassociation rate (nieri et al. 2009; weisman 2009 ). muromonab, igg2a, murine, t-cell cd3 blocade. cd3-specific monoclonal antibodies can re-establish immune homeostasis in treated individuals. this occurs through modulation of the t-cell receptor (tcr)-cd3 complex (also termed antigenic modulation) and/or induction of apoptosis of activated autoreactive t cells, which leaves behind 'space' for homeostatic reconstitution that favours selective induction, survival and expansion of adaptive regulatory t cells establishing long-term tolerance. it is used for early treatment of diabetes type 1 (chatenoud and bluestone 2007; nieri et al. 2009 ). natalizumab (tysabri) humanized, igg4k, anti-a4-integrin (vla-4), in the treatment of sclerosis multiplex (van schouwenburg et al. 2010) . natalizumab blocks both alpha-4 b1 integrin (vcam 1) and alpha-4b7 integrin (madcam 1) interactions (rutgeerts et al. 2009 ). therapy of sclerosis multiplex will be more efficient in combination with interferon (miller et al. 2003a, b; nieri et al. 2009 ). in animal experiments efd g. pig: reduced pregnancy rates in high-dose group; ppnd cyn: increased abortion and stillbirth rates (pentsuk and van der laan 2009). in cynomolgus monkeys, however, the abortion rate had not been increased, but hematopoetic changes were observed. natalizumab had no adverse effects on the general health, survival, development, or immunological structure and function of infants born to dams treated with natalizumab during pregnancy (wehner et al. 2009a, b) . 10% of natalizumab therapy has been stopped because of pregnancy. three of 363 patients treated at least for 24 months developed progressive multifocal encephalopathy (pml; piehl et al. 2011 necitumumab (imc-11f8) anti-egfr human monoclonal antibody (kuenen et al. 2010) . nimotuzumab (theracim) humanised, anti-egfr-1/her-1; apoptosis, adcc; head and neck cancers (hncc), (spicer 2005; nieri et al. 2009 ; reviewed recently by quatrale et al. 2011) . omalizumab (xolair) humanized, igg1k, anti-ige -asthma. causing marked reduction in serum levels of free ige and down-regulation of ige receptors on circulating basophils. effective in monozygotic twins (holgate et al. 2005; just et al. 2007; nieri et al. 2009 ) tolerability (corren et al. 2009 ). onercept, is a recombinant human p55 soluble receptor to tnf, failed in a phase ii trial with crohn's disease and the trial was discontinued (bosani et al. 2009 ). palivizumab (synagis) humanized, igg1k, anti-respiratory syncytial virus "a" epitope of fusion protein. it is used for the prevention of respiratory syncytial virus infection of newborns with different risks for respiratory infections (martin-mateos 2007; nieri et al. 2009; weisman 2009) . panitumumab (vectibix) human, igg2k, anti-human epidermal growth factor receptor binding the catalytic kinase domain of the receptor of colorectal cancer (cc; nieri et al. 2009 ). increased frequency of abortion/fetal death rates were observed in high-dose group (reviewed by pentsuk and van der laan 2009; nieri et al. 2009 ). eyelash trichomegaly in adults were seen (zhang et al. 2007; morris et al. 2011) . racotumomab(1e10), an anti-idiotypic vaccine mimicking the n-glycolyl-gm3 ganglioside (guthmann et al. 2006; hernández et al. 2008 ) effective against breast and lung cancers. ngcgm3 is practically undetectable in healthy human tissues as a result of an alu-mediated inactivation of the gene, the ganglioside is highly expressed in several human cancer cells presumably due to incorporation of dietary ngc . ramucirumab (dc-101) (an antibody to the vegf receptor-2) (tonra et al. 2006; krupitskaya and wakelee 2009) . ranibizumab (lucentis, genentech) humanized, igg1-fab, anti-human vasc. endothel. growth factor-a (vegf-a); for the treatment of choroidal neovascular (wet) age-related macular degeneration (armd) reviewed recently (ferrara et al. 2003 (ferrara et al. , 2006 ; neovascular acute myeloid leukemia (neovascular-aml); (csáky and do 2009; nieri et al. 2009 ). retuximab (epstein-barr virus) anti-cd-20 (sodani et al. 2010) . rituximab (rtx, mabthera k , roche) chimeric, igg1k, anti-cd20 (sulesomab, leukoscan). murine fab, binds to surface granulocyte non-specific crossreacting antigen present on neutrophils. rhinitis, fever, chills and toxic laboratory findings occurred during the treatment (klimm et al. 2005) . hcv cryoglobulinaemia could be also treated (dammacco et al. 2010) . the treatment of pregnants because of bukitt's lymphoma resulted high rituximab concentrations and a transient complete b-cell depletion in the cord blood. b-cell recovery was fast, showing a regular immunophenotype without loss of cd20 antigen, no functional deficits and adequate vaccination igg titers (friedrichs et al. 2006) . administration in third trimester of pregnancy suppresses neonatal b-cell development, but without later neonatal consequences (klink et al. 2008) , in spite of these the prophylactic withdrawal has been recommended before pregnancy (østensen et al. 2008) . human fetal b-cell depletion and lymphocytopenia in cynomolgus, were observed, too (vaidyanathan et al. 2010) . reduction of post-partum microchimerism was documented (rak et al. 2009 ). useful in rheumatoid arthritis therapy (malottki et al. 2011) in combination with chemotherapy depending on human concentrative nucleotide transporter 1 (hcnt1) gene expression rate (rabascio et al. 2010) . non-hodgkin lymphoma, rheumatoid arthritis were the indications (nieri et al. 2009 ). cd137 is a costimulatory molecule expressed on a variety of immune cells after activation, including nk cells. cd137 stimulation by specific igg enhances the antilymphoma activity of anti-cd20 antibodies by enhancing adcc . of 153 pregnancies with known outcomes, 90 resulted in live births. twenty-two infants were born prematurely; with one neonatal death at 6 weeks. eleven neonates had hematologic abnormalities; none had corresponding infections. four neonatal infections were reported (fever, bronchiolitis, cytomegalovirus hepatitis, and chorioamnionitis). two congenital malformations were identified: clubfoot in one twin, and cardiac malformation in a singleton birth. one maternal death from pre-existing autoimmune thrombocytopenia occurred. women should continue to be counseled to avoid pregnancy for 12 months after rituximab exposure; however, inadvertent pregnancy does occasionally occur. practitioners are encouraged to report complete information to regulatory authorities for all pregnancies with suspected or known exposure to rituximab (chakravarty et al. 2011) . due to ongoing bleeding, rituximab was given in the 26th week of pregnancy. the platelet count rose to over 100 â 10(9)/l after 4 weeks. the neonatal b-lymphocyte count normalized at 4 months after delivery. there were no neonatal complications of rituximab therapy (gall et al. 2010) . passenger lymphocyte syndrome has been described by lee et al. (2008a, b) . siplizumab (cd2 or medi-507) is a humanised igglk monoclonal antibody that binds to human cd2 antigen. preclinical studies demonstrated that siplizumab kills target cells by adcc (fanale and younes 2007; watanabe 2011) . teplizumab (cd3-specific, hokt3g1-ala-ala), a humanized fc mutated anti-cd3 monoclonal antibody induced tolerance, on the progression of type 1 diabetes in patients with recent-onset disease even 2 years after the first diagnosis (herold et al. 2002 (herold et al. , 2005 . tocilizumab (toc, roactemra, roche) against receptor of il-6 (mouse anti-human il-6r antibody into human igg1-k chain to create a human antibody with a human il-6r binding site il-6r a-chain or cd126; b-chain or cd130) at a low concentration of 1 microg/ml, tocilizumab (anti-human il-6 receptor monoclonal antibody) inhibited the il-6-induced matrix-metallo-proteinase (mmp) secretion which was shown to be stimulated in preterm premature rupture of membranes (pprm) (sato et al. 1993; mano et al. 2009; malottki et al. 2011; pham et al. 2010) . clinical phase 3 trial for the treatment of rheumatoid arthritis has been approved. inherited autoinflammatory syndrome can be sometimes treated with anakinra and tocilizumab (goldfinger 2009 ). at present reports on abatacept, tocilizumab or anakinra are inconclusive therefore throughout pregnancy cannot be recommended (østensen and f€ orger 2011) . normal pregnancy is characterised by elevated th2 activity and anti-inflammatory cytokines during the first trimester, followed by increased th1 activity and proinflammatory factors near term (challis et al. 2009 ). in contrast, preeclampsia (pe) is marked by an increase in proinflammatory tumor necrosis factor-a (tnf-a) and interleukin 6 (il-6) cytokines as well as a decrease in the anti-inflammatory cytokines il-4 and il-10. in cases of restricted fetal growth, tnf-a is also elevated when compared with normal pregnancy (dávila et al. 2011) . women living at 3,100 versus 1,600 m in colorado had higher proinflammatory (il-6, tnf-a) relative to anti-inflammatory (il-10) cytokines during the second and third trimesters (coussons-read et al. 2002) . multigenerational andean versus shorter duration european high-altitude residents were found to be protected from altitudeassociated fetal growth restriction. higher il-1b might play a role in protection from altitude-associated reductions in fetal growth (coussons-read et al. 2002; dávila et al. 2011) . tocilizumab treatment increased serum levels of il-6 and soluble il-6r (sil-6r; nishimoto et al. 2008 ). in combination with other drugs adult onset of still's disease can be improved using monoclonal antibodies (efthimiou and georgy 2006) . tositumomab anti-cd20 igg, b-cell lymphoma (armstrong and eck 2003; nieri et al. 2009 ). trastuzumab (herceptin) humanized, igg1k, erbb2, anti-her2. induction of cdc or adcc on an fcreceptor g-binding mechanism. human anhydramnion and oligohydramnion will develop because of the caused fetal kidney insufficiency (watson 2005; robinson et al. 2007; katsumi et al. 2008; matsumoto et al. 2009 ). this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich 2011) . transfer using aav-recombinant in mice does not induce anti-idiotypes (wang et al. 2010a, b) . the mechanism of toxicity to the fetal kidneys is proposed to be associated with the different structure of egfr in the fetal renal-tubule epithelial cells (heterodimer of egfr and erbb2 in fetus vs. homodimer of egfr in adults). thus, trastuzumab will have a damaging effect on the fetal renal function, but it does not affect the kidneys of the adult (robinson et al. 2007) . anti-trastuzumab (ladjemi et al. 2011 ): anti-trastuzumab anti-id scfv69, used as a therapeutic or prophylactic vaccine, protects mice from developing her2-positive mammary tumors by inducing both anti-her2 ab1 0 antibody production and an anti-her2 th2-dependent immune response. these results suggest that scfv69 could be used as an anti-id-based vaccine for adjuvant therapy of patients with her2-positive tumors to reverse immunological tolerance to her2. calmodulin inhibitors rescue trastuzumab sensitivity of breast tumours (kulkarni et al. 2010 ). the majority of these patients were able to tolerate therapy; however, oligohydramnios or anhydramnios occurred in 5 out of the 7 patients. this decrease in amniotic fluid seems to be reversible with the discontinuation of trastuzumab (sukumvanich 2011) . tremelimumab (cp-675,206; pfizer); ctla-4 (cd152) t cells; t reg cells; colon and prostatic cancer; . triab 11d10 (triab) breast cancer (reece et al. 2000 (reece et al. , 2001 (reece et al. , 2003 . trigerm (disialoganglioside gd2) melanoma . tositumomab (iodine labelled), murine cd20, cdc, adcc, radio-cytotoxicity non-hodgkin-lymphoma (nieri et al. 2009) veltuzumab is a humanized anti-cd20 antibody with structure-function differences from chimeric rituximab (watanabe 2011) . visilizumab (cd3-specific) for the management of both crohn's disease (cd) and ulcerative colitis (uc). biologics under evaluation or approved for uc that are discussed include monoclonal antibodies to tumor necrosis factor ([tnf] infliximab), inhibitors of adhesion molecules (mln02 and alicaforsen), anti-cd3 antibodies (visilizumab), and anti-interleukin (il)-2 receptor antibodies (daclizumab). biologics under evaluation or approved for cd that are reviewed include three monoclonal antibodies to tnf (infliximab, adalimumab, and certolizumab pegol), monoclonal antibodies against il-12, interferon-g, and il-6 receptors, inhibitors of adhesion molecules (natalizumab, alicaforsen), and growth factors. only the chimeric monoclonal anti-tnf antibody infliximab is currently available worldwide (d'haens and daperno 2006) . 90 yttrium-ibritumomab tiuxetan and 131 iodine-rituximab are anti-cd20 monoclonal antibodies combined with radioactive materials for diagnostic and/or therapeutic applications (watanabe 2011) . zalutumumab anti-egfr mab able to facilitate complement lysis of cancer cells (klausz et al. 2011) . reviewed recently (quatrale et al. 2011 ). expressed by antigen presenting myeloid cells (apc) (magnani et al. 2011 ). cd3 t-cell receptor (tcr)-cd3 complex resulting in the cells becoming 'blind' to antigen, a process that is also known as antigenic modulation (chatenoud and bluestone 2007) . cd20 over 90% (sato et al. 1993 ). cd152 ctla-4 negative regulator of t cell activation (kaufman et al. 1999 ). trail dr5/tnf related receptor (nieri et al. 2009 interleukin 6 (naugler et al. 2007; naugler and karin 2008; voorhees et al. 2009; reinartz et al. 2009 ). pd-1 programmed death protein 1 ). complement factor 5 thomas et al. (1996) breast cancer protein reece et al. (2000 reece et al. ( , 2001 reece et al. ( , 2003 ige immunoglobulin e, hypersensitivity (corren et al. 2009 ). anti-idiotypes carrier of the mimicry of epitopes (ab2) of antigens (k€ ohler 1978) . respiratory syncytial virus (nieri et al. 2009 ). cytotoxic t lymphocyte antigen-4 (ctla-4) and programmed cell death 1 (pd-1) are members of the known t reg -associated molecules. blocking pd-1 abrogate the protective effect of t reg , resulting in a higher median abortion rate in comparison with the t reg / isotype-treated control while ctla-4 blockage did not interfere with the protective effect of t reg . pd-1 as an important mediator in t reg -induced fetal protection in the cba/ j · dba/ 2j murine model (wafula et al. 2009 ). ctla-4 was shown to interact with cd80 and cd86 resulting in termination of immune response (alegre et al. 2001) . mice genetically deficient in ctla-4 expression develop a lymphoproliferative disease which terminates in death by 3-5 weeks of age (tivol et al. 1995; waterhouse et al. 1995) . the cd28 possesses also role in the regulation of t-cells (sansom and walker 2006) . blockade of the interactions between cd28 and their ligands, cd80 and cd86, has been shown to induce antigen-specific peripheral tolerance in organ transplantation. this knowledge has been successfully used in animal models to prevent allograft rejection by blocking cd86 and/or cd80, thereby leading to long-term graft survival. cytokines favoring the maintenance of fetal survival mainly belong to the th2-type (e.g. il-4, il-10, tgf-b), whereas pregnancy failure is associated with the th1-type cytokines (e.g. ifn-g, tnf-a) at the materno-fetal interface and/or the absence of th2-type cytokines. the combined use of anti-cd80 and anti-cd86 mabs in mice was effective in inducing maternal tolerance to the allogeneic fetus. blockade in vivo of cd80 and cd86 costimulation could prevent abortions by shifting cytokines from th1 predominance to th2 bias and expanding peripheral cd4 + cd25 + regulatory t cells (jin et al. 2005) . breakdown of immunologic self tolerance maintained by activated t cells expressing il-2 receptors (cd-25) results in the development of autoimmune diseases (sakaguchi et al. 1995; sakaguchi 2004) , which can be mitigated using anti-cd25 monoclonals. suppressive cd4 + cd25 t reg cells are elevated also during pregnancy (somerset et al. 2004) . intergins: progressive multifocal leukoencephalopathy was observed (pml) probably of polyomavirus etiology after natalizumab (anti-integrin-a4) therapy of crohn's disease (edula and picco 2009 ). tnfalpha blockers were shown to induce autoimmunity on ana and anti-dsdna antibodies in ra and spa patients. autoimmunity was induced more frequently with infliximab than etanercept and to a lesser degree to adalimumab therapy but, more importantly, this emergent autoimmunity was exceptionally associated to clinical manifestations of lupus (bacquet-deschryver et al. 2008) . the effect of infliximab, etanercept or adalimumab on spermatogenesis has been studied in 26 patients with spondylarthritis (villiger et al. 2010) . sperm abnormalities were found in healthy controls. patients on anti-tnf therapy showed significantly better sperm motility and vitality than untreated patients (østensen and f€ orger 2011). antibody products licensed for prevention or treatment of viral diseases include non-immune human immunoglobulin for use against hepatitis a and measles, virusspecific polyclonal human immunoglobulin against cytomegalovirus, hepatitis b, rabies, respiratory syncytial virus (rsv), vaccinia, and varicella-zoster, and the humanized monoclonal antibody palivizumab, fonolizumab and motavizumab (groothuis et al. 2011) . polyclonal immunoglobulin has also been used with various success for diseases caused by other human viruses including parvovirus b19 (pv b19), lassa virus, west nile virus, some enteroviruses, herpes simplex virus, crimean-congo haemorrhagic fever virus (cchfv), junin virus, severe acute respiratory syndrome-associated coronavirus (sars cov) and human immunodeficiency virus (hiv). serum polyclonal antibody preparations have been clinically effective in many cases, problems related to toxicity including a risk for allergic reactions, lot to lot variation and uncertain dosing have limited their use (casadevall 1999 (casadevall , 2006 . the use of rabies and tick-borne encephalitis virus-specific hyperimmune gamma globulins are used in several countries immediately following virus exposure (animal injuries or tick bites). cytomegalovirus-specific hyperimmune gamma globulin is used in the transplantation surgery (schmitz and essuman 1986 ) before the era of gancyclovir preventive therapy. sars cov surface glycoprotein, also called spike glycoprotein, (s protein or s glycoprotein) mediates viral entry into the host cell and has two functional domains s1 and s2. the s1 domain is involved in the binding of the cellular receptor ace2 whereas the s2 domain facilitates the fusion between viral and host cell membranes. infections by many viruses, including coronaviruses, elicit potent neutralizing antibodies (nabs) that can affect the course of infection and help clear the virus; they can also protect an uninfected host exposed to the virus. an improved method for epstein-barr virus (ebv) transformation of human b cells has been developed based on cpg oligonucleotides that increases the b cell immortalization efficiency from 1-2% to 30-100%, and this method was used for selection of human abs specific for sars cov proteins. one of the selected antibodies, which was specific for the s glycoprotein on the viral spikes, was about 500-fold more efficient in neutralization than convalescent serum. nipah virus (niv) and hendra virus (hev) are closely related emerging paramyxoviruses that comprise the henipavirus genus. they are biological safety level-4 (bsl-4) pathogens, and are on the niaid biodefense research agenda as zoonotic emerging category c priority pathogens that could be used as bioterror agents (zhu et al. 2008 ). monoclonal antibodies of enzyme activity have been developed. these can be used in cancer therapy, but the application for the treatment of pregnant women is at present not yet approved (kulkarni et al. 2010; quatrale et al. 2011 ). immunotherapy offers a range of potential treatment options: drug treatment, as well as the treatment of overdose, prevention of brain or cardiac toxicity and fetal protection in pregnant drug abusers. clinical trials, cocaine and nicotine vaccines have been shown to induce antibody titers while producing few side effects (haney and kosten 2004) . plasmin may serve as a major driving autoantigen for some anticardiolipin (acl) in anti-phospholipid syndrome (aps) patients who are positive for igg anti-plasmin ab. one mab displayed the anti-cardiolipin (acl) and the lupus anti-coagulant (lac) activities and induced fetal loss when injected into pregnant mice ). molecular mimicry has been suggested to play a role in the pathogenesis of many autoimmune diseases, such as allergic encephalomyelitis, experimental myocarditis, and experimental autoimmune keratitis and uveitis antigenic molecular mimicry is characterising anti-dna antibodies. these are reacting with different proteins i.e. enzymes (blank and shoenfeld 2004) . in case of schizophrenia, the overall finding has been that, when a monozygotic twin has this serious neuromental disorder (nmd), the other identical twin has a 50% risk; whereas among dizygotic twins, the risk -when one is afflicted -is only 15%. neuromental disorders (nmd) might be caused indirectly by maternal transplacentally-acquired antibodies, to agents with epitope molecular mimicry with the developing nervous system, and cause alterations which will clinically manifest years later (nahmias et al. 2006) . serological evidence of previous exposure to ebv in children with ms supports a role for ebv infection early in ms pathogenesis, as already indicated by prospective studies in adults. higher antibody titers and t-cell responses to ebv in patients compared to healthy ebv carriers indicate possible continuous viral reactivation. ms patients have increased cd4 + and cd8 + t-cell responses to ebv antigens, particularly ebna1. there is some evidence that ebv could break immune tolerance to myelin antigens through molecular mimicry. detection of ebv-infected b-cells in patients' brain raises the possibility that intrathecal b-cell abnormalities and t-cell-mediated immunopathology in ms are the consequence of a persistently dysregulated ebv infection. accordingly, targeting t-cells and/or b-cells with monoclonal antibody therapies ameliorates ms. whether ebv has a causative or pathogenic role in ms can now be addressed in relation to genetic, hormonal and other environmental influences that may affect ebv-host interactions (salvetti et al. 2009 ). functional suppression by cd4 + cd25 + regulatory t cells was also found to be impaired in ms patients (viglietta et al. 2004 ). newborns of pregnants suffering from multiple sclerosis (ms) were impaired by the disease. in case the father of the newborn was suffering from ms, no negative consequences could be documentet i.e. safe paternity characterises ms-patients. the results of mothers does not seem to have an impact on birth weight, however, ms may contribute to a reduced birth weight (hellwig et al. 2010) . the mothers suffering from ms are usually treated with long-term interferon (ifn) beta-therapy in spite of the pregnancy. the foetal exposure to subcutaneous interferon beta-1a therapy before treatment discontinuation was at least 28 days; most pregnancies (199/231; 86.1%) were exposed for 45 days. the rates of spontaneous abortion and major congenital anomalies in live births were in line with those observed in the general population (amato et al. 2010; sandberg-wollheim et al. 2011) . the in vitro susceptibility of bewo cells was increased for toxoplasma gondii 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clinical, serological and immunogenetic study with review of the literature postnatal development in cynomolgus monkeys following prenatal exposure to natalizumab, an alpha4 integrin inhibitor embryo/fetal development in cynomolgus monkeys exposed to natalizumab, an alpha4 integrin inhibitor motavizumab, a second-generation humanized mab for the prevention of respiratory syncytial virus infection in high-risk populations treatment with tumor necrosis factor inhibitors and intravenous immunoglobulin improves live birth rates in women with recurrent spontaneous abortion prevention strategies for type 1 diabetes mellitus: current status and future directions down-regulation of placenta growth factor by promoter hypermethylation in human lung and colon carcinoma mhc class ii tetramers containing influenza hemagglutinin and ebv ebna1 epitopes detect reliably specific cd4+ t cells in healthy volunteers high intra-uterine exposure to infliximab following maternal anti-tnf treatment during pregnancy acquired trichomegaly and symptomatic external ocular changes in patients receiving epidermal growth factor receptor inhibitors rras: a key regulator and an important prognostic biomarker in biliary atresia exceptionally potent cross-reactive neutralization of nipah and hendra viruses by a human monoclonal antibody key: cord-017520-r786yd6i authors: huber-lang, markus; gebhard, florian title: inflammatory changes and coagulopathy in multiply injured patients date: 2015-05-14 journal: the poly-traumatized patient with fractures doi: 10.1007/978-3-662-47212-5_4 sha: doc_id: 17520 cord_uid: r786yd6i severe tissue trauma leads to an early activation of several danger recognition systems, including the complement and the coagulation system, often resulting in an overwhelming almost synchronic proand anti-inflammatory response of the host. although the immune response is associated with beneficial effects at the site of injury including the elimination of exogenous and endogenous danger molecules as well as the initiation of regenerative processes, an exaggerated systemic inflammatory response significantly contributes to posttraumatic complications such as multiple organ failure (mof) and early death. besides pre-existing physical conditions, age, gender, and underlying comorbidities, surgical and anesthesiological management after injury is decisive for outcome. improvements in surgical intensive care have increased number of patients who survive the initial phase after trauma. however, instead of progressing to normal recovery, patients often pass into persistent inflammation, immunosuppression, and catabolism syndrome (pics). the characterization and management of pics will require new strategies for direct monitoring and therapeutic intervention into the patient’s immune function. in this chapter, we describe various factors involved in the inflammatory changes after trauma and aim to understand how these factors interact to progress to systemic inflammation, mof, and pics. multiple trauma results in a significant blood loss and accumulation of necrotic and/or devitalized tissue in an ischemic-hypoxic environment, both of which will become the origin of coagulatory and inflammatory changes. the inflammatory response after polytrauma is a major part of the host's molecular danger response. the acute posttraumatic phase of inflammation consists of two rather synchronically mounted columns: the pro-inflammatory response (systemic inflammatory response syndrome, sirs) and the anti-inflammatory response (compensatory anti-inflammatory response syndrome, cars) [1] . sirs includes changes in the heart rate, respiratory rate, temperature regulation, and immune cell activation (table 4 .1) [2] . in the natural course of the inflammatory response after trauma, the balance of the pro-and antiinflammatory response is in equilibrium, which maintains the biological homeostasis and induces controlled regeneration processes, enabling the patient to recover normally without significant complications. however, the excessive inflammatory response after trauma seems to simultaneously and rapidly involve the induction of innate (both pro-and anti-inflammatory mediators) and suppression of adaptive immunity [1, 3, 4] all of which decisively contribute to the development of the early multi-organ dysfunction syndrome (mods). furthermore, a prolonged and dysregulated immune-inflammatory state is associated with delayed recovery and complications, especially the development of late mods. based on improved intensive care and organ support, there is often a progress to the clinically evident persistent inflammation, immune suppression, and catabolism syndrome (pics) which might have replaced the late mods, but still is associated with a poor outcome, appearing as "silent death" [5] . the steps of an inflammatory reaction to trauma involve fluid phase mediators (cytokines, chemokines, coagulation-and complement activation products, oxygen radicals, eicosanoids, and nitric oxide (no)) and cellular effectors (neutrophils, monocytes/macrophages, and endothelial cells) that translate the trauma-induced signals into cellular responses. these factors are closely interrelated and interconnected by upregulatory and down-regulatory mechanisms. the combination of these factors may cause severe sirs, acute respiratory distress syndrome (ards) and sepsis, acute kidney injury (aki), progressing to mods, depending on the type of injured tissue, the surgical and anesthesiological management after injury, age, gender, genetics, and most importantly, underlying comorbidities and physical conditions (exogenous and endogenous factors) ( fig. 4.1 ). patient survival after severe trauma requires an adequate molecular and cellular danger response. the injured tissues release cytosolic molecules (e.g., atp), organelles (e.g., mitochondria), histones, nucleosomes, dna, rna, matrix, and membrane fragments, all functioning as damage-associated molecular patterns (damps). furthermore, damage of external and internal barriers (e.g., skin, gut-blood barrier, air-blood barrier, brain-blood barrier) facilitates invasion of microorganisms, resulting in additional exposure to microorganisms-derived pathogenassociated molecular patterns (pamps). after multiple injury, the immune system of the injured patient is exposed to both damps (also termed alarmins) and pamps, which are summarized as danger-associated molecular patterns [6] . the "3-r-challenge" for the innate and adaptive immune system is to recognize, respond to, and resolve the "molecular danger". for recognition of the damage, there are effective fluid-phase "master alarm systems", such as the coagulation and complement cascade, and effective cellular "danger sensors", such as the pattern recognition receptors (prr). these systems transfer the damage/danger signals to the cells which in turn mount an acute phase reaction and inflammatory response to resolve the damaged tissue load [7] . within an hour after trauma, inflammation resulting from tissue injury induces an increase in plasma concentration of a number of liverderived proteins (the acute phase proteins, app). pro-inflammatory cytokines (il-1β, tnf, il-6) released locally by kupffer cells can systemically influence other cell types such as hepatocytes to synthesize more apps. proactive apps, sirs can be diagnosed when two or more of these criteria are present such as c-reactive protein (crp), procalcitonin (pct), serum amyloid a (saa), complement activation products (c3a, c5a), activated coagulation proteins (fviia, fxa, fiia), proteinase inhibitors, and metal-binding proteins, are increased during this phase [8] , whereas the production of inhibitory apps, such as albumin, high-density lipoprotein (hdl), protein c, protein s, and atiii are decreased [9, 10] . plasma concentrations of crp are normally below 10 mg/l [11] . hepatic synthesis of crp is regulated mainly by il-6. serum levels of crp can be detected about 12 h after systemic detection of il-6. clinically, the plasma levels of crp are relatively non-specific and may not correlate with injury severity and are not predictive of posttraumatic complications such as infections [12] . in the context of trauma, it is also still unclear whether the native pentameric or the denatured monomeric form of crp is responsible for the crp-induced cellular effects [13] . pct is physiologically produced in the thyroid gland as the precursor molecule of calcitonin [10] . during sepsis, stimulation by endotoxins or pro-inflammatory cytokines such as il-1β or tnf dramatically increases the serum levels of pct up to 1000-fold [14] . in trauma patients, pct has been proposed as a practical biomarker for predicting posttraumatic complications such as severe sirs, sepsis, and mods [14] [15] [16] [17] . the biological immune response after trauma was considered in the past to be divided into an early innate phase and a late adaptive response. however, since multiple intensive interactions between both systems are known (e.g., via the complement cascade), a spatial-or timedependent discrimination of both systems in regard to pathomechanistic changes after multiple injury is irrational. both immune mechanisms contribute to effective recognition, activation, discrimination, regulation, and eradication of invading damage-and pathogen-associated signals [18] . nevertheless, the innate immune response represents the "first line of defense", consisting of a barrier against exogenous nonself antigens and microorganisms. this includes the integrity of epithelial and mucosal cells: skin, respiratory tract, alimentary tract, urogenital tract, brain, and conjunctiva. exogenous pathogens that escape the first barrier are rapidly recognized and removed by the multiple components of innate immune cells such as neutrophils, monocytes/macrophages, natural killer cells, and dendritic cells [19] . the innate immune response is closely accompanied by the specifically acquired immune response after the trauma impact. the adaptive immune response is conducted by the interaction of antigen-presenting cells (apcs), dendritic cells, monocytes/macrophages, t-lymphocytes, and b-lymphocytes. the apcs capture invading pathogens and create peptide-mhc (major histocompatibility complex) protein complexes. t-lymphocytes recognize the peptide-mhc protein complex via t-cells expressing antigen-binding receptors (tcrs) and are thereby activated. in turn, activated t-lymphocytes release cytokines to activate and amplify further cells of the immune system. t-helper lymphocytes (cd4+ t cells) differentiate into two phenotypes according to the cytokine release, the th1 and th2 lymphocytes. th1 cells promote the pro-inflammatory response through the release of il-2, tnf, and interferon-γ (ifn-γ), while th2 cells produce anti-inflammatory cytokines (il-4, il-5, and il-10), which suppress macrophage activity [10] . attention has been focused on the th1/th2-ratio. il-12 secreted from monocytes/macrophages promotes the differentiation of th1 cells by increasing the production of ifn-γ [20, 21] . several studies have shown that a suppressed il-12, il-2, and ifn-γ, and elevated il-4 are observed after major trauma, which correlated with a shift of the th1/ th2 ratio towards the th2-type pattern [22, 23] . this imbalance in th1/th2-type cytokine response (from pro-to anti-inflammation) is not only a compensatory response but also increases the risk of infection by immune suppression [20] . however, other reports do not support this view and question the clinical relevance of the th1/ th2-shift after major tissue injury [24, 25] . bleeding is a leading cause of death following polytrauma, and acute trauma-induced coagulopathy (atic) increases both the risk and severity of bleeding. clinically, there are several routine laboratory parameters which are indicative of coagulopathy development (table 4. 2). around one third of severe polytrauma patients are already coagulopathic upon arrival in the emergency room [27] and coagulopathy belongs together with acidosis and hypothermia to the "lethal triad" of polytrauma. thus, an important diagnostic and therapeutic strategy has been developed proposed as the "stop bleeding campaign" [29] that addresses three major aspects of coagulopathy: fast detection and stopping of relevant bleeding sources; estimation and resuscitation of the lost blood volume; and rapid monitoring for coagulopathic conditions. the major mechanism of activation of the coagulation cascade following trauma is via the extrinsic coagulation system [30] . the extrinsic cascade mediates inflammation by tissue factor (tf). exposure of the fvii to tf (e.g., from injured cells) results in the conversion of fvii to fviia. the fviia-tf-complexes activate fx to fxa, and fxa converts prothrombin to thrombin (fiia). thrombin activates fv, fviii, and fxi, which results in enhanced thrombin formation. thrombin also cleaves fibrinogen, and the fibrin clot is formed following polymerization and stabilization. in normal conditions, small amounts of tf are exposed to the circulating blood. however, under pathophysiological conditions, tf is upregulated on the surface of neutrophils, macrophages, and endothelial cells. endotoxin, activated complement (c5a), and cytokines (il-1β, tnf) induce tf expression [31] . tf is highly thrombogenic, and its upregulation often results in hypercoagulability, leading to an increased tendency of thrombosis [32, 33] . another phylogenetically ancient activation pathway is the rather unknown fsap (fvii activating protease) pathway that is activated by an autocatalytic mechanism promoted by factors released by necrotic or post-apoptotic cells such as nucleic acids, nucleosomes, and polyamines. fsap can regulate coagulation and fibrinolysis by activating factor vii and pro-urokinase, respectively. in polytrauma patients, an early and robust activation of fsap is seen which in turn contributes to the activation of both, the coagulation and complement system [34] . in addition, coagulation mediators (fviia, fxa, and fiia) elicit inflammation with expression of tnf, cytokines, adhesion molecules (mcp-1, icam-1, vcam-1, selectins, etc.), and growth factors (e.g., vegf) [33] . inhibitors to prevent a hypercoagulable state include antithrombin iii (atiii), protein c, protein s and tf pathway inhibitor (tfpi). atiii inhibits fixa, fxa, and thrombin. tfpi suppresses the activity of tf/fviia/fxa complexes [35] . protein c is activated by the thrombin-thrombomodulin complex on endothelial cells, and activated protein c, in combination with free protein s, cleaves and inactivates fv and fviii [36] . therapeutically intervening with the production and/or activity of inhibitors could help to improve outcome by mitigating complications such as ards. for example, the crash2 trial has recently revealed that early application of tranexamic acid (a synthetic derivative of the amino acid lysine) that inhibits fibrinolysis by blocking the lysine binding sites on plasminogen significantly reduces the risk of death in bleeding trauma patients [37] . almost synchronically to the coagulation response, there is an activation of the complement cascade immediately after multiple trauma [38, 39] . the complement system consists of more than 30 proteins. in the resting state, complement proteins circulate as inactive forms in plasma. the activation of the complement system can occur through four pathways (alternative, classical, lectin, and coagulation paths). the classical pathway of complement is activated by antigenantibody complexes (immune-globulin m or g) or crp. the alternative pathway is activated by modified from maegele et al. [27] , brohi et al. [26] , greuters et al. [28] bacterial products such as lipopolysaccharides (lps). the lectin pathway is initiated by lectin binding to mannose, glucose, or other sugars of microorganisms. upon activation of the complement system, there is a generation of biologically active peptides. the cleavage of the central complement components c3 and c5 to the anaphylatoxins c3a and c5a, respectively, also induces the formation of opsonins and the membrane attack complexes (mac, c5b-9) [40, 41] . early after polytrauma, serum levels of the complement activation products c3a and c5a are significantly elevated and correlate with the severity of the injury (e.g., traumatic brain injury), septic complications, and mortality [27, 38] . the circulating soluble mac is also enhanced within the first hours after polytrauma but almost not detectable between 4 and 48 h after polytrauma [38, 39] . regulation of complement activation and protection against complement-mediated tissue destruction is provided by a selection of soluble-and membranebound complement regulatory proteins (cregs). the expression profile of cregs on leukocytes is specifically altered post polytrauma: cd46 (membrane co-factor protein) is significantly reduced in neutrophils, monocytes, and lymphocytes. in contrast, cd55 (decay accelerating factor) seems to be increased on neutrophils early after trauma. a delayed up-regulation of cd55 has been observed in monocytes from trauma patients. an initial enhancement of cd59 (mac inhibitor) expression was measured in neutrophils and monocytes at the time of admission. remarkably, c5a receptor (c5ar), cd59 and cd46 expression on neutrophils reversely correlated with injury severity [42] . the anaphylatoxins c3a and c5a mainly play pro-inflammatory roles, which include the recruitment and activation of phagocytic cells (polymorphonuclear cells, pmns), monocytes/ macrophages, the enhancement of the hepatic acute-phase reaction, stimulation of the release of vasoactive mediators (such as histamine), and promoting the adhesion of leukocytes to endothelial cells and their permeation through injured tissues. c5b forms a complex by the consecutive binding of proteins c6-c9, culminating in the formation of the mac (c5b-9), which leads to the formation of pores in the cellular membrane causing lysis and death of the target cells [43] . furthermore, the inflammatory response of complement activation leads to the production of free oxygen radicals and arachidonic acid metabolites and cytokines. the complement cascade bridges innate and adaptive immunity for defense against microbial pathogens. however, excessive consumption of complement proteins may also cause tissue damage of the host after trauma. within the first 24 h after multiple injuries, there is a massive reduction in complement hemolytic activity (ch50), which recovers only around 5 days after trauma, and can be used to discriminate between lethal and non-lethal outcome. this trauma-induced reduction of global complement function is referred to as trauma-induced "complementopathy" in analogy with "coagulopathy", both of which significantly participate to the impairment of the innate immune response after polytrauma (fig. 4.2 ). the kallikrein-kinin system involves a cascade of plasma proteases and is related to the complement and clotting cascade (intrinsic activation) [44] . this contact system consists of plasma proteins factor xii (hageman factor; fxii), prekallikrein, high molecular weight kininogen (hmwk), and fxi. contact with negatively charged surfaces such as foreign bodies or the membrane fragments of stimulated platelets activates fxii [44] . the active protein fxiia converts prekallikrein into the proteolytic enzyme kallikrein, which in turn cleaves the plasma glycoprotein precursor hmwk to form bradykinin [45] . bradykinin increases vascular permeability and causes dilation of blood vessels by its action on smooth muscle cells. in turn, as a positive feedback loop, kallikrein itself accelerates the conversion of fxii to fxiia. kallikrein can also activate fibrinolysis to counterbalance the clotting cascade activated by fxiia. furthermore, kallikrein also exhibits chemotactic activity, converting c3 and c5 into the chemoattractant products c3a and c5a, respectively [46] . pro-inflammatory cytokines play key local and systemic roles as intercellular messengers to initiate, amplify, and perpetuate the inflammatory response after trauma (table 4. 3). cytokines are produced by many cell types in all organs. they have multiple targets and act in a pleiotropic manner. early after trauma, production and release of pro-inflammatory cytokines such as il-1β, tnf, il-6, and il-8 is initiated by monocytes and macrophages. il-1β and tnf as well as il-6 and il-8 are released early after polytrauma [3, 47] and predominantly function as pro-inflammatory mediators to repair damaged tissue. the release of il-1β and tnf is mainly stimulated by bacterial endotoxins or other microbial products, immune complexes, and a variety of inflammatory stimuli. upon release, il-1β and tnf usually return to baseline levels within 4 h. tnf increases the activity of neutrophils and monocytes by activating the underlying endothelium. tnf promotes the expression and release of adhesion molecules such as icam1 or e-selectin, and increases the permeability of endothelial cells, which facilitates neutrophil migration into the damaged tissue [48] . some studies have proposed tnf as a valid serum marker for complications after trauma. however, the results are inconsistent and to date, no data is available indicating whether tnf correlates to the severity of trauma or trauma outcome [49] [50] [51] [52] [53] [54] . many different cell types produce il-6: in addition to immune cells such as monocytes, macrophages, neutrophils, t cells, and b cells, it is also produced by endothelial cells, smooth muscle cells, and fibroblasts. il-6 upregulates the hepatic acute-phase response, stimulating generation of c-reactive protein (crp), procalcitonin, serum amyloid a, fibrinogen, α1-antitrypsin, and complement activation products (e.g., c5a), which then promote neutrophil activation. there is strong evidence that serum il-6 level correlates with the severity of trauma, trauma pattern (especially in combination with chest trauma), and the risk of subsequent ards, mof, and lethal outcome [47, 55] . therefore, il-6 may be considered as a clinically relevant and feasible parameter to estimate the severity of injury and prognosis after trauma [56, 57] . in addition, for patients requiring second or subsequent surgeries following trauma, il-6 may prove to be an important biological marker in deciding the correct timing of surgery. in trauma patients with high initial levels of il-6 (>500 pg/dl), it is recommended to delay secondary procedures for more than 4 days [58] . the chemokine il-8 is secreted by monocytes/ macrophages, neutrophils, and endothelial cells. il-10 is mainly synthesized by t lymphocytes and monocytes/macrophages. it is the pivotal role of il-10 to inhibit the production of monocyte/macrophage-derived tnf, il-6, il-8, and free oxygen radicals [62] . il-10 plasma levels are proportional to the severity of trauma and to posttraumatic complications [63] [64] [65] [66] [67] ( table 4 .4). in addition to its pro-inflammatory role, il-6 also has anti-inflammatory properties. as an immunoregulatory cytokine, il-6 stimulates macrophages to release anti-inflammatory cytokines such as il-1 receptor antagonists and soluble tnf receptors [8] . moreover, il-6 induces macrophages to release prostaglandin e2 (pge2), the most powerful endogenous immune suppressant. pge2 regulates the synthesis of tnf and il-1β by macrophages and induces the release of il-10 [68] [69] [70] . overall, it has to be emphasized that almost all cytokines may not act strictly in either a pro-or anti-inflammatory manner, but rather may exhibit a "janus-faced behavior" depending on the underlying tissue, local environment, and trauma conditions. furthermore, the categorized pro-and anti-inflammatory cytokines follow not a specific temporal pattern but are rather synchronically and rapidly generated and released [1, 3] , mounting the overall inflammatory response. when the simultaneous cytokine response is excessive, prolonged, and dysregulated, this may lead to severe complications, such as organ dysfunctions [1] or persistent inflammation, immunosuppression, and catabolism syndrome (pics) [5] (fig. 4.3 ). reactive oxygen species are released by leukocytes after exposure to pro-and antiinflammatory cytokines, chemokines, complement factors, and bacterial products. (fig. 4.4) . ros cause lipid peroxidation, cell membrane disintegration, and dna damage to endothelial and parenchymal cells [72, 73] . furthermore, ros secreted by polymorphonuclear leukocytes (pmn) induce cytokines, chemokines [74] , heat shock protein (hsp) [75] , and adhesion molecules (p-selectin, icam-1) [76] leading to cell and tissue damage. early after severe tissue trauma, neutrophils migrate along the chemoattractant gradient of complement activation products, interleukins, and ros to the site of tissue damage and to remote organ tissue. neutrophil mobilization is important for wound healing and protection against invading microorganisms, but their immigration to remote organ tissue contributes to sirs [77] . neutrophil migration is composed of four steps: the first step, generation of leukocyte selectins (e.g., l-selectins) and e-and p-selectins on the endothelium is induced by anaphylatoxins (e.g., c5a), cytokines (e.g., il-6), and toxins [78] . these adhesion molecules are responsible for the rolling of neutrophils. the second step involves expression of integrins on neutrophils such as cd11 and cd18, and intercellular adhesion molecules (icam-1) and vascular cell adhesion molecules (vcam-1) on the surface of endothelial cells, all of which are strongly induced by c5a [79] [80] [81] . the interaction of these upregulated molecules activate neutrophils to reinforce the contact between neutrophils and endothelial cells (sticking). in the next step, migration and accumulation into tissues occur, mediated by chemokines and complement anaphylatoxins. to migrate through cellular barriers, neutrophils undergo significant deformational changes to permeate through small cellular gaps with the help of locally released matrix metalloproteinases. in the final step, activation of neutrophils occurs to protect against dangerous molecules, microorganisms, and cells. neutrophils utilize a large arsenal for forming the "first line of defense" after trauma: chemotaxis, phagocytosis, oxidative burst reaction with release of ros and myeloperoxidase (mpo), generation of no, leukotriens, plateletactivating factor (paf), tissue factor (tf), proteases, and multiple pro-inflammatory cytokines. however, the active substances released from neutrophils may not only harm the invading microorganisms or injured cells but also healthy host cells, especially since neutrophils become "long-lived" after trauma by significant inhibition of neutrophil apoptosis. thus, neutrophils after trauma function as "friend and foe". monocytes/macrophages and neutrophils play a central role for the innate host defense, tissue repair, and remodelling, and for the intermediaries to the antigen-specific adaptive immune response. monocytes are circulating precursors of macrophages. monocytes migrate into the different tissues (liver, spleen, lungs, etc.) even in absence of local inflammation and become tissue macrophages. when monocytes/macrophages are activated by various phagocytotic events in response to trauma, they regulate the activation of t and b lymphocytes, which induce antigen presentation by the major histocompatibility complex ii (mhc ii). monocytes/macrophages also release chemokines, cytokines (il-6, tnf, il-10, il-12, tgf-β), and various growth factors (fibroblast growth factor [fgf], epidermal growth factor [egf], and platelet-derived growth factors [pdgf] ) that initiate the formation of new extracellular matrix and promote angiogenesis and generation of new tissue at the site of injury. the functional phenotype shifts from a pronounced pro-inflammatory m1 type to a more anti-inflammatory and regenerative m2-type macrophage. the monocyte/macrophage cellular response after minor trauma embodies several beneficial effects for the host. however, major trauma induces massive monocyte/macrophage activation. in this state, the effects of the monocyte/macrophage response become systemic and may also induce detrimental effects. systemically, the macrophagemodulated immune response influences microcirculation, metabolism, and triggering and progression of remote organ injury. deactivation of monocytes and decreased expression of mhc ii on their surface are observed after major trauma correlating with the severity of injury [82] . natural killer (nk) cells are antigen-non-specific lymphocytes that recognize pathogen-associated molecular patterns (pamps) of invading microorganisms [83] as well as damaged, transformed, or virus-infected host cells [84] . since they are not dependent on pre-sensitization [85] to mediate their cytotoxic effects and to release excessive amounts of pro-and anti-inflammatory cytokines within minutes of stimulation, nk cells are regarded as part of the "first line of defense" [86] . their ability to release immune-modulatory cytokines may provide important regulatory functions during immune response, especially following severe injury. however, studies addressing the role of human nk cells after severe tissue trauma are rare and contradictory. some studies revealed an increase of nk cells in the early stage after severe trauma [77] , whereas nk cell function is greatly depressed by traumatic injury. however, there was no correlation between the nk cell count or activity and injury severity [85, 87] . concerning the effect of plasma samples from trauma patients on the cytotoxic activity of healthy nk cells in vitro, it has been shown that incubation times of more than 40 h lead to suppressed nk cell function, suggesting that posttraumatic immune suppression is associated with suppression of nk cell activity [85] . vice versa, murine experiments have collectively shown that nk cells as a key source of interferon γ exert harmful pro-inflammatory effects in the posttraumatic immune response and during the pathogenesis of sepsis [88, 89] . in support, early depletion of nk cells results in reduction of liver il-6 expression and a 50 % improved survival rate in a murine polytrauma model. lymphocyte apoptosis in spleen as well as neutrophil infiltration into lungs and liver is also attenuated [88] . furthermore, in various mouse models of sepsis, depletion of nk cells leads to improved survival [89, 90] suggesting that early posttraumatic activation of nk cells promotes amplification of the inflammatory response, and the subsequent loss of cellular functions might contribute to immune suppression manifested in later stages after trauma [87] . mechanisms of the development of organ dysfunction the initial trauma insult activates an inflammatory cascade that stimulates the host immune system. massive initial trauma impact (first hit) causes severe sirs. in this situation, the overwhelming production and release of pro-and anti-inflammatory mediators result in rapid mods and early death. an initial trauma insult of lower severity induces a moderate state of sirs/cars. in this instance, inflammatory and immune cells undergo some "priming". however, some patients develop posttraumatic complications, such as sepsis, aki, ards, and mods. the development of these complications is regulated by various exogenous and endogenous factors. among these factors, it is important to understand the relationship between the biological changes and the anatomical region of initial injury. the central nervous system is a rich source of inflammatory mediators. traumatic brain injuries (tbi) with the disruption of the blood-brain barrier (bbb) allow immune cells to migrate into the subarachnoid space, leading to an accumulation of leukocytes from the periphery [10, [91] [92] [93] . trauma to the chest area, particularly lung contusions, leads to an early increase in plasma mediators, which is associated with systemic inflammatory and anti-inflammatory reactions, such as pneumonia, ards, and mods [94] [95] [96] . patients with severe soft tissue injuries to the extremities with resulting hemorrhagic shock or severe muscle crush syndrome are at risk of developing more serious remote organ injury (e.g., aki). ischemia/reperfusion injury (i/r) leads to the production of large quantities of ros. femoral fractures with soft tissue injuries usually result in alteration of hemodynamic parameters such as increased cardiac output, tachycardia, decreased systemic vascular resistance, and decreased hepatic blood flow [97] . long bone fractures and unstable pelvic fractures are characterized by high blood loss and are associated with severe soft tissue injury, which initiate both a local and systemic inflammatory response [65, [98] [99] [100] [101] [102] . these bodies of evidence suggest that the initial trauma itself predisposes trauma patients to posttraumatic complications. traumatized patients who survive the initial injury ("first hit") may still be at risk of death from sepsis and multiple organ failure. secondary insults following the initial injury amplify the systemic inflammatory response and upset the balance of pro-and anti-inflammatory mediators, pro-and anti-coagulatory factors, pro-and anti-apoptotic events, and pro-and anti-regenerative processes. secondary insults ("second hits") are compounded by endogenous and exogenous factors. endogenous secondary insults include respiratory distress, cardiovascular instability, ischemia and reperfusion injury, and infection. exogenous secondary insults include surgical and anesthesiological interventions [103] [104] [105] , blood transfusions, and -not to forget -missed injuries. clinical studies have revealed that orthopedic surgical intervention can also cause major changes in the inflammatory response, and these changes are in proportion to the magnitude of surgery. for instance, femoral nailing induces an increase in systemic plasma levels of il-6 and il-10. in these patients, human leukocyte antigen-dr expression on monocytes is reduced as well [106, 107] . furthermore, reamed femoral nailing appears to be associated with greater impairment of immune reactivity than un-reamed nailing [107] . blood transfusions are a paramount therapy in the management of trauma/hemorrhagic shock patients. however, various studies have demonstrated that blood transfusions are associated with infection, sirs, ards, and mods after trauma [108] [109] [110] [111] [112] [113] , also representing a "second hit" for the multiply injured patient. ischemia/reperfusion (i/r) injury is a common and important event in clinical situations such as trauma, hemorrhagic shock, cardiac arrest (hypoxemia, hypotension of systemic tissue), contusions, lacerations, vascular injuries, and compartment syndrome (increased pressure in a preformed anatomical compartment with resulting hypoperfusion and hypoxemia of local tissue). inadequate microvascular flow results in the activation of leukocytes and converts local endothelial cells into a pro-inflammatory and pro-thrombotic phenotype. i/r injury consists of two specific stages. during the first stage of ischemia and hypoxemia, oxygen and nutrients are deprived from tissues temporarily by the disruption of blood supply. during the ischemic phase, the lack of oxygen leads to decreased production as well as consumption of adenosine triphosphate (atp). as consumption of atp continues, it is degraded into adenosine diphosphate (adp) and adenosine monophosphate (amp), which is further degraded to inosine and hypoxanthine [114] . atp depletion leads to an alteration in intercellular calcium and sodium concentration. it also results in the activation of cytotoxic enzymes such as proteases or phospholipases, all cumulating to reversible or irreversible cellular damage. the second stage of reperfusion is the revascularization or reestablished supply of oxygen to the ischemic tissue. the hallmark of the reperfusion phase is the generation of byproducts of neutrophil activation, which induces secondary tissue damage and organ dysfunction. on reperfusion with the reintroduction of molecular oxygen into the ischemic tissue, oxygen reacts with leukocytes and endothelial cells promoting the generation of reactive oxygen species and platelet-activation factor. the interactions of neutrophils and endothelial cells have been shown to contribute to massive interstitial edema caused by microvascular capillary leakage after reperfusion injury. the ischemia and reperfusion injury with atp depletion is a major cause for breakdown of physiological organ-blood barriers, such as bloodbrain, blood-gut, and blood-alveolus barrier. broken barriers characterized by diffuse microvascular leakage and tissue edema are thought to be main drivers of bacterial translocation (bt) and sepsis [115] . bacterial translocation is defined as the phenomenon of both viable and nonviable bacteria as well as their products (bacterial cell wall components, lps, and peptidoglycan) crossing the intestinal barrier to external sites such as the mesenteric lymph nodes, liver, and spleen. bt occurs as a result of a loss of integrity of the gut barrier function after trauma, hemorrhagic shock, and burns [116] , and may be associated with posttraumatic complications [117, 118] . although most data on bt and its complications have shown consistent results in animal models of hemorrhagic shock, trauma, and severe burns, its importance in humans is questionable, with variable results in clinical studies. in addition, it is still debatable whether bt is an important pathophysiologic event or simply an epiphenomenon of severe disease [119] . following trauma, acute inflammatory reactions may be triggered by infections (bacterial, viral, fungal, parasitic) and microbial toxins, or by any of several molecules released from necrotic tissue (hmgb1, hyaluronic acid, etc.). pattern recognition receptors (prrs), including tolllike receptors, can detect these stimuli and trigger a signaling pathway that leads to the production of various mediators. in the acute phase of trauma, vasodilatation is induced by vasodilatatory mediators (no, prostaglandins), quickly followed by increased permeability of the microvasculature. vasodilatation and extravasation of plasma result in hemoconcentration, facilitating the peripheral migration of neutrophils. neutrophil migration from the blood stream into interstitial tissue is divided into several steps, which are mediated by endothelial cell adhesion molecules, cytokines produced by monocytes/macrophages and various other cells, chemokines, the complement system, and arachidonic acid. migrated neutrophils produce several mediators such as neutral protease, reactive oxygen species (ros), lipids (leukotriene, paf), and tissue factor (tf). these mediators act as secondary tissue damage mediators and pro-coagulatory factors depending on the degree of initial injury as well as additional insults. during inflammation, the plasmaic cascade, consisting of the complement cascade, the kallikrein-kinin system, and the coagulation cascade, is activated by toxins and inflammatory mediators. activation of the complement system induces generation and depletion of complement activation products, causing an increase in vascular permeability, chemotaxis, opsonization, activation of the coagulation cascade, and trauma-induced complementopathy. excessive activation of the coagulation system results in a hypercoagulable state, leading to an acute trauma-induced coagulopathy (atic). activation of the kallikrein-kinin system results in kinins with vasoactive properties. in addition to its role in stimulating inflammation, the immune system (innate and adaptive) is a main driver for the barrier breakdown, clinically evident as diffuse microvascular leakage syndrome and organ failure. the exact knowledge of the pathophysiological changes after polytrauma is a prerequisite for effective, targeted, and patienttailored future therapies to support the immune and organ functions after severe tissue trauma. a genomic storm in critically injured humans definitions for sepsis and organ 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failure early predictors of postinjury multiple organ failure emerging risks and outcomes of blood transfusion in surgery transfusion of the injured patient: proceed with caution application of heme oxygenase-1, carbon monoxide and biliverdin for the prevention of intestinal ischemia/reperfusion injury broken barriers: a new take on sepsis pathogenesis bacterial translocation: clinical implications and prevention bacterial translocation-related mortality may be associated with neutrophil-mediated organ damage the gut: the 'motor' of multiple organ dysfunction syndrome? curr opin clin nutr metab care bacterial [correction of baterial] translocation in humans key: cord-023393-8nye3nc8 authors: krarup, a.; sørensen, u.; matsushita, m.; jensenius, j. c.; thiel, s. title: mannan‐binding lectin, l‐ficolin and h‐ficolin selectively binds to different bacteria date: 2008-06-28 journal: scand j immunol doi: 10.1111/j.0300-9475.2004.01423al.x sha: doc_id: 23393 cord_uid: 8nye3nc8 mannan‐binding lectin (mbl), l‐ficolin and h‐ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l‐ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type‐1, ‐8, ‐9, ‐11 and ‐12). h‐ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h‐ficolin initiated activation of complement factor c4, whereas l‐ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 μg of mbl/ml, 3.3 μg of l‐ficolin/ml and 18.4 μg of h‐ficolin/ml, respectively. the nuclear receptor heterodimers of liver x receptors (lxrs) are recently identified as key transcriptional regulators of genes involved in lipid homeostasis and inflammation. lxrs and their ligands are negative regulators of macrophage inflammatory gene expression. multiple sclerosis (ms), a demyelinating disease of the central nervous system of unknown cause, is characterized by recurrent inflammation involving macrophages and their inflammatory mediators. sweden belongs to the countries with a high ms incidence. in italy, incidence is lower, with an exception for sardinia where the incidence is even higher than that in sweden. subjects from sardinia are ethnically more homogeneous and differ from swedes, also regarding genetic background and environment. we studied lxrs and their related molecules of blood mononuclear cells (mncs) from female patients with untreated relapsing-remitting ms from sassari, sardinia and stockholm, sweden. sex-and age-matched healthy controls (hcs) were from both areas. mrna expression was evaluated by real-time pcr. lxr-a was lower (p < 0.05) in ms (mean ae sem: 3.1 ae 0.2; n ¼ 37) compared to hc (3.6 ae 0.1; n ¼ 37). lxr-a was lower in ms from stockholm (2.6 ae 0.2; n ¼ 22) compared to corresponding hc (3.4 ae 0.1; n ¼ 22; p < 0.01) and compared to ms (3.8 ae 0.2; n ¼ 15; p < 0.001) and hc (4 ae 0.2; n ¼ 15; p < 0.001) from sardinia. ms patients from stockholm, but not from sassari, also expressed lower (p < 0.05) lxr-b (à4.1 ae 0.4) compared to corresponding hc (à2.9 ae 0.3). ms from stockholm was associated with higher abca-1 (6.1 ae 0.4 versus 5.0 ae 0.3; p < 0.05) and higher estrogen receptor-b-cx (2.4 ae 0.4 versus 0.8 ae 0.4; p < 0.01) compared to corresponding hc. the hc from sassari had higher androgen receptor (2.9 ae 0.2) compared to ms from sassari (1.4 ae 0.3; p < 0.01), ms (1.3 ae 0.4; p < 0.01) and hc from stockholm (1.2 ae 0.3; p < 0.01). ms from sassari had lower cyclooxygenase-1 compared to corresponding hc (5.1 ae 0.4 versus 6.6 ae 0.3; p < 0.01) and lower prostaglandin-e (à0.03 ae 0.5) compared to the hc (1.4 ae 0.5; p < 0.05) and ms (2.7 ae 0.4; p < 0.05) and hc from stockholm (1.9 ae 0.4, p < 0.001). our findings identify lxrs and their related molecules as being involved in ms from stockholm but not from sassari, while sex hormone receptors seem to be involved in ms in sassari. multiple sclerosis: ifn-b induces cd123 + bdca2 -dendritic cells that produce il-6 and il-10 and have no enhanced type i interferon production y. m. huang, 1 s. adikari, 1 u. båve, 2 a. sanna 1,3 & g. alm 4 dc antigens (bdca) and investigate their ability to produce type i ifn in response to virus stimulation. we show that ifn-b induces development of cd123 þ dc from human blood monocytes, which coexpress bdca4 þ but are negative for bdca2 -, a specific marker for plasmacytoid dc. such ifn-b-modulated dc produce large amounts of il-6 and il-10, but no il-12p40 and have no enhanced ifn-b and ifn-b production. the findings indicate that ifn-bmodulated dc represent a myeloid dc subset with diminished cd11c, bdca-1 and cd1a expression, having potent th2-promoting function but lacking antiviral capacity. the association of psoriasis with throat infections by streptococcus pyogenes suggests a potential antigenic target for the t cells that are known to infiltrate dermis and epidermis of psoriatic skin. streptococcal m protein shares an extensive sequence homology with human epidermal keratins. keratins 16 (k16) and 17 (k17) are mostly absent from uninvolved skin but are upregulated in psoriatic lesions. there is increasing evidence that cd8 þ t cells play an important effector role in psoriasis and m proteinprimed t cells may recognize these shared epitopes in skin via molecular mimicry. to identify candidate epitopes, peptides with sequences from k17 were selected on the basis of predicted binding to hla-cw6 and sequence similarities with m6 protein. matched peptides from the sequence of m6 protein and a set of peptides with poor predicted binding were also selected. cw6 þ individuals with psoriasis and cw6 þ healthy controls, having a family history of psoriasis, were recruited. pbmcs were incubated with the peptide antigens. t-cell activation in the cd4 þ , cd8 þ and later the skin-homing cutaneous lymphocyteassociated antigen (cla)-expressing subset of cd8 þ t cells was evaluated by cd69 expression and intracellular ifn-g accumulation using flow cytometry. we demonstrate that cw6 þ psoriasis patients had significant cd8 þ t-cell ifn-g responses to peptides from k17 and m6 protein selected on the basis of sequence homology and predicted hla-cw*0602 binding. these responses were about 10 times more frequent in the skin-homing cutaneous lymphocyte-associated antigen-expressing (cla þ ) subset of cd8 þ t cells. cd4 þ t cells showed only borderline responses. cd8 þ t cells from cw6 þ nonpsoriatic individuals responded to some m6 peptides but very rarely to k17 peptides, and this also applied to the cla þ cd8 þ subset. these findings indicate that psoriatic individuals have cd8 þ t cells that recognize keratin self-antigens and that epitopes shared by streptococcal m protein and human keratin may be targets for the cd8 þ t cells that infiltrate psoriatic skin lesions. autoantibodies directed against citrulline-containing proteins have an impressive specificity of nearly 100% in ra patients and a suggestive involvement in the pathogenesis. the targeted epitopes are generated by a post-translational modification catalysed by the calcium-dependent enzyme peptidyl arginine deaminase that converts the positively charged arginine to polar but uncharged citrullin. the aim of this study was to analyse the presence of citrulline in the joints at different time points of collagen-induced arthritis in da rats by immunohistochemistry and to investigate how immunogenicity and arthritogenicity was affected by citrullination of rat serum albumin (rsa) and collagen type ii (cii). our results indicate that citrulline could be detected in joints of arthritic animals, first appearance at the onset of disease and increasing as disease progressed into a chronic state. unimmunized animals or time points before clinical signs of arthritis were negative. by morphology, we state that some infiltrating macrophages as well as the cartilage surface stain positive for citrulline, while the major source of citrullinated proteins appears to be fibrin depositions. a specific cit-rsa t-cell response was observed in animals challenged by citrullinated rsa, no response was recorded when rsa was used as a stimulus. the igg analysis reveals not only a response towards the modified protein but also cross-reactivity to native rsa. no t-cell or b-cell response was noted in animals injected with unmodified rsa. cit-cii induced a disease with higher incidence and earlier onset than did the native counterpart. we conclude that, in contrast to the human disease, citrulline does not seem to appear before clinical signs. as inflammation proceeds, citrulline is detected specifically in the joints. all other organs investigated were negative. we also conclude that citrullination of a protein can break tolerance and increase its arthritogenic properties. ectopic germinal centers (gcs) can be detected in the salivary glands of approximately 1/5 of patients with sjögren's syndrome (ss) and appear in both primary and secondary ss. previously, ectopic gc have been associated with increased local autoantibody production. the aim of this study was to determine whether gc in primary sjögren's syndrome (pss) defines a distinct seroimmunological phenotype. retrospectively, a material of 130 haematoxylin and eosin-stained paraffin-embedded tissue sections of minor salivary gland tissue from patients with pss was morphologically screened for the presence of ectopic gc. gc-like lesions were detected in 33/130 (25%) of the pss patients. seventy-two pss patients lacking these structures (gc-) were randomly selected for comparison. focus score was significantly increased in the gc þ patients compared to the gcpatients (p ¼ 0.035). in the gc þ group, 54.5% of the patients presented with anti-ro/ssa compared to 43.7% in the gcgroup. anti-la/ssb was detected in 31.3% of the gc þ patients compared to 25.7% of the gcpatients. sixty-one percentage of gc þ patients presented with increased levels of igg, a nonsignificant difference when compared to 39.4% in the gcpatients (p ¼ 0.089). levels of rf, ana, ena, igm and iga were similar in both patient groups, as were esr and crp. in conclusion, patients with ectopic gc have a higher focus score and more often present with autoantibodies and increased levels of igg compared to pss patients with regular focal infiltration (gc -). our findings may indicate a certain seroimmunological phenotype and warrant for further prospective studies. association between mannose-binding lectin and vascular complications in type 1 diabetes complement activation and inflammation have been suggested in the pathogenesis of diabetic vascular lesions. we investigated serum mannose-binding lectin (mbl) levels and polymorphisms in the mbl gene in type 1 diabetic (t1dm) patients with and without diabetic nephropathy and associated macrovascular complications. polymorphisms in the mbl gene and serum mbl levels were determined in 199 t1dm patients with overt nephropathy and 192 t1dm patients with persistent normoalbuminuria matched for age, sex and duration of diabetes as well as in 100 healthy control subjects. the frequencies of high and low expression mbl genotypes were similar in patients with t1dm and healthy controls. high mbl genotypes were significantly more frequent in diabetic patients with nephropathy than in the normoalbuminuric group, and the risk of having nephropathy, given a high mbl genotype, assessed by odds ratio was 1.52 (1.02-2.27), p ¼ 0.04. median serum mbl concentrations were significantly higher in patients with nephropathy than in patients with normoalbuminuria [2306 mg/l (iqr 753-4867 mg/l) versus 1491 mg/l (iqr 577-2944), p ¼ 0.0003], and even when comparing patients with identical genotypes, serum mbl levels were higher in the nephropathy group than in the normoalbuminuric group. patients with a history of cardiovascular disease had significantly elevated mbl levels independently of nephropathy status [3178 mg/l (iqr 636-5231 mg/l) versus 1741 mg/l (iqr 656-3149 mg/l), p ¼ 0.02]. the differences in mbl levels between patients with and without vascular complications were driven primarily by pronounced differences among carriers of high mbl genotypes (p < 0.0001). our findings suggest that mbl may be involved in the pathogenesis of microvascular and macrovascular complications in type 1 diabetes and that determination of mbl status might be used to identify patients at increased risk of developing these complications. neuroimmunology unit, center for molecular medicine, karolinska institutet, stockholm, sweden. e-mail: judit.wefer@cmm.ki.se dna vaccine coding for the encephalitogenic peptide mog 91-108 protects lew.1av1 from subsequent development of experimental autoimmune encephalomyelitis (eae). protection is associated with a type 1 immune response and is dependent on the presence of cpg dna motifs. the mechanisms underlying the observed reduction of eae development in protected rats have not been fully clarified. we investigated immunological characteristics of lymphocytes after dna vaccinaton and subsequent eae induction. we confirm that protection was not associated with suppression of t1 cells, as transcription of the novel molecule rat t-cell immunoglobulin-and mucindomain-containing molecule (tim-3), reported to be exclusively expressed on differentiated t1 cells, was not altered by dna vaccination. we did not note any clonal deletion upon tolerization, but detected an antigen-specific lymphocyte population upregulating ifng upon recall stimulation 3 weeks after protective dna vaccination. in protected rats, we observed (1) no alterations in antigenspecific th2 or th3 responses, (2) reduced mhc ii expression on splenocytes early after eae induction, (3) antigen-specific upregulation of ifnb upon recall stimulation and (4) reduced il-12rb2 on lymphocytes. we thus demonstrate an association of the protective effect of dna vaccination with expression of ifnb. we are currently investigating the cellular mechanisms behind this ifnb-mediated protection. multiple sclerosis (ms) is an autoimmune condition characterized by degeneration of nerve fibre myelin sheets. a candidate autoantigen, myelin basic protein (mbp), has especially attracted attention. the presence of anti-mbp antibodies is a predictor of definite ms, but their role in the pathogenesis remains obscure. t cells have long been known to play a pivotal role in the pathogenesis of ms. recently, an important role for b cells as autoantigen-presenting cells has been demonstrated in other autoimmune diseases, including rheumatoid arthritis and diabetes. the uptake of mbp by b cells and the presentation of mbp-derive peptides to t helper (th) cells by b cells may be promoted by the formation of complement (c) activating immune complexes (ics) between mbp and natural autoantibodies in healthy individuals and disease-associated anti-mbp antibodies in ms patients, respectively. we have investigated the formation of mbp-containing ic, the binding of mbp to b cells, the mbp-elicited induction of th-cell and b-cell proliferation and the cytokine production in peripheral blood mononuclear cells (pbmcs) from healthy donors grown in the presence of intact or c-inactivated serum from healthy donors or patients with ms. while mbp did not induce measurable proliferation of b cells nor cd4 þ t cells, we observed the production of tnf-a, ifn-g and il-10 by pbmc in response to incubation with mbp in the presence of sera from healthy controls as well as sera from ms patients. by contrast, no production of il-2, il-4 and il-5 was detected. we are currently investigating the capability of ms sera to promote the formation of mbp-containing ic and thereby enhance the cytokine responses, by virtue of elevated anti-mbp contents. the phagolysosomally localized acid sphingomyelinase (asmase) activated by proinflammatory cytokines such as tnf and ifn-g generates the signalling molecule ceramide which in turn results in the activation of proteases like cathepsin d. these characteristics of asmase suggest a possible role of this molecule in the phagocytotic uptake and phagosomal degradation processes of antigens or in antigen presentation. we show here that asmase -/mice fail to eliminate the noncytopathic lymphocytic choriomeningitis (lcm) virus as rapidly as littermate wildtype mice. investigation of the immune response revealed a reduced expansion of cd8 þ t cells. the secretion of ifn-g in response to contact with target cells as well as the cytolytic activity of virus-specific cd8 þ t cells was severely impaired. additionally, both phases of the lcm virus-specific dth response, mediated by cd8 þ and cd4 þ t cells consecutively, were diminished in asmase -/mice. however, the secondary memory response of virus-specific ctl was not altered, and the 614 abstracts ................................................................................................................................................................................................. virus was effectively controlled for at least 3 months by asmase -/mice. in conclusion, the results of this study suggest an involvement of the asmase in the activation, expansion or maturation of virus-specific cd8 þ t cells during the acute infection of mice with the lcm virus. novel markers for alternative activation of macrophages: macrophage galactose-type c-type lectins 1 and 2 in parallel with the th1/th2 dichotomy, macrophages are capable of developing into functionally and molecularly distinct subpopulations, due to differences in, for example cytokine environment and pathological conditions. while the best-studied, classically activated macrophage is induced by type i stimuli such as ifn-g, a type ii cytokine environment antagonizes the classical activation of macrophages and is capable of alternatively activating macrophages. however, molecular markers associated with these type ii cytokine-dependent, alternatively activated macrophages remain scarce. besides the earlier documented markers macrophage mannose receptor and arginase 1, we recently demonstrated that murine alternatively activated macrophages are characterized by increased expression of fizz1 and ym. we now report that expression of the two members of the mouse macrophage galactose-type c-type lectin gene family, termed mmgl1 and mmgl2, is induced in diverse populations of alternatively activated macrophages, including peritoneal macrophages elicited during infection with the protozoan trypanosoma brucei or the helminth taenia crassiceps, and alveolar macrophages elicited in a mouse model of allergic asthma. we also demonstrate that, in vitro, interleukin-4 and interleukin-13 upregulate mmgl1 and mmgl2 expression and that, in vivo, induction of mmgl1 and mmgl2 is dependent on interleukin-4 receptor signalling. moreover, we show that regulation of mgl expression is similar in human monocytes and monocyte-derived macrophages. hence, macrophage galactose-type c-type lectins represent novel markers for both murine and human alternatively activated macrophages; thus, paving the way for further characterization of the phenotype of macrophages occurring in th2 conditions. background: human parvovirus b19 (b19) is a ubiquitous pathogen, normally causing a mild self-limiting disease, but also capable of causing both significant pathology and long-term persistence. the small size and stability of the virus makes it suitable for mapping of the full breath and the kinetics of the cellular immune responses following acute viral infection. methods: five patients with acute primary b19 infection were included in the study and followed consecutively for up to 200 weeks. cellular immune responses were mapped by ifng enzyme-linked immunospot to overlapping peptides spanning the whole b19 genome. results: in all five acutely infected patients, we were able to monitor the kinetics of a strong specific cellular immune reaction. responses peaked at levels of 850-1850 sfc/ million pbmcs, roughly corresponding to 0.3-0.6% b19specific cd8 þ cells circulating in peripheral blood at 10-80 weeks post-infection. the responses in individual patients were directed to three or four different peptide pools, and the specificity was confined to the same cd8 epitopes present in the pools throughout the follow-up period. the majority of responses were directed to the virus nonstructural protein, only two patients showed any response to the capsid proteins, elicited by the same epitope in both cases. conclusion: the cellular immune responses to acute b19 infection are surprisingly narrow in distribution and remain at high levels for up to 80 weeks post-infection. the initial epitope specificity is maintained, and the majority of responses target the virus nonstructural protein, which is not included in vaccine preparations, evaluated against the infection. the relationship between malnutrition and malaria is controversial. on one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. we investigated the association between plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of kenya. the study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. we used poisson regression analysis to investigate the association between malaria and nutritional status. compared to baseline (children with a waz or haz score of !à2), the crude incidence rate ratios (irrs) for malaria in children with low haz or waz scores (<à2) during the period prior to assessment were 1.17 (95% ci 0.91-1.50; 0 ¼ 0.21) and 0.94 (0.71-1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of pem. however, we found that age was acting as an effect modifier in the association between malaria and malnutrition. the irr for malaria in children 0-2 years old who were subsequently characterized as wasted was 1.65 (1.10-2.20; p ¼ 0.01), and a significant overall relationship between malaria and low-haz was found on regression analysis when adjusting for the interaction with age (irr 1.89; 1.01-3.53; p < 0.05). although children living on the coast of kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life. a. astrinidou-vakaloudi, 1 s. xytsas, 1 i. diamanti, 1 h. ioannidis 2 & p. pangidis 2 1 microbiology department of general hospital of thessaloniki 'agios pavlos', thessaloniki, greece, and 2 nefrology, 2 nd ika hospital of thessaloniki, thessaloniki, greece. e-mail: stasa@hol.gr aim: renal dysfunction may influence the colonization of gastric mucosa by urea-splitting bacteria such as helicobacter pylori, by increasing urea concentrations in the gastric juice. our aim was to investigate the prevalence of h. pylori in patients with end-stage renal disease (esrd), receiving long-term haemodialysis treatment. methods: this study included 40 sera from patients with esrd (29 male and 11 female) undergoing periodic haemodialysis; mean time of treatment was 42.6 months. using elisa technique, we investigated the presence of igg and iga antibodies against h. pylori as well as igg caga (antibodies specific for caga(þ) strains of h. pylori). sera from 40 healthy blood donors were used as a control group. results: h. pylori igg antibodies were detected in 32 out of 40 (80%) patients in the dialysis group, while 31/40 (77.5%) tested positive for iga. igg caga antibodies were present in 13 out of 40 (32.5%). prevalence of h. pylori igg, iga and caga igg antibodies in the control group was 33, 7 and 15%, respectively. conclusions: although international data suggest that prevalence of h. pylori infection is the same in esrd patients as in healthy individuals, in our study that seems not to be the case. the higher blood and gastric juice urea levels may be a risk factor (among many others), but more studies are required in order to understand the relation of h. pylori infection in this group of patients. flanders interuniversity institute for biotechnology, department of molecular and cellular interactions, free university of brussels, brussels, and 2 pasteur institute of brussels, mycobacterial immunology, brussels, belgium. e-mail: tgartner@vub.ac.be immunity against tuberculosis (tb), caused by mycobacterium tuberculosis, depends largely on activation and maintenance of strong cell-mediated immune responses involving both cd4 þ and cd8 þ t cells and the ability to respond with th1-type cytokines, particularly ifn-g. recent studies suggested that bcg, the only licensed vaccine against m. tuberculosis, may fail to induce t-cell responses in the lung mucosa and may therefore not protect against pulmonary tb. a decrease in tb mortality may be achieved by enhancing immunity in the lung. the present study evaluated the induction of antigen-specific immunity in the lung by intranasal (i.n.) delivery of the lipoprotein i (opri) from pseudomonas aeruginosa. opri has shown to be a toll-like receptor 2/4 agonist that, when given subcutaneously, induces type-1 immune responses against heterologous antigens. here, a fusion of opri to ag85a of mtb (opri-ag85a) was used as a subunit vaccine in homologous prime-boost immunizations. in addition, opri-ag85a was combined with an ag85a-encoding dna vaccine (ag85a dna) or with bcg in heterologous prime-boost vaccinations. intranasal and parenteral delivery with opri-ag85a elicited comparable t-cell responses in the spleen; in addition, i.n. delivery elicited specific t-cell responses in the lung lymph nodes (llns). intramuscular delivery of ag85a dna induced significant systemic th1 immune responses. intranasal boosting with opri-ag85a enhanced this response and in addition induced an antigen-specific ifn-g response in lln. opri may therefore be an efficient adjuvant for mucosal boosting. we continue to evaluate the protection induced by opri-based prime-boost vaccinations against pulmonary tb. results on the immunogenicity and protection against intravenous mtb h37rv infection will be presented. toll-like receptors (tlrs) are pattern recognition receptors of the innate immune system, which recognize molecular structures on pathogens or cellular stress-associated molecules. tlr-ligand interactions trigger activation of inflammatory signal transduction and expression of genes involved in host defense. in this study, we have examined the requirement for different tlr adaptor molecules in virus-induced chemokine expression and are currently trying to identify the tlr involved. we have found that both a herpesvirus [herpes simplex virus (hsv)] and a paramyxovirus (sendai virus) require a functional genome to induce expression or proinflammatory chemokines in human and murine monocytic cell lines. for both viruses, this is independent of the tlr adaptor molecules trif and mal. however, overexpression of the vaccinia virus-encoded inhibitor of tlr-signalling a52r or dominant-negative myd88 totally inhibited hsv-induced rantes expression but only partially prevented sendai virus from inducing this chemokine. this suggests that hsv-induced rantes expression occurs via a tlr pathways, whereas sendai virus utilizes both tlr-dependent and -independent pathways to stimulate expression of rantes. we are currently trying to identify the tlrs involved. data from these studies will also be presented at the meeting. 2 0 -5 0 -oligoadenylate synthetases are interferon-induced, double-stranded rna-activated antiviral enzymes which are the only proteins known to catalyse 2 0 -specific nucleotidyl transfer. this first crystal structure of a 2 0 -5 0oligoadenylate synthetase reveals a structural conservation with the 3 0 -specific poly(a) polymerase that, coupled with structure-guided mutagenesis, supports a conserved catalytic mechanism for the 2 0 -and 3 0 -specific nucleotidyl transferases. comparison with structures of other superfamily members indicates that the donor substrates are bound by conserved active site features while the acceptor substrates are oriented by nonconserved regions. the 2 0 -5 0oligoadenylate synthetases are activated by viral doublestranded rna in infected cells and initiate a cellular response by synthesizing 2 0 -5 0 -oligoadenylates, that in turn activate rnase l. this crystal structure suggests that activation involves a domain-domain shift and identifies a putative dsrna activation site that is probed by mutagenesis. we demonstrated that this site is required both for the binding of dsrna and for the subsequent activation of oas. this rna-binding site is different from known rna-binding site; rather than forming a defined three-dimensional domain, it is located at the interface of the two major domains in oas. this novel architecture ensures that the dsrna helix can make simultaneously contact with both domains of oas and ensure the subsequent structural rearrangement leading to the activation of oas. our work provides structural insight into cellular recognition of double-stranded rna of viral origin and identifies a novel rna-binding motif. bacteria-specific iga antibodies are efficient opsonins for neutrophils and mononuclear phagocytes, provided that the phagocytes express the fca receptor (cd89). expression of cd89 can be stimulated by inflammatory cytokines, activated complement factors and certain microbial components. in one study, unstimulated phagocytes were able to ingest iga antibody-treated pneumococci, but only in the presence of complement, which was found to be activated by the iga antibodies along the alternative pathway. pneumococci produce iga1 protease that cleaves human iga1, but not iga2, molecules in the hinge region. this leaves iga1 as faba (monovalent) deprived of fca which contains the docking site for cd89. iga1 is the vastly predominant subclass of iga in the upper airways and circulation of humans. aims: to examine the effects of iga1 protease activity and complement on phagocytosis of iga antibody-coated pneumococci by an unstimulated human phagocytic cell line (hl60). materials and methods: iga1 and iga2 monoclonal antibodies to serotype 4 pneumococcal capsular polysaccharide (ps) were generated by heterohybridoma technique involving b cells from human vaccinees. isogenic serotype 4 pneumococci with and without iga1 protease activity, respectively, were obtained after inactivation of the iga gene of the tigr4 strain. opsonophagocytosis was quantitated using the assay described by romero-steiner et al. based on enumeration of surviving bacteria by culture. the integrity of iga molecules was examined by western blotting. results: both iga1 and iga2 antibody to type-4 polysaccharide-induced phagocytosis of iga1 protease-deficient type-4 pneumococci equally well in the absence as in the presence of complement. iga1 antibody to type-4 polysaccharide displayed a fourfold higher opsonophagocytosis titer against iga1 protease deficient compared to homologous wildtype target bacteria. a similar effect of iga1 protease activity of the target bacteria was not observed in a parallel experiment where iga2 antibody to type-4 polysaccharide served as opsonin. iga1 antibody extracted from iga1 protease-producing target bacteria was almost exclusively in the form of faba. conversely, iga1 from protease-deficient bacteria and iga2 from both types of bacteria were intact. conclusions: these results indicate that the iga1 protease activity of s. neumoniae may help the bacteria escape iga1 antibody-mediated opsonophagocytosis. besides, in these experiments, iga-mediated opsonophagocytosis was independent of complement. vitamins e and c have been found to increase the cellular and humeral immunity of pigs. vitamin e deficiency has also been found to predispose pigs to different diseases, e. coli infection is one among them. after weaning, the vitamin e status of pigs often decreases to a critical low level. in this experiment, we studied whether vitamin c supplementation would be a possible feeding strategy to optimize the immune status of weaners. the interaction between vitamin e and c is interesting due to the reported sparing action on vitamin e or synergism between these to vitamins. piglets were weaned at day 28 of age from sows fed increasing dietary vitamin e during lactation, and piglets were during the following 3 weeks fed either a control diet or this diet supplemented with 500 mg stay-c per kg. blood sampling was obtained weekly from day 28 and until day 49 of age. on the same days, one piglet per dietary treatment was killed and alveolar macrophages (am) were harvested. vitamin c supplementation increased the concentration of igm in serum of piglets throughout the weaning period. although the vitamin e concentration in am decreased with increasing age of the piglets, the concentration was numerically higher in piglets of sows fed the high dietary level of vitamin e. however, vitamin c supplementation tended to increase the total am concentration of vitamin e after weaning and increased the proportion of the biologically most active isomer of vitamin e [rrr-(a-tocopherol)] in the am. the eicosanoid synthesis by am was not influenced by the vitamin c supplementation, but the synthesis of leukotriene b4 was decreased 2 weeks after weaning compared to other days of am harvesting. in conclusion, dietary vitamin c supplementation improved the immune responses of piglets after weaning. a whole blood stimulation assay with escherichia coli (o111:b4) endotoxin was established to measure the capacity of dairy cows to produce the proinflammatory cytokine tumour necrosis factor-a (tnf-a) ex vivo. initially, a time-and dose-dependent study was carried out to find the optimal stimulation conditions for the tnf-a response. the tnf-a response peaked between 3 and 4 h at 38.5 c. a dose in the range of 5-10 g of e. coli lipopolysaccharide (lps)/ml whole blood was found to give the maximum tnf-a response. thirty-eight danish-holstein dairy cows were investigated for their tnf-a responsiveness ex vivo in the periparturient period. heparin-stabilized blood samples were collected seven times over a period of 4 months (weeks à3, à1, 2, 3, 5, 9 and 13 around calving) and stimulated with 5 g/ml of e. coli lps. indeed, fluctuations in the tnf-a responsiveness occurred over time. moreover, the mean tnf-a responsiveness of 38 cows was found to be significantly increased (p < 0.001) in the weeks close to calving. however, in the more stabile physiological periods, some cows had a consistently low tnf-a response, whereas others had high a tnf-a response. we are currently investigating whether high and low tnf-a responders to e. coli lps also exist in dairy cows in vivo. moreover, the importance of tnf-a responsiveness ex vivo to dairy cows' susceptibility and clinical response to experimental e. coli infections in the udder is being investigated. coelomic cytolytic factor (ccf) is a 42 kda invertebrate pattern recognition molecule isolated from the coelomic fluid of the earthworm eisenia foetida (oligochaeta, annelida). ccf displays a number of similarities with the mammalian cytokine tumour necrosis factor-a (tnfa) as a result of a shared n,n 0 -diacetylchitobiose lectin-like domain. however, these similarities are solely functional and are not based on any (dna or amino acid) sequence homology, thus suggesting a form of convergent evolution. in particular, the lectin-like domain of tnf-a has been shown to induce membrane depolarization in various mammalian cell types, through interactions with endogenous amiloride-sensitive ion channels. this nonreceptor-mediated activity of tnf-a has been reported to be involved in the resorption of oedema. likewise, the lectin-like domain of ccf also induces membrane depolarization in mammalian cells. here, we show that ccf appears to be able to induce oedema resorption in an alveolar epithelial cell line through its lectin-like domain. this lectin-like domain of ccf interacts (directly or indirectly) with endogenous sodium and/or chloride channels, and not potassium channels, on mammalian cells. additionally, we suggest that the jnk/sapk and erk1/2 pathways are involved in ccf-induced macrophage activation. these results further establish the functional analogy between an invertebrate pattern recognition molecule and a mammalian cytokine and, from a more applied point of view, suggest the possibility of utilizing ccf in the treatment of oedema. release of svegf and svegfr1 from white blood cells and platelets during surgery and stimulation with bacterial antigens introduction: the influence of surgery on release of soluble vascular endothelial growth factor (svegf) and the soluble vascular endothelial growth factor inhibitory receptor 1 (svegfr1) is unknown. we studied the effect of major and minor surgery on potential variations in svegf and svegfr1 concentrations in vivo and on bacterial antigen-induced release of svegf and svegfr1 from whole blood in vitro. methods: sixty-one patients with abdominal diseases undergoing five different surgical procedures were included. blood samples were drawn from anaesthetized patients before and after the operation. white blood cells and platelets were counted, and plasma svegf and svegfr1 was determined by an elisa method. whole blood from each blood sample was stimulated in vitro with bacteria-derived antigens (lps or protein-a) and svegf and svegfr1 levels were subsequently determined in the supernatants. stimulation with isotonic saline served as control assay. neither svegf or svegfr1 in plasma changed during surgery. in vitro stimulation of blood samples with bacteria-derived antigens resulted in a significant increase in svegf (p < 0.0001) and a less pronounced but still significant increase in svegfr1. release of svegf due to stimulation was significantly higher after the operation (nonsignificant), whereas svegfr1 release remained largely unchanged after surgery. correlation between bacterial antigen-induced release of svegf and neutrophile cell count was highly significant (p < 0.0001). there was no correlation between svegf and platelet cell count, and bacterial antigen-induced svegfr1 release did not correlate with counts of neutrophils and platelets. conclusions: plasma svegf and svegfr1 concentrations did not change during surgery. in vitro bacterial stimulation led to increased release of svegf and svegfr1, which was not significantly amplified during surgery and which may be related to number of circulating neutrophils. natural killer cell functions and subsets after in vitro stimulation with il-2 and il-12, with special emphasis on intracellular ifn-g and nk-cell cytotoxicity r. nyboe, 1,2 t. rix, 1,2 j. krog, 1,2 e. tønnesen 1 & m. hokland 2 1 department of anaesthesiology and intensive care, aarhus university hospital, and 2 institute of medical microbiology, and immunology, university of aarhus, aarhus, denmark. e-mail: rnsr@studmed.au.dk materials and methods: isolated cryopreserved human peripheral blood mononuclear cells (pbmcs) were stimulated with il-2 and il-12. this stimulation has previously been shown to activate nk cells. cell cytotoxicity was measured by flow cytometry after incubation with k562 cells. this method was compared to the current standard 51cr release assay. cells were treated with bfa to accumulate ifn-g, stained for surface markers, permeabilized and stained for intracellular ifn-g. flow cytometry was then performed to measure intracellular ifn-g production in pbmc, especially in nk cells. results: we have demonstrated that stimulation with il-2 and il-12 is effective in increasing the number of ifn-gpositive cells. there is a distinct difference between the cd3-cd56dim and the cd3-cd56bright subsets, with a much greater proportion of ifn-g-positive cells in the cd3-cd56bright subset. the effects of stimulation with il-2 and il-12 on cytotoxicity will be presented, as will the relation between ifn-g production and cytotoxicity. in addition, we will present results of these assays applied to porcine cells. discussion: in combination, these tests will address nk cell function by combining cytotoxicity with ifn-g production in nk cell subsets. the results will demonstrate whether this could serve as a useful tool in describing nkcell function, which could be of value in clinical and experimental settings. culture of regulatory t-cell lines from bronchial mucosa t lymphocytes play a major role in many immune responses. in the last decade, special focus has been on the function of th1 and th2 effector cells. now the importance of regulatory cd4 þ cd25 þ t cells in maintenance of the immunological homeostasis emerges. sarcoidosis is a multisystem granulomatous disorder often affecting the lungs. the typical sarcoid granulomas consists of epitheloid cells, macrophages and lymphocytes, mainly cd4 þ t cells of th1 phenotype. we have cultured t cells from bronchial biopsies of patients with sarcoidosis as well as from controls in high levels of interleukin 2 (il-2) and il-4 and demonstrate spontaneously arising cd4 þ cd25 þ populations and high concentrations of il-10 in these cultures. the main difference between cultures of sarcoid origin compared to controls is a very much higher concentration of the inflammatory cytokines il-6 and tnf-a in cultures of sarcoid origin. the effects of hyperbaric exposure on human peripheral blood mononuclear cells, with special emphasis on natural killer cell cytotoxicity and subsets materials and methods: as an experimental physiological stress model, we examined the effects of hyperbaric exposure on peripheral blood mononuclear cells (pbmcs) obtained from venous blood drawn from eight divers during a simulated heliox saturation dive. eight persons working in normobar atmosphere outside the pressurized chamber served as control donors. the spontaneous cytotoxicity of the pbmcs was estimated in a 4 h 51cr-release assay using k562 as nk-sensitive target cells. the pbmcs were characterized, using 4-colour flow cytometry, with special emphasis on the nk-cell subsets. the data were statistically analysed using a multivariate regression model (stata 8.2). p values <0.05 was considered statistically significant. results: the estimated cytotoxicity increased significantly in both the group of divers and control donors during the dive (pdivers < 0.01 and pcontrols < 0.01). although the cytotoxicity increased relatively more (p < 0.01) in the group of divers compared to the group of control donors between day 1 and 2. discussion: the increased cytotoxicity of pbmc estimated in the group of divers indicate that parts of the cellular immune system are affected during the extreme physiological conditions induced during the initial phase of the presented experimental hyperbaric setup. the increase in cytotoxicity observed in the group of control donors could hypothetically reflect the stress level in persons working outside the pressurized chamber during the dive. the interaction with adhesion molecules expressed by vascular endothelium is the first step in lymphocyte infiltration into tissues. at both cutaneous and mucosal sites interleukin-10 (il-10), il-12 and transforming growth factor (tgf)-b are important regulators of chronic inflammatory disease, where cutaneous lymphocyteassociated antigen (cla) and ae integrin (cd103) may be expressed. unlike cla, cd103 is not believed to play a role in tissue-specific homing but may help to retain t cells within epithelial layers. we have previously shown that il-12 alone can together with an unknown cofactor increase the expression of cla. stimulation with streptococcal pyrogenic exotoxin c (spec) increased the expression of cd103 by cd8 þ but not cd4 þ t cells. while il-12 increased superantigen-stimulated expression of cla, this cytokine strongly inhibited the cd103 expres-sion, and a combination of il-12 and tgf-b completely abrogated the induced cd103 expression. conversely, il-10 suppressed cla but increased cd103 expression. these findings indicate that, in addition to suppressing the development of th1-mediated inflammatory responses, il-10 may also inhibit the migration of cd8 þ t cells into the skin while il-12 promotes such migration. thus, the expression of cla and cd103 may be antagonistically regulated by il-10 and il-12, and the balance between these cytokines could influence the t-cell migration of inflammatory cells into epithelial tissues. during contact sensitivity reaction, immune cells proliferate. in order to study the histological picture of these proliferation phases, we used a mouse model of contact sensitivity in the oral mucosa and on skin. we also used bromodeoxyuridin (brdu, an analogue to thymidin) that is incorporated into the nucleus during cell replication. the hapten oxazolone (oxa) was used to sensitize and elicit the oral mucosa and/or the ear skin. mice were killed at various times after elicitation, and unsensitized animals were also exposed to the hapten as controls. brdu (25 mg/ kg animal) was injected i.p. 2 h before the kill. specimens from the oral mucosa, ear skin and submandibular and auricular lymph nodes were cut and fixed in 4% paraformaldehyde. they were then treated with acid and biotinylated anti-brdu antibody and developed using abc-kit and dab. the analyses were performed using a leica light microscope and the computer program analysis. in the oral mucosa, the frequency of proliferating cells were increasing during the observation period, 4-24 h after elicitation, regardless of site of sensitization. the proliferating cells were found mainly in the basal cell layer of the epithelium. similar patterns were found in ear skin. the regional lymph nodes demonstrated a few scattered proliferating cells 4 h after elicitation. after 24 h, these cells were found frequently in the whole lymph node. control animals exhibited considerable less proliferating cells at all times. we conclude that most proliferating cells were found 24 h after elicitation locally at the hapten-exposed sites (the oral mucosa or the ear skin) as well as in the regional lymph nodes. the endogenous nucleoside adenosine is released in excess during inflammation or other metabolic stress and is generally known to deliver tissue protective anti-inflammatory effects. adenosine acts via four adenosine receptors of which the a2a receptor is the predominant form in t cells. adenosine levels are elevated in asthmatic lung, and adenosine can directly induce mast cell degranulation and bronchoconstriction in these patients. instead, the role of anti-inflammatory mechanisms of adenosine on t cells in asthma is unclear. aim: to study the a2a receptor expression in peripheral blood cd4 þ t cells in asthmatic and healthy individuals using flow cytometric and quantitative real-time pcr methods. results: unstimulated cd4 þ cells of asthmatic patients expressed significantly lower levels (p < 0.001) of a2a receptor in protein level (mean percentage of cells positive ae sem: 76.8 ae 1.2, n ¼ 6) compared to healthy individuals (90.4% ae 1.9, n ¼ 4). double staining for cd69 expression showed that stimulation of cd4 þ cells decreased a2a expression in both groups but indicated that the detected lower levels of a2a in unstimulated cells of asthmatics was not due to preactivation in these patients. surprisingly, a2a mrna expression in unstimulated cd4 þ cells was significantly higher (p < 0.05) in asthmatics (n ¼ 28) compared to healthy controls (n ¼ 7). the expression did not correlate with serum total ige levels. conclusions: asthmatic individuals express less a2a adenosine receptor on their peripheral cd4 þ t cells. the higher mrna levels instead may point to a negative feedback regulation in the receptor expression. the role of possibly decreased adenosine-mediated anti-inflammatory effects in asthma pathogenesis require further studies on this t-cell mediated disease. the chronic inflammatory skin disease atopic eczema (ae) affects almost 15% of the population in many countries today. the pathogenesis of ae is not fully understood. a combination of genetic predisposition and environmental factors like microorganisms seems to contribute to the symptoms. the yeast malassezia sympodialis is part of our normal skin micro flora but can act as an allergen and elicit specific ige and t-cell reactivity in patients with ae. recently, we identified a novel major m. sympodialis allergen, designated mala s 11 (22.4 kda), with sequence similarity to the mitochondrial enzyme manganese superoxide dismutase (mnsod). interestingly, mala s 11 has a high degree of homology to human mnsod. the aim of this study was to examine the effects of recombinant mala s 11 on antigen-presenting dendritic cells. monocytederived dendritic cells (mddcs) from healthy blood donors were cultured with or without mala s 11 for different time periods. it was found that the maturation marker cd83 and the costimulatory molecules cd80 and cd86 were upregulated on the mddcs exposed to mala s 11 for 24 h, as demonstrated by flow cytometry. furthermore, coculture of mddcs with mala s 11 for 9 h induced an increased production of the inflammatory cytokines il-6 (200-fold), tnf-a (100-fold) and il-8 (sixfold), as detected by the cytometric bead array (cba) analysis. our results suggest that mala s 11 affects the immune response through dc maturation and production of inflammatory cytokines. the potential cross-reactivity with human mnsod needs to be explored and the exact role of mala s 11 in the pathogenesis of ae assessed in clinical studies involving skin prick and atopy patch tests. allergen-specific immunotherapy (sit) is commonly conducted with allergen extracts adsorbed to aluminium hydroxide (alum). drawbacks linked to the use of alum, such as the formation of granuloma at the site of injection, have led to suggestions of novel allergen carriers. an alternative carrier is 2 mm carbohydrate-based particles (cbps). in mouse, allergen-coupled cbps have been demonstrated to skew the allergen-specific immune response towards a th1-like activity (grönlund et al. immunology, 2002) . we here coupled the recombinant major cat allergen fel d 1 to cbps (cbp-fel d 1) by cyanogen-bromide activation, resulting in covalent binding. the effect of cbp-fel d 1 on monocyte-derived dendritic cells (mddcs) from healthy human blood donors was studied. we found that the majority of the cd1a þ mddcs were capable of taking up fitc-labelled cbp-fel d 1, as demonstrated by flow cytometry and confocal laser scanning microscopy. furthermore, incubation with cbp-fel d 1 resulted in an upregulation of the costimulatory molecule cd86 on the mddcs, which was not observed with fel d 1 or cbps alone. finally, cbp-fel d 1 induced a fivefold increase in the release of the pro-inflammatory cytokine tumour necrosis factor (tnf)-a and a fourfold increase in the release of the chemokine interleukin-8 from mddcs. taken together, the effects cbps possess make them interesting as novel allergen carriers for sit. the cysteine protease der p1 from dust mite of the genus dermatophagoides pteronyssinus is a major type i allergen. about 80% of house dust mite (hdm) allergic individuals are reactive to this protease in standard assays for detection of ige. a curative treatment for atopic allergy is immunotherapy (it) with hdm extracts which are complex mixtures occasionally resulting in anaphylactic reactions. novozymes focuses on developing a recombinant variant of der p1 which exhibit lowered risk of ige-mediated allergic reactions, while maintaining its ability to trigger proper th-cell responses. this may provide a safer alternative for specific it of hdm allergy. a secreted recombinant form of pro-der p 1 expressed by saccharamyces cerevisiae was obtained by fusion of the pro-enzyme to a fungal signal peptide. the n-glycosylation site of der p1 was mutated resulting in a deglycosylated pro-enzyme with a molecular mass of 35 kda. protein purification procedure was developed to obtain nearly pure der p1 protein followed by determination of concentration by active-site-titration with the cysteine protease inhibitor e64. the deglycosylated recombinant pro-der p 1 revealed immunologic similarity to the native der p 1 molecule when compared in basophile histamine release, ige-binding assays and t-cell proliferation assays. by in silico epitope mapping of a modelled 3-dimensional structure of der p1, five putative igg and ige epitopes were predicted. by protein engineering, the predicted epitopes were removed one by one in der p1 and screening for hypoallergenic variants was performed. combining inhaled long-acting b-2 agonist (laba) and inhaled corticosteroid (ics) seems to offer asthma control at a lower dose of ics than achieved by ics alone. fine mapping of t-cell surface markers by flow cytometry offers a detailed status of the individual's inflammatory response. the frequency of mt2 (cd4 þ cd45ra -cd62l þ cd11adim) and mt1 (cd4 þ cd45ra -cd62l -cd11abright) cells in peripheral blood, and their ratio, has been shown to differ predictably in atopics and patients with leprosy, where mt2 correlates with a th2 phenotype and mt1 with a th1 phenotype. stable asthmatics, requiring fluticasone propionate (fp) 750-1000 mg daily or equivalent, were randomized to receive, double-blinded, either seretide 1 [salmeterol and fluticasone propionate (sfc, n ¼ 16)] 50 mg/500 mg bd or fp 500 mg bd (n ¼ 17). if asthma was controlled based on lung function and symptoms at clinic visits every 6 weeks, ics dose was tapered until asthma exacerbated or 0 mg was reached. the frequency and ratio of mt2 and mt1 t cells of the patients was monitored at 6 week intervals. as treatment tapered, the frequency of mt2 cells decreased (p ¼ 0038 from first to final visit), whereas that of mt1 cells increased. the ratio of mt2/mt1 decreased (p ¼ 0049 from first to final visit). in patients receiving laba þ ics, the fall in mt2/mt1 ratio appeared to be more pronounced than in patients receiving ics alone. thus, the mt2 phenotype may be associated with stable asthma, whereas an imminent exacerbation may associate with an increase in the mt1 phenotype. laba may allow for a greater effect of fp on the mt ratio. activation of complement pathways, leading to production of c3a and c5a anaphylatoxins, has been postulated in the pathogenesis of asthma and allergic airway inflammation. the present study was undertaken to investigate the role of mannan-binding lectin (mbl), an initiator of the lectin pathway of complement, in asthma and allergic rhinitis. mbl levels and mbl-induced complement activity were determined in 19 patients of bronchial asthma with allergic rhinitis and 20 unrelated, age-matched controls of indian origin. mbl levels and activity were correlated with percent eosinophilia and percent predicted fev1 values of the patients. association of single nucleotide polymorphisms (snps) in exon 1 and intron 1 of the mbl with the disease, clinical markers, mbl levels and mbl-induced complement activity was analysed using standard statistical tools. significantly higher mbl levels and activity were observed in patients of bronchial asthma with allergic rhinitis as compared to the controls. we identified five snps, of which two, a816g in exon 1 and g1011a in intron 1 of the mbl, were novel. snp g1011a was significantly associated with the disease (p ¼ 0.0024, or ¼ 5.8696, 95% ci: 1.7316 < or < 19.8963). individuals with 'a' allele at position 1011 showed increased mbl levels, activity and disease severity. our results suggest that 'a' allele at position 1011 leading to high mbl levels and complement activity may be contributing to the severity of bronchial asthma and allergic airway inflammation. serum resistance of borrelia burgdorferi strains belonging to the b. afzelii and b. burgdorferi sensu stricto genospecies is dependent on binding of complement inhibitor factor h. we recently reported that factor h binding by b. burgdorferi is due to inducible expression of several approximately 20 kda plasmid-encoded, surface-exposed lipoproteins related to ospe (e.g. erpa, erpp and p21). in addition, a second class of factor h-binding proteins of approximately 27-35 kda has been described. the ospe-related lipoproteins are dramatically induced by b. burgdorferi during transmission from its tick vector into the mammalian host. the induction of ospe-related lipoproteins during mammalian infection may play a key a role in the borrelial evasion of the host's immune system. the goal of the present study was to define the factor h-binding regions of ospe-related proteins using mutagenesis, peptide mapping and surface plasmon resonance analysis (biacore). the combined studies revealed that the c-terminal regions of both human and mouse factor h (scrs 18-20) specifically bind to ospe-related lipoproteins. we also found fhr-1, whose c-terminal scrs 3-5 are homologous to scrs 18-20 of factor h, to bind to ospe. peptide mapping revealed five putative regions (designated i-v) in ospe that could directly interact with factor h. deleting the c-terminal 15 amino acid residues from region v of p21 abolished its ability to bind factor h. at the same time, however, synthetic peptides corresponding to the c-termini of ospe, p21 and erpp did not inhibit factor h binding to ospe. thus, the c-terminal-binding region v appears to be necessary but not sufficient for factor h binding. when a more specific mutation strategy was employed, where single amino acid residues in peptides spanning over the factor h-binding regions were mutated to alanines, we observed that lysines in the factor h-binding regions of ospe were required for factor h binding. the combined data have revealed that key lysine residues in ospe-related lipoproteins and ionic interactions are crucial for factor h interactions. furthermore, binding of ospe to the c-termini of both mouse and human factor h suggests that borrelia spirochetes utilize analogous complement resistance mechanisms in both rodents and man. in borrelia garinii strains, which in in vitro analyses have been found to be sensitive to complement killing, differences in the ospe sequences as well as in the expression of factor h-binding proteins may account for their susceptibility to serum lysis. role of yada, ail and lipopolysaccharide in serum resistance of yersinia enterocolitica serotype o:3 mannan-binding lectin (mbl), l-ficolin and h-ficolin are pattern recognition molecules of the innate immune system. we investigated the ability of these molecules to bind to different serotypes and noncapsulated variants of streptococcus pneumonia and staphylococcus aureus. we found that mbl binds to noncapsulated s. aureus strain (wood) but not any of the examined s. pneumoniae serotypes. l-ficolin binds to some capsulated s. pneumoniae serotypes (11a, 11d and 11f) as well as some capsulated s. aureus serotypes (type-1, -8, -9, -11 and -12). h-ficolin does not bind to any of the examined s. pneumoniae and s. aureus serotypes included in this study but did bind to a strain of aerococcus viridans. when bound to bacteria, mbl and h-ficolin initiated activation of complement factor c4, whereas l-ficolin did not. during this study, quantitative assays for the three proteins were developed and the concentration in 97 plasma samples were determined and the median values were estimated at 0.8 mg of mbl/ml, 3.3 mg of l-ficolin/ml and 18.4 mg of h-ficolin/ ml, respectively. the absence of early complement components (c1, c4 and c2 but not c3) is a predisposing factor for systemic lupus erythematosus (sle). recently, we demonstrated that, in c4-deficient (c4 def.) mice, igm-containing immune complexes (igm-ic) are filtered by the splenic barrier of marginal zone macrophages (mzm), resulting in an increased immune response against antigens within these igm-ic, but this could not be observed in wildtype or c3 def. mice. we hypothesized that splenic cd11b þ mzm play an important role in the induction of autoimmunity, and we therefore analysed their cytokine profile after isolation with the help of magnetic antibody cell sorting. mrna was isolated, and real-time pcr was performed with specific primers for murine ifn-g (ifn-g), interleukin-12 (il-12) and ifn-a (ifn-a). we observe a moderate increase of il-12 and ifn-g mrna in cd11b þ cells of c4 def. mice compared to wildtype cells. surprisingly, the concentration of ifn-a mrna is six times higher in c4 def. mice. preliminary results suggest that mrna in cd11b þ cells of c3 def. mice is even lower than that in wt. six hours following i.v. application of 20 mg of a abstracts 625 .................................................................................................................................................................................................. murine monoclonal igm anti-dsdna antibody, production of il-12, ifn-g and ifn-a mrna is increased in cd11b þ cells of both c4 def. and wt mice. several references described increased levels of inf-a in patients with sle. dendritic cells are discussed as a major source of ifn-a. our observation that c4-deficient, sle-susceptible mice demonstrate an increased spontaneous ifn-a production by splenic cd11b þ marginal zone macrophages could be an early sign and a trigger for the development of sle. this is supported by the fact that the absence of c3 is not a predisposing factor for sle and our observation that c3 def. animals display low levels of ifn-a mrna. 200-400 million people worldwide and represents one of the leading causes for liver cirrhosis and hepatocellular carcinoma. control over the hbv infection is achieved mainly by vaccination with hepatitis b surface antigen (hbsag). hbsag contains n-linked glycosylation side and is recognized by both mbl-a and mbl-c in a cadependent manner. hbsag-mbl complexes activate complement and may thus affect humoural immunity. to investigate the role of mbl in humoural responses to hbsag, we immununized mice that lack both mbl-a and mbl-c proteins with soluble hbsag. it has been shown that deficiencies in other complement components like c1q, c4 and c3 result in decreased antibody responses. however, mbl double ko animals mounted dramatically increased humoural responses. after priming, mbl double kos mounted hbsag-specific igm responses, which were threefold higher than wt controls. after boosting the hbsag, total igg was 10-fold higher in mbl ko than in wt control animals. similar to the response to hbsag, other glycosylated soluble antigens (e.g. invertase) induced better humoural responses in mbl double ko animals, suggesting that mbl plays an important role in a negative feedback regulation of adaptive immunity. reconstitution experiments with rmbl partially rescued the ko phenotype. we propose that the clearance of glycoprotein antigens in mbl ko is handled differently from the wt, resulting in better stimulation of humoural responses. alternatively, glycoprotein-ag-mbl-rich complexes inhibit b-cell responsiveness via putative mbl receptors. the complement system is an important part of the innate immune system. the activation of complement proceeds through three different pathways that converge in the generation of c3-activating enzyme complexes. complement activation via the lectin pathway is initiated when recognition molecules, mannan-binding lectin (mbl) or ficolin, bind to carbohydrate structures characteristic for microbial surfaces. in the circulation, mbl and ficolins are found in association with three structurally related mblassociated serine proteases (masp)-1, -2 and -3 and a small, nonenzymatic component, map19. masp-2 has been shown to elicit complement activation through the sequential proteolytic cleavage of c4 and c2 upon binding of mbl/masp-2 complexes to microbial surfaces. we have recently uncovered a polymorphism in the masp-2/map19 gene in a patient shown to be deficient in the lectin pathway of complement activation. the polymorphism results in a single amino acid substitution in the n-terminal part of the masp-2 protein. recombinant wildtype masp-2 and masp-2 containing the amino acid substitution in question was produced, and the ability to activate complement was studied. the mutation had a profound impact on masp-2 function, resulting in the lack of complement activation through the lectin pathway. elisa-based experiments showed that the mutation leads to the impairment of complement activation through influencing the binding of masp-2 to mbl or ficolins. deficiencies in the lectin pathway of complement activation have so far been accounted for only by lack of functional mbl. the mutation described above is the first defect described affecting both activation through mbl and the ficolins. .................................................................................................................................................................................................. th1, th2 and treg cell balance. dcs are present in the gut mucosa and may thus be target for modulation by gut microbes, including ingested probiotics. here, we tested the hypothesis that species of lactic acid bacteria, important members of the gut flora, differentially activate dc. a large panel of human gut-derived lactobacillus and bifidobacterium spp. was screened for dc-polarizing capacity by exposing bone marrow-derived murine dc to lethally irradiated bacteria. cytokines in culture supernatants and dc-surface maturation markers were analysed. substantial differences were found among strains in the capacity to induce interleukin-12 (il-12) and tumour necrosis factor (tnf)-a, while the differences for il-10 and il-6 were less pronounced. bifidobacteria tended to be weak il-12 and tnf-a inducers, while both strong and weak il-12 inducers were found among the strains of lactobacillus. remarkably, strains weak in il-12 induction inhibited il-12 and tnf-a production induced by an otherwise strong cytokine-inducing strain of lactobacillus casei, while il-10 production remained unaltered. selected strains were tested for induction of dc maturation markers. those lactobacilli with greatest capacity to induce il-12 were most effective in upregulating surface mhc class ii and cd86. moreover, l. casei-induced upregulation of cd86 was reduced in the presence of a weak il-12inducing l. reuteri. in conclusion, human lactobacillus and bifidobacterium spp. polarize differentially dc maturation. thus, the potential exists for th1/th2/treg-driving capacities of the gut dc to be modulated according to composition of gut flora including ingested probiotics. the intestinal micro flora is indispensable in developing and maintaining homeostasis of the gut-associated immune system. evidence indicates that lactic acid bacteria (lab), e.g. lactobacilli and bifidobacteria, have beneficial effects on the host. established health effects include increased gut maturation, antagonisms towards pathogens and immune modulation. the objective of this study is to evaluate the immunomodulating properties of a range of lab of human origin. as dendritic cells (dcs) play a pivotal role in the balance between tolerance and immunity to commensal microorganisms, in vitro-generated immature dcs serve as a suitable model for studying the immunomodulating effects of lab. human immature dcs were generated in vitro from monocytes and exposed to lethally uv-irradiated lab. the effect of various species of lab on dcs in direct contact was evaluated. furthermore, the maturation pattern of dcs separated from the bacteria by an epithelial cell layer (caco-2 cells), which should mimic the intestinal environment, was studied. cytokine secretion (il-12, il-10 and tnf-a) and upregulation of maturation surface markers on dcs (cd83 and cd86) was measured. different lab induced diverse cytokine responses. some strains were strong il-12 and tnf-a inducers and others weak. all strains induced il-10. different lab also differentially modulated expression of cd83 and cd86 on dcs. although some variation in the response to lab of dcs generated from different blood donors was observed, general differences in the effect of the various lab was revealed. experiments with the dc caco-2 coculture system are ongoing. different species of lab differentially affect dc maturation; this suggets that the gut flora plays a pivotal role in polarization of the immune response. natural killer (nk) cells are cells of the nonspecific immune system lysing altered self-cells. a noncytolytic subset of nk cells may serve a regulatory role by secreting cytokines. bacteria translocating across the gastrointestinal mucosa are presumed to gain access to nk cells, as consumption of certain lactic acid bacteria has been shown to increase in vivo nk cytotoxicity. here, we investigated how human gut flora-derived lactobacilli affect nk cells in vitro, by measuring proliferation and ifn-g production of human nk cells upon bacterial stimulation. cd3 -cd56 þ nk cells were isolated from buffy coats by negative isolation using non-nk lineage-specific antibodies and magnetic beads. nk cells were incubated with 10mg/ml uv-inactivated bacteria or 10mg/ml phytohemagglutinin (pha) for 4 days. proliferation was assessed by incorporation of radioactive thymidine into nk-cell dna. the ifn-g concentration was measured by elisa. incubation of nk cells with a lactobacillus acidophilus strain increased the proliferation of the nk cells and induced ifn-g production, both to levels comparable to pha stimulation. the proliferative response was further enhanced with autologous monocytes present, probably because cytokines, secreted by monocytes having engulfed bacteria, stimulated the nk cells. in contrast, a lactobacillus paracasei strain caused the nk cells to proliferate only in the presence of monocytes. these results demonstrate that various strains of lactobacilli have the capacity to activate nk cells in vitro, in a monocyte-dependent or -independent way. hence, the encounter of nk cells with lactic acid bacteria will affect nk-cell activation. such activation of nk cells may potentially skew an on-going or subsequent immune response towards a th1 response. lactobacilli are nonpathogenic gram-positive inhabitants of the normal human intestine known for their healthpromoting effects. in our earlier work, it is shown that human monoclonal antibody isolated from sera of a patient with waldenstrom macroglobulinaemia possess innate antibody characteristics and binds to lactic acid bacteria. according to the immune network model, immunization with this bacteria could induce the perturbations in immune system that might result in production of anti-lactobacillus antibodies, human monoclonal antibody like (ab1) and anti-idiotypic antibody (ab2). in this study, balb/c mice were immunized with two doses of bacteria lactobacillus acidophilus in complete and incomplete freund's adjuvant and phosphate-buffered saline (pbs), respectively. seven days after the last immunization, sera from immunized mice were collected and the presence of lactobacillus-specific ab1 and ab2 were determined by elisas. in the sera of immunized mice, antibodies specific to bacteria lactobacillus acidophilus were shown. the concentration of lactobacillus-specific antibodies was higher in the sera of hyperimmunized mice (mice immunized with 1 mg of igm dj) than in sera of mice immunized with 100 times lower doses of immunogen (0.01 mg per doses). moreover, ab1 and ab2 antibodies were detected in the sera of lactobacillus-hyperimmunized mice. in this study, we have shown the idiotypic network interactions in mice immunized with bacteria lactobacillus acidophilus. the normal gastrointestinal flora is crucial for the maturation of the acquired immunity via effects on antigenpresenting cells (apcs). here, we have investigated how two types of apcs, monocytes and dendritic cells (dcs), react to different bacterial strains typical of the commensal intestinal flora. purified monocytes and monocyte-derived dcs were stimulated with uv-inactivated gram-positive (lactobacillus plantarum and bifidobacterium adolescentis) and gram-negative (escherichia coli and veillonella parvula) bacterial strains. monocytes produced higher levels of il-12p70 and tnf, as detected by elisa, in response to l. plantarum than to e. coli and v. parvula. in contrast, dcs secreted high amounts of il-12p70, tnf, il-6 and il-10 in response to e. coli and v. parvula but were practically unresponsive to l. plantarum and b. adolescentis. the lack of response to the gram-positive strains correlated with a lower surface expression of toll-like reseptor 2 (tlr2) on dcs compared to monocytes. the surface expression of tlr4 on dcs was undetectable when analysed by flow cytometry, but blocking this receptor decreased the tnf production in response to v. parvula, indicating that low tlr4 expression on dcs is sufficient to mount an inflammatory response to gram-negative bacteria. ifn-g increased the expression of tlr4 on dcs and also potentiated the cytokine response to gram-negative bacteria. our results indicate that, when monocytes differentiate into dcs, their ability to respond to different commensal bacteria dramatically changes, thereby becoming unresponsive to probiotic gram-positive bacteria. these results may have important implications for the capacity of different groups of commensal bacteria to regulate mucosal and systemic immunity. probiotic bacteria, e.g. lactobacillus spp., may improve diseases such as chronic inflammatory bowel disease. we examined cytokine production and phenotypic change after in vitro stimulation of t cells from healthy volunteers using different probiotic strains. methods: t cells were cultured from colonic biopsies from eight healthy volunteers (agnholt and kaltoft, exp clin immunogenet 2001; 18:213-25) , and dendritic cells were matured from their peripheral blood mononuclear cells. t-cell cultures were stimulated with autologous bacterial sonicate or strains of lactobacillus spp., with and without the addition of dendritic cells. cytokine levels (tnf-a, ifn-g, il-10 and gm-csf) and phenotype (cd3, cd4, cd25 and cd69) were measured on day 4. results: lactobacillus spp. induced higher productions of tnf-a and il-10 than did autologous bacteria. in presence of dendritic cells, the production of all cytokines increased. however, the increases of ifn-g and tnf-a were more pronounced in wells with autologous bacteria than in wells with lactobacillus spp. the addition of dendritic cells upregulated cd25 expression without simultaneous upregulation of cd69. the upregulation was pronounced after stimulation with lactobacillus rhamnosus gg compared with autologous bacteria and other lactobacilli. discussion: in presence of dendritic cells, autologous bacteria induced inflammatory cytokines, while probiotics mainly induced regulatory cytokines. lactobacillus rhamnosus gg induced a regulatory phenotype (cd25 þ ), in part mediated by dendritic cells. future studies will address whether this shift to a cd25 þ phenotype represents a differentiation into competent regulatory t cells. in a clinical context, such cells might be used for treatment of inflammatory diseases. protein microarrays will play a key role in the postgenomic era and offer a unique possibility to perform highthroughput global proteome analysis. a chip can be printed with thousands of protein probes (e.g. antibodies), the biological sample added (e.g. a proteome) and any binding detected. we aim to develop protein microarrays based on human recombinant scfv antibody fragments for global proteome analysis. the concept of comparing proteomic maps of healthy versus diseased samples will allow diseasespecific proteins to be detected. in fact, antibody microarrays will allow us to perform comparative proteome analysis on any sample format in a species-independent manner, as long as a proteome can be isolated. however, the complexity of proteomes, containing several thousands of different proteins, is a problem. here, we have designed antibody microarrays targeting the water-soluble fraction of a proteome. to this end, an anticytokine antibody array was developed and human dendritic cells (aeactivation) was used as model system. the results showed that our antibody microarrays could be used to examine the cytokine profile in complex samples. furthermore, we have taken the first steps towards comparing our results with those of other technologies on both the protein and gene level. due to the complexity of the model proteome, we also examined the possibility to prefractionate the proteome in a simple one-step procedure (based on size) prior to the labelling step. in more detail, the sample proteome was fractionated into two fractions using membrane devices with different molecular weight cut-offs. the results showed that the fractionation considerably enhanced the assay sensitivity allowing cytokines in the pg/ml range to be readily detectable. the immunomodulatory effect of heat shock protein 70: immunization with a dna construct based on the malarial antigen fused with a fragment of hsp 70 primes for a th-1 type of response finding an appropriate adjuvant for human vaccination is crucial. heat shock proteins (hsps) act as adjuvants when coadministered with peptide antigens or given as fusion proteins. however, there is a potential risk of autoimmunity when using the complete molecules, because hsps are evolutionary conserved. to overcome this, we first evaluated the adjuvant effect against two different antigens of a less-conserved fraction of plasmodium falciparum hsp70 (pf70c) and compared it to the whole hsp70 molecule from trypanosoma cruzi (tchsp70). we found that pf70c exhibited similar adjuvant properties as the whole molecule. we later evaluated the adjuvant potential of pf70c against the malarial antigen eb200 in a chimeric dna construct. no appreciable levels of eb200-specific abstracts 629 .................................................................................................................................................................................................. antibodies were detected in mice immunized only with the dna constructs. however, dna primed the immune system, because subsequent challenge with the corresponding recombinant fusion proteins elicited a strong th-1 antibody response. in contrast, no priming effect was observed for ex vivo ifn-g production but stimulation with the hsp-chimeric fusion protein induced a stronger secretion of ifn-g in vitro than other proteins used. these results indicate that the use of hsps is promising in the design of new vaccines. high-throughput proteomics on antibody-based microarrays: the importance of probe and surface design in analogy to dna microarrays, protein microarrays offer a new distinct possibility to perform sensitive highthroughput global proteome analysis. however, the development of the protein microarray technology will place high demands upon the design of both probes and solid supports. the analysis of thousands of heterogeneous proteins on a single microarray requires the use of uniform probes, such as antibodies, directly designed for protein microarray applications. we have recently generated a human recombinant single-chain fv antibody library, genetically constructed around one framework, the ncoder-library, containing 2 â 1010 clones. single framework antibody fragments (sinfabs) selected from this library were successfully applied as probes for microarrays providing sensitive detection in the 600 attomol (mass spectrometry) and the 300 zeptomole range (fluorescence). however, the choice of framework is critical. we have shown that the selected ncoder framework displayed excellent functional on-chip stability and arrayed dehydrated probes retained their activity for several months. furthermore, we have addressed the issues of biocompatibility of the solid support and immobilization strategies for our microarray setup. an in-house-designed substrate, macroporous silicon coated with nitrocellulose (map3-nc7), displayed properties equal to, or better than, those of five commercially available supports used as reference surfaces. we have also evaluated different coupling strategies, such as adsorption, covalent coupling, diffusion and affinity coupling. using a novel affinity tag, the double-(his)6-tag, we increased the binding efficiency of sinfab-molecules to ni2 þ -coated solid supports, thereby allowing nonpurified probes to be directly applied. the mannan-binding lectin (mbl) pathway is part of the innate immune system providing a first line of defence against infections. mbl and ficolins circulate in complexes with mbl-associated serine proteases (masp-1, -2 and -3). after recognition of a microorganism by mbl, activation of the complement system occurs. masp-1 and masp-3 share five domains (making up the so-called a-chain), whereas they have unique protease domains (b-chains). before the identification of masp-3, an assay for masp was presented, based on antibodies against the a-chain of masp-1. with the new knowledge of the three masps, and the sharing of domains by masp-1 and masp-3, assays specific for the protease domains have to be constructed, if one wishes to measure the proteins individually. we present an assay for quantifying total masp-3 in plasma and serum samples. the assay is a sandwich-type assay using as catching antibody a monoclonal antibody against the common a-chain of masp-1/3 and a developing secondary antibody against the c-terminal part of the protease domain of masp-3. we have used this assay for estimating the normal concentration of the protein as well as the concentration in patients and also for characterizing by gel permeation chromatography the masp-3 protein in serum. inducible costimulator ligand (icosl) is a costimulatory molecule related to b7.1 (cd80) and b7.2 (cd86). b cells, monocytes, dendritic cells and endothelial cells express icosl. inducible costimulator (icos) interacts with icosl, and this interaction leads to signals involved in isotype switching and the development of immunological memory. hitherto, no polymorphisms of this gene have been described. the aim of this study was to reveal variation of the icosl gene in normal individuals. all eight exons, except exon 1, were sequenced with flanking introns in 10 healthy blood donors. eight single nucleotide polymorphisms (snps) and two length polymorphisms were found. one of the snps was found in the coding regions of the gene. the base involved was located in exon 3 and caused a conservative amino acid change from valine (gtt) to isoleucine (att). three individuals were heterozygous g/a for the exon polymorphism, while the remaining seven individuals were homozygous for the wildtype g/g. exon 3 encodes the immunoglobulin variable (igv)-like domain of the molecule which is situated outside the cell. this means that the amino acid could be critical for the stability of the molecule or could constitute part of the binding site for icos. the results form the basis for further experiments to find possible associations of the alleles to diseases caused by immune dysregulation. especially, the exon 3 variant is interesting and could play a role for the development of immunological diseases. besides, it would be interesting to see whether both exon 3 alleles are expressed or only the wildtype allele is functional. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. myxovirus a (mxa) is a resistance gtpbinding protein that is specifically induced by treatment with type 1 ifns. for example, ifn-b-induced mxa in blood leucocytes has been used as a biomarker in ifn-btreated patients with multiple sclerosis. however, the degree of specificity of mxa in this regard is unclear, and measurements of mxa protein and/or mrna are not yet suitable for routine clinical use. in an attempt to find new and better reporter genes (and, hopefully, genes and gene products with proven specificity for ifn-a and -b), microarray screenings with u133a genechips (affymetrix) were carried using human blood leucocytes and the human lung carcinoma cell line a549. we studied the simultaneous expression of 22,000 transcripts before and after exposure to human recombinant ifn-a and ifn-b and other antiviral and immunomodulatory cytokines. the results will be presented at the conference. interferon-a/b (ifn-a/b) is increasingly used as antiviral and immunomodulatory therapies. unfortunately, bioavailability varies with ifn species and mode of administration, and all ifn species are potentially immunogenic. assays for antiviral activity (ifn) and antiviral neutralization (antibodies, nab) have been used for some time to monitor patients on ifn biologicals. these assays require laborious titrations making them unsuitable for large-scale clinical use. our laboratories have therefore modified the antiviral assays for ifn bioactivity and nab, so that they are suitable for large-scale screening in specialized laboratories. the read-out is survival of a subcloned a549 cell line in the presence of an otherwise lethal amount of virus. thus, survival increases in the presence of type 1 ifn and decreases in the presence of nab against the ifn added to the cells. mxa is induced by type 1 ifn and can be used for measuring the nab activity. in another assay, the mxa level in the a549 cell line is measured. in an attempt to find a new and better reporter gene for type 1 ifn than mxa and genes specific for either ifn-a or -b, a micro array screen was carried using the u133a chip from affymetrix. the expression of 22,000 genes can be studied simultaneous with this technology. the results will be presented at the conference. in our laboratory, we have developed a database system, which we believe is of immediate interest to the general scientific community. the database represents a computerbased replacement for the laboratory notebooks used in the majority of research laboratories worldwide. in addition, the database provides an effective tool for organizing and managing laboratory information at all levels, spanning from managing and revising standard operating procedures and producing documentation of research activities to keeping track of data and conclusions. using the commercially available database toolkit software filemaker pro, we have developed a relational database solution for management of laboratory information. the system consists of a hierarchy of five interrelated databases, each pertaining to a separate type of information, namely, overall project information, information relating to individual experiment setups, documentation of daily research activity, generated data and descriptions of standard operating procedures. like other databases, each individual database consists of a number of records, each comprised of a set of fields in which information is entered. in each record, a certain field is reserved to specify the relation of the record to a record in another database at a higher level. thus, the database is essentially five databases linked by a hierarchy of one-to-many relations, organizing information in a folder-like structure. importantly, the database system allows multiple users to access and edit records simultaneously, and the data entered in one database immediately becomes accessible through the other databases. the limitations of laboratory notebooks are apparent when looking for information, which is dispersed throughout one or more notebooks, or possibly on loose sheets of paper or printouts 'somewhere'. the often complicated process of gathering laboratory data or results when writing grant applications or research papers is made considerably easier with the database system. thus, the database solution presented should be broadly attractive to researchers, irrespective of their scientific discipline. an effective sars vaccine is likely to include components that can induce specific cytotoxic t-cell (ctl) responses. the specificities of such responses are governed by hlarestricted presentation of sars-derived peptide epitopes. exact knowledge of how the immune system handles protein antigens would allow for the identification of such linear sequences directly from genomic/proteomic sequence information. the latter was recently established when a causative coronavirus (sars cov) was isolated and full-length sequenced. here, we have combined advanced bioinformatics and high-throughput immunology to perform an hla supertype, genome-wide scan for sars-specific cytotoxic t cell epitopes. the scan includes all nine human hla supertypes in total covering >99% of all major human populations. for each hla supertype, we have selected the 15 top candidates for test in biochemical-binding assays. at this time (approximately 6 months after the genome was established), we have tested the majority of the hla supertypes and identified almost 100 potential vaccine candidates. these should be further validated in sars survivors and used for vaccine formulation. we suggest that immunobioinformatics may become a fast and valuable tool in rational vaccine design. rationale: major histocompatibility complex class i (mhc i) molecules monitor the protein content of the cell by binding small derived peptides and presenting them to cytotoxic cd8 þ t cells. the goal of the human mhc project is to predict the binding strength of any given peptide/mhc complex. this prediction allows the design of peptide-based vaccines. the prediction requires representative binding data from mhc alleles from all the nine hla supertypes. here, we describe the genetic construction, protein production and purification as well as the establishment-binding assays for two recombinant mhc supertype alleles, hla-b*1501 and hla-b*5801. methods: using the quikchange multisite directed mutagenesis kit (stratagene), codon-optimized genes encoding hla-b*1501 and hla-b*5801 are created. the two mhc i molecules are fermented and purified by ion exchange chromatography, hydrophobic interaction chromatography and size exclusion chromatography. the binding (kd) of natural t-cell epitopes, as well as predicted peptide ligands, is described by radioactive immunoassays (rias) and enzyme-linked immunosorbent assays (elisas). the mhc molecules are biotinylated during expression. results: the expression of mhc i resulted in multiple disulfide bond isomers, which are separated by hydrophobic interaction chromatography and used in subsequent binding studies resulting in the determination of kd for various peptide ligands ranging from strong binders we have previously demonstrated that bioinformatics tools such as artificial neural networks (anns) are capable of performing pathogen-, genome-and hlawide predictions of peptide-hla interactions. these tools may therefore enable a fast and rational approach to epitope identification and thereby assist in the development of vaccines and immunotherapy. a crucial step in the generation of such bioinformatics tools is the selection of data representing the event in question (in casu peptide-hla interaction). this is particularly important when it is difficult and expensive to obtain data. herein, we demonstrate the importance in selecting information-rich data and we develop a computational method, query-bycommittee, which can perform a global identification of such information-rich data in an unbiased and automated manner. furthermore, we demonstrate how this method can be applied to an efficient iterative development strategy for these bioinformatics tools. methods: a large panel of binding affinities of peptides binding to hla a*0204 was measured by a radioimmunoassay (ria). this data was used to develop multiple first generation anns, which formed a virtual committee. this committee was used to screen (or 'queried') for peptides, where the anns agreed ('low-qbc'), or disagreed ('high-qbc'), on their hla-binding potential. seventeen low-qbc peptides and 17 high-qbc peptides were synthesized and tested. the high-or low-qbc data were added to the original data, and new high-or low-qbc second generation anns were developed, respectively. this procedure was repeated 40 times. the high-qbc-enriched ann performed significantly better than the low-qbc-enriched ann in 37 of the 40 tests. conclusion: these results demonstrate that high-qbcenriched networks perform better than low-qbc-enriched networks in selecting informative data for developing peptide-mhc-binding predictors. this improvement in selecting data is not due to differences in network training performance but due to the difference in information content in the high-qbc experiment and in the low-qbc experiment. finally, it should be noted that this strategy could be used in many contexts where generation of data is difficult and costly. interleukin-18 (il-18), a pro-inflammatory cytokine that is produced by both lymphoid and nonlymphoid cells, has a critical role in modulation of innate and adaptive immunity. its primary function in stimulation of ifn-g production and stimulation of nk-cell-cytotoxic activities makes this cytokine a candidate for cancer immunotherapy. in oral cavity, this cytokine is produced by oral epithelia and carcinoma cells and is related to tumour regression in nude mice bearing salivary adenocarcinoma. however, direct effects of this cytokine on oral cancer cells have not been elucidated. in this project, we investigated il-18 effect on an oral carcinoma (kb) cell line. with rt-pcr technique, kb-cell line was found to express il-18 receptors (il-18ra and il-18rb), indicating that this oral carcinoma line is a target for il-18 study. we showed that recombinant human il-18 inhibited kb-cell proliferation by 17% at concentration of 100 ng/ml (p < 0.05), whereas ldh release by these cells in treatment group and control groups was comparable, indicating that il-18 suppression of cell proliferation was not mediated by the induction of cell death. to further address this hypothesis, we found that il-18 treatment did not induce apoptotic cell death, as studied by dna laddering and tunel assays. in addition, expression pattern of cell death-controlling genes (bcl-2 and bax) was not altered by this cytokine. findings in these studies indicated that suppression of kb-cell proliferation may be attributed to control of cell cycle, growth arrest or induction of cell differentiation. the data presented in this project could provide an insight of how cancer cell directly responds to il-18, as this cytokine is an important regulator of anticancer mechanisms. aloe emodin (ae) is a naturally occurring compound with wide spectrum of biological properties, including antimicrobial, vasorelaxant, immunosuppressive and anticancer actions. this anthraquinone induces apoptosis in several tumour cell lines with special affinity to tumours of neuroectodermal origin. high amounts of nitric oxide (no) released by activated macrophages induce tumour cell death. therefore, we explored the capacity of ae to modulate no-mediated antitumour response in vitro. interestingly, while ae markedly suppressed no release from macrophages alone, it significantly potentiated no production in cocultures of macrophages and c6 cells, after 48 h of cultivation. accordingly, the viability of c6 cells cocultivated with macrophages was reduced in the presence of ae. moreover, the observed ae-imposed potentiation of no production in macrophages was closely related to macrophage culture cell density. according to these data, we proposed that no modulator capacity of ae strongly depended on intercellular contact, indicating that macrophage antitumour response was not compromised but even potentiated by ae. immunotherapy represents an attractive fourth-modality therapeutic approach, especially in the light of the shortcomings of conventional surgery, radiation and chemotherapies in the management of metastatic cancer. to this end, a large number of peptide antigens derived from taa have been applied in immunotherapeutic trials for the treatment of various malignancies, e.g. cancers of the breast, prostate and kidney, in addition to haematological cancers. in some cases the response rates have been impressive and no adverse autoimmunity have been observed. a major strategic difficulty associated with these trials relates to the choice of best-suited peptide antigens. the vast majority of the antigens described thus far is not vital for survival and growth of the tumour cells, and immunoselection of antigen-loss variants may therefore prove to be an additional obstacle for the clinical applicability of most of the known peptide epitopes. in this respect, the development of acquired antigen loss during immunotherapy has been demonstrated in several cases. obviously, the development of loss-variant tumour cells implies that these cells acquire a pronounced growth advantage and are left unaffected by further treatment. ideally, target antigens should be derived from proteins required for survival and growth of tumour cells, as antigens with these characteristics would not be inflicted by the development of loss-variant tumour cells. in this respect, several inhibitors of apoptosis proteins (iaps) are universally expressed among tumours and play an important role in tumour cell escape from apoptosis. we have characterized spontaneous t-cell reactivity against iapderived peptides in cancer patients. from the iap survivin, we have characterized peptides restricted to the class i molecules hla-a1, a2, a3, a11, b7 and b35. furthermore, we have demonstrated that survivin-specific t cells infiltrate metastatic lesions and that isolated survivinspecific ctls are capable of killing hla-matched tumour cells. survivin-derived peptides are now in clinical trial, and continued work in our lab has demonstrated that other iaps are targets for spontaneous t-cell reactivity in cancer patients. we previously reported that in mice with large progressing t-cell lymphoma tumours, dysfunctions in the antitumour ctl activity occur, associated with an accumulation of splenic arginase-producing myeloid suppressor cells (mscs). in this study, we first demonstrate that both the presence and the activation state of these msc depends on tumour evolution. while in tumour regressors hardly any arginase-producing msc can be found, both the amount and the arginase activity of this population expands from early over late progressors. this gradual induction of mscs is paralleled by an increasing suppression of ctl activity and th1, but not th2, cytokine production. upon analysing the molecular repertoire of msc in vitro, we found, besides arginase1, a well-established marker for alternatively activated myeloid cells or m2, a strong upregulation of fizz1 and ym, two additional recently identified markers for m2. further evaluation of molecular markers by microarray analysis in msc yielded genes involved in wound healing (e.g. coagulation factor xiiia), anti-inflammation (e.g. selenoprotein p), immunomodulation (e.g. pd-l2) and fat and sugar metabolism (e.g. leptin receptor). of note, many of these genes are regulated by type 2 cytokines (il-4, il-13 and il-10) and are therefore rather m2 associated. overall, our data provide new markers for msc in cancer and further establish their m2 activation state. study. only sp-a showed a significant expression in normal mucosa which was downregulated in crc. as the absolute signal level was below the noise threshold, these results have to be interpreted with caution and require confirmation by direct measurenment of the proteins. our results suggest that there is no major role for the human collectins in colorectal cancer. tetramerization is visualized by sds-page. conclusion: an effective method for the production of highly pure mhc i molecules has been applied to hla-b*1501 and hla-b*5801, and ria and elisa binding assays for those alleles have been established background: proliferation, differentiation and apoptosis are essential processes in the normal functions of the mammary epithelium. the hypothesis examined in this study is that the transcription factor bcl-6 is critically important not only for regulating b-cell growth and development but also for mammary epithelial apoptosis. methodology: twenty breast cancer cases and 31 healthy controls were used to investigate whether bcl-6 protein in involved in breast cancer (grade iii). full length bcl-6 cdna was retrovirally transduced into eph-4 cell line. we then used flow cytometry of brdurd-stained cells to investigate the cell-cycle duration of the control and transduced cell lines. tunel was used as a marker of apoptosis to find out differences in the frequencies of apoptotic cells in the control and transduced cell lines. finally, immunohistochemistry staining was performed to detect bcl-6 in breast cancer (iii). results: restoration of bcl-6 into eph-4 cells not only inhibits apoptosis but also prolongs the cell cycle and results in increased cell size and protein content. the results also indicated that the cell-cycle time of bcl-6-transduced eph-4 cells is prolonged by about 3 h, presumably as a result of the action of bcl-6 at the bcl-6 at the g1/s transition. we found differences in the frequencies of viable and apoptotic cells in cultures of the parent eph-4 cells, control-transduced eph-4 cells and bcl-6-transduced eph-4 cells. consistently, we demonstrated that bcl-6 is expressed in 90% of high grade of breast carcinoma, which is considered as the most aggressive of tumours. conclusion: together, these results suggest that bcl-6 is likely to be involved in mammary gland development and carcinogenesis. inflammatory cytokines have a critical role in modulation of both innate and adaptive immunity in response to foreign antigen. they also play an important role in anticancer immunity. for example, they can promote cell-mediated immunity against cancer cells. with their immunostimulatory effects, these cytokines are being tested for cancer treatment in the form of dna vaccine or adjuvant or therapeutic cytokines. direct effect of these cytokines on cancer cell, however, is still unclear. in this project, we investigated whether il-1( and il-18 can modulate cancer cell proliferation. we employed a simple nonradioactive proliferation (mtt) assay and detection of lactate dehydrogenase (ldh) to test the effect of these recombinant human cytokines on various cancer cell lines, including breast cancer cell line (mcf-7), oral carcinoma cell line (kb), colon cancer cell line (caco-2) and choriocarcinoma cell line (jar). cytokines used in this study had both inhibitory and stimulatory effect on cell proliferation. findings in this project could provide an insight of cancer cell response to these cytokines and this could lead to a consideration on using cytokine as immunotherapy for cancer treatment.capacity of ae to modulate nitric oxide production depended on intercellular contact donor t cells are involved in the antitumour effects observed after bmt. thus, patients receiving t-celldepleted bmt have a higher risk of leukaemia relapse compared to patients receiving nonmanipulated bmt, and patients experiencing graft-versus-host disease (gvhd) have a lower risk of disease relapse than patients who do not experience gvhd. although the importance of donor t cells for the curative action of bmt has been established, the exact mechanisms and molecules involved in this graft-versus-tumour effect remain largely unknown. in a recently initiated project, we have conducted a longitudinal study of t-cell clonotypes in patients who received peripheral blood stem cell grafts after nonmyeloablative conditioning. peripheral blood samples were obtained sequentially after transplant, and the mononuclear cells (mncs) were isolated and cryopreserved. cd8 þ t cells were isolated from the mncs by use of immunomagnetic beads or facs and analysed for the presence of clonally expanded cells by t-cell receptor clonotype mapping based on rt-pcr and denaturing gradient gel electrophoresis (dgge). using this gel-based methodology, clonally expanded t cells were monitored after transplant and compared to the clinical data of the patients. the preliminary results demonstrates the presence of clonally expanded cd8 þ t cells at all time points analysed. furthermore, a number of clonotypes persisted for more than 6 months, and other clonotypes emerged during this period. the appearance of newly emerged clonotypes which coincided with clinical gvhd could indicate a role for these t cells in the pathogenesis of gvhd. background: deficiency of the mannan-binding lectin (mbl) pathway of innate immunity leads to increased susceptibility to infections. in patients with colorectal cancer, postoperative infection is associated with poor prognosis. the aim of the present study was to evaluate (1) the relation between the mbl pathway and postoperative infectious complications and survival of patients resected for colorectal cancer and (2) the role of mbl as acute phase reactant compared to crp. methods: preoperative mbl concentration, mbl/mblassociated serine protease (masp) activity and crp were determined in serum from 611 patients and 150 healthy controls. the patients were observed for 8 years. postoperative infections, recurrence and survival were recorded. results: the mbl pathway components were increased in the patients (p < 0.0001) compared to healthy controls. low mbl levels were predictive of pneumonia (p ¼ 0.01), and pneumonia (n ¼ 87) was associated with poor survival (p ¼ 0.003, hr ¼ 1.5, 95% ci 1.1-1.9). mbl and mbl/ masp activity could not predict postoperative overall infections. mbl showed no correlation (spearman's r ¼ 0.02, 95% ci à0.06-0.10) with crp. conclusions: low preoperative mbl levels are predictive of pneumonia, which is associated with poorer survival. mbl concentration and mbl/masp activity was not predictive of other postoperative infections or long-term prognosis. mbl apparently is not a surrogate measure of crp. department of surgery, university hospital of erlangen, erlangen, germany. e-mail: michael.siassi@rzmail.uni-erlangen.de introduction: the human collectins, mannan-binding lectin (mbl), surfactant protein-a (sp-a) and surfactantprotein-d (sp-d) play a central role in the innate immune system. immunological responses to malignant transformation of epithelial cells gained increasing interest recently. a former study could demonstrate binding of mbl to certain colorectal carcinoma (crc) cell lines in vitro. we therefore examined the expression of human collectins in normal colon mucosa and in colorectal carcinomas. materials and methods: colon samples from 20 crc patients and 10 normal mucosa samples were collected immediately after surgery. the tissue was microdissected and rna isolated (qiagen, rneasy-kit). gene expression profiles were analysed using gene-chips (affymetrix, hg-u133). we analysed the data for the expression of mbl, its associated serine proteases mannan-binding lectinassociated serine protease 1/2 (masp 1/2), sp-a and sp-d. the signal intensity of the genes of interest was compared using the mann-whitney u-test. results: the expression of human collectins in normal human colon mucosa was generally low. only the expression of sp-a and masp-2 reached the noise threshold of 250 signals. these genes were significantly downregulated in crc specimens. the expression of the other proteins showed no difference in normal mucosa and crc. conclusion: as demonstrated before, the expression of human collectins in normal colon was low in this being the first lymph node to receive drainage from the tumour area, the sentinel node offers a unique possibility to obtain tumour-reactive lymphocytes. we investigated antitumour immune responses in sentinel nodes from patients with bladder cancer, by assaying tumour-specific proliferation and tcr vb repertoires. during tumour surgery, sentinel lymph nodes were identified by peritumoural injection of blue dye. fresh specimens of tumour, sentinel and nonsentinel lymph nodes were obtained, and single-cell suspensions were prepared. cells were assayed for reactivity against autologous tumour extract in [ 3 h]-thymidine incorporation assays and characterized by flow cytometry. parallel analyses of the expression of vb gene families were performed with padlock probes, linear oligonucleotides which upon target recognition can be converted to circular molecules by a ligase. probes were reacted with cdna prepared from magnetically separated cd4 þ cells, and the tcr repertoire was determined by hybridizing the products to oligonucleotide microarrays. dose-dependent proliferation in response to tumour extract could be detected in sentinel lymph nodes. common clonal expansions were detected among tumourinfiltrating lymphocytes and in sentinel lymph nodes. nonsentinel lymph nodes displayed a divergent tcr vb repertoire. these results indicate an ongoing immune response against tumour antigens in sentinel nodes, draining urinary bladder cancer. identification of sentinel lymph nodes makes it possible to obtain tumour-reactive lymphocytes for use in adoptive immunotherapy. key: cord-015910-d9gxew91 authors: grimble, robert f. title: the interaction between nutrition and inflammatory stress throughout the life cycle date: 2005 journal: nutrients, stress, and medical disorders doi: 10.1385/1-59259-952-4:387 sha: doc_id: 15910 cord_uid: d9gxew91 the human race inhabits a world in which it is surrounded by a myriad of different microorganisms—yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately 0.1% of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, 2003). history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, 1999). the human race inhabits a world in which it is surrounded by a myriad of different microorganisms--yeasts, bacteria, protozoa, and viruses. most of these are benign, and some, such as the normal gut flora, play an important part in promoting health via the synthesis of vitamins and stimulation of normal function of gut epithelia. approximately 0.1% of microbes in our environment have catastrophic effects if they penetrate the epithelial surfaces of the body (bryson, 2003) . history reveals many instances in which armies have been defeated and civilizations have collapsed because of encounters between humans and such microorganisms (diamond, 1999) . humans, like all mammals, have evolved with a complex immune system, which is present as specialized organs (spleen, thymus) or cell types (lymphocytes, macrophages, and mast cells) throughout the body. the system can detect and destroy any cell or particle that is not"self," i.e., a normal component of the body. a complex series of events follows from contact between components of the immune system and microbes invading the body ( fig. 1) . the response can be divided into two main categories. the first is the acquired immune response, in which the immune system recognizes specific chemical motifs on the invader and "remembers" the encounter so that a more rapid, specific, and intense response can be produced at any future meeting. the second category is the nonspeciflc response in which the response to each encounter is similar for all invaders of the body. the process of inflammation is a central part of the second category of response. the immune response is also activated by a wide range of adverse events, such as surgery, bums, and trauma. the primary purposes of the response are to kill pathogens and initiate the curative processes that will restore body function to normal. the first purpose is achieved by creating a hostile tissue environment through production of oxidant molecules and activation of t and b lymphocytes. part of the response ensures a supply of substrate, from endogenous sources, for supporting the activity of t and b lymphocytes and enhancement of antioxidant defenses. the latter event is important for protecting healthy tissue from the oxidants produced as part of the inflammatory response (grimble, 2001 a) . the response exerts considerable biological demands and stress on the body. a central part of substrate provision is the release of amino acids into the blood from the breakdown of proteins in skeletal muscle, skin, and bone matrix, and fatty acids released from triglycerides stored in adipose tissue. enhanced gluconeogenesis, catabolic hormone production, and decreased insulin sensitivity occurs to facilitate this redistribution of tissue infective or cellular fig. 2 . diseases and conditions in which inappropriate or excessive amounts of pro-inflammatory cytokines exert adverse or lethal effects on the host. components (fig. 1) . the animal loses the desire to carry out many day-to-day activities. physical weakness ensues, exploratory activity declines, appetite is decreased, and apathy and sleep may occur. the response thus exerts physiological and mental stress upon the body. inflammation comes under the control of signaling proteins (cytokines) that possess hormone-like actions. the pro-inflammatory cytokines interleukin (il)-113, il-6, and tumor necrosis factor (tnf)-t~, are major activators and modulators of the events described above. to modulate the degree of stress imposed on the body, in achieving the essential functions of inflammation, the response comes under the control of powerful anti-inflammatory mechanisms. these will impose their biological effects with increasing vigor as the original stimulus for the inflammatory response (infection, injury) declines in intensity. heat-shock proteins, endorphins, glucocorticoid hormones, and cytokine receptor antagonists are important components of this anti-inflammatory system.this system is essential for closing down the inflammatory response once it has achieved its primary purposes because of the high biological cost it imposes on the body (grimble, 2001 a) . although cytokines play an important role in the response to infection and injury, they can exert damaging and lethal effects on the host. many studies have shown that excessive or prolonged production of cytokines is associated with increased morbidity and mortality in a wide range of acute and chronic inflammatory conditions (fig. 2) . these include sepsis, adult respiratory distress syndrome, malaria, meningitis, cancer, cystic fibrosis, systemic lupus erythematosus, inflammatory bowel disease, rheumatoid arthritis, and asthma. events similar to those seen in the inflammatory response to injury and infection can be observed during the course of overt inflammatory diseases such as rheumatoid arthritis and crohn's disease and in diseases that have a covert inflammatory basis, for example, atherosclerosis and diabetes mellitus (fig. 3) . clearly the inflammatory response in these situations does not have a purposeful nature and contribute to the disease process. recent studies indicate that low-intensity inflammation occurs in elderly and obese individuals (grimble 2002 (grimble , 2003 . thus, the inflammatory response, which has evolved to allow humankind to survive infection and injury, is indiscriminate in both its triggers and targets. as a result, the process is a two-edged sword capable of both defending and damaging its bearer. during the remainder of this chapter we will be exploring the biological and nutritional factors that determine the intensity of, and outcome from, the inflammatory process. various components of the inflammatory response interact to modulate its intensity. predominant among these interactions are the relative amounts of pro-and anti-inflammatory cytokines produced during the response to microbes and injury and the effect of oxidant molecules on cytokine production. early work on cytokines and the response to infection linked excessive pro-inflammatory cytokine production with increased morbidity and mortality in a wide range of conditions, such as malaria, meningitis, and sepsis. however, research in the last 5 yr has shown that the balance in production between pro-and anti-inflammatory cytokines has a more direct bearing on the outcome of infection and injury. for example, in sepsis, plasma il-6 concentrations were higher and il-10 concentrations were lower in patients who died than in those who survived (arnalich et al., 2000; taniguchi et al., 1999) . a survey of over 400 patients admitted to hospital in the netherlands with fever showed that, independently of how the patients were clinically classified (positive blood cultures, presence of endotoxin), those who subsequently died had a higher plasma il-10:tnf-o~ ratio than patients who survived (van dissell, van langervelde, westendorp, kwappenberg, & frolich, 1998) . powerful oxidant molecules (e.g., superoxide, hydrogen peroxide, hypochlorous acid) are produced as part of the inflammatory response. their biological purpose is to destroy invading microbes. however, these molecules also have the capacity to damage host tissues and to increase the intensity of the inflammatory response. clearly both of these biological events can have adverse effects upon the host. the oxidant molecules activate at least two important families of proteins in the host that are sensitive to changes in cellular redox state. the families are nuclear transcription factor k b (nf-rd3) and activator protein 1 (ap1). these transcription factors act as "control switches" for biological processes, not all of which are of advantage to the individual. nf-rd3 is present in the cytosol in an inactive form, by virtue of being bound to an inhibitory unit i-r,b. phosphorylation and dissociation of i-r,b renders the remaining nf-~cb dimer active. the dissociated i-rd3 is degraded, and the active nf-r,b is translocated to the nucleus, where it binds to response elements in the promoter regions of genes. a similar translocation of ap1, a transcription factor composed of the protooncogenes c-fos and c-jun, from cytosol to nucleus, also occurs in the presence of oxidant stress. binding of the transcription factors is implicated in activation of a wide range of genes associated with inflammation and the immune response, including those encoding cytokines, cytokine receptors, cell adhesion molecules, acute-phase proteins, and growth factors (schreck, rieber, & baeurerle, 1991) (fig. 4 ) . activation of nf-rd3 can be brought about by a wide range of stimuli including pro-inflammatory cytokines, hydrogen peroxide, mitogens, bacteria and viruses and their related products, and ultraviolet (uv) and ionizing radiations. the extent of activation of nf-rd3 will depend in part upon the strength and efficiency of the antioxidant defenses of the body. these comprise endogenous components such as glutathione (gsh) and enzymatic components of antioxidant defenses, such as catalase, superoxide dismutase (sod), and gsh peroxidase, and dietary components that have antioxidant properties (e.g., vitamins c and e and polyphenolic compounds). the influence of modulation of inflammation by these dietary factors are dealt with later. an unfortunate side effect of activation of nf-r,b arises from the ability of the transcription factor to activate transcription of the genes of some viruses, such as human immunodeficiency virus (hiv) (fig. 4) . this sequence of events, in the case of hiv, accounts for the ability of minor infections to speed the progression of individuals who are infected with hiv towards acquired immunodeficiency syndrome (aids). thus, if antioxidant defenses are poor, each encounter with general infections results in cytokine and oxidant production, nf-~zb activation, and an increase in hiv replication. it is thus unfortunate that reduced cellular concentrations of gsh are a common feature of infections, including that from the hiv (staal, ela, & roederer, 1992) . oxidant damage to cells will indirectly create a pro-inflammatory effect by the production of lipid peroxides. this situation may also lead to upregulation of nf-tcb activity. as will be seen in later sections, genetic and dietary factors change the intensity of the inflammatory response. thus, although the inflammatory response has evolved to ensure the survival of the human species, individuals may die as a result primarily of the response to invasion rather than from the invasive agent itself. it has recently become apparent that single base changes (single-nucleotide polymorphisms [snps]), usually in the promoter region of genes responsible for producing the molecules involved in the inflammatory process, exert a modulatory effect on the intensity of inflammation. in vitro production of tnf-o~ by peripheral blood mononuclear cells (pbmcs) from healthy and diseased subjects stimulated with inflammatory agents shows remarkable individual constancy in males and postmenopausal females (jacob et al., 1990) . this constancy suggests that genetic factors exert a strong influence. a number of studies have shown that snps in the promoter regions for the tnf-o~ and lymphotoxin table 1 single nucleotide polymorphisms (snps) in cytokine genes associated with altered levels of cytokine production # gene and location of polymorphism in promoter region genotype associated with raised cytokine production and~or altered clinical outcome to inflammation b pro-inflammatory cytokines tnf-t~ -308 lt-t~ + 252 il-l~-511 il-6-174 anti-inflammatory cytokines il-10-1082 c tgf-1 [~ + 915 (arg-25-pro) c tnf-ct -308 a allele (tnf2) lt-t~ + 252 aa (tnfb2:2) ct or tt g allelle gg gg tnf, tumor necrosis factor; lt, lymphotoxin; il, interleukin; tgf, transforming growth factor; c, cytosine; g, guanosine; t, thymidine, a, adenine. athe location of the polymorphism is indicated by the nucleotide position in the promotor region. bpoor clinical outcome for pro-inflammatory cytokines. clmproved clinical outcome for anti-inflammatory cytokines. (lt)-o~ genes are associated with differential tnf-t~ production (allen, 1999; messer et al., 1991; wilson et al., 1993) . the tnf2 (a) and tnfb2 (a) alleles (at -308 and +252 for the tnf-o~ and lt-t~ genes, respectively) are linked to high tnf production, particularly in homozygous individuals. the snp in the lt-ot gene (+252) is found in linkage disequilibrium with major histocompatibility molecules hla-a1, b8, dr3 (messer et al., 1991; wilson et al., 1993) . this genotype has also been reported to define a tnf "high expresser" haplotype (warzocha et al., 1998) , in addition to modifying expression of lt-o~ itself (messer et al., 1991) . a large body of research has indicated that snps occur in the upstream regulatory (promoter) regions of many cytokine genes (bidwell et al., 2001) . many of these genetic variations influence the level of expression of genes and the outcome from the inflammatory response. both pro-and anti-inflammatory cytokines are influenced by the differences in genotype (allen 1999; turner, williams, & sankeran, 1997) . a number of snps that have been implicated in the outcome of inflammatory stress are shown in table 1 . nf-lcb is activated by oxidants and switches on many of the genes involved in the inflammatory response (cytokines, adhesion molecules, and acute-phase proteins). enhancement of antioxidant defenses is important in protecting healthy tissues and in preventing excessive activation of nf-~zb by the oxidative cellular environment during inflammation (schreck et al., 1991) . nf-rd3 upregulates cytokine and adhesion molecule expression, increasing the risk of host damage (jersmann, hii, ferrante, & ferrante, 2001) . genetic factors also influence the propensity of individuals to produce oxidant molecules and thereby influence nf-rd3 activation. natural resistance-associated macrophage protein 1 (nramp1) has effects on macrophage functions, including tnf-ct production and activation of inducible nitric oxide synthase (inos), which occurs by cooperation between the nramp1, tnf-o~, and lt-o~ genes (ables et al., 2001) . there are four variations in the nramp1 gene, resulting in different basal levels of activity and differential sensitivity to stimulation by inflammatory agents. alleles 1, 2, and 4 are poor promoters, whereas allele 3 causes high gene expression. hyperactivity of macrophages, associated with allele 3, is linked to autoimmune disease susceptibility and high resistance to infection, whereas allele 2 increases susceptibility to infection and protects against autoimmune disease (searle & blackwell, 1999) . as indicated earlier, a number of molecules suppress production of pro-inflammatory cytokines and exert an anti-inflammatory influence. these include antioxidant defenses and il-10 (chernoff et al., 1995; espevik et al., 1987) . production is modulated by genetic factors. there are at least three polymorphic sites (-1082, -819, -592) in the il-l0 promoter that influence production (perrey, pravice, sinnott, & hutchinson, 1998) . snps also occur in genes encoding enzymatic components of antioxidant defenses, such as catalase, sod, and gsh peroxidase, which influence levels of activity (chorazy, schumacher, & edlind, 1992; forsberg, lyrenas, de faire, & morgenstern, 2001; mitrunen et al., 2001) . there is circumstantial evidence, that at an individual level, an inflammatory genotype exists that can adversely effect the host. in a study of inflammatory lung disease caused by exposure to coal dust, the tnf2 (lt-a+252 a) allele was almost twice as common in miners with the disease than in those who were healthy (zhai, jetten, schins, franssen, & borm, 1998) . development of farmer's lung from exposure to hay dust was 80% greater in individuals with the tnf2 allele than in those without the allele (schaaf, seitzer, pravica, aries, & zabel, 2001) . the tnf2 allele was also twice as common in smokers who developed chronic obstructive pulmonary disease than in those who remained disease-free (sakao et al., 2001) . in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease. during malaria, children who were homozygous for tnf2 had a sevenfold greater risk of death or serious pathology than children who were homozygous for the tnf1 allele (mcguire, hill, allsopp, greenwood, & kwiatkowski, 1994) . in intensive-care patients the occurrence of 1082"g high-producing allele for il-10 was present in those who developed multiorgan failure with a frequency of one-fifth of that of the normal population (reid, hutchinson, campbell, & little, 1999) . in sepsis, patients possessing the tnf2 allele had a 3.7-fold greater risk of death than those without the allele, and patients who were homozygous for the lt-~ + 252 a allele had twice the mortality rate and higher peak plasma tnf-ot concentrations than heterozygotic individuals (mira et al., 1999; stuber, peterson, bokelmann, & schade, 1996) . the tnf2 allele also been found in increased frequencies in systemic lupus erythromatosus, dermatitis hepetiformis, and insulin-dependent diabetes mellitus and noninsulin-dependent diabetes mellitus (niddm) (jacob et al., 1990 , wilson, clay, & crane, 1995 wilson, gordon, & di giovine, 1994) . thus, it now appears that each individual possesses combinations of snps in their genes associated with inflammation corresponding to inflammatory drives of differing intensities when microbes or tissue injury are encountered. at an individual level this may express itself as differing degrees of morbidity and mortality (fig. 5) . the strength of the genomic influence on the inflammatory process may affect the chances of an individual developing inflammatory disease, particularly if their antioxidant defenses are poor. in addition to disease progression, genetic factors have important effects on mortality and morbidity in infectious and inflammatory disease and following injury (paolini-giacobino, grimble, & pichard, 2003) . there are sex-linked differences in the influence of genotype on the inflammatory processes. in general, males are more sensitive to the genomic influences on the strength of the inflammatory process than females. in a study on lt-a genotype and mortality from sepsis, it was found that men possessing a tnfb22 (lt-o~+ 252 aa) genotype had a mortality of 72% compared with men who were tnfb 11 (lt-o~+ 252 gg), who had a 42% mortality rate. in female patients the mortalities for the two genotypes were 53% and 33%, respectively (schroder, kahlke, book, & stuber, 2000) . in a study on patients undergoing surgery for gastrointestinal cancer, it was found that postoperative c-reactive protein (crp) and il-6 concentrations were higher in men than in women. multivariate analysis showed that males possessing the tnf2 (tnf-o~-308 a) allele had greater responses than men without it. the genomic influence was not seen in females (table 2) (grimble, thorell, et al., 2003) . furthermore, possession of the il-1-511 t allele was associated with a 48% greater length of stay in hospital in old men admitted for geriatric care (table 3) (grimble, anderson, et al., 2003) . women were unaffected by these genetic influences. paradoxically, with improvements in hygiene and vaccination programs against infectious diseases, two major changes in public health and population characteristics have led to a general increase in inflammatory stress in populations of industrialized countries in the last half century. these are, respectively, an increase in the number of overweight and obese subjects and an increase in longevity. we will now examine the mechanisms underlying this phenomenon. it has been recognized for many years that there is a strong link between the "diseases of affluence"--obesity, insulin sensitivity, and atherosclerosis. however, it is only quite recently that the realization came that inflammation provided a link between the three (5) tnf, tumor necrosis factor; il, interleukin; crp, c-reactive protein. a2 d postoperatively. bl d postoperatively. *significantly different from females with same genotype by multivariate analysis allowing for longer operation time and greater blood loss; p = 0.013 andp = 0.027 for crp and il-6, respectively. means + sd, values in parentheses are the number of patients. (28) 27+13 (9) 19 +15 (26) 14+13(16)* 25 +14 (28) tnf, tumor necrosis factor; il, interleukin; c, cytosine; t, thymidine; a, adenine; g, guanine. athe location of the polymorphism is indicated by the nucleotide position in the il-1 ~ and lt-t~ genes, tnfb 11 (gg), tnfb 12(ag), tnfb22(aa). *significantly different from value for same sex possessing the other genotype; p < 0.05 using mann-whitney test. means + sd, values in parentheses are the number of patients. biological phenomena (fig. 3) . many studies have shown a clear link between obesity, oxidant stress, and inflammation (grimble 2002) . the link may lie in the ability of adipose tissue to produce pro-inflammatory cytokines, particularly tnf-t~. there is a positive relationship between adiposity and tnf production. a positive correlation ,i, plasma triglyceride concentrations fig. 6 . interaction between leptin and tumor necrosis factor (tnf) with adipose tissue mass, lipid metabolism, and inflammation. tnf and leptin stimulate the immune system and adipose tissue, respectively. both also act on lipid metabolism and plasma triglyceride concentrations. between serum tnf-~ production and body mass index (bmi) has been noted in niddm patients and healthy women (nilsson, jovinge, niemann, reneland, & lithell, 1998; yaqoob, newsholme, & calder, 1999) . leptin has been shown to influence proinflammatory cytokine production ( fig. 6 ). thus, plasma triglycerides, body fat mass, and inflammation may be loosely associated because of these endocrine relationships. a number of population studies have been conducted to explore the extent and nature of the relationship of inflammation to these diseases of affluence. the studies have examined populations in which there is a high incidence of insulin insensitivity, such as pima indians and individuals with a south asian background. tnf-o~ is overexpressed in adipose and muscle tissues of obese subjects compared with tissues from lean individuals (hotamisligl & spiegelman. 1994) . in a study of a group of nondiabetic pima indians, employing the hyperinsulinemic euglycemic clamp to assess insulin action, strong evidence of the links between inflammation, insulin insensitivity, and obesity emerged. plasma il-6 was found to be related positively to adiposity and negatively to insulin sensitivity. the investigators concluded that the relationship between il-6 and insulin action appeared to be mediated through adiposity (vozarova, weyer, & hanson, 2001) . a number of studies have looked at the extent of the interaction between insulin insensitivity and inflammation by studying the extreme form of diabetes, type 1 diabetes mellitus. a study assessed endothelial cell perturbation by measurement of von willebrand factor and tissue-plasminogen activator (t-pa), in type 1 diabetics who had had the disease for <1 or >1 yr. compared with normal subjects, children with diabetes for <lyear had the highest concentrations of von willebrand factor, indicating that endothelial perturbation represents an early event in type 1 diabetes (romano, pomilio, & vigneri, 2001) . studies that have attempted either to remove the cause of inflammation, to lower plasma lipids, or improve insulin sensitivity have supported the hypothesis that inflammation, insulin sensitivity, and atherosclerosis are intimately interlinked. when a study of the potential anti-inflammatory effect of weight loss was conducted in obese women, it was found that a 1 yr weight-reduction program resulted in lowering of plasma il-6, tnf-cx, and adhesion molecule concentrations (ziccardi, nappo, & giugliano, 2002) . since the 1990s large population studies have indicated that inflammation plays a key role in cardiovascular disease (cvd) (grimble, 1990; ross, 1993) . periodontal disease and other low-grade infections, such as chlamydia pneumoniae infection, have been linked closely with atherosclerosis. development of atherosclerotic plaques to which macrophages have already been recruited occurs by cytokines inducing hypertriglyceridemia and hypercholesterolemia (kol, sukhova, lichtman, & libby, 1998; saldeen & rand 1998) . chlamydial infection induces production of tnf-~. the cytokine inhibits the action of lipoprotein lipase, leading to changed lipid metabolism, elevation of serum triglycerides, and a decrease in serum high-density lipoprotein (hdl) cholesterol, thereby exerting an atherogenic influence (arrnitage, 2000) . in the bruneck study, raised plasma bacterial lipopolysaccharide (lps) was associated with an increased rate of thickening of the coronary artery intima. smoking, a further inflammatory stress, exacerbated this effect (williet & kiechl, 2000) . lps binds in human serum to both low-density lipoprotein (ldl) and hdl cholesterol and makes ldl cholesterol immunogenic or toxic to endothelial cells. thus, the link between specific infections and atherosclerosis may be a nonspecific effect of chronic inflammation on the atherosclerotic process. it is well recognized that alterations in plasma protein concentrations invariably occur among the many metabolic changes that occur during inflammation. proteins that increase during inflammation (positive acute-phase proteins, e.g., crp and fibrinogen) and those that decrease (negative acute-phase proteins, e.g., serum albumin and retinolbinding protein) are used to diagnose inflammation in population nutritional surveys. the early indications that inflammation was involved in atherosclerosis came from the findings that there were links between concentrations of positive and negative acute-phase proteins in blood and cvd (grimble, 1990 ). in the bruneck study of a group of 826 40to 79-yr-old italians, it was found that impaired glucose tolerance and, to a greater extent, type 2 diabetes were strong independent predictors of advanced atherosclerosis (measured by high-resolution ultrasound of the carotid artery) (bonora, kiechl, & oberhollenzer, 2000) . a cross-sectional study of obesity, plasma crp, fibrinogen, and carotid artery intima media thickness (an index of atherosclerosis) in more than 1500 multiethnic subjects showed a positive relationship between plasma crp and body fat. intima media thickness was related to crp and fibrinogen in men. the relationship was attenuated by adjustment for bmi (festa, d'agostino, & williams, 2001) . a cross-sectional study of more than 1800 men and women examined the link between elevated plasma crp concentrations and prevalent cvd, ankle/brachial blood pressure, and carotid artery intima media thickness. after adjustment for age and family type, there was a weak association between crp and intima media thickness in both sexes and with prevalent heart disease in women (folsom, pankow, & tracy, 2001) . a study on a turkish population of 1046 individuals with low cholesterol concentration but a high prevalence of other risk factors for coronary heart disease investigated whether crp acted as a predictor of coronary heart disease. among the risk factors, only crp and systolic blood pressure were independent risk factors for cvd (onat, sansoy, & yildirim, 2001) . in a study in which crp concentrations and conventional risk factors for cvd in 500 healthy indian asians were compared with values in a similar number of healthy european white subjects, crp and cvd risk factors were higher in the former group. however, differences were eliminated when adjustment was made for central obesity and insulin resistance score (chambers, eda, & bassett, 2001) . a study on a brazilian population found that markers of inflammation correlated with components of the metabolic syndrome---cardiovascular and diabetes risk factors, insulin resistance, and central obesity (duncan & schmidt, 2001) . in a study of 574 healthy elderly subjects in the netherlands, acute-phase proteins, soluble adhesion molecules, il-6, and insulin were measured and associated with cholesterol and obesity. the association between insulin, obesity, and cholesterol was as strong as between insulin, acute-phase proteins, and adhesion molecules (hak, pols, & stehouwer, 2001) . insulin insensitivity occurs as part of the normal inflammatory response to pathogens. during inflammation the secretion of catabolic hormones, which enhances muscle protein breakdown and glutamine release, will oppose insulin action. paradoxically, however, although insulin insensitivity may initially exert a beneficial effect during the response to infection and injury, it has an adverse influence in chronic disease processes. glucose and glutamine act as major fuels for immune cells during the normal response to infection. large increases in glucose and glutamine utilization by immune cells occur during the response to infection and injury (spitzer, bagby, meszaros, & lang, 1988 , 1989 . studies in rats given lps and observations in patients with sepsis show that the flow of amino acids into the circulation increases and gluconeogenesis is enhanced under the influence of pro-inflammatory cytokines. an insulin-insensitive state will reduce glucose uptake by tissues in which the process is insulin dependent (muscle), thereby increasing availability to tissues in which the process is not insulin dependent (immune tissue). a study in the united states investigated whether elevated plasma il-6 and crp was associated with the development of type 2 diabetes mellitus in more than 27,000 healthy women. in the 4-yr follow-up period, 188 women developed type 2 diabetes. for these women, baseline il-6 and crp were higher than in controls. the relative risk of future type 2 diabetes in women between the highest and lowest quartiles of these inflammatory markers was 7.5 for il-6 and 15.7 for crp (pradhan, manson, & rifai, 2001) these data suggest a possible role for inflammation in diabetogenesis. furthermore, data collected from the third national health and nutrition examination survey (nhanes iii) in the united states provide further evidence for a possible role of inflammation in insulin resistance and glucose intolerance. more than 2500 men and women were studied for associations between plasma crp, fasting insulin, glucose, and glycosylated hemoglobin (hba 1 c). elevated crp was associated with higher insulin and hba 1 c in both sexes and with raised glucose in women (wu, dorn, & donahue, 2002) . a study on the link between crp, central adiposity, and fasting glucose and insulin in more than 200 healthy italian women showed an independent relationship of adiposity to insulin resistance and crp concentrations (pannacciulli, cantatore, & minenna, 2001) . in a review, nishimura and murayama (2001) discussed the possibility that treatment of periodontal infection may improve insulin sensitivity. their conclusions about the efficacious effect of an anti-infective approach are supported by a study in which 13 type 2 diabetic patients with periodontal disease were given antimicrobial treatment with minocycline. blood tnf-t~ concentrations and glycosylated hemoglobin decreased (iwamoto, nishimura, & nakagawa, 2001) . conversely, improvement in insulin sensitivity exerted an anti-inflammatory effect. a group of 18 hyperlipidemic patients and 20 normolipidemic controls who were insulin resistant and hypertriglyceridemic with low hdl cholesterol concentrations and raised tnf-o~ production and plasma il-6 and fibrinogen concentrations were studied. all subjects were treated with the lipid-lowering drug bezafibrate. the drug normalized all parameters.the drug thus exerts an anti-inflammatory effect associated with its ability to normalize lipid metabolism and insulin sensitivity (jonkers, mohrschladt, & westendorp, 2002) . in an in vitro study on a lung epithelial cell line, the oral hypoglycemic agent thiazolidinedione exerted an anti-inflammatory influence by suppressing production of monocyte chemoattractant protein (mcp-1) (momoi, murao, & imachi, 2001 ). it can be concluded from the studies reported in the above sections that inflammation is closely linked to obesity, cvd, insulin insensitivity, and diabetes mellitus. although this interaction is a relatively novel concept, it has been known for many years that these diseases are interlinked and that insulin insensitivity is a common factor in their pathology. a number of recent studies have examined the mechanisms underlying the link between inflammation and the conditions and diseases in which insulin insensitivity plays a part (fig. 3) . a difficult question to address is whether chronic inflammation leads to a condition of insulin insensitivity and associated diseases, or whether insulin insensitivity, which is associated with obesity, diabetes, and atherosclerosis, brings about a condition of chronic inflammatory stress. many studies suggest that the former is more likely to be the case. reviews have highlighted the risk of inflammation and infection of atherosclerotic plaques associated with poor diabetic control and the importance of elevated nonesterified fatty acid concentrations, glucocorticoids, and low-grade inflammation as causative agents in atherosclerosis and insulin insensitivity (bell, 2000; corry & tuck, 2001) . a study on type 2 diabetes mellitus patients determined the extent to which pbmcs contributed to oxidative stress and inflammation. a linear correlation between hbalc and superoxide release was found. the authors concluded that type 2 diabetes mellitus is accompanied by priming of pbmcs and increased self-necrosis. the necrosis may start a chain of events that results ultimately in oxidant stress and endothelial dysfunction (shurtz-swirski, sela, & herskovitis, 2001) . in a study on pima indians, who are characterized by high incidence of obesity and insulin resistance but not atherosclerotic disease, crp, icam-1, and secretory phospholipase a2 are correlated with body fat but not e-selectin and von willebrand factor. in addition to showing that markers of inflammation increase with adiposity, the study showed that markers of endothelial dysfunction increase in proportion to insulin resistance and inflammation (weyer, yudkin, & stehouwer, 2002) . a further study on pima indians examined whether a raised white blood cell count predicted a worsening of insulin action, insulin secretory function, and the development of type 2 diabetes. a high white blood cell count predicted the development of diabetes with a relative risk of 2.7 when adjusted for age and sex (vozarova, weyer, & lindsay, 2002) . tnf-o~ has been shown to play a key role in mediating insulin resistance as a result of obesity in patients and in numerous rodent models of obesity---diabetes syndromes (hotamiligil & spiegelman, 1994) . multiple mechanisms have been suggested to account for these metabolic effects of tnf-ot. these include the downregulation of genes that are required for normal insulin action, direct effects on insulin signaling, induction of elevated plasma free fatty acids via stimulation of lipolysis, and negative regulation of ppar-7, an important insulin-sensitizing nuclear receptor (moiler, 2000) . the induction of insulin resistance is mediated through its ability to produce serine phosphorylation of insulin receptor substrate (irs)-1, decreasing the tyrosine kinase activity of the insulin receptor (hotamisligil, budavari, murray, & spiegelman, 1994) . neutralization of tnf-t~ in obese fa/fa rats by intravenous administration of a soluble tnf receptor immunoglobulin g chimeric protein substantially improved insulin sensitivity and restored the tyrosine kinase activity in fat and muscle (hotamisligil et al., 1996) . in an in vitro study using 3t3-l 1 adipocytes, tnf-t~ was shown to induce sustained suppressor of cytokine signaling protein 3 (socs-3) production. socs-3 has been shown to decrease insulin-induced irs 1 tyrosine phosphorylation and its association with the p85 regulatory subunit of phosphatidylinositol-3 kinase (emanuelli, peraldi, & filloux, 2001) . these observations therefore suggest that socs-3 may be a key mediator in the development of insulin sensitivity during inflammation. obesity is associated with insulin resistance, particularly when body fat has a central distribution. while elevated plasma leptin concentrations are associated with obesity, some studies have suggested that insulin sensitivity is an additional determinant of circulating leptin concentrations. leptin is produced by adipose tissue in proportion to adipose tissue mass and is a pleiotropic molecule. in addition to playing a role in appetite and adipose tissue regulation, leptin influences immune functions. leptin concentrations increase acutely during inflammation and regulate t-cell responses, polarizing t-helper (th) cells toward a th 1 phenotype. thus, increased leptin production during obesity, may exert a pro-inflammatory influence (faggioni, feingold, & grunfeld, 2001) . further complexity is added to the concept of a link between insulin sensitivity, inflammation, and obesity by the results of a study of 268 individuals selected from the health professionals follow-up study in the united states (chu, spiegelman, & hotamisligil, 2001) . in the study plasma insulin, leptin, and soluble tnf receptor (stnf-r, an index of tnf-o~ production) concentrations were measured and correlated with bmi and the cvd risk factors insulin, triglyceride, t-pa antigen levels, and apolipoprotein (apo)-a1. in a multivariate regression model controlling for age, smoking, alcohol intake, physical activity, and diet, bmi was inversely associated with hdl cholesterol and apo-a1 and positively associated with trigyceride, apo-b, and t-pa antigen levels. the associations between bmi and these cvd risk factors were only slightly changed after adjusting for leptin and/or stnf-r, but were substantially attenuated after controlling for insulin levels. these data suggest that the association between obesity and biological predictors of cvd may be mediated through changes in plasma insulin, rather than leptin or stnf-r levels, and that insulin may be exerting an anti-inflammatory effect. (cnop, landchild, & vidal, 2002) . genetic factors may play a part in the interaction between inflammation, insulin insensitivity, and obesity. nf-~:b is an important mediator of inflammation by increasing transcription of a range of genes central to the inflammatory process (cytokines, adhesion molecules, acute-phase proteins). snps in the nf-r,~b gene were investigated in a group of 217 type 1 diabetic patients and compared with gene frequencies in 111 normal controls. it was found that there was a higher frequency of allele 138 bp (a10) (high bioactivity) and a lower frequency of allele 146 bp (a14) (less bioactive) in diabetics than in controls. genotype may thus contribute to inflammatory stress in diabetes mellitus (hegazy, o'reilly, & yang, 2001) . nf-~:b may also provide an important focus for ppar-7 action, because it has been shown in a number of studies that ppar-~/in combination with retinoid x receptor is able to inhibit nf-~:b activation (wada, nakajima, & blumberg, 2001; fruchart, staels, & durlez, 2001; debril, renaud, fajas, & auwerx, 2001) . it is interesting to note that the n-3 polyunsaturated fatty acids (pufas), found in abundance in fish oil, are ppar-y agonists, raising the possibility that the oil may exert its anti-inflammatory effects partly via this mechanism. in an investigation of cytokine production in 139 healthy males, the author found that in the study population as a whole there were no statistically significant relationships between bmi, plasma fasting triglycerides, and the ability of pbmcs to produce tnf-a. however, individuals with the lt-c~+ 252 aa genotype (associated with raised tnf production) showed significant relationships between tnf production and bmi and fasting triglycerldes (fig. 7) . thus, despite the study population being comprised of healthy subjects, within that population were individuals with a genotype that resulted in an "aged" phenotype as far as plasma lipids, bmi, and inflammation were concerned (paolini-giacobino et al., 2003) . it is well known that the incidence of diseases of affluence and recognizable inflammatory diseases, such as rheumatoid arthritis, increase with aging. are these phenomena an unfortunate side effect of maturity, or is there a common mechanism that determines the appearance of these diseases patterns in the elderly.'? paradoxically, aging is associated with a decline in t-lymphocyte function and an increase in inflammatory stress. a number of elements of the chronic inflammatory response are apparent in otherwise healthy elderly subjects. the elements of the response include muscle protein loss, a rise in plasma acute-phase protein concentrations, and a decrease in plasma zinc. an age-related increase in il-6 concentration has been found in serum, plasma, and supernatants of mononuclear blood cell cultures from apparently healthy elderly people and centenarians (fagiolo et al., 1993; baggio, donnazzan, monti, & marl, 1998; ershler & kerller, 2000) . because il-6 is a pleiotropic cytokine capable of regulating proliferation, differentiation, and activity of a variety of cell types (ershler & kerller, 2000) and plays a pivotal role in neuroendocrlne and immune system homeostasis, it is not surprising that the rise in production of pro-inflammatory cytokines might have long-term pathological effects (bethin, vogt, & muglia, 2000 fasting triglycerides fig. 7 . relationships between tnf production, bmi and fasting triglycerides, and the ability of pbmcs to produce tnf-o~ in the study populations: (a) all subjects irrespective of genotype, (b) individuals with the lt-ct+ 252 aa genotype, associated with raised tnf production. ns, nonsignificant relationship; bmi, body mass index; pbmcs, peripheral blood mononuclear cells. the number of subjects is shown in parentheses; the correlations were examined by spearman' s rank correlation. levels of this cytokine have also been found as early as at 30-40 yr of age (mysliwska, bryl, foerster, & mysliwski, 1998) , particularly in men (young, skibinski, mason, & james, 1999) . population studies have shown that the magnitude of increase in the concentration of il-6 is a reliable marker for functional disability and a predictor of mortality in the elderly (ferrucci, harris, guralnik, & tracy, 1999; harris, ferrucci, tracy, & corti, 1999) . antioxidant status may decline with age (nuttall, dunne, kendall, & martin, 1999) and may thus be linked to increased tnf-~ production (kudoh, katagai, takazawa, & matsuki, 2001; rink, cakman, & kirchner, 1998 ). an enhanced capacity for the release of pro-inflammatory cytokines by white blood cells may contribute to the pathogenesis of ischemic stroke. grau et al. (2001) investigated the lps-induced release of il-1 [3, il-6, il-8, and tnf-0~ in whole blood from patients with a history of ischemic stroke under the age of 50 and age-and sex-matched healthy control subjects. release ofll-8 was significantly higher in young stroke patients than in control subjects (grau et al., 2001) . the question of whether aging is associated with chronic elevation of cytokine production or whether an increased capacity for cytokine production following the normal inflammatory challenges of life develops during aging is an interesting one to consider. insight into this issue can be gained from the response to surgery where an inflammatory stimulus is applied at a defined moment in time, making it easy to follow the subsequent response. ono, aosasa, tsujimoto, ueno, and mochizuki (2001) investigated the agerelated changes in the inflammatory response in patients with gastric cancer undergoing distal gastrectomy. patients were divided into two groups: >75 yr of age (elderly group) and -<75 years of age (young group). serum il-6 concentrations, tnf-ct production and cd 11 b/cd 18 expression by monocytes, and the postoperative clinical course were compared between the two groups to assess the inflammatory response to surgery. tnf-~ production by lps-stimulated monocytes and cd 1 l b/cd 18 expression on monocytes were significantly higher in the elderly than in the young group. moreover, serum il-6 concentrations on the first postoperative day in the elderly group were significantly higher than those in the young group. paradoxically, both loss of body weight and lean tissue and obesity are found in elderly populations. is there, therefore, a link between this phenomenon and increased levels of inflammation? the loss of muscle mass and strength that occurs with aging is described clinically as sarcopenia (rosenberg, 1989; roubenoff, 2001) . it is an important contributor to the development of frailty and functional impairment during aging. it is well established that aging is associated with a significant decline in muscle strength that becomes functionally important by the seventh decade of life. the relationship between chronic inflammation owing to disease during aging and the prevalence of low body mass are well illustrated in rheumatoid arthritis. in a study on patients with rheumatoid arthritis, the loss of body mass was greater for lean tissue than fat, with over 50% of the rheumatoid group falling into the lowest 10th percentile of a reference population for skeletal muscle mass assessed from the upper arm muscle area. in female patients there was a significant correlation between reduced fat-free mass and two indicators of inflammatory stress---erythrocyte sedimentation rate and plasma crp concentration (munro & capell, 1997) . clinical and animal studies show a relationship between raised plasma cytokine concentrations and low muscle mass. visser et al. (2002) investigated whether markers of inflammation are associated with muscle mass and strength over a time course of several years in over 3000 healthy well-functioning black and white elderly persons (70-79 yr). mid-thigh muscle cross-sectional area, appendicular muscle mass, and muscle strength were assessed. plasma concentrations of il-6 and tnf-~ were also measured. higher cytokine concentrations were associated with lower muscle mass and lower muscle strength. the most consistent relationship across the gender and race groups was observed for il-6 and grip strength. when an overall indicator of elevated cytokine production was created by combining the concentrations of il-6 and tnf-o~, with the exception of white men, elderly persons having high concentrations of il-6 (> 1.80 pg/ml) as well as high levels of tnf-~ (>3.20 pg/ml) had a smaller muscle area, less appendicular muscle mass, and lower muscle strength compared to those with low levels of both cytokines. thus, raised plasma concentrations of il-6 and tnf-~ are associated with lower muscle mass and lower muscle strength in well-functioning older men and women as well as those suffering frank inflammatory disease. nutrient intake is clearly another important determinant of lean body weight and fat mass and may play a part in the decline in lean tissue with age as well as an increase in inflammatory stress. a recent survey of 40,000 subjects in 88 communities in nhanes iii in the united states also included a survey of about 5000 elderly people ranging in age from 60 to 69 yr, 70 to 79 yr, and 80+ yr (marwick, 1997) . the report indicated that the median intake of total energy was in general lower than the recommended 2300 kcal for men and 1900 kcal for women (marwick, 1997) . chronic inflammation is either a causative agent or a closely associated process in the pathology of obesity, insulin insensitivity, and atherosclerosis.the incidence of these conditions increases with aging. a fundamental question is which precedes the other-the general increase in inflammation or the development of diseases with overt and covert inflammatory bases? this "chicken-and-egg" question is difficult to answer. however, examination of data from studies conducted in elderly populations may throw some light on the answer to this conumdrum. there are at least two potential mechanisms for the higher level of chronic inflammation observed in elderly than in younger subjects. the first of these is that the elderly are experiencing a higher level of asymptomatic urinary infection. this possibility was studied in 40 consecutive patients (70-91 yr) admitted to the hospital for functional disability. patients were examined for the presence or absence of bacteria in the urine. twenty subjects had a positive urine culture, and 20 sex-and age-matched subjects had a negative urine culture. inclusion criteria were temperature <37.8°c, no clinical signs of infection, and no current antibiotic treatment. patients with asymptomatic bacteriuria had significantly increased levels of tnf receptors and a higher number of neutrophils in the blood compared to the group without bacteriuria. thus, the study provides some support for the hypothesis that asymptomatic urinary infections are associated with low-grade inflammatory activity in frail, elderly subjects (prio, bruunsgaard, roge, & pedersen, 2002) . a second potential mechanism resides in endocrine changes during aging. in aging, dysregulation of secretion of hormones that come under the regulation of the hypothalamic-pituitary-adrenal axis may occur. this may have an effect on the regulation of cortisol secretion, as mentioned earlier. cortisol is important as an anti-inflammatory agent. the effect of aging on glucocorticoid sensitivity of pro-inflammatory cytokine production was examined in elderly men, testosterone-treated elderly men, and young controls. stress-induced increases in cortisol did not differ significantly between experimental groups, but glucocorticoid sensitivity increased significantly in young controls and testosterone-treated elderly men, whereas a decrease was found in untreated elderly men. as the increase in glucocorticoid sensitivity after stress serves to protect the individual from detrimental increases of pro-inflammatory cytokines, the disturbed mechanism in elderly men may result in an increase in inflammatory stress (rohleder, kudielka, hellhammer, wolf, & kirschbaum, 2002) . there is now a large body of evidence suggesting that the decline in ovarian function with menopause is associated with spontaneous increases in pro-inflammatory cytokine production. as mentioned earlier, studies in men and postmenopausal women indicate a remarkable individual constancy in the ability of pbmcs to produce tnf-cz ex vivo, and genetic determinants underlie this constancy. however in premenopausal women production is highly variable at an individual level, indicating how ovarian hormones are able to override the influence of genotype (jacob et al., 1990) . the exact mechanisms by which estrogen interferes with cytokine activity are still incompletely known but may include interactions of the estrogen receptor with other transcription factors, modulation of nitric oxide activity, antioxidative effects, plasma membrane actions, and changes in immune cell function. experimental and clinical studies also strongly support a link between the increased state of pro-inflammatory cytokine activity and postmenopausal bone loss (pfeilschifter et al., 2002) . recent evidence indicates the presence of snps, associated with the strength of the inflammatory response, affects longevity. human longevity may be directly correlated with optimal functioning of the immune system. therefore, it is likely that one of the genetic determinants of longevity resides in polymorphisms for genes influencing the activity of the immune system. it has been estimated that up to 7000 variations in the genome contribute to life span (martin, 1997) . those contributing to loss of muscle and bone mass during aging are related to the inflammatory process and include pro-and anti-inflammatory cytokines and their receptors. studies in mice have shown that the genes controlling the major histocompatibility complex (mhc), known to control a variety of immune functions, are associated with differences in the life span of different strains of mice, but a major difference between observations in mice and humans is that the latter have a lifetime experience of exposure to pathogens, whereas for laboratory animals this exposure is kept to a minimum. thus, although hla studies in mice of different genotypes may be interpreted to support studies of mhc effects on longevity in humans, in mice the association may be by way of altered susceptibility to lymphomas, whereas in human beings the effect on longevity is likely to be via an altered response to pathogens and susceptibility to infectious disease. a number of cross-sectional studies have examined the role of hla genes on human longevity by comparing hla antigen frequencies between groups of young and elderly persons. conflicting findings have been obtained. when this topic was reviewed (caruso et al., 2001) , it was concluded that in humans there may be an association between longevity and some hla-dr alleles or the hla-bs,dr3 haplotype. these genotypes are involved in the antigen nonspecific control of immune response, in other words, the component of immune function associated with inflammation and cytokine biology. recent evidence indicates that presence of snps in certain pro-and anti-inflammatory cytokine genes influences life span. when 700 individuals between 60 and 110 yr of age were studied, it was noted that not only was plasma il-6 concentration positively related to age but individuals with a snp in the promoter region of the il-6 gene, which predisposes to high levels of production of the cytokine (-174 gg), decreased in frequency with age. the effect was seen in men but not in women (bonafe, olivieri, & cavallone, 2001) . although men with snps made up 58% of the 60-to 80-yr-old age group, the percentage fell to 38% in subjects <99 years of age. conversely, one of three snps in the il-10 gene (-1082 gg), which is closely linked to higher production of the anti-inflammatory cytokine il-10 (hutchinson, pravica, hajeer, & sinnott, 1999; turner et al., 1997) , was found in higher proportions in male centenarians than in younger controls (58 vs 34%). in females this genotype exerted no effect upon longevity (lio et al., 2002) . thus, it would appear that genetic characteristics that might influence the balance between proand anti-inflammatory cytokines influence mortality in men but not in women (franceschi et al., 2000) . a study on snps that influence interferon (ifn)-~t production further reinforces the concept that possession of a genotype that predisposes to a raised pro-inflammatory status is not compatible with a long life span (lio et al., 2002) . in women, possession of the a allele, which is associated with low production of ifn-'~, significantly increased the possibility of reaching old age. it might be concluded that possession of high-producing alleles of the il-10 is universally protective against morbidity as well as mortality. possession of a genotype that results in low levels of il-10 production (-1082 aa) increases the risk of developing inflammatory diseases (hajeer, lazanes, & turner, 1998; huizinga, keijsers, & yanni, 2002; tagore, gonsalkorale, & pravica, 1999) . however, as already mentioned, in a large survey of hospitai admission in the netherlands, patients with raised il-10:il-6 ratios had higher mortality rates (van dissel et al., 1998) . not all studies implicate cytokine gene snp in longevity. cytokine gene polymorphisms at il-1 o~, il-1 ~, il-1ra, il-6, il-10, and tnf-o~ were measured in 250 finnish nonagenarians (52 men and 198 women) and in 400 healthy blood donors (18-60 yr) used as controls. no statistically significant differences were found in the distribution of genotype, allelic frequencies, and a2+ carrier status between nonagenarians and younger controls (wang, hurme, jylha, & hervonen, 2001) . in a review on the different impact of genetic factors on the probability of reaching old age, franceschi et al. (2000) concluded from studies conducted in italy that emerging evidence (regarding mtdna haplogroups, thyrosine hydroxylase, and il-6 genes) suggests that female longevity is less dependent on genetics than male longevity and that female centenarians are more likely to have had a healthier lifestyle and more favorable environmental conditions than males. however, a recent study conducted by our group suggests that although a pro-inflammatory genotype may be disadvantageous to elderly males, it may confer a survival benefit in females. subsequent survival was studied in 79 elderly geriatric patients (87 + 7 yr) after a period of hospitalization for a range of conditions necessitating geriatric care. although women possessing a pro-inflammatory genotype (tnf-o~-308 a allele or il-6-174 gg) had improved 3 yr survival rates, men possessing pro-inflammatory genotypes (il-l~-511 t allele or lt-ct +252 aa) had shortened survival rates (grimble, thorell, et al., 2003) (table 3) . as outlined in the preceding sections, the inflammatory response, although essential for survival in the presence of pathogens, can exert deleterious effects on the host.the clear need to find ways of modulating cytokine production and other aspects of inflammation has fostered the research area of immunonutrition. in a clinical context the purpose of immunonutrition is to find nutritional means of altering the patient' s inflammatory response to infection and injury, from the detrimental to the beneficial side of the pivot on which an individual undergoing a response is positioned. while inflammation may be exerting deleterious effects most obviously in patients, people on the borderline of health and disease living in the general population table 4 nutrients commonly used in immunonutrient supplements and their potential mode of action • n-3 polyunsaturated fatty acids: act as anti-inflammatory agents and reverse immunosuppression • sulfur amino acids and their precursors: enhance antioxidant status via gsh synthesis • glutamine: nutrient for immune cells, improves gut barrier function, precursor for gsh • arginine: stimulates nitric oxide and growth hormone production, improves helper t-cell numbers • nucleotides: rna and dna precursors, improve t-cell function may also require nutritional modulation of ongoing inflammatory processes. during the last 20 years the pace of evolution ofimmunomodulatory feeds and intravenous solutions has accelerated. these products contain combinations of a number of components to which various functional attributes are ascribed them (table 4) (grimble, 2001a) . many studies have indicated that n-3 polyunsaturated fatty acids (pufas), glutamine, arginine, sulfur amino acids, and nucleotides are all potentially capable of shifting the balance from a disadvantageous to an advantageous response to infection and injury. the examples used here are illustrative rather than comprehensive. a number of studies indicate that improvement of antioxidant status is associated with an increase in cellular aspects of immune function. meta-analyses have been conducted on the efficacy of immunonutrients that influence antioxidant status. in clinical trials, indices such as infection rates, mortality rates, and length of stay are often measured in the absence of functional and biochemical aspects of the response, such as t-cell function, cytokine production, and antioxidant status, and vice versa, giving a rather incomplete picture of the mechanisms of any observed effects of immunonutrition. however, beale, bryg, & bihari (1999) , in a meta-analysis of 12 studies containing more than 1400 patients receiving enteral immunonutrition, observed that although there was no effect upon mortality, there were significant reductions in infection rates, time spent on a ventilator, and length of hospital stay. while this finding indicates that immunonutrition may be useful in modulating the inflammatory process in patients experiencing severe inflammation, the consistency of the effects observed was disappointing. there are at least three major reasons why it is difficult to demonstrate a consistent effect. first, patients used as the subjects of clinical trials of immunonutrients will constitute a diverse population--different ages, at different stages of a disease process, and undergoing complex clinical treatment in addition to nutrient therapy. second, patients will have differing genetic backgrounds that will influence the intensity of the inflammatory and immune responses they are undergoing. this issue is dealt with below. third, nutrients may exert paradoxical effects, as illustrated by the findings of the first observations of the effects of fish oil on cytokine production in healthy subjects. the findings of endres et al. (1989) that a daily supplement of 18 g/d of fish oil given to nine young men for 6 wk was able to reduce ex vivo production ofll-1 and tnf-~ by lps-stimulated pbmcs aroused great interest in fish oil as an anti-inflammatory nutrient. this perception was supported by a large amount of animal data. however, endres' data showed a wide variability in the effect of the fish oil supplements. the standard deviations of the mean for il-113 and tnf-cx production were 59 and 51%, respectively. this indicates that cytokine production could have risen or fallen as a consequence of taking the supplement. the effects of supplementing 116 healthy young men with 6 g/d offish oil for 12 wk, on tnf-ct production by pbmcs stimulated with endotoxin have been studied in the author' s laboratory. it was found that 51% of subjects experienced a fall in production and 49% a rise. although the ability of fish oil to increase tnf-t~ production is at first sight paradoxical, earlier work of dinarello, bishai, rosenwasser, and coceani (1984) and kunkel, remick, spengler, and chensue (1987) indicated that fish oil could potentially change cytokine production in either direction. what mechanisms could result in this divergent effect? inflammation will result in activation of phospholipase a2, which releases arachidonic acid (aa) (c20:4 n-6) from the cell membrane for prostaglandin e 2 (pge2) or leukotriene b 4 (lt b4 ) synthesis. the in vitro studies (kunkel et al., 1987) showed that pge2 suppressed tnf-t~ production, whereas ltb4 had the opposite effect (dinarello et al., 1984) .. fish oil is rich in eicosapentaenoic acid (c20:5 n-3), which will replace aa in the cell membrane and results in the production of pge3 and lt b5. pge3 and lt b5 are considerably less potent than the corresponding compounds produced from aa, and thus dietary fish oil may lessen the inhibitory influence of pge2 or the stimulatory influence of ltb4 on tnf-o~ production, resulting in a potential increase or decrease, respectively, in production of the cytokine. fish oil could thus result in an inflammatory cytokine response, which could fall on either side of the pivot. the response to bacterial invasion of the body, or injury, contains a paradox. although the inflammatory response and the t-cell response both play a part in defeating the invader, the inflammatory response may in some clinical circumstances exert an inhibitory influence on t-cell function. in severely infected or traumatized patients, an enhanced inflammatory state occurs, which is associated with immunosuppression. in vitro studies support this inverse relationship. pbmcs taken from healthy young subjects and incubated with gsh show decreased pge2 and ltb4 production (reduced inflammation) and an increase in mitogenic index and il-2 production (enhanced immune function) (wu, meydani, sastre, hayek, & meydani, 1994) . thus, enhancement of antioxidant defenses reduces the likelihood of the inflammatory response suppressing t-cell function (grimble, 1997 (grimble, , 2001b . although all antioxidants are important owing to the linked nature of antioxidant defense (fig. 8) , gsh plays a pivotal role as it acts directly as an antioxidant and maintains other components of defense in a reduced state through enzymic conversion between the oxidized and reduced states. various compounds can be used to increase gsh synthesis (fig. 9 ). n-acetyl cysteine (nac) and the gsh prodrug oxothiazalidine-4-carboxylate (procysteine) have been used in a number of clinical studies. tissue gsh content is also influenced by protein and sulfur amino acid intake. unfortunately, surgery, a wide range of diseases that have an inflammatory component, and aging and protein energy malnutrition decrease gsh concentration in blood and other tissues (boya et al., 1999; loguercio et al., 1999; luo, hammarqvist, anderson, & wernerman, 1996; micke, beeh, schlaak, & buhl, 2001; nuttal et al., 1999; reid et al., 2000) (table 5 ). within 24 h of elective abdominal surgery, muscle gsh content falls by >30%. values return to normal 72 h postoperatively. a smaller perturbation in blood gsh occurs over a shorter time course. modification of the gsh content of liver, lung, spleen, and thymus in young rats by feeding diets containing a range of casein (a protein with a low sulfur amino acid content) concentrations changed immune cell numbers in lung (hunter & grimble, 1994 found that in unstressed animals the number of lung neutrophils decreased as dietary protein intake and tissue gsh content fell. however, in animals given an inflammatory challenge (endotoxin), liver and lung gsh concentrations increased directly in relation to dietary protein intake. lung neutrophils, however, became related inversely with tissue gsh content. addition of methionine to the protein-deficient diets normalized tissue gsh content and restored lung neutrophil numbers to those seen in unstressed animals fed a diet with adequate protein content (fig. 10) . why does tissue gsh content have differing effects on immune cell populations depending on whether or not an inflammatory response is occurring? a partial explanation may come from an in vitro study using hela cells and cells from human embryonic kidney. in the study, both tnf-o~ and hydrogen peroxide resulted in activation of nf-~cb and ap1 (wesselborg, bauer, vogt, schmitz, & schulze-osthoff, 1997) . addition of the antioxidant sorbitol to the medium suppressed nf-~fl3 activation as expected, but unexpectedly activated ap 1. thus, the antioxidant environment of the cell might exert opposite effects upon transcription factors closely associated with inflammation (e.g., nf-~fl3) and cellular proliferation (e.g., ap1). evidence for this biphasic effect was seen when gsh was incubated with immune cells from young adults (wu et al., 1994) . a rise in cellular gsh content was accompanied by an increase in il-2 production and lymphocyte proliferation (enhancement of t-cell function) and a decrease in production of the inflammatory mediators pge2 and ltb4 (anti-inflammatory influence). without doubt, a decline in antioxidant status in the presence of oxidant stress will increase inflammatory stress. the interaction between oxidant stress and an impaired ability to synthesize gsh, a situation that stimulates inflammation, is clearly seen in cirrhosis, a disease that results in high levels of oxidative stress and an impaired ability to synthesize gsh . in pena an inverse relationship between gsh concentration and the ability of monocytes to produce il-1, il-8, and tnf-t~ was observed. treatment of cirrhotic patients with the procysteine increased monocyte gsh content and reduced il-1, il-8, and tnf-t~ production. septic patients given an infusion of nac (150 mg/kg bolus followed by infusion of 50 mg/kg over 4-h) showed a decrease in plasma il-8 and soluble tnf receptor p55, had a reduced requirement for ventilator support, and spent 19 fewer days in intensive care than patients not receiving nac (spapen et al., 1998 ). de rosa et al. (2000 showed that nac was able to restore tissue gsh concentrations in individuals with hiv infection. in a study on hiv-positive patients, brietkreutz et al. (2000) showed that a dose of 600 mg/d of nac for 7 mo resulted in a decrease in plasma il-6 (decreased inflammation), an increase in natural killer cell activity, and increased responsiveness of t lymphocytes to tetanus toxin stimulation (improved lymphocyte function). antioxidants might act to prevent nf-r,b activation by quenching oxidants. however, nf-rd3 and ap1 may not respond to changes in cell redox state in the same way. when rats were subjected to depletion of effective tissue gsh pools by administration of diethyl maleate, there was a significant reduction in lymphocyte proliferation in spleen and mesenteric lymph nodes (robinson et al., 1993) . an increase in inflammatory stress would be expected in this study. thus, it can be hypothesized that antioxidants exert an immunoenhancing effect by activating transcription factors that are strongly associated with cell proliferation (e.g., ap 1) and an anti-inflammatory effect by preventing activation of nf-r,b by oxidants produced during the inflammatory response (dr&ouml; ge et al., 1994) .thus, inclusion of antioxidants or substances that increase gsh synthesis in immunonutrient mixes would seem to be beneficial. improvement of antioxidant defenses is also possible by feeding other components of antioxidant defenses. supplementation of the diet of healthy subjects and smokers with 600 iu/d t~-tocopherol for 4 wk suppressed the ability of pbmcs to produce tnf-t~ (mol, de rijke, demacher, & stalenhoef, 1997) . the same dose given to healthy elderly subjects for 235 d increased delayed-type hypersensitivity and raised antibody titers to hepatitis b (meydani et al., 1997 ). an enteral feed enriched with vitamin e, vitamin c, and taurine given to intensive-care patients decreased total lymphocyte and neutrophil content in bronchioalveolar lavage fluid (decreased inflammation) and resulted in a reduction in organ failure rate, a reduced requirement for artificial ventilation, and a reduction of 5 d in intensive-care stay (gadek et al., 1999) . a number of roles have been ascribed to glutamine as an immunonutrient: (a) as an essential nutrient for immune cells, (b) as an important modulator of gut barrier function, and (c) as a substrate for gsh synthesis. a number of reviews have been written about the first two of these roles (newsholme, crabtree, salleh, & ardawi, 1985; elia, 1992) ; we will consider the last one here. could glutamine be exerting an anti-inflammatory influence via an effect on gsh that enhances immune function? in a study in rats, glutamine supplementation resulted in an increased production of gsh by the gut (cao, feng, hoos, & klimberg, 1998) , and total parenteral nutrition (tpn) with glutamine raised plasma gsh concentrations in these animals (denno, rounds, faris, halejko, & wilmore, 1996) . in randomized controlled trials the administration of glutamine, either as a dipeptide during tpn to surgical patients or as a glutamine-enriched enteral feed to trauma patients, resulted, respectively, in improved nitrogen retention (less tissue protein depletion) and a 6.2-d reduction in hospital stay, a concomitant suppression of the rise in plasma-soluble tnf receptors (reduced inflammation), and a lower incidence ofbacteremia, pneumonia, and sepsis (improved immune function) (houdijk et al., 1998; morlion et al., 1998) in the previous section the influence of antioxidants on severe inflammation was considered. do the general findings from this type of study also apply to modulation of low-grade chronic inflammation, such as has been observed in the elderly and obese? because aging is so closely associated with increased oxidative stress, which might both result from and contribute to a stimulation in the level of inflammation in the elderly, antioxidant therapy could produce beneficial effects. the effects would be seen in a decrease in oxidant damage, downregulation of inflammation, and, because of the inverse link between inflammation and immune function, an improvement in t-lymphocyte function. meydani, meydani, & verdon (1986) reported that supplementation of aged mice (24 mo old) with dietary vitamin e (500 ppm) improved several indices of the immune system to levels comparable to those seen in young animals. supplementation of aged mice with this vitamin also increased clearance of influenza virus from the lung to that observed in animals supplemented with other antioxidants such as melathonine, gsh, or strawberry extract, which contains a high level of flavonoids with antioxidant activity . in a double-blind, placebo-controlled study, meydani and colleagues (meydani, barklund, & lui, 1990; meydani et al., 1997) also reported that supplementation of elderly subjects with vitamin e for a short (1 mo) or long (4.5 too) period of time also improved several in vitro and in vivo indices of immune response. the optimal immune response was observed with 200 iu of vitamin e per day in the long-term study. it is worth noting that this level of vitamin e has also been reported to be the optimal level for reducing plasma f2-isoprostane, a reliable index of lipid peroxidation (dillon, vita, & leeuwenburgh, 1998) . improving the immune response in the elderly may result in a lower incidence of infections, which are prevalent among the elderly, and thus may contribute to a longer and healthier life. many observational and clinical trials have also indicated that a high intake or high plasma level of this vitamin is associated with a low risk of cardiovascular disease. the vitamin may be operating at two levels; first, by protecting ldl from peroxidation, thereby reducing its atherogenicity, and second, by lowering the level of chronic inflammation by downregulation of nf-r,b. a reduction in platelet aggregability may also arise out of this action (huang et al., 2001; tanus-santos et al., 2002) . indeed, several lines of evidence indicated that supplements of vitamin e may prevent cardiovascular disease by reducing the susceptibility of ldls to oxidation (jailal, fuller, & huet, 1995) , reducing the expression of chemokines, adhesion molecule expression, and monocyte adhesion (wu, koga, martin, & meydani, 1999) , decreasing smooth muscle proliferation (azzi, boscoboinik, & marilley, 1995) , and decreasing platelet aggregation (steiner 1999) . another anti-inflammatory approach using nutrients would be to supplement diets of the elderly with n-3 pufas. supplementation with n-3 pufas from fish oil, however, has been reported to suppress the immune response (meydani, endres, & woods, 1991; meydani, 1993) , which hampers enthusiasm for the use of n-3 pufas for their benefits in cvd. however, the latter concern could be addressed by including a vitamin e supplement along with fish oil supplements. in a recent study it was found that supplementing elderly persons with (n-3) fatty acids of fish oil in combination with vitamin e while maintaining the anti-inflammatory properties of (n-3) pufas did not reduce immune indices in the elderly . fish oil supplementation is not universally efficacious in the treatment of inflammatory disease (grimble, 1998) . rheumatoid arthritis and inflammatory bowel disease have been the most successfully treated of all inflammatory diseases (calder, 1997) . the antiinflammatory mechanism may be through suppression of tnf-tx production. endres et al. (1989) reported that large doses (15 g/d for 6 wk) of oil in nine healthy volunteers resulted in a small but statistically significant reduction in tnf-o~ and il-1 [3 production from pbmcs. subsequently, fewer than half of 11 similar small intervention studies were able to demonstrate a statistically significant reduction in cytokine production. to understand the differences in response more closely, the author's laboratory conducted a study on 111 young men fed 6 g fish oil daily for 12 wk and measured tnf-tx production by pbmcs before and after supplementation in relation to the snp at -308 in the tnf-o~ and at +252 in the lt-tx genes. no significant effect of fish oil on cytokine production was noted in the group as a whole. however, when data were examined according to tertile of tnf-ct production prior to supplementation, homozygosity for tnfb2 (lt-o~+ 252 a) was 2.5 times more frequent in the highest than in the lowest tertile of production. the percentage of individuals in whom fish oil suppressed production was lowest (22%) in the lowest tertile and doubled with each ascending tertile. in the highest tertile, mean values were decreased by 43% (p < 0.05). in the lowest tertile, mean values were increased by 62% (p < 0.05). tnfb2 (lt-cz+252 aa) homozygotes were strongly represented among unresponsive individuals in the lowest tertile of tnf-cz production prior to supplementation. in this lowest tertile, only tnfb l/b2 (lt-o~+252 ga) heterozygous subjects were responsive to the suppressive effects of fish oil. in the medium tertile, this genotype was six times more frequent than other lt-t~ genotypes among responsive individuals. no relationship between possession of tnf 1 or 2 (tnf-ot-308 g or a) alleles and responsiveness to fish oil was found. clearly, although the level of inflammation determines whether fish oil will exert an anti-inflammatory influence or not and is influenced by the tnfb2 (lt-ct+252 a) allele, the precise genomic mechanism for an anti-inflammatory effect is unclear at present . antioxidant intake also modifies cytokine production. in a study on healthy men and women and smokers, dietary supplementation with 600 iu/d ct-tocopherol for 1 mo suppressed the ability of pbmcs to produce tnf-t~. production was reduced by 22 and 33% in nonsmokers and smokers, respectively (mol et al., 1997) . in a similar dietary intervention study on normolipaemic and hypertriglyceridaemic subjects given 600 iu/ d of o~-tocopherol for 6 wk, reduced tnf-o~, il-113, and il-8 production by lps-stimulated blood mononuclear cells occurred (mol et al., 1997; van tits, demacker, de graaf, hak-lemmers, & stalenhoef, 2000) . a similar effect of cx-tocopherol was noted in a study on normal subjects and type 2 diabetics (devaraj & jialal, 2000) . however, there were large standard deviations in the data from these studies, indicating major intraindividual variability in the ability of vitamin e to suppress production of the cytokine. although a number of studies have shown that o~-tocopherol suppresses superoxide production, the situation with regard to nitric oxide is less clear (mol et al., 1997; van tits et al., 2000) n the a-tocopherol derivative pentamethyl-hydroxychromane inhibited lps-stimulated nf-gb and inos activation in cultured j774 macrophages (hattori, 1995) . at present it is not known whether antioxidants interact differently with snps in the genes associated with oxidant stress and inflammation than they do with the other anti-inflammatory nutrient, n-3 pufa. this topic is currently an area of active research at the author's laboratory. proteomic studies have shown that inos and sod are both influenced by the nramp 1 gene (kovorova, necasova, porkertova, radzoich, & macela, 2001) . the production of oxidant molecules enhancing pro-inflammatory cytokine production via high levels of nf-kb activation may thus be under a genomic influence owing to the aforementioned variations in the nramp1 gene (formica, roach, & blackwell, 1994) . a better understanding of this interaction and of the interaction of n-3 pufas and antioxidants with genotype may allow the better design of nutrient products for the treatment of inflammatory disease. it is clear from the current understanding about the purpose and functioning of the immune system throughout the life cycle that it is a powerful biological entity that profoundly alters body function while it is carrying out its prime purpose of defending the body against invasion by pathogens. however, within the response lie the seeds of disaster at an individual level, for the inflammatory component of the response can turn against the body, particularly as the body ages or becomes obese. the response, which can be devastating when directed against microbes entering the body, also sows the seeds of atherosclerosis, degradation of brain function, and insulin insensitivity and hastens the passage of hiv-infected individuals towards full-blown aids. along with the insights arising from the unraveling of the human genome has come evidence that the inflammatory response is able to protect the human species from invasion by pathogens but not all individuals within the species from ill health. the differing ability of humans, particularly the male of the species, at an individual level to mount an inflammatory response of different levels of intensity owing to genotype can result in widely contrasting outcomes of invasion of the body by pathogens. on the one hand, individuals may effectively fight off invasion provided the immune response follows a normal pathway, whereas other individuals within the same community encoutering the same pathogens will die from the strength and nature of the response rather than from the direct effects of the invader. insights gained from the genomic influences on cytokine production and the response to malaria suggest that the retention of alleles in pro-inflammatory cytokine genes that resulted in enhanced cytokine production within the human gene pool over generations could be a result of the heterozygotes' better capacity for fighting pathogens, whereas homozygotes of the high-producing genotype run an increased risk of a strong adverse inflammatory response. in the case of sickle cell anemia, where heterozygotic individuals reap an advantage in resistance to malaria by possession of only one copy of the anemia allele, homozygous individuals for the sickle cell trait pay the price for possession of two copies of the allele and die young. because of the overall advantage of this situation to the species, the potentially disadvantageous allele will be retained within the human gene pool over generations. with the twin discoveries that nutrients can modulate the inflammatory response and that cytokine genotype can modulate the effectiveness of nutrients in controlling inflammation, nutritional science sits at an exciting moment in its development. the mapping of how pro-and anti-inflammatory cytokine genotypes interact with responsiveness to immunonutrients at an indivividual level will allow tailor-made nutritional treatments of all diseases that have an underlying inflammatory basis. furthermore, a better understanding of how nutritonal therapies and genetics interact will allow the twin adverse biological factors increasing the level of inflammatory stress in populations---obesity and aging--to be tackled by targeted nutritional therapy. the roles of nramp 1 and tnfo~ genes in nitric oxide production and their effect on the growth of salmonella typhimurium in macrophages from nrampl congenic and tumor necrosis factor-alpha-/-mice polymorphism of the human tnf-cz promoter--random variation or functional diversity? molecular immunolology periodontal infections and cardiovascular disease--how strong is the association? oral disease predictive value of nuclear factor kappab activity and plasma cytokine levels in patients with sepsis vitamin e: a sensor and an information 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position 26 and a reduced level of tnfc~ production vitamin e supplementation enhances cell-mediated immunity in healthy elderly subjects oral (n-3) fatty acid supplementation supresses cytokine production and lymphocyte proliferation: comparison between young and older women immunological effects of national cholesterol education panel (ncep) step-2 diets with and without fish-derived (n-3) fatty acid enrichment vitamin e supplementation and in vivo immune response in healthy subjects. a randomized controlled trial vitamin e supplementation suppresses prostaglandin e2 synthesis and enhances the immune response of aged mice oral supplementation with whey proteins increases plasma glutathione levels in hiv-infected patients association of tnf2, a tnf-alpha promoter polymorphism, with septic shock susceptibility and mortality: a multicenter study association between manganese superoxide dismutase (mnsod) gene polymorphism and breast cancer risk plasma levels of lipid and cholesterol oxidation products and cytokines in diabetes mellitus and smokers: effect of vitamin e treatment potential role of tnf-alpha in the pathogenesis of insulin resistance and type 2 diabetes inhibition of monocyte chemoattractant protein-1 expression in cytokine-treated human lung epithelial cells by thiazolidinedione total parenteral nutrition with glutamine dipeptide after major surgery. a double blind controlled study prevalence of low body mass in rheumatoid arthritis: association with the acute phase response increase in il-6 and decrease of interleukin 2 production during the ageing process are influenced by health status glutamine metabolism in lymphocytes. its biochemistry, physiology and clinical importance relation between plasma tumor necrosis factor-alpha and insulin sensitivity in elderly men with non-insulin-dependent diabetes mellitus periodontal inflammation and insulin resistance--lessons from obesity age-dependent oxidative stress in elderly patients with 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synthesis in children with severe protein-energy malnutrition altered cytokine production in the elderly glutathione depletion in rats impairs t-cell and macrophage immune function age and sex steroid-related changes in glucocorticoid sensitivity of proinflammatory cytokine production after psychosocial stress endothelial perturbation in children and adolescents with type 1 diabetes: association with markers of the inflammatory reaction epidemiologic and methodologic problems in determining nutritional status of older persons the pathogenesis of atherosclerosis: a perspective for the 1990s origins and clinical relevance of sarcopenia association of tumor necrosis factor alpha gene promoter polymorphism with the presence of chronic obstructive pulmonary disease interactive role of infection, inflammation and traditional risk factors in atherosclerosis and coronary artery disease reactive oxygen intermediates as apparently widely used messengers in the activation of nuclear transcription 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and outcome of patients with severe sepsis interleukin 10 (il-10) genotypes in inflammatory bowel disease change in the ratio of interleukin-6 to interleukin-10 predicts a poor outcome in patients with systemic inflammatory response syndrome effects of endothelial nitric oxide synthase gene polymorphisms on platelet function, nitric oxide release, and interactions with estradiol an investigation ofpolymorphisms in the interleukin-10 gene promoter antiinflammatory cytokine profile and mortality in febrile patients alphatocopherol supplementation decreases production of superoxide and cytokines by leukocytes ex vivo in both normolipidemic and hypertriglyceridemic individuals relationship of interleukin-6 and tumor necrosis factor-alpha with muscle mass and muscle strength in elderly men and women: the health abc study circulating interleukin-6 in relation to adiposity, insulin action, and insulin secretion high white blood cell count is associated with a worsening of insulin sensitivity and predicts the development of type 2 diabetes ppar-gamma and inflammatory bowel disease: a new therapeutic target for ulcerative colitis and crohn's disease lack of association between human longevity and polymorphisms of il-1 cluster, il-6, il-10 and tnf-alpha genes in finnish nonagenarians genetic polymorphisms in the tumor necrosis factor locus influence non-hodgkin's lymphoma outcome activation of transcription factor nf-kappa b and p38 mitogen-activated protein kinase is mediated by distinct and separate stress effector pathways humoral markers of inflammation and endothelial dysfunction in relation to adiposity and in vivo insulin action in pima indians biology of arterial atheroma comparative genetic association of human leukocyte antigen class ii and tumor necrosis factor-alpha with dermatitis herpetiformis an allelic polymorphism within the human tumor necrosis factor alpha promoter region is strongly associated with hla-a 1, b 8, and dr3 alleles a genetic association between systemic lupus erythematosus and tumor necrosis factor alpha effect of vitamin e on human aortic endothelial cell production of chemokines and adhesion to monocytes effect of dietary supplementation with fish oil in combination with different levels of vitamin e on immune response in healthy elderly human subjects in vitro glutathione supplementation enhances interleukin-2 production and mitogenic responses in peripheral blood mononuclear cells from young and old subjects associations of serum c-reactive protein with fasting insulin, glucose, and glycosylated hemoglobin: the third national health and nutrition examination survey production of tumour necrosis factor--alpha increases with age and bmi in healthy women the influence of age and gender on serum dehydroepiaandrosterone sulphate (dhea-s), il-6, il-6 soluble receptor (il-6 sr) and transforming growth factor beta 1 (tgf-beta 1) levels in normal healthy blood donors polymorphisms in the promoter of the tumor necrosis factor-alpha gene in coal miners reduction of inflammatory cytokine concentrations and improvement of endothelial functions in obese women after weight loss over one year the author is grateful to the bbsrc for funding much of the work reported in this chapter. the author is also grateful to collegues in the united kingdom, sweden, and switzerland for scientific collaboration and advice. key: cord-006828-i88on326 authors: nan title: abstracts dgrh-kongress 2013 date: 2013-09-15 journal: z rheumatol doi: 10.1007/s00393-013-1255-1 sha: doc_id: 6828 cord_uid: i88on326 nan im namen der dgrh, der dgorh und der gkjr begrüßen wir sie ganz herzlich zu unserem diesjährigen kongress visualisierung therapeutischer effekte von vasodilatantien beim sekundären raynaud-syndrom mittels fluoreszenzoptischer bildgebung di.14 stellenwert der gelenksonographie bezüglich diagnose, behandlung und therapiekontrolle der bursitis intermetatarsalis -einer häufig übersehenen differenzialdiagnose. fünf fallbeispiele wegen der deutlich eingeschränkten nierenfunktion konnten therapeutisch keine nsar angewandt werden. wir haben mit 2×0,5 mg colchicin täglich behandelt. die anfänglich schwerkranke bettlägerige patientin konnte innerhalb von 48h mobilisiert werden. um eine abschließende sicherung der diagnose einer uratinduzierten sakroiliitis erreichen zu können ist die patientin mit einem dual-energy-ct (dect) untersucht worden. ergebnisse. mit dieser methode konnten gichttophi in beiden sakroiliakalgelenken dargestellt werden, ebenso an beiden mtp1-gelenken. schlussfolgerung. aktuell liegen bisher noch keine weiteren berichte vor, dass diese methode auch für die diagnostik einer gicht im bereich der sakroiliakalregion zuverlässige ergebnisse liefern kann. zudem zeigt dieser krankheitsverlauf, dass sich die gicht durchaus primär im bereich des achsenskeletts manifestieren kann und nicht in erster linie an den peripheren gelenken zu entsprechenden beschwerden führen muss. a. glimm 1 , s. werner 1 , s. ohrndorf 1 , c. schwenke 2 , g. schmittat 1 , g. burmester 1 einleitung. typische pathologische veränderung bei der rheumatoiden arthritis (ra) ist die synovialitis. auch bei der osteoarthrose (oa) lassen sich entzündliche veränderungen der gelenke finden. diese können mittels fluoreszenzoptischer bildgebung (foi) und dem gelenkultraschall (us) sichtbar gemacht werden. ziel der studie: vergleich der foi mit dem us bei patienten mit ra und oa. methoden. es wurde bei 90 patienten (67 ra, 23 oa) die foi beider hände sowie die us des handgelenks (hg) und der fingergelenke (mcp, pip, dip) der klinisch beschwerdeführenden hand von dorsal und palmar sowohl im b-bild (b-us) als auch mit power-doppler (pd-us) durchgeführt. synovialitis und tenosynovitis im us sowie die intensität des fluoreszenzsignals im bereich der gelenke in der foi wurden qualitativ als auch semiquantitativ nach standardisierten verfahren für den primavistamode (pvm) und drei verschiedene phasen (p1-3; [1] ) bewertet. in der statistischen analyse wurden anschließend sensitivitäten und spezifitäten für die foi bei der ra und oa getrennt für synovialitis und tenosynovitis, dorsal und palmar jeweils für b-us und pd-us als referenzmethode berechnet. ergebnisse. in abhängigkeit von der betrachteten phase zeigen sich für die ra und oa moderate sensitivitäten und spezifitäten. für die ra wurden in der phase 2 des foi die höchsten sensitivitäten mit 68% für b-us und 77% für pd-us berechnet. auch bei der oa ergaben sich die höchsten sensitivitäten in der phase 2 des foi mit 77% für b-us und 89% für pd-us als referenzmethode. die höchsten spezifitäten für beide diagnosen wurden in der foi in phase 3 erreicht. hierbei lag die spezifität bei der ra für b-us bei 96% und für pd-us bei 90%. der höchste spezifitätswert bei der oa sowohl für b-us als pd-us war 88% (. tab. 3 background. pet is a nuclear imaging technique that depicts functional processes within the body with high sensitivity by detecting annihilation radiation from radioactive decay of a positron-emitting radionuclide that was labeled to a biologically active molecule (tracer) and introduced into the body. 18f-fluoride (18f) can be used for pet as a bone-seeking agent reflecting bone perfusion and remodeling. we inaugurated a pilot study with simultaneous pet/mr to examine whether addition of pet provides different and additional information in comparison to mri in axspa patients. methods. eleven axspa patients, median age 39 y, disease duration range 0.5-10 y, mean basdai 5.3, were examined by pet/3-tesla mri 40 minutes after injection of a mean dose of 157 mbq of 18f using a integrated whole-body pet/mr scanner (siemens biograph mmr®). 3t-mris were scored blinded to patient's clinical characteristics by two readers (1 rheumatologist and 1 radiologist/nuclear medicine specialist) using the berlin mri score and also by recording inflammatory lesions on a vertebral edge (ve) level. in a second step pet/mris were read blindly by the same readers also based on the ve involvement of individual vertebral bodies. results. the procedure was successful in all patients. the resulting mean effective radiation dose per patient was 3.76 msv. co-registration of pet/mri fusion images was highly accurate, allowing a precise comparison of mri and pet. in the direct comparison of the mri and pet signal the two readers saw consistent signals in almost 90% of the sites studied. however, there were areas where signals differed, e.g. within existing syndesmophytes where pet signal was increased but conventional mris showed no signal, or the sternum area and lateral or posterior spinal elements such as facets and spinous processes. conclusion. the new technique of integrated pet/mri provides similar imaging signals as conventional mri. however, we observed differences between the two modalities in areas with less inflammatory activity but where bone metabolism seemed to be active or in areas with blurred resolution on conventional mri. the possibility that pet detects osteoblastic activity in areas where no inflammatory signal is detected with mri seems to be of interest. einleitung. sensitiven bildgebenden verfahren wie der hochauflösenden arthrosonographie kommen bei der detektion initial entzündlicher veränderungen im rahmen der frühdiagnostik der psoriasisarthritis (psa) eine große bedeutung zu. die vorliegende prospektive studie untersucht die diagnostische und prognostische wertigkeit der sonographischen befunde im vergleich zur klinischen untersuchung auf ebene einzelner gelenke bei früher psoriasis-arthritis (psa). methoden. rekrutierung von 50 patienten mit therapienaiver früher psa. sonographie von 56 gelenken mit semiquantitativer graduierung (grad 0-3) von b-bild (gsus) und power-doppler-aktivität (pdus; baseline, 12 monate). klinische parameter: anzahl druckschmerzhafter und geschwollener gelenke (tjc68, sjc66), visuelle analogskala, das28-crp, health assessment questionnaire haq. für jede followup-visite erfolgte eine kategorisierung des klinischen ansprechens nach eular-response-kriterien und der für die psa validierten minimal-disease-activity(mda)-kriterien (coates et al.). ergebnisse. baseline 50 patienten, nach 12 monate 19 patienten, erkrankungsdauer (12±17,6 monate). patienten ohne therapie (1), mit nsar (1), steroid i.a. (1) , dmards (10), biologicals (6). bei diagnosestellung zeigte sich eine signifikante korrelation zwischen dem us synovitis score und folgenden klinischen parametern: tjc68 (r=0,52), scj66 (r=0,63), das28-crp (r=0,34). nach 12 monaten zeigte sich eine gute korrelation zwischen der relativen veränderung des us synovitis scores und der relativen veränderung folgender klinischer parameter: tjc68 (r=0,72), haq (r=0,41), pasi (r=0,42), das28-crp (r=0,33). zu baseline waren 407 von 2730 gelenken sonographisch auffällig, davon zeigten 239 kein klinisches korrelat (subklinisch). nach 12 monaten zeigten 29% der initial subklinischen gelenke einen unveränderten befund, 61% waren sonographisch nicht mehr auffällig und 10% wurden klinisch manifest. bei den klinischen respondern war der rückgang deutlicher ausgeprägt. schlussfolgerung. der ultraschall-synovitis-score korreliert mit klinischen aktivitätsparametern sowohl zum zeitpunkt der diagnosestellung als auch im krankheitsverlauf unter immunsuppressiver therapie. die subklinischen veränderungen bilden sich unter immunsuppressiver therapie zu einem großen teil zurück, deutlicher bei klinischen respondern. ein geringer anteil der initial subklinischen gelenke wird im verlauf klinisch manifest, in höherem maße bei klinischen non-respondern. t. diekhoff 1 , k. hermann 1 1 charité -universitätsmedizin berlin, radiologie, berlin einleitung. die gicht ist mit einer prävalenz von 0,4-0,6% insbesondere in den industrieländern eine häufige erkrankungen, die mit gelenkschmerzen einhergeht. bei typischer symptomatik und laborkonstellation ist die diagnose der arthritis urica oft einfach zu stellen, ein atypisches beschwerdebild kann jedoch gelegentlich die abgrenzung zu anderen erkrankungen erschweren. besonders die kalziumpyrophosphat-kristallarthropathie (cppd oder pseudogicht), die selbst mit sehr variabler symptomatik auftreten kann, ist eine relevante differenzialdiagnose, besonders bei älteren patienten. methoden. mit der dual-energy-computertomographie (de-ct) steht ein modernes, innovatives verfahren zur verfügung, das eine detektion von harnsäurehaltigen weichteilverkalkungen ermöglicht und darüber hinaus eine sichere abgrenzung zu kalziumhaltigen verkalkungen gewährleisten kann. das prinzip der de-ct ist relativ simpel und seit längerem bekannt: die messung des untersuchungsvolumens mit zwei unterschiedlichen röhrenspannungen macht es möglich, einen schwächungskoeffizienten zu errechnen, der spezifisch für das untersuchte material ist. allerdings ermöglichten erst moderne cts mit zwei röntgenröhren die klinische anwendung. in jüngster zeit werden jedoch anstrengungen unternommen, die de-ct auch für ein-röhren-systeme verfügbar zu machen. ergebnisse. mit der de-ct können gichttophi sicher vom knochen aber auch von anderen verkalkungen getrennt und zum beispiel farblich kodiert dargestellt werden. im gegensatz zum konventionellen röntgenbild verspricht die de-ct jedoch nicht nur eine höhere sensitivität für tophöse veränderungen, sondern als schnittbildverfahren auch eine bessere abgrenzung und einordnung von anderen morphologischen veränderungen wie zum beispiel von erosionen. schlussfolgerung. dieser vortrag fasst die vor-und nachteile der de-ct in der detektion und abgrenzung von weichteilverkalkungen bei kristallarthropathien zusammen und gibt darüber hinaus einen ausblick auf zukünftige entwicklungen in diesem gebiet. background. anionic glycosaminoglycans interact with a variety of soluble and membrane bound molecules. chondroitin sulfate was shown to have anti-inflammatory properties but its role in arthritis is controversial. methods. we have analyzed the effect of chondroitin sulfate on collagen induced arthritis starting treatment before and after induction of arthritis and in mice with established arthritis. results. in all of these settings chondroitin sulfate significantly reduced the severity of arthritis. it prevented joint destruction, diminished the inflammatory infiltrate and reduced proinflammatory cytokines in joints and plasma. splenocytes restimulated with collagen produced less il-2 and more il-10 and il-13. the beneficial effects of chondroitin sulfate were transient and closely correlated to the suppression of the collagen-specific humoral immune response. chondroitin sulfate, but not other glycosaminoglycans induced a direct btk and syk-dependent proliferation of b cells and markedly expanded the number of plasma cells in the spleen. in immunized mice chondroitin sulfate reduced the number of antigen specific plasma cells in the bone marrow and was able to suppress established humoral immune responses. conclusion. displacement of disease inducing plasma cells from the bone marrow might contribute to the beneficial effects of chondroitin sulfate and could be an attractive strategy to suppress antibody mediated autoimmunity. background. in rheumatoid arthritis a functional deterioration of the hpa-axis in form of inadequately low secretion of glucocorticoids in relation to severity of inflammation can be detected. the reasons for this phenomenon are not known. the purpose of this study was to find possible reasons responsible for adrenal insufficiency during arthritis. methods. da rats were immunized with type ii collagen in incomplete freund adjuvant to induce arthritis. plasma corticosterone was evaluated by ria and plasma acth by elisa. adrenal cholesterol was quan-titatively studied by sudan-iii staining and scavenger receptor class bi (sr-bi, the hdl receptor) by immunohistochemistry. fluorescent nbd-cholesterol uptake kinetics were analysed by flow cytometry. ultrastructural morphology of adrenocortical mitochondria and lipid droplets was studied by electron microscopy. results. initially increased corticosterone and acth levels were reduced to baseline levels in the later phase of the disease. serum levels of corticosterone relative to il-1β were markedly lower in arthritic than control animals (inadequacy). cholesterol storage in adrenocortical cells and expression of sr-bi did not differ between immunized and control rats. however, number of impaired mitochondria largely increased during the course of arthritis (maximum on day 55), and this was paralleled by reduced numbers of activated cholesterol droplets (inhomogenous droplets relevant for generation of glucocorticoids). in addition, number of normal mitochondria positively correlated with serum corticosterone levels. conclusion. this first study on adrenal reasons for inadequate glucocorticoid secretion in arthritis demonstrated impaired mitochondria and altered cholesterol breakdown paralleled by low corticosterone levels in relation to ongoing inflammation. justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, 2 agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main, 3 universitätsklinikum gießen und marburg, orthopädische klinik, labor für experimentelle orthopädie, gießen, 4 universitätsklinikum gießen und marburg, orthopädie und orthopädische chirurgie, gießen, 5 universitätsklinikum erlangen, medizinische klinik 3 , rheumatologie und immunologie, erlangen background. obesity is a risk factor in osteoarthritis (oa), but there is limited information about the interaction between bone formation and adipose tissue-derived factors, the so-called adipokines. adipokines such as adiponectin, resistin or visfatin are associated with the pathogenesis of rheumatoid arthritis (ra) and oa. adipokines are produced also by other cell types than adipocytes in ra and oa joints, for example osteoblasts, osteoclasts or chondrocytes. however, in contrast to their joint-destructive role in ra, their role in oa joint remodeling is unclear. therefore, adipokine expression in osteophyte development and bone forming cells as well as their effect on these cells was analyzed. methods. osteophytes and bone were obtained from oa patients during joint replacement surgery. serial sections of bone tissue were stained (masson trichrome, trap) and scored from grade one (no ossification, mainly connective tissue and cartilage) to five (ossified, mineralized osteophyte, <10% connective tissue, ossified remodeling zones). immunohistochemistry against alkaline phosphatase, collagen-type ii, adiponectin, resistin, and visfatin was performed. oa osteoblasts were stimulated with adiponectin and measurements of il-6, il-8 and mcp-1 were performed in cell culture supernatants. results. adiponectin, resistin and visfatin were detectable in osteoblasts and all osteophyte grades. in non-ossified osteophytes (grade 1), especially adiponectin and to a lower extend resistin and visfatin were localized in connective tissue fibroblasts. in ossified osteophytes (grade 2-5), resistin, visfatin and to a lower extend adiponectin protein expression was co-localized with osteoblasts. resistin and visfatin were expressed by osteoclasts. visfatin was found in chondrocytes of all osteophyte grades (50% of chondrocytes) and adiponectin was detectable in blood vessels. osteoblast stimulation with adiponectin increased the release of the inflammatory mediators il-6 (2.6-fold), il-8 (4.9-fold), and mcp-1 (2.1-fold). zeitschrift für rheumatologie suppl 2 · 2013 | conclusion. the expression of adiponectin and visfatin expression in osteophyte connective tissue and cartilage suggests their involvement in early osteophyte formation. resistin and visfatin expression by osteoblasts and osteoclasts in ossified osteophytes indicates a role in bone remodeling of osteophytes at later stages. osteoblasts respond to adiponectin stimulation with the release of inflammatory mediators. therefore, adipokines are most likely involved in osteophyte formation at different stages affecting different cell types of bone remodeling. free fatty acids contribute to promotion of arthritis k. frommer 1 , a. schäffler 2 , s. rehart 3 , a. sachs 3 , u. müller-ladner 1 , e. neumann 1 1 justus-liebig universität gießen, kerckhoff-klinik gmbh, rheumatologie u. klinische immunologie, osteologie, physikalische therapie, bad nauheim, 2 universitätsklinikum regensburg, klinik und poliklinik für innere medizin i, regensburg, 3 agaplesion markus krankenhaus, akademisches lehrkrankenhaus der johann wolfgang goethe-universität, klinik für orthopädie und unfallchirurgie, frankfurt/main background. obesity is a known risk factor for several arthritic diseases and mechanical stress has been shown not to be the only factor. due to increased levels of free fatty acids (ffa) in obese compared to nonobese individuals and due to the involvement of ffa in inflammatory cardiovascular and metabolic diseases, we hypothesized that ffa play a role in the promotion of arthritic diseases. therefore, we therefore investigated the effect of ffa on various effector cells of arthritis. methods. rheumatoid (ra) synovial fibroblasts (sf), osteoarthritis (oa) sf, psoriatic arthritis (psa) sf, human primary chondrocytes (hch), human osteoblasts (ob), human macrovascular (huvec) and microvascular (hbdmec) endothelial cells were stimulated in vitro with different ffa within their physiological range of concentrations. immunoassays were used to quantify ffa-induced protein secretion. sulfosuccinimidyl oleate sodium (sso) was used to inhibit fatty acid translocase (fat). results. ffa dose-dependently increased the secretion of the proinflammatory factors (il-6, il 8 and mcp-1) as well as matrix-degrading enzymes (mmp-1 and mmp-3) in rasf (e.g. for lauric acid [100 µm] with rasf/il-6: 9.1-fold increase; il 8: 14.9 fold increase; mcp-1: 2.4fold increase; pro-mmp1: 5.1-fold increase; mmp-3: 83.6 fold increase). saturated and unsaturated ffa had similar effects on rasf. however, saturated ffa induced strong secretion of il-6 in chondrocytes, while unsaturated ffa only had a weaker effect on this cell type. at 100 µm, both saturated and unsaturated ffa significantly increased il-6 secretion by osteoblasts to a similar degree as for sf. a high concentration of ffa (100 µm) significantly induced il-6 secretion in huvec and hbdmec, whereas a low concentration of ffa (10 µm) did not have a significant effect (p>0.05) on human endothelial cells. blocking ffa transport into rasf by using sso almost completely abolished the effect of palmitic acid on il-8 secretion. conclusion. ffa are not only metabolic substrates but can also directly contribute to articular inflammation and degradation mediated by various effector cells of arthritis. our data also show that ffa transport into the cell is required for ffa-induced effects in sf. background. chronically inflamed tissues in ra are characterized by local hypoxia and enhanced angiogenesis. the hypoxia inducible factor (hif)-1 and (hif)-2 serve as key regulators of adaptation to hypoxia thereby promoting both angiogenesis and metabolic adaptation of endothelial cells. to investigate the impact of hif-1/hif-2 on the angiogenic and metabolic transcriptome under hypoxia (1% o2) versus normoxia (18% o2) we performed a knockdown of either hif-1α or hif-2α in human microvascular endothelial cells (hmec). methods. specific knockdown of either hif-1α or hif-2α was achieved using shrna-technology. angiogenic and metabolic transcriptome of hmecs was studied by performing an agilent human whole genome microarray under normoxia vs hypoxia. significantly regulated genes were allocated to angiogenic and metabolic processes using panther database. results. in comparison to normoxia the incubation of untransduced hmecs under hypoxia resulted in 73 regulated angiogenesis related genes and 17 regulated cellular metabolism related genes. in both hif-1α and hif-2α knockdown cells, hypoxia was still capable of inducing a differential gene expression pattern, but with a much less pronounced effect compared to control cells. analysis of angiogenesis related processes (vegf-pathway, hif-activation, egfr-pathway) showed that 74% of the differentially expressed genes are controlled by both hif-1 and hif-2. another 14% of the regulated genes are controlled by hif-1. the remaining 12% of regulated genes are under control of hif-2. the differentially regulated genes involved in the cellular metabolism (atpsynthesis, glycolysis, tca-cycle) were found to be to 80% controlled by both hif-1 and hif-2. the remaining 20% are dependent on the presence of hif-1. conclusion. hif-1 and hif-2 are both key regulators of the adaptation of endothelial cells towards hypoxia with overlapping functions. however, they do differ in their capacity to regulate cellular energy metabolism and angiogenesis. this leads us to conclude that hif-1 affects angiogenesis via indirect effects on cellular energy metabolism as indicated by the regulation of metabolic transcriptome to one fifth. hif-2 does more influence angiogenesis directly via regulating the synthesis of proangiogenic factors (as has been previously shown).these findings provide new insights into the divergent regulation of angiogenesis in inflamed (hypoxic) tissues by hif-1 and hif-2 and are, therefore, considered to be of clinical relevance in ra. background. membrane bound glucocorticoid receptors (mgr) play a pivotal role in pathogenesis of chronic inflammatory diseases as indicated by clinical observations. patients with sle show high frequencies of mgr positive monocytes, sometimes even higher than found in patients with active ra. with increasing glucocorticoid dosages expression of mgr on monocytes of sle-patients is downregulated, suggesting a negative feedback loop to control glucocorticoid action. these receptors represent an effective target for diagnosis and monitoring of different inflammatory diseases, but a feasible detection method is still necessary. objectives. we compare two methods of high-sensitive immunofluorescence staining -the well established liposome procedure with the commercialized faser-technique. methods. hek293t cells were cultured for 24 h with/without 5 µg/ml brefeldin a in a humidified incubator at 37°c. human cd4 positive t cells and cd14 positive monocytes were isolated via magnetic-activated cell sorting and subsequently cultured in rpmi 1640. monocytes were incubated for 24 h with/without 2 µg/ml lps. for liposome based highsensitivity immunofluorescence staining cells were incubated with the monoclonal (digoxigenin conjugated) anti-gr antibody, followed by incubation with anti-digoxigenin/anti-biotin matrix. subsequently biotinylated cy5 liposomes were added. faser technique was performed as described by the manufacturer (miltenyi biotec). dead cells were excluded by adding pi before cell acquisition, using a bd facs calibur flow cytometer. the acquired data were analyzed using flowjo 7.6.1 software. results. the human mgr, which cannot be reliably detected with conventional staining methods, is detectable with the liposome procedure as well as with the commercialized faser-apc technique. furthermore, the faser-apc-procedure is more sensitive (94.51% vs 73.2%) and more specific (99.57% vs. 98.93%) compared to the liposome technique. additionally, minor changes of mgr expression can also be demonstrated with the faser technique. the faser procedure shows technical advantages: the commercially available faser-apc-kit is performed according to a standarized protocol and is less time consuming compared to the liposome procedure. conclusion. the human mgr is easily detectable with the commercialized faser kits, which represent an alternative due to a consistent quality and a standardized production. this method facilitates the analysis of the role that mgr play in the pathogenesis of chronic inflammatory diseases and perhaps provoke new insights in glucocorticoid therapy. background. in previous studies we detected th-positive, catecholamine-producing cells in inflamed hypoxic synovial tissue. therefore, the aim of our study was to investigate the influence of hypoxia induced catecholamines on inflammatory responses in arthritis. methods. synovial cells of rheumatoid arthritis (ra) and osteoarthritis (oa) patients were isolated and cultivated under normoxia or hypoxia with/without stimulating enzyme cofactors of th and inhibitors of th. expression of th and release of cytokines and catecholamines was analyzed. the effect of th+ cells was tested by adoptive transfer into dba/1 mice with collagen type ii-induced arthritis (cia). th+ cells were generated from mesenchymal stem cells by defined dopaminergic factors. results. hypoxia increased th protein expression and catecholamine synthesis and decreased release of tnf in oa/ra synovial cells compared to normoxic conditions. this inhibitory effect on tnf was reversed by th inhibition with alpha-methyl-para-tyrosine (αmpt). incubation with specific th cofactors (tetrahydrobiopterin and fe2+) increased hypoxia-induced inhibition of tnf, which was also reversed by αmpt. adoptive transfer of th+ cells reduced cia in mice, and 6 hydroxydopamine, which depletes th+ cells, reversed this effect. conclusion. in summary, this study presents that th-dependent catecholamine synthesis exhibits anti-inflammatory effects in human ra synovial cells in vitro, which can be augmented under hypoxic condi-tions. in addition, the anti-inflammatory effect of th+ cells has been presented the first time in experimental arthritis in mice. background. previously, we demonstrated that long-lived plasma cells contribute to the pathogenesis of antibody-mediated diseases and should therefore be considered as a promising therapeutic target in systemic lupus erythematous (sle). in bone marrow stromal cells expressing the chemokine cxcl12 organize these niches that provide for the plasma cell survival. cxcl12 is the ligand of cxcr4 expressed on plasma cells. in this study we investigated the contribution of cxcl12-cxcr4 interaction to the longevity of plasma cells in the murine model of lupus. methods. plasma cells purified from spleens of nzb/w mice were incubated with the cxcr4 blocker amd3100 and then adoptively transferred to immunodeficient rag1−/− mice. after 14 days we analyzed the number of plasma cells in bone marrow. furthermore, ova immunized nzb/w mice were treated intraperitoneally with amd3100 after boost; anti-ova secreting plasma cells in bone marrow were checked on day 3 and 15 after boost. the effect of plasma cell depletion was investigated in nzb/w mice using amd3100 alone or combined with bortebomib for two weeks. results. two weeks after adoptive transfer the number of plasma cells treated with amd3100 was lowered by 60% in bone. after secondary immunization with ova the amd3100 treatment resulted in a significant reduction of anti-ova secreting plasma cells in bone marrow by 33% on day 3 and by 23% on day 15. after 15 days the number of mhc class ii negative anti-ova secreting plasma cells significantly decreased by 42% in bone marrow of treated mice. amd3100 efficiently depleted plasma cells including long-lived. after two weeks treatment, total plasma cell number was decreased by 69% in spleen and 61% in bone marrow; long-lived plasma cells were reduced by 67% in spleen and 64% in bone marrow. the combination of bortezomib with amd3100 in nzb/w significantly enhanced the depletion of long-lived plasma cells compared to monotherapy. conclusion. cxcr4 blockade with amd3100 can reduce the homing of plasma cells to the bone marrow and the survival of long-lived plasma cells. the combination of bortezomib with amd3100 shows synergistic effects on plasma cell depletion. the findings highlight the importance of the cxcr4-cxcl12 axis for the plasma cell niche. zeitschrift für rheumatologie suppl 2 · 2013 | er.09 tnfr1 expression defines synovial tissue infiltrating cd4+ t cells in patients with rheumatoid arthritis k background. one hallmark of rheumatoid arthritis (ra) is the infiltration of the synovial membrane by cd4+ t cells. it has previously been shown that infiltrating cd4+ t cells differ from non-infiltrating ones in their increased expression of tnfr1. furthermore, tnfr1 is expressed on a fraction of circulating cd4+ t cells from ra patients, but not from healthy controls. aim of the study was the characterization of tnfr1+ cd4+ t cells in patients with rheumatoid arthritis. methods. peripheral tnfr1+ cd4+ t cells from ra patients were analyzed by flow cytometry. the expression of naive and memory t cell markers (cd45ra and cd45ro), markers for t cell activation (cd25, cd71 and cd154) and of icam-1 as well as the frequencies of the positive cells were determined. to identify the t helper cell signature of tnfr1+ cd4+ t cells, intracellular staining of the th1, th2 and th17 master transcription factors t-bet, gata-3 and ror-γt, respectively, was performed. results. peripheral tnfr1+ cd4+ t cells have neither a preferential naive nor a memory phenotype, but showed higher expression of the activation markers cd25, cd71 and cd154 than tnfr1-cd4+ t cells. tnfr1+ cd4+ t cells express higher frequencies of the t-bet and rorγt than tnfr1-cd4+ t cells. there is no difference in gata-3 expression between tnfr1 positive and negative cd4+ t cells. functionally, it has been shown that the cytokine tnf acts as chemokine to attract cd4+ t cells to the rheumatoid joints. beside this direct effect of tnf, there are known indirect effects of tnf including the upregulation of cell adhesion molecules like icam-1. therefore, icam-1 expression of migrating tnfr1+ t cells was investigated. the results show, that migrating tnfr1+ t cells recovered from synovial tissue are more frequently icam-1 positive than non-migrating ones. conclusion. tnfr1+ expression characterizes cd4+ t cells functionally capable of infiltrating the rheumatoid synovium in an icam-1 dependent manner. the results show, that tnfr1 expression defines a pathogenic subset of activated cd4+ t cells with th1 and/or th17 signature in patients with rheumatoid arthritis. hypoxia increased the production of interleukin-1β in lps-primed human monocytes n background. monocytes are major players in the innate immune system and are recruited to sites of inflammation, where the environmental conditions vary extremely compared to the interstitium under physiological conditions. for example, in rheumatoid arthritis the inflamed joints are severely hypoxic. this decreased oxygen level could be a triggering factor for the activation and survival of monocytes. aim of the study was to analyze the influence of hypoxia on lipopolysaccharide (lps)-induced cytokine production in primary human monocytes methods. immunomagnetically separated monocytes from the blood of healthy donors were cultured for 16h under hypoxic conditions (1% oxygen). results. cytokine measurement in the supernatant with elisa showed increased concentrations of interleukin-1β (0.9 ng/ml vs. 4 .35 ng/ ml, p=0.0019) and interleukin-6 (63.8 ng/ml vs. 125.9 ng/ml, p=0.019), but not of tnf, after hypoxia and lps-stimulation. cleavage of the il-1β proform to its active form is dependent on the assembly of the inflammasome and the recruitment of caspase-1 followed by their activation. when inflammasome assembly was blocked with high extracellular k+-buffer or by inhibiting intracellular ca-signalling with the ca-chelator bapta-am, hypoxia induced il-1β release was abrogated. similarly, il-1β release after culture under hypoxia was also abolished in monocytic thp1-cells, which are genetically made deficient for the inflammasome components nlrp3 and asc. one activating signal for the inflammasome was shown to be the release of reactive oxygen species (ros), since mitochondrial ros staining with mitosox revealed an increased mitochondrial ros release under hypoxic conditions. accordingly, the induction of mitochondrial ros through decoupling of the electron transport chain with rotenone also triggered an increase of il-1β release under normoxic conditions. analysis of blood monocyts from ra patients showed no difference in lps and hypoxia induced il-1β release compared to healthy controls (1.84 ng/ml vs. 1.88 ng/ml). conclusion. this study shows, that hypoxia leads to the activation of the inflammasome, the recruitment of caspase-1 and the subsequent cleavage and release of interleukin-1β in human primary monocytes. intracellular calcium mobilization and mitochondrial ros production were shown to be essential mechanisms triggering inflammasome assembly. background. cell-derived membrane-coated microparticles have been identified as important mediators in intercellular communication. during the process of apoptosis, dying cells start to dynamically release microparticles. polymorphonuclear neutrophils are the most abundant type of leukocytes, representing 50-70% of all white blood cells. due to their very short lifespan, they are the source of massive amounts of apoptotic cell-derived microparticles (admps). while the interaction between neutrophils and t lymphocytes has been focus of extensive research, the influence of neutrophil-derived microparticles on t cells has not been analysed yet. in this study, we investigated the effect of membrane-coated microparticles released by apoptotic neutrophils on different t helper cell subsets. methods. different cd4+ t cell subtypes were sorted according to the expression of cd25, cd127, cd45ra and cd45ro and co-cultured with admps or apoptotic cell remnants purified from uv-irradiated neutrophils isolated from the peripheral blood of healthy donors. t cells were stimulated by okt3 and anti-cd28 antibodies and cell proliferation was measured by 3h-thymidine incorporation or pkh26-staining. secretion of cytokines was quantified by elisa. results. admps released by neutrophils selectively suppressed the proliferation of cd4+cd25-cd127+ tc in a dose-dependant manner and prevented the upregulation of cd25 on the t cell surface, while maintaining the expression of cd127. the secretion of tumor necrosis factoralpha (tnfα) by t cells stimulated in the presence of admps was significantly reduced. interestingly, in contrast to admps, the apoptotic cell remnants of neutrophils exerted no effect on t cells. the suppressive effect of admps could be completely abrogated by the addition of interleukin(il)-2 or il-7 or by the presence of cd4+cd25+cd127+ t cells. conclusion. neutrophil admps suppress the proliferation of cd4+cd25-cd127+ t cells under conditions of limiting il-2 and il-7 concentrations. this could represent an important mechanism to prevent inappropriate activation and expansion of resting t helper cells in the absence of sufficient stimulation and cytokine production. t. alexander 1 background. recent reports have shown dysregulated micrornas in murine lupus models, among them increased expression of mirna-182, which has been demonstrated to target the transcription factor foxo1 in activated cd4+ t cells. the loss of foxo1 activity in t cells is associated with spontaneous t cell activation, clonal expansion and autoantibody production, all of which are present in systemic lupus erythematosus (sle). methods. expression levels of microrna-182 and foxo1 were analyzed with rt-pcr in magnetic purified peripheral blood cd4+ t cells from 9 patients with sle and healthy controls (hc). multicolor flow cytometry was performed to analyze cd4+ t cell expression for ccr7, cd45ra, ki-67, foxp3, the interleukin-7 receptor-α and phosphorylated stat-5a (pstat5). analysis of serum il-7 levels was performed with elisa in 27 sle patients and hc (r&d systems). results. mirna-182 was significantly upregulated in cd4+ t cells from sle patients compared to hc (median expression 8.89×10e-7 vs. 3.96×10e-7, p=0.008) while foxo1 mrna levels were decreased, yet without reaching statistical significance. analysis of ki-67 expression revealed an increased percentage of proliferating cd4+ t cells in sle (5.23% vs 2.21%, p=0.006). overall, cd4+ t cellular proliferation in sle was associated with increased frequencies of cd45ra-ccr7-effector memory t cells and enhanced basal pstat5 levels (median mfi 503.5 vs 399.0, p=0.010), suggesting a recent stimulation with common gamma chain(γc)-signaling cytokines. in this regard, tcons from sle samples displayed decreased expression levels for the foxo1 target gene cd127 (mfi 2021 vs. 2553, p=0.049) and serum il-7 levels were significantly higher in sle compared to hc (17.0 pg/ml vs. 10.2 pg/ml, p=0.001). conclusion. mir-182 expression has been shown to be dependent on stat5 activation and to promote clonal expansion of activated cd4+ t cells. our data suggest that enhanced il 7r/stat5 signaling mediates induction of mir 182 expression, which in turn promotes the proliferation of tcons in sle. the relative contribution of il 7r/mir-182/foxo1 axis on the enhanced proliferative capacity of sle tcons remains elusive and merit further investigation. collectively, our data provide new insights in the pathophysiology of t cell hyperactivity in sle and identifies mir-182 as a candidate target for future therapeutic approaches. background. cell activation and apoptotic cell death leads to the formation of membrane-coated vesicles (mcvs). mcvs have previously been identified as mediators of cell-to-cell communication and carriers of microrna. an impaired clearance of apoptotic debris caused by an increased rate of apoptosis or a defect in phagocytic-cell clearance has been observed in sle patients. in this study, we analyzed the microrna content of activated and apoptotic lymphocytes and their corresponding mcvs from both normal healthy donors (nhds) and sle patients. further we investigated the immunomodulatory effect of mcv uptake by monocytes. methods. microrna content of activated and apoptotic lymphocytes and corresponding mcvs of nhds and sle patients were compared in an agilent microrna array and validated by qpcr. apoptosis was induced by uvb-irradiation. mir-155 expression in monocytes after uv-mcvs engulfment was determined by qpcr. expression of mir-155 target protein tab-2 was analyzed by western blot. results. mir-155* levels were decreased after apoptosis induction in lymphocytes and apoptotic mcvs compared to their viable correlates. mir-155, mir-99a and mir-34b were decreased in apoptotic lymphocytes compared to viable ones but increased or not significantly changed in apoptotic mcvs compared to viable mcvs, indicating a directional transport of microrna into mcvs. mir-34a was expressed at higher levels in viable sle lymphocytes and mcvs compared to nhds. mir-34b expression was decreased in uv-lymphocytes and uv-mcvs of sle patients. functional assays confirmed higher mir-155 levels and consecutively decreased target protein levels in monocytes after engulfment of uv-mcvs. conclusion. within this study we could show an unequal distribution of distinct microrna into mcvs released by activated or apoptotic lymphocytes. further the microrna content was regulated in whole apoptotic cells after uvb-irradiation. this suggests a directional transport rather than a random distribution. thus, cells regulate their microrna as well as the microrna content within released mcvs. we could show a microrna and protein expression change in phagocytes after mcv engulfment. hence, our results suggest mcvs could serve as a transport vehicle for microrna to mediate cell-to-cell communication and influence intracellular processes in phagocytes. disturbances of this system might contribute to the pathogenesis of sle. results. we found in the spleens of nzb mice 10-times higher numbers of long-lived plasma cells and megakaryocytes compared to wildtype, in nzw mice equal numbers and in nzb/w mice numbers between those for nzb and nzw or wildtype. moreover, in the spleen a fraction of plasma cells clustered around megakaryocytes. we also detected a missense mutation in the c-mpl gene of nzb mice leading to an amino acid replacement within the essential tpo-binding site. upon tpo stimulation of splenocyte and bone marrow cultures nzb cultures responded significantly stronger resulting in the double amount of megakaryocytes compared to nzw cultures. conclusion. in summary, our data indicate that augmented megakaryopoiesis enables the accumulation of a greater number of autoreactive plasma cells in lupus prone nzb/w mice. thus, we assume that enhanced megakaryopoiesis and higher megakaryocyte numbers are contributing to the development and/or pathogenesis of sle. background. baff is a cytokine important for the stimulation and survival of autoreactive b cells and therefore might play a role in several autoimmune diseases, e.g. autoimmune arthritis. in psoriasis arthritis, baff correlates with disease activity and testosterone, but only in male patients, suggesting a role for sex hormones in the regulation of baff. therefore, we wanted to determine if baff production in rheumatoid arthritis and osteoarthritis fibroblasts was regulated by neuroendocrine mediators. methods. fibroblasts were isolated from synovial tissue of ra (n=10) and oa (n=10) patients and cultured in vitro under different conditions. baff was determined by elisa. results. isolated fibroblasts were cultured in the presence or absence of interferon-gamma (ifn-γ), il-1, lipopolysaccharide (lps), tumor necrosis factor (tnf), cpg, poly i:c, and cortisol in different combinations for 48 and 72 hours to determine the optimal stimulation strategy for induction of baff production (measured by elisa in supernatants) in fibroblasts. ifn-γ best induced baff in ra and oa fibroblasts. ifn-γ-induced baff production in fibroblasts was decreased by dihydrotestosterone in a concentration dependent manner. the effect was specifically inhibited by nilutamid, a testosterone receptor antagonist. furthermore, stimulation of beta-adrenoceptor increased, whereas stimulation of alpha-adrenoceptors did not change inf-γ-induced baff in synovial fibroblasts. in general the effects were more pronounced in ra as compared to oa fibroblasts. conclusion. taken together, inf-γ-induced baff production in synovial fibroblasts is decreased by testosterone and increased by betaadrenergic stimuli. therefore, neuroendocrine regulation of inflammation in the inflamed joint might be in part mediated by regulating baff production in synovial fibroblasts. a. grützkau 1 , c. kyogoku 1 , b. smiljanovic 2 , j. grün 1 , r. biesen 2 , t. alexander 2 , f. hiepe 2 , a. radbruch 1 , t. häupl 2 1 deutsches rheuma-forschungszentrum (drfz), berlin, 2 charité -universitätsmedizin berlin, medizinische klinik mit schwerpunkt rheumatologie und klinische immunologie, berlin background. gene expression profiling experiments using peripheral blood mononuclear cells (pbmcs) revealed a crucial role of type i interferon (ifn) in the pathogenesis of systemic lupus erythematosus (sle). however, it is almost unknown how particular leukocyte subsets contribute to the overall type i ifn signature described for pbmcs. furthermore, a detailed analysis of how ifn signatures differ in autoimmune disease from that observed after viral infection is missing so far. therefore, we compared expression levels of 2442 ifn signature genes in peripheral cd4+ t helper cells and monocyte (mo) subsets isolated from patients with sle, healthy donors (nd) and nd vaccinated against yellow fever by global gene expression profiling. methods. peripheral blood from 8 patients with sle and 4 nd were recruited. same nd were examined before and after immunization by yellow fever vaccine. after sorting cells, isolated rna were applied to affymetrix human genome u133 plus 2.0 array. data analysis was done using bioretis database, genesis software and ingenuity pathway analysis (ipa). results. comparing gene expression profiles of yellow fever immunized individuals and active sle patients it was possible to identify a "common" and an "autoimmune-specific" ifn signature. although major ifn signature genes were commonly expressed in cd4+ t cells and mo of patients with sle and immunized nd, expression magnitudes of them were higher in patients with sle compared to immunized nd. in sle, in addition to the typical "viral-induced" ifn signature, genes that are involved in apoptosis signaling, antiviral pkr signaling, fcγ receptor-mediated phagocytosis and il-10-/il-9-/il-15-mediated jak/ stat signaling pathways were identified by ipa. conclusion. this study demonstrated that ifn signature in autoimmunity and that in viral infection are quite different in the number of ifn-related genes activated and their expression magnitudes. autoimmunity is characterized by a much stronger expression of ifn signature genes and is obviously modulated by a separate set of co-regulated genes defining the "autoimmune-specific" ifn signature. in summary, "common" and "autoimmune-specific" ifn signature genes are of potential interest as clinical biomarkers in sle diagnostics to differentiate between a disease flare and a viral infection. of peripheral blood lymphocytes (pbl). there is currently no data available about nk cells in gpa. the aim of this study was to evaluate the presence of nk cells in gpa granulomas and their proportions in pbl as a basis for a potential role in gpa. methods. paraffin sections of granulomas of 20 gpa, 5 sarcoidosis and 5 tuberculosis patients were stained with a cd56 monoclonal antibody. nk cell (cd3-cd56+) proportions of pbl in 30 gpa patients and 27 healthy controls (hc) were analysed by facs analysis. clinical data was extracted from medical records. results. contrary to granulomas from tuberculosis and sarcoidosis which showed a considerable infiltration by cd56 positive cells, there was not a single cd56 positive cell in granulomas from 20 gpa patients. therefore, the tissue destructive character of gpa granulomas is associated with a lack of nk cells. gpa patients with inactive disease [birmingham vasculitis activity score (bvas) = 0, n=19] possessed a significantly higher nk cell proportion in pbl (mean ± standard deviation: 24.4±13.5%) than both gpa patients with active disease (bvas>0, n=11, mean=10.7±5.6%) (p=0.0026) and hc (n=27, mean=14.4-7.7%, p=0.004). thus, clinical remission is accompanied by an increase in the nk cell proportion in pbl. interestingly, patients with inactive disease that had "normal" nk cell proportions of less than 20% of pbl (n=7) showed a more severe disease course than those with more than 20% of pbl. conclusion. nk cells might, therefore, be helpful to limit granulomatous inflammation. whether nk cell proportion in pbl might be a useful biomarker in gpa, e.g. as predictor for relapses, will be further evaluated in our future studies. v. gerl 1 background. plasmacytoid dendritic cells (pdcs) are considered a crucial element in sle pathogenesis due to their potency to produce high levels of ifn-α. this innate immunological function of pdcs is lost by terminal differentiation into a professional antigen-presenting cell (pdc-derived dc), thereby upregulating costimulatory molecules and downregulating innate characteristics, e.g. bdca-2 and ifn-α expression. pdc-derived dcs have not been described in vivo yet, probably due to the fact that they lose their specific markers during differentiation. furthermore, pdcs can differentiate into myeloid dcs by various stimuli. in sle, where low expression of bdca-2 is commonly seen, this differentiation could be relevant and point to such a lineage switch as well as to an activated state of pdcs. aim. to characterize pdc subsets of differentiation/activation in human peripheral blood and to study their impact on autoimmune inflammation in sle. methods. 8-color-flowcytometric analyses were performed on whole blood of healthy donors and sle patients. pdcs were identified by cd3-/cd19-/cd14-/cd123high//bdca-2+/hla-dr+ expression and characterized for cd11c, bdca-1 and the macrophage-associated siglec-1, expressed on monocytes of active sle patients in an ifn-α dependent manner. cd86 and cd83 expression were measured in parallel. results. we found a small subpopulation of siglec-1 expressing pdcs in human peripheral blood. compared to siglec-1 negative pdcs, siglec-1 positive pdcs express significantly lower bdca-2 and cd123, higher hladr background. agonistic autoantibodies against the angiotensin ii receptor type 1 (at1r) and the endothelin receptor type a (etar) have been identified in patients suffering from systemic sclerosis (ssc). here we examined the expression of at1r and etar in human immune cells and pathological effects mediated through these receptors by corresponding autoantibodies (aabs). methods. at1r and etar protein expression on peripheral blood mononuclear cells (pbmcs) from healthy individuals and ssc patients was analyzed using flow cytometry, mrna expression was examined by real-time pcr in pbmcs from healthy donors. in addition, pbmcs from healthy donors were stimulated in vitro with affinity-purified immunoglobulin g (igg) fractions from ssc patients positive for at1rand etar-aabs, and with igg from healthy donors serving as control. alterations in cell surface marker expression, cytokine secretion and chemotactic motility were analyzed using flow cytometry, elisa, and chemotaxis assays, respectively. results were correlated with characteristics/clinical findings of the igg donors. results. both at1r and etar were expressed on human peripheral lymphocytes and monocytes. protein expression of both receptors was decreased in ssc patients when compared to healthy donors and correlated negatively with disease duration. in addition, igg fractions of ssc patients induced t cell migration in an anti-at1r and anti-etar aab level-dependent manner. moreover, igg of ssc patients was capable of stimulating pbmcs to produce more il-8 and ccl18 than igg of healthy donors. all effects could be significantly abrogated by the application of selective at1r and etar antagonists. statistical analysis revealed a negative correlation between ssc igg-induced il-8 concentrations and disease duration, between ssc igg-induced ccl18 concentrations and time since onset of lung fibrosis as well as an association of ccl18 concentrations with vascular complications of the corresponding ssc igg donors. conclusion. we demonstrated the expression of both, at1r and etar, on human peripheral t cells, b cells and monocytes and found signs for a chronic receptor activation in ssc patients. the inflammatory and profibrotic effects upon aab stimulation in vitro, and their associations with clinical findings suggest a role for autoantibody-mediated activation of immune cells mediated through the at1r and etar in the pathogenesis or even the onset of the disease. the bioenergetic role of hif-1 and hif-2 during angiogenesis of human microvascular endothelial cells background. hypoxia and angiogenesis are features of inflamed and injured tissues. the transcription factors hypoxia inducible factor (hif)-1 and (hif)-2 regulate the cellular and metabolic responses to reduced oxygen tensions thereby promoting angiogenesis with implications on the pathogenesis of ra. we investigated the effects of a knockdown of either hif-1α or hif-2α in human microvascular endothelial cells (hmec) on angiogenesis and bioenergetics under hypoxia (1% o2) versus normoxia (18% o2). methods. specific knockdown of either hif-1α or hif-2α was conducted by shrna-technology. to assess angiogenesis of hmecs both tubuli and node formation under hypoxia versus normoxia were investigated. expression of hypoxia driven genes involved in the metabolic response to hypoxia (gapdh/pgk/glut1/ldha) was quantified by realtime rt-pcr. the bioenergetic status of the cells was quantified via atp/adp measurements. results. knockdown of hif-1α/hif-2α resulted in a loss of hypoxia induced angiogenesis. focusing on bioenergetic aspects, we found hypoxia to significantly induce pgk, ldha and gapdh in control cells. knockdown of hif-1α and hif-2α, respectively, did not affect the hypoxic induction of pgk and ldha. in hif-1α and hif-2α knockdown-cells, hypoxia was still capable of inducing gapdh, with a less pronounced effect in hif-1α knockdown-cells. hypoxia did not significantly up-regulate glut1, neither in control nor in hif-1α or hif-2α knockdown-cells. the knockdown of hif-2α resulted in significantly decreased expression of glut1 under hypoxia. we also found the atp/ adp ratio to be similar in control, hif-1α and hif-2α knockdown-cells under normoxia. under hypoxic conditions hif-1α knockdown-cells showed significantly reduced atp/adp ratios -indicating that less atp is available -compared to hif-2α knockdown-cells. conclusion. hif-1α and hif-2α are both key regulators of angiogenesis. however, they do differ in their potency to regulate cellular energy metabolism. this leads us to conclude that hif-2α does directly influence angiogenesis via regulating the synthesis of proangiogenic factors (as previously shown), whereas hif-1α affects angiogenesis via effects on cellular energy metabolism as indicated by the reduced expression of gapdh and the diminished atp/adp ratio. these findings provide new insights into regulation of angiogenesis in inflamed (hypoxic) tissues and are, therefore, considered to be of clinical relevance in ra. low baseline complement levels, autoantibody persistence and delayed thymic reactivation are risk factors for development of relapses after hematopoietic stem cell transplantation for refractory sle background. our previous research has provided the evidence that an autoreactive immune system can be "reset" into a healthy, tolerant state by immunoablative treatment to eradicate pathogenic effector cells, followed by transplantation of hematopoietic progenitor cells (hsct). nevertheless, disease flares may occur in a subset of these patients posttransplantation. here, we longitudinally analyzed the immune reconstitution of these patients to identify markers for favorable long-term responses. methods. since 1998, 10 patients with refractory sle received a cd34+selected autologous stem cell transplantation after immunoablation with antithymocyte-globulin (atg) and cyclophosphamide as part of a monocentric phase i/ii clinical trial. autoantibody titers were evaluated with elisa, peripheral t-and b lymphocyte subsets immunophenotyped using multicolor flow cytometry. results. clinical remission (sledai ≤3) could be achieved in all patients, despite immunosuppressive drug withdrawal, associated with disappearance of anti-dsdna antibodies and marked reduction of protective antibodies in serum. unfortunately, two patients died due to transplant-related infections. from the remaining eight patients, five patients are in long-term clinical remission for up to 14 years after hsct, while three patients suffered a relapse of sle at 18, 36 and 80 months post-transplantation, respectively. patients with early relapses (≤36 months) had decreased baseline complement levels, showed persistence of antinuclear antibodies (ana), less significant reduction in protective antibody levels and had slower repopulation of cd31+ cd45ra+ thymic-derived cd4+ t cells after hsct (<100/µl at 18 months) when compared to long-term responders. in addition, flow cytometric analyses revealed an expansion of circulating plasmablasts and increased coexpression of siglec-1 on monocytes (as surrogate marker for type-i interferon signature), preceding the clinical flares by ~6 months. conclusion. low baseline complement levels, persistence of ana and delayed thymic reactivity post-transplantation could be identified as risk factors for development of lupus flares after hsct. since atg-mediated cell lysis is complement-dependent, we conclude that low serum complement is directly associated with incomplete depletion of immunologic memory cells in these patients, which provides a rationale for complement substitution before immunoablation. moreover, lupus flares may be predicted individually by flow cytometry with plasmablast expansion and recurrence of type-i interferon signature. background. systemic lupus erythematosus (sle) is a chronic autoimmune disease characterized by the generation of pathogenic antibodies directed against a variety of autoantigens. we have previously shown that long-lived autoreactive plasma cells can contribute to chronicity and refractoriness of sle. our study is aimed to develop new methods for depletion of long-lived plasma cells in nzb/w mice, a model of sle. methods. we studied different treatment protocols on plasma cell survival: irradiation-based and more selective depletive treatments. 10-12 week-old nzb/w f1 mice were exposed to three different irradiation doses (10, 14, and 15 gy in two splitted doses with a 3-h interval). the following protocols were also investigated: 1) two bortezomib (bz) injections (0,75 mg/kg, i.v.) combined with anti-mouse cd20 (10 mg/kg, i.v.), 2) three bortezomib injections combined with anti-mouse cd20, 3) three bortezomib injections combined with anti-lfa-1 and anti-vla-4 antibodies (affecting directly the plasma cell niche; 200 µg, i.p.) in a 2-d interval, plus anti-mouse cd20 and anti-b220 (250 µg, i.v.). the plasma cells were analyzed in spleen and bone marrow by facs and elispot. results. the frequency of remaining plasma cells in bone marrow after 10, 14 and 15 gy irradiation were 52, 14 and 0,7% respectively, and in spleen were almost 50, 10 and 0,2%. short-term treatments with agents that affect plasma cells (bortezomib, anti-lfa1 plus anti-vla4) effectively deplete plasma cells including long-lived plasma cells in spleen and bone marrow of nzb/w mice. because of the b cell hyperactivity in nzb/w mice, we observe a rapid regeneration of autoreactive plasma cells in spleen and bone marrow. therefore, plasma cell depletion protocols were combined with b cell depletion. especially, the combination of plasma cell targeting with bortezomib, anti-lfa1 and anti-vla4 with b cell targeting (anti-cd20 plus anti-b220) interrupted the repopulation of autoreactive plasma cells in spleen and bone marrow. conclusion. very high doses of irradiation result in effective depletion of long-lived plasma cells but lower doses not. depletion of long-lived plasma cells can be achieved by the proteasome inhibitor bortezomib and by targeting both adhesion molecules lfa1 and vla4. the combination with b cell depletion is needed to prevent regeneration of autoreactive plasma cells. varicella-zoster-virus(vzv)-specific lymphocytes and igg antibody avidity in patients with juvenile idiopathic arthritis or rheumatoid arthritis background. varicella zoster virus (vzv) is a herpes virus that establishes a life-long latent infection with risk of reactivation (shingles) particularly in immunosuppressed patients with autoimmune disorders. patients with rheumatoid (ra) or juvenile idiopathic arthritis (jia) have a high risk for disseminating varicella zoster virus (vzv) infection or herpes zoster. this study was aimed to investigate the humoral and cellular immune response to vzv including assessment of igg-anti-vzv avidity and vzv-specific reactivity of lymphocytes in ra (n=56) or jia patients (n=75) on different treatments, including biologic agents, such as anti-tumor-necrosis-factor(tnf)-alpha or anti-interleukin-6 (il-6) receptor inhibition (tocilizumab), compared to 37 healthy adults (ha) and 41 children (hc). methods. igg-anti-vzv concentrations and avidities were quantified by an adapted elisa. vzv-specific interferon-gamma-producing lymphocytes (spot forming units, sfu/1,000,000 cells) were analyzed by elispot. results. no significant differences in the vzv-igg concentrations or avidities were found between the groups. however, lower igg-anti-vzv concentrations were found in tocilizumab-treated ra compared to ha and ra without biologic agents. ra showed lower median sfu (15/1,000,000 cells) than ha (57/1,000,000 cells), with lowest sfu in adalimumab-treated ra (10/1,000,000 cells). sfu were not altered in tocilizumab-treated ra and after incubation with anti-il-6 in vitro. no differences regarding igg-anti-vzv concentrations, rai and cellular reactivity were found between jia and hc. conclusion. our study demonstrated that ra and jia patients are still able to maintain humoral and cellular immune responses to vzv despite immunosuppressive therapy or biologic agents. in ra, the role of lower cellular reactivity for risk of herpes zoster has to be considered for recommendations on vaccination. cmv-specific cd8+ t cells from ra patients contribute to autoimmune disease 41 zeitschrift für rheumatologie suppl 2 · 2013 | 1. increased frequency of lir-1 (also called cd85j or ilt2) on cd8+ t cells has been associated with autoimmune disease. furthermore, it has been shown that latent cytomegalovirus (cmv) infection contributes to the expansion of cd28− t cells. hence we were interested in the influence of cmv infection on the lir1 expression on t cells in ra patients. methods. we were interested in the role of lir1+ t cells in ra patients, which potentially contribute to the autoreactive t cell pool, especially in cmv+ patients. therefore, we investigated the expression and function of lir-1 on cd8+ t cells in peripheral blood mononuclear cells (pbmc) from patients with rheumatoid arthritis by flow cytometry and cytotoxicity assay. results. flow cytometry analysis revealed higher frequencies of lir-1+ cd8+ t cells in cmv seropositive ra (n=49, mean%: 10.4) compared to cmv+ hd (n=51, mean%: 7.5, p=0.043). using hla-a*0201/cmvpp65 dextramers we analyzed cmv-specific cd8+ t cells. patients with ra had higher frequencies of cvm specific cd8+ t cells (n=8; mean%: 3.28) compared to healthy individuals (n=12; mean%: 1.35, p=0.04). phenotypically, cmv-specific cd8+ t cells are mainly cd28 negative and express lir-1. analysis of the cytolytic potential by cd107a expression revealed higher numbers of cd107a+cd8+ t cells in ra patients (n=3, mean%: 0,57) compared to healthy donors (n=3, mean%: 0,17). importantly, we found a significant correlation (p=0.034) of high numbers of cd8+lir-1+ t cells with high disease activity score (das28) in ra patients without immunosuppressive treatment (n=14, r=0,568) . tab.5 . conclusion. this is the first demonstration of significantly increased frequencies of lir-1+cd8+ t cells and of cmv-specific cd8+ t cells in patients with rheumatoid arthritis. these cells are characterized by a terminally differentiated phenotype. the higher cytolytic potential of cmv-specific t cells likely can be attributed to their function in containing latent cmv infection and to prevent cmv disease, but might potentially contribute to disease severity in ra patients. background. systemic lupus erythematosus (sle) is an autoimmune disease characterized by an acquired il-2 deficiency, which leads to a homeostatic imbalance between regulatory t cells (treg) and effector t cells (tcon; humrich et al. 2010). we recently demonstrated that treg homeostasis in lymphoid organs of diseased (nzbxnzw) f1 mice can be restored by treatment with recombinant il-2 (il-2) resulting in an amelioration of kidney disease. the aim of this study was to investigate the impact of il-2 therapy on intrarenal foxp3+ treg and kidney infiltrating conventional cd4+ t cells (tcon) in the (nzbxnzw) f1 mouse model of lupus nephritis. methods. (nzbxnzw) f1 mice with active nephritis were treated with recombinant il-2 either for a short period of 5 days or for a longer period of 30 days in total. absolute numbers, phenotype and proliferation of kidney infiltrating cd4+ t cell subsets were determined by flow cytometry at different time points. results. short-term il-2 treatment resulted in an enhanced proliferation and increased numbers and frequencies of intrarenal cd4+foxp3+ treg compared to untreated control mice. on the other hand, long term il-2 treatment did not result in a persistent expansion of the intrarenal foxp3+ treg population. however, total numbers of kidney infiltrating cd4+ tcon with a memory/effector phenotpye were diminished and cd4+ tcon showed markedly reduced signs of cellular activation. conclusion. our data indicate that short term il-2 treatment is able to expand the size of the intrarenal treg pool. in contrast, long term il-2 treatment decreases the numbers of kidney infiltrating memory/ effector t cells and reduces cellular hyperactivity suggesting that treg suppress the activation and expansion of infiltrating tcon. these results may in part explain the amelioration of disease induced by treatment with il-2 and underline the important role of intrarenal treg for the suppression of kidney disease in lupus mice. these results also provide additional important rationales for an il-2 based immunotherapy of human disease. from transcriptome to protein biomarkers in ra: joint compartment and monocytes outperform serum and whole blood background. a main challenge in disease-management of ra is to establish objective criteria relevant for diagnosis and therapeutic stratification of patients. this study focused on global approaches in dissecting inflammation in ra including transcriptome analyses of synovial tissue and blood monocytes and proteome analyses of synovial fluid and serum. methods. gene-expression profiles from synovial tissues and blood monocytes of ra and osteoarthritis (oa) patients were generated by affymtetrix arrays. elisa and multiplex immunoassays were used for validation of 30 candidate markers at the protein level in synovial fluid (sf) from ra and oa patients and in serum from the same group of patients and healthy donors. results. transcriptome analyses of synovial tissues from ra and oa revealed more than 1000 differentially expressed genes. to avoid difficulties in sampling synovial tissue and to avoid fluctuation in cellular composition of various cell types in blood, the transcriptome analyses from peripheral blood was focused on a specific cell population. monocytes were selected as the favourable cell type involved in the production of cytokines, which are often considered as therapeutic targets in ra. comparisons between ra and oa monocytes disclosed differential expression of more than 100 genes. in total, 30 genes that were up-regulated in synovial tissues and/or monocytes were used for validation at the protein level as potential biomarkers for ra. among these 30 biomarkers, chemokines (cxcl13, ccl18, ip10), adhesion molecules (vcam1, icam1, e-and p-selectins), proteolytic enzymes (mmp1, a1at), and the shedding form of cell surface molecules (cd14, cd163) background. idiopathic membranous nephropathy (imn) is a common cause of nephrotic syndrome in adults and has recently been identified as an autoimmune-mediated disease [1] . autoantibodies directed towards the m-type phospholipase a2 receptor (pla2r) are fairly specific for idiopathic mn and only found to a small percentage in sera from patients with secondary mn [2] . the outcome of patients with imn is quite diverse: about one third of patients have spontaneous remission, another third progress to require dialysis and the last third continue to have proteinuria without progression to renal failure. we performed serological profiles of 162 imn patients in order to compare antibody profiles to antibody frequencies found in the normal healthy population and to hopefully identify factors that help to predict disease course in imn. methods. serum samples of 162 patients with imn were assayed for a variety of autoantibodies by elisa, addressable laser bead immunoassay (albia) and to dsdna by crithidia luciliae assay. results. the prevalence of autoantibodies found in our imn cohort is summarized in tab. 1. anti-pla2r antibodies were found in about 54% of imn patients whereas the frequency of other antibodies was mostly below 2%. the one exception is anti-dfs70 that was found in 16.05% of imn patients. conclusion. the prevalence of anti-pla2r positive patients in our imn cohort matches what has been previously described [3] . the frequency of the other antibodies that we determined is comparable to what has been reported in the normal healthy population. it is important to note that anti-dfs70 antibodies are more prevalent in healthy individuals compared to patients with systemic autoimmune rheumatic diseases (sard; [4] ) whereas anti-ro52 reactivity is often regarded as a marker for sard. the absence of anti-ro52 and the high prevalence of anti-dfs70 confirms that imn is a rather organ specific autoimmune disease. background. activity and the quality of movement belong to the most fundamental diagnostic parameters for neurobehavioural analysis but in the past it has been difficult to include this information into pre-clinical murine disease models. here we tested the applicability of a radiofrequency identification (rfid) based automated tracking system in the experimental murine model of ovalbumin induced arthritis. methods. c57bl/6 mice were immunized twice with cationized ovalbumin in freund's complete adjuvant and onset of arthritis was induced two weeks after the last immunization by direct injection of cationized ovalbumin into the knee joint of the right hind leg. severity of arthritis was assessed through measurement of joint swelling and evaluation of histological changes. additionally mice were implanted with a rfid transponder and throughout the experiment their activity level was monitored by an id-grid sensor plate placed underneath the homecage. results. the joint inflammation in the ovalbumin induced arthritis model showed a quantifiable impact on the activity levels of the mice. our experiments could also show that movement activity correlates with disease severity as evaluated by clinical and immunological parameters. in the past employing behavioral methods was often limiting by group size, observation time and reproducibility and the stress of handling and new surroundings made results difficult to interpret. in our experiments a rfid-based automated tracking system allowed us to monitor individual activity long-term without removal of the mice from their homecage environment. this allowed for the correlation of clinical parameters to behavioral factors and adds another level of analysis to an established murine model. progranulin antibodies in a wide spectrum of autoimmune diseases results. autoantibodies against progranulin, a secreted and direct inhibitor of tnf-α receptors 1&2 were frequently identified in primary vasculitides. in detail, progranulin-antibodies were found during the course of disease in giant cell arteritis/polymyalgia rheumatica (14/65), takayasu's arteritis (4/13), classical panarteritis nodosa (4/10), behcet's disease (2/8), in granulomatosis with polyangiitis (31/81), churg-strauss syndrome (7/31) and in microscopic polyangiitis (6/17). in extended screenings progranulin-antibodies were also frequently detected in autoimmune connective tissue disorders, in rheumatoid and psoriatic arthritis and in inflammatory bowel disorders. in contrast progranulin-antibodies were only detected rarely in healthy controls (1/97), patients with obesity (0/40), residents of nursing homes (1/48), not in patients with cutaneously limited psoriasis (0/100), not in patients undergone sepsis (0/22), and not in patients with melanoma (0/98). a significant association of progranulin-antibodies with active disease states in granulomatosis with polyangiitis suggested a pro-inflammatory activity of progranulin-antibodies. this was supported by an observed neutralizing effect of progranulin-antibodies on the levels of circulating progranulin in elisa and western-blot. moreover, functional assays revealed, that progranulin-antibody containing sera render wehi-s cells far more sensitive to effects of administrated tnf-α, providing evidence for the suspected pro-inflammatory effect of progranulin-antibodies. conclusion. progranulin-antibodies occur in a widespread spectrum of autoimmune diseases and have a pro-inflammatory effect by neutralizing the physiologic tnf-blocker progranulin. background. flow cytometry (fcm) is widely used in research for molecular characterization at single cell level. conventional analysis is a semiautomated process of user defined gating and investigation in 2-d projections. for multiple parameter analysis with hundreds of marker combinations, this manual process is most limiting and impedes high throughput analysis. therefore, we developed a new algorithm for automated and standardized analysis of multiplex fcm data. methods. automation included asinh-transformation of data, cell grouping, population detection and population feature extraction. for grouping of cells, an unbiased unsupervised model based t-mixture approach with expectation maximization (em)-iteration was applied. populations were identified by meta-clustering of several experiments according to position and extension of cell-clusters in multi-dimensional space and by including a general procrustes analysis (gpa) step. for validation, peripheral leukocytes from healthy donors and patients with rheumatoid arthritis (ra) were prepared by hypoosmotic erythrocyte lysis and stained with different sets of lineage-specific antibodies. in parallel, different leukocyte samples were depleted of one of these populations by magnetic beads. qualitative and quantitative characteristics of major populations were compared with conventional manual analysis. results. whole blood leukocytes stained simultaneously with up to 7 markers were correctly distinguished in all major populations including granulocytes, t cells and their subpopulations, monocytes, b cells, and nk-cells. the result was comparable to the "gold standard" of manual evaluation by an expert. the new technology is able to detect subclusters and to characterize so far neglected smaller populations based on the new parameters generated. automated clustering did not require fluorescence compensation of data. cell-grouping is applicable even for large fcm datasets of at least 10 parameters and more than 1 million events. comparing the cell-clusters between ra and healthy controls, differences were detectable in several cell (sub-)populations, stable enough to perform correct classification into controls and disease. conclusion. this new approach reveals promising results for automated and time-saving analysis of large datasets from multiplex fcm. the algorithm avoids operator-induced bias, is able to detect unexpected sub-clusters and to characterize so far neglected populations. this may reveal not only new markers for disease activity but also for therapeutic stratification. background. lasp-1 localizes at focal adhesions, along stress fibres and leading edges of migrating cells regulating metastatic dissemination of different tumors. since rasf have been implicated in the spreading of disease by leaving cartilage destruction sites, migrating via the bloodstream and re-initiating the destructive process at distant articular cartilage surfaces, the underlying mechanisms are of special interest. therefore, we investigated the role of lasp-1 in sf migration and its effects on ra. methods. to identify lasp-1 expression and its sub-cellular distribution in human sf as well as in hind paws of wt and htnftg mice, we performed western blots and immunofluorescence. the migration of sfs derived from wt, lasp-1-/-, htnftg and lasp1-/-/htnftg mice was studied in a modified scratch assay as well as in live cell imaging studies. furthermore, a transmigration assay using sf from all four genotypes and murine endothelioma cells (bend.5) as an endothelial barrier was carried out. sf transmigration under inflammatory conditions was also evaluated by tnf-alpha stimulation of the endothelial cells in vitro. results. lasp-1 expression was up regulated in rasf and in sf from htnftg mice compared to healthy controls and was found at structures of cell adhesion and invasion. the scratch assay as well as the live cell imaging studies showed a significantly reduced migration of lasp(1 ng/ml) was applied. in parallel, il-2 stimulation significantly amplified the expression of anti-apoptotic bcl-2 in sle treg but not tcon. conclusion. in analogy to our previous findings in lupus-prone mice, treg from sle patients show the classical hallmarks of il-2 deficiency with loss of cd25 expression and a homeostatic imbalance between treg and tcon. these findings could be associated with a reduced il-2 expression by cd4+ t cells in sle patients. on the other hand, low-dose il-2 stimulation in vitro could restore these defects, underlying the potential of il-2 as a novel therapeutic option in sle. the glucocorticoid-dependent modulation of immune-mediated inflammatory arthritis by osteoblasts in mice is t cell independent background. at present, the role of adiponectin in rheumatoid arthritis is still controversial. there is some evidence indicating anti-inflammatory effects, for example adiponectin reduces the tnf release by macrophages. in contrast to its anti-inflammatory role, adiponectin also exerts pro-inflammatory effects locally in joints, inducing for example pro-inflammatory factors and matrix-degrading enzymes in ra synovial fibroblasts. moreover, our immunohistochemical analysis of ra bone tissue showed a co-localization of adiponectin with key cells of bone remodelling (osteoblasts, osteoclasts). however, the role of adiponectin in bone remodelling of ra still needs to be defined. in this study, we therefore focussed on adiponectin and its immunomodulatory properties on ra osteoblasts and osteoclasts. methods. human osteoblasts and osteoclasts were isolated from bone tissue and blood samples of ra patients. immunocytochemistry and rt-pcr were used to analyze the expression of adiponectin and its receptors in osteoblasts and osteoclasts. osteoblasts and osteoclasts were treated with adiponectin (10 µg/ml). adiponectin-mediated effects on the cytokine expression in osteoblasts and osteoclasts were analyzed using elisa. results. the expression of adiponectin and its receptors (adipor1, adipor2, and paqr3) by cultured ra osteoblasts and osteoclasts could be confirmed on translational and transcriptional level. stimulation of primary ra osteoblasts and osteoclasts with adiponectin resulted in an alteration of cytokine release. osteoblasts showed a time-and dose-dependent increase in il-6 production. furthermore, adiponectin induced the secretion of il-8 and gro-alpha and significantly increased the il-6 and mcp-1 production (il-6: 5-fold, p=0.004; mcp-1: 6-fold, p=0.004). stimulation with adiponectin resulted in an increase in il-6 production in pre-osteoclasts (5-fold) but not in osteoclasts. the secretion of il-8 was increased in pre-osteoclasts (18-fold) and osteoclasts (2-fold). the results of the present study confirm the pro-inflammatory potential of adiponectin in ra. the cytokines released after adiponectin treatment by osteoblasts and osteoclasts promote osteoclastogenesis or the migratory potential of osteoclasts and monocytes. together with the finding that adiponectin is present in the bone compartment of ra suggest an involvement of adiponectin in articular destruction. acknowledgement: funded by the german research society (spp1468, immu-nobone, ne1174/6-1). novel mechanisms of glucocorticoid therapy in arthritis anti-inflammatory acting glucocorticoids (gcs) are an important component of rheumatoid arthritis (ra) therapy. but their beneficial usefulness, especially in ra therapy, is hampered by severe side effects like glucocorticoid-induced osteoporosis (gio). until now the molecular mechanisms underlying the beneficial and side effects of gc therapy are poorly understood. gcs exert their actions via the glucocorticoid receptor (gr) that alters gene expression by either binding as a dimer to gc response elements in the promoter region of target genes or by interacting with and thus interfering with other transcription factors. for a long time gr dimerization was considered as the molecular base of side effects. interference of pro-inflammatory transcription factors, such as ap-1 and nf-κb by the gr monomer was believed to contribute to the therapeutic effects of gcs. in a model of gio we previously showed that unexpectedly interaction of the gr monomer with ap-1, but not nf-kb in osteoblasts is decisive for bone loss (cell metabolism 2010 11:517). in contrast, in antigen-induced arthritis (aia), we could demonstrate that gcs act in the acute inflammation of ra via the dimerized gr. particularly, gc therapy suppressed th1 and th17 cell derived pro-inflammatory cytokines in a dimerization dependent manner. furthermore th17, rather than th1 cells seem to be the most crucial targets for an efficient gc therapy since il-17-/-mice were resistant to gc therapy whereas ifnγ-/-mice responded as efficient as wild type mice to steroid treatment (pnas 2011 108:19317). in a more chronic arthritis model, the k/bxn serum transfer induced arthritis, we demonstrate now that, unexpectedly, dimerization of the gr in non-hematopoietic cells also contributes to the anti-inflammatory effect of gcs. thus, for immunosuppression of arthritis the gr is required in distinct cell types. taken together, for anti-inflammatory actions the gr dimerization dependent gene regulation is decisive in ra, whereas gio depends on the suppression of ap-1 dependent gene expression. intriguingly for anti-inflammatory activities of gcs immune and non-immune cells are involved. our approaches give new insights into gc action on arthritis and bone that can be translated into new concepts for anti-inflammatory therapies preventing gio. background. new bone formation and ankylosis are a hallmark of ankylosing spondylitis (as). the impact of cytokines and different mechanisms of new bone formation (endochondral vs. membranous) to syndesmophyte formation and joint ankylosis in as are still poorly understood. in order to analyze cartilage hypertrophy -as a potentially important element of endochondral bone formation -and to assess the possible influence of cytokines, we performed an immunohistochemi-cal study of the hyaline articular cartilage of facet joints of as patients in comparison to autopsy controls and patients with osteoarthritis (oa). methods. the cytokines interleukin(il)-6, il-10 and il-22, as well as the marker of cartilage hypertrophy runt-related transcription factor 2 (runx2), matrix metalloproteinase 13 (mmp13) and collagen type 10 (col10) were determined in facet joints from 13 patients with as (undergone correction surgery of rigid hyperkyphosis), 11 oa patients (undergone surgery of the lumbar spine, because of neurological deficits) and 12 controls (autopsies without spinal diseases). immunohistochemistry was performed and the entire cartilage area was analyzed for the frequency of positively stained chondrocytes. background. immunization with glucose-6-phosphate isomerase (g6pi) induces arthritis in susceptible strains of mice. depletion of regulatory t cells (tregs) prior to immunization switches the usually acute, self-limiting course to a non-remitting, destructive arthritis. this provides a possibility to study molecular switches for the transition from acute, self-limiting to chronic, destructive arthritis within one mouse model. to examine the role of fibroblast-like synoviocytes (fls), which are known to modulate immune responses via the production of pro-and anti-inflammatory mediators, the phenotype and function of fls from mice with either acute, self-limiting or non-remitting, destructive arthritis was studied. methods. fls from dba/1 mice that developed either the acute or the chronic form of arthritis were isolated from joints over a time course of 56 days. to investigate the phenotype of fls elisa studies as well as zymography have been performed. for the functional clarification of those cells the matrix-associated transepithelial resistance invasion (matrin) assay and a cartilage attachment assay have been used. furthermore, fls have been transferred in vivo into the knee joints of immunodeficient mice and the joints have been scored histologically. results. fls from treg-depleted mice produced significantly more cytokines (e.g. interleukin 6 (il-6)) upon stimulation with other cytokines, growth factors and tlr ligands. this increased susceptibility to cytokine stimulation in chronic animals compared to acute ones is observable throughout the disease course (56 days). furthermore, the secretion and activity of matrix metalloproteases (mmps) was enhanced in the fls from chronic mice compared to samples from acute ones. additional functional differences include the collagen-destructive potential and the potential to attach and eventually invade wild type cartilage. here, fls from treg-depleted chronic arthritic mice showed a higher invasive and destructive potential. ultimately, fls from treg-depleted mice were able to destroy cartilage in immunodeficient mice. conclusion. our results are compatible with the hypothesis that uninhibited inflammation in the early phase of treg-depleted mice causes the acquisition of an autonomously aggressive phenotype of synoviocytes which contribute to the switch from acute to chronic arthritis even in the absence of late support from t and b lymphocytes. collagen-induced arthritis modulates reactivity to sympathetic neurotransmitter stimuli during osteoclastogenesis of bone marrow-derived macrophages from da rats background. osteoclast(oc)-mediated bone destruction contributes to increased disease burden in rheumatoid arthritis. simultaneously, changes in synovial tissue innervation occur, leading to a reduction in catecholaminergic nerve fibres. studies on sweat gland innervation revealed that catecholaminergic fibres are capable of phenotypic transition to cholinergic nerves. the sympathetic neurotransmitters norepinephrine (ne) and acetylcholine (ach) affect osteoclastogenesis oppositely prompting us to study osteoclastogenesis at different phases of collagen-induced arthritis (cia) in an altered neurotransmitter microenvironment. methods. for induction of experimental arthritis, da rats were immunized with bovine collagen type ii while controls received isotonic nacl solution. to generate oc, bone marrow-derived macrophages (bmm) were isolated and differentiated with recombinant m-csf and rank ligand. the influence of ne and ach stimulation on osteoclast differentiation and activity was compared between arthritic and control animals at the acute (20 days post immunization, pi) and the chronic (40 days pi) disease state. as the nicotinic α7 ach receptor subunit is involved in the cholinergic anti-inflammatory reflex, we also applied a specific agonist, arr-17779. additionally, the gene expression profile for ne and ach neurotransmitter receptors was analyzed. results. ach stimulation generated significantly more osteoclasts in controls (40 days pi). arr-17779 mediated effects were similar to ach. ne decreased osteoclastogenesis via β-adrenoceptors and enhanced via α-adrenoceptor stimulation. cells from arthritic animals were less affected by ne and ach stimulation.oc from arthritic animals showed tendentially decreased activity in an enzymatic cathepsin k activity assay. ach and arr-17779 stimulation decreased cathepsin k activity 20 days pi, but the effect disappeared 40 days pi, representing the chronic arthritis state. ne stimulation significantly inhibited enzyme activity 20 days pi, but has little effect under chronic conditions. the receptor gene expression profile changed in the time course of arthritis. 20 days past immunization muscarinic ach receptors m3 and m5 were significantly upregulated whereas after 40 days adrenoceptors α1d and α2b were significantly downregulated. conclusion. we conclude that cia differentially modulates neurotransmitter influence during oc differentiation and activation but the underlying processes remain still unknown. the observed time pointdependent changes in neurotransmitter receptor gene expression may constitute a regulatory mechanism to counteract alterations in the local neurotransmitter composition. background. the generation of memory t lymphocytes allows effective and fast immune responses during antigen re-challenge and represents a hallmark of adaptive immunity. previous work from our group has demonstrated that murine memory cd4+ t cells reside in specific bone marrow niches and are characterized by the high expression of cd69 and ly-6c. these cells were designated as resting in the context of gene expression and proliferation. here, we aimed to phenotypically and functionally characterize human memory t lymphocytes in peripheral blood and bone marrow of healthy individuals. methods. mononuclear cells were isolated from paired blood and bm samples from individuals undergoing hip replacement surgery. phenotypic analysis and cytokine profile of distinct memory t cell subsets were assessed by flow cytometry. proliferation and cell cycle status were analyzed using ki-67 and propidium iodide (pi) staining, respectively. results. distinct populations of cd69-expressing cd8+cd45ra-and cd4+cd45ra-t cells were detected in bone marrow but not in the periphery. ccr7 and cd62l expression was reduced on bone marrow cd69+cd4+cd45ra-and cd69+cd8+cd45ra-t cells compared to their cd69-counterparts in bone marrow and peripheral blood. cell cycle analysis and ki-67 expression levels demonstrated the nonproliferative state of bone marrow memory t cells. furthermore, bone marrow resident memory t cells showed reactivity against various pathogenic agents, such as tetanus, measles and cmv. conclusion. we have identified a population of cd69-expressing cd8+cd45ra-and cd4+cd45ra-t cells in the bone marrow. despite cd69 expression, which is generally regarded as early activation marker, the cells were resting in terms of proliferation. bone marrow cd69+ memory t cells have downregulated ccr7 and cd62l indicating reduced homing capacity to secondary lymphoid organs. our data underline the role of the bone marrow as a major reservoir for resting memory t lymphocytes. therapeutic methods exerted an influence on satisfaction and future expectations in patients with rheumatoid arthritis (ra). methods. when visiting their rheumatologist, patients with ra were asked to complete a questionnaire at home after the consultation and then return it to an independent opinion research centre, where the data was collected and analysed. the form comprised various areas, namely demography, aspects of the diagnosis, medical care, therapeutic measures, and the illness in a personal context. results. 127 patients (122 females/25 males) from the whole of austria with a particular emphasis on lower austria, upper austria and tyrol completed the questionnaire (of 150 distributed), resulting in a response rate of 85%. 63% of the patients lived in settlements of under 5,000 inhabitants; a further 18% in settlements of under 50,000 inhabitants.. the rheumatologist attended could be reached within one hour for 90% of the patients and within 30 minutes for 41%. in slightly fewer than 30% of the respondents the diagnosis was made within three months, in 44% within six months. in 75%, the diagnosis was made by a rheumatologist. after experiencing the first symptoms, 80% contacted their general practitioner. a high degree of satisfaction appears to originate from the information supplied by the rheumatologist attended. most patients felt they were involved in decisions regarding their therapy. 77% of the respondents were employed prior to their illness; as a consequence of the disease 27% had to leave their jobs. conclusion. the majority of the respondents came from rural areas. the correct diagnosis was made within six months for almost half of the patients questioned. most patients felt well informed by their rheumatologists and involved in therapeutic decision-making. , combinational therapy of synthetic dmards (9.6; 5.1-14.1), and biological-monotherapy (6.07; 0-13). all of these differed significantly on later observation periods. comparing the prescriptions separately by sequential treatments there were no differences in retention rates for the individual dmard classes. regarding retention, in the first treatment synthetic dmards showed the longest retention, but from the second on this was the case for tnfi-combinational treatment and non-tnfi-biologicals (. abb.12) conclusion. traditional dmards are the starting point of therapy and mtx is the anchor drug for the first and all subsequent courses. already at the second sequential course, the combination therapies of mtx+tnfi become numerically more relevant, and their retention is better than mtx. therapie der rheumatoiden arthritis im letzten jahrzehnt -was hat sich verändert? abb.13 | ev.198 anzahl eingeschlossener patienten nach einschlussjahren und therapie sowie zeitlichen entwicklungen im das28 und der eular-response cal features of ra (erosive arthritis with classical radiological features) plus specific laboratory markers (ccp-antibodies). the third patient, a 48-year-old female presented initially with features of ra and sle simultaneously (alopecia, subcutaneous nodules, leucopenia, positive ccp-antibodies, high titres of ana and dna-antibodies). the fourth patient, a 40-year-old patient presented with severe polyarthritis in the upper and lower limbs, subcutaneous nodules, fever and cervical lymphadenopathy, she had high titres of ccp-antibodies (170 u/ml by a normal range of less than 5 iu/ml), ana of 5120 (normal less than 80), and dna of 290 iu/ml (normal less than 100 iu/ml). conclusion. the take home massage of this presentation is to be aware of rhupus if the sle patient develops erosive arthritis or subcutaneous nodules, or if the ra patient develops features of sle like leucopenia, active urine sediment, or clinically significant serositis. rhupus seems to be a distinctive entity and should be kept in mind while dealing with patients having ra or sle as it can affect the treatment and outcome. vorgeschichte. bei der abklärung eines akuten thoraxschmerzes sind auch seltene ursachen, so zum beispiel der einbezug thorakaler organe in eine entzündliche systemerkrankung, zu bedenken. anhand eines besonderen falles möchten wir den weg zur diagnose bei einem schwer kranken notfallpatienten zeigen. leitsymptome bei krankheitsmanifestation. ein 57-jähriger mann wird bereits zum dritten mal mit akuten thoraxschmerzen in die notaufnahme aufgenommen, jeweils war eine kardiale ischämie ausgeschlossen und ambulante diagnostik empfohlen worden. nach der schmerzcharakteristik, der vorgeschichte von rezidivierenden thoraxschmerzen und entsprechenden risikofaktoren wird bei massiver symptomatik jetzt von einem akuten koronarsyndrom ausgegangen und die invasive diagnostik durchgeführt. eine akute koronare ischämie kann jedoch ausgeschlossen werden. fieber, hohe entzündungszeichen, hinfälligkeit und gewichtsverlust von mehr als 10 kg in den letzten wochen lassen dann eine infektbedingte oder tumorbedingte ursache vermuten, abdomensonographie und röntgen-thorax sowie ausführliches labor samt immunologie führen nicht weiter. wegweisende weitere organbefunde finden sich nicht. die behandlung mit antibiotika hat keinerlei effekt auf klinik oder entzündungsparameter. nach 3 tagen wird der krankheitsverlauf kritisch evaluiert, eine nichtinfektiöse ursache der beschwerden wird in betracht gezogen, eine transösophageale echokardiographie wird zum ausschluss einer endokarditis und zur beurteilung der aorta (leitsymptome thoraxschmerzen und fieber!) durchgeführt. dabei zeigen sich die klappen sämtlich unverdächtig, die aorta ascendens weist eine massive echoarme wandverdickung auf. zum sicheren ausschluss eines intramuralen hämatoms wird sofort eine ct der thorakalen aorta durchgeführt, die eine ausgeprägte zirkuläre wandverbreiterung ohne hinweise auf dissektion zeigt. diagnose. riesenzellarteriitis mit aortitis. therapie. steroide, einleitung einer remissionsinduzierenden therapie mit cyclophosphamid boli. weiterer verlauf. innerhalb von 2 tagen ist der patient beschwerdefrei, die entzündungsparameter sind halbiert, der bettlägerige patient lässt sich mobilisieren und verlässt nach 10 tagen die klinik. bei der diffe-renzialdiagnose des akuten thoraxschmerzes sollten eine unklare entzündungsserologie und eine b-symptomatik frühzeitig an eine aortitis denken lassen. in diesem fall fand sich bereits in tee und ct ein ausgeprägter befund, der sich am ehesten durch den langen verlauf vor diagnosestellung erklären lässt. einleitung. die progressive familiär intrahepatische cholestase (pfic) gehört zu einer heterogenen gruppe seltener, autosomal rezessiv vererbter erkrankungen der gallensäurenexkretion mit intrahepatischer cholestase, hohem risiko der leberzirrhose bereits im kindesalter und hepatozellulärem karzinom. das auftreten eines sle bei pfic ist bisher in der literatur nicht beschrieben. methoden. wir berichten über eine jetzt 23-jährige patientin mit genetisch gesicherter pfic-2 (defekt der atp abhängigen gallensalz-exportpumpe bsep), biliostomaanlage im kindesalter, therapie mit udca und kontinuierlicher hepatologischer betreuung. 12/2011 manifestierte sich ein sle mit az-minderung, florider polyarthritis, polyserositis, splenomegalie, anämie und leukozytopenie. die ana waren mit >1:5120 homogen erhöht, die dns-ak im elisa mit 6738 u/ml, ena und antiphospholipid antikörper waren negativ. eine steroidtherapie wurde mit prednisolon 30 mg/tag begonnen. bei guter verträglichkeit und stabilen cholestaseparametern wurde die therapie um chloroquin mit 250 mg an 3 tagen der woche ergänzt. die betreuung wurde interdisziplinär fortgesetzt. ergebnisse. unter der steroidtherapie waren gelenkbeschwerden und serositis gebessert, das crp, die leukozytopenie und anämie normalisiert. die dns-antikörper fielen auf 1977 u/ml, c3 und c4 stiegen um 20%. es persistierten lediglich physiotherapeutisch behandelte muskuläre schmerzen und schonatmung nach pleuritis. bei steroidreduktion unter 10 mg traten die gelenkbeschwerden wieder auf, die dns-antikörper stiegen auf 4549 u/ml und c3/c4 fielen um 10%, das crp blieb normal. die steroiddosis wurde auf 15 mg/tag und die chloroquindosis auf 5×250 mg/woche erhöht. neue organkomplikationen im rahmen des sle sind nicht aufgetreten. die gallensäuren waren mit 47 µmol/l (norm <8) im jahr 2009 (letzte messung vor manifestation des sle) deutlich erhöht, bei manifestation des sle mit 12 µmol/l bereits deutlich gebessert und unter hoch dosierter steroidtherapie mit 4,7 µmol/l nach 1 woche sowie 5,4 µmol/l nach 1 monat normalisiert. unter prednisolon 15 mg/tag stiegen sie bei inaktiviertem sle auf 15 µmol/l nach 4 monaten an, bei steroidreduktion unter 10 mg auf 69 µmol/l. nach erneuter steroiddosiserhöhung auf 15 mg prednisolon/tag fielen sie auf 12 µmol/l ab. schlussfolgerung. die pfic-2 schützt nicht vor der manifestation eines sle. eine behandlung mit steroiden und chloroquin ist auch bei pfic sicher und wirksam. der floride sle und die höher dosierte steroidtherapie senken trotz bestehenden genetischen defekts unabhängig voneinander und synergistisch die gallensäurenspiegel im serum bei genetischer störung der atp-abhängigen sekretionspumpe bsep. augenentzündungen. eine ausgeprägte b-symptomatik mit fieber, nachtschweiß und abgeschlagenheit seit ca. zwei jahren hatte sich jeweils unter der therapie der hörstürze verflüchtigt. die mr-angiographie der aortenwand zeigte den typischen befund eines ausgeprägten wandenhancements der thorakalen aortenwand sowie der supraaortalen gefäße, duplexsonographisch fand sich eine zirkuläre wandverdickung der proximalen und mittleren acc beidseits ohne relevante stenosen. bei fehlendem nachweis zerebraler vaskulitischer oder embolischer veränderungen im kraniellen mrt und bei normalem eeg wurde der cerebrale krampfanfall als gelegenheitsanfall dd im rahmen der hochaktiven grunderkrankung interpretiert. diagnose. takayasu-arteriitis mit assoziiertem cogan-syndrom und rezidivierender polychondritis. therapie. steroidstoß, darunter rasche reduktion der entzündungszeichen und promptes ansprechen der b-symptomatik, beginn einer steroidsparenden therapie mit mtx. bei anhaltender krankheitsaktivität und hohem steroidbedarf wird der patient aktuell mit tocilizumab behandelt. schlussfolgerung. der präsentierte fall zeigt, dass mittels neuer bildgebender verfahren gelegentlich eine großgefäßvaskulitis detektiert werden kann, obwohl das klinische bild (hier: kopfklinik) eine kleingefäßvaskulitis vermuten lässt. das cogan-syndrom ist häufig mit einer großgefäßvaskulitis assoziiert, in diesem fall auch mit einer polychondritis. umgekehrt erweitert sich unser wissen um das befallsmuster der großgefäßvaskulitiden, die zwar überwiegend große, aber auch mittelgroße und kleine gefäße einbeziehen können. in unserem zentrum ist dies der zweite fall einer takayasu-arteriitis mit assoziiertem cogan-syndrom in einem kollektiv von 8 fällen. diagnostik. im rahmen einer vorstellung in unserer rheumatologischen ambulanz zur abklärung eines möglichen sjögren-syndroms, konnte in der lungenfunktionsdiagnostik eine leichte restriktion und überblähung festgestellt werden. in einem auswärtig durchgeführten mrt der halsweichteile zeigten sich die glandula parotis und submandibularis beidseits inhomogen und kräftig kontrastiert, ebenfalls mehrere grenzwertig große lymphknoten. aufgrund der erneut pathologischen lungenfunktion und dem verdacht auf eine lungenbeteiligung bei sjögren-syndrom erfolgte ein ct-thorax. hier zeigte sich eine zysti-sche rarefizierung vor allem der zentralen lungenanteile, differentialdiagnostisch mit einer langerhans-zell-histiozytose vereinbar. auch erschien der diabetes insipidus passend zur histiozytose. die immunologischen untersuchungen waren unauffällig, insbesondere konnten keine ssa-/ssb-antikörper oder eine hypergammaglobulinämie nachgewiesen werden. somit erschien ein sjögren-syndrom unwahrscheinlich. zur weiteren abklärung erfolgte eine bronchoskopie, in den biopsaten konnte immunhistochemisch eine langerhans-zell-histiozytose nachgewiesen werden. in einer pathologischen nachuntersuchung auswärtiger parotis-biopsate bestätigte sich diese, zudem konnte eine mutation des braf-proto-onkogens nachgewiesen werden. im abschließend durchgeführten pet-ct konnte eine vermehrte kontrastmittelaufnahme des hypophysenstiels, eine vermehrte stoffwechselaktivität der parotis beidseits sowie kutan axillär links diagnostiziert werden. abschließend lag somit eine langerhans-zell-histiozytose mit befall von hypophyse, lunge, parotis, haut, lymphknoten sowie einem fraglichen befall des darms vor. therapie. in absprache mit der adulten langerhans-zell-histiozytose studiengruppe erfolgt nun eine therapie mit cytarabin. weiterer verlauf. der weitere verlauf nach geplantem therapiebeginn bleibt abzuwarten. einleitung. die anti-gbm-erkrankung (goodpasture-syndrom) ist eine prototypische autoimmunerkrankung mit ernster prognose, wenn sie als "pulmorenales syndrom" mit der trias rapid-progressive glomerulonephritis, alveoläre hämorrhagie und nachweis von autoantikörpern gegen glomeruläre basalmembranen (anti-gbm-ak) auftritt. über weniger aggressive verläufe ist wenig bekannt. in der literatur wird die bedeutung der frühen diagnosestellung für die prognose der patienten betont. wir berichten über eine 36-jährige patientin, die sich mit abgeschlagenheit, müdigkeit und blutbeimengungen im sputum vorstellt. sie betreibt einen nikotinabusus. methoden. wir sehen eine blasse patientin (hb 4,0 mmol/l, normochrom, normozytär), die keine weiteren auffälligen befunde in der körperlichen untersuchung zeigt. die apparative diagnostik mittels endoskopie und sonographie ergibt keine befunde, die die anämie erklären können; in der bronchoskpie zeigt sich das bild einer alveolären hämorrhagie ohne aktive blutungszeichen. neben der ausgeprägten anämie bestehen eine milde proteinurie mit 300 mg/d sowie eine geringe erythrozyturie. die immunologische diagnostik ergibt gering erhöhte anti-gbm-ak, negative ana und anca. die nierenbiopsie zeigt eine rapid-progressive glomerulonephritis mit halbmondbildung in einem glomerulum, zusätzlich können lineare igg-ablagerungen in der immunfluorenz dargestellt werden. es ist ein glomerulum in der biopsie betroffen, die anderen glomeruli sind unauffällig. es bestehen einzelnen erythrozytenzylinder, diffuse entzündungszeichen lassen sich nicht nachweisen. die nachträglich durchgeführte immunfluoreszenz in der lungenbiopsie bestätigt den befund linearer igg-ablagerungen. ergebnisse. in unserem fall sehen wir eine junge patientin in einem relativ guten allgemeinzustand mit gering erhöhten anti-gbm-antikörpern und einer gering ausgeprägten nierenschädigung mit einem befallenen glomerulum in der biopsie. es wird angesichts des jungen alters der patientin und der geringen ausprägung der erkrankung eine therapie mit cyclophosphamid nach dem "euro-lupus-protokoll" von f. hossiau eingeleitet. im verlauf steigt der hb auf 5,6 mmol/l, die alveoläre hämorrhagie sistiert und die proteinurie ist rückläufig. schlussfolgerung. das goodpasture-syndrom mit einer inzidenz von 0,5-1,0/1 mio. einwohner und jahr ist eine sehr seltene autoimmunerkrankung mit ernster prognose. möglicherweise spielen umweltfaktoren (hier: nikotinabusus) eine rolle für die manifestation der erkrankung. interessant ist, dass die erstbeschreibung im rahmen einer influenza-epidemie -wie auch in diesem jahr bei unserer patientinerfolgte. über die therapie gibt es wenige informationen. die meisten empfehlungen gibt es zum pulmorenalen-syndrom, über weniger aggressiv verlaufende manifestationen gibt es nur wenige informationen. einleitung. wir berichten über einen 46-jährigen raucher mit akut aufgetretener polyarthritis und neu aufgetretenem raynaud-phänomen. auffallend war die diskrepanz zwischen nur geringer systemischer entzündungskonstellation und einer hochaktiven polyarthritis mit schwerem raynaud-phänomen. im verlauf kam es zu einer spontanen thrombophlebitis der v. cephalica. methoden. pathologische werte: crp maximal 1,4 mg/dl (norm <0,6), zellzahl im gelenkpunktat 20.000 leukozyten/µl. im normbereich lagen: bsg 12 mm/1 h, ana, ena, ds-dns-ak, rheumafaktoren, anca, kälteagglutinine, kryoglobuline, tumorsuche initial ohne befund (ct-thorax, bronchoskopie, ct abdomen und becken, coloskopie, gastroskopie, skelettszintigraphie), fdg pet-ct: zwei suspekte lymphknoten rechts cervical sowie suspekte rechte tonsille. ergebnisse. selbst unter 60 mg prednisolon weiterhin hochaktive polyarthritis. nach unauffälliger initialer tumorsuche veranlassten wir ein fdg-pet ct mit nachweis zweier suspekter lymphknoten rechts cervical sowie einer suspekten rechten tonsille. die biopsie der klinisch sich nur in der palpation diskret induriert darstellenden region ergab ein gering differenziertes, gering verhornendes plattenepithel mit 98%iger sequenzhomologie mit hpv typ 16. nach resektion des tumors und radiatio sistierten sowohl die polyarthritis als auch das raynaud-phänomen ohne weiteres rezidiv auch nach absetzen der glukokortikoide. schlussfolgerung. eine akut auftretende hochaktive polyarthritis mit hohem glukokortikoidbedarf in kombination mit einem raynaud-syndrom, auffallend niedriger systemischer entzündungsaktivität und fehlendem autoantikörpernachweis sollte insbesondere bei einem langjährigen raucher anlass zu einer intensiven tumorsuche geben. ein fdg-pet-ct kann bei okkulten tumoren zielführend sein. bemerkenswert ist hier der nachweis von hpv-16 im tumor als weiterer risikofaktor neben dem zigarettenrauch. methoden. bei der klinischen untersuchung fielen ein beidseits positives menell-zeichen und deutlicher klopfschmerz über dem lumbosakralen übergang auf. labordiagnostisch konnte ein erhöhtes c-reaktives protein und ein positiver hla-b27 nachgewiesen werden. der bath ankylosing disease activity (basdai) index betrug 6,875. die beckenübersichtsaufnahme zeigte eine definitive bilaterale sakroiliitis grad 3 gemäß den modifizierten new-york-kriterien. der befund der kontrastmittelunterstützten mrt der iliosakralgelenke bewies das vorliegen einer bilateralen floriden sakroiliitis. bei gesicherter hla-b27 positiver ankylosierender spondylitis wurde die indikation zur einleitung einer biologikatherapie mit einem tnfα-inhibitor gestellt. während der abklärung von kontraindikationen wurde in der konventionellen röntgen-thorax-aufnahme eine rundliche, glatt begrenzte zystische läsion mit flüssigkeitsspiegeln im rechten mittellappen entdeckt. die weitere abklärung mittels nativer thorax-ct, bronchoskopie und biopsieentnahme bestätigte den verdacht auf eine bronchogene zyste. die erregerdiagnostik in der bronchoalveolären lavage zeigte lediglich eine kontamination mit der residenten standortflora. ergebnisse. in anbetracht der geplanten immunsuppressiven therapie, die mit einem erhöhten infektionsrisiko einhergeht, wurde die bronchogene zyste im september 2012 operativ entfernt. zur linderung der beschwerden erhielt die patientin eine schmerztherapie mit nsar. als sich die patientin sechs monate später erneut zur einleitung der biologikatherapie vorstellte, berichtete sie, dass die schmerzen etwa einen monat nach der operativen resektion praktisch verschwunden seien. die klinische untersuchung war unauffällig, der basdai-index lag bei 1,2. auch das zur verlaufskontrolle angefertigte mrt der isg zeigte im vergleich zur voruntersuchung einen deutlichen rückgang der floridität. schlussfolgerung. es handelt sich um den ersten fall einer kompletten klinischen und radiologischen remission einer hla-b27-positiven ankylosierenden spondylitis nach operativer entfernung einer bronchogenen zyste als potenziellen entzündlichen fokus. bei der systemischen verlaufsform der erkrankung treten neben kutanen erscheinungen zusätzlich muskuloskeletale, hämatopoetische (20-60%), renale (ca. 50%), kardiale, cerebrale und pulmonale (4-9%) manifestationen auf. eine polyserositis (11-60%) ist häufig. mit 8-40% werden im sle-schub abdominelle schmerzen beschrieben. nur in seltenen fällen (amerika 0,9%, asien 2,2-9,7% aller sle-patienten) kommt es zum bild einer lupusassoziierten mesenterialen vaskulitis (lmv). die ätiologie der lmv ist weitestgehend unklar, eine genetische präsidsposition sowie auslösende faktoren (bakterielle darminfektionen, medikamente wie nsar, phosphodiesterasehemmer) werden diskutiert. pathogenetisch wird eine mesenteriale ischämie durch eine mikroangiopathie (arteriolen, venolen) bei inflammatorischer immunkomplexpräzipitation sowie thrombembolischen ereignissen angenommen. radiologisch/sonographisch zeigt sich ein segmentales darmwandödem mit darmdilatation. endoskopisch dominieren oberflächliche ulzerationen, perifokale hämorrhagien bis hin zur gangrän. eine erhöhte perforationsneigung wird beschrieben. mikroskopische befunde zeigen eine fibrinoide nekrose subseröser gefäße mit leukozytoklasie der gefäßwand bzw. ein submuköses ödem mit nur diskreter invasion mononukleärer zellen. die prognose der lmv scheint abhängig von genetischer prädisposition, raschem beginn einer immunsuppression sowie restriktivem einsatz operativer interventionen und wird je nach literaturquelle mit einer letalität bis zu 50% angegeben. background. we report a patient with tma in the context of sle treated successfully with the c5 inhibitor eculizumab. the patient had sle with lupus nephritis (ln). before she developed tma with renal failure and neurologic manifestations, she was treated with various immunosuppressive regimens for mucocutaneous and musculoskeletal manifestations and later for ln. the diagnosis of atypical hemolytic uremic syndrome (ahus) was made based on the presence of coombs-negative hemolytic anemia, thrombocytopenia, renal failure, seizures due to cerebral ischemia and signs of tma in the renal biopsy. plasma exchange and hemodialysis were started immediately and could stabilize her condition. six weeks after the beginning of plasmapheresis but still severely compromised renal function and thrombocytopenia, complement inhibition with eculizumab became a therapeutic option. after the first infusions, renal function, anemia and thrombocyte counts markedly improved. dialysis could be stopped. extensive genetic testing of mutations associated with the overactivation of the alternative complement pathway was negative. after 7 months, when the patient was still in remission, eculizumab infusion intervals were widened and it was finally stopped after 12 months of treatment. since then, renal function remained stable with nearly normal glomerular filtration rates. background. il-6 signaling plays an important role in inflammation but is restricted by different regulatory mechanisms. these mechanisms include the decreased availability of gp130, the signal transducing chain of the il 6 receptor, on the cell surface. the aim of this study was to determine whether the inflammatory environment in the arthritic joint has an impact on monocytic gp130 surface expression and the extent to which regulatory processes in the synovial fluid (sf) can be transferred to an in vitro model. flow cytometry and live cell imaging were used to measure the cell surface expression and internalization of gp130. stat3 phosphorylation was monitored by flow cytometry and western blotting. results. the level of cell surface gp130 expression on sf monocytes was reduced compared to peripheral blood (pb) monocytes from patients with juvenile idiopathic arthritis (jia). this reduction could be reproduced by stimulating pb monocytes from healthy donors with sf and was dependent on p38 mapk. the induction of p38 by il-1β in pb monocytes interfered with il-6 signaling due to the reduced cell surface expression of gp130. the results suggest that p38-mediated pro-inflammatory stimuli induce the downregulation of gp130 on monocytes and thus restrict gp130 mediated signal transduction. this regulatory mechanism could be relevant in the inflamed joints of patients with jia. kr.08 sjia patient characteristics of those who successfully discontinued corticosteroids during canakinumab treatment: secondary analysis from a pivotal phase 3 trial background. interleukin-1β (il-1β) is a key driver in the pathogenesis of systemic juvenile idiopathic arthritis (sjia). canakinumab (can), a selective fully human anti-il-1β monoclonal antibody, has been shown to be efficacious in the treatment of sjia [1] . corticosteroids (cs) are a mainstay of therapy for sjia, however due to the well-known long-term side effects, reduction of cs dosage is desirable. objectives. to assess patient features associated with cs discontinuation during can therapy. methods. patients (2-19 years of age) with active sjia received s.c. can (4 mg/kg to 300 mg max) every four weeks during the maximum 20week cs-tapering phase [1] . cs tapering was to be initiated when at least an adapted acr50 was achieved and no fever. a 12-year-old boy presented with nocturnal tingling paresthesia affecting his feet and his calves. no excessive leg movements were noted at night. within a few months, his symptoms worsened. the paresthesia occurred both during the day and at night. moreover, the paresthesia came to be triggered by merely standing up. affecting a sharply demarcated area not corresponding to dermatomes, symptoms resolved promptly with movement. the paresthesia was associated with local skin erubescence in spots that slowly began spreading all over the affected area. symptoms did not occur while the patient was seated. mild painless swelling around both of the ankles was noticed in the evenings. approximately one and a half years after the initial manifestation, painful triphasic color changes of all fingers and toes triggered by cold or stress occurred. the family history was positive only for psoriasis. extensive laboratory studies excluded inflammatory and hematological conditions as well as occlusive arterial diseases known to be associated with secondary raynaud's phenomenon. polyneuropathy and other neurological disorders were excluded as well. inflammatory joint disease suspected from the initial imaging with magnetic resonance of the feet and ankles was not confirmed by repeated investigations and scintigraphy. the only consistent abnormality was a reduced pulse amplitude corresponding to vasospasm, which was revealed by photoplethysmography of toe vessels. additionally, paradoxical amplitude reduction after application of nitroglycerine was seen in finger vessels. placing his hands or feet in cold water did not trigger raynaud's phenomenon. initial treatment with non-steroid anti-inflammatory drugs, topical isosorbiddinitrate and local steroid instillation (suspicion of inflammation of tibialis posterior tendon) was ineffective. systemic therapy with the calcium channel blocker amlodipine was initiated. the initial dosing of 5 mg (0.09 mg/kg/day) was slowly increased to 15 mg (0.27 mg/kg/day) which lead to complete resolution of the patient's ailments. after three years of pharmacotherapy and 1.5 years in remission, a weaning off the treatment is planned. based on the patient's positive response to calcium channel blocker, we conclude that the lower-leg paresthesia was of vascular origin and can be considered an atypical presentation of raynaud's phenomenon. background. the initial treatments of choice for jdm are high-dose corticosteroids and methotrexate. however, no consensus exists about second line therapeutic options in refractory or recurrent cases. results. we present a 9-year-old boy who was diagnosed with jdm due to severe proximal muscle weakness, dysphagia, a heliotrope rash, gottron's sign, nail teleangiectasia and a characteristic muscle biopsy. creatine kinase levels were within normal range and no antinuclear antibodies were present. over a period of seven years, the patient was treated with high-dose corticosteroids, methotrexate, intravenous immunoglobulins, oral steroids, mycophenolate mofetil, rituximab and infliximab. despite all treatment efforts, skin and muscle inflammation persisted and the boy developed severe subcutaneous calcifications, rendering him wheelchair-bound. as il-6 production correlates with disease activity in adult and juvenile dm, treatment with tocilizumab (8 mg/kg every 4 weeks) was initiated, leading to a complete resolution of skin inflammation within 6 months. within 12 months of treatment, the disease activity score (das) decreased from 16 to 9 (out of 20), the childhood myositis assessment scale (cmas) increased from 17 to 27 (out of 52) and the kendall manual muscle test (mmt) increased from 60 to 75 (out of 80). in daily life the wheelchair was no longer necessary. treatment was well tolerated but accompanied by a moderate increase in liver transaminase activities. interestingly, therapy with rituximab was associated with a decline in igm levels only, whereas igg and iga stayed markedly elevated. in contrast, following initiation of tocilizumab treatment, igg levels rapidly declined to normal range, emphasizing the role of the humoral immune system in the pathogenesis of dm. conclusion. taken together, treatment of a severely affected jdm patient with tocilizumab was safe, well tolerated and led to a significant improvement in disease activity. further investigations of il-6-blocking agents as a treatment option in otherwise therapy-resistant jdm patients are warranted. functional capacity of jia patients with an initial adjustment to an anti-tnf-alpha therapy background. thirty three percent of patients with polyarticular jia are treated with biologics [2] . despite substantial improvement achieved by anti-tnf-α treatment according to disease activity [1] patients have joint-specific impairments. this factor should be considered when analyzing the functional effects on joint limitations while performing daily activities. methods. in a prospective study on polyarticular jia patients treated with anti-tnf-α therapies plus functional therapies we study the longitudinal effects on joint function. the measurements include 3-d gait analysis (eight vicon f40 cameras, omg, london, balance control (s3-check, tst, großhoeflein), pedobarography (emed plate (4sensors/ cm², novel, munich), daily activity assessment (step watch, orthocare innovations, ok usa), acr pedi and joint mobility testing. we here present the cross-sectional data of the first 17 patients (15.0±3.1 yrs, 162.7±14.6 cm, 60.0±17.2 kg, pain-level: 4.8±1.7/10 vas, active joints: 6.2±5.9, chaq: 0.4±0.4). results. preliminary results demonstrate that the ability to walk is slightly limited. patients have a reduced push off power generation within the ankle joint (patients: 3.4±1.4 w/kg; healthy controls: 4.5±0.9 w/ kg). further on they show limited sensory motor control and stability in comparison to patients with an inactive disease status while performing balance tests (patients: sensory index: 2.9±1.0, stability-index: 3.7±1.1, patients with inactive status: sensory-index: 2.1±0.8, stability index: 3.0±1.0 (p<0.05). note: lower indices values are better results. conclusion. it is one of the first studies which show functional joint-specific deficits during every day activities in patients who receive an initial anti-tnfα-therapy. the limited stability and motor control might be due to limited joint integrity in the ankle joint. this is supported by the impaired push off function while walking. the next study step will show possible effects of the anti-tnfα-therapy. background. the role of sport as a therapeutic tool in treating patients with jia is becoming more important recently [2] . effects of exercise therapy are reviewed beneath others by takken et al. [3] . they state that short-term effects look promising but the effects of long-term studies remain unknown. methods. the preventive mobility workout (pmw) is a whole body home-exercise-therapy (10 min each day) for patients with an inactive diseases status. it counteracts the deficits which were observed during functional studies in the past [1] . it consists of exercises for muscular strengthening (squads: hamstring to quadriceps ratio), hamstring flexibility (lift and raise), core stability (prone bridge -time-to-failure), shoulder griddle mobility (horizontal extension) and ankle joint integrity (mechanical power while walking . for statistical analysis an anova was calculated and the level of statistical significance was set to p<0.05. results. preliminary results show a group effect of the pmw for the hamstring to quadriceps ratio (h-q-r) for the right side (p<0.05) and a tendency for the left side (p=0.065). the h-q-r for the right side has changed in the tg and cg from 1.6±0.3 to 2.0±0.6 and from 2.1±0.6 to 1.6±0.3, respectively. it has changed for the left side in the tg and the cg from 1.9±0.5 to 1.6±0.3 and from 1.5±0.2 to 1.7±0.3, respectively. all other parameters regarding flexibility or joint integrity show low or no effects. we have re-tested n=18 out of 75 so far and the pmw training show little training-effects. the preliminary results might be a reasonable proof for long-term effects. a possible reason for the little effects might be that patients are supposed to train every day but the training diaries show that they exercise approximately three times a week. the authors would like to thank the "deutsche kinder-rheumastiftung" for supporting the study. conclusion. we will validate these proposed definitions prospectively in a jia associated uveitis cohort. based on the results, we will weight these measures to develop an overall scoring system. background. 6 minute walk is a primary outcome measure in studies in pulmonary hypertension. currently we have a two of sets of data [1, 2] regarding test results in the 6 minute walk test (6mwt) in healthy children with a large span in the norm values in the different age groups. aim of the study was to establish norm values for healthy german children for the 6 minute walk test. methods. the team of an occupational therapist and a study nurse were visiting schools. permission from the parents was give before the test. always just probands from one class were invited to participate. the test were performed according the international guidelines [3] . the demographic data of the probands were collected and the parents filled out a short survey regarding the physical activity and the health condition. children with chronic diseases, which decrease the stamina were excluded. up till now 611probands participated from the age 5 to 14 years. 343 of them were female. the mean 6 minute walk continuously increased with age (. tab background. juvenile idiopathic arthritis (jia) is the most common chronic disease in pediatric rheumatology which often results in foot impairments [1] . patients with jia are reported to have smaller pressure loads underneath the foot while walking [2] . the aim of the study was to analyze the peak plantar pressure distribution of a well described cohort of jia patients with an active symmetrical ankle joint arthritis and no history of foot involvement. [1] ) wurden in bezug auf therapie und outcome anhand der wallace-kriterien beurteilt: "active disease" (ad), "inactive disease" (id), "clinical remission under medication" (crm), "clinical remission off medication" (crom; [2] background. familial mediterranean fever (fmf) is one of the most common autoinflammatory diseases (aid). pathogenomic relevant mutations in the mefv gene show autosomal recessive inheritance, but co-dominant mutations have been described. we aimed to evaluate correlations between ethnic origin, phenotype and genotype for fmf patients in the german aid-net-registry. methods. we used two common scoring systems modified for children (mor et al., pras et al.) to assess disease severity in 243 fmf patients of the aid-net-registry. for the five most frequent mutations, we tested for a correlation of the genotype with the phenotype, mean crp and ethnic origin, respectively. furthermore, we evaluated the applicability of the two severity scores for children. results. among the 243 patients, we detected a total of 433 pyrin mutations and 22 different sequence variants, including one new mutation (p.gly488asp). the five most frequent alterations were p.met694val (55%, n=238), p.met680lle (12%, n=52), p.val726ala (10%, n=44), p.glu148gln (8%, n=34) and p.met694ile (2.3%, n=10). ethnic origin could be determined in 224 cases; the prevailing ancestry was turkish (83%, n=185), 8% (n=18) were lebanese. p.met694val in homozygous form (30%, n=73) was correlated with a more severe disease activity, based on the score by mor, as well as with a higher mean crp (74 mg/l, n=60, 31 mg/l, n=59) compared to patients without this mutation (p=0.01 and p<0.01, respectively). the score suggested by pras did not yield a significant genotype-phenotype correlation; indeed, the two scoring systems were inconsistent with each other (κ<0.07). although a typical distribution of mutations in different ethnic populations was obvious, this trend was not statistically significant, probably due to the divergent number of cases. conclusion. the homozygous p.met694val substitution was associated with a more severe disease activity. there was no origin-genotype correlation in this fmf population. the well-known severity scores for children (mor, pras) are inconsistent. the aid-net is working on a new scoring system. 3. all patients with rp should be investigated by capillaroscopy. capillaroscopy will be classified into "normal", "aspecific changes" or "scleroderma pattern". 4. all patients who have additional symptoms pointing to a definite connective tissue disease should be evaluated according to disease specific guidelines. 5. ana-negative and capillaroscopy-negative patients should be followed-up at least every 6 months. 6. ana positive patients without disease-specific antibodies and with negative capillaroscopy findings should be followed-up at least every 6 months. 7. ana and disease-specific antibody positive patients should have organ specific evaluation according to symptoms, examination and relevant to that particular disease e.g. patients who are ana and scl-70 positive may need organ specific evaluation for jssc as per the juvenile systemic sclerosis inception cohort protocol (www.juvenilescleroderma.com). 8. ana-positive patients, who have no disease specific antibody but have positive capillaroscopy results, should be followed-up at least every 3 months. 9. ana-negative patients with positive capillaroscopy result should be followed-up at least every 6 months. 10. the group could not reach an agreement regarding treatment, due to a lack of data for the paediatric age group. the group agreed that implementation of adult recommendations conclusion. the group made a suggestion for a standard of good clinical practice for rp in children. our aim is that this will facilitate a large multicentre prospective follow-up study of children with rp. background. chronic non-bacterial osteomyelitis (cno) is an inflammatory disorder of the skeletal system of unknown etiology. long-term follow-up and response to treatment data have rarely been reported. the aim of the study was to characterize the clinical, radiological, histological and laboratory data at juvenile cno onset, and to analyze the long term treatment response. methods. the course of disease of 95 juvenile patients with non-bacterial inflammatory bone lesions was evaluated retrospectively. clinical, radiological, histological and laboratory data were assessed at disease onset and for a median time of disease of 40 months. results. the mean age at disease onset was 11.7 years, the mean time between the first symptoms and the diagnosis of cno was 9 months. 84% of the patients had multifocal bone lesions. biopsy was performed in 80 patients. only when bone biopsy was taken within 12 months of symptom onset, cellular infiltrates could be observed. at later time points, fibrosis, hyperostosis and bone edema predominated. the initial treatment consisted of non-steroidal anti-inflammatory drugs (nsaids). 39% of the patients required second line therapy consisting of sulfasalazine and short term oral corticosteroids, 8% of the patients required bisphosphonates or tnf-blocking agents. the number of clinical lesions decreased to 50% within 3.1 months and reached 18.8% after 24 months of treatment. the number of radiological lesions, however, declined to only 66.5% after 24 months of treatment. in detail analysis of the tre-atment response revealed that initiation of sulfasalazine treatment in nsaid non-responders led to a significant and sustained decline of the clinical, as well as the radiological number of lesions. conclusion. the rapid clinical improvement in cno, following initiation of therapy with nsaids, is not accompanied by a likewise decrease of the number of radiological lesions. treatment with sulfasalazine is effective in childhood cno. background. exercise has a wide variety of beneficial health effects. it stimulates bone formation and maintains bone strength as well as decreases the risk of falls. moreover, exercise at regular intervals is also assumed to positively affect immune functions. conversely, in more than 50% of the astronauts during/after space flight and under simulated weightlessness immune functions are suppressed. to assess the effects of simulated weightlessness during the 2nd berlin bedrest study (bbr-2) on immunological parameters. furthermore, to compare the effects of two different exercise performances (resistive vibration exercise and resistance exercise without vibration). methods. 24 physically and mentally healthy male volunteers (20-45 y) experienced 60 days of six degree head down tilt bed rest. they were randomized to 3 groups: resistive vibration exercise (n=7), resistance exercise without vibration (n=8), inactive controls (n=9). blood samples were taken 2 days before bed rest, on day 19 and 60 after beginning of bed rest. composition of immune cells was analyzed by flow cytometry. cytokines and neuroendocrinologic parameters were analyzed by a multiplex suspension array/ elisa in plasma. general changes over time were identified by paired t-test, exercise-dependent effects by 2-group repeated measurements anova. results. for all cases pooled, the number of granulocytes (p<0.05), nkt cells (p<0.01) and hematopoietic stem cells (p<0.01) increased during the study; the concentrations of dhea (p<0.01) and eotaxin (p<0.05) decreased. different impacts of the specific types of exercise on the change over time were shown for lymphocytes, nk cells, nkt cells, tcell subpopulations and the concentrations of ip-10 and rantes. conclusion. we found immobilization/simulated weightlessness to significantly impact immune cell populations, and cytokine and neuroendocrine factor concentrations. exercise was able to specifically influence immunologic parameters. interestingly, these changes resemble those found during the aging process. background. novel therapies have made remission and low disease activity (lda) achievable goals in ra. we assessed the impact of treatment with subcutaneous abatacept or adalimumab on these goals and on functional and radiographic outcomes in ample (abatacept versus adalimumab comparison in biologic-naïve ra subjects with background methotrexate), the first head-to-head trial of biologics in ra patients with inadequate response to mtx (mtx-ir). methods. ample is a 2-year, phase iiib, randomized, investigator-blinded study. biologic-naïve ra patients with mtx-ir were randomized to receive 125 mg abatacept weekly or 40 mg adalimumab biweekly, combined with a stable dose of mtx [1] and radiographic non-progression (defined as change in modified total sharp score ≤2.8) were analysed in patients achieving or not achieving remission or lda at days 85 or 169. results. baseline clinical characteristics of abatacept and adalimumab treatment groups were balanced, as was clinical, functional and radiographic efficacy and safety at day 365 [1] . the proportions of patients meeting each of the remission criteria or lda at day 365 were similar for both groups, but significantly more patients achieved das28 (crp) remission compared to cdai, sdai or rapid3 remission, and the smallest proportion achieved boolean remission. compared to remission, a higher proportion of patients achieved lda. across all definitions of remission or lda, >60% of the patients achieving remission at days 85 and 169 were haq responders at day 365. more than 80% of patients achieving remission or lda at days 85 and 169 were radiographic nonprogressors at day 365. improvement in physical function and radiographic outcomes were consistent between the two treatment groups in both remission and lda populations (. tab.11). conclusion. through 1 year, patients treated with subcutaneous abatacept or adalimumab in ample achieved comparable rates of remission and lda. similar improvements in physical function and radiographic outcomes were observed. these data help to illustrate the relationship between remission, lda and functional and radiographic outcomes independent of treatment with subcutaneous abatacept or adalimumab. background. previous small studies suggest that responses to some immunizations may be attenuated by intravenous abatacept but remain clinically meaningful [1, 2] . we investigated the magnitude of response to pneumococcal and influenza vaccination in a larger number of patients receiving subcutaneous (sc) abatacept therapy. the objective of the study was to evaluate the antibody response to the standard 23-valent pneumococcal polysaccharide vaccine and the 2011-2012 seasonal influenza trivalent vaccine in adult patients with ra on sc abatacept and background dmards. these multicentre, open-label sub-studies of the 23-valent pneumococcal polysaccharide vaccine and seasonal influenza vaccine enrolled patients in the acquire (pneumococcal and influenza) or attune (pneumococcal) studies. patients were enrolled at any point during their sc abatacept treatment cycle after completion of ≥3 months' abatacept treatment. all patients received fixed-dose sc abatacept 125 mg/week with background dmards. a pre-vaccination blood sample was collected and vaccines administered, while continuing background sc abatacept and dmards. after 28±3 days, a post-vaccibackground. in real life, dosage increases are common with biologic agents [1] . intravenous abatacept is administered by patient body weight (10 mg/kg) 2 and 4 weeks after the first infusion and every 4 weeks the-reafter [2] totalling 8 infusions over the first 6 months. no adjustments to this schedule are recommended. abatacept retention rates, efficacy and safety over 12 months in action (abatacept in routine clinical practice) have been reported previously [3, 4] this study was designed to assess adherence to recommended dosing of abatacept over the first 6 months in action. methods. action is an ongoing, 2-year, international, non-interventional, prospective cohort of ra patients treated with intravenous abatacept. all patients on abatacept treatment for ≥6 months, and with infusion data available at initiation and at 6 months, were considered in this analysis. good adherence was defined as correct dose by patient body weight and number of actual-to-recommended infusions within the range 80-120% (i.e. 7-9 infusions). results. in total, 783/1120 (69.9%) patients received abatacept ≥6 months and had the infusion data available. most had established ra and failed ≥1 anti-tnf agent (87.5%). of 774 patients with body weight data available at initiation, 87.6% received the recommended initial dose, 6.5% a lower dose and 5.9% a higher dose than recommended. good adherence to the abatacept treatment schedule was found in 670/783 (85.6%) patients. over 6 months, 34.0% of patients received 7 infusions, 50.1% received 8 infusions and 1.5% had 9 infusions. change in dosage over time was assessed in 680/774 patients with data available at both time points. the majority of patients (86.6%) maintained the recommended dosage. 500/680 (73.5%) patients received abatacept at the recommended dose for body weight and at the recommended treatment schedule over 6 months. conclusion. in the real-world action study, adherence to the recommended abatacept treatment regimen over 6 months was good. few patients received changes in dose and/or frequency of administration over this time period. background. in rheumatoid arthritis (ra), synovial fibroblasts (sf) secrete large amounts of il-6, il-8 and matrix metalloproteinases (mmps) which are crucial for cartilage destruction. rasfs are sensitive to the action of cannabinoids and they express cannabinoid receptors type i and ii (cb1 and cb2), the vanilloid receptor (trpv1) as well as endocannabinoid degrading enzymes. cannabinoids are regarded as antiinflammatory and since anandamide (aea) is found in ra synovial fluid we investigated how this endocannabinoid affects adhesion, proliferation, and production of inflammatory mediators of rasf. methods. adhesion was assessed by the xcelligence system. proliferation was quantified by the amount of incorporated fluorescent dye into cellular dna. mmp-3 and cytokines were detected by elisa. in oasf, aea dose-dependently decreased the il-1β induced production of mmp-3 (by 23%) in a trpv1-mediated manner. il-6 and il-8 levels were only weakly modulated. in rasf however, aea decreased il-1β induced production of il-6 (23%), il-8 (18%) and mmp-3 (20%). the effects of aea were not inhibited by cb1, cb2 or gpr55 antagonists but were blocked by the trpv1 antagonist capsazepine. the inhibitory capacity of aea was enhanced by cyclooxygenase-2 inhibition in rasfs and oasfs, but was unaltered or even slightly reduced by faah inhibition. aea was even more potent in reducing above mentioned mediators when rasfs but not oasfs were incubated under hypoxic conditions and treated with tnf. furthermore aea increased adhesion of oasfs and rasfs to fibronectin. adhesion was modulated by cb1, gpr55, and trpv1 antagonists. combined faah and cyclooxygenase-2 blocked the stimulatory effect of aea on adhesion. proliferation was decreased by aea in rasfs and oasfs via a cyclooxygenase-2 but not via cb1, cb2 or trpv1 dependent mechanism. conclusion. in conclusion, aea promotes an antiinflammatory phenotype of rasfs and oasfs by activating trpv1. cb1, trpv1, and gpr55 act in concert to modulate adhesion of sfs and this is highly dependent on the intracellular concentration of aea. additionally, cyclooxygenase-2 metabolites of aea exert their anti-proliferative effects independent of cb1 and cb2. fc. it has been reported that lower levels of czp, compared to ada or ifx, are transferred from treated mothers to the neonate [1] . this discrepancy may be due to active transport of antibodies across the placenta thought to be mediated by the neonatal fc receptor (fcrn). however, anti-tnf binding to fcrn, and fcrn-mediated transcytosis have not been studied. the objective of this study is to quantify binding of czp, ifx, ada and eta to fcrn and to measure fcrn-mediated transcytosis. a biacore™ assay was used to determine binding of czp, ada and ifx to human fcrn. anti-tnfs were passed over an fcrn-coated chip .mdck-ii cells transfected with human fcrn were used to measure fcrn mediated transcytosis. the anti-tnfs and the control antibody (p146), which possessed a fc modified to prevent binding to fcrn, were biotinylated to allow visualization. the amount of each anti-tnf transcytosed across the cell layer over 4 hours was measured by msd assay. results. ifx (132 nm) and ada (225 nm) had high binding affinity to fcrn while the binding affinity of eta to fcrn was 5-10-fold lower (1500 nm). in contrast, czp did not bind to the fcrn with any measurable affinity. the levels of transcytosis seen with ifx and ada were 249.6 ng/ml and 159.5 ng/ml, respectively (mean of 3 experiments). transcytosis of eta (81.3 ng/ml) was lower than that of ada and ifx. in contrast, the level of czp transcytosis was significantly lower, at 3.2 ng/ml, than that observed with the other anti-tnfs and comparable to the control p146 (5.9 ng/ml). conclusion. czp didn't bind to fcrn and thus no fcrn-mediated czp transcytosis was detected. in contrast, ada and ifx had a relatively high binding affinity to fcrn and were actively transcytosed. eta showed lower binding affinity and transcytosis, but fcrn-mediated transport could still be measured. these results explain the previously observed active transport of anti-tnfs across the placenta seen in patients treated with ifx and ada, whereas only low levels were observed with czp [1]. background. anti-cyclic citrullinated peptide (ccp) status was reported previously as predictive of abatacept response [1] . predictors of retention with abatacept have not been published previously. this study was designed to identify predictors of abatacept retention after failing ≥1 biologic agent. in routine clinical practice) is an ongoing, 2-year, international, non-interventional, prospective cohort including patients with ra treated with intravenous abatacept [2, 3] . patients from canada, germany, greece and italy, where patient numbers were sufficient to explore between-country effects, were included. at data cut-off (february 2012), all patients had 1-year follow-up (interim analysis). abatacept discontinuations were reported by the investigator at any time point during follow-up. socio-demographics, disease characteristics and medical history at abatacept initiation, and previous and concomitant treatments were deemed potential predictive variables. clinically relevant variables and those with p≤0.2 (univariate analysis) were entered into a multivariate cox proportional-hazards regression model, adjusted for clustered data from one investigator. using backwards selection, variables with p≤0.1 were retained in the final model. . there were no interactions or effects of c-reactive protein level, rheumatoid factor status, type of previous anti-tnf failure, infection at initiation and abatacept monotherapy. sensitivity analysis, including all variables significant in univariate analysis, was consistent. conclusion. in this first report of real-world predictors of abatacept patient retention, anti-ccp positivity and failing <2 prior anti-tnf agents were associated with higher retention. differences in retention between some countries may reflect specificities in healthcare systems and populations. abatacept, a biologic agent with no contraindications or special warnings for cardiac comorbidity, seems to be a good option for these patients. weekly subcutaneous abatacept confers comparable onset of treatment response and magnitude of efficacy improvement over 6 months when administered with or without an intravenous abatacept loading dose 109 zeitschrift für rheumatologie suppl 2 · 2013 | methods. patients from the intent-to-treat populations of the acqui-re [2] and ample [3] studies randomized to subcutaneous abatacept plus mtx were included. all patients received fixed-dose subcutaneous abatacept 125 mg/week; in acquire but not ample, patients also received an intravenous loading dose (~10 mg/kg based on weight range) on day 1. for this post-hoc analysis, assessments included acr20 and haq-di response (improvement ≥0.3) over 6 months, with patients who discontinued considered non-responders. mean changes from baseline over 6 months in das28 (crp) were assessed in patients with das28 >5.1 at baseline (last observation carried forward) to account for differences in baseline disease activity between the two studies. results. all patients were biologic naïve at baseline, with mean disease duration of 7.6 and 1.8 years, das28 (crp) 6.2 and 5.5, and haq-di 1.72 and 1.5 in acquire and ample, respectively. efficacy was compared throughout the study. for patients treated with subcutaneous abatacept with and without an intravenous loading dose, acr20 response rates were similar (. tab.18). haq-di response rates were also similar with and without the intravenous loading dose (. tab.18). for the overall populations, mean (standard deviation [sd]) changes from baseline to day 169 in das28 were −2.57 (1.30) and −2.09 (1.38) in acquire and ample, respectively. for patients with baseline das28 >5.1, mean (sd) changes in das28 from baseline to day 169 were −2.65 (1.29) and −2.49 (1.35) in acquire and ample, respectively. conclusion. time to onset and magnitude of acr20 and haq-di responses and das28 improvements were generally similar with subcutaneous abatacept with or without intravenous loading in patients with ra and an inadequate response to mtx. the findings from this posthoc analysis suggest that subcutaneous abatacept can be given effectively without an intravenous abatacept loading dose. background. ra is associated with pain and impairment of physical function, significantly impacting a patient's health-related quality of life (hrqol) and ability to perform daily activities. patient-reported outcomes (pros) related to hrqol and daily activity have become an essential part of assessment in ra. we continue to report here comparative findings from pros assessed with subcutaneous abatacept or adalimumab on background mtx in the first head-to-head study, ample. we compared changes in pros at 1 year in patients with ra treated with abatacept or adalimumab, both on background mtx. methods. ample is a phase iiib, randomized, investigator-blinded study of 24 months' duration. biologic-naïve patients with active ra and inadequate response to mtx were randomized to either 125 mg abatacept weekly or 40 mg adalimumab biweekly in combination with mtx. pros evaluated through day 365 included: hrqol, assessed using short form-36 (sf-36; including physical and mental component summary subscores [pcs and mcs]); activity limitation over the previous 30 days, using the activity limitation questionnaire (alq; [1] ); productivity, using the work productivity and activity impairment questionnaire for ra [2] ; physical and psychosocial independence, captured using items from haq, sf-36 score; and alq [3] . other pros previously reported from ample include: patient pain, patient global assessment, fatigue, and physical function [4] . all efficacy analyses were done using the intent-to-treat population, which included all patients who were randomized and received at least one dose of study drug. baseline characteristics were analysed descriptively and changes in pros from baseline were assessed using ancova. results. baseline demographic and clinical characteristics of the abatacept and adalimumab treatment arms were similar. improvements in all domains of the sf-36, including pcs and mcs observed at day parameter baseline woche16 woche32 itt-gesamt das28, mw ± sd 5,7±1,0 (n=507) 2,6±1,3 (n=423) 2,7±1,4 (n=150) vas da pat., mw ± sd 63,3±20,6 (n=509) 26,3±22,5 (n=429) 28,9±25,3 (n=155) sjc28, mw ± sd 8,3±4,9 (n=509) 3,2±3,6 (n=425) 3,0±3,9 (n=152) vas schmerz, mw ± sd zielsetzung der ole-studie beinhaltete die beurteilung der verträglichkeit und der wirksamkeit von czp. die retentionsraten sowie die wirksamkeit wurden bis woche 280 und die verträglichkeitsdaten bis woche 364 beobachtet. in die verträglichkeitsanalyse wurden alle pat einbezogen, die in die ole-studie eintraten und czp erhielten (n=402; n=276 kombitherapie; n=126 monotherapie), einschließlich der plazebo/czp-patienten, die die ausgangsstudien erfolgreich abgeschlossen/abgebrochen haben. bezüglich der wirksamkeit wurden folgende analysen vorgenommen: 1) czp pat, die die ausgangsstudien erfolgreich beendet haben und zu irgendeinem zeitpunkt während der ausgangsstudien oder ole-studie andere dmards eingenommen haben (n=123; kombitherapie completer); 2) czp pat, die die fast4ward studie erfolgreich beendet haben und zu keinem zeitpunkt andere dmards eingenommen haben (n=48; monotherapie completer). ergebnisse. verteilung und häufigkeit der unerwünschten ereignisse (ue), einschließlich der reaktionen an der injektionsstelle (ereignisse/100 patientenjahre: monotherapie 1,4, kombitherapie 0,9) und der schwerwiegenden unerwünschten ereignisse (sue) waren mit dem vergleichbar, was bisher für czp berichtet wurde (. tab.21) . das auftreten von schwerwiegenden infektionen (si) und malignitätsraten war niedrig. es wurden 11 todesfälle berichtet: 7 kardiovaskuläre ereignis-se, 2 infektionen, 1 unfall und 1 tumorerkrankung. die retentionsraten der pat, die die ausgangsstudien erfolgreich beendet haben, waren zur w280 in der czp monotherapie-(24/48, 50%) und der czp kombitherapie-gruppe (67/123; 55%) vergleichbar. der durchschnittliche das28-3(crp)-wert und dessen abweichung vom baseline-wert der ausgangsstudien zum zeitpunkt des eintrittes in die ole-studie und nach 280 w der monotherapie-completer und der kombitherapie-completer, sowie die zugehörigen haq-werte sind in . tab.22 dargestellt. schlussfolgerung. vorliegende ole-studie konnte das günstige risiko-nutzen-profil der czp-monotherapie bestätigen. die langzeitwirksamkeitsdaten zeigten keine unterschiede zwischen pat, die czp als monotherapie erhielten und pat, die czp in kombination mit anderen dmards erhielten. background. rheumatoid arthritis (ra) is the most common disease of joints that non-or deficiently treated leads to functional loss and premature cardiovascular death within years. but nearly 50% of the ra patients fail to treatment with tnfα-inhibitor (tnfi) indicating a switch to rituximab (rtx). the urgency of personalized promising treatment in time presupposes predictive parameter. rheumatoid factor (rf) and anti-citrullinated protein antibodies (acpas; especially accp) are shown to be better diagnostic than less theranostic biomarkers. in that context we investigated the role of antibody subtypes against mutated citrullinated vimentin (amcv) that determine response outcome in rtx-treatment. methods. a cohort of 50 only amcv igg positive ra patients was tested for amcv subtype igm and iga (additionally for rf igg, igm, iga and accp igg) by elisa at baseline (after failure to first approach with tnfi) and at week 24 (after first rtx cycle). responders were characterized by a difference in their das28 of ≥1.2 (eular good-response) between baseline and week 24. the cohort comprises 37 responders (rr) and 13 non-responders to rtx (nrr). results. amcv igg, igm and iga showed higher treatment related decreases compared to rf and accp ig subtypes and additionally even diverged in both groups depending on response outcome: especially amcv iga exhibited a higher mean titer decline of rr by 67% at lower baseline titers (90.14 to 29.84 u/ml) and a mean titer increase of nrr by nearly 20% at higher baseline titers (182.51 to 218.57 u/ml). at baseline rr displayed relatively more negative iga titers (68%; n=25/37) than nrr, who in return showed more iga positive titers (69%; n=9/13). amcv iga positive patients were more likely to show positively for rf iga (80%) and igm (70%), what could be inversely detected for iga negative patients with seronegativity of rf iga (68%) and igm (60%). conclusion. amcv immunoglobulin subtypes showed treatment dependent changes contrary to already known antibodies (accp). especially amcv iga reflects response outcome: amcv iga negativity at baseline and decreasing titers during treatment are predictive for good eular-response to rtx. einleitung. im rahmen der abklärung eines unter tocilizumab-therapie aufgetretenen arzneimittelexanthems erfolgte die bestimmung von c3c und c4 -beide parameter waren erniedrigt. bei recht geringer und nur kurz andauernder ausprägung des exanthems wurde die therapie komplikationslos fortgeführt. die komplementfaktoren wurden im verlauf bestimmt und blieben erniedrigt. im weiteren verlauf erfolgte die konsekutive messung bei weiteren patienten. methoden. nephelometrische bestimmung von c3c und c4 im serum vor und während der therapie mit tocilizumab (jeweils vor der nach 4 wochen anstehenden infusion) bei patienten mit gesicherter rheumatoider arthritis (rf+, ccp+). ergebnisse. c3c-und c4-komplement wurden bei 13 konsekutiven patienten mit rheumatoider arthritis vor und unter tocilizumab-therapie bestimmt. bei allen patienten fielen sowohl c3c, als auch c4 unter der therapie mit tocilizumab (8 mg/kg kg) ab. 8/13 patienten hatten eine c3c-erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). 4/13 patienten hatten eine c4-erniedrigung (bestimmter wert unterhalb des laborinternen normbereichs). drei patientinnen entwickelten unter der therapie ein exanthem, davon hatten 2 eine komplementerniedrigung. keine "offensichtlich" erhöhte infektneigung in abhängigkeit von komplementspiegeln. bei verlängerten infusionsintervallen aufgrund von infekten zeigte sich, dass der effekt von tocilizumab auf die komplementspiegel reversibel ist. durch blockade des il-6-rezeptors tocilizumab kann ein erworbener komplementmangel induziert werden. ähnliche daten wurden im rahmen einer pilotstudie an sle-patienten erhoben, die mit tocilizumab behandelt wurden. der effekt ist bei der rheumatoiden arthritis nicht vorbeschrieben. der genaue umfang des komplementmangels ist bisher nicht untersucht (andere bestandteile der kaskade?) wurde in der sle-studie ausführlicher untersucht. da die verschiedenen komplementbestandteile erniedrigt waren wurde auf eine synthesestörung und nicht auf einen gesteigerten verbrauch geschlossen, was auch in dieser kohorte der fall zu sein scheint. der erworbene komplementmangel könnte einen teil der infektiösen komplikationen unter der therapie erklären. eine korrelation ist aber aufgrund der geringen fallzahl nicht möglich. schlussfolgerung. anhand dieser studie konnte eine exzellente korrelation zwischen den parametern der dxr und des bx verifiziert werden. mittels der neu entwickelten voll digitalisierten bx-technik ist somit eine quantifizierung der periartikulären demineralisation möglich und als surrogatparameter der radiologischen progression bei einer ra eingesetzt werden. 3 jahre, die ra bestand im median seit 7,8 jahren. 74,7% der patienten waren mit tnf-alpha-blockern vortherapiert, 23,7% ausschließlich mit dmards. der mittlere das28 lag zur baseline bei 5,3. zur woche 76 zeigten 34,0% der patienten eine das28 remission (<2,6) und 29,1% bzw. 56,9% der patienten ein gutes bzw. moderates ansprechen gemäß eular-kriterien. über den beobachtungszeitraum stieg der anteil der tcz-monotherapiepatienten von 40,1% auf 58,4%. die mtx-komedikation sank im gleichen zeitraum um 13,3%. 21,9% der patienten, die tcz zunächst zusammen mit einem dmard erhalten haben, konnten dieses absetzen. tcz zeigte in der mono-und kombinationstherapie eine vergleichbare wirksamkeit: 18,3% bzw. 20,3% der patienten erreichten eine cdai remission (≤2,8). der anteil von patienten ohne glucocorticoid(gc)-begleittherapie stieg über den beobachtungszeitraum um 8,2% auf 25,6% an, der anteil mit einer tagesdosis ≤5 mg auf 76,2%. bei 49,4% war eine reduktion der gc-dosis möglich, nur bei 10,1% war eine erhöhung notwendig. bei 14,5% der patienten, die zur bl mit gc behandelt wurden, konnten diese komplett abgesetzt werden. die mittlere gc-tagesdosis verringerte sich kontinuierlich von 9,2 (bl) auf 5,9 mg/d (w76). schlussfolgerung. diese interimsanalyse der nichtinterventionellen studie ichiban zeigt bei den ersten 490 patienten mit mittelschwerer bis schwerer ra über die bisherige beobachtungsdauer von 76 wochen deutliche verbesserungen der aktivitätsparameter, sowie eine reduktionen der begleitenden dmard-therapien und des bedarfs von glucocorticoiden unter behandlung mit tcz. vergleichbar mit den kontrollierten studien ist die tcz-monotherapie auch unter praxisbedingungen der kombination mit dmards ebenbürtig. diese anhaltende wirksamkeit wird erstmals in rheumatologischen praxisdaten für den langzeitverlauf von 1,5 jahren gezeigt. zeitschrift für rheumatologie suppl 2 · 2013 | einleitung. die arteriosklerose (as) steht als häufigste todesursache im besonderen fokus der medizinischen forschung. neuere erkenntnisse weisen auf einen starken zusammenhang zwischen parametern der systemischen entzündung und der pathogenese der as hin. patienten mit rheumatoider arthritis (ra) haben daher ein stark erhöhtes kardiovaskuläres risiko. ziel: untersuchung des zusammenhangs zwischen verschiedenen ra-krankheitsspezifischen risikofaktoren und dem auftreten einer arteriosklerose bei ra-patienten. methoden. 139 ra-patienten, davon 77% weiblich, 64±11,6 jahre alt, wurden hinsichtlich der krankheitsaktivität (krankheitsdauer 13,8±0,9; das28 3,5±0,1; serum-crp 8,1±0,9 mg/dl,; anti-ccp-antikörper 80,4±8,8 u/ml, radiologisches stadium 1,8±0,1; davon 50,5% mit erosionen), sowie klassischer kardiovaskulärer risikofaktoren der as erfasst, welche durch den score-wert (systematic coronary risk evaluation) zusammengefasst wurden. zur as darstellung wurde eine carotis-duplexsonographie mittels eines 7 mhz-schallkopfes (ge vivid 7 pro) durchgeführt. die mittlere intima-media-dicke (imd) der a. carotis communis wurde durch ein softwaregestütztes messverfahren ermittelt. ergebnisse. plaques waren bei 54 patienten (39%) nachweisbar. diese korrelierten mit einer erosiven form der ra (p=0,05), einer längeren krankheitsdauer (p=0,03) und höheren anti-ccp-antikörpern (p=0,02). die mittlere imd betrug 0,67±0,11 mm. je ausgeprägter die radiologischen veränderungen sind, umso höher war die wahrscheinlichkeit der plagues (p=0,02). mittels altersadjustierter partieller korrelationsanalyse wurde der das28 als altersunabhängiger einflussfaktor auf die imd ermittelt (p=0,02). mittel-und hochgradige stenosen zeigten sich bei fünf ra-patienten (3,4%), welche ausnahmslos eine erosive verlaufsform aufwiesen. normalbefunde stehen in zusammenhang mit einem crp-wert unter 5 mg/dl (p=0,04). auch die traditionellen kardiovaskulären risikofaktoren haben signifikanten einfluss auf as. der score-wert erwies sich als äußerst verlässlicher prädiktor für plaques (p<0,01), imd-verdickung (p=0,01) und stenosen (p<0,01). durch elimination der traditionellen risikofaktoren mittels partieller score-adjustierter korrelationsanalyse bestätigte sich erneut die assoziation von pathologischen ultraschallbefunden mit dem das28 (p=0,03). schlussfolgerung. die erhebung klassischer risikofaktoren bei ra-patienten ist unerlässlich. die nutzung des score-werts als screening-parameter ist besonders effektiv. zusätzlich sollten parameter der krankheitsaktivität von ra zum management von arteriosklerose herangezogen werden. besonders aussagekräftig hierfür sind der das28, ein erosiver krankheitsverlauf, die crp-werte und die erkrankungsdauer background. apremilast, an oral small molecule specific inhibitor of phosphodiesterase-4, works intracellularly to modulate inflammatory mediators. the palace 1-3 trials compared the efficacy and safety of apremilast vs placebo in patients with active psa despite prior dmards and/or biologics. the overall safety and tolerability of apremilast was assessed in a pooled analysis of the palace 1, 2 and 3 placebo-controlled phases. methods. safety data was pooled from 3 phase 3, randomized, placebo-controlled studies; patients with active psa despite prior dmards and/or biologics were randomized 1:1:1 to placebo, apremilast 20 mg bid (apr20), or apremilast 30 mg bid (apr30) stratified by baseline dmard use. at week 16, patients with <20% reduction in swollen and tender joint counts were required to be re-randomized (early escape) to apr20 or apr30 (placebo group) or remained on initial apremilast dose through week 24. stable concurrent dmard therapy was allowed (mtx, sulfasalazine, leflunomide, or combination). the analysis comprises data from the placebo-controlled periods (weeks 0-24). results. 1493 patients were randomized to placebo (n=495), apr20 (n=501), or apr30 (n=497) and included in the safety population. baseline demographic and disease characteristics and prior/concurrent therapy were comparable across treatment groups; 22.4% had prior biologic exposure. adverse events (aes) occurred in 47.5% (placebo), 61.5% (apr20), and 60.8% (apr30) of patients. aes occurring in ≥5% of any treatment group were diarrhea, nausea, headache, and urti (. tab.27); most occurred within the first 2 weeks of treatment and nearly half resolved within 2 weeks. of patients with these aes, most (93-96%) were mild or moderate. rates of discontinuation due to aes were low: 4.2% (placebo), 5.6% (apr20), and 7.2% (apr30). serious aes occurred in 3.8% (placebo), 3.4% (apr20), and 3.8% (apr30) of patients. one death occurred (apr20) due to multiorgan failure not suspected to be treatment-related. no cases of serious systemic opportunistic infections, lymphoma, vasculitis, or reactivation/de novo tb were reported. there were no clinically meaningful differences between apremilast and placebo in terms of major cardiovascular aes, changes in blood pressure, malignancies, or effects on laboratory measurements. conclusion. apremilast was generally well-tolerated with no new safety concerns identified compared with the known profile. the aim of the current study was to investigate the relationship between worsening of functional status, clinical disease parameters and radiographic spinal progression over two years in patients with early axial spondyloarthritis (axspa). methods. in total, 160 patients with early axspa (91 with as and symptom duration ≤10 years, and 69 with non-radiographic axspa (nr-ax-spa) and symptom duration ≤5 years) from the german spondyloarthritis inception cohort (gespic) were included in the current analysis based on the availability of radiographic data and data on the functional status at baseline and after 2 years of follow-up. spinal radiographs were scored according to the modified stoke ankylosing spondylitis spinal score (msasss). functional status was assessed by the bath ankylosing spondylitis functional index (basfi), and clinical disease activity by the bath ankylosing spondylitis disease activity index (basdai). results. basfi worsening in ≥1 point after 2 years (n=44, 27.5%) was significantly associated only with higher basdai worsening over 2 years in comparison to those without functional worsening: 1.2±1.4 vs -0.6±1.6, p<0.001. basfi worsening by ≥2 points (n=20, 12%) was, however, associated not only with basdai change (1.5±1.6 vs -0.3±1.6, p<0.001), but also with a higher rate of radiographic spinal progression measured by the proportion of patients with msasss worsening by ≥2 units (35.0% vs. 13.6% in patients without basfi worsening, p=0.024), or with new syndesmophyte formation (25.0% vs. 6.4%, p=0.018). importantly, in the multivariate analysis both basdai increase and progression of structural damage in the spine remained statistically significantly associated with basfi worsening. no other disease-related parameters (e.g. sex, hla-b27 positivity, symptom duration etc) were found to be significantly associated with basfi worsening over two years. conclusion. in this prospective study we could demonstrate that only 2 factors were significantly associated with worse functional outcome over two years in patients with early axspa: 1) increase of disease activity and 2) progression of structural damage. elevated serum vascular endothelial growth factor is highly predictive for radiographic spinal progression in patients with axial spondyloarthritis who are at high risk for progression background. vascular endothelial growth factor (vegf) is an essential mediator of the endochondral ossification and, therefore, might play a pathogenetic role in the process of syndesmophyte formation in axial spondyloarthritis (axspa). the aim of the study was to investigate the role of serum vegf as a predictor of radiographic spinal progression in patients with axspa. methods. altogether 172 patients with definite axspa [95 with ankylosing spondylitis (as) and 77 with non-radiographic axspa] from the german spondyloarthritis inception cohort (gespic) were included in the current study. radiographic spinal progression was defined as 1) worsening of the modified stoke ankylosing spondylitis spine score (msasss) by ≥2 units after 2 years, and 2) development of a new syndesmophyte or progression of existing syndesmophytes after 2 years. serum vegf levels were detected at baseline. results. mean baseline vegf values were significantly higher in patients with msasss worsening by ≥2 units after 2 years (n=22) as compared to those without progression (562±357 vs. 402±309 pg/ml, respectively, p=0.027) and in patients with syndesmophyte formation/ progression (n=18) as compared again to those without progression (579±386 vs. 404±307 pg/ml, respectively, p=0.041). area under the curve (auc) was 0.646, p=0.027 for the msasss worsening ≥2 units and 0.648, p=0.041 for syndesmophyte formation/progression. importantly, the performance of vegf as a predictor of radiographic spinal progression was clearly in patients who were already at high risk for such a progression due to the presence of syndesmophytes at baseline (n=48): auc was 0.812, p=0.001, and 0.772, p=0.003, respectively. vegf serum level of >600 pg/ml in high-risk patients had a sensitivity of 53%, a specificity of 97%, and an odds ratio (or)=36.6 (95%ci 3.9-341.5) as a predictor of msasss worsening by ≥2 units over 2 years. the same serum level of results. immediately after the second session of plasmapheresis, therapy with infliximab 5 mg/kg resumed. after 2 weeks of hospitalization with repeated administration of infliximab had good dynamics (bas-dai 5.6, asdas (crp) = 3.8, basfi 7.8), significantly reduced pain in the joints and spine, stiffness, increased mobility in the joints and spine. the treatment continued: holding plasmapheresis followed by infliximab. after 6 infusions patient experienced a good effect -basdai 3.6, asdas (crp)=2.2, basfi 6.2. conclusion. plasmapheresis in some patients could be effective by reducing activity and dealing with secondary tnf inhibitors failure, since this procedure deletes the macromolecular blood proteins, including tnf-, igg antibodies, and circulating immune complexes results. there were still signs of osteitis in sacroiliac joints in 5 patients at week 24, in 1 patient the mri-determined sacroiliitis has resolved completely. the patient has improved clinically and fulfilled asas40 improvement criteria. there was a minor decrease in sparcc sacroiliitis score (from 16 to 10) in 5 patients at week 24, indicating reduction of inflammation in sacroiliac joints. sparcc sacroiliitis score stayed the same in the remained patient. conclusion. rituximab may be of some benefit in decreasing mri-evident sacroiliitis in patients with highly active as, even in patients in whom tnf-α inhibitors have failed. background. despite the differences in the pathogenesis of ra and as, neck pain is a frequent clinical symptom in both diseases. we evaluated the correlation between subjective reports of neck pain and objective signs of inflammation as assessed by f bone marrow edema (bme) on mri in ra and as patients. methods. stir-mri of the cervical spine of 40 patients (34 ra, 6 as) were included. mris were scored by two blinded readers using a recently published mri scoring system, with quantification of the extension of bme in the atlantoaxial region, corpus, facet joints and processus spinosus of all cervical vertebrae, ranging from 0-57 points. the presence or absence of degenerative changes was also recorded. conclusion. the majority of patients with ra and as had objective signs of bme but also degenerative changes on mri at different cervical locations. assessment of bme in the atlantoaxial region is important in clinical practice, in addition to degenerative changes, since its presence seems to influence the intensity of neck pain reported by these patients. x. baraliakos , 13 having mri data at w94. of these 13, ten were treated with secukinumab and 3 with placebo in the core study. mris were rescored for this study. asspimri-a scores and the occurrence of vertebral edges (ve) inflammatory and fatty lesions were evaluated by an independent blinded reader. results. all 13 pts completed this exploratory mri substudy. in pts receiving 2×10 mg/kg secukinumab followed by 14×3 mg/kg (n=10) secukinumab, spinal inflammation was reduced compared to bl at w28 -similar to the results of the core study -and this reduction was sustained up to w94 (abb. 1). also in 3 pts who had initially received placebo switching to secukinumab at w28, mri inflammation at w94 was reduced. of the 920 ves evaluated, the proportion of ves with inflammatory lesions was reduced from 9.9% (n=91) at bl to 3.7% (n=34) at w28 and 3.6% (n=33) at w94. in contrast, the proportion of fatty lesions at bl (13.5%, n=124) remained largely unchanged at w28 (14.3%, n=132) and w94 (13.7%, n=126). secukinumab reduced mri inflammation at w28 and w94. conclusion. mri analysis suggests that the il-17a inhibitor secukinumab can reduce spinal inflammation and this effect may be sustained for up to 2 years. unlike reports with tnf blockers, secukinumab appeared to leave the proportion of fatty lesions unchanged. the potential impact of these preliminary findings on radiographic progression under secukinumab therapy will be studied in larger trials. schlussfolgerung. es zeigen sich keine signifikanten unterschiede in der krankheitsaktivität der beiden gruppen (vor einleitung der ada-therapie nach dmard-und nach anti-tnf-versagen). auch bei der patientengruppe mit mehreren vortherapien mit tnf-inhibitoren können keine signifikanten unterschiede in der ausprägung der erkrankung nachgewiesen werden. im trend wurde ein früheres einsetzen der haut-und gelenkmanifestation sowie eine stärkere systemische entzündungsreaktion in den patienten mit vorheriger tnf-therapie festgestellt werden, während die dauer der erkrankung und der bmi mit den charakteristika der patienten mit ausschließlicher dmard-vortherapie vergleichbar sind. long-term (52-week) results of a phase 3, randomized, controlled trial of apremilast, an oral phosphodiesterase 4 inhibitor, in patients with psoriatic arthritis (palace 1) background. apremilast, an oral phosphodiesterase 4 inhibitor, works intracellulary to modulate a network of pro-and anti-inflammatory mediators. the palace 1 study assessed the efficacy and safety of apremilast in patients with active psoriatic arthritis (psa) despite prior dmards and/or biologics. methods. patients were randomized 1:1:1 to placebo, apremilast 20 mg bid (apr20), or apremilast 30 mg bid (apr30). at week 16, patients with <20% reduction from baseline in swollen/tender joint counts were required to be re-randomized (early escape) to apr20 or apr30 (placebo group), or remained on their initial apremilast dose. at week 24, all remaining placebo patients were re-randomized to apr20 or apr30 through week 52. results. 504 patients were randomized. at week 16, significantly more apr20 (31.3%; p=0.0140) and apr30 patients (40.0%; p<0.0001) achieved an acr20 vs placebo (19.4%). at week 52, all patients had a minimum 28 weeks of apremilast exposure. response to apremilast was generally maintained over the treatment period. at week 52, acr20 was achieved by 63.0% (apr20) and 54.6% (apr30) of patients (table) . exposure-adjusted incidence rates for adverse events (aes), severe aes, and serious aes were comparable between 0-24 and 0-52 weeks. the proportion of patients remaining on apremilast to week 52 who first reported the most common gi disturbances (e.g., diarrhea, nausea, and vomiting) after week 24 was low (ranging from 0.6-3% for apr20 and 0-1.8% for apr30). there were no clinically meaningful laboratory findings with exposure up to 52 weeks. no deaths beyond the 1 previously reported in the 0-24 week period were observed in the 24-52 week period. no safety signals with respect to major cardiac events, malignancies, and opportunistic infections were observed, consistent with the 0-24 week period. no cases of lymphoma, tuberculosis, or tuberculosis reactivations were reported for the 52-week period (. tab.30). conclusion. apremilast administered to patients with psa beyond 24 weeks continued to demonstrate meaningful clinical response. for patients who completed 52 weeks of the study, acr20 response rates up to 65% were observed. apremilast continued to be well tolerated with an acceptable longer-term safety profile. methods. to identify how conventional cd4+ and cd8+ t cells and regulatory t cells are recruited into the inflamed kidneys in ln, serum and urine samples of 98 sle patients were analyzed for 18 chemokines using multiplex assays. based on the assay's results a group of 8 corresponding chemokine receptors (ccr1-6, cxcr3 and cxcr6) was chosen, whose frequencies on urinary t cells were subsequently determined in 9 patients with acute ln by flowcytometry. results. 12 chemokines (ccl2, ccl3, ccl4, ccl5, ccl7, ccl8, ccl11, ccl20, cxcl9, cxcl10, cxcl16 and cx3cl1) were significantly elevated in the urine of patients with active ln when compared to the control group. the other 6 chemokines (ccl1, ccl17, ccl22, cxcl1, cxcl5) and cxcl11 showed no significant differences between the groups. ccr5 and cxcr3 were the most prominent receptors on both urinary cd4+ and cd8+ t cells, although cd4+ t cells also expressed high amounts of ccr4 and ccr6. however, when compared to t cells in the blood, urinary cd4+ t cells showed significantly higher expression of all examined chemokine receptors but ccr2 while urinary cd8+ t cells only had higher expression of ccr1 and ccr5. the chemokine receptor expression on cd4+foxp3+cd127-regulatory t cells (treg) differed from conventional cd4+ t cells as well. treg expressed significantly more ccr4 and significantly less cxcr6. conclusion. ccr5 and cxcr3 are the primary receptors in the mechanism of recruiting t cells into the inflamed kidney. key chemokines are ccl3, ccl4, ccl5 and ccl8 as well as cxcl9 and cxcl10. however, at least for cd4+ t cells, there are secondary pathways of recruitment involving ccr4/ccl2 and ccr6/ccl20. also, treg recruitment seems to rely more on ccr4 than that of conventional cd4+ t cells. methods. observe is a multicenter, retrospective medical chart review study. rheumatologists from german academic and non academic centers who treat >10 sle patients annually and have >5 years of practice experience were randomly recruited. physicians identified consecutively all their adult sle patients who had received belimumab as part of usual-care. index date was the first belimumab infusion date. the primary outcome was the change in overall sle disease manifestations 6 months after index date based on physician judgment. the overall response rates as well as reasons for early treatment discontinuation within 6 months were assessed. changes in formal disease area indices, e.g. selena-sledai if available and changes in oral steroid dose are also reported. results. previous analyses from us patients treated with belimumab have described significant clinical improvement across relevant organ systems based on clinical judgment and formal disease activity indices and marked reductions in corticosteroid use in patients that received at least 8 infusions of belimumab. the current study is the first description of patient characteristics and outcomes after 6 months of therapy with belimumab outside of the us. it is also the first time overall responder rates and reasons for discontinuation with belimumab have been described in a real world setting. the study provides insights into the effectiveness and safety of belimumab in an ex-us clinical setting. larger, prospective observational studies are needed to confirm the results. commercial support grant disclosure: research funded glaxosmithkline. background. toll-like receptor 9 (tlr-9) signaling is considered to play an important role in b cell hyperreactivity in sle. b cells from slepatients express significantly more tlr-9 than those from healthy donors (hd), especially if patients have positive dsdna-antibodies and high disease activity. tlr-9 stimulation of b cells is tightly linked to their differentiation into plasma blasts and memory cells. the objective of this study was to analyze in a comprehensive manner the effect of tlr-9 signaling on cytokine production by b cells from sle-patients, in comparison to b cells from hd, and in relation with disease activity. methods. b cells from 19 sle-patients and 13 hd were stimulated in vitro using cpg for 48 hours, and culture supernatants were then tested for 28 cytokines and chemokines (bio-plex). the cytokine responses were compared between both groups. in addition, within sle patients, the patterns of cytokines produced by b cells were compared with indices of disease activity. results. cpg-stimulation significantly increased cytokine production (24 out of 28 parameters; p<0.05) compared to baseline. striking increases were found for il-1ra (94±40 pg/ml), il-6 (431±225 pg/ml), il10 (72±37 pg/ml) and ip-10 (361±289 pg/ml; p<0.001). there was no significant difference between both groups. remarkably, production of il-2, il-4, il-7, il-12p70, il13, il-15, il-17a, eotaxin, basic fgf, g-csf, gm-csf, ifn-γ, ip-10, mip-1α, and vegf correlated inversely with the sledai (p<0.05) and even more (additionally il-1β, il-1ra, mip-1β and tnf-α) with anti-dsdna antibody titers. the frequency of cd27+ memory b cells showed a positive correlation between the production of ip-10 and tnf-α in sle, whereas the levels of il-1β, il-7, mip-1α, and mip-1β showed a positive correlation with cd27+ b cells in hd. conclusion. the current data indicate hitherto unknown perturbations of cytokine/chemokine production by b cells in active sle. the inverse correlation of cytokines/chemokines produced by b cells from sle patients with sledai and anti-dsdna titer suggests that the known enhanced b cell proliferation and differentiation upon tlr9-stimulation possibly diminishes cytokine production. background. several cytokines, including ifn-γ, il-18, il-12, and il-23 have been implicated in the pathophysiology of autoimmune disease. il-18, a potent inducer of ifn-γ, enhances th1 responses that are thought to be synergistic and dependent on il-12. we tested the hypothesis that intra-renal il-18 mediates kidney and systemic disease in mrl-faslpr mice. methods. by constructing il-12p40/il-23-/-mrl-faslpr mice and using an ex-vivo gene transfer to deliver il-18 intra-renally, we determined that il-18, independent of il-12 and/or il-23, incites kidney disease in mrl-faslpr mice. moreover, we provide the novel finding that local intra-renal il-18 mediates systemic disease (lung pathology, systemic auto-abs). results. thus, our data indicate that il-18 is a potential therapeutic target for immune mediated kidney and systemic disease in mrl-faslpr mice. using a caspase-1 inhibitor, that inhibits the release of active il-18 and il-1β, we successfully treated kidney (improved renal function, pathology) and systemic disease (skin lesions, lymphadenopathy, and splenomegaly) in mrl-fas lpr mice, while administration of an il-1 receptor antagonist did not influence disease progression. probing further we found that inhibition of il-18 activation results in an amelioration of lupusnephritis by a reduction of intra-renal infiltrating leukocytes (macrophages and t cells) and reduced activation of these leukocyte populations. moreover, caspase-1 inhibition resulted in decreased inf-y and il-17 production, indicating an altered balance of th17 and th1 cell responses in this model. conclusion. taken together, our findings indicate that il-18, independent of il-1β, il-12 and/or il-23, is the major mediator of kidney and systemic disease mrl-faslpr mice. therefore, caspase-1 inhibition is a potential therapeutic target for autoimmune disease in the mrl-faslpr mice. background. in the treatment of giant cell arteritis (gca) glucocorticoid-related adverse effects occur frequently, particularly in patients with relapsing disease. a 50-year-old woman presented with a 2 month history of fever, chills, arthralgias and cephalgias and markedly elevated serum inflammatory markers. whereas further evaluation including ultrasound of the temporal arteries was unremarkable, a positron emission tomography-computed tomography (pet-ct) demonstrated an intense fluorodeoxyglucose uptake of the aorta, the subclavian, carotid and femoral arteries. gca was diagnosed and treatment with high dose prednisone was begun. results. because of disease flares at prednisone dosages below 20 mg/ day and the occurrence of vertebral fractures, cyclophosphamide and methotrexate (mtx) were added as glucocorticoid-sparing agents. as these treatments had to be stopped because of intolerance and mtxpneumonitis, respectively, we started tcz infusions (8 mg/kg body weight). the clinical status rapidly improved. after 2 infusions of tcz follow-up pet scan showed resolution of the previously seen uptake and we were able to taper the daily dose of prednisone to 5 mg. treatment was well tolerated. however, the patient developed mild hyperthyroidism with a rapid rise of the initially normal levels of anti-thyroid peroxidase and anti-thyroid antibodies, anti-tsh receptor antibodies remained normal. thyroid function normalized and the antibody-levels fell without further treatment in the following months. in conclusion, this case demonstrates the successful treatment of a patient with relapsing giant cell arteritis with tcz. for the first time, we report the occurrence of a transient autoimmune thyreoiditis possibly induced by tcz. klinik für pädiatrie mit schwerpunkt pneumologie und immunologie, sektion rheumatologie, berlin, 4 vestische kinder-und jugendklinik der universität witten/herdecke 13 deutsches zentrum für kinder-und jugendrheumatologie organización médica de investigación arthr care res ar&t in press sp division of rheumatology diagnosesicherung: a + b) mr-morphologisch myositistypische veränderungen (os) histologie + c) generalisierte myalgien und laborchemisch dtl. elevierter ck, sowie positivem nachweis von ana und jo-1-ak, pulmonales ct mit diffusen milchglasinfiltraten, in bronchoalveolärer lavage neutrophile alveolitis. ergebnisse. vormedikationen: a) glukocorticoidmonotherapie, mtx-monotherapie, mtx in kombination mit etanercept, cyclophosphamidboli, und zuletzt intravenöse immunglobuline (ivig) in kombination mit mycophenolatmofetil . b) mtx-monotherapie, mtx in kombination mit glukokortikoiden, cyclophosphamidboli, intermittierend intravenöse immunglobuline, cyclophosphamid per os (fau-ci). c) cyclophosphamidboli jeweils gutes ansprechen des ck-wertes auf jeweilige rituximabgaben mit ebenfalls ansprechen des klinischen bildes mit guter regredienz des aus myalgien resultierenden schmerzniveaus. im fall a keine beatmung mehr notwendig. im fall von c) auch gute regredienz subjektiver dyspnoesymptomatik und besserung wichtiger lungenfunktionsparameter, regredienz ctmorphologischer milchglasinfiltrate, im verlauf fehlender nachweis neutrophilie in bal. weitere rituximabgaben bei a, b und c im verlauf zum remissionserhalt nach jeweiligem klinischem befund production of cytokines by b cells in response to tlr9 stimulation inversely correlates with disease activity in sle-patients berlin zeitschrift für rheumatologie suppl 2 · 2013 | das muskuloskeletale system, eines der am häufigsten betroffenen organsysteme bei sle (bei 53-95% der sle-patienten). das ziel dieser analyse war es um diejenigen parameter zu identifizieren, die zu diesem effekt beigetragen hatten, wurde jeder der 9 einzel-parameter zur untersuchung und symptom-erfassung innerhalb des muskuloskeletalen bilag-organsystems analysiert. die post-hoc-analyse umfasste nur patienten, bei denen ein parameter zu studienbeginn als vorhanden gewertet wurde, und jeder parameter erforderte ≥20 patienten-beobachtungen pro kohorte um einen vergleich zu erstellen dadurch wurde die zahl der patienten mit einer initialen beteiligung des muskuloskeletalen systems aufgedeckt, die eine in woche 52 auflösung der manifestation aufwiesen auch im selena-sledai-score war die rate der verbesserung bei dem arthritis-parameter in der belimumab-gruppe mit 1 mg/ kg (58,3%; n=362) und 10 mg/kg (56,6%; n=364) signifikant höher als die daten weisen darauf hin, dass 10 mg/kg belimumab effektiv auf muskuloskeletale organmanifestationen sind akzeptiert als posterbeitrag auf dem eular klinische forschergruppe für rheumatologie (kfr), freiburg i. br., 10 universitätsklinikum ulm, klinik für dermatologie und allergologie die physikalische therapie (pt) ist ein wesentlicher bestandteil der medizinischen versorgung von ssc-patienten patientenregister des dnss erfasst prospektiv, jährlich klinische verlaufsdaten zur organbeteiligung und therapie von patienten mit systemischer sklerodermie. die mittels freitext erfassten angaben zur verordneten pt wurden ausgewertet hivamat n=50 (3,7%) und hylase n=54 (3,9%) anwendung. die anzahl der verfahren, die die patienten zeitgleich erhielten, variierte zwischen mind. 1 und max. 8. über 50% der patienten erhielten 2 anwendungen gleichzeitig. insgesamt wurden 39 therapiearten genannt. 12,5% der patienten mit gelenkkontrakturen zeigten nach einem jahr physikalischer therapie eine signifikante verbesserung der symptomatik (p=0,027) gegenüber den patienten die keine physikalische therapie erhielten. nach drei jahren waren es 15,7% der patienten (p=0,023). bei den patienten mit muskelschwäche zeigten 11% der patienten eine signifikante symptomverbesserung (p=0,048) dieser studie kann erstmals gezeigt werden, dass pt-symptome wie gelenkkontrakturen und muskelschwäche bei ssc-patienten signifikant verbessern kann. dennoch erhält weniger als die hälfte der ssc-patienten eine physikalische therapie punkten zur kontrolle einer mmf-therapie in der klinischen praxis zu untersuchen bei 15 patienten (12-mal sle, je 1-mal systemische sklerose, sharp-syndrom und primäres sjögren-syndrom) die mmf erhielten, wurde 40, 120 und 180 min nach einnahme von mmf die mpa-konzentrationen im serum per hplc bestimmt. die mpa-auc wurde durch die mathematische methode der bayes 20%) und in der standarddosis von 2 g/tag bei 6 von 9 patienten (66%) eine mpa-auc von >35 µg.h/ml. bei zwei patienten wurde nach der messung die dosis adjustiert: eine patientin mit einem sle mit diffus-proliferativer lupusnephritis hatte trotz einer mmf-dosis von 2 g/tag nur eine mpa-auc von 32,9 µg.h/ ml. die dosis wurde daraufhin auf 3 g/tag erhöht. der mpa-auc stieg danach auf max. 54,9 µg.h/ml und die krankheitsaktivität nahm ab (sledai von 8 auf 0, proteinurie von 550 auf 150 mg/24 h und prednisondosis von 6 auf 4 mg/tag) pharmakokinetic study of mycophenolate mofetil in patients with systemic lupus erythematosus and design of bayesian estimator using limited sympling strategies mycophenolic acid area under the curve correlates with disease activity in pupus patients treated with mycophenolate mofetil colony stimulating factor-1 (csf-1) -neuer aktivitätsmarker der lupusnephritis? brigham and wome's hospital, boston, renal division the authors would like to thank pfizer for supporting the study. furthermore the authors would like to thank the "deutsche kinder-rheumastiftung". einleitung. bei patienten mit früher axialer spondyloarthritis (spa) mit einer krankheitsdauer von<5 jahren und nachweis von akut-entzündlichen veränderungen in der ganzkörper-magnetresonanztomographie (mrt) in der wirbelsäule und/oder den sakroiliakalgelenken (sig) zu baseline [1] untersuchten wir die langzeit-effektivität über vier jahre. methoden. in der esther-studie wurden patienten mit etanercept (eta, n=40) vs. sulfasalazin (n=36) behandelt [1] . ab dem zweiten studienjahr wurden alle patienten mit eta behandelt (einige patienten unterbrachen zwischenzeitlich die therapie (n=12) zur untersuchung der biologika-freien remission und wurden dann (erneut) mit eta behandelt) [2] . klinische, laborchemische und mrt-daten der patienten, die zu den jeweiligen studienzeitpunkten vorhanden waren, wurden im vierten studienjahr analysiert (as-observed-analyse). ergebnisse. von 76 patienten, die zu baseline eingeschlossen wurden, erreichten 52,6% das ende von jahr 4 (n=40). in der gesamtgruppe zeigte sich ein gutes bis sehr gutes ansprechen, wobei etwa 50% eine asas partielle remission und etwa 60-70% eine asdas inaktive erkrankung erreichten (. tab.29). der anteil der patienten mit normalem crp ("crp-remission") stieg von 43,7% zu screening auf 90,2% zu woche 216, während der anteil der patienten mit negativem mrt ("mrt-remission" definiert als fehlen akut-entzündlicher veränderungen in den sig und der wirbelsäule gemäß beider scorer) auf von 0% auf 18,4% anstieg. 10,5% der patienten zu woche 216 waren sowohl in asas-remission, im status einer asdas inaktiven erkrankung als auch in mrt-remission. das ansprechen nach vier jahren war sehr ähnlich in den gruppen unabhängig davon, ob im ersten jahr sulfasalazin gegen wurde oder die therapie im jahr 2 unterbrochen worden war (ergebnisse werden nicht gezeigt).schlussfolgerung. es zeigte sich ein konstantes und anhaltendes ansprechen bei patienten mit früher axialer spa, die mit etanercept behandelt wurden. das ansprechen scheint besser zu als bei patienten mit etablierter ankylosierender spondylitsi mit einer langen krankheitsdauer (>10 jahren; [3] ). einleitung. in einer 12-wöchigen placebokontrollierten studie mit 40-wöchiger offener verlängerung bei 46 patienten mit aktiver nichtröntgenologischer axialer spondyloarthritis (nr-axspa) wies adalimumab eine gute effektivität auf [1] . bei patienten, bei denen es zum wiederauftreten der krankheitsaktivität nach absetzen des medikaments in woche 52 kam, wurde die therapie wiederbegonnen ziel der studie war es, die langzeiteffektivität nach wiederaufnahme der therapie von adalimumab nach stopp über 5 jahre zu evaluieren. methoden. bei 46 ursprünglich in die studie eingeschlossenen patienten wurde die therapie nach 52 wochen beendet und 23 patienten (52% männlich, mittleres alter 32 jahre, range 24-45, mittlere krankheitsdauer vor therapiebeginn 4 jahre, range 1-10, 74% positiv für hla-b27) hatten, definiert durch erreichen eines 40% ansprechens gemäß der assessments in spondyloarthritis society-kriterien (asas40), gut auf die therapie angesprochen. bei wiederauftreten von krankheitsaktivität (definiert durch nicht mehr erreichen von asas40) wurde adalimumab 40 mg alle 2 wochen über 5 jahre (woche r264) weitergeführt. die asas kriterien und der bath ankylosing spondylitis disease activity index (basdai) wurden in form einer completer-analyse berechnet. ergebnisse. 19 der 23 patienten mussten wiederbehandelt werden: 17/19 (90%) erreichten jahr 3, 16/19 (84%) erreichten jahr 4 und 11/19 (58%) der patienten erreichten jahr 5 der wiederbehandlung. nach 3 jahren wiederbehandlung mit adalimumab erreichten 13/17 (77%) wieder asas 40 und 11/17 (65%) erreichten partielle remission gemäß der asas-kriterien. nach 4 jahren erreichten 11/16 (81%) und nach 5 jahren 9/11 (91%) asas 40. asas partielle remission wurde nach 4 jahren von 9/16 (56%) und nach 5 jahren von 7/11 (64%) patienten erreicht. in der completer analyse fiel der mittlere basdai von 5,1±1,8 zum zeitpunkt der wiederbehandlung auf 1,6±1,6 im jahr 3 (p<0,001), 2,2±2,1 (p=0,001) im jahr 4 und auf 1,5±1,8 (p=0,001) im jahr 5 der wiederbehandlung ab. schlussfolgerung. in dieser gruppe von 19 patienten mit aktiver nr-axspa, die ein gutes therapieansprechen über 52 wochen mit adalimumab erreicht hatten und die bei wiederauftreten von krankheitsaktivität nach stopp der therapie in woche 52 weiterbehandelt werden mussten, sprach die mehrheit der patienten, die in der studie verblieben, gut und anhaltend auf das fortsetzen der therapie an. tab. 29 | sp-17 langzeit-effektivität über 4 jahre etanercept-therapie bei patienten mit früher axialer spondyloarthritis. daten zu baseline (bl), jahr 1 (w48), jahr 2 (w108), jahr 3 (w156) und jahr 4 (w216). daten background. secukinumab (ain457) is a new fully human monoclonal antibody (mab) targeting il-17a for the treatment of inflammatory diseases. administration of mabs can be associated with immunogenicity via the induction of anti-drug antibodies (adas). adas can lead to unwanted clinical consequences, such as loss of exposure, loss of efficacy due to altered pharmacokinetics and/or functional neutralization and, in the worst case, anaphylactic reaction and immune complex diseases. the assessment of ada formation is therefore a critical component in the assessment of biotherapeutic safety. methods. the immunogenicity assessment strategy for secukinumab follows a three-tiered approach. first, samples are analyzed for presence of ada in a screening assay which takes a 5% false-positive rate into account. in a second step, screening assay positive samples are tested in a confirmatory assay that identifies true positive responses. finally, true immunogenicity-positive samples are quasi-quantified via titration. a biacore-based assay was used during the early stages of the secukinumab program, and an msd-based bridging assay was applied during the later stages of the program. in addition, pharmacokinetics and clinical efficacy as well as safety data are also evaluated. samples to assess immunogenicity were obtained from 1582 individual subjects encompassing 18 clinical studies in different indications during treatment and during follow-up. dosing regimens included single doses such as 25 mg subcutaneously in psoriasis patients as well as multiple 7×10 mg/kg doses intravenously in ms patients over a six-month period.results. none of the subjects tested for immunogenicity developed sustained adas. in total, 4 subjects met the definition of treatment-related, transient positive immunogenicity showing low ada titers. none of these subjects had evidence of loss of efficacy, deviating pk behavior or reported anaphylactic reaction or immune complex disease.conclusion. based on the available data, secukinumab appears to carry a low risk of immunogenicity. in the very few transient immunogenicitypositive patients identified so far, there has been no indication of altered pharmacokinetics or loss of efficacy, and no adverse event that could be linked to immunogenicity has been detected. more data from the ongoing phase 3 studies are required to strengthen this encouraging finding in a larger patient population. risikofaktoren für eine aa-amyloidose bei entzündlich-rheumatischen erkrankungen und bei der idiopathischen aa-amyloidose methods. we report a case of an 84-year-old woman suffering from ulceration and signs of infection of the ulnar aspect of the right forearm due to subcutaneous calcification in association with crest syndrome.results. this case presents an unusual case of extensive subcutaneous calcification in crest syndrome requiring surgical excision due to secondary ulceration, inflammation and infection. while a surgical approach has already been described for calcification in different connective tissue diseases, only scant data of massive subcutaneous calcification related to a forearm in crest syndrome followed by surgical excision exist. conclusion. in crest syndrome, extensive subcutaneous calcification related to the forearm can occur. surgical excision followed by primary wound closure can lead to an excellent postoperative result. background. the whole blood interferon signature (wbifns) is measured in several clinical trials studying inhibitors of interferon alpha (ifn-α) in sle, but failed repeatedly -in contrast to the less sensitive ifnα -to reflect longitudinal changes in lupus activity and to guide dosage finding of rontalizumab. therefore, better ifn biomarkers reflecting disease activity over time and individual response to the inhibition of ifnα are needed to optimize the risk-benefit ratio of ifn-inhibitors. here, we show that the highly sensitive monocyte restricted ifnα response protein siglec-1, also known as sialoadhesin or cd169, is a useful biomarker to monitor longitudinal changes in disease activity of sle patients. methods. ifn-α and siglec-1 were measured by delfia and flow cytometry, respectively, in 24 accurately characterized lupus patients over a period of up to 12 months (overall 112 visits). changes of biomarker and changes of disease activity (bilag2004) were correlated using spearman rank test (srt). disease courses of selected sle patients were plotted to demonstrate in detail the relations of ifn-biomarkers with disease activity, sle medication and clinical manifestations. background. a 74-year-old woman was admitted because of sudden attack of convulsion and somnolence situation with positive canca and myeloperoxidase antibodies. cerebral magnetic resonance imaging (mri) showed thickening and marked progression of the dura-meningeal enhancement and edematous changes at pre and post central gyrus left side. based on these findings, it was diagnosed as hypertrophic cranial pachymeningitis related to anca-associated vascultis as unusual presentation. there was only temporarily und partial responce to a 7-month therapy with cyclophosphamide 1000 mg i.v and oral glucocorticosteroids . taking into consideration the severe, life-threatening course of the disease in the case of our patient, the decision was made to use rituximab, a chimeric, monoclonal igg1 antibody directed against cd20, leads to destruction of b cells via complement mediated lysis and antibody dependent cellular cytotoxicity. the first administration of the medication was performed according to the pattern for rheumatoid arthritis patients treated with rituximab, i.e. 2 infusions for 1000 mg in 14-day intervals in combined therapy with glucocorticosteroids. a follow-up mri at 6 months after start with rituximab showed significant regression of the meningeal pathology at temporo-occipatel aspects (pachymeningitis) and completely resolution of edematous changes at pre and post central gyres. the complete clinical remission was achieved by introducing rituximab. conclusion. rituximab seems to be successful therapie for the induction and maintenance of remission in patients with anca-associated vasculitis (aav) with cns involvement (hypertrophic cranial pachymeningitis ) , who had previously failed to respond to standard treatment with cyclophosphamide and steroids and a range of alternative treatments [1, 2] . antikörperdiagnostik. mit prednisolon-therapie (1 mg/kgkg, 40 mg/ tag) und zusätzlich methotrexat 15 mg wöchentlich war keine anhaltende normalisierung der entzündungsserologie zu erzielen. infliximab (5 mg/kg) 6-wöchentlich i.v. erbrachte nur kurzzeitig eine normalisierung der entzündungswerte, dann trotz weiterer infusionen einen erneuten anstieg der bsg bis auf 91 mm, crp 58 mg/l. nach umstieg auf tocilizumab (400 mg /8 mg/kgkg) alle 4 wochen konnte nach 6 wochen eine bislang anhaltende normalisierung der entzündungsserologie erzielt werden (crp 1,1 mg/l, bsg 9 mm 1. std.). der allgemeinzustand der patientin besserte sich deutlich, der hb-wert normalisierte sich auf 7,9 mmol/l. in der kontrastverstärkten sonographie fand sich ein abfall in der kontrastmittelaufnahme der a. carotis communis. die maximale intima-media-dicke reduzierte sich bislang auf 1,7 mm. schlussfolgerung. die bisherige standardtherapie der takayasu-arteriitis mit prednisolon und mtx führte auch im vorliegenden fall nicht zur remission. für infliximab fanden wir ein frühzeitiges therapieversagen des sonst erfolgreich beschriebenen ansatzes einer tnf-α-blockade bei riesenzellarteriitis. dennoch gelang mit tocilizumab eine bislang über 12 monate andauernde klinische, sonomorphologische und serologische remissionsinduktion bei monatlicher fortführung der il-6-blockierenden therapie. 4 background. cd56 is the prototypic nk receptor that is also expressed on a unique population of effector cd4+ cells. these cd56-expressing t cells are expanded in rheumatoid arthritis patients and had features of senescent cells. nkg2d is another nk receptor over expressed on effector cd4+ cells in aav patients. cd 56+ as well as nkg2d + t cells seem to be involved in tissue injury as they are capable of mediating tcr-independent immune activation. it is hypothesized that il-15 is able to up regulate the expression of nk cell receptors. interleukin-15 (il-15) is a proinflammatory cytokine that is over expressed in aav and is linked to the expansion of cd4+ effector memory t cells (tem). in aav in remission a persistent expansion of these cd4+ effector memory t cells has been observed. in the present study we assessed the expression cd56 on cd4+ t cells of aav and if expression of these molecules was influenced by il-15. methods. the distribution of cd4+ tem and the proportion of cd56+cd4+ t cells and nkg2d+ cd4+ t cells were analysed in 52 aav-patients and 30 hcs by facs. in vitro effects of il-15 on the expansion of cd4+ tem and up regulation of cytotoxic markers were assessed in the same way. in addition il-15 serum levels were measured in patients and hc by elisa. results. we observed an increased proportion of circulating cd4+cd56+ t cells in aav as well as nkg2d+ cd4+ t cells in patients in remission compared to hc (13.6 vs 0.6 p<0.0001 and 14 vs 0.7 p<0.0001). 80% to 90% of these cells were cd4+ effector memory t cells. the percentages of the cd56+cd4+ t cells and nkg2d+ cd4+ t cells were constant over time. we also observed elevated il-15 serum levels in patients in remission compared to hc (p=0.001). in vitro stimulation of pbmcs with il-15 increased not only the proportion of cd4+ memory cells (cd45ro+) but also the expression of cd56 and nkg2d on these cells. conclusion. the driving force behind the persistent expansion of a cytotoxic subset of cd4+ effector memory t cells expressing cd56 and nkg2d+ and being tcr -independent is likely the increased il-15 expression in aav patients . ergebnisse. unabhängig von regime der remissionsinduktion und der primären erhaltungstherapie lag am ende der nachbeobachtungsperiode bei 69% der patienten eine renale remission vor (45%; 24% pr). 16% hatten eine persistierende proteinurie von >0,5 g/tag bei stabiler nierenfunktion, 3% eine persistierende niereninsuffizienz mit erhöhtem kreatinin bei inaktivem sle, bei 12% wurden eine persistierende aktive ln und/oder renale rezidive beobachtet. vier patienten verstarben. patienten mit langzeit-cr waren gekennzeichnet durch einen niedrigeren tubulointerstitiellen chronizitätsindex in der initialen nierenbiopsie (0,37±0,40 vs. 1,47±1,13; p=0,001), eine hochsignifikant geringere proteinurie nach 6 cyc-pulsen (0,36±0,36 vs. 1,97±1,57 g/tag; p=0,000) und niedrigere dsdna-ak (61±72 vs. 110±89 u/ml; p<0,05) zum zeitpunkt des beginns der erhaltungstherapie. eine proteinurie von <0,35 g/tag nach 6 pulsen cyc zeigte eine sensitivität von 71% und eine spezifität von 94% für eine langzeit-cr. schlussfolgerung. eine proteinurie von <0.35 g/tag nach remissionsinduktion mit 6 pulsen cyc sowie ein geringer tubulointerstitieller chronizitätsindex in der nierenbiopsie sind prädiktoren einer anhaltenden kompletten renalen remission bei ln. background. to evaluate and compare clinical efficacy of three biomarkers for interferon activity (measured directly and indirectly) and six traditional biomarkers to indicate current disease activity in sle. methods. ifn-α (delfia), ip-10 (elisa) and siglec-1 (flow cytometry) was measured in 79 accurately characterized lupus patients and compared to serum titres of anti-dsdna (elisa and ria), anti-dsdna-ncx elisa, anti-nuc elisa, c3 and c4. disease activity was evaluated using bilag-2004 and a modified sledai-2000 (msle-dai-2k). additionally, 31 clinically quiescent patients were monitored for flares over the course of 180 days. results. increased levels of ifn-α, ip-10 and siglec-1 were found in 32%, 50% and 86% of 66 active sle patients. ifnα (r=0.45; p<0.0001) and siglec-1 (r=0.54; p<0.0001) correlated better with bilag-2004 than ip-10 (r=0.38; p=0.0002), farr assay (r=0.40; p=0.0001), anti-dsdna-ncx elisa (r=0.28; p=0.0061), anti-dsdna elisa (r=0.31; p=0.0025), anti-nuc elisa (r=0.25; p=0.0121), c3 (r=-0.43; p<0.0001) and c4 (r=−0.33; p=0.0013). predictors of sle flares were disease duration ≤92 months, mild clinical activity (in contrast to no activity), complement c3≤89 mg/dl and ifn-α ≥20 pg/ml, while only lymphocyte count and age were independent predictors in multivariate analysis. conclusion. ifn-α, ip-10 and siglec-1 emerged as beneficial biomarkers for disease activity in lupus patients. therefore, implementation of ifn biomarkers in standard lupus diagnostics should be reappraised, especially in view of emerging anti-ifn-directed therapies. .0001) and carried significantly more often other antibodies (71.1%; p<0.0001), which were separated into u1rnp-(22.7%), ro-(16.8%), pmscl-(11.5%) antibodies, followed by 10.8% with rheumatoid factors, 7.5% with la-, 5.8% with dsdna-and 2.8% with jo-1-and 2.6% with ku-antibodies. the kaplan-meier analysis of the onset of organ involvement revealed a clear inclined position of overlap patients between patients suffering from lcssc and dcssc, especially regarding lung fibrosis and heart involvement. patients suffering from pah, oesophagus involvement and kidney involvement, overlap and lcssc patients showed nearly similar curve progression (log rank <0.0001). furthermore musculoskeletal involvement was significantly more frequent and more progressive in patients with overlap disease, followed by patients with dcssc and lcssc (log rank <0.0001). conclusion. these data support the current concept, that ssc-overlap syndromes should be regarded as a separate ssc subset, distinct from lcssc and dcssc, due to a different course of the disease, different proportional distribution of specific autoantibodies and skin/organ involvement. methoden. 18 patienten mit gpa (11 mit aktiver und 7 mit in remission befindlicher gpa) wurden durchflusszytometrisch analysiert und mit 17 gesunden verglichen. eine färbung für cd27, cd20, cd19, igd, iga, cd95, mhcii, wurde mittels flowjo-software analysiert. die statistische auswertung erfolgte mit "graph pad prism" und p-werte<0,05 wurden als signifikant angesehen. die studie wurde von der ethik-kommission der charité genehmigt. ergebnisse. deutliche unterschiede (p=0,0018) wurden sowohl für die absolute zahl als auch die frequenz der plasmazellen im peripheren blut der patienten mit gpa mit krankheitsaktivität (6,4±5,06/µl) im vergleich zu denen mit einem bvas von 0 (2,52±1,6/µl) oder gesunden (2,27±1,15/µl) gefunden, ähnlich wie bei sle. bei patienten mit gpa ist außerdem eine signifikante erhöhte anzahl der plasmazellen igapositiv (p=0,0028). die anzahl der plasmazellen sowie die frequenz der plasmazellen an den b-zellen im blut korrelieren mit dem bvas (r=0,9135; p<0,0001). interessanterweise zeigte sich keine expansion der doppelt negativen memory-zellen, die zum beispiel beim sle beschrieben ist. für die naiven b-zellen fand sich ebenfalls ein signifikanter unterschied zwischen patienten mit aktiver erkrankung im vergleich zu gesunden. bei den t-zellen fanden sich nur diskrete veränderungen. schlussfolgerung. die anzahl der plasmazellen ist bei patienten mit aktiver gpa deutlich erhöht, was eine rolle von plasmazell-vermittelten mechanismen in der pathogenese nahelegt. ein großteil dieser plasmazellen ist iga-positiv, diese könnten eine rolle bei der hno-beteiligung spielen. key: cord-006444-eq56zhtd authors: nan title: abstracts of oral presentations and posters date: 1993 journal: ann hematol doi: 10.1007/bf01695978 sha: doc_id: 6444 cord_uid: eq56zhtd nan since the blood cells are primarily concerned with host defense, the introduction of the csfs as therapeutic agents offers the opportunity to develop unique therapeutic strategies designed to enhance overall host defense, particularly with relevance to cancer and aids. administration of csfs is associated with profound changes in cellular function, and treatment strategies will need to consider the potential deleterious effects of heightened host cell activity and potential effects on nonhematopoietic cells. memorial sloan-kettering cancer center, new york, ny 10028, usa a75 3 cytokine the majority of hemopoietic stem cells in the steady state marrow are dormant in the cell cycle. using serial observation (mapping) of blast cell colony formation from bone marrow cells of mice that have been treated with a high dose 5-fluorouracil (5-fu), we have identified a number of cytokines that appear to control the cell cycling of the primitive hemopoietic progenitors. the early-acting cytokines may be divided into three groups. the first group would consist of il-3, gm-csf, and il-4. the second group consists of il-6, g-csf, il-11, il-12, and lif/dia. the third group consists of steel factor (sf). according to our studies in culture, cytokines in each group can interact with those in other groups to initiate cell division in the cell cycle dormant primitive progenitors. while studies using retrovirally-labeled murine stem cells demonstrated unequivocally the presence of lymphohemopoietic progenitors that are capable of producing both lymphoid and myeloid progenies, it has not been possible to identify and quantitate these progenitors in culture. recently, we have developed a two-step methylcellulose culture method to quantitate murine lymphohemopoietic progenitors that are capable of producing myeloid cells and and pre-b-cells. after establishing the primary culture system initially with medium conditioned by pokeweed mitogen stimulated spleen cells, we characterized combinations of cytokines that are able to maintain the b-lymphoid potentials of the primary colonies. we observed that two-factor combinations including sf such as sf plus il-6, sf plus g-csf, sf plus il-11, sf plus il-12 were effective in maintaining the proliferation of b-cell progenitors. somewhat less effectively il-4-based combinations such as il-4 plus il-6 and il-4 plus il-11 also supported the b-cell potentials of the the primary colonies. interestingly, il-3-based combinations were unable to maintain the b-cell potentials of the primary colonies even though the cells in myeloid lineages proliferated strongly. we also found that addition of il-3 to an effective two-factor combination such as sf plus il-11 inhibit the b-cell potentials of the primary colonies. our cell culture for the murine lymphohemopoietic progenitors may provide an important tool for studying the mechanisms regulating the early process of lymphopoiesis. the survival and proliferation of haemopoietic stem cells, induced by growth factors, occurs concomitantly with differentiation and developmeat of the stem cells and their progeny, into mature blood cells. are the growth factors simply permissive for these processes? or do they have an inductive role to play in lineage commitment of stem cells and subsequent maturation into phenotypically mature cells? from various experiments, it is clear that the outcome of the response (that is the types of mature cells produced) is a reflection of the range of growth factors to which the cells are exposed-suggesting that combinations of growth factors may well influence the choice of lineage options taken by multipotent cells. the problem, of course, is that in all systems studied to date, no detailed examination has been made of cell death, and that even in growth factor combinations where, for example, no erythropoiesis is found, it is possible that erythroid progenitors are being produced as a consequence of differentiation of the multipoteni cells but that these cells are then dying due to lack of availability of the appropriate survival (growth factor) stimulus. we have recently been able to circumvent some of these problems and have shown that, orovided the cells receive a survival stimulus, differentiation can occur hi the absence of added growth factors and also that proliferation it not a prerequisite for acquisition of a mature cell phenotype. in other words, the growth factors may act primarily as survival and mitogenic stimuli and not as "inducers" of differentiation. expression of p210bcr/abl by transfection converts interleukin-3 (il-3)-dependent cell lines to factor independence and transforms immature hematopoietic cells in vitro. we tested the hypothesis that p210 bcr/abl may induce factor-independence by constitutively activating signal transduction pathways which are normally regulated by il-3/gm-csf. in both the il-3-dependent murine myeloid cell line, 32dcl3, and the il-3/gm-csf-dependent human line, mo7e, p210 bcr/abl induces rapid factor-independence despite continuous growth in il-3-containing medium. one-and two-dimensional antiphosphotyrosine immunoblotting showed that most proteins tyrosine phosphorylated by p210bcr/abl are different that those phosphorylated in response to il-3. several signaling molecules have been found to be activated or phosphorylated by both il-3/gm-csf and p210bcr/abl, including raf-1, map kinase, shc, vav, and probably pi3k. other signal transducing proteins were found to be phosphorylated only by pz10bcr/abl (p120rasgap, two rasgap associated proteins, and c-fes), or only by . in order to better define the biochemical activities of p210bcr/abl which lead to mitogenesis, a series of cell lines were constructed in which the functional expression of pz10bcr/abl was inducible. the uninduced cell lines had a wild-type phenotype while the induced cell lines displayed markedly reduced apoptosis in the absence of growth factor, and some were hyper responsive to growth factors. the phenotypes of these cell lines have been stable in culture, and the lines should be useful to define biochemical activities of p21 obcr/abl which are important for mitogenesis. r.e. donahue, m.r. kirby, p.d. lawman, s.e. sellers, s.w. kessler, and m.j.p. lawman a novel factor has been purified to homogeneity from a cell line derived from a human mixed germ cell tumor. by western blot analysis, using a polyclonal rabbit antibody raised to the purified native protein, scpf was found to be expressed both as a 32kd secreted and as a 37kd membrane-bound protein. to further evaluate scpfs' ability to support cd34+ cell growth in culture, scpf was used in short-and long-term cultures using immunoselected cd34 + cells. for short-term culture studies, cd34+ cel~s were evaluated prior to and subsequent to a six day exposure to either media alone or media supplemented with il-3, scpf, or il-3+scpf. the greatest expansion of cd34+ cells was in those expressing od38. compared to pre-culture, cultures maintained in scpf, il-3, or il-3+scpf had, respectively, a 0.7, 2.9, or 0.7-fold increase in cd34+cd38+ numbers. there was also a consistent increase in the ratio of large cd34+38+ cells to small cd34+cd38+ cells. presumptively, this change represents an increase in the number of cells in either the g2/m or s phase of the cell cycle. of the cytokine combinations evaluated, only the combination of il-3 + scpf led to a t .8-fold expansion of cd34 + cd38-cells above baseline values. by themselves, scpf and il-3 led to a 0.4 and 0.7-fold reduction in cd34+cd38-numbers. when compared, however, to the number of cd34 + cd38-cells present in media alone after the 6 days in culture, scpf, il-3, and il-3 + scpf hat respectively a 3.0, 5.7, and 12.3-fold greater number of cd34 +cd38-cells. in long-term culture assays in the absence of scpf, the cultures deteriorated rapidly and were test by day 18. in the presence of scpf, cell numbers were maintained over the initial 8-14 days in culture, with proliferation becoming evident 16 days post-culture. at day 21, some of the cells were removed from culture media containing scpf and replatad in culture media alone. after an additional 30 days in culture the cells that were no longer exposed to the scpf had differentiated. interestingly, the cells that were cultured in scpf continued to proliferate. after 50 days in culture, these cells were predominantly cd34 +, cd33 +, cd38 +, cd45 +, cd71 +, and hi.a-dr +, and failed to express thy t, cd4, cd8, cd14, od20, or cd56. karyotypic analysis demonstrated that these cells hat multiple chromosomal aberrations, civin, a. gewirtz, p. rockwell, l. witte we have cloned the cdna for stk-1 (stem cell tyrosine kinase 1), a human growth factor receptor tyrosine kinase, and investigated its expression in bone marrow, leukemias, and leukemic derived cell lines. stk-1 expression is restricted to the cd34+ fraction of normal human bone marrow, the fraction containing all of the hematopoietic stem cell activity of marrow. experiments in which cd34+ cells grown on irradiated bone marrow stromal feeder layers were exposed to stk-1 antisense oligonucleotides resulted in inhibition of colony formation. stk-1 is also expressed in most cases of aml, b lineage all, and t cell all. a number of hematopoietic tissue culture cell lines which express stk-1 have been identified, including kmt2, kgla, kg1, ml-1, hl-60, nalm-16, and reh. ml-1 cells stop growing and differentiate after exposure to phorbol esters and other agents. stk-1 expression is completely shut off by this treatment. antipeptide antibody generated against several regions of stk-1 identifies a doublet of proteins of 155kd and 130kd, probably corresponding to different degrees of glycosylation, in several of the cell lines. these data imply a possible role for the stk-1 receptor in the normal proliferation of hematopoietic stem cells and the abnormal proliferation of leukemic cells. further antisense experiments and the isolation of the growth factor for this receptor will be necessary to fully understand its role in hematopoiesis and leukemia. murine long term repopulation and double transplantation assays have clearly demonstrated irreversible damage to early stem cells by repeated doses of alkylating agents or anti-metabolites. the ability to protect from the short term effects of chemotherapy-induced myelosuppression by administration of hematopoietic growth factors has obscured the potential problem of long term stem cell insufficiency. indeed in murine models involving repeated cyclophosphamide administration, csf administration has been reported to protentiate stem cell damage. the development of techniques for isolation of human hematopoietic stem cells in a cd34 + lin -re fraction of marrow and blood, together with long term culture-initiating assays on marrow stroma or long term ex vivo expansion with cytokine combinations, permits quantitative analysis of human stem cell proliferation potential. it is becoming apparent that extensive chemotherapy treatment gravely compromises the population of primitive hematopoietic stem cells as reflected in their impaired capacity to peripheralize and to be represented in the blood cd34 + population following csf treatment with or without cytoxan. five strategies are currently under evaluation: 1) upfront harvesting of marrow and/or elicited peripheral blood prior to onset of chemotherapy with subsequent "rescue" following chemotherapy; 2)fine tuning of cytokine and chemotherapy administration to take advantage of "rebound quiescence" of stem cells; 3)administration of negative regulators to suppress stem cell proliferation. transforming growth factor 6, macrophage inflammatory protein e and tumor necrosis factor have all proved protective in preclinical models. 4) utilize cytokines, eg il-1, that protect stem cells by increasing drug enzymatic inactivation, decreasing drug influx and/or increasing drug efflux, and inducing dna repair or decreasing dna damage. 5) ut ze gene therapy to introduce into hematopoietic stem cells drug resistance genes such as mutated dihidrofolate reductase that confers methotrexate resistance or enhance the expression of the multi-drug resisting gene (mdr) expression. interleukin-11: a novel hematopoietic cytokine possessing multiple biological activities in vitro and in vivo. j.p. leonard and s.j. goldman, genetics institute, cambridge, ma. interleukin-1 1 is a multifunctional hematopoietic cytokine which was originally identified in the conditioned medium from an il-1 stimulated primate stromal cell line. the human cdna was subsequently cloned from a fetal fibroblast cell line enabling the expression and purification of the human protein. the in vitro biological activities of rhll-11 result predominantly from synergistic interactions with other growth factors. in combination with other cytokines, rht[,-1 1 has been shown to support the formation of primitive human blast ceil coionies from bone marrow, promote erythroid burst formation and stimulate both early and late stages of megakaryocyte proliferation and differentiation. in addition, rhll-11 alone directly increased the size and ploidy of enriched megakaryocytes. although rhll-1 1 has no inherent 13 cell growth factor activity, rhll-1 1 has been shown to stimulate immunoglobulin producing b cells both in vitro and in vivo. rhll-i 1 is biologically active in mice, rats, dogs and primates when administered as a single agent in vivo. the predominant effect of rhll-1 1 in naive animals was on cells of the megakaryocyte lineage, increasing the number of bone marrow megakaryocyte progenitors, stimulating megakaryocyte endoreplication and increasing peripheral platelet counts in a dose dependent fashion. in a variety of murine rtiodels of myelosuppression, the effects of rhtl-1 1 were multitineage, stimulating the recovery of megakaryocyte, erythroid, and granulocyte and macrophage progenitors in the bone marrow, rhll-i 1 administration reduced the platelet and hematocrit nadirs and the overall duration and severity of thrombocytopenia and anemia in these models. in a murine bone marrow transplant model, rhll-1 1 also accelerated neutrophil recovery. the results from ongoing preclinical studies continue to confirm the broad spectrum of biological activities possessed by rhil-1 1 in vitro and suggest this cytokine may be an effective agent in the treatment of myelosuppression associated with cancer chemotherapy and bone marrow transplantation. preclinical biology of human il-10 t.l. nagabhushan, s.k. narula and m.i. siegel human il-10 (hull-10) is a 160 amino acid polypeptide synthesized by a number of different cell types. it is a pleiotropic factor with both immunosuppressive and immunostimulatory activity. recombinant hull-10 expressed in cho cells is not glycosylated, and when expressed in e. coil the protein retains the biological activity of the cho-derived product. in the presence of antigen presenting cells, hull-10 inhibits cytokine synthesis in t cells. hull-10 downregulates ,f-ifn and il-4 induced mhc class ii antigen expression on monocytesmacrophages. in contrast, it has no effect on class ii expression on purified tonsillar and peripheral b cells. hull-10 also inhibits the synthesis of il-1% il-1/l il-6, il-8, tnf-m gm-csf and g-csf at the protein and rna levels in monocytes activated by lps or lps and .r the y-ifn or gm-csf induced phagocytosis of opsonized yeast particles by human peripheral blood-derived macrophages and granulocytes is downregulated by hull-10. il-4 induced ige synthesis by pbmnc is inhibited by hull-10. the protein also potentiates a strong lak activity in human pbmnc. these results will be discussed along with some early in vivo biology in rodents. a monomeric pentapeptide (peedck) inhibits murine hematopoiesis in vitro and in vivo while its dimer counterpart (peedck): formed through a disulphide bridge has a stimulatory effect in the same systems. stable peptides of the rimer have been made by substitution of the disulphide bridge with a dimethylene bridge. in vitro the dimer seems to have no direct effect on gm-cfc or on purified lin-scal + cells in the hpp-cfc assay (400 cells per culture, 15-20% pe). the monomer does not affect gm-cfcs, but inhibits approximately 50% of the purified hpp-cfcs. the dimer stimulates human or mouse stromal cells to produce m-csf and possibly other cytokines which augment colony formation in vitro and also activate human pbl as measured by an increased expression of cd1 lb. in ex vivo experiments it was found that the dimer increases and the monomer decreases cell cycle rate of cfu-gm and cfu-s in the bone marrow. intraperitoneal injection of the dimer (1 to 100 ng/kg) into mice, led to increased progenitor cell number in the bone marrow and also increased survival of mice given a lethal dose of cyclophosphamide (550 mg/kg). at present receptors of the peptides have not been identified. these studies have shown that the dimer has an indirect effect on hematopoiesis as a stimulator of cytokine production, while the monomer seems to act both as an antagonist of dimer action and is also able to directly inhibit early myeloid progenitor cells. the possibility that these two compounds have therapeutic efficacy in diseases involving a myelosuppressed bone marrow is indicated. there is evidence that basic fibroblast growth factor (bfgf) plays a role in the regulation of normal blood cell proliferation and differentiation. basic fgf is produced by and is a potent mitogen for human slromal cells. it is found in megakaryocytes and cells of the granulocyte lineage, in vivo, and it enhances megakaryopoiesis and myelopoiesis in human long term bone marrow cultures. it stimulates progenitor cell growth and augments the proliferation of progenitor cells when added in conjunction with other hematopoietic growth factors. in addition, it counteracts the suppressive effects of transforming growth factor beta. basic fgf also synergizes with stem cell growth factor to augment granulocyte macrophage colony stimulating factor stimulated progenitor cell growth. based on the observations in normal hematopoiesis, the role of bfgf in malignant bematopoiesis is presently an area of active investigation. scf is one of the earliest-acting hematopoietic growth factors. pre-clinical studies have demonstrated its multi-lineage hematopoietic effects. we have conducted a phase i trial of scf and report now on the first 21 patients (pts) with stage iiib (n=7) or iv (n=14) breast cancer. the study was designed to evaluate the toierabllity and biologic effects of scf. pts received scf prechemo (cycle o) and following cycles 2-6 of c/a chemotherapy. cohorts of 5 lots were randomized in a 4:1 ratio to receive either scf (n=17) by subcutaneous injection at dosages of i 0, 25, and so/~g/kg/day for 14 days or no scf as a parallel c/a control group (n=4). various physiologic, biochemical, pharmacokinetic, and hematologic parameters were studied. bone marrow (bm) and peripheral blood (pb) progenitors were assayed. scf administration was associated with pb progenitor mobilization in cycle 0 at all dose levels. absolute neutrophil counts demonstrated modest doserelated increases over baseline ranging from median values of 20% (10 pg/kg/d) to 120% (so/jg/kg/d). no reproducible effects were seen on red blood cells or platelets. the principal adverse events were derrnatologic in nature. they included mild to moderate reactions at the injection site in all pts, and moderate to severe reactions distant to the site in i0 pts (primarily at higher doses). at 50 pg/kg/d, 4/10 pts experienced dose-limiting respiratory symptoms including cough, hoarseness, and laryngospasrn. because of scfs known mast cell effects, prophylaxis with h1/h2 blockers and bronchodilators is being evaluated. no pts developed antibodies to scf. evaluation of effects following chemotherapy is ongoing. we conclude that scf is an active hematopoietin capable of stimulating production of bm and pb progenitor cells as well as peripheral neutrophils. g. trinchieri nksf/il-12 is a heterodimeric cytokine produced by monocytemacrophages, b cells and other accessory cells in response to various stimuli including bacteria and bacterial products. nksf/il-12 acts on t cells and nk cells inducing cytokine production, proliferation, and enhancement of cytotoxic activity. nksf/il-12 is a particularly efficient inducer of ifn-7 production, acting alone or in synergy with other ifn-7 inducers such as il-2, antigens, anti-cd3 or anti-cd16 antibodies, mitogens, and phorbol diesters. nksf/il-12appears to play a major role in regulation of natural resistance: when produced by monocyte-macrophages, it directly activates the cytotoxic activity of nk cells and induces both nk and t cells to produce ifn-7 and other cytokines with important effects on activation of phagocytic cells. the important role of nksf/il-12 in response to bacterial products is clearly demonstrated by the ability of anti-nksf/il-12 antibodies to inhibit in vivo in mice the ifn-y production induced by lps ii~ a toxic shock model. nksf/il-12 is also an important factor in the regulation of adaptive immune response, by inducing the differentiation and growth of t helper cells type 1 (th-1) and by preventing the differentiation of il-4 producing th-2 cells. a possibly obligatory role of nksf/il-12 for th-1 cell differentiation can be demonstrated in vitro in the human lymphocyte response to allergens or bacteria-derived antigens and both in vitro and in vivo in the murine system, e.g. in the immune response to leishmania infection. production of nksf/il-12 by accessory ceils is stimulated by ifn-7, a product of th-1 cells and suppressed by il-10 or il-4, products of th-2 cells. these results suggest that nksf/il-12 represents an important link between natural resistance and adaptive immunity and is at the center of a cytokine network that regulates the equilibrium between cellular and humoral immunity. work with alpha-interferon in cml started at mdacc in 1981 and has evolved over the years to allow us interpretation of response rate in a large number of patients with relatively long follow up. newly diagnosed patients (<i year from diagnosis) were treated with a starting dose of 5 mu/m 2 of interferon. of 313 such patients followed in 6 different studies, we noted a 77% complete hematologic remission; 56% cytogenetic response, and 35% complete and partial cytogenetic response (<35% phi). in long-term analysis of 190 patients, 101 achieved cytogenetic response (53%) of which 49 (26%) were complete; 14 (7%) were partial, and 38 (20%). were minor 56 (55%) of the cytogenetic responses are durable. of major interest is the fact that 92% of the complete cytogenetic remissions are durable (45 of 49) but only a minority of the partial and minor cytogenetic responses are durable (36% and 16%, respectively). this clearly set our goal on improving the incidence of complete eytogenetic remission. median survival ranges between 60-65 months. however, of particular interest is the excellent survival rate of the complete cytogenetic responders (46 of 49 alive at time of follow-up). current studies are focusing on idealizing combination therapies and will be discussed. cml is a paradigm of tumor progression; therefore, we were looking for characteristics associated with disease progression and identified evidence of autocrine growth factor loop formation with disease progression and the overproduction of il-ib. finally, we are also focusing on the study of minimal residual disease in patients with complete cytogenetic remissions and have identified residual ph i progenitor cells in the majority of these patients, including patients with unmaintained remission. the nature of such a remission will be discussed as well. the mxa-protein: a new and excellent marker for endogenously produced type-i-ifn d.jakschies 1, k.u.nolte 1, r.zachoval 2, h.deicher 1, and p.v.wussow 1 the human mxa-protein is a recently identified human ifn-induced protein, which is synthezised by peripheral blood cells specifically and dose-dependently upon stimulation with type-i-interferons both in vitro and in vivo. mononuclear cells (mnc) both from healthy persons and cancer patients (n=55) are usually (in the absence of a virus infection) mx-negative. patients starting an ifn-a-therapy become mxa-positive (n=96) within 24 hours and remain positive throughout their therapy. furthermore, two groups of patients are known to have frequently measurable serum ifn-titer: sle-patients and aids-patients (n=76) studied expressed significant mxa-levels in their mnc. while all patients with measurable ifn-titer had mxa-levels above 1 u/20.000 mnc, also the vast majority of serum ifn-negative patients possessed detectable mxa-levels indicating that also these patients produce endogenous ifn, although due to its rapid clearence from the circulation it may not be measurable in serum. in addition, 55 patients with acute hepatitis were investigated for the mxa-protein. surprhingly large difference in the endogenous tfnproduction were observed. only in the first two weeks after the onset of clinical symptoms hepatitis a patients (n=21) had both high mxaprotein and 2-5-a-synthetases levels, but not thereafter. the levels of both ifn-induced activities reached concentrations found in cancer patients treated with 3-5-mill. iu rifn-c~ per dose. in acute hepatitis b and c (n=20;n=14) only marginally elevated mxa and 2-5-as levels were found. in summary, the mxa-protein is a new and excellent marker for endogenously produced type-i-ifns. m.e. rybak interleukin 4 (il-4) is a cytokine with pleiotropic immunomodulatory activities. these actions include b cell stimulation and co-stimulation, enhancement of il-2 mediated generation of cytotoxic t cells, increase in the expression of hl-a class ii antigens by monocytes and the inhibition of the production of a number of monokines including il-6, il-8 and tnf~. human il-4 has been cloned and expressed in e. coli. the mature protein has 129 amino acids, mw 14,957 d. studies of rhuil-4 in malignancy were prompted by i__nn vitro data demonstrating the ability of il-4 to inhibit the proliferation of chronic myelomonocytic cells, non-hodgkin's lymphoma and myeloma cells in culture, and to inhibit the il-2 induced proliferation of chronic lymphocytic leukemia. inn vivo models utilizing murine il-4 demonstrate an immune response to tumors (e.g lung, sarcoma and melanoma) following systemic administration of il-4 or the administration of tumor cells transfected with the il-4 gene. initial phase i trials of rhuil-4 have been completed in 57 patients. forty-seven patients received daily subcutaneous (s.c.) rhuil-4, 0.25-5.0~g/kg. side effects seen in ~ 10% of patients included headache, fatigue, fevers (<38~ myalgias and reversible elevation of sgot, sgpt and alkaline phosphatase. nausea, pedal and periorbital edema, and rhinitis were also seen. the mtd for rhuil-4 was approximately 2~g/kg/d s.c. activity has been seen in hodgkin's disease, non-small cell lung cancer, chronic lymphocytic leukemia, and multiple myeloma. current studies include phase ii investigations in advanced non-small cell lung cancer, refractory hodgkin's disease, and relapsed multiple myeloma. an update on these studies will be presented. this lecture will review the evidence concerning the possibility that sustained overstimulation by growth factors might provoke or accelerate myeloid leukemia development. the regulatory factors likely to be relevant are the four colony stimulating factors although a possible role for scl and il-6 probably also needs consideration. in general, prolonged overstimulation by growth factors in the mouse 3 either using transgenlc mice or engraftment of hemopoietic cells engineered to produce growth factors leads to hyperplasia but not leukemia. in contrast insertion of csf genes into immortalized, but non-leukemic5 cell lines leads to prompt leukemic transformation. if fdc-pi cells are engrafted in preirradiated animals, these can undergo delayed leukemic transformation often by activation of csf genes by inserted ~ particles. normal cells can be transformed to leukemic cells by retroviral insertion of a combination of a csf gene with fox 2.4, the apparent function of the hox 2.4 being to dysregulate the process of self-generation in early hemopoietic precursor cells. thus two distinct changes appear to be necessary for leukemic transformation -abnormal self-generation and an acquired capacity for autocrine growth factor production. all the above studies involve autocrine growth factor production but we have recently completed a study indicating that sustained stimulation by exogenous growth factors can also accelerate leukemic transformation. this study involved fdc-pi cells engrafted in gm-csf transgenic mice. again the actual transformation process often involved rearrangement of csf genes with autocrine csf production. the latter experiments raise the potential clinical hazard of prolonged csf treatment in myelodysplastic patients but this possibly should not be overinterpreted. since granulocyte (g-csf) and granulocyte macrophage (gm-csf) colony stimulating factors were first introduced in to clinical trials in 1987, there have been a vast number of clinical trials demonstrating the utility of these agents in reducing nentropenia, and therefore infection in patient groups ranging from congenital nentropenia to bone marrow transplantation. the discovery that a large number of progenitors are released into the peripheral blood by these agents either alone or after chemotherapy was not one of the major indications predicted in 1987. the rapid reconstitution of both nentrophils and platelet counts when these peripheral blood progenitors (pbpc) are used as autologous rescue after myeloblative treatment, is encouraging many centres to use this type of rescue instead of abmt. interesting studies are at present indicating that these pbpcs can be expanded in vitro prior to clinical use. attention is now moving to the stimulation of platelet productions with il-3, il-6, and 1l-11. there is increasing evidence from experimental haematologn/ that combinations of hgfs give optimal stimulation often of more primitive cells than previously thought. clinical studies are now being performed, but the logistic problem of testing all the possible comb'mations is daunting. as the use of haemopoietic growth factors successfully protecta the patients against haematological problems, other organs such as the gi tract, become dose limiting, often keeping the patient in hospital after haemopoietic recovery. repeated high dose treatments also deplete the haemopoietic stem cells. great interest is now being found in protecting the normal haemopoietic and other organ stem cells from the effects of radiochemotherapy. this may be possible using molecules such as mip 1 alpha or tgf beta. other workers have used the timing of the use of stimniatory hgfs to induce quiescence of the haemopoietic stem cells. the optimum use of hgfs would probably be to in sequence, prime the haemopoietic progenitors, then protect them from radiochemotherapy, followed by rapid expansion under the influence of probably a mixture of hgfs. crc deft of medical ontology, christie hospital, wilmslow road, withington, manchester, m20 9bx. il-3 is the first cytokine available for human use which was predicted to have both a activity on early hematopoietic progenitor cells, as well as specific action leading an increase in the number of circulating platelets. phase i/ii clinical trials have confirmed these predictions and have demonstrated, in addition, moderate activity on the late myeloid cell lineage leading to increased numbers of circulating neulrophils and eosinophils, events predicted from monkey studies, but not from in vitro and rodent studies. phase i studies provided a clear definition of the maximum tolerated dose (10 #g/kg daily iv or s.c.), since at 15/lg/kg an unacceptable frequency of severe headache, flushing and tissue swelling were observed. at doses between 2.5-10 #g/kg, depending on the bone marrow reserve, cytotoxic chemotherapy and malignant disease, il-3 prevented or reduced the severity of neulropenia and thrombocytopenia, reduced the number of platelet transfusions needed and allowed adherence to myelotoxic chemotherapy. in combination with other cytokines, such as gm-csf or g-csf, enhanced myeloid responses were seen including increased numbers of peripheral blood progenitor cells. these observations have led to now ongoing phase iii clinical trials using il-3 in conjunction with cytoxic chemotherapy in several malignancies and in conjunction with autologous bone marrow transplantation. there appears little doubt at present that il-3 will be an important contribution to management of the patients with oncologic or hematologic diseases. extensive phase ii studies have shown a reduction in the severity and duration of neutropania when filgrastim (r-methug-csf) is administered soon after chemotherapy. two phase iii studies demonstrated that filgrastim, given to small cell lung cancer patients from the day after chemotherapy, reduced the incidence of febrile neutropenic episodes by approximately 50% (crawford et al, 1991 ; trillet-lenoir et al, 1993) . these studies administered filgrastim from the day after chemotherapy, and they suggest that filgrastim is highly effective when used as an adjunct to chemotherapy to prevent fever and neutropenia. in a recent study (maher el al, in press), pilgrastim administration was delayed until after the onset of fever and antibiotic therapy. patients enrolled in lhis study were a median of 10 days after their last dose of chemotherapy. it was found that this later commencement of filgrastim could significantly accelerate neutrophil recovery, produce a shorter duration of febrile neutropenia, and decrease the risk of prolonged hospitalization; however, maximal benefit appears to be derived from adjunctive use of filgrastim prior to the onset of fever and neutropenia. from in vitro and animal studies it is known that il-6 is a stimulator of thrombopoiesis, a stimulatory factor for t and b cells, that it stimulates hepatocytus and can have antitumor activity. an ongoing phase i-ii study with rhll-6 before and after chemotherapy (ct) is performed. patients (pts) with breast cancer or non-small cell lung cancer receive e. coil derived rhll-6 (sandoz, basle) in doses of 0.5, 1, 2.5, 5 and 10 #g/kg/d, with > 3 pts per dose step. before ct rhll-6 is given dl iv and d2-7 sc. ct (mitoxantrone 10 mg/m 2 and thiotepa 40 mg/m 2, iv dl) starts d15, q 3 wks. after ct rhll-6 is given d4-15 se in all cycles (c). before ct 16 pts were treated. rml~ related side effects were: fever who gri-ii (100%), headache (56%), myalgia (25%), local erythema (100%) and starting at 2.5 #g nausea (56%), increase of liver enzymes who gri-ii (44%) and anemia (33%). symptoms were controllable with acetaminophen (n=16) and antiemetics (n=2). upto 10 #g them was a dose response related platelet increase compared to dl (r=0.75, p<0.002), with highest count after rml-6 cessation. at 0.5 /~g max. increase was 25.7+5.6 (se), at 10 ~g 114.0+38.0%. on d3 a leukocyte increase compared to dl was observed starting at 2.5 #g upto 10 /~g (dl 5.80+0.51, d3 8.76+0.45, p<0.001) mainly due to neutrophil increase (p<0.001). at d8 increase of lymphocytes occurred compared to dl (dl 1.02+0.19, d8 1.24+0.19, p<0.05). there was a reversible hemoglobin reduction especially at 5 and i0 ,gg, starting d3 with a max. decrease at dg (5/zg 24.0+7.4 g/l, 10 #g 23.3+5.5 g/l), at d15 initial values were almost reached without intervention. a dose related acute phase response was observed, with max. value of crp (mg/l) 67â�¢ at 0.5 /zg and 602â�¢ at 10 /tg (r=0.87, p<0.002), and max. value of serum amyloid a (rag/l) at 0.5 /zg of 258â�¢ and at 10 #g 838+143 (r=0.80, p<0.002). after ct 2 c (no c=22) were evaluable upto dose 5#g, in these e rhll-6 related side effects were: fever (82%), headache (73%), myalgia (27%), local erythema (68%), nausea (27%) and increase in liver enzymes (14%). uptu c2 there was no difference in platelet nadir, moment and duration of platelets <100 x109/l between 0.5-1 /~g and 2.5-5 #g doses. the same was observed for leukocytes. in conclusion, rml-6 upto 10 #g/kg/d has a acceptable toxicity profile and results, before ct, in dose dependent platelet and acute phase proteins increase. after ct, evaluable now for the first cycle, there is a faster platelet recovery at higher dose steps. university hospital groningen, division of medical oncology, department of internal medicine, oostersingel 59, 9713 ez gronlngen, the netherlands. g-csf in acute lymphoblastic leukemia. d.hoelzer, o.ottmann rhg-csf given in acute lymphoblastic leukemia (all) after induction/consolidation therapy or allogeneic/autologous bmt can shorten the recovery time of neutrophils by 4-9 days, with reductions of the infection rate in some studies. granulocytopenia is the major dose limiting toxicity not only after but also during chemotherapy. for example, severe neutropenia occurs in 66~ of patients during the intensive 8 wk induction regimen of the german all trials. therefore, the effect of parallel administration of g-csf (filgrastim) and chemotherapy was explored, rhg-csf (5~g/kg) was given concurrently with cyclophosphamide, arac, 6-mercaptopurine, prednisone and prophylactic cranial irradiation, rhg-csf was continued until neutrophil counts exceeded 1000/~i, but for at least seven days following the last dose of chemotherapy. in a pilot study of 15 pts, the median duration of granulocytopenia < 500/~i was 6 days compared to 12 days in a historical control group. when rhg-csf was applied throughout the entire 8 wk induction treatment (scherrer et al) in 14 pts, the cr rate was 93%, the tim~ of nm,trophil reeoverv to > ~o~l~l after the end of chemotherapy was reduced from 29,5 days to 14,5 days, and the infection rate was lower in the g-csf treated patients. from these studies it was concluded that the concomitant administration of rhg-csf and intensive chemotherapy in adult all is leasable, well tolerated and without major side effects; in particular, there was no evidence of prolonged neutropenia due to stimulation of hemopoietic stem cells by g-csf and their subsequent elimination by chemotherapy. the clinical effects of rhg-csf given concomitantly to chemotherapy require confirmation in a randomized trial. in an ongoing randomized multicenter trial~ an interim analysis of 40 pts revealed a highly significant reduction of the total duration of severe neutropenia (< 500/~l) in the patients receiving g-csf. further analysis of the potential clinical benefits of this treatment modality is in progress. we performed a phase h trial to assess the ability of g-csf -mobilized pbpc to rapidly and completely restore hemopeiesis after high dose chemotherapy in the absence of bone marrow infusions, with selection for pbpc-only infusions based on yield of granulocyte -macrophage colony -forming cells (gm-cfc) after g-csf treatment. twenty-nine adults with acute lymphoblastic leukemia, non-hodgkin's lymphoma, or hodgkin's disease who were eligible for autologons bone marrow transplantation were treated. g-csf was given at 12 or 24 ixg/kg/d for 6-7 d and mononuclear cells collected by apheresis on days 5,6,7 or 4,6,8. yield of pbpc was assessed by assay for granulocyte-macrophage colony forming cells (gm-cfc). high yield was defined as total gm-cfc collected >30 x 104/kg. high dose bnsulfan (4mg/kg/d x 4d) an& cyclophosphamide (60mg/kgtd x 2(:1) were administered and hemopoiedc cells infusedâ�¢ and recovery of hemopoiesis mouitored. progenitor cell yield was high in 11 of 29 patients. patients given infusions of g-csf-mobilized pbpc, but without bone marrow infusion, experienced recovery of hemopoiesis in all cases. no patient given pbpc alone required bone marrow infusion in up to 24 months of follow up. kinetics of recovery of both the platelet and neutrophil counts were more rapid in the high yield (pbpc-alone) group than in the low yield group (pbpc pins bone marrow). pintelels recovered to >50 x 109/1 at a median of 11 days and neutrophils to >0.5 x 109/1 at 9 days in the high yield group, compared with 37 days and 10 days in the low yield group (p=0.0016 and 0.0308 respectively). fewer platelet transfusions were required in the high yield group (median 11 packs per patient vs. 39, p---0.0138). we conclude that in patients with a high yield of pbpc after g-csf therapy, infusion of g-csf-mobilized pbpc results in rapid and sustained restoration of hemopoiesis. use of g-csf mobilised pbpc to support multiple cycles of hdc for treatment of high-risk stage ii and iii breast carcinoma is now being examined in a phase 1i study pursued by our group. our data on pbpc moblisation without chemotherapy allows consideration of g-csf-mobilised pbpc for haemopoietie ceil allotmnsplantation and for gene therapy trials. our approach for high-dose (hd) chemotherapy is to first treat patients eligible for dose intensification with a standard dose chemotherapy (vip: vp26 = etoposide: 500 mg/m 2, ifosfamide: 4 g/m 2, cis-platinum: 50 mg/m 2) followed by the application of colony stimulating factors (g-csf, gm-csf or il-3 + gm-csf) in order to combine a regimen with broad anti-tumor activity with the recruitment of peripheral blood progenitor cells (pbpcs). pbpcs are reinfused into the patients after high dose intensification chemotherapy (according to underlying disorder: hd-vip, vic-e or beam). highest numbers of pbpc were recruited by the sequential administration of 11_-3 + gm-csf (median of 420 cd34 + cells/iji and 24.000 total progenitors/ml, including multilineage as well as megakaryocytic progenitors). csf-mobilized pbpcs include lineage negative cd34 + cells, 4-hc resistant precursors as well as long term culture initiating cells (ltc-ic). so far, a total of 47 patients were supported with pbpos and csfs following hd-chemotherapy. the period of severe neutropenia (<100//ji) as well as thrombocytopenia (<20.00o//ji) was reduced to a median of 4 or 5days respectively. in order to decrease the number of potentially contaminating tumor cells in the leukapheresis preparations, we have started to positively select cd34 + cells by an avidin-biotin immunoadsorption column (provided by r. berenson, cellpro, usa). in all these patients, recovery data were comparable to the patients who received unseparated cells. to provide sufficient numbers of pbpcs for repetitive use or in patients with low progenitor cell yield as well as to possibly avoid leukapheresis, we investigated the ability of hematopoietic growth factor combinations to expand pbpcs ex vivo. chemotherapy + g-csf recruited cd34 + cells from 18patients were enriched by immunoadsorption (purity: 89.2 -+ 4.8 ) and cultured in suspension. a combination of stem cell factor, erythropoietin, interleukin-16, il-3, and il-6 was identified as the optimal combination for the expansion of clonogenic progenitors. proliferation peaked at day 14 with a mean 190-fold increase (range 46-930) of clonogenic cells. interferon-gamma synergized with the 5-factor combination, whereas the addition of gm-csf or g-csf decreased the number of clonogenic cells. large-scale expansion of cd34 + cells in auto-iogous plasma supplemented with the 5-factor combination resulted in an equivalent expansion. our data indicate the feasibility of large-scale pro+genitor cell expapsion in cancer patients, starting from small numbers of cd34 ceils. given the pressures on health care resources new health technologies need to be assessed to establish whether their benefits justify their costs. g-csf is an important new technology that has profound impacts on cancer chemotherapy and patients outcome. as clinical studies show, the main economic benefits of g-csf come from * a significant reduction of hospitalisation during chemotherapy, * reduced expenditure on inpatient and outpatient i.v. and oral antibiotic treatment, * and reduced platelet and rbc transfusions. in addition, it is expected that a therapy with g-csf during cancer chemotherapy shows intangible and non-monetary benefits: * increase in the rate of treatment as intended (time, number of cycles) * increase of quality of life of patients * reduction of drop-outs * ability to intensify dosage recently coducted economic cost-cost studies show that the cost of managing neutropenia and infection and the cost of chemotherapy are lower for patients receiving g-csf than those who do not. future studies have to focus mainly on the quality-of-life cost-effectiveness of g-csf. the aim of this study is to evaluate the effects of recombinant human erythropoietin (rhu epo) on hematopoietic regeneration after allogeneic or autologous bone marrow transplantation. patients were randomized to receive either 100 u rhu epo/kg body weight or placebo as continuous intravenous infusion from day 1 after bmt until independence from erythrocyte transfusions for 7 days or until day 42. the randomization was performed per each center and stratified according allogeneic or autologous bmt and major abo-blood group incompatibility. at the time of the planned interim analysis with 205 patients treated, the time to erythrocyte transfusion independence after allogeneic bmt was shorter in group a (n = 52) than in group b (n = 55). after autologous bmt no difference between group a (n = 49) and b (n = 49) could be detected so far concerning time to transfusion independence or the number of transfusions applied, considering either erythrocyte or thrombocyte transfusions. there were no major differences in side effects between groups a and b. as of october 1992, the study was finished with a total of 329 patients. since 1978 164 rcc patients have been screened for entry into clinical trials and entered onto surveillance as first management policy until symptoms or serial radiology indicated tumour progression. 3+4(4%) have demonstrated unexplained "spontaneous" cr+pr(median duration 17 mths), and 7(4%) "stable" disease(median 28mths). none of 19 not nephrectomised regressed, while cr+pr was 11% of 40 nephrectomised at diagnosis of metastasis, 1% of 76 developing metastases 1-17 mths after nephrectomy and 10% of 29 developing metastases more than 18mths after nephrectomy. it was 12% of 47 with lung only verses 1% of 117 with other sites. possible evidence for relief of potentially immunosupressive influences have been demonstrated in 4 of 7 patients demonstrating unexplained "spontanous regression. study of hla class i and ii antigen abnormally on these tumours and their influence on tumour infiltrating lymphocytes will be reviewed. 80 patients seen to progress on surveillance were entered into treatment trials, the remainder being too old, sick or having bone or brain metastases needing radiotherapy. 2+6(10%) achieved cr+pr. 2+5 of 14(50%) of patients with lung only verses 0+1 of 66(1.5%) of those with other sites of metastasis. although these results suggest that no harm has come from a period of preliminary surveillance, the fact that the therapeutic benefits including durable complete remission from therapy are confined almost entirely to the good risk small volume asymptomatic patients makes it difficult to justify a policy of surveillance for such patients. auto bmt, as alternative of allo bmt, lack{ immunotherapical effect delivered by allo graft. however immune reconstiturion after auto bmt shows features recalling the post allo setting. futhermore in vitro and in vivo in rll2 is able to stimulate the proliferation and the lyric functions of nk and t cells, major effectors of this gvl effect. for these reasons from 1990, we have so far treated 41 patients with acute leukemia (aml-17;all:24) in cr1 with auto bmt followed with rll2. all conditioning regimen included tbi. median rime between diagnosis and bmt was r~.2 mths (4-9 mths).arll2 was started as soon as platelets reach 50x10~/1 and anl 0.5x10~/1. rll2 was given in 5 cycles of 5 days (c1) and 2 days (c2 to c5), starting on d1,d15,d29,d43,d5~. rll2 was given as a continuous infusion at a median of 16m iu/m~/d (12m-24m). no patient died of rll2 related toxicity. platelet toxicity was obvious during the starting cycle but did not impair the long term hematological recovery. immune stimulation was major and intense for both nk and t cells and both lak and nk activity (all p < 0.05). long term infusion conducts to privilegiate nk stimulation. with a median follow-up of 24 mths, relapse and survival probabilities are respectively 45% and 65% fo all and 27% and 89% for aml comparying favorably with historical control ; these data invited us to set up a randomised study in cr1 aml and all after auto bmt. started in 1991 in france, italy and england, 135 pts have been so far included. we have shown that local, inhalative il-2 application in combination with low dose systemic il-2 and ifna is highly effective and has low toxicity (j urol 147,344, 1992) . here we report about longterm follow-up from 4 to 28 month of an outpatient schedule in 17 patients with pulmonary metastasis of renal cell carcinoma. treatment protocol based on 5 time daily inhalation of il-2 (5 x 200.000 i j) combined with low dose i.v. il-2 (4x106 u/4 days every 2 weeks) in 5 patients or low dose il-2 s.c. injection of 100.000 u per day in 12 patients. all patients received 5 x 106 s.c. ifna three times weekly. median treatment duration was 17 month (range 1-24 month). toxicity of il-2 inhalation was low, no fever, no vasculary leakage. even after up to 24 month of continuous inhalative treatment no evidence for irreversible pulmonary damage due to il-2 induced immunomodulation occured in 17 patients. in pulmonary metastases 1 complete response (6 month), 9 partial response (4,5,8,13, + 18, 18, 24,24,24) and 7 stable disease (1,6,7,10 + 16, 20, 23, +) were achieved. 4 of 17 patients responded with nonpulmonary metastasis. overall tumor response considering both, pulmonary and nonpulmonary metastases was 1 complete response, 7 partial responses 6 stable diseases, 2 mixed responses and 1 progressive disease. while il-2 per inhalation had to be stopped because of grade ii toxicity in 1 patient only, ifna systemically had to be stopped in 10 of 17 and il-2 i.v. cycles in 5 of 5 patients. 13 patients are alive with median actual survival of all patients of 16.9 month which seems to be prolonged compared to risk factors. inhalafive treatment with il-2 combined with low dose systemic cytokines represents a highly valuable model for the low toxicity of lccal il-2 application, resulting in an effective long term treatment schedule with long term responses. recent clinical trials for the biological therapy of solid tumors have used recombinant human cytokines in combination with conventional chemotherapy. in patients with metastatic renal cell carcinoma, we established a 3-drug combination of subcutaneous recombinant human interferon-u (ifn-u), interleukin-2 (il-2) and 5-fluorouracil (5-fu) as outpatient regimen. treatment consisted of eight weeks each of ifn-~ (10 million u/m' x3 per week sq) combined sequentially with il-2 (5-20 million iu/m' x3 per week sq for four weeks) and 5-fu (750 mg/m 2 iv weekly for four weeks). among 39 consecutive patients treated, there were 6 complete (15.4%) and 12 (30.8%) partial responders, with an overall objective response rate of 46.2% (95% confidence interval, 30-63%). median response duration was calculated at 10+ months, and no relapse has occurred among complete responders. systemic toxicity was mild to moderate, with no severe 5-fu related mucositis or diarrhea. there were no dose limiting adverse effects of sq il-2 and no toxic deaths. in summary, this outpatient biochemotherapy was as effective as the most aggressive inpatient iv il-2 regimen; it appeared to significantly improve the therapeutic index in patients with metastatic renal cell carcinoma. medizinische hochschule, d-3000 hannover, germany metastatic melanoma: immunotherapy with ifna and il-2, dtic/ifna as second line treatment, ifne(/il-2 + dtic or cddp. u keilholz, c scheibenbogen, w tilgen, d maclachlan, p brossart, th m0hler, w hunstein. this abstract summarises our 3-year experience in the treatment of metastatic melanoma with sequential or combined chemo-immunotherapy. patients with progressing metastatic melanoma have been treated with ifna and il-2. 10 mill u/sqm ifn(~ were given s.c. on days 1-5, and a new decrescendo regimen of il-2 was used: lmg/sqm/6hours, followed by 1mg/sqm/12 hours, and 1mg/sqm/24 hours, and 0.25mg/sqrrv24 hours x 3. the current response rate is 33% (3 cr, 12 pr, 16 mr/sd, 14 pd). patients not responding to ifnedll-2 (sd and pd) were eligible for subsequent chemotherapy with dtic, 850 mg/sqm day 1, followed by ifna, 3 mill u/day 2-6. the response rate for this second line regimen is 18% (1 cr, 3 pr, 5 sd, 13 pd, n=22). using this sequential approach, the overall response rate in this cohort is 51%, and the median survival is 17 months. in preparation of a randomized trial comparing chemo-immunotherapy and immunotherapy a pilot study was performed. patients not responding to the standard ifnedll-2 regimen received a single dose of dtic, 850 mg/sqm (n=6) or cddp, 100 mg/sqm (n=7) on day one, followed by ifn~il-2 according to the identical protocol as previously without chemotherapy. in the case of cddp, grade 3 nephrotoxicity was observed in 2/7 patients. pharmacokinetics of il-2 were not influenced by previous chemotherapy, except in the patients with cddp-associated nephrotoxicity. induction of secondary mediators (tnfa, ifn'~, neopterin, scd25) by il-2 was not diminished by previous chemotherapy. 3 patients unresponsive to immunotherapy alone showed tumor shrinkage upon chemo-immunotherapy. conclusion~: with initial immunotherapy followed in nonresponders by chemotherapy a response rate above 50% is achieved. combined chemoimmunotherapy is feasible, and the immunologic response to il-2 is not diminished by previous chemotherapy. a randomised trial is necessary to determine, whether combined chemo-immunotherapy is superior to immunotherapy alone. inflammation is characterized by an accumulation of leucocytes at the site of injury. it hab been well established that the disease associated lesions are caused by a plethora of soluble mediators released by both the infiltrated leukocytes as well by tissue cells, including many degrading enzymes, lipid mediators, reactive oxygen species, or cytokines. among those, cytokines play a crucial role in the inflammatory process as their release appears to represent the initial response which mediates the secretion of subsequent effector molecules responsible for the pathophysiological effects (e.g. changes of vascular resistance) in an autocrine or paracrine fashion. this holds true for the acute inflammation during which circulating granulocytes or monocytes are activated directly by a noxious agent; an example being the pivotal function of tumor necrosis factor (tnf) in septic shock. chronic inflammatory diseases are initiated and perpetuated by immune reactions. cytokines represent an important link between immune reactions and the recruitment and activation of blood borne infiltrating inflammatory ceils. thus interferon r and tumor necrosis factor 8, secreted by activated t lymphocytes, are potent stimulators of mononuclear phagocytes. the activated macrophages themselves secrete cytokines such as interleukin-t (il-t), tnf a or interleukin-8 (il-8) which have been termed collectively as inflammatory cytokines, il-1 and tnf again in an autocrine or paracrine way upreguiate the synthesis of the ultimate pathogenic mediators in macrophages-e.g. the secretion of degrading enzymes as in rheumatoid arthritis-. equally important they induce functional changes in vascular and autochthonous tissue cells which are thus recruited to contribute to the inflammatory lesions. il-8, and similarly the lymphocyte derived rantes, chemotactically attracts to the site of lesion and activates granulocytes, which then participate in the inflammatory reaction. while this rapidly recruits circulating phagocytic cells, the secretion of colony stimulating factors by activated t lymphocytes and macrophages also increases the pool of granulocytes and monocytes by stimulating their synthesis in the bone marrow. in turn il-1 an tnf a synthesized by monocytes (and also some tissue cells) are coactivators of t lymphocytes, and thus contribute to al local immune reaction. besides this general role of cytokines in inflammation their involvement in specific situations becomes increasingly apparent, an important example being the allergic inflammation. cytokines synthesized by t lymphocytes such as interleukin-4 or interleukin-5 not only regulate immunogtobulin synthesis of b lymphocytes, including ige, but equally important modulate the functions of mast cells or eosinophilic leukocytes to become effective effector cells of allergic reactions. the detailed functions of cytokines in different acute or chronic inflammatory diseases will be discussed in the subsequent contributions. clinical results e holier, r hintermeier-knabe, b ertl, hj kolb, m kaul, u behrends, s thierfelder, w wilmanns experimental as well as clinical studies suggest dual involvement of proinflammatory cytokines as tnfalpha and ill in complications and gvhd following allogeneic bmt: nonspecific activation of recpient tissues during pretransp~ant conditioning results in early release of tnfa which strongly enhances donor t cell activation; in the effector phase, t cell stimulation is amplified by ifngamma and lps mediated release of tnf and ill. the role of these mediators as critical effectors of gvhd related tissue damage could be confirmed by application of cytokine antagonists early after murine bmt: both, anti-tnfa and ill-receptor-antagonist (illra, as shown by j ferrara, boston) resulted in a >50% reduction of gvhd related mortality in mice, while pentoxifylline proved to be ineffective. in human bmt, phase l/ll studies analyzing anti-tnfa and illra in refractory gvhd report substantial improvement in 50 -70% of patients, though reoccurrence of symptoms after cessation of treatment in most patients indicates a late effector function of cytokines at least in advanced gvhd. contribution of tnfa to recipient related induction of gvhd is further suggested by a recent phase 1/11 study performed in our institution as prophylactic application of anti-tnfa during pretransplant conditioning suppressed occurrence of early gvhd in high risk patients as compared to historical controls. though these data indicate a future role of a variety of cytokine antagonists in management of clinical complications of bmt their impact on long term survival has to be evaluated and compared to classical strategies (e.g.the use of corticosteroids and t-cell-manipulation) in randomized studies. the cytokines il-1 and tnf play central roles in inflammatory responses which can lead to tissue injury, naturally occurring antagonists such as soluble cytokine receptors or receptor antagonists are also produced during inflammatory responses. these soluble receptors and antagonists may act as a buffer system to reduce and limit tissue injury induced by cytokines. however, in some disease states this naturally occurring buffer system may not be sufficient to inhibit the detrimental actions of an inflammatory response. responses to il-1 are mediated via the type 1 receptor (il-1r, type i). the type ii receptor (il-1r, type ii) has never been shown to signal, but instead appears to be shed as an il-1 antagonist. both recombinant soluble il-1r, type i an il-1r, type ii are capable of binding il-1 and inhibiting responses in vitro. in a phase i clinical study evaluating soluble 1l-1r (type 1) in modifying cutaneous allergic responses, il-1r was a potent inhibitor of allergen induced latephase inflammation in the skin, with a high safety profile. responses to tnf are mediated via the ps0 or pt0 tnf receptor. soluble forms of both the p80 and 1960 tnfr occur naturally and are increased in many inflammatory states. dimeric constructs of the p80 tnf have been engineered to form a soluble fc fusion protein with two monomeric tnfr extraceuular portions contained on a truncated ig heavy chain (tnfr:fc). the tnfr:fc possesses higher affinity than a monomeric soluble receptor and the ig-like fc structure imparts a longer half life. animal studies have shown that soluble il-1r, type i and tnfr:fc are effective antagonists of inflammation. in animal models for arthritis and pulmonary inflammation, il-ir and tnfr:fc reduced inflammatory celt infiltration and tissue damage. both molecules are currently in clinical trials and hold promise for treatment of autoimmune and allergic diseases such as rheumatoid arthritis and asthma. recently, evidence was raised that pentoxifylline (pof} is able to suppress the synthesis of tumor necrosis factor-alpha [tnf} in cell cultures, in vivo, and to protect experimental animals against endotoxin shock: it was found that pof selectively inhibits the formation of tnf but not interleukin-6 (il-6]. we could confirm these pharmacological effects of pof in humans under controlled conditions of endotoxlnemia. ten healthy volunteers recieved a bolus injection of endotoxin [100 ng of lps of s.a.e.], which caused a transient increase of circulating tnf and il-6. 3 weeks later the voluntecrs were again injected with endotoxin and pof was also infused. due to pof administration, there was no rise in circulating tnf, whereas il-6 levels rose in parauel with body temperature, comparable to those seen in the first part of the study. treatment of allograft transplant recipients with the murine anti-cd3 monoclonal antibody okt3 leads to an acute cytokine release syndrome. especially tnf seems to play a pivotal role in the pathophysiology of the okt3 first-dose reaction. pretreatment of 8 patients with pof prior to okt3 administration was able to reduce the endogetlous tnf formation significantly as compared to controls and, thus, prevents severe clinical side effects, whereas il-6 formation and febrile response were not affected. severe pulmonary tuberculosis is associated with a chronic cytokine release syndrome [elevated levels of circulating tnf and il-6]. in 8 patients pof treatment inhibited chronic tnf release selectively, and, thus, reduced tnf-dependent caehexia without affecting chronic il-6 formation and related symptoms, such as fever and night sweat. in conclusion, we suggest that pof may improve therapeutic strategies in cases of acute and chronic cytoklne release syndromes. tumor necrosis factor (tnf) plays a central role in the maintenance of the inflammatory events in rheumatoid arthritis. we evaluated the expression of p75 and p55 tnf receptors (tnfr) and of tnf-a and tnf-i~ on the surface of synovial fluid mononuclear cells in patients with rheumatoid arthritis (ra) (n = 9), spondylarthropathy (spa) (n = 11 ), and traumatic effusions (n=3). synovial t-lymphocytes from ra patients express in all cases the p75 tnfr on the cell-membrane, in 4/9 cases also a weak expression of the p55 tnfr is detectable, both mrnas can be detected by polymerase chain reaction (pcr). synovial macrophages also express the p75 tnfr and low amounts of the p55 tnfr. patients with active ra also have circulating p75 tnfr positive t-lymphocytes in their blood. high concentrations of soluble tnfr (stnfr) are found in the joint effusions of ra patients: up to 40 ng/ml of p75 stnfr and ~jp to 54 ng/ml p55 stnfr. significantly lower stnfr levels are found in spa effusions. both receptors are also more elevated in the serum of ra patients (2.59 _+ 0.28 ng/ml p75 stnfr and 4.49 + 0.55 ng/ml p55 stnfr) as compared to spa patients (1.41 _+ 0.09 ng/ml p75 stnfr and 1.78 + 0.08 ng/ml p55 stnfr, p < 0.001). tnf-a could be detected in the synovial fluid of ra patients (up to 140 pg/ml), but not in the serum. the soluble tnfr are biologically active and neutralize the effects of tnf-a in a cytotoxicity assay. the high levels of soluble tnfr in the inflammatory effusions may reflect tnf neutralizing activity in an environment where enhanced tnf synthesis has occured. we have generated several anti-sense-tnf-a oligonucleotides (as-tnf), in order to down-regulate tnf biosynthesis at the mrna level. with as-tnf-3 we could achieve more than 90% inhibition of tnf secretion in pha-stimulated peripheral blood or synovial fluid lymphocytes. the effects of as-tnf-3 on the expression of tnfr and on the synthesis of other cytokines are currently being investigated. to investigate the effects of g-csf (r-methug-csf, aragon, thousand oaks, ca) on neutrophil production, blood distribution, survival and function, daily subcutaneous injections of g-csf were administered to healthy, young (y) (20-30 years) and healthy elderly (o) (70-80 years) subjects for 14 days at three dose levels, 0 mcg (n=7), 30 mcg (n=10), and 300 mcg (n = 10). daily cbcs and serial measurements of neutrophil oxidative activity by chemiluminescence were made. in addition, blood neutrophil kinetics were determined (day 6), transit time of the marrow neutrophil post mitotic pool (ntt) (days 7 to 14), neutrophil migration to skin chambers (days 0 and 5), and blood colony forming cells (cfu-gm) (day 0 and 5), as well as routine marrow morphology studies (days 0, 5 and 15) were performed. baseline neutrophil counts were similar in the y and o subjects and counts increased similarly for the two age groups, with peak counts of 51.6 --+-6.7 for the 300 mcg dose. g-csf significantly shortened the ntt from 5.7 4-1.6 days (control) to 4.2 4-0.3 days (30 mcg) and 2.9 4-0.4 days (300 mcg) (p<0.05). a concomitant of the shortened ntt was a dose dependent increase in the chemiluminescence responses, reflecting higher myeloperoxidase activity. the shortened ntt was also reflected by a decreased proportion of marrow cells in the post mitotic pool (metas, bands and pmns) and apparently lengthened blood half life of the circulating pmns. neutrophil migration to cutaneous inflammatory sites was also decreased in a dose dependent fashion. comparison of the age groups showed the only significant difference to be in the mobilization of blood colony forming cells with blood cfu-gm increasing 80 fold in the young versus 40 fold in the elderly over 5 days (300 mcg). no significant toxicities were observed in these normal subjects. these studies demonstrate the dose dependent stimulation of neutrophil production with g-csf administration, which is not affected by the age of the subjects. the neutrophils released into the peripheral blood have enhanced oxidative responses but somewhat reduced migratory capacities, probably reflecting the accelerated production and early release of the developing neutrophils. these changes are remarkably similar to changes in neutrophil production and function observed with bacterial infections in hematological normal individuals. the systemic regulation of the host response during acute inflammatory states remains poorly understood. among the regulatory systems that are likely to play a role in controlling host responses to bacterial infection is the neuroendocrine axis. the pituitary for example, is ideally situated to integrate central and peripheral stimuli and among other effects initiates the systemic increase in glucocorticoid production that accompanies host stress responses. we studied the secretory response of the murine pituitary cell line att-20 in vitro and whole pituitaries in vivo after endotoxin (lps) stimulation. we identified macrophage migration inhibitory factor (mif), a previously described t-cell cytokine, as a major secreted protein of att-20 cells that were stimulated by sub-nanogram amounts of lps. to study the expression of pituitary-derived mif in vivo, balb/c mice were injected ip with sub-lethal amounts of lps. pituitaries and serum were collected at intervals and pituitary mif mrna and pituitary and serum mif protein were measured. pituitary mif mrna specifically increased with time and reached a plateau 16-24 hr after lps challenge. mif protein, which is present constitutively in the pituitary, decreased within 20 hr. determination of serum mif in normal, athymic and hypophysectomized balb/c mice indicated that the pituitary is an important source of serum mif that appears in the post-acute phase (6-20 hr), whereas t cell mif contributes primarily to the hyper-acute rise in serum mif (2 hr). these data suggest that mif plays a central role in the systemic response to endotoxin and that pituitary mif is likely to reflect the interplay of diverse neurohumoral stimuli that regulate acute and chronic inflammation. pretreatment of mice or rats with granulocyte colony-stimulating factor (g-csf) protected against endotoxin-induced lethal shock or against endotoxin-induced liver injury in galactosamine-sensitized animals. in the animals protected by such pretreatment, the systemic tumor necrosis factor 0r (tnf) bioactivity was significantly suppressed. various macrophage populations taken ex vivo from g-csf-pretreated animals showed an attenuated tnf release following lps stimulation. however, g-csf had no such effects on macrophages when directly added in vitro to the cells. these findings show that g-csf protects against septic shock via tnf suppression in a way requiring the participation of additional circulating cells or factors. pretreatment of rodents with granulocyte macrophage colony stimulating factor (gm-csf) greatly enhanced the susceptibility of the animals to endotoxin and led to a tremendous increase of the systemic tnf found following an lps challenge. an anti-gm-csf antibody significantly protected animals against septic organ failure. considerable amounts of endogenous gm-csf are released following endotoxin chaitenge with a maximum at 2 h. the enhancement of lps-inducible tnf release from rnacrophages takes also place in vitro. it is concluded that gm-csf is a pivotal mediator of sepsis as well as a directly acting enhancer of lpsinducible tnf release. we have previously demonstrated that protein production and mrna expression of gm-csf, g-csf, and il-3 were decreased in activated mnc from umbilical cord (c) compared with adult (a) peripheral blood (cairo, et al, pediatr res 30:362, 1991, and 31:574, 1992) . rhg-csf + recombinant rat stem cell factor (rrscf) administration in newborn rats has, however, resulted in a significant induction of neutrophilia, an increase in bone marrow post-mitotic pool, and is synergistic with antibiotics during experimental group b streptococcal sepsis (cairo, et al, blood 76:1788 , 1990 , and 80:96, 1992 . to assess the toxicity and efficacy of rhg-csf in newborns with presumed sepsis, 42 nb <3 days (26-40 wks) with a diagnosis of presumed sepsis were randomized to either placebo (p), 1.0, 5.0, or 10.0 pg/kg q 24 hrs, 5.0 or 10.0/lg/kg q 12 hrs of iv rhg-csf x 3 days. cbc, differential, and platelet counts were obtained at time 0, 2, 24, 48, 72, and 96 hrs. tibial bone marrow aspirates were performed at 72 hrs and bone marrow nsp, npp, cfu-gm, and cfu-gemm were determined. serum for g-csf levels was obtained before and 2, 6, 12, 14, 16, 18, and 36 hrs after rhg-csf dosing and measured by a sandwich elisa assay. rhg-csf induced significant neutrophilia at 24 hrs vs. placebo (85 + 19%) following both 5.0 (203 + 40%, p<0.04) and 10.0 ijg/kg/d (351 -+ 89%, p<0.02). significant neutrophilia continued at 48 and 72 hrs at both 5.0 (p<0.03 and <0.013) and 10 h'g/kg/d (p<0.0s and <0.014) respectively. rhg-csf also significantly induced a dosedependent increase in the bm post-mitotic pool (p vs. 10 ijg/kg/d) (18 +-3.7 vs. 63 + 7.5%) (p<0.005). t1/z of g-csf in the nb was 4.42 _+ 0.44 hrs (r= -0.9) with peak g-csf levels occurring within 2 hrs. to date there has been no evidence of acute or chronic toxicity secondary to rhg-csf. future studies are required to determine the clinical implications of the biological efficacy of rhg-csf in newborns with presumed sepsis. children's hospital of orange county, orange, california usa depenbrock, t. block, h. vogelsang, ch. fellbaum, j. rastetter, a.-r. hanauske tgf-po is known to function both as inhibitory and stimulatory factor depending on the type of cell line investigated. the purpose of our study was to determine the effects of tgf-~ 1 and tgf-62 (concentrations: 0.1 -1 -10 ng/ml) on soft agar colony formation of freshly explanted i~uman tumors in vitro. of 89 specimens, 6 had to be excluded from further analyses (4 confirmed benign, 2 bacterial/fungal contamination). 50 of the remaining 83 tumors showed evaluable growth in control capillaries (60%). at 10 ng/ml, tgf-po 1 inhibited colony formation (_< 0.5 x control) in 12 specimens (24%): 5/19 renal, 4/6 non hodgkin's lymphoma, 1/7 breast, 1/2 ovarian, 1/2 melanoma (median: 0.37 x control, range: 0.22 -0.49). at 10 ng/ml, tgf-e,= showed inhibition of colony formation in 17 specimens (34%) with a similar spectrum of activity (median: 0.36 x control, range 0.06 -0.48). stimulation (colony formation _> 1.5 x control) was observed in only 1/50 specimens. combination of tgf-po 1 with epidermal growth factor (egf) reversed the inhibitory activity in 4/5 specimens (80%). combination of tgf-p~ with platelet derived growth factor (pdgf) reversed the inhibitory activity in 2/5 specimens (40%). similar results were observed for tgf z. we conclude that tgf-i~ 1 and tgf-i~ 2 inhibit a subgroup of freshly explanted clonogenic tumor cells. their activity, however, appears to be modulated by other growth factors. a korfel. z yon marschall. d ol~erbera. e thiel. and we berdel mip-i~ is a member of a family of proinflammatory cytokines produced by activated macrophages which has been shown to be a negative regulator of early hematopoietic stem cell progenitors. our group investigates the interactions between hematopoietic cytokines and non-hematopoietic malignant cells. here, we describe results testing rhmip-lc~ (rh stem cell inhibitor, sci; kindly provided by dr. wolpe, genetics institute, cambridge, ma, usa) on the clonal growth of different human non-hematopoietic tumor cell lines in vitro. cell lines tested included the following histologies: gtioblastoma 6x, neuroblastoma lx, head and neck carcinoma 2x, lung carcinoma lx, colorectal carcinoma 2x, gastric carcinoma lx, pancreatic carcinoma l x, breast carcinoma l x, bladder carcinoma lx, prostate carcinoma lx, choriocarcinoma lx, ovary carcinoma lx, osteosarcoma lx, melanoma 3x. mip-1 (0, 2, 20, 200 ng/ml) was tested in human tumor cloning assays (htca) in mixtures of methylcellulose and agar. htca has previously been shown to detect positive and negative growth control by cytokines. plating efficacy of the cells in the controls was > 2 % (median 8 %, range 2.2 -46.2 %) in this series of experiments: tumor cells were continuously exposed to the cytokine for the complete assay period. clonal growth of none of the celt tines was significantly and reproducibly stimulated or inhibited by mip-lm since mip-lc~ may enter clinical trials for indications such as protecting hematopoietic stem cells from damage caused by cytotoxic chemotherapy in tumor patients, further experiments should be performed in vitro and in vivo. dfg be 822/4-2 klinikum steglitz, freie universitaet berlin, berlin, germany clinical studies have demonstrated the activity of single hematopoietic growth factors (hgf) in restoring bone marrow function after chemotherapy. these studies have prompted clinical trials using multiple growth factors to promote the maturation of precursor cells at various stages of differentiation. however, hgf also have the capability to stimulate growth of non-hematopoietic tumor ceils at least in long-term cell cultures. we have assessed the growth-modulating activity of combinations of various hgfs on freshly explanted human tumor colony forming units in vitro. a total of 86 tumor specimens were exposed for 21 -28 days to il-3, gm-csf, g-csf, scf (a~l at final concentrations of 100 ng/ml) and il-113 (final concentration: 10 ng/ml) or combinations of these hgfs using a capillary soft agar cloning system. 54 specimens (63%) showed evaluable growth in control capillaries. stimulation of colony growth was observed in 17/1026 tests (1.7%) with 6/54 (11%) specimens expressing sensitivity to individual hgfs, 3/54 (6%) to combinations of two hgf, and 6/54 (11%) to combinations of more than two hgfs. inhibition of colony growth was observed in a total of 24/1026 tests (2.3%) with 1/54 (2%) specimens expressing marginal sensitivity to individual hgfs, 6/54 (11%) to combinations of two hgf, and 7/54 (13%) to combinations of more than two hgfs. for inhibitory effects, median colony survival for combinations > 2 hgfs was 0.46 x control (range 0.31-0.49). for stimulatory effects, median colony survival for combinations > 2 hgfs was 2.0 x control (range: 1.6 -2.5). our data indicate that combinations of hgf will not substantially alter the pattern of clonal proliferation of the majority of freshly explanted tumor cells in vitro. however, growth modulation may occur in a minority of tumors. klinikum rechts der isar der technischen universit&t m0nchen, ismaninger str. 22, 8000 m0nchen 80 supported by grants w41/88/ha-1-2 from the deutsche krebshilfe and 90.055.1 from the withe~m-sander stiftung is granulocyte colony-stimulating factor an angiogenic factor in human glioblastoma? s. corbacioglu, k. welte and t. pietsch granulocyte-colony stimulating factor (g-csf) belongs to a family of glycoproteins winch regulate growth, differentiation and function of hematopoietic ceils of the myelomonocytic lineage. in addition, g-csf induces migration and proliferation of endothelial cells in vitro. to investigate the effects of g-csf on vascularization of glioblastoma in vivo we transplanted the human glioblastoma cell line u-87 mg which is capable of producing g-csf in high amounts. after s.c. inocculation of these cells into the backs of athymic mice, they developped highly vascularized solid tumors. in order to block the effect of g-csf directly or to inhibit the production of g-csf by tumor cells neutrolizing monoclonal antibody (mab) 75a against g-csf or dexamethasone were injected intravenously. at 2-day intervals the tumor volumes were measured. after seven days the mice were sacrificed and the tumors were explanted. blood was collected for differential blood counts and serum was tested for g-csf. fresh frozen sections of the tumors were specifically stained for capillaries using bandeiraea (griffoina) simplicifolia lectin i isolectin b 4 (bsl b4). morphometric studies of the stained sections were performed in order to quantitate the vascularization of the tumors. the differential blood counts showed significantly increased neutropinls due to the effect of human g-csf produced by the glioblastoma cells. tins effect was inhibited by anti-g-csf mabs or dexamethasone. however neither g-csf mabs nor dexamethasone could inhibit tumm growth compared untreated tumor bearing mice. dexamethasone significantly decreased the tumor vascularization whereas anfi-g-csf mabs did not have any effects on tumor vascularization. these findings suggest that g-csf is not an essential angiogenic factor in vivo. pediatric hematology/oncology, medical school hannover, konstanty-gutschow-str. 8, 3000 harmover 61, germany michael martin, torsten spencker, karen welch*, and andreas strasser* the spgm1 cell line was established from a transplantable mouse progranulocytic/promacrophage tumor (d0hrsen u et al. 1989, leukemia 3: 796-803) . extensive phenotyping of this mouse progenitor line revealed the properties of a typical cd5 positive pre-b cell, spgm1 being positive for pb76, b220 (cd45r), and the pre-b cell immunoglobulin receptor complex, comprising p-heavy chains, xs-and vpree-surrogate light chains and the igma and igl~ co-receptor molecules. southern blot analysis revealed clonal rearrangement of the p-heavy chain locus and germline light chain loci. interestingly, spgm1 readily formed blast cell, macrophage and occasional granulocytic colonies in soft agar in the presence of interleukin 3 (il-3). in suspension cultures il-3 also induced macrophage differentiation, the cells becoming larger, adherent and independent of 2-mercaptoethanol in the culture medium. il-3 induced an initial burst of proliferation during differentiation, accompanied by loss of self renewal capacity, subsequently followed by a decrease and cessation of proliferation. the earliest changes were detectable at 24 hours by northern blot analysis. il-3 treatment increased mac1 mrna, induced cfms mrna (m-csf receptor) and down regulated mrna for p, x 5, vpree, and mbl. after 2 to 4 days the cells morphologically, phenotypically and functionally resembled macrophages, expressing strongly mac1 and f4/80, and phagocytosing latex beads (martin met al. 1993 j.immunol. in press). thus, il-3 induced the cd5 positive pre-8 cell line spgm1 to switch its differentiation program in a coordinate fashion from a pre-b cell to a macrophage. igf is known to be mitogenic for a variety of cell lines in vitro. we have studied the effects of insulin-like-growth-factor i [igf-i] and insulin-like-growth-factor ii [igf-ii] on 92 freshly explanted human tumors using a capillary soft agar cloning system. 6 specimens had to be excluded from further analyses (4 confirmed benign, 2 bacterial/fungal contamination). 51/86 specimens (59%) showed adequate growth in controls (17 renal, 7 breast, 7 lymphoma, 6 colorectal, 14 miscellaneous) with a median colony formation of 18.7 colonies/capillary (range: 4.2 -120.0). at final concentrations between 10 lr m and 10 .8 m, igf-i significantly stimulated colony formation (_> 1.5 x control) in 10/51 evaluable specimens (20%) with a median of 2.05 x control (range: 1.57 -3.22) and inhibited colony formation ( _< 0.5 x control) in 4/51 specimens (8%, median: 0.33 x control, range: 0.08 -0.49). igf-ii stimulated 8/51 specimens (16%, median: 2.66 x control, range: 1.50 -3.71 ) and inhibited 8/51 specimens (16%, median: 0.32 x control, range: 0.05 -0.47). the optimal concentration for stimulation was found to be 10 -~ m for igf-i and igf-ii. of 39 specimens not significantly stimulated by either igf-i (10 g m) or epidermal growth factor (egf, 10 1~ m), 4 (10%) were significantly stimulated by the combination of the two factors. 1139 (3%) specimen was stimulated by a combination of igf-ii (10 -~ m) and egf. we conclude that igf modulates the clonogenic growth of a subgroup of freshly explanted human cancer cells in vitro. the myeloid growth factors g-csf and gm-csf are increasingly introduced in therapy trials in neutropenic disorders and in mds. in a series of 4 therapy trials 12.5% up to 36.4% of patients treated with gm-csf displayed a stimulation of blast cells (antin 1988 , estey 1991 , ganser 1989 , vadhan-raj 1987 . recently, we could demonstrate a growth advantage for monosomy 7-cells in gm-csf containing bone marrow cultures (haase, 1990) which is supported by results from others (andreeff 1991). now, further data are available corroborating an association of monosomy 7 and leukemic blast stimulation by myeloid growth factors. in a cytogenetic follow-up study of 13 patients with mds under gm-csf (in preparation) we could observe a cytogenetic, clinical, and cytologic progression in a patient with monosomy 7 within 34 days. in a patient with kostman's syndrome, receiving g-csf, we performed sequential cytogenetic analyses. the patient's disease progressed to mds and finally to aml. he initially had a mosaic karyotypeof normal and monosomy 7 cells but displayed a rapid karyotype evolution with supersession of normal cells and gain of additional abnormalities. a recent publication from a japanese group adds further information to a possible association of monosomy 7 with stimulation of leukemia in 14% of neutropenic children treated with g-csf (kojima, 1992) . besides the need for further cytogenetic follow-up studies in growth factor therapy trials, data are accumulating that monosomy 7 is a risk factor in gm-csf and g-csf therapy. ii-3 is a cytokine with multilineage activity and stimulates proliferation of immature hemopoietic progenitors (yang, 1989) . recently, il-3 has been introduced in clinical trials in mds (dunbar, 1990; ganser, 1990) . as in gm-csf therapy one major concern attributes to an il-3 mediated stimulation of leukemic cells, since a rise in blast counts has been observed in two of 32 patients reported (ganser, 1992) . however, as yet it is not known whether the blasts stimulated belong to the normal or leukemic population. we performed cytogenetic analyses of bone marrow cultures with and without addition of i00 ng/ml il-3 (behringwerke, marburg). the influence of this cytokine on the clonal compostion in 7 patients (3 anll, 4 mds) with mosaic karyotypes was examined. in all 7 patients independent from diagnosis and chromosome abnormality the normal cell population was promoted by il-3 with different intensity. individual data are outlined on the the effect of stem cell factor (scf) on peripheral blood b-cll-celis were studied in vitro by bromodeoxyuridine/propidiumiodide (brdu/pi) cell-cycle-analysis. peripheral blood cells from 41 patients with b-cll were examined with cd-markcrs and prepared as follows: after ficoll centrifugation and lysis of monocytes by leucine-methyl-ester (lme) t-lymphocytes were depleted with a cd3 monoclonal antibody and magnetic cell sorting. the cells were grown in serum free culture medium (cg-medium) containing 10 #mol/l brdu, and 0.1 ng/ml up to 100 ng/ml scf. controls were grown without cytokines. samples were drawn repeatedly at 20, 68, 92, 116, and 140 hours. cell-cycle-analysis was performed after double dna staining with propidiumiodide and anti-brdu-antibodies and determinated by flow cytometry. minimal alterations were observed with t-cell depletion, the b-cll cells from 31 patients were stimulated by scf during the first 92 hours reaching a maximum of -2.77 % compared with the controls after 68 hours in cultures containing 100 ng/ml scf. on the other hand, cultures without t-cell depletion showed no effect for scf. we conclude that scf has only a minimal stimulatory, early effect in inducing the proliferation of b-cll cells. stem cell factor (scf) is known to promote proliferation of hematopoieticprogenitors and mast cells.however, the spectrum ot its biological effects is not completely understood. since scf may be able to accelerate hemopoietic recovery after chemotherapy or in other situations where severe immunosuppression is present, we were interested in its effects on t cells. thus, we have studied the influence of human recombinant scf on t cell proliferation invitro. when cultured for 6 days in serum-f~ee medium, 3h-thymidine incorporation of unstimulated peripheral blood mononuclear ee ells s (pmnc) resulted in 1480-+689 epm without growth factors, 1189+_119 clam with 11.-3 (50ng/ml), 2386+_1149 cpm with scf (lon~/ml), and 3828_+1660 c.m with both 11.3 and scf (n=fi~ addition of scf to one way" mixed lymphocyte cultures (mlc) increased thymidine incorporation by 27% (0-182%); addition of scf plus id3 increased thymidine incorporation by 324% (48-734%; id3 alone 0%, n=5). after depletion of monocytes and the majority of b cells from pmnc, the proliferation of the remaining eeu fraction, which consisted mainly of t cells, was not enhanced by id3, scf or il-3 + scf. we conclude that scf can promote proliferation of unstimtdated or allostimulated t cells in the presence of id3. since this effect requires monoeytes or other accessory ceils, a direct influence of scf on t cells does not become evident from our data. it is, however, still unclear whether ltb 4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. here we report that ltb4 induces synthesis of interleukin (il)-6 by human blood monocytee through transcriptional activation of the il-6 gene. we furthermore demonstrate that this process involves activation of the transcription factor nf-rb and, to a lesser extent, of nf-il6, white the activity of the transcriptior~ factor ap-1, shown to otherwise confer il-6 inducibility, appeared to be unaffected by ltb4. involvement of nf-~:b and nf-il6 in induction of il-6 transcripts by monocytes was demonstrated using deleted forms of the il-6 promoter. activation of the il-6 promoter by ltb4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional il-6 protein as well. in addition, ltb4 mediated transcactivation of a heterologous promoter construct containing the nf-~:b or the nf-il6 enhancer, but not the ap-1 enhancer. the signalling events mediating this effect appeared to involve the release of h202, since ltb4 failed to induce nf-rb or nf-il6 in the presence of the scavenger of h202, n-acetyi-l-cysteine. both interleukin-3 (il-3) and granulocyte-macrophage colony-stimulating factor (gm-csf) have been previously identified to induce rapid phosphorylation of the map-kinase (blood 79:2880 (blood 79: ,1992 . however, little is known about signalling events initiated by il-3/gm-csf which occur downstream of the map-kinase. map-kinase has been shown to phosphorylate the ap-1 transcription factor and to activate two kinases designated insulin-stimulated protein kinase-1 (ispk-1) and mapkapkinase 2. we here report that il-3 and gm-csf induce mapkap-kinase 2 activity in the human megakaryoblastic leukemia cell line mo7e and phos-phorylate the human small heat shock protein hsp27 on serine residues. in contrast, neutrophils failed to phosphorylate hsp27 upon il-3, while gm-csf induced hsp27 phosphorylation in a similar range as observed in mo7e cells suggesting that mapkap-kinase 2 mediated hsp27 activation occurs independently of proliferation. hsp27 phosphorylation is dose-dependent and occurs as early as 5 minutes following exposure to il-3 or gm-csf. moreover, hsp27 phosphorylation is inhibited by tyrosine kinase inhibitors such as genistein or herbimycin a. in addition, we show that protein tyrosine phosphatase and protein phosphatase 2a (ppa2) interfere with the ability of il-3 or gm-csf to induce serine phosphorylation of hsp27. taken together, our findings indicate that tyrosine phosphorylation of map-kinase is a prerequisite for serine phosphorylation of hsp27 mediated by mapkap-kinase 2. hsp27 is localized in the nucleus and has been linked to the cellular stress response. its precise function, however, is largely unknown. our data identify hsp27 as a target of il-3/gm-csf stimulation via map-kinase and mapkap-kinase 2. further-more, our results indicate that hsp27 may also exert phosphorylation-activation functions involved in growth signalling pathways in unstressed cells. in recent years it has been shown that the mechanism of betalactam antibiotic-induced agranulocytosis involves a direct inhibition of the replicative dna polymerases alpha, delta, and epsilon. in this report we examine, as a representative of these antibiotics, the effect of freshly prepared ceftazidime (cef) and degradation products of cef on myelopoiesis. we investigated freshly dissolved cef and cef incubated for 96 hours at 37 + (cefd) on the production of myeloid cells in the supernatant (sn cells) and colony stimulating activity (csa) by the stroma. one week after drug treatment of the mltbmc a significan~ dosedependent inhibition of the myeloid cell production (xl0~ and csa (assessed by cfu-gm assay) occured, as summarized in the following we here report that human lung fibroblasts respond to x-ray treatment (xrt) with release of interleukin (il)-6. synthesis of il-6 upon ionizing radiation is preceded by an increase of il-6 transcript levels resulting from transcriptional activation of the il-6 gene. analysis of deleted fragments of the il-6 promoter indicated that transcriptional activation of the il-6 promoter was due to enhanced binding activity of the transcription factor nuclear factor (nf)-~b. although activation protein (ap)-i did not participate in the rapid induction of the il-6 promoter, its binding activity was also enhanced by xrt. in contrast to binding kinetics observed with nf-~:b, ap-1 binding following xrt was biphasic with the second peak being dependent on de novo protein synthesis. in contrast, however, nf-il-6 activity was not enhanced by xrt in fibroblasts. the introduction of both the nf-~b-and the ap-1 recognition sequence, conferad inducibility by xrt to a heterolgous promoter, with reporter gene activity being maximal 24 hours or 48 hours following xrt, respectively. sequential acitvation of two distinct transcription factors might thus contribute to synchronize transcriptional activation of different genes participating in the x-ray (xr) response. on the basis of study of functional and morphological characteristic of bone marrow stromal tissue of human fetuses, 8-27 week gestation, and of adults aged 16-60 years, and in experiments on animals, the role of the sinusoidal endothelium, reticular, fat and endosteal cells in hemopoiesis regulation has been concretely defined. the endothelium of sinuses forms the histo-hematic barrier "bone marrow-blood', ensures the wanscellular migration of stem cells and mature blood cells, releases hemopoiesis-regulating factors and is involved in the erythroid cell maturation. bone marrow reticular cells participate in the formation of intramedallar supporting frame-work, regulate the transvasal and intramedullar cell migration, form the extracellular matrix, produce humoral regulators of hemopoiesis, effect the cell differentiation by way ot their intercellular contacts with hemopoietic precursors and give origin to adipocytes, lntramedullar adipocytes present an energetic depot of hemopoietic and stromal tissues and in the stage of preadipocytes they effect, by means of contacts, the granuloeyte development. the endosteal cells of bone marrow are the source of intramedullar stromal tissue, they participate in the anchorage of the hemopoietic stem cells and form the microenvironment of the latter, regulate the endosteal ca ion levels and might be a possible source of hemopoietic tissue (population of cells of the residual embryonal mesenchyma). the thesis on the mechanism causative of the impaired regulation of precursor proliferation and differentiation in hematologic diseases is proposed. the self-renewal capacity of cfu-s (spleen colony-forming unit) following the treatment of (cbaxc57b1)f1 female mice with recombinant human granutocyte colony-sffmulating factor (rhg-csf) was investigated. the possibility of synergism between erythropoietin (epo) and rhg-csf in blood cfu-s mobilization was also studied. during the investigation the following observations were made: 25 and 250 mkg/kg/day injection of rhg-csf expended the number of cfu-s-11 in circulation 4-and 32-fold, accordingly. the self-maintenance potential of peripheral blood cfu-s-i1 did not change significantly. the treatment of mice with 250 mkg/kg/day of rhg=csf resulted in two fold increase of spleen cellularity and 15 fold augmentation of cfu-s-11 number, without noticeable changes in their self-renewal capacity. moderate changes in cfu-s-11 number were observed after epo administration in spleen and peripheral blood, however no significant synergistic effect of epo with both doses of rhg-csf was detected. the multifold increase of cfu-s-11 number in peripheral blood following rhg-csf administration with no reduction in their self-maintenance potential suggests that mobilized with rhg-csf blood stem cells provide an appropriate source for reconstitution of the hematopoietic system. we used the brdu-incorporation method to show the effects of 1l-2 (100, 1000, 3000 u/ml),il-4 (0.1, 1, 10 ng/ml) and il-2 (3000 u/nil) plus il-4 (10 ng/ml) on b-cll-ceiis. after ficollseparation, lysis of the erythrocytes (nh4ci) and lysis of monocytes (1-1eucin-methyl-ester), cells were divided into two groups. group 1 was cultured in a serum free medium (+brdu +cytokine) without any t-cell depletion. group 2 was cultured in a serum free medium (+brdu +cytokine) after t-cell (cd3 +) elimination by the macs (magnetic activated cell sorting). samples were taken after 20h, 68h, 92h, l16h and 140h. after staining with anti-brdu fitc and propidiumiodide (pi) proliferation was measured by flow cytomemy (facs scan). .00 p=proliferation n=no effect i=inhibition conclusions: i[,-2 shows a proliferative effect on b-cll-cells independent of t-cells. il-4 shows heterogeneous effects. by itself it has most often no effect on proliferation, but sometimes it inhibits or increases the prolifoation. this effect does not seem to depend on t-cells. it could depend on the dosage or some unknown patients' characteristics. further on il-4 inhibits 11,-2 induced proliferation in nearly all cases independent of t-cells. ii-i and tnf are inflammatory cytokines with overlapping biological activities. human vascular endothelial cells express ii-i and tnf receptors and respond to ii-i and tnf stimulation by elaboration of colony stimulating factors such as g-csf and gm-csf.however quantitative data are required in order to evaluate the contribution of ii-i and tnf to the activation of inflammatory hemopoietic cells such as granulocytes and macrophages by csfs. therefore we quantified the production of g-csf and gm-csf in endothelial cell cultures subsequent to treatment with il-l~ (0.l-200u/ml) and tnf(10-2000u/ml)or ll-1 in combination with tnf.a dosedependent stimulation of g-csf and gm-csf secretion was found following ll-i and tnf treatment of endothelial cells.ll-i was a more potent inducer of g-csf secretion than was tnf using approximately equipotent doses of il-i (100u/ml) and tnf (1000 u/ml) regarding the induction of gm-csf.in addition significant snperadditive stimulation of g-csf and gm-csf production was found with a low dose of il-1 ( iu/ml) and a saturating dose of tnf(10oou/ml) in combination.however the costimulatory effect of ii-l(iu/ml) was significantly more pronounced with g-csf than with respect to gm-csf.in summary the differential modulation of g-csf and gm-csf production ~n endothelial cells by ii-1 and tnf may indicate 'a disparate role of ii-i and tnf in vascular inflammatory processes and atherosclerosis regarding recruitment and activation of inflammatory leukocytes. cytokines are known to be involved in the pathophysiology of graft versus host disease (ghvd) and to effect lymphohematopoetic progenitor cell growth after bone marrow transplantation. the use of t-cell depleted marrow in human transplantation is associated with suppression of gvhd but decreased rates of engraftment. we have shown in rodent models that uvb irradiation (uvb) of donor inoculum inhibits gvhd while preserving engraftment. to determine the effects of uvb on eytokine production by cells associated with gvhd, human marrow mononuclear cells isolated by ficoll density gradient were uvb-irradiated at doses of 10-200 j/m2 and then stimulated with pha/lps or allogeneic cells. elisa assays were used to measure the production of gm-csf, il-3, lif, il-lbeta, il-2, il-6, tnf-alpha, and lfn-gamma by stimulated cells . two week methylcellulose cultures were used to determine viability of cfu-gm, bfu-e and cfu-gemm progenitor cells after uvb. all results were compared to non-uvb-irradiated marrow and to marrow depleted by soybean agglutination and sheep erythrocyte rosetting. progressively increasing doses of uvb produced progressively decreasing levels of all cytokines except il-6, which remained unchanged. t-cell depleted marrow produced decreased levels of all cytokines except il-6. uvb at 75 j/m2 resulted in higher levels of gm-csf and il-3 as compared to t-cell depleted marrow. this same dose of uvb essentially preserved cfu-gm, bfu-e, and cfu-gemm colonies. we conclude that uvb may inhibit the cytokine cascade active in gvhd while preserving progenitor cell growth at uvb 75 jim2. uvb may serve as gvhd prophylaxis in clinical marrow transplantation and in vivo studies on non-human primates are in preparation. the ability to migrate is fundamental for the acquisition of invasive properties by tumor cells. a tumor derived cytokine was identified by its ability to induce direct and random migration via a receptor mediating signal pathway, i.e. autocrine motility factor (amf). we identified the receptor for amf (hamf-r) and found ~at hamf-r plays a role in invasiveness and metastasis in human bladder carcinomas. we investigated the expression pattern of hamf-r in 83 fresh frozen bladder cancer specimens by immunofluorescence technique on the translation level and found a strong correlation (p<0.01) to tumor stage and grade. furthermore we have shown that patients who were found to be hamf-r positive have an increased risk for early tumor progression (p<0.05). a large number of substances from the working place and in the general environment such as quartz and coal mine dusts are causing silicosis and leading to lung fibrosis. alveolar macrophages are the primary target for the noxious effect of quartz dust. quartz dust incubated human macrophages release in vitro a cytokine, which stimulates cell proliferation of human lung fibroblasts (wistar 38). in recent studies we found that beside fibroblasts also epithelial cells of the alveolar unit, such as pneumoeyte type ii cells (a-549) respond to the cytokine with strong proliferation. supernatants of quartz dust exposed macrophages were concentrated by ultrafiltration and thereafter fractionated by gelfiltration (sephadex g150, pharmacia). biological activity of the factor eluted within a molecular range of 75-102 kd. furthermore, the factor was purified by anion exchange chromatography (q-sepharose). fractions revealing biological activity were further analysed by sds-gel eleetrophoresis (page) and showed two protein bands with apparently molecular masses of 76 and 79 kd,respectively. after addition of the supernatant initially spindle shaped pneumocytes were detected, followed by epithelial-like cells when cell proliferation progressed. induction of spindle-shaped pneumocyte type ii cells could also be seen after addition of pure tumor necrosis factora. however, in this case no cell proliferation was observed. we assume that beside the cytokine, which is responsible for the induction of cell proliferation tnf~ is present in supernatant. differences in a variety of immunologic parameters. in peripheral blood however differences to healthy persons have hardly been described. in this investigation we compared serum levels and concentrations of interferon-7 (fin-y) and il-l--ct in stimulated samples of whole blood. 50 ixl whole blood of 38 healthy controls (ref) and 39 patients with sareoidosis, 27 without treatment (pot) and 12 under corticoid medication (put), was incubated in medium at 37~ and 5% co 2. con a was used for stimulation of ifn-y and lps for il-1--m concentrations were determined with an elisa. results: without stimulation no measatalg, e amounts of ifn--y could be found. after stimulation ref showed median coneentrations of 127 ng/ml, pob 10 ng/ml and put 2 ng/ml. the difference between ref and put respectively pot was statistically significant. il-l-m without lps the differences were not significant. under stimulation pot had si~ificantly higher values (220 pg/ml) compared to ref (170 pg/ml) and also to put (155 pg/ml). in conclusion we were able to demonstrate that in conlrast to serum levels stimulation of peripheral whole blood reveals significant differences in concentrations of ifn--y and il-1--ct between patients with sarcoidosis and healthy references. we established a modified polymerase chain reaction protocol for the detection and semiquantitative assessment of mrna-transcdpt levels for il-2, il-4, il-5, il-6, ifn-7, tnf-cl, gm-csf, tgf-13 and il-2-receptor-c{ (il-2r). the method was shown to distinguish 10-fold differences in template concentration after 4 rounds each of amplification in the range from 20 to 40 cycles; the lower threshold of sensitivity was at 10 copies per pcr-reaction. reproducibility was >95% for a positive result after 32 rounds of amplification; it decreased to 80% after 36 rounds. this corresponded to the detection of 1,000 and 100 template copies, respectively, per pcr-reaction. human peripheral blood mononuclear cells (pbmc) and tumor cell lines were evaluated for mrna-expression with or without stimulation and these results were compared to secretion of the corresponding cytokine. for pbmc, constitutive mrna-expression was found positive for tnf-o~, ifn-7, il-4, il-6, tgf-13 and il-2r, whereas detectable expression of il-2, il-5 and gm-csf was induced only after stimulation. using ~-actin as an intemal standard, the pcr could demonstrate relative differences in cytokine transcripts after stimulation with (a) 100 ]u/ml il-2, (b) 10% lymphocyte-culture conditioned media (ccm) and (c) pma (10ng/ml) plus a23187 (100ng/ml). for il-2 transcripts no detectable expression was found without stimulus or after addition of il-2, whereas ccm resulted in a 1,000-and pmna23187 in a 10,000-fold increase. other mrnatranscripts increased t0-fold (tnf-(~) up to 10,000-fold (gm-csf) with or without differences between the stimulating agents. the cell lines caki-1 (renal cell carcinoma) and daudi (burkitt lymphoma) also expressed comparable levels for cytokine transcripts, with a strong induction after stimulation with pmna23187. the relative ifn~ mrnacontent in caki-1 increased from 0 to 1,000, for gm-csf from 0 to 10,000. the influence of different cytostatics on il-6 production by peripheral blood mononuclear cells (pbmc) was studied. pbmc were pre-incubated with or without phytohemagglutinin p (pha-p), then pulse exposed during 1 hr to different cytostatics in their therapeutical concentrations, washed three times and incubated additionally during 24-72 hrs. then the supernatants were collected and il-6 biological activity was measured in mttmodified b9-cells biological assay. though significant individual variations of the il-6 production were found, all the studied drugs can be separated on severa) groups: 1) adriamycine and methotrexate induced late increase of il-6 production ( 48-72 hrs ); 2) cytarabine strongly increased the early as well as the late il-6 production ; 3) the pretreatment with etoposide and rubomycine suppressed subsequent production of il-6 by pbmc during 24-48 hrs, the late production was increased; 4) in contrast, the cyclophosphamide pretreatment stimulated the early production and strongly suppressed the late one. the changes of il-6 production by pbmc was not due to the cellular death. the pha-p stimulated pbmc produced usually more il-6 that unstimulated cells did. these data suggest that the cytostatics possess the different effects on il-6 production by pbmc that could be important in the therapy of malignancies. recent studies implicating a deficiency of interleukins in several diseases have underlined the importance of measuring in vitro the dna synthesis and the cytokine production (il-1, il-2, il-6, tnfalpha) in the same cell system. previously had found that normal peripheral blood mononuclear cells (mnc) of patients suffering from high-malignant non-hodgkin lymphomas showed a decreased capability to proliferate after mitogenic stimulation (pha, con a, pwm). here we have studied the dna synthesis and the production of different cytokines (il-1, il-2, il-6, tgf-i~ and tnf-alpha) by pokeweed mitogen (pwm) stimulated mnc from 15 healthy control subjects and from 14 patients with nhl. the il-2 production of pwm-stimulated mnc of patients with nhl was found to be significantly decreased, wheras the il-1, il-6 and tnf-alpha release were not changed significantly. these data showed a good correlation with the reduced capability of mnc from patients with nhl to proliferate after mitogenic stimulation. the multifunctional cytokine transforming growth factor-ii (tgf-i3) is known to inhibit the dna synthesis, as well as the il-2 production of mitogenstimulated mnc. however, tgf-i~ release was not significantly changed in call culture supernatants from patients with nhl in comparison to healthy controls. we conclude that the suppressed dna synthesis and 11_-2 production of mnc from patients with nhl is not the consequence of a deceased tgf-5 level secreted by these cells. is a co~on problem after chemotherapy and requires supportive care until normal hemopoiesis has recovered. to study the importance of endogenously produced cytokines for regeneration of bone marrow progenitors we measured serum levels of g-csf and ii-6. blood samples were obtained before and 24 hours after chemotherapy, during the stage of leukopenia (<1000/ul) and recovery (>1500/ul)of leucocyte counts. in 6/7 patients we found a more than 5-fold increase in serum g-csf levels at the stage of leukopenia. highest amounts were observed in two patients with lethal outcome. there was no correlation between thrombocytopenia and levels of g-csf or il-6. serum g-csf (pg/ml, mean, range) before (leuko>1500) after (<1000/ul) chemotherapy aml dav 25 (5-i00,n=14) 2000 (50-8000,n=7) all 'h~izer" 100 (10-300,n=4) 1000 ( we measured different hematopoietic cytokines as g-csf, gm-csf and il-6 in amniotic fluid and cord blood to ctearify their physiological and palhophysiological role in fetal and neonatal development amniotic fluid was available from the 15 th to the 21 st week of gestation (n=53), cord blood from the 26 th to the 42 nd week of gestation (n-60). activity levels of cytokines were determined by stimulation of the ceii lines nfs-60, 77d1, and tf-i. which are responding specifically to g-csf, il-6, and gm-csf. specificity has been proved by neutralizing antibodies. calculation of cytokine levels was done by standards of recombinant growth factors. sensivity for g-csf and il-6:5 pg/ml for gm-csf: 1oo pg/ml. in amniotic fluid g-csf tanged from 40 to i23.ooo pg/m[ and il-6 from 20 to 12.000 pg/ml, whereas gm-csf was not detectable. in cord blood g-csf was between 5 and 23.000 pg/ml and [l-6 between 5 and 30 pg/mh in most of the samples (qo%~ gm-csf was beyond the sensivity limit. in 92% (11 of 12 cases) g-csf levels were elevated over 200 pg/ml and associated with amnion infection syndrome, while green amnioflc fluid alone during delivery did not stimulate the production of g-csf, the levels of the hematopoietic cytokines showed no influence by the gastational age. identical twins without maternal infection showed the same g-csf levels. inflammation of the amniotic membrane and maternal sepsis is associated with elevated g-csf levels in cord blood. gm-csf can normally not be detected in cord btcod and amniotic fluid. detectable levels of gm-csf in cord blood and amniotio fluid maybe give a hint for a pathological situation during pregnancy. total number of 110 children with all, 68 boys and 52 girls, aged from 5 to 15 years were included to the study. tnf production was studied acc. the method based on growth inhibition of sensitive to tnf l 929 mice fibroblasts, ill-1 production ace. method based on inhibition of autologous rosette formation by thymoeyteg of cba mouse and 1l-6 production ace. to conventional el1sa genzyme-test. twenty five healthy children served as the control group. it was found that in children with all during the whole period of therapy the il-1 and il-6 production, was significantly lower than that observed in the control group of healthy children (p 0.005). the tnf production in all children before therapy was lower in comparison with the control group values. during cytostafic therapy was higher and grew up above the normal limits after cessation of the therapy. il-1 production grew up after the end of the therapy but never reached the value of the control group. efs at 54 ruth in all children with il-1 production < 10/~ before therapy was higher than that in children with il-1 < 10/~ (efs 90 % v 70 %). the il-1 production seems to be a good prognostic marker. (pts) with active autoimmune disorders as well as with malignant lymphomas. in addition, cd23 and its soluble form (scd23) is thought to be involved in the regulation of b-cell proliferation. therefore, we examined scd8, scd25, and scd23 in pts with b-cell chronic lymphocytic leukemia (b-cll) in order to assess their role as indicators of disease activity. fifty-five pts with b-cll were studied so far. staging was performed according to the classification systems of rat and binet, respectively. serum samples were freshly stored in liquid nitrogen until further processing. levels of scd8, scd25, and scd23 were measured by a sandwich elisa technique using commercially available assays (biermann, germany). advancing rat stages were associated with a progressive increase of all the three serum factors (scds: rat 0 556+165 u/mt vs rat iv 1310+150 u/ml, scd25: rat 0 1326-1-190 u/ml vs rat iv 8850__.1330 u/ml; scd23: rat 0 1265+455 u/ml vs rat iv 6500+_1800 u/ml). this progression was also evident when binet's classification was applied. occurrence of b-symptoms was associated with high levels of scd25 (p<0.001), whereas scd8 and scd23 were found also to be increased but without statistical significance. high levels of all the three factors strongly correlated with a lymphocyte doubling time < 12 months, a lymphocyte count >50.000/tzl, and with the presence of hepato-and splenomegaly. interestingly, occurrence of bulky lymph nodes (i.e. at least one nodule of > 8 cm in diameter) was linked with high levels of scd23 only (p<0.002). in summary, (1) progressive serum levels of scd8, scd25, and scd23 correlate with advancing stages of disease in b-cll. (2) b-symptoms were associated with high levels of scd25. (3) we found scd23 to be the more sensitive marker of the total tumor load than scd8 and scd25. thus, scd23 may be useful in monitoring pts with b-cll. cytosine arabinoside (ara-c) is one of the most active single agenls in the treatment of acute myeloid leukemia (aml). its cytotoxic activity mainly depends on its phosphorylation to ara-ctp and on its incorporation into the dna. based on recent in vitro studies showing that hematopoietic growth factors like gm-csf and il-3 enhance the cytotoxicity of ara-c on clonogenic leukemic cells, the gm-csf priming concept is currently explored in clinical phase ii and iii studies. in an ongoing study at the universities of muenster and goettingen gm-csf (250/~g/m2/d) is started 24 hrs before induction chemotherapy (tad9/ham) until recovery of blood ceil counts. this study provided a means to assess the effect of gm-csf on the intraceunlar ara-c metabolism in vivo in 23 pts with aml. enzyme activities of deoxycytidine kinase (dck), thymidine kinase (tk), dcoxycytidine deaminase (dcd), dna polymerase (pol) and dna polymerase alpha (pola) were determined before therapy, 24 hrs after the administration of gm-csf and 48 hrs after the administration of ara-c. in addition, ara-c incorporation into the dna was measured after 48 hrs ara-c administration. enhancement of enzyme activity was observed in 11/15, 9/17, 10/16, 6/16 and 7/16 cases for pola, pol, tk, dck and dcd, respectively. increases ranged from 13-630% for pola (median 127%), 13-188% for pol (median 77%), 14-820% for tk (median 113%), 10-670% for dck (median 43%) and 29-1350% (84%) for dcd. inadequate blast cell reduction after tad9 (>5% blast cells on day 10 or 21) was associated with significantly higher dcd blast cell activities compared to the dcd activity values obtained for pts with adequate blast cell clearance (median values: 5.12 vs 0.6 nmol/min x mg, p<0.01). cases with dcd activities <5 nmol/min x mg showed significantly higher ara-c incorporation into the dna compared to pts with dcd activities >5 nmol/min x mg (5.2 vs 0.65 ng/107 cells). furthermore, inadequate blast cell clearance was associated with lower ara-c incorporation into the dna (median 0.8 vs 5.3 ng/107 cells) and lower pola activities (median 1.3 vs 3.1 pmol/min x mg). in 11 pts we investigated simultaneously the effect of gm-csf pretreatment on ara-c metabolism in vitro. enzyme activities of pola, tk and pol correlated significantly in vivo and in vitro (rp~ rtk=o.64, rp~ p<0.05). these data demonstrate that gm-csf enl/ances dna synthesizing enzyme activities in vivo and in vitro. furthermore, these data suggest that gm-csf might improve the therapeutic response to induction chemotherapy by increasing dna polymerase alpha activity and thereby increasing the ara-c incorporation into the dna. the effects of interferon-alpha (ifn-alpha), interferon-beta2/ lnterleukin-6 (il-6) and interferon-gamma (ifn-gamma) in inducing megakaryocytic differentiation of blast cells from acute megakaryoblastic leukaemia (amegl) patient determined by the increase in cd41 and cim2b expressions using monoelonal antibodies in apaap technique were investigated in liquid suspension culture. after six days of culture, the percentage of cd41 and cd42b positive cells increased in control cultures from 15,2 % and 10,6 % on day 0 to 32,0 i 4,3 % and 22,1 â�¢ 2,5 %, respectively. the addition of ifn-alpha significantly increased the number of cimi and cim2b positive cells by about two to three fold compared to control cultures, p <0,01 and by about four to six fold compared to day0, p <0,001. similarly, ifn-beta2/il-6 induced a significant increase in cd41 and dc42b positive cells. on the other hand, ifn-gamma failed to increase the number of cim1 and cd42b positive cells in comparison to control cultures on day 6 and instead induced a significant increase in the number of monocytes/macrophages from only 3,7 _+ 1,9 % in control cultures to 47,9 + 2,5 %, 52,3 + 1,4 %, 54,5 â�¢ 2,9 % and 57,5 â�¢ 2,3 % in 1, 10, 100 and 1000 units/nil ifn-gamma-treated cultures, respectively, p <0,001. the present results suggest that megakaryocytic differentiation of blast cells in amegl could be induced by ifn-alpha and il-6 and support a clinical role for il-6 in the treatment of amegl patients. also, the present results showed that monocytic differentiation of blast cells in amegl could be induced by ifn-gamma~ supporting the multipotent stem or progenitor cell origin of the amegl subtype of acute myeloid leukaemia. a monoclonal antibody-based elisa and bioassay were used to measure leukemia inhibitory factor (lif) protein levels, activity and the functional role of lif in superuatants of cultured stromal cells derived from patients with acute and chronic myelogenous leukemia, myelodysplastic syndrome, and hairy cell leukemia and from normal controls. we demonstrate that biologically active lif protein is constitutively produced and secreted by coltured bone marrow stromal cells from all subjects studied. in addition, adherent-layer conditioned-media lif protein levels were significantly elevated in samples from patients with all hematologic malignancies studied as compared to samples from normal controls. confluent adherent layers exposed for 24 hours to interleukin (il) 113 or tumor necrosis factor-9 (tnf-a) showed a significant increase in lif protein levels, whereas exposure to il-4 (sterling drug inc., great valley, pa) resulted in a dose-dependent decrease in lif levels. 3.0 (i.6-8.0) 2.5 (0.9-5.8) 6 0.036 interestingly, neutralizing antibody against lif caused a 25% reduction in normal progenitor proliferation derived from the superuatant but not from the adherent layers, and this effect was reversible by the addition of recombinant lif protein. we conclude that (i) biologically active lif protein is constitutively produced by adherent layers from normal donors, (ii) tnf-ct and il-113 increase and il-4 decreases adherent layers lif protein levels, (iii) the steady state levels of lif protein produced by adherent layers from leukemic patients is significantly elevated, and (iv) lif may participate in the interaction between adherent layers and hematopoietic progenitors to maintain normal hematopoietic colony growth. it is well known that bone marrow stromal cells have crucial impact on haemopoietic cell proliferation. little is known about stromal humoral factors leukemic cell proliferation. the aim of this study is to evaluate the effect of stromal cell conditioned media (sccm) on the 3h-thymidine uptake by normal and leukemic target cells. 21 patients with aml were studied treated with "7+3" based regimens. long term bone marrow cultures were established in non-leukemic and leukemic patients (before and during treatment). target cells for sccm were normal haemopoietic cells and leukemic blasts. the results are the comparison of the effects of leukemic and non-leukemic stromal cells. a part of the patients revealed high stimulative activity upon non-leukemic cells (+125 + 2 %, p<0.001) and inhibited proliferation of leukemic cells (-70 â�¢ 3 %, p<0.001) this group entered complete remission. sccm of another group of patients inhibited proliferation of nonleukemic cells (-84 _+ 3 %, p<0.001) and stimulated blast cell proliferation (+ 146 + 5 %, p<0.01). the magnitude of the figures was even more profound later: during treatment, bone marrow aplasia, recovery. this group of patients failed to achieve remission. it seems that stromal cells has significant on impact on restoration of normal or leukemic hemopoiesis after chemotherapy. peripheral b-cll-cells from 51 patients were investigated upon the proliferating effect of ifn~, tnf~ or combination of both in serum free culture medium (cg-medium). blood cells were drawn from patients and lymphocytes separated by ficoll hypaque and monocyte-lysis (leucinemethyl-ester incubation). t-cells were depleted using a, cd3 mab and macs (magnetic activated cell separat9 r, miitenyi biotec). at each step heterogeniety of the population was controlled by facs analysis with 7 different mab (cd 3, 4, 8, 1o, 14, 19, 20, 56) . the homogeneous population (contamination less than 1%) was co-incubated with both cytokines (0.1 -10 ng/ml) and bromodeoxyuridine (brdu) in cgmedium. after 20, 60, 92, 112 and t40 hours cells were harvested an analysed for brdu-incorporation into the genome. ifnu and tnfc~ measurings (51 in total) were almost similar: 24 patients were non-responder and showed no stimulatory effect on cells; 5 patients showed an inhibitory effect; cells from 21 patients were responding upon cytokine cultivation. the combination of both ifn~ and tnf~ produced in these cells an additive effect (14 out of 21). best results could be observed when the control population (without cytokine) was minimal proliferating compared to no proliferation. a high correlation was observed between cytokine response and pre-treatment: without glucocorticoid treatment of patients prior to measurements the influence of cytokines on resting b-cll-cells was significantly higher (with methyl-prednisolone 15%, without we-treatment 61% were responders). functional defects of the monocyte/macrophage system probably contribute to the increased rate of severe infections in patients with myelodysplastic syndromes (mds). therapeutic trials with hematopoietic growth factors (hpgf) have resulted in substantial improvement of cytopenia, especially neutropenia. however, little is known about the alterations of the monocyte/macrophage system during these therapeutic interventions. it therefore was the aim of the present study to analyze the capacity of monocytes/macrophages from mds patients prior to and after hpgf therapy to secrete il-11~, tnfe, il-6, and il-8 upon in vitro stimulation with lipopolysacharide (lps). sixteen patients were studied: 1t had a refractory anemia, 5 had a refractory anemia with excess of blast cells. prior to therapy, the capacity of adherent monocytes/macrophages to secrete il-11~, tnfc~, il-6, and il8 was significantly reduced by 50-70 percent as compared to normal controls. on the other hand, oxygen radical release was normal in 11 mds patients tested. treatment with gm-csf (15-250/~g/m2/d sq x 7-14; n=7), il-3 (60-500/~g/m2/d sq x 15-18; n=3), and g-csf (1-3 fg/kg/d sq x 84 in combination with all-trans retinoic acid; n=6) normalized the potenital of monocytes/macrophages to secrete il-ti~, tnf~, and il-6. il-8 secretion was only improved by il-3 or gm-csf dosages _> 250 /~g/mz. oxygen radical release was significantly stimulated by both gm-csf and il-3. these results indicate that treatment of mds patients with gm-csf, il-3, and g-csf (the latter in combination with all-trans retinoic acid) can restore deficient monocyte/macrophage secretory function to normal. depletion of cd6 positive t ceils has been used in human patients for prevention of gvhd. we studied depletion of cd6 + cells from canine marrow for induction of gvh-tolerance across a complete dla-haplotype difference. prompt engraftment and fatal gvhd occurs in a littermate combination of dla-homozygous donors and dla-heterozygous recipients when undepleted marrow is given. aiiogeneic marrow depleted with a crossreactive antibody to human cd6 and immunomagnetic beads was given to 6 dogs. one dog died with haemorrhage on day 48 due to thrombocytopenia, 5 dogs showed complete hematopoietic recovery. 4 dogs became tolerant chimeras and one dog died with gvhd due incomplete depletion. chimerism was mixed early after transplantation, became complete later and is still complete in 2/4 dogs after 1-2 years. 4 dogs received cd6 depleted marrow grafts and loijg/kg/d s.c. r-canine g-csf starting on day 2 after transplantation. although all dogs had fast recovery of granulocytes, 2 dogs receiving 0.5 x 108 mnc/kg died of marrow aplasia on days 47 and 62 without recovery of thrombopoiesis. two dogs receiving 1 x 108 mnc/kg had sustained engraftment with delayed recovery of thrombocytes compared to dogs without g-csf. facs analysis of depleted marrow showed complete depletion of cd4 + cells but about 2% of cd8 + cells; cfu-c growth and nk-activity was retained after depletion. cd6 depleted marrow inhibited the generation of cytotoxic cells. these experiments indicate that qualitative t-cell depletion is effective, since cd6 recognizes only subpopulations of t-cells. the application of r-canine g-csf enhanced the recovery of granulocytes but led to graft failure in dogs receiving a low number of marrow cells. gsf-inst. fdr immunologic, marchioninistr. 25, 8000 mqnchen 70 supported by the wilhelm-sander foundation we wished to analyse the factors which may affect the yield of pbpc (cfu-gm) to be collected by leukapheresis following high-dose cyclophosphamide (hd-cyc: 7g/m2). we retropectively studied the following criteria in 31 patients with high-risk mm of which 10 received gm-csf (sandoz sa/ schering-plough) after hd-cyc: time from diagnosis to hd-cyc, number of chemotherapy cycles (ctc) (fermand, 1992) , b2 microglobulin, bone marrow plasma cell count before hd-cyc, administration of gm-csf after hd-cyc, "slow" or "fast" rate of platelet and wbc recovery (jagarmath, schwartzenberg, 1992), "poor" or "good" mobilization ofpbpc (jagannath, 1992), differential wbc count between "day x ~ and "day x-l" during haematopoietic recovery. each variable was studied as continuous (regression analysis) and discontinuous (t-or chisquare tests). when the differential wbc count was < 1300 wbc/ial, 37% of the leukapheresis procedures performed on day x yielded more than 30x 104 cfu-gm vs 90% when it was _> 1300/tjl (p<0.05). the infusion of gm-csf was asaocaated with a higher yield of cfu-gm (bidt, 1993). the patients with "good ~ pbpc mobilization (> 50 x 104 cfu-gm in >_ 2 leukapheresis) could all be transplanted with pbpc alone (vs 47% of those with ~poor" mobilization). they had a shorter duration of aplasia after transplantation than the other patients (p<0.03). the "fast" wbc recoverers had a higher yield of cfu-gm than the other patients (p<0.05). when only the patients who did not receive gm-csf after hd-cyc were considered, a higher yield of cfu-gm was achieved in patients who underwent < 6 crc as compared to those who underwent > 6 ctc before hd-cyc (p<o,04). no other criteria had a significant prognosis value. these criteria will help to define which patients may need the collection of bm or the infusion of gm-csf (i) to collect pbpc and (ii) after transplantation. haemopoietic cells released into the circulation by g-csf (lenograstim) alone or in combination with intense chemotherapy: assessment of progenitors and stem cells. i.baumann*, ng testa*, mehm van hoef ~, c.lange*, e.de wynter*, a.howell $, tm dexter*. we report results of a phase i/ii recombinant human granulocyte colony stimulating factor trial for dose intensification in advanced breast cancer. sixteen patients were studied. seven patients underwent the phase i g-csf trial before entering the phase ii dose intensification study plus chemotherapy. nine patients were entered into the phase ii study with four cycles of dose escalating chemotherapy. the release of progenitor cells into the circulation was determined by clonogenic assays. two stage long term bone marrow cultures were used to assess the repopulating capacity and therefore the stem cell quality of the mobilised cells. normal bone marrow from allogeneic transplant donors (with informed consent) were used as controls. we found equivalent total numbers of progenitors in both cycle 1 and cycle 4 of g-csf plus chemotherapy peaking at day ia-12, respectively. in contrast, we found significant differences between the progenitor cells of cycle 1 and 4 generated from circulating haemopoietic cells when seeded onto preestablished irradiated bone marrow stroma. this indicates that peripheral blood stem collection should be performed in an early stage of chemotherapy due to an increased damage of the stem cell pool with increased levels of chemotherapy. the capacity of haemopoietic cells released by g-csf to repopulate bone marrow stroma was equivalent to normal bone marrow as recently reported. in autologous bone marrow transplantation (abmt) acute phase responses usually occur between day 5 and day 15 following abmt, and increases in bilirubin levels are observed in early endothelial complications such as endothelial leakage syndrome and veno-occlusive disease (vod). il-6 is the central cytokine modulating acute phase responses. therefore we performed a prospective study analyzing il-6, tnf-u, sil-2r and g-csf levels in serum samples of 21 patients undergoing abmt (3 all, 4 aml, 8 hodgkins disease, 5 non hodgkins lymphoma, 1 ewing sarcoma). 10 patients received g-csf post abmt, 11 patients were transplanted without g-csf. during leukopenia <300/gl g-csf levels of 1612 _+ 348 pg/ml (422-3309 pg/ml) were measured in patients not receiving g-csf. during regeneration, when leukocyte counts exceeded 1.000/tal, the endogenous g-csf levels declined to the normal range (<50 pg/ml). severe transplantation associated complications were defined as vod or interstitial pneumonia. il-6 levels increased from 26 +_ 6 pg/ml before therapy to 404 â�¢ 159 pg/ml during febrile leukopenia (p<.01). patients with complicated abmt had significantly higher il-6 levels (1123 -+ 398 pg/ml n=7 vs 99 â�¢ 33 pg/ml n=14) (p<.01). sil-2r-levels increased from 130 + 24 u/ml before abmt to 383 _+ 74 u/ml during leukopenia. in patients receiving g-csf sil-2r-levels further increased to 456 _+ 121 u/ml during regeneration, while in patients not receiving g-csf the sil-2r-levels were 320 â�¢ 71 pg/ml (p<.05). tnf-a levels remained unchanged during the course of abmt; there was no correlation with febrile neutropenia or severe complications. these data suggest a direct feedback regulation of endogenous g-csf release by increases in peripheral granulocyte counts. monitoring of il-6 serum levels during abmt may give insights into pathophysiology of abmt associated complications resulting from activation of monocytes and endothelial cells. we have previously reported that the rate of hematopoietic recovery following autologons blood stem cell transplantation (absct) could be influenced either by the type of conditioning regimen or by the underlying disease. we also reported that peripheral blood stem cell (pbsc) growth was sensitive to the stimulation of allogeneic irradiated stmmal layers. thus we undertook a study to examine the quality of the bone marrow (bm) raiemenvironment of patients who had undergone absct for either malignant lymphoma (ml) or multiple m~,eloma (mm) after either myeloablative chemotherapy or tbi conditmning regimens. thirteen patients with ml and eight patients with mm underwent absct using cells collected during recovery phase after intensive chemotherapy. we used the long term culture system (ltc) to investigate the quahty of the bm microenvimnment from 13 patients (pts) with ml and 8 patients with mm. all bm were collected after absct. twenty-two ltc established with bm from 13 patients with ml were investigated; 10113 pts received myeloablative chemotherapy (cbv) as conditmning regimen and 9 out of ioof these pts developed a complete confluent stmmal layer. three of the 13 ml pts received tbi and 2/3 of these pts developed a complete confluent stromal layer. eight ltc were established with bm from 8 patients with mm, who had att received" till" prior to absct. three ~f the 8 mm pts de~ a complete confluent stromal layer. we evaluated the cfu-gm production by the stmmal layer from all patients. the type of disease (ml vs mm at d7 p=0, 935 and di4 p=0, 294) , the conditioning regime used prior to absct o'b! vs cbv at d7 p=0, 961 and di4 p=0, 126) , the presence or absence of a complete, confluent stromal layer (at d7 p=0,469 and d14 p=0,650) did not influence the production of cfu-gm by bm stromal layers from autografted patients. nor was there a correlation between the development of a confluent stromal layer and the duration between ltc and absct or the date of the cytapheresis as compared with the date of diagnosis or the number of naeleated cells and cfu-gm used to engraft the patients. interestingly, when we examined the ability of confluent and non confluent stmmal layer to support hematopoiesis, we observed no difference in the total cumulative production of nucleated cells or cfu-gm, suggesting that the quality of the bm stromal miemenvironment after absct cannot explain the differences seen in hematopoetic reconstitution. the present study was designed to estimate the immunologic and hematopoietic reconstitution after several consecutive chemotherapy courses (ctx). patients selection: all (n =3/9 ctx-cycles), aml (n = 11/21 ), nhl (n = 6/16), m. hodgkin (n =3/4), and non-seminomatous germ cell tumor pts. (nsgct, n = 7/17). ctx was performed according to the current treatment protocols used at our institution. the nhl and nsgct pts. received g-csf before ctx for mobilization of stem cells for apheresis. g-csf application (5"pg/kg) after ctx was used in all nhl, all and nsgct and 3 aml pts. the collected pbsc were reinfused after each following ctx cycle to all nhl and 5 nsgct pts. the cd3, cd14, cd19, cd25, cd33, cd34, cd56 and hla-dr positive cells in peripheral blood were analyzed, using dual color fluorescence and flow cytometry. cfu-gm from peripheral blood mnc's were used as control for the clonogenic capacity of cd34* cells. samples were obtained before, immediately after ctx and several times after the wbc's reached > lxlog/l. a positive correlation of the rising and decreasing subpopulation counts within the mnc's were noticed (r=.65-.85), however the cd3* were in inversed ratio to the cd14 ⧠cells (r =-.66). the percentages of cd3 * and especially of the cd25 + cells showed an increment immediately after ctx, where the proportions of cd14" and cd34 ⧠cells tended to fall. there was also a correlation between cd34" and cd14 ⧠cells (r=.76, p<.001) and between cd34 ⧠ceils and cfu-gm growth (r= .82, p<.001 ). an increased clonogenity was associated with low numbers of cd34 * cells: cd34~: cfu-gm 38:1 before ctx; 18:1 immediately after ctx; 38:1 during the regeneration phase. the same phenomenon could be seen by intensively pretreated compared to less intensively pretreated patients. the hematopoietic reconstitution parameters (median) were as follows in the pbsc-rescued vs non-rescued pts: platelet transfusions -18 vs 28 (p<.02), rbc transfusions -6 vs 8, days with platelets <50000//11 -6 vs 13 (p<.01), duration of neutropenia with wbc< 1000//ji -5 vs 9, days with fever -6 vs 8.5. the augmentation of cd34 ⧠cells correlated with rising numbers of mnc's and especially of cd14 ⧠but not with cd3*. the correlation between cd34" cells and cfu-gm in peripheral blood was convincing. dose-escalated cytotoxic therapy with stem cell support may be considered for patients with stage ii1 multiple myeloma, because of the poor median survival of only 3.5 years with conventional treatment. a threshold quantity of 5x106 cd 34+ eells/kg bw is necessary for a rapid and sustained engr~ent following myeloablative conditioning therapy. since june 1992 six patients (median age: 46 years, range 31-53) with mm received 5pg g-csf/kg bw (neupogan r, amgen) so. daily at the time of best response with conventional treatment. the content of cd 34+ cells in the peripheral blood was monitored by facs each day. leukapheresis were started when a detectable population of cd 34+ cells appeared. in 2 of 9 steady-state leukaphereses, more than 1,0xl0 ~ cd 34+cells were harvested. after the therapy with high dose eyclophosphamide ( 5 pts 4g/m 2, 1 pt 7g/m 2) plus g-csf more than 1,0xl06 cd 34+ cells were collected in 11 of 19 leukapheresis. to date two pts have undergone myeloablative conditoning therapy with hyperfractioned total body irradiation (14,4 gy) and melphalan (140 mg/mz). one patient received 200 mg/sqm melphalan as eonditoning therapy. after the reinfusion of the g-csf-mobilized pbsc, a rapid engraftment was achieved with median time of 13 days ( range 9-16) to reach 0,5 x 109/1 neutrophils and 9 days (range 5-14) for 20.0x109/1 platelets. no hematopoietic growth factors were given post-transplantation. in this pilot study, high dose eyclophosphamide and g-csf is an effective method for harvesting pbsc. evaluation of the mobilization of hematopoietic stem cells during steady-state hematopoiesis using higher doses of g-csf is planned. were treated with mtx, ifo, ara-c, prednisolone and escalating doses of vp16. pts. (n = 12) with relapsed or advanced non-seminomatous germ cell tumors were treated with cisplatinum, escalating doses of vp16 and ifo. the protocol design was similar: g-csf before ctx (2x12 pg/kg/die s.c.) with pbsc-aphereses at days 5 to 7 (nhl) or days 5, 6 (nsgct} followed by 2 to 4 ctx-courses. cytaphereses were also performed after ctx when the total wbc's recovered above lxl0~ the ctx-cycles were followed by reinfusion of the previously collected pbsc (n = 44) and application of g-csf (5 pg/kg/d.; n = 57) up to the last day of the subsequent stem cell collection. the cd34 ⧠cells, the clonogenic peripheral blood progenitor cells (cfu-gm & bfu-e) and light density cell (ldc) counts were determined in 150 cytapheresis samples. the ctx/g-csf courses contributed to substantially higher progenitor cell amounts than g-csf alone (n=75/21; p<0,01), without a difference in the collected ldc (1.3â�¢ vs 1.7â�¢ ldc; 3.9â�¢ vs 0.8â�¢ ~ cd34 ⧠cells; 10.9+2.1 vs 9.0â�¢ 1.5x104 cfu-gm; 11.6 â�¢ 4.3 vs 6.9 â�¢ 0.9x104 bfu-e/kg/apheresis; n = 102/48; mean + sem), but with approximately two times lower clonogenity. two or three leukaphereses were enough to rescue 3 ctx-courses with a minimal dose of 2x106 cd34 + cells/kg/patient. the optimal time to initiate pbsc-collection after ctx in the studied patient group was proved to be at the l't to 4 ~h day after reaching leukocyte > 1000 pl. the original total leukocyte, light density cell (ldc) and platelet counts in the peripheral blood at start of leukapheresis played an essential role for the efficiency of the procedure (eff), as shown by regression analysis: eff to total leukocyte count correlation was r2=-0.48 (p<0.ot); eff to ldc-count r 2 =-0.55 (p<o.001); and eff to platelet count r2=-0.25 (p=0.056). the results of pbsc retransfusion on hematopoietic recovery were as follows: 8 days to reach leukocytes>1000 pl and 12 days -platelets>50ooo pl; duration of neutropenia was 5 days; and 8 days to become platelet transfusion-independent (median). total number of 25 children, 17 girls and 8 boys, with neoplasia aged from 1-18 years were treated with gm-csf-leucomax sandoz and g-csf filgrastim hoffmann-la roche, during severe myelosupression occuring after intensive polychemotherapy. in 4 children gm-csf was applied twice after 2 consequent courses of chemotherapy. one child received gm-csf four times after 4 chemotherapy courses. twenty children with malignancies served as historical control group. gm-csf was given at dose 5 #g/kg, g-csf 5-10 #g/kg daily s.c. duration of therapy ranged from 2-28 days with median 10 days. after cytokines therapy increase of mean and median numbers of total wbs, neutrophils, monocytes and eosinophils was observed. the median time to hematopoietic recovery was shorter in the group of children treated with cytokines when compared with the control group. (9 v 16 days). in 19 of 25 children signs of infection disappeared even before granulocyte count increase. also shorter median time of febrile days, 4 v 12 days, in comparison with the control group was observed. no serious side effects during cytokines therapy were noticed. only in one patient local erythema in injection place was observed. in two children transient retrosternal pain was seen. our results showed that gm-csf and g-csf administered in children with neoplasia after chemotherapy shortens the period of neutropenia and infection duration. for both hodgkin's disease and non-hodgkin's lymphoma the outcome of chemotherapy has been shown to correlate closely with the dose intensity of treatment. however, dose intensification is limited most often by severe myelosuppression with the subsequent risk of fever and infections. we performed a clinical trial in 17 patients with hodgkin's disease or non-hodgkin's lymphoma to evaluate whether r-methug-csf could facilitate the safe and timely administration of an intensive chemotherapy regimen. patients who developed neutropenia _< 0.5 x 109/l for more than two days and / or fever _> 38.2~ and / or signs of infection after a cycle of chemotherapy (ceboppnim protocol administered at intervals of 21 days), as well as patients in which chemotherapy had to be delayed due to an anc _< 1.5 x 109/l on day 1, were eligible for treatment with r-methug-csf. in the subsequent cycles r-methug-csf was given subcutaneously at a dose of 5 #g/kg/d from day 11 to 20. 16 of 17 patients were evaluable, one patient had received only 1 day of r-methug-csf treatment. 15 of the 16 evaluable patients experienced neutropenia with an anc of less than 0.5 x 109/l during the chemotherapy course preceeding r-methug-csf treatment, whereas only 5 patients had ancs _< 0.5 x 109, l after the subsequent therapy with r-methug-csf (p<0.01).overall analysis showed that the duration of anc nadirs < 0.5 x 109/l was on average 3.27 days in 41 cycles without r-methug-csf compared to 1.78 days in 52 cycles with r-methug-csf treatment. the administration of chemotherapy had to be delayed only for 2.47 days (mean value) during cycles with r-methug-csf. side effects probably related to r-methug-csf, were moderate muscle and joint pain in 3 patients and chills in one patient. in general, r-methug-csf was well tolerated. under this treatment regimen 11 patients reached complete remission, 4 patients reached partial remission and one patient had stable disease. one patient was treated adjuvantly after gastrectomy. in conclusion, r-methug-csf allowed the safe and timely administration of this intensive chemotherapy regimen and reduced myelosuppression for patients with hodgkin's disease and non-hodgkin's lymphoma. based on previous studies (cancer res. 1991 :51, 116, blood, 1992 :80, 1141 we know that many patients (pts) cannot receive ct consisting of carboplatin (cbdca) 300 mg/m 2 and cyclophosphamide (cyclo) 750 mg/m 2 for oc every 4 wks without hematopoietic growth factor support. the desirable dose of rhil-3 based on a phase i/ii study in this setting was 5 or 10 gg/kg/day. a study was designed to determine whether rhil-3 would allow ct administration every 3 wks with 17 pts treated to date. cyclo was administered 750 mg/m 2 and cbdca was dose adjusted to creatinine clearance: 60-80 ml/min: 257 mg/m 2, 80-120 : 300 mg/m 2, 120-140:340 mg/m 2, > 140:385 mg/m 2. a total of 6 cycles (c) was administered. rhil-3 (5 or 10/zg/kg/d) was given sc d2-11 in each c. at 5 and 10 #g doses are 10 (46c) and 7 (33c) pts evaluable for toxicity and i0 (43c) and 7 (27c) pts for efficacy. side effects were fever and headache controllable with acetaminophen. at 5 #g rhll-3 in three c (2 pts) and at 10 #g in six c (5 pts) urticaria occurred. in 4 episodes dyspnea and/or oedema was observed. this reaction only occurred during c34~ and was controlled with antihistamine and prednisolone. ct could be administered every 3 wks in 43/70 c (25/43 c at 5/~g, and in 18/27 c at 10 /zg (ns)), every 4 wks in 13/70 c and > 4 wks in 14/70 c. no platelet transfusions were required. thus, in 61% of c it was possible to give a ct dose intensification of 33%. if full dose ct were to be given every 4 wks it would have been possible to administer in 80% of c in time. conclusion: with rhil-3 ct dose intensification of 33% is possible by reducing ct intervals, while no platelet transfusions were required with rhil-3. dept. of medical oncology. university hospital groningeu. oostersingel 59, 9713 ez groningen. the netherlands. k. mempel, a. reiter, e. yakisan, e. odenwald, m. pfetsch, g. schwab, h. riehm, k. welte. in the multicenter trial all-bfm 90, we have initiated a phase iii study of rhg-csf in children with high risk all. high risk (hr) patients are characterized by at least one of three criteria: 1. prednisone poor response (2 1000/mm 3 absolute blasts number in the blood at day 8 after 7 days' exposure to prednisone), 2. failure to achieve complete remission at day 33 of induction therapy, and 3. t(9;22). the primary objective was to test whether rhg-csf reduces the incidence of febrile neutropenic episodes. the second objective was to examine whether rhg-csf administration allows closer adherence to planned dosing schedule and to determine the overall response to chemotherapy. hr-all pts are randomized to receive either 9 cycles of chemotherapy (hrg i) or 9 cycles of chemotherapy (day 1-6) followed by rhg-csf (day 7-20; hrg li). up to date, 20 pts have been treated according to this protocol (hrg i: 10 pts, hrg i1:10 pts). in hrg ii, rhg-csf is well tolerated without g-csf related adverse events. in each arm, one pt relapsed. the incidence of neutropenia was 37% in hrg i and 10% in hrg i1. more importantly the incidence of febrile neutropenia was 14% in hrg i and 5% in hrg ii. these data demonstrate that rhg-csf allows for reduction of the incidence of febrile neutropenia in hr-all-patients. the patient has experienced complete resolution of stomatitis, fever and malaise. the administration of g-csf in patient with idiopathic neutropenia significantly increased the absolute ueutrophil counts (p < 0.001). g-csf was effective in reducing the severity of neutropenia and infectious complications in our patient. hansen f., stenbygaard l. and skovsgaard t. twenty patients with recurrent metastatic breast cancer treated with high-dose myelosuppressive antjr3eoplastic drugs (cyclophosphamide 2,5 g/m 2 or epirubicin 130 mg/m 2 both q 3. weeks) as first or second line chemotherapy were randomized in a prostective study to gm-csf (n=11) 5 microg/ kg/dag for ten days after cessation of chemotherapy or control (n=9). compared to the control-group highly significant reduction in granulocyte nadir duration (two days (0-5) with gm-csf vs. seven (2-11) days) and severity (wbc 0.4 x 109/i with gm-csf vs. 0.2 x i09/i) was found. no difference in frequency of neutropen fever or antibiotic use could be observed. even though the patients treated with gm-csf at random were more heavily pretreated with chemotherapy, there was a surprisingly higher responserate in these patients as compared to the control-group, namely 64% vs. 22%, resp. no severe side effects were seen, but presumably due to gm-csf one patient developed an allergic type i reaction and one patient developed a possible pericardia[ exudation. both were fully reversible after cessation of gm-csf treatment. keywords: gm-scf, chemotherapy, breast cancer. twelve adult patients with chronic neutropenia, including 7 patients with idiopathic sporadic neutropenia, 2 with idiopathic familial neutropenia and 3 with cyclic neatropenia have been treated with rhu-met-g-csf (amgen, thousand oaks, usa). treatment has been started in all patients with 3 #g/kg/d sc once daily. doses have been modified according to wbc. all patients had a rapid increase of absolute neutrophil counts. data are shown for idiopathic neutropenia (base line, after 1, 2 and 4 weeks). doses required ranged from 0.1 to 9 #g/kg/d. treatment has been continuously given up to three years in patients with severe preceeding infections. the clinical efficacy of the treatment was excellent with abrogation of significant infections. one patient with idiopathic i sporadic neutropenia recovered after 151 days of treatment with an anc of "~ ~o0o >2000/#1 after stop of g-csf. in a 1 patient with familial cyclic neutropenia cycle length shortened from 21 to 14 days. in another patient with acquired idiopathic cyclic neutropenia the cycle length of 120 days remained constant but re t w 2 w 4 w the nadir of anc rose from 0 to 400/#1. this patient was taken off therapy because of urticaria related to g-csf on day 850. there were no further significant adverse events. no loss of effectivity was observed during long-term treatment. we conclude that g-csf is safe for long-term treatment of idiopathic neutropenie with severe preceeding infections. as response to treatment is quic, it may also be an effective interventional treatment in acute infections in these patients. cytokines and growth factors are widely used to promote growth and proliferation ofbematologic cell populations. improvement or wonnu healing by stimulation with g-csf has been relxn'ted in patients suffering from kostmann syndrome, felty syndrome or from neutro-~ nia due to chemotherapy. e report on two patients with mds/ra (ha, female, age 76; sh, male age 82); duration of disease was 3 months and 5 years, respectively. ha was admitted for neutropenia (neuu'ophils: 0.45-0.9 g/l), epistaxis and a growing ulcerous wound in the pubic area (diameter 60 mm) already pretreated with antibiotics for 10 days. surgery was not possible due to poor heart condition and thrombocytopenia refractory to donor platelets. 30 mu g-csf were administered subcutaneously daily for 5 days resulting in neutrophil counts of 3.15 g/1 and effective wound granulation and epithelialisation. the patient died of cardiac failure on day 10. sh was adnutted for infected hematoma of the left thigh. subcutaneous infection progressed due to severe neutropenia (neula-ophils < 0.5 g/l). incision and resection (ulcus diameter 30 ram with deep invasion into the fascia) was performed. 20 days later the defect measured 120 x 50 mm, reaching the knee joint, and the patient underwent a second surgical interventaon. enterococcus, staphylococcus epidermidis and bacterium xerosis could be cultured from direct swabs. therapy with g-csf at a dose of 30 mu s.c. was started on day 61. neutrophils reached 19.5 g/i and g-csf was reduced to every other day. complete wound healing without any further surgical intervention was achieved by day 88 and sh was dismissed. after discontinuation of g-csf the patient is well and has normal differential blood counts. we conclude that g-csf is successful in promotion of wound healing in mds patients due to enhancement of neutrophil production. leukemic conversion of mds during g-csf was not observed. we report on a 44 year old male patient presenting in 5/88 with moderate thrombocytopenia~ transfusion dependent macrocytic anemia and normal., wbc. trephine biopsy showed hyperceuular marrow without fibrosis wzm trilineage dysplasia (mds/ra). cytogenetic analysis was 46,xy. in 10/88 vasculilas ~as diagnosed. from 10/88 to 12/88 three cycles of gm-csf (250 #g/m s.c. -14 days) were administered, resulting in both transient leukocytosis and increased platelet counts_ bone marrow aspirations showed dysplasia but no blast proliferation. in 5/89 vasculitis progressed, splenomegaly and hemolytic anemia developed requiring prednisolon. in 3/90 high dose erythropoietin was started (400 iu/kg i.v. twice weekly) and continued till 6/90. there was no change clinically, bone marrow smears and cytogenetics. in 10/90 the patient complained of pain in the lumbosacral region and neuralgia in both legs developed; a ct scan was negative. both pain and neurological symptoms (paraplegia and sensibility disorders) progressed. act scan and an mrt showed an intraspinal tumor (d2 -d11). "although severe thrombocytopenia refractory to high dose i.v. immnnoglobulin and platelet support (hla class i & lymphocytotoxic antibodies) developed, therapeutic laminectomy was performed in 6/91, but only a part of the tumor could be resected. histologically the tumor consisted only of erythropoiesis with dysplasia without excess of blasts. wound healing was without complicatmns. after surgery gamma irradiation and therapy with ifnc~ (6 #g s.c. 3 times/week) were performed. the patient recovered totally from neurological disorders and is still alive but iransfusion dependent because of severe cytopenia. we conclude: intraspinal extramedullary hematopeiesis is a rare symptom in mds. althoughthis infiltration was diagnosed months after gm-csf a_ad hd-epo therapy, it could be induced by cytokine therapy. ( monoclonal antlbodles elther' with or without gam crosslinking. in addition, we added the cytokines. the phenotypic change of expression of fcy receptors was measured. ~o 2 production and calcium flux using the dihydrorhodamine (dhr) and fluo-3 am methods, resepectively. there were no changes in expression of fcy receptors, but a significant enhancement of pmn activation via fc receptors by all three cytokines. we observed an increase of h202 production 4.5 fold by g-csf, 2 fold by gm-csf and 3.5 fold by il-8. a fcyriii-b specific monoclonal antibody fgr pmn (id3), which was alone unable to mobilize ca i+, together with all three cytokines was a potent stimulator. the effects of gm-csf and g-csf were calcium independent, in contrast, il-8 also enhanced calcium mobilization significantly. in summary, all three cytokines potentiate the fcy receptor activation of pmns and therefore play a significant role in inflammatory granulocyte activation as in leukocytoclastic vasculitis. g-csf is a hematopoetic growth factor required for proliferation and differentiation of hematopoeitic progenitor cells. it is now being successfully used to overcome neutropenias of various etiologies. recently, we demonstrated that rhg-csf induced neutrophils from patients with severe congenital neutropenia showed altered surface marker expression (upregulation of fcyri (cd64} and cd14 and downregulation of fcvriilicd16)) as well as decreased chemotaxis towards a variety of chemoattractants including fmlp. to separate the effects of the underlying disease from those of the rhg-csf therapy, we investigated neutrophils from patients receiving cytotoxic chemotherapy (n = 6) and healthy adults (n = 5) after application of rhg-csf. results: neutrophils from 6 patients receiving daily application of rhg-csf (neupogen ~ 300#g sc.) were studied ex vivo one day before, three times during and 3-5 days after cessation of rhg-csf treatment. expression of fcvri, cd14 and cd54 (measured by flow cytometry) increased during therapy reaching a maximum at 3-5 days after initiation of rhg-csf therapy, whereas expression of fcvrll! decreased to a minimum after 6-9 days. chemotaxis of neutrophils under agarose towards fmlp was also reduced during therapy. investigation of surface marker expression and chemotaxis 3-5 days after cessation of rhg-csf revealed return to levels before therapy. to exclude the possibility that the observed alterations were caused by the underlying disease or chemotherapy, five healthy adults were treated with a single dose of rhg-csf (neupogen', 300pg, sc.). a continuous upregulation of fcvri and cd 14 starting 3h after application with a maximum after 48 hours (fc~l) and 18 hours (cd14) and a downmodulation of fcrriii reaching a minimum at 48 hours was observed. chemotaxis towards fmlp decreased 3 to 4 h after application and returned to normal after 6 h, whereas expression of fcrri, cd14 and f%riii showed baseline values after 96 hours. conclusions: the results obtained from the healthy test subjects clearly demonstrate that neither the malignant disease nor chemotherapy, but rhg-csf induced the profound alterations of fcr receptor and cd14 expression and chemotaxis in neutrophils in vivo. the continuous character of the surface marker alterations without appearance of subpopulations and the increase in cd14 expression suggests that preactivation rather than immaturity of rhg-csf induced neutrophils alone might be responsible for the observed phenomena. fraunhofer institute ita, nikolai-fuchs-sral3e 1, w-3000 hannover 61 severe congenital neutropenia (scn) is a disorder of myelopoiesis characterized by a maturation arrest on the level of promyelocytes with absolute neutrophil counts below 200/pt in the peril~heral blood. in this study we investigated the expression of receptors for the granulocyte colony-stimulating factor (g-csf) on neutrophils from patients with scn during g-csf therapy. the normal g-csf receptor expression on neutrophils is in the range of 480-1200 receptors per cell. neutrophils from scn patients express increased numbers of receptors in the range of 2100-3900 receptors per cell. the dissociation constant of the binding of g-csf to the g-csf receptor is not altered as compared to healthy donors. in contrast neutrophils from patients suffering from cyclic neutropenia express normal g-csf receptor numbers (400-900 receptors per ceil). in addition, we have compared the g-csf receptor cdna of neutrophils from healthy donors and scn patients using the polymerase-chain-reaction technique. we could not detect any major alterations in the g-csf receptor cdna in scn patients. preliminary cdna sequencing data also did not reveal any point mutation. from this data we conclude that there is no defect in g-csf receptor expression and no alteration in the sequence of the g-csf receptor mrna in scn. pediatric hematology and konstanty-gutschow-str. 8, d-3000 hannover 61 oncology, medical school hannover, severe congenital neutropenia is a disorder of myelopoiesis characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmenn's-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median anc below 500/1~1 (cyclic neutropenia). we have treated 32 patients with scn and 4 patients with cyclic neutropenia. thirty of 32 patients with scn and all 4 patients with cyclic neutropenia responded to rhg-csf treatment with an increase of the median anc to above 1000/ixl. the doses needed to achieve and maintain the response varied between 0.8 and 120 i~g/kg/d. long-term treatment did not exhaust the myelopoiesis: the mean anc remained stable up to 5 years of treatment. the increase in anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment episodes, and reduction of hospitalizations. no severe bacterial infections occured in any of the rhg-csf responders during long-term treatment. severe adverse events, most likely associated with the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in 12 patients. these results demonstrate the benefical effects of rhg-csf treatment in severe congenital neutropenia patients. fifty-two patients (pts) (median age -51 years) with philadelphia chromosome positive (ph 1+) chronic myeloid leukemia (cml) have been treated with ifn (5 x 106 units/m 2) within six months of diagnosis (median 1.5 months (mths), range 1 -6 mths). we divided the pts into three groups according to sokars classification: low risk group (n = 24), intermediate risk group (n = 19) and high risk group (n = 9). forty-three pts acheived a complete hematological response (chr) as defined by the houston criteria. the cytological response was evaluable in 40 pts: 23 pts (57.5 %) demonstrated a partial or major eytogenetical response (more than 65 % ph i negative metaphases). the hematological and cytogenetical responses were influenced bsr the risk factors, os the percentage of chr and cytogenetical responses was higher for the pts from the low or intermediate risk groups (88 % and 41% respectively) than for the high risk group (55% and 22 %). transformation occurred in four pts who did not demonstrate a cytogenetical response. the estimated chance of surviving at three years was 83% for the overall population. toxicity was mild but ifn had to be internpted in four pts for cardiac (n = 2), liver (n = t) or neurological (n = 1) tocxic effects. these results confirm that ifn is a very effective treatment for cml. the effect of rhg-csf on platelets was studied in 20 healthy volunteers with the thrombometer, a specially developed device which is described in detail. additionally, conventional aggregation tests were performed low doses of rhg-csf enhance functional platelet activity, as shown by significant acceleration of the occlusion of the thrombometer channel. similar results were found in conventional aggregation tests using collagen for induction. with g-csf concentrations of 0,1 and 1,0 ng/ml the time of response was significantly accelerated and the maximum response was observed in a higher proportion ofplatelets. however, the second phase of aggregation induced by epinephrine was significantly inhibited by 1,0 ng/ml g-csf. the expression of cd41, cd42 and cd 62 on platelets' surface was determined in ten patients before and niter administration of g-csf (facscan flow cytometer).quality controls were done by calculating the events positive for cd41 and cd42, which were expressed in nearly t00 % of the platelets without being changed by the cytokine. the expression of cd62 in the platelets' surface however was significantly enhanced indicating a depletion of the c~-granules. no platelet aggregation was observed. cd62 expressed on thrombocytes' or damaged endothel cells' membrane is a receptor for macrophages. this property facilitates rapid adhesion of leucocytes to endothelium at regions of tissue injury as well as platetetleucocyte interactions at areas of inflammation and hemorrhage.-in contrast cd62 can also have an antiinflammatory function because exposure of tnfa-activated neutrophils to plasmatic cd62 inhibits their cdl8-dependent adhesion to resting endothelium and superoxide production. ifn a has a unique activity in cml leading to complete and partial remissions in 15-30% of the patients. to improve these results, we are currently treating patients with ph'+ cml with a combination of cytosine-arabiuoside at a maximum dose of 20 mg/m z sc on 5 days per week and ifn c~-2b. ifn u-2b is started at a dose of 3 muim 2 sc dally and escalated to the maximum tolerated dose. 48 patients (25 male, 23 female, median age 44 years) have been entered into the trial. 15 patients have been pretreated with other regimen for a median time of 32 months. 33 patients are without pretreatment. the treatment has been well tolerated. besides the ifn a related side-effects some patients experienced gastrointestinal toxicity with nausea and vomiting after prolonged ara-c application. the median observation time in the study is now 8 months and patients are still entered. up to now complete hematologic remissions have been achieved in 24 patients, and partial ones in 15 patients. the rate of complete hematologic remissions was higher in patients without pretreatment (55 %) compared to patients who have been pretreated (40%). five partial eytogenetic remissions have been observed and 3 minor reductions in the ph'+ cell done. all cytogenetic responses have been found in patients without pretreatment. we conclude that a combination of cytosine-arabinoside and ifn u-2b is well tolerated in patients with cml. early results are encouraging. longer follow up times are necessary to evaluate whether combination therpy will give superior results compared to a txeatment with ifn a alone. during rlfn-~2 therapy a minority of patients develops high-titered antibodies neutralizing the injected rlfn-a2. the rlfn-c~-andbodies from six of such patients, who lost their clinical response to rlfn-c~2 and showed a relapse of their leukemia (3 cml, 3 hcl) despite continuous rlfn-c~2a-therapy, as well as ifn-c~-specific antibodies from two patients with systemic lupus erythematusus (sle) were characterized. the anti-ifn activity was purified by sequential protein g -and rifn-c~2 affinity chromatography and was found to consist only of igg-antibodies. these antibodies were further tested for their capacity to neutralize the antiviral and antiproliferative activity of various rifns-c~-subtypes. all six sera tested showed a common pattern of neutralization (mdbk-vsv bioassay) distinct from the sleantibodies. all six neutralized rifn-c~a and rifn-ak consistently with a higher titer against aa. three of the six sera neutralized aa, ak, ~c, ~c/j1 and m, but not m, m1 and some other subtypes. therefore, from the structure of the c/jl-hybrid, it seems that one epitope recognized by these three sera is at the nh2-terminal half of the molecule. in contrast, the sle associated antibed]es neutralized the antiproliferative and antiviral activity of every subtype tested. these data indicate that the therapy-induced antibodies against rifn-c~2 recognize very selected epitopes on the rifn-cd-molecule suggesting that only a part of the rifn-~2 molecule is immunogenic. in vitro experiments indicate higly synergistic effects of combining ifn with cytostatic drugs such as anthracylines (a). in a phase i/ii-study 31 patients (pts) with pro$ressive inoperable hcc were treated with e 20mg/m z weekly x 4 and ifn 3 mio iu/m 2 s.c. 5 x weekly for 4 weeks, followed by one week off treatment. in case of at least no change (nc) and tolerable toxicity the therapy was continued. escalation of e in steps of 5mg/m z per cycle was attempted. pts characteristics: median age 56 years (21 -69); male 20, female 11 pts. pretreated with a 3 pts. toxicity and treatment: total number of cycles 92; median 3 (1-9) per pt. worst toxicity per pt (who): wbc ~ 38%, ~ 6%; platelets ~ 9%, ~ 0%; diarrhoea ~ 5%, ~ ~%; n~isea/vumitinq ~ 5%; ~ 0%; ifn related fever (maximal ~ in 53%. ifn-related wasting syndrome 2 pts, no severe organ toxicity. divisione di ematologia -ospedale san camillo -roma from november 1987 to october 1989 six patients with acute leukaemia, who achieved their first complete remission with standard chemotherapy followed by autologous bone marrow transplantation (abmt), were consecutively treated with r.interferon alfa-2a (ra-ifn). patients (4 all and 2 anll) were from ii to 44 years old, 4 of them (2 anll and 2 all) were reinfused with autologous bone marrow purged with asta-z i00 mcg/ml/ 2 x 107 cells and in the remaining 2 all patients immunomagnetic purging was employed. conditioning regimens were bucy in 4 patients and cy-tbi in the others, ra-ifn started at median time of 6 months (4-11) after abmt when complete consolitated hemopoietic recovery occurred. the ra-ifn dose was 500.000 iu/sqm 3 times a week for 2 years. none of the 6 patients presented significant toxicity and only 3 short suspensions occurred for fever or alt level increase. the incidence of infectious complications were particularly low compared with other autotransplanted groups of patients who received similar antinfectious prophilaxis. one case six months after abmt and 20 days after acyclovir prophilaxis interruption presented a mild herpes-zoster complication which required new acyclovir therapy and resolved i0 days later. the amount of these patients is extremely low because the study was early interrupted to start a new protocol including il-2; but the long duration of the good continous complete remission (3/5 years after abmt) in all these unselected and consecutively treated patients is very interesting. surgical procedures may be associated with an inceased risk of tumor spreading due to surgical mobilisation of the tumor and transient postoperative immunosuppression. recurrences may result either from early growth of micrometastases already present at the time of surgery or from the seeding of malignant cells shed during operative manipulation of the tumor. imrnunomodulators have been proposed to correct the immunological impairement induced by surgical procedures. from 1/92 to 7/92, 23 patients with advanced stage cancer underwent surgical resection with peri-operative interferon-alpha administration. patients received interferon alpha-2a (roferon-a), by daily subcutaneous injection for fourteen days, starting on three days before surgery. incremental doses were 2,3,5,7,9 and 12 x 10 6 iu for 4,4,3,3,3 and 6 patients respectively. peripheral blood lymphocyte (pbl) subset numbers were assessed using flow cytometric analysis the day before injection (d-3), before surgery (d-i), at the day 4 and 12. absolute numbers of total t cell (cd 3+) and nk cell (cd 56+, cd 3-) were determined, as well as auxiliary t cell (cd 4+), activated t cell (cd 3+, dr+), and b cell (cd 19+) counts. short-term cytotoxicity of pbmc against k 562 and daudi target celts in a 4-hour standard chromium release assay were determined. no w.h.o. grade iv toxicity were observed. a significant post-operative fall of the total pbl count, of cd3+; cd4+; cd19+; cd3-56+ occured from d-3 to d4. the decrease were not significant for cd3+dr+. values were not significantly different, between d-3 and d12, only for cd3dr+ and cd3-56+. cytotoxicity against k 562 and daudi target ceils increased significantly from d-3 to d-l, and from d-i to d1, with a significant fall of cytotoxicity against daudi from d-3 to d4. peri-operative interferon alpha administration is well tolerated even at the 12.10 6 iu doses. in spite of treatment and increasing cytotoxicity activity, we observed a post-operative fail for the majority of the imunological parameters. further studies are necessary to compared with a control group, with more patients treated with 12.10 6tiiu daily. patients: six patients have been treated (med. age 29 yrs.; 24-53 yrs.) four patients had acute myeloid leukemia (aml m2: 2, m4: 1, m4eo: 1). two patients had acute lymphocytic leukemia (both t-all). all patients had manifest disease with more than 50% blasts in the bone marrow. patients were selected not to have rapidly progressive diesease allowing the application of the cytokine. treatment and toxicities: ifn a was given for a median of 31 (14-114) days at a dose of 5-10 mu sc daily. the following toxicities _>grade 3 who were observed: fever 5, gpt 2, pulmonal 1, infection 4 (1 pneumonia), pains 1. the patients with aml ware transsusion dependent for platelets and erythrocytes. no significant additional bleeding was observed. results: one aml patients had stable disease and three had disease progression. of the all patients one had disease stabilization and one progression. conclusions: ifn a is well tolerated in patients with refractory aml and all if they ere in a relatively stable condition. the effectivity of ifn a-2b as a single agent is poor in patients with refractory aml or all. freund et al. eds, springer-verlag, 1992) have confn'med the wide range of clinical usefulness of ifn alphabased therapy in cancer patients (pts). we present in this paper a retrospective analyses of 102 cases with advanced neoplasias treated between 1986-1992 by a sequential administration of ifn alpha and standard chemotherapy. there were 71 w, 31 m, aged 18-76 y, with solid cancers 99 pats and lymphomas 3 pts. the treatment consisted of a sequential association of ifn alpha (also with ra all-trans) and cht plus tamoxifen (for appropriate cancers). the ifn schedule was of monthly series, one series consisting of 3 million iu/d for 3 consecutive days. cht, appropriate for each primary cancer also administered in monthly series. the results are grouped according to the status of the disease (subsets of pts) at the onset of ifn-based therapy, and refer especially to long term survival (1-5 y +). for minimal residual disease (mrd) from 39 cases there were 37 cr (24/24 breast ca, 6/6 cr melanomas, each sts and rcc 2/2 rc, 2/4 gastric and head and neck cancers together, 1/t lymphoma. for progressive disease, pre-lfn-based therapy there were 63 pts, and post ifn-cht treatment there were 58 sd and 5 pd. in 3 of failure pts association of ifn alpha and bropirimine (ifn-inducer) appeared an unusual good response. conclusions: 1) ifn alpha-based therapy is a very useful one especially in mrd. 2) the therapy must be individualized for selected subsets of pts and for each patient day to day. authors have used inf alpha 2b in cases of haematological malignancies for three years. the number of cases not too high, and non fo cml cases was so called "early" cml. our cases are: 5cml, 3non-hodgkin malignant lymphoma, 4 myeloma multiplex, 3 essential thrombocythemia. the tnf alpha 2b was used as monotherapy in the cases of low grade non-hodgkin malignant lymphoma, and essential thrombocythemia, and was combined with chemotherapy in the other cases. on the basis of our initial results, we recommend the inf alpha 2b in the treatment of haematological malignancies in suitably selected cases. department of haematology and oncology, hospital of ministry of interior, vftrosligeti fasor 9-11 the influence of low oral doses of human leukocyte derived interferon alpha on the immune system of chronically hbv infected patients with depressed immunological response. low oral doses &the interferon were given to a group &seven children with limphoblastic acute leucaemia in the state of remission, chronically hbv infected. interferon alpha was produced by hayashibara biochemical laboratories inc. okayama, japan, in tablets of 50 iu and 100 iu respectively. immunological response was checked by measuring population and subpopulations of limphocytes, level ofimmunoglobulin and complement c 3 fraction. a distinct stimulation of cellular immune response was observed: the fraction of activated limphocytes t increased significantly, index cd4/cd 8 became normal and population of limphocytes b increased gradually. there was no influence of interferon treatment on immunoglobulin and complement c 3 fraction serum level. the interferon treatment improved the patients' general condition and shortened the period of intoxication after cytostatic treatment. no side effects were observed. none of the children eliminated the hb virus during the six months treatment. cytokines play an important role in activating the immune system against malignant cells. one of these cytokines, il-4 has entered clinical phase i trials because of its immunoregulatory potency. in the present study we report that rhll-4 has direct antiproliferative effects on some human lung cancer cell lines in vitro as measured by a human tumor cloning assay (htca). this activity could be abolished by neutralizing antibody against rhll-4. the biological response of the tumor cells to the cytokine is correlated with expression of receptors for bll-4 on both the mrna level and the protein level. the most responsive cell line ccl 185 secretes il-6 after being incubated with rhll-4. on the other hand, neutralizing antibodies against il-6 showed no influence on the growth modulatory efficacy of rhil-4 in this cell line. furthermore, ccl 185 does not show detectable production ot il-1, tnf-~ or ifn-y alter incubation with rhll-4. thus, the response to rhll-4 is not mediated through autoeriee production of these cytokines triggered by rhll-4. in a next series of experiments the ceillines were xenotransplanted to balb/c nu/nu mice. subsequently, the mice were treated for >12 days with twice 0,5 mg/m2 rhll-4 (rhll-4 was a kind gift from dr. urdal, immunex, seattle, usa) or control vehicle subcutaneously per day. treatment with rhll-4 yielded a significant inhibition of tumor growth versus control in the responsive cell lines ccl 185 and htb 56, but no therapeutic effect in the non-responsive cell lines. plasma levels of rhtl-4 were sufficient for in vitro growth-inhibition in the responsive lines. histology of the tumors in both groups showed no marked infiltration of the tumors with murine hematopoietic and lymphocytic cells consistent with the species specificity of il-4. we conclude that rhll-4 has direct antiproliferative effects on the growth of some human lung tumor cell lines in vitro and in vivo which together with its regulatory effects on various effector cell populations makes this cytokine an interesting candidate for further investigation in experimental cancer treatment. the combination of systemic chemotherapy and immunotherapy comprising interleukin-2 and alpha-interferon leads to significant tumor regressions in patients with advanced malignant melanoma. in contrast to chemotherapy by itself, the combination produces a significantly extended remission duration in the majority of treatment responders. we conducted 2 phase ii studies.to assess the potentially additive or synergistic effects of chemotherapy and immunotherapy in metastatic malignant melanoma patients: the first study comprised two cycles of carboplatin (400mg/m 2) and dacarbazine (750mg/m~); the sesond study included up to four cycles of cisplatin (25mg/m 2 x3 days), dacarbazine (220mg/m 2 x3 days), bcnu (150mg/m 2, cycle i+3) and tamoxifen (20mg daily). chemotherapy was followed by up to 2 cycles of a 6-week immunotherapy comprising interleukin-2 (5-20 million iu/m ~ sc 3x weekly) and alpha-interferon (3-6 million u/m 2 sc 3x weekly). among 25 evaluable patients in study i, there were 9 (12%cr, 24%pr) objective responders; median remission duration was 18+ months for complete, and 10+ months for partial responders. chemotherapy intensification in study ii lead to an increased response rate of 50% (9 out of 18 patients). in both studies, the progression free interval was significantly extended when compared to patients who received chemotherapy, only (historic controls). the role of immunotherapy as maintenance in patients with advanced metastatic malignant melanoma is currently being evaluated in a prospective randomized trial. integrin receptors play a crucial role in cell-cell and cell-matrix adhesive function, and thus are supposed to influence invasion and metastasis. very little is known about the impact of interleukins on integrin regulation in tumor cell lines. therefore, we investigated the expression of 7of and 41i integrin subunits on well (ht29) and poorly differentiated (sw620) human colon cancer cell lines using a panel of specific monoclonal antibodies and cdna probes. ht29 and sw620 expressed similarly high levels of ~1, a2, ~ gt, and f~4 subunits on the cell surface. no a4, ~2, and 1~ 3 was detected on either cell line. while a 5 was not expressed on ht29, sw620 showed higher levels of the laminin receptor ~694. the poorly differentiated cell line sw620 was resistant to il-4, whereas ht29 was sensitive. treatment with il-4 induced a decrease in ~2, a3, '~6, ~v, l~l, and 134 integrin expression. however, ~1 subunit was markedly upregulated. in contrast to il-4, there was no evidence that il-lfi could modulate integrin expression on these celt lines. the function of integrin receptors was assessed by measuring adhesion to collagen, laminin, vitronectin, and fibronectin. il-4 significantly increased the adhesion of ht29 to fibronectin, while attachment to collagen, laminin, and vitronectin remained unchanged. these results suggest differential integrin expression pattern on well and poorly differentiated tumor cell lines. we provide evidence that integrin expression may be selectively regulated by il-4, but not by il-ib. furthermore, il-4 can alter adhesive behavior of tumor cells. since il-4 is currently studied in clinical trials, the metastatic potential of malignant tumors should be monitored thoroughly. immunotherapy with ifncz and il-2 is an active regimen in malignant melanoma and has shown response rates of 20 -30%. in previous studies no prognostic parameters for response could be identified. 72 patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifno~ and high dose il-2 since 1987 with an overall response rate of 30%. 59 patients with mm treated in our phase ii trials could be analysed to identify possible prognostic parameters for response. patients were divided into three groups: responder (3 cr/14 pr), stable disease (16 sd/2mr), and nonresponder (24 pd). all patients had measurable tumor, a karnofsky index of > 70%, no cns metastasis, and no severe cardiorespiratory or renal disease. we examined the following pretreatment parameters for prognostic relevance of response: age, sex, performance status, time from diagnosis to onset of first metastases/ to begin of immunotherapy, tumor toad, number of metastatic sites, organ sites of metastases, ldh, ap, esr, and hla-type. of these several variables were found to significantly correlate with response: tumor load (p=0.023), number of metastatic sites (p=0.045), serum ldh (p=0.005) and ap (p=0.013). tumor load, ldh and ap are no independent parameters. while time from diagnosis to onset of first metastasis is of no prognostic significance for response, the time between first diagnosis and begin of immunotherapy, usually reflecting metastatic disease necessetating systemic treatment, significantly correlates with probability of response (p=0.018). since several hla class i alleles have been shown to function as restriction elements for recognition of melanoma cells by specififc t cells in vitro, namely a1, a2, b44, and cw7, we compared the frequency of these hla antigens between responder and non-responder. we found a1, b44 and cw7 to be increased in responder vs. non-responder. our results indicate that in patients with mm tumor load, number of metastatic sites, ldh, and time from diagnosis to begin of immunotherapy are prognostic parameters for response to immunotherapy. these parameters may be useful to determine patients with good and poor risk for response to immunotherapy and are of relevance for stratification in randomized clinical trials. dept of medicine, university of heidelberg, hospitalstr. 3, 6900 heidelberg, germany surgery of metastatic melanoma following successful il-2 based immunotherapy. u keilholz 1 , e stoelben 2, c scheibenbogen 1 , hd saeger 2, k neumann 3, w hunstein 1 surgery of advanced metastatic melanoma is of limited value and usually not recommended. immunotherapy using high dose il-2 is effective in a substantial proportion of patients, however, the duration of responses is limited, and benefits in survival are not yet proven. this evaluation was done to determine the value ot resection of residual tumor lesions following successful immunotherapy. 72 patients with progressive metastatic melanoma have been enrolled in various immunotherapy trials including ifna and high dose il-2 since 1987. 37 patients showed evidence of antitumor response (4 cr, 15 pr, 18 mr/sd). in patients responding to immunotherapy, residual lesions were resected, whenever technically possible and patients agreed to surgery (13 patients). 22 of the 24 responding patients without surgery relapsed, the median time to progression was 5 months (range 2-14), almost all initial relapses occured locally, 8 patients died so far. 11 of 13 patients who underwent surgery (9 pr, 4 mr/sd) were converted into cr by surgery. 4 of the 11 patients disease-free after surgery relapsed, 3 locally (2, 5, and 9 months after surgery), and one cns 9 months after surgery. 7 patients are still free of recurrence (3+,3+,6+,12+,16+,18+,28+ months after surgery) and 9 of 11 are still alive. in the patient with cns relapse complete resection of this lesion was again possible, and there was no evidence of recurrence for 9 months after this second surgery. histology revealed vital tumor cells in almost all resected specimens, however in 11 of 13 patients profound necrosis of the tumor tissue was observed. of special importance is the observation that 3 patients with minor response or sd according to imaging procedures were found to have an almost complete response histologically. interestingly, almost all metastases resected after immunotherapy had developed a fibrous capsule. surgical reevaluation and resection of residual lesions should be considered in patients with partial response after immunotherapy, and in selected cases also with stable disease. this approach offers the chance for extended disease free survival, and may be curative in certain patients. t-cell-receptor (tcr) vcz~ usage of tumorinfiitrating t-cells in primary, regressing and progressing melanoma metastases following [mmunotherapy with ifn~ and il-2: evidence for a specific t-cell response. mshler, t., willhauck, m., scheibenbogen, c., pawlita, m.#, bludau, h.#, brossart. p.. keilholz. u. the identification and characterization of immunological effector cells mediating tumor regression in immunotherapy with il2 is of great interest for understanding and further development of this therapeutic approach. tumor infiltrating lymphocytes specific for autologous tumor cells can be expanded from certain melanoma tissues. t cells recognizing the same antigen use a limited tcr repertoire with a certain vc~ and [3 variable region, determining the specifity of their receptor. we therefore analyzed t-cell receptor v-regio,q distribution in tumor tissue from melanoma patients prior to and following immunotherapy with il-2. we used a highly sensitive rna-pcr method. after rna-extraction from tissue and subsequent cdna-synthesis semiquantitative pcr with different primers for all known vc~-and vii-t-cell receptor gene families (18 vc~ and 20 v~) was pedormed. 12 tumor tissue samples were analysed including 6 samples of primary malignant melanoma and tumor samples of three patients after immunotherapy. the results were compared to control tissues (peripheral blood, unatfected skin, and liver tissue). the analysis of primary malignant melanoma tissue showed a weak overexpression of different v[3-families. preferential usage of different tcr-v~,genes was more obvious in tumor tissue of patients alter immunotherapy. of special interest is a patient with a mixed response to immunotherapy with progressing and regressing skin metastases, in the regressing lesion we could demonstrate a predominant usage of tcr-v[~11-gene almost lacking in the progressing lesion. this suggests a role of v[311-expressing t-cells in mediating tumor regression in this patient. cloning and sequenzing analysis are currently performed to assess wether this represents a true clonal t-cell proliferation. recently a highly sensitive assay combining reverse transcription and polymerase chain reaction (rt/pcr) to assess for melanoma cells in peripheral blood has been developed. the detection of tyrosinase mrna, a tissue spezific enzyme in melanocytes and melanoma cells in peripheral blood indicates the presence of melanoma cells, we used rt/pcr assay to determine malignant melanoma ceils in peripheral blood of 43 patients with malignant melanoma in different stages of disease. in none of 8 patients with stage i (localised tumor) but in 5 of 14 patients in stage ii (regional lymph node metastases) tyrosinase transcripts were detected. tyrosinase mrna was found in all 21 patients with distant metastases (stage iii). this method may be helpful to define a group of patients at high risk for development of hematogenous metastases, that would be a possible target group to explore adjuvant treatment strategies. we then examined blood samples and bone marrow aspirates of 28 patients with metastatic malignant melanoma for presence of melanoma ceils prior to and after therapy with ifn-a and il-2. 10 patients showed antitumor response to immunotherapy: 3 complete remissions (cr) and ? partial remissions (pr r. hilse, m.meffert, j.grosse, h.kirchner, h.poliwoda, and j.atzpodien we investigated the use of pcr for a semiquantitative estimation of cytokine expression patterns in pbmc before and after administration of il-2 to patients with advanced renal cell carcinoma or malignant melanoma, mrna of 9 cytokines was measured using a modified polymerase chain reaction protocol, which could detect 10-fold differences in mrna-contents of stimulated pbmc in vitro. weekly rna-samples of 7 patients receiving a total of 11 treatment cycles were examined for long term changes, in 2 patients frequent samples were taken immediately after tl-2-administration for transcript-kinetics, mrnaexpression for il-4, il-5, il-6, ifn-)', tnf-c~, gm-csf, tgf-~ and il-2receptor-c~ was clearly detectable in most of the samples, including four healthy donors. however, our method could not detect significant changes in transcript-levels of pbmc during 3 days following injection of (a) 36 mio.lu or (b) 2x18 mioju dl-2 daily. this was in marked contrast to cytokine secretion assayed by elisa. thus, serum il-2 peaked 2-4 hours after administration followed by secondary cytokines with a peak 2-16 hours later. increases for tnf-m ifn-7, il-6 and il-2r serum levels were significant (p<0,05) with the highest response found for il-6, increasing 35-(a) and 32-fold (b) at day 1, or 8-/ 14-fold at day 2. comparing normal individuals to patients, only small differences in constitutive cytokine expression were seen (<10-fold) with no distinct pattern. during therapy, changes could be seen for all cytokines except for il-2 and tgf-i 3. in one patient, a 100-fold increase for il-6, tnf-c~ and ifn-7 transcripts was observed during week 4 of the second treatmentcycle, other changes were approximately 10-fold. abt. h&matologie und onkologie, medizinische hochschule hannover, d-3000 hannover 61, germany regional immunotherapy: perfusion of liver metastases with lak cells u. keilholz, c. scheibenbogen, m. brado, w. tilgen, and w. hunstein a regional approach of adoptive immunotberapy with interleukin-2 and lymphokine activated killer cells for the treatment of liver metastases is reported. the treatment consists of continuous infusion of interleukin-2 i.v. or into the splenic artery, and transfer of ex vivo generated lymplaokine activated killer cells into the portal vein or the hepatic artery. 15 patients with malign ant melanoma, 2 with renal cell carcinoma, and 1 with thyroid carcinoma have been treated. all had progressive liver metastases. trafficking studies using indium-oxine labelled cells revealed that > 80 % of the lak cells remained in the liver after regional adoptive transfer. in 9 patients with liver metastases of cutaneous melanoma, 2 cr (24 and 23+ months), 1 pr (9+ months, converted to cr by surgery), 2 sd (10 and 11 months), ad 4 pd were observed. the lesion in the patient with pr was resectable after two cycles of treatment, and histology revealed almost completely necrotic tumor tissue surrounded by a dense fibrous capsule. no responses were observed in 6 patients with liver metastases of ocular melanoma, suggesting an immunologic difference between these two melanoma subtypes. 1 pr (6 months) and 1 sd (10 months) were achieved in 2 patients with renal cell carcinoma, and 1 sd (6 months) in the patient with thyroid carcinoma. evidence for the crucial role regional cell transfer is provided by the observation in a patient with an anatomic variation of hepatic blood supply in whom we achieved complete and durable tumor regression. in this case anti-tumor responses were only observed in anatomic areas of the liver which were perfused with lak cells. depts. of internal medicine, diagnostic radiology, and dermatology, hospitalstrage 3, 6900 heidelberg, germany in a randomized phase ii study we evaluated the response and side effects of a combined administration of interleukin-2 (il-2) and interferon-alpha 2b (ifn-alpha 2b) versus interferon-gamma (ifn-gamma) in patients with metastatic renal cell cancer. patients in group a received subcutaneous (sc.) 200 meg ifn-gamma once a week. in group b patients were treated with a (sc.) combination therapy of ill-2 (36 x 106 iu/m 2 in week 1 and 4, 18 x' 106 iu/m 2 in week 2,3, 5 and 6, twice a day for 5 days) and ifn-alpha 2b (5 x 106 1u/m 2 ) over 6 weeks once a day 3 times a week. up to now 60 patients were treated, 30 patients in each group. toxicity of ifn-gamma treatment was absent. the therapy with il-2 and ifn-alpha 2b led to sideeffects grade 2 (who): fever, chivering, fatigue and weight-loss. treatment were withheld in 20 %, follow up after 11 months (3-22 months) showed stable disease in 12 patients and progression inl 8 patients in group a in group b there were 4 complete remissions, 2 partial remission and 24 patients with progressive disease. although the combination therapy showed 20 % objective response (p<0.05, fisher-test) no significant improvement on survival was seen (p = 0.342 logrank test). patients with locally advanced renal carcinoma are at high risk of relapse after initial radical surgery. we initiated a clinical phase ii trial using autologous tumor vaccines for the surgical adjuvant therapy of renal cancer patients. seventy-two patients (pts) (25 female, 47 male; median age, 56 yrs; range, 28-77 yrs) with locally advanced renal carcinoma (pt3b-4pn0 or ptxni-2m0) received autologous newcastle disease virus modified and lethally irradiated tumor vaccines in combination with 1.8 million iu of il-2 and 1.0 million u of ifn-~2, once weekly over 10 consecutive weeks. toxicity was very mild with transient flu-like symptoms. among 55 evaluable patients, there were 5 relapses (2pts, pt3ani-2; 3pts, pt3bn0); the median relapse-free survival was 22+ months with a range from 6 to 41+ months; survival probability in this vaccine treated cohort was significantly better than in all historic controls. using western blot analyses, we could demonstrate a vaccine specific in-vivo b-cell response in all patients receiving ndv tumor vaccine. a subset of peripheral blood natural killer (nk) cells has been found to exhibit high density surface expression of the nk associated cd56 antigen; it has been suggested that these nk cells respond to lower concentrations of il-2 when compared to the majority of nk cells expressing cell surface cd56 at low density. we evaluated density of the cd56 antigen on circulating nk cells of 47 patients with advanced renal cell carcinoma by flow cytometry. patients received a combination of low-dose subcutaneous recombinant interleukin-2 (ril-2) at 18 million iu/m2/day on days 1 and 2, followed by 3.6 million iu/m2/day, 5 days per week, over 6 consecutive weeks, in combination with recombinant ~-interferon (rifn-~) at 5 million iu/m 2, three times weekly. antigen density of cd56 before therapy was found 2.2-fold higher (p<0.005) in patients who subsequently achieved a complete or partial tumor remission (n=10) when compared with patients who presented with progressive disease on therapy (n=ll). after a 6-week treatment cycle, nk cells of treatment responders expressed significantly (2.l-fold; p<0.005) more cd56 antigens than nk cells in nonresponding patients. these results suggested a potential role of both pre-and posttreatment nk antigen density levels as a biologic correlate to treatment response in tumor patients receiving low-dose ril-2 and rifn-a. intravesical immunotherapy against superficial bladder tumor recurrences and carcinoma in situ is a recognized and highly effective regimen in urology. to further clarify the mode of action of this approach, the local immune response of patients was investigated: the cytokines il-1, il-2, and tnf were determined in the urine before and after intravesical instillation by elisas and biological assays. furthermore, bladder biopsies taken before and after the treatment course were analysed by means of immunohistology for the presence of mononuclear cell subsets. the results show a significant increase of urinary cytokines with a maximum 4-8 hours after the instillation of bcg which returned to baseline values within 24 hours. this intense locai immune activation was further reflected by the accumulation of activated mononuclear cells, predominated by t cells as demonstrated with bladder biopsies. the local t-helper/t-suppressor cell ratio shifted towards the t-helper subset. these changes persisted for more than 1 year after the initial treatment course. in conclusion, this local immune response may be associated with the therapeutic success of bcg. further analyses will dissect the role of each factor with regard to antitumor cytotoxicity against bladder carcinoma. enhancement of therapeutic effect of intarleukin-2 (il-2) by association with cyclephcsphamide (cy) was studied on el-/+ lymphoma maintained in ascitic form in syngeneic c57bl/6 (h-2 b) mice and lymphoreticulosarcoma (spontaneous origin) maintained in solid form in syngeneic cba (h-2 k) mice. immunotherapy with il-2 (obtained by in vitro stimulation of el-4 lymphoma cells with phorbol myristate-acetate) was applied 24 hours after transplantation of el-4 lymphcma and i0 days after transplantation of lym phoreticulosarcoma be administration i.p. (intratumorel) in el-4 bearing mice and s.c.(peritumoral) in lymphoreticulosarcoma bearing mice for three consecutive days. cyclophosphamide was administered at a dose of 180 mg/kg i.p. six hours before the immune treatment with il-2. the results obtained demonstrated that the prolongation of the survival rate expressed by the median survival time (mst) and the percentage of increasing llfe span (ils) of the groups treated with il-2 associated with cy was significantly higher than that of the groups which receveid a single treatment with il-2 or cy. enhancement of therapeutic effects of il-2 in association with cy on lymphoreticulosarcema was revealed by inhibition of tumor growth with a marked regression in the vol~tme of the established tumors and even resorption in some cases. we conclude that the antitumoral effect of il-2 treatment was enhancemented by association with c2 a well known cytoreductive drug which selectively removed t-suppressor l~mphocytes from the tumor bearers. this could be conside red as an alternative to immune-or chemotherapy in cancer. to investigate the toxicity and clinical efficacy of aerosolized nil-2 (biotest) 15 patients presenting with advanced malignancy were entered into a phase i trial. 13 patients suffered from metastasizing renal cell carcinoma, 2 patients from advanced bronchial carcinoma. at start the patients received either 50000, 150000 or 300000 u nil2 applied as a single dose. if no adverse events were observed, treatment was continued with the same dose 5 times daily for six weeks. in addition to standard invetigations detailed evaluation of the respiratory function was performed once weekly. soluble interleukin 2 receptor serum levels and the effect on numbers and/or phenotype of lymphocytes in the bronchoalveolar lavage fluid were measured for assessment of biological response to inhalative nil-2 treatment. treatment with aerosolized nil-2 was well tolerated. most prominent toxicity appeared to be resistant cough in all patients treated with 5x300000 u/d. no febrile reactions or other constitutional side effects were observed. a dose-dependent increase of the numbers of memory t lymphocytes, macrophages and eosinophil granulocytes could be demonstrated in bal fluid. in addition, the treatment resulted an increased expression of adhesion molecules on lymphocytes. 1 patient suffering from renal cell carcinoma achieved a partial remission after 6 weeks of treatment with 5x50000 u/d. we conclude that treatment with aerosolized nil-2 is biologically active and well tolerated and should be further tested in clinical phase ii trials. divisions of hematology and pulmology of the iiird department of internal medicine, medical center of the johannes gutenberg university, w-6500 mainz, department of urology, univ. hospital eppendorf, hamburg. soluble interleukin-2 receptors (sil-2r) exert a potential role in immunoregulation. we investigated the ex vivo effects of sil-2r on several interleukin-2 (il-2)-dependent activation events. proliferation of the il-2-dependent mouse cell line ctll-2 and isolated human pbmc stimulated with recombinant il-2 (ril-2) was suppressed by sil-2r added to the culture medium in a dose-dependent way. preincubation of sil-2r with ril-2 did not enhance this suppression. cytotoxicity of ril-2-stimulated human pbmc against the human cell lines k562 and daudi was correlated inversely to the concentration of sil-2r in the culture medium during ril-2 stimulation. sil-2r concentrations higher than 4.0 pm produced a significant decrease in cytotoxicity (p<0.01). light microscopy of il-2-stimulated pbmc revealed no signs of cellular activation when high dosages of sil-2r had been added. the effect of different sil-2r concentrations added to cultured human pbmc on secondary il-2 and sil-2r production was tested by elisa. initial supply with high sil-2r dosages yielded weak increase and subsequent slow reduction of il-2 levels. in contrast, strong secondary il-2 production followed by rapid clearance was observed when low sil-2r concentrations had been added. endogenous shedding of sil-2r in response to ril-2 was abrogated by the initial exogenous addition of high amounts of sil-2r whereas low exogenous addition of sil-2r was followed by a continuing endogenous production of sil-2r after five days of culture. our studies may lead to a better understanding of il-2-related immunoregulation in the preclinical and clinical settings. we investigated the effect of interferons (ifn) on expression of il-8 and other cytokines regulating inflammatory responses in various cellular models in vitro and in vivo. in peripheral blood mononuclear cells (pbmnc) of healthy individuals il-8 gene expression which was upregulated in vitro was significantly reduced in presence of ifn-o~. in dose titration experiments a reduction of the il-8 protein was detected at ifn concentrations as low as 30u/ml. in cml patients with constitutive expression of il-8 a reduction of il-8 mrna expression was seen after therapeutic administration of ifn-a. by contrast, in lps stimulated granulocytes ifn failed to inhibit il-8 expression in vitro. we investigated the mechanism of il-8 inhibition in the thp-1 cell line more in detail. nuclear run on assays and rna decay analysis in presence of acinomycin d suggested that the effect was regulated predominant/y at a posttranscriptional level. de novo protein synthesis was not required since the inhibitory effect was also detected in presence of cycioheximide. in addition to il-8 expression we studied the effect of ifn-o~ on the synthesis of il-1, tnf, il-6 and il-1ra in pbmnc and bone marrow stromal cell cultures. these experiments revealed an antagonistic effect of il-1 action by ifn4x at two levels which was most striking in bone marrow stromal cells. expression of il-1 mrna was downregulated whereas the production of il-1ra was enhanced by ifn-c~. in contrast expression of il-6 and tnf was enhanced by ifn. we conclude that ifn-o~ differentially regulates proinflammatory cytokines. the inhibition of il-8 and il-1 action suggest an antiinflammatory role of type i ifns. in a phase i clinical trial of recombinant human interleukin-6 (il-6), 21 patients were entered to receive daily subcutaneous injections of il-6 over 7 days followed by a two week observation period and another 4 weeks of daily il-6 injections. doses varied between 0.5 and 20 pg/kg body weight. 18 patients were evaluable for studying immune functions. at all dose levels il-6 administration led to a marked increase in serum levels of c reactive protein and complement factor c3. natural killer (nk) cell activity was reduced at doses exceeding 5 pg/kg. similarly lymphokine activated killer (lak) cell activity induced by in vitro culture over 4 days in the presence of 200 u/ml interleukin-2 (il-2) was suppressed at 10 and 20 p.g/kg, as was the proliferative response to il-2 in vitro. however no changes were observed in the proliferation induced by phytohaemagglutinin, pokeweed mitogen or fixed staphylococcus aureus. there were no changes in peripheral blood lymphocyte subpopulations as measured by cd4 and cd8, nor in the expression of hla dr. serum levels of immunoglobulins iga, igm and igg remained unaffected by il-6 treatment. in contrast we found consistent elevations in levels of ige all over the dose range. we conclude that il-6 inhibits nk and lak activity in vivo which may be of interest in future studies with cytokine combinations and that the role of il-6 in ige related diseases might be more important than previously thought. one of the most potent stimulatory agents for the induction of cytokines in myeloid cells is the bacterial ceuwallproduct lps (liopopolysaccharide or endotoxin). in the bloodstream it forms a complex with lbp (lps binding protein) and is recognized by effector cells via the cdi4 receptor. here we report on studies performed with human peritoneal macrophages that were stimulated in vitro with lps and a synthetic lps homologue in the presence and absence of serum. as revealed by elisa-based analysis of the cellsupernatants, strong, serum-dependent resonses were seen for tnf-, il-6, il-8 and g-csf production, while unstimulated cells produced basically only il-8. repeated stimulation of the cells with lps resulted in adaptation that was different for certain groups of cytokines. the "adapted" ceils produced much less tnf and il-6 while il-i and g-csf was superinduced. stimulation of the cells with the lipid a anolog mrl 593 showed a similar picture, given that mrl had to be used in higher concentration. "adapatation" of the cells with mrl also resulted in an "adapted" response to a challenge with a high dose of lps so that it might be useful as a therapeutic agent for preventing the septic shock syndrome, e.g. northern blot analysis showed that the differentiated response of the cells towards a low dose lps stimulation regarding cytokine production after an lps challenge occured on transcriptional level, as mrna levels were regulated accordingly. these results give evidence that two different pathways for lps dependent stimulation of myeloid cells for cytokineproduction exist and experiments to further elucidate this phenomenon and possibly discover cdi4 independent and dependent pathways are underway. the neutrophil-activating peptide 2 (nap-2), a member of the "intercrine"-family of chemotactic and reparative host defense cytokines, represents one of several n-terminally truncated cleavage products that originate from platelet-derived 6-thromboglobulin through proteolytic processing. here we present evidence that there exists also a naturally occurring c-terminally truncated form of nap-2 that is about four times more potent in eliciting neutrophil degranulation than the original cytokine. the novel molecule was detected in concentrates of culture supernatants from peripheral blood mononuclear ceils and could be separated from authentic nap-2 by several steps of column chromatography. according to amino acid sequence analysis it had a n-terminus identical to nap-2, whereas electrophoretic analyses indicated a lower molecular weight as well as a higher isoelectric point. immunochemical analyses performed with epitope-characterized antibodies raised against nap-2 c-terminal synthetic peptides identified limited truncation at the c-terminus of the variant molecule. comparison of reactivity patterns of these antibodies in western blots as well as in a nap-2 biologic assay (pmn degranulation assay) confirmed that the variant nap-2 was truncated by at least one and by maximally three residues. thus, there is for the first time evidence that proteolytic processing at the n-terminus is not necessarily the only mechanism regulating the formation of neutrophilactivating peptides, but that modification at the c-terminus may assist in the fine-adjustment of biological activity. cytokines like interleukin-1 -beta (ill), interleukin-6 (il6), interleukin-8 (il8) and tumor-necrosis-factor-alpha (tnf) are involved in the pathogenesis of fever and infection. however, intra-and inter-individual values differ considerably and there is only limited data on early.cytokine serum levels and their evolution in febrile neutropenic patients. therefore. we measured cytokine levels in 14 adults with chemotherapy-induced aplasia and fever. concentrations of ill. il6 and tnf were determined by irma. il8 by elisa in 11 specimens per patient. ill and tnf were elevated in 5 of 10 patients with peak values of 113 pg/ml and 147 pg/ml, respectively. il6 and il8 were elevated in all patients with a maximum of 41.300 pg/ml (median 416 pg/ml, range 104 -41 316 pg/ml) for il6 and 25.6oo pg/ml (median 1100 pg/ml, range 150 -26.000) for il8. both cytokines showed a high correlation (r=0.89). the individual il6concentration-time curve closely paralleled the temperature curve. in 10 of 14 patients il6 was elevated before onset of fever and peaked at or one hour before the temperature maximum in seven cases. so also in the cytopenie patient lacking a main source of cytokine producing cells and cytokine target cells consistently high il6 and il8 serum levels can be detected very early in the course of fever and infection. the biological and clinical significance of this cytokine response and its regulation mechanisms remain to be determined. interleukin 8 (il-8) and the neutrophil-activating peptide 2 (nap-2) are two closely related members of the "intercrine" family of host defense cytokines. the expression of at least two different receptor classes for il-8 on human neutrophils (pmn) exhibiting similar affinities has been demonstrated recently. using iodinated ligands we could directly demonstrate that 12si-nap-2 specifically bound to pmn with two different affinities, characterized by kd-values of about 1.4 nm and 9.6 nm, respectively. cold 72-residue il-8 competed with iodinated nap-2 for binding to the high affinity site(s) with practically equivalent efficacy, while it was significantly more effective in displacing 12%nap-2 from its low affinity site(s) than was cold nap-2 itself. as new findings, unlabeled il-8 could completely displace ~251-nap-2 and vice versa, indicating that there are no distinct binding sites for either cytokine on pmn. in contrast to il-8, nap-2 did not induce pmn degranulation at concentrations (2 nm), engaging solely its high affinity site. however, short-term priming of pmn with the same amount of nap-2 dramatically down-regulated degranulation inducible by higher concentrations of nap-2 as a secondary stimulus. the il-8-induced secondary response was also diminished, but to lower extents. these phenomena correlated with the rapid downregulation and internalization of nap-2 high affinity binding sites from the cell surface. thus, our data provide direct evidence for a regulatory function of nap-2 at very low concentrations, obviously occurring through the modulation of nap-2 and il-8 receptor expression on pmn. determination of cytokine plasma levels possesses many promising features concerning monitoring and studying of an array of different diseases including febrile reactions. detailed analysis of the role of these factors is of crucial importance for the understanding of the complex cytokine network. investigation of cytokine blood levels however is complicated by their short half-fife in circulation, the presence of soluble inhibitors and the ill-defined beginning of fever. looking for a suitable in-vivo-model allowing a sequential and well-defined analysis of cytokine plasma levels we chose the acute toxicity after intravenous amphotericin b (am b) application consisting of fever, chills and hypotension. these side-effects were reported to be mediated by release of pro-inflammatory cytokines such as tnf a and interleuldn 1 (il-1). in order to compare mutual interations and different temporal patterns of liberation we determined a panel of cytokines including tnf a, s-tnfreceptor (s-tnf-r), interleukin-113, interleukin-1-receptor-antagonist (il-1-ra), intefleukin-6 and interleukin-8 from 15 patients suffering from acute leukemia and fungai infections. serial edta-plasma samples were obtained before and up to 8 hours after start of am b infusion. samples were immediately centrifuged and stored at -40 ~ c until analysis by elisa (medgenix and r&d systems). patients experiencing adverse reactions showed tnf c~ peak plasma levels 90 -180 minutes after starting am b infusion reaching maximum concentrations of 750 pg/ml. concentrations declined m base levels within the following 2-3 hours. il-6 as welt as il-8 concentrations showed similar, but delayed changes of plasma concentration. compared to tnf a s-tnf-rlevels peaked about 30 minutes later with a prolonged decrease to basal concentrations. in contrast to tnf c~ no circulating il-i-13 could be detected, while il-1-ra demonstrated up to 5-fold increases in concentration. we conclude that this model of a drug induced acute-phase-reaction offers wide possibilities for studying the behaviour of inflammatory cytokines and their inhibitors by means of plasma level determination. inflammatory processes following severe trauma were found to be associated with an abnormal high secretion of inflammatory cytokines. these cytokines are discussed to be involved in neutrophil activation associated with the release of high amounts of destructive lysosomal proteases into the extracellular space. the task of our investigations was to evaluate the possible regulation of the degranulation of neutrophils by the immunostimulatory cytokine il-6 and the immunosuppressive factor tgf-i.~. we analysed the concentration of the cqantltrypsin-complex of the lysosomal protease elastase as markers for the degranulation of neutrophils as well as the levels of il-6 and tgf-i~ 1 in the plasma of patients with multiple trauma or after severe surgeries. the time courses of the plasma levels of il-6 and the elastase-inhibitor-complex were found to be highly correlated, suggesting a possible regulatory role of this cytokine on the neutrophil degranulation. however the plasma concentrations of tgf-~, were not significantly altered in comparsion to the control group. in additional experiments, the effect of both cytokines on the degranulation of healthy donors was investigated in vitro. pathological high concentrations of rh~l-6 up to 104 ulml (as detected in several probes from the surgical area) were found to be capable to induce a significant degranulation of the azurophilic granules (56,3_+ 20,2% of the total cellular enzyme content) under serum free conditions as detected by measurement of elastase release by elisa technique and enzymatic methods. in contrast to this, the degranulation of neutrophils was found to be uneffected by tgf-i~. in conclusion, these data suggest that the inflammatory cytokine il-6 may contribute to the activation of neutrophil granulocytes in acute inflammatory processes following severe irauma, whereas the immunosupresive factor tgf-fil seems to have no direct regulatory effects beside the described chemotactic effects on neutrophils. we determined serum concentrations of soluble tumor necrosis factor receptor (stnf-rs) in 61 hiv infected individuals. eighty-five percent of these had increased serum concentrations of stnf-r type i (p55) (stnf-r55) and 95% had increased stnf-r type ii (p75) (stnf-r75). the extent of the increase of stnf-r75 was greater in more advanced hiv infection (p=0.046) as it was measured by dividing the 61 individuals into two groups according to the median of the cd4+ t cell count, stnf-r-55 did not differ between these two groups. a strong correlation was found between stnf-r75 and the soluble immune activation markers b2-microglobulin (rs=0.74, p<0.0001) and urinary neopterin rs=0.67, p<0.0001), and a less strong correlation with interferon gamma (rs=0.51, p=0.0001). the correlations observed for stnf-r55 were also significant but were always weaker than that for stnf-r75. a weak inverse correlation was found between the number of cd4+ t cells and stnf-r75 (rs=-0.33, p=0.012), no such correlation was observed with stnf-r55. our findings suggest that increased concentrations of serum stnf-rs in hiv infection are linked to immune activation where synergistic action of interferongamma and the tnf-alpha system are likely to play an important role. to answer the question whether il-6 is of hematogenous origin, immunohistochemistry and in situ hybridization with il-6 specific s 35 labeled rna probes was established to study the frequencies of il-6 expressing peripheral blood mononuclear cells. a two color immunofluorescence assay with antibodies to cd68 and il-6 was utilized to correlate il-6 mrna expression and il-6 protein production in monocytes of patients and control individuals. 60 -70% of circulating monocytes spontaneously expressed il-6 mrna compared to 15% in normal individuals. a strong correlation of il-6 protein production and il-6 mrna was found in monocytes of patients and controls. semiquantification of il-6 mrna and il-11i mrna by pcr demonstrated that about 10 to 60 fold higher amounts of mrna were found in untreated patients compared to normal individuals. in contrast, we did not find evidence for excessive synthesis of tnfa mrna. the overproduction of il-6 and il-111 in the absence of detectable tnfn mrna establishes a specific cytokine pattern in circulating monocytes of pmr and gca patients. we analyzed the il-6 mrna expression in biopsy specimens from gca patients applying in situ hybridization. autoradiographs were analyzed by visual examination and by using an image-analysis system. tissue infiltrating macrophages expressed il-6 mrna, however, the proportion of il-6 mrna + cd68 t was lower than in the peripheral blood. also, immunohistochemistry with an il-6 specific antibody demonstrated that only a small fraction of macrophages produced this cytokine. these data suggest that in pmr and gca, circulating monocytes are activated and produce il-1 and il-6. tissue infiltration of macrophages is not accompanied by a local activation, in contrast, cytokine production is downregulated. our objective was to evaluate the prognostic value of p21 protein levels in patients with advanced hivinfection during cytokine therapy (ifn or ifn/combination). methods: p21 serum levels were measured every two months by elisa in 29 hiv-infected patients during a period of 2 to 15 months (median 8,6) and compared with clinical stage (wr), disease progression and response to therapy. 18 patients suffered from an infection or an inflammation disease during therapy and 6 from kaposi sarcoma (ks). results: in 15 patients with stable disease constant p21 levels (â�¢ 37 ng/ml) were observed. changes of disease correlated with distinct increasing levels of p21 (in 16 patients with acute infection and/or inflammation disease, in 2 patients with progress of ks). after start of azt-therapy, chemotherapy and antibiotic/ antimycotic therapy p21 level decreased. the highest amount of p21 (up to 165 ng/ml) was found in phases with opportunistic infections. the lowest amount of p21 (31 ng/ml) was observed in a clinically stable patient without an evident progress of disease. conclusions: here, we detected increased levels of p21 in hiv-i infected patients under ifn-therapy during acute opportunistic infection. protein p21 may be a useful marker for the activation of the immunesystem and a prognostic marker during the course of ifn-therapy. previous studies have shown that the diacetylated synthetic pentapeptide splenopentin (dac-sp-5) accelerates hemopoietic recovery following sublethal irradiation and nutologons bone marrow transplantation in mice, but the effects of the peptide on human bone marrow cells were still unknown. in this study, human granulocyte-macrophage (rhgm-csf), macrophage (rhm-csf), and granulocyte colonystimulating factor (rhg-csf) as well as interleukin-l~ (rhil-l~) and interleukin-3 (rhll-3) were compared for their stimulatory activity on human granulocytemacrophnge colony-forming cells (cfc-gm) alone and in combination with dac-sp-5. after depletion of accessory cells from bone marrow mononuclear cells (bmmnc) in semi-liquid cultures the combination of rhgm-csf plus splenopentin stimulated the growth of cfc-gm/-m in dose-depended manner. furthermore, similar effects were seen in combination of dac-sp-5 plus rhil-la and and rhil-3, but not in rhg-csf and rhm-csf plus splenopentin. in unseparated as well as in bmmnc enriched for cd34 + cells comparable stimulatory effects of sp-5 alone were missed. additionally, in bmmnc enriched for cd33+/cd34 + population, the preculture with dac-sp-5 for l0 days enhanced the ability of rhgm-csf, rhil-lct and rhll-3 to induce colony formation from this cell source. however, in all these combinations, mainly differentiation to macrophage lineage has been observed. pheuotypie analysis of precultured bmmnc with splenopentin leads to the suggestion that this compound may recruit a cell population being more sensitive to gm-csf, il-la and il-3, because the percentage of cd34 + cells decreased rapidly, whereas the expression of hla-dr + was enhanced. therefore, this potentially interresting molecule might be a candidate as a therapeutic adjuvant with hemopoietic growth factors. although, the examination of gm-csf, il-lcc and 11. the role of t-lymphocyte subsets in the development of aplastie anaemia (aa) remains poorly understood. therefore we analysed by cytophomotry the contribution and proportion of lymphocyte subpopulationa in the peripheral blood (pb) and bone maxrow (ibm3 of patients with aa before, after 6 weeks of treatment and additionally after 12 weeks of therapy with anti-lymphocyte globulin (alg), methylprednisolon, and eyelosporine a (csa). for double labeling immtmofluorescence studies mouoelonal antibodies directed against the following specifities were used: tcrct~-, tcr~5, 5tcs 1, cd2, cd3, cd4, cds, cd16, cd56, cd38, and hla-dr. hi 13 patients with aa a sigmficant decrease of cd3 positive t-cells was observed after 6 weeks of therapy (pb 35,94.12,0; bm 16,2â�¢ as compared with normal controls (pb 75,7â�¢ bm 39,8+3,1). before therapy, the cd4/cd8 ratio in pi3 and lqm did not differ ~om the ratio in the control population; however a reversed ratio (<1) was present in pb and bm after 6 and 12 weeks of therapy. the number of activated t-cells defined by the antigens cd38, hla-dr and cd56 were low or in the normal range,and did not fu~er decrease during therapy in contrast to the non-activated t-cells. ?5-t-cells were significantly decreased betbre and after 6 weeks of therapy as compared with healthy controls. however, the proportion of the ~/~-subpopulation ~tcsi was markedly increased betbre (pb 39+_3,5; bm 31+_3,7 i) after 6 weeks (pb 58, 7+2, 9; bm 45, 1+_3, 4) and esspeciauy after 12 weeks of therapy (pb 94,.t~l7.1) as compared with that in normal subjects (pb 17_+2,0; bm 9,0_+_0,8). these data indicate that tcs1-t-celis are a t-cell population not affected by treatment with alg, methylpredrtisolone and csa . ftwther studies have to show whether tiffs subpopulafion has an effect on the hematopoiesis of patients with aa. il-1j3, il-2, tnf-i3, g-csf and other cytokines are characterised to have proline in the second position of the n-terminal peptide sequence. from this they could be potential substrates of the dipeptidyl peptidase iv (dp iv). using the method of capillary electrophoresis, here we show that purified soluble dp iv is capable of hydrolysing oligopeptides with sequences analogous to the n-terminal part of human il-lb, il-2, tnf-b and mouse il-6 up to a length of 12 amino acids. furthermore, it could be demonstrated that hydrolysis rates are negatively correlated the with chain length of the oligopeptides. glycosylation of threonine in the third position of the il-2 hexapeptide sequence has no effect on the hydrolysis rate of this peptide by the dp iv. in contrast to these results, no degradation was found in the case of rll-lp, rll-2, natural il-2, and rg-csf, using up to 1000-fold higher dpiv concentrations than in the experiments with oligopeptides. after incubation with both, dp iv and aminopeptidase n, also no cytokine degradation was found. possible explanations for this results will be discussed. hemopd~'etic growth factors are now being tested in several institutions, in an effort to reduce the duration of neutropenia after bone marrow trasnplantation (bmt). in the past 3 years, we have conducted or participated to several double blind multicentric studies using gm-csf and g-csf (schering-plough/sandoz, behring/hoechst and roche lab.) in patients (pts) with nhl. we wish to summarize these studies and also report on our own observations. 1-gm-csf post-abmt: double blind international studies and the french national trial that we conducted (blood 1992 (blood , 180, 1149 (blood -1157 have clearly demonstrated the gm-csf infusion post-abmt significantly accelerates neutrophil recovery by 4 to 7 days, both in pts receiving unpurged marrow or marrow purged by mafosfamide. the duration of hospitalization is reduced by 4 days with a possible cost benefit. 2-in pts with documented cmv infection requiring dhpg treatment, myelosuppression was not seen in those who received gm-csf and dhpg concomitantly while in contrast a severe neutropenia developped in those who received dhpg after administration and discontinuation of gm-csf (lancet, 1989 , 1273 . 3-13 pts. in our institution received gm-csf in a compassionate use for delayed enraftment or engraftment failure after autologous (abmt : 12) or allogeneic bone marrow transplantation (bmt : 1). the pretransplant regiment included total body irradiation (tbi) in 10 and consisted of high dose polychemotherapy only in 2. interestingly 9 pts were transplanted for acute myelocytic leukemia (aml), a disease in which the presence of receptors to gm-csf has been detected in vitro in about 50% of cases, 2 pts hat nhl, and 2 all. of the 12 abmt, 10 pts received marrow heavily treated in vitro by mafosfamide and 1 marrow purged by long term marrow culture (ltc). in 7 (aml 4, all 2, nhl 1), granulopoietic recovery occured within 3 days (1-16). this included a pt with refractory all who received ltc marrow, developped cytomegalovirus (cmv) infection and had not engraftment by day 31 : in this pt the absolute nucleated count (anc) peaked to 2.7 10 9/i, 3 days later. an additional pt with aml had not engrafted by day 63 when gm-csf was administered for 12 days with no efficacy. infusion of back up marrow (which has never been effective in our past experience) combined with gm-csf and cyclosporine a was follwoed by sustained engraftment occuring 17 days later. 1 dt (aml) had a minor and transient response and 4 failed. 6 additional pts in the context of entgraftment failure have since received in various sequences gm-csf and cyclosporine a + back up marrow after no response to gm-csf. 3 have recovered sufficient hemopdfesis strongly suggesting a role of cycloporine a in engraftment failure (manuscript in prepration). we conclude that gm-csf is now the first line treatment for poor engraftment and/or engraftment failure. we do not freeze any longer back up marrow in pts autografted after pretransplant regimens not containing tbi. in pts with engraftment failure post tbi, who do not respond to first line gm-csf, we suggest that the combination of back up marrow and cyclosporine a on top of gm-csf should be further evaluated. 4-we administered gm-csf at a dose of 250 #g/m2/day to 5 pts with resistant nhl and bone marrow involvement after the beam myeloablative regimen; reconstitution occurred within the same delays than observed after abmt in 3. we propose that gm-csf may replace abmt in highly selected cases of non-hodgkin's lymphoma with progressive disease and bone marrow involvement (lancet 1991, 338, 601) . 5-we have transplanted since september 1992 8 nhl pts with cd 34 purified stem cells followed by infusion of gm-csf. successful engraftment was obtained in all with recovery to 0.5x109 pmn/i by day 14 (11-25) and to 50x10 9 plts/l by day 23 (13-32). the expansion of cd 34+ cells in short term liquid culture with 6 cytokines (including gm and g-csf) is another approach toward transplantation with total abrogation of neutropenia. overall the introduction of hemopoietic growth factors in transplant units has considerably changed the situation in several aspects. further studies will combine gm-csf and g-csf to other cytokines. dept. of hematology, bone marrow transplant unit -h6pital st-antoine, paris, france alter a pre-phase of vcr 1.4rag/@ (maximum 2rag) i.v. days 1,8 and pradnisolone 60rng/n~ p.o. days 1-10 the high-dose chemotherapy consisted of prednisolone 60rag/n? p.o. days 1-4, ifosfamide days 1-4, methotrexate 5,000 mg/m 2 day 1 as a 24 hour infusion, cytosine-arabinoside 1,000 mg/m 2 i.v. days 3 + 4, and etopeside i.v. days 3 + 4. etoposide has been escalated from 170 mg/rn 2 to 500 rng/ nf at the present time. the dose of itostamide is currently escalated from 1,500 rag/ rlf to 2,500 mg/rtf and finally 3,500 mg/rn 2 as a continuous 24 hour infusion. the high-dose chemotherapy is repeated for a maximum of four times. during the prephase 12pg/kg fiigrastim (recombinant g-csf) are given twice daily. apheresis of apbsc is done on days 5-7. apbsc are reinfused after the high-dose chemotherapy and fiigrastim is given at a dose of 5p.g/kg. with wbc rising to >1,000/p.i aphereses are performed repeatedly. the pre-phase for induction of pbsc was excellently tolerated and a median of 10 we report here the effects of long-term g-csf subcutaneous administration in 48 patients (congenital n=32, cyclic n=4, idiopathic n= 12) treated for 3-5 years. a sustained anc response was seen in 30/32 congenital patients, in 4/4 cyclic patients, and in 10/12 idiopathic patients. the g-csf doses needed to maintain these responses ranged between 1 and 60 #g/kg/d. the anc responses were associated with a significant decrease in the incidence of severe infections and the need for intravenous antibiotics. g-csf has been well tolerated in the majority of patients and resulted in a dramatic improvement in the quality of life. the adverse events noted included: osteopenia (n= 12), vasculitis (n=2), mesangioproliferative glomerulonephritis (n=2), and the development of mds/leukemia (n=2). these adverse events are most likely associated with the underlying disease and not caused by g-csf treatment we have evaluated recombinant human granulocyte-macrophage colonystimulating factor (rhgm-csf) (sandoz-schering/plough) as an adjunct for cop-blam in the primary treatment of high grade malignant non-hodgkin's lymphomas (nhl). patients (n = 182, stage ii-lv, age 15-73 years), were randomized to rhgm-csf (400 #g) or placebo for 7 days s.c. following chemotherapy. efficacy was analyzed for patients receiving at least 70% of study medication (n = 125). the frequency of clinical relevant infection was reduced by rhgm-csf (28 vs 69 infections, 16 vs 30 patients, p = .02) with a cumulative probability of remaining infection free in 70% vs 48% (p = .05, log rank test at 190 days). periods of neutropenia (p _< .01 in 5/8 courses), days with fever (2.1 vs 4.0, p = .04) and days of hospitalization for infection (3.5vs8.0 days, p = .01) were significantly reduced. complete response (cr) rates, assessed by prognostic risk 28 patients (pts) entered an outpatient protocol designed to test the tolerance, and the clinical and biological effects of extended administration of sc low-dose il-2 following high dose chemotherapy with autologous bone marrow rescue (bmt). two il-2 regimens were used. protocol a consisted of a once daily dose administered in 5-day cycles of 3 millions iu/m z sc ril-2 (ru49637 kindly provided by roussel-uclaf) every other week. the pts were treated at home and the treatment was scheduled for 6 months (i.e. 12 cycles for 6 months) in the absence of major disease progression or sideeffects. protocol b consisted of a once daily dose of 6 millions iu/m 2 sc il2 in 5 days/cycles for 6 consecutive weeks (i.e. 6 cycles for 6 weeks). il-2 was started 60 to 90 days following bmt. blood lymphocyte subsets and nk/lak cytotoxic activity were determined monthly. 17 pts received regimen a and 11 pts were given regimen b. in both regimens, inflammatory skin reactions were the main side effect, leading to interruption of treatment in 1 case. no capillary leak was observed. flue like syndrom was occasionny observed in protocol b. hematological parameters were not adversely affected by il-2. at the lowest dose levels (regimen a), long term administration of il-2 did not produce any changes of blood lymphocyte subsets. on the other hand, the administration of 6 millions iu/m 2 il-2 resulted in a significant increase of cd3+, cd25+ cells and cd3-cd56+ ceils, and nk/lak cytotoxic activity of fresh pbls. this study confirms the feasability of long-term administration of sc lowdose il2 following autologous abmt. a dose of 6 millions iu/m2/d resulted in detectable activation of circulating lymphocytes. further studies are needed to assess the clinical impact of prolonged low-dose il-2 in this clinical setting. aim: to study the incidence of infectious episodes (ie) in a single centre series of 7 autologous bmt patients who received il-2 in an attempt to prevent relapse. patients. methods: il-2 was given as a oontinuous i.v. infusion through a hickman catheter with a portable pump: 200.000 iu/m2/d for the first week and 400.000 iu/m2/day thereafter during 12 weeks. il-2 was started when full neutrophil engraftment was achieved and platelet counts remained stable over 50.000 platelets/mm 3 (median: day +64. range: 38-118).median age was 39 years (5-46). 6 were mate. bmt was performed for acute lymphoblastic leukemia (3), lymphoma (2), and myeloma (2). results: il-2 planned therapy was completed in 5 patients (1 early relapse, 1 refusal) in a median of 103 days (90-154). median intensity dose of il-2 was 2.8 x 105 iu/m2/day (1.2 x 105-3.82 x 105).10 infectious episodes who precised admission to the hospital were observed in 5 patients. 7 ol these infections were microbiologically documented: 56% of isolations corresponded to gram negative and 44% to gram positive. first ie was detected at a median of 11 days (4-56) after the starting of il-2. median cumulative tl-2 dose until first infectious episode was 3 x 106 iu/m 2 (0.8 x 10 ~' -15 x loe). catheter were removed in 3 of them. in our experience, low dose continuous i.v. administration ol il-2 is associated with a high incidence of infectious episodes. sr 29009 (sanofi recherche) is a cho-derived r-il2, glycosylated on the threonine in position 3. its specific activity is identical to native il2. dose escalation studies were performed using sr29009 either as iv bolus or iv continuous infusion in advanced-stage cancer patients. treatment schedule consisted in three 5-day cycles with 9-day rest in between. 22 pts received iv bolus q8-12 hrs at 4 dose levels (1;3;6;9 millions iu/m-'/bolus) and 19 pts received iv continuous infusion at 5 dose levels (0.3;0.5; 1;2;3 millions iu/m2/day). mtd were found to be 6 millions iu/m 2 q8 hrs/day and 2 millions iu/m2/day for bolus and continuous iv infusion respectively. toxieities were similar in nature to those described under treatment with non-glycosylated rll-2. dlt were mainly related to capillary leak. 1pr (nhl) was obtained. a significant rise of t-cell and nk cell subsets as well as nk/lak cytotoxicity in blood was observed at day 8. stimulation was already maximum for the lowest dose levels. at 0.5 millions iu/mz/day, the mean number of cd3+ cd25+ cells increased from 113 to 486/mm -~ on day 8. similarly, cd56+ cells increased from 461 to 1345/mm 3, and cytotoxicity of fresh pbl against k562 increased from 10% to 40% (e/t ratio = 25 : 1). in vivo, sr29009 appears to exhibit effects similar to those observed with higher doses (x 4-6 fold) of non glycosylated rll2. although intravesical therapy with bacillus calmette-gutrin (bcg) against superficial bladder cancer recurrences and carcinoma in situ is highly effective, its mode of action is still unclear. the bladder tumor cell lines bt-a, bt-b (grade 3 transitional cell carcinoma), sbc2, and sbc7 (grade 1 transitional cell carcinoma) are nearly resistant to natural killer cell activity in vitro. we could demonstrate that these cell lines are susceptible to lymphokine-activated killer cells generated by interleukin (il)-2 or interferon (ifn)-% we have now investigated whether bcg itself is able to activate a lak cell-like reaction against bladder tumor cells. our results show that activation of pbmc with viable bcg is a potent way of generating cytotoxic effector cells against bladder tumor cells. in contrast, killing of the nk and lak cell-sensitive k562 cell line was not enhanced by bcg-induced pbmc. because of their different target cell pattern and their, compared to lak ceils, distinct way of activation these cytotoxic effector cells were termed "bcg-activated killer (bak) cells". antibodies neutralizing ifn-t activity blocked the induction of bak cell cytotoxicity, indicating that this cytokine is playing a crucial role during this process. with respect to the phenotype of bak cells, our data show that the effector cells belong to the cd8+/cd56 ⧠lymphocyte subset. depletion of either cd8 + or cd56 + cells from bcg-induced pbmc led to a decrease of cytotoxicity against bladder tumor cells. furthermore, positively selected cd8 + cells could maintain the level of cytotoxicity exerted by bcg-induced pbmc whereas the depletion of cd56 ⧠cells from this population also eliminated the cytotoxic effect. a direct involvement of cd4 + cells or macrophages in the killing of bladder tumor cells was not observed. in patients with superficial bladder cancer lak and bak cells might play an important role in the maintenance of the relapse free state. in this report we summarize our experience with high-dose therapy and peripheral blood stem cell (pbsc) autografting in advanced malignant lymphoma. since may 1991,27 patients (19 male / 8 female) were included into this study. the median age was 37 years (range 19-58). 11 patients had hodgkin's disease and 16 non-hodgkjn's lymphoma (9 low-grade / 7 highgrade nhl). four patients received recombinant g-csf (filgrastim) (5 i~g/m2/ day s.c.) during steady-state hematopoiesis, while in the remaining 23 patients recombinant g-csf (filgrastim) was started 24 hours after conventional chemotherapy. for all patients, a target quantity of 0.4 x 109/kg total nucleated cells (tnc) was reached. a wide interindividual range with respect to the level of circulating hematopoietic progenitor cells (325-fold for cfu-gm/ml and 221fold for cd34+ cells/id) was observed. with a median number of 5 leukaphereses (range 2-11), a median of 12.7 x 104 cfu-gm/kg bw and 4.7 x 106 cd34+ celts/kg bw could be harvested, respectively. high-dose therapy consisted of either tbi (14.4 gy, hyper-fractionated)/cyclophosphamide (200 mg/kg) or the beam-protocol. with the exception of 1 patient who died of a respiratory distress syndrome 32 days following autografting, transplant-related toxicity was moderate. the low toxicity is reflected by the kinetics of hematological reconstitution: 15 (median) days for 1.0 x 109/1 pmn and 11 days for 20 x 10~/i platelets. platelet reconstitution was closely related to the number of cd34+ cells reinfused. twenty-six patients are evaluable for response: 5 patients (19%) relapsed after a median of 5 months (range 2-6) posttransplant. the remaining 21 patients are alive in remission with a median follow-up of 9 months (range 3-19). in summary, recombinant g-csf (filgrastim) is highly efficient in mobilizing pbsc capable of restoring hematopoiesis after myeloablative conditioning therapy. the quantity of cd34+ cells harvested was inversely related to the amount of previous cytotoxic chemotherapy. therefore, high-dose therapy with stem cell support should be considered as an upfront treatment modality in poor prognosis nhl patients.department of internal medicine, university of heidelberg, 6900 heidelberg, germany. many of the in vivo effects of the haemopoietic cell growth factors may well have been anticipated based on their known ability to stimulate proliferation and development of multipotent and lineage-restricted progenitor ceils in vitro. what was not predicted from these in vitro studies, however, was the observed ability of at least some of these growth factors to mobillse large numbers of haemopoietic progenitor cells from the bone marrow into the peripheral blood. this was an added "oonus" effect of growth factors and one that is now being widely exploited in a variety of clinical situations.initially these mobilised pbpc were used, in combination with bone marrow cells, to facilitate the more rapid recovery of myeloid cells following transfer into patients receiving high dose cytotoxic therapy. but when experimental studies showed that the mobilised pbpc also contained primitive (repopulating) stem cells -this encouraged the use of pbpc alone as a source of cells for transplantation. in most of these studies however, collection of the pbpc was performed over several cycles of apheresis -and this was somewhat limiting to their widespread use.because of this, our approach has been to define the optimal growth factor regimen for mobillsation of pbpc such that apheresis is not required, i.e. to use whole blood and then to identify groups of patients who may benefit from receiving these cells either for autologous or for allogeneic marrow recenstitution. using a sensitive immunohistochemical technique, 1.3->5.0 logs of breast cancer cell depletion was documented in the positively-selected fractions of marrow (10 patients) and 2->5 logs from the pbpc fractions (3 patients), in whom tumor was initially detected. to date no tumor has been detected in the cd34+ pbpc fractions. the engraftment rates for cohorts 4 and 5 were significantly faster than those of the other cohorts. the preliminary data suggest that since cd34+ pbpcs alone are capable of restoring hematopoiesis following high-dose therapy, a marrow fraction may no longer be needed for this purpose. longer follow-up will be required to assess the ultimate therapeutic effect of the entire treatment program. the principal morbidity and mortality of high dose chemotherapy with autologous bone marrow support (abmt) relates to the infectious complications which occurs during 3-4 week aplasia until the marrow autografi recovers. progenitor cells can be mobilized into the peripheral blood compartment by hematopoietic growth factors, alone or used after chemotherapy. we describe four trials using cytokine-mobilized peripheral blood progenitor cells (pbpc). in the first trial, pbpc collected after gm-csf administration are used to augment marrow. reconstitution of tririneage marrow function occurred quickly, resulting in short hospital stays and fewer platelet transfusions. in a second study, gm-csf/chemotherapy-mobilized pbpc were used as the sole hematopoietic support during high dose chemotherapy. granulocyte and platelet reconstitution was rapid. time to hematopoietie recovery, transfusion requirements and duration of hospital stay were all significantly improved for the patients receiving pbpc compared with similar patients receiving marrow alone. however, some patients had poor platelet engraftment. two recent trials were designed to explore multiple high-dose therapy. in the third trial, pbpc with and without marrow made it feasible to deliver two sequential cycles of high dose therapy. the fourth trial utilizes pbpc in addition to cytokines to deriver four cycles of dose-intensive therapy utilizing doses of chemotherapy that could not be given with cytokine support alone. pbpc appears to make multiple course combination high dose therapy feasible, is particularly useful to support platelets, and may enhance the safety, tolerance and cost of high dose therapy. severe neutropenia and functjonal defects of neutrophils are an increasing problem in treating advanced hiv infected p. neutropenia may result from bone marrow failure due to hiv by itself, or maybe.secondary to drug toxicity (mainly zidovudine, ganciclovir, chemotherapy) or bone marrow infiltration (mycobacterial infection, neoplasms). p. with severe neutropenia are at a higher risk of developing bacterial infections. since both gm-csf and g-csf have been-successfully used in cancer to optimalize dose intensity and to prevent neutropenia and secondary infections in p. receiving myelotoxic agents, clinical trials have recently been conducted in arc and aids p. with neutropenia to determine their potential benefits and toxicity profile. our previous experience in treating p. with non hodgkin's lymphoma (nhl) with a 6-cycle third generation regimen (promnce-cytabom), with gm-csf given at alternate cycles, clearly demonstrated that doses of chemotherapy following the adjunction of gm-csf were significantly higher than doses given without prior gm-csf administration. gm-csf was largely well tolerated in those p., with no sign of active viral replication as measured by hiv-1 p24 antigenemia. we have also performed an open clinical trial in p. with severe neuttopenia to assess the efficacy and tolerance of gm-csf with a three weekly subcutaneous administration in p. who initially responded to a daily dose. 24 out the 28 enrolled p. (median duration : 40 days) showed a rise of the absolute neutrophil count (anc) above the target value of 1000/mm 3 after a median period of 3 day at 3 mcg/kg/d. only 5/28 p. were given antiretrovirals while other concomitant drugs included mainly ganciclovir (8) and pyrimethamine (10). of the 21 p. who entered the maintenance phase at 3weekly doses, 12 (57 %) showed a sustained increase of the anc >1000, while in the remaining 9 p, a new episode of neutropenia was observed.in contrast to p. with nhl, adverse reactions were commonly observed, mostly fever (68%) and flu-like symptoms (50 %). furthermore among the 22p. without antiretroviral therapy who were p24 ag negative at baseline, a detectable p24 antigenemia was found in 9 p. these preliminary data on this original schedule of gm-csl = administration was effective in preserving anc levels at > 1000 in 57 % of the responders. such an approach could enhance p.'s compliance and have a direct impact on the cost/benefit evaluation of such therapy. in addition, antiretrovirals should always be associated with gm-csf to reduce the increased risk of viral replication. attempts to further improving the curative treatment for aml have to overcome the problem of residual disease represented by leukemic cells surviving in a therapy resistant state. a novel approach aims at recruiting those cells into a sensitive state by the use of gm-csf before and together (priming) with chemotherapy. in vitro, the recruitment of leukemic blasts to colony forming cells in presence of gm-csf (blood 67:1448 , 1986 ) is documented by numerous reports. given to patients with newly diagnosed aml gm-csf priming decreased the proportion of leukemic cells in go and increased cells in sensitive cycle phases within 24-48 h (blood 77:700, 1991) . a similar priming with preinfusion of gm-csf for a variable period of 0-8 days before chemotherapy started resulted in significantly inferior outcome and more persistent leukemias than in historical controls suggesting protective effects of gm-csf (blood 79:2246 , 1992 . in a first study we showed that gm-csf following chemotherapy in high risk aml effectively reduced both neutrophii recovery and early mortality and had no adverse impact on leukemic regrowth and remission duration (blood 78:1190 (blood 78: , 1991 . in current randomized study in patients with newly diagnosed aml we give gm-csf from 24 h before chemotherapy and then on to neutrophil recovery which is repeated in each of the initial 5 treatment courses and is compared to chemotherapy alone. 2 1/2 years from study start and after entering 72 patients (median age 50, range 17-75 years) this update in the gm-csf group and controls shows 78% and 81% cr, 2 and 5 persistent leukemias and a clearance of day 16 b.m. blasts to < 5% in 59% and in 38%, and remission duration shows a trend in favour of the gm-csf group. in addition to recruitment, other effects of gm-csf like enhancement of ara-c cytotoxicity (leukemia 3:789, 1989; ch. router et al. leukemia in press) may contribute to this strategy. thus, gm-csf appears not to antagonize antileukemic chemotherapy. whether gm-csf priming and longterm administration ultimately improves the cure rate in aml should be shown some later from the multiple course strategy used in this study.medizinische universit~itsldinika (h~imatologie/onkologie), albert-schweitzer-str. 33, d-4400 mfinster in this multicenter trial was evaluated, whether rhu gm-csf given concomitant and after chemotherapy (ct) can improve the outcome of aml patients by increasing the cytotoxic effect of ct and by reducing the rate of infectious complications. induction and early consolidation therapy included cytarabine (ara-c, 100 mg/m 2, day 1 -8 civi), daunorubicin (60 mg/m 2, iv, day 3 -5,) and etoposide (100 mg/m 2, day 4 -8, 2 h iv infusion) with reduced dosages in the second induction and early consolidation course. late consolidation included one cycle with high-dose ara-c (3g/m 2, 12 doses) and daunorubicin (30 mg/m 2, day 7 -9) for patients aged 50 years and younger, whereas patients over 50 years received a reduced dose of ara-c (0.6g/m 2, 12 doses). patients were randomized after the first induction course to receive either rhu gm-csf (e. coil, 250 pg/m2/day, s.c.) or placebo starting 48 hours prior to the second induction and the subsequent courses and given throughout chemotherapy until absolute neutrophil count had recovered > 500/pl. eighty out of 82 patients (median age = 50 years) included into the study could be randomized to receive either gm-csf (n = 41, median age = 50 years) or placebo (n = 39, median age = 50 years). 64 out of 82 patients (78%) achieved a complete remission (cr). 15 patients (18.5%) were treatment failures. two patients died and in one patient the response to therapy was not evaluable. there was no statistically significant difference in cr rate between patients of the gm-csf (81%) and placebo group (79%) nor between patients aged 50 years or less and those over 50 years old. the proportion of relapse free survival at a median follow up of 12 months is 48 % in the gm-csf versus 33% in the placebo group (p = 0.64). the proportion of survival of the gm-csf group at 22 months is 38% versus 56% in the placebo group (p = 0.64). gm-csf did not significantly shorten the period of critical neutropenia and prolonged the period of critical thrombocytopenia especially in patients aged over 50 years. the overall incidence of infectious complications as well as the non-hematological toxicity was similiar in boths groups. thus gm-csf therapy is feasible in aml therapy while its influence on remission quality and survival remains still open.dept. int. med. iii, university of uim, 7900 uim/d, frg.al17 key: cord-022526-j9kg00qf authors: jones, samuel l.; blikslager, anthony t. title: disorders of the gastrointestinal system date: 2009-05-18 journal: equine internal medicine doi: 10.1016/b0-72-169777-1/50015-9 sha: doc_id: 22526 cord_uid: j9kg00qf nan however, the abdominal wall is too rigid to allow effective palpation of intraabdominal structures. abdominal auscultation is particularly useful for assessing the motility of the large intestine. progressive motility of the small intestine, conversely, is difficult to distinguish by auscultation from nonprogressive motility. the distinct character of the borborygmi produced during propulsive contractions of the cecum and ascending colon allow evaluation of the frequency and strength of retropulsion and propulsion. propulsive contractions of the cecum and ventral colon occur every 3 to 4 minutes and give rise to prolonged rushing sounds heard over long segments of intestine. retropulsive sounds presumably are similar to propulsive sounds, but they occur less frequently. the distinction of propulsion from retropulsion is not important clinically because both types of contractions signify normal motility. inter-and intrahaustral mixing contractions produce nonspecific sounds of fluid and ingesta movement that are difficult to distinguish from other borborygmi, such as small intestinal contractions or spasmodic contractions. 1 auscultation over the right flank and proceeding along the caudal edge of the costal margin toward the xiphoid allows evaluation of the cecal borborygmi. auscultation over a similar area on the left side allows evaluation of the pelvic flexure and ascending colon. typical progressive borborygmi heard every 3 to 4 minutes on both sides of the abdomen indicate normal motility of the cecum and ascending colon. less frequent progressive sounds may indicate a pathologic condition of the large intestine or may result from anorexia, nervousness (sympathetic tone), or pharmacologic inhibition of examination of patients with disease of the gastrointestinal tract must include evaluation of the metabolic and cardiovascular status of the patient, because acute conditions of the proximal or distal intestinal tract have the potential to lead to endotoxemia and sepsis. examination of the cardiovascular system (heart, peripheral pulse, and mucous membranes), lungs, and abdomen is essential to detect clinical signs of systemic inflammation from endotoxemia, coagulation disorders, dehydration, ileus, shock, and other abnormalities resulting from injury to the small or large intestine. chapter 13.7 covers clinical signs of systemic inflammation from endotoxemia and sepsis. one performs the physical examination of the abdomen primarily by auscultation, transabdominal ballottement, and transrectal palpation. abdominal distention often indicates distention of the large intestine; however, small intestinal distention also can cause visible abdominal distention if a large proportion of the small intestine is involved. one can perform abdominal palpation in neonatal foals; after several weeks of age, motility (i.e., α 2 -adrenergic agonists such as xylazine). [2] [3] [4] absolute absence of any auscultable borborygmi suggests abnormal motility and indicates ileus resulting from a serious pathologic condition but is not specific to any segment of the intestine. 3, 5 if borborygmi are audible but progressive sounds are not detectable, determining whether a significant abnormality exists is difficult. 5 borborygmi heard more frequently than normal may result from increased motility following feeding; from excessive stimulation from irritation, distention, or inflammation; or after administration of parasympathomimetic drugs such as neostigmine. large intestinal motility increases in the early stages of intestinal distention regardless of the site. 6 mild inflammation or irritation of the large intestinal mucosa also can stimulate motility. 3 parasympathomimetic drugs stimulate contractions and auscultable borborygmi in the large intestine; however, an increase in parasympathetic tone may result in segmental contractions, which actually inhibit progressive motility. 2 one can detect sand or gravel in the large intestinal ingesta by auscultation behind the xiphoid process. one can hear sand or gravel particles grinding together during progressive contractions of the ascending colon. the presence of sand in the ingesta becomes clinically detectable by auscultation or fecal sedimentation before the amount of sand is enough to produce clinical signs of pain or irritation (diarrhea). 7 if progressive contractions are audible without hearing sand sounds, clinically important quantities of sand likely are not present. if the frequency of progressive contractions is low or absent, detecting sand by auscultation is difficult. percussion of the abdomen during auscultation can reveal gas in the large intestine. the characteristic ping produced by simultaneous digital percussion and auscultation over a gas-filled viscus often is associated with abnormal accumulation of gas under pressure. this technique is particularly useful in foals, ponies, and miniature horses because of the limitations of palpation per rectum. one can use transabdominal ballottement to detect large, firm masses or an abnormal volume of peritoneal fluid. the usefulness of this technique is usually limited to animals too small to palpate per rectum. one can detect soft tissue masses or fetuses by bumping the structures with a hand or fist. if excessive peritoneal fluid is present, one can generate a fluid wave by ballottement; however, this technique is not as useful in horses older than 4 weeks because the abdominal wall is rigid. transrectal palpation is the most specific physical examination technique for investigation of intestinal disease and is particularly valuable when evaluating obstructive diseases. 8, 9 the primary objectives of transrectal palpation are to assess the size, consistency, and position of the segments of the large intestine; to determine the presence of any small intestinal distention; and to detect intraabdominal masses. evaluation of the wall thickness and texture and the mesenteric structures (blood and lymphatic vessels and lymph nodes) also may aid in diagnosis of large intestinal disease. the interpretation of transrectal palpation findings in light of clinical signs and laboratory results is an important diagnostic aid for developing appropriate treatment strategies for intestinal diseases manifested by abdominal pain. enlargement of one or more segments of large intestine detected by transrectal palpation provides evidence of obstruction at or distal to the enlarged segment. by systematically evaluating each segment, one may determine the site of obstruction. obstruction of the pelvic flexure, for instance, results in enlargement of the pelvic flexure and ventral colon, but the dorsal and descending colons are of normal size. enlargement of a segment of the large intestine usually is accompanied by abnormal consistency of the contents. one may distinguish accumulation of gas, fluid, or ingesta and may detect foreign bodies in palpable segments. accumulation of gas and fluid infers complete and acute obstruction, whereas accumulation of ingesta infers chronic and incomplete obstruction. accumulation of fluid usually indicates ileus. one must evaluate the consistency of the contents in light of the size of the segment; ingesta in the ventral colon of a dehydrated patient may be firm, but the size of the ventral colon will be normal. conversely, if the ingesta is firm because of a distal obstruction, the ventral colon will be enlarged. displacement of a segment of the large intestine may create an obstruction detectable by enlargement of the segment and accumulation of gas and fluid, even if the site of obstruction is not palpable. torsion of the ascending colon at the sternal and diaphragmatic flexures results in acute accumulation of gas and fluid proximal to the torsion, causing distention of the left dorsal and ventral colons. depending on the degree of torsion, the position of the ventral and dorsal colons may not be significantly abnormal. displacement of a segment of large intestine often results in incomplete obstruction, and the diagnosis relies solely on detection of the displaced segment in an abnormal position. the position of the displaced segment may not be palpable, and the diagnosis then relies on the inability to find the segment in a normal position. one must take care to ensure that the segment that appears to be displaced is not in a normal position but has become too small to palpate from a decrease in the volume of ingesta. the cecum, right dorsal and ventral colons, pelvic flexure, and descending colon are palpable in most horses. one should palpate the nephrosplenic space to detect the presence of intestine, usually pelvic flexure, entrapped within the ligament. small intestine is not normally palpable in the horse. distention is an indicator of ileus with gas or fluid retention, usually following a strangulating or nonstrangulating obstruction. strangulating obstructions result from conditions such as volvulus or torsion, lipoma, or entrapments. such conditions often are accompanied by severe pain, dehydration, peritoneal fluid changes, and a varying degree of gastric fluid accumulation. the small intestine in these cases is turgid and firm on palpation. one should assess the mesentery and wall thickness as for large intestinal disorders. careful palpation of the inguinal rings in stallions with small intestinal distention is crucial for determining inguinal herniation. evaluation of the wall thickness and mesenteric vessels can reveal venous congestion (mural edema and enlarged blood and lymphatic vessels) or inflammation (mural edema with normal vessels). disruption of arterial blood flow does not cause venous congestion, but the arterial pulse is not detectable. mesenteric tears may not be palpable, but the entrapped ischemic intestinal segment may be thickened with edema. one may detect acute or chronic inflammation with cellular infiltration of the intestinal wall as thickening of the wall without edema and also may note enlargement of mesenteric lymph nodes. one should interpret abnormalities in the wall or vessels in light of the size, consistency, and position of the segment of intestine and the clinical signs. several conditions involving small intestinal strangulating lesions do not necessarily cause abnormal rectal examination findings until the disease has been present for an extended time. these conditions include diaphragmatic herniae and epiploic foramen entrapments. peritoneal fluid analysis may be normal in these cases as well because the fluid is trapped in the thorax or in the cranial abdomen. surgery is usually necessary for diagnosis. nonstrangulating causes of small intestinal distention can be divided further into intraluminal and extraluminal obstructions. ileal impactions are probably the most common cause of intraluminal obstructions, and on rare occasions one can palpate the impaction in the upper right quadrant, near the ileocecal opening. intraluminal masses caused by lymphoma, eosinophilic enteritis, foreign bodies or ascarid impactions often lead to small intestinal distention and are usually indistinguishable from one another based on palpation alone. small intestine in these cases can be moderately to severely distended, depending on the degree of obstruction. extraluminal obstructions include abdominal masses, abscesses or tumors, and large colon displacement. one always should palpate the rest of the abdomen carefully to help rule out these causes. some cases of small intestinal distention result from a physiologic rather than a mechanical obstruction. ileus may result postoperatively or following inflammatory diseases of the bowel (proximal enteritis) or peritoneal cavity (peritonitis). the bowel is usually mildly to moderately distended and almost always is accompanied by significant amounts of accumulated gastric fluid. the small colon is easily distinguishable by the presence of normal fecal balls and an antimesenteric band. in cases of impaction of the small colon, a long, hard, tubelike structure is present in the caudal abdomen, and the band is palpable along the length. fluid stool is often present in the rectum in these cases, as is tenesmus. one can detect and carefully evaluate rectal tears by palpation. one can detect mural masses in palpable segments of intestine or mesentery; however, if a mass causes obstruction, one can detect the result of the obstruction in proximal segments of intestine even if the mass is unreachable. palpation of the mesenteric vessels may reveal thickening and thrombosis, which can lead to ischemia or infarction. one can perform visual inspection of the mucosa of the rectum and descending colon with a speculum or flexible endoscope and also can evaluate rectal tears or perforations, mural masses, strictures, or mucosal inflammation. one also can perform guided biopsy of the mucosa or masses. the obvious limitations are the amount of fecal material interfering with the examination and the distance of the lesion of interest from the anus. these techniques offer little advantage over palpation in many cases unless the patient is too small to palpate. examination of the oral cavity in cases of dysphagia or weight loss is a necessary part of the physical examination. one should adequately sedate the horse and should use a full-mouth speculum to allow palpation and visualization of all parts of the oral cavity. one should examine the area for abnormal dentition, foreign bodies, fractures, abscesses, and ulceration. the presence of fluid accumulation in the stomach indicates a decrease or absence in propulsive motions of the small intestine or obstruction of gastric outflow. decreased small intestinal motility may result from a functional or mechanical blockage. masses, feed impactions, or strictures in the pylorus or in the proximal duodenum may obstruct gastric outflow. one routinely assesses fluid accumulation in the stomach by siphoning off the gastric contents with a nasogastric tube and examining the fluid for amount, color, and any particular odor. normal fluid is green and may contain foamy saliva. the volume obtained by gastric lavage is usually less than 4 l. large volumes of fluid (>8 to 10 l) accumulate in the stomach of horses with proximal enteritis, and the fluid is foul smelling and often has an orange to yellow discoloration. if one suspects proximal enteritis, one can submit the fluid for culture and gram staining. salmonella sp. and clostridium sp. have been cultured in some cases. patients with postoperative ileus also frequently accumulate large amounts of gastric fluid. horses with section 13.1 examination for disorders of the gastrointestinal tract strangulating obstructions or luminal obstructions often accumulate moderate amounts of gastric fluid, but the amount is generally less than in horses with proximal enteritis or postoperative ileus. hemorrhage in the gastric fluid usually indicates devitalized small intestine, stomach wall, or severe gastric ulceration. fluid with large amounts of food material often indicates a gastric impaction, and one should lavage the stomach until obtaining no more ingesta. horses and foals with chronic gastric ulceration in the glandular mucosa of the stomach or in the duodenum may develop strictures and have fluid accumulate in the stomach. endoscopy or contrast radiography aids in diagnosing gastric outflow obstruction. evaluation of the hemogram is essential when one assesses conditions of the gastrointestinal tract. however, hematologic alterations associated with diseases of the gastrointestinal tract are often nonspecific, reflecting systemic response to inflammation, endotoxemia, or sepsis. neutrophilic leukocytosis and normochromic, normocytic anemia with or without hyperfibrinogenemia commonly are associated with chronic inflammatory conditions of the intestine. anemia from chronic blood loss occurs infrequently in adult horses because of the large iron stores and high concentrations of iron in their diet; usually anemia follows chronic inflammation, as do alterations in the leukon and plasma fibrinogen concentrations. plasma protein concentrations vary depending on gastrointestinal losses of albumin and globulin and elevation of globulin concentration from antigenic stimulation. protein-losing enteropathies may manifest predominantly as a hypoalbuminemia or may have a concurrent hypoglobulinemia. immunoglobulin quantification can be useful in selected cases; immunosuppression with low immunoglobulin m concentration has been shown to occur in some cases of lymphosarcoma. 10 parasitic infections, especially strongylosis, may be characterized by elevated serum immunoglobulin g(t) concentration. 11 significant alterations of the hemogram do not accompany acute disease of the intestine unless severe inflammation, dehydration, endotoxemia, or sepsis is present. during the early stages of endotoxemia, elevations in circulating concentrations of inflammatory mediators, epinephrine, and cortisol produce characteristic changes in the hemogram. leukopenia, with neutropenia and a left shift, toxic changes in the neutrophil cytoplasm, and lymphopenia occur commonly. 12 hemoconcentration and hyperfibrinogenemia are also common. thrombocytopenia and other coagulopathies are also features of endotoxemia. indeed, thrombocytopenia may be the earliest indicator of sepsis. 13 endotoxemia and circulating mediators of inflammation activate the coagulation cascade, causing a hypercoagulable state that can lead to consumption of coagulation factors and coagulation defects manifested as elevated prothrombin time, partial thromboplastin time, fibrin degradation products, and bleeding time, and reduced activity of antithrombin iii. [14] [15] [16] neutrophilic leukocytosis occurs during the later stages of endotoxemia. 14 the most common serum biochemical abnormalities with diseases of the large or small intestine are electrolyte imbalances. serum calcium concentrations are often low with strangulating obstructions and acute inflammatory diseases. 17 inflammation of the mucosa can disrupt electrolyte fluxes severely. diarrhea or gastric reflux greatly exacerbates the loss of sodium, potassium, calcium, magnesium and bicarbonate. ischemia of the intestine causing hypoxia and cellular damage may be reflected by an elevated serum phosphate concentration resulting from phosphate leakage from damaged cells. 18 ischemia and cellular hypoxia in any segment of the intestine also causes a shift in energy metabolism to anaerobic glycolysis, resulting in increased production of lactate and elevated serum lactate concentration. reduced perfusion of peripheral tissues from hypotensive shock and intestinal ischemia can cause elevations in serum lactate. however, obstruction of the intestine during ischemia may result in absorption of lactate from the lumen. 19, 20 anion gap is an indirect measurement of organic acid production during states of tissue hypoxia and is a reasonable estimate of serum lactate concentration. 20 metabolic acidosis may accompany lactic acidemia, but an inconsistent association exists between the two, especially when mixed acid-base imbalances are present. 20, 21 elevations of hepatic enzymes, specifically γ-glutamyltransferase, may occur with large colon displacements, duodenal strictures, or anterior enteritis. relative polycythemia from hemoconcentration or splenic contraction and changes in red blood cell deformability from hypoxia or hypocalcemia may increase blood viscosity. blood viscosity increases in patients with acute obstructive disease. hyperviscosity reduces perfusion of capillary beds, thereby exacerbating ischemia and tissue hypoxia. 22 hyperviscosity is one manifestation (along with lactic acidemia, coagulopathies, and clinical signs of shock) of the pathophysiologic events that take place during acute inflammatory or vascular injury to the large intestine. laboratory tests designed to reflect the systemic effects of endotoxemia, ischemia, sepsis, and shock are important to design therapeutic strategies, and monitor response to therapy. abdominocentesis and analysis of peritoneal fluid (pf) is a diagnostic technique performed on many patients with disease of the gastrointestinal tract. one can quantitate cytologic examination of pf; white blood cell and red blood cell counts; protein, fibrinogen, lactate, phosphate, and glucose concentrations; lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity; and ph. the results of pf analysis may help establish a specific diagnosis but more importantly may reflect inflammatory, vascular, or ischemic injury to the intestine requiring surgical intervention. pf reflects a sequence of events that takes place during acute vascular injury to the intestine. the pf protein concentration first increases, followed by an increase in the red blood cell count and fibrinogen concentration. a transudative process resulting from vascular congestion and increased endothelial permeability allows small macromolecules (albumin) to escape into the pf, followed by larger macromolecules (globulin and fibrinogen), and finally diapedesis of cells (red blood cells, then white blood cells). 23, 24 if ischemic inflammation of the intestine and visceral peritonitis occur, an exudative process ensues. severe inflammation of the intestine and visceral peritoneum causes large quantities of protein and white blood cells, primarily neutrophils, to escape into the pf. 24 as damage to the bowel progresses, the protein concentration and red blood cell and white blood cell counts continue to rise. as the degree of irreversible damage to the intestine increases, the pf characteristics become more exudative. 23, 24 eventually, bacteria begin to translocate across the intestinal wall and appear in the pf as the mucosal barrier breaks down. neutrophils predominate, and their cytoplasm becomes granulated, and döhle bodies often are visible. if perforation occurs, bacteria and particles of ingesta appear in the pf, and the neutrophils become degenerate, that is, pyknotic, with karyorrhexis, karyolysis, and smudge cells. 23 elevated pf protein concentration is a sensitive indicator of early inflammation, whereas elevated red blood cell counts in the presence of normal white blood cell counts suggest vascular damage without significant tissue ischemia. 24 elevation of the white blood cell count usually indicates severe tissue inflammation or intestinal injury. 25 the gross color of the pf can be helpful in detecting injury and necrosis of the intestine. a serosanguinous appearance indicates vascular injury, whereas orange or brown-red indicates necrosis with the release of pigments such as hemosiderin. serial samples of pf are most useful in determining the nature and extent of damage to the intestine, but in many cases of ischemia, irreversible tissue damage has occurred by the time pf abnormalities appear. tissue hypoxia and ischemia cause a rapid elevation of pf lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity and lactate concentration. 19, 20, 26 phosphate concentration increases when cellular disruption occurs. 18 pf enzyme activities, phosphate, and lactate concentration increase faster and higher than serum activities. [18] [19] [20] 26 pf ph and glucose concentration tend to decrease during intestinal ischemia, but not as low as in septic peritonitis. 27 although biochemical alterations may provide early indicators of intestinal ischemia and necrosis, they are nonspecific and offer no advantage over conventional methods of pf analysis in many cases. pf alkaline phosphatase has been shown to arise predominantly from degenerating white blood cells, and elevations of other enzyme activities may occur with many inflammatory diseases. 26 thus the specificity of many tests run on pf is questionable. however, in selected cases in which conventional pf analysis and physical examination do not provide sufficient information to develop a treatment plan, biochemical analysis of the pf may be useful. cytologically examined cells of the pf may reflect chronic inflammatory conditions of the large intestine, especially eosinophilic or lymphocytic processes. 28 infectious and inflammatory conditions often cause increases in the neutrophil count and may be indistinguishable unless bacteria are visible. one also may detect neoplastic diseases by pf examination. chronic infection and inflammation may be associated with elevated pf protein and fibrinogen concentrations. culture of pf usually is required to distinguish bacterial infections from noninfectious inflammation unless bacteria are visible on cytologic examination. however, culture of pf is often unrewarding because factors that are found in inflammatory pf inhibit bacterial growth, and leukocytes phagocytose many bacteria in the pf. 29 decreases in pf glucose concentrations (<30 mg/dl) and ph (<7.3) are early indicators of a septic process. the glucose concentration and ph in the pf should approximately equal the blood glucose concentration and ph. a pf fibrinogen concentration greater than 200 mg/dl also indicates bacterial infection. 30 gross examination of the feces can provide information about digestion and transit time in the large intestine. large fiber particles in the feces represent poor mastication or poor digestion in the large intestine. small, mucuscovered, hard fecal balls indicate prolonged transit through the descending colon, whereas increased fluidity implies decreased transit time. feces containing sand or gravel are not necessarily abnormal. however, a significant amount of sand implies that large quantities are present in the colon. frank blood indicates substantial bleeding into the distal colon (right dorsal colon and/or small colon) from mucosal damage. laboratory analysis of the feces is performed frequently in cases of diarrhea. fecal cytologic examination and tests for occult blood detect mucosal inflammation, section 13.1 examination for disorders of the gastrointestinal tract erosion, or ulceration. severe inflammatory diseases in human beings, invasive bacterial infections in particular, have been shown to increase the shedding of leukocytes in the feces. a higher percentage of horses with salmonellosis and diarrhea have fecal leukocyte counts greater than 10 cells per high power field than horses with negative fecal cultures for salmonella. these results suggest that high fecal leukocyte counts indicate salmonellosis in horses with diarrhea. however, the specificity of this test is probably low. low fecal leukocyte counts do not rule out salmonellosis. 31 fecal occult blood tests detect blood in the feces, presumably from erosion or ulceration of the mucosa, but do not distinguish the source of the blood. large volumes of blood (1 to 2 l) given by nasogastric tube were required to produce a positive test for occult blood in the feces, but the amount of blood originating from the large intestine required to produce a positive test is unknown. a positive test implies significant hemorrhage into the gastrointestinal tract. newer, more sensitive tests detect not only occult blood but also degraded blood and may be useful to determine the site and quantity of blood loss. 32 a positive test implies significant hemorrhage into the gastrointestinal tract. bacteriologic examination of the fecal flora has been used to quantitate specific bacterial species in the feces of horses with diarrhea. quantitation of clostridial species may be beneficial in diagnosing clostridial infection of the large intestine. 33 tests to detect clostridial toxins in intestinal contents or feces are important to determine whether clostridia cultured from the feces are causing disease. the most common bacterial pathogens isolated from the feces of horses are salmonella and clostridium. the number of salmonella organisms isolated from the feces of horses with clinical salmonellosis is usually higher than from horses with asymptomatic infections. however, the volume of feces in many cases of acute diarrhea is high, and the concentration of salmonella organisms may be lower than would be expected, accounting for many false-negative fecal cultures. the sensitivity of fecal cultures for detecting salmonella infection may be as low as 20%. culture of five consecutive daily fecal samples is recommended to increase the sensitivity of the test. because salmonellae are intracellular organisms, culture of rectal scrapings or a rectal biopsy sample, along with fecal material, may increase the sensitivity of culture for detecting salmonella infection to 50%. 34 one can perform a polymerase chain reaction assay on fecal samples to detect dna from salmonella sp. the polymerase chain reaction test is more sensitive than culture and is frequently positive in clinically normal horses that continuously shed small amounts of bacteria. polymerase chain reaction or immunologic tests also may detect clostridium perfringens and c. difficile exotoxins in the feces. qualitative fecal examination is a technique to detect nematode and cestode ova, protozoan oocysts, parasitic larvae, and protozoan trophozoites. a direct smear of fecal material is a rapid method to screen feces for ova and oocysts, to detect parasite larvae and trophozoites, and to observe motility of ciliates and parasite larvae. fecal flotation is a more sensitive technique for isolating and detecting ova and oocysts because the eggs are concentrated from the sample. zinc sulfate and sucrose solutions are often used to concentrate less dense ova and oocysts. zinc sulfate produces less distortion of trophozoites and larvae than sucrose solutions. fecal sedimentation is particularly appropriate for ciliates, giardia, and trichomonads. quantitative techniques such as the cornell-mcmaster method allow one to estimate the number of eggs per gram of feces and are most appropriate in monitoring parasite control programs. 35 survey radiography of the normal esophagus is usually unrewarding but may be useful in horses with esophageal obstructions to determine the extent and location of the obstruction. one may detect foreign bodies or soft tissue masses, and in cases of esophageal rupture, one may see free air and ingesta in the tissues surrounding the esophagus and may observe pneumomediastinum. 36 thoracic radiographs may be necessary to detect intrathoracic esophageal obstructions, megaesophagus, or cranial mediastinal masses causing extraluminal obstruction. one may use barium swallows or double-contrast esophagrams after resolution of the obstruction to determine whether a stricture or diverticulum or other underlying disorder is present. 37 barium sulfate is the usual contrast medium and can be administered orally via a dose syringe or by nasogastric tube (50 to 100 ml of a 40% barium sulfate suspension or barium paste). oral administration is preferred for evaluation of swallowing and lesions in the proximal esophagus. administration of contrast using a nasogastric tube (preferably cuffed) allows for delivery of larger volumes of barium (up to 500 ml) but should be performed without sedation if possible. one can follow administration of contrast material with air insufflation to create a double-contrast effect. if one suspects a rupture of the esophagus or if the likelihood of aspiration of the contrast material is high, one should use iodinated organic compounds in an aqueous solution as contrast material. 36 contrast radiography may be the most definitive method for the diagnosis of primary megaesophagus or other functional disorders such as autonomic dysautonomia (grass sickness) affecting the esophagus. 37 one should take care when interpreting esophageal radiographs if the horse is sedated. acepromazine or detomidine administration causes esophageal dilation in normal horses, especially after passage of a nasogastric tube. 38 radiography of the adult equine abdomen is an effective technique in detecting radiodense material in the large intestine, such as enteroliths, sand, and metallic objects. 39, 40 one survey demonstrated that radiography has 76.9% sensitivity and 94.4% specificity for diagnosing enterolithiasis. 39 radiography also can be a useful tool for detecting sand accumulation in the colon that causes diarrhea or impactions ( figure 13 .1-1) and for monitoring resolution in medically treated horses. 41 the large size and density of the adult abdomen precludes evaluation of soft tissue structures because the detail and contrast of the radiographs are usually poor. one is more likely to obtain diagnostically useful abdominal radiographs from small ponies and miniature horses than from full-size adult horses. accumulation of gas is visible on radiographs of adult horses, but distinguishing normal intestinal gas from obstruction is often difficult. horses should be fasted for 24 to 48 hours to reduce the amount of ingesta in the large intestine before radiography. abdominal radiography is more useful in foals than in adult horses. radiographs are more detailed and contrast can be good. radiographic evidence of gas distention in the large intestine may indicate large intestinal obstruction, and radiographic signs of displacement are often diagnostic. one may diagnose impactions, intussusceptions, foreign bodies, and other disorders with the aid of radiography. functional ileus may be difficult to distinguish from mechanical obstruction. 42, 43 administration of contrast (barium sulfate 30% at 5 ml/kg) via nasogastric tube increases the diagnostic capabilities of radiography. 44 gastric ulceration also is recognizable with contrast radiography in the foal, although this is not as accurate a method as endoscopy. 45 contrast administered retrograde via a 24-f foley catheter inserted into the rectum at a dose of up to 20 ml/kg has excellent potential for diagnosing disorders of the small colon, transverse colon, and large colon in foals. 46 ultrasonographic evaluation of the abdomen can add valuable information in cases of acute or chronic gastrointestinal disease. examination of the adult horse requires a 2.5-to 5.0-mhz transducer at minimum. one may use sector, linear, or curved linear transducers. clipping of the hair over the area to be examined, along with the application of isopropyl alcohol and ultrasound coupling gel, enhances evaluation. to evaluate the abdomen adequately, one must know the anatomic location and normal appearance of the individual organs. in the left cranial abdomen, one can assess the greater curvature of the stomach between the eleventh and thirteenth intercostal space, and one can use the spleen and the large splenic vein as landmarks. cases of gastric dilation from gas or impaction appear as an enlargement of the viewing area to cover greater than five rib spaces. 47 one also can evaluate the stomach for intramural or extramural masses such as abscesses or for squamous cell carcinoma. 48 the lesser curvature is not routinely visible. assessment of the small intestine should include evaluation for changes in thickness, motility, location, and visibility. one may find small intestinal loops easily in the left lower quadrant of the abdomen, but these normally are visible in other locations. one can visualize the duodenum consistently on the right side of the abdomen deep to the liver in the tenth to twelfth intercostal space or deep to the right kidney at the fifteenth to sixteenth intercostal space. mural thickening (>4 mm) may occur in cases of infiltrative or proliferative diseases, postoperative cases, enteritis, and paralytic or mechanical ileus. thickening of the small intestinal wall in foals, with or without the presence of gas shadows within the wall, should raise suspicions of clostridial enteritis. one can assess motility by monitoring a specific area for contractions over time. ultrasonography is an accurate method of distinguishing strangulating versus nonstrangulating disorders of the small intestine. strangulated small intestine has thicker small intestinal walls and larger intestinal diameter than in nonstrangulating disorders. strangulating lesions have decreased motility in the incarcerated segments with normal motility elsewhere. 49 cases of paralytic ileus or nonstrangulating obstruction have a diffusely decreased peristalsis, but not to the degree observed with strangulating lesions. 47, 49 one may diagnose some specific lesions of the small intestinal tract using ultrasonography. one may see ascarids in foals in cases of ascarid impaction 47 and epiploic foramen entrapments as edematous loops of small intestine found in the right cranial abdomen. 50 one may note small intestinal intussusceptions as targetlike lesions when viewed in cross sections. 51 the presence of bowel loops, stomach, or liver in the thoracic cavity indicates the presence of herniation through the diaphragm and should be confirmed using radiography or surgical exploration. evaluation of the large intestine may be difficult because of the large amounts of gas within the lumen. however, certain disorders are readily identifiable via ultrasonography. one can assess the nephrosplenic ligament area for bowel entrapment in the left paralumbar fossa. in cases of entrapment, the spleen will be pulled away from the body wall and fluid or gas shadows will be observable dorsal to the spleen, 52 obscuring the kidney, which is normally adjacent and abaxial to the spleen. small colon, small intestine, or pneumoperitoneum also may produce a gas shadow and obscure the kidney from view. 47 sand impactions may appear as hyperechoic bands on the ventral abdominal wall. 47 one may see ileocecal and cecocolic intussusceptions in the upper right paralumbar fossa. 53 in cases of colitis, large, fluid-filled colons may be visible with or without intramural edema. one can find the right dorsal colon consistently abaxial to the liver, within the right thirteenth to fifteenth intercostal space and may be thickened (>5 mm) in cases of right dorsal colitis. evaluation of the abdomen always should include assessment of the peritoneal space for any evidence of an increased amount of pf or increased cellularity of the fluid as indicated by an increase in echogenicity. ultrasonography also can be useful in determining the ideal location for abdominocentesis. one also should evaluate the liver, kidneys, and spleen. one may detect choleliths, nephroliths, masses, abscesses, or enlargement of any of these organs. abscesses or tumors not associated with visceral organs may be difficult to visualize and interpret via ultrasonography. although more commonly used to diagnose lameness and musculoskeletal problems, nuclear scintigraphy has several uses for evaluation of the gastrointestinal tract. scintigraphy is now available at most universities and many private referral hospitals. one must use proper isolation protocols and waste disposal techniques strictly. the procedure requires special gamma cameras and the injection of radioactive materials into the bloodstream. one can use one of two methods: injection of technetium-99m methylene diphosphonate ( 99m tc-mdp) directly into the blood or injection of 99m tc-labeled leukocytes. labeling of leukocytes involves aseptically collecting heparinized blood samples, isolating the buffy coat, and mixing those leukocytes with a radioactive dye ( 99m tc hexamethylpropyleneamine oxime, or 99m tc-hmpao) in vitro. 54 one then reinjects the labeled leukocytes and obtains images. the principle of nuclear scintigraphy then lies in increased uptake of the dye or the white blood cells into areas of inflammation. one of the most common uses of nuclear scintigraphy in evaluating the gastrointestinal tract is diagnosis of dental disease. scintigraphy using 99m tc-mdp proved to be more sensitive in cases of dental disease than was radiography. scintigraphy was slightly less specific, however, and therefore should be used with radiography or computed tomography for ultimate accuracy. 55 scintigraphy using radiolabeled white blood cells can support a diagnosis of right dorsal colitis in the horse. 40 images taken of the abdomen 20 hours after injection showed an increased linear uptake of leukocytes in the region of the right dorsal colon in horses with right dorsal colitis compared with normal horses. this technique also may prove useful for diagnosing intraabdominal abscesses in the horse. other uses of nuclear scintigraphy include evaluation of metastasis of abdominal tumors to bony areas, assessment of biliary kinetics, and determination of gastric emptying times. [56] [57] [58] endoscopy endoscopic examination of the gastrointestinal tract begins with evaluation of the pharyngeal area by examination for any signs of collapse or dysfunction. one should evaluate the ability of the horse to swallow. the floor of the pharynx should be clean and free of feed material and foreign bodies. one can examine the oral cavity with the horse under heavy sedation or anesthesia and with the help of a full-mouth speculum. one can examine the teeth for any irregularities, obvious cavities, sharp points, or hooks and the hard and soft palpate for completeness and any evidence of ulceration, masses, or foreign bodies. one should use a 3-m flexible fiberoptic endoscope to examine the esophagus, which is accomplished best by passing the endoscope into the stomach and viewing the esophagus as one withdraws the endoscope while dilating the lumen with air. the esophageal mucosa normally should be a glistening, light pink color. ulceration can occur with cases of choke, reflux esophagitis or in horses that have had an indwelling nasogastric tube. erosions may be punctate, linear, or circumferential. one should evaluate carefully for any ulcers to ensure that no areas of perforation through the entire thickness of the esophageal wall exist. distinguishing normal peristaltic contractions from areas of stricture requires observation of the area and its motility over time. one also may note diverticula as outpouchings of the mucosa, sometimes associated with a stricture distally. megaesophagus, although rare, appears as a generalized dilation of the esophagus. one may detect food or foreign body impactions of the esophagus via endoscopy. one always should reevaluate the esophagus after removing any obstruction to detect the presence of complications (ulceration, rupture) or initiating causes (strictures, diverticula, and masses). a 3-m flexible endoscope also allows examination of the stomach. the horse should be fasted for at least 12 hours before endoscopy. one can examine the cardia and fundus easily, as well as the margo plicatus. the squamous mucosa should resemble the esophageal mucosa. the glandular mucosa should be glistening red and may have a reticulated pattern. one should carefully examine for evidence of ulceration or masses. one can obtain transendoscopic biopsy material easily from esophageal, pharyngeal, or gastric masses, and because the biopsy size will be small, one should take several samples for histopathologic examination. pharmacologic agents (bethanechol) to empty the stomach and provide complete visualization of the entire fundic region, the pylorus, and the duodenum may be useful. for a complete description of gastroscopy and evaluation of gastric and gastroduodenal ulceration, please refer to chapter 13.10. d-glucose or d-xylose absorption tests are useful in determining malabsorption of carbohydrates from the small intestine in horses. the protocol for absorption tests using either carbohydrate is similar. the horse should be fasted for 18 to 24 hours before testing. increased periods of fasting actually have been shown to decrease absorption of d-xylose and interfere with results. 59 one administers a dosage of 0.5 to 1 g/kg of d-glucose or d-xylose via a nasogastric tube. administration of sedatives may increase the blood glucose levels falsely and interfere with gastrointestinal transit times. one then collects blood samples to measure glucose or xylose concentrations at 0, 30, 60, 90, 120, 150, 180, 210 , and 240 minutes after administration. one may take additional samples up to 6 hours after dosing if the results are questionable. one should measure glucose in blood samples collected with sodium fluoride as an anticoagulant and measure xylose in samples collected in heparinized plasma. a normal d-glucose absorption test, also known as an oral glucose tolerance test, should have a peak between 90 and 120 minutes, and this peak should be greater than 85% above the resting glucose value. 60 complete malabsorption is defined as a peak less than 15% above the resting levels, and partial malabsorption is defined as a peak between 15% and 85% above the resting level. one must keep in mind that gastric emptying, gastrointestinal transit time, length of fasting, cellular uptake and metabolism, and endocrine function influence glucose absorption curves. malabsorption demonstrated by the oral glucose tolerance test is sensitive but not specific. diseases that may cause a lowered or delayed peak include infiltrative lymphosarcoma, inflammatory bowel disease (lymphocytic-plasmacytic or eosinophilic), cyathostomiasis, chronic colitis (salmonella sp.), multisystemic eosinophilic epitheliotropic disease, food allergies, and small intestinal bacterial overgrowth. 61 d-xylose absorption tests have some advantages over the oral glucose tolerance test because xylose is not metabolized in the small intestinal mucosa and insulin does not influence its absorption. gastric and intestinal motility, intraluminal bacterial overgrowth, and renal function still influence xylose absorption, because the kidneys clear xylose. 61 the other main drawback to d-xylose is that it is generally available only in research settings. however, xylose measurements are available at most major universities. a normal d-xylose absorption curve should peak between 20 and 25 mg/dl at 60 to 120 minutes after dosing. 62 decreased xylose absorption can occur in horses with inflammatory bowel disease, lymphosarcoma, multisystemic eosinophilic epitheliotropic disease, cyathostomiasis, extensive small intestinal resections, and any cause of villous atrophy. 61 maldigestion is a common occurrence in foals with diarrhea. bacteria (especially clostridium sp.) and viruses (especially rotavirus or coronavirus) may invade and destroy the villous epithelial cells that manufacture lactase and other disaccharidases, resulting in an inability to digest lactose. in this case, continued ingestion of the mare's milk may cause an osmotic diarrhea, which may exacerbate the underlying enterocolitis. one can perform lactose tolerance testing to assess the degree of maldigestion. one administers d-lactose at 1 g/kg as a 20% solution via nasogastric tube and measures glucose concentrations in the blood at 0, 30, 60, 90, 120, 150, 180, 210 , and 240 minutes. a normal curve shows doubling of glucose levels compared with baseline by 60 minutes after administration. 63 accurate measurement of gastric emptying can be difficult to assess. several methods are currently available. multiple diagnostic imaging techniques have been used to study gastric emptying times. one can use contrast radiography to assess gastric emptying in foals. in the normal foal, barium remains in the stomach for varying amounts of time, but a significant amount should be gone within 2 hours. 44 gastric emptying of solid, nondigestible, radiopaque markers also has been used in adult horses and ponies, but the results were variable and unpredictable even in the normal horse. 64 nuclear scintigraphy is used commonly in human beings to measure gastric emptying and can be used in horses where available. this technique requires oral administration of 99m tc pentenate (10 mci), and serial images taken of the cranial abdomen. the tracer is usually not visible 60 minutes after administration in normal horses. 58 alternatively, if nuclear scintigraphy is not available, one can use acetaminophen absorption testing as an indirect determination of gastric emptying. 58, 65 one performs this test by administering 20 mg/kg of acetaminophen orally and measuring subsequent blood values and calculating the time to reach maximum serum concentrations and the absorption constant. in human beings, the proximal small intestine absorbs almost all of the acetaminophen. 66 the median time to reach peak plasma levels using acetaminophen absorption in horses was 47.7 minutes. 58 one often requires histopathologic examination of tissues from the intestine to diagnose chronic inflammatory, infiltrative, or neoplastic conditions, and such examination can be useful in evaluating the extent of injury after obstruction or ischemia. rectal mucosal biopsies are easy to collect with few complications. however, to collect a full-thickness biopsy of the intestine requires a surgical approach (flank or ventral midline approach). laparoscopy offers a safer technique to observe the large intestine and other abdominal structures. 67 one can obtain biopsies of masses, lymph nodes, mesentery, or intestinal serosa via laparoscopy and mucosal biopsies of the upper gastrointestinal tract via endoscopy. other diagnostics, specifically laparoscopy and computed tomography, are available but require specialized equipment and personnel with specific training. flexible or rigid endoscopes used for laparoscopic evaluation of the abdomen allow for visualization of visceral organs and potentially for collection of biopsy material from masses or organs. full-thickness biopsies of the intestines are not routinely possible through the laparoscope and usually require flank or ventral midline laparotomy. the laparoscopic procedure can be done in the standing or recumbent horse. advantages of this technique over a flank or ventral midline celiotomy include smaller incisions, less healing time, and less procedure time. disadvantages include the large amount of equipment needed, skill involved, and the limitation as a diagnostic modality, rather than a treatment. 68 clinical applications of diagnostic laparoscopy include rectal tears, percutaneous abscess drainage, assessment of adhesions, displacements, and integrity of the serosa of various bowel segments, and biopsy of abdominal masses. 67 computed tomography scans are available at several universities across the country. they have been used frequently to evaluate dental disease and may be useful in evaluating tumors and masses of the head, larynx, pharynx, and proximal esophagus. 69 computed tomography also has promise for evaluating abdominal disorders in foals. most equipment can accommodate up to 400 lb. restrictions to computed tomography as a diagnostic aid include expense, availability, expertise, and weight and size limitation. 12 permeability increases, cells are recruited rapidly into the tissue, plasma protein cascades are activated, and a myriad of soluble products are released that coordinate the response, trigger innate and adaptive immunity, and mobilize reparative elements. although the cellular and vascular response and the secreted mediators of inflammation are important for killing pathogens and limiting invasion of injured tissues by commensal organisms, they can be damaging to host cells and proteins if not tightly regulated. thus if the inciting stimulus is not eliminated quickly, the inflammatory response itself causes significant tissue injury. the mechanism regulating inflammation has been the focus of much research to identify therapeutic targets to modulate the damage to host tissues during many gastrointestinal diseases. recent work has provided some of the molecular and cellular details of this complex physiology and has led to novel therapeutic strategies for treating inflammation. the gastrointestinal epithelium interfaces with a luminal environment inhabited by potentially hostile microbial organisms. the epithelium presents a physical barrier to invasion by the flora of the gastrointestinal tract, consisting of the apical cellular membrane, intercellular tight junctions the permeability of which is highly regulated, and a secreted layer of mucus. breaching of the mucosal barrier by invading pathogens generates potent soluble and neural signals that initiate an inflammatory response. 1 conceptually, the epithelium can be thought of as a sensory organ detecting pathogen invasion to trigger an appropriate host defense and reparative response. noninfectious mucosal injury or invasion of epithelial cells by pathogenic organisms such as salmonella activates synthesis of proinflammatory chemokines (chemoattractants) by epithelial cells and triggers a robust influx of neutrophils into the tissue within hours of the damage. 1 of the chemoattractants produced by epithelium, interleukin-8 (il-8) has a particularly important role in initiating inflammation by recruiting neutrophils from blood [2] [3] [4] and regulating neutrophil migration through tissue matrix adjacent to epithelium. 5, 6 bacteriaderived formylated chemotactic peptides also act as potent chemoattractants that are fully capable of stimulating a robust inflammatory response in the intestine if the epithelial barrier permits the diffusion of the peptides across the mucosa. epithelial cells activated during infection produce cytokines such as tumor necrosis factor α (tnf-α), arachidonic acid metabolites, and other proinflammatory mediators that activate recruited leukocytes. 7 bacterial products, particularly lipopolysaccharide and other cell wall components, are potent activators of leukocytes recruited into the tissue. once the inflammatory response has been initiated, tnf-α, il-1β, and other proinflammatory products of neutrophils, monocytes, mast cells, and epithelial cells amplify the inflammatory response. the enteric nervous system has a key role in sensing and regulating inflammatory responses in the intestine. for example, clostridium difficile toxin a activates a neural pathway that triggers mast cell degranulation and neutrophil influx into the tissue. 8, 9 blockade of this neural pathway is sufficient to abolish the profound inflammatory response induced by toxin a and many of the effects of toxin a on enterocyte secretion. other pathogens and immune-mediated hypersensitivity reactions similarly stimulate inflammation by mechanisms that involve the enteric nervous system. thus the epithelium interacts in a highly complex manner with the intestinal milieu, the enteric nervous system, and inflammatory cells to regulate the tissue response to injury and infection. resident macrophages located in the lamina propria, submucosa, and intestinal lymphoid organs are among the first cells beyond the epithelium to respond to infection or injury. macrophages are activated by bacterial products via pattern recognition receptors and begin to produce proinflammatory molecules important for recruiting and activating neutrophils and monocytes. pattern recognition receptors recognize microbial products ranging from lipopolysaccharide to peptidoglycan and even cpg-containing bacterial dna and signal the invasion by pathogens. of the pattern recognition receptors, the lipopolysaccharide receptor complex is perhaps the best defined. lipopolysaccharide activates macrophages via the cd14-toll-like receptor complex to initiate transcription of the inflammatory cytokines tnf-α and il-1β, which synergize with lipopolysaccharide to amplify the macrophage response. 10 lipopolysaccharide, particularly in concert with inflammatory cytokines, stimulates macrophages to produce copious amounts of nitric oxide, which is microbicidal and vasoactive. nitric oxide and other nitrogen radicals react with reactive oxygen intermediates (rois) to produce some of the most toxic molecules of the host defense system: the peroxynitrites. 11 il-8 is produced as well to recruit neutrophils. as the response progresses, other inflammatory mediators, particularly the arachidonic acid-derived lipids, are produced. these lipids have potent vasoactive effects and are important stimuli of endothelial cells, neutrophils, and platelets. four important changes occur in the intestinal vasculature during inflammation: 1. alteration of blood flow 2. increased vascular permeability 3. increased adhesiveness of endothelial cells, leukocytes, and platelets 4. exposure of the basement membrane and activation of the complement, contact, and coagulation cascades a wide range of mediators alters blood flow during intestinal tract inflammation, from gasses such as nitric oxide (a major vasodilator of the intestinal vasculature) to lipids (prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor), cytokines, bradykinin, histamine, and others. the major sources for these mediators include activated leukocytes, endothelial cells, epithelial cells, and fibroblasts. the primary determinant of blood flow early in inflammation is vascular caliber, which initially decreases in arterioles, but then quickly changes to vasodilation coincident with opening of new capillary beds, increasing net blood flow. the increase in blood flow is short lived, for the viscosity of the blood increases because of fluid loss and tissue edema through leaky capillaries. leukocyte margination, platelet adhesion to endothelial cells and exposed matrix, and areas of coagulation protein accumulation further decrease local circulation. inflammatory mediator actions on the endothelial cells initially increase vascular permeability. histamine, leukotrienes, platelet-activating factor, prostaglandins, bradykinin, and other mediators stimulate endothelial cell contraction, and interendothelial gaps form. 12, 13 this stage of increased vascular permeability is readily reversible. concurrently, mediators such as the cytokines tnf-α and il-1β induce a structural reorganization of the interendothelial junctions, resulting in frank discontinuities in the endothelial monolayer. 14 cytokines also stimulate endothelial cells to express adhesion molecules that support adhesion of leukocytes and platelets, 15 leading to the next and perhaps most devastating event. leukocytes (primarily neutrophils) and platelets adhere to exposed basement membranes and activated endothelial cells. adherent neutrophils and platelets then are exposed to the mediators of inflammation present in the surrounding milieu, which activates the cells to release oxidants and proteases (particularly elastase) that injure the endothelium and have the potential to cause irreparable harm to the microvasculature. [16] [17] [18] marginated neutrophils begin to transmigrate between endothelial cells (as described in later sections), and if their numbers section 13.2 pathophysiology of gastrointestinal inflammation are large enough, they disrupt the integrity of the interendothelial junctions, worsening the vascular leakage. 17 conceptually, these stages of enhanced vascular permeability can be thought of as a mechanism to allow plasma proteins to enter the tissues and to potentiate the critical influx of leukocytes into tissues. however, if not regulated precisely, alterations in hydrostatic and oncotic forces and irreversible damage to the vascular bed may have devastating consequences. moreover, inappropriate activation of plasma protein cascades and leukocytes by activated endothelium and exposed matrix proteins can contribute to systemic inflammation (systemic inflammatory response syndrome; see chapter 13.7 for more information) characterized by hypotension, generalized vascular leak syndrome, and multiorgan dysfunction, which may be fatal. phosphodiesterase inhibitors reduce endothelial permeability in ischemia-reperfusion injury and other models of inflammation-induced vascular leakage 19, 20 by increasing endothelial tight junction integrity and thus may be a viable therapeutic strategy to prevent or reduce the permeability alterations associated with inflammation. endothelial cells respond to products of activated epithelial cells and macrophages in the intestinal tissue to recruit cells and humoral mediators of inflammation into the tissue. activated endothelial cells display a range of molecules critical for neutrophil and platelet adhesion. intercellular permeability increases to expose basement membrane proteins that trigger humoral defense systems (complement, coagulation, and contact system cascades) and to provide access for these macromolecules to the tissue. endothelial cells are an important source of inflammatory mediators that amplify the response and vasoactive substances (particularly nitric oxide) that alter blood flow. infection or injury to the gastrointestinal mucosa causes an influx of leukocytes from the blood that lay the foundation of the inflammatory response. neutrophils, being the first to arrive during inflammation, have a dominant role in the acute response. within minutes, neutrophils are recruited into the tissue where they are activated to release products that not only are proinflammatory and lethal to pathogens but also may damage host cells and tissues. not surprisingly, much attention has been paid to the role of neutrophils in the pathophysiology of many inflammatory conditions. 21 neutrophil depletion is protective in many models of gastrointestinal inflammatory disease. of interest to clinicians, blockade of neutrophil migration into inflamed tissues prevents many of the pathophysiologic events associated with infectious enteritis, ischemia-reperfusion injury, and other gastrointestinal diseases. 16, [22] [23] [24] [25] [26] neutrophil transendothelial migration is a multistep process that is temporally and spatially regulated and has a degree of cell type specificity ( figure 13.2-1) . the predominant sites of neutrophil transendothelial migration are in the postcapillary venules and, in some tissues, the capillaries. endothelial cells in these vessels respond to cytokines and other soluble signals by expressing molecules that promote neutrophil adhesion and transmigration, including selectins and counterreceptors for integrins. as neutrophils flow through these vessels, they are first tethered to activated endothelium. tethering is mediated by selectin molecules expressed on neutrophils (l-selectin) and on activated endothelial cells (p-and e-selectins) that bind to p-selectin glycoprotein ligand-1 (psgl-1), e-selectin ligand-1 (esl-1), and other mucin counterreceptors. 27, 28 tethering functions to increase the exposure of the neutrophil to activating chemokines presented on the surface of the endothelial cells. stimulation of neutrophils by il-8 and other chemokines activates the second step of transendothelial migration. chemokine binding to their receptors on the neutrophil generates signals that activate the binding of integrin adhesion receptors to their ligands, called intracellular adhesion molecules or vascular cell adhesion molecules expressed on endothelial cells in inflamed mucosa. integrin ligation to cellular adhesion molecules arrests the tethered neutrophils, resulting in firm adhesion to the endothelium. of the integrins expressed on neutrophils, the β 2 integrins have a particularly important role in transendothelial migration. calves and human beings with a disorder known as leukocyte adhesion deficiency illustrate the requirement for β 2 integrinmediated adhesion in neutrophil function. leukocyte adhesion deficiency results from an autosomal recessive trait causing the lack of the β 2 integrin expression. the neutrophils from these individuals cannot migrate into most tissues and do not function normally, resulting in poor tissue healing and profound susceptibility to infection, especially at epithelial barriers. 29, 30 other integrins also have a role in transendothelial migration. β 1 integrins mediate transendothelial migration in some cells and seem to be particularly important for mediating emigration of monocytes into many tissues. 31 following this firm adhesion step, neutrophils migrate through the endothelium along a chemotactic gradient of il-8 and other chemoattractants such as leukotriene b 4 . 3, 17, 32 neutrophils migrate across the endothelial monolayer at intercellular junctions via a mechanism involving a series of integrin-ligand interactions mediated by β 2 and β 1 integrins and other adhesion molecules 28 that is generally capable of maintaining the integrity of the endothelial barrier. 33 however, massive flux of neutrophils through the endothelium alters endothelial tight junctions and injures the basement membrane, resulting in increased endothelial permeability to molecules as large as plasma proteins and even endothelial cell detachment from the basement membrane. 17, 18 nonintegrin molecules such as platelet/endothelial cell adhesion molecules (pecams) also are involved in transendothelial migration of neutrophils. 28 homotypic binding of pecams on adjacent endothelial cells form part of the intercellular junction. neutrophils express an integrin of the β 3 family that can bind pecam, and via sequential binding of β 3 integrins to pecam, the neutrophil can , and other proinflammatory mediators. endothelial cells stimulated by inflammatory mediators produce chemoattractants (such as il-8) and display adhesion molecules that promote neutrophil emigration. the three steps of neutrophil (polymorphonuclear [pmn]) emigrationcapture/rolling (mediated by selectins), adhesion (mediated by β 2 integrins), and transendothelial migration (mediated by integrins and platelet/endothelial cellular adhesion molecule [pecam])-occur on activated endothelium. chemoattractant molecules, such as il-8 trigger neutrophil emigration. in inflamed tissues, cytokines (il-1β and tnf-α) and a variety of other proinflammatory mediators stimulate the neutrophil oxidase complex to produce reactive oxygen intermediates (rois; o 2 and h 2 o 2 and their derivatives). activated neutrophils degranulate to release proteases and other hydrolases, cationic peptides (defensins), myeloperoxidase, and other products into the tissue. activated neutrophils synthesize a variety of inflammatory mediators, including prostaglandins (pge 2 ) that modulate the inflammatory response. the products of activated neutrophils (rois, proteases, and mediators) stimulate epithelial secretion and alter tight junction permeability, promoting diarrhea. neutrophils eventually migrate across the infected epithelium by a mechanism that involves integrins, disrupting tight junction integrity and increasing permeability to bacterial products, thus exacerbating the inflammatory response. "unzip" the intercellular junction and migrate through, closing it behind itself. a key feature of neutrophils and other leukocytes is the requirement for integrin-mediated adhesion to extracellular matrix proteins (ecms) or other cells to achieve an optimal effector phenotype. 34 critical components of the ecms in inflamed tissues include fibronectin, fibrinogen, and vitronectin, deposited in tissues as a result of plasma leakage and by synthesis of new proteins by stromal cells and resident macrophages in response to inflammatory mediator activation. the changing composition of the matrix proteins deposited in tissues during inflammation serves as a clue as to the nature of the tissue environment for recruited inflammatory cells as they become activated. individual gene expression studies have demonstrated that adhesion to matrix proteins induces the expression of cytokines and chemokines and their receptors, arachidonic acid-derived lipid mediator synthases, metalloproteinases, growth factors, transcription factors, and other genes that influence the differentiation and activation of inflammatory cells. 35 roi production, phagocytosis, degranulation, and other effector functions stimulated by inflammatory mediators and bacterial products are optimal only when neutrophils adhere to the ecms. 34 adhesion to distinct ecm proteins selectively activates signaling pathways and gene expression of neutrophils, monocytes, and other leukocytes with differing abilities to promote certain functions such that the composition of ecms in many ways controls the development of the ultimate effector phenotype. thus integrin-mediated adhesion provides a mechanism by which neutrophils and other leukocytes can sense the complex tissue environment and respond appropriately. of the activators of neutrophils at sites of inflammation, complement (c3-opsonized particles), cytokines (tnf-α and il-1β), platelet-activating factor, immune complexes, and bacterial products are among the most potent stimuli. other mediators produced during inflammation may modify neutrophil activity, particularly formylated bacterial peptides, chemokines, complement fragments (c5a), leukotriene b 4 , and prostaglandins. activated neutrophils are highly phagocytic; produce large amounts of rois; degranulate to release myeloperoxidase, cationic antimicrobial peptides (defensins), serine proteases (mainly elastase), and metalloproteinases; and secrete inflammatory mediators (tnf-α, il-1β, prostaglandins, leukotrienes, and others) (see figure 13 .2-1). mast cells strategically reside in mucosal tissues, including the submucosa and lamina propria of the gastrointestinal tract, and constitute a crucial first line of defense at epithelial barriers. however, they are also important effector cells of the pathophysiology of inflammatory gastrointestinal diseases. 36 experimental depletion of mast cells, genetic deficiency in the development of mast cells, or pharmacologic stabilization of mast cells to prevent degranulation have a protective effect in a variety of models of gastrointestinal inflammatory disease, including dextran-or trinitrobenzenesulfonic acid-induced colitis, 37, 38 ischemia-reperfusion injury, 39, 40 and immediate hypersensitivity responses. 41 mast cells are activated by a wide variety of microbial products and host-derived mediators. 42 among the activators of mast cells the so-called anaphylatoxins (complement fragments c3a, c5a, and c4a), are potent stimuli causing release of mediators of inflammation. in addition, mast cells are the primary effector cells of immunoglobulin e-mediated anaphylaxis (type i hypersensitivity reactions) by virtue of their high affinity receptors for immunoglobulin e. cross-linking of receptor-bound immunoglobulin e on mast cell surface by antigens (i.e., food antigens) causes rapid degranulation, resulting in the explosive release of granule contents. 43 neural pathways in the intestine also regulate mast cells. mast cells respond to enteric pathogen invasion via neural reflexes that stimulate the release of inflammatory mediators. activated mast cells release preformed histamine, 5-hydroxytryptamine, proteases, heparin, and cytokines from granules. activation also stimulates de novo synthesis of a range of inflammatory mediators, including prostaglandins, platelet-activating factor, and leukotrienes. transcription of a number of peptide mediators, such as the cytokines tnf-α and il-1β among many others, also increases on stimulation of mast cells. mast cell products have profound effects on the vasculature, increasing endothelial permeability and causing vasodilation. 44 moreover, mast cell-derived mediators greatly enhance epithelial secretion by a mechanism that activates neural pathways of epithelial secretion and directly stimulates epithelial cells. 43 mast cell products significantly alter intestinal motility, generally increasing transit and expulsion of intestinal contents. mast cell-derived leukotrienes and tnf-α also have a crucial role in host defense against bacterial pathogens, acting to recruit and activate neutrophils. 45, 46 mast cells have a newly identified role in host defense and inflammatory responses to bacterial pathogens, which in part is caused by the release of proinflammatory mediators during bacterial infection, which is critical for recruiting and activating other innate host defense cells such as neutrophils. however, mast cells are also phagocytic, have microbicidal properties, and can act as antigen-presenting cells to the adaptive immune system. 47 although accumulating evidence was establishing the role of mast cells in innate immunity, a seminal study that unconditionally identified the importance of mast cells in host defense demonstrated that mast cell-deficient w/ w v mice have impaired responses to gram-negative bacterial peritonitis, resulting in a significant increase in mortality. the role for mast cells in host protective responses appears to be as a sensor of bacterial invasion. unlike immunoglobulin e-mediated responses, bacterial products seem to elicit a highly regulated and selective response from mast cells. the complement cascade is a fundamental part of the inflammatory response. activation of the complement cascade by immune complexes (classical pathway) or by bacteria or bacterial products, polysaccharides, viruses, fungi, or host cells (alternative pathway) results in the deposition of complement proteins on the activating surface and the release of soluble proteolytic fragments of several complement components. in particular, activation of either pathway results in the deposition of various fragments of the complement protein c3, which are potent activators of neutrophils and monocytes. 48 opsonization of particles with c3 fragments constitutes a major mechanism of target recognition and phagocyte activation. 49 during the activation of the complement cascade culminating in deposition of c3, soluble fragments of c3 (c3a), c5 (c5a), and c4 (c4a) are liberated. these fragments, termed anaphylatoxins, have potent effects on tissues and cells during inflammation. perhaps most notably, anaphylatoxins are chemotactic for neutrophils (particularly c5a), activate neutrophil and mast cell degranulation, and stimulate roi release from neutrophils. 48 the termination of the complement cascade results in the formation of a membrane attack complex in membranes at the site of complement activation, and if this occurs on host cells such as endothelium, it may cause irreversible cell injury. although the primary source of complement is plasma, epithelial cells of the gastrointestinal tract also produce c3, suggesting that local production and activation of the complement cascade during inflammation occurs in intestinal tissues. clearly, if the regulatory mechanisms of the complement cascade fail, then the inflammatory response may be inappropriate and tissue injury can occur. the role of complement in gastrointestinal inflammation has been most studied extensively in models of ischemia-reperfusion injury. activation of the complement cascades has a major role in altered endothelial and epithelial permeability in these models. several lines of evidence support the importance of complement in intestinal injury. mice deficient in c3 or c4 are protected against ischemia-reperfusion injury. 50 moreover, administration of monoclonal antibodies against c5 reduced local and remote injury and inflammation during intestinal reperfusion injury in a rat model. 51 administration of a soluble form of complement receptor 1, a regulatory protein that halts the complement cascade by dissociating c3 and c5 on host cell membranes, reduced mucosal permeability, neutrophil influx, and leukotriene b 4 production during ischemia-reperfusion injury in rats and mice. 50, 52 although neutrophils and mast cells mediate many of the pathophysiologic effects of the complement cascade, the membrane attack complex may have a primary role in altered vascular permeability during ischemia-reperfusion injury. 53 four components initiate the contact system of coagulation: hageman factor (hf), prekallikrein, factor xi, and high-molecular-weight kininogen. hf is a large plasma glycoprotein that binds avidly to negatively charged surfaces. 54 bacterial cell walls, vascular basement membranes, heparin, glycosaminoglycans, and other negatively charged surfaces in the intestine capture hf and the other three important initiators of the contact system in a large multimolecular complex. of the surfaces that bind hf, the extracellular matrix is a potent activator of the contact system. once bound, hf is converted to hf-α, which cleaves prekallikrein to kallikrein and factor xi to factor xia. the ultimate result is further cleavage of hf by kallikrein and triggering of the contact system cascade, activation of intrinsic coagulation by factor xia, activation of the alternative pathway by hf, and proteolytic cleavage of high-molecular-weight kininogen by kallikrein, releasing biologically active kinins. the products of the contact system, particularly bradykinin, have several important biologic properties that drive many of the vascular and leukocytic responses during inflammation. 54 bradykinin induces endothelial cell contracture and intracellular tight junction alterations that increase vascular permeability to fluid and macromolecules. bradykinin also affects vascular smooth muscle contracture, resulting in vasoconstriction or vasodilation depending on the location. bradykinin also increases intestinal motility, enhances chloride secretion by the intestinal mucosa, and intensifies gastrointestinal pain. in neutrophils, kinins stimulate the release of many inflammatory mediators, including cytokines, prostaglandins, leukotrienes, and rois. 55 kallikrein cleaves c5 to release c5a, a potent chemotactic factor for neutrophils, and thus has a role in recruiting and activating inflammatory leukocytes. the plasma kallikrein-bradykinin system is activated in a variety of acute and chronic inflammatory diseases of the gastrointestinal tract. 56, 57 recent evidence has demonstrated that blockade of the pathophysiologic effects of bradykinin has clinical applications. oral or intravenous administration of the bradykinin receptor antagonist icatibant reduces the clinical signs, onset of diarrhea, and many of the histopathologic changes in experimental models of colitis in mice. 58 inhibition of kallikrein by oral administration of p8720 attenuated the intestinal inflammation, clinical score, and systemic manifestations in a model of chronic granulomatous enterocolitis. 57 thus the contact system is a viable therapeutic target for inflammatory diseases of the intestine. changes in blood flow to the mucosa and other regions of the intestine that reduce perfusion of the tissues can potentiate the initial damage caused by infection or injury. for example, reperfusion of ischemic tissues is associated with platelet and neutrophil clumping in the small vessels of the mucosa, which can impede blood flow. 59 platelets are activated and adhere to exposed basement membrane and activated endothelial cells and provide a surface for leukocyte adhesion. the accumulation of platelets and leukocytes can reduce vessel diameter and blood flow significantly while potentiating local coagulation and thrombus formation. soluble mediators released by activated leukocytes and endothelial cells also affect blood flow. histamine and the vasoactive lipids derived from arachidonic acid (leukotrienes, prostaglandins, thromboxane, prostacycline, and platelet-activating factor) have a prominent role in regulating local perfusion during inflammation and also may have systemic effects on blood flow. procoagulant mediators released by inflammatory cells in response to the inflammatory process (i.e., tissue factor produced by macrophages or endothelial cells), exposed basement membrane proteins, and bacterial components can trigger the contact system and the coagulation and complement cascades, the products of which affect blood flow. nitric oxide, whether produced by endothelial cells or leukocytes (macrophages), is a potent regulator of blood flow and has a significant role in the control of perfusion during inflammation. 60 many of the mediators that affect perfusion also affect endothelial permeability, altering osmotic and hydrostatic balance and tissue edema. in extreme cases, local and systemic coagulopathies initiated by vascular injury and absorption of microbial products and inflammatory mediators induce a hypercoagulable state, leading to microthrombus formation, which can reduce blood flow, or macrothrombus formation, which causes tissue infarction. the cellular mediators of inflammation have the potential to inflict severe injury to intestinal tissues. neutrophils have an important role in the pathophysiology of many intestinal diseases, including ischemia-reperfusion injury, 16, 22 infectious enterocolitis, [23] [24] [25] nonsteroidal antiinflammatory drug-induced mucosal ulceration, 26 and others. depletion of neutrophils, blockade of their emigration into tissues, or inhibition of neutrophil activation reduce the severity of these and other inflammatory diseases. 61 thus many antiinflammatory therapies are emerging that specifically target neutrophil adhesion, migration, and activation. migration of neutrophils through endothelium during emigration into inflamed tissues is remarkable in that the permeability of the endothelial monolayer is preserved under most circumstances. however, a limit exists above which neutrophil migration alters the permeability characteristics of the endothelium. the effect is in part physical in that mere movement of large numbers of neutrophils through the endothelium is sufficient to disrupt the tight junctions mechanically and is caused in part by toxic products of neutrophils that damage endothelial cells and basement membranes. 59, 62 serine proteases (particularly elastase) and metalloproteinases released by degranulating neutrophils destroy tissue matrix proteins and cell-surface proteins that make up endothelial intercellular junctions. these degradative enzymes are particularly damaging to basement membranes and the cellular barriers of the endothelium, thus contributing to vascular permeability (and local tissue edema) and thrombosis. the permeability may be affected to the extent that not only water but macromolecules (albumin, matrix proteins, complement, etc.) leak into the interstitium. blockade of neutrophil adhesion to endothelium with anti-β 2 integrin antibodies has a sparing effect on the microvasculature in experimental intestinal ischemiareperfusion injury, reducing the alterations in vascular permeability and histopathologic evidence of microvascular damage. 59 similar to the endothelium of inflamed tissues, massive neutrophil transmigration occurs across the epithelium in response to infection or injury. neutrophil transepithelial migration increases epithelial permeability by disrupting tight junctions. 62 like the endothelium, neutrophils disrupt the epithelial barrier mechanically as they migrate through (see figure 13 .2-1). proteases, particularly elastase, degrade basement membrane components and tight junction proteins. products of activated neutrophils (tnf-α and interferon-γ) increase tight junction permeability by direct effects on the enterocytes. prostaglandins released by activated neutrophils stimulate epithelial secretion, thus contributing to diarrhea. subepithelial accumulation of neutrophils can lead to deadhesion of the epithelial cells from the basement membrane and mild to severe ulceration. the physiologic results of the effects of neutrophils and their products on the epithelial barrier include protein-losing enteropathy and absorption of bacterial cell wall constituents, which potentiates the local and systemic inflammatory responses. neutrophils in inflamed tissues stimulated by potent host-derived activators (such as il-1β and tnf-α) and bacterial products (lipopolysaccharide) release copious amounts of rois (see figure 13 .2-1). although these oxygen and oxyhalide radicals are important for killing pathogens, they are also potentially toxic to epithelial and endothelial cells and matrix proteins. reactive nitrogen intermediates produced primarily by macrophages during inflammation combine with rois to form peroxynitrites, which are particularly toxic. 11 in addition to injury to mucosal tissues, rois also have an as yet ill-defined role in recruiting and activating neutrophils, thereby potentiating the inflammatory response. 63 in support of the role of rois in inflammatory diseases of the gastrointestinal tract, administration of inhibitors of roi production or pharmacologic roi scavengers can be protective in many models of reperfusion injury or enterocolitis. many therapies are aimed at inhibiting neutrophil activation and effector functions in tissues have been evaluated for use in intestinal diseases. phosphodiesterase inhibitors, by causing cyclic adenosine monophosphate accumulation in neutrophils, are antiinflammatory by virtue of their ability to suppress neutrophil activation and roi production. new phosphodiesterase inhibitors selective for the predominant neutrophil isoform of phosphodiesterase hold promise for use in many inflammatory diseases. subepithelial mast cells also have an important role in altering epithelial permeability in inflamed intestine. during the intestinal hypersensitivity response, subepithelial mast cell release of mast cell protease ii by degranulation increases epithelial permeability via an effect on tight junctions. 41, 64, 65 this alteration in tight junction permeability results in enhanced transepithelial flux of macromolecules, including proteins and bacterial products. cytokines released by mast cells and phagocytes also regulate tight junction permeability. interleukin-4, a product of mast cells and macrophages, increases epithelial permeability. 66 moreover, tnf-α and interferon-γ, products of many inflammatory cells, synergistically increase tight junction permeability. 67 acute equine colitis causes rapid, severe debilitation and death in horses (more than 90% of untreated horses die or require euthanasia). 1 since 1919, several reports have described a number of different acute diarrheal conditions in the horse that appear to share a common characteristic clinical presentation. 2-10 diarrhea associated with acute equine colitis occurs sporadically, is highly fatal, and is characterized by intraluminal sequestration of fluid, moderate to severe colic (abdominal pain), and profuse watery diarrhea with resultant endotoxemia, leukopenia, and hypovolemia. 7, 10, 11 the condition can affect adult horses of all ages but usually occurs in horses between 2 and 10 years of age. disease onset is sudden with a rapid progression and often is preceded by a stressful event. a definitive diagnosis is made in only about 20% to 30% of cases. 7, 11 most ante-and postmortem diagnostic tests remain speculative. treatment of the condition in horses is costly because of the massive fluid therapy required. currently, no curative treatment is available for acute colitis in horses, human beings, or other mammals. treatment regimens provide rehydration, electrolyte and plasma protein replacement, mitigation of the effects of circulating endotoxin, and antimicrobial therapy when indicated. attempts during the past 40 years to develop appropriate treatments (i.e., vaccines or pharmacologic agents) have been hampered by the unavailability of acceptable equine models and have been unsuccessful because of the complex pathophysiology of the intestinal epithelium. although the mechanisms responsible for the fluid losses are not known, inflammatory cells may play an integral role because this condition is characterized by large numbers of granulocytes infiltrating the large intestinal mucosa. [12] [13] [14] [15] [16] equine cecal and colonic tissues collected during the acute stages of experimentally induced acute equine colitis (equine ehrlichial colitis, lincomycin with and without clostridium spp. inoculation, nonsteroidal antiinflammatory drug administration) reveal the presence of numerous neutrophils and eosinophils in the lamina propria and submucosa. 12, 15, 17, 18 granulocytederived reactive oxygen intermediates are crucial to antimicrobial defenses in the gut and stimulate chloride and water secretion by interactions with enterocytes. 19, 20 normal equine intestinal tissue is unique compared with that in most other mammalian species for a preponderance of eosinophils located in the intestinal mucosa and submucosa. 6, 21 production of reactive oxygen intermediates by stimulated phagocytic granulocytes following mucosal barrier disruption may be responsible for the massive fluid secretory response that occurs during the early stages of acute equine colitis. colitis refers to inflammation and mucosal injury of the colon and cecum (typhlocolitis) that may occur in response to a number of causes. 22, 23 the cause of the colonic injury may be well-defined such as in naturally occurring infectious or experimentally induced colitis. however, many cases of human and animal diarrhea have a speculative or unknown diagnosis or no diagnosis. 11, 24, 25 irrespective of the underlying or initiating cause of colonic injury, the colon apparently has a limited repertoire of responses to damage because most forms of colitis demonstrate similarities in histopathologic appearance and clinical presentation. various degrees of mucosal erosion and ulceration, submucosal/mucosal edema, goblet cell depletion, and presence of an inflammatory cellular infiltrate within the mucosa and submucosa are common to many types of human and animal colitis. 21, [23] [24] [25] characteristic clinical manifestations include intraluminal fluid sequestration, abdominal discomfort, hypovolemia, and most often profuse, watery diarrhea. large bowel diarrhea results from abnormal fluid and ion transport by cecal and colonic mucosa. loss of fluid by the large intestine can result from malabsorptive or hypersecretory processes and is often a combination of the two. 26 colonic secretory processes are a function of the crypt epithelium, whereas absorptive processes are limited to surface epithelial cells. 27 under normal baseline conditions, an underlying secretion by crypt epithelium is masked by a greater rate of surface epithelial cell absorption. abnormal forces influencing the rates of secretion and absorption can result in massive, uncontrolled secretion and malabsorption by large intestinal mucosal epithelial cells, leading to rapid dehydration and death. 26, 27 two intracellular processes control colonic secretion: the cyclic nucleotide (cyclic adenosine monophosphate [camp] and cyclic guanosine monophosphate [cgmp]) and the calcium system. 28, 29 agents may activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e 2 [pge 2 ]) or guanyl cyclase (bacterial enterotoxins) and induce increases in camp or cgmp, respectively. this reaction causes phosphorylation of specific protein kinases that induce the actual apical and basolateral membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotide-dependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. 26, 27 calcium may act through calmodulin, which then can activate membranephosphorylating protein kinases. at least four central systems control intestinal secretion: (1) the hormonal system, (2) the enteric nervous system, (3) bacterial enterotoxins, and (4) the immune system. 29, 30 hormonal control of colonic electrolyte transport is exerted primarily through the renin-angiotensinaldosterone axis. 31, 32 the enteric nervous system controls transport through three separate components: (1) extrinsic nerves of the parasympathetic and sympathetic pathways; (2) intrinsic ganglia and nerves, secreting a variety of neurotransmitters including peptides; and (3) neuroendocrine cells (intraepithelial lymphocytes) that reside in the epithelium and release messengers onto the epithelial cells in a paracrine manner. 26, [29] [30] [31] [32] many bacterial enterotoxins can induce intestinal secretion by camp or cgmp signal transduction. 33 bacterial enterotoxins can stimulate enteric neurons, providing evidence for interaction between two controlling systems. 34 preformed inflammatory mediators such as histamine, serotonin, or adenosine and newly synthesized mediators such as prostaglandins, leukotrienes, platelet-activating factor, various cytokines, the inducible form of nitric oxide, and reactive oxygen metabolites can initiate intestinal secretion by directly stimulating the enterocyte and by acting on enteric nerves indirectly to induce neurotransmitter-mediator intestinal secretion. 30 for instance, when added to the t84 colonic cell line, the known mast cell mediators histamine, adenosine, and pgd 2 induce chloride secretion. [35] [36] [37] prostaglandins of the e and f series can cause an increase in chloride secretion in intact tissue and isolated colonic cells. [38] [39] [40] leukotrienes, platelet-activating factor, and a number of cytokines have been shown to have no effect on t84 cell secretion but have a significant effect on electrolyte transport in intact tissue, suggesting that intermediate cell types may be involved in these secretory responses. [41] [42] [43] the epithelial cell chloride secretory response occurs via prostaglandin-and adenosine-mediated increases in cellular camp, whereas histamine acts by h 1 receptor induction of phosphatidylinositol turnover, production of inositol triphosphate, and mobilization of intracellular calcium stores. 30, 33 lipoxygenase products (leukotrienes) are capable of activating a colonic secretory response and do not appear to involve the cyclic nucleotides or calcium ions. 41 phagocyte-derived reactive oxygen mediators (roms) can induce colonic electrolyte secretion in vitro, suggesting that oxidants may contribute directly to the diarrhea associated with colitis. [44] [45] [46] [47] [48] reactive oxygen species initiate the secretory response by increasing cellular camp or stimulating mesenchymal release of pge 2 or prostacyclin, which in turn stimulates the epithelial cell or enteric neuron, respectively. [44] [45] [46] [47] [48] [49] sodium nitroprusside, an exogenous source of nitric oxide, stimulated an increase in chloride secretion in rat colon that was mediated by cyclooxygenase products and enteric neurons. 50 table 13 .3-1 summarizes inflammatory mediator-induced epithelial cell chloride secretion. acute colitis rarely develops by a simple cause or effect phenomenon but is influenced by many extrinsic and intrinsic host and microorganism factors. inflammatory mediators released from mast cells and monocytic or granulocytic phagocytes cause intestinal chloride and water secretion and inhibit neutral sodium and chloride absorption. 29, 30, 67 inflammatory cells, particularly the phagocytic granulocytes, play an important role in mucosal pathophysiology in cases of colitis. 20, 68 large numbers of these cells are observed on histopathologic examination of tissues from human and animal cases of colitis. products of cell activation stimulate direct and indirect secretory responses in intestinal cells and tissues. [28] [29] [30] [45] [46] [47] [48] [49] products of phagocyte secretion may amplify the inflammatory signal or have effects on other target cells in intestine such as enterocytes and smooth muscle cells (table 13 .3-2). the nadph-oxidase system of phagocytes (neutrophils, eosinophils, monocytes/macrophages) is a potent inducer of superoxide radicals used as a host defense mechanism to kill invading microorganisms. 20, 69 during inappropriate stimulation such as inflammation, trauma, or ischemia followed by reperfusion, increased levels of toxic oxygen species are produced, causing damage to host tissues. engagement of any of several receptor and nonreceptor types including phagocytosis mediators, chemotactic agents, various cytokines, and microbial products can stimulate phagocytes. 20 resident phagocytes or those recruited to colonic mucosa early in the disease process are considered to augment mechanisms causing fluid and electrolyte secretory processes, a so-called amplification process. 70, 71 activation of the respiratory burst results in the production and release of large amounts of superoxide anion (o 2 − ) and h 2 o 2 . 69, 72 in addition to these roms, activated phagocytes secrete peroxidase enzyme (myeloperoxidase from neutrophils and eosinophil peroxidase from eosinophils) into the extracellular space. the peroxidases catalyze the oxidation of clby h 2 o 2 to yield hocl, the active ingredient in household bleach products. the peroxidase-h 2 o 2 -halide system is the most cytotoxic system of the phagocytes; hocl is 100 to 1000 times more toxic than o 2 or h 2 o 2 . 69 hocl is a nonspecific oxidizing and chlorinating agent that reacts rapidly with a variety of biologic compounds including dna, sulfhydryls, nucleotides, amino acids, and other nitrogen-containing compounds. hocl reacts rapidly with primary amines (rnh 2 ) to produce the cytotoxic n-chloramines (rnhcl). the mechanisms by which hocl and rnhcl damage cells and tissue remain speculative, but possibilities include direct sulfhydryl oxidation, hemoprotein inactivation, protein and amino acid degradation, and inactivation of metabolic cofactors of dna. 73 peroxidase-derived oxidants have been shown to degrade hyaluronic acid and collagen. 74 in addition, luminal perfusion of specific roms increased mucosal permeability and serosal application caused increases in clsecretion in vitro. 75 tissue myeloperoxidase activity, an index of tissue granulocyte infiltration, is used clinically and experimentally to assess degree of intestinal inflammation. 76, 77 myeloperoxidase activity is elevated in acute flare-ups of human inflammatory bowel disease and various animal models of acute colitis. [76] [77] [78] [79] [80] the acute inflammatory response in these conditions is characterized predominantly by neutrophils, the predominant source of myeloperoxidase activity. however, this assay measures total hemoprotein peroxidase, which includes monocyte and eosinophil peroxidase in addition to neutrophils. 77 moreover, levels of peroxidase activity in equine circulating eosinophils are greater than in circulating neutrophils, 81 and this may apply to resident tissue eosinophils as well. arachidonic acid metabolites are thought to play a role in intestinal inflammation in diarrheal disease. 30, 45, 82 elevated levels of these intermediate metabolites have been demonstrated in natural disease and experimental models of colitis and appear to parallel increases in roms in inflamed intestine. 82 addition of h 2 o 2 or hocl to rat colonic tissue in ussing chambers has been shown to induce pge 2 release and active clsecretion. 47, 48 prostaglandins can stimulate increases in clsecretion in intact intestinal tissue 45, 46, 48 and in isolated colonic t84 cells. 47, 49 interactions between roms and mesenchymal release of pge 2 /pgi 2 may be relevant to the mechanisms producing the diarrheic condition. fibroblasts co-cultured or juxtaposed to colonic t84 cells greatly increased the clsecretory response to h 2 o 2 in vitro through the release of pge 2 . 49 in addition, equine colonic mucosa has an increased sensitivity to endogenously released prostaglandin by exhibiting a significant secretory response under in vitro conditions. 83 endotoxin, the lipopolysaccharide component of the outer cell wall of gram-negative bacteria, is present in large quantities in the large intestine of healthy horses. 81, 83 endotoxins are released into the immediate surroundings when gram-negative bacteria undergo rapid proliferation or die. 84, 85 the intact bowel forms an effective barrier to the transport of significant amounts of these highly antigenic toxins, but the diseased gut absorbs these macromolecules in large amounts, causing the subsequent adverse systemic effects that are often life threatening. 86 disruption of the intestinal barrier (i.e., ischemia, trauma, inflammatory conditions) overwhelms the capacity of the liver to clear endotoxins, and systemic endotoxemia ensues. endotoxins have been shown to be potent activators of the inflammatory process, stimulating the production and release of numerous cytokines by activated macrophages and other immunocytes. 87 in vitro studies suggest that endotoxin activates phagocytic granulocytes to secrete roms, increase release of lysozymes, and enhance the migratory response to chemotactic stimuli. 88 prostacyclin and thromboxane a 2 mediate hemodynamic dysfunction, and lipoxygenase products may induce tissue ischemia. 89 the cytokine interleukin-1 causes a febrile response and initiates the acute phase response. 90 tumor necrosis factor contributes to many of the abnormal physiologic responses, particularly hemostatic functions that potentiate coagulopathy. 91 additional mediators include interleukin-6, platelet-activating factor, procoagulant mediators, and various other speculated substances. 84 endotoxins trigger mucosal immune cells and subsequent release of inflammatory mediators in cases of colitis. the first report of experimentally induced endotoxemia described clinical signs and hematologic findings that closely paralleled those reported for severe colitis in horses. 92 studies in which endotoxin was administered intravenously in human beings and laboratory animals caused significant dose-related gastrointestinal changes, ranging from mild diarrhea to bloody, watery diarrhea. 93, 94 in vitro studies on the effects of endotoxin on intestinal water and electrolyte transport in adult male rats showed a significant decrease in net colonic sodium absorption and increased colonic permeability. 55 in animal models of protein energy deficiency, endotoxin-induced mortality increased compared with that of well-nourished control animals. endotoxin depresses lymphocyte responses to specific mitogens. 95 thus the adverse effects of malnutrition and endotoxin are mutually aggravating. the importance of a normal immune system to the defense of the mucosal surface of the gastrointestinal tract is evident in the immunosuppressed state. primary immunodeficiencies affecting the gastrointestinal tract are well documented. [96] [97] [98] common agammaglobulinemia is the most frequently reported gastrointestinal disease and causes b cell deficiency-associated giardiasis. 99 interestingly, selective immunoglobulin a (iga) deficiency rarely results in intestinal disease because of a speculated increase in mucosal igm response. however, combined iga and igm deficiencies with a higher incidence of intestinal disease occur. a selective deficiency of secretory iga has been associated with intestinal candidiasis. certain mucosal pathogens may enhance their pathogenicity by producing iga proteases. 99 defects in cell-mediated immunity are associated most commonly with intractable diarrhea, and organisms frequently involved include salmonella spp., escherichia coli, and shigella spp. 100 acquired immunodeficiency or immunosuppression in adults can result from infectious diseases (particularly viral), nutritional deficiencies, aging phenomenon, and drugs (corticosteroids, azathioprine, cyclophosphamide). nutrition is a critical determinant of immunocompetence and risk of illness. 101, 102 impaired systemic and mucosal immunity contribute to an increased frequency and severity of intestinal infections observed in cases of undernourishment. abnormalities occur in cell-mediated immunity, complement system, phagocytic function, mucosal secretory antibody response, and antibody affinity. morbidity caused by diarrheal disease is increased particularly among individuals with stunted growth rate because of malnourishment. 102 the critical role of several vitamins and minerals in immunocompetence has been substantiated in animals deprived of one dietary element and findings in human patients with single-nutrient deficiency. tables 13.3-3 and 13 .3-4 summarize nutritional-related immune system abnormalities. in summary, nutritional deficiency can cause increased colonization of the intestine with microorganisms, alter the symbiotic characteristics of resident intestinal bacterial populations, and impair defenses of the gastrointestinal tract, allowing increased risk of systemic spread of infection and absorption of macromolecules (in particular, endotoxin). indigenous microflora greatly impede colonization of the gastrointestinal mucosa by pathogenic organisms. the ability of a potential pathogen to initiate an infection depends on its ability to breach the mucosal epithelial barrier. one mechanism by which the normal flora inhibit establishment of pathogens is by preventing adherence of the pathogen to mucosal cells by occupying the site or by stearic hindrance. [102] [103] [104] [105] resident microbes also produce byproducts such as antibacterial factors that allow symbiosis rather than competition between them. another hindrance mechanism is production of volatile fatty acids by normal microbial digestive processes to create an environment that is toxic to many bacterial populations, particularly the enterobacteriaceae. 106 disturbances in motility patterns occur during inflammatory diseases of the colon, but the role of motility alterations in the pathogenesis of diarrhea remains unclear. invasive bacteria cause characteristic motor patterns in the colon consisting of rapid bursts of motor activity that appear to decrease transit time through the large intestine. the result is reduced clearance of bacteria from the large intestine, which may contribute to the virulence of the organism. 107 absorption of endotoxin and the release of inflammatory mediators such as prostaglandins disrupts the motility patterns of the large intestine, resulting in less coordinated contractions, and may contribute to the alterations in motility seen with invasive bacteria. although the effect of endotoxin and prostaglandins on transit time is not profound, the disruption of coordinated activity may play a role in causing diarrhea. 108 thorough mixing and prolonged retention time of ingesta are important not only in microbial digestion of nutrients but also in absorption of microbial byproducts and fluid. 32, 109 the ingesta is viscous and therefore must be mixed to bring luminal ingesta in contact with the mucosa for absorption. 109 in addition, poor mixing increases the thickness of the unstirred layer, decreasing contact of ingesta with the mucosa and decreasing absorption. 32, 109 progressive motility must be present, however, if a diarrheal state is to occur. 32, 109 ileus may be accompanied by increased fluid in the lumen of the large intestine, pyridoxine, folic acid, impairs cell-mediated immunity and vitamin c, vitamin e reduces antibody response. vitamin b 6 decreases lymphocyte stimulation response to specific mitogens. zinc deficiency affects humoral and cell-mediated immunity. iron inhibits bacterial multiplication. copper depresses antibody response. needed by neutrophils and lymphocytes for optimal function, which may be related to myeloperoxidase and ribonucleotidyl reductase deficiencies. but without progressive motility the fluid is not passed. frequently, acute colitis causes a period of ileus characterized by scant stool. diarrhea is apparent only when motility returns and the ingesta is passed. increased progressive motility has been suggested to cause diarrhea by decreasing transit time and is thought to play a role in irritant catharsis and in the mechanism of action of some laxatives. 110 irritation and distention increase motility and may well decrease transit time, but increased secretion also is thought to contribute to diarrhea caused by these substances. 111 one should direct the clinical investigation of malabsorption at ascertaining and trying to localize the source of the abnormality. in medical practice, impairment of one or more phases of the digestion and absorption of dietary constituents may precipitate clinical signs that are associated primarily with carbohydrate, protein, or fat malabsorption. this level of differentiation is not possible in the horse because of the herbivorous diet and the contribution of large intestinal functions. in human and small animal medicine, disturbances in digestive processes especially from exocrine pancreatic insufficiency or reduced intestinal bile salt concentration are principal determinants of many clinical malabsorption syndromes. the rarity of pancreatic problems in horses and the herbivorous diet makes maldigestion less of a concern and difficult to pursue diagnostically. nevertheless, maldigestion undoubtedly contributes to chronic weight loss conditions in horses, which may be significant with severe infiltrative disease of the small intestine with partial to total villous atrophy and flattened mucosa. impairment of digestive processes may exacerbate diarrhea in the suckling foal through reduced intestinal bile salt concentrations from hepatic or ileal dysfunction. malabsorption is not synonymous with diarrhea, although diarrhea may be a feature. adult horses rarely exhibit diarrhea with small intestinal problems unless large intestinal involvement is concomitant. chronic diarrhea is predominantly a large intestinal disorder that reflects an overload of water and electrolytes and thus may be considered a state of impaired absorption. primary small intestinal disease is more likely to occur in neonates and young foals. for example, acquired small intestinal brush border lactase deficiency may result in increased lactose fermentation in the large intestine and induction of osmotic diarrhea. 1 box 13.4-1 lists conditions that have been or potentially could be associated with malabsorption syndromes and maldigestion in the horse. malabsorption in horses has no pathognomonic clinical syndrome. case recognition derives from the robust investigation of horses with chronic wasting. prevalence is unknown. no strict case definition exists, even for chronic wasting. interest generated by unusual clinical test results and their related pathologic findings has stimulated publication of reports of cases considered as malabsorption in horses. pathologic description of the predominant cellular infiltrate and the pattern of intestinal distribution have resulted in the classification of many conditions as representing examples of chronic inflammatory bowel disease (cibd) (see box 13.4-1), drawing analogies from human medicine. in the affected animal, coexistent enteric protein loss reflecting changes in mucosal integrity from extensive infiltration and inflammation in the intestinal tract is likely to be more debilitating than the malabsorption. the principal concern of the owner is the weight loss and poor condition of the horse. many clinical examination findings, except for body condition, may appear within normal limits. investigation of the weight loss, together with the clinical pathologic findings, helps to eliminate other more commonly encountered causes of wasting. box 13.4-2 lists clinical signs that may be associated with malabsorption syndromes. no characteristic clinical pathologic profile of malabsorption exists. findings relate to the stage of the underlying disease process and intercurrent problems. the syndrome tends to cause anemia (normocytic, normochromic) and neutrophilia. hemolytic or macrocytic anemia and thrombocytopenia have been observed in alimentary lymphosarcoma. lymphocytosis (leukemia) rarely is encountered. eosinophilia is uncommon even with suspected immune-mediated conditions and widespread tissue eosinophilia. many animals are hypoalbuminemic and hypoproteinemic; horses with alimentary lymphosarcoma may exhibit hyperproteinemia and hypergammaglobulinemia. serum or plasma may be lipemic. the clinician may find elevated hepatic and biliary tract enzymes (γ-glutamyltransferase and alkaline phosphatase) in multisystemic conditions; for example, eosinophilic granulomatosis (multisystemic eosinophilic epitheliotropic disease; eg [meed]). abdominocentesis has been of diagnostic value in several alimentary lymphosarcoma cases, but rarely in the granulomatous conditions. ultrasonographic examination of the abdomen can yield infomation on intestinal distention, wall thickness, and unexplained masses detected on rectal palpation. rectal biopsy is easy to perform and may provide an indication of cellular infiltration that could be present at more proximal locations. however, pathologists examine few equine rectal samples, and the interpretation is frequently equivocal. adoption of standardized grading or classification would improve the diagnostic value. a proposed classification system was based on a retrospective study of 130 rectal biopsies from 116 horses ages 1 to 18 years with clinical signs of intestinal disease. necropsy results were studied from 40 horses. biopsy specimens (21 horses) and necropsy rectal tissue (9 horses) from 30 horses ages 4 to 22 years served as controls. simple proctitis, the presence of neutrophils in the crypt or surface epithelium, was an abnormal finding compared with mild scattered neutrophil infiltration in controls. simple proctitis was found in association with malignant lymphoma and other inflammatory disorders. inflammatory bowel disease was diagnosed from rectal biopsy specimens in 6 of 12 eg (meed) cases and 4 of 9 granulomatous enteritis cases confirmed at necropsy. 2 rectal biopsy aided diagnosis for 3 of 7 horses in a series of lymphocytic-plasmacytic enterocolitis cases. 3 eosinophils were demonstrated on impression smears of rectal mucosal biopsies from 1 of 2 horses with eosinophilic enterocolitis. 4 skin biopsies or ultrasound-guided biopsies of liver, lymph node, or lung may reveal evidence of multisystemic disease. one can obtain intestinal and lymph node biopsies via a standing laparotomy. exploratory laparotomy facilitates rigorous inspection of the gastrointestinal tract and associated organs to obtain multiple biopsies from intestinal sites and lymph nodes. the procedure may provide a diagnosis, enabling one to make decisions on potential treatment and management options. cost and potential postoperative complications may limit surgical procedures for diagnosis. laparoscopy may provide an alternative means to facilitate biopsy of certain tissues. however, one should consider surgical exploration as an option early in the process rather than as a last resort. the noninvasive breath hydrogen test used to assess carbohydrate malabsorption in human beings has not proved reliable in equine studies. 5 intestinal function tests can provide a practical and inexpensive means to assess the absorptive capability of the small intestine. for clinical practice purposes this is limited to carbohydrate absorption. abnormality of carbohydrate absorption has become an important precept on which to base a diagnosis of malabsorption in the horse. however, results of the oral glucose tolerance test (ogtt) or d-xylose absorption test require cautious interpretation. pathologic changes in the mucosa and submucosa must be extensive and widely distributed to greatly affect the peak plasma concentration and shape of the curve. the tests are easy to perform in practice and require a baseline blood sample predosing and further samples for up to 6 hours after administration of the solution. many commercial laboratories conduct glucose and xylose assays. the immediate dietary history, gastric emptying rate, intestinal transit, age, and hormonal effects of the horse influence glucose peak and curve shape. higher glucose peaks are recorded from healthy animals eating grass or hay than from those eating concentrates. recent appetite or the level of cachexia may affect test results. maximum plasma glucose level (>85% baseline) is reached by 120 minutes in healthy animals given 1 g glucose per kilogram body mass as a 20% solution. 6, 7 break points below which the probability increases of carbohydrate malabsorption associated with intestinal morphology changes have been proposed. 7 a referral population of 42 mature horses with chronic weight loss was divided into three groups based on ogtt results to determine if any concurrence with the morphologic diagnoses existed. group 1 (n = 5) had a normal ogtt (peak glucose concentration at 120 minutes >85% baseline) and contained animals that had normal small intestinal morphology, and a few with large intestinal lesions. group 2 (n = 25) had partial malabsorption and included 18 horses with small intestinal infiltrative disease that allowed some glucose uptake. diagnoses included lymphosarcoma, villous atrophy, granulomatous enteritis and eg (meed). seven horses had normal small intestinal histologic findings. peak glucose concentrations were less than 85% and greater than 15% of baseline at 120 minutes. seventeen horses in the group had large intestinal pathologic conditions. group 3 horses (n = 12) had total malabsorption; the peak concentration at 120 minutes was less than 15% above baseline. these horses had severe infiltration throughout most of the small intestine that was attributed predominantly to lymphosarcoma or granulomatous enteritis. however, the test is far from definitive; one cannot assume a flat curve indicates malabsorption and a poor prognosis. two horses with chronic weight loss initially diagnosed with malabsorption based on flat ogtt curves subsequently showed more normal ogtt responses. 8 full-thickness intestinal biopsies were unremarkable. one horse had an elevated serum immunoglobulin e to oat allergen. oats and oat straw were removed from access. dexamethasone was given on a tapered protocol, and a repeat ogtt was normal at 18 months. the other horse received oral probiotics to counter suspected small intestinal bacterial overgrowth, was clinically normal in 2 months, and had an improved ogtt with a 60 minute peak. therefore malabsorption, as defined by an absorption test and weight loss, may occur in the horse without significant morphologic changes in the intestine, and the condition may be transient. demonstration of carbohydrate malabsorption in 16 of 24 horses with chronic diarrhea showed poor diagnostic sensitivity for small intestinal involvement. impaired glucose absorption was recorded in horses with predominantly large intestinal problems, cyathostomiasis, chronic colitis, alimentary lymphosarcoma, and meed. 9 although prior dietary history influences peak plasma xylose concentration, xylose is not confounded by hormonal effects or mucosal metabolism. gastric emptying rate, intestinal motility, intraluminal bacterial overgrowth, and renal clearance do affect curve shape. healthy mares not fed for up to 96 hours had flatter curves and a slower decrease in plasma xylose than when deprived for 12 to 36 hours. 10 hence recent appetite or the level of cachexia may influence test results. abnormal d-xylose absorption represented by a flat curve or delayed absorption is considered indicative of significant jejunal disease and has been observed with most examples of cibd, parasitism, and idiopathic villous atrophy. 11, 12 ponies may have lower peak d-xylose concentrations at 60 and 90 minutes than horses, although the range of peak values at the test dosage (0.5 g d-xylose per kilogram body mass in a 10% solution) is wide. potentially diagnostic discriminatory cut off points for peak plasma xylose concentrations have not been determined. abnormal absorption curves have been detected in the absence of small intestinal histologic changes, 13 and interpretation is clouded further by findings from small intestinal resection studies in healthy ponies. nine ponies with 70% distal small intestinal resection and four shamoperated controls were placed on interval feeding for 5 weeks and then turned out to pasture until 6 months after surgery. grazing was supplemented by twice daily (meal feeding) concentrate rations. all ponies gained weight and were clinically normal, and none developed diarrhea. however, the mean peak xylose concentration at 60 minutes declined progressively (at monthly intervals) over the study period in the resection group to 15% of that of controls. lack of clinical malabsorption was attributed to adaptation of the residual 30% of healthy small intestine and of large intestinal function. 14 bacterial overgrowth in the small bowel remnant from refluxed cecal contents (resected ponies had ileocecal valve bypass) may have contributed to the abnormal xylose assimilation. by contrast, xylose absorption decreased over 6 months, associated with substantial weight loss, lethargy, and diarrhea, in an earlier study of extensive (≥60%) small intestinal resection in ponies. 15 an important difference was the feeding pattern; those ponies received pelleted feed twice daily for the entire 6 month follow-up period. consequently, horses with suspected malabsorption may adapt to an interval feeding regimen. the critical factor could be the availability of sufficient unaffected or minimally affected small intestine and large intestinal functional capacity. the outcome for animals with small intestinal disease and some unknown degree of large intestinal pathologic dysfunction may be less successful than shown in the experimental study. abnormal xylose absorption reverted to normal following 35 days of corticosteroid therapy in an adult thoroughbred gelding with a 6-week history of weight loss and diarrhea for 3 weeks; peak xylose concentration at 60 minutes was within normal limits and the horse had gained weight. 4 d-xylose absorption was abnormal in an adult standardbred gelding with a 2-month history of poor performance, weight loss, intermittent fever, mesenteric lymphadenopathy, elevated fibrinogen, and decreased albumin and globulin levels. multiple fullthickness small intestinal biopsies revealed evidence of granulomatous enteritis. the horse received antibiotics postoperatively and then corticosteroids parenterally for 4 to 5 months. after 3 weeks, peak plasma xylose had increased, although absorption was delayed. five months after cessation of corticosteroid therapy, the horse had regained weight and was bright and alert, and d-xylose absorption was normal. 16 diagnostic predictions were made retrospectively by examining d-xylose absorption in horses with granulomatous enteritis compared with those with eg. 17 peak xylose concentrations were much lower in horses with granulomatous enteritis than those with eg, whereas in eg the absorption curve shifted to the right with the peak occurring at 240 minutes. the small intestine is affected predominantly in granulomatous enteritis with extensive villous atrophy and more diffuse lesions in the large intestine, whereas in eg (meed) the large intestine is more involved. hence, the extent and distribution of pathologic changes in the small and large intestines may influence xylose absorption test results. the chronic wasting horse with suspected malabsorption and probable enteric protein loss has at best a guarded to poor prognosis. prognosis may be improved through early and aggressive investigation to achieve a diagnosis, and perhaps assess the stage in the natural progression of the disorder. the owner may elect euthanasia of the animal or may be willing to determine whether the condition can be improved. in the short term, intravenous infusion of plasma or colloids, with or without fluids and electrolytes, may be necessary to stabilize the condition. prognosis is much worse for the horse that is inappetent. prolonged intensive total parenteral nutrition and/or oral alimentation may not be a realistic course of action. the overall therapeutic and management plan can prove to be expensive. the owner must be cognizant from the start that the outcome may not be altered, even after protracted therapy. one cannot make predictions for outcome of therapy without meaningful data because only a few case reports of successful responses with long-term follow up exist. some level of digestive and absorptive capability remains in the diseased small intestine. interval feeding of small quantities of food may be beneficial if the horse continues to eat, and particularly for animals with ravenous appetites that seem able to maintain their reduced state of body condition without further losses. diet should include feeds with a high fiber content to favor large intestinal fermentation, including grass hay and access to pasture complemented by commercial high-fiber rations based on beet pulp and soybean hulls. energy intake can be increased through feeding high-energy dense fats that provide 2.25 times more calories than carbohydrates. most affected horses should tolerate high fat (5% to 10%) processed feeds containing vegetable oils or rice bran (up to 20% of the concentrate mix, equivalent to 8% vegetable oil) to achieve the higher-fat composition. changeover to a higher-fat concentrate should be gradual. even in healthy animals that can eat up to 20% added fat, appetite may decrease as the percentage increases, and fecal consistency may change. clearly, the objective for the horse with suspected malabsorption is to sustain, and preferably increase, dietary intake, value, and efficiency. the owner of an affected horse must be prepared to experiment with feeds, must be patient, and must keep records. no standard procedure exists. exposure to a feed component may contribute to the problem as an allergen eliciting a hypersensitivity reaction. identifying the potential allergen through immunologic testing or by stepwise removal and outcome assessment over a longer period may be difficult. the clinician should give immunosuppressive drugs early in the process. immunosuppressive agents have produced the most promising responses to ameliorate the effects of conditions associated with malabsorption, particularly cibd. short-duration, and in some cases more prolonged and sustained, improvements in body condition, weight gain, demeanor, energy and activity levels have occurred following corticosteroid administration. one should start treatment as early as possible. one should follow initial parenteral (intramuscular or intravenous) loading doses of dexamethasone (sodium phosphate) with a series of depot injections, or orally administered prednisolone or prednisone, on a tapered dose protocol over a period of months. interval low-dose therapy may be necessary if clinical signs return after treatment ends. one uses the lowest dose to control the clinical signs for alternate-day therapy. clinical benefits far outweigh concerns over potential adverse effects. chemotherapeutic agents such as vincristine, cytosine, cyclophosphamide, and hydroxyurea have been tried in a few cases of cibd or lymphosarcoma with no apparent success, probably related to the advanced stage of the disease when treatment was initiated and the dose selected. resection of a segment of intestine that is edematous, hemorrhagic, or constricted is an option in localized forms of cibd, 18, 19 particularly if gross changes are not discernible in adjacent or distant parts of the intestinal tract, that is, malabsorption is not a feature. long-term outcome has been favorable. removal of a substantial proportion of the diseased small intestine may be indicated in a horse with malabsorption, considering that resection of 70% distal small intestine was performed in healthy animals without inducing adverse effects. however, because pathologic changes may exist in normalappearing small or large intestine that is not resected or biopsied, the prognosis remains guarded. two young horses with granulomatous enteritis had the thickened terminal small intestine resected with positive outcomes; one survived 4 months, the other has a follow up extending more than 10 years. 20 anthony t. blikslager to gain an appreciation of the mechanisms whereby the mucosa is injured and subsequently repaired, one must understand how the integrity of the mucosa is regulated physiologically. regulation of mucosal integrity is referred to as mucosal barrier function, which is vital because it prevents bacteria and associated toxins from gaining access to subepithelial tissues and the circulation. however, the mucosa has two conflicting functions: it must serve as a protective barrier and continue to absorb solutes necessary to maintain well-being of the host. this conflict is most notable at the intercellular (paracellular) space, which allows passage of select solutes and water, 1-4 but which does not admit large molecules, including bacterial toxins. 5 the paracellular space is regulated almost exclusively by the tight junction, 6 which is the interepithelial junction at the apical-most aspect of the paracellular space. although these tight junctions originally were viewed as inert cellular adhesion sites, what has become clear in recent years is that tight junction permeability depends on tissue-specific molecular structure and is regulated by a complex array of intracellular proteins and the cytoskeleton. tight junctions consist of a group of transmembrane proteins that interdigitate from adjacent cells. although occludin originally was thought to be the predominant tight junction transmembrane protein, a group of proteins termed claudins appear to be more critical. 7 these transmembrane proteins interact with the cytoskeleton via a series of intracellular proteins, including zonula occludens 1, 2, and 3; cingulin; and others. 8 in addition, local regulatory proteins such as the small guanosine triphosphatase-rho are also critical to tight junction function. in general the relative contractile state of the actin cytoskeleton determines the degree to which tight junctions are open or closed, but the complexities of regulation of this process are understood poorly. 9, 10 the most sensitive measure of mucosal barrier function is transepithelial electric resistance, which is measured by mounting mucosa in an ex vivo system called an ussing chamber, because this measurement is largely a reflection of the permeability of mucosa to ions. 11, 12 ions may follow one of two routes when traversing epithelium: transcellular and paracellular. 5 because cell membranes have a resistance to passive flow of ions 1.5 to 3 log units greater than that of the epithelium as a whole, measurements of transepithelial resistance largely reflect the resistance of the paracellular space, and in particular the tight junctions that regulate paracellular flow of ions. 12 because tight junctions differ in structure from different portions of the mucosa, 13 measurements of transepithelial resistance reflect the net resistance of epithelium of variable permeability within a given tissue. for example, tight junctions in the intestinal glandular structures called crypts are leakier than those in the surface epithelium because of fewer and less organized tight junction strands. 11, 14 conversely, surface epithelium has a greater number of well-organized tight junction strands that result in epithelium with a high resistance. 11 this correlates well with the absorptive function of epithelium located on the mucosal surface and the secretory function of crypt epithelium. structure of tight junctions also varies with the segment of intestine. for example, tight junctions have more strands in the ileum than in the jejunum, which is reflected by a higher transepithelial resistance in the ileum. 15 the stomach has four regions based on the type of mucosal lining (in an orad to aborad order): nonglandular stratified squamous epithelium, cardiac epithelium, proper gastric mucosa, and pyloric mucosa. 16 stratified squamous epithelium has distinct differences in terms of barrier function compared with the remainder of the gastrointestinal tract. this epithelium has baseline transepithelial resistance measurements of approximately 2000 to 3000 ω/cm 2 , which is an order of magnitude higher than the adjacent cardiac mucosa. 17, 18 thus the stratified squamous mucosa is exceptionally impermeable. this in effect is the only mechanism this mucosa has to defend itself against injury. the stratified squamous epithelium consists of four layers: the outer stratum corneum, stratum transitionale, stratum spinosum, and the basal stratum germinativum. however, not all layers contribute equally to barrier function, the barrier being composed mostly of interepithelial tight junctions in the stratum corneum and mucosubstances secreted by the stratum spinosum. 17, 19 the relative impermeability of stratified squamous mucosa can be demonstrated by the effects of hcl on this type of epithelium in vitro, which has little effect until it reaches a ph of 2.5 or lower. 18 thus although most of the literature on equine ulceration pertains to the effects of hcl and inhibitors of hcl secretion, [20] [21] [22] [23] other factors may be critical to the development of gastric ulcer disease. the site of hcl secretion (proper gastric mucosa) also is protected from so-called back-diffusion of h + by a high transepithelial electric resistance (compared with cardiac mucosa), but a number of other critical mechanisms also exist to prevent acid injury. the gastric mucosa secretes mucus and bicarbonate, which together form a hco 3 --containing gel that titrates acid before it reaches the lumen. 24, 25 the mucus layer is formed principally by glycoproteins (mucins) secreted by goblet cells but also includes other gastric secretions and sloughed epithelial cells. mucins consist of core peptides with a series of densely packed o-linked polysaccharide side chains that, once secreted, become hydrated and form a viscoelastic gel. however, the mucus layer does not form an absolute barrier to back-diffusion of acid. thus for acid that does back-diffuse into the gastric mucosa, epithelial na + /h + exchangers are capable of expelling h + once the cell reaches a critical ph. 25 recent studies have renewed interest in the protective mechanisms of mucus because of the discovery of a group of compounds secreted by goblet cells called trefoil peptides. the name of these peptides is derived from a highly conserved cloverleaf structural motif, which confers substantial resistance to degradation by proteases including pepsin. three members of this group are known, ps2, sp, and intestinal trefoil factor, the latter of which is secreted solely by goblet cells in the small and large intestine. ps2 and sp are secreted by goblet cells within the stomach and are believed to intercalate with mucus glycoproteins, possibly contributing to the barrier properties of mucus. 26 these peptides also play a critical role in repair of injured mucosa. an additional mucosal function that serves to reduce the level of injury is adaptive cytoprotection, wherein application of topical irritants to gastric mucosa results in subsequent protection of mucosa in response to repeated exposure to damaging agents. for example, pretreatment with 10% ethanol protected against mucosal damage in response to subsequent application of absolute ethanol, and this effect was abolished by treatment with the cyclooxygenase inhibitor indomethacin. 27 the cytoprotective effect of prostaglandins has been demonstrated directly in studies in which preadministration of prostaglandins protected gastric mucosa from damage by agents such as concentrated hcl and hypertonic saline. 28 prostaglandins appear to be cytoprotective in the stomach at doses less than those used to inhibit gastric acid secretion, ruling out a simple antacid mechanism. 29 although not fully characterized, cytoprotection has been attributed in part to prostaglandin-stimulated mucus production. 30 an associated beneficial effect of prostaglandins is the increased production of bicarbonate, which is trapped within mucus on the surface of the mucosa. 31, 32 interestingly, prostaglandin e 2 (pge 2 ) appears to lose its cytoprotective activity in the presence of the mucolytic agent n-acetylcysteine. attention also has been directed at enhanced mucosal blood flow as a potential mechanism for prostaglandin-mediated cytoprotection. for example, pretreatment with pgi 2 protected against ethanol-induced mucosal damage as a result of increased mucosal blood flow. 33 although pge 2 , which is also cytoprotective, does not increase blood flow, 34 it may prevent vascular stasis associated with irritant-induced vascular damage resulting from inhibition of neutrophil adherence to damaged endothelium. 35 sensory nerves also have been implicated in cytoprotective mechanisms. these nerves are distributed throughout gastrointestinal mucosa. as an example of their importance in mucosal cytoprotection, pretreatment of newborn rats with capsaicin (to which sensory nerves are sensitive) renders the mature rats more susceptible to gastric injury. 36 alternatively, use of a low dose of capsaicin, which stimulates rather than destroys sensory nerves, protects gastric mucosa against injurious agents. 37,38 sensory nerves contain neuropeptides such as calcitonin-gene-related peptide (cgrp) and substance p, which may play a protective role via vascular mechanisms. for instance, cgrp stimulates increased gastric blood flow, which is theorized to reduce injury in much the same way as prostaglandins do. in fact, recent studies suggest that the roles of prostaglandins and cgrp in gastric cytoprotection are intertwined intimately. in particular, pgi 2 is believed to sensitize sensory nerves following treatment with a mild irritant, with resultant increases in cgrp release and mucosal flow. similar studies have shown that antagonists of cgrp inhibit the cytoprotective action of pge 2 . 39 another neural mediator, nitric oxide, also has been implicated in adaptive cytoprotection. interestingly, nitric oxide has a number of actions that are similar to those of prostaglandins, including maintenance of mucosal blood flow. 40 regulation of barrier function in the intestine is not as well characterized as that of the stomach, although mechanisms of barrier function, including secretion of mucus and regulation of mucosal blood flow, are presumed to be similar. the proximal duodenum also has to protect itself from acid damage as it receives gastric contents, and this involves secretion of mucus and bicarbonate in much the same way as the stomach. one other mechanism that helps the stomach and the intestine to maintain mucosal barrier function is the speed with which the mucosa repairs. thus for a defect to develop in the mucosal barrier, injurious factors have to outpace mucosal recovery. such recovery initially involves epithelial migration across denuded regions of basement membrane (restitution), 26 a process so rapid that epithelial defects may be resurfaced within minutes. for example, in bile salt-injured colon, denuded surface mucosa was covered completely by restitution. 41 in the small intestine, villi greatly amplify the surface area of the mucosal luminal surface, which in turn takes far longer to resurface with restituting epithelium once it has become denuded. 42 however, intestinal villi are able to reduce the denuded surface area considerably by extensively contracting. 43 these mechanisms are described in detail under mechanisms of gastrointestinal mucosal repair. although the stratified squamous epithelium is relatively impermeable to hcl, a number of factors can enhance the damaging effects of hcl in this epithelium. in particular, bile salts and short-chain fatty acids are capable of breaking down the squamous epithelial barrier at an acid ph, thereby exposing deep layers to hcl, with subsequent development of ulceration. 18, 44 high concentrations of short-chain fatty acids normally exist within the equine stomach because of microbial fermentation. 17 these weak acids penetrate squamous mucosa and appear to damage na + transport activity principally located in the stratum germinativum. bile salts also may be present in the proximal stomach because of reflux from the duodenum. although such reflux has a high ph, bile salts appear to adhere to stratified squamous epithelium, becoming lipid soluble and triggering damage once the ph falls below 4. 45 diet and management (e.g., periods of fasting) also play crucial roles in the development of conditions conducive to gastric ulceration. typically, a ph gradation in horses exists from proximal to distal compartments of the stomach, with the lowest ph values in the distal stomach. 46 however, fasting disrupts this stratification such that low ph values may be recorded in the proximal stomach. 47 fasting conditions also increase the concentration of duodenal contents within the proximal stomach, particularly bile. 45 proper gastric mucosa is exposed to injurious agents, including pepsin, bile, and acid. parietal cells in the horse secrete acid constantly as an adaptation to near-continuous intake of roughage, 16 but the enterochromaffin-like cells within the proper gastric mucosa and g and d cells within the pyloric mucosa tightly regulate acid secretion. histamine released by enterochromaffin-like cells amplifies acid secretion and interacts with h 2 receptors on parietal cells and g cells, which release the prosecretory hormone gastrin. a combination of histamine and gastrin can have a synergistic effect on parietal cell gastric secretion, because these mediators have distinct receptors and second messengers. however, d cells are sensitive to an acidic environment and release somatostatin, which inhibits acid secretion. 48 nonetheless, gastric mucosa may be exposed to acid for prolonged periods of time, particularly in horses that are extensively meal fed and that do not have the benefit of roughage, which tends to buffer stomach contents. 45, 48 aside from peptic ulceration induced by combinations of acid and pepsin, research in human medicine has revealed the tremendous importance of helicobacter pylori in inducing ulceration. infection with this organism has the effect of raising gastric ph because of disruption of gastric glands and also induces an inflammatory reaction that causes damage. 49 however, little evidence to date indicates that this organism is involved in gastric ulcers in horses. in the absence of a known role for infectious agents in gastric ulceration in animals, ulceration likely develops from injurious factors similar to those found in the proximal stomach, including gastric acid and bile. however, some factors that are important to induction of squamous epithelial ulceration may not be important in development of proper gastric mucosal ulceration. for example, feed deprivation and intensive training reproducibly induce squamous epithelial ulceration in horses but have little effect on proper gastric mucosa in horses. 50 gastric acid likely plays a key role, whereas other factors such as nonsteroidal antiinflammatory drugs (nsaids) serve to reduce gastric defense mechanisms. in particular, inhibition of prostaglandin production reduces mucus and bicarbonate secretion while also reducing gastric mucosal blood flow. 51 some of the nsaids also have a topical irritant effect, although this appears to be of minor significance because the route of administration (oral or parenteral) seems to have little influence on development of ulceration. 52 the source of prostaglandins responsible for gastric protection originally was assumed to be cyclooxygenase 1 (cox-1), because this isoform is expressed constitutively in gastric mucosa, whereas cox-2 is not expressed in the stomach unless induced by inflammatory mediators. however, mice in which the cox-1 gene has been knocked out fail to develop spontaneous gastric lesions, 53 possibly because of compensatory increases in prostaglandin production by cox-2. 54 this concept agrees with recent data indicating that inhibition of both cox isoforms is required to induce gastric ulceration. 55 from a clinical perspective this data indicate that drugs selective for cox-1 or cox-2 may be less ulcerogenic in the horse. because cox-2 elaborates prostaglandins induced by inflammatory stimuli, selective inhibitors of cox-2 may be particularly useful because of their ability to serve as antiinflammatory agents that are less ulcerogenic. 56 the most notable cause of intestinal mucosal injury in horses, particularly those suffering from colic, is ischemia. initially, that a reduction in gastrointestinal blood supply leads to mucosal injury seems intuitive. however, the anatomy of the gastrointestinal tract and the differing structure of the intestinal mucosa at various anatomic locations have a significant influence on the extent of mucosal injury. furthermore, ischemic injury may be induced by several different mechanisms, including occlusion of arterial supply by a thrombus, strangulation of intestinal vasculature, and generalized reduction in blood flow associated with various shock states. in addition, a number of seemingly distinct mechanisms of intestinal injury, such as intestinal distention, also trigger mucosal injury via an ischemic mechanism. finally, reperfusion injury also may influence the extent of mucosal injury following an ischemic episode and has been proposed as a potential site of therapeutic intervention. 57, 58 thus understanding the mechanisms of ischemia-reperfusion injury is critical to developing an understanding of the severity of various clinical conditions and beginning to formulate a therapeutic approach to diseases characterized by this devastating form of injury. the intestinal circulation is capable of closely regulating blood flow during periods of low systemic perfusion pressure. 59, 60 in particular, local regulation of resistance vessels within the microvasculature is particularly prominent, whereby metabolic end products of adenosine triphosphate (atp) result in continued dilation of resistance vessels despite reductions in systemic arterial pressure. dilation results in continued perfusion of gastrointestinal tissues during the early stages of shock, while other organs such as skeletal muscle undergo massive shunting of blood resulting from increased constriction of resistance vessels. the reasons for these differences in regulation are not entirely clear but may relate to the high level of energy required to fuel the intestinal mucosa and the serious systemic effects of breaches in the mucosal barrier. however, as blood flow falls below a critical level, regulatory systems are no longer effective and oxygen uptake by the gastrointestinal tissue decreases, culminating in tissue damage. 59 the tip of the villus is the most susceptible region affected by hypoxia in the equine small intestine, largely because of the countercurrent exchange mechanism of blood flow in the small intestinal villus. 59 this countercurrent exchange mechanism is attributable to the vascular architecture, which consists of a central arteriole that courses up the core of the villus, arborizes at the tip, and is drained by venules coursing down the periphery of the villus. 61 as oxygenated blood flows into the central arteriole, oxygen tends to diffuse across to the adjacent venules, which flow in the opposite direction. this series of events takes place along the length of the villus, resulting in a tip of the villus that is hypoxic even under normal conditions. furthermore, reduced blood flow as occurs in shock exacerbates the countercurrent exchange of oxygen, and the tip becomes absolutely hypoxic. 59 this mechanism might explain why the small intestinal mucosa is more susceptible to ischemic injury, compared with the colon, which has no villi. for example, the duration required to produce severe morphologic damage to the equine colon is approximately 25% longer than in the small intestine. 62 intestinal mucosal epithelium is susceptible to hypoxia because of the high level of energy required to fuel the na + /k + -atpase that directly or indirectly regulates ion and nutrient flux. the first biochemical event to occur during hypoxia is a loss of oxidative phosphorylation. the resulting diminished atp concentration causes failure of the energy-dependent na + /k + -atpase resulting in accumulation of sodium, and subsequently intracellular water. the ph of the cytosol drops as lactic acid and inorganic phosphates accumulate from anaerobic glycolysis. the falling ph damages cell membranes, including lysosomal membranes, resulting in the release and activation of lysosomal enzymes into the cytosol, further damaging cellular membranes. damage to the cell membrane allows the accumulation of high concentrations of calcium in the cytosol, which activates calciumdependent degradative enzymes. 63 these events result in cytoplasmic blebbing of the basal membrane with subsequent detachment of cells from the underlying basement membrane. recent studies on epithelial injury during ischemia suggest that most epithelial cells undergo programmed cell death (apoptosis) during ischemia and reperfusion rather than necrosis, allowing retention of reusable components of irreversibly injured cells. 64 in one study, 80% of detached epithelium during small intestinal ischemia and reperfusion underwent apoptosis. 65 although the most obvious result of apoptosis is loss of surface epithelium, a number of cells on the lower portion of the villus (in the small intestine) and cells within the crypts also may undergo apoptosis that only may become evident up to 24 hours following reperfusion of ischemic tissue. 66 morphologic changes observed in ischemic-injured small intestinal mucosa follow a similar sequence regardless of whether ischemia alone or ischemia and reperfusion induce injury (table 13 .5-1). 67 initially, epithelium separates from the underlying basement membrane, forming a fluid-filled space termed grüenhagen's space ( figure 13 .5-1). the mechanism of fluid accumulation in this space is not understood entirely but may result from continued epithelial absorption of nacl and water before it has detached fully from neighboring epithelial cells. this fluid accumulation likely exacerbates epithelial separation from the basement membrane. subsequently, epithelium progressively sloughs from the tip of the villus toward the crypts, which are the last component of the intestinal mucosa to become injured. [68] [69] [70] injury of crypts likely relates to the vascular architecture, because crypts receive a blood supply separate from the vasculature involved in the villous countercurrent exchange mechanism. the early morphologic changes observed in the equine large colon during ischemia are different from those described in the equine small intestine because of the lack of intestinal villi. however, as might be expected, the more superficially located surface cells are sloughed before those in crypts. 62, 71 the orderly progression of tissue injury has been used by one group of investigators to predict accurately the survival of horses with large colon volvulus. the researchers took biopsies from the pelvic flexure, which has been shown previously to reflect mucosal changes along the length of the colon accurately, 72 and examined them histologically for the width of the crypts and intercrypt interstitial space. they expressed the latter measurements as a ratio of interstitium to crypt width (i:c) and defined nonviable colon as that which has greater than 60% loss of crypt and an i:c ratio greater than 3. using this methodology, researchers correctly predicted survival in 94% of horses. 73 because of the dramatic decline in strongylus vulgarisinduced colic, which was associated frequently with infarction of intestinal arterial blood supply, 74 most ischemic lesions are associated with strangulating obstruction. therefore considering mechanisms of ischemic injury in horses with naturally occurring strangulating lesions is important. the majority of experimental work has assessed complete ischemia (complete occlusion of the arterial blood supply) 62 or low-flow ischemia (during which arterial blood flow is reduced). 75, 76 however, during intestinal strangulation, a disparity between the degree of occlusion of the veins and arteries occurs whereby veins are occluded before arteries because of differences in compliance of vascular walls. thus strangulating lesions are typically hemorrhagic (hemorrhagic strangulating obstruction) as the arteries continue to supply blood to tissues that have little or no venous drainage. the result is ischemic injury, as previously outlined, but also a tremendous congestion of the tissues. such hemorrhagic congestion has two opposing effects: it disrupts tissue architecture, including the mucosa and its epithelium, and continues to provide oxygenated blood to the tissues during much of the ischemic episode. in contrast, when strangulation results in sudden cessation of arterial blood flow (ischemic strangulating obstruction), tissues appear pale, and the mucosa rapidly degenerates because of a complete lack of oxygenated blood. 70 because intestine that may look nonviable (dark red) may in fact have less mucosal injury than that of ischemic strangulated intestine. 77 an additional consideration in clinical strangulating obstruction is the degree of ischemia that intestinal distention may induce. for example, experimental distention (18 cm of h 2 o for 2 hours) and decompression (2 hours) of jejunum resulted in a significant increase in microvascular permeability and a significant decrease in tissue oxygenation similar to that which would be expected with low-flow ischemia. 78, 79 in particular, microscopic evaluation of vasculature revealed capillary endothelial cell damage and local edema formation. 80 this data suggest that distended intestine proximal to an obstruction may undergo mucosal injury despite its normal appearance. indeed, in one study, intraluminal pressures greater than 15 cm h 2 o in naturally occurring cases of colic correlated with a poor prognosis for survival. 81 although that reperfusion of ischemic tissues results in exacerbation of mucosal injury recently has been taken for granted, one should remember that mechanisms underlying intestinal reperfusion injury have been defined largely in laboratory animals under specific conditions. [82] [83] [84] [85] [86] however, studies on reperfusion injury in horses have had some conflicting results. 68, 76, 87 the conflict may be attributable to the way in which the studies have been performed. in particular, the type of ischemia used in most laboratory animal studies has been low-flow ischemia (in which the blood flow typically is reduced to 20% of baseline flow), whereas studies in horses have used a number of different ischemic models, including various types of strangulating obstruction. although strangulating obstruction is of great clinical relevance, this type of ischemic insult is less likely to develop reperfusion injury. 68, 88, 89 conversely, low-flow ischemia appears to prime tissues for subsequent injury once the tissue is reperfused, and considerable evidence supports the presence of reperfusion injury in horses following low-flow ischemia. 75, 76, 80, 90 nonetheless, lowflow ischemia may not be a common clinical entity. in addition to the type of ischemia, other factors are involved in priming tissues for reperfusion injury, including species and anatomic-specific variation in oxidant enzyme and neutrophil levels ( table 13 .5-2). for example, the foal appears to have low levels of small intestinal xanthine oxidase, an enzyme that has been shown to play a critical role in triggering reperfusion injury in laboratory animals, 84, 85, 91 whereas adult levels are much greater, particularly in the proximal small intestine. 92 in addition, horses appear to have low numbers of resident neutrophils in the intestinal mucosa, 93 and this population of neutrophils (rather than those recruited from the circulation) appears to be most critical for induction of reperfusion injury. 86 however, studies demonstrating reperfusion injury in the equine colon following low-flow ischemia have shown significant accumulation of neutrophils within the mucosa. 75 therefore a complete understanding of mechanisms of neutrophilic infiltration and the mechanisms whereby they damage tissue requires further study. reperfusion injury is initiated during ischemia when the enzyme xanthine dehydrogenase is converted to xanthine oxidase and when its substrate, hypoxanthine, accumulates simultaneously because of atp use ( figure 13 .5-2). 57, 94 however, little xanthine oxidase activity occurs during ischemia, because oxygen is required as an electron acceptor. during reperfusion, xanthine oxidase rapidly degrades hypoxanthine in the presence of oxygen, producing the superoxide radical as a by-product. 57 the superoxide radical contributes to oxidative tissue damage and, most importantly, activates neutrophil chemoattractants. 84, 85 thus inhibition of xanthine oxidase in feline studies of intestinal ischemiareperfusion injury prevents infiltration of neutrophils and subsequent mucosal injury. 83, 84 however, inhibition of xanthine oxidase has had no effect on ischemiareperfusion injury in equine small intestine 87 and colon, 95 suggesting that reperfusion injury is simply a continuation of injury initiated during ischemia, as suggested in some equine studies, 63 or that the classic reperfusion injury pathway is activated by alternate sources of reactive oxygen metabolites. the latter has been suggested by studies in feline models of ischemia-reperfusion injury in which the source of a significant proportion of reactive oxygen metabolites is unknown and is independent of xanthine oxidase and neutrophils. 83 a veterinary review of the pathogenesis of intestinal reperfusion injury in the horse suggested the concept of a therapeutic window wherein treatment of reperfusion injury would be beneficial. 57 the basis of this concept is that certain conditions exist under which ischemic injury is minimal and that tissues are damaged severely during reperfusion. 88 thus under conditions of low-flow ischemia, little injury is demonstrated during 3 hours of ischemia, but remarkable injury occurs during 1 hour of reperfusion. [83] [84] [85] however, a therapeutic window may not exist under conditions of strangulating obstruction in which severe injury occurs during ischemia and minimal injury occurs during reperfusion. 96 this in turn greatly reduces clinicians' ability to ameliorate ischemiareperfusion injury with treatments such as antioxidants at the time of reperfusion. mechanisms of gastric repair depend greatly on the extent of injury. for instance, superficial erosions can be covered rapidly by migration of epithelium adjacent to the wound; a process termed epithelial restitution. however, ulceration (full-thickness disruption of mucosa and penetration of the muscularis mucosa) requires repair of submucosal vasculature and extracellular matrix. the formation of granulation tissue initiates repair and supplies connective tissue elements and microvasculature necessary for mucosal reconstruction. connective tissue elements include proliferating fibroblasts that accompany newly produced capillaries that form from proliferating endothelium. recent studies indicate that nitric oxide is critical to both processes, 40, 97 which likely explains the reparative properties of nitric oxide in the stomach. 98 once an adequate granulation bed has formed, newly proliferated epithelium at the edge of the wound begins to migrate across the wound. in addition, gastric glands at the base of the ulcer begin to bud and migrate across the granulation bed in a tubular fashion. 99 repairing epithelium expresses epidermal growth factor, which appears to facilitate these processes. 100 in addition, a mucoid cap facilitates these events and retains reparative factors and serum adjacent to the wound bed. 51 once the ulcer crater has been filled with granulation tissue and the wound has been reepithelialized, the subepithelial tissue remodels by altering the type and amount of collagen. despite the remodeling process, ulcers tend to recur at sites of previous ulceration, and the concern is that this remodeling can result in excessive deposition of collagen and fibrosis. 26 reparative mechanisms are similar in the intestine, except that in the small intestine, mucosal villi contribute to mucosal repair. once intestinal epithelium is disrupted, two events occur almost immediately to reduce the size of the denuded portion of the villus: contraction of the villus and epithelial restitution ( figure 13 .5-3). for example, in porcine ileum subjected to 2 hours of ischemia, villi were 60% of their former height and 50% of the denuded villous surface area was covered in flattened epithelium within 6 hours. 42 enteric nerves appear to regulate villous contraction, because inhibition of enteric nerve conduction prevents villous shortening following injury. the contractile component of the villus is a network of myofibroblasts distributed throughout the lamina propria of the villus and along the central lacteal. inhibition of villous contraction results in retarded epithelial repair because of the larger denuded surface that remains to be covered by migrating epithelium compared with similarly injured villi that have contracted. 43 pge 2 also has been implicated in regulating villous contraction, because application of pge 2 resulted in villous contraction when perfused through normal rat ileum. 101 as villi contract, assuming the basement membrane is intact, epithelium from the margins of the wound migrates centripetally to resurface toward the tip of the villus. 43 the process of restitution is similar in denuded colonic mucosa, except that it may proceed more rapidly because of the lack of villi. 41 epithelial restitution is solely a migratory event that does not depend on provision of new enterocytes by proliferation. cellular migration is initiated by extension of cellular lamellipodia that receive signals from the basement membrane via integrins. intracellular signaling converges on the actin cytoskeleton, which is responsible for movement of lamellipodia. specific components of the basement membrane appear to be critical to the migratory process. for example, application of antibodies to collagen types iii and iv, which are important components of intestinal mucosal basement membrane, impeded epithelial restitution. 102, 103 other elements of the basement membrane, including proteoglycans, hyaluronic acid and noncollagenous proteins such as fibronectin and laminin also may provide important signals. 104 these subepithelial matrix components that facilitate restitution may form the basis for clinical treatments designed to speed up the repair process, analogous to administration of matrix components to horses with articular cartilage damage. although epithelial restitution results in gross closure of previously denuded regions of gastrointestinal mucosa, closure of interepithelial spaces ultimately is required to restore normal epithelial barrier resistance. because the tight junction is principally responsible for regulating the permeability of the interepithelial space, repair and closure of this structure likely is critical to restore intestinal barrier function. recent research indicates that prostaglandins play a vital role in recovery of tight junction resistance, 105 indicating that administration of nonselective cox inhibitors to horses with colic, particularly those recovering from strangulating obstruction, may be deleterious. therefore judicious use of nsaids is appropriate until more selective drugs that allow continued production of reparative prostaglandins are available for use in horse. 56 after restoration of the epithelial barrier, the epithelium must reestablish normal mucosal architecture to allow normal gut absorptive and digestive function. in porcine ileum subjected to 2 hours of ischemia, the epithelial barrier was restored within 18 hours, but villi were contracted and covered in epithelium with a squamous appearance. restoration of normal villous architecture required another 4 days. 42 newly proliferated crypt epithelium replaces the flattened villous epithelium that characterizes restitution. under normal circumstances the dividing stem cells, of which the base of each mucosal crypt has approximately four, form new enterocytes. newly divided enterocytes migrate from the crypt onto the villus. 106 during migration, enterocytes differentiate and acquire specific absorptive and digestive functions. fully differentiated enterocytes reside on the upper third of the villus for 2 to 3 days and then are sloughed into the intestinal lumen. 107 this process accelerates during mucosal repair and requires increased proliferative rates. a variety of locally available gut-derived factors, including luminal nutrients, polyamines, and growth factors, may stimulate increased proliferation within 12 to 18 hours. 42 the return of the normal leaflike shape of the villus occurs following the appearance of normal columnar epithelium. although prostaglandins have been implicated in mucosal cytoprotective function, few studies have assessed their importance in mucosal repair. one study implicated prostaglandins in growth factor-stimulated restitution, 108 but a more prominent role of prostaglandins in mucosal repair is their ability to close interepithelial tight junctions. 105, 109, 110 for instance, ischemic-injured small intestine rapidly recovers barrier function (as measured in vitro as transepithelial resistance) in the presence of pgi 2 and pge 2 , despite the fact that these prostanoids had little effect on villous contraction and epithelial restitution. however, electron microscopic examination of tissues reveals dilation of tight junctions in tissues treated with nsaids, 110 whereas those additionally treated with prostaglandins have closely apposed tight junctions (figures 13.5-3 and 13.5-4). prostaglandins stimulate closure of tight junctions via the second messengers cyclic adenosine monophosphate and ca 2+ , 105 which interestingly were among the first mediators found to modulate tight junction permeability. 111, 112 such tight junction closure is of importance to patients with intestinal injury that are treated with nsaids, because reduced prostaglandin levels may result in increased intestinal permeability. for example, in a study on ischemic-injured porcine ileum, treatment with the nsaid indomethacin resulted in a significant increase in intestinal permeability to inulin and lipopolysaccharide compared with tissues that were treated additionally with pgi 2 and pge 2 . 105 section 13.5 pathophysiology of mucosal injury and repair a b figure 13 .5-4 ultrastructural appearance of repairing ischemic-injured mucosa. a, restituting epithelium 2 hours following a 1-hour ischemic episode in the presence of the nonselective cyclooxygenase inhibitor indomethacin. dilation of the interepithelial space and the apical tight junction (arrows) correlates with a leaky intestinal barrier, b, similar restituting epithelium had been treated additionally with prostaglandins e 2 and i 2 . the close apposition of the tight junction (arrows) and the interepithelial space correlate with normalization of intestinal barrier function. 1-cm bar = 6 µm. the process of restitution absolutely depends on a group of compounds called polyamines. 113, 114 the rate-limiting enzyme in the formation of the polyamines spermine, spermidine, and putrescine is ornithine decarboxylase. in rats with stress-induced duodenal ulcers, systemic administration of the ornithine decarboxylase inhibitor α-difluoromethylornithine significantly reduced polyamine levels and greatly reduced epithelial restitution. furthermore, intragastric treatment of these same rats with putrescine, spermidine, and spermine prevented the delayed mucosal repair induced by α-difluoromethylornithine. 113 interestingly, gastric tissue levels of ornithine decarboxylase increased in rats with stress-induced gastric ulcers, suggesting that tissue injury enhances polyamine production, which may contribute to the normal rapid rate of epithelial restitution. 115 the mechanisms whereby polyamines stimulate epithelial restitution are not clear. mccormack, wang, viar, et al. hypothesized that polyamines increase transglutaminase activity, an enzyme that catalyzes the cross-linking of cytoskeletal and basement membrane proteins. 116 further investigation of the role of polyamines in iec-6 cell migration showed that depletion of polyamines resulted in disruption of the cytoskeleton and reduced the physical extension of lamellipodia. 117 more recent studies have clarified this pathway. in particular, polyamines have been shown to regulate cytoskeletal cellular migration via activation of the small guanosine triphosphatase-rho-a by elevating intracellular ca 2+ levels. these elevations in ca 2+ result from polyamine regulation of expression of voltagegated k + channels and altered membrane electric potential. 118 polyamines also play a role in the normal physiologic regulation of crypt cell proliferation and differentiation. 119,120 polyamines are produced by fully differentiated enterocytes at the tip of the villus and may reach the crypt within sloughed luminal epithelium or via local villous circulation. 121 following intestinal injury, polyamines appear to stimulate enhanced proliferation by increasing the expression of protooncogenes, which control the cell cycle. 122 the mechanism whereby polyamines influence gene expression likely relates to the cationic nature of these compounds, which may influence the tertiary structure of negatively charged dna and rna. 113 locally produced growth factors-including epidermal growth factor (egf), transforming growth factor α (tgf-α), tgf-β, and hepatocyte growth factor-have the ability to modulate mucosal recovery. the most important of these growth factors in early mucosal repair events is tgf-β, which is a potent stimulus of epithelial restitution and modulator of the extracellular matrix. 26 neutralization of tgf-β retards epithelial migration in vitro, and tgf-β apparently may serve as a point of convergence for mediators of restitution, because neutralizing tgf-β also inhibits the effects of other peptides. however, tgf-β paradoxically inhibits epithelial proliferation, thereby reducing the supply of new enterocytes for mucosal repair. conversely, egf, produced by the salivary glands and duodenal brunner's glands, and the related tgf-α, produced by small intestinal enterocytes, are potent stimulants of enterocyte proliferation. these growth factors share approximately 30% of their amino acid structure, bind to the same receptor on the basolateral surface of enterocytes, and are not related to tgf-β. 123 the physiologic role of egf is difficult to discern because it is present in the intestinal lumen, with no apparent access to its basally located receptor. however, egf has been proposed to act as a "surveillance agent" that gains access to its receptor during epithelial injury (when the egf receptor likely would be exposed) to stimulate proliferation. 124 tgf-α presumably has a similar role, but it is present in greater concentrations in the small intestine because it is produced by differentiated villous enterocytes. the mature peptide is cleaved from the extracellular component of the transmembrane tgf-α precursor and released into the lumen. 123 another group of proreparative peptides produced within the gastrointestinal tract are the trefoil peptides. under physiologic conditions, trefoil peptides are secreted by mucus-producing cells at distinct anatomic sites. for example, gastric epithelium produces the trefoil peptide ps2, whereas the small and large intestine mucosa produce intestinal trefoil peptide. 125 however, any of the trefoil peptides may be upregulated within repairing epithelium regardless of anatomic site. 26, 126 in addition, trefoil peptides have the ability to induce their own expression, amplifying the level of these reparative factors at sites of mucosal repair. 127 trefoil peptides are the most potent stimulants of epithelial migration in vitro, and their effects are independent of growth factors, including tgf-β. 128 however, recent evidence suggests that egf receptor activation is required for induction of ps2 and another of the trefoil peptides, termed spasmolytic peptide, in gastric epithelium in vitro. the importance of trefoil peptides to the mucosal repair response in vivo is illustrated by gene knockout studies, in which mice deficient in intestinal trefoil factor have greatly reduced ability to repair intestinal injury. 129 in fact, detergent-induced mucosal injury was lethal because of a lack of restitution compared with wild-type mice that fully recovered from similar mucosal injury. the fact that administration of intestinal trefoil factor restored restitution has important therapeutic implications. the mechanism whereby trefoil peptides stimulate epithelial migration is yet to be characterized fully but appears to involve translocation of the adherens junction protein e-cadherin, thereby allowing cells to become untethered from neighboring cells. 26 the principal metabolic fuel of enterocytes is glutamine and of colonocytes, butyrate. however, recent studies suggest that glutamine and butyrate have more specific proliferative actions aside from their role as nutrients. for example, in the piglet ipec-j2 enterocyte cell line, glutamine enhanced gene transcription by increasing mitogen-activated protein kinase activity. 130, 131 similarly, butyrate stimulated mucosal growth following colonic infusion in the rat. 132 because of such growth-promoting actions, glutamine was shown to prevent intestinal mucosal atrophy and dysfunction that accompanies starvation 133, 134 and long-term total parental nutrition. 135, 136 additionally, glutamine improves function of transplanted small intestine 137, 138 and protects intestinal mucosa from injury if administered before chemotherapy 139 and radiation therapy. 140, 141 intestinal nutrients also may synergize with other proliferative agents. for example, administration of glutamine and tgf-α to porcine ileum that had been subjected to 2 hours of ischemia resulted in a synergistic increase in mitogen-activated protein kinase activity, enterocyte proliferation, and villous surface area. 42 although concern has arisen that such early return to normal surface area may result in dysfunctional mucosal digestive and absorptive function because of resurfacing denuded mucosa with immature epithelium, nutrients and growth factors also appear to promote early differentiation. in the case of glutamine and tgf-α restoration of postischemic small intestine, rapid recovery of digestive enzymes also was documented. 142 effective gastrointestinal motility involves a complex interaction between the enteric nervous system, muscular wall, and luminal contents. additional factors that influence the net transit of digesta include gravity, the volume and viscosity of the contents, and pressure gradients created by simultaneous contraction and relaxation of adjacent segments of bowel. casual use of the term intestinal motility in veterinary medicine often underestimates the complexity of the processes involved in the transit of intestinal contents. this is particularly true when the term is used to describe the frequency and or intensity of intestinal sounds, or borborygmi. the existence of borborygmi does not always equate with progressive movement of intestinal contents. disruption to normal motility occurs commonly in horses for a variety of reasons. examples of diseases in which altered motility may be present include gastroduodenal ulceration, intraluminal obstruction or impaction, excessive wall distention, strangulating obstructions, peritonitis, and inflammatory bowel diseases such as duodenitis proximal jejunits or colitis. ineffective intestinal motility is also a feature of several neonatal diseases, including prematurity, systemic sepsis, and perinatal asphyxia. certain parasitic infections, electrolyte derangements, and endotoxemia can modify digesta transit in horses of all ages. general anesthesia and specific sedatives, such as xylazine, romifidine, or detomidine, also disturb motility. the inhibition of propulsive bowel activity usually is referred to as ileus. ileus is ascribed most frequently to the condition that occurs after laparotomy and is termed simple or uncomplicated postoperative ileus (poi). the term complicated or paralytic ileus describes intestinal motility disturbed for longer periods after surgery. poi in horses is associated most commonly with surgery of the small intestine, particularly after resection and anastomosis, 1,2 is a common complication of small intestinal surgery, and can have a negative effect on short-term postoperative survival. 3, 4 motility dysfunction likely is present in all horses after laparotomy, but many are affected subclinically and require minimal or no specific intervention. in symptomatic animals, clinical signs are apparent shortly after recovery and include colic, tachycardia, dehydration, decreased borborygmi and fecal output, and sequestration of fluid within the stomach. rectal examination and ultrasound reveal small intestinal distention with rare or absent wall movement. the severity and duration of intestinal stasis varies, lasting from minutes to days. a specific motility disorder involving the cecum or ileocecocolic region occurs sporadically in horses. [5] [6] [7] the condition most commonly occurs after general anesthesia and extraabdominal surgery, particularly orthopedic and upper airway procedures, and therefore often is categorized as a form of poi. anecdotally, horses at greatest risk are young male performance animals. other cases occur spontaneously, often in animals with painful primary conditions such as uveitis or septic tenosynovitis. the syndrome is frustrating in that clinical signs are often subtle unless cecal perforation has occurred. in horses with a cecal emptying defect after anesthesia, overt signs are usually apparent 3 to 5 days after the procedure. the earliest detectable signs include depression and a reduction in feed intake and fecal output. ineffective emptying results in overfilling of the cecum with moist contents, which is manifest by signs of mild to moderate colic. if the condition is recognized late or untreated, the cecum may rupture and result in fatal peritonitis. the inherent rhythmicity of electric activity in the intestine is controlled by the interstitial cells of cajal, specialized cells that are electrically coupled to myocytes via gap junctions. 8 these cells are responsible for generating and propagating slow-wave activity and may be critically involved in a range of motility disorders. the enteric nervous system primarily controls and coordinates intestinal contraction. a combination of central and autonomic innervation influences events, but contraction does not require external neural input. the parasympathetic supply to the gastrointestinal tract is via the vagus and pelvic nerves, and the sympathetic supply is through postganglionic fibers of the cranial and caudal mesenteric plexuses. a complex network of interneurons within each plexus integrates and amplifies neural input; the intensity and frequency of resultant smooth muscle contractions are proportional to the amount of sympathetic and parasympathetic input. additional binding sites for a number of other endogenous chemicals, including dopamine, motilin, and serotonin exist within the enteric nervous system and on smooth muscle cells. 9 acetylcholine is the dominant excitatory neurotransmitter in the gastrointestinal tract and exerts its action through muscurinic type 2 receptors on smooth muscle cells. sympathetic fibers innervating the gastrointestinal tract are adrenergic, postganglionic fibers with cell bodies located in the prevertebral ganglia. activation of α 2adrenergic receptors on cholinergic neurons within enteric ganglia inhibits the release of acetylcholine and therefore reduces intestinal contraction. β 1 -, β 2 -, and β-atypical receptors are directly inhibitory to the intestinal smooth muscle. 10 inhibitory nonadrenergic, noncholinergic neurotransmitters include adenosine triphosphate, vasoactive intestinal peptide, and nitric oxide. 11, 12 these neurotransmitters are critical for mediating descending inhibition during peristalsis and receptive relaxation. substance p is a nonadrenergic, noncholinergic neurotransmitter that may be involved in contraction of the large colon. 13, 14 the rate and force of intestinal contractions along the small intestine and large colon of the horse are important determinants of intestinal motility; of even greater importance to the net propulsion of digesta are the cyclical patterns of contractile activity. these patterns are known as the small intestinal and colonic migrating motility (or myoelectric) complexes (mmcs). 15, 16 the colonic complex usually originates in the right ventral colon and variably traverses the ascending and descending colons. many of these complexes are related temporally to a specialized motility event of the ileum, the migrating action potential complex. local inflammation within the intestinal muscularis and inhibitory neural events are important initiators of intestinal ileus. 17, 18 intestinal inflammation not only is important in primary intestinal diseases in horses, such as duodenitis-proximal jejunitis and colitis but also is induced after simple intestinal handling during laparotomy. experimental data from other species suggests that handling of the small or large intestine at the time of surgery activates resident macrophages with resultant increased expression of p-selectin and intercellular adhesion molecule 1 on endothelial cells within the vasculature of the muscularis. the upregulation of associated ligands on leukocytes leads to sequential "sticking and rolling," followed by neutrophil migration into the interstitium. the subsequent release of neutrophil products interferes with cell signaling and results in reduced intensity of smooth muscle contraction. furthermore, the inflamed intestine fails to contract normally in response to putative prokinetic agents. another key factor in the development of intestinal stasis after inflammation is the local overproduction of nitric oxide caused by the upregulation of inducible nitric oxide synthase (inos) by resident macrophages. nitric oxide is a key inhibitory neurotransmitter of the nonadrenergic, noncholinergic system. 12 nitric oxide synthase inhibition has been a pharmacologic target in the treatment of experimental ileus. the inhibitory effects of α 2 -agonists such as xylazine and detomidine on cecal and large colon motility are well described. [19] [20] [21] [22] [23] [24] intravenously administered xylazine inhibits cecal and large colon motility for 20 to 30 minutes without seriously disrupting small intestinal myoelectric activity, and detomidine can reduce large intestinal myoelectric activity for up to 3 hours. the α 2 -antagonist yohimbine has a weak but positive effect on cecal emptying in normal ponies, suggesting that normal motility is under constant α 2 -adrenergic tone. 24 atropine is a postganglionic blocking agent that binds to muscarinic receptors. when administered at 0.04 mg/kg, atropine inhibits individual small intestinal, cecal, and colonic contractions for about 120 minutes but supresses small intestinal and colonic migrating complexes for up to 8 hours. 25 neural reflexes also may mediate inhibition of motility associated with peritoneal inflammation. 26, 27 the afferent segment is composed partly of capsaicin-sensitive visceral afferent c fibers that terminate in the dorsal horn of the spinal cord, where they can activate inhibitory sympathetic fibers or synapse directly on the sympathetic ganglia. consequently, the efferent limb of the reflex expresses increased sympathetic outflow, primarily mediated through stimulation of α 2 -adrenoreceptors, and inhibition of acetylcholine release, which provides the rationale for α 2 -blockade in treating ileus. intraluminal infusion of capsaicin before abdominal surgery ameliorated the severity of poi in experimental rats. this finding highlights the importance of visceral afferent fibers in the development of poi. 28 ileus also can occur in association with intestinal obstruction or displacement. mild to moderate distention of the bowel, such as that occurring in the early stages of an intraluminal obstruction, evokes an increase in local contractile activity. 29, 30 excessive distention results in inhibition of motility within the distended segment of bowel. intestinal stasis is not always detrimental and under certain conditions may be protective. endotoxemia is a clinical feature of many diseases of the equine gastrointestinal tract, and endotoxins independently can exert a negative effect on intestinal motility and transit. 31 a variety of mediators likely are involved, but activation of α 2 -adrenoreceptors and production of prostanoids appear to be important, for pretreatment with yohimbine or nonsteroidal antiinflammatory drugs (nsaids; phenylbutazone or flunixin), respectively, ameliorates the inhibitory effects of experimental endotoxin infusion. 32, 33 endotoxin infusion induced an inflammatory response in the intestine of rats that mimicked the response induced by handling during laparotomy. 34 the similarity of the responses were highlighted in a recent study that demonstrated that prior exposure of the muscularis to endotoxin protected the intestine from the effects of manipulation. 35 the pathophysiology of cecal emptying defect is not known. this syndrome may best mimic poi in human beings and generally is considered a large intestinal disorder. an important difference in horses is that laparotomy is a rare predisposing factor, and most cases occur in horses undergoing routine extraabdominal surgical procedures. general anesthesia itself is a potent inhibitor of gastrointestinal motility in horses, but these effects are short-lived and reversible within hours of anesthetic withdrawal. 15 the return of normal motility in horses after experimental ileus was most delayed in the cecum, suggesting that this may be a common site of ileus in horses. 36 a link between routine postoperative medications, such as phenylbutazone and aminoglycoside antibiotics, has been suspected but not established. an inhibitory effect of nsaids on large colon contractility has been demonstrated using in vitro techniques. 37 primary sympathetic overstimulation could be involved, for many of the affected animals are young, male horses or animals with painful diseases. the duration of surgery influences the development of small intestinal poi, but not cecal emptying dysfunction. 7,38 technique may have a weak influence on small intestinal poi after jejunojejunostomy. the duration of intestinal ileus was shorter in animals that received a sideto-side stapled anastomosis than those that had a hand sewn end-to-end procedure. 3 the duration of ileus after stapled end-to-end anastomosis was not different from that after either procedure. reported risk factors for the development of poi in horses include age (>10 years), small intestinal resection and anastomosis, breed (arabians had a greatest risk than other breeds), and duration of surgery. 38 interestingly, performing a pelvic flexure enterotomy and emptying the colon had a protective effect against poi. the diagnosis of ileus is based on history and physical examination findings. important tests include determination of pulse rate and rhythm, auscultation and percussion of the abdomen, rectal palpation, and passage of a nasogastric tube. a complete blood count with fibrinogen estimation and cytologic analysis of peritoneal fluid may improve the accuracy of diagnosis. affected animals may be colicky because of accumulation of fluid in the upper gastrointestinal tract (classical poi) or cecal contents (cecal emptying defect). decompression of the stomach is important diagnostically and therapeutically in horses with poi after small intestinal surgery. failure to relieve pain with gastric decompression could point toward mechanical obstruction, severe inflammation of the intestine, or peritonitis. most animals with ileus are depressed and have reduced fecal output and intestinal borborygmi. one should interpret intestinal sounds with caution, however, because the presence of borborygmi does not always equate to progressive intestinal motility and merely may reflect local, nonpropagated contractions. rectal palpation findings in cases of persistent poi or duodenitis-proximal jejunitis are usually nonspecific but may reveal dilated, fluid-filled loops of small intestine. the clinician occasionally can palpate roughened peritoneal surfaces if peritonitis is present. one can palpate cecal distention with digesta in horses with advanced cecal dysfunction. distinguishing functional ileus from mechanical obstruction is important and can be difficult, but horses with mechanical obstruction typically have sustained high volumes of gastric reflux that vary little over time. the management of intestinal ileus depends on the segment of gastrointestinal tract involved. therapy for ileus of the proximal gastrointestinal tract involves a combination of gastric decompression, fluid and electrolyte therapy, and antiinflammatory drug therapy. electrolyte therapy is critical, particularly for maintaining adequate extracellular concentrations of potassium, calcium, and magnesium. calculation of the volume of fluid to be administered should include maintenance requirements (40 to 60 ml/kg/day) plus an estimate of losses, especially those lost through gastric decompression. one should consider parenteral provision of calories when feed has been withheld for more than 96 hours, particularly after the horse has had surgery. a combination of amino acids and dextrose with or without lipids effectively provides these calories. hand walking also may provide some benefit to these animals but is not likely to have a direct effect on intestinal motility. one should avoid drugs that may impair normal intestinal motility, including the anticholinergics such as atropine and opiate receptor agonists such as morphine and meperidine. butorphanol appears to have little or no adverse effect on small or large intestinal motility. 39, 40 one should use α 2 -agonists sparingly because of their inhibitory effects on large intestinal motility. fluid therapy is the key component in managing cecal emptying defect, usually in combination with lubricants or laxatives, such as mineral oil or magnesium sulfate, and with careful use of antiinflammatory drugs. horses with primary cecal impaction or impaction caused by an emptying defect frequently require surgery to prevent fatal rupture. the surgical management of these cases is controversial and may include typhlotomy alone, typhlotomy with a bypass procedure such as ileocolic or jejunocolic anastomosis, or a bypass without typhlotomy. 41 most horses that undergo simple typhlotomy have an uneventful recovery, 42 although a small number experience impaction again and require a second laparotomy. experimental and anecdotal evidence provides a strong rationale for using antiinflammatory drugs to prevent and treat gastrointestinal ileus, particularly in animals that may have endotoxemia. 43 flunixin meglumine is used widely in equine practice as an analgesic and antiinflammatory agent, and it ameliorates many of the adverse systemic effects of endotoxin, particularly those on the cardiovascular system. a potential negative effect of nsaids on large intestinal contractility has been suggested. broad-spectrum antimicrobials are indicated when one suspects sepsis or for the compromised immune system, as in cases of moderate to severe endotoxemia. theoretical concerns have been raised regarding the use of aminoglycoside antibiotics in animals with ileus. high concentrations of aminoglycoside antimicrobials inhibited intestinal contractions in exposed sections of intestine in vitro, but this inhibitory effect is unlikely to occur at clinically relevant doses. 44 motility-enhancing drugs have been advocated to treat gastrointestinal ileus. unfortunately, information directly pertinent to horses is limited and must be extrapolated cautiously from that of other species because of the differences in intestinal anatomy and physiology. prokinetic drugs potentially can shorten the length of hospitalization, thereby reducing the cost of treatment and the number of potential complications such as weight loss, thrombophlebitis, and laminitis. experimental evidence indicates that prokinetic drugs can minimize the development of postoperative abdominal adhesions. 45 most prokinetic drugs require a healthy gut wall to enhance intestinal contraction. therefore one should not assume that many of these drugs would be effective in the presence of an inflammatory injury such as that which can occur after intestinal manipulation at surgery or that associated with duodenitis-proximal jejunitis. bethanechol is a parasympathomimetic agent that acts at the level of the myenteric plexus and directly on intestinal smooth cells through muscarinic receptors. bethanechol is a synthetic ester of acetylcholine and is not degraded by anticholinesterase. bethanechol has cholinergic side effects, including abdominal discomfort, sweating, and salivation, although these are minimal when the drug is administered at 0.025 mg/kg body mass subcutaneously or orally. bethanechol has efficacy in diseases that involve abnormal gastric emptying and delayed small intestinal transit and has been shown to increase gastric contractility and hasten the emptying of liquid and solid phase markers from the stomach of normal horses. [46] [47] bethanechol also increases the strength and duration of wall contractions in the cecum and right ventral colon and consequently speeds up cecal emptying. neostigmine increases receptor levels of acetylcholine by inhibiting cholinesterase. the drug (0.022 to 0.025 mg/kg intravenously) promotes cecal and colonic contractile activity and hastens the emptying of radiolabeled markers from the cecum. 24 neostigmine has been used to manage small intestinal ileus, but it significantly delayed the emptying of 6-mm beads from the stomach of normal adult horses. 48 metoclopramide acts principally as a 5-hydroxytryptamine 4-receptor (5ht-4) agonist and 5ht-3-receptor antagonist. in contrast to newer generation benzamides, metoclopramide is also an antagonist at dopamine 1 (da 1 ) and 2 (da 2 ) receptors. antagonism of prejunctional da 2 receptors facilitates acetylcholine release and smooth muscle contraction. metoclopramide crosses the blood-brain barrier, where its antagonist properties on central da 2 receptors can result in extrapyramidal signs, including seizure. these signs were responsible for poor acceptance of the drug in equine practice. most investigators have failed to demonstrate significant effects of metoclopramide in experimental animals, but constant intravenous infusion (0.04 mg/kg/hr) in a population of postoperative horses significantly decreased the volume and duration of gastric reflux over control and intermittent drug infusion groups. 49 infusion was well tolerated and appeared to be superior to intermittent infusion or no treatment at all. cisapride is a second-generation benzamide that acts as a 5ht-4 agonist and 5ht-3 receptor antagonist but is without antidopaminergic action. stimulation of 5ht-4 receptors within the enteric nervous system enhances release of acetylcholine from the myenteric plexus. several reports suggest the efficacy of cisapride in managing intestinal disease in horses, including the resolution of persistent large colon impaction, treatment of equine grass sickness, and as a preventative for poi in horses after small intestinal surgery (0.1 mg/kg body mass intramuscularly during the postoperative period). [50] [51] [52] [53] the horse erratically absorbs tablets administered rectally, but a method for preparing a parenteral form of the drug from tablets has been described. 54 cisapride has the potential to cause adverse cardiac side effects mediated through blockage of the rapid component of the delayed rectifier potassium current that include lengthening of the qt interval and development of torsades de pointes, a potentially fatal arrhythmia. these adverse effects have resulted in withdrawal of the drug in the united states. domperidone acts as a competitive antagonist at peripheral da 2 receptors. the drug is a therapeutic agent (1.1 mg/kg/day) for mares grazing endophyte-infected tall fescue, principally because of drug-enhanced prolactin release. the potential prokinetic effects of domperidone have not been studied extensively in horses, but a modest efficacy of domperidone (0.2 mg/kg intravenously) has been demonstrated in experimental ileus in two ponies. erythromycin is a direct motilin receptor agonist on smooth muscle cells and also may act within the enteric nervous system to facilitate the release of acetylcholine and motilin. erythromycin enhances gastric emptying in normal horses but has a more pronounced effect on the hindgut. 47, 55 erythromycin lactobionate (1.0 mg/kg intravenously) hastens cecal emptying in normal animals and induces colonic mmc-like activity across the colon. administration often is associated with defecation and abdominal discomfort. the drug may help prevent cecal impaction in horses after anesthesia, although its effectiveness on cecal motility in the immediate postoperative period may be reduced. 36 high doses, constant infusion, or prolonged use of erythromycin induces receptor downregulation and inhibition of activity. erythromycin can induce diarrhea in adults, therefore one should avoid dosing over many days. naloxone (0.05 mg/kg intravenously) induces contractile activity in the cecum and left colon. 56 defecation commonly follows administration of naloxone within 15 to 20 minutes. α 2 -adrenoreceptor antagonists such as yohimbine or tolazoline counteract increased sympathetic outflow in response to nociceptive stimulation. yohimbine infusion (75 µg/kg) also may attenuate the negative effects of endotoxin on motility. 32 intravenous infusion of lidocaine may suppress primary afferent neurons, thereby limiting reflex efferent inhibition of motility. an infusion dose of 15 to 20 mg/min over 5 to 6 hours has been recommended for horses. lidocaine infusion is associated with reversible side effects that include muscle fasciculations, ataxia, and seizure. consequently, the rate of infusion requires close monitoring. katharina l. lohmann, michelle henry barton endotoxemia is defined as the presence of endotoxin in the bloodstream. most often, however, the term is used to refer to the associated clinical manifestations caused by an overshooting inflammatory reaction. in its pathophysiologic consequences the innate immune response to endotoxin (lipopolysaccharide) is similar to the response to other stimuli; for example, overwhelming bacterial infection, viral infection, or severe trauma. the term systemic inflammatory response syndrome therefore was introduced to describe a general systemic inflammatory process independent of cause. sepsis is defined as the "systemic inflammatory response to infection," and septic shock as "sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction." 1 according to these definitions the diagnosis of sepsis requires documentation of infection by culture in addition to two or more of the following findings: hypo-or hyperthermia, tachycardia, tachypnea or hypocapnia, and leukocytosis, leukopenia, or an increased proportion of immature leukocyte forms. organ failure is a common sequela of endotoxic or septic shock, and the term multiple organ dysfunction syndrome describes insufficiency of two or more organ systems, as evident by clinical or clinicopathologic changes. in horses one should include the laminae of the feet in the list of organs susceptible to failure. german scientist richard pfeiffer (1858-1945), in working with vibrio cholerae, first described endotoxin as a toxin "closely attached to, and probably integral of, the bacterial body." 2 he observed this toxin to be distinct from the actively secreted, heat-labile, and proteinaceous bacterial exotoxins. endotoxin later was found to be a heat-stable lipopolysaccharide structure, and the terms endotoxin and lipopolysaccharide now are used interchangeably. lipopolysaccharide is a major structural cell wall component of all gram-negative bacteria, including noninfectious species (figure 13 .7-1). with 3 to 4 × 10 6 molecules per cell, lipopolysaccharide makes up about 75% of the outer layer of the outer cell membrane and is a key functional molecule for the bacterial outer membrane, serving as a permeability barrier against external noxious agents. the lipopolysaccharide molecule consists of four domains, which are essential for the virulence of gram-negative bacteria. 3 three of the domains (inner core, outer core and o-specific chain) represent the hydrophilic polysaccharide portion of the molecule, whereas the lipid a portion represents the hydrophobic lipid portion ( figure 13 .7-2). combined, these domains confer the overall amphiphilic properties of the molecule that lead to the formation of micellar aggregates in aqueous solutions. o-specific chains (also called o-antigen polysaccharides or o-chains) are characteristic of any given type of lipopolysaccharide and show enormous structural variability between bacterial serotypes. 4 o-chains are synthesized by addition of preformed oligosaccharide blocks to a growing polymer chain and therefore have a repetitive structure. o-specific chains determine part of the immunospecificity of bacterial cells 5 and, on interaction with the host immune system, serve as antigens for the production of species-specific antibodies. 6 o-specific chains are further responsible for the smooth appearance of gram-negative bacterial colonies on culture plates, 3 and lipopolysaccharide molecules containing an o-chain are termed smooth lipopolysaccharide. the inner (lipid a-proximal) and outer (o-chainproximal) core oligosaccharide portion is more conserved between different strains of gram-negative bacteria than the o-specific chain. 4 the core of all lipopolysaccharide molecules contains the unusual sugar kdo (3-deoxy-dmanno-oct-2-ulopyranosonic acid), which links the core region to the lipid a molecule. synthesis of a minimal core is essential for the survival of bacteria, 7 and the smallest naturally occurring lipopolysaccharide structure consists of lipid a and kdo. 8 in contrast to the s-form colonies, colonies of gram-negative bacteria with lipopolysaccharide molecules that lack the o-specific chain but contain a core region show a rough appearance on culture plates. rough lipopolysaccharide molecules are denoted further as ra, rb, etc. to indicate the length of the core region. in re-lipopolysaccharide (also called deep rough lipopolysaccharide), the core region is reduced to a kdo residue. remutants often are used to raise antibodies against the core region in an attempt to provide cross-protection against a variety of bacterial species. the lipid a portion serves to anchor the lipopolysaccharide molecule in the bacterial outer membrane and has been identified as the toxic principle of lipopolysaccharide, 9 and its structure is highly conserved among gramnegative bacteria. the common structure shared by lipid a molecules is a 1,4'-bisphosphorylated β1,6-linked d-glucosamine disaccharide backbone (lipid a backbone), which is acylated by up to six fatty acids. 4 figure 13 .7-3 shows the acylation pattern for escherichia coli lipopolysaccharide. variation in the lipid a structure between gramnegative bacteria affects the number, length, and position of fatty acids and the backbone structure and the substitution of phosphate by other polar groups. 6 according to its nature as a structural cell wall component, the presence of endotoxin implies the presence of gram-negative bacteria as a source. depending on the nature of the underlying disease, these bacteria may circulate in the bloodstream in their intact form (i.e., bacteremia), may be confined to a localized infectious process, or may be part of the endogenous bacterial flora colonizing the gastrointestinal tract. in any of these scenarios, endotoxin molecules are released as a by-product of bacterial growth and in large numbers on bacterial cell death. 10 common infectious conditions associated with endotoxemia in horses include neonatal gram-negative sepsis, bacterial pneumonia and pleuropneumonia, endometritis, peritonitis, and infectious colitis with bacteria such as salmonella spp., that are not part of the normal intestinal flora. in one study, for example, endotoxin was detectable in plasma of 50% of foals evaluated for presumed sepsis. 11 the term translocation describes entry of endogenous bacteria and bacterial products from the gastrointestinal tract into tissues and the systemic circulation. 12 the natural intestinal flora of horses consists mainly of gramnegative, anaerobic bacteria, and thus large amounts of endotoxin normally exist in the lumen of the equine intestinal tract. 13 even in health, small amounts of endotoxin cross the intact mucosal barrier and reach the portal circulation and the liver. these molecules are cleared, however, by the mononuclear phagocytic system in the liver and only lead to a localized and restricted activation of the host immune system. for endotoxin translocation to become detrimental, excessive amounts have to cross the intestinal barrier and overwhelm the mononuclear phagocytic system or the capacity of the liver to detoxify lipopolysaccharide must be compromised. the latter may be a concern in conditions such as hepatitis, cholangiohepatitis, or portosystemic shunting of blood. permeability of the intestinal mucosal barrier frequently increases in cases of acute gastrointestinal disease. colic patients are prime candidates to development endotoxemia, and plasma endotoxin was detectable in 10% to 40% of colic patients on admission. 14,15 a higher percentage of horses tested positive for endotoxin when only patients presented for surgical intervention were evaluated. 15 aside from gastrointestinal rupture, increased permeability to intact bacteria or free endotoxin molecules is thought to be associated most commonly with ischemic insults such as strangulating obstruction and bowel infarction, severe inflammation as in proximal enteritis and colitis, bacterial overgrowth, and intraluminal acidosis, which occurs with grain overload. 16, 17 one study, however, found no difference in plasma endotoxin detection between disease groups, therefore emphasizing the fact that any disease of the abdominal cavity can induce endotoxemia in horses. in the same study, endotoxin was approximately 3 times more likely to be detected in peritoneal fluid as opposed to plasma samples. similarly, higher cytokine concentrations have been measured in peritoneal fluid than in plasma. the likely explanation for these findings is a local inflammatory response in the peritoneal cavity elicited by translocated bacteria and/or lipopolysaccharide molecules before their absorption into the systemic circulation. 14 although certainly the most important factor in horses, conditions other than gastrointestinal disease may result in translocation of endotoxin and bacteria. in experimental studies using laboratory animals, entry of gutassociated bacteria into the lymphatic system was demonstrated after hypovolemic shock, burn injuries, trauma, malnutrition, and starvation. [18] [19] [20] furthermore, endotoxin itself caused bacterial translocation into mesenteric lymph nodes after intraperitoneal administration to mice. 21 these findings have received much attention in the literature concerning human patients because they serve to explain cases of endotoxic shock in the absence of demonstrable bacterial infection. one should keep in mind the possibility of translocation when evaluating cases of presumed systemic inflammatory response syndrome in horses, in which one cannot demonstrate bacterial infection or gastrointestinal disease. endotoxin translocation also may be associated with strenuous exercise, which results in reduced splanchnic blood flow, hypoxemia, and a higher body temperature. in fit racehorses a significantly increased mean plasma lipopolysaccharide concentration was found after racing, whereas antilipopolysaccharide immunoglobulin g levels were decreased. fit horses showed significantly higher antilipopolysaccharide immunoglobulin g concentrations at rest than sedentary controls, suggesting leakage of small amounts of endotoxin from the intestinal lumen during training and racing. 22 the clinical significance of these findings requires further investigation. the initiating event in the pathophysiology of endotoxemia is the activation of lipopolysaccharideresponsive cells by endotoxin, resulting in altered cellular functions and increased expression of inflammatory mediators. immune cells such as macrophages, which are the first to encounter endotoxin, respond to minute amounts of lipopolysaccharide, which usually allows them to eliminate gram-negative bacteria and free lipopolysaccharide molecules efficiently. an important factor in the exquisite sensitivity to lipopolysaccharide is the presence of lipopolysaccharide-binding protein (lbp). 4 lbp is an approximately 60-kd plasma glycoprotein 23 synthesized by hepatocytes 24 and belongs to the group of acute phase proteins. under the control of inflammatory agents and cytokines, lbp concentration in plasma increases approximately 100-fold within 24 hours of an inflammatory stimulus. 25 the main function of lbp is to transfer lipopolysaccharide to endotoxin-responsive cells, which include mononuclear phagocytes, neutrophils, lymphocytes, and endothelial cells. the importance of a highly sensitive response to lipopolysaccharide for protection against gram-negative bacterial infection is demonstrated in experiments using lbp "knock-out" mice (mice that lack the lbp gene and are therefore unable to synthesize lbp). although these animals are resistant to the effects of isolated lipopolysaccharide, they are unable to control bacterial infection and rapidly succumb. 26 despite its crucial importance for an effective host defense, lbp is not essential for lipopolysaccharide-receptor interaction per se, because high concentrations of lipopolysaccharide can activate cells in the absence of lbp. 27 aside from its role as a catalyst of cellular activation by lipopolysaccharide, lbp has opsonizing activity 28 and participates in the phagocytosis of lipopolysaccharide by macrophages and neutrophils. 29, 30 although phagocytosis of lipopolysaccharide is receptor dependent, it appears to be uncoupled from intracellular signaling events and occurs in the absence of cell activation. 31 lbp further catalyzes transfer of lipopolysaccharide to lipoproteins such as high-density lipoprotein, which neutralizes lipopolysaccharide activity. 32 this detoxifying effect may become important when large amounts of lipopolysaccharide are present. a protective effect of lbp against lipopolysaccharide challenge and infection has been demonstrated in a murine model. 33 the most important lipopolysaccharide receptors known to date are cluster differentiation antigen 14 (cd14) 27 and toll-like receptor 4 (tlr4). 34 both are classified as pattern recognition receptors, 35 which means that they recognize lipopolysaccharide as a pattern common to all gram-negative bacteria. cd14 is a 53-kd protein that in its membrane-bound form (mcd14) is inserted into the cell membrane via a glycosylphosphatidyl-inositol anchor. 36 cd14 is expressed primarily on monocytes and tissue macrophages and to a lesser extent on neutrophils. 37 cd14 also is found in a free, or soluble, form (scd14) 38 that can bind to cell types lacking cd14, such as endothelial cells, and make them lipopolysaccharide-responsive. in addition to this proinflammatory effect, high concentrations of scd14 can sequester and neutralize lipopolysaccharide. 39 the amount of circulating scd14 greatly increases during inflammation, which makes it a useful marker of acute and chronic inflammation. 37 although cd14 is known to be crucial for cellular activation, it cannot transmit signals to the inside of the cell because it lacks a transmembrane domain. the missing link between cd14 and the cytosolic environment is a toll-like receptor in association with a molecule named md-2. 40 the name toll-like receptor stems from the homology of the mammalian receptor with a receptor type in drosophila (toll) that is important for dorsoventral orientation and immune responses in the fly. a number of toll-like receptors have been identified in mammalian species so far, but tlr4 appears to be the receptor subtype most important for lipopolysaccharide signaling. 34 the importance of cd14 and tlr4 in the cellular response to lipopolysaccharide has been demonstrated in a number of experiments. mice deficient in cd14 are incapable of mounting a normal inflammatory response to lipopolysaccharide, 39 whereas mutation or deletion of the gene encoding for tlr4 causes lipopolysaccharide hyporesponsiveness. [41] [42] [43] after binding of lipopolysaccharide to cellular receptors, a multitude of signaling events takes place within the cell and results in the alterations of cellular metabolism known as cell activation. signaling pathways are characterized by sequential phosphorylation and thereby activation of enzymatic activities. a typical end result of intracellular signaling is the activation of transcription factors; for example, proteins that bind to dna and promote gene transcription. translational mechanisms are activated in a similar manner. among the bestcharacterized pathways in endotoxin-induced cell signaling are the mitogen-activated protein kinase (mapk) pathways and the activation of transcription factor nuclear factor κb (nfκb) (figure 13.7-4) . 44, 45 in the nfκb pathway the intracellular domain of tlr4 associates with the adapter protein myeloid differentiation factor 88 and recruits interleukin-1 receptor-associated kinase to the complex. activation of interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor, nfκb-inducing kinase, and iκb-kinase follow, and lastly, iκb is phosphorylated. iκb is an inhibitor protein complex that sequesters and inactivates nfκb in the cytoplasm. on phosphorylation, iκb is ubiquinated and degraded, and nfκb is translocated to the nucleus where it unfolds its activity. 46 nfκb is a dimeric protein complex with several isoforms of which the p65/p50 heterodimer is the most important inducible complex in mammals. 47 proteins of importance for the pathogenesis of septic shock, the genes of which contain promoter elements for nfκb, include cytokines, inducible nitric oxide synthase, and cyclooxygenase 2 (cox-2). 44 three groups of mapks known to be crucially important for lipopolysaccharide-induced signal transduction are extracellular signal-regulated kinase, c-jun-terminal kinase, and p38. final effects of signaling through mapk pathways include the activation of several transcription factors, translation initiation factors, and cytosolic enzymes such as phospholipase a 2 , as well as an increase in the expression of adhesion molecules on the cell surface. 44 despite the characterization of seemingly separate pathways, one should recognize that interaction and synergy between pathways is likely to occur. for example, simultaneous activation of p38, c-jun-terminal kinase, and extracellular signal-regulated kinase results in much higher levels of tumor necrosis factor (tnf) reporter expression than activation of a single pathway. 44, 48 aside from the mechanisms described here, pathways involving atypical protein kinase c 49,50 and receptor-independent integration of lipopolysaccharide into the cell membrane and ceramide-like second messenger activity of lipopolysaccharide 37 have been proposed. additional pathways are likely to be uncovered in the ongoing investigation of intracellular signaling mechanisms and their in vivo significance. although endotoxin can exert some direct effects, cytokines are a primary mediator of lipopolysaccharide effects. cytokines are glycoprotein molecules that regulate inflammatory and immune responses by acting as a signal between cells. 51 cytokines of major interest in the pathogenesis of endotoxemia include tnf, the interleukins, chemokines, and growth factors such as granulocyte-monocyte colony-stimulating factor. tnf is thought of as the most "proximal" cytokine released in response to lipopolysaccharide. studies corroborate this by showing that administration of recombinant tnf mimics the effects of lipopolysaccharide, 52 and that antibodies directed against tnf protect against the lethal effects of endotoxin. 53 increased plasma activity of tnf is associated with increased mortality in equine patients with acute gastrointestinal disease and in septic neonates. 14 despite being a structurally diverse group of proteins, cytokines share several characteristics that allow them to execute their complex functions in the inflammatory response. 51 any individual cytokine generally is produced by several different cell types, can act on different cell types, and has multiple effects on any given cell. furthermore, cytokine effects are redundant, meaning that different cytokines can share the same effect. in endotoxemia, this is particularly true for the effects of interleukin-1 (il-1) and tnf. 54 many of the biologic activities of cytokines in vivo result from synergistic or antagonistic actions involving two or more cytokines. 55 within itself the cytokine response is highly regulated: cytokines induce or suppress synthesis of other cytokines including their own (feedback regulation), regulate expression of cytokine receptors, and regulate cytokine activities. additional regulatory mechanisms include the release of specific cytokine inhibitors such as soluble il-1 and tnf-α receptors, cytokine receptor antagonists such as il-1 receptor antagonist, and antiinflammatory cytokines including il-10, il-4, il-13, and transforming growth factor β. glucocorticoids also are produced increasingly in response to endotoxin and inhibit the production of cytokines. 56 during a controlled inflammatory response, therefore, cytokine secretion is a selflimited event, whereas excessive stimulation of cytokine release can lead to the perpetuation of the inflammatory response even after the initial stimulus has been removed. conversely, the compensatory antiinflammatory reaction can become severe enough to cause anergy of the immune system and increased susceptibility to infection, which has been termed the compensatory antiinflammatory response syndrome. overall, excessive and unbalanced stimulation of the immune system therefore may result in predominantly proinflammatory (systemic inflammatory response syndrome), antiinflammatory (compensatory antiinflammatory response syndrome), or combined (mixed antagonist response syndrome) responses. 57 interestingly, tolerance to endotoxin develops after repeated exposure to lipopolysaccharide. 58 tolerance can be demonstrated in vitro and in vivo and encompasses decreased production of cytokines and a diminished clinical response. 58, 59 mechanisms that likely are responsible for the development of endotoxin tolerance include receptor downregulation and inhibition of intracellular signaling pathways. 60, 61 cytokines such as tnf are important mediators in the development of endotoxin tolerance. 62 the development of endotoxin tolerance in horses has been reported. 63, 64 aside from cytokines, a number of other molecules function as inflammatory mediators in the pathogenesis of endotoxemia, the synthesis and release of which are stimulated by endotoxin and by cytokines. these mediators include the arachidonic acid metabolites or prostanoids, platelet-activating factor (paf), oxygenderived free radicals, nitric oxide (no), histamine, kinins, and complement components. table 13 .7-1 summarizes the origins, targets, and effects of the most important inflammatory mediators involved in the pathogenesis of endotoxemia. figure 13 .7-5 shows the pathways of arachidonic acid metabolism by cox and lipoxygenase. cox products are the prostaglandins (pgs), prostacyclin (pgi 2 ) and thromboxanes, and the lipoxygenase produces the leukotrienes. the innate immune response to lipopolysaccharide is an efficient defense mechanism that provides maintenance of homeostasis and therefore health in the face of an almost continuous exposure to microorganisms and their products. 65 detrimental consequences of this immune response only occur if excessive and uncontrolled mediator output results in endothelial damage, neutrophil-mediated tissue damage, and uncontrolled activation of the coagulation and fibrinolytic cascades and the complement system. ultimately, the combination of these events culminates in cardiovascular instability, impaired hemostasis, organ failure, shock, and death. the following discussion addresses the various pathophysiologic events in the development of endotoxemia and shock and the role of inflammatory mediators. normal endothelium plays an important role in regulating blood pressure and regional tissue perfusion and provides an anticoagulant surface. endothelial dysfunction and damage result in a decreased responsiveness to vasoactive agents (vasoplegia), increased vascular permeability, and a tendency for clot formation in the microvasculature. if the basement membrane and underlying matrix are compromised, further microvascular hemorrhage can occur. endothelial cell damage is primarily neutrophilmediated. more specifically, damage is caused by oxygen-derived free radicals, which are produced within endothelial cells via reactions involving neutrophil-derived elastase and hydrogen peroxide molecules, endothelial cell enzymes such as xanthine oxidase, and endothelial cytosolic iron. the hypochloric anion radical (ho˙) is thought to be responsible most directly for endothelial cell cytotoxicity. no˙produced by constitutively expressed nitric oxide synthase in endothelial cells may afford protection from oxygen radical-induced endothelial cell damage. no is able to scavenge superoxide radicals and react with them to form peroxynitrite. variations in the ability to produce no may explain why vascular beds in different organs vary in their susceptibility to neutrophilmediated damage. 66 excessive production of no by an inducible form of nitric oxide synthase (i nos), however, contributes to tissue damage, and increased peroxynitrite concentrations may be responsible in part for paf-induced increases in vascular permeability. 67 in addition to oxygen-derived free radicals, activated neutrophils release matrix metalloproteinases, which contribute to tissue damage. 56 vascular endothelial cells are further susceptible to direct effects of various cytokines, most prominently tnf-α and il-1. these cytokines are thought to cause damage via the induction of cox activity and production of prostanoids and through generation of free radicals. neutrophil activation by lipopolysaccharide and cytokines results in stimulation of phagocytosis and the respiratory burst, release of lysosomal enzymes and inflammatory mediators, and expression of adhesion molecules. perhaps the single most specific clinicopathologic indicator of endotoxemia is pronounced neutropenia, 68 which temporally correlates with peak plasma concentrations of tnf. 69 neutropenia is caused primarily by margination of neutrophils in the vasculature, whereas significant loss through active migration into peripheral tissues likely is limited to the presence of a localized source of infection. margination is made possible by adhesion molecules on endothelial cells and leukocytes that interact and allow sticking of leukocytes to the endothelial lining of blood vessels. endotoxin or cytokines such as tnf and il-1 can initiate expression of adhesion molecules. 70 subsequent transmigration of cells into tissue spaces is aided by the production of leukocyte collagenase, which allows enzymatic destruction of the vascular basement membrane. margination and transmigration of neutrophils occurs in three phases. 70, 71 in the first phase of tethering and rolling, endothelial cells are stimulated to express p-selectin and e-selectin, which bind to p-selectin glycoprotein ligand-1 and sialylated lewis-x-like structures on leukocytes, respectively. whereas p-selectin is stored preformed in weibel-palade bodies of endothelial cells, e-selectin is expressed only following stimulation by cytokines. additionally, constitutively expressed l-selectin on neutrophils can bind to endothelial glycoproteins and glycolipids. during the second phase, firm adhesion is mediated by binding of neutrophil integrins (lfa-1 and mac-1, also known as cd11a/cd18 and cd11b/cd18) to intercellular adhesion molecule 1 (an immunoglobulin structure) on endothelial cells. although integrins are expressed constitutively on the leukocyte surface, activation signals are necessary to induce a high-affinity state and interaction with the endothelial surface. transmigration finally requires the expression of yet another adhesion molecule, namely platelet/endothelial cell adhesion molecule 1, which is located at the intercellular junction of endothelial cells. 71 chemotactic factors including activated complement factor c5a and the cxc chemokines control transmigration. 66 the latter group includes il-8, which is expressed by endothelial cells in response to activation. rebound neutrophilia, which is observed frequently following episodes of endotoxemia, is caused by neutrophil release from the bone marrow reserve pool and by stimulation of myeloid cell proliferation via granulocyte-macrophage colony-stimulating factor and is mediated primarily by tnf and il-1. 54 in health, coagulation and fibrinolysis underlie stringent control mechanisms that allow appropriate clot formation and their resolution. coagulopathies frequently are observed in horses with colic 16, 72, 73 and foals with sepsis 11 and are likely attributable to endotoxemia. disseminated intravascular coagulation (dic) results from a widespread activation of the coagulation and fibrinolytic systems and failure of their control mechanisms. ultimately, this leads to disseminated fibrin deposition in the microvasculature, consumption of platelets and clotting factors, and accumulation of fibrin degradation products (fdps). depending on the underlying disease process and the impairment of the systems, dic can manifest as a diffuse thrombotic syndrome leading to ischemic organ failure, a fibrinolytic syndrome with uncontrolled hemorrhage, or a combination of both. 74 a procoagulant state in which one can detect clinicopathologic abnormalities precedes dic. activation of the coagulation cascade culminates in the cleavage of fibrinogen to fibrin by the serine protease thrombin. thrombin deposition on endothelial cell surfaces leads to platelet adherence and their activation by surface-bound paf. 56 the intrinsic and extrinsic arms of the coagulation cascade are activated in endotoxemia. the intrinsic pathway is initiated by activation of coagulation factor xii (hageman factor), prekallikrein, and highmolecular-weight kininogen, which compose the contact system. 75 although direct activation of coagulation factor xii by endotoxin has been demonstrated, 76 the extrinsic pathway likely is more important for the development of coagulopathy in endotoxemia and sepsis. 75 activation of the extrinsic pathway depends on the interaction of coagulation factor vii with tissue factor, which is the only coagulation factor not constitutively present in blood. tissue factor is present in subendothelial tissues and is exposed on vascular injury but also is expressed on endothelial cells and mononuclear phagocytes in response to lipopolysaccharide. 77, 78 increased expression of monocyte tissue factor (also described as increased procoagulant activity) was found to be associated significantly with coagulopathy and poor prognosis in horses with colic. 79 furthermore, lipopolysaccharide-induced tissue factor expression by equine peritoneal macrophages may be associated with the development of intraabdominal adhesions. 63 regulatory mechanisms of the coagulation cascade include tissue factor pathway inhibitor, antithrombin iii (at iii), and the protein c system. 75 protein c acts as an anticoagulant by inactivating clotting factors v and viii and promotes fibrinolysis by inactivating plasminogen activator inhibitor (pai). 80 protein c activation by thrombin-thrombomodulin complexes is important for the anticoagulative properties of normal endothelium, 75 and downregulation of endothelial thrombomodulin expression by tnf and il-1 and decreased expression of at iii and tissue factor pathway inhibitor by damaged endothelial cells contribute to the procoagulant state in endotoxemia and sepsis. [81] [82] [83] in addition, activation of vascular endothelial cells leads to a loss of prostacyclin and no production and an increased release of thromboxane a 2 (txa 2 ). as a result, platelets are stimulated to aggregate and release txa 2 and paf, thereby further promoting clot formation. 17 the crucial step in the fibrinolytic cascade is the conversion of plasminogen to plasmin, a fibrin-degrading enzyme. 75 tissue-type (tpa) and urokinase-type (upa) plasminogen activator are the major initiators of fibrinolysis, whereas pai and α 2 -antiplasmin are the main regulatory components. 84, 85 tnf and il-1 have been shown to induce the release of upa and tpa and the synthesis of pai. 75 activation of fibrinolysis leads to consumption of α 2 -antiplasmin and accumulation of fdps, which if present in high concentrations can interfere with platelet aggregation, fibrin polymerization, and thrombin formation and can promote bleeding. additionally, fdps mediate an increase in vascular permeability. lipopolysaccharide infusion in rabbits 86 and human beings 87 resulted in an early increase in plasma tpa activity, followed by a later profound rise in pai activity and fall in tpa activity. increased plasma pai concentrations also were found in horses with colic compared with controls. 88, 89 thus although fibrinolysis may compensate initially for accelerated coagulation, its subsequent inhibition contributes to clot formation. activation of the complement system in endotoxemia occurs via the alternative pathway through interaction with lipopolysaccharide. increased concentrations of plasmin and kallikrein (caused by activation of the fibrinolytic and contact system) further promote this pathway by directly activating complement factors c3a and c5a. aside from being key molecules in the complement cascade, c3a and c5a are anaphylatoxins and cause an increase in vascular permeability via mast cell degranulation. c5a further activates the lipoxygenase pathway in neutrophils and monocytes, acts as a chemotaxin for leukocytes and monocytes, and promotes neutrophil adhesion to endothelial cells. in response to acute inflammation, synthesis and secretion of a number of proteins called the acute phase proteins increases in hepatocytes, whereas synthesis of albumin decreases. the primary function of this acute phase response may be to suppress and contain inflammatory responses. 56 il-6 and il-1 are the most important cytokines that induce the acute phase response, 90 which typically begins within a few hours of the insult and subsides within 24 to 48 hours, 91 unless the initiating cause persists. in horses, fibrinogen (the most commonly evaluated), haptoglobin, transferrin, ferritin, ceruloplasmin, coagulation factor viii:c, serum amyloid a protein, c-reactive protein, α 1 -acid glycoprotein, and phospholipase a 2 are considered part of the acute phase response. 92 one must consider the effect of acute inflammation on the serum concentration of several coagulation factors when evaluating coagulation profiles. serum fibrinogen concentration is determined primarily by the acute phase response, although fibrinogen is consumed increasingly on activation of the clotting cascade. shock is characterized by a loss of homeostasis attributable to breakdown of hemodynamic control mechanisms, decreases in cardiac output and the effective circulating volume, and inadequate perfusion of vital organs. shock caused by endotoxemia is classified as distributive shock 93 and is largely initiated by vascular dysfunction in the periphery. peripheral vascular beds are of major importance for the regulation of local tissue perfusion and affect systemic blood pressure by regulating total peripheral resistance. normally, vascular smooth muscle tone is regulated by endothelin-1 (vasoconstriction), no, and prostacyclin (vasodilation) released from vascular endothelial cells. 94 as mentioned before, detrimental effects of no are attributable to induction of i nos in macrophages and other cell types, rather than endothelialderived no. peripheral vasomotor effects of endotoxin manifest as vasodilation and vasoplegia and are mediated by pgi 2 , no, and mediators such as bradykinin. widespread vasodilation leads to vascular blood pooling, decentralization of blood flow, decreased venous return, and in effect decreased effective circulating volume and cardiac output. 93 compensatory responses in the form of an initial hyperdynamic phase include tachycardia, increased cardiac output and central venous pressure, pulmonary hypertension, peripheral vasoconstriction, and increased peripheral vascular resistance. 93, 95, 96 the early vasoconstrictive phase corresponds to an increased serum concentration of txa 2 , 17 but additional vasoconstrictors such as arginine vasopressin, angiotensin ii, serotonin, endothelin, and norepinephrine likely are implicated in the pathogenesis of shock and organ failure. 56 with progression of disease, the animal enters a stage of decompensated shock and progressive systemic hypotension, which correspond to increased plasma concentrations of prostacyclin, pge 2 and bradykinin. 17, 56 inadequate blood flow and oxygen delivery to tissues caused by hypotension is confounded by direct myocardial suppression via no, 93 increased vascular permeability, 17 intravascular microthrombosis, and impaired tissue oxygen extraction 93 and results in progressive metabolic acidosis and inhibition of normal cellular metabolism. quantification of endotoxin in plasma samples is possible. the limulus amebocyte lysate assay is an activity assay based on the endotoxin-sensitive hemolymph coagulation cascade in the horseshoe crab limulus polyphemus. in limulus this reaction is thought to be a defense mechanism against gram-negative infection. 97 although frequently used as a research tool, the assay is not convenient enough to become a routine clinical test. the clinician therefore must appreciate the primary disease processes associated with a high risk of endotoxemia and rely on clinical signs and clinicopathologic data to achieve a diagnosis. in some cases, endotoxemia may be the first indication of disease or may be the most overt of otherwise subtle clinical manifestations. with colitis or proximal enteritis, for example, one may detect signs of endotoxemia before the development of colic, diarrhea, or gastric reflux, which more specifically indicate the nature of the primary illness. diseases such as peritonitis or pleuritis, however, may show nonspecific clinical findings including fever, anorexia, and depression. findings such as neutropenia, which indicate endotoxemia, should urge the clinician to search for a septic process. in vivo experiments in horses clearly show that many of the clinical signs associated with acute gastrointestinal disease and sepsis are attributable to the activities of lipopolysaccharide and cytokines such as tnf. on administration of sublethal doses of lipopolysaccharide the clinical response can be divided into the early hyperdynamic and the later hypodynamic or shock phases. clinical signs during the first phase, which begins within 15 to 45 minutes after lipopolysaccharide administration, include anorexia, yawning, sweating, depression, evidence of abdominal discomfort, muscle fasciculation, and recumbency. heart and respiratory rates increase, and decreased borborygmi suggest ileus. hyperemia of the mucous membranes and an accelerated capillary refill time indicate the hyperdynamic state. 68 if one administers large amounts of lipopolysaccharide or if exposure is ongoing, depression worsens progressively, anorexia persists, and feces develop diarrheic character. signs of colic typically abate after the initial stage. fever develops as a result of direct action of tnf on the thermoregulatory center and il-1-induced local production of pge 2 in or near the hypothalamus. 98, 99 because of compromised peripheral perfusion, mucous membrane color changes to brick red or purple, a dark "toxic" line appears, and capillary refill time is prolonged. 68 inadequate peripheral perfusion and compromised organ function finally characterize the hypodynamic shock phase. body temperature may become subnormal and the skin, especially on extremities, is cool to the touch. the arterial pulse weakens and venous fill is decreased. vascular endothelial damage and increased capillary permeability result in a muddy mucous membrane color and diffuse scleral reddening. hemostatic abnormalities can manifest in the form of thrombosis, such as of the jugular vein, or increased bleeding tendency with mucosal petechiation or ecchymoses and prolonged bleeding from venipuncture sites. 74 bleeding also may occur in spontaneous epistaxis or prolonged hemorrhage after nasogastric intubation. 17 additional clinical signs typically reflect the development of organ failure. renal failure and laminitis appear to be common complications of endotoxemia in horses. renal failure results from ischemic cortical necrosis and acute tubular necrosis caused by coagulopathy-induced afferent arteriolar obstruction. clinical signs may include oliguria, anuria, or hematuria caused by renal infarction. laminitis may lead to lameness, increased digital arterial pulsation, increased warmth of the hoof wall, and sensitivity to hoof tester pressure. other signs of organ failure include icterus, anorexia and depression caused by liver failure, 74 tachypnea and dyspnea caused by pulmonary failure, colic and bleeding caused by ischemia-induced gastrointestinal ulceration and abnormal motility patterns, 17 and persistent tachycardia or cardiac arrhythmia in cases of cardiac failure. in pregnant mares, fetal death and abortion can occur because of increased production of pgf 2α and decreased serum progesterone concentrations. 100, 101 alterations in the hemogram and serum biochemical profile are nonspecific and mainly reflect the underlying disease process and the occurrence of organ failure. leukopenia caused by neutropenia may be the most specific indicator of acute bacterial sepsis or endotoxemia. 68 in prolonged cases, an increased proportion of immature neutrophil forms (bands) and toxic changes are observable. toxic changes resulting from neutrophil activation include vacuolation, cytoplasmic granulation, basophilic cytoplasm, and döhle's bodies. because neutropenia occurs early in the development of endotoxemia, it also may be a useful parameter for monitoring horses at risk. 68 on recovery, neutropenia typically is followed by a pronounced rebound neutrophilia. an elevated hematocrit and total serum protein concentration are evidence of dehydration; however, splenic contraction caused by increased sympathetic stimulation and protein losses also influence these parameters. a normal or only slightly decreased serum protein and albumin concentration in the face of an elevated hematocrit and clinical signs of dehydration should alert the clinician to the possibility of protein loss. hypoproteinemia and hypoalbuminemia can occur because of loss via the gastrointestinal or urinary tract or with pleural or peritoneal cavity effusion. increased vascular permeability and edema formation contribute to hypoproteinemia. serum electrolyte abnormalities primarily depend on the nature and duration of underlying disease processes and need to be evaluated individually. common sources of electrolyte loss are gastrointestinal secretions, urine, and sweat; however, severe effusion into body cavities may contribute. in anorexic patients, lack of dietary intake is a confounding factor that warrants consideration. in human patients, gram-negative sepsis frequently is associated with hypocalcemia, more specifically a decrease in serum ionized calcium concentration. endotoxin is thought to be a causative factor, and proposed mechanisms include acquired parathyroid gland insufficiency, dietary vitamin d deficiency, impaired calcium mobilization, and renal 1α-hydroxylase insufficiency leading to decreased 1,25-hydroxylation of vitamin d. hypocalcemia in septic human patients was found to be associated with hypotension and poor outcome. 102 in horses with surgically managed gastrointestinal disease, decreased serum ionized calcium concentration was a common finding and was most severe in patients with strangulating or nonstrangulating infarctions. in some horses, ionized calcium concentration decreased further throughout surgery. treatment with calcium gluconate resulted in normalization of serum ionized calcium concentrations in all cases. 103 septic neonatal patients are frequently hypoglycemic. aside from decreased oral intake and generally increased metabolism, glucose use by the infecting bacteria, inhibition of gluconeogenesis by endotoxin, and insulinlike activity produced by macrophages are responsible for hypoglycemia. 17 interestingly, experimental endotoxin administration results in transient hyperglycemia in adult horses, 95 whereas profound hypoglycemia occurs in foals. 104 one should evaluate coagulation parameters if coagulopathy is suspected. the most significant changes can be expected with severe inflammatory disease such as colitis, 72,73 devitalized intestine as with strangulating obstruction, 73, 105 and with increased duration of disease. in 30 horses with acute gastrointestinal disease, coagulation profiles were considered normal in only 2 horses. 72 although coagulation times may be shortened during the procoagulant state, commonly observed abnormalities with developing dic include an increased concentration of fdps and soluble fibrin monomer, prolonged prothrombin time indicative of factor vii consumption, prolonged activated partial thromboplastin time indicative of factor viii:c and ix consumption, prolonged thrombin time, decreased at iii activity, thrombocytopenia, and decreased protein c and plasminogen activities. fibrinogen concentration frequently increases and reflects the acute phase response rather than coagulation abnormalities. 79 some clinicians make a diagnosis of dic if three or more coagulation parameters (specifically at iii, fdps, platelet count, prothrombin time, and activated partial thromboplastin time) are abnormal, 105 whereas others require overt clinical signs of hemorrhage and concomitant thrombosis in addition to classic laboratory findings. 73 the prognostic value of coagulation parameters has been evaluated. 16, 73, 89 overall, persistence or worsening of abnormalities in the face of treatment appears to be more indicative of poor outcome than alterations in any specific parameter. in one study, decreased serum at iii concentration was the parameter most commonly associated with fatal outcome in mature horses with colic. 72 one should further evaluate the serum biochemical profile regarding compromise or failure of specific organ systems. increases in serum creatinine and urea nitrogen concentration can have prerenal, renal, or postrenal causes. in cases of endotoxemia and sepsis, prerenal azotemia caused by dehydration and decreased renal blood flow and renal azotemia caused by organ failure are most likely to occur. one can use urine specific gravity and the response to fluid therapy to differentiate renal from prerenal causes of azotemia. although ideally one should perform urinalysis before initiating fluid therapy, one should never delay treatment to obtain a sample and instead should use the first available urine sample. with prerenal azotemia, urine specific gravity is increased, urinalysis is normal in other respects, and azotemia resolves with adequate fluid therapy. azotemia in the face of normal or decreased urine specific gravity, however, indicates compromised renal function. depending on the extent of renal damage, proteinuria and hematuria also may be present. bacteriuria and an elevated urine leukocyte count may occur if urinary tract infection is the underlying cause for the development of endotoxemia. in these cases, urine culture and sensitivity testing are indicated to aid appropriate antimicrobial therapy. increased serum activity of liver enzymes (aspartate aminotransferase, γ-glutamyltransferase, sorbitol dehydrogenase, alkaline phosphatase) are common in endotoxemic patients; however, liver failure caused by endotoxemia is rare. sorbitol dehydrogenase is the most liver-specific of the enzymes and a sensitive indicator of acute hepatocellular necrosis; however, sorbitol is unstable and routine assays may not be available. one should evaluate liver enzymes and function tests (serum indirect and direct bilirubin concentration, serum bile acids and blood ammonia) in cases of prolonged and profound depression to rule out hepatoencephalopathy. one should evaluate arterial blood gases in patients with primary respiratory disease or with clinical evidence of respiratory failure and in profoundly depressed, recumbent patients, especially neonates. hypoxemia observed in response to endotoxin infusion is thought to be caused by an increase in ventilation-perfusion mismatch rather than pulmonary edema as occurs in human patients with acute respiratory distress syndrome. the lung is not a major shock organ in horses; however, pulmonary edema may occur in patients with associated sepsis or complications such as dic. 106 the ideal treatment for endotoxemia is prevention. if one possibly can recognize and closely monitor patients at risk, one can provide treatment proactively and may reverse the effects of endotoxin before the inflammatory response has developed a dynamic of its own. unfortunately, endotoxemia can develop rapidly, and horses are exquisitely sensitive to the effects of endotoxin; therefore, many equine patients are not evaluated until reaching more severe stages of endotoxemia or shock. prognosis and patient outcome then frequently depend on the severity of complications associated with endotoxemia. 17 treatment of endotoxemia involves multiple aspects, and the following strategies have been proposed 107 : • inhibition of endotoxin release into the circulation • scavenging of lipopolysaccharide molecules to prevent direct effects and interaction with inflammatory cells • inhibition of cellular activation by lipopolysaccharide • inhibition of mediator synthesis • interference with the effects of inflammatory mediators • general supportive care in addition, complications such as renal failure, one also must address liver failure, cardiac failure, laminitis, and abortion in pregnant mares. when evaluating reports concerning the efficacy of any one treatment, one should keep in mind differences in underlying disease processes and the complexity of the inflammatory cascade. a "one for all" treatment most likely will not be found, and similarly, any one treatment can only address one or few pathophysiologic aspects of endotoxemia. understanding the rationale for different treatment strategies is important to be able to tailor treatment to the needs of the patient. inhibition of endotoxin release requires identification and removal of its source. therefore whenever endotoxemia is evident, the clinician should strive to reach a diagnosis of the underlying disease and ascertain whether ischemic or inflamed bowel or a gram-negative septic process is present. aside from history, physical examination, and routine laboratory tests, evaluation may include exploratory laparotomy in colic patients, roentgenologic and ultrasonographic evaluation of the pleural and peritoneal cavity and organs, ultrasonographic evaluation of umbilical remnants in neonatal foals, evaluation of passive transfer and calculation of a sepsis score in foals, and repeated culturing of blood or other specimens. if one suspects an infectious process, one should pursue identification of the responsible microorganisms and their antimicrobial sensitivity spectrum; however, one should not delay treatment to obtain culture results. specimen containers with removal devices for antimicrobials are available and are useful in cases for which one has initiated treatment before specimen collection. once one reaches a diagnosis, one must take appropriate measures to correct the primary disease process. examples are removal of devitalized sections of bowel or infected umbilical remnants, drainage of infected pleural or peritoneal fluid, and gastric lavage followed by administration of mineral oil and/or activated charcoal in cases of grain overload to prevent further absorption of endotoxin. one must address any septic process with appropriate antimicrobial therapy. initially, broad-spectrum coverage of the most likely organisms is recommended; one then should specify therapy according to results of culture and sensitivity testing. sepsis in foals is caused predominantly by gram-negative organisms, of which e. coli, actinobacillus spp., klebsiella spp., salmonella spp. and pasteurella spp. frequently are identified. 108 the reader is referred to other texts for review of general principles of antimicrobial therapy. regarding endotoxemia specifically, antimicrobial therapy has been suggested to increase the amount of circulating endotoxin by inducing endotoxin release on cell death of gram-negative bacteria. a recent in vitro study compared endotoxin release and inflammatory mediator activity between antimicrobials commonly used to treat e. coli septicemia in foals and specifically evaluated amikacin, ampicillin, amikacin plus ampicillin, ceftiofur, and imipenem. although these antimicrobials showed no difference in the ability to kill bacteria, amikacin and the amikacin/ampicillin combination resulted in the lowest and ceftiofur in the greatest release of endotoxin. endotoxin release appeared to be dose-dependent in that lesser amounts were released at higher antimicrobial concentrations. 109 based on these results and clinical experience, combining antimicrobial therapy with endotoxin-binding agents such as polymyxin b may be beneficial, especially when using β-lactam antimicrobials. antimicrobials frequently are given perioperatively to colic patients to lower the risk of peritonitis, incisional infection, and generalized sepsis and endotoxemia. because antimicrobial therapy has been implicated in the development of colitis, the duration of treatment should be minimal. unless evidence of sepsis, such as fever or changes in the leukogram, is present, perioperative administration of antimicrobials should not exceed a 24 to 48 hours. conversely, antimicrobial therapy frequently is used in cases of infectious colitis to treat the inciting cause and to prevent sepsis from translocation of bacteria. endotoxin typically has a short plasma half-life and is removed rapidly by mononuclear phagocytes or neutralized by binding to serum proteins and lipoproteins. many conditions responsible for the development of endotoxemia in horses, however, are associated with an ongoing release of endotoxin. examples include severe gastrointestinal inflammation as in proximal enteritis or colitis, grain overload, or uncontrolled sepsis. therapy directed against endotoxin itself may be able to interrupt the continuous activation of the inflammatory cascade in these cases. further benefits of antiendotoxin treatment may be derived if large amounts of endotoxin have been released before the inciting cause can be addressed. an important consideration regarding the efficacy of immunotherapy is the region of the lipopolysaccharide molecule against which antibodies are raised. the o-chain of lipopolysaccharide acts as a potent antigen on infection with gram-negative bacteria 6 ; however, antibodies directed against the o-chain are serotype specific and cannot afford significant cross-protection against heterologous bacterial strains. the core and lipid a region, both of which show a much higher degree of homology between lipopolysaccharide derived from different bacterial strains, offer a more promising target for immunotherapy. active immunization against endotoxin has been reported for horses. vaccination with a bacterin/ toxoid vaccine prepared from rough mutants of salmonella typhimurium or s. enteritidis protected horses against homologous and heterologous endotoxin challenge 110, 111 and carbohydrate overload. 111 despite these encouraging results and the current availability of a vaccine for use in horses (endovac-equi, immvac inc., columbus, missouri), active immunization against endotoxin does not appear to be a common practice. in comparison, passive immunization with antilipopolysaccharide antibodies is used widely. rough bacterial mutants, most commonly j5 of e. coli o111:b4 and s. minnesota re595, are used to immunize donor horses and subsequently prepare serum or plasma products. proposed mechanisms of action after binding of the antibodies to lipopolysaccharide include steric blockade of lipid a interaction with cellular receptors and enhanced bacterial clearance by opsonization. [112] [113] [114] studies concerning the efficacy of antibody administration in equine patients vary in their results. beneficial effects have been described in experimental models of endotoxemia, acute gastrointestinal disease, and neonates with sepsis, 111, [115] [116] [117] whereas in other studies, antibodies failed to protect foals and horses against endotoxin effects. 118-120 furthermore, administration of a s. typhimurium antiserum to foals was associated with an increased respiratory rate and higher serum activities of il-6 and tnf. 118 various equine serum and plasma products are currently commercially available. an antiserum raised against the lipopolysaccharide-core of s. typhimurium (endoserum) is available for administration to endotoxemic horses at a recommended dose of 1.5 ml/kg body mass. diluting the serum ten-to twentyfold in crystalloid intravenous solutions, administering it slowly over 1 to 2 hours, and monitoring the patient for adverse reactions is advisable. although the product is marketed for use in foals with failure of passive transfer, adverse effects have been reported, 118 and one should use caution when administering it to neonates. plasma from donors inoculated with j5 (e. coli) and s. typhimurium (re mutant) is available under a restricted license (polymune-j, vet dynamics, inc., san louis obispo, california). the manufacturer recommends administration of at least 1 to 2 l in cases of endotoxemia. in addition, hyperimmune plasma, which has a guaranteed minimum immunoglobulin g content but does not contain specific antiendotoxin antibodies (hi-gamm equi, lake immunogenics, inc., ontario, new york; polymune and polymune-plus, vet dynamics, inc.), is marketed for treatment of failure of passive transfer, and many clinicians use it to treat endotoxemia and sepsis. in addition to antibodies and protein, plasma contains active constituents such as complement components, fibronectin, clotting factors, and at iii 116 and therefore may be particularly useful in patients with endotoxemia-induced coagulopathy. volumes of 2 to 10 ml/kg body mass of hyperimmune plasma have been recommended for use in endotoxemic patients. 56, 121 polymyxin b polymyxin b is a cationic polypeptide antibiotic that binds to the anionic lipid a portion of lipopolysaccharide and neutralizes its endotoxin capacity. 122 at dosages required for antimicrobial activity, polymyxin b carries the risk of respiratory paralysis and ototoxic, nephrotoxic, and neurotoxic side effects; however, a much lower dose is required for endotoxin-binding activity. the effects of polymyxin b in horses have been evaluated in different experimental models. 118, 122, 123 in an in vivo study in foals, treatment with polymyxin b at a dosage of 6000 u/kg body mass before infusion with s. typhimurium lipopolysaccharide resulted in significantly less severe elevations of body temperature, respiratory rate, and serum activities of tnf and il-6 compared with untreated controls. 118 similarly, polymyxin b treatment of adult horses given endotoxin significantly ameliorated clinical signs and decreased plasma tnf activity. 124 in the latter study, benefits of treatment were also evident at lower dosages of polymyxin b (1000 and 5000 u/kg body mass) and administration of polymyxin b 1 hour after the start of endotoxin infusion. conversely, polymyxin b failed to ameliorate clinical signs of endotoxemia or prevent the development of coagulopathy, acidosis, lameness, and shock in experimental carbohydrate overload. 125 side effects suggestive of neurotoxicity appeared after repeated administration of 5 mg/kg body mass (36,000 u/kg) and in milder form, 2.5 mg/kg body mass (18,000 u/kg) polymyxin b. nephrotoxicity was not observed. currently, use of polymyxin b in equine patients is recommended at dosages of 1000 to 5000 u/kg body mass every 8 to 12 hours. 126 one should initiate treatment as early in the disease process as possible, because the beneficial effects of lipopolysaccharide scavenging are limited to the first 24 to 48 hours after the onset of endotoxemia, before tolerance develops. side effects in the form of neuromuscular blockade and apnea, which necessitate slow infusion of the drug in human patients, have not been observed in horses. one therefore can administer the entire dose as a slow bolus. if one uses polymyxin b in horses with hypovolemia, dehydration, or azotemia, one should attempt to improve peripheral tissue perfusion, minimize the polymyxin b dose, and closely monitor patients for nephrotoxicity. close monitoring is also important if one administers medications such as aminoglycoside antibiotics, which share a similar spectrum of potential side effects, concurrently. azotemic neonates have been reported to be more susceptible to the nephrotoxic effects of polymyxin b than adult horses. 124 in an attempt to decrease the risk for adverse effects while preserving lipopolysaccharide-neutralizing ability, a conjugate of polymyxin b with dextran has been developed. 127 in conjugated form, polymyxin b is prevented from extravasation into tissues, where it exerts toxic effects by interaction with cell membranes. in addition, conjugation increases the residence time of polymyxin b in the circulation and therefore should prolong the antiendotoxic effect. the polymyxin b-dextran combination was evaluated at a total dose of 5 mg/kg body mass of polymyxin b in 6.6 g/kg body mass dextran given 15 minutes before administration of endotoxin in horses. 128 treatment was found to block the development of tachycardia, tachypnea, fever, and neutropenia completely and to prevent increases in serum concentrations of tnf, il-6, txb 2 (a txa 2 metabolite), and the prostacyclin metabolite 6-keto-pgf 1α . although mild adverse effects in the form of tachypnea, sweating, and increased systolic blood pressure were observed, these were transient and could be prevented by pretreatment with ketoprofen. the polymyxin b-dextran combination is not commercially available at this time. natural endotoxin-binding proteins such as lbp, lipoproteins, and scd14 have been evaluated experimentally. results of these studies are controversial, and detrimental effects occurred in some cases. 129 a protein receiving much attention regarding potential therapeutic efficacy is the bactericidal permeability-increasing protein (bpi). this protein is structurally similar to lbp but is expressed exclusively in myeloid precursors of polymorphonuclear leukocytes. 130 bpi is stored in primary granules of mature neutrophils and during inflammation is expressed on their cell membranes and secreted into the extracellular environment. 131 bpi has an even higher affinity for lipopolysaccharide than lbp 132 and shows antibacterial activity specific for gram-negative bacteria. 65 binding of bpi to the gram-negative bacterial membrane results in growth arrest and is an important factor in the antibacterial activity of intact neutrophils. furthermore, bpi binding disrupts normal membrane organization and makes bacteria more susceptible to hydrophobic substances, including antimicrobials. 133 experimentally, recombinant bpi has been shown to protect against the toxic and lethal effects of isolated lipopolysaccharide and intact gram-negative bacteria, and clinical trials in human patients show promising results concerning its therapeutic use. 134 the biology and potential use of bpi in horses has not been evaluated. treatments aimed at inhibiting lipopolysaccharide interaction with cells or turning off intracellular signaling pathways are under investigation. nontoxic lipopolysaccharide or lipid a structures can act as endotoxin antagonists, if they competitively inhibit binding to lbp or cellular receptors or inhibit cellular activation by other mechanisms. of the potential antagonists that have been evaluated experimentally, lipopolysaccharide and lipid a from the phototrophic bacterium rhodobacter sphaeroides, and a synthetic compound (e5531) the structure of which is based on r. sphaeroides lipopolysaccharide, have been most promising. [135] [136] [137] [138] [139] unfortunately, species differences exist regarding cellular response to these structures, and r. sphaeroides lipopolysaccharide acts as a potent inducer of cytokine expression in equine cells. 140 based on results of receptor transfection studies in other species, 141,142 tlr4 is likely responsible for this phenotypic variation. additional compounds including lipopolysaccharide derived from nitrogen-fixing plant bacteria of the species rhizobium are being evaluated to reveal further insight into structural requirements for endotoxin antagonists in horses. nonsteroidal antiinflammatory drugs (nsaids) are probably the most commonly used drugs to treat endotoxemia. the rationale for their use is inhibition of prostaglandin endoperoxide h synthase, that is, cox, and thereby inhibition of prostanoid production (see figure 13 .7-5). additional beneficial effects may include scavenging of oxygen-derived free radicals and iron chelation; however, side effects may occur at dosages required to achieve these effects. 143 prostanoids have been identified as important mediators in the inflammatory response in a number of studies, and inhibition of their synthesis is associated with beneficial effects. two cox isoforms are recognized: constitutively expressed cox-1 and inducible cox-2. upregulation of cox-2 expression results from various proinflammatory stimuli, including lipopolysaccharide, tnf-α, and il-1. 144 constitutively expressed cox products are likely important for maintenance of homeostasis, whereas increased production of prostanoids by cox-2 is thought to be responsible for detrimental effects during inflammation and shock. research has focused on the development of selective cox-2 inhibitors; however, none of these products are currently available for use in horses. in horses the most commonly used nsaid to treat endotoxemia is flunixin meglumine. beneficial effects of flunixin meglumine have been described in experimental models of endotoxemia [145] [146] [147] and in clinical cases. in equine colic patients, treatment with flunixin meglumine before exploratory surgery resulted in reduced plasma concentrations of txb 2 and pge 2 and had a favorable effect on cardiovascular parameters. 148 flunixin meglumine was shown further to maintain cardiac output and systemic arterial blood pressure, improve blood flow to vital organs, reduce pulmonary endothelial damage, and improve survival on endotoxin challenge. [149] [150] [151] [152] nsaid use in horses carries the risk of side effects, most importantly the development of gastrointestinal ulceration and renal papillary necrosis (renal crest necrosis). in a study comparing the side effects of flunixin meglumine (1.1 mg/kg body mass), phenylbutazone (4.4 mg/kg body mass), and ketoprofen (2.2 mg/kg body mass) given 3 times daily for 12 days, lesions of the gastric glandular mucosa occurred most commonly. phenylbutazone resulted in the most severe side effects, which included small intestinal edema, erosions, and ulcers in the large colon and decreased serum albumin concentration. renal crest necrosis occurred more frequently in horses treated with phenylbutazone but also occurred with flunixin meglumine treatment. 153 despite the higher risk of side effects, use of phenylbutazone has been suggested for certain cases. in colic patients, phenylbutazone may provide analgesia and ameliorate endotoxin-induced ileus without masking cardiovascular effects of endotoxin, which are used to determine the necessity of surgical exploration. 154 for similar reasons and to minimize side effects a reduced dose of flunixin meglumine (0.25 mg/kg body mass) has been suggested and is used widely in horses. 155 at this dosage, flunixin meglumine was shown to inhibit eicosanoid synthesis efficiently in an in vivo model of endotoxemia. 156 reduction of clinical signs, however, was dose dependent, and therefore one should choose the appropriate dose based on the circumstances of each case. ketoprofen has been suggested to have superior effects because of a proposed dual inhibitory effect on cox and lipoxygenase and may carry a decreased risk of side effects compared with flunixin meglumine and phenylbutazone. a comparison of cytokine and eicosanoid production by lipopolysaccharide-stimulated isolated monocytes in vitro, however, showed no significant difference between horses pretreated with flunixin meglumine (1.1 mg/kg body mass) or ketoprofen (2.2 mg/kg body mass), respectively. 157 eltenac has been evaluated in an experimental endotoxemia model in horses. 158 given 15 minutes before lipopolysaccharide infusion, eltenac at a dose of 0.5 mg/kg protected against changes in clinical, hemodynamic, and hematologic parameters and blunted the lipopolysaccharide-induced rise in plasma cytokine concentrations in comparison with controls. some parameters, however, including heart rate, leukocyte count, lactate concentration, and plasma tnf activity, were not improved. ibuprofen may have beneficial effects superior to the other nsaids, because it may be possible to achieve tissue concentrations safely that allow iron chelation to occur. according to a study in healthy foals, dosages of ibuprofen up to 25 mg/kg every 8 hours can be given safely for up to 6 days. 143 the use of corticosteroids for antiinflammatory therapy in sepsis and endotoxemia has been controversial in human and equine patients, and beneficial effects superior to the ones achieved by nsaids have not been demonstrated consistently overall. corticosteroids inhibit the activity of phospholipase a 2 and the release of arachidonic acid from cell membrane phospholipids, as well as the production of tnf, il-1, and il-6 in response to a lipopolysaccharide stimulus. experimentally, beneficial effects of dexamethasone in equine endotoxemia have been demonstrated. 159, 160 to inhibit tnf production by equine peritoneal macrophages, however, the required concentration of dexamethasone was high and corresponded to an in vivo dosage (approximately 3 mg/kg body mass) greatly exceeding current recommendations. 159 although single doses of corticosteroids are unlikely to carry a disproportionate risk of side effects, one should consider the suggested association of laminitis with corticosteroid use in horses. in cases of sepsis, further immunosuppressive effects could be detrimental. in human patients with certain types of septic shock, dysfunction of the hypothalamic-pituitary-adrenal axis has been recognized and successfully treated with hydrocortisone replacement therapy. 161 use of corticosteroids for this indication has not been evaluated in horses. pentoxifylline, a methylxanthine derivative and phosphodiesterase inhibitor, has been suggested for use in endotoxemia because of its effects on neutrophil function and its ability to inhibit the production of various cytokines, interferons, and thromboplastin. decreased production of tnf, il-6, txb 2 , and thromboplastin in response to endotoxin was shown in an equine ex vivo model. 162 in horses given endotoxin followed by treatment with pentoxifylline (7.5 mg/kg body mass followed by continuous infusion of 3 mg/kg/hr for 3 hours), however, only minimal beneficial effects were observed. 163 treatment significantly improved body temperature, respiratory rate, and whole blood recalcification time, but no effect was observed regarding heart rate, blood pressure, leukocyte count, plasma fibrinogen concentration, and serum cytokine concentrations. the conclusion was that benefits of treatment with pentoxifylline might be restricted to administration of high bolus doses or continuous infusion early in the pathophysiologic process. in an in vivo endotoxemia model in horses, combination of pentoxifylline (8 mg/kg body mass) and flunixin meglumine (1.1 mg/kg body mass) was found to have greater benefit than each treatment on its own. 164 the currently recommended dosage for oral administration of pentoxifylline is 8 mg/kg every 8 hours. because of its rheologic properties, that is, the ability to increase erythrocyte deformability and microvascular blood flow, pentoxifylline may be particularly useful in endotoxemic patients showing evidence of laminitis. an intravenous preparation of pentoxifylline is not commercially available. dimethyl sulfoxide (dmso) is used frequently in an attempt to scavenge oxygen-derived radicals. the treatment may be most appropriate in cases of ischemia-induced intestinal damage and associated reperfusion injury. however, dmso failed to show beneficial effects in an experimental model of intestinal ischemia when administered on reperfusion of the ischemic intestine. 165 dmso at the commonly used dose of 1 g/kg body mass was shown to increase mucosal loss after ischemia and reperfusion of the large colon, 166 and hence a reduced dose of 0.1 g/kg body mass has been proposed for cases of intestinal ischemia. for intravenous administration, dmso needs to be diluted in polyionic solutions to a concentration not exceeding 10%. oral administration of a 10% to 20% solution via nasogastric intubation is also possible. aside from dmso the xanthine oxidase inhibitor allopurinol has been suggested as a treatment to prevent oxygen radical-induced tissue damage. during periods of ischemia, tissue xanthine dehydrogenase is converted to xanthine oxidase, which on reperfusion catalyzes the generation of superoxide radicals. 167, 168 evaluation in horses showed beneficial effects of 5 mg allopurinol per kilogram body mass administered 12 hours before endotoxin challenge. 169 in another study, mucosal damage attributable to oxygen-derived free radicals was not attenuated by allopurinol in an experimental ischemia-reperfusion model. 166 lidocaine given intravenously has been suggested as an antiinflammatory, analgesic, and prokinetic agent, and some clinicians use it to treat colic and laminitis in horses. in an experimental endotoxemia model in rabbits, lidocaine was found to inhibit hemodynamic and cytokine responses to endotoxin profoundly if given immediately following lipopolysaccharide infusion. 170 use of lidocaine therefore may have additional merit in endotoxemic patients. a common regimen for lidocaine use in horses is administration of an initial bolus (1.3 mg/kg body mass) section 13.7 endotoxemia followed by continuous infusion at a rate of 0.05 mg/ kg/min. one should monitor patients for toxic neurologic effects associated with a lidocaine overdose. high concentrations of ω-3 fatty acids can alter the phospholipid composition of cellular membranes toward a decreased ratio of ω-6 to ω-3 and thereby can affect membrane functions such as phagocytosis, receptor binding, and activities of membrane-bound enzymes. 68 most importantly for the treatment of endotoxemia, ω-3 fatty acid incorporation into cell membranes decreases the availability of arachidonic acid (an ω-6 fatty acid) for eicosanoid synthesis 171 and provides alternative substrates. metabolism of ω-3 fatty acids via the cox and lipoxygenase pathway leads to the production of 3-series prostaglandins and 5-series leukotrienes, which have less biologic activity than their 2-series and 4-series counterparts derived from arachidonic acid. aside from these mechanisms, ω-3 fatty acids prevent lipopolysaccharideinduced upregulation of cd14 in monocytic cells and therefore may be able to block transmembrane signaling of lipopolysaccharide. 172 cells from horses given linseed oil (high in ω-3 fatty acids) for 8 weeks before blood collection showed significantly decreased expression of procoagulant activity, txb 2 , and tnf in response to lipopolysaccharide stimulation. 173, 174 in an in vivo experimental model of endotoxemia in horses, treatment resulted in prolonged activated partial thromboplastin time and whole blood recalcification time, suggesting an anticoagulant effect; however, a significant beneficial effect on clinical response and serum eicosanoid concentrations was not observed. 175 because dietary addition of ω-3 fatty acids requires several weeks of treatment, intravenous infusion was evaluated and shown to alter the composition of cell membrane phospholipids rapidly. 176 further evaluation of this treatment for use in horses is necessary before dosage recommendations can be made. monoclonal and polyclonal antibodies against equine tnf have been evaluated. [177] [178] [179] administration of a monoclonal antibody preparation before lipopolysaccharide infusion resulted in significantly reduced plasma tnf-activity, improved clinical abnormality scores, lower heart rate, and higher leukocyte count compared with controls. 178 furthermore, plasma concentrations of lactate and 6-keto-pgf 1α were reduced significantly, whereas txa 2 production was not affected. 177 in another study, 179 administration of a rabbit polyclonal antibody against recombinant human tnf was unable to improve clinical and hematologic parameters when given shortly (15 minutes) after lipopolysaccharide infusion, although inhibition of tnf activity was present in vitro. 179, 180 findings in horses are in agreement with studies in other species and suggest that beneficial effects of tnf inhibition may be limited to administration before lipopolysaccharide exposure. widespread clinical use therefore is unlikely to become feasible. clinical trials in human patients have not shown significant benefits of tnf antibody treatment. 181, 182 the effects of selective paf receptor antagonists have been evaluated. paf is implicated in the development of systemic hypotension, 183 lipopolysaccharide-induced platelet aggregation, 184 ileus, 185 and increased vascular permeability 186 and may mediate recruitment of leukocytes to inflamed tissues. 187, 188 a study in horses using the paf receptor antagonist sri 63-441 before lipopolysaccharide infusion showed significant decreases in heart rate and shorter elevation of lactate concentrations in response to the treatment. although not statistically significant, additional beneficial effects included delayed onset of fever, a shortened period of neutropenia, and reduced maximal platelet aggregation. 189 whenever possible, the clinician should correct volume and electrolyte deficits, or at least improve them, before anesthetizing a patient for a surgical procedure. for initial resuscitation, polyionic solutions such as lactated ringer's solution given at rates of 10 to 20 ml/kg/hr are appropriate. patients with severe hypovolemia and shock may require higher fluid volumes. a viable alternative to large-volume resuscitation with isotonic fluids is the use of small volumes of hypertonic solutions, which transiently raise plasma osmolality, thereby causing a fluid shift from the interstitial space into the vasculature and rapidly restoring circulating volume. hypertonic saline solution (7.5% sodium chloride) is the most commonly used hypertonic solution and has been shown to have beneficial effects in endotoxemic horses. 190 a dose of 4 ml/kg is recommended, which one should give as a bolus infusion over 10 to 15 minutes, followed by administration of an isotonic solution to restore total body fluid volume. one should use hypertonic saline with caution in patients with sodium and/or chloride derangements and should monitor serum electrolyte concentrations in the case of repeated administration. improvement of the cardiovascular status in response to fluid therapy is indicated by normalization of heart rate, mucous membrane color, and capillary refill time. failure of urination to occur despite appropriate fluid resuscitation should result in critical evaluation of renal function. once one has stabilized the patient, one should choose a maintenance fluid rate to maintain adequate hydration and plasma volume. for adult horses, the maintenance fluid rate is approximately 2 ml/kg/hr, whereas neonatal foals that are not nursing may require larger volumes (4 ml/kg/hr). one should monitor fluid administration carefully in endotoxemic patients, because lowered plasma oncotic pressure caused by hypoproteinemia along with an increased vascular permeability increase the risk of tissue edema formation. furthermore, a rapid increase in total body fluid volume may be detrimental in patients with compromised cardiac and peripheral vasomotor function and may increase the severity of vascular pooling in peripheral organs. in these patients, hypertonic saline or colloids may be more appropriate means of stabilization than large volumes of crystalloid solutions. plasma is an ideal colloid and should be administered to maintain a serum total protein concentration above 4.2 g/dl. 121 to raise plasma protein concentration and colloid osmotic pressure significantly, however, horses often require large volumes of plasma (7 to 10 l or more in a 450-kg horse), and one should consider alternative colloids. furthermore, high-molecular-weight polymers are thought to provide superior oncotic effects in cases of sepsis and endotoxemia, when vascular permeability is increased. hetastarch, or hydroxyethyl starch, (hespan) is commercially available as a 6% solution in 0.9% sodium chloride. hetastarch molecules have very high molecular weight, and degradation must occur before renal excretion. 191 these properties result in a longer plasma half-life and prolonged oncotic effects compared with other colloids; persistence of the oncotic effect for 24 hours was found in hypoproteinemic horses. 192 a dosage of 5 to 15 ml/kg given by slow intravenous infusion along with an equal or greater volume of crystalloid fluids is recommended. 191, 193 in human patients, prolonged activated partial thromboplastin time, decreased factor viii activity, and decreased serum fibrinogen concentration have been described in association with hetastarch use. 194 in the limited number of equine studies, bleeding times were not affected 195, 196 ; however, one should monitor patients treated with hetastarch for coagulopathy. one should base correction of serum electrolyte concentrations on the results of laboratory evaluation. ideally, one should evaluate serum electrolyte concentrations of patients receiving fluid therapy daily. one should take ongoing losses and lack of dietary intake into account, especially when serum concentrations, as in the case of potassium, poorly reflect total body electrolyte stores. potassium supplementation is recommended in patients experiencing prolonged (greater than 48 hours) periods of anorexia. one can add calcium in the form of calcium gluconate, which is available as a 23% solution. based on a study in healthy horses, rates of administration for calcium gluconate in the range of 0.1 to 0.4 mg/kg/min are recommended, 197 and as a guideline, one should administer 0.5 to 1 ml/kg body mass per day of a 23% solution. one can add potassium in the form of potassium chloride or potassium gluconate to intravenous solutions at a dose of 20 to 40 meq/l given at a maintenance rate. administration of potassium should not exceed a rate of 0.5 to 1 meq/kg/hr. metabolic acidosis in endotoxic shock is attributable to lactic acidemia and inadequate tissue perfusion. 198 acid-base balance often improves considerably after fluid resuscitation (preferably with alkalinizing solutions such as lactated ringer's solution) alone; however, additional sodium bicarbonate may be required in cases in which serum bicarbonate concentration remains below 15 meq/l. for adult horses, the bicarbonate deficit (in meq hco 3 ) is calculated as 0.3 × body mass (kg) × base deficit, whereas for foals one should use a factor of 0.5. as a general rule, one should administer half the required amount as a bolus followed by the remaining half over 12 to 24 hours. because endotoxemia is a dynamic process and losses are ongoing, one should reevaluate acid-base status at least once daily. foals with sepsis are frequently hypoglycemic, and 5% dextrose solutions are useful as initial resuscitation fluids. one should reduce the glucose concentration of intravenous solutions according to the blood glucose concentration to avoid prolonged hyperglycemia. administration of hyperimmune plasma (20 to 40 ml/kg body mass) is highly recommended in foals with evidence of partial or complete failure of passive transfer. one should consider positive inotropic and vasomotor agents in patients with persistently inadequate tissue perfusion. lower dosages of dopamine (0.5 to 2 µg/kg/min) result in vasodilation of the renal, mesenteric, coronary, and intracerebral vasculature via dopaminergic effects, whereas higher dosages (up to 10 µg/kg/min) also exert stimulation of β 1 -adrenergic receptors, resulting in increased myocardial contractility and heart rate. 199 dobutamine is a direct β 1 -adrenergic agonist and does not appear to have significant vasodilator properties. dosages for dobutamine of 1 to 5 µg/kg/min as continuous intravenous infusion have been recommended for use in horses. in addition, norepinephrine was evaluated in hypotensive critically ill foals that were refractory to the effects of dopamine and dobutamine. 200 at dosages up to 1.5 µg/kg/min administered concurrently with dobutamine, six out of seven foals showed an increase in mean arterial pressure, and all foals had increased urine output. because of the risk of cardiac side effects, close monitoring of heart rate and rhythm should accompany infusion of inotropes. indirect blood pressure measurements using a tail cuff may be used to monitor the effects of treatment. more frequently than overt thrombosis or bleeding attributable to dic, hemostatic abnormalities occur in the form of alterations in the coagulation profile. a procoagulant state with shortened bleeding times or prolonged bleeding times caused by consumption of clotting factors may be evident. one should address abnormalities in the coagulation profile as early as possible but especially if they persist more than 24 hours after initiation of therapy. because of the complex interactions of coagulation and fibrinolysis during endotoxemia, one should combine anticoagulant therapy with the administration of fresh frozen plasma to replace clotting and fibrinolytic factors. heparin acts as an anticoagulant by activation of at iii and subsequent inhibition of thrombin, release of tissue factor pathway inhibitor from endothelial cells, and inhibition of platelet aggregation. 201 because endogenous at iii levels frequently are decreased in patients with coagulopathy, addition of heparin to fresh frozen plasma may be the most effective route of administration. an initial dose of 100 iu/kg body mass followed by 40 to 80 iu/kg body mass 3 times daily has been recommended. 121 anemia caused by erythrocyte agglutination occurs in some patients during therapy with unfractionated heparin 202,203 but typically resolves within 96 hours if therapy is discontinued. 121 because of the risk of microthrombosis associated with erythrocyte agglutination, use of low-molecular-weight heparin (50 iu/kg body mass subcutaneously every 24 hours) has been recommended 204 but may be cost-prohibitive. one may give aspirin orally (10 to 20 mg/kg body mass, every 48 hours), which irreversibly inhibits platelet cox activity, to inhibit platelet aggregation and microthrombosis. platelet hyperaggregability has been implicated in the pathogenesis of carbohydrate-induced laminitis, 205 and heparin and aspirin have been recommended to prevent development of laminitis. in an in vitro study, however, aspirin did not inhibit endotoxin-induced platelet aggregation. 206 luteolysis caused by increased concentrations of pgf 2α leads to pregnancy loss in endotoxemic mares before day 55 of pregnancy. 207 daily administration of altrenogest (regu-mate, hoechst-roussel agri-vet, somerville, new jersey) at a dose of 44 mg orally consistently prevented fetal loss in mares if administered until day 70 of pregnancy. 100 treatment with flunixin meglumine, by blockade of pgf 2α release, 101 also may contribute to the maintenance of pregnancy in endotoxemic mares. the pathogenesis of fetal loss and abortion caused by endotoxemia, surgery, or systemic disease later in gestation is not understood completely. proposed mechanisms include direct effects on the fetus, placental function, or placental progesterone production. 208 the pathophysiology of laminitis caused by endotoxemia is understood incompletely; however, decreased digital blood flow 209, 210 and intravascular microthrombosis have been implicated. decreased no production by vascular endothelial cells in response to endotoxin has been suggested as a mechanism for vasoconstriction and decreased blood flow 211 ; however, use of no donors remains controversial. maintenance of adequate peripheral perfusion and anticoagulant and antiinflammatory therapy may be helpful in preventing and treating laminitis caused by endotoxemia. although the innate immune response to endotoxin (lipopolysaccharide) is crucially important for the preservation of homeostasis and health, large amounts of endotoxin can evoke an excessive and uncontrolled inflammatory response and result in a dysfunction of hemostatic and circulatory control mechanisms, loss of vascular integrity, and finally tissue damage. conditions commonly associated with the development of endotoxemia in horses are acute gastrointestinal diseases, especially of ischemic and severe inflammatory nature, and localized or generalized infections. although measuring endotoxin concentrations in equine plasma is possible, this is not feasible in a clinical setting, and one typically reaches a diagnosis of endotoxemia based on clinical signs and clinicopathologic data. successful treatment of endotoxemia requires resolution of the primary disease process in addition to neutralization of circulating endotoxin, interference with the activities of inflammatory mediators, and general supportive care. newer treatments, such as blockade of endotoxin-interaction with cells or interruption of cell signaling pathways, are under investigation. possible sequelae of endotoxemia include dic, multiple organ failure, circulatory failure, and death. frequently, the outcome of conditions associated with endotoxemia in horses depends on the severity of associated complications; for example, renal compromise, laminitis, and abortion. resting state the lateral margins of the vesicle, that is, the buccal mucosa, are in close contact with the cheek teeth. caudally, the external space communicates with the pharynx through the aditus pharyngis. the mucous membrane of the mouth is continuous at the margin of the lips with the skin and during life is chiefly pink but can be more or less pigmented, depending on the skin color and the breed type. the lips are two muscular membranous folds that unite at angles close to the first cheek teeth. each lip presents an outer and an inner surface. the upper lip has a shallow median furrow (philtrum); the lower lip has a rounded prominence or chin (mentum). the internal surface is covered with a thick mucous membrane that contains small, pitted surfaces that are the openings of the ducts of the labial glands. small folds of the mucous membrane called the frenula labii pass from the lips to the gum. the free border of the lip is dense and bears short, stiff hairs. the arteries of the mouth are derived from the maxillary, mandibular, labial, and sphenopalatine arteries of the major palatine artery. the veins drain chiefly to the lingual facial vein. sensory nerves originate from the trigeminal nerve (cranial nerve v) and the motor nerves from the facial nerve (vii). the cheeks spread back from the lips and form both sides of the mouth and are attached to the alveolar borders of the bones of the jaws. the cheeks are composed of skin and muscular and glandular layers and then the internal mucous membrane. the skin is thin and pliable. in contrast, the oral mucous membrane is dense and in many areas of the oral cavity is attached firmly to the periosteum so that construction of oral mucosal flaps can be achieved only by horizontal division of the periosteal attachment. such a feature is important in reconstructive techniques applied to the oral cavity. the blood supply to the cheeks comes from the facial and buccal arteries and the sensory nerves from the trigeminal and motor nerves from the facial nerve. the hard palate (palatum durum) is bounded rostrally and laterally by the alveolar arches and is continuous with the soft palate caudally. the hard palate has a central raphe that divides the surface into two equal portions. the word mouth is used commonly to signify the first part of the alimentary canal or the entrance to it. 1 the mouth is bounded laterally by the cheeks, dorsally by the palate, and ventrally by the body of the mandible and by the mylohyoideus muscles. the caudal margin is the soft palate. the mouth of the horse is long and cylindric, and when the lips are closed, the contained structures almost fill the cavity. a small space remains between the root of the tongue and the epiglottis and is termed the oropharynx. the cavity of the mouth is subdivided into sections by the teeth. the space external to the teeth and enclosed by the lips is termed the vesicle of the mouth, and in the enter the nose from the glands of the vomeronasal duct. to what extent these secretions aid in pheromone reception is not known. 2 that portion of the palatine mucosa immediately behind the incisor teeth frequently is swollen (lampas) during eruption of the permanent teeth. this swelling is physiologic and not pathologic. the tongue is situated on the floor of the mouth between the bodies of the mandible and is supported by the sling formed by the mylohyoideus muscles. the root of the tongue is attached to the hyoid bone, soft palate, and pharynx. the upper surface and the rostral portion of the tongue are free; the body of the tongue has three surfaces. the apex of the tongue is spatulate and has a rounded border. the mucous membrane adheres intimately to the adjacent structure and on the dorsum is dense and thick. the lingual and sublingual arteries supply the tongue from the linguofacial trunk and matching veins. the linguofacial trunk drains into the linguofacial vein. the lingual muscles are innervated by the hypoglossal nerve (xii) and the sensory supply is from the lingual and glossopharyngeal (ix) nerves. the formula for the deciduous teeth of the horse is 2 times i3-3 c0-0 p3-3 for a total of 24. the permanent dental formula is 2 times i3-3 c1-1 p3-3 or p4-3 m3-3 for a total of 40 or 42. in the mare the canine teeth are usually small or do not erupt, hence reducing the number to 36 or 38. the first premolar tooth (wolf tooth) is often absent and has been reported as occurring in only 20% of the upper dentition of thoroughbred horses. 3 the teeth of the horse are complex in shape and are compounded of different materials (dentin, cementum, and enamel). they function as grinding blades to masticate and macerate cellulose food in the important first stage of the digestive process. the cheek teeth in the horse are a well-documented feature of the evolution of equus caballus. the first incisor is present at birth or the first week of life. the second incisor erupts at 4 to 6 weeks of age; the third incisor, at 6 to 9 months of age; the first and second premolars, at birth to 2 weeks of age; and the third premolar, 3 months of age. the eruption times for the permanent teeth are as follows: first incisor, 2 1 / 2 years of age; second incisor, 3 1 / 2 years of age; third incisor, 4 1 / 2 years of age; the canine tooth, 4 to 5 years of age; the first premolar (wolf tooth), 5 to 6 months of age; the second premolar, 2 1 / 2 years of age; the third premolar, 3 years of age; the fourth premolar, 4 years of age; the first molar, 10 to 12 months of age; the second molar, 2 years of age; and the third molar, 3 1 / 2 to 4 years of age. this eruption sequence clearly indicates that the eruption of the second and third permanent premolar teeth give the potential for dental impaction. the modern horse has six incisor teeth in each jaw that are placed close together so that the labile edges form a semicircle. the occlusal surface has a deep enamel invagination (infundibulum) that is filled only partially with cementum. as the incisor teeth wear, a characteristic pattern forms in which the infundibulum is surrounded by rings of enamel, dentin, enamel, and crown cementum in a concentric pattern. each incisor tooth tapers from a broad crown to a narrow root so that as the midportion of the incisor is exposed to wear, the cross-sectional diameters are about equal; that is, at 14 years of age, the central incisor tooth of the horse has an occlusal surface that is an equilateral triangle. observations on the state of eruption, the angles of incidence of the incisor teeth, and the pattern of the occlusal surfaces are used as guides for aging of horses. the canine teeth are simple teeth without complex crowns and are curved. the crown is compressed and is smooth on its labial aspect but carries two ridges on its lingual aspect. no occlusal contact occurs between the upper and lower canine teeth. when erupted, the six cheek teeth of the horse function as a single unit in the mastication of food. each arcade consists of three premolar and three molar teeth. the maxillary arcade is slightly curved, and the teeth have a square occlusal surface. the occlusal surfaces of the mandibular teeth are more oblong, and each arcade is straighter. the horse is anisognathic, that is, the distance between the mandibular teeth is narrower (one-third) than the distance between the upper cheek teeth. this anatomic arrangement affects the inclination of the dental arcade as the jaws slide across each other in the food preparation process. the unworn upper cheek tooth presents a surface with two undulating and narrow ridges, one of which is lateral and the other medial. on the rostral and lingual side of the medial style is an extra hillock. the central portion of these surfaces is indented by two depressions that are comparable with, but much deeper than, the infundibula of the incisor teeth. when the teeth have been subjected to wear, the enamel that closed the ridges is worn through and the underlying dentin appears on the surface. thus after a time the chewing surface displays a complicated pattern that may be likened to the outline of an ornate letter b, the upright stroke of the b being on the lingual aspect. dentin supports the enamel internally, cementum supports the enamel lakes, and the peripheral cementum fills in the spaces between the teeth so that all six teeth may function as a single unit, that is, the dental arcade. transverse ridges cross each tooth so that the whole maxillary arcade consists of a serrated edge. the serrations are formed so that a valley is present at the area of contact with adjacent teeth. these serrations match fitting serrations on the mandibular arcade. the true roots of the cheek teeth are short compared with the total length of the tooth. cheek teeth have three roots: two small lateral roots and one large medial root. by custom, that portion of the crown embedded within the dental alveolus is referred to as the reserve crown, and the term root is confined to that area of the tooth that is comparatively short and enamel free. wear on the tooth gradually exposes the reserve crown, and the roots lengthen. in an adult 1000-lb horse the maxillary cheek teeth are between 8.0 and 8.5 cm in length. dental wear accounts for erosion and loss of tooth substance at a rate of 2 mm/yr. the pulp chambers of the teeth are also complex. the incisors and canines have a single pulp chamber. the mandibular cheek teeth have two roots and two separate pulp chambers. the maxillary cheek teeth, although they have three roots, have in fact five pulp chambers. as occlusal wear proceeds, deposition of secondary dentin within the pulp chambers protects the chambers (e.g., the dental star, medial to the infundibulum on the incisor teeth). in the mandibular cheek teeth the transverse folding of the enamel anlage (during morphogenesis of the tooth) does not take place, and the occlusal surface is a simple surface of central dentin surrounded by enamel. each tooth then is conformed to a single arcade by the presence of peripheral crown cementum. the oral cavity and oropharynx are subject to a variety of diseases. however, many conditions affecting the first portion of the alimentary system produce the same clinical signs, regardless of their cause. the clinical signs may include inappetance or reluctance to eat, pain on eating or swallowing, oral swelling, oral discharge, and fetid breath. affected animals may show some interest in food but hesitate to eat it. salivation may be excessive and may be contaminated with purulent exudate or blood. the occurrence of bruxism (i.e., grinding of teeth) can indicate discomfort in other areas of the alimentary tract; for example, bruxism and frothing oral saliva are characteristic features of gastric ulceration in the horse. the clinician needs to be aware that considerable weight loss can occur rapidly with inability to feed and swallow. diseases that result in denervation of the pharynx and inappropriate swallowing can have the complication of inhalation pneumonia. after a complete physical examination and ascertaining the history, the clinician should approach examination of the mouth systematically in all cases. one can examine a considerable portion of the mouth and teeth from the outside by palpation of the structures through the folds of the cheek. most horses allow an oral examination without sedation or the use of an oral speculum. in many cases, however, one best achieves the detailed oral examination by sedation and the use of an oral speculum and a light source. one should irrigate the mouth to wash out retained food material so as to be able to inspect and palpate the lips, cheeks, teeth, and gums. the classic signs of dental disease in the horse include difficulty and slowness in feeding, together with a progressive unthriftiness and loss of body condition. in some instances, the horse may quid, that is, it may drop poorly masticated food boluses from the mouth, and halitosis may be obvious. additional problems reported by owners include bitting and riding problems and headshaking or head shyness. facial or mandibular swelling may occur. nasal discharge can result from dental disease associated with maxillary sinus empyema. mandibular fistulae frequently are caused by lower cheek tooth apical infections. some correlation exists between the age of the animal and clinical signs (table 13 .8-1). ancillary aids for a complete examination of the oral cavity of the horse may include radiology, endoscopic examination, fluoroscopy, biopsy, and culture. one should take care always during endoscopic evaluation of the oral cavity using a flexible endoscope. the author recommends sedation and the use of an oral speculum to prevent inadvertent mastication of the endoscope. if one uses general anesthesia as part of the diagnostic workup, then endoscopic evaluation of the oral cavity is much easier. in selected cases, advanced imaging technologies such as computed tomography, magnetic resonance imaging, or nuclear scintigraphy may be beneficial. the lips of the horse are mobile and prehensile. in many ways they function like the tip of the elephant's trunk in that they test, manipulate, and sample the environment for potential nutritive value. consequently, loss of motor function (e.g., facial palsy) affects the efficiency of the prehensile system. the lips grasp food in grazing or browsing, and the incisor teeth section the food. with mastication and lubrication with saliva, the bolus of food forms and is manipulated from side to side across the mouth, assisted by the tight cheeks of the horse and the palatine ridges. swallowing begins as the food bolus contacts the base of the tongue and the pharyngeal walls. during swallowing, the soft palate elevates to close the nasopharynx, the base of the tongue elevates, and the hyoid bone and the larynx move rostrally following contraction of the hyoid muscles. during this process, the rima glottidis closes and the epiglottis tilts dorsally and caudally to protect the airway so that food is swept through lateral food channels around the sides of the larynx into the laryngoesophagus. fluoroscopic studies in nursing foals in the dorsoventral view showed that contact occurs between the lateral food channels in the midline so that in outline the food bolus achieves a bow tie shape. 4 dysphagia is defined as a difficulty or inability to swallow. anatomic classifications for dysphagia include prepharyngeal, pharyngeal, and esophageal (postpharyngeal) dysphagias. the site of the cause for dysphagia influences the clinical signs. prepharyngeal dysphagia is characterized by dropping food (quidding) or water from the mouth, reluctance to chew, hypersalivation, or abnormalities in prehension. pharyngeal and esophageal dysphagias are characterized by coughing; nasal discharge containing saliva, water, or food material; gagging; anxiousness; and neck extension during attempts to swallow. the following section describes esophageal dysphagia in more detail. causes of dysphagia can be divided into four types: painful, muscular, neurologic, or obstructive (table 13 .8-2). pain and obstruction cause dysphagia by interfering with the mechanics of prehension, bolus formation and transfer to the pharynx, and deglutition. muscular and neurologic causes of dysphagia impede prehension and swallowing by affecting the motor function of the lingual or buccal musculature, muscles of mastication (temporal and masseters), and pharyngeal and cranial esophageal muscles. sensory loss to the lips, buccal mucous membranes, pharynx, or tongue also may cause dysphagia. neurologic causes of dysphagia may affect the forebrain, brainstem, or peripheral nerves that control prehension (cranial nerves vm, vs, vii, and xii), transfer of the food bolus to the pharynx (cranial nerves vs and xii) and swallowing (cranial nerves ix and x). diagnosis of the cause of dysphagia is based on physical examination including a careful oral examination, neurologic examination, clinical signs, and endoscopy of the pharynx, esophagus, and guttural pouches. radiology may be useful to assess the bony structures of the head and throat. ultrasonography is valuable for examining the retropharyngeal space and esophagus to detect and evaluate masses. one may detect pharyngeal or esophageal causes of dysphagia with routine endoscopic examination or with contrast radiography. although one also can use endoscopy to assess deglutition, one must remember that sedation adversely affects the deglutition mechanism. one may assess deglutition using fluoroscopy 4 or manometry, 5 but these techniques require specialized equipment. specific diagnostic procedures for nonalimentary causes of dysphagia are covered elsewhere in this text (see chapter 3). specific treatments aimed at resolving the underlying disorder causing dysphagia are discussed in detail elsewhere. one should avoid feeding roughage with long fiber length (hay or grass) to most horses with dysphagia. dietary modifications that promote swallowing such as feeding slurries made from complete pelleted feeds may be sufficient to manage some cases of partial dysphagia. one must take care to prevent or avoid aspiration pneumonia in horses with pharyngeal or esophageal dysphagia. one can manage foals by feeding mare's milk or a suitable substitute through a nasogastric tube. one also may administer pellet slurries or formulated liquid diets via nasogastric tubes to older horses. prolonged nutritional management of dysphagic horses may require extraoral feeding using a tube placed through an esophagostomy. 6 formulated pelleted diets are often easy to administer through a tube as slurry and are balanced to meet the nutritional requirements for healthy horses. one must feed sufficient quantities to deliver adequate calories (16 to 17 mcal/day for a 500-kg horse). adjustments may be necessary for horses that are cachectic or have extra metabolic demand (such as pregnancy). adding corn oil to the ration (1 cup every 12 or 24 hours) is a common method of increasing fed calories. liquid diets also have been used for enteral feeding 7 but may not be tolerated as well as pelleted diets. regardless of the method of nutritional management, one must monitor and replace salivary losses of electrolytes. saliva contains high concentrations of na, k, and cl. a group of ponies with experimental esophagostomies 8 and a horse with esophageal squamous cell carcinoma 9 were fed a complete pelleted diet through esophagostomy tubes but developed metabolic acidosis, hyponatremia, and hypochloremia apparently because of salivary losses. surprisingly, salivary losses of potassium did not result in hypokalemia in these cases, presumably because of replacement in the diet. however, if the diet is deficient in potassium, hypokalemia may result. one often can accomplish electrolyte replacement by adding nacl and kcl to the diet. one can maintain horses for months with frequent feedings through an esophagostomy tube. 9 parenteral nutrition (total or partial) may be useful in the short term but is not often feasible for long-term management. tooth eruption is a complex phenomenon involving the interplay of dental morphogenesis and those vascular forces responsible for creating the eruption pathway. these changes are responsible for osteitis and bone remodeling within the maxilla and mandible. young horses frequently show symmetric bony swelling resulting from these eruption cysts. in some cases, additional clinical signs of nasal obstruction with respiratory stridor or nasal discharges may be apparent. pathologic problems associated with maleruption include a variety of dental diseases. oral trauma can displace or damage erupting teeth or the permanent tooth buds. as a result, teeth may be displaced and erupt in abnormal positions or may have abnormal shapes. supernumerary teeth, incisors and molars, can develop, as well as palatal displacement of impacted teeth (maxillary p3-3, or third cheek tooth). in almost all of these conditions some form of surgical treatment is necessary. significant evidence from the location of apical osteitis in diseased teeth (table 13 .8-3) confirms that dental impaction is a major cause of dental disease in the horse. in a series of 142 extracted teeth, 63 were p3-3 or p4-4 (cheek tooth 2 or 3, respectively). 10 early observations had indicated that the first molar (m1, or cheek tooth 4) was the most commonly diseased tooth, and an "open infundibulum" in this tooth has been suggested as the cause. 11 studies on cementogenesis of the maxillary cheek teeth have shown, however, that in fact most maxillary cheek teeth have a greater or lesser degree of hypoplasia of cementum within the enamel lakes and that this "lesion" rarely expands into the pulp. the central infundibular hole is the site of its vascular supply to the unerupted cement lake. on those occasions in which cases one can use apicoectomy and retrograde endodontic techniques to save the diseased tooth. one must take care, however, in selection of patients. in most cases of apical osteitis in the horse that result from dental impaction, immature root structures make achieving an apical seal of the exposed pulp difficult. gingival hyperemia and inflammation occur during the eruption of the permanent teeth and are common causes of a sore mouth in young horses (particularly 3-year-olds as the first dental caps loosen). such periodontal changes usually resolve as the permanent dental arcade is established. during normal mastication, the shearing forces generated by the occlusal contact of the cheek teeth essentially clean the teeth of plaque and effectively inhibit deposition of dental calculus. wherever occlusal contact is ineffective, periodontal changes and calculus buildup occur; for example, the deposition of calculus on the canine teeth of mature geldings and stallions is common. routine dental prophylaxis forms an important component of maintaining normal occlusal contact, and for this reason one should remove arcade irregularities that result in enamel point formation on the buccal edges of the maxillary cheek teeth and the lingual edges of the mandibular cheek teeth. one should remove these edges annually in horses that are at grass and twice yearly in young horses, aged horses, and stabled horses. horses at grass have been shown to have a greater range of occlusal contact and therefore better periodontal hygiene than stabled horses. in stabled horses the range of occlusal contact is narrower and the formation of enamel points occurs more frequently with subsequent buccal ulceration and the initiation of a cycle of altered occlusal contact and hence irregular arcade formation. this process leads to severe forms of periodontal disease and wave mouth formation. periodontal disease occurs with abnormal occlusal contact and initiation of the cycle of irregular wear and abnormal contact. such changes progress to loss of alveolar bone, gross periodontal sepsis, and loss of tooth support. in this sense periodontal disease truly is the scourge of the equine mouth and results in tooth loss. 13 palatine clefts may result from an inherited defect and are caused by failure of the transverse palatal folds to fuse in the oral cavity. harelip accompanies few palatine clefts in the horse. the degree of palatine clefting depends on the stage at which interruption in the fusion of the 852 part ii disorders of specific body systems caries of cementum occurs, that is, secondary inflammatory disease and acid necrosis of the cementum, apical osteitis may develop. pulpitis is key to the pathogenesis of dental decay in the horse. the initiation of inflammatory pulp changes may be a sequela to dental impaction or dental caries or may result from fracture of a tooth. if the onset of the inflammatory process is slow, then formation of secondary dentin within the pulp chambers may protect the pulp and the tooth. secondary dentin formation occurs from stimulation of odontoblasts within the pulp chamber. such changes are the normal process of protection during dental wear and attrition as crown substances wear away and the reserve crown comes into wear. in acute disease, however, this defense mechanism is ineffective, and the changes that occur and that are sequelae to pulpitis reflect the location of each affected tooth. for example, pulpitis and apical osteitis of the third mandibular cheek tooth most commonly results in the development of a mandibular dental fistula. pulpitis of the third maxillary cheek tooth, however, results in an inflammatory disease within the rostral maxillary sinus and in development of chronic maxillary sinus empyema (figure 13.8-1) . oblique radiographs greatly assist the diagnosis of dental decay by demonstrating sinus tract formation, sequestration of bone, mandibular osteitis, hyperplasia of cementum, and new bone formation (so-called alveolar periosteitis). 12 the management of dental decay in the horse usually involves surgical extraction of the diseased tooth. in some palatopalatal folds occurs. toxic or teratogenic effects are documented in other species, but little data are available in the horse. in recent years, treatment for repair of uncomplicated palatine defects has been recommended but prognosis is generally poor because of the considerable nursing care required and the high incidence of surgical failures. one should emphasize early surgery and the use of mandibular symphysiotomy in affording surgical exposure. the combination of mandibular symphysiotomy and transhyoid pharyngotomy to approach the caudal margins of the soft palate affords surgical access, and one can construct mucosal flaps to repair the defects. however, the incidence of surgical breakdown is high, and healing by first intention is the exception rather than the rule. a recent surgical report documented the successful closure of a median cleft of the lower lip and mandible in a donkey. 14 foals born with a severely deviated premaxilla and palate have a wry nose. one can achieve surgical correction of the deviated premaxilla by submucosal division of the premaxilla across the nose at the line of the first cheek tooth. circumstantial evidence indicates that such a defect has a genetic cause, and the defect occurs most frequently in the arabian breed. other developmental abnormalities are subepiglottic cysts resulting from cystic distortion of remnants of the thyroglossal duct, which may cause dyspnea and choking in foals. surgical removal of these cysts results in normal function. the most significant developmental defect of dental origin is a maxilla that is longer than the mandible, that is, the horse is parrot-mouthed. an overbite of 2 cm in the incisor arcade may be present in a horse with a mismatch of less than 1 cm between the first upper and lower cheek teeth. parrot mouth and monkey or sow mouth are thought to be inherited conditions. some correction of minor incisor malocclusion occurs up to 5 years of age. recognition and detection of parrot mouth are important in the examination of potential breeding stock. surgical attempts to inhibit overgrowth of the premaxilla by wiring or by the application of dental bite plate procedures have been documented in recent years. 15 as has been indicated, the horse is by nature a curious animal and uses its lips as a means of exploring a variety of objects. wounds of the lips, incisive bone, and the mandibular incisor area occur commonly in the horse and usually result from the horse getting the lips, jaw, or teeth caught in feeding buckets, in fence posts, or in halters or having a segment of tongue encircled with hair in tail chewing. as the horse panics and pulls away from its oral entrapment, considerable trauma can occur to the lips, teeth, and gums. most wounds repair satisfactorily, provided one finds them early and observes the basic principles of wound hygiene, excision of necrotic tissue, and wound closure. one must ensure that oral mucosal defects are closed and that effective oral seals are made before external wounds are closed. in some cases, offering specially constructed diets or even feeding the horse by nasogastric tube or esophagostomy during the healing processes may be necessary. foreign body penetration of the tongue, cheek, or palate has been reported in grazing and browsing horses and in particular in horses that have certain hay sources that contain desiccated barley awns or yellow bristle grass. 16 other plant material and grass awns also occasionally may penetrate the tongue, gingiva, or cheek, causing inflammation or abscesses. ulcerative stomatitis also results from the toxicity of phenylbutazone therapy. 17 vesicular stomatitis is a highly contagious viral blistering disease described in more detail elsewhere. treatment of glossitis and stomatitis primarily aims at removing the inciting cause. actinobacillus lignieresii, the causative agent of actinobacillosis, has been isolated and identified from ulcers on the free border of the soft palate and oral and laryngeal granulomata. the bacterium also was reported in a sublingual caruncle in a horse with a greatly swollen tongue. 18 therapy with 150 ml of 20% sodium iodide and 5 g of ampicillin every 8 to 12 hours effected a clinical cure. saliva is important for lubricating and softening food material. the horse has paired parotid, mandibular, and polystomatic sublingual salivary glands. the parotid gland is the largest of the salivary glands in the horse and is situated in the space between the ramus of the mandible and the wing of the atlas. the parotid duct is formed at the ventral part of the gland near the facial crest by the union of three or four smaller ducts. the duct leaves the gland above the linguofacial vein, crosses the tendon of the sternocephalicus muscle, and enters the mouth obliquely in the cheek opposite the third upper cheek tooth. the parotic duct orifice is small, but some dilation of the duct and a circular mucous fold (the parotid papillae) exist at this point. the mandibular gland is smaller than the parotid gland and extends from the atlantal fossa to the basihyoid bone. for the most part, the mandibular gland is covered by the parotid gland and by the lower jaw. the mandibular duct is formed by union of a number of small duct radicles that emerge along the concave edge of the gland and run rostral to the border of the mouth opposite the canine tooth. the orifice is at the end of a sublingual caruncle. the mandibular gland possesses serous, mucous, and mixed alveolar glandular components. the parotid gland is a compound alveolar serous gland. the parotid salivary gland can secrete saliva to yield rates of 50 ml/min, and a total daily parotid secretion can be as much as 12 l in a 500-kg horse. parotid secretion only occurs during mastication, and administration of atropine or anesthesia of the oral mucosa can block secretion. parotid saliva is hypotonic compared with plasma, but at high rates of flow, concentrations of sodium, chloride, and bicarbonate ions increase. parotid saliva of the horse has a high concentration of calcium, and occasionally calculi (sialoliths) form within the duct radicles of the parotid salivary gland. 19 congenital parotid duct atresia, acquired stricture from trauma to the duct, or obstruction by plant material (sticks or foxtails and other seeds) also may occur. the clinical signs of sialolithiasis or other forms of ductule obstruction include a fluid swelling in the form of a mucocele proximal to the stone and occasionally inflammation of the parotid gland. ultrasonography is useful to diagnose salivary mucoceles and to detect foreign bodies or sialoliths. measurement of electrolyte concentrations in aspirates from suspected mucoceles might be helpful to distinguish them from hematomas. salivary potassium and calcium concentrations are higher than plasma. treatment may require surgical removal of the stone or plant material in the case of sialolithiasis or foreign body obstructions. other causes of obstruction may require resection of the affected portion of the duct or chemical ablation of the gland. 20 primary sialoadenitis is unusual but can occur in one or both glands. the condition is painful and may be associated with a fever and anorexia. secondary sialoadenitis is more common and usually is associated with trauma. infectious sialoadenitis from corynebacterium pseudotuberculosis 21 or other bacterial pathogens also may occur. diagnosis is by physical examination and by finding an enlarged edematous parotid gland tissue on ultrasonographic examination. culture and cytologic examination of aspirates may be useful for diagnostic purposes. treatment in usually palliative, consisting of nonsteroidal antiinflammatory drugs. appropriate antibiotic therapy is indicated as directed by culture and sensitivity results. chemical irritation, glossitis, stomatitis, or other causes of prepharyngeal dysphagia cause ptyalism or excessive salivation in horses. specific therapy for the ptyalism usually is not required as long as salivary losses are not excessive, resulting in dehydration and electrolyte imbalances. ingestion of the fungal toxin slaframine also causes hypersalivation in horses. 22 the fungus rhizoctonia leguminicola, which produces slaframine, causes black patch disease in red clover. slaframine is a parasympathomimetic compound that stimulates exocrine secretion in the parotid gland. slaframine toxicosis most commonly occurs in the spring or early summer and rarely requires treatment other than removal from the pasture. mowing removes the source in most cases because regrowth in pastures often has less fungal contamination. 23 15. the esophagus has no digestive or absorptive functions and serves as a conduit to the stomach for food, water, and salivary secretions. the esophageal mucosa is a keratinized stratified squamous epithelium. 1 the submucosa contains elastic fibers that contribute to the longitudinal folds of the esophagus and confer elasticity to the esophageal wall. a transition occurs in the muscle type composing the tunica muscularis from striated skeletal muscle in the proximal two thirds of the esophagus to smooth muscle in the distal third. in the proximal esophagus the skeletal muscle layers spiral across one another at angles. within the smooth muscle layers of the distal esophagus the outer layer becomes more longitudinal, whereas the inner layer thickens and becomes circular. the wall of the terminal esophagus can be 1 to 2 cm thick. deep cervical fascia, pleura, and peritoneum contribute to the thin fibrous tunica adventitia of the esophagus. motor innervation to the striated skeletal muscle of the esophagus includes the pharyngeal and esophageal branches of the vagus nerve, which originate in the nucleus ambiguus of the medulla oblongata. parasympathetic fibers of the vagus nerve supply the smooth muscle of the distal esophagus. sympathetic innervation of the esophagus is minimal. passage of ingesta through the esophagus can be considered part of the swallowing process, which consists of oral, pharyngeal, and esophageal stages. the oral stage is voluntary and involves transport of the food bolus from the mouth into the oropharynx. during the involuntary pharyngeal stage the food bolus is forced through the momentarily relaxed upper esophageal sphincter by simultaneous contractions of the pharyngeal muscles. in the esophageal phase of swallowing the upper esophageal sphincter closes immediately, the lower esophageal sphincter opens, and esophageal peristalsis propels the bolus into the stomach. 2 unlike a food bolus, liquids do not require peristalsis to reach the lower esophageal sphincter and may precede the food bolus during swallowing. the upper esophageal sphincter prevents esophagopharyngeal reflux during swallowing and air distention of the esophagus during inspiration. upper esophageal pressure increases in response to pressure from a food bolus and to increased intraluminal acidity, as would occur with gastroesophageal reflux. the lower esophageal sphincter is a smooth muscle located at the gastroesophageal junction that is morphologically ill defined but forms an effective functional barrier. 2 normally the lower esophageal sphincter is closed in response to gastric distention to restrict gastroesophageal reflux. relaxation of the lower esophageal sphincter permits passage of ingested material from the esophagus to the stomach. distention of the stomach with ingesta mechanically constricts the lower esophageal sphincter. gastric distention also triggers a the esophagus is a musculomembranous tube that originates from the pharynx dorsal to the larynx and terminates at the cardia of the stomach. 1 in adult thoroughbred horses the esophagus is approximately 120 cm long. the cervical portion is approximately 70 cm long; the thoracic portion, approximately 50 cm long; and the short abdominal portion, only approximately 2 cm long. the cervical esophagus generally lies dorsal and to the left of the trachea in the cervical region. in the thorax the esophagus courses through the mediastinum lying dorsal to the trachea and crosses to the right of the aortic arch dorsal to the heart base. impactions. intramural causes of esophageal obstruction include tumors (squamous cell carcinoma), strictures, diverticula, and cysts. [3] [4] [5] [6] [7] [8] [9] [10] mediastinal or cervical masses (tumors or abscesses) may cause extramural obstructions. congenital anomalies are covered in detail later. the clinician must perform a thorough physical examination, including complete oral and neurologic examination, to help rule out causes of dysphagia and nasal discharge other than esophageal obstruction. the clinical signs associated with esophageal obstructions are related primarily to regurgitation of food, water, and saliva caused by esophageal (postpharyngeal) dysphagia. 11 horses with esophageal obstruction are often anxious and stand with their neck extended. one may note gagging or retching, particularly with acute proximal obstructions. bilateral frothy nasal discharge containing saliva, water, and food material; coughing; odynophagia; and ptyalism are characteristic clinical signs, the severity of which varies with the degree and location of the obstruction. distention in the jugular furrow may be evident at the site of obstruction. one may observe other clinical signs related to regurgitation of saliva, water, and food material, such as dehydration, electrolyte or acid-base imbalances, weight loss, and aspiration pneumonia. in extreme cases, pressure necrosis from the impaction or trauma to the esophagus may cause esophageal rupture. if the rupture is in the cervical esophagus, crepitus or cellulitis may be evident along with signs of systemic inflammation. thoracic auscultation is important to determine whether aspiration pneumonia is present. intrathoracic esophageal rupture may result in pleuritis and its associated clinical signs. passage of a nasogastric tube is an effective way to detect and localize an obstruction but provides little information about the nature of the obstruction or the condition of the esophagus. the most direct method for diagnosis of esophageal obstructions is endoscopic examination. most cases of esophageal obstruction occur at sites of natural narrowing of the esophageal lumen, such as the cervical esophagus, the thoracic inlet, base of the heart, or the terminal esophagus, thus one may need an endoscope longer than 1 m for complete evaluation. endoscopic evaluation is useful before relief of an impaction to localize the obstruction and to investigate the nature of the impaction if one suspects a foreign body. foreign bodies may be retrievable via transendoscopic tethering. 12 one can obtain critical diagnostic and prognostic information following resolution of the impaction. assessing the affected esophagus for mucosal ulceration, rupture, masses, strictures, diverticula, and signs of functional abnormalities is important (figure 13.9-1) . ultrasonography of the cervical region is useful not only to confirm a cervical esophageal impaction but also 856 part ii disorders of specific body systems vagal reflex that increases lower esophageal sphincter tone, a safety mechanism against gastroesophageal reflux. the mechanical and vagal mechanisms that promote lower esophageal sphincter tone prevent spontaneous decompression of the stomach, which along with a lack of a vomiting reflex in the horse, increases the risk of gastric rupture during episodes of severe distention. esophageal obstruction has many causes (table 13 .9-1) and most often is manifested clinically by impaction of food material and resulting esophageal dysphagia. esophageal obstruction may be caused by primary impactions (simple choke) of roughage, particularly leafy alfalfa hay, coarse grass hay, bedding, and even grass. prior esophageal trauma or poor mastication caused by dental abnormalities may predispose horses to primary esophageal impaction. 3 wolfing or gulping food may precipitate primary impactions, particularly if the horse is exhausted or mildly dehydrated after a long ride or is weakened from chronic debilitation. impactions also may result from disorders that physically impede the passage of food material and fluid by narrowing the luminal diameter, reduce the compliance of the esophageal wall, or alter the conformation of the esophageal wall such that food material accumulates in a pocket or diverticulum. foreign bodies, intra-or extramural masses, or acquired or congenital anomalies cause these so-called secondary to provide critical information about the location and extent of the impaction and esophageal wall thickness and integrity. ultrasonography may provide information about the cause. 13 radiographic assessment of the esophagus can confirm the presence of esophageal obstruction in cases in which one cannot view the affected area adequately using endoscopy. one can detect impacted food material in the esophagus by a typical granular pattern and often can observe gas accumulation proximal to the obstruction. air or barium contrast radiographic studies are most useful for evaluating the esophagus following relief of the impaction if one suspects a stricture. one often can detect esophageal dilation, diverticula, rupture, functional disorder (megaesophagus), or luminal narrowing caused by extraluminal compression more easily using contrast radiographic studies instead of endoscopy ( figure 13 .9-2). 14-16 one should take care when interpreting radiographic studies in sedated horses, particularly after passage of a nasogastric tube or other esophageal manipulations that may contribute to esophageal dilation. 17 the primary goal of treatment for esophageal impaction is to relieve the obstruction. parenteral administration of acepromazine (0.05 mg/kg intravenously), xylazine (0.25 to 0.5 mg/kg intravenously) or detomidine (0.01 to 0.02 mg/kg intravenously), oxytocin (0.11 to 0.22 iu/kg intramuscularly), and/or esophageal instillation of lidocaine (30 to 60 ml of 1% lidocaine) may reduce esophageal spasms caused by pain or may decrease esophageal tone. [17] [18] [19] [20] some clinicians advocate parasympatholytic drugs such as atropine (0.02 mg/kg intravenously) to reduce salivary secretions and lessen the risk of aspiration. however, undesirable effects of atropine including excessive drying of the impaction and inhibition of distal gastrointestinal motility may preclude its use. resolution of an impaction may require physical dispersal of the material. 18 one can use a nasogastric tube to displace the impacted material along with external massage if the obstruction is in the cervical region. often, carefully lavaging the esophagus with water via an uncuffed or a cuffed nasogastric tube while the head is lowered is necessary to aid in breaking up the impaction. some clinicians advocate a dual tube method whereby a tube is placed through each nasal passage into the esophagus for ingress and egress of the lavage fluid. because of the risk of aspiration of water and food material, esophageal lavage sometimes is done under general anesthesia with a cuffed nasotracheal tube. in refractory cases, intravenous administration of isotonic fluid containing 0.9% nacl and kcl (10 to 20 meq/l) for 24 hours at a rate of 50 to 100 ml/kg/ day along with esophageal relaxants such as oxytocin may promote hydration and softening of the impaction and help prevent or alleviate any electrolyte or acid-base imbalances resulting from salivary losses of chloride, sodium, and potassium. 21 one should note that the effects of oxytocin on esophageal tone occur in the proximal two thirds of the esophagus and may not be effective for mucosa has recovered as assessed by endoscopy, one can feed the horse soft food (moistened pellets and bran mashes). one can return the patient gradually to a highquality roughage diet over 7 to 21 days, depending on the degree of esophageal damage induced by the impaction and the nature of any underlying disease. the prognosis for survival is good (78%), but some horses may require permanent dietary modification if persistent chronic obstruction is a problem. 3 aspiration pneumonia and perforation are potential complications of severe or prolonged esophageal obstructions. if aspiration is suspected, administration of broad-spectrum antibiotics that are effective against gram-positive and gram-negative organisms, including metronidazole (20 mg/kg orally every 8 hours) for anaerobes is advisable. a subsequent section describes treatment of esophageal perforation or rupture. esophagitis refers to a clinical syndrome of esophageal inflammation that may or may not be ulcerative. the major protective mechanisms of the esophageal mucosa include salivary and food material buffers, normal peristaltic motility, and the barrier formed by the gastroesophageal sphincter. reflux esophagitis is caused by repeated episodes of gastric fluid regurgitation into the distal esophagus and subsequent chemical injury to the mucosa ( figure 13 distal obstructions. 19, 20 rarely, esophageal obstruction ultimately may require esophagotomy to relieve the impaction. one must enforce strict restriction of food and water, including access to bedding material, until the obstruction is resolved and the esophagus has regained function. systemic effects of dysphagia associated with esophageal impaction include dehydration, hyponatremia, hypochloremia, and metabolic alkalosis from prolonged loss of salivary free water and electrolytes. 21 if the duration of a complete esophageal obstruction is 48 hours or longer, one should correct dehydration and electrolyte and acid-base imbalances. one can restore fluid and electrolyte balance with oral electrolyte solutions if the patient is less than 6% to 7% dehydrated and the esophageal obstruction is resolved. horses that are greater than 6% to 7% dehydrated or those that have a refractory obstruction or moderate to severe electrolyte imbalances may require intravenous fluid therapy with solutions containing 0.9% nacl and kcl (10 to 20 meq/l). one should perform esophageal endoscopy after relief of the impaction to determine whether any complications of the impaction have developed or if a primary cause of the obstruction is present. endoscopic examination is critical to determine the postobstruction treatment plan and for follow-up evaluation of esophageal healing. one should reevaluate the horse every 2 to 4 weeks following resolution of the impaction if one notes esophageal dilation or mucosal injury. additional evaluation via radiography may be warranted to assess motility and transit times. dilation proximal to the site of obstruction, mucosal injury from trauma, stricture formation, formation of a diverticulum, megaesophagus, and esophagitis are sequelae to esophageal obstruction that predispose patients to reobstruction. the rate of reobstruction may be as high as 37%. depending on the duration of the obstruction and the degree of trauma or dilation, the risk of reobstruction is high for 24 to 48 hours or longer, thus one should withhold food for at least 24 to 48 hours after resolution of the obstruction. sucralfate (20 mg/kg orally every 6 hours) may hasten healing if esophageal ulceration is evident, but the efficacy of sucralfate for this purpose is not established. some clinicians suggest that administration of a nonsteroidal antiinflammatory drug (nsaid) such as flunixin meglumine (1 mg/kg orally or intravenously every 12 hours) or phenylbutazone (1 to 2 mg/kg orally or intravenously every 12 to 24 hours) for 2 to 4 weeks after resolution of the impaction may reduce the development of strictures. judicious use of nsaids is recommended to prevent nsaid-induced worsening of esophageal mucosal injury. one should avoid orally administered nsaids if esophagitis is present. after 48 to 72 hours or when the esophageal duodenal strictures caused by chronic ulceration commonly have reflux esophagitis. diagnosis requires endoscopic examination of the esophagus. one may note diffuse, patchy, linear, or coalescing erosion or ulcerations (see figures 13.9-3 and 13.9-4). one also may observe significant edema or hyperemia. determining whether an underlying disease, such as infection, neoplasia, esophageal strictures, or diverticula, is present is important. in addition, one must examine the stomach to determine whether the esophagitis is associated with gastritis, gastric obstruction, or gastric ulcer disease. contrast radiography may be helpful to detect esophageal ulceration and is useful to assess esophageal motility and transit time. 14 the principles of therapy for reflux esophagitis include control of gastric acidity, mucosal protection, and correction of any underlying disorder contributing to gastroesophageal reflux. reduction of gastric acid production with h 2 histamine receptor blockers such as ranitidine or proton pump antagonists such as omeprazole is critical for resolution of the esophagitis. some clinicians advocate using sucralfate to promote healing of ulcerated esophageal mucosa. however, the ability of sucralfate to bind ulcerated esophageal mucosa is not proven, nor is the efficacy of sucralfate for hastening esophageal ulcer healing. horses with reflux esophagitis following delayed gastric outflow caused by gastroduodenal ulcer disease, gastric paresis, or proximal enteritis may benefit from prokinetic drugs that act on the proximal gastrointestinal tract. metoclopramide (0.02 to 0.1 mg/kg subcutaneously every 4 to 12 hours) reduces gastroesophageal reflux by increasing lower esophageal sphincter tone, gastric emptying, and gastroduodenal coordination. one should exercise caution when giving metoclopramide to horses because they are prone to extrapyramidal neurologic side effects of the drug. cholinergic drugs such as bethanechol (0.025 to 0.035 mg/kg subcutaneously every 4 to 24 hours or 0.035 to 0.045 mg/kg orally every 6 to 8 hours) may improve gastric emptying and are effective for treating reflux esophagitis. for esophagitis from trauma or pressure injury after esophageal impaction, judicious use of nsaids may be warranted to reduce esophageal inflammation and pain. dietary modification may be necessary for patients with esophagitis, depending on the degree of ulceration or if motility is impaired. one should feed horses with mild esophagitis frequent small meals of moistened pellets and fresh grass. severe esophagitis may necessitate withholding food and complete esophageal rest for several days. although the prognosis for esophagitis is good in the absence of underlying disease, the risk of esophagus is delayed, such as in functional disorders of the esophagus. like ulceration of the squamous portion of the stomach in horses, gastric acid and bile salt chemical injury is a major mechanism of esophageal squamous epithelial ulceration. 22, 23 reflux esophagitis may occur along with gastric ulcer disease, motility disorders, increased gastric volume from gastric outflow obstructions, gastric paresis, intestinal ileus, or impaired lower esophageal sphincter function. 7, 22 other causes of esophagitis in horses include trauma (foreign bodies, food impactions, nasogastric tubes), infection (mural abscesses), or chemical injury (pharmaceuticals, cantharidin) ( figure 13 .9-4). [24] [25] [26] [27] the clinical signs of esophagitis are nonspecific and similar to esophageal obstruction and gastric ulcer diseases. gagging or discomfort when swallowing may be evident, and hypersalivation and bruxism are signs of esophageal pain. esophageal (postpharyngeal) dysphagia may be evident. one may note partial or complete anorexia such that horses with chronic esophagitis may have significant weight loss. esophageal hypomotility dysfunction caused by the inflammatory process may result in esophageal impaction. clinical signs of underlying diseases that predispose to esophagitis may predominate or mask the signs of esophagitis. horses with gastrointestinal motility disorders such as proximal enteritis or gastric outflow obstruction are at a high risk of developing reflux esophagitis because of the presence of gastric acid and bile salts in the fluid reflux. foals with gastric, pyloric, or stricture formation is high if severe circumferential or coalescing ulcerations are present. esophagitis from severe trauma or infection may be prone to stricture formation. motility dysfunction of the equine esophagus is caused most commonly by hypomotility resulting in esophageal dilation (ectasia) or megaesophagus. although megaesophagus in horses most commonly is acquired, reports indicate idiopathic megaesophagus in young horses may be congenital. [28] [29] [30] [31] acquired megaesophagus in horses may be a consequence of chronic or recurrent esophageal obstruction. 3, 7 esophageal impactions of a short duration cause a proximal dilation of the esophagus that is generally reversible. 14 however, if the duration of the obstruction is long enough, the motility of the esophagus proximal to the site of obstruction may be impaired permanently. other causes of acquired megaesophagus include extraesophageal obstruction by tumors or abscesses, pleuropneumonia, and vascular ring anomalies. 3, 8 acquired megaesophagus also may result from neurologic, neuromuscular, and muscular disorders. neurologic diseases that cause vagal neuropathy-such as equine protozoal myeloencephalitis, equine herpesvirus myeloencephalitis, and idiopathic vagal neuropathy-have been associated with megaesophagus in horses. pleuropneumonia may be associated with a vagal neuropathy resulting in megaesophagus. megaesophagus is an early sign of equine dysautonomia 32 and may be observable in patients with botulism. myasthenia gravis is a well-known cause of megaesophagus in nonequine species but has not been reported in horses. also in other species, electrolyte disorders, cachexia, primary myopathies, myositis, and addison's disease may affect esophageal motility but have not been associated with megaesophagus in horses. one can induce iatrogenic megaesophagus by the α 2adrenergic agonist detomidine, but this is transient and reversible. 17, 33 nonetheless, the use of this drug may complicate clinical evaluation of esophageal motility. esophageal inflammation, particularly reflux esophagitis, may affect motility and cause megaesophagus. however, because esophageal hypomotility affects the tone and function of the lower esophageal sphincter, reflux esophagitis also may be a complication of a primary functional disorder. thus assessing esophageal motility in horses with esophagitis that is not responding appropriately to treatment is important. along with a complete physical examination one should include a careful neurologic examination to help rule out primary neurologic causes of megaesophagus. because esophageal hypomotility is a functional obstruction, the clinical signs of esophageal hypomotility or megaesophagus are similar to esophageal obstruction. unlike mechanical obstruction the onset of clinical signs is insidious rather than acute. the clinical signs include those associated with esophageal dysphagia. 7, 8, [28] [29] [30] [31] the cervical esophagus may be dilated enough to be evident externally. weight loss is a common sign. signs attributable to an underlying disease may be evident. diagnosis of esophageal hypomotility requires transit studies. one can measure the transit time of a bolus from the cervical esophagus to the stomach by fluoroscopy or contrast radiography. 14,32 other signs of esophageal hypomotility and megaesophagus include pooling of contrast material and an absence of peristaltic constrictions. 7,14,28,32 endoscopy may reveal a dilated esophagus and an absence of peristaltic waves. 7,28 one may observe evidence of underlying disease causing obstruction or esophageal dilation. 3, 7 one should evaluate the esophagus for evidence of esophagitis that is causing esophageal motility dysfunction or is a result of impaired esophageal clearance of gastric fluid. esophageal manometry may be useful to document abnormal postdeglutition contraction pressures, contraction time, and propagation times but is not often available for routine clinical application. 28, 34 one should perform other diagnostic tests such as a complete blood count and chemistry to help determine a possible underlying cause. cerebral spinal fluid analysis may be indicated to rule out neurologic disorders. specialized testing such as electromyography to detect neuromuscular disorders may also be indicated. treatment of esophageal hypomotility or megaesophagus should aim at treating the underlying cause. dietary modification should aim at improving esophageal transit of food. one should feed the horse slurries of pellets, and feeding from an elevated position to promote transit may be beneficial. metoclopramide or bethanechol may benefit patients with reflux esophagitis associated with megaesophagus by increasing lower esophageal tone, gastric emptying, and reducing gastroesophageal reflux. the prognosis depends on the underlying cause and the degree of dilation. although many cases of megaesophagus associated with reflux esophagitis respond well to treatment, many other forms of megaesophagus including congenital megaesophagus have a poor prognosis. strictures most commonly are caused by pressure necrosis from esophageal impactions that induce circumferential erosion or ulceration of the esophageal mucosa, although esophageal injury caused by oral administration of corrosive medicinal agents and trauma to the neck may also result in stricture formation. 35 congenital strictures also have been reported. 36 strictures caused by mucosal and submucosal trauma are termed esophageal webs or rings. strictures may also originate in the muscular layers and adventitia of the esophagus (mural strictures) or in all of the layers of the esophagus (annular stenosis). 36, 37 horses with these lesions have a presentation similar to those with simple obstructions, because strictures result in partial obstruction and impaction of food material in the lumen. one can detect esophageal webs or rings with endoscopy (see figure 13 .9-1), whereas identification of mural strictures or annular stenosis may require a double-contrast esophogram (see figure 13 .9-2). in a retrospective study of horses with esophageal stricture following simple obstruction, maximal reduction in esophageal lumen diameter occurred within 30 days of the esophageal obstruction. although surgery has been used to relieve such strictures, initial medical management is warranted because strictures may resolve with conservative therapy, and the esophagus continues to remodel for up to 60 days following ulceration. in one report, seven horses with esophageal obstruction-induced stricture were treated conservatively by feeding a slurry diet and administering antiinflammatory and antimicrobial medications, and five of seven were clinically normal within 60 days. 35 one of the five successfully treated horses had a 10-cm area of circumferential ulceration, suggesting that the potential exists for extensive mucosal injury to resolve without permanent stricture formation. if resolution of strictures within 60 days is insufficient, one should investigate other methods to increase esophageal diameter. bougienage has been used successfully in small animal patients and human beings. the technique involves passage of a tubular dilatable instrument down the esophagus and stretching of the stricture. one may perform the technique by passing a nasogastric tube with an inflatable cuff. however, one has to perform the procedure frequently to have any success, and horses do not tolerate it well. 36 alternatively, a number of surgical techniques have been used to resolve strictures, including resection and anastomosis, 38,39 temporary esophagostomy with fenestration of the stricture, 37 esophagomyotomy for strictures of the muscularis and adventitia, 40, 41 or patch grafting with local musculature. 42 however, such surgeries are fraught with complications, largely because of the propensity of the traumatized esophagus to restricture. 3, 35 the esophagus lacks a serosal layer and does not rapidly form a fibrin seal as does the remainder of the intestinal tract, so anastomoses tend to leak. 38 in addition, tension on the esophagus during swallowing and movement of the neck impairs healing of anastomoses. 37, 39 in spite of these difficulties, the long-term prognosis for horses with chronic esophageal strictures treated surgically is better than for those treated nonsurgically. 3 two types of diverticula are traction (true) diverticula and pulsion (false) diverticula. traction diverticula result from wounding and subsequent contraction of periesophageal tissues, with resultant tenting of the wall of the esophagus. pulsion diverticula arise from protrusion of esophageal mucosa through defects in the muscular wall of the esophagus and usually result from trauma or acute changes in intraluminal pressure. 36 traction diverticula appear as a dilation with a broad neck on contrast esophagography, whereas pulsion diverticula typically have a flask shape with a small neck on an esophagram (see figure 13 .9-2). 10, 43 although traction diverticula are usually asymptomatic and of little clinical significance, pulsion diverticula may fill with feed material, ultimately leading to esophageal obstruction. [43] [44] [45] a movable mass in the midcervical region may be noticeable before onset of complete obstruction. 36 pulsion diverticula may be corrected surgically by inverting or resecting prolapsed mucosa and closing the defect in the wall of the esophagus. 10, 43, 45 inversion of excessive mucosa may reduce the diameter of the esophageal lumen and predispose horses to esophageal obstruction and therefore should be reserved for small diverticula. 10 congenital disorders of the esophagus are rare. reported congenital abnormalities include congenital stenosis, 46 persistent right aortic arch, 8 esophageal duplication cysts, [47] [48] [49] intramural inclusion cysts, 9, 50 and idiopathic megaesophagus. 28, 30, 31 in the one report of congenital stenosis, double-contrast radiography revealed concentric narrowing of the thoracic esophagus in the absence of any vascular abnormalities at the base of the heart. successful treatment included having the foal stand with the forelimbs elevated off the ground following each feeding. 46 persistent right aortic arch is a congenital anomaly in which the right fourth aortic arch becomes the definitive aorta instead of the left aortic arch, which results in constriction of the esophagus by the ligamentum arteriosum as it extends between the anomalous right aorta and the left pulmonary artery. clinical signs may include those associated with esophageal (postpharyngeal) dysphagia, drooling, and distention of the cervical esophagus resulting from partial obstruction of the thoracic esophagus. 8, 51 endoscopic examination typically reveals dilation of the esophagus cranial to the obstruction section 13.9 esophageal diseases with evidence of diffuse esophagitis. successful surgical treatment of persistent right aortic arch has been reported in one foal. 51 esophageal duplication cysts and intramural inclusion cysts cause typical signs of esophageal obstruction, including salivation, esophageal dysphagia, and swelling of the cervical esophagus as the cysts enlarge. 47, 49, 50 such signs can make them difficult to differentiate from other forms of esophageal obstruction (choke). endoscopic examination may reveal compression of the esophageal lumen and communication with the esophageal lumen if it exists. ultrasonographic examination may be the most useful method of antemortem diagnosis if the cyst is in the cervical esophagus. examination of an aspirate of the mass may aid in the diagnosis by revealing the presence of keratinized squamous cells. 47, 50 surgical treatments have included complete surgical resection and surgical marsupialization. 47, 49, 50 the latter appears to be more successful and results in fewer complications. 49, 50 complications of surgical resection have included laryngeal hemiplegia following surgical trauma to the recurrent laryngeal nerve in the region of the esophagus and esophageal fistula formation. 50 perforation typically occurs in the cervical region in response to external trauma, necrosis of the esophageal wall caused by a food impaction, or rupture of an esophageal lesion such as an impacted diverticulum. the esophagus is particularly vulnerable to external trauma in the distal third of the neck because only a thin layer of muscle covers it at this point. 52 iatrogenic perforation may occur in response to excessive force with a stomach tube against an obstruction or a compromised region of the esophagus. 24 esophageal perforations may be open or closed and tend to cause extensive cellulitis and necrosis of tissues surrounding the wound because of drainage of saliva and feed material within fascial planes. systemic inflammation associated with endotoxemia from septic cellulitis may occur. closed perforations of the esophagus are particularly troublesome because food material, water, saliva, and air may migrate to the mediastinum and pleural space via fascial planes. 24, 52 because of the leakage of air into the tissues surrounding the rupture, extensive subcutaneous and fascial emphysema frequently develops and is usually evident clinically and on cervical radiographs. pneumomediastinum and pneumothorax are potentially fatal complications of esophageal ruptures. treatment should include converting closed perforations to open perforations if possible, 53 extensive debridement and lavage of affected tissues, broadspectrum antibiotics, tetanus prophylaxis, and esophageal rest. the clinician may achieve the latter by placing a feeding tube into the esophagus via the wound. alternatively, one may place a nasogastric tube using a small tube (12-f diameter) . 24 for open perforations, once the wound has granulated and contracted to a small size, one may attempt peroral feeding. 52 extensive loss of saliva via esophageal wounds may lead to hyponatremia and hypochloremia. in addition, transient metabolic acidosis occurs because of salivary bicarbonate loss, followed by progressive metabolic alkalosis. 21 although reports of esophageal wounds healing well by second intention exist, healing takes a prolonged time. 54 in addition, some perforations never completely heal and form permanent esophagocutaneous fistulae that may require surgical correction. the development of esophageal strictures is not common because wounds are usually linear and not circumferential. however, traction diverticula may develop. other complications of esophageal wounds include horner's syndrome and left laryngeal hemiplegia. 52 in a retrospective study on esophageal disorders, only 2 of 11 horses with esophageal perforations survived long-term 3 ; in a report of esophageal trauma following nasogastric intubation, 4 of 5 horses were euthanized. 24 the prognosis is therefore poor in horses with esophageal perforations, largely because of the extent of cellulitis, tissue necrosis, shock, and local wound complications. 14 specialized endoscopic equipment allowing visual inspection of the entire adult equine stomach has become increasingly available to veterinarians in academia and private practice. thus gastric disease in horses recently section 13 .10 diseases of the stomach has gained increasing awareness among veterinarians, owners, and trainers. peptic ulcer disease is defined as erosions or ulcers of any portion of the gastrointestinal tract normally exposed to acid. 1 mucosal damage can include inflammation, erosion (disruption of the superficial mucosa), or ulceration (penetration of the submucosa). in severe cases, fullthickness ulceration can occur, resulting in perforation. the proximal (orad) portion of the equine stomach is lined by stratified squamous mucosa similar to the esophageal lining. the distal (aborad) portion of the stomach is lined with glandular mucosa, and the distinct junction between the two regions is deemed the margo plicatus. ulceration can occur in either or both gastric regions, although different clinical syndromes and pathophysiologic mechanisms apply. as a result, the broad term equine gastric ulcer syndrome (egus) has been used to encompass the wide array of associated clinical syndromes. egus develops in horses of all ages and continues to be of major clinical and economical importance. 2 the prevalence of gastric ulceration has been reported for a variety of breeds and types of horses; however, most current data involve thoroughbreds in race training. the prevalence of squamous ulceration in horses in race training varies from 70% to 94% 3-8 and can be as high as 100% when limited to animals actively racing. 4 in a survey of 50 active show horses, 58% had gastric ulceration, with only 1 horse having ulceration of the glandular fundus. 9 in one large retrospective study (3715 adult horses from 1924 to 1996) evaluating incidence of gastric ulceration identified at necropsy, an overall prevalence of 10.3% was found. the highest prevalence was found in thoroughbreds (including arabians) and standardbred trotters, and cold-blooded horses were affected significantly less. lesions were located most commonly in the squamous mucosa along the margo plicatus, followed by the glandular body, proximal squamous mucosa, and antrum. 10 many studies investigating prevalence of gastric ulceration do not differentiate between squamous and glandular lesions or evaluate only squamous disease. in a recent study in which the gastric antrum and pylorus were evaluated in 162 horses in a hospital setting, 58% had antral or pyloric erosions or ulcerations, 58% had squamous mucosal lesions, and 8% had lesions involving the glandular body. 11 a correlation between the presence or severity of squamous disease and antral/pyloric disease was not identified. the reported prevalence of gastric ulceration in foals varies from 25% to 57%. [12] [13] [14] an imbalance between inciting and protective factors in the mucosal environment can result in ulcer formation. 15, 16 the major intrinsic factors promoting ulcer formation include hydrochloric acid, bile acids, and pepsin, with hydrochloric acid being the predominant factor. various intrinsic factors protect against ulcer formation such as the mucus-bicarbonate layer, maintenance of adequate mucosal blood flow, mucosal prostaglandin e 2 and epidermal growth factor production, and gastroduodenal motility. in human beings, extrinsic ulcerogenic factors include nonsteroidal antiinflammatory drugs, helicobacter pylori, stress, changes in diet, or gastrointestinal disorders, especially those resulting in delayed gastric emptying. 1 in human neonates, physiologic stress associated with a major primary illness seems to be associated strongly with gastric ulcers. 17 many of the other factors mentioned previously are believed to be important in horses, but clear evidence of an infectious agent has not yet been identified in horses or foals with egus. 18, 19 recently, the possibility of helicobacter infection in horses has reemerged with the identification of polymerase chain reaction products from urel, a protongated urea channel unique to gastric-dwelling helicobacter species, in the squamous epithelium of three horses, two of which had squamous erosions. 20 the specific factors involved in injury and the protective mechanisms vary between regions of the proximal gastrointestinal tract. the pathophysiology of squamous mucosal ulceration in the horse appears similar to that in gastroesophageal reflux disease in human beings and ulceration of the nonglandular mucosa in pigs. excess acid exposure is the predominant mechanism responsible for squamous mucosal ulceration, although many details remain unclear. 21 hydrochloric acid is secreted by parietal cells in the gastric glands via a hydrogen-potassium adenosine triphosphatase (h + ,k + -atpase) pump on the luminal side. horses secrete acid continuously, and measured ph of equine gastric contents varies from less than 2 to greater than 6 depending on the dietary state of the horse (fed or fasted). 22,23 a protocol of repeated 24-hour periods of fasting and feeding has been shown to induce squamous erosion and ulceration. 24 because this protocol results in periods of prolonged gastric acidity (ph <2.0) and because concurrent administration of the histamine 2 (h 2 ) receptor antagonist ranitidine reduces lesion severity, the protocol supports the role of acid exposure in the pathogenesis of squamous ulcer disease. several peptides can stimulate or inhibit parietal cell secretion of acid. the predominant stimuli for hydrochloric acid secretion are gastrin, histamine, and acetylcholine via the vagus nerve. 1 g cells release gastrin within the antral mucosa, whereas mast cells and enterochromaffin-like cells release histamine in the gastric gland. histamine binds to type 2 receptors on the parietal cell membrane, causing an increase in cyclic adenosine monophosphate and resulting in phosphorylation of enzymes that activate the proton pump. gastrin and acetylcholine can act via calcium-mediated intracellular pathways and also stimulate histamine release directly. 25 isolated equine parietal cells respond maximally to histamine stimulation and only minimally to carbachol and pentagastrin. 26 gastrin release is controlled primarily by gastrin-releasing peptide, which is stimulated by gastric distention and increased luminal ph, but the interaction between gastrin and histamine has not been elucidated fully in the horse. somatostatin, released by fundic and antral d cells, is the primary inhibitor of gastric acid secretion by parietal cells. the inhibitory effect of somatostatin is primarily paracrine, but plasma levels of somatostatin negatively correlate with gastric luminal acidity. 27 epidermal growth factor, a peptide produced in saliva, also inhibits gastric acid secretion. 28 foals can produce significant amounts of gastric acid by the second day of life, with consistent periods of acidity (ph <2.0) in clinically normal animals. 29, 30 in one study, foals tended to have a high gastric ph at day 1 of age, 30 but in a study of critically ill foals, some foals demonstrated periods of gastric acidity on the first day of life. 31 suckling was associated with an immediate rise in gastric ph, whereas periods of rest in which foals did not suck for more than 20 minutes were associated with prolonged periods of acidity. 29 whereas premature human infants are capable of gastric acid production at 28 weeks of gestation, 32 only 1 of 7 premature foals demonstrated an acidic ph recording in a study of gastric ph profiles in critically ill foals. 31 however, multiple factors likely were involved in critically ill foals of this study, and the true ontogeny of gastric acid production in foals is currently unknown. equine squamous mucosa is thin at birth but becomes hyperplastic and parakeratotic within days. 33 the parallel between decreasing ph and proliferation of squamous epithelium correlates with that observed in other species. 34 the combination of a thin gastric epithelium with a high acid output may leave neonatal foals susceptible to ulcer formation at a young age. in addition, one must remember the difference in normal appearance of the squamous mucosa when interpreting gastric endoscopy in a neonatal population. in esophageal squamous mucosa, intercellular tight junctions and bicarbonate secretion are the major factors involved in protection against acid injury in other species, although squamous bicarbonate secretion had not been documented in the horse. [35] [36] [37] the principal barrier is a glycoconjugate substance secreted by cells in the stratum spinosum, with a contribution from the tight junctions in the stratum corneum. 37 this barrier function is considered weak at best, and thus a functioning lower esophageal sphincter, normal salivary flow, and salivary mucins contribute to the prevention of acid injury in human gastroesophageal reflux disease. in horses a mechanical barrier like the lower esophageal sphincter is not available to protect the gastric squamous mucosa from acid exposure. the normal gastric fill line rests just below the cardia, so only the squamous mucosa along the lesser curvature adjacent to the margo plicatus should receive exposure to acidic gastric contents regularly. not surprisingly, this correlates with the most common location of squamous mucosal ulceration. bile salts and pepsin have been implicated as contributing factors to ulcer disease in many species. in rabbit esophageal mucosa, bile salt absorption occurs and is correlated directly with mucosal barrier disruption. the unconjugated bile salts cholate and deoxycholate have a pk a (negative logarithm of the ionization constant of an acid) of 5 and 5.3, respectively, and therefore cannot remain in solution and cause mucosal damage in the presence of acid. alternatively, the conjugated bile salt taurocholate (pk a 1.9) can cause mucosal injury in the ionized salt form at ph 7 or the un-ionized acid form at ph 1 to 2. 38 in the pig, bile salts or acid alone cause squamous mucosal damage, whereas a combination of the two result in extensive damage in vitro. 39 in the horse a similar synergistically damaging effect was found with the addition of bile salts and acid (ph 2.5) to stratified squamous mucosa in vitro in one study. 40 in addition, the investigators were able to document levels of bile salts and acid sufficient to cause mucosal damage in gastric contents within 14 hours of feed deprivation. this is not surprising, given that duodenogastric reflux occurs normally in the horse. 41 in a separate in vitro study of equine squamous mucosa, prolonged exposure to acid alone (ph 1.5) had a damaging effect, and synergism with exposure to a combination of acid and pepsin or taurocholate was not found. 42 the lack of synergism likely is caused by the lower ph used in this study and stresses the importance of acid exposure in squamous ulcer disease. pepsinogens are secreted primarily by chief cells, although secretion by neck cells, cardiac glands, and antral pyloric glands also occurs. 43 in an acidic environment (ph <3.0), pepsinogen is converted to the active pepsin. although the proteolytic activity of pepsin normally is directed toward dietary protein, it also can act on the gastric mucosa. 44 thus acid remains the major contributing factor to squamous mucosal damage, although other factors such as pepsin and bile salts may play an important role as well in the initiation or perpetuation of disease. several mechanisms help protect the glandular mucosa from acid injury. the mucus-bicarbonate layer serves to titrate h + ion from the gastric lumen to co 2 and h 2 o. cellular restitution and prostaglandins of the e series, which enhance mucosal blood flow and secretion of mucus and bicarbonate in the glandular mucosa have not been documented in squamous epithelium. 21, 36 of these mechanisms, mucosal blood flow is likely the most important contributor to overall gastric mucosal health. nitric oxide is a key regulator of mucosal blood flow and prostaglandin synthesis and thus may play a role in mucosal protection. 45 dietary factors also have been implicated in ulcer disease. horses in race training have a high incidence of gastric ulceration and frequently are fed high-concentrate, low-roughage diets. in one study, higher volatile fatty acid (acetic, propionic, and isovaleric acid) concentrations, higher gastric juice ph, and lower number and severity of nonglandular ulceration were documented after feeding an alfalfa hay-grain diet compared with a bromegrass hay diet. 46 however, many factors differed between the diets, such as digestible energy, bulk, crude protein, and mineral content (especially calcium). thus dietary factors represent an important area of further investigation in the pathophysiology of egus, particularly squamous ulceration. the pathophysiologic correlation between exercise and squamous ulcer disease has not yet been defined despite the high prevalence of ulceration in performance horses. preliminary work suggests that gastric compression occurs during treadmill exercise, presumably because of an increase in intraabdominal pressure. 47 such contracture could result in increased acid exposure to the squamous mucosa by raising the fill line of gastric contents. further studies in this laboratory have provided support for this theory by demonstrating a high ph in the proximal stomach, immediately distal to the lower esophageal sphincter, during resting conditions that decreases during treadmill exercise (m. lorenzo-figueras and a.m. merritt, personal communication, 2002) . risk factors associated with gastric ulceration include gender and age, and the reported prevalence of gastric ulcers has increased over time. in one study, ulcers were found more commonly in stallions, and the prevalence of gastric ulceration decreased with age, independent of gender, although this trend was only significant in the population of standardbred trotters. 10 interestingly, the frequency of gastric ulceration increased from less than 6% before 1945 to approximately 18% after 1975. in a study of thoroughbred horses in race training, an increase in squamous ulcer severity was noted in horses 3 years old or older and in those horses that had raced. 4 in the same study, severity of glandular lesions did not change between examinations, and age (>3 years) was the only factor associated with glandular lesion severity. several studies have failed to document a correlation between nonsteroidal antiinflammatory drug (nsaid) administration and naturally occurring ulcer disease. 3, 4, 6, 7, 10 however, nsaid administration is a well-known cause of gastric ulceration under experimental conditions. [48] [49] [50] [51] [52] nsaid-related ulceration typically is described as predominantly glandular, although nonglandular ulceration also can occur by a mechanism that has not yet been characterized fully. nsaids cause a decrease in prostaglandin e 2 synthesis because of inhibition of the cyclooxygenase pathway. therefore a resultant decrease in glandular mucosal protection, most notably via decreased mucosal blood flow and mucus production, is the most likely mechanism of action. in one study, however, phenylbutazone administration resulted in ulceration of the glandular mucosa at the pyloric antrum but did not alter mucosal prostaglandin e 2 concentration significantly. 52 clinical signs typically associated with gastric ulceration in foals include poor appetite, diarrhea, and colic. many foals probably never exhibit clinical signs, and some do not exhibit clinical signs until ulceration is severe or fatal perforation has occurred. glandular ulceration typically is considered the most clinically significant type of disease in this population. the physiologic stress of a concurrent illness has been associated with gastric ulceration in foals. retrospectively, 14 (23%) of 61 foals up to 85 days of age with a clinical disorder were found to have lesions in the gastric glandular mucosa, 13 and prospectively 8 (40%) of 20 foals up to 30 days of age with a clinical disorder had glandular ulceration. 53 by contrast, only 4% to 9% of clinically normal foals examined in endoscopic surveys had lesions observed in the gastric glandular mucosa. 14, 54 critically ill neonatal foals can have a greatly different ph profile compared with that in clinically normal foals, potentially because of alterations in gastric motility and acid secretion. 31 gastric ulceration was not identified in any animals at necropsy in that study; however, ulceration has been documented in a similar population. 12 thus factors other than acid exposure, most notably mucosal blood flow, may play an important role in the stressrelated ulceration in neonates. subjectively, gastric ulceration and rupture in the hospitalized neonatal population occurs less commonly now than in previous reports. advances in overall neonatal care, especially supportive care, likely have contributed to this decline. in suckling foals less than 50 days old, lesions typically originate in the squamous mucosa adjacent to the margo plicatus along the greater curvature. such lesions can occur in foals as young as 2 days of age and have been observed in 50% of foals less than 50 days old. histologic examination of these lesions has revealed disruption of the epithelial layers of the mucosa and a neutrophilic infiltration. another phenomenon that occurs in young foals is the shedding, or desquamation, of squamous epithelium, which appears as flakes or sheets of epithelium. desquamation occurs without ulceration in up to 80% of foals less than 35 days of age, and this process typically is not associated with clinical signs. 13, 14, 54 in older foals, lesions become more prevalent in the squamous mucosa, particularly along the lesser curvature. 53 lesions also are found in the squamous mucosa of the fundus and adjacent to the margo plicatus. these lesions can be severe and often are associated with clinical signs such as diarrhea, poor appetite, and poor growth and body condition. diarrhea is the most frequent sign in symptomatic foals with squamous mucosal lesions and is associated with more diffuse erosion or ulceration of the squamous mucosa than that which occurs in asymptomatic foals. in some foals, poor growth, rough hair coat, a potbelly appearance, or all of those occur along with moderate to severe squamous mucosal ulceration. in horses with severe or diffuse squamous ulceration, bruxism or colic may occur. gastroduodenal ulcer disease occurs almost exclusively in suckling and early weanling foals. clinical signs of duodenal ulceration are similar to those described for gastric ulceration (bruxism, colic, salivation, diarrhea), but the consequences are often more severe. lesions occur primarily in the proximal duodenum and range from diffuse inflammation to severe ulceration. foals with duodenal ulceration often have delayed gastric emptying and may have gastroesophageal reflux. complications can include gastric or duodenal rupture, duodenal stricture, and ascending cholangitis. severe squamous and esophageal ulceration and aspiration pneumonia can occur following gastroesophageal reflux. 15, [55] [56] [57] [58] the gastroduodenal ulcer disease syndrome can occur in outbreaks and most commonly is identified in intensive breeding operations. the cause of duodenal lesions in foals is not known. one theory is that the problem begins with diffuse duodenal inflammation that can coalesce down to a focal area of ulceration (g.d. lester and a.m. merritt, personal communication, 2002) . a temporal relationship between gastroduodenal ulcer disease and rotaviral diarrhea has been suggested, but an infectious cause remains unproven. although lesion location and severity associated with rotaviral infection varies among species, duodenal ulceration has not been reported. 59 clinical signs attributable to egus in older horses vary and classically include anorexia and chronic or intermittent colic of varying severity. 60 many horses with endoscopic evidence of disease may appear to be clinically normal or have vague signs that include decreased consumption of concentrates, postprandial episodes of colic, poor performance or failure to train up to expectations, poorquality hair coat, and decreased condition or failure to thrive. diarrhea typically is not associated with gastric ulceration in adult horses, although ulceration can occur concurrently with other causes of diarrhea. horses actively racing are more likely to have squamous ulceration than those solely in training. 4 lesions occur predominantly in the squamous mucosa, particularly adjacent to the margo plicatus ( figure 13.10-1) . in more severe cases, lesions can extend dorsally into the squamous fundus. clinically relevant lesions typically affect a greater portion of the squamous mucosa and can be deep enough to cause bleeding. however, bleeding from ulcers in the gastric squamous mucosa typically is not associated with anemia or hypoproteinemia. according to a recent study, the incidence of glandular lesions, particularly within the pyloric region, may be higher than previously reported, 11 which emphasizes the importance of a thorough endoscopic examination and proper documentation of lesion location when reporting or discussing egus, especially the differentiation between squamous and glandular disease. although one may suspect a diagnosis of egus based on clinical signs and response to treatment, the only current method of confirmation is via gastroendoscopy, which one can perform easily in the standing horse or foal with mild sedation. in adult horses a 3-m endoscope allows for visual inspection of the entire stomach, pylorus, and proximal duodenum. shorter scopes permit examination of the gastric body and fundus, but not the pyloric antrum in most cases. one should use an endoscope with a maximum external diameter of 9 mm for neonatal foals. numerous scoring systems for lesion severity have been described, but a recent consensus has been published by the equine gastric ulcer council (table 13 .10-1). 2 duodenal ulceration can be difficult to confirm. duodenoscopy is the most specific means of diagnosis, although the procedure is more difficult than gastroscopy. additionally, an endoscope at least 200 cm in length is needed for foals up to 5 to 7 months old, and a longer endoscope usually is required for older animals. diffuse reddening or inflammation may be the only recognizable lesion in cases of early duodenal disease. excessive enterogastric reflux of bile through the pylorus suggests duodenal dysfunction. however, the pylorus frequently appears open, and some degree of enterogastric reflux is common under normal conditions. ulceration at the pylorus or pyloric antrum also suggests the presence of a duodenal lesion. if one can perform gastroendoscopy, but not duodenoscopy, the severity of lesions, particularly in the glandular mucosa and in the squamous mucosa of the lesser curvature dorsal to the pyloric antrum, usually will be severe when duodenal ulcers are present. multiple pharmacologic treatments have been suggested for treating egus. because acid has been implicated as the most important pathophysiologic component of squamous ulcer disease, most antiulcer therapy centers on suppression or neutralization of gastric acid. severity and location of gastric lesions and severity and duration of clinical signs, as well as medication cost, can play a role in the therapeutic management of egus (table 13 .10-2). if gastroendoscopy is unavailable, some guidelines to therapy can be used, but the efficacy of the treatment is based on clinical signs, which are often vague or nonspecific. signs of colic or diarrhea that result from gastric ulcers often resolve within 48 hours. one can note improvements in appetite, bodily condition, and attitude within 1 to 3 weeks. if one does not observe improvement in clinical signs, treatment has not been effective or gastric ulceration was not the primary problem. the principal therapeutic options for ulcer treatment include h 2 antagonists (cimetidine, ranitidine, famotidine, nizatidine), proton pump blockers (omeprazole, pantoprazole, rabeprazole, esomeprazole), the mucosal adherent sucralfate, and antacids. the h 2 antagonists suppress hydrochloric acid secretion through competitive inhibition of the parietal cell histamine receptor that can be overcome partially with exogenous pentagastrin. 61 use of h 2 antagonists has been successful in raising gastric ph and resolving gastric lesions in foals and adult horses. 29, 55, 62 clinical and experimental evidence has demonstrated greater individual variability with lower dosages of h 2 antagonists. 63 thus dosage recommendations are based on levels necessary to increase gastric ph and promote ulcer healing in a majority of horses. commonly recommended dosages are 20 to 30 mg/kg orally every 8 hours or 6.6 mg/kg intravenously every 6 hours for cimetidine and 6.6 mg/kg orally every 8 hours or 1.5 to 2 mg/kg intravenously every 6 hours for ranitidine. famotidine has been used less extensively in the horse, but a dose of 10 to 15 mg/kg/day has been recommended. because gastric perforation caused by glandular ulcer disease has been reported in hospitalized neonates, many clinicians routinely use prophylactic antiulcer therapy in this population. although clinically normal foals respond predictably to ranitidine, 29 sick neonates have shown variability in ph response to intravenously administered ranitidine, with a much shorter duration of action and in some cases no noticeable response. 31 thus currently used dosing schedules for hospitalized foals may be inadequate. because some critically ill foals have a predominantly alkaline gastric ph profile and because gastric acidity may be protective against bacterial translocation in neonates, the need for prophylactic ulcer therapy is controversial. in critically ill human neonates, intravenous administration of ranitidine raises gastric ph and gastric bacterial colonization but does not increase the risk of sepsis. 64 in a retrospective study of 85 hospitalized foals less than 30 days of age, no difference in the frequency of gastric ulceration at necropsy was found between those foals that received prophylactic treatment for gastric ulcers and those that did not. 65 because the study was retrospective, specific details regarding lesion location and severity were not available; however, none of the foals in the study died because of gastric ulcer disease. h 2 antagonist therapy should continue for 14 to 21 days, but complete ulcer healing may take 30 to 40 days. if an animal is kept in race training during therapy, clinical signs may resolve but the lesions may not. currently, cimetidine and ranitidine are available in injectable, tablet, and liquid forms. famotidine and nizatidine are available in tablets. proton pump inhibitors block secretion of h + at the parietal cell membrane by irreversibly binding to the h + ,k + -atpase proton pump of the cell. these agents have a prolonged antisecretory effect, which allows for once-daily dosing. omeprazole, the first proton pump inhibitor to be developed, is the only currently approved agent for the treatment of egus. several studies have documented the safety of orally administered omeprazole in foals and adult horses. 66, 67 omeprazole has demonstrated efficacy in the healing of nsaid-induced ulcers in horses and in naturally occurring cases of egus. 68, 69 more importantly, omeprazole has been shown to eliminate or reduce the severity of gastric ulcers in thoroughbreds maintained in race training. 70 the available equine preparation of omeprazole (gastrogard, merial, ltd., duluth, georgia) is recommended at a dose of 4 mg/kg orally every 24 hours. initial reports suggested that 3 to 5 days of omeprazole therapy were necessary to achieve maximum acid suppression; however, an increase in gastric ph and a decrease in acid output are evident 5 to 8 hours after omeprazole paste administration. 71 after initial treatment (28 days), treatment with 2 or 4 mg/kg every 24 hours has been shown to decrease or prevent the recurrence of disease in animals maintained in training. 72 the powder form of omeprazole degrades rapidly in an acidic environment, thus one must use an enteric-coated capsule (as used in the human preparation) or a specially formulated paste (such as gastrogard) to allow delivery of the active drug to the small intestine for absorption. many compounding pharmacies prepare omeprazole in liquid or paste formulation for use in horses, but their efficacy has not been evaluated to date. other proton pump inhibitors have been developed recently for use in human beings, including rabeprazole, lansoprazole, esomeprazole, and pantoprazole. in gastroesophageal reflux disease treatment in human beings, esomeprazole has demonstrated a higher rate of healing at 4 and 8 weeks compared with omeprazole, but rabeprazole, lansoprazole, and pantoprazole have similar efficacy. 73 an intravenous formulation of pantoprazole recently became available commercially and may prove beneficial for patients in need of antiulcer therapy that cannot be treated orally. research regarding the pharmacokinetics and efficacy of other proton pump inhibitors in horses is not currently available. sucralfate is effective in treating peptic ulcers and preventing stress-induced ulcers in human beings. the mechanism of action likely involves adherence to ulcerated mucosa, stimulation of mucus secretion, enhanced prostaglandin e synthesis, and increased concentration of growth factor at the site of ulceration, although the prostaglandin effects may not play an important role in ulcer healing. 74 these are factors relevant to glandular mucosa, and the efficacy of sucralfate in treating ulcers in the equine gastric squamous mucosa remains undetermined. sucralfate may be effective in preventing stress-induced ulcers in neonatal foals, because these occur in the glandular mucosa, although no clinical evidence directly supports this concept. in human beings, sucralfate provides protection against stress-induced ulcers with a decreased risk of pathogenic gastric colonization. 75 one should give sucralfate at a dosage of 10 to 20 mg/kg every 6 to 8 hours. the efficacy of sucralfate in an alkaline ph is controversial but appears likely. [76] [77] [78] moreover, at the time of administration of an section 13. 10 diseases of the stomach h 2 antagonist, the gastric ph likely will have returned to an acidic ph since the last dosage and will remain so for 30 to 60 minutes depending on the route of administration; thus one likely can administer the agents simultaneously if so desired. the use of antacids to treat gastric ulcers has not been examined critically in the horse. research in horses has shown that 30 g aluminum hydroxide per 15 g magnesium hydroxide results in an increase in gastric ph above 4 for approximately 2 hours. 79 thus although antacids may be useful for treating ulcers in horses, a dose of approximately 180 to 200 ml at least every 4 hours is necessary for a standard adult horse. the use of synthetic prostaglandin e 1 analogs, such as misoprostol, has been effective in treating gastric and duodenal ulcers in human beings, and the proposed mechanism of action involves inhibition of gastric acid secretion and mucosal cytoprotection. 80 frequently reported adverse effects of intestinal cramping and diarrhea in human beings have precluded the use of misoprostol in horses. one should consider prokinetic drugs in foals with duodenal disease and gastroesophageal reflux and when one suspects delayed gastric emptying without a physical obstruction. the cholinergic drug bethanechol has been shown to increase the rate of gastric emptying in horses. 81 in cases of acute gastric atony, bethanechol 0.025 to 0.030 mg/kg administered subcutaneously every 3 to 4 hours has been effective in promoting gastric motility and emptying, followed by oral maintenance dosages of 0.35 to 0.45 mg/kg 3 to 4 times daily. adverse effects can include diarrhea, inappetance, salivation, and colic, but at the dosages stated, adverse effects have been infrequent and mild. a complete review of ileus and prokinetic therapy is available in chapter 13.6. for foals with severe gastroduodenal ulcer disease that have developed duodenal stricture, surgical therapy is necessary. 57, 82 these animals require a serious financial commitment because intensive perioperative medical therapy is critical for a successful outcome. even with surgical therapy, these foals often warrant a guarded prognosis. pyloric stenosis is a structural resistance to gastric outflow. congenital pyloric stenosis has been reported in foals and one yearling and results from hypertrophy of the pyloric musculature. [83] [84] [85] acquired pyloric stenosis can result from neoplasia or duodenal ulceration. [86] [87] [88] [89] clinical signs depend on the degree of obstruction and include abdominal pain, salivation, and teeth grinding. complete or near complete obstruction can result in gastric reflux and reflux esophagitis. in foals with congenital pyloric hypertrophy, clinical signs may begin with the consumption of solid feed. in foals one can make a presumptive diagnosis via gastric endoscopy and radiography (plain and contrast studies). depending on the cause and severity of disease, gastric endoscopy may provide a presumptive diagnosis in the adult horse. measurement of gastric emptying can aid the diagnosis. several methods of measurement are currently available, including nuclear scintigraphy, acetaminophen absorption, and postconsumption [ 13 c] octanoic acid blood or breath testing. 81, 90, 91 exploratory laparotomy shows a distended stomach and thickened pylorus accompanied by a relatively empty intestinal tract. if complete obstruction is not present, medical therapy with a prokinetic such as bethanechol can increase the rate of gastric emptying. 81 phenylbutazone and cisapride also have been shown to attenuate the delay in gastric emptying caused by endotoxin administration. 90, 92 surgical repair is necessary for definitive treatment of complete or near-complete obstruction and consists of gastroenterostomy or pyloroplasty. 57, 82 gastric dilation can be classified as primary, secondary, or idiopathic. causes of primary gastric dilation include gastric impaction, grain engorgement, excessive water intake after exercise, aerophagia, and parasitism. 86, 93 secondary gastric dilation occurs more commonly and can result from primary intestinal ileus or small or large intestinal obstruction. time to development of gastric reflux is proportional to the distance to the intestinal segment involved, with duodenal obstruction resulting in reflux within 4 hours. 94 clinical signs of gastric dilation include those associated with acute colic and in severe cases, ingesta appearing at the nares. associated laboratory abnormalities include hemoconcentration, hypokalemia, and hypochloremia. 86 the most common reported cause of gastric rupture in horses varies between reports. in a retrospective study of 54 horses, gastric rupture occurred most commonly as a secondary phenomenon (65%), usually because of small intestinal obstruction, with primary gastric dilation and idiopathic rupture occurring almost equally (15% and 17%, respectively). 93 in another retrospective study of 50 horses in combination with a search of the veterinary medical database (vmdb), 60% of the gastric rupture cases were classified as idiopathic. 95 risk factors for gastric rupture include feeding grass hay, not feeding grain, gelding, and a nonautomatic water source. 93, 95 nasogastric intubation does not preclude the possibility of gastric rupture, and the amount of reflux obtained before rupture varies greatly. 93 because these reports were retrospective, one cannot rule out confounding factors with certainty. regardless of the initiating cause, gastric rupture usually occurs along the greater curvature. in horses with rupture caused by gastric dilation, tears in the seromuscular layer are frequently larger than the corresponding tears in the mucosal layer, indicating that the seromuscularis likely weakens and tears before the mucosa. 93, 95 in contrast, horses with gastric rupture following gastric ulceration usually demonstrate full-thickness tears of equal size in all layers. gastric rupture is usually fatal because of widespread contamination of the peritoneal cavity, septic peritonitis, and septic shock. initial clinical signs vary with the primary disease; however, when rupture occurs, a previously painful animal can exhibit signs of relief. subsequent signs are consistent with peritonitis and shock, including tachypnea, tachycardia, sweating, and muscle fasciculations. surgical repair is thus limited but has been reported for partial-thickness tears, 96 and in one case of a combined tear of the mucosa and muscularis with only a focal serosal tear, a full-thickness repair was performed with a favorable outcome. 97 gastric impaction can result in acute or chronic signs of colic in the horse. although a specific cause is not always evident, ingestion of coarse roughage (straw bedding, poor-quality forage), foreign objects (rubber fencing material), and feed that may swell after ingestion or improper mastication (persimmon seeds, mesquite beans, wheat, barley, sugar beet pulp) have been implicated. possible predisposing factors include poor dentition, poor mastication and rapid consumption of feedstuffs, and inadequate water consumption. clinical signs can vary from anorexia and weight loss to those consistent with severe abdominal pain. in severe cases, spontaneous reflux may occur, with gastric contents visible at the nares. in cases of acute severe abdominal pain, one often makes a diagnosis during exploratory celiotomy. in animals not exhibiting signs of colic warranting surgical intervention, an endoscopic finding of a full stomach after a normally adequate fast (18 to 24 hours) often can confirm the diagnosis. abdominal radiographs are reserved for smaller horses and ponies. in addition to pain management, specific treatment consists of gastric lavage via nasogastric intubation or massage and injection of fluid to soften the impaction during laparotomy. [98] [99] [100] nonulcerative gastritis rarely occurs in the horse; however, a single case of emphysematous gastritis caused by clostridium perfringens has been reported. 101 mimic that of a strangulating or nonstrangulating small intestinal obstruction, so distinguishing between the two syndromes is important because appropriate treatment of small intestinal obstruction usually requires surgical intervention. studies suggest that the survival rate for horses with dpj that endured surgical exploratory laparotomy was poor compared with those treated medically, although differences in disease severity may have accounted for the results in these early reports. 1, 2 the clinical syndrome of dpj was well described in the 1980s, and although recognized by its classical presentation, varying degrees of focal intestinal and systemic illness may occur. 1-4 dpj usually occurs alone but can occur along with gastritis, ileitis, typhlitis, and or colitis. typical pathologic findings in horses with dpj include involvement of the duodenum and usually the proximal jejunum. 3 the ileum and large colon usually are determined to be grossly normal. gastric distention is a common finding and is thought to be caused by hypersecretory mechanisms in the proximal small intestine and a functional ileus of affected enteric segments. the small intestine may be 5 to 7 cm in diameter because of fluid distention with malodorous, red to brown-red intralumenal fluid accumulation. duodenal (and jejunal) serosal surfaces may have varying degrees and distribution of bright-red to dark-red petechial and ecchymotic hemorrhages and yellow to white streaks. the enteric mucosal surfaces are usually hyperemic and have varying degrees of petechiation and ulceration. microscopically, the most severe lesions have been located in the duodenum and proximal jejunum but may extend proximally to the gastric mucosa and aborally to the large intestinal mucosa and submucosa. 3 microscopic lesions consist of varying degrees of mucosal and submucosal hyperemia and edema. more severe lesions include villus degeneration with necrosis and more severely, sloughing of villous epithelium. the lamina propria, mucosa, and submucosa may have varying degrees of granulocyte infiltration (predominantly neutrophils), and the muscular layers and serosal surfaces contain small hemorrhages. proximal small intestinal serosal fibrinopurulent exudate is a common finding in the more severe cases; therefore the term hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis has been suggested as a more descriptive name for this syndrome. horses with dpj often have evidence of multiple organ involvement such as hepatic changes including congestion and varying degrees of biliary duct hyperplasia. additional systemic involvement likely is caused by endotoxin absorption, metabolic imbalances such as acidemia, and circulatory changes. the cause of this syndrome remains an enigma (much like the cause of other inflammatory conditions affecting the intestinal tract). several microorganisms have been implicated as playing a role in triggering dpj, including clostridium spp., salmonella spp., and some mycotoxins, but efforts to reproduce the syndrome experimentally have been futile. 5 a recent dietary change with an abrupt increase in dietary concentrate level has been suggested to predispose a horse to developing dpj because of intraluminal microbial imbalances. two intracellular processes control intestinal secretion, the cyclic nucleotide (cyclic adenosine monophosphate and cyclic guanosine monophosphate) and the calcium systems. 6 agents (inflammatory mediators, microorganisms, toxic agents) can activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e 2 ) or guanyl cyclase (bacterial enterotoxins) and induce increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate, respectively. this reaction causes phosphorylation of specific protein kinases, which induce the actual mucosal membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotidedependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. 7 calcium may act through calmodulin, which then can activate membrane-phosphorylating protein kinases. the net effect is increased movement of sodium and chloride into the mucosal cell from the interstitium, with secretion of sodium and chloride into the intestinal lumen. water follows the directional flux of sodium and chloride through highly permeable intercellular spaces. several bacterial toxins and endogenous mediators can cause active secretion and contribute to a synergistic mucosal secretory response. passive secretion of protein-rich fluid into the lumen occurs following damage to the mucosal epithelium, capillary endothelium, and submucosal inflammation in the proximal small intestine. the clinically relevant events that result from active and passive fluid secretion are proximal small intestinal distention and nasogastric reflux, dehydration, and circulatory shock. the concentration of protein in the peritoneal fluid from horses with dpj is usually higher than in horses with small intestinal obstruction. a disproportionate increase in total protein concentration relative to nucleated cell count occurs probably by leakage of blood or plasma into the peritoneal cavity without a significant stimulus for leukocyte chemotaxis. suggested mechanisms for increased abdominal fluid protein concentration include serositis associated with inflamed intestine and small intestinal distention causing passive congestion and increased capillary hydrostatic pressure of visceral peritoneal vessels. 8 small intestinal ileus is another hallmark sign of dpj and the pathophysiology is complicated, involving primary and secondary dysfunction of the central, autonomic, and enteric nervous systems and their purported roles in governing intestinal motility. 9 primary role-players in dpj-associated ileus include peritoneal inflammation, inflammatory cell migration/activation within the muscularis, small intestinal mechanical distention, and effects of endotoxin absorption. the use of prokinetic agents for treating ileus and gastric/small intestinal distention in horses with dpj is becoming more common, but veterinarians should realize that a potential restriction on their use is the need for normal intestinal integrity. in spite of that, one may use motility modifiers judiciously. the veterinarian has the challenge of differentiating horses with dpj from horses with small intestinal obstructive lesions so as to avoid surgical intervention (table 13 .11-1). horses with dpj typically show signs of acute abdominal pain initially, and then after gastric decompression, volume replacement, and analgesic therapy, the colic signs subside, but signs of lethargy and malaise become more apparent. in contrast, horses with obstructive lesions of the small intestine usually show signs of abdominal pain until the affected viscus is repaired via surgical intervention or the viscus ruptures. another differentiating characteristic is the large volume (>4 to 20 l with each decompressive effort) of nasogastric reflux that is often malodorous and orange-brown or red-brown. dpj-affected horses have moderate to severe small intestinal distention palpated on rectal examination, temperature of 38.6°to 39.1°c (101.5°to 102.5°f), dehydration, brick-red mucous membranes, lethargy and absent borborygmi, prolonged capillary refill time, tachycardia (>60 beats/min), and tachypnea. although the signs of abdominal pain usually resolve after gastric decompression, most horses remain severely lethargic. without periodic removal of the fluid that accumulates in the proximal intestinal tract, signs of abdominal pain usually recur. horses with dpj often require gastric decompression at 2-hour intervals, with 2 to 10 l of fluid recovered each time. nasogastric tubes left in place for long periods of time cause varying degrees of pharyngitis, laryngitis, and esophagitis. typical clinical laboratory findings include an increased packed cell volume and total plasma protein reflective of volume depletion, a metabolic acidosis (with elevated anion gap) in longstanding or severe cases, an increased peritoneal fluid protein concentration (often >3.5 g/dl), and a mild to moderate elevation of the peritoneal white blood cell count, although the count usually is less than 10,000 cells per microliter. 3, 4 the peritoneal fluid is usually yellow and turbid, but in severe cases diapedesis occurs resulting in a serosanguinous color. the white blood cell count in the peripheral blood may be normal, decreased, or increased. in addition, hyponatremia, hypochloremia, hypokalemia, and acid-base alterations (elevated anion gap) are often evident. the loss of enteric bicarbonate through evacuation of enterogastric reflux and poor tissue perfusion from hypovolemia can lead to metabolic acidosis. one makes a definitive diagnosis of dpj in most cases by gross examination of the duodenum and proximal jejunum at surgery or at necropsy. some equine practitioners have observed an apparent geographic relationship in the incidence and severity of the syndrome, with more cases occurring in the southeastern united states. horses with dpj appear to share a common characteristic clinical presentation, and the mechanisms leading to electrolyte imbalances, fluid loss, ileus, and endotoxemia and septicemia are similar. treatment regimens are supportive and aim at plasma volume replacement (usually in the form of crystalloid fluid replacement), analgesia and antiinflammatory therapy, gastric decompression, antiendotoxin therapy, antimicrobial therapy if indicated, nutritional support, and nursing care. one should institute aggressive intravenous polyionic fluid therapy immediately in a horse with dpj. one should calculate the total fluid deficit based on clinical assessment of dehydration (e.g., for 8% or moderate dehydration, 0.08 × 450 kg body mass = 36 l) and should administer replacement fluids rapidly (up to 6 to 10 l per hour per 450-kg adult horse). administering intravenous hypertonic saline (7%) may be useful to treat hypovolemic shock in horses with severe circulatory shock. the use of 1 to 2 l of hypertonic saline (7% nacl) improved systemic blood pressure and cardiac output in horses with hemorrhagic shock and in a model of equine endotoxemia. 10 if one chooses this treatment option, intravenous administration of replacement isotonic fluids must follow immediately to maintain tissue integrity. one should not allow horses with significant volumes of gastric reflux to ingest foodstuffs or liquids orally. once one has administered replacement fluids and the horse is well hydrated, one should administer maintenance fluid amounts, which may be as high as 120 ml/kg/day. unfortunately, the intravenous fluid therapy itself may accelerate the flux of fluid from the vasculature into the intestinal lumen because of a reduction in intravascular oncotic pressure and an increased capillary perfusion pressure, which can result in an increased volume of gastrointestinal reflux. however, the veterinarian should not consider reducing the volume of intravenous fluid therapy because excessive fluid losses continue to occur. one should monitor plasma protein concentration, overall hydration, and the volume of reflux and then determine the rate of intravenous fluid administration. during the initial hours of therapy, even aggressive intravenous fluid administration results in only moderate clinical improvement. the clinical response, as evidenced by improved hydration status, decreased nasogastric reflux, improved attitude, and improvement in values reflecting kidney function (decreased blood urea nitrogen and creatinine), correlates with improvement of intestinal damage. horses with dpj that continue to reflux large volumes of enterogastric fluid frequently for more than 36 to 48 hours most likely will experience protein loss from the inflamed and disrupted intestinal mucosal barrier and from systemic protein catabolism. decreased colloid oncotic pressure leads to decreased effective circulating fluid volume and edema. total plasma protein may decline to below 4 g/dl and the albumin may decrease to below 2.0 g/dl. fresh or thawed frozen plasma is ideal for replacement of functional proteins. one should consider treatment with intravenous plasma therapy or a combination of plasma and synthetic colloid (e.g., synthetic amylopectin) as soon as one sees evidence of a consistent decline in total plasma protein or albumin (<2.0 g/dl) or if the horse is developing dependent edema. fresh plasma (preferred) or fresh frozen plasma is the treatment of choice if coagulation disorders accompany protein loss. an average-size horse (450 kg) requires 6 to 10 l of plasma (albumin 3.0 g/dl) or synthetic colloid to improve plasma oncotic pressure. administration of additional aliquots of 2 to 10 l of a balanced colloidal solution may be necessary if the dpj crisis continues. in addition to albumin (the major colloid component), plasma contains other components that provide overall systemic support (e.g., fibronectins, complement inhibitors, elastase and proteinase inhibitors, antithrombin iii). one may administer a 6% solution of hydroxyethyl starch (hetastarch (6%), abbott laboratories, north chicago, illinois), a synthetic colloid, at 5 to 10 ml/kg. because of the large size of the starch molecules, this solution is an effective plasma volume expander, resulting in sustained dosedependent decreases in packed cell volume and plasma protein concentration with increased oncotic pressure. the cost of an appropriate amount of commercial plasma or synthetic colloid solution for treatment of adult horses with dpj may be prohibitive but can be life-saving. horses with enteritis frequently absorb large amounts of endotoxin from the disrupted intestinal mucosal barrier, therefore putting these horses at a high risk for laminitis. one should monitor digital pulses every 4 to 6 hours until systemic signs of enteritis have abated (fever, leukopenia, etc.). treatment to combat endotoxemia is critical, and several therapeutic approaches are available. choice of treatment options is based on severity of disease, renal function, hydration status, and economics. the reader is referred to chapter 13.7 for a thorough discussion of endotoxemia pathophysiology, treatment, and prevention. nonsteroidal antiinflammatory drugs are the most frequently used group of drugs for treatment of abdominal pain in horses (flunixin meglumine 1.1 mg/kg intravenously every 12 hours or phenylbutazone 2.2 mg/kg orally or intravenously every 12 hours). the clinician must weigh the benefit of the analgesic effect of nonsteroidal antiinflammatory drugs with the possibility of further damage to the intestine by potentially blocking the protective effects of intestinal mucosal prostaglandins. one should consider other classes of drugs for treating colic associated with dpj. butorphanol (torbugesic; an opioid analgesic) at 0.06 to 0.1 mg/kg with detomidine (dormosedan; an α-agonist) at 0.01 to 0.02 mg/kg given intramuscularly every 6 to 8 hours is a useful combination that has minimal effects on gastrointestinal motility. because clostridium spp. are suspected as a causative agent of dpj, penicillin often is administered to affected horses. however, one should consider broad-spectrum antimicrobial coverage for horses with dpj. one can add an aminoglycoside (gentamicin, amikacin) or thirdgeneration cephalosporin (ceftiofur [naxcel], upjohn co., kalamazoo, michigan) to the penicillin therapy, keeping in mind the potential adverse effects of these drugs on renal function. effective antisecretory medications targeting the equine small intestine have not been identified. one should consider the nutritional needs of horses with dpj. most horses have a total body protein loss from cachexia and a protein-losing enteropathy. total parenteral nutrition may be indicated in horses that remain anorectic for more than 3 to 4 days. parenterally administered solutions containing glucose, balanced amino acid solutions, lipid emulsions, balanced electrolyte and trace minerals, and vitamins have been administered to adult horses with small intestinal ileus or enterocolitis. based on a small number of horses, this therapy has proved promising in terms of minimizing protein losses and decreasing the duration of illness. providing for part of the nutritional requirements of the horse (8000 to 12,000 kcal/day) is possible with glucose-amino acid solutions, which are of moderate cost. one may suppose reasonably that providing nutritional support to an anorectic, severely ill horse will facilitate the healing process and even shorten the duration of illness. thus the overall cost of providing parenteral nutritional supplementation to horses with dpj may well be offset by quicker recovery and diminished requirements for other, expensive treatments. normal (healthy) intestine is necessary for optimum performance of prokinetic agents in horses. many motilitymodifying agents likely are ineffective in cases of dpj. however, some benefit may come of the judicious use of prokinetic agents in inflammatory conditions of the equine intestine, particularly if the agent provides additional effects such as analgesia. for example, lidocaine infusion has several actions that may be beneficial in the treatment of ileus, including suppression of primary afferent firing, antiinflammatory properties, an observed analgesic effect, and direct stimulation of smooth muscle. 9 an infusion dose of 15 to 20 mg/min over 5 to 6 hours has been recommended. the reader is referred to chapters 13.6 and 13.15 for a complete description of motility modifying agents. medical therapy is sufficient in most cases of dpj, but in those cases in which the horse continues to produce copious enterogastric reflux, one may consider surgery as an option. refractory cases have been observed to improve with surgical intervention; however, some horses with refractory dpj have been observed to recover with supportive medical care alone even after 20 days of refluxing large amounts of fluid every 2 to 4 hours (personal observation). the decision of when to intervene surgically often is difficult. one may elect surgery to determine the extent of gross pathologic condition and intestinal distention and to perform intestinal bypass so as to direct enterogastric reflux toward the cecum and colon, where the fluid can be reabsorbed. allen and clark 5 have described two approaches for surgical therapy in such cases. a standing right flank laparotomy with resection of the last rib has been used to approach the duodenum and cecal base. using this approach, one makes a small stoma between the duodenum and cecum using a handsewn 1.0-to 1.5-cm side-to-side anastomosis. the stoma may act as a shunt to decompress the proximal small intestine and deliver the small intestinal fluid to the cecum for reabsorption. following recovery, the stoma likely will close. when a veterinarian is confronted with a horse exhibiting abdominal discomfort, with small intestinal distention palpable per rectum, and greater than 2 l of gastric reflux, the veterinarian should recommend referral of the horse to a facility capable of performing abdominal surgery. the chance that such a horse has an intestinal obstruction is too great to decide to treat it as if it may have dpj. surgery on such horses is not unusual, even though dpj is possible, to rule out an obstruction. at present, the survival of horses with dpj that undergo surgery is much greater than previously described, and certainly greater than that of horses with small intestinal obstruction that do not have surgery. horses with dpj that receive appropriate therapy have a reasonably good chance of making a full recovery. horses that continue to have frequent episodes of voluminous nasogastric reflux and systemic signs of endotoxemia and septicemia have a poorer prognosis for recovery. frequent complications of dpj include laminitis, thrombophlebitis, and weight loss. the clinical signs of chronic wasting and poor body condition, although nonspecific for a diagnosis of malabsorption antemortem, can be attributed to proliferative or inflammatory intestinal disorders, often collectively referred to as chronic inflammatory bowel diseases. 1 clinical signs include alimentary lymphosarcoma, granulomatous enteritis, multisystemic eosinophilic epitheliotropic disease (meed), and lymphocyticplasmacytic enterocolitis-conditions affecting young and adult horses. proliferative enterocolitis, 2 a transmissible disease of foals 3 to 7 months of age characterized by significant small intestinal pathologic changes, will be included in this group. however, several other primarily small intestinal conditions described from a morphologic perspective, such as chronic postinfarctive inflammation and mycobacterial infections, 3 will not be discussed. in addition, a single case of aa amyloid-associated gastroenteropathy in an 18-year-old morgan stallion that had evidence of severe malabsorption based on poor d-xylose absorption is included. 4 for comparative purposes, table 13 .12-1 lists the clinical and clinicopathologic features of the diseases, and tables 13.12-2 and 13.12-3 present the gross morphologic and histopathologic findings, respectively. the extent of small intestinal disease is the key to determine whether one can demonstrate malabsorption based on abnormal carbohydrate absorption. as described in chapter 13.4, this is not an all-or-nothing situation. in the same animal the staging of the pathologic changes differs in different regions of the small and large intestines, thus influencing severity of clinical signs and absorption findings. furthermore, the extent of pathologic changes in different animals with ultimately the same morphologic diagnosis affects absorption studies and progress of the disease. early diagnosis remains a challenge, and even multiple intestinal biopsies taken at exploratory laparotomy may prove unhelpful. by contrast, intestinal infiltration with the predominant cell types can be found in grossly normal appearing intestinal tissue. alimentary lymphosarcoma of the horse may represent a primary neoplasia of the gut associated lymphoid tissue with significant cellular infiltration of the small intestine and associated lymph nodes with minimal large intestinal or systemic involvement. case series and pathology reports indicate that young horses 2 to 4 years of age primarily are affected, although the age range can be broad. [5] [6] [7] no breed or sex predilection exists. prevalence is unknown. despite the progressive nature of lymphomata, onset of clinical signs can be rapid and the animal may become acutely ill. as with all adult cases of chronic inflammatory bowel disease, antemortem diagnosis is by a process of exclusion and usually is confirmed post mortem. frequently, the horse has anemia, thrombocytopenia, neutrophilia or neutropenia, hypoalbuminemia, normal serum protein or hyperproteinemia, and hypergammaglobulinemia. lymphocytosis is rare. one may palpate intraabdominal masses, mainly enlarged mesenteric lymph nodes, rectally. abdominocentesis has been of diagnostic value. carbohydrate absorption tests usually reveal partial to total malabsorption indicative of the severely reduced surface area resulting from significant villous atrophy and the extensive mucosal or transmural infiltration. rectal biopsy has aided diagnosis. early confirmation of a suspected diagnosis necessitates exploratory laparotomy to obtain multiple intestinal and lymph node biopsies. in the future, markers of cancer cells may become available and may be cost-effective to aid diagnosis. prognosis is poor. natural progress of the disease is unknown. most horses are presented in an advanced state of disease. immunosuppressive drugs or chemotherapy may afford temporary improvement. however, outcome is unaffected. the chronic wasting condition granulomatous enteritis was first described in 1974 8 ; 9 of 10 horses were young standardbreds. most affected horses are 2 to 3 years of age. case reports from many countries revealed a predominance of standardbred over thoroughbred horses by three to one. 9,10 some of the standardbreds were related, implicating a genetic predisposition. prevalence is low. the condition is sporadic and has an insidious onset, and the course can be protracted. significant diagnostic features include anemia, slight increases or decreases in white blood cell counts, hypoalbuminemia, normal serum protein or hypoproteinemia, occasional increases in serum alkaline phosphatase activity, normal serum γ-glutamyltransferase activity, and enlarged mesenteric lymph nodes on rectal palpation. reduced carbohydrate absorption to the level of partial to total malabsorption is reported frequently, consistent with the severe morphologic changes throughout the small intestine. one can attribute the low proportion of horses exhibiting diarrhea 9,10 to the preferential distribution of inflammatory infiltration in the small intestine, 11 with lesser involvement of the large intestine. rectal biopsy can be a useful aid to diagnosis. 12 treatment of horses with granulomatous enteritis with a variety of drugs, particularly corticosteroids, has not affected the outcome except in the short term. 13 one successful response has been reported. prolonged corticosteroid administration produced clinical remission in a 6-year-old standardbred gelding based on improvement in clinical signs and in d-xylose absorption. 14 five months after cessation of approximately 5 months therapy, d-xylose absorption was normal and the horse was bright, alert, and resumed a level of athletic performance. parenteral administration of dexamethasone sodium phosphate was tapered to achieve a minimal effective dose to reduce intestinal inflammation and abolish clinical signs. adverse effects were not reported. the outcome of this single case is encouraging. surgery may be indicated if the disease is localized. two young horses underwent resection of the thickened terminal small intestine to confirm a diagnosis and provide a means of treatment; one horse died 4 months after surgery, and the other has remained clinically normal for at least 10 years. 10 the cause of granulomatous enteritis is unknown. several infectious agents have been implicated, including mycobacterium avium. 15 the condition may represent a granulomatous hypersensitivity reaction. immunemediated responses to dietary, parasitic, or bacterial antigens may be important initiating factors. 1 recently, six cases purported to represent granulomatous enteritis were linked to environmental contamination with aluminum. 16 although the case definition was flawed and problems existed with the data and interpretation, 17 the report nevertheless raised the possibility that a toxicologic basis may exist for some equine inflammatory bowel disorders. lumen from parasitic, bacterial, or dietary sources. infectious agents have not been identified. 18, 19 widespread use of the avermectins has tended to reduce parasite loads and composition to favor small strongyles (cyathostomes). eosinophilia is a feature of parasitism in the equine intestinal tract, although nematodes rarely have been identified in any lesions of meed. 3, 18 however, failure to detect larval structures in these lesions may be attributable to chronicity of the disease and destruction of the parasites in tissue. 10 biopsies of the rectal mucosa 12 or of the skin, liver, intestinal tract, and lymph nodes may assist in diagnosis. treatment has been attempted with a variety of drugs, including antibiotics, corticosteroids, and anthelmintics with larvicidal activity. immediate improvement has not been borne out in the long term. prognosis is poor. the clinical objective is to reach a tentative diagnosis early in the course of the disease for intervention to be more than transient. unlike the other conditions (see table 13 .12-1), meed has definitive liver and pancreatic involvement, and thus maldigestion may make a significant contribution to the wasting disease. for example, the lowered albumin and protein could result in part from impaired pancreatic enzyme digestion, and the effects of inflammatory lesions in the liver and ileum may decrease bile salt concentrations. the morphologic findings in lymphocytic-plasmacytic enterocolitis reflect the predominant infiltrative cellular elements of this rarely encountered condition. a retrospective study of 14 horses 23 provided the information presented in the tables. no specific clinical or clinicopathologic features differentiate this condition antemortem from other inflammatory diseases of adult horses. carbohydrate absorption was abnormal or delayed in 9 of 12 horses, consistent with the predominance of small intestinal pathologic changes. rectal biopsies were abnormal in 3 of 7 horses, two of which were reported as having lymphocytic-plasmacytic proctitis. prognosis is poor. treatment has been unsuccessful, probably because of the advanced nature of the condition at the beginning of treatment. proliferative enteropathy has not been associated with abnormal carbohydrate absorption based on three horses subjected to carbohydrate absorption tests. however, the florid mucosal lesions in the jejunum and ileum undoubtedly contribute to impaired digestive function and potential malabsorption of vitamins, minerals, and amino acids in the distal small intestine. the condition meed encompasses disorders characterized by a predominant eosinophilic infiltrate in the gastrointestinal tract, associated lymph nodes, liver, pancreas, skin, and other structures and accompanied by some degree of malabsorption and enteric protein loss. the disorders include chronic eosinophilic gastroenteritis, 18 eosinophilic granulomatosis, 9 chronic eosinophilic dermatitis, 19 and probably basophilic enterocolitis. 20 the condition differs from idiopathic eosinophilic enterocolitis, 10 in which segmental lesions in the small or large intestine induce signs of colic requiring surgical intervention 21,22 without evidence of malabsorption or multisystem involvement. although prevalence is low, meed appears to be more common than granulomatous enteritis based on the accumulated published reports and personal experience in australia and the united states. most affected horses are 2 to 4 years of age, and standardbreds and thoroughbred are reported to predominate. the condition is sporadic and has an insidious onset, and the course is protracted with a duration of 1 to 10 months. diarrhea is common in contrast to granulomatous enteritis. severe skin lesions with exudative dermatitis and ulcerative coronitis are prominent and frequently are the principal reason for veterinary attention being sought. despite extensive tissue eosinophilia, systemic eosinophilia is rare. hematologic values are usually unremarkable. notable features include hypoalbuminemia and normal serum protein or hypoproteinemia, and because of liver involvement, serum γ-glutamyltransferase and alkaline phosphatase activities may be increased. most reports of carbohydrate absorption test findings (glucose or d-xylose) indicate retarded absorption and a reduced or normal peak concentration delayed to at least 180 minutes. one can interpret this pattern as the existence of sufficient small intestinal absorptive capacity to enable moderate absorption with possibly delayed gastric emptying or ileocecal ejection. morphologic changes are less pronounced in the small intestine than in the large intestine, 9 and small intestinal lesions predominate segmentally in the proximal duodenum and distal ileum. furthermore, significant hyperkeratosis of the fundic region may contribute to gastric muscle contractile disruption. diarrhea can be a consequence of the severe segmental or multifocal granulomatous lesions in the large intestine with mucosal and transmural thickening and extensive ulceration. abundant fibrosis is a feature of all affected tissues (see table 13 .12-3). the cause of meed is unknown and could represent a chronic ongoing immediate hypersensitivity reaction against undefined antigens ingested or excreted into the affects foals 3 to 7 months of age, particularly those that have been weaned recently. the disease is caused by lawsonia intracellulare, an obligate intracellular bacterium found in the cytoplasm of proliferative crypt epithelial cells of the intestine. the condition in a foal was described first as intestinal adenomatosis, 24 because of similarity to the swine disorder of the same name. later, molecular studies showed that intestines from an affected foal contained l. intracellulare sequences as determined by polymerase chain reaction analysis and confirmed by southern blot hybridization. 25 recently, studies of a cluster of affected foals on three breeding farms in canada provided much information on the clinical syndrome, laboratory investigations, and response to treatment. 2 two of the three farms bred arabians, hence a demographic predominance of arabian foals exists. clinical signs included depression, rapid and significant weight loss, edema, diarrhea, and colic. poor body condition, a rough hair coat, and potbelly appearance were common findings. other problems often were concurrent, including respiratory tract infection, dermatitis, intestinal parasitism, and gastric ulceration. significant laboratory findings were anemia, transient leucocytosis, hypoalbuminemia, hypoproteinemia, and elevated serum creatine kinase concentrations. diagnosis was confirmed by identifying characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells using silver stains and by results of polymerase chain reaction analysis and immunohistochemical testing. antemortem diagnosis relied on clinical signs, hypoproteinemia, and exclusion of common enteric infections. one can confirm diagnosis in live animals by fecal polymerase chain reaction analysis (positive in 6 of 18 foals tested) and serologic testing; 7 foals with proliferative enteropathy were evaluated serologically and had antibodies against lawsonia intracellulare. 2 treatment is effective. most foals received erythromycin estolate (15 to 25 mg/kg per os every 6 to 8 hours), alone or with rifampin (7 to 10 mg/kg per os every 12 hours) for 2 to 4 weeks. foals frequently needed supportive therapy at the outset for stabilization. response to therapy has been excellent. 2 rapid improvement in clinical signs even within 24 hours preceded the rise in plasma protein concentration. the source of the infection was undetermined. no apparent link existed between the three farms and a swine operation or solid and liquid waste disposal on pasture. however, one cannot exclude airborne spread of dried fecal material over distances. comparisons of epidemiologic findings from the swine disease indicated that overcrowding, feed changes, antibiotic usage, and mixing and transportation were potential risk factors at two of the farms. recent weaning appeared to be a key element in the pathogenesis. 24 samuel l. jones acute diarrhea caused by colitis in adult or young horses is a potentially life-threatening disorder of a variety of causes (table 13 .13-1) characterized by hypersecretion of fluid, motility disturbances, altered microbial flora in the colon, and an impaired mucosal barrier caused by direct injury or inflammation. many of the clinical and clinicopathologic features are similar regardless of the cause. severe dehydration with profound electrolyte abnormalities is common, as is systemic inflammation from absorption of endotoxin or other bacterial products through the compromised mucosa. gastrointestinal protein loss may result in reduced colloid oncotic pressure from hypoproteinemia, leading to tissue edema. colitis is a highly catabolic disorder, and weight loss may be rapid and severe. some cases of colitis may be complicated by extensive mucosal ulceration, serosal inflammation, or mural ischemia/infarction extending from the inflammation or resulting from coagulopathies. thus diagnostic measures aimed at determining the cause necessarily must be accompanied by clinical and laboratory assessment of hydration, electrolyte and acid-base balance, plasma protein concentration and colloid oncotic pressure, organ function, and evaluation of the degree of systemic inflammation and of the integrity of the intestinal wall. although therapeutic strategies are similar for many causes of colitis, consisting primarily of control of local and systemic inflammation, maintenance of fluid and electrolyte balance, promotion of tissue perfusion, replacement of plasma protein, preservation of colloid oncotic pressure, promotion of mucosal repair, restoration of the microbial ecology of the colon, and nutritional management, some causes of acute colitis have specific therapies aimed at eliminating the cause. a variety of infectious organisms has been identified as causes of acute colitis in adult horses. the clinical syndromes associated with these infections are indistinguishable in most horses. however, appropriate diagnostic tests including fecal bacterial culture, fecal bacterial toxin analysis, pcr, and/or serology may identify specific infectious organisms. salmonella is a genus of gram-negative facultatively anaerobic bacteria that are common gastrointestinal pathogens in horses. many serotypes of salmonella have been reported to infect horses, but those classified in group b appear to be associated more commonly with disease than those in other groups. group b includes s. typhimurium and s. agona, two of the species most frequently isolated from horses. 1-3 s. typhimurium is the most pathogenic serotype in horses and is associated with a higher case fatality rate than other species of salmonella. 1 the number of horses that are infected inapparently with and actively shed salmonella in their feces has been reported to be as high as 10% to 20%, but actual prevalence of salmonella-shedding in the general horse population is likely to be much lower, less than 1% to 2%. 4 horses shedding salmonellae are a potential source of infection to susceptible horses, 1,5 as are environmental reservoirs. [6] [7] [8] for these reasons, salmonellosis is one of the most common nosocomial diseases in horses. nosocomial salmonellosis significantly affects morbidity and mortality in hospitalized horses. 9 the emergence of multidrug resistance in equine salmonella isolates has been a cause of concern because of the importance of salmonellosis as a nosocomial disease and because salmonella represents a significant zoonotic pathogen. 7, [10] [11] [12] the virulence of the bacteria varies tremendously with serotype and even among strains of the same serotype in part because of the important role of host susceptibility in the pathogenicity of particular organisms. the infective dose is generally millions of organisms inoculated orally, but various environmental and host factors can reduce the infective dose to a few thousand or even hundreds of organisms. [13] [14] [15] environmental factors or stresses that increase susceptibility to salmonella infection are not well defined, but high ambient temperature, for example, is known to increase the prevalence of salmonellosis in horses greatly. indeed, the peak incidence of salmonellosis in horses occurs in late summer and fall. 6, 14, 15 other environmental and host factors that increase the risk of salmonella infection include transportation, antibiotic administration, gastrointestinal surgery, general anesthesia, preexisting gastrointestinal disease, change in diet, and immunosuppression. 1, 8, 15 host factors that restrict gastrointestinal colonization and invasion by pathogens include gastric ph, commensal gastrointestinal flora, gastrointestinal motility, the mucosal barrier and mucosal immunity. 1, 16 gastric acidity is an important defense mechanism preventing live organisms from reaching the intestine. altering the gastric ph with histamine 2 receptor antagonists, for example, may increase susceptibility to infection. gastrointestinal flora inhibits the proliferation and colonization of salmonella by secreting bacteriocins, short-chain fatty acids, and other substances that are toxic to salmonella. in addition, elements of the normal flora compete for nutrients and space, especially on the mucosa. 16 being predominantly anaerobic, the normal flora maintain a low oxidationreduction potential in the environment of the large intestine, which inhibits the growth of many bacterial pathogens. 17 the importance of normal host gastrointestinal ecology is illustrated by the fact that disturbances of the colonic flora with antibiotics, changes in feed, ileus, or other underlying gastrointestinal disease greatly increase the susceptibility of the host to infection by salmonella, often resulting in serious disease. the immune status of the host may be one of the most important factors determining not only the susceptibility to salmonella infections but also the degree of invasion and subsequent outcome of the infection. local immunity, such as mucosal antibody secretion and enterocyte-derived cationic peptides, prevents colonization of the mucosa. 16, 18, 19 opsonizing antibodies and activation of the complement cascade are important in fighting systemic invasion by salmonella by increasing the efficiency of phagocytosis and by direct bactericidal activity. humoral immunity, however, is often ineffective in preventing disease and dissemination once invasion occurs and salmonella has established in its intracellular niche. following invasion, salmonella is capable of surviving and multiplying within macrophages, rendering humoral (noncellular) immune systems ineffective. 20, 21 specific cellular immunity may be the most effective defense mechanism in the host arsenal against dissemination and systemic infection by salmonella. 21, 22 oral inoculation with small numbers of virulent organisms may induce protective immunity in horses and calves, but the duration of the immunity is not known. 23, 24 oral and parenteral vaccines using killed or attenuated organisms and bacterial products have been promising but are effective only against homologous organisms and are usually not cross-protective among different serogroups. [23] [24] [25] in adult horses, salmonella primarily infects the cecum and proximal colon, causing enterocolitis, and the ability to disseminate beyond the intestine and cause enteric fever is limited. in foals, however, salmonellosis often is associated with septicemia. the ability of salmonella to cause enterocolitis depends on the ability of the bacteria to invade the gastrointestinal mucosa. 16, 20 invasion of the gastrointestinal mucosa occurs preferentially through specialized enterocytes called m cells that overlay intestinal lymphoid tissues such as peyer's patches in nonequine species. a variety of enteric pathogens exploit m cells during infection of intestinal tissue. 26 invasion of the epithelium occurs by self-induced uptake via the apical membrane of the m cell, often killing the cell in the process. 20 salmonellae then invade neighboring cells via the basolateral membrane, eventually spreading the destruction of the epithelium beyond the principle area of attack. virulent salmonellae have a well-developed invasion mechanism involving generation of an apparatus called a type iii secretory system that enables virulence gene products to be injected directly into enterocytes. 27 virulence proteins injected by salmonellae into enterocytes engage the cellular machinery and induce the cell to engulf the bacteria by macropinocytosis. salmonella virulence gene products also induce enterocyte chloride and fluid secretion and upregulate enterocyte transcription of inflammatory cytokines (tumor necrosis factor α and interleukin-1β) and chemokines that trigger a mucosal inflammatory response. 20, 27, 28 after salmonellae invade the mucosa, they are phagocytosed quickly by macrophages and dendritic cells in the lamina propria and lymphoid tissues. the ability of salmonellae to disseminate systemically and cause enteric fever is associated with the ability to survive and proliferate in macrophages. indeed, phagocytes have an important role in dissemination of the pathogen to blood, lymph nodes, liver, and spleen. most salmonellae in the blood and tissues of infected animals that are competent to cause enteric fever are within phagocytic cells. 29 in adult horses with salmonellosis, dissemination appears to be limited to the intestine and mesenteric lymph nodes, and salmonella rarely is cultured from blood. however, in foals and in some adults, salmonella causes an enteric feverlike disease with dissemination to mesenteric lymph nodes, liver, spleen, and blood. salmonella organisms require specific virulence gene clusters encoded on the chromosome or on plasmids for intracellular survival in macrophages. 20 some of these genes are sensors that signal the bacteria that it has entered an intracellular environment and turn on genes required for intracellular survival. others, like invasion genes, are transported from the bacteria and injected into macrophage cytosol by a type iii secretory system to prevent phagosome/lysosome fusion and subvert other essential macrophage killing mechanisms. salmonellae also possess multiple genes that confer resistance to reactive oxygen and nitrogen metabolites, perhaps the most lethal antimicrobial mechanisms of macrophages. 30 diarrhea associated with salmonellosis has multiple causes. a salmonella cytotoxin inhibits protein synthesis in mucosal cells, causing morphologic damage and altered permeability. 31 virulent salmonellae also produce an enterotoxin similar to the heat-labile toxin (lt) produced by escherichia coli. 32, 33 the enterotoxin contributes to but is not required in the pathogenesis of diarrhea. 34, 35 salmonella enterotoxin increases secretion of chloride and water by colonic mucosal cells in many species, including horses, by increasing intracellular cyclic adenosine monophosphate concentrations. the ability of virulent salmonellae to cause diarrhea appears to be associated most closely with the ability to invade enterocytes and to trigger an inflammatory reaction in the intestinal tissue. 20, 36 gene products injected into enterocyte cytosol by the type iii secretory system of invading salmonellae stimulate chloride and fluid secretion. 27 salmonella invasion of enterocytes is also a potent activator of inflammatory chemokine and cytokine production, resulting in the recruitment of leukocytes, particularly neutrophils, and activation of resident macrophages and mast cells. products of these activated leukocytes, including prostaglandins, leukotrienes, reactive oxygen metabolites, and histamine, are potent stimulators of chloride secretion in the colon of many species. 16, [37] [38] [39] the enteric nervous system integrates the diverse processes of pathogen recognition, triggering of the inflammatory response, and induction of enterocyte fluid secretion. 39 many of the inflammatory mediators studied stimulate colonic secretion by prostaglandin-dependent mechanisms, resulting in increased intracellular cyclic adenosine monophosphate or calcium concentrations or both in mucosal cells. 37 in addition, these mediators and the enteric nervous system may stimulate secretion by prostaglandin-independent mechanisms, inhibit sodium and water absorption, cause motility disturbances, and potentiate tissue injury, all of which enhance the pathogenicity and dissemination of salmonella and contribute to the pathogenesis of diarrhea. 37, 39 neutrophils recruited to the mucosa by signals generated by the infected enterocytes physically contribute to mucosal injury by producing a variety of products that are lethal to pathogens but are also toxic to host cells. 40, 41 moreover, neutrophils attracted to infected epithelial cells accumulate beneath the monolayer, lifting it off the basement membrane in sheets. neutrophils also migrate across the epithelial monolayer in potentially massive numbers. transepithelial migration of neutrophils increases the permeability to macromolecules, bacterial products, and even bacteria. 41 potentially massive losses of electrolytes, water, and protein can occur depending on bacterial and host factors. perhaps most devastatingly, mucosal injury and altered permeability allow systemic absorption of bacterial products and dissemination of bacteria, resulting in systemic inflammatory responses such as occur with endotoxemia and septicemia. four syndromes of salmonella infection have been described clinically and reproduced experimentally in horses: (1) inapparent infections with latent or active carrier states; (2) depression, fever, anorexia, and neutropenia without diarrhea or colic; (3) fulminant or peracute enterocolitis with diarrhea; and (4) septicemia (enteric fever) with or without diarrhea. inapparent infections can be activated to clinical disease in compromised horses, such as horses with colic or horses being treated with antibiotics, causing mild to severe enterocolitis. in addition, latent infections (nonshedding) can become active infections (shedding) under certain conditions, such as transportation stress and antibiotic treatment. horses with depression, anorexia, fever, and neutropenia without diarrhea generally have a good prognosis and recover in several days without specific treatment. 42 the septicemic form is restricted mostly to neonatal foals and is uncommon in adult horses. this discussion focuses on acute enterocolitis. acute enterocolitis is characterized by severe fibrinonecrotic typhlocolitis, with interstitial edema and variable degrees of intramural vascular thrombosis that may progress to infarction. 1 severe ulceration of the large intestinal mucosa may occur with serosal ecchymoses and congestion. the earliest signs of enterocolitis are usually fever and anorexia. 1, 15 signs of colic may be apparent early in the course of the disease, especially if ileus is present. clinical signs of endotoxemia are common and range from fever, elevated heart and respiratory rates, poor peripheral perfusion, and ileus to fulminant and rapidly progressive signs of endotoxemic shock. oral mucous membranes are often pale with perigingival hyperemia (a toxic rim) but may be brick red or cyanotic, with prolonged capillary refill time. one may note weakness, muscle fasciculations, cold extremities, and other signs suggestive of hypotensive shock; synchronous diaphragmatic flutter; abdominal pain; and significant metabolic and electrolyte abnormalities in severe cases of enterocolitis. one also may note signs of mild dehydration before diarrhea is apparent. once diarrhea is evident, dehydration may become severe rapidly. occasionally, horses die peracutely, without developing diarrhea. diarrhea may not be apparent for several days but usually occurs by 24 to 48 hours after the fever begins. 1, 15 the duration of the diarrhea may be days to weeks. the character of the first diarrheal feces is usually watery with particles of roughage but may become fluid rapidly without solid material. finding frank blood and fibrin in the feces is unusual. the volume of feces is often large, with frequent defecation. one may note straining or signs of colic when the patient is defecating, and rectal prolapse may occur occasionally. persistent straining and rectal prolapse may be a sign of colonic infarction. abdominal borborygmi are often absent early in the course of the disease because of ileus but become evident later, usually when diarrhea begins. fluid and gas sounds are commonly audible, but normal progressive motility is less frequently audible than normally. transrectal palpation may reveal edematous rectal mucosa and colon and fluid-filled colon and cecum. one may obtain gastric reflux, especially early in the course when ileus is evident. hematologic abnormalities early in the course of the disease include moderate to severe neutropenia, lymphopenia, and leukopenia, a mild to moderate left shift, and toxic changes in the neutrophils. 1, 15 thrombocytopenia, moderate to severe hemoconcentration, and hyperfibrinogenemia are also common. neutropenia is an early but nonspecific indicator of salmonellosis, often occurring concurrently with the onset of fever. 1 later in the course of disease, one may see neutrophilic leukocytosis, indicating recovery. a degenerative left shift, with metamyelocytes and myelocytes in the peripheral blood, is a poor prognostic sign. serum biochemical analysis may reveal azotemia, elevations in serum sorbitol dehydrogenase and γglutamine aminotransferase activity, and elevated serum lactic acid concentration. azotemia is often prerenal, but acute hemodynamic renal failure may occur in severely dehydrated, endotoxemic, or septicemic patients. indeed, elevation of creatinine concentration is a poor prognostic indicator in horses with acute colitis. 43 hemodynamic renal disease may be complicated by toxic injury caused by administration of nephrotoxic drugs. hyponatremia may also contribute to prerenal azotemia. elevations in hepatocellular enzymes are usually mild and reflect damage to the hepatocytes from absorbed toxins such as endotoxin and from poor perfusion caused by hypotensive shock or dehydration. lactic acidemia may be present, reflecting poor tissue perfusion. plasma protein rapidly drops as protein is lost in the gastrointestinal tract, causing moderate to severe hypoalbuminemia and hypoglobulinemia. peripheral or organ edema (vascular leak syndrome) may occur if hypoproteinemia is severe, coupled with increases in endothelial permeability induced by systemic inflammation. hypokalemia, hyponatremia, hypochloridemia, and hypocalcemia are common electrolyte abnormalities in patients with enterocolitis. metabolic acidosis also may be present. coagulopathies, such as decreased antithrombin iii activity and disseminated intravascular coagulation, may occur. urinalysis may reveal isosthenuria, proteinuria, hematuria, cylindruria, and glucosuria if hemodynamic or toxic renal injury is present. the number of leukocytes in the feces usually is elevated, and occult blood may be detectable. peritoneal fluid is usually normal except when severe mural inflammation or colonic infarction occurs. routine detection of salmonellae in feces involves five daily cultures of large samples (10 to 30 g) of feces using enrichment techniques. 1, 44, 45 however, the sensitivity of fecal culture can be as low as 30% to 50%, even if one cultures several fecal samples collected daily. concurrent culture of rectal biopsy specimens and feces increases the sensitivity of culture techniques to 60% to 75%. 45 currently, the polymerase chain reaction (pcr) is the most sensitive and rapid test for detecting salmonella in feces. a single pcr test applied early in the course of disease is a more sensitive test for the presence of salmonella than repeated fecal cultures. 46, 47 detection of salmonellae in feces does not prove a diagnosis of salmonellosis, but the positive predictive value of a positive pcr or culture results is high in horses with compatible clinical signs. culture of peripheral blood may allow isolation of the organism if bacteremia or septicemia is present, but blood cultures are not a sensitive test for salmonellosis in adult horses. although foals are more likely than adults to become septicemic, culture of blood is recommended in all cases with signs suggestive of septicemia. increased numbers of fecal leukocytes suggest an invasive process in the colon but are not specific for salmonellosis. early in the course of the disease, dehydration, electrolyte and acid-base imbalances, endotoxemia, and sepsis may be life threatening. aggressive treatment during the acute stages to replace fluids lost in the diarrhea and to control sepsis and endotoxemia is often effective in controlling the primary disease. weight loss and hypoproteinemia are often severe. complications such as multiorgan dysfunction, vascular leak syndrome with peripheral and organ edema, laminitis, acute renal failure, venous thrombosis and septic phlebitis, irreversible protein-losing enteropathy or chronic malabsorption, pulmonary aspergillosis, and gastrointestinal infarction can occur. in many instances, horses recover from acute salmonellosis with aggressive treatment, only to succumb to complications of the disease, partially explaining the high fatality rate of equine salmonellosis compared with human salmonellosis. chronic, mild to moderate diarrhea occasionally occurs in horses after a bout of severe salmonellosis, usually with protein-losing enteropathy. if the chronic diarrhea persists beyond 4 to 5 weeks after the onset of signs, the prognosis for recovery is poor. 15 potomac horse fever (equine monocytic ehrlichiosis) is caused by the obligate intracellular rickettsial organism neorickettsia risticii. [48] [49] [50] [51] the disease is most common from late summer to early fall, with a peak incidence in july and august. potomac horse fever was described first in the northeast but since has been diagnosed in most areas of the continental united states, with a particularly high prevalence in the northeast and midwest. the geographic distribution is characterized by a significantly higher percentage of cases found along waterways and rivers. 48, 49 the disease occurs sporadically, temporally and geographically, and can affect any age group of horses. the case fatality rate is 5% to 30%. 48 transmission of n. risticii has been reproduced experimentally by oral, intramuscular, intradermal, subcutaneous, and intravenous routes. 48, 52 however, the natural route of infection has remained a mystery until recently. the epidemiologic data, the fact that many other rickettsial diseases are transmitted by insect vectors, and the finding that the disease can be transmitted via whole blood have implicated insect vectors in the natural transmission of the organism. attempts to transmit the disease experimentally with ticks (dermacentor variablis) or biting flies (stomoxys calcitrans) have been unsuccessful. 53, 54 recently, n. risticii has been found to infect virgulate cercariae larval stages of trematodes that use operculate freshwater snails (juga spp.) as part of their life cycle in northern california. 55 infected virgulate cercariae have been identified in aquatic snails collected in other parts of the country as well. virgulate cercariae are part of the life cycle of trematodes that are common parasites of many species and use freshwater snails and aquatic insects as intermediate hosts. although the trematode species infected with n. risticii remains to be identified definitively, at least two species are considered potential vectors. 52 during the trematode life cycle, aquatic snails release large numbers of infected cercariae into water, where they seek their next intermediate host. infected metacercaria have been identified in a variety of aquatic insects. 56 preliminary studies suggest that n. risticii in fact may be transmitted via ingestion of mature caddis flies containing infected metacercariae. 57 possibly horses are infected by drinking water containing infected cercaria released from snails or by ingesting infected metacercariae in other aquatic insects. 52 the number of pcr-positive snails in endemic regions corresponds to the seasonal incidence of potomac horse fever and may be as high as 26%. 58 the pathogenesis of n. risticii is not understood completely. the organism infects and survives in monocytes and monocyte-derived leukocytes and can be found in blood monocytes during natural infections, but the sequence of events resulting in enterocolitis remains speculative. the organism appears first to infect blood monocytes in experimentally infected horses, which may be the vehicle of organ infection. 50, 59 however, in naturally infected horses, whether leukocytes of monocytic lineage or epithelial cells are infected first is unclear. the target organ is the gastrointestinal mucosa, with the most severe lesions found in the large intestine. 59, 60 infection of human colonic cells in vitro does not cause major cytopathologic effects for several days. disruption of the microvilli in the region of the plasma membrane where sodium chloride channels are located has been observed in human colonic cell cultures. 61 infection in horses is associated with variable degrees of morphologic damage. 59, 60 mild morphologic damage and mononuclear cell infiltration of the lamina propria occur early during the infection, but fibrinous, necrotizing typhlocolitis with severe mucosal ulceration and inflammation of the lamina propria may occur later in the disease. vasculitis and intravascular coagulation are consistent features in the large intestine, with perivascular edema. 60 one can observe n. risticii in mucosal cells and macrophages and mast cells of the lamina propria. 59,60 n. risticii can survive and multiply in macrophages by inhibiting the production of reactive oxygen intermediates and by avoiding lysosomal digestion by blocking phagosome-lysosome fusion. [62] [63] [64] evidence of impaired sodium chloride absorption in the colon has been suggested to contribute to diarrhea in infected horses and may be related to destruction of the enterocyte membrane structure in the region of sodium chloride channels. 61, 65 direct injury to the mucosa by n. risticii and colonic inflammation are likely to be prominent features leading to diarrhea, especially later in the disease. 60 fluid, protein, and electrolyte loss likely is caused by mucosal injury and effects on enterocyte fluid secretion caused by the inflammatory response. like other inflammatory conditions of the colon, systemic inflammation caused by absorption of bacteria and bacterial products is a potential complication of n. risticii infections if mucosal injury is severe, which contributes to the clinical signs seen during the disease. n. risticii infection is clinically similar to other forms of enterocolitis and is characterized by anorexia, depression, and fever. 48, 60, 66 experimental infections produce a biphasic fever occurring 6 to 7 days apart. decreased gastrointestinal motility, manifested as reduced borborygmi, occurs during the early stages before the onset of diarrhea. diarrhea occurs in 75% of cases and occurs 2 days after the second fever episode during experimental infections. 66, 67 the diarrhea can be moderate to severe and dehydrating. ileus can develop at any stage of the disease and can cause signs of moderate to severe colic. systemic signs of endotoxemia, shock, and peripheral edema may occur and are similar to those described for salmonellosis. experimental and natural infection with n. risticii can cause abortion of infected fetuses in pregnant mares. 68, 69 laminitis is a complication in 20% to 30% of naturally occurring cases and is often severe. other complications include protein-losing enteropathy, thrombosis, and renal failure, as described for salmonellosis. hematologic abnormalities reflect endotoxemia, dehydration, and sepsis and are essentially identical to those described for salmonellosis. neutropenia with a left shift is a consistent feature and occurs concurrently with or soon after the onset of diarrhea. thrombocytopenia is common and often severe. 67 neutrophilic leukocytosis occurs later in the course of the disease. hyperfibrinogenemia is usually more pronounced than that with salmonellosis. serum electrolyte, acid-base, and biochemical abnormalities are also similar to those described for salmonellosis. coagulopathies are common during n. risticii infection and reflect activation of coagulation pathways. disseminated intravascular coagulation is common and may be related to the high frequency of laminitis associated with n. risticii infection. 70 one cannot base diagnosis of n. risticii infection solely on clinical signs because the disease is clinically similar to other forms of enterocolitis. however, in endemic areas, acute colitis is likely to be caused by n. risiticii, and thus the clinical signs of acute inflammatory colitis in fact may have a high predictive value. serologic evidence of infection, such as rising antibody titers to n. risticii detected by indirect immunofluorescence or enzymelinked immunosorbent assay in paired serum samples may be helpful in establishing a diagnosis. 49, 71 one should take care when interpreting the indirect immunofluorescence serologic test for n. risticii because the test appears to have a high false-positive rate. 72 culture of the organism from blood is possible but is difficult and is generally useful only in the research laboratory. recently developed polymerase chain pcr tests for n. risticii dna are rapid, highly sensitive (as sensitive as culture), and specific for n. risticii infection and can be applied to blood or feces. [73] [74] [75] prevention prevention of the disease by reducing exposure to the causative organism is difficult because the mode of transmission is not known. a killed vaccine has been developed that is effective in preventing clinical illness other than fever in 80% of experimentally challenged horses using the vaccine strain. however, field studies suggest the vaccine has limited benefit for prevention or decreasing the severity of natural infection. vaccine failures have been attributed to strain differences in antigenicity or to poor antibody responses to the vaccine. 76, 77 clostridiosis is an important cause of acute enterocolitis in foals and adult horses. clostridium perfringens and c. difficile are associated most commonly with intestinal clostridiosis in horses, but other clostridial species, including c. septicum, c. cadaveris, and c. sordellii also have been isolated from horses with enterocolitis. [78] [79] [80] [81] [82] [83] in horses of all ages, clostridial enterocolitis appears to be a common antibiotic-associated and nosocomial cause of enterocolitis. 82, 84, 85 however, clostridiosis in neonatal foals is a distinct clinical entity and is discussed in more detail elsewhere. this chapter focuses on adult intestinal clostridiosis. clostridia are obligate anaerobic to aerotolerant spore-forming gram-positive rods that are ubiquitous in the environment in the spore form. 83 clostridia are elements of the normal flora of horses of all ages and are among the first bacteria acquired after birth. however, clostridia inhabiting the gastrointestinal tract normally are found in low numbers and do not produce enterotoxins. clostridiosis is associated with an increase in the number of a particular species of clostridia in the gastrointestinal tract and, perhaps most importantly, exotoxin production. although the conditions resulting in exotoxin production are not understood fully, several factors increase clostridial numbers in the gastrointestinal tract. dietary factors are known to affect the numbers of clostridium species shed in the horse feces. 78 experimental induction of colic increases fecal shedding of clostridium species in the absence of diarrhea. 86 antibiotics, particularly administered orally or recycled via the enterohepatic system, are well documented to increase the recovery of clostridia colony-forming units (cfus) in equine feces and may result in clinical clostridiosis. 79, 81, [87] [88] [89] [90] indeed, clostridiosis associated with c. perfringens or c. difficile is likely to be the most important cause of antibiotic-induced enterocolitis in the horse. clostridium perfringens c. perfringens is made up of many genetically distinct strains of variable virulence that produce one or more of a large group of exotoxins. the pattern of exotoxin production is used to classify c. perfringens into five types, a, b, c, d, and e. c. perfringens type a is the most common clostridium isolate from healthy horses of all ages and adults and foals with diarrhea worldwide. c. perfringens types a, b, c, and d have been associated with hemorrhagic enteritis in foals less than 10 days of age, with type c being the most common cause in north america. the primary toxin produced by c. perfringens type a is α-toxin (phospholipase c), which interferes with glucose uptake and energy production and activates arachidonic acid metabolism and signaling pathways in enterocytes. 83 oral administration of α-toxin does not cause tissue necrosis but causes increased secretion by small intestinal mucosal cells in human beings. 91, 92 the β-toxin of types b and c is a cytotoxin that causes enterocyte necrosis, ulceration, and ultimately severe intestinal inflammation and hemorrhage. 93 a novel toxin designated β2 may also have a role in c. perfringens enterocolitis. 94 the biologic activity of the β2-toxin is similar to β-toxin, but β2-toxin is not related to β-toxin in sequence. the β2-toxin was prevalent in two groups of horses with acute enterocolitis but not in healthy horses. 95 the β2-toxin appears to be associated predominantly with c. perfringens that would have been classified otherwise as type a but that in fact may represent a previously undescribed type. virulent strains of c. perfringens type a and to a lesser extent type c also may produce enterotoxin. enterotoxin is a cytotoxin that inserts into cell membranes to form pores, which alter permeability to water and macromolecules and ultimately lead to cellular necrosis. 96 massive desquamation of the intestinal mucosa resulting from enterotoxin cytotoxicity triggers an inflammatory response, intestinal edema, mural hemorrhage, and systemic inflammation. 97 enterotoxin also alters tight junction integrity, resulting in increased paracellular permeability by a noncytotoxic mechanism. 98 clostridium difficile c. difficile produces several toxins, but only two, toxin a and toxin b have been studied in detail. toxin b is a potent cytotoxin in vitro, but its role in enterocolitis is less clear than the role of toxin a. toxin b does not induce fluid secretion, inflammation, or characteristic alterations in intestinal morphology. c. difficile induces an inflammatory response with hypersecretory diarrhea that is induced in large part by the enterotoxin toxin a. 99 toxin a induces neutrophil influx into intestinal tissue, mast cell degranulation, and secretion of prostaglandins, histamine, cytokines, and 5-hydroxytryptamine by these activated leukocytes. 100, 101 the products of neutrophils and mast cells have a prominent role in the vasodilatory and secretory responses in the intestine during c. difficile infection. the enteric nervous system is central to the induction of intestinal inflammation and mucosal secretion by toxin a. a model for toxin a-induced secretory diarrhea has emerged in which toxin a stimulates substance p-containing afferent sensory nerve fibers, which in turn stimulate mast cell degranulation, recruitment and activation of polymorphonuclear leukocytes, and vasodilation. [102] [103] [104] toxin a-induced stimulation of enterocyte secretion can occur via secretomotor neuronal stimulation by substance p-containing sensory neurons or products of mast cells and polymorphonuclear leukocytes. neural blockade or depletion of substance p abolishes mast cell degranulation, polymorphonuclear leukocyte influx, and enterocyte secretion. how toxin a triggers the sensory component of the enteric nervous system is not known yet, but toxin a-induced necrosis of enterocytes likely exposes afferent neurons to the noxious milieu of the intestinal contents. equine intestinal clostridiosis is clinically similar to other forms of acute enterocolitis in horses. 78, 83 although the clinical course is usually acute, peracute colitis with rapid death may occur. occasionally, a milder, more prolonged clinical course occurs. one may note fever, anorexia, and depression before the onset of gastrointestinal signs, but more commonly no prodromal signs are apparent. signs of endotoxemia and shock may accompany acute signs of colic and severe, dehydrating diarrhea. diarrhea may not be profuse but is usually dark and foul smelling. like the clinical signs, hematologic and serum biochemical abnormalities are similar to those associated with other forms of enterocolitis and reflect fluid, protein, and electrolyte loss and systemic inflammation from endotoxemia. neutropenia, leukopenia, and hemoconcentration are common. hypoproteinemia may be profound. one often may note hyponatremia, hypokalemia, hypochloremia, hypocalcemia, and a mixed prerenal/renal azotemia, as well as metabolic acidosis and coagulopathies. serum concentrations of hepatocellular enzymes such as sorbitol dehydrogenase may be elevated, and liver function may be reduced. preliminary diagnosis of equine intestinal clostridiosis caused by c. perfringens is based on the isolation of greater than 100 cfus of c. perfringens type a per gram of feces from patients with diarrhea and signs suggestive of toxemia. similar criteria are used to screen human patients for c. perfringens type a infection. normal horses shed fewer than 100 cfus/g of feces, and usually horses with intestinal clostridiosis shed greater than 1 × 10 6 cfus/g. 78, 105 however, identification of increased numbers of clostridium organisms in the feces does not prove infection. detection of c. perfringens toxins in feces or intestinal contents in horses with increased numbers of fecal cfus and clinical signs of enterocolitis is more conclusive evidence of an enterotoxigenic infection than culture alone. immunoassays are available that are primarily designed to detect c. perfringens enterotoxin. 83 however, the reliability (specificity) of some immunoassays for diagnosis of c. perfringens infection has come into question. recently, pcr multiplex and gene probe assays have been developed to detect the major lethal toxins in isolates or fecal samples to determine the pattern or toxin production and are currently the preferred methods of detection. [106] [107] [108] as for c. perfringens, diagnosis of c. difficile infection consists of culture of the organism from feces and identification of toxins in the feces. bacterial culture of c. difficile may be difficult and may be an insensitive test in horses. [109] [110] detection may require enrichment techniques and culture of multiple fecal samples. 110, 111 detection of toxins a and b in feces is regarded as the preferred test for diagnosis of c. difficile infection in human beings. 83 commercial enzyme-linked immunosorbent assays are available for toxins a and b that are specific and appear to be more sensitive than a single culture for identifying c. difficile infection in adult horses. 109, 110 one also may induce toxin production by c. difficile isolates. sensitive pcr methods also have been developed for application to fecal samples and isolates to detect the genes for toxins a and b. 83 proliferative enteropathy is a chronic hyperplastic disorder of the small intestine and has been described in a variety of mammalian and avian species. 112, 113 the only causative agent identified to date that induces proliferative enteropathy is the obligate intracellular pathogen lawsonia intracellulare. 113, 114 the pig is the most frequently naturally affected species. however, the reports of equine proliferative enteropathy associated with l. intracellulare have increased in recent years. [115] [116] [117] [118] the relatedness of the strains of l. intracellulare causing proliferative enteropathy in pigs and horses or even among other affected species is not known. no host restriction is apparent because hamsters and other rodents can be infected with porcine strains of l. intracellulare. before the year 2000, reports of proliferative enteropathy in the literature describing isolated cases were sporadic. [116] [117] [118] however, since 2000, outbreaks on breeding farms have been documented on farms in canada, suggesting that a new strain has emerged. 115 the mode of infection is fecal-oral, and infected animals can shed large numbers of organisms in feces. 113 affected animals shedding the organism in the feces serve as a source of infection for herdmates. possibly nonequine species serve as reservoirs contributing to outbreaks on horse farms. factors that increase the risk of proliferative enteropathy in pigs include overcrowding, ration changes, transport, and weaning. 113, 114 like pigs, horses are affected as weanlings. factors associated with weaning and other stresses may affect immunity and increase susceptibility to infection. the incubation period is 2 to 3 weeks in nonequine species and is presumed to be similar in horses. experimental l. intracellulare infection produces characteristic pathologic lesions in pigs and hamsters that are identical to lesions in horses with proliferative enteropathy. 113, 114 a profound hyperplasia of the mucosa predominantly caused by proliferation of crypt epithelium and crypt hyperplasia is induced locally in infected islands of tissue and eventually extends to the entire distal jejunum and ileum. l. intracellulare preferentially infects proliferating cells, thus the tropism for the crypt epithelium. infected cells proliferate far more rapidly than uninfected cells, suggesting that l. intracellulare directly induced the proliferative response. however, the molecular basis for the enhanced proliferation is not known. l. intracellulare penetrates epithelial cells in a membrane-bound vesicle but eventually escapes the vacuole and is found free in the cytoplasm concentrated at the apical pole of the cell. the gross pathologic lesions found in equine proliferative enteropathy are characteristic. [115] [116] [117] [118] lesions may be segmental and typically are found in the ileum and terminal jejunum in horses. however, lesions also may affect the duodenum. severe mucosal hypertrophy often occurs but may wane during the chronic stages of the disease. the mucosa may become corrugated with focal erosions or ulcers. one often can identify submucosal edema easily on cut sections of affected segments. moderate to severe crypt hyperplasia with atrophy of the intestinal villi is a consistent feature. the hyperplastic crypts are branched and may herniate into the submucosa. necrosis, edema of the submucosal and lamina propria, hemorrhage, mononuclear inflammation and muscular hypertrophy have been reported in affected intestinal segments but are not consistent. special stains such as silver stain are required to detect intracellular organisms. the organisms are curved or comma-shaped rods found clustered in the apical cytoplasm of hyperplastic crypt epithelium. the proliferative response of the intestinal mucosa alters absorption of nutrients and fluid secretion by disrupting the architecture of the villi and by altering the maturation of epithelial cells into absorptive cells, accounting for the secretory diarrhea and often severe weight loss. 112, 114 the combined effects of the inflammatory 892 part ii disorders of specific body systems response and malabsorption may account for the protein-losing enteropathy. proliferative enteritis most commonly affects weanling foals ages 4 to 6 months. the clinical signs of proliferative enteropathy include ill thrift, weight loss, peripheral edema, diarrhea, and colic. [115] [116] [117] [118] the diarrhea is usually in the form of soft feces but may be profuse and watery. some foals with mild diarrhea have black, tarry feces. secondary complications such as gastric ulceration, bronchopneumonia, or parasitism may occur concurrently with the proliferative enteropathy. clinicopathologic features include mild to moderate anemia, moderate to severe hypoalbuminemia (often <2 g/dl), hypoglobulinemia, neutrophilic leukocytosis, and hyperfibrinogenemia. creatine kinase activities may be elevated in affected foals. prerenal azotemia and electrolyte imbalances such as hyponatremia may be associated with diarrhea. peritoneal fluid analysis is usually unremarkable. ultrasonographic examination of the small intestine often reveals significant thickening of the intestinal wall ( figure 13 .13-1). intestinal edema may be evident as a hypoechoic appearance to one or more layers of the intestinal wall. methods for antemortem diagnosis include serologic analysis of l. intracellulare antibodies and pcr analysis of feces. 115 serologic analysis using an indirect immunofluorescent antibody test may be the most useful single test available. the pcr test is specific but the sensitivity may be low. by the time clinical signs appear, 90% of pigs are serologically positive for anti-lawsonia intracellulare immunoglobulin g (igg). in contrast, only 37% of pigs had positive fecal pcr tests. 119 of the seven foals tested in an outbreak of equine proliferative enteropathy, 115 four foals with confirmed disease and three with suspected proliferative enteropathy had serologic titers against l. intracellulare of 1:30 or greater. in contrast, serum samples collected from 72 foals before the outbreak were negative for l. intracellulare antibodies. fecal pcr for l. intracellulare was positive in 6 of 18 foals tested, and half of the serologically positive foals had negative fecal pcr tests. many clinicians combine serologic analysis with fecal pcr testing to increase the sensitivity and specificity of these diagnostic methods. isolation and culture of the organism requires cell culture techniques that are not widely available. thus no practical method exists for culturing the organism from feces or tissues that is available for clinical use. definitive diagnosis requires histopathologic examination of affected tissues. 112 diagnosis is based on typical histopathologic findings of mucosal hypertrophy and submucosal edema and identification of small, curved, rod-shaped intracellular bacteria at the apical pole of epithelial cells in affected segments of intestine. special stains such as warthin-starry silver stain are required to detect the bacteria in histopathologic specimens. pcr analysis of affected intestinal tissue is a specific test for the presence of l. intracellulare and, unlike fecal pcr analysis, appears to be sensitive. 120 strongyle infections in horses are caused by two groups of nematodes: large and small strongyles (see the section cyathostomiasis). large strongyles that are pathogenic in horses include strongylus vulgaris, s. edentatus, and s. equinus. of these species, s. vulgaris is by far the most important cause of disease in the large intestine and in fact is the most pathogenic parasitic infection in horses. s. vulgaris infection in horses is manifested clinically by two forms: acute and chronic disease. 121 the age and resistance of the host, the infective dose, and the size and function of the affected arteries influence the type and degree of disease that occurs. sudden ingestion of large numbers of infective larvae by a naïve host causes acute strongylosis, whereas ingestion of fewer infective larvae over a long period of time by an older, more resistant host causes chronic strongylosis. acute strongylosis is more likely to cause colic than diarrhea and may be rapidly fatal. chronic strongylosis tends to cause debilitation and signs of colic but may also cause diarrhea. diarrhea associated with acute strongylosis occurs within several days of infection and is likely to be caused by migration of the larvae through the intestinal wall. fourth-stage larvae migrate through the mucosa and submucosa into the arterioles of the intestine, causing mural edema, hemorrhage, and infiltration with inflammatory cells into the intestinal wall. 121,122 increased secretion and decreased absorption of fluid and electrolytes, stimulated by inflammatory mediators such as prostaglandins and histamine, may play a role in the diarrhea induced by s. vulgaris. interstitial edema and damage to the interstitial matrix and mucosa may result from inflammation and migration of the parasites, causing increased secretion of fluid and albumin loss. abnormal gastrointestinal motility also may play a role in the development of diarrhea. migration of larvae through the intestinal wall early in the course of infection affects myoelectric activity and motility in the large intestine and may affect retention of ingesta and absorption of fluid. 123,124 the cause of death in acute strongylosis has not been addressed but may be related to massive migration through the vasculature, causing thrombosis with ischemia and infarction of the intestine. chronic strongylosis causes typical verminous arteritis and is associated more commonly with natural infections in horses than with acute strongylosis. 121 lesions of the large intestinal vasculature caused by migration of larvae through the intima are characterized by thrombus formation, narrowing of the arterial lumen, fibrosis, and thickening of the arterial wall. 121,122 embolization may occur, causing acute segmental infarction of the large intestine, but more commonly reduced blood flow without embolization causes ischemia and occasionally infarction. 122,125 postmortem examination of horses with colonic infarction failed to reveal embolization as the cause in most cases. 125 reduced blood flow in the tissues of the intestine usually results from narrowing of the arterial lumen by the thrombus and formation of microthrombi at sites independent of the parasites. release of vasoconstrictive inflammatory mediators such as leukotrienes from platelets, neutrophils, and eosinophils and elaboration of parasitic antigens or toxins may cause vasoconstriction and ischemia. 126 horses with experimental strongylosis were found to have a 50% reduction of blood flow in the colonic vasculature. 127 clearly, reduced blood flow is an important effect of chronic strongylosis, but the relationship between blood flow and diarrhea is unclear. disrupted motility resulting from ischemia may lead to diarrhea by reducing the retention of ingesta and absorption of fluid. acute infarction and mucosal ulceration have been found to cause severe chronic diarrhea in naturally infected horses. 128 release of inflammatory mediators such as prostaglandins, histamine, and kinins from inflammatory cells associated with thrombi and inflamed intestine also may affect secretion, absorption, and motility, leading to diarrhea. the clinical signs of acute strongylosis caused by s. vulgaris infection are characterized by depression, moderate to severe colic, and fever. 129 diarrhea is less often a feature of acute strongylosis than is colic. 121 most cases of acute strongylosis occur in young, naïve horses that are introduced to an infested environment or are inoculated experimentally with infective larvae. this form of strongylosis often is not recognized naturally. chronic strongylosis, however, occurs most commonly as a natural syndrome. weight loss or poor weight gain; chronic, intermittent colic; fever; poor appetite; and diarrhea are frequent signs. 121,122 diarrhea may be profuse and watery, or the feces may be soft but of normal volume. transrectal palpation may reveal thickening and fremitus in the cranial mesenteric artery. young horses are most commonly affected, but older horses also may be affected. horses with acute infarction or large intestinal ulceration following chronic strongylosis may have signs of severe abdominal pain, sepsis, and endotoxemia, and profuse, watery diarrhea is common. hematologic abnormalities associated with strongylosis include neutrophilic leukocytosis and eosinophilia. [129] [130] [131] neutrophilia appears to be an early event during the course of the disease, and eosinophilia tends to appear later. 129, 131 hyperfibrinogenemia also may occur, especially later in the course of the disease. serum αand β-globulin and igg(t) concentrations are characteristically elevated. [130] [131] [132] horses with chronic ulcerative colitis following strongylosis may exhibit severe hypoalbuminemia. 128 peritoneal fluid analysis may reveal an elevated protein concentration and eosinophilia. 130, 131 tentative diagnosis is based on clinical signs, hematologic abnormalities, and peritoneal fluid analysis. elevated serum αand β-globulin concentrations and igg(t) concentration support the diagnosis. 132 fecal analysis may reveal strongyle eggs, but fecal egg counts are often unreliable because nonpatent larvae cause the disease. appropriate preventive measures are important in controlling this disease, including management procedures such as preventing overcrowding, reducing exposure of susceptible individuals, and instituting proper deworming schedules. ivermectin is the preferred anthelmintic used to control strongylosis in horses. monitoring fecal egg counts as a means of evaluating the efficacy of parasite control measures is recommended. infection with small strongyles (cyathostomiasis) is well recognized as a cause of diarrhea and large intestinal disease in horses of all ages. [133] [134] [135] [136] [137] [138] clinical disease is caused by intramural larval stages. the cyathostome life cycle requires migration by fourth-stage larvae through the mucosa of the large intestine and may include a period of hypobiosis during which the larvae remain encysted within the mucosal layer of the large intestine. after a period of hypobiosis the larvae emerge in response to a largely unknown stimulus. most cases occur when larval emergence takes place, classically in the late winter and spring in the northern temperate zones when larvae are expected to emerge and in the late fall or winter months in the southeastern united states and subtropical regions. 133 sudden emergence of encysted larvae causes the mucosal injury, ulceration, and inflammatory reaction responsible in large part for the clinical disease. 133, 139 however, migration of the larvae as they penetrate the mucosa affects motility patterns and can cause inflammation that may contribute to diarrhea. 139 chronic, eosinophilic, granulomatous colitis and diarrhea with histopathologic evidence of hypobiotic cyathostome larvae in the large intestine have been reported in two horses during a period in which emergence of larvae would not be expected to occur (early winter). 133 natural emergence of cyathostome larvae causes fibrinous inflammation of the large intestine, focal necrosis, mural hemorrhage, and ulceration of the large intestinal mucosa, which even may result in bleeding into the lumen. mild to moderate eosinophilic and mononuclear inflammation of the lamina propria occurs, and moderate to severe interstitial edema frequently occurs. 122, 139 colonic inflammation and interstitial edema may contribute to the diarrhea, along with the loss of the mucosal barrier, by causing increased active and passive secretion of fluid, electrolytes, and protein. protein loss is often significant, resulting in profound hypoalbuminemia and interstitial edema of skin and other organs. chronic, granulomatous colitis has been reported to occur in response to encysted larvae and may cause diarrhea by increased secretion following granulomatous inflammation or disruption of the interstitium by granulomatous infiltration. administration of an anthelmintic to horses with a heavy load of encysted larvae also may cause rapid larval death and acute and often severe inflammation similar to natural emergence. cyathostomiasis may be the most commonly identified cause of chronic diarrhea in the horse. [140] [141] [142] however, an acute syndrome also has been associated with cyathostomiasis. 138 clinical signs of cyathostomiasis are characterized by moderate to severe weight loss or poor weight gain, ill thrift, ventral edema, intermittent fever, and intermittent, mild colic. acute onset of diarrhea is typically profuse and progresses to chronic diarrhea that is often mild, is the consistency of bovine feces, and may be intermittent. [133] [134] [135] [136] [137] [138] 142 appetite is usually normal, but affected horses occasionally have a ravenous appetite. transrectal palpation usually does not reveal any abnormalities. cyathostomiasis may affect any age of horse, and clinical signs are more common during periods of emergence of larvae, corresponding to late winter and spring in northern temperate zones. the deworming history may appear to be adequate. neutrophilic leukocytosis is typically evident, but the white blood cell count may be normal. [133] [134] [135] [136] [137] [138] profound hypoalbuminemia is a characteristic feature of cyathostomiasis, manifested clinically by ventral edema. plasma αand β-globulin concentrations may be elevated, which can result in a normal total plasma protein concentration in spite of hypoalbuminemia. [134] [135] [136] the serum igg(t) concentration, however, has been reported to be normal, which may help distinguish cyathostomiasis from s. vulgaris infection. 133, 135, 136 in addition, peritoneal fluid analysis does not usually reveal any abnormalities, in contrast to horses with s. vulgaris infection. fecal analysis may be unrewarding because the infection is often not patent when clinical signs are apparent. measurement of plasma fructosamine may provide a measure of protein catabolism or protein loss in the absence of hypoalbuminemia. plasma fructosamine concentrations are significantly lower in horses with experimental cyathostomiasis than in normal controls, 142, 143 suggesting that this test may be a useful diagnostic tool. however, the test has not yet been validated in naturally occurring cases, and neither the specificity nor the sensitivity is known. rectal scrapings or rectal mucosal biopsies may reveal evidence of cyathostome larvae. 133, 136 definitive diagnosis usually requires microscopic examination of biopsy specimens of the cecum and ascending colon, collected by laparotomy. examination of biopsy specimens collected from the small intestine is recommended to rule out other causes of weight loss and diarrhea. one should include appropriate diagnostic tests, such as culture of feces for pathogenic bacteria, in the workup to rule out other causes. preventive measures are appropriate for other horses on premises known to have a problem with cyathostomiasis, particularly frequent deworming (every 6 weeks) during times of high infectivity (spring and summer in the north and fall, winter, and early spring in the south) to eliminate parasites before they become patent. 133 because of high levels of resistance to benzimidazoles, avermectins (ivermectin or moxidectin) are the drugs of choice. [144] [145] [146] resistance to ivermectin has been demonstrated, but the prevalence of ivermectin resistance appears to remain low. 144 although daily pyrantel pamoate administration also has been reported to reduce worm burdens and pasture infectivity in young and mature horses effectively, 147 cyathostome resistance has been reported and is a concern for the use of this drug as a routine preventive anthelmintic. 145, 148 diarrhea in adult horses may also occur secondary to administration of antimicrobial or antiinflammatory medications or after ingestion of toxic compounds. affected horses exhibit clinical signs that may be indistinguishable from signs exhibited by horses with diarrhea of infectious etiology. antibiotic-associated diarrhea has been reported in many species, including horses. 149 certain antibiotics-such as trimethoprim-sulfonamide combinations, erythromycin, penicillins, tetracyclines, clindamycin, and lincomycinare associated with naturally occurring and experimental enterocolitis syndromes in horses. 79, [149] [150] [151] [152] in some cases, such as with trimethoprim-sulfonamide combinations, the geographic incidence of antibiotic-associated diarrhea appears to differ considerably. clostridium perfringens, c. difficile, and salmonella spp. are apparently the most common causes of antibioticassociated diarrhea in horses. outbreaks of c. difficile have been reported in hospitalized horses being treated with antibiotics. 81, 85 in sweden, accidental erythromycin ingestion has been associated with c. difficile enterocolitis in mares in which their foals were being treated for rhodococcus equi. 89, 151, 153 interestingly, this phenomenon has not been reported in other areas of the world. foals being treated with erythromycin are at a higher risk for diarrhea than foals being treated with other antibiotics. tetracycline administration has been shown to be associated with an increase in the numbers of gram-negative enteric bacteria and c. perfringens in the feces of horses and reactivation of salmonellosis and prolongation of fecal shedding of salmonella. 78, 154 the most common mechanism by which antibiotics cause diarrhea is by disrupting the gastrointestinal flora. the normal large intestinal flora, comprised of mainly obligate anaerobes and streptococci, protects the host from pathogenic bacteria by colonization resistance. 17 ecologic factors play an important role in colonization resistance. for example, surface bacteria in the large intestine interact with receptors on the mucosal cells, facilitating adherence to the mucosa. 17, 155 in doing so, the normal organisms compete more successfully for this important niche. competition for space and nutrients is an important means of preventing colonization and proliferation of pathogenic bacteria. in addition, anaerobic bacteria produce short-chain fatty acids (scfas) and other metabolites that are toxic to facultative anaerobic bacteria, especially in the conditions of the large intestine. 16, 17, 155 organisms of the normal flora also produce bacteriocins that inhibit growth of potential pathogens. 16 antibiotics that deplete the population of obligate anaerobes and streptococci efficiently decrease colonization resistance. 16 production of fatty acids diminishes, thus reducing competition for space and nutrients. as a result, gram-negative enteric bacteria such as salmonella are able to proliferate. in addition, pathogenic anaerobes normally found in low numbers can proliferate. antibiotic-resistant strains of bacteria, especially gram-negative enteric bacteria and possibly clostridia, may be selected by antibiotic administration, allowing proliferation of pathogenic bacteria resistant to many antibiotics. 156 obligate anaerobic commensal organisms, perhaps the most critical group of microbes for maintaining colonization resistance, are usually susceptible to macrolides, tetracyclines, β-lactams, and lincosamides, perhaps explaining the high incidence of diarrhea associated with the administration of these antibiotics. 83 in addition to reduction of colonization resistance, depletion of the normal anaerobic microbial population in the intestine decreases carbohydrate fermentation and production of scfas, which contributes to the pathogenesis of antibiotic-associated diarrhea by decreasing absorption of sodium and water by the colonic mucosa. 157 ampicillin decreases colonic fermentation of carbohydrates in human beings. 158 human patients with antibioticassociated diarrhea have greatly impaired colonic fermentation and low production of scfas. erythromycin, ampicillin, or metronidazole treatment is associated with decreased production of scfas in patients with and without diarrhea. 88 absorption of sodium and water is stimulated by absorption of scfas in the equine colon, suggesting that reduction of colonic scfa content by antibiotic-induced depletion of anaerobic flora has similar effects in horses as in human beings. 157 broad-spectrum antibiotics exert a more profound effect on the gastrointestinal flora than narrow-spectrum antibiotics. antibiotics administered orally, especially those that are poorly absorbed, are more likely to cause diarrhea than are parenterally administered antibiotics. for instance, clindamycin is less likely to cause diarrhea in human beings when administered intravenously than when administered orally. antibiotics with extensive enterohepatic circulation, such as tetracyclines and erythromycin, are excreted in high concentrations in the bile and are associated more commonly with diarrhea than antibiotics that do not undergo enterohepatic circulation. 159 antibiotics may cause diarrhea by means other than by disrupting the normal flora. direct toxic effects may play a role in producing irritation, increasing secretion, and disrupting motility patterns. tetracyclines are irritating to the gastrointestinal mucosa and may cause inflammation and increase secretion. 159 erythromycin has been shown to interact with smooth muscle cells, stimulating gastrointestinal motility. 159, 160 normal peristalsis plays an important role in suppressing the population size of potentially pathogenic bacteria. normally, bacteria that are prevented from adhering to the mucosa by colonization resistance are swept aborally by peristalsis and are excreted in the feces. disruption of normal motility patterns may prevent clearance of pathogenic bacteria, contributing to the colonization of mucosal surfaces. diarrhea induced by antibiotics usually occurs within 7 days of antibiotic administration or can occur several days after cessation of antibiotic treatment. the clinical syndrome of antibiotic-associated diarrhea can vary from mild diarrhea to fulminant enterocolitis with severe diarrhea. mild cases of diarrhea are common, especially in foals receiving erythromycin, trimethoprim-sulfonamide combinations, or rifampin. 151, 161 mild cases of diarrhea are usually not clinically significant. however, acute, severe enterocolitis can occur in all ages of horses receiving antibiotics and can be life threatening. clinical signs are identical to other causes of acute enterocolitis. severe, dehydrating diarrhea, endotoxemia, sepsis, and shock may occur. hemoconcentration, neutropenia, hypoproteinemia, and electrolyte and acid-base imbalances are common. severe hyponatremia may occur in foals with antibiotic-associated diarrhea, especially if trimethoprimsulfonamide and rifampin combinations are the cause. 161 more detailed descriptions of the clinical and laboratory findings were given previously. diagnosis is presumptive, because definitive diagnosis of antibiotic-associated diarrhea is impossible. fecal culture and pcr testing may reveal salmonella or clostridium infection. toxicity resulting from nonsteroidal antiinflammatory drug (nsaid) administration has been well documented in several species, including horses. [162] [163] [164] [165] [166] [167] [168] in horses and human beings, nsaid toxicity is manifested by renal and gastrointestinal disease. elderly human patients are more susceptible to nsaid toxicity, but the effects of age on nsaid toxicity in horses are less well defined. foals are considered to be more susceptible than adult horses to gastrointestinal disease following nsaid administration, and ponies may be more susceptible than horses. all nsaids are capable of inducing gastrointestinal and renal damage at toxic concentrations, and the toxicity is not significantly different among products. aspirin is potentially more toxic than other nsaids because it irreversibly inactivates cyclooxygenase by acetylation, whereas other nsaids reversibly inhibit cyclooxygenase. 162 however, phenylbutazone is the drug most commonly reported to cause toxicity in horses, perhaps because of its widespread use by veterinarians and horse owners. phenylbutazone toxicity in horses is characterized by mucosal ulceration throughout the gastrointestinal tract, oral ulceration, renal papillary necrosis, vasculopathy, thrombosis, and protein-losing enteropathy with hypoalbuminemia. [164] [165] [166] this discussion focuses on the toxic effects of nsaids on the large intestine but necessarily includes elements of upper gastrointestinal and renal disease. horses with large intestinal disease resulting from nsaid toxicity generally are receiving inappropriately large doses. the dosage regimen recommended for phenylbutazone (4.4 mg/kg twice in 1 day and then 2.2 mg/kg twice daily) is considered to be safe. experimental studies in horses, however, have shown toxicity to occur when greater than the recommended dosage (6.6 mg/kg/day) is administered for several days. 164, 165 in most reported cases of phenylbutazone toxicosis horses were receiving higher than recommended dosages. 166, 168, 169 regardless, administration of phenylbutazone at the recommended dosage has been reported to cause a significant decrease in plasma protein concentration and gastrointestinal disease. 165, 170 moreover, signs of nsaid toxicity have been reported in normovolemic horses treated with appropriate doses of phenylbutazone. 170, 171 dehydration, sepsis, and endotoxemia exacerbate the renal and gastrointestinal toxicity of nsaids. 162 clearly, the margin of safety is narrow for phenylbutazone and probably for other nsaids used in horses as well. gastrointestinal disease induced by nsaids is manifested by mucosal ulceration, inflammation, bleeding, and protein-losing enteropathy. 164, 165, 168, 170 in addition to direct effects on the mucosal barrier, nsaid administration has been shown to cause an acute relapse of preexisting colonic inflammatory disease and worsen colonic inflammation in human beings. 164, 165, 170 whether this occurs in horses is not clear. the mechanism by which nsaids induce mucosal damage is probably multifactorial. direct irritation may play a role in oral and gastric irritation and ulceration; however, parenteral administration of nsaids produces oral and gastric ulceration as well. inhibition of prostaglandin synthesis by inhibition of cyclooxygenase 1 (the constitutive cox) and cyclooxygenase 2 (the inducible cox) appears to be the most important mechanism of mucosal injury. prostaglandins, particularly pge 2 and pgi 2 , are critical for mucosal health. 172,173 pge 2 has been shown to increase mucosal blood flow; increase secretion of mucus, water, and bicarbonate; increase mucosal cell turnover rate and migration; stimulate adenyl cyclase activity; and exert other protective effects in the gastric mucosa of several species. 162, 172, 173 perhaps most importantly, pge 2 and pgi 2 have a role in maintaining epithelial tight junction integrity, which is indispensable for mucosal barrier function and repair after mucosal injury. 172 in spite of the overwhelming amount of information about the role of prostaglandins in maintaining the mucosal barrier in other species and clear clinical and experimental evidence that nsaids injure the equine colonic mucosa, the role of prostaglandins in mucosal protection in the equine colon is not yet well defined. inhibition of cox-1 and cox-2 in equine colonic mucosa with flunixin meglumine resulted in reduced electric resistance of the mucosa and increased permeability to macromolecules in vitro (a.t. blikslager and s.l. jones, 2002) , suggesting that flunixin treatment disrupts the epithelial tight junctions in the equine colon. mucosal changes were correlated with a profound inhibition of pge 2 and pgi 2 concentrations in the treated tissues. in other studies, administration of a pge 2 analog prevented the gastrointestinal manifestations of phenylbutazone toxicosis in ponies. 165 recent development of nsaids specific for cox-2 have greatly reduced the frequency and severity of gastrointestinal side effects in human beings taking nsaids for chronic musculoskeletal conditions. 174 thus cox-2-specific nsaids hold promise for use in horses to treat arthritis and reduce the incidence of toxicity. for example, the cox-2-specific inhibitor etodolac was less harmful to equine colonic mucosa than flunixin meglumine in vitro (a.t. blikslager and s.l. jones, 2002) . moreover, etodolac was significantly more permissive than flunixin for recovery of the mucosa in equine ischemic-injured intestinal tissues, and in fact, recovery was no different than control tissues. 175 however, their use is at present limited because the specificity of the so-called cox-2selective inhibitors and their efficacy as analgesics have not been demonstrated in the horse. nsaid-induced mucosal injury is associated with a significant inflammatory response to microbial products exposed to the lamina propria. 176 this inflammation exacerbates mucosal dysfunction and injury associated with nsaid toxicity. for example, depletion of neutrophils or blockade of neutrophil influx into gastrointestinal tissues or inhibition of neutrophil activation and release of toxic products prevents many of the pathophysiologic effects of nsaid toxicity in the gastrointestinal tract. [177] [178] [179] [180] the inflammatory response alone may result in moderate to severe gastrointestinal ulceration, mural vascular thrombosis and edema, fluid secretion, protein-losing enteropathy, and mucosal hemorrhage. nsaid colitis manifests as two clinical syndromes: right dorsal colitis (rdc) and generalized nsaid toxicity. rdc is an ulcerative disorder isolated to the right dorsal segment of the large intestine. 167, 168, 171 the most prominent clinical signs of rdc are anorexia, lethargy, and colic. anorexia, depression, diarrhea, fever, and signs of section 13.13 inflammatory diseases of the gastrointestinal tract causing diarrhea endotoxemia also may be features. if the rdc is chronic, weight loss, intermittent colic, lethargy, anorexia, and ventral edema are common clinical signs, along with soft and unformed feces. severe ulceration of the right dorsal colonic mucosa results in proteinlosing enteropathy and significant hypoproteinemia attributable mainly to hypoalbuminemia. hypoproteinemia may be severe enough to cause peripheral (usually ventral) edema.in some cases, one may note dehydration, electrolyte abnormalities, neutropenia or anemia, azotemia, and biochemical abnormalities if the ulceration and diarrhea are severe or if systemic inflammation is present. clinical signs of generalized nsaid toxicity may vary from mild diarrhea with no systemic signs to severe dehydrating diarrhea with anorexia, fever, depression, peripheral edema, oral ulceration, and colic. 165, 166, 169 clinical signs of systemic inflammation caused by endotoxemia may occur, manifested as poor peripheral perfusion, tachycardia, tachypnea, weakness, trembling, and cyanotic or hyperemic oral mucous membranes. hematuria or oliguria may be present if renal involvement is present. complications associated with other forms of severe enterocolitis, such as laminitis, thrombophlebitis, and severe weight loss, may occur. hematologic abnormalities of generalized nsaid toxicity are nonspecific and include neutropenia with a left shift or leukocytosis and hemoconcentration. serum biochemical analysis is characterized by profound hypoproteinemia, hyponatremia, and metabolic acidosis. 169, 170 hypocalcemia, hypokalemia, hypochloremia, and elevated hepatocellular enzyme activities also may occur. hypoproteinemia may occur without signs of diarrhea. azotemia may be prerenal from dehydration but frequently is caused by renal failure resulting from a combination of hemodynamic and toxic renal injury. urinalysis frequently reveals hematuria, proteinuria, cylindruria, and isosthenuria. fecal occult blood is frequently detectable. diagnosis of either form of nsaid colitis is often presumptive, with a history of overdose of nsaids being strong evidence of nsaid toxicity. but as discussed earlier, toxicity may occur with dosage regimens that are not considered inappropriate, particularly if the horse experiences a concurrent period of dehydration. one can use ultrasonographic examination of the right dorsal colon to confirm a diagnosis of rdc, but the sensitivity of this method is questionable. ultrasonography (3.5-to 5-mhz transducer at the right twelfth to fifteenth intercostal spaces below the margin of the lung axial to the liver) may reveal a thickened right dorsal colon (>0.5 cm) and evidence of colonic edema in horses with rdc. 181 however, the sensitivity of this method of diagnosis is questionable. one can use nuclear scintigraphy of horses after infusion with technetium 99-labeled white blood cells to document inflammation of the right dorsal colon. 182 diagnosis of rdc may require one to perform laparotomy or laparoscopic examination of the right dorsal colon. one must rule out other causes of enterocolitis, such as salmonellosis, potomac horse fever, clostridiosis, and antibiotic-associated diarrhea. cantharidin is the toxic principle found in beetles of the genus epicauta, commonly known as blister beetles. [183] [184] [185] ingestion of the beetles in contaminated alfalfa hay causes release of the toxin from the tissues of the beetle and absorption through the gastrointestinal tract. transcutaneous absorption may occur but appears to be rare in horses. 184 blister beetles feed on the flowers of alfalfa and may be incorporated into processed alfalfa hay if the hay is cut and processed simultaneously, as by crimping. [183] [184] [185] the beetles often swarm, and one may find large numbers of beetles in small portions of hay. the lethal dose of cantharidin is less than 1 mg/kg, but the concentration of cantharidin varies from species to species of blister beetles and between sexes. 183, 184 as many as 100 to as few as 6 to 8 beetles may be lethal. usually, only one or a few horses fed contaminated hay ingest beetles because the beetles are concentrated in a small portion of the hay. however, outbreaks involving many horses on a farm have occurred. most cases occur in texas and oklahoma, but horses in other states may be affected as well, especially if hay is imported from states where blister beetles are common. peak incidence is in late summer and fall. 186 the fatality rate may be 50% or greater, 183, 187 but if the patient survives several days, recovery is probable. cantharidin is absorbed from the gastrointestinal tract and excreted via the kidney. cantharidin is a potent irritant, causing acantholysis and vesicle formation when applied topically. 183, 185, 187 the chemical is thought to disrupt oxidative metabolism in the mitochondria, causing mitochondrial swelling, plasma membrane damage, and changes in membrane permeability. 183 the mucosa of the gastrointestinal tract is affected most commonly in horses because they ingest the toxin. cell swelling and necrosis occur, resulting in mucosal ulceration. oral, esophageal, gastric, and small and large intestinal ulceration have been observed in natural and experimental canthariasis. 183, 185, 187 severe fibrinous to pseudomembranous inflammation and submucosal edema of the intestine also have been reported. diarrhea probably results from the severe ulceration and inflammation of the large intestine, causing increased secretion of water, electrolytes, and protein and decreased absorption of fluid. large volumes of fluid and protein are lost in the gastrointestinal tract, causing hemoconcentration and profound hypoalbuminemia in some cases. 183, 184, 187 cystitis and myocarditis occur in natural and experimentally produced cases of cantharidin toxicity. 183, 185, 187 cystitis occurs because renal excretion of cantharidin results in high concentrations in urine. occasionally, hemorrhagic cystitis may occur, with hematuria or frank hemorrhage into the bladder. 183 the cause of the myocarditis and myocardial necrosis is unknown but also may be a direct effect of the toxin on the myocardium. elevated plasma creatine kinase activity often occurs and has been postulated to arise from the damaged myocardium. 183, 184 horses have a characteristically stiff gait, but histopathologic evidence of skeletal muscle injury that explains the elevated plasma creatine kinase activity has not been observed. 184 the kidneys are often pale, swollen, and moist, with occasional infarcts. 185 hypocalcemia and hypomagnesemia are biochemical features of cantharidin toxicity in horses that have not been explained. 183, 184, 187 hypocalcemia may occur from hypoalbuminemia, but the ionized calcium concentration often is decreased along with the total calcium concentration, indicating that hypoalbuminemia is not responsible for the hypocalcemia. 184 in addition, clinical signs of hypocalcemia, such as synchronous diaphragmatic flutter, are often associated with hypocalcemia from cantharidin toxicity. hypocalcemia associated with hypoalbuminemia alone does not produce clinical signs. cantharidin toxicity can cause a range of clinical signs from mild depression and abdominal discomfort to fulminant signs of toxemia and rapid death, depending on the ingested dose of toxin. most commonly, clinical signs include depression, sweating, irritability, abdominal pain, elevated heart and respiratory rates, fever, polyuria, polydypsia, and profuse diarrhea. 183, 184, 187 blood is rarely visible in the feces. affected horses frequently posture to urinate; indeed, stranguria and pollakiuria are characteristic of cantharidin toxicity. 183 signs of hypocalcemia include synchronous diaphragmatic flutter and tremors. a stiff and stilted gait may be evident. one may note neurologic signs such as head pressing, swaying, and disorientation. 187 signs of systemic inflammation from endotoxemia may be apparent in severe cases. some horses develop severe depression and toxemia and may die within hours after ingestion of cantharidin without developing diarrhea. 183, 187 hematologic abnormalities include hemoconcentration and neutrophilic leukocytosis. occasionally, neutropenia and leukopenia may accompany endotoxemia. serum biochemical analysis usually reveals elevated creatine kinase activity, hypocalcemia, and hypoalbuminemia. 183, 184 biochemical abnormalities include hypocalcemia (ionized and total calcium concentrations), hypomagnesemia, and azotemia. 183, 184, 187 urine specific gravity is characteristically in the hyposthenuric range. 183, 184 microscopic hematuria and mild proteinuria may be evident. fecal occult blood is often present, but hematochezia is unusual. one can make a tentative diagnosis based on clinical signs and the finding of blister beetles in the hay. determining the species of the insects may be necessary to estimate the amount of cantharidin ingested. all species of epicauta contain cantharidin, but some have small amounts. definitive diagnosis requires the measurement of the cantharidin concentration in gastric or intestinal contents and urine. 183, 186 measurement of cantharidin concentration in the beetles is often done but is not necessary. arsenic toxicosis is an unusual cause of diarrhea in horses, resulting from ingestion of arsenic-containing herbicides, insecticides, and other pest control products contaminating water or roughage used as a food source. 188 the toxicity of arsenic depends on the valence of the element. 188, 189 arsenate may be reduced to arsenite in mammalian systems, and arsenite is thought to be more toxic than arsenate and less rapidly excreted in urine. arsenate and arsenite uncouple oxidative phosphorylation, leading to breakdown of energy metabolism in the cells of many tissues. 189 widespread cellular injury and death occur rapidly during acute arsenic toxicosis. multiorgan failure usually results. in fact, cardiomyopathy and pulmonary disease are common causes of death in human beings. 190 damage to the large intestine is probably caused in part by direct cellular toxicity and corrosion by the compound. however, vasculitis is a hallmark of the disease in human beings and horses and is thought to be the most important mechanism of large intestinal disease in human beings. 181, 191 acute hemorrhagic colitis is a feature of arsenic toxicosis, with severe mural edema and mucosal ulceration. 188 profuse, hemorrhagic diarrhea and abdominal pain result. chronic arsenic toxicity can occur but appears to be rare in horses. acute depression, weakness, abdominal pain, hemorrhagic diarrhea, and shock are characteristic of acute arsenic toxicosis in horses. death may occur before diarrhea is evident. initial clinical signs may be difficult to distinguish from other peracute forms of colitis and are related to endotoxic shock, metabolic disturbances, and dehydration. later, cardiac arrhythmias, pulmonary edema, acute renal failure, and neurologic deficits (ataxia and stupor) may develop. 188 one may observe anuria or polyuria. hemolytic anemia caused by preferential binding of arsenic compounds to red blood cells is a feature of arsenic poisoning in human beings. hematologic abnormalities may be apparent after the peracute stages from injury to bone marrow cells and ongoing hemolysis. leukopenia and thrombocytopenia have been described in human patients. 190 serum biochemical analysis may reveal azotemia, hepatocellular enzyme activities higher than generally attributed to endotoxemia, and elevated creatine kinase activity. 188 urine specific gravity may be in the isosthenuric range, with hematuria, cylindruria, and proteinuria evident by urinalysis. diagnosis may be possible by measuring blood and urine arsenic concentration, but these tests may not be diagnostic. postmortem diagnosis is by measuring the arsenic concentration in liver and kidney samples. 188 history of exposure and clinical signs remain the primary means of diagnosis. other disorders associated with diarrhea in adult horses include anaphylaxis, carbohydrate overload, and sand enteropathy. careful evaluation of history, environment, and management will assist the clinician in arriving at an accurate diagnosis. severe intestinal anaphylaxis is a syndrome in horses characterized by peracute, rapidly fatal colitis. 192 the severe syndrome is clinically and pathologically similar to other known causes of peracute colitis, such as salmonellosis, clostridiosis, and antibiotic-associated diarrhea. some cases are less severe and manifest as mild to moderate diarrhea or colic. an ige-mediated type i hypersensitivity or an ige-independent anaphylactoid reaction can produce the syndrome of intestinal anaphylaxis. 193, 194 local gastrointestinal exposure to a food, environmental contaminant, drug, or other allergen usually induces intestinal anaphylaxis, 193, 195 but anaphylaxis also may occur with systemic exposure to an allergen. [196] [197] [198] [199] massive mast cell degranulation, secretion of inflammatory mediators, and activation of enteric neural reflexes in the intestine causes profound alterations in blood flow, increased vascular permeability and interstitial edema, recruitment of neutrophils, altered motility, mucosal injury, absorption of microbial products, and mucosal hypersecretion. [200] [201] [202] [203] [204] systemic signs may be caused by the anaphylactic reaction or may be associated with systemic inflammation triggered by microbial products (endotoxin) absorbed through the injured and hyperpermeable mucosa. intestinal anaphylaxis in horses may be a peracute, fulminant enterocolitis with endotoxemia that may be fatal. 192, 205 this form is characterized by severe intramural edema and hemorrhagic inflammation of the large intestine, often producing submucosal thickening on the order of many centimeters. vascular thrombosis may be widespread with mucosal and serosal petechia and ecchymoses. less severe forms of intestinal anaphylaxis may manifest as patchy areas of intestinal edema and congestion. 196 diarrhea results from intestinal inflammation initiated by the type i hypersensitivity response. many of the mediators of type i hypersensitivity, such as histamine and 5-hydroxytryptamine, have well-documented stimulatory effects on mucosal secretory activity, vascular and epithelial permeability, and motility [200] [201] [202] in the intestine. systemic inflammation from endotoxemia may be overwhelming once the mucosal barrier breaks down. infarction of intestinal segments and other organs may occur from intravascular coagulation. ileus, abdominal distention, and moderate to severe abdominal pain may result from motility disturbances and infarction of the large intestine. the clinical signs are similar to those described for other forms of peracute colitis. however, the severity may vary, manifesting as colic or moderate diarrhea. characteristically, severe shock, signs of systemic inflammation from endotoxemia, and severe metabolic disturbances are observable. 192, 205 heart and respiratory rates may be elevated greatly, with other signs of cardiovascular collapse such as weak and thready peripheral pulses and peripheral vasoconstriction. however, peripheral vasodilation may occur later in the course of disease. dark red, muddy, or cyanotic mucous membranes with a prolonged capillary refill time signify sepsis. borborygmi are usually absent, and abdominal tympany may be heard on percussion, following ileus. moderate to severe colic may accompany ileus. severe diarrhea may occur, but death may occur before diarrhea is evident. multiorgan failure from disseminated intravascular coagulation is not unusual. rapid onset of weakness, staggering, and trembling commonly precedes death. the syndrome may cause death in 4 to 24 hours. hematologic abnormalities include severe neutropenia and leukopenia, thrombocytopenia, and hemoconcentration. 192 serum biochemical alterations include hyponatremia, hypokalemia, hypocalcemia, and severe metabolic acidosis. blood urea nitrogen and creatinine may be elevated from prerenal or renal azotemia. if acute renal failure accompanies the colitis, hyperkalemia may result. hepatocellular enzyme activity may be elevated in the serum from endotoxemia. severe coagulopathies are common, resulting in prolonged coagulation times, elevated fibrinogen, decreased antithrombin iii activity, and elevated plasma concentration of fibrin degradation products. analysis of peritoneal fluid may be valuable because infarction of the large intestine is not unusual. protein concentration and the white blood cell count may be elevated. red blood cell counts are less likely to be elevated, because infarction and not strangulation of the intestine occurs. diagnosis is based on clinical signs, postmortem findings, and exclusion of other causes. cultures and toxicologic analysis of fecal samples and gastrointestinal tissues fail to demonstrate a clear cause. other diagnostic tests are also inconclusive. if an antigen is suspected as the trigger of the anaphylaxis, a prausnitz-küstner passive cutaneous anaphylaxis sensitization test can confirm the presence of antigen-specific ige in the patient serum. 196 overeating of soluble carbohydrates, especially so-called hot grains such as corn, overwhelms the digestive capability of the small intestine, resulting in a high percentage of the soluble carbohydrates entering the large intestine. the amount of soluble carbohydrates that produce diarrhea varies according to the previous dietary history of the individual. horses fed diets higher in soluble carbohydrates are more resistant to the deleterious effects of carbohydrate overload. gradual accommodation to a diet high in carbohydrates can be accomplished over several weeks. however, horses fed an unusually large amount of grains or other form of soluble carbohydrates often develop diarrhea and may, depending on the amount ingested, develop severe colitis, systemic inflammation from endotoxemia, metabolic acidosis, and laminitis. [206] [207] [208] [209] the pathogenesis of colitis from carbohydrate overload is caused primarily by the toxic effects on the microbial flora in the large intestine. 207 a sudden delivery of soluble carbohydrates to the large intestine causes rapid fermentation by gram-positive lactic acid-producing bacteria and a sudden increase in organic acid production. the cecal ph rapidly decreases, and the lactic acid concentration rapidly increases. rapid organic acid production overwhelms the buffering capacity of the large intestine not only by directly depleting the buffers found in the contents but also by reducing the efficiency of buffer secretion. bicarbonate secretion is linked to absorption of volatile fatty acids, which are produced in low amounts by fermentation of soluble carbohydrates. the contents of the large intestine become profoundly acidic, resulting in unfavorable conditions for the microbial flora. lactic acid-producing bacteria flourish, while the gram-negative bacteria, especially the enterobacteriaceae, are killed in large numbers by the acids. large quantities of endotoxin are released from the dying bacteria. 208 the osmotic load from the lactic acid produced in the large intestine is an important factor in the development of diarrhea because organic acids such as lactic acid are absorbed poorly. mild cases of carbohydrate overload may result purely from osmotic diarrhea. in more severe cases, the acidic contents of the large intestine are toxic to the mucosa, causing necrosis of the mucosal tissues, similar to that occurring in ruminal acidosis. mucosal ulceration allows absorption of large quantities of endotoxin and lactic acid produced by the massive die-off of acid-intolerant microbes and fermentation of soluble carbohydrates, normally poorly absorbed by intact mucosa. 209 systemic inflammation from endotoxemia may be overwhelming, and profound metabolic acidosis may occur. secretory diarrhea caused by the direct effects of acid luminal contents on the mucosa, as well as the effects of inflammatory mediators on enterocyte secretion, worsens the acidosis and dehydration. systemic inflammation from endotoxemia, along with intestinal inflammation, adversely affects intestinal motility, and ileus develops. ileus and gas production from fermentation of the carbohydrates may cause severe distention of the large intestine and signs of abdominal pain. laminitis is a frequent complication of endotoxemia and lactic acidosis. in fact, carbohydrate overload is used to induce laminitis as an experimental model because of the consistency of the laminitis produced. [207] [208] [209] clinical signs of colitis from carbohydrate overload can vary according to the amount of carbohydrates ingested and accommodation of the flora to a high-carbohydrate diet. mild cases may result in a transient osmotic diarrhea with no systemic effects. more severe cases are characterized by signs similar to those described for other forms of colitis, including abdominal pain, moderate to severe diarrhea, and dehydration. signs of endotoxemia and sepsis are frequently present in severe cases. elevated heart and respiratory rates are common, with peripheral vasoconstriction early in the disease, followed by peripheral vasodilation as the disease progresses. depression may be profound from metabolic acidosis and endotoxemia. abdominal auscultation and percussion may reveal ileus and intestinal tympany. nasogastric intubation may yield significant gastric acidic reflux. one may note particles of grain in the gastric reflux and the feces, if grain overload is the source of the carbohydrate overload. laminitis may complicate mild and severe cases of carbohydrate overload, especially if the animal has had previous bouts of laminitis. hematologic abnormalities include neutropenia and leukopenia. severe dehydration may result in profound hemoconcentration. protein loss later in the course of disease may result in hypoproteinemia. serum biochemical abnormalities include azotemia, elevated hepatocellular enzyme activity, hyponatremia, and hypokalemia. severe hypocalcemia and metabolic acidosis are characteristic of the disease. serum lactate concentrations are elevated in the absence of evidence of intestinal strangulation or infarction. peritoneal fluid analysis often reveals no abnormalities. sand enteropathy is described in more detail under the heading of obstructive diseases, because acute obstruction is often associated with abnormally large amounts of sand in the large intestine. 210 however, chronic sand-induced diarrhea is a distinct syndrome that can occur at any age from abnormal accumulation of sand in the large intestine. 211, 212 chronic diarrhea and signs of colic may occur without obstruction. the pathogenesis of sand accumulation in individual horses, other than simple ingestion of large quantities, is unclear. presumably the sand causes irritation and may disrupt motility, leading to diarrhea. the diarrhea is usually not severe and dehydrating and may be intermittent. weight loss is characteristic and can be severe in some cases. complications may occur such as peritonitis and acute obstruction. 211 diagnosis usually is based on finding abnormal amounts of sand in the feces. because sand-induced chronic diarrhea is associated primarily with sand accumulation in the ventral colon, auscultation of the ventral abdomen immediately behind the xiphoid process may reveal characteristic sand sounds. 213 this technique is only sensitive if peristalsis is present. ultrasonography also may be useful to identify sand in the ventral colon but is not useful to quantitate the amount of sand. occasionally, radiography may be required to detect sand in the colon. 211 the principles of therapy of acute diarrhea from colitis are similar regardless of the cause and include replacement of fluid and electrolyte losses, control of colonic inflammation and reduction of fluid secretion, promotion of mucosal repair, control of endotoxemia and sepsis, and reestablishment of normal flora. this section focuses on a review the principles of therapy with references to specific therapies for particular causes as they arise. replacement of fluid and electrolyte losses is of primary concern in treating horses with salmonellosis. depending on the severity of the disease, fluid losses may be minimal or massive. one can administer fluid and electrolytes orally or intravenously. some horses with mild to moderate diarrhea may maintain hydration and electrolyte balance by consuming water and electrolytes voluntarily. freshwater and water containing electrolytes should be available in all cases. in many instances, periodic nasogastric intubation and administration of water and electrolytes via the tube may be sufficient to maintain hydration. 214 in more severe cases, one can maintain indwelling nasogastric tubes and can administer up to 4 to 8 l of fluid by the tube every 20 to 30 minutes, if ileus is not evident. however, intravenous administration of fluids is preferred in most cases, requiring significant quantities of fluid to replace and maintain hydration and electrolyte balance. 215 for patients with severe diarrhea to require large volumes (50 to 100 l/day) of intravenous fluids to maintain hydration is not unusual. frequent monitoring of packed cell volume, serum electrolyte concentration, venous blood gases or total serum carbon dioxide, blood urea nitrogen and creatinine, urine protein and cytologic findings, and body weight is important to monitor hydration, electrolyte and acid-base balance, and renal function. isotonic sodium chloride or lactated ringer's solution frequently is used to restore and maintain fluid and electrolyte balance. one can add potassium chloride to the fluids and administer it at a rate up to 0.5 to 1.0 meq/ kg/hr. generally, a rate of less than 0.5 meq/kg/hr is used. hypertonic nacl solutions (1 to 2 l of 3% to 5% nacl) have been used in horses that are severely hyponatremic (<120 meq/dl). one should not administer hypertonic solutions to severely dehydrated horses, but such solutions have been used clinically without complication and with considerable beneficial effect in patients with endotoxemia. the beneficial effects of hypertonic nacl are short-lived (30 to 60 minutes). one should administer isotonic solutions concurrently or immediately following administration of hypertonic nacl solutions. isotonic (1.3%) or hypertonic (5.0%) sodium bicarbonate solutions are used to correct metabolic acidosis. prolonged administration of sodium-containing fluids may promote diuresis and renal water loss or accumulation of peripheral edema and should be used conservatively when one notes a free water loss. administration of isotonic dextrose (5%) or 2.5% dextrose/0.45% nacl solutions may be beneficial when free water loss (sodium excess) is evident. many horses with acute colitis are concurrently hypoproteinemic because of gastrointestinal losses and are absorbing bacterial products that induce a systemic inflammatory response. thus plasma oncotic pressures are abnormally low in the face of increased vascular permeability. interstitial edema formation is a clinical problem in these patients and contributes to organ dysfunction. crystalloid fluids, although critical for replacing water and electrolyte losses from diarrhea, actually may contribute to a drop in plasma oncotic pressure because of hemodilution. 216, 217 administration of colloid solutions are important for volume expansion and to maintain plasma oncotic pressures, which improve tissue perfusion and oxygenation and organ function in hypovolemic, hypotensive, and hypoproteinemic patients with or without systemic inflammatory response syndrome. 218 colloids are more effective than crystalloid fluids at expanding plasma volume and thus require smaller volumes. moreover, the effect of colloid volume expansion is longer lasting than crystalloid fluid volume expansion, because colloids are retained in the vasculature better. 217, 218 natural colloids, such as plasma and purified albumin are used commonly. in addition to its beneficial colloidal properties, plasma harvested from donor horses immunized with rough mutants of escherichia coli (j5) or salmonella typhimurium may have other benefits for treatment of endotoxemia from gastrointestinal disease. 219, 220 the horse may require large volumes (6 to 8 l/day) to increase and maintain plasma protein concentration significantly. synthetic colloids such as dextrans, starches, or polymerized hemoglobin are also available for use in the horse. hetastarch (5 to 10 ml/kg of a 6% solution) increases colloidal oncotic pressures for up to 24 hours in hypoproteinemic horses and has beneficial effects on cardiac output and other cardiorespiratory parameters, vascular permeability, interstitial fluid content, and tissue perfusion in models of hypoproteinemia and systemic inflammatory response syndrome. when one administers synthetic or even natural colloids, monitoring plasma oncotic pressure may be more relevant than monitoring plasma protein concentrations as a means of assessing the need for plasma or other colloid administration. 216 hetastarch may prolong bleeding times by altering von willebrand's factor function; thus one should use this synthetic colloid cautiously in horses with suspected coagulopathies, active hemorrhage, or other bleeding problems. 217 control of colonic inflammation and secretion is a difficult and poorly studied aspect of equine acute colitis. the role of inflammation and mediators such as prostaglandins as causes of fluid loss is well known for salmonella and clostridium infections. cox inhibitors (nsaids) have antisecretory effects in the equine colon and in models of salmonellosis that appear to extend to clinical management of salmonellosis. 16, 36, [221] [222] [223] indeed, nsaids commonly are administered to horses with salmonellosis. however, prostaglandins such as pge 2 and pgi 2 are also cytoprotective to gastrointestinal mucosa and critical for mucosal repair. 172 the doses of nsaids used pharmacologically to inhibit colonic inflammation and secretion in fact may be detrimental to the mucosa if not used judiciously. nsaids have been shown to exacerbate colonic inflammation in human beings with inflammatory colitis, impede mucosal healing in several models of mucosal injury, and have well-documented detrimental effects on colonic mucosa in horses. 164, 172, 224 in addition to toxicity to the colonic mucosa, gastric ulceration is not unusual in horses with enterocolitis and may be related to treatment with nsaids. in addition to nsaids, other drugs occasionally are used as antiinflammatory or antisecretory therapy. metronidazole has beneficial effects in experimental models of gastrointestinal inflammation, including nsaid toxicity 176 and may be useful for treating horses with colitis, but evidence supporting its use is lacking. bismuth subsalicylate solutions administered orally often are used to decrease inflammation and secretion in the colon. in adult horses the volume of solution necessary to be beneficial is large (3 to 4 l every 4 to 6 hours). often the solution is administered twice daily instead of 4 to 6 times daily. if one does not achieve a beneficial effect within 3 to 4 days of treatment, one should discontinue administration of bismuth subsalicylate solution. one can administer the treatment more frequently in foals, and clinical improvement occurs more often in foals than in adult horses. in light of the role of reactive oxygen metabolites in colonic inflammation, free radical scavengers have been advocated to reduce the effects of these molecules. sulfasalazine metabolites have been shown to reduce reactive oxygen metabolite-induced colonic inflammation in other species, 176 and sulfasalazine has been used to treat chronic inflammatory disease in horses but has not been used to treat acute colitis. the only free radical scavenger used commonly in horses with colitis is dimethyl sulfoxide, which at a dosage of 0.1 to 1.0 g/kg intravenously every 12 to 24 hours in a 10% solution has been used in clinical cases of colitis, but evidence of efficacy has not been established. systemic inflammatory response syndrome associated with endotoxemia frequently occurs in patients with salmonellosis. the principles of therapy for endotoxemia are covered in detail elsewhere in this chapter. oral administration of activated charcoal and mineral oil is used commonly to reduce absorption of endotoxin in horses with colitis. low doses of nsaids (such as flunixin meglumine at 0.1 to 0.25 mg/kg intravenously every 6 to 8 hours) inhibit eicosanoid synthesis induced by endotoxin. in addition, administration of nsaids prevents laminitis from endotoxemia, a devastating complication of salmonellosis. one must remember that prostaglandins are important for mucosal healing and may worsen mucosal injury in colitis. although the benefits of low doses of nsaids administered to horses with systemic inflammatory response syndrome are believed to outweigh the risks of worsening gastrointestinal damage, judicious use is recommended. sucralfate (20 mg/kg orally every 6 hours) has been advocated to aid in healing the colonic mucosa, but the efficacy in the large intestine is questionable. 94 misoprostol (2 µg/kg orally 3 to 4 times daily) and other synthetic pge analogs have been shown in several species including horses to enhance mucosal healing in the intestine and promote recovery in experimental models of colitis. 225 misoprostol may be particularly useful for treating nsaid toxicity, the generalized form or rdc. however, the efficacy of misoprostil for hastening mucosal healing is clinically unproven in equine colitis. the primary drawbacks of prostaglandin analogs such as misoprostol are the side effects of the drug, including abdominal cramping, diarrhea, sweating, and abortion in pregnant mares. one can add psyllium mucilloid to the diet (5 tablespoons once or twice daily) to increase the production of scfas in the colon. amylase-resistant fermentable fiber such as psyllium is hydrolyzed by colonic bacteria to scfas such as butyrate, which represent a major energy source for colonocytes. butyrate and other scfas hasten epithelial maturation and stimulate salt (and thus fluid) absorption in the colon, improve the clinical course of ulcerative colitis, and hasten colon healing. 226 psyllium is itself a source of butyrate in the colon and also promotes the movement of amylase sensitive carbohydrates into the distal colon, which then are fermented to scfas. thus psyllium is thought to be clinically useful for promoting mucosal healing in colitis. many horses with salmonellosis or other forms of colitis have mild to severe signs of abdominal pain from gas and fluid distention of the colon, colonic ischemia, or infarction. one can accomplish analgesia with nsaids such as flunixin, but the potential for worsening mucosal injury or nephrotoxicity may prevent the use of analgesic doses, especially in horses with suspected nsaid toxicity. newer nsaids that specifically target cox-2 (the inducible cox) but have little activity against cox-1 (the constitutive cox) may be useful analgesics that spare the gastrointestinal mucosa. for example, etodolac (10 to 15 mg/kg intravenously or orally once daily) has analgesic properties in horses and may spare the intestinal mucosa from the detrimental effects associated with nonselective cox inhibitors (a.t. blikslager, personal communication, 2002) . however, the specificity for cox-2 in horses is unproven. thus avoiding the use of any nsaids in horses with rdc or other forms of nsaid toxicity is advisable. xylazine or detomidine may provide temporary relief of pain. butorphanol is a useful analgesic that one can administer intramuscularly (0.1 mg/kg every 6 hours) or as a continuous infusion. an infusion of 13.2 µg/kg/hr in isotonic crystalloid fluid such as lactated ringer's solution has been suggested. 227 continuous lidocaine infusions (1.3 mg/kg intravenous loading dose administered slowly over 5 minutes and followed by 3 mg/kg/hr infusion in isotonic crystalloid fluids) can provide profound visceral analgesia and may have added prokinetic benefits if ileus is present. broad-spectrum antibiotic treatment often is recommended in neutropenic horses or horses with signs of septicemia. neutropenia is associated with an increased risk of septicemia and septic complications such as septic phlebitis and infection of surgical site. 1 septicemia is a potentially life-threatening complication of enterocolitis and may be caused directly by salmonella, clostridium, other invasive enteric bacteria, or indirectly by toxic injury to the colonic mucosa that breaks down the barrier to luminal microbes. neutropenia possibly may weaken host defenses enough to render horses susceptible to organisms that breach the mucosal barrier. although most attempts to culture bacteria from the blood of adult horses with colitis fail to isolate organisms, no detailed studies have been undertaken to determine the prevalence of bacteremia or septicemia in these patients. disseminated aspergillosis has been reported in horses as a complication of acute colitis, demonstrating the potential for systemic infections with rarely pathogenic organisms stemming from colonic mucosal injury in the face of potential immunosuppression from neutropenia. 228, 229 broad-spectrum antibiotics lessen septic complications in human patients. however, evidence supporting this principle in horses with colitis is lacking. treatment with antibiotics is controversial in horses with salmonellosis and is not thought to alter the course of the enterocolitis. antibiotics directly targeted at the salmonella are reserved for patients with the enteric fever (septicemia) form of salmonellosis, documented with positive blood cultures. lipid-soluble antibiotics are suited ideally for salmonella infections, because the bacteria persist intracellularly. trimethoprim-sulfadiazine or other potentiated sulfa drugs, enrofloxacin, and chloramphenicol are preferred antibiotics for the enteric fever form of salmonellosis for this reason. as with other causes of enterocolitis, the use of antibiotics for equine monocytic ehrlichiosis is controversial. fear of inducing salmonellosis or other forms of antibiotic-induced diarrhea and the difficulty of diagnosing the disease early have caused most authors to recommend judicious use of antibiotics. 49 however, in patients with a high suspicion of neorickettsia risticii infection, treatment with antibiotics often is indicated before definitive diagnosis. lipid-soluble drugs are desirable because the organism can live within cells. oxytetracycline (6.6 mg/kg intravenously every 24 hours), doxycycline (10 mg/kg orally every 12 hours), trimethoprim-sulfadiazine (5 mg/kg trimethoprim orally or intravenously every 8 to 12 hours and 25 mg/kg sulfadiazine every 8 to 12 hours), or erythromycinrifampin (30 mg/kg and 5 mg/kg, respectively, orally every 12 hours) have been used effectively to treat clinical cases. 49, [230] [231] [232] the tetracyclines appear to be the most effective antibiotics for treatment of potomac horse fever. treatment is most successful if initiated before the onset of diarrhea. 49, 231 clostridiosis if one has administered antibiotics since the onset of enterocolitis, one should discontinue administration as soon as possible. specific treatment with metronidazole (15 to 25 mg/kg orally every 8 hours) is effective for treating clostridiosis in human beings and appears to be effective in horses. 83, 233 metronidazole resistance in clinical isolates of clostridium difficile has been reported in one outbreak but appears to be rare in most human and equine cases. 234 metronidazole-resistant isolates were sensitive to vancomycin, which may be effective for treating clinical cases if one suspects metronidazole resistance. however, metronidazole remains the treatment of choice. some authors describe the off-label use of c. perfringens type c antitoxin in cases of neonatal clostridiosis, described in more detail elsewhere. 235 antitoxin preparations generally are not advocated for use in adult horses with clostridiosis. lawsonia intracellulare is susceptible to a variety of antibiotics in vitro, including chlortetracycline, erythromycin, penicillin, difloxacin, and ampicillin. 236 lipid-soluble antibiotics with a large volume of distribution usually are chosen to treat proliferative enteropathy because l. intracellulare is an intracellular organism. erythromycin estolate (15 to 25 mg/kg orally every 6 to 8 hours) alone or with rifampin (5 mg/kg orally every 12 hours) is the most commonly reported efficacious treatment for proliferative enteropathy. chloramphenicol (50 mg/kg orally every 6 hours) has also been reported to be effective if erythromycin worsens the diarrhea. 115 anecdotal reports suggest that oxytetracycline and doxycycline also may be effective. supportive care including maintenance of hydration and electrolyte balance and plasma or colloid administration to increase colloid oncotic pressure in hypoalbuminemic patients is also indicated. one should treat affected foals until clinical signs, hypoproteinemia, and ultrasonographic evidence of intestinal thickening resolve. the prognosis depends on the duration of the disease and the degree of fibrosis and destruction of the intestinal architecture. hypercoagulability is a common complication of enterocolitis, associated with systemic inflammation from endotoxemia. administration of heparin (20 to 80 iu/kg subcutaneously or intravenously every 6 to 12 hours) may prevent thrombosis in these patients, provided antithrombin iii concentrations are adequate in the plasma. concentrated sources of antithrombin iii are not available for use in horses, but whole plasma may provide an important source. treatment with heparin is thought to decrease thrombosis, especially of the jugular vein, a serious complication of salmonellosis. low-dose aspirin treatment (15 mg/kg orally every 24 to 48 hours) along with heparin treatment may provide added benefit by irreversibly inhibiting platelet function. 237 heparin and aspirin may have protective effects on the digital lamina. 237, 238 heparin also may enhance the phagocytic activity of the reticuloendothelial system by enhancing the efficiency of opsonins such as fibronectin and immunoglobulin, thereby stimulating phagocytosis of products of coagulation and possibly other particles, including bacteria. 239, 240 maintenance of the bacterial flora and antagonism of pathogenic bacteria such as salmonella in the gastrointestinal tract are important defense mechanisms preventing colonization by pathogenic bacteria. the use of probiotic preparations containing beneficial bacteria has been shown to prevent colonization of pathogenic bacteria, including salmonella, in poultry. 241 little work has been done to investigate the efficacy of these products in preventing salmonellosis in horses, but ongoing studies may provide important information. probiotic and other preparations designed to restore normal flora to the gastrointestinal tract, such as fecal suspensions, sour milk, and yogurt, have been used clinically to shorten the course of salmonellosis, with variable results. therefore prevention of infection by using probiotic agents and other means is important. exposure of susceptible horses to salmonella should be avoided, but the task is difficult, especially because asymptomatic infections are common and the bacteria are ubiquitous in the environment. prophylactic use of probiotic preparations, judicious use of antibiotics in susceptible horses, control of environmental conditions such as temperature, and restricted exposure to pathogenic bacteria are important for control of salmonellosis. because altered large intestinal flora appears to play an important role in the pathogenesis of equine intestinal clostridiosis or any antibiotic-associated diarrhea, probiotic preparations have been advocated to treat affected horses. sour milk, a product containing lactose-producing streptococcus species, appears to improve the clinical course greatly in horses suspected of having clostridium perfringens type a infection. sour milk may benefit the patient by altering the flora and antagonizing enterotoxigenic c. perfringens type a but also is reported to be bactericidal against c. perfringens type a. 78 preparations of saccharomyces boulardi are effective for reducing diarrhea and the frequency of c. difficile recurrence in human beings. 83 however, whether relapse is a problem in horses with c. difficile colitis is not clear. lactobacillus preparations have a protective effect in human beings and decrease the severity and duration of antibioticassociated diarrhea. 242, 243 however, evidence of their clinical usefulness in horses is lacking. good nursing care and adequate nutrition are vital to the treatment of horses with salmonellosis. salmonellosis is a severely catabolic disease, increasing caloric requirements greatly. normal intake of roughage to provide energy may be inadequate; however, one should avoid feeding of grains to prevent carbohydrate overload. dietary management usually consists of restricting or eliminating long-stem roughage (hay) from the diet and feeding exclusively a complete pelleted diet (at least 30% dietary fiber). the rationale behind this recommendation is to reduce the mechanical load on the colon. frequent meals (4 to 6 times a day) are recommended. one can add corn oil (1 cup every 12 to 24 hours) to the pellets to increase the caloric intake without adding roughage or grain. one should note that if a horse with colitis refuses to eat pelleted feed, then one should feed good-quality grass hay. in anorectic or severely catabolic patients, enteral and parenteral nutrition (total and partial) has been used successfully to provide calories and nutritional support. strongylus vulgaris infection requires treatment of the migrating parasite larvae and the lesions produced by the parasite. fenbendazole (10 mg/kg orally every 24 hours for 3 days or 10 mg/kg orally every 24 hours for 5 days) and ivermectin (200 mg/kg orally) are effective in killing fourth-stage larvae. 121 other anthelmintics also may be 906 part ii disorders of specific body systems effective when given at higher doses than those required to kill adult worms. the efficacy of these anthelmintics against larvae within thrombi is not known. thrombolytic and antithrombotic therapy has been advocated in horses with suspected strongylosis. 121, 128 heparin (20 to 80 iu intravenously or subcutaneously every 6 to 12 hours) is often administered as an anticoagulant. aspirin (10 to 30 mg/kg orally every 12 to 48 hours) is usually combined with heparin to inhibit platelet adhesion. aspirin also may inhibit release of platelet products such as thromboxane that affect the motility of the large intestine. low-molecular-weight dextrans have been advocated as antithrombotics that act by inhibiting platelet function and coagulation. 128, 218 the clinical efficacy of dextran administration appears to be good, but no controlled studies have been performed. anthelmintic administration is usually the only treatment necessary for mild to moderate cases of cyathostomiasis treated early in the course of the disease (within 1 to 3 weeks of onset). fenbendazole is effective against many larval stages, but resistance is increasing. although the reported efficacy of ivermectin varies against certain stages, 244 one study reported an overall efficacy of 75%. 245 currently, fenbendazole (7.5 to 10 mg/kg orally every 24 hours for 5 days) followed on day 6 by ivermectin (200 mg/kg orally) is the most commonly advocated treatment regimen. 133, 246 moxidectin (400 µg/kg orally once daily) also may be effective against adults and l 3 and l 4 larval stages 247 and may be useful for treating cyathostomiasis. antiinflammatory therapy also may be beneficial, especially in severe or refractory cases. nsaid administration may have limited value, but dexamethasone appears to be efficacious in refractory cases when used with larvicidal anthelmintics. 133, 136 pretreatment with dexamethasone or prednisolone is indicated before anthelmintic administration if heavy larval loads are suspected to prevent an acute exacerbation of the disease by rapid death of encysted larvae. bismuth subsalicylate often is administered orally as an antisecretory agent in young animals. supportive care may be necessary in severe cases, particularly if hypoproteinemia is severe. horses occasionally require administration of intravenous crystalloid fluids and plasma or other colloids. proper nutritional support is also important. supportive care is the most important principle of therapy for cantharidin toxicity. intravenous fluid administration; maintenance of electrolyte balance, especially calcium; and prevention of further renal and urinary tract damage is important. 183, 187 diuresis by intravenous fluid administration is often sufficient to prevent renal failure. furosemide often is administered after rehydration of the patient to further promote diuresis and to decrease the concentration of the toxin in the urine, which may ameliorate some of the effects on the urinary tract mucosa. diuresis also has been suggested to increase clearance of the toxin, but no evidence for this has been found. judicious use of nsaids may be necessary to control abdominal pain but should be reserved until the patient is rehydrated and renal failure has been ruled out. cantharidin is lipid-soluble; therefore oral administration of mineral oil may prevent further absorption of the toxin. 183 activated charcoal often is administered with the mineral oil. to reduce arsenic absorption, one should initiate administration of cathartics such as mineral oil and magnesium sulfate slurries and activated charcoal by nasogastric tube immediately. chelation therapy with sodium thiosulfate 20 to 30 g in 300 ml of water orally and dimercaprol (bal) 3 mg/kg intramuscularly every 4 hours is indicated. 188 dimercaprol is a specific antidote for trivalent arsenicals, but its efficacy in horses is questionable. intravenous fluid administration may help treat shock, replace fluid lost in feces, and promote diuresis but should be monitored carefully because pulmonary edema is a frequent complication. the horse may require more specific treatment of renal, cardiac, pulmonary, or neurologic disease. treatment of intestinal anaphylaxis is in principle similar to treatment of other forms of colitis but is often unsuccessful because of the rapidly progressive nature of the syndrome. inclusion of heparin in intravenous fluids (20 to 80 iu/kg intravenously every 8 to 12 hours) may help prevent vascular thrombosis. administration of hypertonic saline solutions or colloids may prove to be useful during initial periods of shock. early treatment with prednisolone succinate (10 to 20 mg/kg intravenously) or dexamethasone (0.1 to 0.2 mg/kg intravenously) may be essential for successful treatment. 192 mild cases of carbohydrate overload may not require treatment other than exclusion of grains from the diet for several days to weeks and gradual reintroduction of grain into the diet later if the horse needs the extra energy. patients showing signs of colic or diarrhea without other systemic signs may benefit from administration of mineral oil, charcoal, and fluids via nasogastric tube. one also may lavage residual carbohydrates from the stomach with the nasogastric tube. nsaids such as phenylbutazone (2.2 to 4.4 mg/kg/day intravenously) or flunixin meglumine (1 mg/kg intravenously every 12 hours) often are administered to prevent laminitis. phenoxybenzamine and heparin given before the onset of laminitis may prevent or decrease the severity of laminitis. 238, 248 more severe cases with dehydrating diarrhea, systemic signs of endotoxemia, or metabolic acidosis require intravenous fluid support to maintain water, electrolyte, and acid-base balance in addition to the previously mentioned treatments. large amounts of bicarbonatecontaining solutions may be required. one should take care when administering hypertonic bicarbonate solutions, because many patients already may be hyperosmotic from lactic acidemia. isotonic sodium bicarbonate 1.3% may be useful in the hyperosmotic patient. careful attention to calcium balance is also important, because severe hypocalcemia may occur. one should institute aggressive therapy for systemic inflammation from endotoxemia. one should administer broad-spectrum antibiotics intravenously to combat bacteremia and septicemia, which frequently complicate colitis induced by carbohydrate overload. in extreme cases, especially if the patient has ingested a large quantity of grain, surgical removal of the grain from the large intestine may be indicated, especially if one can accomplish surgery before the onset of severe clinical signs. however, administration of oral cathartics, such as magnesium sulfate slurries or mineral oil, or a combination of these, is often sufficient to clear the carbohydrates from the large intestine before fermentation, mucosal damage, and absorption of endotoxin and lactic acid occur. oral administration of activated charcoal may prevent absorption of endotoxin by binding the molecules in the lumen of the bowel. in any case, one should discontinue feeding of the source of the soluble carbohydrates, such as grains. one should feed the horse low-carbohydrate and low-protein roughage such as grass or oat hays until the microbial flora recovers. oral administration of probiotic preparations containing lactobacillus is contraindicated; however, other sources of normal equine large intestinal microbial flora, such as fecal extracts from normal feces, may be useful to reintroduce appropriate microorganisms. complications from laminitis and sepsis are common and often cause death. treatment of sand enteropathy requires removal of the sand from the gastrointestinal tract using psyllium products and magnesium sulfate slurries administered orally. analgesics may be required initially to relieve pain and stimulate appetite. a diet high in roughage often stimulates further passage of sand. treatment may require several weeks to remove as much sand as possible. prevention of the disease is important, and recurrence is not unusual. lumen results in clinical signs similar to those of simple obstruction, occlusion of the blood supply results in a more rapid deterioration of the intestinal mucosa and subsequent onset of endotoxemic shock. although a great deal of interest in the relevance and treatment of intestinal reperfusion injury has arisen recently, 1-3 the lesion that develops during strangulation is often severe, leaving little viable bowel for further injury during reperfusion. 2 although extensive lengths of strangulated small intestine may be resected, strangulation of the large colon presents a much greater treatment dilemma because strangulated intestine usually extends beyond the limits of surgical resection. 4 therefore horses with large intestinal strangulation often recover with extensive intestinal injury left in place. thus subtle degrees of reperfusion injury may be important in horses with large colon disease, warranting further work in this area in an attempt to reduce mortality. 3 strangulating obstruction may be divided into hemorrhagic and ischemic forms. 5, 6 hemorrhagic strangulating obstruction, which is most common, involves initial occlusion of veins before occlusion of arteries because of the greater stiffness of arterial walls. this lesion is characterized by a darkened appearance to affected bowel and increased thickness as blood is pumped into the lesion. ischemic strangulating obstruction occurs if the intestine is twisted tightly enough to occlude arteries and veins simultaneously. in the case of the colon, such strangulation has been suggested to be determined by how much ingesta is in the colon, because intestinal contents may prevent the intestine from twisting tightly. 7 tissue involved in ischemic strangulating obstruction appears pale and of normal or reduced thickness because of a complete lack of blood flow ( figure 13 .14-1). bowel peripheral to strangulating lesions also may become injured because of distention, which reduces mural blood flow once it reaches critical levels. furthermore, as this intestine is decompressed, it also may undergo reperfusion injury. [8] [9] [10] small intestinal strangulation horses with small intestinal strangulating obstruction typically have moderate to severe signs of abdominal pain that are only intermittently responsive to analgesic medications. during the latter stages of the disease process, horses may become profoundly depressed rather than painful as affected intestine necroses. horses have progressive signs of endotoxemia, including congested mucous membranes, delayed capillary refill time, and an elevated heart rate (>60 beats/min in most cases). in addition, one typically obtains reflux following passage of a stomach tube, and one usually can detect loops of distended small intestine on rectal palpation of the abdomen. 11 however, these latter findings vary depending on the duration and location of the obstruction. for example, horses with ileal obstructions tend to reflux later in the course of the disease process than horses with a jejunal obstruction. furthermore, a horse that has an entrapment of small intestine in the epiploic foramen may not have palpable loops of small intestine because of the cranial location of these structures. 12 abdominocentesis can provide critical information on the integrity of the intestine and is indicated in horses in which one suspects strangulation of the small intestine. 13 a horse that has signs compatible with a small intestinal obstruction and additionally has serosanguinous abdominal fluid with an elevated protein level (>2.5 mg/dl) is likely to require surgery, although one must differentiate these a b figure 13 .14-1 ischemic strangulating obstruction of the small colon by a mesenteric lipoma. a, the lipoma (arrow) has encircled a segment of small colon tightly. b, following resection of the lipoma, a pale area of strangulated small colon clearly is demarcated (arrows), the appearance of which is consistent with ischemic strangulating obstruction. cases from proximal enteritis. in general, horses with small intestinal strangulation show continued signs of abdominal pain, whereas horses with proximal enteritis tend to be depressed after initial episodes of mild abdominal pain. in addition, horses with small intestinal strangulation continue to deteriorate clinically despite appropriate medical therapy and will likely begin to show an increased white blood cell count (>10,000 cells/µl) in the abdominal fluid as the duration of strangulation increases. however, cases occur in which the differentiation between small intestinal strangulation and proximal enteritis is not clear, at which point one may elect surgery rather than risking delay of abdominal exploration of a horse with a potential strangulating lesion. 14 the prognosis for survival in horses with small intestinal strangulating lesions is generally lower than for most forms of colic. 15 however, recent studies indicate that in excess of 80% of horses with small intestinal strangulating lesions are discharged from the hospital. 16 nonetheless, veterinarians should warn owners that the long-term survival rates are reduced substantially to below 70%, 17 in part because of long-term complications such as adhesions. 18, 19 in addition, the prognosis is particularly low for some forms of strangulation, including entrapment of small intestine within a mesenteric rent. 20 the epiploic foramen is a potential opening (because the walls of the foramen are usually in contact) to the omental bursa located within the right cranial quadrant of the abdomen. the foramen thus is bounded dorsally by the caudate process of the liver and caudal vena cava and ventrally by the pancreas, hepatoduodenal ligament, and portal vein. intestine may enter the foramen from the visceral surface of the liver toward the right body wall or the opposite direction. studies differ as to which is the most common form. 12, 21 in the case of entrapments that enter the foramen in a left-to-right direction, the omental bursa ruptures as the intestine migrates through the epiploic foramen, which may contribute to intraabdominal hemorrhage often seen with this condition. clinical signs include acute onset of severe colic with examination findings compatible with small intestinal obstruction. the condition tends to be more prevalent in older horses, 12 possibly because of enlargement of the epiploic foramen as the right lobe of the liver undergoes ageassociated atrophy. 22 however, the disease also has been recognized in foals as young as 4 months of age. 23 one makes a definitive diagnosis at surgery, although ultrasonographic findings of distended loops of edematous small intestine adjacent to the right middle body wall suggest epiploic foramen entrapment. 12 in general, thickened, amotile intestine on ultrasonographic examination is highly predictive for small intestinal strangulating obstruction. 24 small intestine entrapped in the epiploic foramen may be limited to a portion of the intestinal wall (parietal hernia), 25 and the large colon may become entrapped within the epiploic foramen. 26 in treating epiploic foramen entrapment, one must not enlarge the epiploic foramen by blunt force or with a sharp instrument, because rupture of the vena cava or portal vein and fatal hemorrhage may occur. prognosis has improved substantially over the last decade, with current short-term survival rates (discharge from the hospital) ranging from 74% 27 to 79%. 12 preoperative abdominocentesis has been found consistently to be the most predictive test of postoperative survival. 12,27 lipomata form between the leaves of the mesentery as horses age and develop mesenteric stalks as the weight of the lipoma tugs on the mesentery. the stalk of the lipoma subsequently may wrap around a loop of small intestine or small colon causing strangulation. one should suspect strangulating lipomata in aged (>15 years old) geldings with acute colic referable to the small intestinal tract. 28, 29 ponies also appear to be at risk of developing disease, 29 suggesting alterations in fat metabolism may predispose certain horses to development of mesenteric lipomata. one usually makes the diagnosis at surgery, although on rare occasions one can palpate a lipoma per rectum. 30 treatment involves surgical resection of the lipoma and strangulated bowel, although strangulated intestine is not always nonviable. 28 studies indicate that approximately 50% 29 to 80% 28 of horses are discharged from the hospital following surgical treatment. a volvulus is a twist along the axis of the mesentery, whereas torsion is a twist along the longitudinal axis of the intestine. small intestinal volvulus theoretically is initiated by a change in local peristalsis or the occurrence of a lesion around which the intestine and its mesentery may twist (such as an ascarid impaction). 11 volvulus is reportedly one of the most commonly diagnosed causes of small intestinal obstruction in foals. 31, 32 the theory is that young foals may be at risk of small intestinal volvulus because of changing feed habits and adaptation to a bulkier adult diet. onset of acute, severe colic, a distended abdomen, and radiographic evidence of multiple loops of distended small intestine in a young foal suggest small intestinal volvulus. however, one cannot differentiate volvulus from other causes of small intestinal obstruction preoperatively. in adult horses, volvulus frequently occurs in association with another disease process, during which small intestinal obstruction results in distention and subsequent rotation of the small intestine around the root of the mesentery. although any segment of the small intestine may be involved, the distal jejunum and ileum are affected most frequently because of their longer mesenteries. 11 one makes the diagnosis at surgery by palpating a twist at the origin of the cranial mesenteric artery. treatment includes resection of devitalized bowel, which may not be an option because of the extent of small intestinal involvement (similar to large colon volvulus). prognosis is based on the extent of small intestine involved and its appearance following surgical correction of the lesion. in general, horses with greater than 50% of the small intestine devitalized are considered to have a grave prognosis. 33 a number of structures, when torn, may incarcerate a segment of intestine (typically the small intestine), including intestinal mesentery, 20 the gastrosplenic ligament, 34 the broad ligament, 35 and the cecocolic ligament. 36 horses with such incarcerations have signs typical of a horse with strangulating small intestine, including moderate to severe signs of abdominal pain, endotoxemia, absent gastrointestinal sounds, distended small intestine on per rectal palpation, nasogastric reflux, and serosanguinous abdominal fluid. however, the prognosis for many of these horses appears to be lower than for horses with other types of small intestinal strangulations. for example, in horses with small intestine entrapped in a mesenteric rent, only 7 of 15 horses were discharged from the hospital, and only 2 of 5 horses for which follow-up information was available survived long term (>5 months). 20 poor outcome may result from the difficulty in unentrapping incarcerated intestine, the degree of hemorrhage, and the length of intestine affected. inguinal herniae are more common in standardbred and tennessee walking horses that tend to have congenitally large inguinal canals. 11 inguinal herniae also may occur in neonatal foals but differ from herniae in mature horses in that they are typically nonstrangulating. the nature of the hernia (direct versus indirect) is based on the integrity of the parietal vaginal tunic. in horses in which the bowel remains within the parietal vaginal tunic, the hernia is referred to as indirect, because strictly speaking the bowel remains within the peritoneal cavity. direct herniae are those in which strangulated bowel ruptures through the parietal vaginal tunic and occupies a subcutaneous location. these direct herniae most commonly occur in foals and should be suspected when a congenital inguinal hernia is associated with colic, swelling that extends from the inguinal region or the prepuce, and intestine that may be palpated subcutaneously. 37, 38 although most congenital indirect inguinal herniae resolve with repeated manual reduction or application of a diaper, surgical intervention is recommended for congenial direct herniae. 37 historical findings in horses with strangulating inguinal herniae include acute onset of colic in a stallion that recently had been used for breeding. a cardinal sign of inguinal herniation is a cool, enlarged testicle on one side of the scrotum (figure 13 .14-2). 39, 40 however, inguinal herniae also have been reported in geldings. 41 one also can detect inguinal herniae on rectal palpation, and one can use manipulation of herniated bowel per rectum to reduce a hernia, but this is generally not recommended because of the risk of rectal tears. in many cases, the short segment of herniated intestine greatly improves in appearance after reduction and in some cases can be left unresected. the affected testicle will be congested because of vascular compromise within the spermatic cord, and although the testicle may remain viable, resection generally is recommended. 42 the prognosis in adult horses is good, with up to 75% of horses surviving to 6 months. 40 horses that have been treated for inguinal herniae may be used for breeding. in these horses, the remaining testicle will have increased sperm production, although an increased number of sperm abnormalities will be noticeable following surgery because of edema and increased temperature of the scrotum. although umbilical herniae are common in foals, strangulation of herniated bowel is rare. in one study, 6 of 147 (4%) horses with umbilical herniae had incarcerated intestine. 43 clinical signs include a warm, swollen, firm, and painful hernia sac associated with signs of colic. the affected segment of bowel is usually small intestine, but herniation of cecum or large colon also has been reported. in rare cases, one may find a hernia that involves only part of the intestinal wall, called a richter's hernia. in foals that have a richter's hernia, an enterocutaneous fistula may develop. in one study, 13 of 13 foals with strangulating umbilical herniae survived to discharge, although at least 3 died of long-term complications. 44 an intussusception involves a segment of bowel (intussusceptum) that invaginates into an adjacent aboral segment of bowel (intussuscipiens). the reason for such invagination is not always clear but may involve a lesion at the leading edge of the intussusception, including small masses, foreign bodies, or parasites. in particular, tapeworms (anoplocephala perfoliata) have been implicated. 45 ileocecal intussusceptions are the most common intestinal intussusceptions in the horse and typically affect young animals. in one study evaluating 26 cases of ileocecal intussusception, the median age of the horses was 1 year old. acute ileocecal intussusceptions are those in which the horses has a duration of colic of less than 24 hours and involve variable lengths of intestine that ranged in one study from 6 to 457 cm long. in acute cases the involved segment of ileum typically has a compromised blood supply. chronic ileocecal intussusceptions typically involve short segments of ileum (up to 10 cm long), and the ileal blood supply is frequently intact. 46 abdominocentesis results vary because strangulated bowel is contained within the adjacent bowel. obstruction of the small intestine often is evident, including nasogastric reflux and multiple distended loops of small intestine on rectal palpation. horses with chronic ileocecal intussusceptions have mild, intermittent colic, often without evidence of small intestinal obstruction. in one study, a mass was palpated in the region of the cecal base in approximately 50% of cases. 45 transabdominal ultrasound may be helpful in discerning the nature of the mass. the intussusception has a characteristic target appearance on cross section. 47 other segments of the small intestine also may be intussuscepted, including the jejunum (figure 13.14-3) . in one study of 11 jejunojejunal intussusceptions, the length of bowel involved ranged from 0.4 to 9.1 m. 48 attempts to reduce intussusceptions at surgery are usually futile because of intramural swelling of affected bowel. one should resect jejunojejunal intussusceptions. for acute ileocecal intussusceptions, one should transect the small intestine as far distally as possible and perform a jejunocecal anastomosis. in horses with particularly long intussusceptions (up to 10 m has been reported), one may attempt an intracecal resection. 49 for horses with chronic ileocecal intussusceptions, one should perform a jejunocecal bypass without small intestinal transection. the prognosis is good for horses with chronic ileocecal intussusceptions and guarded to poor for horses with acute ileocecal intussusceptions, depending on the length of bowel involved. 46 herniation of intestine through a rent in the diaphragm is rare in the horse and may involve any segment of bowel, although small intestine is herniated most frequently. diaphragmatic rents may be congenital or acquired, but acquired herniae are more common. congenital rents may result from incomplete fusion of any of the four embryonic components of the diaphragm: pleuroperitoneal membranes, transverse septum, and esophageal mesentery. in addition, abdominal compression of the foal at parturition may result in a congenital hernia. 50 acquired herniae are presumed to result from trauma to the chest or a sudden increase in intraabdominal pressure, such as might occur during parturition, distention of the abdomen, a sudden fall, or strenuous exercise. 51 herniae have been found in a number of different locations, although large congenital herniae are typically present at the ventral most aspect of the diaphragm, and most acquired herniae are located at the junction of the muscular and tendinous portions of the diaphragm. 50 a peritoneopericardial hernia has been documented in at least one horse. 52 figure 13 .14-3 jejunojejunal intussusception in a horse presented for colic. the intussusceptum has become ischemic because of invagination of intestine and its mesenteric blood supply into the intussuscipiens. clinical signs usually are associated with intestinal obstruction rather than respiratory embarrassment. 51 however, careful auscultation may reveal an area of decreased lung sounds associated with obstructed intestine and increased fluid within the chest cavity. 53 such signs may prompt thoracic radiography or ultrasound, both of which one can use to make a diagnosis. auscultation also may reveal thoracic intestinal sounds, but differentiating these from sounds referred from the abdomen typically is not possible. in one report, two of three horses diagnosed with small intestinal strangulation by diaphragmatic hernia had respiratory acidemia attributable to decreased ventilation. 54 treatment of horses with diaphragmatic hernia is fraught with complications because of the need to reduce and resect strangulated bowel and the need to repair the defect in the diaphragm. 55, 56 because dorsal defects in the diaphragm are among the common forms of diaphragmatic defect, closing the diaphragmatic hernia via the approach used for abdominal exploration may not be possible. however, because herniation is likely to recur, 55 scheduling a second surgery using an appropriate approach to resolve the diaphragmatic defect is appropriate. horses with large colon volvulus have rapid onset of severe, unrelenting abdominal pain, most often in postpartum broodmares. 4 once the large colon strangulates (≥270-degree volvulus), gas distention is significant, leading to gross distention of the abdomen, compromised respiration as the distended bowel presses up against the diaphragm, and visceral pooling of blood as the caudal vena cava is compressed. horses with this condition are frequently refractory even to the most potent of analgesics. these horses may prefer to lie in dorsal recumbency, presumably to take weight off the strangulated colon. an abbreviated physical examination is warranted in these cases, because the time elapsed from the onset of strangulation to surgical correction is critical. under experimental conditions, the colon is irreversibly damaged within 3 to 4 hours of a 360-degree volvulus of the entire colon. 57 despite severe pain and hypovolemia, horses may have a paradoxically low heart rate, possibly related to increased vagal tone. in addition, results of abdominocentesis often do not indicate the degree of colonic compromise 4,58 and in many cases are not worth attempting because of extreme colonic distention. 59 palpation per rectum reveals severe gas distention of the large colon, often restricting access to the abdomen beyond the pelvic brim. one may make the diagnosis tentatively based on signalment, severity of pain, and degree of distention. at surgery, the volvulus typically is located at the mesenteric attachment of the colon to the dorsal body wall and the most common direction of the twist is dorsomedial using the right ventral colon as a reference point. however, the colon may twist in the opposite direction, twist greater than 360 degrees (up to 720 degrees has been reported) or twist at the level of the diaphragmatic and sternal flexures. 4 in all cases, one should decompress the colon as much as possible, and in many cases a colonic evacuation via a pelvic flexure enterotomy greatly aids correction of the volvulus. one must determine after correction of the volvulus whether the colon has been injured irreversibly and should base the determination on mucosal color and bleeding (if an enterotomy has been performed), palpation of a pulse in the colonic arteries, serosal color, and appearance of muscular motility. if one judges the colon to be damaged irreversibly, one can consider the feasibility of a large colon resection. although 95% of the colon can be resected (that part of the colon distal to the level of the cecocolic fold), damage from the volvulus usually exceeds that which can be resected. in these cases, surgeons may elect to resect as much damaged bowel as possible or may advise euthanasia. 7 the prognosis is guarded to poor because of the rapid onset of this disease. in one study the survival rate was 35%. 58 in a more recent report the survival rate was 36% for horses with 360-degree volvulus of the large colon compared with 71% for horses with 270-degree volvulus. 4 however, one study in central kentucky documented a high success rate, possibly because of early recognition of the disease and the proximity of the hospital to the surgical caseload. 60 postoperative complications include hypovolemic and endotoxic shock, extensive loss of circulating protein, disseminated intravascular coagulation, and laminitis. in addition, large colon volvulus has a propensity to recur. although one study documented a recurrence rate of less than 5%, 58 some authors believe recurrence may be as high as 50%. 7 therefore one should consider methods to prevent recurrence in patients at risk of recurrence, particularly broodmares that tend to suffer from the disease recurrently during the foaling season. 61, 62 the most common intussusceptions of the large intestine are cecocecal and cecocolic intussusceptions. 63, 64 both are likely attributable to the same disease process, with variable inversion of the cecum. these conditions doughnut-shaped prolapse of rectal mucosa and submucosa. type ii prolapses involve full-thickness rectal tissue, whereas type iii prolapses additionally have invagination of small colon into the rectum. type iv prolapses involve intussusception of proximal rectum or small colon through the anus in the absence of prolapse of tissue at the mucocutaneous junction at the anus. 73 one can differentiate type iv from other forms of prolapse by their appearance and a palpable trench between prolapsed tissue and the anus. type i prolapses occur most frequently in horses with diarrhea, in which the rectal mucosa becomes irritated and protrudes intermittently during episodes of tenesmus. if tenesmus persists, rectal mucosa can remain prolapsed. rectal mucosa rapidly becomes congested and edematous under these conditions, which one should treat with osmotic agents such as glycerin or magnesium sulfate and by massaging and reducing the prolapse. 74 a purse-string suture may be required to keep the mucosa inside the rectum. topical application of lidocaine solution or jelly, epidural anesthesia, and sedation may help reduce tenesmus that incites and exacerbates rectal prolapse. one can apply similar treatments to type ii rectal prolapses. however, these more severe prolapses may not be reducible without surgical resection of mucosa and submucosa from the prolapsed bowel. 70, 74 type iii and iv rectal prolapses are more serious injuries because of involvement of small colon. 75 in horses with type iii prolapses, one should perform an abdominocentesis to determine if injured small colon has resulted in peritonitis. one should reduce the small colon component manually if possible, although prolapsed rectal tissue typically requires mucosal/ submucosal resection. one should perform surgical exploration of the abdomen to determine the status of the small colon, although one can use serial abdominocenteses in lieu of surgery to detect progressive necrosis of bowel. type iv prolapses occur most commonly in horses with dystocia. 73 these prolapses are almost always fatal because of stretching and tearing of mesenteric vasculature, with subsequent infarction of affected bowel. therefore euthanasia usually is warranted tend to occur in young horses (63% were less than 3 years old in one study) and may be associated with intestinal tapeworms. horses show highly variable clinical signs, including acute severe colic, intermittent pain over a number of days, or chronic weight loss. 64 these variable presentations likely relate to the degree to which the cecum has intussuscepted. initially, the cecal tip inverts, creating a cecocecal intussusception, which does not obstruct flow of ingesta. as the intussusception progresses, the cecum inverts into the right ventral colon (cecocolic intussusception), obstructs flow of ingesta, and often causes severe colic. the cause of abdominal pain is often difficult to differentiate in these cases, although detecting a mass on the right side of the abdomen by per rectal palpation or ultrasound examination sometimes is possible. 63, 64 treatment involves manual surgical reduction by retracting the intussusceptum directly 63 or via an enterotomy in the right ventral colon. 65 however, a number of cases occur in which one cannot reduce the cecum readily because of severe thickening or in which surgical procedures result in fatal contamination. for example, one report stated that 8 of 11 horses were euthanized in the perioperative period because of complications, 63 and another report stated that 12 of 30 horses were euthanized before or during surgery. the latter included all of the horses with chronic disease because of irreversible changes to the cecum. 64 however, one recent report on cecocolic intussusceptions indicated that seven of eight horses that underwent right ventral colon enterotomy and cecal resection survived long-term, 65 suggesting that continued improvements in surgical techniques may improve the prognosis. colocolic intussusceptions are rare but have been reported to affect the pelvic flexure and the left colons. [66] [67] [68] [69] although the condition is reported to be more common in young horses, 67-69 the condition may affect older horses. 66 clinical findings may include a palpable mass on the left side of the abdomen. 67 ultrasonography also may be useful. treatment requires manual reduction of the intussusception at surgery, 67, 69 or resection of affected bowel. 66 because the left colons may be exteriorized extensively and manipulated at surgery, 66-69 the prognosis is fair. rectal prolapse may occur following any disease that causes tenesmus, including diarrhea, rectal neoplasia, and parasitism, 70 or prolapse can occur following elevations in intraabdominal pressure during parturition or episodes of coughing. 71, 72 rectal prolapses are classified into four categories (table 13. 14-1) based on the extent of tissue prolapsed and the severity. type i rectal prolapse is most common and is characterized by a intussusception of rectum and poor small colon through the anus based on physical examination findings. however, confirmation of severe small colonic injury requires abdominal exploration via a midline approach or laparoscopy. 76 a horse with compromised small colon conceivably could undergo a colostomy of the proximal small colon, but the compromised small colon typically necroses beyond that which can be resected via a midline abdominal approach. 74 nonstrangulating infarction occurs following cranial mesenteric arteritis caused by migration of strongylus vulgaris and has become a rare disorder since the advent of broad-spectrum anthelmintics. although thromboemboli have been implicated in the pathogenesis of this disease, careful dissection of naturally occurring lesions has not revealed the presence of thrombi at the site of intestinal infarctions in most cases. 77 these findings suggest that vasospasm plays an important role in this disease. 78 clinical signs vary greatly depending on the extent to which arterial flow is reduced and the segment of intestine affected. any segment of intestine supplied by the cranial mesenteric artery or one of its major branches may be affected, but the distal small intestine and large colon are more commonly involved. no clinical variables exist that one can use to differentiate this disease from strangulating obstruction reliably. in some cases, massive infarction results in acute, severe colic. 77 occasionally, one may detect an abnormal mass and fremitus on palpation of the root of the cranial mesenteric artery per rectum. one should consider this disease a differential diagnosis in horses with a history of inadequate anthelmintic treatment and the presence of intermittent colic that is difficult to localize. although one should perform fecal parasite egg counts, they are not indicative of the degree of parasitic infestation. in addition to routine treatment of colic, dehydration, and endotoxemia, medical treatment may include aspirin (20 mg/kg every 24 hours) to decrease thrombosis. 78 definitive diagnosis requires surgical exploration. however, these cases are difficult to treat because of the patchy distribution of the lesions and the possibility of lesions extending beyond the limits of surgical resection. in addition, further infarction may occur following surgery. the prognosis is fair for horses with intermittent mild episodes of colic that may be amenable to medical therapy but is poor in horses that require surgical intervention. 77, 78 surgical exploration of a horse with on-going intestinal injury exacerbates shock induced largely by endotoxin traversing damaged mucosa, and this in turn correlates with mortality. 3 the initial clinical step in the workup of horses with colic is taking a thorough history. however, one may have to delay taking a complete history until after the physical examination and initial treatment, because management of abdominal pain may take precedence. if possible, one should obtain the vital components of the history before examination and treatment: the duration and severity of colic symptoms, analgesics already administered, and a history of any adverse drug reactions. the two most critical factors from a history that would support a decision to explore a horse with colic surgically are the duration of signs and the extent of pain. one deduces the latter from asking the owner about the presence and frequency of pawing, looking at the flanks, rolling, repeatedly going down and getting back up, posturing as if to lie down or urinate, among other clinical evidence of pain. 4 table 13 .15-1 lists other important components of the history one should obtain to try to ascertain why colic has occurred. just as the history necessarily may need to be brief to allow rapid treatment of colic, so the clinician must be able to alter the extent of the physical examination to treat the horse in a timely fashion. the most critical examination finding is the heart rate of the horse, because it provides an excellent assessment of the cardiovascular status of the horse. 4 the heart rate is likely the single most reliable predictor of the need for surgery and survival. 4, 5 because analgesics can alter the heart rate dramatically, if possible, one should obtain the heart rate before administering analgesics. other components of the examination are designed specifically to gather information about the cardiopulmonary status of the horse (quality of the pulse, mucous membrane color, capillary refill time, respiratory rate, and full auscultation of the chest), and the nature of the intestinal obstruction (auscultation of gastrointestinal sounds, per rectal palpation of the abdomen, and presence of nasogastric reflux). although classic presentations exist for horses with obstructions of the small or large intestine (table 13. clinical management of colic is distinctly different from management of many other clinical syndromes because the initial focus is often not on defining the definitive diagnosis but rather on deciding whether a horse requires surgical exploration. therefore the clinician must collect historical, physical examination, and clinicopathologic information and make a decision whether these findings warrant medical management or whether to perform surgical exploration of the abdomen because of a suspected obstructive or ischemic lesion. for example, one may examine a horse with signs of severe abdominal pain, poor cardiovascular status, and abdominal distention that may be compatible with an extensive list of differential diagnoses but that more importantly indicate the need for abdominal exploration to minimize the extent of intestinal injury. the speed with which one can make this clinical decision has a tremendous effect on the well-being of the patient, 1,2 because delaying gastric fluid accumulation because of direct compression of the small intestine by distended colon or via tension on the duodenocolic ligament. the most useful diagnostic test for determining the type of intestinal obstruction is rectal palpation of the abdomen. 4 however, one can reach only approximately one third of the abdomen via the rectum, and this percentage may be substantially less in large horses or heavily pregnant horses. nonetheless, attempting to determine the type of obstruction present (small intestine versus large intestine, and simple obstruction versus strangulating obstruction) is worthwhile; this information directly affects prognosis. in one study, interns and residents at a veterinary teaching hospital were able to predict the type of lesion with a specificity exceeding 90%. 6 findings from palpation are helpful in educating the client about the potential findings in surgery and the likelihood of survival for the horse. before considering how to manage signs of colic, one should remember that such signs are poorly localized. therefore although colic is most frequently associated with intestinal disease, one should consider dysfunction of other organ systems, including urinary obstruction, 7,8 biliary obstruction, 9 uterine torsion or tears, 10,11 ovarian artery hemorrhage, 10 and neurologic disease as differential diagnoses. 12 however, the duration and severity of colic the most immediately useful clinicopathologic information in horses with colic are the packed cell volume and total protein, because one can use them to substantiate clinical estimates of dehydration and they correlate strongly with prognosis. 20, 21 a serum biochemical profile is useful for assessing electrolyte imbalances, tissue perfusion (anion gap or lactate), and kidney and liver function. one can use serum biochemical or blood gas analysis to assess acid-base status. horses with colic most frequently show evidence of metabolic acidosis associated with poor tissue perfusion caused by hypovolemia or endotoxemia, but one may note other abnormalities such as metabolic alkalosis in association with extensive loss or sequestration of stomach chloride. metabolic acidosis has been investigated further in horses with colic by measuring blood lactate, although this test is not offered routinely in many laboratories. lactate levels also have been inferred from measurement of the anion gap, although one study noted that lactate in horses with colic did not account for the entire anion gap. 22 lactate levels and anion gap closely correlate with prognosis for survival. 20, 23, 24 other key components of assessment of the horse with colic are abdominocentesis and complete blood count. the total white blood cell count and differential can provide crucial evidence of systemic inflammation associated with endotoxemia stemming from colic attributable to colitis (leukopenia, neutropenia, and a left shift) rather than an obstruction (highly variable complete blood count findings). peritoneal fluid may be helpful in determining the integrity of the intestine. specifically, as the intestine becomes progressively devitalized, the peritoneal fluid becomes serosanguinous as red blood cells leak into the abdomen, followed by an elevation in the total protein (>2.5 g/dl) and progressive increases in total nucleated cell count (>10,000 cells/µl). however, these findings do not always correlate well with the condition of the intestine, particularly in horses with large colon volvulus. for example, in a study of 57 horses with large colon volvulus, the average total protein (2.5 g/dl) and total nucleated cell count (1000 cells/µl) were normal despite the fact that only 36% with a 360-degree volvulus survived. 4, 25 these measures may appear normal because the development of severe mucosal injury following large colon volvulus is rapid and may not allow enough time for protein and leukocytes to equilibrate with the abdominal fluid. 26 investigators have taken all the variables routinely assessed during evaluation of horses for colic and have attempted to develop models to predict accurately the need for surgery and the prognosis for life. [27] [28] [29] [30] none of these predictor models has taken the place of clinical decision making, although these studies have added 924 part ii disorders of specific body systems signs are excellent predictors of whether a horse requires surgical exploration of the abdomen. in fact, refractory pain supersedes all other predictors of the need for surgery in the colic patient. once signs of colic have been recognized and categorized as to their severity, rapidly and effectively relieving the pain is critical for the well-being of the horse and to reduce the owner's anxiety. in addition, pain is best managed before it becomes severe. 13 several classes of analgesics are readily available to treat horses with colic (table 13. 15-3), including α 2 -agonists (xylazine, detomidine), opiates (butorphanol), and nonsteroidal antiinflammatory drugs (nsaids, such as flunixin meglumine). although much of this information is familiar to most practitioners, several principles deserve emphasis. the short-duration drugs xylazine and butorphanol, which provide analgesia for 30 to 45 minutes, allow the veterinarian to determine if pain is recurrent within the time period of the typical examination. in contrast, flunixin meglumine is not as potent as an analgesic but has a much longer duration of action. to avoid deleterious effects on gastrointestinal mucosa and the kidneys, one should not administer flunixin meglumine more frequently than recommended. 14, 15 the recent discovery of two isoforms of cyclooxygenase (cox), the enzyme inhibited by nsaids, has resulted in discovery of drugs that can more selectively inhibit proinflammatory cox-2 while permitting continued constitutive production of prostanoids. such specificity may be advantageous in horses with colic, particularly when one considers recent evidence of reduced intestinal recovery from an ischemic event with flunixin compared with a drug that is more selective for cox-2. 16 one should reserve the α 2agonist detomidine for horses with severe, unrelenting pain because of its tremendous potency. 17 in addition, one should remember that α 2 -agonists reduce the heart rate associated with a transient increase in blood pressure, 18, 19 thereby reducing the predictive value of the heart rate and pulse pressure. tremendously to understanding of the importance of some prognostic factors, particularly those reflecting cardiovascular function. simple obstruction involves intestinal obstruction of the lumen without obstruction of vascular flow. however, because a tremendous volume of fluid enters the small intestinal lumen daily, 31,32 the obstructed intestine tends to become distended, which in turn may reduce mural blood flow. 33 ultimately, such distention may result in necrosis of tissues, particularly in the immediate vicinity of the obstruction. 34 few are the causes of simple obstruction in the small intestine, and the incidence of these obstructions is low (approximately 3% of all referred horses in one large hospital-based study). 5 however, in some geographic regions, this type of obstruction is prevalent. for example, in the southeastern united states, ileal impactions are common. 35 ileal impactions most commonly occur in adult horses in the southeastern united states. although feeding of coastal bermuda hay has been implicated in the regional distribution of the disease, separating geographic location from regional hay sources as risk factors has been difficult. nonetheless, feeding coastal bermuda hay likely places horses at risk of ileal impaction, particularly if the coarse fiber content of the hay is high. furthermore, sudden changes in feed from an alternate type of hay to coastal bermuda hay likely places a horse at risk of ileal impaction. 38 studies in england have revealed tapeworm infection as another important risk factor for ileal impaction. based on risk analysis, the data suggested that in excess of 80% of the ileal impaction cases studied were associated with serologic or fecal evidence of tapeworm infection. 39 because of the poor sensitivity of fecal analysis for tapeworms, proudman and trees have developed a serologic test (enzyme-linked immunosorbent assay) with a sensitivity of approximately 70% and a specificity of 95%. 40 clinical signs of horses with ileal impaction are typical for a horse with small intestinal obstruction, including onset of moderate to severe colic and loops of distended small intestine palpable per rectum as the condition progresses. because the ileum is the distal most aspect of the small intestinal tract, nasogastric reflux may take a considerable time to develop and is found in approximately 50% of horses requiring surgical correction of impacted ileum. 35, 41 one usually makes the diagnosis at surgery, although on occasion one may palpate an impacted ileum per rectum. multiple loops of distended small intestine frequently make the impaction difficult to palpate. ileal impactions may resolve with medical treatment 36 but frequently require surgical intervention ( figure 13.15-2) . at surgery, one can infuse fluids directly figure 13 .15-1 appearance of roundworms that have been retrieved within the nasogastric reflux from a foal with an ascarid impaction. the large size of these ascarids (bar = 1 cm) contributes to the risk of impaction following sudden kills of these parasites by broad-spectrum anthelmintics. into the mass, allowing the surgeon to breakdown the impaction. the surgeon may include dioctyl sodium sulfosuccinate in the infused fluid to aid in disruption of the mass. extensive small intestinal distention and intraoperative manipulation of the ileum may lead to postoperative ileus, 42 but recent studies indicate that this complication is less frequent as the duration of disease before admission decreases. 35 recent studies indicate that the prognosis for survival is good. 35, 36 ileal hypertrophy is a disorder in which the muscular layers (circular and longitudinal) of the ileum hypertrophy for unknown reasons (idiopathic) or following an incomplete or functional obstruction. for idiopathic cases, proposed mechanisms include parasympathetic neural dysfunction resulting in chronically increased muscle tone and subsequent hypertrophy of the muscular layers of the ileal wall. such neural dysfunction possibly could result from parasite migration. alternative hypotheses include chronic increases in the muscular tone of the ileocecal valve, leading to muscular hypertrophy of the ileum as it contracts against a partially occluded ileocecal valve. the jejunum also may be hypertrophied, alone or with the ileum. clinical signs include chronic intermittent colic as the ileum hypertrophies and gradually narrows the lumen diameter. in one study, partial anorexia and chronic weight loss (1 to 6 months) were documented in 45% of the horses, most likely because of intermittent colic and reduced appetite. because hypertrophy does not affect the ileal mucosa, no reason exists to believe that these horses experience malabsorption of nutrients. one usually makes the diagnosis at surgery, although one may palpate the hypertophied ileum per rectum in some cases. for treatment, one performs an ileocecal or jejunocecal anastomosis to bypass the hypertrophied ileum. without surgical bypass, intermittent colic persists and the thickened ileum ultimately may rupture. 43 the prognosis is fair with surgical treatment. 44 secondary ileal hypertrophy is most commonly notable in horses that previously have had colic surgery and that may have a partial or functional obstruction at an anastomotic site. for example, in one case report, a horse developed ileal hypertrophy after surgical correction of an ileocecal intussusception. 45 ileal hypertrophy also was noted in a horse with cecal impaction in which an ileocolic anastomosis was oriented incorrectly. 46 horses are typically re-presented for recurrence of colic in these cases. surgical therapy is directed at addressing the cause of small intestinal obstruction and resecting hypertrophied intestine. meckel's diverticulum is an embryonic remnant of the vitelloumbilical duct, which fails to atrophy completely and becomes a blind pouch projecting from the antimesenteric border of the ileum. 47, 48 however, similar diverticula also have been noted in the jejunum. 49 these diverticula may become impacted, resulting in partial luminal obstruction, or may wrap around an adjacent segment of intestine, causing strangulation. 47 occasionally, an associated mesodiverticular band may course from the diverticulum to the umbilical remnant and serve as a point around which small intestine may become strangulated. mesodiverticular bands also may originate from the embryonic ventral mesentery and attach to the antimesenteric surface of the bowel, thereby forming a potential space within which intestine may become entrapped. clinical signs range from chronic colic for an impacted meckel's diverticulum to acute severe colic for intestine strangulated by a mesodiverticular band. one makes the diagnosis at surgery, and treatment requires resection of the diverticulum and any associated bands. the prognosis is good for horses with simple impaction of a meckel's diverticulum and is guarded for horses with an associated small intestinal strangulation. 50 adhesions of one segment of bowel to another or of a segment of intestine to other organs and the body wall most typically occur following abdominal surgery and may be clinically silent, cause chronic colic attributable to partial obstruction, or result in acute obstruction. these differing clinical syndromes are attributable to the type of adhesions that develop. for example, a fibrous adhesion that does not by itself obstruct the intestinal lumen might serve as the pivot point for a volvulus, whereas an adhesion between adjacent segments of the intestinal tract may create a hairpin turn that causes chronic partial obstruction. 51 the number of adhesions that develop also may vary greatly from horse to horse. some horses may develop a single adhesion adjacent to an anastomotic site or a discrete segment of injured intestine, whereas other horses may develop diffuse adhesions involving multiple segments of intestine, likely because of widespread inflammation of the peritoneum at the time of the original surgery. the mechanism whereby adhesions develop is complex but likely involves initial injury to the serosa initiated by intestinal ischemia, reperfusion injury, and luminal distention. 52 importantly, such injury involves infiltration of neutrophils into the serosa accompanied by loss of mesothelial cells. in one study assessing the margins of resected small intestine, extensive neutrophil infiltration was documented in the serosa, particularly in the proximal resection margin that had been distended before correction of a variety of strangulating lesions. 53 regions of serosal injury and inflammation subsequently undergo reparative events similar to any wound, including local production of fibrin, de novo synthesis of collagen by infiltrating fibroblasts, and ultimately maturation and remodeling of fibrous tissue. unfortunately, during this process, fibrin may result in injured intestinal surfaces adhering to adjacent injured bowel or an adjacent organ. once a fibrinous adhesion has developed, new collagen synthesis may result in a permanent fibrous adhesion. alternatively, proteases released by local phagocytes may lyse fibrinous exudate, thereby reversing the adhesive process. thus one can view formation of adhesions as an imbalance of fibrin deposition and fibrinolysis. 54 prevention of adhesions depends on inhibition of the mechanisms involved in adhesion formation, including reduction of serosal injury with early intervention and good surgical technique, reduction of inflammation by administration of antiinflammatory medications, physical separation of inflamed serosal surfaces (e.g., carboxymethylcellulose and hyaluronan), [55] [56] [57] and pharmacologic modulation of fibrinous adhesion formation (e.g., heparin). 58 in addition, early return of motility in the small intestine after surgery may reduce contact time between inflamed surfaces of intestine, thereby reducing the chances of adhesion formation. 54 horses at greatest risk of developing adhesions after colic surgery appear to be those that have small intestinal disease. 51, 59 in one study of horses undergoing surgical correction of small intestinal obstruction, 22% developed a surgical lesion associated with adhesions. foals appear to have an increased incidence of adhesions compared with mature horses regardless of the nature of the abdominal surgery. 51 one study indicated that 17% of foals developed lesions attributable to adhesions regardless of the type of initial surgery. 60 studies conflict as to whether the degree of surgical intervention influences adhesion formation, 51 but in one study, horses that require enterotomy or resection and anastomosis were at greatest risk of developing adhesions. 59 as an indication of the importance of postoperative adhesion formation, adhesions were among the most common reasons for repeat laparotomy in postoperative colic patients. 59, 61 clinical signs of horses with adhesions vary greatly depending on whether the adhesion is causing partial obstruction or complete luminal obstruction or involves intestinal vasculature. adhesions would be an important differential diagnosis for intermittent colic in the postoperative period, particularly if such colic was not relieved by nasogastric decompression of the stomach. continued intermittent colic should prompt abdominocentesis to determine if septic peritonitis is present, which may contribute to adhesion formation. placement of a large bore drain and peritoneal lavage ( figure 13 surgery should prompt immediate nasogastric intubation to decompress the stomach. treatment should include attempts at obtaining reflux from the horse at frequent intervals rather than relying on passive flow of reflux. in addition, administration of intravenous fluids should account for the maintenance requirement (50 ml/kg/day, about 1 l/hr in the average horse) and fluid losses via reflux. in practice, this requires frequent monitoring of packed cell volume and total protein to ensure that the horse remains well hydrated. although concerns have arisen that overhydrating horses may contribute to increased nasogastric reflux, 42 keeping horses well-hydrated to avoid hypovolemic shock is critical. additionally, one should monitor electrolytes frequently, particularly considering their potential role in smooth muscle contraction and nerve excitability. because of the important role of inflammation in postoperative ileus, including elaboration of cox-2-produced prostanoids, 70 administration of nsaids is indicated. nsaid administration is particularly necessary if postoperative ileus is associated with endotoxemia, because lipopolysaccharide-induced prostanoid production disrupts propulsive motility in horses. 71, 72 interestingly, phenylbutazone is more effective than flunixin meglumine at reducing the deleterious actions of lipopolysaccharide on intestinal motility. 73 however, one should use caution when administering nsaids to patients with postoperative ileus in light of research suggesting that complete inhibition of prostanoid production can alter motility patterns in normal equine intestine. 68 the advent of selective cox-2 inhibitors may provide optimal antiinflammatory treatment in the future. 74 other treatments aimed at specifically modulating intestinal motility include lidocaine (bolus of 1.3 mg/kg followed by 0.05 mg/kg/min for 24 hours), erythromycin (0.5 to 1.0 mg/kg slow intravenous infusion in 1 l saline every 6 hours), and metoclopramide (0.04 mg/kg/hr). 66, 75, 76 the mechanism of lidocaine is presumed to be inhibition of sensory nerve activity within the wall of the intestine, thereby reducing reflex sympathetic inhibitory activity. in addition, intravenously administered lidocaine appears to be an effective analgesic. thus an important feature of intravenous lidocaine therapy may be to control postoperative pain-induced reduction of gastrointestinal motility and mucosal secretory activity. 77 metoclopramide may stimulate intestinal motility by several mechanisms, including dopamine receptor blockade, cholinergic stimulation, and adrenergic blockade. 66 although metoclopramide has been shown to be beneficial for reversing postoperative ileus in clinical patients and research animals, it has central nervous system excitatory side effects in the horse that make its use difficult. nonetheless, administration of metoclopramide to horses with postoperative ileus resulted in elect repeat laparotomy or laparoscopy. in one study of adhesions, 70% of repeat laparotomies were performed within 60 days, suggesting that surgical colic attributable to adhesions typically occurs within 2 months of an initial surgical procedure. unfortunately, the prognosis for horses with colic attributable to adhesions is low, with only 16% of horses in one study surviving from adhesion-induced colic. 51 the definition of ileus is intestinal obstruction, including physical and functional obstructions. however, in veterinary medicine, the term typically is used to designate a lack of progressive aboral propulsion of ingesta resulting in functional obstruction. 62 one typically bases the diagnosis of postoperative ileus on the presence of excessive gastric fluid accumulation (reflected as excessive nasogastric reflux). postoperative ileus may occur following any abdominal exploratory procedure. however, horses undergoing surgery for strangulating small intestinal lesions or small intestinal obstructive lesions such as an ileal impaction are at greatest risk. 42 recently, the syndrome of postoperative ileus in horses has been broadened to include those horses that may have delayed transit of ingesta through the large intestine following surgery. this large intestinal ileus may follow any type of surgery, particularly horses that have had orthopedic surgery, and is characterized by reduced fecal output (fewer than three piles of manure per day) rather than excessive nasogastric reflux. 62 however, horses with excessive nasogastric reflux are unlikely to have normal fecal output, so the distinction between these two manifestations of ileus is not absolute. mechanisms involved in precipitating postoperative ileus characterized by small intestinal dysfunction likely involve local inflammation, reduced coordination of progressive motility, and increased sympathetic tone. a recent series of studies in the rat has shown that surgical manipulation of intestine results in delayed transit time associated with infiltration of neutrophils into intestinal longitudinal muscle [63] [64] [65] and upregulation of inducible nitric oxide synthase and cox-2. the mechanisms in the horse may be similar in that extensive manipulation of the intestine resulted in abnormal intestinal motility in ponies, 66 and prostanoids and nitric oxide alter or reduce intestinal motility in horses. [67] [68] [69] clinical signs of postoperative ileus following colic surgery include evidence of abdominal pain, increased heart rate, reduced gastrointestinal sounds, and reflux of gastric fluid via a nasogastric tube. of these signs, heart rate is critical because it appears to be a more sensitive indicator of pain in the postoperative period than overt evidence of colic. therefore a sudden increase in the heart rate of a postoperative patient following colic a significantly reduced duration of reflux and shorter postoperative hospital stays compared with horses not receiving this drug. 76 in the same study, constant infusion of metoclopramide was superior to intermittent infusion. recent in vitro studies indicate that metoclopramide effectively increases smooth muscle contractile activity throughout the small intestine. similarly, the motilin agonist erythromycin had stimulatory effects on equine small intestine, although the results were not uniform throughout the small intestine. erythromycin stimulates contractile activity in the longitudinal muscle of the pyloric antrum but inhibits contractile activity in circular smooth muscle in this segment of the gastrointestinal tract. 75 the latter may be attributable to activation of motilin receptors on inhibitory nerves and may result in enhanced gastric emptying. in vivo studies on erythromycin confirmed the stimulatory action of this drug on the distal small intestine and indicated this drug also stimulates contractile activity in the cecum and pelvic flexure. however, the stimulation depends on the temporal association with surgery. erythromycin stimulated contractile activity in the postoperative period in the ileum and pelvic flexure but not the cecum, 78 suggesting this drug may be useful for treating select cases of postoperative ileus. for horses with presumed ileus of the large colon, signs included reduced fecal output (fewer than three piles of manure per day), reduced gastrointestinal sounds, variable presence of colic, and on occasion a palpable impaction of the cecum or large colon. risk factors for this syndrome include orthopedic surgery, length of the operative period, and most importantly inadequate treatment with phenylbutazone, presumably resulting from insufficient control of postoperative pain. although treatment of large colon impaction in the postoperative period typically is uncomplicated, onset of cecal impaction is fatal in many cases because of the difficulty in recognizing horses that have cecal dysfunction. therefore one should pay close attention to fecal production and optimal analgesic treatment in any horse following an orthopedic procedure. 62 other painful procedures, including ophthalmologic procedures, also likely place horses at risk of developing ileus of the large intestine. simple obstructions of the large intestine such as impaction tend to have a more gradual onset than those of the small intestine, although horses may become acutely and severely painful with some forms of colon displacement. in fact, some of these cases mimic and may progress toward large colon volvulus. medical therapy is frequently successful in correcting large colon impactions. however, cecal impactions present much more of a dilemma because of the greater propensity of this organ to rupture, the relative difficulty of surgically manipulating the cecum, and the onset of cecal dysfunction that may prevent the cecum from emptying following surgical resolution of impaction. cecal impaction may be divided into two syndromes: primary cecal impactions that result from excessive accumulation of ingesta in the cecum and secondary cecal impactions that develop while a horse is being treated for a separate problem. 79, 80 although primary impactions typically consist of impacted, relatively dry fecal material and secondary cecal impactions tend to have fluid contents, considerable overlap exists between the two syndromes, and one must approach each case carefully. in horses with primary cecal impactions, onset of abdominal pain occurs over a number of days, reminiscent of the development of a large colon impaction. one should differentiate cecal impactions from large colon impactions on the basis of rectal palpation findings. cecal impactions have a propensity to rupture before the development of severe abdominal pain or systemic deterioration and therefore must be monitored closely. 79 secondary cecal impactions typically develop following unrelated surgical procedures that result in postoperative pain (particularly orthopedic surgeries). secondary cecal impactions may be even more difficult to detect because one may attribute postoperative depression and decreased fecal output to the operative procedure rather than to colic. by the time horses with secondary cecal impactions show noticeable signs of colic, the cecum may be close to rupture. in many cases, no signs of impending rupture are evident. 80 therefore all horses that undergo surgeries in which considerable postoperative pain may develop should have feed intake and manure production closely monitored. a recent study indicated that horses that produce fewer than three piles of manure daily in the postoperative period are at risk of developing a large intestinal impaction. furthermore, horses that underwent prolonged (>1 hour) orthopedic surgery that received inadequate treatment with phenylbutazone were at considerable risk of reduced postoperative fecal output. 62 these results are in contrast to statements indicating that nsaids may place horses at risk of impaction, statements that appear to be based largely on clinical impressions rather than on risk analysis. 80 the diagnosis of primary cecal impaction is based on palpation of a firm, impacted cecum per rectum. in some cases, cecal impactions may be difficult to differentiate from large colon impactions. however, careful palpation reveals the inability to move the hand completely dorsal to the impacted viscus because of the attachment of the cecum to the dorsal body wall. treatment for horses with primary cecal impactions may include initial medical therapy, including aggressive administration of intravenous fluids and judicious use of analgesics. 80 however, if the cecum is distended grossly or if medical therapy hasno effect within a reasonable period of time, surgical evacuation of the cecum via a typhlotomy is indicated. 79 in addition, performing an ileocolostomy to bypass the cecum is advisable, because postoperative cecal motility dysfunction with recurrence of the impaction is common. 46, 81 in horses that develop secondary cecal impactions, diagnosis is based on palpation of a greatly distended cecum filled with semifluid intestinal contents. the nature of the contents likely is related to the more rapid progression of this disease compared with primary cecal impaction. one should not delay surgery because of the risk of cecal rupture. 82 however, if the cecum appears healthy following typhlotomy and evacuation, bypass of the cecum is not as critical as it is for primary impactions as long as one can control the inciting cause of the impaction (such as orthopedic pain). the prognosis is guarded for surgical treatment of all cecal impactions because of the potential for the cecum to rupture during prolonged medical treatment or during surgical manipulation, the possibility of abdominal contamination during surgery, and the extensive surgical procedures required. in a recent report, seven of nine horses for which cecal impaction was treated by typhlotomy and ileocolostomy or jejunocolostomy lived long term. 46 however, a separate report indicated that all horses with cecal impaction following another disease process had cecal rupture without any signs of impending rupture. 80 ingesta impactions of the large colon occur at sites of anatomic reductions in luminal diameter, particularly the pelvic flexure and the right dorsal colon. 83 although a number of risk factors have been reported, most have not been proved. however, a sudden restriction in exercise associated with musculoskeletal injury appears frequently to be associated with onset of impaction. 84 another consideration is equine feeding regimens, which usually entail twice daily feeding of concentrate. such regimens are associated with large fluxes of fluid into and out of the colon, associated with readily fermentable carbohydrate in the colon and subsequent increases in serum aldosterone, respectively. one may prevent these fluid fluxes, which may cause dehydration of ingesta during aldosterone-stimulated net fluid flux out of the colon, with frequent small feedings. 32 amitraz, an acaricide associated with clinical cases of colon impaction, can induce impaction of the ascending colon. 85, 86 this effect may provide some clues as to the pathogenesis of large colon impaction. in particular, amitraz appears to alter pelvic flexure pacemaker activity, resulting in uncoordinated motility patterns between the left ventral and left dorsal colon and excessive retention of ingesta. absorption of water from the ingesta increases with retention time, dehydrates the contents of the colon, and results in impaction. conceivably, parasite migration in the region of a pacemaker may have a similar action. 87 other factors implicated in large colon impaction include limited exercise, poor dentition, coarse roughage, or dehydration. clinical signs of large colon impaction include slow onset of mild to moderate colic. fecal production decreases, and the feces are often hard, dry, and mucuscovered because of delayed transit time. the heart rate may be elevated mildly during episodes of pain but is often normal. signs of abdominal pain are typically well controlled with administration of analgesics but become increasingly more severe and refractory if the impaction does not resolve. the diagnosis is based on palpation of a firm mass in the large colon per rectum. however, one may underestimate the extent of the impaction by rectal palpation alone because much of the colon is out of reach. adjacent colon may be distended if the impaction has resulted in complete obstruction. one should attempt initial medical treatment. administration of analgesics (e.g., flunixin meglumine at 0.5 to 1.1 mg/kg intravenously every 8 to 12 hours; butorphanol at 0.04 to 0.1 mg/kg intramuscularly every 4 to 6 hours; or xylazine at 0.3 to 0.5 mg/kg intravenously as needed) controls intermittent abdominal pain. administration of oral laxatives such as mineral oil (2 to 4 l by nasogastric tube every 12 to 24 hours) and the anionic surfactant dioctyl sodium sulfosuccinate (6 to 12 g/500 kg diluted in 2 to 4 l of water by nasogastric tube every 12 to 24 hours) are used commonly to soften the impaction. saline cathartics such as magnesium sulfate (0.1 mg/kg in 2 to 4 l by nasogastric tube) also may be useful. one should not permit access to feed. for impactions that persist, one should institute aggressive oral and intravenous fluid therapy (2 to 4 times the maintenance fluid requirement). if the impaction remains unresolved, the horse becomes uncontrollably painful, or extensive gas distention of the colon occurs, surgery is indicated. in addition, one can monitor abdominal fluid serially to determine the onset of intestinal compromise. 83 at surgery, one evacuates the contents of the colon via a pelvic flexure enterotomy. the prognosis is good for those horses in which impactions resolve medically (95% long-term survival in one study) and fair in horses that require surgical intervention (58% long-term survival in the same study). 84 enteroliths are mineralized masses typically composed of magnesium ammonium phosphate (struvite). 88 however, magnesium vivianite also has been identified in enteroliths, along with variable quantities of sodium, sulfur, potassium, and calcium. the formation of magnesiumbased minerals is puzzling because of the relative abundance of calcium in colonic fluids, which would favor the formation of calcium phosphates (apatite) rather than struvite. 89 however, elevated dietary intake of magnesium and protein may play a role. many horses that develop enteroliths are located in california and are fed a diet consisting mainly of alfalfa hay. analysis of this hay has revealed a concentration of magnesium approximately 6 times the daily requirements of the horse. 90 furthermore, the high protein concentration in alfalfa hay may contribute to calculi formation by increasing the ammonia nitrogen load in the large intestine. enteroliths most commonly form around a nucleus of silicon dioxide (a flintlike stone), but nidi have included ingested nails, rope, and hair. 88 enteroliths usually are found in the right dorsal and transverse colons. 90 although enterolithiasis has a wide geographic distribution, horses in california have the highest incidence. in one california study, horses with enterolithiasis represented 28% of the surgical colic population, and arabians, morgans, american saddlebreds, and donkeys were at greatest risk of this disease. 91 in a study of enterolithiasis in texas, risk factors also included feeding of alfalfa hay and arabian breed. however, in that study, miniature horses were also at risk. 92 horses with enteroliths are rarely under 4 years old, 90 although an enterolith in an 11-month-old miniature horse has been reported recently. 93 enterolithiasis is characterized by episodic, mild to moderate, intermittent abdominal pain. 90 progressive anorexia and depression may develop. the amount of pain depends on the degree of obstruction and amount of distention. partial luminal obstruction allows the passage of scant, pasty feces. heart rate varies and depends on the degree of pain. in some cases, an enterolith is forced into the small colon, where it causes acute small colon obstruction. one may diagnose enteroliths by abdominal radiography or at surgery. on rare occasions, one may palpate an enterolith per rectum, particularly if it is present in the distal small colon. in general, these cases require surgery, although enteroliths being retrieved per rectum have been reported. in fact in one study, 14% of horses presented for treatment of enterolithiasis had a history of passing an enterolith in the feces. however, enteroliths typically are located in the right dorsal colon, transverse colon, or small colon. at surgery, one gently pushes the enterolith toward a pelvic flexure enterotomy, but removal frequently requires a separate right dorsal colon enterotomy to prevent rupture of the colon. following removal of an enterolith, one must conduct further exploration to determine if other enteroliths are present. solitary enteroliths are usually round, whereas multiple enteroliths have flat sides. the prognosis is good (92% 1-year survival in one study of 900 cases), unless the colon ruptures during removal of an enterolith. in one recent study, rupture occurred in 15% of cases. 91 sand impactions are common in horses with access to sandy soils, particularly horses eating feed placed on the ground. some horses, especially foals, deliberately eat sand. fine sand tends to accumulate in the ventral colon, whereas coarse sand may accumulate in the dorsal colon. 94, 95 however, individual differences in colonic function may contribute to accumulation of sand, because some horses can clear consumed sand, whereas others cannot. distention from the impaction itself, or gas proximal to the impaction, causes abdominal pain. in addition, sand may trigger diarrhea, presumably because of irritation of the colonic mucosa. 96 in horses with sand impactions, clinical signs are similar to those of horses with large colon impactions. 94 one may find sand in the feces, and auscultation of the ventral abdomen may reveal sounds of sand moving within the large colon. 97 however, unlike sand-induced diarrhea, one may not hear sand impactions easily because of the lack of colonic motility. to determine the presence of fecal sand, one places several fecal balls in a rectal palpation sleeve or other container, which subsequently is filled with water. if sand is present, it accumulates at the bottom of the container. in addition, one may detect mineral opacity within the colon on abdominal radiographs, particularly in foals, ponies, and small horses. abdominal paracentesis typically yields normal fluid and poses some risk because large quantities of sand in the ventral colon make inadvertent perforation of the colon more likely. 95 peritoneal fluid is often normal but may have an elevated protein concentration. initially, medical therapy is warranted. administration of psyllium hydrophilic mucilloid (0.25 to 0.5 kg/500 kg in 4 to 8 l of water by stomach tube) may facilitate passage of sand. one should administer the solution rapidly because it will form a viscous gel. an alternative method of administration is to mix psyllium with 2 l of mineral oil, which will not form a gel and can be pumped through a nasogastric tube easily. one then pumps 2 to 4 l of water through the tube. the psyllium separates from the oil phase and mixes with the water, forming a gel within the gastrointestinal tract. psyllium is thought to act by stimulating motility or by agglutinating the sand. however, a recent experimental study failed to show a benefit of this treatment for clearing sand from the colons of otherwise normal horses. 98 if a severe impaction is present, one should not give the psyllium until softening the impaction by administrating intravenous or oral fluids and other laxatives. perforation is a potential complication in horses with sand impactions because the sand stretches and irritates the intestinal wall and causes inflammation. therefore if colic becomes intractable, one should perform surgical evacuation of the large colon. the prognosis is generally good. 94, 95 displacement of the ascending colon is a common cause of large intestinal obstruction. the ascending colon is freely movable except for the right dorsal and ventral colons. contact with adjacent viscera and the abdominal wall tends to inhibit movement of the ascending colon from a normal position; however, accumulation of gas and fluid or ingesta may cause the colon to migrate. 99 feeding practices, including feeding of large concentrate meals, likely plays a role in initiating displacement of the large colon. large concentrate meals increase the rate of passage of ingesta, allowing a greater percentage of soluble carbohydrates to reach the large intestine, 31 which in turn increases the rate of fermentation and the amount of gas and volatile fatty acids produced. the production of large amounts of volatile fatty acids stimulates the secretion of large volumes of fluid into the colon. 100 the association between feeding concentrate and development of displacements of the large colon is illustrated by studies indicating that ascending colon displacement is more prevalent in horses fed a highconcentrate, low-roughage diet. 101 abnormal motility patterns of the ascending colon also have been suggested to contribute to the development of colonic displacement. feeding stimulates colonic motility via the gastrocolic reflex, but large meals may alter normal motility patterns and concurrently allow rapid accumulation of gas and fluid from fermentation. 31, 102 migration of parasite larvae (strongyles) through the intestinal wall also has been shown to alter colonic motility patterns. other experimental studies also have shown that strongylus vulgaris infection results in reduced blood flow to segments of the large intestine without necessarily causing infarction. electric activity of the colon and cecocolic junction increases after infection with s. vulgaris and cyathostome larvae, probably reflecting a direct effect of migration through the intestine and an early response to reduced blood flow. 103 displacements of the ascending colon generally are divided into three types: left dorsal displacement, right dorsal displacement, and retroflexion. left dorsal displacement is characterized by entrapment of the ascending colon in the renosplenic space. the colon often is twisted 180 degrees such that the left ventral colon is situated in a dorsal position relative to the left dorsal colon. the entrapped portion may be only the pelvic flexure or may involve a large portion of the ascending colon, with the pelvic flexure situated near the diaphragm. the colon may become entrapped by migrating dorsally between the left abdominal wall and the spleen or may migrate in a caudodorsal direction over the nephrosplenic ligament. occasionally, one can palpate the ascending colon between the spleen and abdominal wall, lending support to the first mechanism of displacement. gastric distention is thought to predispose horses to left dorsal displacement of the ascending colon by displacing the spleen medially, allowing the colon room to migrate along the abdominal wall. right dorsal displacement begins by movement of the colon cranially, medial (medial flexion) or lateral (lateral flexion) to the cecum. according to one author, the proportion of right dorsal displacements with medial versus lateral flexion is approximately 1:15. 104 in either case the pelvic flexure ends up adjacent to the diaphragm. retroflexion of the ascending colon occurs by movement of the pelvic flexure cranially without movement of the sternal or diaphragmatic flexures. displacement of the ascending colon partially obstructs the lumen, resulting in accumulation of gas or ingesta and causing distention. secretion of fluid in response may exacerbate the distention. 105 tension and stretch of the visceral wall is an important source of the pain associated with colonic displacement. tension on mesenteric attachments and the root of the mesentery by the enlarged colon also may cause pain. 99 ischemia rarely is associated with nonstrangulating displacement of the colon. however, vascular congestion and edema often occur in the displaced segments of colon, resulting from increased hydrostatic pressure from reduced venous outflow. morphologic damage to the tissues is usually minor. clinically, displacement of the ascending colon is characterized by intermittent signs of mild to moderate abdominal pain of acute onset. however, one also may note an insidious onset of colic. 104 one may note dehydration if the duration of the displacement is prolonged. the heart rate may be elevated during periods of abdominal pain but is often normal. abdominal distention may be present if the colon is enlarged by gas, fluid, or ingesta. fecal production is reduced because progressive motility of the large intestine is absent. one often diagnoses left dorsal displacements by palpation per rectum. one can feel the left ventral colon in a dorsal position; it often is filled with gas. one can trace the ascending colon to the nephrosplenic space, and the spleen may be displaced medially. alternatively, one can reach a tentative diagnosis using abdominal ultrasonography. the spleen is visible on the left side of the abdomen, but the gasdistended bowel obscures the left kidney. evaluation of this technique indicates that false positives occur in few instances, although false negatives occasionally may occur. 106 a definitive diagnosis therefore may require surgery. right dorsal displacements are characterized by the presence of the distended ventral colon running across the pelvic inlet and may be felt between the cecum and the body wall if a lateral flexion is present. the pelvic flexure is usually not palpable. retroflexion of the ascending colon may produce a palpable kink in the colon. if the displaced colons are not distended by gas in the instance of right dorsal displacement and retroflexion, the ascending colon may not be palpable and is conspicuous by its absence from a normal position. peritoneal fluid may increase in amount, but the color, protein concentration, and white blood cell count are usually normal. however, as the displaced segment becomes edematous, fluid leaking through the serosa into the peritoneal fluid increases the protein concentration. surgical correction of colon displacement is the most effective means of resolving this disorder. however, nonsurgical intervention has been successful in select cases of nephrosplenic entrapment of the large colon. [106] [107] [108] before attempting such manipulations, the clinician must be certain of a diagnosis. one anesthetizes the horse and places it in right lateral recumbency, rotates the horse up to dorsal recumbency, rocking it back and forth for 5 to 10 minutes, and then rolls the horse down into left lateral recumbency. 109 one should palpate the nephrosplenic space per rectum to determine whether the entrapment has been relieved before recovering the horse from anesthesia. one may administer phenylephrine (3-6 µg/kg/min over 15 minutes) to decrease the size of the spleen. 110 more recently, phenylephrine has been used successfully with 30 to 45 minutes of exercise to reduce nephrosplenic entrapments in four of six horses. 26 the authors suggested that the technique be used on horses with mild to moderate colonic distention, particularly when financial constraints are severe. a number of cases occur in which nonsurgical interventions do not correct the problem and others in which nonsurgical manipulations correct the entrapment but result in large colon volvulus or displacement. 111 one should take horses in such condition to surgery promptly. the prognosis for horses with large colon displacement is good. in one study on horses with nephrosplenic entrapment of the large colon, survival exceeded 90%. 108 the horse, particularly young horses, may ingest foreign material that can cause obstruction, such as bedding, rope, plastic, fence material, and feedbags. these foreign bodies may result in impaction with ingesta and distention of the intestine, typically in the transverse or descending colon. young horses usually are affected. in one study the obstructing mass could be palpated per rectum in three of six horses. 112 fecaliths are common in ponies, miniature horses, and foals. 113 older horses with poor dentition also may be predisposed to fecaliths because of the inability to masticate fibrous feed material fully. fecaliths commonly cause obstruction in the descending colon and may cause tenesmus. 112 other clinical signs are similar to those of enterolithiasis. abdominal radiography may be useful in smaller patients to identify the obstruction, especially if gas distention around the foreign body or fecalith provides contrast. the horse usually requires surgical treatment. mural masses such as abscesses, tumors (adenocarcinoma, lymphosarcoma), granulomata, and hematomas ( figure 13 .15-4) can cause luminal obstruction and impaction, typically in older horses. impaction may result from obstruction of the lumen or impaired motility in the segment of intestine with the mass. abscesses may originate from the lumen of the intestine or may extend from the mesentery or mesenteric lymph nodes. intramural hematomas form most commonly in the descending colon and cause acute abdominal pain. 114 once the acute pain from the hematoma subsides, impaction proximal to the hematoma develops because of impaired motility through the affected portion of the colon. trauma, ulceration of the mucosa, and parasitic damage are speculated causes of intramural hematomas. 114, 115 stricture of the large intestine occurs when fibrous tissue forms in a circular pattern around or within the intestine, reducing the luminal diameter and the ability of the wall to stretch. strictures may be congenital or may follow peritonitis, previous abdominal surgery, or inflammatory bowel disease. in a report of 11 horses with inflammatory bowel disease, 6 horses had strictures, four of which were in the small intestine and two of which were in the large colon. 116 clinical signs vary according to the degree of luminal obstruction. partial obstruction and impaction tend to produce mild to moderate abdominal pain of insidious onset. mural hematomas tend to produce signs of acute abdominal pain. 114, 115 per rectal palpation of the abdomen may reveal the presence of a mass or simply the impacted segment but not the mass itself. one may note fever, weight loss, and anorexia if an abscess or tumor is the cause. an elevated white blood cell count; hyperfibrinogenemia; hyperglobulinemia; or normocytic, normochromic anemia may occur with abscesses or tumors. peritoneal fluid may reflect the cause of the mass. tumor cells may occur infrequently. one may note evidence of inflammation with bacteria if the cause of colic is an abscess or granuloma, in which case one should culture the fluid. hematomas may cause hemorrhage into the peritoneal fluid. treatment usually requires surgical resection of the mass. one may treat abscesses with appropriate antibiotics if the impaction can be resolved medically with oral or intravenous analgesics and laxatives. streptococcus spp, actinomyces pyogenes, corynebacterium pseudotuberculosis, rhodococcus equi, anaerobic bacteria, and gram-negative enteric organisms commonly are involved in abscesses. atresia of a segment of the colon is a rare congenital abnormality in horses. the heritability and causes of the condition are unknown. one potential mechanism for development of the lesion is intestinal ischemia during fetal life, which results in necrosis of a segment of intestine. clinical signs include a failure to pass meconium and colic within the first 12 to 24 hours of life. secondary abdominal distention results from complete intestinal obstruction, and abdominal radiographs may reveal gas-distended colon. one makes the diagnosis at surgery. any portion of the colon may be absent, but the distal segment of the large colon or the proximal small colon usually is affected most severely. if sufficient tissue is present, one may attempt anastomosis to the proximal blind end of the colon. 117 the prognosis depends on which segment of the colon is absent but is usually poor because of an absence of distal colon. neoplasia in the alimentary tract of the horse is uncommon. 1 primary and metastatic neoplasia can affect multiple locations within the oral cavity and gastrointestinal tract (boxes 13.16-1 and 13.16-2). neoplasia is not limited to older horses. the average age of horses with squamous cell carcinoma is 8.6 to 14.6 years. 2, 3 the alimentary form of lymphoma occurs most commonly in horses less than 5 years of age. 4 identification of benign versus malignant tumors is imperative to justify treatment and predict survival. clinical signs associated with alimentary neoplasia are related to the tumor location. clinical signs of oral neoplasia can include enlargement or ulceration of the mandible or maxilla. 48 neoplasia of the tongue results in weight loss, quidding, prepharyngeal dysphagia, halitosis, and nasal discharge containing feed material if the oropharynx is involved. [6] [7] [8] tumors of the esophagus cause signs typical of esophageal dysphagia, ptyalism, choke, intermittent colic, fever, weight loss, and halitosis. 10, 49, 50 gastric neoplasia can be associated with abnormal chewing and swallowing behavior, anorexia, weight loss, chronic intermittent colic, abdominal distention, and intermittent fever. 16 abdominal neoplasia has been implicated in 4% of horses with intermittent or chronic colic. 51, 52 altered stool character, weight loss, ventral edema, and recurrent fever have been associated with intestinal neoplasia. 4 acute signs of abdominal discomfort can occur in intestinal obstructions from malignant and benign neoplastic disease. paraneoplastic syndromes may occur in the horse. the most common syndromes are cancer cachexia, ectopic hormone production, anemia, leukocytosis, thrombocytopenia, hypergammaglobulinemia, fever, and neurologic abnormalities. 53 horses with cancer cachexia have profound weight loss despite adequate consumption of calories. diagnosis of alimentary neoplasia can be challenging. data collected from a complete blood count, biochemistry panel, and urinalysis may support the diagnosis of neoplasia but rarely confirms it. normocytic normochromic anemia indicates chronic disease and is the most likely cause of anemia associated with neoplasia. blood loss anemia (via gastrointestinal tract) and immune-mediated hemolytic anemia (lymphoma) 54 are less frequent causes of anemia associated with abdominal neoplasia. peripheral eosinophilia has been reported in association with multisystemic eosinophilic, epitheliotropic disease with lymphoma. 14 leukocytosis and hyperfibrinogenemia are common findings. serum chemistry can confirm hypoalbuminemia caused by inflammation of the bowel wall. hyperglobulinemia can be characterized with serum electrophoresis, which is nonspecific and can reveal chronic inflammation. a few cases of lymphoma have been identified with monoclonal hypergammaglobulinemia. 55 ectopic hormone production may result in hypercalcemia (calcium >14 mg/dl), which is associated with alimentary neoplasia such as lymphoma, multiple myeloma, carcinomata, and ameloblastoma. 2, 56 hypoglycemia (blood glucose <70 mg/dl) can occur with neoplasia of the pancreas or liver. 2 rectal examination may detect an abdominal mass, thickening of the intestinal wall, lymph node enlargement, or a gritty texture in horses with carcinomatosis. 2 rectal biopsy can reveal lymphoma in some cases. 42 fecal occult blood test is nonspecific for neoplastic disease but can reveal blood loss through the gastrointestinal tract. occasionally, abdominocentesis can identify neoplasia if the tumor exfoliates cells into the abdomen. one can diagnose squamous cell carcinoma, adenocarcinoma, and mesothelioma from peritoneal fluid. 10, 45, 46, 57 characterization of peritoneal fluid as an inflammatory exudate or modified transudate without any neoplastic cells present is common. cytologic analysis of peritoneal fluid samples collected by abdominocentesis accurately predicted the presence of neoplasia in 11 of 25 cases in one study. 58 cytologic examination of two or more peritoneal fluid samples increases the sensitivity of this test for detecting abdominal neoplasia. measurement of peritoneal fluid glucose concentration and ph is valuable to differentiate inflammation in the peritoneum caused by neoplasia from bacterial peritonitis. abdominal neoplasia typically is associated with peritoneal glucose concentrations similar to blood and ph higher than 7.3. d-xylose absorption tests can reveal malabsorptive diseases that include lymphoma. 42, 59 immunoglobulin m deficiency is associated with lymphoma in some young adult horses, but the prevalence of immunoglobulin m deficiency in horses with lymphoma and the value of measuring serum immunoglobulin m concentrations for the diagnosis of lymphoma have not been evaluated. 60 dna cell cycle analysis of suspect neoplastic cells has been used to detect lymphoma in equine patients confirmed with the disease. this method of evaluating fluid or tissues aspirates may increase the accuracy for diagnosing neoplasia in the future. 61 a complete evaluation of the oral cavity may include using a full-mouth speculum, radiographs, and endoscopy of the pharynx. evaluation of the esophagus and stomach with a 3-m endoscope can reveal intralumenal masses. 11 pleuroscopy has been used to obtain biopsy samples of extralumenal masses surrounding the esophagus. 49 contrast radiography can assist in the diagnosis of neoplasia within the wall or outside of the esophagus. 49, 62 ultrasonography of the stomach, small intestine, cecum, and large colon is useful in detecting intestinal wall thickness, abdominal masses, and excessive peritoneal fluid. 63 identification of neoplasia in the liver, kidney or spleen may support metastasis to other parts of the gastrointestinal tract or lymph nodes. laparoscopy and exploratory laparotomy often are required to obtain a final diagnosis. 64 lymphoma is the most common neoplasia in the horse and has been divided into four categories. this section covers only the intestinal/alimentary form. in the past, lymphoma has been called lymphosarcoma, but the preferred term by oncologists is lymphoma because no benign form of this disease exists. 2 lymphoma originates from lymphoid tissue and predominantly affects the small intestine and intestinal lymph nodes. chronic weight loss from malabsorption, intermittent colic, and fever are the most common clinical findings. 27, 65 chronic diarrhea has been reported in some cases. 66 paraneoplastic pruritus and alopecia have been identified in one case of diffuse lymphoma. 67 one generally does not note peripheral lymphadenopathy, but one may palpate enlargement of the intestinal lymph nodes on rectal examination. 4 large colon resection for treatment of lymphoma in horses has increased short-term survival in two horses. 35 chemotherapy in two mares that were pregnant extended their lives long enough to foal normally. 68 long-term prognosis for intestinal lymphoma is poor. squamous cell carcinoma (scc) is a malignant tumor of the gastrointestinal epithelium. scc is the second most common neoplasia in the horse and is the most common oral neoplasia. however, the incidence of scc is rare. 10, 16, 50 in the oral cavity scc may affect the lips, tongue, hard palate, pharynx, and mucosa. 69, 70 treatment for scc in the oral cavity may involve surgical resection, iridium-192 brachytherapy, 5-fluorouracil, or intralesional cisplatin. 5, [71] [72] [73] the prognosis for survival is good if complete removal of the tumor is possible. metastasis beyond the regional lymph nodes is rare for oral scc. squamous cell carcinoma is the most common tumor of the stomach and esophagus 11, 16 and can invade these areas and metastasize to the lymph nodes and lungs. abnormal masses were palpated on rectal examination in four of five cases of gastric scc. 16 treatment by surgical resection is not possible in most cases and the horses die or are euthanized. 2 adenocarcinoma is a malignant tumor that can occur in the small intestine, cecum, and large colon. 18 the tumor arises from the glandular crypts of the gastrointestinal tract and has been reported in middle-aged and older horses. metastasis to the lymph nodes, liver, and lungs can occur. intestinal adenocarcinoma has been reported to metastasize to the bone and was diagnosed using nuclear scintigraphy following injection of technetium-99m hydroxymethylene diphosphate. 29, 32 no reports of successful surgical resection have been published. leiomyosarcoma is a malignant tumor of the smooth muscle lining the gastrointestinal tract and has been reported in the stomach, small intestine, and rectum. 13, 22, 23, 40, 58 in one case report, gastroscopy could not identify the mural mass in the stomach that was found during exploratory surgery. another report describes a favorable outcome for surgical resection of a leiomyosarcoma that was protruding from the anal sphincter in a 4-year-old quarter horse. 40 prognosis for survival is favorable if surgical resection is possible. leiomyoma is a benign tumor of the smooth muscle of the gastrointestinal tract that can occur in the stomach, small intestine, and small colon. 37, 38 clinical signs are consistent with intestinal obstruction. surgical resection and anastomosis of the affected portion of the intestine have been performed without complications. lipoma is a benign tumor that occurs in older horses (10 to 26 years) and arises from mesenteric adipocytes. the tumor grows on a stalk that wraps around the intestine, causing a strangulating lesion manifested clinically by acute obstructive colic. intestinal injury caused by pedunculated lipomata may occur in the small intestine, small colon, and rectum. long-term survival with surgical resection and anastomosis of the affected segment has been reported to be 40% to 50%. 26, 74 oral neoplasia oral cavity neoplasia may involve the dental tissue (odontogenic tumors), bone (osteogenic tumors), or soft tissues (see box 13.16-1). ameloblastoma occurs in horses greater than 10 years old and mainly affects the mandible. ameloblastic odontoma affects younger horses and usually involves the maxilla. both are benign but locally invasive. radiographs may distinguish the difference between an ameloblastoma (radiolucent lesion) and ameloblastic odontoma (radiolucent lesion with partially mineralized density). the best treatment option is surgical resection and radiation therapy regardless of the type. 48 juvenile mandibular ossifying fibroma occurs in the rostral mandible of young horses between the ages of 2 months and 2 years. the fibroma may cause significant distortion of the bone. with early diagnosis and surgical excision of the mass, the horse has a good prognosis. 75 melanomas, sarcoids, and oral papilloma occur on the mouth and lips. melanomas rarely metastasize, but they commonly are found in the parotid salivary glands and lymph nodes. sarcoids are the most common skin tumor that can involve the mouth. ulcerations of the buccal mucosa are difficult to treat. intralesional cisplatin, cryosurgery, radiation, and laser excision have been tried with limited success. 5 equine papilloma virus is responsible for the common skin wart found on the lips and muzzle of young horses. these lesions are usually selflimiting but may be removed successfully by cryosurgery or excision. a number of detailed and informative reviews are available describing the anatomy, physiology, and pathophysiology of the equine peritoneum. [1] [2] [3] [4] [5] the peritoneum consists of a single layer of mesothelial cells lining the peritoneal cavity and serosal surfaces of the intraabdominal viscera. the mesothelial lining of the diaphragm, abdominal walls, and pelvic cavity is termed parietal peritoneum. the visceral peritoneum includes the serosal surfaces of the intraabdominal organs. the parietal and visceral portions of the peritoneum are contiguous with each other through the omentum, mesenteries, and ligaments. caudally, the peritoneum reflects over the surfaces of the pelvic organs (portions of the urogenital tract and rectum), excluding them from the peritoneal space, and thus much of the pelvic cavity and contents are described as retroperitoneal. the peritoneal space communicates with the lumen of the uterus (and thus the external environment) via the fallopian tubes in females. in males the peritoneum forms a true blind sac. the vascular supply and nervous innervation of the visceral peritoneum are supplied by the splanchnic vessels and visceral autonomic nerves, respectively. branches of the intercostal, lumbar, and iliac arteries supply the parietal peritoneum, and the phrenic and intercostal nerves provide nervous innervation. the clinical relevance is that inflammation of the parietal peritoneum is perceived as somatic pain, resulting in a splinted abdominal wall, pain on external palpation, and reluctance to move. visceral pain is mediated by small type c sensory fibers, which are believed to be stimulated by bowel distention, smooth muscle spasms, tension on the mesentery, and ischemia. the peritoneal lining functions as a semipermeable barrier to the diffusion of water and low-molecular weight solutes between the blood and the abdominal cavity. 1 the peritoneum secretes a serous fluid that lubricates the abdominal cavity, inhibits adhesion formation, and has minor antibacterial properties. 1,2 macrophages, mast cells, mesothelial cells, and lymphocytes provide immune function within the peritoneum. 2,3 peritoneal macrophages impart antibacterial activity via complement receptors, phagocytic activity, interaction with t lymphocytes, neutrophil chemotaxis, and fibroblast activation. the peritoneal surface maintains a high level of fibrinolytic activity through the production of plasminogen activators by mesothelial cells. this function, together with the lubricant properties of the peritoneal fluid, helps to maintain gliding surfaces within the peritoneum and prevent adhesion formation. in quadrupeds, peritoneal fluid produced by the mesothelium tends to move ventrally and cranially, aided largely by diaphragmatic movement. peritoneal fluid, waste products, and foreign material (including bacteria) exit the peritoneal cavity to enter the lymphatic system through diffusely distributed subendothelial pores or via the large diaphragmatic stomata, depending on particle size. large molecules and particles greater than approximately 40,000 mw (such as bacteria) exit through the diaphragmatic stomata and ultimately enter the thoracic duct. peritonitis is inflammation of the mesothelial lining of the peritoneal cavity and is characterized by desquamation and transformation of mesothelial cells; chemotaxis of neutrophils; release of several soluble mediators of inflammation; exudation of serum, fibrin, and protein into the peritoneal cavity; and depression of fibrinolytic activity. peritonitis occurs in association with a variety of disorders that result in mechanical, chemical, or infectious insult to the peritoneal lining. [1] [2] [3] [4] any process resulting in disruption or irritation of the peritoneal lining, inflammation or infection of abdominal organs, or compromise of the intestinal wall can result in peritonitis (box 13.17-1). common mechanical injuries include blunt or perforating trauma to the abdominal wall, breeding and foaling accidents, and abdominal surgery. a variety of traumatic insults of iatrogenic origin can cause peritonitis, such as the pathogenesis of peritonitis as a series of stages, as reviewed and described by trent, is useful. 2 the contamination stage, lasting 3 to 6 hours, involves introduction of bacteria into the peritoneum and initiation of the acute inflammatory response previously described. if the organisms are not eliminated and infection is established, the process evolves to the stage of acute diffuse peritonitis. although the overall movement of contaminants is toward the diaphragmatic stomata and into the thoracic duct, the nature of the peritoneal circulation is such that regardless of the location of the initial contamination, bacteria spread throughout the peritoneum within several hours. the stage of acute diffuse peritonitis lasts up to 5 days. the inflammatory response persists and escalates with continued exudation of proteinaceous fluid and influx of inflammatory cells. offending organisms are delivered to the lymphatic system and may be eliminated by the immune system. organisms, however, may gain access to the systemic circulation in sufficient numbers to result in clinically relevant septicemia. in human beings and laboratory animals having undergone polymicrobial contamination of the peritoneum, the organisms causing septicemia at this stage are usually coliforms, e. coli in particular. this stage of the disease process has the highest mortality because of the effects of severe peritoneal inflammation, endotoxemia, and septicemia. if the animal survives this stage but fails to eliminate the infection in the peritoneal cavity, the disease enters a transitional phase referred to as the acute adhesive (or localizing) stage. this stage occupies a time frame of perhaps 4 to 10 days after the initial insult. neutrophils are still active, macrophages are increasing in numbers, and fibrin aggregates are being organized or lysed. in human beings and laboratory animals, selective reduction and synergism continue such that anaerobes and gram-negative aerobes predominate. if infection persists beyond this point, organization of fibrin proceeds and organisms become isolated from host defenses. at this point, the disease process enters the stage of chronic abscessation. this stage can begin as early as 8 days after inoculation and persists indefinitely. clinical signs of peritonitis depend on the primary disease process, the duration of the problem, and the extent of peritoneal inflammation. localized peritonitis may have few or no systemic manifestations, whereas severe localized or generalized peritonitis often is accompanied by severe toxemia or septicemia or both. septic peritonitis usually causes more severe clinical signs because of the inflammatory mediators released in response to bacterial toxins and because of the presence of endotoxin when gram-negative organisms are involved. most clinical signs are nonspecific and include fever, depression, inappetance, decreased borborygmi, and dehydration. additional signs, reported in 30 horses (ages 2 months to 16 years) with peritonitis, were colic, ileus, weight loss, and diarrhea. 14 horses with peracute peritonitis, as occurs with rupture of the bowel or rectal tear, have severe toxemia, weakness, depression or severe colic, tachycardia, tachypnea, and circulatory failure. fever may not be present depending on the degree of shock. typical clinical findings include sweating, pawing, muscle fasciculations, weak peripheral pulses, red to purple mucous membranes, prolonged capillary refill time, and decreased skin elasticity. parietal pain, characterized by reluctance to move, splinting of the abdominal wall, and sensitivity to external abdominal pressure occur in some acute cases. urination or defecation may be painful for the horse, and urine and fecal retention may be evident on rectal examination. palpation of the abdomen externally may elicit flinching, aversion movements, or groaning. with extensive abdominal fecal contamination, rectal examination may reveal a gritty feeling of the serosal and parietal surface of the peritoneum because of fibrin deposition and a dry texture of the peritoneum. in horses with more chronic peritonitis, rectal examination findings can include pain on palpation of fibrinous or fibrous adhesions, intestinal impaction or distention following ileus and dehydration, an abdominal mass (abscess or neoplasia), or an impression of bowel floating in fluid. in many cases, one can detect no abnormalities on rectal examination. horses with localized, subacute, or chronic peritonitis may have signs of chronic or intermittent colic, depression, anorexia, weight loss, intermittent fever, ventral edema, exercise intolerance, decreased or absent intestinal sounds, and mild dehydration. heart rate and respiratory rate may be normal. fecal output may be normal; however, horses with chronic diarrhea and weight loss have been reported. 14 foals with peritonitis usually exhibit signs of colic (acute or chronic) and are febrile, depressed, and inappetant. in some foals with primary peritonitis, pleural effusion occurs. in young foals, peritonitis can cause rapid metabolic deterioration, and determination and correction of the primary problem requires immediate attention. in older foals, peritonitis may occur insidiously, as occurs following s. equi or r. equi infections. clinicopathologic abnormalities vary depending on the time of onset and severity of peritonitis. horses with acute, septic peritonitis can have leukopenia, hemoconcentration, metabolic acidemia, azotemia, and electrolyte imbalances reflective of systemic inflammation from endotoxemia and hypovolemia. horses with peritonitis of a few days' duration may have leukocytosis and hyperfibrinogenemia. plasma protein levels vary depending on the hydration status, degree of exudation into the peritoneum, and type of underlying problem. in chronic peritonitis, hyperproteinemia with hyperglobulinemia may be present. neonates with uroperitoneum caused by urinary bladder rupture or urachal disease tend to develop azotemia, hyponatremia, hypochloremia, hyperkalemia, and acidosis. foals with peritonitis following septicemia, severe enterocolitis, severe meconium impaction, intussusception, small intestinal volvulus, gastric or duodenal rupture, or ascarid impactions usually have clinicopathologic findings reflective of systemic inflammation, such as inflammatory leukogram or leukopenia, hemoconcentration, and acidosis. chronic abscessation, as occurs in foals with r. equi and streptococcal infections, results in clinicopathologic findings reflecting chronic inflammation (anemia, hyperfibrinogenemia, hyperglobulinemia). abnormalities in the composition of peritoneal fluid occur with peritoneal inflammation, and peritoneal fluid analysis is principal to the diagnosis of peritonitis. one collects peritoneal fluid through puncture of the abdomen on the ventral midline. one should clip and prepare an area aseptically. usually, the lowest point of the abdomen, 5 to 10 cm caudal to the xiphoid cartilage, is prepared for puncture; although in some cases one may perform paracentesis more caudally, particularly when one suspects a specific area of sequestered fluid or abscessation. in addition, one may choose a site to the right of midline in an attempt to avoid the spleen. one can perform peritoneal puncture using a 1 1 / 2 -inch, 18-gauge needle or, following local anesthesia and a stab incision with a no. 15 scalpel blade, using a sterile cannula. one collects fluid by gravity flow and should collect fluid in a tube containing anticoagulant, preferably edta for cytologic examination, and in a sterile tube without anticoagulant for visual inspection and, if desired, for culture. one should fill the edta tube to half its volume, because the edta will alter the refractive index of the fluid, resulting in a falsely elevated value for total solids when one collects only a small volume and tests it with a refractometer. one should evaluate peritoneal fluid routinely as to color, turbidity, total protein, white blood cell (wbc) count and differential, and the presence of bacteria as determined by gram stain. normal peritoneal fluid is clear and straw-colored and does not coagulate spontaneously. peritoneal fluid becomes turbid when increased numbers of white blood cells and concentration of protein are present. pink or red fluid indicates free hemoglobin or hemorrhage. blood introduced into the peritoneal fluid iatrogenically in some cases may be differentiated from blood from internal hemorrhage based on the presence of platelets and hematocrit. fluid with iatrogenic blood contamination contains platelets, whereas fluid with blood following internal hemorrhage or diapedesis often does not have platelets. blood contamination resulting from splenic puncture often results in the packed cell volume of the sample being greater than that of the blood. large volumes of dark brown or green fluid with a fetid odor obtained from several sites strongly suggest bowel rupture, but one should perform cytologic examination for confirmation. the distribution of polymorphonuclear and mononuclear cells varies widely, and one should interpret the results of cell counts and differentials as supporting a number of disorders rather than a specific diagnosis. normal equine peritoneal fluid contains fewer than 5000 nucleated cells per microliter. 2, 15 wbc counts in acute peritonitis (>100,000/µl) are reported to be higher than those in chronic peritonitis (20,000 to 60,000/µl) [14] [15] [16] ; however, this is not always the case, and the wbc count depends most on the cause of the peritonitis. the wbc level does not always correlate with severity of peritonitis or the prognosis. the peritoneal fluid wbc count can be greater than 100,000/µl following enterocentesis, with no clinical signs or problem. 17 conversely, peritoneal wbc counts of fewer than 100,000/µl may be found in foals or horses with intraabdominal abscesses. 18 the peritoneal wbc count can increase to greater than 150,000/µl following celiotomy 19 and can be higher if an enterotomy is done. postoperatively, the wbc count normally continues to decline and returns to near normal by 5 to 7 days. failure of the wbc count to decrease suggests peritonitis resulting from a postoperative complication. finally, peritoneal fluid wbc counts greater than 500,000/µl indicate severe focal or generalized peritoneal sepsis. the distribution of polymorphonuclear and mononuclear cells varies in normal peritoneal fluid, 2,15 but polymorphonuclear cells usually predominate. with acute peritonitis, polymorphonuclear cells typically increase to a greater degree than mononuclear cells, but this depends on the cause. in horses that have bowel disease accompanied by endotoxemia, the number of peritoneal mononuclear cells increases, as does transformation of mesothelial cells to macrophages. in chronic cases, one easily may mistake transforming mesothelial cells for neoplastic cells, which can make diagnosis difficult, particularly when the presenting problem is compatible with a neoplastic process. in such cases, consultation with a clinical pathologist regarding cytologic findings is prudent. factor, and interleukin-6 have been measured in the peritoneal fluid of horses with abdominal disorders, but the diagnostic and prognostic implications of the presence or absence of these enzymes and analytes is limited. [20] [21] [22] one should submit peritoneal fluid samples in appropriate media (port-a-cul vial, bbl microbiology system) for aerobic and anaerobic cultures in an attempt to identify the pathogenic organism(s). obligate anaerobic bacteria such as bacteroides are difficult to culture, because one must collect, transport, and culture the sample under strict anaerobic conditions. frequently, bacterial cultures are negative when bacteria are present in peritoneal fluid. to enhance recovery of bacteria, one can inoculate peritoneal fluid into blood culture medium (septi-chek columbia, hoffmann-laroche inc., nutley, new jersey), and if the horse has received antimicrobial treatment, one first should pass fluid through an antimicrobial removal device (a.r.d., becton dickinson & co., cockeysville, maryland). early and aggressive therapy is required if treatment of peritonitis is to be successful. the goals of treatment are to resolve the primary problem, minimize the inflammatory response, and prevent long-term complications. in the acute phase, one gives primary consideration to the arrest of endotoxic, septic, or hypovolemic shock; correction of metabolic and electrolyte abnormalities and dehydration; and management of pain. in the absence of blood gas and electrolyte determinations, adequate volumes of a balanced electrolyte solution are required to correct dehydration and support the cardiovascular system. if the plasma protein concentration of the horse is less than 4.0 g/dl, one should consider administration of plasma or synthetic colloids. one should administer flunixin meglumine (banamine) for its local and systemic antiinflammatory effects. dosages vary depending on the severity of peritonitis, degree of toxemia, severity of pain, and hydration status of the horse and range from 0.25 mg/kg intramuscularly or intravenously every 6 to 10 hours to 1.0 mg/kg intramuscularly or intravenously every 12 hours. the higher dosage provides greater visceral analgesia, whereas the lower dosage is effective in modifying the effects of experimental endotoxemia. 23 in addition to analgesic and general antiinflammatory effect, flunixin meglumine may be effective in retarding adhesion formation when administered early in the acute, diffuse stage of septic peritonitis. 2 heparin therapy has been recommended to prevent adhesion formation and to render bacteria more susceptible to cellular and noncellular clearing mechanisms. in experimental models using laboratory animals, normal peritoneal fluid protein concentration is less than 1.5 g/dl. 15 protein levels between 1.5 g/dl and 2.5 g/dl can be difficult to interpret, but one should consider levels greater than 2.5 g/dl to be elevated abnormally. fibrinogen concentration increases with inflammation, and levels greater than 10 mg/dl in the peritoneal fluid suggest that an acute inflammatory process is present. 20 fibrinogen content will also increase from blood contamination. the presence of free and phagocytosed bacteria in peritoneal fluid indicates generalized suppuration, abscessation, or compromised bowel. if one observes numerous microorganisms of mixed types free in the peritoneal fluid or if one observes plant material, massive bacterial contamination of the abdomen following bowel rupture likely has occurred. the presence of toxic or degenerate neutrophils and bacteria within polymorphonuclear cells helps to distinguish peritoneal fluid from intestinal contents in such cases. enterocentesis yields discolored fluid containing mixed microorganisms and plant material and that is largely devoid of white blood cells. bacterial contamination of a sample can occur during collection of the sample, and iatrogenic contamination of a sample can result in free and intracellular bacteria in peritoneal fluid, particularly if processing is delayed. in such cases the bacterial numbers are few and the neutrophils appear healthy. in some cases of gastrointestinal perforation the luminal material, inflammatory cells, and protein may be sequestered by the omentum and further contained by fibrinous adhesions. abdominal fluid obtained via standard ventral paracentesis may have low cellularity and protein content but large numbers of mixed bacteria indicating bowel rupture. 5 examples include gastric rupture along the greater curvature of the stomach between the omental layers (omental bursa) and perforated gastric or duodenal ulcers in foals. correlating all cytologic findings with clinical and clinicopathologic findings is important for interpreting the results of peritoneal fluid cytologic examination. biochemical analysis of peritoneal fluid may be useful in detecting sepsis when cytologic examination and culture are negative or otherwise unavailable. in a prospective study by van hoogmoed, rodger, spier, et al., peritoneal fluid ph and glucose concentrations from horses with septic peritonitis were significantly lower than horses with nonseptic peritonitis and healthy horses. 21 peritoneal fluid ph less than 7.3, glucose less than 30 mg/dl, and fibrinogen concentration greater than 200 mg/dl were considered highly predictive of septic peritonitis. serum to peritoneal glucose concentration differences of greater than 50 mg/dl was considered the most diagnostically useful test for septic peritonitis in the study. increased activities of alkaline phosphatase, lactic dehydrogenase, creatine kinase, aspartate aminotransferase, tumor necrosis heparin therapy was associated with decreased adhesions in septic peritonitis. 24 heparin has not yet been demonstrated clearly to have similar efficacy in horses, although it may. suggested dosages range from 20 to 40 iu subcutaneously every 12 hours for 48 hours 4 to 40 to 80 iu/kg subcutaneously every 8 hours. 5 one should note that heparin induces red blood cell aggregation in horses, 25 which may adversely affect capillary blood flow. one should initiate antimicrobial therapy after making a diagnosis of peritonitis and before the results of peritoneal culture are available, because isolating an organism may take several days and often culture fails to isolate the organism(s). intravenous administration of antimicrobials is preferred over oral or intramuscular routes in acute, diffuse, septic peritonitis because more reliable levels of drug are achieved in the tissues and peritoneal fluid than otherwise would be obtained in horses with hypovolemia or decreased intestinal motility. 26 the combination of a β-lactam antibiotic with an aminoglycoside is appropriate in most circumstances and certainly in the acute diffuse stage of septic peritonitis. these drugs act synergistically to provide a broad spectrum of activity against a variety of gram-positive and gramnegative aerobic and anaerobic bacteria. 27 potassium penicillin (22,000 to 44,000 iu/kg intravenously every 6 hours) combined with gentamicin (6.6 mg/kg every 24 hours) is an appropriate regimen. in most cases, peritonitis will have resulted from bowel contamination, and thus one should presume a mixed infection with gram-negative aerobic bacteria and gram-positive and gram-negative anaerobic bacteria. 2 one also should presume the same in many cases of traumatic peritonitis, as occurs with foreign body puncture, breeding trauma, or foaling trauma. therefore a strong possibility exists of infection involving penicillin resistant bacteroides fragilis, so that adding metronidazole (15 mg/kg orally every 6 to 8 hours) to the regimen is prudent. combination therapy with β-lactam and aminoglycoside antibiotics (and metronidazole when indicated) is a standard and generally effective protocol. one can modify this antimicrobial regimen when culture and antimicrobial sensitivity results become available. aminoglycosides and nonsteroidal antiinflammatory drugs have the potential to induce acute renal tubular damage, particularly when dehydration and decreased renal perfusion are present. therefore adequately rehydrating the patient and ensuring that renal function is intact before initiating treatment with these drugs are important. furthermore, maintaining hydration and monitoring renal function during the course of treatment are important. monitoring serum creatinine concentration; performing serial uninalysis observing for pigment, red blood cells; and casts; determining the ratio of γ-glutamyltransferase to creatinine in the urine; and therapeutic drug monitoring 26 of aminoglycoside levels are useful in this regard. sodium ampicillin and ceftiofur sodium are β-lactam antibiotics that may be useful in combination therapy. these drugs have an extended gram-negative spectrum compared with penicillin. however, as a third-generation cephalosporin, ceftiofur is less effective against anaerobes than penicillin. one also may consider ceftiofur, trimethoprim-potentiated sulfonamides, amikacin, and enrofloxacin for treatment of gram-negative infection based on culture and sensitivity results. enrofloxacin is a quinolone drug with excellent activity against gramnegative pathogens, including pseudomonas, 27 and also can be effective against resistant staphylococci (personal observation). such staphylococci may be involved in infections caused by traumatic puncture of the abdominal wall. enrofloxacin has a variety of potential toxic effects, including cartilage damage in young growing animals. 29 however, a recent study concluded the drug was safe when administered to adult horses intravenously at 5 mg/kg every 24 hours for 3 weeks. 30 one probably should avoid using the drug in young, growing animals until the issue of cartilage damage is resolved. administration of enrofloxacin to horses constitutes off-label usage. one should treat horses with acute, diffuse, septic peritonitis with antibiotics until the white blood cell count, plasma fibrinogen, and abdominal fluid analysis are normal. in horses that respond to therapy, this process takes a variable amount of time depending on the offending organisms and stage of disease at the time treatment is initiated. horses with abdominal abscessation resulting from polymicrobial infection may require many months of antibiotic treatment. abdominal abscesses caused by streptococci and corynebacterium pseudotuberculosis also may require long-term treatment (weeks to months). long-term antibiotic treatment generally necessitates the use of oral antibiotics, and the options are limited. trimethoprim-potentiated sulfonamides are administered orally and are effective against a variety of gram-positive and gram-negative organisms, although some streptococci are resistant. metronidazole is an orally administered drug effective against anaerobic bacteria, as previously discussed. other orally administered antimicrobials one may consider for long-term use include doxycycline (broad spectrum), erythromycin (gram-positive spectrum), and enrofloxacin (mostly gram-negative spectrum). importantly, rifampin, when used with other drugs, can be effective in penetrating and resolving abscesses. combination therapy with erythromycin and rifampin is the standard treatment for rhodococcus equi infection in foals. peritonitis caused by actinobacillus equuli usually is manifested as a diffuse, supporative peritoneal exudate. 6 the same is true for some cases involving streptococci (personal observation). these infections generally respond well to combination therapy with penicillin and gentamicin. if streptococci are involved as the sole pathogen, then penicillin alone should be effective. streptococci potentially can be involved in mixed, synergistic peritoneal infections in horses. 2 drainage or lavage of the peritoneal cavity may be of benefit in removing toxic bacterial by-products and products of inflammatory cells. 31 high numbers of inflammatory cells and release of their mediators can persist even after the primary stimulus of the inflammatory response has resolved. infusing large volumes of isotonic, warmed fluid into the peritoneal cavity also dilutes the inflammatory mediators, possibly reducing their deleterious effects. when successful, peritoneal lavage decreases the peritoneal fluid wbc count and total protein, potentially reflecting a decrease in diffuse inflammation. the benefits of peritoneal lavage are controversial, and a positive effect may be more likely during the acute, diffuse stage of disease. 2, 4 some studies suggest peritoneal lavage, along with heparin therapy, may reduce the incidence of adhesions. 2 one should perform peritoneal drainage and lavage using a drain of no less than 24f diameter. foley-type catheters can be used, but "mushroom" drains provide a larger area for fluid to enter the drain. two approaches to peritoneal lavage are (1) retrograde irrigation through a ventrally placed ingress-egress drain and (2) placement of ingress catheter(s) in the paralumbar fossa(e) for infusion of fluids, with a drain placed ventrally for removal of infused fluid. one must recognize that thorough peritoneal lavage can be achieved only via ventral midline laparotomy. complications associated with the use of abdominal drains or repeated peritoneal penetration to drain fluid include retrograde infection, local irritation, pneumoperitoneum, and subcutaneous seepage around the drain and resultant cellulitis. if the patient is hypovolemic or hypoproteinemic, one should consider volume replacement and administration of plasma before removing large quantities of fluid from the abdomen. in horses with suspected parasite involvement, such as verminous arteritis, one should give larvicidal doses of an anthelmintic once the condition of the horse is stabilized. ivermectin, fenbendazole, and thiabendazole have been recommended as larvacidal therapies. visualization of auscultation sounds of the large intestine small intestinal betagalactosidase activity in the horse rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of 116 cases equine 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paste against mucosal stages of cyathostomes elimination of mucosal cyathostome larvae by five daily treatments with fenbendazole moxidectin: spectrum of activity and uses in an equine anthelmintic program references 1. laws eg, freeman de: significance of reperfusion injury after venous strangulation obstruction of equine jejunum how important is intestinal reperfusion injury in horses? clinical relevance of intestinal reperfusion injury in horses strangulating volvulus of the ascending colon in horses mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies histologic findings in the gastrointestinal tract of horses with colic large colon resection evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal diseases of the small intestine small intestinal herniation through the epiploic foramen: 53 cases (1987-1993) determining the diagnosis and prognosis of the acute abdomen duodenitis-proximal jejunitis risk factors and clinical signs associated with cases of equine colic short-term survival and prevalence of postoperative ileus after small intestinal surgery in horses short-and longterm survival and prevalence of postoperative ileus after small intestinal surgery in the horse survival after small intestine resection and anastomosis in horses abdominal adhesions after small intestinal surgery in the horse mesenteric rents as a source of small intestinal strangulation in horses: 15 cases (1990-1997) small intestine incarceration through the epiploic foramen of the horse right hepatic lobe atrophy in horses: 17 cases incarceration of the jejunum in the epiploic foramen of a four month old foal use of diagnostic ultrasonography in horses with signs of acute abdominal pain parietal hernia of the small intestine into the epiploic foramen of a horse transection of the pelvic flexure to reduce incarceration of the large colon through the epiploic foramen in a horse incarceration of the small intestine in the epiploic foramen: report of 19 cases pedunculated lipomas as a cause of intestinal obstruction in horses: 17 cases (1983-1990) an analysis of 75 cases of intestinal obstruction caused by pedunculated lipomas strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma abdominal surgery in foals intestinal surgery in the foal effects of extensive resection of the small intestine in the pony incarceration of small intestine through rents in the gastrosplenic ligament in the horse jejunal displacement through the mesometrium in a pregnant mare strangulating obstruction caused by intestinal herniation through the proximal aspect of the cecocolic fold in 9 horses congenital inguinal hernias associated with a rent in the common vaginal tunic in five foals ruptured inguinal hernia in new-born colt foals: a review of 14 cases acquired inguinal hernia in the horse: a review of 27 cases surgical treatment of acquired inguinal hernia in the horse: a review of 51 cases different types of inguinal herniation in two stallions and a gelding surgery of the small intestine complications of umbilical hernias in horses: 13 cases strangulated umbilical hernias in horses: 13 cases (1974-1985) surgical management of intussusception in the horse ileocecal intussusception in horses: 26 cases ultrasonographic diagnosis of small-intestinal intussusception in three foals jejunal intussusception in adult horses: 11 cases ileocecal intussusception corrected by resection within the cecum in two horses diaphragmatic hernias in horses and cattle diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases peritoneopericardial hernia in a horse diaphragmatic herniation as a cause of lethargy and exercise intolerance in a mare diaphragmatic hernia repair in three young horses surgical repair of a diaphragmatic hernia in a racehorse diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases morphologic alterations observed during experimental ischemia of the equine large colon equine large intestinal volvulus: a review of 124 cases diseases and surgery of the large colon large colon volvulus: surgical treatment of 204 horses colopexy in broodmares: 44 cases cecocolic intussusception in horses: 11 cases (1979-1989) cecocolic and cecocecal intussusception in horses: 30 cases (1976-1996) cecal amputation via a right ventral colon enterotomy for correction of nonreducible cecocolic intussusception in 8 horses resection of intussuscepted large colon in a horse intussusception of the large colon in a horse intussusception of the left dorsal colon in a horse intussusception of the colon in a filly rectal prolapse in the horse rectal prolapse and cystic calculus in a burro rectal prolapse in a foaling mare management of rectal injuries rectum and anus disruption to the blood supply to the small colon following rectal prolapse and small colon intussusception in a mare laparoscopic diagnosis of ischemic necrosis of the descending colon after rectal prolapse and rupture of the mesocolon in two postpartum mares intestinal infarction associated with mesenteric vascular thrombotic disease in the horse diseases of the large colon diagnostic and prognostic procedures for equine colic surgery examination of the horse with colic detection of endotoxin in cases of equine colic determining the diagnosis and prognosis of the acute abdomen risk factors and clinical signs associated with cases of equine colic accuracy of clinicians in predicting site and type of lesion as well as outcome in horses with colic struvite urethral calculus in a three-month-old thoroughbred colt urolithiasis in 68 horses cholelithiasis in horses: ten cases review of 115 cases of colic in the pregnant mare surgical management of uterine torsion in the mare: a review of 26 cases rabies in horses: 21 cases mechanisms of pain and their therapeutic implications gastrointestinal pharmacology nonsteroidal anti-inflammatory drugs the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse alpha 2 adrenoceptor agonists in the horse: a review cardiovascular effects of medetomidine, detomidine and xylazine in horses cardiovascular effects of xylazine and detomidine in horses prognosis in equine colic: a study of individual variables used in case assessment a: study of variables commonly used in examination of equine colic cases to assess prognostic value clinical evaluation of blood lactate levels in equine colic use of clinical pathology in evaluation of horses with colic the anion gap as a prognostic indicator in horses with abdominal pain strangulating volvulus of the ascending colon in horses diseases and surgery of the large colon multivariable prediction model for the need for surgery in horses with colic prognosis in equine colic patients using multivariable analysis prognosis in equine colic: a comparative study of variables used to assess individual cases prognostic index for acute abdominal crisis (colic) in horses feeding and digestive problems in horses: physiologic responses to a concentrated meal effect of meal feeding on plasma volume and urinary electrolyte clearance in ponies evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression morphologic effects of experimental distention of equine small intestine surgical reduction of ileal impactions in the horse: 28 cases medical treatment of horses with ileal impactions: 10 cases ascarids: recent advances the purported role of coastal bermuda hay in the etiology of ileal impactions: results of a questionnaire (abstract) tapeworm infection is a significant risk factor for spasmodic colic and ileal impaction colic in the horse use of excretory/secretory antigens for the serodiagnosis of anoplocephala perfoliata cestodosis ileal impaction in the horse: 75 cases evaluation of factors associated with postoperative ileus in horses: 31 cases idiopathic muscular hypertrophy of the equine small intestine: 11 cases (1980-1991) obstruction of the ileum in the horse: a report of 27 clinical cases ileal muscular hypertrophy and rupture in a pony three years after surgery for ileocaecal intussusception jejunocolostomy or ileocolostomy for treatment of cecal impaction in horses: nine cases (1985-1995) small intestinal strangulation caused by meckel's diverticulum in a horse volvulus associated with meckel's diverticulum in the horse congenital jejunal diverticulum in a foal mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse abdominal adhesions after small intestinal surgery in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal the characteristics of intestinal injury peripheral to strangulating obstruction lesions in the equine small intestine current concepts in management of abdominal adhesions one percent sodium carboxymethylcellulose prevents experimentally induced abdominal adhesions in horses effect of carboxymethylcellulose and a hyaluronate-carboxymethylcellulose membrane on healing of intestinal anastomoses in horses intraperitoneal use of sodium carboxymethylcellulose in horses undergoing exploratory celiotomy prevention of intraabdominal adhesions in ponies by low-dose heparin therapy retrospective analysis of the results of 151 exploratory laparotomies in horses with gastrointestinal disease surgical treatment for colic in the foal retrospective evaluation of repeat celiotomy in 53 horses with acute gastrointestinal disease risk factors for reduced postoperative fecal output in horses: 37 cases (1997-1998) role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model the effect of prostaglandin e1 on motility of the equine gut in vitro investigation of the effect of prostaglandins and nonsteroidal antiinflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon prostanoid production via cox-2 as a causative mechanism of rodent postoperative ileus the action of low dose endotoxin on equine bowel motility sir frederick hobday memorial lecture: all wind and water-some progress in the study of equine gut motility antagonism of endotoxin-induced disruption of equine bowel motility by flunixin and phenylbutazone cyclooxygenase inhibitors in equine practice in vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: 70 cases (1989-1992) role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period cecal impaction in the horse caecal disease in equids ileocolostomy: a technique for surgical management of equine cecal impaction diseases and surgery of the cecum treatment of impaction colics large colon impaction in horses: 147 cases (1985-1991) effects of amitraz, several opiate derivatives and anticholinergic agents on intestinal transit in ponies experimental studies of druginduced impaction colic in the horse retropulsion-propulsion in equine large colon clinical and structural features of equine enteroliths petrographic and geochemic evaluation of equine enteroliths enteroliths in horses evaluation of enterolithiasis in equids: 900 cases risk factors for enterolithiasis among horses in texas obstructive enterolith in an 11-month-old miniature horse surgical treatment of sand colic in equids: 48 cases (1978-1985) surgical treatment of sand colic: results in 40 horses diarrhea associated with sand in the gastrointestinal tract of horses abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation failure of psyllium mucilloid to hasten evaluation of sand from the equine large intestine nonstrangulated colonic displacement in horses functions of the equine large intestine and their interrelationship in disease comparisons of age, breed, history and management in 229 horses with colic motor functions of the intestine the effect of strongylus vulgaris larvae on equine intestinal myoelectrical activity displacement of the large colon reversal of colonic net absorption to net secretion with increased intraluminal pressure use of ultrasound in horses for diagnosis of left dorsal displacement of the large colon and monitoring its nonsurgical correction renosplenic entrapment of the large colon in horses: 33 cases renosplenic entrapment of the large colon in horses: 57 cases further experiences with non-surgical correction of nephrosplenic entrapment of the left colon in the horse effect of phenylephrine on hemodynamics and splenic dimensions in horses displacement of the large colon associated with nonsurgical correction of large-colon entrapment in the renosplenic space in a mare foreign body obstruction of the small colon in six horses fecalith impaction in four miniature foals obstruction of the small colon by intramural haematoma in three horses submucosal haematoma as a cause of obstruction of the small colon in the horse: a review of four cases inflammatory bowel disease in horses: 11 cases intestinal atresia in horses clinical survey of tumours and tumourlike lesions in horses in south east queensland abdominal neoplasia (excluding urogenital tract) clinical manifestation of squamous cell carcinoma in horses lymphoma (lymphosarcoma) in horses oral and dental tumors in equine denistry t cell-rich b cell lymphosarcoma in the tongue of a horse multiple myeloma in a horse rhabdomyosarcoma of the tongue in a horse paraneoplastic bullous stomatitis in a horse gastric squamous cell carcinoma in three horses use of esophagoscopy in the diagnosis of esophageal squamous cell carcinoma in a horse gastric hyperplastic polyp in a horse gastric leiomyosarcoma in a horse multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse an immunohistochemical study of equine b-cell lymphoma squamous cell carcinoma of the equine stomach: a report of five cases six cases of squamous cell carcinoma of the stomach of the horse small intestinal adenocarcinoma in a horse ganglioneuroma as a cause of small intestinal obstruction in the horse: a case report intestinal carcinoid in a mare: an etiologic consideration for chronic colic in horses leiomyoma of the small intestine in a horse jejunal intussusception associated with leiomyoma in an aged horse duodenal leiomyoma associated with colic in a two-year-old horse leiomyosarcoma of the duodenum in two horses pedunculated lipomas as a cause of intestinal obstruction in horses: 17 cases (1983-1990) clinical aspects of lymphosarcoma in the horse: a clinical report of 16 cases multiple peripheral nerve sheath tumors in the small intestine of a horse equine adenocarcinomas of the large intestine with osseous metaplasia intestinal myxosarcoma in a thoroughbred mare gastrointestinal stromal tumors of the equine cecum colonic adenocarcinoma with osseous metaplasia in a horse intestinal adenocarcinoma causing recurrent colic in the horse equine colonic lipomatosis large colon resection for treatment of lymphosarcoma in two horses colic in a mare caused by a colonic neurofibroma small colon intussusception associated with an intralumenal leiomyoma in a pony leiomyoma of the small colon in a horse an analysis of 75 cases of intestinal obstruction caused by pedunculated lipomas rectal leiomyosarcoma in a horse strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of 116 cases standing rectal and tail surgery disseminated peritoneal leiomyomatosis in a horse ascites as a result of peritoneal mesotheliomas in a horse a case of peritoneal mesothelioma in a thoroughbred mare omental fibrosarcoma in a horse neoplasia of the mouth and surrounding structure pleuroscopic diagnosis of gastroesophageal squamous cell carcinoma in a horse recurrent esophageal obstruction due to squamous cell carcinoma in a horse chronic colic in the mature horse: a retrospective review of 106 cases recurrent colic in the mature horse: a retrospective review of 58 cases paraneoplastic syndromes lymphosarcoma and associated immune-mediated hemolytic anemia and thrombocytopenia in horses differentiation of chronic lymphocytic leukemia in the horse: a report of two cases hypercalcemia associated with malignancy in a horse adenocarcinoma of intestinal origin in a horse: diagnosis by abdominocentesis and laparoscopy differentiation between intra-abdominal neoplasms and abscesses in horses, using clinical and laboratory data: 40 cases (1973-1988) malabsorption in the horse associated with alimentary lymphosarcoma immunodeficiency associated with lymphosarcoma in a horse flow cytometric methods to diagnose selected equine immune-mediated disorders pleural effusion associated with squamous cell carcinoma of the stomach of a horse use of diagnostic ultrasonography in horses with signs of acute abdominal pain the indications for equine laparotomy: an analysis of 140 cases equine lymphosarcoma diarrhoea in the horse as a result of alimentary lymphosarcoma paraneoplastic pruritus and alopecia in a horse with diffuse lymphoma lymphoma in the horse. proceedings of the twelfth annual meeting of the american college of veterinary internal medicine squamous cell carcinoma of the pharyngeal wall in a horse squamous cell carcinoma of the oral, pharyngeal and nasal mucosa in the horse treatment of superficial ulcerative squamous cell carcinoma in three horses with topical 5-fluorouracil intratumoral chemotherapy with cisplatin in oily emulsion in horses excision of oral squamous cell carcinoma in a horse extensive resection and anastomosis of the descending (small) colon in a mare following strangulation by a mesenteric lipoma section 13.17 peritonitis references 1. hosgood g: peritonitis. 1. a review of the pathophysiology and diagnosis the peritoneum and peritoneal cavity large animal internal medicine diseases of the peritoneum and mesentery equine internal medicine peritonitis associated with actinobacillus equuli in horses: 51 cases veterinary gastroenterology peritonitis and other intra-abdominal infection antibiotic susceptibility of bacterial pathogens from horses peritonitis in horses: 67 cases biologic reactions to endotoxin effect of dietary alpha-linoleic acid on equine monocyte procoagulant activity and eicosanoid synthesis review of 30 cases of peritonitis in the horse reference values for equine peritoneal fluid diagnostic cytology in the equine species: overview of effusions (peritoneal, pleural, and synovial joint) and transtracheal wash effects of enterocentesis on peritoneal fluid constituents in the horse internal abdominal abscesses in the horse: a study of 25 cases equine peritoneal fluid analysis following celiotomy analysis of equine peritoneal fluid evaluation of peritoneal fluid ph, glucose concentration, and lactate dehydrogenase activity for detection of septic peritonitis in horses tumor necrosis factor and interleukin-6 activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxemia in horses heparin in the treatment of experimental peritonitis erythrocyte agglutination associated with heparin treatment in three horses therapeutic strategies involving antimicrobial treatment of large animals with peritonitis effect of long-term administration of injectable enrofloxacin solution on physical and musculoskeletal variables in adult horses peritoneal lavage in the horse pharmacologic principles therapeutic drug monitoring: a tool for rational drug therapy. proceedings of the seventh american college of veterinary internal medicine forum toxicity of quinolones although a number of potential causes of peritonitis exist, sepsis is a common and serious complication, and the identification and control of bacterial sepsis is critical for a successful outcome. bowel leakage (as well as external trauma) results in contamination of the peritoneum with large numbers of many types of bacteria. the intestinal tract contains a mixed population of bacteria, and the quantity of bacteria and prevalence of anaerobic species increase in the distal segments. [1] [2] [3] [4] [5] [6] [7] there are approximately 1 × 10 9 anaerobic and 1 × 10 5 aerobic bacteria per milliliter of cecal and colonic fluid, thus the potential for bacterial contamination of the peritoneum is great. high mortality is associated with contamination from the lower bowel because of the large numbers of bacteria present. 8 hirsch and jang 9 reported isolation of an infective agent from equine peritoneal fluid in approximately 25% of attempts. obligate anaerobic bacteria were cultured most frequently, followed by members of the enterobacteriaceae family (escherichia coli). penicillin-resistant bacteroides fragilis was isolated from 10% to 20% of cases. in another study in which bacteria were identified in equine abdominal fluid by cytologic examination or culture, e. coli was the organism most commonly isolated. 10 in human beings and laboratory animals the well-established fact is that despite the variety of organisms initially introduced subsequent to these events, established infections are characterized by only a few types of bacteria, which are often gram-negative aerobes and anaerobic bacteria. 2 this selectivity occurs through the processes of selective reduction of bacterial populations and bacterial synergism. a well-known example of synergism in human beings and laboratory animals is peritonitis involving e. coli and b. fragilus. the presence of each organism is beneficial to the survival of the other, and each is important in the overall pathogenesis of the disease. e. coli is associated with septicemia and early mortality, whereas b. fragilis infection tends to result in chronic abscessation with delayed morbidity and mortality. some evidence suggests that in horses, in addition to coliforms and anaerobes, streptococci and perhaps c. psuedotuberculosis may survive selective reduction and participate in synergistic infection following polymicrobial contamination.biologic events resulting from contamination of the abdomen or injury to the mesothelial cells have been described [1] [2] [3] [4] and include release of catecholamines, histamine, and serotonin from peritoneal mast cells; vasodilation and hyperemia; increase in peritoneal vascular permeability; secretion of protein-rich fluid into the peritoneum; transformation of mesothelial cells into macrophages; and influx of polymorphonuclear cells, humoral opsonins, natural antibodies, and serum complement into the peritoneal cavity. additionally, depression of the peritoneal fibrinolytic activity, fibrin deposits on the peritoneal surface, and sympathetic-mediated ileus of the gastrointestinal tract can occur.these processes benefit the animal by confining contamination and infection, and indeed, with clean, minimally invasive procedures such as enterocentesis or trocharization, this is effective. however, with greater severity of peritoneal contamination or irritation, these processes are magnified and become deleterious, resulting in problems such as hypovolemia, hypoproteinemia, ileus with resultant bowel distention, ischemia of the bowel wall with subsequent absorption of bacteria and toxins, and ultimately adhesion and abscess formation. additionally, systemic responses to bacterial toxins, particularly lipopolysaccharide, 11, 12 can compromise the metabolic condition of the patient further. equine peritoneal macrophages release several mediators when exposed to bacterial lipopolysaccharide, 13 undoubtedly an important component of septic peritonitis.pathologic description of peritonitis includes origin (primary or secondary), onset (peracute, acute, chronic), distribution (localized versus diffuse), and presence of bacteria (septic versus nonseptic). 3, 4 clinically, viewing key: cord-020643-0yzkqykg authors: müller-werdan, u.; buerke, m.; christoph, a.; flieger, r.r.; loppnow, h.; prondzinsky, r.; reith, s.; schmidt, h.; werdan, k. title: schock date: 2006 journal: klinische kardiologie doi: 10.1007/3-540-29425-2_6 sha: doc_id: 20643 cord_uid: 0yzkqykg nan pathophysiologie des schocks -347 therapie der metabolischen azidose -375 6.4.6 volumentherapie -375 6.4.7 inotrope und vasoaktive substanzen -379 6.4.8 optimierung des intravasalvolumens -383 6.4.9 neue therapieansätze zur schockbehandlung -383 6.4.10 gerinnungsaktive substanzen -383 6.4.11 hypothermie/cooling -384 6.4.12 pharmakotherapie des kritisch kranken -384 6.5 multiorgandysfunktionssyndrom (mods) -384 6.5.1 hypothesen zur entstehung -384 6.5.2 schweregradeinteilung durch score-systeme -386 6.5.3 organdysfunktion der lunge -389 6.5.4 organdysfunktion der niere (akutes nierenversagen) -390 6.5.5 organdysfunktion des gehirns -393 6.5.6 organdysfunktion des peripheren und autonomen nervensystems sowie der skelettmuskulatur -393 6.5.7 organdysfunktion des herzens und des kreislaufs -394 6.5.8 organdysfunktion des gastrointestinaltrakts -394 6.5.9 dysfunktion des gerinnungssystems -395 6.5.10 dysfunktion des stoffwechsels und künstliche ernährung -397 6.5.11 dysfunktion des endokrinium -397 6.5.12 dysfunktion des immunsystems -398 6.6 spezifi sche schockformen -398 6.6.1 kardiogener schock -398 6.6.2 septischer schock -409 6.6.3 hypovolämischer schock -418 6.6.4 traumatischer schock -419 6.6.5 anaphylaktischer schock -419 die reduktion der effektiven gewebeperfusion kann dabei hervorgerufen werden durch eine abnahme der herzleistung, durch störungen der makro-und mikrozirkulation mit verminderter perfusion, durch eine kritische abnahme des blutvolumens, eine beeinträchtigung der o 2 -aufnahme in der lunge und der o 2 -abgabe im gewebe. zur beeinträchtigung essenzieller zellfunktionen kommt es im gefolge einer exogenen intoxikation, einer endogenen überschwemmung mit mediatoren (7 abschn. 6.2.6) oder endokrin/metabolischer krisen. klar zu trennen vom schockbegriff sind die synkope und der kollaps. eine passagere kritische herabsetzung der gehirndurchblutung mit gleichzeitiger bewusstlosigkeit wird als synkope bezeichnet (task force on syncope, european society of cardiology 2001). nicht immer ist damit eine allgemeine kreislaufi nsuffi zienz vergesellschaftet. bei einem kollaps über-wiegen vagale reaktionen, das ereignis ist von kurzer dauer, organschäden infolge o 2 -mangels treten nicht auf. von den zahlreichen schockklassifi kationsversuchen (adams et al. 2001; kumar u. parrillo 2001) erscheint die schockeinteilung nach hämodynamischen gesichtspunkten für den kliniker am hilfreichsten (. tabellen 6.1, 6.2 und . abb. 6.1): hypovolämischer schock infolge eines im verhältnis zur gefäßkapazität verminderten zirkulierenden blutvolumens, charakterisiert durch erniedrigte diastolische füllungsdrücke und -volumina des herzens; kardiogener schock infolge eines pumpversagens, hervorgerufen durch eine eingeschränkte myokardkontraktilität, einen verlust an funktionsfähiger myokardmasse oder eine intrinsischstrukturelle bzw. -mechanische behinderung der herzfunktion, charakterisiert durch erhöhte diastolische füllungsdrücke und -volumina des herzens; extrakardial-obstruktiver schock infolge einer flussobstruktion im herz-kreislauf-system, charakterisiert entweder durch eine behinderung der diastolischen füllung oder eine exzessive nachlasterhöhung; verteilungsschock infolge eines verlustes der vasomotorenkontrolle mit dilatation von arteriolen und venolen, charakterisiertnach adäquater volumensubstitution -durch einen erhöhten herzindex und einen erniedrigten systemischen gefäßwiderstand. f f f f abb. 6.1. zusammenspiel von schockursachen bei verschiedenen schockformen. im falle des kardiogenen, hypovolämischen und obstruktiven schocks resultiert die hypotonie im wesentlichen aus der abnahme des herzindex, mit konsekutivem anstieg des systemischen gefäßwiderstands. im falle des distributiven -insbesondere des septischen -schocks ist die hypotonie v. a. auf die erniedrigung des systemischen gefäßwiderstands zurückzuführen mit sekundärem anstieg des . herzindex. bei zahlreichen schockformen wird das hämodynamische profi l durch komponenten der hypovolämie, der myokarddepression (ischämisch und infolge anderer ursachen) und der gefäßdysfunktion (mit einfl uss auf die nachlast) mitbeeinfl usst. bei einem volumenverlust von 15-35% (stadium ii) beginnen die kompensationsmechanismen zu versagen, es treten eine milde bis mäßige hypotonie, eine abnahme des herzindex und eine orthostase auf. der systemische gefäßwiderstand nimmt beträchtlich zu, und das serumlaktat kann ansteigen. traumapatienten, die 35-40% ihres blutvolumens verloren haben (stadium iii), weisen eine ausgeprägte tachykardie, hypotension und bewusstseinsstörungen auf. ein hypovolämischer schock manifestiert sich bei einem verlust von über 40% (stadium iv) des zirkulierenden volumens, es resultiert ein abfall des herzindex und der gewebeperfusion auf weniger als die hälfte; die auftretende laktatazidose kündigt eine ungünstige prognose mit in der regel irreversiblem hämorrhagischem schock an. besteht ein blutverlust von 40% oder mehr über einen zeitraum von 2 h oder sogar länger, so muss bereits mit der erfolglosigkeit der dann eingeleiteten maßnahmen gerechnet werden. prognostisch ungünstige faktoren beim hypovolämischen schock sind die geschwindigkeit des auftretens des volumenverlustes (je rascher, desto ungünstiger) und eine vorbestehende einschränkung der herzfunktionsreserve (z. b. bei zustand nach herzinfarkt). povolämischen schock die eingeleitete therapie trotz adäquater volumensubstitution nicht mehr erfolgreich ist. blutverluste, plasmaverluste und exogene flüssigkeits-/wasserverluste zählen zu den häufi gsten ursachen dessen, was sich in komplizierter verkettung der sekundären reaktionen und regulationen klinisch-symptomatologisch als »hypovolämischer schock« manifestiert. der kardiogene schock ist die häufi gste todesursache von infarktpatienten in der krankenhausphase (hollenberg 2003; prondzinsky et al. 2004) . klinisch fi ndet sich ein zentralisierter kreislaufschock mit low-output-syndrom (hypotonie, oligurie, blasse, kühle haut) und den symptomen der akuten links-und rechtsherzinsuffi zienz mit 3. herzton, halsvenenund lungenstauung bis hin zum lungenödem. hämodynamisch (. tabelle 6.2) imponieren die zunahme der vorlast (zunahme von ventrikelvolumina, linksventrikulärem füllungsdruck, pulmonalkapillardruck und zentralem venendruck) und eine abnahme von herzindex, schlagvolumenindex und schlagarbeitsindex, verbunden mit einem anstieg des systemischen gefäßwiderstandes. als kriterien des infarktbedingten kardiogenen schocks (hochman et al. 1999; prondzinsky et al. 2004) myokardfunktionsverlust. die häufi gste ursache eines kardiogenen schocks (. tabelle 6.4) ist ein mindestens 40%iger myokardfunktionsverlust im rahmen einer khk: beim akuten, großen myokardinfarkt -häufi g bei proximalem verschluss des r. interventricularis anterior (riva) der linken koronararterie oder der lad (»left anterior descendens«)aber auch bei rezidivinfarkten und in der stunned-myocardium-phase. akute mitralinsuffi zienz. eine weitere ursache eines kardiogenen schocks ist die akute mitralinsuffi zienz infolge einer sehnenfadenruptur oder einer papillarmuskeldysfunktion/ruptur bei myokardischämie, akutem herzinfarkt (häufi g infolge eines lad-verschlusses), endokarditis, stumpfem thoraxtrauma, mitralklappenprolaps oder klappenprothesendysfunktion. die ischämische papillarmuskelruptur tritt häufi g 3-7 tage nach akutem herzinfarkt auf; ein neu aufgetretenes apikales holosystolikum kann der ruptur vorausgehen. akute aorteninsuffi zienz. als schockursache ist die akute aorteninsuffi zienz meistens auf eine fl oride endokarditis zurückzuführen, weiterhin auf eine prothesendysfunktion oder eine aortendissektion. > tabelle 6.4. ursachen des infarktbedingten kardiogenen schocks; daten von 1422 patienten des shock trial und shock registry a (hochman et al. 2000; mod. nach prondzinsky et al. 2004) . häufi gkeit (%) ventrikelruptur. die infarktbedingte ventrikelseptumruptur tritt ebenfalls wenige tage nach dem infarkt, häufi g bei lad-verschluss, auf. bei einem therapiefraktären hinterwandinfarkt ist auch an das vorliegen eines begleitenden (selten eines isolierten) rechtsherzinfarkts zu denken, der in 10-20% der fälle zum kardiogenen schock führt (zehender et al. 1993 ). weitere ursachen. weitere auslöser des auftretens eines kardiogenen schocks sind akute myokarditis, terminalstadien von kardiomyopathien, dekompensiertes hochdruckherz und vitien, maligne brady-und tachykarde rhythmusstörungen sowie traumatisch bedingte myokardkontusionen und intoxikationen mit negativ-inotropen pharmaka. die klinische symptomatik und die hämodynamischen befunde sind geprägt durch die kombination krankheitsspezifischer befunde mit einem low-output-syndrom. auch bei dieser schockform ist der zeitverlauf prognosebestimmend. bei einer akuten perikardtamponade durch ischämische ruptur der freien ventrikelwand (in der regel 5-7 tage nach herzinfarkt) oder infolge einer blutung bei thoraxtrauma bzw. nach thrombolysetherapie reichen bereits 150 ml blut im perikardbeutel zur schockentwicklung aus; bei einem chronischen malignen oder entzündlichen perikarderguss können dagegen 1-2 l ergussfl üssigkeit ohne schockentwicklung toleriert werden. bei einer akuten lungenembolie ohne vorbestehende kardiopulmonale erkrankung führt der verschluss von 2 oder mehr lappenarterien und damit von 50-60% des lungengefäßbettes zum schock; im falle chronischer, rezidivierender lungenembolien mit adaptativer rechtsherzhypertrophie werden teilweise wesentlich höhere anteile an lungenstrombettverschlüssen ohne schocksymptomatik toleriert. der gemeinsame nenner des verteilungsschocks (. tabelle 6.1) ist der verlust an peripherem gefäßwiderstand. neben den allgemeinen schockzeichen mit arterieller hypotonie, tachykardie, tachypnoe, oligurie und bewusstseinsstörung imponieren warme, relativ gut durchblutete extremitäten und ein erniedrigter diastolischer blutdruck. anamnese und charakteristische befunde können häufi g eine rasche ätiologische einordnung ermöglichen: eine urtikaria spricht für einen anaphylaktischen, eine rückenmarksverletzung für einen neurogenen und zeichen der infektion für einen septischen schock, der weitaus häufi gsten form des verteilungsschocks. hämodynamisch imponiert beim verteilungsschock eine ausgeprägte erniedrigung des systemischen gefäßwiderstandes, wobei jedoch in den einzelnen organgefäßbetten der widerstand erniedrigt, unverändert und auch erhöht sein kann. initial (vor volumentherapie) können herzindex und ventrikuläre füllungsdrücke erniedrigt sein, danach sind sie in der regel erhöht (. tabelle 6.2). der septische schock, die häufi gste todesursache auf der intensivstation, ist folge einer toxin-/mediatorkaskade, die sowohl durch gramnegative als auch durch grampositive bakterien ausgelöst werden kann, ebenso polymikrobiell und in seltenen fällen auch durch pilze oder viren (7 abschn. 6.2.6). 64% der patienten mit einer sepsis entwickeln innerhalb von 24 h (median) eine schwere sepsis, und 23% der patienten mit schwerer sepsis innerhalb der nächsten 28 tage (median) einen septischen schock (moerer et al. 2005) . die letalität der sepsis (ca. 10-15%) steigt bei der schweren sepsis auf ca. 30% und beim septischen schock auf 50-60% an. circa 50% aller sepsistodesfälle sind auf ein intraktables mods zurückzuführen, 40% auf ein therapierefraktäres kreislaufversagen, und bei 10% ist eine nicht beherrschbare septische kardiomyopathie (7 abschn. 6.6.2) die ursache. an der verbesserung der sepsisdefi nitionen und -klassifizierung (. tabelle 6.3) wird permanent gearbeitet: die piro-klassifi zierung ist ausdruck des bemühens um eine bessere charakterisierung der sepsis ähnlich der tumorklassifi zierung (levy et al. 2003) . wesentlich aussagekräftiger als die qualitative sepsisdiagnose (. tabelle 6.3) sind quantitative parameter zur beschreibung des schweregrads der sepsis (z. b. der sepsis-score nach elebute u. stoner, . abb. 6.22), des septischen multiorganversagens (z. b. der apache-ii-schweregrad-score, . abb. 6.15), des ausmaßes der septischen gefäßschädigung (abfall des systemischen gefäßwiderstands) und der septischen myokarddepression (abnahme des linksventrikulären schlagarbeitsindex). sie erlauben nicht nur eine abschätzung der prognose der sepsispatienten, sondern auch des ansprechens auf die therapie (müller. . tabelle 6.5 zeigt das charakteristische profi l von sepsispatienten im vergleich zu patienten mit fieber, aber lediglich lokaler infektion und im vergleich zu patienten mit kardialem pumpversagen. die sepsispatienten sind durch einen hohen sepsis-score, eine hyperdyname kreislaufsituation mit hohem herzindex bei erniedrigtem systemischem gefäßwiderstand und schlagarbeitsindex und durch störungen der o 2 -verwertung gekennzeichnet. die inzidenz der schweren sepsis liegt bei 6/1000 krankenhaus-und 24-136/1000 intensivstationaufnahmen, die inzidenz des septischen schocks bei 1,4/1000 krankenhausaufnahmen und 61-87/1000 intensivaufnahmen (moerer et al. 2004) . im verständnis der sepsis und des septischen schocks ist allerdings in den letzten jahren ein erheblicher wandel eingetreten (. abb. 6.2). stand früher die bakterielle oder pilzinfektion mit einschwemmung von keimen in die blutbahn ganz im vordergrund der betrachtungsweise, so sind es heute vielmehr die durch die mikrobiellen toxine via aktivierung von mediatorzellen induzierten zytokin-und mediatorkaskaden, die für das mods und den septischen schock verantwortlich gemacht werden (7 abschn. 6.2.6). diese einschätzung wird unterstützt durch die tatsache, dass nur bei jedem 2. oder 3. patienten mit sepsis eine positive kultur gefunden wird und diese auch nicht prognosebestimmend ist. auch dass nichtinfektiöse noxen (trauma, pankreatitis, herzchirurgische operationen mit der herz-lungen-maschine) zu einem ganz ähnlichen klinischen bild wie bei bakteri-ell ausgelöster sepsis und septischem schock führen können, spricht für eine mehr oder weniger gemeinsame zytokin-/mediatorendstrecke als verantwortliche schädigungskaskade sowohl bei infektiösen als auch bei nichtinfektiösen (sirs, . tabelle 6.3) systemisch-entzündlichen schockformen. der anaphylaktische schock ist ein akut eintretender, »warmer« schockzustand, der durch anaphylaktische und anaphylaktoide reaktionen ausgelöst wird: der blutdruckabfall infolge vasodilatation mit relativer hypovolämie kann einhergehen mit larynxödem, bronchospasmus, angioödem, urtikaria, erythemen und myokarddepression. tabelle 6.5. herz-kreislauf-veränderungen und o 2 -metabolismus bei patienten mit sepsis, fieber und kardialem pumpversagen. (nach werdan et al. 1991; boekstegers et al. 1994) . sepsis fieber kardiales pumpversagen (n=14) (n=11) (n=7) sepsis-score (elebute) 22 (4) 13 (4) 13 (3) multiorganversagen-score (apache ii) 32 (6) 26 (4) 29 (3) hämodynamik herzindex (l/min/m 2 ) 4,8 (1,4) 3,0 (0,5) 2,3 (0,2) linksventrikulärer schlagarbeitsindex (g · m/m 2 ) 37,5 (12,5) 36,6 (6,7) 21,4 (4,3) systemischer gefäßwiderstand (dyn · s · cm -5 ) 597 (149) klassische anaphylaktische reaktionen sind ige-vermittelte allergische ereignisse in reaktion auf ein antigen, entsprechend einer typ-i-reaktion nach gell u. coombs, die perakut und generalisiert systemisch ablaufen. antibiotika, insekten-und schlangengifte, impfstoffe, seren, jodhaltige kontrastmittel und nahrungsmittel gehören zu den typischen auslösenden allergenen. ige-spezifi sche effektorzellen der immunantwort sind im wesentlichen mastzellen und basophile, die nach stimulation eine vielzahl proinfl ammatorischer mediatoren freisetzen und damit das klinische erscheinungsbild der anaphylaxie hervorrufen. davon abzugrenzen sind ige-unabhängige unverträglichkeitsreaktionen ohne vorausgehende sensibilisierung mit einem sehr ähnlichen oder identischen klinischen erscheinungsbild: bei anaphylaktoiden reaktionen (typischerweise ausgelöst z. b. durch röntgenkontrastmittel, salizylate und opiate) kommt es durch chemische, physikalische oder osmotische stimuli zur mediatorfreisetzung aus mastzellen und basophilen. eine idiopathische anaphylaxie kann typischerweise bei jungen erwachsenen auftreten, häufi g nachts oder postprandial; auslösefaktoren und effektorzellen sind unbekannt. die anaphylaxis factitia wird dem münchhausen-syndrom zugerechnet. daneben wurden auch immunreaktionen vom typ iii nach gell u. coombs bei anaphylaktischen reaktionen beschrieben, bei denen komplexe aus igg und spezifi schem antigenkomplement komplement aktivieren und über die anaphylatoxine c3a und c5a die mediatorfreisetzung aus mastzellen und basophilen stimulieren. charakteristischerweise tritt diese reaktion bei patienten mit hereditärem iga-mangel, dem häufi gsten angeborenen defekt des immunsystems (1:500-700), im rahmen von bluttransfusionen auf: präformierte antikörper gegen iga im serum dieser patienten können die mediatorkaskaden auslösen. der begriff »anaphylaktoide reaktion« kann auch als überbegriff verwandt werden für akute unverträglichkeitsreaktionen mit den symptomen einer anaphylaxie, ohne damit eine aussage zum pathomechanismus zu implizieren walther u. böttiger 2004) . genaue zahlen zur inzidenz anaphylaktischer und anaphylaktoider reaktionen sind aufgrund deren unvorhersehbarkeit und unberechenbarkeit nicht bekannt walther u. böttiger 2004) . entsprechend einer retrospektiven epidemiologischen studie muss mit etwa 21 anaphylaxiefällen pro 100.000 einwohner gerechnet werden, wobei zu 36% nahrungsmittel, zu 17% medikamente, diagnostika oder immuntherapeutika, zu 17% insektenstiche und zu 15% unklare ursachen zur anaphylaxie führten (yocum et al. 1999 ). antibiotika, jodhaltige kontrastmittel. etwa bei 1 von 2700 hospitalisierten patienten kommt es nach einer älteren studie zur medikamenteninduzierten anaphylaxie. nach schätzungen ist bei verabreichung von penicillin bei einem von 10.000 patienten mit einer anaphylaxie zu rechnen, die in 9% der fälle tödlich verläuft, so dass jährlich in den usa mit mehreren hundert todesfällen zu rechnen ist. auch die einnahme von cephalosporinen, neueren β-laktamantibiotika und fluoro-chinolonen kann zur anaphylaxie führen. es wird geschätzt, dass etwa 3-7% der patienten mit penicillinallergie kreuzreaktionen gegen ein cephalosporin aufweisen. etwa 200-800 todesfälle gehen in den usa jährlich zu lasten jodhaltiger kontrastmittel. tierische gifte. typische verursacher der anaphylaxie sind insektengifte, übertragen durch stiche der tiere der ordnung hymenoptera (u. a. bienen, wespen, hornissen), und schlangengifte (z. b. klapperschlangen, mokkassinschlangen), die neben toxischen auch schwere allergische reaktionen hervorrufen können. etwa 0,5-5% der bevölkerung haben schon eine schwere allergische reaktion auf einen insektenstich durchgemacht, und 1% dieser reaktionen kann in eine lebensbedrohliche anaphylaxie münden. nahrungsmittel. in der praxis ist bei akuten reaktionen nach mahlzeiten die abgrenzung allergischer reaktionen zu nahrungsmittelintoleranzen und bakterientoxinerkrankungen schwierig. nahrungsmittelallergien bestehen bei etwa 1-6% der kinder und sind im erwachsenenalter seltener anzutreffen. schwere anaphylaktische reaktionen auf nahrungsmittel sind eher selten, sind jedoch gehäuft für erdnüsse, sojabohnen, eiweiß und schalentiere beschrieben worden. latex, exotische früchte. zunehmend ist die inzidenz latexallergischer anaphylaxien. diese können durch die benutzung von latexhandschuhen ausgelöst werden, es sind aber auch bei einführen von kathetern aus latex und bei kondomen anaphylaxien beschrieben worden. auch wenn keine exakten epidemiologischen daten vorliegen, so ist davon auszugehen, dass etwa 7-18% der ärzte und des pfl egepersonals auf latex allergisch reagieren. gefährdet sind neben arbeitern aus der latex-verarbeitenden industrie v. a. patienten, die sich mehreren operativen eingriffen unterzogen haben, ganz besonders spina-bifi da-patienten, die zu >50% sensibilisiert sind. ein hohes allergiesierungspotenzial haben auch schleimhautkontakte, z. b. bei urogenitalen katheterisierungen oder bariumkontrasteinläufen. inzwischen stehen kommerzielle tests zur verfügung, die präoperativ innerhalb weniger stunden eine aussage darüber zulassen, ob bei einem patienten latex-spezifi sche ige-antikörper im serum vorhanden sind. überzufällig häufi g ist die latexallergie vergesellschaftet mit nahrungsmittelallergien, häufi g gegen exotische früchte wie avocado, banane, kiwi, passionsfrucht oder auch kastanien. das risiko unerwünschter und multipler arzneimittelwirkungen erhöht. allergien. häufi g erlebt man anaphylaktische reaktionen bei allergikern und häufi ger nach intravenöser als nach oraler allergenzufuhr, weshalb der anaphylaktische schock nicht selten iatrogen verursacht wird. narkosemittel, muskelrelaxanzien. anaphylaktoide narkosezwischenfälle bis hin zum herzstillstand treten nach einer umfangreichen französischen studie mit einer inzidenz von 1:4500-1:6000 allgemeinanästhesien auf. in ca. 6% der fälle kam es trotz adäquater therapie zum tod des patienten. auslösende agenzien waren in 60-70% der fälle muskelrelaxanzien, in ca. 18% latexprodukte und in ca. 5% kolloidale volumenersatzmittel. aufgrund struktureller besonderheiten (quartäre ammoniumgruppe) können alle muskelrelaxanzien unverträglichkeitsreaktionen hervorrufen. aber auch opioide, lokalanästhetika und narkotika wurden als potenziell anaphylaktoid wirkende agenzien herausgestellt. kumulativer mediatoreneffekt. der kumulative effekt der freigesetzten mediatoren besteht im wesentlichen in einer erhöhten gefäßpermeabilität, einer ausgeprägten vasodilatation und einem bronchospasmus. autoptisch wurde bei tödlich verlaufenden anaphylaxien ein ödem der lungen mit oftmals fl üssigkeitsgefüllten alveolen, ein ödem der oberen atemwege, einschließlich des larynx und der epiglottis, der haut und der viszeralen organe gefunden. im zusammenhang mit ödemen der oberen atemwege kommt es oft zu einer pulmonalen überblähung oder auch zu einer ausgeprägten bronchokonstriktion. der neurogene schock wird durch einen verlust der peripheren vasomotorenkontrolle infolge einer dysfunktion oder einer verletzung des nervensystems hervorgerufen. das klassische beispiel dafür ist der meist mit einer rückenmarkverletzung assoziierte spinale schock (hund u. abel 2002) . dem verlust des venentonus mit erhöhter venöser kapazität scheint hier die entscheidende bedeutung zuzukommen, wobei auch der arteriolentonus reduziert sein kann. in letzterem falle kommt es nach flüssigkeitssubstitution zu einer steigerung des herzindex. eine -allerdings selbstlimitierende und vorübergehende -neurale fehlregulation fi ndet sich auch als ursache der vasovagalen synkope und bei der spinalanästhesie. die addison-krise kündigt sich mit unspezifi schen symptomen an wie anorexie, übelkeit, erbrechen, diarrhöen, abdominellen schmerzen, myalgien, gelenkschmerzen, kopfschmerzen, schwäche, verwirrtheit und agitation oder delir. bei exsikkose kann es zu hohem fieber kommen. das initiale hämodynamische profi l ähnelt dem des hypovolämischen schocks; nach adäquater volumensubstitution demaskiert sich ein hyperdynamer, vasopressorrefraktärer schock (claussen et al. 1992; parkar u. taylor 2001) , der einem septischen schock ähneln kann. überlagern sich beide schockformen, so sind das erkennen der nebenniereninsuffi zienz (kortisolbestimmung, acth-test) und die eingeleitete kortikoidbehandlung oft lebensrettend. zur addison-krise (parkar u. taylor 2001) kann es einerseits bei patienten mit chronischer, substituierter nebennierenrindeninsuffi zienz bei besonderen krankheits-oder operationsbelastungen ohne ausreichende steigerung der kortikoidmedikation kommen; andererseits kann auch eine beidseitige nebennierenblutung im gefolge einer sepsis (meningokokken, gramnegative bakterien), einer hiv-infektion, einer pilzinfektion, einer malignen infi ltration, eines anaphylaktischen schocks (lefevre et al. 1996) oder auch eine einblutung unter antikoagulation zur akuten nebenniereninsuffi zienz führen (rao et al. 1989 ). seltene schock-und synkopenformen sind in der . übersicht 6.1 aufgeführt. mit dem begriff »vasalperipherer schock« werden jene akuten zustände von kreislaufi nsuffi zienz bezeichnet, de-. der verlust von myokardmasse beim akuten herzinfarkt mit minderung der kontraktilität führt zur abfl achung der frank-starling-kurve und zur zunahme des vorhofdrucks (. abb. 6.4a, a-b). durch das positiv-inotrop wirkende dobutamin (ohne wesentlichen einfl uss auf den venösen rückfl uss) kann die kontraktionskraft des herzens gesteigert und damit der herzauswurf erhöht werden (. abb. 6.4a, b-c). im gegensatz zum dobutamin würden die katecholamine noradrenalin und dopamin nicht nur positiv-inotrop wirken, sondern auch die venöse kapazität reduzieren und damit den mittleren zirkulationsdruck erhöhen. hypovolämie vermindert das zirkulierende blutvolumen und damit auch den mittleren zirkulationsdruck pcm (. abb. 6.4b, a-b). die resultierende abnahme des venösen rückstroms und damit des herzauswurfs kann durch volumensubstitution ausgeglichen werden (. abb. 6.4b, b-c). das hämodynamische bild des hämorrhagischen hypovolämischen schocks ist nicht nur durch den blutvolumenverlust geprägt, sondern auch durch eine initiale kompensatorische vasokonstriktion, die im weiteren verlauf von einer therapeutisch schwer beeinfl ussbaren vasodilatation gefolgt wird. für letztere wird zum einen eine hochregulation der spinalen stickoxidsynthase verantwortlich gemacht (lu et al. 1999 ; 7 abschn. 6.2.6), zum anderen eine aktivierung der poly(adp-ribose)-synthetase (pars), einem schlüsselenzym der apoptose (7 abschn. 6.2.7). der septische schock (thiemermann 2000a) vor adäquater volumensubstitution reicht die abnahme des rv allerdings nicht aus, um die reduktion des pcm auszugleichen: der herzauswurf bleibt vermindert (. abb. 6.4c, a-b). durch eine adäquate volumensubstitution lässt sich der pcm normalisieren; aufgrund des weiterhin erniedrigten venösen widerstands (rv) führt dies, bei fehlen einer myokarddepression, dann sogar zu einem supranormalen venösen rückstrom und einem supranormalen herzzeitvolumen (. abb. 6.4c, b-c). die graphik verdeutlicht, dass die rv-abnahme bis zu einem gewissen grad eine meist vorhandene mäßige myokarddepression maskieren kann. erst bei einer ausgeprägten kontraktilitätsminderung wird diese als einschränkung des herzauswurfs manifest (. abb. 6.4c, c-d), was bei ca. 20% aller sepsispatienten zutrifft (kumar u. parrillo 2001) . die mikrozirkulation und deren erkrankungsbedingte störungen können heutzutage beim patienten mit der orthogonalen polarisations-spektral-bildgebung (»orthogonal polarization spectral imaging«, ops; groner et al. 1999 ) sichtbar gemacht werden (. abb. 6.5). eine regelrechte durchblutung auf mikrozirkulationsebene (gefäße bis 100-150 µm durchmesser) ist voraussetzung für eine adäquate gewebeperfusion; ein normales herzzeitvolumen und ein normaler blutdruck sind dafür noch nicht ausreichend. die regionale durchblutung auf mikrozirkulationsebene wird durch lokale intrinsische (autoregulation) und durch extrinsische faktoren (autonomes nervensystem und humorale faktoren) geregelt (holtz 2000) . der blutfl uss zu den einzelnen organen wird durch den tonus der präkapillären arteriolen und prä-und postkapillären sphinktergefäße sowie durch lokale veränderungen der metabolischen aktivität gesteuert. mit der ops-methode (groner et al. 1999 ) lässt sich bei schwerer sepsis zeigen, dass die sublinguale mikrozirkulation in ihrer gefäßdichte reduziert -4,5(4,2-5,2)/mm bei sepsis vs. 5,4(5,4-6,3)/mm bei gesunden -und der anteil der perfundierten kleinen gefäße (<20 µm) vermindert ist -48(33-61)% vs. 90(89-92)%. diese veränderungen sind bei versterbenden ausgeprägter als bei überlebenden. die topische applikation von azetylcholin kann diese veränderungen wieder rückgängig machen (de backer et al. 2002; ince 2002) . die durchblutung der einzelnen organe kann über einen weiten blutdruckbereich organspezifi sch konstant gehalten werden (. tabelle 6.6), für den menschen wird diese autoregulation für einen bereich von 60-100 mmhg angegeben (bond 1993 bei hypovolämie und anderen hypodynamen schockformen wird der blutfl uss in gehirn und herz autoregulatorisch aufrechterhalten, in allen anderen organgefäßbetten ist er jedoch teilweise erheblich eingeschränkt, damit der systemische blutdruck möglichst stabil gehalten werden kann (. tabelle 6.6). für diese vasokonstriktion sind v. a. ein erhöhter sympathikotonus und die freisetzung von katecholaminen aus der nebenniere verantwortlich. diese adaptativen mechanismen reichen aus, um bei gering bis mäßig eingeschränktem herzzeitvolumen lebenswichtige organe adäquat zu perfundieren; bei ausgeprägter hypotonie kommt es jedoch zur organischämie und zum organversagen. selbst nach wiederherstellung stabiler herz-kreislauf-verhältnisse können die störungen auf mikrozirkulationsebene über tage persistieren, v. a. im gehirn, in den nieren, in der leber und in anderen splanchnikusorganen (wang et al. 1990 ). die irreversible phase eines schweren hämorrhagischen schocks ist aufgrund experimenteller befunde durch die vasodilatation präkapillärer sphinkter charakterisiert. die splanchnikusperfusion beim kritisch kranken variiert sehr stark in abhängigkeit von der grunderkrankung, kompensatorischen mechanismen und therapeutischen interventionen (jakob u. takala 2000) : im falle einer systemischen hypoperfusion oder hypoxämie wird die splanchnikusperfusion reduziert, wobei der leberblutfl uss auch unter diesen bedingungen aufgrund intrinsischer kontrollmechanismen relativ hoch gehalten wird (jakob et al. 2002) . bei sepsis und septischem schock fi nden sich bereits bei noch relativ adäquaten blutdruckwerten störungen der organdurchblutung, die eine primäre schädigung auf mikrozirkulationsebene nahe legen (thiemermann 2000a sie beruht auf einer sepsisinduzierten aktiven vasodilatation und dem verlust der extrinsischen vasomotorenkontrolle (bond 1993) , wodurch die durchblutung ausschließlich vom herzzeitvolumen abhängig wird. eine ausnahme von dieser regel stellt die hirndurchblutung dar, die in der sepsis weiterhin die fähigkeit zur autoregulation beibehält: bei patienten mit sepsis ist die hirndurchblutung bereits vor der ausbildung des schockzustandes um ein drittel reduziert, wobei diese durchblutungseinschränkung jedoch nicht als ursache der septischen enzephalopathie angesehen wird. im koronargefäßsystem fällt dagegen der widerstand noch stärker ab als in den anderen organen und demzufolge ist die koronarperfusion bei patienten mit septischem schock sogar häufi g erhöht (dhainaut et al. 1993 schock. die systemische und regionale minderperfusion spielt als verursacher der ischämisch bedingten zellschädigung bei den meisten schockformen eine prägende rolle. sind die neurohumoralen adaptationsmechanismen (. abb. 6.9) nicht mehr in der lage, die schockinduzierte organischämie und hypoxie zu kompensieren, so sind die hemmung des aeroben zellstoffwechsels, die abhängigkeit der energieproduktion von der allein nicht ausreichenden anaeroben glykolyse, die daraus resultierende zelluläre energieverarmung mit laktatanstieg und azidose die folge. diese schwerwiegende ischämisch bedingte zellschädigung ist jedoch keine simple hypoxiefolge auf die mitochon-driale atp-produktion allein: die mitochondrien zeigen auch noch bei sehr niedrigem o 2 -partialdruck (michaelis-konstante <1 µm) eine adäquate atp-produktion. demzufolge garantiert diese hohe o 2 -affi nität auch eine regelrechte funktion der atmungskette, selbst in der hypoxie, sieht man von extrembedingungen einmal ab. der so gestörte substrat-und energiestoffwechsel verursacht zahlreiche zellschäden, die sich als destruktion von mitochondrien, als beeinträchtigung der strukturellen und funktionellen zellmembranintegrität, in form zytotoxischer effekte und schließlich als zelltod manifestieren. leber und niere scheinen auf einen abfall des zell-atp besonders empfi ndlich zu reagieren. sepsis und septischer schock. die bedeutung eines energiemangels in der sepsis ist noch nicht eindeutig geklärt. zumindest lässt sich nachweisen, dass der atp-gehalt im skelettmuskel bei nichtüberlebenden stärker vermindert ist als bei überlebenden (brealey et al. 2002) . zytokine (zell et al. 1997) , exzessive produktion von stickoxid und reduzierte zelluläre glutathionreserven (brealey et al. 2002) scheinen ursächlich für die der verminderten energieproduktion in der sepsis zugrunde liegenden hemmung der atmungskette zu sein, wie für den skelettmuskel (brealey et al. 2002) und den herzmuskel (gellerich et al. 1999) gezeigt. bei der diskussion dieser ergebnisse darf allerdings nicht vergessen werden, dass nicht nur ischämie und hypoxie zur beeinträchtigung der mitochondrialen funktion und atp-produktion führen können, sondern auch mediatoren, insbesondere zytokine (. übersicht 6.3; . tabelle 6.7; zell et al. 1997 endotoxin ist zweifellos ganz entscheidend für die schlechte prognose bei gramnegativem schock mitverantwortlich. darüber sollte jedoch nicht vergessen werden, dass es in niedrigen konzentrationen über eine nur moderate mediatorfreisetzung bei der infektabwehr durchaus günstig wirken kann (mäßiges fieber, steigerung der immunabwehr, abtötung der keime). die durch endotoxin induzierte hypotension scheint mit der zeit eine abschwächung zu erfahren (mailman et al. 1999) . auch bei der entstehung und perpetuierung der herzinsuffi zienz scheint endotoxin eine rolle zu spielen (rauchhaus u. müller-werden 2001) . aus kardiologischer sicht bietet das endotoxin aber auch überraschungen: neben der deletären direkten und indirekten kardiodepressiven wirkung (7 abschn. 6.2.8) lassen sich auch protektive effekte auf das herz nachweisen (mcdonough et al. 1995; song et al. 1994; yao et al. 1993 ). grampositive erreger verursachen mindestens ebenso viele sepsiserkrankungen wie gramnegative keime. da grampositive bakterien keine lipopolysaccharide synthetisieren und in ihre zellwand integrieren, scheidet endotoxin als induktor einer grampositiven sepsis aus. hierfür kommen einerseits peptidoglykane (. abb. 6.6), die wie endotoxin an cd14 binden können, und die lipoteichonsäuren der zellmembran in frage und andererseits toxine, die entweder als superantigene, als porenbildner oder als adp-ribosylierende toxine ihre zytotoxische wirkung entfalten. peptidoglykane und lipoteichonsäuren wirken pyrogen, aktivieren komplement und b-lymphozyten. dabei werden die makrophagen aktiviert und sezernieren zytokine, teils in größeren mengen als nach endotoxinstimulation. toxine mit superantigeneigenschaften, wie z. b. das toxinschocksyndromtoxin 1 bestimmter staphylokokken, besitzen wie konventionelle antigene die fähigkeit, t-lymphozyten zu aktivieren. unter umgehung bestimmter kontrollmechanismen stimulieren sie jedoch nicht nur 0,01% der zellen, wie konventionelle antigene, sondern 2-10-25% aller t-lymphozyten gleichzeitig. die folge ist eine überaktivierung der t-zellen mit massiver zytokinfreisetzung, insbesondere tnf-α, und evtl. letalem schockverlauf. die bedeutung der superantigene bei der pathogenese der sepsis kann gegenwärtig noch nicht ausreichend abgeschätzt werden (visvanathan u. zabriskie 2000) . endotoxin und toxine grampositiver keime können sich in ihrer wirkung verstärken und über die toll-like-rezeptoren miteinander kommunizieren (li et al. 2003 das pseudomonas aeruginosa exotoxin a ist ein adp-ribosylierendes toxin, das hochselektiv den ribosomalen elongationsfaktor 2 der proteinsynthese adp-ribosyliert und damit inaktiviert. das toxin stellt einen wesentlichen virulenzfaktor der pseudomonassepsis dar. an kardiomyoyzten führt exotoxin a über eine partielle proteinsynthesehemmung zu einer störung der neusynthese von β-adrenozeptoren und damit zur kontraktilitätsabschwächung auf katecholamine (müllerwerdan et al. 1997) . das peptidoglykan ist hauptbestandteil der zellwand grampositiver bakterien. es wird ebenso wie endotoxin von cd14-und toll-like rezeptoren gebunden; die bindung an letztere bewirkt eine zellaktivierung. weitere bakerielle toxine wie die lipoteichonsäure, lipoarabinomannan von mykobakterien oder nannuronan führen ebenfalls über einen cd14-und toll-like rezeptor-abhängigen weg zur aktivierung von leukozyten. bestimmte bakterielle dns-sequenzen sind in der lage, antigenpräsentierende zellen (monozyten) zu aktivieren. diese dns-abschnitte sind reich an nichtmethylierten cpg-motiven. oligodeoxynukleotide mit cpg-motiven werden von toll-like-rezeptoren 9 erkannt; diese dns-abschnitte werden in die zellen aufgenommen und führen nach einer azidifi zierung in den endosomen zu einer aktivierung sowohl von mitogen aktivierten proteinkinasen als auch dem transkriptionsfaktor nf-κb. das verständnis um die bedeutung von toxinen und mediatoren im schockgeschehen fußte zunächst auf erkenntnissen, die bei der aufklärung der pathogenese des septischen schocks gewonnen wurden (. abb. 6.6). heutzutage kann jedoch davon ausgegangen werden, dass die mediatorkaskade die gemeinsame endstrecke nicht nur des septischen schocks darstellt (. abb. 6.6), sondern dass auch ein hypovolämischer, traumatischer, anaphylaktischer und sogar der kardiogene schock komponenten dieser kaskade als auslösende oder unterhaltende ursachen beinhalten. dieses konzept spiegelt sich auch in der aktuellen terminologie von sepsis und sirs (»systemic infl ammatory response syndrome«, . tabelle 6.3) wider: ausgehend von infektiösen oder nichtinfektiösen stimuli (. abb. 6.6) kommt es zur aktivierung von mediatorzellen, die primäre mediatoren wie den tumornekrosefaktor α freisetzen. diese primären mediatoren beeinfl ussen weitere zielzellen, die einerseits geschädigt werden können, andererseits (wie z. b. die neutrophilen granulozyten) fi nale mediatoren auf diesen stimulus (wie z. b. reaktive o 2 -verbindungen oder stickoxid) freisetzen. die mediatorfreisetzung soll zwar eigentlich zur bekämpfung der bakterien, zur neutralisierung von bakterientoxinen und zur schadensbegrenzung nichtinfektiöser insulte dienen, die aggressiven verbindungen schädigen dabei jedoch auch das herz-kreislauf-system und weitere vitale or zytokine, insbesondere tnf-α und il-1, stehen am anfang des mediatornetzwerks von schock und sepsis. bakterielle toxine, aber auch sirs-stimuli, können monozyten und makrophagen zur bildung und freisetzung insbesondere von tnf-α aktivieren (. abb. 6.6; loppnow 2001). als resultat der direkten und indirekten zytokinwirkungen stehen beim septischen schock die häufi g irreversible herz-kreislauf-schädigung und das mods. die bedeutung der zytokine beschränkt sich jedoch nicht nur auf den septischen schock: auch beim hämorrhagischen schock fi nden sich erhöhte tnf-serumspiegel; beim kardiogenen schock sind die interleukin-6-spiegel vergleichbar hoch wie beim septischen schock , und das akute lungenversagen bei hypovolämischem und traumatischem schock lässt sich durch den einsatz von anti-tnf-α-antikörpern bessern. erhöhte zytokinserum-und -plasmaspiegel fi nden sich darüber hinaus auch bei zahlreichen infektiösen und nichtinfektiösen herz-kreislauf-erkrankungen (. tabelle 6.7). derzeit muss allerdings noch häufi g die frage offenbleiben, ob zytokine dabei kausale bedeutung haben, ein sekundäres phänomen von krankheitswert oder nur ein unwichtiges epiphänomen darstellen (ausführliche diskussion in müllerwerdan et al. 1996; rauchhaus u. müller-werdan 2001) . das verstehen der zytokinwirkmechanismen (loppnow 2001; rauchhaus u. müller-werdan 2001 ) -gezeigt am beispiel des tnf-α -bietet die möglichkeit einer -bisher leider weder beim septischen schock noch bei herzinsuffi zienz erfolgreichen -kausalen therapie, z. b. mit anti-tnf-α-antikörpern. an der zielzelle bindet tnf-α (als trimer) an einen der beiden tnf-rezeptoren (tnfr1 mit einem mg von 55.000; tnfr2 mit einem mg von 75.000) und löst damit verschiedene zelluläre signale aus (7 abschn. 6.2.8). unterschiedliche stimuli (fieber, endotoxin, tnf-α selbst und andere zytokine) können zur proteolytischen spaltung der tnf-rezeptoren und ihrer freisetzung als lösliche tnf-rezeptoren ins plasma führen. deren bedeutung ist jedoch bisher noch unklar: in niedrigen konzentrationen scheinen sie ein reservoir für das gebundene tnf-α darzustellen, in höheren konzentrationen das tnf-α und damit seine zytotoxische wirkung zu neutralisieren. zytokine sind nicht nur schädlich; es ist vielmehr die massiv gesteigerte produktion und damit die dysbalance von proinfl ammatorischen mediatoren wie tnf, il-1, il-2, il-8, ifn und dem plättchenaktivierenden faktor (paf) mit den antiinfl ammatorischen substanzen und mechanismen (il-10, interleukin-1-rezeptorantagonist, lösliche tnf-rezeptoren, glukokortikoide und auch fieber), die letztlich bei schock und sepsis die ungünstige prognose determiniert. so zeigt eine hohe interleukin-6/interleukin-10-relation bei kritisch kranken eine ungünstige prognose an (taniguchi et al. 1999) . die tatsache, dass niedrige zytokinkonzentrationen (z. b. tnfα) bei sepsis durchaus zur abwehrreaktion in protektiver weise beitragen können, belastet das kausale therapiekonzept der unterbrechung dieser zytokin-/mediatorkaskade. > > zytokine und gerinnung. bei schwerer sepsis und bei septischem schock kommt es regelhaft auf endothelzellebene zu störungen der gerinnung und fibrinolyse im sinne einer disseminierten intravasalen gerinnung (dic, verbrauchskoagulopathie), allerdings individuell in jeweils sehr unterschiedlichem ausmaß: gerinnungsvorgänge sind gesteigert, fibrinolyseaktivitäten vermindert, und das verarmen an gerinnungsfaktoren wie des aktivierten protein c trägt zur proinfl ammation bei (dempfl e 2003). zytokin-genpolymorphismen. die prognose eines schockpatienten hängt wahrscheinlich nicht nur von seinem phänotyp, sondern auch von seinem genotyp ab, wie bisher v. a. für patienten mit sepsis und septischem schock gezeigt worden ist. männliche sepsispatienten, die homozygot sind für das allel tnfb2 des tnf-polymorphismus (ncoi-restriktionsfragmentlängenpolymorphismus), produzieren höhere tnf-plasmaspiegel und haben eine ungünstigere prognose als die sepsispatienten mit dem genotyp tnfb1/b1 bzw. tnfb1/b2. verknüpft mit dem tnf-polymorphismus und ohne eigenständige prognostische aussagekraft sind polymorphismen der hitzeschockproteine hsp70-hom c/t und hsp70-2 g/a bei patienten mit schwerer sepsis. während sich für die genotypverteilung des interleukin 1β keine unterschiede zwischen sepsispatienten und gesunden zeigen, fi ndet sich das allel a2 des interleukin-1-rezeptorantagonisten (il-1ra) bei patienten mit schwerer sepsis häufi ger als bei gesunden. dies könnte auf eine erhöhte sepsisempfi ndlichkeit bei dieser allelkonstellation hinweisen. nicht nur genpolymorphismen proinfl ammatorischer zytokine, sondern auch die antiinfl ammatorischer scheinen relevanz zu besitzen: sepsispatienten mit spezifi schen interleukin-10-genpolymorphismen sezernieren geringere mengen dieses antiinfl ammatorischen zytokins und haben eine erhöhte sterblichkeit (lowe et al. 2003 ). reaktive o 2 -verbindungen stellen im schockgeschehen eine wichtige gruppe terminaler mediatoren dar (de vega et al. 2002; motoyama et al. 2003) . dabei handelt es sich um moleküle mit einem ungepaarten elektron in der äußeren hülle. ursprungsort von freien radikalen sind zum einen aktivierten mediatorzellen, wie neutrophile granulozyten, makrophagen und endothelzellen, sowie andererseits (in der reperfusionsphase der schockbehandlung) das hypoxanthin als abbauprodukt des atp. das superoxidanion (o 2 -), das hydroxylradikal (oh · ), das wasserstoffperoxidmolekül (h 2 o 2 ), die hypochlorige säure (hocl) neutrophiler granulozyten und das peroxinitritradikal (onoo -) sind dabei die wichtigsten verbindungen (. abb. 6.7). peroxinitrit -gebildet aus dem stickoxid-und dem superoxidradikal -ist ein potentes und hochreaktives oxidans mit ausgeprägter zytotoxischer wirkung, das besonders zur endothelschädigung führen kann, die thrombozytenaggregation steigert und die ansprechbarkeit der koronargefäße auf vasodilatatoren vermindert (lamy et al. 2001) . treten die so entstandenen sauerstoffradikale bzw. reaktiven verbindungen in kontakt mit einer zielzelle, so können sie auf verschiedene zelluläre strukturen wirken: auf die zellmembran, das zytosol, den zellkern und die mitochondri-> 6.2 · pathophysiologie des schocks en. schließlich wird durch diesen oxidativen stress eine reihe von genen aktiviert (»stress-response genes«), die zur bildung von antioxidanzien, o 2 -radikalabbauenden enzymen, zytokinen und transkriptionsfaktoren führen. sie dienen einerseits der reparatur von zellschäden durch die sauerstoffradikale und führen, falls dies nicht möglich ist, zum gerichteten zelltod, der apoptose. ein beispiel des schädigungsmusters reaktiver sauerstoffverbindungen bei ischämie und reperfusion im herzen gibt . abb. 6.7. das zunächst als »endothelial-derived relaxing factor« klassifi zierte stickoxid (no) wird enzymatisch aus arginin gebildet. die cnos-isoformen sind ständig vorhanden. sie können sofort aktiviert werden und produzieren geringe mengen an no, das dann zahlreiche physiologische funktionen erfüllt. die cnos-isoenzyme können weiterhin in die neuronale form (nnos oder nos i, lokalisiert im zytosol von zentralen und peripheren neuronen) und in die endotheliale form (enos oder nos iii, überwiegend membrangebunden) eingeteilt werden. die enos-isoform besitzt sowohl parakrine als auch autokrine aktivität; sie wird überwiegend durch den scherstress der gefäße aktiviert, das gebildete no diffundiert in die glatten gefäßmuskelzellen und führt zur erschlaffung. die enos ist nötig, um die gefäße in einem partiell relaxierten zustand zu halten, der durch noradrenalin und endothelin antagonisiert wird. sie reguliert damit den gefäßtonus, die organperfusion und den blutdruck. weiterhin hemmt das von der enos produzierte no die adhäsion von thrombozyten und neutrophilen an endothelzellen und möglicherweise auch die proliferation von gefäßmuskelzellen. die induzierbare form der stickoxidsynthase (inos oder nos ii) wurde zuerst in makrophagen gefunden, sie kommt jedoch in vielen zelltypen vor. ihre induktion durch zahlreiche triggersubstanzen (. tabelle 6.8) dauert einige stunden, das enzymmolekül ist relativ langlebig (halbwertszeit mehrere stunden), und es kann durch die inos-aktivität in kurzer zeit lokal sehr viel stickoxid gebildet werden. demzufolge besteht die möglichkeit, dass das zur verfügung stehende enzymsubstrat arginin die produktion von stickoxid durch inos limitiert, was im falle der wesentlich geringeren no-produktionsraten durch die cnos-aktivität nicht der fall ist. reaktive stickstoff-sauerstoff-verbindungen (rnos) -insbesondere das aus dem stickoxid-und dem superoxidradikal entstehende peroxinitritradikal -wirken, überschießend produziert, im zellstoffwechsel hochtoxisch (7 abschn. 6.2.6; lamy et al. 2001) . ein dritter, für das schockgeschehen möglicherweise sehr bedeutungsvoller bildungsweg ist die nichtenzymatische reduktion von nitrit zu no in minderdurchblutetem gewebe. im ischämischen, azidotischen myokard fi ndet sich nitrit in mikromolaren konzentrationen. mittels elektronenspinresonanzspektren ließ sich nachweisen, dass in diesem hochreduktiven stoffwechselmilieu nitrit in großen mengen nichtenzymatisch zu stickoxid reduziert wird (zweier et al. 1995) . das entstandene stickoxid kann entweder am ort der bildung reagieren (autokrine wirkung) oder in benachbarte zellen diffundieren (parakrine wirkung). ein großteil der stickoxidwirkungen (vasodilatation, kardiodepression) beruht auf der aktivierung der löslichen guanylatzyklase durch stickoxid, mit bildung des zyklischen guanosinmonophosphats (cgmp), des gegenspielers des zyklischen adenosinmonophosphats (camp). bildung und wirkung des stickoxids können auf verschiedenen stufen blockiert werden: die induktion der inos kann durch glukokortikoide unterdrückt werden; die stickoxidsynthasen lassen sich durch argininanaloga wie l-nmma (ng-monomethyl-l-arginin; thiemermann 2000b) hemmen, und die aktivität der guanylatzyklase kann durch methylenblau blockiert werden. in der schockauslösung und der schocktherapie ist stickoxid ein entscheidender mediator (. tabelle 6.8; thiemermann 2000a). die hemmung der zellulären o 2 -verwertungs-> störung in den organen durch no-inhibition einer reihe mitochondrialer enzyme (aconitase, nadh-ubichinon-reduktase, succinat-ubichinon-oxidoreduktase; brealey et al. 2002; thiemermann 2000a ) könnte dabei eine ursache für die erhöht gefundenen o 2 -partialdrücke im skelettmuskel von sepsispatienten sein (. tabelle 6.5). natriuretische peptide wie das atriale natriuretische peptid (anp) und das b-typ-natriuretische peptid (»brain natriuretic peptide«, bnp) sind indikatoren einer mit einer ungünstigen prognose gekoppelten herzfunktionseinschränkung. darüber hinaus können zytokine der interleukin(il)-6-familie die sekretion von anp und bnp induzieren. im septischen schock steigen plasma-anp (82,7±9,9 vs. 14,9±1,2 pg/ml) und plasma-bnp (12,4±3,6 vs. 5,5±0,7 pg/ml) an. der anp-anstieg korreliert dabei mehr mit dem il-6-anstieg und weniger mit der kardiovaskuären dysfunktion, während der bnp-anstieg die pumpfunktionseinschränkung des herzens (abnahme des herzindex) widerspiegelt (witthaut et al. 2003) . in die schockpathogenese sind noch zahlreiche weitere hormone und mediatoren -endothelin, vasopressin (7 abschn. 6.4.7), adrenomedullin, adhäsionsmoleküle, hitzeschockproteine, arachidonsäurederivate u. a. -involviert (thiemermann 2000a; müller-werdan et al. 1996 burchardi et al. 2000) . schockart (dhainaut et al. 1993 ). apoptose -der gerichtete zelltod -fi ndet sich bei patienten mit sepsis v. a. in lymphozyten und intestinalen epithelzellen, nicht dagegen in nennenswertem maße im herzen und anderen parenchymatösen organen. bei nichtseptischen schockzuständen ist die apoptose ein noch selteneres phänomen wie bei der sepsis (hotchkiss et al. 1999; moldawer 1999 ). zahlreiche der im schock und in der sepsis gebildeten mediatoren (übersicht und literaturangaben in werdan 2000; krishnagopalan et al. 2002; müller-werdan et al. 1996; können eine myokarddepression hervorrufen und damit zum bild der akuten septischen kardiomyopathie beitragen. welche von den zahlreichen beschriebenen wirkungen auf die herzmuskelzelle jedoch tatsächlich klinische relevanz besitzen, ist bisher nur mit einschränkung zu beantworten. die z. z. am meisten favorisierte »negativ-inotrope kaskade« ist die endotoxin-tnf-α/il-1-no-cgmp-kaskade (. abb. 6.8). zirkulierendes endotoxin stimuliert mediatorzellen zur systemischen und myokardialen freisetzung von tumornekrosefaktor α (tnf-α) und interleukin 1 (il-1). tnfα und (wohl von geringerer bedeutung) il-1 induzieren dann die bildung der induzierbaren stickoxidsynthase (inos) im herzen. das von ihr gebildete stickoxid (no) stimuliert die lösliche guanylatzyklase des kardiomyozyten, und der daraus resultierende anstieg des cgmp führt schließlich über eine hemmung des ca 2+ -einstroms in die zelle oder über eine desensibilisierung der myofi lamente gegenüber ca 2+ zur kardiodepression (kumar u. parrillo 2001; . alle einzelschritte sind weitgehend belegt. die gabe von endotoxin führt bei probanden zur hyperdynamischen > herz-kreislauf-situation mit blutdrucksenkung, vasodilatation und steigerung des herzindex bei gleichzeitiger abnahme des schlagarbeitsindex als klinischem inotropieparameter (suffredini et al. 1989) . auch der anstieg des zirkulierenden tnf-α nach endotoxinapplikation ist bei probanden gezeigt worden (michie et al. 1988) , und erhöhte tnfα-plasmaspiegel fi nden sich auch im septischen schock. bei therapeutisch mit tnf behandelten tumorpatienten kann es zur ausgeprägten myokarddepression kommen. im tierexperiment führt die gabe von tnf-α zu einer bis mehrere tage anhaltenden myokarddepression (müllerwerdan et al. 1996) . der nachweis der direkten depressorischen wirkung des tnf-α auf die funktion des kardiomyozyten lässt sich im zellkulturexperiment führen: tnf-α hemmt in klinisch relevanten konzentrationen nach mehrstündiger einwirkungsdauer die »positiv-inotrope« wirkung des β-adrenozeptoragonisten isoproterenol und auch die hoher ca 2+ -konzentrationen. die depressorische tnf-α-wirkung kann durch anti-tnf-α-antikörper neutralisiert werden. auch die induktion einer induzierbaren stickoxidsynthase in kardiomyozyten durch tnf-α und il-1 ist experimentell gut belegt, ebenso wie bei patienten mit septischem schock eine erhöhte aktivität der inos und ein cgmp-anstieg im herzen. es spricht also vieles dafür, dass die diskutierte »negativinotrope« kaskade für die myokarddepression in der sepsis und im septischen schock wesentlich mitverantwortlich ist. dennoch scheint dies nicht der einzige kardiodepressive mechanismus zu sein: einerseits kann tnf-α im experiment auch stickoxidunabhängig negativ-inotrop wirken, andererseits wird durch tnf-α nicht nur die positiv-inotrope wirkung von β-adrenozeptoragonisten, sondern auch die von α-adrenozeptoragonisten gehemmt. letzteres ist über eine alleinige induktion der stickoxidsynthase nicht zu erklären; so hemmt endotoxin, das in kardiomyozyten die stickoxidsynthase stimuliert, zwar auch die positiv-inotrope wirkung von β-adrenozeptoragonisten, nicht aber die von α-adrenozeptoragonisten (müllerwerdan et al. 1996) . aufbauend auf experimentellen befunden dieser art kann davon ausgegangen werden, dass neben der stickoxid-cgmp-kaskade auch noch andere mechanismen für die negativ-inotrope wirkung von tnf-α verantwortlich sein müssen (. abb. 6.8). die belegte hemmung des phosphoinositolstoffwechsels durch dieses zytokin erklärt die hemmung der positiv-inotropen wirkung von α-adrenozeptoragonisten, und die ebenfalls dokumentierte abschwächung des ca 2+ -transients durch tnf schwächt alle inotropen effekte ab. ebenfalls belegt ist die hemmung des ca 2+ -einwärtsstroms durch einen kardiodepressiven peptidfaktor, der sich in hämofi ltraten von patienten mit kardiogenem und septischem schock in konzentrationen nachweisen lässt, die diese hemmwirkung in humanen herzmuskelzellen hervorrufen ). die gewebeschäden sind nicht allein durch den mangel an sauerstoff während der ischämiephase, sondern auch durch die reperfusion und reoxygenation der ischämischen organe bedingt (abella u. becker 2002) . bei der enzymatischen reaktion der in der ischämiephase akkumulierten atp-abbauprodukte hypoxanthin und xanthin mit sauerstoff kommt es zur bildung freier sauerstoffradikale (wasserstoffperoxid, superoxidradikal, hydroxylradikal), die durch lipidperoxidation die aktivierung des prostaglandinsystems und eine denaturierung von zellmembranproteinen bewirken. katalysiert wird diese radikalproduktion durch das enzym xanthinoxidase, das in der ischämie durch umwandlung aus der xanthindehydrogenase (nicht zur radikalbildung befähigt) entsteht. dieser umwandlungsprozess läuft in den geweben bei ischämie unterschiedlich schnell ab (darm: 10 s; herzmuskel: 8 min; leber, milz, niere, lunge: 40 min), was die unterschiedliche empfi ndlichkeit der einzelnen gewebe auf reperfusionsschäden mit erklären soll freie sauerstoffradikale rufen darüber hinaus eine verstärkte akkumulation polymorphkerniger leukozyten hervor, begünstigen das leukozytensticking sowie über den »respiratory burst« die bildung weiterer sauerstoffradikale aus polymorphkernigen leukozyten (. abb. 6.7). demzufolge kommt der leukozyten-endothel-interaktion -leukozyteneinwanderung, -rollen, feste adhäsion, gefäßtransmigration -via adhäsionsmoleküle -selektine, integrine, immunglobulinsuperfamilie, cadherine -bei dem ischämiereperfusionsschaden eine große bedeutung zu (finney et al. 2002) . exzessive stickoxid-und damit peroxinitritbildung, komplementaktivierung (ciurana u. hack 2002) und apoptoseinduktion (abella u. becker 2002) tragen wahrscheinlich ebenfalls entscheidend zum komplexen ischämiereperfusionsschädigungsmuster bei. ein weiterer kandidat ist das kernenzym poly(adp-ribose)-polymerase (parp; synonym: par-synthetase, pars; liaudet et al. 2001; szabo u. liaudet 2002; . abb. 6.7): proinfl ammatorische zytokine im kreislaufschock produzieren reaktive sauerstoffverbindungen via stimulation der xanthinoxidase, de-novo-expression der induzierbaren stickoxidsynthase, induktion einer mitochondrialen dysfunktion und rekrutierung von neutrophilen granuloyzten mit expression der nadph-oxidase. als folge davon werden die sauerstoffradikale peroxinitrit, wasserstoffperoxid und hydroxylradi-kale gebildet, durch interaktion von superoxidanionen, stickoxid und der eisenkatalysierten oxidation von superoxidanionen. der so entstehende oxidative zellstress induziert die bildung des transkriptionsfaktors ap-1 und löst dns-einzelstrangbrüche aus. die dns-schäden aktivieren die parp. die parp-aktivierung verbraucht atp sowie redoxäquivalente (nadph), induziert damit eine endothelzellschädigung sowie den zelltod und potenziert die aktivierung der transkriptionsfaktoren ap-1 und »nuclear factor kappa b (nfκb)«. letzteres hat die verstärkte transkription und translation der ap-1-und nfκb-abhängigen gene für inos, icam, mip-1α, tnf-α und komplement-c3 zur folge. das aus c3 gebildete c5 und die gesteigerte endotheliale expression des adhäsionsmoleküls icam-1 führt zu einer verstärkten einwanderung von aktivierten leukoyzten in den infl ammatorischen fokus und bewirkt damit eine intensivierung der bildung reaktiver sauerstoffverbindungen. der teufelskreis wird mit der triggerung weiterer dns-strangbrüche durch den oxidativen stress geschlossen. da natürliche schutzstoffe gegen die effekte der sauerstoffradikale (scavenger-substanzen, superoxiddismutase (elsakka et al. 2001) , glutathion, glutathionperoxidase, katalase) im intrazellulärraum nur in geringer konzentration vorliegen, können sie den als folge der reperfusion und reoxygenation eintretenden reperfusionsschaden, der durch endothelläsion, intrazelluläres ödem und letztlich deletären einstrom von kalziumionen in die zellen charakterisiert ist, nicht verhindern. der reperfusionsschaden wird als wichtiger pathogenetischer faktor bei der entwicklung des mods im schock gesehen. noch ausstehend ist der nachweis der klinischen wirksamkeit einer antioxidanzientherapie (mullan u. mccloskey 2002) , der in zahlreichen studien für folgende substanzen nicht erbracht werden konnte: superoxiddismutase, katalase, glutathionperoxidase, ascorbinsäure, glutathion, harnsäure, α-tocopherol, karotinoide, der xanthinoxidasehemmer allopurinol, lazaroide, pyruvat (fink 2003) , der hemmung der komplementaktivierung (ciurana u. hack 2002) , von parp-inhibitoren (szabó u. liaudet 2002; liaudet et al. 2001 ) oder der inhibition der map-kinasen (tulleken et al. 2001 ). hrv-td zeitdomäne der herzfrequenzvariabilität; hrv-fd frequenzdomäne der herzfrequenzvariabilität zusätzlich zu den intrinischen anpassungsmechanismen der neurohumoralen und auto-/parakrinen regelkreise im schock können bakterientoxine (endotoxin) und möglicherweise auch mediatoren eine fehlstellung der stellglieder induzieren (schmidt et al. 2001; schmidt u. müller-werdan 2003) : sowohl die baro-als auch die chemorefl exsensitiät und ebenso die herzfrequenzvariabilität zeigen bei kritisch kranken im schockbedingten mods ausgeprägte, prognosebestimmende einschränkungen (. tabelle 6.9; 7 abschn. 6.5.1). auf welcher ebene toxine und mediatoren diese autonome dysfunktion bewirken -zentralnervensystem, autonomes nervensystem, zielzelle (. abb. 6.10) -bleibt noch zu klären; experimentell ist bei spontan schlagenden neonatalen kardiomyozyten (»schrittmacherzellen«) durch züchtung der zellen in endotoxin eine einschränkung der schlagfrequenzvariabilität zu erzielen, ohne dass dabei das autonome nervensystem involviert ist (schmidt et al. 2001 ). ) schock bedeutet für den patienten immer eine lebensbedrohliche notfallsituation. er erfordert ein sofortiges handeln, beginnend in der prähospitalphase (christ u. lackner 2004) , fortgesetzt in der notaufnahme (laggner 2004 ) und schließlich auf der intensivstation. bei der erstversorgung müssen basisdiagnostik und basistherapie zum frühestmöglichen zeitpunkt begonnen werden. diese basisversorgung dient primär der kreislaufstabilisierung und gilt für alle schockformen. sie mündet nach feststellung der schockform in eine schockformspezifi sche behandlung. je kürzer die schockdauer ist, je schneller die stabilisierung des kreislaufs erreicht wird und je früher die schockformspezifi sche behandlung begonnen werden kann, um so günstiger die prognose. patienten, bei denen ein kreislaufschock vermutet wird, sollten auf der intensivstation weiter betreut und behandelt werden. die in der . übersicht 6.4 aufgeführten technischen untersuchungen schließen sich an. patienten mit unklarer schockätiologie oder mit einer sich nach volumensubstitution nicht rasch stabilisierenden kreislaufl abilität sollten baldmöglichst einem invasiven hämodynamischen monitoring unterzogen werden (7 abschn. 6.3.4). extremitäten. blutungen und thromboembolische komplikationen können bei diesen patienten auf eine verbrauchskoagulopathie hinweisen. kranke mit einem septischen fieber und neutropenie weisen häufi g keinen fokus auf; der septische schock kann sowohl mit hohem fieber als auch mit hypothermie einhergehen. das klinische bild des schockpatienten ändert sich mit der behandlung (laggner 2004) : der unbehandelte patient mit septischem schock in der notaufnahme ist ein patient mit zentralisiertem, »kühlem« schock, der behandelte patient mit septischem schock auf der intensivstation zeigt das »klassische« bild des »warmen«, hyperzirkulatorischen schocks (. tabelle 6.10). das vorgehen bei klinischem schockverdacht im rahmen der erstversorgung zeigt die . übersicht 6.4. empfehlungen zur klinisch-hämodynamischen basisdiagnostik bei kardiogenem schock sind der . übersicht 6.5 zu entnehmen. die höhe der troponin-und ck-mb-anstiege und das flächenintegral korrelieren zwar mit der infarktausdehnung und der ungünstigen prognose, ein cut-off-wert für den infarktbedingten kardiogenen schock kann allerdings nicht angegeben werden. ein nicht unerheblicher anteil (>50%) der patienten mit schwerer sepsis und septischem schock zeigt erhöhte troponinserumspiegel und weist damit auf eine myokardschädigung in der sepsis hin. patienten mit erhöhten troponinwerten haben eine ungünstigere prognose (spies et al. 1998 bei schwerer sepsis und septischem schock scheint das serielle pro-nt-bnp-monitorig innerhalb der ersten 7 tage nach diagnosestellung prognostische bedeutung zu haben: während bei den später versterbenden die pro-nt-bnp-spiegel stark erhöht bleiben oder sogar weiter ansteigen, sinken sie bei den überlebenden signifi kant ab. gerinnungsparameter. da patienten mit akuten kardialen erkrankungen in der regel antithrombotische, antithrombozytäre und fi brinolytische substanzen singulär oder in kombination erhalten, ist die standardmäßige bestimmung von parametern der blutgerinnung obligat: thrombozyten, aptt, prothrombinzeit, fibrinogenkonzentration, d-dimere (bei verdacht auf beinvenenthrombose, lungenembolie). sobald die umstände dies gestatten, sollten vorrangig ein ekg und eine thoraxröntgenaufnahme angefertigt werden. bildgebende (janssens 2001; und invasive verfahren (s. unten), evtl. kombiniert mit interventionellen maßnahmen, ergänzen die diagnostik und bieten erste therapeutische ansätze. das monitoring und entsprechende therapeutische konsequenzen sind in den . übersichten 6.4 und 6.6 wiedergegeben (müller arterielle druckmessung. bei allen patienten mit kreislaufschock sollte eine kontinuierliche, blutige arterielle druckmessung erfolgen, da die manuelle messung mit der manschette oder mit nichtinvasiven oszillometrischen techniken aufgrund der zentralisierung und vasokonstriktion unzuverlässig. ist. in der regel wird dafür die a. radialis kanüliert; bei schwerster zentralisation sollte allerdings die a. femoralis bevorzugt werden. zentraler venendruck. die messung des zentralen venendrucks ist bei kritisch kranken, insbesondere schockpatienten, für das hämodynamische monitoring normalerweise nicht genügend, eine abschätzung der linksventrikulären vorlast kann damit nicht ausreichend sicher durchgeführt werden, ebenso wenig wie mit der klinischen einschätzung allein. für die therapieoptimierung des patienten im kardiogenen shock ist die kenntnis des herzzeitvolumen (hzv) zwingend erforderlich. das hzv ist die regelgröße des herz-kreislauf-systems und wird im wesentlichen durch vorlast, nachlast und kontraktilität sowie die herzfrequenz bestimmt. klassische klinische zeichen wie blutdruck, urinausscheidung, halsvenenfüllung, hautperfusion und hautturgor erlauben keine zuverlässige einschätzung der hämodynamik beim schwerkranken intensivpatienten (eisenberg et al. 1984) . pulmonalarterienkatheter. der pulmonalarterienkatheter (pak) ist weiterhin ein grundpfeiler der erweiterten hämodynamischen überwachung bei patienten mit kardiogenem schock (flieger et al. 2004; janssens 2001 bruch et al. (2003) f für das prognoserelevante monitoring weniger geeignet (müller-werdan u. werdan 2005) als anfangs gedacht. der grad der regionalen gewebeoxygenierung im schock ist spektroskopisch durch messung der hämoglobin-o 2 -sättigung und des cytochrom-aa 3 -redoxstatus möglich (beilman et al. 1999) . während die kontinuierliche messung der co 2 -atemgaskonzentration (kapnographie) v. a. in der notfallmedizin und in der kardiopulmonalen reanimation zur beurteilung von kreislauf-und respiratorischer funktion zum einsatz kommt, ist die sublinguale kapnometrie (messung des sublingualen co 2 -partialdrucks) ein neues, nichtinvasives verfahren zur beurteilung des schweregrades und der prognose von schockzuständen (weil et al. 1999 ). bei patienten mit akutem koronarsyndrom wurden klinische modelle entwickelt, die zuverlässig das auftreten eines kardiogenen schocks im weiteren verlauf vorhersagen können. für den patienten mit st-strecken-elevations-myokardinfarkt (stemi) erfolgte dies auf der grundlage der gusto-i-und -iii-studiendaten (. tabelle 6.11). dieser risiko-score, der innerhalb weniger minuten in der notaufnahme erhoben werden kann, erlaubt auf schnelle und unkomplizierte weise, eine frühzeitige abschätzung des risikos einer schockentstehung im weiteren klinischen verlauf (hasdai et al. 2000b das grundsätzliche ziel der symptomatischen schocktherapie ist die wiederherstellung einer suffi zienten durchblutung der vitalorgane und gewebe, ehe sich ein zellschaden ausbilden kann. dies erfordert einen ausreichenden herzindex und blutdruck. kurze perioden einer ausgeprägten minderperfusion werden besser toleriert als gravierende blutdruckabfälle. die aufrechterhaltung eines mittleren blutdrucks von mehr als 60-65 mmhg hat initial priorität vor dem anheben des herzindex auf werte von >2,1 l/min/m 2 im falle des kardiogenen und obstruktiven bzw. >4,0-4,5 l/min/m 2 beim septischen und beim volumensubstituierten hämorrhagischen schock. die durchblutung sollte zumindest so gesteigert werden, dass der arterielle laktatspiegel unter 2,2 meq/l verbleibt, eine weitgehende garantie dafür, dass der stoffwechsel weiter ae nicht länger als 30 s sollte die intubation als sicherste beatmungsmethode in anspruch nehmen. die intubation (klasse-i-empfehlung) sollte nur durch sehr gut trainierte personen praktiziert werden. im zweifelsfall ist (auch aus juristischen gründen) die leichter zu beherrschende maskenatmung (beatmung immer mit 100% sauerstoff) anzuwenden. larynxmaske oder kombi-tubus gelten als klasse-ii-option. der (peripher-)venöse zugang ist ebenfalls eine klasse-i-intervention. die anfl utzeit der medikamente beträgt 1-2 min. aufgrund vielfältiger komplikationsmöglichkeiten wird die anlage eines zentralvenösen zugangs nur in ausnahmefällen als indiziert angesehen. alternativ besteht insbesondere bei kindern, aber auch bei erwachsenen die möglichkeit, medikamente über eine intraossäre kanüle zu applizieren; dies wird unterhalb der tuberositas tibialis in der markhöhle platziert und ermöglicht neben der gabe von medikamenten auch die infusion von flüssigkeit. f f f f > adrenalin, atropin und lidocain können in notfällen auch endobronchial über den tubus gegeben werden (7 abschn. 6.4.8). hierbei muss dann die dosis mindestens verdoppelt (2-bis 2,5fach) in 10 ml 0,9%igem nacl appliziert werden. adrenalin ist das einzige routinemäßig empfohlene medikament, alle anderen, z. b. antiarrhythmika, natriumbikarbonat, kalzium-und magnesiumzubereitungen, sind speziellen reanimationssituationen vorbehalten. adrenalin. die standarddosis ist 1 mg i.v. alle 3-5 min bis zur kreislaufstabilisierung, gefolgt von jeweils 20 ml i.v.-spülvolumen. dosen über 1 mg haben bestenfalls den primären, nicht jedoch den langfristigen reanimationserfolg verbessern können (gueugniaud et al. 1998 ). höhere adrenalindosen korrelieren sogar mit einem ungünstigen neurologischen defi zit (behringer et al. 1998) . dennoch sehen die empfehlungen im verlauf der reanimation eine eskalation der adrenalindosis auf bis zu 0,2 mg/kgkg alle 3-5 min im sinne einer klasse-ii-empfehlung vor. vasopressin. ermutigend, aber noch nicht standardempfehlung, ist die wirkung von vasopressin (40 ie i.v.), das in vergleichsstudien mit adrenalin zumindest gleichwertig, bei nachweis einer asystolie sogar vorteilhaft war (wenzel et al. 2004) . im falle eines adrenalinrefraktären schocks infolge kammerfl immerns bei erwachsenen gilt vasopressin als klasse-iia-empfehlung. atropin. in einer dosierung von 1 mg als bolus i.v. alle 3-5 min bis zu einer gesamtdosis von 0,04 mg/kgkg ist atropin indiziert bei asystolie und pulsloser elektrischer aktivität. die dosis von 1 mg atropin sollte nicht unterschritten werden, da es bei niedrigeren dosen zu paradoxen effekten (bradykardien) kommen kann. bei symptomatischen bradykardien tritt die atropingabe hinter die transvenöse oder behelfsweise transkutane stimulation zurück. amiodaron. amiodaron wird empfohlen bei persistenz von kammertachykardie oder kammerfl immern nach defi brillation und adrenalingabe (dorian et al. 2002) . initial wird eine rasche 300-mg-infusion in 20-30 ml kochsalz-oder glukoselösung gegeben, ggf. gefolgt von einer infusion mit 1 mg/ min für 6 h und anschließend 0,5 mg/min bis zu einem täglichen maximum von 2 g. die gabe von lidocain ist generell nicht als routinemaßnahme zu sehen, auch nicht bei patienten mit akutem myokardinfarkt! in einer dosis von 1,0-1,5 mg/kgkg i.v. kann lidocain bei kammerfl immern/pulsloser kammertachykardie nach erfolgloser defi brillation und adrenalingabe verabreicht werden (wirksamkeit als unsicher eingestuft), ggf. gefolgt von weiteren 0,5-0,75 mg/kgkg innerhalb von 3-5 min (maximaldosis 3 mg/kgkg oder 200-300 mg/h). lidocain stellt z. z. allerdings bei dieser indikation im vergleich zu amiodaron nur ein antiarrhythmikum der zweiten wahl dar. > ! > 6.4 · therapieprinzipien bei schock zur rezidivprophylaxe nach erfolgreicher defi brillation oder kardioversion kann lidocain in einer dosierung von 1,0-1,5 mg/kgkg gegeben werden, gefolgt von 20 ml nacl 0,9%, mit einer anschließenden dauerinfusion von 1-2 mg/ kgkg/h. vor einer defi brillation angewandt, erhöht lidocain eher die defi brillationsschwelle. monomorphe kammertachykardien nicht primär ischämischer genese können mit lidocain nur zu etwa 10% unterbrochen werden. dagegen ist ajmalin (1,0 mg/kgkg i.v.) mit einer 60%igen erfolgsrate wesentlich effektiver. natriumbikarbonat. da eine bedeutsame arterielle azidose während der kardiopulmonalen reanimationssituation in der regel durch eine unzureichende ventilation begründet ist und nahco 3 selbst zu einem paradoxen intrazellulären co 2 -anstieg führen kann, ist die gabe von natriumbikarbonat üblicherweise nicht indiziert. in speziellen situationen kann die gabe von bikarbonat allerdings hilfreich sein: bei vorbestehender metabolischer azidose, bei hyperkaliämie und bei intoxikationen mit trizyklischen antidepressiva oder phenobarbital. nach protrahiertem herz-kreislauf-stillstand oder langdauernden wiederbelebungsbemühungen -nach ineffektiver defi brillation, herzmassage, intubation, beatmung und vasopressorentherapiekann bikarbonat (1 meq/kgkg als initiale dosis) von nutzen sein. andere puffersubstanzen haben bisher keinen eingang in die offi zielle empfehlung gefunden. elektrolyte: kalzium, magnesium. eine kalziumgabe als routinemaßnahme kann nicht empfohlen werden. nur im falle einer hypokalzämie, einer hyperkaliämie oder einer intoxikation mit kalziumantagonisten ist die gabe von kalzium bedingt indiziert (klasse-iib-empfehlung): 10%ige kalziumchloridlösung in einer dosis von 2-4 mg/kgkg, wiederholung in 10-minütigen intervallen möglich. die applikation von magnesium (intravenöse gabe von 1-2 g magnesiumsulfat in 1-2 min) ist indiziert bei bestätigter hypomagnesiämie und refraktärem oder rezidivierendem kammerfl immern. ebenfalls indiziert ist die intravenöse gabe von 1-2 g magnesiumsulfat bei torsade-de-pointes-tachykardien. enttäuschend war bisher der einsatz der transkutanen antibradykarden stimulation in einer studie mit 1056 patienten mit herz-kreislauf-stillstand. weder bei asystolie noch überbrückend nach defi brillation mit nachfolgender asystolie konnte dadurch der ausgang der prähospitalen kardiopulmonalen reanimation entscheidend effi zienter gestaltet werden (literatur in . in mehreren studien konnte gezeigt werden, dass nach kardiopulmonaler reanimation durch die induktion einer milden therapeutischen hypothermie mit temperaturen zwischen 32 und 34°c eine verbesserung des überlebens und der neurologischen funktion erreicht werden kann (bernard et al. 2002; group thacas 2003) ; aufgrund der guten ergebnisse ist das > »cooling« nach cpr bei herzstillstand infolge kammerfl immerns als klasse-i-maßnahme in die ilcor-empfehlungen eingegangen (nolan et al. 2003) . nach eventueller durchführung von reanimationsmaßnahmen erfolgt die stabilisierung des herz-kreislauf-systems und der lungenfunktion. zur herz-kreislauf-stabilisierung dienen kristalloide und kolloide plasmaersatzlösungen sowie der einsatz von katecholaminen (7 abschn. 6.4.6 und 6.4.7). dazu dienen auch anxiolyse und analgesie, relaxierung zur einsparung von sedativa und die beseitigung von fieber (hyperthermie steigert den o 2 -verbrauch um 7%/°c). ebenfalls beachtet werden müssen die negativen auswirkungen der peep-beatmung auf die herzfunktion (zunahme der rechtsventrikulären nachlast, zunahme des rechtsventrikulären durchmessers und abnahme der linksventrikulären diastolischen dehnbarkeit, direkte myokarddepressive wirkung), ebenso bei kontrollierter mechanischer (»controlled mechanical ventilation«, cmv) oder intermittierender ventilation (»intermittent mandatory ventilation«, imv). die langwierige und schwierige weaning-off-phase gerade bei herzkranken und schockpatienten kann durch einhalten standardisierter protokolle erleichtert werden (lehmann et al. 2003; seige et al. 2001) . die myokarddepression kann weiterhin verstärkt werden durch anästhetika und barbiturate. während benzodiazepine und opioide für sich keine relevante myokarddepression hervorrufen (mit ausnahme von meperiden), können sie in kombination einen additiven negativ-inotropen effekt induzieren. > > evidenzbasierte empfehlungen zur beatmung des schockpatienten, speziell des patienten mit kardiogenem schock, gibt es z. z. noch nicht (kontoyannis et al. 1999; lesage et al. 2004 ). bei überwiegen der vorteile der maschinellen beatmung bei patienten mit kardiogenem schock (reduktion der kardialen vor-und nachlast, abnahme der pulmonalen stauung, reduktion der atemarbeit) erscheint eine eher großzügige indikationsstellung bei diesen patienten gerechtfertigt. ein engmaschiges kardiopulmonales monitoring und ein rasches anpassen der beatmung an änderungen der herzfunktion ist angebracht (lehmann et al. 2001; seige et al. 2001 ). antiarrhythmische therapie. ein spezifi sches muster an rhythmusstörungen ist für den schockpatienten nicht dokumentiert. dennoch muss häufi g mit potenziell malignen rhythmusstörungen gerechnet und entsprechend behandelt werden. die im kardiogenen schock meist vorhandene hochgradige pumpfunktionseinschränkung reduziert die frequenztoleranz des patienten beträchtlich und birgt so die gefahr einer weiteren verschlechterung der schockzustandes, falls die hämodynamisch relevante rhythmusstörung nicht rasch beseitigt wird. abhängig von der klinischen situation (z. b. kardiogener schock bei herzinfarkt) kann eine schmerzbehandlung und eine analgosedierung erforderlich werden (ruß et al. 2005) . intravenöse morphinbolusgaben (2-5 mg i.v. alle 5-30 min bis zu einer gesamtdosis von 2-3 mg/kgkg) sind, z. b. in den ersten stunden eines herzinfarktes, das mittel der wahl. auf eine mögliche blutdrucksenkung infolge einer direkten und indirekten hemmung des sympathikus muss geachtet werden, v. a. bei relativer hypovolämie. im kreislaufschock kann die morphinclearance durch die minderperfusion der leber eingeschränkt sein. morphin hemmt nicht nur den schmerz, sondern trägt auch zur senkung des o 2 -verbrauchs bei. der wichtigste parameter der metabolischen azidose ist der laktatanstieg. die anhebung des extrazellulären ph-werts mit bikarbonat führt nicht immer zum gewünschten erfolg des intrazellulären ph-anstiegs, der zudem eine linksverschiebung der hämoglobindissoziationskurve und damit eine erschwerte o 2 -abgabe an das gewebe bewirkt. die therapie der laktatazidose kann aber zu einer besserung der systemischen und hepatischen zirkulation führen, die ihrerseits zum abbau verbleibenden laktats beiträgt. der therapeutische einsatz von bikarbonat sollte auf ph-werte von 7,10-7,15 beschränkt bleiben. klinisch wurde die hämodynamik von schockpatienten durch bikarbonat auch bei ausgeprägter azidose nicht verbessert (cooper et al. 1990 ); eine isolierte stimulation der pyruvatdehydrogenase mit dichloracetat senkte bei laktatazidose die hohe letalität von 70% nicht (stacpoole et al. 1992 ). ob kollodiale oder kristalloide lösungen zur volumensubstitution besser geeignet sind, wird seit langem kontrovers diskutiert; die art der lösung scheint allerdings für den therapieerfolg nur eine untergeordnete rolle zu spielen. unterschiede bezüglich morbidität und letalität konnten für verschiedene flüssigkeitsregimes bisher nicht eindeutig gezeigt werden (kreimeier u. prückner 1998; ; task force of the american college of critical care medicine, society of critical care medicine 1999). kristalloide lösungen sind kostengünstig, leicht zu lagern, steigern ausreichend die diurese und können zusätzlich extravasale flüssigkeitsverluste bei dehydratationszuständen ersetzen. nachteilig sind das auftreten ausgeprägter peripherer ödeme und die relativ kurze hämodynamische wirksamkeit (. tabelle 6.13). am häufi gsten kommen physiologische (0,9%ige) kochsalzlösung und vollelektrolyte (z. b. ringer-laktatlösung) zum einsatz, die sich beide gleichermaßen im intravasalraum und im interstitium verteilen; nach 1 h fi nden sich aber nur noch weniger als 25% des infundierten volumens in der zirkulation. beide lösungen senken den kolloidosmotischen druck. im vergleich zu kolloidalen lösungen muss etwa das 2-bis 4fache des intravasalen flüssigkeitsdefi zits an kristalloider flüssigkeit zur erzielung einer vorübergehenden normovolämie infundiert werden. 5%ige glukoselösung fi ndet sich 1 h nach infusion nur noch zu 8% im intravasalraum; sie erhöht neben dem volumen des extrazellulärraums unerwünschterweise auch das des intrazellulärraums (infolge des wassereinstroms in die zellen zum ausgleich des infusionsbedingten osmotischen gradienten). sie sollte deshalb zur volumentherapie nicht verwendet werden. zu den kollodialen lösungen zählen albumin, hydroxyäthylstärke, dextran und gelatine (. tabelle 6.13). diese verbleiben zunächst vorwiegend im intravasalraum und stellen dort den plasmaonkotischen druck wieder her. sie führen demzufolge in geringerem ausmaß zu peripheren ödemen, und es genügen kleinere volumina zur substitution als beim einsatz von kristalloiden lösungen. nachteile sind neben den hohen kosten die bekannten, substanzeigenen nebenwirkungen (s. unten). dass kolloidale lösungen v. a. bei der sepsis mit ihrer erhöhten kapillarpermeabilität das auftreten eines lungenödems fördern, ist viel diskutiert, aber bisher nicht gesichert worden. von den kolloidalen substanzen wird in deutschland am häufi gsten hydroxyäthylstärke eingesetzt. für die initiale volumentherapie mit kolloidalen lösungen erscheinen 6%ige lösungen von mittelmolekularer hydroxyäthylstärke (hes 200.000/0,5 oder 200.000/0,62) sowie die 6%ige dextran-60-lösung (macrodex) am besten geeignet. nicht nur wegen der limitierten tagesdosen können die kolloidalen lösungen mit kristalloiden (z. b. im verhältnis 1:1) kombiniert werden. die derzeitige datenlage spricht dafür, dass das kolloid der zukunft eine stärkepräparation der dritten genera-> 6.4 · therapieprinzipien bei schock tion (hes 130/04; . tabelle 6.13) sein wird (boldt 2003; dieterich 2001) . albumin. albumin fi ndet als 5%ige (kolloidosmotischer druck ca. 20 mmhg) und als 20-bis 25%ige lösung (kolloidosmotischer druck ca. 80-100 mmhg) verwendung und verbleibt relativ lange im intravasalraum (>90% nach 2 h). bei hypovolämie ist initial eher die 5%ige lösung angebracht; bei ödematösen patienten kann die verwendung der hyperonkotischen albuminlösung eine erwünschte flüssigkeitsverschiebung aus dem interstitium in den intravasalraum bewirken. beim direkten studienvergleich der volumensubstitution kritisch kranker erbrachten die gabe von albumin im vergleich zu kochsalzlösung keine unterschiede hinsichtlich letalität und morbidität (safe study investigators 2004). kristalloide und synthetische kolloidale lösungen haben das humanalbumin bei der volumentherapie weitgehend ersetzt; geblieben ist beim erwachsenen als indikation eine hypalbuminämie <2,5 g/dl bzw. ein gesamteiweiß <4,0-4,5 g/dl. es ist jedoch nicht sinnvoll, eine hypalbuminämie als folge eines kapillären lecks (wie bei der sepsis) mit albumin vollständig auszugleichen, da dieses mit einer halbwertszeit von 1-6 h aus dem intravasal-in den extravasalraum abwandert. der absolutwert des kolloidosmotischen drucks hat sich als parameter zur albuminsubstitution nicht durchgesetzt. unerwünschte, in der regel milde nebenwirkungen (fieber, schüttelfrost, urtikaria) treten mit einer häufi gkeit von ca. 0,5% auf; die blutgerinnung wird nicht beeinträchtigt. bei der infusion großer albuminmengen kann es zu einer senkung des ionisierten plasmakalziums kommen. > dextrane. dextrane sind hochmolekulare lineare polysaccharide mit vereinzelten seitenketten, gelöst in physiologischer kochsalzlösung. sie werden entsprechend ihrer molekularmasse entweder direkt (mg <50.000) oder nach enzymdegradation, bevorzugt renal, eliminiert. durch den hohen kolloidosmotischen druck füllt die 10%ige dextran-40-lösung den intravasalraum durch einen ausgeprägten einstrom aus dem interstitium auf, was im schock mit gestörter mikrozirkulation erwünscht, bei einem depletierten extrazellulärraum jedoch eher unerwünscht ist. dextran 40 reduziert die geldrollenbildung der erythrozyten, und es soll auch die gewebeoxygenierung verbessern. hydroxyäthylstärkepräparationen. im deutschsprachigen raum ist hydroxyäthylstärke (hes) sicherlich die am häufi gsten eingesetzte substanz zur therapie der hypovolämie. hes-lösungen werden aus kartoffel-oder maisstärke produziert innerhalb der letzten jahre ist es zur entwicklung deutlich verbesserter hes-präparationen (1. generation: 450/07; 2. generation: 70/05, 200/05, 200/0,62; 3. generation: 130/04; . tabelle 6.13) hinsichtlich intravasalem hes-abbau und nebenwirkungsprofi l -res-speicherung oder nephrotoxität (s. unten) -gekommen (boldt 2003 in einem cross-over-vergleich bei kritisch kranken erzielte die gabe von 100 ml albumin 25% eine zunahme des plasmavolumens 45 min nach infusionsende um 465 ml, nach 1 l ringer-laktatlösung waren es dagegen kurzfristig maximal 194 ml. wesentliche verbesserungen der hämodynamik und des o 2 -transports korrelieren eindeutig mit der plasmaexpansion. sie sind nach gabe kristalloider lösungen entweder gar nicht oder wesentlich schwächer als nach infusion kolloidaler lösungen nachweisbar. die wirksamkeit der einzelnen kolloidalen lösungen untereinander scheint vergleichbar (. tabelle 6.13), die wirkdauer der hes-lösungen dagegen länger als die der 5%igen albuminlösung. lungenfunktion. bei der diskussion um die ideale volumenersatzlösung spielt die potenzielle gefahr der auslösung eines lungenödems eine entscheidende rolle. verfechter des einsatzes kolloidaler lösungen führen an, dass kristalloide flüssigkeiten den kolloidosmotischen druck (kod) nachhaltig erniedrigen und damit diese gefahr hervorrufen. befürworter des einsatzes kristalloider lösungen fürchten dagegen bei der anwendung kolloidaler lösungen einen verstärkten abstrom kolloidosmotisch wirksamer moleküle durch die geschädigte alveolokapilläre membran ins interstitium, verbunden mit einem anstieg des extravaskulären kod und damit der gefahr der ausbildung oder verstärkung eines lungenödems. eindeutige vorteile hinsichtlich der organperfusion sind weder für kristalloide noch für kolloidale lösungen bei der volumensubstitution kritisch kranker überzeugend belegt (müller-werdan u. . als zielkriterien einer adäquaten flüssigkeitssubstitution des schocks dienen zunächst klinische parameter wie herzfrequenz, diurese und blutdruck, die sich in den physiologischen bereichen bewegen sollten. bei ausbleiben einer raschen hämodynamischen stabilisierung und bei patienten mit bereits vorbestehender eingeschränkter herzfunktion empfi ehlt sich das invasive monitoring mittels pulmonalarterienkatheter (7 abschn. 6.3.4). während der volumensubstitution muss mit dilutionsbedingtem abfallen von hämatokrit und hb (1-3 g/dl) gerechnet werden, die in der regel auch akzeptiert werden können: bei einem hämatokritwert von 30% liegt eine maximale o 2 -transportkapazität ohne gefahr einer gewebehypoxie vor. während der primären volumentherapie -in der initialen phase der hypovolämie -ist aus diesem grund ein hb-wert von 10-11,5 g/dl bzw. ein hämatokrit von 30-35% als ausreichend anzusehen. nachfolgend -bei normovolämie, erzielt durch volumengabe -ist eine substitution von erythrozytenkonzentraten erst ab hb-werten <8-10,0 g/dl indiziert > kritisch kranke. bei kritisch kranken ist eine mäßige anämie infolge eines okkulten blutverlustes und einer supprimierten erythropoese nicht selten. übereinstimmung besteht darüber, dass patienten mit akuter anämie und einem hämoglobinwert von ≤7 g/dl und darunter entsprechend den allgemeinen empfehlungen (simon et al. 1998 ) mit erythrozytentransfusionen substituiert werden sollten. dagegen konnte bei kritisch kranken mit hb-werten von 7-10 g/dl durch erythrozytentransfusionen die prognose nicht verbessert werden (hebert et al. 1999) . mögliche günstige effekte der transfusion könnten durch ungünstige reaktionen -wie rheologische störungen gealterter transfundierter erythrozyten, störungen der immunfunktion infolge der transfusion nicht leukozytengefi lterter erythrozyten (blumberg u. heal 1998 ) -wieder zunichte gemacht werden. sepsispatienten. die gabe eines erythrozytenkonzentrates erbrachte bei anämischen (hb <10 g/dl) sepsispatienten keine verbesserung der o 2 -utilisation, weder global (vo 2 ) noch regional (magentonometrie). beobachtet wurde eine 10%ige zunahme des linskventrikulären schlagarbeitsindex, aber auch eine ungünstige, 15%ige zunahme des pumonalvaskulären widerstands (fernandes et al. 2001) . bei patienten mit schwerer sepsis und septischem schock mit einem hämatokrit <30% und gleichzeitig einer auf <70% erniedrigten zentralvenösen sauerstoffsättigung (s cv o 2 ) wird die gabe von erythrozytenkonzentraten zur anhebung des hämatokrits auf ≥30% empfohlen, zumindest in der frühphase der sepsis innerhalb der ersten 6 h auf der notaufnahme (rivers et al. 2001) . ansonsten gilt die empfehlung, erythrozytenkonzentrate bei einem hämoglobinwert <7,0 g/dl (<70 g/l) zu geben und das hb auf einen wert von 7,0-9,0 g/dl anzuheben (dellinger et al. 2004 ). kritisch kranke. die verminderte erythropoetinbildung beim anämischen kritisch kranken und der hohe anteil von knapp 40% transfundierten intensivpatienten bilden die rationale für die erprobung einer erythropetingabe (epo) bei intensivpatienten: im rahmen einer studie mit 1300 intensivpatienten wurden wöchentlich -bis zu 4-mal -40.000 einheiten rhuepo bzw. placebo gegeben. die epo-gabe reduzierte den prozentsatz transfusionspfl ichtiger patienten von 60,4 auf 50,5% und die zahl der transfusionen um 19%, wobei der hb-anstieg mit 1,32 g/dl stärker ausfi el als in der placebogruppe (0,94 g/dl). die letalität der epo-gruppe war mit 14% nicht unterschiedlich im vergleich zu der der placebogruppe (corwin et al. 2002) . der nutzen dieser epo-gabe bei intensivpatienten wird z. z. kritisch gesehen (eckardt 2003) . sepsispatienten. bei patienten mit schwerer sepsis wird erythropoetin zur behandlung einer anämie nicht empfohlen (dellinger et al. 2004 ). mit ausnahme des hypovolämisch-traumatischen schocks mit versorgungsengpässen gibt es bei allen anderen schock-formen z. z. für den einsatz hyperton-onkotischer lösungen keine indikation (meier-hellman u. burgard 2004). im wesentlichen sind es die katecholamine dobutamin, dopamin, noradrenalin und adrenalin (. tabelle 6.14) und die phosphodiesterasehemmstoffe amrinon, milrinon und enoximon, (. tabelle 6.15), deren positiv-inotrope wirkung ausgenutzt werden kann . sie werden eingesetzt, um die herz-kreislauf-schädigung zu kompensieren und damit die durchblutung und die o 2 -versorgung der vitalorgane sicherzustellen. bei den klinischen symptomen eines kardiogenen schocks ist dobutamin (positiv-inotrope wirkung, mittel der 1. wahl bei > akutem myokardinfarkt; 2-20 µg/kgkg/min) und ggf. zusätzlich noradrenalin (positiv-inotrope und vasokonstriktorische wirkung; 0,05-0,3 µg/kgkg/min) indiziert; burchardi et al. 2000; . es handelt sich dabei um eine symptomatische, nicht um eine kausale therapie. bei vergleichbarer positiv-inotroper wirkung beeinfl ussen katecholamine herzfrequenz, blutdruck und gefäßwiderstand sowie den linksventrikulär-enddiastolischen füllungsdruck am gesunden herzen in unterschiedlicher weise (. tabelle 6.14; burchardi et al. 2000; : adrenalin wirkt am stärksten, noradrenalin am wenigsten positiv-chronotrop. vor allem noradrenalin erhöht den gefäßwiderstand und damit den blutdruck; in höheren konzentrationen tun dies allerdings auch dopamin und adrenalin. bei sepsis und schock können desensibilisierungsprozesse und toxin-und mediatorschädigungen das ansprechen auf katecholamine ganz erheblich beeinträchtigen (7 abschn. 6.4.8). . dosisbereich ( (hochman et al. 2000) . fragt man sich dann aber, wie denn die wirksamkeit der katecholamintherapie gesichert sei, so wird man enttäuscht: weder für den kardiogenen noch für den septischen schock gibt es hinsichtlich einer möglichen letalitätssenkung evidenzbasierte daten! lediglich für die behandlung der akuten herzinsuffi zienz fi ndet sich in der literatur eine metaanalyse, die sich mit der wirksamkeit von intravenös applizierbaren, über den adrenergen signalweg wirksamen inotropen substanzen -vorwiegend dobutamin, dopamin, dopexamin und phosphodiesterasehemmer -beschäftigt (thackray et al. 2002) . das ergebnis der kleinen studien mit geringer statistischer aussagekraft (21 studien mit insgesamt 632 patienten) ist enttäuschend: nicht eine letalitätssenkung, sondern eine im trend höhere sterblichkeit wurde bei dem einsatz der inotropen substanzen gefunden (odds-ratio 1,50; 95%iges konfidenzintervall 0,51-3,92). wegen der potenziell unerwünschten und gefährlichen nebenwirkungen der katecholamintherapie -wie die infl ammationsinduktion sowohl systemisch als auch im herzen selbst schwertz et al. 2004 ) -sollte man beim einsatz von katecholaminen im schock eher zurückhaltend sein. während dobutamin den linksventrikulär-enddiastolischen druck entweder unbeeinfl usst lässt oder ihn sogar geringfügig senkt, wird er durch dopamin meistens etwas gesteigert. die ursache dafür dürfte ein erhöhter venöser rückstrom durch eine α-adrenozeptorvermittelte venokonstriktion sein. bei der volumensubstitution des septischen schocks mit dobutamin können deshalb größere flüssigkeitsmengen erforderlich werden als im falle des dopamins müller-werdan u. werdan 2004 ; task force of the american college of critical care medicine, society of critical care medicine 1999). dobutamin ist das katecholamin der wahl zur therapie der eingeschränkten pumpfunktion der septischen kardiomyopathie! dagegen ist nach der empfehlung einer deutschen expertenkommission dopamin kein katecholamin der ersten wahl bei der therapie des septischen schocks in kontrollierten studien konnte durch dopamin in »nierendosis« (0,5-2-3 µg/kgkg/min) keinerlei nephroprotektiver effekt und auch keine prognoseverbesserung dokumentiert werden. der routinemäßige einsatz von niedrigdosiertem dopamin zur nephroprotektion bei schock-, mods-und sepsispatienten kann demzufolge nicht empfohlen werden dellinger et al. 2004; . bei patienten mit septischem schock zeigen katecholamine (dobutamin) eine geringere positiv-inotrope wirkung als bei patienten mit sepsis ohne schock (silverman et al. 1993) . diese katecholamintoleranz ist zumindest partiell auf eine dysregulation des β-adrenozeptor-adenylatzyklase-systems zurückzuführen (down-regulation der β 1 -adrenozeptoren; hochregulation der inhibitorischen g-proteine), hervorgerufen durch endogene und pharmakologisch applizierte katecholamine, sowie durch zytokine u. a. sepsismediatoren (. abb. 6.13a,b; müllerwerdan et al. 1996; reithmann et al. 1993; silverman et al. 1993) . sie lässt sich zumindest partiell durch eine steigerung der katecholamindosis ausgleichen. da diese β 1 -adrenozeptor-adenylatzyklase-desensibilisierung jedoch alle am myokardialen β 1 -adrenozeptor angreifenden katecholamine in gleichem maße betrifft, resultieren daraus keine differenzialtherapeutischen konsequenzen. über den erfolgreichen einsatz von β-rezeptorenblockern zur verbesserung der katecholaminansprechbarkeit wurde bisher beim patienten mit schock noch nicht berichtet. auch die gefäße zeigen im septischen schock eine katecholamintoleranz mit einer abgeschwächten bis fehlenden vasokonstriktion auf α-adrenozeptoragonisten. in diesem falle scheint jedoch nicht die im tierexperiment bei sepsis und endotoxinämie gefundene abnahme der zahl der gefäß-α-adrenozeptoren die entscheidende rolle zu spielen, sondern vielmehr das vermehrt gebildete stickoxid. durch hemmstoffe der stickoxidsynthase lässt sich im sepsisund endotoxin-tiermodell die stark abgeschwächte vasokonstriktorische katecholaminwirkung wieder restaurieren (. abb. 6.13a,b). die zahl der gefäß-β 2 -adrenozeptoren ist im tierexperiment bei sepsis und endotoxinämie als nicht verändert beschrieben. insbesondere bei kardiogenem schock nach herzoperationen scheinen sich phosphodiesterasehemmstoffe (pde-hemmstoffe; . tabelle 6.15) als katecholaminbegleittherapie zu bewähren . bei akuter herzinsuffi zienz konnte der pde-hemmer milrinon in einer randomisierten studie lediglich bei patienten mit nichtischämischer herzinsuffi zienz, nicht jedoch im gesamtkollektiv eine letalitätssenkung erzielen, wohingegen bei patienten mit akuter ischämischer herzinsuffi zienz sogar eine letalitätserhöhung erwartet werden muss (cuffe et al. 2002) . digitalis spielt bei sinusrhythmus in der therapie des kardiogenen schocks als inotropikum keine wesentliche rolle; da-> gegen ist es bei tachykardem vorhoffl immern und vorhoffl attern zur frequenznormalisierung bei schockpatienten das antiarrhythmikum der wahl. als neues therapieprinzip bei akuter herzinsuffi zienz scheint der kalziumsensitzer levosimendan erfolgreich (follath et al. 2002; kivikko et al. 2003; moiseyev et al. 2002) . levosimendan (in deutschland nicht zugelassen) sensibilisiert kalziumabhängig troponin c für kalzium und verbessert damit die kontraktilität des herzmuskels, wobei die diastolische relaxation entweder unbeeinfl usst bleibt oder sogar verbessert wird. durch öffnung atp-sensitiver kaliumkanäle wirkt levosimendan darüber hinaus vasodilatierend. bei akuter herzinsuffi zienz (herzindex 1,9 l/min/m 2 ) war levosimendan (bolus von 24 µg/kgkg über 10 min, danach 0,1 µg/ kgkg/min für 24 h) hämodynamisch effektiver als dobutamin (180-tage-letalität in der levosimendangruppe: 26%, in der dobutamingruppe 38%; follath et al. 2002) . ähnlich günstige effekte von levosimendan wurden für patienten mit akutem herzinfarkt und linksherzinsuffi zienz beschrieben (moiseyev et al. 2002) . zur senkung der vor-und nachlast dient bei akutem herzinfarkt in 1. linie nitroglyzerin und in 2. linie nitroprussidnatrium. nitroglyzerin wird in einer infusionsdosierung von 0,3-0,5-4 µg/kgkg/min gegeben; die nitroprussidnatriuminfusi-on wird mit 0,3 µg/kgkg/min begonnen und bis zum maximal erwünschten effekt alle 2 min bis zu einer dosis von 1-6 µg/ kgkg/min gesteigert; sie kann mit positiv-inotropen pharmaka kombiniert werden (alpert u. becker 1993) . nesiritide, das endogene b-typ-natriuretische peptid (in deutschland nicht zugelassen), senkt bei dekompensierter herzinsuffi zienz den pulmonarkapillardruck rascher und effektiver als nitroglyzerin. im vergleich zur behandlung mit dobutamin scheint nesiritide (i.v.-bolus 0,3 µg/kgkg, anschließend infusion mit 0,015 µg/kgkg/min bzw. i.v.-bolus 0,6 µg/kgkg, anschließend infusion mit 0,030 µg/kgkg/min) rascher eine rekompensation zu bewirken und möglicherweise sogar die prognose zu verbessern (silver et al. 2002 klinzing et al. (2003) infundierten vasopressin in einer dosis von 0,06-1,8 u/min, um die gabe von noradrenalin vollständig beenden zu können. ihre ergebnisse zeigen eine deutliche umverteilung des intestinalen blutfl usses zu lasten der magenschleimhaut. im vergleich zu anderen arbeiten wurde allerdings in dieser studie vasopressin nicht im sinne einer substitution bei vasopressinmangel, sondern in einer sehr hohen dosierung eingesetzt. dieselbe arbeitsgruppe hat auch die effekte einer niedrig dosierten (0,04 u/kgkg/h) vasopressininfusion auf die globale und intestinale hämodynamik untersucht. in dieser untersuchung wurden 12 patienten im septischen schock (noradrenalinbedarf: 0,13-1,4 µg/kgkg/min) mit arginin-vasopressin behandelt. ein adäquate splanchnikusperfusion blieb trotz eines signifi kantem abfalles des herzindexes erhalten. allerdings kam es auch bei dieser niedrigen dosierung zu einer redistribution des blutfl usses zu lasten der gastralen mukosa. auch eine niedrigdosierte vasopressintherapie kann mit einer bedeutsamen gastrointestinalen minderperfusion einhergehen. es gibt hinweise darauf, dass eine restriktive flüssigkeitstherapie kombiniert mit der infusion von vasopressin bei der behandlung einer unkontrollierbaren intraabdominellen blutung zu einer verbesserung der überlebensrate führt. wer dieses -im tierexperiment überaus erfolgreiche -konzept in die praxis einführen will, muss jedoch wissen, dass es zur zeit noch keine klinischen beweise seiner wirksamkeit gibt. beim septischen schock besteht ein vasopressinmangel. in mehreren untersuchungen konnte gezeigt werden, dass vasopressin sowohl in niedriger als auch in hoher dosierung bei septischen patienten zu einer deutlichen stabilisierung der hämodynamik führt. signifi kante nebenwirkungen traten dabei nicht auf. allerdings gibt es andere untersuchungen, in denen störungen der intestinalen und der hepatischen perfusion und generell der mikrozirkulation nach vaso-> pressininfusion nachgewiesen wurden. solange es so deutlich widersprüchliche erkenntnisse gibt, kann unseres erachtens die gabe von vasopressin im septischen schock nicht generell empfohlen werden, auch wenn die surviving sepsis campaign (. tabelle 6.20; dellinger et al. 2004 ) bei therapierefraktärem septischem schock die zusätzliche gabe von vasopressin (0,01-0,04 u/ml) für vertretbar ansieht. der kardiogene schock führt häufi g zu lungenstauung/lungenödem und zum prärenalen nierenversagen und damit zur oligurie. neben der kreislaufstabilisierung können diuretika und hämofi ltration erforderlich werden (7 abschn. 6.5.4). das im schock via induktion der inos überschießend produzierte stickoxid (no) zeigt neben seinen erwünschten wirkungen im rahmen der infektabwehr unerwünschte deletäre effekte (7 abschn. 6.2.6). insofern erscheint die blockade der überschießenden no-produktion mittels inos-hemmer (argininanaloga wie l-name) sinnvoll. klinische ergebnisse zu diesem therapieprinzip liegen für den septischen und den kardiogenen schock vor. gesicherte relevanz besitzen diese substanzen bei kardiogenem und bei septischem schock. kardiogener schock. der einsatz von thrombolytika und weiteren gerinnungsaktiven substanzen im kardiogenen schock wird in 7 abschn. 6.6.1 besprochen. gp iib/iiia-rezeptor-antagonisten scheinen neben ihrer thrombozytenaggregationshemmenden auch antiinfl ammatorische eigenschaften zu besitzen (bonz et al. 2002; köster et al. 2003; lincoff et al. 2001) , deren therapeutischer stellenwert aber noch nicht ausreichend abgeschätzt werden kann. nach größeren notfalloperationen muss in ca. 8% der fälle mit dem auftreten eines einorganversagens, in 4% mit einem zweiorganversagen, in 2% mit einem dreiorganversagen und in 1% mit einem vierorganversagen gerechnet werden; lungen-, nieren-und leberversagen sind dabei mit 7-9% etwa gleich häufi g. die prognose der patienten mit mods ist um so ungünstiger, je mehr organe geschädigt sind und je länger das organversagen anhält. die sterblichkeit nach 1-, 3-und 7-tägiger dauer eines einorganversagens liegt bei 20, 30 bzw. 40%, im falle eines zweiorganversagens bei 50, 60 bzw. 70%, und bei einem dreiorganversagen bei 80, 90 bzw. fast 100%. die aufgeführten zahlen gehen auf untersuchungen zurück, die vor 25 jahren durchgeführt worden sind. bei der ungünstigen prognose des mods hat sich seit 1973 mit einer berichteten letalität von 94% bis 1994 mit 60% zwar eine verbesserung gezeigt, die sterblichkeit ist jedoch weiterhin sehr hoch und weitgehend unabhängig von der art des geschädigten vitalorgans. ein lebensalter >65 jahre erhöht die letalität auf das doppelte. die mediatoren-/zytokin-hypothese (. abb. 6.2, 6.3 und 6.6) postuliert eine exzessive oder prolongierte produktion von zytokinen durch entzündungszellen, z. b. granulozyten und makrophagen, sowie durch dazu fähige körperzellen, z. b. kardiomyozyten. zu diesen zytokinen gehören interleukin 1, tumornekrosefaktor α, interleukin-6, das auch antiinfl ammatorische teilwirkungen hat, interleukin-8 u. a. diese zytokine induzieren die produktion fi naler mediatoren wie stickoxid, arachidonsäuremetaboliten, bradykinin und histamin, die neutrophile granulozyten und endothelzellen aktivieren und damit die gewebeschädigung induzieren. die induktion dieser überschießenden infl ammation kann sowohl durch infektiöse stimuli (infektion, sepsis) als auch durch nichtinfektiöse stimuli (sirs; 7 abschn. 6.1) induziert werden. die mikrozirkulations-und ischämie-/reperfusionshypothese propagiert die ischämie und/oder die gefäßendothelschädigung als ursache des mods, mit inadäquater o 2 -versorgung von geweben und zellen (alleinige ischämie), mit einer ischämie-/reperfusionsschädigung und der generie-! rung von toxischen o 2 -radikalen und/oder gewebeschädigung infolge der endothel-leukozyten-interaktion. diese hypothese favorisiert auch die annahme mehrerer toxischer stimuli in sequenz als auslöser des mods (»two-hit model of mods«). diese hypothese geht von der darmwandtranslokation von bakterien und endotoxinen als mods-ursache aus. bei patienten mit septischem und nichtseptischem mods fi ndet sich eine erhebliche autonome dysfunktion (»uncoupling of biological oscillators«; godin u. buchman 1996), die sich z. b. als einschränkung der herzfrequenzvariabilität messen lässt (. abb. 6.14a-d). im gegensatz zur autonomen dysfunktion herzkranker mit gesteigerter sympathikusaktivierung scheint diejenige des mods-patienten von einer abschwächung sowohl der sympathikus-als auch der parasympathikusaktivität geprägt zu sein (heinroth et al. 1999 ob es allerdings tatsächlich nur eine einheitliche ursache eines einheitlichen mods gibt, muss z. z. noch offen bleiben! viel wahrscheinlicher ist eine multifaktorielle mods-genese unter beteiligung der aufgeführten mechanismen. die komplexität des mods macht es verständlicherweise schwierig, den prognosebestimmenden schweregrad dieses krankheitssyndroms zu beschreiben. score-systeme stel-> len eine möglichkeit dazu dar (graf et al. 2003) . zwei versuche dieser art sollen hier vorgestellt werden: der schweregrad-der-erkrankung-score apache ii (»acute physiology and chronic health evaluation«) und der sepsisbezogene organversagen-score (sofa-score). in verschiedenen bereichen der medizin werden score-systeme seit längerem zur quantitativen erfassung von befunden eingesetzt. so dient z. b. die glasgow-coma-scale (. tabelle 6.17) zur abschätzung des schweregrades einer bewusstseinstrübung. bei patienten einer intensivstation können score-systeme eingesetzt werden, um objektive, quantifi zierbare parameter zu gewinnen zur (graf et al. 2003; scores (wie der schweregrad-der-erkrankung-score apa-che ii oder der sepsis-score nach elebute u. stoner) kön-f f f f nen am patientenbett innerhalb von 5-10 min mit einem auf einem mikrocomputer installierten programm einfach bestimmt und dokumentiert werden (müller. fortentwicklungen mit noch besserer prognostischer aussagekraft wie der mpm-score, der apache-iii-score und das supportsystem bedürfen noch der praktikabilitätsumsetzung und haben noch keine sehr weite verbreitung (graf et al. 2003; janssens et al. 2002) . bei einem schweregrad-der-erkrankung-score wie dem apa-che-ii-score (knaus et al. 1985; . abb. 6.15) korreliert die score-höhe mit der letalität, und zwar sowohl des gesamtkollektivs kritisch kranker als auch bestimmter subkollektive (z. b. patienten mit sepsis). ein mögliches problem bei der anwendung von score-systemen stellt jedoch die abhängigkeit der score-werte von der grundkrankheit dar. zusätzlich zu den bereits bestehenden organversagen-scores wurde von der europäischen intensivmedizinischen gesellschaft der sofa-score (»sepsis-related organ failure assessment score«) entwickelt (. tabelle 6.17). er erfasst die wichtigsten organdysfunktionen mit jeweils einem einzelnen pa-tabelle 6.17. schweregradklassifi zierung des mods-sofa-score. (nach vincent et al. 1996) . sepsisbezogener organversagen-score (sofa-score) punktzahl augen öffnen: 4 (spontan), 3 (bei aufforderung), 2 (bei schmerz), 1 (nicht); beste motorische antwort: 6 (gezielt nach aufforderung), 5 (gezielt nach schmerz), 4 (ungezielt nach schmerz), 3 (beugemechanismen), 2 (streckmechanismen), 1 (keine); verbale antwort: 5 (orientiert), 4 (verwirrt), 3 (inadäquat), 2 (unverständlich), 1 (keine) rameter und teilt den schweregrad der organdysfunktion entsprechend der abweichung dieses parameters von der norm ein. der sofa-score wird zunehmend auf bei nichtsepsispatienten zur abschätzung des schweregrades des mutiorganversagens eingesetzt, auch bei kardiologischen patienten. auch die prognose von patienten mit herzerkrankungen auf einer coronary care unit (ccu) kann mit score-systemen prognostiziert werden (7 abschn. 6.5.2): die aussagekraft des initial erhobenen saps-ii-score (»simplifi ed acute and physiology score«) ist bei ccu-patienten mindestens so prägnant wie bei patienten auf einer intensivstation (. abb. 6.16a,b; schuster et al. 1997) . dies unterstreicht eindrücklich, dass nicht nur die schwere der herzerkrankung, sondern v. a. das daraus resultierende multiorgandysfunktionssyndrom über das überleben des herzpatienten entscheidet. bei patienten nach herzoperationen erlaubt der apache-ii-score die identifi zierung der patienten mit eskalierendem systemischen infl ammationsreaktionssyndrom nach kardiopulmonalem bypass (eskalierendes cpb-sirs; . tabelle 6.4). es ist charakterisiert durch eine -im vergleich zum unkomplizierten postoperativen verlauf -überschießende systemische entzündungsreaktion infolge des operationstraumas und des einsatzes des kardiopulmonalen bypass, mit hohen tumornekrosefaktor-α(tnf-α)-und tnf-rezeptor-, leukozytenelastase-und neopterinplasmaspiegeln als ausdruck einer leukozyten/makrophagen-aktivierung, einem sepsisähnlichen multiorgandysfunktionssyndrom mit myokarddepression und hoher letalität (müller. dieses nur bei wenigen patienten (<10%) auftretende eskalierende cpb-sirs kann bereits am ersten postoperativen tag anhand eines apache-ii-score≥24 identifi ziert werden (. abb. 6.17). aufgrund der ergebnisse von beobachtungsstudien in den zeiträumen 1988 zeiträumen -1990 zeiträumen und 1996 pathophysiologie nach kreislaufschock kann es in abhängigkeit von der schockform und der zugrunde liegenden erkrankung zu 2 formen der lungenschädigung mit daraus resultierender insuffi zienz kommen: ein kardiogen bedingter kreislaufschock induziert primär eine erhöhung des lungenkapillardrucks, während die übrigen schockformen eine gesteigerte permeabilität der lungenkapillaren bedingen (engelmann 2000a ). das lungenödem bei erhöhtem kapillardruck hat als häufi gste ursache einen gesteigerten hydrostatischen druck in den lungenkapillaren, der wiederum in erster linie folge eines linksherzversagens ist. die filtration von flüssigkeit und protein wird als folge der erhöhten kapillarpermeabilität so gesteigert, dass der abtransport über die lymphwege bei weitem nicht ausreicht, um dies zu kompensieren. aufgrund der gestörten barrierefunktion kann auch kein nennenswerter osmotischer druckgradient aufgebaut werden, so dass sich diese ödemform mit rascher progredienz entwickelt. die störung der barrierefunktion des endothels wird nach gegenwärtiger auffassung durch mediatoren ausgelöst. die uniformität der pulmonalen reaktion bei sehr unterschiedlichen klinischen ausgangskonstellationen wird dabei > auf das limitierte repertoire des organismus an effektorsystemen zurückgeführt. es wird postuliert, dass alle formen des kreislaufschocks, die zu einem ards führen, letztlich ein ischämie/reperfusionssyndrom darstellen, wobei endotoxineinschwemmung, gewebehypoxie, makrophagen-und leukozyteneinwanderung, -adhäsion (mittels adhäsionsmolekülen) und -aktivierung mit freisetzung von zahlreichen entzündungsmediatoren als dominierende faktoren angesehen werden. bei 50% aller schockpatienten fi ndet sich eine akute respiratorische insuffi zienz. die frühesten veränderungen der lungenfunktion im rahmen eines kreislaufschocks basieren auf reaktionen des zentralen atemantriebs oder der atemmuskulatur. sowohl der gesteigerte atemantrieb infolge der stimulation der pulmonalen j-rezeptoren und der chemorezeptoren des karotissinus als auch die minderperfusion des medullären atemzentrums führen zur steigerung des atemminutenvolumens (tachypnoe, hyperpnoe), zur hypokapnie und zur initialen respiratorischen alkalose. ventilations-perfusions-störungen infolge des gesteigerten atemminutenvolumens bei gleichzeitiger reduktion des herzzeitvolumens können die folge sein. der lungenwiderstand ist initial unverändert oder nur minimal erhöht, falls nicht bereits schockbedingt eine arterielle hypoxämie besteht. die erhöhte atemarbeit bei gleichzeitiger minderperfusion der atemmuskulatur und des zwerchfells kann zur frühzeitigen respiratorischen insuffi zienz führen. falls der schockzustand nicht rasch behoben werden kann, sind kardiales lungenödem bzw. ein ards zu befürchten. zur klinik des lungenödems mit erhöhtem kapillardruck (kardiales lungenödem) 7 kap. 4.2. das lungenödem bei erhöhter kapillarpermeabilität bedingt ein klinisches syndrom, für das der begriff des »adult/acute respiratory distress syndrome« geprägt worden ist. die letalität des ards beträgt nach wie vor ca. 50-60%, wobei fortschritte auf dem gebiet der beatmungstechniken (engelmann 2000a, b) ten patienten mit ards nach unterschiedlichen auslösern zu einer vermehrten mikrobiellen belastung der lunge, für die im wesentlichen eine retrograde keimaszension aus dem gesamtintestinaltrakt mit mikroaspirationen in verbindung mit gestörten host-defense-mechanismen der lunge verantwortlich gemacht wird. in abhängigkeit von der dauer des ards kommt es zunehmend zu nosokomialen pneumonien, die bei über 10 tägiger beatmung mehr als 70% der ards-patienten betreffen können. die übergänge zwischen pneumonie und ards sind fl ießend und entziehen sich häufi g einer exakten klinischen defi nition (american thoracic society et al. 1999 bei zentraler atemdepression und bei pulmonal bedingter respiratorischer insuffi zienz mit erniedrigtem po 2 und erhöhtem pco 2 ist eine endotracheale intubation und eine assistier-> > te oder kontrollierte beatmung angezeigt (artigas et al. 1998; lehmann et al. 2003; seige et al. 2001) . für den beatmungspfl ichtigen patienten mit kardiogenem schock wurde in einer studie eine letalität von 51% angegeben und als wesentliche prognostische risikofaktoren ein apache-ii-sore >29, ein serumkreatinin >180 µmol/l und eine linksventrikuläre auswurffraktion <40% gefunden (lesage et al. 2004 vorbedingung für prophylaxe und therapie (kierdorf 2001 der routinemäßige einsatz von niedrigdosiertem dopamin zur nephroprotektion bei schock-, mods-und sepsispatienten kann nicht empfohlen werden (7 abschn. 6.4.7). diese substanz ist für die verbesserung der nierendurchblutung besonders attraktiv. ein signifi kanter überlebensvorteil durch die gabe von diuretika bei intensivpatienten mit akutem nierenversagen konnte bisher nicht gezeigt werden. eine retrospektive untersuchung erbrachte sogar im zusammenhang mit einer diuretikagabe eine letalitätssteigerung und eine erhöhung der inzidenz einer terminalen niereninsuffi zienz (mehta et al. 2002 bei freisetzung von chromoproteinen (hämolyse, myolyse), bei paraproteinämie und aminoglykosidinduziertem anv wirkt eine alkalisierung mit bikarbonat protektiv. sie wurden bislang unter der vorstellung, eine zelluläre schädigung zu verhindern oder abzuschwächen, bei folgenden formen des anv zur nephroprotektion eingesetzt: bei ischämieinduziertem anv nach nierentransplantation und bei nephrotoxininduziertem anv (kontrastmittel, aminoglykoside). zur differenzialtherapie stehen verschiedene verfahren zur verfügung: hämofi ltration, hämodiafi ltration, high-fl ux-dialyse, ultrafi ltration, plasmapherese, hämoperfusion (kierdorf 2001). bei der hämofi ltration liegt die standardhämofi ltrationsrate im anv bei 1-2 l/h. nicht bestätigt hat sich die hoffnung, damit relevante mengen deletärer mediatoren eliminieren zu können: zwar können immunmodulatorische substanzen wie zytokine und kardiotoxische faktoren hämofi ltrationsmembranen passieren, ein wesentlicher abfall der plasmakonzentrationen dieser substanzen scheint jedoch dadurch nicht zustande zu kommen, obwohl eine vorübergehende kreislaufstabilisierung -messbar als anstieg des systemischen gefäßwiderstandes -beschrieben ist (hoffmann et al. 1996 (hoffmann et al. , 1999 . auch die »high-volume-hämofi ltration« mit durchsatzraten von 6 l/h kann diesbezüglich trotz einsparung von vasopressoren kaum überzeugendere ergebnisse liefern (cole et al. 2001) . eine effi ziente endotoxinelimination bei patienten mit gramnegativer sepsis und mods sowie mit peritonitis ist mit dem matisse-adsorber möglich (reinhart et al. 2004 ). an makroporöse trägerkügelchen gebundenes immobilisiertes humanes serumalbumin bindet während eines hämoadsorptionzyklus endotoxin und bewirkt auf diese weise einen deutlichen abfall der serumendotoxinkonzentration. in einer phase-ii-studie mit 145 patienten mit vermuteter gramnegativer sepsis, davon 104 mit peritonitis, führte die tägliche endotoxinadsorption in den ersten 4 tagen nach diagnosestellung im trend zu einer vorübergehenden besserung des mods (stärkerer abfall des apache-ii-und des sofa-score während der ersten 4 tage) und zu deutlicheren senkung des serumendotoxins; die letalität -nicht primäres zielkriterium (!) -war mit und ohne endotoxinabsorptionstherapie nicht unterschiedlich (reinhart et al. 2004) . für aphereseverfahren (formica et al. 2003; kellum 2003; samtleben et al. 1998; shehata et al. 2002; stegmayr 2001; stegmayr et al. 2003) und die gekoppelte plasmafi ltrationadsorption sind bei patienten mit septischem mods günstige effekte bisher nur in fallberichten und in relativ kleinen kontrollierten studien beschrieben. die schwere leberinsuffi zienz ist charakterisiert durch eine, möglicherweise stickoxidbedingte, hyperdyname systemische kreislaufsituation mit erhöhtem herzzeitvolumen und erniedrigtem systemischem gefäßwiderstand. reaktiv dazu kommt es zur konstriktion der nierenarterien und damit zur na + -retention und aszitesbildung. diese hochgradige renale vasokonstriktion bei leberschädigung wird durch eine aktivierung des renin-angiotensin-aldosteron-systems und eine noradrenalinfreisetzung bewirkt und als hepatorenales syndrom bezeichnet. es ist charakterisiert durch eine oligurie (<300 ml/24 h), einen anstieg des serumkreatinins trotz adäquatem blutdruck und eine erniedrigte urin-na + -konzentration von <10 mmol/ l. im initialstadium ist es reversibel, im weiteren verlauf treten jedoch tubulusschäden auf. eine gesicherte therapie ist nicht bekannt, ggf. muss hämodialysiert werden. obwohl neurone sehr ischämieempfi ndlich sind, kommt es durch die protektive autoregulation der hirndurchblutung erst in relativ späten schockstadien zu ausgeprägteren zerebralen durchblutungsstörungen. ohne vorbestehende zerebrovaskuläre insuffi zienz fi nden sich erst bei einem abfall des arteriellen mitteldrucks auf 50-60 mmhg irreversible störungen der ischämieempfi ndlichsten kortex-und rückenmarksareale. bereits vorher können jedoch reversible bewusstseinsstörungen in form von konfusionen bis zur bewusstlosigkeit auftreten, je nach grad der durchblutungsstörung, ggf. verstärkt durch begleitende störungen des säure-basen-und des elektrolythaushalts. das elektroenzephalogramm zeigt dabei unspezifi sche veränderungen (zauner et al. 2000) . septische enzephalopathie. die septische enzephalopathie ist charakterisiert als eine reversible dysfunktion des zentralnervensystems ohne erkennbare strukturelle schäden, als deren ursachen toxinwirkungen oder ein geändertes neurotransmittermuster diskutiert werden (papadopoulos et al. 2000) . sie kann sich bereits bei blutdruckwerten >60 mmhg manifestieren und geht mit einer erhöhten letalität einher (briegel 2003; eggers et al. 2003; green et al. 2004) . sie äußert sich als irritabilität, agitation, desorientiertheit, konfusion, stupor und koma. eine spezifi sche therapie der septischen enzephalopathie ist z. z. noch nicht etabliert. bei erfolgreicher behandlung der sepsis kann in der regel mit einer raschen besserung der septischen enzephalopathie gerechnet werden. über 50% aller patienten mit länger bestehender sepsis und multiorganversagen scheinen eine axonale sensorische und motorische neuropathie zu entwickeln, die charakterisiert ist durch abgeschwächte oder fehlende sehnenrefl exe und schlaffheit der extremitätenmuskulatur (hund 2001) . die hirnnerven sind intakt; im liquor fi ndet sich bei manchen patienten eine erhöhung des proteingehalts, die zellzahl ist normal. eine spezifi sche therapie ist nicht bekannt. eine retrospektive analyse (33 patienten mit multiorganversagen) erbrachte erste hinweise auf eine mögliche günstige wirkung einer frühzeitigen immunglobulin-gma-gabe bei der polyneuropathie kritisch kranker mit gramnegativer sepsis (mohr et al. 1997) ; als gesicherte therapie kann dieses konzept z. z. noch nicht empfohlen werden. septisches und nichtseptisches mods sind durch eine autonome dysfunktion geprägt, die sich als einschränkung der herzfrequenzvariabilität sowie der baro-und chemorefl exsensitivität bemerkbar macht, im ausmaß mit dem schweregrad des mods korreliert und auch bei intensivpatienten verlässlich und praktikabel messbar ist (schmidt et al. 2001; schmidt u. müller-werdan 2003) . eine gezielte therapie der autonomen dysfunktion des mods-patienten existiert bisher nicht. generalisierte muskelschwäche und abgeschwächte oder fehlende muskeleigenrefl exe kennzeichnen auch das klinische bild der myopathie des kritisch kranken (hund 2001) . diese ist durch eine typ-i-und v. a. durch eine typ-ii-faseratrophie gekennzeichnet und geht nur selten mit einer erhöhung der serumkreatinkinasewerte einher. differenzialdiagnostische schwierigkeiten kann die abgrenzung zur polyneuropathie des kritisch kranken (muskelbiopsie), zur myopathie durch glukokortikoide und muskelrelaxanzien sowie zur -sehr seltenen -septisch-metastatischen pyomyositis bereiten. das klinische bild von polyneuropathie und myopathie ist durch eine schwäche der extremitäten, hyporefl exie, verzögerte respiratorentwöhnung und durch eine komplikationsreiche, verlängerte motorische rehabilitiation mit erhöhter letalität gekennzeichnet. muskelrelaxanzien und kortikosteroide können zusätzliche schädigende effekte am neuromuskulären system hervorrufen, die sich als transiente neuromuskuläre blockade, axonale motorische neuropathie oder myopathie der dicken filamente zeigen. wegen der eingeschränkten klinisch-neurologischen beurteilung eines intensivpatienten kommt den elektrophysiologischen untersuchungen (elektromyo-und elektroneurographie), der messung der kreatinkinase im serum, der muskel-und nervenbiopsie, der liquorpunktion und gelegentlich der kernspintomographie eine große diagnostische und differenzialdiagnostische bedeutung zu. nach schwerem krankheitsverlauf fi nden sich bei mehr als 90% der patienten noch nach jahren muskelschwäche und neurologische defi zite (fletcher et al. 2003) . die schockverursachte sympathikusaktivierung manifestiert sich am herzen als tachykardie, selten bei hämorrhagischem schock als eine vagusvermittelte bradykardie, und in form von tachykarden supraventrikulären und ventrikulären rhythmusstörungen. bei koronarkranken patienten kann sie über die steigerung des myokardialen o 2 -verbrauchs (bei gleichzeitiger hypotonie) ein defi zit der koronarperfusion hervorrufen und damit eine myokardischämie provozieren. eine sympathikusvermittelte gefäßkonstriktion wird an den koronararterien durch die gefäßautoregulation (7 abschn. 6.2.3 und . tabelle 6.8) weitgehend verhindert. die sympathikusaktivierung ist auch für die bei manchen schockformen (septisch, hypovolämisch, traumatisch) beschriebene steigerung der myokardkontraktilität verantwortlich, die jedoch durch zirkulierende kardiodepressive schockfaktoren auch vermindert werden kann. die akute septische kardiomyopathie wird in den 7 abschn. 6.2.8 und 6.6.2 dargestellt. zur komplexen organdysfunktion des kreislaufs bei den verschiedenen schockformen 7 abschn. 6.2. im schock reagieren die splanchnikusgefäße auf die sympathikusaktivierung mit einer raschen und intensiven vasokonstriktion. demzufolge ist der darmtrakt sehr ischämiegefährdet; typische schädigungsmuster sind in . tabelle 6.16 aufgeführt. aufgepfropft auf die darmischämie können die in der reperfusionsphase gebildeten o 2 -radikale eine schädigung der darmbarriere bewirken (bahrami et al. 1998; stallmach u. zeitz 1998) . alle symptome treten nur inkonstant auf. übelkeit, erbrechen und schmerzen im rechten oberbauch gehen dem fieber häufi g voraus; die allerdings nur selten palpable raumforderung im rechten oberbauch sichert weitgehend die diagnose. laborparameter helfen diagnostisch häufi g nicht weiter; wegweisend ist die sonographie. die therapie besteht in der gallenblasenentfernung (letalität ca. 13%) oder in der perkutanen, transhepatische gallenblasenpunktion mit und ohne drainageeinlage (cholezystotomie). wenige stunden nach einem akuten stressereignis können erosionen und ulzera der schleimhaut vorwiegend im magenkorpus, weniger im antrum oder duodenum, auftreten. die pathophysiologie dieser läsionen ist komplex und unterscheidet sich von der der durch säure und helicobacter pylori hervorgerufenen ulzera. durch eine mukosale minderversorgung mit oxygeniertem blut als folge von minderperfusion oder hypoxie wird ein schleimhautschädigender circulus vitiosus, bestehend aus venöser stase, sludge, vasospasmus, gewebehypoxie, mediatorenfreisetzung, radikalbildung und autokongestion der gefäße in gang gesetzt. die magensäuresekretion ist bei den meisten kritisch kranken reduziert und nur bei patienten mit sepsis, verbrennungen, erhöhtem intrakraniellem druck oder schädel-hirn-trauma erhöht (ruß et al. 2005 ). eine gastrointestinale blutung des kritisch kranken kann 2 schweregraden zugeordnet werden (ruß et al. 2005) : zum einen die offenkundige blutung, nachgewiesen durch hämatemesis oder aspiration aus einer gastralen sonde, und zum anderen die klinisch relevante blutung, bestehend aus einer offenkundigen blutung mit arterieller hypotension und transfusionsbedarf von 2 erythrozytenkonzentraten. die rate der offenkundigen blutung liegt bei beatmeten intensivpatienten bei 9% und im gesamtkollektiv der intensivpatienten bei 4,4%. die häufi gkeit klinisch relevanter gastrointestinaler blutungen bei kritisch kranken auf der intensivstation ist in den letzten 20 jahren von bis zu 20% ohne prophylaxe auf z. z. ungefähr 1,5% gesunken. dafür sind neben der ulkusprophylaxe überwiegend die verbesserten intensivmedizinischen therapiemöglichkeiten verantwortlich. für das auftreten klinisch relevanter stressulkusblutungen im gesamtkollektiv der intensivpatienten konnten in einer großen prospektiven studie nur 2 der vermuteten risikofaktoren statistisch signifi kant bestätigt werden: in der vorbeugung einer stressulkusblutung des kritisch kranken sind h 2 -rezeptorantagonisten, sucralfat und antazida hocheffektiv gegenüber placebo (ruß et al. 2005) . bei einem ph-wert <4 ist der magensaft von 90% der intensivpatienten steril. dies gilt nur noch für 15% bei einem intragastralen ph-wert >4 unter säurehemmender medikation, bedingt durch eine ph-wert-abhängige kolonisation des magensaftes mit gramnegativen keimen. diese erreger können u. a. durch regurgitation und/oder weitere kolonisation des oropharyngealen raumes und anschließender mikroaspiration in das bronchialsystem gelangen und nosokomiale, besonders beatmungsassoziierte pneumonien hervorrufen. der vergleich von ranitidin (3-mal 50 mg i.v./tag) mit sucralfat (4-mal 1 g intragastral/tag) zeigte in der prophylaxe einer klinisch relevanten blutung bei für mehr als 48 h beatmeten intensivpatienten eine signifi kant niedrigere blutungsrate für ranitidin (1,7 vs. 3,8%). im gegensatz zu früheren untersuchungen und metaanalysen konnte kein signifi kanter unterschied der sterblichkeit (23,5 vs. 22,8%) und nur ein trend in der reduzierung der gesamtrate beatmungsassozierter pneumonien (19,1 vs. 16,2%) unter sucralfat nachgewiesen werden. die für h 2 -rezeptorantagonisten, antazida und sucralfat beschriebene wirksamkeit bei der stressulkusprophylaxe kritisch kranker gilt auch für die protonenpumpeninhibitoren, wobei für diese substanzgruppe ähnlich umfangreiche untersuchungen und analysen bisher nicht vorliegen (weitere dosierungsempfehlungen s. ruß et al.2005 ). die leber reagiert empfi ndlich auf hypotonie und minderperfusion. dennoch ist die »schockleber« -massive ischämische nekrosen mit sehr hohem transaminasenanstieg -ohne vorbestehende lebererkrankung ein seltenes ereignis (böker u. manns 1998). viel häufi ger dagegen ist eine zentrilobuläre schädigung mit einem milden anstieg der transaminasen, der ldh, des bilirubins und der alkalischen phosphatase. der transaminasenanstieg erreicht am 1.-3. tag sein maximum, er normalisiert sich über die folgenden 3-10 tage. trotz der hepatischen synthese von akute-phase-proteinen ist die proteinsynthese der leber im kreislaufschock eingeschränkt; dies trifft v. a. für präalbumin, albumin und gerinnungsfaktoren zu. auch nach erfolgreicher schockbehandlung kann die biliäre stase mit erhöhten werten für bilirubin und alkalische phosphatase lange persistieren. die septische hepatopathie imponiert klinisch durch den ikterus, funktionell ist die eingeschränkte syntheseleistung das entscheidende. eine spezifi sche therapie ist nicht bekannt. therapierefraktärer aszites, hepatorenales syndrom, hyponatriämie und hepatopulmonales syndrom können bei patienten mit chronischen lebererkrankungen und mods/ sepsis den sepsisverlauf komplizieren (ruß et al. 2005) . pathophysiologie mods, sirs und sepsis verursachen eine aktivierung des gerinnungssystems sowie eine initiale aktivierung und anschließende hemmung der fibrinolyse (huhle 2003; dempfl e 2003; riess 1998 )! diese veränderungen führen schließlich zur disseminierten intravasalen gerinnung (dic) -ausdruck einer mikroangiopathischen hämolyse, verbrauchsthrombozytopenie, verbrauch an gerinnungsfaktoren und mikrothromben. an der pathophysiologischen bedeutung der thrombozyten bei verschiedenen klinischen und experimentellen schockzuständen ist nicht mehr zu zweifeln. bei einer reihe von noxen, die direkt (z. b. biogene amine, thrombin usw.) oder indirekt (z. b. kollagen) nach freisetzung aus subendothelialen gefäßstrukturen auf die thrombozyten einwirken, kommt es zu deren aggregation. diese aggregation ist zunächst reversibel. ihr kann unter geeigneten bedingungen die spontane desaggregation folgen. untersuchungen an patienten mit quantifi ziertem schweregrad einer sepsis (elebute-score) und eines multiorganversagens (apache-ii-score) sprechen dafür, dass die sepsis einen hyperaggregiblen, aber noch reversiblen zustand des thrombozyten induziert (erhöhte fibrinogenrezeptoraktivität, gemessen als libs1-expression), während der zunehmende schweregrad des multiorganversagens zur irreversiblen thrombozytendegranulierung führt, erkenntlich an der verstärkten oberfl ächenexpression der thrombozytenadhäsionsmoleküle gmp-140 und thrombospondin mit zunehmendem schweregrad des multiorganversagens (gawaz et al. 1995) . diese normalerweise in den plättchengranula gespeicherten adhäsionsmoleküle verstärken durch ihre oberfl ächenaggregation die plättchenmikroaggregation und führen somit zur irreversiblen thrombozytendegranulation im sinne der dic. die dic ist klinisch durch die kombination von blutungen (petechien, purpura, ekchymosen, verstärkte blutungen nach gefäßpunktionen) und thrombosen (gangrän, akrale zyanose, hautnekrosen, tiefe venenthrombosen) charakterisiert (dempfl e 2003). sie fi ndet sich bei unterschiedlichen erkrankungen, wobei sepsis und septischer schock im rahmen des mods im vordergrund stehen. die dic bei gramnegativem schock (wo sie am häufi gsten gesehen wird) wird mit dem endotoxin als auslöser in verbindung gebracht (7 abschn. 6.2.5); die fulminant verlaufende dic bei meningokokkensepsis (waterhouse-friderichsen-syndrom), die mit sehr hohen endotoxinspiegeln einhergeht, liefert dafür ein gewichtiges argument. allerdings fi ndet sich die dic auch bei grampositiver sepsis, wie z. b. der pneumokokkensepsis. beim hämorrhagischen schock ist das auftreten einer dic ein seltenes ereignis; hier steht die verdünnungsthrombozytopenie nach adäquater volumensubstitution im vordergrund. abfall von thrombozytenzahl, fibrinogen und antithrombin, anstieg von ptt, prothrombinzeit, thrombinzeit, d-dimeren, fibrinmonomeren, thrombin-antithrombin-komplex, fibrinopeptid a und prothrombinfragmenten (f 1 + f 2 ). im sofa-score (. tabelle 6.17) dient die thrombozytopenie als maß für den schweregrad der gerinnungsstörung in der sepsis. kontrollierte studien zur prophylaxe der dic bei mods, schock und sepsis gibt es bisher nicht. die gesicherten therapieempfehlungen zur therapie der klinisch manifesten dic bei mods, schock und sepsis sind in der . übersicht 6.9 zusammengefasst (riess 1998; . gerinnungshemmende medikamente werden bei sepsis nicht nur bei klinisch manifester dic, sondern häufi g auch unabhängig davon eingesetzt (kujath et al. 2003; werdan et al. 2005) . dieses vorgehen beruht zum einen auf der vorstellung einer »prophylaxe« der ingangsetzung der pathogenetisch gefährlichen gerinnungskaskade. zum anderen wird zunehmend evident, wie eng gerinnungsaktivierung und proinfl ammation in der sepsis verknüpft sind; darauf beruht das konzept, mit der bremsung der gerinnungskaskade -v. a. der thrombinaktivierung -auch das überschießende proinfl ammatorische potenzial des körpers zu dämpfen. lange zeit war heparin ein nachdrücklich propagierter ansatz bei sepsis, wahrscheinlich aber ein wenig oder sogar nicht wirksamer und dazu noch ein potenziell gefährlicher. in den letzten jahren konzentriert sich das interesse v. a. auf den ersatz endogener inhibitoren einer unerwünschten generalisierung der gerinnungsabläufe, die in der sepsis erniedrigte serumspiegel zeigen und meist mit einer ungünstigen prognose verknüpft sind: antithrombin, protein c, protein s und gewebsthromboplastininhibitor (tissue-factor-pathway-inhibitor, tfpi). und schließlich darf nicht vergessen werden, dass in der sepsis thrombozytopenie und thrombozytendysfunktion ebenfalls gerinnungsprobleme machen können. wenig bekannt ist, dass thrombozyten auch mikrobiozide peptide sezernieren und über kontaktabhängige mechanismen zur bakterienabtötung beitragen können (. übersicht 6.10). in der prowess-studie (»effi cacy and safety of recombinant human activated protein c for severe sepsis) wurden patienten in der frühphase einer schweren sepsis (sepsis mit organversagen, . tabelle 6.4) eingeschlossen und entweder mit aktiviertem protein c (drotrecogin alfa aktiviert, xigris) oder mit placebo behandelt (bernard et al. 2001) . nach einschluss von 1690 patienten wurde die studie wegen der überlegenheit des verumpräparates vorzeitig abgebrochen: die 28-tage-letalität der patienten mit standardtherapie lag bei 30,8%, die der patienten mit aktiviertem protein c bei 24,7% (p =0,005). dies entspricht einer letalitätssenkung absolut von 6,1% und relativ von 19,4% (nnt 16). der günstige effekt war dabei unabhängig von der höhe des initialen plasma-protein-c-spiegels. nicht alle sepsispatienten scheinen in gleicher weise zu profi tieren: pneumonie, patientenalter >50 jahre, apache-ii-score ≥25, mehrorganversagen und schock sind faktoren, bei denen eine besonders erfolgreiche behandlung erwartet werden kann; die prognostisch günstige urosepsis und die nichtabominelle chirurgische sepsis scheinen dagegen eher weniger anzusprechen. demzufolge ist die indikation in den usa auf den erwachsenen patienten mit schwerer sepsis und einem hohem letalitätsrisiko (wie einem apache-ii-score ≥25) und in europa auf den erwachsenen sepsispatienten mit mindestens 2 organversagen ausgerichtet. der gestörte stoffwechsel bei patienten mit mods, schock und sepsis und die erforderlichen ernährungsmaßnahmen besitzen für die prognose des kritisch kranken eine wesentlichere bedeutung als lange angenommen . bei patienten mit mods ist -wie bei allen kritisch kranken -eine möglichst frühzeitige enterale ernährung anzustreben (ruß et al. 2005) . trotz des ausgeprägten katabolen zustands kritisch kranker und insbesondere der schock-, mods-und sepsispatienten kann der gesteigerte abbau nicht einfach durch ein mehr an zugeführten kalorien ausgeglichen werden, da diese patienten, insbesondere mit septischem schock, gar nicht in der lage sind, ein übermaß an kalorienangebot zu verwerten. in der an die akute katabole krankheitsphase anschließenden anabolen erholungsperiode muss dann durch eine überdurchschnittliche kalorienzufuhr dem gesteigerten bedarf rechnung getragen werden (ruß et al. 2005) . immunonutrition. der einsatz der immunonutrition -die enterale zufuhr von mehrfach ungesättigten fettsäuren, nukleotiden und arginin sowie die parenterale/enterale gabe von glutamin -zur verbesserung des immunstatus kritisch kranker scheint bei intensivmedizinischen risikopatienten -ards (pacht et al. 2003) , mods-und sepsisgefährdete patienten in der perioperativen phase -günstige effekte zu haben; bei manifester sepsis sprechen studiendaten eher für eine ungünstige wirkung (bertolini et al. 2003 , heyland u. samis 2003 suchner 2002) . welche »drüsen« sind mods-anfällig? derzeit richtet sich das »endokrinologische augenmerk« bei mods, schock und sepsis auf den insulin-glukose-stoffwechsel einerseits (7 abschn. 6.5.10) und die hypothalamus-hypophysen-nebennieren-achse (»hypothalamo-pituitary-adrenal axis«, hpa-achse) andererseits (beishuizen u. thijs 2003; werdan et al. 2005) . endotoxin kann die hpa-achse auf unterschiedlichen ebenen beeinfl ussen (beishuizen u. tijs 2003 über verschiedenene mechanismen kann es im septischen schock zu einer relativen nebennierenrindeninsuffi zienz kommen . bei diesen patienten wird in unerwünschter weise die antiinfl ammatorische wirkung des endogenes cortisols abgeschwächt. die folge ist ein vermindertes ansprechens des kreislaufs auf vasopressorische katecholamine und wahrscheinlich auch eine erhöhte sterblichkeit (annane u. cavaillon 2003) . im prolongiert verlaufenden septischen schock geht die negative feedbackkontrolle der hypothalamus-hypophysen-nebennierenrinden-achse (hpa-achse) verloren, mit einem verlust des pulsatilen sekretionsverhaltens des cortisols. ursache dafür sind eine tnf-bedingte sekretionshemmung von corticotropin-releasing-hormon und acth sowie eine abschwächung der acth-stimulierbarkeit der cortisolproduktion der nebenniererenrinde. infolge der down-regulation zellulärer glukokortikoidrezeptoren mit abnahme von rezeptorzahl und affi nität bildet sich im septischen schock eine glukokortikoidresistenz aus. wahrscheinliche ursache dafür ist eine hohe lokale konzentration proinfl ammatorischer zytokine, die über die bildung von transkriptionsfaktoren wie activator-protein 1 (ap1) und »nuclear factor kappa b« (nfκb) zu einer komplexbildung mit aktiviertem glukokortikoid-rezeptorelementen führt. da die glukokortikoid-rezeptorzahl in den gefäßen mit dem mittleren arteriellen blutdruck korreliert, trägt die glukokortikoid-down-regulation entscheidend zur hämodynamischen instabilität im septischen schock bei (zonghai et al. 2003) . bei zwei dritteln aller patienten mit septischem schock lässt sich anhand des acth-tests die diagnose einer relativen nebennierenrindeninsuffi zienz stellen. bei diesen patienten können stress-hydrocortison-dosen von 200-300 mg/tag die erworbene glukokortikoidresistenz überwinden und die proinfl ammation im septischen schock dämpfen. für den behandelnden intensivmediziner ist es beeindruckend, wie unter dieser hydrocortisongabe die katecholaminansprechbarkeit der gefäße wiederhergestellt und damit innerhalb weniger tage eine drastische einsparung von katecholaminen möglich wird (annane et al. 2002) . die in einer relativ kleinen studie mit 300 patienten gefundene letalitätssenkung durch hydrocortison bei patienten mit septischem schock und relativer nebennierenrindeninsuffi zienz (annane et al. 2002) wird z. z. in der europäischen corticus-studie mit geplanten 800 patienten überprüft. nicht nur bei septischem schock, sondern auch bei eskalierendem sirs nach herzchirurgie scheinen stressdosen von hydrocortison die überschießende infl ammationsreaktion zu dämpfen und die prognose zu verbessern (kilger et al. 2003 ). die hohe infektanfälligkeit kritisch kranker und insbesondere der patienten im schock ist folge einer komplexen schädigung des humoralen und zellulären immunsystems bei schock und sirs (. tabelle 6.4, . abb. 6.4a-c und 6.6). von den möglichkeiten einer infektions-/sepsisprophylaxe bei intensivmedizinischen risikopatienten sind neben allge-> meinen hygienemaßnahmen und der erzielung einer normoglykämie mittels hochdosierter insulingabe (7 abschn. 6.5.10) v. a. die möglichkeiten einer prophylaxe mit immunglobulinen zu nennen (werdan 2001b; werdan et al. 2005) . durch prophylaktische gabe von immunglobulinen (ig) kann das auftreten von infektionen bei verschiedenen intensivpatientenkollektiven gesenkt werden, v. a. bei »infektions-hochrisiko-patienten« und bei »infektions-hochrisiko-operationen« (werdan 2001b) : anerge patienten (ca. 8%) nach herzchirurgischen operationen mit der herz-lungen-maschine: die prophylaktische gabe von iviggma senkt die im vergleich zu normergen patienten höhere inzidenz postoperativer infektionen auf werte unter diejenige normerger patienten (kress et al. 1999 einheiten, schweres abdominelles oder retroperitoneales trauma mit transfusionsbedarf von mehr als 10 einheiten und intubationspfl ichtigkeit länger als 24 h. bei patienten mit schwerem trauma (injury severity score 16-50) senkt die prophylaktische gabe von ivigg (0,25 g/kgkg an den tagen 1,2,3,6) zwar die häufi gkeit von infektionen, insbesondere pneumonien (um mehr als 50%), die infektionsverursachte morbidität und letalität wird allerdings nicht vermindert (douzinas et al. 2000) . spezifi sche schockformen k. werdan, r. prondzinsky, m. buerke, u. müller-werdan unabhängig von der art des kardiogenen schocks (. tabellen 6.1 und 6.2) besteht die initialbetreuung des patienten mit kardiogenem schock (. übersicht 6.11) zunächst in der hämodynamischen stabilisierung und ausreichenden oxygenierung, dem adäquaten hämodynamischen monitoring und der möglichst raschen klärung der schockursache. anschließend lassen sich, soweit möglich, kausale behandlungsmaßnahmen einleiten, die im infarktbedingten myogenen pumpversagen ischämischer genese in der möglichst raschen wiedereröffnung des verschlossenen infarktgefäßes besteht (. abb. 6.18). die schockbehandlung ist ein wettlauf mit der zeit! jede »schockminute« erhöht das risiko des auftretens des prognosebestimmenden multiorgandysfunktionssyndroms (7 abschn. 6.5)! die behandlung des kardiogenen schocks kann in symptomatische (medikamentöse therapie, mechanische unterstützungssysteme) und in kausale (interventionelle oder chirurgische koronarreperfusion, verschluss von shunts oder rupturstellen) maßnahmen eingeteilt werden. ziel der medikamentösen, symptomatischen (!) behandlung (bengur u. meliones 1998; goldberg et al. 1999; hollenberg 2003; hollenberg et al. 1999) ist die steigerung der myokardkontraktilität und die situationsbezogene optimierung von vor-und nachlast (volumenentzug, vasodilatatoren, blutdruckstabilisierung), um so -gesteuert mittels invasivem monitoring -einen adäquaten herzindex und blutdruck und damit eine ausreichende perfusion der vitalorgane aufrechtzuerhalten (7 abschn. 6.4.1, cotter et al. 2003a ). im wesentlichen sind es die katecholamine dobutamin, dopamin, noradrenalin und adrenalin und die phosphodies-! terasehemmstoffe amrinon, milrinon und enoximon, deren positiv-inotrope wirkung ausgenutzt werden kann (7 abschn. 6.4.7; . tabellen 6.14 und 6.15). digitalis. digitalis spielt bei sinusrhythmus in der therapie des kardiogenen schocks als inotropikum keine wesentliche rolle; dagegen ist es bei tachykardem vorhoffl immern und vorhoffl attern zur frequenznormalisierung das antiarrhythmikum der wahl. levosimendan. als neues therapieprinzip bei akuter herzinsuffi zienz scheint der kalziumsensitzer levosimendan erfolgreich (7 abschn. 6.4.7). der kardiogene schock führt häufi g zu lungenstauung/lungenödem und zum prärenalen nierenversagen und damit zur oligurie. neben der kreislaufstabilisierung können diuretika und hämofi ltration erforderlich werden (7 abschn. 6.5.4). zur senkung der vor-und nachlast dient bei akutem herzinfarkt in erster linie nitroglyzerin und in zweiter linie nitroprussidnatrium (7 abschn. 6.4.7). bei dekompensierter herzinsuffi zienz ist nesiritide, das endogene b-typ-natriuretische peptid (in deutschland nicht zugelassen) ein neuer therapieansatz (7 abschn. 6.4.7). zum experimentellen einsatz von stickoxidsynthetaseinhibitoren (l-name/l-nmma), inhibitoren der komplementkaskade und von pyruvat 7 abschn. 6.4.9. antiarrhythmische therapie (7 kap. 3) die konservativ nicht beherrschbare schocksymptomatik kann ein chirurgisches eingreifen erforderlich machen. die akutherztransplantation dieser patienten wird zwar weiterhin die ausnahme bleiben, mit den ventrikulären assist-systemen (wie z. b. dem novacor) stehen dem chirurgen jedoch über wochen einsetzbare bridging-maßnahmen zur verfügung (martin et al. 2003) , die auch dem patienten mit kardiogenem schock zugute kommen könnten. bei kardiogenem schock infolge eines myokardinfarkts ist aufgrund der guten erfolge der akut-pci eine aortokoronare bypass-notfalloperation nur noch selten nötig. als mögliche indikationen gelten hierfür: fortbestehende ischämie bei koronarographisch nachgewiesener mehrgefäßerkrankung und/ oder lca(linke koronararterie)-hauptstammstenose; nach pci-komplikationen bzw. nicht erfolgreicher pci. die operation sollte dann aber notfallmäßig erfolgen, da die präoperative ischämiedauer der entscheidende prädiktor der perioperativen letalität ist. auch bei der perkutanen koronarintervention (pci) muss mit einem akuten koronarverschluss und mit einer letalität von 0,4-1,0% rechnen, vorwiegend als folge eines sich entwickelnden kardiogenen schocks. vor allem bei der hochrisiko-pci (hochgradig eingeschränkte linksventrikuläre pumpfunktion, dreigefäßkrankheit, das zu dehnende gefäß als das . übersicht 6.11. basisbehandlungskonzept des kardiogenen schocks katecholamine, (phosphodiesterasehemmer) ggf. ca ++ -sensitizer vom typ des levosimendan vor-und nachlastsenkung (cave: hypovolämie): vorlast: nitrate (0,3-0,5-4 µg/kgkg/min bzw. 0,3-6,0 mg/h) vorlast + nachlast: nitroprussidnatrium (0,1 einzige noch perfundierte koronargefäß) kann das risiko der koronardehnung durch prophylaktisch oder im stand-by genutzte mechanische unterstützungssysteme (. übersicht 6.12) reduziert werden (ferrari u. figulla 2005) . in der wartephase auf die herztransplantation können auch hier ventrikuläre assist-systeme als bridging-verfahren eingesetzt werden. bezüglich der pci bei myokardinfarkt s. unten. die mechanischen unterstützungssysteme im kardiogenen schock reichen von der kardiopulmonalen reanimation (7 abschn. 6.4.2) über die wiederherstellung einer suffi zienten koronarperfusion bis hin zu mehr oder weniger komplexen kreislaufunterstützungssystemen. die jährlich 270.000 patienten mit einem akuten herzinfarkt in deutschland, von denen ca. 5-10% einen kardiogenen schock entwickeln, unterstreichen das quantitative ausmaß, das solche verfahren bereits haben oder haben könnten. mechanische kreislaufunterstützungssysteme fi nden beim akuten herzstillstand (ferrari et al. 1998) , im kardiogenen schock und bei der hochrisiko-ptca anwendung (martin et al. 2003) . beim herzstillstand wird ein komplettes kreislaufunterstützungssystem benötigt, das in 15 min unter fortlaufender kardiopulmonaler reanimation möglichst unter röntgenkontrolle installiert werden kann (ferrari et al. 1998) . im kardiogenen schock ist eine partielle kreislaufunterstützung mittels der intraaortalen ballonpulsation (iabp) für einen zeitraum bis zu etwa 10 tagen möglich. bei der hochrisiko-pci kommt die mechanische kreislaufunterstützung mittels iabp oder kardiopulmonalem bypass je nach befundkonstellation auf der stand-by-basis im bedarfsfall oder elektiv vor beginn der pci zum einsatz. da 90% der akuten reokklusionen innerhalb der ersten 6 h nach pci auftreten, sollten diese verfahren zumindest für diese zeit sofort verfügbar sein (ferrari u. figulla 2005) . methodik, komplikationen, kontraindikationen. ein ballonkatheter (8 f) von ca. 70 cm länge (z. b. ballonlänge 26 cm, insuffl ationsvolumen 40 ml) wird perkutan transfemoral über eine schleuse (z. b. 9 f) eingeführt und so platziert, dass der ballon in der thorakalen aorta distal des abgangs der linken a. subclavia zum liegen kommt. mit einem ekg-gesteuerten augmentierungsmodus von 1:1-1:4 wird der ballon in der diastole aufgeblasen (füllung mit heliumgas) und am beginn der systole abrupt desuffl iert (. abb. 6.19a-c) . die komplika-tionsrate (zumeist ischämie der unteren extremität, arterielle thrombose, infektion, persistierende blutung inguinal; bei längerer anwendung gelegentlich beeinträchtigte leber-und nierenfunktion) liegt aktuell bei 2,7%, bei einer mittleren liegezeit von 3 tagen; eine vorzeitige beendigung der iabp-therapie ist bei etwa 2% der patienten erforderlich (gregg et al. 2003) . kontraindikationen sind schwere iliofemorale gefäßveränderungen, aortendissektion, höhergradige aortenklappeninsuffi zienz und eine blutungsdiathese. wirkprinzip und erzielbare wirkungen. durch das streng auf die diastole beschränkte aufblasen des ballons nimmt der aortendruck phasisch zu (diastolische augmentierung; »kamelhöckerform« der blutdruckkurve; . abb. 6.19a) und bewirkt damit eine zunahme der koronarperfusion. die abrupte desuffl ierung des ballons am beginn der systole führt über eine sogwirkung zur absenkung des bei pumpversagen erhöhten diastolischen ventrikeldrucks (vorlastsenkung) sowie des systolischen aortendrucks (nachlastsenkung) und damit zu einer, allerdings nicht sehr ausgeprägten, senkung des myokardialen o 2 -verbrauchs. im transösophagealen echokardiogramm zeigte sich eine verbesserung der linksventrikulären pumpfunktion, und bei der dopplersonographischen koronarfl ussmessung steigerte sich die maximale sowie die integrale flussgeschwindigkeit um etwa 115% bzw. um ca. 80%. hypotensive patienten mit einem systolischen blutdruck ≤90 mmhg zeigen dabei den größten anstieg der koronaren flussgeschwindigkeit. der fluss im poststenotischen anteil (cohen et al. 2003) . der vorteilhafte effekt der intraaortalen gegenpulsation im zusammenhang mit perkutaner revaskularisation (brodie et al. 1999; lindholm et al. 2003) wie auch unter thrombolytischer therapie (barron et al. 2001; kovack et al. 1997 stomel et al. 1994) ist bei patienten mit infarktbedingtem kardiogenem schock gut belegt. amerikanische kliniken mit hoher iabp-implantations-frequenz (>35/jahr) erzielen bei schockpatienten eine um 30% niedrigere letalität (barron et al. 2001) . bei akutem koronarverschluss nach pci und bei pci-hochrisikopatienten mit mehrgefäßerkrankung und erheblich eingeschränkter pumpfunktion (im mittel 10% aller patienten) lässt sich die iabp mit gutem primärergebnis und ausreichend sicher einsetzen. bei patienten mit kardiogenem schock kann der iabp-einsatz bereits in der notaufnahme sicher durchgeführt werden (bur et al. 2002) . ein früher einsatz der iabp bei nichtherzchirurgischen krankheitsbildern scheint nach den daten der benchmark-registry mit weltweit über 19.636 patienten die prognose zu senken (cohen et al. 2003) . limitationen der iabp-anwendung sind die notwendigkeit eines mindestherzindex von 1,3 l/min/m 2 und eines weitgehend stabilen rhythmus sowie der eher bescheidene erfolg mit einer einsparung des myokardialen o 2 -verbrauchs um nur 10-20%. bei herzchirurgischen patienten mit »low-output-syndrom« kommt die iabp häufi g zum einsatz. ein prädiktiver score erlaubt die identifi zierung derjenigen patienten, bei denen die iabp-behandlung des low-output-syndroms nicht ausreicht und ein wechsel auf ein herzunterstützungssystem notwendig wird (hausmann et al. 2002) . die katheter können entweder perkutan oder nach chirurgischer gefäßeröffnung in die femoralgefäße eingeführt werden. im falle der »supported angioplasty« bevorzugen wir als anästhesieverfahren die elektive intubationsnarkose. das system kann von einem erfahrenen team innerhalb von 15 min installiert werden. indikationen. indikationen sind die überbrückung akut vital bedrohlicher mechanischer infarktkomplikationen wie der ventrikelruptur, der herzstillstand und die hochrisiko-pci. in den usa erleiden jährlich 70.000-100.000 patienten einen herzstillstand im krankenhaus; diese patienten könnten somit möglicherweise ohne zeitlichen verzug wiederbelebt werden. bei erfolgloser konventioneller reanimation wären diese patienten kandidaten für eine notfall-pcps (ferrari et al. 1998) . die zu erwartenden ergebnisse wären günstig, wenn man die resultate einer multizentrischen studie mit 218 pcps-reanimierten patienten zugrunde legt (34% der mit pcps reanimierten patienten konnten aus dem krankenhaus entlassen werden, im vergleich zu 14% der konventionell reanimierten eines historischen kontrollkollektivs). 7 von 9 patienten mit fulminanter myokarditis und kardiogenem schock überlebten bei vorübergehendem einsatz des perkutanen kardiopulmonalen bypass-systems für 3-10 tage ohne nachfolgende transplantation (kato et al. 1999) . natürlich müssen indikationsrichtlinien für die anwendung des perkutanen kardiopulmonalen bypass-systems vorliegen, soll dieses verfahren mit einer gewissen aussicht auf erfolg eingesetzt werden und nicht nur den irreversiblen schockzustand und damit das leiden des patienten verlängern. als richtlinien können gelten (ferrari u. figulla 2005): keine spontanzirkulation trotz optimaler reanimation über 5 min bei patienten unter 60 jahren mit dokumentiertem plötzlichem herzstillstand, sofort eingeleiteter reanimation und ohne neurologische defi zite; der einsatz der perkutanen kreislaufunterstützungssysteme als überbrückung bis zur diagnostik, bis zur koronarrevaskularisation (ptca), bis zur implantation eines biventrikulären unterstützungssystems (»bridging to bridging«). die bisher beschriebenen mechanischen unterstützungssysteme sind für den einsatz von stunden bis tage gedacht; sie sind geeignet, eine akute schockphase zu überwinden. jüngst konnte auch mittels eines perkutan einsetzbaren mechanischen unterstützungssystems (tandem-heart) ein günstiger verlauf für patienten im protrahierten kardiogenen schock aufgezeigt werden (thiele et al. 2001) , ebenso wie für das impella-system (ferrari u. figulla 2005) . komplexere ventrikuläre unterstützungssysteme, wie z. b. das linksventrikuläre system novacor oder biventrikuläre künstliche herzen, dienen für terminal herzinsuffi ziente patienten zur überbrückung der wartephase auf ein spenderherz (martin et al. 2003) . bei der konventionellen herzdruckmassage (hdm) wird ein herzzeitvolumen von nur 25-30% des normalwertes erreicht. diastolische drücke von 40 mmhg und systolische drücke von 60-80 mmhg werden selten überschritten. unter den bedingungen der manuellen herzdruckmassage reichen zerebraler und myokardialer blutfl uss nicht aus, um auf dauer einen hypoxischen schaden abzuwenden. um höhere blutfl üsse zu erreichen, werden alternative bzw. zusätzliche methoden zur hdm erprobt, wie die sequenzielle thorakoabdominelle kompression, die cpr mittels pneumatischer weste, die aktive kompressions-dekompressions-reanimation und die sog. »inspiratory threshold valve«. während die pneumatische weste v. a. den kompressionsvorgang automatisiert, haben die anderen genannten verfahren eine steigerung des venösen rückfl usses zum ziel. nach den aktuellen empfehlungen können mechanische hilfsmittel zur cpr verwendet werden. die herzdruckmassage mit interponierter abdomineller kompression (iac, »interposed abdominal compression cardiopulmonary resuscitation«) stellt ein vielversprechendes verfahren dar. bei der herzdruckmassage wird von einem zusätzlichen helfer mit der handfl äche eine kompression von 100±20 mmhg auf die nabelgegend ausgeübt, und zwar alternierend mit der kardiopulmonalen kompression im verhältnis 1:1. dadurch kommt es zu deutlich höheren blutfl üssen als mit konventioneller hdm. die aktive kompressions-dekompressions-pumpe (acd-cpr, »active compression-decompression cardiopulmonary resuscitation«) ist ein vielversprechendes und leicht anwendbares konzept (. abb. 6.21a,b). ein griff wird mittels eines saugnapfes (z. b. cardiopump) auf der brust des patienten fixiert. so kann sowohl druck auf den thorax ausgeübt als auch durch zug an dem griff ein unterdruck erzeugt werden (mit förderung des venösen rückfl usses und einem höheren aortalen blutfl uss bei der nächsten kompression). in mehreren randomisierten prähospitalstudien zeigte der einsatz der cardiopump leider keinen überlebensvorteil. bei patienten, die außerhalb des krankenhauses mit acd-cpr reanimiert worden waren, ließ sich jedoch eine verbesserte langzeitüberlebensrate nachweisen (plaisance et al. 1999 ). > 6.6 · spezifi sche schockformen während in den vergangenen jahren die dokumentierte infarktsterblichkeit seit der einführung der intensivüberwachung, der thrombolyse, effektiver antithrombotischer therapien sowie der perkutanen koronarintervention (pci) von ca. 30% um 1960 auf zwischenzeitlich 6%-7% deutlich gesenkt werden konnte, ist die historisch zwischen 70 und 80% angesiedelte sterblichkeit bei kardiogenem schock infolge eines akuten myokardinfarktes trotz moderner therapiemaßnahmen nicht in diesem ausmaß verbessert worden. unverändert stellt der kardiogene schock die hauptursache der infarktsterblichkeit im krankenhaus dar. ein kardiogener schock entwickelt sich bei 5-10% aller patienten mit akutem herzinfarkt (hochman et al. 1999 , jacobs et al. 2000 menon et al. 2000c) , und zwar am häufi gsten dann, wenn mindestens 35% der linksventrikulären muskelmasse akut oder sukzessive infarziert (menon et al. 2000a; prondzinsky et al. 2004) abb. 6.21a,b. aktive kompressions-dekompressions-pumpe (acd-cpr). a das system besteht aus einem saugnapf (radius 6,5 cm), einem gleitkolben und einem horizontalen griff (gesamthöhe 13,5 cm). b in die oberfl äche des griffes ist eine messskala (kg oder pounds) eingearbeitet. die aktive kompression/dekompression wird mit der mitten auf dem sternum platzierten pumpe mit 80-100 kompressionen (unabhängig vom atemzyklus) durchgeführt. der kompressionsdruck beträgt dabei (abhängig von der steifheit des brustkorbs) 29,5-50,0 kg, entsprechend einer kompressionstiefe von 3,8-5,1 cm. die gleichlange dekompressionsphase mit einem sog von im mittel 9,1 kg dient dazu, den brustkorb ohne kontaktverlust maximal zu expandieren. (nach cohen et al. 1994) . primäre pumpstörung mit schock bei ausgedehntem infarkt die daten der prähospitalen thrombolysestudie (captim; bonnefoy et al. 2002) legen nahe, dass der frühzeitige prähospitale einsatz von thrombolytika tendenziell zur verringerung nachfolgend auftretender schocksituationen führt. diese prospektiv erhobenen daten, die einen günstigen trend zu gunsten der thrombolyse mit einer annähernden halbierung der schockrate im weiteren hospitalverlauf aufwiesen, werden durch eine reihe retrospektiver daten positiv gestützt. verschiedene kleinere studien wie die daten des shock-trial-registry weisen auf einen deutlichen behandlungsvorteil für patienten mit kardiogenem schock in folge linksventrikulären pumpversagens mittels thrombolyse und begleitender intraaortaler gegenpulsation hin, insbesondere bei nachfolgender revaskularisation mittels pci oder aortokoronarer bypass-op. die autoren schlussfolgern, dass für krankenhäuser ohne möglichkeiten zur unmittelbaren koronarintervention eine strategie mit früher systemischer thrombolyse und gleichzeitiger intraaortaler gegenpulsation eine frühestmögliche vorbehandlung darstellt, bei nachfolgendem transfer des patienten in ein entsprechendes herzzentrum mit möglichkeiten zur pci oder aortokoronaren bypass-op. diese daten stehen interessanterweise in einem deutlichen kontrast zu den daten der danami-studie (andersen et al. 2003) , in der bei herzinfarktpatienten auch bis zu 2 h zeitverzögert interventioneller koronartherapie ein eindeutiger behandlungsvorteil zu gunsten der mechanischen koronarrevaskularisiation gegenüber der thrombolyse aufgezeigt werden konnte. hierbei ist jedoch einschränkend zu bemerken, dass die gesamtsterblichkeit in beiden gruppen überdurchschnittlich niedrig war und sich diese daten primär nicht auf schockpatienten übertragen lassen dürften. darüber zeigte sich in der isolierten betrachtung des kombinierten endpunktes nach 30 tagen (tod, reinfarkt, schlaganfall) nur für den einzelnen endpunkt »reinfarkt« ein signifi kanter vorteil für die patienten mit primärer pci. erst die wiederherstellung eines effektiven blutfl usses durch die sofortige wiedereröffnung des verschlossenen koronargefäßes mittels akut-pci (meyer et al. 1982 ) erbrachte nahezu eine halbierung der sterblichkeit. die shock-studie (hochman et al. 1999; ryan 1999) überprüfte die wirksamkeit der akutrevaskularisation (überwiegend akut-pci) bei 302 patienten mit kardiogenem schock nach herzinfarkt im sinne der evidenzbasierten medizin (. tabelle 6.19). primäres zielkriterium war dabei die senkung der 30-tage-letalität, sekundäres zielkriterium u. a. die senkung der 6-monate-letalität. die 17%ige senkung der 30-tage-letalität im gesamtkollektiv war nicht signifi kant unterschiedlich, wohl aber die der 6-monate-letalität, und -in einer nachuntersuchung -die 12-monate-sterblichkeit. besonders die patienten <75 jahren profi tierten von der akutrevaskularisation, ebenso die patienten mit einem zeitintervall <6 h von schmerzbeginn bis randomisierung und die mit einem zweitinfarkt, weiterhin männer mehr als frauen. zu ähnlichen aussagen kommt das alkk-schockregister in deutschland (zeymer et al. 2004 während im shock-trial (hochman et al. 1999 ) der eindeutige behandlungsvorteil auf die altersgruppe der unter-75-jährigen begrenzt war, konnten weitere arbeiten den behandlungsvorteil der pci auch für patienten im höheren alter aufzeigen (antoniucci et al. 2003; dzavik et al. 2003) . das alter stellt zwar bei kardiogenem schock einen eigenständigen risikoprädiktor dar; bei frühzeitiger und technisch erfolgreicher intervention ist allerdings der relative nutzen vergleichbar. die möglichkeit zur stent-implantation und der einsatz von gp iib/iiia-hemmern hat den effi zienzgrad und die sicherheit der akut-ptca bei kardiogenem schock noch weiter gesteigert. die effektivität des glykoproteinrezeptorantagonisten abciximab konnte sowohl bei perkutanen koronarhochri 6.6 · spezifi sche schockformen sikointerventionen als auch insbesondere bei infarktbedingtem kardiogenen schocks aufgezeigt werden (zahn et al. 2003; zeymer et al. 2003) . so wird in einer pci-serie (antoniucci et al. 1998 ) mit 66 schockpatienten über die implantation eines stents bei 47% berichtet; die primäre erfolgsrate lag dabei bei 94%, ein optimales angiographisches ergebnis wurde zu 85% erzielt, die 6-monate-überlebensrate lag bei 71%; und 80% der patienten hatten nach 6 monaten einen nyha-herzinsuffi zienzschweregrad von nur i-ii. von besonderem interesse war, dass gestentete koronarien eine geringere 6-monate-restenose-/reokklusionsrate (20%) hatten als dilatierte, aber nichtgestentete herzkranzgefäße (67%). ebenso ließ sich auch ein langfristiger überlebensvorteil bei erfolgter stent-implantation und verabreichung von abciximab gegenüber einer strategie mit alleiniger ballonangioplastie, alleiniger stent-implantation oder ptca sowie abciximabgabe ohne stent-implantation belegen. der initial signifi kant verbesserte timi-iii-fluss ließ sich auch nach 2,5 jahren als signifi kanter überlebensvorteil für die patienten, die mit stent und abciximab behandelt wurden, nachweisen (antoniucci et al. 2002; chan et al. 2002; giri et al. 2002) . die akut-pci kann bei kardiogenem schock infolge herzinfarkt als das verfahren der wahl angesehen werden (hamm et al. 2004) der überzeugende erfolg der pci im vergleich zur medikamentösen therapie mit i.v.-thrombolyse dürfte darauf zurückzuführen sein, dass mittels pci in einem sehr viel höheren prozentsatz ein timi-grad-3-koronarfl uss (komplette reperfusion) wiederhergestellt werden kann, als dies mit der thrombolyse möglich ist. wegen der günstigen ergebnisse der akut-pci bei infarktbedingtem schock scheint die empfehlung gerechtfertigt, infarktpatienten mit manifestem oder beginnendem schock (katecholaminpfl ichtigkeit) möglichst rasch in ein zentrum mit der möglichkeit zur akut-pci durch ein erfahrenes herzkatheterteam zu verlegen (hamm et al. 2004) . dabei kann durchaus vorab eine i.v.-thrombolyse durchgeführt werden, sie stellt für die akut-pci keine kontraindi-> kation dar. falls verfügbar, sollte der transport mit liegender intraaortaler gegenpulsation (iabp) erfolgen, ein beträchtlicher nutzen der iabp bei den so behandelten patienten (n =46) ist in einer retrospektiven analyse aufgezeigt: 1-jahres-letalität mit iabp-behandlung 33%, 1-jahres-letalität ohne iabp-behandlung 68% (p =0,019; kovack et al. 1997 ). die akute mitralinsuffi zienz (bei 1% aller infarktpatienten; 0,4-5% aller infarkttodesfälle; 2-7 tage, im mittel 4 tage nach hinterwandinfarkt) kann entweder als folge einer papillarmuskeldysfunktion oder einer papillarmuskelruptur entstehen; meistens ist die papillarmuskeldysfunktion von transienter natur und führt nur zu einer milden mitralinsuffi zienz, während der papillarmuskelabriss in der regel ein refraktäres lungenödem und eine schwere beeinträchtigung des kreislaufs hervorruft. die mitralinsuffi zienz infolge papillarmuskelabriss fi ndet sich typischerweise bei älteren patienten mit erstmaligem, oftmals eher kleinem infarkt und nur leicht eingeschränkter auswurffraktion; eine insuffi zienz infolge dysfunktion des halteapparates -ohne papillarmuskelabriss -tritt eher bei jüngeren patienten mit koronarer mehrgefäßerkrankung und eingeschränkter auswurffraktion auf (calvo et al. 1997) . frauen und ältere infarktpatienten sind von der akuten mitralinsuffi zienz, die gemeinsam mit der ruptur des ventrikelseptums etwa 8% aller schockinfarktpatienten ausmacht, häufi ger betroffen; möglicherweise führt die i.v.-thrombolyse zu einem früheren auftreten dieser komplikation (hochman et al. 2000) . bei der mehrzahl der patienten ist die rechte kranzarterie oder der r. circumfl exus das infarktgefäß. bei bis zu 50% aller patienten mit akutem myokardinfarkt lässt sich auskultatorisch oder echokardiographisch eine mitralinsuffi zienz nachweisen. eine höhergradige mitralinsuffi zienz fi ndet sich hingegen nur bei 3% aller infarktpatienten, wobei die hälfte dieser patienten ein lungenödem oder einen manifesten kardiogenen schock entwickelt (tcheng et al. 1992) . das systolikum -in typischer weise holosystolisch über dem apex zu auskultieren und in die axilla ausstrahlend -ist tabelle 6.19. shock-studie: akutrevaskularisation bei patienten mit kardiogenem schock nach herzinfarkt. (nach hochman et al. 1999 (zotz et al. 1993) . das hämodynamische monitoring zeigt eine abnahme des herzzeitvolumens und das auftreten einer v-welle in der pulmonalkapillare (. tabelle 6.2). die letalität innerhalb der ersten 24 h kann bis zu 50% betragen. nach initialer stabilisierung mit vasopressoren, nachlastsenkung (typischerweise mit dem gut steuerbaren nitroprussidnatrium) und implantation der intraaortalen ballongegenpulsation muss bei der papillarmuskelruptur zum frühestmöglichen zeitpunkt ein klappenersatz (ggf. mit anlegen eines aortokoronaren bypasses) durchgeführt werden. die ergebnisse der frühoperation sind mit einer letalität von 44% deutlich besser als die der spätoperation (letalität 78%; thompson et al. 2000) . falls eine papillarmuskelruptur sicher auszuschließen ist und »nur« eine papillarmuskelischämie vorliegt, kann eine pci des den betroffenen papillarmuskel versorgenden koronargefäßes in betracht gezogen werden. eine ventrikelseptumruptur (birnbaum et al. 2002; menon et al. 2000b ) tritt bei 1-3% aller infarkte innerhalb der 1. woche auf und ist etwa gleich auf alle infarktlokalisationen verteilt. die ruptur kann mit neuerlichen herzschmerzen einhergehen. typisch dafür ist das laute holosystolikum mit punctum maximum am linken sternalrand, gelegentlich vergesellschaftet mit einem schwirren. die diagnose lässt sich echokardiographisch stellen (ballal et al. 1993) , der o 2 -sättigungssprung zwischen rechtem vorhof und rechtem ventrikel lässt eine quantitative shunt-abschätzung zu. neben der shunt-größe ist prognostisch auch die lokalisation wichtig: die hämodynamische beeinträchtigung durch den shunt wird v. a. durch die gestörte compliance des rechten ventrikels geprägt, wenn dieser, wie bei einem hinterwandinfarkt, ebenfalls infarziert ist (held et al. 1988) . die hälfte der patienten entwickelt einen kardiogenen schock. die prognose wird in erster linie vom ausmaß der links-und rechtsventrikulären infarzierung und weniger von der größe des defektes bestimmt (cummings et al. 1997) . bei hämodynamischer instabilität besteht nach ansicht der meisten autoren eine dringliche bzw. notfallmäßige operationsindikation, da sich unter konservativer therapie bei diesen patienten häufi g ein multiorgandysfunktionssyndrom entwickelt (lemery et al. 1992; cox et al. 1996; killen et al. 1997) . therapeutisch bietet nur die rasche operative deckung des defektes nach vorheriger implantation der intraaortalen ballongegenpulsation (thiele et al. 2003 ) und medikamentöeser stabilisierung (nachlastsenkung mittels nitroprussidnatrium zur shunt-reduktion, ggf. in kombination mit katecholaminen) aussicht auf erfolg. aber selbst bei frühzeitiger operation muss mit einer sterblichkeit von 73% im vergleich zur sterblichkeit von 95% bei abwartender haltung gerechnet werden (hochman et al. 1997) . der perkutane translu-minale verschluss einer septumruptur gilt vorerst als experimentelle methode. die ruptur der freien ventrikelwand (figueras et al. 2001; slater et al. 2000) geht mit einer letalität von 90% einher. sie tritt am häufi gsten zwischen dem 2. und 8. tag nach infarkt auf, aber jede 3. ruptur fi ndet bereits innerhalb der ersten 24 h statt. sie betrifft häufi ger frauen, den erstinfarkt, den vorderwandinfarkt, den älteren patienten und diejenigen mit hochdruck. sie wird gehäuft gesehen bei vorausgegangener medikation mit kortikoiden und antiphlogistika. der frühzeitige einsatz von thrombolytika scheint die inzidenz zu senken, der späte einsatz sie zu erhöhen. die ruptur der freien wand führt zum hämoperikard und zur raschen perikardtamponade. oft kommt es zu neuerlichen starken herzschmerzen mit sofortiger elektromechanischer entkopplung. am häufi gsten führt der einseitige einriss zum tode; subakute einrisse können jedoch ebenfalls als akute tamponade, als großer perikarderguss oder als chronisches pseudoaneurysma imponieren. der akute schock nach infarkt muss immer an die diagnose einer ventrikelruptur denken lassen. eine sofortige perikardpunktion kann zur vorübergehenden stabilisierung führen, die zur zügigsten diagnostik (echokardiogramm: perikarderguss; swan-ganz-katheter: tamponadedruckprofi l) genutzt werden muss. sehr selten wird eine ventrikelruptur überlebt, nur dann, wenn der patient schnellstens (richtwert: 1 h) an die herz-lungen-maschine angeschlossen wird und der einriss erfolgreich übernäht werden kann. das geronnene blut im perikardbeutel verstopft sehr schnell eine perikarddrainage, was für die reanimation am offenen thorax spricht. zahlreiche erkrankungen sehr unterschiedlicher ätiologie können in einen kardiogenen oder extrakardial-obstruktiven schock münden (. tabelle 6.1). nach entsprechender diagnostik wird sich die therapie zunächst darauf konzentrieren, einerseits eine besserung der pumpfunktion des herzens zu erzielen, andererseits versuchen, kardiale und extrakardiale obstruktionen zu beseitigen (z. b. punktion eines tamponierenden perikardergusses, thrombolyse bei massiver lungenembolie) oder schockauslösende rhythmusstörungen zu beherrschen (z. b. kardiopulmonale reanimation). man wird dann bestrebt sein, die therapie in eine kausale münden zu lassen, soweit dies möglich ist. bei aktueller inoperabilität eines patienten mit dekompensierter aortenklappenstenose und schock kann auf der intensivstation bei sorgfältigem invasivem hämodynamischem monitoring eine nachlastsenkung mit nitroprussidnatrium zur entlastung des ventrikels versucht werden (khot et al. 2003) , und auch die aortenklappenvalvuloplastie kann im einzelfall zur rekompensation beitragen und damit die voraussetzungen für den späteren klappenersatz schaffen (melzer et al. 2001 ). > 6.6 · spezifi sche schockformen intoxikationen mit kardiodepressiven und vasotoxischen substanzen (schmidt et al. 1998) . trotz der potenziell akut lebensbedrohlichen situation ist die langzeitprognose dieser patienten nach erfolgreicher akutbehandlung in der regel sehr günstig. dies rechtfertigt auch den einsatz eines kardiopulmonalen bypasses für einige stunden, falls die konventionellen detoxikationsmaßnahmen und die symptomatische therapie zu keiner herz-kreislauf-stabilisierung führen. herzverletzungen (redling et al. 1998 ). offene, aber auch stumpfe thoraxtraumen mit schockentwicklung sollten immer an eine herzverletzung denken lassen; nach rascher diagnosestellung ist hier die sofortige herzoperation häufi g lebensrettend. perioperativer herzinfarkt mit schockentwicklung. wegen der häufi g nur kurz zurückliegenden operation muss bei auftreten eines herzinfarktes häufi g auf die i.v.-thrombolyse verzichtet werden, obwohl wegen des meist kurzen zeitintervalls zwischen infarktbeginn und infarktdiagnostik die erfolgschancen der thrombolyse günstig wären. in solchen fällen sollte -wann immer möglich -die akut-ptca auch des beatmeten patienten in erwägung gezogen werden. zwischen 1980 zwischen und 1987 f − − − f − − in deutschland werden z. z. jährlich 65.000 operationen mit der herz-lungen-maschine durchgeführt mit einer durchschnittlichen letalität von 3,3%. wesentliche todesursachen sind dabei septischer schock und mods. gründe für das erhöhte sepsisrisiko sind die kardiale vorschädigung der patienten, die folgen der thorakotomie mit einer erhöhten inzidenz an pneumonien sowie bakteriämie, endotoxinämie und vorwiegend pulmonalen mikrozirkulationsstörungen als folge der extrakorporalen zirkulation. ein weiterer wesentlicher aspekt ist die kontaktaktivierung des blutes in der herz-lungen-maschine: sie löst eine ausgeprägte systemische entzündungsreaktion (»post pump infl ammatory response«) aus. als folge davon kommt es zur aktivierung von blutzellen mit freisetzung von aggressiven mediatoren und proinfl ammatorischen zytokinen, die z. t. in höheren serumkonzentrationen gefunden werden als in der sepsis. diese entzündungsreaktion wird für das auftreten des mods nach herzchirurgischen operationen mitverantwortlich gemacht (prondzinsky et al. 2005 ). die möglichkeiten einer positiv-inotropen intervention sind bei dem muskelschwachen rechten ventrikel relativ begrenzt (maisch u. christ 2004) . insbesondere digitalis sollte vorsichtig dosiert werden, um intoxikationen zu vermeiden. die senkung der zur dekompensation führenden pulmonalen hypertonie steht im vordergrund. rechtsherzdekompensation bei ards und sepsis: 7 abschn. 6.6.2 in der therapie des rechtsherzversagens und der pulmonalen hypertonie nach operativen, insbesondere herzchirurgischen eingriffen, bewirkt inhalativ appliziertes stickoxid (no, 0,5-40 ppm) eine selektive pulmonale vasodilatation ohne systemvaskuläre nebenwirkungen rinne u. zwissler 2004) . die pulmonale vasodilatation geschieht ohne beeinträchtigung der hypoxischen pulmonalen vasokonstriktion, so dass es weder zu einer erhöhung des intrapulmonalen shunts noch zu einer reduktion der arteriellen oxygenierung kommt. die wirkung von inhalativ appliziertem no kann jedoch variieren. prinzipiell denkbare effekte auf koronarien und myokard scheinen nicht sehr ausgeprägt bis fehlend zu sein (cheifetz et al. 1996) . bedenken gegen den einsatz von inhalativem no bei akuten koronaren syndromen bestehen nicht (lindenfeld 1998) . zunehmend setzt sich zur intraoperativen anwendung neben inhalativem no das inhalativ applizierbare, stabile prostaglandin-i 2 (pgi 2 )-analogon iloprost (ilomedin) aufgrund seiner längeren hwz und seiner technisch einfacheren anwendbarkeit durch (rinne u. zwissler 2004 (bowers et al. 1998; jacobs et al. 2003; pfi sterer 2003; seyfarth u. schömig 2004) . hinweisend dafür sind st-strecken-hebungen in den rechtspräkordialen ableitungen v 3r und besonders v 4r . der rechtsherzinfarkt -charakterisiert durch einen schockzustand (hypotonie, niedriges herzzeitvolumen) ohne lungenstauung -lässt sich echokardiographisch als großer, hypokinetischer rechter ventrikel und kleiner linker ventrikel kenntlich machen. wegweisend für die diagnose sind neben klinik und echokardiographie auch der niedrige pulmonalkapillardruck trotz schockzustand. der rechtsherzinfarkt disponiert zu bradykarden rhythmusstörungen in form höhergradiger av-blockierungen. vorgehen der wahl bei rechtsherzinfarkt mit oder ohne begleitenden schockzustand ist die akut-ptca (bowers et al. 1998) : in einer untersuchung an 53 patienten mit rechtsventrikulärem infarkt konnte durch die akut-ptca bei 41 patienten (77%) eine wiedereröffnung der verschlossenen rechten koronararterie erzielt werden, mit einer prompten und beeindruckenden erholung der rechtsventrikulären pumpfunktion innerhalb einer stunde und einer niedrigen hypotonie-(12%) und letalitätsrate (2%). bei den patienten, bei denen die wiedereröffnung nicht gelang, persistierte die rechtsventrikuläre funktionsstörung; die hypotonierate lag bei 83% und die letalität bei 58% (bowers et al. 1998 trotz des gewachsenen verständnisses des krankheitsablaufs und neuer therapieansätze konnte die hohe letalität des septischen schocks von 40-60% bisher noch nicht wesentlich gesenkt werden. die sepsishäufi gkeit ist zunehmend, in den vereinigten staaten erkranken jährlich etwa 750.000 patienten an einer sepsis, und 200.000 versterben daran. wesentliches zur klassifi kation und ätiologie (. abb. 6.22, 7 abschn. 6.1.5), zur pathophysiologie (7 abschn. 6.2.3), zur basisdiagnostik (7 abschn. 6.3.2) und basistherapie (7 abschn. 6.4) und zum mods (7 abschn. 6.5). die typischen herz-kreislauf-veränderungen bei gramnegativem und grampositivem septischen schock sind: blutdruckabfall infolge der ausgeprägten vasodilatation, erniedrigung des systemischen gefäßwiderstandes bis auf 30% der norm, kompensatorische zunahme des herzzeitvolumens, des schlagvolumenindex und des schlagarbeitsindex. fehlt eine relative kardiale vorschädigung, so liegen die gemessenen herzfunktionsparameter dabei auch beträchtlich höher als die gesunder probanden mit einem normalen systemischen gefäßwiderstand um 1100 dyn · s · cm -5 (. abb. 6.23). die beiden toxinschocksyndrome werden durch das toxicshock-syndromtoxin 1 (tsst-1) von staphylococcus aureus bzw. durch das streptococcus-pyogenes-exotoxin (spe) von streptokokken produziert. beide toxine haben superantigeneigenschaften (7 abschn. 6.2.5), was den dramatischen klinischen verlauf über eine massive zytokinfreisetzung erklären könnte. bei verdacht auf eine der genannten erkrankungen müssen die erkrankten auf die intensivstation gebracht werden, wo schnellstmöglich mit der antibiotika-und der symptomatischen schocktherapie und engmaschigen überwachung begonnen werden muss, da sich sehr rasch ein schock entwickeln kann. die toxische vasodilatation mit massivem blutdruckabfall könnte ein gesundes herz durch einen drastischen anstieg des herzzeitvolumens kompensieren (. abb. 6.23). eine so weitgehende kompensation der erniedrigten nachlast, d. h. ein wesentlicher anstieg des herzzeitvolumens wie in . abb. 6.23 beschrieben, wird im septischen schock nur selten beobachtet, v. a. nicht bei protrahierten verläufen: die pumpfunktionsparameter des herzens sind dabei zwar im vergleich zu gesunden probanden mit normalem systemischem gefäßwi-> derstand meist nicht erniedrigt oder sogar leicht erhöht, berücksichtigt man jedoch die inverse korrelation mit dem systemischen gefäßwiderstand (. abb. 6.23), so wird die eingeschränkte pumpleistung des herzens bei vielen patienten bereits in der hyperdynamen phase des septischen schocks und sogar bereits bei septischen patienten mit noch normalem blutdruck (raper et al. 1989 ) rasch evident. die erklärung für die nur inadäquate steigerung der herzförderleistung ist eine potenziell reversible, multifaktorielle herzschädigung in der sepsis, die durch klinische befunde belegt werden kann. als charakteristika der akuten septischen kardiomyopathie (müllerwerdan et al. 1996; (redl et al. 1993) . insgesamt ist jedoch die diskussion um das »ideale« katecholaminregime bei primär rechtsventrikulärer septischer herzschädigung noch in vollem gange; die möglichkeit zur detaillierten quantifizierung des therapieerfolgs wird im einzelfall sicherlich hilfreich sein. der septische schock stellt ein bereits weit fortgeschrittenes krankheitsstadium dar! er ist in der regel hyperdynam (herzindex >5,5 l/min/m 2 ; systemischer gefäßwiderstand 600 dyn·s·cm -5 ) und nur in der spätphase oder bei nicht ausreichender volumensubstitution hypodynam (<2,5; 1200). die z. z. noch weitgehend symptomatische behandlung beinhaltet zunächst die rasche und adäquate volumensubstitution, ggf. gefolgt und unterstützt durch den einsatz von katecholaminen. die mikrobielle diagnostik (blutkulturen und organbezogener keimnachweis), die fokuselimination und der beginn einer antibiotischen therapie leiten bereits zu den kausalen maßnahmen über. sepsis und septischer schock können ein facettenreiches mods induzieren, das die prognose des patienten entscheidend prägt. das konzept der therapie des septischen schocks besitzt mehrere eckpfeiler (czock u. keller 2002; dellinger 2003; dellinger et al. 2004; freeman et al. 2001; werdan et al. 2005 bei der wahl des richtigen präparates zur kalkulierten und gezielten antiinfektiösen therapie sind die empfehlungen der paul-ehrlich-gesellschaft sehr hilfreich (bodmann u. vogel 2001) , wobei die zunehmende resistenzentwicklung ein ernsthaftes problem darstellt. mit einer innerhalb der ersten 6 h auf der notaufnahme begonnenen, an hämodynamischen zielkriterien orientierten volumen-und katecholamintherapie (. abb. 6.25) lässt sich bei patienten mit schwerer sepsis und septischem schock die 28-tage-sterblichkeit im vergleich zur »konventionellen intensivtherapie« von 49 auf 33% senken (rivers et al. 2001) . neu an diesem im vergleich zu früheren »sauerstoff-zielorientierten«, bisher erfolglos gewesenen ansätzen ist der sehr frühe beginn der behandlung. dennoch darf dieses »erfolgsrezept« nicht kritiklos von der notaufnahme auf die intensivstation übertragen werden: der noch nicht anbehandelte »notfallsepsispatient« unterscheidet sich hämodynamisch beträchtlich von dem klassischen »intensivstationsepsispatienten« (dan-nino et al. 2002; . einen die früh-und die stabile sepsisphase integrierenden vorschlag zur herz-kreislauf-therapie gibt . abb. 6.26. sepsispatienten sind durch ein absolutes und ein relatives intravasales volumendefi zit charakterisiert. die sofortige und adäquate flüssigkeitstherapie ist der entscheidende erste schritt zur behandlung des septischen schocks (. tabelle 6.20; müller-werdan u. . bei einem hämatokrit <30% und gleichzeitig einer auf <70% erniedrigten zentralvenösen sauerstoffsättigung (s cv o 2 ) wird die gabe von erythrozytenkonzentraten zur anhebung des hämatokrits auf ≥30% empfohlen (. abb. 6.25), zumindest in der frühphase der sepsis innerhalb der ersten 6 h auf der notaufnahme (rivers et al. 2001) . ansonsten gilt die empfehlung, erythrozytenkonzentrate bei einem hämoglobinwert <7,0 g/ dl zu geben und das hb auf einen wert von 7,0-9,0 g/dl anzuheben. eythropoetin wird nicht zur behandlung der anämie im rahmen einer schweren sepsis empfohlen (. tabelle 6.20; dellinger et al. 2004 ). berücksichtigt man weiterhin die auswirkungen der katecholaminbehandlung auf die leber-splanchnikus-perfusion, den magenmukosa-ph und den laktatspiegel, so schneiden dobutamin und noradrenalin hinsichtlich unerwünschter wirkungen günstiger als dopamin und wesentlich günstiger als adrenalin ab , so dass auch aus diesen gründen der einsatz von noradrenalin und dobutamin empfohlen werden kann. phosphodiesterasehemmstoffe und dopexamin sind im septischen schock eher pharmaka der 2. wahl. digitalis ist bei tachykardem vorhoffl immern und bei vorhoffl attern indiziert, bei sinusrhythmus ist seine bedeutung als positiv-inotrope substanz eher gering einzustufen. selten kommen vasopressin sowie -noch wesentlich seltener -angiotensin, vasodilatatoren, n-acetylcystein, glukagon und kalzium als positiv-inotrope und direkt sowie indirekt vasoaktiv wirkende pharmaka bei patienten mit septischem schock zum einsatz dellinger 2003; dellinger et al. 2004; . eine prognosebesserung ist nicht belegt. zu ernährung und stoffwechsel . tabelle 6.20 und 7 abschn. 6.5.5. die gabe von hydrocortison hat eingang in die offi ziellen therapieempfehlungen gefunden (. tabelle 6.20). hydrocortison bessert den vaskulär bedingten schock, mit einem anstieg des erniedrigten systemischen gefäßwiderstandes und einer verkürzung der behandlungsdauer mit vasopressorischen katecholaminen; die myokarddepression der septischen kardiomyopathie lässt sich durch hydrocortison allerdings nicht bessern: der erniedrigte linksventrikuläre schlagarbeitsindex steigt nicht an (briegel et al. 1999) . die hinweise auf eine letalitätssenkende wirkung (annane et al. 2002) werden z. z. in der corticus-studie überprüft (7 abschn. 6.5.11). auch die gabe von aktiviertem protein c (drotrecogin alfa aktiviert; xigris) ist bestandteil der offi ziellen therapieempfehlung bei schwerer sepsis in der frühphase (. tabelle 6.20; 7 abschn. 6.5.6). wie in einer cochrane-analyse beschrieben haben i.v.-immunglobuline -das iviggma-präparat pentaglobin -je 0,25 g/ kgkg an 3 aufeinanderfolgenden tagen -mehr noch als ivigg-präparate eine letalitätssenkung in mehreren kleinen studien gezeigt (septischer schock mit endotoxinämie, abdominelle sepsis; zitiert in werdan 2001b; werdan et al. 2005) ; die datenlage reichte der survival sepsis campaign jedoch nicht aus, um die gabe von iviggma in ihre therapieempfehlungen aufzunehmen (dellinger et al. 2004) . derzeit nicht zu empfehlen ist der einsatz von antiendotoxintherapien, von steroiden in hoher dosierung und prostaglandin e, von anti-tnf-α-antikörpern, interleukin-1-rezeptorantagonisten, paf-antagonisten, n-acetylcystein und antioxidanzien. die peripheren venen sind kollabiert und der jugularvenendruck ist niedrig, es sei denn, der hypovolämische schock ist assoziiert mit einer einfl ussstauung, z. b. durch ein myokardiales pumpversagen, eine perikardtamponade oder einen pneumothorax. die akren sind kühl, und v. a. bei älteren patienten kann es zu einem absinken der körpertemperatur kommen. die kapilläre füllung ist vermindert. weitere klinische zeichen des hypovolämischen schocks sind periphere zyanose, verminderter hautturgor und trockene schleimhäute. die patienten klagen über durst, schwitzen und kurzatmigkeit, sie sind ängstlich. im schweren schock werden die patienten zunehmend apathisch oder verwirrt. der blutdruck kann nichtinvasiv nicht mehr messbar sein, oder es kann ein großer gradient zwischen blutig und unblutig gemessenem blutdruck nachweisbar sein. die hypotension kann im sitzen oder orthostatisch verstärkt sein. die atemabhängigen schwankungen des systolischen arteriellen blutdrucks sind intensiviert. die typischerweise zu beobachtende tachykardie kann durch die vorausgegangene einnahme von β-rezeptorenblockern ausbleiben. gelegentlich kommt es zu einer bradykardie. der zur abschätzung des volumen-/blutverlustes berechenbare schockindex -quotient aus puls und systolischem blutdruck; normal 0,5 (blutverlust <10%); drohender schock 1 (blutverlust <20-30%); manifester schock 1,5 (blutverlust >30-50%) -ist keine große hilfe und kann zu fehleinschätzungen führen (große variabilität; interferenz mit vormedikation (β-blocker) und begleiterkrankungen (arterielle hypertonie). sind trauma und äußerliche blutung die ursache des schocks, so haben die kontrolle des blutverlustes, die bereitstellung von gekreuzten blutkonserven und die infusion von flüssigkeit und blutprodukten eine höhere priorität als weitergehende diagnostische maßnahmen (groneveld 2001). nach stumpfem abdominellem trauma sollte eine diagnostische peritoneallavage durchgeführt werden. eine computertomographie des abdomens gehört zur weiterführenden diagnostik; diese ist zwar zeitaufwendiger und weniger sensitiv als die peritoneallavage und nur bei relativ stabilen patienten durchführbar, sie ist aber diagnostisch spezifi scher. ein rupturiertes bauchaortenaneurysma kann sonographisch oder, falls der zustand des patienten es erlaubt, angiographisch erfasst werden. ein stumpfes thoraxtrauma kann kompliziert sein durch eine aortenruptur, einen spannungspneumothorax, hämatothorax, hämoperikard oder eine tamponade. eine thoraxröntgenaufnahme kann hier diagnostisch weiterführen. bei gastrointestinalen blutungen sollten weiterführende endoskopisch-diagnostische maßnahmen auch erst nach der initialen flüssigkeitstherapie erfolgen. eine magensonde sollte gelegt werden, um die aspiration von mageninhalt bei erbrechen zu verhindern und um eine magenblutung erkennen zu können. > > laboruntersuchungen umfassen neben der blutgruppenanalyse und bereitstellung gekreuzter blutkonserven die bestimmung des blutbildes, der elektrolyte, der laktatkonzentration, des serumkreatinins, der blutgase einschließlich des ph-werts und der glukose. unmittelbar nach einer blutung können der hämoglobingehalt und hämatokrit des blutes noch normal sein. ein akuter hypovolämischer schock kann mit einer leichten leukopenie und anschließenden leukozytose einhergehen. die überwachung der patienten erfordert neben einer kontrolle des arteriellen blutdrucks und der urinausscheidung ggf. die messung des zentralen venendrucks und ein hämodynamisches monitoring mittels pulmonalarterienkatheter zur überwachung der flüssigkeitstherapie. die messung des pulmonalkapillären verschlussdrucks ist besonders wichtig bei funktions-und compliance-störungen des linken ventrikels, etwa bei einer vorbestehenden herzerkrankung. umgekehrt kann bei schwerer pulmonaler hypertonie und rechtsherzinsuffi zienz der druck im linken herzen anhand des zentralen venendrucks unterschätzt werden. durch kontrolle der kardialen füllungsdrücke kann die entstehung eines lungenödems bei der flüssigkeitssubstitution vermieden werden. therapieziel ist neben der substitution des verlorengegangenen volumens die optimierung der o 2 -zufuhr an die peripheren gewebe (christ u. lackner 2004; groeneveld 2001) . der therapieplan beim hypovolämischen schock erfolgt nach den aufgeführten gesichtspunkten: lagerung und schmerzstillung, volumenersatz, kausaltherapie, zusätzliche (fakultative) maßnahmen, prophylaxe. aufgrund der mikrozirkulationsstörung und der sekundären volumenverluste sind oft weit höhere volumina erforderlich, um die makro-und mikroperfusion sicherzustellen, als aufgrund des initialen volumenverlustes bzw. schockereignisses zu erwarten wäre. seit jahrzehnten ist gegenstand der diskussion, ob die volumenersatztherapie mit kristalloiden oder kolloiden lösungen erfolgen soll (7 abschn. 6.4.6). in der regel werden bei der behandlung des hypovolämischen schocks kolloide und kristalloide in einem fi xen verhältnis (z. b. 1 teil kolloid: 2, evtl. 3 teile kristalloid) verabreicht. hyperton-onkotische lösungen zeigen beim hypovolämischen schock keine medizinischen vorteile (meier-hellman u. burgard 2004). vasoaktive pharmaka werden im hypovolämischen schock dann eingesetzt, wenn die therapieziele mit adäquater volumensubstitution nicht zu erreichen waren, oder als überbrückende maßnahme bis zur einleitung der volumentherapie. in abhängigkeit vom klinischen bild sind begleitende therapiemaßnahmen wie sedierung, analgesie und frühzeitige beatmung anzustreben. zur beherrschung schwerster hypovolämischer schockzustände kann der einsatz von antischockhosen (mast, »medical/military anti-shock trousers«) hilfreich sein. dabei werden durch getrennte pneumatische kammern unterschenkel, oberschenkel und abdomen mit zentripedal abfallenden drücken komprimiert. nach wie vor ist trauma die häufi gste todesursache bei personen im alter von 2-40 jahren. verkehrsunfälle sind die häufi gste ursache des stumpfen traumas. statistisch sind in der frühphase nach einem trauma verbluten oder schwere schädelverletzungen die häufi gsten todesursachen; einige tage nach dem ereignis kommt es zu einer häufung von todesfällen durch schwere schädel-hirn-traumen; im abstand von mehreren wochen zum trauma sind sepsis und multiorganversagen die häufi gsten todesursachen. die traumatische verletzung ist ein komplexes, multifaktorielles geschehen, das ein weites spektrum an verschiedenen autonomen endokrinen und zellulären reaktionen auslöst. schmerz, angst, blutverlust, gewebeverletzung, hypoxie und bakterielle kontamination wirken als neuroendokrine stimuli; zusätzliche einfl ussfaktoren sind der individuelle bewusstseinszustand und die gabe von medikamenten, v. a. anästhetika. meist handelt es sich beim traumatischen schock jedoch um die pathogenetische konstellation eines hypovolämischen schocks, der durch wiederholte blutverluste aggraviert verlaufen kann (shapiro 2001 ). beim hämorrhagischen schock fi nden sich die klinischen zeichen der hypovolämie (7 abschn. 6.6.3) und das bild der äußeren oder inneren blutung oder andere zeichen der gewalteinwirkung (shapiro 2001) . der traumatische schock ist nicht immer ein blutungsschock. viele weitere ursachen sind zu nennen und bestimmen das klinische bild, z. b. spinaler schock (hund u. abel 2002) , hirnödem, kardiale kontusion, herztamponade, spannungspneumothorax, hypothermie, flüssigkeitsverluste durch verbrennungen und crush-verletzungen mit einer massiven freisetzung von myoglobin. bei allen patienten, die ein trauma erlitten haben, ist nach zeichen des schocks zu fahnden. der genaue unfallhergang ist ebenso zu erfragen wie eventuelle internistische vorerkrankungen. gerade bei älteren patienten können auch scheinbar geringfügige gewalteinwirkungen zu schweren verletzungen führen, etwa aufgrund einer osteoporose. der vollständig entkleidete traumapatient sollte nach überprüfung der vitalfunktionen mit größter sorgfalt körperlich untersucht werden. zur weiterführenden diagnostik 7 abschn. 6.3.3. da das ausmaß der verletzungen oftmals nicht anatomisch genau feststellbar ist, kommen zur objektivierung des schweregrades des traumas score-systeme zur anwendung, häufi g wird der »revised trauma score« genutzt. parallel zur überprüfung der vitalfunktionen (atemwege, atmung, herz-kreislauf-funktion) sollte sofort eine rasche volumensubstitution erfolgen. bleibt der patient trotz volumengabe hypotensiv, so können zusätzlich erythrozytenkonzent-> rate transfundiert werden. die zentrale frage bei der weiteren behandlung des traumapatienten ist, ob eine sofortige operative therapie erforderlich ist. ein fehlendes ansprechen der schocksymptomatik auf die flüssigkeitsverabreichung ist verdächtig auf eine andauernde blutung. aktive externe blutungen können oft schon durch direkten druck unterbrochen werden. sind äußere blutungen kontrolliert, so muss nach versteckten blutungsursachen gefahndet und diese ggf. operativ angegangen werden. gerade in notaufnahmen muss darauf geachtet werden, dass die patienten im traumatischen schock nicht außerdem noch in eine hypothermie geraten, die den patienten zusätzlich gefährdet und die gerinnung beeinträchtigen kann. eine massive transfusion von erythrozytenkonzentraten kann zu einer schweren koagulopathie führen durch den prozentual zu geringen anteil an gerinnungsfaktoren im vergleich zu den blutzellen, durch hypothermie und durch die sekundäre gerinnungsstörung im schock. die koagulopathie wird durch gabe von ffp (»fresh frozen plasma«) und thrombozytenkonzentraten behandelt. eine klinische unterscheidung zwischen anaphylaktischer und anaphylaktoider reaktion gelingt nicht. das klinische erscheinungsbild (haupt 2001; walther u. böttiger 2004) variiert interindividuell stark, auch in abhängigkeit vom antigeneintrittsort, der absorptionsrate und dem ausmaß der sensibilisierung. initial können daher gastrointestinale symptome, übelkeit, erbrechen, durchfälle, kolikartige beschwerden, unwillkürlicher abgang von stuhl und harn, selten darmblutungen, hauterscheinungen oder beschwerden von seiten des respirationstraktes im vordergrund stehen. das zeitliche intervall bis zum auftreten von beschwerden kann minuten bis mehrere stunden betragen; ganz überwiegend treten die symptome innerhalb der ersten stunde nach antigenexposition auf. die sich meist rapide entwickelnde systemische reaktion geht sehr oft (mehr als 90%) einher mit hauterscheinungen wie pruritus, flush, erythem, urtikaria und in schweren fällen angioödem. häufi g sind juckreiz und schwellungen der nasen-, augen-und mundschleimhaut sowie ödeme der lippen, der augenlider und der zunge. häufi g und bedrohlich sind atemwegsobstruktionen, extrathorakal durch ödeme im larynx-und pharynxbereich, intrathorakal durch bronchialobstruktion. oft kommt es zum lungenödem. in schweren fällen einer anaphylaxie, etwa bei intravenöser antigenexposition, kann es ohne hauterscheinungen und atembeschwerden unmittelbar zum schock kommen. die hämodynamischen veränderungen des anaphylaktischen schocks sind aus kasuistischen beschreibungen bekannt (hanashiro u weil 1967) . im vordergrund stehen hypovolämie aufgrund von flüssigkeitsverschiebungen ins interstitium bei erhöhter gefäßpermeabilität und peripherer vasodilatation, tachykardie und erniedrigte kardiale füllungsdrücke (carlson et al. 1981; silverman et al. 1984) . initial im verlauf einer anaphylaxie wurden erhöhte herzzeitvolumi-! 6.6 · spezifi sche schockformen na beobachtet, die einerseits durch die erhöhten katecholamin-und histaminspiegel zustande kommen, andererseits die folge des erniedrigten systemischen gefäßwiderstands sein könnten (moss et al. 1981; hamberger et al. 1980) . im weiteren verlauf sinkt das herzzeitvolumen ab bei einem erniedrigten venösen rückstrom. niedrige pulmonalkapilläre verschlussdrücke, wie sie im anaphylaktischen schock gefunden werden, sprechen gegen eine kardiale verursachung des häufi g gefundenen lungenödems; vielmehr ist dies am ehesten die folge der erhöhten gefäßpermeabilität (carlson et al. 1981) . jedoch wurden im anaphylaktischen schock ekg-veränderungen gefunden im sinne von ischämiezeichen und arrhythmien (booth u. patterson 1970; sullivan 1982; austin et al. 1984) . auch über eine reversible myokarddepression wurde bereits berichtet (raper u. fisher 1988) , wenn auch ältere studien nur eine geringe beeinträchtigung der herzfunktion fanden. für die notfalltherapie (haupt 2001; waltheru. böttiger 2004) spielt die unterscheidung zwischen anaphylaktischer und anaphylaktoider reaktion keine rolle. im folgenden ist daher nur von der anaphylaxie als überbegriff die rede. bei verdacht auf eine anaphylaxie ist sofortiges handeln erforderlich (. tabelle 6.21), so dass eine rasche evaluation der situation unter berücksichtigung möglicher differenzialdiagnosen erfolgen muss (haupt 2001) . schwere anaphylaktische reaktionen können auch unter adäquater therapie progredient verlaufen, oder es kann nach einer vorübergehenden besserung zu einem erneuten einbruch der symptomatik kommen. daher ist oftmals eine intensivmedizinische überwachung dieser patienten angezeigt. grundpfeiler der sofortbehandlung bei hypotension und hypoxie sind: ausschalten des mutmaßlichen auslösers, offenhalten der atemwege, 100%ige o 2 -zufuhr, intravaskuläre volumenexpansion und katecholamine. differenzierte empfehlungen zur schweregradabhängigen (stadium 0-iv) akuttherapie anaphylaktoider reaktionen unter berücksichtigung der führenden klinischen symptomatik (kutan, pulmonal oder kardiovaskulär) wurden in einer interdisziplinären konsensuskonferenz erarbeitet (ahnefeld et al. 1994) . hinsichtlich der differenzialtherapie mit volumenersatz, katecholaminen, histaminantagonisten, glukokortikoiden und theophyllin wird auf die detaillierte abhandlung der konsensuskonferenz verwiesen. allgemeine therapiemaßnahmen und -prinzipien entfernung des auslösenden agens. das auslösende agens muss an der eintrittspforte entfernt (z. b. insektenstachel) oder die weitere systemische absorption vermindert (z. b. anlegen eines tourniquets bei eintrittspforte an einer extremität) bzw. die antigenzufuhr gestoppt werden. in bestimmten situationen (z. b. insektenstich) kann die subkutane injektion von adrenalin (0,1-0,2 mg) -möglichst in nähe der einstichstelle -sinnvoll sein. freie atemwege. freie atemwege müssen sichergestellt sein -schon ab stadium i (leichte allgemeinreaktion) o 2 -zufuhr über eine maske, bei bedrohlicher hypotension und/oder dyspnoe endotracheale intubation und 100%ige o 2 -beat-mung. ein larynxödem kann die intubation erschweren oder sogar unmöglich machen: in solchen fällen kann die koniotomie lebensrettend sein. entwickelt sich eine obstruktion der oberen atemwege, so wird eine intubation des patienten erforderlich, die dann meist schwierig ist. eine kontrollierte mechanische beatmung mit positivem endexspiratorischem druck wird häufi g notwendig, wenn sich hypoxie und lungenödem entwickelt haben. lagerung. die flachlagerung des patienten, möglichst trendelenburg-lagerung (ausnahme: lungenödem) wird empfohlen. volumen. schon ab stadium i (leichte allgemeinreaktion) sollte über einen zuverlässigen, möglichst großlumigen venösen zugang, möglichst rasch volumen (elektrolyt-und kolloidale lösungen) substituiert werden. die kausale therapie der relativen hypovolämie ist die adäquate volumensubstitution. schwere anaphylaktoide reaktionen erfordern nicht selten die zufuhr größerer flüssigkeitsmengen innerhalb kurzer zeit (2-3 l in 20-30 min). dies ist nur über einen großlumigen zugang möglich. auch nach primärer kreislaufstabilisierung können im verlauf der nächsten stunden infusionen von mehreren litern erforderlich werden. gelingt die zufuhr ausreichender volumina in kürzester zeit, sind häufi g keine weiteren therapeutischen maßnahmen erforderlich. dies gilt offenbar v. a. für anaphylaktoide reaktionen in der perioperativen phase, die sich primär oder ausschließlich am kardiovaskulären system manifestieren. bei kardial grenzwertig kompensierten patienten sollte die zufuhr großer volumina unter erhöhter vorsicht erfolgen, um eine akute kardiale dekompensation zu vermeiden. im stadium iii (bedrohliche allgemeinreaktion: anaphylaktischer schock) ist die alleinige gabe von elektrolytlösungen unzureichend. höhermolekulare lösungen sind zu bevorzugen. albumin bietet dabei gegenüber den künstlichen plasmaersatzmitteln keine vorteile. hydroxyäthylstärke (hes) mit einem mittleren molekulargewicht (hes 200.000) kann als volumenmittel der wahl zur soforttherapie anaphylaktoider reaktionen angesehen werden. begrenzt wird der einsatz durch die maximal zu verabreichende menge von etwa 20-30 ml/kgkg/tag (ca. 1,5 l beim erwachsenen). eine darüber hinaus erforderliche volumenzufuhr sollte bevorzugt mit elektrolytlösungen erfolgen. adrenalin. die pharmakologische behandlung der anaphylaxie beruht in erster linie auf dem einsatz von adrenalin, womit sowohl die hypotension als auch die bronchokonstriktion wirksam bekämpft werden kann. adrenalin kann intravenös, intramuskulär (sofortige selbsttherapie von patienten mit bekannter allergie nach allergenexposition im stadium ii -ausgeprägte allgemeinreaktion -mit kommerziell erhältlichen fertigspritzen, fastjekt), sublingual, endotracheal oder als dosieraerosol verabreicht werden. eine eindeutige indikation zur parenteralen verabreichung von adrenalin besteht im stadium iii (bedrohliche allgemeinreaktion: schock), jedoch kann der einsatz bei zunehmender hypotension trotz adäquater volumengabe schon im späten stadium ii erwogen werden. die intravenöse verabrei-tabelle 6.21. differenzialtherapie anaphylaktischer/anaphylaktoider reaktionen. (mod. nach ahnefeld et al. 1994; . kutane um eine ausreichend genaue dosierung zu erreichen, wird hierbei 1 mg (1 ml) adrenalin in einer 10-ml-spritze mit 9 ml nacl 0,9% aufgezogen. eine maximaldosis von 1 mg adrenalin (med nach dab) sollte in der regel nicht überschritten werden. steht kein intravenöser zugang zur verfügung, kann adrenalin endobronchial appliziert werden. in diesem fall sollte adrenalin etwa 2-bis 3-mal höher als bei intravenöser gabe dosiert werden (ca. 0,3 mg) und mit nacl 0,9% oder aqua bidest. auf ein volumen von etwa 5 ml verdünnt werden; evtl. erforderliche wiederholungsgaben sollten möglichst intravenös erfolgen. die wirkung von adrenalin hält bei endobronchialer gabe länger an als bei intravenöser verabreichung. eine pulmonale symptomatik (bronchospasmus) im stadium ii oder iii kann durch inhalative applikation von adrenalin oder -bei nichtverfügbarkeit -mit den zur asthmatherapie verwandten β 2 -sympathomimetika wirksam behandelt werden; die dosierung richtet sich nach den nebenwirkungen. die maximaldosis ist erreicht, wenn tachykardie und etwas später tremor auftreten. die therapie kardiovaskulärer reaktionen mittels adrenalininhalationen ist jedoch nicht gesichert. man sollte jedoch an diese möglichkeit denken, wenn keine parenteral applizierbaren katecholamine zur verfügung stehen. besondere aufmerksamkeit und vorsicht erfordert der einsatz von adrenalin bei patienten mit khk oder arrhythmien. in diesen fällen kann adrenalin zu einer akuten koronarinsuffi zienz bis hin zum myokardinfarkt bzw. kammerfl immern führen. andererseits muss gerade bei patienten mit khk der perfusionsdruck ausreichend hoch gehalten werden. dies gelingt im stadium iii häufi g nur durch gleichzeitige gabe von volumen und einem vasokonstriktor in ausreichender dosierung. stationäre aufnahme. alle patienten mit einer anaphylaktischen reaktion müssen stationär aufgenommen und kontinuierlich überwacht werden, auch dann, wenn die symptome rasch auf eine adäquate 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anti-infl ammatory agent cell adhesion molecules and leucocyte traffi cking in sepsis hinds: persistent neuromuscular and neurophysiologic abnormalities in long-term survivors of prolonged critical illness pulmonalarterienkatheter-monitoring bei kritisch kranken -eine momentaufnahme effi cacy and safety of intravenous levosimendan compared with dobutamine in severe low-output heart failure (the lido study): a randomised double-blind trial hemodynamic response to coupled plasmafi ltration-adsorption in human septic shock critical care medicineprinciples of diagnosis and management in the adult, 2 nd edn severity of multiple organ failure (mof) but not of sepsis correlates with irreversible platelet degranulation impaired energy metabolism in hearts of septic baboons: diminished activities of complex i and complex ii of the mitochondrial respiratory chain exzessive gewebespeicherung von kolloiden im retikuloendothelialen system results of primary percutaneous transluminal coronary 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the neurologic outcome after cardiac arrest for the european epinephrine study group (1998) a comparison of repeated high doses and repeated standard doses of epinephrine for cardiac arrest outside the hospital endotoxin-induced cross-tolerance to gram-positive sepsis cardiopulmonary resuscitation by chest compression alone or with mouth-to-mouth ventilation cardiodepressant factors anaphylaxis and plasma catecholamines anaphylactic shock in man: report of two cases with detailed hemodynamic and metabolic studies herausgegeben vom vorstand der deutschen gesellschaft für kardiologie -herz-und kreislaufforschung teil 2: akutes koronarsyndrom mit st-hebung (myokardinfarkt) cardiogenic shock complicating acute coronary syndromes predictors of cardiogenic shock after thrombolytic therapy for acute myocardial infarction platelet glycoprotein iib/iiia blockade and outcome of cardiogenic shock complicating acute coronary syndromes without persistent st-segment elevation critical care medicine 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infarctions complicated by cardiogenic shock cardiogenic shock complicating acute myocardial infarction-etiologies, management and outcome: a report from the shock trial registry. should we emergently revascularize occluded coronaries for cardiogenic shock? ventricular septal rupture causing cardiogenic shock: clinical profi le, timing and outcome der akute myokardinfarkt -ein unterschätzter und oft unerkannter mortalitätsfaktor bei septischen patienten? effect of hemofi ltration on hemodynamics and systemic concentrations of anaphylatoxins and cytokines in human sepsis hemofi ltrate from patients with severe sepsis and depressed left ventricular contractility contains cardiotoxic compounds cardiogenic shock cardiogenic shock hrsg) expertenforum: hämodynamisch aktive substanzen in der intensivmedizin apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction neuromuscular sequelae of critical illness themenheft: sepsis und gerinnung neurological complications of sepsis: critical illness polyneuropathy and myopathy spinaler schock the microcirculation unveiled prophylactic intravenous administration of standard immune globulin as compared with core-lipopolysaccharide immune globulin in patients at high risk of postsurgical infection for the shock investigators (2000) cardiogenic shock with non-st-segment elevation myocardial infarction: a report from the shock trial registry cardiogenic shock caused by right ventricular infarction splanchnic hemodynamics in critical illness splanchnic vasoregulation during mesenteric ischemia and reperfusion in pigs mortality probability model (mpm ii 0-72) bei patienten einer kardiologischen intensivstation use of percutaneous cardiopulmonary support of patients with fulminant myocarditis and cardiogenic shock for improving prognosis hemoadsorption therapy for sepsis syndromes stress doses of hydrocortisone reduce severe systemic infl ammatory response syndrome and improve early outcome in a risk group of patients after cardiac surgery early repair of postinfarction ventricular septal rupture sustained hemodynamic effects of intravenous levosimendan nitroprusside in critically ill patients with left ventricular dysfunction and aortic stenosis high-dose vasopressin is not superior to norepinephrine in septic shock apache ii: a severity of disease classifi cation system ) 40jähriger patient ohne vorerkrankung mit akuter schocksymptomatik und schwerer verbrauchskoagulopathie akutes cor pulmonale bei lungenembolie -entscheidender prognostischer faktor und kritischer parameter für die auswahl der therapeutischen strategie mechanical ventilation in conjunction with the intra-aortic ballon pump improves the outcome of patients in profound cardiogenic shock effects of tirofi ban on hemostatic activation and infl ammatory response during cardiopulmonary bypass thrombolysis plus aortic counterpulsation: improved survival in patients who present to community hospitals with cardiogenic shock volumentherapie bei hypovolämie und schock reduced incidence of postoperative infections following intravenous application of an iga-and igm-enriched immunoglobulin preparation in anergic patients undergoing cardiac surgery myocardial dysfunction in the patient with sepsis improved outcome of apache ii score-defi ned escalating systemic infl ammatory response syndrome in patients post cardiac surgery in 1996 compared to 1988-1990 aktiviertes protein c, antithrombin shock: classifi cation, pathophysiology and approach to management der schockpatient auf der notaufnahme und der intensivstation reactive nitrogen and oxygen species: role and evidence of their production in humans bilateral massive adrenal haemorrhage complicating anaphylactic shock: a case report beatmung beim herzkranken prognosis in rupture of the ventricular septum after acute myocardial infarction and role of early surgical intervention complicated acute myocardial infarction requiring mechanical ventilation in the intensive care unit: prognostic factors of clinical outcome in series of 157 patients sccm/esicm/accp/ats/sis international sepsis defi nition conference distinct post-receptor alterations generate gene-and signal-selective adaptation and cross-adaptation of tlr4 and tlr2 in human leukocytes poly(adp-ribose) synthetase as a novel therapeutic target for circulatory shock abciximab suppresses the rise in levels of circulating infl ammatory markers after percutaneous coronary revascularization safety and effectiveness of inhaled nitric oxide and tirofi ban for acute coronary syndromes -a report from the cardiovascular and renal advisory panel of the food and drug administration effect of early revascularisation in cardiogenic shock complicating acute myocardial infarction. a single center experience multiple-center, randomized, placebo-controlled, double-blind study of the nitric oxide synthase inhibitor 546c88: effect on survival in patients with septic shock zytokine: klassifi kation, rezeptoren, wirkungsmechanismen infl uence of interleukin-10 polymorphisms on interleukin-10 expression and survival in critically ill patients spinal nitric oxide participates in the control of the blood pressure during graded hemorrhage in the conscious rat time-and surgery-dependent effects of lipopolysaccharide on gut, cardiovascular and nitric oxide functions mechanische kreislaufunterstützung bei akuter und chronischer herzinsuffi zienz -eine standortbestimmung unter besonderer berücksichtigung der entwicklung in deutschland effects of endotoxin on the guinea pig heart response to ischemia reperfusion injury diuretics, mortality and nonrecovery of renal function in acute renal failure neue therapieansätze bei der prähospitalen und hospitalen schockbehandlung -hyperton-onkotische lösungen und vasopressin severe aortic stenosis and reduced ejection fraction: intensive care treatment the clinical profi le of patients with suspected cardiogenic shock due to predominant left ventricular failure: a report from the shock trial registry outcome and profi le of ventricular septal rupture with cardiogenic shock after myocardial infarction: a report from the shock trial registry acute myocardial infarction complicated by systemic hypoperfusion without hypotension: report of the shock trial registry successful treatment of acute myocardial infarction shock by combined percutaneous transluminal coronary recanalization and percutaneous coronary angioplasty detection of circulating tumor necrosis factor after endotoxin administration sepsis -epidemiologie und ökonomische aspekte effects of early treatment with immunoglobulin on critical illness polyneuropathy following multiple organ failure and gram-negative sepsis on behalf of russlan study investigators (2002) safety and effi cacy of a novel calcium sensitizer, levosimendan, in patients with left ventricular failure de to an acute myocardial infarction organ apoptosis in the septic patient: a potential therapeutic target? cardiogenic shock hormonal and hemodynamic profi le of an anaphylactic reaction in man possible role of increased oxidant stress in multiple organ failure after systemic infl ammatory response syndrome yearbook of intensive care and emergency medicine acc expert consensus document. present use of bedside right heart catheterisation in patients with cardiac disease septischer schock und systemisches entzündungsreaktions-syndrom anaphylaxie und allergie -empfehlungen für die notfalltherapie septic cardiomyopathy cytokines and the heart -molecular mechanisms of septic cardiomyopathy fortschritte in der therapeutic hypothermia after cardiac arrest. an advisory statement by the advancement life support task force of the international liaison committee on resuscitation terlipressin for norepinephrine-resistant septic shock enteral nutrition with eicosapentaenoic acid, γ-linolenic acid, and antioxidants reduces alveolar infl ammatory mediators and protein infl ux in patients with acute respiratory distress syndrome pathophysiology of septic encephalopathy: a review critical care medicine -principles of diagnosis and management in the adult, 2 nd edn benefi cial effects of short-term vasopressin infusion during severe septic shock right ventricular involvement in myocardial infarction and cardiogenic shock a comparison of standard cardiopulmonary resucitation and active compression-decompression resuscitation for out-of-hospital cardiac arrest esc-erc recommendations for the use of automated external defi brillators (aeds) in europe kardiogener schock: pathomechanismen, klinischer verlauf, therapeutische ansätze und perspektiven surgical trauma affects the proinfl ammatory status post cardiac surgery to a higher degree than cardiopulmonary bypass pulmonary artery consensus conference: consensus statement bilateral massive adrenal hemorrhage: early recognition and treatment profound reversible myocardial depression after anaphylaxis relative myocardial depression in normotensive sepsis the effects of coronary artery disease on cardiac function in nonhypotensive sepsis toll receptors and sepsis right ventricular function in early septic shock states open randomized phase ii trial of an extracorporeal endotoxin absorber in suspected gram-negative sepsis desensitization of rat cardiomyocyte adenyl cyclase stimulation by plasma of noradrenalinetreated patients with septic shock hämostasestörungen im umfeld von sepsis und sirs intraoperatives anästhesiologisches management bei patienten mit pulmonaler hypertonie early goal-directed therapy in the treatment of severe sepsis and septic shock allgemeine intensivtherapie early revascularization in cardiogenic shock -a positive view of a negative trial a comparison of albumin and saline for fl uid resuscitation in the intensive care unit help apheresis in the treatment of sepsis impact of thrombolysis, intra-aortic balloon pump counterpulsation, and their combination in cardiogenic shock complicating acute myocardial infarction: a report from the shock trial registry. should we emergently revascularize occluded coronaries for cardiogenic shock? warum sollte der intensivmediziner der autonomen dysfunktion seiner patienten beachtung schenken? lebensbedrohliche akute intoxikationen durch kardio-und vasotoxisch wirkende medikamente und drogen autonomic function in the icu patient pulmonary and left ventricular decompression by artifi cial pulmonary valve incompetence during percutaneous cardiopulmonary bypass support in cardiac arrest the ability of the simplifi ed acute physiology score (saps ii) to predict outcome in coronary care patients katecholamine im kardiogenen schock: hilfreich, nutzlos oder gefährlich critical care medicine -principles of diagnosis and management in the adult, 2 nd edn mortality in emergency department sepsis (meds) score: a prospectively derived and validated clinical prediction rule a review of randomized controlled trials using therapeutic apheresis effect of nesiritide versus dobutamine on short-term outcomes in the treatment of patients with acutely decompensated heart failure impaired β-adrenergic receptor stimulation of cyclic adenosine monophosphate in human septic shock: association with myocardial hyporesponsiveness to catecholamines hemodynamic changes in human anaphylaxis practice parameter for the use of red blood cell transfusions -developed by the red blood cell administration practice guidelines development task force fo the college of american pathologists cardiogenic shock due to cardiac free-wall rupture or tamponade after acute myocardial infarction: a report from the shock trial registry attenuation by dexamethasone of endotoxin protection against ischaemia-induced ventricular arrhythmias serum cardiac troponin t as a prognostic marker in early sepsis a controlled trial of dichloroacetate for treatment of lactic acidosis in adults der darm als immunologisches organ apheresis as therapy for patients with severe sepsis and multiorgan dysfunction syndrome plasma exchange as rescue therapy in multiple organ failure ncluding acute renal failure treatment strategies for acute myocardial infarction complicated by cardiogenic shock in a community hospital enterale immunonutrition: wann, für wen, welche zukunftsperspektiven gibt es? the cardiovascular response of normal humans to the administration of endotoxin cardiac disorders in penicillin-induced anaphylaxis low-dose vasopressin in the treatment of septic shock in sheep myocardial ischemia-reperfusion injury: role of the peroxynitrite-poly(adp-ribose) polymerase pathway change in the ratio of interleukin-6 to interleukin-10 predicts a poor outcome in patients with systemic infl ammatory response syndrome task force of the american college of critical care medicine, society of critical care medicine (1999) practice parameters for hemodynamic support of sepsis in adult patients in sepsis outcome of patients sustaining acute ischemic mitral regurgitation during myocardial infarction the effectivenes and relative effectiveness of intravenous inotropic drugs acting through the adrenergic pathway in patients with heart failure -a meta-regression analysis reversal of cardiogenic shock by percutaneuos left atrial to femoral artrial bypass-systems short-and long-term hemodynamic effects of intra-aortic balloon support in ventricular septal defect complicating acute myocardial infarction role of nitric oxide in sepsis and ards (update in intensive care and emergency medicine 24) hrsg) expertenforum: hämodynamisch aktive substanzen in der intensivmedizin hrsg) expertenforum: hämodynamisch aktive substanzen in der intensivmedizin cardiogenic shock due to acute severe mitral regurgitation complicating acute myocardial infarction: a report from the shock trial registry nephritis epidemica auf einer interdisziplinären notaufnahme -fallsammlung im rahmen einer endemie-erfassung der ostalbregion und literaturübersicht hemodynamic and metabolic effects of low-dose vasopressin infusions in vasodilatory septic shock inhibition of 38 mitogen activated protein kinase: a novel strategy in sepsis? yearbook of intensive care and emergency medicine hemodynamic and metabolic therapy in critically ill patients outcome benefi t of intensive insulin therapy in the critically ill: insulin dose versus glycemic control on behalf of the working group on sepsis-related problems of the european society of intensive care medicine (1996) the sofa (sepsis-related organ failure assessment) score to describe organ dysfunction the role of bacterial superantigens in sepsis and treatment implications anaphylaktoide reaktionen in der prähospitalphase hemorrhage produces depression in microvascular blood fl ow which persists despite fl uid resuscitation sublingual capnometry: a new noninvasive measurement for diagnosis and quantitation of severity of circulatory shock a comparison of vasopressin and epinephrine for out-of-hospital cardiopulmonary resuscitation hrsg) expertenforum: hämodynamisch aktive substanzen in der intensivmedizin mechanische und elektrische therapie kardialer arrhythmien assessment of ivig for prophylaxis and therapy of sepsis akute septische kardiomyopathie: bestandteil des multiorganversagens in der sepsis? plasma atrial natriuretic peptide and brain natriuretic peptide are increased in septic shock: impact of interleukin-6 and sepsis-associated left ventricular dysfunction cardioprotective effects of monophosphoryl lipid a, a novel endotoxin analogue in the dog epidemiology of anaphylaxis in olmsted county: a population -based study effectiveness of the glycoprotein iib/iiia antagonist abciximab during percutaneous coronary interventions (pci) in clinical practice at a single high-volume center metabolic encephalopathy in critically ill patients suffering from septic or nonseptic multiple organ failure right ventricuar infarction as an independent predictor of prognosis after acute inferior myocardial infarction tnf-a and il-1a inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: evidence for primary impairment of mitochondrial function prospective evaluation of early abciximab and primary percutaneous intervention for patients with st elevation myocardial infarction complicated by cardiogenic shock: results of the reo-shock trial predictors of in-hospital mortality in 1333 patients with acute myocardial infarction complicated by cardiogenic shock treated with primary percutaneous coronary intervention (pci) -results of the primary pci registry of the arbeitsgemeinschaft leitende kardiologische krankenhausärzte (alkk) study on glucocorticoid receptors during intestinal ischemia shock and septic shock diagnosis of papillary muscle rupture after acute myocardial infarction by transthoracic and transesophageal echocardiography enzyme-independent formation of nitric oxide in biological tissues transfundieren sie thrombozytenkonzentrate bei einem thrombozytenabfall auf <5000/mm 3 (5×10 9 /l), unabhängig vom vorliegen einer blutung, weiterhin bei thrombozytenzahlen von 5000-30.000/mm 3 (5-30×10 9 /l) und vorhandenem signifi kanten blutungsrisiko. höhere thrombozytenzahlen wie ≥50.000/mm 3 (50×10 9 /l) sind für chirurgische und invasive prozeduren erforderlich (empfehlungsgrad e) key: cord-004879-pgyzluwp authors: nan title: programmed cell death date: 1994 journal: experientia doi: 10.1007/bf02033112 sha: doc_id: 4879 cord_uid: pgyzluwp nan it is widely held that all developmental cell death is of a single type (apoptosis) and that neuronal death is primarily for adjusting the number of neurons in a population to the size of their target field through competition between equals for target-derived factors. we shall draw on our research and on that of others to criticize these views and replace them by the following. at least three types of neuronal death occur, only one of which resembles apoptosis; a neuron can choose between several self-destruct mechanisms depending on the cause of its death. the purpose of the death is to regulate connectivity, not neuron number. competitors for trophic factors are unequal, and many losers have made axonal targeting errors. a neuron's survival and differentiation depend on multiple anterograde and retrograde signals. activity affects retrograde signals and some but not all anterograde ones. the pattern of activity is more important than the overall amount. in rodents, the period of naturally occuring cell death of motoneurons is followed by a period of supersensitivity to axonal injury. thus, in newborn rodents lesion of the facial nerve leads to a rapid degeneration of the injured motoneurons. we have tested whether overexpression, in rive, of the bcl-2 proto-oncogene was capable of preventing death of axotomized motoneurons. to address this question we used transgenic mice whose motoneurons overexpress the bcl-2 protein. one of the two facial nerves of newborn mice was transected on the 2nd-3rd post-natal day. seven days after the lesion, the morphology of the facial nuclei was analyzed. in control mice, and when compared to the intact nucleus, 70 to 80 % of axotomized motoneurons had disappeared. in contrast, in the transgenic animals, the number of motoneurons on the lesioned side remained unchanged when compared to the eontralateral nucleus. furthermore, their axons remained visible up to the distal lesion site. these experiments show that, in rive, motoneurons overexpressing the bcl-2 protein survive after axotomy, and suggest that, in rive, bcl-2 protect neurons from experimentally induced cell death and could be a target for treatment of motoneurons degenerative diseases. messmer s., mattenberger l., sager y., blatter-garin m-c., pometta d., kate a., james r.w. drpt de mrdeeine, drpt. de pharmacologie, div. de neurophysiologie clinique, facult6 de mrdecine, gen~ve. clusterin is a widely expressed glycoprotein, highly conserved across species. numerous functions have been postulated for this protein. the most important are roles in lipid transport, as elusterin is associated with apolipoprotein ai in hdl, complement regulation and tissue remodelling, in particular during cell death and differentiation. using cultures of rat spinal cord neurones (90% neurons and 5-10% non-neuronal cells), we have studied the expression of clusterin and ape e in glutamate-induced neuronal cell death to examine potential roles in lipid management. up-regulation of the two proteins was observed. clusterin and ape e appear in the conditioned medium respectively 15h and 7.5h after incubation with glutamate. control studies, in the presence of a noncompetitive nmda receptor agonist showed the secretion of clusterin and ape e to be diminished by >60%. no up-regulation of either protein was observed in complementary studies with exclusively non-neuronal cell cultures. the cellular origin of the 2 secreted proteins is presently under investigation. programmed cell death and tissue remodelling are consequences of hormonally induced restructuring of the rat ventral prostate after castration and the rat mammary gland after weaning. we used the "differential display"-method (liang and pardee, 1992, science 257:967) to detect and isolate edna fragments whose corresponding rnas are regulated either coincidentally, or in an organ specific fashion during mammary gland involution and postcastrational prostate regression. partial sequencing of 12 clones revealed high, but not absolute homology of 5 fragments with sequences, previously characterized in different biological contexts. these five encode functions which could be anticipated to be important for cell growth and/or programmed cell death, we are presently investigating the functions of several of these transcripts in cell culture and in rive. antisense oligos are being employed in vivo to determine whether these genes contribute to the phenotype of programmed cell death. b epitopes derived from the envelope gp52 glycoprotein (ep3) or from the viral superantigen of mmtv have been incorporated into inert or live vaccines. the inert vaccine consists of purified chimeric proteins which contain the b epitopes alone or fused to multimeric promiscuous t helper epitopes from tetanus toxin. mice were immunized subcutaneously with these chimeric proteins. the live vaccine consists of an avirulent strain of salmonella typhimurium which expresses the mmtv epitopes in the form of chimeric proteins fused to the nucleocapsid protein of hepatitis b virus. this vaccine is given to mice in one oral dose. the level, duration and isotype of the immune response generated by each vaccine have been measured and compared. the level of protection has been investigated by systemically challenging immunized mice with the relzovims. a reduced binding of oxytocin (ot) occurs with aging in some, but not all, areas of the rat brain (arsenijevic et al., experientia 1993, 49, a75) . the candate putamen showed the most impressive loss of ot receptors. two other regions, the hypothalamic ventromedial nucleus (vmh) and the islands of caueja (icj) had also an important deficit of ot binding sites. on the other hand, these two regions were known to be sensitive to sex steroids. in the present work, we treated from 20 month old rats during one month with testosterone propionate (2 #g/kg s.c., once every 3 days) dissolved in oil. three rats of the same age injected with oil only served as controls. we labelled ot receptors throughout the brain of old rats using a 125i-labelled ligand specific for ot receptors. analysis of autoradiograms by an image analyzer revealed that the testosterone treatment increased ot binding sites in the vmh, in the icj, and, to a lesser extent, in the bed nucleus of the stria terminalis, a region also sensitive to sex steroids, by contrast, in the caudate putamen, the disappearance of ot receptors was not compensated. in conclusion, the decrease of ot receptors occurring in vmh and icj with aging can be reversed by administration of gonadal steroids. in contrast, the loss of ot receptors in the striatum appears to depend on another mecanism. vasopressin (avp) receptors are expressed transiently in the facial nucleus during development (tribollet et el., 1991, dev. brain res., 58, 13-24) . avp may therefore play a role in the maturation of neuromuscular connexions in the neonate rat, and possibly in the restanration of these connexions after nerve lesion in the adult. in order to investigate the latter proposition, we have sectionned the facial nerve in adult rats and used quantitative autoradiography to look at avp binding sites in the facial nucleus at various postoperative times. we observed a massive and transient increase of avp binding sites on the operated side. the number of facial avp binding sites reaches a maximum about one week after nerve section, remains stable during 2-3 weeks, then begin to decrease towards control level. the induction of avp receptors is markedly delayed if the proximal stump of the nerve is ligated. to assess whether other motor nuclei would also react to axotomy by up-regulating the expression of avp receptors, we have sectionned the hypoglossal nerve and the sciatic nerve. in both cases, the binding of avp receptor ligand increases massively in the respective motor nuclei, with a time-course similar to that found in the facial nucleus. altogether, our data suggest that central avp could be involved in the process of nerve regeneration. cytotoxic t-cell mediated apoptosis schaerer,e, karapetian,o.,adrian,m. and tschopp,j. inst.de biochimie, univ.de lausanne, 1066 epalinges. an apoptotic cell death mechanism is used by cytolytic t cells (ctl) to lyse appropriate target cells. ctl harbor cytoplasmic storage compartments, containing the lytic protein perforin and serineproteases (granzymes), whose content is released upon target cell interaction. we show that these granules are multivesieular bodies and that degranulation releases these intragranular vesicles (igv) having granzymes, t-cell receptor and yet undefined proteins associated. isolated igvs and perforin induce dna breakdown in target cells within 20 minutes. microscopic analysis demonstrates that igv specifically interact with target cell via the t-cell receptor and that their contents is taken up by the target cell. already 15 min. after interaction, 3 distinct igv proteins are found in the nucleus of the target cell.one of the molecules has been identified to be granzyme a, previously reported to be involved in apoptosis. we propose that lymphocytes transfer apoptosisinducing proteins to the nucleus of the target cells using vesicles as vehicles for delivery. cytotoxic t cells kill their targets by a mechanism involving membranolysis and dna degradation (apoptosis). recently, two sets of proteins have been proposed as dna breakdown-inducing molecules in t cells: granzyme a, b and tia-i. in this study, we cloned and further characterized the tia-i mouse homologue. aa sequence comparison with the human tia-1 showed an overall identity of 93%. devoid of a signal peptide, tia is yet localized to cytotoxic granules, probably targeted via a gly-tyr-motif. as tia-i, its mouse homolcgue contains three rnabinding domains. expression of tia during development shows a very strong signal in the brain and weaker signals in thymus, heart and other organs. during embryonic development several structures that contribute to organogenesis form transiently and are later eliminated by apoptosis. this pattern of tia expression could indicate its involvement in apoptosis. prostate involution occurs after castration in rats and is associated with the death by apoptosis of a large fraction of the epithelial cells. we have isolated several genes from a prostate involution bacteriophage lambda library using differential screening methods. among these clones, one d~monstrated an especially strong signal when used as a probe against northern blots of prostate mlhna obtained before, and at different times after castration. this gene is down-regulated after castration by 40-fold within 5 days. intramuscular injection of a testosterone depot resulted in complete restoration of expression within 24 hours. upon sequencing it became apparent that this clone has a high degree of homology to a known ndah dehydrogenase encoded in mitochondrial dna. the clone failed to hybridize to any transcripts from rat organs other than prostate. we are now in the process of isolating the htm~n hc~olog to this gene for use as a biomarker in study of benign hyperplasia and developing carcinoma. this gene is a possible indicator for testosterone-independent cell populations or of cells lacking ftl~ctional testosterone receptor. during the first three postnatal weeks the rat lung undergoes the last two developmental stages, the phase of alveolarization and the phase of microvascular maturation. the latter involves a decrease of the connective tissue mass in the alveolar septa and a merging of the two capillary layers to a single one. speculating that programmed cell death may play a role during this remodeling, we searched for the presence of apoptotie cells in rat lungs between days 10 and 24. lung paraffin sections were treated with y-terminal transferase, digoxigenin-dutp, and anti-digoxigeninfluorescein-f(ab)-fragments, and the number of fluorescent nuclei was compared between sections at different days. while the number of apoptotie ceils was low until the end of the second week and at day 24, we observed an about eight fold increase of fluorescent nuclei towards the end of the third week. we conclude that programmed cell death is involved in the structural maturation of the lung. brunner, a., wallrapp, ch., pollack, i, twardzik, t. and schneuwly, s. lehrstuhl genetik, biozentrum universit~t w~rzburg, mutants in the giant lens (g/l) gene show a strong disturbance in ommatidial development. in the absence of any gene product, additional phetoreceptors, cone cells and pigment cells develop. opposite effects can be seen in flies in which the gene product of the giant lens gene can be ectopically expressed by heat shock. a second very typical phenotype is the disturbance of photoreceptor axon guidance. molecular analysis of gil shows that it encodes a secreted protein of 444aa containing three evolutionary conserved cystein-motives very similar to egf-like repeats. we propose that gil functions as a secreted signal, most likely a lateral inhibitor for the development of specific cell fates and that gil, either directly or indirectly, is involved in targeting photoreceptor axons into the brain. the decrease in cellularity during scar establishment is mediated through apoptosis desmouliere, a., redard, m., darby, i., and g. gabbiani department of pathology, cmu, 1 rue michel server, 1211 gen~ve 4 dudng the healing of an open wound, granulation tissue formation is characterized by replication and accumulation of fibroblastic cells, many of which acquire morphological and biochemical features of smooth muscle cells and have been named myofibroblasts (sch0rch et el., histology for pathologists, t992). as the wound evolves into a scar, there is an important decrease in ceuuladty, including disappearance of myofibroblasts. the question adses as to which process is responsible for myofibroblast disappearance. during a previous investigation on the expression of (z-smooth muscle actin in myofibroblasts, we have obsewed that in late phases of wound healing, many of myofibroblasts show signs of apoptosis end suggested that this type of cell death is responsible for the disappearance of myofibroblasts (darby et al., lab. invest. 63:21, 1990) . we have tested this hypothesis by means of electron microscopy and morphometry and by in situ end-labeling of fragmented dna (wijsman et al., j. histochem. cytochem. 41:7, t 993) . our results show that the number of apoptotic cells increases as the wound closes and suggest that this may be the mechanism for the disappearance of myofibroblasts as well as for the evolution of granulation tissue into a scar. (supported by the swiss national science foundation, grant n~ s01-16 r. jaggl, a. marti and b. jehn. universit~t bern, akef, tiefenaustr. 120, 3004 bern at weaning the mammary gland undergoes a reductive remodelling process (involution) which is associated with the cessation of milk protein gene expression and apoptosis of milk-produclng epithelial cells. this process can be reversed by returning the pups to the mother within 1 day. elevated nuclear protein kinase a (pka) activity was observed from one day post-lactation, paralleled by increased c-los, junb, ]und and to a lesser extent c-]un mrna levels. ap-1 dna binding activity was transiently induced and the ap-1 complex was shown to consist principally of cfos/jund. oct-1 dna binding activity and oct-1 protein were gradually lost from the gland over the first four days of involution, whereas oct-1 m_rna levels remained unchanged. comparing nuclear extracts from normal mammary glands with nuclear extracts from glands which had been cleared of all epithelial cells three weeks after birth revealed that pka activation, ap-1 induction and oct-1 inactivation are all dependent on the presence of the epithelial compartment. the increased fos/jtm expression and the inactivation of oct-1 may be consequences of the increased pka activity. when involution is reversed, both, pica activity and ap-1 dna binding activity (and fos andjun mrna levels) are reduced to basal levels. our data suggests a role for pka and ap-1 on progranlmed cell death of manlnmry epithelial ceils. bcl-2~ does not require membrane attachment for its survival activity c. borner*, i. martinout, c. mattmann*, m. irmler*, e. sch&rrer*, j.-c. martinou-j-, and j. tschopp*. * institute of biochemistry, university of lausanne, 1066 epalinges, 1 institute of molecular biology, glaxo inc.,1228 plan los ouates. 8cl-2(z is a mitochondrial or perinuclear-associated oncoprotein that prolongs the life span of a variety of cell types by interfering with programmed cell death. how it exerts this activity is unknown but it is believed that membrane attachment is required. to identify critical regions in bcl-2o~ for subcellular localization and survival activity, we created by site-directed mutagenesis, various mutations in regions which are most conserved between the different bcl-2 species. we show here that membrane attachment is not required for the survival activity of bcl-2o< a truncation mutant of bcl-2(z lacking the last 33 amino acids (t3) including the hydrophobic domain is soluble, yet fully active in blocking apoptosis of sympathetic neurons induced by ngf deprivation or l929 fibroblasts induced by tnfc~ treatment. we further provide evidence for a putative functional region in bcl-2 which lies in the conserved domains 4 and 5 upstream of the hydrophobic cooh terminal tail. the breakdown of nuclear dna is considered to be a hallmark of apoptosis. we previously identified the perinuclear membrane localized dnase i as the endonuclease involved in the formation of oligonucleosomal-sized fragments (dna ladder). it is not clear how the nuclease is activated and has access to the dna. we show that in thymocytes induced to undergo apoptosis, lamin breakdown preceded dna laddering. by transfeeting hela cells with a constitutively active cdc2 mutant, nuclear envelope breakdown and typical apoptotic features (ehromatin condensation) were observed. moreover, co-transfection with cdc2 mutant and dnase i led to dna degradation. we propose that apoptosis can be induced by wrongly timed and hence abortive mitosis leading to uncontrolled nuclear membrane disintegration. s02-01 s02-04 platelet-derived growth factor (pdgf) is thought to play an active role in fibrosing diseases. bronchiolitis obliterans-organizing pneumonia (boop) is a condition characterized by intraluminal proliferation of connective tissue inside distal air spaces. to evaluate pdgf expression in boop we performed immunohistoehemistry on lung biopsies from 20 patients and 10 controls free of fibrosis. sedal sections were stained with an antibody against either pdgf or the monoeyte/macrophage marker cd68, in both groups the pdgf ~9 cells were essentially tissue macrophages. using point counting to measure volume fraction (vv) , pdgf-pesitive cells represented 4.65+1.63% (mean+sd) of the volume occupied by lung tissue in the boop cases, and 2,12+0.65% in the controls (!0<0,001). similarily, 10.73+4.69% of the lung tissue was occupied by cd68 e~ macrophages in the boop cases, compared to 5.37:~3.73% in the controls (p<o.005). a positive linear correlation was found between the v v of pdgf ~ cells and the v v of cd68@ cells on the 30 cases (r=g.50 p<0.0o5). we conclude that boop is characterized by a recruitment of tissue macrophages and that a significant proportion of them express pdgf. as pogf can also induce the macrophage chemo-attractant mcp-1, we speculate that maerophages are involved in a positive feed back regulation, and may play a pivotal role in the pathogenesis of boop. barrier lesions in hydrostatic pulmonary edema d.wu, h.bachofen, u.gyger and e.r. weibel department of anatomy, university of bern, ch 3012, bern pulmonary edema induced in isolated perfused rabbit lungs by moderate elevation of perfusion pressure (14 tort ) results in the formation of characteristic bleb-like extensions of the thin cytoplasmic leaflet of alveolar epithelium ( bachofen et al. am rev resp dis. vo1.147. pp989-996,1993) . in this study, we show by means of electron microscopy and morphometry that the frequency of the blebs correlate with the distribution of alveolar edema fluid. we find that 5% of the epithelial surface is involved in the formation of blebs. three-dimensional reconstruction showed that blebs had near-spherical shape and that their opening toward the interstitium is irregular. these finding demonstrate a great plasticity of epithelial cell extensions when subjected to moderate pressure stress. at high pressure the cell linings demonstrate clear breaks. two major roles have been defined for no: cellcell communication mediated by the stimulation of cgmp synthesis, and cytotoxicity by direct interaction of the free radical no with cellular target. to investigate these effects in human epithelia, which constitute a major source of no in man, we introduced the murine inos sequence into sv40-t immortalized human bronchial epithelial cells (beas-2b). inos activity was higher than 100 pmol citrulline/min/ mg prot in the first passages of inos transfected cells, but it decreased by subculturing. no inos activity was detected in cells transfected with control vector. in inos transfected cells, no stimulated a cgmp synthesis and c-los expression which could be inhibited by addition of lnma. thus, in human epithelial cells no is able to significantly alter signal transduction pathway. partial pneumoneetomy, i.e. the resection of lung tissue, is known to initiate compensatory growth in the remaining lobes. under appropriate conditions this results in a complete restoration of lung parenchymal structure and function. unfortunately the molecular mechanisms controlling the onset of this compensatory growth are poorly understood. the aim of our study was to find and eharacterise genes which are up or down regulated during this time. for isolating such genes, we chose the differential display approach (p.liang and b.pardee 1992, science 257:p 967). therein a 3'end fragment of a subset of reverse transcribed mrnas is amplified by the polymerase chain reaction. the fragments can be separated on a dna sequencing gel. genes differentially expressed in pneumonectomised and sham-treated rats show different patterns on these gels. the fragments of such genes can be isolated from the gel and cloned into vectors in order to provide tools to characterise their gene. a set of such genes, some up-regulated and others downregulated following pneumonectomy, have been isolated, partially sequenced and characterised. time periods. our new plasma marker consisting of highly concentrated gold nanospheres was infused via a femoral vein catheter into the right atrium of anaesthetised and ventilated rabbits. five or 10 seconds after start of infusion, the blood flow was stopped and the lung was fixed by instillation. silver enhanced paraffin sections revealed labeled and unlabeled blood vessels. electron-microscopic sections showed various plasma gold particle concentration. morphometric analysis of electron micrographs showed an increase of the portion of marked capillaries from shorter to longer circulation times. based on our results, we conclude the existance of inhomogeneous plasma flow velocities within the pulmonary microvascular bed. (supported by snsf grant 31-30946.91) in schizosaccharomyces pombe pairing of meiotic chromosomes is not accompanied by the formation of a tripartite synaptonemal complex (sc), a structure which connects the homologs in most other eukaryotes. meiotic nuclei revealed novel structures ("linear elements") that might be related to the axial elements ($c precursors) of other organisms and function in meiotic chromosome pairing, recombination, and segregation (baler et al. (1993) jcb 121:241). to get further insight into the behavlour of meiotic chromosomes, we performed fluorescence in situ hybridizations on spread nuclei with probes from different chromosomal regions. interestingly, all centromeres and telomeres become clustered during meiotic prophase. the centl:omeres are paired already before meiosis, whereas the telomeres initiate pairing specifically during meiotic prophase, followed by pairing of interstitial regions. painting of whole chromosomes revealed that the homelogs occupy together a specific territory in the nucleus. an expression vector containing the vai gene read by rna polymerase ill, injected into xenopus zygotes, yields high levels of rna in virtually all embryonic cells. anti sense fina is produced from a segment of the xhoxla gene inserted in opposite orientation into the vai gene. immunological staining of whole mount embryos shows that target mrna level and xhoxla protein expression of xhoxla protein is diminished in about half of the antisense treated individuals. accordingly, nearly half of the embryos develop typical malformations in regions where the xitoxla gene is normally expressed. somites el the anterior trunk are heavily disorganized and mesodermal denvalives surrounding the intestinal tract are reduced or missing. antisense inhibition, through production of rna in situ from expression vectors, thus represents a useful tool to study the functional role of zygotic genes in xenopus development. cloning and characterization of the mouse homolog to s.cerevisiae sep1 encoding a dna strand exchange and homologous pairing protein vladimir bashkirov and wolf-dietrich heyer. institute of general microbiology, university of bern, baltzer-strasse 4, 3012 bern. homologous pairing and dna strand exchange are the central steps postulated in the current models of genetic recombination. to date no mammalian gene encoding a homologous pairing protein has been cloned. to get insight into the mechanism of recombination in higher eukaryotes we were interested to clone such genes. protein sequence comparison of the s.cerevisiae homologous pairing protein, sepi, and its homolog from s.pombe, p140 ex~ revealed several conserved stretches of amino acids in their nh2-terminal regions. using fully degenerate primers and mouse testes edna as a template for the polymerase-chain-reaction a product with the expected length has been amplified. this 360 bp dna was shown to encode a putative polypeptide with high degree of homology to both proteins from the two yeasts. the mouse pcr-product was used as a hybridization probe for screening a 1 gtlo mouse testis cdna library. after several rounds of successive screening a total of about 4 kb of mouse edna (msep1) was cloned and sequenced. the putative translation product revealed high homology throughout the entire sequence to its s.pombe and s.cerevisiae counterparts, 38 % and 41% of amino acid identity, respectively. the msep1 sequence was shown to be unique in the genome and the chromosomal gone is likely to contain many introns. we want to establish a functional full length edna clone of msepi and characterize the gone and protein product. we have earlier developed a cell free system to study recombinational repair of dna double strand breaks and deletions. the method allowed us to purify and characterize a high molecular weight multiprotein complex (rc-1), which catalyzes dna recombination. a dna polymerase and dna ligase as well as other enzymatic activities copurify with rc-1.we have commenced an investigation of homologous dna recombination in nuclear extracts which were prepared fzom normal and mutated mouse thymocytes. the scid mutation in mice causes an aberrant v(d)j joining reaction, an elevated sensitivity to ionizing radiation and a defect in recombinational repair. moreover, the normal mouse thymocytes were sorted into the cd4/cd4 double negative (dn), double positive (dp), and single positive (sp) subclasses and their nuclear extracts, and extracts from rag-2 -/-mice, were tested. only the scid mice and the cd4/cd8 sp thymocyte extracts were inactive in the recombinational repair reaction. this indicates a development stage specific activity and a scid specific defect of recombinational repair in thymocytes. restoration of the recombination activity was observed in thymocyte extracts derived from scid, which express a wansgene for the t-cell receptor i~ chain. a protein could be purified from normal thymus cells to near homogeneity which specifically rescues the recombination activity in scid extracts. this protein, srsp ~cid recombination stimulatory ~otein), migrates as a single band of app. 7 2 kda in sds polyacrylamide gel electrophoresis. the ade6-m26 mutation in the ade6 gene of the fission yeast schizosaecharomyces pombe gives rise to a hot spot for meiotic homologous recombination. to address whether transcription plays a role in m26 activity, the effect of the deletion of the ade6 promoter in combination with either the m26 and m375 mutations on recombination frequency was studied at the tetrad level. deletion in eis of the promoter abolishes m26 hot spot activity relative to the m375 control deletion strain. it is possible that deletion of the promoter has eliminated a sequence necessary for m26 hot spot activity, rather than lack of transcription being responsible for the loss of m26 activity. the role of transcription in m26 hot spot activity is currently being examined. we are analysing meiosis in a temperature sensitive top2 mutant. complementary temperature-shift experiments showed that topoisomerase ii is required for both meiotic divisions; early meiotic events are not impaired. in a meiotic time course, dapi staining revealed that the cells are blocked before the first meiotic division. interestingly, in meiotic spreads we found that the spindle pole body (spb) cycle is not affected by the block: the final arrest point showed 1 nucleus with 4 completely separated spbs. we want to analyse the number of spbs and spindle formation with immunofluorescence. we are planning to analyse the course of meiosis in a top2 rec7 double mutant (rec7 is recombination deficient) to see if topoisomerase ii activity is necessary for separation of recombined chromosomes. analysis of meiosis in a top2 rec8 double mutant (rec8 is recombination deficient and does not form linear elements) could give us a clue about the function of the linear elements. genetic recombination is able to induce cancer and to mediate its further progression. in cells from cancer predisposed individuals, mitotic recombination can lead to loss of heterozygous tumor suppressor genes as is e.g. observed in the hereditary form of retinoblastoma. one mechanism for gene loss is inter-or intrachromosomal recombination involving short duplicated sequences. our aim is to identify xenobiotics that are able to induce mitotic recombination and to elucidate the involved molecular mechanisms. for that purpose we have constructed transgenic d. melanogaster (1). rn.). cell lines. cells were stably transfeeted with an extrachromosomal dna element providing a putative recombination substrate+ "l%e constructed element contained two ta'uncated fragments of the neomycin resistance gene (nee r) interrupted by a hygremycin resistance marker (hygr). the hyg r insert was flanked on both sides by identical 352 bp fragments of nee r, different d. m. promoters shown to be constitutively active in various d. m. tissues were inserted 5' to both e. coil genes. restoration of a functional nee r gone may occur by deletion of the hyg r after recombination between the homologous regions. stably transfected schneider line 2 cells were obtained and preliminary results concerning chemical induction of genetic rearrangements will be shown. fleck, 0., sch~r, p. and kohli, j., institute of general microbiology, baltzerstr. 4, 3012 bern to detect mismatch-binding proteins of s. pombe we performed band shift assays with radiolabeled oligonucleotides containing a defined mismatch. we found two distinct mismatch-binding activities. one shows high affinity to most of the mismatches, but is absent for c/c. this protein might belong to the general mismatch-repair system, which is thought to be related to the bacterial muts dependent pathway. the second activity preferentially binds to those mismatches containing a cytosine and therefore represents a novel type of mismatch-binding protein. baur m., sch~r p. and kohli j., institute of general microbiology, baltzerstrasse 4, ch-3012 bern homologs of the salmonella typhimurium (mutl, s and 59 and streptococcus pneumoniae (hexa, b) genes involved in mismatch repair have been identified in several distantly related organisms. so far no mismatch repair gone is known in schizosaccharomyces pombe. in order to get more insight into the ~chanisals of mismatch repair in s. pombe we started the cloning of a mutl homolog. degenerated oligonucleotide primers based on conserved regions of mutl homologs were used in the polymerase chain reaction (fcr) to amplify a corresponding dna fragment from s. pombe. a dna fragment of about 220 bp was amplified whose deduced amino acid sequence shared a high degree of homology with paiql of saccharomyces cerevisiae and mutl, hexb. with the help of this dna fragment the gone was isolated from a genomic dna library by colony hybridization. sequence analysis of this mlhl gene (mlh = mut~ homolog) shows an orf of the expected size with three putative introns. while the deduced amino acid sequence of the 5' region shares strong homology with the procaryotic genes mutl, hexb and the eucaryotic gene pmsi, the amino acid sequenoe of the 3' region shows homology only to pmsi and hexb. the central region of the protein seems to be conserved only weakly. parisi s., and kohli j., institute of general microbiology, university of bern, ch-3012 bern rec7 and rec8 are supposed to be genes with major functions in meiotic recombination since mutations in these genes reduce the frequency of meiotic intragenic recembination ~1000 fold. these genes have been isolated and sequenced but no enzymological activity could be assigned to the protein. a first step toward enzymological and biochemical characterization of rec7 and rec8 is to make beth protein products available in sufficient quantities and in reasonable pure form. for this purpose both genes will be cloned in a s. /x~nbe over-expression system using the strong and inducible nmtl promoter.in parallel both genes will also be expressed in e. coli as gst fusion proteins in order to produce polyclonal antibodies in rats and rabbits. these antibodies can then be used for protein purification in s. pombe as well as for immunofluorescence experiments. induction of expression in e. coli produces a band of the expected size of 56kda for rec7 and of 73kda for rec8. to remove the gst carrier, the rec7 and rec8 fusion proteins can successfully be cleaved by thrombin or factor xa respectively. production of antibodies is in progress. d6bbeling, u., nobi, r,, berchtold, m.w. and kuenzle, c.c. institut fdr veterinarbiochemie, universit6t zurich, wintertharerstrasse 190, normally, only pre b-and pre t-cells can rearrange their immunoglobulin genes by v(d)j recombination, but when other cell types are transfected with the rag-i and rag-2 genes they also become able to perform v(d)j recombination. by transiently transfeeting nih 3t3 fibroblasts with both intact rag genes we found da recombination rate of 0.06%. no recombination was detected, when we transfected the intact rag-1 gene with a mutant rag-2 gene, which lacks 89 c-terminal amino acids, or the intact rag-2 gene with a rag-i gene containing a deletion in the topoisomerase iz homology region. these experiments show that these two regions of the rag genes are essential for v(d)j recombination. when the l4 fibroblast cell line, which has been rendered recombination competent by stable transfection with the rag genes was overtransfected with both wild type rag genes, we found in contrary to an expected increase of v(d)j recombination a reduction by a factor of 2-2.5. this decrease was not observed with the mutant rag genes.this result indicates, that there exists an optimal intracellular rag concentration for efficient v(d)j recombination. m. fischer, a. sailer, h. blieler, t. riilicke*, a. aguzzi +, p. autenried and c. weissmann; 1nstitut fiir molekularbiologie l universitfit ziirich, h6nggerberg, 8093 ziirich, *biologisches zentrallabor and + lnstitut fiir neuropathologie , universitdtsspital ziirich, 8091 zfirich, switzerland the infectious agent of transmissible spongiform encephalopathies such as creutzfeldt-jakob disease in man or scruple in sheep is thought to be prpsc, a posttranslationally modified form of the normal host protein prpc we have shown that mice devoid of prpc (prn-polo) are completely resistant to scrapie. here we report that also heterozygous prn-p o/+ mice with reduced levels of prp c show enhanced resistance to scrapie, as manifested by a long delay in the onset and slow progression of clinical disease. conversely, prn-po/o animals overexpressing prp transgeues exhibit shortened incubation times and accelerated progression of the disease. propagation of the scruple agent is completely abolished in prnpolo animals. high titers of infectivity, about 108 logld50 units/g of brain, are detected in prn-po/+ mice 33 weeks after inoculation (the earliest time point measured), 24 weeks or more before the animals die of scrapie. in contrast, wildtype animals survive no more than 16 weeks after reaching similar titers of infectivity. a practical implication of our findings is that moderate inhibition of prpc synthesis could mitigate disease progression in spongiform encephalopathies. the ruvb protein has been shown, biochemically and genetically, to be involved in the process of homologous recombination in e. coli. one of its functions is to promote branch migration within holliday junctions formed during the process of recombination. to investigate the mechanism by which ruvb promotes branch migration we have used conventional electronmicroscopy, cryo-electron microscopy, scanning transmission electronmicroscopy, and image analysis to study the interaction of ruvb protein with dna. we observed that ruvb forms multimeric rings on dsdna which encircle the dna. we also observed that such rings tend to localize at the holliday junctions. we propose that ruvb rings can actively move along dna using the energy of atp hydrolysis.this movement continues upon encountering a holliday junction, thus forcing branch migration along the dna. gschwind m. and huber g., pharma division, preclinical research, in some families with early onset aizheimer's disease mutations on the 8-app gene have been identified which cosegregate with the disease. so far all these pathogenic mutations have been identified on exons 16 and 17 in humans framing the sequence of the 8-amyloid itself.a genomic clone was isolated from mouse strain c57 black/6 containing the coding regions of the last three exons (16) (17) (18) according to the human sequence. sequencing of all three exons including the large 3' non coding region showed a 100% homology to the previously described balb/c mouse cdna. exon-intron boundaries were determined at the same positions as in human and rabbit genome and the flanking regions in the introns are also very similar, not only is the homology between the human and mouse f$-app very high (97.6%) but also the genomic organisation of their genes is nearly identical. therefore homologous recombination can be applied to introduce molecular 8-app changes and to study their functional consequences on the protein level. we systematically investigated the ability of reca protein to push the dna strand exchange reaction through heterologous regions. we observed that reca can push the strand exchange through substantial heteroiogy (up to 150 bp) when the heterologous insert was placed on the so called proximal end of the linear duplex. in the case of the medial beterology the strand exchange reaction could overcome heterologies of up to about 50 bp. heterology at the distal end was most difficult for reca to resolve, and already 22 bp long insert was completely blocking the progress of the strand exchange. these results, together with earlier electron microscopy studies, allow to propose a molecular mechanism by which reca can exchange strands between partially heterohigous dna molecules. institut for veterin~r-virologie, universit~t bern, ch-3012 bern bovine viral diarrhae virus is a positive-stranded rna virus of the pestivirus group. two biotypes, cytopathic and non-cytopathic in cell culture, can be distinguished. all cytopathic strains characterized so far carry insertions in their genomes. they probably result from a strand-switching activity of the viral rna polymerase during viral replication. to study the switching capacity of the viral enzyme, rna isolated from cells infected with two different strains of bovine viral diarrhea virus were analysed for homologous recombination in an rt-pcr assay. performing the assay we found that fragmented rna present during reverse transcription, the reverse transcriptase enzyme itself and viral rna present in the pcr reaction result in the production of artificial recombination during the assay. it was not possible to detect a homologous recombination activity of the viral polymerase above the background recombination frequency of the rt-pcr assay. our results strongly question the use of the rt-pcr assay for the study of recombination of rna viruses. transpositional rearrangements mediated by insertion sequence (is) elements represent an important source of genetic variation within populations 1. we analyze dna rearrangements at the level of the complete genome by rflp, using 7 is elements as probes for hybridisation to southern blots samples taken at different time points from 12 independent cuttures of e coli b grown by serial transfer for 10'000 generations 2 and stored at -70~ are used to study structure and evolution of genetic diversity within populations. the seven is elements contribute differently to genetic variation and lead to a succession of mutants affecting the genetic structure of the population. we now search for correlations between rflp patterns and the increase of relative fitness over time observed previously 2 naas t., 81o~ m, fitch w.m and arber w, (1993) the complete dna sequence of the locusta migratoria mitochondrial genome has been derived from four cloned mtdna fragments. the sequence is 15.2 kb in length, contains 13 peptide coding sequences, and genes for 22 trnas and two rrnas. the organisation of the genome shows a strong resemblance to drosophila, the gene order and orientation being the same except for the positions two trnas. this contrasts with the only other non-dipteran insect mtdna to have been sequenced, apls mellifera, which shows differences in the positions of ii trnas. this finding is discussed in relation to the at content of the different genomes. the sequences of the three mtdna genomes are also compared directly and the results related to existing knowledge concerning the evolution of insects. udem, new jersey medical school, newark, nj 07103-2714, usa nonsegmented negative strand rna viruses are so far not amenable to reverse genetics. the genomes must associate with nucleocapsid (n), phosphoprotein and polymerase proteins to form a functional rnp. as a step towards reverse genetics of measles virus (mv), plasmid vectors were constructed allowing synthesis of rnas corresponding precisely to a mv genome in which all coding and intercistronic regions are replaced by the chloram-phenicol acetyl transferase (cat) coding region, transfection of these negative polarity transcripts into mv infected hela or 293 cell lines gave rise to cat activity which could be serially transferred and massively amplified together with progeny helper virus in cell cultures. both expected mv/cat chimeric mrna and replication product were identified in the progeny. preliminary experiments indicate that only rnas in which the total number of nucleotides is a multiple of six replicate and transcribe efficiently, consistent with the data on natural and modified copyback di rna of sendal virus (calain and roux, j. virol. 67 4822 (1993) ). precise end to end encapsidation of rna, each n protein contacting six nucleotides, might be required. the embryonic tissue specificity of the human cytomegalovirus immediate-early (hcmv-ie) promoter/enhancer (-522 to +13) was examined by l~-galactosidase staining of transgenic mouse embryos carrying hcmv-ie regulated lacz genes. embryos of three transgemc lines were analyzed between 8.5 -13.5 dpc. for all lines, hcmv-ie transcription was primarily confined to subsets of neural and vascular cells. however, extents of transgene expression along the embryonic primary axis were slightly and greatly restricted in two of the lines relative to the third line at all stages analyzed. these differences most likely represent integration site-dependent chromatin constraints that affect levels of hcmv-ie-directed lxanscripfion. together, the three lines can be taken to define a graded potential for hcmv-ie transcription along the anteriorposterior axis of the embryo with greatest permissivity in mid-axial structures which include the dorsal neural tube at the level of the hindbraln and upper cervical spinal cord, the inner ear and vestibular acoustic ganglia, branchial arteries, aortic sac, and lung buds. the cell typespecificity and axially graded permissivity for hcmv-ie transcription in the mouse embryo provide a basis for understanding the pathology and relative frequencies of different types of birth defects caused by congenital hcmv infection in humans. stauffer, y., 0fford, e. and beard, p., swiss institute for experimental cancer research (isrec), ch-i066 epalinges, switzerland. hpvi8 is associated with genital carcinoma. like other human papillomaviruses, hpvi8 cannot be easily propagated in cell culture, because the viral growth cycle is dependent on the differentiation of its host cell, the keratinocyte. moreover, very little viral capsid protein is found in vivo in infected tissues. the hpvi8 l1 and l2 genes code for the major and minor capsid proteins, respectively. to obtain the viral capsid proteins we made constructs with the l1 and l2 genes for expression in e. coli (the pqe system) and in recombinant vaccinie virus (a modification of the phgs-i system) infected cells. the l1 and l2 proteins expressed in e. cell contained a histidine tag encoded by the vector. this allowed us to purify the proteins on a sickelaffinity column in order to raise antibodies. these will permit us to detect the presence of capsid proteins expressed from recombinant vaccinia viruses. we expect the l1 and l2 proteins expressed together from the vaccinia-based vector to selfassemble to form virus-like particles. to papain-like proteinases. the role of these putative catalytic residues in the activity of the proteinase was studied by site-directed mutagenesis. a minimal proteinase has also been constructed by n-and c-terminal deletion to determine the boundaries of the proteolytic domain, sequence analysis of the c-terminal cleavage product should indicate the site of cleavage. cell entry, uncoat~ng and nuclear transport of adenovirus z . u. f. greber and a. helenius. yale university school of medicine, new haven, ct, u.s.a. to enter cells, viruses must achieve three major goals: transfer their genome across the plasma membrane into the cytosol, target the genome to the replication site and decondense it for replication. adenovirus, an icosahedral nonenveloped particle with a double stranded genomic dna and ii to 15 structural proteins~ enters into human kb cells by a sequential disassembly program during receptor-mediated endocytosi~, acid-dependent penetration into the cytosol, and targeting to the nucleus. during internalization, the fibers, primarily responsible for virus binding to the cell surface, are detached. in early endosomes, the capsid-stabilizing proteins ilia and viii are released. fenton base, a vertexassociated protein implicated in penetration across the endosomal membrane, is removed in endosomes, if penetration is blocked with lysosomotroplc reagents. in the absence of inhibitors, a large fraction of penton base stays associated with the cytoplasmic capsid. the viral dna is disconnected from the shell after proteolysis of the internal protein vi~ a capsid and dna binding component. removal of protein ix, a capsid binding factor, further destabilizes the coat, and the remaining 'stripped-down' virus particles associate with nuclear pore complexes to release their dna-genomes into the nucleoplasm. this stepwise dissociation program is thought to provide the high efficiencies of the entry process needed to successfully deliver the viral dna across multiple barriers to the cell nucleus, the site of replication. ch. schmidt and w. arber; biozentrum der universit~t basel, abt. mikrobiologie, klingelbergstr. 70, ch-4056 basel bacteriophage p1, carried as a single copy plasrnid in e. col~, is widely known for its horizontal gene transfer ability (transduction) in enterobscteria. as a temperate phage, it can lysogenize its host and thereby provides it with accessory functions (restriction of foreign dna by ecop1, functions expressed by transposons carried on the p1 genome or integrative suppression). these properties reflect both symbiotic and evolutionary functions of pi. p1 regulates expression from its genes by two different mechanisms: trans(:ription of early regulatory genes is repressed by the phage encoded repressors c1 and bof, whereas transcription of its morphogenetic genes from p1 specific late promoters is induced by the early expressed gpl0. by site-directed mutagenesis the late promoter ps, composed of the -22 inverted repeat aagttactt and the -10 box, was functionally characterized. p5 and its derivatives compare well with several other late promoters of pt with regard to their sequence dependent strengths: the strong promoters ps, pdar and lpr2b all share the perfect inverted repeat around position -22, whereas the weaker promoters lpr91, lpr82a, lpr82b and lpr83 contain mismatches in their inverted repeats or the inverted repeat is positioned mere upstream relative to the transcription initiation site. homologies. however, the phages formed two immunity groups. the results may suggest a common genetic trait providing a selective advantage to lysogenic aa. by using a dna substrate with defined gap size we found that human immunodeficiency virus type 1 reverse transcriptase (hiv~rt) was able to perform strand displacement dna synthesis. this activity was not affected first by calf thymus proliferating cell nuclear antigen and replication factor c and second by escherichia coli single-stranded dna binding protein~ which together allow dna polymerase ~ to perform strand displacement dna syrlthesis (podust , v. and h~bscher, u. (1993) nucl. acids res. 21, . 3'-azido-2',3'-dideoxy-thymidine tripbosphate inhibited displacement completely indicating that dna synthesis is required for this reaction. the hiv-rt p66 polypeptide alone could perform limited strand displacement dna synthesis, whereas the hiv-rt p51 polydeptide was completely inactive, likely due to its inability to replicate extensively on a mi3 dna template, on the other hand the hiv-rt psl polypeptide e~hanced the strand displacement activity of the hiv-rt d68 subunit at a molar ratio of 4:1 mainly by chasing short products into longer ones. furthermore kinetic experiments after complementation of hiv=rt p66 with kiv-rt psl indicated that hiv-rt psl can restore rate and extent of strand displacement activity by hiv-rt p66 compared to the hiv-rt heterodimer d66/d51, suggesting a function of the 51 kda polypeptide, the mouse mammary tumor virus proviral dna contains an open reading frame in the 3' long terminal repeat which can code for a 36 kda polypeptide with a putative transmembrane sequence and five n-linked glycosylation sites. this gene is known to code for a superantigen which deletes a specific subset el co4 + t-lymphocytes in vivo, the superantigen encoded by the exogenous mouse mammary tumor virus of the gr strain acts specifically on v[314 bearing t-cells. we produced recombinant vaccinia viruses to express either the complete or a truncated orf protein after infection of primate cells in culture. the complete err gene in mammalian cells leads to the production of a 47 kda protein which is specifically detected with an anti-orf-peptide antiserum. the 47 kda protein can be labeled with d-[2-3h]mannose and its synthesis is inhibited by tunicamycin, an n-linked glycosylation inhibitor, indicating that it is a glycoprotein. the truncated orf protein beginning at the second atg of the open reading frame is also modified, but the c-terminal half of orf, starting at the fifth atg (orf5), has the expected size of the non modified polypeptide. pulse-chase experiments indicate that the 47 kda orf g}ycoprotein has a short half-life of about 1.5-2 hours in this system whereas the orf5 truncated protein is even less stable; its half-life is about 20 minutes. the cellular localization of the orf protein and the role it could play in vivo are currently under investigation. conclusion was based on a restriction fragment length polymorphism (rflp) with probes of is-elements. the mutants displayed different growth behavior. the evolutionary stability of 32 of these mutants was further tested by competing a mixture of them for ten days. polymorphism was maintained during this competition but two fitter mutants took over 60% of the population. implications of these results will be discussed in terms of generation and selection of genetic variants for the adaptation of microbial population. mouse mammary tumor virus (mmtv) is produced in the mammary gland of infected females and transmitted by the milk to suckling new-boms. the passage of mmtv from the gut to the mammary gland is still poorly understood, the nature of the tissue tropism of mmtv has not been elucidated, a single receptor present on mammary epithelial and lymphoid cells might serve for the entry of the virus. a cell-type specific entry mechanism could in part be responsible for this phenomenon. alternatively different surlace molecules might be involved in its uptake. we have studied the cell susceptibility to mmtv using a sensitive method of detection of newly acquired exogenous proviral dna sequences, prior to viral integration and transcription. thereby we confirm that mmtv infects mouse, rat and monkey cell lines of different tissue origin in vitro demonstrating the lack of a strict host range specificity in tissue culture as previously described. we have also observed the infection by mmtv of various human cell lines. furthermore various infection susceptibilities are observed for cell lines of a same species. r. cattaneo, p. spielhofer, k. kaelin, m.a. billeter mo/eku/arbio/ogie /, universit~t zorich, hsnggerberg, 8093 zorich subacute sclerosing panencephalitis (sspe), a rare, lethal disease is caused by clonally selected defective measles viruses (dmvs) characteristically mutated. this emerged from sequencing of five mv genes from sspe cases and by functional tests of the envelope glycoproteins hemagglutinin (h) and fusion protein if). these expressed h and f variants migrate poorly to the cell surface but maintain some cell fusion activity whereas the envelope associated matrix protein (m) appears dispensable. thus, the spread of dmv genomes through the brain seems to occur by membrane fusion at local cellular contacts, essentially hidden from the massive immune responses. in more than half of sspe cases, the propagated dmvs contain m genes in which up to 50% of u residues are replaced by cs. such "biased hypermutations" are likely due to the simultaneous conversion of many a residues to inosine in double stranded rna regions accidentally formed, mediated by an ubiquitously occurring adenosine deaminase. similar events at less spectacular degrees have now been observed in functional genes of several negative strand rna viruses. thus, this enzyme might be an important evolutionary driving force for mononegavirates. pull, i., wieland, s., nieder~st, b., keist, r., walter, e., and blum, h.e. department of medicine, university hospital z'tirich infection by hbv-positive organ donors remains a serious problem in transplant surgery. here we present a case of a 58-year-old woman who received a heart transplant from a hepatitis b surface antigen (hbsag) positive donor. the patient was negative for all hbv markers prior to transplantation. two months after transplantation she became hbsag positive and died from subacute liver failure about seven months later. cloning and sequencing of hbv dna revealed several hbv mutants in the serum of the recipient, each with a 108 bp in-frame deletion in the pre-s 1 region. transfection studies in huh-7 cells with the predominant pre-s 1 deletion mutant showed a higher replication competence compared to wild-type hbv. sequence analysis of pcr products from serum of the donor showed mainly wild-type hbv dna in the pre-s region without the pre-s 1 deletion. these data demonstrate that mutant viruses accumulate in immunesuppressed patients. specific mutants may play a critical role in the severe or fatal clinical course of hbv-infection in transplanted patients. in non-neuronal cells herpes simplex virus (hsv) establishes a productive lyric infection starting with transcription of its immediate early (ie) genes by corecruitment of a viral protein (vpi6) and a cellular factor (oct-l) onto characteristic oetamer-like sequence motifs ftaatgarat) in the promotors of the ie genes. in neurons, however, hsv establishes latent infections whereby transcription of the ie genes is prevented and the lyric program aborted. our efforts are focused towards determining which neuronal factors are involved in the regulation of hsv ie gene transcription and whether they may be involved in hsv latency or reactivation in neurons. in electrophoretic mobility shift assays (emsa) using mouse brain nuclear extracts we found that neuronal oct factors (noct-2, noet-3 and noct-4) bind to taatgarat sequences from the viral icp0 and icp4 promoters. an additional brain protein also binds to these sequences but not to a consensus octamer sequence (atgcaaat) as do neuronal oct factors. emsa assays with mutated taatgarat probes show that the taat sequence is crucial for this binding and we have tentatively named this brain protein taat-1. we are currently testing the noct factors in in vivo and in vitro transcription assays to study their involvement in hsv ie promoter regulation. biochemical and functional analysis of a vaccinia virus recombinant containing two copies of the p37k gene. schmutz, c., and wittek, r. institut de biologie animate, univei-sit~ de lausanne, ch-1015 lausanne. the most abundant protein of the vaccinia virus envelope is the palmitated 37k protein. this protein is required for envelopment and subsequent release of virus from infected cells. the amount of extracellular enveloped virus (eev) produced varies considerably depending on the virus strain and host cell line but remains in any case low (<30% of the virus production). if p37k is a limiting factor for envelopment, its overexpression might be a taeans to increase the production of eev. for this purpose a vaccinia virus recombinant containing a second copy of the p37k gene was enginee,'ed. western blot analysis clearly showed increased levels of this protein. titration assays revealed slightly lower yields of both intracellular naked virus (inv) and extracellular enveloped virus (eeu in ceus infected with recombinant virus. unexpectedly, with recombinant virus, the ratio of eev versus inv was significantly lower when compared to wild-type virus infection, despite the higher level of p37k expression. we are currently testing whether this is a consequence of a lower production, or a reduced infectivity of eev particles in cells infected with recombinant virus. hanspeter stalder, christian hertig and ernst peterhans institut f~r veterin~r-virologie, universit-st bern, ch-3012 bern bovine viral diarrhoea virus (bvdv) causes acute transient infection in animals exposed to virus postnataly and persistent infection in animals infected in utero in the early phase of gestation. the latter is associated with specific immunotolerance to the infecting viral strain. animals infected in utero may be born normal and remain healthy despite the persisting infection with a noncytopathic biotype of bvdv. mutation to the cytopathic biotype, or superinfection with an antigenically similar cytopathic biotype, results in lethal mucosal disease. we have pcr amplified and sequenced parts of the region coding for the surface glycoprotein e2 and p80, a nonstructural protein associated with enzymatic activities. over a time of one year, virus obtained from an immunotolerant persistently infected animal remained identical in its nucleotide sequence in the analyzed regions. contrasting with this remarkable evolutionary stasis over some 600-800 virus generations is the heterogeneity of viral isolates obtained from different persistently infected animals. we propose that the muitilineage distribution of bvdv is the result of antigenic drift in the population of acutely infected animals, combined with evolutionary stasis in immunotolerant animals. in 20 to 30% of naturally infected goats, the sevedty of disease in infected animals correlates with the antibody titers to the viral envelope protein (enw) and especially to the gp 38 transmembrane podion (tm). in order to analyze linear b epitopes of env, we produced a random library of caev env polypeptides fused to ~-galactosidase and expressed in e. coli. the complete env gene obtained from an infectious clone of caev-co was subcloned in a puc18 plasmid and dnase digested, random fragments of 200 bp were inseded in ~,gtl 1 dna and expressed in e. coil y1090. as the first step of b epitope mapping and in order to identify immunodominant group epitopes we used high titer sera from naturally infected swiss goats to screen the library. six immunoreactive clones were obtained and subsequently sequenced. all six mapped to the tm region of env. a fine mapping of these immunoreactive regions was performed using pepscan technology and elisa with synthetic peptides. four epitopes could be identified including the eysteine loop corresponding to an immunodominant epitope in other lentiviruses. these peptides were used in vitro to test the reactivity of sera from naturally and experimentally caev-infected goats and in rive to modulate the immune response of goats to tm. the minute virus of mice (mvm) has a 5kb linear single stranded dna genome. at both termini, short selfcomplementary sequences are folded into stable hairpin structures. the 5' (right) hairpin is a single stem structure with only three unpaired bases within the stem. these unpaired form a bubble structure. we examined the requirement in mvm lytic infection for the bubble structure in the 5' terminal hairpin of mvmp. mvmp rf dna containing the entire 3' extremity and extending to the hha i site downstream of the axis of symmetry of the 5' extremity was cloned into pgem-szf(+). this construct contains more than half of the 5' palindrome, however it lacks the sequence information required to form a bubble. murine fibroblast a9 cells were transfected with this dna and the 5' terminal sequence of resulting virus was analysed. a bubble was seen to be generated, however the sequence of nucleotides contributing to the bubble was of n0n-viral, plasmid origin. the nucleotides in question were removed from the original plasmid construct and the experiment repeated. virus was obtained as before. sequence analysis of the 5' end indicated that both flip and flop forms were present. the corresponding 5' hairpin deduced from these two forms contains no unpaired bases at the position where the bubble is normally located in wild type mvmp. the immediate early lie) gene promoters of herpes simplex virus type-1 share a similar sequence which in some cases overlaps with a binding site for octamer binding proteins. this sequence confers responsiveness to a viral transaetivator, vp16. on these promoters, vp16 combines with multiple cellular proteins to form an activating complex. we set up a reconstituted in vitro system consisting of hela nuclear extract and recombinant oct-1/pou domain protein (without the activation domain of oct-1 ) and vp16 where we showed that the activation of ie genes is not mediated by oct-i itself. rather, oct-1 serves to recruit vp16 that has no intrinsic dna binding capacity. vp16, however, possesses a stong acidic c terminal activation domain of -80 amino acids which is indispensable for the in vivo function of the protein. as this and other acidic activation domains possess only a relatively weak activating capability when we test them in in vitro transcription assays we are interested in investigating the role of this acidic activation domain in vitro i.e. under condition of cell free extracts and on naked template dna. we are using recombinant oct-1/pou domain protein and recombinant vp16 that has been truncated at the c terminus. additionally, we are also testing different truncations of vp16 in vitro that have been produced in cos cells. furthermore, we are also trying to see whether these different truncations already affect the assembly of the actvating multiprotein complex. we have studied the kinetics of synthesis of transcripts initiated from the vaecinia virus 7.5 kd gene early promoter. unexpectedly, after a first burst of rna synthesis early in infection, the promoter showed a second peak of activity in the late phase.reactivation of transcription was neither dependent on the presence of the sequences upstream of the early promoter, nor on the location of the promoter in the genome. reactivation seems to be dependent on virus assembly since it is prevented by rifampicin, that specifically inhibits an early step of viral morpho-g~nesis. analysis of several other early genes showed that reactivation is not unique to the 7.5 kd early promoter. analyzing, by in situ hybridization using digoxigenin-labelled riboprobes, the expression of the viral genome relative to that of an array of cytokines including tnf-(z; ifn-% il-i~, il-2, il-8 and gm-csf. initial experiments suggest that viral genome expression is restricted in the inflammatory infiltrates whereas certain cytokines, particularly tnf-oq are strongly expressed in the synovial membranes. overall, the results point to a prominent role of macrophage activation in the maintenance of chronic inflammation and possibly also in the development of arthritic lesions. the core protein of the hepatitis b virus (hbv) interacts with the viral rna pregenome and the reverse-transcriptase.jdna-polymerase to form a core particle which allows for replication of the viral genome. deletion analysis of the core protein revealed, that the arg-rich carboxyterminal domain is required for rna encapsidation and productive viral dna synthesis. we demonstrate, that the duck hepatitis b virus (dhbv) core protein can be derided into two domains comprising amino acids 1-67 (n-ter) and 67-262 (c-ter), respectively. the co-expression of these two core subdomains leads to the formation of replication competent core particles. furthermore, we demonstrate that the carboxy-terminal domain of dhbv core (c-ter) can be functionally replaced by the corresponding domain of the human hbv. the complete hbv core protein, however, does not form replication competent core particles with the dhbv pregenome and rt. these results imply that the carboxy-terminal part of the hbv core proteins define an exchangeable, functionally conserved domain. influenza ha mediates the adhesion and penetration of host cell membranes. acylation of three conserved cysteine residues in the membrane spanning region of several ha subtypes has been shown. the biological significance of this hydrophobic modification remains to be elucidated. a possible role for the membrane fusing activity has been controversially discussed in the past. removal of covalently bound fatty acid from protein by incubation with hydroxylamine is a commonly applied method. as we shall show for a number of different envelope viruses with acylated glycoproteins, this treatment leads to quite different consequences in a functional seiase depending on the virus used. as an alternative approach to study the biological significance of palmitoylation we expressed wild-type influenza ha and an hamutant lacking the fatty acid binding sites using the baculovirns system. both wild type and fa--mutant ha, after expression in insect cells bind to red blood cells, induce hemolysis, and fuse efficiently with fluoreseently labeled erythrocyte ghosts. hydroxylumine treatment of both recombinant wt-and mutant-ha leads to a loss of hemolysis and of fnsion activity. at least in the ease of fa--i-ia its inhibitory action must be related to some other effect than fatty acid cleavage. these results indicate that hydroxylamine treatment may not always be suitable for the generation of fatty acid free glycoproteius or virus particles for functional studies. gesemann and j.g. nicholls, biocenter univ. basel, 4056 basel, switzerland depolarization of leech neurons leads to growth cone collapse and neurite retraction. this response to depolarization is influenced by the substrata on which the cells are growing and is mediated by the influx of calcium (s. grumbacher-reinert and nicholls, 1992, j. exp. biol. 167:1-14; neely, 1993 , i. neurosci. 13:1292 -1301 . the morphologieal changes induced by depolarization of leech neurons growing on leech extracellular matrix extract is accompanied by a loss of microfilaments in the growth cones. leech neurons growing on a different substrate, the plant lectin coneanavalin a (coma), did not retract their neudtes or lose rnierofilaments, but continued to grow after depolarization. we attribute this to the reduced calcium influx during depolarization of neurons on cona (ross et al., 1988, pnas 85:4075-4078) . increasing the intraeellular calcium concentration by incubating neurons with the calcium-ionophore a23187, led to cessation of motility and disruption of microfilaments but not microtubutes in growth cones on coma. to investigate the mechanism by which calcium affects the organization of microfilaments we stained neurons with antibodies against gelsolin, a protein that severs microfilaments when it is activated with calcium. we have observed that these antibodies colocalize with microfilaments in leech growth cones, we are now analyzing the nature of this antigen that cross-reacts with the anti-gelsolin antibodies with the goal of establishing its potential role in the calcium-induced disruption of microfilaments in leech neuronal growth cones. this work was supported by a grant from the swiss nationalfond no. 3127814.89 to j.g. nicholls. activation of metabotropic glutamate receptors (mglurs) was studied in voltage-clamped ca3 cells in rat hippocampal slice cultures using the whole-cell pateh-clamp configuration. in saline containing 16 mm k +, 1s,3r-acpd (50 ~tm), a mglur agonist induced an inward current associated with an increase in membrane conductance. the reversal potential, assessed with a voltage ramp protocol from -80 to -60 mv and calculated by linear regression, was -15.7 â�¢ 18.7 mv, and was shifted to -53.4 â�¢ 6.4 mv when the concentration of external monovalent cations was halved. these results indicate that the conductance underlying this current is selective for cations (mainly k + and na+). the steady-state i/v curve for the 1s,3r-acpd-induced inward current showed a slight inward rectification. in most of the cells, this inward current was followed (or overlapped) by an outward current concomitant with a decrease in a k + conductance (ere v = -51.2 ~ 5.6 mv in [k+]o = 16 mm and shifted to -74.0 :t: 2.8 mv in [i4+]o = 8 ram). this k + current was voltage-independent in the membrane potential range of-120 to -20 mv. similar results were obtained with methacholine, a cholinergic muscarinic agonist. the non-specific cationic current, seen only in the presence of elevated extracellular k + concentration, could play an important role during intense neuronal activity. the c-myc protein has been implicated in the regulation of cell growth and differentiation, c-myc specifically interacts with max which in turn forms heterodimers with mad and mxil and homodimera with itself. we present evidence that this network o! proteins is regulated both at the level of relative protein concentration as well as by post-translational mechanisms. in the hematopoietic cell lines u-937, ml-1 and hl-6o e-myc mrna and protein are rapidly downregulated after induction o| differentiation whereas max expression is not significantly altered. in contrast the expression of mad is induced with properties similar to immediate early genes, taxi1 is induced to a lesser degree and with slower kinetics, in an effort to study the regulation of o-myc we have mapped all the major in vivo phosphorylation sites in both c-mye and max. two sites in the n-terminal transactivation domain are important for the regulation of the transforming potential el c-myc both in established cell lines as well as in primary rat embryo tibroblasts. however, no difference in the transactivating potential of corresponding mutants was observed. phosphorylatien of max at sites close to the basic region increases both on-and off-rates of either myc-max hetero-as well as max homodimers to dna. brain research institute, university of z~rich, ch-8029 z~rich, switzerland gaba b and adenosine receptors act via pertussis toxinsensitive g-proteins of the gi/g o class to mediate longlasting inhibition in the cns. this class of g-protein is negatively coupled to the enzyme adenylyl eyclase. our aim was to determine whether the inhibition of adenylyl nyclase mediated by these receptors has functional consequences for electrical signaling in hippocampal neurons. the calciumdependent k + current, i~, underlying adaptation of cell firing, was used as an electrophysiological bioassay for adenylyl cyclase activity, as it is very sensitive to intracellular levels of camp. accordingly, the ~-adrenergic agoniat iaoproterenol (5-500 nm), that activates g,-linked receptors, reduced the amplitude of i~ by 51.5 â�¢ 3.6% in voltage-clamped ca3 pyramidal cells in ttx (n=20). reduction of imm by isoproterenol was curtailed to 27.3 â�¢ 2.9%, (p < 0.001) in the presence of baclofen (i0 ~m), acting at gaba b receptors, or adenosine (50 nm). thus, the inhibition of adenylyl cyclase activity due to activation of gaba a and adenosine receptors can modulate responses to other neuretransmitters by an interaction occurring at the level of intracellular signal transduction. furthermore, stimulation of these receptors will tend to enhance i~, suppressing repetitive cell firing. this represents a further mechanism which will result in prolonged inhibition of hippocampal neurons. a study of the possible modulation by nitric oxide (no) of endogenous dopamine (da) release was performed in bovine retina in vitro. isolated half retinas were incubated in kkg at 37 ~ for 3 successive 30-rain incubations, and da was measured in the incubation medium by hplc with ec detection. when retinas were incubated in the presence of the no-generating compound hydroxylamine (300 ttm), a decrease in either basal or k +-(50 ram) stimulated da release was observed during the 2 nd 30-rain period. tested under similar conditions, dibutyryl cgmp (1 mm) was ineffective. the no synthase inhibitor l-ng-nitro-arginine methyl ester (l-name, 100 gm) increased basal da release during the 1 st and 2 nd incubation, whereas it did not affects the k+-stimulated da release. in addition, its effect were potentiated in calcium-free medium. finally, we have also shown in cell-free experiments that retinal no synthase activity was totally calcium-dependent and blocked by l-name. taken together, these results suggest that endogenous no regulates da release via a cgmp independent mechanism. division of endocrinology, university hospital, ch-1211 geneva 14 while the stimulation of aldosterone biosynthesis by angiotensin ii (angii) in bovine glomerulosa cells clearly depends upon a sustained influx of ca 2+, the pathway for ca 2 ⧠entry is not yet accurately defined. at least two mechanisms seem to be involved: 1) the opening of voltage-operated ca z ⧠channels, and 2) the stimulation of a "capacitative" ca 2+ entry regulated by intraocllular ca 2 ⧠stores; however the relative contribution of these pathways to the stimulation of steroidogenesis needs to be determined. two pharmacological tools were used: nieardipine, a blocker of to and l-type ca 2+ channels, and thapsigargin, which releases ca ~+ and therefore induces a capacitative ca ~+ influx. nicardipine (1 lam) completely blocked the cytosolic ca 2+ response to k +, which exclusively activates voltage-operated ca 2. channels, but only partially (22 %) the response to angli. in the presence of nicardipine, angll was unable to stimulate a ca 2+ influx aiter depletion of ca 2 ⧠stores with thapsigargin, demonstrating that angli principally stimulates a capacitative ca 2+ entry pathway. in addition, nicardipine completely blocked the steroidogenic response to k +, but only 30% of the response to angll. thapsigargin-induced aldosterone formation, as the response to angll, was markedly potentiated by low concentrations of k +. in conclusion, this study shows that in bovine glomerulosa cells, angli activates a sustained ca 2+ influx, principally through a capacitativc pathway, leading to aldostcrone formation. this finding could partially explain the poor efficiency of dihydropyridine ca 2 ⧠antagonists for preventing aldosterone secretion. harald holder and john m. lucocq*. institute of anatomy, university of berne, ch 3012 berne *depanment of anatomy and physiology, university of dundee, uk dundee dd1 4hn. subjection of hela cells to 45~ for 30 minutes leads to a complex activation pattern of map-kinases as revealed with renaturadon gels. exponentially growing hela cells show activallon of a myelin basic protein (mbp) phospborylating kinase migrating with an apparent molecular mass of around 60kda upon heat shock induction. in contrast, in cells arrested in go by serum starvation for 24h, essentially three different proteins phosphorylafing mbp are activated subsequent to heat shock treatment. these kinases migrate with apparent molecular masses of around 32kda, 44kda and 55kda. western blot analysis with antibodies recognizing the human boraologues of erkl and erk2 revealed that considerable pools of erkl and erk2 show retarded elec/rophnretic migration behavioar indicating that they are phosphorylated and probably activated in heat subjected and growth-arrested hela cells. down-regulation of protein kinase c (pkc) by a prolonged treatment with 12-o-tetradecanoyl-phothol-13-acetate (tpa) did neither change the major activation pattern observed upon separation of cell lysates on renaturation gels nor that one observed on western blots decorated with anti-erkl or anti-erk2 antibodies. these results suggest that heat shock induced activation of at least erkl, erk2 and the 55kda mbp-kinase is not mediated via activation of pkc. this work was supported by swiss nsf grant no. 31-27636.89. effects of dominant negative glucocorticoid receptor mutants in cell cultures and transgenic animals stefano brenz verca, stefan schneider, sandro rusconi, institut fiir molekularbiologie h der universitat zf~rich, winterthurerstr. 190, switzerland the glueocorticoid receptor (gr) is involved in a large number of developmental and biochemical processes. recently, we obtained a trans-dominant negative gr mutant (tdn) -called gr[aia] -by a frame shift of the cag-repeat of the rat gr (see poster by hug et al.) . the gr[ala]-mutant shows normal llgand-binding and dna-binding but cannot stimulate transcription (lanz et al., steroids; in press) . modulatable versions of the tdn-mutant have also been created by substituting the hbd of the grmutant with the hbd of sex-hormone receptors. these constructs have only been tested so far in transient cotransfection assays. therefore, we are currently trying to express them permanently in cell lines (rat hepatoma cell line fto-2b) in order to verify whether they can affect the transcription level of a known endogenous gr-dependent target-gene, like the tyrosine aminotransferase (tat) gene or a permanently transformed mtv-iacz reporter. so far no rapid test-system has been described, that allows the quantitative analysis of such effects in transient expression assays. we are about to set up such a rapid and generally applicable transient test-system. in the future we plan to express dominant negative gr mutants in a tissuespecific manner in transgenic mice. it will be particularly interesting to investigate their effects on the immune system or on liver functions. these studies investigated growth cone responses m brief local encounters with surface molecules implicated in neuronal pathfinding using chick drg neurons. we tested two interrelated hypotheses: 1) discrete points of information (guideposts) override sustained surface information and dominate growth cone behavior and 2) guidance molecules provide such information by activating second messengers instead of simply increase adhesivity. the potential guidance molecule lamialn was covalentiy coupled to polystyrene beads in order to mimic guidepost cells. encounters with laminin model guideposts significantly altered the behavior and morphology of growth cones advancing on fibronestin or polylysine. growth cones steered towards theses beads in a saltatory medea once a long lasting contact with a single filopodium had been established. subsequent to a pause at the bead, growth cones advanced with a significantly increased growth rate for the next llmin. the average increase in rate stimulated by bead contact was 5 times the basal rate on polylysine and 2.5 times that on fibronectin. positioning of multiple model guideposts along a path and within filopodial reach maintained this bead stimulated rate of outgrowth and directed growth cone advance. the laminin induced pattern of events was totally blocked in the presence of either h7 or sphingosine, two pkc inhibitors, or neomycin, a plc inhibitor. neither of these inhibitors affected neurite outgrowth on the substrata alone. our results demonstrate the necessity of filopodial contacts, the importance of spatially restricted stimuli and the activation of transducing pathways as key mechanisms in neuronal pathfinding. mechanisms of glucocorticoid receptor desactlvation by homopolymeric alanines martin hug, manuela h&fferer and sandro rusconi, institut fiir molekularbiologie 11 der universitat uz zart'ch, winterthurerstrasse 190, 8057 ziirich, switzerland the rat glucocorticoid receptor (gr) edna contains in its y-portion a (cag-repeat that encodes a siring of 22 gin residues interrupted by 1 arg. a mutant with the frame-shifted repeat which codes now for poly-ala residues (called gr[aia]) is completely trans-inactive, is more cytoplasmitally located in absence of ligund and exhibits dominant-negative properties in presence of ligand (lanz et al., steroids, in press) . we generated gr recombinants with increasing numbers of cag-triplets in three possible reading frames (oligo-gln, -ser or -ala). the gr-oligo[ala]~-~.23 mutants showed an activity comparable to wild type gr, whereas the groligo[ala]>_25 were inactive. the severity of the effect of oligo a.la su'ings seems to be dependent on the position of the repeat along the primary sequence (e.g. progressive decrease of the effects by moving toward more earboxy-position). we have also examined the effects of the oligo[ala]21, 23, 25, 27, 31 on gal4 chimeric factors and found that the inhibition by ala repeats is probably factor-specific. our results made us speculate that possible differences in post-translational modifications may be at the root of the negative effects by the ala repeats. we believe that these effects are due to transient association of the nascent gr[aia] mutant proteins with intraceuular membranes. these hypotheses are currently tested. the activation of steroidogenesls in calcium-clamped bovine glomerulosa cells and its modulation by angiotensin ii and camp, python cp, laban op, rossier mf, vallotton mb and capponi am division of endocrinology, university hospital, geneva, switzedand. the ca2+-messenger system plays a crucial role in the regulation of steroid secretion in adrenal zona glomerulosa cells, as it is known to mediate the action of angiotensin ii and potassium ion. in the present study, we used intact isolated glomerulosa cells in which the cytosolic free ca 2+ concentration ([ca2+]c) was clamped at various levels with the ca2+-ionophore, ionomycin, in order to locate the sites of action of ca 2+, to determine their sensitivity towards ca 2+ and the modulation by other physiological factors. by measuring simultaneously steroid synthesis and [ca2+]c, we show that ca2+-clamping (50-860 nm) induced a concentration-dependent increase in the production of both pregnenolone (506___70 % of the basal level) and aldosterone (255+27 % of the basal level, ec50 = 273 nm). by contrast, ca 2+ did not influence the conversion of 1 t-deoxycorticosterone into aldosterone at concentrations up to 600 rim, although at higher concentrations we observed a modest but significant inhibition. in addition, ca2+-clamping did not affect the formation of pregnenolone from a freely diffusible analogue of cholesterol, 25hydroxycholesterol, indicating that ca 2+ acts at a step upstream of cholesterol side-chain cleavage. both angiotensin ii and a membrane-permeant surrogate of camp, dibutyryl cyclic amp, potentiated the steroidogenic response to increases in [ca2+]c . in summary, these studies reveal an exquisite sensitivity of the glomerulosa cell steroidogenic pathway toward very small physiological changes in [ca2+]c. radtke, rainer heuchel, oleg georgiev, gerlinde stark#, michel aguet# & walter schaffner institut fiir molekularbiologie 11, universilat ziirlch, 8057 z'6rich # lnstitut rir molehdarbiologie i, universitiit ziirich, 8093 ztirich we have previously described and cloned a factor (mtf-1) that binds specifically to metal-responsive dna sequence elements in the enhancer/promoter region of metallothionein genes. mtf-1 is a protein of 72.5 kda that contains six zinc fingers and multiple domains for transcriptional activation. here we report the disruption of both alleles of the mtf-1 gene in mouse embryonic stem ceils by homologous recombination. the resulting null mutant cell line fails to produce detectable mounts of mtf-1. moreover, due to the loss of mtf-1 the endogenous metalinthionein-i gene is silent, showing that mtf-i is required for both basal and metal-induced transcription. accordingly, reporter genes containing either synthetic or natural metal-reslxmsive promoters were not transcribed after transfection into the null mutant ceils. cotramsfection of the mtf-i expression vector rescued both basal and heavy metal-induced transcription. these results demonstrate that mtf-1 is essential for normal metallothionein gene regulation. rat pheochromocytoma cells (pc12) were differentiated with ngf for 3-4 days, a time sufficient for the formation of growth cones and elongation of neurites. addition of the phosphatase inhibitor calyculin a (cl-a) (0.2.50.0 nm) led to a concentrationdependent collapse of growth cones and retraction of the neurites within 15 minutes. after the retraction, the cell bodies showed the appearance of a grape-like domain opposite to the cell nucleus. they recovered to normal shape within about 30-60 minutes if the inhibitor was washed out. the neurite retraction was further analysed using both low-light level fluorescence video-microscopy and fura-2 single cell microfluorimetry. the onset of retraction started in the central part of the growth cone prior to a complete collapse of filopodia. the basal intracellular free calcium concentration ([ca2+] i) remained constant during neurite retraction. as a measure for the viability of cl-a-treated cells, agonist-isduced responses of [ca2+] i were analysed. ca2+ entry across voltage-activated ca 2-~ channels was inhibited by 50% after 30 minute treatment with cl-a, while the atp-induced ca2+ entry via ca2*-permeable cation channels was not significantly reduced. however, the onset of the atp-induced rise in [ca2+]i started at the grape-like domain. the speed of the ca 2+ wave across the cell body was slower than in control cells. the addition of 0.5 gm okadaie acid, a different type of phosphatase inhibitor, caused no cell shape change or neurite retraction within 24 hours. taken the specificity of the two phesphatase inhibiters into account, the observed effects seem to be induced by inhibition of a ppl-class phosphatase. myosin in nematocytes of hydra michel nakano & robert p. stidwill dept. zoology, university of ztirich, switzerland the functions of nonmuscle myosins during cell locomotion are not clear. several models have been presented reaching from very active participation of the molecules in different parts of cells to only minor or no roles at all. there is increasing evidence that the functions of myosin ii (capable of forming bipolar filaments) are quite distinct from the ones of myosin i and generally that the different members of the growing family of myosin-like proteins may not all have identieal functions. we have attempted to demonstrate the occurrence and determine pattern of myosins in tissue of hydra and especially in migrating nematoeytes mainly for two reasons. first, nematocytes are fairly rapidly moving cells which can be studied in vitro and, second, the question is of interest from a evolutionary point of view. we have utilized several antibodies against vertebrate and invertebrate myosins for immunofluoreseenee (confocal laser scanning microscopy) and western blotting studies. in addition to these results findings on the screening of a ~.zap c-dna library are presented. the jaki protein tyrosine kinase is a member of a family of intracellular kinases characterized by having two kinase catalytic domains: a bona fide tyrosine kinase domain and an amino terminally positioned kinase-related domain. recently it has been shown that jaki and the other members of this family (jak2, tyk2) are intimately involved in cytokine and growth factor signalling. the presence of two putative nuclear localizing sequences in the amino terminus of the protein prompted us to examine the subcellular localization of this protein. immunohistochemical and biochemical analysis revealed that a part of jaki was localized to the nucleolus. metabolic labelling showed that (a) the jaki protein partitioned rapidly into the nucleus and (b) the nuclear jaki became smaller with time. the size difference was not due to either co-translational glycosylation or phospherylation. these results suggest that part of the jaki protein is rapidly translocated to the nucleoli where it becomes processed. revealed that this clone is expressed predominantly in spleen, mammary gland and thymus as two transcripts, a major species of approximately 5kb and a minor species of approximately 4kb. nucleotide sequence analysis revealed a 84% homology to the porcine syk gene. the syk gene is expressed as a major 3.0kb transcript and has been reported to be lymphoid specific. due to the discrepancy of transcript size and number, we suggest that our cdna clone represents a novel member of this family of ptks. we are currently isolating a full length sequence from a mouse spleen cdna library. in addition, we want to characterize which cell(s) in the mammary gland are responsible for the observed expression. expression and function of g-protein isoforms were studied in differenflaring (6c8) and nondifferentiaflng ((33) subclones of red-1 rauscher murine erythroleukemia cells. erythroid differentiaflon induced by the combined action of erythropoietin (epo) and dmso was associated with a selective loss (>70%) of immunoreactive gai3. simultaneously, a iruncated form of g~2 accumulated in the cytosol, while the membrane concentrations of gai2, gets and gaq/11 remained unchanged. nondifferenfiating (33 cells contained sigrtificanfly higher levels of most get subunit isoforms that remained unchanged upon epo/dmso treamaent. changes in adenylyl cyclase activity and in intracellular ca ion levels ([ca]i) resulting from activation of g-protein-coupled receptors were monitored for differentiation-dependent effects on transmembrane signaling. adp-mediated changes of [ca]i in native and differenflaflng 6c8 cells were consistent with p2t receptor coupling to gai3. thrombin receptors seemed to couple preferentially to gai in g3 cells and to gaq in 6c8 cells. our results document substantial alterations in g-protein-mediated signaling during erythroid differentiation. to investigate the involvement of protein tyrosine kinases (ptks) in the growth control of the mammary gland, we have used pcr based cloning to identify ptks expressed during normal mammary gland development. this approach led to the isolation of three members of the eph-related family of receptor ptks: myk-;, a novel member expressed predominantly in lung, heart and mammary gland; m-eck, the murine homologue of the human eck gene and mek5, the murine homologue of the chicken cek5. all three ptks were expressed in a distinct and narrow window of mammary gland development: puberty and estrus cycle. no expression was found either in late pregnancy or in the end differentiated state of lactation. 0vet-expression was found in the undifferentiated and invasive tumors of transgenic mice expressing the h-ras oncogene. in contrast, no elevated expression of all three genes was detected in the well differentiated, non-invasive mammary tumors characteristic of c-myc expressing transgenic mice. these results suggest that members of this family of ptks are involved in the control of proliferation of the mammary gland but are incompatible with its differentiation. np-tcii is a lymphocyte specific transcription factor (lattion a.-l. et el., mol. cell. biol., 1992, 12:5217-5227 a nucleotide receptor that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases c and d, respectively. here we report that atp and utp potently stimulate mesangial cell proliferation. both nucleotides stimulate phosphorylation and activation of map kinase and the up-stream map kinase kinase. activation of map kinase by both nucleotides is dose-dependently attenuated by the p2 receptor antagonist suramin. stimulation of protein kinase c (pkc) by phorbol ester increased map kinase phosphorylation and activation, and downregulation of pkc partially inhibited atp-and utp-induced map-kinase activation. inhibitors of pkc which display a selectivity for the ca2+-dependent isoenzymes (c~, [3, 7) , as compared to the ca2+-independent isoenzymes (5, e, ~, rl) such as staurosporine and the specific pkc inhibitor cgp 41251 did not inhibit nucleotide-stimulated map kinase phosphorylation, thus implicating the involvement of a ca2+-independent pkc isoform. in conclusion, these data suggest, that atp and utp trigger the activation of the map kinase signalling cascade in mesangial cells and this may be responsible for the potent mitogenic acitivity of both nucleotides. subcellular ion-gradients in ca signaling p. lipp & e. niggli, department of physiology, univexsity of bern recent experimental evidence has indicated an important role for submicroscopic concentration gradients during ca-signaling in various cell types. in heart muscle cells influx of ca via l-type ca channels triggers ca release from the sarcoplasmic reticulum (sr) and links electrical excitation to mechanical contraction (ec-coupling). we used a ratiometric confocal method (fluo-3 and fura-red) to follow the intraceuular ca-concentration while ha-and ca-currents (inn, ica) were elicited in the whole cell mode of the patch-clamp technique. both, icand in, were able to trigger ca release from the sr. kinetic differences between ini and ic -induced ca-transients, li-substitution experiments and the existence of a residual inn-induced ca transient in the absence of sr function suggested that these ca signals were mediated by the ha-ca exchange running in the ca influx mode. control experiments showed that ins-induced ca transients did not result from spurious activation l-type ca channels. the significant increase of the na concentration required to activate the ha-ca exchange can only occur provided cytosolic diffusion of na is restricted in the vicinity of the exchangers. this limited diffusion results in significant ha-gradients in the subsarcolemmal space. in conclusion, i~iinduced ca influx may represent a safety factor for ec-coupling while ic, ensures sustained ca-release and is the source for refilling the sr with ca. supported by snf. the regenerative ca-induced ca release (cicr) mechanism is an important amplifier in cardiac signal transducilon. the cicr contributes to the ca transient responsible for mechanical activity, but also generates ca waves propagating within the cytosol. we investigated subcellular ca signals in neonatal rat and adult guinea-pig ventricular myocytes using ratiometric confocal microscopy with a mixture of two fluorescent ca indicators. individual resting myocytes exhibited spontaneous ca release events from the sarcoplasmic reticulurn (sr) that were attributable to three distinct patterns: i) local ca release events without propagation; ll) planar ca waves propagating across the cell; iii) spiral ca waves spinning around a nucleus or shifdng along a subcellular region without entering it. transitions between these patterns were frequently observed, suggesting that a particular release pattern is not the property of an individual cell but reflects the functional state of the sr. the existence of focal release and refractory zones within a single cell indicates that the sr is not uniform on the subcellular level. the sr seems to he a network of elements with distinct functional properties. a suhcellular variability in the positive feedback of the cicr mechanism can account for the observed features of ca signaling. the degree of positive feedback exhibited by a single sr element may depend on its ca content. to characterise the intracellular signalling and regulatory properties of the cloned parathyroid hormone (pth) receptor we have stably transfected two renal epithelial cell lines with cdna's encoding the pth receptor (provided by dr g. segre). the proximal tubular, porcine (llc pk1 [clone 4]), and the distal tubular, madine darby canine (mdck) cell lines were transfected since the culture of either cell line on permeant filter supports leads to the development of polarised epithelial cell layers that mimic the epithelial organisation in vivo. transfection of the cloned pth receptor into each of these cell lines allowed us to examine, independently, the functional properties of the receptors expressed at the apical and/or the basolateral cell surface. while the basolateral application of pth produces a large increase in camp production in both cell lines, the relative efficacy of an apical application of pth differs between the cell types. the transfected llc pk1 cells exhibit a greater response to apical pth compared to the transfected mdck cells. pth-mediated regulation of intrinsic phosphate transport was also examined in polarised, transfected mdck cells. neither apical or basolateral application of pth affects intrinsic apical or basolateral ha-dependent or phosphate transport activities. similar experiments are being performed with the transfected llc pk1 cells. a. and preiss, a. biozentrum der universit&t basel, ch 4056 basel hairless, a recessive larval lethal mutation, causes dominant defects in sensory organs of adult drosophila flies.manifold genetic interactions indicate that hairless antagonizes neurogenic gene function suggesting an involvment also in neurogenesis. in order to gain insight into its function we have cloned the hairless gene which encodes an extremely basic, very serine rich protein of ii0 kd calculated molecular weight. no significant homologies to proteins of known function could be identified. therefore, we are concentrating by various means on a structure -function analysis of the hairless protein. firstly, we are testing a series of hairless deletion constructs for rescue capacity and generation of anti-hairless phenotypes after overexpression compared with the wild type gene. secondly, we are analysing hairless protein distribution throughout development, also at the subcellular level. heat induced hairless protein runs at ~ 150 kd, possibly due to phosphorylation which might be intrinsic to hairless function, a hypothesis we are currently scrutinizing. thy-1 and cd26 surface glycoproteins are both capable of delivering proliferative signals to t cells upon cross-linking with appropriate antibodies. the tyrosine phosphatase cd45 is a key regulator of t cell proliferation as it controls the activity of src family kinases p56/~k and p59fy n. associations of thy-1 with cd45, and of cd26 with cd45 have been reported after chemical cross-linking of intact cells and it is likely that such associations constitute part of the structural basis for the t-cell stimulating capacity of thy-1 and cd26 accessory molecules. we report that thy-1, cd26 and cd45 can be specifically crosslinked at the cell-surface and isolated as multimolecular complexes containing a 78 kda protein that is recognized by an anti-bip (grp78) antibody. in vitro kinase assays show the selective association of p56 ick and p59fy n with thy-1 and cd45 contained in multimolecular complexes. the multimolecular structure we describe could representa transducing unit incorporating surface receptors and regulators of tyrosine phosphorylation that are stabilized through interaction with the bip protein in the plasma membrane. structure-function-analysis of hairless, a gene involved in drosophila nervous system development maier, d., functional coupling of ppars and trs ijpenberg, a., wahli, w., desvergne, b., institut de biologie animale, 1015 lausanne we are interested in a possible functional coupling of thyroid hormone (tr) and peroxisome proliferator-activated receptors (pfar). to this end, we performed cotransfection experiments in nih 3t3 cells using cat reporter constructs containing the promoter of either the t3 responsive malic enzyme (me) gene, or the ppar regulated acyl coa oxidase (aco) gene. the me reporter construct showed very moderate response to the activated mouse ppar (mppar), whereas combination of try1 and unactivated mfpar potentialized the t3 induction. a putative ppar response element was identified within this promoter and will be further investigated. the aco reporter constructs were not responsive to trs. in constrast, a tr and t3 dependent inhibition of the fpar mediated activation was observed. the mechanism of functional coupling of these receptors, which may possibly involve the 9-cisretinoic acid receptor rxr, will be further investigated by molecular analysis. unliganded steroid receptors form a complex with hsp90 via their hormone binding domain (hbd). the receptor activation involves a hormone-induced release of hsp90. we genetically addressed the role of hsp90 in budding yeast by analyzing three kinds of hsp82 (the essential yeast homologue of hsp90) mutants. these mutations affect either the quantity of the protein (low versus normal levels), its integrity (deletion analysis) or its nature (hsp82 homologues from other species). hsp82 mutant are examined for their ability to complement a hsp82 deletion mutant. moreover, they are tested for functional interaction with a hbd. interestingly, a viable deletion of a charged region, suspected to be involved in hsp82-hbd interaction, only slightly interferes with hormonal activation of both estrogen or glucocorticoid receptors. furthermore, a short deletion in the last third of hsp82 leads to the production of a dominant negative mutant which prevents ceil growth at elevated temperature. the ability of various hsp82 homologues to palliate or not the absence of the yeast protein allows us to define, using chimeras, the regions of the protein which are necessary and sufficient to support cell life. zhang,h.,reynaud, s. and spohr,g. d~partement de biologie cellulaire, sciences iii, 30, quai ernest-ansermet, ch-1211 gen&ve 4 id cdnas expressed during early xenopus development have been isolated and sequenced. the encoded protein is homologous to the proteins described in higher vertebrates. northern blot analysis reveals that transcription starts soon after mid blastula and decreases to some extent after tailbud-stage. lower levels of transcription are detected also in adult frog tissues such as liver and heart. microinjection into oocytes of in vitro-synthesized myodor/and id-mrna, along with a cat reporter gene whose expression is under the controll of an a3-actin promoter supplemented with four e-boxes shows that myo d function is repressed by id. to investigate the importance of negative regulatory sequences we have isolated and characterised the id promoter. as a strategy for identifying cis-regulatory elements, chimeric genes consisting of different 5' elements of the promoter were attached to the cat coding sequences and microinjected into embryos. we are studying the tissue distribution of the rat peroxisome proliferators activated receptor (ppar~), a member of the nuclear hormone receptors superfamily, during development and in adults. high levels of ppar~ mrnas are detected in adult liver, kidney and heart. muscle, stomach, and intestine show moderate amounts of the ppar~, whereas brain, testis and lung present a very low expression. during postnatal development, we observe a continuous increase of the ppar~ expression in the kidney, in contrast to a steady level in the liver. anjard, c., groux, b ., gamboni, s. and reymond, c.d., institut d'histologie, unil, rue du bugnon 9, 1005 lausanne. the camp dependent protein kinase (capk) from dictyostelium is composed of a single catalytic (c) and a single regulatory (r) subunit. the c subunit we characterised, pkac is about twice the size of its mammalian counterpart. we showed that it possesses all properties of a catalytic subunit. it associates with the r subunit in absence of camp, it copurifies with capk activity and an increased activity is observed in cells over-expressing pkac. furthermore, we dissected its role during development and cell differentiation. overexpressing pkac under cell type specific promoters allowed to show the requirement for its activity during spore differentiation. pkac seems to play a more complex role in prestalk cells. we are now dissecting the functional regions of the pkac protein, making use of the known tertiary structure of the c subunits of mammalian enzymes. dna binding properties and stimulation of gene transcription of baculovirus expressed xppar~. hihi, a.k, mermod, n., medin, j., ozato, k. and wahli, w., inst. de biol. animale, uni lausanne, ch-1015 lausanne. lab. of mol. growth reg., nih, bethesda, md, 20892 usa. the peroxisome proliferators activated receptors (ppars) are members of the steroid/thyroid nuclear receptor superfamily. so far, they have been found in amphibians, rodents and humans. in xenopus laevis, 3 subtypes (xppara,~.y} have been isolated. in order to study the mode of activity of the ~ form, the xppar~ gene product was overexpressed using a baculovirus expression system. the nuclear protein obtained has the correct molecular weight of 46 kd. gel shift assays showed that nuclear extracts containing the recombinant protein are able to specifically bind the 'ppar response element' which is a direct repeat of the core aggtca motif. this dna binding activity is potentiated by another nuclear receptor, the mouse rxr~ and is inhibited by phosphatase, suggesting a role for phosphorylation in ppar binding to dna. a functional approach, using an in vitro transcription system, was chosen to define ppar and rxr action on transcription stimulation, as well as the role of their respective activators. arachidonic acid (0.5 -20 ram) induces various patterns of ca2+i signal in bronchial smooth muscle cells, as revealed by single cell dynamic video imaging of fura-2 loaded cells. most frequently, ca2+i oscillations were observed, although other patterns (a single ca2+i transient; a single peak followed by a sustained elevated level, or a sustained ca2*i elevation solely) were occasionally seen. the amplitude of ca2*i oscillations was related to the concentration of arachidonic acid. the frequency histogramm was noticeably shifted to higher frequencies by nordihydroguaiaretic acid (ndga, 0.2 rtm, a lipoxygenase inhibitor), whereas it was markedly shifted to lower frequencies by indomethacin (50 pm, a cyclooxygenase inhibitor) and by 5, 8, 11, 14-eicosatetraynoic acid (etya, 3 ~tm, a lipoxygenase and cyclooxygenase inhibitor). these observations suggest that: (1) arachidonic acid per se elicits ca2+ i oscillations in these ceils; (2) cyelooxygenase activity products modulate the frequency of these oscillations. promoter analyses of a growth factor regulated gene t. triib, m. kalousek, r. kessler, and r. klemenz dept. of pathology, university hospital, 8091 ziirich stimulation of quiescent cells to enter the cell cycle results in altered gene expression. some of the first genes to be induced (immediate early (i.e.) genes) encode transcription factors which are thought to pass on the mitotic signal to the delayed early (d.e.) genes. we have analysed the promoter elements required for growth factor mediated induction of the d.e. mouse gene t1 which encodes a secreted glycoprotein of the immunoglobulin superfamily. a 448 bp region located between 3.6 and 4.0 kb upstream of the transcription start site is sufficient for growth factor mediated inducibility. it contains an ap-1 binding site which is absolutely required for t1 gene expression. it is surrounded by 3 e-boxes. at least 2 of them are essential for efficient gene induction. thus the i.e. proteins encoded by the fos and jun gene family which form the ap-1 complex can activate the d.e. gene t1 in collaboration with helixloop-helix transcription factors binding to the e-boxes. the increase of radiolabelled inositol phosphates ([3h]ips) production in agonist-stimulated human airway smooth muscle cells (asmc) was determined by hplc. in order to observe the role of calcium, pkc and pla2 on plc activity during agonist stimulation, the effects of pharmacological agents (able to alter calcium, pkc and pla2) were tested on ip production elicited by a 5 sec stimulation with histamine. the inhibitor of pkc staurosporine increased, while pma (an activator) decreased the histamine-stimulated production of [3hi 1,4,5-ip3 and of its degradation products. an inhibition was also observed with the two pla2 inhibitors, quinacrine and p-bromophenacyl bromide. in calciumfree buffer, no difference in the ip increase was shown, but an inhibition was observed when thapsigargin, an inhibitor of the ca 2+, mg 2+-atpasc of the sarcoplasmic reticulum, was added in the same conditions. the calcium ionophore ionomycin increased the lp production in presence of external calcium, whereas it completely blocked the stimulation in calcium-free buffer. the results suggest that plc activity, in human asmc, is modulated by a positive feedback of calcium and by a negative feedback of pkc and pla2. regulation of hormone-stimulated calcium signals m. boolman, afrc, laboratory of molecular signalling, dept. zoology, cambridge, uk stimulation of cells with hormones that activate inositol 1,4,5-trisphosphate (insp~) formation, leads to an increase in the intracellular ca 2. concentration ([ca2+]~). these hormone-stimulated [ca2 ⧠signals have a complex temporal and spatial regulation, with the [ca2*]~ increase often taking the form of a series of repetitive spikes or oscillations, arising from a steady basal level. the spatial counterpart of a [ca2+]~ spike is a wave, where [ca2 ⧠is first elevated in a localised region of a cell, and then spreads throughout the cell in a regenerative manner. the [ca~*]~ spike frequency is modulated by several environmental factors including hormone concentration, extracellular calcium and thiol reagents. current evidence suggests that a cell can interpret these complex [ca2+]~ changes to regulate a diverse range of cellular activities. although many of the biochemical steps between hormonereceptor activation and the [ca2 ⧠response are known, the precise mechanism generating these complex [ca2+]~ signals is unclear. in order to understand this mechanism more clearly, we have investigated the regulation of insp~-mediated ca ~ ⧠release from intracellular stores in intact cells. our current data suggests that under certain conditions, the insp3-sensitive intracellular stores behave as functionally discrete units, and that during a ca 2 ⧠spike these stores become functionally coupled by the diffusion of ca z+ from one store to the next, triggering a regenerative ca 2+ release. phenylarsenoxide as a tool for identifying proteins involved in the secretory process wiedemann. c., schaefer, t.,*gitler, c., burger, m. m. friedrich-miescher institut, p.o.box 2543, ch-4002 basel "department of membrane research and biophysics, weizmann institute of science, rehovot 76110, israel phenylarsenoxide (pao) at 20btm blocks exocytosis in chromaffm cells of the bovine adrenal medulla. this inhibition can be reversed by small dithiois, such as dithiothrcitol or 2,3~limercaptopropanol. because trivalent arsenicals interact specifically with dithiol4containing proteins, we conclude that dithiol-proteins do play an important role in regulated secretion in chromalym cells. to identify dithiol-proteins putatively involved in the secretory process, we compare pao-binding proteins from chromaffm and pci2 cells and from fibroblasts as well as from sulx:ellular fractions by 2-dimensional gelelectrophoresis. the proteins are identified by radioactive pao bound to the dithiol in a complex that witltslands sds-page. affinity chromatography on pao-scpharose with various protein fractions is used to purify the dithiol-proteins of interest. enzymatic activity, e.g. kinase or phosphatase activity, can be measured in the eluates of such a column to give further hints at the possible function of dithiol-proteins. rac-pks (~elated to pka_ and pkcc protein kinnses) represent a serine/threonine kinase family whose catalytic domain is closely related to those of protein kinases a and c. they contain a ph (pleckstrin homology) domain n-terminal to the catalytic domain. in addition, rac-pk~ was shown to be a proto-oncogene. in order to investigate the role of rac-pk in cellular signallimg we undertook genetic and biochemical analysis of the drosophila homolog (drac-pk). the drac-pk gene encodes multiple classes of transcripts. antisera raised against the drac-pk recombinant protein specifically recognize 3 distinct molecular weight forms (68, 85 and 120 kda). all forms are expressed throughout development and are both maternally and zygotically regulated. the drac-pk gene is localized at 89b4-i0 region of the third chromosome, betwee~ the pannie~ and the stubble genes. universit6 de lausanne, rue du bugnon 9, 1005 lausanne when spinal meninges homogenates were incubated with reduced glutathione (gsh), a cofactor of prostaglandin (pg) biosynthesis, [14c] arachidonate (aa) was bioconverted into a major and a minor product which comigrated on tlc with pge2 and pgd 2 respectively. in absence of gsh: 1) pgd2 could not be detected; 2) the level of pge2 was dramatically reduced but surprisingly counterbalanced by accumulation of an unusual [i4c] aa metabolite which exhibits particular traits of migration on tlc and of retention time on hplc. this aa metabolite: 1) does not correspond to a degradation product of pge2; 2) crossreacts with pge 2 antibody; 3) fits the conditions required for a 15 hydroperoxy pge2 (chemical degradation into pge2 by hydroperoxyde reducing reagents such as snc12 or gsh-hemin). it is therefore inferred that depletion of gsh favours accumulation of hydroperoxyprostaglandins which would participate to oxidative injury. the ecdysteroid receptor (ecr) and ultraspiracle (usp) from both, chironomus tentans and drosoplu'la melanogaster, were expressed in e. coil as fusion proteins with glutathione-s-trsusferase. the identity of the expressed recombinant proteins was confirmed by western blot analysis. the drosophila ecr binds to its cognate hormone response element as a heterodimer with usp as a partner. we now want to compare the capabilities of the two receptor partners from both insect species in regard to dna binding and heterodimerization by means of gel mobility shift assays. the use of naturally occurring andartificial hormone response elements will allow to define a hormone response element consensus sequence for ecr-usp heterodimers and, if existing, for ecr and usp homodimers, respectively. for other members of this receptor family it was shown that the dna binding domain and adjacent sequences alone sufficiently defines response element specificity for both homo-and heterodimers. the dna binding domains of ecr and usp from the two species show a high similarity (92% and 85% identity, respectively). the corresponding sequences were selected for overexpression in e. coli. in combination with immtmoprecipimtion of receptor-dna complexes and pcr amplification steps, the expressed proteins will be used for the detection of naturally occurring hormone response elements within genomic dna. university of fribourg, ch-1700 fribourg. various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to parallel the synthesis of ptdins(3,4,5)p 3. but the importance of this rise is not clear. we show here that wortmannin blocks pdgf-induced production of ptdins(3,4,5)p 3 in human foreskin fibroblasts with an ic50 of about 5 nm. a similar inhibition was observed in in vitro assays (ics0=lnm) with pi 3-kinase ii~nunoprecipitated by antibodies directed against the 85 kd subunit (p85). on the other hand, wortmannin did not affect pdgf-mediated phosphorylation of p85, indicating the correct interaction of p85 with the pdgf-~ receptor. the pl10/p85 complex of the pi 3-kinase remained intact in the presence of ~m concentrations of wortmannln. these results are consistent with a direct, specific inhibition of pi 3kinase by wortmannin at concentrations relevant for its previously reported effects on cellular responses. when stimulated with pdgf, human foreskin fibroblasts form circular membrane ruffles rich in filamentous actin. the fact that wortmannin inhibits these pdgf-mediated aotin rearrangements suggests the need of pi 3-kinase activity as a signal for this cell response. caicineurin is a calcium/calmodulin-regulated protein phosphatase which is comprised of a catalytic subunit a and regulatory subunit b. although calcineurin was discovered in brain, the calmodulin stimulated protein phosphatase which has caicineurin like structure has been described in other tissues. the protein structure show that the calcineurin b is very conserved in different tissues and species, using anti-calcineurin b monocicnal antiserum, the tissue extracts from rat have been explored. immunoreactivity was obverved in all tissues, although its intensity was different. the highest intensity was found in brain. spleen, heart and thymus had an intermediate level intensity. the results were supported by the quantitative measurement of calcineurin. the caicineurin concentration was 10 to 50 fold higher in brain than that in other tissues. furthermore, the distribution of the calcineurin activity in rat tissue was studied using dephosphorylaticn assay of calctneurin. the highest caicineurin activity was found in brain too, but was only 5 to 15 fold more than that in other tissues. the specific activity of calcineurin was different in tissues. these results indicate the caicineudn activity might be regulated in tissue specific fashion. burst and cytoskeleton arcaro a. and wymann m.p., institute of biochemistry, university of fribourg, ch-1700 fribourg chemoattractants like n-formyl-met-leu-phe (fmlp) stimulate in neutrophils a rapid and transient increase in the levels of ptdins(3,4,5)p3 (pip3) which is due to the activation of ptdlns(3)-kinase. pip 3 has been proposed to be a second messenger molecule, but its cellular targets are presently unknown.here we report that pretreatment of human neutrophils with wortmannin inhibits the fmlp-etimulated production of pip 3 in a dose-dependent manner (ic50 = 5 nm). furthermore, ptdins(3)-kinase activity immunoprecipitated from resting cells is totally abolished by i0 nm wortmannin (ic50 = 1 nm). these results show a potent and direct effect of wortmannin on ptdins(3)-kinase. wortmannin also inhibits the respiratory burst of neutrophils at nanomolar concentrations, which suggests that pip3 is involved in the signalling pathway controlling activation of the nadphoxidase.when pretreated with wortmannin and stimulated with fmlp, neutrophils display oscillatory changes in factin content that correlate with oscillations in cell ~hape. this, and the inhibition of ptdins(3)-kinase, suggest a modulatory role for pip 3 in cytoskeletal rearrangements.we are currently investigating the effects of pip 3 on the functions of gelsolinl a protein that is proposed to mediate actin polymerization and can be regulated by polyphosphoinositides and ca 2+. matthias gstaiger, walter schaffner and christopher hovens institut f'tir molekularbiologie ill der universitat z'tirich, ch 8057 ziirich the octamer motif atgcaaat plays a central role in mediating the activity of a number of both lymphoid specific and ubiquitous promoters and in the immunoglubulin heavy chain intronic enhancer. the activity of thils motif in lymphoid cells is presumably mediated by the lymphoid cellspecific factor oct-2a. when ectopically expressed in non-lymphoid ceils, oct-2a is not sufficient to mediate enhancer activity of a multimerized 50 bp enhancer core element derived from the immunoglobulin heavy chain intronie enhancer, a segment, which is however highly active in b-cells. this and other findings support the supposition that additional b-cell specific factor(s) can account for the specificity and extent of the octdependent transcription observed in lymphoid cells. to further our understanding of the molecular mechanisms involved in mediating lymphoid-specific expression, we have employed the yeast two hybrid system to identify human proteins capable of physically associating with human oet-2a. to this end, a oct-2a expressing yeast strain has been constructed which bears two integrated reporter genes, his3 and lacz under the control of the octamer motif. transformation of this strain with a cdna library has allowed us to isolate clones interacting with oct-2a. we are currently analysing these candidates to determine the veracity of these interactions in higher enkaryotic cell background. in general, untransformed nuclear hormone receptors are predominantly confined to either the nucleus or the cytoplasm of a cell. receptors of the latter class are translocated to the nucleus upon interaction with ligand. our studies of the use of the ecdysteroid receptor complex from insects, consisting of a heterodimer formed between ecr and usp, as a tool for target gene activation in vertebrate cells have revealed a novel mechanism of nuclear translocation of ecr that does not depend on hormone action. after transient transfection of respective expression plasmids into hela or cv-1 cells, the subeellular distribution of the individual receptor types was analyzed by indirect immanofluorescence staining. while ecr alone was detected in both cellular compartments, nucleus and cytoplasm, usp was predominantly found in the nucleus of either host cell. cooxpression of both ecr and usp resulted in an efficient transloeation of ecr into the nucleus. usp appears to trap ecr in the nucleus, presumably by the formation of a heterodimeric complex. thus, heterodimer formation of ecr and usp not only appears to be required for high affinity binding to cognate hormone response dements (and subsequently for transactivation of an adjacent target gene) and for binding to ecdysteroid but in addition for an enhanced translocafion of ecr into the nucleus. activation of rodent macrophages with cytokines or bacteria is accompanied by the induction of a ca2+-independent nitric oxide (no) synthase which plays a key role in antimicrobial and antitumoral activity. firm evidence for expression of a similar enzyme in other mammals or in man has been lacking. we now show that bovine bone marrow-derived macrophages produce nitrite in an l-arginine-dependent manner upon stimulation with heat-killed grampositive or gram-negative bacteria. homologous interferon-7 and tumor necrosis factor-~ induced little no 2-production, but primed macrophages for enhanced n0 2-production induced by salmonella dublin or lps. this is one of the first demonstrations of no 2-production by nonrodent macrophages and encourages a search for an involvement of reactive nitrogen species in the killing of parasites or tumor cells by macrophages from mammals other than rodents. lectins are used in many analytical methods to study the carbohydrate structure of glycoproteins. we report here a technique which combines the high resolution of two-dimensional gel electrophoresis (2-dpage) to separate plasma proteins, the specificity of lectins to bind carbohydrates and the sensitivity of chemiluminescence. this method is compared with that of a commonly used colorimetrio reaction. since the reference plasma protein map obtained by 2-d page is available (i), the technique described here allows a quick and specific identification of plasma glycoproteins. therefore any ma j or glycosylation modification which may happen in some diseases can be detected. (i) electrophoresis 1992; 13:707-714. this work was supported in part by fcar (quebec, canada). in our laboratory we use genetic and biochemical approaches to study translation initiation, in particular the initiation factor eif-4a in yeast. this factor from higher eukaryofic cells is known as an rna dependent atpase and functions as a rna helicase together with another initiation factor, elf-4b. it belongs to a family of putative rna helicases, the d-e-a-d proteins. to find new genes involved in translation initiation, suppressors of a temperature sensitive eif-4a mutant were isolated. one of the suppressors (stm1) is a single copy gene which encodes a protein resembling the human translation initiation factor eif-4b. disruption of the gene is not lethal to the cell, but affects growth. polysome analysis of strains with a deleted stm1 gene have shown that this new gene is also involved in translation initiation. it carries six repeated sequences of 25 amino acids of unknown function and has -like the human eif-4b -a rna binding domain. the second suppressor (stm2) is also a novel gene. it encodes a large protein which shows no homology to other proteins present in the data libraries. it contains high portion of charged amino acids and is rich in glutamines and serines. its function in translation initiation is currently under investigation. tobacco translationini~ationfactore~4aisencoded by a large anddiverse genefamily karl brander, therese mandel, isabelle lutziger, george owttrim and cris kuhlemeier, institute of plant physiology, university of berne, altenbergrain 21, ch-3013 berne using heterologous probes we isolated cdnas coding for translation initiation factor eif-4a from tobacco, eif-4a is encoded by a large multigene family of which at least nine members are expressed in leaves and most if not all other organs of the tobacco plant. while screening genomic libraries we found several additional genes. two of them code for genes highly homologous with eif-4a throughout the coding region, but with changes in the gkt, ptrela and dead-box motifs. a third gene is expressed exclusively in the developing male gametophyte. this eif-4a-related gene may have a role in the well-documented regulation of translation in pollen. in order to study the function of these genes we have begun altering their expression in transgenic tobacco plants. seauence reauirements for a non-aug protein initiaf~jon non-aug initiation codons are still infrequent and were initially observed in animal viruses, but a number of cellular examples have now also been reported. several years ago we reported the use of an acg in the p/c mrna of sendal virus (sen) to initiate the non-structurar c' protein. we recently described a c' protein in the human parainfluenza virus type 1 (hpiv1), which is initiated from a gug. in both cases, the c' protein is an n-terminally ebngated form of the c protein which is initiated at the second aug, in the +1 reading frame relative to the first aug. this aug is used to initiate the p protein, an essential component of the viral rna polymerase. unlike the acg in sen, the gug in hplvt is clearly not a weak start codon since c' is the most abundant protein expressed from this mrna both in rive and in vitro. it appears that not any upstream gug in an optimal context will function as a start codon. for instance, the p gene of hpiv3 cannot express a c' protein (the orf is closed by stop codons) but it does contain a gug in an optimal context upstream of the p protein aug, which could encode a p' protein. nevertheless, we have been unable to demonstrate that this gug functions as a start cedon. with the use of chimeric mrnas between hpivl and 3, we have shown that positions +5 and +6 (the first g of the gug triplet being +1) are the major determinants of the efficiency of gug as an initiaton codon for protein synthesis. we are currently investigating the possibility that these nucleotides may increase the initiation rate of weak non-augs such as the acg of sen. biological activity of the heterodimeric subunit srp9/14 of the signal recognition particle is retained in 2 fusion proteins fabrice bovia & katharina strub, d6partement de biologie cellulaire, universit6 de gen~ve, ch-1211 gen~ve 4 the targeting of secretory proteins to the membrane of the endoplasmie reticulum is mediated by a cytoplasmic ribonucleoparticle, the signal recognition particle. it is composed of 6 proteins and f rna molecule (srp rna). the 2 smallest proteins srp9 and srp14 are required for the translational control function of srp. it effects a specific arrest in the biosynthesis of er-targeted proteins. these 2 polypeptides bind to the srp rna exclusively as a heterodimer (srp9/14). we have constructed single-chain variants of this dimer using a short peptide linker of 17 aa to connect the c-terminus of the 14 kd subunit to the n-terminus of the 9 kd subunit (14-9) and vice versa (9-14). we found that both proteins bind srp rna with a similar efficiency as the wild type protein. glutaraldehyde cross-linking experiments and sedimentation analysis indicate that the 2 fusion proteins fold into a similar structure as srp9/14 and bind srp rna as a monomer. furthermore, they can functionally replace srp9/14 in the elongation arrest and translocation assay. since the 2 fusion proteins are circular permutations of each other, we can conclude from this results that n-and c-termini of both proteins have no essential role in folding, rna-binding and in mediating their biological activity. furthermore, the possibility to express an oligomeric protein as a single polypeptide facilitates the analysis of its functions as well as its structure. a24 s06-08 studer, r. and hunziker, p. e., 8iochemisches institut der universit~t z0rich, ch-8057 z0rich metallothioneins (mts) are small cys-rich proteins which are inducible by a variety of agents such as bivalent transition-metal ions, tumor promoters, cytokines and hormones. the biological role of mts is still unclear. initially postulated to serve in the sequestration of the toxic heavy metals such as cadmium and mercury, they are now believed to play a major role in the cellular metabolism of essential metals such as zinc and copper. to explore this function further we have now established a method to quantify basal mt production in several human cell lines. thus, cellular isomt contents were monitored by h.pj.c, and the rate of synthesis was followed by the incorporation of ~ss-cysteine. the results show that maximum mt synthesis and accumulation occurs when the cells enter the exponential growth phase. with increasing celldensity the mt content decreases progressively until confluence is reached. in correlation with preliminary measurements our results suggest a close relationship between the mt content, the cell growth rate and the intracellular abundance of zinc. vincent bernard, adrian etter, heinz tobler and fritz mtiller. institute of zoology, university of fribourg, 1700 fribourg (switzerland). the nematode ascaris lumbricoides undergoes chromatin diminution which causes a loss of dna in all somatic ceils. we have identified a ribosomal protein (rp) gene (coding for alep1 = ascaris lumbrieoides eliminated protein 1) which is located within the germqine specific material. alep1 shows strong homologies with the ribosomal protein s19 (rps19). the transcription of its gene takes place only in the germ-line. 2d-gel electrophoresis on germ-line and somatic purified ribosome fractions shows that the alep1 protein (rps19) is present only in the germ-line ribosome. does the alep1 germ-line specific protein have a homologue in the somatic ribosomes? we have cloned a homologue of alep1 from ascaris somatic tissue named rps19', we are currently studying the expression pattern of this rps 19'. these results indicate the existence of a developmentally regulated ribosome heterogeneity between the germ-line and somatic ceils. what is the function of rps 19? since ascaris is not suitable for genetic analyses, we isolated rps19 from the nematode caenorhabditis elegans. the c. elegans rps19 cdna shows only 60% homology at the nucleic acid level. rps19 is located on the c. elegans chromosome iv. the identification of a rps19 mutant in c. elegans will indicate its function. favre, d, l, pavlovic, j2 and michel, m.r. t 1 institute of medical microbiology, university of beme, ch-3010 berne. 2 institute for immunology and virology, ch-8006 ziirich we have successfully purified the recombinant c (recc) protein from spodoptera frngiperda (sf9) insect cells which were infected with acnpv-c (a). the recc protein retained the biological activities of native c protein obtained from wild type sfv (b). our results suggest that the c protein is responsible for the cellular shut-off of host protein synthesis by inhibiting the initiation of translation. we are currently investigating the molecular mechanisms involved in this shut-off. tif3 is a new eukaryotic initiation factor which shows 26% identity with the sequence of mammalian translation initiation factor eif-4b. the tif3 gene is not essential for growth; however, its disruption results in a slow growth and coldsensitive phenotype. in vitro translation of total yeast rna in an extract from a tif3 gene-disrupted strain is reduced as compared to a wild-type extract. the translational defect is more pronounced at lower temperatures and can be corrected by the addition of purified yeast tif3,or mammalian eif-4b. in vivo translation of ~galactosidase reporter mrnas with varying degree of rna secondary structure in the 5' leader region in a tif3 gene-disrupted strain show preferential inhibition of translation of mrna with more stable secondary structure. chloroplast protein synthesis requires the import of elongation factors ef-tu (tuf-gene) and ef-g (fusgene). it seems that the expression of both genes is light regulated. we recently cloned and sequenced a chloroplast specific nuclear fus gene in soybean (biochim.biophys acta 1174 : 191-194, 1993 . further analysis revealed that a second very similar (sequence, anatomy) nuclear fus-2 gene exists which, however, shows strong sequence diversion in the 3'trailer region. several cdna clones were partially sequenced but sofar only transcripts from the fusi gene were retrieved. we currently study the promoter regions of either gene to test possible differential expression of fus genes. comparative mapping and sequencing studies of the two genes in the 3"-region indicate that a major dna insertion occurred distal to the coding part of both fus genes what may also influence gene expression. the rna-binding domains in the 9kd and 14kd subunits of the signal recognition part~clehavea putative~-h~licalstructure. nazarena buiand katharina strub dpt de biologie cellulalre, science ill, universit4gen~ve the signal recognition particle (sr2), a cytoplasmic ribonucleoprotein is important for targeting nascent secretory proteins to the endoplasmic reticulum. srp9and srpl4are constituents of srpandare, together with the alu sequences of srp rna, required for the elongation arrest activity of the particle. srp9 and srpi4 form a heterodimer in the absence of the rna and bind the rnaexclusively as a heterodimer. the primary sequence of srp9 and srpi4 revealed that both proteins are highly basic and lack structural similarities to other characterized rna-binding proteins. we have therefore been interested in characterizing the molecular nature of rna-protein interactions. the analysis of n-and c-terminal deletion mutants of the proteins have shown that both proteins contribute to the formation of an rnabinding pocket, the rna-binding and dimerization domains in both proteins, are found in regions that have a predicted ~~helical structure. in sp~pi4, this u-helical region is located betwen aa 72and i00 at the c-terminus; it contains the rna binding and dimerization domains adjacent to each other. our results support the model that the hydrophobic face of the putative u-helix is implicatedinprotein-protein interactions and the positively charged face in rna-protein interaction. in sp~pg, the two rna-binding domains found n-and c~ terminally, also have a predicted amphipatic s-helical structure. it has been proposed that the decline in protein synthesis observed in aging organisms may result from a decrease in the protein synthesis elongation factor ef-la. john shepherd's finding that transgenic drosophila melanogaster carrying an additional copy of the ef-la gene have an extended lifespan further indicated that the ef-la gene may play an important role in determining longevity (shepherd et al., 1989) . in order to test this hypothesis, we have quantitated ef-la mrna, ef-la protein, as well as catalytic ef-la activity in john's flies. young and aging ef-hx transgenic (ef) and control lines ((2) were examined. because the the additional ef-lc~ copy is under the control of the hsp70 promoter, flies were aged either at 25~ or at 29~ the results show that transgenic ef flies do not express more ef-lct than the control flies, neither at the rna nor at the protein level. the question arises, whether the lransgene can be induced at all. this was tested by doing rnase protection assays. transgenlc message can be detected only in ef flies heat shocked at 37~ but not in ef flies grown at 29~ nor in control flies at 37~ from this experiment we conclude that the transgene is indeed inducible, but is not expressed in detectable amounts at 29~ we conclude that the difference in the lifespan does not result from the overexpression of the ef-la transgene. phenobarbital (pb) induces the expression of liver enzymes involved in the metabolism of drugs and other foreign compounds (xenobiotics). these enzymes include several cytochromes p450, nadph:p450 reductase, aldeyde dehydrogenase, epoxide hydrolase, udp-glucoronyltransferases and glutathione-s-transferases. many other proteins not yet recognized may however be induced by pb. our first approach therefore is aimed at identifying gene products which respond to pb treatment. as model systems we are using either chicken embryos (induction in ovo) or primary cultures of chicken embryo hepatocytes (induction in culture) (althaus et al., jbc 254 pp 2148 , 1979 . to differentially display mrna of induced vs non-induced cells we are using a rt-pcr technique with sets of random primers. electrophoretic separation of amplification products allows to identify mrna species which are induced (or decreased) following treatment. data obtained from these experiments and sequence information revealed from extracted pcr products indicate that pb treatment results in the transcriptional avtivation (or repression) of a variety of so far unknown genes. antithrombin -binding heparan sulfates from ovarian granulosa cells hosseini g., de agostini, a., clinique de st6rilit6, hcug, gen~ve. at the time of ovulation, several serine proteases of the plasminogen activator and coagulation cascades are activated in the ovary. these proteolytic activities are tightly controlled in time and space, and the heparin-activated serpins plaminogen-aetivator-inhibitor-1, protease nexin-1 and antithrombin (at) are present in the follicular environment. we have observed that granulosa cells produce a subset of heparan sulfate proteoglycans that bind and activate at. these at-binding heparan sulfates (ahspgs) endow the vascular endothelium with antithrombotic properties. we are now examining the role of ahspgs in the extravascular compartment formed by the ovarian follicle. using 125i-at binding assays, we have detected ahspgs on granulosa cell surfaces and culture media, in amounts comparable to those found for endothelial ceils. after 48h of stimulation by the gonadotropin fsh (1-100 ng/ml) granulosa cells increased the amounts of ahspgs they released in culture media by 2-6-fold, while keeping their cell surfaceassociated ahspgs constant. analysis of 35s-labelled hspgs revealed that heparan sulfates constitute about 40 % of the surface-associated and 10% of the soluble granulosa cell glycosamineglyeans. finally, using 125i-at autoradiography on rat ovary cryosections, we have localized ahspgs on the granulosa cell layers of large antral follicles, while ahspgs could not be detected in smaller follicles. these data suggest that granulosa cell ahspgs could be critically located to modulate proteolytic activities in the changing environment of the growing follicle. division, cibabasel, switzerland. the development of chelators which can mobilise excess storage iron (fe) and permit its excretion is necessary in the treatment of fe overloaded diseases. although oral administration of such a pharmaceutical is highly desirable, no orally active fe chelator is so far in regular clinical use. searches for orally active and safe fe chelators require appropriate animal models of fe overload in order to evaluate the potential of these drugs. an animal model is particularly useful for testing the capacity of new fe chelating ce~oounds in enhancing the mobilisation of clinically relevant storage iron. iron loading in animals was achieved by dietary supplementation with carbonyl fe, fe fumarate and 3,5,5-trimethylhexanoyl ferrocene (tmh-ferrocene). liver fe concentrations were increased 6-21 times above normal levels. tmh-ferrocene was the most efficient compound to induce stable liver fe overload. the orally given con'pounds induced a predominantly hepatocellular iron distribution and was found distributed among reticuloendothelial cells only at a later stage. this pattern of liver fe storage is similar to that observed in the early stages of primazy ha6~ochromatosis and late stage of transfusional hae~ochromatosis. eeeently, we and ethers have cloned and characterized novel transcription factors called peroxisome proliferator-actirated receptors (ppak). they belong to the superfamily of nuclear hormone receptors as the steroid, thyroid and retinoid hormone receptors. ppars are activated, beside xenobiotic peroxisome proliferators, by natural fatty acids and induce, together with the receptor for 9-cis retinoic acld (rxr), the transcription of genes involved in the ~-and ~-oxidation of fatty acids. ppar and rxr bind as heterodimers to the ppar response element (ppre), consisting of a direct repeat of aggtca-iike sequences with one intervening nucleotide. now, we show by gel retardation analysis and transient transfection assays that ppar/rxr heteredimers also bind to and transactivate gene transcription through estrogen response elements (ere). the ere is a palindrome with aggtca half-sites separated by three nucleotides. the selectivity and specificity of the transactivation through an ere was demonstrated by the testing of various related palindromic and inverted palindromic repeats of aggtca sequences, which were all inactive. the possible activation of estrogen responsive genes by fatty acids via ppars in vivo is discussed especially with regard to a debated role of fatty acids in breast cancer. the purpose of this study was to evaluate whether intermittent doxorubicin (dox) treatment in vitro selects for multidrug resistance in rpmi 8226 myeloma cells and, if so, to determine the underlying mechanism(s). cells were exposed to constant doses of dox for 4 days alternating with growth in dox-free medium for 17 days. after > 9 months of treatment, the cells developed an atypical mdr phenotype with 4-to 6-fold resistance to topo ii poisons. cross-resistance to vincristine and taxotere was < 2-fold, sensitivity to cisplatin and melphalan remained normal. efflux of mdr1 drugs was incresed with no effect of verapamit; subcellular dox distribution was normal. expression of topo ii a was found to be reduced by around 50 and 60-70% at the rna and protein level, respectively. minimum mdrt rna overexpression was detected when using rt-pcr; p-glycoprotein was not detectable. mrp rna expression was increased by 60-90% relative to the wild type cells without detectable p190. this data suggests that atypical mdr might play a role in acquired dox resistance in human myeloma. mucosal surfaces cover a vast area in the human body. they satisfy its need to communicate with its environment, e.g. in terms of nutrient uptake and sexual reproduction. however, at the same time they offer a vulnerable gate for invasion by pathogenic microorganisms. for immunoprotection of these regions, large amounts of iga antibodies are produced in the bloodstream and transported across the epithelial barrier to the lumen by means of polymeric ig receptor. there the receptor is cleaved, and its extracellular portion remains associated as secretory component (sc) to the secretory iga complex. in the context of a project aiming to mass production of secretory iga as a passive vaccine, we evaluated a series of eukaryotic expression systems for production of sc. the cdna encoding polymeric ig receptor was engineered in a way that only its extracellular domains corresponding to sc were expressed in yeast, in insect cells infected with recombinant baculovirus, and in mammalian cells infected with recombinant vaccinia virus. furthermore, a c-terminal poly-histidine tag was introduced for simple purification of the products. we will show optimization of expression levels for each of the systems as well as a quantitative and qualitative comparison of the products. recombinant vaccinia viruses (rvv) that express gone products from other organisms has become a popular and widely used laboratory expression system. our current interest is directed toward the production of secretory component (sc), a subunit of secretory iga antibody complex involved in mucosal immunity. toward this goal, we constructed rvv governing sc transcription under the control of two viral-based and the t7 bacteriophage promoters. expression of the protein was assessed in several mammalian cell lines, such as human tk-, human hela (grown in suspension or as a monolayer) and monkey cv-i. optimization of infection parameters and culture conditions were also performed. institut de biologic animale de runiversit~ de lausanne protection of mucosal surfaces is mediated by secretory iga ant/bodies (alga) constituted of dimeric iga and secretory component (sc) . toward the aim of reconstituting slga complexes in vitro and using them for passive oral immunization, sc has been produced in yeast, insect and mammalian expression systems. the gone encoding sc has been engineered so that a) the c-terminus of the protein carries a 6xhis tag and b) the newly synthesized polypeptide is released in the culture medium. culture conditions have been established to permit recovery of sc in serum-free medium. purification procedures based on nickel chelate and classical chromatographies have been optimized to yield sc under native conditions. finally, in vitro reassociation with purified dimeric igas obtained from hybddomas has been used to assess biological activity of recombinant sc. using a magnet-assisted subtraction-hybridisation technique (mast), 17 cdna clones were isolated that are expressed in nqrm~l l~nq %issue but n__d_i expressed in the corresoondina nsclc tissue, ii of the 17 edna clones are highly homologous to edna sequences for the following proteins: pulmonary surfactant proteins sp-a and sp-b; receptor for advanced glycosylated endproducts of proteins (rage); calmodulin-like protein; natural killer gone 5 (nkg5); matrix gla protein (mgp); glutamine synthetase; ubiquitin; vascular smooth muscle alpha-actin; cytoskeletal beta-actin; vimentin. the remaining 6 edna clones may represent parts of still unknown genes. the mast edna clones were derived from a patient who developed squamous adenocarcinoma. northern blot analysis of normal/tumour rna from three other nsclc patients showed that the lack of gone expression was independent of nsclc type and stage. enzymatic methylation of cytosines in mammalian dna is believed to play a fundamental role in basic gene regulation. methylation in the vertebrate genome is restricted to cpg dinucleotides at the c-5 position of cytosine. in vitro studies have shown that methylation can drastically influence the dna structure. cpg islands remain unmethylated in normal cells, whereas several immortalised, transformed cell lines contain partially methylated cpg islands. transcription of genes can be inhibited by methylation of cpg's. this modification contributes e.g. to the stable repression of genes on the inactivated x chromosome of females. the collagen vi genes show typical cpg islands in their promoter region and they were shown to be down regulated at the transcdptional level in src or myc transformed cells. given these facts we dee~ded to investigate the effects of methylation in the collagen vi gwene. e could demonstrate that the promoter activity of (x2(vi) chicken collagen gone was strongly diminished by methylation in vitro. furthermore dna methylation completely prevented binding of a novel transcription factor to the promoter, whereas binding of sp1 was not affected. to show evidence of methylation in vivo, we are currently iocalising the exact positions of methylated cpg's in normal and transformed cells by genomic sequencing. alterations of putative tumour suppressor genes in human non-small cell lung carcinoma (nsclc). r. shipman and c.u. ludwig, molecular oncology, lab 405 (zlf), basel, ch-4031 chromosomal subregion llp13 displayed non-random allelic loss in 61 nsclcs. llp13 contains the genetic loci for catalase (cat), the wilms' tumour gene (wti) and the follicle-stimulating hormone ~-chain (fsh~). 76% of the nsclcs were deleted at cat suggesting that loss of a gene(s) near cat may be involved in the progression of nsclc. yac clones containing the cat locus are being prepared for use in a direct selection procedure for cdnas encoded at this region. although 38% of the nsclcs were deleted at wti, no mutations were detected in the remaining wti allele. wti expression was subsequently demonstrated in fetal lung, fetal hematopoietic tissue, normal adult lung and nsclc. allelic deletion at 17p13, immuno-histochemical and sequence analysis of the p53 gene were also examined in our nsclcs. these analyses support the contention that aberrant expression of the p53 gene is a pivotal event in the progression of nsclc. $08-11 recently, a number of advances have been made which enable the experimentalist to study the effects of low levels of ionizing radiation. the results suggest a threshold to radiation damage exists and that above a critical level, recovery mechanisms in the cell are induced and the biological response falls. thus low levels of radiation cause greater than expected levels of biological response. this has been demonstrated in studies of mutations, cell survival and cancer induction. because the low dose-rates and the low doses per fraction result in the total doses being small, the magnitude of the biological effects induced is insufficient to warrant alarm. identification of genes associated with the revertion of the malignant phenotype of a rhabdomyosarcoma cell line. michele genini, sabina solinas toldo*, ruedi fries* and beat w. schafer, dept. of pediatrics, university of ziirich, steinwiesstr. 75, 8032 ziirich, and *institut for nutztierwissenschaften, eth ztifich, 8092 ztirich. the human rhabdomyosarcoma cell line rd is tumorigenic and differentiates very poorly despite the expression of the myogenic factors myf3 and myf4. therefore it can be regarded as natural mutant. to identify genes which are involved in suppression of tumorigenicity or enhance differentiation we applied two different approaches. since the development of human embryonal rhabdomyosarcoma has been associated with abnormalities of chromosome 11 band p15, in the first approach we transferred a normal human chromosome 11 into rd ceils via microcell fusion. the microcell hybrids did not show a higher degree of differentiation. suppression of tumorigenicity was observed in one clone but is probably independent of chromosome 11, as it was shown by chromosome 11 specific painting. ' in the second approach we applied a subtractive hybridization procedure between primary myoblasts and rd cells to find genes specific for the normal phenotype. these genes will be tested in vivo for induction of differentiation and/or tumorsuppression. deficient synthesis of the gpi anchor is the molecular basis of idiopathic paroxysmal nocturnal hemoglobinuria ("pnh") and a similar phenotype can accompany aplastie anemia cpnh-iike"). the population expressing cd48, a gpi-anehored membrane glycoprotein, was quantified by flow cytometry in samples from patient p.g. (pnh) and patient m.m. (pnh-like) and amounted to 90% (p.g.) and 78% (m.m.), respectively. t lymphocytes from both patients were isolated, expanded and cloned. from both patients, clones expressing the cd48 marker (cd48+) and cd48 deficient clones (cd48-) were obtained. takeda et al. (cell 73, 1993, 703-11) showed that in some (cd59-) bcell clones, a deletion of the p1g-a gene product is responsible for the expression of the pnh-phenotype. pcr analysis of t cell mrna from extracts of both cd48+ and cd48-clones of patient m.m. (pnh-like) using pig-a specific primers revealed a band pattern from which can he concluded that no deletion is present in the pig-a gene product. this result suggests that cd48-t lymphocytes of the pnh-like patient express a normal pig-a gene product; a disorder in regulation of pig-a expression or an unrelated biosynthetic defect may explain the cd48phenotype in these t cells. further work is aimed at defining the differences in biosynthetic defects of pnh and pnh-like cd48-t cell clones. supported by grant 31-30757-91 of the snsf to egb. w@ have recently employed an experimental approach to turn the function of "nuclear messengers" such as c-fos (and c-jun) on and off at will in polarized me/nmary epithelial cells. to this end we used constructs encoding the c-fos (and c-jun) genes fused to the hormone-binding domain of the human estrogen receptor, designated c-foser (and c-juner), we could show that short-term activation (30 mins.) of c-foser by estradiole (e2) led to the disruption of epithelial cell polarity within 24 hours, as characterized by the expression of apical and basolateral marker proteins. during the next 3-5 days, however, the cells fully regained their polarized phenotype. conversely, long-term activation (24-48 hours) caused the irreversible loss of epithelial cell polarity, leading ultimately to an epithelial-fibroblastoid cell transition. these cells underwent dramatic changes in gene expression including the complete loss or reduction of epithelial markers such as e-cadherin/uvomorulin, zo-i and cytokeratins, and the de novo expression of mesenchymal proteins like vimentin, type i collagen and fibronectin. expression of the v-ha-ras oncogene (acting upstream of c-fos) did not markedly influence epithelial cell organization when the cells were grown on plastic. however, when cultured within type i collagen gels in the presence of serum, or when injected into the mammary glands of nude mice, rapid cell growth and a clear epithelial-fibreblastoid cell transition was observed. growth properties determine the organ colonization ability of a metastatic murine melanoma cell line. the ability of metastatic tumor cells to specifically colonize distant organ sites strongly depends on the interaction of the metastatic cells with the local organ environment. we have selected a b16 routine melanoma cell fine (b16-ls9) which has an increased ability to colonize the liver, and compared its behavior with either its parental (b16-f1) or inng-specific (b16-f10) cell line. we have found that adhesion in vitro and organ lodgement in vivo were not different for ls9 and f10. in contrast, b16-ls9 grew at slower rates than the other lines both in vitro and in vivo after subcutaneous or intra-foot-pad injection. analysis of tyrosine-phosphorylated proteins indicated a higher phosphorylation activity in b16-ls9 cells, which might suggest an altered regulation of some kinase(s) in this cell line. intercellular contact with hepatocytes in vitro, or the liver in vivo could restore an efficient growth of b16-ls9, enabling thus these cells to colonize this organ much better than others. hepatocytes or liver plasma membrane (p.m.) extracts conserved the growth stimulatory activity towards b16-ls9 ceils. chromatographic fractionations of these extracts showed that a major growth sfimulatory protein in this fiver p.m. fraction turns out to be ttansferrin, which in the liver appears to exist in at least two different mature forms. protein import into mitochondria requires atp in two locations: outside the organelle, and in the matrix space. depletion of matrix atp arrests translocation of precursors across the inner membrane, but does not prevent precursors from crossing the outer membrane. as a result, proteins that reach the inner membrane or intermembrane space without passing through the matrix are often sorted normally in atp-depleted mitochondria. external atp is used to maintain precursors in a translocationcompetent state. however, some precursors can fold and still remain translocation-competent, and import of these precursors is independent of external atp. with the folded cytochrome b2 precursor, it is matrix atp rather than external atp that drives translocation across the outer membrane. thus matrix atp generates a pulling force that can drive both the translocation and the unfolding of precursor proteins. mitochondrial hsp70 probably functions as the atp-dependent import motor. arsenijcvic d, dulloo ag & girardicr l, dpt of physiology, cmu. geneva, ch. the relationship between body weight, daily energy expenditure (assessed by indirect calorimetry), and immune status (determined by lymphocyte proliferation tests) was investigated in swiss webster mice maintaining a stable body weight several weeks after infection with toxoplasma gondii(me49). following an initial period of weight loss (infection-induced cachexia), the surviving mice could be differentiated into two main groups: (i) those showing a partial regain in body weight (the infected gainers) and eventually stabilizing al a mean body weight 25% below a non-infected control group, and (if) those showing no weight regain (the infected non-gainers) and eventually stabilizing at 45% below control levels. immune function was found to be markedly suppressed in the infected non-gainers, whereas it was normal (and similar to control levels) in the group of infecled gainers. daily energy expenditure (and food intake), after normalisation for differences in body weight, was found to be the same in both infected gainers and non-gainers, but lower by 15% when compared to controls (p<0.01); thereby indicating that the infected groups were able to increase their overall efficiency of food utilization in response to the low food inlake. in conclusion, this study conducted during a weight stable phase of chronic infection shows a clear-cut association between the inability to regain body weight and impaired immune function. in contrast, no relationship was found between metabolic rate and body weight nor between metabolic tale and immune function, but the chronically infected mice exhibited the same phenomenon of enhanced metabolic efficiency characteristic of chronic underfeeding per se. identification and functional analysis of chaperonin 10, the groes homolog of yeast mitochondria. biozentrum, university of basel, klingelbergstrasse 70, ch-4056 basel, switzerland. phone ++41-61-2672171, fax ++41-61-2672148. the mitochondrial chaperonin system consists of chaperonin 60 (cpn60; also termed hsp60), which is homologous to e. cull gruel, and chaperonin 10 (cpnlo), which is homologous to e. coil groes. we have used a functional assay to identify cpnl0 of yeast mitochondria. when dimeric rubisco is denatured and allowed to bind to yeast cpn60, subsequent refolding of the enzyme is strictly dependent upon yeast cpnl0. in the presence of mgatp, yeast cpn60 and yeast epnl0 form a stable complex that can be isolated by gel filtration. the atpase activity of hsp60 is not inhibited by complex formation with cpnl0. a different result is obtained with the e, coil system, where groes is able to completely inhibit the atpase activity of gruel (1). to test the in vivo role of cpnl0, we have cloned, sequenced and disrupted the corresponding nuclear gene cpnio. this gene encodes a protein of 11,372 da that is imported into the mitochondrial matrix without detectable cleavage. haploid cells lacking a functional copy of cpnio fail to grow at temperatures between 23 ~ c and 37 ~ c (2). (1) rospert, s. et al. (1993) proc. natl. acad. sci. 90, in press. (2) rospert, s. et al. (1993) febs lett., in press. characterization of yeast mitochondrial heat shock protein 70 , l. bolliger, b. s. glick and g. schatz, biocenter, university of basel, 4056 basel. mitochondrial hsp70 (mhsp70) is thought to use the energy of atp hydrolysis to pull precursor proteins across the mitochondrial membranes. to facilitate the purification of yeast mhsp70, we used the cloned gene (a gift of dr. n. morishima) to modify mhsp70 with a six-histidine tag at its c-terminus. this construct supports growth of yeast cells lacking wild-type mhsp70. we developed a purification procedure that allows us to recover this tagged protein with a purity of about 95%. experiments are in progress to characterize the nucleotidedependent interaction of purified mhsp70 with unfolded proteins. in addition, we are looking for partner proteins that may modulate the action of mhspt0. we have copurified a protein of about 20 kd together with mhsp70. the 20 kd protein could be released from mhsp70 by atp or adp. when tryptic fragments of this protein were subjected to microsequencing, one fragment showed strong homology to the grpe protein of e.coli. the transmembrane electrical potential of mitochondria (awmit) can be assessed in single hepatocytes by using epifluorescence microscopy. for this end the rhodamine 123 fluorescence of a mitochondria-rich (fr) and a mitochondria-poor region (fp) are measured. the ratio of these signals depends on the awmit according to a modified nernst equation (reber b .f.x., somogyi r. & stucki j.w. (1990) , bba, 1018, 190-193) . to calibrate this method the cell membrane was permeabifized for monovalent cations with nystatin and gramicidin. then the cytosolie k + was replaced by na + from a k+-free bath solution. subsequent addition of valinomycin made the mitochondrial membrane permeable for k + ions. by the addition of different k + concentrations to the bath, the awmit could be clamped to defined values. the relation between the ratio fr/fp and the awmit could be well fitted to our modified nernst equation. however, the calibration curve flattens out at potentials higher than about 110 inv. therefore the detection limit of changes of a~mit within the physiological range is about 10-20mv. maximal stimulation of the na+/k + atpase by addition of nystatin to the na + containing bath medium caused a drop of awmit of about 20inv. subsequent addition of ouabain resulted in an almost complete reversal of this drop. this shows that changes in atp consumption can be assessed via changes of audmit. schneiter ph., di vetta v., j6quier e. and tappy l. institut de physiologie de l'universitg bugnon 7,1005 lausanne. the metabolic effects of an exercise performed in the fasting or fed state were studied in 8 subjects over a 8 hour period in a respiratory chamber. a mixed meal containing 13c labeled carbohydrates was ingested at 9:30 am and an exercise session (walking at 5 km/h 45 rain, slope 10 %) was performed at 8:15 am (fasting) or 11 am (postprandial). net carbohydrate (net cho ox) and lipid oxidation (indirect calorimetry) and oxidation of exogenous carbohydrates (exo cho ox, breath 13co2) were measured. glycogen breakdown was calculated as net cho ox -cho exo ox, and glycogen synthesis as cho in -cho exo ox. energy expenditure over 8 hours was similar but net cho ox was 16 % lower, lipid ox 61% higher and exo cho ox 39 % lower when exercise was performed in fasting vs fed state; moreover, glycogen synthesis was increased by 40 % (p<0.0001), while glycogen breakdown was increased by 12 % (p=0.06). in conclusion, a 45 min exercise in the fasting vs fed state a) increases utilization of endogenous lipids, b) enhances muscle glycogen turnover over a 8 hour period. hormonal regulation of e-abl tyroslne kinase by fusion to a steroid binding domain t. mattioni, o. 8chini hooft van huijsduijnen, p.k. jackson and d. picard d4partement de bielogie cellulaire, universit4 de geneve, ch-1211 geneve 4 the activities of a wide variety of proteins can be subjected to hormonal control by fusion to the hormone binding domain of steroid receptors. we show that this strategy can also be applied to a tyrosine kinase. in particular, transforming derivatives of c-abl become hormone-inducible oncogenes by fusion to a steroid binding domain. it is noteworthy that the hormone-inducible transformation of nih3t3 cells is completely reversible upon removal of the specific ugand. reversion experiments reveal another facet of abl: its ability to inhibit cell proliferation. 48 hours after hormone depletion, cells are not only morphologically reverted, but the cell cycle appears to be blocked in g1. a cytostatic effect of abl overexpression has been reported by several groups but, until today, it could only be examined by comparing different abl derivatives or the same derivatives in different cellular contexts. in contrast, our hormone-regulable system has the advantage that the two distinct functions of abl (transformation versus growth inhibition) can be studied using the same derivative expresed in the same cell line. we are using the hormone inducible system to investigate the mechanisms regulating the transtorming and the cytostatic effects of abl in a variety of cell lines. jiewu yang and herbert zuber institute for molecularbiology and biophysic, eth, 8093 z'tirich enzymes from thermophilic organisms show higher thermostability and temperature optima than those of mesophilic organisms. it has been shown that these properties are based on a specific structure of the protein. the primary structure of thermophilic (b. stearothermophilus) and mesophilic(b, megaterium) triosephosphate isomerase(tim) have been determined by cloning and sequencing of the tim genes. comparison of the primary structures of the thermophilic and rnesophilic enzyme showed specific amino acid substitutions, particularly in the c~-helices of the tim barrel, which could play a critical role with respect to thermostability. we have consmacted mutants by exchanging (x-helices between tim of b. stearothermophilus and b. megaterium. helix h1, h2 and h7 of tim from b. megaterium, corresponding to amino acid residues 13-36, 44-55. and 220-227 respectively, have been exchanged. the properties (thermostability and temperature optimum) of the hybrid-mutants were compared with the wild type enzymes. in a comparative investigation of structural and functional parameters related to the oxidative substrate metabolism in skeletal muscle cells of dogs and goats, a close relationship was found between intracellular lipid stores and mitochondria. as revealed in serial sections with subsequent em analysis, each lipid droplet was in direct contact to one or several mitochondria. quantitatively, 23% of the lipid droplet surface in goats and 40% in dogs were in direct contact to outer mitochondrial membranes or -in other terms -1% of outer mitochondrial membranes in goats and 2% in dogs were in direct contact to lipid deposits. these findings were in good agreement with the physiological data showing a 2 times higher oxidation rate of fatty acids drawn from intracellular stores for dogs than for goats. human muscle adapts to altered load with changes in the expression of enzymes involved in energy metabolism. endurance exercise in humans is known to increase the oxidative capacity of skeletal muscles (hoppeler, int. j. sports med. (1986), 7, 187) . at the ultrastructurai level, a higher mitochondriai content contributes to the higher oxidative capacity of skeletal muscles. little is known about the molecular mechanisms that lead to such adaptations in humans. the expression of mitochondriai components involves complex regulation of both mitochondriai and nuclear encoded genes. we have developed a pcr approach to quantify dna and rna from human skeletal muscle cryostat sections and addressed the question, whether the structural adaptations in endurance trained athletes are accompanied by concomitant changes in respective rna steady state levels. preliminary data indicate a higher concentration of coxiv and coxl (1.8 and 1.6 times, respectively) in trained subjects. no differences were observed for mtdna, despite a two fold higher mitochonddal content. human skeletal muscle contains three main fiber types which are based on the myosin heavy chain composition of the individual fiber, the isoforms of other contractile proteins within a given fiber type can vary leading to a continuum of different phenotypes. we are studying the fiber type specific expression of the mrnas of myosin alkali light clmins (mlc) using in situ hybridization, especially the rarer slow form mlc lsa. differences were found between shoulder muscles and leg muscles: m. deltoideus yielded stronger signals for mlc lsa and mlc lsb in the least glycolytic type i fibers (ia), weak but detectable signals for mlc lsb and mlc lf/f mrna in more glycolytic type i fibers (ib) and strong signals for mlc lf/3f in type ii fibers. in m. vast. lat. mlc lsb was strongly expressed in type ia as well as ib fibers, mlc lsa mrna was only found in some ia fibers. mlc isa has been shown to be expressed at a particular time point during muscle development and regeneration. in a biopsy of a becker dystrophy patient, we found very strong signals in some small fibers displaying the morphology of regeneralmg fibers. thus this probe could be useful to dete~t regenerative processes in pathological muscle samples. the aim of this study was to validate the complementarity of two new non-invasive methods for the assessment of autonomic regulations. reactions to a moderale exercise (75 w on a cycloergometer) were compared in cardiac transplant recipients (ctr) who modulate their heart rate (hr) by means of circulating calecholarnines, their heart being donervated, and in normal subjects where such exercise level doesn't stimulate the sympathetic system. the methods used were: 1) the spectral analysis of hr variability where the high frequency component (hf, above 0.15 t4z, related to the respiratory frequency) is a marker of the vagal activity, the low frequency component (lf; at about 0.1 hz) depends on both sympathetic and parasympathetic activities and the lf/rf ratio indicates changes in the sympatbo-vagal balance; and 2) a pure sympathetic index obtained from the amplitude of the t-wave of the ecg (twa) which is decreased by adrenergic stimulation of the myocardium. our results indicate that in both groups hr was increased by the exercise. in ctr, a net decrease in twa (-39_+ 10%, n=6) was measured. contrasting with this, in normal subjects, no change in twa (+2_+12%, n=6) but a decrease in beth hf (-67_+13%, n=7) and lf (-85_+5%, n=7) without change in lf/hf ratio (-19+_.26%, n=7) were observed. these results indicate that ctr subjects increase their hr by an adrenergic stimulation, whereas the normal subjects regulate their hr for such moderate exercise only by redudng their vagal tone. there was a significant relationship (r=0.79, p<0.0005) between ps and the ree. the slope of the regression line indicated a net cost of ps of 3.6 kcal/g. we conclude that obesity in children is associated with an absolute increase in whole-body protein turnover consistent with the increased ffm and ree. the "glucose paradox" in the anoxic and reoxygenated embryonic myocardium raddatz, e., tran, l., rochat, a.-c., kucera, p. and de ribaupierre, y. institut de physiologie de l'universitd, ch-1005 lausanne. the hearts of 4-day-old chick embryos explanted in vitro react rapidly, reversibly and reproducibly to anoxia and reoxygenation. this work deals with the effects of exogenous glucose on the mechanical activity of the hearts submitted to strictly controlled normoxia-anoxia-normoxia transitions. the anoxic periods lasted from 10 to 60 seconds. instantaneous heart rate, amplitude of contraction, velocities of contraction and relaxation of the ventricle wall were determined optically. in presence of 8 and 20 mmol/1 of glucose 1) the arrest of cardiac activity under anoxia was delayed by 60 and 110 %, respectively, 2) the duration of the postanoxic cardioplegia was reduced and 3) recovery was faster. paradoxally, these concentrations of glucose induced arrhythmias both during anoxia and reoxygenation. the incidences of such arrhythmias increased with the duration of anoxia. the absence of glucose or blockade of glycolysis suppressed arrhythmias. image analysis showed that the observed myocardial dysfunctions originated in the atrium and were sometimes associated with disturbances of propagation. the microinjeetion of okadaie acid into incompetent oocytas leads to the release of the prophase block with entry into m-phase. these g2/m-like alterations include chromatin condensation, germinal vesicle breakdown (gvbd), microtubules reorganization and formation of an abnormal (often multipolar) spindle of meiosis i, with chromosomes not aligned on the metaphase plate. the polar body is never extruded. moreover okadaic acid microiujection induces the emergence of phosphorylated cytolasmic foci as detected by the monoclonal antibody mpm-2, known to react with mitotic phosphoproteins associated with centrosomes. the kinetics of gvbd following mieroinjection is extremdy retarded (24-48hrs) when compared to spontaneous maturation (30ran-lhr) and depends on the oocyte diameter. interestingly, when inc~ompetent oocytes are cultured during 48hrs and then microinjeeted with okadaic acid meiosis resumes within a short delay (3-rhrs). mpm-2 epitopes are present on ecntrosomes only after okadaic acid injection. following gvbd one of the step depending on protein synthesis, the expression of tissue type plasminogen activator, is triggered. indeed oa-injected incompetent oocytes produce small amounts of this activator. altogether these results argue for an effect of okadaic acid favoring entry into mphase including microtubule organization, centrosomes phosphorylatiou and the recruitment of one of the masked mrnas (tpa). st~rillt~, ncb~, c~-121l g~. ~t4rs-inse~, monf~llier, france. meiotic reinitiation of the mouse oocyte is caracterized by a slow entry into metaphas~ i, b6~jinnin9 with germinal ve~ir break@own (gvbd) and ending with spindle formation. it is accompanied by a cascade of protein kinases and phosphatases increasir~g protein phosphorylation. the activation of histone hi kinase (maturation promoting factor, mpf) and of the 1342 hapk have been compared during spontaneous or okedalc acid (oa)-induced rhetoric reinitiation. in spontaneously maturing oocytes, hist6ne hi kinase activity increases before gvbd (2x) and is a~sociated with the disappearance of the upper (tyrosine phosphorylated) migrating form of p34 cdc2, following gvbd, histone hi klnase activity culminates (8x) in metaphase i. activation by phesphorylation of p42 mapk occurs after gvfld. in contrast, no increase of histone hi kinase is detec~cable in oocytes induced to reinitiate meiosis by a transient exposure %o oa, neither before gvbo nor during the following 7 hours of culture. the upper migrating form of p34 cdc2 is present for 8 houra. the activation of p42 mapk b~lins before gvbd. furthermore, when oa is applied on coclrces microinjected with p13 sucl, neither iocrease of histone hi kinase activity nor p34 cdc2 dephosphorylation are observed although gvbd is induced; p42 mapk is activated. altogether these results suggest that gvbd may or may not be associated with a detectable activation of histene hi kinase, depending on the experimental conditions. activation of ps4 cdc2 and p42 mapk are separable events. we propose that, in the mouse c~yte, oa might be able to aetivaf8 an alternative pathw~ leading to gvbd that is cdc2-independent and that involves p42 mapk activation ensuing mpf-independent phosphorylations. s 10-04 urich, k., 4002 basel, switzerland transformation of cells by polyomavirus is mediated by middle-t antigen. this viral protein is able to form complexes with src family tyrosine kinases (e.g. c-src), phosphatidylinositol-3-kinase (pi 3-k) and phosphatase 2a (pp2a). c-src associated with middle-t is constitutively dephosphorylated at tyrosione 527, a site negatively regulating the activity of this enzyme. this results in high tyrosine kinase activity in interphase and mitotic polyoma-transformed cells. middle-t is transiently hyperphosphorylated during mitosis as reflected by an increase in the apparent lvl, on sds acrylamide gels. two putative phosphorylation sites for a cyclin-dependent kinase present in middle-t, threonine 160 and threonine 291, are also phosphorylated by purified p34 ~a~ in vitro. we constructed mutant middle-t genes where individual phosphorylation sites were converted to alanine residues. mutants lacking threonine 160 were still able to associate with all cellular enzymes described above yet defective for gell transformation. interestingly, the defect of this mutation could be compensated by additional changes in the middle-t protein sequence introduced further downstream in a domain suspected to play a role in the targeting of this protein to intracellular membranes. $10-05 schmidt, s., fa~khauser, c., and viesturs, s. isrec, 1066 epalin~es, switzerland little is understood of the mechanisms which regulate cytokinesis, or how it is integrated with mitosis. we have cloned a number of genes from the fission yeast s. po~be which are required for the initiation of septation and are characterising them. defects in the cdc7 and cdcll genes result in continued nuclear division without cytokinesis, while cdcl6 mutants undergo multiple rounds of septum formation without cell cleavage. our analysis suggests that phosphorylation will play an important role in the regulation of cytokinesis and its integration with mitosis. a functional cdcl6 product is required for maintenance of p34 r activity in mitosis and the primary sequence of the cdc7 protein indicates that it encodes a protein kinase. data concerning the role of the cdc7, cdcll and cdcl6 genes will be presented. rfc was originally identified in human cell extracts as one of the cellular factors essential for the replication of sv40 origin-containing dna in vitro. it is a five subunit protein with one large subunit of 100-i40 kda and four small subunits of 36-40 kda. rfc binds specifically to primertemplate structures at the 3'-end of the primer. together with proliferating cell nuclear antigen (pcna) it serves as a primer recognition factor for dna polymerase ~ and ~. to show the invobement of rfc in cellular dna replication we are currently cloning s. cerevisiae rfc. the amino acid sequences of a number of tryptic peptides have been obtained. this information was used to amplify parts of the s. cerevisiae genes by per and to screen a genomic library with these probes. three genes were cloned so far. the large subunit, rfc1, codes for a 95 kda polypeptide that is 36% identical to the large subunit of human rfc (lirfc). the sequences for two of the small subunits were also obtained. rfc2, coding for a 40 kda polypeptide, is 50% identical to the hrfc 37 kda subunit. rfc3, coding for a 38 kda polypeptide, is 51% identical to the hrfc 36 kda subunit. there is also considerable similarity between the different subtmits. rfcj, rfc2 and rfc3, as well as the hrfc subunits, have consensus sequences for a atp/gtp binding site. all three genes are essential in s. cerevisiae. p53 is a major tumor suppressor gene which spefically interferes with the onset of e phase. we have therefore cxa~ined the possibility that p53 modulates the normal functions of enzymes and proteins directly involved in dna replication. we have shown that human wtp53 protein binds physically to calf thymus single stranded dna binding protein a, rp-a, we have also found that p53 blocks dna helicases in a typical strand displacement assay. this may reflect the fact that p53 is able to reanneal complementary dna single strands. since both wt and some mutant forms of p53 share this function, they are unlikely to be related to the p53 tumor suppressor activity. to our surprise, among the helicases which were tested we determined that the p53 inhibition could be released by rp-a in the case of cellular but not in the case of viral dna helicases. additionally, we found that p53 can partially inhibit dna polymerase ~ and s holoenzymes on singly primed mi3 dna. this inhibition, however, was only evident when e. coli ssb was substituted for rp-a. our data thus show that p53 exerts an inhibitory effect on several enzymes involved in dna replication and dna repair. the p53-rp-a interaction may function to protect replication enzymes from inhibition by p53 under certain physiological conditions. catalytic subunit showed that pol ~ is the most conserved replicative pol (cullmarm g., hindges r., berehtold m.w. and htibscher u., gene, in press). we synthesized pcr primers and cloned three fragments spanning the whole catalytic subunit. the resulting pcr products, called n-term, middle and c-term have a size of 1600, 1570 and 1280 bp, respectively. the primer for the n-term and middle fragments contained a histidin tag in order to purify the recombinant proteins by using a ni-nta column. these fragments were cloned into the expression vector pgemex174. because of the natural termination codon in the c-term fragment, the histidin tag was placed directly in the vector in front of the fusion protein, generating a new vector, prhh1s. all three fragments were expressed in e.coli. the fusion proteins could be purified in a single step and were used to raise polyclonal antibodies. finally, the entire pol 8 sequence was joined together and could be expressed. data on the characterisation of the antibodies and the proteins will be shown. hsp 70 from calf thymus can influence dna polymerase under heat shock conditions. we have generated antibodies against dnak protein, the hap70 homolog from escherichia coil, and used them for detecting of hsp70 protein during its purification from calf thymus tissue. the apparently purified protein has a molecular weight of 70kda, and has been recognized by antibodies against dnak and eucaryotic hsp72/73, it possesses a very week atpase activity, which can be stimulated by different poly-and oligopeptide substrates. results will be presented showing the influence of hsp70 on dna polymerase r from calf thymus under heat shock conditions. identification of a vertebrate cdc2 mutant which is unable to complete the g1/s transition. nicole sr and viestars simanis. chemin boveresses 155, 1066 epalinges,.isrec. s. pombe strains have been constructed which should permit the generic and molecular analysis of the events which occur in late gi when cells become committed to the mitotic cell division cycle and the initiation of dna synthesis. a number of mutants of the chicken cdc2 gene were eonsuueted during the course of analysis of phosphorylation sites some of which lead to the interesting phenotype of cold sensitivity when expressed in a cdc2 null background, when the only source of p34 cde2 is the chicken homologue of the gene, expressed from a muldcopy plasmid. in order to create a genetically tractable strain, the wild-type chicken cdc2 gone and one mutant into the s. pombe genome to create a strain in which cell cycle progression is dependent upon the chicken cdc2 gene. analysis of this strain indicates gnat the cells block predominantly before replication of dna. in order to determine whether the cells were arrested before or after the execution of the start control their ability to conjugate at the arrest point was assessed. consistent with a block before the traverse of start and contmim~ent to s-phase, cells were able to conjugate with high efficiency. further support for the view that this mutant is defective only for the g1-s transition is provided by the observation that it is able to complement a cdc2a2l mutation in trans. this mutant is defective only for (32 function and not traverse of start. tl~e double mutant is viable at the restrictive temperature of either of tile parents alld is no longer cold sensitive. further work will concentrate on cloning genes involved in the traverse of gi into s phase in fission yeast. (masson and kreis, 1993, j. cell biol. 123:357) where it is localized on a subset of microtubulea. when caco-2 cells are grown to confluency and become polarized, e-map-115 expression levels increase concomitant with a higher microtubule stability. transfection of fibroblastic cells (veto), which do not contain any detectable e-map-115, with cdna encoding this protein, results in stabilization of microtubules to nocodazole treatment. these data suggest that e-map-115 may be involved in the stabilization and reorganization of microtubules in polarizing epithelial cells. since e-map-115 is a stabilizing protein, its interaction with microtubulen should presumably be modified at the onset of mitosis to allow disassembly of the interphase microtubule network and formation of the mitotic spindle. indeed, immunolabeling for e-map-t 15 varies during mitosis. in early prophase, no e-map-115 can be detected on the microtubules of the forming mitotic spindle. the protein is observed on the spindle microtubules of metaphase cells and staining becomes bright on the new interphase microtubules of telophase cells. analysis of the protein in cells blocked in mitosis by low concentrations of nocodazole indicates that e-map-115 is hyperphosphorylated. this hyperphosphorylated form of e-map-115 is no longer able to co-sediment with microtubules in in vitro microtubule-binding assays. phosphopeptide map and phosphoamino acid analysis show the existence of novel phosphorylation sites in e-map-115 during mitosis compared to the protein during interphase, we are currently characterizing the kinases involved in the modulation of e-map-115 activity. little is known about the specific functions of calcium-binding proteins in epithelial cells ~md in particular in tumoral cells of epithelial origin. calretinin (cr) and parvalbumin (pv) are supposed to be involved in cell proliferation. we observed the endogenous expression of cr in several cell lines from human colon adenoearcinomas, and we obtained stably transfected cells for pv in a human ovarian adenocarcinoma cell line. we studied the cell cycle in correlation with the endogenous or ectopic expression of cr and pv, respeetiveiy. g1 and mitotic celts are strongly labelled with the cr-antiserum 7696, while pv immunoreactivity is dominant in interphasic, but not in mitotic cells. in cr expressing cells, the cell cycle seems to be normal and the rate of proliferation to be proportional to the percentage of positive cells. the cell cycle is inhibited at mitosis after the addition of cr antisense oligo nucleotides to the culture medium. the cell cycle is blocked in gi/s and g2 in the cells eetopically expressing pv. the results support the hypothesis that these two calcium.binding proteins, cr and pv, could intervene at different moments and probably with different mechanisms on the mechanism of the cell cycle in the tumoral cells studied. production of polyclonal antibodies against calretinin cr-22k gander, j.-ch., gotzos, v., cello, m.r. and schwaller, 8. institute of histology and gen. embryol., university; 1700-fribourg a mrna for an alternatively spliced form of calretinin, ealretinin-22k (cr-22k) was detected in widr cells (a cell line from a human adenocarcinoma). we therefore decided to produce antibodies specific for this protein. it is directed against the 14 amino-acids localized at the c-terminus of the truncated protein which are unique for this protein and not present in full-length calretinin. two different approaches have been chosen to obtain a specific antiserum. we have produced a chimeric protein containing the cterminal region of cr-22k and the (h)6(nanp)19 polypeptide as carrier. the fusion protein was expressed in e. coil and purified on a nickel chelate column. as a second method, a synthetic peptide (corresponding to the last 15 amino acids of cr-22k) was crosslinked with the carrier protein klh (keyhole limpet hemocyanin). the antigens were used to immunize two rabbits. after the first boost, the 2 sera were tested. we have observed that both antisera recognize besides the protein used for immunization also cr-22k (expressed in e. coil) in a western blot specifically. no other protein bands of either e.coli extracts or from eytosolic extracts of widr cells crossreact with the 2 antibodies. both antisera detected the protein cr-22k in immunohistochemieal stainings of widr cells confirming the presence of cr-22k in widr cells. the tctp (p23) is a growth related tumour protein which is expressed under translational control. homologous acidic proteins have been found in higher plants and in saccharomyces cerevisiae (ykl312) . the striking conservation of this polypeptide between these very divergent organisms suggests some important but still, unknown cellular function. to address this question, a null mutation was constructed in yeast, by deleting almost all the ykl312 structural gone. the deleted strain shows no detectable phenotype. therefore, we conclude that, in yeast, this gene is dispensable. the protein ykl 312has been overproduced in e. coil and injected to rabbits to raise antibodies. we are now characterizing the ykl312 expression at rna and protein level. ellenrieder,c., forrer,p., kosovsky,j., rischle, m., nueller, r. and jaussi, r., institut f~r medizinische radiobiologie, paul scherrer institut & universitgt zh, ch-5232 villigen radiation therapy of tumors could be improved by influencing cellular radiation sensitivity. yeast strains with defective cell cycle checkpoint genes (e.g. radl, rad3, radg) show increased radiation sensitivity. cross-hybridization of a probe from the tad9 gene on human mrna northern blots yielded a single prominent signal. experiments to clone the corresponding 4 kb cdna are underway. we have cloned a hamster cdna encoding the homologue of human cdk2. probably, this cdna encodes the p40 protein from hamster whose phosphorylation responds to radiation checkpoint signals and who does not bind to p13 sucl beads. treatment of cells with cdk2 antisense oligonucleotides is hoped to modulate the radiation sensitivity. proliferation of smooth muscle cells (smcs) is a major event in vascular development and atheromatous plaque formation. in order to characterize smc replicative potential, newborn rats have been injected with 3h-thymidine (3h-tdr) during their first week of life when most of smcs were proliferating; then, 3h-tdr labelling was evaluated in adult rats. depending on the number of mitosis that smcs had accomplished after the first week of life, four different smc subpopulations could be defined indicating that rat aortic smcs are heterogeneous in their replicative activity. 5-bromo-2'-deoxyuridine (brdu) incorporation after balloon induced endothelial denudation of rat aorta in adult rats showed that smcs entering into the cell cycle were mainly devoid of 3h-tdr labelling, this category could derive from two distinct smc subpopulations: smcs which were arrested in their proliferation before or just after birth or smcs which had actively replicated during development. thus, one (or two) subpopulation(s) of rat aortic smcs, characterized by a particular replicative activity during development, is (are) selectively activated after balloon induced endothelial denudation. the situation in vivo of dermal fibroblasts embedded within the connective tissue can be emulated in vitro by growing the cells in collagen gels. we have investigated how fibroblasts cultivated within a matrix react upon wounding of this dermal equivalent. human skin-derived fibroblasts (kd-cells) growing within attached collagen matrices were monitored over a period of 8 days. fluorescently labeled cytoskeletal elements (tubulin, vimentin, acfin) and fibronectin served as phenotypica/ markers. the 3d-arrangement of fibroblast microtubules, intermediate filaments and microfilaments, as well as of fibronectin was visualized by clsm and optical sectioning. cells at the wound margin, adjacent to the wound, and in non-wounded controls, up to a depth of about 100gm were analyzed. upon wounding, i.e. upon excision of a 1 mm 0 "punch biopsy" from the center of the collagen matrix, fibroblasts in the wound region oriented towards the matrix defect. layer-like structures of increased cell density evolved over time at the wound margin. the tissue defect was covered by fibroblasts, reminescent of the situation in vivo. global yeast genome expression analyzed by 2-d page after tctp homolog gene (ykl312) disruption. sanchez a translationally controlled tumor protein(tctp) was identified and microsequenced from a human liver sample. previous publications described the presence of a gene related to tctp in plants, mouse erythroleukemia and ascetic tumor, human mammary carcinoma and more recently in the yeast. tctp has no known function but its high degree of homology from plants to human underlines its probable crucial role in cell function. in order to study multifactorial chan~es in protein expression as a function of the ykl312 gene disruption m the yeast, we used high resolution two-dimensional gel electrophoresis combined with an efficient computer system (melanie). two groups of gels (ykl312 +(n=10) and ykl312 -(n=l 0)) were analyzed, compared and classified using correspondence analysis. there was no significant difference between the two populations of gels according to either qualitative and quantitative spots expression except for two spots which completely disappeared. the first one corresponded to ykl312 gsne product. the second one corresponded to a 28 kda peptide with an isoelectric point of 4.6. this ykl312 associated peptide still needs to be characterized and linked to the yeast genome. demyelinating and neurodegenerative diseases are associated with altered cellular responses including processes mediated by receptor protein tyrosine kinases. using a pcr cdna cloning approach we have identified a novel member of the eck/elk/eph genefamily in myelinating serum-free brain cell cultures. alternatively spliced cdnas encoding complete open reading frames for putative transmembrane proteins have been isolated from both rat and human brain. a recombinant protein derived from the rat cdna was demonstrated to have protein tyrosine kinase activity. an affinity purified antiserum to this protein was used to identify a glycoprotein of 120 kd molecular weight in brain cultures, and developping and adult brain tissue. the protein expression is most prominent during embryonal development and during the first three weeks of postnatal life. by in-situ mrna hybridisation the transcripts were found predominantly in restricted neuronal populations. in order to investigate systematically the effects of geometrically defhied abrupt tissue expansion on the characteristics of impulse propagation, we constructed expanding cellular structures in vitro using patterned monolayer cultures of neonatal rat heart cells (photolithographically patterned substrates.) the preparations, which consisted of narrow cell strands (40-80 tim wide) inserting into large reetangular cell areas (side lengths >1500 ~m), were stained with the voltage-sensitive dye di-g-anepps and were imaged onto a 12 x 12 photodiode matrix array (maximal spatial resolution 151.tin x 151am in the object plane). while impulse propagation was uniform in linear cell strands (average conduction velocity 0.35 m/s), a transient decrease in conduction velocity hi the transitional region was invariably observed in the suddenly expanded structures. this decrease was accompanied by marked distortions of the local action potential shapes due to bidirectional electrotonic interactions across the transition: upstream from the expansion, the repolarization phase was interrupted, a few ms after its inception, by a small secondary depolarization ("spike and dome" configuration), while, downstream, the action potential upstrokes were preceded by prominent prepotentials. in those cases where conduction failed at the tlansition, action potential durations upstream were dramatically abbreviated. these findings showed that phenomena similar to those recorded in intact purkinje-fiber-ventricular preparations can be observed in cell ensembles in vitro having comparable geometlins but consisting of cells with uniform active membrane properties. supported by swiss nsf grant 823a-028424 (sr) and usphs grant ns 16824 03ms). it is a noncollagenous glycoprotein with a molecular mass of 148 kda consisting of three identical subunits. cmp is selectively extracted with edta-containing buffer what indicates a divalent cation dependent anchorage of the protein in cartilage matrix. electron microscopy revealed the presence of three compact globular subdomains and sequence analysis indicated the presence of a coiled-coil c~-helical assembly domain formed at the c-terminal end of each subunit. the trimeric structure was retained after complete reduction under native conditions which reveals that the subunit structure of cmp is not only stabilized by disulfide bridges but also by the coiled-coil assembly domain. tissue distribution studies in mouse revealed that cmp is selectively expressed in some but not all cartilages. adult rat cardiomyoeytes (arc) in culture degenerate the myofibrillar apparatus and after attachment to the substrate they grow and reassemble new rnyofibrils. the appearance of new, well organized rnyofibrils can be observerd in several distinct regions. they appear close to the membrane proximal to the culture substrate and in the perinuclear region close to the substrate. previous studies have shown that embryonic cultured cells assemble new myofibrils which insert in adherens junctions at sites of cell-cell contacts and in sub-sarcolemmal adhesions plaques (saps) at sites of cell-substrate contacts. this saps are thought to function as the nucleation sites for myofibril formation. we have investigated the formation of this saps in cultured adult rat cardiomyocytes. using confocal light microscopy we can show that the interaction between rnyofibrils and membrans occurs close to the membrane proxirnal to the substrate and that this saps are flanking both nascent myofibrils and sfls, the latter structures being believed to serve as scaffold for myofibrillogenesis, additionally we have used electron microscopy to investigate the formation of this structures at higher resolution. $11-05 muscle satellite cells (sc) are quiescent myoblasts, ready to be activated and to regenerate new muscle fibers in case of muscle damage. in vitro, single human muscle sc, cultivated as clones, give rise in proliferating conditions to two subpopttiations of cells, cells expressing both ~-striated muscle actin and desmin (c~-sr+dsm+), and cells expressing desmin alone (dsm+). in culture conditions promoting differentiation, a clone .typically gives rise to a fusing progeny, yielding c~-sr+dsm + myotubes, and to non fusing myoblasts (nfmb). the latter are dsm +, a phenotype similar to quiescent sc found in situ. in order to determine whether nfmb are the in vitro equivalent of in situ quiescent sc, this first generation of nfmb were selectively collected and subcultured. in proliferating conditions, nfmb were able to resume proliferation and to give rise to a progeny with both c~-sr+dsm + and dsm + cells. when cultured in differentiating conditions, nfmb formed ct-sr+dsm + myntubes, and a new generation of dsm + nfmb. when nfmb of the second generation were again selectively collected and subculture& they resumed proliferation and ~re again able to yield both myetubes and a third generation of nfmb. nfmb of the first generation were also cultivated as clones, and 5 to 25% of them were able to resume extensive proliferation, yielding in their progeny both c~-sr+dsm + and dsm + cells, which differentiated into myotubes as well as nfmb. these results strongly suggest that sc have the stem cell property, of selfrenewal, yielding in their progeny both cells ready to differentiate into muscle and ceils similar to themselves. extra cellular matrix (ec~ regulates the expression of b-casein in cultured mouse mammary epithelial cells. we have developed a functional ~ epithelial cell strain, which expresses high level of milk proteins, forms alveolar-like structures when plated onto a reconstituted basement membrane and secretes casein unidirectior~lly into a it~aen. we have further shown that fflv~dependent regulation of b-casein occurs mainly at the transcriptional level and that 5' sequences play an inloortant role in this regulation. we have located a 160bp transcriptional enhancer (bcei) within the 5' flanking region of the b-casein gene. using functional assays, we show that bcei contains responsive elements for ~ and prolactin-dependent regulation. bcei placed upstream of a truncated inactive b-casein promoter reconstitutes a promoter even more potent than the intact prcmoter, which contains bcei in its natural context more than 1.5kb upstream. this small fusion promoter reconstitutes the nonaal regulation pattern. we show that bcei mediates f/24 dependent regulation even when linked to a het~rologous viral promoter. purified satellite cells (sc) were obtained from human skeletal muscle biopsies following cell sorting by flow eytometry. a chemiluminescent assay for acetylnholine (ach) revealed the presence of 1,3 nmoles/weu of 100.000 sc. in elonal cultures of proliferating sc, this intracellular level of ach was 0.l nmoles/well. when cells were cultured in the presence of an esterase inhibitor (10 ixm phospholine), the ach amount was enhanced 2 fold. conversely, cultivating the cells in presence of a potent inhibitor of choline acetyl transferase (2 gm bromoach), gave a 50% decrease of ach content finally, the release of ach was detected in the supernatant of cultured sc, following a 15 min incubation, and the measured level of ach was 3 pmoles/well/min. the presence of an ach-like compound in myogenic cells in both freshly isolated and in proliferating sc suggests that sc may contain ach in situ. in additiou, the fact that ach was present in the extracellular medium suggests that ach can be released spontaneously by the cultured sc. type xii collagen is an extracellular matrix protein associated with collagen fibrils in vivo. as observed for several other extracellular matrix proteins, the synthesis of type xii collagen by chick fibroblasts cultured in 0.1% fcs is stimulated by tgf-i~i. two splice variants of type xii collagen are known, with subunits of either 220 kda or 350 kda. by using antibodies specific for the large (350 kda) form of type xii collagen, we could show a more restricted distribution of the large variant compared to the smaller form in the embryo. while only the large variant carries chondroitin sulfate, both variants are identical in their collagenous domain. it is thought that via this domain, type xii collagen binds to collagen type i fibrils. we demonstrate here that type xii collagen can bind to collagen i fibrils also in vitro. by neutralizing acid soluble collagen type i, we could coprecipitate type xii collagen together with newly formed collagen type i fibrils. this interaction occurs in physiological saline but is highly salt dependent. in further studies we investigated wether 35s-methionine labeled type xii collagen treated with chondroitinase abc, ct-chymotrypsin, or collagenase can still be precipitated together with type i fibrils. in addition, we found that type xii collagen also affects the rate of type i collagen fibril formation. h.u. keller department of pathology, university of bern, ch-3010 bern locomoting blebbing cells colchicine (10-sm) can induce locomotion associated with blebbing in walker carcinosarcoma cells. blebs expand at a rate (about 2~m/sec) which is much faster than known rates of actin elongation. this suggest that the membrane is directly pushed forward by forces other than actin elongation, possibly by hydrostatic pressure. this interpretation is suggested by the observation that there is significant f-actin staining all along the cell membrane but not with the cytoplasmatic content the blebs. blebbing is suppressed by 0.5 m sorbitol. the finding that cytochalasin d suppresses blebbing shows that actin polymerization is nevertheless indirectly instrumental in bleb formation, possibly by generating a high intracellular pressure. a tentative model explaining locomotion of blebbing cells is presented. osteopetrosis encompasses a family of diseases with different causes and the common phenotype of impaired bone resorption. the osteopetrotic mouse mutant oo/oo is deficient in csf-1, the growth factor for the cells of the mononuclear phagocytic system. the phenotype of oo/ou mice is characterized by a low number of peritoneal macrophages and peripheral monocytes, and a virtual absence of osleoclasts, leading to the osteopetrotic phenotype. injections of csf-1 into op/oo mice reversed the osteopetrotic phenotype, proving the requirement for csf-1 during the process of osteoclastogenesis. by in situ hybridization, expression of csf-1 was demonstrated during bone development. osteoclast precursors and mature osteoclasts were identified as putative target cells for the growth factor, since these cells express csf-1 receptors, which is encoded by the proto-oncogene c-fm~. subsequently, specific binding of csf-1 to osteoclasts was shown. expression of c-fms parallels the expression of csf-1 in bone both in time and place, suggesting a local action of this growth factor in osteoclast formation, but also in the modulation of osteoclastic activity. different transcripts, raised by alternative splicing of a commmon nuclear rna precursor, have been found to encode a secreted and a membrane associated forms of csf-i. the secreted form can be modified by attachment of a glycosaminoglycan side chain, which serves as an anchor to integrate the peptide into the extracellular matrix. investigation of the role the different csf-1 forms play during recruitment of osteoclasts and regulation of their activity will provide further insights into the mechanisms governing these processes. comparative sequence alignments of known voltage-gated k + channels revealed two conserved cysteine residues in the putative transmembrane segments $2 and $6. if the cysteines were connected by a disttifide bridge, it would put a structural consllaint on possible channel models by placing $2 next to $6. we used site-directed mutagenesis to study systematically the potential roles of these invariant cysteines. fourteen of 17 substitutions in $2 (c232), and 7 of 19 substitutions in $6 (c393) maintained channel function in xenopus oocytes microinjected with mutant crna. therefore, the conserved cysteines are not essential for k + channel expression in xenopus oocytes. however, electrophysiological characterization of the cysteine replacement mutants revealed distinct roles for the two cysteines. inactivation, deactivation, and ion permeation did not considerably change in $2 mutants. in $6, in contrast, cysteine replacement by leucine, asparagine, glycine and valine accelerated inactivation and deactivation kinetics substantially, whereas serine and threonine showed opposite effects. furthermore, the voltage dependence of deactivation was differently affected. in summary, it appears that the side-chain at position 393 in $6 of kv2.1 is involved in channel gating by participating in the transitions from the open to the closed and inactivated state of the channel. (supported by grants from the nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) the segment between $5 and $6 of voltage-gatedk + channels, called h5 or p region, has been implicated to form part of the ion conduction pathway. little is known about the conforroation of this region although various models have been proposed including a j3 barrel-like structure formed by four adjacent antiparanel j3 sheets. to gain insight into the secondary structure of this region, we used cysteine substitution mutagenesis and sulfhydryl-specific, membrane-impermeant reagents to probe the accessibility of amino acid side chains in and around the p region of kv2.1 (drki). twemy-eighi positions from k356 to t383 were each mutated to a cysteine. after expression of mutant k + channels in xenopus oocytes, cysteine side chain accessibilities were probed by superfusion with ch3so2sch2ch2nme3 + (mtset), mtset can form a mixed disulfide with an accessible cysteine, and this may lead to current reduction if the covalently modified side chain is in or close to the ion conduction pathway. thus far, we have identified four mutations that showed k + current reduction after superfusion with 3 mm mtset. the mutants p361c, i379c, y380c, and k382c showed 87%, 99%, 39%, and 21% reduction in current amplitude, respectively. in coutrast, mutants $363c, a367c, t368c behaved like wild type kv2.1 with less than 5% current reduction. our results suggest that the side chains of p361,1379, y380, and k382 are directly accessible from the extracellular environment. taken together, these residues may, therefore, face the lumen of the ion channel pore. furthermore, the degrees of inhibition are in agreement with a model in which the ion conduction pathway narrows from residue k382 to y380 to i379. (supported by grants from nih and mda to rhj, and from the snf and the swiss foundation for medical-biological grants to rdz.) motejlek k., h,,iuselmann r., and liig:her b.; pharmakologisches institut der universitiit ziirich, winterthmtr. 190, ch-8057 zlirich comparison of 5' flanking sequences of three gabaa-receptor subunit genes (atl, ~, 8) revealed a novel conserved purine sequence dement with the consensus sequence gagaggggagaoga gagag(gg/aa)g. this element is present once each in the (zl and 72 subunit gene promoters and in seven tandem copies in the 8 gene promoter. a novel dna binding activity (bsf1) was identified that binds to various versions of this purine element. bsf1 was found ordy in brain but not in any other tissues tested. furthermore, the factor is distinct of beta, another brain-specific dna binding .protein with a purine-rich dna recognition sequence. the expression of bsf1 during differentiation of eerebeuar granule cells in vitro correlates with the expression of gabaa-receptor subunit genes. this correlation suggests a role for bsf1 as a transcriptional activator of neuronal genes. therefore, bsf1 may be important for cell ty~specific regulation of gabaa-receptor gone expression. we also found that bsf1 binds to wheat germ agglutinin, suggesting that this protein is glycosilated. taking advantage of this property, we are in the course of purifying bsf1, the question, whether bsf1 is indeed a transcription factor is being investigated in vivo and in vitro. to produce a simple method of estimating the free magnesium concentration ([mgzq) in solution, we have manufactured dip cast mg 2+ macroelectrodes using the neutral mg carrier eth 7025.we have used the elecmides to measure the dissociation constant (k~) for mgatp in a background solutions mimicking the intracellniar milieu. for the mcasmement of the big atp dissociation constant, the background solution contained 2 mmol/l n%atp. this solution was titrated with mgci 2 and the changes in [mg ~] above 0.05 retool/1 monitored with the maeroelectrode. during the titration the ph was maintained constant. fitting such titration curves with the standard hyperbolic binding equation, after correction for zero drift, gave the following mean+sd values for the ka (pmol/1 at 37~ ph 6.4, 103.3-+-25,3 (n=9); ph 7, 59.7:k-27.4 (n=6) and ph 7.4, 30.g~15.4 (n=7). elevation of the [naq to 50 m~l/1 and reducing the [kq to 1(30 mmol/l was without effect at ph 6.4 (n=6). reducing the temperature to 25~ increased the k a at 7.4 to 49.0:k21.6 (n= 5). mg =+ macroelcctrodes provide an easy way of measuring [mg z ⧠in solution. [mg ~] can also be measured in a background solution containing i mn~i/l ca, although the electrode response is reduced. interference by protein to date prevents their use in plasma. similarily to the block by zn 2+ and ca 2+, protons only partially block the channel. the ability for all mutated channels to. conduct na + current, and the partial block induced hy zn 2+, ca 2+ or h + indicate that y401 is not located deep in the pore but rather in the extracellular mouth of the channel. progesterone modulates the activity of glycine receptor expressed in colliculli neurons of neonatal rats. maury, k. & bertrand, d. dpt of physiology, cmu, 1211 geneva 11. steroids are potent narcotics and have been shown to act on ligand-gated channels activated by gaba or nicotine. however, little is known about their possible actions on other receptor types. for instance, while it was demonstrated that progesterone inhibits glycine receptors expressed by spinal cord neurons isolated from chick embryos, nothing has yet been reported for brain receptors. in this study, we have determined the properties of glycine channels expressed by brain neurons of neonatal rats using the whole-cell voltage-clamp technique. in inferior colliculi neurons, glycine elicited little-desensitizing inward currents which reversed around -40 mv. these currents were blocked by strychnine in the nanomolar range. surprisingly, however, we found that progesterone, in the micromolar range, potentiated the glycine evoked currents. these results, contrasting those obtained in chick spinal cord neurons, suggest that progesterone enhances or antagonizes glycine currents depending on the subtype of the receptor. the mammalian auditory organ (organ of corti) relies on two groups of sensory cell for its normal hearing: inner hair cell & outer hair cell. ihcs are mainly innervated by afferent fibers while the ohcs essentially receive efferent fibers. the ohcs possess unique electromofile properties which are thought to enhance the motion the basilar membrane and to refine the mapping of the sound frequency. we found, using whole ceil recordings, that 5-10% of the fleshly dissociated ohcs displays fast inward currents. the magnitude of peak currents which can be as large as 2na vary from cell to cell. further experiments have revealed that this current is sensitive to ttx and strongly reduced when extracellular sodium is replaced by choline. its kinetic is relatively slow compared to that of sodium current of typelspiral ganglion cell, and has a more negative inactivation. other voltage activated currents and ligand-gated currents are under inverstigation. these results will provide further insights in the hearing transduction mechanism. the formation and the properties of homotypic and heterotypic gap junctions were studied using two types of insect cells, c6/36 and sf9. two single cells were pushed against each other and the subsequent de novo formation of gap junction channels was assessed by means of the dual voltage-clamp method (bukauskas and weingart: pfl~g. arch. 423:152, 1993 it was symmetrical for homotypic junctions. the vj-sensitivity was less pronounced in sf9 cells. hence, the relationship for heterotypic junctions was asymmetrical. all pairs examined revealed a s-shaped relationship between gj(ss) and v, which was virtually superimposable (gj(ss) declined upon depolarization). each channel contains a vm-gate and two ~-gates. the vj-gates are operated independently. they close when their intracellular aspect is made positive. shaped curve compatible with a two-state boltzmann process. half maximal inactivation was reached at -77 mv and +66 mv, implying an asymmetrical gating behavior. in case of an asymmetrical protocol (~ and vewas stepped simultaneous, but in opposite direction), the q-dependent asymmetry disappeared (half maximal inactivation at -59 mv and +60 mv). the asymmetry in gj-gating, attributable to the pulse protocol, was also reflected in the time constants of ij inactivation (r177 cerebellar granule cell cultures were used to study the regulation of the nmda receptor expression. we have shown previously that chronic membrane depolarisation (25 him k +) or treatment with nmda {i00 ~m) promotes the functional expression of nmda receptors, as assessed by nmda-evoked 45ca2+ influx. we have now developed a rnase protection procedure for the quantitative determination of the mrna levels of the nmda receptor subunits known so far (nr-i,2a,2b,2c,2d). the growth conditions mentioned above failed to alter the mrna levels of the different subunits. at the protein level, nmda receptors were evaluated quantitatively by the labeling pattern obtained by the new photoaffinity ligand 125i-cgp55802a. the intensity of the phctolabeled bands, thought to represent nr-i and nr-2a subunits, was increased by treatment with high k + or nmda compared with control cultures. this result suggest an increase in receptor protein by high k + or nmda treatment. thus, posttranscriptional mechanisms seem to play an essential role in the modulation of the nmda receptor activity. the gene encoding the pore-forming subunit of the rat epithelial na + channel (~renac) was cloned recently and shown to belong to a novel gene family coding for cation channels (nature 361, p467-470 (1993) . transport of na + ions through these channels is the rate-limiting step of na + absorption and thereby controls osmotic balance of body fluids and secretions. in order to study the implication of ~renac in regulating sodium balance, blood volume and blood pressure and therefore its involvement in hypertension, we expressed ~renac under the control of the human cmv promoter in transgenic mice. several lines of mice showing expression in different tissues were obtained. progress in the analysis of these mice will be discussed. activation of metabotropic glutamate receptors increases the excitability of neurones in the lateral septum of the rat. to elucidate the mechanism(s) of this action, we used whole-cell patch-clamp recordings from coronal brain slices of young animals. in voltageclamped cells, the selective agonlst (is,3r)-acpd, at 1-50 /~m, had the following effects, a) it evoked a sustained inward current, which was resistant to ttx and to cadmi~am and which persisted in neurones loaded with bapta, a calcium chelator. this current displayed inward rectification and was reduced, or suppressed, when extracellular sodium was partially replaced with n-methyl-d-glucamine. b) it elicited a ttx-insensitive, voltage-dependent inward current, which activated at -50/-40 mv and which reversed at aound 0 mv. this current was suppressed in a low-calcium/high-magnesium perfusion solution and was undetectable in the presence of intracelhilar bapta. we suggest that acpd exerts a dual action on lateral septal neurones. it causes a steady depolarization by generating a sodium-dependent current and it triggers transient plateau potentials by inducing a calcium-dependent cationic current. interaction between these currents results in a powerful, self-reinforcing neuronal excitation. we have investigated the effects of protein kinase c (pkc) activators (4[~-pma, oag) and of phosphatase inhibitors (okadalc acid, calyculin a) on voltage-gated ca 2+ and k + channels in ngf-differentiated pc12 ceils. whole-cell ba 2+ and k + currents were recorded with the patch-clamp technique. by using specific ca 2+ channel blockers (co-conotoxin (cgtx), isradipine) we found 3 types of ba 2+ currents (iba): a) a ~cgtx-sensitive iba; b) an isradipine-sensitive iba; and c) a t0-cgtx plus isradipineresistant iba. 4[~-pma and oag specifically down-modulated the isradipine-sensitive iba. the inhibition of iba was prevented by staurosporine and pkc (19-31) (2 pk inhibitors). the delayed rectifier k + current was unaffected by pkc activators. applied externally, okadaic acid and calyculin a inhibited the total iba by affecting several components of the ba 2+ currents. however, the 0~-cgtx plus isradipine-resistant iba was unaffected by okadaic acid. in conclusion, our results suggest a differential modulation of voltage-gated ca 2+ and k + channels by the pkc signalling pathway in ngf-differentiated pc 12 cells. agrin, a protein isolated from basal lamina extracts of the electric organ of the marine ray, is thought to mediate the motor neuron-induced aggregation of acetylcholine receptors (achrs) in muscle fibers at their neuromuscular junction. recent cloning in the marine ray, rat and chick has revealed that agrin can be alternatively spliced at two sites in its c-terminal half. site a encodes either 0 or 4 amino adds and site b encodes either 0, 8,11 or 19 (8+11) amino acids. studies of agrin mrna expression indicate that early in synaptogenesis, chick motor neurons contain high levels of a4b19. in contrast, non-neuronal cells and muscle calls contain a4b0 and aob0 mrna. in the adult ray, electric lobe motor neurons that innervate the electric organ contain a4b8, whereas agrin mrna in the electric organ again lacks both sites (mcmahan et al. (1992) , curr. up, cell biol. 4:869-874). to investigate the functional properties of agrin isoforms in more detail, we have now compared their specific activity to aggregate achrs on cultured chick myotubes. heterologous expression of the c-terminal half in cos-7 and 293 cells revealed that chick agrin isoforms a4b8 and a4b19 were highly active, while a4bll was up to 40 fold lower in activity. no activity could be detected for the a4b0 and aob0 isoforms. in agreement with these findings the a4b8 isoform of the marine ray also showed high achr-clustering activity. therefore, we conclude that motor neurons throughout development synthesize highly active agrin isoforms while the postsynaptic target ceils do not play a primary inductive role in the formation of synapses. we have used whole-cell patch-clamp recordings and hypothalamic slices in order to characterize the effect of n-methyl-d-aspartate (nmda) on suprachiasmatic neurones. in ceils clamped at or near their resting membrane potential, nmda (50-100 #m) generated an inward current of 10-60 pa, which was insensitive to ttx and which reversed at about 0 inv. the nmda current-voltage (i-v) relation contained a region of negative slope conductance. the nmda current was reduced or suppressed by d(-)-2-amino-5-phosphonopentanoic acid (d-aps) or by mk-801. it was potentiated by reducing the extracellular magnesium concentration from 1 to 0.01 ram, or by adding glycine (10 /~m) to the perfusion solution. in a majority of neurones, lowering the extracellular calcium concentration from 2 to 0.01 mm caused a 1.5to 4-fold enhancement of the nmda current. i-v relations indicate that in the low-calcium solution, the region of negative slope conductance was attenuated. this effect was due to extraceuular calcium, since it persisted in neurones loaded with the calcium chelator bapta. we conclude that nmda channels present in suprachiasmatic neurones may be modulated by extraeellular calcium. institut, and abt. pharmakologie*, biozentrum, basel. during innervation of skeletal muscle, the myonuclei underlying the synapse begin to express acetylcholine receptor (achr} ~-subunit gene and they remain activated after the nerve is removed. our experiments show that this is due to a factor in the synaptic portion of the muscle fibre basal lamina (bl). one candidate for this factor is agrin, which regulates ache clustering in the synaptic membrane: i) unlike in untreated muscle, the level of s at synapses was strongly reduced in cultured rat muscle fibres after proteolysis of synaptic bl. 2) conversely, when rat myotubes were cultured on bl isolated from adult muscle, they preferentially expressed achr accumulations and ~-mrna at sites where they contacted the synaptic portions of the isolated bl. 3) when various substrates were impregnated locally with agrin a4big, an isoform expressed by motor neurones in fetal spinal cord, it locally induced in the myotubes the expression of s-mrna. art increase of functional nicotinic acetylcholine receptors precedes the fusion of cultured human muscle satellite cells. r. m. kranse, c.-r. bader and l. berrtheim. department of physiology and division of clinical neurophysiohigy, university medical center, 1211 geneva 4, switzerland nicotinic acetylcholine receptors (nactlr) are expressed on embryonic myoblast and acb may play a role in mechanism of fusion. our study focuses on the appearance of functional nachr in freshly isolated and cultured satellite cells (sc) from normal human muscle biopsies. sc cart be conditioned to either proliferate or fuse to form myotubes depending upon culture media. presence of ach-activated current was investigated using the whole-cell and single.channel patch-clamp technique. in freshly isolated sc (whose properties should be close to the quiescent in vivo sc/no nachr were observed (n=16). in the proliferating state, 54% (n=35) of the satellite ceils (which are also called myoblasts) displayed a small ach-activated current (10 pa/pf) and, as expected, after fusion of salellite cells into myotabes, 100% of the ceils displayed an ach-activated current (n=6; 26 pa/pf). to examine, whether the increased expression of nachr precedes sc fusion, sc were cultured in a medium which promotes fusion, but were prevented from fusing by keeping them at a low density. in this culture condition, 93% of the sc (n=15) displayed an ach-activated current and, in addition, these cells expressed a 4 times higher ach-current density. (40 pa/pf) than the proliferating sc. we also observed that, in high density cultures, a small population of sc do not fuse even atter 3 months in the medium promoting fusion. these non-fusing myoblasts (nfmb) had no nachr. our results suggest that the appearance of nachr may be related to the process of sc fusion as i) quiescent sc in vivo do not express nachll ii) nachr expression increases in conditions promoting fusion and iii) no nachr were observed in nfmb. the latter cells may be equivalent of quiescent sc in vivo. $12-20 we have investigated the sensitivity of neuronal nicotinic acetylcholine receptors (nachrs) of known subunit composition to various cholinergic antagonists. neuronal nachrs were expressed in xenopus oocytes after injection of pairwise combinations of (x2 or c~3 with either 1~2 or 1~4 crnas. the two-electrodes voltage-clamp technique was used to measure currents induced by rapid application of low concentrations of acetylcholine (ach) together with increasing concentrations of antagonists. the response of ct31~4 neuronal nachrs to ach was halfmaximally inhibited by following antagonist concentrations (ic50): hexamethonium (0.3 i.tm), mecamylamine (0.2 p.m), pentolinium (0.2 i.tm) and trimetaphan (1.2 ~tm). with (x3132 nachrs the ic50s of the same antagonists were about 10 times higher. finally, (+)-tubocurarine was a conventional competitive inhibitor of ach in ~3132 and t~2~2 nachrs. in contrast, low concentrations of (+)-tubocurarine increased the response to low ach concentrations in a3(34 and t~2~4 nachrs. these results further underline the importance of [l subunits in determining the functional properties of neuronal nachrs. supported by the hochschnlstiftung and nf grant 31.31018.91 to abc. previously, the role of the transglial tubular system was shown for the squid giant axon: in na + free (tris) solutions the series resistance increased in correlation with a decrease of the tubular opening density (tod). in the crayfish giant axon, which show similar tubules, we correlate the change in tod, measured in freeze-fracture preparations, witl] the conduction velocity. the control to'd was estimated to be 16 per #ms. after 30 and 75 rain tris-inkubation the tod decreased to 10,4 and 1,5, respectively. this reaction was reversible when these axons were reincubated in na~-c41ntaining solution. after 120 rain reincubatiou the tod was 11.8 per tam'z. to determine the influence of decreased tod on the impulse conduction velocity, the nerve was incubated for 30 min in tris solution followed by reincubation. impulse propagation then recovered in two phases. during an initial fast phase with a short time constant of 1-2 rain na + diffused back into the periaxonai space and allowed impulses to occur with a low conduction velocity. in the second phase with a long time constant of 7 min, conduction velocity recovered slowly to normal values. this time course was comparable to the recovery of tod, as estimated from recent freeze-fracture preparations after short reincubation. these results indicate that the normal tod is a necessary condition for the normal excitability of the axon and determines its conduction velocity. most spinal cord neurons respond to the inhibitory action of gaba and glycine, suggesting co-expression of gaba a-and glycine receptors 4n individual ceils. while glycine-receptors are exclusively found in post-synaptic densities, the cellular localization of gaba areceptors has not yet been characterized. in this study, the distribution of gabaa-receptors in the spinal cord was analyzed with an antiserum recognizing the (xl-subunit. double-and tripleimmunofluorescence staining were employed to identify synaptic receptors apposed to gabaergic terminals (immunoreactive for glutamic acid decarboxylase) and to assess their co-localization with glycine-receptors (visualized with an antibody to the 93 kda protein gephyrin). staining for the gabaa-receptor ctl-subunit decorated the soma and dendrites of numerous neurons in laminae iii-viii and x, revealing their morphology in clear detail. by contrast, laminae 1i and ix contained little immunoreactivity for these gabaa-receptors. most gabaa-receptor-positive cells also exhibited a prominent glycine-receptor immunoreactivity. both types of receptors had very similar distribution patterns and were frequently co-localized in sites apposed to gabaergic boutons. these results indicate that gaba aand glycine-receptors may co-exist within single post-synaptic densities, suggesting a possible synergism between gaba and glycine neurotransmission in spinal cord neurons. prenatal benzodiazepine (bdz) exposure changes both behavioral and neuiochemical parameters of developing iats. these changes are not a consequence of remaining bdz in brain tissue, but rather indicate alterations in bdz receptol binding and function. since the bdz binding site is located on the gaba~ receptor complex, it is possible that prenatal bdz treatment influences the expression of gaba~ receptor subunits. to evaluate effects of pienatal bdz exposure on mrnas expression specific for several gaba~. receptor subunits (~i, ~5, ~ & y2), we treated plegnant rats with diazepam (l.25mg/kg/d; s.c.) from gestational day 14 to 20. we then analyzed specific mrna levels in offspring at four diffeient developmental stages (gd20, pns, pni5 & adult) by in situ hybridization. pleliminary data flom optical density measurements indicate a deciease in expression of mrna in particular for ~-subunit of gab~ receptors in different brain reglons of plenatally diazepam-treated zats. processing of sensory information within the auditory system has been well characterized using histological and eleetrophysiologlcal techniques. however, relatively little is known about the neurotransmitters involved. the present study was performed to elucidate the possible role of excitatory (eaa] and inhibitory (i.e., gaba) amino acids in neurotransmission within the primary auditory cortex (all the main afferent to the ai, the ipsilatera} medial geniculate body (mgb), was electrically stimulated and evoked responses were recorded intracellularly in the ai region in halothane anesthetized cats. a seven-barrelled iontophoresis pipette glued alongside the recording electrode allowed localized application of eaaergic and gabaergic compounds onto the recorded neuron. in most ai neurons, mgb stimulation evoked a short latency, short duration epsp followed by a long-lasting ipsp. pattern, duration, and amplitude of synaptic potentia{s were highly variable and strongly dependent on stimulation intensity, reduction of gabaa receptor-mediated inhibition by iontophoretic application of either bicuculline or sr95531 markedly enhanced mgb stimulation-evoked epsps. only the late component of this enhanced epsp was reversibly blocked by the competitive nmda receptor antagonistl ap7 at currents which selectively blocked neuronal excitation induced by iontophoretic application of nmda, our results demonstrate that in the eat 1) nmda receptors in ai are activated following mgb stimulation and that 2) a dominant gaba~ receptor mediated inhibition usually masks this nmda receptor activation under our experimental conditions, a highly purified and specific cell wall degrading endo-l,5-u-l-arabinanase was isolated from an a. niger pectinase preparation by low and medium (fplc) pressure column chromatographic separation methods. the isolated enzyme was most active on linear 1,5-~-l-arabinans (-90 i.u./mg), whereas branched arabinans from sugar beets were degraded to a lesser extent (-14 i.u./mg). the enzyme was shown to be electrophoretically pure after silver staining (sds-page) and its identity was confirmed through specific binding to an antiserum directed against endo-l,5-~-l-arabinanase. the major physico-chemical characteristics of the enzyme were the following: mr 42'000, iep ~ 3.0, ph optimum 4.8, temperature optimum 55~ ph stability 3.5-8.0, temperature stability s 45oc, km = 0.205 mg/ml, vmax = 17.7.10 -3 ~unol/min. zn and hg showed potent inhibitory effects. 3.2) is a specific enzyme of the glyoxylate cycle, which plays a key role in the initiation of gluconeogenesis in germination oilseeds, this pathway is localized in glyoxysomes, single membrane organelles which are converted into peroxisomes when lipid reserves are depleted. the glyoxylate cycle enzymes have been the subjects of numerous conflicting reports typically addressing the problems of their synthesis (on free or bound ribosomes), targetting and suborganelar localization (membrane bound or matrix proteins). recent biochemical studies have shown that ms from germinating soybean cotyledons is an interconvertible enzyme (membrane bound, aggregated or soluble fomls) which is significantly affected by its ionic environment (henry et al., 1992, plant science 82:21-27) . microscopy studies have now been carried out using immunofluorescence staining or immunogold-silver staining for light microscopy, and immunogold labelling for electron microscopy. all results consistently characterize ms as a glyoxysomal mawix enzyme. chorismate mutase (ec 5.4.99.5) catalyzes the first step in that branch of the shikimate pathway which leads to the aromatic amino acids phenylalanine and tyrosine. we have isolated a cdna for this enzyme from the higher plant arabidopsis thaliana by complementing a yeast strain (aro7) with a cdna library from a. this is the first chorismate mutase cdna isolated from a plant. the a. thaliana chorismate mutase expressed in yeast revealed allosteric control by the three aromatic amino acids as previously described for plastidic chorismate mutase isozymes. an attachment of rubisco to chloroplast membranes under cu++-stress has been described for wheat (mehta et al., j. biol. chem. 267, 2810 -2816 . the displacement to the membranes has been discussed as a possible step in the degradation of this predominant stromal protein, e.g. during leaf senescence. in our recent experiments it became evident that such interactions are not restricted to rubisco. several other stroreal, and even a peroxisomal enzyme (glycolate oxidase), were also detected in the membrane fraction after 6 to 30 hours of oxidative stress induced in wheat seedlings by a treatment with 10 mm cuso 4. the velocity and the degree of membrane attachment varied for different enzymes. based on these results it was interesting to check the solubility of rubiseo and its previously described 45 kd fragment which accumulates during the incubation of bean leaf discs under oxygen deficiency (hildbrand and feller, experientia 47, a67) . neither rubisco nor the breakdown product were found to accumulate in the membrane fraction. thus, at present, the steps involved in rubiseo catabolism cannot be generalized. different mechanisms depending on the metabolic situation and on environmental conditions should be considered. overexpression of the four parsley phenylalanine ammonia-lyase (pal) isoenzymes in e. coli. characterization of the enzymes and kinetic analysis of the inhibition by 2-aminoindan-2-phosphonic acid. christoph appert, j/irg schmid, jerzy zorn* and nikolaus amrhein institute of plant sciences, eth-z/jrich, 8092 zijrich. *technical university wroclaw, 50-370 wroclaw, poland. all four phenylalanine ammonia-lyase (ec 4.3.1.5) isoenzymes of parsley (petroselinum crispum nym.) were expressed as glutathione s-transferase fusion proteins in e. coil after affinity purification, the glutathione stransferase moieties were cleaved off with factor xa and the phenylalanine ammonia-lyases were characterized. the proteins form enzymatically active tetramers, even as fusion proteins. the four isoenzymes have comparable km values for l-phenylalanine (15gm to 24.5/.tm), similar temperature (58~ and ph optima (8.5). the aminooxy-and phosphonic-analogues of l-phenylalanine are competitive inhibitors of the enzymes. 2-aminoindan-2-phosphonic acid (j. zo{a & n. amrhein, liebigs ann. chem. 1992,625) was found to be a potent slow-binding inhibitor of these and other phenylalanine ammonnia-lyases, both in vivo and in vitro. chlorophyll a fluorescence has been used to compare the drought resistance of two varieties of solanum tuherosum (avr/i)c-12st-19 and sibtema). fast fluorescence rise kinetics were measured during the first second of illumination with a time resolution of l0 ms and 1z bits in fluorescence intensity. the rise kinetics shows the typical steps called fo -j -i -p. with this values different indexes have been defind with the goal to compare the behaviour of these two varieties of potato. salicylic acid (sa) was proposed as a putative signalling molecule for the induction of systemic acquired resistance (sar) in infected plants. we studied its biosynthesis as follows. cotyledons of cucumber plants were injected with a solution containing pseudomonas lachr~ans and fed with radioactive sa precursors by injection of 14c-benzoic acid (14c-ba) or 14cphenylalanine (14c-phe). alternatively, leaf 1 of cucumber plants were inoculated with tobacco necrosis virus (tnv) and leaf disks were then vacuum-infiltrated using 14c-ba or 14c-phe. free and bound 14c-phenolies were quantified by hplc. incorporation of 14c into sa was found in the two plant-pathogen systems. i~c-ba gave mostly 14c-sa and an unknown polar compound. 14c-phe gave also some 14c-sa, in addition to 14c-labelled lignin precursors like ferulic and p-coumaric acids. the biosynthetic pathway of sa from phe through cinnamic acid and ba will be discussed. after infection with a necrotizing pathogen, systemic acquired resistance (sar) is often observed in plants. in cucumber, salicylic acid (sa) increases in the lower infected leaf as well as in the upper uninfected leaves. sa was also found in the phloem sap of infected cucumber plants and was proposed as a signalling molecule for the induction of sar. we intend to clarify whether sa is translocated from the site of infection to the upper leaf, or wether it is made in the phloem tissue upon the action of a primary systemic signal. one cotyledon of cucumber plants was first infected with pseudomonas lachrymans and then fed with 14c-sa, 14c-benzoic acid (ba) or 14c-phenylalanine. after different times, cotyledons and first leaves were collected and the radioactive ~henolics were analyzed by hplc. in some cases, 4c-sa was found in the first leaf. in addition, two unknown radioactive compounds were detected in leaf 1 after treatment with 14c-sa and 14c-ba respectively. we will discuss signalling processes for the induction of sar in cucumber. one modern approach of creating virus resistant grapevine plants is the introduction of resistance genes into existing grapevine varieties by genetic engineering. the goal of this work is to use the coat protein (cp) mediated strategy to induce resistance to nepovirus in vitis spp. as a first step, several chimeric nepovirus cp genes, were constructed by addition of various promoter regions upstream of the gflv and armv cp regions. their ability of conferring resistance to nepovirus infection in transgenic nicotiana benthamiana and n. tabacum plants will be presented. the best constructions will be used to transform several grapevine varieties in order to create nepovirus resistant grapevine plants. $13-12 the bctalains axe a class of natural pigments found only in plants of the order caryophyllales and in some fungi. the first step in betalain biosynthesis is the conversion of tyrosine to dopa. subsequently , dopa is transformed to betalamic acid, the bctalain chromophorr through the action of dopa-4,5-dioxygenase. we have purified and characterised a copper-containing enzyme of -38kda from amanita muscaria pileus that catalyses the hydroxylation of tyrosine to dopa. considering that the enzymatic activity was restricted to the coloured parts of the mushroom, we postulate that it is involved in betalain biosynthesis. the enzyme featured a broad substrate specificity, and also oxidised the diphenols to their corresponding ortho-quinones, a reaction typical for tyrosinases. the implications of our findings on betalain biosynthesis axe discussed. chlorophyll a fluorescence was measured under steady state conditions of pea and tomato leaves adapted to low light intensity (30 wm-2s -i) at different temperatures (havaux and strasser, z. naturforsch. 45c, 1133 -1141 , 1990 ). when leaves were exposed for a short period (i0 min) to heat (25 to 45~ in darkness, the level of variable fluorescence decreased. when the heat stress was imposed in presence of low light, the variable fluorescence was much less affected and virtually no effect of heat treatment was observed until 37~ this protecting mechanism by low light was absent in algae and mostly absent in submerged water plants but fully present in free floating plants on the water surface. we conclude that a mechanism has been developed for higher land plants during evolution which protects the plants against heat damage on warmer days. caryophyllales, e.g. portulaca grandiflora, and in a few fungal species, e.g. amanita muscaria. we analysed the similarity of a key enzyme of the pathway, dopa-4,5-dioxygenase, in plants and fungi both at the protein and the dna level. antibodies against purified dopa-4,5-dioxygenase from the fungus a. muscaria crossreacted with protein from p. grandiflora petals and two cdna clones were obtained from this plant. their similarity with fungal sequences and with other dioxygenases is discussed. kinetics of prollne hydroxylauonj intrucellnlur transport and c-terminal processing of the tobacco vacuolar cbltlnase. ernst frcydl, thomas boiler and jean-marc neuhaus botenisehes instimt, abt. pflanzenphysiologie, hehoistxasse 1, ch-4056 basel the vacuolar chitinase a of tobacco (nicotlana tabacum) is syndietlzed as a preproprotein with ma n-teaminal signal peptide which causes its synthesis in the er mad a c-termlnal extension, which has been idcmified as the vacuolar targeting peptide (vtp) (1) and whleh is cleaved off from the maybe chitinase. it has recently been shown that mature chltin:~e a contains several hydi'oxyprolines within the short peptide spacer that links the n-terminal cysteine-rieh chitln-bindlng domain to the catalytic domain (2). we received specific antibodies against mature chitinasr (a kind gift from dr. f.meins. f/vii) and raised antihodiea against a synthetic vtp. we performed pulse-chase experiments and cell [ractlonation with 1) stably transformed tobacco plants expressing chitinase a or mutants lacking either the chitin-binding domain and spacer (chitah) or the vacuolar targeting poptid 9 (chitavtp) and 2) transient expression of the same conswacts in nicotiana plumbaginifolia protoplasts. in both systems, proline hydroxylatino in chltinase a was detectable after 30 vain. and complete after 90-120 rain. the ehltinase intermediate forms were detected in the mlcrosomal and soluble fractions for up to 90-120 rain. the mature chitinase continuously accumulated only in the soluble fraction. in both systems chitah showed the same kinetics of intraceilular transport and processing. whereas the transport of cbitavtp seemed more rapid.these results indicate that in vivo cbitinase a is synthetized as a proprotein that is than modified by proline hydroxylation. this second intermedinte form is then transported to the oolgi apparatus and sorted to the vacuole. c-terminal processing occurs late in this pathway or in the vacuole. infection of bean roots with the soil-borne phytopathogenic fungus fusarium solani f.sp. phaseoli leads to a rapid increase of chitinase activity (~five-fold). part of this increase in enzyme activity is due to transcriptional activation of the basic class i chitinase isoenzymes. semi-native polyacrylamide gels stained for chitinase activity using the substrate glycol-chitin revealed the appearence of three additional chitinase isoenzymes that are absent from uninfected control roots. conventional protein purification techniques showed that fusarium solani infected bean roots contain two basic and two acidic chitinase isoenzyrnes. their physiochemical properties and possible biological function will be discussed. proteasomes are multicatalytic protease complexes that function as a major nonlysosomal proteolytic system. they exhibit multiple endopeptidase activities that promote the intracellular turnover of abnormal polypeptides and short-lived regulatory proteins. moss proteasome has been purified from protonemata by successive chromatography on macroprep q, bio-gel a-1.5, bio-gel a-5 and ha. the molecular mass was estimated to be 1,300 kd by gel filtration. gel electrophoresis of proteasome under nondenaturing condition gave a single band and two-dimensional gel electrophoresis identified the moss proteasome to be composed of 14 different subunits with molecular weight in the range 22-35 kd. electron microscopy in aqueous solution showed that it is a cylindrical particle composed of four stacked rings with a diameter of 9 nm and a height of 14 rim. end-on projection established a 7-fold symmetry of the outer disks. substrate specificity of proteasome indicates that it contains endopeptidase activity against substrates bearing hydrophobic, basic, acidic and glycine residues immediately preceding the cleavage site. antibodies raised against moss proteasome reacted with proteasomes of other eukaryotes, including human. $14-01 hunger r.e., hess m.w., laissue j,a,, mueller c. the nod (non obese diabetic) mouse, a widely used animal model for insulin dependent diabetes mellitus, shows in addition to mononuclear cell infiltration of the islets of langerhans, massive cellular infiltrates of the submandibular and lacrimal glands with considerable tissue damage. several lines of evidence suggest that both insulitis and sialadenitis represent autoimmune disorders, to investigate a possible role of tumor necrosis faetor-a (tnf-c0 and activated cytotoxic cells (nk cells, t cells) in the inflammatory process, tissue sections of nod mice were hybridized with radiolabeled rna probes specific for the detection of mrna for tnf-r and the serine proteases granzyme a and b (gra and gr8) and perforin which are expressed in activated cytotoxic cells. tnf-e expressing cells were mainly located in infiltrates and are absent in non affected glands, whereas gra, grb and perforin expressing cells were distributed over the whole section with highest densities in the zones adjacent to parenchyma. the finding that activated cytotoxic cells are present in early stage of disease development indicates that cell mediated cytotoxicity may play a crucial role in initial tissue destruction. the role of tnf-a in autoimmune diseases is not known yet. the appearance, however, of cells expressing this gene in the infiltrate indicates a possible role of this cytokine in the progression of the inflammatory reaction, e.g. by increasing the lymphocyte traffic to this organ. we further demonstrated that the induction of inos was at the transcriptional level. in order to understand the underlying mechanisms we characterized the promoter region of the rat nos gene. a 9enomic rat library was screened using a cdna-fragment coding for the if-end of the inos cdna (nucleotides 1-817). a 1,8 kb hinc-ii fragment containing the 5"-flanking region of the inos gene was characterized by dna-sequencing. the transcriptional start site was determined by primer extension. sequencing analysis revealed a multitude of possible cis-acting elements homologues to consensus sequences for the binding of different transcription factors including ifn~' response element (ire), nuclear factor-kb (nf-~b), tumor necrosis factor response element (tnf-re), 7f-activated sites (gas) and one x-box. nf-kb, a transcription factor involved in signaling and immediate early gene activation during inflammatory processes was induced by treatment of mesangial cells with 2 nm of il-113. this was shown by the appearence in nuclear extracts of the dna binding activity of nf-~b using electrophoretic mobility shift assays (emsas) with a 32p-labeled ~r dna probe. pyrrolidinedithiocarbamate (pdtq), an inhibitor of nf-kb, suppressed the expression of inos mrna in mesangial cells without affecting the dibutyryl-camp-triggered increases in nos mrna levels. this data suggest that the il-i ~ signalling pathway responsible for nos expression requires nf-k8 activation and is definetly different from the signalling cascade directed by camp. recent findings indicate that the multifunctional cytokine il-6, which plays a key role in irm-nune and inflammatory responses, also exerts specific effects in the central nervous system (cns). for example, il-6 promotes neuronal survival, induces differentiation and modulates neurotrophin production. using the very sensitive technique of reverse transcription combined with polymerase chain reaction the developmental profile of il-6 and its receptor mrnas was analyzed in various rat brain regions. our results indicate that both genes are expressed in a region-specific manner and are developmentally regulated. these findings support the hypothesis that il-6 is involved in differentiation and maintenance of neuronal subpopulation in the cns. interleuldn 2 (il-2) expression is strictly dependent on a signal transmitted by the t cell receptor upon antigen stimulation. this signal can be mimicked in vitro with phorbol esters and calcium ionophores. we have found that stimulation with ca-ionophore alone induces expression of il-2 mrna, but does not induce secretion of il-2. in polysome gradient fractionation of cells stimulated with phorbol esters and ionophore, the fractions containing il-2 rrtrna are found at the bottom of the gradient, bound to polysomes. however, in ionophorestimulated cells the fractions containing il-2 mrna are clearly shifted to the top of the gradient, and therefore are not lzanslated. this effect is specific for il-2, as other mrna's like 1~2-microglobulin are not regulated. this data suggests that il-2 gene is translationally regulated. in addition, in vitro experiments have shown that the lack of translation of il-2 mrna in ionophore-stimulated cells is due, most likely, to the presence of a translational regulator that specifically inhibits translation of il-2 mrna. assuming the presence of a translational regulator that specifically represses il-2 mrna translation, we can predict that, if the represser is in excess, even upon a second stimulus that is by itself able to induce secretion of il-2, there will be no translation of il-2 mrna. the resuhs of such an experiment confirmed our prediction. polysome gradients showed that after ionophore stimulation, il-2 mrna gets "stacked" with one ribosome, and no further ribosome loading takes place. if malaria is highly endemic, every febrile child is a presumptive malaria case, and there is no differential diagnosis based on clinical, immunological or parasitological grounds. we evaluated the diagnostic usefulness of interleukin-2 receptor (il-2r), tumor necrosis factor-receptors 55 (tnf-r55) and 75 (tnf-r75) . sera came from tanzanian pediatric fever patients and controls, all enrolled in the kilombero malaria project. we found that all 3 receptors marked pediatric fever episodes, whereas stability of observed levels was highest for tnf-r75 and lowest for tnf-r55. tnf-r55 levels reflected severity of the fever attack. regardless of the presence of a febrile illness, tnf-r75 levels were strongly associated with parasite density. immunological markers can be applied in rapid pre-and post-intervention morbidity assessments, validation of health interviews and monitoring of convalescence. we have recently shown that human eosinophils produce mrna for interlenkin (il)-8 when stimulated with calcium ionophom (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was released into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or inwacellular calcium mobilization am necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinophil derived il-8 in asthma. since the eosinophil is the predominant cell in the asthmatic airways, we determined 1l-8 concentrations in bronchoalveolar lavage (bal) fluids from normal individuals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supematants released by activated eosinophils. the role for 1i-,-8 in asthma remains to be determined. to study the effect of canthaxanthin in vitro, the hydrophobic compound was introduced in vivo into chick high density lipoproteins (hdl) by canthaxanthin feeding. the effects of hdl and hdl associated canthaxanthin on two types of cultures have been tested: flat sedimented cell cultures of embryonic chick neuronal retina, retinal figment epithelium (rpe), brain and meninges, and in reaggregate cell cultures of the neuronal retina. at high canthaxanthin concentrations the formation of colored, birefringent entities were induced in the neuronal retina in vitro. in line with human data, parameters of cellular function and cell differentiation remained unaffected by canthaxanthin at these concentrations. by contrast, fidl was found to induce various cell type dependent effects. proliferation of meninges cells was decreased at low hdl concentrations as concluded from.light microscopic examination and indicated by protein content, and lysosomal and mitochondrial activity of the cultures (ic50:15-30 mg hdl apoprotein/l medium). these parameters, however, were increased in brain cell cultures, while they remained unaffected in cell cultures of neuronal retina and rpe. concentrations above 0.3 g. hdl apoprotein/l caused a reduction of glial cell differentiation. $14-08 tnf-c~ had close-dependent antiproliferative activity on hormone-dependent human breast cancer cells that represent an early stage of the disease, whereas hormone-independent cells representing a late stage were not affected. however, in the presence of 1000 u/ml interferon ?(inf), high concentrations of tnf-c((lnm) were able to inhibit the growth of hormone-independent cells. using specific monoclonal antibodies in ftow cytometry, no significant changes of either the p75 or the p55 tnf receptors were observed upon inf treatment of hormone-independent cells. this indicates that the increased responsiveness of these cells for tnf-c~ is not due to a change in available tnf receptors. the affinity of the receptors for tnf-r are one order of magnitude different for the two cell types. inf treatment shifted the ecs0 of hormone-independent cells towards the ecs0 of hormone-dependent cells. thus, the basis for the increased tnf-sensitivity of hormone-independent cells in the presence of inf might be the interconversion of tnf receptors from low-to high-affinity. the adhesion of tumor cells to endothelium is the first step in the processus of metastasis, and is frequently dependent of cytokinemediated activation of endothelial cells. cytokines are also involved in nitric oxide (no) production by various cells. these experiments were designed to evaluate no production during tumor cell adhesion to endothelium. rat brain-derived endothelial and rat colon carcinoma cell lines were used during these experiments. no was evaluated with the griess reagent. activation of endothelial cells with cytokines (tnf-d, and ifn-~ ), only slightly increased (10-20 %) the adhesion of tumors cells to endothelium. activation of endothelial cells with tnf-ol and ifn-~, induced a 4-8 fold increase of no production. however, addition of tumor cells to the endothelial monolayer, either directly or in a semi-permeable membrane, decreased the cytokine-induced no production. these results indicate that no production is modulated during the proeessus of adhesion of tumor cells to endothelium. is induced in rat mesangial cells by inflammatory cytokines such as interleukin 1 ]3 (il-1 ~) and requires bh 4 as a cotactor. 2,4-diamino-6-hydroxypyrimidine (dahp), a selective inhibitor of gtp cyclohydrolase i, the rate-limiting enzyme for bh 4 synthesis, potently suppressed il-i~induced nos activity, measured as nitrite production. inhibition of no synthesis by dahp was reversed by sepiapterin, which provides bh 4 via a salvage pathway. sepiapterin dose-dependently augmented il-1 i],-stimulated no synthesis, indicating that availability of bh 4 limits the production of no in cytokine-stimulated mesangial cells. n-acetylserotonin, an inhibitor of the bh 4 synthetic enzyme sepiapterin reductase, completely abolished il-lj~-induced no formation whereas methotrexate, which inhibits the pterin salvage pathway, displayed only a moderate inhibitory effect, thus suggesting that mesangial cells predominantly synthesize bh 4 tom gtp. in conclusion, these data demonstrate that bn 4 synthesis is an absolute requirement for cytokine induction of nos in mesangial cells. inhibition of bh 4 synthesis may provide new therapeutic approaches to the treatment of pathological conditions mediated by no. -1 [3) can induce a macrophage-type of nitric oxide synthase (inos). northern-blot analyses of inos mrna-levels and nuclear run-on experiments of control and il-1 ~ stimulated mesangial ceils revealed that the induction of inos was at the transcripuonal level. here we demonstrate that platelet-derived growth factor bb (pdgf-bb) can suppress the il-113 dependent inducuon of the inos gene. nortllern-blot analyses of cellular rna isolated from mesangial ceils that were coincubated with il-1 [3 (2nm) and pdgf-bb (10 ng/ml, 100 ng/ml and 300 ng/ml) revealed a dosedependent suppression of inos mrna-levels. using run-on experiments with nuclei from mesangial ceils after stimulation with il-i~ (2nm) and pdgf-bb (100 ng/ml) we demonstrate that the inhibition occurs at the transcriptional level. basic fibroblast growth factor (bfgf), on the other hand, potentiates the il-1 dependent stimulation of inos. coincubation of mesangial cells with il-113 (2nm) and bfgf (3ng/m[, 10ng/m[, 30ng/m[, 100ng/m0 dose-dependently superinduces inos mrna levels as shown by northern-blot analyses of total cellular rna form control and stimulated cells. run-on experiments with nuclei from mesangial cells stimulated with il-113 (2nm) and bfge (100ng/ml) revealed an enhanced transcriptional activity of the inos gene. thus, pdgf-bb and bfgf differentaity modulate the il-i~ dependent expression of the inos gene and are therefore an excellent model system to study the crosstalk between signal transductjon pathways. we report that ifnyr-/-mice have an increased resistance to lipopolysaccharide-induced toxicity (lps). lps-induced lymphopenia, thrombocytopenia and weight loss seen in wild type mice were attenuated in ifntr-/-mice. ifntr-/mice survived in the d-galactosamine-lps model when conditions were 100% lethal for wild type mice, correlating with serum tnf levels of up to 10 fold higher in wild type mice. bone marrow and splenic macrophages from ifnyr-/-mice had a 3 to 5 fold decreased lpsbinding capacity, with serum from these mice lowering macrophage lpsbinding by a further 50%. thus, depressed tnf synthesis, diminished expression of lps receptors and low plasma lps-binding capacity in the mutant mice likely combine to manifest in the resistant phenotype of ifny r-/mice to endotoxin. in a complementary approach we have screened the 5' portion (-7.8 kb/+4.1 kb) of the gene for tissue specific and/or inducible dnasei hypersensitive sites. in chromatin from fibroblasts or kidney epithelial cells this segment of the il2ra gene does not contain any dnasei hypersensitive sites. in contrast, in resting t-and b-lymphocytes, as well as in activated t cells, two sites (dhi, -0.05 kb; dh3, -5.8 kb) were found. a third site (dh2, -1.35 kb) is detectable in t cells that have been activated with concanavalin a and il2. this site maps to the same position as cisacting regulatory sequences that are required for the response of the il2ra gene to signals from the il2 receptor. interleukin-6 (il-6) production in cultured human dermal fibroblasts can be stimulated by interleukin-i (il-i} added to the culture medium. absence of fetal calf serum and of growth factors (insulin, egf, t3) reduced the stimulation by more than 50 %. egf and t3 and even more choleratoxin increased the il-6 production in absence of fetal calf serum. the effect was dose dependent. 2% human ab serum substituted for i0 % fetal calf serum. pretreatment of the cultures with serum or growth factors prior to stimulation with il 1 had a stimulatory effect (serum, t3, egf) or an inhibitory effect (hydrocortisone, choleratoxin). our results suggest that the il-i signal transduction to il-6 is modulated by serum components and growth factors as well as through c-amp produced by choleratoxin. interleukin-6 (il-6) is produced by a variety of cells and plays a central role in host defense mechanisms. its function include induction of il-2 and il-2 receptor expression, proliferation and differentiation in t-cells. in cocultures of human dermal fibroblasts and allogenic human t-cells a cell number dependent 10 to 100 fold increase of the il-6 production could be demonstrated after 24h and 48h. conditioned medium from tcells and from fibroblasts failed to induce il-6 secretion in fibroblast and t-cell cultures, respectively. no up-regulation of il-6 production was found in cocultures of fibroblasts with tcells killed by ethanol and in t-cell culture incubated with recombinant hll-lc~. after separation of the cells il-6 production in fibroblast and t-cells returned to precoculture levels. our results indicate that cell-cell contact more than soluble factors must be involved in the up-regulation of il-6 synthesis in cocultures of t-cells and fibroblast. using cocultures of autologes human t-cells and fibroblasts, we are currently investigating whether the cell contact essential for the il-6 production depend on immunolcical interactions of t-cells and fibroblasts. rapid growth of tumor often results in oxygen and nutritional deprivation leading to extensive necrosis, which is one of the characteristics of glinblastomas (gbl). neoplastic cells surrounding necrotic areas often present a peculiar arrangement of cells called "pseudo-palisading" -as a hallmark of this entity, together with abundant neovascularization. a special biological milieu is probably formed in these palisading areas by certain unknown cytokines and/or growth factors induced by the necrotic process. plate et al. (1992) reported the expression of vascular endothelial growth factor (vegf) in the palisading cells. we previously demonstrated that gbl cells produce il-8, which is known to be an angiogenic factor. the aim of this study is first to assess if il-8 is expressed in the palisading ceils, and second to determine if hypoxia can trigger il-8 gene expression. in rive observation using in siru hybridization and immunohistuchemistry showed that il-8 is highly expressed specifically in the palisading cells. we also demonstrated the mrna expression of il-8 receptor in the endothelial cells adjacent to necrotic area. to simulate the in vivo condition, two in-vitro models are currently developed to determine if il-8 is induced by hypoxic insult on cells. first, gbl cell lines are cultured in the absence of oxygen and il-8 expression is assessed by northern blot analysis. preliminary results have demonstrated the induction of il-8 by hypoxia. secondly gbl cells are grown in spheroids until development of central necrosis. the il-8 expression will be assessed by in situ hybridization combined with immunohistochemistry to determine if the ceils surrounding necrosis express il-8. it has been proposed by taniguchi and his colleagues that activation of the interferon (ifn)-i3 gene by virus or double-stranded rna involves induction and modification of irf-1. overexpression of irf-1 can induce expression of the ifn-b gene in certain cells. a role of irf-1 in the activation of ifn-a genes has also been proposed. furthermore, irf-1 has been claimed to play the role of a tumor suppressor gene. we have generated mice in which both irf-1 alleles were disrupted and found them to develop normally. no spontaneous tumors have been observed so far. injection of poly(i)-poly(c) resulted in the same levels oflfn in blood of wildtype and null mice, and equal ifn-ct mrna levels in spleen. induction of wild type and irf ~ embryo fibroblasts (mefs) with virus resulted in the same levels of ifn-a and ifn-i] mrna respectively. in the case of polyo)-poly(c) induction, the levels of both 1fn mrnas were higher in wild type than in mutant cells; this difference was abolished if the cells were first primed with type i ifn. we conclude that irf-1 does not play an essential role in the induction oflfn genes. mitsuhiro tada, annie-claire diserens, isabelle desbaillets, marie-france hamon, nicolas de tribolet, erwin van meir; chuv, lausanne there is increasing evidence that a variety of cytokines produced by glioblastoma (gbl) cells modulate the tumor growth by affecting the host's immune response and by stimulating neovascularization. cytokines such as il-i, il-6, il-8, mcp-i, il-10 and tgf-132, affect each other's production and receptor system and seem to form a cytokine network. among them, il-1 may play a pivotal role, since il-i can induce or increase the production of other cytokines (il-6,il-8,mcp-i,tgf-132) and modulate their receptor expression. to test this hypothesis, we investigated co-expression of cytokines and their receptors in 15 gbl cell lines and 15 gbl tissues using rt-pcr, northern blot analysis, immunnhistnchemistry and elisa quantification. a majority of the gbl cell lines and gbl tissues expressed il-i~, il-113, type i and type ii receptors, indicating the presence of an il-1 autocrine loop. il-1 stimulated growth of some of the cell lines. a positive correlation of il-6 and il-8 expressions with the presence of an il-i autocrine loop in the cell lines was found. immunohistochemical studies showed the in rive co-expression of the il-i family and the secondary cytokines (il-6, il-8) in gbls. antisense oligonucleotide to il-lc~ and/or il-113 partly suppressed this secondary cytokine expression. these results demonstrate that the il-1 autocrine loop plays a central role in the formation of the cytokine network in gbls. double-stranded rna-dependent protein kinase (pkr) is induced by type i interferon (ifn) and is implicated in the establishment of the antiviral state. upon activation, pkr phosphorylates the eukaryotic initiation factor eif-2, thereby throttling protein synthesis. it was suggested that pkr may be essential for the induction of ifn-13 gene expression by both virus and double-stranded rna and for the activation of at least some ifninducible genes. recently it was proposed that pkr is a tumor suppressor gene because 3t3 ceils overexpressing inactive human pkr gave rise to tumors in nude mice (koromilas et al., 1992; meurs et al., 1993) . we have produced homozygous pkr knockout (pkr ~176 mice and found that they develop normally and have no striking phenotype. induction of ifn-c~ and ifn-13 gene transcription by virus was unimpaired in fibroblasts derived from pkr ~ mice, as was the induction of several ifn-stimulated genes. so far, no spontaneous tumor formation was observed. pkr o/o embryonal stem ceils showed no increased malignancy in nude mice as compared to their wild type counterparts. we conclude that pkr does not play an essential role in viral induction of the ifn genes and that its tumor suppressing effect, if any, is redundant. tgf-13 plays an important role as a negative regulator of hepatocyte proliferation in liver regeneration. exposure of rodents to nongenotoxic carcinogens like cyproterone acetate (cpa), thioacetamide (ta) and phenobarbital (pb) causes abnormal hepatocyte proliferation and liver tumors after long term treatment. this suggests that these chemicals have either a direct mitotic activity or impair the negative regulatory system for growth. therefore the inhibitory activity of tgf-6 on dna-synthesis induced whith cpa was investigated in cultured rat hepatocytes. after a 48h exposure to cpa, a dose dependent increase in dna synthesis (3h-tdr incorporation) was observed. the maximum effect was attained with 12 pm cpa (up to 4.3 fold) which was stronger than with 10 ng/ml egf (up to 2.8 fold). tgf-81 and tgf-132 inhibited this response dose dependently (idso = 0.1 ng/ml) and reduced it to control levels at 1 ng/ml. these findings show that the tgf-6 mediated pathway for negative growth control in hepatocytes is still responding after cpa treatment. mouse mxl protein is an interferon (ifn) induced gtpase with intrinsic antiviral activity against influenza a viruses. it accumulates in the cell nucleus and inhibits the transcription of influenza virus genes, recently, we have shown that mxl exerts its inhibitory activity by interfering with the function of the viral polymerase subunit p82. pb2 is responsible for recruiting 5" ends of cellular mrnas as primers and for the elongation of nascent viral transcripts. to elucidate which pb2 dependent step of viral mrna synthesis is the target of mxl action, we examined the activity of recombinant mxl protein in an in vitro transcription system of influenza virus. first, we expressed wildtype and mutant mxl proteins, carrying a hlstidinetag at their n-terminus, in e. coil and purified them under nondenaturing conditions by ni-chelate affinity chromatography. mxl efficiently inhibited viral transcription in vitro, while mutant mxl proteins, lacking antiviral activity in vivo ,were inactive. moreover, the use of capped synthetic rna primers revealed, that mxl almost completely abrogated the elongation of viral transcripts, whereas the the cap-binding and endonuclease activity of pb2 was not affected. a50 $14-28 neutralization rates of tnf-a from eight animal species by a monoclonal antibody against human tnf: implication for receptor action? we have recently developed a sensitive bioassay for measuring porcine tnf-a based on homologous pk(15) cells. this bioassay is 100-to 1000-fold more sensitive than the widely used l929 bioassay, also, human tnf-a and human tnf-8 were detected with a slightly higher sensitivity in the new test compared to the l929 bioassay, we now show that also canine, ovine, bovine, equine and caprine tnf-a can be detected with the pk(15) bioassay. we tested a murine monoclonal anti-human tnf-a antibody for its capacity to neutralize tnf-a from eight different animal species. the action of this antibody revealed that neutralization of tnf bioactivity is a complex phenomenon, indicating species-specific differences in the interaction with tnf receptors. to study this differential neutralization, we are cloning the porcine tnf receptors for expression in a heterologous cell system. administration.of high dose r-tnf(~ in combination with ifn 7 in isolation and perfusion of the limbs in melanoma patients has shown to be very promising with a response rate greater that 80%. this observation prompted us to investigate the possible expression of the tnf receptors in melanoma cells using the monoclonal antibodies utr-1 specific for the tnf type a (55 kda) receptor and htr-9 specific for the tnf type b (75 kda) receptor. flow cytometric analysis of cultured melanoma cells showed the presence at low level of the tnf type a and to a slightly higher level of the type b receptor. similar results were obtained in vivo by immunohistochemistry on fresh tumor raateriai, treatmem of melanoma cells in culture with the-amp induced up to a 2 fold increase in the number of type a tnf receptors with only a minimal change in the number of type b receptors. this increased expression of tnf receptors is conflrmed by direct binding experiments using 125i-labeled tnftx. the number of cpm's bound on dbc amp treated cells was about 0.5-3 fold higher than on untreated control calls, likewise incubation of melanoma cell lines with ifny increased the specific binding of subsequently added 125i-labeled tnfct. from flow cytometric analysis using the two anti-tnf receptor antibodies it became evident that flint was able to increase the expression of both type a and b receptors depending on the cell line used. characterization of a murine tumor necrosis factor (x-lacz reporter construct tumor necrosis factor ot (tnfcq is a prominent mediator of a variety of different pathologies such as endotoxic shock syndrom and rheumatoid arthritis. it also plays a crucial role in host defence against bacterial and viral infection. up to now, only few data about signal pathways leading to tnfo~ induction are available. an easy to handle tnfc~ -lacz reporter assay will represent a useful tool to study signal transduction on the one hand and provide data about compounds with potential tnfo~ inducible activity on the other hand. in order to develop a routine macrophage cell line capable to easily report tnfc~ induction, different tnfe-lacz reporter constructs were assembled. data about functionality of the different constructs in routine macrophages will be presented in the context of tnfot expression. cloning of a novel protein binding to lymphokine, fos and myc mrna with unexpected enzyme activity j. nakagawa, h-p. waldner, s. meyer-monard, and ch. moroni inst. medizinische mikrobiolegie, univ. basel an au-rich sequence in the 3' untranslated region of lymphokines, c-los, and c-myc proto-oncogene mrna is responsible for their rapid decay. we have purified a 32 kd protein by an auuua affinity column from human brain. partial amino acid sequence was obtained and the corresponding edna has been cloned. unexpectedly the edna sequence exhibited significant homology to enoyl coa hydratase and bacterially expressed recombinant protein indeed showed the activity. while rat hydratase did not show rna binding activity, the recombinant protein bound to cfos, c-myc, auuua cluster of il-3 mrna, and adeno virus iv, but not to mutated ad-iv, nor irrelvant transcript. the binding was competed out by poly u. interestingly the protein seems to be processed from a larger precursor. we suspect that the protein plays a role in mrna turnover and perhaps in oncogenesis. institute for medical microbiology, university of basel in t cells, the immunsuppressive agent cea is known to inhibit ca 2+ dependent induction of lymphokine mrna transcription. in contrast, the immunsuppressive drug rapamycin inhibits the lymphokine induced proliferation. we have analysed the effect of these drugs on a v-h-ras induced autocrine mast cell tumor line which expresses il-3 constitutively. csa and rapa inhibited autocrine growth by different mechanisms,, because added il-3 could antagonize inhibition by csa, but not by rapa. whereas csa acted by downregulation of il-3 mrna expression, rapa blocked il-3 induced proliferation. cea also inhibited il-3 superinduction by ca-ionophore, whereas rapa did not. nuclear run on assay indicated that the mechanism is posttranscriptional. therefore, we have introduced il-3 transgenes with and without the au-rich region in the 3' utr of the mrna into a tumor line. in contrast to the wild type, the expression of the mutant construct was insensitive to csa. since the mutant lacks sequences known to regulate rapid mrna decay, we suggest that csa affects the regulation of il-3 mrna stability. some non-hematopoietic cell lines produce both scf and its receptor, the c-kit protein (c-kit). testing the hypothesis that scf may be involved in secondary growth of nb bone marrow metastasis, we found and previously communicated (beck d. et al, proc aacr, 34, 52, 1993) a low or absent expression of c-kit in nb cells. the mrna and surface expressions of scf gene in nb cell lines grown in chemically-defined medium were evaluated by northern blot analysis and flow cytometry. the scf antigenic concentrations in culture supernatants were determined by elisa and growth-inhibition experiments performed by pre-incubating nb cells with anti-scf antibodies prior to 3h-thymldine uptake assay. the scf mrna was expressed in 4 of 5 tested lines but no membrane-bound antigen could be detected on those cells. detectable levels of scf in the supernatants were found in 5/7 lines (range : 39-96 pg/ml). blocking experiments resulted in a decrease of dna synthesis in i out of 7 lines only. from these and previous results, we conclude that scf is synthesized and released by many nb cells in vitro but the functional ~ole of it remains undetermined since scf does not appear to be usually involved in autocrine or paracrine growth stimulation of nb cells (supported by swiss fnrs grant 3200-031005). expression of the v-h-ras oncogene in the il-3 dependent pb-3c mast cells leads to the generation of two classes of il-3 autocrine tumors in vivo. autocrine il-3 expression in class-1 tumors results from increased il-3 mrna stability due to the loss of negative trans-acting posttranscriptional control. this defect can be corrected by somatic cell fusion to the nontumorigenic parental pb-3c resulting in downregulation of oncogenic il-3 expression and concomitant tumor suppression. expression of the v-h-ras oncogene remains unaffected in class-1 hybrids. in contrast, class-2 tumors are characterized by transcriptional activation of the il-3 gene due to the insertion of an endogenous retroviral element (intracisternal a-particle) which cannot be overcome by ceil fusion (see hirsch et al, 1993, j.exp.medicine 178, 403-411) . although v-h-ras is required for generation of either class of tumors, expression of anti-sense ras constructs inhibited only the proliferation of class-i tumor cells. experiments are underway to characterize the target and the nature of the class-1 alteration. nitric oxide (no) promotes vasorelaxatlon of renal vessels in vitro. the purpose of the present study was to assess the role of no in regulating renal hemodynamics in vivo. renal blood flow (rbf) and glomerular filtration rate (gfr) were studied in anesthetized mechanicallyventilated newborn rabbits both before and after the administration of a no synthesis inhibitor, ng-nitro-larginine methyl ester (l-name), at a dose of 50 pg/kg followed by 10 jzg/kg x rain. such a dose of l-name did not modify mean arterial pressure or pulse rate. by contrast, the renal vascular resistance increased by 31 + 9% (p < 0.005) while rbf decreased by 20-+6% (p < 0.02). gfr and urine flow rate remained constant. the overall results suggest that in normal conditions no may play a role in decreasing the renal vascular resistance of the immature kidney, without altering systemic blood pressure. interleukin-4 (il-4) is a t-cell derived lymphokine which, in addition to its effects on the immune system, is also able to suppress the growth of certain tumor cells. il-4 reduced the growth rate of four human colon tumor cell lines up to 70% in a dose-dependent manner. three other cell lines showed no significant alteration of 3h-thymidine incorporation or colony formation in the presence of 100 u/ml il-4. responder and nonresponder tumors were immunopositive for il-4 receptor (il-4r) expression in flowcytometric analysis, using monoclonal antibodies raised against recombinant il-4r and bound biotinyated il-4. responsive tumors expressed more receptors. 11-4 binds with high affinity to a 130 kda transmembrane receptor (il-4r), through which the extracellular signal has been shown to be transduced. coimmunoprecipitation and chemical crosslinking studies have enabled us to demonstrate il-4r target proteins. tyrosine phosphorylation of various proteins between 70 and 170 kda appears to be initiated during il-4mediated signaling events in colon tumor cells. the cloning of human il-4r target proteins will contribute to the understanding of the il-4 signal transduction pathway at the initial stage. the renal effects of endothelin-1 were investigated in 16 anesthetized and meehanleally-ventilated newborn rabbits. each animal acted as its own control. in 8 newborn rabbits, a bolus injection of 5 nmol.kg "1 of endothelin-1 caused an initial fall in mean blood pressure (mbp) followed by a gradual but significant increase in mbp that lasted for 45 rain. the dramatic increase in renal vascular resistance (+ 28 -+ 4%) induced by endothelin led to a fall in glomerular filtration rate (-12 -+ 4%) and renal blood flow (-16 _+ 3%). in spite of the reduction of gfr and rbf, urine flow and sodium excretion rates increased significantly (+ 20 _+ 5% and + 49 _+ 9%, respectively). in 8 additional newborn rabbits, a bolus injection of lnmol.kgaof endothelin-1 -a dose that usually induces marked renal and systemic vasoconstriction in adult models -did not affect systemic or renal hemodynamics. in conclusion, endothelin induces renal and systemic vasoconstriction, and affects water and sodium homeostasis during the neonatal period. however, these effects occur under higher doses than those used in adult animals, possibly reflecting receptor immaturity and/or interference of high levels of counteracting hormones. ontogeny of the endocrine pancreas in xenopus laevis c, maake 1 , w. hanke 2 and m. reine~ke 1 , 1 institute of anatomy, university of z0rich, switzerland and department of zoology ii, university of kaflsruhe, f.r.g. the pancreatic islets of anurans show insulin (ins), glucagon (gluc), somatostatin (som) and pancreatic potypeptide (pp) containing cells. since only limited information exists on the ontogeny and on the presence of insulin-like growth factor 1 (igf-1), we studied the development of the gastro-entero-pancreatic (gep) system in xenopus laevis using immunohistochemical techniques. singular ins-immunoreactive (-ir) cells were observed in the pancreas as early as on stage 41. at stage 43, the first pp-ir cells were found in the gep system followed by som-ir and gluc-ir cells. the first pancreatic islets consisting of ins-ir and gluc-ir cells occurred around stage 50. between stage 54 and 60, i.e. after the onset of metamorphosis, the endocrine cells decreased in number and the islets partly disintegrated. starting with stage 61, the islets reformed and the amount of endocrine cells increased reaching the adult status at stage 63. around stage 52, igf-l-immunoreactions appeared. igf-1immunoreactivity was found in pp-ir and gluc-ir cells but not in ins-ir and som-ir cells. it is assumed that islet-derived igf-1 may p~ay an important role during metamorphosis in xenopus. t. cathomen and r. cattaneo, institut for molekularbiologie i, universit~t zorich, hfnggerberg, ch-8093 zorich the signals used for synthesis of the measles virus fusion (f) protein are poorly understood: a long (>570 bases) untranslated region is followed by three in frame augs at codons 1,4 and 15. f is a type i transmembrane protein with a postulated signal sequence of 31 amino acids. we confirmed that the 46 aminoterminal residues contain a signal peptide by transfering them to another protein. with the other envelope protein hemagglutinin, f protein induces virus-cell and cell-cell fusion. mutagenesis of the three augs indicates that proteins initiated at codon 1, 4 or 15 are all functional, but show slightly different fusion efficiencies. forms translated from aug 1 or 4 appear larger in electrophoresis than forms initiated at aug 15, suggesting that two different signal peptide cleavage sites are used. we are currently investigating whether both f protein forms are incorporated in viral particles. the production of protein isoforms with staggered amino-termini is common in cytoplasmic and type ii transmembrane proteins, but signal sequences usually preclude a similar organization of type i transmembrane proteins. beguin p., beggah a.t., rossier b.c., jaisser f. and geering k. institut de pharmacologie et de toxicologie de l'universit6, ch-1005 lausanne recent experimental evidence suggests that na,k-atpase might be a candidate for regulatory phosphorylation by protein kinases a and c (pka and pkc). to verify this hypothesis, we have mutated several serine and threonine residues in consensus phosphorylation sequences of the ~subunit of bufo marinus na, k-atpase. the mutants were expressed in x~nopus ooeytes and phosphorylation was studied in oocyte homogenates upon stimulation of oocyte, pka and pkc. our results indicate that a unique phosphorylation site for pka is located at serine 943 in the c-terminus of the ~-subunit but its phosphorylation can only be revealed in the presence of detergent. on the other hand, mutations of several serine and/or threonine residues located in consensus sequences for pkc phosphorylation did not or only partially abollsh phosphorylation by pkc. in particular, mutations close to the catalytic phosphorylation site of the ~-subunit influenced phosphorylation by pkc, suggesting that either this region contains a real phosphorylation site or is part of a conformational domain necessary for phosphorylation by pkc. the heat-stable antigen (hsa or mouse cd24) gene is differentially regulated but has a housekeeping promoter. roland h. wenger*, georges kshler and peter j. nielsen. max planck institut f(jr immunbiologie, st0beweg 51, d-79108 freiburg i. bsg. "present address: physiologisches institut der universit&t zi.)rich, winterthurerstrasse 190, ch-8057 z0rich, expression of the gpi-anchored murine glycoprotein heat-stable antigen (hsa) shows tissue-specific as well as developmental regulation. during the maturation of several hematopoietic lineages, hsa expression is generally high in immature precursor ceils and low or absent in terminally differentiated cells. we present evidence suggesting that this regulation of the hsa gene (cd24a) occurs at the transcriptional level. in addition, sequence and methylation analysis of the cd24a promoter revealed characteristics of both "housekeeping" and tissue-specific promoters, including a methylation-free, hpall tiny fragment (htf) island, multiple putative sp1 and ap-2 consensus binding sites and a tata box. functional analysis of a 0.6 kb dna fragment containing these elements fused to the cat reporter gone in transient transfection experiments showed activity in both hsa expressing and non-expressing cell lines with a strength similar to that of the hsv tk promoter. large fragments from the flanking region of the cd24a promoter did not influence the ubiquitous nature of this promoter, finally, the cd24a, cd24b and cd24c genes were mapped to mouse chromosomes 10, 8 and 14 respectively. we have cloned, sequenced and characterized the gone for the m__itochondriat nadh-cytochrome b5 reductase (mcri) of saccharomyces cerevisiae. surprisingly, this gone encodes two mitochondrial isoforms of the flavoenzyme: a 34 kda integral protein exposed on the outer surface of outer mitochondrial membrane (mcrlp34), and a 32 kda soluble protein of the intermembrane space (mcrlp32). the smaller intermembrane space isoform is generated from the larger form by the action of the inner membrane protease i (impi) on the outer surface of the inner membrane. this is the first demonstration that a single gone encodes proteins which are located in different compartments of the same organelle. with special interest in any with a mitochondrial localization. degenerate primers were designed on the basis of high homology between members of the abc family, and pcr was performed on yeast genomic dna. ten dna fragments bearing significant homology to the abc family were amplified, nine of which were previously unidentified. disruptions of five of the corresponding genes were performed. disruption of one gene led to markedly impaired growth on rich medium and cessation of growth on minimal medium. the gene was cloned and found to encode a protein of 694 amino acids, a "half-transporter" which likely forms a dimer. ibi~tlnofluorescence on yeast expressing the gene cmyc-tagged at the c-terminus reveals co-localization of the protein with porin and with mitochondrial dna, visualized by dapi staining. nycodenz-purified mitochondria are enriched for the protein by immunoblotting. additionally,the c-myc epitope is protease-protected in mitoplasts, indicating an inner membrane sub-localization and a probable orientation with the c-terminus in the matrix. the gene, termed atmi for abc transporter of mitochondria, thus encodes the first member of the large abc transporter superfamily to be localized to this organelle. present work is aimed at revealing the substrate of this transporter. cycle in cardiac myocyte. the molecular study of this protein has been difficult even after its over-expression was made possible by the cloning of the corresponding cdna. the chief problem is the lack of convenient domains which could be used in the purification of the molecule. we have previously reported the efficient expression of the cardiac na+/ca 2+ exchanger in mammalian cell lines using the t7 rna polymerase-hybrid vaccinia virus system. we have now constructed the recombinant virus for the exchanger with a 6xhis-tag at the c-terminal. this tag has enabled the purification of the protein through a ni-nta agarose column. the expressed tagged protein induced na-dependent ca uptake which was not different from that in intact cells, i.e., cells in which the exchanger did not have the tag, the most important aspects of the purification produced and those of the reconstitution of the expressed protein will be described. $15-08 the neural cell adhesion molecule l1 is involved in the formation and specification of cell contacts in the central and peripheral nervous system during development, and thus may also play a role in synaptic plasticity of the adult central nervous system, using extracellular recordings of evoked field potentials in the rat hippocampal slice, we found that locally applied polyclonal anti-l1 and fusion proteins containing the ig-domains i-vi of l1 specifically block the stabilization of ltp in the ca1 region. baseline synaptic transmission was not affected by the presence of the anti-l1 antibodies. the anti-ll-induced effect was dependent on the antibody concentration and anti-l1 antibodies had no effect on previously established ltp. ltp was also reduced by an antiserum against the recombinantly expressed ig-domains i-vi of l1, whereas controt antibodies against liver cell membranes, which bind to neuronal membranes in the hippocampus, exhibited no effect on ltp. the contribution of l1 to stabilization of ltp within the ca1 synapses suggests that members of the ig-supertamily of cell adhesion molecules are involved in the structural modifications which are associated with synaptic plasticity in the mammalian central nervous system. an increasing number of surface proteins are described as being attached to the membrane by a glycosyl-phosphatidylinositol (gp1) anchor instead of the conventional transembranous hydropbobic domain. they are initially synthesized with a coohterminal polypeptide extension which is cleaved in the e.r. and replaced by the preformed glycolipid. this processing event is directed by a signal, located in the c-terminal region of the protein, which requires a group of three small amino acids positioned 10-12 residues upstream of an hydrophobic c terminal sequence. we previously transformed the transmembfanous glycoprotein d of herpes simplex type i into a gpi-anchored molecule by deleting its cytoplasmic domain and thus creating a molecule, gdww63, which meets the requirements for anchoring via a gpi structure. we now demonstrate, using gpi-deficient cell lines and biochemical studies, that a hybrid protein consisting of the thy-i ectoplasmic domain and of the c-terminal region of gdww63 can be alternative[y expressed in a gpi-anchored or in a transmembranous form. service de p6diatrie, chuv, ch-1oll lausanne proteolipid protein (plp) is a major myelin protein in the central nervous system (cns). a number of x-linked dysmyelinating disorders have been identified as mutations in the pip gene. we have identified a novel plp mutation in paralytic tremor (it) rabbit, characterized by body tremor, spastic limb paresis and hypomyelination in the cns. reduced levels of plp protein and its mrnas were observed in the cns as well as in the pns. plp sequence analysis revealed a single nucleotide change in exon 2 which results in the substitution of a histidine by a glutamine at position 36. histidine 36 is localized on the boundary between the first transmembrane region and the intracellular part of the protein. therefore, its position can be crucial for the efficient plp interaction with other proteins and lipids, and correct incorporation of the plp molecule into the membrane. histidine 36 is also part of a short fragment of ten amino acids which is conserved in all species studied. the same amino acid is altered in another hypomyelinated mutant, shaking pup, stressing its functional importance. furthermore, the pt mutation affects the plp "premyelin" functions, including oligodendrocyte maturation. equilibrium constants for octamer dissociation as well as dissociation rates in vitro increased in correlation to the number of charged residues eliminated, e.g. mutant 4-7, in which all four charged residues in the n-terminal heptapeptide are substituted with uncharged amino acids, showed a 100-fold higher equilibrium constant for octamer dissociation and a 145-fold increased dissociation rate compared to wild type. this strongly suggests that the charged amino acids in the n-termlnal heptapeptide of mib-ck, and therefore an ionic interaction, play an important role in forming the oetameric molecule. gangliosides and neutral glycosphingolipids (gsls) cell surface molecules are involved in a variety of biological processes and are assumed to play an important role in the mechanisms of hiv entry into lymphoid and epithelial cells. the total lipid-bound sialic acid (lbsa) content and the composition of gangliosides and gsls were investigated on pbmn cells from hiv+ve and normal donors. lbsa level was quantified by a fluorimetric assay, while gangliosides and gsls were assayed with high performance thin layer chromatography (hf'tlc) after multistep lipid extraction of 100 0013 x g membrane fractions. in normal pbmn cells the lbsa content accounted for 2.6/~g/108 cells or 20 nmol/mg protein whilst in pbmn cells from hiv+ donors the lbsa level decreased to 1.6 ,ug/108 cells or 13 nmol/mg protein. the qaantitation of neutral gsls and gangliosides was carried out by scanning spots revealed on hptlc plates and the integrated areas computed with a dedicated software. our findings showed that the most relevant ganglioside present in normal pbmnc was gm3 (65-80 % of total gangliosides) followed by small amounts of od3 (5 -15 %) and other acidic glycosphingolipids. neutral gsls included gl1, gl2, gl3 and gl4-hexosylceramide. hiv+ pbmn cells were characterized by a decrease of neutral glycosphingolipids as well as gm3 content which represented only 55 % of the ganglioside fraction, indicating an alteration in the glycolipid composition in the plasma membrane of infected cells. the neuropeptide y (npy) is one of the most abundant peptides of the mammalian brain. upon stimulation of cell surface receptors, npy generates diverse physiologic responses. npy acts on the cardiovascular system. it is a vasoconstrictor by itself and in addition, it potentiates the action of vasoactive substances such as angiotensin ii or norepinephrine. npy also inhibits glucose induced insulin secretion and is a potent inducer of appetite. to facilitate the development of npy antagonists we are investigating the interaction of npy with its cell surface receptor.we first constructed a series of mutants in which negatively charged residues present in putative extracellular domains of the y1 receptor were systematically replaced by alanines. the mutant cdnas were transiently expressed in hela ceils using a vaccinia virus derived expression system and the abi}ity of the encoded proteins to bind npy was evaluated. the capacity of the mutant proteins to be recruited to the cell surface was assessed by confocal microscopy. we identified initially 4 spacially clustered extracellular acidic residues of the human y1 npy receptor essential for npy binding. subsequently, we mutated 36 additional residues. based on these data a detailed computer model of the interactions between npy and its receptor was built. functional activity to energy metabolism. l. pellerin and p.j. magistretti. institut de physiologie, universit6 de lausanne, switzerland. astrocytes play a central role in brain energy metabolism. for example glucose, the exclusive brain energy substrate, is avidly taken up by astrocytes (pnas 90:4042, 1993) . application of glutamate (glu), the main excitatory neurotransmitter, stimulates in a concentration-dependent manner 3h-2-deoxyglucose (2-19(3) uptake by astrocytes in primary culture with an ec50 of ~ 100 ~m. the effect is not receptor-mediated since it can not be reproduced by glutamatergic agonists such as (nmda, kainate, quisqualate, t-acpd) nor prevented by antagonists (apv, cnqx, ap3). instead, it involves glutamate uptake since the effect is blocked by dl-threo-j3hydroxyaspartate, a potent inhibitor of the glu transporter. replacement of na + in the medium by choline also prevents the effect, suggesting a na+ driven process. finally ouabain, a selective inhibitor of the na+/k + atpase, completely prevented the effect of glu. these observations suggest that when glutamatergic synapses are active, glutamate, taken up by astrocytes, stimulates 2-dg uptake by astrocytes whose end-feet are known to surround capillaries, i.e. the source of glucose. they also provide a simple explanation for the mechanism linking functional activity to energy metabolism. cloning and molecular characterization of the mouse astrocyte glycogen synthase. g. pellegri, c. rossier, s. van berchem, p.j. magistre~i and j.-l. martin. institut de physiologie, universit6 de lausanne, switzerland. in the brain glycogen is predominantly localized in astrocytes, although its presence has been detected in certain large neurons, in ependymal and choroid plexus cells. glycogen is synthesized by the enzyme glycogen synthase (glys) from udp-glucose. out of the different glycogen synthase isozymes, only the muscle and the liver forms of glys have been cloned. we have isolated and characterized from a mouse astrocyte ~.zapii cdna library, a cdna clone encoding the mouse cerebral cortical astrocyte glys. the 3.5 kb clone isolated contains an open reading frame of 2214 bp encoding a protein of 738 amino acids. the coding region of the mouse astrocyte glys cdna shares 87% and 86% identity with the nudeotide sequences of the human muscle and the rabbit muscle giys cdna respectively. the cellular distribution of the glys mrna studied by northern blot shows the presence of a single transcript of 4 kb in cultures of astrocytes and, to a lesser degree, of neurons. the distribution of glycogen phosphorylase, which was also examined by northern blot shows the presence of a 4.2 kb transcript both in cultured astrocytes and neurons with however higher levels expressed in astrocytes. the functional molecular size of the vacuolar h+-pyrophosphatase (h +-ppase ec 3.6.1.1) from maize (zea mays l.) roots was estimated by radiation inactivation, both for substrate hydrolysis and for proton transport. light microsomal vesicles enriched in tonoplast membranes were prepared from young (2 days) maize roots using sucrose step gradients. these vesicles were still competent for ppi-dependent proton transport after freeze-drying and rehydration of the membranes. frozen native or freeze-dried samples were irradiated with 6~ for various periods of time. after thawing the samples, the activities of glucose-6phosphate dehydrogenase (added as an internal standard) and h+-ppase (hydrolysis of pyrophosphate (ppi) and ppi-dependent proton transport) were tested. by applying target theory, the functional size for hydrolysis was found to be around mr 155 000 when the native or freeze-dried samples were irradiated. no ppi-dependent proton transport activity was measurable after irradiation of the native samples. on the contrary, the freeze-dried membranes were still pumping protons after irradiation and a target size of around mr 250 000 was measured for the transport activity. these experiments suggest that the native h+-ppase from maize roots may exist as a dimer (at least) of the mr 80 800 catalytic subunit. $15-20 the na,k-pump moves 3 na + out and 2 k + ions into the cells. the domains of the protein involved in ion translocation have not been determined. we have studied the role of the n-terminal region in the na + translocation by measuring the kinetics of charge movements under na/na exchange conditions in wild type (wt) and two n-terminally truncated forms of the bufo ~ subunit with deletions of the 31 (t31) and 40 (t40) first amino acids expressed in xenopus oocytes. we have developped a new technique to perform fast (< i ms) voltage clamp on whole oocyte. the membrane on one side of the oocyte is permeabilized by digitonin allowing to measure current flowing across the other half. we observed presteady state ouabain-sensitive currents similar to that reported by other techniques (j.gen. physiol. 101:117,1993) . the estimated forward charge translocation rate was lower in both n-truncated mutants (wt 430 â�¢ 14, t32 215 z 8, t41 124 z 3 s-l). the midpoint potential of the charge translocation (best fit to boltzman equation) was -79 , -45, and -28 mv in the wt, t31 and t40 groups, respectively. the highly charged nterminus of the q subunit has a significant role in the forward translocation of na + ions. deletion mutants of human small intestinal lactase-phlorizin hydrolase (lph) were constructed to determine the role of protein subdomains in the intracellular transport of lph. the mutant lph1646mact (236 aa deletions), which contains the membrane anchor (ma) and the cytoplasmic tail (ct), was transported to the cell surface in a fashion similar to wild type lph. by contrast, lph1646 lacking the transmembrane domain persisted as a mannose-rich polypeptidc. this suggests that the membrane anchor and/or cytoplasmic tail are implicated in the er-egress of lph. another mutant, lph1559mact (323 aa deletions), was not efficiently transported to the golgi apparatus. epitope mapping with monoclonai antibodies as well as protease-sens{tivity assays provided an evidence that the tertiary structure of lph1646mact is very similar to that lph1559mact. in view of the variations in the proportions of the complex glycosylated molecules of the two mutants we conclude that the domain between lph1646mact and lphi559mact may play an essential role along the secretory pathway of lph. containing tyrosine. hussein y. naim, and michael g. roth; dept, of biocherni~try, ut-southwestem medical center, at dallas, dallas,texas-usa we have investigated an artificial internalization signal created by site-directed mutagenesis of the short cytoplasmic sequence of the influenza virus hemagglutinin (ha). mutation of cysteine 543 to tyrosine converted ha from a protein essentially excluded from coated pits to one that internalized at rate similar to some cellular endocytic receptors. to determine whether or not the ha-y543 signal is a degenerate form of the internalization signal found in proteins such as the transferrin receptor and the mannose-6-phosphate/igfii receptor, we have mutated amino acid positions in the ha-y543 shown to be important for internalization of the two receptors. we have changed amino acids on either side of y543 to those that either fit, or break the pattern of aromatic and hydrophobic residues proposed as consensus sequence. our results indicate that the ha-y543 mutant contains a sub-optimum sequence for a tyrosine-based internalization signal similar to those found in the receptors for transferrin, ldl, and mannose-6-phosphate/igfil. however, amino acids with side chains having different chemical properties functioned well in positions that are important for the internalization signal, indicating that the consensus sequence for this signal is more variable than previously proposed. n-linked glycesylation is an essential protein modification occuring in all eukaryotic cells. core-oligosaccharides are transferred by the enzyme n-oligosaccharyl-transferase frc~ the donor glc man gicnac -dolichol to selected asparagine 9 2 . resldues of t~e nascent polypeptlde chazn. the donor is build up in a stepwise fashion at the er-membraneby the products of the alg-genes (asparagine-linked-~lycosylafion) mutants defective in this pathway are available, but often lack a phenotype suitable for isolation of the corresponding genes. we found that double mutants of alg5 or alg8 with a wbpl mutation (defective oligosaecharyl-transferase) are severely growth retarded at 30oc. the double mutant phenotype allowed the isolation of both alg5 and alg8 genes which have been refractory to cloning so far. both genes encode potential trarsmenbrane proteins and are able to complement the biochemical defects of the corresponding alg5 or alg8 mutants. the na,k-atpase produces the driving force for the regulated transport of na across epithelial ceils. it is composed of an c~-subunit which carries the catalytic and ouabain binding sites and a b-subunit. to test the role of the usubunit in transport regulation, we transfected a6 cells with the b. marinus al-subunit (txl-tbm). this subunit confers a 300-fold higher resistance to ouabain (k i = 50/~m) compared to the endogenous pump. taking advantage of this difference, stable transfectants were selected with ouabain. expression of the cd-tbm subunit was visualized by western blotting. approximately half of the positive cell lines showed a high electrical resistance similar to untransfected cells, when cultured on filters. na-transport activity, measured as isc, was lower even in the absence of ouabain, but the relative increase in response to aldosterone was maintained. functional pumps containing the exogenous or endogenous al-subunit were quantified by equilibrium ouabain binding. interestingly, cells cultured on plastic dishes had a large fraction of low affinity binding sites (txi-tbm), while most sites were of the high affinity type when the cells were grown on filters. we conclude that the ouabain-resistant na,k-atpase cd-subunit of b. marinus can be expressed in a6 cells and used as a selective marker. however, the results suggest that the expression of functional ouabain-resistant pumps containing the al-tbm subunit could be influenced by the degree of cellular differentiation. in infected cells fully functional na/pi-cotransport was found in a time dependent manner; up to a 50-fold stimulation of na/p;-cotransport compared to uninfected cells was observed aft6r 3-4 days. transport of phosphate by infected cells was highly dependent on the presence of sodium, exhibited a kmtd~ of around 0.16 mm and was dependent on extraceuular i~h'. in agreement with expressed transport of phosphate, infected cells produced an immunoreactive polypeptide of 66 kda that represents a non-glycosylated form of the 80 to 90 kda mature na/p;-cotransporter. we conclud$ that the protein napi-2/rat when expressed in sf9 cells exhibits na/p-eotransport with similar kinetic characteristics as observdd in prox ma tubular apical na/pcotransport. therefore the napi-2 protein as expressed h sf9 cells can be used for further structural and functional studies. talin interacts in vitro with actin, vinculin, integrins and bilayers of acidic phospholipids, and nucleates actin filament growth at the bilayer surface. it is therefore believed to be a key protein mediating aetin-membrane linkage. we have analyzed functional properties of proteolytic fragments of purified platelet talin. using limited proteolysis two fragments of 47 and 190 kd were generated. preliminary results, obtained using viscometry and f-aetin-nucleating assays, suggest that the 190 kd fragment contains the actin binding site. we also assessed the lipid-binding capacity of the fragments. when a mixture of the two fragments was centrifuged in the presence of large multilamellar liposomes containing phosphatidylserine, only the 47 kd fragment, but not the 190 kd domain, co-sedimented with the liposomes, suggesting the presence of a specific lipid-binding site in the 47 kd domain. interestingly, this fragment contains the n-terminus of talin which shows homologies to the membrane-binding regions of protein 4.1. we are now analyzing membrane-binding properties of talin domains in more detail using hydrophobic photolabelling. recent studies suggest a role of the neural cell adhesion molecules l1 and ncam in mechanisms of memory formation (doyle e, et al, j. neurochem. 59:1570 -1573 , 1992 scholey a.b. et al, neuroscience 55:499-509, 1993; lijthi a. et al. unpublished data and see usgeb 94) . in the present study we analyzed the effect of chronic intraventricular infusion of polyclonal antibodies against l1 (anti-u) or ncam (anti-ncam) on the performance of male wistar rats during the acquisition and retention of a spatial learning task. briefly, rats had to remember the position of a submerged escape platform in a water tank (morris water-maze). when the escape platform was removed after acquisition training (= retention test), the performance of animals chronically injected with anti-l1 showed an impaired search pattern when compared with that of salineqnjected animals (p>0.05, mann-whitney). whereas control animals spent up to 45% of their time searching for the platform in the correct (= training) quadrant, the time anti-l1 animals swam in the correct quadrant was closer to chance level (31%). although monitoring penetration of antkncam into the hippocampus was almost as efficient as that of anti-l1, the performance of the anti-ncam-group was highly variable and did not differ from the performance of controls. these results agree with recent findings on the involvement of neural cell adhesion molecules in other forms of learning. we have isolated cdnas from mouse and from xenopus encoding the ~-subunit of the gastric h,k-atpase, which is expressed in the parietal cells of the stomach mucosa. the gastric h,k-atpase transports h + into the stomach lumen with the counter transport of k +, all at the expense of atp hydrolysis. when xenopus oocytes are injected with crna encoding ~h,k from either species, as well as a gastric ~h,k subunit, the two subunit proteins are synthesized, assemble, and form functional pumps located in the plasma membrane as demonstrated by 86rb+ uptake. the sensitivity to the gastric h,k-atpase inhibitor sch28080 was determined, with the ki for beth species found to be in the low ~m range. spodoptera frugiperda ceils are often used to overexpress eucaryotle proteins using baculovirus vectors. the atomic force microscope (afm) is a device which allows the observer to obtain high resolution images of biological samples immersed in a fluid medium; this instrument has therefore the potential to visualize overexpressed proteins on the surface of living cells. however, the interpretation of the images is sometimes difficult and requires a good knowledge of the specimen topography, i.e. the "normal" plasma membrane of the cell used for the overexpressisn. on our poster we present afm images of the plasma membrane of both fixed and living spodoptera frugiperda ceils. in additon, we discuss the possibility of achieving high resolution in the imaging of dynamic changes which affect /iving systems. the deduced protein sequence consists of 295 amino acids with about 55% amino acid sequence identicy when compared to mammalian ~h,k-subunits. data from other species and from the structurally similar na,k-atpase has shown that the ~-subunit is necessary for the formation of a functional pump, which consists of an ~/~ heterodimer. following co-injection of xenopus oocytes with crna for both the ~-and ~subunits, we have observed by rb + uptake h,k-atpase activity in the plasma membrane. we are presently studying the role of the ~-subunit in the maturation and function of the h,k-atpase. the atomic force microscope (afm) is a new type of microscope which allows visualisation of inorganic and organic samples with a very high resolution. a sharp tip fixed at the end of a cantilever is used as a topographic sensor. by scanning the sample with this device, a computer records the minute deformations of the cantilever and computes the topography of the sample. the instrument allows also the measurement of the interaction between the tip and the sample as a function of the distance. the knowledge of these interactions allows us to compute several mechanical properties like indentation, elasticity and stiffness. on this poster we report the results of these measurements on cells (sfg, c131), bacteria (e. coli, b. subtilis) and proteins (actin). in addition we discuss the use of the afm as a sensor for probing the mechanical properties of biological systems at a nanometric scale. neonatal thymectomy of balb/c mice induces aig. we have cloned the ~-and ~subunits of the balb/c h,k-atpase and expressed the proton pump in crna injected oocytes. both and ~ proteins are made, form functional pumps, and are immunoprecipated using auto-immune sera. the ~-subunit can also be in~unoprecipated independent of the ~-subunit when oocytes are injected with ~ crna alone. balb/c mice will be immunized with oocyte membranes containing either the ~/~ or ~ antigens in order to study the role of each h,k-atpase subunit in triggering aig. the prion protein is expressed in both astrocytes and oligodendrocytes of the neonatal brain and is down-regulated in adulthood. m. moser, colello, r, , pott, u, and b. oesch institut for hirnforschung der universit~t zorich, august forel str. 1, 8029 zorich the infectious agent causing transmissible spongiform encephalopathies consists largely or entirely of prp sc, a modified isoform o| normal prp (prpc). prpc is most abundant in the brain. the development of the disease strictly depends on the presence of prpc. and incubation times are shortened by overexpression of prpc. the normal function of prpe is not known and its cellular iocalisation in the cns is poorly characterised, except for its evident presence in neurons. here we describe the expression of prp c mrna in both oligodendrocytes and astrocytes during neonatal development of hamster and rat. in adult animals, expression is down-regulated in gila but not in neurons. our findings provide a link to the previously described accumulation of prpsc in astrocytes and the apparently faster course of prion disease in neonatal hamster. our results argue for a major involvement of non-neuronal cell types in prion diseases. such structures we raised mab against membrane protein fractions. a ~lab against a vitronectin receptor containing fraction was found, which if applied in vitro to bi6fi mouse melanoma cells, influenced growth, adhesion and morphology. ~tnen bi6fi cells prior to tail vein injection were treated, 2 different outcomes were observed. short treatment (lh) resulted in 85% inhibition of lung colonization whereas long-ter~ treatment ( 2weeks) resulted in 10-15 times more lung colonies. a candidate molecule likely involved in these observed effects could be identified as a 150 kda cell surface protein. sequence information from peptides revealed in several cases 100 % homology to mouse centrosomin a, i.e.a 32 kda cytosolic protein involved in the organization of the spindle apparatus. these data suggest that we may have found a protein which may participate in a link between the extra-cellular space and the microtubular system. we are currently trying to clone this 150 kda protein from a bi6fi c-dna library. wahlberg, j.m., and spiess, m. dept.biochemistry,biozentrum, university of basel. signals for correct basolateral sorting of subunit hi of the human asialoglycoprotein receptor are located in the cytoplasmic tail, involving a tyrosine at position 5. removal of this residue results in nonpolarized transport of the protein to the surface in mdckii-celis. a deletion mutant of hi, lacking 30 of the normal 40 residues of the cytoplasmic tail, including the tyrosine, has been found not to be transported to the cell surface, but to be accumulated intracellularly. the mutant protein is complex glycosylated and sialylated, indicative of passage into or through the trans-golgi. immunofluorescence analyses show defined juxtanuclear, tubular structures, which do not colocalize with markers for er, early endosomes or lysosomes, but which show similar staining pattern as markers for late endosomes and tgn. to define the determinants responsible for the intracellular accumulation a series of deletions and fusions have been constructed. the results suggest that the length of the cytoplasmic domain is one important determinant for missorting of hi. salerr~ n., medilansld, j., pellegrinelli, n. and eder-colli, l. departement of pharmacology, cmu, 1211 geneva 4 in chollnergic neurons the enzyme choline acetyltransferase (chat) exists as cytosolic(80-90%) and membrane-bound activity (10-20%}. are these two activities encoded by a single mrna? is membranebound enzyme preferentially associated with a given cellular membrane? in order to investigate these questions we isolated a fulllength cdna (4.2 kb) encoding drosophila chat and used it to transfect xenopus oocytes, rat fibroblasts and human neuroblastoma cell lines. triton x-114 fractionation of transfected cells showed that hydrophilic and amphiphilic chat activity were produced. the sp act. of hydrophflic enzyme reached 2, 0.8 and 0.25 ~mol ach/h/rng protein respectively in oocytes, fibroblasts and neuroblastoma whereas the sp. act. of amphiphilic enzyme was 1.8, 0.4 and 0.125 hinol ach/h/mg protein, respectively. amphiphilic chat was less sensitive to inhibition by the product acetylcholine and to heat denaturation than was hydrophilic enzyme. similar results were observed for the native drosophila chat. amphiphillc enzyme sedimentation was affected by the type of detergent present in linear density gradients of sucrose. transfected oocytes were subjected to subfractionation. besides a large amount of soluble chat activity, a significant proportion of enzyme was found associated with a fraction enriched in endoplasmic reticulum (er). preliminary results using transfected mammalian cells indicate that apart cytosolic chat, plasma mernbranes+golgi enriched fractions as well as er-enriched fraction contain chat activity. we have characterized a drosophila melanogaster gene encoding a very hydrophobic protein. the amino acid sequence shows considerable homology (33-42% identity) to neurotransmitter transporters. the gene is, however, not expressed in neural tissue, but in the male germline. transcription and translation occurs in early spermatocytes. antibody staining data suggest the following pathway of the protein during spermatogenesis: the protein is synthesized in early spermatocytes and is transported into the nucleus during prophase of rt i. at meiosis it associates with the mitochondria and it stays associated while they fuse to the nebenkern. during individualization it is stripped off in the cystic bulge. inspection of the putative protein sequence shows that it possesses alle the necessary targeting signals to allow its transfer from the cytoplasm through the nucleus into the mitochondria: three nuclear targeting signals, a mitochondrial import signal and, moreover, two pest-sequences for rapid protein degradation are present. we shall present a, currently tested, working hypothesis. in hg-treated toad skins, water permeability is high or low depending on whether the major anion of the inner bathing medium is so~ or ci. we report here the results of experiments designed to determine if, in skins bathed with so4-ringer, net water flow (j~,) could be diminished by agents added to the outer medium. toad skins were mounted inbetween glass chambers and exposed to a standard osmotic gradient of 200 mosm. jw was continuously monitored by automatic, volumetric techniques. exposure to lmm hgclz or chzcihg (external side) led to the typical anion-induced j,, changes. re-exposure to hg caused a mild (10%), although significant (n=8, p<0.001), fall in jw. since cuso4 is a sh-reagent like hg, we looked at the effects of cuso4 (lmm). there was a 68% reduction of j, (n=7, p<0.001), an effect totally reversible by simple washing of the skins. likewise, addition of dithiothreitol (dtt, 5mm) in the presence of cu, caused a 90% reversal of the cu effect ; there was no change in jw if dtt was given before cu, or if both agents were added simultaneously. finally, in hg-treated, glutaraldehyde-fixed skins, lmm and 5ram cuso4 caused 51% and 92% jw decrements, respectively, that were totally reversible. in conclusion, cu causes a dtt-sensitive inhibition of jr, in hgtreated skins. further work is needed to establish if the cu effect is exerted on sh-groups of apical aquapories of toad skin. $15-44 the amino acid sequences of the pc-645 e-subunits and other cryptomonad biliprotein ~-subunits pose the most intriguing question about the phylogenetic origin of these unique chromopeptides. cryptomonad pc-645 ~-subunits seem not to be closely related to the known phycobiliproteins, linker polypeptides (lpps), bpe or any other light-harvesting chl polypeptides from the thylakoid. for most of them the number of identical amino acid residues was 15%-20%. sequence alignment of c-pe associated lpps with t-subunits from cyanobacteria and red algae and the cryptophytan ~-subunits however show that the identity between c-pe associated lpps and cyanobacterial x-subunits is still significant, whereas it reaches the limit of significance between cyanobacteril and eucaryotic x-subunits. nevertheless it has to be assumed that x-subunit derive from cyanobacterial c-pe associated lpps as well as cryptomonad ~-subunits may derive from t-subunits. whereas the the phycobiliprotein ~-and 8subunits remained structurally conservative during evolution (e.g. -70% identity between cyanobacterial and red algal pe-subunits) the lpps and t-subunits show drastical changes in their chromophore binding domains. $15-45 functional and evolutionary relationships of the components of the primary events in photosynthesis have been traced using amino acid sequence comparison. basically, the question was posed regarding the interrelationship of the apoproteins of different photosynthetic organisms which gather light and/or separate charges across membranes. do their protein-chemical structure and organisation decipher us certain clues and parts of the path by which they have been selected and derived from a possible primordial reaction centre polypeptide? within that context the light-harvesting-and energy transfer systems of purple bacteria offered as ideal model components. a data set of more than 70 apoprotein sequences has pointed to some keystructural elements which have been partially verified as such by limited proteolysis and genetic engineering. a careful inspection of the determined bacterial antenna-structures revealed some remarkable similarities to eukaryotic antenna and reaction centre apoproteins indicating possible basic structural motifs for complexing pigment molecules, like chlorophylls or bacteriochlorophylls in very distant representatives of phototrophs. a number of glycoproteins of the nervous and/or immune system that are involved in cellular recognition and/or adhesion share the common lz/hnk-1 carbohydrate structure. in a large proportion of patients with peripheral demyelinating neuropathy associated with paraproteinemic gammopathy (ppn), monoclonal igm antibodies (m-igm) react with the lz/hnk-1 determinant of mag and p0. we plan to test the hypothesis that anti-mag m-igm autoantibodies may alter the expression of hnk-1 bearing myelin proteins such as mag, p0, mog, ncam or pmp22. by using monoclonal antibodies to myelin proteins on tissue sections, preliminary results indicate that mbp protein content seems to be strongly downregulated, whereas galc and $100 protein do not appear to be affected by the loss of myelin in ppn nerves. gfap protein expression appear to be upregulated in demyelinating biopsy specimens from ppn patients with anti-mag m-igm gammopathy. in conclusion, the expression of some l2/hnk-1 negative proteins might be disregulated by the presence of circulating anti-mag m-igm in the serum of ppn patients. we have isolated a human liver na+-taurocholate cotransporting polypeptide (ntcp) by screening a human liver edna library with a rat edna probe derived from ntcp (pnas 88, 10629, 1991) . the cdna codes for a protein of 349 amino acids which shows 77% amino acid homology with the rat ntcp. expression of ntcp in x. laevis oocytes resulted in na+-dependent bile acid uptake which exhibited saturability with an apparent km for taurocholate of 6 ~m. ntcp mediated taurocholate uptake into oocytes was inhibited by all major bile acid derivatives, bumetanide and bromosulphophthalein. southern blot analysis of genomic dna from a panel of human/hamster somatic cell hybrids mapped the human ntcp-gene to chromosome 14. in conclusion, these studies supplement the previously suggested selective mammalian distribution of the ntcp gene family and provide the possibility for direct molecular characterization of human bile acid transporter genes. diverse cell surface molecules of the nervous system play an important role in specifying ceil interactions during development. with the aid of a polyclonal antibody against l1, a 1.skb edna clone closely related to l1 (ch-l1) was isolated from a ~. gtl 1 library derived from poly(a)* l~qa of day 8 mouse brain. using this edna as probe the remaining cdi'ia 5' sequences were cloned out of a plasmid library constructed from postnatal day 6-14 mouse brain poly (a)+ p, na. two independent full length clones of 4.2 and 4.4 kb encompassing the entire coding region of chl] were isolated. these represent putative alternatively spliced mp, nas. the deduced primary structure of chl1 reveals that, like the cell adhesion molecules (cams) mouse l1, chicken ng-cam, and nr-cam (bravo), chl1 is composed of six ig-like domains, a transmembrane domain, and a short cytoplasmatic region. in contrast to the other l1 family members, only four and a half instead of five fibronectin type hi-like repeals are found in chl1. the fibronectin type [h-like repeats were expressed in e. col[ and the protein fragment was used for immunization. as observed for li, ng-cam, and nr-cam, chl1 is susceptible to proteolytic cleavage. bands of 185kd, 165kd, 125kd, and 50kd could be detected bywestern blot analysis. by in situ hybridization, a predominant expression by neurons was found in the adult mouse brain. western blot analysis showed expression of chl1 protein in brain and heart, but not lung, kidney and intestine. in liver, a 50 kd immunoreactive band was found. the relationship of chl1 to molecules known to be involved in cell adhesion and neurite outgrowth, combined with its pattern of expression, suggests a role for this molecule in cell interactions dndng neural development. work from this laboratory revealed that in hg-treated, glutaraldehydefixed toad bladders, the water permeability coefficient (pf) is high in sod-ringer and low in ci-ringer ; in all these studies the osmolality of the serosal medium was 220mosm. as an attempt to determine the mechanism(s) underlying such pf changes, experiments were carried out with bladders whose serosal surface was exposed to [so-, hypo-or hyperosmotic media ; the outer surface was always exposed to a hyposmotic solution (22 costa). net water flows were measured continuously with automatic, volumetric techniques. after exposure to 1 mm chzcihg (10', outer medium) followed by fixation with 0.25% glutaraldehyde (5', serosal medium), the bladders were thoroughly washed. in sod-ringer, pf (/~m/sec) was as follows (n=6) we wanted to determine the molecular weight (mw) of ntcp and its topology in the plasma membrane (pm) of rat liver. a rabbit antiserum generated against the c-terminus of ntcp was used to determine its mw on western blots and its subcellular distribution in liver and in hepatocytes by immunofluorescene (if). site directed mutagenesis and deletion experiments were used to determine the n-linked glycosylation sites. ntcp encodes a 50 kd protein in rat liver pm vesicles. the basolateral localization of ntcp in liver was confirmed by if. ntop could only be immunostained in permeabilized hepatocytes suggesting an intracellular localization of the cterminus, cnda constructs showed the ntcp is glycosylated at positions 5 and ii. the data show a selective basolateral expression of ntcp in rat liver and they suggest that the two ends of ntcp are located on opposite sides of the pm. zymogen granule membranes (zgm) are known to contain proteins with nucleoside phosphatase activity, but their exact nature and function are unknown. the activities require ca ++ and mg ++ and are sensitive to atpase inhibitors but not to inhibitors of na/k-atpase activity. the aim of this study was to isolate individual proteins of pig pancreatic zgm, to attribute enzymatic activity to individual proteins and to characterize them with various substrates (gtp, atp, amp etc.), inhibitors (amp-cp, amp-cpp, gtpgs etc.) and lectins (maa, dsa, gna etc.). we have subjected highly purified zgm solubilized in chaps to ief on immobiline dry strips | (pharmacia) with s ph range 3--10.5 (ist dim.). these strips were then electrophoresed in a polyacrylamide gradient gel containing 0.2% chaps and tris/hci at ph 9.5 (2nd dim.). nucleoside phosphatase activity was detected histochemically in the 2d gel by incubation with substrate in the presence of lead nitrate. focussed strips were also subjected to sds-page for comparison. several proteins showed strong activity to gtp, atp and itp. the role of these phosphatases are discussed in the context of the role of gtp-binding proteins and the de-/phosphorylating events on the zgm in intracellular targeting and exocytosis. the disease-specific isoform of the priori protein (prp sc ) is an essential part of the infectious particle causing scrapie or bse. prp sc differs from prp of normal animals (prp c ) by its relative protease resistance. the physical nature of this difference is still unknown. here we analyzed the protease-resistance of prp sc quantitatively. prp sc was rendered completely protease-sensitive at alkaline ph or in guanidinium thiocyanate (>1.5 m). denaturation in 2m guanidinium thiocxanate (gdnscn) completely abolished protease resistance of prp bc within 15 min while denaturation in 6 m urea showed a much slower time course. denaturation cuwes were used to calculate the gibbs free energy (as d ) in dependence of gdnscn or urea at different concentrations. the linear relationship between agd and the denaturant concentration is suggestive of a two state model involving the conformational change of a single protein domain. this is also reflected in the small number of side chains (11,6) additionally exposed to the solvent upon conversion of prp sc to its proteasesensitive isoform. our results suggest that minor rearrangements of the structure of prp sc are sufficient to abolish its protease resistance. of physiology, university of zurich, winterthursrstr. 190, ch-8057 zt~rich in contrast to mammalian kidneys where inorganic phosphate (pi) is reabsorbed unidirectionally, flounder renal epithelial cells both re.absorb and secrete pi. in order to investigate on a molecular level the cellular pi handling we have cloned a re,absorptive nalp i transport system from flounder renal tubules. based on homology with the cloned na/p i cotransporter from rat we have isolated a edna clone (flounder napi-ii, 2424 bp) encoding for a protein of 637 amino acids. in the eight transmembrane spanning domains the protein is 78% identical with the corresponding system from rat kidney, in the hydrophilic regions only 30%. expression of in vitro transcribed rna in xenopus oocytes specifically stimulated na-dependent pi transport. binding properties of na and pi as well as the ph-dependency were similar to the ones found in mammalian systems (rat, human, ok cells). by northern analysis three transcripts, 1.9, 2.7, and 4.2 kb in length, could be detected. rna from flounder renal tubules contained all of the transcripts, intestinal rna only the two lower bands, and non-epithelial tissue from kidney expressed only the 1.9 kb transcript. mammalian systems share only the 2.7 kb transcript but not the related species. the close functional relationship of flounder napi-ii with the known najp i transport systems and the pronounced differences in primary structure provide the basis for structure function analysis of na/p i cotransport. the amyloid 13/a4 protein precursor (app) is a transmembrane protein expressed in different isoforms throughout the organism. our work has consisted in a detailed study of the structure and biological function of app. specifically, we have shown that the secreted form (sapp) of app-695 is involved in the growth regulation offibroblast% and also stimulates neurite extension in b 103 neurnblastoma cell line. functional mapping studies have shown that the domain responsible for both the growth-regulating and the neurotrophic activities of sapp-695 is the rerms site, arg-328 to ser-332. the presence ofsapp binding sites on the surface of b 103 ceils (through the active rerms site) indicate that the neurotrophic activity of sapp is mediated by a receptor, and the subsequent activation of a signal transduction mechanism. in addition, we have shown that the rerms site is also involved in the neuronal survival properties of sapp-695. finally, we have initiated an in vivo study of app biological function, and shown that a peptide representing the neurotrophic domain ofsapp-695 can strengthen memory retention in rat through stabilization of synaptic structures in the frontal cortex. this work was supported by grants from the swiss national science foundation (823a-028366), and nih (ag 05131). we previously showed that in smooth muscle cells, the (~i-ar is rapidly phosphorymted following exposure to agonist. to understand this biochemical event, we investigated the phosphorylation of the c(1b adrenergic receptor stably expressed in rat1 fibroblasts (3.3 pmol/mg prot.). the =(1b-ar was solubilized from 32p-labeled rat cells and immunoprecipitated with antibodies raised against the n-terminus part of the receptor. treatment of ceils with 100 ~.m epinephrine showed a rapid and significant increase in receptor phosphorylation above basal. treatment of cells with a specific pkc inhibitor (ro-318220) did not affect agonist.promoted phosphorylation while it completely abolished phorbol esterinduced receptor phosphorylation. this strongly suggests that pkc is not involved in agonist-induced c{1b-ar phosphorylation. the elb-ar contains several putative phosphorylation sites in both its third intracellular loop and c-terminus tail. to investigate the role of these domains, a trunoated receptor (lacking its last 147 amino-acids) was constructed and stably expressed in rat cells (1.3 pmol/mg prot). this receptor mutant displayed similar ligand-binding properties and abirity to activate phospholipase c as compared to the wild-type receptor. phosphorylation experiments revealed that this mutant is not at all phosphorylated, neither by agonist nor by phorbol esters. agonistinduced decrease in cell surface receptors, measured by binding of the antagonist 3h-prazosin at 4~ was however similar for both the wild-type and mutant receptor. this suggests that loss of phosphorylation sites does not affect receptor internalization. human intestinal lactase-phlorizin hydrolase (lph) is synthesized as a precursor molecule (pro-lph), which undergoes proteolytic processing during maturation. lph expressed in cos-1 cells was enzymatically active, the electrophoretic properties of lph-species synthesized by transfected cos-1 cells correspond to those in organ cultured human intestinal explants, in caco-2 ceils and in transfected mock ceils. they included the pro-lph and a proteolytically processed form, which had similar electrophoretic properties to the mature enzyme. the appearance of the putative mature form was inhibited by brefeldin a, ammonium chloride and chloroquine. in addition, only complex glycosylated pro-lph (pro-lphc) was inserted into the cell surface membrane. these data indicate that in cos-1 cells pro-lph is inserted into the cell membrane, is internalyzed and enters lysosomes where proteolytic events lead to the appearance of a mature-like enzyme. $15-59 assembly and transport of paba peptide hydrolase alpha and beta subunits in cos-1 cells eldedng, j.a., gr0nberg, j. and sterchi, e., institut for biochemie und molekularbiologie, universit&t bern. paba peptide hydrolase (pph) (ec 3.4.24.18), a metalloendopeptidase from epithelial cells of human small intestinal mucosa, consists of two subunits, ~ and 13, which form homodimers and oligomers, cdna cloning has revealed homologies with developmentally important proteins from different species and has led to the definition of a new family of metalloendopeptidases, the astacin family (j. biol. chem. 266, 21381, 1991) . here, we report on the expression of the cdnas of pphcz and pphj3 in cos-1 cells. when expressed on its own, pph(z is synthesized in a core glycosylated form only, suggesting that it is transport-incompetent and not able to exit the er. immune-fluorescence studies have confirmed that pph~ is not expressed on the surface of cos-1 cells. pphl3, on the other hand, matures and is transported to the cell surface. when the two subunits are coexpressed, ppho~ can also be detected on the cell surface, suggesting that a heterooligomeric assembly is necessary for the surface expression of pphtz. truncated forms of both pphc~ and pphi~ could be detected in the medium of transfected cos-1 cells. in our previous work, we demonstrated by immunocytochemical reaction that sensory neurons expressed nuclear triindothyronin receptors (nt3r) in embryonic as well as adult rats. in contrast, in sciatic nerve, schwann cells exhibited only transient nt3r immunoreactivity from e17 to postnatal day 10. in the present work, we attempt to demonstrate by radioautography that the detected nt3r are effectively the binding sites of [125i] labeled triiodothyronin. cryostat sections of dorsal root ganglia (drg) or sciatic nerve were incubated with 0.1 nm of l, 3,5,3'-[t25i]triiodothyronin 2200 ci/m mol (nen), while the controls were incubated with a large excess (llxm) of unlabeled trfiodothyronin (t3). in aduit rat drg, radioautographs revealed that silver grains were exclusively restricted to neuronal cell bodies, while the schwann cells ensheathing the axons were free of significant label. in newborn rat sciatic nerve, silver grains accumulated over schwann cells; in contrast in adult rat sciatic nerve, schwann ceils were free of label. in control drg or sciatic nerve sections incubated in large exceas of unlabeled "1"3, all the radioautographs exhibited only scattered silver grains. in conclusion, our results show that sensory neurons and schwann cells are able to express functional nt3r which bind thyroid hormones (snf 31-33671-92). characterization of the maturepreeursor glycophospholipid for glycosylphophatidylinositoi anchors of saccharomyces cerevisiae. many attempts to isolate the mature yeast precursor gpi glycophospholipid ready to be attached to newly translated proteins for gpi-anehoring have failed although such precursors were previously identified in mammalian cells and protozoa. here we show that metabolically labeled glycolipids of the expected structure can be observed if the incorporation, their accumulation and their extraction are optimized. two very polar, (3h)-mannose-labeled glyeolipids named cpi and cp2 were identified and purified. they were structurally characterized using phnspholipase treatments, partial deacylation with methanolie ammonia, hydrofluoric acid dephosphorylation, nitrous acid deamination, acetolysis, exoglycosidase treatments and combinations thereof to produce labeled fragments which could be analyzed by paper chromatography or thin layer chromatography. cp1 and cp2 both contain the identical core oligosaccharide mangl,2(x->po4-6)manal,2mamxl,6(y->)mana-glcn-lansitol, x and y being hydrofluoric acid-sensitive substituents (most likely ethanolamine and phospheethanolamine, respectively). the inositol is acylated although no acyllaositol is present on protein-bound yeast gpi-anchors. cp1 and cp2 can also be observed after lableling with (3h)myo-inositol, the lipid moieties of cp1 and cp2 can be completely removed by mild alcaline hydrolysis altough the protein-bound gpi-anchors made by the pmi210 cells under identical labeling conditions are completely mild base resistant. this finding reinforces the notion that the ceramide moiety typically found on the majority of yeast gpi-proteins are added only after addition of the gpi precursor glycolipid to proteins. the distributions of the mannose-6-phosphat e/igfll-recept or and a2,6slalyltransferase in hepg2 cells: no evidence for co-localization by confocal laser scanning microscopy. the organization of the trans golgi apparatus (ga) with respect to (recycling) man-6-p/igfii receptor (mpr) and (resident) c~2,6sialyltransferase (st) has been investigated by double immunofluorescence laser scanning confocal microscopy in hepg2 cells. they were chosen because biochemical evidence suggested sialylation of mpr upon recycling (duncan & kornfeld, j. cell biol. 1988, 106, 617-28) implicating colocalization of both proteins. in the steady state st was confined to the ga whereas total (endogenous) mpr was localized to coarse vesicles without evidence for colocalization by confoeal microscopy. endocytosis of surface-labeled mpr was monitored in presence and absence of brefeldin a (bfa): without bfa, early endosomes (2' of warming up) showed no, late endosomes (20') little if any colocalization with st. in presence of bfa, the st-compartment disappeared whereas late endosomes persisted with some tubular emanations. under similar conditions, in "absence of bfa, endogenous mpr concentrated in the ga region suggesting colocalization with st. in presence of bfa the mpr compartment formed a redculum whereas the st compamnem disappeared. in conclusion, within the limits of the techniques used, exogenous mpr was not detectable in the st compartment whereas endogenons mpr concentrated in the ga region but obvious co-localization was exceptional. supported by grant 31-30757.91 of the snsf to egb. cultures of dissociated striatal neurons prepared from fetal rats were grown in the presence of neurotrophin-4/5 (nt-4/5) as well as the other known neurotrophins, ngf, bdnf and nt-3. we found that acute administration of nt-4/5 to seven days old cultures stimulates the hydrolysis of phosphatidyllnositol, an event involved in neurotrophin signal transduction. growth of stdatal cultures in presence of nt-4/5 resulted in increased cell survival as indicated by elevations in total cell number, protein content and number of viable cells. nt-4/5 exposure increased gaba uptake and staining intensity in gaba immunocytochemistry in these cultures indicating atrophic action on gabaergic neurons. to further identify responsive cell populations we analyzed for calretinin, a calcium binding protein known to colocalize with gaba in a number of neuronal cells. nt-4/5 strongly increased the number of calretinin positive cells in cultures prepared from rats of embryonic day 15, as we]l as calretinin levels determined by western blot analysis. when cultures were prepared from embryonic day 18 rats, nt-4/5 very strongly increased the morphological differentiation of calretinin positive cells. while all effects produced by nt-4/5 were mimicked by bdnf with similar potency, nt-3 was found to be only marginauy effective, our findings identify nt-4/5 as a potent neurotrophie factor for striatal gabaergic neurons. fusion of cells and organelles is a general patho-biological phenomenon: muscle cells, transport vesicles, langhans cells and virus induced fusion from within (ffwi, during replication) and without (ffwo, by the particles). in vivo and after virus isolation only little fusion can be seen, only after cultivation fusing hsv can be detected; a selective pressure must be released. 6 syn loci are known on the genome; the syn 3 locus is located inside of the cell in the carboxy terminal part of glycoprotein b, all other fusion domains are outside the ceil. fusion inducing mutations are in aa 816, 853, 854, 856, 857 and 872 and were correlated to fusion from within and without as well as to selective cyclosporin a effects. a model is presented for the 3 syn locus. -by recombination the fusion capacity could be transfected to fusion negative viruses. -hsv penetrates the cellmembrane by fusion by 2 ligand-recepter interactions. ffwo+-strains penetrate at 0~ cell/cell fusion was analysed and found to need also 2 receptor-ligand interactions if induced by syn 3 mutations. timing analysis of fusion events and by certain inhibitors allow further conclusions. quenching of lhcii isolated from dark-adapted (lhcii-d) or preilluminated (lhcii-l) rye leaves was similar in both preparations, but reversibility of this process within two hours of darkness was slower in lhcii-d. no differences in fluorescence quenching and full reversibility was observed for both lhcii preparations in reverse micellar solution. xanthophyll/chl concentrations in lhcii were consi-derably decreased under this conditions. excitation difference spectra (before and after illumination) of lhcii in asolectin liposomes showed typical changes of energy transfer between carotenoids and chl. a model is proposed according the xanthophyll cycle controls reversibility of light-driven excitation quenching as well as the xa~a~q~_x~tentof ~,,~nchi~g i/~ lhc~z. myelinogenesis is a complex, developmentally regulated process involving coordinate expression of myellnafion-related proteins. the myelin forming cells are the oligodendrocytes in the central nervous system (cns) and the schwann cells in the peripheral nervous system (pns). we differentially screened a rat postnatal day 16 (p16) spinal cord edna library with probes derived from normal (plus probe) and from myelin-free (minus probe) p16 spinal cord mrnas. we succeded in the isolation of several novel edna clones whose corresponding mrnas were expressed either selectively by oligodendroeytes or by oligodendroeytes and sehwann cells. here we describe one of the edna clones (tentatively denominated ns 2) whose mrna is specifically expressed by oligodandroeytes and schwann cells. the 3.5 kd transcript is expressed at highest level during myellnation and at much lower levels in the adult as it is known for other myelin-specific mrnas. sequence analysis of the full length edna sequence showed a 50% identity to the rat liver udpglucuronosyltransferase family, involved in the detoxificafion system. the 541 amino acid open reading frame encodes a 62 kd protein with a putative signal peptide and two transmembrane domains, whether this ns 2 protein is involved in a detoxification reaction or in glycosilation of proteins and lipids with glueuronie acid is under investigation. neuroblastoma (n8) are sympathetic tumors of childhood characterized by mycn amplification in advanced stages. cd44 standard (cd44s) and splice variants (cd44v) represent a group of surface glycoproteins whose expression has been recently linked to metastasis. we analysed the expression of cd44s and cd44v on n8 tumors and cell lines by immunohistology and northern blot. results showed high levels of cd44s on 33/44 nb, 6/10 n8 cell lines and 4/4 ganglioneuromas (mature, benign sympathetic tumors). lack of cd44s staining was observed on stage iv, mycn amplified tumors only. in cell lines, expression was not detected on cells with a neuronal phenotype while high levels of cd44s were expressed by cells with a glial differentiation, independently of mycn amplification. up-regulation of cd44s was obtained with agents that induce a glial differentiation, while neuronal differentiation by retinoic acid was not accompanied by a change in cd44s expression. no gd44v were detected on tumors or cell lines. we conclude that cd44s expression is down-regulated in a subset of nb ceils and tumors and that mycn product and additional mechanisms responsible for control of differentiation are involved in this regulation. the glycoprotein gc of herpesviruses is known to be non-essential for virus replication. non-essential herpesvirus proteins are suggested to perform "luxury functions" which may influence the outcome of an infection. we are aiming to analyse the function(s) of ibc specified by bovine herpesvirus 1 (bhv1), bovine herpesvirus 5 hv5) and caprine herpesvirus 1 (caphv1). these viruses are closely related but differ in their pathogenic potential. in a first step we have cloned and sequenced the individual genes. the nucleotide and the deduced amino acid sequence homologies of two 8hv1 reference strains was found to be >98%. the sequence homologies between bhv1 and bhv5 were >75% and those between bhv1 and caphvl were >65%. comparisons on the amino acid sequence level revealed that the differences were most prominent in the n-terminal parts, whereby the potential signal sequence region might be concerned. the putative glycosylation sites, however, were not affected, and the transmembrane sequences were similar in size and location. in order to characterize the significance of these differences, we are constructing gc-deletion mutants and recombinant viruses carrying a foreign gc gene. the in vitro and in vivo behaviour of these viruses will be compared to the wildtype virus. $15-67 o. moullet and j.-l. dreyer, instimt de biochimie, unlversit6 de fribeurg, ch-1700 fribourg camp has been recently shown to promote a cell survival response by retarding apoptosis (berridge, m.v& el. (1993) exp. hematol. 21,269-276) . on the other hand quinones are widely distributed substances of often potential toxicological significance. we have tested the effects of quinones on adenylate cyclase. the enzyme is rapidly inhibited by pbenzoquinone (ic50=40-45 ktm) or dicnlorophenol-indopbennl (/6'50=200 ilm), but 2-substituted quinones are inactive. the lc50's are decreased 3-10 times after membrane soinbilization but are not affected by gtp, gdp or analogues nor by cholera and pertussis toxins. the inhibition is not mediated by a g-protein or by the activation of a defined receptor. quinone inhibition stoichiometrically competes with forskolin activation of adenylate cyclase, equimolar concentrations of beth quinone and forskolin restoring the enzyme activity to its basal value. the cytotoxicity of benzoquinone, tested in vivo on hep-g2, a human bepatocellular carcinoma cell line, could be prevented with forskolin. in plasma membranes quinones tightly bind to only one major and two minor proteins that were purified by page electrophoresis under native conditions. together these observations indicate that quinone action can be attributed to targetting to a limited number of proteins at tile plasma membrane in a highly selective way. by affecting adenylate eyclase, a modulation of the camp pool induces apoptosis as a result of the cytotoxicity via. t.congolense is an african,unicellular hemoflagellate, pathogenic for domestic animals such as cattle.within the host bloodstream,parasites adhere to the wall of the microvasculature,eausing severe organ damage.we have set up and characterized an in vitro assay in order to study the metabolic requirements,reeeptor-ligand interactions and the ultrastructure of this adhesion phenomenon.our findings suggest that adhesion is an active process requiring metabolic energy on part of the parasite, but is independent of the target cells.we also show that t.congolense possess a leetin-like domain localized at specific sites along the surface of the anterior end of the flagellum, which interacts with specific carbohydrate receptors, probably sialic acid and/or n-aeetylglueosamine residues,on the endothelial cell surface. this suggests that adhesion is likely to be mediated by a trypanosomal surface component distinct from the variable surface glycoproteins(vsgs). we also suggest that the cytoskeletal protein aetin is involved in this process. local immune response based upon intestinal parasitespecific t and b cells to giardia lamblia may be impomrant in parasite clearance. experimental infections qf mice (cr:nih:s) with g. lamblia (clone gem-h7 which e~presses a major 72kd antigen on its surface recognized b~ mab g10/4) have demonstrated that the parasite undergoes surface antigenic variation in rive. experimental i~fection of immu/%odeficient mice (nu/nu mice and sci~d mice) and oontrol nu/+ littermates provided insights into associated immunological mechanisms: dominant surfade epitope-specific slga plays a major role in modulatin6] surface antigenic variation, and thymus-dependent t-lyn~phecytes in the induction of selfcure. athymio nu/nu mioe (zu. icr-strain) were reconstituted with immune peyer:~ patch lymphocytes obtained from self-healed littermat~ nu/+ mice. intestinal b-cells from these nu/+ mice @s well as from reconstituted athymic nude mice synthesized in vitro parasite-specific iga. this iga showed a predominant immunoblet reaction with the major surface antigen (a mr 72,000 polypep-tide) characterizing the giardia clone in question. nu/nu mice reconstituted wit~ immune cells acquired the potential to decrease significantly their intestinal parasite mass. thus the hypothesis on the causative role of intestinal iga in tl~ control of intestinal g. lamblia infection has found further experimental support. stephan jentsch, heinz tobler and fritz m011er, institute of zoology, university of fribourg, p4rolles, "1700 fribourg the process of chromatin diminution in the parasitic nematode ascaris lumbricoides leads to the formation of somatic cells that contain less dna than the germ-line cells. during this process, which occurs between the third and the fifth embryonic cleavage divisions in five different blastomeres, the termini of the chromosomes are cut off and eventually degrade in the cytoplasm. to analyze this process at the molecular level we constructed a xzap somatic telomere library. the analysis of three cloned telomeres and their corresponding germ-line genomic regions revealed that chromosomal breakage takes always place within short specific chromosomal regions (cbrs) and is followed by the addition of the telomeric sequences (ttaggc)n to the breakage sites. the different cbrs do not crosshybridize to each other. a comparative nucleotide sequence analysis is now being performed to screen for the existence of conserved sequence motifs. in a parallel approach we analyze the chromatin structure of the cbrs and their flanking regions in developmental stages before and during the elimination process, putative recognition or regulation sequences important for the elimination process might be recognized as dnasel hypersensitive sites. once such sequences will be identified, it should be interesting to establish their role in the elimination process and to look for specific binding factors. chromatin diminution takes place in all presomatie ceils of the early embryo of ascaris lumbricoides and is followed by the addition of many repeats of the telomeric sequence ttaggc. chromosomal fragmentation is developmentally regulated and occurs within specific chromosomal regions (cbrs). one of these cbrs (cbr1) was analyzed in detail. agene located close to the cbr1 encodes a putative gtp-binding protein, whose promoter region is located within a distance of only 2 kb from the telomeric ttaggc repeats of the corresponding somatic chromosome. a homologous gene was isolated from c. elegans. most interestingly, the c. elegans gtp-binding protein gene is also located at a telomeric position, namely at the end of chromosome v. in ascaris, transcripts of this gene are present in all developmental stages, though they seem to be stronger expressed in oocytes and early embryos than in later developmental stages and somatic tissues. a high degree of sequence conservation of these genes between the two nematode species and man (a corresponding partial cdna clone was identified in a human heart cdna library) suggests an important biological function. currently, we are using genetic methods to determine the possible function of this gene in c. elegans and to test whether its telomeric localization in both nematode species has any functional importance. it encodes a nuclear protein which is associated with she chromatin together with other probeins of the poiyoomb group. the mechanism of gene regulation caused by pelycomb seems to be similar to that of the modifier genes of the position variegation effect which also encode structural proteins of the heterochromatin. one of these proteins is hpi which shares with polycomb a region of similarity at the amino terminus called the ~omatin ~rganisation m~difier domain (chromo domain). the particular function of the chromo domain is not yet understood, but it seems to be important for the protein-protein interactions in chromatin associated complexes. furthermore, chromobox cdntai~ing genes were found in several species, suggesting that they are widespread in the animal and plant kingdoms. here we show chat ~. clemens contains at least one chromobox containing gene (cdna: cm08h7). length and szrusture of the putative ~. elegs~ chromo domain protein are similar to those of the chromo domain containing prozeins of other species. the expression pattern of cm08h7 is s:udied with antibodies raised against the putative chromo domain protein and by injecting a 5-ga! fusion construct inns the germ line of ~. eleman$. chromatin diminution in the parasitic nematode ascaris lumbricoides is a complex molecular mechanism, involving cleavage of the chromosomes at specific breakage regions, degradation of the eliminated chromatin and new telomere formation in all presomatic cells during the early embryonic development. the aim of the present project is to reproduce this dna elimination process in vitro, step by step or all in one, by using cell-free extracts of a. lumbricoides eggs. therefore, 4-8 cell extracts were established and their quality was assessed by the ability to transcribe 5s rrna genes (pol iii) and sl rna genes (pol li). faithful extracts were then tested for cleavage activity on dna substrates derived from different chromosomal breakage regions. finally, preliminary results revealed a possible non processiv rnase sensitive telomerase activity in the in vitro extracts, capable of adding specific nucleotide sequences to an oligonucleotide primer containing four repeats of the telomeric sequence traggc. as de novo synthesis of several kilobases of somatic telomere is likely to require a strong telomerase activity, we assume that this a. lumbricoides in vitro system is a good tool to isolate the telomerase protein and its corresponding gene or other implicated factors, s16-07 university of berne, ch 3010 berne a common feature of all mycobacteria -whether pathogenic or not -is their richness and variety of lipids. among numerous lipids widely distributed, pathogenic mycobacteria exhibit a group of sulfated surface lipids thought to be determinants of virulence. therefore, we studied sulfolipid-bioslrnthesis in pathogenic and apathogenic (opportunistic) mycobacteria species by monitoring the kinetics of [35s]sulfate incorporation into cellular lipids, pathogenic mycobaeterla (two virulent m. tuberculosis strains) showed a marked sulfolipid-synthesis with highest rates during exponential growth, while apathogenic ones (an avirulent m. tuberculosis strain and an opportunistic species) manifested negligible incorporation of the label. the partial characterization of the [35s]-sulfated lipids of pathogenic mycobacteria by thin layer chromatographic separation, combined with autoradiographic detection, revealed the presence of several radiolabeled lipid components of different polarities and with altering expressionpatterns during growth. these results strengthen the hypothesis, that sulfolipids are involved in the pathogenesis of mycobacterial disease. maiada (1,2) . some features of human pf maiada are observed in mudne malaria, although the pattern of sequestration changes due to the different plasmodial species involved, in mice, we showed by immunohistocbemistry that the expression of vcam-1 and icam-1 is upregulated in brain, lung and heart of p/asmodium bergheiinfected baib/c and also in lps-pdmed 8cid mice. in $gid mice injected with labeled human erythrocytes (e), a higher rate of sequestration to brain was observed with pfe than with uninfected e. lps-pdming increased the retention of pfe in the brain and lungs significantly (t test), but decreased it in the spleen. we conclude that pfe express surface structures which allow them to adhere to the vasculature of the brain more efficiently than uninfected e. lps increases the number of adhesive molecules on the host endothelium. interestingly, sequestration in the 8cid mouse resembles that of pfe in man. f. roth, f. riniker, k. schopfer and t. burkart inst. med. microbiology, univ. of berne, ch 3010 berne it has recently been shown that bacterial lipids cancarry antigenic determinants. the study of antibody specificity to such lipid epitopes in vitro turns out to be difficult since hydrophobic antigens have to react with watersoluble molecules. we have developed a solide phase lipid antigen immunoassay (laia) that allows to quantify the antibody reactivities with an array of lipid antigens derived from mycobacterial cells. defined amounts of extracted lipids were immobilized as equidistant lines on a nitrocellulose sheet. small strips of nitrocellulose, each carrying the same sets of lipids were incubated with different human sera. bound antibodies were then reacted with [125-i]-iodine labelled antihuman antibodies and visualized by autoradiography. the reaction was quantified by densitometry. assay conditions were optimized for antigen and antibody concentrations and appropriate incubation times. in addition, the effects of different ingredients (salts, proteins, detergent) on antigen immobilization and epitope accessability were studied. the presented laia-system is rapid, sensitive and reproducible. it allows to compare the specificities of different bacterial lipid antigens and to quantify their reactivity with serumantibodies. the atomic force microscope is a very convenient tool for studying hard and flat samples with atomic resolution. biological objects, usually soft and rough, are sometimes difficult to image using this technique. in contrast bacteria, which are too small to be observed with high resolution using the optical microscope, are hard enough to be observed with an afm at a relative good level of magnification. this kind of microorganisms can be prepared for afm imaging using very rapid and simple techniques. this method could be a convenient tool to observe the effect of bioactive substances such antibiotics. we show in this poster an exemple with the action of penicillin on b. subtilis. within the family trypanosomatidae, leishmania is a protozoan pathogen producing a broad spectrum of human disease. its life cycle is divided in two stages. promastigote is the flagellated form which colonizes the gut of the sandfly vector, and amastigote is the non-flagellated form that is specialized for growth and survival in the vertebrate host macrophage. to understand gene regulation during the transition between the promasttgote and the amastigote stage, we were interested in the characterization of stageregulated genes. we isolated a gene, sw3, from leishmania major whose mrna is more abundant in amastigote compared to promastigote. structural analysis of this gene showed that an additional polypeptide could be encoded by the opposite strand of sw3. we confn'med the presence of an open reading frame on the opposite strand by in vitro translation of the sw3 antisense rna. in vivo, an amastigote sw3 antisense transcript and the corresponding polypeptide were also detected suggesting that the sw3 is transcribed in both directions and that stage specific transcripts and polypeptides could be produced. analysis of other gene segments tend to indicate that an intensive usage of the leishmania genome to produce various polypeptides could be a general phenomenon in trypanosomatidae. the four variants and / or posttranslational modifications of histone h1 of procyclic trypanosoma brucei brucei (protozoa, kinetoplastida) were purified by hplc reversed phase chromatograpy and characterized by their amino acid composition and partial amino acid sequence. differences in sequence of up to 45% as compared to histone h1 of higher eukaryotes exist. alkaline phospatase digestion and analysis of h1 by means of hpce was used to demonstrate the presence of phosphorylated modifications. the unique biochemical properties of h1 of t. b. brucei contribute to the structural differences seen in the chromatin of the parasite as compared to that of the higher eukaryote host. larvae of the parasitic wasp chelonus inanitus (braconidae, hymenoptera) develop inside embryonic and larval stages of the host spodoptera littoralis (noctuidae, lepidoptera). parasitism induces in the host a precocious onset of metamorphosis and developmental arrest in the precocious prepupa. the wasp injects, along with the egg, pdv and venom into the host egg. injection experiments with pdv and venom revealed that pdv play a crucial role in altering host development and in suppression of the host's immune system. the genome of the pdv of c. inanitus consists of at least i0 segments of double-stranded circular dna ranging in size from 7-30 kb. we analyzed the expression of viral genes in the course of parasitization with cdna made from mrna at various developmental stages of s.littoralis. we also investigated the localization of the expressed viral genes on the various segments. in addition, these bacteria were isolated and cultured from brain tissue. to identify the infectious agent we used, among other techniques, an atomic force microscope (afm). when the. isolation fluids derived from the cortex of three alzheimer's cases were examined with afm and scanning electron microscopes, we observed bacteria whose size and morphology fit to the order of spirochaetales. these images are presented and discussed on our poster. ~ottstein bruno, institute of parasitology, ch-berne. %lveolar echinococcosis ae is due to infection with the metacestode (larval stage) of echinococcus multilocularls. an immunodominant antigen em2 of e. multilocularis was characterized by mab-gii, respective studies revealed, that (a) antigen expression metacestode stage-sdeci-fic and (b} that expression was restricted to the laminated layer. the em2-antigen i s a lectin-binding carbohydrate linked to a lipid residue. the "em2positive" laminated layer proved to be a crucial factor in protecting the parasite from host immune-effector mechanisms: only e. multilocularis larval structures exp ressing em2 were able to induce secondary alveolar chinococcosis in rodents. subsequent experimental studies in resistant (c57bl/10) versus susceptible (c57bl/ 6j and akr) mouse strains showed that resistance was as-sociated with synthesis of anti-em2 igg 3 and igg i. sus-ceptibility of c57bl/6j-mice was associated anti-em2 s and no igg 3 . the parasite-specific in vitro lym-~hoproliferative i~une response remained stationary ~ith time after infection in c57bl/10 and decreased in r and aki< mice. day 30 p.i. cd4 ⧠-lymphoblast cells dominated in all meuse strains; day 90 p.i. an in-creased nu/0ber of cd4-/cd8 + and cd4+/cd8 + cells were observed. only susceptible mice exhibited a significantly ~eereased response of lymph node cells to cona-stimula-~ien with time p.i. in conclusion, subtypes of parasitespecific cellular and humoral host immune responses seem leishmania species are protozoan parasites causing a spectrum of clinical manifestations in man ranging from cutaneous lesions to morbidity and death. the insect stage (promastigote) and intracellular stage (amastigote) differ in terms of morphology, physiology, antigenicity and gone expression. it is clear that the amastigote stage is uniquely adapted for survival within the inimical environment of the macrophage phagolysosome and that mechanisms regulating these adaptations must be called into play during the early stages of infection when the parasite undergoes transformation. in an effort to elucidate these regulatory mechanisms, we have constructed a "subtracted" cdna library which is specifically enriched for parasite transcripts expressed during the first 7 hours of infection ("switch" genes). several clones have been characterized by sequence, northern, and southern analysis. three clones, sw3, sw44, and sw20, are expressed at a several-fold greater level in the amastigote stage and potentially encode proteins which interact with nucleic acids. sw3 predicted protein is homologous with histone h1 proteins and sw44 and sw20 potentially encode ribosomal proteins. analyses of transcripts from this library are yielding clues not only to mechanisms underlying the regulation of parasite transformation, but also insights into post-transcriptional and translational control of gene expression in leishmania. $16-17 microbiology, university of geneva, medical school, c.m.u., 9 avenue de champel, 1211 geneva 4. a viral rna representing the sequence of a defective interfering rna, naturally selected for efficient replication, i.e. only containing the replication promoter, was cloned under the control of the t7 rna polymerase promoter. the expressed 17 transcript was then replicated in rive by supplying in trans the replication functions, equally expressed from plasmids. this system, completely accessible to genetic manipulations, allowed us to define a basic rule directing sendal virus replication, the "rule of six" (calain and roux, j.virol. 67 : 4822-4830, ig93). a second defective rna, representing a shorter version of the viral rna, in that it contains not only the replication but also the transcription promoters, was then cloned and replicated in the same system, although with lower efficiency. the replication of this "transcribing" viral rna was found to obey the "rule of six" as well. replicationitranscdpfion promoters were then achieved to define the cissequence requirements needed for efficient replication or for replication /transcdption. results obtained with these chimerical viral rnas will be presented and their contdbufion to the comprehension of the paramyxovirus replication and transcription processes bovine t-cells that become infected with the intracellular parasite theileria parva undergo lymphoblastoid transformation and acquire the ability to proliferate continuously. they cease to require specific antigenic stimulation, become independent of exogenous growth factors and cause tumors in nude mice. the il2 and il2r genes are constitutively expressed and the transcriptional activator nfwd3 which regulates these two genes is continuously activated. these processes are all strictly dependent on the presence of the parasite in the host cell cytoplasm and cease upon the specific killing of the parasite by the theilericidal drug bw720c. we have shown that the presence of the parasite results in the activation of the protein tyrosine kinases fyn and ick, which can participate in early t cell receptor signalling, suggesting that the parasite may use this signal transducti0n pathway to induce continuous proliferation. cysteine proteinases of leishmania as targets for chemotherapy. jacques bouvier*t and judy sakanari*. *department of pathology, ucsf school of medicine, san francisco, ca, and tanimal health, ciba-geigy, ch1566 st. aubin, switzerland. the overall goal of the study is to apply the structure-based approach to develop lead compounds and design novel enzyme inhibitors as potential therapeutic agents against leishmaniases. in this regard, we have identified and characterized the cysteine proteinases of leishmania major, and showed that they consist of excellent targets for drug design. we have purified a membrane-boand cysteine pmteinase not previously described in other trypanosomatids or protozoans and obtained amino acid sequence information from the purified enzyme. in addition, we have also characterized and purified the soluble cysteine proteinases of the parasite. consequently, we have isolated two genes encoding the soluble and membrane proteinases. the soluble cysteine proteinase gene occurs in multiple copies of 2.9 kb with flanking regions of 1.2 and 2.2 kb and is localized on one chromosome of approximately 440 kb. the membranebound enzyme gene is 3.1 kb and occurs as a single copy on a chromosome of approximately 1100 kb. a three dimensional computer model of both genes wiu be generated and enable us to scan the vast chemical database for new lead componds. we akeady have identified several cysteine proteinase inhibitors that are effective compounds in killing both promastigote and amstigote stages of the parasite in the micromolar range in vitro without affecting the host cells. dollenn~ier, g., cassinotti, p. and weitz, m., institute for clinical microbiology and irananology, 9000 st .gallen/swit zerland hav is a ~r of the picornaviridae. its rna gencrne rf~y be divided into 3 coding regions (pi, p2 and p3), qhe p3 region contains a protease 3cp ro that generates mature proteins by cis-cleavage of the initial polyprotein precursor. catalytic activity of 3cp r~ has been d6~aonstrated in a vacciniavirus/t7 ~qa-polymerase hybrid expression system. (balouter aided ali~ts of hav 3cp r~ sequence with that of other picornaviruses imply that amino acid residues his-44 and/or h) the complete hav open reading frame, hav specific expression products were identified by radioj_rmmnoprecipitation and lyy sds-page. the analysis with an antibody specific for putative nonstructural protein 2b revealed a 27,5 kda peptide. this is in contrast to its predicted size (12 kda). however, analysis with anti-(putative)2a antiserum confirmed an upstream location of the 2b n-terminus. the new 2bpeptide was also identified in lyeates of hav infected mrc-5 cells. hence, recombinantly expressed hav polyprotein underwent authentic processing and the predicted p2 organization has to be modified. we are interested in the development of animal models for one of the two major pathological changes found in the brains of patients with aizheirnar~s disease, the formation of fleuroitbrillary tangles, a main component of which is abnormally phosphorylaled tau-protaln. (tau itself is a protoin associated with mlcrotubuli.) two fundamental questions may be answered with our models: first of nit we woald like to reproduce the formation of tangles, and secondly we hope to answer the question, whether the formation of these pathological structures is sufficient for the bevalopment of senile dementia ot the alzheimer type (sdat). at present there are only a tow putative candidate genes known which may be in-voned in the hyperphosphor~lation ot tau in v/vo, one of which is the map-kinase erk2. we have cloned this candidate kinase from rat txaln rna via an rt-pcr approach and expressed the cona under,the co.rat of tour different promoters in transgenis mice. these promoters were tested for maximal expression. a similar approach was chosen to express the human taut0 isofonn in the brain ot transganic mice. we have stated to analyze different tau an~ erk2 expressing lines, northern blot analysis has revealed high levels of expression for all transgenes (up to fnetoid {n comparison to endocjenous levels). the neuronal spectficry of the transgenio erk2expression was c~nfirmsd by in situ hybridization analysis. ir~ addition several tauexpi'esalog tines were analyzed in wealernblots to determine the relative amount of htau= protein in the brain. sections of the brain were stained with tau-sp~c~c ardib~iies to identify the neurons expressing tau. from intemrosses of tau-with erk2-expressing lines, we have already identified double-positive mice which we currer~y analyze by immunohistocbemistry and in western nots to examine the phosphorylaiton status of tau. we are well aware of the possibility that only animals trans~enic for both tau and erk 2 might beveiop tang{es, whereas single transgenics mlsht not. pairs of individual neurons were recorded with sharp electrodes or in whole-cell configuratio~ in hippocampal slice cultures of rat. synaptic connections were studied between pre-and postsynaptie cells in ca3 and ca1 hippneampal fields by eliciting single aetinn potential in the presynaptic cell. monosynapfic excitatory connections were highly probable between ca3 and ca1 pyramidal ceils (p=0.76, n=82) with almost no feed-forward inhibition (p=0.01). by contrast, disynaptic ipsps, blocked by cnqx or bieuculline were observed with a very high probability (9--0.6) between ca3 pyramidal cells. monosynaptic excitation was found ha 56% of cases between ca3 neurons (n=43) but only in 27% of cases (n=ll) between two ca1 cells. monosynaptie ipsps with very short lateneies (<lms) and blocked by bicuculline, but not by cnqx, were recorded within area ca3 (5/6) and within cai (2/3), but not between ca3 and cat fields (2/2). probability of transmitlzr release, studied in recordings where failures could be unambiguously distinguished from spontaneous or small psps, was found to be close to 100% at both excitatory and inhibitory synapses. specific inhibitors of presynaptie release at excitatory and hahibitory terminals (i.e. adenosine and opioid peptides, respectively) gradually reduced the anaplitude of the psps indicating that the release at excitatory and inhibitory unitary synapses is multiquantal. the cns of a neonatal opossum, monodelphis domestic& shows profuse growth of fibers across a lesion. as in other mammals, such repair is not seen in adults. experiments have been made to define the age of the animal and stage of development at which repair ceases to occur. the entire cns was removed from animals aged 3-6 days or 11-14 days after birth. the spinal cord was crushed and growth of fibers assessed 5 days later by electrical recording of impulses conducted through the crush. repair after lesions to spinal cords of younger animals occurred in 38% of the trials (45 animals, aged 3-6 days). in nervous systems from older animals (11-14 days after birth) the success rate was low (only 4 out of 38 preparations). similarly, the carbocyanine dye dil labeled numerous fibers in spinal cords of younger animals (12 out of 21), whereas only one preparation out of 15 showed scant outgrowth into lesions in the older animals. our results suggest that there is a critical period for neuronal repair; at about 12 days nerve fibers lose the ability to grow across lesions. it is an advantage that at this later stage the nervous system is still small enough to survive in vitro. features correlated with these changes are oligodendrocyte differentiation and myelin formation. with immunofluorescent staining as well as electron microscopy myelination becomes apparent at 8 days after birth. we are now using time lapse-videomicroscopy to analyze molecular mechanisms that could play a part in promoting or preventing repair. supported by grants to j.g.n from the swiss nationalfonds (31-3626292) and from the internat. res. inst. for paraplegia. i. impulse conduction in axons has integrative functions. the dorsal root ganglion (drq) of the embryonic rat put into culture forms a monolayer and is thus accessible to eonventionalmieroelectrode technique, which allows testing the above hypothesis. we have chosen a dual approach by correlating ext~rimental el~ctrophysiological data from the chlture with results obtained from a compartrhental computer model. ii. the safety factor for conduction was found to be low. thus failures of ap invasion of the drg cell soma could occur at sites of impedance mismatch when a second stimulus felt into the relative refractory period of the first or when the axon was repetitively stimulated. the electrotonic resiuues (e.rs) of the failed aps had discrete amplitude levels, suggesting that the failures were always caused at the same site along the axon. these sites of low safety factor were found to be the branch point in the unipglar drg cell and the entrance of the stem piece into the soma in both cell types, the bipolar as well as the unipolar. a systematic comparison of 15oth cell types showed that the ap conduction through the latter is more reliable.'the length of this stem piece is inversely' correlated to the delay caused at the branch point, as tile electrical load of the soma is more efficiently masked by a long stem piece. the dendrites of motoneurons (and subsequently many other neurons) have been assumed to be electrically passive since the work of coombs, ecc]es and fatt in the 1950's. however, direct evidence has been impossible to come by until recently with the advent of appropriate dyes and equipment for measuring very small and very fast optical signals. we examined the propagation of action potentials, both spontaneous and evoked, in the dendritic trees of spinal cord neurons using a 12 â�¢ 1 2 array of photodiodes and a ccd camera as weft as conventional microelectrode techniques. organotypic slice cultures of embryonic rat spinal cord were stained with a voltage-sensitive dye (di-8-anepps) or a calcium dye (fluo-3) and recordings were made from motoneurons identified by their location and morphology. the results indicate that there is in fact an increase in calcium in the dendrites that accompanies action potentials whether synaptically evoked or evoked by current injection at the soma. spikes in the dendrites up to 1 60 i~m from the soma are much larger than could be explained by models assuming passive dendritic trees. work is currently underway to test the possibility that sodium conductances might contribute to the propagation of action potentials in the dendrites. cat auditory cortex contains multiple cochlear representations in both hemispheres that are interconnected through the corpus callosum (cc). the distribution of auditory callosal axons was studied on the mediosagittal level after injections of biocytin at electrophysiologically identified locations. single injections were done in ai (2 cats), aaf, aii and paf; multiple injections in ai & aaf in one cat. an additional cat received 1 injection in sii. the mediosagittal image of cc was subdivided into 30 segments along a rostro-caudal axis. labelled axons crossing the midline were counted in coronal or horizontal sections. axons from auditory fields were found in the posterior 2/3 and those from the somatosensory cortex in the anterior third of cc. axons from a discrete injection (ca imm in diameter) spread over as much as i/5 of cc. the rostrocaudal distribution of axons at the midline reflected roughly the rostrocaudal position of the field of origin, but apparently not the tonotopic order of a given field. the major efferent projections of substantia nigra pars reticulata (snr) convey information to the thalamus, to the tegmentum, to the superior colliculus, and to the spinal cord. such a range of targets suggests a highly organized activity within snr and this study investigates the temporal organization of spike trains. in the portion of snr between +2.7 and +4.5 mm interaural levels, we recorded 40 single units along 6 electrode tracks in 4 young female wistar rats. most units (n=32) fired in a non-regular poisson process, although only a small number (7/32) was characterized by an average burst size with more than 3 spikes. crosscorrelograrns were computed for 25 pairs of units simultaneously recorded from the same electrode and for 26 pairs simultaneously recorded from distinct electrodes. most crosscorrelograms (27/51) showed a symmetrical peak centered on time zero, which indicates a tendency to synchronous firing. when units were recorded from the same electrode tip we observed that 1/8 to 1/40 of their spikes were synchronous. about half of the significant interactions were characterized by a relatively long duration (>250 ms) and are likely to correspond to a different mechanism of synchronization. these results support the hypothesis that snr cells receive common afferences of two different types and their fine temporally organized activity yields coordinated activity between the target structures. the neuronal microtubule-associated protein map2 binds to microtubules via a domain containing 3 or 4 repeats of an 18 amino acid sequence. we have previously shown that when cultured nonneuronal cells are transfected with map2 their microtubules become stiffer and are then capable of supporting process outgrowth when their cortical actin cytoskeleton is depolymerised. we hypothesise that this stiffening effect of map2 is caused by the 18 amino acid sequences binding to neighbouring tubulin subunits in the walls of the microtubules thus reducing their freedom of movement relative to one another. to investigate this further we have created map2 mutants in which 1 or 2 repeats of the tubulin-binding motif were deleted. these were tested by transfection and expression in cultured cells. the results confirm that the number of repeats affects the stability, stiffness and the cytoplasmic arrangement of cellular microtubules. we were previously able to demonstrate the presence ot kinin-like immunoreactive structures in the mouse brain. in order to characterize them, we have now performed a donble-immunofluorescence study using both polyclonai auti-srudykinin (bk) and mouse monocloual anti-neeron-specificenolase (nse) antibodies. the latter is considered to be a specific neuronal marker. after fixation with bonin-hollande solution, the brains were removed, embedded in paraffin and serially sectioned at 10 am. the sections were incubated with the antibodies, which were visualized by an indirect immunofluorescence technique using fitc for nse and by an avidin-biotin technique using texas red for bk. double-immunofluorescent cells were found in the thalamus ventralis, in the nucleus cechleuris ventralis and in the nucleus mesencephaficns nervi trigemini (v). bk-positive cells in the nucleus principalis v were nse-negative ih spite of their typical neuronal aspect. the other bk-positive structures (pia, ependyma, hnic~es) also were alwa~ nse-negatlve. most of the nsepbsifive bk-negative cells were seen in the cortex. the neuronal nature of many kinin-like immunoreacfive structures in the mouse brain is thus ascertained. in contrast to the amphibian optic nerve (on), on of adult mammals is unable to regenerate after injury. astrocytes disappear from and macrophages accumulate at the lesioned site (blaugrund et al., j. neurocytol, 118, 105, 1992) , we have followed the distribution of astrocytes and microglial cells in xenopus tadpole ons over 10 d after mechanical crush. astrocytes were stained for cytokeratin and vimentin, and microglial cells for griffonia isolectin b4. soon after crush, immunoreactivity for intermediate filament proteins was strongly increased. astroeytic processes extended into the lesion from both proximal and distal stump. at ,5 d, astrocytes had crossed the injured region. in the distal stump, some of them contained vacuoles indicating phagocytic activity. while in the normal on microglial cells were ramified and scarce, 2.5 d after crush they were roundish and vacuolized, and scarce within the lesion but numerous distal to it. at 10 d, their number was decreased and most of them had reacquired ramifications. conclusively, in the tadpole on, astrocytes are virtually never absent from the lesion site but rather extend rapidly into it. astrocytes may thus provide a guiding support for outgrowing axons, microglial cells that are frequent in the distal stump, do not accumulate at the lesion. the behaviour of the two cell types differs profoundly from that in mammals and is likely to be one of the factors that may contribute to successful neuronal regeneration of the amphibian on. ci4mence, j. f. 1, ranieri, j. p.2, aebischer, p.2, and sigrist, h. 1, 1institute of biochemistry, university of berne, ch-3012 berne; 2division autonome de recherche chirurgicale, chuv, ch-1011 lausanne. small peptidic sequences of laminin (yigsr, ikvav) are known to promote attachment and/or neurite outgrowth of various neuronal cell lines (pc12, nb2, ng108-15). new and established heterobifunctional photo crosslinkers were used to immobilize these peptides in a topically defined fashion onto biocompatible surfaces such as hydroxylated fluorinated ethylene propylene (fep-oh) and pely(vinymlcohol) (pva) . n-[m[(3-trifluoromethyl) diazirine-3-yl]phenyl]-4-maleimido-butyramide (mad) was thermochemically linked to the synthetic peptide cdpgyigsr via its n-terminal cysteine, leaving the bioactive part (yigsr) free for cell receptor interaction. mad-cdpgyigsr was radioactively labeled by reductive methylation and purified by reversed phase hplc. patterned (20 and 300 #m) peptide immobilization was attained by photocoupling onto pva and fep-oh and visualised by autoradiography. light-structured biomaterial surfaces with molecularly oriented nerve growth promoting peptides were investigated for spatially controlled neuronal cell attachment and differentiation. it is striking to observe that these inhibitory or stimulatory effects were corroborated by binduced decrease or increase in 2-deoxyglucose uptake, respectively. this gives b a putative role in the limbic regulation. the sheet-like processes of oligodendrocytes wrap themselves spirally around central axons, thus formtng the myelin sheath. a single oligodendrocyte may donate internodal segments of myelin to each of 20-30 or more adjacent axons. it is not known, if one oligodendrocyte picks out axons randomly or if it prefers axons with a certain diameter or with a certain chemical specificity. we have studied this last possibility by combining intracellular injection of a fiuorochrome, and immunohistological detection of calciumbinding proteins (cabp's), markers for the axons ef certain subpopulafions of nerve ceils. lucifer yellow was injected into oligodendrocytes of the optic nerve, of living rat brain slices. the oligodendrocytes were identified by their electrophysiologieal properties. slices containing the iniected cells were immunostained /or parvalbumin or calretinin using second antibodies labelled with texas red. using co.focal laser-scanning microscopy combined with a three*dimensional reconstruction programm, the relationship between oligodendrocyte processes and axons positive for one of the cabp's was determined. for ultrastructural confirmation, single, lucifer-yellow injected, oligodendrocytes were photoconverted, and the cabp's-positive axor~ were revealed by gold-labelled antibodies. the few oligodendrocytes injected show the classic morphology, that is of a small cell body and few processes running parallel to the course of the axons. the proximity between glial processes and positive axons can be easily appreciated in confocal laser-scanning images. a preferential assodation of oligodendrocyte processes with parvatbumin-positive axons has not as yet been found. thus, the confirmation of the hypothesis that oligodendrocytes choose their axonal targets according to chemical cues awaits further work including the injection of a lar~er number of these cells. to initiate a molecular genetic analysis of brain development in insects, we are characterizing the mechanisms of embryonic brain development in the fly drosophila. early in embryogenesis, a small number of molecularly diverse brain neuroblasts generates the neurons of the brain. subsequently, pioneering brain neurons initiate axonal outgrowth along glialbound brain compartments. these pioneering neurons establish the primary brain tracts. the pioneering brain axons express the cell-cell adhesion molecule fasciclin i dynamically during outgrowth and initial fasciculation; a subset of the pioneering axons expresses fasciclin ii. the axonal framework of the embryonic brain tracts is used for outgrowth and fasciculation by subsequently differentiating axons and, thus, prefigures the tracts of the mature brain. a comparison of early brain development in drosophila and in vertebrates reveals common mechanisms for brain development. supported by the swiss nsf. institute ot histology, university of fribourg, p~rolles, ch-1700 fribourg calretinin (cr), an ef-hand ca2+-binding protein, is expressed in cajai-retzlus cells during development of the rat cerebral cortex. these cells are located in the marginal zone and are the ear{iest cortical neurons to be generated. they have been consldered as unusual on account of their morphology and their fate during corticogenesis. the functional significance of cajai-retzius cells and their destiny in adulthood is still controversial. in order to approach these questions we have applied organotypic culturing methods. slices of neocortex from brains of 0-6 days-old rat pups were cultured for 10-21 days applying the interphase technique (stoppini etal, j. neurosci. methods 37, 1991) or the roller-tube technique (g&hwiler, j. neurosci. methods 4, i981) . the fixed cultures were immunolabe0ed with an antibody against cr. the obtained results showed that cr positive structures develop in vitro like in vivo. however, in organotyp[c cultures their distribution corresponded to a more mature stage than the situation on the day of the explantations. many cr-positive cells were seen in layer l they were horizontally oriented or had a pyriform shape. based on their morphology these ceils were identified as cajai-retzius cells. orpositive cells were numerous in layer i1-l[i and showed mostly a bipolar morphology. some muripo[ar cells could also be observed. a fine net of crpositive fibers and puncta was found in layers [ and iv. the demonstration that cajai-retzius celts are detectable in these cultures will allow us to follow the fate of these cells dynamically and [nte~ene in their function by applying exogenous factors. cervical primary afferent input to vestibule-spinal neurones projecting to the dorsal horn: a double labelling study in the rat. wheat germ agglutinin-horseradish peroxidase (wga-hrp) was injected by pressure into cervical spinal ganglia c2 or c3. in the same experiments fluorogold (fg) was iontophorefically injected into the ipsilateral dorsal horn of different cervical spinal cord segments. anterogradely labelled fibers (wga-hrp) were present mainly in the external cuneate nucleus, but also to a lesser extent in the caudal part of the medial and descending vestibular nuclei (mvn, dvn) . the fg injections, restricted to the cervical dorsal horn, revealed retrogradely labelled cells in the central part of the caudal mvn. a double exposure (fluorescent and polarisadon optics) under the microscope showed primary afferent fibers surrounding like baskets retrogradely labelled fg cells. the projection from cervical ganglia cells to the mvn represents a pathway for direct afferent information from neck receptors (in particular muscles) to vestibular nuclei and supplies the mvn with afferent information which could enable the vestibulospinal nenrones, projecting to the dorsal horn, to influence the local information processing. in the sciatic nerve, all myelinated and non-myelinated axons were snap-ir. in peripheral tissues, all nerve endings were positive. the preterminal schwann cells of motor axons were also snap-25-ir. after sciatic nerve section, many myelinating sehwann cells also expressed snap-25-ir. in conclusion, snap-25 is expressed by neurons in the pns. it seems to be also expressed by preterminal schwann cells and by myelinating schwann cells during regeneration. $17-19 repair of completely transected spinal cord of neonatal opossum in culture h.a. vischer, dept. pharmacology, 8iocenter, uni. of basel, switzerland woodward et al. (j. exp. biol. 1993 , 1993 have shown that fibers grow rapidly and profusely across a lesion made by crushing the spinal cord of the neonatal opossum monodelphis domestica. in such experiments careful controls must be made to establish that fibers were in fact broken by the lesion. tests have now been made to determine whether similar repair can occur after a more drastic lesion involving complete transection of the cord. after dissection of the entire gns from animals aged 3-8 days, the cord was cut into two with scissors at the c5 -c7 level. the two-ends were glued together with matrigel on a sylgard mold, which encompassed and held the cns. the rostral end was labeled with the fluorescent carbocyanine dye dil in order to visualize growing fibers. in 33 preparations fibers grew over the cut into the separated part of the spinal cord. such growth could extend to 500 ixm. fibers grew straight, branched and multidirectionally, or in fascicles. in another set of experiments cords were combined from different animals of the same or different ages. again, fibers could grow across the cut but they did so less frequently. these results set the stage for investigating fiber growth after rotating the two ends at a defined angle and for analyzing factors that influence the direction of growth. map la is a microtubule associated protein of 360 kd. in rat brain two monoclonal antibodies, a and bw6, recognized map la in neurons and their axonal and dendritic processes. in mouse cerebellum, monoclonal a recognized purkinje ceils and granule cells uniformly, as already described for rat brain. in contrast, monoclonal bw6 stained selectively some dendrites of purkinje cells, forming bands in the molecular layer. we compared this striation with that obtained with a monoclonal antibody, anti-zebrin ii, which recognized aldolase c. in the mouse vermis, zebrin stained in a striated pattern, but complementary to bw6. these results demonstrate that map la is present in forms which are differentially distributed in the mouse cerebellum. these observations may be explained either by differences in metabolic states of neurons, or by differences in their regional functions, or by differences in the regional stability of microtubules. this work was supported by grant no 31-33447.92 from the swiss national science foundation. serum free aggregating brain cell cultures prepared from 16 day old fetal rat telencephalon are used as a model to study brain development. in these cultures it is possible to distinguish ontogenetic events such as cell proliferation, differentiation and myelination, in the present study we examined synaptogenesis by analyzing the expression of synaptic proteins such as synaptophysin, snap-25, synapsin i, and brain spectrin. different developmental stages were analyzed by western blotting. immunoreactivity for synaptic proteins was detectable already at day 12 in culture, suggesting that the first synapses are already present at this early stage. the expression of synaptic proteins strongly increased between day 20 and day 30 in culture, reflecting a period of intense synaptogenesis. treatment of the culture with an elevated concentration of kci (30mm) increased the expression of synaptic proteins, suggesting a stimulation of synaptogenesis. these findings demonstrate the utility of this approach to study brain synaptogenesis, and possibly synaptic plasticity. drenhaus, u.i gunten, a.v., rager, g. ; institute of anatomy, university of ch-1700 freiburg the retina of tupaia comprises three types of ganglion cells (rgcs) analogous to x-, y-, and w-cells found in other mammals. these cells differ in size and in their distribution pattern: large rgcs are frequent in temporal, small rgcs in nasal retina. as the fiber diameter correlates with the size of its respective rgc, it can serve as a parameter for the topographic representation of rgcs in the nerve. to study this we analyzed the optic nerves of three tupaia. using the electron microscope we recorded the density, diameter, and the position of fibers in the nerves. we found, that fibers with the largest diameter are located dorso-temporally and close to the center of the nerve. they are surrounded by zones of smaller fibers. the diameter decreases gradually towards the periphery and is smallest in the ventro-nasal region of the nerve. since the fiber diameter distribution corresponds to that of the size of rgc-types in the retina, we assume that the topographic relationships in the nerve and the retina are similar. (supported by s 17-23 stoppini luc, s. duport, l. parisl, c. oropes~, departement of pharmacology, cmu, 1211 geneva 4 the micro environment of the central nervous system is important for neuronal function. in this context, the blood-brain (bbb) provides and maintains the extracellular medium that is compatible with normal neuronal and synaptic activity. due to the difficulties inherent using whole animal as an experimental model to study permeability and metabolic processes at the cellular level, major efforts have been engaged in recent years, in order to bring up suitable /n vitro models. we are presently developping an in vitro bbb by interfacing a coculture of endothelial cells monolayem and nervous organotypic cultures. the main idea of this study is based on the premise that the complex intercellular interactions between the different cell types pertaining to the nervous tissue and the neighbeurlng endothelial cell is of fundamental importance to promote a realistic bbb system. we expect that erganotypic culture of nervous tissue, combined to endothelial cell monolayers, will initiate and maintain a bbb phenomena/n vilro. we will test more specifically the hypothesis that neuronal activities [spontaneous or elicited) will influence gila] cell response which will in turn modify endothelial properties. preliminary results clearely show a good nervous tissue and endothelial cell survival. tight junction llke structures could be identified using freezefracture or conventional transmission electron microscopic thechniques. {work supported by chemodyne gent~ve ). the process of myelination is a prerequisite for the proper function of the brain since it enables rapid saltatory conduction of axons. in the central nervous system (cns) myelination is performed by oligodendrocytes. a differential screening approach was used to isolate new rat cdna clones that are expressed in this glial cell type. here we report the further characterization of two of these novel cdnas which appear to be expressed specifically in oligodendrocytes. the two characterized cdna clones (tentatively called cns1 and cop-25.1) share an approximately 420 bp region of sequence identity which suggests that they are dedved from the same gene by alternative splicing, within this area a putative open reading frame is located. we demonstrated by northern blot analysis and in situ hybridization that the two mrnas of the novel gene are expressed exclusively in oligodendrocytes. however, the mrnas show a different specific spatial localization within the cell. we compared mrna expression of myelin basic protein (mbp) and proteolipid protein (plp), with that of cns1 and cop-25.1 in brain and optic nerve during development. it appeared that the mrnas of the novel gene were delayed significantly as compared to mbp and plp mrnas. streit. phyaiologisches institut, btihlplatz 5, 3012 bern in organotypic slice cultures of the embryonic spinal cord, rhythmic spontaneous activity arises when the inhibitory synaptie transmission is blocked. the rhythmic activity consists of bursts of regular oscillations of activity at 4 -5 hz. this study investigates the origin of the regular oscillations. when stimulated electrically at 5hz, the synaptic transmission in the cultures showed strong depression by about 50% on average. the synaptie depression was highly variable among individual connections and tended do be less pronounced in frequently used connections. when random depression ratios with an average of 50% at 5hz were implemented in a computer model consisting of 100 randomly connected excitatory cells, regular oscillations of activity did not arise, even in the presence of synchronous pacemaker cells. however, when individual depression ratios were adapted according to the rate of activity of individual connections, regular oscillations at 4 -5 hz arose in the presence of few unsynchronized pacemakers. this finding suggests that the regular oscillations in the cultures originate in a network formed by the plasticity of synaptic depression. maier a., schlumpf m., beer h.f., sehubiger p.a. and lichtensteiger w., inst. of pharmacology, univ. of zurich, the ontogeny of expression of dopamine d 1 receptor mrna in the rat brain and binding to the receptor was studied at 12 time points from gestational day (gd) 13 to postnatal day (pn) 60 by quantitative receptor autoradiography and in situ hybridization.long evans rat pups and fetuses of time pregnant rats were frozen and sectioned coronally and sagitally at i0 um. d 1 recepto~o~inding , determined with the selective antagonist ~ji-sch 23982 was first noted on gd 18 in the developing striatum, basal ganglia and the olfactory tubercle, reaching adult levels at around pn 14. the expression of d i receptor ~na was studied by using a mixture of 3-~js-labelled oligonu-specific cleotides. on gd 13 messages were noted in the developing stiatum, olfactory tubercle and retina. this study shows a good regional correlation between the development of receptor binding and mrna, however, mrna precedes detectable binding to the receptor by several days. earlier work had shown that q~ replicase forms complexes with q~, plus strand rna via interactions at two internal binding sites, the s-and the m-site, while binding of the 3'-end is mediated by host factor. in contrast, the 3'-end of the minus strand appeared to be directly accessible by replicase. we have prepared a series of plus and minus strand variant rnas containing either internal deletions or terminal deletions extending from the 5'-end. the template activities of the variants were determined by single-round replication assays. for the plus strand we find that while deletion of the s-site remains without effect (as expected from previous results), deletion of the m-site reduces template activity to 25 % or less compared to wild-type rna; the residual activity shows a decreased dependence on the presence of host factor. the results agree with an important role of the m-site interaction both with replicase and with host factor for the formation of productive initiation complexes. the template activity of the minus strand was unexpectedly found to be strongly dependent on the presence of a segment between nt 76 and 210 from the 5'-end. this shows that recognition of the minus strand by rep(icase does not only involve interactions near the 3'-end but requires a previously unknown structural feature near the 5'-end of this template. $18-02 karsten graning and angela kr&mer d~partement de biologie cellulaire, universite de gen~ve, 30, quai emest-ansermet, 1211 gen~ve 4 splicing of nuclear pre-mrnas takes place within multicomponent complexes (spliceosomes) that are assembled by interactions between the pre-mrna, snrnps and protein splicing factors. we have purified two splicing factors that function in the formation of the pre-splicing complex. sf3a consist of three subunits of 60, 66 and 120 kda and corresponds to three proteins present in the functional 17s but not in the 12s u2 snrnp. it binds to the 12s u2 snrnp only in the presence of sf3b, suggesting a two step assembly pathway of 17s u2snrnp: binding of sf3b to the 12s u2 snrnp generates a 15s intermediate particle that is converted to the active 17s u2 snrnp by the subsequent association of sf3a. comparison of proteins enriched in the sf3b fraction with polypeptides found in the purified 15s u2 snrnp suggests that sf3b comprises at least five of the other 17s u2 snrnp specific proteins and together with sf3a promotes the u2 snrnp/pre-mrna interaction. by edna cloning, two of the subunits of sf3a have been identified as homologs of well characterized yeast proteins that function at the same stage of spliceosome assembly in yeast. splicing factor sf1, a heat-stable 75-kd protein, functions early during spliceosome assembly. tryptic peptides of sf1 were sequenced and cdna libraries were screened with degenerate oligonucleotides. the information obtained by comparing the sequences of three overlapping cdnas suggests the existence of at least three mrnas that could be derived from a common pre-mrna by alternative splicing. in agreement with this assumption mrnas of the expected sizes that are differentially expressed depending on cell line or tissue are detected by northern blotting. in theory, three polypeptides could be generated that differ by the presence or absence of 7 internal amino acids and in their ctermini. the deduced amino acid sequence is rich in prolines and contains several possible phosphorylation sites and a putative leucine zipper. proline-rich domains, which are also present in splicing factors psf and sap62, as well as leucine zippers have been found in transcription factors and could mediate protein/protein contacts, suggesting that sf1 could function during splicing by interaction with other spliceesomal proteins. pre-mrna splicing is catalyzed by a multicomponent complex (the spliceosome) that consists of small nuclear ribonucleoprotein particles (snrnps) and non-snrnp protein factors. the spliceosome is assembled in a stepwise fashion on the pre-mrna. chromatographic fractionation of hela cell nuclear extracts and subsequent reconstitution of splicing in vitro has allowed the separation and isolation of snrnps and several protein factors. sf4 triggers the transition between splicing complex b, which contains all spliceosomal snrnps but is not competent for catalysis, and complex c, the active spliceosome. this transition involves a conformational change that leads to altered base pairing interactions between snrnas and pre-mrna. the purification of sf4 has been impeded by its low abundance; however, a correlation between sf4 activity and a polypeptide of 50 kd could be established in the most purified fractions. we are currently investigating whether this protein represents sf4. interestingly an atp-dependent rna-helicase cofractionates with sf4 through several purification steps. whether or not this activity is relevant for the splicing process is under investigation. selection of iron-responsive element rnas with high affinity for iron regulatory factor henderson, b.r., menotti, e., bonnard, c. , and kith_n, l.c., isrec, ch-i066 epalinges iron regulatory factor (irf) post-transcriptionally regulates iron homeostasis via binding to mrna iron-responsive elements (ires). the ire loop sequence (5'-cagugn-3') and "bulge" cytosine are phylogenetically conserved. we prepared a pool of 16,384 ire molecules randomized at these 7 nucleotide positions, and employed in vitro selection to identify optimal sequences which bind human irf. we define two major classes of high affinity rna ligand, the optimal loop sequences of each are 5'-cagugn-3' (wild-type) and 5'-uaguan-3'. this novel finding predicts base-pairing within the loop between positions i and 5. all nucleotide substitutions in the "bulge" or at loop positions 2,3 and 4 decreased binding by 36% to 99%. in addition, binding specificity of rat irf differed with that of the related rat ire-binding protein, irf b. in vitro analysis of ire-like stem-loops identified by computer search has not yet revealed any new ire-containing genes. a73 $18-08 3' processing of histone pre-mrnas: a phylogenetic comparison reto kohler, petra duda and daniel schiimperli. zoologisehes institut der universtit/it bern, abteihing fiir entwicldungsbiologie, baltzerstr. 4, ch-3012 bern, switzerland. we are using a phylogenetic approach to study the biochemical reaction by which animal histone pm-mrnas are processed at their 3' ends. in mammals, the efficiency and specificity of this reaction is known to depend absolutely on the u7 snrna which interacts with conserved spacer sequences downstream of the proc~sing site. a highly conserved hairpin element which precedes the cleavage site serves as a binding site for an additional processing factor, but its importanea for efficient processing varies greatly between different historic genes and between extracts of different mammalian cell lines. to determine whether the relative importance of the hairpin and spacer dements have been conserved during evolution, we analysed the processing of histone genes from three different animal phyla in vitro, i.e. vertebrates (mouse), arthropods (drosophila melanogaster) and nematodes (c. elegans). in the mouse system, processing was strongly dependent on the presence of a mouse spacer element. in nuclear extracts of drosophila ke cells, processing occurred exclusively when a drosophila spacer element was present in the rna. processing efficiency was not reduced by foreign (mouse, c. elegans) hairpins. in whole cell extracts of ascaris lumbricoides embryos, an inverted situation was observed. 3' processing products were only produced by rnas that included an upstream segment derived from a c. elegans histone gene, irrespective of the spacer sequences present. these surprising results correlate with certain unique features in the c. elegans upstream element. we are currently in the process of cloning ascaris lumbricoides histone genes, which will allow us to verify our results in a homologous system. in xenopus laevls ooeytes, wild type mouse u7 rna gets assembled into functional u7 snrnps, both when transcr~ed from an injected gene or when injected as/n vitro synthesized rna. if the sm binding site of u7 rna is converted into the canonical sm sequence (u7 sm opt), the rna assembles into a particle which accumulates more efficiently in the nucleus but wbach is nonfunctional. this u7 sm off particle inhibits the function of preformed endogenous u7 snrnps, most likely by nonproductive binding to histone pre-mrnas, histone pre-mrna processing can be demonstrated in c/s if u7 rna is placed 3' of hlstone pre-mrna and injected into oocytes. only u7 sm wt sequences are active in this c/s processing. three proteins can be photoaffinity cross]inked to u7 sm wt rna, the common snrnp proteins g and b/b' and a u7-specific protein of 40 kd (50 kd in mouse). these crosstinks map to closely spaced positions within the sm binding site with the u7-specific crosslink being located most 3'. u7 sm opt rna does not become crosslinked to the u7specific protein and is also more tightly associated with sm proteins. we now investigate the in vitro assembly of u7 snrnps using these characterized u7 rnas and a preparation of highly enriched snrnp proteins. u7 sm wt and u7 sm opt rnas associate with common sm proteins in this system, but the interaction with the u7-speciflc protein could not be demonstrated. the double-stranded (ds)rna modifying enzyme, which can convert adenine to inosine, was first identified in xenopus laevis, but has since then been detected in different species and in mammalian cells. the enzyme is a specific adenine deaminase which uses dsrna as substrate and recently, it has been postulated to be responsible for a specific mrna editing reaction. we have purified this enzyme to homogeneity, approximately 400,000 fold from calf thymus whole cell extracts. the protein was purified primarily by ion exchange chromatography over six columns, with the final step being chromatography on a dsrna affinity column. the purified protein has a molecular weight of 115 kd and is the only protein present when enzymatically active fractions are visualized on an sds-polyacrylamide gel stained with silver. the dsrna modifying enzyme is a very low abundant protein. we are in the process of further characterizing the purified enzyme and of cloning cdnas coding for it. detailed mutational analysis of histone rna 3' end formation carmen spycher, andr6 furger, adrian streit, daniel albrecht, d. schiimperli, zoologlsehes iustitut der universtit/it bern, abteilung thr entwicklungsbiologie. baltzerstr. 4, ch-3012 bern, switzerland histone rna 3' ends are formed by cndonucleolytic cleavage resulting in poly(a)" mrnas with a highly conserved 3'-terminal hairpin loop structure. for the mouse h4-12 gene major and minor processing sites are located 3 and 5 nucleotides downstream of the hairpin, respectively. for the cleavage reaction, the u7 snrnp interacts with the spacer element of histone pre-mrna by base-pairing through the 5' end of its rna moiety, u7 rna. we have observed that this base-pairing potential extends further than previously recognised in either direction. we have made site-directed rnutagenesis in fltis potential hybrid region and around the processing site. rna processing and complex formation with the u7 surnp were analysed by incubation in nuclear extract from mouse k 21 cells. our results indicate that base-pairing with u7 rna both in the classical spacer element and downstream of it are very important for histone rna processing. in contrast, sequences upstream of the spacer element do not seem to be involved in base-pairing with u7 rna, but mutations in this region may affect processing by other mechanisms. systematic mutations of the highly conserved four nucleofides immediately following the hairpin show no qualitative or quantitative effects in the processing reaction. alteration of nucleotides 5 to 7 around the minor processing site, however, result in a dramatic decrease in processing efficiency and alter the specificity of the cleavage site. in further experiments we will introduce specific nuclcetidc modifications at the two processing sites, which should allow us to characterize potential cleavage factors by chemical crosslinking. evolution callaerts p., glardon s., j.,oosli f., quiring r. and gehring w. cell biology, biozentmm, basel. the pax genes encode a family of dna binding factors which share the paired-box and play an important role in the control of development. they can be subdivided into four different classes which differ in the presence or absence of other conserved sequence motifs coding for a paired type homeobox or an octapeptide. the pax-6 gene, which contains a paired-box and a homeobox, has been cloned from humans, mice, rats and zebra fish. recently, we have cloned the pax-6 homologue of drosophila melanogaster. the five genes share a very high degree of sequence identity both in the paired-domain and the homeodomain. similar to its mammalian counterparts, the drosophila gene is expressed in the eye primordia and the nervous system. in addition, mutations in pax-6 affect eye development: aniridia in humans, small eye in mouse and rat, and probably eyeless in drosophila. this would imply that the pax-6 homologues share a conserved function in eye morphogeuesis in both vertebrates and invertebrates. furthermore, this suggests that pax-6 may have been present in a common ancestor. therefore, we are now trying to isolate pax-6 homologues from other, more primitive organisms by means of the polymerase chain reaction, and to determine whether they are implicated in eye development. putative candidates have been identified from a number of species and are being characterized. their sequence similarity and some evolutionary implications will be discussed. keegan liam and gehrin~ walter j., biozentrum, university of basel, klingelbergstr. 70, ch-4056 basel, switzerland using an enhancer map screen we have identified two genes that may be direct targets of antennapedia involved in forming the adult leg. spalt major is repressed in the leg imaginal disc and tea, shirt is activated by antennapedia. we wish to determine whether the control of these genes by antennapedia is direct, spalt major is expressed in a ring in the antennal disc and is repressed by antennapedia in the leg disc. the antennal ring is repressed by ectopie antennapedia expression that transforms the antenna to a leg. we are defining an enhancer at spalt major that is required for antennal ring expression and repression by antennapedia. we are using various approaches to show that antennapedia binds to the antennal ring enhancer at spalt major. these include polytene chromosome banding studies using antibodies to antennapedia as well as in vitro dna-binding and genetic experiments. in search for novel regulators potentially involved in myogenesis, we identified a homeobox of the paired type in human muscle. subsequent cdna cloning revealed that we had cloned the full length coding region of human pax7. our human sequence extends the known mouse edna both on the 5' and the 3' end. as a first step in order to test for a functional role of pax7 in myogenesis, we analyzed its expression pattern during chicken development. transcripts are present in the neural tube and in the dermamyotome of the developing somites. therefore, the pattern of expression of pax7 is conserved between mouse and chicken. next, we assessed the expression of pax7 in different mouse and human cell lines. we found pax7 transcripts specifically in myogenic cells and not in any other cell types. interestingly, pax7 is already present at the myoblast stage. moreover, 10ti/2 fibroblasts converted to myoblasts by either transfection of myod or treatment with 5-azacytidine expressed pax7, whereas the parental cells did not. while myod was able to activate pax7 in 10t1/2 cells, expression of pax7 was not sufficient to induce the myogenic phenotype. nevertheless, our expression data are consistent with a role of pax7 during the mesodermal commitment to the myogenic lineage. the segmental organization of the drosophila head is achieved by a flow of positional information from maternal gene products to the zygotic gap genes. recent genetic analysis has identified three new gap genes involved in head development. one of these genes is the empty spiracles (ems) gene. mutations in eras cause severe defects in the head and the filzk~rper at the posterior end are missing. the eros gene encodes a putative transcription factor with a homeodomain. the eros rna is expressed in two phases of embryonic development. first expression is seen at the blastoderm stage in a single anterior band, correlating with its function as an anterior gap gene. later during embryogenesis eros is expressed in the posterior spiracles as well as lateral regions of each segment where the tracheal pits form and lateral neuroblasts originate. using g-gal fusions we could identify at least five different regulatory elements in the eros promotor region responsible for tissue specific expression of the gene. a 300 bp element responsible for the early expression which is dependent on the maternal gene bicoid, the key gene of the anterior system, and the terminal gene tailless was identified and studied in detail. this element contains two bicoid and two tailless binding sites. the effects of muations in these binding sites on eros expression will be discussed.this analysis should allow us to elucidate the molecular mechanisms by which the morphogen bicoid regulates subordinate target genes like ems in the drosophila embryo. the pax-6 gene of the mouse encodes a transcription factor with both a paired-and a homeodomain. pax-6 is affected by several allelic mutations in the small eye (sey) gene. sey mutations cause a reduction of the eyes in heterozygotes, and homozygotes lack both eyes and nasal openings and die as newborns. the corresponding mutation aniridia in humans affects eye and cranlofacial development in a similar manner. we have isolated a pax-6 homolog from drosophila (dpax-6), which shares more than 90% sequence identity in the paired-and the homeodomain with the mammalian genes. also outside of these domains we find considerable sequence conservation arguing for a true pax-6 orthologue. the drosophila gene spans ~16 kb and is expressed in the brain and the ventral nervous system of the embryo, and in the eye imagjnal discs and the brain of the larva. the gene was mapped to the eyeless (ey) locus. mutations at the ey locus cause either the partial or complete absence of the compound eyes or embryonic lethality. in one ey allele we have identified a transposable element in the first intron of dpax-6, which affects dpax-6 transcription in the eye discs, suggesting that dpax-6 is encoded by the ey gene. this implies that pax-6 (sey) in the mouse is homologous to ey in drosophila. thus, despite of the different modes of eye morphogenesis, the same gene seems to control eye development in insects and vertebrates. the drosophila homolog of the vertebrate serum response factor (srf) was isolated by low stringency-hybridization. nucleotide sequence analysis revealed that the drosophila srf homolog (dsrf) codes for a protein which displays 93% of sequence identity with human srf in the mads domain, the region required for dna binding, dimerization and interaction with accessory factors. the dsrf gene is expressed during several phases of embryonic development. both the rna and the protein are maternally provided to the egg and slowly decrease in their levels during gastrulation. after germ band retraction, high levels of zygotic expression were observed in a distinct subset of peripheral tracheal cells distributed throughout the embryo. many of these cells are at the tip of tracheal branches and are in direct contact with the target tissues these branches tracheate. the dsrf gene was mapped to position 60c on the second chromosome, and overlapping deficiencies which remove the gene were identified. analysis of tracheal development in embryos carrying these deletions revealed a degeneration of most of the major branches of the tracheal system. although the initial migration of tracheal cells was not affected in those deficiency embryos, many tracheal cells appeared not to maintain their formerly correct position and continued to migrate. thus, the dsrf gene might play a role in the proper formation and maintenance of the major branches of the trachea. s 19-08 our experiments would be expected to provide insight into such problems as the evolution and function of transcription factors with multiple dna binding domains. ideally, we would be able to mimic "gene splitting" which occurred during evolution. the poan gene plays an important role during drosophila neurogenesis. it is expressed in specific subsets of sensory mother ceils (smcs) and neuroblasts. the smcs that express poxn differentiate into polyinnervated external sense (p-es) organs. in the absence ofpoxn, smcs differentiate into mono-innervated rather than poly-innervated external sense organs. as the poxn gene contains a paired-box, it probably encodes a transcription factor. since the function ofpoxn in the central nervous system is not known and no po~n mutants are available, an ems mutagenesis screen was initiated to isolate such mutants. based on the assumption thatpoxn null alleles are lethal, we screened for lethals uncovered by the deficiency df(2r)wmg, which includes poxn, but survive over the deficiencies df(2r)xte-18 and df(2r)kl-32 flankingpoxn. in a further test, such lethals are screened for the absence of embryonic p-es organs. ifpoxn mutants are not lethal, they will escape this screen. therefore, we are inducing local hopping of p-elements that have inserted in the vicinity of poxn. the offspring of flies that display an altered eye phenotype are screened for p-element insertion into poxn. we hope that these approaches will generate mutants that will be crucial for future studies ofpoxn function in neurogenesis. chromatin structures and transcription of rdna in yeast saccharomyces eerevisiae reinhard dammaun, renzo lucchini, thee keller and jest m. sogo; institute of cell biology, eth-honggerberg, ch-8093 ziirich the chromatin structure of yeast ribosomal dna was analyzed in rive by cross-linking intact cells with psoralen. we found that in exponentially growing cultures the regions coding for the 35s rrna precursor fall into two distinct classes. one class was highly accessible to psoralen and associated with nascent rnas, characteristic for transcriptionally active rrna genes devoid of nucleosomes, whereas the other class showed a cross-linking pattern indistinguishable from that of bulk chromatin and was interpreted to represent the inactive rrna gene copies. by cross-linking the same strain growing in complex or minimal medium, we have shown that yeast ceils can modulate the proportion of active (non-nueleosomal) and inactive (nucleosomal) rrna gene copies in response to variations in environmental conditions which suggests that yeast can regulate rrna synthesis by varying the number of active gene copies, in contrast to the vertebrate ceils studied so far. whereas intergenie spacers flanking inactive rrna gene copies are packaged in a regular uucleosomal array, spacers flanking active genes show an unusual cross-linking pattern suggesting a complex interaction of regulatory factors and histories with dna. $20-04 r. felleisen & b. gottstein; institute of parasitology, university bern, bern, switzerland most patients suffering from alveolar echinococcosis (ae) respond to infection with a marked igg synthesis directed against e.multilocularis metacestode antigen ii/3, which represents a novel member of the family of cytovillin related proteins. although the respective gene basically is also present and expressed in e.granulosus, most of the cystic echinococcosis (ce) patients do not recognize the antigen. this phenomenon was tackled by generating cdna derived from full length ii/3 genes from both echinococcus species, performed by rt-pcr. sequence analysis revealed a very high degree of conservation of the primary sequence (98.6% homology), cdna fragments were expressed as recombinant proteins and were comparatively assessed in elisa respective to antibody-binding characteristics. sera from patients suffering from ce were showing no significant differences in reactivity with the antigens derived from both species. therefore, parameters others than minor differences in the primary sequence seem to be responsible for the lack of antigen recognition with respect to ce. $20-02 patrick rigoni, sandro rusconi, institut f molekularbiologie h der universitat zarich, winterthurerstr. 190, 8057 z~rich, switzerland. trinuclcotide repeats are often found in the coding sequence of transcriptional regulators in both mammals and yeast, however, their function is yet unclear and data collected in our laboratory on the gheocorticoid receptor even suggest that they may not have any, and that their presence is probably the result of a selfish replicative behavior. nonetheless, we believe that these motifs can be used as tags to identify flexible protein domains typical of modular proteins such as transcription factors. another kind or repeat (coding for a poly-glutamine/alanine) has been discovered in the yeast regulators gall1 and ssn6. from northern blots, we know that such caggcn repeats are present also in higher eukaryotes and we want to isolate them by constructing an enriched edna library using a 5' race protocol. so far, no mammalian protein bearing the motif glrdala has been characterized. among the positive clones we hope to find the homologous or paralogous of gall1 and ssn6 that have escaped so far conventional cloning techniques. meanwhile, we have been testing the effect of coexpressed yeast gall 1 on different reporter-activator combinations in mammalian cells. preliminary results show that galll but not the mutant form galllp can inhibit the activation properties of some (not all) gal4 chimeras. we are also producing antibodies directed against oligo-ala and oligo-gln and oligo-gln/ala motifs in order to better characterize natural proteins containing these repeats. we are interested in the early development of c. elegans, particularly in the contribution of genes whose homologues in vertebrates and d. melanogaster mediate positional infolzzations during development. therefore, we are working on ceh-13 which belongs to a cluster of at least 4 homeobox containing genes. this cluster is considered to be equivalent to the homeotie cluster of drosophila and to the hox clusters in vertebrates. by generating ~-galactosidase transgenie lines, we have observed that ceh-13 is expressed very early during embryogenesis, embryonic expression appears to be restricted to the posterior half of the embryo, which is rather surprising, since the ceh-13 hemologues in drosophila and vertebrates are anterior-specific. during larval stages, ceh-13 is expressed in neuronal cells. these results are now in the process of being confirmed by immunolocalization of the ceh-13 protein product with a polyclonal antiserum. in order to clarify the function of ceh-13 in the c. elegans development, we have isolated a ceh-13 mutant from a tcl insertion library, in collaboration with r. h. a. plasterk from amsterdam. from this worm, which looks wt, we are now trying to obtain a deletion derivative. previous studies have shown that bovine herpesvirus 1 (shv-1) infected cell pratein (bicp) 0 acts -depending on the promoter -as a strong transactivator or as a repressor in transient expression assays. the 81cp0 polypeptide contains a cysteine-rich zinc finger domain (c3hc4) which is conserved in a number of viral and cellular regulatory proteins including the 8icp0 homologs icr3 of herpes simplex virus type 1 and protein 61 of varicella zoster virus. this type of zinc finger (the so-called ring tinge0 was shown to bind zinc ions but functional requirement for zinc has not yet been demonstrated, a gap which we aimed to fill with the present study. transient expression assays were performed in oocytes which had been microinjected with thionein to chelate and deplete the intracellular pool of zinc. 81cp0-induced cat activity of a promoter-cat construct was 72-fold higher than the basal cat activity of the reporter plasmid, in the presence of thionein, bicp0-induced transaotivatlon was reduced 54old. with a set of control experiments we excluded that thionein might affect transcription and protein synthesis in general, to our knowledge this is the first demonstration of zinc-dependence for a member of the ring finger family. we have identified by pcr and edna cloning five pou genes expressed during early embryogenesis of zebrafislx four of these genes show extended homology to the brn-1 class of pou genes. preliminary evidence suggests that the brn-1like pou genes overlap with most of their expression domains.the gene studied in most detail so far (zp-50) is first expressed on the ventral side of the future fore-and midbrain. slightly later, expression is also found in rhombomeres 3 and 5 in the hindbraln. these rbombomeres were identified by the specific expression of the krox-20 marker using a novel in situ hybridization protocol which allows the simultaneous detection of two different transcripts using different color substrates. in the 24 hours old embryo a complex expression pattern is found involving a variety of brain structures and the spinal cord. the distribution of the transcript suggests that zp-50 is mostly expressed in glia cells. another pou gene we have identified (gp-9) defines a novel class of pou proteins. as a maternal mp, na it is initially ubiquitously present. after gastrnlation its transcript is found most notably in the developing hindbrain in rhombomeres 2 and 4 for which so far no good molecular markers were known. we are investigating the roles the identified pou genes may have in zebrafish hindbrain segmentation. we are currently attempting to manipulate gene expression in developing embryos by injection of rna or by the production of transgenic fish. we are also screening the zebrafish genome for new developmental marker genes. progress about these experiments will be presented. $20-07 willimann, t. and trueb, b. to gain insight into the regulatory mechanisms of collagen vi synthesis we have characterized the cis-acting elements of the chicken ctl(vl) collagen promoter. footprinting experiments with nuclear extracts from chicken embryos revealed three distinct elements, designated a, b and c, that were protected from dnase i digestion. the nuclear proteins that interact with the three sites were identified by gel retardation assays in combination with the use of various oligonucleotide competitiors as well as specific antibodies raised against well-characterized transcription factors. site a was found to be a target for transcriptional activator apf, whereas sites b and c were shown to be recognized each by two distinct nuclear proteins which belong to the spl multigene family. to address the question whether the three sites alone are able to direct transcription, a minipromoter construct was created in which the sequences of sites a, b and c were placed in front of a reporter gene. after transfection into chicken fibroblasts, this construct exhibited a high relative promoter activity when compared to a large genomic fragment containing the basic ctl(vi) collagen promoter. thus, the three sites are sufficient to induce transcription of this gene. octamer transcription factors (oct or otf) are a subset of the pou family of transcription factors which regulate expression of cellular and viral genes by binding to the octamer sequence atgcaaat. neurons and astroglial cells harbour, in addition to the ubiquitous oct-i factor, brain specific factors termed n-oct 2, 3, 4 and 5. in the present study we determined the chromosomal localization of the gene encoding the n-oct 3 transcription factor and characterized the structure of isolated n-oct 3 genomie clones. the chromosomal mapping was performed by analysis of somatic cell hybrid panels and radioactive and fluorescence in situ hybridization of human metaphase chromosomes. it is interesting that the 5' end of the n-oct 3 coding sequence contains repetitive cag and ggc residues. several disorders have been discovered to be related to expansion of trinucleotide repeats. we are presently investigating if the n-oct 3 cag or ggc clusters are hot spots for such mutation mechanisms and if so, which diseases could be associated with it. two dna regions upstream of the distal glucocorticoid receptor binding site interact with nuclear proteins that are tissue-specific (region a) or ubiquitous (region c). the characteristics of the dna-protein interactions have been studied in vitro. these sequences, in different combinations with the natural mmtv promoter, were tested in transfection assays of fibrcblast or mammary cell lines. we show that they are able to modulate the level of glucocorticoid-stimulated transcription. the proximal region of the mmtv promoter has binding sites for the transcription factors ctf/nf-i and oct-1. p~asmids with a deletion or mutations in the oct-1 site were tested in stable transfections, that are most representative of the state of proviral dna with respect to both number of integrated dna templates and chromatin organization. we show that the oct-1 site is important for the basal level of promoter activity. the data further indicate that the functional outcome may depend beth on the relative ratio of factors to dna templates and on the relative affinity of binding sites, as determined by oligonucleotide competition footprinting. besides the fact that telomeres represent specialised structures important for chromosome stability, the particular significance of cloned c. elegans telomeres is that they will contribute to the completion of the physical mapping of chromosomes and that they provide one set of elements for the construction of artificial c. elegans chromosomes. the cloning of c. elegans telomeres, however, is impeded by the fact that tandem repeats of the sequence ttaggc are not only located at the ends of the c. elegans chromosomes, but also at many internal chromosomal sites. therefore, we have developed a special protocol for the construction of a c. elegans endlibrary in 2~ zap. the resulting telomeric library was screened for recombinants containing ttaggc repeats and yielded about 70 positive clones. so far, 30 were analysed by partial sequencing and twelve of them satisfy all criteria required for telomeric clones. their ttaggc tandem repeats are located at the expected position, namely just adjacent to the blunt end cloning site in the polylinker. furthermore, the g-rich strand of the repeats is oriented 5' to 3' towards the end of the fragment, thus corresponding exactly to the defined orientation of eukaryotic telomeric sequences. finally, hybridisation data with one of these putative telomeric clones show that a subtelomeric fragment hybridises to bal31-sensitive fragments on a southern blot with c. elegans dna, indicating that this clone represents a c. elegans telomere. $20-09 to investigate whether chromatin structure or transcription can interfere with replication, derivatives of the yeast trp1ars1 minichromosome were constructed that contain either the ded1 or pet56 promoter firing against the origin of replication ars1. while the pet56 constructs transformed yeast at high frequencies and were maintained as high copy number minichromosomes, the ded1 constructs transformed at low frequency and the constructs were integrated in the genome suggesting that ars function was impaired. insertion of the h4-ars, a second origin of replication, rescued a ded1 construct as a minichromosome (yrpdh3).the chromatin structure mapped at low and high resolution of the ars1 region in yrpdh3 was indistinguishable to that of the pet56construct yrpft61. ananlysis of rna showed that transcription was going through ars1 in yrpdh3 and in yrpft61, but the levels of transcripts in yrpdh3 were much higher. we conclude that transcription through are1 interferes with replication and prevents extrachromosomal maintenance. dna sequence of a repeated dna segment on circular and linear plasmids of borrelia burgdorferi w.r. z%ckert and j. meyer, abt. pzmom, zahn&rztliches institut der universit~t basel a plasmid-associated repeated dna segment of b. burgdorferi strain b31 was cloned. at least two copies of this segment appear to be present on the 29 kb circular plasmid, whereas one copy is carried on the 50 kb linear plasntid of this strain. dna sequence analysis revealed a region containing a novel 906 bp putative open reading frame (orfl) on the 50 kb linear plasmid, orfl displayed extensive sequence homology (95%) to putative open reading frames present on the clones obtained from the 29 kb circular plasmid. heterogeneity is mainly caused by 3rd-base-wobbllng. flanking sequences share 85-95% homology, including the 5' end of an additional putative open reading frame irmaediately downstream of orfl. whether orfl and/or the related sequences are being transcribed and yield, in the case of orfl, an approximately 36.5 kd, lysine-rich polypeptide, is yet unknown. the repeated sequence seems to be specific for b. burgdorferi sensu lato and therefore may be useful in nucleic acidbased diagnostics of lyme disease. $20-16 similar to type ib oculocutaneous albinism in humans, overall production of pigment is greatly reduced in dark eyed albino and only obvious in eyes. we have studied the molecular basis of the c 44h mutation and show that expression of the tyrosinese gene is not affected. after sequencing tyrosinase cdna isolated from c44h/c44h homozygotes we uncovered a single base alteration from wild type leading to a serine to isoleucine exchange. the importance of this mutation was demonstrated by generating transgenic mice containing a mutated tyrosinase minigene. this showed that the single base change is sufficient to severely depress pigment production in transgenic mice. we therefore conclude that the point mutation is responsible and sufficient to generate the dark eyed albino phenotype. members of the pou-family of transcription factors are involved in developmental and differentiation processes. using a pcr-based cloning strategy with degenerated oligonucleotides we identified several pougenes from the lactating and involuting mouse mammary gland. three of these cdna clones which contained as yet unknown sequences were chosen for further investigations: clone 5.80 which has a high homology to pit-l, clone 5.54 which is related to the brn-3 gene and clone 5.48 which belongs to the class iii of pou-proteins. the expression levels were analyzed in different mouse organs and in several developmental stages of the mammary gland, including the postlactational process of involution. because of the low abundance of the specific transcripts we used a semiquantitative, reverse transcriptase-mediated pcr assay to investigate the mrna levels of the three novel pou-family members. distinct and specific expression patterns for all three members were obtained in the different investigated organs. interestingly, the expression of the pit-1 related cdna clone 5,80 was elevated in all developmental stages of the lactogenic-hormone dependent mouse mammary gland and in sceletal muscle, whereas its expression was low in other organs. repetitive sequences in dna can allow ectopic (outof-register) homologous recombination, which is often undesirable and can even lead to disease in humans. to prevent this, long homologies should often be interrupted during evolution. accelerated mutation of repetitive sequences by so-called ripping may be one of the mechanisms used to accomplish this in fungi. we are investigating if introns in vertebrate genes have an undiscovered role as interrupters of homology within and/or between genes, in addition to their established role in exon shuffling. by inserting homology-poor introns in an otherwise homology-rich region the genome could prevent ectopic homologous recombination. such a mechanism could be especially useful where there is an advantage in encoding highly repetitive protein sequences. it appears that within the collagen gene family there is indeed a correlation between repetitiveness and intron density. the influence of prenatal diazepam exposume (i,25 mg/kg/d, s.c.) from gestational day 14 to 20 on the development of the ~-opioid binding sites in striaturn, nucleus accumbens and midbrain of pn 14, pn 28 and 8 week old male and female rats was studied. 8 week old prenatally diazepam exposed male rats showed a significant decrease of k-opioid binding sites in the nucleus accumbens. a sex-dependent difference in k-opioid binding site densities could also be detected between pn 28 prenatally diazepam exposed male and female rats. we now investigate by in situ hybridisation whether changes in the level of mrna encoding fo~ prodynorphin, the precttrsor for various endogenous ~-opioid agonists, could be responsible for the decrease of ~-opioid binding sites in the nucleus accumbens of 8 week old prenatally diazepam exposed male ~ats. $21-04 distribution and morphology of nitric oxide-positive neurons in the marmoset cerebral cortex during pre-and postnatal development j.p. hornung* and j. schultz** *institute of anatomy, university of lausanne, 1005 lausanne, and **university of fdbourg, 1700 fribourg nitric oxide, synthesized by the enzyme nitdc oxide synthase (nos), is a neuroactive substance and a limited number of neurons were shown to express this enzyme either by histochemistry or by immunocytochemistry. both techniques were performed on brains of marmosets with ages ranging from 6 weeks before birth to adult. the morphology and distribution of nos-positive neurons were analyzed in all lobes of the cerebral cortex. the same pattern was revealed by histochemistry or immunocytochemistry. three populations of nos-positive neurons were revealed. first, a transient population of neurons in the molecular layer of the embryonic cortex, many having the morphology of the retzjus-cajal cells. a second population was made of large multipolar neurons in the deep layers of the cortex and the underlying white matter, which persisted from embryonic to adult life. a third, permanent, population was made of small, weakly reactive neurons in the supragranular layers appearing postnatally, this study demonstrates that no can exert its actions in the cerebral cortex through several pathways at different developmental stages. trimethyltin (tmt), a well-known neurotoxicant, was used to study the sensitivity of the different brain cell types (i.e. neurons, astrocytes, oligodendrocytes and microglial cells) to a neurotoxic insult. aggregating brain cell cultures of fetal rat telencephalon were treated during 10 days with tmt at different concentrations, ranging from 10 -10 m to 10 -6 m. microglia were found to be the most sensitive cell type, since already at 10-10m of tmt an increased number and clustering of griffonia simplicifolia-positive ceils could be observed. at 10 -8 m of tmt astrocytes showed increased staining for glial fibrillary acidic protein, characteristic of gliosis. neurons and oligodendrocytes appeared to be the least sensitive cells, since a decrease in the activity of cell type-specific enzymes was observed only at 10 -6 m of tmt. these results show that microglial cells and astrocytes respond to a toxic insult before any neuronal changes could be detected, and that the microglial reaction may be the first target of tmt. this reaction could then trigger the astrocytic response. vif is widely distributed in brain with suggested functions related to development. vip expression was studied during rat brain development using hybridization histochemistry with a 48met probe. vip mrna was found in thalamic nuclei on el7, later recognized as the ventrolateral and reticular nuclei after further maturation during prenatal period, vip mrna was found in hypothalamus, especially suprachiasmatic nucleus on el9 and its expression matured over the next days. few cortical neurons contained vip mrna on e21, they continuously increased in number and signal intensity over the perinatal period. vip gradually matured in the first three postnatal weeks and adult-like patterns were found on p 22, when cerebral cortex, ventrolateral and reticular thalamic nuclei, hypothalamus, esp. suprachiasmatic nucleus, were the regions with most prominent vip expression. these results demonstrate the relatively late appearance of vip gene expression in the rat forebrain, as compared with peptides like srif and cck, not suggesting a major role in earlv brain maturation. in primary cultures of mouse cerebral cortical astrocytes, a rapid glycogenolysis followed by a massive glycogen resynthesis ( six-to ten-fold over basal levels after 9 hr) are induced by vasoactive intestinal peptide (vip) or noradrenaline (na). both actions of these neurotransmitters are mediated by camp. since the induction of glycogen resynthesis triggered by vip or na is abolished by inhibition of transcription and translation, we applied the 2-d gel electrophoresis technique to search for astrocytic proteins induced by vip or na. the comparison of 35s-labeled proteins from primary astrocyte cultures treated or not with vip 1 ~tm revealed two newly synthesized proteins: a 36 kda protein (pi=5) and a 23/24 kda protein (pi=6). only the latter is visible on a silver stained 2-d gel, which is a prerequisite to consider its purification and microsequencing. induction of the 23/24 kda protein by vip was time-dependent with a maximum at -12 hr. preliminary results indicate a similar induction by na and isoproterenol. in humans, the central analgesic effect of tramadol (t) is only weakly reversed by the ~t opioid antagonist naloxone (nx) (br j clin pharmacol 1993; 35:73p). t analgesia may thus result from an action on monoaminergic pathways as suggested by in vitro and animal data. we therefore investigated the effect of ct2-adrenoeeptor antagonism by yohimbine (y) on t analgesia. according to a randomised double-blind crossover and placebo (p) controlled design, healthy volunteers (n=10) received t (100mg orally), followed (+ 3 h) by y (0.1mg/kg intravenously), and y + nx (0.8mg intravenously). analgesia was assessed over 8 h by subjective pain threshold (numerical scale -ns-) and objective pain threshold (rill nociceptive reflex -riii-) monitoring. peak analgesic effect was observed at ca. 3.25 h (riii +8.8 +semi.6 ma and ns +8.4 +1.8) vs p (rih -0.3 +1.2 and ns +2.3 +1.6, p<0.05) and lasted ca. 6 h. y reversed t analgesic effects during ca. 1 h (r/ii -2.9 +1.8, p<0.05 and ns +2.8 +1.5, p<0.05), whereas y + nx abolished t effects thoughout the study period (rill -1.9 4-1.1 and ns +0.6 +1.3, p<0.05), y alone tended to lower pain thresholds (rill -4.1 +1.5 and ns -1.9 +1.4, p>0.05 anova). yohimbine, an r antagonist, reverses tramadol effects, thus pointing to the major role of monoaminergic modulation in tramadol anfinociception. a monoclonal antibody (in-l)was raised against neurite growth inhibitory proteins present in rat cns myelin (caroniand schwab, neuron 1: 85, 1988) . in-1 neutralizes the inhibitory ettect of cns tissue in vitro and in vivo, thus permitting regeneration of certain lesioned cns fiber systems. we have used this antibody to immunostain cryostat sections of the nervous system of the rat. we found that in-1 stains white matter and myehnated fiber tracts in the cns. the staining pattern corresponds to that of the known myelin specific antigens mbp (myelin basic protein), mag (myelin associated glycoprotein) and mog (myelin oligodendrocyte glycoprotein). the staining of in-1 in the cns is found in regions of the adult nervous system which have been shown to be inhibitory for axonal growth. interestingly, the regions which show a high abundance of in-1 antigens are low in staining for gap-43, a marker for growth and plasticity in the developing and adult brain. in contrast, gap-43 is strongly expressed in unmyelinated fiber tracts or nuclei. prevention of oligodendrocyte development in rat spinal cord resulted in suppression of in-1 expression and elevated gap-43 levels. this suggests that inhibitory myelin proteins may negatively influence plasticity in the rat cns. to date, the existence of anp and npy in the adrenal medulla has been shown only for a few mammalian and anuran species. thus the present immunohistochemical investigation attempts to localize anp-like and npy-like peptides n the adrena gland of representative mammals, birds, reptiles, amphibia and bony fish by the use of antisera specific for mammalian anp and npy. furthermore, the catecholamine-containing cells were characterized by antisera against enzymes of catecholamine synthesis. anp-and npy-immunoreactivies were detected in adrenal chromaffin cells of all vertebrates studied while no immunoreactivities were observed in the adrenal cortex or in its homolog, the interrenal. the representatives of the different vertebrate classes exhibited marked differences in the proportions of anp-immunoreactive and npy-immunoreactive cells, in the presence of anp-and npy-immunoreactivities in noradrenalineand/or adrenaline-containing cells and in the amount of of cells containing both anp-and npy-immunoreactivities. our results give evidence for a long phylogenetie history of adrenal anp-and npy-like peptides. thus, they stress the physiological impact of these peptides as adrenal paracrine and/or endocrine hormones. human neuronal growth inhibitory factor (gie) is involved in the regulation of neuronal growth and is deficient in the brains of alzheimer's disease victims. this brain-specific metalloprotein consists of 68 amino acids out of which 20 are cys and has been found to contain copper and zinc. proteins with similar primary structure were isolated from bovine and equine brain. the amino acid sequence of these proteins shows about 70 % homology to those of mammalian metallothioneins (mt), with two conserved inserts of 1 thr and a glutamic acid rich hexapeptide in the n-and c-terminal regions, respectively. in contrast to mts, which usually contain only zn(i1), the presence of cu(i) and zn(ii) (6-7 moles total) in all gif isolations suggests the functional importance of both metals ions. to spectroscopically probe the native zn-binding sites, the zn(ii) ions were replaced by cd(ii). both bovine and equine cd/cu-gif derivatives exhibit similar absorption and cd spectra with features characteristic of cd-thiolate clusters. the release of 3h-arachidonic acid evoked by glutamate was characterized in cerebral cortical neurons in primary culture grown under conditions that prevent glial cell proliferation. in these cultures, 99% of the total ceils are immunocytochemicauy characterized as being positive for specific neuronal markers such as neuron-specific enolase and neurofilament. specific markers for astrocytes, oligodendrocytes or microglia were not detected. glutamate induces a concentration-dependent release of 3h-arachidonic acid with a ec50 of 3 pm. the pharmacological profile of this glutamate response shows the involvement of two receptor types: the nmda and the ampa ionotropic receptors. the glutamate-evoked release of 3h-arachidonic acid is phsensitive. when the incubation buffer is alkalinized from 7.25 to 7.65, both basal and glutamate evoked release of 3h-arachidonic acid are enhanced. the glutamate response is also enhanced as intracellular ph is alkalinized by pharmacological treatments: such as inhibition of c1-/hco3-antiport by dids. these results further stress the role of intracellular ph in glutamate-mediated neurotransmission. $21-09 multiceli culture system for the study of drug kinetics wechsler d., *schittny j.c. and honegger u. e., depts. of pharmacology and *anatomy, university of bern, ch-3010 bern a cell culture system was developed for the investigation of cellular drug kinetics in cultures with up to 4 cell types. it was used for the study of uptake, competitive uptake, release and redistribution of drugs. individual cell types (human skin fibroblasts, rat astrocytoma cells (c6) and rat hybfidoma ceils (oligodendrocyte x astrocytoma, roc)) were separately grown to confluency on glass circle sectors. in the same petri dish 4 sectors in various combinations were exposed to media containing radiolabelled chlorpromazine (cpz). single and repetitive uptake and release of cpz were measured in each cell type after individual exposure or exposure in any combination of cell types: in 2 hour competitive uptake studies fibreblasts reached 1.7 and 2.6 times the concentrations of c6-and roc-cells, :respectively. in redistribution experiments the exchange of cpz from preloaded to unloaded cells was tested. the exchange rate was dependent on cell type and loading period. it decreased with increasing time of exposure suggesting the formation of more stable cpz stores. we have previously demonstrated that certain neurotransmitters such as vip, noradrenaline (na) and adenosine promote glycogenolysis in mouse cerebral cortical astrocyte cultures (brain res. 563: 227-233, 1991) . the question that we then addressed was the nature of the metabolic substrate released by astrocytes following glycogenolysis. we therefore determined the presence of various metabolic substrated released from astrocytes under static conditions and in the superfusate of a laminar flow system developed in our laboratory. neither glucose nor any intermediate of the tficarboxylic acid cycle could account for the decrease in glycogen stores; astrocytes however were shown to release great quantities of pyruvate and lactate at a rate of 50 nmol/mg protein/minute. noradrenaline but not vip promotes a significant increase (42%) in lactate release. the effect of noradrenaline is mimicked by isoproterenol and inhibited by propranolol, suggesting a i~adrenergic mediation. when astrocytes are incubated in an isotonic solution containing no energy suhstrate, the glycogen stores are rapidly depleted and lactate, but not glucose, is released in the medium. in conclusion, lactate appears to play a major role in cerebral energy metabolism homeostasis, as substrate released by astrocytes to prey!de energy fuel for neurones and other neighboring cells. it is known so far that calcium-binding proteins (cabps: calbindin, pan, albumin and ca/retinin) are expressed in some cells of the pineal gland of laboratory mammals. the exact phenotype of these cells is unknown. nevertheless, according to recent studies, it seems that some of them are ganglion cells. therefore we studied the pineal gland of different species (gerbils, rats, goats, cows and humans) using calretiain (cr) antibody which is considered as marker for neurons (andressen & al, cell & tissue rss, 27t:181, 1993) , the cr-expressing cells were found in all oar species: in gerbils, rats, cows and humans they were scattered throughout the parenchyrna, whereas in goats they lined mostly the pericapillary spaces. according to our findings, it seems that most of the reactive ceils are rather neuron-like elements since morphological criteria for true neurons (axosomatic synapses, nissl bodies, bundles of neurofilaments) are missing. however one cannot exclude that among these cells some are intrapineal ganglion cells, because of ranvier's nodes found along the processes of some crpositive cells. thus, it is possible that these cells are involved in the modulatory control mechanism of the pineal neurohumoral activity and/er by accumulation of intracellular calcium in the formation of pincal calcifications. besides the excitatory amino acid transmitter glutamate, its sulfur containing analogue homocystcic acid (hca) could play a role in excitatory processes in the central nervous system. hca is present in and released from nervous tissue and is a potent neuronal excitant, predominantly activating n-methyl-d-aspartate receptors (do et al., 1992) . these properties are suggestive of a classic synaptic neurotrausmitter role. on the other hand, hca seems to be localized not in neurones but in glial cells (grandes et al., 1991; tschopp et al. 1992) . the efflux of hca is temporally delayed following activation of specific pathways (klancnik et at., 1992) ; these latter observations are not in accordance with the classic concepts of synaptically-mediated neurotransmission. a laminar flow superfnsian system of monolayer cultures was developed and the released materials from mouse cortical astrocytes, previously preincubated with [35s]-methioulne, were investigated. during application of either noradrenaline or the b-adrenergie agonlst isoproterenol, the extracellalar level of [35s]-methianine was decreased and that of labelled hca was increased. this effect was not observed with the -adrenergic agonist methoxamine, and could be blocked by the ll-adrenergic antagonist propanolol these results, combined with the delayed hca release, the glial localisatian of hca and its neuroactivity, strongly suggest a potential role of hca as a gliotransmitter, modulated by noradrenaline inputs. vasoactive intestinal peptide (vip) induces long lasting metabolic effects in the mouse cns. we have investigated a shorter neuromodulatory action of vip on the synaptic responses in coronal slices of adult mouse cingulate cortex by means of intracelhilar current clamp recordings. electrical stimulation of the underlying white matter evoked a multiphasic synaptic potential consisting of early epsp~ early ipsp, late epsp and late ipsp mediated by non-nmda, nmda, gaba a and gaba b receptors, respectively. vip (0.1 ~d~l) superfused for 5 rain. had no effect, whereas concentrations over 0.2 ixm selectively and reversibly depressed the nmda-medlated responses in 12/16 cells. in the presence of cnqx (10 [tm) and biencuuine (10 ~tm), the isolated nmda-mediated late epsp was still attenuated by 0.3 ~m vip in 8/10 cells (mean:-42% al~er 30 rain.). in 3 other cells vip induced a spreading depression (sd) and a strong, reversals inhibition of the late epsp (mean:-64 % after 10 min.).these results indicate that in the absence of gabaergic inhibition vip can induce two types of effects in the mouse cingulate cortex, a prolonged decrease of the nmdamediated syaaptic response and a striking sd. interaction between vip and glutamate on c-fos mrna expression. |.-l. martin, d. gasser and p.] . magistretti. institut de physiologie, universit4 de lausanne, lausanne, switzerland. the regulation of c-los mrna expression by vip and glutamate was examined in primary cultures of neurons originating from the mouse cerebral cortex. in primary cultures of cortical neurons, vip increases camp levels and stimulates c-los mrna expression. interestingly vip stimulation is completely blocked by mk-801, an nmda receptor antagonist but not by cnqx or ap3, which antagonize ampa/kainate and metabotropic receptors respectively, c-los expression stimulated by glutamate shares the same pharmacology. these results suggest an involvement of endogenously released glutamate in the stimulation of c-fos mrna expression evoked by vip. furthermore, when added together, vip and glutamate interact synergistically to increase cfos mrna levels. consistent with the pharmacology of vip and glutamate, the synergistic interaction between vii' and glutamate on c-fos mrna expression is also inhibited by mk-801. interestingly, this synergism is completely inhibited by h-89, a protein kinase a inhibitor, whereas staurosporin and kn-62 which block protein kinases c and ca++/calmodulin dependent respectively exhibit smaller inhibitory effects. presynaptic proteins involved in neurotransmitter release (i.e. synapsin, b-50) and some postsynaptie gaba a receptor subunits possess phosphorylation sites for camp-dependent protein kinase (pka) and/or protein kinase c (pkc). the functional consequences of phosphorylation by these kinases is not known. we have studied the action of specific activators of pkc and pka, phorbol ester (pdbu) and forskolin, respectively, on gabaergic synaptic transmission in area ca3 of hippocampal slice cultures. pdbu significantly enhanced the amplitude of evoked monosynaptic ipscs (in cnqx and ap5), and both pdbu and forskolin increased the frequency of spontaneous miniature ipscs (m[pscs) in the presence of cnqx, ap5 and ttx. this effect by pdbu was antagonized by staurosporin, a protein kinase inhibitor. pdbu and forskolin did not change the amplitude or decay of mipscs, suggesting that only presynaptic kinases are involved. furthermore, pdbu enhanced mlpsc frequency by a comparable amount in the presence of the ca 2+ channel blocker cd 2+, indicating that pdbu did not enhance gaba release from presynaptic endings by facilitating ca 2+ influx into axon terminals. valiunas, v., sc~ilinsky fluri, g. and weingart, r. arachidonic acid (aa) reversibly impairs the gap junction conductance (g~) in cardiac cells. now, we examined whether aaacts directly or via its catabolites. experiments were performed on pairs of neonatal rat myocytes using a dual voltage-clamp method. we used blockers which interfere with enzymes of various catabolic pathways. pretreatment with i0 ~mpoca (blocks carnitine acyltransferase i) did not prevent the decline in g9 by i0 ~m aa; i.e. intermediates of p-oxidation are not involved. similarly, preexposure to i0 ~m indomethacin (blocks the cyclooxygenase pathway) did not prevent aa from uncoupling; this rules out an involvement of prostaglandins and thromboxanes. exposure to 10 ~mndga (blocks the 5-1ipoxygenase pathway) per se produced a decrease in gj. likewise, exposure to i0 ~m etya (blocks the 15-1ipoxygenase pathway) also impaired gj. these effects cannot result from accumulation of endogenous aa because preexposure to 50 ~m 4bpb (blocks phospholipase a 2) did not prevent ndga-or etya-induced uncoupling. exposure to 75 ~m skf 525-a (blocks the epoxygenase pathway) also produced a decrease in gj. hence, ndga, etya and skf 525-a, compounds with different biochemical actions and of different chemical structure, act on g~ either directly or via an unknown mechanism. $21-19 potential changes associated with electrical activity in nonmyelinated nerve fibres a. robert and p. jirounek d~partement de pharmacologie, cmu, 1211 gen~ve 4 simuheneous measurements of the extracellular concentration of k + ([k+]e), and of the concomitant changes in the membrane potential (era) were measured in the rabbit vagus nerve during and after a period of activity. during a 15 hz stimulation there was a large increase in the [k+]e , accompagned by a change in era, which roughly followed the depolarization expected from the increase in [k+] e . at the end of the stimulation, although the [k+]e was still elevated, the nerve developed a posttetanic hyperpolarization (pth). the pth was generated by the electrogenicity of the na+-k + pump, but its amplitude and time-course were strongly affected by two depolarizing currents: (i) an inward ba2+-sensitive current of k + and (ii) an outward current of ci-. we hypothesize that during activity and during the early phase of recovery there was a ba2+-sensitive uptake of potassium by the schwann cells. the ci" current, by short-circuiting the na+-k ⧠pump, contributes to the control of the e m, and by this way to the driving force for the inward k ⧠current. overdose of ifosfamide has ooo/red in a canc~c patient (25g i.v. iy0/24 hours with 20 g as ~utectarfe). urir~ collece{on was obtaj/3ed in 24 hour ~ fcr t~o days. c~ was measured ~ a gas liquid d~romatogr~ method of the u5~ ~ (s~@pl. v, pag.2627-262g) ~sir~ a ~e~s ar~ ni~, sensitive ths~mi(~3ic d~. ~ u~s p~mt ~ ~ ~ ~ ~ ~ 1500 m~/24h and 207 r~/24h at day one ard day res~ec~vely. 9~ _h=~=mmtly ~ has also bsm foa~ in mti~s ~ mgh e~e ~ ~ (~/o~e). w~n ~ ~s am/nist~ to rats (i~ m~/~ ~c 200 m~f~ intraperit~neally), no ~ omild be detected in urine within 24 ht~r~ after drug administl-4(cicn. from these ~ data we conclude that c~ is extensi~_ly metabolized in rats and ~ mscbard_sm of ~ el ~mlnaticn in pati~rfc t=cj_ne m~cjlr[c reflect a flmicticrsl der~,~t of c~ m~t~0olisn in man to ifo~=~de ~tion. there are numerous reports of improved memory functions in rats following a dietary supplementation with choline. in some cases, this improvement can be related to enhanced cholinergic transmission (mack et al., behav neurosci., 103, 1234 -1241 , 1989 ). but it is not clear whether there is a general improvement in memory or whether specific aspects of learning and memory are affected. this work was aimed at analysing the effects of perinatal cholinergic supplementation on the development of spatial abilities and upon adult performance. choline supplementation (3.5 g./l. in 0.02 m saccharine solution in tap water) was maintained during two weeks before birth. additional supplementation was maintained from the 5th to the 1oth week postnatal. spatial learning capacities were studied at the ages of 26, 65 or 80 days in a cimular swimming pool (morris place navigation task) and at the age of of 7 months on a homing board. adult and aged rats were tested on a radial maze. selective aspects of the performance were markedly improved. this treatment had no specific effect upon spatial memory per se, but, instead, affected learning abilities by promoting efficient behavioural strategies hypothetically based on active representation and anticipation processes. the effects of the treatment remained evident up to 6 months following the supplementation. barcelona, e-08193 bellaterra females of both lines (n=b/grp) were injected sc daily between days 15-20 of pregnancy with vehicle (v), i(di) or 3(d3) mg/kg diazepam, or not(c). in these studies(a,b) their 5-7 mo old offspring were subjected to a 50-run session in a shuttlebox(a:12 males, 6 females of each line/condition) or a 6min session in a hexagonal tunnel maze with strategically-placed barriers and a lit central arena(b_:6-7 males of each line/condition). a: the freezing behavior of ld3 rats was reduced ~s lc, this behavior reverting more to escapes in males & avoidances in females, although v had an effect in the latter also. no within-line differences in inter-trial responses were seen, but pre-session activity was increased in female lvs and ldis, returning to lc levels for ld3s. no differences existed among the h groups. b: whereas h maze activity exceeded that of l, no wtthin-line differences appeared. hd3s entered the lit arena less than hcs or hvs, indicating an increased timidity after pnd, and a trend in the same direction existed for ldi/ld3 vs lv. two groups of abstinent female smokers, performing either a fixed rate or a subject paced version of a rapid information processing task were compared. both groups participated in two test sessions (in which the task was performed twice) to compare two different types of cigarettes which were smoked before and during the second task trial: a) the subject's habitual cigarette with a nicotine yield of at least 0.7 mg/cigarette and b) a nearly nicotine free test cigarette with a tar yield comparable to that of the habitual cigarette. reaction times to correct detections (series of three odd or even digits in a pseudorandom sequence of single digits) were longer with the subject paced version and not affected by the type of cigarette but shorter with the fixed rate version and even more decreased with the smoking of the habitual cigarette. on the other hand, the pre-to posttreatment increase in the subject paced processing rate was greater with the habitual than the test cigarette. it was concluded that the two task versions assess different cognitive functions. whereas the fixed rate version primarily assesses vigilance performance and seems to be more suitable to measure changes in reaction time, the subject paced version is more sensitive to changes in speed capabilities in rapid information processing. heart rate, physical activity, and cigarette consumption were continuously recorded under field conditions, whereas subjective craving was assessed six times per day and saliva cotinine was measured once daily. abstinence, habitual cigarettes (with a nicotine yield of at least 0.7 mg/cigarette), and nearly nicotine free test cigarettes but with a tar yield comparable to that of the habitual cigarettes were compare~) in a sample of twelve female smokers for two days each. whereas physical activity was similar for the three conditions, heart rate was highest with the habitual cigarettes, lowest on abstinence days and in between with the test cigarettes. cigarette consumption was similar for both types of cigarettes and subjective craving was higher on abstinence than on smoking days but no differentiation between the two cigarette types was obtained. saliva cotinine values were highest on the days with the habitual cigarettes but lower and similar with the test cigarettes and on abstinence days. it was concluded that heart rate and saliva cotinine depended only on the amount of nicotine absorbed, whereas subjective craving was reduced by smoking independently of the actual nicotine yield of the cigarette. (ptps) is the rate limiting enzyme in the synthesis of bh, in man, a cofactor for several hydroxylases involved in catecholamine and serotonin biosynthesis. the human and rat liver cdnas encoding the 16 kda subunit of ptps were expressed and the recombinant enzymes purified to homogeneity. the apparent k~ for the substrate, pi and heat stability of the re-combinant enzymes were similar to the native enzymes. the rat enzyme was crystallized and the x-ray structure showed a hexameric native form arranged as a sandwich of two trimers. three histidine, a glutamic acid, and -also shown by site-directed mutagenesis -a cysteine residue characterize the active center of the enzyme thought to be mg2+-dependent. according to the proposed ligands we replaced mg 2* by fe 2 ⧠or mn ~+ and observed an enhanced activity up to 100%. m. neerman-arbez, v. cirnlli and p. halban. laboratoires/eantet. centre m4dical universitaire, 1211 gen~ve 4. pci(3) and pc2, members of the mammalian family of pro-protein convertases homologous to the yeast kex 2, are both expressed in pancreatic islets of langerhans and are thought to be responsible for the conversion of proinsulin to insulin and c-peptide in beta cells. however, the insulin secreting beta ceils are not the only ceils present in these complex microorgans, prompting us to evaluate the expression of pc1 and pc2 in islet beta and non-beta cells. rat islet cells were sorted by autofluorescence activated flow cytometry to separate beta cells from non-beta ceils and conversion cndoprotease levels were analysed by western blotting. in beta cells, pc1 levels were higher than pc2 (pc1/pc2=2.6+0.2) whereas the opposite was found for non-beta cells (pc1/fc2=0.05â�¢ confirming that pc1 is important for proinsulin conversion while suggesting a role for pc2 in the conversion of proglucagon, prosomatostatin and propancreatic polypeptide. transformed murine cell lines (insulin-producing beta-tc and glucagonproducing alpha-tc) were found to faithfully reflect the primary rat cells in terms of their pc1/pc2 ratios. finally, post-translational modification of the convertases themselves was found to differ between cell-types, in particular, a precursor 75 kd form of pc2 accumulated in beta cells whereas only the fully processed 67 kd form was detected in the non-beta cells. the kringle (2+3) domains (k2+3) were successfully expressed in e. coil a n-terminal hexahistidine tag was fused to insure the isolation of k2+3 by affinity chromatography on a ni-2+-ntaagarose-column. to be able to remove the hexahistidine tag a factor xa cleavage site was introduced between the 6 histidine residues and the n-terminus of the kringle structures. the correct arrangement of the disulfid bonds was determined by amino acid-and sequence analysis. the lysine binding site was proven by affinity chromatography on iysine-bio-gel, as previously shown for k2. the dissociation constant of k2+3 was measured by intrinsic fluorescence titration with 6-aminohexanoic acid. a replication protein a copurifying dna helicsse from calf thymus anthl georgakl, narendra tuteja, blrglt sturzenegger and ulrich hobscher department of veterinary biochemistry university of z0rich-lrchel winterthurerstrasse f 90 ch-8057 z0rich, switzerland a dna helicase from calf thymus copurified with replication protein a through several steps of purification including deae-sephacel, hydroxyapatite and single stranded dna cellulose. it is finally separated from replication protein a on fplc mono q where the dna helicase elutes after replication protein a. we named this new calf thymus enzyme dna helicase f. characterization of the helicase f by affinity labeling with [a32p]atp indicated that the enzyme has a catalytic subunit of 72 kda. gel filtration experiments suggested that dna helicase f can exist both in a monomeric and an oligomedc form. the enzyme unwinds in the 5' ->3' direction in relation to the dna strand it binds. all eight deoxyribonucleoside-and ribonucleosidetriphosphates could serve as an energy source. testing a variety of dnndna substrates indicated that the dna heitcase f preferentially unwinds very short substrates and is slightly stimulated by a single stranded 3'-tail replication protein a allowed the dna helicase f to unwind longer dna substrates up to 400 bases suggesting that copurification of replication protein a with the dna helicase might be of functional relevance. 2,4,5,2',4',5'-hexachlorobiphenyl (6-cb) shows limited elimination and extensive storage in adipose tissue in vivo (lipophilie unmetabolizable compound) while chlorpromazine (cpz) is known for its lysosomal storage. uptake and release of 6-cb and cpz were studied in 3t3-preadipocytes (p) and 3t3-adipocytes (a). 3t3-cells were cultivated in dulbecco's minimal essential medium supplement with 10% fetal calf serum and grown to eonfluency. differentiation was stimulated by dexamethason and bezafibrate exposure for 48h. radiolabelled 6-cb (20~tm) or cpz (5~tm) was added to the medium. following a single dose 70-80% of cpz were taken up within an hour into p and a. in contrast, 6-cb reached an intracellular plateau of 30-50% of the dose after lh in p whereas dependent on the triglyceride content a accumulated up to 95% of the dose. 6-cb uptake into a was temperature dependent and not saturable with repetitive daily doses up to 1 week. 6-cb uptake into p was fully reversible while the release from a was limited to 10% of the accumulated drug. in contrast, the release of cpz was higher from p than from a as a consequence of the different lysosomal activities of the respective cells. the use of 3t3 cells allowed to show a remarkable difference in cellular handling of neutral and basic lipophilic drugs and may be helpful to further elucidate basic mechanisms involved. chronic granulomatous disease: an attempt to reconstitute the function of the x-linked form of the disease. chronic granulomatous disease (cgd) is a group of congenital immunodeficiencies that predispose patients to recurrent severe bacterial and fungal infections. the cause of the disease is lack or impairment of 02-production in phagocytic cells due to mutations in any of the four components of the superoxide producing system. approximately 70% of all cgd patients suffer from an x-linked form of the disease where the phagocyte oxidase gp91phox is affected. this project concentrates on the reconstitution of the deficient gene in phagocytic patient cells. expression of recombinant gp91 was assayed in vitro and in vivo. attempts to "tag" gp91 with a foreign epitope were unfortunately so far unsuccessful. the target cells for gene reconstitution are non-dividing monocytes. thus, we have chosen an adenovirus over other viral systems because of: a) its ability to infect nondividing cells, b) its genetic stability and, c) lack of human adenovirus related malignancies or side-effects. we constructed two recombinant adenoviruses: one recombinant expresses a lacz gene (control), another the gp9 lphox gene. we are trying the expression of recombinants adeno/lacz and adeno/gp91 in normal and patient-derived cells (for example ebvtransformed normal and patient b-cell lines), as well as the reconstitution of nadph oxidase function in peripheral blood monocytes of cgd patients. kinetics of molecular chaperone action schmid, d., gehring, h., *baici, a. and christen, p., biochemisches institut der universit&t z~rich, ch-8057 zorich; *rheumaklinik, universit&tsspital, ch-8091 z%rich molecular chaperones of the hsp70 type transiently sequester unfolded segments of proteins and promote their correct folding. target peptides labelled with an environmentally sensitive fluorophore allowed to follow their binding to the molecular chaperone dnak of escherichia coli in real-time. the two-step process is characterized by relaxation times z~ = 27 s and z2 = 200 s at 2 ~m dnak and 0.i ~m ligand at 25~ in the presence of atp, the formation of the complex is greatly accelerated and follows a single-exponential process with z : 0.4 s. the rate of dissociation of the complex was even more increased than that of association resulting in a decreased net affinity for ligands in the presence of atp. the binding-release cycle of dnak thus occurs in the time range of polypeptide chain elongation and folding and is too fast to be stoichiometrically coupled to the atpase activity of the chaperone (ko~ ~ = 0.13 min i at 30"c). supported by the olga mayenfisch stiftung, z%rich, and the hartmann m~ller-stiftung, z~rich. comparison of the activities of native bovine seminal ribonuclease and that secreted from e. coil schein, ch* (siat, technopark, pfingstweidstr. 30, ch8005 ziirich) and haugg, m (lab. org. chemistry, e.tm., . seminal ribonuclease (bs-rnase) differs from the pancreatic rnase a in that it forms a cysteine-linked dimer and has a relatively higher specific activity in the cleavage of double-stranded (ds-) rna substrates. we expressed bs-rn in a secretion system similar to that described previously (biochem. j. 283:377-344, 1992) using the signal sequence of routine pancreatic ribonuelease. about i mg bs-rn was isolated per liter culture supernatant. human and bovine recombinant interfcron-~ (ifn-r) inhibit the degradation of ss-and ds-rna by bovine pancreatic rnase while they activate the activity of bs-rn on the same substrates (febs letters, 270:229-332, 1990) . recombinant bovine or routine pancreatic rnase (produced in our secretion system) are inhibited by ifn-y, while the recombinant bs-rn or a cysteine-linked dimer of rnase a is also activated by ifn-y. ifn-y activates bs-rn even in the presence of inhibitory concentrations (0.1-1 ram) of mononueleotides. thus the mode of activation cannot be attributed to alleviation of product inhibition. kinetic studies using 300 bp ds-rna as substrate suggest that ifn-~ interacts directly with the bs-rn to increase the rate of highmolecular weight producffsubstrate release, as the apparent ks increases proportionately to the increase in kcat. significantly smaller movements were observed in the simulation with the liganded enzyme. the structural differences between the protein in the crystal and the ensuing calculated structures might arise from the absence of lattice forces in solution. evolutionary relationships among pyridoxal-5'-p-dependent amino acid decarboxylases sandmeier, e., hale, t.i. and christen, p. biochemisches institut der universit~t zgrich, 8057 z~rich. the pyridoxal-5'-p (plp)-dependent amino acid decarboxylases (de) can be subdivided into four apparently unrelated groups. group i is represented by glycine dc, group ii comprises glutamate, histidine, tyrosine and aromatic-l-amino acid dc, group iii procaryotic ornithine and lysine dc as well as the procaryotic biodegradative type of arginine dc, group iv eucaryotic ornithine and arginine dc as well as the procaryotic biosynthetic type of arginine dc and diaminopimelate dc. (n-l) profile analysis, a more stringent application of profile analysis [gribskov et al. (1990) methods enzymol. 183, 146-159] established the homology among the enzymes of each group. a search with the profile of group ii indicated a distant relationship with aminotransferases and thus with the ~ family of plp-dependent enzymes. no evidence was obtained that groups i, iii and iv are related with other plp-dependent enzymes or any other protein in the database. apparently, the amino acid dc are of multiple evolutionary origin, in some cases even if they have the same substrate specificity. a. lo russo, a.c. passaquin, u.t. r~eg~, ecole de pharmacie, %hiv. lausanne, c~-1015 lallsaraqe drug-induced local vasoccmstricticn appears to be respmnsible for the hypertensive side effect of the imn/nosti~pressant cyclosporin a (csa). we have therefore ex~rnir~ the [ca 2+] i increase caused by the vasoccr~trictor hormcme [ar~]vascpressin (avp) in vascular ameoth ~uscle cells (vsmc) treated with csa. [ca2+] i levels were m~nitored either with a fluoresc~qce analyser using fura 2 or by 45ca 2+ efflux. pretreatment of v~mc with csa increased the avp induced rise in [ca2+]i. a leftward and upwsxd shift of the ccncentraticm-respmnse curve of the avp-induced rise in 45ca2+ efflux was also observed after csa treatxr~%t. ilzg/cticn of csa dote~tiaticn w-as detectable after 3 rain, took 1 to 2 h to become fully established and was reversed after washout. csa potentiaticm was cc~centraticn-deperdent with a threshold at 10 -7 m. %]]is potentiating effect of csa may be the underlyin~ cause for csa-ind/ced hypert~3sicn. 92) barja, f. 1 and turian, g. i, ilab. gen. microbiology, 2dept. biochemistry, 3station exp. zoology, university of geneva, 30 quai ernest-ansermet, 1211 geneva 4 the actin has been found from the simplest forms of cell to the highly evolved ones. in higher organisms it is transcribed by multigene families but in yeast and several filamentous fungi there appears to be only one actin gene. it was therefore of interest to study the expression of actin gene in n. crassa in which three actin isoforms have been characterized (barja et al., 1991, fems left. 77:19-24) . after isolating genemic dna from wild type n. crassa a pcr was performed with primers corresponding to actin consensus sequences and an amplified fragment of the gene (verified by sequencing) was obtained. this fragment was 'used as a probe in a southern to assess the number of aetin gene(s). our results suggest that n. crassa has alsc only one actin gene. the blocking effect of the n-terminal decapeptide of msmooth muscle actin (sma) (ac.eeedstalvc) on the monoclonal antibody anti-asm-1 was compared with that of synthetic peptides modified by changing the n-terminal acetyl group or by substituting a single amino acid in position 1 to 5. using immunofluorescence or immunoblotting techniques, anti-c~sm-1 activity was abolished after incubation with the native peptide and peptides modified in position 1 (only when e~a) or 5. on immunoblots running bsa crosslinked peptides on denatured gels, only the natural peptide and peptides with substitution in position 1 or 5 were detected by the antibody. our results indicate that ac.(e)eed is the epifope for anti-c~sm-l. binding of anti-~sm-1 to sma increased actin polymedzation by decreasing the critical concentration. as control this action was not exerted on skeletal actin. this is the first example of the role of a precise n-terminal sequence in the polymerization of a single actin isoform. double immunofluorescence for msma and total actin on cultured aortic sm cells microinjected with the native peptide showed a selective disappearance of msma staining suggesting that this peptide traps a protein involved in msma polymerization. work is in progress to search for the putative actin-binding protein(s) physiologically interacting with the n-terminal sequence of o~-sma. ( within mitochondria, the mechanism of selective protein degradation is poorly understood. while the bulk of mitochondrial proteins are long-lived, some are degraded rapidly. the selective degradation of mitochondrial proteins is likely to be mediated by proteases similar to those found in bacteria; this is based on the hypothesis that mitochondria have evolved from bacterial endosymbionts, in conjunction with the finding that mammalian mitochondria contain an atp-dependent proteolytic activity similar to that in bacteria. one atp-dependent protease from bacteria, lon, is of particular interest because it is involved in regulated protein turnover. degenerate oligonucleotide primers corresponding to two regions of the bacterial lon gene were used to pcr amplify a 1 kb fragment from yeast dna that encoded an open reading frame with striking similarity to the bacterial lon protease. by screening a yeast cdna library, we isolated a 3.6 kb clone potentially encoding a protein of 1133 amino acids. haploids bearing a disruptedlon gene were unable to respire or to grow on ethanol/glycerol. fractionation of rnjtochondrial matrix from wild-type cells revealed a peak of aip-dependent proteolytic activity which was absent in the disruptant. furthermore, we have identified matrix proteins that are degraded with a half-time of ~60 min in intact wild-type ceils but are stable in the ion disruptants. electron microscopy of lon disruptants revealed the presence of many electron dense bodies in the mitochondrial matrix which are not found in lon + cells. hydrolysis of high molekular weight kin1nogen by plasma kallikrein benner, s. and duffer, h., laboratory for organic chemistry, eth h6nggerberg, ch-8093 z/inch high molecular weight kininogen (hmwk) is cleaved by the serineprotease plasma kailikrein to generate the hypotensive peptide bradykinin in human blood. we established a measuring system for the time course of the hydrolysis of hmwk in its native and deglycosylated form and in the presence or absence of c~-inhibitor. analysis of the bradykinin release and the yielded protein fragments lead to the three following results: 1. the hydrolysis did not follow plane michaelis-menten kinetics due to a hmwk-fragment operating as a competitive inhibitor. 2. addition of the ci-inhibitor stopped the hmwk-hydrolysis immediately, which is in contrast to the physiological role of plasma kallikrein in blood. 3. so far we have no evidence that the high content of o-glycosylated side-chains of the hmwk-light chain has an effect on the kinin-liberation as proposed in literature. ( tetrahydrobiopterin (bh4) is the essential cofactor for the hepatic phenylalaoine hydroxylase, but also for tyrosine and tryptophan hydroxylases that are responsible for the production of nettrofransmitter precttrsors. furthermore, bh4 is required for the deavage of giyceryl ethers aztd is i~vo[ved in the biosynthesis of nitric oxide by the nitric oxide synthase. a vazia~t type of hyperphenylalani~emia is caused by a deficiency of bh4. the most frequent form of this cofactor deficiency is due to lack of 6-pyruvoyl-tetrahydropterin synthase (ptps) activity. the human cdna for ptps was isolated, and the recombinant protein was found to be active when expressed in e. coil, thus allowing us to perform hmctional t e,3ting of mutant proteins. primary skin fibroblasts derived from ~everal ptps deficient patieilts were investigated for the molecular nature of this autosomal recessive disorder. all fibroblasts had f1"ps mrna expressed, and subsequent cdna sequence analysis revealed different mutatkons in the coding region of ptps (codon replacemenls, deletions, or nonseme codons) responsible for a lowered or no enzyme activity. in order to potentially correct ptps activity (and restore bi-i 4 biosynthesis) we are establishing a recombhtant rel~oviral-mediated gene transfer system to efficiently introduce the wild-type human ptps-cdna in these fibroblasts. the thylakoid membrane (tm) contains i0 molecular species of phosphatidylglycerol (pg). the relative amount of these species depends on the plant species and growth conditions. using phospholipase a2 (pla2) at 0~ to digest preferentially pg in the outer monolayer of spinach tm, we have shown previously that this monolayer was enriched in pg (ca 70~). the purpose of this investigation was to determine the transmembrane distribution of each pg molecular species. to this aim, we have studied the hydrolysis kinetics of each species in the presence of pla 2 in spinach and squash tm containing different relative proportion of molecular species. the hydrolysis rate and extent of the molecular species depended on the unsaturation degree of the fatty acid at the sn-i position as well as on the presence or absence of 16:1(3t) at the sn-2 position. for instance, the hydrolysis rate of pg ig:3/16:l(3t) was greater than that of pg 16:0/16:0. these results will be discussed in terms of the thylakoid transmembrane distribution of each pg molecular species. (supported by the snsf 3100-33693.92). from previous electrophysiological evidence, we concluded that metal ions (hgz+,cd2+,ag +) at low concentrations (i0-6m) increase rapidly (10-60 s) cation (na + , k + ) conductance at the apical membrane of epithelial cells. we investigated here the effects of these metals on [ca2+]i in mdck-i cultured cells, for a potential correlation between functional membrane changes and [ca2+]i . metals (10-4-10 -6 m) were added at the apical side of the epithelium, and [ca2+]i was measured with fura-2. hg 2+ induced a rapid (peak 5-10 s) and marked increase in [ca2+]i, whereas cd 2+ entailed a slower (peak 2-5 min) and marked response. the time-course of effects for both metals was similar to the electrophysiological changes. in contrast, the pattern of effects of ag + on [ca2+]i differed markedly from that on apical membrane conductance. thus, the effects of metals on [ca2+]i are conspicuous but not tightly associated with changes in membrane conductance. c. bulliard and l-l. dreyer, institut de biochimie, universit6 de fribourg, ch-1700 fribourg trans-plasma membrane oxydoreductases (pmo) play a significant role in energy transduction and post-receptor signal transmission, but the enzymes have not been characterized so far. we have achieved the purification of pmo from rat brain synaptic vesicles and from synaptic plasma membranes. the membrane enzymes were extracted by 1% lubrol xl tm, precipitated with ammonium sulfate (between 40 and 70% saturation) and purified by means of hydrophobic chromatography on butyl-agarose and gel f'fltration on superose-12, followed by page under native conditions. specific enzymatic staining of native page gels yields two homogenous diaphoraselike activities, pmo-i and pmo-ii . sds-page of the enzymes showed that pmo-i is made of two subunits of 52 kda and 40 kda whereas pmo-ii consists of a single sub-unit of 52 kda. the 52 ki)a subunits from pmo-i and pmo-[i are probably identical from their respective properties. partial n-terminal sequencing (13 aa) of the 40 kda subunit from pmo-i showed 84% homology with rat brain fructose-l,6-bisphophate aldolase. these data indicate that pmo is closely associated with the glycolytic enzyme that co-purifies in all steps under suitable conditions. the biochemical functions of pmo's remain to be established. ferredoxin:thioredoxin reductase (ftr) is the key enzyme in the ferredoxin/thioredoxin system, the light-dependent regulatory system in oxygenic photosynthesis. it is a nucleus encoded ironsulfur protein, composed of two dissimilar subunits, one of which is the catalytic subunit containing a redox-active disulfide bridge functional in the reduction of thioredoxins and a 4fe-4s cluster. using pcr we have isolated and characterized a cdna clone of 738 bp from spinach and a clone of 911 bp from corn coding for the catalytic subunits including the transit peptides. the first 31 (spinach) resp. 38 (corn) amino acids after the start exhibit typical features of chloroplast transit peptides. following the transit peptides are 113 (spinach) or 114 (corn) ami[~o acids representing the catalytic subunits. the primary structures are highly conserved between these two species with 91% similarity and 81% homology. seven of the eight cys residues found in the spinach subunit and important for the catalytic activity are at conserved positions. (snf 31-28811.90 and 31-37725.93) $23-20 r. zurbriggen and j.-l. dreyer, institut de biochimie. univarsit6 de fribourg, ch-1700 fribourg most mammalian cells display transplasma membrane oxidoreductase activity (pmo). the enzymes use intracellniar reduced pyridine nucleoticle to reduce an unspecific extracellular electron accepter as substrate. in order to set up a methodology for purifying, characterizing and quantifying pmo's, the plasma membrane from a neuroblastoma cell line, nb41a3, has been biotinylated. subsequently the biotinylated proteins were purified by immanoprecipitation with avidin, antiavidin-antibodies and insoluble protein a. the protein recovery of an immunopurified membrane preparation was <0.15% of the protein content in the cell extract. specific pmo activity was increased 15 to 20 fold compared to the activity in whole cells. this approach demonstrates the presence o1' pmo within the cell plasma membrane. this activity accounts for about one third of the total cellular diaphorase activity and cannot be attributed to an increased perraeabilization of the plasma membrane induced upon biotinylation nor to intracelluiar activity from lysed cells. pmo also promotes cell growth at low substrate concentrations (0.1-ll.tm). native gel electrophoresis of iarinobiotinylated and affinity purified plasma membrane extracts displays two diaphorase-positive bands, indicating that a homogeneous cell population may express several pmo activities at the plasma membrane. fluvastatin (fs) is a new hmg-coa reductase inhibitor. its affinity for 3 major human drug metabolizing p450 monooxygenases (cyp2c9, cyp2d6 and cyp3a4) was determined in liver microsomes. fs showed low affinity (ki > 50 ~tm) for cyp2d6 and cyp3a4 whereas cyp2c9 was selectively and competitively inhibited (ki = 0.1 ~tm). the potential for in vivo fs interactions with cyp2c9 substrates was therefore investigated in 14 volunteers using dicinfenac (df) as a probe (25 mg orally) on days 0, 1 and 8. df and 4'-ho-df kinetics were determined by hplc/uv. df cmax increased over time (0.28 [sd 0.12], 0.38 [0.20] and 0.45 [0.4] mg/l on day 0, 1 and 8, resp.). oral clearance was reduced on days 1 and 8 (14 % and 15 %, resp.). a time dependent decrease in urinary metabolic ratio (4'-ho-df/df) was noted (1.07 [0.34], 0.90 [0,23] and 0.70 [0.18] on day 0, 1 and 8, resp. (p < 0.0001)) only over the first 4 hours. fluvastatin therefore exhibits a two-phase interaction in vivo: an early transient and a late cumulative inhibition. the transient phase matches inhibition profiles predicted by quantitative models integrating in rive pharmacokinetics and in vitro inhibition data (q-dips). simple competitive inhibition by fs cannot explain the late phase. other mechanisms (i.e. fs or metabolite cumulation, mi complexation) are hypothesized. in some situations fs is expected to cause interactions with cyp2c9 substrates, which are partially predicted by quantitative modeling of in vitro data (snsf project n o 32-36600.92). 1211 gen~ve, switzerland. previously, a parvalbumin mutant, pvf~02w, was constructed with a unique reporter trp in the middle of the hydrophobie center. in the present study three new parvalbumin mutants, derived from pvft~w and containing alterations essential for ca2+-binding in either one, pv.câ�¢ and pv_m, or both, pv-co~, of the two ca2+-binding sites, were analyzed in order to study the metal binding properties of individual ca~+-binding domains and their contributions to the structure and stability of the protein. both, pv_ct, and pv.~, bind i ca ~ ⧠with affinity constants, kc,, of 1.1,107 and 3.2-106 m "~ in the absence of mg 2+. mg 2+ shifts the ca~+-binding isotherms of pv.co to higher free [ca z+] according to the competition equation with km~ = 2 9 103 m t. pv.m~ is much less effected by mg z* (kmg = 80 m "t) and can be considered as possessing a ca2+-specific site. nevertheless, in the absence of ca 2 ⧠both, pv.co and pv~, bind i mole of mg 2+ with much higher affinity than predicted from the competition studies. pv_cd;~ binds neither ca 2. nor mg 2+. trp-fluorimetry and uv-difference spectroscopy revealed that the ca~*-loaded conformations of pv_co, pv.ef and pvr~o~w are similar, with the trp-102 deeply buried in the hydrophobie core. however, striking differences between the mutants are found in the metal-free and mg~+-loaded forms. the conformation of pv~t~;~ is not affected by ca 2~ or mg z*. our results indicate that destroying the ca2+-binding of the ef loop, but not of the cd loop, strongly destabilizes the protein in the absence of ca z ⧠. biochemlsches institut der unlversitfit zfirich, switzerland, ch-8057. the sea urchin, strongylocentrotus purpuratus, metallothionein gene, mta, was constructed and expressed under the control of a heat shock promoter in a protease-deficient strain of e, coll. the nascent recombinant protein was stabilised by the addition of exogenous cd. the cd-containing protein was precipitated with ethanol and subsequently purified to homogeneity by gel permeation and ionexchange chromatography. the purified protein was identified as the desired gene product by tryptic peptide map analysis, sequence analysis and mass spectroscopy. typical yields of protein were 2mg per litre of culture. the presence of 7 cd ions per molecule was confirmed by the sh/cd ratio and by the existence of 7 h3cd nmr resonances. the apparent association constants of sea urchin mt with cd, which has a charge of -8, were found to be approximately 15 times weaker than for rabbit mt, at ph 7. (mt) are small metalloproteins displaying as their main feature clusters of d i~ metal ions bound to the thiolate ligands of the abundantly occurring cysteine residues (cys). from a set of over 85 sequences a general multialignment of 62 class i mt has been obtained on the basis of the needleman & wunsch algorithm. all cys are conserved in the man%mals and over 83% of them in other vertebrates and invertebrates. on the basis of similarity/identity scores we can isolate groups of sequences. evolutionary relationships between species and groups have been calculated with the maximum parsimony method of felsenstein and with the distance matrix method of fitch and margoliash. the divergences observed among the mammalian mts indicate a partition into four distinct subforms which all may have arisen before the separation of the species in evolution and which in part may differ in function. $23-28 bourque, a., singh, a., dykeman, a., macmahon*, a., and foster*, w. atlantic veterinary college, charlottetown, canada, and *reproductive toxicology section, environmental health centre, tunney's pasture, ottawa, canada. hexachlorobenzene (hcb) is a known environmental pollutant that is produced as an industrial by-product of certain chemicals. when administered in high dosage, hcb induces alterations in the rhesus monkey ovary. a purpose of the study was to determine a no observable adverse effect level (noael) for the compound. twenty, cynomolgus monkeys, 6-13 years old were placed in five groups. hcb was given orally in concentrations of 0.01, 0.1, 1.0, and 10 mg/kg b.w. in gelatin capsules, daily, for 13 weeks. one group of animals fed glucose only, served as the control. ovary specimens fixed in 2% buffered glutaraldehyde were prepared by conventional methods for transmission electron microscopy. ultrastructural alterations were revealed in the developing ova and follicular cells in all the ovaries from hcb-treated animals in a dose-related manner. clinical chemistry was unaffected by hcb. a noael for this reproductive toxicant is yet to be determined. a.b. was a recipient of the nserc undergraduate student award. an acidic haem-and zinc-containing protein has been isolated from bovine brain. native and sds-polyacrylamide-gel-electrophoresis reveal a monomeric protein with an apparent molecular weight of 47,2 kda. the absence of co-staining with tetramethylbenzidine implies that the haem group is non-covalently bound. the electronic absorption spectrum, with a soret-band absorption maximum at 412 nm and c~-and i~-bands at 540 and 575 nm, respectively, is similar to that of fe(ll)-myoglobin, consistent with the presence of haemiron in the ferrous oxidation state. in air the protein was easily oxidized to the fe(lll) form with a subsequent shift of the soretband maximum to 405 nm. atomic absorption measurements indicate approximately 1 mole of zinc and 1 mole of iron per mole of protein. the amino acid composition is similar to that of indoleamine-2,3-dioxygenase, although no such enzymic activity has been detected. in this study, we looked for this substance and its metabolites in young and old bovine lenses, because of their possible role in cataract formation. the substances were detected by hplc analysis. the kynurenine aminotransferase (kat) activity was determinated by the method of tobes (methods enzymol 1987~ 142:217) . the fluorescent substance formed from 3-hk was characterized by thin layer chromatography followed by reaction with rtinhydrin, uv and fluorescence spectrometry, and atom bombardment for molecular mass determination. 3-hk at concentrations of 0.08-+0.01, 0.2_+0.04, and 1_+0.4 btg/g of tissue was detected in iris/ciliary body, retina, and calf lens respectively. this substance is deaminated by kat. the activity of this enzyme was 2.6_+ 0.3, 3.7_+0.2, and 9.6+_2 ~tmol/g of tissue/hour in retina, iris/ciliary body, and lens respectively of old bovine eyes, but it was not found in calf eyes. the deamination of 3-hk resulted in the formation of a fluorescent substance which was identified as oxo-xanthurenic acid (oxa) with a molecular mass of 205 daltons. the accumulation of oxa acid possibly interacting with lens proteins could induce cataract formation. snf 32-36058.92, emdo, and hartmann-mtiller. the goal of covalent immobilization of oligonucleotide capture probes to solid supports has been accomplished by light-dependent, photolinker polymer mediated immobilization strategies. synthetic oligonucleotides were immobilized by facile layer coating procedures. the procedure does not require functionalization of the oligonucleotide probe or the matedal surface. oligonucleotides were linked to polystyrene with a biopolymer which was multiply substituted by photoactivatable diazirines. capture probe photoimmobilization (350 nm irradiation) was strictly light and diazirine dependent. using radiolabeled oligonucleotides as capture probes and polystyrene as material surface, the light dependent coupling efficiency was 40%. nonspecific oligonucleotide binding was below 2 %. photojmmobilized oligonucleotides retained the ability to form stable complexes with complementary dna strands, and target oligonueleotides of varying length were captured as well as dna fragments produced by pcr techniques. indoleamine 2,3-dioxygenase (ido), a superoxide radical scavenger, is present in transparent human lenses and produces 3-hydroxykynurenine, a uv-b filter. the synthesis of 3-hydroxykynurenine by ido and its degradation by kynurenine aminotransferase (kat) were measured in transparent lenses and cataracts. post-mortem eyes of elderly persons between 72 and 84 years of age with transparent lenses were obtained from the eye-bank of the department. fresh cataracts were obtained from cataract surgery patients. ido activity was measured by the method of yamazaki et al (biochem j 1985; 230:635) adapted for hplc. kat activity was measured by the method of tobes (methods enzymol 1987; 142:217) . ido activity found in post-mortem transparent lenses (cortex and nucleus) was 0.7_+0.3 nmol/mg protein/hour. in cataracts a low 1do activity of 0.3_+0.2 nmol/mg protein/hour was found in the cortex but not in the nucleus. kat activity, found in the eight cataracts examined, was 1.6_+0.9 nmol/mg protein/hour. inhibition of ido activity and induction of kat activity seems to be involved in human cataract formation snf 32-36058.92, emdo, and hartmarm-m011er photolinker polymer mediated covalent immobilization of antibodies and f(ab') fragments has been achieved by newly established lightdependent coupling procedures. anti alpha-l-fetoprotein monoclonal antibodies and f(ab') fragments derived from anti prostate specific antigen monoclonal antibodies were covalently linked to microplates. diazirine derivatized bovine serum albumin served as multifunctional light activatable linking agent. both immunoreagents, monoclonal antibodies and f(ab') fragments remained immunologically active after 350 nm irradiation (irradiance 0.7 mw cm -2 for 20 minutes). covalency of antibody binding was inferred from i) the photoreagent dependence, ii) the light dependence of the immobilization process, and iii) the reversibility of immunocomplexation after acid treatment. $23-33 two ~ of extracelluiar elecirical resistance in v~kwricular myocardil~ fleischhauer j.c., kl~ber a.g., dept. of physiol. bern in myocardial tissue, the electrical resistive properties of the extracellular space are important for local current flow during e~citation and therefore influence impulse conduction and the magnitude of the extracellular electrical field. to assess the r~tlti~t nature of the resistance of the extracellular space, electrical cable analysis was applied on an arterially perf~-sed rabbit papillary mascle. ~he resistivity of the intravascular space was changed (70 to 220~i~n) by variation of hematocrit frcrn 0 to 60% in the perfusate. in order to change the voltm~ and hence the electrical resistance of the interstitial space, colloidosmotic pressure in the perfusate was varied by changing dextran {m r 70000) concentration (i0 to 80g/l). altering the interstitial space volume had a marked influence on the extracellular resistance (ro) , conduction velocity and the magnitude of the extracellular field. variations of the resistive properties of the intravascular space had no significant influence on ro, conduction velocity and the magnitude of the extraeellular electrical field. our results suggest that the microvascular tree is insulated from the interstitial space and local electrical current flow during excitation in heart muscle is confined to the narrow interstitial clefts. s04-25, s 15-69 s05-18, s05-19 s 11-05, s11-06 bchini hooft van huijsduijnen s08-12 s05-30 s15-45 s08-13 s08-05, s08-06 s01-08 s15-67, $23-19 s13-03 nook, ek s01-15, s10-15 s05-02, s05-05 s 15-03, s15-29, s15-30 s19-03, s19-04, $19-05 s16-02, $16-15 s12-22 s15-48 s05-24 s03-01, s03-02, s03-03 s03-01 lrmler s05-28, s09-06 s03-07, s03-10, s03-11 s18-05 lipp, p. s05-26 so 1-02 s15-15, s15-16, $21-03, $21-11, $21-12, $21-14 $23-29,$23-30 s01-17 s 15-29, s15-30 s12-12, s12-24 molar s15-19 s08-05, s08-06 e s09-06, s09-07 s15-03, s15-29, s15-30 e. s03-05 s05-09, s05-10, s05-51 s05-52 s10-16 s08-14 s04-23, s04-29, s05-13, s05-53, $20-15 s10-16, s10-19 s09-01, s09-02 s15-68, s 17-24 stalder h. s03-19 s10-21 s09-09, s13-10, s13-13 s10-21 teheesen s01-10, s01-11, s01-18 s15-16 van dillewyn s05-32, s05-37, s05-38 s08-05, s08-06 s05-18, s05-19 huber d., ojha m. and turian g. laboratory of general microbiology, university of geneva, sciences iii, 30 quai ernest-ansermet, 1211 geneva 4, switzerland. ca2+-dependent protease in allomyces is predominantly localized in the apical region of the exponentially growing hyphae (huber and ojha, submitted) . many cytoskeletal proteins like actin and tubulin are also mainly localized in this growing region (heath, l~91). it is known that the ca2+-dependent protease proteolyzes a number of cytoskele~ ton proteins in order to regulate the plasticity of the cell (mellgren, 1987) . we have studied the proteolysis of some cytoskeletal proteins by this protease purified from the aquatic fungus allomyces erbuscula. actin, tubulin and desmin, were found to be rapidly proteolyzed in vitro at a high substrate to enzyme ratio. immunofluorescence studies of proteolyais made in situ in exponentially growing hyphae showed modification of apieally localized cytoskeletal proteins. this colocalization of the ca2+-dependent protease and cytoskeletal proteins in the same apical region is inferred in relation to hyphal elongation. jbiological databases grow exponentially. in order to provide access, as much data las needed and as little volume as possible shall be returned upon a query. the icurrentiy available dna and protein sequence databases are updated in a sophisticated network of procedures and expose a considerable amount of redundancy. research is performed on optimizing the creation of non-redundant data sets. the resulting data are disttibuted in switzerland, or made accessible to researchers who cannot utilize local resources. transparent access to the swiss resources, as well as paneuropean services, is provided with a job scheduling system which was developed in basel, the hierarchical access system for sequence libraries in europe ("hassle"), and various other computer programs which allow comprehensive browsing such as "gopher" and "www". training courses to use the resource effectively are running throughout the year. key: cord-336432-tu00gffr authors: wang, zhiyu; wang, yanfei; vilekar, prachi; yang, seung-pil; gupta, mayuri; oh, myong in; meek, autumn; doyle, lisa; villar, laura; brennecke, anja; liyanage, imindu; reed, mark; barden, christopher; weaver, donald f. title: small molecule therapeutics for covid-19: repurposing of inhaled furosemide date: 2020-07-07 journal: peerj doi: 10.7717/peerj.9533 sha: doc_id: 336432 cord_uid: tu00gffr the novel coronavirus sars-cov-2 has become a global health concern. the morbidity and mortality of the potentially lethal infection caused by this virus arise from the initial viral infection and the subsequent host inflammatory response. the latter may lead to excessive release of pro-inflammatory cytokines, il-6 and il-8, as well as tnf-α ultimately culminating in hypercytokinemia (“cytokine storm”). to address this immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. however, despite the obvious clinical utility of such biologics, their specific applicability to covid-19 has multiple drawbacks, including they target only one of the multiple cytokines involved in covid-19’s immunopathy. therefore, we set out to identify a small molecule with broad-spectrum anti-inflammatory mechanism of action targeting multiple cytokines of innate immunity. in this study, a library of small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. this has enabled us to identify the loop diuretic furosemide as a candidate molecule. to pre-clinically evaluate furosemide as a putative covid-19 therapeutic, we studied its anti-inflammatory activity on raw264.7, thp-1 and sim-a9 cell lines stimulated by lipopolysaccharide (lps). upon treatment with furosemide, lps-induced production of pro-inflammatory cytokines was reduced, indicating that furosemide suppresses the m1 polarization, including il-6 and tnf-α release. in addition, we found that furosemide promotes the production of anti-inflammatory cytokine products (il-1ra, arginase), indicating m2 polarization. accordingly, we conclude that furosemide is a reasonably potent inhibitor of il-6 and tnf-α that is also safe, inexpensive and well-studied. our pre-clinical data suggest that it may be a candidate for repurposing as an inhaled therapy against covid-19. covid-19, a potentially lethal infection caused by the sars-cov-2 virus, has emerged as a public health crisis of global concern. currently, there are no effective curative treatments for covid-19, affording little patient recourse beyond supportive care (cortegiani et al., 2020) . the morbidity and mortality of covid-19 arise from two competing pathological processes, the initial viral infection and the subsequent host inflammatory response, the latter of which may lead to excessive release of pro-inflammatory cytokines (interleukins (e.g., il-6, il-8) or non-interleukins (e.g., tnf-a)) and may culminate in hypercytokinemia ("cytokine storm"), a self-targeting inflammatory response syndrome (conti et al., 2020; mehta et al., 2020; qin et al., 2020; huang et al., 2020) . reflecting these dichotomous disease processes, current therapeutic development strategies may be categorized into two broad groups: anti-viral and anti-inflammatory. arguably, truly effective treatments may require both agents, since an antiviral will fail to suppress uncontrolled pro-inflammatory cytokine release once the process has been triggered. to address the covid-19 immuno-inflammatory pathogenesis, multiple clinical trials have been proposed to evaluate anti-inflammatory biologic therapies targeting specific cytokines. agents under consideration include tocilizumab (peking university first hospital, 2020; tongji hospital et al., 2020; national cancer institute naples, 2020) and sarilumab (regeneron pharmaceuticals & sanofi, 2020) , both monoclonal antibodies that target the il-6 pathway (sebba, 2008; boyce et al., 2018) , as well as adalimumab, which binds with specificity to tnf-a (furst et al., 2003) . data from a chinese study in which tocilizumab was given to 21 patients with severe covid-19 reported that the patients "improved remarkably" with 13 patients being discharged within 14 days or less following treatment, six patients within 14-21 days and only two patients stayed in the hospital for more than 21 days . however, despite the obvious clinical utility of such biologics for many disorders, their specific applicability to covid-19 is hampered by various issues: they target only one of the multiple cytokines implicated in covid-19's immunopathy; if administered systemically, they can predispose patients to secondary infections or other toxicities, such as hepatoxicity; and they may be expensive to mass produce and distribute. therefore, they are of limited utility in the context of a global pandemic. accordingly, we set out to identify a small molecule with the following properties: broad-spectrum anti-inflammatory mechanism of action targeting cytokines of innate immunity; low toxicity and excellent safety profile; chemically stable; easily stored and administered; able to be rapidly adopted in clinical settings worldwide; and, widespread availability with inexpensive and efficient means of production. as described herein, a systemic series of in silico and in vitro studies have enabled us to identify furosemide as a candidate molecule. in silico studies molecular docking simulations on the endogenous molecule (1,136, created in-house through literature search) and known drug datasets (1,768, taken from drugbank.ca) against il-6 and tnf-a were carried out in two steps. the first step involved placement of the endogenous or ligand molecule in an identified binding pocket (docking) of il-6 and tnf-a. in the second step, the calculation of the binding energies of the docked molecules (scoring) was completed. preparation of tnf-a and il-6 3d protein structures the tnf-a structure (pdb code: 2az5) and il-6 structure (pdb code: 1alu) were selected for molecular docking simulations in this work. we have employed these x-ray crystallographic structures because of three underlying motivations: (1) these x-ray crystallographic structures are solved for human il-6 and tnf-a proteins. (2) both of these x-ray structures have been solved at high resolution that is, 1 alu for il-6 at 1.90 å and 2az5 for tnf-a at 2.10 å, which minimizes the error while solving the structure from electron density maps. (3) the ligand molecules did not involve any metal atoms, which can otherwise bias the active site binding residues. the downloaded pdb structures were loaded into molecular operating environment (moe 2019.0101) software (molecular operating environment (moe), 2019). the loaded crystal structure was prepared employing the "quickprep panel" in moe, which contains the "structure preparation" feature. the "protonate3d" function was used to optimize ionization states of the added hydrogen atoms. water molecules, which were present 4.5 å away from ligand or receptor, were deleted. the mff94x force field, with rms gradient of 0.05 kcal/å, was used for energy minimization (panigrahi & desiraju, 2007) . a ramachandran plot was used to confirm the geometric and stereochemical qualities of the tnf-a and il-6 protein structures. the datasets of endogenous molecules (1,136) and drugs (1,768) were prepared using moe software. two functions 'wash at dominant ph of 7.4′ and "energy minimize" were used to obtain an energy minimized molecular form of molecules present at physiological ph of 7.4. the active site of il-6 and tnf-a was identified using the "moe-site finder" module. the "triangle matcher" placement method and "london dg" scoring functions were used for docking simulations. the poses from ligand conformations were generated in the placement phase. after placement of poses, the refinement was done with the "gbvi/wsa dg" scoring method using "induced fit" option. induced fit refinement allows both the ligand molecule and protein active site to move freely to facilitate residue alignment during docking simulations. thirty docked poses of the placement retention simulation and 10 poses of the refinement step were retained. the docking pose corresponding to the highest score for an endogenous or a drug molecule was considered for comparison of binding affinity within datasets. further details of descriptor calculation and docking simulations are provided in our recent publications (gupta et al., 2019 (gupta et al., , 2020 and in supporting information. validation of the docking protocol was carried out by re-docking the co-crystallized ligand in the active sites of the il-6 and tnf-a protein receptors. the following parameters were confirmed for validating the accurate docking protocol: 1. molecular superposition: the lowest energy docked conformation of a native ligand for il-6 and tnf-a is overlaid relative to the co-crystallized ligand present in the x-ray structure of the protein; the root-mean-square deviation (rmsd) has been calculated and is taken as an evaluation criterion for the validation of docking simulations. an rmsd value of <2.0 å between docked and crystallographic ligand conformation confirms successful docking (plewczynski et al., 2011; ramírez & caballero, 2018) . the docking protocol is validated if the docked ligand interacts with the active site of the protein similar to the corresponding co-crystallized ligand conformer and exhibits the same interactions with active site amino acid residues (plewczynski et al., 2011; ramírez & caballero, 2018) . inflammation activation on raw264.7 macrophage raw264.7 cells were purchased from atcc and maintained in dulbecco's modified eagle's medium (dmem) containing foetal bovine serum (fbs) at a final concentration of 10%. raw264.7 cells were seeded in 12-well plates at 0.25 × 10 6 cells/well seeding density, one day before experiments. to activate the cells, cell culture medium was changed to a lipopolysaccharide (lps) and interferon γ (ifnγ) containing medium with dimethyl sulfoxide (dmso) or furosemide at required concentrations, followed by 24 h incubation at 37 c; conditioned media and lysates were harvested for analysis. inflammation activation on thp-1 monocytic cells thp-1 cells (atcc) were maintained in rpmi 1,640 medium supplemented with 2-mercaptoethanol at a final concentration of 0.05 mm and fbs at a final concentration of 10%. thp-1 cells were seeded in each well of a 12-well tissue culture plate at a density 0.5 × 10 6 cells/ml, one day before experiments. thp-1 monocytes were differentiated by 150 nm pma (phorbol 12-myristate 13-acetate) for 24 h. cells were then treated with lps+ifnγ with dmso or furosemide at the required concentration, followed by 48 h incubation; conditioned media and lysates were collected for analysis. nitric oxide production from the conditioned media of raw264.7 and sim-a9 cell cultures was examined by a griess assay. conditioned medium and sulfanilamide were mixed in a microwell plate to form a transient diazonium salt. then, n-naphtylethylenediamine was added to all wells to form a stable azo compound by incubating for 5-10 min in the dark at room temperature. the absorbance was measured between 520 nm and 550 nm. the concentration of no production was quantified by being plotted against a standard curve. cytokines were quantified using elisa kits following the manufacturer's instructions. briefly, the high-binding plates were coated at 100 ml/well with diluted capture antibodies (1:250) at 4 c overnight. the coated plates were then blocked with the diluent for 1 h before assay. each sample was diluted accordingly and added to the plates for a 2 h incubation period at room temperature. plates were then washed with 250 ml/well pbs with 0.05% tween-20 and incubated with detection antibodies (1:250 in assay diluent) for 1 h at room temperature. after another washing step, 1:250 diluted avidin-hrp (horseradish peroxidase) was added and incubated for 30 min. next, 100 ml tmb-substrate (3,3′,5,5′-tetramethylbenzidine) was added and the plate was incubated in the dark until the signal was sufficiently developed. the final concentration of tmb substrate solution was 200 µg/ml. the reaction was stopped with 50 ml of 2 n sulfuric acid. absorbance was measured at 450 nm with a correction of 570 nm using a plate reader. cells were washed twice with ice-cold pbs and harvested in ripa buffer supplemented with a protease inhibitor cocktail. the whole-cell extracts were then centrifuged at 22,000×g for 20 min at 4 c to remove cell debris. protein concentrations were quantified using a micro bca protein assay kit. the absorbance was measured at 595 nm using a microplate reader. equal amounts of cellular protein were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) and transferred onto polyvinylidene difluoride (pvdf) membranes at 100 v for 90 min. the sds-page was performed with 10% polyacrylamide gel for inos and 12.5% for pro-il-1β. the membranes were blocked for 1 h in tris-buffered saline (tbs), ph 7.4, with 0.1% tween-20 (tbs-t) containing 10% skim milk. the membrane blot was then incubated overnight at 4 c with primary antibodies against inos (1:1,000), il-1β (1:1,000), actin (1:5,000) and gapdh (1:5,000) in tbs-t containing 5% skim milk. the membrane was washed with tbs-t 3 × 10 mins and incubated with goat anti-rabbit igg-horseradish peroxidase (1:5,000) for 1 hour. after the washing step, the immunoblotting was visualized by chemiluminescence hrp-substrate. cells were harvested and re-suspended with staining buffer. cells were stained with antibody (1:100) by incubating at 4 c for 30 min in the dark. stained cells were centrifuged, and the supernatant was discarded. the cell pellets were then re-suspended in cell flow buffer, transferred to facs tubes and analyzed by flow cytometry within 48 h. to all cells in the experiment, fc blocker was added. sim-a9 (atcc) cells were maintained in dulbecco's modified eagle medium: nutrient mixture f-12 (dmem-f12) with 10% foetal bovine serum, 5% horse-serum and antibiotic-antimycotic. sim-a9 cells were seeded 24 h before the experiment. culturing medium was replaced with dmem-f12 medium containing 5% fbs + 2.5% horse serum with required lps concentration (final volume was one ml/well). the conditioned media and lysates were harvested for cytokine and cell marker examination. data are presented as mean ± sd or ±sem. statistical analysis was performed with graphpad prism software version 6.01c, applying a two-tailed unpaired t-test. a p-value of >0.05 was considered significant. a screening strategy was devised for identifying an initial compound with potential broad-spectrum anti-inflammatory activity targeting relevant cytokines of the pulmonary innate immune system. since immunologically-mediated inflammatory responses in the human body are subject to tight homeostatic regulation, there exist multiple endogenous compounds capable of either up-or down-regulation of innate immune processes. accordingly, we sought to identify an endogenous compound as an initial molecular platform around which to devise a therapeutic. a library of 1,136 small molecules endogenous to the human body was assembled, subjected to in silico molecular docking simulations and a focused in vitro screen to identify anti-pro-inflammatory activity via interleukin inhibition. multiple compounds within the tryptophan metabolic pathway were identified, both indoleamine and anthranilate metabolites. in particular, 3-hydroxyanthranilic acid (3-haa) was found to demonstrate significant anti-inflammatory potential (in accord with previous studies by others, such as lee et al. (2013) , who have shown significant activity of 3-haa in inhibiting il-6 and tnf-a). regrettably, 3-haa is a small polar molecule with poor drug-like properties and is not an approved therapeutic for human use. the task of converting 3-haa to a drug or drug-like compound can be addressed via two approaches: (1) synthesis of new chemical entities with drug-like properties based on the 3-haa scaffold, or (2) identification of known drugs with structural properties similar to the 3-haa scaffold with the goal of repurposing. because of the urgency imposed by the unfolding pandemic, approach 2 was selected to provide a short-term solution. accordingly, 1,768 known drugs were computationally evaluated for anthranilate structural components. this screen identified mefenamic acid (n-(2,3-xylyl)-anthranilate) and furosemide (4-chloro-5-sulfamoyl-n-furfuryl-anthranilate) as the two strongest leads. mefenamic acid is known to provide significant protection against elevated levels of tnf-a and il-1β in radiation-induced genotoxicity of human lymphocytes (armagan et al., 2012; hosseinimehr et al., 2015) ; furosemide is known to significantly reduce production of il-6, il-8 and tnf-a in bronchial inflammation of asthma (prandota, 2002; yuengsrigul, chin & nussbaum, 1999) . this screen also identified other loop diuretics structurally related to 3-haa and furosemide, including bumetanide, piretanide and azosemide (fig. 1) . arising from the observation that furosemide can reduce bronchial inflammation, can be administered by inhalation (vide infra, discussion) (prandota, 2002; inokuchi et al., 2014; waskiw-ford et al., 2018) and is a widely available drug, furosemide was selected to be explored for repurposing in the treatment of covid-19. furosemide has been used worldwide for decades as a loop diuretic working via the renal na + /k + -atp shuttle pathway. to pre-clinically evaluate furosemide as a putative covid-19 therapeutic, a series of in vitro efficacy assessments was performed to investigate its anti-inflammatory properties. first, we investigated if furosemide could reduce the release of pro-inflammatory cytokines induced by lps in macrophage cell line. raw264.7 macrophages were stimulated with lps in the presence or absence of furosemide and the levels of tnf-a and no were measured from the conditioned media using elisa and griess assay, respectively. the results in figs. 2a and 2b show that lps induces the production of no and tnf-a, indicating macrophage polarization to an m1 pro-inflammatory phenotype. when cells were treated with lps in the presence of 25 mm of furosemide, the production of no and tnf-a significantly decreased. to further investigate the reduction of no production by furosemide, we determined the expression level of inducible nitric oxide synthase (inos) which produces no in response to inflammatory stimulations. therefore, we stimulated raw264.7 macrophages with lps and ifnγ. the expression of inos was significantly induced by stimulation with lps and ifnγ, as shown by the western blot results in fig. 2c . we found that furosemide was able to suppress the figure 1 screening approach with chemical structures of important compounds. with the goal to find a small therapeutic with anti-inflammatory properties and endogenous to the human body, a library of 1,136 small molecules was screened by docking simulations and further investigated by in vitro experiments. this intensive screen yielded 3-hydroxyanthranilic acid (3-haa) as initial hit. since 3-haa is not approved for medical applications, 1,768 known drugs were screened computationally for a 3-haa substructure. besides mefenamic acid, which is not displayed in this figure, the diuretic furosemide was the strongest lead directly followed by bumetanide, azosemide and piretanide. full-size  doi: 10.7717/peerj.9533/ fig-1 expression of inos during lps and ifnγ induced stimulation. densitometry analysis showed that normalized inos/gapdh ratio was reduced from 1 to 0.88 by furosemide. lps is recognized by the cell surface pattern-recognition receptors such as the toll-like receptor 4 protein (tlr4) and triggers downstream signaling pathways. lps induces expression of the pro-inflammatory cytokine ifnγ. ifnγ is known to increase tlr4 expression which may then promote the response to lps stimulation. we investigated the effect of furosemide on lps-induced tlr4 expression in raw264.7 macrophage cells using flow cytometry. as shown in fig. 3 , lps increased the tlr4+ cell population significantly, whereas il-4, an anti-inflammatory cytokine, barely induced tlr expression from the cells. interestingly, furosemide completely blocked lps-induced tlr4 expression, suggesting the possible involvement of furosemide in the lps-or ifnγinduced inflammation. as the next stage of macrophage activation, we studied the activity of furosemide on the expression of il-1β which plays a key role in modulating inflammatory response, as a downstream pro-inflammatory marker of the tlr4 signaling pathway by using differentiated thp-1 monocytes. we analyzed the expression of il-1β precursor protein (pro-il-1β) from thp-1 macrophage cells by using western blot analysis. furosemide, as shown in fig. 4 , significantly decreases the expression of pro-il-1β in differentiated thp-1 cells, demonstrating yet another inflammatory cytokine targeted by furosemide. to further explore the effect of furosemide on different macrophages, we next tested it on sim-a9 cells. we stimulated sim-a9 macrophages with lps and analyzed the release of pro-inflammatory markers such as no, il-6 and tnf-a. the results in fig. 4 show that lps induced the production of no, il-6 and tnf-a from sim-a9 cells. similar to raw264.7 cell results, furosemide significantly reduced the production of all these pro-inflammatory markers from sim-a9 cells as well. we have tested three different macrophage cell lines for the effects of furosemide on pro-inflammatory markers. furosemide consistently reduced pro-inflammatory markers such as no production, secretion of il-6, tnf-a and expression of pro-il-1β from different macrophage cell lines, implying that furosemide has broad inhibitory activity against pro-inflammatory cytokines. then, we evaluated if furosemide exhibits any effects on anti-inflammatory cytokines. we measured the levels of anti-inflammatory cytokines from the conditioned medium of differentiated thp-1 cells after 48 h of stimulation with lps and ifnγ by multiplex assay using flow cytometry. furosemide induces the expression of il-4, interleukin-1 receptor antagonist (il-1ra) and arginase, which are anti-inflammatory markers, suggesting the polarization of thp-1 macrophage to an m2 phenotype (fig. 5) . together with the experiments discussed above, these results show that furosemide inhibits the expression of m1 pro-inflammatory markers and promotes the expression of m2 anti-inflammatory markers. thus, furosemide is a broad-spectrum anti-inflammatory drug candidate targeting multiple cytokines. the molecular mechanism of action whereby 3-haa and furosemide inhibit il-6 and tnf-a activity is unknown at this time and lies outside the scope of the present study. interestingly, in an in silico screen of possible binding sites for 3-haa and related drug anthranilates, we noted favorable interactions with both the il-6 and tnf-a proteins. whilst it is improbable that such interactions are completely responsible for the observed anti-inflammatory effects, this notable observation is worthy of future experimental evaluation. the docking protocol needs to be validated before any conclusive in silico results can be obtained. to validate the docking methodology, co-crystalized ligands for tnf-a and il-6 have been re-docked into the active sites of the corresponding proteins. table 1 shows the re-docking parameters that have been evaluated for confirmation of accuracy of the employed docking protocol. il-6 (pdb code: 1alu) has been re-docked with tartaric acid. after re-docking simulations, the docked conformer of tartaric acid is overlaid with the co-crystallized ligand in the pdb structure. figure 6a represents the overlay of the docked pose and the co-crystallized ligand conformer. the rmsd between the docked conformer and co-crystallized ligand is 0.6114 å, which is <2.5 å as recommended for successful docking method confirmation. furthermore, all the amino acid residues which are interacting with the native il-6 binding pocket (arg179, arg182 and gln175) are shown to be interacting with the docked conformer of the tartaric acid in the re-docking simulation (as shown in fig. 6b ). this further confirms that the re-docking method is able to accurately predict the binding mode and the active site of il-6. the tnf-a dimer inhibitor has also been re-docked with tnf-a for validation of the docking method. the overlay of the docked ligand with the co-crystallized ligand conformation is presented in fig. 7a. from fig. 7a it can be seen that the tnf-a dimer inhibitor 307 is superposed with the co-crystallized ligand conformer with an rmsd value of 0.1677 å, which validates the docking simulation methodology for tnf-a. the ligand interaction diagram of the docked conformer of tnf-a, is shown in fig. 7b . the amino acid residues present within 4 å of the active site are labeled i.e. leu57, tyr59, ser60, leu120, gly121, gly122, tyr151. these residues are present in the active site of the tnf-a dimer in the vicinity of co-crystallized ligand. the re-docking simulations of co-crystallized ligands for il-6 and tnf-a confirm the accuracy of the docking methodology employed in this study. the computational simulations of 3-haa and furosemide with tnf-a and il-6 are briefly outlined below. the binding of 3-haa in il-6 and tnf-a active sites is presented in fig. 8 . the docking score (s) of minimum energy pose of 3-haa in il-6 is −4.620 and in tnf-a is −4.487. in figs. 8a and 8c, the ligand 3-haa is shown in yellow and amino acid residues of binding pocket have been labeled. figures 8b and 8d represent the ligand interaction diagrams of 3-haa with active site residues of tnf-a and il-6. 3-haa interacts with the arg179 and arg182 in il-6 and docks in the tnf-a dimer site with residues tyr119, tyr59, ser60, leu120, gly121 and tyr151. this confirms that 3-haa interacts with il-6 and tnf-a in the same binding site as observed in re-docking simulations of co-crystallized ligands for the corresponding proteins. figure 9 presents docking simulations of furosemide with tnf-a and il-6, respectively. in figs. 9a and 9c, furosemide is shown in yellow and the amino acid residues of the binding site are labeled. the tnf-a protein complex (pdb: 2az5) contains four identical chains having 148 amino acids each and two bound ligands. the a and b chains were retained (coloured orange and purple) and the co-crystalized ligand was removed while preparing the protein structure for docking. figure 9a shows the minimum energy docking pose of furosemide in the tnf-a active site. the amino acid residues leu b57, tyr b59, gly b121, tyr a151, tyr a119 and leu a120 are involved in forming hydrogen bonding and π-π interactions (arene-arene, arene-h, arene-cation) with furosemide in minimum energy docked poses. the docking score of the minimum energy pose is −6.0854. the similar mode of binding in the active site of tnf-a as of co-crystallized ligand (as shown in fig. 7b ) and favorable binding energy indicates that furosemide fits well into the active site of tnf-a and inhibits its activity. figures 9c and 9d show the minimum energy docking pose of furosemide in the protein structure of human il-6 (pdb: 1alu), having 186 amino acid residues and a co-crystallized ligand (tartaric acid). the co-crystallized ligand had been removed during the structure preparation step of the il-6 protein. furosemide interacts with arg 182, gln 175, leu 33 and arg 179 in most of the minimum energy poses, which agrees well with the il-6 co-crystallized ligand tartaric acid binding mode. the docking score (s) of the minimum energy pose of furosemide with il-6 is −5.1343, which computationally supports the ability of furosemide to inhibit il-6 activity. the pathogenic mechanisms of covid-19 morbidity and mortality are diverse, though immuno-inflammatory contributions are likely a central player. it is now appreciated that covid-19 afflicted individuals with major respiratory symptoms have pathologically elevated levels of pro-inflammatory cytokines including il-6, il-8 and tnf-a (conti et al., 2020; mehta et al., 2020; qin et al., 2020; huang et al., 2020) . a logical therapeutic approach to the management of covid-19 thus includes a need to modulate immunotoxicity. tryptophan and its metabolites, particularly via the indoleamine-2,3-dioxygenase initiated pathway, have a previously described role as endogenous modulators of innate immunity. of these various metabolites, 3-haa has been identified to exhibit a significant anti-inflammatory ability to suppress inflammation mediated via multiple pro-inflammatory interleukin cytokines, including il-1β, il-6, il-8 and tnf-a (lee et al., 2013) . this motivated our search for an anthranilate-based 3-haa-like agent from a library of known drugs; furosemide emerged as a possible candidate from this search. in this study, as part of its pre-clinical evaluation, we studied furosemide's anti-inflammatory activity on multiple macrophage cell lines involved in innate immunity. as pattern recognition receptors, tlrs contribute to the recognition of the molecules that are commonly shared by pathogens, such as lps and viral nucleotides (alexopoulou et al., 2001) . the activation of tlrs triggers downstream signaling through the myd88-dependent pathway and eventually induces the activation of nuclear factor-кb (nf-кb) (oeckinghaus, hayden & ghosh, 2011) . nf-кb has been shown to play an important role in coronavirus infections. for instance, nf-кb activation was identified in the lungs of sars-cov infected mice and triggered the production of pro-inflammatory cytokines, such as tnf-a (dediego et al., 2014) . however, the mechanism of sars-cov pathogenesis is debated. an in vitro study suggested the nucleocapsid protein of sars-cov was crucial in the pathogenesis, although this correlation is cell-specific (liao et al., 2005) . on the other hand, sars-cov lacking the envelope protein (e protein) attenuated nf-кb activation and associated pro-inflammatory responses (dediego et al., 2014) . the pandemic outbreak of covid-19 is caused by the infection of sars-cov-2 that shares 79.5% identity to sars-cov . to investigate the potential use of furosemide as a therapy for covid-19, we developed in vitro assays using lps as exogenous stimulation to induce inflammatory responses. lps-induced tlrs activation is well studied: it interacts with tlrs and triggers nf-кb activation followed by pro-inflammatory responses (lu, yeh & ohashi, 2008) . in our in vitro assays, we aimed to ascertain if lps induces a similar "cytokine storm" as sars-cov-2 infection and if furosemide inhibits the production of pro-inflammatory cytokines or promotes the secretion of anti-inflammatory cytokine products. broadly conceptualized stimulated macrophages can be polarized to either an m1 pro-inflammatory phenotype or an m2 anti-inflammatory phenotype. we investigated if furosemide inhibits the production of pro-inflammatory cytokines (m1) or promotes the secretion of anti-inflammatory cytokines (m2) using various macrophage cell lines including raw264.7, thp-1 and sim-a9. upon stimulation, these cell lines initiated an immune response by producing cellular stress signals and secreting pro-inflammatory cytokines. these pro-inflammatory markers were reduced upon treatment with furosemide, indicating that furosemide suppresses the m1 polarization, including no, il-6 and tnf-a. more importantly, our study results demonstrated furosemide promotes the production of anti-inflammatory cytokine products (il-1ra, arginase), indicating m2 polarization. all these results strongly suggest the potential application of furosemide as an immunomodulating agent for such disease conditions in which the inflammatory burden of patients increases suddenly. it also suggests that furosemide can be used for treating in which the sudden increase of pro-inflammatory cytokines is part of the disease pathogenesis. furosemide is a small molecule with a molecular weight of 330.75 g/mol and relatively low lipophilicity (logp = 2.03) (hardman, goodman & gilman, 2001) . although the drug has low water solubility at ph 7, furosemide can be formulated in weakly basic buffer solution (ph 8) to achieve 10 mg/ml solutions suitable for intravenous administration. due to the presence of a primary sulfonamide and carboxylic acid group, furosemide is highly bound to albumin with a human plasma protein binding value of 98.6 ± 0.4%. the drug has a very low volume of distribution (v d = 0.13 ± 0.06 l/kg) and a relatively short half-life of 1.3 ± 0.8 h (hardman, goodman & gilman, 2001) . these pharmacokinetic parameters along with high plasma protein binding equates to low tissue distribution with furosemide being retained with the blood. alveolar macrophages are front line innate immune cells, playing a crucial role in the maintenance of lung homeostasis and lung tissue defense through various immune responses. tissue-resident alveolar macrophages are derived from the foetal liver and populate the alveoli shortly after birth. these macrophages are self-renewing and persist over the life span. however, exposure to environmental challenges and injury induces recruitment of monocyte-derived alveolar macrophages from circulating monocytes. the intrinsic molecular properties of furosemide are ideal for targeting macrophages recruited from the blood stream prior to their tissue distribution. furosemide exhibits a large therapeutic window and is listed on the who's list of "essential medicines"; it is readily available worldwide, is easily manufactured, and has a long record of safety and efficacy when given orally or intravenously. more importantly, furosemide may also be administered safely by inhalation. more than 20 years ago, the concept of inhaled furosemide was explored as an approach to reduce dyspnea, primarily based on the rationale that edematous airway mast cells would be reduced in size following diuresis (prandota, 2002) . however, further investigations established that the mechanism of action was not related to local diuretic effects or engagement of the na + /k + -atp shuttle. more recent studies have reported reduction in pulmonary il-6, il-8 and tnf-a levels upon administering inhaled furosemide to patients with conditions including tachypnea (armed forces hospital pakistan, 2016; university of cologne, 2012), bronchopulmonary dysplasia (university of north carolina et al., 2015) and chronic lung disease (beth israel deaconess medical center, 2014; mcgill university, 2016; oxford brookes university, 2015) . a 2018 double-blind, placebo-controlled trial by grogono et al. (2018) evaluated inhaled nebulized furosemide (40 mg furosemide in 4 ml 0.9% saline) in healthy adults, demonstrating effective relief of experimentally induced air hunger during dyspnea after multiple dosing per day with no untoward effects. therefore, accumulated data indicate that furosemide is a cytokine-targeting anti-inflammatory, which may be safely administered by inhalation multiple times per day. our in silico screening identified other loop diuretics with structural similarities to furosemide. bumetanide exhibits anti-inflammatory properties in lps stimulated raw264.7 cells, reduces lps-induced production of cytokines following direct pulmonary administration, and lowers levels of tnf-a production in lung-injured mice (hung et al., 2018) . bumetanide however failed to inhibit sodium metabisulfite induced bronchoconstriction in asthmatic subjects (o'connor et al., 1991) . piretanide and azosemide have also been variously studied in models of cytokine-mediated inflammation and bronchoconstriction (yeo et al., 1992) . since none of these agents are in widespread clinical use, we have elected to pursue the development of furosemide in covid-19 because of its worldwide availability in the time of a global pandemic. the potential use of furosemide in the anti-inflammatory treatment of covid-19 has strengths and weaknesses. furosemide is inexpensive and available in every country in the world; it is safe and has profound anti-inflammatory cytokine activity, particularly against il-6 and tnf-a. if administered orally, furosemide can produce a profound diuresis which would be a clinical detriment in a febrile and potentially dehydrated person. when administered orally, the pharmacokinetics of furosemide indicate that it would have primarily intra-vascular distribution, suggesting greater utility early in the course of the disease, but less so in later-stage ventilator-supported individuals in whom macrophage migration from bloodstream to pulmonary tissue has occurred. as the disease progresses, direct administration of furosemide to the lungs by nebulized delivery adequately addresses the need to have furosemide reach intra-alveolar macrophages. arguably, truly effective treatments may require both anti-viral and anti-inflammatory agents since the early stages of the infection are dominated by high viral replication. however, application of an anti-inflammatory agent only, especially in the current situation where an effective anti-viral is still missing, may have the potential to reduce the number of patients requiring mechanical ventilation and reduce cough as a symptom which could help limiting the spread of the virus. care must be taken to prevent viral contamination and bystander exposure during the aerosolized administration of the drug, but this can be achieved with appropriate cautions in place. we are currently pursuing a clinical study of inhaled furosemide in people with covid-19. covid-19 is a pandemic threatening global health. the need to identify innovative therapeutics which may be deployed rapidly and efficiently is a pharmacological priority. furosemide is a safe, inexpensive, well-studied small molecule which is a reasonably potent inhibitor of il-6 and tnf-a and may be administered locally to the lungs; pre-clinical data from in silico and in vitro experiments suggest that it may be a candidate for repurposing as an inhaled therapy against the immunopathologies of covid-19. a list of physiochemical descriptors used as initial screening test of the dataset; a list of promising drug candidates along with corresponding physiochemical descriptors; docking score 's' of promising drug candidates with il-6 and tnf-a; figures and ligand interaction diagrams of mefenamic acid, bumetanide, piretanide and azosemide docking into binding site of tnf-a and il-6. this information is available free of charge on peerj. this work was supported by the canada research chair program (donald f. weaver, canadian research chair tier 1). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. recognition of double-stranded rna and activation of nf-κb by toll-like receptor 3 neuroprotection by mefenamic acid against d-serine: involvement of oxidative stress, inflammation and apoptosis role of salbutamol and furosemide in ttn aerosol inhalation treatment for dyspnea-patients sarilumab: review of a second il-6 receptor antagonist indicated for the treatment of rheumatoid arthritis induction of pro-inflammatory cytokines (il-1 and il-6) and lung inflammation by coronavirus-19 (covi-19 or sars-cov-2): anti-inflammatory strategies a systematic review on the efficacy and safety of chloroquine for the treatment of covid-19 inhibition of nf-κb-mediated inflammation in severe acute respiratory syndrome coronavirus-infected mice increases survival adalimumab, a fully human anti tumor necrosis factor-alpha monoclonal antibody, and concomitant standard antirheumatic therapy for the treatment of rheumatoid arthritis: results of star (safety trial of adalimumab in rheumatoid arthritis) inhaled furosemide for relief of air hunger versus sense of breathing effort: a randomized controlled trial the origin, transmission and clinical therapies on coronavirus disease 2019 (covid-19) outbreak-an update on the status the brain exposure efficiency (bee) score the blood-brain barrier (bbb) score goodman & gilman's the pharmacological basis of therapeutics radioprotective effect of mefenamic acid against radiation-induced genotoxicity in human lymphocytes clinical features of patients infected with 2019 novel coronavirus in bumetanide attenuates acute lung injury by suppressing macrophage activation effectiveness of inhaled furosemide for acute asthma exacerbation: a meta-analysis the tryptophan metabolite 3-hydroxyanthranilic acid suppresses t cell responses by inhibiting dendritic cell activation activation of nf-kappab by the full-length nucleocapsid protein of the sars coronavirus lps/tlr4 signal transduction pathway inhaled nebulized furosemide & physical activity-related breathlessness covid-19: consider cytokine storm syndromes and immunosuppression integrated computer-aided molecular design platform. montreal: chemical computing group ulc. national cancer institute naples. 2020. tocilizumab in covid-19 pneumonia (tocivid-19) crosstalk in nf-κb signaling pathways specificity of dyspnoea relief with inhaled furosemide effect of inhaled furosemide and bumetanide on adenosine 5'-monophosphate-and sodium metabisulfite-induced bronchoconstriction in asthmatic subjects strong and weak hydrogen bonds in the protein-ligand interface favipiravir combined with tocilizumab in the treatment of corona virus disease 2019 can we trust docking results? evaluation of seven commonly used programs on pdbbind database furosemide: progress in understanding its diuretic, anti-inflammatory, and bronchodilating mechanism of action, and use in the treatment of respiratory tract diseases dysregulation of immune response in patients with covid-19 in wuhan, china is it reliable to take the molecular docking top scoring position as the best solution without considering available structural data? evaluation of the efficacy and safety of sarilumab in hospitalized patients with covid-19 tocilizumab: the first interleukin-6-receptor inhibitor hubei xinhua hospital, wuhan no.1 hospital, wuhan central hospital. 2020. tocilizumab vs crrt in management of cytokine release syndrome (crs) in covid-19 the emmes company llc. 2015. safety of furosemide in premature infants at risk of bronchopulmonary dysplasia (bpd) effect of inhaled nebulized furosemide (40 and 120 mg) on breathlessness during exercise in the presence of external thoracic restriction in healthy men effective treatment of severe covid-19 patients with tocilizumab protective effect of loop diuretics, piretanide and frusemide, against sodium metabisulphite-induced bronchoconstriction in asthma immunosuppressive and cytotoxic effects of furosemide on human peripheral blood mononuclear cells the authors declare that they have no competing interests. the following information was supplied regarding data availability:the raw measurements are available in the supplemental files. supplemental information for this article can be found online at http://dx.doi.org/10.7717/ peerj.9533#supplemental-information. key: cord-017639-wtc8bml5 authors: el-radhi, a. sahib title: pathogenesis of fever date: 2019-01-02 journal: clinical manual of fever in children doi: 10.1007/978-3-319-92336-9_3 sha: doc_id: 17639 cord_uid: wtc8bml5 the generation of fever involves the following steps: numerous substances from outside the body, exogenous pyrogens, initiate the fever cycle. endotoxin of gram-negative bacteria, with their pyrogenic component lipopolysaccaride, is the most potent exogenous pyrogen. fever is also a common finding in children without obvious evidence of infection, for example hypersensitivity reaction, autoimmune diseases and malignancy. exogenous pyrogens initiate fever by inducing host cells (primarily macrophages) to produce and release endogenous pyrogens such as interleukin-1, which has multiple biological functions essential for the immune response. endogenous pyrogens are transmitted to the hypothalamic thermoregulatory centre, specifically organum vasculosum of the lamina terminalis (ovlt), where they induce synthesis of prostaglandins, of which pge2 is the most important. these raise the thermostatic set point to initiate the febrile response. the hypothalamic thermoregulatory centre accomplishes heat production by inducing shivering and heat conservation through vasoconstriction. at an established degree, fever is regulated (even at a temperature of over 41.0 °c) and heat production approximates loss, as in health, though at a higher level of the set point. therefore fever does not climb up relentlessly. in addition to the function as an endogenous pyrogen, il-1 activates t-lymphocytes to produce various factors, such as inf and il-2, which are vital for immune response. the production of fever simultaneously with lymphocyte activation constitutes the clearest and strongest evidence in favour of the protective role of fever. the induction of fever results in inhibition of bacterial growth, increased bactericidal effects of neutrophils, production of acute-phase protein synthesis and other physiological changes such as anorexia and somnolence. these changes suggest that fever has an adaptive role in the host’s survival during infection. • although infection is the most common cause of fever, fever is also a common finding in hypersensitivity reaction, autoimmune diseases and malignancy. • febrile response is mediated by endogenous pyrogens (cytokines) in response to exogenous pyrogens, primarily micro-organisms or their direct products (toxins). • these endogenous pyrogens act on thermo-sensitive neurons in the hypothalamus, which ultimately upgrade the set point via prostaglandins. • the body reacts by increasing the heat production and decreasing the heat loss until the body temperature reaches this elevated set point. • fever, in contrast to hyperthermia, will not climb up relentlessly because of an effective central control of the hypothalamic centre. • cytokines play a pivotal role in the immune response by activation of the b cells and t-lymphocytes. the production of fever simultaneously with lymphocyte activation constitutes the clearest and strongest evidence in favour of the protective role of fever. • the protective processes of the immune response are optimal at high temperature (around 39.5 °c). • not all effects resulting from fever generation benefit the host; some are harmful and even lethal. this occurs mainly by overproduction of the cytokines or imbalance between cytokines and their inhibitors, such as severe and fulminate infections and septic shock. research in fever has been centred on the hypothesis that fever results from physiological processes that are set in motion by an external stimulus. egyptian scholars recognized that local inflammation was responsible for fever. in 1868, billroth (1829-1894) attempted to confirm this ancient observation by injecting pus into animals, thereby producing a febrile response. in 1943, menkin carried out similar experiments and isolated a product termed "pyrexin" [1] . beeson in 1948 isolated a fever-inducing substance from a leukocyte, leukocyte pyrogens, which later became known as endogenous pyrogen (ep). interleukin-1 (il-1) was first identified as a cytokine by gery and waksman and proved to be identical with ep [2] . • fever (pyrexia) is a regulated body temperature above the normal range occurring as a result of il-1-mediated elevation of the hypothalamic set point. once fever is established, body temperature is regulated, as in health, by a net balance between heat production and loss. • hyperthermia is an unregulated elevated body temperature above the normal range due to imbalance between heat production and loss. interleukins are not involved and therefore the hypothalamic set point is normal. • a pyrogen is a substance (infectious organisms or their product toxins or cytokines) that provokes fever. • exogenous pyrogens are substances, which originate outside the body and which are capable of inducing interleukins. • endogenous pyrogens are substances, which originate inside the body and which are capable of inducing fever by acting on the hypothalamic thermoregulatory centre. il-1, tumour necrosis factor (tnf) and interferon (inf) are endogenous of albumin and transferrin decreases. characteristically there are a decreased concentration of iron and zinc and an increased copper concentration. the low iron is the result of reduced intestinal assimilation of iron and increased liver storage of iron. these changes contribute to host defence by depriving invading micro-organisms of essential nutrients, such as iron and zinc. the process is referred to as nutritional immunity. • cytokines are proteins produced throughout the body, mainly by activated macrophages, monocytes and t cells to regulate the immune responses within the body, control inflammatory and haematopoietic processes and may induce fever. as they enter the circulation and act on distant organs, they are considered as hormones. pro-inflammatory cytokines (e.g. il-1, il-6, tnf-α, inf-γ, granulocytes-macrophages colony stimulating factor, gm-csf) are responsible for initiating an effective defence against exogenous organisms (e.g. activating neutrophils). their overproduction may be harmful by causing shock, multiple organ failure and death. anti-inflammatory cytokines (e.g. il-1 receptor antagonist, il-4, il-10) antagonize the pro-inflammatory cytokines and thus promoting healing and reducing inflammation. • monokines are cytokines that are produced by mononuclear phagocytic cells. • chemokines are cytokines that attract cells to the site of infection using chemical message (chemotaxis). typical chemokine is cxcl-8 that attracts neutrophils. • interleukins are cytokines, acting specifically as mediators between leukocytes, hence their name. their number known nowadays is enormous: at least 37 interleukins have been identified. if their amino acid sequence is known, they are assigned an interleukin number. if their sequence is not known, then they are named according to the biological property. il-1 and il-6 play a major part in the pathogenesis of fever. • lymphokines are cytokines that are secreted by lymphocytes to regulate the immune response. important lymphokines are il-2, il-3, il-4, il-5, il-6, il-9, il-10, il-13, il-14 and tnf-gamma. • prostaglandins are lipids that are made at sites of infection and tissue damage to produce inflammation and fever as part of the healing process. • acute-phase response is the term used for haematological, endocrinological and metabolic changes that follow (within hours or days) the onset of fever in response to infections or local damage to a tissue. these changes are induced by several cytokines (il-6 being the primary inducer), which are beneficial to the host. during the response, various acute-phase proteins, notably c-reactive protein (crp) and serum amyloid a, are synthesized by hepatocytes and released into circulation in large amounts. crp plays a role in complement activation, opsonization and increasing platelet aggregation. although acutephase response is closely associated with fever, crp levels can be normal in viral infections and high in diseases without fever (e.g. tumours). syntheses of albumin and transferrin and the concentration of iron and zinc decrease, while copper concentration increases. the low iron is the result of reduced intestinal assimilation of iron and increased liver storage of iron. these changes contribute to host defence by depriving invading micro-organisms of essential nutrients, such as iron and zinc. the process is referred to as nutritional immunity. exogenous pyrogens (e.g. bacteria, viruses, toxins) initiate fever, usually within 2 h of exposure, by interacting with macrophages or monocytes, leading to cytokine induction. other mechanisms to initiate fever include: • some endotoxins, produced by bacteria, act directly on the hypothalamus to alter the set point. il-1 is not involved. radiation of the hypothalamus, ddt (dichlorodiphenyltrichloroethane), poisoning and scorpion venom may also induce fever by a direct effect on hypothalamus. • exp may activate lymphocytes to secrete lymphokines, particularly inf-y, which in turn stimulate macrophages and monocytes to produce il-1. • some bacteria produce exotoxins, which stimulate macrophages and monocytes to release il-1. this mechanism operates in scarlet fever and toxic shock syndrome. in toxic shock syndrome, the shock is due to the toxin. diseases involving exotoxins produced by gram-positive bacilli are less fever-inducing than those produced by pyrogenic gram-positive cocci. • borrelia spirochetes (the cause of relapsing fever) do not contain endotoxin, and the attachment of these bacteria to the mononuclear cells induces il-1. • other bacteria, such as pneumococci, have no endotoxin or other pyrogens, and the mechanisms responsible for fever are presumably immunological • gram-negative bacteria. the pyrogenicity of gram-negative bacteria (e.g. escherichia coli, salmonella) is due to a heat-stable factor, endotoxin. the active components of endotoxin are lipid and carbohydrate (lipopolysaccharide, lps), which are the major components of the outer membrane of these bacteria. endotoxin causes a dose-related progressive increase in temperature. in severe cases, it causes shock with vasodilatation, capillary leakage and hypotension. septicaemia caused by gram-negative endotoxin does not elicit fever in certain situations such as neonates, young infants or children with fulminating infection or with malnutrition. these children may present with normal temperature or even hypothermia in response to severe infection. mortality is significantly higher in septic children [3] and adult [4] patients due to reduced capacity to release tnf-α and il-β upon infection with lps. • gram-positive bacteria. the main pyrogen of most bacteria is peptidoglycan that forms the cell wall. penicillin works by inhibiting the biosynthesis of peptidoglycan, which results in cell lysis. this explains why penicillin is more effective against gram-positive bacteria. • viruses. it is well known in clinical practice that viruses cause fever. mechanisms by which viruses may produce fever include direct invasion of macrophages, immunological reaction to viral components involving antibody formation, induction by inf and necrosis of cells by viruses. • fungi. live or killed fungal products are exogenous pyrogens that induce fever. the induction of fever mainly occurs when the fungi are in the bloodstream. children with neoplastic diseases who develop fever associated with neutropenia are at high risk for developing invasive fungal infection. • phagocytosis is largely responsible for fever in blood transfusion reactions (once an infection is excluded) and immune haemolytic anaemia. • antigen-antibody complexes. an exogenous antigen may react with circulating, sensitized antibodies to form a complex, which induces il-1 production (immune fever). examples of immunologically mediated fever include systemic lupus erythematosis and adverse drug reactions. fever associated with penicillin hypersensitivity results from interaction of antigen-antibody complexes with leukocytes, which release il-1. • most steroids are endogenous antipyretics, which suppress fever through their inhibitory effects on il-1 and tnf-α production as well as inhibition of prostaglandin synthesis. certain steroids, however, are pyrogenic in human such as etiocholanolone, a major metabolite of testosterone and 17-ketosteroid, which induce the release of interleukin-1. etiocholanolone produces fever only when injected intramuscularly (not intravenously). etiocholanolone fever is characterized by recurrent fever for few days, in association with arthralgia, abdominal pain, leukocytosis, high esr and etiocholanolone level. fever does not respond to antipyretics but to steroids, e.g. prednisolone. etiocholanolone is responsible for fever in some patients with adrenogenital syndrome. • other nonmicrobial pyrogens include some hormones, drugs and intracranial lesions such as bleeding and thrombosis mononuclear cells are leukocytes (3-8% of the leukocytes) and are largely responsible for the production of il-1 and fever induction. polymorphonuclear granulocytes are no longer thought to be responsible for il-1 production because fever may occur in their absence, e.g. agranulocytosis. the mononuclear cells are either circulating monocytes in the peripheral blood or tissue macrophages (histocytes) scattered in organs such as lung (alveolar macrophages), lymph nodes, placenta, peritoneal cavity and the subcutaneous tissue. the origin of both monocytes and macrophages is the granulocyte-monocyte colony-forming unit (gm-cfu) in the bone marrow. monocytes enter the circulation either to remain there for a few days as circulating monocytes or to migrate to the tissue where they undergo functional and morphological transformation into macrophages, when their life span is several months. these cells play an important role in: • host defence, including engulfing and destroying the microbe (phagocytosis) recognition of antigen and presenting it to attached lymphocytes. • activation of t-lymphocytes and tumour cell destruction. situations associated with reduced function of the mms include newborn infants, corticosteroid and other immunosuppressive therapy, systemic lupus erythematous, wiskott-aldrich syndrome (immune deficiency involving b and t cells, eczema and thrombocytopenia) and chronic granulomatous disease. the two major monocytemacrophage products (cytokines) are il-1 and tnf. endogenous pyrogens (ep) il-1 consists of three structurally related polypeptide, two agonists (il-1α and il-1β) and an antagonist (il-1 receptor antagonist = il-1ra), which inhibit the activities of the two powerful agonists. anakinra is a naturally il-1ra. il-1α is produced in: • cells of healthy people including in all epithelial cells of mucosal membranes. • blood monocytes and tissue macrophages. • hepatic kupffer cells, keratinocytes and pancreatic langerhans cells. • astrocytes in the brain tissue, which may contribute to the immunological responses within the cns and the fever secondary to cns bleeding. • cells from certain malignant tumours (e.g. hodgkin's disease, acute leukaemia and renal carcinoma). this explains the frequent association of fever in these conditions in the absence of infection. il-1β is not present in cells of healthy people and is mainly produced by monocytes, macrophages and dendritic cells. interleukin-1 has important roles in the following conditions ( fig. 3. 3): • induction of fever by acting on the hypothalamus to raise its set point. • induction of hepatic acute-phase proteins (see above). • induction of inflammatory response and lymphocyte activation factor. • appetite suppression. il-1 is a potent anorexic cytokine (more potent than the hunger inhibitor leptin), which explains the reduction of food intake commonly seen in febrile illness. il-1ra reverses the decrease in food intake. circulating il-1ra, along with il-18, are increased in obese and type 2 diabetes [5] . • il-1β (along with tnf-α) regulates sleep by promoting non-rapid eye movement sleep. this cytokine is produced in astrocytes of the brain. the action of il-1β may explain the observation of increased sleep in febrile illnesses. • joint diseases such as rheumatoid arthritis (ra) and ankylosing spondylitis. ra is an autoimmune mediated by il-1. treatment with anakinra prevents the migration of inflammatory cells into the joint. • inflammation of the blood vessels (vasculitis). • stimulating the liver to produce acute-phase proteins (see above). • congenital deficiency of il-1 receptor antagonist is associated with overwhelming inflammation of the skin, joints and bones and large infiltration of neutrophils. infants with this condition die early in life unless anakinra is given that rapidly reverses the inflammation and prevents the death [6] . • auto-inflammatory diseases are characterized by periodic fever due to recurrent episodes of systemic and local inflammation. anakinra has been successful in treating conditions such as familial mediterranean fever (see chap. 6). • il-1-mediated inflammation is contributing to the acute ischaemic conditions such as myocardial infarction, stroke, liver and renal failure. • hiv replication. il-1ra has suppressive effects on the virus. • macrophage activation syndrome is a life-threatening disease in patients who suffer from eb-virus or cytomegalovirus. anakinra causes rapid recovery. • diabetes (type 1 is an autoimmune disease mediated by t-lymphocytes; type 2 is associated with obesity and physical inactivity causing insulin resistance). both types produce high glucose concentration that stimulates il-1β causing destruction of the insulin-producing β-cells of the pancreas. tnf-α, discovered in 1975, is a pro-inflammatory cytokine produced by immune cells, e.g. monocytes and macrophages (tnf-α), lymphocytes (tnf-β), natural killer cells, kupffer cells, astrocytes and microglia of the cns, in response to invasive or injurious insults. tnf-α is an endogenous pyrogen acting on the hypothalamus to induce fever. unlike il-1, tnf has no direct effect on stem cell and lymphocyte activation. tnf-α has diverse beneficial biological effects, including: • sharing many biological properties with il-1, e.g. early enhancing host defence against infection, promoting normal tissue remoulding, including wound healing, enhancing chemotaxis of macrophages and neutrophils as well as increasing their phagocytic and cytotoxic activity. • stimulant (along with il-6) for acute-phase response. • crucial physiological processes in the cns such as learning and memory, sleep and water and food intake. the initial enthusiasm to use tnf-α as a systemic antitumour treatment has waned because of its significant toxicity and lack of therapeutical benefit. however, tnf-α blockers (monoclonal antibodies, infliximab, adalimumab and etanercept) have altered the outcomes for children with inflammatory bowel disease, such as crohn's disease and ulcerative colitis and rheumatoid arthritis. according to a systematic review of literature [7] , the treatment has resulted in a variety of infections in the treated children including bacterial, fungal, viral and tb. few children died. the antigen-specific cells of the immune system are lymphocytes, of which there are two main types: • b cells are responsible for antibody production. • t cells are the master regulators of the antigen-specific adaptive immune response. they regulate antibody synthesis and mediate cytotoxic function as well as inflammatory response of delayed-type hypersensitivity. t cells are either: -th1 cells which produce inf-γ, il-2 and tnf-β and promote cell-mediated immunity and phagocytic activity. -th2 cells which produce il-4, il-5, il-6, il-9 and il-10. these promote antibody production and play a crucial role in allergic responses (immediate-type hypersensitivity). il-1 has an essential role in the activation of lymphocytes. the t-lymphocyte recognizes antigen only after the antigens are processed and presented to them by macrophages; only then do t-lymphocytes become active. interferons are known for their ability to "interfere" (hence the name) with viral replication in infected cells. in addition, these cytokines have pyrogenic effect, antitumour and immuno-regulatory functions. there are three types, type 1 (ifn-α, ifn-β), type ii (ifn-γ) and type iii (ifn-λ). only type i is used as therapy. type i and iii are produced by a variety of cells (such as leukocytes, dendritic cells, fibroblasts and macrophages), whereas synthesis of type ii is restricted to t-lymphocytes. the functions of the interferons include: • key mediators of both innate and adaptive immune responses, stimulating b cells to increase antibody production, and increasing the efficiency of natural killer cells. • inhibition of viral replication including hiv-1 replication. • interferon-γ release assays (igra) serve as a useful blood test in patients with tb. the result cannot distinguish between latent and active tb, and it is not affected by bcg vaccination status. type i inf is used as a treatment for a variety of diseases, including: • various viral infections, in particular hepatitis b and c. • upper respiratory tract infection. inf-α in a nasal spray is capable of significantly reducing symptoms due to rhinoviruses, but not those due to influenza viruses, parainfluenza viruses or coronaviruses. • thrombocytosis associated with myeloproliferative disorders. toxic effects of inf preparations are numerous and include fever, chills, arthralgia, myalgia, severe headaches, somnolence and vomiting. fever may occur in over 50% of the patients who receive inf and may reach 40.0 °c. these side-effects are responsive to paracetamol and prednisolone. severe side-effects include hepatic and cardiac failure, neuropathy and pancytopenia. inf therapy is contraindicated in pregnancy owing to its anti-proliferative effect. il-2 is probably the second most important lymphokine (after inf), which is released by activated t-lymphocytes (in particular cd4 + and cd8 + ) in response to exogenous pyrogen. it has a crucial effect on the growth and function of t cells, natural killer cells and b cells and for the development of cd4 + t cells. cases of severe congenital combined immunodeficiency due to a specific defect in the production of il-2 have been reported. effects of il-2 include: • stimulating the release of other cytokines, including il-1, tnf and inf-γ. • as il-2 is also produced by mast cells, it controls the severity of chronic allergic dermatitis. • antitumour cytotoxicity (e.g. melanoma, including metastatic melanoma, renal cell carcinoma, acute myelogenous leukaemia) as a result of proliferation and activation of activated cytotoxic t lymphocytes. il-6 is the third most studied cytokines that has the following characteristics: • a pro-inflammatory, multifunction cytokine, which is secreted by macrophages and t-lymphocytes to stimulate both b and t cell function and immune response against infection. • acting on hepatocytes to induce acute-phase proteins such as crp, amyloid and haptoglobin. • an early marker of infection (preceding the increase of crp), responding within 3-4 h of bacterial infection, e.g. in early-onset neonatal bacterial infection. • increased in many diseases, e.g. sepsis, autoimmune diseases (e.g. systemic lupus erythematosus), kawasaki disease, tumours (e.g. multiple myeloma, renal cell carcinoma), brain disorders (e.g. astrocytoma, glioma, psychosis), and autoimmune and chronic inflammatory diseases. • is markedly elevated in many rheumatic diseases including systemic juvenile rheumatoid arthritis (jra) and ankylosing spondylitis. anti-il-6 receptor antibody, tocilizumab, has successfully been used to treat patients with jra. other cytokines with their main effects are shown in table 3 .1. of the four haematopoietic colony-stimulating factors (erythropoietin, granulocytecolony-stimulating factor (g-csf), macrophage colony-stimulating factor (m-csf), granulocyte-macrophage colony-stimulating factor (gm-csf)), gm-csf appears to have the most potential clinical benefits. it is a pro-inflammatory cytokine, which is produced mainly by lymphocytes, although monocytes, macrophages and mast cells are also capable of producing it. gm-csf's principal functions and potential therapeutic uses are: • to stimulate haematopoietic progenitor cells to proliferate and differentiate into granulocytes and macrophages, enhancing phagocytosis and promoting leukocyte chemotaxis and adhesion. • as a treatment in sepsis-associated immunosuppression. • it has a central role in the pathogenesis of autoimmune inflammatory diseases such as rheumatoid arthritis, multiple sclerosis and crohn's disease [8] . • approved to treat chemotherapy-induced neutropenia, myelodysplasia and aplastic anaemia associated with stem cell transplantation. • gm-csf alone or with il-4 have been used in cancer treatment, including melanoma, renal cell carcinoma and glioma. the administration of gm-csf may be associated with the development of fever, which is blocked by non-steroidal anti-inflammatory drugs such as ibuprofen. thermoregulation requires intact peripheral mechanisms, which balance heat production and loss, and a functioning hypothalamic thermoregulatory centre regulating these mechanisms. this centre receives thermoreceptors from the temperature of the blood as it passes through the brain (the core temperature) and thermoreceptors from the skin via the dorsal horn of the spinal cord. both thermoreceptors have cold and warm receptors. the activation of warm receptors causes inhibition of cold receptors. the aim of the thermoregulation is to maintain a relatively constant body temperature at 37 °c. heat production occurs by various mechanisms: • at rest as many organs such as the brain, muscles, viscera, liver, heart, thyroid, pancreas and adrenal glands contribute to heat production at the cellular level involving adenosine triphosphate (atp). • the newborn infants have no shivering due to skeletal muscle immaturity, and they rely on non-shivering thermogenesis to produce heat to protect newborns against cold exposure. brown adipose tissue (bat), localized mainly in the neck and scapular area, is highly vascularized and contains a large quantity of mitochondria. fatty acid oxidation in these mitochondria can increase heat production to twofold in response to cold. • in older children and adults, the first response to cold is behavioural (e.g. curling up, putting more clothes). if this response is insufficient, then the hypothalamic centre is stimulated to conserve heat by vasoconstriction and generate heat by shivering. the predominant stimulus for shivering is the skin rather than the core temperature. the energy produced is released as heat. • bat was, until recently, thought to be only functional in neonates and some animals. bat as a non-shivering thermogenesis has now emerged as a significant component of thermoregulation in elevating body temperature. this process induces and activates mitochondria, which uncouples protein to release chemical energy as heat. the bat-metabolic thermogenesis is regulated by norepinephrine which is secreted by bat-sympathetic nerve terminals. pathological uncontrollable increase of heat production occurs in malignant hyperthermia (see chap. 2). in response to a rise in body temperature above 37 °c (or ambient temperature above 30-31 °c), heat is lost from the body via the four physical modalities: evaporation, radiation, convection and conduction. when the body core temperature rises (e.g. fever), heat loss through evaporation (causing sweating) becomes the primary mechanism of heat loss. this is associated with cutaneous vasodilatation via acetylcholine-mediated relaxation of the vascular smooth muscles. the following are the mechanisms by which heat loss occurs at rest: • about one-quarter is lost by evaporation from the skin and lungs, which occurs as water is converted from liquid to gas (58 kcal is lost for every 100 ml of water). • in general, 60% of the total heat is lost by radiation (transfer of heat from the skin surface to the external surroundings not in contact), through electromagnetic waves. • convection (12% of the heat loss) is increasing blood flow to body surfaces to maximize heat loss. • conduction (3% of the heat loss) is the heat transfer between two objects in direct contact and at different temperatures. this is the primary mode of heat loss from the core to the surface. in a warm environment or when core temperature is elevated, the hypothalamic thermoregulatory centre activates efferent fibres of the automatic nervous system to produce vasodilatation. the increased blood flow to the skin causes heat loss from the core through the skin surface to the surroundings in the form of sweating. the hypothalamus stimulates vasodilatation to increase insensible loss (for every 1 °c elevation of body temperature, there is a 10% insensible loss) and activates the sweat glands to increase perspiration production. physical factors obviously affect the ability to respond to temperature changes. the greater heat loss in the newborn infant is mainly due to a greater surface area compared to that of an older child. failure of heat loss occurs in anhidrotic ectodermal dysplasia and during anticholinergic drug overdose. in the classical model of pathogenesis, fever induction includes the following stages: • pyrogenic endogenous cytokines (e.g. il-1, tnf, il-6 and interferons) are released into the bloodstream in response to exogenous pyrogens (e.g. viruses, bacteria, toxins). • these endogenous pyrogens act on a specific preoptic area of the anterior hypothalamus, which contains clusters of thermo-sensitive neurons localized within the rostral wall of the third ventricle. the site is called organum vasculosum of the lamina terminalis (ovlt), which has emerged as an interface between circulation and brain. the firing rate of these thermo-sensitive neurons changes according to the temperature of the area's blood supply and the input from the skin and muscular thermoreceptors. warm-sensitive neurons have firing rates that increase with warming and decrease with cooling, whereas the firing rates of cold-sensitive neurons increase with cooling or decrease with warming. • endogenous pyrogens enter the perivascular space of the ovlt through the fenestrated capillary wall to stimulate cells to produce prostaglandin e2 (pge2), which diffuses into the adjacent preoptic area to upturn the temperature set point and cause fever. • another structure termed circumventricular organs (cvos), which are situated in the anterior wall of the third ventricle. these organs are characterized by extensive vasculature and lack of blood-brain barrier allowing direct exchange between blood and nervous tissue. when circulating pyrogenic cytokines are detected by the cvos, pge2 is induced. • the ultimate result of these complex mechanisms is an upward shift of the thermostatic set point to a febrile level that signals efferent nerves, especially sympathetic fibres innervating peripheral blood vessels, to initiate heat conservation (vasoconstriction) and heat production (shivering). this is aided by behavioural means aimed also to increase body temperature, such as seeking a warmer environment or covering up with a blanket. the resulting temperature increase continues until body temperature approximates to the temperature of the elevated set point. • the raised set point is reset back to normal if the concentration of the cytokines falls or if antipyretics are administered that block prostaglandin synthesis. the normalization of temperature is initiated by vasodilatation and sweating through increased skin blood flow controlled by sympathetic fibres. prostaglandin e2 has been found to exert a negative feedback on the release of the cytokines, thus terminating the mechanisms that initially induced the fever the peptide angiotensin 11 has been shown to lower body temperature at the final step of fever. it is involved in maintaining body temperature at the set point. in addition, arginine vasopressin (avp) acts within the cns to reduce pyrogensinduced fevers. a decrease in hypothalamic calcium concentration or an increase in sodium concentration elevates body temperature. the generation of fever involves the following steps: • numerous substances from outside the body, exogenous pyrogens, initiate the fever cycle. endotoxin of gram-negative bacteria, with their pyrogenic component lipopolysaccharide, is the most potent exogenous pyrogen. fever is also a common finding in children without obvious evidence of infection, for example hypersensitivity reaction, autoimmune diseases and malignancy. • exogenous pyrogens initiate fever by inducing host cells (primarily macrophages) to produce and release endogenous pyrogens such as interleukin-1, which has multiple biological functions essential for the immune response. • endogenous pyrogens are transmitted to the hypothalamic thermoregulatory centre, specifically organum vasculosum of the lamina terminalis (ovlt), where they induce synthesis of prostaglandins, of which pge2 is the most important. these raise the thermostatic set point to initiate the febrile response. • the hypothalamic thermoregulatory centre accomplishes heat production by inducing shivering and heat conservation through vasoconstriction. at an established degree, fever is regulated (even at a temperature of over 41.0 °c), and heat production approximates loss, as in health, though at a higher level of the set point. therefore fever does not climb up relentlessly. • in addition to the function as an endogenous pyrogen, il-1 activates t-lymphocytes to produce various factors, such as inf and il-2, which are vital for immune response. the production of fever simultaneously with lymphocyte activation constitutes the clearest and strongest evidence in favour of the protective role of fever. • the induction of fever results in inhibition of bacterial growth, increased bactericidal of neutrophils, production of acute-phase protein synthesis and other physiological changes such as anorexia and somnolence. these changes suggest that fever has an adaptive role in the host's survival during infection (see chap. 9 for detail). chemical basis of fever potentiation of the lymphocyte response to mitogens: cellular source pf potentiating mediators infection in neonatal hypothermia risk factors, host response and outcome of hypothermic sepsis il-1 family members in the pathogenesis and treatment of metabolic disease: focus on adipose tissue inflammation and insulin resistance treating inflammation by blocking interleukin-1 in a broad spectrum of diseases infections in children and adolescents with juvenile idiopathic arthritis and inflammatory bowel disease treated with tumour necrosis factor alpha inhibitors: a systematic review of literature dual role of gm-csf as a pro-inflammatory and a regulatory cytokine: implications for immunotherapy key: cord-015147-h0o0yqv8 authors: nan title: oral communications and posters date: 2014-09-12 journal: inflamm res doi: 10.1007/bf03353884 sha: doc_id: 15147 cord_uid: h0o0yqv8 nan the drosophila host defense is a multifaceted process which involves reprogramming of gene expression, activation of proteolytic cascades in the blood and uptake of microrganismsby professionalphagocytes. most of the recent studies have focused on challenge-induced expression of antimicrobial peptides and have addressed the following questions : (1) which genes are upregulated during various types of bacterial, fungal or viral infections and what are their functions? (2),what is the nature of the intracellular signalling cascades which lead togene transcription during these infections; (3) how does drosophila recognize infections and does it discriminate between various types of aggressors (e.g. fungal versus bacterial or viral) to mount an appropriate response. over the last ten years we have gained significant insights into these various aspects and the presentation will review our current understanding of the drosophila immune response and put it into a phylogenetic perspective, namely by drawing some stringent parallels with the mammalian innate immune response. there is strong evidence that autoimmunity to myelin antigens plays a major role in the development of multiple sclerosis. several myelin-derived autoantigenic targets have been described and include myelin basic protein (mbp), proteolipid protein and myelin oligodendrocyte glycoprotein. there has been a particular focus on mbp for at least two reasons: mbp-specific cd4+ tcell receptors (tcrs) have been found in multiple sclerosis brains, and cells presenting an immunodominant mbp(85-99) peptide in complex with hla-dr2b have been shown to be present in multiple sclerosis lesions. also, humanized mice expressing the hla-dr2b gene and a human t-cell receptor (tcr) that recognises the mbp85-99 peptide in the context of hla-dr2b either spontaneously or after immunization with mbp85-99 develop experimental autoimmune encephalomyelitis, which has several features in common with multiple sclerosis. this talk will focus on, how humanized mice recently has been used to study the interplay between genetic and environmental risk factors in multiple sclerosis. to resolve the pathogenic mechanisms of rheumatoid arthritis (ra) we need to identify the causative genetic and environmental factors. this has however proven to be complex, with many factors and genes interacting. inbred animals are useful for studies of the identification of genes associated with complex traits and diseases such as ra. animal models offer a possibility to better define the traits, and to segregate the genes in a controlled way enabling linkage analysis. there are several arthritis models, which each may reflect various variants of the heterogeneity of ra in humans. examples are collagen induced arthritis (cia) and pristane induced arthritis (pia), which both fulfills the clinical diagnostic criteria for ra. both diseases are genetically complex and the susceptibility is, as ra, dependent on many polymorphic genes operating in concert. so far 2 genes in this concert have been identified; the mhc class ii ab gene in the mouse (1) and the ncf1 gene in the rat (2) and in the mouse (3) . the ncf1 protein is a part of the nadph oxidase complex mediating oxidative burst. the discovery of the ncf1 polymorphism led to a new proposed pathway in which oxygen radicals modify antigen presentation and the resulting activation of autoreactive t cells. mice with the deficient ncf1 allele are more susceptible to cia, and also developed a chronic form of arthritis. interestingly, the immune response to cii was enhanced by the ncf1 deficiency linking the ncf1 pathway to the adaptive immune response. oxidation of t cell membranes seem to be a key event in the pathogenic mechanism as reduction of t cell membranes induces arthritis in rats (4). on the basis of these findings a new type of therapy myasthenia gravis is a prototypic autoimmune disease, caused in most cases by autoantibodies to the muscle acetylcholine receptor (achr) at the neuromuscular junction. the antibodies reduce the number of achr leading to failure of neuromuscular transmission and muscle weakness. the achr antibodies as measured in conventional immunoprecipitation assays are igg, high affinity, polyclonal and specific for human achr. they reduce the numbers of achr by antigenic modulation and by complement-mediated damage to the neuromuscular junction. myasthenia gravis has a very intriguing relationship with the thymus gland. in many younger patients, the thymus is hyperplastic with immune cell infiltrates and germinal centre formation. around the germinal centres, within the medulla, there are rare muscle-like cells called myoid cells that express achrs. there are many b cells and plasma cells and thymic lymphocyte preparations synthesise achr antibodies. the possibility that the thymic achr induces the germinal centre formation and achr antibody synthesis is supported by much evidence. some patients, however, have thymic tumours and in these the role of the thymus is less clear. moreover, older patients with typical myasthenia usually have thymic tissue which is normal for their age. there are up to 20% of myasthenia patients that do not have the typical achr antibodies. some of these have instead antibodies to muscle specific kinase, a receptor tyrosine kinase that is restricted to the neuromuscular junction. the pathogenic mechanisms by which the antibodies cause disease are not yet clearly identified and the evidence will be discussed. finally, among the patients who have neither achr nor musk antibodies by conventional testing, we have evidence for lower affinity antibodies to achr which can now be detected using molecular approaches which will be described. arry-886 (azd6244) is an inhibitor of mek1/2 currently in development for cancer. phase 1 determined the msd (100 mg) and the safety of the compound given continuously. in decreasing frequency, common treatment-related side effects were rash, diarrhea, nausea, peripheral edema, and vomiting. paired pre-and postdose tumor biopsies showed a reduction in perk (-83%) and proliferative index (-46%). the trough plasma concentration (400 ng/ml) corresponded to~40% inhibition of perk. about 45% of pts had stable disease after 2 months. these results demonstrate that arry-886 (azd6244) is well-tolerated, hits its target, and produces a high incidence of long-lasting stable disease. there are several on-going phase 2 studies, in melanoma, colorectal, lung and pancreatic cancer. arry-162 is another potent, selective mek 1/2 inhibitor, currently in development for inflammatory diseases. when human whole blood was stimulated with tpa, arry-162 inhibited tnfa, il-1b and il-6 (ic50s of 23, 21 and 21 nm, respectively). 50% inhibition of perk required 280 nm. arry-162 was highly efficacious in cia and aia rat models, with ed50s of 3 and~10 mg/ kg, respectively. in normal volunteers arry-162 was well-tolerated and there was a dose-proportional increase in drug exposure. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpa-induced tnfa and il1b. an on-going multiple ascending dose clinical study is further exploring the pharmacokinetics, pharmacodynamics and tolerability of arry-162 monotherapy. in addition, we have initiated a clinical trial designed to evaluate arry-162 in combination with methotrexate in patients with rheumatoid arthritis. rho kinases (rock) are involved in many physiological and pathological processes including smooth muscle contraction, cytoskeletal arrangement, cell adhesion, migration and proliferation.rocks prominent role in cytoskeletal architecture suggests that rock inhibitors should have therapeutic impact in oncology and fibrotic diseases which require cytoskeletal rearrangement to progress.we have synthesized small molecule inhibitors of rock which are specific for the rock-2 isoform.these rock-2 inhibitors, typified by slx-2119 and slx-3060, are potent (ic50 < 100 nm), selective for rock-2 (>100 fold selectivity for rock-2 over rock-1) and exhibit good oral bioavailability.this talk will focus on several areas in oncology and fibrotic disease where the ability to demonstrate an in vitro effect on the cytoskeleton translates into activity in the disease model in vivo.slx-2119 inhibits cell proliferation and migration in several tumor cell lines including ht-1080, panc-1 and mda-mb-231. moreover in xenograft studies using nude mice, slx-2119 significantly inhibited tumor growth with these same cell lines. in liver fibrosis, slx-2119 prevents the differentiation of rat primary hepatic stellate cells into myofibroblasts and inhibits the proliferation of myofibroblasts as well as inhibiting hepatic steatosis in an atherosclerosis model.slx-3060 is an effective antifibrotic agent in the kidney unilateral urethral obstruction model and inhibits renal fibroblast differentiation and proliferation.these data suggest that rock-2 selective inhibition of cytoskeletal modification in key cell types (e.g. tumor cells, stellate cells and fibroblasts) by compounds such as slx-2119 and slx-3060 will provide effective treatment for oncology and fibrotic disease. cyclooxygenases (cox) catalyze the first step in the synthesis of prostaglandins (pg) from arachidonic acid.cox-1 is constitutively expressed.the cox-2 gene is an immediate early-response gene that is induced by variety of mitogenic and inflammatory stimuli.levels of cox-2 are increased in both inflamed and malignant tissues.in inflamed tissues, there is both pharmacological and genetic evidence that targeting cox-2 can either improve (e.g., osteoarthritis) or exacerbate symptoms (e.g., inflammatory bowel disease).multiple lines of evidence suggest that cox-2 plays a significant role in carcinogenesis.the most specific data that support a cause-and effect relationship between cox-2 and tumorigenesis come from genetic studies.overexpression of cox-2 has been observed to drive tumor formation whereas cox-2 deficiency protects against several tumor types.selective cox-2 inhibitors protect against the formation and growth of experimental tumors.moreover, selective cox-2 inhibitors are active in preventing colorectal adenomas in humans.increased amounts of cox-2-derived pge2 are found in both inflamed and neoplastic tissues.the fact that pge2 can stimulate cell proliferation, inhibit apoptosis and induce angiogenesis fits with evidence that induction of cox-2 contributes to both wound healing and tumor growth.taken together, it seems likely that cox-2 induction contributes to wound healing in response to injury but reduces the threshold for carcinogenesis. (1), k hagihara(2), t nishikawa(1), j song(1), a matsumura (2) (1) health care center, osaka university, japan (2) osaka university graduate school of medicine, japan it is still less known about the actual pathogenic role of il-6 in the inflammatory status. to know the pathogenic role of il-6 and the efficacy of il-6 blockade in inflammation, a humanized anti-il-6 receptor antibody, tocilizumab, was used for the treatment in chronic inflammatory diseases, such as castlemans disease, rheumatoid arthritis and crohns disease. since il-6 blocking therapy improved the clinical symptoms and the laboratory findings, the il-6 function in inflammation could be analyzed for the induction of inflammatory molecules, such as serum amyloid a (saa). saamrna induction, saa promoter activity and assembling of transcriptional factors on saa promoter were analyzed by the real time rt-pcr, gel shift assay and dna affinity chromatography in hepatocyte stimulated with the proinflammatory cytokines, il-6, il-1 and tnf-alpha. in result, il-6 was an essential cytokine in induction of saamrna through the activation of stat3 which constructed the complex with nf-kappab p65 and a cofactor p300. although there was no stat3 consensus region on saa promoter, stat3 bound at the 3 site of nf-kappab re. the above research proved that il-6 signal is essential on the synergistic induction of saa via newly discovered stat3 transcriptional mechanism, suggesting the presence of this stat3 mechanism in inflammation, and confirming the normalization of serum saa level by il-6 blocking therapy in inflammatory diseases. this research method develops a subsequent therapy for serious aa amyloidosis by inhibition of saa production, and elucidates the cytokine mechanism on immunopathogenesis of chronic inflammatory diseases. takashi wada(1), k matsushima(2), s kaneko (1) (1) kanazawa university, japan (2) university of tokyo, japan accumulating evidence indicates that chemokine/chemokine receptor system plays a key role in the pathogenesis of various renal diseases via leukocyte migration. pathophysiological impacts of chemokines have shed light on molecular mechanisms of leukocyte trafficking and their activation in the inflammatory aspects of progressive renal injury.locally expressed chemokines are proven to be capable of inciting leukocyte migration to the kidney, resulting in initiating and promoting chronic kidney diseases.the possible positive amplification loop from cxc chemokines to cc chemokines may contribute to progressive renal injury, resulting in sclerosis/fibrosis.it is of note that monocyte chemoattractant protein (mcp)-1/ monocyte chemotactic and activating factor (mcaf)/ ccl2, a prototype of cc chemokines, promotes and escalates chronic kidney diseases with any etiologies via the infiltration and activation of monocytes/macrophages, proteinuria and collagen synthesis.interactions between infiltrated inflammatory cells and resident renal cells eventually lead to the progression of fibrosis. new insights into renal fibrosis have been uncovered by the regulation of fibrocytes dependent on chemokine system. in addition, recent studies demonstrate that chemokines have been expanding their universe beyond leukocyte migration to the kidney, including homeostasis, development and repair of the kidney.the selective intervention of chemokines might have the therapeutic potential to alter inflammatory responses, thereby halting the progression of renal injury. we focus on recent progresses on the role of chemokines and their cognate receptors in renal injury in this symposium. (1), p lacamera (1), b shea (1), g campanella (1), b karimi-shah (1) , n kim (1), z zhao(2), v polosukhin (3), y xu(2), t blackwell (3) aberrant wound-healing responses to injury have been implicated in the development of pulmonary fibrosis, but the mediators directing these pathologic responses remain to be fully identified.here we demonstrate that lysophosphatidic acid (lpa) is induced by lung injury in the bleomycin model of pulmonary fibrosis, and that mice deficient for one of its receptors, lpa1, are dramatically protected from pulmonary fibrosis and mortality following bleomycin challenge. the absence of lpa1 markedly reduced fibroblast responses to the chemotactic activity present in the airspaces following bleomycin, and attenuated the subsequent accumulation of fibroblasts in the lung.the increase in vascular permeability caused by lung injury was also markedly reduced in lpa1-deficient mice, whereas bleomycin-induced leukocyte recruitment was preserved.these results demonstrate that lpa1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak, two of the wound-healing responses that are thought to be inappropriately excessive when injury leads to fibrosis rather than repair.lpa1 therefore represents a new target for lung diseases in which aberrant responses to injury contribute to the development of fibrosis, such as idiopathic pulmonary fibrosis and the acute respiratory distress syndrome. we have reported that inflammation is detrimental for survival of new hippocampal neurons early after they have been born. our data now show that microglia activation, as an indicator of inflammation, is not pro-or antineurogenic per se but the net outcome is probably dependent on the balance between secreted molecules with pro-and antiinflammatory action. we have found that a substantial fraction of the new hippocampal neurons formed after status epilepticus survive despite chronic inflammation. we have started to explore the role of tnf-alpha for adult neurogenesis. infusion of an antibody to tnf-alpha was shown to reduce survival of new striatal and hippocampal neurons generated after stroke, probably by interfering with action of the ligand on the tnf-r2 receptor. we have shown that tnf-r1 is a negative regulator of progenitor proliferation in basal and insult-induced hippocampal neurogenesis. we have also used patch-clamp technique to explore whether a pathological environment influences the synaptic properties of new granule cells. rats were exposed to either a physiological stimulus, i.e., running, or a brain insult, i.e., status epilepticus which is associated with inflammation. we found that new granule cells in runners and status epilepticus animals had similar intrinsic membrane properties. in contrast, the new neurons which had developed in the physiological and pathological tissue environments differed with respect to tonic drive and short-term plasticity of both excitatory and inhibitory afferent synapses. the role of inflammation for these differences is currently being explored. proteinase-activated receptor-2 (par-2) is cleaved within its aminoterminal extracellular domain by serine protei-nases such as trypsin, unmasking a new aminoterminus starting with sligkv that binds intramolecularly and activates the receptor. par-2 is implicated in innate defense responses associated with lung inflammation. we showed that par-2 is expressed by human alveolar (a549) and bronchial (16hbe) epithelial cell lines, and is activated by trypsin and by the activating synthetic peptide sligkv-nh2. in cystic fibrosis patients, airspaces are invaded by polymorphonuclear neutrophils that release elastase and cathepsin g, two serine proteinases, and by pseudomonas aeruginosa that secretes an elastolytic metalloproteinase.we demonstrated that these three proteinases do not activate par-2, but rather disarm this receptor, preventing its further activation by trypsin but not by sligkv-nh2. preincubation of a par-2 transfected cell line with either proteinases leads to the disappearance of the cleavage/activation epitope. proteolysis by these three proteinases of synthetic peptides representing the aminoterminal extracellular domain encompassing the cleavage/activation sequence of par-2, generate fragments that would not by themselves act as receptor-activating ligands and that would not yield receptor-activating peptides upon further proteolysis by trypsin. our data indicate that neutrophiland pathogen-derived proteinases can potentially silence the function of par-2 in the respiratory tract, thereby altering the host innate defense mechanisms. caspase-3 -dependent killing of host cells and to disrupt intestinal barrier function, which, at least in the case of giardiasis, ultimately causes lymphocyte-dependent intestinal malfunction, and the production of diarrheal symptoms. ongoing research is investigating whether par agonists and microbial pathogens may cause epithelial apoptosis, increased permeability, and overall epithelial malfunction in the gastrointestinal tract, via common or intersecting pathways. the intestinal epithelium is exposed to a variety of proteases in both health and disease, including digestive proteases such as trypsin.given that protease-activated receptor 2 (par2) responds to trypsin and is expressed on intestinal epithelial cells, we investigated the effect of trypsin on intestinal epithelial barrier function.scbn, caco-ii and t84 epithelial cells were grown to confluence on filter supports and mounted in ussing chambers to study short circuit current (isc) and transepithelial resistance (rte).cell monolayers were incubated with inhibitors of transcellular ion transport in order to isolate the contribution of the paracellular pathway to rte.apical exposure to serine proteases including trypsin, elastase and chymotrypsin caused a rapid and sustained increase in rte and decreased the transepithelial flux of a 3000mw dextran.interestingly, the effect of trypsin could not be replicated by activators of pars 1, 2 and 4, suggesting that the effect on rte was not due to activation of pars.subsequent experiments showed that trypsin activated phosphatidylinositol-dependent phospholipase c.a trypsin-induced increase in intracellular calcium was not involved.inhibition of pkc-zeta, but not of typical pkcs, also blocked the response.our data point to a role for postprandial trypsin that extends beyond that of a digestive enzyme; it is also a participant in cellular pathways that control tight junction permeability. physiologically, the trypsin-induced increase in resistance could augment transcellular transport by reducing passive paracellular back-diffusion of ions. further studies will assess how these pathways might be disrupted in the barrier dysfunction characteristic of intestinal inflammation. clustering of inflammatory bowel disease in large families and the observation of an increased concordance between monozygotic twins suggests heritable components in these disorders. the high concordance in monozygotic twins (>55%), which is not seen in dizygotic twins (<5%) points to strong contribution of genetic susceptibility to the overall risk for disease. ibd represents a "complex disease" and may involve a large number of interacting disease genes. crohns disease has become an example for the successful molecular exploration of a polygenic etiology. crohns disease was not known before 1920. incidence has increased since now leading to a lifetime prevalence of up to 0.5% in western industrialized countries. the current hypotheses propose unknown trigger factors in the life style of western industrialized nations that interact with a polygenic susceptibility. it appears that increased expression and production of tnf and an enhanced state of activation of the nfkb system are main drivers of the mucosal inflammatory reaction. the exploration of inflammatory pathophysiology of crohns disease using full genome, cdna and oligonucleotide based arrays, respectively, has generated large sets of genes that are differentially expressed between inflamed mucosa and normal controls. while this may lead to new targets for a pathophysiology oriented therapy, it appears, however, that the dissection of the inflammatory pathophysiology does not allow to identify the multifactorial etiology of the disease. genome-wide linkage analysis has demonstrated eight confirmed susceptibility regions with the one on chromosome 16 being most consistent between different populations. in 2001 three coding variations in the card 15 gene were identified that are highly associated with development of the disease. all variants affect a part of the gene that codes for the leucin rich part of the protein, that appears to be involved in bacteria induced activation of nfkb in macrophages and epithelial cells. interestingly, the three disease associated snps are never found on the same haplotype. in compound heterozygotes or homozygotes they result in a rr of > 35 to develop crohns disease as an adult. a particular subphenotype with localization of the disease in the ileocecal region is highly associated with the variants in the card 15 gene. variations in the card 15 gene do not fully explain the linkage finding in the pericentromeric region of chromosome 16. after stratification for card 15 variants, the broad linkage peak is reduced to two more defined peaks on 16p and 16q, respectively. while the exploration of these regions has led to several association signals that are subject to further fine mapping a further disease gene progress has been greater in the other linkage regions (i.e. on chromosomes 10 and 5, respectively). dlg-5 is the example of a low-risk susceptibility gene with a modest associated odds ratio (1.2-1.5) . interestingly, the associa-tion signal appears to be confined to young males. slc22a4/5 which encode the kation-transporters octn1 and 2 have been suggested to represent the disease gene in the 200+ kb haplotype block on chromosome 5q31. mdr1 has also been implicated as a disease gene in ibd. although the human association studies have resulted in highly controversial findings a knockout mouse with a colitis phenotype makes mdr1 likely as a low risk susceptibility gene. with the advent of high-density, genome wide association studies enormous progress has been made to discover the remaining disease genes. recently a 330k illumina scan has been published identifying il-23r as a further disease gene. we used a genome wide candidate gene approach (with appr. 20.000 csnps) to identify atg16l1 as a further disease genes. both genes were confirmed and a further regulatory snp involving ptger4 was annotated by a belgian genome wide scan. by the time of presentation three further genome wide snp scans in crohn disease will most likely have entered the public domain. the further exploration of crohns disease (and other inflammatory conditions of barrier organs) will have to annotate the function and pathophysiologies based on genetic risk maps that are completed with amazing speed. the creation of a medical systems biology of disease will lead to new models and eventually new therapies. the chemokine receptor ccr9 plays a pivotal role in mediating the migration of t cells to the gastrointestinal mucosa. the ligand for ccr9, teck, s highly expressed in the gi tract. the pathogenicity of intestinal ccr9+/ cd4+ t cells has been demonstrated in animal models and this cell population is substantially increased in the peripheral circulation of crohns and celiac disease patients. ccx282-b is a highly selective and potent, orally bioavailable, small molecule antagonist of ccr9.the compound proved to be highly efficacious in the tnf-aare and mdr1a-/-murine models of inflammatory bowel disease (ibd). in phase i trials, ccx282-b was well-tolerated, and no drug-related saes were reported.a 28-day placebo-controlled phase ii study was recently completed in patients with moderate to severe crohns disease. ccx282-b was shown to be both safe and to have encouraging clinical results: 56% of patients on ccx282-b (cdai !250, crp >7.5mg/l) exhibited a 70-point drop in cdai compared to 29% on placebo. furthermore, a crp decrease of 11 mg/l was seen in the ccx282-b group compared to placebo. colonic biopsy samples were analyzed for expression of several pro-inflammatory cytokines. a mean decrease from baseline in the concentrations of tnf-alpha, il-12 p70, ifn-gamma, and the chemokine rantes was shown in the ccx282-b group while levels remained stable in the placebo group. ccx282-b is the first chemokine-based inhibitor of leukocyte trafficking to be tested in ibd. the compound shows anti-inflammatory activity and encouraging evidence for clinical benefit in the treatment of crohns disease. the activating receptor nkg2d seems to be implicated in the pathogenesis of several autoimmune diseases in humans and in animal models for type 1 diabetes and multiple sclerosis. the aim of this study was to asses the role of nkg2d in a model of inflammatory bowel disease, where cd4+cd25-t cells from balb/c mice are adoptively transferred to scid mice, and to evaluate the therapeutic effect of an anti-nkg2d antibody therapy. the expression of nkg2d was evaluated by flow cytometry, immunohistochemistry and by pcr. we found a marked up-regulation of nkg2d on the cell surface as well as increased levels of nkg2d mrna in cd4+ t cells from colitic scid mice as compared to normal balb/c mice. we next studied the effect of anti-nkg2d antibody (cx5) treatment initiated either before onset of colitis, when the colitis was mild, or when severe colitis was established. cx5 treatment decreased the cellsurface expression of nkg2d and prophylactic administration of cx5 attenuated the development of colitis significantly. a moderate reduction in the severity of disease was observed after cx5 administration to mildly colitic animals, whereas cx5 did not attenuate severe colitis. thus, nkg2d may be involved in the pathogenesis of colitis in this model, particularly in the early phases, since the expression of nkg2d in cd4+ t cells increased markedly during the development of disease and since administration of cx5 early but not late in the course attenuated the disease severity. proteins are used already for more than a century in the treatment of disease. the first generation were proteins derived from animals such as antisera used to treat infectious diseases as diphtheria and tetanus and later bovine and porcine insulin for the treatment ofdiabetes. the second generation were natural proteins from human source like the plasma derived clotting factors and human growth hormone. the development of the recombinant dna and cell fusion technology in the seventies of the 20th century opened up the possibilities to produce human proteins and monoclonal antibodies in unlimited amount in microbial and mammalian host cells. in 1982 human insulin was introduced as the first recombinant dna derived biopharmaceutical and since than more than 160 have gained approval. the pipeline contains many more potential biopharmaceuticals and at present 1 in 4 new drug applications concerns a biotechnology derived product. a major problem of therapeutic proteins is the induction of antibodies. for foreign proteins such as the murine derived monoclonal antibodies thisimmunogenicity was to be expected. however the humanization of monoclonal antibodies has reduced but not solved the problem of immunogenicity. and also the proteins which are homologues of endogenousfactors such as gm-csf, interferons etc. induce antibodies, sometimes even in the majority of patients. by definition we are immune tolerant to products which are copies of endogenous proteins. the products not necessarily need to be exact copies of the natural proteins to share this immune tolerance. when human therapeutic proteins induce antibodies, they are breaking b cell tolerance, which starts with the activation of autoreactive b-cells. presenting the self-epitopes in an array form is very potent activator of these b-cells. this explains why aggregates of human proteins are the most important factor in induction of antibodies. these aggregates may not be immediately present in the product, but may appear during storage making stability and formulation an important issue in predicting the immunogenicity. there are only a few studies in experimental model systems on the properties of the aggregates which break b-cell tolerance, indicating that only multiple order aggregates (>trimers) are involved. we study the capacity of a protein product to break b-cell tolerance in mice made transgenic for the specific protein. these mice are immune tolerant and there is a good correlation of an immune response in these mice and in patients. although these models have helped to identify the factors important for breaking b-cell tolerance and also have been useful in improving the formulation of products, there is not yet enough experience to use them as absolute predictors of immunogenicity of human proteins. they also allow to study the involvement of tcells in breaking b cell tolerance. all data obtained untilnow indicate this process to be t-cell independent. contact information: dr huub schellekens, central laboratory animal institute, utrecht university, utrecht, the netherlands e-mail: h.schellekens@gdl.uu.nl biomonitor aps, and institute for inflammation research (iir), rigshospitalet, copenhagen, denmark using recombinant technology, one can now produce protein drugs which are almost identical with naturally occurring human proteins, including antibodies (abs). many have assumed that these drugs may be administered with little or no risk of triggering specific t-and/or b-lymphocyte reactivities, because patients according to immunological dogma are tolerant towards their own proteins. unfortunately, this is not the case, and even socalled 100% human biologicals are potentially immunogenic (1) (2) . i shall discuss two examples: 1) recombinant human cytokines (ifn-beta-1a and -1b), and 2) anticytokine ab constructs (anti-tumor necrosis factor (tnf)-alpha). ifn-beta has been used for treatment of patients with multiple sclerosis since the early nineties. though initially neglected as a clinical problem, ifn-beta like many other human proteins is indeed immunogenic, especially those produced by recombinant gene technologies. the reported frequencies and titers of anti-ifn-beta ab vary considerably depending upon ifn-beta preparations and administration, and the types of assays being used (2-4). it took more than 10 years of clinical use before abmediated decrease in bioactivity of ifn-beta, a condition in which the clinical effect of continued injection of rec. ifn-beta is minimized or abrogated, was universally recognized (5, 6). 2) anti-tnf-alpha human ab constructs tnf-alpha is an inflammatory cytokine of central pathogenic importance in many immunoinflammatory conditions, and measures to diminish production and/or effects of tnf-alpha have long been a goal in the treatment of these conditions. currently, there are three approved and two other anti-tnf-alpha biopharmaceuticals in clinical use. unfortunately, response failure is frequently encountered. thus, 30-40% of patients are primary non-or lowresponders to the anti-tnf constructs, and secondary response failure is commonplace, mostly due to induction of anti-abs. several different methods have been used to assess circulating levels of anti-tnf drugs as well as anti-abs. most of these have been based on elisa technology, with their inherent problems of false positivity, susceptibility to nonspecific interference, etc. interferon beta (ifnbeta) has been an important step forward in the treatment of multiple sclerosis(ms), an inflammatory disease of the human central nervous system. however, one of the problems of ifnbeta is its immunogenicity; a substantial percentage of ms patients treated this recombinant protein develop anti-ifnã� antibodies, primarily of the igg class. the level of these antibodies tends to be low in the first month or two and peaks by six to eighteen months after initiation of therapy. most studies of these antibodies have measured their ability to neutralize ifnbetas effect in vitro, using assays in which sera from ms patients inhibit the protective effect of ifnbeta on viral killing of target cells. this antibody population is called neutralizing antibodies (nabs). tests measuring binding of antibodies to ifnã� in vitro are called binding antibody (bab) assay. anti-ifnbeta antibodies detected by bab assays are present in a high percentage of ms patients, and can occur at low levels without any apparent adverse effect on ifnbeta bioactivity. the distinction between babs and nabs is artificial, and all binding antibodies are likely neutralizing, if the neutralizing assay system is adequately sensitive; i.e., the development of babs and nabs is a continuum with the assay systems simply measuring the strength of the antibody response. in many treated patients, the anti-ifnbeta antibody response is strong, despite the resemblance of the injected protein to the human homologue, and high levels of neutralizing antibodies develop. high levels of anti-ifnbeta antibodies with high affinity results in loss of ifnbeta bioactivity, a phenomenon which has been called antibody-mediated decreased bioactivity or adb. adb can be considered the in vivo correlate of the neutralizing effect of the anti-ifnã� antibody population, while the nab assay measures the in vitro neutralization of this population of immunoglobulins in the serum. the three ifnbeta preparations have different incidences of nabs and different patterns of appearance and disappearance over time of nabs. because there is no direct correlation between nab levels and bioactivity at moderate levels of nab, in vivo bioactivity assays for ifnbeta have become increasingly utilized. in a large multicenter study in the us, called the insight study, bioactivity as measured by ifnbeta induced upregulation of the ifn-response genes mxa, viperin, and ifit1, was shown to be highly correlated with nab levels, confirming a single center study (pachner, a.r., pak,e., narayan, k., multiplex analysis of expression of three ifnbeta-inducible genes in antibody-positive ms patients, neurology, 66:444-446, 2006) . multiple studies, including a large multicenter study in denmark and a recent study from our center using high resolution mri of the brain once a month, have demonstrated that nabs abolish the salutary effects of ifnbeta on clinical aspects of ms, especially inflammation. recent guidelines for european neurologists recommend stopping ifnbeta in nab-positive patients. in order to maintain bioactivity of this important medication for ms, some neurologists have attempted to use immunosuppressives either to prevent the development of nabs, or to treat them once they have developed. however, at this point in time, there is no clearly optimal way to treat nabs. major efforts have been underway to decrease the immunogenicity of ifnbeta and a new formulation of one of the higher immunogenicity products has been recently developed and tested. proteinase-activated receptors (pars). endogenous serine proteinases such as thrombin, mast cell tryptase, trypsins, kallikreins, cathepsin g, for example, as well as exogenous proteases released by mites or bacteria are involved in cutaneous inflammation, host defense or tumor cell regulation. thus, the expression of pars on keratinocytes, endothelial cells, nerves, and immune cells suggest important role of pars as a part of the communication system in the skin during inflammation and the immune response. for example, par2 activates nf-kb in keratinocytes and endothelial cells, stimulates the release of chemokines and cytokines, and is involved in proliferation and differentiation. on sensory nerves, this receptor controls neurogenic inflammation by modulating edema and extravasation via release of neuropeptides into the inflammatory site. par1 and par2 also modulate leukocyte-endothelial interactions in the skin, thereby regulating inflammatory responses such as leukocyte trafficking through the vessel wall. they also stimulate signal transduction pathways involved in cutaneous inflammation. in sum, this novel receptor family requires a paradigm shift in thinking about the role of proteases in cutaneous biology and disease. novel compounds regulating protease and par function may be beneficial for the treatment of several skin diseases such as atopic dermatitis, psoriasis or pruritus. serine proteinases are upregulated in arthritic joints where their enzymatic activity participates in the destruction of articular soft tissues. in addition to their degradative functions, serine proteinases can also act as signalling molecules by activating members of the gprotein coupled receptor family called the proteinase activated receptors (pars). these receptors are known to regulate tissue inflammation and pain, although their function in joints is unclear. our study examined the effect of par4 activation in joint inflammation and pain. male c57bl/6 mice received an intra-articular injection of either the par4 activating peptide aypgkf-nh2 or the inactive peptide yapgkf-nh2 (100 mg) into the right knee. knee joint blood flow was then measured in these mice by laser doppler perfusion imaging while joint diameter measurements gave an indication of tissue oedema. mechanical allodynia was also assessed in these animals by application of von frey filaments onto the plantar surface of the ipsilateral hindpaw and a pain score was calculated. intra-articular injection of the par4 activating peptide caused knee joint blood flow to gradually increase by up to 25% over the succeeding 2hrs. knee joint swelling was also observed as well as the development of a mechanical allodynia. all responses could be blocked by pre-treatment with the selective par4 antagonist pepducin p4pal10 (100 mg i.p.). the control peptide yapgkf-nh2 had no discernible effect on joint inflammation or pain. these experiments show that peripheral activation of par4 receptors in mice knees causes joint inflammation and pain. vincent lagente (1), e boichot (2) (1) air liquide, centre de recherche claude-delorme, jouy en josas, france (2) inserm u620, universitã� de rennes 1 matrix metalloproteinases (mmps) are a major group of proteases known to regulate the turn-over of extracellular matrix and so they are suggested to be important in the process of lung disease associated with tissue remodelling. these led to the concept that modulation of airway remodeling including excessive proteolysis damage of the tissue may be of interest for future treatment. among metalloproteinases (mmps) family, macrophage elastase (mmp-12) is able to degrade extracellular matrix components such as elastin and is involved in tissue remodeling processes in chronic obstructive pulmonary disease (copd). pulmonary fibrosis has an aggressive course and is usually fatal for an average of three to six years after the onset of symptoms. pulmonary fibrosis is associated with deposition of extracellular matrix (ecm) components in the lung interstitium. the excessive airway remodeling as a result of an imbalance in the equilibrium of the normal processes of synthesis and degradation of extracellular matrix components could be in favor of anti-protease treatments. indeed, the correlation of the differences in hydroxyproline levels in the lungs of bleomycin-treated mice strongly suggests that a reduced molar pro-mmp-9/timp-1 ratio in broncholaveolar lavage fluid is associated with collagen deposition, beginning as early as the inflammatory events at day 1 after bleomycin administration. finally, these observations emphasize those effective therapies for these disorders must be given early in the natural history of the disease, prior to the development of extensive lung destruction and fibrosis. in addition to their degradative properties, proteases can act as signalling molecules that send specific messages to cells.recent work has demonstrated that proteases are able to signal to peripheral sensory neurons, thereby participating to neurogenic inflammation processes and to the transmission or inhibition of pain messages. serine proteases cleaving specifically at an arginin site are able to activate protease-activated receptors (pars), which then send specific messages to cells. we have demonstrated that 3 members of the par family (par1, par2 and par4) are present on peripheral sensory neurons, where they can be activated by different proteases.the activation of par1 and par2 in isolated sensory neurons provokes calcium mobilization and the release of substance p and cgrp, while the activation of par4 inhibited bradykinin-and capsaicin-induced calcium signal, and neuropeptide release.thrombin and pancreatic trypsin caused inflammation respectively through a par1 and par2-dependent mechanism involving the release of neuropeptide.the extrapancreatic form of trypsin (mesotrypsin or trypsin iv) also caused neurogenic inflammation through a par2 and par1dependent mechanism, and causes inflammatory hyperalgesia and allodynia, through a par2-dependent mechanism.in contrast, activation of par4 on peripheral sensory neurons inhibited inflammatory hyperalgesia and allodynia. taken together, these results provide evidences that proteases can interfere with inflammatory and pain mechanisms through the activation of pars on peripheral sensory neurons.determining the role of each individual proteases and their receptors in sensory neuron signalling and above all inflammatory and pain mechanisms constitutes an important challenge to raise new anti-inflammatory and analgesic drugs. introduction: a scoring system for disseminated intravascular coagulation (dic) in humans has been proposed by the international society on thrombosis and haemostasis (isth). it was the objective of this study to develop and validate a similar scoring system for dic in dogs in order to establish the dog as a spontaneous animal model. methods: for the developmental study, 100 consecutive dogs admitted to the intensive care unit (icu) were enrolled prospectively (group a). blood samples were collected daily and a broad panel of coagulation assays performed. diagnosis of dic was based on the expert opinion of one human physician and two veterinarians. a multiple logistic regression model was developed with the coagulation parameters as explaining variables for the diagnosis of dic. integrity and diagnostic accuracy was subsequently evaluated in a separate prospective study according to the stard criteria. the validation study prospectively enrolled 50 consecutive dogs (group b). results: 37 dogs were excluded from group a where 23/63 dogs (37%) had dic. final multiple logistic regression model was based on aptt, pt, d-dimer and fibrinogen and had a very high diagnostic sensitivity and specificity. diagnostic accuracy of the model was sustained by prospective evaluation in group b. conclusion: based on generally available assays, it was possible to design an objective diagnostic model for canine dic, which has both a high sensitivity and specificity. such a model will provide basis for treatment optimization and make it possible to conduct multicentered therapy studies with a minimum risk of systematic misclassification of patients. in 1997, a coagulation independent change in light transmittance (biphasic waveform [bpw] ) was reported in automated activated partial thromboplastin time assays (aptt) in patients with disseminated intravascular coagulation (dic). a calcium-dependant precipitate of creactive protein and very-low-density-lipoprotein was causing the bpw. our group recently identified this phenomenon in dogs also. initially, bpw was introduced as a complementary tool to assist diagnosing dic. however, recent studies reported that bpw may have a stronger potential as a prognostic marker for survival. the aims of the study were to prospectively investigate (a) the diagnostic significance of bpw regarding dic and (b) the significance of bpw to outcome, in dogs with diseases known to predispose for dic. the study was performed as a prospective, observational study including 50 consecutive dogs with a final diagnosis known to predispose for dic (20% were finally diagnosed with dic). outcome was 28-day survival. bpw was assessed by means of a hirudin-modified aptt assay (kjelgaard-hansen et al., jvim 2006:20; 765-766) . relative risk according to bpw (rr [95% confidence interval]) for (a) a dic diagnosis and (b) 28-day mortality, were assessed. 28-day mortality in the study population was 44%. 28% were bpw positive. bpw was not a significant diagnostic factor for dic (rr=0.64 [0.16; 2.67] ), but strongly so for outcome (rr=2.57 [1.46;4.52] ) with a 79% (11/14) mortality amongst bpw positive dogs. in conclusion, bpw was observed in dogs predisposed to dic, with a strong potential as a risk factor for outcome, a finding in line with recent findings in humans. (1), b hideo (2) (1) department of molecular pathology, kumamoto university, japan (2) department of gastroenterological surgery, kumamoto university, japan aeromonas species are facultative anaerobic gramnegative rods that are ubiquitous, waterborne bacilli, most commonly implicated as causative agents of gastroenteritis. aeromonas infections often develop sepsis and disseminated intravascular coagulation syndrome (dic) is a life-threatening complication of sepsis patients, causing multiple organ failure.however, a mechanism leading to coagulation induction in the bacterial infection has not been known. to study the dic induction by aeromonas species infection, we investigated coagulation activity of a serine protease (asp) from aeromonas sobria, predominantly isolated in patients blood. proteolytically active asp shortened both activated partial thromboplastin time and prothrombin time of human plasma in a dose-dependent manner starting at an enzyme concentration of 30 nm. asp activated human prothrombin, releasing hydrolytic activity for thrombinspecific substrate boc-val-pro-arg-mca, but no enzymatic activity was produced from coagulation factors ix and x. analysis by sds-page revealed that asp released a prothrombin fragment with a molecular weight identical with that of fâ¿-thrombin in an incubation timedependent manner. western blotting using biotinylated phe-pro-arg-chloromethylketone, a thrombin inhibitor, showed that asp produced an enzymatically active fragment whose molecular weight was same as that of fâ¿-thrombin. prothrombin incubated with asp but not the protease itself caused platelet aggregation. these results indicate that asp activates prothrombin, producing fâ¿-thrombin that converts fibrinogen to fibrin clot, and suggest that asp coagulation-inducing activity contributes to dic development in sepsis caused by aeromonas sobria infection. the present study shows a link between inflammation and coagulation mediated by a bacterial protease. hemolytic episodes are often associated to high amounts of free heme in circulation (up to 20 um) and the development of an inflammatory response that may develop to a chronic inflammation. our group has shown that free heme is a prototypical proinflammatory molecule, able to induce neutrophil migration, actin cytoskeleton reorganization and nadph oxidasederived reactive oxygen species (ros) generation, as well as pkc activation and interlukin-8 expression (graã�a-souza et al., 2002) . moreover, free heme inhibits human neutrophil spontaneous apoptosis, a feature that is closely related to the impairment of resolution of inflammation and consequent promotion of chronic inflammatory status. heme protective effect requires nadph oxidase-derived ros and involves the activation of mapk, pi3k and nf-kb signaling cascades as well as heme oxygenase (ho) activity (arruda et al., 2004) . more recently, we have shown that heme antiapoptotic effect is closely related to the maintainance of mitochondrial stability, inhibition of bax insertion into mitochondria and a dramatic increase on bcl-xl/bad protein ratio in a ros-dependent manner, requiring the same signaling pathways that regulate heme anti-apoptotic effect. these findings attest to a prominent role of free heme in the onset of inflammation associated to hemolytic episodes as well as the statement of chronic inflammation related to these disorders. the recent advance on the study of free heme as a proinflammatory molecule brings up hope for the development of new strategies to ameliorate acute and chronic inflammation found during hemolytic episodes.financial support: faperj, cnpq, capes. (1) (1) university of melbourne, victoria, australia (2) monash university, victoria, australia we have previously demonstrated that mice lacking the anti-oxidative enzyme, glutathione peroxidase (gpx1), show significantly larger infarcts after stroke. recent studies have demonstrated that adhesion molecule-mediated leukocyte recruitment is associated with increased tissue damage in stroke, while mice lacking key adhesion molecules conferred neuro-protection. nevertheless, the involvement of oxidative stress in leukocyte recruitment and subsequent regulated cell injury is yet to be elucidated. to explore this, gpx1-/-mice were subjected to transient mid-cerebral artery occlusion (mcao) followed by cerebral intravital microscopy, for assessment of leukocyte-endothelium interactions in intact cerebral microvasculature. after 1hr mcao, leukocyte-endothelium interaction was significantly reduced in gpx1-/-mice compared to wt counterparts during the second hour of reperfusion. laser doppler and direct measurement of blood flow in pial postcapillary venules revealed a reduction of reperfusion in gpx1-/-mice following transient mcao. this suggests that the reduction in nutritive blood flow following stroke in gpx1-/-mice may explain the enhanced injury in these mice as well as the reduced leukocyte-endothelium interaction. furthermore, matrix metalloproteinase-9 (mmp9) which has previously been shown to be implicated in endothelial dysfunction and the pathogenesis of stroke was found to be up-regulated in gpx1-/-mice to a greater extent than in wt mice after mcao, suggesting a role for oxidative stress in cerebral microvascular injury. the data present here suggests oxidative stress may be one of the factors that contribute to reduced post-ischemic perfusion, via the disruption of the endothelial function as indicated by the increased level of mmp9. chris bolton(1), c paul(2), s barker (3), r mongru (3) (1) william harvey research institute, london, uk (2) university west of england, bristol (3) queen mary university of london adrenomedullin (am) acts as a vasodilator in many vascular beds including the cerebral circulation where the peptide is produced in larger amounts than in the periphery.in vitro work has shown that am beneficially regulates blood-brain barrier (bbb) characteristics including transendothelial electrical resistance, permeability and p-glycoprotein pump activation.our preliminary studies in acute experimental autoimmune encephalomyelitis (eae), a model of the human disease multiple sclerosis (ms), have demonstrated significant elevations in am peptide levels corresponding with am mrna changes during late, neurological disease where am production may be linked to the restoration of bbb function. however, am is not exclusively produced as result of am gene upregulation. furthermore, am peptide levels do not always match am mrna changes during other disease phases of eae.the current study has investigated, more closely, the relationship between am gene expression and subsequent levels of associated peptides. am mrna levels were determined, by rt-pcr, in the cerebellum, medulla-pons and spinal cord of normal and eae-inoculated lewis rats at the height of disease. am and proadrenomedullin peptide (pamp) levels were measured in the tissues by radioimmunoassay.all tissues examined showed an increase in am gene mrna compared to control levels.am and pamp changes were observed in the samples and differences between the peptide profiles were recorded.an understanding of alterations in the generation of am and related peptides during neuroinflammation may provide insight into mechanisms affecting bbb permeability and be of relevance to the changes in neurovascular function seen during ms. platelet-activating factor (paf) contributes to the robust inflammatory responses in acute phase and spread of secondary injury. although, paf is believed to be a potent edematous but non-painful mediator in peripheral tissues, we recently demonstrated that paf may be a mediator of noxious signaling in spinal cord in case of neuronal injury. paf-induced tactile allodynia may be mediated by atp, glutamate and the generation of nitric oxide (no). the present study elucidated down-stream signaling pathway for paf-induced tactile allodynia. paf-and glutamate-induced tactile allodynia was blocked by the pretreatment with no scavengers and inhibitors of no synthase, soluble guanylate cyclase or cgmp-dependent protein kinase (pkg). recent evidence attributes the generation of pain to specific disfunctions of inhibitory glycinergic neurotransmission. to explore the target molecule for induction of tactile allodynia, the effect of knockdown of glycine receptors containing the a3 subunit (glyr a3) by sirna spinally transfected with hvj-e vector was examined. in mice spinally transferred with sirna for glyr a3, the reduction of glyr a3 was demonstrated in superficial layer of dorsal horn by immunohistochemical analysis. pcpt-cgmp, paf, glutamate failed to induce tactile allodynia in mice spinally transferred with sirna of glyr a3, while these compounds produced tactile allodynia in mice transferred with mutant sirna of glyr a3 as a control. glycine tranporter inhibitors ameriolated paf-and pcpt-cgmp-induced allodynia. these results suggest that glutamate-no-cgmp-pkg pathway plays a key role for paf-induced tactile allodynia in spinal cord and glyr a3 may be a target molecule for pkg to induce allodynia. (1), r leite(2), ys bakhle (3) (1) federal university of minas gerais, belo horizonte, brazil (2) medical college of georgia, augusta, usa (3) imperial college, london, uk selective cyclooxygenase 2 inhibitors (coxibs) induce a characteristic increase in mechanical nociceptive threshold, referred to as "hypoalgesia", in inflammatory pain induced by carrageenan in rat paws.we have here assessed the role of the cytoskeleton in this hypoalgesia induced by celecoxib (cx). male holtzman rats (150-200g; 3-5 animals/group) were injected in the right hind paw (ipl) with a range of cytoskeletal inhibitors (selective inhibitors of microtubules (taxol, nocodazole, colchicine), of actin microfilaments (latrunculin b, cytochalasin b) or of intermediary filaments (acrylamide) (pico to nanomoles per paw) and 30 min later given cx (12mg/kg, s.c.). after a further 30 min, rats were injected (ipl) with the inflammatory stimulus, carrageenan (250 mg/paw). mechanical pain threshold was hourly measured over the next 4 h, using the randall-sellitto method. the cxinduced hypoalgesia was reversed by low doses of latrunculin b or cytochalasin (latrunculin 100% reversal = 1.26 nanomoles), higher doses of microtubule inhibitors (taxol 100% reversal = 23.4 nanomoles) with no effect of acrylamide (7 up to 141 nanomoles).we conclude that 1) local changes in (paw) cytoskeleton occurred during cxinduced hypoalgesia and 2) actin microfilaments were the cytoskeletal components most critically involved in this hypoalgesia.financial support: cnpq, fapemig and capes there are reports regarding the up-regulation of cyclooxygenase isoenzyme particularly inducible isoform i.e. cox-2 in brain during neurodegenerative or neuropsychiatric disorders.in the present study, we examined the effect of nimesulide (a preferential cox-2 inhibitor) in subchronic immobilization stress. mice were subjected to immobilization stress for 6 hrs daily for a period of seven days. nimesulide (2.5 mg/kg, i.p.) was administered daily for 7 days before challenging them to immobilization stress. behavioral analysis revealed the hyperlocomotor activity and increased anxious response. subchronic stress decreased % retention of memory and also caused hyperalgesic response in mice. biochemical analysis revealed that chronic immobilization stress significantly increased lipid peroxidation and nitrite levels and decreased the reduced glutathione and adrenal ascorbic acid levels. chronic treatment with nimesulide significantly attenuated the immobilization stress-induced behavioral and biochemical alterations. these results suggested that the use of nimesulide could be a useful neuroprotective strategy in the treatment of stress. there is accumulated evidence for ngf role as a peripheral pain mediator. ngf is upregulated in diverse inflammatory conditions and evokes hyperalgesia when injected in humans and rats. ngf increase was also observed in temporomandibular join (tmj) after cfa injection, indicating its possible involvement in local hyperalgesic states. therefore, the objective here was to evaluate if ngf participate in the tmj nociception. to test this hypothesis, the ngf was injected into the tmj alone or after carrageenan (cg) and the spontaneous nociceptive behavior of head flinches was counted for up 30min. further evidence for the ngf nociceptive activity was obtained quantifying the local production of ngf after cg injection, by elisa, and the fos-like immunolabeling in the trigeminal sensory nucleus (including the caudalis, interpolaris and oralis) after ngf injection. injections were performed in 0.25ul. ngf (0.2, 1 and 5ug) injected in the tmj challenged 1h prior by cg (100ug) induces a dose-dependent increase in the number of head flinches. this increase was reduced by k252a (1 and 2ug), indicating a trka receptor-mediated effect. we detected a significant increase in the ngf production 1 and 3h after the tmj cg (300ug) injection. the tmj injection of ngf (5ug) alone did not induce detectable spontaneous nociceptive behavior. however, the ngf (5ug) injection induces a significant increase in the fos like immunolabeling (fli) in the sensory trigeminal nucleus compared to the saline injection. these results indicate that the ngf participates in the nociceptive activity in the tmj, specially in inflammatory conditions. mif was reported as a key cytokine in the pathogenesis of rheumatoid arthritis (ra) several years ago, but it now clear that mif is also involved in the pathogenesis of systemic lupus erythematosus (sle) and atherosclerosis. mif-deficient lupus-prone mrl/lpr mice exhibit prolonged survival and reduced renal and skin disease compared to mif-expressing mice. similarly, mif-deficient atheroma-pone ldlr-deficient or apoe-deficient mice are significantly protected from disease and antimif mab therapy is beneficial. ra and sle are each characterised both by an increased prevalence of atherosclerotic vascular disease and by overexpression of mif. given the effects of mif on atherosclerosis it can be hypothesised that mif overexpression participates in the risk of atherosclerotic vascular disease in ra and sle. recent data have provided insights into mechanisms of action for mif relevant to all these concepts. firstly, the newly described role of mif in the selective recruitment of monocyte-macrophage lineage cells is of particular relevance to ra, sle, and atherosclerosis, with evidence that mif mediates macrophage recruitment in sle and atherosclerosis. secondly, glucocorticoid (gc) therapy is possible risk factor for atherosclerosis in patients with ra and sle, and it is now clear that gc increase the expression and release of mif, potentially implicating mif in gc-related increases in atherosclerosis in ra and sle. specific therapeutic targeting of mif in ra and sle may address not only primary disease pathways but also the increased risk of atherosclerosis in these diseases. to enter inflamed tissues, leukocytes must undergo adhesion molecule-mediated interactions with the endothelial surface of vessels at the site of inflammation.cytokines such as tumour necrosis factor (tnf) are established as important mediators capable of promoting leukocyte-endothelial cell interactions.however, in inflammatory diseases such as atherosclerosis and rheumatoid arthritis, elevated expression of another cytokine, macrophage migration inhibitory factor (mif) occurs, yet the role of this cytokine in leukocyte recruitment is unknown.therefore we explored the ability of mif to regulate leukocyte recruitment.this was achieved using intravital microscopy to examine the intact microvasculature in mice following local mif treatment. these experiments showed that mif induced leukocyte adhesion and transmigration in vivo, resulting in accumulation of predominantly cd68+/f4/80-ve/cd11c-ve monocyte/ macrophage lineage cells.mif did not induce upregulation of adhesion molecules p-selectin and vcam-1, although their constitutive expression contributed to recruitment.in contrast, mif-induced recruitment was blocked by antibodies to the monocyte-specific chemokine, ccl2/mcp-1, and its receptor ccr2, and in response to anti-cxcr2.this was supported by in vitro experiments showing that mif induced ccl2/mcp-1 release from cultured murine endothelial cells.finally, mice lacking cd74, the putative mif binding molecule, did not respond to mif.these data demonstrate a previously unrecognized function of this pleiotropic cytokine: induction of monocyte migration into tissues, and indicate the involvement of a pathway involving a complicated chemokine/chemokine receptor pathway with contribution from cd74.this function may be critical to the ability of mif to promote diseases in which macrophages are key participants. gm-csf and m-csf (csf-1) can enhance macrophage lineage numbers as well as modulate their differentiation and function. of recent potential significance for the therapy of inflammatory/autoimmune diseases, their blockade in relevant animal models leads to a reduction in disease activity. what the critical actions are of these csfs on macrophages during inflammatory reactions are unknown. to address this issue, adherent macrophages (gm-bmm and bmm) were first derived from bone marrow precursors by gm-csf and m-csf, respectively, and stimulated in vitro with lps to measure secreted cytokine production, as well as nf-kb and ap-1 activities. gm-bmm preferentially produced tnfa, il-6, il-12 and il-23 while, conversely, bmm generated more il-10 and ccl2; strikingly the latter population could not produce detectable il-12 and il-23. following lps stimulation, gm-bmm displayed rapid ikba degradation, rela nuclear translocation and nf-kb dna binding relative to bmm, as well as a faster and enhanced ap-1 activation. each macrophage population was also pre-treated with the other csf prior to lps stimulation and found to adopt the phenotype of the other population to some extent as judged by cytokine production and nf-kb activity. thus gm-csf and m-csf demonstrate at the level of macrophage cytokine production different and even competing responses with implications for their respective roles in inflammation including a possible dampening role for m-csf. granulocyte macrophage-colony stimulating factor (gm-csf), initially discovered for its role in the differentiation of haematopoietic cells into granulocytes and macrophages, can also affect mature cell function and may be considered proinflammatory. gm-csf is able to prime macrophages for increased pro-inflammatory responses, including the increased release of tnfa and il-12 following stimulation with, for example, lps. in addition, gm-csf has been shown in vivo, using murine disease models, to play a key role in a number of inflammatory diseases. gm-csf-/-mice have been shown to be resistant to several diseases, including arthritis, and, most notably, blockade of gm-csf with a neutralizing monoclonal antibody was effective at ameliorating arthritis when given either prophylactically or therapeutically. t cells appear to be the major cell type responsible for gm-csf production required for arthritis, and gm-csf appears important in the effector phase of disease, subsequent to t cell activation. blockade of gm-csf results in fewer inflammatory cells, particularly macrophages, and cytokines such as tnfa, at the site of inflammation. these findings suggest that blockade of gm-csf may be an effective treatment in a range of inflammatory diseases. the autoimmune disease type 1 diabetes mellitus (t1dm) is thought to be mediated by autoreactive t cells recognizing islet autoantigens, including gad65, ia-2 and proinsulin. this disease arises on a distinctive genetic background, mapping most notably to the mhc, and is also open to strong environmental influence. to investigate the pathogenesis of the disease, and in particular the prevailing paradigm that islet autoreactive t cells are important, we have developed an approach to epitope identification that is mhc allele and autoantigen specific, and operates for both cd4 and cd8 t cells. utilizing this, we have uncovered populations of islet antigen-specific t cells that have the immunological credentials to be both pathogenic (eg th1, tc1) and protective (treg) in the disease. we have cloned some of these cell types, enabling us to analyse their function and provide an insight that will be important for an understanding of disease mechanisms, as well as guiding novel therapeutic interventions. tcr transgenic targeting b:9-23 cause diabetes 2. knockouts of the insulin 2 gene (expressed in thymus as well as islets) accelerates diabetes while knockout of insulin 1 gene (islet expression) prevents 90% of diabetes 3. dual insulin knockout with transgenic insulin with altered peptide (b16:a) prevents all diabetes 4. islets with native b:9-23 sequence, but not altered sequence when transplanted into knockouts restore anti-insulin autoimmunity and diabetes transfer by t cells 5.anti-b9-23 t cells have conserved valpha and jalpha chain usage but no conservation n region or beta chain 6. alpha chain as transgene sufficient to engender anti-insulin autoantibodies 7. kay and coworkers demonstrate insulin reactivity "upstream" of igrp and igrp reactivity nonessential.future studies in nod directed at deleting specific conserved alpha chains to test diabetes prevention and develop therapeutic.in man we can now identify at birth genetic risk as high as 80% of activating anti-islet autoimmunity with mhc analysis and restricted heterogeneity suggesting dominant target.insulin autoantibodies in prospective studies such as daisy usually appear initially and levels are related to progression to diabetes.analysis of cadaveric donors is underway to elucidate primary targets. (t1d) is an autoimmune disease in which genes and environment contribute to cell-mediated immune destruction of insulin-producing beta cells in the islets of the pancreas. the holy grail of autoimmune disease prevention is negative vaccination against autoantigens to induce disease-specific immune tolerance. this has been achieved in rodents by administering autoantigen via a tolerogenic route (mucosal), cell type (stem cell or resting dendritic cell), mode (with blockade of t-cell co-stimulation molecules) or form (as an altered peptide ligand). compelling evidence demonstrates that proinsulin is the key autoantigen that drives beta-cell destruction in the non-obese diabetic (nod) mouse model of t1d, and possibly in humans. proinsulin/ insulin dna, protein or t-cell epitope peptides administered in a tolerogenic manner to the nod mouse can delay or prevent the development of diabetes, via one or more mechanisms (deletion or anergy of effector t cells, induction of regulatory t cells). administration of autoantigen via the mucosal route, which induces anti-diabetic regulatory t cells in the rodent, is the most immediately translatable approach to humans. initial human trials of vaccination with oral autoantigens lacked evidence of bioeffect, probably due to inadequate dosage in end-stage disease. recently, however, the first evidence for a therapeutic effect of mucosal autoantigen has been seen in trials of oral and nasal insulin in islet autoantibodypositive individuals at risk for t1d. combination autoantigen-specific vaccination also shows promise in combination with non-specific immunotherapy in established t1d. leukocyte extravazation is an integral process both physiologically (immunosurveillance) and pathophysiologically (inflammation). the initial paradigm of a 4-step process comprising tethering/rolling, activation, firm adhesion, and diapedesis, each involving specific adhesion molecules, has repeatedly been modified in the light of more recent findings. additionally, organ-specific differences regarding the role of distinct molecules were established. finally, the skin became a good "model" to study due to its accessability and availability of powerful animal models. in-vitro adhesion assays, flow-chamber systems, intravital microscopy, animal models for delayed-type hypersensitivity, and transplantation approaches have successfully been employed to investigate leukocyte extravazation. numerous molecular interactions such as the cutaneous lymphocyte-associated antigen and sialyl-lewisx, or icam-1 and lfa-1, have been proven sufficiently relevant to make them candidates for potential therapies. with the anti lfa-1 antibody efalizumab, approved for the treatment of psoriasis, the first therapeutic agent specifically targeting leukocyte extravazation is already on the market; other compounds are under development. moreover, novel data suggest that well-established anti-inflamamtory therapies such as fumarates also influence this process, thus contributing to their clinical efficacy. ongoing research aks for adopting a more "dynamic" view on leukocyte extravazation as several molecules obviously perform multiple tasks throughout this process rather than being limited to just one step of this multi-step cascade; this is particularly true for the so-called junctional adhesuã­on molecules which obviously mediate more than just diapedesis. finally, similarities between leukocyte extravazation and hematogenic metastases are emerging. consequently, certain anti-inflammatory compounds may turn out to also exhibit striking anti-metastatic efficacy, and vice versa. department of dermatology, heinrich-heine-university, dã¼sseldorf, germany atopic dermatitis, psoriasis vulagaris and cutaneous lupus erythematosus represent chronic inflammatory skin diseases showing distinct clinical phenotypes but sharing one aspect. the recruitment of pathogenic leukocyte subsets into the skin represents a prerequisite for their initiation and maintenance. during recent years, our knowledge of the immunopathogenesis of chronic inflammatory skin diseases increased significantly. with regard to the recruitment pathways of leukocytes, a superfamily of small cytokine-like proteins so called chemokines has attracted significant attention. here the complex interactions within the chemokine ligand-receptor network are introduced, the involvement of chemokines in memory t and dendritic cell trafficking is outlined and current concepts of their role in the immunopathogenesis of atopic dermatitis, psorasis vulgaris and cutaneous lupus erythematosus are summarized. the skin serves as a unique organ for studying general principles of inflammation because of its easy accessibility for clinical evaluation and tissue sampling. a network of pro-inflammatory cytokines including il-1 and tnf-a is known to play a key role in the pathogenesis of cutaneous inflammatory diseases through activation of specific signalling pathways. recently, progress in understanding the underlying mechanisms regulating inflammatory signalling pathways in the immunopathogenesis in skin carcinomas, psoriasis vulgaris and atopic dermatitis has been made. kinases have been identified to play a crucial role in regulating the expression and activation of inflammatory mediators in these inflammatory skin diseases. mitogen-activated protein kinases (mapks) are a family of serine/threonine protein kinases that mediate a wide variety of cellular behaviours in response to external stress signals. increased activity of mapks, in particular p38 mapk, and their involvement in the regulation of synthesis of inflammatory mediators at the transcriptional and translational level has recently been demonstrated. progress in our understanding of inflammatory signalling pathways has identified new targets for treating inflammatory diseases, but the challenge is to place a value on one target relative to another and to evolve strategies to target them. a careful examination of different signalling pathways in various inflammatory conditions is therefore needed. this presentation gathers recent advances in signal transduction in skin inflammation focusing interleukin-1, tnf-âµ, p38 mapk, msk1/2, mk2, nf-kappab and ap-1. histamine is an important inflammatory mediator in humans, and despite their relatively modest efficacy antihistamines are frequently used to treat allergic conditions, as well as other histamine-mediated reactions such as pruritus. in contrast, antihistamines are of very limited use for controlling other conditions where histamine production is abundant, including asthma. the discovery of the histamine h4 receptor (h4r) prompted us to reinvestigate the role of histamine in pulmonary allergic responses, as well as in pruritus. h4r deficient mice and mice treated with h4r antagonists exhibited decreased allergic lung inflammation in several models, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in th2 responses. ex vivo restimulation of primed t cells showed decreases in th2 cytokine production, and in vitro experiments suggest that decreased cytokine and chemokine production by dendritic cells after blockade of the h4r was responsible for the the t cell effects. the influence of h4r on allergic or histamine-induced pruritus was explored in mice using selective histamine receptor antagonists and h4r deficient mice. the h4r was found to mediate the majority of histamine-mediated and allergic itching, while the contibution by the h1r was minor. surprisingly the h4r effect was independent of mast cells or other hematopoetic cells. this work suggests that the h4r can modulate both allergic responses via its influence on t cell activation, and pruritus through mechanisms that are independent of hematopoetic cells. the studies show that the h4r mediates previously uncharacterized effects of histamine and highlight the therapeutic potential of h4r antagonists. (1), bsp reddy (2) (1) nizams institute of medical sciences, hyderabad; india (2) genix pharma, india rupatidine, carries the majority of the histamine h1receptor -blocking activity and has been introducedfor the treatment of allergic rhinitis and urticaria. objectives: the aim of this study was to compare the effect of two by measure of inhibition of histamine induced wheal and flare response. methodology: 12 male volunteers were enrolled after written informed consent before to ethic committee approved protocol. in this randomised, double-blind, single oral dose, cross overstudy, they were randomized to receive either 10 mg rupatidineformulation after overnight fast. washout was 10 days. wheal and flare were induced on the forearm of the trial subjects, by histamine intradermally injection while the subject was lying comfortably with arm resting on the bed. ten minutes later, wheal and flare were visualized under a bright lamp. histamine induced wheal and flare skin test was performed before and regularly to 24hours after drug administration. results: administration of reference and test formulations of rupatidine, significantly inhibited the histamine induced cutaneous response in all the subjects. the least square mean ratio (%) t vs r for peak activity imax-% (maximum inhibition of histamine induced wheal and flare response); area under the activity time curve (auc0-24 mmsq/hr and auc 0-24 %/hr) both for untransformed and log transformed data were found to be within 80-125% of90% ci limits both formulations well tolerated. conclusions: it can thus be concluded that be concluded that test formulation of rupatidine tablet is bioequivalent to reference rupatidine tablet (1), h yoshimura(1), k ohara (1), y mastui (1), h hara (1), h inoue (1), h kitasato (1), c yokoyama(2), s narumiya (3), m majima (1) (1) kitasato university school of medicine, kanagawa, japan (2) tokyo dental medical colledge, tokyo, japan (3) kyoto university school of medicine, kyoto, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported the magnitude of cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitorsconstitute revascularization. we hypothesized that txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregainflamm. res., supplement 3 (2007) tion.to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb model. recovery from acute hind limb ischemia, as assessed by the ratio between the treated ischemic limb and the untreated control right limb was assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out(ipko) and thromboxane (tx) a2 receptor (tp) knock out(tpko). blood recovery in tp-/-significantly delayed compared to wt and ipko. immunohistochemical studiesrevealed that tp-/-mice were less stained against pecam positive cells compared to wt and ipkoplasma sdf-1 and vegf concentration were significantly reduced in tp-/-mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. purpose: the s100 calcium-binding proteins, a8, a9 and a12 are constitutively expressed in neutrophils and induced in activated macrophages. high levels are found in sera from patients with infection and several chronic inflammatory diseases. the calgranulin complex, a8/a9 is anti-microbial; a8 has oxidant-scavenging functions. a12 is chemotactic for monocytes, and recruits leukocytes in vivo by activating mast cells (mc). effects of these mediators on mc and monocyte function were compared. methods: human pbmc or murine mc were activated in vitro with s100 and mediator release and cytokine induction (assessed by quantitative rt-pcr/elisa), determined. a cys41 to ala41 a8 mutant was used to determine whether effects on mc are mediated by redox. immunohistochemistry was used to demonstrate s100s in asthmatic lung. the s100s were expressed in asthmatic lung, particularly in eosinophils and alveolar macrophages. strong reactivity occurred with an antibody recognising predominantly the hypochlorite-oxidised from of a8. a8, a9 or the a8/a9 complex had relatively low ability to induce il1, tnf, il6, and chemokines mrna from pbmc compared to a12. only a12 induced significant levels of il13; none induced il4 or gm-csf mrna compared to lps. in contrast to a12 which is activating, a8 significantly inhibited mc degranulation provoked by ige cross-linking; suppression was dependent on cys41. conclusions: the cytokine profile generated by a12 in mc and monocytes strongly supports a role the pathogenesis of asthma. in contrast, results strongly support a role from a8 in oxidant defence, particularly to hypobromite generated by activated eosinophils. (1), d mankuta(2), g gleich (3), f levi-schaffer (1) (1) hebrew university of jerusalem, israel (2) hadassah university hospital, israel (3) university of utah, usa the onset, amplitude and termination of allergic responses is regulated at the mast cell/eosinophil interface. eosinophil major basic protein (mbp), which activates mast cells in the late-chronic phase of allergic inflammation, is a central determinant in this interface. characterized more than two decades ago, the exact nature of this activation has not been clarified as yet. here we demonstrate that mbp exerts its activating effect on human mast cells and basophils through cd29 and hematopoietic cell kinase (hck). a genome-wide analysis showed that hck displays shifts in mrna levels specifically upon mbp-induced mast cell activation. hck also shows a unique priming pattern prior to this activation. cd29 is phosphorylated specifically upon activation with mbp and deploys a signaling complex that critically depends on hck. extracellular neutralization of cd29 interferes with mbp entry into the cell, and this as well as rna silencing of hck results in defective mbp-induced activation. finally, cd29 neutralization abrogates mbp-induced anaphylaxis in-vivo. these findings picture for the first time a chronic-phase specific pathway mediating eosinophil-induced mast cell activation with critical consequences for the therapy of chronic allergic inflammation. alexander robinson (1), d kashanin (2), f odowd(2), v williams(2), g walsh (1) (1) cellix ltd, institute of molecular medicine, dublin, ireland (2) university of aberdeen, scotland, uk leukocyte adhesion to endothelial cell bound proteins, such as icam-1 and vcam-1, is an initial step of the inflammatory response. we have developed an in vitro microfluidic system which mimics conditions found in blood vessels in vivo during an immune response. using this system, we can record leukocyte adhesion levels under physiologically relevant flow conditions (e.g. 0-5 dynes/cm2). the adhesion profiles of resting and pmastimulated peripheral blood lymphocytes (pbls) were recorded, with respect to vcam-1, icam-1, and bsa. images at each shear stress level were captured using a digital camera, and analysed using our in-house ducocell software package. distinct morphological changes in pma-stimulated pbls, compared to non-stimulated cells, can be observed. these include a less rounded appearance of the pma-stimulated pbls, and evidence of "uropod" formation, which anchor the t cell to the endothelium as part of the migration process. levels of adhesion to vcam-1 are high (80-90%, compared to control), but there appears to be little difference between the adhesion profiles of non-stimulated and pma-stimulated pbls.however, there is a distinct difference between the adhesion levels of non-stimulated and pma-stimulated pbls to icam-1, with pma-stimulated cells showing a higher affinity for icam-1 than nonstimulated cells (approx. 70% and 40%, respectively).pbl adhesion to bsa is negligible. we present a novel in vitro microfluidic pump system that can simulate leukocyte adhesion to the endothelium under flow conditions. this platform is a more efficient and economical system compared to those currently available, due to reduced material costs and style of construction. introduction: pulmonary aspiration of gastric contents is a common complication observed in icu patients and a potential trigger of ards. in this study we evaluated the course of lung inflammation induced by intranasal instillation of gastric juice (gj). methods: gj was obtained from donor rats (ph 1.6). male c57bl/6 were instilled with 2 ml/kg of gj. after 2 or 24 h, the animals were sacrificed and lung and balf were collected. control group consisted of non-manipulated mice. 287.3ae18.6, sg24h: 277.8ae63.8; pg/ml). discussion: gj aspiration induced an initial adherence of pmn to lung tissue that is correlated with increased tnf-a/il-10 ratio in balf at the 2nd h. the reduction of mpo activity is correlated with the decrease in tnf-a/il-10 ratio. the late increase of pmn in balf might be a consequence of the early production of tnf-a. the results are suggestive that the treatment of patients exposed to acid aspiration should be focused in the initial period of the insult and in the blockage of tnf-a. objectives: intestinal i/r is implicated as a prime initiating event in the development of acute respiratory distress syndrome (ards) after trauma and hemorrhagic shock. we investigated the effects of lps challenge to mice previously submitted to i-i/r, a two-hit model of acute lung injury. methods: male c57bl/6 mice were subjected to 45 min of intestinal ischemia and challenged with 0.1 mg/kg of intranasal lps at the 4th hour of reperfusion (two-hit). balf and culture of lung explants were performed 20 h after lps challenge. mice subjected to i-i/r or lps alone were used as controls. results: two-hit mice showed marked increase in lung evans blue dye leakage compared to i-i/r (375.5ae53.9 vs 48.8ae3.5, mg/mg). lung mpo was increased (0.30ae0.03 vs 0.15ae0.03; od 450nm) whereas the neutrophil recruitment to balf was inhibited in the two-hit group compared to lps group (11.5ae2.6 vs 28.5ae3.4; x 10e3cells/mouse). the levels of nox-in the two-hit group were significantly increased when compared to i-i/ r controls in balf (10.6ae0.5 vs 6.5ae0.8; mm) and in lung explants (3.1ae0.3 vs 1.3ae0.1; mm/mg of tissue). conclusions: intestinal i/r predisposes the animal to an exacerbated response to a low dose lps insult. the exacerbated production of nitric oxide observed in the two-hit group may cause endothelial damage, thereby explaining the major increase in vascular permeability in the two-hit group. the results are suggestive that patients exposed to systemic inflammatory response might develop ards when in contact with secondary inflammatory stimuli. nitric oxide may play an important role in this process. (1) (1) novartis institutes for biomedical research, horsham, uk (2) university of michigan, usa obligatory for using oxygen in energy transfer pathways was the simultaneous co-evolution of enzymes that detoxify the reactive species formed as by-products. thus, we hypothesized that individuals with low aerobic function will have reduced anti-oxidant capacity and, therefore, be more susceptible to smoking-related lung diseases like copd. to test this hypothesis, we exposed high capacity runner (hcr) and low capacity runner (lcr) rats to 3 months of whole-body smoke exposures.the animals, bred over successive generations on the same background strain for high or low running capacity, differ by over 500% (p<1x10-37) for exercise capacity, measured by running on a treadmill.after 3 months of exposures, inflammatory cells in bronchoalveolar lavage fluid were increased in both the hcr-and lcr-smokeexposed(se) animals compared to air-exposed controls (p<0.001); however there was a 2-3-fold increase in the number of neutrophils and lymphocytes in the lcr-over the hcr-se group (p<0.001).histopathology revealed there was greater inflammation and lung damage present in the lcr-versus hcr-se group (p<0.05). metabonomic (metabolite profiling) analysis revealed that while peroxidation of lung lipids occurred for both se groups, oxidative damage to the lung surfactant layer was significantly more extensive for the lcr-se. systemic oxidative damage was also more apparent in the lcr-se group, with metabolic profiling suggesting a reduced capacity to regenerate muscle glutathione. the metabolic data suggest that repair processes maybe more effective in the hcrs. in summary, these data support the concept that aerobic capacity may be central to ones susceptibility to developing smoking-related lung disease. (1), ap ligeiro-oliveira(1), jm ferreira-jr(4), sr almeida(1), w tavares de lima(1), shp farsky (1) (1) university of sâ¼o paulo, brazil (2) regional integrated university of alto uruguai and missã°es, brazil methods: male wistar rats were exposed to vehicle or hq (50 mg/kg; ip.;daily, 22 days, two-day interval every five days). on day 10, animals were ip sensitized with ovalbumin (oa). assays were performed on day 23. results: hq-exposed rats presented reduced number of leukocytes in the bronchoalveolar fluid and by impaired in vitro oa-induced tracheal contraction. the latter effect suggests reduction on mast cell degranulation, and it was corroborated by in vivo decreased mesenteric mast cell degranulation after topical application of oa. the oa-specificity response was confirmed by normal ability of mast cells to degranulate in both groups of animals after topical application of compound 48/80. in fact, lower levels of circulating oa-anaphylactic ige antibodies were found in hq-exposed rats. this latter effect was not dependent on number or proliferation of lymphocytes, nevertheless reduced expressions of costimulatory molecules cd45 and cd6 on oa-activated lymphocytes indicated the interference of hq exposure on signaling of the humoral response during an allergic inflammation. contact information: ms sandra manoela dias macedo, regional integrated university of alto uruguai and missã°es / university of sâ¼o p, department of clinical and toxicological analyses, sâ¼o paulo, brazil e-mail: smdmacedo@yahoo.com.br (1) (1) radboud university nijmegen, medical centre, nijmegen, the netherlands (2) university hospital, zã¼rich, switzerland toll-like receptors (tlr) are essential in the recognition of invading microorganisms. however, increasing evidence shows involvement of tlr in autoimmunity, such as rheumatoid arthritis (ra), as well. here we investigated whether synovial expression of tlr3 and tlr7 was associated with the expression of ifna, tnfa, il-1b, il-12, il-17, and il-18 and studied in what way these receptors and cytokines were associated in vitro. using immunohistochemistry, we found that tlr3 / tlr7 expression in synovial tissue was associated with the presence of ifna, il-1b and il-18, but not tnfa, il-12 and il-17. to investigate whether ifna, il-1b and il-18 could induce tlr3 / tlr7 upregulation in vitro, we incubated separate lymphocyte populations with these cytokines and subsequently determined tlr3/7 mrna expression. ifna incubation resulted in significant tlr3 /tlr7 upregulation, whereas il-1b and il-18 did not. pre-incubation with ifna and subsequent stimulation of tlr3 /tlr7 significantly enhanced il-6, tnfa and ifna/b production, indicating that the ifn-induced tlr upregulation was functional. low amounts of biologically active il-1b were produced upon stimulation with atp, but not upon tlr3 / tlr7 stimulation, although mrna levels were high. interestingly, ifna-priming significantly increased the atp-induced il-1b production. here, we demonstrated a dual role for ifna in vitro, which could explain the association between tlr and il-1b / il-18 in synovial tissue. first, involvement in tlr3 /tlr7 regulation and second, involvement in atp-induced production of biologically active il-1b. these results suggest involvement of anti-viral immune responses in ra and ifna as a key player in chronic inflammation. the pathogenesis of chronic joint inflammation remains unclear although the involvement of pathogen recognition receptors (ppr) has been suggested recently. here, we described the role of two members of the nacht-lrr (nlr) family, nod1 (nucleotide/ binding oligomerization domain) and nod2 in model of acute joint inflammation induced by intraarticular injection of tlr2 (toll-like receptor) agonist streptococcus pyogenes cell wall fragments. we found that nod2 deficiency resulted in reduced joint inflammation and protection against early cartilage damage. in contrast, nod1 gene deficient mice developed enhanced joint inflammation with concomitant elevated levels of proinflammatory cytokines and cartilage damage. to explore whether the different function of nod1 and nod2 occurs also in humans, we exposed pbmcs carrying either nod1frameshift or nod2frameshift mutation with scw fragments in vitro. both tnfa and il-1b production was clearly impaired in pbmcs carrying the nod2fs compared to pbmcs isolated from healthy controls. in line with the nod1 gene deficient mice, human pbmcs bearing the nod1 mutation produced enhanced levels of proinflammatory cytokines after 24h stimulation with scw fragments. these data indicated that the nlr family members nod1 and nod2 have a different function in controlling tlr2-mediated pathways. we hypothesize that intracellular nod1-nod2 interactions determine the cellular response to tlr2 triggers. whether lack of controlling tlr2-driven pathways by nod1 signalling is involved in the pathogenesis of autoinflammatory or autoimmune disease, such as rheumatoid arthritis (ra), remains to be elucidated. leukocyte immunoglobulin-like receptors (lilrs) are a family of receptors with potential immune-regulatory function. activating and inhibitory receptors play a role in maintaining immunological equilibrium and an imbalance may lead to the onset of autoimmune diseases such as rheumatoid arthritis (ra). ra is a chronic inflammatory disease of joints caused by mediators (i.e. tnf-a) produced by activated leukocytes. we recently demonstrated expression of activating lilra2 in synovial tissue macrophages from ra patients. the aim of this study was to determine expression and function of lilra2 in monocytes and macrophages. peripheral blood mononuclear cells (pbmc) were prepared by standard density gradient separation and in vitro-derived macrophages were generated by differentiating thp-1 cells with vitamin-d3. lilra2 expression was measured by flow cytometry before and after modulation with cytokines. differentiation to macrophages significantly up-regulated lilra2 expression (p=0.0239). treatment of macrophages with lps, tnf-a, il-1b and ifn-g but not il-10 caused significant down-regulation of lilra2 (p<0.01). function of lilra2 was assessed by cross-linking with plate-immobilised lilra2-specific mab. soluble tnf-a was measured by elisa. activation of cells elicited tnf-a production in a dose-dependent manner while time-course analysis shows maximal production at 18h. correlation between lilra2 expression and response to cross-linking indicates that level of expression may relate directly to the degree of activation. decrease expression in response to acute-phase cytokines suggests controlled regulation during inflammation. in ra, abnormal regulation of lilra2 could potentially exacerbate inflammation by inducing uncontrolled production of proinflammatory cytokines. pharmacological blocking of lilra2 could potentially provide therapeutic benefit. (1) (1) university of valencia, spain (2) northwick park institute for medical research, uk co-releasing molecules (co-rms) mimic the biological actions of co derived from heme oxygenase activity. in the present work we studied the effects of a water-soluble co-releasing molecule (corm-3) on an animal model of human rheumatoid arthritis. dba-1/j mice were treated with corm-3 (5, 10 or 20 mg/kg/day, i.p.) from day 22 to 31 after collagen-induced arthritis (cia) and sacrificed on day 32. administration of corm-3 resulted in a significant improvement of the clinical profile of this disease since it markedly reduced joint swelling and redness. histological analysis of the joints in control arthritic mice indicated the presence of granulocytes and mononuclear cells, cartilage erosion, chondrocyte death and proteoglycan depletion. all these parameters were significantly reduced by corm-3 treatment with the most pronounced protective effect observed at 10 mg/kg. the levels of pro-inflammatory mediators (pge2, il-1beta, tnfalpha, il-2 and il-6) in the hind paw homogenates were significantly inhibited by corm-3. in addition, comp levels in serum, a marker of cartilage degradation, was reduced by the co-releasing agent. our studies show that therapeutic administration of corm-3 alleviates the clinical features of murine cia at the late phase of this response. the beneficial action of co liberated from corm-3 appears to be associated with a decrease in inflammatory cytokines and reduction of cell infiltration into the synovial tissues ultimately leading to a protective effect on the cartilage. aim: to setup a bovine model for cytokine-induced articular cartilage collagen degradation, and characterize the model using a variety of compounds targeting different disease mechanisms relevant to arthritis. methods: full thickness bovine articular cartilage punches were cultured with or without 10 ng/ml il-1a, tnf-a and oncostatin m. after three weeks the cartilage and culture medium were analyzed for weight changes, water content, dna content, glycosaminoglycans (gag), hydroxyproline (hyp), damaged collagen molecules, mmp activity, ctx-ii and comp. diclofenac, dexamethasone, pioglitazone, remicade, risedronate, galardin and a77-1726 were tested for their effect on cartilage degradation. results: exposure of articular cartilage to cytokines resulted in a decreased cartilage weight, increased proteoglycans degradation, increased collagen degradation, increased percentage of denatured collagen, increased water content and increased levels of active mmps (all p < 0.01). comp release during the first week of culture showed a trend towards up regulation during the first week of culture for all three donors, this was however not significant due to the small number of donors. most of the described processes were modulated by one or more of the drugs tested, indicating that this model for articular cartilage destruction is sensitive to treatment. discussion: stimulation of bovine articular cartilage explants with a cocktail of il-1a, tnf-a and osm results in clear and consistent changes in the cartilage, highly reminiscent of cartilage destruction during arthritis. further research needs to establish whether the model is also sensitive to anabolic factors that potentially could repair the damage. toll-like receptors (tlrs) may contribute to the progression of rheumatoid arthritis through recognition of hostderived damage-associated ligands that have repeatedly been found in arthritic joints. involvement of tlr2 and tlr4 activation in the expression of arthritis was studied using interleukin-1 receptor antagonist deficient (il-1ra-/-) mice, which spontaneously develop an autoimmune t-cell mediated arthritis. spontaneous onset of arthritis was dependent on tlr activation by microbial flora, as germ-free mice did not develop arthritis. after crossing with tlr knockouts, il-1ra-/-tlr2-/-mice developed more severe arthritis compared to il-ra-/-tlr2+/+ littermates; whereas, tlr4-/-il-1ra-/-mice were protected against arthritis. to clarify the mechanism by which tlr2 and tlr4 differentially regulated the disease expression, we studied the role of these tlrs in il-23 production and th17 development, both important in il-1ra-/-arthritis. wild type bone-marrow-derived dendritic cells (bmdcs) produced similar levels of il-23 upon stimulation with tlr2 and tlr4 ligands; however, il-1ra-/-bmdcs produced less il-23 than wild type dcs upon tlr2 stimulation and more il-23 than wild type dcs upon tlr4 stimulation. furthermore, il-1ra-/-t cells produced lower amounts of il-17 when cultured with tlr2-activated apcs and higher amounts of il-17 when cultured with tlr4-stimulated apcs, both in combination with cd3 stimulation. facs analysis of th17 (cd4+/il-17+) cells from both spleen and draining lymph nodes revealed 70% reduction in il-1ra-/-tlr4-/-mice compared to il-1ra-/-tlr4+/+ littermates. specific cd3/cd28 stimulation of non-adherent splenocytes confirmed lower il-17 production in il-1ra-/-tlr4-/-. these findings suggest important roles for tlr2 and tlr4 in regulation of th17 development and expression of arthritis. prostaglandine2 (pge2) stimulates the transactivational activity of p53 through p38map kinase-dependent ser15 phosphorylation (jbc 2006) .p53 controls cell-cycle progression, in part, by differential regulation of ap-1 proto-oncogenes (jun/fos).currently we studied pge2 control of cyclin d1 promoter activity with particular attention to the role of ap-1 oncogenes.pge2 induced a 7.6 fold increase in junb mrna expression (northern blot), a 2.1-fold increase in junb promoter activity (luciferase assay), and increased ser259junb phosphorylation in human synovial fibroblasts (hsf) (western blot).c-jun was strongly inhibited while jund, c-fos, fra1/ 2, and fosb expression were upregulated by pge2.in cell-cycle experiments, transformation with a constitutively active ha-ras construct (ras g12v) resulted in a 3.9 fold increase in cyclin-d1 promoter activity, cyclin-d1 synthesis, thr202/tyr204 phosphorylation of erk1/2 (6.2 fold) and ap-1 (c-jun)-dependent transactivity (3.1 fold); cyclin d1/cdk4-6 inhibitor p16ink4a synthesis was suppressed. addition of excess rass17n dominant negative mutant construct to the plasmid mix abrogated the aforementioned processes.ectopic expression of c-jun, c-fos and especially jund expression constructs stimulated cyclin d1 promoter activity/protein synthesis, blocked p16ink4a synthesis; the latter effects were reversed by the addition of excess junb.pge2 exerted temporal and bi-phasic dose-dependent control of the cyclin d1 promoter activity, largely through differential ap-1 activation and promoted cell cycle arrest and apoptosis in hsf at high physiological concentrations.the results provide further insight into the biology of the cpla2/ cox/pges biosynthetic axis and highlight the complexity of pge2 action in terms of cell-cycle progression. di-glucopyranosylamine (diga) is an antikeratitic (roberts et al., 1990 , acvo conference, scottsdale) immunomodulatory pyranosyl disaccharide with parenteral anti-rheumatic activity (bolton et al., 2005 , inflammation res. 54(s2) s121) and unknown mechanism of action.interestingly, anti-tnf therapy is anti-keratitic.-diga hydrolyses to monoglucosylamine (mga) and glucose, which is prevented by n-acetylation (nacdi-ga).lider (1995, pnas. 92: 5037-41) showed that sulphated disaccharides are orally active, inhibit tnf synthesis and the dth reaction.we have investigated the anti-tnf and anti-rheumatic activity of the sulphated and free digas.human whole blood (hwb) was stimulated with pha (5mg/ml) to synthesise tnf.antigen induced arthritis (aia) was induced in methylated bovine serum albumin (mbsa) sensitised c57bl/6 mice challenged i.a. into the stifle joint.collagen arthritis (cia) induced in dba mice by sensitisation to bovine collagen, were boosted i.p. with collagen at day 21. hwb tnf synthesis was inhibited by diga, mga and nacdiga(ic50 <0.1mm). diga (10ml, 1mm) i.a. prevented 24 hour aia (-0.32+/-0.06mm).diga at 100mg/kg reduced aia when administered i.v. (-0.40+/-0.13mm, p<0.05) and i.p. (-0.34+/-0.13mm, p<0.05), but is hydrolysed p.o. (0.24+/-0.08mm ns). polysulphated diga (diga8s) was unstable, but stabilised by n-acetylation (nacdi-ga8s).tnf synthesis was potently inhibited by both nacdiga and nacdiga8s (ic50 <0.1mm).nacdiga8s (100mg/kg p.o.) inhibited aia (-0.43+/-0.13mm), and nacdiga8s with lower degrees of sulphation (mw 800 and 1200 kda) inhibited the development of mouse collagen induced arthritis as assessed by clinical score. sulphated diglucosylamines represent a new class of heparinoid which are potent inhibitors of tnf synthesis and possess oral anti-rheumatic activity. excessive no appears to play a key role in the pathogenesis of chronic inflammatory diseases. in this study we aimed to evaluate no synthesis in rheumatoid arthritis (ra) before and after therapy. it was performed on 92 persons, divided into 6 groups: a negative control group of healthy volunteers, a positive control group with ra, a group with ra and physiotherapy (phys), a group with ra and low doses of cimetidine (cim) + doxycycline (dox), a group with ra and combined treatment phys + cim + dox, and a group treated with usual doses of ibuprofen (ibu). serum nitrite/nitrate (griess) was measured in order to evaluate no synthesis. results: compared to the positive control group, in all the treated groups no synthesis decreased significantly. there was no significant difference between phys and cim+dox effect alone. the combined treatment, phys + cim + dox had a much better inhibitory effect on no synthesis. between the phys, cim + dox and phys + cim + dox groups and that treated with ibuprofen, there was no significant difference in reducing no synthesis. conclusions: 1) in ra phys + cim + dox treatment was as efficient as ibuprofen in reducing no synthesis. 2) the low doses of cim and dox may allow a longer treatment due to the lower side effects risk enhanced socs1 expression following exposure of murine macrophages to lps implicated socs1 in the control of lps-mediated signaling. socs1 regulates nfkb signaling in murine macrophages, blocking at the level of mal or ikba phosphorylation. we investigated the role of socs1 in regulating the production tnf by lps and pam3csk4-activated primary human monocytes. blood monocytes were isolated by centrifugal elutriation and either infected with an adenoviral vector expressing socs1 (adv-socs1), control vector (adv-gfp) or left untreated. adv-socs1 monocytes were exposed to tlr4 and tlr2 ligands, lps (500 ng/ml) or pam3csk4 (300 ng/ml). facs analysis demonstrated infection efficiencies of 50ae4% and 67ae4% (n=16, mean ae sem) of monocytes expressing adv-gfp or adv-socs1 at moi 50. adv-socs1 blocked lps and pam3csk4 induced tnf mrna and protein production in a dose-dependent manner. in contrast, il-6 and il-10 production by adv-socs1-infected monocytes was not blocked. adv-socs1 also blocked lps and pam3csk4induced tnf production by macrophages isolated from synovial fluid. infection efficiencies of 67ae1% or 72ae1% were obtained. quantitative western blot analysis revealed that the classically defined nfkb pathway was not altered at the level of ikba or p65 activation. furthermore, the kinetics of lps and pam3csk4induced ikba phosphorylation and degradation in adv-socs1 monocytes remained unaffected (n=5 and 2 donors, respectively). further, analysis of parallel mapk pathways demonstrated no block in p38 or erk mapk pathways. these data suggest that socs1 regulation of lps and pam3csk4-induced tnf production by human monocytes occurs downstream of tlrs, possibly at the level of transcription. recently, beta-nad+ has emerged as a novel extracellular player in the human urinary bladder. beta-nad+ is the natural substrate of cd38 which catalyzes the conversion of beta-nad+ to cadpr. under normal conditions in vivo, there is no or only very small quantities (submicromolar range) of extracellular beta-nad+ compared to intracellular levels (200-1000 mm). during inflammation cell lysis may cause bursts of high local beta-nad+ levels. however, the effect of beta-nad+ on the human detrusor smooth muscle cells (hdsmc) was unknown. the effect of beta-nad+ on cultured (explant technique) hdsmc was determined by: 1) measuring cytosolic free calcium ([ca2+] i) in fura-2 loaded hdsmc using spectrofluorometry and 2) force measurements in 10-20 mg detrusor strips. hdsmc responded to beta-nad+ (40-100 mm) with an immediate and transient increase in [ca2+] i. the ca2+ transient was followed by one or two much slower and transient increases in [ca2+] i, indicative of beta-nad+ enzymatic conversion into cadpr. the ca2+ responses persisted in the absence of extracellular calcium. the ca2+ responses to beta-nad+ were not affected by exposure of hdsmc to atp supporting the notion that the effects of beta-nad+ were not mediated via p2x7 purinoceptors. furthermore, beta-nad+ caused a concentration-dependent detrusor muscle relaxation. this is the first study to report that extracellular beta-nad+ affect intracellular calcium homeostasis and force in hdsmc. these powerful actions of beta-nad+ suggest a role for beta-nad+/cadpr system as a novel extracellular player in the human detrusor during inflammation. aids remains a worldwide threat more than two decades after identification of hiv as the etiological agent. its wide dissemination can be partly attributed to its successful suppression of immunity resulting in disease progression and concomitant opportunistic infections including mycobacterial and cytomegalovirus infections. hiv trans-activator (tat) is one of the regulatory proteins that mediates hiv replication and dysregulates cellular functions such as apoptosis and cytokine expression. for example, tat induces tumor necrosis factor (tnf) and enhances gp120-induced neurotoxicity. we recently showed that tat induces the overexpression of il-10 via cellular kinase pkr and activation of transcription factor ets-1. in this study, we examined whether tat plays a role in perturbing interferon-& (ifn&) signal transduction. we showed that tat impaired ifn&-induced stat1 tyrosine phosphorylation, but had no effects on the serine residue of stat1 and jak kinases in primary human blood monocytes. furthermore, we found that the nuclear translocation of phospho-stat1 was abrogated by tat. the inhibition of phospho-stat1 led to the deformation of stat1 homodimers and subsequent stat-dna complex. to investigate the cellular consequences, we measured the expression of ifn&-stimulated genes including human leukocyte antigen (hla) and 2,5oligoadenylate synthetase (2,5oas), a key enzyme in the activation of latent ribonuclease l. the results showed that tat inhibited transcriptional activation of 2,5oas and hla. taken together, we identified a new role for tat in which it impairs ifn& signal transduction and suppresses inflammation, thus crippling the immune system and contributing to hiv persistence, opportunistic infections and disease progression. caspase-1 belongs to the group of inflammatory caspases and is the activating enzyme for the pro-inflammatory cytokine interleukin-18 (il-18), a cytokine known to play an important role in the pathogenesis of psoriasis. the purpose of this study was to determine the expression of caspase-1 in psoriatic skin and the signaling mechanisms involved in stress induced activation of caspase-1 and il-18. interestingly, increased caspase-1 activity in lesional compared with nonlesional psoriatic skin was seen as determined by western blotting. in vitro experiments in cultured human keratinocytes demonstrated anisomycin induced, p38 mapk dependent increased secretion of procaspase-1 and active caspase-1. furthermore, anisomycin increased the mrna expression of il-18 through a p38 mapk dependent but caspase-1 independent mechanism, reaching a maximum level after 12 hours of stimulation. finally, anisomycin caused a rapid (4 hours) increase in the secretion of proil-18 and active il-18. secretion of active il-18 was mediated through a p38 mapk/caspase-1 dependent mechanism, whereas secretion of proil-18 was mediated by a p38 mapk dependent but capsase-1 independent mechanism. these data demonstrate that the activity of caspase-1 is increased in psoriatic skin and that il-18 secretion is regulated by a p38 mapk/caspase-1 dependent mechanism, making caspase-1 a potential target in the treatment of psoriasis. prostaglandin e2 (pge2) regulates the stability of cyclooxygenase-2 (cox-2) mrna through adenylate/uridylate-rich elements (ares) in the 3untranslated region (3utr) by a positive autocrine/paracrine feed-forward loop. the principal objective of this study was to elucidate the molecular mechanisms involved in the pge2dependent stabilization of cox-2 in human synovial fibroblasts (hsfs). transfection of well-known are binding proteins (aubps) demonstrated that tristetraprolin (ttp) potently destabilized a [luciferase-cox-2 3utr] reporter fusion mrna (71ae5.7% decrease in luciferase activity vs. control). ttp protein levels in hsfs remained constant despite il-1b-induced changes in ttp mrna levels, thus suggesting translational regulation of its expression. pge2 did not affect the transcription or translation of this gene in hsfs. western blot analysis of hsf ttp demonstrated the existence of a specific, covalent~55kda heterocomplex containing ttp (ttphcx). although ttphcxs exact composition and stoichiometry is yet to be defined, pge2 selectively regulated the amount of this heterocomplex in a time-dependent manner. furthermore, protein shuttling studies performed using real-time confocal microscopy revealed that pge2 can induce export of a small nuclear pool of ttp-gfp. finally, transfection of ttp into hsfs also influenced cox-2 gene transcription, thus enabling ttp to regulate cox-2 gene expression at both the transcriptional and post-transcriptional level. in conclusion, we have demonstrated that ttp is an rna binding protein capable of influencing cox-2 mrna stability and transcription and whose localization and interaction with other factors is regulated by pge2. these data can provide important insight into deciphering the role of pge2 in fine-tuning physiological and pathophysiological gene regulation. (1) (1) chinese academy of sciences, shanghai, pr china (2) ohio state university, usa mitogen-activated protein (map) kinases play a critical role in innate immune responses to microbial infection through eliciting the biosynthesis of proinflammatory cytokines. map phosphatases (mkp)-1 is an archetypical member of the dual-specificity phosphatase family that deactivates map kinases. induction of mkp-1 has been implicated in attenuating the lipopolysaccharide (lps) and peptidoglycan (pgn) responses, but how the expression of the mkp-1 is regulated is still not fully understood. here, we show that inhibition of p38 map kinase by specific inhibitor sb 203580 or rna interference (rnai) markedly reduced the expression of mkp-1 in lps or pgn-treated macrophages, which is correlated with prolonged activation of p38 and jnk. depletion of mapkap kinase 2 (mk2), a downstream substrate of p38, by rnai also inhibited the expression of mkp-1. the mrna level of mkp-1 is not affected by inhibition of p38, but the expression of mkp-1 is inhibited by treatment of cycloheximide. thus, p38 mapk plays a critical role in mediating expression of mkp-1 at a posttranscriptional level. furthermore, inhibition of p38 by sb 203580 prevented the expression of mkp-1 in lpstolerized macrophages, restored the activation of map kinases after lps restimulation. these results indicate a critical role of p38-mk2-dependent induction of mkp-1 in innate immune responses. the i-kb kinase (ikk) complex regulates the activation of nf-kb a key transcription factor in inflammation and immunity. whilst ikka activity is necessary for proinflammatory and anti-apoptotic gene expression, ikka has distinct roles in lymphorganogenesis and b cell maturation. here we describe a role for ikka in cell mediated immunity (cmi). paw inflammation in methylated bsa-induced cmi was significantly reduced in transgenic mice expressing a mutant ikka protein that cannot be activated (ikka aa/aa ) compared to wild-type (wt). antigen-induced il-2 and ifng production by ikka aa/aa splenocytes and ikka aa/aa t cell:dc cocultures were also significantly reduced ex vivo. this could be normalised by using wt t cell: ikka aa/aa dendritic cell (dc) but not ikka aa/aa t cell:wt dc combinations. this suggests that reduced cmi in ikka aa/ aa mice is due to a defect in ikka aa/aa t cells not dcs. this is not due to a requirement of ikka in tcrmediated activation of t cells, since anti-cd3/cd28 mediated activation of ikka aa/aa t cells was unaltered. however, lps-induced production of the important th1 cytokine il-12 is impaired in ikka aa/aa dcs. we are currently addressing the hypothesis that ikka activity may be required for the generation and maintenance of antigen-specific t cells in vivo. recently we described a role for ikkâµ in the negative regulation of innate immunity and acute inflammation, which is in contrast to its role shown here in promoting adaptive immunity and antigen-driven inflammation. ikka may represent an alternative target for the treatment of autoimmune disease which would not compromise host defence. as a latent transcription factor, nf-kb translocates from cytoplasm into nucleus upon stimulations and mediates expression of genes important in immunity, inflammation and development. although extensive studies have been done regarding how nf-kb is triggered into nucleus, little is known about how it is regulated inside nucleus. by twohybrid approach, we identify a prefolding-like protein snip that is expressed predominantly and interacts specifically with nf-kb inside nucleus. we show that rnai knockdown of snip leads to impaired nucleus activity of nf-kb and dramatically attenuates expression of nf-kb dependent genes. this interference also sensitizes cells to apoptosis by tnf-a. furthermore, snip forms a dynamic complex with nf-kb and is recruited to the nf-kb enhancesome upon stimulation. interestingly, snip protein level correlates with constitutive nf-kb activity in human prostate cancer cell lines. the presence of nf-kb within nucleus of stimulated or constitutive active cells is significantly diminished without endogenous snip. our results reveal that snip is an integral component of nf-kb enhancesome and essential for its stability in nucleus, which uncovers a new mechanism of nf-kb regulation. bone remodeling is a tightly regulated process that couples resorption of old bone by osteoclasts and the deposition of new bone by osteoblasts. an imbalance between bone formation and bone resorption can result in various metabolic bone diseases, such as rheumatoid arthritis and osteoporosis. osteoclasts are terminally differentiated cells that arise from a haematopoietic stem cell lineage, which also gives rise to monocytes and macrophages. osteoclast differentiation and regulation of this process to maintain bone homeostasis are central to the understanding of the pathogenesis and treatment of bone diseases, such as osteoporosis. in vitro, osteoclast formation from bone marrow macrophages is induced by rankl (receptor activator of nf-kappa b ligand) in the presence of m-csf (macrophage colony stimulating factor). osteoclastogenesis is markedly enhanced in bone marrow macrophages from ifnar1-/-and ifnar2-/-mice and results in increased number of multinucleated cells positive for osteoclast marker, trap (tartrate-resistant acid phosphatase). consequently, the mutant mice develop osteoporotic phenotype, characterised by reduced bone density. these findings suggest that the ifn alpha/beta system is critical for the negative feedback regulation of osteoclastogenesis and that rankl signaling is essential for the induction of osteoclast differentiation. atp acting on p2x7 receptors in macrophage is one of the main physiological signals that lead to the processing and release of the pro-inflammatory cytokine, interleukin-1beta (il-1b), their activation also leads to rapid opening of a membrane pore permeable to dyes such as ethidium. here we identify pannexin-1, a recently described mammalian protein that functions as an hemichannel when ectopically expressed, as this dye-uptake pathway and show that signalling through pannexin-1 is required for processing of caspase-1 and release of mature il-1b induced by p2x7 receptor activation. furthermore, maitotoxin and nigericin, two agents considered to evoke il-1b release via the same mechanism were studied. maitotoxin evoked dye uptake whose kinetics were similar to a slow pannexin-1-independent phase induced by p2x7 receptor activation, and this was unaltered by pannexin-1 inhibition.nigericin did not induce dye uptake.inhibition of pannexin-1 blocked caspase-1 and il-1b processing and release in response to this two stimuli.thus, while pannexin-1 is required for il-1b release in response to maitotoxin, nigericin and atp, a mechanism distinct from pannexin-1 hemichannel activation must underlie the former two processes. introduction: saa is a classic acute-phase protein upregulated during inflammatory response. saa is active on leukocytes and modulates inflammation and immunity through the induction of cytokines, including the chemokine il-8. here we verify the effect of saa on the mrna expression and release of mip-3alpha, a chemokyne involved in the recruitment of dendritic cells. methods: peripheral blood mononuclear cells (pbmc) isolated from peripheral blood by density gradient were cultured in rpmi medium in the presence of saa. mip-3alphaconcentration was determinated in the supernatant of cell cultures by elisa. mrnawas analyzed bythe ribonuclease protection assay (rpa). results: pbmc stimulated with saa (20 ug/ml) induced the expression of mip-3alpha mrna at 2, 4 and 12 hours. mip-3alpha protein was found in the suppernatant of 24 and 42 hours cultures (p<0,05) and the addition of sb 203580 (p38 inhibitor) and pd 98059 (erk 1/2 inhibitor) completely abolished the release of mip-3alpha. conclusions: saa is an inducer of mip-3alpha expression in pbmc and p38 and erk1/2 are important pathway signaling to this effect. saa is one of the factors responsible by the recruitment of dendritic cells. the p38 pathway is activated in numerous inflammatory conditions, including ra, ibd asthma, acute coronary syndrome, and copd, and its activation helps drive the production of inflammatory mediators. inhibitors of p38 decrease mediator production and therefore can produce profound anti-inflammatory effects. arry-797 is a potent inhibitor of p38 enzyme (ic50 < 5nm) with a novel pharmacophore and physiochemical properties distinct from those of other p38 inhibitors, being very water soluble. it is extremely potent in human whole blood, blocking lps-stimulated tnf production with an ic50 < 2nm.in animal models of rheumatoid arthritis (cia and aia) the compound significantly normalized histologic endpoints, such as inflammation, bone resorption and cartilage damage (ed50~3 mg/kg). a phase i single ascending dose clinical study was run in healthy volunteers. after an oral dose of 50, 100, 200, 300 or 400 mg), blood was drawn at various times, stimulated ex vivo with lps, and analyzed for cytokines and inflammatory mediatorsfj arry-797 was well-tolerated and drug exposure was proportional to dose. in ex vivo samples, there was both a time-and concentrationdependent inhibition of il1, pge2 and tnffz with >80% inhibition observed at the 50 mg dose level. the plasma concentrations of drug peaked at~20 ng/ml at the 50 mg dose and cytokine inhibition was sustained for >4 hours, showing that low doses of arry-797 produced profound effects on clinical biomarkers. further evaluation of arry-797 in patients with inflammatory diseases is planned. introduction: we demonstrated that in vivo chronicle blockage of nos (l-name, 20mg/kg; oral route; 14 days) impairs leukocyte-endothelial interactions and neutrophils migration into inflammatory focus. these effects may be depending, at least in part, on decreased expression of l-selectin on leukocytes and pecam-1 on endothelium. aimed to clarify the mechanisms involved on these inhibitory effects, we now investigated the role of l-name treatment on secretion of tnf and il-1b; by circulating leukocytes and migrated peritoneal neutrophils. methods: male wistar rats were treated with l-name (20mg/kg; oral route; 14 days) or sterile saline (control). circulating leukocytes were isolated from blood collected from abdominal aorta and migrated neutrophils were obtained 4 hours after i.p. injection of oyster glycogen (1%;10ml). no (griess reaction) and cytokines (elisa) were quantified in supernatants of 1 x 106 cultured cells before and 18 hours after lps stimulation (5m;g/ml). results: levels of no, tnf and il-1b; were reduced in circulating leukocytes from l-name-treated rats in both basal and lps stimulated conditions. on the other hand, only secretion of il-1b; was impaired by migrated neutrophils. conclusions: results show that in vivo l-name treatment, which partially reduces no production, decreases the secretion of pro-inflammatory cytokines by circulating leukocytes. however, the same pattern of inhibition is not detected if neutrophils are in vivo primed. objectives: to investigate the ability and mechanism of ifn-g to suppress interleukin-1 (il-1)-induced mmp-13 expression in articular chondrocytes. methods: human chondrocytes were treated with ifn-g or il-1beta alone or in combination. mmp-13 mrna was analyzed by rt-pcr. mmp-13 protein, phospho-stat1 and p44/42 mapk levels were measured by western blotting. mmp-13 promoter-luciferase, cmv-cbp/p300 plasmids and stat1 sirna were transfected by calcium phosphate method. ap-1 activity was monitored by elisa. stat1-cbp/p300 interaction was studied by immunoprecipitation. results: ifn-gpotently suppressed il-1-induced expression of mmp-13 and promoter activity. blockade with neutralizing ifn-gr1 antibody revealed that mmp-13 inhibition by ifn-â¼ was mediated by the ifn-â¼ receptor. ifn-beta-stimulated activation of stat1 was directly correlated with mmp-13 suppression. knockdown of stat1 gene by specific sirna or its inhibition with fludarabine partially restored the il-1 induction of mmp-13 expression and promoter activity. ifn-g did not alter activator protein (ap-1) binding ability but promoted physical interaction of stat1 and cbp/p300 co-activator. p300 overexpression reversed ifn-g inhibition of endogenous mmp-13 mrna expression and exogenous mmp-13 promoter activity. conclusions: ifn-g through its receptor activates stat1, which binds with cbp/p300 co-activator, sequesters it from the cell system and thus inhibits transcriptional induction of mmp-13 gene in chondrocytes. ifn-g and its signaling pathways could be targeted therapeutically for (1), p asmawidjaja (1), r hendriks(2), erik lubberts (1) (1) erasmus medical center, department of rheumatology, rotterdam, the netherlands (2) erasmus medical center, department of immunology, rotterdam, the netherlands the objective of this study was to identify the role of il-23 in th17 polarization in the prone autoimmune dba-1 mice with and without collagen-induced arthritis and to evaluate th17 specific cytokine and transcription factor expression. il-23 induced th17 cells in vitro from spleen cells of naã¯ve and collagen-type ii (cii) immunized dba-1 mice. the percentage of th17 cells is markedly higher in cii-immunized versus naã¯ve dba-1 mice. adding il-23 to tgf-beta/il-6 stimulated cd4 + t cells did not significantly increase the percentage of th17 cells. tgfbeta/il-6 in contrast to il-23 induced a relatively high percentage of il-17+/ifn-gamma-cells and low il-17-ifn-gamma+ cells. tgf-beta/il-6 did not increase il-23 receptor expression, which may explain why adding il-23 directly or two days after tgf-beta/il-6 did not result in an increase in the percentage of th17 cells. elevated expression of il-17a and il-17f as well as the th17 specific transcription factor rorgammat was found under il-23 as well as tgfbeta/il-6 conditions. interestingly, il-23 but not tgf-beta/il-6 is critical in the th17 cytokine il-22 expression in t cells from ciiimmunized dba-1 mice. these data show that il-23 was more pronounced in inducing il-17+/ifn-gamma-(th17) cells under cii-immunized conditions. furthermore, il-23 did not markedly increase the percentage of th17 cells induced by tgf-beta/il-6. however, il-23 is critical for the induction of il-22 expression, suggesting a unique role for il-23 in the induction of specific th17 cytokines ebi3 was initially discovered as a transcriptionally activated gene in epstein-barr virus-infected human b lymphocytes, and similar to p40 of il-12. ebi3 protein has been shown to form heterodimers with p28. p28/ebi3 termed il-27, can influence the function of multiple t cell subsets, including naive, effector, regulatory and memory t cells. however, previous studies showed that the overlapped expression of ebi3 and p28 is very limited. these data lead to the hypothesis that ebi3 may play a role independently from its association with p28. thus, to define the function of ebi3, we generated ebi3 transgenic (tg) mice expressing in multiple tissues. ebi3 tg mice exhibited no histologic abnormalities in various organs and normal numbers of naive and memory cd4+, cd8+ t cells, b cells, nk cells and nkt cells. cd4+t cells isolated from spleens of ebi3 tg mice, however, produced less ifn-g than cells from wt (wild type) control mice after in vitro stimulation with anti-cd3 and anti-cd28 antibodies. in vivo studies, delayed-type hypersensitivity (dth) and contact hypersensitivity (chs) responses were significantly reduced in ebi tg compared with wt mice. moreover, the chs responses in ebi3 tg mice were recovered with anti-ebi3 polyclonal antibody. notably, chs reaction in wt mice was increased by anti-ebi3 antibody. in contrast, anti-p28 antibody suppressed chs responses in wt mice. these data suggest that ebi3 acts in different from il-27, and reduces th1 responses. (1), o thaunat(2), x houard (1), o meilhac (1), g caligiuri(2), a nicoletti (2) (1) inserm u698 and university denis diderot-paris 7, chu xavier bichat, paris, france (2) inserm umr s 681, universitã� pierre et marie curie-paris 6, centre de recherche des cordeliers, paris, france arteries are composed of three concentric tissue layers which exhibit different structures and properties. because arterial injury is generally initiated at the interface with circulating blood, most studies performed to unravel the mechanisms involved in injury-induced arterial responses have been focused on the innermost layer (intima). in contrast, the role of the outermost tunica, the adventitia, has attracted relatively little attention and remains elusive. in the present review, we focus on involvement of the adventitia in the response to various types of arterial injury leading to vascular remodeling. several lines of evidence show that the initial insult and the early intimal response lead to the genesis of (neo-) mediators that are centrifugally conveyed by mass transport towards the adventitia. these mediators trigger local adventitial responses including angiogenesis, immuno-inflammation, and fibrosis. we propose that these three processes sequentially interact and that their net balance participates in producing each specific pathological condition. hence, an adventitial adaptive immune response predominates in chronic rejection. inflammatory phagocytic cell recruitment and initiation of a shift from innate to adaptive immunity characterize the adventitial response to proteolysis products in abdominal aortic aneurysm. centripetal adventitial sprouting of neovessels, leading to intraplaque hemorrhages, predominates in atherothrombosis. adventitial fibrosis mediated by low inflammation characterizes the response to mechanical stress and is responsible for constrictive remodeling of arterial segments and initiating interstitial fibrosis in perivascular tissues. these adventitial events thus impact not only on the vessel wall biology but also on the surrounding tissue. atherosclerosis has many of the characteristics of an inflammatory disease, and thus would classically involve endothelial cox-derived prostaglandins such as pge2 and prostacyclin acting on ep and ip receptors, respectively.activation of vascular ip receptors is especially important in limiting the atherogenic properties of thromboxane a2 acting on tp receptors.more recently, expression of ghrelin receptors has been shown to be increased in atherosclerotic plaques, and ghrelin itself has anti-inflammatory properties in addition to its classical role as a hunger hormone.as well as the complex crosstalk between g-protein-coupled receptors (gpcrs), recent evidence indicates that many gpcrs exist constitutively as homodimeric complexes, and that the formation of heterodimers not only influences the classical cell signalling pathways used by these receptors, but also affects their subcellular distribution.we have found that ep3-i, tp and ghrelin receptors readily form homodimers, but that co-transfection of hek293 cells with these receptors results in the formation of heterodimers with unpredictable effects on receptor distribution and cell signalling properties.since inflammatory conditions are thought to change the relative expression levels of gpcrs in the vasculature, and since varying the expression levels of gpcrs will affect their ability to form heterodimers, then one might predict that gpcr heterodimerization would indeed influence the reactivity of vascular tissue during inflammation. [this work was fully supported by grants from the research grants council of the hong kong special administrative region (cuhk4267/02m and 2041151 vascular inflammation leads to formation of leukotrienes through the 5-lipoxygenase pathway of arachidonic acid metabolism. leukotriene forming enzymes are expressed within atherosclerotic lesions and locally produced leukotrienes exert pro-inflammatory actions within the vascular wall by means of cell surface receptors of the blt and cyslt receptor subtypes. recent mechanistic studies have supported the notion of a major role of leukotriene signaling in atherosclerosis. leukotriene b4 (ltb4) is for example one of the most potent chemotactic mediators formed within the atherosclerotic lesion, inducing migration of a number different cell-types of both hematopoietic and non-hematopoietic origin. initially identified on neutrophils, blt receptor activation is involved in monocyte chemotaxis and adhesion as well as in vascular smooth muscle cell migration and proliferation, providing examples of potential mechanisms in ltb4-induced atherogenesis. targeting ltb4-induced activation of vascular smooth muscle cells has beneficial effects in models of intimal hyperplasia and restenosis after vascular injury. furthermore, blt receptor expression has been demonstrated on t-cells, suggesting ltb4 as a potential link between innate and adaptive immunological reactions. taken together, the local formation of leukotrienes within the atherosclerotic lesion and the potent pro-inflammatory effects of leukotriene receptor activation in target cells of atherosclerosis provide a rationale for a role leukotrienes in this disease. further experimental and clinical studies are however needed to develop therapeutic strategies of treatments targeting leukotriene signaling in atherosclerosis. in normal physiological conditions, the prostanoid (prostaglandin (pg) and thromboxane (tx)) synthesis is dependent on the constitutive isoform of cyclooxygenase (cox-1). this synthesis and release happen few minutes after cell or tissue stimulation. in vascular preparations submitted to pro-inflammatory conditions for some hours, the inducible isoform of cyclooxygenase (cox-2) and other prostanoid synthases can be observed. as an illustration of the previous experimental results, there is an increased presence of cox-2 and the inducible enzyme responsible for pge2 synthesis (mpges-1) detected by immunocytochemistry in the carotid atherosclerotic plaque with strong inflammation. in vascular cells in culture, pgi2 is the major biological active prostanoid produced in the normal physiological conditions. however, when cox-2 is induced, pgi2 and pge2 are equally produced. the role of cox-1, cox-2 and mpges-1 activities is also dependent on the expression of the various prostanoid receptors in the considered vessel. there is increasing evidence for the presence and a role of the ep receptor subtypes (ep1, ep2, ep3 or ep4) preferentially stimulated by pge2 in the vascular wall. for these reasons, we have characterized the receptors activated by pge2 in human mammary arteries. in these vessels incubated with a pro-inflammatory cytokine (interleukin-1ã¢) and lipopolysaccharides a reduced contractility to norepinephrine has been observed. this effect is abolished by treatment of the vascular preparations with a selective cox-2 inhibitor, suggesting that prostanoid synthesis and/or prostanoid receptors could be involved. rheumatoid arthritis is a syndrome which probably consists of a number of diseases for which the risk factors differ. two major processes were identified: the generation of the anti-citrullinated antigens immune response (highly sepcific for ra).we show that the different hla class ii alleles contribute to the development of anti-ccp-positive and anti-ccp negative ra.the se alleles do not independently contribute to the progression to ra, but rather contributed to the development of anti-ccp antibodies. next we determined the effect of smoking and observed that smoking only conferred risk to contract ra in the ccp-positive group and not in the anti-ccp negative group. for the risk factor ptpn22 (a gene that regulates treshold of lumphocyte activation) the allele c1858t only contributed to ccp-positive ra. in contrast to hla two other risk factors were found to be associated with both ccp-positive and ccp-negative ra. the risk factor in the fcrl-gene as has been identified in the japanese population was also tested in 931 dutch ra cases and 570 unrelated dutch controls. carrier analysis of the snp (rs7528684) revealed association of cc genotype with higher risk of developing ra as compared to tt & tc carriers (p =0.039 and or =1.31). in a meta-analysis of all studies comparing 9467 individuals, the or for the cc genotype to develop ra was 1.2 and the p-value < 0.001. in conclusion, different steps in pathogenesis of the syndrome ra can be delineated this talk will focus on recent advances in understanding primary genetic factors predisposing to inflammatory bowel disease (crohns disease and ulcerative colitis). proven genes containing genetic variants predisposing to crohns disease include ibd5/5q31, card15/nod2 and il23r. data is suggestive but not yet as convincing for many other genes. a common theme is of genetic variants influencing early innate immune responses to intestinal bacterial components, and subsequent adaptive immune responses, leading to intestinal inflammation. only for card15/nod2 is there (partial) understanding of how genetic variation influences biological function to cause chronic disease. some mouse models (gene targeted) of card15 appear to show opposite effects to other models and human systems. in humans, card15 mutations impair responses to bacterial components (muramyl dipeptide) mainly at low dose sensing. it is likely this receptor system normally maintains intestinal crypt sterility and protection from invasive infection. pathogen-recognition receptors (prrs) are key components of immune systems and are involved in innate effector mechanisms and activation of adaptive immunity. since their discovery in vertebrates, toll-like receptors (tlrs) have become the focus of extensive research that has revealed their significance in the regulation of many facets of our immune system. recently a new family of intracellular prrs, the nod-like receptors (nlrs), which include both nods and nalps have been described. mutations within the nalp3/cryopyrin/ cias1 gene are responsible for three autoinflammatory disorders: muckle-wells syndrome, familial cold autoinflammatory syndrome, and cinca/nomid. the nalp3 protein associates with asc and caspase-1 (thereby forming a molecular machine termed inflammasome that displays high proil-1beta-processing activity. macrophages from muckle-wells patients spontaneously secrete active il-1beta. increased inflammasome activity is therefore likely to be the molecular basis of the symptoms associated with nalp3-dependent autoinflammatory disorders. here we will emphasis on the ability of this protein complex to promote the development of autoinflammatory syndromes. allergic inflammation (ai) is a complex phenomenon initiated by allergen binding to ige sensitized mast cells and consequent mast cell activation. this causes the symptoms of the early phase of ai and the onset of the late phase characterized by the penetration in the inflamed tissue of inflammatory cells, notably the eosinophils. their subsequent activation is believed to cause tissue damage and to be the main responsible for the tissue remodeling, especially when the ai becomes a chronic process. we defined a novel functional unit that we termed the allergic synapse formed by mast celleosinophil couples. in the synapse these two old cellular players of ai have a cross talk via soluble mediators and receptor-ligand interactions. this results in mast celleosinophil functional synergism that consequently amplifies and prolongs the inflammatory response. in addition, mast cells and eosinophils are influenced and influence as in a sort of allergic niche the surrounding structural cells, i.e. fibroblasts and endothelial cells. we propose to view the allergic synapse/niche as a specialized effector unit worthy to be blocked for the treatment/prevention of allergic inflammation. (1) (1) erasmus mc, rotterdam, the netherlands (2) department of immunology weizmann institute of science, rehovot, israel allergic asthma is one of the most common chronic diseases in western society, characterized by reversible airway obstruction, mucus hypersecretion and infiltration of the airway wall with th2 cells, eosinophils, and mast cells. if we are to devise new therapies for this disease, it is important to elucidate how th2 cells are activated and respond to intrinsically harmless allergens. dendritic cells (dcs) are the most important antigen presenting cells in the lung and are mainly recognized for their exceptional potential to generate a primary immune response and sensitization to aeroallergens. we have shown that intratracheal injection of ovalbumin (ova) pulsed dcs induces sensitization leading to eosinophilic airway inflammation upon ova aerosol challenge. we investigated the role of dcs in the secondary immune response in a murine asthma model. ova aerosol challenge in ova-dc sensitized mice, induced an almost 100 fold increase in the number of airway dcs as well as an increase in eosinophils and t cells. to investigate the functional importance of dcs for the induction and maintenance of airway inflammation in response to allergen challenge, we conditionally knocked-out endogenous dcs in sensitised cd11c-diphtheria toxin (dt) receptor (cd11cdtr) transgenic mice by airway administration of dt 24 h before ova aerosol (4x) challenge or during an ongoing inflammation (depletion after 3x ova aerosols continued with 3 additional ova aerosols). numbers of balf eosinophils, th2 cytokine production by mediastinal lymph nodes and peribronchial and perivascular inflammatory infiltrates were dramatically decreased, illustrating an essential role for airway dcs during secondary challenge. karolinska institute, stockholm, sweden nk cells are innate lymphocytes with potent immunoregulatory functions. they are potent producers of several cytokines and chemokines, and also respond to similar molecules in the body and at inflammatory sites. even though traditionally best characterized for their role in anti-viral and anti-tumor immunity, they influence several other types of immune responses. for example, they are involved in, and affect, acute as well as chronic inflammatory responses. in the present talk, a general overview on our current knowledge of nk cell biology will be provided, with a special emphasis on the role of these cells in allergic inflammation. basophils are major effector cells in allergic reactions due to their ability to release substantial quantities of histamine and eicosanoids following activation of high affinity ige receptors (fc<epsilon>ri) with allergens. although these attributes are shared with their tissuefixed mast cell compatriots, basophils are unique in their ability to also rapidly elaborate th2-type cytokines (e.g. il-4 and il-13), subsequently supporting ige synthesis and underlying atopy. importantly, these mediators are additionally secreted following primary exposure to certain parasites (e.g. s. mansoni) and immunoglobulin superantigens, suggesting a role for basophils in innate immunity and in assisting developing th2-type adaptive immune responses. while we are beginning to understand the potential physiological functions of these cells regarding host defence, blocking their activity with respect to treating symptoms of allergic disease has remained an enigma. recent advances, however, have shed light upon the major intracellular signal transduction processes involved in fc<epsilon>ri activation and may lead to novel therapeutic strategies for inhibiting mediator secretions. an important discovery in this regard is the phosphatase ship, which downregulates pi 3-kinase signalling in both basophils and mast cells. recent data shows that ship expressions in basophils are reduced from donors with active allergic disease but that these levels may be increased, and the activity of basophils subsequently inhibited, by targeting receptors associated with ship recruitment (cd200r, cd300r). identifying the natural ligands for these inhibitory receptors may therefore pave the way for new therapies for the treatment of allergic inflammation. mitogenesis and proliferation of vsmc play an important role in atherogenesis. pro-inflammatory secretory phospholipases a2 (spla 2 ) hydrolyse glycerophospholipids of hdl and ldl and release pro-inflammatory agents, lyso-lipids, oxidized and non-oxidized fatty acids and isoprostanes.spla 2 s lipolysis products localize in vascular wall in vicinity of vsmc.we have tested the impact of spla 2 , hdl and ldl and of their hydrolysis products on mitogenesis and pge2 and ltb4 release from vsmc.mitogenesis was significantly enhanced by native hdl, and ldl, and by group v spla 2 . spla 2 hydrolysis of hdl and ldl enhanced mitogenic activity in order v>x>iia.the release of pge2 from vsmc was enhanced by group x spla 2 s but not iia or v.the greatest effect was seen for hdl hydrolysed by group v and x spla 2 .native ldl and its spla2 hydrolysis products enhanced the release of pge2 in order x>v>iia.the release of ltb4 from vsmc was markedly increased by native ldl and hdl, and hydrolysis products of group v and x, but not iia spla2.migration of vsmc was significantly enhanced by spla 2 iia and inhibited by hdl.this study demonstrates a complex interaction of hdl and ldl with pro-inflammatory spla2s, which affects mitogenesis, eicosanoid release and migration of vsmc.study of biocompatible spla2 blockers in the therapy of atherosclerosis is indicated. contact information: professor waldemar pruzanski, university of toronto, department of medicine, toronto, ontario, canada e-mail: drwpruzanski@bellnet.ca (1) (1) ipmc-cnrs umr6097, valbonne, france (2) university of washington, seattle, usa (3) inserm umrs 525, paris, france (4) university of naples, italy the superfamily of phospholipase a2 comprises at least 9 intracellular enzymes and up to 12 secreted pla2s (spla 2 s). elucidating the biological roles of each pla2 member is currently the most challenging issue in the pla2 field. the different spla 2 s are not isoforms and are likely to function either as enzymes producing key lipid mediators (eicosanoids and lysophospholipids) or as ligands that bind to specific soluble or membrane-bound proteins (like cytokines). increasing evidence suggests that spla 2 s iia, iii, v, and x are involved in inflammatory diseases including atherosclerosis. among spla2s, the human group x (hgx) enzyme has the highest enzymatic activity towards phosphatidylcholine, the major phospholipid of cellular membranes and low density lipoproteins (ldl). on human alveolar macrophages, hgx spla 2 can trigger secretion of tnf alpha, il1 and il6 in a non-enzymatic manner. on colorectal cancer cells, hgx spla 2 stimulates cell proliferation, produces potent eicosanoids including pge2, and activates the transcription of key genes involved in inflammation and cancer. the enzyme can also hydrolyze pc and platelet-activating factor (paf) of ldl particles very efficiently. finally, hgx spla 2 is present in human atherosclerotic lesions and converts ldl into a proinflammatory particle that induces macrophage foam cell formation, as well as map kinase activation, arachidonic acid release, and expression of adhesion molecules in huvec cells. some other key molecular features of spla 2 s including hgx will be presented. we have reported preferential release of polyunsaturated fatty acids during hydrolysis of lipoprotein phosphatidylcholine (ptdcho) by spla 2 s, but the mechanism of this selectivity is not known. since both sphingomyelin (sm) and lysoptdcho inhibit the activity while increasing fatty acid specificity of other pla2s, we have examined fatty acid release by spla2siia, v and x in relation to relative increases in proportion of endogenous sm and lysoptdcho during lipoprotein digestion. the analyses were performed by normal phase liquid chromatography with on-line electrospray mass spectrometry (lc/esi-ms) and lc/collision induced dissociation (cid)/esi-ms using conventional preparations ofldl and hdl. the highest preference for arachidonate release from ldl by group x spla 2 was observed when the residual sm/ pdcho molar ratio had reached 1.2 compared to a starting ratio of 0.4.group v spla 2 showed preferential release of linoleate at residual sm/ptdcho molar ratio 1.5, while at intermediate ratios, both arachidonate and linoleate were released at more comparable ratios. the relative increases in lysoptdcho and sm during the digestion with spla 2 iia were much more limited, and a preferential hydrolysis of polyunsaturated fatty acids was not observed. these results suggest a lipid phase separation as a likely basis for a differential hydrolysis of molecular species of ptdcho. the residual sm/ptdcho ratios reached during group v and x spla 2 digestion are similar to those observed for lesional ldl, which promote release of ceramides by smase leading to ldl aggregation. the above findings support a potential role of sphingomyelins in atherogenesis. although sphingomyelin (sm) is one of the most abundant phospholipids in lipoproteins and cell membranes, its physiological significance is unclear. because of its localization in the outer surface of the cells, and its structural similarity to phosphatidylcholine (pc),we proposed that it competitively inhibits phospholipolysis of cell membranes by external phospholipases (pla). we showed that sm inhibits several lipolytic enzymes including secretory pla2 iia, v, and x, and hepatic and endothelial lipases, all of which hydrolyze pc. treatment of sm in the substrate with smase c not only relieved the inhibition but also activated the pla2 reaction further, suggesting that ceramide, the product of smase c, independently stimulates pla2, possibly by disrupting the bilayer structure. smase d treatment, which produces ceramide phosphate, did not stimulate the spla2. the fatty acid specificity of pla2 is significantly affected by sm. thus spla2x exhibited enhanced specificity for the release of arachidonic acid (20:4) in presence of sm, due to a preferential inhibition of hydrolysis of other pc species. in contrast, sm inhibited the release of 20:4 by spla2v. ceramide selectively stimulated the release of 20:4 by both enzymes. only the long chain ceramides (> 10 carbons) were effective, while ceramide phosphate did not stimulate spla2 activity. sm-deficient cells released more 20:4 in response to spla2-treatment than normal cells, and pretreatment of normal cells with smase c increased their susceptibility to spla2 attack. these studies show that sm and ceramide regulate the activity and specificity of pla, and consequently the inflammatory response. secretory phospholipase a2 (spla2) types iia, v, or x, have been associated with inflammatory diseases and tissue injury including atherosclerosis in humans and mice.given the link between spla2 and atherogenesis, a mouse model of atherosclerosis (apoe-/-) was used to study the effects of a-002, an inhibitor of spla2 enzymes, on atherosclerosis and cholesterol levels over 16 weeks of treatment. mice were fed with a high-fat, high cholesterol diet alone during the study (21% fat; 0.15% cholesterol, 19.5% casein) and were treated with vehicle or a-002 bid at 30 mg/kg or 90 mg/kg by oral gavage. total cholesterol was significantly decreased after one month of treatment and remained lower throughout the study.treatment with a-002 significantly reduced aortic atherosclerotic plaque formation in apoe-/-mice fed a high fat diet when compared to the untreated control by approximately 40 %. in a different model that used angiotensin ii in conjunction with a high fat diet in a background of apoe-/-deficient mice for 4 weeks, oral dosing of a-002 (30 mg/kg bid) significantly reduced aortic atherosclerosis and aneurysm rate when compared to vehicle. these data suggest that a-002 is a potential novel therapeutic agent for the treatment of atherosclerosis. (1), s doty(2), c antonescu (3), c staniloae (1) (1) saint vincents hospital manhattan, new york, usa (2) hospital for special surgery, new york, usa (3) sloan-kettering institute for cancer research, new york, usa tnf-stimulated gene 6 (tsg-6) is induced by tnf-a during inflammation and its secreted product tsg-6 glycoprotein is involved in immune-mediated inflammatory diseases and fertility. it regulates cox-2 and prostaglandin synthesis, and participates in extracellular matrix remodeling. considering the chronic inflammatory nature of atherosclerosis we hypothesized that tsg-6 is expressed in atherosclerotic plaques and investigated tsg-6 protein expression and cellular distribution on 12 superficial femoral artery endarterectomy specimens from 6 diabetic and 6 non-diabetic patients with peripheral vascular disease. six histologically normal radial artery specimens were analyzed as control. paraffin embedded samples were studied by immunohistochemistry using a goat polyclonal anti-human-tsg-6 antibody. tsg-6 expression was consistently present in all atherectomy specimens but not in control specimens. a distinct, strong cytoplasmic staining pattern was uniformly detected in the endothelial lining of the intima, as well as in the neo-vessel proliferation of the plaque. cytoplasmic staining was also identified in the smooth muscle cell proliferation of the neo-intima. patchy tsg-6 expression was noted in the extracellular matrix. within the inflammatory plaques from diabetic patients, tsg-6 stained the foamy macrophages. tsg-6 expression was also confirmed and quantified by qrt-pcr that showed a significant up-regulation of tsg-6 gene (more that 100 fold induction compared to housekeeping genes). our study identifies for the first time the preferential expression of tsg-6 in atherosclerotic lesions and characterizes its distribution within the cellular and matrix components of the plaque. tsg-6 is a novel inflammatory mediator of atherosclerosis and a potentially new marker of endothelial / smooth muscle cell activation. (1), r krohn (1), h lue (1), jl gregory(2), a zernecke (1), rr koenen (1), t kooistra (3), p ghezzi(4), r kleemann (3), r bucala(5), mj hickey (2), c weber (1) (1) university hospital of the rwth aachen, germany (2) centre for inflammatory diseases, monash university, melbourne, australia mediators, which cannot be classified into chemokine subfamilies but share functional patterns, e.g. signaling through chemokine receptors, constitute a group termed chemokine-like function (clf)-chemokines. the pleiotropic cytokine macrophage migration inhibitory factor (mif) plays a critical role in inflammatory diseases and atherogenesis. the underlying molecular mechanisms are poorly understood, but, interestingly, mif displays structural features resembling chemokines. we have identified the chemokine receptor cxcr2 as a functional receptor for mif. mif triggered galphai/integrin-dependent arrest and chemotaxis of monocytes specifically through cxcr2, inducing rapid integrin activation. mif directly bound to cxcr2 with high affinity (kd of 1.4 nm). monocyte arrest mediated by mif in inflamed or atherosclerotic arteries involved cxcr2 as well as cd74, a recently identified membrane receptor moiety for mif. accordingly, cxcr2 and cd74 were found to occur in a receptor complex. in vivo, mif deficiency impaired monocyte adhesion to the aortic/arterial wall in atherosclerosis-prone mice, as evidenced by intravital microscopy. thus, mif displays chemokine-like functions by acting as a non-cognate ligand of cxcr2, serving as a regulator of inflammatory and atherogenic recruitment. these data harbor an intriguing novel therapeutic prospect by targeting mif in atherosclerosis and add a new dimension to mif and chemokine receptor biology. (1), r toes (3), h van bockel(2), paul quax (1,2) (1) tno bioscienses, leiden, the netherlands (2) department of vascular surgery, leiden university medical center, the netherlands (3) department of rheumatoly, leiden university medical center, the netherlands the immune system is thought to play a crucial role in regulating collateral circulation (arteriogenesis), a vital compensatory mechanism in patients with arterial obstructive disease. here, we studied the role of lymphocytes in a murine model for artiogenenesis after acutehind limb ischemia. arteriogenesis was impaired in c57bl/6 mice depleted for natural killer (nk)-cells by anti-nk1.1 antibodies and in nk-cell-deficient transgenic mice. arteriogenesis was, however, unaffected in jâµ281knockout mice that lack nk1.1+ natural killer t (nkt)cells, indicating that nk-cells, rather than nkt-cells are involved in arteriogenesis. furthermore, arteriogenesis was impaired in c57bl/6 mice depleted for cd4+ tlymphocytes by anti-cd4 antibodies, and in major histocompatibility complex (mhc)-class-ii-deficient mice that lack mature peripheral cd4+ t-lymphocytes. this impairment was even more profound in anti-nk1.1treated mhc-class-ii-deficient mice that lack both nkand cd4+ t-lymphocytes. finally, collateral growth was severely reduced in balb/c as compared with c57bl/6 mice, two strains with different bias in immune responsiveness. correspondingly, fewer cd3-positive lymphocytes accumulated around collaterals in balb/c mice. these data show that both nk-cells and cd4+ t-cells play an important role in arteriogenesis. moreover, our data hold promise for the development of novel clinical interventions as promoting lymphocyte activation might represent a powerful method to treat ischemic disease. post-interventional vascular remodeling in venous bypass grafts, seen as intimal hyperplasia (ih) and accelerated atherosclerosis, often causing graft failure. inflammation is an important trigger for these processe. complement is an important part of the immune system and participates in regulating inflammation. although involved in several other inflammatory diseases, the role of the complement cascade in vein graft remodeling is unknown. the involvement of the complement system in vein graft disease was studied here using a model in which caval veins are grafted in carotids arteries of hypercholesterolemic apoe3leiden mice. in these veins ih and accelerated atherosclerotic lesions develop within 28 days, consisting mainly of foamcells and smc. to study the functional role of complement in vein graft remodeling, cobra venom factor (cvf:20u daily) was used to deplete complement starting one day prior to vein graft surgery. cvf-treatment reduced vein graft thickening by 64% (p=0.02), when compared to saline treated controls (n=6).to confirm that the reduction by cvf was due to hampered complement function and not a direct effect of cvf, complement activation was blocked using crry-ig (inhibiting c3 convertases). crry-ig (3mg every other day) led to 50% decrease in vein graft thickening (p=0.03) compared to controls receiving non-relevant control igg. these data prove that complement activation plays a major in intimal hyperplasia and accelerated atherosclerosis in vein grafts. (1), m-c koutsing tine(1), p borgeat(2), h ong (1), sylvie marleau (1) (1) universite de montreal, quebec, canada (2) centre de recherche en rhumatologie et immunologie, canada we have previously shown that ep 80317, a growth hormone-releasing peptide (ghrp) analogue binding selectively to the scavenger receptor cd36, elicits a striking reduction in atherosclerosis development in apolipoprotein-deficient (apoe-/-), a condition associated with increased circulating numbers of primed/activated leukocytes. we investigated the effect of ghrp analogues on i/r-elicited remote lung injury in 18week-old apoe-/-mice fed a high fat high cholesterol (hfhc) diet from 4 weeks of age. at 18 weeks old, mice were treated daily with a s.c. injection of ep 80317 (300 mg/kg) for 14 days and were then subjected to unilateral hindlimb ischemia (by rubber band application) for 30 minutes, followed by 180 minutes reperfusion. our results show that ep 80317 significantly reduced leukocyte accumulation by 56% in the lungs, from 3.6 (ae 0.7) in vehicle-treated mice to 1.6 (ae 0.6) x 107 leukocytes/g lung in ep 80317-treated mice (n = 6-7 per group), as assessed by myeloperoxidase assay. this was associated with a 56% reduction of opsonized zymosan-elicited blood chemiluminescence. in contrast, neither blood chemiluminescence, nor leukocyte accumulation in the lungs were signicantly modulated in apoe-/-/cd36-/-deficient mice, from 1.9 (ae 0.5) in vehicle-treated mice to 1.9 (ae 0.8) x 107 leukocytes/g lung in ep 80317-treated mice. we conclude that ep 80317 protects i/r-elicited circulating leukocyte priming/activation and remote lung injury, possibly through a cd36-mediated pathway. glycogen synthase kinase 3beta (gsk-3beta) is a serine/ threonine protein kinase that has recently emerged as a key regulatory switch in the modulation of the inflammatory response. dysregulation of gsk-3beta has been implicated in the pathogenesis of several diseases including sepsis. here we investigate the effects of two chemically distinct inhibitors of gsk-3beta, tdzd-8 and sb216763, on the circulatory failure and the organ injury and dysfunction associated with hemorrhagic shock. male wistar rats were subjected to hemorrhage (sufficient to lower mean arterial blood pressure to 35 mmhg for 90 min) and subsequently resuscitated with shed blood for 4 h. hemorrhage and resuscitation resulted in an increase in serum levels of (a) creatinine and, hence, renal dysfunction, and (b) alanine aminotransferase and aspartate aminotransferase and, hence, hepatic injury. treatment of rats with either tdzd-8 (1 mg/kg, i.v.) or sb216763 (0.6 mg/kg, i.v.) 5 min before resuscitation abolished the renal dysfunction and liver injury caused by hemorrhagic shock. the protection afforded by these compounds was confirmed by histological observations of lung, kidney and liver samples. in addition, tdzd-8, but not sb216763, attenuated the increase caused by hemorrhage and resuscitation in plasma levels of the proinflammatory cytokine interleukin 6. neither of the gsk-3beta inhibitors however affected the delayed fall in blood pressure caused by hemorrhagic shock. thus, we propose that inhibition of gsk-3beta may represent a novel therapeutic approach in the therapy of hemorrhagic shock. (1), y ito (1), h yoshimura (1), h inoue (1), n kurouzu (1), h hara (1), y mastui (1), h kitasato (1), s narumiya(2), c yokoyama (3), m majima (1) (1) kitasato university school of medicine, japan (2) kyoto university school of medicine, japan (3) tokyo medical and dental university, japan thromboxane (tx) a2 is a potent stimulator of platelet activation and aggregation and vascular constriction. we have reported cytokine-mediated release of sdf-1 from platelets and the recruitment of nonendothelial cxcr4+ vegfr1+ hematopoietic progenitors constitute the major determinant of revascularization. we hypothesized txa2 induces angiogenic response by stimulating sdf-1 and vegf which derived from platelet aggregation. to evaluate this hypothesis, we dissected the role of the txa2 in angiogenesis response using mouse hind limb ischemia. recovery from acute hind limb ischemia, as assessed in wild type mice (c57bl/6 wt) , prostaglandin i2 receptor (ip) knock out mice (ipko) and thromboxane (tx) a2 receptor (tp) knock out mice (tpko) by using lase doppler. blood recovery in tpko significantly delayed compared to wt and ipko. immunohistochemical studies revealed that the number of cd31positive cells in the ischemic quadriceps were less stained in tpko compared to wt and ipko.plasma sdf-1 and vegf concentration were significantly reduced in tpko mice. we observed during in vivo fluorescence microscopic study that compared to tpko, ipko and wt significantly increased platelet attachment to the microvessels around ligated area. tpko translpanted wt bone marrow cells increased blood recovery compared to tpko transplanted tpko bone marrow cells. in addition, mice injected with txa2 synthase c-dna expressing fibroblast increased blood flow recovery compared to control mice. these results suggested that tp signaling rescues ischemic condition by inducing angiogenesis by secreting sdf-1 and vegf from platelet aggregation. administration of selective tp agonist may open new therapeutic strategy in regenerative cardiovascular medicine. during renal ischemia/reperfusion (i/r) injury, apoptosis has been reported as a very important contributor to the final kidney damage. the determinant role of cytoskeleton derangement in the development of apoptosis has been previously reported, but a clear description of the different mechanisms involved in this process has not been yet provided. the aim of the study is to know the role of peroxynitrite as inductor of cytoskeleton derangement and apoptosis during the inflammatory process associated to renal ischemia-reperfusion. based in a rat kidney i/r model, by experiments in which both the actin cytoskeleton and peroxynitrite generation were pharmacologically manipulated, results indicate that the peroxynitrite produced during the i/r derived oxidative stress state, is able to provoke cytoskeleton derangement and apoptosis development. thus, the control in the peroxynitrite generation during the i/r could be an effective tool for the improvement of cytoskeleton damage and reduction apoptosis incidence in the renal i/r injury. metabolomics, the global profiling of metabolites, may inform about the multiple interacting processes involved in inflammatory disease. using nmr spectroscopy we analysed metabolite fingerprints in serum from early arthritis, and at a site of inflammation, in the posterior segment of the eye. serum from 101 patients with synovitis of "t 3 months duration whose outcome was determined at clinical follow-up was used. vitreous samples were from 31 patients undergoing vitrectomy for vitreoretinal disorders. one dimensional 1h nmr spectra were acquired. principal components analysis (pca) of the processed data was conducted along with a supervised classification. with the arthritis serum there was a clear relationship between each samples score in the pca analysis and the level of crp. supervised classification of the initial samples was able to predict outcome, whether rheumatoid arthritis, other chronic arthritis or self-limiting arthritis, with high specificity and sensitivity. a similar approach using the eye fluids was able to give a clear discrimination between two pathologically similar conditions lens-induced and chronic uveitis. in this case differences were not due to a straightforward relationship with inflammatory markers (il-6, ccl2), which did not correlate with pca in these samples. similarly, certain molecules, such as lactate, were associated with ocular disease, but not rheumatoid arthritis. these results suggest that underlying inflammatory processes may differ in these conditions or may reflect predisposing metabolic patterns in individual patients. 1h-nmr-based metabolomics may provide a useful measure of outcome in inflammatory diseases and give novel insights into the pathological processes involved. (1), am artoli(2), a sequeira(2), c saldanha (1) (1) instituto de medicina molecular,faculdade de medicina de lisboa, portugal (2) cemat, instituto superior tã�cnico, universidade de tã�cnica de lisboa, portugal the recruitment of leukocytes from the blood stream and their subsequent adhesion to endothelial walls are essential stages to the immune response system during inflammation. the precise dynamic mechanisms by which molecular mediators facilitate leukocyte arrest are still unknown. in this study combined experimental results and computer simulations are used to investigate localized hydrodynamics of individual and collective behaviour of clusters of leukocytes. leukocyte-endothelial cell interactions in post-capillary venules of wistar rats cremaster muscle were monitorized by intravital microscopy. from these experiments the haemorheologic and haemodynamical measured parameters were used in time dependent three-dimensional computer simulations, using a mesoscopic lattice boltzmann solver for shear thinning fluids. the dynamics of leukocyte clusters under non-newtonian blood flow with shear thinning viscosity was computed and discussed. in this paper we present quantified distributions of velocity and shear stress on the surface of leukocytes and near vessel wall attachment points. we have also observed one region of maximum shear stress and two regions of minimum shear stress on the surface of leukocytes close to the endothelial wall. we verified that the collective hydrodynamic behaviour of the cluster of recruited leukocytes establishes a strong motive for additional leukocyte recruitment. it was found that the lattice boltzmann solver used here is fully adaptive to the measured experimental parameters. this study suggests that the influence of the leukocytes rolling on the increase of the endothelial wall shear stress may support the activation of more signalling mediators during inflammation. macrophages are essential for host defence, but when excessively and persistently activated, these cells contribute to the initiation and progression of inflammatory diseases such as rheumatoid arthritis. investigating the function of inflammatory genes in macrophages may identify novel therapeutic targets for inflammatory diseases. one family of transcripts that are highly expressed in activated macrophages are members of the schlafen (slfn) gene family; a recently identified family whose function is still unknown. this study examined the mrna expression of slfn in activated bone marrowderived macrophages in vitro, and in collagen-induced arthritis (cia) in vivo. real-time pcr expression analyses of bone marrow-derived macrophages stimulated with lipopolysaccharide (lps) over a time course, revealed differential expression of individual slfn family genes. in particular, slfn-1, slfn-2, and slfn-4 were maximally induced after 24 hours. the maximal induction of slfn-8 and slfn-9 was observed after 4 hours of lps treatment. individual members of the slfn family were also differentially expressed in cia, a model of rheumatoid arthritis. mrna levels of slfn-1, slfn-2, slfn-4 and slfn-5 were elevated in joints affected by cia. to investigate the role of slfn-4, we have generated a transgenic mouse line, which over expresses slfn-4 specifically in cells of the mononuclear phagocyte system, by using a novel binary expression based on the c-fms promoter and gal4. further characterisation of the slfn-4 over expressing mouse line will be used to assess the function of slfn-4 in macrophage biology and inflammation, and its potential as a therapeutic target. macrophages play an important role in resolving inflammation. it is known that the resolution of inflammation requires alternative activation of macrophages. but the precise events of phenotype switching in macrophages remain poorly understood. we show that lipocalin 2, lcn-2, is able to provoke a switch in macrophage activation. in an in vitro co-culture model for renal epithelial cells and macrophages, we detected by sirna technique that the presence or absence of lcn-2 determines proliferation processes in damaged renal epithelial cells. the proliferative response was dependent on proinflammatory or anti-inflammatory environment. as lcn-2 is an acute phase protein synthesized during inflammation and unregulated in a number of pathological conditions, it may play an important role in survival and regeneration. we anticipate here that our results could be relevant for further research on the mechanisms of the phenotype switch induced by lcn-2. (1), y cao (1), s adhikari (1), m wallig (2) (1) national university of singapore, department of pharmacology, singapore (2) university of illinois at urbana champaign, usa it has earlier been shown that the extent of apoptotic acinar cell death is inversely related to the severity of acute pancreatitis. our previous works have demonstrated that induction of pancreatic acinar cell apoptosis by crambene protects mice against acute pancreatitis. the current study aims to investigate the role of phagocytic receptors and the anti-inflammatory effect of phagocytosis in protecting mice against acute pancreatitis by crambene. acute pancreatitis was induced in the mouse by administering hourly injections of caerulein (50 mg/kg) for 3, 6 and 10 hours respectively. neutralizing monoclonal anti-il-10 antibody (2.5 mg/kg) was administered either with or without crambene (70 mg/kg) 12 hours before the first caerulein injection. rt-pcr, western blotting and immunostaining were performed to detect cd36 expression. apoptosis in pancreatic sections was visualized by tunel. severity of acute pancreatitis was evaluated by estimation of serum amylase, pancreatic myeloperoxidase (mpo), water content, and morphological examination. pancreatic levels of inflammatory mediators were examined by elisa. the protective effect of crambene is mediated by reducing production of pro-inflammatory cytokines such as mcp-1, tnf-a and il-1㢠and up-regulating anti-inflammatory mediators like il-10. phagocytotic clearance in mouse acute pancreatitis may be essentially through macrophage surface receptor cd36.the anti-inflammatory mediator il-10 plays an important role in crambene-induced protective action against acute pancreatitis. the release of anti-inflammatory mediator il-10 is downstream of phagocytosis. these results show that induction of pancreatic acinar cell apoptosis by crambene treatment protects mice against acute pancreatitis via induction of anti-inflammatory pathways. (1,2) (1) northern ontario school of medicine, thunder bay, ontario, canada (2) lakehead university, canada integrin receptors and their ligands are involved in adhesion and internalization of several human pathogens, including pseudomonas aeruginosa. we have recently established that beta1 integrins in lung epithelial cells (lec) provide co-stimulatory signals regulating inflammatory responses (ulanova et al, am j physiol, 2005, 288: l497-l507). we hypothesized that lec integrins serve as receptors to recognize pathogen-associated molecules and mediate the innate immune response to p. aeruginosa. to determine molecular mechanisms of integrin involvement in innate immunity, we used an in vitro model of p. aeruginosa infection of a549 cells. to investigate interactions of bacteria with lec, p. aeruginosa strain pak was chromosomally labeled with a green fluorescent protein gene using a mini-tn7 delivery system.using several fluorescence-based detection systems, we established that the natural beta1 integrin ligand, fibronectin, mediates bacterial adhesion to lec.p. aeruginosa infection caused rapid transcriptional upregulation of alphav and beta4 integrin expression followed by the increased cell surface protein expression. the surface expression of integrin beta1 increased shortly following bacterial exposure without alterations of mrna expression, suggesting rapid protein redistribution within the cells. the data indicate that p. aeruginosa are capable to modulate integrin gene/protein expression in lec, potentially using fibronectin to alleviate bacterial binding to beta1 integrins. upon their engagement, integrin receptors can initiate intracellular signaling involved in innate immune and inflammatory responses to the pathogen. integrin receptors in lec may represent significant therapeutic targets in pulmonary infection caused by p. aeruginosa. the purine nucleoside adenosine has a major modulatory impact on the inflammatory and immune systems. neutrophils, which are generally the first cells to migrate toward lesions and initiate host defense functions, are particularly responsive to the action of adenosine. through activation of the a2a receptor (a2ar) present on neutrophils, adenosine inhibits phagocytosis, generation of cytotoxic oxygen species, and adhesion. also, recent work showed that adenosine can transform the profile of lipid mediators generated by neutrophils, inhibiting leukotriene b4 formation while potentiating that of prostaglandin e2 through the up-regulation of the cyclooxygenase (cox)-2 pathway. moreover, our laboratory determined that a2ar engagement can dramatically modulate the generation and secretion of neutrophil-derived cytokines/chemokines, including tnf-and mips. in mice lacking the a2ar, migrated neutrophils expressed less cox-2 than their wild type counterpart while displaying higher mrna levels of tnf-and mip-1. mononuclear cells from a2ar knock out mice, which eventually replace neutrophils into the air pouch, also displayed a more pro-inflammatory phenotype than those from wild-type animals. signal transduction experiments, aiming to delineate the intracellular events leading to the modulation of neutrophil functions following a2ar engagement, implicate pivotal metabolic pathways such as intracellular cyclic amp, p38 and pi-3k. together, these results indicate that adenosine may have a profound and multi-pronged influence on the phenotype of neutrophils and present this cell as being pivotal in mediating adenosines anti-inflammatory effects. the newest developments regarding adenosines effects on neutrophil functions will be presented.this work is supported by the canadian institutes of health research (cihr). human skin serves not only as a physical barrier against infection, but also as a "chemical barrier" by constitutively and inducibly producing antimicrobial proteins (amps). to identify human skin amps, we analysed extracts of healthy persons stratum corneum by reversed phase-hplc and purified a novel 9 kda amp that showed sequence similarity to mouse hornerin. suggesting that it originates from the human ortholog, we cloned it. human hornerin encodes a 2850 amino acid protein that contains a s100 domain, an ef-hand calciumbinding domain, a spacer sequence and two types of tandem repeats, suggesting that it represents a novel member of the fused s100 protein family. strongest constitutive hornerin mrna expression was seen in differentiated keratinocyte cultures. to follow the hypothesis, that hornerin fragments represent amps, we recombinantly expressed three hornerin peptides, rhrnr2 (tandem repeat unit b), rhrnr3 (tandem repeat unit a) and rhrnr4 (c-terminus) and subsequently analysed their antimicrobial activity using the microdilution assay system. the rhrnr3 peptide, containing the sequence motif found in the purified natural hornerin fragment isolated from stratum corneum, exhibited antimicrobial activity at low micromolar concentrations against escherichia coli, pseudomonas aeruginosa and candida albicans. the other peptides were found to be not or nearly not antimicrobially active. our results suggest that hornerin may have a yet unknown protective function in healthy human skin as part of the "chemical barrier" with preformed amps, which are generated from parts of the tandem repeats of a hornerin precursor molecule by a yet unknown cleavage mechanism. (1), n lu(1), r jonsson(2), d gullberg (1) (1) department of biomedicine, university of bergen, norway (2) the gade institute, university of bergen, norway a11ã�1 is the latest addition to the integrin family of heterodimeric receptors for the extracellular matrix. previously, it has been shown that this collagen receptor takes part in processes such as cell migration and matrix contraction. in this study we investigated the factors that regulate mouse integrin a11ã�1 expression. specifically, we have analyzed the influence of cell passage, growth factors and the 3-d microenvironment. using sv40 immortalized as well as primary fibroblasts, we show that a11ã�1 integrin is up-regulated when these cells are cultured within stressed collagen type i lattices. however, a11ã�1is downregulated when the collagen gels are made under relaxed conditions, allowing cells contract the lattice diameter. we also show here that a11 is upregulated by tgf-a on planar substrates. these findings suggest that mechanical tension and tgf-a are important factors in the regulation of a11ã�1 that need to be to taken into consideration when evaluating the role of a11ã�1 in wound healing and fibrotic disorders. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada the objective of this study was to investigate the effects of par2 deficiency in various models of colonic inflammation in order to elucidate the role of endogenous par2 in the process of inflammation in the gut.colonic inflammation in c57bl6 wildtype and par2 -/-mice was induced by treatment with 2.5% dss (in drinking water) or tnbs (1mg or 2mg in 100ul of 40% ethanol, single intracolonic injection) or pre-sensitizing mice with 3% oxazolone (in olive oil) applied to the skin of the abdomen, and 7 days later, a single intracolonic injection of 1% oxazolone (dissolved in 50% ethanol).intravital microscopy was performed, 7 days (tnbs/dss) or 4 days (oxazolone) after induction of colitis on the colonic venules to assess changes in leukocyte rolling, adhesion and vessel diameter.lastly, various parameters of inflammation were assessed following the intravital microscopy.par2 -/-mice showed significantly lower leukocyte adherence and vessel dilation compared to the wildtype mice in dss, and tnbs challenge. in all three challenges, mpo activity, macroscopic damage score and bowel thickness were significantly higher in wild-type mice, compared to par2 -/-.our evidences indicate that deficiency in par2 attenuates inflammatory responses in the experimental models of colitis associated with either th1 (tnbs/dss) or th2 (oxazolone) cytokine profile.therefore, par2 deficiency in the gut exerts antiinflammatory properties that are independent of th1 or th2 cytokine profile.the present study further highlights par2 as a potential target for inflammatory bowel diseases. (1), n vergnolle (1), p andrade-gordon (2) (1) inflammation research network, university of calgary, canada (2) rw johnson pharmaceutical research institute, canada in a previous study, inflammatory responses induced by three different models of colitis (tnbs/dss/oxazolone) were significantly attenuated in mice deficient for par2 (par2-/-). among the inflammatory parameters observed, infiltration of granulocytes to the colon was consistently reduced by par2 deficiency. aim of this study was to assess the effects of par2 deficiency (via par2-/-mice) on the recruitment of leukocyte in colonic venules. in anaesthetized animals, leukocyte rolling/ adherence and vasodilation were induced, by topical administration of fmlp (100 mm) or paf (100 nm) or by intraperitoneal injection of tnf-a; (0.5 mg -given 3 hours before the intravital microscopy). using intravital microscopy, we evaluated the ability of various leukocyte stimuli to induce leukocyte trafficking and vasodilation in colonic venules of par2 -/-versus par2+/+ mice. fmlp and paf as well as tnf-a; induced significant vasodilation and an increase in rolling/adhesion of leukocytes in mouse colonic venules. par2 -/-mice showed significantly lower leukocyte rolling compared to the wildtype mice in response to fmlp topical administration. leukocyte adherence induced by fmlp and tnf-a; was significantly lower in par2 -/-mice compared to wild types as well. no difference was observed between par2 -/-and wildtype for leukocyte rolling/adherence-induced by paf. the lack of functional par2 attenuated leukocyte trafficking in response to fmlp and tnf-a; but not to paf. the involvement of par2 activation in mouse colon leukocyte trafficking highlights par2 as an important mediator of inflammatory cell recruitment and thereby a potential target for the treatment of inflammatory bowel diseases. (1), kk hansen(2), k chapman(2), n vergnolle (2), ep diamandis (1), md hollenberg (2) (1) advanced center for detection of cancer, mount sinai hospital, university of toronto, toronto, on, canada (2) proteinases and inflammation network, university of calgary, calgary, ab, canada kallikreins (klks) are secreted serine proteinases identified in many cancers and multiple sclerosis lesions. we have recently shown that klks can activate proteinaseactivated receptors (pars), a family of g-protein coupled receptors associated with inflammation. we hypothesized that like trypsin, kallikreins can trigger inflammation via the pars. we studied the ability of klks 6 and 14 to activate pars 1, 2 and 4 in vitro and to cause oedema in a mouse model of paw inflammation in vivo. we found that klk14 is able to activate both of pars 2 and 4 and to prevent thrombin from activating par1. on the other hand, klk6 was a specific activator of par2. kallikrein administration in vivo resulted in a paw oedema response comparable in magnitude and time to that generated by trypsin. the oedema was accompanied by a decreased threshold of mechanical and thermal nociception. our data demonstrate that by activating pars 2 and 4 and by inactivating par1, kallikreins, like klks 6 and 14, may play a role in regulating the inflammatory response and perception of pain. (1), d park (1), b short(2), n brouard(2), p simmons(2), s graves (1), j hamilton (1) (1) melbourne university, melbourne, victoria, (2) peter maccallum cancer institute. melbourne, australia mouse mesenchymal stem cell enriched populations can be isolated from bone tissue by employing lineage immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cyto-kines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. using are-driven and nf-kb-targeted reporter genes, transfection of the nf-kb p65 subunit and nrf2 into hepg2 or other cells, as well as sirna technique to knockdown endogenous p65 in cells, we found that nf-kb p65 subunit repressed the anti-inflammatory and anticarcinogenetic nrf2-are pathway at transcriptional level. in p65-overexpressed cells, the are-dependent expression of heme oxygenase-1 was strongly repressed. in the cells where nf-kb and nrf2 were simultaneously activated, p65 unidirectionally antagonized nrf2 transcriptional activity. the p65-mediated are inhibition was independent of the transcriptional and dna-binding activities of p65. co-transfection and rna interference experiments revealed two mechanisms which coordinate the p65-mediated repression of are: (1) p65 selectively deprives creb binding protein (cbp) from nrf2, but not mafk, by competitive interaction with the ch1-kix domain of cbp, resulting in inactivation of nrf2 transactivation domain and concomitant abrogation of the nrf2-stimulated coactivator activity of cbp; (2) p65 promotes recruitment of histone deacetylases 3 (hdac3) to are by enhancing the interaction of hdac3 with either cbp or mafk, leading to inactivation of cbp and deacetylation of mafk. this study may establish a novel pro-inflammatory and pro-carcinogenic model for the transrepression of the are-dependent gene expression by p65 subunit. since various inflammatory and tumor tissues constitutively overexpresses p65 in their nuclei, the finding in this study implies a strong repression of are-dependent gene expression must take place in those tissues. in this regard, the findings in this study may help to explain why oxidative stresses and toxic insults usually occur in those pathological loci. dendritic cells (dc) play a pivotal role in the induction of immune response and tolerance. it is less known that dc accumulate in atherosclerotic arteries, where they might activate t-cells and contribute to the progression of disease. the serine protease thrombin is the main effector protease of the coagulation cascade. thrombin is also generated at sites of vascular injury and during inflammation. hence, thrombin generation is observed within atherosclerotic and other inflammatory lesions including rheumatoid arthitis. thrombin activates various cells via protease-activated receptors (pars). immature dc do not express pars. upon maturation with lps, tnfalpha, or cd40l, only lps-matured dc expressed par1 and par3 on their surface. stimulation of dc with thrombin, par1-or par3-activating peptides elicited actin polymerization and concentration-dependent chemotactic responses in lps-, but not in tnf-alphamatured dc. the thrombin-induced migration was a true chemotaxis as assessed by checkerboard analysis. stimulation of pars with thrombin or respective receptoractivating peptides led to activation of erk1/2 and rho kinase i (rock-i) as well as subsequent phosphorylation of the regulatory myosin light chain 2 (mlc2). the erk1/2-and rock-i-mediated phosphorylation of mlc2 was indispensable for the par-mediated chemotaxis as shown by use of pharmacological inhibitors of rock, erk and mlc kinases. in addition, thrombin significantly increased the ability of mature dc to activate proliferation of naive t-lymphocytes in mixed leukocyte reactions. in conclusion, our work demonstrates expression of functionally active thrombin receptors on lps-matured dc. we identified thrombin as a potent chemoattractant for mature dc, acting via rho/ erk-signaling pathways. data concerning the role of circulating modified low density lipoproteins (modldl) in atherogenesis and other pathologies are scarce. one reason for this is the lack of suitable radiolabeling methods for direct assessment of metabolic pathways of modldl in vivo. we report a novel approach for specific labeling of human native ldl (nldl) and modldl (iron-, hypochloriteand myeloperoxidase-oxidized, nitrated, glycated, and homocysteinylated ldl) with the positron emitter fluorine-18 (18f) by either nh2-reactive n-succinimidyl-4-[18f]fluorobenzoate or sh-reactive n-[6-(4-[18f]fluorobenzylidene)-aminooxyhexyl]maleimide (radiochemical yields, 15-40%; specific radioactivity, 150-400 gbq/ mmol). radiolabeling itself caused neither additional oxidative structural modifications of ldl lipids and proteins nor adverse alterations of their biological activity and functionality in vitro. the approach was evaluated with respect to binding and uptake of 18f-nldl and 18f-modldl in cells overexpressing various lipoproteinrecognizing receptors. the metabolic fate of 18f-nldl and 18f-modldl in vivo was delineated by dynamic small animal pet studies in rats and mice. the in vivo distribution and kinetics of nldl and modldl correlated well with the anatomical localization and functional expression of ldl receptors, scavenger receptors, and receptors for advanced glycation end products. the study shows that ldl modification, depending on type and extent of modification, in part or fully blocks binding to the ldl receptor, and reroutes the modldl to tissuespecific disease associated pathways. in this line, 18flabeling of modldl and the use of small animal pet provide a valuable tool for imaging and functional characterization of these pathways and specific sites of pathologic processes, including inflammatory processes, in animal models in vivo. the p38 mapk signaling pathway, which regulates the activity of different transcriptions factors including nuclear factor-ã�b (nf-ã�b), is activated in lesional psoriatic skin. the purpose of the present study was to investigate the effect of fumaric acid esters on the p38 mapk and the down stream kinases msk1 and 2 in cultured human keratinocytes. cell cultures were incubated with dimethylfumarate (dmf), methylhydrogenfumarate (mhf) or fumaric acid (fa) and then stimulated with il-1b before kinase activation was determined by western blotting. a significant inhibition of both msk1 and 2 activations was seen after pre-incubation with dmf and stimulation with il-1b whereas mhf and fa had no effect. also, dmf decreased phosphorylation of nf-kb / p65 (ser276), which is known to be transactivated by msk1. furthermore, incubation with dmf before stimulation with il-1b resulted in a significant decrease in nf-kb binding to the il-8 kb and the il-20 kb binding sites as well as a subsequent decrease in il-8 and il-20 mrna expression. our results suggest that dmf specifically inhibits msk1 and 2 activations and subsequently inhibits nf-kb induced gene-transcriptions which are believed to be important in the pathogenesis of psoriasis. these effects of dmf explain the anti-psoriatic effect of fumaric acid esters. a humanized model of psoriasis was successfully established by transplanting non-lesional skin biopsies from psoriasis patients onto bg-nu-xid mice lacking b, t and nk cells. in this system, a psoriatic process is triggered by intradermal injection of activated autologous peripheral blood lymphocytes. inflammation is associated with the expression of activation markers and inflammatory medi-ators such as tnf-alpha, hla-dr and cd1a and this results in increased proliferation and differentiation of keratinocytes, demonstrated by increased expression of ki-67 and ck-16. epidermal hyperplasia is a typical readout in this model. in a series of studies, this model was found to be sensitive too a wide range of compounds, including inhibitors of tnf-alpha, antibodies directed against growth factors, mmp-inhibitors, calcipotriol, metothrexate, betamethasone and cyclosporine a.in addition, we showed that inhibition of fatty acid oxidation had an anti-psoriatic effect in this model (caspary et al. brit j dermatol 2005; 153, 937-944) . employing lesional skin it was demonstrated that inhibition can also be performed in a therapeutic setting.due to its humanized nature this model represents a powerful tool for the identification or validation of compounds with potential for the treatment of psoriasis. kristian otkjaer (1), e hasselager(2), j clausen(2), l iversen (1), k kragballe (1) (1) aarhus university hospital, denmark (2) novo nordisk a/s, denmark interleukin-20 (il-20) is assumed to be a key cytokine in the pathogenesis of psoriasis. increased levels of il-20 are present in lesional psoriatic skin compared with nonlesional skin where it is barely detectable. whether il-20 is derived from antigen-presenting cells or keratinocytes remains unsolved. the aim of the present study was, therefore, to characterize il-20 expression in non-lesional psoriatic skin ex vivo. 3 mm punch biopsies from nonlesional psoriatic skin were collected. biopsies were transferred to cacl enriched keratinocyte basal media and cultured with vehicle or il-1beta (10ng/ml) for 0, 1, 2, 4, 6, 12 and 24 hours, respectively. the samples were analyzed by in situ hybridisation, qrt-pcr, immunofluorescent staining and elisa. incubation with il-1beta rapidly induced il-20 mrna expression in the biopsies. the highest level of il-20 mrna was detected after 4 hours and in situ hybridisation revealed that basal as well as suprabasal keratinocytes throughout the epidermis were the only cellular source of il-20 mrna. increased levels of il-20 protein were detected in the supernatant of the il-1beta stimulated biopsies. immunofluorescent staining of the biopsies showed no il-20 protein in the keratinocytes, whereas the il-20 protein was present in epidermal cd1a positive dendritic cells. our data emphasize the keratinocyte as the cellular source of il-20 expression in human skin. interestingly, immunofluorescent staining of our cultured biopsies showed il-20 protein in epidermal dendritic cells whereas no il-20 was detected in the keratinocytes. this indicates that epidermal dendritic cells are the target for keratinocytederived il-20. one response of epidermal keratinocytes to inflammatory stress is the induction of matrix metalloproteinases (mmps) that participate in tissue remodeling. excessive proteolytic activity is associated with chronic wounds and tissue damage during persistent inflammation. calcitriol, the hormonally active form of vitamin d, is known to have beneficial effects during cutaneous inflammation. we hypothesized that one way in which calcitriol exerts its effect on inflamed skin is by attenuation of damages caused by excessive mmp proteolytic activity. our experimental model consists of hacat keratinocytes cultured with tnf to simulate an inflammatory state. pro-mmp-9 was quantified by gelatin zymography and mrna by real-time pcr. the levels and activation of signaling proteins were determined by immunoblotting. the increase in pro-mmp-9 activity and mrna levels induced by tnf was inhibited by~50% following 48h treatment with calcitriol. using specific inhibitors we established that the induction of mmp-9 was dependent upon the erk pathway, while p38-mapk and pkc inhibited, and jun-kinase, pi-3-kinase and src did not affect it. levels of c-fos, a component of ap-1 transcription complex known to mediate mmp-9 induction, were elevated by tnf and further increased by calcitriol. the induction of mmp-9 by tnf was abolished by inhibition of the egfr tyrosine kinase attesting to the requirement for egfr trans-activation. calcitriol also inhibited the induction of mmp-9 by egf. we conclude that calcitriol inhibits the induction of mmp-9 gene expression by tnf in keratinocytes by affecting an event downstream to the convergence of the egfr and the tnf signaling pathways. (1), p verzaal (1), t lagerweij (1), c persoon-deen (1), l havekes (1), a oranje (2) (1) tno pharma, department of inflammatory and degenerative diseases, leiden, the netherlands (2) erasmus medical center, department dermatology and venereology, rotterdam, the netherlands mice with transgenic overexpression of human apolipoprotein c1 in liver and skin display a strongly disturbed lipid metabolism. moreover, these mice show a loss of skin-barrier function evident from increased trans epidermal water loss. these mice develop symptoms of atopic dermatitis, i.e. scaling, lichenification, papules, excoriation and pruritus. both hyperplasia of epidermis and dermis are observed. histological analysis shows increased numbers of cd4+ t cells, eosinophils, mast cells and ige-positive cells in the dermis. serum levels of ige are increased as well. cytokine profiling of draining lymph nodes is in favor of a th2-mediated disease. development of atopic dermatis in this model was found to be sensitive to topical treatment with triamcinoloneacetonide, fluticasone-proprionate and tacrolimus. moreover, oral treatment with dexamethasone successfully inhibits the development of disease in this model. impairment of the skin barrier is most likely the underlying cause of the development of atopic dermatitis in this model.this model is useful for identifying new therapeutic strategies and obtaining new insight into the pathogenesis of atopic dermatitis. topical immunosupppressants such as elidel and protopic are highly efficacious therapeutics for the treatment of atopic dermatitis and other dermatological conditions.-when delivered topically, these calcinuerin inhibitors offer several advantages over topical steroids; however, these marketed drugs have received a controversial "black box warning" because of a potential cancer risk. we speculated that systemic exposure of these drugs over long term use may contribute to the cancer risk.accordingly, we have designed and discovered a series of "soft" cyclosporin a (csa) derivatives as potentially safer alternatives.in general, soft drugs are engineered, via medicinal chemistry, to be effective upon local delivery but upon systemic exposure they are rapidly inactivated by metabolic pathways.in this way, exposure of active drug to distal organs is greatly minimized resulting in a significant enhancement in therapeutic index.the results or our drug discovery efforts around soft csa derivatives will be presented. (1), y sawanobori (2), u bang-olsen (3), c vestergaard(4), c grã¸nhã¸j-larsen (1) background: a strain of japanese fancy-mice, nc/nga, serves as a model for atopic dermatitis. under specific pathogen-free conditions, the mice remain healthy, but when kept under non-sterile conditions, they exhibit pruritic lesions like atopic dermatitis. scratching behaviour of the mice precedes the development of dermatitis, and a correlation between registered scratching counts and expression of il-31 mrna has been shown. also, transgenic mice over-expressing il-31 exhibit increased scratching behaviour and develop severe dermatitis. consequently we decided to explore the therapeutic effect of an anti il-31 antibody on scratching behaviour and dermatitis in nc/nga mice. methods: prior to clinical manifestation of dermatitis, we commenced treatment of nc/nga mice with il-31 ratanti-mouse 10 mg/kg intraperitoneally every fifth day for seven weeks. clinical dermatitis, scratching behaviour and weight gain, was assessed throughout the intervention period. serum analysis for ige and il-13 as well as histopathological and immunohistochemistry analysis on skin biopsies were also performed at end-point. results: taken over the entire intervention period, treatment with anti il-31 antibody in nc/nga mice from age seven weeks did not meet the primary end points, which were scratch, dermatitis and body weight. however, post hoc analysis revealed a significant reduc-tion of scratch by the anti il-31 antibody treatment in the time interval day 22-43. our results suggest an anti pruritic role for il-31 antibody in an atopic dermatitis-like animal model. anti il-31 antibody is therefore a new therapeutic opportunity for the treatment of pruritus in atopic dermatitis and perhaps other pruritic diseases. (1), p ferro (1), hm asnagli (1), v ardissone (1), t ruckle (2), f altruda (3), ch ladel (1) (1) rbm merck serono/university of torino, italy (2) merck serono pharmaceutical research institute, geneva, switzerland (3) university of torino, dipartimento di genetica, biologia, biochimica, italy class-i phosphoinositide 3-kinases (pi3ks) play a critical role in modulating innate and adaptive immune responses, as they are important transducers of external stimuli to cells, such as granulocytes and lymphocytes. since pi3k-g plays a pivotal role in mediating leukocyte chemotaxis and activation, as well as mast cell degranulation, the pharmacological blockade of pi3k-g might offer an innovative rationale-based therapeutic strategy for inflammatory skin disorders. in our study the inhibitory properties of a selective pi3k-g inhibitor as605858 on inflammation was applied to murine models modeling skin diseases like psoriasis and dermatitis. two mouse models were used: the first, irritant contact dermatitis (icd), is an innate inflammatory skin condition arising from the release of pro-inflammatory cytokines in response to haptens, usually chemicals. the second, contact hypersensitivity (chs) is a t-cell dependent model, modeling in part t-cell-mediated skin diseases such as psoriasis. we demonstrated the therapeutic effect of pi3kg inhibition and subsequent inhibition of chemotaxis in models of skin diseases and showed that a selective pi3k-g inhibitor can excert an important therapeutic efficacy (dose-dependent) in models of innate immunity (icd) -effective dose 0,3mg/kg p.o. once -as well as in t-cell mediated skin pathology (chs)effective dose 1mg/kg p.o. bid. we conclude that the mechanism of action related to inhibition of pi3k-g are demonstrable after oral administration of selective inhibitors like as605858 in models of acute and chronic skin inflammation and are mediated by modulation of innate and acquired immunity. introduction: high mobility group box 1(hmgb1) has recently been identified as a late mediator of endotoxin lethality. we newly developed an extra corporeal hmgb1 absorber. the purpose of this study was to test the hypothesis that hmgb1 removal could prevent or reduce endotoxin induced lethality or tissue injury of rats. methods: all experiments were conducted in accordance with the institutional care and use committee.male wistar rats were randomly allocated into three groups; hmgb1 absorber group (group i),hmgb1 nonabsorber group (group ii), and vacant column group (group iii).we applied these columns for each groups at 6 hours after lps injection. the rats were sacrificed 48 hours after lps injection for pulmonary histology. we statistically analyzed survival rate with kaplan-meier and the levels of hmgb1 with anova. results: survival rate was 67% in the group i at 48 hours after lps injection, as compared with 0% in the group ii and 11% in the group iii. the pulmonary histology in both group ii and group iii showed acute inflammatory injuries, whereas group i showed less inflammatory changes.the level of hmgb1 in the group i was significantly lower than those of group ii and iii. discussions:these results demonstratethat specific absorption of endogenous hmgb1 therapeutically reverses lethality of established sepsis indicating that hmgb1 inhibitors and absorber can be treated in a clinically relevant therapeutic window that is significantly wider than for other known cytokines. contact information: dr hideo iwasaka, oita university, anesthesiology and intensive care unit, yufu city, japan e-mail: hiwasaka@med.oita-u.ac.jp (1) (1) department of pharmacology, national university of singapore, singapore (2) dso national laboratories, singapore hydrogen sulfide (h2s) is increasingly recognized as a proinflammatory mediator in various inflammatory conditions. in this study, we have investigated the role of h2s in regulating expression of some endothelial adhesion molecules and migration of leukocytes to inflamed sites in sepsis. male swiss mice were subjected to cecal ligation and puncture (clp) induced sepsis and treated with saline, dl-propargylglycine (pag, 50 mg/kg i.p.), an inhibitor of h2s formation or sodium hydrogen sulfide (nahs) (10mg/kg, i.p.), a h2s donor. pag was administered either 1 hour before or 1 hour after induction of sepsis while nahs was given at the time of clp. using intravital microcopy, we found that in sepsis, prophylactic and therapeutic administration of pag significantly reduced the leukocyte rolling and adherence in mesenteric venules coupled with decreased mrna and protein levels of adhesion molecules (icam-1, p-selectin and e-selectin) in lung and liver. in contrast, injection of nahs significantly upregulated leukocyte rolling and attachment as well as tissue levels of adhesion molecules in sepsis. on the other hand, normal mice were given nahs (10mg/kg i.p.) to induce lung inflammation with or without pretreatment of nf-xã�b inhibitor, bay 11-7082. h2s treatment enhanced the pulmonary level of adhesion molecules and neutrophil infiltration in lung. these alterations were reversed by pretreatment with bay11-7082. moreover, expression of cxcr2 in neutrophils obtained from h2s treated mice was significantly upregulated leading to an obvious elevation in mip-2 directed migration of neutrophils. therefore, h2s act as an important endogenous regulator of leukocyte trafficking during inflammatory response. transient receptor potential vanilloid 1 (trpv1) is primarily found on sensory nerves. we have demonstrated its pro-inflammatory potential in arthritis and now present evidence that it is protective in an endotoxininduced model of sepsis. selective trpv1 antagonists are not available for use in the mouse in vivo, thus established trpv1 knockout (-/-) mice were used. c57bl6 wt and trpv1-/-mice were matched for age and sex and injected intraperitoneally (i.p.) with lipopolysaccharide (lps). the response was monitored for 4h. blood pressure, measured before and at intervals after lps in conscious mice via a tail cuff was reduced in both wt and trpv1-/-mice, with trpv1-/-mice showing an enhanced drop at 4h. in a separate group temperature, a proposed pre-mortality marker was also reduced by 4h, again with a significantly increased drop in trpv1ko. furthermore higher levels of two inflammatory markers tnfa and nitrite (as an indicator of no) were measured in peritoneal lavage and higher levels measured intrpv1-/-as compared with wt samples. finally aspartate aminotransferase (ast) levels also enhanced in trpv1-/-versus wt mice, although markers for kidney and pancreatic damage were similar in both genotypes. we conclude that trpv1 plays a protective role in sepsis. trpv1 is known to be present on nonneuronal (e.g. vascular components) and their relative involvement in sepsis is unknown. (1), da souza-junior(2), l de paula (1), mc jamur (2), c oliver (2), sg ramos (2), cl silva (2), lucia helena faccioli (1) ( h37rv) . infected balb/c mice developed an acute pulmonary inflammation and higher levels of tnf-a, il-1, kc, mcp-1 and mip-2 were detected in the lungs by day 15. in vivo degranulation of mast cells by c48/80 led to a reduction of the inflammatory reaction associated to a marked decline proinflammatory cytokine and chemokine levels in the lungs. the magnitude of cellular immune response was also partially impaired in infected mice and treated with c48/80. histologically, the exacerbated granulomatous inflammation shown in the lung parenchyma of infected mice was attenuated in infected mice and treated with c48/80. of interest, the number of mycobacterial bacilli recovered from the lungs was 1 log higher after treatment of infected mice with c48/80. these findings suggest that mcs participate in host defense against m. tuberculosis infection through of the modulation of cytokines and chemokines, which are important for the recruitment and activation of inflammatory cells. (1), ms chadfield (2), db sã¸rensen (1), h offenberg (1), m bisgaard (1), he jensen (1) (1) department of veterinary pathobiology, faculty of life sciences, university of copenhagen, denmark (2) novo nordisk a/s, cell biology, gentofte, denmark introduction: pasteurella multocida is an important cause of pneumonia in several animal species and may spread systemically. the aim of this study was to evaluate initial inflammatory reactions and the inocula effect due to strains of p. multocida of different origin in an aerogenous murine model. materials and methods: 30 female balb/ c-j mice (app. 20 g, taconic, denmark) were infected intranasally with two clinical isolates of p. multocida of avian (vp161) and porcine (p934) origin, at three different levels of inocula concentration. after euthanasia, specimens of lung and liver tissue were collected for bacteriological and histopathological evaluation. furthermore, lung tissue samples were taken for measurement of expression of metalloproteinase mmp-9 and metalloproteinase inhibitor timp-1. results: all mice infected with the avian strain were euthanized after 24 hours. viable counts recovered from lung and liver tissue were high, and histopathology revealed pronounced acute bronchopneumonia. in the liver, disseminated necrosis with formation of microabscess was also seen. on the contrary, a dose response was observed with the porcine strain with regard to recovery of viable counts and development of lesions was apparent after 24, 48 and 72 h. furthermore, differences were seen in the nature of the lesions caused by the two strains. there was a difference in expression of mmp-9 and timp-1 between infected and noninfected mice. the model proved suitable for the evaluation of pulmonary inflammatory reactions between the two different host-derived strains as demonstrated through viable counts, histopathology and expression of mmp-9 and timp-1. (1), r molinaro (1), a franã�a (1), m bozza (1), f cunha(2), s kunkel (3) (1) universidade do rio de janeiro, brazil (2) universidade de sâ¼o paulo, brazil (3) university of michigan, usa introduction and objectives: studies reveal that regulatory t (treg) cells control immune responses; therefore these responses must be controlled to enable the effective protection against infections and cancer. ccr4 knockout mice (ccr4 -/-) are more resistant to lps shock. so, our aim is to study the mechanisms involved in the resistance of ccr4-/-subjected to severe sepsis by cecal ligation and puncture (clp) and how tregs modulate this effect. results: c57/bl6 mice were subjected to clp model, whereby the cecum was partially ligated and puncture nine times with a 21g needle. sham-operated mice were used as control. mice subjected to clp and sham surgery were treated with antibiotic since 6h after and until 3 days. ccr4 -/-mice subjected to clp presented an increase in survival rate (78%) compared with wildtype mice (17%), and a marked improvement in the innate response concern to neutrophil migration to peritoneum and lung, bacteria load and cytokine levels compared to wild-type mice. besides, tregs from ccr4-/-clp mice did not inhibit proliferation of t effector cells as observed for treg from wild clp mice, at a proportional ratio of teffector:treg. interesting, treg from ccr4-/-clp mice did not inhibit neutrophil migration to bal when co-injected with fungal challenge as secondary infection, while the treg from wild clp mice did, as expected. conclusions: these results suggest that treg cells from ccr4-/-mice did not present suppressive response and it could be an important factor in their survival. inflammation and oxidative stress are known to be one of the important causes that are responsible for many diseases. inflammation has been associated with diseases like cancer, diabetes and many other. proinflammatory cytokine (tnf -âµ and il-1ã¢) and no are considered as pivotal mediators in inflammatory conditions like rheumatoid arthritis, sepsis and cancer etc. thus inhibition of pro-inflammatory cytokines and no production are important targets for treatment of inflammatory disorders. nowadays due to the emerging side effects of cox inhibitors, these targets have been paid more attention for the treatment of these conditions. some medicinal plants such as curcuma longa (1), commiphora mukul (2) in humoral memory, antibodies secreted into serum and other body fluids protect an individual against repeated challenges of previously encountered pathogens. antibody-secreting plasmacells are mostly considered to be shortlived, terminally differentiated b lymphocytes, eliminated after a few days or weeks by apoptosis. however, in secondary lymphoid organs and in the bone marrow, plasma cells can survive for months and years, without dna-synthesis and refractory to signals from antigen or antigen-antibody complexes. the lifetime of these longlived plasmacells depends on an intrinsic competence to survive in the distinct environment of those organs, which defines a specific survival niche. the niche provides survival signals like il-6, cxcl12 and tnf. within a functional niche, the lifetime of a plasma cell is apparently not limited intrinsically. the number of niches in the body has to be limited, in order to maintain physiological concentrations of serum immunoglobulins. thus recruitment of new plasmacells to the pool of old memory plasma cells has to be competetive. this competition is probably controlled by a simple molecular mechanism, namely the dual functionality of chemokines like cxcl12, which attract newly generated plasmablasts to a survival niche and at the same time are a survival signal for the plasma cell. plasmablasts and plasma cells express cxcr4, the receptor for cxcl12. while plasmablasts migrate in response to cxcl12, plasma cells depend on it for survival in the niche, but are no longer migratory. thus once disloged from their niche, they will die. plasmablasts newly generated upon systemic secondary immunization, upon concommittant stimulation with interferon-gamma, can also express cxcr3, the receptor for the interferon-gamma-induced chemokines cxcl9, 10 and 11, which may lure the plasmablasts into inflamed tissue. the switch in the potential to migrate provides also an efficient means to eliminate plasma cells of the peak of an immune response, which as plasmablasts had migrated to the tissue inflamed in that pathogenic challenge. inflamed tissue contains survival niches for plasma cells. in the inflamed tissue, plasma cells provide high local antibody concentrations while the tissue is inflamed. upon resolution of the inflammation the plasma cells will be dislodged and die. longlived plasma cells provide longlasting antibody titers (protective memory) and leave memory b cells a role in reactive memory, generating memoryplasmacells in secondary challenges, and if serum titers are not sufficient to protect. in chronic inflammation, this mechanism can contribute to pathogenesis. thus in the nzb/w model of lupus, longlived autoreactive plasma cells are generated early in pathogenesis, which survive in bone marrow and spleen. later, in established disease, autoreactive plasma cells are shortlived and continuously generated. they do not compete with the longlived plasma cells, and both populations coexist as prominent populations. interestingly, longlived plasmacells are resistant to therapeutic immunosuppression, while the generation of shortlived plasma cells is blocked. this may be the reason for the failure to cure antibodymediated immunopathology, e.g. in autoimmunity and allergy, by conventional immunosuppression, (1), h lee (2) c5a is a potent inflammatory mediator produced during complement activation. unregulated c5a signalling through its receptor (c5ar) on neutrophils and other leukocytes is implicated in the pathogenesis of autoimmune diseases including rheumatoid arthritis and systemic lupus erythematosus. considerable effort has gone into development of c5ar antagonists for human therapy. we took neutrophils from genetically modified human c5ar knock-in mice in which the mouse c5ar coding region was replaced with human c5ar sequences and immunized wild-type mice to generate high affinity antagonist monoclonal antibodies (mabs) to human c5ar. these mabs inhibit c5a-induced neutrophil migration and calcium-flux, and bind to a region of the 2nd extracellular loop of c5ar loop that seems to be critical for receptor activity. this study investigated the effectiveness of these mabs in the k/bxn serum-transfer model of inflammatory arthritis. human c5ar knock-in mice were given 1-10 mg/kg mab intraperitoneally, before or after inflammatory arthritis developed. mice treated with anti-c5ar mab one day before serum transfer did not develop swelling or clinical signs of arthritis in contrast to controls. histopathology of the joints in anti-c5ar mab-treated mice revealed a complete block of the massive influx of leukocytes and cartilage erosion seen in controls. furthermore, and most significantly, a single 1 mg/kg dose of anti-c5ar mab given 5 days after initiation of disease completely reversed inflammation. in the collagen-induced arthritis (cia) model, injection of anti-c5ar mab after development of inflammation also reversed inflammation to baseline. these potent new antibodies to human c5ar are in preclinical development. the cytokine macrophage migration inhibitory factor (mif) participates in fundamental events in innate and adaptive immunity. the profile of activities of mif in vivo and in vitro is strongly suggestive of a role for mif in the pathogenesis of many inflammatory diseases, including rheumatoid arthritis (ra), asthma, and sepsis. mif also has a unique relationship with glucocorticoids, in that despite antagonizing their effects, the expression of mif is in fact induced by glucocorticoids. thus, mif functions as a physiological counter-regulator of the anti-inflammatory effects of glucocorticoids. therapeutic mif antagonist may therefore provide a specific means of steroid sparing. since mif are highly conserved among different species, it is hard to develop high affinity antibodies due to immune tolerance.we developed a proprietary technique to break the immune tolerance and selected high affinity mouse monoclonal antibodies against mif.the antibody can neutralize mif activity in cell based assays, and is very effective in a lps induced mouse sepsis model. using this antibody as a tool, we are studying the function of mif in comparison with the function of lps.we found that lps induced inos expression and no secretion are dependent on the secretion of mif.we also found that although both lps and mif induce g1 arrest in macrophage cell line raw264.7, their functions are independent to each other. structure-based small molecule drug design. an effective agent would be the first orally active cytokine antagonist. methods: collagen-induced arthritis (cia) was induced in dba-1 mice by immunisation with bovine type ii collagen/adjuvant on day 0 and 21. cor100140, synthesized on the basis of computer modeling of mif protein x-ray crystallographic data, was administered by daily oral gavage from day 21. etanercept (8mg/kg ip q2d) was used as a positive control. the mek-erk pathway is activated in numerous inflammatory conditions, including ra, ibd and copd. arry-162 is a potent (ic50 = 12 nm), selective, atp-uncompetitive mek1/2 inhibitor.arry-162 is highly efficacious in cia and aia rat models, with ed50s of 3 and 10 mg/kg, respectively, equal to or better than standard agents. addition of arry-162 to methotrexate, etanercept, ibuprofen or dexamethasone regimens in these models results in improved efficacy that is at least additive, if not synergistic. in tpa-stimulated human whole blood, this compound inhibited tnf, il-1 and il-6 production (ic50s of 23, 21 and 21 nm, respectively). in contrast, inhibition of perk required 280 nm to achieve a 50% reduction, demonstrating that inhibition of pro-inflammatory processes is very sensitive to perk inhibition.in clinical studies, healthy volunteers were administered a single oral dose of 10, 20, 30, 40 or 80 mg); blood was drawn at various times after dosing and stimulated ex vivo with tpa. arry-162 was well-tolerated and drug exposure was dose-proportional. in ex vivo blood samples, there was a dramatic time-and concentration-dependent inhibition of tpainduced il1 and tnffz with >80% inhibition observed at plasma concentrations of 50 and 150 ng/ml, respectively. similar inhibition of perk required 300 ng/ml. a multiple ascending dose clinical study has confirmed the pharmacokinetics and pharmacodynamics of arry-162 and helped define tolerability. clinical evaluation of arry-162 in combination with methotrexate in patients with rheumatoid arthritis is on-going. p38 (a mitogen-activated protein kinase) has been shown to play a key role in the release of cytokines such as tnfand il-1a from monocytes in signaling cascades that are initiated due to extra cellular stress stimuli. inhibition of p38 activity is expected to regulate the levels of tnf-a and il-1b thereby alleviating the effects of inflammation in ra. a new class of p38 inhibitors based on the naphthyridininone scaffold have been discovered. x-ray crystallography and site directed mutagenesis studies were critical tools that aided the evolution of the naphthyridinone lead class starting from a pyrido-pyrimidinone template. this presentation will discuss the derivation of key benchmark pre-clinical candidates in these novel scaffold classes (shown below) as influenced by structural biology studies, mutagenesis data and molecular modelling. efficacy studies in animal models for benchmark compounds will also be presented. (1), h aaes (1), w-h boehncke(2), j pfeffer(2), t skak-nielsen(1), i teige (1), k abell (1), ph kvist (1), e ottosen (1), tk petersen (1), lars svensson (1) (1) discovery, leo pharma, 55 industriparken, ballerup, denmark (2) department of dermatology, johann wolfgang goethe-university, frankfurt am main, germany p38 map kinase plays an important role in mediating an inflammatory response in mammalian cells. as a consequence of activation, several inflammatory mediators are released including il-1b] and tnfa. both cytokines have a central role in the pathogenesis of inflammatory conditions such as psoriasis. approximately 30% of psoriasis patients develop psoriatic arthritis. leo15520 is a member of newly developed class of selective p38 map kinase inhibitors. the compound was tested orally in in vivo models relevant for psoriasis and psoriatic arthritis. the in vivo models selected include the cia arthritis model, the human psoriasis xenograft scid mouse model, the uvb-induced dermatitis model, the lps induced tnfa model and a local gvh model. treatment with leo15520 led to an amelioration of the ongoing inflammation in all investigated in vivo models. in the cia model, a clear dose response effect was observed on the developing arthritis in both rats and mice (65 % reduction in mice and 77 % in rats at 10 mg/kg p.o.). in the humanised psoriasis model, leo15520 at a dose of 20 mg/kg, had an effect on both the hyperplastic epidermis (epidermal thickness reduced by 41%) and on the infiltrating inflammatory cells. the anti-inflammatory effect of leo15520 was even close to the effect of systemically delivered steroids in both models. we believe that the new highly selective class of p38map kinase inhibitors has a strong potential as an orally delivered therapy for systemic inflammation diseases such as psoriasis and arthritis. slx-2119 is a potent, selective, orally bioavailable inhibitor of the rho-kinase rock-2. its ic50 for rock-2 and rock-1 inhibition is 100 nm and >10 mm respectively. the ability of slx-2119 to inhibit septic liver injury was investigated in c57bl/6j mice challenged with lipopolysaccharide (lps) and d-galactosamine (d-gal). mice were given lps (10 mg/mouse) and d-gal (18 mg/ mouse) i.p and 5.5 hours later were sacrificed for analysis of liver injury. mice challenged with lps/d-gal had a >10 fold increase in serum alt and ast levels. this increase was reduced by >90% in mice pretreated with slx-2119 either orally (100 mg/kg, 2 and 20 hrs prechallenge) or i.p. (10-100 mg/kg, 15 min pre-challenge). slx-2119 inhibited the increase in hepatic levels of tnffalpha produced by lps/d-gal by >50%. to assess the kinetics of slx-2119s benefit, slx-2119 (100 mg/kg i.p.) was given 15 min prior to, or 15 or 60 min after the lps/ d-gal. slx-2119 was effective at inhibiting the rise in alt and ast levels at all 3 time points suggesting that inhibiting rock-2 even after the initiation of the lps/d-gal driven cascade protects against septic liver injury. in a survival study, 9 out of 10 mice given lps/d-gal were dead by 8 hrs whereas in mice given slx-2119 (100 mg/kg orally) 1 animal died at 10 hours and the remaining 9 mice were alive 48 hrs later. these results show that specific inhibitors of rock-2 may have therapeutic utility in the treatment of sepsis and subsequent liver injury. theta through three key hydrogen bond mediated interactions.they potently inhibit protein kinase c activity in vitro as demonstrated by inhibition of il-2 secretion in human purified t-cells stimulated with anti-cd3 and anti-cd28 or whole blood seb challenge.the pkc-theta inhibitors are orally bioavailable and demonstrate immunosuppressive activity in a mouse model of human delayed-type hypersensitivity responses. (1), j zhang (1), k henley (1), m white (1), d hilton (1), b kile (1) ( to investigate pathogenesis of rheumatoid arthritis, we used mice transgenic for the uniquely human fcgam-mariia in inducible and passively transferred models of arthritis. transgenic mice developed severe ra-like disease in both model systems, indicating that the transgene played a major role in arthritis pathogenesis. disease could be reduced by the administration of either specific monoclonal antibodies to fcgammariia or small chemical entities (sce) designed to bind to the fcgam-mariia dimer. to investigate the cause of this enhanced sensitivity to auto-immune stimuli, the phagocytic capacity of transgenic mice compared to c57bl/6 control mice was examined using phagocytosis of fluorescent beads coated with ova or ova/anti-ova immune complex or opsonised sheep red blood cells. in both assays macrophages from fcgammariia transgenic mice showed significantly increased phagocytosis comapred with cells from control mice at 4 hours.this difference diminished over time andwas only seen where particles were opsonised. treatment of macrophages with specific fcgammariia blocking monoclonal antibody fragments 8.7 f(ab)2 or iv.3 f(ab) reduced phagocytosis to background levels . macrophages from transgenic mice also showed significantly greater production of inflammatory cytokines tnf-alpha and il-1beta when stimulated in vitro with heat aggregated immunoglobulin (hagg). moreover, this response was also blocked by specific fcgammariia monoclonal antibody fragments or sce. thus, expression of the fcgammariia transgene in these mice leads to increased uptake of and reactivity to immune complexes, resulting in enhancement of inflammatory sequelae in the form of increased th1 cytokine secretion andamplification of the pro-inflammatory response leading to arthritic disease. harald burkhardt (1), u hã¼ffmeier (2), i kã§nig (3), j lacorz (2), a reis (2), k reich (4) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander-unisversity of erlangen-nuremberg, germany results: whereas the earlier described strong association of allele tnf*-238a with psoriasis could be confirmed, our study revealed that this association was completely dependent on carrying the psors1 risk allele. for psa, but not psoriasis vulgaris without joint involvement strong association with the allele tnf*-857t was detected (or=1.956, 95% ci 1.33-2.88; pcorr=0.0025) also in patients negative for the psors1 risk allele. our results indicate genetic differences between psoriasis vulgaris patients with and without joint manifestation. while the previously reported association between tnf*-238a and psoriasis seems to primarily reflect ld with psors1, tnf*-857t may represent a risk factor for psa independent of psors1. (1), n modi (1), m stanford (1), e kondeatis (1), r vaughan (1), f fortune (2), w madanat (3), c kanawati(4), p murray (5) methods: dna was obtained from 167 patients with bd, 61 from the uk and 106 from the middle east (me), and 159 controls individuals, 129 from the uk and 30 from me. dna was prepared by proteinase k digestion, and salt extraction and -318 and 49 snp were detected by a pcr-ssp. results: there was no significant difference in expression of -318c/t or 49a/g when all bd patients were compared to all control individuals, (p=0.3 and 0.9, respectively). as we have previously shown differences in snp expression in different patient groups we tested the uk and me patients separately. however, there was no significant difference in either snp in uk bd patients, (p=0.15, p=0.39) or me bd patients (p=0.7, p=0.6) when compared to the appropriate controls. (1), da brown (1), h johnen (1), mr qiu (1), t kuffner (1), pgm curmi (1), l brown (1), m mazzanti (2), sn breit (1) ( introduction: cerebral palsy (cp) is a nonprogressive motor disorder caused by white matter damage in the developing brain. it is often accompanied with neurocognitive and sensory disabilities. the cause and pathogenesis of cp is multifactorial and continues to be poorly understood. chorioamnionitis, clinically silent or manifest, has been reported to be a risk factor for cp both in term and preterm infants. il-16 gene is single copy gene located on chromosome 15q26.1-3 in humans. interleukin-16 is synthesized by a variety of immune (t cells, eosinophils and dendritic cells) and non-immune (fibroblasts, epithelial and neuronal) cells. it is also detected in organ-specific secretions in a number of inflammatory processes. amniotic fluid interleukin-16 concentrations decreased with advancing gestational age. but, women with preterm labor and women with chorioamnionitis have higher interleukin 16 amniotic fluid concentrations than those who delivered at term or those with sterile amniotic fluid. the aim of our study was to estimate allelic frequency for regulatory il16 -295 snp in the children with the cp. methods: dnas obtained from peripheral blood of 46 cp patients and 182 unrelated healthy volunteers were genotyped for the il16 -295 snp pcr-rflp method. results and conclusions: il16 -259 genotype fncc was more common in the population with cerebral palsy in the comparison to healthy volunteers. the significance of the association between il16 fngene polymorphisms and cerebral palsy has to be investigated in the future studies. (1), d delbro (1), e hansson (2) (1) kalmar university, sweden (2) gã§teborg university, sweden background: acetylcholine (ach) is a major signalling molecule, binding partly at nicotinic receptors (nachrs; a family of ions channels with nicotine as a selective ligand). one subtype, the alpha7nachrs, has antiinflammatory effects by way of down-regulation of tnfalpha release from macrophages. the alpha7nachrs have been demonstrated in neuronal as well nonneuronal tissues, e.g. astrocytes and microglia. aim. in rat astrocytes in primary culture, and in astrocytes cocultivated with primary microvessel cultures study: 1. the expression of alpha3nachrs and alpha7nachrs by immunofluorescence and western blot. 2. intracellular ca2+-transients spread within the astroglial networks after stimulation with nicotine. 3. pro-inflammatory cytokines, il-1b, il-6, and tnfalpha, released from microglial cells after stimulation with nicotine. results: alpha7nachrs expression was more evident in astrocytes co-cultivated with endothelial cells, suggesting that endothelial cells release factors, which increase the maturity of astrocytes. the ca2+ transient evoked by nicotine was also more pronounced in the co-cultured astrocytes. these ca2+ responses were blocked by the alpha7nachr antagonist alpha-bungarotoxin. the release of pro-inflammatory cytokines was down-regulated after stimulation by nicotine. conclusion: alpha7nachrs appear to be involved in some of the effects of nicotine administration to rat astrocytes. an anti-inflammatory action of cholinergic nerves on the astrocytes via nachrs seems probable, which may have therapeutic implications. university, department of natural sciences, kalmar, sweden e-mail: ann.pettersson@hik.se gedeon richter plc., budapest, hungary tramadol, an atypical opioid analgesic, is increasingly used for the treatment of osteoarthritis because it does not produce typical side effects of nsaids. review of clinical data show that it produces symptom relief and improves function, but these benefits are small and adverse events often cause participants to stop taking the medication (cohran database 2006). efficacy of tramadol in animal models of inflammatory pain is well established; however complex characterization of the drug regarding analgesic and side effects is missing in rats. our aim was to assess oral efficacy of tramadol at side effect free doses in rats. anti-hyperalgesic effect was determined in complete freunds adjuvant (cfa) induced thermal hyperalgesia test (th) and in model of cfa-induced knee joint arthritis. effect on inflammation was characterized in carrageenan induced paw edema test (et). for the characterization of side effect accelerating rotarod assay (arr) was used. significant impairment in arr was noted at 55 mg/kg dose, and above. in the th test weak 35% effect was seen at side effect free dose (40 mg/kg). in the arthritis model b.i.d. 40 mg/kg dose of tramadol given on days 3-7 after cfa caused a maximum 86% effect on weight bearing incapacitance (day 4). however, its effect seemed to diminish (34% on day 7) upon repeated treatment. in et tramadol had significant anti-inflammatory effect at 100 but not at 30 mg/kg dose. these results show that efficacy of tramadol in rat inflammatory pain models is limited by its side effects, in accordance with clinical data. chronic relapsing experimental allergic encephalomyelitis (cr eae) is a disease that bears striking similarities to the human condition multiple sclerosis (ms). in particular, cr eae and ms have major inflammatory events in the central nervous system (cns) that culminate in demyelination and disruption of axonal function.one group of mediators involved in the progressive cns inflammation are the prostaglandins (pgs).pg generation is regulated in experimental non-immune conditions of the cns by n-methyl-d-aspartate (nmda) receptor activation.nmda receptor-mediated events are also evident in eae and may therefore have the potential to influence pg production.the study was designed to profile pge2 and pgd2 in cns tissues during the development of cr eae and to examine the role of the nmda receptor in pg production through the use of the specific antagonist mk-801.biozzi mice were inoculated for cr eae and cns tissues were sampled during the course of disease.enzyme immunoassay of processed samples revealed pge2 and pgd2 levels within normal limits during the acute and subsequent remission phases of cr eae.in contrast, dramatic changes in pg concentrations were observed with a relapse of symptoms and a remission of disease.mk-801 was therapeutically administered to cr eae-diseased mice at the onset of relapse and changes in cns pg levels were recorded.the relapse phase of cr eae, but not the acute stage of disease, is characterised by an increase in cns pg production that may be influenced by nmda receptor activation. livia l camargo(1), lm yshii (1) (1), sk costa (1) (1) university of sâ¼o paulo, brazil (2) king's college, london, uk (3) butantan institute, sâ¼o paulo, brazil objectives: the neuropeptide substance p (sp) released by capsaicin-sensitive nerves (csn) plays a pivotal role in neurogenic inflammation. despite the prevalence of arthritis, the contribution of sp to the progression of arthritis has not been established. this study investigated the effect of csn ablation and sr140333, a sp antagonist, on knee joint inflammation and pain induced by intraarticular (i.a.) injection of kaolin (10 %, 5 h time course) in female wistar rats. the kaolin-injected knee (ipsilateral -ipsi) of vehicle-treated rats exhibited a significant, timedependent oedema as compared to the contralateral knee. in addition, increased pain score and high levels of myeloperoxidase (mpo, marker of neutrophil accumulation) and both pro-inflammatory (il-1b and il-6, but not tnfa) and anti-inflammatory (il-10) cytokines was detected in the ipsi synovial fluid of these animals. both destruction of knee joint csn fibres by neonatal capsaicin treatment and i.a. injection of rats with sr140333 (1 nmol/cavity) significantly attenuated the kaolin-induced pain score and knee oedema, suggesting that kaolin is acting, at least partially, via a neuronal mechanism. in contrast, the same treatment caused increased mpo activity and cytokine concentrations measured 5 h post kaolin injection. conclusions: peripheral release of sp after kaolin injection acts to increase pain generation, oedema formation and inflammatory cell influx. however, chronic tachykininergic depletion by capsaicin treatment up-regulates the production of pro-inflammatory cytokines that are important in triggering cell influx in the synovial cavity. (1) (1) university of sâ¼o paulo, sâ¼o paulo, brazil (2) butantan institute, sâ¼o paulo, brazil objectives: previously, intra-tracheal (i.tr.) injection of dep or 1,2-naphthoquinone (1,2-nq) evoked plasma extravasation and cell influx in rat airways. we now investigated whether simultaneous injection of these pollutants had a synergistic inflammatory action. we also determined the ability of dep and 1,2-nq-induced airway inflammation to evoke changes in the rat isolated thoracic aorta (rta) and corpus cavernosum (rcc) reactivity using organ bath assays. results: capsaicin-or vehicle-treated male wistar rats received i.tr. injection of dep (1 mg/kg) and 1,2-nq (35 nmol/kg). after 15 min, dep and 1,2-nq produced a potent (additive) plasma extravasation in the trachea and bronchi, but not lung, compared with each compound alone. in capsaicin-treated rats or treated with tachykinin antagonists, the response was inhibited, suggesting an important role for c-fibres, primarily tachykinins. increased mpo and cytokine levels were detected in bronchi of capsaicin-treated rats following 3 h treatment with pollutants. this treatment contributes to augment the ach (10-9-10-4 m)-induced relaxation in the rta but not in the rcc. in capsaicin-treated rats, the rta response to the pollutants was not affected but it was capable of evoking a marked relaxation in rcc in animals challenged with pollutants. conclusions: 1,2-nq exacerbates dep-induced plasma extravasation and mpo activity in the airways, indicating a neurogenic mechanism through tachykinins. the exposure to dep and 1,2-nq affects the endotheliumdependent response in rta without interfering with rcc. neuropeptides are unlikely to affect the pollutantsinduced changes in the rta. acknowledgements: capes, cnpq, fapesp. we thank ma alves for technical assistance. trans-resveratrol (rv) is a naturally occurring polyphenolic compound present in certain foods that has anticancer and anti-inflammatory properties. the purpose of this study was to determine the effect of rv on the production of proinflammatory cytokines and reactive oxygen species stimulated by lipopolysaccharides (lps) in glial cells. rt-pcr showed that rv (5, 25, 50 mm) dose-dependently inhibited 500 ng/ml lps induced tnfa, il-1b, il-6, mcp-1 and inducible nitric oxide synthase mrna expression. rv also inhibited lps-induced production of these cytokines (elisa), nitric oxide and reactive oxygen species in a dose-dependent manner. western blot analyses showed that resveratrol could inhibit lps-stimulated phosphorylation of erk1/2 and jnk but not p38. nf-kb reporter assay showed rv could inhibit nf-kb activation by lps in microglia and astrocytes. these results suggest that rv may inhibit lps-induced microglial and astrocyte activation through erk1/2, jnk and nf-kb signaling pathways. therefore, rv is a natural product with therapeutical potential against disease conditions in cns that involve an overproduction of proinflammatory cytokines and reactive oxygen species. (1), cs patil(2), sv padi (1), vp singh (1) (1) university institute of pharmaceutical sciences, panjab university, chandigarh, india (2) pharmacology r & d, panacea biotec ltd. lalru, punjab, india persistent stimulation of nociceptors and c-fibers by tissue injury causes hyperalgesia and allodynia by sensitization of nociceptors and facilitation of synaptic transmission in the spinal cord. the important participant in the inflammatory response of injured peripheral nerve may be nitric oxide (no). the aim of the present study was to test the sensitivity of pde5 inhibitor sildenafil in chronic constriction injury (cci) model, a rat model of neuropathic pain. sciatic nerve injury is associated with development of hyperalgesia 14 days after the nerve ligation. sildenafil (100 and 200fã� g/rat, i.t.) produced a significant decrease in pain threshold, which in lower dose did not alter the nociceptive threshold. the hyperalgesic effect of sildenafil was blocked by l-name and methylene blue (mb), which on per se treatment showed antinociceptive effect in nerve ligated rats. the results from the present study indicated that the major activation of no cgmp pathway in the chronic constriction injury model of neuropathic pain. the aggravation of hyperalgesic response might be due to the increased cgmp levels resulting in pkg-i activation and its upregulation. glycine transporter (glyt) 1 and glyt2 are expressed in glia and neurons, respectively. glyt1 make clearance of glycine released from glycinergic neuron in synapse and thus terminates the neurotransmission and also regulates over-stimulation by glycine spilled over to nmda receptors, while glyt2 supplies glycine into synaptic vesicles in glycinergic neurons. therefore, glyt inhibitors could modulate inhibitory glycinergic or excitatory glutamatergic neurotransmission. the present study examined the effects of glyt inhibitors on pain in animal models. inhibitors of glyt1 (sarcosine and org25935) and glyt2 (alx1393 and org25543) by intrathecal (i.t.) injection reduced formalin-induced nociceptive behaviors. glyt1 inhibitors reduced allodynia score and reversed the reduction of paw withdrawal threshold in complete freund adjuvant (cfa)-induced inflammation mice but the antiallodynia effects appeared after latent period. on the other hand, glyt2 inhibitors produced antiallodynia effects immediately after the i.t. injection in cfa-treated mice. these inhibitors produced the similar antiallodynia effects in the partial sciatic nerve ligationinjury or streptozotocin-induced diabetic neuropathic pain models, either by i.t. injection or i.v. injection.pretreatment of specific antagonists of glycine site of the nmda receptors disappeared the latent periods of glyt1 inhibitors and potentiated the antiallodynia effect. glycine receptor antagonist, strychnine by i.t. injected reversed the antiallodynia effect of i.v. injected glyt inhibitors. these results suggest that both glyt1 and glyt2 inhibitors by enforcing glycinergic inhibitory neurotransmission in spinal cord produce potent antinociceptive effect and may be novel candidate for medicament of pain control. the tumour microenvironment in particular tumour associated macrophages (tams) play a role in determining tumour outcome. despite strong causative links between inflammation and human gastric cancer progression, little is known of the role of tams in this disease. we have utilized our mouse model of gastric tumourigenesis, the gp130757ff mouse to assess the effect of the gp130757ff mutation on macrophage function and ascertain the role of macrophages in tumor formation. this mouse has a knockin mutation in the il-6 family cytokine receptor gp130 preventing shp2/ras/erk signaling and leading to constitutive stat3 transcriptional activation. tumour development is inflammation dependent, there is a requirement for il-11, and development is inhibited by nsaid treatment or microbial eradication, however an adaptive immune response is s410 inflamm. res., supplement 3 (2007) posters dispensable for tumorigenesis. in gastric antrum the gp130757ff mutation results, decreased erk1/2 activation and constitutive phosphorylation of stat3, abnormal signaling is replicated in macrophages. antral stat3 activation is unaffected by depletion of il-6. however in macrophages an absence of il-6 results in higher stat3 activation demonstrating the anti-stimulatory role of il-6 on macrophages. the gp130757ff mutation results in macrophages with decreased il-6 and increased inos mrna expression reflecting a more basally activated phenotype. manipulations of the gp130757ff mouse that reduce tumor size (eg. antibiotic or nsaid treatment or stat3 hemizygosity) coincidently result in reduced macrophage infiltration of antral mucosa. macrophages of gp130757ff mice display aberrant gp130 signaling potentially resulting in exaggerated response to stimuli. the key difference between mutant gastric mucosa and macrophages are changes in transcription of target genes. (1), y riffo-vasquez(2), s brain(2), s costa (1) (1) university of sâ¼o paulo, brazil (2) king's college london, uk objectives: we have previously shown that simultaneous intra-tracheal injection of diesel exhaust particles (dep) and 1,2-naphthoquinone (1,2-nq) caused a potent inflammation in rat airways partially dependent on neurogenic-mediated mechanism. this study investigates the mechanism of action of thee pollutants using inflammatory assays and histopathological approach in the lung and trachea of wild type (wt) and trpv1 knockout (ko) mice exposed to dep and 1,2-nq. dep (1 mg/kg) and 1,2-nq (35 nmol/kg)-induced airway inflammation was assessed via 125i-labelled albumin 1 h post pollutants injection. mpo assay and histopathology were performed 3 h after treatment. staining of lung and trachea specimens with h&e provided a profile of cell infiltration in both wt and ko animals. results: injection of dep and 1,2-nq evoked a potent plasma extravasation into the trachea and lung of wt, but not trpv1 ko, suggesting these pollutants act via a trpv1-mediated mechanism. in contrast, mpo activity in the airways of trpv1 ko mice was exacerbated compared to wt mice data. likewise, the histopathology revealed high numbers of leukocytes and macrophages infiltrated into the lungs and trachea of trpv1 ko mice compared to wt. conclusions: inhibition of increased microvascular permeability in the airways of trpv1 ko mice treated with pollutants suggests that these receptors are the predominant mechanism involved in the inflammation. however, neutrophils/macrophages accumulate more in trpv1 ko, indicating that the lack of trpv1 receptors up-regulates production of inflammatory cells in response to pollutants, thus supporting a protective role for trpv1 receptors. acknowledgements: capes, cnpq, fapesp. (1), n sato(1,2), y endo (1), s sugawara (1) (1) division of oral immunology, department of oral biology, tohoku university graduate school of dentistry, sendai, japan (2) division of fixed prosthodontics, department of restorative dentistry, tohoku university graduate school of dentistry, sendai, japan biotin is a water-soluble vitamin of the b complex and functions as a cofactor of carboxylases.biotin deficiency causes alopecia and scaly erythematous dermatitis.moreover, serum biotin levels are significantly lower in atopic dermatitis patients than in healthy subjects, indicating that biotin deficiency is involved in inflammatory diseases.however, immunological effects of biotin on allergic inflammation remain unclear.in this study, we investigated the effects of biotin-deficiency on metal allergy using nickel (ni)-allergy model mouse and a murine macrophage cell line j774.1. female balb/c mice (4 weeks old) received a basal diet or a biotin-deficient diet for 8 weeks.ten days after sensitization with intraperitoneal injection of lps and nicl2, the mice were challenged with intradermal injection of nicl2 into the pinnas.allergic inflammation was measured by ear swelling.the ethical board for nonhuman species of the tohoku university graduate school of medicine approved the experimental procedure followed in this study.j774.1 cells were cultured in biotin-sufficient or deficient medium for 4 weeks. ear swelling was significantly higher in biotin-deficient mice than biotin-sufficient mice.il-1 beta productions by splenocytes were significantly higher in biotin-deficient mice than in biotinsufficient mice.moreover, biotin-deficient j774.1 cells produced il-1 beta significantly higher than biotinsufficient j774.1 cells.to investigate the therapeutic effects of biotin-supplementation, biotin-deficient mice received biotin contained-water for 2 weeks.ear swelling was significantly lower in biotin-supplement mice than biotin-deficient mice.these results indicated that biotindeficiency deteriorates allergic inflammation.the augmentation of il-1 beta production is probably involved in those deteriorations. one of the most important targets of cytokine action is the blood vessels, which undergo some structural and functional changes that result in activation of endothelium. applying the elisa technique, levels of il-18, icam-1 and e-selectin were studied in 77 patients with acute pancreatitis. mediators levels were studied in arterial, venous and pancreatic ascites samples. according the atlanta criterion the mild pancreatitis was established in 33 patients and severe -in 44 patients. the highest levels of il-18 were noted in ascites and lowest in arterial samples. the highest concentration of adhesion molecules was in venous samples and lowest in ascites. it was a clear correlation between levels of il-18 and adhesion molecules and severity of pancreatitis. during first week the levels of il-18 gradually increased in patients with severe pancreatitis, while in patients with the edematous pancreatitis its levels decreased starting from the third day. icam-1 levels gradually increased during first three day with the following decrease after this term. the highest levels of e-selectin were noted at the time of admission. the clear correlation between il-18 and adhesion molecules was noted in both groups of patients. besides that, the clear strong correlation was observed between il-18 and quantity of circulating granulocytes and between e-selectin and hematocrite in patients with necrotizing pancreatitis. our study confirms the importance of activation of endothelium as a part of the systemic inflammatory response in patients with acute pancreatitis. subsequently eos infiltrate the tissues, are activated, and release mediators inducing the late phase response. this is characterized by tissue damage and repair, and in chronic reactions, tissue remodeling and fibrosis. mc/eos cross-talk by physical and non-physical contact is an essential feature of late-phase and chronic allergic reactions. we have previously described an mc/eos interaction facilitated by soluble mediators and shown to enhance allergic inflammation. still, pathways that mediate mc/eos cross-talk in allergy are not fully characterized. methods: human cord-blood derived mc were cocultured with peripheral blood eos, and activated with anti-ige. mc/eos couples in co-culture and in human nasal polyp tissue sections were specifically stained and counted using microscopy. expression of surface molecules was analyzed by facs. mc activation was measured by chromogenic assays for ã¢-hexosaminidase. relevant surface molecules were neutralized using antibodies, to assess interference with couple formation and activation. results: mc and eos physically interact, forming welldefined couples in-vitro and in-vivo. in the presence of eos, mc are more releasable under baseline or igeactivating conditions. this effect is partially mediated by cd48 and dnam-1 on mc, and 2b4 and nectin-2 on eos. we describe a novel physical interaction between mc and eos that we name "the allergic synapse". this synapse may upregulate allergic reactions, thus serving as a target for therapeutic intervention in allergic and inflammatory diseases. methods: mc were obtained from cord blood mononuclear cells (6-8 weeks with scf, interleukin-6 and prostaglandin e2, cbmc). cbmc were activated, after their culture for 5 days with myeloma ige (2mg/ml), by rabbit anti-human ige antibodies (5mg/ml), for 1, 3 and 6h at 378c. activation was measured by b-hexosaminidase release, determined by enzymatic-colorimetric assay. the expression of flip, mcl-1, bcl-2, bcl-xl, bak and bax was assessed by immunoblot analysis. results: two anti-apoptotic proteins were found to be upregulated: flip, which is involved in the extrinsic apoptotic pathway and mcl-1 that is mainly implicated in the intrinsic apoptotic pathway. in contrast, the expression of two other anti-apoptotic proteins that we have examined (i.e. bcl-2 and bcl-xl) was not altered. likewise, the expression of pro-apoptotic proteins from the bcl-2 family (i.e. bak and bax) was either undetectable or unchanged. conclusions: our findings reveal that ige-dependent activation of human mc mainly induces the selective increase in the expression of two pro-survival molecules. this may be one of the mechanisms that underlay mc hyperplasia in the chronic allergic inflammation. (1), c armishaw(2), z yang (1), h cai (1), p alewood(2), c geczy (1) (1) university of new south wales, faculty of medicne, sydney, australia (2) university of queensland, institute for molecular biosciences, st lucia, australia purpose: human s100a8, s100a9 and s100a12 are closely related proteins associated with inflammation. s100a12 is expressed in the human genome, but not in the mouse. s100a12 is a potent monocyte chemoattractant and mast cell (mc) activator. mouse (m) s100a8 and human (h) s100a8 share 58% structural identity, but the hinge domains are more divergent. the ms100a8 hinge (ms100a842-55) is a potent chemoattractant for leukocytes whereas this sequence in hs100a8 (hs100a843-56) is inactive. methods: s100a12 hinge domain and its alamine scan mutants were synthesized and their activities were tested by using thp1 cells or murine mc in vitro and mouse footpad injection. results: s100a12 hinge domain (s100a1238-53) was chemotactic for monocytes and mc and provoked mc degranulation in vitro and oedema, and leukocyte recruitment in vivo. in contrast to s100a12, the hinge domain only weakly induced cytokine production (il6, il8) by macrophages. residues essential for oedema were hydrophobic in nature (leu40, isoleu44, isoleu47 and isoleu53). n42 and i44 were essential for responses provoked by 10-12m s100a1238-53 whereas mutation of k38, l40, n46, i47, d49, k50 and i53 significantly reduced migration with 10-9m s100a1238-53. conclusions: s100a12 and ms100a8 may be functional chemattractant equivalents; s100a12 may have arisen by duplication of human s100a8. isoleucine residues in s100a12 hinge domain are essential for its proinflammatory properties. (1), g kiriakopoulou (1), e tsimara(2), a voultsou(2), k zarkadis (1) (1) general hospital of zakynthos, greece (2) medical centre of katastari, zakynthos, greece schistosome is a disease which pests in 74 tropical countries. the endemic areas are south america, far and middle east, and africa. our aim is to present an incident which concerning a parasitic infection, not endemic in greece. patient: man 30 years old who immigrated from pakistan (at the side of the aparkenar river) in greece, a year ago. he turned out with anlage, headache and weight loss. he is a swimmer. he mentioned also a fever before migrating to greece. clinically: largely-scaled paleness, stomach -mild and diffused sensitivity, with also lightly increased enteric sounds and a small degree of hepatomegaly, when palpated deeply. laboratory examination: leucocytes 9.030 eo 24.8%, hb 8.2g/dl, ht 27.2%, mcv 65,6fl, mch 19.1pg, thrombocytes 419.000, blood sedimentation 10mm, fe 10mg/ml, ferritin 2.98ng/ml. normal biochemical examinations. u/s: livers size lightly increased 16.5mm, hepatoportal vein with normal amplitude. parasitology of feces: in the immediate confection with lugol of the second sample, there were observed: big scattering oval ovules (130 -60mm) with a thin wall and quills on the side, as well as three imago worms (>10 mm): schistosoma mansoni. treatment: praziquantel was given. recheck in a months time: blood examinationleucosites 11.230, eo 9.8%, hb 10,2g/dl, ht 33.6%. parasitology of feces is negative. in the diagnosis of anemia combined with fever, to patients who are immigrants, schistosomiasis posters inflamm. res., supplement 3 (2007) should be taken into account, especially when sideropenic anemia is accompanied with intense eosinophile, because the disease is mostly a reaction of retardate supersensitivity. bronchial asthma is a chronic airway inflammatory disease caused by immune cells such as t lymphocytes and eosinophils. recently, high-sensitivity crp (hs-crp) has become available for detecting small changes in crp levels within the normal range, allowing for assessment of subclinical inflammation. this study was undertaken to investigate the relationship between hs-crp and bronchial asthma. we collected blood samples from 109 patients with bronchial asthma with or without attacks and measured serum eosinophil cationic protein (ecp) and pulmonary function as well as serum hs-crp. serum crp levels in patients without attacks (average 0.473 mg/ l; p < 0.001) and with attacks (average 0.908 mg/l; p < 0.001) were significantly higher than those of normal controls (average 0.262 mg/l). serum hs-crp levels were inversely correlated with fev1.0% in asthmatic patients (r = -0.4915, p < 0.01). in conclusion, these results indicate that serum hs-crp as well as ecp may be related to the state of asthma exacerbation and allergic inflammation. objectives: the pshr assay can be used to test the biologic activity of allergens since it mimics the effector phase of a type i hypersensitivity reaction. in this study we tested methods for removing ige-antibodies (stripping) from donor basophils. moreover a method was developed for improving the antigen specificity profile in pshr. proof-of-concept was provided using absorption with complete allergen extracts. methods: buffy coats were screened to exclude reactivity against relevant allergens. subsequently pbmcs were purified and basophils were stripped with either lactate or a phosphate buffer. cells were passively sensitized with patient sera and challenged with allergen extracts in various concentrations. released histamine was measured spectrofluorometrically (hr-test, reflab). cutoff was 10% hr. for absorption experiments patient sera (from a peanut allergic and a codfish allergic patient) were incubated with streptavidin-coated sepharose beads coupled with biotinylated allergens (peanut, and bsa as control). after centrifugation the supernatant was used to passively sensitize stripped basophils. hr was measured as described above. results: stripping experiments using the two buffers only partially removed surface ige but passive sensitization of stripped basophils was equally effective enabling determinations of sub-nanogram quantities of peanut allergen. absorption experiments showed that it was possible to specifically remove peanut specific ige from patient serum. removal of specific ige reactivity to peanut extract was verified by western blotting. conclusions: using peanut extract as a model it was demonstrated that in pshr antigen specificity can be modified. (1), sk bk(1), v sharma(2), rp bhandari (3) (1) nepal medical college teaching hospital, nepal (2) all india institute of medical sciences, new delhi, background: nepal has one of the highest maternalmortality rates in the world. this study was to evaluate the incidence, disease pattern, and risk-factors for thromboembolism in pregnant nepalese women. methods: women with thromboembolic diseases were identified and their case records retrieved and reviewed s414 inflamm. res., supplement 3 (2007) posters from january 1998 to december 2006. demographiccharacteristics were compared between women with and without thromboembolism. the total number of deliveries over the study period was 16,993, giving an incidence of 1.88 per 1000 deliveries. there were two cases of pulmonary-embolism and one resulted in a maternal-death. the others had deep-vein-thrombosis of which over 80% were limited to calf veins only. the ultrasound-examinations requested for suspected deep-venous-thrombosis before and after the event of maternal-death were 1.62 and 10.7 per 1000 deliveries (p <.001);the corresponding cases of deepvenous-thrombosis diagnosed were 0.29 and 2.94 per 1000 deliveries, respectively (p <.001). the majority (75%) of cases were diagnosed in the postpartum period, mainly after cesarean-delivery. women with venous-thromboembolism were older, had higher bmi, and a higherincidence of preeclampsia. there were approximately twice as many postpartum as antepartum events. bloodgroup a, multiple-pregnancy, caesarean-section, cardiacdisease, delivery at gestational-age of <36 weeks, a bmi of25, or more and maternal-age of 35 or over were all found to increase incidence of venous-thromboembolism. the long-standing belief that thromboembolism is rare among nepalese is at least partly because of undiagnosis most of these events are deep-veinthromboses occurring in the postpartum period and it is very essential for primary prevention developing country like nepal. (1), ch ladel (1), t ruckle(2), c rommel(2), r cirillo (1) (1) rbm-merck serono, colleretto giacosa, italy (2) serono pharmacological research institute, geneva, switzerland rheumatoid arthritis (ra) is a severe articular disease. massive leukocyte activation and infiltration into joints result in cartilage and bone destruction. blockade of pi3k signalling pathway has been demonstrated to be curative in a murine model of ra, collagen-induced arthritis (cia). in this study we explored the molecular mechanisms by which pi3k signalling inhibition resulted in clinical amelioration of disease symptoms. as605858, a novel isoform non-selective, yet specific class-i-pi3k inhibitor, administered at 15 mg/kg twice-a-day for 8 days, to mice showing signs of arthritis, paw swelling and inflamed digits, induced a significant amelioration of disease course. at the end of treatment, post-arthritic paws were removed and phosphrylation levels of akt (p-akt), downstream target in pi3k-mediated signal, were determined by semi-quantitative immunohistochemistry, also immunophenotyping of circulating cells by flowcytometry was conducted. akt phosporylation resulted to be significantly enhanced by disease induction and as605858 was able to decrease its levels down to values comparable with naã¯ve animals. controls and as605858treated mice were bled before treatment, after two days of treatment and at treatments end. no changes in cellular composition (morphology and hematology parameters) between experimental groups were observed. t cell number was not affected, however a significant decrease of natural-killer, memory and regulatory t cells was observed after as605858 administration. finally, a non-significant, moderate reduction of bcell number was also observed. these data demonstrate that efficacy of as605858 in arthritis models is mediated by direct modulation of the target resulting in a mixed anti-inflammatory (via pi3kg) and immunosuppressive (via pi3kd/a/b) activity. (1) (1) institute of biomedical science, university of sao paulo, brazil (2) ibilce -sao paulo state university, brazil epidemiologic data suggest that female sex hormones are involved in the pathophysiology of allergic asthma. we investigated in rats the immunomodulatory potential of estradiol and progesterone on the expression of allergic asthma. seven days after being ovariectomized (ovx), groups of rats were sensitized with ovalbumin (oa). fourteen days after sensitization, the animals were oachallenged and used 1 day thereafter. allergic,shamoperated animals were used as controls. some ovx animals were treated with estradiol (280 ã¬g/kg) or progesterone (200 ã¬g/kg) 24 h before being challenged. mast cell degranulation was quantified in samples of isolated, oa-challenged bronchi. the airway reactivity of inner bronchi to methacholine and the functional activity of cells we analysed. ovx caused reduction of the allergic lung inflammation and bronchial hyperresponsiveness with regard to intact female rats. estradiol reverted the reduced cellular recruitment into lungs, whereas progesterone reduced the pulmonary inflammatory response and reverted the bronchial hyperresponsiveness. cultured bal and bone marrow cells from allergic rats increased posters inflamm. res., supplement 3 (2007) s415 the release of il-10 and reduced that of il-1 and tnf. the release of il-4 by bone marrow cells was significantly reduced. these effects were reverted by estradiol, and progesterone reduced il-4 and increased il-10 production in bal and increased that of il-1 and tnf in bone marrow cells. bronchial mast cell degranulation upon direct contact with oa in ovx rats was less than in controls. it is suggested that female sex hormones can modulate the allergic lung inflammation in rats by acting on cellular migration/activity and airway responsiveness. objectives: to study the participation of fsh on modulation of e-and l-selectin, icam-1 and mac-1 expression in ovariectomized (ovx) rats made allergic. methods: female rats were sensitized (oa/alumen) after 1 (ovx-1) or 7 days (ovx-7) of ovx or sham-operated (sh). fourteen days thereafter animals were challenged (oa, 1%; aerosol) and sacrificed 24 h after. bronchoalveolar lavage (bal) was collected and flow citometry analyses oficam-1, mac-1 and l-selectin expression was studied. in parallel, lungs were frozen and sections were analysedfor e-selectin expression by immunohistochemistry. results: at day 1, e-selectin expression increased(ovx-1=1,8ae0,08) and at day 7, decreased (ovx-7=1,3ae0,1) as compared to respective controls (sh-1=1,45ae0,08; sh-7=1,9ae0,08). estrogen treatment reverted this profile in both groups (ovx-1+e=1,37ae0,09; ovx-7+e=1,8ae0,1). mean fluorescence intensity of bal cells showed increase of mac-1 expression (ovx-1=19ae0,5 vs sh-1=15,5ae1,0), icam-1 (ovx-1=18,1ae1,1 vs sh-1=13,3ae0,2) and l-selectin (ovx-1=11,2ae0,8 vs sh-1=9,5ae0,12) at day 1 i.e., ovx-1 group. on the other hand, we observed a decrease in icam-1 (ovx-7=14,2ae0,75 vs sh-7=19ae1,4) and mac-1 expression (ovx-7=20,7ae1,4 vs sh-7=27,5ae1,5) was seen in ovx-7 group. conclusions: oscillation of hormone levels during immunization with oa increased (ovx-1) and decreased (ovx-7) expression of adhesion molecules. estradiol treatment reverted this effect. this results suggest that fsh modulates the ali in rats by acting on cell (1), s lim (1), y lin (1), bp leung (1), c thiemermann (2), wsf wong (1) (1) national university of singapore (2) the william harvey research institute, london, uk glycogen synthase kinase 3b (gsk-3b) is known to regulate various cellular functions including inflammatory responses. we hypothesized that inhibition of gsk-3b may have anti-inflammatory effects in a mouse asthma model. balb/c mice were sensitized with ovalbumin (ova) and challenged with aerosolized ova. tdzd-8, a non-atp competitive gsk-3b inhibitor, was administrated by i.v. injection one hour before ova challenge. tdzd-8 significantly reduced the ova-induced eosinophilia in a dose-dependent manner and inhibited the levels of il-5, il-13 and eotaxin in bronchoalveolar lavage (bal) fluid. tdzd-8 also suppressed the mrna levels of icam-1, vcam-1 and chitinase proteins in the lung. histological studies revealed that tdzd-8 substantially reduced the inflammatory cell infiltration and mucus secretion in the lung tissue. tdzd-8 also decreased ova-specific ige level in the serum. in addition, ova-induced increase in airway resistance and reduction in dynamic compliance were attenuated by tdzd-8. our findings suggest that inhibition of gsk-3b may have therapeutic potential for the treatment of allergic airway inflammation. (1), a yildirim (1), f ercan (2), n gedik (3), m yuksel(4), inci alican (1) ( materials and methods: sprague-dawley rats (200-250 g) were exposed to 90 oc (burn group) or 25 oc water bath (control group) for 10 s under ether anesthesia. adm (100 ng/kg; s.c) was administered 10 min before burn and all rats were decapitated 24 h after the burn insult. trunk blood was collected for the measurement of tnf-alpha level and the lung, ileum and kidney samples were stored for microscopic scoring and for determination of lipid peroxidation (lp), myeloperoxidase (mpo) activity and formation of reactive oxygen metabolites (roms) using chemiluminescence assay. results: burn resulted in severe morphologic damage in tested tissues. lp increased in lung and kidney (p<0.01-0.001) and mpo activity showed a marked increase in all tested tissues (p<0.001) of the burn group. adm reversed these parameters effectively (p<0.05-0.001). luminol chemiluminescence levels showed increases in both ileum and lung (p<0.01-0.001) whereas lucigenin chemiluminescence levels increased in ileum and kidney (p<0.01) of the burn group. adm treatment was also beneficial in reducing chemiluminescence levels near to controls (p<0.01). adm reduced plasma tnf-alpha level (p<0.01) which showed a significant increase in burned animals compared to controls (p<0.001). conclusions: adm is beneficial in remote organ damage following burn insult via decreasing neutrophil infiltration, rom generation, lp, and the release of proinflammatory cytokine tnf-alpha. kalpana panday (1), sd joshi (2), kr reddy (3) ( we determined the crystal structure of human hematopoietic prostaglandin (pg) d synthase (h-pgds) as the quaternary complex with glutathione (gsh), mg2+, and an inhibitor, hql-79, with anti-inflammatory activities in vivo, at 1.45 resolution. hql-79 was found to reside within the catalytic cleft between trp104 and gsh in the quaternary complex. hql-79 inhibited h-pgds competitively against the substrate pgh2 as well as noncompetitively with respect to gsh. surface plasmon resonance analysis revealed that hql-79 bound to h-pgds with an affinity that was 10-fold higher in the presence of gsh and mg2+ than in their absence. hql-79 inhibited selectively pgd2 production by human h-pgds-expressing megakaryocytes but only marginally affected the production of other prostanoids, suggesting the tight functional engagement between h-pgds and cyclooxygenase. orally administered hql-79 inhibited antigen-induced production of pgd2, and airway inflammation in mice without affecting the production of pge2 and pgf2fâ¿. knowledge about this quaternary structure should accelerate the structure-based development of novel anti-inflammatory drugs that inhibit pgd2 production specifically. introduction: it has been proved that high levels of mechanical ventilation produce lung injury as well as local inflammation. this study was designed to evaluate how the generation of inflammatory mediators by an over-stretched lung affects the non-hyperventilated lung. methods: male wistar rats (250-275g) were anesthetized and paralyzed, and the two lungs were independently intubated. differential ventilation was applied for 3h (70 breath/min). one lung was subjected to hyper-ventilation (20ml/kg/lung) and the other was ventilated with a normal volume (5ml/kg/lung). in a control group, both lungs were ventilated with a normal volume. after sacrifice, samples of lung, plasma and liver were collected. the expression of the pro-inflammatory chemokine mip-2 was evaluated by rt-pcr and the edema was assessed by the ratio between the wet and dry weight of the lung. systemic inflammation was estimated in liver by measuring the expression of tnfa by rt-pcr as well as its levels in plasma. the hiper-ventilated lung showed an increase in the ratio between the wet and dry weight and in mip-2 expression compared to the normal ventilated lung. no differences were found in edema, neither expression of mip-2 between the normally ventilated lung and control lungs. no significant changes were observed in liver expression and plasma levels of tnfa as a consequence of unilateral lung hyper-ventilation. the over-straining caused to the hyperventilated lung leaded to a local inflammatory response without systemic effects. the normal ventilated lung is not affected by the inflammatory process triggered on the over-strained lung. (1), j-y gillon(2), v lagente (1), e boichot (1) (1) university of rennes 1, rennes, france (2) serono international s.a., geneva, switzerland macrophage elastase (mmp-12) is a metalloproteinase involved not only in emphysema but also in the inflammatory process associated with copd (chronic obstructive pulmonary disease). the mechanism of action of mmp-12 in the development of pulmonary inflammatory process is still unknown. in the present study, we investigated the effect of recombinant human mmp-12 (rhmmp12) on il-8/cxcl8 release from alveolar epithelial cell line a549 and we explored the underlying mechanisms. a549 cells were stimulated with rhmmp-12 (1.10-3-2.10-1 u/ml) during 6 hours and il-8/cxcl8 level in supernatant was determined by elisa. involvement of map (mitogen activated protein)-kinases was studied by western-blotting and also using chemical inhibitors. nfkb activation was examined with the transam nfkb p65 kit. we observed that mmp-12 elicited il-8/cxcl8 release in a dose-dependent manner. this production could be prevented by a pretreatment for 1hour with a selective mmp-12 inhibitor (as111793 1-30 mm) or with the nonselective inhibitor batimastat (1-30mm) . the il-8/cxcl8 production was also inhibited by actinomycin d (5mg/ml), erk1/2 inhibitors (u0126 5mm and pd98059 10mm) and nfkb inhibitors including bay11-7082 (10mm) and nfkb activation inhibitor (10nm), whereas p38 kinase inhibitor (sb203580) had no effect. stimulation with mmp-12 was rapidly followed by a phosphorylation of erk1/2 (5min) and an nfkb nuclear translocation and activation (1h). the nfkb activation was not inhibited by a treatment with u0126. these data suggest that alveolar epithelium is a target of mmp-12 since it upregulates gene expres-s418 inflamm. res., supplement 3 (2007) posters sion and release of il-8/cxcl8, via erk1/2 and nfkb activation, but these two pathways appears to be distinct. agents which are associated with lung inflammation, such as cigarette smoke and lipopolysaccharide (lps), induce the production of pro-inflammatory chemokines in lung epithelial cells in vitro, and the induction of interleukin (il)-8, in particular, is often used a measure of relative toxicity.in this study we have compared mrna expression and mediator release in nci-h292 human lung epithelial cells exposed to lung toxicants, namely: cigarette smoke total particulate matter (tpm), lps, bleomycin, diesel exhaust particles, residual oil fly ash (rofa), carbon black and vanadyl sulphate.polystyrene, poly(methyl-methacrylate) and the tpm vehicle, dimethyl-sulphoxide were used as negative controls.confluent monolayers of h292 cells were exposed to serial dilutions of test agents in serum-free medium for 24 hours.the conditioned medium was then removed and assayed for a range of pro-inflammatory cytokines and other selected mediators by luminex technology.the levels of gene expression of il-8, matrix-metalloprotease-1 (mmp-1), the gel-forming mucin muc5ac, heparinbinding epidermal growth factor-like growth factor (hb-egf) and the cytochrome p450s cyp1a1 and cyp1b1 were determined by quantitative-polymerase chain reaction.all of the toxicants induced similar responses whereas the negative controls were largely ineffective.-such a panel of biomarkers may enable an in vitro assessment of the potential to cause lung inflammation.-moreover the use of several biomarkers could give a more accurate picture of toxicity than the determination of il-8 alone, particularly in the case of agents such as tpm, where the conventional vehicle is found to have some biological activity. respiratory tract infections are a major public health issue. prevention in high risk populations relies mainly on vaccination. vaccination is highly recommended in decrease absenteeism. immunomodulating drugs are important tools in the treatment of infectious diseases. immunomodulatory agents are probably contributive in decreasing exacerbation rates. the authors present different classes of immunomodulators that are currently in use. the vaccine, created from a bacterial protein, reeducates the immune system to stop inflammation. by preventing infections, vaccines prevent the development of a strong t helper (th1) response. the challenge is now to inform about new possibilities of an optimal prevention respiratory tract infections. deoxypodophyllotoxin (dpt) is a medicinal herbal product that is isolated from anthriscus sylvestri. that inhibits cyclooxygenase-2 (cox-2) and cox-1dependent phases of prostaglandin d2 (pgd2) generation in bone marrow-derived mast cells (bmmc) in a concentration-dependent manner with ic50 values of 1.89mm and 65.3mm, respectively. this compound inhibited cox-1 and 2-dependent conversion of the exogenous arachidonic acid to pgd2 in a dose-dependent manner with an ic50 values of 0.01mm and 12.1mm, respectively. however, dpt did not inhibit cox-2 protein expression up to a concentration of 30mm in the bmmc, indicating that dpt directly inhibits cox-2 activity. furthermore, this compound consistently inhibited the production of leukotriene c4 (ltc4) in a dose dependent manner, with an ic50 value of 0.37mm. these posters inflamm. res., supplement 3 (2007) s419 results clearly demonstrate that dpt has a dual cox-2/5-lox inhibitory activity in vitro. therefore, this compound might provide the basis for novel anti-inflammatory drugs. in order to determine anti-allergic and antiasthmatic activity of dpt in vivo, we used rat pca model which was activated by anti-dinirophenyl ige and ovalbumin/alum induced mouse asthmatic animal model, respectively. as a result, dpt strongly inhibited pca reaction as well as it reduced infiltrated eosinophil numbers in bronchoalveolar lavage fluid. furthermore, dpt decreased the mrna levels of the th2 cytokines in a murine asthmatic model. in addition, northern blot analysis showed that dpt also reduced both the eotaxin and arginase i mrna levels in a dose dependent manner. these results suggest that dpt may be beneficial in regulating various inflammatory diseases. an imbalance of proteases and anti-proteases in the lung has been implicated in the pathogenesis of chronic obstructive pulmonary disease (copd), a smokingrelated disorder associated with accelerated lung function decline.in particular, the activity of matrix metalloproteases (mmps) have been implicated in driving both the inflammation and parenchymal destruction observed in copd patients.here, we tested whether a broad spectrum mmp inhibitor, pkf-242484, could block the inflammation induced by an acute exposure to cigarette smoke in 2 strains of mice, balb/c and c57/bl6.animals were administered the compound (1-10 mg/kg) either per os (p.o.) or intranasally (i.n.) 1 hour before and 5 hours after exposure to smoke on three consecutive days.bronchoalveolar lavage (bal) was performed and lungs were flash frozen for inflammatory marker analysis.pkf-242484 dose-dependently reduced lung neutrophilia in balb/c mice when dosed either p.o. (~45% at 10 mg/kg; p < 0.01)or i.n. (~60% at 10 mg/kg; p < 0.01).however, the compound had no clear effect on bal neutrophil infiltration in c57/bl6 mice by either route of administration.interestingly, in both strains bal macrophages dose-dependently trended towards an increase when the compound was dosed p.o. and decreased when dosed locally (p < 0.05). examination of lung tissue cytokine levels revealed that while smoke-exposure increases il-1beta, kc, and mip-2, pkf-242484 had little effect on these cytokines.these data suggests the ability of broad spectrum mmp inhibitors to inhibit smoke-induced acute neutrophil inflammation is strain-dependent, while its ability to limit macrophage infiltration may be route dependent. to investigate the role of seh in the regulation of the pulmonary inflammatory response, we have used seh deficient mice in a locally administered lps model. male seh deficient mice (ko) and their wildtype (wt) littermates were exposed to inhaled lps.four hours later they were sacrificed and bal was performed. differential counts and cytokine levels in bal were evaluated. results: lps induced a significant increase in total cell number and neutrophil number in bal in the seh deficient mice and in wt mice;no significant differences between the groups were seen (table 1 ) cytokine analysis showed significant increases in tnfalpha, il-6, kc, gm-csf, mcp-1, il-1beta and rantes in the lps-exposed wt mice. no significant differences were seen between lps exposed wt and ko mice except for a significant increase in tnfa in ko mice. our results show that seh has no pivotal role for the regulation of the acute inflammatory response to lung administered lps. fam.liliaceae. based on literature data which signaled the presence of steroidic saponins in the rhyzomes, isolation, identification and quantitative determination of these compounds were done. the antiinflammatory activity was tested in non-immune chronic inflammation model:the cotton-pellets granuloma test in rats, and in an immune chronic inflammation model:arthritis test induced by freunds adjuvant in rats. in the first test, the antiinflammatory effect of steroidic saponins 600 mg/kg is weak, statistically insignificant. in the arthritis test, steroidic saponins 600 mg/kg proved an antiinfalammatory activity, influencing especially the primary response, but also the secondary one, in the last part of the experiment (after 16 days). in both tests, the suprarenal glands weight was modified. objectives: dendritic cells (dcs) are professional antigen presenting cells. many types of dc with subtle difference in phenotype have been reported in several organs. existence of dc was reported in synovial tissue of rheumatoid arthritis (ra), however, the details in ra dc still remains unclear. in this study, we generated a new lineage of dc with gmcsf (+tnfa) and investigated their functions. furthermore the ability of osteoclastgenesis was examined. methods: monocyte-derived dcs or macrophage were generated in (1) tnfa + gm-csf (2) gm-csf (3) il-4 + gm-csf (4) mcsf. the phenotypes of these cells were analyzed by morphological examination and flow cytometry (facs calibur). cell proliferation was examined by wst-8 assay. dc functions were assessed in antigen presenting ability (mlr assay), cytokine production (elisa), and endocytosis (fitc-dextran uptake). concerning osteoclastgenesis, monocyte-derived dcs were incubated with rankl and mcsf. trap stain was performed and the resorption ability was assessed on osteologic cell culture system and dentine slices. results: these cells were dendritic-like and their surface markers were cd1a low cd11b + cd11c + cd14 + cd86 + cd83 low hla-dr + dc-sign low and different from conventional dc or macrophage. they had an antigen presenting ability to induce na*ve cd4+ t cell proliferation, il-12 production and endocytosis. in the presence of rankl and mcsf, they differentiated multinuclear trap-positive cells with bone resorption ability, which was strengthened by tnfa. we generated a new lineage of dc with gm-csf (+ tnfa). the dc seemed to play a pivotal role in inflammatory arthritis under tnf immunity. the clinical effectiveness of rituximab and other b cellattenuating rheumatoid arthritis (ra) therapies has increased interest in understanding the role of b cells in ra pathogenesis.the possible mechanisms underlying the effectiveness of rituximab were investigated by performing biosimulation research in the entelos ra physiolab platform, a mathematical model of the joint of an ra patient.the platform dynamically integrates the contributions of immune cells (t cells, b cells, and macrophages), resident cells (fibroblast-like synoviocytes and chondrocytes) and mediators to the joint inflammation and structural damage observed in ra.the b cell lifecycle is represented in the platform, as well as effector functions such as antigen presentation, mediator and autoantibody production, and immune complex formation.the dynamics of these b cell properties were calibrated to reproduce reported experimental behaviors and clinical outputs from ra patients (e.g., acr score and radiographic progression rates).an assessment of the contribution of individual b cell functions to clinical outcome suggests that plasma cell-derived immune complexes are key modulators of inflammation in ra patients. in contrast, proinflammatory cytokine production by b cells contributes minimally to synovial hyperplasia, but plays a role in the progression of structural damage.immune complex formation leads to monocyte activation, increased mediator production by macrophages and an increase in antigen availability to t cells.biosimulation research in the ra physiolab platform is advancing our understanding of the mechanisms underlying effective b cell-targeting ra therapies, and may guide the development of improved second-generation therapeutic approaches. methods: the hmscs were obtained at the operation.the mononuclear cells were extracted and the colony forming assay were performed after 2weeks. the hmscs were cultured and in passage1,the cell surface antigens of both groups were analyzed by flow cytometory.in passage2, in control group, the cells were cultured with beta glycerophosphate(bgp) and in osteogenic group, the cells were cultured with bgp and ascorbic acid(aa) and dexamethasone(dex).after 2weeks, alp staining and activity were measured in each group.after 3weeks, alizarin red s assays were performed.rna was extracted from the cells cultured with bgp and aa and dex for 2weeks and 3weeks and the gene expressions of bone formation markers were examined by real time pcr. the mann-whitney test was used for the statistical analyses. the colony forming assays showed no significant differences in oa and ra. in flow cytometory, the cell surface antigens in oa and ra were almost same.in alp activity,there were no significant differences in oa and ra.in alizarin red s, there were significant differences.in real time pcr,the gene expressions showed no significant differences in oa and ra. conclusions: the hmscs of ra will be able to use for regenerative therapy. silje vermedal hã¸gh (1), hm lindegaard (2), gl sorensen (1), a hã¸j (3), c bendixen (3), p junker (2), u holmskov (1) ( cytosolic phospholipase a2 (cpla2) plays a crucial role in eicosanoid production, by releasing an arachidonic acid from membrane phospholipids. in addition, cpla2 regulates the phagocyte nadph oxidase-releasing superoxides and that is the only isozyme responsible for the production of eicosanoid in phagocytic cells. collageninduced arthritis (cia) in mice is an experimental model of auto-immune diesease with several clinical and histological features resembling rheumatoid arthritis in humans. previous studies show that cpla2-deficient mice are resistant to cia. thus we aimed to study whether cpla2 is up-regulated during the development of cia and to detect its exact location in the inflamed joints. immunoblot analysis revealed an increase in the level of joint cpla2 protein during the development of the disease which correlates with the severity of the inflammation, as examined by paw thickness. immunohistochemistry with specific anti-cpla2 antibodies revealed low positive cpla2 protein levels in skeletal muscles, sebaceous glands and skin (epidermis, dermis) tissues of healthy paws. in the joints of the cia mice, large amounts of inflammatory infiltrate containing cpla2 were detected. in addition, robust cpla2 protein expression in the skeletal muscles surrounding the joints and strong cpla2 positive staining in sebaceous glands were detected. the high correlation between the severity of inflammation and the elevated cpla2 protein, due to an inflammatory infiltrate and increased cpla2 expression, suggests an important role of cpla2 in the development of arthritis. rheumatoid arthritis (ra) is the complex disease depending on environmental as well as genetic factors. in spite of the large research efforts we know in fact only small number of genes involved in this disease. animal models are useful tools for better understanding of the pathogenic mechanisms and genes leading to the disease process. the aim of the current project is to identify the genes and their functional role importance for arthritis. two loci associated with arthritis were identified using a cross between the b10.q (intermediate susceptible) and nod.q strains (resistant to arthritis). one on chromosome 2 a disease protective locus (cia2) and another on chromosome 1 a disease-promoting locus (cia9). nod.q allele at cia9 promotes arthritis whereas cia2, has a protective effect in contrast to the b10.q allele on chromosome 2. a promising candidate gene in cia2 locus is complement factor 5 (c5) as the nod.q allele produced defective c5 protein. cia9 locus contains several genes of potential importance for disease such as fc riib, fc riii, fc riv ncf2, fh. the results of cia (collagen induced arthritis) and caia (collagen antibody induced arthritis) experiments using the subcongenic mice generated that contains fc r region showed a significant difference in incidence and severity of arthritis. the disease is controlled in a recessive pattern. the fragment devoid of fc r region seems to be protective. the subcongenic for the cia2 locus has been generated recently which will be tested for cia and caia susceptibility. the results from these experiments will be discussed in detail. angela pizzolla, ka gelderman, r holmdahl ncf1 is a component of the nadph oxidase complex, which produces reactive oxygen species (ros) upon activation into phagosomes and extracellularly. polymorphisms in the ncf1 gene that impair the capacity to produce ros, enhance susceptibility of both mice and rats to arthritis. activation of autoreactive t cells drives arthritis development but neither ncf1 expression nor oxidative burst have been detected in t cells. we hypothesize that antigen presenting cells influence t cell activity by producing ros during antigen presentation. we aimed to clarify the role of ros produced by dendritic cells (dc) on t cell activation. dc were grown from bone marrow from ncf1 mutated and wildtype mice. we could show that ncf1 mutated dc proliferated and differentiated better, had higher expression levels of costimulatory molecules and mhc ii upon stimulation as compared to ncf1 wildtype dc. in addition, ncf1 mutated dc induced higher levels of il-2 production by hybridoma t cells. to analyze the role of ncf1 in dc on arthritis, mice were developed expressing functional ncf1 restricted to dc (b10.qdcn). these mice are characterized for burst, ncf1 expression and ability to present antigen. we published that immunization with myelin oligodendrocyte glycoprotein (mog) protein resulted in higher disease severity than immunization with mog peptide in ncf1 deficient mice. this suggests that ncf1 plays a role in the uptake and processing of posters inflamm. res., supplement 3 (2007) s423 antigens, probably by dc. this will be further investigated with the b10.qdcn mice using in vitro assays as well as in vivo models for arthritis and multiple sclerosis. purpose: macrophage migration inhibitory factor (mif) is a pro-inflammatory cytokine involved in both innate and adaptive immune responses. it is expressed in human ra synovial tissue and its suppression inhibits t or b cell dependent animal models of ra. we investigated the role of mif in k/bxn serum transfer arthritis. methods: arthritis was induced by injection of k/bxn serum on days 0 and 2 in littermate wt and mif-/-mice. arthritis was scored clinically, ankle thickness was measured using microcallipers, and joints collected for histology. sections were scored for synovitis, synovial fluid exudate, cartilage degradation and bone damage. results: wt mice exhibited arthritis as early as day 1 and 100% incidence was observed on day 7. mif -/-mice exhibited delayed arthritis, with onset on day 3 and 100% incidence on day 9. mif -/-mice exhibited significantly reduced disease severity as measured by clinical disease score ( methods: osteoarthritis was induced by bilateral transection of the medial meniscus in dunkin-hartley guinea pigs using minimal invasive surgery to avoid cartilage damage due to inflammation and/or intra-articular bleeding. results: the first signs of osteoarthritis development were macroscopically observed four weeks after meniscal transection. twelve weeks after surgery the lesions were still restricted to the medial side of the joint and did not reach into the subchondral bone. cartilage destruction due to meniscal transection was also histologically detected. however, biomarkers for cartilage destruction (ctx-ii, hp/lp ratio, comp) were not increased. treatments aiming at different processes in osteoarthritis, such as bone destruction (risedronate), inflammation (pioglitazone and anakinra), and cartilage destruction (galardin) were not effective in this model. the early degenerative changes in this transection model are probably too mild to be measured in the systemic circulation using classic biomarkers. further research into new biomarkers is needed to detect and monitor the early stages of osteoarthritis. the ineffectiveness of the compounds tested in this model underscores the urgent need for new strategies to treat the disease. the meniscal transection model might prove to be useful tool for identifying new biomarkers and treatments. livia l camargo (1), a denadai-souza (1), lm yshii (1), a schenka (2), ma barreto (1), d boletini-santos (3), c lima (3), v rioli (3), mn muscar (1), e ferro (1), sk costa (1) ( methods: aia was induced via intraarticular (i.a.) injection of methylated bovine serum albumin in immunized male rats. knee oedema and pain score were assessed daily in controls and animals treated with hemopressin (10 or 20 ã¬g/day; i.a.). histopathological changes, cell number and cytokines in the synovial fluid of aia rats were determined at day 4. results: aia rats developed a severe mono-arthritis characterized by a joint oedema and pain. at day 4, there was marked cellular infiltration, hyperplasia, pannus formation and destruction of bone and cartilage, but pro-inflammatory cytokines were undetectable by elisa. both doses of hemopressin significantly reduced the knee oedema, but only 20 mg of hemopressin attenuated the pain score. acute joint inflammation was significantly reduced by hemopressin, but failed to significantly affect chronic histopathological signs (hyperplasia, pannus etc.). conclusions: hemopressin has potential for treating acute signs of aia by reducing synovial plasma protein extravasation, alleviating pain and reducing acute joint histopathological changes, thus providing an alternative strategy for treatment of oedema and pain in arthritis. calcitriol, the hormonal metabolite of vitamin d and it synthetic analogs exert an anti-inflammatory action on psoriatic skin lesions while eliciting mild inflammation on healthy skin. the map kinase erk plays an important role in the induction of chemokines, cytokines and adhesion molecules in keratinocytes that maintain the epidermal inflammatory response.we hypothesized that the dual effect of calcitriol may be partially attributed to differential effects on erk activation in the presence or absence of inflammatory mediators. our experimental model was immortalized non-tumorigenic human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators. inflammation was mimicked by exposure to tnf. level and activation of signaling molecules were determined by immunoblotting. by using the specific egf receptor (egfr) tyrosine kinase inhibitor, ag1487, we established that tnf activates erk in an egfr-dependent and egfrindependent modes. the egfr-dependent activation resulted in the induction of the transcription factor, c-fos, while the egfr-independent activation was of a shorter duration and did not affect c-fos expression. treatment with calcitriol alone increased erk activation and c-fos induction. pretreatment with calcitriol enhanced egfr-dependent erk activation and tyrosine phosphorylation of the egfr, but completely abolished egfr-independent tnf-induced erk activation. pretreatment with calcitriol increased the rate of de-phosphorylation of activated erk, accounting for the inhibition of egfr-independent erk activation by tnf. it is possible that effects on the erk cascade underlie the dual action of calcitriol and its synthetic analogs on cutaneous inflammation. christina barja-fidalgo (1), r saldanha-gama (1), ja moraes (1), r zingali (2), c marcienkewicz (3) (1) universidade do estado do rio de janeiro, rio de janeiro, brazil (2) universidade federal do rio de janeiro, rio de janeiro, brazil (3) temple university, philadelphia, usa neutrophils adhere on vascular endothelium and directly migrate toward inflamed tissue to exert their primary defense function. integrin are receptors that drive cell adhesion and motility and interfere with cell activation, functions and survival.acting as both anchoring molecules and signaling receptor, transducing signals outsidein and inside-out, integrins are potential targets for therapeutic and diagnostic opportunities. disintegrins are a family of cystein-rich low-molecular weight peptides that usually contain an rgd sequence, a cell attachment site of ecm and cell surface proteins recognized by integrins. they are considered selective and competitive antagonists of integrins, being potent inhibitors of platelet aggregation and cell-cell/cell-ecm interactions. we reported that rgd-disintegrins, selectively interact with integrin amb2, a5b1 and/or avb3) on human neutrophils, interfering with cell functions through the activation of integrin-coupled intracellular signaling pathways. recently showed that, a selective ligand of a9/a4b1 integrins, vlo5, induces neutrophil chemotaxis, cytoskeleton mobilization and potently inhibiting neutrophil spontaneous apoptosis. these effects are mediated vlo5 interaction with a9b1 integrin, activating the focal adhesion cascade. vlo5 effects on the delay of neutrophil is modulated by pi3k, erk-2 mapk and nf-kb pathways that seems to interfere with the balance between anti-and pro-apoptotic bcl-2 family members and with mitochondrial membrane potential. data emphasize mechanistic details of the role of a9b1 integrins interactions on human neutrophils and support the use of disintegrins as prototypes to develop logical combinations of drugs to optimize or minimize the susceptibility of a selected target cell population to apoptosis during therapeutic interventions. (faperj, cnpq, capes, ifs-sweeden) (1), h serezani (2), m peters-golden(2), sonia jancar (1) '(1) university of sâ¼o paulo, brazil (2) university of michigan, usa it is been shown that leukotriene (lt) b4 and cysteinyl lt (ltc4, ltd4 and lte4) enhances fcr-mediated phagocytosis in alveolar macrophages (am), dependent on protein kinase c (pkc). in contrast, ltb4 but not cyslt effects are exclusive on syk activation. in the present study we investigated the role of specific pkc isoforms and its upstream and downstream targets involved in lt-enhanced fcr-mediated phagocytosis. to this purpose, ams were pretreated or not with inhibitors of pkc-d (rottlerin -6 um), pkc-a (ro-32-0432-9 nm), pi3k (ly290042-10 um and wortmannin-10nm), erk 1/ 2 (pd98059 -2 um), cpla2 (aacocf3-10 um), p38mapk (sb20190-10 um) and ca++ (bapta/am-10 um), before stimulation with ltb4 or ltd4 and addition of igg-opsonized red blood cells for 90 min. activation (phosphorylation) of signaling molecules by lts were analyzed by western blot. our results demonstrate that ltb4 -enhanced phagocytosis is dependent on pkc-a, while ltd4 effects are mediated by pkc-d. cell proliferation and differentiation, adhesion, cell activation and apoptosis. while galectin-1 mainly acts as an anti-inflammatory and pro-apoptotic molecule, galectin-3 is known as a strong pro-inflammatory and anti-apoptotic signal. we have recently recognized galectin-3 as a new molecular target of immunomodulatory drugs in monocyte/macrophage-like cells. in this study we investigated the effects of immunomodulatory drugs (aspirin, indomethacin, hydrocortisone and dexamethasone), applied in therapeutic ranges on the expression of galectin-1 at gene and protein level in monocytic thp-1 cells. we have also tested the effects of these drugs on both galectins in the cells activated by lipopolysaccharide from e. coli (lps). the targeted mrna level was evaluated using quantitative rt-pcr technique and the expression of both galectins in cell homogenates was determined by western-immunoblot analysis. the results showed that immunomodulatory drugs affected the expression of galectin-1 on both, gene and protein level, and that the effects were dependent on drug type and applied concentration as well as time of the exposure. the modulatory effects of the applied drugs on galectin-1 and -3 expressions were also observed in the cells activated by lps. these findings represent important step in the understanding of the effects of immunomodulatory drugs on galectin-1 and-3 expressions, as well as the role of these lectins in the physiology of monocytes. introduction: pancreatitis-associated protein (pap) has been recently described as an endogenous mechanism involved in the regulation of inflammation. in the present study, we show some of the molecular mechanisms implicated in the intracellular signaling pathways modulated by pap. the pancreatic human cell line panc1 was incubated with pap (500ng/ml) and/or tnfa (100ng/ml). total rna was obtained and the expression of tnfa was examined by rt-pcr. in addition, the effect of pap on nfkb activation was measured by inmunofluorescence in cells. western blot analysis was used to determine the expression of nfkb mediators: phosphorylated ikk, ikba and p65. results: we observed that pap administration to cells prevented nfkb translocation to the nucleus as well as the tnfa-induced tnfa gene expression. when tnfastimulated cells were treated with cycloheximide in order to block protein synthesis, the induction of tnfa gene expression was completely restored. on the other hand, pap had no effect on ikk phosphorylation or ikba degradation. conclusions:in this study we have provided evidence that pap modulates the inflammatory response by blocking nfkb translocation to the nucleus. this pap-induced nfkb inhibition requires a jak/stat-dependent de novo protein synthesis. objectives: this study investigated the inhibitory mechanism of hyaluronan (ha) on lipopolysaccharide (lps)stimulated production of proinflammatory cytokines in u937 macrophages. methods: ha was added to u937 macrophage cultures in the presence of lps, with or without pretreatment with anti-intercellular adhesion molecule-1 (icam-1) antibody. secreted levels of tumor necrosis factor a (tnfa), interleukin (il)-1b, and il-6 were determined by enzyme-linked immunosorbent assay. the phosphorylation of nuclear factor (nf)-kb, ikba, and mitogenactivated protein kinases (mapks) was analyzed by immunoblotting. results: lps stimulated production of tnfa, il-1b, and il-6. in contrast to 800 kda ha, 2700 kda ha at 1 mg/ ml inhibited lps-induced cytokine production. anti-icam-1 antibody blocked the effects of ha on the lps actions on u937 cells. lps activated nf-kb and mapk pathways, whereas ha down-regulated p65 nf-kb and ikba phosphorylation by lps without affecting mapks. inhibition studies revealed the requirement of nf-kb for lps-stimulated cytokine production. anti-icam-1 antibody reversed the inhibitory effects of ha on phosphorylation of p65 nf-kb and ikba. conclusions: ha of intrinsic molecular weight suppresses lps-stimulated production of proinflammatory cytokines via icam-1 through down-regulation of nf-kb and ikb. exogenous ha into arthritic joints could act as an anti-nf-kb agent by the mechanism demonstrated in the present study. the principal eicosanoid product of endothelial cox-2 is prostacyclin (pgi2), which is a potent vascular patency factor. induction of endothelial cox-2 under hypoxic conditions is well documented. this response, along with associated pgi2 release, is likely an important protective homeostatic response. in order to explore the role of candidate signalling agents in cox-2 expression in response to hypoxia, studies were undertaken using luciferase reporter constructs of the cox-2 promoter region in huvec. cox-2 induction under hypoxic conditions was confirmed with the wild type construct. strategic mutations of transcription factor binding sites showed that sites for hypoxia inducible factors (hifs) were more important for cox-2 expression than those for nfkb. furthermore, expression of cox-2 was increased under normoxic conditions by transfection of huvec with normoxia-stable hif mutants. emsa showed hif binding in nuclear extracts from untransfected hypoxic huvec. under these hypoxic conditions, increased release of pgi2 but not vaso-occlusive thromboxane a2 was seen. thus the putative protective induction of cox-2 in endothelial cells in response to hypoxia involves signalling by hifs. crescentic glomerulonephritis is characterized by crescent formation and rapid progress to renal failure, where the predominance of th1 immune response plays a crucial role. however, the therapeutic efficacy of the regulation of th1-predominant immune responses remains to be investigated. therefore, the effects of a th1 selective inhibitor tak-603 were investigated in a model of crescentic glomerulonephritis in wky rats. methods: tak-603 was administered orally, starting at the time of induction of glomerulonephritis. in group 1, the drug was administered daily for the initial 6 days. tak-603 was administered on day 0 only in group 2, and from day 3 to 5 in group 3. in each group, nephritic rats were killed on days 6 and 56. results: in group 1, glomerular damage, including crescent formation, was improved on day 6, with reductions in the numbers of cd4, cd8 and ed-1 positive cells, as well as in urinary protein excretion. protein and transcript levels of th1 cytokines in the diseased kidneys were markedly decreased by tak-603 treatment. renal pathology, including glomerulosclerosis and interstitial fibrosis, was ameliorated and proteinuria was markedly decreased. elevated levels of serum creatinine showed concomitant improvement. in group 3, in which treatment was initiated shortly after the appearance of glomerular abnormalities, glomerular damage was also diminished, resulting in a decrease in urinary protein excretion. treatment only on the first day in group 2, partially rescued renal dysfunction. conclusions: these results suggest that the initial inhibition of th1 immune response has an appealing therapeutic potential for crescentic glomerulonephritis. parasitic nematodes and their hosts are now known to produce a wide range of galectins. whilst host galectins have been shown to modulate the recruitment and effector function of inflammatory cells including mast cells, neutrophils and eosinophils, the role of secreted parasitic galectins is less well defined. studies at moredun have demonstrated that both the endoparasitic helminth, haemonchus contortus, and the ectoparasitic mite, psoroptes ovis, produce galectin-like factors which, in vitro, directly influence the migration and survival of eosinophils from their natural sheep host. excretory-secretory extracts from both parasites contained potent chemokinetic activity and were also able to promote eosinophil survival in the presence of dexamethasone. separation by affinity chromatography, as well as specific sugar inhibition and mass spectrometric profiling, revealed the active components to be galectins. in the case of h. contortus, there was homolgy with known est sequences, which allowed subsequent cloning and expression studies to be undertaken. a functional in vivo role for these parasitederived galectins awaits confirmation, but the possibility is raised that they could directly influence the host immune response following infection. this may have particular significance in mite infections in which exudates from the associated eosinophilc lesion appear s428 inflamm. res., supplement 3 (2007) posters to provide the primary nutrient source for their survival. moreover, the observation that two very different parasites may have evolved similar mechanisms for manipulating the host inflammatory response to their benefit, raises the further possibility that parasite galectins may provide potentially novel therapeutic targets. the aim of the study was to investigate the time course of cytokine gene expression in liver and lungs of mice with lipopolysaccharide (lps)-induced septic shock and to assess the effect of three different immunomodulatory agents on cytokine mrna levels in these tissues. male cd-1 mice were injected intraperitoneally with 50 mg/kg lps alone or concomitantly with an intravenous dose of pentoxifylline (100 mg/kg), lisofylline (100 mg/kg) or prednisolone (10 mg/kg). the tissues were harvested 0, 1, 4, 6, and 24 h following lps administration and stored at -808c. relative quantification of tumor necrosis factoralpha (tnf-alpha), interleukin-1beta (il-1beta), interleukin-6 (il-6), and interferon-gamma (ifn-gamma) mrna levels was performed by real-time rt-pcr. the highest levels of cytokine mrna were observed at 4 or 6 h after lps administration, whereas the expression of tnf-alpha and il-1beta in lungs and il-6 in liver reached the peak values at 1h and then decreased gradually. in addition, the lps effects on cytokine mrna were more pronounced in liver when compared to lungs. all administered compounds inhibited the lpsinduced tnf-alpha mrna expression (by up to approximately 50%), whereas lisofylline significantly increased ifn-gamma mrna levels in both tissues at most investigated time points. for other cytokines, the observed differences did not reached statistical significance. in conclusion, with the exception of ifn-gamma, the time course of cytokine mrnas differed considerably depending on the type of tissue. in the murine model of lps-induced septic shock, only tnf-alpha gene expression was suppressed by all compounds under investigation. maria sanz(1), m losada (1), c company (1), c lope-gines(1), l piqueras(2), j cortijo (3) the migration of leukocytes into inflamed tissues involves a cascade of molecular events finely regulated by cell adhesion molecules and chemokines. fractalkine/ cx3cl1 (fr) is a membrane-bound chemokine that functions as a mononuclear leukocyte chemoattractant and as an adhesion molecule. clinical studies and animal disease models have shown that fr is also involved in the pathogenesis atherosclerosis. we have demonstrated that angiotensin-ii (aii) has proinflammatory actions inducing the initial attachment of mononuclear cells to the arteriolar endothelium. in the present study we have investigated whether aii can cause the synthesis and expression of fr on human umbilical arterial endothelial cells (huaecs). huaecs were stimulated with ang-ii 1 microm or with tnfalpha (20 ng/ml) for 1, 4 and 24 h. fr was determined in the culture supernatants by conventional sandwich elisa. fr was only detected after 4 h and 24 h stimulation with tnfalphafnwhereas aii was unable to provoke the cleavage of the chemokine. semiquantitative rt-pcr analysis of huaecs showed increased fr mrna expression in aii-stimulated cells for 1 and 4 h. these effects were caused by the interaction of aii with its at1 receptor since they were abolished by losartan (at1 receptor antagonist). tnfalpha also increased fr mrna. immunohistochemical analysis of the cultured endothelial cells showed a clear expression of fr in huaecs stimulated with aii or tnfalpha for 4 and 24 h. these results suggest that fr could be a key chemokine in the selective adhesion of mononuclear leukocytes to the arterial endothelium elicited by aii. the lipophilic yeast malassezia is an exacerbating factor in atopic dermatitis (ad).among organisms of the malassezia species, m. globosa and m. restricta are particularly dominant on the skin of ad patients. our previous study has demonstrated that human keratinocytes responded to the two malassezia species with different th2-type cytokine profiles, i.e. m. globosa induced il-5, il-10, and il-13 secretion from the keratinocytes, whereas m. restricta induced il-4 secretion.these findings suggest that m. globosa and m. restricta play a synergistic role in triggering or exacerbating ad by stimulating the th2 immune response. pattern recognition receptors (prrs) of human keratinocytes play an important role in the induction of inflammatory and innate immune responses. in this study, we assessed the role of prrs for cytokine production by human keratinocytes in response to malassezia species. human keratinocytes were pretreated with various anti-prr monoclonal antibodies (mabs) and stimulated with m. globosa or m. restricta. cytokine secretion from keratinocytes was measured by using fast quant elisa kit. exposure of human keratinocytes to m. globosa and m. restricta resulted in enhanced secretion of il-5 and il-4, respectively. the m. globosainduced increase in il-5 secretion was inhibited by mabs against cd14 and cd91. in case of m. restricta, mabs against toll-like receptor 2 (tlr2) and cd14 suppressed significantly il-4 secretion from keratinocytes. these findings suggest that the distinct prrs interactions with fungal pathogen-associated molecular patterns (pamps) are key factors in differential cytokine secretion from keratinocytes stimulated with malassezia species. atopic dermatitis (ad) is a chronic, relapsing inflammatory skin disease associated with allergy. mdc (macrophage-derived chemokine/ccl22) and tarc (thymus and activation-regulated chemokine/ccl17) are th2type cytokines, and it has been reported that serum mdc and tarc levels are associated with ad disease. in present study, we investigated the effect of prunus yedoensis matsum barks on the inflammatory chemokines (mdc and tarc) and jak-stat pathway in hacat keratinocytes. as a result, etoac fraction and e5 sub-fraction inhibited the mrna expression and protein level of mdc and tarc in a dose-dependent manner. also, e5 sub-fraction showed inhibitory activity on the stat1 protein level. these results suggest that p. yedoensis may have an anti-atopic activity by suppressing the inflammatory chemokines (mdc & tarc). the il-1 family now consists of 11 members, most of which have assigned functions.there are 10 members of the il-1 receptor family (including the decoy receptor type ii il-1r).many of the il-1 family members possess neither a signal peptide nor an apparent prodomain, but nevertheless manage to exit the cell.the il-1 family members il-1f6, f8 and f9 signal through a complex of the il-1r family member rp2 in association with il-1r acp to activate common inflammatory pathways.the specific activity is is low, on the order of 1-10ug/ml ec50.we have found that removal of a few n-terminal amino acids from il-1f5, f6, f8 and f9 can increase their bioactivity approximately 1000-fold. the location of the n-terminus leading to increased specific activity is quite specific; removal of one more or one fewer amino acid eliminates the effect.in addition, n-terminally truncated il-1f5 is capable of antagonizing signaling via il-1rrp2, but full-length f5 is inactive. (1) university of ulsan, japan kyoto university, japan inflammation plays a pathogenic role in the development of obesity-related complications such as type ii diabetes and atherosclerosis. tumor necrosis factor alpha (tnfa) is closely associated with the enhanced inflammatory responses in obesity and the obesity-related pathologies. tr2 (hvem/tnfrsf14), which is a member of the tnf receptor superfamily and the receptor for lymphotoxins-related inducible ligand that competes for glycoprotein d binding to herpesvirus entry mediator on t cells (light/tnfsf14), is a potent mediator of inflammatory responses. the purpose of this study is to examine the hypothesis that obesity-induced inflammatory responses can be attenuated by inhibiting tr2 pathway. c57bl/6 tr2 knockout mice and their wild-type control were fed a high-fat diet for 12 weeks and the obesity phenotypes were determined in the obese tr2 knockout mice and the control. the obese tr2 mice fed a high-fat diet elicited the attenuation of body weight gain and insulin resistance relative to wild-type control mice. expression levels of inflammatory genes significantly decreased in the adipose tissue of the obese tr2 knockout mice compared with those of the control. our results demonstrate that obesity-induced inflammatory responses and insulin resistance can be attenuated in obese tr2 knockout mice fed a high-fat diet. objectives: the present study was undertaken to investigate the role of insulin on allergic airway inflammation. methods: diabetic male wistar rats (alloxan, 42 mg/kg, i.v.) and controls were sensitized with ova (100 ã¬g) and al(oh)3 (8 mg, s.c.) 10 days after alloxan or saline injection. the animals were challenged 14 days later by the intratracheal instillation of ova (1 mg/0.4 ml). the following analyses were performed 6 h thereafter: (a) total and differential cell counts in bronchoalveolar lavage (bal) fluid; (b) quantification of tnf-alpha and il-1 beta in the bal by elisa; and (c) immunohistochemistry for p-and e-selectins on lung vessels. results: compared to the control animals, diabetic rats exhibited reduced number of neutrophils (90%) and mononuclear cells (50%); reduced levels of tnf-alpha (45%) and il-1 beta (30%), and reduced p-selectin expression (30%) in response to ova challenge. these abnormalities were corrected after treatment of diabetic rats with a single dose of nph insulin (4 iu, s.c.), 2 h before ova challenge. although we did not find differences in e-selectin expression between diabetic rats and controls, expression of this molecule was amplified by insulin. conclusions: data presented show that insulin controls neutrophils migration during allergic airway inflammatory possibly by modulation of tnf-alpha and il-1 beta production and selectin expression. supported by fapesp and pronex. hormonally active vitamin d derivatives are beneficial in the treatment of cutaneous inflammatory disorders, particularly in psoriasis. their anti-inflammatory effect is usually attributed to inhibition of the activity of infiltrating immune cells.we examined whether vitamin d also interferes with the pro-inflammatory action of the keratinocytes themselves. human hacat keratinocytes cultured in the absence of exogenous growth factors or active mediators were exposed to tnf to simulate an inflammatory challenge and their response was monitored by assessing mrna levels of the cytokine tnf, the chemokine il-8 and the adhesion molecule icam-1 by real-time pcr. icam1 and il-8 were induced rapidly peaking after 2h, their mrna levels increased again from 8h to reach a plateau between 16h to 24h after exposure to tnf, whereas tnf mrna levels increased steadily between 2h and 24h. 24h pretreatment with calcitriol, the hormonal form of vitamin d, inhibited induction of il-8 but did not affect that of icam-1 or tnf 2h following exposure while calcitriol markedly inhibited the induction of all 3 pro-inflammatory genes 16h after the tnf challenge.calcitriol inhibits the activation of jun kinase (jnk) and p38 by tnf. this action was mimicked by the posters inflamm. res., supplement 3 (2007) s431 jnk inhibitor sp600125 and the p38 inhibitor sb203580.the combination of the two inhibitors fully reproduced the time and gene dependent modulatory effect of calcitriol. we conclude that vitamin d attenuates the active contribution of keratinocytes to cutaneous inflammation and that this modulatory effect is explained by inhibition of the jnk and p38 cascades. (1), cm lotufo (2), p borelli (1), zs ferreira (2), rp markus (2), shp farsky (1) (1) department of clinical and toxicological analyses, school of pharmaceutical sciences, university of sâ¼o paulo, brazil (2) department of physiology, bioscience institute,university of sâ¼o paulo, braziil introduction: we showed that endogenous glucocorticoids (eg) control neutrophil mobilizations from the bone marrow and peripheral compartment by modulating the expression of l-selectin on segmented cells. aims: we evaluated the role of eg on endothelial cells (ec) and the molecular mechanisms responsible for hormonal actions in neutrophils and ec. methods: neutrophils were collected from blood, segmented leukocytes from femoral bone marrow and ec were cultured from testis vessels. cells were obtained from adrenalectomized (adx), ru 38486-treated, shamoperated, vehicle-treated and non-manipulated (nm) wistar rats. results: circulating neutrophils and segmented cells from ru 38486-treated rats presented elevated and decreased expressions of l-selectin vs cells from control animals, respectively. the effects were not dependent on alterations of l-selectin mrna levels. ec from adx animals presented more ability to adhere neutrophils from nm rats and enhanced mrna levels and membrane expressions of icam-1, vcam-1 and pecam-1. participation of the glucocorticoid cytosolic receptor(gcr) on these effects was shown by similar results in cells from ru 38486 treated rats. nfkappab translocation in neutrophils was equivalent in all groups of animals, but it was enhanced in ec from adx or ru 38486-treated rats. conclusions: data show the participation of the gcr on events involved in neutrophil mobilizations, but nfkappab transcription is only involved on ec. (1), y naito(2), t okuda (3), k mizushima (3), t okayama (3), i hirata (3), h tsuboi (3), t suzuki (3), o handa (1) background: despite the inhalation of co at high concentrations had been considered as a toxic gas, the inhalation of co at low concentration has recently been shown the cytoprotective and anti-inflammatory effect against various animal models. however, it is unclear whether the direct exposure of co to the intestinal inflamed mucosa is effective or not. in this study, we investigated the therapeutic efficacy of the rectal co administration for rat colitis model. acute colitis was induced with trinitrobenzene sulfonic acid (tnbs) in male wistar rats. co(200ppm-10ml) was intrarectally administrated twice a day after the induction of colitis. rats were sacrificed at 3 days after the administration of tnbs. the distal colon was removed, and the ulcer lesions were measured. thiobarbituric acid (tba)-reactive substances and tissueassociated myeloperoxidase (mpo) activity were measured in the colonic mucosa as indices of lipid peroxidation and neutrophil infiltration, respectively. moreover, we evaluated the expressions of cinc-1 mrna/protein and p-p38 mapk protein. the intracolonic administration of co ameliorated tnbs-induced colonic ulceration. the increases in tba-reactive substances and mpo activity after tnbs administration were both significantly inhibited by treatment with co. moreover, the rectal administration of co significantly inhibited the increased expression of cinc-1 mrna/protein and p-p38 mapk protein after the induction of tnbs-induced colitis. the rectal administration of co protected from the intestinal inflammation in rats. based on these data, the beneficial effects of co on the intestinal mucosal injury may be attributed to its anti-inflammatory properties. alessandra gambero (1), m marã³stica (1), m saad(2), j pedrazzoli jr (1) (1) sâ¼o francisco university medical school, brazil (2) state university of campinas, brazil recent studies have shown that adipocytes produce and secrete several bioactive molecules like adipocytokines. the adipose tissue can also present short and long-term changes during inflammation and infectious pathologies. in this study, the alterations of mesenteric and perinodal mesenteric adipose tissue during experimental colitis induced by repeated intracolonic tnbs instillations were evaluated. the adipocyte size was measured after collagenase digestion. the basal lipolysis (glycerol release) and adipocytokine production was monitored after short time culture of adipose tissue. the colitis animals showed higher mesenteric fat mass (1.02+-0.06 and 0.78+-0.09 % of body weight for colitis and control, respectively; p<0.05) in despite of the lower body weight. the mesenteric adipocytes from colitis animals presented reduced diameter (31.67+-0.28 and 35.69+-0.37 um for colitis and control, respectively; p<0.01), higher basal lipolysis (41.67+-6.89 and 18.77+-3.66 ug.mg-1 for colitis and control, respectively; p<0.05) and tnf-alpha production (8.64+-0.77 and 5.51+-0.86 ng.mg-1 for colitis and control, respectively; p<0.05). perinodal mesenteric adipocytes presented normal diameters, higher basal lipolysis (72.48+-15.38 and 25.96.77+-3.05 ug.mg-1 for colitis and control, respectively; p<0.05), increased tnfalpha (46.34+-5.82 and 15.71+-2.15 ng.ml-1 for colitis and control, respectively; p<0.01), leptin (400.41+-77.10 and 201.01+-52.01 pg.ml-1 for colitis and control, respectively; p<0.05) and adiponectin production (15029+-2340 and 5918+-498 ng.ml-1 for colitis and control, respectively; p<0.01). the mesenteric adipose tissue was modified during the experimental inflammation, but some alterations were site specific. perinodal adipose tissue retained the ability to produce anti-inflammatory and pro-inflammatory cytokines, while mesenteric adipose tissue only the pro-inflammatory one. this work was financially supported by fapesp. inflammatory bowel disease (ibd) is a group of chronic inflammatory disorders of the intestine. the role of the pro-inflammatory p38 mapk signalling cascade in the pathogenesis of ibd is highly controversial. we therefore aimed to investigate the role of p38 mapk in chronic dextran sodium sulfate (dss) induced colitis, an experimental model of ibd. chronic intestinal inflammation was induced by oral cyclic administration of 3 % dss in sjl mice. clinically, the dss treatment produced episodes of colitis manifested by diarrhoea, gross intestinal bleeding, marked loss of body weight, and shortening of the colon. at the molecular level, this was accompanied by an up-regulation of tnfa, il-1ã¢, il-6, il-17, kc, cox-2, igg heavy chain, and phospho-stat3 in the dss treated mice.the clinical and molecular features described above recapitulate findings reported in human ibd. in order to assess the role of p38 mapk, the activation of the p38 mapk signalling cascade was analysed by western blot analysis. the expression and phosphorylation levels of both p38 mapk and of mk2 and hsp27, two down-stream targets, were not increased in dss treated animals compared to controls. leo 15520, a potent inhibitor of p38 activity in vivo, was dosed as pretreatment and after completion of dss treatment. pretreatment had a deteriorating effect on all measured cytokines, whereas treatment after disease induction had no effect on any measured parameters. collectively, these results strongly suggest that the p38 kinase pathway only plays a minor role, if any, in the dss model. (sp) were gmcsf differentiated, dcs purified through gr1+ cell depletion, and spleen tcells isolated by pan tcell negative selection. spdcs or bmdcs were stimulated +/-100ng/ml lps. for mlr, balb/c tcells were added for 5 days. cells were incubated with sb203580 (4-(4-fluorophenyl)-2-(4-ethylsulfinylphenyl)-5-(4-pyridyl)1h-imidazole, sb) or ml3403 ((rs)-{4-[5-(4-fluorophenyl)-2-methylsulfanyl-3h-imidazol-4-yl]pyridine-2-yl}-(1 -phenylethyl)amine, ml) and washed prior to lps stimulation (bmdcs) or cell mixing (t cells). 3hthymidine incorporation was measured, cell viability by mtt assay, tnfa and il-12 production by elisa. mlr tcell proliferation inspdcs or bmdcs was inhibited by sb (ic50 spdc 0.06mm, bmdc 1.0mm) and ml (ic50 spdc 0.03mm, bmdc 0.03mm). preincubation with dcs had no effect, despite reduced lps stimulated il-12 and tnfa synthesis by sb (ic50 il-12 1.2mm, tnf 0.16mm) and ml (ic50 il-12 0.9mm, tnf 0.11mm). preliminary data shows that preincubation of t cells with sb and ml modifies the mlr response. p38 plays a role in the interaction of dcs and t cells in antigen recognition. however, pre-incubation of drugs with dcs was ineffective. the role of t cell p38 mapk in the mlr is under investigation. p38 inhibitors may possess disease modifying properties because of reduced tcell antigen reactivity to dc antigen presentation. (1), s luik(2), s laufer(2), m seed (3), v holan(4), s fiorucci (5) (1) synovo gmbh, tã¼bingen, germany (2) university of tã¼bingen, germany in vivo anti-inflammatory activity of certain p38 kinase inhibitors is limited by bioavailability. however, it is possible that they may be useful in the therapy of ibd should it be possible to mediate there uptake in and around bowel lesions. we reasoned that activity could be especially increased if drug physical properties were altered to allow preferential partition into macrophages and neutrophils (wbc) associated with lesions. we prepared prodrugs of p38 inhibitors and screened them using whole human blood, murine spleenocytes and peritoneal macrophage. pharmacologically inert macrocycle (azilide) conjugates were assessed for enhanced efficacy in murine collagen induced arthritis either therapeutically (after onset of signs) or prophylactically (2 d post boost) or in a dss or tnbs model of ibd in the mouse. in both types of models, the prodrugs achieved improved suppression of arthritis and inflammatory score in colon sections at tolerated doses with optimal activity at 15mmolkg-1d-1. we propose that the prodrugs increase efficacy via improved pharmacokinetics partly related to biased disposition of the prodrug toward immune cells. despite the potent anti-inflammatory and immunosuppressive properties of glucocorticoids its applying in the management of severe necrotizing pancreatitis is still controversial. the plasma levels of interleukins (il-8, il-18) and adhesion molecules (e-selectin and icam-1) were measured in 36 patients with necrotizing pancreatitis. the measurement was performed immediately after admission, at the 3, 7, and 14 day. all patients were divided on two groups: first group compiled 20 patients, in which dexamethasone (16 mg/day during 7-8 days) was applied in the complex management of acute pancreatitis, and control group -16 patients that did not receive corticosteroids. all patients received the initial therapy. the increased levels of il-6, il-8, il-18, icam-1, and eselectin were noted in both groups of patients at the time of admission. the gradually increase of all proinflammatory mediators plasma levels up to seventh day was noted in patients of the control group. its levels clear correlated with the severity of mods and spreading of necrotic processes confirmed by ct. starting from the third day the gradually decrease of mediators levels were noted in the patients of the first group. the incidence of contamination of necrotic foci had no difference in both groups of patients. the ability of glucocorticoids to inhibit expression of proinflammatory mediators due to the glucocorticoids-mediated repression of nf-kappa b pathway provide the pathogenetic substantiation for the applying of glucocorticoids in the complex management of necrotizing pancreatitis. the objective of this study was to examine whether t cell specific overexpression of the th2 transcription factor gata-3 can inhibit th1/th17 cell mediated experimental mbsa arthritis. mbsa-immunized wild type mice developed joint inflammation which gradually increased in time with a maximum at day 7. at day 1, t cell specific gata-3 tg mice did not show any difference in arthritis score compared to wild type mice. however, at day 7, wild type mice had developed severe joint inflammation having the maximum arthritis score. in contrast, gata-3 tg mice showed only mild joint inflammation, suggesting that t cell specific overexpression of gata-3 protects against development of severe joint inflammation. facs analysis revealed low levels of il-17 +/ifn-gammacells in wild type as well as in gata-3 tg mice at day 1. as expected, il-4 positive cells were higher in gata-3 tg mice compared with wild type mice. interestingly, at day 7, the percentage of il-17 +/ ifn-gamma-cells were markedly increased in wild type mice but not in gata-3 tg mice, suggesting prevention of th17 expansion under gata-3 overexpression in vivo. these data revealed that t cell specific overexpression of the th2 transcription factor gata-3 protects against progression of severe joint inflammation during mbsainduced arthritis. furthermore, il-17 +/ifn-gammacells play a critical role in the progression of joint inflammation in this model and gata-3 overexpression in t cells prevents expansion of the il-17 +/ifn-gamma-t cell subset. pingping jiang (1), pt sangild(2), t thymann (2), hh-y ngai (1), w-h sit (1), k-l chan (3), jm-f wan (1) ( necrotizing enterocolitis (nec) is a severe intestinal inflammatory disease for which the disease etiology and progression remain unclear. preterm delivery and enteral milk formula feeding are factors predisposing to nec. to understand the pathophysiology of nec, two-dimensional gel electrophoresis (2d page) proteomic approach was applied in studying changes in intestinal protein pattern in preterm piglets with spontaneous nec occurring in response to 2 days of parenteral feeding followed by 1 day of enteral formula feeding. the intestinal proteomes of pigs with clinical symptoms of nec (n = 3) were compared with corresponding pigs remained healthy (n = 4). syproruby staining was used and differently expressed proteins were identified by maldi-tof ms or maldi-tof/tof ms. the proteins with significantly different expression between nec and healthy pigs involve in energy metabolism (sorbitol dehydrogenase, mitochondrial aldehyde dehydrogenase 2 and chain a, medium-chain acyl-coa dehydrogenase with 3-thiaoctanoyl-coa etc.), inflammation (peptide-binding protein 74 (pbp74) and snail homolog 3), signal transduction proteins (thyroid hormone binding protein precursor, park7 protein and chain b, structure of the rho family gtp-binding protein cdc42 in complex with the multifunctional regulator rhogdi etc.) and anti-oxidation (manganese-containing superoxide dismutase(sod)). these data underscore the significant impact of intestinal proteomics in unraveling nec pathophysiology and biomarker discovery. blood are used to monitor the progression of inflammation. the aim of our study were to investigate systemic markers of disease in a rat model of lps induced pulmonary inflammation to provide a link between preclinical in vivo research and early clinical research. animals were exposed to bacterial lipopolysacharide (e.coli 026:b6) by inhalation. the lungs were lavaged 6 hours post provocation and the level of cell influx was determined. relevant mediators of acute pulmonary inflammation were analysed with standard elisa technology in bronchoalveolar lavage fluid and in blood. in addition, measurement of changes in body temperature were performed at different time-points post provocation in order to monitor the systemic inflammatory responses to the local pulmonary inflammation manifested as alteration in body-temperature. results showing the effects of lps challenge on local and systemic parameters will be presented and the possible link to lps responses in man discussed. pulmonary inflammation models are widely used in pharmacological research. however, provocations and treatments aimed directly at the lung are often invasive which limits the possibility to perform repeated administrations of test agents and compounds. also, results derived from bronchioalvelar (bal) fluid are subject to variability if the retrieval techniques are non standardized. here we describe a non-invasive standardized method for retrieval of bal fluid to be used in mice. we present the characterisation of these techniques using the inflammatory response to lps and propose that this non invasive method can be used to refine lps and other challenge models. the objectives were to evaluate the dynamic response after a single intra-tracheal administration of of 5mg (50ml/animal) of lps (p.aeruginosa) to c57bl/6j mice. control animals received a single dose of sterile 50ml 0.9% saline/animal. the mice were terminated 4, 24, 48, 72 and 96h after instillation using a non invasive and operator independent lavage technique. results showing the effects of single lps challenge on bal parameters, excised lung gas volume and lung weight will be presented showing reliable dynamic responses. these techniques open the possibility to run repeated treatments and chronic provocations and are not subject to variability from bal fluid retrieval. the human psoriasis xenograft scid mouse model is one of the most accepted and well characterized models for screening of novel anti-psoriatic compounds. the model has primarily been applied for testing novel treatment principles via systemic or intradermal administration routes. in order to evaluate the model for topical treatment, psoriatic keratome biopsies were grafted to immune-deficient scid mice. transplanted mice were treated with daivonex /dovobet (calcipotriol) and bms (betamethasone). the results show a strong antipsoriatic efficacy after treatment with bms (epidermal thickness reduced by 68%). treatment with daivonex / dovobet also showed an anti psoriatic effect with a 31% reduction in epidermal thickness. serum did not contain test compounds, indicating that the observed effect were not due to systemic exposure. the observed effects are in concordance with clinical results of treatment of psoriasis. it is concluded that the model is useful for testing topical treatments. we have demonstrated that adult rats offspring of dams submitted to protein restriction during early lactation, presented impaired acute immune responses probably related to an imbalance in glucocorticoids and insulin secretion (barja-fidalgo; inflamm res 52 (11): 2003) . here, we evaluated the innate immunity mediated by neutrophils and host defense against infection in adult rats offspring of dams fed with either a protein free diet (un-group) or 22% protein diet (c-group) during the first 10 days of lactation. un rats showed lower number of blood pmn, though no difference in bone-marrow neutrophils number was observed. blood neutrophils from un-group presented a significantly reduced phagocytic activity against opsonized zymosan, constitutively expressed inos and spontaneously produced o2-, no and tnf-alpha. in vivo treatment with lps, at non-lethal doses, significantly increases tnf-alpha and superoxide production by neutrophils, compared with controls. lps increased no production by neutrophils from both groups, inducing inos expression in control cells, but no further increase in inos expression in un rats. nucleare nf-kb is constitutively augmented in un rats. un animals presented a higher survival rate in a model of clp-induced severe sepsis. these results indicate that a metabolic programming induced during early lactation affects the innate immune responses in adult rats, which are unable to properly mount an inflammatory response, may predispose to chronic diseases in adult life. transgenic mice over-expressing vascular endothelial growth factor (vegf) in the epidermal basal layer under the human keratin 14 (k14) promoter have previously been reported to develop a psoriasis-like inflammatory condition in the skin. important hallmarks of psoriasis are epidermal hyperplasia in association with infiltration of t-cells in the dermis and epidermis and also increased dermal angiogenesis. the aim of this study was to describe the epidermal hyperplasia and the infiltration of the skin with t-cells in transgenic k14/vegf mice. we induced a cutaneous inflammation in the ear skin by repeated topical treatments with 12-o-tetradecanoylphorbol-13-acetate (tpa), in order to investigate the inflammatory response. the in vivo pharmacological effect of topical treatment with a number of reference compounds, including betamethasone-17-valerate, was also investigated. the ear thickness was significantly increased in transgenic animals compared to wild type animals following tpa-induction. the epidermal thickness measured in histological sections of biopsies from the ear skin was also significantly increased in transgenic animals. furthermore, increased dermal vascularisation was observed in the histological sections of the ear skin. a marked infiltration with cd3-positive cells was observed in both dermis and epidermis, and this was highly correlated with the increase in epidermal thickness. finally, topical treatment with betamethasone-17-valerate significantly reduced the ear swelling and epidermal thickness. we conclude that over-expression of vegf in the epidermal basal layer plays an important role in skin inflammation and for the development of important psoriatic hallmarks. the model may furthermore be used as an in vivo screening tool for novel anti-psoriatic compounds. background and aims: the diabetes-prone bb (bbdp) rat spontaneously develops insulin-dependent diabetes resembling type 1 diabetes (t1d) in man. the bbdp rat is t-cell lymphopenic with a profound lack of regulatory t cells. the recent thymic emigrants in bbdp rapidly undergo apoptosis unless rescued from apoptosis by tcr stimulation. the increase in apoptosis is due to a frameshift mutation in gimap5 which causes a severe truncation of the protein. the mutation is the strongest genetic factor for rat t1d. we aim to detect how gimap5 affects the lifespan of t cells. results: overexpression in c58 cells of both wt gimap5 and gimap5 with the bbdp mutation causes an increase in apoptosis -the latter with a very rapid onset. reduction of human gimap5 by rna-interference in jurkat cells did not affect the number of apoptotic cells. overexpression of human gimap5 causes apoptosis in jurkat cells and primary naã¯ve t cells but not in activated t cells. finally, gimap5-mrna is upregulated in in vitro activated human primary t-cells (detected by rt-pcr). conclusions: based on the phenotype of the bbdp, rat gimap5 was expected to be anti-apoptotic. however, we report here that overexpression of both mutated and wt gimap5 causes rapid death of the cells. this suggests that gimap5 is pro-apoptotic. the results with human wt gimap5 support this conclusion: recently, much focus has been on the cellular cd38/ cadpr signaling system during inflammatory processes. the cd38/cadpr system has been shown to be regulated by interferon, estrogen and the proinflammatory cytokine il-8. to our knowledge, the expression and function of the cd38/cadpr signaling system in the human detrusor muscle have not been described. cd38 protein expression in cultured (explant technique) human detrusor smooth muscle cells (hdsmc) was demonstrated by western blot (wb) and confocal microscopy (cm). cytosolic free ca2+ concentration ([ca2+] i) in hdsmc and isometric force in human detrusor strips were measured by spectrofluorometry and myograph technique, respectively. wb and cm showed that hdsmc expressed cell surface cd38 which could be upregulated by il-8 (20 ng/ml). in hdsmc briefly activated with il-8 (20 ng/ml) cadpr induced a rapid, transient dose-dependent increase in [ca2+]i. cyclic adpr-mediated ca2+ increase was greatly reduced in ca2+ free medium suggesting ca2+ entry as well as ca2+ release. cyclic adpr -elicited ca2+ increase was mimicked by 3-deaza-cadpr, and blocked by 8-bromo-cadpr, a cadpr antagonist, but not by nifedipine or verapamil. in the presence of il-8, cadpr caused concentration-dependent relaxations of detrusor muscle. we report for the first time that 1) hdsmc express cell surface cd38, 2) the expression and function of cd38 are augmented by il-8, 3) externally added cadpr elicited a rapid, il-8 -dependent, and 8-bromo-cadpr-inhibitable ca2+ mobilization, 4) cadpr induces relaxation of human detrusor muscle. the study indicates a role of cd38/cadpr in human urinary bladder inflammation. miao lin is a formulation of sen miao san and lingzhi that consists of cortex phellodendri, atractylodisa rhizoma, radix achyranthis bidentatae, and ganoderma lucidum. these ingredients are reported to have anti-inflammatory and analgesic effects. in this study, we have investigated the anti-arthritic property of miao lin in an animal model of arthritis induced by unilateral injection of freunds complete adjuvant (fca) into rat knees. contents of the miao lin capsules were dissolved in saline and administered to the rats daily by intraperitoneal or oral route for 7 days before induction of arthritis and 7 days after. extension angle, size and blood flow of the rat knee joints were measured to give indexes of algesia, oedema, and hyperaemia, respectively. assessments of the extent of cell infiltration, tissue proliferation, and erosions of cartilage and bone provided additional indexes of the arthritis condition. single unilateral injection of fca into rat knees produced significant oedema, algesia, hyperaemia, immune cell infiltration, synovial tissue proliferation, and erosions of cartilage and bone in the ipsilateral knees compared with the contralateral saline-injected knees. intra-peritoneal injection of miao lin (50 mg/kg/day) suppressed oedema, pain and hyperaemia in the inflamed knees, and oral administration (500 mg/kg/day) suppressed oedema and hyperaemia. histological examination showed that both routes of administrations of miao lin reduced immune cell infiltration and erosions of cartilage and bone, and intraperitoneally administered miao lin also attenuated synovial tissue proliferation. these findings suggest treatment with intra-peritoneal or oral miao lin could provide significant anti-arthritic effects. an extract of the anti-arthritic thermalife cream contains 13 trace elements. diffusion studies were undertaken to assess the permeability of human epidermis to the trace elements. non-penetrating trace elements were discarded from the test formula (t2), and compared with the original formula (t1) for in vitro anti-inflammatory efficacy (tnf-a secretion in lps-challenged human monocytes). methods: human epidermis was mounted in vertical franz type diffusion cells (stratum corneum facing up). t1 cream (n=4) or no cream (n=4) was applied to the donor compartment of diffusion cells, with pbs in the receptor compartment (3.0ml ; stirred continuously at 37 c). 240 min after administration the receptor fluid was analysed for presence of metal ions by icp-ms. a replication study used a different skin donor. subsequently, human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4,) or not in the presence of 10% t1, 10% t2, or no treatment.24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). statistical analyses used a treat by lps anova (p < 0.05). results: zinc was the only trace element to penetrate the human epidermis significantly. both formulations strongly suppressed lps-induced tnf-a secretion. t2 with zinc only was more effective than t1 (treat:f2,12 = 57.13, p < 0.0001; lps:f1,12 = 245.47, p < 0.0001; treat by lps:f2,12 = 70.01, p < 0.0001). conclusions: anti-tnf efficacy from thermalife extracts was retained with zinc chloride as the only trace element. (1) (1) osprey pharmaceuticals limited, canada (2) probetex, inc., texas, usa the ccl2/ccr2 chemokine/receptor axis, infiltrating monocytes/macrophages (m/m), th1 cells and mast cells play a pathological role in tissue damage and fibrosis in kidney diseases. the eradication of the supernumerary activated leukocytes should curb the production of inflammatory mediators and modulate chemokine communications, thus ameliorating disease. a recombinant fusion protein comprised of the human ccl2 chemokine fused to a truncated form of the enzymatically active a1 domain of shiga toxin has been developed. the ccl2 portion binds specifically to ccr2-bearing leukocytes and enters the cells, where the sa1 portion inhibits protein synthesis. the compound was tested in a model of anti-thymocyte serum (ats)-induced mesangioproliferative glomerulonephritis. male rats were injected with ats on day 0 and treated intravenously with vehicle, 50 or 100mg/kg of the recombinant protein q2d from day 2 until day 8. urine and blood collections were made prior to ats injection and on days 5 and 9. animals were sacrificed on day 9. no treatment related effects on body weight or signs of clinical toxicity were observed. urine protein levels were decreased in treated animals. histopathological analyses of kidney sections revealed maximum reductions of 40, 36, 38, and 28% for glomerular lesions, m/m count, fibronectin and âµ-smooth muscle actin, respectively. the latter two proteins are markers for extracellular matrix synthesis and mesangial cell activation, respectively. these results indicate a significant renal-protective effect in this model of nephritis. further observations suggest that different chemokine-ligand toxins may be used in the treatment of diseases modulated by other chemokine/receptor axes. inflamm. res., supplement 3 (2007) posters immuno-depletion followed by fluorescence-activated cell sorting based on the cell surface expression of the sca-1 antigen. such isolated cells can subsequently be cultured and differentiate towards the osteogenic, adipogenic or chondrogenic linage in vitro. using this model we investigated the influence of the proinflammatory cytokines, tnfa or il-1b, on early osteogenesis in vitro. under osteogenic conditions, il-1b was found to inhibit cell proliferation in a dose dependent manner, whereas tnfa exhibited no effect. histochemical examination revealed the presence of either tnfa or il-1b to dramatically decreased mineralization in a dose dependent manner. q-pcr analysis indicated that in the presence of il-1b, despite increased expression of bone-specific alkaline phosphatase (akp2) mrna, levels of other osteogenesis markers (runx2, col1a and sp7) were decreased. in the presence of tnfa, levels of akp2, runx2 and sp7 were all decreased. our findings indicate that the influence of early mesenchymal progenitor cells on bone remodelling may be substantially altered in the presence of proinflammatory cytokines. coronary artery disease (cad) is characterized by enrichment of inflammatory cells in the vessel wall. we hypothesized that an altered transmigration and activation of monocytes may contribute to plaque build up. in vivo transmigration was studied by use of a skin blister model. blisters are raised by suction and cells are analysed the following morning (0h blister) and after additional ten hours of incubation with pbs or autologous serum, corresponding to intermediate and intense blister. monocytes were analysed by flow cytometry for the expression of cd11b, before and after in vitro fmlp stimulation. chemokines in serum and blister fluid was analysed in parallel. cd11b expression on resting monocytes harvested from 0h blister was lower in patients as compared to controls (p=0.001). lower expression of cd11b in patients was also observed in the intermediate and intense blisters after stimulation with fmlp (p=0.04 and p= 0.005, respectively). the number of transmigrated cells was similar in both groups and increased with the intensity of inflammation. serum concentration of mip-1âµ was higher among patients (p=0.01) and similar levels were seen in blister fluids. concentration of mcp-1 was similar in both serum and blister fluid. we demonstrate that monocytes from patients with cad have a reduced expression and ability to up-regulate the adhesion molecule cd11b at sites of inflammation. these differences may modulate events related to the transmigration process and indicate a changed activation pattern. to which extent this feature might contribute to monocyte entrapment at the atherosclerotic site needs further studies to be delineated. in inflammation, nitric oxide (no) is produced by inducible nitric oxide synthase (inos) induced by bacterial products and cytokines, and no acts as a regulatory and proinflammatory mediator. one of the anti-inflammatory mechanisms of glucocorticoids is the inhibition of no production. the aim of the present study was to investigate the mechanisms how glucocorticoids inhibit inos expression and no production. dexamethasone and a dissociated glucocorticoid ru24858 inhibited no production, and inos protein and mrna expression in murine j774 macrophages exposed to bacterial lipopolysaccharide (lps). in the presence of a glucocorticoid receptor (gr) antagonist mifepristone, the effects of dexamethasone and ru24858 on no production were reduced. the role of histone deacetylation in the glucocorticoid effect was studied by using three inhibitors of histone deacetylases (hdacs); non-selective trichostatin a and apicidin, and hdac1 selective mc1293. hdac inhibitors reversed the effects of dexamethasone and ru24858 on inos expression or no production. stably transfected a549/8 cells containing human inos promoter were used in promoter-activity studies. cytokine-induced inos promoter activity was inhibited by dexamethasone and the inhibitory effect was reversed by trichostatin a. these results suggest that glucocorticoids inhibit inos expression and no production by a gr-mediated and gre-independent manner possibly through histone deacetylation and transcriptional silencing. we are investigating mechanisms involved in tnfainduced hyperalgesia in the mouse paw. previously, we have seen that tnfa causes a trpv1-dependent bilateral hyperalgesia. here we investigate the role of cox in this process. female cd1 mice (25-30g) were given intraplantar injections (i.pl.) of tnfa (10pmol/50microl) and tyrode (as vehicle, contralateral paw; 50microl). thermal hyperalgesic thresholds were measured using the hargreaves technique before and 1h after injection. indomethacin (20mg/kg) was co-injected with tnfa whilst contralateral paw was injected with tyrode and corresponding amounts of indomethacin vehicle (5% nahco3). another group of mice were injected with tnfa i.pl. plus 5% nahco3 with the contralateral paw injected with tyrode plus indomethacin (20mg/kg). results are expressed as mean ae s.e.m and statistical analysis performed using students t-test. tnfa (10pmol) leads to significantly reduced (p<0.05 compared to baseline values) paw withdrawal latency in both paws 1h after injection i.e bilateral hyperalgesia. however, local injection of indomethacin (20mg/kg) with tnfa prevented this reduction in paw withdrawal latency in both paws suggesting that prostaglandins are important in the development of hyperalgesia. interestingly, indomethacin co-injected with tyrode in the contralateral paw did not prevent the reduction in paw withdrawal latency in both paws. the same results were seen using the selective cox-2 inhibitor, nimesulide. in conclusion, cox-2 derived prostaglandins are important in the development of hyperalgesia. local cox-2 inhibition at the site of tnfa-induced inflammation prevents the bilateral hyperalgesia suggesting that local prostaglandin production is sufficient to cause hyperalgesia in the contralateral paw. hydrogen sulfide (h2s) is synthesized naturally in the body from cysteine by cystathionine g lyase (cse). h2s has been variously reported to exhibit both pro-and antiinflammatory activity. in an attempt to obtain further information about the role of h2s in inflammation we examined the effect of dexamethasone on lipopolysaccharide (lps)-mediated endotoxic shock. male sprague dawley rats (240-280g) were administered dexamathasone (1 mg/kg, i.p.) either 1 h before or 1 h after lps (4 mg/kg, i.p.) injection. animals were killed 3 h after lps administration and plasma and tissues harvested. as expected, lps injection significantly increased plasma tnfa and il-1b as well as liver and lung myeloperoxidase (mpo) activity. lps also increased plasma nitrate/ nitrite (nox), h2s concentration and liver and kidney h2s synthesis from exogenous cysteine indicative of upregulation of cse in these tissues. either pre-or post treatment of animals with dexamethasone reduced signs of inflammation and also reduced the increase in plasma h2s and tissue h2s synthesizing activity. in separate in vitro experiments, exposure of rat peripheral leucocytes to lps (100 ng/ml, 3 h, 37oc) resulted in upregulation of both cse and inos (measured by western blot). dexamethasone (100 nm) significantly (p<0.05) reduced expression of both cse and inos. these data provide further evidence that h2s is synthesised during endotoxic shock and suggest, for the first time, that at least part of the anti-inflammatory effect of dexamethsone may be related to inhibition of h2s production. (1), u jalonen (1), h kankaanranta (1), r tuominen(2), e moilanen (1) (1) the immunopharmacology research group, medical school, university of tampere and tampere university hospital, tampere, finland (2) the division of pharmacology and toxicology, faculty of pharmacy, university of helsinki, helsinki, finland tristetraprolin (ttp), also known as nup475, tis11, g0s24 and zfp36, is a factor that binds to 3utr of mrna of some transiently expressed inflammatory genes and regulates mrna stability. ttp has been implicated in the posttranscriptional regulation of e.g. tumor necrosis factor a and inducible nitric oxide synthase. however, the regulation of the expression of ttp itself is largely unknown. in the present study, we investigated the role of classical protein kinase c (cpkc) isoenzymes in the regulation of ttp expression. in j774 macrophages ttp expression is induced by lipopolysaccharide (lps) and this can be further enhanced by addition of 100 nm phorbol myristate acetate (pma). this additive effect of pma on ttp was abolished by a prolonged preincubation with a higher s442 inflamm. res., supplement 3 (2007) posters concentration of pma for 24 h, which also downregulated the expression of pkca, pkcbi and pkcbii isoenzymes. pkc inhibitors ro318220 (inhibits pkcb, & and e), gã�6976 (inhibits pkca, b and &) and cgp53353 (inhibits pkcbii) reduced lps + pma -induced ttp protein and ttp mrna expression. pkcbii inhibitor cgp53353 did not affect ttp mrna half-life and therefore we measured the effects of cgp53353 on the activation of transcription factors involved in ttp expression. cgp53353 had no effect on the activation of nf-kb, egr1 or sp1. in contrast, cgp53353 reduced the activation of transcription factor ap-2, which may explain its inhibitory action on ttp expression. the results suggest that pkcbii is involved in the regulation of ttp expression in activated macrophages, possibly through the activation of transcription factor ap-2. the most widespread gracilaria verrucosa in the sea of korea is the attached form of red algae growing on a rockly substrate. in this study, we isolated fourteen compounds from g. verrucosa and investigated their inhibitory effect on the production of inflammatory markers (tnf-a il-6, il-1 and no) in raw264.7 cells. among them, 10-oxooctadec-8-enoic acid and 11-oxooctadec-9-enoic acid inhibited the production of tnf-a, il-6, il-1 and no at the concentration of 20 mg. also, these two compounds showed inhibitory activity on the mrna expression and protein level of inflammatory markers (tnf-a il-6, il-1 and inos) in a dose-dependent manner. these results suggest that g. verrucosa may have anti-inflammatory activity through the inhibition of inflammatory cytokines and inos. lene jensen(1), p hjarnaa (1), j fensholdt (1), p-p elena(2), k abell (1), tk petersen (1) (1) discovery, leo pharma, ballerup, denmark (2) iris pharma, la gaude, france angiogenesis is known to play an important role in many inflammatory diseases including arthritis. additionally, inflammation is known to play a role in the angiogenesisdriven disease age-related macular degeneration (amd). we have synthesized a potent angiogenesis inhibitor, leo-a, targeting kinases related to angiogenesis, e.g. vegfr-2. additionally, leo-a has potent effects on a broad panel of other kinases, whose normal functions are related to inflammation and immunity. the compound was tested systemically in inflammatory in vivo models in mice and rats. the in vivo models selected include the cia arthritis model (mice and rats), the local gvh rat model, the lps induced tnfa model (mice and rats), the anti-cd3 induced il-2 response mouse model and the rat argon laser-induced choroidal neovascularisation (chnv) model, a model for amd. the following results were obtained after systemic treatment with doses of up to 30 mg/kg i.p. or 50 mg/kg p.o. once daily: in the local gvh model, leo-a significantly inhibited the growth of the local lymph node by 50 %. in the cia model, leo-a had a significant inhibitory effect on the progress of arthritis both in mice and in rats when dosed early (pretreatment). in the lps induced tnfa model in mice, high doses of leo-a were found to inhibit the tnfa release. in the chnv model, a significant effect was obtained following systemic treatment. in conclusion, leo-a has an interesting profile for the treatment of diseases in which inflammation and angiogenesis are involved. mice lacking pi3kg and d isoforms display severe impairment of thymocyte development, but the outcome of this developmental defect has not been investigated. we show here that mice harbor pi3kg gene depletion and pi3kd kinase-inactive mutation, pik3cgd koi, exhibited thymus atrophy, similar to previously reported pi3kg and d double knockout (p110g/d-/-) mice, and profound peripheral lymphoid depletion, markedly reduced lamda chain production and seemingly lymphopenia-provoked effector/memory t cell activity. in particular, serum igg1/ igg2a ratio and ige level were elevated in pik3cgd koi mice corresponding to a skewed th2 profile in vitro. histological analysis revealed eosionophil-and t celldominated inflammation in stomach and salivary gland as well as occasionally other organs of pik3cgd koi mice, but organ-specific auto-antibody was not detected in circulation. on the contrary, when mature wt t cells were treated with pi3k d or together with pi3k g selective inhibitors, while th1 cytokines were suppressed th2 cytokines were not augmented in vitro. thus, t cell development, but not peripheral t cell proliferation or cytokine production, requires cooperativity of pi3kg and d. genetic inactivation of these two isoforms leads to the development of severe lymphopenia, skewed type 2 ig and t cell response, and increased susceptibility to eosinophilic multiple organ inflammation; whereas pharmacological inhibition at the adult stage would probably not promote th2 reaction but attenuate th1 medicated disorders. platelet activating factor (paf) is an important mediator in several pathophysiological processes. paf receptor activation can causes a series of cellular and tissue modifications and can lead to the production and/or release of diverse molecules, including cytokines, chemokines and receptors, amongst others, which are capable of amplifying the inflammation. paf can up-regulate kinin b1 receptor expression by various mechanisms. our aim was to investigate the role for kinases in paf-induced kinin b1 receptor up-regulation. wistar rats were treated with paf, or left untreated as controls, 6h before i.d. injection of 0.1ml pbs containing des-arg9-bradykinin (dapk, 100nmol right hind paw) and 0.1ml pbs (for control, left paw). various kinase inhibitors were administered to the rats after paf treatment and oedema was measured by the use of a plethysmometer (ugo basile) 10-120 minutes after dapk-injection. oedema was expressed in ml as difference between right and left paws.additionally paw samples were taken for western blot analysis for total and phosphorylated forms of jnk and erk1/2. dabk-induced paw oedema after pafinjection is significantly inhibited by the selective jnk sp600125 and erk1/2 pd98059 inhibitors. western blot analysis shows that phosphorylation of jnk and erk1/2 is important in the up-regulation of b1 receptors. our results clearly show that the phosphorylation of both erk1/2 and jnk mapkinases is an important step for the in vivo up-regulation of b1 receptors by paf. however, the exact mechanisms (transcriptional and post-transcriptional) by which paf can trigger kinase phosphorylation and then up-regulate the b1 receptor require further investigation. continued interest in development of small molecule inhibitors of p38 mitogen-activated protein (map) kinase is based on the central role this enzyme plays in inflammatory cell signaling. activation of p38 leads to increase production of pro-inflammatory cytokines such as tnf-a and il-1b making it an prominent target for antiinflammatory drug discovery. a virtuell screening approach identified 3-(2-chlorophenyl)-6-((4-methoxyphenoxy)methyl)[1, 2, 4] triazolo [3,4-b] [1, 3, 4] thi adiazole as a potential hit. this was confirmed by synthesis and testing. to explore further sar, a first set of derivates was prepared by cyclization of the 5-substituted-4-amino-3-mercapto-4h-1,2,4-triazoles with carboxylic acids in presence of phosphorus oxychloride. the synthetic strategy used allows both variation at position 3 and 6. synthesis and sar will be presented. cytokines like il-1b and tnfa play central roles in inflammatory diseases like rheumatoid arthritis. production of cytokines in monocytes, macrophages and other cells is triggered by factors such as lps, uv-light, osmotic and cellular stress or physical and chemical attraction. in particular il-1b and tnfa are key regulators as they amplify inflammatory stimuli in cells by induction and upregulation of further cytokines. involved in this signal pathway, p38mapk as a pivotal enzyme is considered to be a validated drug target and therefore, p38mapkinhibitors are of therapeutical interest. in this study, we developed and validated an economic in vivo whole blood assay for optimization and characterization of small molecule p38mapk-inhibitors with promising in vitro activity. the assay procedure involves defined blood cell stimulation by lps and isolation of tnfa or il-1b, which are subsequently quantified by tmb-elisa technique via photometric measurement. the validation of the assay conditions involved well characterized p38mapk inhibitor sb203580 and a highly active compound developed in our lab. a data set was generated by determining 18 whole blood samples consisting of in each case three male and female individuals on three different days. statistical methods were used to analyze specificity, baseline-peak correlations, repeatability, robustness as well as gender specific intra-and interindividual differences. p38 mitogen-activated protein (map) kinase is required for the biosynthesis and release of pro-inflammatory cytokines il-1 and tnf a. inhibition of p38 map kinase could reduce the expression of these cytokines and is therefore a promising target for the treatment of many inflammatory disorders, like rheumatoid arthritis and inflammatory bowel disease. trisubstituted pyridinylimidazoles are potent inhibitors of the p38 map kinase. scope of this work was to investigate 2-thio-ether moiety as a position to link the inhibitors to macrocyclic drug carriers. we synthesised 2-alkylsulfanyl, 4-(4-fluorophenyl), 5-(2-aminopyridin-4-yl) substituted imidazoles as p38 map kinase inhibitors. as substitution at this pyridinyl moiety allows both increase the anti-inflammatory activity as well as selectivity. the synthesis and biological testing of effective the 2-aminopyridin-4-yl imidazoles with low inhibitory concentrations are described. biological data demonstrate both the imidazole derviates and the linked imidazoles lead to highly efficient inhibitors.variation at the 2-thio-ether moietywhich interacts in the phosphate binding region of the enzyme -with polar groups shows no loss of activity. studies underscored the importance of hydrogen bonding with the backbone nh group of met109, for inhibitory activity. less clear is the importance of the hydrogen bond between n3 of the imidazole ring and lys53 of p38 map kinase.to investigate the role of lys53 in interacting with the scaffold we prepared two sets of 4,5diaryl-substituted isoxazoles. these data suggest a dynamic interaction of the core heterocycle with lys53, contrary to the observation on the compound vk-19911 and p38 map kinase, that a nitrogen atom bearing a lone pair in position 3 of the imidazole ring could be necessary to avoid a repulsive interaction with the positively charged side chain of lys53 rather than to form an attractive interaction with p38 map kinase. to complete our study, we focused on the interdependency of biological effects exerted by substitution at the pyridine ring for a series of 3-substituted and unsubstituted 4,5-diarylisoxazoles investigating the interaction with the hydrophobic pocket ii of p38. these data indicate that the isoxazole has better scaffold properties compared with imidazoles, suggesting that heterocycles that are stable as regioisomers, such as isoxazole (in contrast to tautomeric imidazoles), are worthy of further investigations. despite of the intensive research effort, sepsis is still the leading cause of death in critically ill patients. it is a consequence of acute inflammatory response to lipopolysaccharide (lps), a major component of the outer membrane of gram-negative bacteria. natural products are known sources of bioactive components exerting antioxidative and anti-inflammatory effects. in this study, we investigated the effect of ferulaldehyde (fa), a natural compound of red wine, on lps-induced endotoxic shock in mice and on lps-stimulated murine macrophage-like raw 264.7 cells. treatment of c57bl/6 mice with fa significantly attenuated the lps-induced inflammatory response in the gastrointestinal tract, and decreased the level of the two major pro-inflammatory cytokines tnf-a and il-1b in the serum. the serum level of the anti-inflammatory cytokine il-10 was higher in mice treated with fa and lps compared to lps treatment alone. lps-induced phosphorylation and thereby activation of akt, and jnk was also strongly inhibited by fa treatment whereas the phosphorylation level of erk1/ 2 and p38 mapks remained unaltered. activation of nuclear factor-kappab (nf-kb) in liver of fa-treated mice were significantly suppressed. although fa had no effect on the production of inflammatory cytokines, or on inhibition of signal transduction pathways in raw 264.7 cells either, it decreased the lps-induced ros and nitrite production in a dose-dependent manner. our results suggest that fa has antioxidative and anti-inflammatory activities by enhancing antioxidative defense systems, which in turn decrease inflammatory cytokine response and suppress nf-kb activity via the down-regulation of akt and jnk. myeloperoxidase (mpo), stored in the azurophilic granules of the neutrophil granulocyte, is a heme enzyme with the unique property of oxidising chloride ion to the powerful reactant hypochlorous acid in the presence of hydrogen peroxide. therefore, it plays an important role at inflammatory loci in killing invading micro-organisms. on the other hand, hypochlorous acid reacts with a variety of biomolecules as amino acids or membrane lipids and causes therefore host tissue damage resulting in widespread diseases like atherosclerosis or rheumatoid arthritis, e.g. the formation of chloramines from taurine or ammonium ions is one possibility to reduce tissue toxicity while maintaining bactericidal properties. membrane charge alterations during apoptosis provide docking sites for the kationic enzyme myeloperoxidase and this close contact to the membrane lipids opens the possibility for lipid alteration pathways even though these reactions will normally not take place because of their slowness. we investigated alterations in phospholipids after reaction with hypochlorous acid or the myeloperoxidase-hydrogen peroxide-chloride system by matrixassisted laser desorption and ionisation time-of-flight mass spectrometry (maldi tof ms). specific reaction products play an important role in the modulation of the immune response. comparative pathobiology of the disease is also discussed within the context of current human and animal reoviral disease models. objectives: to study the safety and efficacy of infliximab plus leflunomide combination therapy in adult rheumatoid arthritis (ra). methods: twenty patients with active ra received leflunomide 100 mg for 3 days followed by 20 mg daily for 32 weeks. at week 2 all patients started infliximab 3 mg/kg, and received a further four infusions at weeks 4, 8, 16 and 24. results: the commonest adverse event was pruritis associated with an eczematous rash. there was no relationship between the serum concentration of a77 1726, the active metabolite of leflunomide, and adverse events. the mean disease activity score (das28) fell from 7.18 at week 0 to 5.18 (p<0.0001) at week 4 and remained between 3.85 and 4.85 up to week 32. in those patients remaining on treatment, more than 80% achieved an acr20 response from week 8 to week 28, and up to 46% achieved an acr70 response. conclusions: infliximab plus leflunomide combination therapy appears to be highly efficacious in the treatment of adult ra. however, widespread use may be limited by adverse events, which were common and in some cases severe. objective: the transcription factor nuclear factor-kb (nfkb) regulates the expression of proinflammatory cytokines such as tnfa and il-1 those play pivotal roles in pathogenesis in rheumatoid arthritis. parthenolide, a sesquiterpene lactone, was reported to inhibit the dna-binding of nfkb. the objective of this study is to investigate the potential of parathenolid to inhibit the pathogenesis of collagen-induced arthritis. methods: mice were injected i.p. with a cocktail of 4 anticollagentype ii mabs on day 0, followed by i.p. injection of lps on day 3 to induce anti-collagen mab-induced arthritis. the mice were orally administrated with parathenolide (50 mg/kg/day) starting on the day of first immunization (day 0) in prophylactic treatment group and after the onset of arthritis (day 4) in the therapeutic treatment group. clinical disease score, radiographic and histological scores were evaluated. mrna expression of il-1b and tnfa in the affected joints were measured by real-time pcr. results: clinical disease scores were significantly reduced both in prophylactic treatment group (7.4ae3.7) and therapeutic treatment group (7.25ae3.1) compared to untreated group (13.6ae2.7, p = 0.0163 and p = 0.0084 respectively). histological scores of joint destruction were significantly reduced in prophylactic treatment group compared to untreated mice (p<0.05). steady state mrna levels of il-1b and tnfa in isolated joints were significantly decreased in prophylactic treatment group compared to untreated mice (p<0.05). the results in this study suggest that nfkb is an important therapeutic molecular target for treatment of inflammatory arthritis. fibrinogen is a soluble plasma glycoprotein, multifunctional, that participates in haemostasis and has adhesive and inflammatory functions through specific interactions with other cells. the concentration of this glycoprotein increase in inflammatory conditions. a fundamental paradigm involved in the acute inflammatory response is neutrophil migration to the affected tissues to mount an initial innate response to the aggression. the objective of this study is to characterize how fibrinogen modulates the pattern of neutrophil activation. neutrophils from healthy donors were isolated from peripheral venous blood and loaded with the fluorescent probe dihydrorhodamine 123 (1ã¬m) to detect oxygen free radical production. the cells (1,0x 106 cell/ml) were then incubated with a range of concentrations of fibrinogen (0-400mg/dl) for 15 minutes. our results show that posters inflamm. res., supplement 3 (2007) s449 fibrinogen leads to an increase in neutrophil activation as measured by free radical production. this effect becomes evident at borderline-high concentrations (300-400mg/ dl), and in some of the individuals it was possible to differentiate two subpopulations of low-responsive and high responsive neutrophils to activation by fibrinogen. we hypothesize that, in this regard, the concentrations of fibrinogen identified as a risk factor might promote the setting of an inflammatory microenvironment in the circulation and facilitate cardiovascular disease progression. cyclooxygenases (cox-1 and cox-2) are isoenzymes involved in the first steps of the biosynthesis of prostanoids. the constitutively expressed isoform cox-1 is mainly involved in homeostatic processes, while the inducible isoform (cox-2) is associated with inflammatory reactions. various in vitro assays have been developed in order to define the selectivity against cox-1 and cox-2 of nonsteroidal anti-inflammatory drugs (nsaids). however, these in vitro assays can give discordant results related to several parameters. the aim of this study was to optimize and standardize two distinct in vitro methodologies to evaluate new nsaid candidates. first, in an enzymatic cox assay, the arachidonic acid concentration (aa; cox substrate), and the species of cox enzymes tested (ovine vs. human), two factors able to conceal the anti-cox activity of nsaids, have been evaluated and optimized. next, we developed an in vitro cell-based assay using human whole blood depleted from plasma and reconstituted in saline solution. this cell-based assay allows concomitant measurement of anti-cox-1 and anti-cox-2 effects by prostaglandin e2 (pge2) measurement after a23187 (calcimycin) and bacterial lipopolysaccharide (lps) stimulations, respectively. both assays have been calibrated and compared by testing 7 reference nsaids, selective or not for cox-1 or cox-2. fifty % inhibitory concentration (ic50) values against cox-1 and cox-2 and cox-2:cox-1 ratios obtained were in accordance with the previously described nsaid specificity and coherent between both assays (r= 0.93). in conclusion, both in vitro assays are optimized to determine the efficiency and the selectivity of new nsaid candidates against human cox-1 and cox-2. for increasing the success of preclinical drug candidate molecules, there is a need for translatable animal models. the human serum skin chamber technique and the rodent carrageenan induced air pouch model are two wellestablished methods for measuring interstitial inflammation in respective species. we aimed to study the translational aspects of these models. material and methods: in humans, epidermal skin chambers were stimulated with autologous serum for 10 hours. in rats, a dorsal subcutaneous air pouch was stimulated with autologous serum on day 6. the inflammatory response was measured after 4, 8 and 24 hours. the cellular distribution of in vivo transmigrated cells, the expression of cytokine receptors, adhesion molecules and inflammatory mediators was investigated. results: at 8/10 hours the cellular distribution was similar in air pouch and skin chambers. the major population constituted of granulocytes, followed by monocytes/macrophages and lymphocytes. both in human and rats the concentrations of mpo and mcp-1 were increased. furthermore, transmigrated cells displayed a different chemokine receptor pattern. in rats transmigrated cells expressed cd11b, were cd45lo, ssclo and rp-1+ (granulocyte marker). in humans, transmigrated granulocytes expressed cd16 and cd11b. these cells had a significantly higher cd11b expression compared to corresponding cells in peripheral circulation. our results indicate that the serum induced human skin chamber technique and rodent air pouch model translate well to each other. these models may be useful for bridgingpreclinical and clinical drug discovery. furthermore, they may work as translatable proof of mechanism (pom) models for drug candidates targeting different inflammatory components. objectives: to analyse if neopterin (a by-product of activated macrophage metabolism) is elevated in patients with systemic inflammatory insult at the time of ischemic stroke. material and methods: we investigated 86 consecutive patients with mean age 67ae7.8 years who were admitted within 24 h after ischemic stroke. a control group of 37 patients with mean age 58ae4.9 years without ischemic stroke was also tested. measurement of serum neopterin levels were performed using enzyme linked immunosorbent assay. results: patients with acute ischemic stroke had significantly higher serum levels (mean value+sd) of neopterin than those without acute ischemic stroke: 9.6ae1.2 and 7ae0.8 nmol/l. correlation analysis revealed p<0.01. discussion: immune mechanisms contribute to cerebral ischemic injury. the finding of higher serum levels of neopterin, which is regarded as a humoral component of the immune-mediated inflammatory response sustains the hypothesis that patients with ischemic stroke may show higher levels of inflammatory markers like neopterin. our results indicate increased macrophage activation after ischemic stroke. in patients with stroke it has been shown that neopterin was determinant of endothelium-dependent vascular dysfunction. however, these preliminary results need be confirmed by controlled studies. produced a marked (p<0.01) reduction in the number and duration of ventricular tachycardia (vt) during both ischemic and reperfusion phases. the total number of ischemic ventricular ectopic beats (vebs) reduced from 667ffae116 in the control to 66ffae22 at the concentration of 50 ffmg/ml (p<0.001). in the ischemic phase, cynodon dactylon (50 ffmg/ml) also decreased the incidence of vt from 100% (control) to 33%. in addition, incidences of reperfusion-induced vt, total vf and reversible vf duration were significantly lowered by the same concentration (p<0.01 for all). the results show that cynodon dactylon has a protective effect against i/r-induced cardiac arrhythmias in isolated rat hearts. regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their potential role in the scavenging of oxygen free radicals, it seems that the cardioprotective effects of cynodon dactylon probably is due to its anti-inflammatory properties.key words: cynodon dactylon; arrhythmias; anti-inflammatory; isolated heart; rat objectives: intestinal ischemia-reperfusion (iri) is well known to be associated with distant organ dysfunction; but no evidences to date have focused either the brain or skeletal muscle. we thus decided to investigate the effects of iri on nos and cox isoforms, neutrophil infiltration (mpo), lipoperoxidation (tbars) and protein tyrosine nitration (nt) in different brain areas and diaphragm muscle of wistar rats. methods: iri comprised the occlusion of superior mesenteric artery during 45 min followed by 2 h of reperfusion. sham animals were submitted to the surgical procedure with no interference on the blood flow. results: iri resulted in increased expression of mrna for nnos (cortex) and cox-2 (hypothalamus) associated to a marked reduction of ca2+-dependent nos activity in cortex, hypothalamus and hippocampus (but not in cerebellum). tbars contents were also reduced in cortex and hypothalamus. neither mpo activity nor nt was altered by iri in the brain. diaphragms from animals with iri exhibited increased mpo and ca2+-dependent nos activities, as well as tbars content and nt. in contrast, enos protein expression and both gene and protein nnos expression were reduced. no effects were observed on cox isoforms or enos gene expression. conclusions: these findings suggest that, within the first 2 h of reperfusion following intestinal ischemia, an oxidative response is observed in diaphragm, involving both lipid and protein modifications. in the cns, distinctive susceptibility to the iri seems to occur in the different areas, probably as a defensive strategy aimed to counteract the iri-mediated systemic injury. anne-sofie johansson (1), h qui (2), m wang (2), i vedin (1), jz haeggstrã§m (2), j palmblad (1) ( (1), r carnuccio(2), p romagnoli (3), f rossi (1) (1) second university of naples, italy (2) university of naples, italy (3) university of florence, italy we previously found that several inflammatory markers, e.g., nuclear factor-kb (nf-kb), were increased and a neointima was formed in a model of carotid surgical injury (1) . the purpose of the present study was to determine if chronic treatment with rosiglitazone protects rat carotid artery from surgical injury induced by an incision of the vascular wall. to this aim we measured cox-2, nf-kb, platelet aggregation and neointima formation in rats administered rosiglitazone (10 mg/kg/ die, by gavage) for 7 days before carotid injury and 21 days after injury. control rats received physiological solution. 14 days after injury cox-2 expression, evaluated by western blot, was significantly lower in the treated carotid versus controls (p<0.0001). rosiglitazone also caused a significant decrease of nf-kb/dna binding activity, evaluated by electrophoretic mobility shift assay, in nuclear extracts of treated carotids at all time points considered. platelet aggregation was reduced by 30% in treated versus control carotids (p<0.0005). the influx of inflammatory cells in response to injury, monitored by electron microscopy and immunohistochemistry, was lower in treated than in control carotids starting 7 days after rosiglitazone treatment. the results indicate that rosiglitazone inhibits molecular and cellular inflammatory events induced by vascular injury. the aim of the present study was to investigate the relevance of peripheral macrophage activity for the susceptibility to the induction of experimental allergic encephalomyelitis (eae). rats of eae-susceptible dark agouti and eae-resistant albino oxford strain were immunized with guinea pig spinal cord homogenate (dagpsc and aogpsc), while non-immunized rats served as controls (danim, aonim). on day 15 after immunization rat peritoneal macrophages were tested for adherence capacity, zymosan phagocytosis and respiratory burst. macrophages from aonim rats exhibited lower adherence capacity and higher phagocytosis and h2o2 production then macrophages from danim rats. immunization decreased adherence and phagocytosis and increased h2o2 production in macrophages from ao rats, but did not influence these activities in macrophages from da rats. our results suggest that inflammatory activities of macrophages from ao rats could be considered as regulatory mechanisms connected with the resistance to eae induction ( (1), b sehnert (2), h lanig(2), s pã¤ã�ler(2), r holmdahl (3), h burkhardt (1) (1) johann wolfgang goethe university, frankfurt, germany (2) friedrich-alexander university of erlangen-nuremberg, germany (3) lund university, sweden objectives: the aim of the present study was to characterize the interaction sites between the prototypic arthritogenic murine igg mab ciic1that is highly somatically mutated and its epitope on type ii collagen (cii, aa359-369). methods: the establishment of a dynamic simulation modelling of a ciic1 single-chain fragment (scfv) in complex with the triple helical ciic1 epitope permitted structural insights into immune complex formation. the computer-based data were experimentally tested by mutations of predicted critical residues into alanine in the c1scfvs and the respective ciic1 epitope that were produced as recombinant constructs. the binding affinities of the mutated scfvs were determined by elisa and surface plasmon resonance measurements. the mutation experiments confirmed the predicted interaction sites of cii in the cdr2 and cdr3 regions of both heavy andlight chain. surprinsingly also the model prediction, that the conversion of the c1scfv sequence into the respective germline does not affect cii binding affinity (kd 3x 10-8) could be confirmed experimentally by the mutagenesis of 13(!) positions. our data indicate that potentially harmful cartilage specific humoral autoimmunity is germline encoded. the molecular modeling further demonstrate that the rigid collagen triple helix restricts the likelihood of molecular interactions with the corresponding cdrregions of the antibody considerably compared to globular antigens. these sterical constraints might provide an explanation why somatic mutations have no obvious impact on cii recognition by the arthritogenic autoantibody. moreover, the structural insights into cii-autoantibody interaction might be useful in future developments of collagenomimetic ligands for therapeutic and diagnosistic purposes. we observed a significant association between the mbp-elicited cd4+ t-cell proliferation and active brain lesions, on the one hand, and il-4, il-5 and ifn-gamma, on the other. when grown in the presence of standard serum from a healthy donor, pbmc from healthy individuals responded to mbp with a higher il-10 production than pbmc from ms patients. thus, normal pbmc respond to mbp with production of tnf-alpha, ifn-gamma and il-10, but ms is associated with enhanced tnf-alpha-, ifn-gammaand decreased il-10 responses, and disease activity is associated with mbp-induced proliferation of cd4+ t cells. (1), k goula (1), p georgakopoulos (2) (1) renal unit, st. anrdew hospital, patras, greece (2) intensive care unit, st. anrdew hospital, patras, greece background: urethritis is an infection of the urethra. most cases are sexually transmitted. haemodialysedpeople seem more prone to all kinds of urinary tract infectionsthan others. patients with underlying diabetes are also a specific population at risk. urethritis may be caused by some sexually transmitted diseases (chlamydia, gonorrhea, and ureaplasma urealyticum infections) and by the same organisms that cause urinary tract infections (e. coli or klebsiella). viral causes of urethritis include herpes simplex virus and cytomegalovirus. neisseria gonorrhoeae and c trachomatis account for most cases of urethritis in men (70%). the aim of our study was to determine all cases of urethritis of haemodialysed patients at our unit during the last five years. we also determined diabetes as a coexisting factor in the infected patients. we retrospectively reviewed all cases of urethritis of 72 maintenance haemodialysis patients at our center over the past 5 years. the diagnosis was made according to patients symptoms and signs but also using urine specimens for culture.12 patients (16.6%) from the study group were diabetic. results: 4 cases of urethritis were determined. all infected patients were diabetic. isolated microorganisms were e. coli (2 cases), enterobacter aerogenes (1 case objectives: to explore the ability to use paquinimod as a steroid sparing drug in an animal model for sle. methods: mice were initially treated with a high dose of prednisolone (2 mg/kg/day). thereafter the amount of steroid was reduced to 0.5 mg/kg/day and a low dose of paquinimod (0.2 mg/kg/day) was added. the development of glomerulonephritis was measured as hematuria during the experimental period. serum was collected for analysis of anti-dsdna antibodies. kidneys were collected and histopathological observations were performed. organ weight and lymphocyte sub-populations were assessed in the spleen. results: when treatment with high dose prednisolone was replaced by low dose prednisolone andpaquinimod a steroid sparing effect was seen in a number of variables. a significant reduction in the level of hematuria, in spleen enlargement and in the total number of cd4+, cd8+ and on cd4-cd8-t cells was observed in mice treated with paquinimod and low dose of prednisolone compared to mice treated with high dose prednisolone alone. the development of glomerulonephritis was also significantly reduced in these mice. an almost complete inhibition of anti-dsdna in serum was seen in all treated groups. conclusions: when high dose prednisolone was replaced by low dose prednisolone and paquinimod a steroid sparing effect was seen when a number of variables e.g., hematuria, t-cell sub-populations and development of glomerulonephritis were examined. this setting could be of great importance in future treatment of human sle in order to reduce the steroid dose needed in the treatment of this disease. and la(ssb). the purpose of this study was to screen for novel antibodies against cell surface antigens in primary sjs. proteins (mp) were isolated from cell membranes (hela cells), and were tested with sera from sjs patients or healthy blood donors individually in western blot (wb) at 1:100. mp were separated on 2-d gels and tested in wb (1:100) to locate the appropriate spots for mass spectrometry (ms) analysis. paraformaldehyde fixed hela cells were incubated with sera from patients or blood donors and examined by fluorescence microscopy. antigens were isolated at around 35, 50, 75, 100 kda (64 total positive/79 tested patients). the dominant antigen was at 50 kda. large quantities of endogenous proteins were obtained and the membrane fraction was enriched. one of the main obstacles to further study possible surface antigens as m3 muscarinic receptor was overcome. proteins were separated on 2d-gels and tested in wb to locate the relative spots for ms. the correct localization of the patients antibodies on the cell surface was confirmed by fluorescence microscopy. in conclusion, membrane or membrane-associated antigens were recognised by sera from sjs patients. one of them might correspond to m3 muscarinic receptor. this identification might help in developing a diagnostic assay for sjs. osamu handa, s kokura, k mizuahima, s akagiri, t takagi, y naito, n yoshida, t yoshikawa aim: various additives and preservatives are used in cosmetics, foods and medicines in order to prevent deterioration. however the precise mechanism of cytotoxicity of these additives are not known. in this study, we investigated the effects of ultraviolet-b (uvb) exposure on additives-treated human normal skin keratinocytes (hacat). most popularly used additives in cosmetics such as methylparaben (mp), octandiol (od) and phenoxyethanol (pe) were used. hacat keratinocyte was cultured in mp-containing medium for 24 h, exposed to uvb and further cultured for another 24 h. subsequent cellular viability was evaluated by fluorescent microscopy and flow cytometry using double staining method with hoechst 33342 and propidium iodide or annexin-v. same experiments were done using od and pe respectively instead of mp under same condition. in addition, gene chip analysis was performed in each group. results: uvb exposure enhances cytotoxicity of these additives even at low concentration. gene chip analysis showed that the expression of apoptosis-related genes, oxidative stress-related genes and transcription related genes were significantly upregulated in each group. these results indicate that some additives, which have been considered safe preservatives in cosmetics, may have harmful effects on human skin when exposed to sunlight. these kinases in the pathogenesis of psoriasis. recently, increased focal activation of the downstream target mitogen-and stress-activated protein kinase 1 (msk1) was demonstrated in psoriatic epidermis. the purpose of this study was to investigate msk2 and the transcription factor camp-response-element-binding protein (creb) in psoriatic skin and in cultured normal human keratinocytes. keratome and punch biopsies were taken from patients with plaque-type psoriasis. normal human keratinocytes were cultured and stimulated by interleukin-1㢠(il-1ã�) or anisomycin. some of anti-inflammatory plant flavonoids as a form of whole plant extracts have been used topically for skin inflammatory disorders. on human skin inflammation, matrix metalloproteinase-1 (mmp-1) plays a pivotal role on unbalanced turn-over or rapid breakdown of collagen molecules. in the present study, for establishing a therapeutic potential against skin inflammatory disorders, the effects of natural flavonoids on mmp-1 activity and mmp-1 expression were examined. from the results, the flavonols including quercetin and kaempferol were revealed to be strong inhibitors of human recombinant mmp-1 with the ic50s of 39.6 -43.7 ã¬m, while the flavones such as apigenin and wogonin showed weak inhibition. when the effects of flavonoids on mmp-1 induction were studied, it was found that quercetin, kaempferol, apigenin and wogonin (12.5 -25.0 ã¬m) strongly inhibited mmp-1 induction from tpa-treated human dermal fibroblasts, but naringenin (flavanone) did not. by gel shift assay, these flavonoids were also found to inhibit the activation of the transcription factor, ap-1, whereas naringenin did not. among mapks, quercetin inhibited the extracellular signal-regulated protein kinase (erk) and p38 mapk activation, and kaempferol inhibited the p38 mapk and c-jun n-terminal kinase (jnk) activation. on the contrary, the flavones and naringenin did not inhibit the activation of these three mapks. all these results indicate that the capacity of mmp-1 inhibition and mmp-1 down-regulation of flavonoids may block collagen breakdown in certain pathological conditions and certain flavonoids are useful to treat skin inflammation, especially by topical application. (1) (1) university of valencia, spain (2) istituto di chimica biomolecolare cnr, napoli, italy avarol is a marine sesquiterpenoid hydroquinone with several pharmacological properties including antioxidant, anti-inflammatory, and antipsoriatic effects. recently, its derivative avarol-3-thiosalicylate (ta) also demonstrated interesting perspectives as anti-inflammatory drug in vitro and in vivo.it is interesting to note that avarol and ta inhibited nf-ã�b activation in hacat keratinocytes. now, the effect of avarol and ta was investigated in the tpa-induced hyperplasia murine skin model, which presents some similarities with psoriatic lesions. topical treatment with ta (20 mg/ml) produced a 97 % inhibition of oedema and a strong reduction of pge2 (100 %), ltb4 (100 %) and mpo activity (65 %) in skin homogenates. the inhibitory effect of avarol at the same dose was 79 % for oedema, 90 % for pge2, and 50 % for ltb4 and mpo activity. histological study for both compounds showed a decrease in epidermal hyperplasia as well as leukocyte infiltration respect to tpa treatment. besides, the reduction of cutaneous tnf-a by avarol and ta was also detected by immunohistochemical analysis. these compounds were also capable of suppressing nf-ã�b nuclear translocation in mouse skin. in summary, our results suggest that inhibition of proinflammatory metabolites by ta and avarol might be beneficial for the treatment of the inflammatory component of psoriasis. its mechanism of action is related to the inhibition of nf-ã�b activation and can be mediated by the downregulation of intracellular signal-transduction (1), ams silva(2), cmm santos(2), dcga pinto(2), jas cavaleiro(2), jlfc lima (1) (1) requimte, departamento de quã­mica-fã­sica, faculdade de farmâµcia da universidade do porto, porto, portugal (2) departamento de quã­mica, universidade de aveiro, aveiro, portugal 2-styrylchromones are a novel class of chromones, vinylogues of flavones (2-phenylchromones), which have recently been found in nature. several natural and synthetic chromones have demonstrated to possess biological effects of potential therapeutic applications. however, the anti-inflammatory potential of 2-styrylchromones has not been explored so far. thus, the aim of this work was to evaluate the putative anti-inflammatory properties of several synthetic 2-styrylchromones by studding their influence on different systems that are related to the inflammatory process. the putative inhibitory effects of several 2-styrylchromones on the proinflammatory enzymes cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2) and 5-lipoxygenase (5-lox) was evaluated in vitro and compared with structurally related flavonoids. the capacity of the studied 2-styrylchromones to scavenge reactive oxygen (ros) and nitrogen species (rns) was also assessed by different in vitro assays, which allowed to identify the influence of those compounds in each reactive species, separately. from the tested 2-styrylchromones, those having a catecholic bring where shown to be the most effective scavengers of ros and rns, being, in some cases, more active than flavonoids. no considerable correlation was found between the scavenging profile of these compounds and their interactions with pro-inflammatory enzymes. the results obtained from the present study indicate that some of the tested compounds are promising molecules with potential therapeutic value. the usefulness of 2-styrylchromones in the prevention or control of inflammation can only be clarified with additional studies concerning their influence on other relevant mechanisms of this pathology. the importance of tumor-associated inflammatory cells, able to affect different aspects of neoplastic tissue, is a current matter of debate. primarily monocytes are recruited from the circulation into solid tumors and metastases where they differentiate into macrophages with several phenotypes and, e.g., may significantly contribute to uptake of certain radiotracers. we therefore sought to characterize the uptake of various radiotracers used for positron emission tomography (pet) in a well characterized in vitro model of human monocytes/macrophages in comparison with that in various human tumor cells. uptake of radiotracers 18f-fluorodeoxyglucose (fdg), 3-o-methyl-6-18f-fluoro-l-dopa (omfd), and 18f-labeled native/oxidized low density lipoproteins (nldl, oxldl) in single-or cocultivated human myeloid (monocytic) leukemia cell line thp-1 was compared with that by squamous cell carcinoma (fadu), mamma (mcf-7) and colorectal adenocarcinoma (ht29) cell lines (without or in the presence of specific inhibitiors). several thp-1 phenotypes along the monocytic pathway (monocytes, differentiated macrophages, retrodifferentiated cells) were studied before, during and after incubation with phorbol myristate acetate. differentiated thp-1 cells show, when compared with tumor cells, a comparable fdg accumulation, a considerably lower omfd uptake, and a significantly higher oxldl uptake. on the other hand, during differentiation and retrodifferentiation thp-1 cells obviously establish a distinct sequence of biological processes also reflected by considerable alterations in radiotracer uptake. the observed differences in uptake of several radiotracers in vitro in-between thp-1 phenotypes and between thp-1 phenotypes and tumor cells, respectively, stimulate studies on the contribution of macrophage radiotracer uptake to the overall uptake in neoplastic or inflammatory lesions in vivo. genomic and full-length cdna sequences provide opportunities for understanding human gene expression. determination of the mrna start sites would be the first step in identifying the promoter region, which pivotally regulates transcription of the gene. although the mrna start sites of most genes show heterogeneity, this may reflect physiological, developmental, and pathological states of the particular cells or tissues. recently, we have developed a 5-end sage (5sage) that can be used to globally identify the transcriptional start sites and frequency of individual mrnas. a strong association exists between states of chronic inflammation and cancer, and it is believed that mediators of inflammation may be responsible for this phenomenon. another important factor in tumor development seems to be the epigenetic effects on tumor suppressor genes. because of its ability to suppress tumor cell proliferation, angiogenesis, and inflammation, the epigenetic drug such as histone deacetylase (hdac) inhibitor is currently in clinical trials. however, how epigenetic drugs mediate its effects is poorly understood. to assess the effects of epigenetic drugs, the gene expression by 5sage in colon cancer cell lines treated with epigenetically affecting agents, 5-aza-2deoxycytidine, a potent inhibitor of genomic and promoter-specific dna methylation and trichostatin a, a hdac inhibitor was investigated. epigenetic modification induced not only the change of expression of several inflammation-associated genes and the cell cycle progression-associated genes in human colon cancer cells but also the gene expression with aberrant start sites. colon cancer is one of the most frequently diagnosed cancers in western societies. interleukin-6 (il-6) is a potent, pleiotropic, inflammatory cytokine that contributes to a multitude of physiological and pathophysiological processes. il-6 is produced by many different cell types. the main sources in vivo are stimulated monocytes, fibroblasts, and endothelial cells. a variety of studies have demonstrated that over expression of il-6 contributes to the pathogenesis of various inflammatory diseases as well as cancer. it has been reported that human colorectal cancer cells display a wide heterogeneity in their potential to express and produce il-6. serum levels of il-6 are elevated in patients with colorectal cancer, however serum levels of il-6 were found to be independent of il-6 mrna expression in tumor tissue. in this study we analyzed il-6 mrna expression by real-time pcr in 50 sporadic colon cancer tissue as well as corresponding normal mucous tissue. il-6 mrna expression in tumor tissue was lower than in the corresponding normal mucous tissue (p=0,0074). there was no correlation betweenil-6 mrna expression and tumor grade or stage. thus we can conclude that il-6 produced at the tumor site is not involved in sporadic colon cancer progression. (1), t aiamsa-ard (1), v chinswangwatanakul (2), ki techatraisak (3), s chotewuttakorn (1), a thaworn (1) ( material and methods: huvec were cultured as standard techniques and grown to confluence until use. serum was obtained from cholangiocarcinoma patients and normal healthy subjects. huvec were treated with 10% of serum and incubated for 24 hours. cells were analyzed by using [3h] thymidine and immunoblotting assay for cell proliferation and cox-2/nos-2 protein expression, respectively. results: serum of cca patients trend to have more effect on proliferation of endothelial cell than healthy control subjects. on the protein expression, cca serum significantly increased the expression of cox-2 but not nos-2 in hevec. however, the proliferate effect on endothelial cells by cca sera did not correlate with the expression of cox-2. conclusions: this result suggested that some factors in serum of cancer patients could induce cox-2 protein expression in huvec. the increasing of cox-2 might be one of various factors involve in the proliferation process. aim: superoxide is responsible for the neutrophil-mediated tumoricidal activity. the aim of our work was to monitor the changes of superoxide production from neutrophil attributed to tumor development from the early phase to the advanced stage, and to investigate the effects of ok-432@on neutrophil-derived superoxide production and tumor growth. methods: ah109a rat hepatocellular carcinoma cells were implanted into the hind leg of male donryu rats. pmns were harvested from rat peritoneal cavity 6 h after intraperitoneal injection of oyster glycogen. superoxide production were measured by the method of cladependent chemiluminescence, which has high sensitivity and specificity to superoxide. the counts of peripheral leukocytes were significantly increased during tumor progression, and there are significant difference between that of controls and tumor-bearing rats after 18 days of tumor inoculation. both pma and oz-induced superoxide generation derived from neutrophils became significantly reduced in the advanced stage of cancer. the suppression of neutrophil-derived superoxide generation was accompanied with tumor progression and an increased number of neutrophils in the peripheral blood. the subcutaneous administration of ok-432, a biological response modifier, prevented the suppression of neutrophil-derived superoxide generation during tumor progression, which might induce the tendency of tumor growth suppression. our results suggested that the decreased superoxide generation as well as the high leukocytes concentration in the peripheral blood could be considered as indicators of an advanced stage of cancer. furthermore, the effect of ok-432 on neutrophil-derived superoxide production in cancer-bearing rats may provide pharmacological evidence to the therapeutic effects of ok-432. (1), m jokic (2), v zjacic-rotkvic(1), s kapitanovic (2) (1) university hospital sestre milosrdnice, bucharest, romania (2) division of molecular medicine rudjer boskovic institute, bucharest, romania introduction: il-6 is a pleiotropic cytokine mapped to chromosome 7p21-24. its promoter snp -174 g/c is associated with high serum cytokine production, and according to current investigation can play a role in the development and progression of different gastrointestinal malignancies. we tested its genotypes in the gastrointestinal and pancreatic neuroendocrine tumors (gep-nets). patients and methods: dnas from 80 patients diagnosed with gep-net and 162 age and sex-matched volunteers were analyzed for -174 g/c snp of the il-6 gene. to analyze il6 -174 c/g polymorphism we used pcr -nlaiii rflp method. for statistical analysis ã�2 test and fishers exact test were used. the level of significance was 0.05. results: there were no differences observed in the frequencies of the -174 high expression (gc and gg) genotypes between the patients and healthy volunteers (p=0.5518), as well as between patients with gastrointestinal or pancreatic endocrine tumors (p=0.3762). -174 g/c genotype was statistically more frequent among patients with non-functional pancreatic endocrine tumors (pets) than in those with functional pets (p=0.0246). conclusions: high expression genotypes of il-6 -174 snp are more frequent in non-functional pets and may be a marker for the mentioned malignancies. are important in inflammation, are found around and within a variety of human tumors. their number correlates with tumor vascularity and aggressiveness and is a negative indicator for patient survival. how mast cells influence tumor growth is not well understood. the neuroendocrine peptide, neurotensin (nt) is a potent secretagogue of mc that has tumor-promoting effects in animals and promotes the growth of a variety of human cancer cells, acting via its gpcr nt-type 1 receptor (nts1). here we show that hmc-1 human mc express nt-precursor (pront) mrna and protein, and secrete immunoreactive nt when stimulated. rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to pront and a band corresponding to the pront processing enzyme, pc5a.immunocytochemistry on hmc-1 cells showed specific staining for pront. stimulation of hmc-1 cells with a23187 + pma, pge2, c48/80 or mastoparan released immunoreactive nt.rt-pcr on hmc-1 cell rna yielded a band with 99% sequence identity to human nts1. western blotting gave bands corresponding to unglycosylated (49 kda) and glycosylated (55 kda) nts1.immunocytotochemistry on hmc-1 cells showed specific staining for nts1. these finding have significance for the role of mast cells in tumor growth. (1), j buddenkotte (1), mp schã§n(2), m steinhoff (1) (1) university hospital mã¼nster, germany (2) university hospital wã¼rzburg, germany the proteinase-activated receptor par-2 has been demonstrated to modulate tumor growth, invasion and metastasis in various tissues. however, the role of par-2 in cutaneous cancerogenesis is still unknown. here we could show a protective role of par-2 in the development of epidermal skin tumors: we established a mouse skin tumor model using chemically induced carcinogenesis. to this end, par-2-deficient and wild-type mice were painted once with dmba (7,12-dimethylbenz[a]anthracene) for sensibilization, followed by topical application of the phorbol ester pma (phorbol myristate acetate (12-o-tetradecanoylphorbol-13-acetate)) twice per week at the same sites. tumors started to appear after eight weeks. after 13 weeks, par-2-deficient mice showed a significantly increased number of skin tumors (14 per animal on the average) in contrast to the wild-type (eight tumors per mouse). analysis of possible signal transduction pathways activated upon par-2 stimulation in hacat keratinocytes showed an involvement of extracellular signal regulated kinase 1/2 (erk1/2) and profound epidermal growth factor receptor (egfr) transactivation, leading to secretion of the tumor-suppressing factor transforming growth factor-beta 1 (tgf-ã¢1). thus, our results provide the first experimental evidence for a tumor-protective role of par-2. (1), ma arbã³s (1), a fraga (1), i de torres(2), j reventã³s (1), j morote (3) ( pathogenesis of benign prostatic hyperplasia (bph) and prostate cancer (pca) is still unresolved, although chronic inflammation may play a significant role in disease progression. prostate stromal fibroblasts may be contributing to the inflammatory process through the expression and secretion of pro-inflammatory mediators, in particular proteoglycan-bound chemokines and other chemoatractants, and the interaction with inflammatory cells such as monocytes. to better understand molecular mechanisms underlying functional differences among prostate fibroblast populations, our primary objective was to characterize proteoglycan and chemokine gene expression in human fibroblasts of different histological/ pathological origin cultured in normal and monocyteconditioned media. we analysed 20 primary human fibroblast cultures from normal transition zone (tz), normal peripheral zone (pz), benign prostatic hyperplasia (bph), and pathologically confirmed prostate cancer (ca). cells of different origin displayed distinct mrna expression profiles for the core proteins of proteoglycans and both sdf1/cxcr4 and mcp1/ccr2 chemokine axis. when incubated with monocyte-conditioned medium all four cell types significantly changed sdf1/cxcr4 and mcp1/ccr2 expression in a fibroblast population dependent manner. monocyte-fibroblast cell adhesion and the chemotactic response of fibroblasts to human peripheral blood monocytes were investigated in a coculture system. monocytes adhered rapidly to fibroblasts and preferentially to bph and pz cells. in addition, chemotaxis was significantly induced in both fibroblast cultures after incubation with monocytes. our results suggest that prostatic fibroblasts have a key inflammatory role associated to a distinctive proteoglycan gene expression and chemokine induction, which is dependent on their histological and pathological source. supported by the spanish urology society (madrid, spain). we have recently shown that paf-receptor is involved in phagocytosis of apoptotic and necrotic but not viable cells, possibly through its interaction with paf-like molecules present on the surface of these cells. removal of altered cells by macrophages could modify the microenvironment at an inflammatory site, and thus influence tumor growth. in the present study we investigated the impact of apoptotic cells or treatment with paf-r antagonist on ehrlich ascitic tumor (eat, ip) and melanoma b16f10 (sc). paf-r antagonist, web2170 (5mg/kg, ip) was given daily for 6 days. we found that eat growth was significantly reduced by pretreatment with web2170, and that inoculation of apoptotic cells (thymocytes) before tumor implantation stimulated tumor growth, an effect reversed by web2170 pretreatment. eat growth was accompanied by increased production of prostaglandin e2, vegf and no which was reduced significantly by web2170 treatment. in b16f10 melanoma, web2170, alone or in association with an apoptosis inducer chemotherapeutic agent, dacarbazin (dcb, 40 ug/kg,ip) significantly reduced tumor mass volume and the number of intratumoral small vessels. in association with dcb, web-2170 reducedactive caspase-3 expression in the tumor andmarkedly increased the survival of tumor-bearing mice. the data obtained here show that during tumor growth, activation of paf-r by molecules present in the surface of apoptotic/necrotic cells, or by paf produced in the milieu, favors tumor growth and suggests that pafantagonists could be useful in tumor treatment, particularly when in association with chemotherapy. financial suport by fapesp and cnpq. (1), mt quiles (1), a figueras(2), r mangues(2), f vinals(2), jr germa(2), g capella (2) (1) institut de recerca vall de hebron, barcelona, spain (2) translational research laboratory, idibell -institut cataladoncologia, spain the malignant potential of tumor cells may be influenced by the molecular nature of k-ras mutations. we have previously shown that codon 12 mutations associate with an increased resistance to apoptosis. we hypothesized that their different malignant potential in vivo could be also related to the generation of a distinct angiogenic and inflammatory profile including vascular structure, macrophage infiltration and expression of angiogenic modulators, proteolytic mediators and the cxcl12(sdf-1)/ cxcr4 chemokine axis. to do so we have combined in vitro and in vivo studies using stable cys12 and asp13 nih3t3 transfectants. cys12 tumors showed a higher microvessel density associated with shorter latency period. prominent vessels with âµ-smooth muscle actin positives cells surrounded by f4/80 macrophages were only observed in asp13 tumors associated with a shorter growth period. asp13 tumors displayed increased vegf expression both at the rna and protein levels, mainly produced by tumor cells. tsp-1 protein levels were similarly diminished in both transfectants. higher mmp9 and mmp7 activities and expression were observed in asp13 tumors probably produced by macrophages or stromal cells. total and active mmp2 levels were higher in cys12 tumors. the expression of sdf-1 and cxcr4 remained unchanged while sdf-1g isoform was selectively induced in cys12 tumors, suggesting sdf-1a or b are induced in asp13 tumors. these results show distinct k-ras mutations induce specific angiogenic phenotypes. the differential stimulation of vegf expression, metalloprotease activities and sdf-1 expression observed is the result of the joint action of tumor cells and the local microenvironment. contact information: dr maria a arbos via, institut de recerca vall de hebron, unitat de recerca biomedica, barcelona, spain e-mail: maarbos@ir.vhebron.net incisional hernias (ihs) represent a common complication of laparotomies, involving remarkable healthcare costs. representative ih animal models are lacking and characterization of human tissue resources is scant. this limited understanding of fundamental mechanisms regulating the destruction of the abdominal wall currently limits the prevention and treatment of ihs. here, we compared tissue specimens (carefully obtained > 5cm of the defect) and primary fibroblasts cultures from fascia and skeletal muscle of subjects with/without ih hernia. the most prominent morphologic characteristics of ih tissue were: alterations of the microstructure of the connective tissue and loss of extracellular matrix (ecm), and a paucity of fibroblasts. in ih muscles, inflammatory infiltrates were observed. other significant changes were: decreased collagen type i/iii ratio; differential proteoglycans mrna expression; enhanced metalloproteinases/ endogenous inhibitors ratio (mmps/timps); and upregulation of apoptosis effectors (caspase-3 and substrates; tnf-alpha; il-6). in vitro, hernia fibroblasts (ihfs) exhibited significantly higher (2-fold) cellular proliferation and migration rates and decreased strength of adhesion as compared to control fibroblasts, even after several passages. moreover, ihfs ultrastructure analysis revealed accumulation of autophagic vacuoles, autophagolysomes-like structures and multilayered lamellar and fingerprint profiles, as well as mitochondrial swelling. based on these descriptive results in human tissues, a novel hypothesis emerges regarding ih formation. specifically we propose that inflammation-related mechanisms triggering proteolytic and apoptotic effectors regulate cell turnover and eventually contribute to atrophy and progressive tissue insuffiency. overall, this may be causally involved in the mechanisms of ecm destruction yielding ih (supported by fis pi_ 030290 and gencat_agaur_ xt_0417). (1), m spinola(2), c pignatiello(2), w cabrera (1), og ribeiro (1), n starobinas(1), t dragani (2) (1) butantan institute, sao paulo, brazil (2) istituto nazionale tumori, milan, italy airmax and airmin mice are phenotypically selected for maximal or minimal subcutaneous acute inflammatory response, respectively, and display high inter-line differences in protein exudates and neutrophil infiltration, as well as in bone marrow granulopoiesis, inflammatory cytokines, and neutrophil apoptosis. in a combined experiment of urethane-induced lung inflammatory response and lung tumorigenesis, airmin mice developed a persistent subacute lung inflammation and a 40fold higher lung tumor multiplicity than airmax mice, which showed a transient lung inflammatory response. we have analyzed gene expression profiles of these outbred lines in comparison to the lung cancer resistant c57bl/6 and lung cancer susceptible a/j mouse strains. gene expression profile analysis of urethane-treated and untreated animals was performed using the applied biosystems mouse genome survey microarray containing 32,000 mouse transcripts. mrna expression of candidate differentially expressed genes was validated by quantitative real-time pcr and the over-represented biological themes were analyzed with the ease software. urethane treatment modulated the gene expression profile in all four lines. among the confirmed genes, vanin3 (vnn3) and major histocompatibility antigen e alpha (h2-ea) resulted common to both mouse models. the most represented gene categories in air model were acute phase response, immunoresponse, electron and lipid transport, complement activation and tissue repair. mhc/antigen process and presentation and immunoresponse were the major themes in the inbred model. moreover, a gene cluster in chromosome 3 (45.0 cm) was observed. the study suggests that the expression of a subset of genes may show a strain-and line-specific modulation pattern during inflammatory response and lung tumorigenesis. inhibition of tumour induced angiogenesis constitutes very attractive anti-cancer therapeutic approach.it is well established that the vegf signal transduction pathway is one of the key drivers of deregulated angiogenesis and selective inhibition can lead to inhibition of tumour growth. however, multiple angiogenic growth factors and pathways are involved, leading to a phenomenon of redundancy and overcoming of an inhibition of vegf signalling only. we have developed a nanomolar inhibitor (compound a) of the receptor tyrosine kinase vegfr-r2 (kdr), which was subsequently shown to be a potent inhibitor of closely related kinases (vegfr-1 and -3, pdgfr, kit, csf-1r) but also unrelated soluble tyrosine kinases (src-familily of kinases and raf). compound a potently inhibits vegf stimulated endothelial cell proliferation but has no effect on non-ec proliferation, which is suggestive of a selective antiangiogenic potential. the unique kinase inhibitory profile of compound a combined with excellent oral bioavailability (87%) has translated into superior in vivo antiinflamm. res., supplement 3 (2007) posters tumour efficacy when compared to the relatively selective kdr inhibitor ptk787. thus, treatment of nude mice implanted with either commercial atcc derived tumour cells (a431 and du-145) or low passage patient derived tumors (cxf 1103; colon cancer, rxf 631; renal cancer) with compound a resulted in inhibition of tumour growth which was significantly better than for ptk787 treated mice. compound a is fairly well tolerated by rodents and extended toxicological studies have been initiated to determine the therapeutic index, which also may allow for exploration of other non-cancer indications. (1), p bobrowski(2), m shukla (3), t haqqi (3) (1) albany medical college, usa (2) rainforest nutritionals, inc, usa (3) case western reserve university school of medicine, usa background: the amazonian medicinal plant sangre de grado (croton palanostigma) has traditional applications for wound healing and inflammation. we sought to characterize an extract (progrado) in terms of safety, proanthocyanidin profile, antioxidant activity and anabolic/catabolic actions in human cartilage explants. methods: acute oral safety and toxicity was tested in rats according under oecd protocol #420. proanthocyanidin oligomers were quantified by hplc and progrados antioxidant activity assessed by the orac, norac and horac assays. human cartilage explants, obtained from surgical specimens, were treated with il-1b (10 ng/ ml) to induce matrix degradation and glycosaminoglycans (gag) release. progrado (2-10 mg/ml) was tested for its ability to maintain optimal igf-1 transcription and translation in cartilage explants and cultured chondrocytes. results: progrado displayed no evidence of toxicity (2000 mg/kg po) leading to gsh safety rating of 5/unclassifiable. oligomeric proanthocyanidin content was high (158 mg/kg) with the majority of oligomers > 10mers.progrado was a remarkably potent antioxidant and in an ex vivo model of inflammation-induced cartilage breakdown, progrado was exceptionally effective in reducing both basal and il-1b induced glycosaminoglycan release from human cartilage explants. progrado prevented il-1b induced suppression of igf-1 production from human cartilage explants as well as stimulating basal igf-1 production (p<0.05). comparable changes in igf-1 gene expression were noted in cultured human chondrocytes. conclusions: progrado has a promising safety profile, significant chondroprotective and antioxidant actions, and promotes the production of the cartilage repair factor, igf-1. this suggests that progrado may offer therapeutic benefits in joint health, wound healing and inflammation. the solvent extracts from korean fermented soybean (chungkukjang) were evaluated for their protective effects against the generation of free radicals and lipid peroxidation. the activities of chungkukjang were compared with several antioxidants and soybean isoflavones including genistein and daidzein. in addition, the protective effects against h 2 o 2 -induced cytotoxicity and oxidative dna damage in the nih/3t3 fibroblasts line were examined. the extracts from chungkukjang and soy isoflavones inhibited the generation of 1,1-diphenyl-2picryl hydrazine (dpph) radicals, and had an inhibitory effect on ldl oxidation. the extracts from chungkukjang and soy isoflavones strongly inhibited h 2 o 2 -induced dna damage in the presence or absence of endonuclease iii and fpg. furthermore, they showed cytoprotective effects against h 2 o 2 , without cytotoxicity except for the hexane extract at high concentrations (> 450 mg/ml). the ethanol and n-butanol extracts appeared to have most potent antioxidant activities. these in vitro results show that the extracts of chungkukjang may be a useful antigenotoxic antioxidant by scavenging free radicals, inhibiting lipid peroxidation and protecting against oxidative dna damage without having cytotoxic effect. moreover, the extracts of chungkukjang inhibited mda formation in the liver, dna damage assessed by comet assay and the microucleated reticulocyte formation of peripheral blood in kbro 3 -treated mice. these in vivo results were similar to those of in vitro experiments. therefore, chungkukjang containing soy isoflavones is a promising functional food that can prevent oxidative stress. (supported by bk21 project from korea research foundation). sirt1 is a histone deacetylase, involved in oxidative stress and aging. because the role of aging and exercise on sirtuins activity in rats is unknown, we investigated the effects of exercise on age-related changes in the sirt1 activity, comparing heart (h) and adipose (a) tissue of sedentary young (n10), sedentary old (n10) and trained old (n10) rats. the trained old rats performed a 8-weeks moderate training on treadmill. on h and a tissue of all rats sirt1 activity was evaluated by assay kit, peroxidative damage measuring malondialdehyde (mda) and protein-aldehyde adducts 4-hydroxynonenal (4-hne), mnsod, catalase and foxo3a by western blot, and gadd45a, cyclin d2 and foxo3a mrna by rt-pcr. aging reduced sirt1 activity in h (p<0.0001) without effects in a, producing an increase of mda (h, p<0.0005; a, p<0.0001) and 4-hne (h, p<0.005; a, p<0.0005), and a decrease of mn-sod (p<0.02) and catalase (p<0.0001) expression in both h and a. aging did not affect foxo3a protein expression in h, and foxo3a mrna in a. exercise produced an increase in h foxo3a protein expression (p<0.02) and in a foxo3a mrna, associated to higher mn-sod (h, p<0.01; a, p<0.005) and catalase (h, p<0.0001; a, p=0.01) levels in both h and a of aged rats. in heart exercise-induced higher sirt1 activity bring on decrease in cyclin d2 and increase in gadd45a mrna expression. in a we found a similar decrease in cyclin d2, without changes in gadd45a mrna expression. these findings suggest that exercise is able to increase sirt1 activity in aged rats. (1), t horiguchi(1), k abe(2), h inoue(2), t noma (1) (1) institute of health biosciences, the university of tokushima graduate school, tokushima, japan (2) minophagen pharmaceutical co. ltd, japan objectives: glycyrrhizin (gl) is a major component of glycyrrhizae radix (licorice) that is generally used for treatment of hepatitis. gl has a regulatory activity on arachidonic metabolism, immunological function, and anti-viral effects. however, the molecular mechanisms of the effects remain unclear. to analyze the molecular basis of gl signaling, we performed the microarray analysis using ccl4-induced mouse hepatitis models. methods: eight-week-old icr male mice were treated intraperitoneally with 100 fã�l/ kg bw of ccl4 w/wo 250 mg/ kg bw of gl. after 24 hours and 72 hours, livers and serum were collected and analyzed. for microarray analysis, the expression patterns of genes between 72 hour-treated-livers (ccl4 or ccl4 and gl) and no treated-livers were compared. results: gl-treatment dramatically decreased the gpt activity in plasma at 24 hours compared to that in ccl4treated plasma. however, the levels of mrna expression of inflammatory genes such as phospholipase a2, hsp47, and procollagen were still very high in gl-treated liver. after 72 hours, the mrna levels of them were significantly reduced in gl-treated mice compared to those of ccl4-treated liver. then, we screened 41,000 genes by microarray and found that 71 genes were up-regulated and 63 genes were down regulated in ccl4+gl compared to ccl4 treatment. interestingly, ros scavenger-related genes were significantly up-regulated in ccl4 + gl. detail analysis is currently ongoing. we found the unique relationship between gl activity and ros regulation. this finding suggests a novel way to treat inflammatory diseases including hepatitis. objectives: experimental autoimmune encephalomyelitis (eae) is a demyelinating autoimmune disease that results from an immunological reaction against different myelin components at the cns. it is widely employed as an animal model of human multiple sclerosis. interestingly, the number of studies relating these diseases with peripheral organs is limited. we thus investigated the consequences of eae on the degree of lipoperoxidation (tbars) and mpo activity in different rat peripheral organs (eg. lung, spleen, liver, stomach, duodenum, colon, ileum, kidney and bladder). university of waikato, hamilton, new zealand mitochondria play a fundamental role in the life and death of all eukaryotic cells. cells with dysfunctional mitochondria are known to have higher levels of a molecular stress protein (cpn60). this protein is increasingly being implicated to play a role in modulating cellular inflammation. we have developed an in vitro model cell system using thp-1 monocyte cells with compromised mitochondrial bioenergetic functions to investigate the relationships between mitochondrial dysfunction, cpn60 expression and modulation of proinflammatory cytokine responses. we have found that the ability of cpn60 to modulate tnf-a expression was strongly correlated with the loss of mitochondrial bioenergetic functions in our thp-1 cells. we also demonstrate that such modulation involves both erk1/2 and p38mapk pathways. the significance of these results in relation to the role of mitochondria as modulators of inflammation will be discussed. (1), b arnold(2), g opdenakker (3) (1) jagiellonian university, department of evolutionary immunobiology, krakow, poland (2) german cancer research center, department of molecular immunology, heidelberg, germany (3) rega institute for medical research, university of leuven, laboratory of immunobiology, leuven, belgium we showed that in mice genetically deprived of metalloproteinase 9 (mmp-9-/-) at least one compensatory mechanism operates as there are elevated levels of pge2 of cox-1 origin expressed by peritoneal macrophages during zymosan peritonitis; and this leads to increased early vascular permeability observed in those animals. also infiltration of peritoneal cavity by inflammatory neutrophils is changed in mmp-9-/-mice as at 6 hrs of inflammation, when otherwise highest numbers of neutrophils are detected in peritoneum, the cell numbers are significantly lower in the mice in comparison to their controls. in contrary, at 24 hrs of peritonitis, when normally resolution of peritonitis takes place, no decrease in neutrophil counts is observed. thus the aim of the present study was to evaluate if impairment of neutrophil apoptosis could account for this latter phenomenon in mmp-9-/-mice. for this numbers of apoptotic (annexin v) and necrotic (7-aad) peritoneal leukocytes were evaluated and levels of active caspases were tested by application of either caspase detecting antibodies or fluorochrome-labelled inhibitors; all analyses were performed by flow cytometry. the results revealed that both, numbers of apoptotic cells and levels of active caspase 3 were significantly lowered in mmp-9-/-mice while levels of caspase 7, 8 and 9 were significantly elevated in comparison to control animals. we conclude that an impairment of apoptosis is observed in mmp-9-/mice during zymosan peritonitis and it is due to the decreased levels of active caspase 3. the increased activity of other examined caspases is most probably independent of apoptosis. (1), h james (1) the selective inhibition of nitric oxide generation by inhibiting the activity of nitric oxide synthase(nos) isoforms represents a novel therapeutic target for the development of anti-inflammatory agents. the aim of this study was to evaluate the activity of nos inhibitors in experimentally induced inflammation, pain and hyperalgesia. the effect on acute inflammation was studied in carrageenan-induced paw edema in rats. the effects on carrageenan-induced hyperalgesia, tail flick response to radiant heat and acetic acid-induced writhing were also studied. nos inhibitor ng-nitro-l-arginine methylester (l-name), 10 and 20 mg/kg produced a dose-dependent inhibition of paw edema (42% and 57% at 2h; 45% and 57% at 4h). a marked reduction in paw edema was observed with ng-monomethyl-l-arginine acetate (l-nmma), 10 mg/kg(79% at 2h; 91% at 4h). selective inducible nos(inos) inhibitor aminoguanidine hemisulfate inhibited the paw edema at a dose of 20 mg/ kg(59% at 2h; 64% at 4h) but not with a dose of 10 mg/kg . the effects were comparable to nonselective cox inhibitor indomethacin 5 mg and 10 mg/kg (40% and 77% at 2h; 29% and 72% at 4h respectively) and selective cox-2 inhibitor rofecoxib, 5 mg/kg (49% and 78% respectively). nos and inos inhibitors significantly increased the pain threshold latency in the tail-flick test. these inhibited the acetic acid-induced writhes, the effect being comparable to indomethacin. however, carrageenan-induced paw hyperalgesia was not inhibited. the results suggest that nitric oxide plays a role in carrageenan-induced acute inflammation and both nosand inos inhibitors have a potential anti-inflammatoryand anti-nociceptive activity. (1), p hart(2), j edwards (1), c quirk (1) (1) molecular pharmacology limited (usa), australian division, perth, western australia (2) telethon institute for child health research, perth, western australia thermalife cream, an anti-arthritic biological product, has been successfully used off-label for sun burn recovery. a novel product, derived from thermalife, was assessed on its therapeutic potential in oxsoralen-uvb burns. as a possible mechanism for the sunburn efficacy, suppression of tnf-a and il-1㢠production by human monocytes was assessed in vitro. methods: sunburn: four sites were marked on the arm of the subject. three sites were exposed to oxsoralen (1%) plus uva/uvb light, one site was exposed to oxsoralen only. cream was applied at 5min, or at 4hrs after injury. a third injury site was not treated. photographs were taken before, 24hrs, and 7weeks after injury. cytokines: human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0% or 10% active ingredient.24hrs after incubation, culture media was collected, centrifuged, and assayed (cytokine elisa). results: at 24hrs after oxsoralen-uv, the 5min treatment site showed slight erythema, the 4hr treatment site had pronounced erythema and slight blister formation, whereas the untreated site had pronounced erythema and strong blister formation. 7 weeks after injury, the 5min site was normal, the 4hr site was a dark colour, whereas the untreated site had a significant scar. oxsoralen alone had no effect on the skin. the novel product suppressed lps-induced tnf-a and il1㢠secretion by 48.7% and 71.1%, respectively. conclusions: a novel thermalife-derived product reduced total injury after oxsoralen enhanced uva/ uvb burns, which is possibly related to cytokine suppression. (1), p hart(2), j snowden (1), maud eijkenboom (1) (1) molecular pharmacology limited (usa), australian division,perth, western australia (2) telethon institute for child health research, perth, western australia a mixture of bovine plasma protein fractions and zinc chloride (bov-zn) was assessed for its ability to regulate cytokine production by lps-stimulated monocytes. dosereponse curves for tnf-a suppression were generated. further, competition with fcs in the culture medium and the metabolism of monocytes under influence of bov-zn were assessed. in all experiments the culture medium environment was similar. human monocyte cultures (10% fcs, 5% co2) were either stimulated with 500ng/ml lps (e.coli 0111:b4) or not in the presence of 0%, 2.5%, 5%, 7.5%, 10%, 20% or 40% bov-zn (two pooled experiments). 24 hours after incubation, culture media were collected, centrifuged, and assayed (cytokine elisa). a competitive inhibition design for the standard tnf-a assay was set up for 0%, 1%, 5%, 10% fcs against 0%, 2.5%, 5%, 10% bov-zn. the culture media were treated as above. metabolism of non-proliferating monocytes was measured via accumulation of bioreduced formazan (promega celltiter 96) in treated and untreated cell cultures over 0-45 hrs at intervals. the ic50 for tnf-a suppression was reached at 2.5% bov-zn in each of two experiments. fcs did not compete with bov-zn in suppressing tnf-a in lpsstimulated monocytes. at low fcs concentrations bov-zn stimulated tnf-a production in the absence of lps. this tnf-a increase was countered with increasing concentrations of fcs. metabolism of cells was not affected by 10% bov-zn. conclusions: bov-zn could reliably and effectively reduce tnf-a secretion in vitro, without competing with the fcs in the culture medium, and without disturbing the metabolism of monocytes. inflammatory diseases such as rheumatoid arthritis (ra) result from overproduction of cytokines including tnf-â£\ and il-1fã�. these cytokines are known to be regulated by the stress-activated p38fnfnmap kinase pathway. because of this, inhibition of p38 map kinase has been one of the most compelling targets for the treatment of inflammatory disease. over the last 10 years, numerous groups have reported on the development of p38 map kinase inhibitors. x-ray co-crystallization with the enzyme suggests a propensity to accommodate structurally diverse molecules. regions of the binding site are known to be unique to p38 vs other kinases, enabling the development of p38 selective molecules. inflamm. res., supplement 3 (2007) posters anti-inflammatory activities. a series of 11 labdane-type diterpenoids (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) with various patterns of substitution were tested for potential anti-inflammatory activity.of these compounds, 4 and 11 were selected to evaluate their influence in targets relevant to the regulation of the inflammatory response. these derivatives displayed good in vivo anti-inflammatory activity, and maximum inhibitions of 50 and 90 % were noted in the 12-o-tetradecanoyl-phorbol-13-acetate (tpa)-induced ear oedema in mice. in addition, inhibition of myeloperoxidase activity, an index of cellular infiltration, was also observed. the diterpenoids also reduced the production of nitric oxide, prostaglandin e2, and tumour necrosis factor-alpha in bacterial endotoxin-activated raw 264.7 macrophage cells with ic50 in the range 1-10 mm. inhibition of these inflammatory mediators was related to reductions in the expression of inducible nitric oxide synthase, and cyclooxygenase-2, as determined by western-blot analysis and rt-pcr. since nuclear factor-kappab (nf-kb) plays a central role in the transcriptional regulation of these proteins, we investigated the effects of these diterpenoids on this signalling pathway. our results indicate that both compounds interfere with the phosphorilation of ikbalpha and ikbã�, resulting in inhibition of their degradation. in summary, the anti-inflammatory effects of these labdane diterpenoids are related to the inhibition of inflammatory mediators by blocking nf-kb activation and provide potentially therapeutic perspectives in inflammatory conditions. the aim of the study was a research of mechanisms of inflammatory action of a new drug fepolen at a dosage of 100 mg/kg prepared from bee products (bee pollen and phenolic hydrophobic extract of propolis) for prostatitis treatment. to fulfill above mentioned task a model of zimozan induced oedema whose dynamics gives a possibility to estimate influence of a drug on both routs of arachidonic acid metabolism -via cyclooxiginase and lipooxigenase was used. a comparison with diclofenacum at a dosage of 8 mg/kg and substance ffw-755ã� (inhibitor of cyclooxygenase and lipooxygenase and has a high antiinflammatory effect (41%) at a dosage of 16 mg/kg) was made. the results of influence of drugs dynamics of zimozan inflammation show that anti-inflammatory action of fepolen is based on decrease of release of biogenic amines and activity of lipooxigenase and is higher then effect of diclofenacum. fepolen revealed the highest effect during first 30 min and 1 hour of inflammation that was higher then action of diclofenacum. these data proves that fepolen decreases lipooxygenase activity that is in charge for the inflammation during this period. during next hours therapeutic effects of fepolen and diclofenacum were at the same level. fepolen showed the same dynamics of anti-inflammatory action as ffw-755ã� that demonstrates a property to influence both routs of arachidonic acid metabolism. in summary with previous results in conditions of carageninic inflammation we can conclude that anti-inflammatory action of fepolen is based mostly on influence on lipooxygenase then cyclooxygenase. the aim of this study was to establish a method by which probiotic bacteria can be selected for their immuno modulatory properties, especifically the ability of certain strains to suppress an inflammatory response. the gastrointestinal inflammatory condition crohns disease involves a th1-response with increased levels of proinflammatory cytokines like tnfa and il12, and mouse models of crohns disease show that the balance of il10/12 is crucial for disease progression. we have used mouse bone marrow derived dendritic cells (bmdc) to model a proinflammatory crohns disease like condition in vitro with cocktail-induced bmdc secretion of il12, il6 and tnfaf jthe model was validated using anti-inflammatory molecules like dexamethasone and prostaglandin d2, which were able to suppress the cocktail induced il12 secretion. further validation of the model is confirmed by the fact, that probiotic strains which are able to suppress tnbs induced colitis in mice in preventive studies, also show potent anti-inflammatory activity in our model. among clinically relevant as well as novel probiotic strains, we have selected strains with potent anti-inflammatory properties, and are currently investigating the possible mechanism of action of these strains. in summary, our established model is suitable for identification of anti-inflammatory activity of probiotic strains and potentially other immune suppressing components, for rational selection of candidates for further preclinical and clinical evaluation and development. p11-12 inflammation and human incisional hernia pathophysiology maria antonia arbos via(1) eae was induced by immunization of female lewis rats with guinea-pig myelin basic protein (mbp) in complete freunds adjuvant (cfa) and the animals were studied at the stage iii of the disease (characterized by complete paralysis of the hind-limbs) compared to cfa rats, eae resulted in increased mpo activity (u/mg tissue) in kidney (280 ae 75 vs. 121 ae 14 179 ae 35; p<0.001), and higher tbars contents (nmol mda/mg tissue) in liver acknowledgements: capes, cnpq, fapesp. contact information: ms simone teixeira, university of sâ¼o paulo, department of pharmacology, campinas, brazil e-mail: mone@usp.br tolerability, investigator and subject global assessments and rescue medication consumption supported by bk21 project from korea research foundation) contact information: mr young hoon kim here we report on the development of the pge2 mimetic combination therapy (dp-837953) that inhibits basal and lps/tlr4 induced tnf-?, il-1ã�, and mmp-1, 8, 9, 13 in human and murine synovial membranes and peripheral macrophages.in a murine model of chronic synovitis (dorsal skin air pouch), dp837953 dramatically inhibited il-1ã�, tnf-?, mip2, mcp-2 and il-8 expression, delayed the profile of leukocyte/neutrophil extravasation and reduced exudate volume.in a model of inflammatory arthritis (collagen induced arthritis, cia), dp837953 markedly reversed the inflammatory pathology by reducing synovial hyperplasia, cartilage erosion and articular inflammation.in addition, tnf-?, il-1ã�, mmp-8 and to a lesser extent mmp-9 expression/synthesis levels were strongly suppressed as judged by rt-pcr and elisa measurements we have developed two rias, one for functional blood levels of the above mentioned anti-tnf-alpha constructs, and one for anti-abs (all isotypes), and we have used these methods to monitor patients treated with infliximab/remicade and etanercept/enbrel ; i shall present some of these data (7, 8 anatomy-physiology, faculty of medicine, laval university, quebec, canada neutrophils, which are often the first leukocytes to migrate at inflamed sites, can generate ltb4 from the 5-lipoxygenase pathway, pge2 through the inducible cyclooxygenase (cox-2) pathway and cytokines/chemokines as tnf-alpha, il-1beta, il-8, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha. engagement of the adenosine a2a receptor (a2ar) blocks the in vitro synthesis of ltb4 while it potentiates the cox-2 pathway in fmlp-treated neutrophils. in addition, it selectively prevents the expression and release of tnfalpha, mip-1alpha, mip-1beta, mip-2alpha and mip-3alpha in toll-like receptor-4-stimulated human neutrophils. little effect was observed on il-1beta and il-8. using the murine air pouch model of inflammation with a2ar-knockout mice, we observed that the activation of a2ar positively impacts the expression of cox-2 in vivo, with particular magnitude in inflammatory leukocytes. in mice lacking the a2a receptor, neutrophils that migrated into the air pouch 4h following lps injection expressed higher mrna levels of tnf-alpha, mip-1alpha and mip-1beta than neutrophils from wild type mice. together, these results indicate that neutrophils are important mediators of adenosines protective effects. given the uncontrolled inflammatory phenotype observed in a2ar-knockout mice and in view of the potent inhibitory actions of pge2 on inflammatory cells, an increased cox-2 expression and a prevented release of tnf-alpha, mip-1alpha and mip-1beta caused by a2ar activation, observed particularly in neutrophils, may take part in an early modulatory mechanism promoting anti-inflammatory activities of adenosine. sepsis induced by endotoxins including lipopolysaccharide (lps) is a big problem in clinical medicine. for a better insight into the molecular pathways and to assess markers of endotoxin-induced sepsis, we applied thetwo dimensional gel electrophoresis (2d-page) and maldi-tof to follow the changes of significant proteins in a murine macrophage cell line -raw 264.7 after challenged with lps (escherichia coli 026:b6) for 12, 18 and 24 hours. we identified 21 proteins from approximately 500 detected protein spots with either increased or decreased in relative abundance as a result of lps treatment. the proteins identified with increased expression are the retinoblastoma binding protein 7, capg protein, poly(rc) binding protein 1, isocitrate dehydrogenase 3 (nad+) alpha, lactate dehydrogenase 1, a chain, guanine nucleotide binding protein (g protein), beta polypeptide 2 like 1, triosephosphate isomerase 1 and proteasome alpha 5 subunit); and ones with decreased expression are the acidic ribosomal phosphoprotein p0, malate dehydrogenase, soluble, proliferating cell nuclear antigen, proteasome (prosome, macropain) subunit, alpha type 1 and rho, gdp dissociation inhibitor (gdi) beta). many of these altered proteins have interesting functions in inflammation. with the information obtained with the proteomic approach, it is possible to improve current methods of monitoring endotoxemia and to identify new therapeutic targets. the ubiquitous mitogen-activated protein (map) kinases are important enzymes in signal-tranduction cascades which regulates diverse cellular events such as cell transformation, proliferation, differentation, and apoptosis. they are therefore potential drug targets for therapeutic intervention in the treatment of inflammation, cancer, and other immune diseases. based on a virtual screening approach we identified 6-amino-1-benzyl-4-(4-bromophenyl)3-methyl-1,4-dihydropyrano[2,3c] pyrazole-5-c arbonitrile as a potential novel lead structure as p38 map kinase inhibitors. a set of compounds were prepared starting from different substituted pyrazol-5(4h)-ones via a base-catalyzed condensation with aldehydes and ch acids, such as malononitrile, to provide them for biological tests in a p38 enzyme assay. first structure-activity relationship confirm the value of this novel lead. this study was conducted to determine the physiological c-reactive protein (crp) and alpha 1-acid glycoprotein (aag) levels for two groups of beagle dogs: healthy dogs of various ages and pregnant dogs. serum crp levels were measured by elisa and aag levels were measured in healthy beagles of various ages by tia, and then separately -in pregnant beagles -by srid. serum crp levels ranged from 1.5 to 16.0 ã¬g/ml in male, and from 1.8 to 18.9 ã¬g/ml in female dogs. no significant sex-related differences were observed in the values. further, there were no significant age-related differences either. serum crp levels increased during pregnancy and peaked at 70.2-90.4 ã¬g/ml 30 or 45 days after ovulation, demonstrating two characteristic features of crp levels change in pregnant dogs. serum aag levels ranged from 40 to 960 ã¬g/ml in male, and from 47 to 833 ã¬g/ml in female dogs, without any significant sex-or age-related variation. serum aag levels increased in all pregnant beagles and peaked in the middle of gestation at 250-1,000 ã¬g/ml. despite a high value of 1,210 -1,360 ã¬g/ml being observed for serum aag levels in 3 pregnant beagles inoculated with staphylococcus aureus, its levels in umbilical cord blood were below the detection limit of srid (40 ã¬g/ml). no significant sex-/-age related differences were observed in serum both crp and aag levels and these levels increased during pregnancy. the results of aag levels in umbilical cords were below the detection levels suggest aag is not transported to the placenta. polymorphonuclear neutrophils (pmns) play a key role in the inflammatory response against infectious agents.however, they can elicit significant tissue damage and in this respect, anti-inflammatory drugs are of interest.in this study, we examined the effect of pbi-1393, a low molecular weight immunomodulatory molecule, on pmn activation by lps both in vitro and in vivo. we measured by elisa the production of tnf-a by human lps-activated pmn in the presence or absence of pbi-1393.the ability of pbi 1393 to modulate pmn activation and recruitment in vivo was assessed using a rat air pouch model of inflammation.exudates from different groups of animals (controls and pbi-1393 treated animals, n=6) were used to assess leukocyte infiltration and to measure by elisa tnf-âµ, mcp-1 and pge2 production.in vitro, pbi-1393 is able to significantly decrease by 28.8% ae 0.08% (p<0.05), tnf-a production by human lpsactivated pmn.in vivo, pbi-1393 significantly decreased the production of tnf-a (41.4% ae 7.2%; p<0.005), mcp-1 (16.3% ae 8.4%; p<0.05) and pge2 (29.2% ae 13.5%; p<0.05) induced by lps injection.however, pbi-1393 did not significantly inhibit leukocyte infiltration.these results show that pbi-1393 is able to modulate pmn activation and inflammatory response and suggest potential use as anti-inflammatory agent. (1), lj lowenstine,(2), aj norris(2), t spangler(2), lm woods (2) (1) zoological society of san diego, usa (2) department of pathology, microbiology, and immunology, university of california, davis, usa this study investigated the role of a novel reovirus in a 2004 outbreak of necrotizing typhlocolitis in american crows in california. included is a detailed characterization of the necrotizing and inflammatory characteristics of the disease, as well as a discussion of the implications of these findings upon proposed mechanisms of pathogenesis. complete histopathology including stains for lesion characterization and potential concurrent etiologic agents was performed on all outbreak crows. feces and ceca were submitted for culture, parasitology, and negative contrast electron microscopy. two control groups (n=10 each) were selected for parasitology and em (group 1), and gross and histopathology (group 2). all outbreak cases and group 2 controls tested negative for west nile virus by pcr.all outbreak crows had marked, necrotizing heterophilic typhlocolitis, fibrinonecrotizing splenitis, and variable intestinal lamina proprial necrosis and hemorrhage.two cases had multifocal hepatic necrosis. negative contrast em revealed reovirus particles in 100 % (4/4) of outbreak cases and in 0% (0/ 10) of controls. supplemental tests failed to suggest other concurrent or confounding etiologic agents.overall, the findings suggest association between the reovirus and the outbreak of typhlocolitis, and the absence of reovirus in controls suggests that it is not ubiquitous in the crow population.there was a noteable absence of similar typhlocolitis in 65 archived crows submitted to the vmth from 1994-2004, suggesting an emerging corvid disease in california, which bears further investigation. mitogen-activated protein kinase (mapks) pathways play an important role in the signalling system activated by proinflammatory cytokines. among the most important cascades the activation of erk1/2 by mek1/2 is reported to be responsible for inflammatory responses and degradation of osteoarthritic cartilage. as701820, a selective mek1 inhibitor, demonstrated anti-inflammatory properties in reducing tnf-alpha production induced by lps injection (ic50 2 mg/kg). therefore, primary aim of the present study was to assess the therapeutic strength of the as701820 in a mouse model of collagen-induced rheumatoid arthritis (cia) assessing the effect of the compound on structural changes related to the cartilage. cia is characterized by severe polyarthritis affecting peripheral joints, synovial hyperplasia with persistent inflammation and cartilage erosion. as701820 treatment was initiated when signs of arthritis were clinically visible (in terms of paw swelling and redness) and was continued for 7 days (twice daily), by oral route at the doses of 10, 30 and 100 mg/kg. as701820 at 30 and 100 mg/kg significantly reduced clinical arthritic read-outs such as clinical score and paw swelling. at histology, vehicle-treated animals showed severe inflammation and joint surface erosion. administration of as701820 significantly decreased inflammatory infiltrates and treated cartilage surfaces that presented normal levels of proteoglycan content. in conclusion, the results obtained in this study clearly demonstrate that the selective blockade of mek1 could be considered as an innovative therapeutic approach to treat rheumatoid arthritis. experimental evidences have shown that the toxicity of ni salts may involve inflammatory processes, with a subsequent overproduction of reactive oxygen species (ros) and carcinogenicity. neutrophils are the most abundant leukocytes of blood, and participate actively in the inflammatory innate host defense response. however, relatively little is known about the potential of nickel salts in activating human neutrophils.thus, the aim of the present study was to evaluate the putative stimulation of oxidative burst in isolated human neutrophils by nickel nitrate. the measurement of neutrophil burst was undertaken in vitro, by chemiluminescence, by monitoring the oxidation of luminol by neutrophil-generated ros and reactive nitrogen species (rns). enzymatic inhibitors and specific reactive species scavengers were used to evaluate which species were involved in neutrophils activation by nickel nitrate. the obtained results showed that nickel nitrate stimulates human neutrophils burst in a concentration-dependent manner, within levels that may be attained in vivo. in the present experimental conditions, the reactive species involved in neutrophils activation by nickel nitrate were superoxide radical (o2-.), hydrogen peroxide (h2o2), hydroxyl radical (ho.) and perchloric acic (hocl). the observed activation of isolated human neutrophils burst by nickel nitrate and subsequent tissue damage due to a sustained formation of reactive species may contribute for the deleterious effects attributed to this transition metal, though this assumption needs to be confirmed in vivo. (1), h spalteholz (1), u reibetanz (1), p salavei (1), m fischlechner (1), h-j glander(2), j arnhold (1) (1) university of leipzig, medical faculty, institute for medical physics and biophysics, germany (2) university of leipzig, derpartment of dermatology, andrology training centre of the european academy of andrology, germany unintentional childlessness often caused by common reasons as inflammation affects 15 -20 % of german couples. inflammations of the male genital tract lead to an infiltration of polymorphonuclear granulocytes (pmn), respectively induce a restricted spermatozoa quality associated with early triggered acrosome reaction (ar) and apoptosis as well as changes in the lipid structure and reduced mobility. stimulated pmn release the strongly cationic heme protein myeloperoxidase (mpo), which is able to bind to negatively charged membrane surfaces, e.g. apoptotic cell membranes with externalized phosphatidylserine (ps). a population of freshly prepared spermatozoa shows only a very small amount of cells with mpo binding ability as well as externalization of ps. the number of spermatozoa able to bind mpo raises considerably in samples containing predamaged cells or introducing the ar as could be observed with rhodamine b isothiocyanate (ritc)labelled mpo and antibody techniques by fluorescence microscopy as well as flowcytometry. the activation ofmpo with its substrate hydrogen peroxide (h2o2) in the presence of chloride ions generates the powerful oxidizing and chlorinating species hypochlorous acid (hocl) and enhanced markedly the number of annexin v positive and non-vital cells. components of seminal plasma as well as serum albumin can protect spermatozoa for the deleterious effects of mpo. the coincidence of ps externalization and mpo binding to spermatozoa surfaces indicates an up to now unknown role of this enzyme in recognition and removal of apoptotic cells during inflammation. recent findings suggest a crucial role of proteinaseactivated receptor-2 (par2) in inflammation and innate immunity. par2 is the second member of a novel g protein-coupled receptor subfamily with seven putative trans-membrane domains. this subfamily is characterized by a unique mechanism of receptor activation. accessible serine proteases cleave the receptor to expose a new, previously cryptic, n-terminal sequence ("tethered ligand") which further interacts with the same receptor and activates it. tryptase, trypsin, and bacterial serine proteases are capable of directly activating par2. par2 is expressed by human neutrophils, however its functions on these cells remained unclear. the data of our present study indicate that par2 agonists enhance interferon gamma (ifna)-induced up-regulation of cell surface fca;ri, one of the key receptors involved in neutrophil phagocytic activity. moreover, par2 agonists (serine proteases as well as synthetic activating peptide) and their receptor represent an additional system which controls neutrophil transendothelial migration and apoptosis in vitro. additionally, there is a significant increase of par2 expression on the neutrophil cell surface in the case of septic patients as compared to cells from healthy volunteers. together, our results indicate that par2 may be involved in the pathophysiology of acute bacteria-induced human diseases (sepsis or septic shock, for example) potentially by regulating neutrophil apoptosis, transendothelial migration and fca-receptor expression. aim: to ascertain the role of macrophages as direct inducers of regeneration after renal ischemia/reperfusion, and to establish whether inflammatory conditions contribute to the process. we determined whether adoptive transfer of macrophages at different stages of kidney inflammation after mouse renal i/r could restore reparation and assessed the influence of inflammation in the process.results: i/r provoked the increases in renal regeneration, as evaluated by inmunohistochemistry and pcr mrna of stathmin and pcna. the cytokine profile revealed the influence of the inflammatory environment on kidney repair. regeneration was macrophage-dependent, decreasing when depletion was provoked, and increasing with adoptive transfer of macrophages; however, administration of resting macrophages did not induce repair at the time points in which tissue was inflamed, and was only able to promote regeneration in the absence of inflammation (72 hours). pro-inflammatory cytokines increased at the early stages of reperfusion, coinciding with low regeneration, and anti-inflammatory cytokines increased during the longer periods of reperfusion when regeneration was more evident.conclusions: macrophages directly induce renal regeneration after ischemia/reperfusion in an inflammationdependent manner. (1), k bendtzen (1), f sellebjerg (2), ch nielsen (3) ( antibodies against myelin basic protein (mbp) are present in sera from patients with multiple sclerosis (ms), but the role of these antibodies is controversial. we collected sera from 22 ms patients and 17 healthy individuals and found that both groups contained igm anti-mbp antibodies, while ms sera contained small amounts of igg anti-mbp. however, the two groups of sera did not differ significantly with respect to the content of either antibody subclass. addition of mbp to the various sera and subsequent addition of the mixtures to normal peripheral blood mononuclear cells (pbmc) resulted in a significant deposition of igm on cd14+ monocytes, indicating that formation of mbp/igm complexes had occurred. this deposition was strongly inhibited by addition of 10 mm edta to the sera, indicating that it was complement dependent. the pbmc produced significant amounts of il-10, tnf-alpha and ifn-gamma upon stimulation with mbp, and the extent of the cytokine production did not depend upon whether sera from ms patients or from healthy controls were present. however, disruption of the tertiary structure of mbp by boiling significantly reduced the production of all three cytokines, supporting a role for antibodies in the induction of cytokine responses to mbp. we propose that natural igm autoantibodies may form complexes with mbp, facilitating the uptake of mbp by antigenpresenting cells (apc). since sera from ms patients did not enhance this uptake and the subsequent cytokine production, the mechanism may be part of an appropriate peripheral regulation of self-reactivity. we currently investigate this possibility. loredana postiglione(1), g tarantino(2), a spanã²(2), p ladogana (1), fl perrone(1), s padula(2), a riccio (2) (1) federico ii university medical school of naples, department of molecular and cellular biology and pathology l.califano, naples, italy (2) federico ii university medical school of naples, departmentof clinical and experimental medicine, naples, italybackground: hepatitis c virus (hcv) infection can induce immunological disorders with different clinical expression such asarthritis, sjogren sindrome and various form of vasculitis.aim: to study the prevalence of anti-cyclic citrullinated peptides antibodies (anti-ccp) in a group of patients affected by hcv-related arthritis and the eventual correlations with rheumatoid factor (rf) and/orantinuclear antibodies (ana), and articular involvement. study design: 30 patients with arthritis were selected in a population of 380 subjects affected by hcv infection. each patients was evaluated by clinical examination (23 denoted poliarticular and 7 mono-oligoarticualr involvement), by x-graphic aspects of joint involvement (8 patients presented join erosions), by ana, rf and anti-ccp positiveness.results: 33,3% of patients presented positivenessfor anti-ccp, without significant correlation between suchparameter and ana, rf and articular involvement. anti ccp resulted positive in 4 out of the 8 patients with joint erosions, and only in 6 out of the 22 patients without joint erosions. such frequency analyzed by chi square ended up in no significant differences. our patients presented an interesting prevalence of the positiveness for anti-ccp. these data suggest a consideration about the specificity, commonly attributed to this parameter in the diagnosis of rheumatoid arthritis. expression of nkg2d on cd4+ t cells is generally rare in both mice and humans, but has been reported in a number of inflammatory diseases, including rheumatoid arthritis, crohns disease and an animal model of type 1 diabetes. the monoclonal antibody cx5 recognizes murine nkg2d and has been shown to block ligandbinding and mediate internalization of nkg2d. furthermore, cx5 can inhibit and/or ameliorate disease in animal models of type 1 diabetes and inflammatory bowel disease. thus, it is very likely that nkg2d plays an important role in the development of inflammatory and autoimmune diseases. since little is known about the pharmacokinetics and pharmacodynamics of the cx5 antibody, we decided to study this in both regular balb/ c mice and immunodeficient cb17.scid mice. different doses of cx5 antibody was injected intraperitoneally and pk and pd was measured by elisa (anti-cx5 elisa in serum) and flow cytometry (down-regulation of nkg2d on cd49b+ nk cells) for up to two weeks after administration. we found that cx5 very efficiently down-regulate nkg2d on cd49b+ nk cells and that the effects of the antibody can be seen for more than two weeks after one single injection. finally, we propose a model which may be helpful in predicting the effects of different doses of cx5 antibody in vivo. (1), k mehta(2), n deo(2), j chaudhary(2), p bobrowski (3) (1) albany medical college, usa (2) vedic lifesciences, usa (3) rainforest nutritionals, inc, us background: the efficacy and safety of reparagen, in treating osteoarthritis was compared to glucosamine sulfate in a mumbai-based multi-center, randomized, double-blind study.methods: subjects (n=95) were screened and randomized to receive glucosamine sulfate (n= 41, 1500 mg/day) or reparagen (n=38, 1800 mg/day), a polyherbal consisting of vincaria (uncaria guianensis) and rni 249 (lepidium meyenii) administered orally, twice daily. primary efficacy variables were womac scores, visual analog score (vas) for pain, and response to treatment defined as a 20% improvement in womac pain, with assessments at 1, 2, 4, 6 and 8 weeks. secondary variables were results: subject randomization was effective and both treatments showed significant benefits in primary outcomes within one week (p<0.05), with a similar, progressive improvement over the course of the 8 week treatment protocol (42-49% reduction in total womac or vas scores). the response rate was substantial for both glucosamine (88%) and reparagen (92%), which exceeded placebo responses (55%, p <0.01) and supported by investigator and subject assessments. tolerability was excellent and safety parameters were unchanged. rescue medication use was significantly lower in the reparagen group (p <0.01), and serum igf-1 levels were unaltered.conclusions: both reparagen and glucosamine sulfate produced substantial improvements osteoarthritis symptoms. response rates were high and the safety profile was excellent, with significantly less rescue medication use with reparagen. we speculate that the high response rate to glucosamine sulfate may reflect higher baseline pain levels or synergy with dietary curcumin. inflammation accompanies and aggravates progression of all modern human chronic pathological conditions. growing evidence indicates the beneficial role of proper nutrition in controlling inflammation. we investigated the effects of selected essential nutrients in experimental inflammation and the molecular mechanisms involved. tested nutrient mixture (nm) consisted of green tea catechins, citrus flavonoids hesperidin, naringenin and quercetin, ascorbate, lysine, proline, arginine and cysteine. systemic inflammation in mice challenged with bacterial lipopolysaccharide (lps) was monitored by blood plasma levels of fourteen key inflammatory cytokines. two week supplementation with 250 mg nm/kg body weight prior to lps challenge provided significantly greater protection than did supplementation with ibuprofen. induction of interleukin-6 (il-6) and monocyte chemoattracting protein-1, two cytokines especially responsive to lps challenge, was reduced in nmsupplemented animals by 58% and 86%, respectively. corresponding reduction in ibuprofen group was 34% and 43%. protective mechanisms involved were assessed in human cultured u937 macrophages stimulated with lps.the cytokines most responsive were tumor necrosis factor alpha (71% and 17% reduction by supplementation with nm and ibuprofen, respectively) and il-12 (66% and 15% in corresponding reduction). nm supplementation dramatically reduced prostaglandin e2 secretion by stimulated macrophages along with cyclooxigenase-2 (cox2) cellular protein expression. mrna levels forcox2 and inflammatory cytokines were also dramatically reduced. quercetin was the most effective nutrient when tested individually. however, nm appeared to surplus the combined effect of individual components. we conclude that the tested combination of essential nutrients demonstrates strong beneficial effects in experimental inflammation by targeting responsible gene expression. (1), hp kim (1), kh son (2) (1) college of pharmacy, kangwon national university, south korea (2) department of food and nutrition, andong university, south korea chalcones belong to flavonoid family from plant origin and some of them possess anti-inflammatory activity. recently, several natural and synthetic chalcones were reported to inhibit inducible nitric oxide synthase (inos)-catalyzed no production in cell cultures. in the present study, to find the optimal chemical structures and to elucidate their action mechanisms, 41 synthetic chalcones having the substituent(s) on a-and b-rings were prepared and their effects on inos-catalyzed no production were evaluated using lps-treated raw 264.7 cells. among the tested compounds, 2-methoxy-3,4-dichlorochalcone (ch15), 2-hydroxy-6-methoxychalcone (ch29), 2-hydroxy-3-bromo-6-methoxychalcone (ch31) and 2-hydroxy-4,6-dimethoxychalcone (ch35) potently inhibited no production (ic 50 s, 7.0 -9.9 mm). the favorable chemical structures were found to be a methoxyl substitution in a-ring at adjacent position (2 or 6) to carbonyl moiety with/without 2-(or 6-)hydroxyl group and 3-halogen substitution in b-ring. when the cellular action mechanisms of ch15, ch31 and ch35 were further examined, it was revealed that ch15 and ch31 clearly down-regulated inos expression while ch35 did not. moreover, ch15 and ch31 were proved to suppress the nuclear transcription factor-kb activation. from the results, it is suggested that certain chalcone derivatives potently inhibit inos-catalyzed no production by the different cellular mechanisms, inos down-regulation or inos inhibition, depending on their chemical structures. these chalcone derivatives may be possibly used as lead compounds for developing new anti-inflammatory agents. an oligomeric stilbene alpha-viniferin (avf) was isolated from root of carex humilis (cyperaceae) as an inhibitor of cyclooxygenase (cox)-2 activity by bioassayguided fractionation. the avf was later found to downregulate lipopolysaccharide (lps)-induced cox-2 expression as well as to inhibit nuclear factor (nf)-kb activation, in addition to its inhibitory effect on cox-2 activity. furthermore, the compound exhibited antiarthritic effect in vivo. avf is a trimer of resveratrol and contains benzofuran moieties in its central part. starting from benzofuran and its related chemicals, 2cyclohexylimino-6-methyl-6,7-dihydro-5h-benzo [1, 3] oxathiol-4-one (lyr-64) was discovered to inhibit lpsinduced nf-kb transcriptional activity in macrophages raw 264.7. the lyr-64 reduced lps-induced dna binding activity and nuclear translocation of nf-kb as well as inhibited lps-induced degradation and phosphorylation of inhibitory kb (ikb) protein. these results suggest that lyr-64 could suppress lps signaling molecule, putatively ikb kinase (ikk) complex, upstream ikb degradation in nf-kb activating pathway. lyr-64 inhibited in vitro kinase activity, gst-ikb phosphorylation, of wild type ikkbeta or a constitutively active ikkbeta mutant (c/a, cys-179 to ala) but did not affect that of another constitutively active ikkbeta mutant (ss/ee, ser-177 and 181 to glu). therefore, lyr-64 could inhibit lps-induced nf-kb activating pathway by targeting ser-177 and/or 181 residues on the activation domain of ikkbeta. as pharmacological actions, lyr-64 prevented nf-kb-dependent expression of inducible nitric oxide synthase, cox-2, or inflammatory cytokines at the transcription level in lps-stimulated macrophages raw 264.7. furthermore, lyr-64 protected lpsinduced septic shock in vivo. faculty of medicine, institute of pharmacology, ljubljana, slovenia a part of anti-inflammatory action of antidepressants can arise from their effect on histamine elimination from the side of inflammation. in mammals histamine is mainly degraded by two enzymes: histamine-n-methyltransferase (hnmt) and diamine oxidase (dao). the aim of present investigation is to establish whether antidepressants amitriptyline and sertraline can affect histamine metabolism. their effects on enzyme activity and mrna expression were studied in guinea pig tissues. plasma and tissue homogenates were incubated with saline (control) and different antidepressant concentrations. specific enzymatic activities of dao and hnmt were determined by radiometric assay. in addition, guinea pigs were treated with saline or amitriptyline (4 mg/kg, ip), afterwards dao and hnmt mrnas were detected by pcr in different tissues. results showed thatamitriptyline, 100 nm, 50, 100 and 500 mm, increased guinea pig plasma dao activity by 5, 7, 17 and 11 %, respectively, while sertraline increased it at 30 mm (by 15 %). at higher concentrations (100 and 500 mm) sertraline decreased dao activity. in the guinea pig tissues hnmt activity changes were found only when incubated with amitriptyline; sertraline had no effect. at 10 and 50 nm amitriptyline the activity of hnmt increased by 20 and 9 %, respectively. in animals treated with amitriptyline an induction of dao and hnmt mrna expression was noticed in several tissues. our results suggest that in guinea pigs due to higher histamine metabolism antiinflammatory effects can be expected at lower concentrations of antidepressants. the effect might be the opposite with higher amitriptyline concentrations. steven hefeneider(1,2), c macarthur (1), d trune (1), s mccoy (2) (1) oregon health and science university, portland, oregon, usa (2) targeted gene delivery, inc., portland, oregon, usa engagement of toll-like receptors (tlrs) by bacterial components such as lps and dna initiates inflammation.the current study examines a novel anti-inflammatory peptide, termed p13, for treatment of inflammation induced by either lps or bacteria.peptide p13 was derived from an immunoregulatory protein of vaccinia virus, and interferes with tlr signaling.in this study we examined the efficacy of p13 to limit inflammation in a mouse model of sepsis and a model of middle ear inflammation, termed acute otitis media (aom).we demonstrate in the sepsis model, that in vivo treatment of mice with p13 inhibited lps-induced production of serum inflammatory mediators.moreover, p13 treatment, administered after initiation of inflammation, significantly increased survival of mice injected with lps.in the aom model, peptide p13 significantly reduced in vivo middle ear inflammation and fluid accumulation initiated by h. influenza.assessment of route of administration and delayed treatment studies demonstrated the efficacy of peptide p13.simultaneous injection of bacteria and peptide p13 resulted in a significant reduction in fluid accumulation, infiltrating cells, and tympanic membrane thickness.fluid accumulation within the eustachian tubes was also significantly reduced following p13 treatment.-subcutaneous and oral administrations of p13, but not intravenous administration, were also efficacious in reducing inflammation. administration of p13 after initiation of an ongoing inflammatory response was effective at reducing inflammation and fluid development.taken together, these results demonstrate the therapeutic potential of peptide p13 to limit an inflammatory response and suggest a possible new treatment strategy for bacterial-induced inflammation. (1), c zhou(2), y zhang(2), m sun(2), x wan (1), h yu(2), x yang(2), rd ye (3), j-k shen (1) formyl peptide receptor-like 1 (fprl1) is a structural homologue of fpr, which binds chemotactic peptides of as small as 3 amino acids (e.g., fmet-leu-phe, fmlf) and activates potent bactericidal functions in neutrophils. in comparison, fprl1 ligands include peptides of 6-104 amino acids, such as trp-lys-tyr-met-val-[d]met (wkymvm) and other synthetic peptides. to determine the core peptide sequence required for fprl1 activation, we prepared various analogues based on wkymvm and evaluated their bioactivities in an fprl1-transfected cell line. although substitution of d-met6 resulted in loss of activity, removal of val5 together with d-met6 produced a peptide that retained most of the bioactivities of the parent peptide. the resulting peptide, wkym, represents a core structure for an fprl1 ligand. further substitution of lys2 with nle slightly improved the potency of the tetrapeptide, which becomes a dual agonist for both fprl1 and fpr. based on these structure-activity studies, we propose a model in which the modified tetrapeptide trp-nle-tyr-met (wnleym) binds to fprl1 through aromatic interactions involving the side chains of trp1 and tyr3, hydrophobic interaction of nle2, and the thio-based hydrogen bonding of met4, with the respective residues in fprl1 which have not been identified. the identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for fprl1-related disorders.there is a growing awareness of the interaction of food constituents with the immune system. the present study aims to evaluate immunomodulatory effects of two of these nutritional components, i.e. glycine and lactoferrin. mice orally supplemented with glycine, lactoferrin or a combination were injected intradermal (in the ear) with zymosan. ear swelling, as a measure for inflammation, as well as il-1, tnf-a and il-6 levels in the ear and the number of tnf-a producing spleen cells were analyzed.-glycine and lactoferrin were able to decrease the zymosan induced inflammatory response locally (decreased ear swelling and pro-inflammatory cytokine levels) as well as systemically (reduced number of tnf-a producing spleen cells).glycine effects (20, 50 and 100 mg/mouse/day) were concentration dependent whereas for lactoferrin only the lowest doses (0.1 and 1 mg/mouse/ day) inhibited the inflammatory response significantly. surprisingly higher doses of lactoferrin (5 and 25 mg/ mouse/day) failed to influence the inflammatory reaction. a combination of both nutrients (lactoferrin 0.1mg/ mouse/day in combination with glycine 20 or 50 mg/ mouse/day) inhibited the zymosan induced ear swelling synergistically. additionally an additive effect of both components was seen on the number of tnf-a producing spleen cells. the present data show anti-inflammatory activity of glycine and lactoferrin using the zymosan induced inflammation model.moreover a combination of both components demonstrated a synergistic effect on inflammation of the skin and an additive effect on the number of tnf-a producing spleen cells. (1), p sambrook(1), k fukudome(2), m xue (1) (1) university of sydney, st leonards, nsw, australia (2) saga medical school, saga, japan objectives: to investigate the i) expression of endothelial protein c receptor (epcr) in synovial membrane and peripheral blood monocytes from patients with rheumatoid arthritis (ra) and osteoarthritis (oa) and ii) role of epcr and its ligand, activated protein c (apc), on the function of monocytes from ra patients.methods: epcr, cd68 and pc/apc in synovial tissues were detected by immunostaining and in situ pcr. monocytes were isolated from peripheral blood of patients with ra and treated with apc, lipopolysaccharide (lps), and/or epcr blocking antibody, rcr252. cells and supernatants were collected to analyze the expression/activation of epcr, nuclear factor nf-kb and tumour necrosis factor tnf-a.results: epcr was expressed by both oa and ra synovial tissues but was markedly increased in ra synovium. epcr was colocalized with pc/apc mostly on cd68 positive cells in synovium. in ra monocytes, apc upregulated epcr expression reduced monocyte chemoattractant protein-1-induced chemotaxis of monocytes by approximately 50%. apc also completely suppressed lps-stimulated nf-kb activation and attenuated tnf-a protein by more than 40% in ra monocytes. the inhibitory effects of apc were reversed by rcr252, indicating that epcr modulates the inhibitory effects of apc.conclusions: our results demonstrate for the first time that epcr is expressed by synovial tissues, particularly in ra, where it co-localizes with pc/apc on monocytes/ macrophages. in addition, apc inhibits the migration and activation of ra monocytes via epcr. these inhibitory effects on ra monocytes suggest that pc pathway may have a beneficial therapeutic effect in ra. key: cord-006230-xta38e7j authors: nan title: deutsche gesellschaft für experimentelle und klinische pharmakologie und toxikologie e.v. date: 2012-02-22 journal: naunyn schmiedebergs arch pharmacol doi: 10.1007/s00210-012-0736-0 sha: doc_id: 6230 cord_uid: xta38e7j nan nucleoside diphosphate kinases (ndpks) are multifunctional enzymes involved in a variety of cellular processes including cancer metastasis and heart diseases. the plasma membrane content of the three major ndpk isoforms ndpk a, b and c is increased in human heart failure. we have previously shown that the ndpk b isoform regulates camp levels and cardiac contractility through a receptor-independent gprotein activation involving direct g protein β subunit phosphorylation. the precise role of ndpk c in the heart is unknown and was the object of this study. ndpk c function was assessed in neonatal (nrcm) and adult (arcm) rat cardiomyocytes with real-time pcr, immunoblotting, and quantification of camp content. heart failure was induced by chronic treatment with isoproterenol (iso, 2.4 mg/kg/d 4 days) via minipumps. chronic iso increased mrna levels of ndpk c by 9.4±1.9-fold and its protein levels by 2.1±0.11-fold. immunoprecipitation of the g protein β subunit resulted in coimmunoprecipitation of ndpk c and the stimulatory gαs subunit: iso enhanced this interaction. upon iso stimulation, ndpk c translocated from the cytosol to the plasma membrane within 3 hours in both nrcms and arcms. adenoviral overexpression of ndpk c in nrcms caused a 1.5-fold increase in basal and iso induced camp synthesis, whereas sirna mediated knockdown of endogenous ndpk c decreased camp levels by ~50%. our results establish ndpk c as a novel and critical regulator of camp synthesis and gs signaling in the heart. the up-regulation of ndpk c and the increased responsiveness to iso in failing hearts point to ndpk c as a potential counterregulatory factor in the onset of heart failure. uptake and metabolism of methylated myricetin derivatives: studies in cell culture and c. elegans ackermann d. 1, 2 , büchter c. secondary plant compounds like flavonoids that are ubiquitary present in fruits and vegetables are believed to exert health protective effects in terms of lowering the incidence of widespread diseases such as cardiovascular diseases and cancer. besides their antioxidative effects, flavonoids may also modulate cell signaling pathways and thereby performing their disease-protective actions. though this class of dietary polyphenols has become increasingly popular as dietary supplements, only little is known about their metabolic fate in vivo. therefore, we investigated the absorption and metabolism of several flavonoids such as myricetin and its methylated derivatives laricitrin, syringetin, and myricetin-3',4',5'-trimethylether in the human colon carcinoma cell line hct116 and the human hepatoma cell line hepg2 as well as the model organism caenorhabditis elegans. all flavonoids were rapidly taken up by both cell lines as shown by hplc analyses. the intracellular amount of myricetin and laricitrin did not increase with time and was only half of that of syringetin and myricetin-3',4',5'-trimethylether. interestingly, no metabolites of these flavonoids could be detected which might at least in part be due to their low intracellular amounts. absorption as well as intracellular distribution was also evidenced by using the fluorescent dye "naturstoff reagent a" nsra) . fluorescence microscopy indicated a predominant cytosolic distribution of the employed flavonoids. in the model organism caenorhabditis elegans, the flavonoids were exclusively distributed in the intestine as visualized by nsra. the antioxidative capacity of the four flavonoids (measured by using the cell free teac assay and the h 2dcf-da assay in hct116 cells) decreased with increasing methyl groups in the b-ring. myricetin-3',4',5'-trimethylether (three methyl groups) was the least effective radical scavenging flavonoid in both the cell free system and in hct116 cells compared to myricetin (no methyl groups). in conclusion, for exerting their biological effects, uptake and distribution as well as metabolism of flavonoids in certain organs such as liver and gut are important and more research in that field is warranted. munich heart alliance, münchen, germany signaling through g protein-coupled receptors is affected by receptor polymorphisms, yet the molecular basis for the functional differences of individual receptor variants is unclear. to investigate the impact of the frequent gly389arg variant of the β1-adrenergic receptor (β1ar) on receptor conformation we used β1ar-sensors capable of fluorescence resonance energy transfer (fret). these sensors retained the pharmacological and functional characteristics of the native receptors. upon stimulation of the sensors we determined the activation characteristics of the polymorphic receptors in real time and in living cells. we found the β1ar variants to behave similar upon a single stimulation with an agonist, but to differentially respond with a change of their activation kinetics during subsequent stimulations. while the arg389-β1ar did not show altered activation kinetics after prestimulation, the gly389-β1ar became slower compared to the initial stimulation suggesting that β1ars possess a memory of previous activation. we then permeabilized β1ar-sensor-expressing cells with saponin to remove soluble cytosolic factors. upon permeabilization the β1ar variants did not display receptor memory, suggesting that the β1ar memory depended on the interaction of the receptors with soluble cytosolic factors upon their initial activation including the phosphorylation of agonist-bound receptors by protein kinase a or g protein-coupled receptor kinases. our findings suggest an intrinsic, polymorphism-specific property of βars that alters activation kinetics upon continued stimulation and that might account for individual drug responses. micro-rna replacement therapy: nanoparticle-mediated in vivo delivery of mirna-145 or mirna-33a exerts antitumor effects in colon carcinoma xenograft mouse models weirauch u. 1 micro-rnas (mirnas) control the expression of various genes, and under pathological conditions several mirnas are up-or downregulated. previous in vitro studies have established a pro-apoptotic and anti-proliferative role of mir-145, which shows decreased levels in colon carcinoma. in contrast, while mir-33a is only weakly expressed in several tumors as well, its role in cancer has not been analysed so far. in this study, we demonstrate the tumor-relevance of mir-33a and identify the protooncogenic kinase pim-1 as a target of mir-33a. pim-1 harbours a highly conserved mir-33a binding site within its 3'-utr, and seed mutagenesis of this target sequence abolishes the mir-33a-mediated downregulation of pim-1. the knockdown of pim-1 by rnai or mirna transfection inhibits proliferation in leukemia and in colon carcinoma cells by decelerating cell cycle progression, thus establishing a tumor inhibitory function of mir-33a. we furthermore introduce polyethylenimines (peis) for the therapeutic application of mirnas in vivo, which is critically dependent on the development of appropriate delivery tools. peis are able to form non-covalent complexes with mirnas, leading to mirna protection after systemic application in combination with an attractive biodistribution profile and the efficient uptake in target organs/cells. therapeutic effects of pei-mediated mirna delivery were demonstrated in subcutaneous colon carcinoma xenograft mouse models. the in vivo application of mirna-145 through systemic or local injection of pei/mirna complexes resulted in efficient mirna delivery and in antitumor effects, based on the concomitant repression of erk5. likewise, tumor growth inhibition was observed upon treatment of tumor-bearing mice with pei-complexed mir-33a. this is due to the mir-33a-mediated downregulation of pim-1 expression and resembles the pim-1 knockdown through rnai / pim-1 sirnas. taken together, in tumor xenograft mouse models we establish mirna replacement therapy through the pei-complexation of mirnas as a novel therapeutic strategy and demonstrate that mir-145 and mir-33a may be promising mirnas in colon carcinoma therapy. md 288, a hybrid of chloroquine and primaquine, is a potential drug against infectious diseases such as malaria. since one moiety of the hybrid, the known antimalarial drug chloroquine, is a known intercalator, the potential of md 288 to intercalate into dna was determined. due to the ability of intercalators to cause frame shift mutations, the mutagenic potential of md 288 was also investigated. the potential of md 288 to intercalate into dna was investigated by means of fluorescence based micro plate assay using ethidium bromide (eb) and isolated calf thymus double stranded dna. as a positive control chloroquine was used. the potential of md 288 to cause gene mutations was determined using the hypoxanthine-guanine phosphoribosyltransferase (hprt) test in chinese hamster v79 lung fibroblasts (v79 cells). v79 cells were treated with 0.4 µm, 0.8 µm and 1.5 µm md 288 or the positive control, the direct mutagen 4-nitroquinoline-n-oxide (nqo, 1 µm) for 24 h. on day 6, mutants exhibiting loss of hprt function were selected with 6-thioguanine . whereas at 1 µm chloroquine, a 38% decrease in fluorescence intensity of eb (indicating dna intercalation) was observed, 10 µm md 288 were needed to observe a similar decrease (28%) in fluorescence intensity of eb. therefore, md 288 is 10fold less potent to intercalate into dna than its moiety chloroquine. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells was 9 ± 2. as expected, 1 µm nqo caused a significant increase in the mutant frequency (mf, 168 ± 9) . in contrast, mf was not significantly affected by treatment with md 288 at both noncytotoxic (0.4 µm: 5 ± 4) and cytotoxic (0.8 µm: 9 ± 7) concentrations. in conclusion, md 288 is a less potent intercalator than the known antimalarial drug chloroquine. furthermore, md 288 does not cause gene mutations in the hprt test. since current studies show that various metabolites of md 288 are formed in vitro, their mutagenic potential is currently under investigation as well. -conducting channels but depends critically on the membrane potential. trp channels form cation entry channels thereby either contributing to ca 2+ entry or depolarisation. recently, we showed that trpm4 acts as a ca 2+ -activated non-selective cation channel and critically determines the driving force for ca 2+ influx in mast cells following fcεri-stimulation (1) . in addition to trpm4 we also identified the expression of other trp transcripts in bone marrow derived mast cells (bmmc) including those encoding trpc2, trpc3, trpc5, trpc6 and trpm7. in peritoneal mast cells (pmc), rt-pcr indicated expression of trpc1, trpc4, trpc5, trpc6, trpm2, trpm4 and trpm5. to identify the functional role of those trp channel proteins for mast cell activation we analysed ca 2+ signaling using microfluorimetry in bmmcs and pmcs after stimulation with substances known to activate trpc6 channels in other cell systems such as the diacylglycerol analogue oag, the hyperforin analogue hyp-9 and flufenamic acid (ffa), but could not evoke a rise in the [ca 2+ ]i in both pmc and bmmc. sphingosine 1phosphate and lysophosphatidylcholine, which were reported to activate trpc5 channels, induced only minor rise in [ca 2+ ]i in bmmcs, respectively. here, we will present our analysis of ca 2+ signaling following stimulation of the fcεri receptor and application of secretagogues that are supposed to affect ca 2+ -dependent mast cell activation such as adenosine, endothelin-1, substance p and compound 48/80 in bmmcs and pmcs derived from mouse lines with inactivation of trpc1, trpc3, trpc4, trpc5 or trpc6 since specific antagonists are still lacking for these trp channels. the α2a-ar is the main ar in the central nervous system and it plays a crucial role in regulating norepinephrine (ne) release from nerve terminals via presynaptic feedback inhibition. it is also associated with a number of physiological effects, including hypotension, pain perception, sedation and modulation of mood. ne, once released in the synaptic space, binds to α2a-ars and induces a rearrangement of the receptors from the inactive state into an active conformation. this allows the binding and activation of the cognate gi-protein and, hence, the transduction of the transmembrane signal to the downstream effectors. generally, α2a-ar activation has been deduced from the stimulation of a receptor-mediated biological response that could be easily followed experimentally. however, most of these approaches do not employ living cells and are normally applied under equilibrium conditions that need prolonged incubation periods incompatible with the physiological temporal dynamics of ne. here, we monitored the ne-mediated α2a-ar and gi-protein activation by using a fluorescence resonance energy transfer (fret)-based approach in living cells. to examine the effects of increasing concentrations of ne on the speed and extent of α2a-ar activation with very high temporal resolution, we took advantage of the previously described α2a-ar flash/cfp sensor [1] . the results indicate that in our system the efficacy of ne in eliciting α2a-ar flash/cfp activation increases in a time-dependent way and reaches the maximum with a half-life of ~ 70 ms. the ec50 values decrease in an exponential manner and arrive at ~ 2 µm with a half-life of ~ 330 ms. next, we analyzed the ability of increasing concentrations of ne to trigger a downstream intracellular response after α2a-ar stimulation by monitoring the kinetics and amplitude of gi activation in living cells. we applied the previously well characterized gi cfp/yfp sensor [2] . the results show that both the efficacy and the potency of ne in inducing gi activation reach the steady state slower compared to receptor activation (half-life ~ 700 ms and ~ 3,000 ms respectively). in conclusion, we were able to monitor ne-mediated events occurring in the millisecond time scale and reaching the equilibrium in a time interval compatible with physiological conditions. sphingosine-1-phosphate (s1p) is an immune modulator produced by sphingosine kinase 1 (sphk1) and sphingosine kinase 2 (sphk2) and de-phosphorylated or degraded irreversibly by s1p phosphatases and a lyase, respectively. we recently showed that tlr4-induced il-12p70 is selectively counter regulated by sphk1, s1pr1 and its extracellular ligand s1p. on the other hand, spiegel et al. have demonstrated that specific, sphk1-dependent, binding of s1p to traf2 enhances the tnf-alpha signaling. therefore we were interested whether the tlr/tir and tnf-alpha-signaling pathways are interfered with each other and are modulated by s1p. in a first approach we focused our investigations on sphk1 effects on both traf2 and traf6 stimulatory signals and cytokines produced downstream. experimentally, with gm-csf expanded, bone marrow-derived dcs we first desensitized the lps-tlr4 or cpg-tlr9 signal by a defined time period of costimulation with tnf-alpha. the initial results showed a partial decrease of il-12p70 secretion in tnf-alpha-co-stimulated dcs in contrast to lps stimulation alone. this might indicate that traf2 activated via tnf-alpha interacted with the traf6 pathway to reduce il-12p70. further series with dcs derived from sphk1-deficient mice confirmed our former results that il-12p70 in contrast to other cytokines is specifically sensitive to sphk1-s1p feedback, but did not change the effects of tnf-alpha on wt dcs il-12p70 release. in comparison, cpg-tlr9-induced il-12p70 release reached only 20% of lps-induced il-12p70 levels and was less sensitive to tnf-alpha costimulation. however, sphk1-deficiency strongly augmented cpg-dependent il-12p70 production. in ongoing experiments we started to analyze the details of traf2/rip1 and traf6/tak1 activation by ubiquitination blots in wt, sphk1-and s1plyase-deficient dcs. in conclusion, we hope to unravel possible mechanisms of the observed differential effects of s1p and its enzymes on inflammation and cancer-relevant cytokines. identification of the kh type splicing regulatory protein (ksrp) as a new important mediator of the anti-inflammatory effects of resveratrol art j. 1 , besche v. 2 , bros m. 2 , li h. 1 , handler n. 3 , bauer f. 3 , erker t. 3 , behnke f. 4 , mönch b. 5 , förstermann u. 1 , dirsch v. m. 6 , werz o. 5 , kleinert h. 1 , pautz a. university of vienna department of pharmacognosy, althanstr. 14, 1090 wien, austria resveratrol, a polyphenol derived from different plants, possesses multiple pharmacological functions such as anti-oxidative, anti-diabetic, cardioprotective, anticancer, neuroprotective and anti-inflammatory properties. many of these effects have been attributed to its anti-oxidative activity but resveratrol also modulates signal transduction pathways like the p38 mapk pathway or the activity of different transcription factors like nf-κb redox-independently. moreover, the histone deacetylase sirtuin 1 (sirt1) is an important mediator of resveratrol effects. nevertheless the direct molecular target of resveratrol remains unclear. in target fishing experiments we identified the rna-binding protein ksrp as direct resveratrol binding partner. ksrp is an rna-binding protein that controls proinflammatory gene expression on the post-transcriptional level by modulation of mrna stability. moreover, it is involved in the biogenesis of mirnas. resveratrol treatment of human dld-1 cells resulted in a decreased mrna expression of a number of well known ksrp target mrnas and enhanced mirna-155 function. downregulation of ksrp expression by sirna prevented the mrna destabilizing effect of resveratrol. as the activity of ksrp is mainly regulated on the post-translational level by phosphorylation of different serine and threonine residues we analyzed whether resveratrol changes ksrp activity by altering the phosphorylation of the protein. indeed, our immunoprecipitation experiments demonstrated that resveratrol reduces the p38 mapk-mediated phosphorylation of threonine residues in the ksrp protein and thus leads to an increase of ksrp activity. interestingly, resveratrol does not block p38 mapk activation or activity. in addition we have evidence that sirt1 is not involved in the resveratrol mediated activation of ksrp. so we believe that activation of ksrp by resveratrol is the major mechanism mediating the anti-inflammatory effects of resveratrol. in vitro testing of oecd reference nanomaterials (nm-series) in rat precision cut lung slices aumann a. 1 the oecd has defined reference nanomaterials (nm) to be tested in different endpoints concerning human health and environmental safety (1) in order to evaluate if the toxicity of nanomaterials can be linked to their physico-chemical properties. for nanomaterials, inhalation presents the major exposure route of concern and can be assessed using acute inhalation toxicity studies in rodents. however, these in vivo studies are resource intensive and animal consuming. the oecd working party on nanomaterials has named several alternative methods as being of particular interest for testing of nanomaterials; among them is the precision-cut lung slices model (pcls) to estimate respiratory toxicity. we have tested all 16 nm in pcls measuring cytotoxicity, apoptosis, oxidative stress and inflammatory response of the tissues as well as observing them histological. for in vitro exposure of pcls the test material was dispersed in medium. since it is the nature of these materials to change their surface characteristics and agglomeration state in different environments, a standardized dispersion method (nanocare) using bovine serum albumin as a stabilizing agent, was used. particle size-distributions of the nanomaterial dispersions were characterized via analytical ultracentrifugation and found the nanomaterials well dispersed. silver and zinc oxide but none of the other nm showed cytotoxicity to the lung tissue in the tested concentrations. however, differences in cytokine profiles among the nm were observed and showed several correlations to the results obtained in in vivo inhalation or instillation studies. universität des saarlandes institut für molekulare zellbiologie, gebäude 61, 66421 homburg, germany tmem2 proteins show similarities in their primary sequence to motifs that are conserved amongst various members of the trp protein family. based on hydropathy analysis these proteins exhibit 6 to 10 membrane spanning domains. in contrast to trp channels there is no evidence that these proteins form ion channels in the plasma membrane following overexpression of their cdna in hek293 cells. tmem2 -/mice are viable and show no obvious signs of disease, but exhibit increased pancreatic amylase and lipase plasma levels. microfluorimetric measurements using fura-2 revealed that the elevation of the cytosolic ca 2+ concentration after stimulation with carbachol and the cholecystokinin analogue caerulein is unchanged in tmem2-deficient acinar cells. tmem2 is expressed in several cell types including pancreatic acinar cells, cardiac myocytes, cardiac fibroblasts, but their subcellular localization is still unkown. we generated several constructs encoding tmem2 fusion proteins with fluorescence protein tags by fusing eyfp, mcherry and tagrfp-t to the n-and c-terminus of the protein, respectively. based on western blot experiments and expression in hek293 cells the tmem2-eyfp construct was most suitable for further colocalisation analysis and generation of viral vectors including adenovirus and semliki forrest virus. in contrast to the prediction by the psort ii algorithm tmem2-eyfp could not yet be identified in the plasma membrane of fibroblasts, cardiac myocytes or acinar cells but showed a vesicular subcellular localization pattern. we localized tmem2-eyfp in acidic compartments and predominantly in lysosomes (pearson coefficient (pcc) 0,79 ± 0,03, n= 4 using lysotracker ® dye). analysis of subcellular localization with independent tmem2 fusion constructs and additional vesicular markers will be presented as a framework to get insights towards the cellular function of tmem2 and to reveal the mechanisms underlying increased amylase release from acinar cells of tmem2 -/mice. the small molecule bcl-2/mcl-1 inhibitor tw-37 shows single-agent cytotoxicity in neuroblastoma cell lines bachmann h. s. 1 , akdeli n. high-risk neuroblastoma (nb) remains a therapeutic challenge in paediatric oncology. pro-survival bcl-2 family proteins critically regulate apoptosis, and may represent important therapeutic targets in nb. primary nb tumours heterogeneously express mcl-1 or bcl-2, with high expression correlating to high risk phenotype. co-expression can be detected in approximately 10% and is correlated to reduced survival. recent studies with two inhibitors that predominantly target bcl-2 and other proteins, but not or to a lesser extend mcl-1, elucidated the importance of mcl-1 inhibition for cytotoxicity in nb. tw-37 is a small molecule inhibitor that showed almost equal affinity to bcl-2 and mcl-1. to explore the effect of combined bcl-2/mcl-1 inhibition on neuroblastoma cells, four cell lines (sk-n-as, imr-5, sy5y and kelly) were treated with tw-37 and changes in growth properties were determined. furthermore, nude mice with kelly (human neuroblastoma cell line) xenografts were treated with tw-37. using sirna, we investigated the functional relevance of mcl-1 and bcl-2 in kelly cells. for in vitro cell viability we observed ic50 values of 0.59 ± 0.39 µmol/l. on treatment with 1 µmol/l dose of tw-37, all neuroblastoma cell lines analyzed showed significantly reduced proliferation and increased apoptosis rates. bcl-2 as well as mcl-1 knockdown induced apoptosis in kelly cells. interestingly, tw-37 was able to reduce, but not to abrogate growth of kelly neuroblastoma xenografts in nude mice. in conclusion, combined inhibition of bcl-2 and mcl-1 using tw-37 exhibits strong single-agent antitumor activity on human neuroblastoma cells in vitro, but limited single-agent activity in vivo. therefore, inhibition of bcl-2/mcl-1 may represent an interesting therapeutic strategy, most likely in combination with conventional chemotherapy and other specific inhibitors. localization and functional characterization of membrane transporters for sulfated steroid hormones in the human testis bakhaus k. 1 , wapelhorst b. circulating sulfated steroid hormones like estrone sulfate (e1s) or dehydroepiandrosterone sulfate (dheas) are delivered to the testis via membrane uptake carriers such as the sodium-dependent organic anion transporter (soat). inside the cell these sulfated steroids can be metabolized to active steroid hormones by the catalytic activity of the steroid sulfatase (sts), which shows high enzymatic activity in the testis ("sulfatase pathway"). in addition to soat, other candidate carriers like the organic solute carrier protein 1 (oscp1) and the organic anion transporting polypeptides oatp6a1 and oatp1c1 are predominantly expressed in the human testis and demonstrate transport activity for sulfated steroids. we aimed to evaluate the cellular expression of soat and the other steroid sulfate carriers and their co-localization with the steroid sulfatase (sts) in human testis. furthermore we want to perform functional transport studies with the steroid sulfate carriers in stably transfected hek293 cells. we detected soat by rt-pcr and western blot analysis in the human testis. single cell analysis and in situ hybridization revealed pachytene primary spermatocytes to express the soat mrna. soat expression in specimens showing maturation arrest at the level of early round spermatids seems to be severely reduced or absent. sts mrna was detected by rt-pcr in testis homogenates. preliminary immunohistochemical data showed that sts may be expressed in germ cells and interstitial leydig cells. hek293 cells stably expressing the soat carrier protein showed significant transport activity for dheas. this was demonstrated by using a radiolabeled [ 3 the hepato-intestinal induction of the detoxifying enzymes cyp3a4 and cyp3a5 by the xenosensing pregnane x receptor (pxr) constitutes a key adaptive response to oral drugs and dietary xenobiotics. in contrast to cyp3a4, cyp3a5 is additionally expressed in several, mostly steroidogenic organs, which creates potential for induction-driven disturbances of the steroid homeostasis. using cell lines and mice transgenic for a cyp3a5 promoter we demonstrate that the cyp3a5 expression in these organs is noninducible and independent from pxr. instead, it is enabled by the loss of a suppressing yin yang 1 (yy1)-binding site from the cyp3a5 promoter which occurred in haplorrhine primates. this yy1 site is conserved in cyp3a4, but its inhibitory effect can be offset by pxr acting on response elements such as xrem. taken together, the loss of yy1 binding site from promoters of the cyp3a5 gene lineage during primate evolution may have enabled the utilization of cyp3a5 both in the adaptive hepato-intestinal response to xenobiotics and as a constitutively expressed gene in other organs. our results thus constitute a first description of uncoupling induction from constitutive expression for a major detoxifying enzyme. they also suggest an explanation for the considerable tissue expression differences between cyp3a5 and cyp3a4. serum albumin adducts as biomarkers for systemic bioavailability of active metabolites of various glucosinolates in animal models and humans barknowitz g. 1 , engst w. glucosinolates (gls) are natural pesticides of brassicales, which comprise many important food and feed plants. upon physical damage to the plant, the enzyme myrosinase can convert gls to reactive metabolites (e.g. isothiocyanates). the same reaction can also be catalyzed by enzymes of the intestinal microbiota. modification of sensor proteins (e.g. keap-1) by some gls metabolites leads to adaptive responses, resulting in enhanced detoxification of reactive metabolites and other protective reactions. at least in experimental models, this mechanism can be exploited for chemoprevention of carcinogenesis induced by various chemical carcinogens. however, own research revealed that certain reactive gls metabolites can covalently bind to dna in vitro and in vivo, involving possible genotoxic and carcinogenic risks. animal models are useful for studying beneficial and adverse effects of gls. however, it has to be taken into account that the toxicokinetics of gls may differ between rodent and humans and that the active metabolites are short lived and thus difficult to detect and quantify. likewise, exposure of humans to gls and their breakdown products may enormously vary depending on (i) food preferences, (ii) cultivars, growth conditions and preparation of plants consumed and (iii) variations in human xenobiotic metabolizing system and composition of intestinal microbiota. in order to estimate individual levels of systemic exposure to reactive gls metabolites, blood protein adducts may be useful. we have developed lc-ms/ms methods for quantifying serum albumin adducts formed by glucoraphanin, glucotropaeolin and neoglucobrassicin. the method involves digestion of the protein to amino acids and the usage of isotope-labelled amino acid adducts as internal standards. serum albumin adducts were detected in mouse models after feeding broccoli and pak choi respectively, as well as after administration of purified gls and breakdown products. likewise, gls adducts were detected in human blood plasma after consumption of broccoli or cress. this work was financially supported by the bundesministerium für bildung und forschung (grant 0315370d) . barlow s. harrington house, 8 harrington road, brighton, bn1 6re, great britain values for cancer endpoints for the application of the ttc approach have been derived by linear extrapolation of results from animal carcinogenicity studies to calculate "virtually safe doses" (vsds). a vsd is defined as an exposure that represents an estimated upper bound increase in risk of 1 in a million of developing cancer during a lifetime. in 1995, from consideration of a range of vsds for carcinogens, the us food and drug administration (fda) proposed and adopted a value of 0.5 micrograms/kg of diet (0.5 ppb) as a threshold of regulation to be used for substances present in food contact materials. the fda considered that if exposure to a substance in the diet was below this value, consumers would be protected "with reasonable certainty of no harm" and no toxicological data on the substance need be submitted. the value of 0.5 ppb is equivalent to 1.5 micrograms/person per day, assuming that 1500 g of food and 1500 g of fluids diet might be consumed daily. this value was subsequently incorporated into the ttc approach to be used for assessment of substances without a structural alert for genotoxicity. in 2004, kroes and colleagues further explored cancer as an endpoint and recommended a lower ttc value of 0.15 micrograms/person per day for substances with a structural alert for genotoxicity and exclusion from the ttc approach of certain groups of high potency carcinogens (with vsds below this value). in this presentation, the data underpinning these ttc values will be discussed from the perspective of their reliability for risk assessment of substances with low exposures, for which there are no toxicity data, but which may in fact be genotoxic or non-genotoxic carcinogens. stable conjugates which are recognized by the immune system. in the current investigations, the ability of chemical pre-treatment to interfere with antibody-protein binding has been investigated using ovalbumin (ova) as the model protein and naturally occurring anti-ova igg from healthy human donors. preparation of conjugates: ova (1 mg/ml) was dissolved in sodium borate buffer (0.1m, ph 9.4) . for chemical treatment various amounts (50-200 mg) of 1-fluoro-2,4dinitrobenzene (dnfb; sensitizer) or 2,4-dichloro-1-nitrobenzene(dcnb; non-sensitizer) was added and stirred for 2 hrs at room temperature. unbound compound was removed by consecutive dialysis against phosphate buffered saline (pbs) and distilled water. inhibition elisa: plates were coated with 100 µg/ml ova and blocked with 10% fcs in pbs. ova samples (native or conjugated; 0.6 -10 µg/ml) were pre-incubated with polyclonal antibodies (ab) from pooled normal human serum for 30min and then added to the plates. human anti-ova igg abs were detected by colorimetric analysis using ortho-phenylendiamine as a substrate. the concentration of soluble native ova or conjugate required to displace 50% of ab to plate-bound ova (ic50) was calculated (minimum of n=3 independent experiments). treatment of ova with the chemical sensitizer and protein reactive dnfb resulted in increased ic50 value, whereas mock treatment resulted in comparable ic50 values to native ova. treatment with dcnb showed that the presence of a chemical per se (and possible denaturation) was not sufficient to alter the ic50 value; conjugation of the compound to the protein was required. the analysis is not test chemical specific (specific ab against compound-protein conjugates are not required) and the colorimetric analysis is unaffected by absorbance of the compound itself. thus, this method may have utility for the identification of chemical sensitizers which are directly protein reactive. 2´-deoxy-camp in human cell lines: another second messenger? beckert c. 1 , hinz c. we have shown that 2´-deoxy-camp (dcamp) is synthesized by recombinant human soluble adenylyl cyclase (sac). here, we report that dcamp can be detected and quantified by liquid chromatography coupled to mass spectrometry (lc-ms) in various human cell lines. in most cells a ratio of camp : dcamp of ~10 was observed. as a remarkable exception, in hl-60 promyelocytic leukemia cells, the dcamp concentration exceeded the camp concentration more than 3-fold. a differential regulation of camp versus dcamp was determined upon replacement of the incubation medium (proliferating condition with serum / serum-free resting condition). for example, camp was dramatically reduced in hek293 cells after 24 hours under resting conditions whereas dcamp was significantly increased. in cellular subfractions of hek293 cells ac assays (mn 2+ /forskolin-or mn 2+ /bicarbonate-stimulated) with either atp or datp as substrate revealed that comparable amounts of camp and dcamp accumulated. in addition to sac, membranous acs such as ac v were capable of forming dcamp with vmax and km values for datp comparable to those for atp. we also analyzed the substrate-specificity for several human phosphodiesterases. pde3 and pde4 hydrolyzed dcamp more effectively than camp. taken together, these data point to a putative second messenger role of dcamp in human cells. we are currently investigating the regulatory role(s) of camp and dcamp in apoptosis of hek293 cells. as a novel tool for these studies, we will use the cellpermeant dcamp-acetoxymethylester which penetrates the plasma membrane and releases dcamp intracellularly. the biogenic amine histamine is recognized by target cells via four different histamine receptors subtypes (h1r -h4r), which all belong to the family of g-protein-coupled seven-transmembrane receptors. histamine plays a crucial role in allergic reactions such as rhinitis or conjunctivitis and also in allergic asthma. previously, we showed an interaction of the effects of antagonists at the h1r and h4r in a mouse model of allergic asthma. however, not much is known about the signaling pathway activated by murine h1r and h4r. in order to analyze these signaling pathways, we established a cellular model using transfected hek 293 cells which stably express recombinant mh1r or mh4r. proper expression of the receptors was verified by western blot analysis and flow cytometry. in functional assays we demonstrated that histamine stimulation results in the increase of intracellular ca 2+ concentration ([ca 2+ ]i) in cells expressing either of both, mh1r (pec50 = 8.2) or mh4r (pec50 = 6.9). as a second readout, we analyzed the modulation of forskolin-induced camp-accumulation. in mh1r-expressing cells the intracellular camp concentration was increased by stimulation with histamine, while in mh4r-expressing cells forskolin-induced camp accumulation was reduced. the histamine-induced effects in h1r-expressing cells were blocked by the h1r antagonist mepyramine ([ca 2+ ]i: pkb = 8.6) and those in the h4r-expressing cells by the h4r antagonist jnj7777120 ([ca 2+ ]i: pkb = 8.8) or by pertussis toxin, which selectively blocks receptor gi-protein coupling. jnj7777120, which behaves as a partial mh4r agonist in the steady-state gtpase assay using membranes of infected sf9 cells, was without effect on [ca 2+ ]i and forskolin-induced camp-accumulation in the mh4r-expressing cells. currently, we are investigating mitogen-activated protein (map)-kinase pathways activated by the h1r and the h4r. using a phospho-map-kinase array, histamine dependent phosphorylation of erk1/2, p38, jnk, creb, pkb (akt), and mkk 3/6 were detected in cells expressing either of both, mh1r and mh4r. in summary, the hek 293 cell lines stably expressing selective histamine receptors are very useful tools to investigate hxr signaling pathways in-vitro. enhanced fibroblast motility in the absence of the β3 regulatory subunit of voltage-activated calcium channels belkacemi a. 1 cavβ subunits of voltage-activated ca 2+ channels are required for trafficking the poreforming cavα1 subunit to the plasma membrane and modulate the kinetics of its current. mouse embryonic fibroblasts (mefs), acutely isolated cardiac fibroblasts (cfs) and nih 3t3 fibroblasts do express cavβ2 and cavβ3 subunits, but we could not detect any voltage-activated ca 2+ influx. whereas in mouse cardiomyocytes or hek 293 cells coexpressing cavβ3 and cavα1 subunits a dihydropyridin-sensitive voltage-activated ca 2+ influx was readily detectable. apparently, cavβ subunits serve functions in fibroblasts unrelated to voltage-activated ca 2+ influx. among the proteins potentially interacting with cavβ3 are the inositol 1,4,5-trisphosphate receptors (ip3rs) [1, 2] . we therefore coexpressed mouse cavβ3 and mouse ip3r type 1, 2 or 3 in cos-7 cells and found coimmunoprecipitation of ip3rs using an antibody for cavβ3 and vice versa. to study the release of ca 2+ from ip3-sensitive stores we performed fura-2 measurements on fibroblasts isolated from wild type and cavβ3-deficient mice either in the presence of thapsigargin or after stimulation of gq-coupled receptors by par-1, lpa or bradykinin. receptor-activated ca 2+ release was more pronounced in β3-deficient mefs and cfs, whereas thapsigargin-induced ca 2+ release was the same in cells from both genotypes. in addition, ip3 production measured by a radioreceptor assay was already increased in β3-deficient cells under basal conditions. fibroblasts are migrating cells and involved in various physiological and pathophysiological processes. we therefore started in vitro assays for proliferation, migration and angiogenesis as well as in vivo assays for skin wound healing. angiogenesis and proliferation were apparently not different in both genotypes but migration (measured as transwell migration and in scratch assays) and wound healing were affected in different ways. fluorescent staining of cytoskeleton and quantification of the f-actin/g-actin ratio show similar results in both genotypes, suggesting that the increased migration rates and wound repair in β3 knockout may result, in part, from the increased amount of ip3-releasable ca 2+ . [1] berggren, yang, murakami, et al., removal of ca channel β3 subunit enhances caoscillation frequency and insulin exocytosis. cell 119, (2004) 273-284. [2] müller, haupt, bildl, et al., quantitative proteomics of the cav2 channel nanoenvironments in the mammalian brain. pnas 107, (2010) 14950-14957. bender-sigel j., closs e. i. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany human cationic amino acid transporters (hcat) are a family of multimembrane spanning proteins that mediate the transport of cationic amino acids through the plasma membrane. our earlier results have demonstrated that activation of either protein kinase c (pkc) by pma or cdc42 by egf leads to an internalization of these transporters. in addition, in a recent collaboration with the group of alexander sorkin (university of colorado denver) we found that ubiquitination and clathrin-dependent endocytosis are necessary for the down regulation of hcat-1-mediated arginine transport by pma (vina-vilaseca et al, j biol chem 2011 286:8697) . this mechanism requires nedd4 e3 ligases, but hcats do not contain a ppxy motif to bind the ligases, suggesting that an adaptor protein takes part in this process. however, an involvement of the adaptor protein beta-arrestin in this mechanism could be excluded. using sirna against pkc alpha we now show that pkc alpha is the major isoform that induces the reduction of arginine transport in human u373 glioblastoma cells overexpressing hcat-2a-egfp. in addition, sirna-mediated knock down of cdc42 prevented the decrease of hcat-2amediated transport induced by pma. taken together pkc seems to negatively influence the constitutive cycling of cats by activation the ubiquitination machinery and clathrinmediated endocytosis. cdc42 is part of this pathway. converging of the classical mitochondria-related pathway in parkinson and nuclear dna-repair signaling? scherr a. -l. 1 parkinson disease is the second most neurological disorder worldwide. despite the fact that most cases are idiopathic and only few can be traced back to specific genes, general progression between both tracks of the disease is comparable. the variety of clinical symptoms in motor control like tremor, rigor and postural problems all originate from loss of dopaminergic neurons in the substantia nigra pars compacta of the brain. several proteins mutated in pd are involved in surveillance pathways, monitoring functionality and integrity of proteins and organelles either by the proteasome degradation machinery or by clearance of mitochondria via autophagy (mitophagy). disturbed calcium-and redox-homeostasis seems to play a major role in susceptibility to cell death signals in dopaminergic neurons, but if this is a preceding or successive event in cell death related to pd progression is not known. on the other hand, experimentally elicited parkinsonism by oxidative stress inducers like paraquat or rotenone and mptp (inhibitors of mitochondrial complex i) lead to damage in nuclear dna and activation of the dna repair protein poly(adp-ribose) polymerase 1. by consuming substantial amounts of its substrate nad + , this enzyme can drastically decrease energy levels and disturb the redox balance within a cell, also sensitizing it to stress induced cell death. inhibition of poly(adp-ribose) polymerase 1 has been proven to be beneficial to some extent in cell culture models as well as in experimental pd in mice. our research focuses on a putative crosstalk mechanism we recently discovered between the two pathways of experimentally induced cell death in culture models, i.e. mitochondrial signaling and parp1-dependent poly(adp-ribosyl)ation. both converge on two mitochondrial chaperones, mortalin and trap1. whereas mutations in mortalin have been reported recently to be responsible for some parkinson disease cases in humans, trap1 is a specific target of the kinase pink1 (pten induced putative kinase), which is often mutated in autosomal-recessive forms of the disorder. pink1 is a central regulator of the mitophagy process, tagging mitochondria with dissipated membrane potential for destruction. we could show now that both chaperones bind to short-chain poly(adp-ribose) specifically synthesized by poly(adp-ribose) polymerase 1. we will present our most recent findings about regulation of these two chaperones after application of parkinson-inducing toxins. aldrich). the effect was reversible within ten minutes when cells were re-incubated in regular cell culture medium. stimulatory effects were not due to osmolarity or cell stress due to medium exchange. analysis of different components of both media (table 1) revealed that bicarbonate stimulates accumulation of ccmp and cump besides cgmp and camp in a time-and concentration-dependent manner. bicarbonate is known to activate soluble adenylyl cyclase (sac) and particulate guanylyl cyclase g (pgc-g), regulating sugar metabolism, sperm motility and olfaction by synthesis of camp and cgmp, respectively. in order to identify a responsible cyclase for ccmp and cump generation after bicarbonate treatment, we are currently analyzing transiently and stably transfected hek293 cells overexpressing various known adenylyl and guanylyl cyclases (sac, mac1, mac3, mac9, soluble guanylyl cyclase, pgc-g, pgc-a, and pgc-d) for their pyrimidinyl and purinyl cyclase activity in vivo and their regulation by bicarbonate. in addition, cell fractions will be analyzed for the detection of specific cyclase compartments. question: long term ventricular pacing, especially at the right ventricle (rv), results in left ventricular (lv) failure. there are several lines of evidence that disturbed ca 2+ homeostasis is involved in the pathophysiology of human heart failure. in this study we examined if ventricular pacing affects the na + -and ca 2+ -channels and the expression of ca 2+ -handling proteins and investigated if there is a differential effect between right ventricular free wall (rvfw) pacing and left ventricular apex (lva) pacing. methods: after av-node ablation 14 minipigs underwent ventricular pacing at 120 beats/min (ddd mode) for one year. 7 minipigs were paced from the rvfw and 7 minipigs from the lva, respectively. 7 minipigs with normal sinus-rhythm served as control group. patch-clamp-experiments were studied to measure na + -and ca 2+ currents. western-blots were carried out to investigate the expression of the ca 2+handling proteins l-type ca 2+ -channel, serca2 and phospholamban. results: both rvfw-and lva-pacing led to significant decreased ca 2+ -currentdensities in cardiomyocytes of the lv compared to the control group. the plateau phase of the action potential was significantly shortened after ventricular pacing in relation to control minipigs. furthermore cardiomyocytes of rvfw-and lva-paced minipigs had significant lower na + -current-densities than control minipigs. the action potential amplitude was significantly decreased after rvfw-and lva-pacing whereas the diastolic potential remained unchanged. the expression of the l-type ca 2+ -channel was significantly reduced after ventricular pacing, regardless of the pacing site. in contrast rvfw-and lva-paced minipigs showed significant increased serca2-expression. the expression of phospholamban remained unchanged after rvfw-and lva-pacing compared to control minipigs. conclusion: in a chronic animal model ventricular pacing leads to remodeling of ionchannels and ca 2+ -handling-protein-expression, regardless of the pacing site. investigation on metabolic competence of dermal systems: native human skin, in vitro skin models and keratinocytes blatz v. 1 the implementation of reconstructed human skin equivalents (rhes) as an alternative method for dermal toxicity testing became very prominent in the last decades. their advantages are e. g. the human cell origin and an organ-like 3d structure. already regulatory accepted methodologies are widely in use for testing the skin corrosion (oecd 431) and irritation (oecd 439) within rhes. but there are still some questions open, one of them the metabolic competence of such dermal systems. in this context, enzyme activities of oxidizing (cyp; fmo; adh; aldh) and conjugating enzymes (nat; ugt) were investigated in subcellular fractions of in vitro systems such as keratinocytes and rhes (epidermis model epiderm tm (mattek), full-thickness skin models epiderm tm ft (mattek) and phenion ® ft (henkel ag)) and compared to those of native human skin. activities of cyp 1a, 2b and 3a isoenzymes were measured fluorometrically by oxidative desalkylation of alkoxyresorufines. fmo 1/3 activities were evaluated by hplc/fld detection of n-oxygenated product of benzydamine [1] . adh and aldh activities were investigated by photometrical detection of nadh generation during ethanol (adh) [2] or propanal (aldh) oxidation [3] . nat1 activity was followed by hplc/uv detection of acetylated p-aminobenzoic acid. ugt1 activity was quantified fluorimetrically by glucuronidation of methylumbelliferone [1] . during the course of this study the following results were observed: (loq = limit of quantification) since the metabolic competence of rhes is confirmed, these in vitro systems are estimated as suitable for further toxicity tests (e. g. genotoxicity by comet assay), where metabolic activation of substances may play a crucial role. however, for the data assessment, the determined metabolic profiles should be taken into account. we acknowledge bmbf funding this project (0315226d). signalling pathway indicates that in b104 cells the adenine receptor couples to a gqprotein followed by activation of phospholipase c pathway. these findings represent a new signalling pathway of the adenine receptor and allow the assumption that different adenine receptor subtypes exist in the rat brain. in the scope of the project lexukon ("foodborne exposure to environmental contaminants -data analysis to support and standardise exposure assessments based on nvs ii") exposure to the heavy metals cadmium (cd), lead (pb) and mercury (hg) via food consumption has been assessed for the german adult population based on the national nutrition study ii ( the updated intake assessments show that especially foods regularly consumed such as vegetables and grain contribute mainly to exposure of cd that is about 1.5 µg/kg body weight (bw) per week for average consumers over all food groups. this corresponds to 58% of the tolerable weekly intake (twi) of 2.5 µg/kg bw for cd defined by the european food safety authority (efsa) in 2009. beverages and vegetables are the food groups most relevant for exposure to pb. about 3.7 µg pb/kg bw is taken up by average consumers that is below the benchmark dose for renal toxic effects (4.41 µg/kg bw) defined by efsa for the weekly pb intake. for hg the intake amounts for all population groups examined were significantly below the toxicological reference values. for average consumers the weekly intake of hg is 0.49 µg/kg bw that is primarily taken up by eating fish and fishery products. however, individual population groups and high consumers reach and/or exceed the toxicological reference values for the daily intake amounts for cd and pb. high consumers almost reach the twi for the cd with 94%. for pb a weekly intake of 5 µg/kg bw was estimated for high consumers that exceeds the benchmark dose for renal toxic effects for the weekly pb intake. the results show that data collection should also focus on highly consumed and not only on highly contaminated foods. further, uncertainties in concentration levels should be reduced e.g. by lowering and standardizing the analytical limits. it's recommended to consider further measures in view of the reduction of contents of environmental contaminants in foods. however, other sources can also contribute to the intake of the mentioned heavy metals (e.g. smoking). major cell biological processes are regulated by rho-gtpases, actin-mediated processes in particular. amongst others, rho-gtpases are stimulated by the receptormediated activation of gα12/13 and gαq via specific rhogefs. the p63rhogef is activated by gαq and plays a major role in the acute response of vascular smooth muscle cells to angiotensin ii treatment. the aim of the present study was to establish a fret-assay between gαq-cfp and venus-p63rhogef and characterize the dynamics of p63rhogef-gαq-interaction in single living cells. the fusion of p63rhogef with venus resulted in a functional gαq-regulated p63rhogef-protein as determined by means of rho-luziferase-assays. whereas no specific fret signal was observed between the two interaction partners in the absence of receptor stimulation, a robust and rapid fret signal developed in response to stimulation of histaminergic h1and cholinergic m3-recptors. the onset of this signal after rapid application of agonist paralleled gαq activation kinetics. similar to the kinetics of gαq-protein deactivation the dissociation of p63rhogef and gαq after withdrawal of agonist was slow (tens of seconds). the specificity of the fret signal between gαq-cfp and venus-p63rhogef was verified by introducing point mutations rendering p63rhogef unable to bind to active gαq. furtehrmore we observed a robust acceleration of the dissociation of p63rhogef and gαq upon cotransfection of rgs2, suggesting a very short lifetime of the p63rhogef-gαq-complex or the ability of rgs2 to bind to p63rhogef-associated gαq. taken together, fret-based imaging of the interactions between p63rhogef and gαq revealed fast interaction kinetics closely resembling g-protein activation kinetics, both of which can be regulated by rgs2. toxicity of silver nanoparticles in intestinal cells boehmert l. 1 the rapid development of nanotechnology has been accompanied by an increased concern for the safety of nanomaterials. especially silver nanoparticles are used in many manufacturer identified consumer products including silver coated food contact materials or hydrosol silver supplements. these products lead to an intentional or unintentional oral uptake of silver nanoparticles and hence to a contact with the intestinal barrier. the human cell line caco-2 is a well established model system in studying effects on human enterocytes. although these cells are colon carcinoma cells and exhibit typical features of cancer cells when they are kept sub confluent, these cells have the capability to differentiate into polarized cells with morphological and biochemical properties of small enterocyte cells. we investigated the effects of silver nanoparticles on these colon carcinoma (proliferating) and small intestinal epithelium like (differentiated) caco-2 cells. the silver nanoparticles agpure were commercially available from rent-a-scientist gmbh. the behaviour of these silver nanoparticles in cell culture medium were characterised using asymmetric flow-fild flow fractionation (a4f), small ankle x-ray scattering (saxs) and dynamic light scattering (dls). we investigated the particle toxicity on both cell states using cell titer blue assay, xcelligence impedance measurements, annexin-v and caspase measurements, diclorofluorescein assay and antioxidant pre incubated cells. the agpure stock solution is an aqueous suspension of silver particles with a metal core radius of 7.2 nm stabilised with tween-20 und polyoxyethylenglycerol trioleate. agpure silver nanoparticles were toxic for proliferating as well as differentiated caco-2 cells in a time and concentration depending manner. the presence of foetal calf serum in the incubation medium has a minor influence on the toxicity. prior to cell death, morphological abnormal adherence characteristics and morphological changes in the cells were observed using microscopy and quantified by xcelligence impedance measurement. it is concluded that cell death is caused by an oxidative stress related mechanism rather than apoptosis. the release of sphingosine-1-phosphate from human platelets during acute coronary syndrome is attenuated by aspirin böhm a. 1 , polzin a. in addition to atherosclerosis ttp knock out mice develop more cardiovascular dysfunctions. in tail vein bleeding assays we monitored a significant difference in the bleeding times of ttp deficient mice in comparison to wildtype mice, triggered by a stronger granulopoeisis. our results leed us to the assumption that the chonic inflammation seems to be more improtant for the development of cardiovascular diseases in ra patients than the traditional risk factors. differentially expressed cardiac genes in a mouse model with heart-specific overexpression of pp2a bollmann p., makarova e. a., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06112 halle (saale) , germany in transgenic (tg) mice with cardiac myocyte-specific overexpression of the catalytic subunit of protein phosphatase 2a (pp2a) reduced cardiac protein phosphorylation, cardiac hypertrophy and impaired cardiac contractility were noted compared to wild type (wt) littermates. the hearts of tg mice also suffered from ventricular dilatation and a diminished response to β-adrenergic stimulation. analyses of mrnas expressed in tg and wt hearts (n=3) using affimetrix mouse genome microarray chips resulted in several candidate genes possibly differentially regulated. in this study, we focussed on verifying the mrna data of selected genes important for stress response and signal transduction on protein level in cardiac homogenates by western blotting. hearts from wt littermates were used as control. compared to wt heat shock protein 25 (hsp25) and calcium calmodulin dependent protein kinase type ii (camkii) mrnas were upregulated in tg but only hsp25 protein was increased (p<0.05, n=7-8) but not camkii (p>0.05). protein phosphatase type 5 (pp5) and superoxide dismutase (sod) were downregulated on mrna level in tg but on protein level this could be found only for sod (p<0.05, . in contrast, pp5 protein was upregulated (p<0.05, in tg compared to wt. for comparison the regulatory a-subunit of pp2a and hsp90 were studied. both genes were unchanged on mrna level in tg: western blotting revealed the same results for the corresponding proteins. in summary, mrna expression data could only partially be confirmed on protein level elucidating the importance of western blotting studies. these data indicate that increased pp2a activity is associated with modified gene expression in tg hearts possibly affecting stress response and regulation of cell signalling. (supported by the deutsche forschungsgemeinschaft) center for regenerative therapies, technische universtität, dresden, germany cell therapy in the form of beta cell replacement to cure diabetes has been practiced for decades without become a routine clinical therapy. more widespread clinical application is hindered by the scarcity of suitable organ donors, a dramatic loss of transplanted cells within the first days post-transplant, the requirement of long term immunosuppression to maintain graft survival, and despite this, a loss of graft function from a recurrence of autoimmunity in some patients. research is currently dedicated to overcome each of these limitations. additional beta cell sources investigated include embryonic stem cell derived insulin producing cells, human insulin producing cells lines, and xenogeneic beta cells. parallel to these efforts are the development of encapsulation devices to protect these sources from immune and inflammation mediated destruction, and transplantation into new sites such as the muscle and bone marrow to infuse beta cells. additional therapy to reduce immune suppression includes the infusion of t regulatory cells to control autoimmune and alloimmune response, and cytokine and chemokine receptor directed compounds aimed at blocking early inflammation or autoimmunity. these efforts are likely to lead to an expansion of clinical activity to replace beta cells in diabetes, and to novel pharmaceutical therapies that may be more generally applicable in patients with diabetes. pdgf-bb induces the h2s producing enzyme cystathionine-γ-lyase via a rosdependent mechanism in rat renal mesangial cells boosen m. 1 , hassan m. there is increasing evidence that hydrogen sulfide (h2s) that is endogenously produced in several cell types serves as a potent gasotransmitter in a wide variety of physiological processes involving vascular homeostasis and inflammation. in the present study we investigate the expression and the regulation of the hydrogen sulfide synthesizing enzyme cystathionine γ-lyase (cse) in cultured rat renal mesangial cells. as demonstrated by qpcr and western blot experiments, mesangial cells showed a marked time-and dose-dependent upregulation of cse mrna and protein levels after treatment with platelet-derived growth factor (pdgf-bb). the cse upregulation by pdgf-bb is accompanied by a marked increase in reactive oxygen species (ros) formation. interestingly, co-administration of the ros scavenger n-acetylcysteine, the glutathione peroxidase mimetic ebselen and the nadph oxidase inhibitor diphenylen iodonium chloride (dpi) drastically reduced pdgf-induced cse expression, indicating a role for endogenously produced ros in mediating regulation of cse. as demonstrated by electrophoretic mobility shift (emsa) experiments pdgf-bb induces binding of the redox-sensitive transcription factor nf-e2-related factor 2 (nrf2) to a consensus antioxidant response element and this effect was also diminished by co-administration of antioxidants (dpi, nac, ebselen) . furthermore, lps/ifnγ-as well as pdgf-bb-induced cse upregulation was nearly completely abolished in nrf2 -/spleen macrophages and mesangial cells, respectively. as a consequence of the elevated cse levels we could demonstrate increased h2s levels and a higher cse enzyme activity in mesangial cells after stimulation with pdgf-bb by using the colorimetric methylenblue method and a cse activity assay. importantly, in a rat model of anti-thy-1-induced proliferative glomerulonephritis we observed a marked upregulation of cse protein during the course of the disease paralleled by a stabilization of nrf2 protein. from our data, we hypothesize that pdgf-bb-mediated regulation of cse via a redox-mediated activation of nrf2 may constitute a protective mechanism during glomerular inflammatory disease. rac1 knockout protects from acute hepatic damage following doxorubicin treatment bopp a., wartlick f., fritz g. heinrich-heine-universität institut für toxikologie, universitätsstr. 1, 40225 düsseldorf, germany rac1 belongs to the best characterized members of the ras-homologous (rho) family of small gtpases, which are key regulators of the actin cytoskeleton. furthermore, rac1 is part of the activation of the nadph oxidase, which produces reactive oxygen species and regulates the activity of stress kinases (e.g. sapk/jnk) and transcription factors such as nf-κb and ap1. anticancer drugs cause dna damage, which in turn stimulates the dna damage response (ddr) regulating dna repair, cell cycle progression and, in case of non-repairable dna damage, triggers apoptosis. so far, a role of rac1 in the ddr has not been reported. based on its exceptional function as a regulator of transcription and because of its recently found ability to translocate to the nucleus, we hypothesize that rac1 may be involved in the ddr. to study the in vivo function of rac1 we used an inducible cre-based knockout mouse model (rac1 flox/flox/mxcre ). mice were treated with different doses of doxorubicin for different periods of time. we monitored gh2ax foci formation as a marker of dna strand breaks, used the masson-goldner staining for the detection of collagen accumulation, analyzed phosphorylated histone 3 as a marker of mitotic events and performed a tunel assay to detect apoptotic cells. in the absence of rac1 the basal mrna expression of pro-fibrotic ctgf was decreased. collagen levels were increased and mmp1 mrna expression was reduced in the liver of rac -/animals as compared with rac1 proficient animals. in addition we found more apoptotic cells in rac1 -/mice. 96 hours after treatment with the anthracycline derivative doxorubicin the number of gh2ax foci in rac1 -/animals was reduced in comparison to rac1 +/+ animals. we also found lower level of ctgf mrna expression and reduced amount of collagen in rac1 -/mice. none of these protective effects resulting from rac1 deficiency could be detected after administration of three consecutive doxorubicin injections over a time period of 21 days. there were no significant differences in the number of gh2ax foci or collagen accumulation. the mrna expression of ctgf was even higher in rac1 -/animals. furthermore the number of mitotic events was almost two times higher in the rac1 -/mice compared to the rac1 +/+ mice. summarizing, our findings show that impaired hepatic expression of rac1 protein is hepatoprotective against acute damage following doxorubicin exposure, but does not protect against doxorubicin-induced subacute toxicity. in vitro cytotoxicity of tbhq (tert-butyl-hydroquinone) braeuning a., vetter s., schwarz m. institut für experimentelle und klinische pharmakologie und toxikologie toxikologie, wilhelmstrasse 56, 72074 tübingen, germany at high concentrations, tert.-butyl-hydroquinone (tbhq), a phenolic antioxidant frequently used as a food preservative, exerts cytotoxic effects, which are closely linked to its ability to form reactive oxygen species as a consequence of redox cycling processes. here we describe that treatment of murine 3t3 cells with tbhq in 96-well culture plates induces the death of untreated cells in neighboring wells on the same plate. the mechanisms underlying that effect were investigated. death of the seemingly untreated neighboring cells was caused by a more toxic and volatile tbhq oxidation product which was formed in a non-enzymatic process involving metal ions and oxygen. the unexpected perturbation of cytotoxicity testing by the volatile tbhq metabolite shows that not only metabolic processes, but also non-enzymatic mechanisms have to be considered as important parameters for in vitro assays. furthermore, our data show that even cells several wells distant from the site of treatment do not necessarily constitute proper "untreated" controls when cells are treated with tbhq, e.g. in assays aimed to analyze the activity of the tbhq-inducible nrf2 pathway. s14 056 angiotensin ii causes oxidative stress and dna damage in mouse kidneys via the angiotensin ii type 1 receptor brand s. 1 , amann k. 2 , schupp n. 1 1 universität würzburg institut für pharmakologie und toxikologie, versbacherstr. 9, 97078 würzburg, germany 2 universität erlangen-nürnberg institut für pathologie, krankenhausstraße 8, 91054 erlangen, germany angiotensin ii (ang ii), the reactive peptide of the renin-angiotensin-system, causes vasoconstriction and, in higher levels hypertension, which is connected with an increased cancer risk in the kidney. treatment of male c57bl/6 mice with ang ii results in the formation of superoxide radicals and dna damage in the kidney as well as in the heart. to answer the question if the dna damage is caused by hypertension or by elevated ang ii concentrations, mice were treated with different compounds: the angiotensin-converting-enzyme blocker ramipril, the ang ii receptor blocker ramipril, the ang ii receptor candesartan, the antioxidant tempol and the vasodilator hydralazine. the effect on blood pressure and renal function of ang ii-treated c57bl/6 mice was examined. treatment with ang ii led to a significant increase in blood pressure. candesartan and hydralazine led to a decrease, whereas intervention with ramipril and tempol had no effect. equal conditions could be found by examining renal function regarding the excretion of urinary albumin, which was ameliorated by candesartan and hydralazine. in addition, histopathological changes were investigated. there was significant glomerular damage and tubulointerstitial damage in ang ii-treated animals compared to control animals, which was significantly improved by candesartan and tempol. hydralazine and ramipril mitigated the observed renal damage but were less effective than candesartan. furthermore, the ang ii-induced formation of superoxide radicals in the kidney and the heart was slightly affected by all interventions. genomic damage, in the form of double strand breaks was prevented by the ang ii receptor antagonist candesartan and the antioxidant tempol. to sum up, the results from this study show that ang ii induces the elevation of markers of kidney failure and dna damage, which is prevented by substances lowering blood pressure like candesartan, showing the receptor responsibility for the induction of dna damage. actually by substances not lowering blood pressure like tempol, the oxidative stress and dna damage was ameliorated, showing the involvement of reactive oxygen species. optimization of the balb/c-3t3 cell transformation assay by coupling a drug metabolizing system brauneis m. d., steinberg p. stiftung tierärztliche hochschule hannover institut für lebensmitteltoxikologie und chemische analytik, bischofsholer damm 15, 30173 hannover, germany the analysis of the carcinogenic potential of chemicals plays an important role in toxicology. up to now the acquisition of such data requires a large amount of animal experiments. the aim of this study is to reduce the number of experimental animals being used by further optimizing the balb/c-3t3 cell transformation assay, an already well-established in vitro method. this method, which is also well suited for high throughput screening applications, allows a quantitative analysis of the aforementioned carcinogenic potential. the incubation of balb/c-3t3 cells (murine embryonic fibroblasts) with mutagenic compounds leads to a loss of contact inhibition between these cells, which results in the development of so-called foci. these foci can be distinguished by characteristic changes in cell growth behaviour, a result of the treatment with carcinogenic compounds, and their number is therefore directly related to the genotoxic potential of the latter. a major disadvantage of the "classic" balb/c-3t3 cell transformation assay is that a number of compounds initially require a metabolic transformation to gain their full genotoxic potential. hence, without prior metabolic transformation many chemicals are not detected as carcinogenic in the abovementioned test system. to overcome this drawback the balb/c-3t3 cell transformation assay has been coupled to a drug metabolizing system, in this case the so-called liver s9. in a first step the well-known genotoxic agents benzo[a]pyrene, aflatoxin b 1 and nnitrosodimethylamine were tested in this assay. all three compounds led to a concentration-dependent increase in the number of foci formed, whereby this concentration-dependent increase was observed in a non-cytotoxic concentration range. in a next step the balb/c-3t3 cell transformation assay will be coupled to further drug metabolizing systems as well as to the soft agar assay. this study is being financially supported by the stiftung set and the doerenkamp-zbinden foundation. adenylyl cyclases (acs) synthesize the second messenger camp. the family of acs consists of nine membranous and one soluble isoforms with ac5 and ac6 being the predominantly expressed isoforms in the heart. in the heart, acs integrate β-adrenergic (β-ar) signaling as the main physiological mechanism to improve cardiac performance. although ac5 and ac6 share high sequence homology, opposing effects on cardioprotection have been reported, where disruption of ac5, as well as overexpression of ac6 both exerting beneficial effects in heart failure. prospective pharmacological treatment of heart failure on the level of ac is under investigation. our study explored the impact of ac5 ko on ac-activities in the heart at a functional level. complementary, mrna expression studies of the β-ar-g-protein-ac signaling cascade were performed to detect possible compensatory alterations. hearts from 16-20 week old homozygote ac5 knockout and wild-type male littermates were examined in this study. ac activities where measured in cardiac membrane preparations from left ventricles. ac activities were assessed under β-ar and g-protein (g s) stimulation by isoproterenol, guanosine 5'-triphosphate (gtp) and 5'-o-(3thiotriphosphate) (gtpγs) as well as for direct activation by forskolin. relative mrna expressions for ac1-9, gs-, gi-a and β1-, β2-ar where measured by quantitative realtime pcr. surprisingly, assessment of basal, β-ar and g-protein-mediated ac-stimulation as well as direct activation by forskolin revealed no changes in ac activities. besides from detection of the ac5 knockout, mrna expressions analysis of ac1-9, gs-, gi-a and β1-, β2-ar did not detect any compensatory alteration. these findings suggest that proximal adrenergic signaling in the heart does not necessarily require ac5. whether physiological integration of beta adrenergic signaling in the heart is mediated by both isoforms ac5 and ac6, or can be attributed to one main isoform remains to be elucidated. melanocortin-promoted pka activation decreases ampk activity via erk-1/2 and lkb-1 in hypothalamic gt1-7 cells breit a., ellen d., gudermann t. goethestrasse 33, 80336 münchen, germany α-melanocyte stimulating hormone (α-msh)-induced activation of the melanocortin-4 receptor (mc4r) in hypothalamic neurons increases energy expenditure and inhibits food intake. intrahypothalamic injection of melanocortins decreased food intake due to the inhibition of amp-activated protein kinase (ampk) that has recently been reported to enhance food intake in rodents. until now, it is not clear if α-msh affects ampk via direct intracellular signaling cascades or if the release of paracrine factors is involved. herein, we used a murine, hypothalamic cell line (gt1-7 cells) and monitored ampk phosphorylation at thr 172 which has been suggested to increase ampk activity. we found that α-msh dephosphorylated ampk at thr 172 and consequently decreased phosphorylation of the established ampk substrate acetyl-coa-carboxylase at ser 79 . inhibitory effects of α-msh on ampk were blocked by specific inhibitors of protein kinase a (pka) or extracellular-regulated kinases-1/2 (erk-1/2), pointing to an important role of both kinases in this process. since α-msh-induced activation of erk-1/2 was blunted by pka inhibitors, we propose that erk-1/2 serves as a link between pka and ampk in gt1-7 cells. furthermore, down-regulation of liver kinase b-1 (lkb-1), but not inhibition of calcium-calmodulin-dependent kinase kinase-β or transforming growth-factor-beta-activated kinase-1 decreased basal phosphorylation of ampk and its dephosphorylation induced by α-msh. thus, we propose that α-msh inhibits ampk activity via a linear pathway including pka, erk-1/2 and lkb-1 in gt1-7 cells. given the importance of the melanocortin system in the formation of adipositas detailed knowledge about this pathway might help to develop drugs targeting obesity. autism spectrum disorder (asd) is a complex neurodevelopmental disorder with dysfunction of social interaction and communication. a hitherto unknown complexgenetic principle of origin probably underlies asd. so far, more than 100 candidate genes were identified in literature. the patients affected with the monogenic timothy syndrome show multiorgan dysfunction including lethal arrhythmias, immune deficiency, skeleton-dysplasia, syndactylia and autism. this single gene disorder serves as a model disease for asd, giving insights in a possible pathophysiology. here, a point mutation in a highly-conserved region of the pore-forming subunit of the voltage-dependent calcium channel (ca v) cav1.2 gene (cacna1c) results in incomplete inactivation of the l-type calcium currents (splawski et al., cell 2004; 119:19-31) . functionally similar biophysical effects can be induced by structural variation β1-and β2-subunits of the voltagedependent calcium channels (herzig et al., faseb j. 2007; 21:1527-38; jangsangthong et al., pflugers arch. 2010; 459:399-411) . supported by findings in a meta-analysis of linkage data of asd patients (trikalinos et al., mol psychiatry. 2006; 11:29-36) , we are investigating a function-based candidate gene hypothesis linking the β2 subunit gene (cacnb2) with asd. we performed a case control study sequencing all exons and flanking intronic regions of cacnb2 in 155 patients with asd. we found three rare missense mutations in asd patients, but not in 259 unaffected controls. all three mutations occur at highly conserved positions and might alter protein function; additionally results one amino acid substitution highly probable in a post-translational modification by phosphorylation. so far, we characterized two of these mutations and also a phosphorylation-mimicking mutant in electrophysiological studies. all variants show a decelerated and incomplete time-dependent inactivation of the co-transfected cav1.2 subunit. furthermore, two variants exhibit a significant increased slope factor of voltage-dependent steady-state inactivation. we here present mutations in the β2 subunit gene of asd patients that result in a retardation of inactivation behavior, thus phenocopying the monogenic timothy syndrome mutations of cav1.2. β2 subunit mutations may influence neuronal function or development in some asd patients. jangsangthong, w., kuzmenkina, e., khan, i.f., matthes, j., hullin, r., and herzig, s. (2010) . inactivation of l-type calcium channels is determined by the length of the n terminus of mutant beta (1) subunits. pflugers arch 459, 399-411. herzig, s., khan, i.f., grundemann, d., matthes, j., ludwig, a., michels, g., hoppe, u.c., chaudhuri, d., schwartz, a., yue, d.t., et al. (2007) . mechanism of ca(v)1.2 channel modulation by the amino terminus of cardiac beta2-subunits. faseb j 21, 1527-1538. splawski, i., timothy, k.w., sharpe, l.m., decher, n., kumar, p., bloise, r., napolitano, c., schwartz, p.j., joseph, r.m., condouris, k., et al. (2004) . ca(v)1.2 calcium channel dysfunction causes a multisystem disorder including arrhythmia and autism. cell 119, 19-31. trikalinos, t.a., karvouni, a., zintzaras, e., ylisaukko-oja, t., peltonen, l., jarvela, i., and ioannidis, j.p. (2006) . a heterogeneity-based genome search meta-analysis for autism-spectrum disorders. mol psychiatry 11, [29] [30] [31] [32] [33] [34] [35] [36] background: brain serotonin (5-ht) has been implicated in the regulation of food-intake. the ingestive effects of 5-ht are mediated by a number of different receptor subtypes under which the 5-ht1a-receptor plays a central role. former in vivo studies have shown an increased intake of food, elicted by 5-ht-receptor agonists. the aim of this behavioural pharmacologic project was to determine if the hyperphagic effect is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei or by postsynaptic 5-ht1a heteroreceptors in serotonergic terminal structures. methods: the effect of the 5-ht1a receptor agonist 8-oh-dpat (0.1, 0.5 or 1.0mg/kg) was investigated on feeding behaviour in non-food-deprived young-adult and adult nmri and transgenic l35 mice. l35 mice are characterized by an overexpression of postsynaptic 5-ht1a receptors. results: the administration of the 5-ht1a receptor agonist induced hyperphagia in all groups of mice, except for the adult transgenic mice which showed no drug effect. conclusion: the results confirm a key role of the 5-ht1a receptor in food intake. further, we make the assumption that the hyperphagic effect of 8-oh-dpat is mediated by presynaptic 5-ht1a autoreceptors in the raphe nuclei which decreases 5-ht function in the central nervous system. it can be speculated that the aberrant feeding behaviour of the adult transgenic mice refers to a possible opposite role of the postsynaptic 5-ht1a receptors. these receptors might affect the release of neuropeptides in the hypothalamus. the efflux transporter abcc2 (mrp2) expressed at different compartment barriers is important for the elimination of various endogenous and exogenous compounds. with some evidence inflammatory processes regulate abcc2 expression and cause changes of absorption, distribution and clearance of a number of xenobiotics. the investigation of the influence of interleukin (il) 1β on abcc2 mrna and protein expression in various cell lines representing specific tissues is the aim of our study. a further aim is to characterize the signaling pathways regulating abcc2 expression while inflammation. three different cell lines a) hepg2 cells (liver tissue) and b) caco2 (colon tissue) both without naïve il1β expression and c) skhep1 cells representing physiological liver tissue with naive il1β expression, were stimulated with different concentrations of il1β (range 10 pg/ml to 10 ng/ml). over a period of 48h samples were taken at defined time points. abcc2 mrna and protein expression were quantified by qrt-pcr and western blot analysis, respectively. by using small molecule kinase inhibitors for signal transduction proteins (p38 mapk, akt, erk1 and jnk) we analysed the signal transduction pathways associated with il1β-mediated transcriptional abcc2 regulation. on abcc2 mrna level an up-regulation in caco2 cells (1, 35 fold) and hepg2 cells (3, 6 fold) within the first hour after stimulation with 1 ng/ml il1β was shown. in contrast skhep1 cells demonstrated a decreased abcc2 mrna expression (0,59-0,62 fold) in comparison to unstimulated controls. the abcc2 protein expression exhibited a time and il1β dependent regulation as well. the analysis for the signal transduction showed for p38mapk a moderat time dependet down regulated phosphorylation (15%) in hepg2 cells whereas it showed no effect in caco2 cells. concluding, the expression of abcc2 is regulated moderately by il1b in a concentration and time-dependent manner. interestingly, the effects are strongly tissue-dependent concerning abcc2 expression and signal transduction pathways and show partly contradictory results. the regulation of the different signaling pathways is currently subject of ongoing investigations. introduction: despite the remarkable success of imatinib treatment of chronic myeloid leukemia (cml), therapy resistance emerged as a major clinical problem. the aim of this study was to identify micrornas, which may serve as biomarkers for therapy response or predict pathways involved in pharmacoresistance of imatinib treatment. methods: blood was collected from 21 cml-patients, ten of whom responded to imatinib therapy. after rna extraction from leukocytes, we performed a taqman low-density array screen to determine the expression of 667 micrornas. statistical analysis using the 2 -∆ct method was performed. micrornas showing a p-value<0.01 and a fold change>2 were considered to be significantly differently expressed. in addition, by using microrna target prediction databases (targetscan 1 , mirdb 2 , pictar 3 , microcosm 4 , diana microt 5 ), selected putative target genes were further functionally investigated by the david bioinformatics database 6 . results: comparing treatment-naïve responders and non-responders four micrornas were identified to be deregulated that were predicted to target 97 genes, especially transcription regulators (21%). pathway analysis showed that six of the predicted genes are relevant in cancer pathways, four of which play a role in cml (smad4, nras, rb1, raf1). when comparing patients' expression profiles before and under treatment, seven micrornas were identified to be deregulated in responders and five micrornas in nonresponders. ninety-nine targets of the latter include transcription regulators (19%), but also cellular transporters (18%, especially uptake transporters of the slc-family). most target genes are involved in mapk signalling or endocytosis pathways. conclusion: analysis of microrna expression profiles revealed four micrornas involved in imatinib-response and 12 micrornas deregulated during imatinib treatment. predicted target genes code mainly for transcription factors as well as oncogenes relevant for cml and are involved in transporter expression and endocytotic processes. dissociations in the effects of beta2-adrenergic receptor agonists on camp formation and superoxide production in human neutrophils brunskole i. 1 , buschauer a. activation of the β2-adrenergic receptor (β2ar), a classically gs-coupled receptor, in neutrophil granulocytes results in an inhibition of inflammatory responses [1] , which could be further therapeutically exploited. the aim of the present study was to evaluate the effects of various β2ar ligands on cyclic adenosine 3',5'-monophosphate accumulation (camp assay) and n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced superoxide anion production (o2 •assay) in isolated human neutrophils, which are a physiologically relevant native test system. camp concentration in neutrophils was determined by hplc/tandem mass spectrometry, and o2 •formation was assessed by monitoring the superoxide dismutase-inhibitable reduction of ferricytochrome c. (-)-isoproterenol, (-)-adrenaline, salbutamol and dobutamine were more potent in inhibiting fmlp-induced o2 •production than in stimulating camp accumulation. (-)-ephedrine and dichloroisoproterenol were devoid of any agonistic activity in the camp assay, but could partially inhibit fmlp-induced o2 •production at higher concentrations. moreover, (-)-adrenaline and dobutamine were equi-efficacious in both assays whereas the efficacy of salbutamol was more than two fold higher in the o2 •assay. this suggests that salbutamol is able to stabilize a different receptor conformation than the other two ligands. thus, ligand-directed signaling via β2ar can also occur in human neutrophils. in addition, differences between the data from neutrophils and recombinant test systems [2, 3] were noticed, pointing to the problem of insufficient comparability of effects in recombinant and native test systems. the investigation of β2ar antagonists on neutrophil granulocytes is subject of ongoing work, in order to find out whether pkb values of β2ar antagonists in the camp assay and the o2 •assay are different. such differences were previously reported for β2ar antagonists in other test systems [4] . moreover, studies with protein kinase a inhibitors should give deeper insight into the signaling events in neutrophils that result in inhibition of fmlp-stimulated o2 •production and clarify how camp increase interferes with this events. agonist-selective internalization of the human 5-ht2a receptor buchborn t., kahl e., höllt v., koch t. otto-von-guericke-universität magdeburg institut für pharmakologie und toxikologie, leipziger straße 44, 39120 magdeburg, germany the serotonin 2a (5-ht2a) receptor is a g protein coupled receptor and the molecular target of lsd-like hallucinogens. downregulation of 5-ht2a receptors is an adaptive process considered relevant for the therapeutic action of diverse serotonergic antidepressants, such as ssris. since the antidepressant targeting of 5-ht2a receptors, however, is largely restricted to indirect agonists and/or antagonists, little is known about the mechanisms and implications of their regulation by direct agonists. in the present study we, therefore, investigated the capacity of various agonists to regulate the human ha-tagged 5-ht2a receptor by internalization. using immunocytochemical techniques in stably transfected hek293 cells, we show that agonists differ in their capacity to internalize the receptor. serotonin, quipazine and doi are the agonists most efficaciously internalizing the receptor, dmt and methysergide, on the other hand, hardly internalize; other agonists like psilocin, ergotamine and lsd induce low to intermediate internalization. the specificity of the agonistic effect was demonstrated by the 5-ht2a selective antagonist ketanserin, which blocked the agonist-induced internalization. in additional experiments, we show that the internalized 5-ht2a receptors colocalize with a488-labelled transferrin receptors, and that the internalization can be blocked by high molar sucrose; these results are indicative of a clathrin associated sequestration of 5-ht2a receptors in recycling endosomes. also, we demonstrate that the proteinkinase c activator pma efficaciously induces 5-ht2a internalization in the absence of an agonist, and that the doi-induced internalization can be blocked by the proteinkinase inhibitor staurosporine. we, thus, confirm previous findings that the activation of proteinkinases seems to be necessitated for the 5-ht2a internalization to occur. overall, we conclude that the internalization of the human 5-ht2a receptor is agonist-selective, and employs a proteinkinase (possibly pkc) dependent, clathrincoated endosome associated pathway. as there is recent evidence that the regulation of 5-ht2(a) receptors by agonists might have antidepressant (-like) properties, knowledge about the agonist-selective processing of 5-ht2a receptors could help to identify agonists most promising for future (pre-)clinical research. non-clinical safety assessment of homeopathic medicinal products: criteria for establishing a first safe dilution buchholzer m. -l., werner c., knoess w. bfarm bundesinstitut für arzneimittel und medizinprodukte zulassung 4, kurt-georg-kiesinger-allee 3, 53175 bonn, germany like all human medicinal products the homeopathic medicinal products for human use must demonstrate adequate safety. in general, they are regulated according to the analogue non-clinical safety principles (points to consider on non-clinical safety of homeopathic medicinal products of botanical, mineral and chemical origin, adoption by hma 2007). one particular approach is the recently introduced concept of a first safe dilution (fsd; introduction to the list of first safe dilutions, adoption by hma 2010) . this contribution summarizes the first experiences in establishing fsds of a selection of given homeopathic preparations by bfarm. for a given preparation the major toxicological concern and available data set is identified. this determines the safety assessment route: food regulation, permitted daily exposure (pde), threshold of toxicological concern (ttc) or lowest human recommended dose (lhrd/100). finally the acceptable amount/tolerable daily intake is derived and the respective fsd is calculated. for example the draft evaluation for reserpinum (ph. eur. method 4.1.1) and for atropine (ph. eur. method 3.1.1 or 4.1.1) based on lhrd leads in each case to a suggested fsd of d7 related to 10 g of preparation. furthermore, the draft evaluation for potassium iodide (ph. eur. method 3.1.1 or 4.1.1) based on food legislation emerged a proposed fsd of d6 related to 10 g of preparation. the concept of fsd combines a scientific and at the same time pragmatic approach in differentiated risk assessment of homeopathic medicinal products. impact of myricetin and its methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether in c. elegans büchter c. 1 , ackermann d. polyphenolic compounds ubiquitously present in herbal food are discussed to contribute to the health beneficial effects of a diet rich in vegetables and fruits. additional to a strong antioxidative activity of various flavonoids, most of these substances display a variety of other pharmacological properties. we investigated the flavonoid myricetin found in several species of berries, as well as the methylated derivatives laricitrin, syringetin and myricetin-3`,4`,5`-trimethylether. in this study caenorhabditis elegans was used as a model to explore the impact of myricetin and its methylated derivatives in vivo and to investigate molecular modes of action. myricetin (100 µm) caused an increase in mean and median adult lifespan of c. elegans. this longevity effect was associated with a decrease of the aging marker lipofuscin as well as a decrease in ros induction, measured by using the h 2dcf-da assay. however, myrictin failed to improve heat stress resistance, an attribute often associated with longevity in c. elegans. the methylated myricetin derivatives (100 µm) showed a decrease in lipofuscin accumulation and ros induction and they further improved the heat stress resistance. in order to elucidate the basis of the life prolonging action of myricetin, we investigated its influence on factors known to have important functions in stress response and the regulation of aging, namely the foxo homologue daf-16, the nad + -dependent protein deacetylase sir-2.1 and the heat-shock transcription factor hsf-1, respectively. lifespan extension by myricetin disappeared in daf-16 and sir-2.1 loss of function mutant strains, showing the effect is at least partially dependent on these signaling molecules. by using a hsf-1 loss of function mutant strain of c. elegans, it was further shown that the life prolonging effect of myricetin is independent of hsf-1. in conclusion, our results indicate that the life prolonging effect of myricetin is at least in part dependent on daf-16 and sir-2.1, probably due to a modified expression of target genes. stimulatory and inhibitory control of phospholipase c-gamma 2 bühler a., walliser c., becker l., gierschik p. universität ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany activation of phospholipase c-γ2 (plcγ2) upon b cell antigen receptor (bcr) stimulation has been implicated to be a critical step in the bcr-mediated calcium signaling. therefore it is important to understand the mechanisms of how the activity of plcγ2 is stimulated and inhibited. the mammalian plcs are divided into six subfamilies, designated β, γ, δ, ε, ζ, and η. within the plcγ subfamily, the two plcγ isoforms share a number of features that are distinct from those of the other plc subfamilies. the most striking difference is the insertion of additional domains between the catalytic subdomains x and y. this specific array (sa) contains a second, split pleckstrin homology (spph) domain, consisting of two halves separated by two src homology 2 (sh2) domains and one src homology 3 (sh3) domain. there is abundant evidence in the literature that plcs are autoinhibited in their basal state by structural elements within their x/y linker, pointing to a conserved role of the x/y linker in autoinhibitory regulation of plc isozymes. data from our group show that plcγ 2 is also regulated by autoinhibitory elements within its specific array (walliser et al., 2008; everett et al., 2011) . our recent data demonstrated that plcγ2 is negatively regulated by its sa. specifically, within the sh domain tandem, the c-terminal sh2 (sh2c) and the sh3 domain in combination, but not either one alone, cause the strongest autoinhibitory control of plcγ2. plcγ2 has been shown to be phosphorylated at tyrosine residues 753 and 759 upon bcr stimulation (kim et al., 2004) . both tyrosines are located in the linker between the sh2c and the sh3 domain, which we have shown to be the major elements involved in autoinhibitory regulation of plcγ2. interestingly, a novel phosphorylation site in plcγ2 was found in non-small cell lung cancer (nsclc) tissue which is located at tyrosine residue 733 (rikova et al., 2007) . in this work, we demonstrate, for the first time, the activation of plcγ2 by phosphomimetic mutations in these three positions and the functional interplay of the three tyrosine phosphorylation targets. most interestingly, mimicking phosphorylation of tyr733 is critical to fully activate the enzyme. the results not only point to a crucial role of plcγ2 in pulmonary tumorigenesis, but also prompt and stimulate the search for the protein kinase involved in phosphorylating plcγ2 at tyr733. molecular characterization of hepatotoxic effects of perfluorooctanoic acid (pfoa) buhrke t., scharmach e., lampen a. bundesinstitut für risikobewertung (bfr) lebensmittelsicherheit, max-dohrn-str. 8-10, 10589 berlin, germany perfluorooctanoic acid (pfoa) is an industrial chemical that is used for the fabrication of numerous products with oil-, dirt-and water-repellent properties. pfoa is resistant to chemical, thermal and biological degradation and has become a global contaminant of soil, water, air and food in the meantime. the toxicological data of pfoa give cause for concern as the substance was shown to damage the liver of rodents and to impair embryo development. currently, the hazard potential of pfoa for humans is controversially discussed. in this study the human liver cell line hepg2 was employed to analyse the hepatotoxic effects of pfoa on the cellular and on the molecular level. pfoa was shown to stimulate cellular proliferation at concentrations in a range between 5 µm and 25 µm. at concentrations higher than 25 µm the substance was cytotoxic to the cells (ic 50 47µm). cytotoxicity was not due to apoptotic mechanisms as no increase of caspase activity was detected up to a level of 100 µm pfoa. on the molecular level pfoa is known to act as an agonist of the peroxisome proliferator-activated receptor alpha (pparα), and the observed hepatotoxic effects in rodents are associated with pfoa-mediated pparα activation. here we show that pfoa has the capacity also to activate the human isoform of pparα. additional human nuclear receptors were tested for activation by pfoa, and pparγ as well as the pregnane x receptor (pxr) were shown to be activated at high concentrations of pfoa whereas pparδ and the liver x receptor alpha (lxrα) were insensitive to activation by pfoa. notably, we observed a significant inhibition of the activity of the hepatocyte nuclear factor 4α (hnf4α) by incubating the cells already with moderate concentrations of pfoa at a level of about 1 µm. these findings indicate that additional, pparα-independent mechanisms may contribute to the observed hepatotoxicity of pfoa. the elucidation of novel modes of action of pfoa is relevant for the ongoing risk assessment of the substance. s17 070 human breast stem cells as a toxicological model for endocrine disruptors, such as soy isoflavones stempin s. 1 , bumke scheer m. (kao et al., 1995) . two daughter cell lines were developed from m13sv1 after x-ray irradiation (m13sv1 r2) and an additional transfection with a mutated erbb2 oncogene (m13sv1 r2-n1), resulting in high and low tumorigenicity respectively and showing a change in estrogen response after growth in minimal media (wang et al., 2010) . isolated isoflavones are currently widely used in the treatment of postmenopausal symptoms of women. according to the stem cell theory of carcinogenesis, breast stem cells are the ideal target for the proposed research. however, the epidemiological data to the effect of isoflavone intake on breast cancer is contradictory. therefore, we want to develop a toxicological model using these human breast stem cell lines to test the effect of endocrine disruptors, such as soy isoflavones in a human relevant model. in the present study we analyzed the effect of the phytoestrogen genistein, the most intensively studied soy protein, on the differentiation of the 3 hbec lines. the expression of different luminal epithelial cell markers, estrogen receptors and stem cell markers was measured on the mrna level by quantitative real time pcr. the analysis of several of these markers was also performed on the protein level using western blot. additionally, a broad number of genes related to breast cancer and estrogen receptordependent signal transduction were studied using a commercial pcr-array. in parallel we are also analyzing the changes on the protein level using 2d gel electrophoresis. we want to use this panel of different markers to establish a toxicological model that can be used in the future to analyze a wide range of different endocrine disruptors. kao cy, nomata k, oakley cs, welsch cw, chang cc. (1995) bronchial asthma is a common inflammatory disease of the airways whose occurrence has increased dramatically over the past decades. histamine plays an important role in mediating the inflammatory response leading to characteristic symptoms like wheezing, coughing, chest tightness, and shortness of breath. since antagonizing the histamine h1-receptor (h1r) shows no ameliorating effects on asthmatic symptoms, h4r antagonists may be new drugs for asthma therapy. in addition to the h3r-and h4rselective antagonist thioperamide, the selective h4r antagonist 1-[(5-chloro-1h-indol-2yl)carbonyl]-4-methylperazine (jnj7777120) is used in pharmacodynamic studies. a correct interpretation of the collected data requires the detailed knowledge of the pharmacokinetics of the applied substances. for this reason, we developed a fast and robust method based on high performance liquid chromatography coupled to tandem mass spectrometry (hplc-ms/ms) which allows the simultaneous quantitation of thioperamide and jnj7777120 as well as the selective h3r antagonist 1-({4[3-(piperidin-1-yl) propoxy]phenyl}methyl)piperidine (jnj5207852) in murine plasma and lung tissue. the treatment of plasma samples based on protein precipitation performed with a mixture of methanol and 0.2 m znso4 using 30 µl of plasma. analyte extraction from lung tissue was achieved by treating 150 -200 mg of tissue with a mixture of ethanol and water followed by rigorous mixing using a fastprep-system. ten µl of the extracted samples were transferred to a synergy polar-rp 80a mercury column (10 x 2mm; 4µm) connected to a polar-rp security guard. chromatographic separation was performed via a gradient using an acetate buffer (ph 5) and methanol at a flow rate of 0.4 ml/min. the analytical run-time was 5 minutes. for plasma samples the assay was linear over a concentration range of 0.078 -40 µg/ml for jnj7777120 and jnj5207852, and 0.313 -40 µg/ml for thioperamide, respectively. in tissues, thioperamide could be quantified in a concentration range of 0.008 -2 µg/sample, jnj7777120 and jnj5207852 in a range of 0.004 -2 µg/sample. our results show that the developed hplc-ms/ms method is suitable for the quantitation of all tested histamine-receptor ligands in murine plasma and lung tissue. the functionality of the heart greatly depends on strict homeostasis and interplay of a range of signalling cascades. deregulation of either one is always harmful and eventually detrimental for life. some of the most relevant signals in the adult heart are triggered by the stimulation of g protein-coupled receptors such as adrenergic or angiotensin receptors. those in turn are modulated by a small subset of kinases, the g protein-coupled receptor kinases (grks). interestingly, grks, which for the longest time were believed to regulate only g protein-coupled receptors were shown to modulate also non-receptor-mediated signalling pathways. by now it is well documented that grk5 plays important roles in both the physiological as well pathological setting of the adult heart. in spite of the important functions of grks in the adult heart, it must be assumed that grk5, one of the two main cardiac grks, may also be involved in signal modulation in the course of heart development. deregulation of grk5-dependent pathways may very well be causal for impaired cardiac development up to congenital heart disease. in fact, grk5 is already expressed during embryogenesis and we can detect it in the developing heart. however, grk5 has not been studied yet for a potential function during embryonic development in general or heart formation in particular. we have established zebrafish as a very time and cost efficient vertebrate model to investigate the role of grk5 on cardiac signalling and development. tools for grk5 specific loss-as well as gain-of-function analyses have been developed in our lab. we revealed an unexpected role of grk5 in the development of left-right asymmetry in zebrafish. clinically, this has been associated with disorders such as heterotaxy and other syndromes linked to ciliary dysfunction. many of those disorders are known to affect proper heart development resulting for example in septum defects or in detrimental translocation of the outflow tract. precisely, depletion of the close homolog of human grk5 in zebrafish mirrors the human syndrome called heterotaxy by displaying randomized placement of inner organs, aberrant heart looping and disrupted valve formation in the heart. in addition, loss of zebrafish grk5 results in a lower heart rate as well as dilatation of the embryonic heart at later stages of development. therefore, we believe that grk5 may potentially serve as a candidate gene for congenital heart disease. identification of a hcn3 interacting protein in mouse brain cao-ehlker x., hammelmann v., zong x., fenske s., biel m. department of pharmacy, center for drug research ludwig-maximilians-universität, munich center for integrated protein science cipsm, butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization activated cyclic nucleotide-gated cation channels (hcn) pass a depolarizing current (ih) that is involved in cardiac pacemaking and the control of numerous basic functions in neuronal circuits. the four hcn channel types (hcn1-4) display specific expression pattern in brain suggesting that each channel fulfills a distinct physiological function. while hcn1, hcn2 and hcn4 channels have been studied in quite some detail there is only little information on the particular role of the hcn3 channel. as an important step towards achieving a better understanding of hcn3 function we set out in this study to identify proteins that are assembled with hcn3 in brain tissue. to this end we performed a yeast two hybrid screen with a mouse cdna library using the hcn3 c-terminal domain as bait. several proteins were obtained and confirmed for interaction with hcn3 using heterologous coexpression in hek293 cells. here, we provide an in-depth analysis of the functional interaction between hcn3 and one of the identified interacting proteins (hip3.1). we show that hip3.1 physically binds to hcn3 channels in vitro and in vivo. still several open issues remain to be clarified i.e. the precise function of tpc1 and its tissue-specific and subcellular distribution. therefore we established a mouse model with a general deletion of tpcn1 and generated a series of tpc1 antibodies. using these tools we investigate the closer molecular and vesicular environment by different biochemical approaches i.e. affinity purification from native tissue derived from wild-type and as a control from knock-out mice, density gradient based vesicle separation, fluorescence activated organelle sorting (faos), total internal reflection fluorescence (tirf) and confocal microscopy. so far, we confirmed the tpc1 knock-out model by our self-generated tpc1 antibodies. tpc1 knockout mice are viable and do not show any obvious deficits. to isolate tpc1 containing vesicles or protein complexes, tissue or cell culture derived material was prepurified by sucrose density gradient centrifugation. for a subsequent mass spectrometric analysis this preparation was taken as a source material for coimmunoprecipitation or faos respectively. in another approach the migration pattern of tpc1 containing endosomes on linear density gradients was compared with a series of endolysosomal markers i.e. different rab-, er-, golgi-and lysosomal antibodies. potentially interesting markers were then in turn analyzed for their co-localization with tpc1 by confocal microscopy/tirf. by combining these results with that from mass spectrometric analysis of faos samples we collect data to get detailed information on the precise endolysosomal distribution pattern of tpc1. foxos are involved in a wide spectrum of cellular functions, including cell proliferation, apoptosis and regulation of oxidative stress. in order to identify novel target genes of foxo transcription factors and to achieve further insight into their role in cancer cells, dna microarray analysis was performed using wild type mcf-7 breast cancer cells and mcf-7 cells overexpressing foxo. we found that several genes involved in the tnf receptor/nf-κb pathway were differentially regulated. one of the genes that was identified to be up-regulated in foxo4 overexpressing cells was a20 a negative regulator of nf-κb signaling pathway. at both mrna and protein level foxo4-dependent up-regulation of this ubiquitin modifying enzyme was confirmed. to determine whether a20 is a direct target of foxo4, a luciferase reporter containing a 1.2 kb of the a20 promoter was co-transfected with different amounts of foxo4 wild-type expression construct. foxo4 induced a dosedependent increase in a20 promoter activity, supporting the assumption that a20 is a direct transcriptional target of foxo4. overexpression of foxo4 led to decreased activity of nf-kb signaling pathway as confirmed by reporter gene and nf-kb specific elisa assays. in addition, mcf-7 cells can be sentisized to tnf-α mediated cytotoxicity which is assocciated with a dimineshed activation of nf-κb. altogether, we identified a20, an ubiquitin modifying enzyme, as a novel foxo4 target gene. our data implicate that sustained foxo4 expression may be involved in regulation of tnf receptor/nf-κb pathway and leading to reduced cell survival. trpm7 is a bi-functional protein consisting of a transient receptor potential ion channel segment linked to an α-type protein kinase domain. trpm7 is essential for motility, proliferation and cell growth. up-regulation of trpm7 function is involved in anoxic neuronal death, cardiac fibrosis and tumor cell proliferation. recently, we have demonstrated that the recombinant trpm7 channel is inhibited by the known modulators of sk1-3 channels such as antimalarial plant alkaloid quinine, cyppa, dequalinium, ns8593, ska31, ucl 1684. the most potent of these compounds, ns8593 (ic50 1.6 µm), interferes with the regulation of trpm7 by cytosolic mg 2+ . here we show that ns8593 (10 µm) fully and reversibly inhibits native trpm7-like currents in hek 293 cells, freshly isolated smooth muscle cells, primary podocytes and ventricular myocytes. furthermore, we examined whether targeting of the native trpm7 currents by ns8593 would impact cellular processes known to be affected by a genetic inactivation of trpm7. we found that ns8593 (10-30 µm) suppressed motility of hek 293 cells without a detectable effect on cell viability. taken together, our findings indicate that ns8593 is a potent and reversible inhibitor of endogenous trpm7 currents and may be a good candidate drug for pharmacological targeting of trpm7. sulfur mustard (2,2'-dichlorodiethylsulfide; sm) is a highly toxic and mutagenic warfare agent classified as a weapon of mass destruction. as soon as sm was first used as a warfare agent, research aimed at the development of an effective antidote was launched. early studies with first-generation inhibitors of poly(adp-ribose) polymerases (parp) have revealed promising therapeutic potential in sm-induced skin injury, but the underlying mechanism remains elusive. the current renaissance of parp inhibitors in cancer chemotherapy has revived the discussion on their use for treatment of sm injury. thus we established a comprehensive study aiming the elucidation of the role of parp in sm pathology based on model substance 2-chloroethyl ethyl sulfide (cees), which is not classified as warfare agent. we have recently demonstrated that parp becomes rapidly activated in living human keratinocytes (hacat) after treatment with cees. the maximal parp activity was observed 10 minutes after treatment with 3 mm cees. the activation was transient and dose dependent. to our knowledge this is the first demonstration of parp activation after treatment with mustards in the context of live cells. an important question is how parp-1 becomes activated upon treatment with mustards. parp-1 is a first-line protein involved in the cellular response to dna strand breaks. however, mustards do not directly induce large numbers of such lesions. one possibility is that parp-1 is activated by dna breaks incorporated as base excision repair (ber) or nucleotide excision repair (ner) intermediates. thus, we performed knockdown experiments of ape1 and ercc1, i.e. endonucleases involved in ber and ner, respectively. the reduction of ape1 expression had no effect on parp activity. surprisingly, the knockdown of ercc1 almost completely abolished the cellular par production after cees treatment. the functional consequence of the errc1-parp cross-talk with regards to adduct removal is under investigation. however, our present data indicate that parp activity is not obligatory for the survival of cells upon cees treatment, as revealed by the lack of effect of the potent parp inhibitor abt-888. expression and activity of g protein coupled receptor kinase 2 (grk2) are elevated in several conditions of compromised heart function. although grk2 inhibition has been characterized as a promising therapeutic strategy in heart failure, a specific grk2inhibitor is not available. raf kinase inhibitor protein (rkip) inhibits raf1 but it also acts as a physiological inhibitor of grk2 upon phosphorylation by pkc at serine153. a detailed understanding of the rkip/grk2 interaction may help to identify inhibitory compounds for grk2. since phosphorylation often induces homo-oligomerization of proteins, we investigated whether this could be implicated in switching rkip from a raf1-into a grk2-inhibitor. co-immunoprecipitation assays showed that rkip self-association was substantially increased after pkc-mediated phosphorylation of rkip. rkip mutants either lacking or mimicking s153 phosphorylation confirmed that this phosphorylation is indeed a prerequisite for rkip/rkip association. cross-linking experiments with myc-tagged rkip in living cells or with purified rkip revealed that rkip phosphorylation by pkc promotes rkip dimers -not oligomers. to test whether dimerization is a critical step for the association of rkip with grk2, we generated a peptide to inhibit rkip dimerization. intriguingly, the peptide did not only prevent rkip dimerization but also attenuated rkip/grk2 association. this implicates, that dimerization of rkip is essential to bind grk2. to determine whether rkip dimers consequently inhibit grk2 activity, we established rkip mutants with high tendency to form dimers. subsequent functional analyses demonstrated that enhanced dimerization of rkip indeed translates into increased grk2 inhibition. we conclude that pkc-mediated phosphorylation of rkip is important for dimerization and that these dimers are essential for grk2 binding and inhibition. our results reveal new insights in the molecular mechanism of rkip/grk2 interaction and will help to develop specific grk2 inhibitors. expression and function of trpm3 ion channels in epithelial mdck2 cells dembla s., meiser j., philipp s. university of saarland institute for experimental and clinical pharmacology and toxicology, kirrberger str. 1, 66421 homburg, germany trpm3 proteins build ca 2+ permeable cation channels [1] activated by steroids [2] and sensitive to increased temperatures [3] . trpm3 channels are expressed in pancreatic ßcells as well as neurons of the dorsal root ganglion, where they act as mediators of insulin release [2] or as nociceptors of noxious heat, respectively [3] . however, northern blots and in situ hybridization experiments revealed that trpm3 is also expressed in epithelial cells of the choroid plexus and the ciliary body [1] as well as in the kidney. pcr analysis of different epithelial cell lines indicated that trpm3 is also expressed in madin-darby canine kidney2 (mdck) cells. quantitative analysis of trpm3 expression by qrt-pcr revealed a ~ 5 fold upregulation in mdck2 cells grown in confluency compared to well separated, proliferating cells. in contrast the level of expression of trpm7, a related ion channel described as regulator of proliferation in other cell types, remained constant. hek293 cells overexpressing trpm3 channels did not proliferate in the presence of the trpm3 agonist pregnenolone sulphate. however, as indicated by impedance analysis, the proliferation of mdck2 cells in the presence pregs was only slightly affected. when we analysed the transepithelial resistance (ter) of mdck2 epithelial cells in transwells as a measure for the formation of tight junctions, we found that the ter of cells grown in the presence of pregs was reduced. interestingly, ca 2+ imaging experiments using fura2 revealed that pregnenolone sulphate induces ca 2+ entry in well separated mdck2 cells but not in cells growing in confluency. we hypothesize that trpm3 might act as a regulator of cell proliferation and/or the formation of tight junctions in mdck2 cells. inhibition of grk2 by rkip improves cardiac contractility and structure in a transgenic mouse model of heart failure denzinger s., schmitt j. p., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany the raf kinase inhibitor protein (rkip) has been identified as a physiological inhibitor of g-protein coupled receptor kinase 2 (grk2). grk2 initiates g protein coupled receptor (gpcr) desensitization. since expression and activity of grk2 are upregulated in human heart failure, it has been proposed that grk2 inhibition may resensitize badrenergic receptor activity in heart failure patients. in this study, we evaluated chronic grk2 inhibition by rkip as a potential strategy to improve cardiac function in heart failure. to analyse the effect of rkip on heart failure, rkip transgenic mice were crossed with mice carrying a mutation in phospholamban (pln r9c ). pln r9c causes severe heart failure and premature death in humans and transgenic mice. cardiac function was significantly improved in the presence of rkip as shown by left ventricular catheterization and echocardiography. expression of heart failure marker genes anf and bnp was indistinguishable between wild-type mice and mice co-expressing rkip and pln r9c . in line with these findings, the life span of double transgenic mice was significantly prolonged compared to pln r9c transgenic mice. slow calcium transport into the sarcoplasmatic reticulum was characterised as cause for dilatated cardiomyopathy of pln r9c transgenic mice. since western blot analyses of rkip transgenic heart lysates showed increased phosphorylation of important regulators of cardiomyocyte relaxation, we analysed calcium transients and contractility of isolated cardiomyocytes as possible mechanism of the rkip mediated rescue. in the presence of rkip, calcium reuptake into the sarcoplasmatic reticulum was accelerated and cardiomyocyte relaxation improved. furthermore, coexpression of rkip significantly attenuated pathological cardiac remodelling. interstitual fibrosis and apoptotic cells were quantified in histological sections after sirius red-and tunel-staining. this study revealed a protective function of rkip in a genetic mouse model of human dilated cardiomyopathy by improving cardiac contractility and attenuating interstitial fibrosis and apoptosis. a detailed understanding of this rescue may help to find a new therapeutic strategy to improve cardiac contractility in heart failure. gαi-proteins comprise a group of three highly related members characterized by specific expression patterns. based on previous work of gi-mediated signaling pathways in cardiomyocytes and platelets, we checked gαi expression in mouse heart and platelets. the analysis revealed the presence of gαi2 and gαi3 with gαi2 as the predominant isoform. gene-targeted mice lacking either gαi2 or gαi3 were analyzed to unravel the physiological role of gαi-proteins in the cardiovascular system. extraordinarily prolonged bleeding times in gαi2-deficient animals were an obvious phenomenon. detailed analysis using isolated platelets gαi2-deficient mice exhibited reduced platelet activation and attenuated aggregation in response to stimulation by various agonists accompanied by reduced thrombus formation and diminished stability on a collagen-coated surface. employing in vivo injury/thrombosis models revealed abrogated thrombus formation selectively in gαi2-deficient mice. comparable results were obtained in experiments using mice with megakaryocyte/platelet-specific gαi2deficiency. to assess the pathophysiological consequences of platelet gαi2 function, we challenged these mice in experimental models of myocardial and cerebral ischemia. the results clearly show that platelet-gαi2-deficient mice were protected from both, myocardial and cerebral ischemia. in contrast, conventional gαi2-deficient mice subjected to the heart ischemia/reperfusion model exhibited a significantly increased susceptibility to ischemic injury as compared to wild type controls. in contrast, gαi3deficient mice were strongly protected from injury. thus we suggest that gαi2 and gαi3 play distinct roles in major cardiovascular disorders pointing to specific, non-redundant functions of these two highly related gαi isoforms. the cgmp signaling pathway is activated by nitric oxide (no), natriuretic peptides (anp, bnp & cnp), and cgmp-elevating drugs. it regulates several physiological functions such as smooth muscle relaxation, platelet inhibition, and cell growth and differentiation. recent studies indicate that cgmp signaling might also play a role in tumorigenesis, but the cellular and molecular mechanisms of cgmp's potential pro-and/or anti-tumor activities are not well understood. this study has examined the expression and function of components of the cgmp pathway in melanoma cells of murine and human origin. we have found that mouse b16 melanoma cells specifically express the alpha isoform of the cgmp-dependent protein kinase type i (cgkialpha) but not the beta isoform. treatment of intact cells with the membrane-permeable cgmp analog 8-br-cgmp induced the phosphorylation of cgki substrates, vasodilator stimulated phosphoprotein and phosphodiesterase 5. anp and cnp, ligands of the membrane-bound guanylyl cyclase gc-a and gc-b, respectively, did also activate the endogenous cgmp/cgki pathway. however, b16 melanoma cells did not respond to dea-no, which stimulates no-sensitive soluble guanylyl cyclases. interestingly, activation of cgmp/cgkialpha signal transduction was associated with an increase in erk1/2 and p38 phosphorylation, growth and migration of b16 melanoma cells. similar results were obtained with wm1205 human melanoma cells. in conclusion, we have identified a gc-a/gc-b/cgmp/cgkialpha pathway in melanoma cells, which stimulates tumor cell growth and migration in vitro. pharmacologic inhibition of cgmp signaling may offer a promising strategy for the treatment of melanoma. an increasing body of evidence supports important roles for voltage-gated calcium channels in idiopathic generalized epilepsies (iges), however which calcium channels participate in ige pathogenesis and how has yet to be fully understood. recently, it has been proposed that cav2.3 (r-type) and t-type calcium channels jointly contribute to oscillatory bursting in the reticular thalamus (rt) 1 , which is associated with absence epilepsy. cav3.2 is one of the two t-type calcium channels known to be expressed in the rt 2 . it has been demonstrated that ablation of either cav2.3 or cav3.2 reduces susceptibility to experimentally induced epilepsy 3;4 and in addition that both channels share several pharmacological properties [5] [6] [7] . to gain further insight into interacting mechanisms of these two channels in epilepsy, we tested cav2.3(-|-), cav3.2(-|-) and cav3.2(-|-)xcav2.3(+|-) mice side-by-side in the kainic acid model of epilepsy. we provide first in vivo data supporting a synergistic mode of action for cav2.3 and cav3.2 calcium channels in epileptogenesis. the deubiquitinase cyld regulates mechanisms of rip1/rip3-dependent necroptosis in neuronal cells diemert s. 1 , krieg s. vivo model of cerebral ischemia, we found, that cyld -/-mice exhibit significantly reduced infarction volume compared to control littermates. overall, these data reveal a role for cyld in rip1/3-dependent mechanisms of necroptosis in a model of glutamate toxicity in neuronal cells and further suggests cyld-mediated mechanisms of neuronal cell death as a potential therapeutic target after acute brain injury in vivo. cyanamide-mediated inhibition of n-acetyltransferase 1 dierolf d., bonifas j., blömeke b. university trier department of environmental toxicology, universitätsring 15, bldg. n, 54286 trier, germany cyanamide has been used for decades for medical purposes in the treatment of alcoholism and for agricultural purposes as a plant growth regulator and bud-breaking agent. its therapeutic effect is mediated by reversible inhibition of aldehyde dehydrogenase and it was reported to be metabolised in vivo mainly via coenzyme a dependent n-acetylation by n-acetyltransferases. reported to be a substrate for n-acetyltransferases, cyanamide has a different molecular structure to arylamines and hydrazines, the preferred substrates for nacetyltransferases. therefore a more detailed investigation of its interrelations with nacetyltransferases was performed. we analysed nat1 enzyme activities after incubation of thp-1 cells with cyanamide for 24h, and found that the metabolic conversion of the classic substrate para-aminobenzoic acid was significantly reduced at physiologically relevant concentrations. in detail a significant dose-and time-dependent nat1 protein inhibition was observed for 100 and 1000 µm cyanamide using over-expressed human recombinant nat1 (insect cell cytosol containing recombinant human nat1*4). however, no inhibition was found in the presence of recombinant nat2*4. as we also provide evidence that cyanamide is not metabolised via coenzyme a dependent nacetylation in vitro by human nat1 or nat2 cytosol, by thp-1 cells or by human liver cytosol, we can conclude that this inhibition is not based on substrate-dependent downregulation of nat1. further mechanistic and kinetic studies indicated that cyanamide reacts with the active site cysteine residue of nat1, leading to its rapid inhibition. since the presence of the reduction agent dithiothreitol did not reverse the results it could be that it is possibly not caused by oxidative processes. in sum these data indicate that cyanamide is able to interact with cysteine residues of human nat1, which causes its inhibition but not by a substrate-dependent mode of action. taken together our results show, that cyanamide is not n-acetylated by human nats, but might modulate nat1 dependent detoxification and activation of arylamines. dissecting the signal transduction pathway of acute hypoxic vasoconstriction (hpv) in precapillary pulmonary arterial smooth muscle cells ( low levels of oxygen in the pulmonary airways induce acute hypoxic pulmonary arterial vasoconstriction (acute hpv) redirecting blood flow to normoxic areas of the lung to assure optimal uptake of oxygen during ventilation. acute hpv lasting several minutes occurs predominantly in the precapillary region of the pulmonary vascular tree [1] . therefore, precapillary pulmonary arterial smooth muscle cells (pasmc) have been suggested as sensor as well as effector cells and trpc6 a member of the classical transient receptor potential (trpc) ion channel family was identified to be essential for the initiation of ca 2+ influx and the subsequent contraction of pasmc [2] . however, the underlying oxygen sensor and the exact signal transduction pathway(s) in pasmc have not been fully elucidated yet. by using gene-deficient mouse models as well as downregulation of potential candidate proteins by specific small interfering rnas (sirnas), we aim to dissect signaling cascades of trpc6 channel activation in acute hpv. for pasmc isolation and culture from mice we use a technique based on magnetic separation of intrapulmonary arteries originally developed in rats [3] . trpc-expression in freshly isolated and passaged pasmc cultured in low (5%) and high fetal bovine serum (25%) was analyzed. interestingly higher passage numbers resulted in a significant down-regulation of trpc1 and trpc6 the most predominantly expressed channel monomers in pasmc, while different serum concentrations resulted in no significant changes in their expression rates. sirnas were designed, transfected and successfully tested in hek293 cells and pasmc. initial results of the dissection of the signal transduction pathway activating trpc6 and inducing acute hpv in pasmc will be presented. references [1] staub, n. c. (1985) . site of hypoxic pulmonary vasoconstriction, chest 88, 240s-245s. [2] weissmann, n. et al. (2006) . classical transient receptor potential channel 6 (trpc6) is essential for hypoxic pulmonary vasoconstriction and alveolar gas exchange, proc. natl. acad. sci. u.s.a. 103, 19093-19098 trpv5 is a highly selective calcium channel expressed in various tissues amongst others in placenta. the channel may be involved in transcellular calcium transport in epithelial tissues thereby playing some role in calcium homeostasis of the body. in the placenta trpv5 is assumed to contribute to the maternal-fetal calcium transport. most probably trpv5 is part of a multiprotein channel complex but most of the components of this complex are unknown so far. our aim is to find interaction partners of the trpv5 protein in the placenta that might contribute to the regulation of the trpv5 protein function. therefore we expressed the intracellularly located n-and c-terminal parts of trpv5 (aa 1-330 and 571-723) as trpv5-gst (glutathione-s-transferase)-fusion proteins and used the purified recombinant proteins for pulling down proteins from human placenta cell extracts. the proteins pulled down by this approach were analysed by mass spectrometry. we identified several potential trpv5 interacting proteins which were not associated with the gst protein only. one of the proteins which was highly enriched with the n-terminal part of the trpv5 protein is calpain 6. in contrast to the classical calpains (calcium activated cystein proteases), calpain 6 is unique in that it lacks the cysteine residue in the active site. calpain 6 is mainly expressed during embryogenesis and is reported to be involved in cytoskeleton stabilisation but with unknown function in placenta. the interaction between trpv5 and calpain 6 was confirmed in reciprocal pulldown experiments and the trpv5 binding region for calpain 6 was narrowed down by using overlapping n-terminal trpv5-gst fusion proteins. after injection of trpv5 crna into xenopus laevis oocytes calcium uptake into the oocytes was measured; this uptake was largely reduced after co-injection of the calpain 6 crna. in further experiments we want to study potential regulatory effects of the trpv5 protein on the calpain 6 function in cell culture models. comparative studies on the effects of the human carcinogen inorganic arsenite and its recently identified thiolated metabolite thio-dma v on human urothelial cells ebert f., leffers l., unterberg m., schwerdtle t. universität münster institut für lebensmittelchemie, corrensstr. 45, 48149 münster, germany it has been demonstrated that chronic ingestion of 50-200 µg/day inorganic arsenic is associated with an increased risk for cancers of the skin, the lung and the bladder, but until now the underlying toxic modes of action are still under debate. in this context, in the last five years one main focus has been given to the role of human inorganic arsenic metabolism and nowadays it is generally accepted that human biomethylation contributes to inorganic arsenic induced carcinogenicity. due to further improvements in arsenic speciation techniques recently a new thiolated arsenite metabolite, the thio analogue of the well known metabolite dimethylarsinic acid (dma v ), the so called thiodimethylarsinic acid (thio-dma v ), has been discovered in human biological samples. after synthesizing and analytically characterizing this metabolite (bartel et al. 2011 , j toxicol. 2011 we investigated its toxic effects in direct comparison with ias iii in human urothelial cells. thereby cell cycle studies revealed a g2/m-and s-phase arrest as well as subg1 peak formation in case of thio-dma v . moreover, thio-dma v induced apoptosis (subg1, caspase 3 activity) at lower concentrations and earlier time points as compared to ias iii . most likely this is partly due to the higher cellular bioavailability of thio-dma v (aas/icp-ms). regarding genotoxicity, a generation of dna single strand breaks (alkaline unwinding technique) as well as an increased formation of reactive oxygen species (ros, dcfh-da-fluorescence) occurred only at high cytotoxic concentrations. however, thio-dma v strongly increased h2o2 induced ros formation at very low nanomolar concentrations, which might result in cogenotoxic effects. since our earlier studies have shown a strong inhibition of h2o2 induced poly(adp-ribosyl)ation especially by trivalent methylated arsenic metabolites, actual studies investigate the impact of thio-dma v on cellular poly(adp-ribosyl)ation, parp-1 gene expression, protein level and cellular cleavage, which might hopefully give further hints regarding the mode of action behind thio-dma v induced apoptosis. mitochondrial dysfunction in models of alzheimer´s disease eckert a. universitäre psychiatrische kliniken basel neurobiology laboratory, wilhelm klein strasse 27, 4012 basel, switzerland the histopathological characteristics of alzheimer's disease (ad) are amyloid-ß (aß) containing plaques and neurofibrillary tangles (nfts) as well as neuronal and synaptic loss. until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. there is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several app and tau transgenic mouse models. recently, we examined mitochondrial function in a novel triple transgenic mouse model (pr5/app/ps2) -triplead mice -that combines both pathologic features of the disease in brain. using comparative, quantitative proteomics (itraq) and mass spectroscopy we found a massive deregulation of 24 proteins, of which one-third were mitochondrial proteins mainly related to complexes i and iv of the oxidative phosphorylation system (oxphos). remarkably, deregulation of complex i was related to tau, whereas deregulation of complex iv was aß dependent, both at the protein and activity levels. the triplead mice showed synergistic effects of aß and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. at 12 months, the strongest defects on oxphos, synthesis of atp and reactive oxygen species were exhibited in the triplead mice, again emphasizing synergistic, ageassociated effects of aß and tau in impairing mitochondria. evidences from ad post-mortem brain as well as cellular and animal ad models indicate that aß and tau protein trigger mitochondrial dysfunction through a number of pathways, such as impairment of oxidative phosphorylation, elevation of reactive oxygen species production, alteration of mitochondrial dynamics, and interaction with mitochondrial proteins. moreover, recent reports indicate that aß may also interact directly with intracellular proteins such as the mitochondrial enzyme abad (aß binding alcohol dehydrogenase) in executing its toxic effects. mitochondrial dysfunction occurs early in ad, and aß's toxicity seems to be in part mediated by inhibition of abad. in total, a vicious cycle as well as several vicious circles within the cycle, each accelerating the other, can be drawn emphasizing the synergistic deterioration of mitochondria by tau and aß. olesoxime is a novel mitochondrial-targeted compound that is orally active and crosses the blood brain barrier. the cholesterol-oxime targets proteins of the outer mitochondrial membrane and represents a promising drug candidate for neurodegenerative diseases 1 . we evaluated olexoxime's neuroprotective effects against mitochondrial dysfunction in an animal model for alzheimer`s disease (ad). dissociated brain cells (dbc) and mitochondria were isolated from brains of c57/bj6-thy1-appsl (ad-mice) mice that were fed with 600 mg olesoxime/kg feed for 3 months. drug plasma levels reached approx. 600 ng/ml. respiration of isolated mitochondria were significantly diminished in ad-mice due to reduced complex i, i+ii and cox activities. consequently, mitochondrial membrane potential (mmp) was significantly reduced in dbc from ad-mice. olesoxime normalized respiration chain complex activities and the mpp. to further evaluate the beneficial effects of olesoxime on complex i activity, we challenged dbc with rotenone ex vivo and observed that olesoxime treatment was protective. to further clarify the mode of action, we analyzed the ability of olesoxime to prevent opening of the mitochondrial permeability transition pore (mptp) in vitro using energized brain mitochondria by measuring ca 2+ -and atractyloside (atr) induced swelling. the opening of mptp precedes apoptosis and can be induced by mitochondrial dysfunction due to calcium overload, oxidative stress, elevated phosphate concentrations or adenine nucleotide depletion. olesoxime prevented ca 2+ -as well as atr induced mitochondrial swelling. atr inhibits the adenine nucleotide translocase (ant) that requires appropriate membrane properties to mediate mitochondrial permeability transition (mpt). since cholesterol (cho) and its derivates represent potent modulators of membrane viscosity, we related the effects of cho and olesoxime on mptp opening to membrane properties. both, cho and olesoxime reduced the flexibility of membrane acyl-chains in energized mitochondria and prevented atr induced mptp opening. however, cho didn`t prevent ca 2+ -induced mptp opening, indicating a different mode of action for olesoxime. our data confirm olesoxime as drug candidate against mitochondrial dysfunction, which is considered to play a pivotal role in neurodegenerative diseases. the work was supported by the european union under the 7th framework program for rtd -project mitotarget -grant agreement health-f2-2008-223388. several inflammatory glomerular kidney diseases are accompanied with a massive production of reactive oxygen species (ros) that may attack the glomerular filtration barrier by affecting podocyte function and may contribute to apoptotic or necrotic cell death of mesangial cells. otherwise, ros also trigger fine-tuned signaling processes that may result in cell proliferation or cell migration. to define such redox-driven signaling devices, we performed a non hypothesis-driven proteomic approach, to identify homo-or heteromeric protein complexes induced by ros. to this end, protein lysates of human podocytes were treated with or without hydrogen peroxide (250 µm) for 10 min. thereafter, the cell lysates were subjected to diagonal 2d gel electrophoresis and putative redox-affected proteins were analyzed by ms/ms-analysis. by this approach, we could identify a series of proteins that form interprotein-disulfide bonds in a redoxdependent manner. one of those proteins could be characterized as the regulatory subunit of protein kinase a (r-subunit of pka), which belongs to the family of serine/threonine kinases. to evaluate whether ros is capable to activate pka also in a more physiological setting, we treated rat mesangial cells with pdgf-bb to induce ros formation and we could demonstrate that pdgf-bb induces dimerization of r-subunits in a redox-dependent manner. to demonstrate whether pdgf-bb induces pkadependent pathways, we analyzed the effects of pdgf-bb on phosphorylation of serine 157 of vasodilater stimulated protein (vasp) a classical target of pka. in fact, pdgf-bb induced vasp phosphorylation independently of intracellular camp levels. moreover, elevating camp levels via activation of adenylate cyclase with forskolin did not change the dimerization state of r-subunits. pdgf-bb-induced dimerization of the r-subunits and subsequent phosphorylation of vasp was blocked by diphenyljodonium (dpi), indicating activation of a nadph oxidase is essential for pka activity. taken together, we demonstrate a redox-dependent activation of pka by pdgf-bb and this may hint also for a probably protective role of ros in rat mesangial cells. testing the potential sensitizing capacity of chemicals is currently done by using the murine local lymph node assay (llna). animal welfare and eu cosmetics directive demands alternative methods to animal tests. the purpose of this study was to establish an in vitro assay for the prediction of skin sensitizers. based on the finding that the majority of skin sensitizers are electrophilic or have the potential to be metabolized to electrophilic substances, it is assumed that they can activate the nrf2-keap1-antioxidant response element (are) regulatory pathway. here, we report the results obtained from the lusens assay that detects electrophilic chemicals using the nrf2 pathway. the cell line lusens was derived from immortalized keratinocyte hacat cells and carries a luciferase reporter gene under the control of an are-element from the rat nadph quinone reductase nq1. the lusens assay was in house validated with a panel of 54 chemicals and cosmetic ingredients including the 22 performance standard substances of the local lymph node assay. the predictivity of this assay was compared to the predictivity of the murine llna and to human patch test data and can be considered as reliable screening approach (accuracy of 83% compared to human data). however, in order to cope with the complex multi-step mechanism of skin sensitization, an integrated approach of in vitro assays mimicking several steps was designed; thereof, the are-dependent gene activation represents one module. time-resolved fluorescence ligand-binding assays for parathyroid hormone receptors emami-nemini a. 1 ligand-binding studies represent essential tools for pharmacological research on g protein-coupled receptors. in recent years, time-resolved fluorescence gained significant relevance as readout for ligand binding studies. however, ligand-binding assays for parathyroid hormone receptors (pthrs) utilizing fluorescent parathyroid hormone (pth) were missing. therefore, we generated various fluorescent pth analogues which exhibit properties of native pth in terms of affinity, potency and internalization. for the purposes of academic and commercial research, we utilized labeled pth to set up three time-resolved fluorescence assay formats: (i) classical separation binding assay, based on time-resolved fluorescence and suitable for native receptors; (ii) homogeneous timeresolved fluorescence resonance energy transfer (htrf) based on tag-lite technology for high through-put screening; (iii) htrf based on antibodies, a synergistic approach using htrf with minimized receptor modification. this work will facilitate the development of new drugs directed to the pthr as well as fundamental research on the pthr. anandamide production in eosinophilic granulocytes is independent of il-5 and eotaxin stimulation engeli s., reinke j., zörner a., tsikas d., jordan j. medizinische hochschule hannover institut für klinische pharmakologie, carl-neuberg-straße 1, 30625 hannover, germany introduction: some animal and in vitro studies suggest that endocannabinoids exert anti-inflammatory effects. specifically, inhaled anandamide reduced the obstructive effect of leukotrien d4 in airways, and a specific cannabinoid receptor agonist significantly reduced pulmonary inflammation in guinea pigs. we have recently shown that segmental bronchial allergen challenge is associated with significant increases of anandamide concentrations in bronchoalveolar fluid of patients with asthma. the concomitant increase in eosinophilic counts, eotaxin and il-5 concentrations in bronchoalveolar fluid led us to hypothesize that anandamide is produced by eosinophilic granulocytes in response to chemotactic stimuli. peripheral eosinophilic granulocytes were isolated from whole blood by means of percoll gradient centrifugation and magnetic separation employing cd16antibodies conjugated to magnetic beads. isolated cells were counted and anandamide measurements were typically performed in whole cell lysates of 1.5x10 6 eosinophils. we stimulated eosinophils with varying concentrations of il-5, eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26). to prevent anandamide degradation, a specific fatty acid amide hydrolase (faah) inhibitor (oloxa) was employed. anandamide concentrations and faah activity were determined by stable isotope dilution using lc-ms/ms protocols. results: first, we confirmed the ability of eosinophilc granulocytes to synthesize anandamide. however, cellular anandamide content could only be measured when faah was effectively blocked with oloxa, and strong faah activity was demonstrated in eosinophils. with oloxa, typical anandamide concentrations were in the range of 2-4 pm/1.5x10 6 eosinophils. neither il5 (50-500 pg/ml), nor any of the eotaxins (50 ng/ml either alone or in varying combinations) did stimulate anandamide production after 30min of incubation. our results suggest that chemotactic molecules like eotaxin and il-5 are not responsible for increased anandamide formation in eosinophils during allergen challenge. in a next step, the effects of anandamide on eosinophils and bronchial epithelial cells need to be determined. the suprisingly high faah activity in eosinophils may point to an alternative pathway facilitating prostaglandin and leukotriene synthesis by production of arachidonic acid. screening methodology for estimatation of dermal absorption in vitro fabian e., goth c., guth k., mehling a., van ravenzwaay b., landsiedel r. basf se experimentelle toxikologie, carl-bosch-strasse 38, 67056 ludwigshafen, germany dermal absorption is used in the evaluation of the effectiveness of pharmaceutical or cosmetic formulations, but often dermal absorption is a critical parameter in risk assessment of pesticides or chemicals. therefore, knowledge of dermal absorption is e.g. helpful in formulation development. skin absorption is routinely measured in vivo or in vitro following oecd tg 427 or 428. however, these tests are complex, time consuming and expensive. therefore, a method was developed to allow simple and rapid screening. the experiment uses dermatomized skin in modified franz type diffusion cells. 10 µl of test substance preparation are applied to the skin preparation. after 6h, the skin is washed and the amount of penetrated substance is quantified. the receptor fluid and the washing solutions are optimized for subsequent analyses by lc-ms. we performed dermal absorption screenings in parallel to our routine guideline studies and demonstrated a good correlation of the results of both study types: the total recovery found in the screening studies is somewhat lower than in the corresponding guideline studies but is always in an acceptable range above 80%. the efficacy of the skin washing procedure is lower than under routine conditions, most probably due to the change to an lc-ms-compatible washing solution. overall, the dermal absorption screening is an easy, fast and cost-effective screening method for the estimation of dermal absorption of a wide variety of test substances and formulations. the p53 tumor suppressor protein is frequently inactivated in human cancers by diverse mechanisms. owing to its fundamental role in the maintenance of genomic stability and cancer prevention, p53 is an attractive target in cancer therapy and several approaches were pursued to restore p53 function in tumor cells. polyamidoamine (pamam) dendrons are positively charged molecules with a systematically branched structure that interact with the negatively charged cell membrane, inducing cellular uptake via endocytosis. in the present study, biotin-labeled p53 protein was attached to a dendronized streptavidin nanocarrier to facilitate its internalization into different tumor cell lines. first, biotin-substituted pamam dendrons were conjugated with streptavidin to allow formation of the dendronized streptavidin nanocarrier. the nanocarrier displayed uptake into hela cervix carcinoma and a549 lung adenocarcinoma cells without detectable cytotoxicity. biotin-labeled p53 was then conjugated to the dendronized streptavidin, preserving its specific dna-binding in vitro. immunoblot analysis revealed efficient internalization of biotin-p53 into hela cells in the presence of dendronized streptavidin. in line with this finding, specific cellular uptake of biotin-p53 was observed by confocal microscopy, which showed a cytoplasmic and peri-nuclear localization in hela, a549 and saos osteosarcoma cells. the internalized biotin-p53 also partially co-localized with early endosomes. importantly, the delivery of biotin-p53 into p53-deficient saos cells resulted in impaired cell viability and upregulation of caspase 3/7 activity, demonstrating its biological functionality. this study intriguingly demonstrate the efficient delivery of functional biotin-p53 into different tumor cell lines using the novel streptavidin nanocarrier, which can be further modified to allow cell-type specific targeting and combined with cytotoxic drugs such as doxorubicin. identification of novel ahr target genes in rat liver oval cells faust d. 1 , vondracek j. the aryl hydrocarbon receptor (ahr) is a transcription factor involved in physiological processes, but also mediates most, if not all, toxic responses to 2,3,7,8tetrachlorodibenzo-p-dioxin (tcdd), some polycyclic aromatic hydrocarbons (pahs) and dioxin-like polychlorinated biphenyls (pcbs), such as pcb126. activation of the ahr by these ligands leads to its dimerization with arnt and transcriptional activation of several phase i and ii metabolising enzymes. while it is generally accepted that many pahs are thereby transformed to genotoxic metabolites, this classical signalling pathway so far failed to explain the tumour promoting effects of the nongenotoxic compounds tcdd and pcb126. thus, there is an urgent need to define genetic programmes orchestrated by ahr to unravel its role in physiology and toxicology. we have recently shown that treatment of rat liver oval cells with tcdd or pcb126 leads to a release from contact-inhibition involving activation of the ahr, elevation of jund protein levels and transcriptional activation of cyclin a (1, 2) . loss of contact-inhibition is one hallmark of tumour promotion. to better understand ahr-driven pathways we identified the transcriptional programme using high density microarrays in response to pcb126. already 6 h after treatment, 69 genes were found to be upregulated and 76 genes downregulated indicating that these are direct ahr-dependent target genes. david analysis revealed that these genes are involved, for instance, in drug and lipid metabolism, cancer pathways, tgf-b signalling and cell-cell communication. ten of the 69 genes were selected for confirmation by semi-quantitative rt-pcr. using the ahr inhibitor ch-223191 and sirna directed against ahr and arnt, we further demonstrated that ahr-and arnt-function is required for transcriptional activation of the selected genes. finally, we identified the transcription factor foxq1as a novel ahr target protein in rat liver oval cells. although the function of foxq1 is poorly understood, it has been shown very recently that foxq1 is overexpressed in colorectal cancer and is involved in epithelial-mesenchymal transition in breast cancer cells. its function in ahrmediated tumour promotion, however, remains to be determined. the practical relevance of histamine h1 and h3 receptors in the brain can be easily deduced since h1 receptor antagonists of the first generation have a sedative effect and an inverse h3 agonist, pitolisant (close to its introduction to the market), is active against excessive daytime sleepiness associated with narcolepsy. in this context the question arises whether also h2 and h4 receptors possess a practical relevance in the brain. to this end, we examined whether the electrically evoked 3 h-noradrenaline release in superfused human cerebral cortex slices is affected by agonists at the above receptors. the h2 agonist impromidine 10 µm failed to affect noradrenaline release in human cortex slices although it facilitated noradrenaline release in guinea-pig cortex slices; the maximum extent of facilitation was 50 %, the pec50 was 6.9 and the pa2 of the h2 antagonist ranitidine against impromidine was 7.2. with respect to h4 receptors there is some controversy in the literature whether they occur in the brain at all. however, we were able to detect h4 receptor mrna in the human and mouse cortex by the reverse transcriptase polymerase chain reaction. in cortex slices of either species, noradrenaline release was not affected by the h4 agonist 4-methylhistamine 10 -30 µm but inhibited by histamine 10 µm via h3 receptors by 28 and 55 %, respectively. in mouse cortex membranes, 4-methylhistamine 30 µm also failed to affect 35 s-gtpγs binding although r-α-methylhistamine 3 µm, acting via h3 receptors, increased it by 20 %. in conclusion, h2 receptors facilitating noradrenaline release are detectable in the isolated guinea-pig but not human cortex. despite the presence of h4 mrna in the brain, functional readouts of this receptor, i.e. modulation of noradrenaline release (humans, mice) and modulation of 35 s-gtpγs binding (mice), could not be shown. murine cx40 promoter activity is dependent on the transcription factor creb fels b., nunes f., schmitz w., müller f. u. westfälische wilhelms-universität münster institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany connexin 40 (cx40) is a gap junction protein expressed in atrial myocytes and the ventricular conduction system, mediating the electrical intercellular communication in the myocardium. alterations in cx40 function were linked to the pathophysiology of atrial fibrillation and heart failure. in the heart, camp dependent gene transcription is regulated by members of the creb/crem/atf family which bind to camp responsive elements (cres). similar to the human cx40 gene promoter, the murine promoter contains one cre. cardiomyocyte-specific overexpression of a crem-isoform (crem-ib∆c-x) led to cx40 down-regulation, suggesting that creb related transcription factors are involved in cx40 gene regulation. in order to study the functional role of creb in the regulation of the cx40 promoter we have generated a luciferase based murine cx40 promoter reporter gene construct and monitored its activity in a permanent cell line upon overexpression of constitutive-active creb (cacreb) and a non-phosphorylatable dominant-negative creb (dncreb) isoform. surprisingly, overexpression of cacreb and dncreb both lead to a reduction of cx40 promoter activity (cacreb 46% ± 5% vs control 100% ± 3%; p<0.05 vs control , n=15), dncreb 45% ± 2% vs control 100% ± 3%; p<0.05 vs control, n=18). the activity of the murine connexin 40 promoter is modulated by creb. both cacreb and dncreb led to cx40 down-regulation, which could be explained by induction of inhibitory transcription factors creb/crem/atf1 transcription factor family, which in turn could suppress cx40 promoter activity. (supported by the dfg) results: fxa increased par-2 mrna, protein and cell-surface expression and augmented par-2-mediated mitogenesis. par-1 expression was not influenced. the regulatory action of fxa on par-2 was concentration-dependent and mimicked by a par-2 selective activating peptide. the thrombin inhibitor argatroban or par-1 gene silencing did not influence fxa-stimulated par-2 expression. fxa increased oxidative stress and expression of the nadph oxidase subunit nox-1 in smc. nox-1 gene silencing prevented fxa-stimulated par-2 regulation, as did ebselen and catalase. exogenous hydrogen peroxide increased par-2 expression and mitogenic activity. fxa induced nuclear translocation and par-2 dna binding of nuclear factor kb (nfkb). inhibition of nfkb prevented fxa-stimulated par-2 expression. in separate studies, fxa promoted par-2 mrna stabilisation through increased human antigen r (hur)/par-2 mrna binding and cytoplasmic shuttling. hur gene silencing abolished fxa-stimulated par-2 expression. conclusion: expression and mitogenic activity of vascular par-2, but not par-1, is upregulated by fxa. this action involves transcriptional and post-transcriptional mechanisms mediated through nox-1-containing nadph oxidase and its downstream effectors hydrogen peroxide, nfkb and the mrna stabilising protein hur. continued generation of fxa by the mural thrombus, and the autoregulatory feedback control of par-2 may maintain the inflammatory and proliferative state of the injured vessel, thereby promoting vascular remodeling. the mrna stabilising factor hur is a critical regulator of human proteaseactivated receptor 4 aim: we recently reported that functional expression of par-4 thrombin receptors is induced in human saphenous vein smc exposed to high glucose. this effect could be attributed in part to transcriptional mechanisms mediated through nfkb but the contribution of post-transcriptional effects such as mrna stabilisation is not known. this study explored the potential role of the mrna stabilising factor human antigen r (hur) in the regulation of par-4. methods: human saphenous vein smc were serum-deprived prior to study. gene expression was determined by realtime pcr, protein expression by western blotting. gene silencing utilized commercially available sirna. hur binding to par-4 mrna was determined by immunoprecipitation ("pull-down") pcr. results: high glucose (25 mm vs 5.5 mm) slowed par-4 mrna degradation in the presence of actinomycin d. par-1 mrna decay was not affected. hur binding to par-4 mrna and nucleo-cytosolic shuttling was enhanced by high glucose, total hur protein expression was not affected. hur sirna abolished the high glucose-stimulated induction of par-4 mrna. hydrogen peroxide (h 2o2) also induced cytosolic hur shuttling and increased par-4 mrna and total protein expression. the role of endogenously generated h2o2 in the regulatory effect of high glucose on par-4 expression was investigated with the nadph oxidase inhibitors apocynin/diphenyliodonium (to prevent h2o2 generation) and cell-permeant catalase (to degrade cellular h2o2). both approaches prevented the stimulatory effect of high glucose on par-4 expression. cyclic amp has been reported to suppress hur activation and in the present study, the cyclic amp stimuli forskolin and cicaprost (prostacyclin analog) suppressed basal hur shuttling and par-4 transcript stability. cicaprost also attenuated high glucose-induced hur binding to par-4 mrna and as a consequence normalised par-4 expression and inflammatory signalling in high glucosetreated cell. conclusion: the regulation of par-4 thrombin receptors in human vascular smc is critically dependent on the mrna stabilising actions of hur. through activation of hur, high glucose and other hur stimuli such as ang ii and exogenous h2o2, increase par-4 expression, while cyclic amp agonists such as prostacyclin oppose this effect. such interactions could potentially represent a fine-tuning mechanism to control par-4 expression and ultimately also the mitogenic and inflammatory actions of thrombin in the vessel wall. nucleoside diphosphate kinase b (ndpk b) is a member of a family of ubiquitously expressed enzymes required for nucleoside triphosphate synthesis. thus, they are involved in the regulation of a variety of cellular processes, e. g. g protein mediated signal transduction. however, whether ndpk b has a specific role in the regulation of angiogenic processes in endothelial cells is unknown. therefore, we studied the function of ndpk b in the vasculature in a developmental, an ischemia-induced and an in vitro model of angiogenesis. firstly, depletion of ndpk b expression was achieved by morpholino-mediated knockdown in zebrafish embryos in which the developing vasculature can be visualized by egfp expression in the endothelium. 72h post fertilization, ndpk b knockdown larvae showed a dramatic inhibition of intersegmental and dorsal longitudinal anastomatic vessel formation compared to control injected fish. this phenotype could be rescued by early re-expression of ndpk b. secondly, ischemia driven angiogenesis was studied in ndpk b-depleted mice and wildtype littermates after excision of the left femoral artery. hind limb blood flow was assessed by laser doppler perfusion imaging immediately before and after ligation (day 0) and on postoperative days 3, 7, 14, 21, 28, and 35 . a significant reduction of recovery was observed in the ndpk b depleted mice at days 3 and 7. thirdly, in vitro-sprouting angiogenesis was analyzed in human umbilical vein endothelial cell (huvec) spheroids with and without sirna-mediated ndpk b knockdown. vascular endothelial growth factor (vegf)induced sprouting was significantly attenuated by ndpk b knock down by more than 50% in comparison with control transfected huvec. we conclude from these results that ndpk b is an essential regulator of angiogenesis. the loss of ndpk b may specifically interfere with the vegf-induced migration and proliferation during endothelial sprouting. ethylene oxide in blood of ethylene-exposed volunteers ethylene (et) is a commercially important high volume industrial chemical. inhaled and endogenous et is metabolized in mammals to ethylene oxide (eo), which is carcinogenic in rats and mice. until now, no data on the oxidation of et in et-exposed humans has been published. in the present study, we investigated the formation of eo in four male adult volunteers exposed for 4 hours to constant atmospheric et concentrations of 5, 20, or 50 ppm by means of a breathing mask. during exposure, et concentrations were measured in inhaled and exhaled air by gc/fid and eo concentrations in venous blood by gc/ms. rates of et metabolism were obtained from the product of the differences in the et concentrations with the pulmonary ventilation. in each subject, linear correlations were found between the et exposure concentration and the rate of et metabolism or the eo concentration in blood. mean rate of et metabolism was 5.5 ± 1.4 nmol/h/ppm/kg body weight. steady-state concentrations of eo in blood differed by a factor of 2 between the volunteers. these inter-individual differences likely reflect the polymorphism of glutathione s-transferase theta, the main eo metabolizing enzyme in human liver. mean eo concentration in blood at steady state was 1.5 ± 0.08 nmol/l blood per ppm of et. the data will be used for validating a physiological toxicokinetic model which will describe the et related eo tissue burdens in rodents and humans. the model predictions will support risk evaluations of et. financially supported by the lower olefins sector group of cefic. in vitro effect of stw11 on human dendritic cells fink c. 1 , bonaterra g. a. extracts of echinacea (purple coneflower) are used in the prevention and therapy of infectious diseases. the medicinal product stw 11 contains the extract from purple coneflower, and in addition, extracts of monkshood, venom of honey bee and bushmaster snake in homeopathic dilutions. previous studies showed a stimulation of the cellular and humoral immune response. dendritic cells (dcs) are antigen presenting cells that act at the interface of the innate and adaptive branches of the immune system. during stages of dc differentiation, the ability to internalize antigens varies and decreases during maturation. in this study, we determined the influence of stw11 on the expression of maturation related genes (cd1a, cd83), cytokines (tnfα, il-4, il-12), chemokines (ccr7), major histocompatibility complex ii(mhc-ii) and toll-like receptors (tlr2, tlr4). in mature (mdc) and immature dc (idc) using real-time rt-pcr. peripheral blood mononuclear cells (pbmcs) were isolated from buffy coats of human volunteers by densitygradient (ficoll ® ) and seeded in 6 well plates. non-adherent cells were eliminated. to induce idc development, 50 ng/ml il-4 and 50 ng/ml granulocyte macrophagecolony stimulating factor (gm-csf) were added. at day 6, maturation was induced by addition of lipopolysaccharide (lps) at a final concentration of 1µg/ml and cultured for additional 3 days. after incubation with different concentrations (0. in idc, compared to control, we found a significantly increased expression of cd83 (2.1-2.6 fold) and tnfα (2.6-3.3-fold) genes after treatment with 0.1-5% stw11, respectively, but no effect was found on the expression of cd1a, il-4, il12, adam19, cd11c, cd40,tlr2, tlr4, mhc-ii and ccr7. in summary, these data demonstrate a stimulatory effect of stw 11 in idc and especially in mdc, concerning an increase of various genes related to maturation (cd83), immunomodulation (tnfα, cd40), adhesion (ccr7) and antigen presentation (mhc-ii) and are in accordance with the therapeutic use in infectious diseases. waterproofing sprays are widely used consumer products containing for example fluorinated polymers or silicon based compounds dissolved in alcohols or volatile petroleum distillates. there have been repeated reports on cases of severe acute respiratory disorders especially when using products that newly entered the market. it is hypothesized that impairment of the pulmonary surfactant by deposition of inhaled respirable particles of the active compound is one of the main causes of the acute lung injury. since the inhalation toxicity cannot be predicted a priori based on the physical and chemical properties of the formulation, proper test strategies are required to ensure consumer safety. we propose to combine screening tests addressing both, exposure and acute lung toxicity. the exposure potential of the spray product is characterized by determining the release fraction of the active compound in the respirable particle size range under conditions relevant for the product application. this is carried out by spraying defined quantities of the product into a control volume and measuring the concentration of health related size fractions. this procedure takes into account spray ageing, especially size reduction of the droplets due to solvent evaporation. the isolated perfused lung is used as a model for testing acute toxicity. ventilated rat lungs are exposed to aged aerosols with proper particle size of approximately 1 µm mmad generated from the liquid spray formulation. lung compliance and lung resistance are continuously monitored during exposure. dose dependent deviations from the normal values (without exposure) are used as read-out parameters. using the combined procedure, different sprays could be ranked according to their realistic exposure risk and, most importantly, sprays with known lung toxicity could be uniquely distinguished from those that have been shown to be safe. in its current stage of development the simple test method is recommended for screening of substances only. induction of oxidative damage in calf thymus dna by the fusarium mycotoxin zearalenone after metabolic activation with liver microsomes fleck s. c., pfeiffer e., metzler m. kit -institute of applied biosciences chair of food chemistry, adenauerring 20a, 76131 karlsruhe, germany zearalenone (zen) is an estrogenic mycotoxin produced by fusarium species. the adverse effects of zen and its reductive metabolite zearalenol (zel) are often compared to those of 17-beta-estradiol (e2) and estrone (e1). these endogenous estrogens are associated with an increased risk for cancer, which may be mediated by two mechanisms, i.e. (i) hormonal activity and (ii) genotoxic effects by p450-catalyzed metabolic activation to catechols (wang et al., chem res toxicol 23, 1365 . like e2 and e1, zen and zel exhibit marked estrogenicity and also undergo aromatic hydroxylation to catechol metabolites (pfeiffer et al., mol nutr food res 53, 1123 . the aim of the present study was to examine the formation of catechol metabolites of zen by liver microsomes of various species and their potential for redox cycling. catechol metabolites are frequently associated with the generation of reactive oxygen species and subsequent oxidative damage of dna, for which 8-oxo-7,8-dihydro-2'deoxyguanosine (8-oxo-dg) is a common biomarker. the propensity of the catechol metabolites of zen and zel to cause the formation of 8-oxo-dg in isolated calf thymus dna was determined using a lc-esi-ms/ms method. to this end, zen was incubated with microsomes from human, rat, mouse, bovine and porcine liver as well as with human cyp1a2, and the incubations were extracted with ethyl acetate. the extract was analyzed with lc-ms and then added to a solution of calf thymus dna in the presence of copper(ii) ions and nadph. the formation of 8-oxo-dg could be demonstrated with each extract. the levels of 8-oxo-dg correlated directly with the extent of catechol formation, which increased from steer to swine to human to mouse to rat microsomes. 15-hydroxylated zen/zel, which is the major catechol, was more reactive than 13-hydroxylated zen/zel to form 8-oxo-dg. in conclusion, our study has shown that the catechol metabolites of zen are highly reactive and give rise to oxidative dna damage in vitro. in addition, recent research from our laboratory revealed that the catechols of zen are less efficiently inactivated by catechol-o-methyl transferase than the catechols of e2 and e1. the genotoxic potential of zen may constitute another biological activity in addition to the well-known estrogenicity. supported by deutsche forschungsgemeinschaft (grant me 574/32-1) and "food and health" of kit. thrombin regulates expression of sphingosine kinase-1 (sphk-1) in human vascular smooth muscle cells -inhibition by dabigatran reduces vascular sphk-1 expression and atherosclerotic burden in vivo flößer a. 1 results: thrombin induced a time-and concentration-dependent (1-100 nmol/l) increase in sphk-1 mrna and protein expression in human saphenous vein smc, n=6-7. this was mimicked by a synthetic par-1 ligand. inhibition of sphk-1 attenuated thrombin-induced smc proliferation but not smc migration (n=5). the regulatory action of thrombin on sphk-1 expression was suppressed by sirna against the mrna stabilisiserhur. in thrombin-stimulated smc, hur binding to sphk-1 mrna and subsequent nucleo-cytosolic shuttling was enhanced. accordingly, thrombin induced sphk-1 mrna stabilisation in smc in the presence of actinomycin d. in apoe-deficient mice, long-term treatment with the direct thrombin inhibitor dabigatran significantly reduced aortic sphk-1 expression by 50% (n=5) and plaque size by 35% compared to control animals (n=10). conclusions: thrombin induces sphk-1 expression and s1p synthesis in vascular smc via the mrna stabilising protein hur. this leads to increased smc proliferation. inhibition of thrombin by dabigatran treatment in vivo attenuates progression of plaques possibly by reducing sphk-1 expression. mycotoxin contamination and cytotoxicity of grain mill products typical grain mill products from north-rhine westphalia, i.e. grains, flour, wholemeal flour and bran (from wheat, rye and spelt) as well as typical by-products (outsourced fractions) from the milling process were analysed for their mycotoxin content by lc-ms/ms. the cytotoxicity of sample extracts with known mycotoxin composition was then assessed in v79 cell cultures by means of the neutral red uptake assay, in parallel with pure reference mycotoxin mixtures. extracts from flour and wholemeal flour samples with low levels of deoxynivalenol and enniatin b (from not detectable to 0.4 µg/g don and 0.5 µg/g ennb) induced no measurable cytotoxicity. on the other hand, although mycotoxin contamination levels were also rather low in bran, these samples induced strong cytotoxicity: extracts of bran derived from rye and spelt were more cytotoxic than those of wheat bran. the cytotoxic effects of the bran samples cannot be related to their mycotoxin content as comparable concentrations of pure mycotoxins and mycotoxin combinations tested in parallel were not cytotoxic. by-products from certain stages of the milling process (sorting and waste fractions) were found to contain mycotoxins at rather high levels: enniatin b was detected in nearly all samples, and also t-2 toxin, ht-2 toxin, ergotamine, ergocornin and deoxynivalenol were present. waste sample extracts with notable mycotoxin levels (up to 6 µg/g don, 7 µg/g ennb, 16 µg/g ergotamine, 50 ng/g ht-2 toxin) exert pronounced cytotoxicity in v79 cells. the cytotoxicity of these samples was somewhat stronger than expected when compared with mixtures of reference mycotoxins tested in parallel. in summary, the tested flour and wholemeal flour extracts contained only low levels of mycotoxins and were not cytotoxic. in contrast, bran samples showed cytotoxicity which cannot be explained solely by their mycotoxin content. this unexpected observation in real samples and combination effects of mycotoxin mixtures require further studies. the ahr is a ligand-activated transcription factor that mediates the toxicity of dioxins and related compounds. upon ligand binding the ahr translocates into the nucleus and dimerizes with arnt to modulate gene expression, e.g. of cyp1a1. recently, we have shown that uvb irradiation of human keratinocytes results in activation of the ahr and associated egfr signaling leading to an enhanced expression of cyp1a1 and proinflammatory cox-2, respectively. the initial step is the uvb induced intracellular formation of the tryptophan photoproduct 6-formylindolo [3,2b] carbazole (ficz), a high affinity ahr ligand. thus, the ficz activated ahr is an important mediator of the dna damage independent part of the uvb response. our current study aims to identify further aspects of ahr mediated uvb responses. therefore, we analysed changes in protein expression, proliferation and apoptosis in ahr+/+ and ahr-/-keratinocytes (nctc 2544) by western blot, flow cytometry and brdu incorporation. uvb exposure of nctc cells led to a dose-dependent increase in apoptosis. compared to ahr+/+ cells, ahr-/-cultures exhibited an increased amount of apoptotic cells. this finding was confirmed in irradiated ahr+/+ cells, pretreated with the ahr antagonist 3'methoxy-4'-nitroflavone. moreover, the proliferation of sham as well as uvb irradiated ahr-/-cells was significantly decreased. in ahr-/-cells we found a reduced expression of checkpoint kinase 1 (chk1), an important cell cycle regulator that arrests the cell in g2/m upon dna damage. interestingly, uvb exposure led to a higher net phosphorylation of chk1 in ahr-/-cells, indicating that this pathway is responsible for the observed ahr-dependent differences in proliferation and apoptosis. further expression analyses of chk1 client proteins emphasize our hypothesis. in conclusion our study identifies the ahr as an anti-apoptotic player in uvb irradiated human nctc cells. therefore, we propose that the ahr is a suitable target to prevent uvb induced skin diseases. synthetic progestins exert divergent effects on thrombosis in a murine model of atherothrombosis background: medroxyprogesterone acetate (mpa), a synthetic progestin often used in postmenopausal hormone replacement therapy, has previously been described to be pro-thrombotic in a murine model of accelerated atherosclerosis. however, nothing is so far known about effects of progestins with receptor profiles different from mpa (i.e. agonism or antagonism of mineralocorticoid-or androgen-receptors), such as drospirenone, levonorgestrel and norethisterone acetate. methods: apo -/mice were bilaterally ovariectomized (ovx) and substituted subcutaneously with mpa, drospirenone, levonorgestrel and norethisterone acetate as well as the respective placebo pellets for 90 days on a western-type diet. subsequently, thrombosis was induced by photochemical injury to the right carotid artery using rose bengal and a green light laser. results: compared to placebo, animals substituted with mpa showed significantly shortened times to occlusion of the right carotid artery (placebo mpa: 50.0 ± 5.8 min. vs. mpa: 33.4 ± 4.2 min., n = 6 -9, p < 0.05). in contrast, drospirenone, levonorgestrel or norethisterone acetate did not alter thrombotic responses. however, at least drospirenone (placebo drospirenone: 53.7 ± 5.4 min. vs. drospirenone: 47.5 ± 4.8 min., n = 7 -8) and levonorgestrel (placebo levonorgestrel: 47.0 ± 3.2 min. vs. levonorgestrel: 41.8 ± 2.1 min., n = 6) showed a trend towards shorter times to stable occlusion. furthermore, analysis of aortic gene expression revealed that in aortas of mpa-treated mice expression of matrix-metalloproteinase 9 (mmp-9) was induced as compared to placebo-treated mice. conclusion: mpa, a progestin with glucocorticoid effects, exerts a pro-thrombotic effect that is either progesterone-or glucocorticoidreceptor-dependent while progestins with receptor profiles different from mpa do not show a significant pro-thrombotic effect. furthermore, the pro-thrombotic effect exerted by mpa may be associated with increased expression of mmp-9, a metalloproteinase being known to destabilize atherosclerotic plaques and make them more prone to rupture. rapid screening for mitochondrial toxicity in vitro using an oxygen-sensitive phosphorescent probe freyberger a. bayer healthcare bph gdd ged toxikologie -p & cp, aprather weg 18, 42096 wuppertal, germany impaired mitochondrial function has been implicated with disease, aging, and druginduced toxicities. analyzing mitochondrial respiration (mr) rates is one of the most informative ways to assess mitochondrial function as it provides information on the the bioenergetic capacity of a tissue, however, previously measurements using polarography (clark electrode) were cumbersome with only low throughput. in this work we explored luxcel's water-soluble phosphorescent oxygen-sensitive probe mitoxpress tm for the assessment of mitochondrial toxicity in freshly isolated male rat liver mitochondria (rlm) in a 96-well plate format using glutamate/malate (20 mm/0.5 mm) and succinate (25 mm) as respiratory substrates. inhibition of mitochondrial complexes i to iv, adenosine triphosphate synthetase and the adenosine diphosphate (adp) / adenosine triphosphate (atp) antiporter by rotenone, thenoyltrifluoracetone (ttfa), antimycin a, potassium cyanide, oligomycin, and atractyloside was readily detected in adp-stimulated rlm. use of the two different substrates in parallel allowed to discriminate complex i inhibition by rotenone from complex ii inhibition by ttfa, whereas downstream of these complexes inhibition by the other model inhibitors occurred independent of the substrate used. decoupling of mr from oxidative phosphorylation by carbonylcyanid-p-trifluormethoxyphenylhydrazone (fccp) was detected best in the absence of adp. compared to polarographic measurement, the use of an oxygen-sensitive probe is superior with regard to assay cycle time and sample throughput and offers new opportunities to characterize and screen for mitochondrial toxicity, but also to support studies on mitochondria-mediated modes of action of new chemical entities. the murine local lymph node assay (llna) and the guinea pig maximization test (gpmt) have been used to study the sensitization potential of a series of unsaturated compounds by kreiling et al. (2008) . we have examined the same substances in the loose-fit coculture-based sensitization assay (lcsa), developed by our working group (schreiner et al., 2007) . eight unsaturated compounds [oleic acid (oa), linoleic acid (la), linolenic acid (lna), undecylenic acid (ua), fumaric acid (fa), maleic acid (ma), squalene (sq), 1-octyn-3-ol (oc)] and succinic acid (sua) were investigated using a coculture of keratinocytes and dendritic cell-related cells (dcrc). sensitization potential was quantified by flow cytometry measuring the increase of cd86 expressed on dcrc (ec50 = half maximal effective concentration). a pronounced induction of cd86 at low concentrations was seen with la, lna and oa (ec50: 3, 5 and 5 µmol/l, respectively). ua exhibited an intermediate response (ec50: 307 µmol/l). with oc and ma, we observed effects at higher concentrations only (ec50: 1340 and 2293 µmol/l). no significant increase of cd86 was observed with fa, sua and sq. because of poor solubility, sq could not be studied adequately. induction of cd86 was generally observed at concentrations which did not cause a major impairment of cell viability. our results show a high degree of concordance with those obtained by the gpmt, except for oa. in comparison with the results of the llna, those compounds which showed a strong effect in the llna (oa, la, lna) also induced an increase of cd86 at low concentrations, whereas those with low stimulation indices in the llna induced no significant increase of cd86 (fa, sua) or only at higher concentrations (ua). we observed a discrepancy between the tests with ma and oc, causing a strong stimulation of the murine lymph nodes, while the expression of cd86 was increased at high concentrations only. we assume that ma and oc might be false-positives in the llna, because they were also negative in the gpmt. background: the first step in elimination of many cationic drugs is their uptake from the blood into hepatocytes and renal proximal tubular cells by the organic cation transporter 1 (oct1) and oct2, respectively. the pivotal role of octs in the excretion of cationic drugs raises the possibility of drug-drug interactions in which one drug reduces octmediated elimination of a second drug. although many psychoactive drugs are cationic at ph 7.4 and some of these have already been recognized as oct inhibitors, a systematic screen of this class of compounds is missing. methods: we screened a drug library of 50 most frequently prescribed psychoactive drugs (inpatient prescriptions in germany, at least 4 million ddd each) for their inhibitory interaction with oct1 and oct2. human embryonic kidney (hek) cells stably overexpressing oct1 or oct2 and the prototypical oct substrate 1-methyl-4phenylpyridinium (mpp+) were used as a test system. cells transfected with the empty vector were used as controls. results: at 20 µm, 59% and 51%, respectively, of the tested compounds significantly decreased oct1-and oct2-mediated uptake of mpp+. the most potent inhibitors (inhibition >75%) of oct1 were chlorprothixen and clomipramine, whereas olanzapine, clomipramine and doxepin were the most potent inhibitors of oct2. in contrast, neither at 20 µm nor at 200 µm carbamazepin, haloperidol, lithium, moclobemide and valproic acid did significantly inhibit mpp+ uptake into hek-oct1 or hek-oct2 cells. there was a significant correlation between the degree of oct1 and oct2 inhibition (p conclusions: our results demonstrate that inhibition of oct function by psychoactive drugs has to be considered as a potential mechanism underlying drug-drug interactions. considering estimated peak sinusoidal concentrations e.g., of chlorprothixen and clomipramine between 30 and 80 µm in humans, inhibitory interactions of these compounds with hepatic oct1 have to be taken in account. our data will help to create a chemoinformatic model to predict potential oct-dependent interactions of psychoactive drugs with the hepatic or renal elimination of coadministered drugs. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. cardiac gene expression is altered during the development of hypertrophy and heart failure compared to the healthy heart. the molecular mechanisms controlling gene expression in cardiac failure are only partially known. dna methylation is one epigenetic mechanism that regulates long-term changes in gene-expression. to elucidate whether dna methylation is altered during the development and progression of chronic heart failure, genome-wide dna methylation profiles were determined in myocardial biopsies from control patients and patients with cardiac hypertrophy or failure. cardiac biopsies were obtained from patients with aortic aneurysm who served as control and did not show clinical signs of chronic heart disease (ef: 58±3 %, n=3) and from patients with aortic stenosis. the latter group was subdivided according to the ejection fraction into hypertrophic (ef: 63±7 %, n=3) and failing patients (ef: 28±1 %, n=3). after bisulfite conversion of extracted dna, the methylation status of genomic dna was quantified using the infinium® humanmethylation450 beadchip (illumina). this microarray allows analysis of more than 485,000 methylation-sites throughout the whole genome at single-base-pair resolution. these experiments identified 1280 cpg sites in hypertrophic samples and 1365 cpg sites in failing samples which were differentially methylated compared to control specimens (delta >15%; p<0.05). 523 cpg sites were significantly altered in both aortic stenosis groups compared with control hearts. from these cpgs, 496 sites were altered concordantly in hypertrophic and failing samples. analysis of regions harbouring distinct cpg densities revealed that most changes occured in shelf regions of cpg islands whereas the methylation status in the cpg islands was more stable. further analysis showed that differences in methylation were most frequent in gene body, enhancer and 3`utr regions. specifically 9 probes spanning a cpg-island at the promotor region of the muscle-specific serine kinase 1 (srpk3) showed diminished cpg-methylation in hypertrophic (-11.5±0.02%) and failing (-11±0.02%) as compared to control biopsies. remarkably, no alterations of dna-methylation were observed in loci of classic marker genes of chronic heart failure like nppa, serca, ctgf, myh6 or myh7. these results indicate that dna methylation is specifically altered in chronic heart disease but does not affect classic marker genes of chronic heart failure. gliomas are the most abundant type of primary brain tumor in the central nervous system in adults. the current standard of glioblastoma multiforme (gbm) therapy is surgery followed by radiotherapy and chemotherapy. however the morbidity and mortality of gbm remain very high and the median survival period is only 15 months even with treatment. therefore it is important to identify novel drugs to reduce gbm cell proliferation. purine-analogues (pa) are well known for their anti-proliferative effects on eukaryotic cells. in this study novel pa were synthesized and the library of substance-derivatives was tested using different gbm cell lines namely ln18, u87-mg and gl261. the effect on proliferation and viability was assessed by using brdu and resazurin assays. using these in vitro methods we were able to identify several compounds with cytotoxic and anti-proliferative effects in vitro showing ic50 values in the deeper µm range. cytotoxicity of selected compounds was further analyzed by assessment of caspase 3 and propidium iodide based cell cycle facs analysis to discriminate between apoptosis and cell cycle arrest. based on these data purine-derivatives might inhibit proliferation and induce apoptosis in glioma cells. as a result we hypothesize that these compounds could be potentially interesting for the drug-development of gbm therapy and therefore a clue for chemical modifications. further studies are required to identify the exact underlying mechanism of action of the tested purine-analogues. the biological role of adenosine receptors in brown adipose tissue gnad t. 1 brown adipose tissue (bat) is responsible for basal and inducible energy expenditure in mammals. bat contains large amounts of mitochondria and is highly vascularized. bat lipolysis and thermogenesis are stimulated by sympathetic neurons. importantly, recent findings indicate that adult humans possess metabolically active bat 1 . here, we analyzed the expression and function of adenosine receptors in bat. adenosine receptors (ador) are members of the superfamily of g protein-coupled receptors. there are four subtypes of adors in humans referred to as adora1, a2a, a2b and a3. they are widely expressed in tissues and mediate a variety of cellular functions, mostly due to their regulation of camp levels within cells. interestingly, it has been shown that adenosine can either inhibit or stimulate lipolysis in white adipocytes through adora1 or a2a, respectively 2 . however, the role of adenosine in the differentiation of brown preadipocytes to adipocytes and in bat function is not clear. to analyze the role of adors in bat, we use preadipocytes isolated from bat of newborn mice and subjected them to a differentiation protocol. 3 abundance of adora1, a2a, a2b and a3 mrna was measured using qpcr. all four receptor subtypes are present in preadipocytes with adora2b being the most abundant. adora1, adora2a and adora3 are significantly transcriptionally upregulated -albeit at varying degree -during differentiation. adora1 is upregulated 4.4 fold (+/-0.3 fold) and 11 fold (+/-0.49 fold) at day 4 and at day 7, respectively, as compared to preadipocytes (n=5). adora2a is 23 fold (+/-1.96 fold) upregulated at day 4 and 57 fold upregulated (+/-2.48 fold) at day 7, respectively (n=4). adora3 was found upregulated 3.6 fold (+/-0.46 fold) at day 7 (n=4). in contrast to this, ador2b was downregulated to 0.87 fold (+/-0.09 fold) at day 4 and to 0.69 fold (+/-0.04) day 7 compared to preadipocytes (n=5). to investigate the functional role of ador in bati cells, we analyzed lipolysis in mature cells after acute treatment with specific agonists and antagonists. we observed that adora2a activation by cgs21680 significantly increased lipolysis by 88% (+/-0.33%) compared to untreated control. moreover, adora1 antagonist psb36 increased lipolysis by 35% (+/-0.05%) (n=4). in conclusion, ador are highly regulated during brown fat cell differentiation. lipolysis of mature brown fat cells is significantly increased by adora2a agonist or adora1 antagonist, respectively. munich heart alliance, münchen, germany activation of the sympathetic nervous system and the subsequent activation of βadrenergic receptors (βars) through catecholamines represents the strongest mechanism to increase cardiac function. however, long-term activation of cardiac βars is clearly detrimental and β-blockers have been introduced as an effective treatment modality in cardiac failure. despite their central role in cardiac physiology and disease, our knowledge about the intracellular mechanism of βar stimulation is confined to a few targets and is likely incomplete. here, we report a functional proteomics approach to directly assess the entire phosphoproteome of βar-stimulated mouse hearts in vivo. to identify proteins that are phosphorylated in response to β-adrenergic stimulation in vivo, we treated mice with isoproterenol or, as a control, with propranolol. after lysis of hearts and tryptic digest, phosphopeptides were enriched by tio 2 or immobilized metal ion affinity chromatography (imac). subsequent analysis of eluated peptides by tandem mass spectrometry (ms/ms) mapped several phosphopeptides to cardiac proteins, among which known mediators of βar signaling such as phospholamban, troponin i and myosin binding protein c. we then employed multiple reaction monitoring (mrm) as a quantitative approach to assess changes of phosphorylation after βar stimulation. using this combination of ms approaches, we identified 39 peptides with pka consensus phosphosites that were more abundantly detected under βar stimulation. among those, we found myozenin-2 (myoz2) and g protein signaling modulator 1 (gpsm1, also termed ags3) as proteins previously not related to βar signaling. we validated the βar-dependence of phosphorylation at these sites in isolated cardiomyocytes by in vivo labelling or phosphoepitope-specific antibodies. current efforts aim at the functional characterization of these novel candidate mediators of βar signaling in the heart. taken together, we report the β-adrenergic phosphoproteome of the mammalian heart in vivo. we have identified several new targets of βar signaling that may represent essential factors in cardiac physiology and disease. background: drug measurement in autopsy material is normally used to investigate the cause of death. in our study it was possible to measure concentrations of drugs that were part of a regular treatment without connection to the cause of death. metamizole is used as an analgetic and spasmolytic agent. the active metabolite maa (4-methyl-aminoantipyrin) is metabolized by the liver and eliminated by the kidney. hepatic and renal dysfunction can therefore influence maa clearance. methods: maa concentrations were measured in different samples of the autopsy material (heart blood, venous blood, urine, liver, kidney and brain) using an hplc-ms/ms method. information about the dosage and time of drug application as well as information about existing renal or hepatic disorders were taken from the corresponding patient records. because of the low number of cases an explorative single-case study was necessary. results: 10 cases with oral intake of metamizole in a customary continuous dosage could be indentified. the maa distribution into body liquids and organs depended on the time between last oral intake and death. in two cases without renal or hepatic diseases maa blood levels were below 10 µg/ml. five cases with combined renal and hepatic disorders showed either increased blood levels of 40-50 µg/ml or prolonged maa elimination half-life of up to 12 hours. in one case with manifest hepatic insufficiency an maa concentration of more than 200 µg/ml was measured in venous blood. two cases with renal insufficiency alone had maa venous blood levels of less than 10 µg/ml. (pet) . pet detects the positron emission of neutron-deficient radioactive nuclides and allows their external localization in vivo. fet, a modified amino acid, is not incorporated in proteins but accumulates in glioblastomas. one pathway responsible for its accumulation is the preferential transport into the tumor cells, probably via amino acid transporters. we investigated in more detail (a) which individual, cloned amino acid transporters accept fet as substrate and (b) which transporter is responsible for the major fet transport into glioblastoma cells. studies with xenopus laevis oocytes, expressing individual human amino acid transporters, revealed that system l, y + l and b 0+ amino acid transporters recognize fet as substrate (lat1 and 2, y + lat2, and b 0+ at, respectively). in contrast, y + lat1 and atb 0,+ did not transport fet. rna expression studies using qrt/pcr revealed that lat1 is the dominant amino acid transporter in all glioblastoma cells investigated (ln229/u373/u87mg/u251/a172/t98g). a strong lat1 expression was also shown on the protein level. to find out whether lat1 is the main transporter responsible for fet accumulation, we first studied transport of the parent amino acid l-tyrosine in ln229 glioblastoma cells. [ 3 h] tyr uptake was completely na + -independent and inhibited by leu, phe and trp, but not by arg, pro or ser. sirna-mediated down-regulation of lat1 in ln229 cells led to a concomitant decrease of lat1 mrna and tyr transport (down to 3% and 20%, respectively). these results indicate that tyr is exclusively transported by lat1 in ln229 cells. we are currently performing transport studies using [ 18 f]fet to investigate whether fet transport is also exclusively mediated by lat1 in glioblastoma cells. a further question is if lat1, a sodium-independent transporter, can be responsible for the accumulation of fet observed in glioblastoma cells. if true, other amino acid derivatives that are lat1 substrates might also proof useful in cancer diagnosis. telmisartan reduces adipose tissue inflammation and biglycan accumulation in diabetogenic ldl-receptor knockout mice grandoch m., nagy n., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany in addition to lowering blood pressure some of the angiotensin ii at1 receptor antagonists (arb) such as telmisartan have additional beneficial effects on the onset of type 2 diabetes mellitus and obesity. this was contributed mainly to peroxisome proliferator activated receptor (ppar)γ modulating activity. hyaluronan (ha), a high molecular weight polysaccharide and the small leucine rich proteoglycans, decorin and biglycan, are known to be involved in atheroprogression. mechanistically these matrix components contribute to inflammatory processes via toll-like receptor-signalling and are supposed to modulate lipid retention. the aim of this study was to elucidate the effects of telmisartan in comparison to valsartan, an arb without pparγ activity, on extracellular matrix remodelling and inflammation in atherosclerosis and the interrelationship with adipose tissue inflammation using the ldlr-/-model of accelerated atherosclerosis. male ldlr-/-mice were fed either a diabetogenic diet alone or in combination with telmisartan (10 mg/kg), valsartan (25 mg/kg) or valsartan (50 mg/kg) from 8 weeks of age for 17 weeks. all treatment groups except of the lower valsartan dose showed significant effects on reducing the aortic plaque score. the content of ha, collagen and decorin in the aortic root were not changed. however, telmisartan reduced the content of biglycan in the aortic root significantly in contrast to valsartan. in addition, a trend towards decreased mac2-positive macrophages in abdominal adipose tissue was detectable after telmisartan treatment as well as a strong reduction in the adipose tissue mrna expression of biglycan. finally, telmisartan reduced the expression of hyaluronan catabolizing enzymes potentially leading to an increase of high molecular weight ha in the adipose tissue, which is thought to be homeostatic and antiinflammatory. in summary, the results of this study underline the pronounced anti-inflammatory capacity of telmisartan on atherosclerosis and adipose tissue inflammation in comparison to valsartan and strongly suggest that biglycan might be an additional target of telmisartan not only concerning matrix composition of atherosclerotic lesions but also concerning the structure of adipose tissue and metabolic effects of the compound. human primary malignant cancer cells derived from peritoneal effusions of a patient with colorectal carcinoma, as assessed by comet assay. the primary cancer cells were more efficient in dsb repair than ht-29 cells, and their doxorubicin ic50 was four times higher. comparative protein expression levels showed that the primary cells had less rad 51 and 52 as well as less topoiiα, while ku70 and 80 levels were similar. another very interesting protein is the mrn (mre11-rad50-nbs1) complex that initializes the phosphorylation of atm and thereby starts the signalling cascade. the newly described mrn-atm pathway inhibitor mirin interrupts mrn activity by inhibiting the exonuclease activity of mre11. the toxicity of mirin in ht-29 cells was measured using a luminescence-based assay detecting the amount of atp, which is correlated with cellular viability. mirin did not show any toxic effects up to a concentration of 100 µm and incubation times of 24 hours, indicating that mirin can be used under these conditions without detrimental effects. we are currently investigating the effect of mirin on the toxicity of topoiiα inhibitors. the inhibition of dna repair may be a valuable strategy to enhance the effect of dnadamaging anticancer drugs. since tumours (even of the same entity) are not only heterogeneous, but also polyclonal, a broad selection of response modifiers of anticancer drugs would be helpful to individually enhance chemotherapeutic effectiveness. evidence has been provided that diet and environmental factors directly influence epigenetic mechanisms associated with cancer development in humans. epigenetics play an important role in the control of gene expression. epigenetic mechanisms comprise modulation in dna methylation, histone modification and non-coding rna. several polyphenols have been reported to possess histondeacetylase (hdac) inhibitory properties [1] . histone deacetylation is generally linked to transcription repression. furthermore, hdac belongs to the group of small ubiquitin-related modifier (sumo) substrate proteins. sumoylation of hdac is associated with a modulation of its biological activity [2] . little is known so far about the mechanism by which hdac sumoylation mediates inhibition of gene transcription. we addressed the question whether sumo e1 and hdac 1 expression and whether potential hdac-sumoylation will be affected by polyphenols such as chlorogenic acid, genistein and (-)epigallocatechin-3-gallate (egcg). chlorogenic acid, genistein and egcg decreased sumo e1 protein level in the human colon carcinoma cell line ht29 after 24h of incubation measured with western blot analysis. egcg exhibited the most pronounced effect at concentrations ≥ 50 µm. hdac 1 expression was also affected by these polyphenols. the direct impact of polyphenols on the hdac sumoylation is detected by co-immunoprecipitation experiments with the respective antibodies against hdac-1 and sumo e1. these experiments are still under investigation. in conclusion, chlorogenic acid, genistein and (-)-epigallocatechin-3-gallate influenced the sumo and hdac expression in vitro. in further studies the direct impact on subtract-sumoylation will be investigated. these studies contribute to a better understanding of potential chemopreventive effects of dietary polyphenols on specific epigenetic alterations may provide chemopreventive strategies for reducing cancer risk. the no/cgmp cascade is thought to be essential for penile erection. within the smooth muscle of corpus cavernosum, nitric oxide activates the no-sensitive guanylyl cyclase (no-gc) which raises the intracellular concentration of cgmp. this second messenger activates the cgmp-dependent protein kinase i (pkgi) and subsequent phosphorylation of target proteins leads to relaxation of cavernosal smooth muscle. knock out of key enzymes of the no/cgmp cascade has led to discrepant results: the deletion of pkgi in the mouse has been shown to lead to erectile dysfunction whereas mice lacking neuronal no synthase are fertile. to investigate the role of the no receptor in fertility we have generated mice lacking no-gc (gcko), a bottleneck enzyme of the no/cgmp cascade. we have shown that lack of no-gc resulted in arterial hypertension concomitant with a totally abolished no responsiveness of vascular and gastrointestinal smooth muscle. in addition, we generated a mouse line in which no-gc was specifically deleted in smooth muscle cells (sm-gcko). using these ko strains we here examined the role of no/cgmp signaling with regards to the smooth muscle relaxation of corpus cavernosum. no failed to affect corpus cavernosum from gcko in organ bath experiments: neither exogenously produced no by no donors nor endogenous no release from neurons induced by electrical field stimulation led to relaxation. similar results were observed in the corpus cavernosum of sm-gcko mice. to our surprise, the gcko animals were fertile and produced offspring albeit at a reduced rate compared to wt animals. our data show that interruption of no/cgmp signaling results in complete absence of no-induced relaxation of penile corpus cavernosum in mice and reduces the ability to produce offspring but does not abolish fertility. novel modes of invasive cell motility regulated by the formin class of actin nucleators khan j., grosse r. philipps-universität marburg, pharmakologisches institut, karl-von-frisch-str. 1, 35034 marburg, germany pathological invasive cell migration essentially reqires actin polymerization. formins are the largest group of rho-gtpase effectors involved in actin nucleation and assembly as well as microtubule dynamics. here we studied the role of formins in cytoskeletal regulation during homotypic cancer cell invasion. we identified the actin-dependent steps and structures involved for this process. using live cell analysis we characterize the distinct actin dynamics controlled by formin-like 2 and rho function. the specific involvement of this signaling module will be discussed. formin-driven nuclear actin assembly controls mal/srf activity baarlink c., wang h. polymerization of actin in the cytoplasm is tightly linked to transcriptional activation of the srf cofactor mal (also known as mrtf-a) through release of actin/mal interactions and subsequent nuclear accumulation of mal. formins directly promote assembly of actin filaments thereby efficiently regulating mal-dependent transcription for cell shape, adhesion and motility. here we show that formins assemble f-actin and promote mal activation inside the mammalian nucleus. the rho-gtpase effector mdia2 rapidly enters the nucleus in a signal-dependent fashion and an active mdia confined to the nucleus potently promotes release of g-actin from mal to specifically activate srf. live cell imaging reveals formin-mediated nuclear actin dynamics. moreover, using actin assembly assays we find that inhibition of endogenous mdia formins controls f-actin turnover in isolated nuclear extracts. thus, formin activity is dynamically compartmentalized to the mammalian nucleus to potently regulate actindependent mrtf function. in women the placenta becomes the main source of maternal estrogens during pregnancy. placental estrogen biosynthesis is located in the syncytiotrophoblast, a syncytium that builds the main part of the placental barrier and limits the transfer of substances between the fetal and maternal compartment. since the human placenta is unable to convert cholesterol into 17-oh-pregnenolone, the placenta tissue highly depends on the supply of c-19 steroids for their conversion into c-18 estrogens. in contrast to lipophilic unconjugated steroids that penetrate the cell membrane passively via diffusion, circulating sulfated steroid hormones are delivered to the placenta via carrier-mediated transport, followed by their reactivation via the catalytic activity of the steroid sulfatase (sts). dheas of maternal and fetal origin contributes about equally to the placental formation of estrone (e 1) and estradiol (e2), while 16αoh-dheas supplied by the fetus contributes to over 90% of placental estriol (e3) synthesis. soat, a member of the slc10 family with highest expression in hormone-responsive tissues such as testis, placenta, and mammary gland has been shown to transport the sulfoconjugated steroid hormones dehydroepiandrosterone sulfate (dheas), estrone sulfate (e1s), and pregnenolone sulfate (pregs) [1] . aim of this project is to investigate the role of soat for placental estrogen synthesis by means of the choriocarcinoma cell line jeg-3 as in vitro model for the human syncytiotrophoblast. therefore, we characterized a jeg-3 cell line that transformed dhea into e2 and 16αoh-dhea into e3. by qrt-pcr we found expression of sts and aromatase, both essential for estrogen synthesis in these cells. upon transient transfection of soat the carrier was located in the cell membrane of transfected jeg-3 cells. currently we investigate the transformation of dheas of these soat-jeg-3 cells by lc-ms-ms. we could demonstrate transport of 16αoh-dheas for stably transfected soat-hek293 cells. in situ hybridization and immunohistochemistry showed coloured syncytiotrophoblasts and vascular endothelial cells in late term placenta. in conclusion, soat-mediated transport of sulfated steroids could play a pivotal role for placental estrogen synthesis from sulfated steroid hormones. developing non-animal test systems for evaluation of toxicity was important in the past and will remain essential in the future. here we present a toxicity test using the chicken yolk sac area vasculosa (cav) of fertilized white leghorn chicken eggs [1, 2] and compare it to hen's egg test on chorioallantoic membrane (het-cam) [3] for polymer toxicity testing. fertilized chicken eggs were incubated and after 72 h explanted shell less into sterile petri dishes. test substances were applied on the cav and the appearance of different effects (vascular lysis, haemorrhage, aggregation of blood components, lethality) was determined by light microscopy after 1 -48 h (fig.1 ). these effects were combined to a cav test score based on the irritation score calculation used for het-cam evaluation. different polymers like poly(ethylene glycol) (peg; neutral), poly(ethylene imine) (pei; cationic) and dextran sulphate (ds; anionic), as well as guideline-conform (recommended het-cam protocol from the interagency coordination committee on the validation of alternative methods) negative (0.9 % nacl) and positive controls (1 % sodium dodecyl sulphate (sds) and 0.1 n naoh) were investigated. additionally ld 50 values for different cationic polymers have been determined. within the selected incubation times (1 -48 h) , effects such as vessel lysis and blood component aggregation could be detected. additionally to het-cam, lethality as well as recovery of the cav could be observed. differences between neutral, positively and negatively charged polymers were obtained. pei showed strong vessel lysis and aggregation of blood components whereas ds and peg showed none of these effects. lethality was found to increase from peg < ds < pei and is concentration and time dependent. the results demonstrate that differences, regarding the toxicity of the used polymers, can be shown with this test. these findings in the cav test can be well correlated with already existing data. in summary, the cav test provides same data (testing control substances) and more information (recovery and lethality) compared to het-cam and could be a suitable model for toxicity testing of polymers. risk characterisation of chemicals consists of three steps (1) hazard identification and characterisation, based on substance-specific toxicological hazard data, (2) estimates of the level of exposure toward the substance and (3) the comparison between the toxicologically safe level and the exposure level. in contrast to the classical risk assessment approach, the threshold of toxicological concern (ttc) approach is developed as a tool to assess the risk of substances without toxicity data. its application requires (1) information on human exposure, for which it is essential that exposure is fully captured and (2) knowledge of the chemical structure to assess whether the chemical is not excluded from the application of the ttc concept. instead of chemical specific no observed (adverse) effect levels (noels/noaels), the ttc approach utilises knowledge on the empirical distribution of several hundreds of noels/noaels, originally 613, based on toxicological testing in animals (munroe et al., 1996) . with the basis on noaels, the ttc concept builds on the fundamental principle of toxicology, that toxicity is a function of dose and that a dose exists, below which no adverse effects of the substance can be detected. it is assumed that exposures below this level will not result in health risks. three separate ttcs were derived (munroe et al., 1996) by classifying the chemicals into three toxicity classes using a decision tree based on a series of 33 questions related to chemical structure, and on natural occurrence in food and in the body (cramer et al., 1978) . the ttc values are derived from empirical distribution of the noels/noaels in the class taking the 95th percentiles and dividing them by the default uncertainty factor of 100. it is assumed that the probability is very low that the unknown noael of a not tested chemical will be lower than the value of the 95th percentile in the distribution of the known noels/noaels. hence, at exposures below the ttc values, the probability of adverse effects on human health is considered to be very low. introduction: kibra, mainly expressed in kidney and brain tissue, is involved in brain development and memory formation as a postsynaptic scaffold protein. in podocytes, kibra is proposed to regulate cell motility as a linker between components of the cytoskeleton and polarity protein complexes (duning et al, jasn 2008) . furthermore, kibra has been identified as key regulator of the hippo pathway, which is involved in organ size control and tumorigenesis. in the current study, we focused on the identification of kibra gene expression regulation and functional promoter characterization. methods: serial promoter deletion constructs were generated by cloning 3627 bp of the 5'flanking region of kibra into the pgl3-vector system. deletion constructs were transiently transfected into human neuroblastoma cells (sh-sy5y) and immortalized human kidney epithelial (ihke) cells. potential transcription factors (tfs) were investigated in cotransfection experiments. transcriptional start sites (tss) were determined by rapid amplification of 5'cdna ends (5'race) . tss utilization between cell lines was assessed by semiquantitative pcr. transcriptional activity (ta) of the kibra promoter p1 was separated by ~1060 bp into two distinct regions, promoter p1a and p1b. deletion constructs harbouring promoter p1b were transcriptionally active only in ihke cells. 5'race revealed two alternative tss in both cell lines upstream of the annotated tss (nm_015238). exclusively in ihke cells, two additional tss were detected in intron 1, resulting in two alternative exons. deletion constructs harbouring the putative regulatory regions (p2 and p3) of both exons were transcriptionally active only in ihke cells. overexpression of full length tcf7l2 (transcription factor 7-like 2 [t-cell specific, hmgbox]) resulted in a ~3-fold increase of promoter p1a and intron promoter p2 ta. kibra gene expression is driven by a complex alternative promoter system comprising the constitutional promoter p1 and three alternative promoters p1b, p2 and p3. the tss utilization is cell type-specific. subsequent usage of an alternative translation start site within exon 3 could result in truncated kibra protein isoforms. tcf7l2 is involved in the differential kibra gene expression regulation. resulting kibra protein isoform and their cellular function will be assessed in further studies. skin absorption in vitro based on the study of human/animal skin ex vivo or reconstructed human epidermis, respectively, is an alternative method which is accepted by the oecd. guideline tg 428 and a corresponding technical guidance document (gd 28) give technical guidance how to perform valid experiments 1, 2 . the requirements include integrity evaluation tests for the skin samples. different tests are proposed to ensure an exclusively use of undamaged skin. to decide which test suites best to our routine test strategy, we investigated the correlation between integrity test results and absorption profiles of various penetrants (logp range: -0.07 -6.2). finite dose experiments using rat and human skin were performed with 14 c-labeled testosterone, caffeine, mcpa (4-chloro-2-methylphenoxyacetic acid) and its 2-ethylhexyl-ester mcpa-2ehe. for each experiment at least three of the five following integrity tests were conducted: transepidermal electrical resistance (teer), transepidermal water loss (tewl), transepidermal tritiated water flux (³h2o), transepidermal absorption of methylene blue (blue) , transepidermal absorption and flux of a ³h-labeled internal standard (istd); ³h-testosterone or ³h-mannitol was used as istd. the applied radioactivity of the ³h-istd was selected to show no analytical interference with the 14 c-penetrants. teer, tewl and ³h2o represent pre-study, istd concurrent and blue post-study tests. calculated maximal permeability constants (kp) and absorbed doses (ad) of the penetrants were compared to the results of the integrity tests. individual linear regression analysis was used to evaluate the correlation the correlations varied over a wide range for all five methods and four penetrants. the best correlations in average were achieved with the istd. no inverse correlations were obtained for the istd, but partly for tewl, teer, ³h2o and blue. in conclusion, the istd represents best the achieved absorption profiles of the test compounds and is based on that the most suitable integrity test for our dermal absorption studies. we will further confirm its effectiveness and generate a sufficient historical database in order to include the istd in our routine test protocol. investigation of mirna expression and dna methylation in focal and non-focal brain tissue of therapy-resistant epilepsy patients haenisch s. 1 background: resistance to anticonvulsants affects one third of all epilepsy patients. limited bioavailability of the drug at the target site caused by increased expression of efflux transporters on the blood brain barrier or alterations of target genes are potential mechanisms for therapy resistance. however, these mechanisms alone cannot completely explain the observed resistance and it is likely that multifactorial alterations lead to pharmacoresistance. there is increasing evidence that expression of micrornas probably caused by dna modifications is deregulated in many neuronal diseases. we hypothesize that mirna regulation of target genes is involved in drug resistance in epilepsy. methods: hippocampal focal and cortical non-focal brain tissue samples from 13 patients diagnosed with mts (mesial temporal sclerosis) who underwent neurosurgery have been screened for mirna expression using taqman low density arrays. in silico approaches for both a hypothesis-based (efflux-transporter and target gene) as well as a hypothesis-free approach were used to identify potential phenotype-relevant target genes. using the program r (bioconductor) a mann-whitney-u test was performed to compare mirna expression between brain regions. pyrosequencing was performed to investigate methylation status 5'-upstream of dna regions encoding for selected candidate mirnas. results: out of 754 mirnas, 150 were detected in both tissue types. the expression of one mirna was 7.2 fold higher (q=0.01) and another was 3.8 fold lower (q=0.01) in the hippocampus relative to the cortex. evidence could be found that down-regulation of the latter is possibly caused by hypermethylation of 5'-flanking region of its encoding dna locus. bioinformatic analysis has identified eight genes important for neuronal regulation and signal transmission (e.g. sox11, mecp2, bsn), as well as one abc effluxtransporter, as potential targets for these differentially regulated mirnas. conclusion: differential regulation of two mirnas could contribute to an altered function of several genes resulting in an imbalance between neuronal excitation and inhibition that is independent from mechanisms presently targeted by anticonvulsants. this work was supported by a fellowship from dfg and nih grant gm61390. recently, it has been reported that human b cells express and secrete the cytotoxic protease granzyme b (grb) after the combined stimulation of the il-21-and the b cell receptors. grb produced by b cells is enzymatically active and b cells deliver grb to sensitive cancer cell lines, thereby inducing apoptosis. to date, there is little experimental evidence on the mechanisms involved in grb expression, or its function in b cell biology. as experimental transgenic murine systems should enable us insights into these issues, we assayed for grb in c57bl/6 b cells using an extensive array of physiologically relevant stimuli, but were unable to detect either grb expression or its proteolytic activity, even when antigen specific transgenic b cell receptors were cross-linked. similar results were also obtained with b cells from dba/2, cba or balb/c mice. in vivo, infection with either influenza virus or murine γ-herpesvirus induced the expected expression of grb in cytotoxic t lymphocytes, but not in b cell populations. we also investigated a possible role of grb on the humoral immune response to np-klh, but grb-deficient mice produced normal amounts of antibody with typical affinity maturation and heightened secondary response, demonstrating conclusively the redundancy of grb for antibody responses. our results highlight the complex evolutionary differences that have shaped the immune systems of mice and humans and demonstrate the need to develop novel in vivo systems to study human humoral immune responses. investigations of the cholinergic neurotransmitter system in dyt1 mice hamann m. 1 early-onset torsion dystonia is an autosomal dominant inherited movement disorder associated with the dyt1 gene defect with deletion of a glutamic acid residue in the protein torsina. despite the gene defect, the pathophysiology is poorly understood. animal models can help to understand the underlying mechanisms and thereby to develop new therapeutic strategies. sharma et al. (2005, j. neurosci. 25 [22] , 5351-5355) initially described a transgenic mouse model (dyt1 mice) with overexpression of mutant torsina. previous studies in these mice pointed to alterations in the cholinergic system. to investigate the functional relevance of these in-vitro findings, we carried out pharmacological in-vivo experiments and determined the density of striatal cholinergic interneurons as well as the expression of choline acetyltransferase in different brain regions. the acute intraperitoneal administration of the cholinomimetic drug pilocarpine (75, 100 and 125 mg/kg) as well as a long-term treatment over 21 days (100 mg/kg/d) did not induce pronounced effects in dyt1 mice compared to wildtype controls. the higher incidence of epileptic seizures in dyt1 mice compared to controls after repeated local striatal applications of pilocarpine (25 and 50 µg/0.5 µl/hemisphere) let presume an altered synaptic plasticity in dyt1 mice. the immunohistochemical investigations revealed a moderately reduced density of striatal cholinergic interneurons in the dorsomedial subregion of dyt1 mice compared to wildtype controls, while significant differences in other striatal subregions were not detected. western blot analysis did not show clear differences in the expression of choline acetyltransferase between dyt1 and wildtype control mice. these results indicate that the cholinergic system seems not to play a key role in this line of dyt1 mice. ongoing receptor autoradiographic analysis of binding to different muscarinic receptors subtypes have to further clarify the existence of possible alterations within the cholinergic system of these dyt1 mice. inhibitors direct against cell cycle-regulatory kinases are being tested in clinical trials as anti-proliferative agents. thus, the atp-competitive kinase inhibitor pd332991 which inhibits cdk4 and cdk6 is currently tested in patients with solid tumors such as glioma. we found that pd332991 suppressed il-1-induced expression of il-8 suggesting that cdk4 or cdk6 may have unknown anti-inflammatory properties. to study the effects of cdk6 on the il-1-signaling network, we established a bidirectional doxycyline-inducible system to express a constitutively active mutant of cdk6, cdk6 s178p, in asynchronized hela cells. cdk6-expressing cells were identified by gfp which was expressed from the same promoter, isolated by laser-microdissection and analysed for mrna expression using a down-scaled rt-qpcr assay. compared to the uninduced state, cdk6 s178p enhanced il-1-induced il-8 and il-6 mrna expression. moreover, shrna-mediated suppression of endogenous cdk6 confirmed a role of this kinase in regulation of maximal il-1-induced gene expression of il-8. these data also revealed that the contribution of cdk6 to inflammatory gene expression is highest in g1, when activity of endogenous cdk6 is activated by d-type cyclins. these findings were corroborated in hela cells expressing fluorescent ubiquitin-dependent cell cycle indicator (fucci) proteins. hela-fucci cells from g1, g1/s, g2 or mitotic states were isolated by laser-microdissection and analyzed by rt-qpcr for tnf-inducible gene expression. stable knockdown of cdk6 in hela fucci or inhibition by pd332991 suppressed inducible il-8 expression. microarray experiments identified many additional genes that required active cdk6 for maximal il-1-or tnf-inducible gene expression. we also found that cdk6 co-immunoprecipitated with p65 nf-κb, colocalized with p65 in the nucleus and was recruited together with the p65 subunit to the proximal il-8 promoter as assessed by chip and re-chip experiments. collectively, these results suggest an unexpected control of inflammatory gene expression through a classical cell cycle regulatory pathway. these results also imply that pharmacological targeting of cdks may have effects and side-effects on the immune system in addition to inhibition of cell cycle progression. trp channels form a heterogeneous family of calcium-permeable channels, which play major roles in physiological functions ranging from sensory reception to cellular signal transduction. members of the trpc subfamily (classic transient receptor potential channels) are downstream targets of hormone receptors. of particular interest is the biological role of trpc6 channels. they are directly activated by diacylglycerol due to phospholipase c-driven signalling pathways which are involved in smooth muscle contractility, neuronal plasticity, keratinocyte differentiation and renal function. their impact in renal function became evident from analyzing patients suffering from familial forms of focal segmental glomerolusclerosis (fsgs) which could be linked to trpc6 mutations. since the first descriptions at least 17 different pathogenic mutations have been identified in humans to cause fsgs. in order to study the underlying pathophysiological mechanisms of trpc6 mutations, we have analysed all mutated trpc6 channels known to date heterologously expressed cells. one set of mutations showed a gain-of-function phenotype which has been previously suggested to cause an increased intracellular calcium load and subsequent cell death. hence, gain of function mutations fit to the current paradigm of fsgs pathophysiology. however, another set of mutations found in the patients showed a loss-of-function phenotype. our results enable a change in the current paradigm for the role of trpc6 in renal pathophysiology and may provide a basis for our understanding of the pathophysiology of loss-of-function mutations in familial focal segmental glomerolusclerosis. karlsruher institut für technologie (kit) institut für angewandte biowissenschaften, abteilung lebensmittelchemie und toxikologie, adenauerring 20a, 76131 karlsruhe, germany risk assessment for genotoxic carcinogens is an important challenge in toxicology. even though manifold attempts have been made to substitute carcinogens and to reduce exposures, their complete elimination appears to be not possible. thus, low concentrations of known or suspected genotoxic carcinogens are present at workplaces, in the environment and in food. in order to deal with this situation and to set priorities for risk management, different concepts have been established such as the alara principle (as low as reasonably achievable) and the margin of exposure (moe), based on the ratio between concentrations being carcinogenic in experimental animals and the actual exposure of humans for example via foodstuff. while usually linear doseresponse-relationships have been used as default assumption, analytical methods are now available to assess the induction and repair of dna lesions on low exposure conditions, including environmental background exposure, and to relate the extent of exposure-induced dna lesions to endogenous dna damage. this may be an important prerequisite to establish health-based limit values for selected genotoxic carcinogens. within this workshop, different examples will be discussed and research need will be identified. dendritic cells from h4r-deficient mice lose their ability to properly stimulate t lymphocytes hartwig c., seifert r., neumann d. mhh pharmakologie, carl-neuberg str. 1, 30625 hannover, germany the incidence of allergic airway diseases is increasing throughout the world, especially in western countries. although histamine (ha) is found at high concentrations in asthmatic lungs, a role for ha in bronchial asthma is still a neglected topic in clinical research. in particular, the capacity of ha to modulate the underlying immune reaction is far from being understood. the histamine h4-receptor (h4r) is involved in acute inflammation and th2 cytokine production. consequently, we intended to analyze the role of h4r in a murine th2 lymphocyte transfer-based model of asthma. specifically the ability of h4r expressed on dendritic cells (dcs) to modulate t cell function was analyzed. ova-specific cd4 + t cells were polarized in vitro under th2-favoring conditions with ova peptide-pulsed dcs, obtained either from wild-type or h4r -/mice. analysis of the polarized t cells after in vitro restimulation revealed a marked decrease of il-4 production in t cells polarized in the presence of h4r -/-dcs compared to those polarized in the presence of wild-type dcs. thus, on dcs, the h4r is essential for proper stimulation of spleen t cells and for directing their polarization towards a th2 phenotype. the transfer of in vitro polarized t cells into recipient mice and subsequent provocation elicited an asthma-like disease. the h4r on dcs not only affects in vitro polarization of t cells, but also the in vivo function of the obtained polarized t cells. a parameter indicating allergic inflammation is the enhanced influx of inflammatory immune cells into the lung tissue, mainly driven by eosinophils, which are virtually absent in non-asthmatics. when analyzing the number of eosinophils, a dramatic difference due to the polarizing conditions of t cells occurs. in bal fluids of mice that received t cells polarized in the presence of wild-type dcs, about 40% eosinophils were detected. in contrast, the transfer of t cells polarized in the presence of h4r -/-dcs yielded only about 10-20% eosinophils in bal fluids. in summery, the h4r on dcs plays an important role for t cell polarization and consequently affects the allergic reaction during sensitization. since the lack of the h4r on dcs reduced their ability to stimulate proper th2 polarization of cd4 + t cells, we conclude that ha via the h4r significantly affects the manifestation of asthmatic inflammation. antioxidant polyphenols and their effects on nrf2 (skn-1) signalling in a cell culture system and the model organism c. elegans havermann s., wätjen w. heinrich-heine-universität düsseldorf institut für toxikologie, p.o. box 101007, 40225 düsseldorf, germany oxidative stress has been connected with a variety of diseases, (e.g. alzheimer`s and parkinson´s disease), cancer and ageing over the last years. certain polyphenols were shown to have an antioxidant capacity as well as being able to activate the protective nrf2 signalling pathway. compared to direct radical scavengers modulators have the advantage of building up a permanent defense against oxidative insults whereas scavengers do not protect any more after consumption or may even cause stress due to redox cycling. we have employed cell culture based assays (dcf, western blot, gfp reporters) to analyse the effects of polyphenols. further we tested the coumpounds in vivo in the nematode c. elegans where skn-1 is the nrf2 homologue. baicalein and caffeic acid phenethylester (cape) protected cells and the nematode from ros accumulation after application of stress (shown by dcf assay). activation of nrf2 signalling is correlated with translocation of the transcription factor into the nucleus. in both systems nrf2::gfp accumulation in the nuclei could be observed after incubation with baicalein (fluorescence microscopy). but while cape is a potent activator of nrf2 in cells, it has no effect on skn-1 localisation. further the effect on the nrf2 protein amount was investigated by western blot analysis. the expression of target genes can be investigated by differing means: while pcr methods and western blotting are standard for in vitro studies, the vast number of available gfp reporter strains offers opportunities for research using c. elegans. we have performed congruent assays in a cell culture system and the model organism c. elegans to compare antioxidative capacity and effects of polyphenols on nrf2 signalling. therefore, depending on the substance tested, c. elegans is a suitable model system to investigate effects of natural compounds in an organism. being associated with adverse health effects, the human exposure to dehp is subject to concern. quantifying the population's exposure and determining the contributions of different exposure routes is a key task of environmental health risk assessment. the study presented comprises a review of the available data on dehp levels in foods, consumer products, and house dust. extensive survey data, e.g. from the current national nutrition survey ii and the eu rapex system were processed for modeling the exposure by the oral, inhalative and dermal path of the population in germany. the study also included analytical analyses of dehp levels in selected foods and consumer goods (incl. migration rates for mouthing). probabilistic techniques allowed elucidating the exposure's variation and the relevance of different routes. mean exposure estimates for german children and adults to dehp are 36 and 26 µg/(kg d), resp. for children, food accounts for 36% of the total exposure, followed by mouthing (30%) and house dust (19%). the adult exposure is almost entirely (82%) due to food. as dietary exposure is a result from concentration and consumption, foods exhibiting high contamination e.g. butter (8%) and dressings (mayonnaise) (14 %) as well as highly consumed foods e.g. bread and bakery (16%) and vegetables (6,8 %) contributed significantly. the mean estimate of children's dehp exposure via mouthing revealed 0,9 µg/(kg d). high exposures were estimated (95th percentile) up to 10,8 µg/(kg d). on average people in germany are exposed to dehp below the current tdi of 50 µg/(kg d). however, individual exposures exceeding the tdi still cannot be excluded. current data on dehp and other plasticizers in foods are scarce, which warrants broader monitoring. our findings highly facilitate further exposure modeling focusing on dehp substitutes and risks of combined exposure. this study was funded by the federal ministry for the environment, nature conservation and nuclear safety in the frame of the environmental research plan (umweltforschungsplan, förderkennzeichen (ufoplan) 3707 61 201). on the basis of the available measurements of dehp, the exposure assessment has been focused on 37 food categories characterising a selection of the most important food groups covering all major food classes of the german population. based on an extensive literature survey, the analysis considered the available data of dehp measurements in food, as well as the official german food control data taken from the national food consumption survey. the high amount of considered data allowed the consideration of several exposure assessment tiers (deterministic and probabilistic by using monte carlo simulation). a quantitative evaluation of the uncertainties of the estimate of the 37 food categories groups has been performed by means of a sensitivity analysis by using the methodology proposed within the 2008 who ipcs guidance document of characterising and communication uncertainty in exposure analysis. qualitative uncertainty analysis (tier 1) was applied to determine the most important sources of uncertainty, i.e. concentration of dehp in all food categories. the probabilistic monte carlo simulation (tier 2) was then used to rank the cumulative probability distributions of the exposure assessments of 37 food categories on the basis of the food categories that appear to dominate. sensitivity analyses were applied to prove the impact correlation of food groups for uncertainties. by a scenario based concept, the aggregation of the 37 food groups to 10 groups has been evaluated, as well as the sensitivities by characterising particular scenarios. for this purpose, particular "meals" have been described as fixed combined scenarios and. the aggregation leads to a considerable higher exposure estimate which can be explained by the combination of high contaminated foods with others of high consumption. the evaluation confirms the considerable role of possibly high contaminated foods e.g. fats, or mayonnaise. the evaluation shows that quantitative probabilistic sensitivity analysis is a suitable and pragmatic tool for uncertainty analysis in exposure assessment. the transcription factor camp response element (cre)-binding protein (creb) plays a critical role in regulating gene expression in response to activation of the campdependent signaling pathway, which is implicated in the pathophysiology of heart failure. we observed creb knock-out cardiomyocytes to be larger than wildtype cardiomyocytes (cell area in µm 2 ; mean±sem; creb-ko 4871±258 vs. wt 3396±171; n=68/69 cells, n=4 mice, p<0.01 vs. ctr.). the nuclear factor of activated t-cells c3, nfatc3, is another transcription factor involved in the development of heart failure and also a known positive regulator of hypertrophy. hence, we investigated whether inhibition of the cre-dependent transcriptional activation has an impact on the nfatc3 signaling pathway. we first studied the effects of an overexpression of a dominant negative creb mutant (dncreb) or of nfatc3 on the activity of a nfat-dependent model promoter in a permanent cell line. overexpression of dncreb evoked an 8.0±1.5 fold increase of the nfat model promoter activity (n=36; n=6 transfections), but had no impact on kv4.2 promoter activity which is known to be regulated by nfatc3 (1.2±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). nfatc3 overexpression led to a 4.5±0.7 fold increase of the nfat-dependent model promoter activity (n=12; n=2) and to an inhibition of kv4.2 promoter activity (0.8±0.1 fold; n=18; n=3; p<0.05 vs. ctr.). we conclude that creb is a negative regulator of nfat-mediated gene transcription and that activation of nfatc3 might contribute to the observed hypertrophy of creb-ko cardiomyocytes. neuroleptika der perazin-klasse sind potente modulatoren des p2x7-rezeptors -perazine-type neuroleptic drugs are potent modulators of p2x7 receptors hempel c., nörenberg w., urban n., sobottka h., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany p2x7 receptors belong to a family of atp-gated, non-selective cation channels, which play an important role in immune cell activation, inflammatory hyperalgesia and neuropathic pain. they differ from other p2x family members by the low atp affinity, and by the ability to form or recruit dilated pores in the sustained presence of atp. owing to its involvement in many diseased states, p2x7 is a promising target for pharmacological intervention. accordingly, p2x7 blockers are currently tested in phase ii clinical trials. in an attempt to identify p2x7-modulating properties of approved drugs or natural compounds, we performed a medium-throughput screen, using an appropriate compound library (spectrum collection) and a stably transfected hek293hp2x7 cell line. with ic50 values of 1-2 µm, the tricyclic antipsychotics prochlorperazine and trifluoperazine showed a high potency and efficacy to block the atp (1 mm)-triggered increases in the intracellular ca 2+ concentration ([ca 2+ ]i) that was mediated by human p2x7 (hp2x7). the closely related phenothiazine-class neuroleptic drugs, such as chlorpromazine or triflupromazine did not have an appreciable effect on hp2x7mediated ca 2+ influx. whole-cell inward currents, measured at -60 mv, were blocked by more than 60% by 3-10 µm prochlorperazine. the inhibitory effects of perazines developed within about 200 ms, hinting to a direct mode of action by binding to the p2x7 protein. prochlorperazine added intracellularly via the patch pipette did not substitute for the extracellularly applied drug, indicating that its binding site is accessible from the extracellular side. in addition, both compounds blocked yo-pro-1 uptake when preincubated before p2x7 stimulation with 1 mm atp or when applied subsequent to the agonist. interestingly, when added to a hek293 cell line expressing the rat p2x7, perazines potentiated the atp-induced increase in [ca 2+ ]i. measurements in human monocyte-derived macrophages confirmed the ability of prochlorperazine and trifluoperazine to inhibit atp-evoked increases in [ca 2+ ]i, changes in yo-pro-1 permeability and whole cell currents. taken together, we conclude that perazine-type neuroleptics impede on p2x7 activity in a species-specific manner, presumably by binding to an extracellularly accessible binding site of recombinant or natively expressed p2x7. similarly, pre-treatment with lov also lowered dox-induced stabilisation of p53 and phosphorylation of chek1 and sapk/jnk. while lov had no influence on ir-induced initial dna damage formation in huvec and rat cardiomyoblasts (h9c2), it decreased dox-and eto-induced phosphorylation of histone h2ax, which is a surrogate marker of dna-double strand breaks. this indicates that lov specifically protects against the genotoxicity of topoisomerase type ii poisons. in an acute and subacute balb/c mouse model lov protected from ir-induced toxicity. this effect rested on inhibition of pro-inflammatory and pro-fibrotic processes as measured via quantification of mrna levels of il6, ctgf and tnfα. the same was true for dox-induced toxicity, i.e. heart and liver damage. similar to the in vitro experiments, dox-induced hepatic dna-damage was attenuated by lov treatment. overall, liver and heart toxicity were reduced by lov as mirrored by the serum levels of gldh/gpt and ctn-i, respectively. both in liver and in heart we observed collagen rich perivascular areas following dox treatment. under situation of lov-co-treatment these areas occurred more rarely and were less pronounced, pointing to a lowered level of fibrosis. pcr-array-based mrna analyses showed inhibitory effects of lov on dox-triggered expression of genes involved in oxidative stress response, drug transport, dna repair, cell cycle progression and cell death. for instance, up-regulation of p21, wee1, cjun/fos and hmox-1 following dox administration was attenuated by lov. altogether, we suggest that including lov in current cancer therapeutic regimen might widen the therapeutic window of anticancer therapeutics by lowering normal tissue damage. the p values. in addition, array data underwent cluster analysis for identification of substantial differences of gene regulation among the three different types of biopsies. results: of particular interest in our study was the expression of genes coding for metabolism and transport proteins. therefore 42 genes from the 156 significant differentially regulated genes, were selected for the qrt-pcr analysis. genes coding for abcb1 and abcg8 transport proteins showed higher expression in the jejunal tissue one year after surgery compared to the duodenal tissue (fold change 1.80 and 1.73). moreover, cyp7a1 mrna involved in metabolic processes is higher expressed in postoperative jejunum than in the jejunum tissue taken during the surgery (fold change 1.9). in conclusion roux-en-y gastric bypass operation leeds a change of mucosal gene expression profile in the jejunum during one year. there was also a significant differential gene expression between the original duodenum and jejunum one year after surgery. these results give strong evidence that jejunum not exposed to pancreatic but only to gastric fluids may change its gene regulation. background. numerous genome-wide association studies (gwas) identified polymorphisms located in transporter genes such as slc2a9, abcg2, npt1, and urat1 as predicitive for the serum levels of urate 1 . these genes encode membrane proteins expressed in the apical membrane of human kidney proximal tubule cells and are assumed to facilitate tubular exchange of urate 2, 3, 4, 5 . importantly several single nucleotide polymorphisms (snp) located in vicinity of slc2a9 have been identified as highly associated with serum urate levels. little is known about the transcriptional regulation of slc2a9. therefore, the aim of our study was to investigate which sequences in the slc2a9 gene harbour ciselements and regulate its gene expression. we also asked whether intronic snps influence gene expression at the transcriptional level. methods and results. performing dual luciferase reporter gene assays we found gene regulating modules in the slc2a9 gene. dna from human kidney samples was then genotyped for rs6449237 being part of this region. next total slc2a9 mrna-expression levels of the samples were determined using real-time quantitative rt-pcr assay. male samples with two minor alleles of snp rs6449237 showed lower slc2a9 mrna levels than samples with the wild type alleles. the effect was not seen in females. reporter gene constructs with either minor or major allele of rs6449237 were then used in luciferase assays, however showing no significant difference in activity. furthermore mrna-expression levels of other urate transporter genes were determined in kidney samples. after linear regression a positive correlation of mrna-expression of slc2a9, urat1, npt1, and oat10, respectively was observed. conclusion. our data suggest that the slc2a9 snps rs6449237 and rs74794351 might influence slc2a9 mrna-level without controlling the transcriptional activity. it needs to be elucidated whether those snps alter mrna stability. however, the mrna coexpression of slc2a9 and other urate transporter might be attributed to a common gene regulating pathway of an "transportosome" controlling urate homeostasis. sulfotransferases mediate the bioactivation of methyleugenol to a reactive sulfate ester binding to dna in vitro and in vivo herrmann k. 1 methyleugenol (me) is a secondary metabolite occurring in many herbs and spices. although me is hepatocarcinogenic in rodents, standard genotoxicity tests were negative. this may be due to the lack of critical activating enzymes responsible for the terminal bioactivation of me to a genotoxicant. me is initially hydroxylated by cytochrome p450 enzymes yielding 1´-hydroxymethyleugenol (1´-ohme). this alcohol can be further activated by sulfotransferases (sults) to an electrophilic sulfate ester that can be easily attacked by dna. the dna adducts formed could lead to mutation and further carcinogenicity observed in animals. the aim of the present study was to clarify whether individual human (h) and murine sult forms are involved in the activation of me to a genotoxicant. in order to identify critical sults, mutagenicity tests including bacteria expressing different sult forms were conducted. (±)-1´-ohme (separated into its enantiomers) served as test compound. we could show that hsult1a1, standing out due to its high expression level in many tissues, can efficiently activate both enantiomers even at low concentrations. furthermore, dna adduct formation in hsult1a1-proficient and sult-deficient bacteria was examined after incubation with 7 µm of (+)-or (-)-1´-ohme. for selective detection and quantification of me-derived 2´-deoxyadenosine (da) and 2´-deoxyguanosine (dg) adducts we developed a sensitive tandem mass spectrometry method including stable isotope dilution analysis. adduct formation was only observed in bacteria expressing hsult1a1. the concentration dependence of adduct formation in hsult1a1-proficient bacteria was examined for (+)-1´-ohme. both adducts turned out to be concentrationdependent. to check the extent and organ specificity of adduct formation in vivo we administered 54 mg/kg bw (±)-1´-ohme (i.p.) to mice carrying the hsult1a1/1a2 gene cluster. mice getting only the vehicle served as controls. animals were sacrificed and dna from eight organs was extracted. by means of tandem mass spectrometry adducts were measured and quantified. da and dg adduct formation was observed in all tissues studied, but not in untreated animals. furthermore, adduct levels were higher than in experiments using wild-type mice. altogether, we herein could show that sulfo conjugation leads to bioactivation of me to a dna-binding intermediate in vitro and in vivo. this work was financially supported by bundesinstitut für risikobewertung. objective: the soluble adenylyl cyclase (sac) activates the na + /k + -atpase in renal epithelial collecting duct cells. nuclear sac constitutes a functional complex with camp response element binding protein (creb), suggesting a more general role of sac in overall gene regulation. we determined the chromatin binding capacities of sac at cre sequences and its influence on genes, which play a role in aldosterone signalling. furthermore, we functionally characterised expression relevant promoter portions of sac and the influence of aldosterone and camp mediated signalling pathways on sac gene regulation. design and methods: in vascular endothelial cells (ea.hy926) and in human kidney cell lines (hek293t; ihke), we performed chromation immunoprecipitation (chip) assay with antibodies against sac and creb. we conducted transfection with a cre luciferase reporter vector and sac promoter constructs, following treatment with sac inhibitors and aldosterone. total rna of ea.hy926 cells, which were treated with sac inhibitors and aldosterone, was isolated and subsequently analysed by real-time pcr for expression of genes involved in aldosterone signalling. in vivo binding of sac at cre motifs was shown using cre consensus sequences in chip experiments. specific pharmacological inhibition of sac led to a significant decrease of transcriptional activity of the cre control vector in endothelial and kidney cell lines. furthermore, we were able to show the different effects of sac on the expression of downstream targets of aldosterone signalling, e.g. mineralocorticoid receptor and na + /k + -atpase alpha1 and beta1 and sac itself. regulation of sac itself is mediated by two different promoter portions, which are influenced by aldosterone and inhibition of sac and differentially accessed in kidney and endothelial cells. sac has transcriptional trans-acting properties as it interacts with cre sites and potentially influences the expression of genes, which play a role in aldosterone signalling. transcription of sac is regulated via aldosterone and camp. the location of promoter ta is cell type-and stimulation-specific. the role of the sodium-calcium exchanger (ncx1) in cardiac pacemaking herrmann s., stieber j., ludwig a. friedrich-alexander-universität erlangen-nürnberg institut für experimentelle und klinische pharmakologie und toxikologie, fahrstrasse 17, 91054 erlangen, germany the mammalian heart is driven by the sinoatrial node, the primary cardiac pacemaker. the unique feature of sinoatrial node (sn) cells is the ability to generate a spontaneous diastolic depolarization that periodically initiates action potentials which set the heart rhythm. the molecular origin of this cardiac pacemaker activity is still a matter of debate. recent findings point to a coordinated interplay between intracellular ca 2+ -cycling processes and plasma membrane-localized ion channels which determines the origin, periodicity and rate modulation of pacemaker potentials. in this study, we investigated the contribution of the cardiac sodium-calcium exchanger (ncx1) to pacemaking. ncx1 is a key sarcolemmal protein for the maintenance of calcium homeostasis in the heart. it was speculated that the membrane depolarizing current incx, whose activity is dependent on intracellular ca 2+ -fluctuations, represents a main determinant of the spontaneous diastolic depolarization. we used an inducible and sinoatrial node-specific cre-transgene to delete ncx1 in the murine pacemaker system. the successful creation of a cardiac pacemaking and conduction system specific ncx1 knockout (cpncx1ko) was demonstrated by transcript quantification as well as immunofluorescence experiments. telemetric ecg recordings of cpncx1ko displayed a distinct cardiac phenotype. mutant animals were deeply bradycardic and lost their capability of maintaining a stable heart beat as demonstrated by various ecg abnormalities like sn arrhythmia, sn pauses, av block and ventricular tachycardia. analysis of the spontaneous activity of isolated sn preparations showed a slower and arrhythmic contraction rate of the mutant tissues strips confirming that the bradycardia and arrhythmia induced by deletion of ncx1 results from a slower and arrhythmic intrinsic pacemaker activity. a battery of experiments using different heart rate lowering as well as increasing drugs revealed an altered heart rate modulation in cpncx1ko animals as compared to controls. in conclusion, these initial results establish ncx1 as a major contributor to cardiac pacemaking. a wide variety of contaminants are ingested through food, among them the procarcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (bp) which is resorbed and partially metabolized in the enterocytes of the small intestine. previous in vitro studies revealed that bp phenols are excreted as phase ii metabolites including bp glucuronides and bp sulfates. this export is mediated by the breast cancer resistance protein (bcrp/abcg2). the ultimate carcinogenic phase i bp metabolite anti-bp-7,8dihydrodiol-9,10-epoxide (bpde) can be detoxified by glutathione conjugate formation catalyzed by various glutathione s-transferases. in the present study, differentiated human intestinal caco-2 cells were used as a model for the human small intestine to investigate the detoxification of bpde and the subsequent transport of the stereoisomeric glutathione conjugates in the presence of an inhibitor (acivicin) of the glutathione-cleaving enzyme gamma-glutamyl transpeptidase (ggt) at the surface of the cells. the results indicate that the glutathione conjugates of bpde are formed and excreted mainly to the apical and to a minor extent to the basolateral side of the polarized caco-2 monolayer. to stimulate the transport rate several inducers known to enhance gene expression of xenobiotic-metabolizing enzymes as well as transport proteins were used (quercetin, oltipraz, butyrate). however, solely oltipraz substantially increased the efflux of bpde glutathione conjugates after inhibition of ggt. inhibition studies revealed that the multidrug resistance-associated proteins (mrps/abccs) are involved in the transport of the bpde glutathione conjugates. stable abcc1, abcc2 and abcc3 knockdown cell lines were generated allowing to demonstrate that abcc1 mediates the basolateral, abcc2 the apical excretion of the bpde glutathione conjugates. in conclusion, the ultimate carcinogen bpde is detoxified via glutathione conjugation and subsequently excreted by caco-2 cells in both apical and basolateral directions. .this finding is equivalent to a transport into the feces as well as blood system in the in vivo situation. signaling via irag regulates store operated calcium entry (soce) in aortic vsmc hieke b., hüttner j., schlossmann j. university of regensburg department of pharmacology and toxicology, universitätsstr. 31, 93053 regensburg, germany the mechanisms involved in the activation of store operated calcium entry (soce) through depletion of intracellular stores and their regulation are not yet fully understood. we examined the effect of inositoltriphosphate-receptor associated cgmp-kinase substrate (irag) on soce. aortic vascular smooth muscle cells (vsmc) from wild type (wt) and irag-knock-out (ko) mice were loaded with the calcium indicator fura 2-am and soce was measured as a change in the intracellular calcium concentration. in experiments with vsmc from wt mice soce was attenuated by the application of 8-br-cgmp. this effect was not observed in vsmc isolated from irag-ko mice. these differences in the strength of the soce-signal were abolished by the replacement of extracellular sodium with n-methyl-d-glucamine. the observed sodium dependence of the soce regulation via irag suggests, that an alternated sodium conductance might be responsible to some extent for the differences detected in wt and irag-ko vsmc. as a change in sodium conductance might result in a changed membrane potential we tried to track these changes with the flipr membrane potential assay kit while executing the soce protocol with and without 8-br-cgmp. no significant differences in membrane potential could be detected in the various stages of soce. in conclusion, our results indicate that irag exhibits a dual action on calcium regulation as it inhibits not only the intracellular calcium release but also the extracellular calcium influx through soce. induction of the icer promoter in vascular smooth muscle cells hildebrandt i. 1 tokyo metropolitan institute of gerontology, tokyo japan several transcription factor isoforms are encoded by the crem (camp response element modulator) gene. one prominent isoform is the inducible camp early repressor (icer), which acts as a transcriptional repressor on so called camp responsive elements (cres) in its target gene promoters. the icer mrna expression is regulated by an intronic promoter of the crem gene. in vascular smooth muscle cells (vsmcs) crem/icer is involved in the regulation of cell proliferation and apoptosis with physiological consequences in vivo. for instance crem-knockout mice, in which none of the known isoforms can be expressed, exhibit an increased neointima formation after carotid ligation as well as an increased atherosclerotic plaque formation after high fat diet on an apoe background. these observations were associated with an increased proliferation rate in isolated crem deficient vsmcs. on this background we wanted to clarify the specific role of icer isoforms in the vasculature. in first experiments we examined the inducibility of icer in primary vsmcs and smooth muscle cell lines. reporter luciferase assays showed that the activity of the icer promoter is induced at the maximum of fourteen fold after 4 hours of stimulation with forskolin (fsk) in immortalized rat vsmcs (13.7 ± 0.93; n=12 from 3 isolations). in a7r5 rat smooth muscle cells the icer promoter showed a maximum stimulation of 3.2 ± 0.16 fold after two hours of fsk stimulation (n=20 from 4 isolations). these pilot experiments showed that the icer promoter is inducible in vsmcs by camp dependent pathways. further experiments have to be carried out to elucidate the role of icer in the vascular system for example by stimulation of primary vsmcs and analysis of icer knockout mice. (supported by the dfg) overexpression of transmembrane channel-like proteins (tmcs) uncouples receptor-mediated calcium mobilisation hill k., urban n., straub i., schaefer m. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany the family of transmembrane channel-like proteins (tmcs) consist of 8 members (tmc1-tmc8) all tmc genes are predicted to encode transmembrane proteins with at least six membrane-spanning helices. mutations of tmc1 cause deafness in human and mice whereas tmc6 and tmc8 (also referred to as ever 1 and 2) are linked to epidermodysplasia verruciformis (ev), a skin disorder, which is characterised by an enhanced susceptibility towards cutanous infections by human papillomaviruses. the cell biological and physiological functions of tmc proteins still remain elusive. we have overexpressed tmc6 and tmc8 in hek293 cells to get insights into their physiological function. all tmcs were located within the endoplasmic reticulum (er) after overexpression of yfp or cfp-tagged constructs. ratiometric calcium imaging revealed that after overexpression of tmc6 or tmc8, stimulation of gq-coupled receptors with carbachol and atp resulted in a greatly reduced amount of calcium release from the er. moreover, challenging tmc6-or tmc8-expressing cells with the serca pump inhibitor thapsigargin was also not followed by a release of er-based calcium within the cell. to test whether the lack of calcium release was caused by a reduced calcium content within the er, we investigated calcium dynamics within the er using an er-targeted fret-based calcium indicator (d1er cameleon). the experiments revealed that the amount of calcium within the er was reduced upon overexpression of tmc6 or tmc8. recently, it has been reported that tmc6 and tmc8 might influence intracellular zinc distribution in human keratinocytes. we could confirm the presence of tmc6 and tmc8 mrna in a human keratinocytes cell line (hacat). upon overexpression of tmc8, hacat cells revealed the same phenotype as described above for the hek293 cells with an uncoupling of the receptor-mediated calcium mobilisation due to a depletion of the er calcium store. the mechanism by which overexpression of tmc proteins causes a reduced calcium concentration within the er remains unclear. considering that the presumed topology of the tmc proteins distantly resembles those of other ion channel superfamilies such as anoctamins, one might speculate that a conductance through the tmc protein itself leads to a calcium leak from the er. terahertz radiation is defined as radiation between 0.1 thz and 10 thz. a number of applications are currently being developed using radiation in this frequency range. these applications will lead to exposure of the general public, making it very important to study potential effects on biological systems. historically, only a few studies on effects caused by terahertz radiation have been conducted because of the lack of suitable generators and detectors. during the last decade, a number of studies on effects caused by radiation with frequencies around 100 ghz have been published. the present study investigated the genotoxic potential of terahertz radiation at three different frequencies, 0.106 thz, 0.380 thz and 2.520 thz. two skin cell types were used, primary human dermal fibroblasts (hdf) and a keratinocyte cell line (hacat). the cells were irradiated applying different exposure times and different power intensities. two genotoxicity tests were applied: the comet assay quantifies dna strand breaks as well as alkali-labile sites whereas the micronucleus test quantifies chromosomal damage. all experiments were performed and evaluated under blinded conditions as three independent replicate experiments. positive (mms-treated) and negative (untreated, sham-exposed) controls were included. in the comet assay no dna damage was observed as a consequence of the exposure under all experimental conditions. the same was true for the chromosomal damage investigated with the micronucleus test. the latter finding was particularly interesting for the experiments at 0.106 thz, because this type of radiation had been reported to cause mitotic disturbances. therefore these experiments were extended, applying higher power intensities and longer exposure periods. also with these modifications, no genomic damage was observed in the form of micronucleus formation. all in all, terahertz radiation did not induce genomic damage under the applied experimental conditions. this result is in line with published findings on genotoxicity of low-frequency terahertz radiation around 0.1 thz. the question, why the reported mitotic disturbances do not lead to manifest genomic damage remains open and requires further research. introduction: tea flavonoids derived from camomile and green tea such as apigenin and epigallocatechin gallate (egcg) can inhibit intestinal neoplasia. recurrences of adenomas and cancers were reduced in patients with resected colorectal cancer by treatment with tea bioflavonoids after tumor operation [1] . to clarify the biomolecular pathway for suppression of neoplasia we investigated the anti-inflammatory effect of a nutritional supplement flavo natin® (fn) which had been used in the clinical study on tertiary tumor prevention and of egcg in a colon tumor cell line. the aim of our study was to investigate if tea flavonoids are capable to suppress the inflammatory markers produced by tumor cells after cytokine stimulation. method: we studied the cytotoxicity of fn in the colon cancer cell line t-84 by resazurin fluorescence and compared it with the placebo supplement. additionally, the t-84 cells were incubated with fn, egcg or placebo and stimulated with tnf-alpha, if-gamma and il-1-beta. after the cytokine stimulation the mrna expression of ip-10, il-8 and tnf-alpha was measured by quantitative real-time pcr (qrt-pcr). results: stimulation of t-84 cells increased the expression of ip-10 (gamma-interferon inducible protein 10), tnf-alpha and il-8. by preincubation with fn at 10 µm the mrna expression of ip-10 was strongly reduced (log2-ratio -14). the tnf-alpha mrna was also but less decreased by fn. egcg displayed an inhibition pattern similar to fn. placebo did not influence the mrna expression of the chemokines and tnf-alpha. discussion & conclusion: clinically useful dietary tea bioflavonoids inhibit the expression of inflammatory genes in a colon cancer cell line. down-regulation of inflammatory gene products could be achieved in vivo by botanicals without clinically relevant side effects. [1] h. hoensch, b. groh, l. edler, w. kirch (2008) . prospective cohort comparison of flavonoid treatment in patients with resected colorectal cancer to prevent recurrence. world j gastroenterol, 14, 2187-2193. the cxcr7 c-terminal domain mediates efficient cxcl12 uptake and degradation hoffmann f., müller w., schütz d., schulz s., stumm r. universitätsklinikum jena institut für pharmakologie und toxikologie, drackendorfer str. 1, 07747 jena, germany cxcl12-signaling mediated by the g protein-coupled cxcr4 receptor plays a key role during embryonic development and disease states including cancer and inflammation. the second cxcl12-receptor cxcr7 modulates cxcl12/cxcr4-signaling by acting as a cxcl12-scavenger. given the distinct functions of cxcr4 and cxcr7, we hypothesized that trafficking and receptor stability are differently regulated by the distinct c-terminal domains. here, we examined epitope-tagged wild type and c-terminal mutant receptors expressed in human embryonic kidney cells (hek293) with respect to trafficking, stability, 125 i-cxcl12 radioligand degradation, and g protein-coupling. we found that the 24 c-terminal residues of cxcr7 were sufficient for cxcr7 to undergo rapid spontaneous internalization. replacement of the cxcr7 c-terminal domain with that of cxcr4 (cxcr7-4tail mutant) abolished spontaneous internalization but permitted ligand-induced internalization in conjunction with c-terminal phosphorylation. conversely, replacement of the cxcr4 c-terminal domain by that of cxcr7 caused ligand-independent internalization of cxcr4. receptor-mediated 125 i-cxcl12-uptake, release of 125 i-cxcl12-degradation products, and degradation of the receptor protein itself were accelerated with receptors bearing the cxcr7 c-terminus. while the cxcr7 c-terminus was sufficient to abolish g protein coupling in the cxcr4-7tail mutant, replacement of the cxcr7 c-terminus, cxcr7 second intracellular loop or both domains with the corresponding cxcr4 domain did not generate a g protein-coupled cxcr7 chimera. taken together, we provide evidence that the cxcr7 c-terminal domain influences the ligand-uptake/degradation rate, g protein-coupling, and stability of the receptor. this suggests that heterologous regulatory pathways targeting the cxcr7-c-terminal domain may effectively control cxcr7 functions. soluble guanylyl cyclase is a key mediator of brown adipocyte differentiation hoffmann l. s. 1 brown adipose tissue (bat) uses energy to produce heat by inducible thermogenesis. recent studies show that active bat is present in adults and involved in human energy balance, suggesting that the energy consuming property of bat might be exploited to fight obesity and related diseases. the no/cgmp signaling pathway is a key player in diverse physiological processes. recently, we have shown in bat that cgmp signaling is connected with insulin signaling and abrogation of cgmp signaling leads to impaired bat differentiation and function (haas, b. et al., sci signal, 2009 ). here we investigated the role of the cgmp generating enzyme soluble guanylate cyclase (sgc) in bat differentiation in vitro. mesenchymal stem cells isolated from bat of newborn sgcβ 1 -/mice and wt littermates were differentiated in vitro into brown adipocytes in the presence or absence of cgmp. abundance of sgc isoforms was determined by qrt-pcr and western blotting. bat differentiation was assessed by redo staining of accumulated intracellular lipids, measurement of triglyceride (tg) content, determination of expression of bat marker proteins pparγ, c/ebpα, ap2 and bat marker genes ucp1, pgc1α, cidea. the α2 and β1 isoforms of sgc were highly expressed in bat whereas α1 sgc could not be detected. redo staining of wt brown adipocytes showed basal differentiation which was increased upon addition of 8-pcpt-cgmp. in contrast, staining was lower in sgcβ1 -/cells compared to wt under control conditions and increased in the presence of cgmp. tg measurement showed that sgcβ1 -/brown adipocytes contain approximately 50% less lipids than wt cells under basal conditions. addition of cgmp doubled tg content in both genotypes. similar results were observed for marker protein expression. deletion of sgc resulted in 56-40% decrease in c/ebpα, pparγ and ap2 expression compared to wt. again, cgmp roughly doubled protein expression in sgcβ1 -/and wt cells compared to control. under basal conditions, bat marker gene expression was decreased by approximately 80% in sgcβ1 -/cells compared to wt cells. this decrease was prevented by addition of cgmp. these results show that sgc deletion leads to dysfunctional bat differentiation and emphasize the central role of cgmp signaling in bat differentiation. further investigation of sgc/cgmp signaling in bat might reveal new drugable targets bringing bat-dependent pharmacological therapy to treat obesity and related disease into closer reach. comparative inhalation toxicity of carbon-nanomaterials (multi-wall carbon nanotubes, graphene and carbon black) hofmann t. 1 carbon black is a spherical carbon anomaterial whereas multi-wall carbon nanotubes (mwcnt) are cylindrical and graphene is a laminar allotrope of carbon. processing and handling as well as abrasion processes can set free inhalable cnt particles. results of rodent studies collectively show that regardless of the process by which cnts were synthesized and the types and amounts of metals they contained, cnts were capable of producing inflammation, epithelioid granulomas, fibrosis, biochemical and or toxicological changes in the lungs (lam et al. 2004 , muller et al. 2005 , ma-hock 2009 , pauluhn 2010 . graphene possess similar physical properties as cnt but may different toxicological property. we performed short-term inhalation studies in rats to compare the toxic potency of four different cnt, two graphenes and one carbon black. the materials are characterized thoroughly according to the oecd list. the four mwcnt caused morphological changes as descriped above. several biochemical and cytological parameters in the broncho-alveolar lavage fluid were strongly increased consistent with the histological findings. two mwcnt exhibited a higher toxic potency than two other mwcnts and findings caused by one graphene typ were even less severe. the graphene with lower surface area as well as low surface area carbon black did not cause any adverse effects up to 10 mg/m 3 . the short-term inhalation studies were able to descriminate different toxic potencies of carbon-based nanomaterials and is hence used for the selection of less toxic materials for further product development as well as to define and prioritize higher-tier toxicological testing of nanomaterials. 165 synthesis and analytical assessment of possible dna adducts formed after activation of the tobacco alkaloid myosmine högg c., zwickenpflug w., gudermann t. walther-straub-institut abt.: toxikologie, nußbaumstraße 26, 80336 münchen, germany myosmine represents one of the minor tobacco alkaloids and its effective uptake from smokeless tobacco or tobacco smoke, as well as by consumption of food is not understood in detail. myosmine can be activated by peroxidation and n-nitrosation yielding 4-hydroxy-1-(3-pyridyl)-1-butanone (hpb) which is well known as reactive intermediate during activation of a variety of tobacco specific n-nitrosamines (tsna). therefore, myosmine may be a potential candidate for possible mutagenic or carcinogenic risk to human health. furthermore, myosmine n-nitrosation yields the tobacco specific nitrosamine n-nitrosonornicotine (nnn), which is classified as carcinogenic to humans. considerable efforts have been undertaken, especially in organic synthesis, to verify and elucidate the significance of the hpb precursor the pyridyloxobutyl (pob) intermediate and its dna-adducts, which were analysed only in animal experiments till now. the formation of 3-pyridylmethanol was observed under myosmine peroxidation and identified as a metabolite in rat urine after application of myosmine to rats. the formation of the 3-pyridylmethanol intermediate, might provide for the reactive electrophilic picolinium ion which could interact with dna. these possible adducts might be of special interest to elucidate the role of myosmine concerning its uptake by smokers and passive-smokers in contrast to non smokers ingesting the substance by consumption of food. dna adducts may help to obtain more information about possible risk assessment of myosmine and to differentiate between the activation from the other nicotinoids and tsna. the specific dna adducts, 5-methyl-1,3-bispyridin-3-ylmethyl-1h-pyrimidin-2,4-dion and 3-(3''-picolyl)thymidine have been synthesised as reference substances. the former was prepared by addition of diisopropylazodicarboxylate (diad) to a mixture of 3-pyridylmethanol, thymine and triphenylphosphine. the reaction product was identified using nmr ( 1 h, 13 c, cosy, hmqc, hmbc). for synthesis of 3-(3''-picolyl)thymidine the hydroxyl groups of thymidine were initially acetylated using acetic acid anhydride and 4dimethylaminopyridine (dmap). the existence of this thymidine adduct was confirmed using nmr. this adduct was labelled with 5-([4,6,-dichlorotriazin-2-yl]amino)fluorescin (dtaf) to enhance the sensitivity using hplc-fluorescence chromatography and used as reference substances for analysis of dna samples from biological tissue. supported by dfg grant (ty 81/1-1). cancer and cardiovascular diseases such as atherosclerosis are the most important causes of death in western societies. common to both diseases is a deregulation of cell death, with significant contribution of inflammatory processes. enhanced oxidative stress plays a dominant role in such events as it forms a vicious cycle with inflammation and controls multiple forms of cell demise. therefore, anti-oxidative enzyme systems gained considerable interest since control of reactive oxygen species (ros) has the capacity to regulate cell death in either direction. the human enzyme family of paraoxonases consists of three members, known as pon1, pon2 and pon3. while pon1 is found predominantly in the circulation, pon2 and pon3 are intracellular enzymes with established anti-oxidative functions. it has been shown that both pon2 and pon3 are protective against atherosclerosis. underlying mechanisms of their protective and antioxidative functions however remained elusive. here we demonstrate that both enzymes locate to the endoplasmic reticulum (er) and mitochondria where they fulfill vital functions in the control of ros generation. in particular, pon2 and pon3 were shown to interact with coenzyme q10 which diminishes mitochondrial ros formation. as a consequence, these enzymes reduce execution of mitochondrial apoptosis, such as cardiolipin peroxidation, cytochrome c release and caspase activation. moreover, pon2 and pon3 reduced er stress-triggered cell death, i.e. by diminishing jnk signaling and chop expression. while these results elucidate their protective role in cardiovascular diseases, it also establishes a relevant function in survival of tumor cells. in accordance, we demonstrate that both enzymes are frequently found overexpressed in various tumors. in cancer cell culture studies, overexpression of both enzymes granted considerable resistance against chemotherapeutics. in turn, knock-down of pon2 caused spontaneous apoptosis of several cancer cell lines. finally, our analyses also revealed that pon2-knockout mice show severe alterations of the hematopoetic stem cell compartment, which implies a significant role in leukemias where these enzymes are frequently found overexpressed. together, our results propose pon2 and pon3 as new putative anti-tumor candidates and demonstrate the efficacy of interventions targeting cellular redox-balance. steigerwald arzneimittelwerk gmbh wissenschaftliche abteilung, havelstr. 5, 64295 darmstadt, germany stw 5 (iberogast ® ), a multi-component herbal drug, is successfully used in the therapy of functional dyspepsia and irritable bowel syndrome (ibs). previous studies revealed effects of stw 5 on disturbed motility and inflammatory processes. although the antiinflammatory properties of stw 5 are well examined, the contribution each of the individual herbal constituents to the anti-inflammatory effect remains unclear. therefore, we studied the effects of stw 5 and its components on inflammation-induced cell death and on the release of the pro-inflammatory cytokine tnf-α. the aim of these investigations was to analyse additive or synergistic effects of the components. the experiments were carried out on caco-2 cells after stimulation with lps (10 ng/ml) for 2 hours. cytotoxicity was evaluated using a commercially available ldh (lactate dehydrogenase)-assay. furthermore, the release of tnf-α after lps (100ng/ml) stimulation of differentiated thp-1 cells was measured using a commercially available elisa. stw 5 (62.6 -500.5 µg/ml) reduced lps (10 ng/ml)-induced cell death in a concentration-dependent manner with a maximum inhibition of 50.0 %. the herbal components in equivalent concentrations contributed to the inhibitory effect of stw 5 to different extents. the maximum inhibition differed in a wide range between the components. stw 5 (500.5 µg/ml) reduced significantly the release of tnf-α by 87 % in lps (100 ng/ml)-stimulated differentiated thp-1 cells while having no effect in untreated cells. in concentrations equivalent to stw 5 caraway, milk thistle, lemon balm and greater celandine had no effect on the lps-induced increase in tnf-α release. bitter candytuft, peppermint, chamomile, liquorice and angelica reduced the tnf-α release, though less pronounced as compared to stw 5, indicating a possible synergistic effect. our results indicate a multi-target effect of stw 5. the anti-inflammatory effect may be due to a reduction of the cytotoxic effect on intestinal mucosa cells and to an inhibition of the release of the pro-inflammatory cytokine tnf-α from immune cells. the individual herbal components seem to contribute by different mechanisms of action to the overall effect of stw 5. immune cell-induced local steroidogenesis in the lung: implications for asthmatic disease and therapeutic intervention hostettler n. 1 , brunner t. glucocorticoids are steroid hormones with potent anti-inflammatory properties. synthetic glucocorticoids are frequently used for the therapeutic treatment of inflammatory disorders, such as asthma. endogenous glucocorticoids are predominantly produced in the adrenal glands in response to emotional, physical and immunological stress. recent years, however, revealed several alternative sources of these immunoregulatory steroid hormones. thus, we have found that the intestinal epithelium is a rich source of glucocorticoids and intestinal glucocorticoids contribute to the maintenance of local immune homeostasis (1) . as the intestinal and the pulmonary epithelium have much in common, i.e. barrier functions and transport of nutrients, resp. gases, we wondered whether the lung mucosa is also capable of synthesizing immunoregulatory glucocorticoids in response to immune cell activation. the murine lung was found to expresses all enzymes required for the synthesis of corticosterone from cholesterol. while most enzymes where expressed in a constitutive manner, cyp11a1, encoding p450ssc, was strongly induced in response to immunological tress. treatment of mice with t cell-activating anti-cd3 antibody of macrophage-activating lipopolysaccharides induced strong local glucocorticoid synthesis, which was effectively blocked by the corticosterone synthesis inhibitor metyrapone, indicating that glucocorticoids measured were produced bona fide in the lung tissue. surprisingly, allergen-induced allergic airway inflammation failed to trigger local glucocorticoid synthesis despite the massive infiltration of neutrophils, eosinophils and t cells. in contrast to that in the intestinal epithelium local glucocorticoid synthesis in the lung was found to be dependent on the presence of adrenal glands as adrenalectomy abolished pulmonary steroidogenesis. in line with the notion that the lung metabolizes steroid precursors we found that ex vivo cultured lung tissue metabolized 3 hduring the last decade small rna molecules has been identified as important regulators for gene expression. these micrornas (mirna) are single-stranded transcripts, which are expressed in many cell types, where they modulate rna stability and translation and, therefore, controlling various cellular mechanism and tissue development. against this background, in the present study the mirna expression and potential target genes were studied in the human placenta as a tissue demonstrating various developmental changes in a limited period of time. taqman®array microrna cards for profiling of 365 mirnas in placentas of different gestation times revealed a significant expression of 277 mirnas by comparing placentas of early gestation (<10.week), preterm (<30.week) and term (>37.week). when comparing the analyzed groups, 106 of these mirnas expose a continuous up-(including mir-20a, mir-379) or downregulation (including mir-9, mir-200a). while comparing placentas of early gestation to term placentas, 30% (e.g. mir-9, mir-429) were up-and 70% (e.g. mir-126, mir-373) were downregulated. by focusing on the latter group with early preterm placentas the ratio is opposed. here, 60% of mirnas showed higher (e.g. mir-107) and 40% lower expression (e.g. mir-627, mir-191). with emphasize on these mirnas, a computational prediction algorithm using the mirò database predicted a potential interaction between corticotropin-releasing hormone (crh) and the mirnas mir-9 and mir-429. specific expression analyses validate an inverse expression between mirnas and target with a reduced expression of mir-9 (93%) and mir-429 (94%) in term placentas, while crh is upregulated 114fold. this interaction was verified on functional level by reporter gene assay. a significantly suppressed luciferase activity of the reporter plasmid containing the 3´-utr sequence complementary to crh was exhibit for the predicted mir-9 binding site. here, the mirna-mrna-interaction reduces the luciferase activity by ~40%, whereas the decreased luciferase activity for the predicted mir-429 binding site is not significant. the results demonstrate a gestation dependent expression of placental mirnas, which may help to explain gestational changes in gene expression and highlight the potential of mirnas as biomarker for pregnancy-related pathologies. since homo sapiens era wound treatment by early civilizations was based on the use of local flora. scilla indica knuth liliaceae (s.i) is a perennial herb with a pear shaped, tunicated bulb, bearing fibrous roots, white flowers and leaves on the stem resembling u. maritima. it is used as cardiac tonic, against inflammation, ulcers and sinus diseases. traditionally the powdered bulb is used topically for warts treatment, while roasted and crushed bulbs are applied to corns of the feet soles. the plant contains steroid glycosides (bufadienolides 1-3%), glucoscillarene a, proscillaridine a, scillarene a, scillcyanoside ,scilliglaucoside ,mucilage and alkyresorcinol derivatives exerting skin healing properties similar to wheat bran products. the study is focused on the investigation of the restoration quality of a skin dorsal incision wound in wistar rats in three groups, control (a1) and experimental (a2,a3 ) of rats weighing 350-450g local application of dichloromethane extract of s.i in vehicle olive oil preparations of 50 and 100mg were used. animals were anaesthetized ( ether pro narcosi ), trauma 2 cm long and 2mm deep was performed by lancet on depilated dorsal area skin until the muscular aponeurosis and were treated for 4 days with 60λ of each preparation. group a3 showed increased remodelling of trauma area (limited width). granulomatous tissue was more pronounced in length, width and surface in group a2. the macroscopic ulcus dimensions were better in group a3 while the microscopic view were similar in a2 and a3 and better in comparison to control a1. a tendency to trauma remodelling was observed, without statistical significance (t =0.3) in recent years, it has been suggested that nanoparticles generated from combustion processes (e.g. diesel engine exhaust particles), may contribute to the pathogenesis of neurodegenerative diseases such as alzheimer's disease (ad). the aim of our current study was to investigate the effects of subchronic exposure to diesel engine exhaust (dee) in the 5xfad mouse model, which is characterised by progressive behavioural deficits as well as amyloid plaque formation and neuron loss. ten weeks old female 5xfad mice and their nontransgenic littermates were exposed by whole body inhalation to diluted dee (~1 mg particles/m 3 ) or clean air (controls) for 3 or 13 weeks (5 days/week and 6 hour/day). subsequently, all animals were subjected to a series of behavioural tests. at ten days post-exposure, mice were sacrificed to investigate lungs and brain tissues for pathological and biochemical and molecular-biological changes. in line with the expectations, the 5xfad mice displayed typically age-dependent behavioural deficits and amyloid plaque formation in cortex and hippocampus. a significant dee exposure-related effect was observed for the string suspension test, representing a measure of motor coordination/grip strength. dee exposure was also associated with mrna expression changes of markers of inflammation and oxidative stress in specific brain regions, including the olfactory bulb. histopathology of plaqueload in cortex and hippocampus (from a limited number of animals investigated so far) did not reveal clear evidence for increased plaque formation due to the dee exposures. further research is needed to evaluate the effects of long term exposure to nanoparticles in the central nervous system. this work is supported by funds from the research committee of the medical faculty of the university of düsseldorf (9772-365), the dfg graduate school grk1033 and the rivm -centre for environmental health, bilthoven, netherlands. mono-glucosylation of (h/k/n)ras by clostridium sordellii lethal toxin (tcsl) blocks critical survival pathways, including the pi3k/akt, the ralgef/ral, or the raf/erk, and results in apoptosis. in this study, ras glucosylation is presented to result in expression of the cell death-regulating small gtpase rhob based on transcriptional activitation. rhob expression depends on k-ras inhibition, as sirna-mediated knock-down of specifically k-ras (neither h-ras nor n-ras) provokes rhob expression. rhob expression further depends on inhibition of pi3k/akt, as activation of pi3k/akt using a pi3k activator prevents rhob expression downstream of inactivated ras. newly synthesized rhob is gtp-loaded and rapidly degraded in a proteasome-and a caspase-dependent manner, providing first evidence for caspase-dependent degradation of a rho family protein. although often characterised as a pro-apoptotic protein, rhob suppresses caspase-3 activation in tcsl-treated fibroblasts. conclusions: 1. rapid and efficacious ras inactivation by tcsl turns out to be particularly useful in the characterisation of ras inactivation-induced rhob expression as an immediate-early gene response. 2. the finding on the cytoprotective activity of rhob in tcsl-treated cells re-enforces the concept that rhob exhibits cytoprotective rather than pro-apoptotic activity on cellular background of inactive ras. the activity of the rhob promoter is suppressed by rhoa (through a not yet identified pathway or ras (through the pi3k/akt pathway. ras glucosylation by tcsl results in de-suppression of the rhob promoter and rhob expression. the calcium-binding protein annexin a4 (anxa4) is involved in diverse cellular processes including e.g. vesicular transport, ion channel regulation and transcriptional regulation. upon ca2+ binding anxa4 undergoes conformational changes, which lead to oligomerization of anxa4 to homotrimers and cause an increased affinity for membrane phospholipids, which in turn provokes the translocation from the cytosol and the nucleoplasm to plasma and nuclear membranes. in order to examine the effect of anxa4 on cre-and creb-(camp response element-binding protein) dependent transcription, a series of transient transfections using a luciferase reporter gene driven by the creb target promoter of the inducible camp early repressor (icer) was carried out. when the luciferase reporter construct was co-transfected with anxa4, there was significant reduction of basal (dmso) luciferase activity ( the implantation of drug eluting stents (des) after coronary artery intervention was an important step treating coronary artery disease. indeed, cytotoxic compounds like sirolimus used on des are responsible for reduced in-stent restenosis in comparison with bare metal stents. pimecrolimus, a potent anti-inflammatory drug has also been investigated for its efficacy on des. preclinical studies in pigs revealed promising antiproliferative effects of pimecrolimus on neointima formation. however, in humans, pimecrolimus coated stents exerted adverse effects. we hypothesize that compared to the highly active sirolimus, pimecrolimus may influence additional cellular processes leading to the worse outcome. in order to identify those processes we conducted in vitro studies in human coronary artery endothelial cells (hcaec) and smooth muscle cells (hcasmc). brdu in vitro assays of hcaec treated with pimecrolimus examined an ic50 value of 6.787 µm [5.745 to 8 .017] which is much higher than ic50 values of sirolimus described in literature [0.1nm to 1nm]. genechip array analysis comparing gene expression in pimecrolimus and sirolimus treated hcaec and hcasmc showed significant induction of several genes involved in interferon signaling. in detail, the expression of ifnβ activated genes like irf9 and ifitm1 was up regulated in cells treated with pimecrolimus while no or oppositional effects were observed with sirolimus. gene regulatory effects were validated by real time pcr. incubation with ifnβ itself showed similar effects in up regulation of genes involved in interferon signaling. furthermore, we were able to demonstrate a significant increase of ifnβ secretion in hcasmc and hcaec treated with pimecrolimus. however, comparison of ifnβ and pimecrolimus on proliferation of hcaec and hcasmc revealed different cellular responses. while ifnβ significantly decreased hcasmc and increased hcaec proliferation, treatment with pimecrolimus lead to anti-proliferative effects on both cell types. in conclusion, pimecrolimus activates pathways involved in interferon signaling but exerts different pharmacological effects, compared to the endogenous compound suggesting that infβ secretion is not the major factor contributing to the difference in pimecrolimus function. identification of novel phospho acceptor sites of the mu opioid receptor regulating receptor internalization illing s., just s., doll c., schulz s. uniklinikum der fsu jena pharmakologie und toxikologie, drackendorfer straße 1, 07747 jena, germany the opioid alkaloid morphine is among the most potent clinically used analgesics. however, the clinical utility of morphine to treat chronic pain is limited by its rapid development of tolerance and dependence. the stimulation of the mu opioid receptor (mor) with damgo or morphine results in different patterns of receptor phosphorylation and trafficking. so far, the three major phosphorylation sites of the mu opioid receptor namely serine 363 (s363) threonine 370 (t370) and serine 375 (s375) have been identified using phosphosite-specific antibodies. however, mutations of these three residues to alanine (s363a/t370a/s375a) did not prevent agonist-dependent internalization. in the present study, we have constructed a series of phosphorylationdeficient mutants showing that mutation of at least six residues (s363a/t370a/s375a/t376a/t379a/t383a) was required to completely block agonistdriven endocytosis. consequently, we generated phosphosite-specific antibodies to t376 and t379 which enabled us to provide direct evidence for agonist-dependent phosphorylation of these sites. our analysis of time-and dose-dependent phosphorylation of mor revealed that s375 was the primary phosphorylation site, whereas t370, t376 and t379 were secondary sites. moreover, the partial agonist morphine induced only the phosphorylation of s375 but not of t370, t376 of t379. our results show, that mor phosphorylation occurs in a hierarchical and agonist-selective manner directly regulating receptor sequestration. assessment of mda effects from toxicity studies with regard to endocrine modulation jäger r. 1 methylene dianiline (mda; cas no. 101-77-9) is on the usepa list 2 for endocrine disruption screen testing. in a yeast androgen screening (yas) assay (in vitro assay on receptor binding or blocking) mda revealed a significant anti-androgenic activity at a concentration of 0.01 mm. to assess the biological relevance of this observation under in vivo conditions the existing data from animal toxicity studies with mda were reviewed for indications of possible endocrine modulating (em) effects (weight-of-evidence approach). in addition, literature was searched for relevant studies on mda and structural analogues using the american chemical society scifinder client-server software. structural analogues indicated several drivers for em activity, notably the diphenolic ring structure. however, in vitro receptor binding assays showed mda had no androgenic or anti-estrogenic activitiy. one report described a weak estrogenic binding at 0.2 mm mda, while another described no effect at similar concentrations and a rat estrogen receptor binding study found no effect at 0.2 mm. overall it is concluded that mda does not bind to the estrogen receptor. endocrine related effects of mda were investigated in several species and dose routes in unvalidated research-type protocols. guideline subchronic and chronic toxicity studies with rodents revealed neither adverse organ weight changes nor histopathological alterations of sex organs. the main systemic effects from the oral 13-week studies were body weight reduction and histopathological changes of the thyroid and bile duct (lowest loael: 7.5 mg/kg bw). mda was carcinogenic to rats and mice (thyroid and liver) after oral administration over 2 years. non-neoplastic effects in the thyroid (rats and mice) were observed (lowest loael for systemic toxicity: 9 mg/kg bw). mda inhibits iodide oxidation which with concomitant decreased thyroid hormone formation is known to induce thyroid tumors. in summary, in vitro screening tests revealed no consistent endocrine related effects of mda. in vivo, there was an effect on the thyroid gland, possibly by enzyme inhibition. there were no histopathological changes of gonads and accessory sex organs and no evidence of sex hormone related em. the evidence from the full dataset on mda does not indicate androgenic or estrogenic effects. overall, based on a weight of evidence assessment there are insufficient alerts for em activity to suggest further testing should be done. the gtpase arfrp1 controls the assembly of apoa-i to and the lipidation of chylomicrons in the golgi of intestinal epithelium jaschke a. 1 background: the uptake and processing of dietary lipid by the small intestine is a multi-step process that involves luminal digestion, cellular uptake of fatty acids by the mucosa, and subsequent synthesis and export of chylomicrons. the gtpase adpribosylation factor related protein 1 (arfrp1) is a member of the arf-family and controls the arf-like 1 (arl1)-mediated golgi recruitment of grip domain proteins which in turn bind several rab-gtpases. the aim of the study was to define the role of arfrp1 in intestinal nutrient absorption. methods: for the generation of intestine-specific null mutants arfrp1 flox/flox mice were crossed with mice expressing the cre recombinase under the control of the intestinespecific villin promoter (vil-cre) and arfrp1 expression was suppressed by sirna in caco2-cells. the phenotype in respect to lipid absorption and chylomicron production in the intestinal epithelium and in caco2-cells, respectively, was analyzed. results: arfrp vil-/mice were viable but showed an early postnatal growth retardation (mean body weight of arfrp1 vil-/was 43.3±5% lower than that of control mice at the age of 28 days) arfrp1 vil-/mice displayed reduced triglycerides, free fatty acids and glucose plasma levels indicating that the growth retardation is the result of a malabsorption. uptake of glucose and amino acids were unaffected by the deletion of arfrp1. in contrast, lipid uptake as elucidated by oral fat tolerance tests was impaired in arfrp1 vil-/mice. arfrp1 vil-/enterocytes as well as arfrp1 mrna depleted caco-2 cells absorbed fatty acids normally but secreted chylomicrons with a markedly reduced triglyceride content. in addition, while the release of apolipoprotein a-i (apoa-i) was dramatically decreased apoa-i accumulated in the arfrp1 vil-/epithelium and was predominantly colocalized with rab2. our results demonstrate that the growth retardation of arfrp vil-/mice is a consequence of impaired intestinal fatty acid absorption. we suggest that arfrp1 is required for the assembly of aopa-i to the chylomicrons and for the further lipidation of chylomicrons in the golgi of intestinal epithelial cells. this finally leads to an secretion of chylomicrons with a markedly reduced triglyceride content. rhoa influences adhesion and spreading of cardiac fibroblasts via complex regulation of cytoskeletal proteins jatho a. 1 the monomeric gtpase rhoa is thought to be involved in the pathology of heart diseases, however, its role in cardiac cells is not well defined. therefore we intended to analyze the effect of rhoa in cardiac fibroblasts by using a lentivirus-based knockdown approach. by doing so, we could show that the knockdown of this gtpase by about 50% resulted in a massive change in cell morphology, but displayed no effect on cell viability. the appearance of the cells in the 2d-culture changed from a fibroblast-typical stretched morphology with intracellular stress fibers to a more epithelial-like cell morphology. by morphometrical analyses we demonstrate that fibroblasts with reduced rhoa expression display an increase in cell surface by 2.2-fold and in perimeter by 1.5fold. moreover, the depletion of rhoa significantly influences the adhesion velocity, as within the first hour after cell detachment about 20% of the rhoa knockdown cells reattach, but only 5% of the respective control cells. based on these findings, we investigated the distribution and composition of different cytoskeletal proteins by immunofluorescence stainings and immunoblot analysis. we found, that the amount of b-actin is not reduced in rhoa knockdown cells, however, the distribution is markedly changed. in these cells internal star-shape bundles of actin could be found instead of the commonly appearing stress fibers in control cells. in contrast, the cortical actin fibers, mainly consisting of g-actin, were not affected. in addition, smooth muscle-actin, which is characteristic for myofibroblasts, was clearly reduced in rhoa knockdown cells compared to control cells by 33%. this reduction might be responsible for the more relaxed cell shape. in summary, the knockdown of rhoa influences cell adhesion and the morphological characteristics of cardiac fibroblasts, without obviously affecting cell proliferation and viability. using site-specific fluorescence labeling to study uptake of pasteurella multocida toxin into eucaryotic cells jehle d., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abteilung 1, albertstraße 25, 79104 freiburg, germany pasteurella multocida is an opportunistic pathogenic bacterium living in the nasal pharyngeal space of animals. p. multocida is of particular importance in the livestock management of pigs. under special conditions infection of pigs with p. multocida leads to an atrophic rhinitis, which is characterized by the atrophy of nasal turbinate bones accompanied by a shortening and twisting of the snout. the causative agent of the atrophic rhinitis was found to be the bacterial protein toxin pmt (pasteurella multocida toxin). after entering the cell the 146-kda toxin activates various signal transduction pathways by stimulating heterotrimeric g proteins of the gαq, gα13 and gαi family. the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. the uptake of pmt into cells is not comprehensively understood. therefore, we utilized a recently described technique, called "sortagging" (popp mw et al. nat chem biol. 2007; 3: 707) , to specifically couple fluorescence tags to the n-or c-terminus of pmt. the enzyme sortase a (srta) from staphylococcus aureus attaches proteins to the bacterial cell wall. the substrates are recognized by an lpxtg motif. srta cleaves the peptide bond after the threonine and adds a glycine-containing nucleophile. we introduced these motifs into pmt to express srta-recognized toxin and coupled the toxin with fluorescence tags, respectively. fluorescently labeled pmt was used to study the uptake of the toxin into eucaryotic cells by laser scanning microscopy. munich heart alliance, münchen, germany cells within the myocardium communicate by secreted factors and this has been suggested to contribute to cardiac remodeling. to identify novel factors secreted by the myocardium, we have previously reported a genetic yeast screen which led to the identification of protease inhibitor 16 (pi16). here we report the generation of a mouse line where pi16 can be deleted globally or conditionally using the cre/loxp system. after electroporation of the pi16 floxneo targeting vector in embryonic stem cells and injection into murine blastocysts we gained a mouse line that carried the targeted modification of the pi16 allele. global pi16 deficiency (pi16 lox/lox ) per se did not lead to a cardiac phenoytpe (hw/bw (mg/g): pi16 +/+ = 5.4 ± 0.2 (n = 6), pi16 lox/lox = 5.9 ± 0.7 (n = 4), p > 0.05; fibrosis (%): pi16 +/+ = 3.3 ± 0.2 (n = 7), pi16 lox/lox = 3.9 ± 0.8 (n = 5), p > 0.05; fractional shortening (%): pi16 +/+ = 29.8 ± 0.7 (n = 7), pi16 lox/lox = 26.4 ± 2.5 (n=5), p > 0.05). in addition we carried out an immunohistochemical analysis of pi16 expression using pi16-deficient mice as negative controls. pi16 localized to cardiac fibroblasts, to the epididymis and the trachea. in the failing heart we detected accumulation of pi16 preferentially in fibrotic areas. we are currently applying cardiac stress models to gain a broader understanding of the function of pi16 and its potential as a therapeutic target molecule. enantioselective determination of r-and s-hyoscyamine in mammalian plasma and urine samples john atropine (atr) is the racemic mixture of the tropane alkaloids s-and r-hyoscyamine (hyo). s-hyo acts as a competitive acetylcholine antagonist at the muscarinic receptors (eutomer) inducing mydriasis, excitations, hallucinations, coma and ultimately death, whereas r-hyo does not (distomer). atr is used for clinical intervention of poisoning with organophosphorus (op) pesticides or nerve agents. despite well known differences in pharmacological behavior, individual pharmacokinetics of both atr enantiomers have rarely been addressed in the literature [1, 2, 3] . therefore, we initially developed a nonchiral liquid-chromatography-electrospray tandem mass spectrometric method (lc-esi ms/ms) that allows quantification of atr and additional natural and synthetic tropane alkaloids from plasma after simple deproteinization [4] . to discriminate both atr enantiomers the sample preparation step was expanded by an enzymatic pretreatment. samples were incubated either with diluted human serum (not containing atropinesterase, atre, procedure a) or with diluted rabbit serum (procedure b). rabbit serum contains atre (ec 3.1.1.10) which is suitable for stereospecific hydrolysis of shyo into tropine and tropic acid while r-hyo remains unaffected. after sample precipitation, hyoscyamines were quantified by the lc-esi ms/ms method. following procedure a the concentration of total hyo and following procedure b remaining r-hyo were determined. s-hyo was calculated by the difference between these concentrations [3] . the impact of potential matrix ingredients that may appear in samples from oppoisoned patients under atr therapy were evaluated (oximes, op agents, carbamates) [3] . the assay was applied to diverse toxicological and pharmacological samples. i) measurement of natural s-hyo in an extract of atropa belladonna leaves as well as in plasma and urine of a female patient who was poisoned after ingestion of such leaves revealed that no biotransformation to r-hyo occurred. ii) analysis of plasma obtained from an op-poisoned female patient under atr therapy revealed faster elimination of shyo when compared to r-hyo [3] . iii) in contrast, no enantioselective differences were obvious in healthy anaesthetized swine after intravenous injection of atr [5] . data indicate that the enzymatic enantioselective procedure represents a useful tool to characterize in vivo behavior of r-and s-hyo allowing to reveal individual kinetics. failing hearts are unable to adequately supply the body with blood and oxygen. common therapeutic strategies interfere with cardiac remodelling, reduce cardiac preand afterload or aim at direct improvement of cardiac contractility. cardiac contractility is mainly controlled by β1-adrenergic receptors. resensitization of b-adrenergic receptors by inhibition of g-protein coupled receptor kinase 2 (grk2) is discussed as a potential strategy to treat heart failure. we characterized raf kinase inhibitor protein (rkip) as a physiological inhibitor of grk2 and found rkip to increase contractility of neonatal cardiomyocytes. the present study evaluated the role of rkip in heart failure. we assessed its effects on cardiac function in pressure overload induced heart failure and determined the expression patterns of rkip in failing hearts of humans and mice. transverse aortic contriction (tac) was performed on 7-week-old c57/bl6 mice to induce heart failure by pressure overload of left ventricles. after three weeks of tac, echocardiography showed distinct signs of decreased cardiac function in wild-type mice. fractional shortening was reduced and left ventricular diameters were increased. histological analyses revealed increased interstitial fibrosis, caspase-and tunelassays indicated myocyte apoptosis. western blot analysis showed significant upregulation of rkip expression in failing hearts compared to non-banded control hearts. interestingly, this upregulation of rkip expression was also detected in failing human hearts and in samples of patients with aortic valve stenosis but not in healthy control samples. to assess the effects of rkip overexpression on heart failure, we analysed heart function and structure of rkip transgenic mice and wild-type mice 3 weeks after tac. while left ventricular hypertrophy was increased to similar extents in wild-type and rkip trangenic mice, rkip mice did neither develop dilatation of the left ventricle nor a decrease in fractional shortening. in contrast to wild-type mice, the expression of the heart failure markers bnp and anp was not upregulated in banded rkip transgenic mice after 3 weeks of tac. taken together, cardiac overexpression of rkip prevented left ventricular dilatation and loss of contractile function in a mouse model of pressure overload induced heart failure. therefore, increased rkip expression may be an interesting target to prevent detrimental effects from increased left ventricular pressure. the main focus of this study was the chromatographic separation. the most frequently prescribed antibiotic drugs clarithromycin, erythromycin, clindamycin, cefuroxim, doxycyclin, amoxicillin, levofloxacin, and ciprofloxacin were selected for the screening method. method: a relatively short operation time and a sufficient separation were reached by column, eluent, and gradient optimization with poplc (phase optimized liquid chromatography). in the first step columns with the five stationary phases c18 eps 2, c18 sh 2, phenyl 2, cn 2, and c30 were used to determine the retention times of the drugs in an isocratic mode. the stationary phases, the column length and the retention times were fed in the poplc software and the optimal column was calculated. this column contained different stationary phases and was compared with customary columns. results: using the optimal column a sufficient chromatographic separation of the eight antibiotic drugs was reached. that was not possible with the customary columns. with the optimal column the time of measurement was too long. using a mobile phase gradient the measuring time could be reduced. discussion: with lc-ms/ms a complete chromatographic separation of all analytes is not necessary. but when measuring many transitions in a biological matrix two problems should be excluded: ion suppression and a too small number of measurement points per peak. especially when using positive and negative ionization in the ms a good separation is mostly necessary. to determine only the eight antibiotic drugs an optimized column is not necessary, but for a screening method with more than twenty drugs the poplc system is very helpful. we have investigated the uptake mechanism of cdt and in particular the intracellular membrane translocation of cdta. our data indicate that cdt requires acidification of the endosomal lumen for translocation of cdta across endosomal membranes into the cytosol. bafilomycin a1, an inhibitor of endosomal acidification protects vero (african green monkey kidney) cells from intoxication with cdt. consistently, translocation of cdta was observed when the acidic conditions of the endosomal lumen were mimicked at the cytoplasmic membrane of intact cells. next, we tested whether host cell factors are involved in membrane translocation of the toxin. radicicol, a specific pharmacological inhibitor of the chaperone heat shock protein hsp90 as well as cyclosporine a, an inhibitor of cyclophilins delayed the intoxication of cells with cdt but not with toxins a and b [1] . this result was confirmed by analyzing the adp-ribosylation status of actin from such cells in the presence or absence of the inhibitors. in addition, we excluded that the inhibitors of hsp90 and cyclophilins have any effect on receptor binding, endocytosis or enzyme activity of cdt. the data strongly suggest that the participation of hsp90 and cyclophilin is crucial for translocation of cdta into the cytosol. comparable results were obtained for the related binary iota toxin of c. perfringens. in vitro purified immobilized hsp90 and cyclophilin a specifically bound to the enzyme components of cdt and iota toxins. in conclusion, the results imply a common hsp90/cyclophilin a-dependent translocation mechanism for the family of binary actin-adp-ribosylating toxins. our current investigations focus on the participation of fk506-binding proteins (fkbps), another group of peptidyl-prolyl cis/trans isomerases in the membrane translocation step of these toxins. [1] kaiser, e., kroll, c., ernst, k., schwan, c., popoff, m. r., fischer, g., buchner, j., aktories, k. and barth, h. (2011) . complex multicellular in vitro coculture models represent a promising tool regarding e. g. cellular interactions with nanoparticles, since they more closely mimic the cellular composition of the body. therefore, we used our developed coculture model of the alveolar-capillary barrier composed of lung epithelial cells (nci h441) on top and microvascular endothelial cells (iso-has-1) on the bottom side of a filter-membrane to study nanoparticle cytotoxicity and cellular uptake. with a coculture period of about 10 days the cells achieve a more differentiated and polarized state and develop a tight barrier, which can be measured via ter (transepithelial electrical resistance). regarding cellular uptake of fluorescently labeled amorphous silica nanoparticles (asnps, 30 nm) the coculture took up much less asnps than conventional monocultures. besides, we could not verify a specific uptake mechanism (e. g. clathrin-, caveolae-mediated endocytosis) via immunofluorescence staining of the cells. however, we detected asnps incorporated in flotillin-1 and -2 labelled vesicles. former studies concerning cytotoxicity (lactate dehydrogenase assay) of amorphous silica nanoparticles (asnps, 30 nm) revealed that our coculture behaved much more robustly compared to conventional monocultures. however, regarding inflammatory responses (e. g. sicam, il-8 increase) the coculture responded more sensitively than conventional monocultures. in a further development we added a third cell type, the alveolar macrophage (am), to our coculture. since ams embody the front-line of alveolar defense against inhaled pathogens and particles, they play a central role in regulating lung immunity. as model we applied the human acute monocytic leukemia cell line, thp-1 (prestimulated with 8 nm pma for 4d) apically to the epithelial monolayer of the coculture. our preliminary studies concerning inflammatory responses of the tripleculture (h441/iso-has-1 with thp-1) revealed a higher sensitivity of the triple-culture compared to the double coculture. the triple-culture responded with an increased il-8 release upon lps or tnf-a stimulation. in conclusion, this triple-culture model offers a promising prospect to mimic more closely realistic cell interactions with nanoparticles in the distal lung. ethanol is a component of many herbal fluid preparations [1] , since it is an excellent extraction solvent for the phytochemical components of herbal drugs and contributes to the stability of these medicines. toxicological and pharmacokinetic evaluations [2] have shown that the small amounts of ethanol applied with therapeutic doses are safe even in children. despite that these medicines have been used safely since many decades, they have occasionally been subject of discussion in the public, triggered by the increasing problem of recreational abuse of alcoholic beverages by children and young persons [3, 4] . therefore, there is a growing need of a systematic evaluation of pharmacovigilance data on these medicines. for evaluating the experience gained from the therapeutic use of these medicines, 16 pro-and retrospective studies with 10 herbal medicinal products containing ethanol at doses of 40 to 240 mg per single dose, depending on the age group, have been analyzed, covering 49 816 patients of 0-12 years of age. in these studies, altogether 15 adverse drug effects have been described, none of which was attributable to the ethanol content of the medicines. in a survey of the worldwide use of these and other 6 herbal medicinal products it was shown that during the past few years, more than 764 mio daily doses have been sold, corresponding to more than 33 mio of patients (data obtained from manufacturers; figures available partly from 1993 onwards, partly from 2003/4 onwards). from the packages sold in germany in the years between 2005 and 2009, 48.1 mio were attributable to self-medication, 10.8 mio to prescriptions reimbursed by health insurance (ims, frankfurt). as non prescription medicines are reimbursed in germany only in children, at least the latter part of the prescriptions can be attributed mainly to children. all of these medicines are registered or licensed by regulatory authorities. adverse effects are covered by the pharmacovigilance system, and no adverse effects attributable to the ethanol content have been reported. this set of data supports the conclusion drawn from the experience of a safe use over decades, i.e. that the ethanol content of herbal medicinal products does not give any causes for concern regarding their safety even in children. dedication: this contribution is dedicated to prof. dr. hilke winterhoff, institute for pharmacology and toxicology, university of münster, who died on 9 may 2010. she has initiated this work. prucalopride was introduced as a new selective 5-ht4 receptor agonist that is approved for treating obstipation. whereas one could expect -due to the fact that 5-ht4 receptors are functionally expressed in the human heart -in clinical trials prucalopride did not show cardiac effects. this is quite surprising because other 5-ht4 receptor agonists have been withdrawn from the market just for that reason. in this study we used prucalopride for in vitro studies with atrial preparations from transgenic (tg) mice with cardiac myocyte-specific overexpression of the human 5-ht4a receptor. isolated electrically driven (1 hz) left and spontaneous beating right atria of tg mice were compared with those of wild type (wt) littermates. moreover, we used isolated electrically driven (1 hz) human right atrial preparations from patients undergoing cardiac surgery. finally gr113808, a 5-ht4 receptor antagonist, was added. prucalopride exhibited a dose dependent positive inotropic effect in left atria and a positive chronotropic effect in right atria of tg mice with a logec50 of -6.8 and -7.1, respectively (p<0.05 vs. wt, n=3). in human atrial tissue prucalopride also acts as an agonist, leading to an inotropic effect. all effects could be antagonized by 10 µm gr113808. we could demonstrate that prucalopride acts as an agonist at the 5-ht4 receptor in our transgenic mice model and also in human right atrial preparations. these findings suggest that tachycardia and arrhythmias are possible side effects, which should be carefully looked for. the involvement of psychological factors, especially stress, are known to play an important role in functional gastrointestinal diseases (1, 2) probably by affecting the brain-gut axis. based on the good correlation between stress & functional dyspepsia (fd), many animal models for fd have been developed where animals are subjected to various kinds of psychological stress either during the neonatal period or in adulthood. this stress was found to induce gastric motor dysfunction resembling symptoms of fd. two models for stressinduced fd were performed in order to choose the more adequate one for studying sensitivity changes of the fundus to various mediators as well as changes in some relevant hormone levels in the blood for subsequently testing the efficacy of treatment with stw 5 (a 9 component herbal preparation of proven clinical efficacy in fd and in irritable bowel syndrome (3, 4) ). in one model, maternal separation (5) was performed on weanling rats starting from postnatal day 2 for 3 h each day for 3 weeks. rats were then allowed to mature to an adult age. the other model was that of restraint stress (rs) (6, 7) . adult animals were restrained for 90 min/ day for 1 week. the animals of both models were eventually sacrificed, the stomach fundus was isolated and its sensitivity in vitro to carbachol, potassium chloride, serotonin and adrenaline was tested. blood samples were taken to assess levels of ghrelin, corticosterone releasing factor (crf) and corticosterone. the sensitivity of fundus strips from restrained rats towards the agents tested, partly representing autonomic responsiveness, was more depressed than those from maternally separated ones. levels of ghrelin, crf and corticosterone were also more elevated in the rs model. that model was therefore chosen to test the efficacy of stw 5 in restoring the deranged parameters. a group of animals received stw 5 orally once daily starting treatment 1 week before exposing them to rs and continuing treatment for a further week during subjection to rs. stw 5 was effective in normalizing the depressed stomach fundus responses exhibited by animals subjected to rs and to normalize to a large extent the deranged blood levels of ghrelin, crf and corticosterone. the findings lend further evidence to the role of the brain-gut axis in fd and gives supportive evidence for the first time for the clinical usefulness of stw 5 in this condition. there is good evidence that oxidative damage to dna leads to down-regulation of transcription of affected genes and epigenetic gene silencing in a mechanism dependent on the 8-oxoguanine dna glycosylase (ogg1), which generates harmful repair intermediates [1, 2] . we have recently shown that the magnitude of inhibition of transcription of an egfp reporter gene by single 8-oxo-7,8-dihydro-deoxyguanosine (8-oxodg) varies between the opposing dna strands of the gene [3] . we now have addressed the question, to which extent the transcription inhibitory potential of 8-oxodg depends on its position in the gene and on the dna microsequence surrounding the modified nucleobase. to investigate the effect of position, we produced plasmid vectors containing single 8-oxodg or dg (underlined) in the second position of an agc trinucleotide. measurements of egfp expression in transfected mammalian host cells showed that a single 8-oxodg caused a strong inhibition of gene transcription. the magnitude of this effect for 8-oxodg situated in the transcribed dna strand of the gene was the same as in the non-transcribed dna strand. similarly, there was no quantitative difference between the effects of 8-oxodg present in the 5′-versus 3′-utr of the gene. the results thus indicate that inhibition of transcription by this base modification does not depend on the position in the gene. further comparisons were done between the effects of 8-oxodg nucleobases localised in the same dna strand but within different sequence contexts. gene expression analyses in the repair proficient host cells showed that the degree of inhibition of transcription caused by single 8-oxodg was dependent on the neighbouring nucleotides. among three tested sequence motifs, the minimal effect on the gene transcription was found to correlate with a significantly less efficient base excision by the purified human ogg1 protein. the results thus support the initiatory role of ogg1 in the mechanism of transcriptional repression. in addition, the finding of the effect of dna sequence on the base excision activity of ogg1 suggests that repair rates of single base modifications in genome could also be heterogeneous. allgayer j., kitsera n., epe b., khobta a. johannes gutenberg university of mainz institute of pharmacy and biochemistry, staudingerweg 5, 55128 mainz, germany interference of dna base modifications with gene transcription is an important biological consequence of genotoxic damage [1] . an efficient method for incorporation of a single 8-oxo-7,8-dihydroguanine (8-oxog) at a defined position in the egfp gene in a plasmid vector was recently developed in our lab. the method relies on the availability of adjacent sites for a sequence-specific nicking endonuclease, which allow the insertion of a synthetic oligonucleotide containing 8-oxog in a chosen position. we further showed that this single lesion inhibits transcription after excision by ogg1 [2] . in order to determine to which extend the observed effect depends on the position of the modified base in the gene, we constructed several new plasmid vectors which allow incorporation of the same dna oligonucleotide containing 8-oxog or g in different positions in different dna-strands. dna sequence cassettes were designed to contain a 5′-cattgcttcgctagcacg nucleotide sequence in different orientations, which was flanked by two unidirectional bsrdi recognition sites (5′-gcaatgnn). adapters containing the restriction sites for directional cloning into the 5′-or the 3′-utr of the plasmid-borne egfp gene were added. the produced plasmid vectors thus allow the insertion of a single 8-oxog in the same sequence context into opposing dna strands in the 5'utr and in the 3'utr. keratinocytes are the major cell type of epidermis and are responsible for the formation of an outer barrier, the statum corneum, to protect an organism against harmful environmental influences. for generation of this barrier, keratinocytes pass through a complex differentiation program that is accompanied by synthesis of lipids, like cholesterol and ceramides. finally, the differentiation of keratinocytes leads to apoptosis. another function of keratinocytes is to sense environmental factors, some of which are decoded by members of the transient receptor potential (trp) ion channel family. trpv3, for example, is predominantly expressed in keratinocytes, and decodes different chemical and physical stimuli like the terpenoid-derived ligands camphor and thymol or temperatures above 33°c. less is known about the influence of cholesterol on trpv3 signalling. we modified the cholesterol content of hek293 cells stably transfected with trpv3 and performed flipr-based calcium measurements. these experiments revealed that cholesterol enrichment robustly potentiates trpv3 by sensitizing it to lower agonist concentrations. we verified these results with whole-cell patch-clamp measurements. in contrast, trpv2, another heat-sensing channel, was not affected by cholesterol modification. since former studies showed a defective formation of epidermal barrier in trpv3 -/mice, our results imply that a cholesterol-regulated trpv3 signalling may contribute to the progression of differentiation or initiation of apoptosis of keratinocytes. ischemia-reperfusion injury causes severe problems in the early period after lung transplantation. since transient receptor potential (trp) channels are important regulators of vascular permeability and tone, we investigated the influence of a trpc6 blocker on pulmonary function after simulated transplantation, using an ex vivo model of isolated perfused and ventilated rabbit lungs. to this end, heart-lung blocks were excised and mounted in an artificial thorax chamber. negative pleural pressure ventilation was initiated, and lungs were perfused with albumin-containing tyrode-solution (100 ml min -1 ). after equilibration in a stable perfusion and ventilation mode for 30 min, lungs were flush-perfused with perfadex ® solution, followed by an ischemic storage for 4 h on ice. subsequently, ventilation and perfusion were re-initiated to simulate a transplantation situation. in the oxygenated group, pulmonary artery po2 was adjusted to 120 mmhg, in the deoxygenated group, the perfusate inflow was gassed with nitrogen to achieve a pulmonary artery po2 of 50 mmhg. both transplantation conditions were conducted in the absence or in the presence of the trpc6 blocker larixol acetate (5 µm). hemodynamic and ventilatory parameters were continuously monitored. the weight of deoxygenated lungs steadily increased during 2 h of reperfusion from 22.1 ± 1.32 g to 35.4 ± 4.23 g. this weight gain was inhibited by supplementation of the trpc6 blocker (from 21.4 ± 0.91 g to 27.4 ± 2.42 g 2 h after reperfusion). in contrast, oxygenated lungs showed no marked weight gain after reestablishment of perfusion (from 17.5 ± 1.51 g to 21.5 ± 2.29 g 2 h after reperfusion), and the trpc6 blocker had no additional effect (initial 19.5 ± 1.28 g, 2 h reperfusion 21.3 ± 1.22 g). we conclude that a trpc6-blocking compound to the lung perfusate during simulated transplantation counteracts the endothelial permeability increase and the resulting post transplant weight gain. the results indicate a role for trpc6 in the regulation of pulmonary vessel permeability and support the concept that perfusion of donor lungs with trpc6 blockers may prevent edema formation caused by ischemiareperfusion injury shortly after lung transplantation. regulation of human inducible nitric oxide synthase (inos) expression by noncoding rnas (ncrnas) kleinert h., schmitz k., koch k., hahn s., pautz a. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher str. 67, 55101 mainz, germany the transcriptome analyses of human cells showed that additionally to the 30.000 protein coding sequences the human dna codes for much more (450.000 ?) non-coding rnas 1 . beside the ribosomal, and transfer rnas (rrna and trna) involved in the mechanism of translation there are also short (snrna, sorna, mirna) and long noncoding rnas (ncrnas) implicated in regulation of gene expression (splicing, translation, chromatin packaging etc.). matsui et al. described regulation of il-1β-induced inos expression by an antisense rna (as-3-utr-rna) in rat hepatocytes 2 . a promoter located on the antisense strand 3' to the last exon (exon 27) of the rat inos gene drives the expression of an as-rna complementary to the 3'-utr of the rat inos mrna. using different sense primers with homology to the 3'-utr sequences of the human inos gene for specific rt-pcr reactions (detecting only an as-rna) we were not able to detect such an as-3-utr-rna in human cells. in addition, transient transfection analyses using constructs containing a 2.7 kb fragment of the 3'-flanking genomic sequences (as used by matsui et al. in the rat system) of the human inos gene in front of a luciferase reportergene into dld-1 cells revealed no promoter activity of these sequences. korneev et al. described the expression of a 2 kb anti-inos-ncrna in different brain tumors and showed reciprocal expression to the inos mrna in human embryonic stem cells 3 . analyzing the expression of this anti-inos-ncrna in cytokine-treated dld-1 cells also showed a basal expression of this as-rna and an enhancement of the expression by cytokine-treatment. downregulation of the anti-inos-ncrna by sirnas reduced whereas overexpression enhanced cm-induced inos expression in human dld-1 cells. this indicates that this anti-inos-ncrna regulates cytokine-induced inos expression in a positive manner. rna 14, 2030 rna 14, -2037 rna 14, (2008 . using directed evolution to improve functional expression of class b g-protein coupled receptors klenk c., scott d. j., plückthun a. universität zürich institut für biochemie, winterthurerstrasse 190, 8057 zürich, switzerland the class b of g-protein coupled receptors (gpcrs) comprises 15 peptide hormonebinding receptors which regulate important endocrine and neuroendocrine functions of the human body. several of these receptors are implicated in the pathogenesis of severe diseases such as diabetes, osteoporosis, growth disorders and depression, which makes them attractive targets for drug therapy. to develop new compounds targeting these receptors a detailed understanding of the molecular structure is required which has not been succeeded to date. structural studies of proteins by x-ray crystallography or nmr spectroscopy generally require large and homogenous quantities of protein. for gpcrs this prerequisite is difficult to achieve as the vast majority of gpcrs exhibits low endogenous expression and is very unstable in solution. therefore, improved expression conditions are necessary for the efficient characterization of new gpcr structures. here we present a method to optimize class b gpcrs for improved heterologous expression and increased thermostability by means of directed evolution. libraries of class b gpcrs were obtained by random mutagenesis and were expressed in a heterologous expression system in which functional gpcr is targeted to the inner membrane of e. coli. mutants that display increased receptor expression levels and ligand binding were selected by flow cytometry using fluorescently labeled ligands. repetitive cycles of randomization and selection allow to gradually increasing the level of protein expression and stability. with this evolutionary approach key residues within the receptor sequence can be identified rapidly that are responsible for improved biophysical properties without affecting the pharmacological features of the receptor. such gpcr mutants will become a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies. pasireotide (som230) is currently under clinical evaluation as a successor compound to octreotide for the treatment of acromegaly, cushing's disease and carcinoid tumors. whereas octreotide acts primarily via the sst2 somatostatin receptor, pasireotide was designed to exhibit octreotide-like sst2 activity combined with enhanced binding to other somatostatin receptor subtypes. somatostatin and octreotide stimulate the complete phosphorylation of at least six carboxyl-terminal serine and threonine residues namely s341, s343, t353, t354, t356 and t359, which in turn leads to a robust endocytosis of the sst2 receptor. surprisingly, pasireotide failed to phosphorylate the four threonine residues and induced only a detectable phosphorylation of s341 and s343. somatoprim and ke108 are recently developed somatostatin analogs capable of binding to four of five somatostatin receptor subtypes. here, we performed an in vitro study comparing the effects of pasireotide, somatoprim and ke108 on sst2 somatostatin receptor binding, phosphorylation, internalization and signaling. further somatostatin, octreotide, pasireotide, somatoprim and ke108 were tested for functional selectivity at sst5 receptor mutants, which possess a carboxyl-terminal sst2tail. this approach allows detection of receptor activation by phospho-specific sst2 antibodies. compared to octreotide, somatoprim activates sst2 but has a higher activity on sst5. ke108 and pasireotide are partial agonists at the sst2 receptor. pasireotide, ke108 and somatoprim show comparable effects on sst5 receptor. however, none of these new pan-somatostatin analogs behaves like natural somatostatin on the sst5 receptor. cadmium modulates ahr-associated gene expression in the rat intestine after oral exposure kluxen f. m. 1, 2 , höfer n. cadmium has been shown to mimic steroid estrogen effects in vivo and in vitro. we have recently identified cross-talk of estrogen receptor (er) and aryl hydrocarbon receptor (ahr) in the rat uterus where 17beta-estradiol (e2) and cdcl2 modulate ahrassociated genes via er after i.p. injection in a similar fashion (kluxen et al., arch toxicol doi 10 .1007/s00204-011-0787-x). however, the predominant route of exposure to cadmium in non-smokers is via diet. moreover, uterus expresses mainly the receptor subtype eralpha, whilst small intestine and colon express mainly erbeta. thus, we now investigated by real-time rt-pcr the effects of cadmium (2mg/kg b.wt cdcl2 (cd2)) or steroid estrogen (0.1 mg/kg b.wt. 17alpha-ethinylestradiol (ee2)) on ahrassociated gene expression (i.e., ahr, cyp1a1, gsta2, nqo1) in the small intestine of rats after oral exposure (3 days gavage). the animals were also co-treated with cd2 and pure anti-estrogen (2.5 mg/kg b.wt zk191703 (zk)) or ee2, to asseess whether ermediated processes are involved. we also measured cyp1a1 mrna expression in two estrogen receptor negative colon cancer cell lines (ht-29 and caco2) treated for 5 days with cdcl2 (1µm) and e2 (0.01µm). the dose-dependency of cadmium induced ahr target gene modulation was studied in a second animal experiment, with administration of cadmium in drinking water for 28 days (0.4-9 mg/kg b.wt cdcl2 equivalent to 5, 50, and 150 ppm) and ee2 (0.08 mg/kg b.wt) as steroid reference. in summary we present two major results: the metalloestrogen cadmium modulates dose-dependently the ahr-associated gene expression in the intestine after oral exposure. yet, since the cadmium induced modulation of ahr target genes was not antagonized by anti-estrogen in the small intestine in vivo and was also found to occur in er-negative colon cells in vitro, we propose that er-independent mechanisms might play a role in this effect. meg) is the most cytotoxic lesion. if not repaired by o 6 -methylguanine-dna methyltransferase (mgmt), o 6 meg/t mismatch is recognized by the mismatch repair system (mmr) that performs futile repair cycles. during this process secondary lesions (i.e. dna single-strand brakes) are formed, which block dna replication in the next replication cycle, leading to dna double-strand breaks (dsbs). these dsbs eventually signal to apoptosis and other genotoxic endpoints. here, we wished to address the question whether autophagy is part of the cellular response triggered by o 6 meg. we also assessed whether autophagy influences apoptosis induced by tmz in glioma cells. we show that tmz induces autophagy in u-87 mg and ln-229 glioma cell lines. the maximum amount of autophagy was observed several days (96 h) after tmz treatment and mgmt proficient cells did not display significant autophagy. thus, the data show that mgmt protects against tmz induced autophagy, pointing to o 6 meg as the critical lesion responsible for induction of autophagy. using colon cancer cell lines proficient and deficient in mmr, we show that mmr is required for tmz-induced autophagy. because dsbs, which emerge during the processing of o 6 meg, are repaired preferably by homologous recombination (hr) ln-229 cells stably down-regulated for hr were tested for autophagy induction. the data indicate that dsbs are involved in tmz-induced autophagy. because autophagy following tmz treatment occurs earlier than apoptosis we hypothesize that autophagy protects glioma cells against apoptosis. using an early stage autophagy inhibitor 3-methyladenin we have shown, that autophagy inhibition sensitized glioma cells to tmz-induced apoptosis. taken together our data point out that tmz induces autophagy in glioma cells and that autophagy protects glioma cells against tmz-induced apoptosis. o 6 meg is the lesion responsible for autophagy induction. furthermore, the data also shows that mmr and dsbs are involved in the induction of autophagy after tmz treatment. work was supported by bmbf (02nuk016). retinitis pigmentosa (rp) is a severe human retinal disease characterized by a progressive degeneration of rod photoreceptors and a secondary loss of cone function. in most cases, rp finally leads to legal blindness. mutations in the regulatory subunit of the rod cyclic nucleotide-gated (cng) channel (cngb1a) have been found in patients suffering from rp. we used cngb1-deficient (cngb1 -/-) mice to establish a gene replacement therapy as a potential treatment for rp by means of recombinant adenoassociated viral (raav) vectors. the packaging limitations of raav vectors required a capacity-optimized vector of the large cngb1a cdna (approx. 4 kb). therefore, we replaced regulatory elements within the expression cassette and used a short mouse rhodopsin promoter element for rod-specific expression. after injection of therapeutic raavs (serotype 8) into the subretinal space of 2-week-old cngb1 -/mice, we assessed the restoration of vision by analyzing i) protein expression and localization, ii) retinal function and morphology and iii) vision guided behavior. we found that treated cngb1 -/mice expressed full-length cngb1a and cnga1, which were previously downregulated. both proteins co-localized in rod outer segments and formed regular cng channel complexes in the treated area of the cngb1 -/retina. using electroretinography (erg) we observed a distinct rescue of rod-driven responses. moreover, cngb1 replacement significantly preserved outer segment morphology and delayed retinal degeneration. finally, treated cngb1 -/mice performed significantly better than untreated mice in a modified water maze task designed to test for rod-mediated, vision-guided behavior. in summary, this work provides a proof-of-concept for the treatment of rod channelopathyassociated retinitis pigmentosa by raav-mediated gene replacement. most endocrine disruptors interact with hormone receptors or steroid biosynthesis and metabolism, thereby modifying the physiological function of endogenous hormones. here, we present an alternative testing paradigm for detection of endocrine modes of actions that replace and reduce animal testing through refinement. receptor mediated endocrine effects were assessed using the yeast based receptor mediated transcriptional activation yes/yas assays and effects on steroid hormone biosynthesis were assessed using the human cell line h295r in the steroidogenesis assay. in our testing paradigm we propose to complement the in vitro assays with a single in vivo repeated dose study in which plasma samples are analyzed for their metabolome profile. the combination of these methods does not only contribute to refinement and reduction of animal testing, but also has significantly increased the efficient allocation of resources and allows for a sound assessment of the endocrine disruption potential of compounds. thus, this proposal constitutes a potentially attractive alternative to epa's endocrine disruptor screening program. data on 14 reference substances for which the in vitro yes/yas and steroidogenesis assays and the in vivo metabolome analysis were performed to assess their putative endocrine mode of action is presented here. the bovine corneal opacity and permeability (bcop) test has been adopted by oecd for the identification of corrosive and severe ocular irritants (ghs category 1) for single component substances and multi-component formulations. eye irritation tests (eit) using human reconstructed tissue models (such as epiocular) have been described to predict ocular non-irritants (ghs no category). thus the ultimate repaltement of the draize rabbit eye irritation test (oecd tg 405) by a combined or tiered testing strategy could be possible. the purpose of this study was to evaluate whether the bcop with additional corneal histology together with the eit could be used to predict eye irritancy of agrochemical formulations according to different classification schemes including un ghs and epa systems. we have performed the bcop (plus histology) and the eit of 50 agrochemical formulations for which in vivo eye irritation data were already available (for registration purposes). using the oecd tg guideline evaluation scheme for opacity and permeability in the bcop was not predictive for the agrochemical formulations assessed here, while corneal histology grades and the epiocular tissue viabilities were useful predictors of eye irritancy potencies and could be applied for the different classification schemes. the nanomaterials offers extraordinary opportunities. the nano-structure can change the physical and chemical properties, and often also alters the biological effects. hence the toxicity of a nanomaterial can differ from its larger-scale material; but as of today, no new quality of a general nano-specific toxic effect has been observed. therefore the established testing methods are generally suitable. it is, however, difficult to apply the nanomaterials to in vitro test systems, since it is the nature of these materials to change their surface properties and agglomeration stateand the uptake and distribution in the body may differ from their larger-scale materials. while the methods for topical effects may be used for nanomaterials without further modification, the in vitro methods for genotoxicity testing require the dispersion in culture media. the use of reproducible and well-documented dispersion protocols andthe characterization of its particle size distribution is de rigueur . [1] . for many nanomaterials published genotoxicity studies did not give a consistent picture [2] and therefore there are rather effects of individual nanomaterials than nano-genotoxicity per se. modern toxicology is based on the insight into toxic pathways. for nanomaterials a testing strategy will include testing for their primary effects (which might be only a handful: particle effects, catalyzing the formation of reactive molecules and ion release) and their uptake, distribution and clearance. the use of dermal penetration studies in vitro for the risk assessment of sunscreen nanomaterials has been demonstrated [3] . in vitro methods for specific effects (such as inflammation, pulmonary toxicity, sensitization) are currently awaiting validation (for both chemicals/molecules as well as nanomaterials). in the meantime alternative short-term in vivo studies with optimized biological readouts can deliver information on the toxic pathways as well as the biokinetics and dose-response relation of nanomaterials in the body. a short-term inhalation test for nanomaterials has already been used successfully [4] . testing strategies based on those methods engage less animals and provides more significant data than classical testing. moreover data from these methods will serve as a benchmark and a validation for the in vitro models under evaluation. nanoparticles (np) are increasingly used in various field of industry which necessitates evaluation of their safety. also in the food industry, nps have gained strong interest, for example as food additives or to improve food packing. however, the potential risks of ingested np have been rarely investigated. inhalation studies have revealed that inflammation and oxidative stress may represent unifying mechanism for the induction of adverse health effects of toxic np. in the present study, a co-incubation model of human polymorphonuclear neutrohils (pmns) and caco-2 human intestinal epithelial cells was used as a model to address potential genotoxic effect of np during intestinal inflammation. oxidative dna damage induction (measured by the fpg-modified comet assay) was induced in the caco-2 cells by activated pmn and this effect increased with increasing pmn to caco-2 cell ratio. the crucial involvement of the phagocyte nadph oxidase complex could be demonstrated using treatment of caco-2 cells with bone marrow-derived pmn from nadph oxidase deficient mice. dna damage by pmn as well as h 2o2 was increased in buthionine sulphoximine (bso) pre-treated caco-2 cells, illustrating the importance of the cellular glutathione (gsh) status in these target cells. gsh depletion in caco-2 cells could also be shown upon treatment with various types of np. our data suggests that ingested np may increase the susceptibility of the colon mucosa to genetic damage during the occurrence of intestinal inflammation. the ingestion of seafood contaminated by acute toxic doses of the marine toxin okadaic acid (oa) is responsible for diarrhetic shellfish poisoning. it is recently known that both the rat and the human hepatic cytochrome p450 monooxygenases (cyp) are able to metabolize this toxin. currently, there is a lack of data about the toxicity and mode of action of oa after xenobiotic metabolism. the aim of our study was the measurement of the toxicity and oxidative stress status in hepg2 cells incubated with oa in the absence and presence of s9 mix. pure oa, as well as oa pre-activated with liver homogenisates (s9 mix) were used to treat human hepg2 cells that have nearly undetectable levels of functional cyp but express phase ii enzymes. the experiments were performed with both human and rat s9 fraction plus cofactors of phase i enzymes. the cell viability was measured after 4 h using mtt-test and xcelligence real time cell monitoring system. furthermore, levels of intracellular reactive oxygen species (ros) were detected by 2´,7´-dichlorofluorescein diacetate and additionally by measuring the intracellular glutathione content. in the presence of both human and rat s9 mix oa showed a higher toxicity than the parental substance. oa pre-incubated in rat s9 mix was toxic at 75 nm oa. strong effects could be observed when oa was pre-activated with human s9-mix at a concentration of 50 nm oa. pure oa was non-toxic in that concentration range. we could also detect an increase of oxidative stress in hepg2 cells treated with oa in the presence of all investigated s9-mix. these results suggest that oa is activated after oxidative xenobiotic metabolism into metabolites which possess a higher cytotoxic activity and increase the amount of intracellular ros in hepg2 cells. ballast water treatment -emerging health risks werschkun b., banerji s., krätke r. bundesinstitut für risikobewertung, max-dohrn-strasse 10, 10589 berlin, germany the introduction of invasive marine species into new environments by ships' ballast water, via ships' hulls and other vectors has severe impacts on the oceans. in 2004 the international maritime organisation (imo) launched the international convention for the control and management of ships ballast water and sediments which requires ballast water to be treated in order to eliminate alien aquatic species. ballast water treatment may include mechanical, physical or chemical measures. any ballast water management system using active substances needs imo approval. therefore identification of active substances, relevant chemicals and submission of specified datasets on their physical, chemical and toxicological properties is required in order to assess the safety for the aquatic environment and for human health. the bfr is the german federal agency responsible for health risk assessment and has been involved in more than twenty assessment and approval processes so far. the majority of imo approved systems are based on oxidative principles such as chlorination and ozonation. these methods can generate disinfection by-products (dbps), which are a mixed group mostly of halogenated organic substances like trihalomethanes, haloacetic acids and haloacetonitriles. the formation of dbps is well known from the disinfection of drinking water. some dbps are regulated under drinking water directives because of their long-term toxicity but many are unregulated and have unknown toxicological properties. the formation of dbps may vary significantly depending on the treatment system as well as on environmental parameters like temperature, ph and composition of the organic matter within the aquatic environment. in sea water, sources for dbp formation besides ballast water treatment are aquaculture and the cooling systems of coastal power plants. in order to address possible health and environmental risks from dbps formed during ballast water treatment a conference on emerging risks from ballast water treatment was held at bfr in october 2011. here we summarise the main conference findings and identify areas for future research. two presentations corroborated that significant amounts of dbps can be formed in sea water and a presentation on the toxicological properties of dbps pointed out that many have genotoxic properties. accordingly, the determination of dbp species and generated concentrations under different ballast water treatment conditions was seen as a mayor task. different approaches for health and environmental risk assessments were also discussed. appropriate human exposure scenarios and methods for exposure assessment, taking into account common approaches used in risk assessment were presented during the conference. a suitable approach based on derived pec-values for exposure quantification was proposed in order to improve the procedure available for risk assessment of chemical agents used for ballast water treatment. agonist-selective signaling of µ-opioid receptors in t lymphocytes kraus j., börner c., lanciotti s., koch t., höllt v. inst. für pharmakologie und toxikologie, leipzigerstr. 44, 39120 magdeburg, germany opioids are the most potent analgesics and irreplaceable for the treatment of severe pain. in addition to their central effects, opioids modulate a great variety of immune effector cell functions, which may result in unwanted side effects during opioid treatment. the effects of most of the commonly used opioids are mediated by µ-opioid receptors, which belong to the superfamily of g protein coupled receptors. recent data support the concept that g protein coupled receptors function as dynamic entities, which may occupy multiple conformations and activate multiple signaling pathways in a ligand-dependent manner. consequently, different ligands activating the same receptor may have different cellular effects, which has been termed "agonistselective signaling". little is known about agonist-selective signaling of µ-opioid receptors in immune effector cells. in a first attempt to understand if and why such different profiles among different opioids occur we investigated effects of different opioids in human jurkat t cells. we report that the µ-opioid receptor ligands fentanyl, methadone, loperamide and betaendorphin induce internalization of a µ-opioid receptor-green fluorescent reporter construct, whereas morphine and buprenorphine did not induce internalization. the internalization was dependent on p38 mapk and phospholipase d2. in line with this, we observed marked phosphorylation of p38 mapk and activation of phospholipase d2 induced by the internalizing opioids, but no or little such activity by morphine and buprenorphine. as a physiological result, fentanyl, methadone, loperamide and betaendorphin treatment of primary human t cells and jurkat t cells resulted in a strong, up to 100 fold induction of il-4, which was dependent on p38. in contrast, morphine and buprenorphine only showed a weak, approximately one order of magnitude lower induction of il-4. by inducing il-4 opioids significantly modulate the t helper cell balance into the type 2 direction, which influences various immune responses, e. g. the antiviral, t helper cell type 1-mediated response. considering the vital necessity of opioid use in humans, it is an intriguing goal to identify analgetically feasible opioids that have little or no immunosuppressive or -modulatory effects. modulation of cgmp signals by phosphodiesterases in smooth muscle cells krawutschke c., koesling d., russwurm m. ruhr-universität bochum pharmakologie und toxikologie, universitätsstrasse 150, 44780 bochum, germany within the cardiovascular system, cgmp mediates smooth muscle relaxation and inhibition of platelet aggregation. cgmp is formed by particulate guanylyl cyclases and nitric oxide-sensitive guanylyl cyclases, that are activated by natriuretic peptides or nitric oxide (no), respectively. besides the cgmp-forming enzymes, the cgmp-degrading phosphodiesterases strongly determine amplitude and shape of cgmp signals. in vascular smooth muscle cells, three phosphodiesterases are considered to be responsible for cgmp degradation: pde5, the cgmp-specific phosphodiesterase is activated directly by cgmp binding to its gaf domains; this activation if further stabilized by cgmp-dependent protein kinase-mediated phosphorylation. pde1, the ca2+/calmodulin-stimulated pde constitutes the majority of cgmp-hydrolyzing activity in smooth muscle cells at least in the presence of high intracellular ca2+ concentrations. and lastly pde3, the cgmp-inhibited pde displays some cgmp-degrading activity, although cgmp binding to its catalytic domain is primarily thought to inhibit the campdegrading activity of pde3. cgmp signals measurable in radioimmunoassays require stimulation with cgmpincreasing vasodilator concentrations that are orders of magnitudes higher than those required for relaxation. thus, we developed fluorescent sensors for real-time measurement of cgmp signals in single cells. by using these indicators, we analyzed the contribution of different cyclic nucleotide-degrading phosphodiesterases to the modulation of cgmp signals elicited by physiologically relevant vasodilator concentrations. hyaluronan (ha) is a major component of extracellular matrices and is thought to control cellular phenotypes such as proliferation and migration. therefore, ha synthesis may play an important role in the pathophysiology of atherosclerosis. there are three major ha-synthase isoenzymes (has1-3). the has3 gene is alternatively spliced. has3 transcript variant 2 (has3v2) encodes the smallest has isoenzyme which has a different c-terminus and contains only two transmembrane domains compared to has3 transcript variant 1. the aim of the present study was to investigate whether has3v2 is expressed by vascular cells, how it is regulated and where it is localized in cells and whether it indeed synthesizes ha. has3v2 mrna expression was monitored by quantitative real-time rt-pcr. protein expression was determined by western blotting using a polyclonal antibody that was raised in rabbit. an n-terminal eyfp-has3v2 fusion protein and a ddk-tagged has3v2 construct were expressed for subcellular localization studies and co-immunoprecipitation (co-ip). endogenous has3v2 mrna was expressed in both vascular smooth muscle cells (vsmc) and endothelial cells. furthermore, western blotting revealed has3v2 protein expression in vsmc and platelets. in vsmc has3v2 mrna expression was strongly up-regulated in response to interleukin-1β (il-1β, 10 ng/ml) whereas stimulation with interleukin-10 (10 ng/ml), platelet-derived growth factor-bb (10 ng/ml), transforming growth factor β (10 ng/ml), tumor necrosis factor α (10 ng/ml) and interferon-γ (10 ng/ml) had no effect. transfection of hek cells with eyfp-has3v2 fusion protein revealed localization to the endoplasmic reticulum but not to the plasma membrane. furthermore, co-ip experiments showed that tagged has3v2 proteins were precipitated together suggesting formation of multimeric has3v2 complexes. transfection of has3v2 did not cause increased secretion of ha into the cell culture supernatant in hek cells. in conclusion, has3v2 is present in vascular cells and responds to inflammatory cytokines such as il-1ß. because of the intracellular localization and the lack of ha secretion in has3v2 transfected cells, has3v2 may serve intracellular functions apart from ha synthesis. the tubulin antagonist pretubulysin shows strong vascular-disrupting properties in vitro several epidemiological studies indicate a correlation of human exposure to ultrafine particulate air pollution caused by incomplete combustion processes and an increase in the incidence of pulmonary immune diseases like asthma. as a possible mechanism behind this pathological phenomenon, the adjuvant effect of lung inflammation induced by poorly soluble environmental particles has been hypothesised. the aim of our study was to investigate the causal link between carbon nanoparticle-induced lung inflammation and modulations of immune cell populations during processes leading to sensitization, and allergic immune responses of the airways. therefore mice were treated with ovalbumin (ova) alone or in combination with carbon nanoparticles (cnp) by pharyngeal aspiration. the induction of inflammation and the immune adjuvant activity were studied in the lungs and lung draining peribronchial lymph nodes (pbln) at the level of sensitization, and at the level of the immune response. ova-specific ige antibodies were measured in blood serum, and the development of allergic airway inflammation was studied after ova challenge. results at the level of sensitization showed that cnp-induced immediate airway inflammation had immune adjuvant activity resulting in an increase of specific cell populations in pbln and in a stimulation of asthma-specific th2 cytokines. a specific reduction of the neutrophilic lung inflammation by application of the compatible solute ectoine significantly reduced the adjuvant effects of cnp. in ova-sensitized mice, application of cnp 12 hours prior to allergen challenge, led to a significant increase in inflammatory cell infiltrate and respective cytokines in broncho-alveolar lavages. coapplication of 1 mm ectoine together with cnp reduced the particle-induced effects. our data show a link between neutrophilic lung inflammation and adjuvant effects of cnp. a specific reduction of neutrophils by the application of ectoine attenuated this np induced adjuvant effect, indicating that particle-induced lung inflammation rather than the direct interaction of nanoparticles with immune cells is the critical step in environmentally modulated pulmonary immune diseases like asthma. introduction: drug-eluting stents (des) are commonly used in the treatment of acute artery occlusion. however, even if released cytotoxic drugs reduced neointimal proliferation significantly there is still the risk of in-stent thrombosis. it is presumed that this is due to reduced reendothelialization. it has been suggested that coating the stent with biomolecules may provide a new approach to circumvent the lack of healing of the endothelial layer. one approach would be the use of biomolecular signals, such as (poly)peptides and growth factors. rgd and redv are peptide motifs, known to enhance cell attachment and spreading. aim: the aim of our study was to evaluate the efficacy of proteins, derived from elastin-like proteins (elp) and artificial modified by incorporating with the amino acid motifs rgd,redv and p15, in terms of endothelial healing on stents and other cardiovascular devices. results: in this work, we generated vectors encoding for different biopolymers consisting of various bioactive signal molecule sequences. the peptides, e.g. based on the elastinlike matrix (vpgig)2-vpgkg-(vpgig)2, were synthesized using heterologous expression. after optimizing culture conditions and extraction procedures their biological activity was assessed using human umbilical vein endothelial cells (huvec). elastin like proteins with differently incorporated bioactive signals (redv, rgd and a small p15 peptide) were linked covalently via carbodiimide coupling to poly(l-lactide) (plla) films. huvec growth was determined on these modified surfaces using the brdu assay (cell proliferation) and resazurin assay (cell viability). the chemically modified plla surfaces conferred higher cell viability after 1 h adhesion (60%) and an enhanced proliferation (63%, 1 h adhesion, 24 h cultivation) in comparison to the unmodified plla. these results indicate that the synthesized elp incorporated with amino acid motifs promote an accelerated endothelialization of the biodegradable stent material plla. discussion: in summary, we were able to generate elastin-like proteins modified by bioactive sequences. those sequences enhanced endothelial cell proliferation and adhesion. further studies are warranted to determine the activity on smooth muscle cells of these peptides. (1) the failing heart is characterized by excessive extracellular matrix production by myofibroblasts (myocfs) causing fibrosis and myocardial stiffening. myocfs represent phenotypically transformed cardiac fibroblasts (cfs) and are characterized by the expression of contractile proteins like a-smooth muscle actin (α-sma) and enhanced secretion of growth factors (e. g. ctgf). identification of intracellular enzymes that modulate this transformation process is desired to therapeutically modulate pro-fibrotic progression in heart failure. we show that pde2a, a phosphodiesterase isoform, that is able to hydrolyse cgmp and camp, is markedly upregulated in failing hearts from patients with end-stage heart failure (2-3-fold, p<0.05, n=8). notably, pde2a protein abundance is 4-fold higher in myocfs compared to cardiomyocytes from neonatal rat hearts (p<0.05, n=7). to this end we tested whether pde2a modulates the transformation of cfs isolated from neonatal rat hearts to myocfs. indeed, as assessed by immunoblotting and fluorescent microscopy (α-sma, phalloidin, dapi), adenoviral pde2a overexpression induced α-sma expression (3.2-fold p<0.05, n≥8) and to a lower extent ctgf synthesis (1.5-fold, p<0.05, n≥5). mechanistically, pde2a showed preferential subsarcolemmal localisation with diminished total cgmp levels (-56%, p<0.05, n≥5). consistently, parallel stimulation with atrial natriuretic peptide (anp), a selective activator of membrane-bound guanylyl cyclase, normalized ctgf synthesis indicating that pde2a controls cgmp in a discrete subdomain near the plasma membrane. moreover pde2a overexpression diminished the protein levels of vasodilator-stimulated phosphoprotein, a membrane cytoskeletal component (-60%, p<0.05, n≥5). these data implicate pde2a-dependent subsarcolemmal cgmp regulation in myofibroblast formation and potentially cardiac fibrosis. therefore, targeting pde2a may lead to regression of the fibrotic remodeling associated with heart failure. several anorganic nanoparticles (np) causedhigher inhalation toxicity than the corresponding coarse particles (oberdoerster et al. 2005) . we examined an organic pigment and a polymer dispersion each as nanomaterial and as the chemical identical coarse material in short-term inhalation studies in malerats. the polymer was an anionic acrylic ester copolymer containing free carboxylic groups. three different particle sizes were synthesized by varying polymerization conditions: 12, 80 or 250 nm. although polymeric acrylic ester was reported to be irritating to the respiratory tract at 3 mg/m3, all three tested polymers -including the np (12 and 80 nm) -did not cause any changes in lavage fluid and in histopathology at 10 mg/m3. the organic pigment was a poorly soluble pyrrol with an intense orange color. the np (10 to 50 nm width and 30 to 400 nm length) and the coarse pigments (70 to 200 nm width and 0.3 to 3 µm length) are both needle-like. they were tested at 1, 3, 10 and 30 mg/m3 for the np and 3, 10 and 30 mg/m3 for the coarse materials. mild and partly reversible morphological changes were observed in lung and lymph nodes at the highest concentrations, but the more pronounced effect were found in rats exposed to the coarse material. likewise there was an increase of lavage parameters in rats exposed to thecoarse material but not to the np. these data demonstrate that inhalation of finer np is not necessarily associated with higher toxicity compared to the coarse material. the results were obtained with two organic particles of rather different size and composition but are in contrast to the more severe effects seen with several anorganic np when compared to the corresponding coarse particles. within the nanocare project a standard short-term inhalation test to examine the toxicity of inhaled aerosols from nanomaterials has been developed. the inhalation toxicity of nano-andpigmentary materials was studied: baso4, zno, ceo2, al-doped ceo2, zro2, amorphous silica, surface-coated amorphous silica, titania, carbon black and three multi-wall carbon nano tubes, all with complete phys-chem-characterization as planned for the oecd sponsorship program. quartz dust tio2 and zno were tested as pigmentary materials. rats were exposed nose-only to three concentrations of one of these materials, 6 h a day for five consecutive days. positive controls were exposed to quartz dust or pigmentary zno, negative controls to clean air. a wide range of endpoints for pulmonary toxicity were evaluated immediately after the last exposure and after 3 days and 3 weeks after the last exposure. among these parameters, polymorphnuclear granulocyte count in bronchoalveolar lavage fluid is the most sensitive early parameter indicating inflammation process in lung, while histological examination reveals the type and localization of inflammation. among these substances, we identified baso4 as having the lowest toxicity. all mwcnts were most potent in producing progressive inflammation in the lung; granulomas in lung and lung associated lymph nodes were observed without indication for fibrosis. the noaecs of the 16 substances ranged between < 0.1 and >50 mg/m 3 . generally the material was only found in the lung (surface and macrophages) and in the draining lymph nodes. surface modified amorphous silica was also found in the spleen. the data demonstrate that the method is able to differentiate the toxic potential of different nanomaterials and to indicate regression or progression of the effects effects. moreover the lung burden and potential translocation to other tissues was detecable. comparing the material properties and effects of the 16 materials, no general relationship between the toxicity and either particle size, specific surface area or aerosol particle number concentration was found. hence we must not expect to find a gerneral "nanotoxicology" or a unifying dosimetry for all nanomaterials. we must rather be prepared to test individual nanomaterials for their effects. and to develop grouping concepts not only based on material properties but also on biopersistence, biokinetics and biological effects. part of this studes has been sponsored by bmbf (nanocare). endpoint-centric search for toxicological information and data to support the information retrieval for regulatory programs landsiedel r. 1 , wächter t. the eu reach regulation no 1907/2006 requires industry to ensure the safety of chemical use and manufacturing. all substances manufactured or imported in quantities above one tonne per year must be registered. information requirements for the dossiers increase with increasing tonnage or once hazards are suspected. searching for substance specific literature and the compilation of hazard data for safety assessments are highly challenging procedures. the novel web-based search engine go3r, accessible free of charge at www.go3r.org, has been created to allow quickly finding relevant hazard information and data. go3r provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information. furthermore, go3r specifically highlights information on animal testing alternatives. search results are presented automatically linked to an "intelligent table of contents" which enables the user to sort the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. retrieved documents are automatically organized in categories relating to the iuclid 5 chapters. hereby, the user can browse directly through the entire 21 million documents without even having to start the search with an initial query. the semantically enriched platform supports the user during query formulation, allows for bibliographic analysis, and specifically highlights information related to the replacement, reduction, and refinement of animal experiments. search results in go3r are shown in an dynamic table of contents (left) making them browsable for the contained information on animal testing alternatives and toxicologogical endpoints. towards a differentiation therapy of acute myelogenous leukemia with histamine h2-receptor agonists laue s., burhenne h., seifert r. hannover medical school institute of pharmacology, carl-neuberg-str. 1, 30625 hannover, germany acute myelogenous leukemia (aml) is a devastating malignancy characterized by a differentiation block of myeloid progenitor cells. recently, histamine dihydrochloride in combination with interleukin 2 has been approved as orphan drug for the consolidation treatment of aml (1) . it is assumed that histamine exerts its effects by activating the histamine h2-receptor (h2r) in human neutrophils, resulting in improved anti-tumor function of t killer cells by inhibiting nadph oxidase-catalyzed superoxide formation. previous studies had shown that histamine also induces myeloid differentiation (2) . considering the fact that all-trans-retinoic acids constitutes a powerful differentiation therapy of acute promyelocytic leukaemia, a specific subtype of aml (3), we initiated a study to explore the possibility that h2r-mediated myeloid differentiation provides an alternative or complementary strategy to treat leukemias associated with differentiation blocks. as model system, we used hl-60 cells. in hl-60 cells, histamine and various h2-receptor agonists induced concentrationdependent increases in camp levels. interestingly, ligands differentially increased cytosolic calcium concentration and extracellular receptor kinase (erk) pathways, indicative for ligand-specific h2r conformations. h2r activation resulted in myeloid differentiation as assessed by enhanced formyl peptide receptor-mediated increases in cytosolic calcium concentration. h2r agonists showed no signs of cytotoxicity. intriguingly, following h2r activation, the majority of the formed camp was exported into the extracellular space via multi-drug resistance protein (mrp) 4, indicating that export is a more important pathway for signal termination than cleavage of camp by phosphodiesterases. despite effective camp export, even a short-term exposure (30 minutes) of cells was sufficient to induce expression of functionally active formyl peptide receptors. these data indicate that in contrast to previously held dogma, induction of myeloid differentiation does not require continuous presence of a camp signal. from a therapeutic point of view this is very important since "spike" therapy with campincreasing substances may be sufficient to induce a therapeutic effect in aml, thereby also reducing toxic side effects. currently, we are systematically exploring the effects of h2r agonists on signal transduction pathways and differentiation in various myeloid cell types to identify highly efficacious compounds. introduction. activation of gαq/11 protein-coupled receptors of postsynaptic neurons can elicit the production of endogenous cannabinoids (endocannabinoids), which in turn inhibit transmitter release from axon terminals by activating presynaptic cb1 receptors. the aim of the present experiments was to study the mechanism of the endocannabinoid production. specifically, we wanted to clarify the role of ca 2+ release from intracellular stores in triggering endocannabinoid production. methods. patch-clamp-and ca 2+ imaging experiments were performed on purkinje cells in mouse cerebellar brain slices. glutamatergic excitatory postsynaptic currents (eepscs) were elicited by electrical stimulation of parallel fibers. the gαq/11 proteincoupled metabotropic glutamate receptor 1 (mglur1) was activated by superfusion of (rs)-3,5-dihydroxyphenylglycine (dhpg) or -more physiologically -by burst stimulation of the parallel fibers. results. both dhpg superfusion and burst stimulation of parallel fibers elicited an increase in intracellular ca 2+ concentration in the postsynaptic purkinje cells. dhpg superfusion and burst stimulation suppressed eepscs, and this suppression was abolished in the presence of the mglur1 antagonist cpccoet. the suppression of the eepscs was also sensitive to the cb1 receptor antagonist rimonabant, pointing to involvement of endocannabinoids and cb1 receptors. the suppression of the eepscs was attenuated after depletion of the endoplasmic reticulum ca 2+ stores by thapsigargin, cyclopiazonic acid and ip3. the results indicate that after activation of the gαq/11 protein-coupled metabotropic glutamate receptor 1 (mglur1) of the postsynaptic neuron ca 2+ is released from the endoplasmic reticulum. this ca 2+ release significantly contributes to the production of endocannabinoids. the endocannabinoids diffuse in the synaptic cleft retrogradely to the terminals of afferent axons and inhibit transmitter release there through presynaptic cb1 receptors. the guanine nucleotide exchange factor dock9 controls reelin dependent cdc42effects on radial migration pichler m. 1 the regulation of blood glucose levels is under tight control of a complex system including hormone and neurotransmitter signalling. many of these cellular signalling pathways are initiated by binding of the ligand to a g-protein coupled receptor (gpcr), e.g. noradrenaline inhibits insulin secretion upon binding to a gi-coupled receptor. upon gpcr activation the heterotrimeric g-protein is activated and both the α-subunit and βγdimers are released and interact with their specific target proteins. by the usage of bordetella pertussis toxin (ptx) as a common gαi inhibitor gαi-dependent signalling pathways are interrupted which leads to increased insulin secretion, and significantly improves glucose tolerance. since the gαi-isoform specific roles in the regulation of glucose homeostasis are still debated we studied the glycemic control in gαi2-deficient mice. surprisingly and in contrast to the ptx data, glucose tolerance was unchanged in the gαi2-deficient mice compared to wild type controls. however, the plasma insulin levels were significantly reduced upon glucose challenge. these findings point to disturbed islets function and improved peripheral insulin sensitivity. analysing gαi2deficient islets we show that islet size and number of nuclei are reduced. nevertheless, in vitro insulin secretion is improved at low (3 mm) and high (16 mm) glucose concentrations and can be further stimulated upon ptx-treatment. these data indicate that gαi2 proteins influence islet development and inhibit insulin secretion. in addition, these findings support our hypothesis that gαi2-deletion influences peripheral insulin sensitivity. therefore, we investigated glucose homeostasis and pakt-levels after two hours feeding ad libitum in gαi2-deficient mice. under feeding conditions no differences in plasma insulin levels were visible although blood glucose levels were significantly reduced in gαi2-targeted mice. pakt-levels of liver and skeletal muscle were unaltered, whereas akt phosphorylation in white adipose tissue was significantly increased, indicating improved glucose uptake of adipocytes. in conclusion, gαi2 is a negative regulator of both insulin secretion and peripheral insulin sensitivity and important for the maintenance of glucose homeostasis. 11689, 1992) in a radioligand binding test and to determine their functional effects with a membrane potential test using the dye r7260 (molecular probes, 0.125 mg/ml, excitation 505 nm, emission 530 nm). the affinity of compounds in radioligand binding was slightly higher in sur2b than in sur2a-type channels, but the enantiomeric ratio in sur2a channels matched that one determined for the sur2b-type indicating some conformity of the binding pockets of sur2a and sur2b-proteins. surprisingly, however, the membrane potential tests revealed that the (3r,4s)-enantiomer acted as agonist (a) whereas the (3s,4r)-enantiomer acted as antagonist (b): (3r,4s)-bms-191095 induced membrane hyperpolarisation whereas (3s,4r)-bms-191095 repolarised cells prestimulated with submaximally effective concentrations of diazoxide. concluding, bms-191095 is not selective for sur2a as compared to sur2b-type k atp channels. its enantiomers activate and block sur2-type katp channels in a stereospecific manner. thieno-thiadiazine derivatives with full agonistic activity at sur2b-type katp channels act as partial agonists at cardiac sur2a-subtypes oldenhage c., grittner d., schmidt c., lemoine h. heinrich-heine universität, inst. für lasermedizin, mol. wirkstoff-forschung, universitätsstr. 1, 40225 düsseldorf, germany new potassium channel openers (kco) of the thieno-thiadiazine(ttd)-type initially developed as agonists for the sur1-type katp channels (nielsen et al., j med chem 45: 4171, 2002) were characterized in sur2b-type katp channels as agonists and antagonists, if r contains a quaternary (methyl-cycloalkyl) and a tertiary (r = cycloalkyl) carbon, respectively (lemoine et al., this journal 375, r45, 2007) . to investigate the selectivity of ttd-derivatives for myocardial katp channels the membrane potential actions of compounds were tested in hek 293(kir6.2/sur2a)-cells and compared to hek 293(kir6.1/sur2b)-cells as a model for smooth muscle-type katp channels. membrane potential was measured by fluorescence (excitation 505 nm, emission 530 nm) using 0.125 mg/ml of the dye r7260 (molecular probes). standard-kco induced hyperpolarisation with ~10-fold smaller potency (pec50) in sur2a. ttd-compounds with ch3-cycloalkyl residues not only lost potency but also intrinsic activity for channel activation (emax) in sur2a. possibly, this loss of emax would be much greater in native heart cells with a normal channel density. in contrast, ttd-compounds with cycloalkyl residues acted as antagonists of cells pre-hyperpolarized with diazoxide with similar affinity in sur2a and sur2b-type katp channels. concluding, selectivity of kco for katp channel-subtypes cannot only be achieved by a different affinity but also by a selective stimulation of the channel of interest. small-conductance calcium activated potassium (kcnn/sk/kca2) channels maintain neuronal calcium homeostasis, shape synaptic functions and prevent excitotoxic neuronal death. so far, little is known about the function of kca2 channels in nonneuronal cells. the aim of this study was to investigate the expression of kca2 channels in microglial cells and their potential function in microglial activation and maintenance. expression of kca2 channel subtypes in microglial cells was assessed by mrna analysis, western blots and immunocytochemistry. lipopolysaccharide (lps)-induced microglial proliferation was evaluated by the xcelligence impedance-based system and mtt assays, and immunogenic activation of microglia was determined by measuring cytokines and nitric oxide (no) release into the cell culture medium. the kca2.2 and kca2.3 channel activator cyppa (25 µm) and specific inhibitory peptides (50 µm) were applied to distinguish effects mediated by the kca2 channel subtypes. all kca2 channel subtypes were detected on mrna and protein levels in resting and in lps-activated microglial cells. xcelligence real-time measurements and mtt assays demonstrated that lps (200 ng/ml) induced microglial proliferation. the kca2.2/kca2.3 channel activator cyppa reduced lps-induced microglial proliferation in a concentration-dependent manner. specific peptide inhibitors of kca2.3 channels, but not of kca2.2 channels, reversed the cyppa-effects on lps-induced microglial proliferation. cyppa alone did not alter the production of tnf-alpha or il-6, but strongly reduced the lps-dependent cytokine production. interestingly, chelation of extracellular calcium by edta induced differential cytokine kinetics by decreasing lps-dependent il-6 production while tnf-a production was not affected. moreover, using inhibitory sk3/kca2.3 channel peptides, we demonstrated that sk3/kca2.3 channels modulate lps-induced cytokine il-6 production in a calcium-dependent manner, while the tnf-a release was independent of extracellular calcium. in summary, the present study revealed that kca2.3 channel stimulation reversed microglial activation. thus, kca2.3 channels may serve as a therapeutic target for reducing microglial activity and related inflammatory responses in cns diseases. (3r,4s)-(3s,4r)intracellular amyloid beta (aß) oligomers and extracellular aß plaques are key players in the progression of sporadic alzheimer disease (ad). still, the molecular signals triggering aß production are largely unclear. we asked whether mitochondria-derived reactive oxygen species (ros) are sufficient to increase aß generation and thereby initiate a vicious cycle further impairing mitochondrial function. complex i and iii dysfunction were induced in a cell model using the respiratory inhibitors rotenone and antimycin resulting in mitochondrial dysfunction and enhanced ros levels. both treatments lead to elevated levels of aß. presence of an antioxidant rescued mitochondrial function and reduced formation of aß demonstrating that the observed effects depended on ros. conversely, cells overproducing aß showed impairment of mitochondrial function such as comprised mitochondrial respiration, strongly altered morphology, and reduced intracellular mobility of mitochondria. again, the capability of these cells to generate aß was partly reduced by an antioxidant indicating that aß formation was also ros-dependent. moreover, mice with a genetic defect in complex i, or ad mice treated with a complex i inhibitor, showed enhanced aß levels in vivo. several lines of evidence show that mitochondria-derived ros result in enhanced amyloidogenic amyloid precursor protein processing, and that aß itself leads to mitochondrial dysfunction and increased ros levels. we propose that starting from mitochondrial dysfunction a vicious cycle is triggered that contributes to the pathogenesis of sporadic ad. comparison of methods to derive health-based guidance or limit values for chemicals licht o., voss j. -u., mangelsdorf i. fraunhofer item chemikalienbewertung, nikolai-fuchs-str. 1, 30625 hannover, germany health-based guidance or limit values are derived for chemicals to compare measured or estimated exposure concentrations with these values. if the exposure is below the limit value, adverse effect for human health can be regarded as negligible, e.g. the exposure is expected to be tolerable. in germany such values have been derived since years for chemicals that can be found in soil, water and air as well as human blood and urine (biomonitoring). in a research project sponsored by the german umweltbundesamt several methods used by the agency are compared to the method laid in reach guidance document r.8 to derive a derived no effect level (dnel). the aim was to identify possibilities for standardization as well as to figure out specific elements in individual methods. in addition to extrapolation factors the public availability of guidance and specific derivations as well as procedures for consensus on the limit value were evaluated. the comparison of extrapolation factors revealed that, although they are named differently such as extrapolation, safety or assessment factors, they are used in a comparable manner. factors for interspecies and intraspecies extrapolation are presented in more detail. the standard factor for such extrapolation is 10 in most cases. in the reach guidance this factor consists of a part for allometric scaling and remaining differences. other factors are only defined in some methods, like a factor for extrapolation from loael to noael, data quality or data gaps. a factor for data quality is not laid down in the basic scheme for setting of indoor air guidance values, but is used in some of the recent derivations of limit values. also the who guidelines for drinkingwater quality use comparable factors to account for adequacy of studies or database and nature and severity of effect. a transparent and documented derivation is necessary for acceptance of the value. the derivation methods as well as the evaluation document on a specific substance are available through publications or the internet in nearly all cases. for the dnel only the numeric value is available at the echa website, but not any information on starting point and extrapolation factors. although all guide or limit values are derived in a comparable way, differences, however, exist in some details. in most cases detailed explanation is lacking when deviating from standard or default assumptions. often such deviation is based on expert judgement. hepatocellular carcinoma (hcc) is the fifth most common cancer in the world and has a poor prognosis with limited therapeutic options. up to now, no curative systemic therapy exists emphasizing the high clinical importance of new therapies for hcc. therefore, the identification and characterization of novel drugable targets is a relevant goal. cyclin-dependent kinase 5 (cdk5) is well characterized for its function in cns development and disease. recently, few reports indicate functions of cdk5 in cancer. cdk5 was shown to regulate tumor growth, and our group discovered that cdk5 regulates angiogenesis. since hcc is a highly vascularized tumor and anti-angiogenic treatment (sorafenib) has shown some therapeutic benefit, we hypothesize that cdk5 is an interesting target for hcc therapy. the aim of this study was to characterize the function of cdk5 in hcc. histology of tissue micro arrays indicates an increased expression of cdk5 in human hcc tissue in comparison to healthy liver tissue of the same patient. to investigate the function of cdk5 in hcc, we analyzed the impact of both pharmacological inhibition of cdk5 and specific downregulation of cdk5 with rna interference on hcc cells. pharmacological inhibition of cdk5 with the small molecule roscovitine (r-roscovitine, seliciclib) decreased proliferation and clonogenic survival, induced g2/m cell cycle arrest and cell death, and reduced motility of huh7 and hepg2 cells. transient downregulation (sirna) and stable knockdown (shrna) of cdk5 also reduced proliferation, clonogenic survival, migration and invasion of huh7 cells. in a subcutaneous hcc xenograft model, treatment with roscovitine reduced tumor growth and angiogenesis, indicated by decreased tumor weight and volume, and reduced vessel density. moreover, cotreatment of hcc cells with roscovitine and tumor necrosis factor related apoptosis inducing ligand (trail) resulted in an over-additive additive effect on the induction of apoptosis. this coincided with reduced phosphorylation and activity of the anti-apoptotic transcription factor stat3 at ser727 that is directly phosphorylated by cdk5, and tyr705. in line with this, the expression of the antiapoptotic protein mcl-1 is reduced by inhibition of cdk5. our results point to an important function of cdk5 in hcc and suggest cdk5 as an interesting pharmacologically druggable target for hcc therapy. delivery of mono-biotinylated rnasea into macrophages with streptavidinconjugated clostridium botulinum c3 toxin lillich m. 1 , chen x. clostridium botulinum produces the adp-ribosyltransferase c3, which modifies and thereby inactivates exclusively the small gtp binding proteins rho-a,-b and -c. recently, we discovered a specific endocytotic internalization of c3 toxin in macrophages and myeloid leukaemia cells, but not in epithelial cells [1] . thus, c3 toxin provides a tool to target cells of the monocyte/macrophage lineage, which are involved in various diseases and are of great clinical interest. we used a biochemical crosslinking approach to design a delivery system based on an enzymatic inactive c3bot mutant (c3mut) and streptavidin. the c3 portion mediates uptake of the transporter into monocytes/macrophages and streptavidin allows for binding of biotinylated cargo molecules to the transporter. in vitro, the generated c3mut-streptavidin bioconjugate showed specific and concentration dependent binding to biotinylated oligonucleotides as demonstrated by electrophoretic mobility shift assay. cell fractionation experiments indicated an uptake of the bioconjugate into the cytosol of j774a.1 macrophages. in the next step, mono-biotinylated bovine pancreatic ribonuclease a (rnasea) was used as a model cargo for the delivery of macromolecules by the bioconjugate. rnasea is a highly stable, well studied protein which catalyzes the degradation of rna. mono-biotinylated rnasea interacts in a specific and concentration dependent manner with the c3mut-streptavidin bioconjugate in vitro as analysed with dot blot technique. the c3mut-streptavidin bioconjugate efficiently mediates the internalization of biotinylated rnasea into j774a.1 macrophages as analyzed with laser scanning microscopy in fixed cells. this finding was also confirmed by live cell imaging. furthermore, cell fractionation showed a cytosolic delivery of biotinylated rnasea in the presence of c3mut-streptavidin. as expected we could not observe a cytotoxic effect of biotinylated wild-type rnasea on j774a.1 macrophages, which is attributable to the presence of ribonuclease inhibitor protein in mammalian cells. in summary, the c3mut-streptavidin bioconjugate mediates the efficient internalization of biotinylated (macro)molecules into macrophage like cells, and therefore represents a useful tool for the transduction of exogenous molecules into macrophages. in addition, cytotoxic rnasea mutants are available and will be used in further studies. organometal compounds such as cisplatin or the second generation complexes carboplatin and oxaliplatin have become more and more important as antitumor agents. nevertheless there is still an increasing demand for novel metal-based compounds. this is necessary due to severe side effects and the occurence of resistent tumour cells. in this context we investigated the cytotoxic effects of imidazole-based phosphane gold(i) complexes as potential agents for cancer treatment. initially we have used the mtt-assay to examine the toxic potential of the gold complexes in h4iie rat hepatoma cells. in this context cw60 (a diphosphane ligand with azoyl substituents r2p(ch2)2pr2, r= thiazol-2-yl) turned out to be the compound with the highest cytotoxic potential with an ic50 value of 6,5mm (24h incubation). further investigations revealed that cw60 induced an apoptotic cell death in h4iie demonstrated by the activation of caspase 3/7 (48h incubation with 10mm cw60). in addition the induction of apoptosis was confirmed by the dna ladder formation (24h incubation with 5mm cw60). in connection with the molecular mechanisms of apoptosis induction we used the comet assay to analyse the generation of dna strand breaks as well as the dcf-assay to detect the formation of reactive oxygen species. however neither dna strand breaks nor increased levels of reactive oxygen species were detected after 1h of incubation. furthermore we analysed if the compound influences intracellular signalling pathways such as the jnk pathway and the pi3k/akt but after 24h of incubation neither pakt nor jnk were influenced. the imidazole based phosphane gold (i) complex cw60 shows strong toxic effects in h4iie cells and turned out to be a promising compound as a potential agent for cancer treatment. the high and inappropriate intake of loop diuretics in hypertensive elderly reported in former studies has again been confirmed. remembering that inappropriate intake of loop diuretics can lead to exsiccosis and electrolyte loss especially in elderly, better medical education has to follow these alarming results to improve the pattern of diuretic prescription. furthermore, our results lead us to assume a high estimated number of unreported cases of torasemide use in uncomplicated arterial hypertension in elderly. this loop diuretic agent shows a longer duration of action compared with furosemide (elimination half-life: 3-4 hrs vs. 1 hr) and is effective in decreasing blood pressure in subdiuretic doses. it must be pointed out that loop diuretics are still frequently inadequately prescribed because current guidelines recommend loop diuretics only in complicated arterial hypertension. the role of cgmp/cgki signaling and trpc channels in regulation of vascular tone loga f., domes k., hofmann f., wegener j. pharmakologie und toxikologie for 923, biedersteiner str 27, 80802 münchen, germany signaling by intracellular cgmp and cgmp-dependent protein kinase i (cgki) is the major pathway in vascular smooth muscle, by which endothelial no regulates vascular tone. recent evidence suggests that trpc channels are targets of cgki in smooth muscle and mediate, at least partially, the relaxant effects of cgmp. we tested this concept by investigating the role of cgmp/cgki signaling on vascular tone and peripheral resistance using cgki-, trpc6-, and trpc3-, and trpc3/c6-double knock-out mice. we found larger contractile responses to α-adrenergic stimulation in intact aorta from cgki-, trpc6-, and trpc3/c6-double knock-out mice as compared to aorta from ctr and trpc3-knock-out mice indicating a functional link between cgki and trpc6 channels. no differences were found if the vasodilator tone, provided by the no generation in the vascular endothelium, was inhibited by l-name. likewise, no differences were observed in the increase in peripheral resistance by α-adrenergic stimulation using the hind limb perfusion system. activation of cgki by 8-br-cgmp diminished aortic tone and peripheral resistance to a similar extent in control, trpc6-, trpc3-, and trpc3/c6-double knock-out mice. no effect of 8-br-cgmp was observed in preparations from cgki -/mice. to test the co-localization of cgki and trpc channels, we performed immunocytochemistry on isolated smooth muscle and endothelial cells from aorta of ctr, trpc3-, and trpc6-knockout mice. trpc3 could be detected in both smooth muscle and endothelial cells whereas trpc6 was only detected in endothelial cells. the results suggest that absence of cgki or trpc6 impairs the vasodilator tone induced by endothelial no production but that cgki and trpc6 channels are not functionally coupled in vascular smooth muscle. we thank profs birnbaumer (nih) and freichel (homburg) for providing us with trpc6 -/-, and trpc3 -/mice and prof. flockerzi (homburg) for the antibodies against the trpc channels. whole genome microarray analysis of the effects of tcdd and pcb 153 in human hepatic cell models lohr c. 1 , neser s. after the treatment with tcdd, however, a total of 281 genes were more than 2-fold up regulated in hepg2 e.g. cytochrome p450 1a1 (cyp1a1) (32-fold) a sensitive marker for ahr activation. additional up regulated genes in hepg2 were; arylhydrocarbon receptor repressor (ahrr) 15-fold, aldehyde dehydrogenase 3 a1 (aldh3a1) 11-fold, and cytochrome p450 1b1 (cyp1b1) 10-fold. 44 genes were more than 2-fold down regulated in hepg2 cells e.g. -proprotein convertase subtilisin/kexin type 9. markedly different findings were obtained in hheps, i.e., 117 genes were up regulated, the highest up regulated gene was cyp1b1 with a 95-fold increase in gene expression, followed by cyp1a1 (41-fold) and aldh3a1 (21-fold). only a small group of genes were significantly down regulated (17 in total), e.g., solute carrier family 2 (facilitated glucose transporter). comparing both human cell types, there was an unexpected small overlap of genes being up or down regulated. interestingly, in both cell types, only 30 in common genes were up regulated, including cyp1a1, cyp1a2, cyp1b1 and aldh1a3. only platelet-derived growth factor receptor, beta polypeptide, was down-regulated in both hepg2 and hheps. in conclusion, our data indicate pronounced differences in the patterns of tcdd-regulated genes between hepg2 cells and hheps. detection of redox modified proteins in nociceptive processing lorenz j. e. 1 , kallenborn-gerhardt w. recent data indicate that redox modifications of proteins induced by reactive oxygen species (ros) contribute to sensitization of pain pathways during persistent pain. however, little is known about the targets of ros in pain processing, because the relatively unstable nature of many reversible protein oxidation states hampers the reliable detection and identification of modified proteins. here, we used the quantitative thiol trapping technique termed oxicat to identify proteins which are redox modified during nociceptive processing. we investigated spinal cords of untreated mice, after zymosan injection into a hindpaw (inflammatory pain model) and after spared nerve injury (neuropathic pain model). we identified several proteins with marked changes in their redox states after nociceptive stimulation. our results show that the oxicat method is an efficient method to detect redox modifications in proteins and that redox modifications seem to play a role in pain processing. supported by the deutsche forschungsgemeinschaft (sfb 815/a14). additive antinociceptive effects of a combination of vitamin c and vitamin e after peripheral nerve injury lu r., kallenborn-gerhardt w., geisslinger g., schmidtko a. pharmazentrum frankfurt/zafes institute of clinical pharmacology, goethe university, frankfurt am main, germany accumulating evidence indicates that increased generation of reactive oxygen species (ros) contributes to the development of exaggerated pain hypersensitivity during persistent pain. in the present study, we investigated the antinociceptive efficacy of the antioxidants vitamin c and vitamin e in mouse models of inflammatory and neuropathic pain. we show that systemic administration of a combination of vitamins c and e inhibited the early behavioral responses to formalin injection and the neuropathic pain behavior after peripheral nerve injury, but not the inflammatory pain behavior induced by complete freund's adjuvant. in contrast, vitamin c or vitamin e given alone failed to affect the nociceptive behavior in all tested models. the attenuated neuropathic pain behavior induced by the vitamin c and e combination was paralleled by a reduced p38 phosphorylation in the spinal cord and in dorsal root ganglia, and was also observed after intrathecal injection of the vitamins. moreover, the vitamin c and e combination ameliorated the allodynia induced by an intrathecally delivered ros donor. our results suggest that administration of vitamins c and e in combination may exert synergistic antinociceptive effects, and further indicate that ros essentially contribute to nociceptive processing in special pain states. -206, -213 and -214) replaced by leucine residues. both amino acids are comparable in terms of hydrophobicity, volume and the preference for forming α-helices, but only methionine is oxidizable to a sulfoxide, in contrast to leucine. in the present study we examined the protein-protein interaction (ppi) of recombinant ac 1, expressed in sf9 insect cell membranes, with cam, cam-206, -213, -214 and -215 by measuring the catalytic activity of ac 1. cam-mutants show a 3-4-fold lower potency than cam, but they are more efficacious than cam. most prominently, cam-215 was 133 % more efficacious than cam. such striking differences between cam and cam-mutants have not yet been observed for other mammalian effector proteins. as a result of the exchange of all methionine against leucine residues in cam-215, it is more hydrophobic than cam and this leads to a better ppi with ac 1. in future studies we will examine the effects of cam inhibitors, antidepressants and antipsychotics on cam/ac 1 interaction. furthermore we will analyze the effects of oxidized cam and cam-mutants on the catalytic activity of ac 1. because oxidative stress is of great importance in aging, it is important to know more about the abovenamed interaction in view to the demographic change. taken together, our data point to a unique cam/ac 1 interaction that may be selectively targeted by small molecules. in particular, enhancers of these interaction could be useful to improve memory and learning. gender differences in fat distribution and diabetes prevalence in nzo mouse lubura m., scherneck s., zucker a., schürmann a. deutsches institut für ernährungsforschung experimentelle diabetologie, arthur-scheunert-allee 114-116, 14558 potsdam, germany background: excessive fat accumulation in visceral but not subcutaneous fat depots as well as ectopic fat storage in liver, skeletal muscle and pancreas are associated with an increased risk for the development of type 2 diabetes in humans. in this study we aimed to examine the influence of early fat distribution on onset of type 2 diabetes in mice. methods: nzo mice are regarded as insulin resistant model in which only males become diabetic. we used male and female mice fed with high-fat and standard diet. we determined fat distribution by computed tomography for three times and conducted oral glucose tolerance tests on two different time points. besides we assessed body weight and blood glucose levels on weekly basis. results: contrary to previous findings, we observed that not only male nzo mice on high-fat diet develop diabetes. blood glucose levels at the 16 th week of age and total pancreatic insulin content indicated diabetes prevalence of 68% in males and 25% in females these results lead to the conclusion that high-fat diet counteracts protective action of estrogens against diabetes. inversely to the findings in humans, female mice tend to store more fat in abdominal region than males. there was no relationship between early accumulation of fat in abdominal region and onset of type 2 diabetes. however, visceral fat was associated with liver fat in males as well as in females. furthermore, at the age of ten weeks hepatic fat content correlated with blood glucose levels (r² = 0.69) indicating that the early hepatosteatosis is a predictor for hyperglycemia. however, there was no correlation between hepatic insulin sensitivity (indicated by quantitative insulin sensitivity index-quicki) and amounts of hepatic fat we conclude that early hepatosteatosis does not predict for glucose intolerance in nzo mice. in the nzo mouse, the amount of liver fat but not the early fat distribution predicts for the later onset of type 2 diabetes. further experiments are needed to examine the gender dependent differences in the diabetes prevalence of this mouse strain. with a prevalence of about 20-30% non-alcoholic fatty liver disease (nafld) represents the most common liver disorder in europe. nafld manifestation ranges from steatosis through steatohepatitis (nash) to fibrosis and cirrhosis, followed in some cases by liver failure and hepatocellular carcinoma. fatty degeneration of liver cells, increased oxidative stress with concomitant lipid peroxidation and an induction of pro-inflammatory cytokines are proposed as possible causes for developing inflammation and fibrosis, but the exact pathogenesis of the progression of nafld into nash is still unknown. thus, besides life style modifications and weight reduction interventions, no established pharmacological therapy exists so far. to gain further insights into the pathogenesis of nafld and nash and to develop new therapeutic strategies, appropriate animal models are essential. thus, in the present study three different dietary animal models for nafld were evaluated and compared to the biochemical and metabolic alterations seen with nafld and nash in man. male adult lewis rats were given standard food or one of three different diets: fatty liver diet [fld] , methionine/choline deficient diet [mcd] or methionine/choline deficient plus high fat diet [mcd+hf] . after 1, 2, 4, 6 or 12 weeks of treatment, animals were sacrificed and body and liver weights, laboratory parameters (asat, alat) as well as histopathological changes in the livers and different parameters indicating oxidative stress or representing the biotransformation capacity of the livers were analyzed. with fld and mcd+hf a normal body weight gain was observed, whereas with mcd body weight gain was strongly impaired. liver weights were mainly increased after mcd+hf. elevation of asat and alat values and hepatic steatosis were more pronounced after mcd and mcd+hf than after fld. all three diets caused an increase in the oxidative stress in liver tissue, but especially with mcd a tremendous elevation in the hepatic levels of lipid peroxidation products was seen. with regard to liver biotransformation capacity, with all three diets mainly an induction of the cytochrome p450 2e1 and 4a1 isoforms expression and activity was observed, which was most pronounced after mcd and mcd+hf. in summary, the changes induced by mcd or mcd+hf most closely resemble the alterations described in literature for nafld in man and thus should be preferred over fld in future investigations on nafld and nash. ep3 receptors for prostaglandin e2 convey stimulatory and inhibitory effects. e.g., their stimulatory effect leads to vasoconstriction in the human pulmonary artery and their inhibitory activity to reduction of neurotransmitter release from neuron endings. the aim of our study was (1) the pharmacological characterization of ep3 receptors in human pulmonary arteries and (2) the examination of the involvement of these receptors in the regulation of the neurogenic tachycardia in pithed rats. l-826266 served as the ep3 antagonist. experiments were performed in human pulmonary arterial rings isolated from patients undergoing lobectomy during resection of lung carcinoma and in pithed and vagotomised rats. the ep1/ep3 agonist sulprostone (1 nm -100 mm) concentrationdependently contracted human pulmonary artery rings (pec50 and emax; 6.89±0.12 and 106.5±5.2%, relative to the contraction induced by kcl 60 mm). the concentrationresponse curve of sulprostone was not affected by the ep1 antagonist sc 19920 (10 µm) but shifted to the right by l-826266 (10 µm) (apparent pa2 6.22). extending the exposure time to l-826266 from 0.5 to 3 h increased its antagonistic potency to 7.39 (schild plot-based pa2; concentrations 0.1, 1 and 10 µm). in pithed rats electrical stimulation (0.66 hz, 1 ms, 50 v for 5 s) of the preganglionic sympathetic nerve fibers or intravenous isoprenaline (0.15 nmol/kg) increased heart rate (hr) by 55 beats/min. sulprostone (10 -1000 nmol/kg) did not affect the isoprenaline-induced increase in hr but inhibited the neurogenic tachycardia dose-dependently, maximally by 80%. l-826266 (3 µmol/kg) diminished the inhibitory effect of sulprostone 1000 nmol/kg on the neurogenic tachycardia by 20%. in conclusion, ep3 receptors (1) located postsynaptically strongly contract human pulmonary arteries and (2) located presynaptically on sympathetic nerve fibres supplying the heart of rats strongly inhibit the neurogenic tachycardia. -5-bromo-n[3-(5voltage-gated ca 2+ channels of the central nervous system control a multitude of ca 2+ dependent processes such as neurotransmitter release, neuronal excitability, neurite outgrowth, synaptogenesis, plasticity and neuronal survival. the cav2.1 ca 2+ channelalso known as p/q-type channel -belongs to the subfamily of high voltage activated ca 2+ -channels. ca 2+ influx via cav2.1 ca 2+ channels located at presynaptic nerve terminals triggers vesicle fusion and transmitter release at brain synapses and at the neuromuscular junction. thus, cav2.1 ca 2+ channels play a crucial role in synaptic transmission. the global cav2.1 knock-out phenotype is characterized by severe ataxia, dystonia and lethality during the first postnatal weeks and is therefore an unsuitable model to analyze the importance of cav2.1 ca 2+ channels for learning and memory. therefore, we crossed a floxed cav2.1 mouse line with nex-cre transgenic mice to establish a viable, forebrain specific knock-out mouse line (fbko-mice). results from western blot analysis confirmed an efficient knock out of cav2.1 in hippocampal and cortical preparations, whereas the expression level in the cerebellum was not altered. to investigate the specific role of cav2.1 channels in hippocampus and neocortex dependent behavior, we performed tests for motor functions and sensory abilities and in particular learning and memory tasks. mice with a forebrain specific cav2.1 knock-out show significant deficits in spatial learning & reference memory and a significant reduced recognition memory as revealed by the morris water maze and an object recognition task. the fbko-mice exhibit no obvious locomotor deficits during behavioral tasks in the open field test and elevated plus maze. some fbko-mice demonstrate episodes of seizures in the morris water maze and during different rotarod tasks. to assess motor-function of fbko-mice in a stress reduced environment, we performed home cage based running-wheel motor-learning tasks. in summary, the diverse phenotypes of the forebrain specific knock-out mouse line emphasize the critical importance of cav2.1 for learning and memory. helicobacter hepaticus-infected rag2 -/mice emulate many aspects of human inflammatory bowel disease (ibd), including the development of colitis and colon cancer [erdman et al., 2009 , pnas 106: 1027 -1032 . toward the goal of elucidating mechanisms of inflammation-induced carcinogenesis and developing biomarkers of inflammation, we undertook a comprehensive analysis of macromolecular damage products during disease progression in h. hepaticus-infected rag2 -/mice. infected mice developed severe colitis and hepatitis, accompanied by infiltration of myeloperoxidase-positive neutrophils and f4/80-positive macrophages, by 10 wks postinfection (pi), progressing into colon carcinoma by 20 wks pi. qpcr array-based gene expression profiling revealed that pathophysiological changes were associated with characteristic alterations in the expression of genes related to inflammation, dna repair, and oxidative stress response. to study inflammation-related macromolecular damage, colon and liver tissues were analyzed by isotope-dilution chromatography-coupled mass spectrometry to quantify a battery of 16 different dna, rna and protein damage products thought to represent the full spectrum of inflammation-related chemistries. our data revealed a significant predominance of chlorinated dna-, rna-, and protein damage products by 20 weeks pi. in contrast, levels of damage products arising from oxidation, nitration and nitrosation changed only modestly or remained unchanged. our analyses also revealed higher levels of damage products in rna than in dna and demonstrated organ-specific differences of oxidative damage products, such as 8-oxo-dg and its oxidation products spiroiminodihydantoin and guanidinohydantoin. collectively, these results suggest that neutrophil and myeloperoxidase-induced chlorination chemistry may serve as a biomarker of ibd and may play important roles in the pathophysiology of ibd and colitis-associated cancer. characterization of a membrane protein expressed in mouse heart and brain mannebach s. 1 recently, a novel membrane protein in drosophila was shown to be localized in presynaptic vesicles. it appears to mediate a ca influx after vesicle fusion with the plasma membrane. disruption of the corresponding gene leads to endocytic defects in drosophila [1] . apparently, this protein plays a role in exo-and endocytosis and could serve as a ca channel supplying ca required for endocytosis. we have identified a protein in mouse, c90rf7, which shares 26,3% amino acid sequence identity with the drosophila protein. it covers 171 amino acid residues. using rt-pcr the full length transcripts could be identified in brain, kidney, pancreas, heart, spleen, thymus and mast cells. coexpression of c90rf7 and the ca v2.2 channel in xenopus oocytes reduced the amount of the α1b and cavβ3 subunits of the ca 2+ channel in the plasma membrane but did not affect the gating properties of the cav2.2 channel. expression of c90rf7 alone did not yield any channel activity. we therefore started to produce recombinant protein using the his-sumo-prokaryotic expression vector. the protein was efficiently expressed as his-sumo-c9orf7-fusion in e.coli (yield 2mg at 1mg/ml). we are currently preparing the c9orf7 part of the his-sumo-c9orf7fusion protein by ulp1-protease digestion followed by various chromatographic steps. the purified recombinant protein will be used to immunize rabbits to get antibodies. in parallel we generated antisera by immunizing rabbits with peptide fragments derived from the c9orf7 sequence. we could not identify any homologues of c9orf7 in the mouse genome and to analyze its function we are currently generating c9orf7 deficient mouse lines by gene targeting. we have chosen a strategy for conditionally inactivation of the gene with the option to study the cellular localization of c9orf7 by expression of the bgalactosidase gene under the control of the endogenous c9orf7 promoter. by southern blot analysis we´ve already identified 210 homologous recombinant embryonic stem cell clones out of 300 analyzed ones and we will proceed with blastocyst injection to get chimeric mice and finally mice carrying the introduced mutations in the c90rf gene. parps are involved in various biological processes such as regulation of dna repair, cell cycle progression, and cell death. consequently, several parp inhibitors are currently in clinical development as chemo-and radiosensitizers as well as monotherapeutic agents following the concept of synthetic lethality. pharmacological and toxicological studies call for an accurate analysis of parp activity in terms of a detailed knowledge of the structure of par and a reliable method for its quantification. we have developed a sensitive, precise, and accurate bioanalytical method based on liquid chromatography coupled to electrospray tandem mass spectrometry (lc/ms-ms) to characterize and quantify par with femtmol sensitivity: par is extracted from cells and hydrolysed to specific monomeric units, i.e., ribosyladenosine, which is characteristic for linear par, diribosyladenosine, which is characteristic for branching points, and adenosine, which represents the terminal part of the polymer. using this method, we are currently analyzing par levels in different cell lines and in primary human peripheral blood mononuclear cells (pbmcs) both under physiological conditions as well as upon genotoxic stress and in the presence of potent parp inhibitors. we expect that after completing method validation this assay will be useful for a wide range of applications in pharmacology and toxicology. gene mutagenic potential and metabolite profile of 17β-estradiol in cultured v79 cells expressing human cytochrome p450 1a1 martínez jaramillo d., lehmann l. university of wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany oxidative metabolism of the female sex hormone 17β-estradiol (e2) is considered to play a major role in the initiation of hormone-induced carcinogenesis. in extrahepatic tissues, e2 undergoes metabolic activation by cytochrome p450-dependent monooxygenase (cyp) isozyme 1a1 to 2-hydroxy-(2-ho) and to a lesser extent to 4-ho-e2. if not conjugated, these catecholestrogens (ce) can further oxidize to electrophilic quinones (q), which may react with dna and induce thereby mutations. conjugation of these ce in extrahepatic tissues is mainly catalyzed by catechol-omethyltransferase. in order to identify possible mutagenic metabolites (i) the induction of gene mutations by e2 was determined in male chinese hamster lung fibroblasts (v79 cells) expressing human (h) cyp1a1 and (ii) the metabolite profile of e2 in these cells was analyzed via gas chromatography/mass spectrometry after solid phase extraction of the cell suspension in the culture medium. (i) gene mutations were assessed using the hypoxanthine-guanine phosphoribosyltransferase assay. the promutagen benzo[a]pyrene (bap) served as positive control requiring metabolic activation by hcyp1a1 and dimethylsulfoxide as solvent control. v79 hcyp1a1 were treated with 100 nm e2 for 3 weeks and the resulting 6-thioguanine (6-tg) resistant mutants selected at weeks (w) 2 and 3. the frequency of spontaneous 6-tg resistant mutants per 10 6 colony-forming cells ranged from 5 ± 1 (w2) to 9 ± 4 (w3). as expected, 0.25 µm bap induced a significant increase in mutant frequency (mf, 266 ± 13, w2 and 132 ± 46, w3) . treatment with 100 nm e2 resulted in a 3-fold (17 ± 2, w2) and a 2-fold (23 ± 4, w3) increase in mf, suggesting slight mutagenic activity. in culture medium of v79 hcyp1a1 treated with 100 nm e2, 2-ho-e2, 2-methoxy-(meo)-e2, 3-o-methyl-2ho-e2 and 4-meo-e2 (suggesting intracellular formation of 4-ho-e2) were detected. while 4-meo-e2 concentration remained constant over the exposure period, the concentration of the other metabolites increased in a timedependent manner. the maximum concentration increase was reached at w3 for methylcatechols and at w2 for 2-ho-e2, correlating with the maximum increase in mf, observed after 2 weeks as well. in conclusion, e2 possessed a slight mutagenic potential after hcyp1a1-mediated activation to 2-, 4-ho-e2 and their corresponding methylcatechols. cumulative effects of three triazole fungicides in a broad dose range in vitro rieke s., kneuer c., bumke scheer m., lampen a., hirsch-ernst k., marx-stoelting p. bundesinstitut für risikobewertung chemikaliensicherheit, max-dohrn-str., 10589 berlin, germany consumers are exposed to multiple residues of different pesticides via the diet. this raises questions concerning potential cumulative effects, especially for substances causing toxicity by a common mode of action. the aim of this work was to investigate potential combination effects of the three triazole fungicides epoxiconazol, tebuconazol and flusilazol for selected parameters in a broad dose range in vitro. parameters investigated were cytotoxicity, hormone synthesis (17-β estradiol, progesterone and β-hcg), expression of a panel of androgen-or estrogen-responsive genes in the human placental choriocarcinoma cell-line jeg-3 and transactivation via estrogen receptors α and β in stably-transfected hek 293 cells. the ability to inhibit steroidogenesis was analysed by measuring the concentrations of 17β-estradiol and progesterone in cell culture supernatants of jeg-3 cells. additionally, the placental peptide hormone β-hcg was measured. while no change in β-hcg and 17β-estradiol concentrations were observed, all triazoles induced a dose-dependent decrease in progesterone concentration and a cumulative effect was observed implying dose additivity at individual doses of >1.7 µg triazole/ml. significant activation of erβ by the three triazoles, especially by flusilazol, was observed at 5 µg triazole/ml and combined exposure showed additive effects, while no significant activation of erα was observed. based on the data, our findings suggest dose-additivity of triazole pesticides with the same mode of action for selected parameters in vitro. no significant effects were observed at lower doses [1ng -1µg triazole/ml] neither for substances applied individually nor in combination. transient receptor potential channels as mediators of catecholamine release mathar i. 1 trp proteins form cation channels that are regulated through strikingly diverse mechanisms. recently, genetic association studies identified many trp genes including trpm4 as risk factors for disease states such as arrhythmias, hypertension and cardiomyopathy. the melastatin trp channels trpm4 and trpm5 have distinct properties within the trp channel family; they form non-selective cation channels activated by intracellular calcium ions and are expressed in heart, aortic endothelial cells, kidney and adrenal gland. disruption of the trpm4 gene in mice leads to increased basal blood pressure without evidence for impairment of endothelium-or smooth muscle-dependent regulation of contractility of peripheral resistance vessels, the renin angiotensin aldosterone system, basal cardiac output or body fluid homeostasis. instead, trpm4-deficient chromaffin cells exhibit increased acetylcholine-induced exocytosis of catecholamines which is associated with elevated level of epinephrine in the plasma and its metabolites in the urine. this indicates that trpm4 serves as an inhibitory regulator of exocytotic catecholamine release, at least in chromaffin cells. whether catecholamine release is also regulated by trpm4 in other cells of the sympathetic nervous system such as perivascular neurons still needs to be clarified as well as the molecular mechanism underlying how trpm4 regulates catecholamine release. besides trpm4 we recently identified transcripts encoding additional trp channels including trpc5 and trpc6 in chromaffin cells isolated by laser capture microdissection but their functional role in these cells is still unknown. measurements of the time course of the intracellular calcium concentration before and during acetylcholine stimulation (10µm) of catecholamine release as well as the analysis of the number of released vesicles in chromaffin cells relvealed no changes in trpc1/c5/c6 triple knock out mice compared to wildtype controls. although it seems that these trpc proteins are not directly involved in catecholamine release from chromaffin cells induced by acetylcholine application in our hitherto existing experiments, their contribution to the modulation of catecholamine release by agonists of gq-coupled receptors still needs to be analysed. aims: sulfonylureas (sus) are among the most widely used oral hypoglycaemic drugs that stimulate insulin secretion. in addition, sus have pleiotropic effects on other tissues. regarding the effects of sus on adipocytes conflicting findings were reported. we have now investigated the actions of glimepiride and glibenclamide (=glyburide) in primary human adipocytes. methods: primary cultured human white pre-adipocytes were differentiated in vitro according to a standard protocol. lipid accumulation was assessed by oil red o staining and determination of triglyceride content; gene expression was measured by real-time pcr and western blotting. results: we initially characterized the genes regulated during human preadipocyte differentiation by a global microarray analysis. treatment with glimepiride and glibenclamide caused a strong accumulation of lipid droplets and an increase in triglyceride content. genes involved in lipid metabolism were induced, chemokine expression was decreased. interestingly, the effects of sus were over all qualitatively and quantitatively similar to pioglitazone. in direct comparison glibenclamide was more potent than glimepiride in respect to the induction of fabp4 (ec 50 0.32 vs. 2.8 µm), an important adipocyte marker gene. su-induced differentiation was virtually completely blocked by the pparγ-antagonist t0070907 but not affected by diazoxide, indicating pparγ activation by sus. repaglinide, causing insulin liberation like sus but being structurally different, had no effect on adipocytes. conclusions: in primary human pre-adipocytes, glibenclamide and glimepiride strongly induced differentiation, apparently by activating pparγ . thus, sus but not repaglinide may be used to influence insulin resistance beyond their effect on insulin liberation. the role of at1a and at1b receptors as mechanosensors in myogenic vasoconstriction blodow s. 1 , schneider h. arterial myogenic tone denotes the intrinsic property of vascular smooth muscle cells to constrict in response to an elevated intraluminal blood pressure. this physiological reaction is more distinct in small resistance arteries than in large conduit arteries. understanding the underlying mechanisms should provide useful information for the treatment of diseases like anaphylactic shock and systemic hypertension in which this reaction is altered. whereas the underlying signaling cascade has been extensively studied, the molecular identity of the mechanosensory elements still remains elusive. recent studies at the cellular level suggest a sensory function for a subgroup of gprotein coupled receptors (gpcrs) coupling to gq/11-proteins. by determining mrnaexpression levels of selected gpcrs in consecutive pairs of resistance and conduit vessels, we could identify a subset of gq/11-coupled receptors such as angiotensin ii at1b, vasopressin v1a, endothelin eta and etb and α1a adrenoceptor significantly enriched in resistance vessels. by pharmacological blocking of those highly expressed gpcrs by different antagonists and inverse agonists, we evaluated their influence on the formation or the intensity of myogenic tone, as measured in isolated murine mesenteric arteries ex vivo. while blocking of v1a receptor and α2a and α2ab adrenoceptors showed no differences of myogenic tone, blocking of at1a and at1b receptors by losartan and candesartan, eta receptor by bq123 and α1a adrenoceptor by prazosin caused significant reductions of the vascular response. analyzing the myogenic response of at1a -/mice with and without additional blocking of at1b receptors by candesartan suggested that especially at1b receptors play a dominant role for mechanosensitivity in mice. this was further supported by investigating the myogenic response of at1b -/mice. these findings suggest that mechanosensitive gq/11-protein coupled receptors, especially at1b receptors, play a dominant role for the development of myogenic vasoconstriction. trpm3 ion channels are activated by steroidal compounds and noxious heat and are considered to be involved in insulin secretion and pain perception. the expression of the trpm3 gene generates a variety of different transcripts which arise by alternative splicing and the use of different promoters [1] . they encode a substantial variety of isoformes and so far we have identified more than 20 distinct trpm3 proteins in mouse and rat each varying in exons 1, 2, 8, 13, 15, 17 and 24 . these variants differ enormously in their biophysical properties. for example splicing within exon 24 affects the channel pore and causes significant changes of the ionic selectivity of trpm3 channels [2] , whereas splicing of 54 nucleotides encoded by exon 13 leads to dormant trpm3 proteins. however, the frequency of these different isoformes in trpm3 expressing tissues is completely unknown. to get insight into the significance of the different trpm3 isoformes we investigated the abundance of alternative trpm3 transcripts in different tissues and cell types by reverse transcription quantitative pcr (rt-qpcr). we found that the frequency of splicing within exon 13 ranges from 5 up to 20 % in different cell types and tissues. furthermore we analyzed the trpm3 transcriptome in the choroid plexus of the brain and the pituitary gland, tissues in which trpm3 transcripts are most abundant. for that purpose we sequenced more than 120 clones, each. corresponding to our rt-qpcr result, we found a significant number of transcripts lacking exon 13. in cells of the choroid plexus nearly all (124 /126 clones) carried the short ca 2+ permeable pore. furthermore, we identified seven variants spliced in exon 20 encoding truncated trpm3 proteins. however, the composition of the trpm3 transcriptome in the choroid plexus and pituitary gland differed enormously, indicating the importance of alternative splicing for trpm3 function in different tissues. the concept of "thresholds of toxicological concern" (ttc) defines tolerable dietary intakes for chemicals without toxicity data and is widely applied to chemicals present in food in low concentrations such as flavorings. based on a statistical evaluation of the results of many toxicity studies and considerations of chemical structures, the ttc concept derives a maximum daily oral intake without concern of 1800, 540 or 90 µg/person/day for non-genotoxic chemicals depending on the allocation to so-called cramer classes i, ii or iii. for substances with a structural alert for genotoxicity a ttc value of 0.15 µg/person/day might be used. recently, it has been investigated, whether the ttc values, which were derived based on mostly chronic oral dietary rodent studies would cover all relevant toxicities (neurotoxic, repeated dose, reproductive and developmental, immune effects and endocrine-related effects). several authors using different specific databases have confirmed that the ttc values derived using cramer classes are also covering immunotoxic, neurotoxic, reproductive and developmental effects. a respective decision tree is going to be presented, also considering substances or substance classes which shall be excluded from the ttc approach. there are several areas in which the ttc concept is already used, or a ttc approach is considered useful, to assess low-level human exposures, or help in prioritizing toxicological testing; as for example the assessment of plant metabolites and degradates of pesticide active substances, feed and food additives, chemicals with a low exposure profile under reach, residues, metabolites and impurities in plants, chemicals, plant protection products or pharmaceuticals. if no structural alert for genotoxicity is given or standard genotoxicity tests are negative the cramer class iii value of 90 µg/person/day, which corresponds to a dose of 1.5 µg/kg bw is considered to represent a chronic tolerable daily intake of the test substance. examples for current and future uses of the ttc concept in regulatory toxicology are presented. objective: hyaluronan (ha), synthesized by three ha-synthases (has1, -2, -3), is a prominent matrix component of atherosclerotic lesions. the aim of the present study was to identify the has isoenzyme that is associated with ha-matrix remodeling in inflammatory regions of atherosclerotic plaques. furthermore the underlying regulatory pathways were determined and functional aspects of this regulation in vascular smooth muscle cell (vsmc) were addressed. methods and results: during atherosclerosis in apoe deficient mice the peak of macrophage invasion at 14 weeks coincided with ha deposition and induction of has3 in aortic root plaques. in human symptomatic carotid artery plaques has3 was by far the most prominent has isoenzyme as determined by quantitative real time rtpcr. in vitro, in human vascular smooth muscle cell (vsmc) has3 was specifically induced via activation of nfkb by interleukin-1β (il-1β) and tumor necrosis factor alpha (tnfa) as shown by chip assay and utilization of nfkb inhibitor bay 11-7082. has3 was also upregulated in a co-culture system by activated macrophages via paracrine release of tnfa and il-1β as verified by neutralizing antibodies. in human atherosclerotic lesions nfkb positive vsmc were frequently detected in close proximity with ha and f4/80 positive macrophages as shown by immunohistochemistry. to study the effects of has3 mediated ha synthesis in human coronary vsmc, lentiviral overexpression and knockdown of human has3 were employed. overexpression of has3 resulted in increased migration and proliferation whereas knock down had the opposite effect. the effects of has3 were mediated by both pi3k signaling and mapk signaling via hyaluronan receptors cd44 and rhamm. conclusion: the present results suggest that has3-dependent ha synthesis is induced in human vsmc by inflammatory cytokines released from activated macrophages. moreover, has3-mediated ha production induced phenotypic activation of vsmc. pulmonary inflammation and airway remodeling are major features of chronic obstructive lung disease (copd). in addition, pulmonary hypertension is a common comorbidity, which is associated with a poor prognosis of the disease. recent studies in a guinea pig model of allergic asthma have shown that increased arginase activity, which converts larginine into l-ornithine and urea and competes with nitric oxide synthases for the common substrate, contributes to allergen-induced airway inflammation, hyperresponsiveness and remodeling. there is evidence that cigarette smoke and lipopolysaccharide (lps), both involved in the pathogenesis of copd, increase the expression of arginase, however, its role in the pathogenesis of copd is currently unknown. this study aimed to investigate the role of arginase in pulmonary inflammation and remodeling, using a guinea pig model of lps-induced copd. to this aim, guinea pigs were instilled intranasally with lps or saline twice weekly for 12 weeks and were pretreated by inhalation of the arginase inhibitor (2)s-amino-boronohexanoic acid (abh) or pbs. repeated lps exposure increased lung arginase activity, resulting in increased lornithine/l-arginine and l-ornithine/l-citrulline ratio's. both ratio's were reversed by abh treatment. repeated lps exposure also induced increased il-8 levels, neutrophils, goblet cells, hydroxyproline and airway collagen content in the lung, which were all abrogated by abh. moreover, repeated lps exposure increased right ventricular mass, indicative of pulmonary hypertension, which was similarly prevented by abh. in conclusion, increased arginase activity contributes to pulmonary inflammation, airway remodeling and right ventricular hypertrophy in a guinea pig model of copd, indicating that arginase inhibitors may have therapeutic potential in the treatment of this disease. (supported by msd). behavioral abnormalities in hcn3-deficient mice michalakis s., schöll-weidinger m., mader r., cao-ehlker x., fenske s., wahl-schott c., biel m. center for integrated protein science munich (cipsm) department of pharmacy -center for drug research, ludwig-maximilians-universität münchen, butenandtstr. 5-13, 81377 münchen, germany hcn3 encodes a hyperpolarization-activated and cyclic nucleotide-gated channel, which is expressed in various brain regions including thalamic, hypothalamic and habenular nuclei as well as brain stem and olfactory bulb. in this study we performed a comparative analysis of hcn3 -/and hcn3 +/+ mice using a battery of behavioral tests and telemetric biopotential measurements to evaluate a potential role of hcn3 in central nervous system function. in general, the knockout mice showed normal motor function as assessed by the rotarod and open field tests. telemetric home cage activity and core body temperature measurements confirmed a normal circadian behavior, but revealed a lower basal activity that concurred with decreased body temperature during the light phase and the light-dark transition phase. hippocampus-dependent spatial learning was normal. by contrast, hcn3 knockout mice showed more immobility than control mice on day two of the porsolt forced swimming test, which could reflect increased depressionlike behavior. however, center exploration in the open field test as well as performance in the light-dark transition and the elevated-plus maze tests was normal in hcn3 -/mice. this suggests that general anxiety was not changed in the knockout mice. in addition, hcn3 knockout mice were less active on the second day of the open field test, which supports a habituation phenotype. finally, hcn3 -/mice had higher burying scores in the marble-burying test, which is a test for certain aspects of obsessive compulsive disorder in rodents. taken together, genetic deletion of hcn3 in mice results in distinct behavioral abnormalities related to behavioral despair and expression of repetitive behaviors in response to mild stressors. mielke h. 1 , gundert-remy u. alcohol consumption when breast feeding is discussed controversially. some groups recommend breast pumping before alcohol consumption and feeding the stored milk instead of breast feeding after drinking alcohol. this study was performed to simulate the blood concentration in the breastfed baby and to assess the health impact. method: we established a physiologically based kinetic model. its parameters were calculated (partition coefficients tissue/blood ; schmitt, 2008) silva et al.,1993) . we simulated 1. the alcohol concentration in a breastfed neonate and a 3-month-old suckling infant after the nursing mother had consumed alcohol,2. the alcohol concentration in utero/fetal compartment during pregnancy assuming the identical alcohol consumption of the pregnant woman 3. the alcohol concentration during infant´s treatment of bloating by an approved herbal drug containing alcohol. results: peak maternal alcohol concentration was 0.59 ‰ after consuming 0.25 l of wine, peak concentration was 0.0033 ‰ in the newborn, 0.0038 ‰ in the 3-month-old infant and 0.38 ‰ in the utero/fetal compartment. the peak concentration after herbal drug treatment was 0.015‰ in the neonate and 0.015‰ in the 3-month-old infant, respectively. we discuss the results of the simulations and compare it with doses and published concentrations measured in experimental animals or in vitro studies. conclusions: we conclude that the recommendation "1 to 2 glasses of wine on occasion" (agence nationale d'accréditation et d'évaluation en santé, assante 2002) is in accordance with the simulation results presented here whereas stricter rules are not scientifically sound. (2002) http://www.has-sante.fr/portail/upload/docs/application/pdf/ breastfeeding_guidelines.pdf da silva et al. (1993) adenylyl cyclases (ac) mediate physiological responses in virtually all cells, where their regulation through receptors and g proteins results in the modulation of camp. in the present study we focused on the kinetics of interactions between the alpha-subunit from inhibitory g protein type 1 (gαi1) and adenylyl cyclase type v (ac5). these proteins were labeled with cfp and yfp, respectively. the dynamics of their interactions was monitored by means of high temporal resolution fret imaging in hek cells expressing unlabeled a2a-receptor and gβg subunits. to activate the signaling pathway, we applied agonist using a rapid superfusion device. application of norepinephrine resulted in the development of a fret signal, indicating interaction between gai1-cfp and yfp-ac5. after withdrawal of agonist the fret signal recovered with a remarkably slow time course compared to the deactivation kinetics of gi proteins reported previously (bünemann et al. 2003) . to further analyze the properties of the dissociation between gai1 and ac5 we measured in parallel the offset kinetics of the interaction between gai1-yfp and gβg-cfp (gi1-fret) after agonist withdrawal under comparable conditions. in addition we tested to what degree the coexpression of rgs4 accelerated the deactivation of gi proteins and the dissociation of gai1-cfp from yfp-ac5. these experiments revealed that in the absence of rgs4 the dissociation of gai1 from ac5 after agonist withdrawal takes about 3 times longer than the deactivation of gi proteins. in the presence of rgs4 this difference is even larger due to the pronounced acceleration of g protein deactivation. the dissociation of gai1 from ac5 was only marginally accelerated by rgs4. these observations lead us to hypothesize, that ac5 might trap activated g protein-subunits and thereby affect the g protein cycle by shifting the equilibrium towards activated g proteins. if this hypothesis is true, it should result in a left-shifted dose response curve compared to g protein activation dose response. in support of this hypothesis we found that the concentration response curve for gai1-ac5 interaction was several-fold leftward-shifted compared to the concentration-response curve of gi-protein activation under very similar conditions. influencing the dynamics of the g protein cycle by effectors may represent a novel and powerful mechanism for finetuning the sensitivity of receptor evoked responses in an effector-specific manner. obesity, the excessive accumulation of white adipose tissue (wat), has reached pandemic dimensions. the factors that determine fat mass are not fully understood, but adipocyte hypertrophy and adipokine secretion are thought to be important. in present study, we investigated the role of the cyclic gmp (cgmp) signaling pathway focusing on cgmp-dependent protein kinase i (pkgi) in white adipocytes. pkgi is expressed in wat, preadipocytes and differentiated adipocytes as demonstrated by real-time pcr, western blot and immunochemistry. differentiation of pkgifl/fl preadipocytes, using an optimized protocol, resulted in an enhanced lipid accumulation as evidenced by oil red o staining. deletion of pkgi in pkgifl/fl adipocytes infected with a cre lentivirus (lv-cre, pkgi0/0) exhibited reduced differentiation. analysis of the triglyceride (tg) content revealed a significant decrease of tg levels by 65% ± 1% in pkgi0/0 as compared to pkgifl/fl adipocytes. western blot analysis of white adipocytes showed a significant decrease of c/ebpalpha (19% ± 4.8%), ppargamma (66% ± 2.9%) and ap2 (37% ± 10.6%) expression in pkgi0/0 cells as compared to pkgifl/fl. treatment of 3t3-l1 cells with cgmp resulted in increased lipid accumulation and enhanced expression of fat marker genes. lentiviral overexpression of pkgi further increased differentiation. importantly, pkgi significantly induced mitochondrial biogenesis in 3t3-l1 cells. concomitant activation of pkgi in 3t3-l1 preadipocytes and treatment with the demethylating agent 5-aza-deoxycytidine significantly increased expression of uncoupling protein-1 (ucp-1) -a unique protein of brown fat cells. we found rhoa as major target of pkgi signaling with increased phosphorylation of rhoa at ser-188 in pkgi overexpressing cells. moreover, pkgi-dependent phosphorylation counteracts the effects of rhoa on insulin signaling as well as adipokine expression. taken together, pkgi is a key player in white adipocyte differentiation that regulates cell size and has an anti-inflammatory effect. pkgi decreases the secretion of proinflammatory adipokines via inhibition of rhoa signaling. in addition, activation of pkgi can establish a brown fat cell like phenotype during white adipocyte differentiation if the ucp-1 promoter is accessible. the rag gtpases, raga, ragb, ragc, and ragd form a subfamily gtpases of the ras-related superfamiliy. rag proteins are characterized by a modified ras-like gtpbinding domain and a unique c-terminal region lacking a lipid modification motif. interestingly, rag proteins have been proposed to function as heterodimeric complexes consisting of raga or ragb associated with ragc or ragd. rag gtpases have been implicated in the control of mammalian target of rapamycin (mtor) function, in particular in regulation of the nutrient-stimulated and/or hormone-regulated mtor activity. the protein kinase mtor is found as the catalytic subunit of two larger protein complexes referred to as mtor complex 1 and 2, mtorc1 and 2. under amino-acidrich conditions, activated mtorc1 promotes protein synthesis and inhibits autophagy, while under starvation autophagy inhibition is released. increasing evidence suggests that activation of rag gtpases contributes to mtorc1 function. thus, rag proteins were found to be associated with a protein complex termed ragulator, a major regulatory protein of mtorc1 function and guanine nucleotide exchange of rag gtpases within the rag-ragulator-complex were described to promote mtorc1 translocation to its functional lysosomal compartment. however, the guanine nucleotide exchange properties of rag proteins are poorly characterized, and it is currently unknown, how amino acids promote rag proteins to facilitate the formation of the active, raptor-binding state of the rag heterodimers. to characterize the guanine nucleotide exchange properties of the rag gtpases as momomers or heterodimers in more detail, recombinant rag proteins were expressed in bacteria and purified from this source to near homogeneity. first, the parameters of gdp/gtp exchange of each of these proteins were compared using the non-hydrolysable gtp analogon gtpgs. the results showed that the rag isoforms are distinct in their guanine nucleotide exchange activities. in particular, nucleotide exchange on raga and ragc, but not on ragb and ragd, was only observed at low concentrations of gdp and mgcl 2 in extraction and assay buffers, i.e. conditions favoring the gdp/gtp exchange. these findings may indicate that guanine nucleotide exchange on raga and ragc is controlled by guanine nucleotide exchange factors and suggest specific functions of the individual rag gtpases within individual rag heterodimers. in in-vitro studies on rat and canine mast cells and human mast cell leukemia cells hmc1.2 bz at micromolar concentrations inhibited mediator release which appeared to be related to an inhibition of the intracellular camp pathway. in order to identify potential targets on/in mast cells at which bz may cause an inhibitory effect on mast cell activation, the 1,4-bz flunitrazepam (flu), clonazepam (clo) and 4chlorodiazepam (4-cd) were selected because of their different affinity and selectivity to/for the gaba-a-receptor and the translocator protein (tspo): flu and clo bind with nanomolar affinity to gaba-a receptors, whereas 4-cd is a selective ligand at tspo with nanomolar affinity to tspo but only micromolar affinity to gaba-a receptors. flu also possesses nanomolar affinity to tspo, whereas clo has no or only micromolar affinity to tspo. after incubation of hmc1.2 cells with 4-cd, flu and clo for 1, 6 and 24 hours up to 712 genes were significantly differently expressed in a substance-specific and timedependent manner. comparison of the genes differently expressed at 6 hours revealed that the expression of 217 genes was regulated by both flu and clo but only 8 genes were regulated by both 4-cd and flu suggesting that flu and clo induce gene expression by acting at a target site different from that of 4-cd. the difference between the gene regulation by flu and clo on the one hand and that of 4-cd on the other hand is also reflected in pathway analysis. since it was conceivable that the beneficial effects of the 1,4-bz could be mediated by the recognition sites targeted by the 2,3-bz, i.e. the gria2-encoded ionotropic glutamate receptor ampa2, we investigated by quantitative pcr whether hmc1.2 cells express gria2, tspo, the genes encoding the subunits of the gaba-a receptor and the gaba-forming enzyme glutamic acid decarboxylase. tspo, gabra3, gabrb3, gabre and gabrd were moderately expressed. in addition, there was a week or very week expression of gabra2, gabra4, gabrb2, gabrg3 and gabrr2. expression of gria2 was not detectable. taken together, it cannot be decided yet from our data whether the inhibitory effect of benzodiazepines on mast cell activation is due to an action at tspo or at gaba-a receptors of a novel subunit composition. monien b. h., glatt h. german institute of human nutrition (dife) department of nutritional toxicology, arthur-scheunert-allee 114-116, 14558 nuthetal, germany 5-hydroxymethylfurfural (hmf) and furfuryl alcohol (ffa) are common constituents of foodstuffs in which they are formed by heat-and acid catalyzed reactions from carbohydrates. hmf and ffa have been reported to induce the formation of hepatocellular adenomas in female mice and renal tubule neoplasms in male mice, respectively. we studied whether the carcinogenic effect of these hydroxymethylsubstituted furans may originate from sulfotransferase (sult)-catalyzed formation of electrophilic esters. hmf was inactive in in vitro mutagenicity tests using standard activating systems. in contrast, it was mutagenic in v79 cells genetically engineered for expression of human sult1a1 suggesting that hmf is converted into the reactive 5sulfooxymethylfurfural (smf). following incubation of mutagenic smf with porcine liver dna in vitro, specific methylfurfural adducts were detected using liquid chromatography tandem mass spectrometry (lc-ms/ms), i.e., n 6 -((5-formylfuran-2-yl)methyl)-2'deoxyadenosine (n 6 -ffmda) and n 2 -((5-formylfuran-2-yl)methyl)-2'-deoxyguanosie (n 2 -ffmdg). these adducts were also detected in dna from v79-sult1a1 cells incubated with hmf. in order to determine sulfo conjugation of hmf in mice in vivo, we conducted pharmacokinetic measurements showing that about 500 ppm of the hmf dose was converted to smf and reached the circulation. like hmf, ffa was negative in the standard ames test and various other in vitro genotoxicity tests. we showed that ffa is mutagenic in salmonella typhimurium ta100 engineered for expression of human sult1a1. the putative mutagen 2-sulfooxymethylfuran was synthesized and incubated with porcine liver dna, in which various nucleoside adducts were found. the main adducts, -mfda were detected in dna of ffa-exposed salmonella strain ta100-sult1a1 and in dna of liver, lung and kidney of fvb/n mice that had received about 390 mg ffa/kg body weight per day via the drinking water for 28 days. in summary, both furan derivatives form mutagenic sulfate esters in vitro and in vivo. in the future, we will use genetically engineered mice to characterize the role of single murine and human sult forms in the bioactivation of the furan derivatives and the contribution to tumor induction. background: micrornas are small non-coding rnas that can negatively regulate gene expression on a post-transcriptional level and have been shown to interact with epigenetic mechanisms like dna methylation. mecp2 (methyl cpg binding protein 2) is a protein that binds methylated dna cpgs in the promoter region of genes and can thus regulate their expression. otherwise, mecp2 is known to be a target gene for several micrornas including the cluster mir-132/212 in the brain. recently, our group could show that mecp2 expression is downregulated in human heart failure suggesting that mecp2 might be involved in cardiac pathogenesis. the aim of this project is to study the upstream regulation of mecp2 by the cluster mir-132/212 in the heart during cardiac hypertrophy in-vitro and in-vivo. methods and results: to test whether hypertrophic stimuli can induce mir-132/212 expression, we treated cultured nrcms with 40 µm norepinephrine for 72 hours. this induced cardiomyocyte hypertrophy and expression of the hypertrophy marker nppa, but also of mir-132 (1.79 ± 0.14-fold of untreated cells, p<0.01) and of mir-212-3p (1.73 ± 0.26-fold of untreated cells, p<0.05) and downregulated mecp2 mrna and protein levels (0.80 ± 0.04-fold of untreated cells, p<0.05). to check whether mecp2 downregulation also occurs by direct mir-132/212 activation we increased levels of mir-132 and mir-212 in cardiac myocytes by transfecting precursor mir-132 and mir-212-3p molecules. again, we observed nrcm hypertrophy, nppa mrna upregulation and mecp2 mrna and protein downregulation (0.75 ± 0.05-fold of control, p<0.05) after mir-132 overexpression. similar results were obtained by overexpression of mir-212-3p. to test the effects of adrenoceptor activation on the mir-132/212-mecp2 axis in-vivo, wild-type mice received isoprenaline and phenylephrine via osmotic minipumps (30 mg/kg/day each). after 7 days, cardiac ventricles were analyzed. nppa gene expression (2.81 ± 0.32 -fold of control animals), mir-132 and mir-212-3p levels (3.67 ± 0.5 and 2.62 ± 0.4 -fold of control animals, p<0.01 and p<0.05, respectively) were increased while mecp2 protein levels decreased to 77% (p<0.05) conclusion: these results suggest that in-vitro and in-vivo adrenoceptor stimulation leads to the activation of mir-132/212 expression and to downregulation of mecp2 in cardiac myocytes in-vitro and in-vivo. leopold-franzens-universität, innsbruck, austria at-1 receptor antagonists block the angiotensin ii-enhancing effect on noradrenaline release from sympathetic neurons. in a cell-free assay the binding affinity of the at-1 receptor antagonists telmisartan and valsartan to the gamma peroxisome proliferatoractivated receptor (pparγ) is close to that of the pparγ selective agonists thiazolidinediones (tzds). we tested whether the tzds rosiglitazone and pioglitazone would also modify the prejunctional facilitatory effect of angiotensin ii. left ventricular slices of rats were incubated with tritiated noradrenaline, perifused and electrically stimulated. the negative logarithm of the drug concentration that caused a 30% increase of control (pec30%) was calculated. angiotensin ii caused a concentration-dependent increase of tritium overflow induced by electrical stimulation [pec30%=8.6±0.2 (mean±sem, n=18); maximum increase=110±8%]. neither rosiglitazone nor pioglitazone (0.3-3 µm) had a direct effect. the concentrationresponse to angiotensin ii in the presence of fixed concentrations of rosiglitazone was shifted to the left with increase of the maximum (pec30%=8.8±0. 2, 9. 2±0.2 and 9.3±0.3; maximum increase=118±14%, 146±13% and 148±16%, in the presence of 0.3, 1 and 3 µm of rosiglitazone, respectively, n=4-6, each). in contrast, pioglitazone in concentrations up to 3 µm had no effect on the release-enhancing effects of angiotensin ii. results show that rosiglitazone but not pioglitazone potentiates the noradrenalinerelease enhancing effect of angiotensin ii. this action might contribute to the risk for myocardial infarction from rosiglitazone use but not from pioglitazone use. deleted in liver cancer 1 (dlc1) is a tumor suppressor whose allele is lost in 50% of liver, breast, lung and 70% of colon cancers. despite its significance, the molecular mechanisms that drive cancerous transformation upon dlc1 loss remain unclear. we found that the transcriptional coactivators megakaryoblastic leukemia 1 and 2 (mkl1/2) are constitutively localized to the nucleus in hepatocellular and mammary carcinoma cells that lack dlc1. moreover, dlc1 loss and mkl1 nuclear localization correlated in primary human hepatocellular carcinoma. nuclear accumulation of mkl1 in dlc1-deficient cancer cells was accomplished by activation of the rhoa/actin signaling pathway and concomitant impairment of erk-mediated mkl1 phosphorylation. dlc1 loss led to constitutive activation of the mkl-dependent, tumor-relevant target genes ctgf, cyr61, myl9 and myh9. furthermore, we identified a novel target gene, integrin a5, with a key role in cell migration and metastasis, that exhibited a dlc1-and mkldependent regulation. depletion of mkl1/2 suppressed not only cell migration, but also cell proliferation and anchorage-independent cell growth induced by dlc1 loss. our data provide insight into the mechanism by which dlc1 loss initiates tumorigenesis. as mkl1 and 2 have a key role in this process, this pathway may provide promising pharmacological targets for cancer therapy. universität bonn, pharma-zentrum bonn pharmazeutisches institut, pharm. chemie i, an der immenburg, 53121 bonn, germany membrane receptors activated by purine and/or pyrimidine nucleotides ("p2 receptors") are widely distributed in the body and constitute novel (potential) drug targets. they are subdivided into g protein-coupled p2y receptors (p2y1, 2, 4, 6, 11, 12, 13, 14) , and homo-or heterotrimeric ligand-gated ion channel or p2x receptors (subunits: p2x1-7). we have been interested in the identification and development of potent and subtype-selective ligands -as tool compounds and potential drugs -for the various p2y and p2x receptor subtypes. our strategy involves (i) establishment of a proprietary compound library consisting of synthetic small molecules and natural products; (ii) development of screening assays suitable for medium throughput screening; (iii) careful analysis of structure-activity relationships at each target and systematic optimization of the lead structures; (iv) pharmacological evaluation of selected compounds. this approach has led to new biological tools for several targets, including p2y and p2x receptors [1] [2] [3] [4] [5] [6] . fine particles in particulate matter (pm) are effective vehicles to transport toxicants into the lung; depending on their size, smaller particles may reach the bronchiolar or alveolar space. in recent years the pm fraction pm2.5 has especially been correlated with both pulmonary and cardiovascular diseases. in order to better characterize pm emission and distribution of environmental tobacco smoke (ets) from cigarettes (reference cigarette (rc), brand cigarette (bc)) we have developed an ets emitter to simulate human smoking emission and measured pm2.5 concentration in a telephone booth (1,75 m3 volume) as an example for small indoor spaces like cars. fine particulate matter was measured using an aerosol spectrometer with 6 sec time resolution; laser scatter allowed a size resolution from 0,25 µm to 32 µm. for the pm2.5 concentration the following values were calculated: cumulative pm2.5 concentration as auc-pm2.5 (µg/m3/sec), peak pm2.5 concentration as cmax-pm2.5 (µg/m3) and average pm2.5 concentration cmean-pm (µg/m3). in closed door condition both cigarettes produced particulate auc-pm2.5 values of 59 000 ± 15 000 µg/m3/sec (rc) to 85 000 ± 31 000 µg/m3/sec (bc after myocardial infarction (mi) inflammatory cells and cardiac fibroblasts (cf) determine the remodeling response. interleukin-6 (il-6) is induced in the ischemic myocardium and is known to stimulate the differentiation of fibroblasts to myofibroblasts. hyaluronan (ha) is an extracellular matrix component synthesized by ha-synthase isoenzymes (has 1-3) and is also known to control fibroblast phenotypes. however, it is presently unknown whether il-6 participates in the remodeling of the ha-matrix or whether the ha-matrix modulates the responses to il-6. therefore, the aim of the present study was to elucidate whether il-6 regulates the expression and function of ha-matrix in cfs. cells were isolated from c57bl/6j mice and used during passage 2-3 for experiments. cfs were stimulated with il-6 or hyper-il-6 which is a fusion protein of il-6 and soluble il-6 receptor (sil-6r). after 10 and 30 min signal transducer and activator of transcription 3 (stat3) was phosphorylated in response to hyper-il-6 but not in response to il-6. rt-pcr revealed rapid upregulation of has 1 (5.94 ± 2.49 fold of unstimulated control, 1h) in response to hyper-il-6. has2 was induced to a lesser degree (2.04 ± 0.55 fold of unstimulated control, 1h) whereas has3 was not responsive (1.49 ± 0.42 fold of unstimulated control, 1h). in contrast, il-6 had no effect on transcript levels of has isoenzymes. in turn, expression of has1 and has2 in response to hyper-il-6 was inhibited by ag490, which indicates the involvement of stat signaling. interestingly, despite induction of has1 and has2 the amount of secreted ha as determined by an elisa-like assay was not affected by hyper-il-6. this may indicate that il-6 regulates the cell surface associated ha-matrix of cfs. in conclusion, the present data demonstrate that cardiac fibroblasts respond to il-6 trans-signaling (hyper-il-6) via the soluble il-6r and subsequent stat3 signaling with increased ha-synthesis. the fact that il-6 had no significant effect suggests that the expression of the non-signaling membrane-bound il-6 α-receptor (il-6r) in cultured murine cardiac fibroblasts is not sufficient to induce has 1 and -2 gene expression. therefore, il-6 trans-signaling mediated by il-6 and the circulating sil-6r might be necessary to mediate the il-6-induced has expression in vivo. mrgprd receptor endogenously expressed in dorsal root ganglia: evidence for an activation by 3-aminoisobutyric acid müller s., hoffmann k., von kügelgen i. universität bonn institut für pharmakologie und toxikologie, sigmund-freud-straße 25, 53127 bonn, germany the gpcr mrgprd (mrgd) is highly expressed in small diameter dorsal root ganglion (drg) neurons and has been implicated to play a role in nociception. the receptor was previously shown to respond to β-alanine. in the present study we searched for agonistic activity of structural analogues of β-alanine. for further characterization of the receptor we used fura-2 fluorimetry, a nfat luciferase reportergene assay and the determination of the inhibition of forskolin-induced camp production ([ 3 h]-camp affinity assay). first, we confirmed the activation of the receptor by β-alanine and gaba. in reportergene experiments we then identified 3-dlaminoisobutyric acid as an agonist, with similar potency but weaker affinity when compared to β-alanine (ec50 165 µm). fura-2 fluorimetry showed an increase in intracellular ca 2+ levels by 3-dl-aminoisobutyric acid (300 µm). moreover, 3-dlaminoisobutyric acid reduced the forskolin-induced camp production by up to 65 % (ec50 195 µm). in addition to 3-dl-aminoisobutyric acid, we identified 3-dl-aminobutyric acid as a weak agonist acting at the mrgprd. other closely related substances failed to show significant responses. next to the agonists we further characterized antagonists inhibiting the response to βalanine mediated by mrgprd. chlorpromazine shifted the concentration-response curve of β-alanine to the right with an apparent pkb of 4.9 (nfat assay), thioridazine with an apparent pkb of 5.4 (nfat assay) and 5.3 (camp assay) and rimcazole with an apparent pkb of 5.2 (nfat assay) and 5.2 (camp assay). in conclusion we show for the first time that 3-dl-aminoisobutyric acid is an agonist at the mrgprd and that the structure-activity relationship of agonists at mrgprd is very close. the sdf-1-chemokine receptor cxcr4 plays a key role during embryogenesis and regulates functions of immune and stem cells in adult life. furthermore, cxcr4 is involved in disease states including inflammation and cancer. it is well established that sdf-1-stimulated cxcr4 receptors activate gi protein-dependent signal transduction pathways and undergo c-terminal phosphorylation and internalization. because the cxcr4 c-terminal domain contains 15 serine and 3 threonine residues, it is incompletely understood which of the potential phosphorylation sites contribute to homologous and heterologous regulation of cxcr4. here, we analyzed the phosphorylation pattern of cxcr4 at 3 c-terminal sites after stimulation of the receptor with sdf-1 and after pma-induced activation of the pkc pathway as a model for heterologous receptor phosphorylation. using phospho-specific antibodies against s324/325, s338/339 and s346/347 in immunoblot analyses, we showed that the 3 sites were phosphorylated after stimulation with sdf-1 or pma. stimulation with egf or forskolin did not induce phosphorylation at these sites. sdf-1-induced phosphorylation at s324/325, s338/339 and s346/347 was reversible after wash out of the ligand. time course analyses revealed that phosphorylation occurred first at s346/347 and then at s324/325 and s338/339. taken together, these results indicate that the c-terminus of cxcr4 is phosphorylated at multiple sites by homologous and heterologous pathways and that phosphorylation at the different sites may be hierarchically organized. human milk represents the best form of nutrition for infants early in life. however, it can also contain toxic contaminants that may adversely affect infant's development. the nephrotoxin ochratoxin a (ota) is present in human milk (tab. 1 in [1] ), but information on transfer from maternal blood to milk is scarce: published data [2] indicate that levels of ota in milk are roughly one tenth (0.1) of those in blood. but, the efficiency of the ota-transfer at various stages of breastfeeding may vary since studies in animals revealed that transfer of ota is apparently time-and dose-dependent. thus, the aim of this study was to assess the ota transfer from blood to milk at different stages of breastfeeding in humans. in a small chilean cohort, 18 lactating women were asked to provide blood and milk on the same day. these samples were collected on four different occasions within the first months after delivery and analyzed using hplc with fluorescence and/or tandem mass spectrometric detection [1] . the transfer of ota from blood to milk was quantitatively assessed by measuring the milk to plasma ratio (m/p). the average ota level in blood plasma was 235 ± 129 ng/l, and no major variations were observed over time (p = 0.19). on the other hand, ota levels found in colostrum (85 ± 60 ng/l) were higher than in mature milk (p < 0.05). in line with these data, higher m/p ratios (table) were obtained with samples collected in the first six days after delivery. this study showed that the transfer of ota from blood to milk was more efficient with colostrum (m/p 0.43 ± 0.26) than with mature milk. thus, a higher exposure to ota can be expected for neonates than for infants at later stages of breastfeeding. moreover, the lactating women have lower average ota levels in plasma than non lactating women from chile [3] , indicative of milk as additional excretion route. acknowledgement: this work has been supported by a stipend from conicyt/daad to km. exposure of infants to ochratoxin a (ota) deserves particular attention since ota is nephrotoxic, and one of the most potent rodent renal carcinogen studied to date [1] . moreover, infants may be more vulnerable to the toxic effects of contaminants than adults. ota-levels in plasma of infants are indicative of an early exposure in life [2] . but blood sampling is an invasive method not readily applicable for breastfed infants. thus, the aim of this study was to implement a non invasive biomonitoring method to assess ota-exposure in this group. to assess the exposure to ota, breast milk and infants' urine specimens were collected, from two different cohorts: chile (n= 28) and turkey (n=10, only urine). analysis of the samples was performed using enzymatic hydrolysis prior to extraction and hplc-ms/ms [3] . the magnitude of infants' exposure was assessed by calculating the ota-daily intake with human milk and relating it also to urinary ota levels. calculations of the daily intake with human milk [4] showed that infants may be exposed to ota at high levels, exceeding the tolerable daily intake (tdi) of 5 ng/kg-bw/day set for adults [1] . in both cohorts, most of the urine samples tested positive for ota (chile 72%, turkey 80%). ota levels observed in urine samples from the turkish infants (range: 116 -1,361 ng/l) were 10 fold higher than levels found in chilean samples (range positive samples: 17 -320 ng/l). further analysis of phase ii metabolites in urine confirm the excretion of ota as conjugate (glucuronide) in highly exposed infants. in conclusion, ota exposure of infants early in life was documented. given that otaintake by several infants exceeded the tdi for adults, further biomonitoring in this vulnerable group is advised including also suitable biomarkers of effect. a mixture of (e)-and (z)-clomiphene citrate is the first line therapy of female infertility. however, up to 30% of patients do not respond. (e)-clomiphene is structurally closely related to another selective estrogen receptor modulator, tamoxifen which is frequently used for the treatment of hormone receptor-positive breast cancer. like tamoxifen, clomiphene is extensively metabolised by the cytochrome p450 system. using the estrogen receptor response assay (e)-4-hydroxyclomiphene and (e)-4-hydroxy-n-desethylclomiphene (ec50: 2.5 and 1.4 nm, respectively) turned out to be 100 times more active at the er compared to the parent drug isomers and de-ethylated metabolites. using recombinant expressed human cyp isoforms and inhibitory antibodies, cyp2d6 revealed to be the major isoenzyme involved in the formation of 4-hydroxlated metabolites. n-deethylation was catalysed by cyps 3a4/5, 2d6, 2c19 and 2c8. rates of 4-hydroxylation in microsomes from 30 human liver donors correlated with the number of functional cyp2d6 genes. these in vitro results were confirmed in a pharmacokinetic study with female healthy volunteers receiving a single dose of clomiphene. in carriers of two non-functional cyp2d6 genes (poor metabolizers) cmax of (e)-4-hydroxyclomiphene and 4-hydroxy-ndesethylclomiphene was 8 and 12 times lower, respectively, when compared with subjects with at least one fully functional cyp2d6 allele. in contrast, half-life of (e)-clomiphene and (e)-n-desethylclomiphene was 10 and 50-fold higher, respectively, in poor metabolizers. our data provide first evidence of a pharmacogenetic rational for the variability in the response to clomiphene treatment. among the tested compounds, compound 1 proved to be the most active derivative, showing a significant toxicity at a concentration of 2,5 µm. compounds 2 and 3 showed significant toxic effects at a concentration of 5 µm. the compound 4 showed no toxicity up to a concentration of 100 µm. all derivatives 1, 2 and 3 have a ec50 between 10 and 15 µm. we further proved the induction of apoptosis by apo-one assay (caspase 3/7 activity) and life/dead-assay (fluorescence microscopy). in conclusion, these gold complexes exhibit an example of interesting potential candidates for future anticancer pharmaceuticals due to relatively high cytotoxicity. gene regulating effects in mouse liver subsequent to treatment with selected dioxin-like compounds and pcb 153 using whole genome microarray analysis neser s. 1 , lohr c. 1 , van ede k. i. 2 , andresen k. 3 interaction with the aryl hydrocarbon receptor (ahr), with 2,3,7,8-tetrachlorodibenzo-pdioxin (tcdd) being the most potent congener amongst the ahr agonists. recent risk assessments have employed the toxic equivalency factor (tef) concept. the current eu-project systeq aims at developing, validating, and implementing human systemic tefs as indicators of toxicity for dl-compounds. at present, the best known parameter of ahr mediated effects is the induction of cytochrome p450 isoenzymes (cyps), i.e., cyp1a1, 1a2, and 1b1. one of the major objectives of the systeq project is the identification of novel quantifiable biomarkers. in a three day study, female c57bl/6 mice were treated with single doses of six dl-congeners (tcdd, pcb 118, pcb 126, and pcb 156) , and the 'non-dioxin-like' (ndl) pcb 153. quality tested (agilent ® bioanalyzer) mrna isolated from livers was analyzed using the agilent ® mouse whole genome array (4x44k) system. the quantity of genes affected (≥ 2fold) was highly heterogeneous amongst the dl-compounds. whereas tcdd-treatment upregulated 89 genes, and down-regulated 66, 4-pncdf-treatment had impact on 3208 (up), and 2396 (down). treatment with pcb 118 led to marginal numbers of 16 up and 6 down-regulated genes. with 64 (up), and 38 (down) genes shared, the most extensive overlap occurred between tcdd-and 1-pncdd-treatment. no overlap was found due to treatments with the ndl pcb 153 (21 up, 1 down) and tcdd. when comparing the effects of all dl-congeners, minor numbers of genes of 19 up, and 5 down-regulated remain, most of them being related to drug metabolism. while pcb 118 regulated only genes involved in drug metabolism, omission of pcb 118-regulated genes resulted in consistently 51 (up), and 24 (down) regulated among dl-compounds. in conclusion, our findings suggest that the pattern of gene regulation in mouse liver elicited by pcb 153 was strictly different from tcdd, while a very limited coincidence of genes was found even among dl-compounds. comparison of these 'core' genes with data from human models is required with respect to determination of novel biomarkers. introduction: proper use of antibiotics is essential with regard to effective treatment of bacterial infections. providing adequate information for patients can contribute to achieve this aim. materials and methods: data was collected from the relational database of the drug information service at dresden university of technology. the patients, who used the service, were interviewed concerning socio-demographic characteristics, reason for enquiry, number and kind of drugs taken, and diseases. possible contact paths were phone, e-mail or letter. in the present evaluation, all enquiries from the years 2009 and 2010 were analyzed descriptively focussing primarily on systemic antibiotics as reason for the enquiry. results: in the evaluated period, 4454 enquiries were registered in total. in 4.7% of those enquiries systemic antibiotics were named with a total number of 283 drugs. 50.9% of those antibiotics were found to be the direct reason for the enquiry. most common information requested by patients corresponded to adverse drug reactions (50.7%), diagnosis/treatment (35.4%), drug application (29.2%) and drug-druginteractions (19.4%). the majority of the requesting patients (61.5%) was born before 1970. a correlation between incidence of enquiries especially concerning antibiotics and quarterly statistics could not be detected. conclusion: mainly patients aged 40 years or more seem to need or search for further information about antibiotic medication. advice is required especially regarding adverse drug reactions and diagnosis or treatment. in order to this, the advisory service can help patients to lose their insecurity and to gain more confidence in handling antibiotic drugs. colon cancer is one of the most frequent cancers in the industrialized nations. epidemiological studies show a correlation between highly processed meat and the development of colorectal tumours. it is assumed that the risk of developing colorectal cancer, among various different factors, is related to the uptake of toxic substances contained in food such as heterocyclic aromatic amines that arise during the processing of fish and meat. phip is the most abundantly formed heterocyclic compound, and therefore has the biggest impact. in a previous study, we measured the absorption of phip in different intestinal segments of the rat. in the present study we focussed on the potential mechanisms by which phip is reabsorbed. the unidirectional phip transport from the mucosal to the serosal compartment (j ms) and in the opposite direction (jsm) was examined using the ussing chamber technique and 14 c-phip as a radiotracer. the proximal jejunum and distal colon of male fischer 344 rats in short-circuit current chambers was clamped, so that mucosal and serosal compartments were built. the phip flux rates were determined at defined intervals over 120 min. the experimental conditions were selected in such a way that negative net flux rates (jnet = jms-jsm) were indicative of an active secretion. both intestinal segments showed large differences. while in the jejunum jms and jsm of phip were not significantly different, there was an active secretion in the colon. in a next step the transport proteins involved in this process should be examined. introduction: human organic anion transporter 2, oat2 (slc22a7), is abundantly expressed in kidney and liver and mediates the sodium-independent uptake of clinically relevant drugs like 5-fluorouracil, paclitaxel, bumetanide, tetracycline, and zidovudine. while immunohistochemical studies have localized human oat2 to the basolateral membrane of kidney proximal tubules, its hepatic localization is currently unknown. we, therefore, firstly determined oat2 localization in human liver. because interindividual variability of oat2 expression may affect hepatobiliary drug uptake and elimination, we next systematically investigated the influence of genetic and non-genetic factors on hepatic oat2 expression. methods: an expression profile of oat2 for 20 human tissues was determined by realtime quantitative polymerase chain reaction (taqman). oat2 mrna expression was analyzed in well-characterized human liver samples from 150 caucasians that were accompanied by detailed demographic and clinical data. oat2 was localized in human liver cryosections using a commercial rabbit polyclonal antibody and hepatic oat2 protein levels were determined. resequencing, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and genome-wide single nucleotide polymorphism microarray technology served to genotype variants in the slc22a7 gene region. results: oat2 mrna was expressed in several human tissues, including liver. moreover, a new alternatively spliced variant of oat2 was identified in human liver. hepatic expression of full-length oat2 mrna and oat2 protein varied 37-fold and 41fold, respectively. oat2 mrna and protein levels did not correlate with each other. oat2 was localized to the sinusoidal membrane of human hepatocytes. no novel variants in the 9 exons, the 5'-flanking region, or the 3'-untranslated region of the slc22a7 gene were identified. univariate analysis showed that oat2 mrna is reduced in patients diagnosed for cholestasis (p=0.0012) and is affected by genetic variants. whereas the influence of genetic variants on hepatic oat2 expression appears to be limited, cholestasis significantly contributes to the variable interindividual oat2 expression. this indicates consequences for hepatic drug elimination of and response to oat2 drug substrates such as paclitaxel or tetracycline. the life threatening toxicity of organophosphorus (op) nerve agents is caused by the inhibition of the acetylcholine esterase (ache). oximes were shown to be potent reactivators of inhibited ache, but in poisoning by some compounds, e.g. soman, they have only a small therapeutic effect. for such cases, an alternative new strategy may be the intervention at nicotinic acetylcholine receptors (nachr). previous studies with the bispyridinium non-oxime mb327 demonstrated therapeutic effects against soman in vitro and in vivo which was partly attributed to its direct interaction with nachrs [1] . we investigated the interaction of mb327 and several structure analogous at the orthosteric binding site of human α7 nachr (hα7 nachrs), a subtype which appears to be widespread in the human body, and compared the results with data obtained from torpedo-nachrs, which show a high degree of homology with human muscle-type nachrs. interaction of compounds with the orthosteric binding site of hα7 nachrs were investigated with radioligand binding experiments performed as high-throughput method [2] . membrane preparations of gh 4c1 cells stably expressed hα7 nachrs were incubated with the nachr agonist [³h] epibatidine and appropriate concentrations of the unlabelled competitors e.g. bispyridinium compounds. after incubation, bound and free [³h] epibatidine were separated by rapid vacuum filtration. ki values of the competing compounds were calculated with nonlinear regression. three bispyridinium compounds, mb442, mb456 and mb770 exhibited ki values at micromolar concentrations while three other compounds, mb327, mb583 and the pharmacological inactive mb424 (negative control) did not show any interaction with the orthosteric binding site of hα7 nachrs. with torpedo-nachrs, ki values were in similar orders of magnitude -except mb442 which indicated significant subtype selectivity. interestingly, the affinity of monomeric pyridinium derivates did not correlate with their bispyridinium structure analogues. obviously, no correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission exists, although species-related differences cannot be excluded. in this study, we analysed the cytotoxic and clastogenic effects of the anticancer drug nimustine (acnu) in cells deficient in repair proteins involved either in homologous recombination (hr) or non-homologous end-joining (nhej). we show that hr mutants are extremely sensitive to acnu as measured by the induction of apoptosis and colony formation as well as the induction of chromosomal aberrations. the nhej mutants were slightly sensitive to acnu and differed in their sensitivity, with the ku80 mutants being moderately sensitive and the dna-pkcs mutant resistant, comparable to the wild-type (wt). cell death was mostly executed via the caspase-dependent apoptotic pathway with involvement of caspase-3 and -7, and necrosis was also induced. further, we investigated the kinetics of dna double-strand break (dsb) formation that resulted from the repair of acnu-induced interstrand cross-links by means of γh2ax and 53bp1 foci analysis in wt and mutant cells. cells mutant in hr did not repair dsb and went into the apoptotic or necrotic pathway, whereas wt cells were able to repair most of the dsb. cells deficient in ku80 formed at early times after acnu treatment less γh2ax and 53bp1 foci compared to the corresponding wt, which might be due to a reduced capacity of recognising dsb. at later times after treatment, ku80 mutant cells show foci levels similar to the wt indicating restitution of h2ax phosphorylation. we also analysed whether dsb formation after acnu treatment was replication-dependent using synchronised cells. we determined the formation of γh2ax and other dsb marker in wt cells that passed through the first cell cycle after demecolcine synchronization. the level of γh2ax foci increased significantly in the s-phase and remained at a high level during g2 where a fraction of cells remained arrested. rad51, atm, mdc-1 and rpa-2 foci were also formed and shown to co-localize with γh2ax. these foci were ameliorated significantly in s-and g2-phase, which was similar to the time course of γh2ax foci formation. in western blots, we confirmed a higher phosphorylation level of atm and chk2 and less phosphorylation of chk1 in hr mutants. the data indicate that acnuinduced dna cross-links give rise to cyto-and genotoxicity via the formation of dsbs that activate the cellular dna damage response. the endocannabinoid system has been established as a mediator of numerous central and peripheral biological functions. cannabinoids have emerged as attractive alternatives or supplements to therapy with opioids for chronic pain states. however, in human the activation of cannabinoid receptors is associated with side effects. for clinical exploitation of the analgesics properties of cannabinoids, a major challenge is to devise strategies that reduce or abolish their adverse effects on cognitive, affective and motor functions without attenuating their analgesics effect. in animal studies, the anti-nociceptive efficacy of cannabinoids has been unequivocally demonstrated in several models of inflammatory and neuropathic pain. however, there are marked inconsistencies between different reports with respect to the locus of these pain-protective effects. we are working towards establishing the contribution of cb1 receptors expressed on the peripheral terminals of nociceptors to cannabinoid-induced analgesia. using cb1 globally knock-out animal as background, we induce the expression of cb1 specifically in nociceptive neurons localized in the peripheral nervous system and test the analgesic effects of cannabinoid systemical delivery in these mice. our results support the development of peripherally acting cb1 analgesic agonist with reduced central side effects. furthermore, we are utilizing proteomics approach to identify protein complexes that interact with cb1 receptor which hold promise in understanding cannabinoid signaling in health and disease. most chemoattractants, including chemokines, complement c5a, fmlp, and leukotriene b4 are signaling through heterotrimeric g proteins of the pertussis toxin (ptx)-sensitive gi family. the functional inactivation of all gαi proteins with ptx leads to a fulminant decompensation of the immune system, whereas the constitutive inactivation of a single gαi coding gene results in mild phenotypes in mice. we are mostly interested in the nonneuronally found gαi2 and gαi3 isoforms and their redundant and specific roles in immune function and infection. for this purpose cellular in vivo and ex vivo models and in vivo infection model with listeria monocytogenes are being used. macrophages were isolated from the peritoneal cavity of wild type (wt) and gαi-deficient mice 4 days after i.p. injection of 4% thioglycolate that induces peritonitis in vivo. we confirmed previous observations that in gαi2-deficient mice the migration of macrophages into the peritoneal cavity was reduced after induction of peritonitis. regarding the expression levels of gαi and gβ isoforms in the lavage samples, the predominant gαi isoform gαi2 was upregulated in gαi3-deficient macrophages. vice versa gαi3 was upregulated in gαi2-deficient macrophages. concerning gβ isoforms, both gβ1 and gβ2 were strongly reduced in the gαi2-deficient macrophages which resulted in a reduced total amount of gβ. surprisingly, the gαi3-deficient macrophages showed reduced gβ1 protein levels only which caused a change in the gβ2/ gβ1 quotient in favour of gβ2. we are currently establishing an in vivo infection model with l. monocytogenes in gαi2and gαi3-deficient mice. our previous in vitro infection studies in mice embryonic fibroblasts provided us with information about possible distinct roles of these two isoforms as far as the uptake of l. monocytogenes in the cells is concerned. challenging the immune system of gαi-deficient mice with this pathogenic organism will give us new insights into the systemic immune response in these mice upon bacterial infection. our data indicate that we may surmount the redundancy between these two isoforms and focus on their distinct and specific roles in pathogen defense. fret-based β-arrestin2 biosensors reveal conformational changes upon binding to the β2-adrenergic receptor in real time and living cells nuber s., zabel u., ziegler n., hoffmann c., lohse m. j. institut für pharmakologie und toxikologie pharmakologie, versbacherstr.9, 97078 würzburg, germany β-arrestins are multifunctional adapter proteins that regulate seven transmembranespanning receptor (7tmr) signaling and initiate also alternative signaling pathways. studies have shown that β-arrestins undergo conformational changes upon receptor stimulation, which are thought to be necessary for its downstream actions. to investigate these conformational changes in living cells we constructed fret based biosensors of β-arrestin2, in which cfp was fused to the c-terminus and the flashbinding motif (ccpgcc) was inserted to different positions within the n-or c-domain of β-arrestin2. upon β2-adrenergic receptor (β2ar) stimulation we observed a decrease of the intramolecular fret signal between cfp and flash at the n-domain (β-arrestin2flash2), indicating a conformational change moving the c-terminus and the ndomain of β-arrestin2 relative to each other. kinetic analysis revealed that this conformational change immediately follows β-arrestin2/β2ar interaction on a timescale of seconds. a β2ar mutant that was previously shown not to interact with β-arrestin2 was utilized as control and did not induce a conformational change in the β-arrestin2 molecule. our data provide evidence that β-arrestin2 changes it`s conformation upon binding to the activated β2ar in living cells. the β-arrestin2flash2 sensor could serve as universal biosensor for gpcr activation. studies on the physiological role of annexin a4 in the heart nunes f. 1 the calcium binding protein annexin a4 has been examined in the context of heart failure in the past. annexin a4 expression level was found to be elevated in ventricles of human failing heart in comparison to expression levels in non-failing ventricles. furthermore the intracellular localization pattern in atrial cardiomyocytes was found to be altered in the failing human heart (moravec and matteo, cardiovasc res 2000). in order to gain insight into the possible physiological significance of these findings we utilized an annexin a4 gene trap model (gt) in which the annexin a4 protein content was not detectable in ventricles and atria. measurements of sarcomere shortening and calcium transient kinetics in isolated ventricular cardiomyocytes revealed a prolonged calcium transient decay at stimulation frequencies of 0.5 hz, 1hz and 2 hz as well as an increased sarcomere shortening at 1 hz and 2 hz in anxa4 gene trap animals in comparison to wild type (wt) ( the effects of the β-adrenoreceptor agonist isoprenaline (iso) on the shortening of ventricular cardiomyocytes was increased in gt as compared to wt (0,2±0.013 vs. 0.17±0.012, *=p<0.05 vs. wt; n=14-18/5). western blot analyses indicated that the expression of the sarcoplasmic reticulum (sr) ca 2+ -atpase (serca2a) and the phosphorylation status of its regulator protein phospholamban (plb) did not differ between groups (n=7). however, co-immunoprecipitation experiments suggest, that anxa4 is able to interact with hax1, which acts as a repressor of serca2a (n=4). we performed force measurements in isolated and electrically stimulated left atria in response to rising isoprenaline concentrations (10 -9 m-10 -5 m). the positiv inotropic effect of isoprenaline was significantly increased in gt atria (rel. force at 10 -4 m iso [%]: wt: 433±76; gt: 699±63 *= p<0.05 vs wt; n=8-10). in conclusion, annexin a4 contributes to the regulation of cardiomyocyte contractility. the anxa4 up-regulation might therefore contribute to diminished cardiac performance in heart failure. matteo rg, moravec cs. immunolocalization of annexins iv, v and vi in thefailing and non-failing human heart. cardiovasc res. 2000 mar;45 (4) background: pregnane x receptor (pxr) is considered the most important sensor of natural and anthropogenic xenobiotics in vertebrates. in contrast, the amphibian ortholog is involved in neural development and irresponsive to xenobiotics. instead, the xenopus laevis constitutive androstane receptor (car) was recently found to possess pxr-like properties, featuring low basal activity and a pronounced ligand spectrum. thus a structural and functional characterisation of x. laevis car may provide further insights into human car basal and ligand-induced activity. methods: the time-point of origin of car genes was determined by macrosynteny analyses of car, pxr, and vdr (vitamin d receptor) gene loci, which form the nr1i subfamily of nuclear receptors. based on a 3-dimensional protein model of xenopus laevis car, docking studies with structurally diverse agonists were conducted. proteinligand-interactions as well as sequence comparisons were performed in order to select amino acids to be mutated towards human car. the organ response to car activators was determined in xenopus laevis using rna microarrays. results: car emerged together with pxr and vdr from an ancestral nr1i gene in early vertebrates via two whole-genome duplications. this was followed by losses of car from the fish lineage and of pxr from sauropsida (reptiles and birds). amino acids important for ligand binding were identified. structural features responsible for the pronounced basal activity in human constitutive androstane receptor are not present in x. laevis car. in human pxr the inter-helical loop in front of helix 3 is part of the ligandbinding pocket and supposed to be responsible for the wide substrate spectrum. in amphibian car this inter-helical loop plays no role in ligand binding. car agonists resulted in a pronounced induction of antimicrobial peptides in the ovary. conclusions: car emerged already in early vertebrates and it is conserved in land vertebrates, whereas xenosensing pxr is found only in the fishes and mammals. we provide a comprehensive modeling and mutational analysis of this first reported amphibian xenosensor. the induction of antimicrobial peptides by car activators suggests a link between xenosensing and innate immunity. the latter one may play a previously unrecognized role in the amphibian reproduction. background: retigabine belongs to a novel class of potent anticonvulsant drugs and is currently being investigated in clinical routine. the therapeutic range of retigabine serum concentration is unknown. a therapeutic drug monitoring (tdm) is used for most other anticonvulsant drugs. the aim of this study was to develop a method for the determination of retigabine in serum of patients and to compare the effect and the side effects of retigabine with the blood levels of the drug. method: a hplc method with tandem mass spectrometric detection for the sensitive determination of retigabine was developed. solid-phase extraction (spe) of 250 µl serum with oasis hlb cartridges allowed a reliable quantification down to 5 ng/ml. in order to develop an assay with high sample throughput and to obtain maximum response for the analytes we required the shortest possible retention time. to implement the determination of retigabine in a second step in the routine tdm of anticonvulsant drugs the corresponding hplc method was selected: a purospher rp18 column (55 mm x 2 mm; 4 µm, merck) and a mobile phase with a steep acetonitrile gradient. results: the great advantage of having analytes with different molecular masses and similar retention times in combination with ms/ms detection enabled us to aim at a minimum separation that might remove some salts or matrix components that can suppress or interfere with the analyses from the target components, while maintaining good sample throughput. the method was validated. the assay is precise, accurate, fast, sensitive, and selective. discussion: the developed method is suitable for therapeutic drug monitoring of retigabine. the correlation of the serum concentration and the effect of the drug and thus the necessity of tdm have to be tested. targeting inflammatory t lymphocytes with conditional chemokine receptor antagonist expression for a tissue-specific therapy of chronic inflammatory disorders ogrissek n., giegold o., pfeilschifter j., radeke h. h. uniklinikum der goethe-universität pharmazentrum / zafes, theodor-stern-kai 7, 60590 frankfurt am main, germany chemokines and their receptors are known to be involved in the pathogenesis of chronic inflammation and autoimmune diseases. several approaches tried to use chemokine receptor antagonists as therapeutics to reduce exagerrated immune response, however, due to compensation and systemic side effects clinical trials often failed. in previous experiments our group identified three promising antagonists. cxcl11(4-79) has antagonistic function for cxcr3, cxcl12(p2g2) is able to inhibit cxcr4 and the herpesvirus 8 encoded protein vmip-ii interferes with ccr1, -2 and -5 as well as with cxcr3, -4 and cx3cr1. their expression and secretion was confirmed in pichia pastoris and antagonistic function has been proven by a reduction of t cell migration. the aim of this project is to develop a cell-based therapy for chronic inflammation with a treatment that is based on the collective effect of cxcl11(4-79), cxcl12(p2g2) and vmip-ii. with targeting of stable transduced memory t cells these antagonists should be conditional expressed and secreted directly in the centre of inflammation, resulting in inhibition of further inflammatory t cell accumulation. to realize this project we first cloned constitutive lentiviral constructs containing these antagonists and optimized transduction of t cells, such as the ova-specific memory th-1 cell clone if12 with the potential to initiate antigen specific nephritis in scid mice. next we investigated expression and secretion of cxcl11(4-79), cxcl12(p2g2) and vmip-ii with pcr, western blot and elisa. at the moment we want to measure the inhibition efficiency of t cell migration in vitro with chemotaxis and flow chamber assays. construction of an inducible lentiviral vector plasmid to ensure expression of the antagonists only upon t cell activation, is also part of our current work. finally we would like to test the chemokine receptor antagonists in vivo in two relevant mouse models of type-1-diabetes and contact dermatitis. small heterodimer partner 1 (shp-1) is a member of the superfamily of nuclear receptors (nrs). in contrast to other nrs this orphan receptor lacks the dna binding domain. however, shp-1 is known to inhibit activity of several nrs by direct proteinprotein interaction. importantly several of the interacting nrs have been shown to directly regulate shp-1 expression, suggesting that shp-1 is involved in negative feedback loops of various metabolic pathways, such as cholesterol-, bile acid-and drug metabolism and glucose homeostasis. recently binding sites for nrs were identified in the promoter region of shp-1, including hnf4α, lrh1, lxr, fxr, srebp1c and pparγ. the aim of our study was to identify single nucleotide polymorphisms (snps) in the promoter region of shp-1 and to determine their impact on the transactivation of shp-1. 120 dna samples from subjects of the population based cohort study of health in pomerania were analyzed by sanger sequencing, thereby we identified four snps namely -594t>c (rs71636795), -413g>c, -423 c>t (rs78182695) and del-195ctga (rs145613139). subsequently those polymorphisms were tested for their functional consequence performing cell based reporter gene assays testing all above mentioned modulators (lrh1, lxr, fxr, srebp1c and pparγ) of shp-1 expression. only the transactivation by hnf4α was decreased in the presence of the -423 c>t polymorphism to 69% and the -413g>c polymorphism to 75%. in conclusion we described snps with impact on transactivation. it will be aim of future studies to determine the potential impact on physiological processes or disease development. autosomal recessive polycystic kidney disease (arpkd) is a rare genetic disease, afflicting about 1 in 20.000 individuals. arpkd is characterized by cystic fusiform dilatations of the renal collecting ducts leading to massive enlargement of the kidneys and ultimately loss of renal function. in addition, the patients suffer from congenital hepatic fibrosis (chf), possibly leading to portal hypertension and liver enlargement. so far, there is no cure for arpkd. therapy is focussing on controlling the disease symptoms [1] . mutations in the pkhd1 gene cause arpkd. more than 300 different mutations in this gene have been reported, all leading to the same phenotype, though there are differences regarding the severity of the disease [2] . in animal models of autosomal dominant polycystic kidney disease (adpkd) as well as arpkd elevated levels of camp were shown [1] [2] [3] . in isolated kidney cells camp stimulates cl-secretion and activates the b-raf /mek/erk pathway. these both are important factors for cyst development and disease progression [2, 3] . intracellular camp regulation is based on conversion of atp to camp by adenylyl cyclases (acs) and degradation by phosphodiesterases . referring to this, we asked the question if there are differences in the activation and expression pattern of acs in pck rats, an animal model of arpkd [4] and in sprague dawley rats. therefore, we examined membranes in a radioactive ac activity assay using various stimulatory compounds, e.g. forskolin, a direct ac activator, or hormones like glucagon and vasopressin to characterize acs. furthermore, we examined ac isoform expression on the mrna level via rt-pcr. we observed that in pck rats ac activity was decreased in general in comparison to sprague dawley rats. in future experiments we are aiming to obtain further knowledge about the influence various hormones exhibit on pck rat acs and to biochemically characterize acs. the major pathogenicity factors tcda and tcdb from clostridium difficile monoglucosylate and thereby inactivate small gtp-binding proteins of the rho subfamily after entering host cells via receptor-mediated endocytosis. although the intracellular mode of action of the toxins is well understood, far less is known about binding structure and internalization pathway of tcda and tcdb. since antibodies directed against the c-terminal located clostridial repetitive oligopeptides (crops) are able to neutralize toxin cytotoxicity the crop domain is acknowledged to mediate receptor binding. however, we recently demonstrated that crop deletion mutants of tcda (tcda 1-1874 ) and tcdb (tcdb 1-1852 ) enter host cells and exhibit full cytopathic potency though lacking the proposed receptor binding domain. we therefore refute the accepted opinion of a solely crop-mediated toxin uptake and re-evaluate the role of the crops in toxin endocytosis. tcda 1-1874 and tcdb 1-1852 induced time and concentration dependent cell rounding and rac1-glucosylation. however, depending on the cell line, truncated toxins exhibit up to 10-fold reduced potency towards host cells compared to the respective full length toxin. the observed difference in toxin potency might reflect the recognition of different receptor structures or the use of various endocytotic routes. interestingly, pre-incubation of cells with the isolated crop domain enhances binding as well as cytotoxicity of subsequent applied truncated tcda indicating that the crops primarily determine toxin uptake. in fact, competition experiments revealed that tcda and tcda 1-1874 predominantly use different receptor structures corroborating the notion of alternative internalization processes utilized by tcda. different routes for cellular uptake might enable the toxins to enter a broader repertoire of cell types leading to the observed multifarious pathogenesis of c. difficile. thus, characterization of alternative endocytotic pathways used by the c. difficile toxins might therefore be the basis to investigate the opportunity of toxin uptake inhibition as therapeutic option. in neurodegenerative diseases, such as alzheimer´s disease and parkinson´s disease, mitochondrial pathways of apoptosis are considered as major features of the underlying neuronal cell death. such mitochondrial mechanisms of apoptosis are mediated by the bh3-only protein bid, a member of the bcl-2 family that triggers mitochondrial permeabilization and the subsequent release of death-promoting proteins into the cytosol. the pivotal role of bid in apoptotic cascades of neuronal cells has been shown in our previous studies showing a neuroprotective effect of bid sirna and small molecule bid inhibitors such as bi6c9 in vitro. in vivo, however, the available bidinhibitors failed to protect brain tissue likely because the compounds were not bioavailable or did not cross the blood brain barrier. therefore, chemical modifications of bi-6c9 were generated resulting in new structures and molecules with different pharmacophors. the aim of the present study is to identify novel potent bid inhibitors available for applications in model systems of brain damage in vivo. for the first screening of 80 compounds we used a model of glutamate toxicity in immortalized mouse hippocampal neurons (ht-22 cells). in this model system, glutamate induces a decrease of intracellular glutathione levels resulting in lipoxygenase activity and enhanced formation of toxic reactive oxygen species (ros). to investigate the compounds' ability to prevent glutamate induced cell death, we first analyzed the cell viability by the mtt assay. in addition, we examined the cell survival by using real time monitoring of cell impedance (xcelligence system) to determine the neuroprotective potency of the new structures. using these assays, we identified 10 novel molecules that significantly prevented glutamate-induced toxicity in ht-22 cells. further we were able to express and to purify recombinant bid in a high amount. in the ongoing study the purified bid protein will be used for co-crystallization with the identified neuroprotective structures for further optimization of novel bid inhibitors for therapeutic applications in experimental models of neurodegenerative diseases in vivo. polymorphic enzymes, urinary bladder cancer risk and structural change in the local industry ovsiannikov d. 1 , selinski s. in the 1990s, an uncommonly high percentage of glutathione s-transferase m1 (gstm1) negative bladder cancer cases (70 %) was reported in the greater dortmund area (golka et al., 1997) . the question arose whether this uncommonly high percentage of gstm1 negative bladder cancer cases was due to environmental and occupational exposure decades ago. thus, 15 years later, another study on bladder cancer was performed in the same area after the coal, iron and steel industries had finally closed in the 1990s. in total 196 bladder cancer patients from the st.-josefs-hospital dortmund-hörde and 235 controls with benign urological diseases were investigated by a questionnaire and genotyped for gstm1, gstt1 and the n-acetyltransferase 2 (nat2) tag snp rs1495741. the frequency of the gstm1 negative genotype was 52 % in bladder cancer cases and thus much lower, compared to a previous study performed from 1992-95 in the same area (70%). nat2 genotypes were distributed equally among cases and controls (63% slow acetylators). less gstt1 negative genotypes were present in cases (17%; controls 20%). apoptosis inducing factor (aif) has been identified as a key factor in intrinsic pathways of caspase-independent neuronal death in model systems of acute brain injury and neurodegenerative diseases, such as alzheimer's disease and parkinson's disease. aif is a mitochondrial intermembrane flavoprotein with the capacity to translocate to the nucleus where it induces chromatin condensation and large-scale dna fragmentation. previous studies revealed that aif deficiency leads to protective effects in different models of neuronal death in vitro and in vivo. however, aif also plays an important physiological role for the integrity and function of the mitochondrial respiratory chain. thus, aif deficiency may significantly alter mitochondrial functions and metabolic homeostasis thereby preconditioning the cells to tolerate subsequent stress stimuli. the present study addresses this hypothesis and investigates whether neuroprotection by aif depletion was attributed to a preconditioning effect, i.e. protecting mitochondrial function and integrity. as model system we use glutamate induced oxytosis in immortalized mouse hippocampal ht-22 neurons. silencing of aif expression by sirna (20nm) protected mitochondrial morphology and integrity against glutamate induced damage. microscopy analysis of the mitochondrial morphology revealed that aif sirna prevented mitochondrial fission. furthermore, facs analysis confirmed that mitochondrial membrane potential was stable in cells with aif silencing. this protection of mitochondrial morphology and integrity by aif depletion was associated with preserved atp levels and inhibition of cell death as detected by an mtt assay. pronounced formation of lipidperoxides as another indicator of mitochondrial damage was also attenuated in cells preconditioned by aif sirna. these protective effects of aif sirna were highly similar to effects obtained with low doses of rotenone (20nm), which was applied as an inhibitor of complex i and mediated comparable preconditioning effects in the ht-22 cells. overall, these findings support the conclusion that aif depletion mediates a preconditioning effect protecting neuronal cells from a subsequent glutamate toxicity. in order to link these preconditioning effects to complex i functions, protein expression and functional analysis of complex i are being analysed to identify the molecular mechanisms of aif dependent control of neuronal life and death. -dependent inactivation and display very slow voltage-dependent inactivation. both properties are of crucial importance in ribbon synapses of retinal photoreceptors and bipolar cells, where sustained ca 2+ influx through cav1.4 channels is required to couple slowly graded changes of the membrane potential with tonic glutamate release. mutations in the gene coding for cav1.4 cause severe impairment of retinal circuitry function and have been linked to congenital stationary night blindness type 2a (csnb2), aland island eye disease (aied) and cone-rod dystrophy type 3 (cordx3). the clinical phenotypes of these eye diseases vary substantially regarding the ratio of rod to cone functional impairment. the reasons for this variability are not known. to gain more insights into the pathophysiology caused by loss of cav1.4 function we analyzed the visual phenotype of cav1.4-deficient mice. to this end, we combined immunohistochemistry, electroretinography (erg) and vision-dependent behavioral testing. immunohistochemical analysis using synaptic and postsynaptic markers revealed severe synaptic defects in cav1.4-deficient mice. heterozygous cav1.4 mice showed mosaic synaptic defects most probably caused by random x-chromosomal inactivation of the healthy allele. electroretinography revealed a loss of scotopic and photopic photoreceptor function. this loss of retinal network function resulted in impaired performance of cav1.4 knockout mice in a water maze-based behavioral test of rod and cone function. in conclusion, loss of cav1.4 channels strongly impairs rod and cone retinal function and vision in mice. lsr is the host cell receptor for clostridial iota-like toxins papatheodorou p., aktories k. albert-ludwigs-universität freiburg institut für experimentelle und klinische pharmakologie und toxikologie, albertstr. 25, 79104 freiburg, germany the human enteric pathogen clostridium difficile is the most serious cause of antibioticassociated diarrhea and pseudomembranous colitis. hypervirulent strains of the pathogen, associated with more severe disease and increased death rates, produce the binary actin-adp-ribosylating toxin cdt (c. difficile transferase) in addition to the rho glucosylating toxins a and b. cdt is member of the family of clostridial iota-like toxins, including c. spiroforme toxin (cst) and the eponym c. perfringens iota toxin. the toxins induce depolymerization of the actin cytoskeleton and the formation of microtubulebased cell protrusions that increase adherence and colonization of clostridia. using a haploid genetic screen, we identify the lipolysis-stimulated lipoprotein receptor (lsr) as the target molecule for entry of cdt into host cells. in addition, we present evidence that lsr is shared as a cell entry point by all members of the iota-like toxin family. identification of the toxin receptor provides a most valuable basis for antitoxin strategies. bisphenol a (bpa) is a chemical of high interest due to its endocrine activity. controversy exists concerning the blood concentration due to normal exposures. some authors claimed to have measured concentrations in the ng/ml range which is in contrast to kinetic properties of bpa. bpa is excreted in the urine as glucuronide and sulfate metabolites. recently, data on the in vitro metabolism of bpa by recombinant udpglucuronyltransferase 2b15 enzymes (ugt2b15) revealed that ugt2b15.2 and ugt2b15.5 had markedly lower intrinsic clearance as compared to ugt2b15.1 (hanioka et al., 2011) . using the in vitro metabolism data, we scaled the kmand vmaxvalues in an established human physiologically based toxicokinetic (pbtk) model (mielke and gundert-remy, 2009, mielke et al., 2011) to the values of the variants. for oral doses at relevant exposure levels, the maximum blood concentration (cmax) for the ugt2b15.2 variant (v2) was 5 fold and those of the ugt2b15.5 variant (v5) was 7 fold higher than that of the ugt15.1 variant. with dermal exposure at a relevant exposure level, the cmax values were 1.4 (v2) and 1.6 fold (v5) of ugt15.1 variant. a combined exposure of oral and dermal exposure, an exposure scenario, which occurs in daily life, resulted in 2.4 fold (v2) and 3.2 fold (v5) higher cmax values as compared to ugt15.1 variant. the values for the area under the blood concentration time curve (auc) were for a relevant oral dose 5.7 fold (v2) and 8.6 fold (v5), for relevant dermal exposure 1.4 fold (v2) and 1.6 fold (v5), and for combined exposure 1.9 fold (v2) and 2.5 fold (v5) of ugt15.1 variant. from the results we conclude: (1) polymorphism of udpglucuronyltransferase (2b15.2 and 2b15.5) has an impact on the blood concentrations which, however, is less than 10 fold for cmax and for auc. the effect is more pronounced for oral as compared to dermal or combined exposure. (2) polymorphism of metabolism does not explain the blood/plasma concentrations in the ng/ml range measured by some authors. hanioka n, oka h, nagaoka k, ikushiro s, narimatsu s. effect of udpglucuronosyltransferase 2b15 polymorphism on bisphenol a glucuronidation. arch toxicol. 2011, 85(11) :1373-81. mielke h, gundert-remy u. bisphenol a levels in blood depend on age and exposure. toxicol lett. 2009 8; 190(1) :32-40. mielke h, partosch f, gundertthe heart responds to maladaptive pro-hypertrophic stimuli by stimulating intrinsic signals that contrast and dampen the onset and development of hypertrophy. cyclic guanosine monophosphate (cgmp) and its downstream effector cgmp kinase i (cgki) have been suggested to be an important anti-hypertrophic signaling pathway (1) . intracellular levels of cgmp can be raised by the action of nitric oxide (no) and natriuretic peptides (anf, bnf), or by inhibiting cgmp-degrading phosphodiesterases (pde). a growing body of evidence suggests that the pde5 specific inhibitor sildenafil (sil) prevents and reverses hypertrophy and chamber remodelling in the heart of mice subjected to thoracic aorta constriction (tac) by elevating cgmp levels and cgki activation (1) . in contrast, using a mouse model that lacks cgki expression in every cell type except smooth muscle cells (βres mice; see ref. 2), we recently showed that the absence of this kinase does not alter the onset of hypertrophy induced by tac or isoproterenol infusion (2) . sil is believed to increase cardiac cgmp levels, although it is unclear, if its target (pde5) is expressed in cm (2). sil may act on other pdes, such as pde1c which is abundant in cms. it is also unclear if sil effects are mediated by other cardiac cell types, in particular by cardiofibroblast. to answer these questions, we are currently investigating whether sil is able to prevent hormone induced cardiac hypertrophy in the absence of cgki in cm. preliminary results on βres mice show that even in the case of chronic angii infusion, lack of cgki in cm does not alter the induction of hypertrophic response, at least in the initial phase (7days of angii infusion at 2mg/kg/day). interestingly, βres mice showed impaired cardiac function, as indicated by decreased fractional shortening. sil was able to partially block the onset of cardiac hypertrophy in wt animals, but not in βres mice, indicating a requirement of cgki in this process. in particular, sil was able to block the transcription of pro-fibrotic genes such as tgfβ, ctgf, collageni and fibronectin. the overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of the stable somatostatin analogs octreotide and pasireotide. whereas the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (t333) and 347 (t347), which enabled us to selectively detect either the t333-or the t347-phosphorylated form of sst5. we show that agonist-mediated phosphorylation occurs at t333, whereas t347 is constitutively phosphorylated in the absence of agonist. we further demonstrate that the pan-somatostatin analog pasireotide and the sst5-selective ligand l-817,818 but not octreotide or ke108 were able to promote a clearly detectable t333 phosphorylation. however, none of these compounds was able to stimulate t333 phosphorylation and sst5 internalization to the same extent as the natural somatostatin. agonist-induced t333 phosphorylation was dose-dependent and selectively mediated by g protein-coupled receptor kinase 2 (grk2). like that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. however, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were complete within minutes. we also identify protein phosphatase 1g (pp1g) as sst5 receptor phosphatase. together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal t333. in addition, we identify grk2-mediated phosphorylation and pp1g-mediated dephosphorylation of t333 as key regulators of rapid internalization and recycling of the human sst5 receptor. termination of signaling of activated g protein-coupled receptors (gpcrs) is essential for maintenance of cellular homeostasis. although the regulation of agonist-induced phosphorylation has been studied in detail for many gpcrs, the molecular mechanisms and functional consequences of receptor dephosphorylation are far from understood. recent studies have shown that phosphatase inhibitors, such as okadaic acid and calyculin a, can block the dephosphorylation of a number of gpcrs including the ß2 adrenergic receptor, d1 dopamine receptor, parathyroid hormone receptor 1, thromboxane a receptor and the vasopressin receptor 1. however, a specific phosphatase has not been identified so far. in present studies, we have examined the mechanism and function of receptor dephosphorylation using the sst2a somatostatin receptor and the µ-opioid receptor (mor) as models. within those analyses, we have identified protein phosphatase 1beta (pp1ß) as the phosphatase for the cluster of phosphorylated threonines (353ttetqrt359) within the sst2a somatostatin receptor carboxylterminus using sirna knock down screeening. those phosphorylation sites mediate ß-arrestin binding. we have also identified protein phosphatase 1gamma (pp1γ) as mor phosphatase that catalyzed t370 and s375 dephosphorylation at or near the plasma membrane within minutes after agonist removal. here, we show the different activated phosphatases with functional selective mutants. we examined tailswap mutants which specify the different phosphatase activities. therefore we produced a mor-rsst2a chimera with the rsst2a c-terminal tail and a rsst2a-mor chimera with a mor c-terminal tail. detoxification by conjugation of glutathione? formations of dna adducts of patulin activated by glutathione pfenning c., lehmann l. university wuerzburg, institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany as a frequent contaminant in apple juice, the mycotoxin patulin (pat) has shown mutagenic potential in cultured mammalian cells at concentrations which are equivalent to those found in marketable foods. this fact is in contrast with the assumption that conjugation to the major intracellular nucleophile glutathione (gsh) leads to detoxification of the electrophile pat. although pat reacts readily with gsh, previous studies showed that co-incubation of pat with model thiols and amine compounds increased the reactivity of pat towards amines forming mixed-type adducts. thus, we hypothesise that the potential to react with dna bases after being activated by gsh might contribute to the mutagenicity of pat. adduct formation of dna bases (adenine, guanine or cytosine) with pat in the presence and absence of gsh was studied under neutral conditions. liquid chromatography coupled with electrospray ionization tandem mass spectrometry was applied for identification and structure elucidation of putative adducts. besides published as well as hitherto unknown pat-gsh adducts, several pat-dna base adducts were formed both in presence and absence of gsh. in addition, with each of the three dna bases one product exhibiting a mass to charge ratio and fragmentation pattern suggesting a mixed thiol-amine adduct was detected. based on the fragment ions of adducts formed with gsh and chemically modified derivatives, we postulate a cyclic structure of the pat-gsh-dna base adducts, resulting from the reaction of the α-amino group of the glutamic acid residue with the c7-carbonyl function of pat. the exocyclic amino group of the dna base is linked to c1 of the pat backbone by an amid bond. thus, the present study demonstrates the reactivity of pat towards dna bases and the participation of gsh in adduct formation. the postulated structures of dna adducts could be used as biomarkers for the determination of the internal exposure to pat in humans. prescribing information detailed in the summary of product characteristics (spc) forms the officially approved basis for safe prescribing of drugs. in a project funded by the german federal ministry of education and research (bmbf) we aimed to derive an internationally valid data set for safe prescribing of psychiatric drugs and therefore analyzed and compared the content of internationally available prescribing information. a team of pharmacists and clinical pharmacologists performed an in-depth comparison of the german, swiss, british and us-american spcs of 10 top prescribed psychiatric drugs. for 7 drugs (of identical pharmaceutical form) the spcs from the same manufacturer were available for all countries, whereas for three drugs spcs were only available from different companies. in these cases the most recent prescribing information from each country was included in the comparison. in 40 spcs 2220 individual data points (55.5±17.4 per individual spc) were compared. between countries the timeliness of prescribing information for an individual drug varied by a median of 18.5 (range: 6-134) months. the respective spcs covered on average 71.4±30.3% (range: 12.5-100%) of all mentioned indications and 70.1±24.4% (range 15.4-100%) of all mentioned contraindications. the warnings and precautions section of an individual spc covered on average 59.5±17.1% (range: 12.5-93.3%) of all mentioned warnings and precautions for that drug. the variation observed was only marginally improved when restricting the analysis to the 28 spcs of the 7 drugs available in all four countries from the same manufacturer. across countries, the summary of product characteristics of individual psychiatric drugs show substantial variation in crucial prescribing information. as different manufacturers are unlikely to explain much of the observed variation, these data argue for a better international cooperation and standardization of the content of summary of product characteristics. this project is supported by the german federal ministry of education and research (bmbf), project grant no. 01 ex1015b. protein kinase ck2 (former name 'casein kinase 2') is a highly conserved serine/threonine kinase, which acts as a component of regulatory networks implicated in many cellular processes but is also linked to various types of human cancer [1, 2] . elevated ck2 activity has been associated with aggressive tumor behavior and results in growth advantage, enhanced survival and dynamic adaption to stress of cancer cells [3] . ck2 is a heterotetramer consisting of two catalytic subunits (ck2α) attached to a dimer of regulatory subunits (ck2β) [4] . ck2β stabilizes ck2α against denaturation and modulates the substrate specificity of the catalytic subunit [5] . due to the relatively small and hydrophobic ck2α-ck2β interface (832 å²) [4] , low molecular weight inhibitors are able to interfere with the ck2 subunit interaction and thus affect the kinase activity [6] . such inhibitors might exhibit an increased specificity in comparison to those compounds interacting with the conserved atp binding site [3] . we have developed an elisa-based ck2α-ck2β binding assay using recombinant human ck2 subunits. different blocking reagents were analyzed to minimize nonspecific binding. the optimized binding assay was then applied to screen for inhibitors of the ck2 subunit interaction. primary hits were further characterized by determination of the parameters ic50 and ki as well as by comparing the results from the binding assay with literature-known or recently obtained crystal structures. numerous epidemiological studies have shown associations between exposure to ultra fine particles and an increase in cardiovascular diseases such as atherosclerosis, coronary heart disease and myocardial infarction. ultra-fine particles have an aerodynamic diameter of < 0.1 µm and are highly diverse with impurities of transition metals and organic compounds (e.g. polycyclic aromatic hydrocarbons). posttranslational modification (ptm) of proteins, particularly phosphorylation, is a key element in the regulation of cell function and any disturbance can lead to multiple diseases. the present study focused on the proteomic-based identification of phosphorylated proteins to understand the mechanism behind ultrafine particle exposure and cardiovascular disease development. as one of the major sources for ufp emissions are diesel exhaust, therefore to mimic the diesel particles, carbon black (cb) and benzo[a]pyrene loaded carbon black (cb+) were used in the present study. cells of the endothelial cell line ea.hy926 were exposed for 14 days to 100 ng/ml cb and cb+. phosphoprotein extraction of whole cell lysates was carried out by the method developed in our lab 1 . the obtained proteins were then separated by two-dimensional gel-electrophoresis followed by maldi-tof-ms (matrix-assisted laser desorption/ionization time-of-flight mass spectrometry) analysis of differently expressed proteins. to further validate the results invasive potential of cells were monitored by plating exposed cells for 24 hrs on top of matrigel-coated inserts. differential expressions of 20 phosphoproteins were found in cb-treated cells while an altered expression of 33 phosphoproteins was observed in cb+-treated cells. the maldi-tof analysis revealed proteins involved in the regulation of the endothelial permeability and the cellular plasticity such as vimentin, actin and transitional endoplasmic reticulum atpase. further, the invasion assay supported these results as the cb-exposed cells showed a high invasive potential as compared to control. [1] pink m. et. al. , precipitation by lanthanum ions: a straightforward approach to isolate phosphoproteins, j.protomics, 75, 2011, [375] [376] [377] [378] [379] [380] [381] [382] [383] 302 application of the ttc approach in cosmetics platzek t. bundesinstitut für risikobewertung, max-dohrn-straße 8-10, 10589 berlin, germany regulatory toxicologists in europe have been discussing the ttc approach since more than a decade, e.g. the previous scientific committee on food in 1996. since then, the concept was further developed and is now applied in the eu for the assessment of flavouring substances by efsa. two committees are discussing possible applications: the efsa scientific committee prepared an opinion exploring options for the application in food and feed, e.g. for impurities of food additives, thermal reaction products, food contact materials, contaminants etc. in addition, an eu non-food expert committee consisting of members of sccs, scher and schenir discussed the ttc concept in general as well as additional possible fields of application with the focus on cosmetics and an opinion was already published for public consultation. the proposal to apply the ttc approach also for cosmetic ingredients was introduced by a paper published in 2007 by a colipa (the european cosmetics association) supported working group. major aspects to be considered are the following: 1. applicability domain. the chemical space of the ttc dataset (> 600 compounds) has to be compared with that of cosmetic ingredients (> 10 000 compounds). route to route extrapolation. since no adequate dermal toxicity database is available both data on oral intake used in the ttc approach and on dermal exposure to cosmetic ingredients have to be transfomed to internal exposure figures. gastrointestinal and dermal bioavailability as well as route specific differences in metabolism have to be integrated. exposure. the reliability of exposure estimation is the second pillar of the ttc approach. compared to food, data on exposure to substances in cosmetics and consumer products is scarce. a pragmatic step forward is the comparison of ttcs and noael-derived safe exposure levels for cosmetic ingredients. this work was already done with substances in food contact materials and chemicals from the elincs list. for cosmetic ingredients a similar european project is ongoing. further refinement of the ttc approach is needed taking into account the up-to-date toxicological knowledge. with cosmetics specific problems may arise in praxi: according to the new eu cosmetic legislation the safety of cosmetic products available on the market has to be assessed by the manufacturer or importer and also assessors with limited toxicological experience may apply the ttc approach, e.g. by running the toxtree software. plöttner s., marczynski b., käfferlein h. u., welge p., groth h., engelhardt b., schmitz k., erkes a., brüning t. institut für prävention und arbeitsmedizin der deutschen gesetzlichen unfallversicherung -institut der ruhr-universität bochum (ipa) toxikologie, bürkle-dela-camp-platz 1, 44789 bochum, germany polycyclic aromatic hydrocarbons (pahs) comprise several hundred compounds with different carcinogenic potentials, typically occurring in complex mixtures. due to a lack of data risk estimations for pah mixtures are usually based on those for benzo[a]pyrene (b[a]p). the aim of our present study was to explore the suitability of a permanent human lung cell line as tool for future studies on genotoxicity of pah mixtures. in this pilot study we investigated the time-and concentration-dependent generation of specific anti-benzo[a]pyrene -7,8-diol-9 ,10-epoxide (anti-bpde)-dna adducts as well as cytochrome p450 (cyp) 1a1/1b1 enzyme activities after b[a]p-incubation in vitro. we used metabolically competent a549 lung carcinoma cells which display several characteristics of alveolar epithelial type ii cells. after 24 h and 48 h incubations with different b[a]p-concentrations cytotoxic effects were assessed with the neutral red assay and cyp1a1/1b1 activities using luminescent tests. the formation of specific anti-bpde-dna adducts was determined by hplc with fluorescence detection. a time-and concentration-dependent formation of anti-bpde-dna adducts was observed with maximum rates of 340.5 ± 39.1 and 599.6 ± 132.4 anti-bpde/10 8 nucleotides after incubation with 1 µm (24 h) and 3 µm b[a]p (48 h), respectively. however, the mean adduct rates decreased at higher b[a]p-concentrations. the reduction was more pronounced after 24 h than after 48 h. increased cyp1a1/1b1 activities were observed at > 0.1 -1 µm (24 h) and > 0.1 -3 µm (48 h). a clear decrease of enzyme activities was observed at higher concentrations for both incubation times. in the neutral red assay no more than 10% cytotoxicity in relation to the negative control were found after 24 h incubation with ≥ 10 µm b[a]p and after 48 h with ≥ 1 µm b[a]p. overall, incubation of a549 cells with b[a]p resulted in a time-and concentration-dependent increase of cyp-activities and anti-bpde-dna adducts. this clearly shows that a549 cells are able to generate mutagenic dna-adducts. thus, the in vitro model used in the present work appears suitable for genotoxicity studies of individual pahs and pah mixtures, and therefore may be a useful tool for research on syncarcinogenesis. the β subunit of the cav1.2 channel complex has been shown to play a key role in cav1.2 channel trafficking and channel characteristics like opening probability. furthermore, the last exon of the cacnb2 gene coding for cavβ2, exon 14, contains several potential phosphorylation sites, e.g. for protein kinase a or ca 2+ /calmodulin dependent camkii. pka-dependent phosphorylation mediates β-adrenergic stimulation of cav1.2. potential phosphorylation sites are ser478 and ser479 (in the β2 subunit in rat, perez-reyes et al. 1992 a ) and ser1928 in the c-terminus of the poreforming α1c subunit (lemke et al. 2008 b ) . in cardiomyocytes camkii regulates ca 2+ release and reuptake from and into the sr and is involved in the facilitation of the calcium channel. potential interaction sites between the cav1.2 channel complex and camkii are thr498 in the β2 subunit (in rat, grueter et al. 2006 c ) and ser1512 and ser1570 in the cterminus of the α1c subunit (blaich et al. 2010 d ) . however, the exact pathways remained widely unclear up to now. to clarify these mechanisms and to identify the relevant phosphorylation sites for pka and camkii we established a mouse line carrying a stop codon in exon 14 after aa pro501. this mutation prevented translation of the cavβ2 c-terminus containing the corresponding potential phosphorylation sites mentioned above (cavβ2stop mouse). these mice were viable, showed unaltered expression of the truncated of cavβ protein and unchanged ecg and echocardiography. electophysiological analysis of isolated cardiomyocytes showed no differences in current density, the effect of isoproterenol, the time course of inactivation and the facilitation property when compared to cells isolated from littermate controls. for further investigations we bred the cavβ2stop mice with s1928a mice (lemke et al. 2008 b ) lacking the pka phosphorylation site s1928 in the α1c subunit (s1928aβ2stop mouse) or with sf mice (blaich et al. 2010 d ) lacking the camkii phosphorylation sites s1512 and s1570 in the α1c c-terminus (sfβ2stop). both mouse lines were viable and showed unchanged echocardiography recordings compared to their control littermates and unaltered ecg for s1928aβ2stop mice. electrophysiological investigations on cardiomyocytes of s1928aβ2stop mice showed unchanged β-adrenergic stimulation with isoproterenol compared to littermate controls. these results suggest that β adrenergic regulation of the cardiac cav1.2 channel is not mediated by these phosphorylation sites. introduction. chronic atrial fibrillation (caf) is marked by increased fibrosis which contributes to the perpetuation of the disease. in addition to the role of fibrosis in structural remodeling of cardiac tissue, fibroblasts can couple with cardiomyocytes via gap junction thereby altering the electrophysiological properties of the later and potentially participating in atrial electrical dysfunction. in order to understand the importance of fibroblasts in the pathophysiology of caf, we compared the electrical properties of atrial fibroblasts isolated from patients in sinus rhythm (sr) and caf. methods. fibroblasts were isolated by outgrowth culture from right atrial biopsies and cultivated in medium containing 10% fetal calf serum. we used whole-cell patch clamp techniques to investigate ion currents and membrane potential. results. sr and caf fibroblasts showed similar capacitance (sr: 43.6 ± 4.6 pf, n=33; caf: 54.7 ± 5.1 pf, n = 17) and membrane potential (sr: -21.0 ± 4.3 mv, n = 14; caf: -27. 4 ± 4.8 mv, n = 16) . in both groups, we observed fast activating outward currents with a mean threshold at -20 mv. interestingly, current amplitude was significantly larger in sr than caf cells (sr: 23.8 ± 4.2 pa/pf, n = 15; caf: 6.1 ± 1.0, n = 6; p < 0.05). when maintained in culture for 3-5 weeks, cells from both groups developed na + currents. surprisingly, the fraction of caf cells displaying such currents was larger than the sr counterpart (caf: 38%; sr: 15%). furthermore, na + current amplitude was significantly larger in caf fibroblasts (sr: 6.1 ± 2.0 pa/pf, n = 5; caf: 17.4 ± 4.4 pa/pf, n = 6; p < 0.05). na + currents were not altered by 100 nm tetrodotoxin (ttx), but 10 µm ttx reduced current amplitude to 42% of control, suggesting that the channel involved is the cardiac ttx-resistant isoform nav1.5. conclusion. in the context of caf, fibroblasts undergo electrophysiological changes which need to be thoroughly described. understanding whether those changes contribute to the af substrate might provide new therapeutic targets for the treatment of caf. the initial recruitment of gi to the alpha2a-adrenergic receptor is affected by gprotein-coupled receptor kinases and arrestins prokopets o. s., krasel c., 35043 marburg, germany most g-protein-coupled receptors undergo homologous desensitization after agonist stimulation. in this process, agonist-activated receptors are phosphorylated by gprotein-coupled receptor kinases (grks), followed by binding of arrestins to the still agonist-occupied, phosphorylated receptors. it is assumed that arrestin competes with heterotrimeric g-proteins for the receptor molecule and thereby causes desensitization of g-protein-mediated responses. we tested this idea by investigating the effect of grks and arrestins on the recruitment of g-proteins by the alpha2a-adrenergic receptor. hek293t cells were transfected with yfp-tagged alpha2a-adrenergic receptor and the three subunits of gi1. the g(beta) subunit was cfp-tagged. upon stimulation with noradrenaline, a very rapid, robust increase in fret between the receptor and the g(beta) subunit was observed, confirming previous observations that the alpha2aadrenergic receptor recruits gi with a half-life of around 150 milliseconds. when arrestin3 and grk2 were also co-transfected, the half-life of gi recruitment was substantially delayed, increasing to around 2 seconds. there seemed to be no effect of grk2+arrestin3 cotransfection on a second stimulation; neither the kinetics nor the extent were altered compared to the first stimulation. interestingly, grk2 alone seemed to cause a similar but slightly less pronounced delay of g(beta) recruitment to the alpha2a-adrenergic receptor. in corresponding experiments, we measured the recruitment of arrestin3-cfp to yfp-tagged alpha2a-adrenergic receptors in the presence of grk2. upon stimulation with noradrenaline, we observed a robust increase in fret between the receptor and arrestin3, confirming previous observations that the alpha2a-adrenergic receptor recruits arrestin3. co-transfection of the three subunits of gi1 had no effect on the kinetics of arrestin3 binding to the alpha2a-adrenergic receptors. based on previous results with other receptors, an attenuation of gi recruiting to the alpha2a-adrenergic receptor is quite unexpected. our data suggest that the relation between receptors, g-proteins, grks and arrestins may be more complex than previously postulated. introduction: human urinary bladder expresses mrna of the three known badrenoceptor (b-ar) subtypes (b1, b2, b3) in detrusor and urothelium. we have shown previously that only b3-ar is involved in human detrusor relaxation. to investigate the urothelium-induced modulation of b-ar-mediated relaxation, we have examined systematically whether other b-ar subtypes are involved. human detrusor tissue samples were obtained from patients undergoing radical cystectomy for the treatment of bladder cancer. detrusor strips were studied with and without an intact mucosa layer. muscle strips were precontracted with 1 µm carbachol and relaxation was studied in response to the b-ar agonist ne. a-ar mediated processes were blocked with the a-ar antagonists phentolamine and prazosin. selective b-ars antagonists were used to investigate b-ar mediated relaxation. at the end of each experiment 10 µm forskolin was used to determine maximum camp-mediated relaxation. the presence of intact urothelium reduces potency but not effectivity of ne indicating involvement of urothelium-mediated processes not only during detrusor contraction but also during relaxation. ne-mediated detrusor relaxation is mediated through b3-ar in the absence of urothelium. but in intact detrusor strips b2-ars seem to have an additional inhibitory effect. affinity of the selective b3-ar antagonist l748,337 is unchanged, therefore the intact urothelium does not interact with the function of b3-ars. aldosterone causes oxidative stress and dna damage independent of blood pressure in vivo queisser n., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany background: an inappropriate increase of the mineralocorticoid aldosterone (ald) can be induced by a stimulated renin-angiotensin-aldosterone system. epidemiological studies exploring the connection between hypertension and cancer found higher cancer mortality and an increased risk to develop kidney cancer in hypertensive individuals. we recently showed that ald produces oxidative stress, activates transcription factor nf-kb and is genotoxic in kidney tubule cells. objectives: this study investigated the capacity of ald to induce oxidative/nitrosative stress, dna damage, dna repair, apoptosis, cell proliferation and the activation of nf-κb in rat kidneys. methods: mineralocorticoid-dependent hypertension was induced by ald/salt in sprague dawley rats. dna damage and oxidative/nitrosative stress markers were detected immunohistochemically. results: ald/salt treatment caused increased blood pressure compared to untreated rats. tempol, an antioxidant, and hydralazine, a vasodilator acting independent of the renin-angiotensin-aldosterone system, could lower the blood pressure, while the mineralocorticoid receptor (mr) antagonist spironolactone was administered in a subtherapeutical dose not lowering the blood pressure. ald/salt treatment caused oxidative and nitrosative stress, structural dna damage, double strand breaks, dna repair and nf-κb activation. spironolactone decreased these markers significantly. tempol was also able to reduce these markers, while hydralazine had no effect. ald/salttreated kidneys showed a tendency to lower apoptosis and to increased cell proliferation compared to control rat kidneys. discussion: this study provides a first hint of blood pressure-independent effects of ald. the mr and the production of ros seem to be crucial for the damaging effects of ald. an aberrant or long-term activation of nf-κb by persistently high ald levels could support resistance to apoptosis and the survival of cells with damaged dna, and increase cell proliferation. these actions could contribute to the increased cancer incidence in hypertension by initiating carcinogenesis. grant support by the dfg is gratefully acknowledged. creb regulating transcriptional coactivator 1 (crtc1) is a transcriptional coactivator of the transcription factor creb. we have recently shown its expression in cardiomyocytes and its activation by beta-adrenergic signaling. beta-adrenergic signaling contributes to the pathogenesis of cardiac hypertrophy, leading to heart failure, as evidenced by the therapeutic success of the beta-adrenoceptor antagonists. in order to investigate if crtc1 is involved in this process, we investigated the expression of crtc1 in hypertrophied myocardium from mice and humans. methods: protein lysates from mouse and human samples were investigated for crtc1 protein expression. we distinguished between an acquired and an inherited form of cardiac hypertrophy. acquired cardiac hypertrophy is an adaptation of the heart to an increased cardiac workload and can be found in patients with an aortic valve stenosis. in mice this kind of hypertrophy can be evoked by transverse aortic constriction (tac). the inherited form of cardiac hypertrophy is caused by mutations in genes coding for proteins of the sarcomeric apparatus and is referred to hypertrophic cardiomyopathy (hcm). as a model for hcm, transgenic mice with a mutation in the mybpc3 gene, coding for the sarcomeric protein cardiac myosin-binding protein c (cmybp-c), were used. these transgenic mice were characterized by a hypertrophied heart. in case of human samples we distinguished between patients with an acquired form of hypertrophy due to an aortic valve stenosis, and patients with a hypertrophic obstructive cardiomyopathy (hocm), a special form of hcm. results: in the tac mice and in the myppc3 mutant mice the expression of crtc1 was significantly higher than in the controls (2.1-and 1.9-fold, respectively; n=3). in both forms of human hypertrophy, we found a significantly 5-fold upregulation of crtc1 protein level in comparison to human samples from non-failing myocardium or samples from patients with a dilatative or ischemic cardiomyopathy (n=7-10 for each group). conclusions: crtc1 is upregulated in cardiac hypertrophy with acquired or geneticbased origin. the evaluation of the role of crtc1 in the heart may help to elucidate the role of beta-adrenergic signaling in the development of cardiac hypertrophy. microarray gene expression profiling reveals up-regulation of the cardiac lipid metabolic process at the onset of heart failure fu x., abd alla j., quitterer u. eth zürich molekulare pharmakologie, winterthurerstrasse 190, 8057 zürich, switzerland atherosclerosis and chronic pressure overload are major cardiovascular risk factors for the development of heart failure in patients. to mimic those risk factors in experimental models we used atherosclerosis-prone apolipoprotein e (apoe)-deficient mice, and chronic pressure overload was imposed by abdominal aortic constriction (aac). cardiac function was monitored by echocardiography. severe atherosclerosis in aged apoedeficient mice or chronic pressure overload induced signs of heart failure as evidenced by a significantly reduced cardiac ejection fraction (<30%). to investigate pathomechanisms underlying the development of heart failure, microarray gene expression analysis and qt-rt-pcr were performed of failing heart tissue relative to age-matched controls. gene ontology analysis of the microarray data revealed that the onset of heart failure, in two different experimental models, was characterized by a strong up-regulation (≥2-fold) of the cardiac lipid metabolic process and lipid overload. lipid metabolism genes were involved in lipid synthesis, storage and oxidation. the major palmitate-synthesizing enzyme, fatty acid synthase, was causally related to the development of cardiac dysfunction by enhancing cardiomyocyte apoptosis. taken together the data support that the onset of experimental heart failure is characterized by a dysfunction of the cardiac lipid metabolism promoting cardiomyocyte death. at1-receptor blockers (arbs) are established for the treatment of high blood pressure and new onset of diabetes is reduced by arbs. in the past years evidence increased that body weight may also be lessened particularly in rats and mice. however, less data are available whether arbs still reduce weight, when treatment was initiated not until animals became obese by diet. prior to drug treatment, spontaneously hypertensive rats were fed for 6 months with chow but also with a cafeteria diet (cd) to develop obesity. controls received only chow (conchow). cd-fed shr were treated for 3 months with telmisartan (tel 8mg/kg/d) or amlodipine (aml, 12 mg/kg/d), whereas controls received vehicle (concd). systolic blood pressure (sbp), feeding behaviour, body weight, abdominal fat mass (by mrt) and energy expenditure (by indirect calorimetry) was monitored. leptin sensitivity was assessed by measuring energy intake and expenditure after repetitive injections (s.c.) of leptin. insulin sensitivity was functionally determined by glucose and insulin tolerance tests. due to cd feeding body weight was increased after 6 months by more than 60 g. tel normalized sbp whereas it remained >200 mmhg in concd and conchow. tel additionally reduced cd-induced increase of body weight and abdominal fat mass. food intake was diminished during the first 4 weeks, but raised beyond control levels during the last 4 weeks of treatment. the shift of the respiratory index to lower levels indicated improved energy expenditure. in response to exogenous leptin, the food intake of concd was higher compared to conchow, indicating a leptin resistance. this assumption is further supported by high triglyceride concentrations of concd. after tel, leptininduced food intake was reduced and energy expenditure was increased compared to concd, indicating that leptin sensitivity was at least partially restored. accordingly, triglycerides were reduced. compared to concd, the insulin sensitivity was improved by tel since maximal increases in plasma concentrations of glucose and insulin in response to glucose challenge were reduced, but glucose response to insulin challenge was diminished. even though reduction in blood pressure was almost similar between tel and aml, metabolic and antiobese efficacies of aml were markedly attenuated. we conclude that telmisartan reveals wide efficacies in improving all symptoms of the metabolic syndrome. the pleiotropic effects are not related to the hypotensive action of tel. a β2-adrenoceptor -camp mediated, immediate stimulation of β2-adrenoceptor gene expression in human lung fibroblasts is opposed by a delayed up-regulation of inhibitory factors racké k., lamyel f., kämpfer n., schütz i., warnken m. univ. bonn dept. pharmacol. &toxicol., 53105 bonn, germany based on their bronchodilatory effects, β2-adrenoceptor agonists constitute essential elements in the treatment of bronchial asthma and copd. however, treatment with long-acting β2-adrenoceptor agonists has been associated with possible worsening of airway hyper-reactivity, possibly because of loss of β-adrenoceptor function. therefore, the molecular regulation of β2-adrenoceptor expression was addressed here. mrc-5 human lung fibroblasts were cultured for up to 48 h in absence or presence of test substances, followed by β2-adrenoceptor mrna determination by qpcr. β2-adrenoceptor mrna decreased with a half-life of 25 min after inhibition of mrna synthesis with actinomycin d (30 µm), but increased by 333±85%, 502±52% and 640±165% (means±sem) within 1.5, 4 and 6.5 h, resp. after inhibition of protein synthesis by cycloheximide (30 µm). the β2-adrenoceptor agonists formoterol and olodaterol (1-100 nm) induced a rapid increase in β2-adrenoceptor mrna (maximally within 1 h by 100±19% and 110±19% at 10 nm, resp.). however, after 4 h exposure to 10 nm formoterol or olodaterol a reduction in β2-adrenoceptor mrna by 59±8% and 58±6%, resp., was observed. both, the stimulatory and inhibitory effects of β2adrenoceptor agonists were mimicked by forskolin (10 µm, increase by 88±14% and inhibition by 49±4%) and cholera toxin (5 ng/ml, increase by 76±12% and inhibition by 77±7%). the formoterol-induced up-regulation of β2-adrenoceptor mrna was blocked by actinomycin d, but not by cycloheximide. moreover, in presence of cycloheximide, β2adrenoceptor agonist induced inhibition was converted into a marked stimulation. in conclusion, expression of β2-adrenoceptors in human lung fibroblasts is highly regulated at transcriptional level. the observations with cycloheximide indicate that the β2-adrenoceptor gene is under strong inhibitory control of short-living, not yet identified suppressors. although both, the time-dependent up-and down-regulation of the β2adrenoceptor gene expression by β2-adrenoceptor activation appears to be mediated via adenylyl cyclase -camp signalling, only the stimulatory effect appears to be a direct action on the β2-adrenoceptor gene. et-1 appears to be involved in the pathogenesis not only of pulmonary hypertension, but also in fibrotic remodeling associated with chronic obstructive airway diseases. since human lung fibroblasts (hlf) are a source of et-1 and have been shown to be controlled by muscarinic receptors and β-adrenoceptors, a possible muscarinic and βadrenergic modulation of et-1 expression in hlf was explored. mrc-5 hlf were cultured for up to 24 h in absence or presence of test substances, followed by prepro-et-1 (ppet-1) mrna determination by qpcr. the muscarinic agonist oxotremorine (10 µm) induced an increase in ppet-1 mrna by 180%, an effect prevented by 10 nm tiotropium. the β2-adrenoceptor agonist olodaterol (up to 100 nm) caused a reduction of ppet-1 mrna expression by 45%. the effect of 10 nm olodaterol was prevented by ici 118,551 (1 µm), but not affect by cgp 20712 (3 µm) . the pka agonist 6-bnz-camp (500 µm) caused a reduction in ppet-1 mrna expression by 65%, whereas the epac agonist 8-cpt-2'-o-me-camp (100 µm) caused only a marginal inhibition by 22%. olodaterol (10 nm) strongly opposed the stimulatory effect of 10 µm oxotremorine. an increase in ppet-1 mrna expression by 185% caused by 0.3 ng/ml tgf-β was effectively opposed by 10 and 100 nm olodaterol, resulting in an inhibition comparable to that in absence of tgf-β. however, the increase in ppet-1 mrna caused by a maximally effective concentration of tgf-β (1 ng/ml, increase by 620%) was not significantly affected by 10 or 100 nm olodaterol. likewise, the pkaagonist 6-bnz-camp (500 µm) opposed the increase in ppet-1 mrna expression caused by 0.3 ng/ml tgf-β, but not that caused by 1 ng/ml tgf-β. tgf-β caused, with an ic 50 of 0.3 ng/ml, a marked down-regulation of β2-adrenoceptor mrna expression, maximally by 90% within 6 h. et-1 expression in hlf is stimulated by muscarinic receptors and inhibited by β2adrenoceptors. the effect of β2-adrenoceptors may be mediated via pka. et-1 expression in hlf is markedly up-regulated by tgf-β, but only effects of sub-maximally effective concentrations of tgf-β are opposed by the β2-adrenoceptor -pka pathway, in part because of tgf-β-induced down-regulation of β2-adrenoceptors. since et-1 can promote pro-fibrotic features in hlf, inhibition of et-1 expression could contribute to long-term beneficial effects of long-acting β2-adrenoceptor agonists such as olodaterol and long-acting muscarinic antagonists such as tiotropium. cardiac hypertrophy leads to up-regulation of dipeptidyl aminopeptidase-like proteins in human and rat radicke s., hutschenreuther a., schaefer m. universität leipzig rudolf-boehm-institut für pharmakologie und toxikologie, härtelstr. cardiac hypertrophy is a major risk factor for heart failure and associated morbidity and mortality. functional down-regulation of k + currents is a prominent feature of cells isolated from failing ventricles. a marked decrease in the transient outward potassium current ito has been shown in various models. changes in the k + channel expression differ depending on the species, and the mechanism of induction of heart failure. to study the regulation of ito channel subunits we compared the hypertrophic responses in human ventricular tissues from failing hearts with doxorubicin-induced hypertrophy in rats and in h9c2 embryonic rat cardiac cells. specifically, we quantified mrna expression of the pore-forming subunits kv4.3 and kv4.2, the cytosolic β-subunit kchip2 and the transmembrane subunits dpp6 and dpp10 using rt-pcr. treatment with doxorubicin (2 µm) induced hypertrophy and increased the mrna expression of the hypertrophy marker genes anf, bnp and beta-mhc in h9c2 cardiac myoblasts. while kv4.3 was detected in h9c2 cells and hearts from human and rat, kv4.2 mrna was only expressed in adult rats. during hypertrophy kv4.3 was downregulated in human tissue as well as in doxorubicin-treated h9c2 cells compared to the controls. in rat hearts kv4.2 expression was increased after doxorubicin treatment. interestingly, kv4.2 was also found to be up-regulated in rat heart tissues and h9c2 cells after treatment with doxorubicin. as previously shown, kchip2 mrna expression was significantly reduced in tissues of failing hearts. in contrast, kchip2 mrna was upregulated in hypertrophic rat hearts and h9c2 cells. the expression of dpp6 and dpp10 was observed only in human hearts. but mrna levels of both were significantly increased in failing tissues. dpp6 and dpp10 were not expressed in the adult rat heart or h9c2 cells, whereas in rats with doxorubicin-induced cardiac hypertrophy and in doxorubicin-treated h9c2 cells, the mrna of dpp6 and dpp10 was up-regulated. h9c2 cells showed almost identical hypertrophic responses to those observed in human ventricles and rat hearts. this finding validates h9c2 cells as a model for in vitro studies of cardiac hypertrophy. in further studies we will investigate the consequences of a knock-down of dpp6 and dpp10 in doxorubicin-induced hypertrophic h9c2 cells. in preliminary experiments specific short-hairpin rna, targeting dpp6 and dpp10, has been designed and tested in heterologous expression systems. the nonsynonymous c.521t>c germline genetic variation in the liver-specific organic anion transporter slco1b1 is associated with methotrexate pharmacokinetics in pediatric acute lymphoblastic leukemia radtke s. 1 background: methotrexate (mtx) plasma concentration is related to its clinical effect. transport proteins, such as abcc2, slco19a1, and slco1b1, have been implicated in the disposition of mtx. here we investigated whether common reduced-function variants in abcc2, slco19a1, and slco1b1 contribute to the interindividual variability in methotrexate pharmacokinetics in children with acute lymphoblastic leukemia (all). we analyzed mtx pharmacokinetics (mtx plasma concentration at the end of infusion c24h, mtx auc24-48h, and mtx clearance cl) in an unselected population of 419 children with all from the all-bfm 2000 trial (clinicaltrials.gov: nct00430118) who received 1676 courses of mtx at 5 g/m 2 as 24 h infusions. the contribution of genes (genetic component, rgc) to the interindividual variability in mtx pharmacokinetics was estimated according to the method of kalow et al. (1998) . abcc2 c.-24c>t (rs717620), slco19a1 c.80g>a (p.his27arg, rs1051266), slco1b1 c.521t>c (p.val174ala, rs4149056) and slco1b1 388a>g (p.asn130asp, rs2306283) genotypes were analyzed by taqman polymerase chain reaction. there was substantial interpatient variability in average (± sd) mtx c24h (50.95 ± 24.15 µmol/l), auc24-48h (57.44 ± 37.52 h*mg/l), and cl (390.72 ± 223.95 ml/min/m²). the rgc values of c24h, auc24-48h, and cl ranged from 0.61-0.71 suggesting that variation in mtx pharmacokinetics has a substantial genetic component. after adjustment for age and sex by multiple regression, the slco1b1 c.521t>c snp was significantly associated with c24h (p<0.001), auc24-48h (p<0.001), and cl (p=0.011) of mtx. compared with the wildtype genotype, in patients with the tc genotype c24h and auc24-48h increased by 18% (p=0.009) and 28% (p=0.003), respectively, whereas cl significantly decreased by 15% (p=0.012). pharmacokinetic variables significantly changed with increasing number of variant c.521t>c alleles (p<0.02, jonckheere-terpstra), suggesting a per allele effect consistent with a co-dominant model of association. in contrast, the abcc2 c.-24c>t, slco19a1 c.80g>a, and slco1b1 388a>g polymorphisms did not show an association with mtx pharmacokinetics. conclusions: the nonsynonymous c.521t>c polymorphism in slco1b1 contributes to the variability of mtx pharmacokinetics in this study of high-dose mtx in pediatric all. this project is supported by the johannes und frieda marohn-stiftung. the antitumorigenic mechanism of the selective cyclooxygenase-2 (cox-2) inhibitor celecoxib is still a matter of debate. using human lung cancer cell lines (a549, h358, h460), the present study investigates the contribution of cox-2 and peroxisome proliferator activated receptor γ (pparγ) to apoptosis elicited by celecoxib. celecoxib was found to cause apoptotic cell death in a concentration-dependent manner (10 -50 µm), whereas structurally-related cox-2 inhibitors (etoricoxib, rofecoxib, valdecoxib) were inactive in this respect. apoptotic cell death by celecoxib was suppressed by preincubation of tumor cells with the selective cox-2 inhibitor ns-398, the pparγ antagonist gw-9662 and by sirna targeting cox-2 or pparγ. celecoxib-induced apoptosis was paralleled by a time-and concentration-dependent upregulation of cox-2 and pparγ at both mrna and protein level. using an established cox-2 activity assay monitoring immediate conversion of exogenously added arachidonic acid to the respective prostaglandins (pgs), ns-398 was shown to suppress celecoxib-induced cox-2 activity when added prior to arachidonic acid. among the cox-2-dependent pgs analyzed, pgd 2 and its dehydration product 15-deoxy-∆ 12,14 -pgj2 were found to induce cytosol-to-nucleus translocation of pparγ as well as pparγ-dependent apoptosis. celecoxib-elicited translocation of pparγ was inhibited by preincubation of cells with ns-398 which itself did not alter celecoxib-induced total pparγ protein expression. finally, a cox-2-and pparγ-dependent proapoptotic mechanism of celecoxib was confirmed in primary tumor cells obtained from brain metastases of two lung cancer patients. together, our data demonstrate a proapoptotic mechanism of celecoxib involving initial upregulation of cox-2 and pparγ and a subsequent nuclear translocation of pparγ by cox-2-dependent pgs. uncoagulated ppp contained 40-60 nm thrombin throughout. fxa was initially measured in clots at 10 nm. this level declined over time while clot-conditioned pbs accumulated fxa. exposure of human aortic smc to clots or native unclotted ppp for 24h only marginally influenced smc apoptosis but increased mitogenesis over 15-fold. this was reduced by all 4 inhibitors. clot-stimulated induction of tnfα and interleukin-6 mrna was also attenuated by the inhibitors. denatured ppp (no protease activity) increased smc mitogenesis to a level seen in smc exposed to clot and combined hirudin + dx9065a, reflecting the well-known mitogenic actions of serum alone. conclusion: coagulation of human plasma generates nanomolar amounts of thrombin and fxa, sufficient to stimulate the proliferative and inflammatory properties of adjacent smc. our observations validate the use of purified thrombin and fxa at nanomolar concentrations for in vitro studies, and support the individual and coagulationindependent roles of these proteases in cell proliferation and inflammation. antithrombotic therapy with argatroban or rivaroxaban may limit the cellular effects of clot-derived thrombin and fxa, while normal anti-platelet therapy would not. this aspect should be considered in the clinical use of these agents, specifically in healing processes after vessel injury. rhogef17 mediates cgmp/cgk induced adherence and relaxation of vascular smooth muscle cells rauch j. 1 , stephan-schnatz k. the guanine nucleotide exchange factor rhogef17 is the only gef known so far to be directly activated by cgmp-dependent kinase. it is expressed in various types of smooth muscle cells and has been shown to play a role in the regulation of cell integrity. in a previous investigation we showed that the knockdown of rhogef17 by a shrna approach caused a loss of actin stress fibers and a subsequent change of smooth muscle cell morphology that finally resulted in cell rounding. we now provide evidence that the expression level of rhogef17 influences the re-attachment of cultured rat aortic smooth muscle cells (rasmc) to a surface after detachment. although rhogef17 depleted rasmc were still able to adhere and spread, their cell surface area remained considerably smaller than that of control cells in the first 24 hours after seeding. cell counting revealed that 6 to 12 hours after seeding the percentage of adherent cells was significantly lower in the rhogef17 knockdown group compared to the control group. these data indicate a delay in attachment. interestingly, the knockdown of rhogef17 was paralleled by a loss in rhoa and cadherine expression. as rhogef17 mediated a cgmp-induced activation of the small gtpase rhoa in rasmc, we studied the effect of a stable cgmp analogon (8-pcpt-cgmp) on the adhesion process. in accordance with previously published data, cgmp treatment accelerated the attachment of rasmc to the surface within the first 12 hours. in contrast, the adhesion of rasmc after rhogef17 knockdown was no longer stimulated by cgmp. as these data indicate that rhogef17 mediates cgmp-dependent signalling in a physiological process we wondered whether this protein might also play a role in the regulation of cgmpdependent relaxation of vascular smooth muscle cells. thus, we performed a collagenbased contraction assay with rhogef17 depleted rasmc. while the contraction in response to serum was not affected by the depletion of rhogef17, we observed a slight decrease in basal contractility. interestingly, cgmp was not able to counteract the serum-driven contraction of rhogef17 knockdown cells. there was no cgmp-induced relaxation in these cells. we conclude that rhogef17 is involved in the cell adhesion of vascular smooth muscle cells and likely promotes the expression of specific proteins. its activation is required to mediate cgmp-induced signalling in terms of vascular smooth muscle cell adherence and relaxation. human eosinophil and neutrophil granulocytes are cells of the innate immune system. they both express formyl peptide receptors (fpr) und histamine h2 receptors (h2r). activation of fpr leads to a release of reactive oxygen species (ros). h2r activation results in an increase of intracellular 3'-5'-cyclic adenosine monophosphate (camp) concentration and inhibition of fpr-mediated ros release via adenylyl cyclase activation. in this study we compared the effects of various h2r ligands on camp accumulation and formyl peptide n-formyl-l-methionyl-l-leucyl-l-phenylalanine (fmlp)induced ros release in isolated eosinophils and neutrophils. camp concentration was determined by hplc/tandem mass spectrometry, and ros release was assessed by monitoring superoxide dismutase-inhibitable reduction of ferricytochrome c. in eosinophils, histamine, amthamine and 5-methylhistamine exhibited similar potencies and efficacies with regard to camp accumulation and inhibtion of ros release. in marked contrast, in human neutrophils, we observed dissociations in potencies and efficacies of ligands at increasing camp accumulation and inhibition of ros production. our data suggest that in human eosinophils, but not neutrophils, camp mediates inhibtion of ros production. in a broader context our data provide a compelling example of the context-dependency of the pharmacological properties of g-proteincoupled-receptors. specifically, one has to be cautious when extrapolating experimental observations from one cell type to another, even when they are very closely related to each other. comonomers and monomers are used as dental restorative materials (e.g. in dental composites). unconverted compounds can be released from dental composites and can enter the body in humans. comonomers can induce various side effects in humans. this study was evaluated to qualify and to quantify eluted compounds from various dental composites. following composites were tested (producer in parentheses): els extra low shrinkage (saremco), synergy duo shade (coltène), grandio (voco), tetric evo ceram (vivadent), venus (kulzer), gradia (g.c.), and premise (kerr).polymerized composites (100 mg) were incubated in gc vials with 1 ml dest. water or 1 ml methanol, each at 37 °c for 72 hours. aliquots were taken, and eluted compounds were analyzed with the method of gas chromatography/mass spectrometry (gc-ms) and liquid chromatography/mass spectrometry (lc-ms).from all composites 18 different compounds were found. following comonomers were quantified (µg/ml; mean ± s.d.; n=4)( fig. 1 ).following range of the eluted and detected comonomers from dental composites was found (dest. water; decreasing elution): venus > gradia > synergy duo shade > tetric evo ceram premise > grandio > els extra low shrinkage. * n.d. = not detectable (below limit of detection). triphenylstibane was detected in tetric evo ceram (5 ± 2 µg/ml). reimann c., lupp a., schulz s. institut für pharmakologie und toxikologie universitätsklinikum jena, drackendorfer str. objective: cxcr4 is a plasma membrane chemokine receptor which is involved e.g. in organogenesis, hematopoesis and inflammation. in the adult organism cxcr4 is physiologically expressed on various cell types, in particular on lymphocytes. with respect to neoplastic tissues, in the current literature an over-expression of cxcr4 is described in different types of tumors, especially in breast and prostate cancer. additionally, an involvement of cxcr4 in tumor metastasis is discussed. subsequently, detection of cxcr4 expression in a given human tumor sample would provide a valuable predictive information on disease prognosis and possible therapeutic intervention. however, previous attempts to localize cxcr4 using poorly characterized mouse monoclonal or rabbit polyclonal antibodies have yielded predominant nuclear and occasional cytoplasmic staining, but did not result in the identification of cell surface receptors. thus, the aim of the present study was to reassess the cxcr4 expression in a panel of formalin-fixed and paraffin-embedded human normal and neoplastic tissue samples by means of immunohistochemistry using the well characterized novel rabbit monoclonal anti-cxcr4 antibody umb-2. methods: in comparison to negative and positive control samples (cxcr4-knockout and wild-type mouse embryos) the extent of staining in the different normal and neoplastic tissue specimens was scored from zero (no expression) to three (high expression). results: cxcr4 was found to be expressed in all neoplastic tissue entities analyzed. in many cases, the receptor was predominantly localized at the plasma membrane of the tumor cells. however, in all cxcr4-expressing tumor entities a huge interindividual variability both in the percentage of positive cells and in the intensity of staining was noted which strongly differed also between the various types of cancer. the most intense (score three) staining was found in the samples of (small cell) lung cancer, ovarian cancer and of pheochromocytoma. additionally, lymphatic organs such as lymph nodes, spleens and tonsils were cxcr4 positive, with mainly b cells displaying a distinct staining of the plasma membrane. the rabbit monoclonal antibody umb-2 may prove to be of great value in the assessment of cxcr4 expression in different human tumor entities and of the mechanisms underlying the formation of metastases, thus helping to find new targets and strategies in cancer therapy. link between β2-adrenoceptor-mediated inhibition of formyl-peptide-induced o2 β2-adrenoceptor (adrb2)-agonists are in daily use for asthma therapy. although most cases of asthma are controlled by standard medication, a subpopulation of asthmatics remains difficult to treat. the adrb2 gene contains a total of 49 polymorphisms. this variability could cause part of the ~70 % genetically-determined differences in therapy response. genetic and corresponding functional data on adrb2 can help to understand the complex disease and, in cases of severe asthma, optimize therapy with adrb2agonists for each individual. (ortega et al. 2007; chung et al. 2011) our present study connects sequence data with pharmacological data of prototypical adrb2-ligands, namely, (r)-isoproterenol, (r,r)-and (s,s)-fenoterol, (r)-and (s)salbutamol and (r,r)-formoterol. as a pathophysiologically relevant cell type, we analysed human neutrophils from peripheral blood of healthy volunteers. formyl-peptide-induced o2 .production and its inhibition by the agonists are examined in a 96-well cytochrome-c assay. characteristic pharmacological values (pic50, emax) are obtained for each individual. the data-set for each individual is supplemented by a differential blood cell analysis and an asthma-related questionnaire. most importantly, each volunteer's adrb2-sequence variant is determined by sanger-sequencing. complete determination of a 1,490 bp sequence, including the entire adrb2 exon and part of the flanking 5'-and 3'-untranslated regions, allows mapping of the most common, but also of new or rare polymorphisms. first data demonstrate the inter-day and intra-individual robustness of the functional data. in the next step, we will link sequence variants and functional differences within a population of sixty volunteers with sufficient statistical power. collectively, this study represents a straightforward approach to link functional and genetic data of a clinically relevant receptor. cyclic nucleotide phosphodiesterases (pdes) are classified into eleven families and are essential for second messenger metabolism in human cells. 1 recently, we have shown that several human pdes possess a much broader substrate-specificity than previously assumed, being capable of hydrolyzing not only the purine nucleotides cyclic adenosine 3',5'-monophosphate (camp) and cyclic guanosine 3',5'-monophosphate (cgmp), but also pyrimidine nucleotides such as cyclic uridine 3',5'-monophosphate (cump). 2 these data were obtained using a highly sensitive hplc mass spectrometric assay which is quite expensive and whose technical requirements are available only in few laboratories. in our present study we developed a fluorimetric pde activity assay using 2'-o-(n'methylanthraniloyl) (mant)-substituted cyclic purine and pyrimidine nucleotides that can be used more broadly in the scientific community. human pde3a is important for the regulation of platelet aggregation, oozyte maturation, vascular smooth muscle relaxation and contractility of cardiac myocytes. 1 moreover, this pde shows a broad substrate-specificity, hydrolyzing camp, cgmp, cump and cyclic inosine 3',5'-monophosphate (cimp). 2 using this enzyme, here, we demonstrate that various mant-substituted cyclic nucleotides are substrates of pde3a and undergo a significant change in fluorescence whilst being hydrolyzed, thus allowing a quantitative analysis of catalysis via fluorescence detection. in fact, not only native cump but also mant-cump is a substrate of pde3a. this finding is consistent with data published by hardman and sutherland 3 who described a cump-degrading pde activity in homogenates from beef and dog heart, leading to the assumption that the pde activity described there could be attributed to pde3a. as cump has furthermore been proven to be present in mammalian cells 4 , to differentially activate camp-and cgmp-dependent protein kinases 5 and to be synthesized from utp by mammalian soluble guanylyl cyclase 6 , our present study supports the hypothesis that this cyclic nucleotide could play an important role in cell metabolism. the newly established fluorescence assay with mant-cump facilitates future studies on pde3a and the assumed second messenger function of cump. rhoa is reportedly involved in stat-dependent transcription. however, the pathway connecting the gtpase and stat signaling has not been characterized. we made use of bacterial toxins, which directly activate rho gtpases to analyse this pathway. cytotoxic necrotizing factors (cnfs) are produced by pathogenic escherichia coli strains and by yersinia pseudotuberculosis. they activate small gtpases of the rho family by deamidation of a glutamine, which is crucial for gtp hydrolysis. we show that rhoa activation leads to phosphorylation and activation of stat3 and identify signal proteins involved in this pathway. rhoa-dependent stat3 stimulation requires rock and junkinase activation as well as ap1-induced protein synthesis. the secretion of one or more factor/s activate/s the jak-stat pathway in an auto/paracrine manner. we identify ccl1/i-309 as an essential cytokine, which is produced and secreted upon rhoa activation and which is able to activate stat3-dependent signaling pathways. the knowledge about the connection between rhoa and stat signaling is crucial for understanding several deseases, especially cancer. acid sphingomyelinase-deficient mice are protected from the lethal cardiovascular effects in tnf-induced septic shock reiss l. k. 1 christian-albrechts universität institut für immunologie, michealisstrasse 5, 24105 kiel, germany introduction: the cytokine tumor necrosis factor (tnf) is a mediator of septic shock. sepsis is a major cause of acute respiratory distress syndrome (ards), a heterogeneous lung disease with a mortality of about 50%. the present study was designed to investigate the effects of high systemic tnf-levels on the lung and on the systemic circulation in wildtype and acid sphingomyelinase-deficient (asm -/-) mice. the enzyme acid sphingomyelinase generates the signaling molecule ceramide that plays a critical role in edema formation and vasodilatation. material and methods: asm -/and wildtype mice were ventilated mechanically at vt=8ml/kg and f=180min -1 with fio2=0.3 and peep=2cmh2o while lung mechanics were followed. half of the mice received 50µg of murine tnf intravenously. blood pressure was stabilized by intra-arterial fluid support and body temperature was kept at 37°c to prevent lethal shock and to allow investigation of blood gases, lung histopathology, pro-inflammatory mediators and microvascular permeability 6 hours after tnf application. results: tnf induced septic shock in wildtype mice, as indicated by metabolic acidosis, high serum levels of the sepsis marker procalcitonin, decreasing blood pressure and reflex tachycardia. interestingly, asm -/mice were protected from the tnf-induced cardiovascular effects and mortality. in the present study, circulating tnf failed to cause lung injury. lung mechanics stayed stable during ventilation in all groups and also pulmonary histopathology, cytokine levels and microvascular permeability were unaffected. conclusion: circulating tnf alone is not sufficient to cause acute lung injury. we conclude that the cardiovascular effects in tnf-induced septic shock are partly mediated by acid sphingomyelinase. cyclophilin a sirna provides mitoprotection and prevents aif-dependent neuronal cell death reuther c. 1 cyclophilin a (cypa) is a peptidyl-prolyl-cis-trans isomerase which is localized in the cytosol. recent data suggested that neuronal cell death involved cytosolic cypa translocation to the nucleus, where it formed a pro-apoptotic complex with apoptosis inducing factor (aif). this cypa-aif complex induced caspase-independent chromatin condensation, dna degradation and cell death in various paradigms of apoptosis. on the basis of these data, the selective inhibition of the aif-cypa complex was proposed as a potential strategy to prevent aif-dependent cell death in neurons. therefore, the aim of this study was to determine effects of cypa silencing in a model of glutamate toxicity in immortalized hippocampal ht22 neurons. first, we addressed the interaction of aif and cypa by immunoprecipitation and their translocation to the nucleus by immunohistochemistry and confocal fluorescence microscopy. after exposure of ht-22 cells to glutamate the translocation of aif and cyp a occurred prior to cell death. cypa sirna attenuated glutamate-induced cell death as detected by the mtt-assay, impedance measurements (xcelligence system), and by facs analysis after annexin v/ propidium iodide staining. most intriguingly, cypa sirna also preserved the mitochondrial membrane potential as shown by facs analysis after tmre staining. further, confocal microscopy showed that cypa silencing prevented mitomorphology alterations and blocked the release of mitochondrial aif to the nucleus. the inhibition of the aif translocation to the nucleus was also shown by western blot analysis. in summary this study demonstrates that silencing of cypa prevents mitochondrial disruption and attenuates glutamate toxicity in vitro. thus, cypa is a promising target for mitoprotection as a basis for novel strategies of neuroprotection. up to now the question is unresolved how the ingested dose influences the absorption and metabolism of chlorogenic acids (cga) from food. so far no studies have been performed on the impact of the dose on cga absorption, circulation and excretion. recently we performed a dose-response study in a randomized, double-blinded, crossover design with five ileostomy subjects. in three trials the volunteers consumed after a two day polyphenol free diet coffee with varying cga content (high 4525 µmol; medium 2219 µmol; low 1053 µmol). the cga concentrations in plasma, ileal effluent and urine were subsequently identified and quantified by hplc-esi-ms, hplc-esi-ms/ms and hplc-dad. the results showed that the consumption of higher cga concentrations lead to a faster ileal excretion measured in the ileal effluents. this corresponded to the renal excretion of 8.0 ± 4.9% (high), 12.1 ± 6.7% (medium) and 14.6 ± 6.8% (low) of total cga and metabolites. we found that cgas with a caffeic acid moiety are predominantly sulphated and those with a ferulic acid moiety are predominantly conjugated via glucuronidation prior renal excretion. furthermore, in the ileal effluents, sulphation of both structural units dominated. in plasma samples (after enzymatic deconjugation) the auc values were determined by the major cga classes in coffee, the caffeoylquinic acids: 551.5 ± 93.8 nm*h -1 (high); 299.3 ± 79.6 nm*h -1 (medium) and 222.8 ± 91.3 nm*h -1 (low). no major differences in the metabolic pattern were observed. additionally, we were able to identify new metabolites of cga in urine and ileal fluids. we conclude that the consumption of high concentrations of cga via coffee might influence the gastrointestinal transit time and consequently affect cga bioavailability. this study was supported by the nestlé research centre (lausanne, switzerland). interaction of antagonists with the atp binding pocket at the human p2x3 ion channel helms n., riedel t., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany the homomeric p2x3 receptor (p2x3r) is a rapidly activating and desensitizing cation channel, gated by extracellular atp. it consists of three homomeric subunits. this representative of the p2x receptor family is highly expressed on sensory afferent neurons and plays a significant role in chronic pain, bladder reflexes and taste sensation. therefore, the development of selective antagonists for p2x3 receptors and knowledge about the binding of these antagonists are of great significance for future pain therapy and therapy of urge incontinence. to simulate the shape of the rapidly desensitizing agonist-induced current responses via p2x3 receptors, we created a specific markov model to describe the binding of agonists and competitive antagonists. this model can be used to prove the competitive character of inhibition and to calculate the association and dissociation constants of the antagonists. furthermore we use this model to fit current responses at p2x3 wild type receptors and their mutants to α,βmethylene atp in the presence of different antagonists. whole-cell patch-clamp recordings were performed on hek 293 cells, heterologously expressing the human p2x3 receptor, to determine the concentration-response relationship of different antagonists. by applying increasing concentrations, differences of antagonist potency could be observed at the wild type receptor. afterwards, we chose amino acid residues for replacement by alanine, which seem to be important for agonist binding and should be so for competitive antagonist binding as well, based on our homology model, developed from the zebrafish p2x4r crystal structure and previous mutagenesis studies. we intend to identify those amino acids which are important for competitive antagonist binding by monitoring the altered antagonist potency on the mutated receptor when compared with the wild-type receptor. analysis of the p2x3 agonist binding site by double mutant cycles riedel t., wiese s., illes p. rudolf-boehm-institut für pharmakologie und toxikologie, härtelstraße 16-18, 04107 leipzig, germany purinergic p2x receptors belong to the family of ligand-gated ion channels. they are non-selective cation channels, activated by extracellular atp. one of the seven members of the p2x receptor family, the p2x3 receptor, is localized at the plasma membrane of sensory neurons and is involved in pain perception. therefore, this receptor is a possible target for new drugs in pain treatment. the development of such drugs can be supported by an exact knowledge of the receptor structure and function. there are many hints to the atp binding site, but the interaction of the p2x receptor with its agonists and antagonists remains still unknown. in this study, we investigated the effects of single alanine substitutions of amino acid residues in the supposed atp binding site of the homomeric human p2x3 receptor on the effect of nucleotide analogues. the mutant receptors were expressed in hek293 cells and the nucleotide effects were measured by means of the whole-cell patch-clamp method. modifications in the receptor binding site changed the concentration-response dependency as well as the current kinetics during fast pulsed agonist applications. based on this fact, we were able to distinguish binding from gating, conductance, and desensitisation, using a markov model that describes the complete channel behaviour by a matrix of rate constants. the results were also checked for consistency with a structural hp2x3 model that we developed from the known zebra fish p2x4 crystal structure in the closed state. voltage-dependent modulation of alpha2a adrenergic receptor signaling rinne a., birk a., bünemann m. philipps-universität marburg institut für pharmakologie und klinische pharmazie, karlvon-frisch str. 1, 35034 marburg, germany g protein-coupled receptors (gpcrs) are proteins that regulate numerous signaling pathways by activation of intracellular g proteins. gpcrs are activated by extracellular stimuli, such as light, hormones and neurotransmitters. recent evidence suggests that some gpcrs exhibit voltage-sensitivity leading to a modulation of their activity by the membrane potential (vm). we used a fret-based biosensor of the α2a adrenergic receptor to analyze receptor activation at defined membrane potentials in hek 293 cells by means of voltage-clamp recording. the biosensor was stimulated either with the partial agonist clonidine or with the full agonist norepinephrine (ne) and receptor activation was measured as decrease in the ratio of acceptor-/donor-fluorescence. receptor stimulation by ne was inhibited at depolarizing membrane potentials but enhanced by hyperpolarization. inhibition of ne activated receptors was strong at low concentrations (500 nm: 60 % inhibition) but almost absent at saturating agonist concentrations (100 µm: 9 % inhibition). both agonist-induced and hyperpolarizationinduced receptor activation exhibited a similar monoexponential time course and speed of activation was primarily dependent on agonist concentration for both activation modes. the latter indicates that depolarization lowers the apparent affinity of the ne receptor interaction and thus causes receptor deactivation by means of ne release. application of clonidine (1 µm, vm=-90 mv) resulted in a fret response that was inhibited by 40 % at +60 mv. in contrast to ne, strong receptor inhibition at +60 mv was present even at super-saturating concentrations of clonidine (100 µm), suggesting that voltage alters the equilibrium between active and inactive conformations of the receptor. voltage-dependence of the a2a adrenergic receptor also modulated downstream receptor signaling: g protein activation or the recruitment of arrestins, which we determined in fret assays that directly detect gαi protein activation or receptor-arrestin interactions, were both substantially inhibited at vm = +60 mv. therefore we conclude that negative membrane potentials promote active conformations of the a2a adrenergic receptor, increase affinity of full agonists and enhance receptor signaling. in conclusion the present data show that scc cells extrude more ha, possibly related to increased levels of has3, in comparison to keratinocytes. increased amounts of ha appear to be essential for the uvb induced tumourgenesis of sccs in mice. this effect might be related to the pro-proliferative property of high molecular weight ha. furthermore biological active ha fragments derived from ha degradation by hyaluronidases (hyal1,2) are thought to be pro-angiogenetic, anti-apoptotic and proinflammatory, thus possibly also promoting tumour growth and malignancy. estradiol induced paracrine release of egf from keratinocytes protects the dermal hyaluronan/versican matrix during photoaging röck k. 1 , meusch m. hyaluronan (ha) and versican are key components of the dermis and are responsive to uvb induced remodeling. the aim of the present study was to investigate the molecular mechanisms of estrogen (e2) mediated effects on ha-rich ecm during actinic aging. 10 weeks of uvb irradiation (3 x 1 med (80 mj/cm 2 ), weekly) of hairless skh-1 mice caused a marked decline of dermal ha, which was aggravated by ovariectomy (ovx). subcutaneous substitution of estrogen (e2) by means of controlled release pellets abolished these effects confirming the stimulatory role of e2. the increase of dermal ha correlated with induction of ha synthase has3 by e2. in addition the ha-binding proteoglycan versican was induced by uvb and further increased by e2. however in cultured skin fibroblasts e2 reduced the expression of versican and had no effect on has3. therefore, direct upregulation of has3 and versican in fiborblasts by e2 was excluded. however, e2 increased the expression of egf in uvb irradiated skin in vivo and in keratinocytes in vitro. egf in turn upregulated the expression of has3 and versican in dermal fibroblasts. furthermore the supernatants of estradiol treated keratinocytes led to the same effects in dermal fibroblasts, which could be abolished by previous treatment of the supernatant with neutralizing egf antibody or treatment of the fibroblasts with egf receptor blocker erlotinib. functionally, dermal ha and versican induction by e2 correlated positively with proliferation and negatively with accumulation of inflammatory macrophages in the dermis. collectively these data suggest that e2 treatment increases the amount of dermal ha and versican via paracrine release of egf which may be implicated in the pro-proliferative and anti-inflammatory effects of e2 during photoaging. differential pro-and eukaryotic toxicity of silver released from nanocomposite surfaces increases the therapeutic window of silver in antibacterial treatments röhl c. 1 , hrkac t. silver has been used since ancient times as antimicrobial agent. recently, silver gained new attention due to its higher effectiveness in its nanoform. this led to new developments of silver nanomaterials, e.g., for medical devices and consumer products. though, it is generally assumed that silver is less toxic for eukaryotes than for prokaryotes, concern is raised, if nanosilver at the same time might also increase mammalian cytotoxicity. in our study we examined the toxicity of silver released from nanocomposite surfaces with that of silver from agno3 solutions for adherent bacteria and mammalian cells. therefore, we established an in vitro reference system which enabled us to compare the therapeutic window between prokaryotic toxicity and eukaryotic integrity in both exposure settings. we focussed especially on the comparability of the bacterial and mammalian cell systems and the development of characterized ag/tio2 nanocomposite coatings with well-defined silver filling factors and silver surface release, which could be varied over a wide concentration range. as reference cells the e. coli sar 18 strain and human dermal fibroblasts, which are of special relevance in the context of medical devices like implants or wound dressings, were chosen. bactericidal effects were determined by direct growth visualization of the gfp-producing e. coli strain by epifluorescence microscopy. mammalian cell growth and toxicity was determined by the mtt assay, protein measurements and phase contrast microscopy. the ag/tio2 samples were prepared by sputter co-deposition from two separate magnetron sources. the silver surface concentration release was determined by xps and the silver release by icp-ms. in solution a concentration-dependent constant silver concentration could be determined between 2 and at least 72 hours at the surface. while lowest bactericidal and cytotoxic concentrations of ag + from agno3 solutions with 0.64 and 0.95 mg/cm 2 , respectively, differed only slightly, the therapeutic window increased significantly if ag + was released from the nanocomposite surface. while the toxicity on the fibroblasts was unchanged the bactericidal potency increased at least one order of magnitude. taken together, it can be concluded that local exposure factors i) can be modulated by silver nanocomposites and ii) play an important role for the differential toxicity of surface silver on bacteria and mammalian cells. charite -universitätsmedizin institut für integrative neuroanatomie, funktionelle zellbiologie, philippstr. 2, 10115 berlin, germany c3 exoenzyme (c3bot) a clostridial adp-ribosyltransferase does not possess a cellbinding/-translocation domain. nevertheless, c3 is able to efficiently enter intact cells, including neuronal cells but the mechanism of uptake is not yet understood. in the present work, binding of c3bot to the hippocampus-derived ht22 cell line was characterized by means of binding and blot overlay assays as well as mass spectrometry analysis to identify binding partners of c3bot. the binding assays established that c3bot bound in a concentration-dependent manner to ht22 cells. in the overlay assay we detected one clear band of 55 kda. to elucidate whether glycosylation is important for the c3bot-protein interaction, ht22 cells were incubated with glycosidase f resulting in a decreased binding of c3 to the 55 kda band. to explore the involvement of phosphorylation in the binding of c3 to the putative binding protein, blot was pre-treated with cip (calf intestinal phosphatase) before overlay with c3bot. pretreatment greatly reduced the c3bot-protein interaction. moreover, inhibition of dephosphorylation by vanadate before in intact cells showed an increased level of c3botprotein interaction in the following overlay. thus, interactions between c3bot and ht22 cell proteins may require phosphorylation. to further characterize the 55 kda band as binding target of c3bot, the 55 kda band was digested with trypsin and then subjected to lc-orbitrap mass spectrometry analysis. from this 55 kda single gel band 141 proteins were identified. further analysis of the identified proteins will provide a possible interaction partner of c3bot. in sum, protein overlay assays revealed that phosphorylation and glycosylation are critical for efficient c3bot-protein interaction. s76 335 solvent effects on enzyme kinetics in vitro rokitta d., pfeiffer k., gerwin h., streich c., fuhr u. uniklinik köln institut für pharmakologie, gleueler str. 24, 50931 köln, germany kinetic parameters provide essential quantitative information for characterisation of drug metabolising enzymes. such enzymes are located in an a partially queous environment, but to solve potential lipophilic substrates for in vitro measurements organic solvents are regularly needed. to preserve the enzymes from denaturation and other solvent related effects, the concentration of these solvents must be kept low. data on nature and extent of such solvent effects is sparse. in this study, we investigated the effects of methanol, ethanol, acetonitrile and dimethylsulfoxide (1% to 4%) on the assessment of k m, vmax and clint with regard to the 1-hydroxylation of midazolam via cyp3a4 and the cyp1a2 catalyzed metabolism of caffeine to paraxanthine in vitro. the presence of acetonitrile showed the highest vmax value for paraxanthine formation but the lowest values for 1-hydroxymidazolam formation. the km value for midazolam showed no systematic effects of organic solvents, while for caffeine km was up to eightfold lower for solvent free samples compared to solvent containing samples. the present example suggests that the presence of organic solvents may considerably influence enzyme kinetic parameters beyond a mere change in apparent activity. these effects are differing between enzyme-substrate systems and solvents. it remains to be determined to which extent such effects compromise in vitro -in vivo extrapolations, and which solvents are most appropriate. atrial remodeling and arrhythmia induced by the transcription factor er81 rommel c. 1 , rösner s. introduction: the transcription factor er81 (ets related 81) belongs to the large family of ets-transcription factors that are involved in developmental processes and in the pathogenesis of cancer. er81 is activated by gq-and gs-coupled receptors leading to a phosphorylation of the transcription factor by map-kinases and protein kinase a, respectively. cardiac er81 mrna expression is increased in failing human hearts. however mechanical unloading by a left ventricular assist device leads to normalization of er81 expression. thus, the aim of the present study was to investigate the cardiac function of er81 in genetically modified mouse models. we previously generated transgenic mice overexpressing er81 under control of the cardiomyocyte-specific α-myosin heavy chain gene (αmhc) promoter by pronuclear injection and established independent transgenic lines. electrocardiography (ecg) was assessed in mice at day 5 after birth (p5) and in adult mice (3 months) during isoflurane anesthesia and by ecg telemetry in awake mice, respectively. ecg analysis revealed no differences between the genotypes at day 5 after birth. however, we found a decreased heart rate, a replacement of regular p-waves by an undulating baseline and frequent supraventricular extrasystoles in adult er81 αmhc transgenic mice. next, isometric contractile force measurements on isolated left atria were carried out in organ baths. while wt left atria responded to increasing concentrations of isoprenaline, nkh477 and calcium with an increase in contractility, the maximal positive inotropic responses to these substances were severely blunted in er81 αmhc atria. we performed western blots to identify potential aberrations of calcium handling and regulatory proteins. phosphorylation of serine 16 of phospholamban (pln) was reduced in er81 αmhc mice. in addition, protein phosphatase 1 (pp1) expression was significantly increased in er81 αmhc mice, which is consistent with the increased dephosphorylation of phospholamban. furthermore, we found a decreased expression of calsequestrin and serca2a protein in er81 αmhc atria. electron microscopy revealed the significant structural remodeling of er81 αmhc atria at 3 months of age. conclusion: increased cardiac expression of the ets-transcription factor er81 leads to structural and electrical remodeling of the atria. thus, er81 may play an important role in the pathogenesis of cardiac arrhythmias in chronic heart failure. signal transduction pathway of atp and utp in neonatal rat cardiac myocytes rothkirch d., gergs u., neumann j. institute for pharmacology and toxicology medical faculty, magdeburger str. 4, 06097 halle (saale), germany extracellular atp and utp can be released from the heart during pathological conditions such as ischemia or hypoxia. in humans, atp and utp levels are increased during myocardial infarction. atp and utp can act via p2-purinoceptors which are further divided in p2x1-7 and p2y1-14-receptors. as previously shown atp and utp can induce inotropic effects in cardiac preparations of mice and man. for rat articular chondrocytes and human intestinal cells it has been demonstrated that the mapk cascade can be activated by atp and utp. therefore, the cardiac effects of atp and utp on force of contraction probably occur via the mapk pathway. to investigate the signal transduction pathway involved, we studied the effects of atp and utp on mapk phosphorylation in isolated neonatal rat cardiac myocytes using phosphorylation-specific antibodies. 100 µm atp as well as utp transiently increased phosphorylation of erk 1/2 and p38 mapk with a maximum effect at 5 to 10 minutes after application of atp and utp in neonatal cardiac myocytes (n=3 preparations each). the maximum phosphorylation of p38 increased with atp to 284% ± 68% (p<0.05) at 10 minutes and with utp up to 204% ± 21% (p<0.05) at 5 minutes. the phosphorylation with erk 1/2 mapk increased with atp to 234% ± 46.5% (p<0.05) at 5 minutes and to 337% ± 27% (p<0.05) with utp at 10 minutes of basal values, respectively. after 20 minutes, predrug values of mapk phosphorylation were reached again. in summary, we noted an atp-and utp-induced phosphorylation of erk 1/2 and p38 mapk in isolated neonatal rat cardiac myocytes. the involved receptor subtype(s) and the link between mapk phosphorylation and inotropic effect of atp and utp need to be elucidated. hameel: use of elearning in teaching pharmacology and toxicology -the halle experience rulf k. 1 , gergs u. introduction: during the past three years, our faculty has started to integrate items of elearning into the standard curriculum of a classical medical school: the "hallesches medizinisches elearning -hameel". our hypothesis was that these new elearning tools would improve the willingness of students to spend more time into learning and this would lead to an improved outcome (in multiple choice tests). methods: hence, we offered medical students (5 th or 10 th semester) additional learning environments. the courses for students (experimental pharmacology and toxicology or clinical pharmacology) were existed of a weekly lecture and in addition tutorials (problem-based-learning style, paper cases) or classical seminars. furthermore, we offered the possibility to use an online multiple choice quiz (involving 8 -12 previously used tests) and/or an online module on heart failure each week. we used the learning management system ilias software in combination with the content management system stud.ip. all students were subjected to an introductory test (to assess knowledge prior to our teaching section and allowing us to exclude a conceivable bias due to previous knowledge, involving basic items from prior teaching opportunities), a mid-term test and a final test to assess gain of knowledge. a maximum of 60 points could be obtained as a sum of both tests. results: in the means 40% of students used the new elearning tools (quizzes, heart failure module). however, there was no association between the use of self-assessment quizzes and examination results. the usage of the online quizzes increased in the periods before the exams. however, usage of the heart failure module was accompanied by significantly increased scores in exams. moreover, in a formalized evaluation system, students positively commented on our elearning efforts. conclusions: while usage of our quizzes did not improve test marks, another more sophisticated clinically oriented elearning module seemed to be improving test outcomes marginally. targeting of erk thr188 phosphorylation attenuates cardiac hypertrophy but preserves the anti-apoptotic effects of erk1/2 ruppert c., vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany background and aims: the extracellular regulated kinases 1 and 2 (erk1/2) play an important role in cardiac hypertrophy and cell survival. erk1/2 are phosphorylated at the so-called tey motif, which in turn activates erk1/2. hypertrophic stimuli lead to an additional autophosphorylation threonine188 (thr188). this autophosphorylation of erk1/2 stimulates activation of nuclear erk targets, which are known to induce hypertrophy. the aim of this study is to investigate whether specific targeting of erk thr188 phosphorylation affects both erk functions -erk mediated hypertrophy and cardioprotective cell survival. methods and results: for the analysis of cardiomyocyte hypertrophy in vitro, we stimulated cardiomyocytes with phenylephrine and measured the incorporation of tritiated isoleucine. cardiac hypertrophy was assessed by echocardiography before and after transverse aortic constriction (tac). for analysis of cell survival, caspase activity and dna fragmentation was determined upon hydrogen peroxide stimulation in vitro and in response to tac in vivo. to differentiate between inhibition of erk1/2 activity and prevention of erk thr188 phosphorylation, we either inhibited erk activity with pd98059 or overexpressed a mutant of erk2, which cannot be phosphorylated at thr188 phosphorylation. while inhibition of overall erk activity with pd98059 attenuated cell survival and hypertrophy in vitro, specific targeting of erk thr188 phosphorylation by overexpression of the phosphorylation deficient mutant (erk2 t188a ) attentuated phenylephrine induced hypertrophy, but preserved the anti-apoptotic effects of erk. cardiac overexpression of erk2 t188a significantly reduced tac-induced hypertrophy compared to wild-type erk2 overexpressing mice. in line with the in vitro experiments, erk thr188 inhibition only prevented hypertrophy in the tac model without promoting apoptosis. conclusions: these results show that blockade of erk thr188 phosphorylation attenuates cardiomyocyte hypertrophy but preserves anti-apoptotic effects of erk1/2. therefore, specific targeting of erk thr188 phosphorylation might be a promising strategy for the treatment of pathological hypertrophy. intracellular camp levels are determined by interplay of camp formation by adenylyl cyclases and camp degradation by phosphodiesterases (pde). eleven families of pdes are known. one of the most recently identified pdes is pde10, a pde in principle capable of hydrolysing camp as well as cgmp. pde10 contains a tandem of so called gaf domains in its n-terminal regulatory domain that mediate activation by camp. because current knowledge about the tissue distribution of pde10 was mostly based on the analysis of mrna distribution, we generated antisera against pde10 to analyze tissue distribution of the protein level. using these antibodies, we found a prominent occurrence of the enzyme in testis and in brain, where it was confined to the striatum. thus, pde10 displays a comparably restricted tissue distribution which is in contrast to that of many other pdes. low camp levels in so called medium spiny neurons of the striatum have been implicated in schizophrenia. furthermore, studies using the nonspecific pde10 inhibitor papaverine as well as specific pde10 inhibitors suggest pde10 as a target for the treatment of schizophrenia. here we set out to analyze the contribution of pde10 to camp degradation in striatum, to identify the physiological pathways pde10 is involved in and to clarify the functional impact of the proposed phosphorylation of the enzyme. identification of cgmp-dependent kinase i substrate complexes salb k., schlossmann j. universität regensburg lehrstuhl pharmakologie, universitätsstrasse 31, 93053 regensburg, germany the cgmp-dependent kinases (cgks) are components of the no/cgmp/cgk-signalling pathway and have a great physiological importance in a multitude of tissues and organs such as smooth muscles and platelets. two isoforms of the cgki and the cgkii are known. cgkiα and cgkiβ differ only in their first ~ 100 amino acids which constitute the leucine zipper and the autoinhibitory domains. the n-terminal leucine zipper domains mediate homodimerization of the kinase and the interaction with diverse substrate proteins. since cgkiα and cgkiβ express different n-termini they interact with different substrates. the cgkiβ isoform is assembled in a macrocomplex at the endoplasmic reticulum (er) with the intracellular calcium release channel inositoltrisphosphate receptor i (insp 3r-i) and the inositol-trisphosphate receptor associated cgmp kinase substrate (irag). we investigated, whether irag also interacts with the insp3r-ii and the insp3r-iii in murine platelets and tissues. additionally, we analyzed the interaction between the 52 amino acid peptide phospholamban (plb), which is also located at the er and regulates the er calcium reuptake by the sarco/endoplasmic reticulum ca 2+ -atpase (serca), and the two cgki isoforms. we performed cgmp-agarose experiments with murine wt and irag-ko platelets to examine the irag-insp3r interactions. the insp3r-ii isoform was neither bound to cgmp-agarose nor detected in the anti-irag immunoprecipitate. on the other hand, insp3r-iii from wt but not from irag-ko platelets was bound to cgmp-agarose. hence, insp3r-iii interacts directly with irag but not with cgkiβ in murine platelets. however, in colon smooth muscle lysate, insp3r-iii not only interacted with the irag protein but was also detected in the anti-cgkiα-immunoprecipitate. phospholamban from wt and irag-ko platelets was also bound to cgmp-agarose. subsequent immunoprecipitation experiments with the respective antibodies against the two cgki isoforms revealed that plb interacted both with cgkiβ and cgkiα. these results were supported by analysis of colon smooth muscle tissue from wt and irag-ko mice. in conclusion, irag interacts with insp3r-i and insp3r-iii but not with insp3r-ii in murine platelets and colon smooth muscle tissue. moreover, phospholamban is an interacting partner of both the cgkiα and the cgkiβ isoform. the human immunodeficiency virus type 1 enhancer binding protein 1 (hivep1) is regulated by proinflammatory stimuli and statins salomon a. 1, 2 , schmitz b. objective: hivep1 binds nf-ĸb and other proinflammatory consensus sequences, and is suggested to be involved in inflammatory processes. we recently identified two tagging snps, one positioned 90 kb upstream (rs169713) and another in exon 4 (rs2228220) of the hivep1 gene, to be replicatively associated with venous thrombosis in gwas and follow-up studies (ajhg, 2010; plos one, 2011) . methods: total rna isolation was performed after treatment of vascular endothelial cells (ea.hy926) with proinflammatory cytokines or statins (24h). serial hivep1 promoter deletion constructs were cloned into the pgl3-basic vector, a potential enhancer fragment, harbouring rs169713c/t, into the pgl3-promoter vector. in ea.hy926 cells and thp1 monocytes, reporter gene assays were performed by transient transfection and overexpression of transcription factors. chip and bandshift assays were performed to identify candidate transcription factors. results: in ea.hy926 cells, endogenous hivep1 expression was increased by proinflammatory cytokines tnfα and il-1β. simvastatin (1.2 and 2.4 µm) and atorvastatin (9 µm) -but not pravastatin or aspirin -both dose-dependently decreased basal and tnfα-stimulated hivep1 expression. the construct harbouring rs169713t exerted significantly higher transcriptional activity (ta) compared to rs169713c (p<0.001). for an intronic modulator, reporter gene assays demonstrated a regulatory effect on hivep1 expression in ea.hy926 and thp1 cells. cotransfection of sp1 and egr1 led to an increase in ta, while wt1 exclusively upregulated ta of constructs comprising the intronic modulator. chip and bandshift assays combined with specific antibody detection revealed binding of sp1 to the 5'-flanking region and the intronic modulator of hivep1. conclusion: increased hivep1 expression during inflammatory conditions can be repressed by simvastatin and atorvastatin, and not by pravastatin or aspirin. basal hivep1 expression is regulated by sp1 combined in a transcription factor module with egr1 and wt1 under basal and/or inflammatory conditions. the rs169713 site harbours potential activational capacity for hivep1 gene transcription and may communicate with the sp1/egr1/wt1 module. to date, the treatment of various movement disorders of the central nervous system is still insufficient. in most cases this is due to the sparse knowledge of the pathophysiology. l-dopa-induced dyskinesias (lid) represent a severe complication of long-time pharmacotherapy in parkinson's disease that deserves novel therapeutics. an increased activity of striatal projection neurons, which express kv7.2/3 channels, seems to be involved in the pathophysiology of these spontaneous involuntary dystonic and choreatic movements. previous studies demonstrated an antidyskinetic effect of the kv7.2-7.5 channel opener retigabine after acute and chronic treatment in a rat model of lid. in order to clarify if this effect was based on the modulation of kv7.2/3 channels, we examined the acute effects of the preferred kv7.2/3 channel opener ica 27243 on lid in this animal model. four weeks post 6-ohda lesioning of the left forebrain bundle, dyskinesia was induced by chronic treatment with 10 mg/kg l-dopa and 15 mg/kg benserazide for 20 days. three subtypes of dyskinesia (limb, axial and orolingual) were rated according to a score system from 0 to 4 over 180 min. for drug testing, ica 27243 (5, 10 and 15 mg/kg) was administered intraperitoneal additionally to l-dopa (or vehicle). effects of drug action in comparison to vehicle controls were detected by adding up the severity scores of each observation time. additionally, effects on parkinsonian symptoms were examined 20 min after drug administration using the block and the stepping test. ica 24273 reduced the severity of dyskinesia significantly at all doses while no negative impact on the antiparkinsonian effect of l-dopa was observed. whereas the antidyskinetic effect was restricted to the first 20 min after the application of 5 mg, it lasted up to 110 min in rats treated with 10 mg ica 27243. a higher dose of 15 mg did not further enhance the antidyskinetic effect. the results of our study suggest that the antidyskinetic effect of the k v7 channel opener retigabine was based on its action on striatal kv7.2/3 channels. in line with the results of previous studies with retigabine, this action does not seem to interfere with the antiparkinsonian effect of l-dopa. this study was supported by the micheal j. fox foundation. background: skeletal muscle toxicity is the major side effect of hmg-coa-reductase inhibitors (statins) and can be simulated in engineered skeletal muscle. statins are known to exert "pleiotropic" effects, e.g. reducing endothelial dysfunction by inducing no synthases and no production. the role of no synthases in skeletal muscle under statin treatment is largely unknown. interestingly, some skeletal muscle pathologies (e.g. duchenne muscular dystrophy) may be exacerbated by increased inos activity. here we tested whether or not statin-induced skeletal muscle toxicity would be associated with enhanced no synthesis. we generated engineered skeletal muscle (esm) from rat skeletal muscle cells, matrigel and collagen. esms displayed typical skeletal muscle properties (differentiated muscle fibres, tetanic contractions). under baseline conditions esm expressed enos most abundantly, followed by inos and nnos (n=4-5). myotoxic cerivastatin (0.01, 0.1, 1 µm for 5 days) caused a concentration-dependent decrease of contractile force (p<0,05, n=17-20) paralleled by an increase in inos transcript (mean±sem: 0.01 µm 4±0.9-fold, n=3 p<0.05; 0.1 µm 9.3±2.8-fold, n=3 p<0.05) and protein (0.01 µm 5.1±2-fold, n=4 p<0.05; 0.1 µm 7.8±0.8-fold, n=3 p<0.05). mevalonic acid fully prevented the inos increase suggesting that the induction is hmg-coa reductase-dependent. to test whether inos may contribute to the decrease in contractile force we co-treated esm with 1400w, a specific inos inhibitor. we applied 5 µm of 1400w, a concentration found to potently reduce lipopolysaccharide (lps)induced no-production in cultured myotubes. however, we did not observe a rescue effect (n=9-15). also, l-name (10 mm), an unspecific nos inhibitor, did not improve contractile function, instead we observed increased myotoxictiy (n=6-13, p<0.05). to further investigate the role of no for muscle function we treated the esms with increasing concentrations of the no-donor snp. only high concentrations of snp (10 µm) caused a reduction of contractile force. combined treatment with cerivastatin and 0.1 µm snp showed a tendency towards improved force development in esm. conclusions: statins increase inos activity in our skeletal muscle model (esm). however, this does not seem to functionally contribute to myopathy in esm. increased production of no may in fact be a protective measure. esm may help to dissect clinically relevant functional changes in statin myotoxicity. characterization of primary skin fibroblasts of patients with 3m syndrome and mutations in the cul7 gene meyer k., hieber m., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany 3m syndrome is an autosomal-recessive disorder characterized by pre-and postnatal growth retardation (< -4 sd), facial dysmorphism and skeletal anomalies. the majority of patients harbor missense mutations of the cul7 (76%) or obsl1 (16%) gene, respectively. cul7 constitutes an e3 ubiquitin ligase that is involved in the regulation of the insulin-like growth factor 1 (igf-1) signaling pathway via ubiquitin mediated degradation of insulin receptor substrate 1 (irs-1). to investigate the role of cul7 mediated irs-1 degradation in the pathogenesis of 3m syndrome. primary skin fibroblasts of seven 3m syndrome patients (six with cul7 mutations, one with a obsl1 mutation) and control fibroblasts were analyzed for proliferation rate (cell counter), cell cycle profile (facs), cell morphology and cellular senescence (histochemistry), irs-1 protein concentrations and activation of the igf-1 signaling pathway (western blot). the proliferation rate of 3m patient fibroblasts was significantly increased when compared to control cells. in contrast, irs-1 protein levels and activation of the pi3k/akt and erk mapk pathway were only increased in a subset of 3m cells that carried cul7 mutations, but not in cells from a patient with the obsl1 mutation. no significant differences in cell cycle profile, cell morphology or cellular senescence were observed in 3m patient fibroblasts when compared to control cells. to determine the pathogenetic contribution of increased irs-1 levels to the observed phenotype, human imr90 fibroblasts were stably transfected with retroviral vectors encoding irs-1. despite 20-fold overexpression of irs-1 compared to empty vector controls, no significant effect of igf-1 stimulation on proliferation rate or pi3k/akt and erk mapk signaling was observed. skin fibroblasts of 3m patients with cul7 mutations displayed an increased proliferation rate and enhanced activation of the igf-1 signaling pathways. despite accumulation of irs-1 in fibroblasts from a subset of 3m patients with cul7 mutations, no pathomechanistic role for irs-1 could be demonstrated. collectively, our data indicate that a dysregulated igf-1 signaling may contribute to the pathogenesis of 3m syndrome, yet in an irs-1 independent manner. pharmacases.de -a student-centered elearning project of clinical pharmacology zollner b., berg c., gros n., muß n., oestreicher d., engelhardt s., sarikas a. technische universität münchen institut für pharmakologie und toxikologie, biedersteiner str. 29, 80802 münchen, germany pharmacases.de is a novel e-learning website of clinical pharmacology that presents clinically relevant aspects of pharmacology and toxicology in an interactive and multimedial manner. the aim of the project pharmacases.de was to develop an innovative concept for creating high quality elearning content that i) integrates and promotes the theoretical and cooperative skills of final year medical students and ii) is easily adoptable by cooperating institutes and hospitals. a peer-teaching concept was developed in which final year medical students with the elective pharmacology (pj wahlfach pharmakologie) independently researched and wrote elearning lessions ("pharmacases"). subject-specific expertise was acquired by consulting elective students of other disciplines. at present (11/2011) , this "peer network" consists of elective students of nine cooperating institutions (pathology, microbiology, radiology, cardiology, psychiatry, dermatology, neurology, ophthalmology, pediatrics) at the technische universität münchen. the average time for the generation of one elearning lession by the peer network was 10 days. to date, the website consists of 49 pharmacases that are available to all students online (http://www.pharmacases.de). the website also contains a discussion forum and evaluation form for direct feedback. on average, pharmacases.de has 1000 visitors per month with the following evaluation results: "excellent": 76%, "good": 15% and "satisfactory": 9% (n=33). the didactic concept of pharmacases.de enabled the efficient generation of high quality elearning content in a student-centered and interdisciplinary manner. the peer-teaching approach supports the collaborative skills of final year medical students and facilitates the transfer of theoretical pharmacological knowledge into clinical practice. improved glucose tolerance, less chronic adipose tissue inflammation and reduced adipose tissue mass in mice with adipocyte-specific loss of tak1 sassmann a., offermanns s., wettschureck n. max-planck-institut für herz-und lungenforschung pharmakologie, ludwigstr. 43, 61231 bad nauheim, germany tgf-β activated kinase 1 (tak1) is known to be involved in numerous inflammatory processes by linking receptors for inflammatory stimuli like lps, interleukin-1 or tnfa to ikk, p38 and jnk activation. chronic inflammation of white adipose tissue is one of the major causes for the development of insulin resistance and impaired glucose tolerance in states of obesity. to investigate the role of tak1 in white adipose tissue, we crossed the tamoxifen-inducible white adipocyte-specific cre mouse line adipoqcreer t2 with animals carrying floxed alleles of the tak1 gene. adipoqcreer ; tak1 fl/fl animals and cre negative control littermates are viable and fertile and do not show any developmental defects. after tamoxifen induction and high fat diet feeding adipocytespecific tak1 knockout mice show improved glucose tolerance and lower fasting insulin levels compared to control animals. in line with this, serum levels of the adipose tissuespecific hormone resistin are reduced in adipocyte-specific tak1 knockout mice. these findings are accompanied by a lower state of chronic inflammation of adipose tissue as indicated by a dramatic reduction of adipose tissue macrophage number and lower serum levels of tnfα and interleukin-6. stimuli like tnfα, interleukins and tgf-β released from macrophages and adipocytes are known to promote obesity-related adipose tissue inflammation. when stimulated with these substances tak1 deficient adipocytes show reduced activation of jnk and p38 which both play an important role in the development of insulin resistance. interestingly, we observe a lean phenotype in adipocyte-specific tak1 knockout mice when fed a high fat diet which reflects a reduction of white adipose tissue mass. currently we are investigating the molecular mechanisms underlying the reduced adiposity and lower state of chronic inflammation in adipose tissue. growth of small cell lung cancer (sclc) cells is regulated via the autocrine stimulation of g protein coupled receptors (gpcrs), i. e., neuropeptide and muscarinic acetyl choline (ach) receptors. the activation of gq/11 and calcium-dependent gpcr pathways results in the stimulation of erk signaling which is necessary for the mitogenic effects of neuropeptides or ach on sclc cells. in contrast, the role of calcium-independent gpcr signaling and its interplay with gq/11-regulated pathways in sclc cells are less well defined. the aim of our studies was to characterize the molecular make-up and the interaction of these pathways, and to delineate the phenotypic effects of calciumdependent and -independent signaling cascades in sclc cells. using a panel of sclc cell lines, we found that the stimulation of neuropeptide receptors led to an increase of calcium which was independent of extracellular calcium and could be prevented by depleting internal calcium stores. this calcium increase was sufficient to activate the tyrosine kinase pyk2 and subsequently the erk1/2 cascade. the role of pyk2 for the growth of sclc cells was further supported by the fact that inhibition of pyk2 using a sirna approach or a novel specific inhibitor, pf431396, exerted pronounced cytotoxic effects on sclc cells, whereas non-sclc cells were less sensitive. interestingly, the inhibition of g 12/13 signaling by sirna-mediated g(alpha)12 or g(alpha)13 knockdown also markedly reduced the growth of sclc in vitro or in subcutaneous tumor xenografts, and increased the sensitivity of sclc cells towards certain cytostatics. to further define the role of calcium-dependent signaling via pyk2 versus the role of calcium-independent signaling via g12/13, we tested the effect of pyk2 inhibition in cells with impaired g12/13 signaling. notably, pyk2 and g12/13 double inhibition led to an even increased proliferation. thus, we propose that dysbalanced g protein signaling favoring either pyk2 activation or g12/13-dependent cascades inhibits the growth of sclc cells, whereas the parallel inhibition of both pathways restores again the balance and the growth capacity in this tumor entity. dendritic cells (dcs) are essential for the initial immune response and for the defence against inhalated pulmonary toxins and carcinogens in lung. to differentiate dcs, the cell line thp-1 were used for 7 days and stimulated with various cytokines (il-4, gm-csf, tnf-a, ionomycin). the dcs were characterized by flow cytometry with different typical dendritic cell markers (for example cd11c, cd209, cd83) and by immunfluorescence compared to monocytes. the bronchial tract contains up to 800 dcs per mm² and therefore we established a triple culture model to mimic the situation in vivo. the triple culture consists out of primary human epithelial cells from small bronchi (hbec) and lung fibroblasts which are cultured under air-liquid conditions on filter membranes for 4 weeks and dcs which were added after the differentiation phase of the bronchial cells. during the cultivation time the hbec formed an epithelial layer expressing both tight and adherens junctions. they also produced mucus, formed functional cilia with a beat frequency of between 16 to 20 hz and the transepithelial resistance values were stable between 600 to 800 ω·cm². pathomechanisms of pulmonary toxicity in vivo are difficult to investigate, so the tripleculture model is the basis for investigations of the toxic effects at cellular level. lungtoxic substances such as organophosphates are usually absorbed through inhalation. organophosphates are dangerous nerve agents for the human organism. at high concentrations organophosphates damage in the coculture without dcs the cell-cell contacts of the epithelial layer. in the triple culture dcs firstly respond to inhaled organophosphates and seem to compensate effects on the other cells. in summary, it is very important to understand the pathogenic mechanisms of lung injury in relation to the role of dendritic cells in lung. they could play an essential role in therapy against damage of organophosphates in the lung. co-purification of arf gtpase-activating protein git1 and cavb3 schalkowsky p., wissenbach u., fecher-trost c., flockerzi v. universität des saarlandes institut für experimentelle und klinische pharmakologie und toxikologie, kirrbergerstraße, 66421 homburg, germany high-voltage activated ca channels are assembled from pore-forming α1 subunits and two distinct types of auxiliary subunits, cavβ1-β4 and, maybe, α2δ1-δ4. by a cavβ3-specific antibody based affinity chromatography the cavβ3 protein was highly enriched from rat brain microsomal membranes. proteins associated with cavβ3 were identified by mass spectrometry (lc-esi-ms/ms) and include α1-subunits, α2δ-subunits and β-subunits. in addition to these expected interacting proteins additional proteins were co-purified with the cavβ3 protein, including the g protein-coupled receptor kinase-interactor 1 (git1). the 770aa git1 is a ubiquitously expressed multidomain protein which may serve as a scaffold to bring together molecules to form signaling modules controlling, for example, vesicle trafficking, cytoskeletal organization and cell migration. in rat brain lysates the git1 and cavβ3 proteins were co-immunoprecipitated by the antibodies for cavβ3 and git, respectively. we cloned the git1 cdna from mouse brain and co-expressed it with the cavβ3 subunit in hek cells. like in brain lysates the git protein was retained by cavβ3 precipitated by the antibody for cavβ3 and cavβ3 was retained by the git1protein precipitated by the antibody for git1. both proteins, cavβ3 and git1 are endogenously co-expressed in mouse embryonic fibroblasts (mef). we could not observe potassium-induced voltage-activated ca influx in these acutely prepared cells. accordingly, mefs can be used as a model system to study the impact of cavβ3-git1 interaction in the absence of functional cav channels. in addition, using mefs from cavβ3-deficient mice enables us to control the impact of cavβ3 on git1 function. vice versa down-regulation of git1 by specific sirnas might allow to control the impact of git1 on cavβ3 function. as read-outs we use cell migration assays and monitor receptor-dependent and receptor-independent calcium signaling in these cells. effects of sphingosine-1-phosphate and fty720 on epidermal hyperproliferation and inflammation in an imiquimod induced mouse model of psoriasis the sphingolipid sphingosine 1-phosphate (s1p) is a mediator that modulates various physiological functions of skin cells. s1p has distinct direct effects on keratinocytes as it diminishes proliferation and induces differentiation which is a classical goal of psoriasis therapy. furthermore, s1p modulates the function of various immune cells, mainly to an anti-inflammatory direction. thus, the strategy of targeting immune cells with locally acting s1p was explored in an experimental animal model of psoriasis vulgaris, the recently established imiquimod induced psoriasis mouse model and in the mouse tail test. topical administration of imiquimod onto back and ear skin led to a distinct inflammatory response characterized by epidermal hyperproliferation, scaling and redness which was scored with a modified pasi (psoriasis area and severity index). the positive control diflorasone diacetate and s1p, but not fty720 reduced the epidermal hyperproliferation by topical administration onto ear skin, indicating a mode of action for s1p via the s1p2 receptor, which is not activated by fty720. there was also a moderate reduction of inflammatory cell influx and edema formation in ear skin by s1p treatment, which was even more pronounced by treatment with diflorasone diacetate. the pasi determined on back skin was, however, only significantly reduced by diflorasone diacetate. the discrepancy between outcome on ear and back skin remains elusive. in the mouse tail assay, the influence of s1p in stratum granulosum formation (orthokeratosis) was tested compared to the positive control calcipotriol. whereas topical administration of calcipotriol led to the expected significant increase of stratum granulosum in mouse tail epidermis, s1p lacked such an effect, indicating a different mode of action in epidermal differentiation. taken together, these results imply that topical administration of s1p might be a new option for the treatment of mild to moderate psoriasis lesions. inhalation of toxicants such as sulphur mustard (sm), an alkylating chemical warfare agent, cause pulmonary complications like respiratory failure, pulmonary edema and secondary pneumonia. in order to investigate pathomechanisms of pulmonary toxicity, an in vitro alveolar-capillary co-culture model has been established recently by our group. in this model the human lung adenocarcinoma epithelial cell line (h441) is mimicking the epithelial site of the alveoli while the human hemangiosarcoma cell line (iso-has) represents the endothelial site. acute respiratory injuries are accompanied by disruption of the alveolar-capillary barrier that can be detected by the use of biochemical markers (e.g. ldh) and electrochemical indicators (e.g. transepithelial resistance). sm-mediated pulmonary injury is characterized by the increased secretion of proinflammatory mediators (e.g. il-6). a shortcoming of this model is the missing inflammatory component in the lung. aim of the present project is the addition of macrophages to the established co-culture model to improve the model and to investigate the relevance of inflammatory processes in toxic lung injury. the effect of sm on this triple-culture model is characterized with special regard to the interaction of epithelial cells and macrophages. the human acute monocytic leukemia cell line (thp-1) was stimulated to allow differentiation into macrophages. validation of the cellular differentiation was checked by specific clusters of differentiation (e.g. cd206) using flow cytometric analysis. after successful differentiation into macrophages, these inflammatory cells were added to the co-culture model before and after exposure with sm, respectively. the cytotoxicity of sm on the triple-culture model was evaluated by xtt assays and ter measurements. furthermore, immunohistochemical staining of tight junction proteins (e.g. zo-1) and of adherens junction proteins (e.g. e-cadherin) was conducted to enhance the knowledge of the function of the intercellular junction in injured and rejuvenated regions as well as the interaction of epithelial cells and macrophages. for the contact allergen para-phenylenediamine (ppd) we showed that concentrations above 50 µm are accompanied with inhibition of nat1 activity in human keratinocytes [1] . in the following we investigated the impact of ppd on nat1 activity in antigenpresenting cells using dendritic cell-like cells, namely the monocytic thp-1 cells. measured nat1 activity of thp-1 was comparable to those found in primary keratinocytes. a 24h treatment of thp-1 cells with physiologically relevant concentrations of ppd (10-200 µm) led to a 47% reduction of nat1 activity. comparable results were found for mono-acetylated ppd (mappd) whereas di-acetylated ppd demonstrated no inhibition. time-dependent studies found a significant decrease in enzyme activity already 8h after application of ppd or mappd while nat1 mrna levels were not modified. these results are indicative for a substrate-dependent inhibition. further investigations concentrated on the restoration of nat1 activity after treatment with ppd or mappd. here we found that n-acetylation capacities were restored after 24h cultivation of the treated cells in fresh medium. independent of the enzymatic activity, certain compounds are known to oxidise the catalytic cysteine or form adducts with the nat1 protein. therefore we studied whether ppd and/or oxidised ppd including the trimer bandrowski´s base interact additionally with recombinant nat1 protein itself in the absence of acetyl-coenzyme a. we found that all compounds but mappd bind to nat1 protein after 2h. the greatest inhibition was found for oxidised ppd (up to 50%). due to the greater inhibition by oxidized ppd we propose that oxidation products interact with the protein whereas ppd itself modulates nat1 enzyme activity in a substrate-dependent mode of action. overall we demonstrated that ppd can inhibit nat1 in two different ways. the work was partially financed by federal office of public health (foph), switzerland and stiftung zur förderung begabter studierender und des wissenschaftlichen nachwuchses objective: fabry's disease is a rare progressive multisystem disorder resulting from deficiency of the lysosomal enzyme alpha-galactosidase a (gla, ec 3.2.1.22). we hypothesize that genetic gla variants, especially those in its promoter region are of pathophysiological relevance for the development and progression of fabry's disease phenotypes. this study focuses on the characterization of the gla promoter, identification of functional genetic variants and impact of transcription factor eb (tfeb), a regulator of lysosomal genes. we screened 4011 bp of the 5'-flanking region of gla in 60 patients with fabry's disease and 60 controls for genetic variants. serial promoter deletion constructs for reporter gene assays were designed and identified genetic variants were introduced by site-directed mutagenesis. constructs were transiently transfected into immortalized human kidney epithelial (ihke) cells and human vascular endothelial cells (ea.hy926) to determine transcriptional promoter activity (ta). sequencing of patients' dna revealed five genetic variants in the 5'flanking region of gla, significantly more frequent in fabry's patients compared to control group (rs2071225; rs3027580; rs3027579; rs59647857; rs3027575; all minor alleles p<0.0027). we identified two regions, a proximal one between -110 and -425 and a distal region between 1106 and -1421 with significant ta, in both cell lines. cotransfection with tfeb activated ta of both regions significantly up to 5.3-fold (p<0.001). in ihke cells, insertion of the minor t allele (rs2071225) significantly enhanced basal ta of the proximal promoter region (p=0.0006), while insertion decreased basal ta (p<0.0001) of the distal promoter portion. the combined insertion of the minor c alleles (rs3027580; rs3027579), which were in complete linkage disequilibrium, significantly increased basal ta of the distal promoter region (p=0.0037). our results indicate that three genetic variants, overrepresented in fabry's patients, are located within transcriptionally active regions, possibly altering tf binding sites and therefore, affecting gla expression. future analysis will assess the impact of gla promoter variants and gla regulation by tfeb with respect to fabry's phenotypes. multiple sclerosis (ms) and its animal counterpart experimental autoimmune encephalomyelitis (eae) have a major inflammatory component that drives and orchestrates both diseases. ceramides (cer) are known as mediators of inflammatory processes, but until now their role in ms was not elucidated. we measured the ceramide levels in the cerebrospinal fluid of ms patients and control patients using lc-ms/ms. interestingly, the c16:0-cer levels were 1.9 fold increased in ms patients. this translates into the finding that c16:0-cer levels were also significantly elevated in the lumbar spinal cord of eae mice. the raised c16:0-cer levels in the lumbar spinal cord were caused by a transiently increased expression of ceramide synthase (cers) 6 in macrophages. nitric oxide (no) and tumor necrosis factor alpha (tnf-α) secreted by interferon gamma (infγ ) induced macrophages play an essential role in the development of ms. astonishingly, rnai experiments reveal that cers6 and its product c16:0-cer are mediators of inf-γ induced no/tnf-α release in raw macrophages. moreover, treatment of eae mice with l-cycloserine prevented the increase of c16:0-cer and of inos/tnf-α expression and caused a remission of the disease. in summary, cers6 plays a critical role in the initial phase of ms, most likely by regulating the no and tnf-α synthesis. this let us speculate, that a substance designed to inhibit cers6 and therefore to limit the inflammatory effects of c16:0-cer may represent a new drug in ms therapy. role of cgmp-dependent protein kinase i for kidney fibrosis schinner e. 1 cgmp is synthesized via nitric oxide-or natriuretic peptide-stimulated guanylyl cyclases and exhibits pleiotropic regulatory functions also in the kidney. hence, the integration of cgmp signaling via cgmp-dependent protein kinases (cgk) might play a critical role for renal physiology. both isozymes were detected in arterioles, mesangium and within the cortical interstitium. in contrast to cgkiα, the β isoform was not detected in the juxtaglomerular apparatus and medullary fibroblasts. here, we focused on the function of cgki in the renal interstitium emphasizing a functional differentiation of both isoforms. the interstitium exists mainly of fibroblasts playing a prominent role in the interstitial fibrosis. accordingly, cgki could also be involved in this pathophysiological process. therefore, we studied whether cgki influences renal fibrosis which was induced by unilateral ureter obstruction (uuo). at first we analysed the role of the no/cgmp signaling by application of cgmp increasing yc1 or isdn. thereby we detected antifibrotic effects of these substances. subsequently we tested whether these effects are mediated by cgki by using mutant mice. on the one hand we examined αsm-rescue mice (expressing cgkiα only in smooth muscle under the control of the sm22 promotor with a cgki-ko background) and cgki-ko mice (expressing no cgki). on the other hand we used tgtg mice expressing more cgkiα in smooth muscle than wt mice (transgenic cgkiα under the control of the sm22 promotor). due to the steeply increased use of nanomaterials for commercial and industrial applications, toxicological assessment of their potential harmful effects is urgently needed. moreover, the continuous development of novel materials requires the implementation of hazard-predicting models to prevent potential health effects resulting from human exposure. in the present study, we studied the toxic potential of a set of nanoparticles (np) with varying physicochemical properties in human a549 lung epithelial cells, hepg2 liver epithelial cells and hk-2 proximal tubule epithelial cells. the used nanomaterials incorporated five tio 2 samples, two zno samples (i.e. uncoated and coated), two multi-walled carbon nanotube (mw-cnt) samples and a nanoparticulate ag sample. cells were treated with np at doses ranging from 0.3 to 80 µg/cm 2 for cytotoxicity and from 06 to 40 µg/cm 2 for genotoxicity. dna damage was evaluated using the alkaline comet assay while concurrent cytotoxicity was determined by the wst-1 assay. marked contrasts in cytotoxic and dna damaging properties were observed among the different materials. the overall strongest responses were observed with the uncoated zno-np sample and with ag-np, although effects were found to depend on the cell type. notably, the dna damaging effect of ag-np could at least partly be attributed to its dispersant. present results form part of a growing data set which are generated in the framework of the eu fp7 project enpra (fp7-nmp) to establish dose-response relationships and a mathematical model to predict the hazard of nanoparticles. increased spontaneous hprt mutant frequency in v79 cells expressing human cytochrome p450 1b1 schlechtweg a., esch h. , martínez jaramillo d., lehmann l. university of wuerzburg/institute of pharmacy and food chemistry section of food chemistry, am hubland, 97074 wuerzburg, germany the hypoxanthine-guanine phosphoribosyltransferase (hprt) assay in chinese hamster v79 lung fibroblasts (v79 cells) represents a widely-used mammalian test system to detect gene mutations. since v79 cells do not express any cytochrome-p450dependent monooxygenase (cyp) isozymes, usually an activating system has to be added. therefore, v79 cells expressing human (h) cyp isozymes have been commercialized. to test these v79 cells for their use in the hprt test, v79 h1a1 and h1b1 cells were characterized regarding (i) spontaneous frequency of 6-thioguanineresistant clones per 10 6 clonable cells (smf), (ii) the stability of which over 4 weeks (w), and (iii) the mutational spectrum (ms) of cdna from mutant clones. ms of cdna was determined by isolation of total rna, reverse transcription/amplification of the coding region by polymerase chain reaction and sanger sequencing of the amplification product. activity of cyp isozymes was verified by ethoxyresorufin-o-deethylase (erod) assay. (i)/(ii) whereas the smf of v79 cells (w2:13±4; w4:2±1) and v79 h1a1 (w2:17±4; w4:3±0) only varied within the range of historical controls, smf of v79 h1b1 increased continuously over time (w2: 36±6; w4: 68±13). (iii) although the smf of v79 and v79 h1a1 were similar, the mutational spectrum of v79 cells was characterized by as many transversions as transitions and deletions of exon 4 or exon 4+5, whereas the mutational spectrum of v79 h1a1 was characterized exclusively by transversions and deletion of exon 7+8. surprisingly, with 57 out of 59 cdnas derived from v79 h1b1 mutant clones, no amplification product was detected. first results indicate that there is at least one gene mutation in the untranslated region before and behind the coding region precluding amplification with the original primers. (ii)to reduce the smf of v79 h1b1, cells with wildtype hprt activity were cloned and one clone with an erod activity which did not differ significantly from the original cell population was further characterized. initially, smf of the clone varied between 0.7±0.6 and 1.2±0.0. yet its smf was unstable reaching up to 104±6. in conclusion, the mutational spectrum differed between the v79 cell lines. furthermore, h1b1 expression seemed to enhance smf in v79 cells. even though a temporary reduction of the smf by cloning was possible, smf of v79 h1b1 cells was unstable. we wanted to investigate the possible antithrombotic effects and elucidate the chemical identity of the active principles involved in inhibitory effects against adp-induced aggregation of human platelets by wild garlic, allium ursinum l. method: bioassay-guided isolation procedure was used followed by spectrometric identification of pure active compounds. for the bioassay, blood was taken from healthy human volunteers and platelet rich plasma (prp) was prepared for turbidimetric platelet aggregation tests. prp, stimulated with 20µm adp, was treated with extracts of different polarities, fractions and isolated single compounds from allium ursinum. the extracts were investigated by thin layer chromatography, hplc, mass spectroscopy, esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. for references the adt-antagonist mes-amp was used. result: fresh allium ursinum leaves were extracted with ethanol, which was the potent form that effectively inhibited adp-induced aggregation of human platelets. this ethanolic extract was subjected to liquid-liquid partition. whilst the aqueous phase containing the moiety of cysteine sulphoxide and thiosulphinate derivatives showed only weak activity on platelet aggregation, the ethyl acetate and especially the chloroform partitions showed highest aggregation inhibiting potency. thus, in our bioassay effects of alliins/allicins could be neglected. the chloroform phase, possessing the strongest activity, was separated into 28 fractions by gradient elution open cc on silica gel. the most active fractions 11-17 were separated again yielding 10 subfractions. this afforded 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol and β-sitosterol-3-o-β-dglucopyranoside, the structures of which were determined by esi-ms and 1d/2d 1h/13c-nmr spectroscopic techniques. furthermore, the diminutive amounts of volatile oil of a.ursinum leaves obtained by steam distillation according to ph.eur. could be evaluated as a third aggregation inhibiting principle. conclusions: at the first time two active, non-sulphur-containing constituents of wild garlic, namely a galactolipid and a phytosterol, could be identified exhibiting inhibitory action on adp-induced aggregation in human blood platelets. as a major constituent, the galactolipid 1,2-di-o-α-linolenoyl-3-o-β-d-galactopyranosyl-sn-glycerol, not yet found in allium spec., appears as a new, highly useful marker substance for a.ursinum drugs, or their pharmaceutical preparations. in recent years, public attention focused more and more on risk factors which may impair sperm quality and thereby human reproduction. in this context, for example pesticides, alcohol, cigarettes, and even mobile phones are discussed. a variety of parameters exists including sperm counts as well as sperm motility, which are considered to be two of the most important parameters to evaluate sperm quality in animal models with the final aim to assess human risk. in recent years computer assisted sperm analysis (casa) devices mostly replaced the formerly used manual counting and manual motility assessment. however, although casa offers multiple opportunities and can allow for an objective and more detailed evaluation, several pitfalls exist which can alter the results profoundly and consequently compromise the quality of the data and ultimately the validity of a study. the aim of the present study was to establish and validate the casa device tox ivos sperm analyzer from hamilton thorne and thereby to gain detailed knowledge about the practical advantages but also intricacies which may alter the obtained results. in this regard healthy adult male rats (10-12 weeks old) were used. ultrasonic sound resistant sperm heads were isolated from the testis and in addition, sperms were isolated from the cauda epididymis. testicular sperm head counts and sperm motility were assessed using different isolation procedures and/or instrument settings. results different instrument settings modulate both -sperm motility and testicular sperm counts. in this regard, a wide range of results including slight changes as well as false positive/negative results were obtained. in addition, the modification of the isolation procedure can lead to variable results especially for sperm motility. conclusion isolation procedures as well as instrument settings can alter the results. consequently, in an experimental setting, potential adverse effects can be confounded with methodologically mediated apparent findings exerted via inappropriate use of the device -depending on the respective conditions in the test laboratory. this study demonstrates the relevance of standardization of testing conditions adopted for computer assisted sperm analysis and the need for a robust validation prior to use in experimental settings. orai and stim proteins have been identified as central components of the highly ca 2+ selective, store-operated current in immune cells (icrac). the molecular basis of selective orai-mediated activation of the calcineurin/nfat pathway and the crosstalk with other channel and scaffold molecules of the trpc family are still incompletely understood. using patch clamp recordings complemented by fluorescence and tirf microscopy we investigated interactions between orai1 and trpc3 in plasma membrane microdomains of rbl-2h3 mast cells. orai1-mediated crac currents, activated by passive store depletion, were found significantly reduced by over-expression of trpc3. this negative impact of trpc3 on icrac was independent of channel function as the trpc3 pore dead mutant (e630k) inhibited icrac to a similar extent as wild type trpc3. importantly, despite a reduction in icrac, nfat translocation in trpc3 overexpressing rbl cells remained unchanged, or was even slightly promoted. store depletion-induced nfat translocation in rbl cells was as well unaffected by trpc3e630k but substantially reduced by trpc3 mutants with either i) eliminated fkbp12/calcineurin binding (p704q) or ii) deficiency in pkc phosphorylation (s712a). moreover, inhibition of pkc phosphorylation by (gfx109203x; 3 µm) strongly suppressed nfat signaling. we suggest trpc3 as a scaffold that links orai-mediated ca 2+ -entry to nfat/calcineurin signaling within plasma membrane microdomains. neurally-induced bronchoconstriction in human and guinea pig precision-cut lung slices schlepütz m. 1 , rieg a. d. introduction: precision-cut lung slices (pcls) are well suited to study peripheral airway responses in different species. airway tone is under close control of the autonomic nervous system and dysregulation may contribute to airway hyperresponsiveness as observed in human lung diseases such as asthma. hence, the aim of the present study was to characterize neurally induced bronchoconstriction (bc) in guinea pigs (gp) and to compare the results with those in human pcls. methods: pcls were prepared from gp or human lung tissue. nerve endings in pcls were activated by electric field stimulation (efs) or capsaicin addition. cholinergic nerve responses were proven by atropine. capsaicin was used to show excitatory nonadrenergic non-cholinergic (enanc) responses. ruthenium red or skf96365 were used to confirm transient receptor potential (trp) channel contributions upon enanc activation. results: gp and human pcls were both sensitive to efs and airways contracted to 39±26% of the initial airway area (%-iaa) and 63±21%-iaa, respectively. in frequency response curves half maximal response was found at 7.0±1.2 hz for guinea pig pcls and 8.1±0.8 hz for human pcls. efs-induced bc was inhibited by atropine in both species. capsaicin contracted gp to 18±15%-iaa. 60% of human pcls were responsive to capsaicin and airways contracted to 72±10%-iaa, respectively. in gp ruthenium red and skf96365 blocked capsaicin-as well as efs-induced bc. conclusion: gp and human pcls contain atropine sensitive cholinergic and capsaicin sensitive enanc nerve endings. since gp pcls were sensitive to trp channel inhibitors, the involvement of those channels can be characterized with respect to lung diseases. in conclusion, gp pcls resemble the human distal lung innervation and represent a useful model to study neural airway pharmacology. the erk1/2-pathway is involved in pkc-induced nox4 up-regulation schlufter f., xia n., förstermann u., li h. universitätsmedizin mainz institut für pharmakologie, obere zahlbacher straße 67, 55131 mainz, germany nadph oxidases (nox) are major producers of reactive oxygen species in the vascular wall and nox4 is the most abundant nox isoform in human endothelial cells. we have previously shown that treatment of human ea.hy 926 endothelial cells with phorbol 12myristate 13-acetate (pma) for 48 h leads to an up-regulation of nox4 expression. this effect of pma is mediated by protein kinase cα, because it is preventable by the pkc inhibitor gö 6983 and by pkcα-sirna. the present study is aimed to investigate the signal transduction cascade downstream of pkcα. pma-induced nox4 up-regulation can be attenuated by pd 98,059 (an erk1/2 inhibitor), but not by sp 600125 (a jnk inhibitor), indicating in the involvement of erk1/2. consistently, pma treatment leads to a sustained activation of erk1/2, and sirnamediated knockdown of erk1/2 markedly reduces the pma-induced nox4 up-regulation. h89, an inhibitor of the mitogen-and stress-activated protein kinases (msks) has no effect on the pma-stimulated nox4 expression, indicating that msks are not the target molecules of erk1/2 in this scenario. on the contrary, knockdown of the transcription factor elk-1 by sirna significantly reduces the pma-induced nox4 up-regulation. in conclusion, erk1/2 and elk-1 are involved in the pkcα-induced nox4 up-regulation. determination of spontaneous mutation frequencies in normal human mammary gland tissue using the random mutation capture technique schmalbach k., lehmann l. university of wuerzburg section of food chemistry, am hubland, 97074 wuerzburg, germany annually, over 57,000 women develop breast cancer in germany. the accumulation of genetic mutations in mammary gland tissue during lifetime may be reasonable for developing breast cancer. in particular mutations in tumor suppressor genes, e.g. p53, seem to play an important role in developing cancer. up to now, lack of a method sensitive enough to determine the expected very low spontaneous mutation frequency (smf) in normal mammary gland tissue precluded the investigation of the role of spontaneous mutations acquired in the p53 gene in epidemiological studies. the only test with the potential to determine low smfs was the random mutation capture (rmc) assay, a genotype selective method which detects mutants that render the mutational sequence non-cleavable by the taqi restriction enzyme after accumulation of the target sequence. therefore, the suitability of the rmc assay to determine smf in p53 gene in normal human mammary gland tissue was evaluated. thus, the rmc assay was optimized concerning (i) dna isolation, (ii) pcr conditions, and (iii) amount of mammary gland tissue. (i) genomic dna from normal human mammary gland tissue, obtained from healthy women who underwent mamma reduction surgery for cosmetic reasons, was isolated using an extended proteinase k digestion prior to chloroform extraction. (ii) the target sequence in intron 6 of p53 gene was captured by hybridization with a complementary uracil-containing dna-probe synthesized via polymerase chain reaction (pcr), followed by magnetic separation from the remaining genomic dna. the copy number of the target sequence was quantified by competitive pcr. the number of mutants was detected after cleavage of the target dna with taqi by means of pcr with a primer set flanking the restriction site. (iii) with 2 g of normal mammary gland tissue a smf of 2.2±1.4x10 -7 per base pair was determined indicating the rmc assay suitable for smf determination. in conclusion, the smf in the p53 gene in normal human mammary gland tissue was determined for the first time, enabling the future investigation of factors influencing the smf during breast cancer development. cigarette smoke-induced release of pro-inflammatory cytokines including interleukin-8 (il-8) from inflammatory as well as structural cells in the airways, including airway smooth muscle (asm) cells, may contribute to the development of chronic obstructive pulmonary disease (copd). despite the wide use of pharmacological treatment aimed at increasing intracellular levels of the endogenous suppressor cyclic amp (camp), little is known on its exact mechanism of action. we report here that next to the β2-agonist fenoterol, direct and specific activation of either exchange protein directly activated by camp (epac) or protein kinase a (pka) reduced cigarette smoke extract (cse)-induced il-8 mrna expression and protein release by human asm cells. cse-induced iκbαdegradation and p65 nuclear translocation, processes that were primarily reversed by epac activation. further, cse increased extracellular signal-regulated kinase (erk) phosphorylation, which was selectively reduced by pka activation. cse decreased epac1 expression, but did not affect epac2 and pka expression. importantly, epac1 expression was also reduced in lung tissue from copd patients. in conclusion, epac and pka decrease cse-induced il-8 release by human asm cells via inhibition of nf-κb and erk, respectively, pointing at these camp effectors as potential targets for antiinflammatory therapy in copd. however, cigarette smoke exposure may reduce antiinflammatory effects of camp elevating agents via down-regulation of epac1. polycyclic aromatic hydrocarbons (key marker substance benzo[a]pyrene (bap)) have been assumed to play a role in the development of bladder cancer. the objective of the present study was to unravel cellular and in particular cytoskeletal response to bap. to follow the sequential steps of chemical carcinogenesis the differential proteomic profile was analyzed at early and late time points. the study was carried out in a superficial human bladder cancer cell line (rt4) exposed to 0.5 µm bap, a subacute concentration based on results of proliferation (brdu) and dna damage (tunel) tests. cells of a human bladder cancer cell line (rt4) were exposed to 0.5 µm bap for 24 h (n=5), 4 wk (n=7) and 8 wk (n=3). proteins of whole cell lysate were separated by twodimensional electrophoresis (fig. 1) . differentially expressed proteins were identified by matrix-assisted laser desorption/ionization-time of flight analysis. cortactin, actin and tubulin were immunohistochemical stained. changes in migration and colony forming ability were assessed by scratch wound-healing assay and soft-agar colony formation. results: by using several databases (uniprot, reactome, panther) the identified proteins were categorized into different functional classes such as mrna processing, translation, protein metabolic process, or several areas associated with the organization of the cytoskeleton. 45 % of the differentially expressed proteins after 24 h of treatment are involved in the processing of pre-mrna (20 %) and protein metabolism (25 %). this pattern changed after 8 wk of treatment. then, 48 % of the proteins affected the cytoskeleton whereas still 26 % were categorized to protein metabolism and only 7 % to pre-mrna processing. in the immunhistochemical staining, the treated cells appeared after 8 wk of exposure larger and rounder predominantly due to the modifications of the actin cytoskeleton. merged images of actin and cortactin revealed that both proteins colocalized in invadopodiae. after 8 wk, a higher number of treated cells moved toward the centre of the wound and they formed more soft-agar colonies compared to vehicle conditions, suggesting a transformation of the cells. in conclusion, bap exposure causes in an early phase an activation of the spliceosome which can led to an epithelial-tomesenchymal transition. two coordinators of a cell-type-specific splicing program, epithelial splicing regulatory proteins 1 and 2, are currently being validate by pcr. fused master gel : representative 2-de gel of rt4 cells exposed to 0.5 µm bap for 8 wk. protein spots which were differentially expressed compared to control and identified were marked. cannabinoids stimulate mesenchymal stem cell migration via a mitogen-activated protein kinase pathway schmuhl e. 1 , ramer r. mesenchymal stem cells (mscs) are known to be involved in various regenerative processes such as cardiac, ocular, skin and bone tissue healing. however, little is known about the pharmacotherapeutical options aiming at tissue healing steps such as the mobilization and homing of mscs. here, we show that cannabidiol (cbd), a nonpsychoactive cannabinoid, stimulates the migration of human adipose-derived mscs in both boyden chamber and in vitro scratch wound assays. in boyden chambers cbd (0.01 -3 µm) was shown to promote cell migration in a time-and concentration dependent manner. this promigratory action was inhibited by am-630 (cb2 receptor antagonist) and by o-1602 (g protein-coupled receptor [gpr] 55 agonist). moreover, cbd activated the mitogen-activated protein kinase (mapk) pathway as evidenced by increased phosphorylation of extracellular signal-regulated kinase (erk) 1/2. blockade of erk activation by pd98059 prevented cbd-stimulated msc migration, whereas inhibition of p38 mapk by sb203580 was inactive in this respect. furthermore, am-630 and o-1602 were found to attenuate cbd-induced erk activation. an erk-dependent promigratory action was likewise demonstrated for the phytocannabinoid ∆ 9 tetrahydrocannabinol and for the hydrolysis-stable anandamide analogue r(+)methanandamide. we conclude that cbd promotes msc migration via receptordependent erk activation, possibly contributing to tissue healing. the duffy antigen receptor for chemokines (darc) binds promiscuously many inflammatory chemokines without showing intracellular signal transduction. it is mainly expressed on endothelial cells of postcapillary venules and on red blood cells, where it acts as a transendothelial transporter of chemokines and as a chemokine sink, respectively. surprisingly, as shown for human and mouse brain, darc is also expressed at high density in the cerebellum. however, nothing is known about the function of darc in this location. we addressed this question by subjecting c57bl/6 wildtype and darc-deficient mice to a series of behavior experiments including morris water maze-, elevated plus maze-, rotarod-and actometer tests. while the results from the water maze experiments are ambiguous, elevated plus maze trials show a strong aversion of darc -/mice to walk to the end of the open arm, which is consistent with anxiety-like behavior. moreover, darc -/mice show greatly reduced locomotor activity, which is at least partly caused by episodes of reduced mobility occurring more frequently than in the corresponding wildtype controls (elevated plus maze, actometer). finally, darc -/mice spend a significantly reduced time on the rotating rod compared to c57bl/6 wildtype controls, which may indicate an impaired cerebellar function. we conclude that darc in fact modulates brain function. surprisingly, this appears to be happening under homeostatic conditions, although darc binds for the most part to inflammatory chemokines. it remains to be elucidated, how this effect can be caused by a non-signaling chemokine receptor. it may be an indirect consequence of altered brain chemokine concentrations or of as yet unknown signaling pathways activated by darc. transporter gene expression in human head-neck squamous cell carcinoma and epigenetic regulation mechanisms schnepf r. 1 hals-nasen-ohren-klinik, kopf-und halschirurgie, friedrich-alexander-universität erlangen-nürnberg, waldstraße 1, 91054 erlangen, germany background: membrane transporters may affect the disposition and thereby treatment efficiency of anticancer drugs in human head-neck squamous cell carcinoma (hnscc). the gene expression profile of transporters in hnscc, however, is unknown and was evaluated in this study. moreover, we evaluated mechanisms by which transporters are regulated in hnscc. we focused on the role of the nuclear pregnane x receptors (pxr, nr1i2) and epigenetic mechanisms. methods and results: real-time rt-pcr revealed a significantly increased mrna expression of slco1a2 and slco1b3 and a significantly decreased expression of transporters such as slco2b1, slco2a1 and abcc3 in human hnscc tissue samples compared to adjacent normal mucosa. moreover, an association between slco2b1 mrna levels in tumor tissues and five-year survival of hnscc patients was observed (χ 2 =6.59; p=0.010; n=34). bisulfite sequencing revealed that promoter cpg islands of abcc3 and slco2a1 were not methylated and thus these genes were not epigenetically silenced in hnscc tissues. in the hnscc-derived umscc-1 and scc-15 cell lines, transcript expression of transporters (e.g., abcc3, slco2a1; p<0.001) and pxr (nr1i2; p<0.001) was markedly induced by the dna methyltransferase inhibitor decitabine. cotreatment with the prototypical pxr activator rifampicin significantly reversed decitabine-induced abcc3 and slco2a1 expression. conclusions: transporter expression profiles significantly differed between hnscc and normal mucosa and expression levels of slco2b1 may serve as a marker for prognosis. modulation of the epigenome with the anticancer drug decitabine substantially affects transporter expression in umscc-1 and scc-15 cells, suggesting epigenetic regulation mechanisms. moreover, interactions between epigenetic and nuclear receptor-mediated mechanisms in transporter regulation occur. this work was in part supported by the johannes und frieda marohn foundation. the role of hcn2 in neuropathic and inflammatory pain schnorr s. 1 , eberhardt m. the pacemaker current ih is carried by hyperpolarization-activated cyclic nucleotidegated cation channels (hcn1-4) and contributes to cellular excitability in the heart and the nervous system. hcn1 and hcn2 are the two most abundant hcn subunits in peripheral sensory neurons with hcn2 being the prevalent isoform in nociceptive small sized c-fibre dorsal root ganglion (drg) neurons. we examined the role of hcn2 for peripheral sensitization and spontaneous neuronal activity in neuropathic and inflammatory pain. we generated a conditional deletion of hcn2 by using a nociceptor specific cre-transgene driven by the nav1.8 promoter. the nociceptor-specific knockout of hcn2 in drg neurons (nosphcn2ko) was confirmed by quantitative rt-pcr and western blot. immunohistochemical staining revealed that the deletion of hcn2 was mainly restricted to small sized drg neurons. the conditional loss of hcn2 resulted in a significant reduction of ih positive small diameter drg neurons pointing to a central role of this isoform to the hcn current in nociceptive neurons. behavioral studies showed that the lack of hcn2 did not influence basal pain responses but led to a significant reduction in mechanosensation in both neuropathic and inflammatory pain models. however, thermosensation of the mutants was only decreased in neuropathic pain conditions. in wild-type animals, intraperitoneal, intraplantar and even intrathecal injection of the hcn channel blocker zd7288 nearly eliminated tactile allodynia caused by inflammation in contrast to thermal hyperalgesia which remained unaffected. in contrast, pain thresholds in nosphcn2ko mice did not significantly increase after pharmacological block of ih. additionally, experiments revealed that the inflammatory condition induced an upregulation of hcn2 protein in the spinal dorsal horn compared to saline injected mice. our results suggest that hcn2 might be a new target in the treatment of neuropathic and inflammatory pain. the proper functioning of the central, as well as the peripheral nervous systems is of outstanding importance to the survival and well-being of humans. yet, the integrity of neuronal systems is constantly challenged by a plethora of environmental and occupational toxins. some of these toxins preferentially target neural cells. these neurotoxins can exert their devastating effects by very different modes of action. neurotoxins may induce apoptosis or necrosis of neurons, or interfere with axon growth and elongation. these processes can be identified by specialized in vitro tests. furthermore, neurotoxins have been described to alter glial function which may compromise the viability of surrounding neurons. as another important mode of action, several neurotoxins act on neurotransmitter receptors, thereby altering signal propagation within neuronal networks. countless natural and synthetic substances have been characterized for their effects on neurotransmitter receptors and today can be used for detailed studies of receptor function. however, environmental toxins of anthropogenic origin and occupational toxins that both represent constant sources for human exposure are still poorly studied with respect to their effects on neurotransmitter receptors. thus, the need for a better understanding of the susceptibility of neurotransmitter systems for toxic effects exerted by these substances is of outstanding importance for the protection of human health. here, we introduce an imaging-based approach for the screening of the effects of potential and known neurotoxins on neurotransmitter receptors of intact cells in vitro. different neuronal cells were tested for their sensitivities for classical neurotransmitters using life-cell imaging experiments. in more detail, we examined the proportion of responding cells and determined the ec50 values for the most prominent neurotransmitters in cell lines widely used for in vitro neurotoxicity studies on the one hand, namely sh-sy5y and lumes cells, and primary mouse neurons on the other hand. with these data at hand, we are now able to identify and characterize the effects of neurotoxins on receptor function in chronic, as well as acute exposition paradigms. the use of an in vitro imaging-based physiological test system is at the interface between non-functional in vitro approaches and in vivo toxicity tests, thus, giving mechanistic insight into neurotoxic processes without requiring animal experiments. apomorphine acts on trpa1 channels scholze a., schaefer m., hill k. universität leipzig -universitätsmedizin rudolf boehm-institut für pharmakologie und toxikologie, härtelstr. 16-18, 04107 leipzig, germany apomorphine is a non-narcotic derivative of morphine which acts as a dopamine agonist and is clinically used to treat "off-states" in patients suffering from parkinson´s disease. adverse effects of apomorphine treatment include dopaminergic effects such as nausea, but also ulceration and pain at the injection site. we wanted to test whether an activation of trp (transient receptor potential) channels in sensory neurones contributes to the perception of pain after apomorphine injection. while the warm/heat receptors trpv1, trpv2, trpv3, and trpv4 and the cold receptor trpm8 were insensitive towards apomorphine treatment, trpa1 could concentration-dependently be modulated by apomorphine. low micromolar apomorphine concentrations potently activated heterologously expressed trpa1 channels in a stably transfected cell line (hek293-trpa1), as well as natively expressed trpa1 in cultured dorsal root ganglion neurones. on the other hand, when using higher concentrations of apomorphine, we observed inhibition of trpa1 activity. previous studies have shown that subcutaneously administered apomorphine produces a biphasic dose response relationship in rats, inducing hyperalgesia at low doses whereas high doses of the substance cause antinociception. from our studies we conclude that such in vivo effects of apomorphine are presumably mediated by activation/inhibition of trpa1 expressed in sensory neurones agonist binding to a g protein-coupled receptor (gpcr) induces a conformational change of the receptor protein, which results in the activation of receptor-associated heterotrimeric g proteins [1] . in radioligand binding studies, conducted to investigate ligand binding to specific gpcrs, receptors are usually probed with radioantagonists. as in other gpcrs [2] , agonists of the muscarinic m2 receptor exhibit biphasic kinetics and biphasic competition curves with radioantagonists, indicating a more complex situation probably caused by g protein interactions. here, we present a detailed study of the binding of agonists to muscarinic m2 receptors including the novel super-high affinity agonist iperoxo and a differential chemical knockout of g proteins. in addition to membrane homogenates living cells were employed. we demonstrate that the high affinity fraction in biphasic curves does not differ between selected full agonist and is sensitive to pertussis toxin, thus indicating that this receptor population is associated with gi proteins. however, despite promiscuous signalling properties of m2 receptors, the low affinity fraction is not associated with any other g protein, since low affinity binding is insensitive to high concentrations of guanylnucleotides and cholera toxin. moreover, high affinity agonist binding appears solely in membrane homogenates but not in experiments conducted with living cells, probably due to their high intracellular concentration of guanylnucleotides. taken together the chemical knock-out of g proteins revealed that the high affinity binding of agonists in membrane homogenates is associated with the interaction of the muscarinic m2 receptor with gi proteins. the low affinity binding cannot be related to another g protein, although the muscarinic m2 receptor exhibits promiscuous g protein signalling properties. interestingly data obtained with living cells do not reveal any high affinity binding of agonists. prolonged stress leads to a dysregulation of the hypothalamus-pituitary-adrenal (hpa)axis and may affect the sensitivity of pain perception. however, it is not yet known whether the alterations of hpa-axis and increased pain sensitivity are related. to create a long lasting stressful situation, male wistar rats were exposed to a restraint-stress for 1 h daily over a period of two weeks. the effect of stress on the hpa-axis was determined by adrenal morphology and stress hormone levels, the influence on mechanical pain sensitivity was evaluated by the randall-selitto paw pressure test. on day 15 the animals exhibited a significant mechanical hyperalgesia. they also showed increased acth and corticosterone plasma levels and an enlarged zona fasciculata of the adrenal gland, indicating a dysregulation of the hpa-axis. for testing the correlation of hpa-axis dysregulation and hyperalgesia a persistent increase in plasma corticosterone in wistar rats was generated by the administration of corticosterone via the drinking water for two weeks. these animals also showed an increased mechanical nociceptive sensitivity with an accompanied decrease of the adrenal glands and reduced acth levels. the results show that chronic stress leads to a dysfunction of the hpa-axis with an accompanied mechanical hyperalgesia which can be mimicked by oral administration of corticosterone. thus, this in-vivo test system may provide a new animal-friendly pharmacological model for stress-related pain disorders. the alternaria mycotoxins aoh and ame induce cyp1a1 and apoptosis in murine hepatoma cells dependent on the aryl hydrocarbon receptor mycotoxins are secondary metabolites of fungi including the genus alternaria (black mold). alternaria fungi are known to infest different types of foodstuffs and produce diverse toxins amongst them the mycotoxins alternariol (aoh) and alternariol methyl ether (ame) which are potential carcinogens. as planar compounds, aoh and ame are preferentially metabolized by cytochrome p450 (cyp) 1a1 and 1a2. the most prominent regulator of cyp1a1 is the dimeric transcription factor complex ahr/arnt, which is activated by planar ligands. therefore we studied the activation of ahr/arnt by aoh and ame and monitored cyp1a1 induction in murine hepatoma cells (hepa-1c1c7). indeed, aoh and ame enhanced the levels of cyp1a1 in hepa-1c1c7 cells but not in cells with inactivated ahr (hepa-1c1c12) or arnt (hepa-1c1c4). furthermore, we studied the cytotoxicity of aoh and ame. by using a fluorescence-based microscopic readout we measured effects on cell counts, apoptosis, senescence and micronuclei formation. both aoh and ame reduce the cell number and the cell nuclei show drastic morphological changes e.g. enlargement after aoh treatment or micronuclei formation. the observed effects where, except for the induction of apoptosis, independent of ahr/arnt. in summary, aoh and ame activate the ahr/arnt pathway to induce cyp1a1 expression and apoptosis. however, the predominant cytotoxic effect of aoh and ame in hepatoma cells is a profound reduction in cell numbers, which is independent of the ahr/arnt pathway. special purpose databases are the first place for researchers in the life sciences to obtain expert curated data. naturally, such resources are limited in terms of timeliness and comprehensiveness. the literature database pubmed alone lists more than 20,000,000 scientific abstracts, and 700,000 are newly added every year. the protein sequence database uniprotkb stores over 10,500,000 sequences, a hundred times more than ten years ago. turning these data into meaningful information and making it accessible to both humans and computers have become an essential part of biological discovery and biomedical research. text mining techniques have proven useful to extract the missing links in areas such as drug-target and drug-disease prediction, the extraction of mutation-phenotype associations, or the prediction of protein-protein and protein-ligand interactions. by systematically extracting information from available literature, reports or patents, text mining techniques can help to refine existing categorical knowledge stored in databases, and hence will support drug repositioning, the discovery of novel cancer biomarkers, or help to understand causes of hereditary diseases. in the area of regulatory toxicology we developed go3r, the first knowledge-based search engine for alternative methods to animal experiments. the system not only helps retrieving information on the availability of alternative methods that allows for replacing, reducing or refining animal experiments, but also provides an endpoint-centered literature search to all scientists and regulatory authorities seeking for toxicological information and data. the up-to-date taxonomicstructured "table of contents" provided by go3r allows for search in the literature listed in pubmed or the toxicology data network (toxnet) in a fast and comprehensive manner. the semantically enriched platform supports the user during the query formulation, allows for bibliographic analysis, and reveals existing relations to replacement, reduction, and refinement of animal experiments. impaired cardiac excitation-contraction-coupling in mice with complete inactivation of the crem gene schulte j. s., tekook m., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany the structurally related transcription factors camp response element binding protein (creb) and camp response element modulator (crem) mediate a regulation of gene transcription in response to camp and are expressed in the heart. mice with complete inactivation of crem display impaired cardiac contraction and relaxation, decreased expression of serca and down-regulation of β1-adrenoceptors. to elucidate the underlying functional mechanisms on the cellular level we here investigated cellular electrophysiology and ca 2+ -cycling in ventricular cardiomyocytes from crem ko mice (ko). adult ventricular cardiomyocytes were isolated from ko and wildtype (wt) mice (age 16-20 weeks) and subsequently used for experiments within 6 hours after isolation. action potentials (aps) were recorded from ventricular cardiomyocytes (perforated patch, whole cell current clamp inactivation of crem seems to have no consequences for ap duration and possibly associated ion channels but leads to impairment of ca 2+ cycling and sarcomere shortening under basal conditions well explaining the previous findings in vivo. our results show that crem is essential for a regular excitation-contraction coupling in the mouse heart. (supported by the izkf münster) new mechanistic insights in no/cgmp actions in the vasculature schulte k., koesling d., universitätsstr. 150, 44781 bochum, germany hypertension, a major risk factor for cardiovascular diseases, is associated with vascular changes resulting in increased vascular contractility und vascular peripheral resistance. a prominent factor in the development and maintenance of hypertension is the reninangiotensin-aldostrerone system. angiotensin ii (ang ii) is the main mediator of this system and a powerful vasoconstrictor. ang ii increases the intracellular ca 2+ concentration thereby activating myosin light chain (mlc) kinase, which enhances mlc phosphorylation and subsequent vascular contraction. opposite to angii-induced vascular contraction, no/cgmp pathway promotes vascular relaxation by decreasing ca 2+ concentration and lowering mlc phosphorylation. responsible for mlc dephosphorylation is the mlc phosphatase (mlcp), whose activity is regulated by different phosphorylations. phosphorylation of mlcp via rhoa-activated rho-kinase enhances phosphatase activity while phosphorylation via the cgmp-dependent protein kinase has been proposed to decrease enzymatic activity. to investigate the interplay of angii with the no/cgmp pathway, we treated wild-type and ko mice lacking the cgmp forming no receptor, no-gc1, with angiotensinii (1.44 mg / kg bw / d) for two weeks. in addition to various cardiovascular parameters, physiological changes in vascular reactivity of aortic rings of angii-treated wt and no-gc1 ko mice were assessed in organ bath experiments and correlated with biochemical parameters as the phosphorylation state of mlc, mlcp and rho-kinase activities examined by immunoblot analysis. analysis of cgmp levels revealed that angii treatment decreased cgmp in wt mice to levels comparable to those of the ko mice which were unaltered by the treatment. our study will provide further mechanistic insights in the molecular interactions between constrictor and dilator stimuli in the vasculature. nanomaterials are already used today and offer even greater use and benefits in the future. the progress of nanotechnology must be accompanied by investigations of their potential harmful effects. for airborne nanomaterials, lung toxicity is a major concern and obviously the particle size is discussed as a critical property directing adverse effects. while standard toxicological test methods are generally capable of detecting the toxic effects, the choice of relevant methods for nanomaterials is still discussed. we have investigated two genotoxic endpoints -alkaline comet assay in lung tissue and micronucleation in polychromatic erythrocytes of the bone marrow -in a combined study 72 hours after a single instillation of 18 µg gold nanoparticles (np) into the trachea of male adult wistar rats. the administration of three test materials differing only in their primary particle size (2-, 20-and 200-nm) did not lead to relevant dna damage in the mentioned tests. the measurement of clinical pathology parameters in bronchoalveolar lavage fluid (balf) and blood indicated neither relevant local reactions in the animals' lungs nor adverse systemic effects. minor histopathology findings occurred in the lung of the animals exposed to 20-nm and 200-nm sized nanomaterials. in conclusion, under the conditions of this study the different sized gold np tested were non-genotoxic and showed no systemic and local adverse effects at the given dose. platelet dense granule secretion mediates platelet-dependent enhancement of tumor cell transmigration and formation of metastases schumacher d., strilic b., wettschureck n., offermanns s. mpi für herz-und lungenforschung offermanns, ludwigstr. 43, 61231 bad nauheim, germany tumor cell metastasis to distant organs is the primary cause of mortality in cancer patients. tumor cells leave the primary tumor, intravasate, survive in the circulation and extravasate through the endothelial cell layer to grow in the target organ. it has long been known that blood platelets play an important role in tumor cell survival and dissemination, but the mechanism by which platelets promote metastasis remained unclear. given that platelets are found closely associated with tumor cells shortly after vascular arrest, we explored whether platelets can facilitate the transmigration of tumor cells through the endothelium and thereby promote extravasation of tumor cells into the organ parenchyma. the ability of various mouse and human tumor cells like lewis-lung carcinoma cells (llc1), b16f10 melanoma cells or human neuroblastoma cells (sh-sy5y) to transmigrate through an endothelial cell layer was strongly enhanced by seeding tumor cells together with mouse or human platelets onto the endothelial cell layer. this indicates that platelets facilitate tumor cell transmigration in vitro. we found that platelet granule secretion is involved in this process as supernatant from platelets incubated with tumor cells but not from resting platelets was sufficient to enhance tumor cell transmigration. additionally, no platelet-mediated increase of tumor cell transmigration was observed in dense granule secretion-defective platelets of munc13-4 deficient mice. thus, dense granule secretion is required for platelet-dependent tumor cell extravasation in vitro. while the growth and weight of primary tumors after subcutaneous injection of llc1 and b16 cells was indistinguishable between wild-type mice and animals lacking munc13-4, the number of metastases in the lung was strongly reduced in munc13-4-deficient animals. the strong decrease in formation of metastases in munc13-4 deficient mice was also observed after i.v. injection of llc1 and b16f10 cells. thus, platelet dense granule secretion plays a critical role in tumor cell metastasis by enhancing tumor cell transmigration through the endothelial cell layer. formation of dna adducts in mouse tissues by the brassica ingredient 1methoxy-3-indolylmethyl glucosinolate and its break-down product 1-methoxy-3indolylmethyl alcohol schumacher f. 1 , engst w. glucosinolates are secondary metabolites present at substantial levels in cruciferous vegetables, such as broccoli and cabbage. after injury of plant tissue they are activated by the enzyme myrosinase to form various electrophilic degradation products like isothiocyanates. we previously showed that 1-methoxy-3-indolylmethyl (1-mim) glucosinolate (or neoglucobrassicin) is a potent genotoxicant in bacterial and mammalian cells after activation by myrosinase. the induction of mutations could be correlated with the formation of dna adducts [1] . we have identified and synthesized the major dna adducts n 2 -(1-mim)-dg and n 6 -(1-mim)-da. moreover, we developed a highly sensitive uplc-esi-ms/ms method for selective mrm quantification of these adducts using stable-isotopic labeled adducts as internal standards. while the plant enzyme myrosinase is probably almost completely inactivated after cooking the vegetables, the glucosinolates reach the gut mostly intact due to their good heat and ph stability. enzymes of individual intestinal bacteria are able to cleave the glycosidic bond of the glucosinolates, which leads to the formation of reactive metabolites within the gut lumen. we were able to detect significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in a dose-dependent manner in the large intestine of mice treated orally with isolated 1-mim glucosinolate. the peak levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da in the murine large intestine were reached 8 h after a single administration of 600 µmol 1-mim glucosinolate/ kg body weight. the oral application of the relatively stable metabolite 1-mim alcohol to mice led to the formation of identical dna adducts. this benzylic alcohol can be activated by sulfotransferases to an electrophilic sulfo conjugate. in contrast to the intact glucosinolate the orally administered 1-mim alcohol generated significant levels of n 2 -(1-mim)-dg and n 6 -(1-mim)-da not only in the large intestine but also in other tissues, such as the liver, of mice. [1] h. glatt, c. baasanjav-gerber, f. schumacher, b. h. monien, m. schreiner, h. frank, a. seidel, w. engst, chem.-biol. interact., 192 (2011) human pregnane x receptor genotype of the donor but not of the recipient is a risk factor for delayed graft function after renal transplantation schwab m. 1, 2 , schaeffeler e. delayed graft function (dgf) is an important immediate complication in renal transplantation significantly contributing to decreased long-term allograft survival. in addition to donor-and recipient-related risk factors altered renal excretion of xenobiotics by membrane transporters may influence dgf as well. using recipients' and donors' dna, we assessed the impact of genetic variants on dgf for the transporter proteins, pglycoprotein (abcb1) and multidrug resistance protein 2 (abcc2), and the nuclear pregnane x receptor (pxr/nr1i2), regulating the transcription of drug metabolizing enzymes and membrane transporters. in our local cohort of transplant patients (n=178) dgf occurred in 27.5 %. logistic regression analysis using four different genetic models (i.e. co-dominant, dominant, recessive and log additive) indicates that only the donor's pxr rs2276707 8055tt genotype was significantly associated with dgf (recessive model: or, 9.0; 95%ci, p=0.004 unadjusted) , even after correction for multiple testing (p=0.035 holm-adjusted). when we performed multivariate analysis including genetic and 16 clinical co-variates (i.e. age, gender, hla mismatches, panelreactive antibodies, immunosuppression using cni, t cell-depleting agents, anti-il-2 receptor antibody and steroids, cold or warm ischemia time, living vs deceased donors or graft loss) again dgf was significantly associated only with the pxr rs2276707 tt genotype of the donor (recessive model: or, 16.08; 95% ci, 2.0-129.46; p=0.0046 unadjusted) which held true after correction for multiple testing (p=0.04). for abcc2 variants only the donor rs17222723 3563t>a genotype correlated with dgf by univariate (or, 4.65; 95%ci, p=0.005 unadjusted) as well as by multivariate analysis (or, 5.5; 95%ci, p=0 .01; table 4 ) but not after correction for multiple testing (p=0.12). for variants in the abcb1 gene no significant associations with dgf were detected for both the donor's and the recipient's genotype. in summary, our findings suggest for the first time that pxr may be a risk gene for the development of dgf independently from previously identified risk factors. supported by the robert-bosch foundation, stuttgart, germany, the bmbf grant 03is2061c (berlin, germany) and by the ferdinand eisenberger grant of the german society of urology (id krs1/fe-10). formation, morphology and structural requirements for formation of microtubule protrusions by clostridium difficile toxin cdt schwan c., kruppke a. s., nölke t., aktories k. institut für experimentelle und klinische pharmakologie und toxikologie i, albertstr. 25, 79104 freiburg, germany clostridium difficile is an anaerobe, gram-positive pathogen. it causes antibioticassociated diarrhoea and pseudomembranous colitis by production of the rho gtpaseglucosylating toxins a and b. recently emerging hypervirulent clostridium difficile strains additionally produce the binary adp-ribosyltransferase toxin cdt (clostridium difficile transferase). cdt is taken up via receptor-mediated endocytosis after binding to the lipolysis stimulated lipoprotein receptor (papatheodorou et al., pnas 2011) . in the cytosol, cdt adp-ribosylates actin at arg 177, thereby actin polymerization is blocked, resulting in disruption of the f-actin network. cdt and other binary actin-adp-ribosylating toxins induce redistribution of microtubules in the cell interior and formation of long (>150 µm) microtubule-based protrusions at the surface of intestinal epithelial cells which increase bacterial adherence (schwan et al., plos pathog 2009 ). the clostridial actin-adp-ribosyltransferases influence the dynamicity of microtubules and their capture at the cell cortex by indirectly affecting different microtubule regulating proteins like clasp2 and acf7. besides the influence of cdt on microtubule regulatory proteins, the formation of protrusions depends on plasma membrane composition. depletion of cholesterol, the breakdown of sphingomyelin or inhibition of sphingolipid-synthesis reduce the formation of microtubule-based protrusions. surprisingly, most of the cdt-induced processes contain membrane-tubules derived from the endoplasmatic reticulum (er). the remodeling of the er is microtubule dependent and is mainly mediated by stim1 that usually functions as a calcium sensor in the er and activates the store operated orai1 calcium ion channels in the plasma membrane. the data suggest that toxin-induced changes of the microtubule system including alterations of the er, may affect trafficking and er-dependent signalling. bilobalide is a neuroprotective constituent of ginkgo biloba with an unknown mechanism of action. in the present study, we first used microdialysis in mice to evaluate changes in the extracellular fluid of the brain during and after stroke. microdialysis probes were implanted into the striatum of cd-1 mice, and dialysates were obtained while a monofilament was inserted for 60 min via the common carotid artery (cca) to block perfusion through the middle cerebral artery (mca). while glucose levels dropped immediately upon middle cerebral artery occlusion (mcao), glutamate concentrations in the microdialysates -as measured with a cma 600 analyzer -rose extensively during ischemia to more than 2000% of baseline level. both glucose and glutamate levels recovered rapidly when mcao was terminated after 60 min. when bilobalide (10 µm) was perfused into the striatum through the microdialysis probe during mcao, glucose levels dropped but the neurotoxic rise of glutamate was significantly attenuated and reached only 500% of baseline level (p<0.01). in the following experiments, we investigated the activity of mitochondria in ischemic brain. ischemia was induced by mcao, and ischemic as well as "healthy" tissue from the opposite hemisphere was obtained. mitochondria were isolated and mitochondrial respiration was monitored using the oroboros ® oxygraph. significant deficits of respiration were observed after ischemia. in the healthy hemisphere, the respiratory states (leak i+ii, complex i+ii+iv, oxidative phosphorylation (oxphos) and electron transport system (ets) capacity) showed a decrease of oxygen consumption to 60-70% of sham-operated mice. in the ischemic hemisphere, several values were lower at 40% of sham-operated mice (leak i+ii, complex ii+iv,oxphos and ets) whereas complex i showed a remarkably low respiratory capacity of 16% of baseline. direct addition of bilobalide (10 µm) to post-ischemic mitochondria caused a 2-fold increase of complex i activity in vitro. pretreatment of mice with bilobalide (10 mg/kg i.p.) one hour before mcao caused a significant, 3-fold improvement of complex i respiration when measured ex vivo. these data clearly indicate that bilobalide targets mitochondrial processes within the respiratory chain, preserving complex i function during ischemia. this action likely explains its neuroprotextive activity in vivo. unreacted resin monomers such as 2-hydroxyethyl methacrylate (hema) are environmental stressors released from dental composites after incomplete polymerization. the production of reactive oxygen species (ros) is a major response of cells to monomer exposure. moreover, adverse effects of monomers including delayed cell differentiation or mineralization processes, dna damage or apoptosis are associated with increased ros production. the intracellular redox homeostasis is controlled by the major non-enzymatic antioxidant glutathione (gsh), and antioxidant enzymes. here, we hypothesized that cells exposed to hema responded by a differential expression of antioxidant enzymes such as superoxide dismutase (sod-1), catalase (cat) or glutathione peroxidase (gpx1/2). raw246.7 mouse macrophages were exposed to hema (0-8mm) for 24h, and protein expression was analyzed by western blotting. to study the influence of intracellular gsh on enzyme expression, gsh synthesis was reduced by the inhibitor buthionine sulfoximine (50µm bso), or enhanced by 2-oxothiazolidine-4-carboxylate (5mm otc) and n-acetyl cysteine (10mm nac). expression of sod-1 found in untreated cultures was decreased in the presence of hema and even further reduced by bso. in contrast, nac counteracted hemainduced inhibition of sod-1 expression. cat expression was not detected in untreated cells, however, the enhanced expression of cat in cells exposed to hema indicated the decomposition of abundant levels of hydrogen peroxide. the minor influence of bso or otc showed that expression of cat was independent of gsh levels while a decrease of hema-induced cat expression in the presence of nac indicated reduced oxidative stress. gpx1/2 was expressed in untreated cultures, and its down-regulation by bso indicated that this enzyme was primarily responsible for h2o2 decomposition. the inhibitory effect of hema on gpx1/2 expression was enhanced by bso but counteracted by otc or nac. these findings indicate that h2o2 is the predominant reactive oxygen species generated in the presence of dental resin monomers like hema. abundant h2o2 production leads to the activation of cat expression and a feed-back inhibition of sod-1 expression. the hema-induced reduction of gpx1/2 expression is most likely a consequence of reduced gsh levels because of the formation of glutathione disulfide (gssg) or by gsh-hema adducts. the life-threatening effects of certain organophosphorus compounds such as soman or fenamiphos cannot be antagonized adequately by the treatment with atropine and oximes. alternative approaches are necessary. since the adequate restoration of disturbed muscle function is considered to be life-saving, a model is needed for screening of potentially therapeutic substances. an established model for the development of such new therapies is the diaphragm preparation. however, this model requires a large number of animals and experimental available time frame is limited to some hours. here, the organotypic nerve-muscle co-culture may be an appropriate alternative, because a large number of specimens with low numbers of animals and a long period of investigation over several days is possible. in the present study, the restoration of vx paralysed muscle function with obidoxime was investigated by using both models. slices of spinal cord and muscle tissue were dissected from mice embryos (e 14-15) , fixed to coverslips and incubated in roller tubes for about 2-4 weeks. spontaneous muscle activity was recorded by video microscopic techniques and was quantified offline. muscle force production in mice diaphragm preparations was elicited by indirect field stimulation technique in a 12 chamber organ bath and quantified as time-force diagrams (auc) that were expressed as relative changes of the muscle force compared to the control data. application of the nerve agent vx (0.75 µm) resulted in a strong reduction of muscle activity in the co-culture and of muscle force production in the diaphragm muscle. after obidoxime (10 µm) was added spontaneous muscle activity in the co culture recovered from 0.45 ± 0.24 hz to 1.67 ± 0.24 hz (control 1.79 + 0.22 hz) . muscle force remained stable over the next days. the vx-blocked muscle force of diaphragm was restored to 69.9 ± 21.3 % by obidoxime compared to control. muscle force production after indirect stimulation was stable for 6 hours only. our results suggest that the organotypic nerve-muscle co-cultures may be an appropriate tool for the screening of new therapeutic approaches in restoration of blocked neuromuscular transmission. moreover, the model offers an additional advantage as long-term experiments may be performed and pre-and postsynaptic effects may be assessed directly. additionally, the number of experimental animals could be reduced. the modulation of gene expression by the transcription factor crem (camp responsiveelement modulator) represents a fundamental mechanism of gene control in response to elevation of intracellular camp levels. in vascular smooth muscle cells (vsmcs) crem is involved in the regulation of cell proliferation and apoptosis supporting its relevance for vascular proliferative diseases. mice with a global inactivation of crem (cko) showed a significant increase in neointima formation after ligation of the carotid artery and an increase of atherosclerotic plaque formation after high fat diet on an apoe knockout background compared to wildtype controls (wt). on the cellular level a crem deficiency was associated with a 1.3 fold increased proliferation rate of primary vsmcs after stimulation with the platelet-derived growth factor (pdgf; n=10 from 6 isolations). microarray analysis and subsequent realtime-rt-pcr validation revealed that the alpha-type platelet-derived growth factor receptor (pdgfra) the regulator of g-protein signaling 5 (rgs5) and peptidylprolyl isomerase a (ppia) were 1.5-2.5 fold upregulated in pdgf treated cko vsmcs compared to wt vsmcs (n=6). transcripts of rgs5 (2.6 fold, ) and ppia (1.8 fold, were also upregulated in the carotid artery of cko mice in comparison to wt mice (n=10-12). we conclude that crem deficiency is associated with transcriptional changes of rgs5, pdgfra, ppia, which might explain the increased proliferation rate in cko vsmcs and the increased responsiveness to pathophysiological conditions. (supported by the izkf münster). the role of non-catalytic p101 and p87 subunits in regulating phosphoinositide 3kinase γ by gβγ and h-ras shymanets a. 1 phosphoinositide 3-kinase γ (pi3kγ) controls a plethora of cellular responses. pi3kγ, a heterodimer formed by non-catalytic p101 or p87 and catalytic p110γ subunits, is regulated by gβγ and ras. earlier we speculated that p101 binds to gβγ to translocate cytosolic pi3kγ to the plasma membrane, enabling direct activation of p110γ (brock et al., j. cell biol. 2003) . however, the p87 subunit does not function as gβγ adapter (kurig et al., pnas 2009) . since the impact of each non-catalytic subunit in regulating p110γ by gβγ and ras still remains elusive, we studied their role in detail. gβ1γ2 variants harbouring mutations in positions involved in interaction with gα subunit were purified from sf9 cells and tested for their ability to activate pi3kγ. we observed that p101, but not p87, was able to rescue the stimulatory activity of gβ1γ2 mutants incapable to activate p110γ (shymanets et al., biochem. j. doi:10 .1042/bj20111664). to further study the functional impact of the non-catalytic subunits on pi3kγ regulation, we have designed phospholipid vesicles containing similar amounts of recombinant pi3kγ variants. although p87/p110γ exhibited stronger sensitivity to gβ1γ2 than p110γ, the activity of vesicles-associated p101/p110γ was significantly higher as compared to vesicles-associated p87/p110γ or p110γ in the absence and in the presence of gβ1γ2. to study an effect of ras proteins on pi3kγ variants, recombinantly expressed h-ras was purified from sf9 cells. the posttranslational processing and lipidation of the protein was verified by mass spectrometry analysis. the impact of h-ras on regulation of p87/p110γ and p101/p110γ differed, which may explain integration of pi3kγ variants in different signalling pathways. taken together, p87 and p101 subunits implement discrete functions in respect to (i) membrane recruitment of pi3kγ and (ii) regulation of enzymatic activity by gβγ and h-ras. preparation of consolidated exposure scenarios for mixtures under reach sica m., dorn s., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany under reach, formulators of mixtures need to include substance-related information into extended safety data sheets (esds), if mixtures are classified as dangerous according to the dangerous preparation directive (directive 1999/45/ec). one way to add information on substances into esds of mixtures is to generate exposure scenarios (ess) for mixtures. in order to fulfil this task, two approaches have been developed for the identification of the risk-determining substances (lead substances) in the mixtures: the critical component approach (cca) relies on dnels and pnecs for all substances, their concentrations in the mixtures as well as substance and use-specific availability parameters (echa, 2008) . in contrast, the dpd+ method is based on the legislation for classification of preparations (directive 1999/45/ec). the dpd+ method defines a lead substance for each route of human exposure and for the aquatic environment (cefic, 2010) . however, each of these methods has certain limitations. the aim of the present work is to improve the preparation of consolidated ess for a number of mixtures and provide information about their safe use. to this end, we first adopted information on risk management measures (rmms) and operational conditions (ocs) of the lead substances using the dpd+ methodology. at the same time, we considered the specific conditions of use of the mixtures (e.g. spraying, brush painting). we then conducted risk assessments by deriving dnels for the mixtures and using exposure modelling tools recommended under reach (e.g., ecetoc tra, riskofderm, art). we compared the outcome of these assessments with results obtained from the application of the dpd+ methodology. the work presents the results of application of dpd+ approach and the cca and indicates possible improvements for the risk assessment of mixtures. to check for seasonal and weather dependent influences in the prescription rate of drugs used to treat cardiovascular and respiratory diseases (atc codes c and r) a survey covering all prescriptions of a specimen german general regional health insurance (aok plus, data for saxony, largest health insurance service, approx. 50 % of all saxonian citizens are inscribed to this service) for the years 2005 to 2007 was analysed on a monthly basis. the number of prescriptions for cardiovascular drugs changed approximately +/-20 % around the mean for the different month without a clear seasonal pattern. for respiratory drugs only the systemic anticholinergics and drugs used to treat obstructive lung disease displayed a distinct seasonal pattern with a 50 -70 % above average prescription figure during spring time (february to may) and a 25 to 40 % trough in late summer/autumn (july to october). the data have to be analysed for further cofounders (e.g. influenza prevalence, environmental conditions etc.) to fully understand the fluctuations observed. the prescription rate for cardiovascular drugs and respiratory drugs seems to be influenced by multiple factors aside seasonal influences. pasteurella multocida toxin prevents osteoblast differentiation by activation of heterotrimeric g proteins siegert p., aktories k., orth j. institut für experimentelle und klinische pharmakologie und toxikologie abt. i, albertstr. 25, 79104 freiburg i. br., germany pasteurella multocida toxin (pmt) is a major virulence factor of pasteurella multocida causing pasteurellosis in man and animals and is responsible for atrophic rhinitis in pigs. the toxin modulates various signaling pathways by acting on the heterotrimeric g proteins gαq, gα12/13 and gαi. pmt activates gq to increase inositol phosphate production via phospholipase cβ and alteration of gene expression via the jak/stat pathway. the toxin also activates rhoa via gαq and gα12/13 family proteins. we showed that the underlying mechanism of the activation of heterotrimeric g proteins is a deamidation of an essential glutamine residue leading to a constitutive activation of the g protein. because pmt is the causative agent to induce progressive atrophic rhinitis in pigs, which is characterized by loss of nasal turbinate bones leading to a twisting and/or shortening of the snout, we studied the effect of pmt on bone cells. here we studied the effect of the toxin on osteoblast differentiation in st-2 cells and in primary osteoblasts from rat calvaria. st-2 cells are stromal derived cells, which can be differentiated into osteoblasts or adipocytes. the toxin inhibits the differentiation of st-2 cells into osteoblasts studied by determination of specific osteoblast markers. additionally, pmt represses the induction of transcription factors essential for osteoblast differentiation. moreover, the principal pathways activated by pmt to induce these effects were investigated. ventilator-induced lung injury (vili) is a serious problem in intensive care medicine. its mechanisms are only incompletely understood, although it is widely accepted that ventilation-induced inflammation (biotrauma) makes an important contribution. the isolated perfused mouse lung (ipl) is a valuable tool to investigate the mechanisms of vili. several studies have shown considerable differences between various mouse strains with respect to lung mechanics and inflammatory responses. therefore, we hypothesized that the pulmonary responses to mechanical ventilation differ between c57bl/6 and balb/c mice. in addition, this study introduces the novel half lung technique that allows to obtain lung tissue from the same mouse at two different time points. isolated perfused mouse lungs from c57bl/6 or balb/c were subjected to high (25cmh 2o) or low pressure (8cmh2o) ventilation for 240 minutes. after 180 minutes the left lung was removed and used for western blot analysis. the right lung was ventilated for another 60 minutes. by the end of experiment the right lung was removed and qrt-pcr performed. during the whole experiment perfusate sample were taken from the venous catheter and used for protein quantification by elisa. it was possible to remove half of the lung and to further ventilate the other half without acute changes in lung mechanics. in both strains high pressure ventilation elicited a significantly higher cytokine release than low pressure ventilation. c57bl/6 mice showed higher tnf, il-1β and amphiregulin levels after high pressure ventilation, whereas balb/c exhibited increased production of cxc chemokines (cxcl-1, cxcl-2) and il-6. kinase activities (jnk, akt, erk1/2, p38 map kinase) were increased in high pressure ventilated animals, but were strain independent. the novel half lung technique builds on the well established ipl method. it permits to separately analyze the left and the right lung at different time points during continual ventilation. this method reduces animal numbers by 50% and allows statistical within subject analysis. using this method, the present study showed that inflammatory response to mechanical ventilation differ between c57bl/6 and balb/c. these findings show that the biotrauma response in mice depends on the strain that is studied. macrophages play an important role as an integral part of the first line of immune defense. two different macrophage populations have been described. m1 macrophages produce proinflammatory cytokines and are involved in inflammatory processes. by contrast, m2 macrophages release anti-inflammatory cytokines and extracellular matrix components. they can enhance wound repair and angiogenesis, but they can also promote tumor progression. recently, industrial nanoparticles have raised concerns because of their putative toxic effects. on the other hand, specifically designed nanoparticles can be used as clinical diagnostics and as drug carriers for pharmacotherapy. thus, investigations on the interactions of engineered nanoparticles with living cells and organisms are of great importance. macrophages as phagocytosing cells scavenge nanoparticles circulating in the bloodstream. therefore, we analyzed how nanoparticles with different surface functionalization might affect functions of human macrophages. monocytes were isolated from buffy coats and differentiated to macrophages with macrophage colony-stimulating factor. carboxy-(ps-cooh) and amino-(ps-nh 2) functionalized polystyrene nanoparticles were produced by the miniemulsion polymerization process and the average particle size, the polydispersity index and their zeta potential were determined by dynamic light scattering. the macrophages were cultured in the presence or absence of different concentrations of ps-cooh and ps-nh2 nanoparticles for up to 6 days. analysis of cell viability revealed that ps-nh2 but not ps-cooh concentration-and time-dependently reduced the macrophage viability. by annexin v/propidium iodide double staining we could show that ps-nh2 trigger apoptosis in macrophages. we further polarized macrophages to either m1 or m2 using ifn-γ and lps or il-4, respectively. these macrophage populations were characterized by their expression of extracellular markers by flow cytometry and their production of cytokines by elisa. the effects of functionalized polysterene nanoparticles on the cytokine production and surface marker expression of m1 and m2 macrophages were analyzed. our data indicate that surface functionalization is a critical parameter in the nanoparticle-induced toxicity in human macrophages. this work was supported by the dfg spp1313. loos c., lunov o., syrovets t., simmet t. ulm university institute of pharmacology of natural products & clinical pharmacology, helmholtzstr. 20, 89081 ulm, germany nanoparticles are currently used for various medical applications including imaging, diagnosis and drug delivery. due to particle size and surface area, their fundamental properties differ significantly from those of corresponding bulk materials. nanoparticles circulating in the blood are mainly sequestrated by the reticuloendothelial system that consists predominantly of phagocytic macrophages. macrophages express a variety of cellular receptors for sensing and internalizing particular material like viruses, microorganisms, and foreign particulate matter including nanoparticles. therefore, a detailed understanding of the intracellular fate and processing of the nanoparticles by macrophages is indispensable for controlled biomedical applications of nanoparticles. introducing distinct surface modifications, one might control nanoparticle uptake by different cell types and thereby target specific tissues and cellular compartments. tumor cell lines are frequently used as models for primary cells to analyze the effect of nanoparticles on cells. here we show that carboxy-(ps-cooh) and aminofunctionalized (ps-nh 2) polystyrene nanoparticles of ~100 nm in diameter are internalized by human macrophages, thp-1 monocytic leukemia cells, and by pmadifferentiated thp-1 cells via different mechanisms. in buffer, macrophages and thp-1 rapidly internalize both types of nanoparticles, yet, the carboxy-functionalized particles were taken up to a higher extent. the uptake of both nanoparticles was drastically reduced in media containing serum. using pharmacological and antisense in vitro knockdown approaches, we showed that the specific interaction between cd64 receptors and the particles determines the macrophage uptake of particles by phagocytosis, whereas particle internalization by thp-1 cells occurred via dynamin iidependent endocytosis. by contrast, pma-differentiated thp-1 cells took up the particles via macropinocytosis. in line with the in vitro data, more intravenously applied ps-cooh particles accumulated in liver tissue, whereas ps-nh 2 were preferentially targeted to tumor tissue. these data show that the amount of particle internalization, the uptake mechanisms, and kinetics differ significantly among primary cells and model tumor cells, whether differentiated or not, and that they are further critically dependent on the particle opsonisation by serum proteins. this work was supported by the dfg spp1313. specifically designed and functionalized nanoparticles hold great promise for a variety of biomedical applications. to ensure their safe application, such particles require a rigorous analysis of their effects on cell functions. here we demonstrate that aminofunctionalized polystyrene nanoparticles (ps-nh2) of ~100 nm in diameter in contrast to carboxy-(ps-cooh) and nonfunctionalized (ps) particles induce an nlrp3 inflammasome activation and the subsequent release of il-1β in human macrophages. amino-functionalized ps nanoparticles induced time-dependent lysosomal destabilization followed by release of lysosomal enzymes. this resulted in mitochondrial damage and formation of reactive oxygen species. accumulation of mitochondrial reactive oxygen species was accompanied by oxidation of thioredoxin, a protein playing a central role in maintaining the cellular redox balance. upon oxidation, thioredoxin dissociated from the thioredoxin-interacting protein (txnip). liberated txnip, in turn, interacted with the nlrp3 protein resulting in a conformational change of the pyrin domain of the nlrp3 protein as predicted by molecular modeling. txnip interaction with nlrp3 led to assembly of the nlrp3 inflammasome complex, to caspase-1 activation, and release of il-1β. using an in vitro knockdown approach, we showed that ps-nh 2 induced activation exclusively of nlrp3, whereas other inflammasomes remained unaffected. treatment of macrophages with n-acetyl-l-cysteine, a scavenger of reactive oxygen species, abolished both, the caspase-1 activation and the subsequent release of il-1β caused by ps-nh2 nanoparticles. these data reveal a novel mechanism of the nlrp3 activation induced by amino-functionalized nanoparticles and provide a strategy as to how such an effect can be functionally antagonized by supplementation with a radical scavenger. this work was supported by the dfg spp1313. the semi-permeable barrier of the endothelial cell lining of the blood vessels has important synthetic and metabolic functions including transport of cells and biomolecules, regulation of vascular smooth muscle tone, and control of hemostasis. plasmin is a serine protease, which is generated from its zymogen plasminogen under physiological and pathological conditions. small amounts of plasmin are produced in the context of contact activation during inflammation. consistently, increased generation of plasmin has been reported during atherosclerosis. we have shown previously that plasmin, in addition to its role in fibrinolysis, could induce proinflammatory activation of various cells including monocytes, macrophages, and dendritic cells. therefore, we analyzed how plasmin might affect the functions of endothelial cells, which could be relevant during inflammation and atherosclerosis. using flow cytometry, western immunoblotting and fluorescent microscopy, we show that endothelial cells of different origin express the plasmin receptor complex composed of annexin a2 and s100a10. addition of plasmin to human umbilical vein endothelial cells (huvec) induced timeand concentration-dependent cytotoxic effects in the cells. in addition, within 30 min plasmin triggered a rapid and prolonged expression of free radical oxygen species (ros) in endothelial cells as analyzed by microscopy and fluorometry using the rossensitive dye carboxy-h 2dcfda. the ros production in endothelial cells was accompanied by cell detachment. fluorometric and western blot analysis of caspase 3 activation in the cells treated with plasmin showed that plasmin induced apoptotic cell death in endothelial cells, which was evident already several hours after exposure to plasmin. thus, plasmin might induce production of ros in endothelial cells, their detachment and apoptosis, events which might be relevant for the development of atherosclerosis. this work was supported by the dfg. sesquiterpene lactones (stl) comprise a large group of secondary plant metabolites that constitute the active principle of a number of traditional anti-inflammatory phytomedicines. specifically helenalin and parthenolide have recently gained considerable attention as lead compounds or putative therapeutics for the treatment of inflammation and possibly cancer. both compounds have been shown to interfere with the signal transduction through inhibition of the nuclear factor κb (nf-κb). whereas the inhibitory effects of the stl on nf-κb are undisputed, their molecular mechanism of action remains a matter of debate. surface plasmon resonance (spr) analysis allows label-free measurement of molecular interactions. yet, analysis of the interaction of immobilized recombinant proteins with small molecular ligands remains a technically challenging task. in the present study we used spr technology to investigate the molecular interaction of the stl helenalin with putative intracellular target proteins such as the nf-κb protein p65/rela, the catalytic subunits of the ikk complex, namely ikkα and ikkβ, and the intracellular antioxidant glutathione (gsh). at physiological ph 7.4, helenalin interacts with rela (k d = 4.8 µm), yet it failed to bind either ikkα or ikkβ. hence, when dna with nf-κb binding consensus sequence was immobilized on sensor chips, the binding of rela was inhibited by helenalin with an ic50 of 5.0 µm. moreover, we provided several lines of evidence that stl may modify rela on cysteine 38 by a michael-type addition. this interaction was confirmed by molecular docking that identified the best matching interaction between rela and helenalin with predicted hydrogen bonding interactions between helenalin and residues arg35, lys37, gly44 and ile118 of rela. consistent with our hypothesis that helenalin interacts with sulfhydryl groups at ph 8.0, helenalin was also able to interact with reduced, but not oxidized, glutathione with a kd of 24 µm, though no significant interaction was observed at ph 7.4. thus, we showed that the sesquiterpene lactone helenalin interacts with the nf-κb protein rela but not with ikkα or ikkβ. moreover, at physiological ph, helenalin does not interact with glutathione to any significant extent. direct interaction of helenalin with rela leading to inhibition of rela-dna binding and transactivation might present the molecular mechanisms underlying the anti-inflammatory effects of stls. although nanosized materials are quickly taken up by macrophages, our understanding of the involved processes is still rather limited. therefore, we analyzed the uptake of diagnostically used carboxydextran-coated iron oxide nanoparticles of two different sizes, superparamagnetic iron oxide nanoparticles of 60 nm (spio) and ultrasmall superparamagnetic iron oxide nanoparticles of 20 nm (uspio), by human macrophages. by pharmacological and in vitro knockdown approaches, the principal uptake mechanism of macrophages for both particles was identified as clathrin-mediated, scavenger receptor a-dependent endocytosis. further, we created a mathematical model of the nanoparticle uptake by macrophages that permitted determination of key parameters of endocytotic process, such as the uptake rate, the mean uptake time, the number of particles taken up by a cell, and the correlation between the number of internalized particles and their extracellular concentration. the model also provided information on the individual and collective wrapping time of the nanoparticles and described the relation between biophysical parameters such as cytoskeletal forces, membrane elasticity, and the uptake time. finally, we gained information on the minimal linear spacing between simultaneously acting neighboring endocytotic pits that contain single nanoparticles and govern the collective uptake process. the calculated parameters were further confirmed experimentally using spinning disc confocal microscopy. thus, the new model provides important insights into the biophysical processes involved in endocytosis of nanoparticles by human macrophages. this work was supported by the dfg spp1313. prostaglandins (pg) are hormones which are formed during inflammatory processes from arachidonic acid by cyclooxygenases and prostaglandin synthases [4] . in the subsequent metabolism, in which the five-membered ring is dehydrated, α,β-unsaturated carbonyl compounds are generated [2, 3] . these come along with mercapto groups of amino acids in a michael addition reaction associated with activation of cellular enzyme cascades [1] that potentially contribute to their possessed antiinflammatory, antineoplastic and antiviral effects [5] . however little is known so far about possible adverse health effects.we addressed the question whether selected cyclopentenone prostaglandins (cypg) exhibit potential mutagenic and genotoxic properties in the hamster lung fibroblast cell line v79. induction of dna damage was investigated by single cell gel electrophoresis assay (scge). the impact of cypg on cellular redox status was detected by total glutathione (tgsh) assay. the induction of micronuclei and apoptosis was determined by staining with 4',6-diamidino-2-phenylindole (dapi). furthermore the hypo-xanthine-guanine phosphoribosyltransferase (hprt) assay was used for mutagenicity testing. , followed by prostaglandin a2 (pga2), showed the most distinctive genotoxicity, i.e., induction of micronuclei, and apoptotic effects. furthermore, the 15dpgj2 and pga2 -induced significant decrease in the tgsh level in v79 may contribute to the observed increase in oxidative dna-damage. however, none of the tested cypg exhibited mutagenic properties in the hprt assay. in conclusion, a potential in vitro genotoxicity of cypg has been observed which may be involved in carcinogenesis associated with chronic inflammation. parabens and methylisothiazolinone are used as preservatives in personal care products. sensitization to parabens and methylisothiazolinone is relatively rare considering their wide use in cosmetics, but only few quantitative or clinical data exist. therefore, we have tested methyl-, ethyl-, propyl-, isopropyl-, butyl-, isobutyl-, pentyl-, phenyl-and benzylparaben , and methylisothiazolinone in the loose-fit coculture-based sensitization assay (lcsa) developed by our working group. the coculture of primary human keratinocytes and allogenic dendritic cell-related cells (dc-rc) in this assay emulates the in vivo situation of the human skin. sensitization potential of the test substances was determined by flow cytometric analysis of the dc-rc maturation marker cd86. determination of the concentration required to cause a half-maximal increase in cd86-expression (ec 50) allowed a quantitative evaluation. the irritative potential of the substances was assessed by 7-aad (7-amino-actinomycin d)-staining. the concentration required to devitalize 50 % of the examined cells compared to a zero control was termed ec50%. parabens exhibited weak (methyl-, ethyl-, propyl-and isopropylparaben) or strong (butyl-, isobutyl-, pentyl-and benzylparaben) sensitizing potential, phenylparaben was found to be a moderate sensitizer, with ec50-values ranging from 16.67 µmol/l (pentylparaben) to 325.36 µmol/l (methylparaben). due to a pronounced cytotoxicity (ec50% = 70.94 µmol/l), we could not estimate an ec50-value for methylisothiazolinone. sensitization potential of parabens correlated with side chain length. parabens showed no (methyl-and ethylparaben) or weak irritative potential (propyl-, isopropyl-, butyl-, isobutyl-, phenyl-and benzylparaben) , only pentylparaben was rated to be irritative. apart from phenyl-and benzylparaben, irritative potential also correlated with side chain length but did not correlate strictly with the sensitization potential. overall, we were able to demonstrate and compare the sensitizing potential of parabens in this in vitro test. it was weak for methyl-and propylparaben, the most commonly used parabens. furthermore, we showed an irritative potential for most of the perservatives. thus the lcsa is a useful in vitro test to compare the sensitizing potential of xenobiotics. phosphorylation of the neurodegeneration-related septin 4 by protein kinase dyrk1a at serine 107 affects protein stability soppa u. 1 septins are gtp-binding proteins forming heterooligomeric complexes and filaments by interactions of the 13 family members. these complexes have important functions by building scaffolds for proteins involved in cell cycle or cell polarity but their subcellular distribution as well as their regulation remain largely unclear. septin 4 (sept4) was found in neurodegeneration related protein aggregates and is associated with migration of cortical neurons. here we first describe a potential mechanism for regulation of sept4 by phosphorylation via dual specificity tyrosine-phosphorylation-regulated kinase 1a (dyrk1a). dyrk1a is overexpressed in down syndrome and supposed to be involved in neurodevelopment and neurodegeneration. by site directed mutagenesis of flag tagged mouse sept4 and overexpression in hela cells we identified serine 107 as the major phosphorylation site of dyrk1a and generated a phosphospecific antibody. transient coexpression of sept4 and dyrk1a in hela cells increased phosphorylation of serine 107 by 50% in relation to basal phosphorylation. in contrast, cotransfection of the kinase deficient dyrk1a mutants k188r and d287n did not increase serine 107 phosphorylation. moreover we could show, that inhibition of kinase activity by the dyrk1a inhibitor harmine reduced phosphorylation of exogenous sept4 at serine 107 about 25% in hela cells. furthermore, down regulation of dyrk1a by rna interference lead to decreased phosphorylated serine 107. these results indicate that endogenous dyrk1a contributes to sept4 phosphorylation in hela cells. finally we analyzed protein stability of wild type sept4 compared to the phosphorylation resistant s107a mutant in hela cells by inhibition of translation with cycloheximide. we found that in living cells the sept4 s107a mutant is more stable than wild type sept4. in summary, our results suggest phosphorylation at serine 107 by dyrk1a as a novel mechanism to regulate sept4 stability and indicate a possible link of these proteins in cellular processes. is formaldehyde a good example for a "genotoxic carcinogen" with a threshold mode of action? speit g. institut für humangenetik, universität ulm, 89069 ulm, germany formaldehyde (fa) induces toxic and genotoxic effects in directly exposed cells (site of contact). several studies in which fa was administered to rats by inhalation showed evidence of tumor induction in nasal epithelium. there is also some epidemiological evidence that fa causes nasopharyngeal cancer in humans. although fa is a known mutagen, it is still a matter of discussion whether carcinogenesis is primarily mediated via a mutagenic mode of action. there is evidence that cytotoxicity and induced proliferation are the main causes for tumor formation. however, a decisive role of mutagenesis cannot be excluded and a mutagenic mode of action has to be considered for risk estimation. the basic assumption is that mutagens have a non-threshold mode of action. a threshold mode of action for a chemical is likely when a substance with a known mutagenic potential does not induce mutations at low concentrations due to a specific type of reaction with the genetic material and / or physiological protective mechanisms. because fa is a directly acting dna-reactive substance, a threshold mode of action may only be considered because of physiological protective mechanisms. after inhalation, pre-lesion protection occurs by unspecific binding to mucus, cellular proteins and glutathione. furthermore, fa is efficiently inactivated by enzymatic pathways. if fa reaches and damages the nuclear dna, dna repair mechanisms act as efficient postlesion protection mechanisms. in vivo inhalation studies with rats indicated that fa induces primary dna damage in the nasal epithelium but increased mutation frequencies were not measured. data are now available to show the relative distribution of endogenous versus exogenous dna adducts in different locations of the nose and other organs. considering the fa concentrations present in every living cell and the background levels of endogenous dna adducts, appropriate risk assessment and the identification of practical thresholds for fa-induced genotoxicity become feasible. fraud and misconduct in clinical trials steffen c. formerly federal institute for drugs and medical devices clinical trials unit, kurt-georg-kiesinger-allee 3, 53175 bonn, germany plagiarism in scientific publications has been the subject of public ("guttenplag") and scientific debate. plagiarism violates, however, "only" the intellectual property of its authors. in clinical trials, misconduct may also endager the health of patients. the suppression or falsification of data in clinical trials may mislead patients and doctors to use worthless treatments. clinicians may be provoked to repeat these trials, thereby wasting time and money. in clinical trials, misconduct includes everything from suppression or their repeated publication, the "correction" of unwanted results to the complete invention of the data, their intentional or negligent misinterpretation leading to the publication of biased conclusions from otherwise correctly performed clinical studies. the peer review system cannot protect against fraud, as few reviewers will have the means and the time to reevaluate original data. they will have to rely on these data, the calculations and statistics that are presented to them. some kinds of fraudulent behavior, such as double publications, can be found in the review process, but this is also time-consuming and depends on a helpful librarian. other kind of fraud, as the suppression of patient data in clinical trials (the deletion of "non-responders", according to cinderella: the good ones go into the pot, the bad ones go into your crop) can only be detected by the national competent authorities performing an inspection according to good clinical practice. even if the results of such an inspection become public as in the case of ukrain (gansauge et al. 2002) , there is no institution that will further analyze and publish the misconduct or initiate the retraction of the incriminated paper. a german agency similar to the us office of scientific integrity could foster good clinical practice in germany. although it is sometimes very difficult to decide whether improper results arise from fraud or error, a bias for the source of funding is obvious. trials funded by for-profit organizations were significantly more likely to recommend the experimental drug as treatment of choice (als-nielsen et al. 2003) . medicinal products with disputed efficacy such as orally applied enzymes for systemic action, bacterial preparations for irritable bowel syndrome and food supplements are to be reviewed with special attention. further examples from the author's experience will be presented. ]i favors further kca opening, kca may establish a feed-forward regulation of ca 2+ influx. in the present study we analyzed whether kca channels of sk4 and bk type play a role in sustaining ca 2+ oscillations at g1-to s-phase transition of primary mouse tumor cells and in human breast cancer cells, aiming towards a better understanding how kca modulate tumor cell proliferation. methods: kca expression was quantified by qpcr in human breast cancer biopsies, transgenic mmtv/c-neu + mouse mammary tumors and primary tumor cells derived thereof. the identity of the tumor cells was verified by gliolan staining. proliferation of the primary mammary tumor cells in the presence or absence of bk and sk4 modulators was tested using a real time cell monitoring system. the cellular dna content as a measure for cell ploidity was determined by propidium iodide staining and flow cytometry. changes in [ca 2+ ]i oscillations and peak amplitude were determined using ca 2+ indicator fura-2am. results: sk4, but not bk, expression is detectable in human and mouse breast cancer biopsies and in primary tumor cells derived from the mmtv/c-neu + mouse model. sk4 inhibition by tram-34 dose-dependently (0,1 to 10 µm) inhibits the growth of primary mammary tumor cells probably by a g1 cell cycle arrest. ca 2+ oscillations in proliferating mmtv/c-neu + tumor cells were ablated upon pharmacologic inhibition of sk4 channels. -dependent cell cycle progression is dependent on sk4 activity. blocking sk4 disrupts a feed-forward loop that coordinates ca 2+ influx via trp or crac channels in tumor cells. the consequences of sk4 inhibition in mammary tumors in vivo will be discussed. synthesis of a triphenylphosphonium substituted derivative of 5-hydroxymethyl-5-methylpyrroline n-oxide stolze k. 1 esr combined with spin trapping is a well-known analytical approach to detect free radicals formed in various biological systems, e.g. superoxide, hydroxyl and a series of carbon-centered free radicals, which are involved in oxidative stress. our aim was to modify the established spin trap 5,5-dimethyl-pyrroline n-oxide (dmpo) with a functional side chain, which can be used further as anchor for moieties enabling the spin trap to penetrate mitochondrial membranes, such as the positively charged triphenylphosphonium substituent. several synthetic routes were tested to introduce a 4-carboxybutyltriphenylphosphonium-substituent to the spin trap 5-hydroxymethyl-5-methylpyrroline noxide (hmmpo). while the activation of the carboxy group via the corresponding chloride was not successful, the use of a mixed anhydride with acetic acid appeared to be a promising way, although the reaction is considerably slower. preliminary spin trapping experiments have been performed with model systems generating superoxide, hydroxyl-, and carbon-centered radicals. othman e. m., stopper h. universität würzburg toxikologie, versbacher str. 9, 97078 würzburg, germany type 2 diabetes mellitus (dm2) is a growing health problem affecting more than 150 million people worldwide. it is associated with severe acute and chronic complications that negatively influence both the quality of life and survival of affected individuals. epidemiological studies clearly indicate that the risk of several types of cancer (including pancreas, liver, breast, colorectal, urinary tract and female reproductive organs) is increased in diabetic patients. diabetic patients are exposed to oxidative stress which plays a pivotal role in the pathogenesis of both micro-and macro-vascular complications. this is due to a decreased antioxidant capacity and chronic exposure to increased levels of reactive oxygen species (ros). since the insulin resistance in dm2 leads to hyperinsulinemia we studied the cellular consequences of the elevated insulin level and showed that it generates superoxide anions (o 2-) and dna damage by a nadph oxidase dependent mechanism in cultured cells. in addition, we found elevated genomic damage in the lymphocytes of diabetic patients as well as oxidative stress and genomic damage in kidneys of diabetic rats. this effect of insulin may contribute to the pathogenesis and progression of dm2 complications including the elevated cancer risk. the classical transient receptor potential (trpc) channel subfamily is regarded as nonselective, calcium permeable cation channels involved in a wide range of physiological events that require calcium signaling. until now, the specific roles of trpc channels in neuronal function are still elusive. given that trpc1 is able to form receptor-operated heterotetrameric channel complexes with other trpc channel subunits, we investigated the role of trpc1 for receptor-operated calcium influx in the heterologous expression system as well as in neurons. for this electrophysiological whole-cell measurements, fluorimetric calcium measurements, mn 2+ quenching and qpcr analysis were applied. furthermore, the effect of trpc1 knock-down on neuronal migration was monitored performing scratch assays, videomicroscopy and g-actin/f-actin assays. employing these techniques, we found that recombinant trpc1 was not able to function as a homomeric channel. instead, trpc1 subunits formed functional receptor-operated heteromeric channel complexes with trpc3, 4, 5, 6, and 7. heteromers containing trpc1 subunits showed significantly decreased calcium permeation in heterologous cell systems. mutation of amino acids in the putative pore forming region of trpc1 further reduced calcium permeability. in gnrh neurons endogenously expressing trpc1, 2, 5, and 6, downregulation of trpc1 by shrna resulted in increased basal cytosolic calcium concentrations and elevated calcium permeability. trpc1 was not involved in store-operated cation influx in gnrh neurons. moreover, trpc1 suppressed the migration of gnrh neurons without affecting cell proliferation. these findings suggest a novel regulatory mechanism relying on the expression of trpc1 and the subsequent formation of heteromeric trpc channel complexes with reduced calcium permeability, thereby fine-tuning neuronal migration. the transient receptor potential melastatin-3 (trpm3) is a calcium permeable nonselective cation channel that can be activated by the neurosteroid pregnenolonesulfate (pregs) or heat. trpm3 is expressed in various tissues, including insulin-secreting βcells and a subset of sensory neurons from dorsal root (drg) and trigeminal ganglia. the ability of pregs to evoke trpm3-like currents in pancreatic β-cells and to induce insulin secretion indicated its involvement in blood glucose regulation. however, trpm3 -/mice show so far no metabolic deficits but further investigations are recommended to evaluate its function in insulin secretion. further studies showed that trpm3 is a nociceptor channel involved in sensing heat and inflammatory thermal hyperalgesia. we performed a calcium-based screening of a compound library (spectrum collection) that identified several natural compounds as trpm3 blockers. the most potent blockers were the citrus fruit flavonoids hesperetin and naringenin as well as ononetin, a chalcon from ononis spinosa. the ic50 values of the substances are in the low micromoles ranges. electrophysiological whole cell measurements as well as calcium measurements confirmed the potency of the trpm3 blockers. furthermore, we could show that these blockers are effective on endogenous trpm3 in drg neurons from mice and isolated β-cells. by drinking grapefruit juice naringenin could be consumed in concentrations that are sufficiently high enough to block trpm3 activity in vivo. in sensory neurons, trpm3 may exert similar functions as trpv1. thus, trpm3 blocker could bear a therapeutic potential for analgesic treatment. xtt-based cell viability assay was used to determine the half-maximum effect concentration (ec50) for the investigated composite components in hgf. following concentrations of substances were used to determine the induced double strand dna breaks (dsbs): 1 /25× ec50, 1 /10× ec50 , 1 /3× ec50, and 1× ec50. each experiment was performed at least four times. hgf were incubated with various concentrations of substances for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative γh2ax analysis foci in cell nucleus were counted by eye down using a fluorescence microscope. each experiment was performed at least four times. in the xtt test following ec50 values of substances were found (mmol/l;mean +/-sem): tmp(eo)9ta a, b 0.087 ± 0.011; 1,6-hddma b, c 4.500 ± 0.700; etma a, c ; 12.000 ± 1.100; a significantly (p < 0.05) differently to 1,6-hddma, b significantly (p < 0.05) differently to etma, c significantly (p < 0.05) differently to tmp(eo)9ta. after six hours of exposure with tmp(eo)9ta at 0.00348 mm there were induced 0.55 γ-h2ax foci-formations in hgfs, at 0.00870 mm 0.67 foci, at 0.02900 mm 0.86 foci and at 0.08700 mm 0.97 foci. after exposure with 1,6-hddma at 0.180 mm there were induced 0.45 γ-h2ax foci, at 0.450 mm 0.64 foci, at 1.500 mm 0.94 foci and at 4.500 mm 1.32 foci. after exposure with etma at 0.48 mm there were induced 0.43 γ-h2ax foci, at 1.2 mm 0.50 foci, at 4.0 mm 0.61 foci and at 12 mm 0.71 foci. the negative controls dmso and medium cultures displayed 0.31 -0.34 γ-h2ax foci/cell. it was found that the induction of foci/cell were concentration-dependet for all xenobiotics in the order of: 1,6-hddma > tmp(eo)9ta > etma. these results show that dental composite components can induce dsbs in primary oral cells and therefore these substances demonstrate a genotoxic potential. effects of antioxidants on the dna-toxicity of dental (co)monomers in human gingival fibroblasts styllou p. 1 , scherthan h. unreacted (co)monomers can be released from restorative dental materials and may show biologic activity after ingestion in the human organism. in previous studies the mutagenic/carcinogenic effect of dental monomers/co-monomers (e.g. methacrylates) on the human dna was demonstrated. in this study the effects of the antioxidants vitamin c and n-acetylcysteine on the dna toxicity of the (co)monomers triethylenglycol-dimethacrylate (tegdma) and 2-hydroxyethyl methacrylate (hema) was investigated. the induction of dna double-strand breaks with (co)monomers alone and in combination with antioxidants was investigated in human gingival fibroblasts (hgf). hgf were incubated with substances without or with antioxidants for a period of 6 hours. induced dna double-strand breaks (dsbs) were tested by the γh2ax focus assay, which is a direct marker for dsbs using anti γh2ax antibodies. for quantitative analysis of the γ-h2ax test, foci were counted by the same investigator by eye down the fluorescence microscope. each experiment was performed at least four times. the halfmaximum effect concentration ec 50 (mmol/l) of triethylenglykol dimethacrylat (tegdma) and 2-hydroxyethyl methacrylat (hema) was taken from of a previous study after using xtt-based cell viability assay. tegdma induced significantly (p < 0.05) higher dsbs compared to hema (1.91 ± 0.04 vs 1.66 ± 0.02). the mean number of cells scored and the standard deviation (sd) were calculated. when cells were exposed to tegdma in combination with the antioxidant vitamin c an increase of dsbs was observed (2.02 ± 0.06), compared to tegdma alone. when cells were exposed to hema in combination with vitamin c an increase of dsbs was observed (1.89 ± 0.07), compared to hema alone. when cells were exposed to tegdma in combination with the antioxidant nacetylcysteine a decrease of dsbs was observed (1.64 ± 0.04), compared to tegdma alone. when cells were exposed to hema in combination with n-acetylcysteine a decrease of dsbs was observed (0.76 ± 0.02), compared to hema alone. these results show that dental (co)monomers can induce dsbs in primary oral cells. it also shows for the first time that the genotoxic potential may be reduced by the addition of the antioxidant n-acetylcysteine. purpose: we aimed to investigate the role of superoxide and peroxynitrite generated by genetically destabilized enos for the development of endothelial dysfunction and vascular remodelling. methods: a mutant of bovine enos in which cys 101 was replaced by ala (c101a) resulting in destabilization of enos has been generated (enos-c101a). transgenic mice carrying c101a were generated on a c57bl/6 background using the endotheliumspecific tie-2 promoter. by breeding these mice with enos knockouts (enos-ko), mice that express enos-c101a (enos-ko/enos-c101a-tg) exclusively in the endothelium were obtained. unilateral common carotid artery ligation experiments were performed in c57bl/6, enos-ko, and enos-ko/ enos-c101a-tg to study a role of destabilized enos for vascular lesion formation. results: western blot analysis confirmed the expression of enos in enos-ko/enos-c101a-tg in aorta (37.1±8.4%, n=9), skeletal muscle (45.4±5.3%, n=10) and myocardium (17.4±4.9%, n=7) and revealed an increased phosphorylation of enos on ser1176/79 (470±47%) as compared to c57bl/6 (p<0.05, n=8). endothelium-specific overexpression of destabilized enos induced a large increase in superoxide and peroxynitrite formation in the aorta and the heart of enos-ko/enos-c101a-tg (p<0.05, n=5-8), which was abolished by nos-inhibitor l-nitroarginine (l-na) suggesting enos-c101a as a source of elevated radical generation. endothelium-specific introduction of enos-c101a at ~ 35% of c57bl/6 level almost completely restored aortic endotheliumdependent relaxation. experiments with l-na, soluble guanylyl cyclase inhibitor odq, peg-catalase and no-scavenger fe(detc)2 indicated that endothelium-dependent relaxation in enos-ko/enos-c101a-tg is nos-and cgmp-dependent and nomediated. four weeks after the carotid artery ligation, neointima formation, media thickening and luminal narrowing were observed in the ligated arteries of all studied genotypes (p<0.05, n=4-7). consistent with vasoprotective roles of enos, neointima formation was accelerated in enos-ko (n=4-7, p<0.05). despite significantly higher vascular levels of nitrotyrosine and peroxynitrite, neointima formation in enos-ko/enos-c101a-tg was substantially lower then in enos-ko and tended to be similar to c57bl/6. conclusions: increased vascular superoxide and peroxynitrite formation caused by destabilization of enos does not induce endothelial dysfunction in healthy mice and has negligible effect on neointima formation. fenton reactivity as a determining parameter for the interaction of manganese oxide nanoparticles with lung epithelial cells sydlik u. 1 , bieschke c. nanoparticles consisting of manganese oxide have been suggested for several innovative technological approaches, including the use in nanomedicine and diagnostics. therefore, the interaction of such nanoparticles with human target cells is of particular interest for the success of nanomedical approaches but also with regard to unintended side effects. to address this problem, we tested different kinds of manganese nanoparticles (mnnp) in an in vitro system which we earlier evaluated for proliferative, apoptotic, and pro-inflammatory endpoints induced by carbon nanoparticles (cnp). mnnp were synthesized by hydrothermal treatment of manganese salt solutions. the particles were subsequently characterized by scanning electron microscopy and dynamic light scattering. biological and toxic effects of the generated particles were studied in comparison to carbon nanoparticles (cnp) in experiments with rat and human lung epithelial cells (rle-6tn and 16hbe14o-). cytotoxicity was determined as measures of membrane damage (lactate dehydrogenase release) and metabolic activity (water soluble tetrazolium conversion). the oxidative capacity of the particles as well as the generation of intracellular oxidative stress was monitored using dichlorofluorescein diacetate in cell free experiments and flow cytometry assays (facs), respectively. the particle-specific phosphorylation of src family kinases (sfk) and mitogen activated protein kinases erk1/2 were investigated using western blot techniques. after physico-chemical characterization, a set of three mnnp consisting of mn3o4 or mno2 with significant differences in size and shape were selected. according to the different oxidation stages of manganese, the particles showed significant differences in fenton reactivity in the cell free system. these data did not reflect the capacity of the particles to induce intracellular oxidative stress. the characteristic to trigger membranedependent signaling processes, however, was correlated to the intrinsic oxidative capacity of mnnp than to the ability to induce intracellular ros. furthermore, the metabolic activity (wst) was negatively correlated with intracellular ros, indicating a link between mitochondrial activity and ros generation. none of the particles had effects on the membrane integrity of the cells. the data demonstrate that mnnp, unlike other poorly soluble nanoparticles (e.g. cnp), mainly trigger adverse health effects through ros production via the fenton reaction. acute ozone induced airway inflammation does not effect resting human sympathetic nerve traffic tank j. 1 numerous mediators released in inflammatory and neuropathic pain states activate gprotein-coupled receptors (gpcrs) and modulate nociception via activation of gs, gi/o, g12/13, or gq/11 g proteins. each of the g protein-coupled receptor pathways is involved in nociceptive modulation and pain processing, but the relative contribution of the individual signaling pathways in vivo has not yet been worked out. the gq/11 signaling branch is of particular interest in pain research because it leads to the activation of phospholipase c, protein kinase c, and the release of calcium from intracellular stores. using a conditional gene-targeting approach we generated double-deficient mice lacking gaq and ga11 selectively in nociceptors to investigate the contribution of the entire gq/11signaling pathway in nociceptors towards the regulation of pain. we observed that mice lacking gq/11 in nociceptive neurons show normal development of the nociceptive circuitry. the nociceptor-specific loss of gq/11 results in reduced pain hypersensitivity following paw inflammation or spared nerve injury. surprisingly, our behavioral and electrophysiological experiments also indicated defects in basal mechanical sensitivity in gq/11 deficient mice, suggesting a novel function for gq/11 in tonic modulation of acute nociception. patch-clamp recordings revealed changes in voltagedependent tetrodotoxin-resistant and tetrodotoxin-sensitive sodium channels in nociceptors upon a loss of gq/11, whereas potassium currents remained unchanged. our results indicate that the functional role of the gq/11 branch of g-protein signaling in nociceptors in vivo not only spans sensitization mechanisms in pathological pain states, but is also operational in tonic modulation of basal nociception and acute pain. provocation of arrhythmic events in single primary isolated adult mouse ventricular cardiomyocytes tekook m., fehrmann e., schulte j. s., schmitz w., müller f. u. westfälische wilhelms-universität institut für pharmakologie und toxikologie, domagkstraße 12, 48149 münster, germany ap duration and ca 2+ cycling are altered in cardiomyocytes of different genetic mouse models. here, we systematically tested various protocols to study the inducibility of arrhythmic events in mouse cardiomyocytes. adult ventricular cardiomyocytes were isolated from wildtype (wt) mice by enzymatic digestion and subsequently tested to trigger arrhythmic events within 6 hours after isolation. in patch clamp experiments (perforated patch, whole cell current clamp) aps were stimulated for 1 second (5hz; 10hz; 5hz + s2-stimulus after 50-80ms) followed by a 4s rest period. the resting-membrane-potential (rmp) was observed over 30 cycles. we observed rmp-fluctuations of different length (amplitude <5 mv; % of 13 observed cardiomyocytes, mean events/cell; <1s: 100%, 17.1; <3s: 92%, 11.5; >3s: 69%, 5.8), spontaneous depolarizations (>5 mv; 92%, 5.1) and spontaneous aps (62%, 1.9). intracellular ca 2+ transients and sarcomere shortening were measured after loading cardiomyocytes with indo-1/am. after preconditioning (10 min/1 hz) cells were measured under basal and continuous isoprenaline (10 -6 m) stimulation (iso). a 4 min pacing period was followed by a 1 min interval of no pacing. pacing frequency was reduced after each cycle (1 hz, 0.5 hz, 0.25 arrhythmic events were provocable with stimulation/rest protocols both by field stimulation and direct stimulation via patch pipette. however, low stimulation frequencies seem to lead to distinct destabilization of cardiomyocytes probably due to ca 2+ overload. we conclude that the tested stimulation protocols are able to provoke arrhythmic events even in wt single adult mouse ventricular cardiomyocytes and may serve as a tool to test for the relevance of potential proarrhythmic substrates in mouse models. ruhr-universität bochum pharmakologie ma nord1, bochum, germany cgmp is a second messenger involved in many (patho-)physiological processes such as smooth muscle relaxation, platelet inhibition, and the development and plasticity of the nervous system. however, it is not fully understood how cgmp regulates these and other processes on a mechanistic level. in particular, the existence and functional relevance of global and local cgmp signaling domains is not clear. recently, highly specific genetically-encoded optical biosensors for cgmp have been developed. these cgmp indicators are either based on fluorescence resonance energy transfer (fret), with cgmp-binding domains sandwiched between fluorescent proteins with overlapping spectra, or they consist of a single fluorescent protein fused to cgmp-binding domains. with these cgmp indicators, the spatiotemporal dynamics of cgmp signals, which result from the interplay between cgmp-producing guanylyl cyclases, cgmp-binding effectors, and cgmp-degrading phosphodiesterases (pdes), can be monitored in living cells. here, we report the generation of transgenic mice expressing the fret-based cgmp indicators cgi500 and cgi6000 with apparent cgmp affinities of 500 nm and 6000 nm, respectively. one mouse line expresses cgi6000 driven by a cmv promoter in neural cells. fret experiments were performed with isolated cerebellar granule neurons, hippocampal neurons, and astrocytes. we observed nitric oxide (no)-induced cgmp transients and analyzed the capability of other agents (natriuretic peptides, glutamate) to induce cgmp responses. in another mouse line, the sm22alpha promoter directs cgi500 expression specifically to smooth muscle cells (smcs). fret experiments have been performed with smcs isolated from aorta, bladder and colon, as well as with intact vessels in the retina and cremaster muscle of transgenic animals. in primary smcs we studied responses to no, atrial and c-type natriuretic peptide (anp,cnp). in different smc types we observed differences in the overall ability to react to these stimuli and in the kinetics of the induced cgmp transients. we also studied the effects of pde inhibitors on the no-, anp-, and cnp-induced cgmp signals. importantly, we were able to detect cgmp transients upon no stimulation in intact vessels of the retina and cremaster. we conclude that the cgi transgenic mouse lines are valuable tools to visualize cgmp signals in living cells in vitro and, possibly, also in vivo in the intact animal under physiological and pathophysiological conditions. current research data dealing with pharmacotherapy of α-ama intoxication shows a particularly high variability regarding the protective effect of silibinin. the aim of this study was therefore to evaluate the influence of the frequently used clinical antidotes benzylpenicillin, silibinin and their combination in human hepatocyte culture intoxicated with α-ama. cytotoxicity and apoptosis testing were performed after two and five days of simultaneously exposure to α-ama and/ or tested antidotes. to quantify apoptosis, necrosis and cell viability, we used cell death detection elisa plus®, toxilight® bioluminescence assay and cell proliferation kit ii (xtt). furthermore, we analysed the ways of apoptosis by using immunohistochemistry (differential detection of caspase 3, 8 and 9, activated caspase 3, and aif). exposure of hepatocytes to α-ama at concentrations of 0,2 µm, 0,5 µm and 1 µm resulted in disorder of cell cultures, apoptosis and reduction in cell viability compared with unexposed hepatocytes. in hepatocyte cultures treated with benzylpenicillin at concentrations of 30 µm and 1mm, silibinin at 50 µm and 100 µm and a combination of both (30 µm benzylpenicillin and 50 µm silibinin, 1mm benzylpenicillin and 100 µm silibinin), toxilight® values in the supernatant and xtt values were not significantly different from untreated cultures. simultaneous exposure to α-ama (at all tested concentrations) and benzylpenicillin, silibinin or combination of both showed higher cell viability and lower values of necrosis compared to the cultures exposed to α-ama alone (exept 50 µm silibinin at 0,2 µm α-ama); however, in both groups dosed with benzylpenicillin the highest hepatocyte viabilitiy was observed. this protective effect was particularly revealed at high α-ama concentrations (0,5 µm and 1 µm). in conclusion, our data suggest that benzylpenicillin in monotherapy is more effective than in combination with silibinin or silibinin alone. glucocorticoids (gcs) are important hormones in the regulation of metabolic homeostasis. synthetic gcs, such as dexamethasone (dex), play a fundamental role in the treatment of inflammatory diseases. there are numerous side effects of a dex therapy, e.g. the development of hypertension. in the pathogenesis of hypertension oxidative stress is a crucial factor. glucocorticoid-induced hypertension has been shown to be associated with an imbalance between nitric oxide (no) and superoxide. however, the source of this elevated superoxide production is unknown. we hypothesize that an uncoupling of the no synthase (enos), a key mediator of vascular homeostasis, may contribute to dex-induced oxidative stress. incubation of human endothelial cells (ea.hy 926) with dexamethasone led to a decrease in enos expression at mrna and protein levels. this effect of dex was timeand concentration-dependent. since the major cause of enos uncoupling is a deficiency of its co-factor tetrahydrobiopterin (bh 4), we analyzed the amount of bh4 in ea.hy 926 by hplc. a concentration-dependent reduction of bh4 and also bh2 (dihydrobiopterin) could be demonstrated in response to treatment with dexamethasone. bh4 can be synthesized endogenously by two different pathways -the de novo pathway (from gtp with gtp cyclohydrolase i, gch1, acting as the rate-limiting enzyme) and the salvage pathway (conversion of sepiapterin to bh4 involving dihydrofolate reductase, dhfr). treatment of ea.hy 926 cells with dex decreased mrna and protein expression of both gch1 and dhfr. because bh4 is the major "coupling switch", an enos uncoupling is likely to occur in dex-treated cells. in summary, we showed that dex treatment led to a reduced availability of the important co-factor bh4 which could lead to enos uncoupling. the uncoupled enos may possibly contribute to glucocorticoid-induced vascular oxidative stress. the cellular oncoprotein c-fos is a major component of the heterodimeric transcription factor ap-1 and has been commonly found over-expressed in tumors and cancer cells of different origin. previous work showed that mouse cells lacking the immediate-early gene c-fos are hypersensitive to ultraviolet (uvc) light. here we demonstrate that in human telomerase-immortalized vh10tert foreskin fibroblasts (behaving like primary cells) and sv40-immortalized gm637 fibroblasts, uvc-triggered induction of c-fos protein is a delayed and long-lasting event. sustained up-regulation of c-fos went along with transcriptional stimulation of the nucleotide excision repair (ner) gene xpf, carrying an ap-1 binding site in the promoter. c-fos mrna was induced in a biphasic manner. an immediate c-fos mrna expression (30-90 min after exposure) was not translated into the protein, the second wave of transcription (4-24h after uvc exposure) resulted in c-fos protein expression, 18-48h post-uv. the stress-activated/mitogen-activated protein kinases (jnk, p38k and erks) were immediately induced upon uvc exposure and stayed active for at least 24h. inhibitor experiments revealed that c-fos was phosphorylated by erks and jnk. the activation of c-fos preceded re-synthesis and the induction of xpf mrna, which was observed 24-40h post-uvc, resulting in the increased expression of the xpf protein. cells over-expressing c-fos showed an accelerated induction of xpf mrna, and consequently a faster repair of cyclobutane pyrimidine dimers (cpds). sirna-mediated silencing of c-fos (transient c-fos knockdown) resulted in abrogated uvc-triggered induction of xpf, attenuated repair of cpds and increased apoptosis. finally, we observed that the removal of cpds but not of photoproducts was significantly faster when cells were pre-exposed to a low uvc dose, indicative of an adaptive response to dna damage. the work was financed by deutsche forschungsgemeinschaft (dfg ch 665/2-1). the addition of clopidogrel to aspirin reduces ischemic events in patients with acute coronary syndrome and in those undergoing percutaneous coronary intervention (pci). however, recurrent ischemic event occurrence during dual antiplatelet therapy remains a major concern. variability in the pharmacodynamic response to clopidogrel is well recognized, and patients with higher platelet reactivity while receiving clopidogrel are at increased risk of ischemic cardiovascular events. clopidogrel is an inactive prodrug requiring biotransformation to form the platelet inhibiting metabolite. interindividual differences in clopidogrel metabolism are the major source of variability in antiplatelet response. polymorphically expressed cytochrome p450 (cyp) enzymes play a critical role in the metabolism of clopidogrel. these findings gave rise to the concept of personalized antiplatelet therapy -i.e. individual platelet function testing and correction of insufficient platelet inhibition to reduce ischemic events in patients with high on-clopidogrel platelet reactivity (hcpr). gravitas was the first study to test this concept by comparing double-dose clopidogrel to standard-dose clopidogrel in patients with hcpr. gravitas failed to correct hcpr consistently in the study arm, which coupled with a low overall event rate precluded demonstrating a substantial benefit from improved platelet inhibition. the trigger-pci trial tested the effectiveness of the more potent thienopyridine prasugrel versus clopidogrel in patients with hcpr after elective pci with implantation of drug-eluting stents (des). switching from clopidogrel to prasugrel in patients with hcpr afforded effective platelet inhibition. however, given the low rate of adverse ischemic effects using contemporary des after pci in stable ischemic heart disease, the clinical utility of this strategy could not be demonstrated and the study was terminated prematurely for futility. multiple studies have shown that both heterozygotes and homozygotes for loss-offunction cyp2c19 alleles have higher rates of adverse cardiovascular events as compared with noncarriers on approved maintenance dosing of clopidogrel (75mg qd), albeit carriage of cyp2c19 loss-of-function alleles accounted for only a minor proportion of the variability in on-clopidogrel platelet reactivity. results of ongoing studies with antiplatelet treatment stratified by cyp2c19 genotyping are awaited to assess the clinical benefit of this approach. the organic cation transporter novel type 2 (octn2/slc22a5) represents a high affinity uptake system for carnitine. besides metabolic disease like severe system carnitine deficiency, genetic variants within the slc22a5 gene have been associated with inflammatory diseases like colitis ulcerosa. against this background, we characterized octn2 expression in peripheral blood cells thereby identifying its expression in all cell types. in the present work we studied octn2 expression in monocytes and thp-1 cells as an in vitro model for this cell type. in addition we examined transcriptional regulation of the carnitine transporter in lps activated thp-1 and investigated the effect of carnitine and its analog mildronate on the respective cytokine response. octn2 expression was characterized on monocytes and thp-1 cells on mrna and protein level. transporter mrna expression could be shown in both cell types by realtime pcr. however, the protein expression was analyzed by western blot, flow cytometry and immunofluorescence microscopy demonstrating octn2 specific signals as well as a localization in the plasma membrane. following thp-1 cells were activated using lps (10ng/ml) for up to 6h, thereby indicating the expected cytokine response as demonstrated by increased tnfα (24fold induction) and il-1β (37fold induction) mrna levels. in addition, octn2 expression was analyzed identifying an initial reduction of around 60% compared to untreated cells. in parallel activated thp-1 cells were coincubated with increasing concentrations of the octn2 substrate carnitine or its analog resulting in reduced cytokine release as shown by elisa for tnfα. here, the tnfα effect was diminished by 64% in the presence of 50mm carnitine. this effect does not rely on a direct neutralization of lps by carnitine since it was also present in cells only preincubated with carnitine. in the present work we could show that thp-1 cells represent a useful model to study octn2 expression and function. in addition, we demonstrate immunosuppressive effects of octn2 substrates like carnitine. further experiments will be necessary to identify the underlying mechanism of this observation. castor oil has been used for more than 3000 years for its laxative effects and also to induce labor in pregnant women. despite its wide-spread use, the mechanism of action remained unknown. the active metabolite of castor oil is ricinoleic acid which is released from castor oil by intestinal lipases. we have found that exposure of meg-01 cells to ricinoleic acid caused an increase in [ca 2+ ]i, an effect which was dose-dependent and abolished by pretreatment of cells with pertussis toxin, suggesting the involvement of a g-protein coupled receptor. to search for a putative receptor, we determined ricinoleic acid-induced [ca 2+ ]i increases in cells transfected with a sirna library directed against human gpcrs. in this way, we identified prostaglandin e2 receptors ep3 and ep4 as mediators of ricinoleic acid-induced effects. to test if ep3 and ep4 receptors mediate pharmacological effects of castor oil in vivo, we analyzed laxative effects induced by castor oil in wild-type (wt) mice, ep3-deficient (ep3 -/-) or ep4-deficient mice (ep4 -/-). while ep4 -/mice responded similarly to the wt mice, ep3 -/animals were totally insensitive to castor oil-induced laxation. moreover, mice lacking the ep3 receptor only in the smooth muscle cells did not respond to castor oil, in contrast to mice which lack ep3 receptor only in epithelial cells of the intestinal mucosa. similarly, ricinoleic acidinduced contractions of isolated ileal segments were absent in segments lacking ep3, consistent with a preferential expression of the ep3 receptor in the longitudinal muscle layer of the intestine. also, ricinoleic acid-induced contractions of isolated uteri were dependent on the expression of ep3 receptor in the myometrium. these findings identify the cellular and molecular mechanism underlying the effects of castor oil and indicate a role of the ep3 receptor as a pharmacological target to induce laxative effects. introduction: patients seek health information from various sources. they are facing the challenge to differentiate between reliable and untrustworthy sources and at the same time identify the best drug therapy for them. furthermore generalised health information confuses more than they benefit or rather unsettle. patients are not necessarily qualified to assess the evidence of statements properly. there is thus a need for providing competent drug information, which is offered by the independent drug information service at the institute of clinical pharmacology in dresden, germany. for the present descriptive evaluation we selected 5 drugs (arimidex ® , cipralex ® pentalong ® , onbrez ® and pradaxa ® ), that were affected by new referenceprice formation, generic registration, warnings or directions in 2011. in specified time frames we assessed the increase in and the cause of enquiries. deductively we draw conclusions for a perspicuous presentation of patient information. since generic registrations of the aromatase inhibitor arimidex ® enquiries on side effects of this drug were stable, but 17 additional consultations were held on generic changeovers. the antidepressant cipralex ® as well as the long-acting β-agonist onbrez ® were assigned to reference-price groups, which resulted in an 8-times (cipralex ® : 5 → 40) and 5-times (onbrez ® : 2 → 10), respectively, increase in enquiries. main aspect was to give background information on reference prices and point out therapeutic alternatives (cipralex ® : 34 of 40; onbrez ® : 10 of 10). an additional amount of 22 conversations were carried on the fictive registered drug pentalong ® after health insurance companies advised practitioners to avoid recourse by not prescribing this organic nitrate. notable insecurity was aroused by media reporting on lethal bleeding after taking pradaxa ® for anticoagulation. every tenth enquiry in the evaluation period was focussing on these instigative reports (21 of 227). patients are confronted by current changes, but often do not get enough background information from their health care providers to become acquainted with the tidings. health seekers may find eligible data from media coverage. however the individual assessment as well as the risk-benefit-relation may not be feasible for them. the drug information service for patients is a convenient helpline to reduce lack of knowledge and uncertainties and therefore support shared decision making. insulin effects on hyaluronan production -a possible link between diabetes and cancer? twarock s., fischer j. w. institut für pharmakologie und klinische pharmakologie, universitätsklinikum der heinrich-heine-universität düsseldorf, moorenstraße 5, 40225 düsseldorf, germany background: epidemiological studies have shown an elevated incidence of certain tumor entities in diabetes type 1 and type 2 patients. to reveal the underlying mechanisms we focused on the effects of increased glucose uptake in cancer cells with respect to matrix production. abundant production of hyaluronic acid (ha) in the vicinity of gastrointestinal cancer cells is a hallmark in tumor development. esophageal cancer is a rare but severe kind of gastrointestinal cancer which is differentiated in adenocarcinoma and squamous cell carcinoma (scc) of the esophagus. we studied the effects of increased glucose levels on ha production in an scc cell line (osc1). in starving and full media, elevated glucose concentrations increased the production of ha secreted to the medium in 24h as measured by an ha-binding protein linked assay (starved, 0g/l glucose: 100±4.7%; 1g/l: 185.7±44.8%; 4g/l: 362.6±58.3%; full medium 100±15.2%; 155.3±32.3%; 422.7±32.0%). surprisingly, total ha concentrations were about 2.2-2.8 fold higher under starved conditions. to investigate whether this effect might be due to insulin actions, starved cells were treated with 10 mg/l insulin for 24h. we observed a dosedependent decrease in ha production following insulin treatment (control vs insulin, 1g/l glucose: 100±6.5% vs 67.83±4.4%; 4g/l: 100±2.2% vs 71.8±6.6%). this finding might suggest that insulin directs glucose usage to the glycolytic pathway thereby diminishing ha synthesis. a premise to this assumption is the ability of osc1 cells for insulin independent glucose uptake. to verify this thesis, mrna expression levels of insulinindependent and insulin-dependent glucose transporters (glut1, glut4) were analyzed by qrt-pcr. the relative abundance was 24.32±3.49 in favor of glut1 indicating the presence of insulin-independent glucose transport. conclusion: in osc1 the absence of insulin actions caused increased ha production which might be due to diminished insulin driven glycolysis, thus leading to the use of early glucose metabolites for ha production instead of energy gain. this finding could be important in the context of diabetes type 2, where insulin actions are also diminished because of insulin resistance. since increased ha production is of critical importance for cancer growth and spread, the cellular shift in glucose usage from glucose catabolism to ha anabolism could therefore indicate a possible link between diabetes type 2 and cancer progression. schwarz m., unterberger e. universtität tübingen, institut für klinische und experimentelle pharmakologie und toxikologie abteilung toxikologie, wilhelmstraße 56, 72074 tübingen, germany chemical hepatocarcinogenesis is a multi-stage process triggered by an intitiating mutation in a gene encoding an important cell-regulatory protein. tumour initiation may be caused by genotoxic substances which directly interact with the dna, causing mutations. cells carrying permanent mutations experience clonal expansion which may be accelerated by exposure of the experimental animals to tumour promoters during the following step of tumour promotion. it has been shown that substances which constantly activate certain nuclear receptors act as tumour promoters in rodent liver, such as the model tumour promoter phenobarbital which, amongst others, activates constitutive androstane receptor (car). since these tumour promoters do not seem to directly interact with dna causing mutations they can be regarded as non-genotoxic carcinogens. however, the molecular mechanisms of non-genotoxic carcinogenesis are still widely unknown which also poses a major problem in preclinical drug-development. the aim of the marcar (biomarkers and molecular tumour classification for nongenotoxic carcinogenesis) project is to establish early biomarkers for non-genotoxic carcinogenesis by creating a comprehensive molecular profile of tumours generated by a regimen including model tumour promoters such as phenobarbital. the ultimate aim is to differentiate spontaneous liver tumours from tumours generated by non-genotoxic carcinogens. this molecular profile includes mutational analyses, immunostaining for known tumour-specific markers, phospho-proteome analyses, genome wide and promoter-specific dna methylation analyses, as well as mirna analyses. mutation analyses were carried out with mouse and rat tissue from phenobarbital promoted liver tumours to identify mutations which phenobarbital provides a growth advantage for. furthermore, real time pcr measurements show that the expression of a particular non-coding rna and mirna precursor is up-regulated in tissue isolated from phenobarbital promoted mouse liver tumours. additional in-situ-hybridisation experiments demonstrated the localisation of this transcript in ctnnb1-mutated tumours. larch-derived diterpenes are potent and selective trpc6 blockers urban n. 1 , kübler w. the transient receptor potential channel trpc6 is a poorly ca 2+ -selective cation channel that is activated by the membrane-resident second messenger diacylglycerol (dag). consistent with the major sites of trpc6 expression, its activation has been implicated in pulmonary and renal diseases, such as pulmonary hypertension, lung edema, chronic obstructive lung disease, allergic airway disease, and focal segmental glomerulosclerosis. amongst various plant extracts, conifer oils and resins are traditionally used to treat pulmonary ailments. therefore, we reasoned that they may contain constituents with a biological activity to modulate trpc6 activity. the true turpentines, oils and resins of various coniferous genera were tested with respect to a possible inhibition of dag-or receptor-induced activation of trpc6 and trpc3. indeed, turpentines and resins, but not coniphere oils blocked trpc6 and trpc3 in a concentration-dependent manner. interestingly, the larch-derived turpentine exerted a trpc6-prevalent inhibition. we identified larixol and its mono-and diacetates as the specific compounds that are contained in larch resin and give rise to a trpc6-selective block. larixol acetates displayed an ic50 towards the dag-or receptor-stimulated trpc6 activity of about 0.3-1 µm, but did not strongly inhibit a number of other trp channels, including trpv1, trpm2, trpm3, trpm8, or trpa1. selectivity for trpc6 compared to its closest relative, trpc3, was about 30-fold. unlike conipherous oils, which contain toxic pinenes, the resin constituent larixol ant its acetates exerted no significant cellular toxicity at concentrations that are required to block trpc6. electrophysiological analysis confirmed the highly potent block, which was voltageindependent and reversible. in a murine hypoxia-induced pulmonary vasoconstriction (hpv) model, larixol acetate abrogated the euler-liljestrand mechanism and, thus, mimicked the phenotype of trpc6 -/mice. we conclude that trpc6 blockers and, more specifically, larixol-related derivatives may provide novel therapeutic strategies to treat or prevent pulmonary diseases. dioxin is an environmental contaminant, believed to affect basic biological equilibria such as calcium and iron homeostasis. however, the molecular mechanisms underlying these effects are still largely unknown. this strongly hampers the estimation of the hazard to humans associated with dioxin exposure and necessitates further studies aimed at the clarification of these mechanisms. it has been suggested that nearly all biological and biochemical processes are mediated by protein complexes. the most commonly used technology for monitoring changes in the expression of complex protein mixtures is still 2d gel electrophoresis, but this method suffers from poor expression of low or moderately abundant proteins. blue native page and subcellular fractionation form an ideal partnership when it comes to enrichment and analysis of intracellular organelles and low abundant multiprotein complexes. the aim of the study is to identify and characterize multiprotein complexes by blue native page to elucidate the network of protein-protein interactions that regulate protein function after dioxin exposure. sample preparation and subcellular fractionation rt4 cells were cultured in mccoy's 5a medium. cells at confluence were harvested and fractionated into cytosolic, membrane/organelle and nuclear fraction by using the proteoextract subcellular proteome extraction kit. first dimension (bn-page) 50 mg of protein sample was mixed with 5% of coomassie blue g-250 (cbb g-250) and loaded in each lane of 4-15% polyacrylamide native gradient gels. the lanes from the first dimension were cut into individual strips and were placed into a 12% sds gel. the gels were stained with coomassie and the spots were picked up for mass spectrometry. bn/sds-page combined with ms led to the identification of proteins involved in the regulation of both calcium and iron homeostasis in dioxin-exposed cells. these results demonstrate for the first time that dioxin exposure simultaneously affects calcium and iron metabolism. since important iron and calcium requirement changes occur during the regulation of cell growth, the protein expression changes observed in our study may be associated with dioxin-dependent cell-fate decisions. the murine protease inhibitor serpina3n inhibits mechanical allodynia in a model of neuropathic pain vicuna l. 1, 2 , simonetti m. several chronic diseases are accompanied by strong, long-lasting pain. a majority of chronic pain diseases are not well understood yet and cannot be controlled by conventional analgesics or non-pharmacological approaches. therefore, there is a major need to develop novel therapeutic principles. using a genetic screen, we identified serpina3n, a serine protease inhibitor, which is homologous to human a1-antichymotrypsin, to be a determinant of low neuropathic pain. we found that serpina3n is expressed in the dorsal root ganglia (drg) and spinal cord and that it is upregulated in these tissues in mice developing neuropathic pain. importantly, we observed that spinal delivery of recombinant serpina3n inhibits mechanical allodynia in a mouse model of neuropathic pain. we identified a novel serine protease substrate for serpina3n, which is upregulated in the spinal cord in mice undergoing neuropathic pain ('enzyme e'). recombinant enzyme e delivered intrathecally to the spinal cord of mice elicited rapid and long-lasting allodynia, which was fully blocked by concomitant administration of serpina3n. our results suggest that serine protease-serpin signaling modulates spinal neuronal and glial cell networks involved in processing pain and that activity-induced spinal release of serpina3n constitutes an endogenous defence mechanism against establishing chronic pain hypersensitivity. these data have important implications for the pathophysiology of pathological pain and potentially hold therapeutic relevance. gβγ subunits are involved in β-adrenergic receptor induced cardiac hypertrophy vidal m., lohse m. j., lorenz k. institut für pharmakologie und toxikologie pharmakologie, versbacher str. 9, 97078 würzburg, germany introduction. activated β1-adrenergic receptors and their g protein gαs induce the development of cardiac hypertrophy. however, the hypertropic effects of direct activation of downstream effectors, such as adenylyl cyclase, camp or pka, are controversely discussed. recently, a hypertrophic pathway involving a g protein βγ subunit induced phosphorylation of the mitogenic kinases erk1/2 at threonine 188 (erk thr188phosphorylation) has been described to mediate erk-induced hypertrophy. this study aims to investigate whether erk thr188 -phosphorylation is involved in cardiac hypertrophy triggered by β-adrenergic receptors. -phosphorylation was detected in hek cells overexpressing β1-receptors, murine hearts and neonatal rat cardiomyocytes after isoprenaline treatment. we performed [ 3 h]-isoleucine incorporation assays to assess cardiomyocyte hypertrophy in vitro. neonatal rat cardiomyocytes (nrcms) overexpressing wild-type erk2 showed a significant increase in [ 3 h]-isoleucine incorporation after isoprenaline treatment. in contrast, nrcms transfected with erk thr188 -phosphorylation deficient mutants (erk2 t188a and erk2 t188s ) or pretreatment with the erk inhibitor, pd98059, significantly attenuated cardiomyocyte hypertrophy. for in vivo studies, isoprenaline was given subcutaneously for 14 days to wild-type mice and transgenic mice overexpressing either wild-type erk2 t188t or erk2 t188s . echocardiography and histological analyses revealed that erk t188s mice developed less left ventricular hypertrophy than control mice. hypertrophic target proteins of erk (e.g. elk1) are located in the nucleus. western blot and confocal microscopy analyses showed that overexpressed erk2 t188a or erk2 t188s are retained in the cytosol and prevented elk1-phosphorylation after isoprenaline stimulation. co-immunopreciptation assays in hek cells and nrcms underlined the direct involvement of g protein βγ/erk interaction upon isoprenaline stimulation. in line with this finding, direct activation of adenylyl cyclase by forskolin did not lead to gβγ induced erk thr188 -phosphorylation. conclusion. taken together, gβγ-subunits participate in β1-adrenergic receptor mediated hypertrophy by enhancing erk thr188 -phosphorylation. these findings add important insight to the molecular signaling of g proteins in cardiac hypertrophy. the protein tyrosine kinase src and its role upon alpha-toxin stimulation of human platelets vogel k. 1 , burke m. introduction: alpha-toxin, a 34 kda calcium pore forming exotoxin, is a major virulence factor in the pathogenesis of staphylococcus aureus infections. alpha-toxin affects human blood cells such as platelets and induces aggregation that is accompanied by multiple changes in platelet protein tyrosine phosphorylation and dephosphorylation (1). in the present paper, we focused our interest on the protein tyrosine kinase src, the most abundant member of the src-family kinases present in platelets (2) . by the use of various inhibitors, we studied src and its role in α-toxin-induced platelet aggregation. methods: isolated human platelets from healthy volunteers were stimulated with α-toxin in the presence or absence of the src-family member inhibitors pp1, pp2 or su6654 (referred as src inhibitors). src and autophosphorylation of src were analyzed by sds-page and western blotting using specific antibodies against src and tyr-416-phospho-src from calbiochem and cell signaling, respectively (3). furthermore, calpeptin, an inhibitor of the calcium-dependent protease calpain, was used. platelet aggregation was measured by the method of born. staphylococcal α-toxin induced platelet aggregation in a concentration-dependent manner (0.18 -3.0 µg/ml of toxin). pre-incubation with 3 src inhibitors (pp1, pp2 or su6654) reduced α-toxin-induced platelet aggregation by about 50%. similar inhibitory effects have been observed by the use of calpeptin that acts as an inhibitor of the src degrading protease calpain. with respect to src itself, a-toxin induced autophosphorylation at tyr-416 followed by a fast and complete dephosphorylation within 10 min. while calpeptin modified the time course of dephosphorylation, only little effect of the src inhibitors has been seen on tyr-416 phosphorylation/dephosphorylation. the typical calpain-dependent degradation of src can be blocked by calpeptin (1µm), but also by depletion of extracellular calcium indicating that it is a calcium-dependent process. conclusion: taken together, our data demonstrate that α-toxin of staphylococcus aureus induces platelet aggregation accompanied by src degradation and autophosphorylation at tyr-416 typically observed in activated platelets. inhibition of the cellular tyrosine kinase src as well as the protease calpeptin reduces aggregation indicating an important role of src and/or other src-family members in α-toxin-induced platelet stimulation. the five subtypes of muscarinic acetylcholine receptors belong to the superfamily of gprotein coupled receptors. the even-numbered subtypes m2 and m4 prefer coupling to gi proteins, whereas the odd-numbered receptors m1, m3 and m5 prefer coupling to gq proteins. with respect to ligand binding and m2 receptor activation, the conserved epitope trp 7.35 at the beginning of tm7 displays remarkable functional features. it is located at the junction between the orthosteric and the allosteric binding site of the m2 receptor [1] . in the inactive m2 receptor, it provides subtype-independent baseline affinity for allosteric antagonists [1] . in the active receptor, m2 trp 7.35 affords binding affinity for the full agonist acetylcholine and intrinsic efficacy for the partial agonist pilocarpine [2] . to study the role of trp 7.35 for m3 receptor activation, agonist-induced formation of dmyo-inositol-monophosphate was measured in cho-cells transfected with the respective human receptor-cdna. surface receptor expression measured by radioligand binding was similar in hm3 wild-type-cells and hm3 trp 7.35→ala-cells, amounting to 0.07 and 0.11x10 6 receptors per cell, respectively. the intrinsic efficacy of acetylcholine was not influenced at m3 trp 7.35→ala relative to m3 wild-type, whereas potency was reduced about tenfold. these findings resemble those made previously in m2 and the corresponding mutant. in the case of pilocarpine, replacement of trp 7.35 by alanine in m3 did not reduce intrinsic efficacy. this finding is in contrast to m2, where the corresponding mutation induced a loss of pilocarpine's intrinsic efficacy. the potency of pilocarpine was diminished about tenfold at the m3 trp 7.35→ala mutant relative to m3 wild-type. this finding is also in contrast to m2, at which pilocarpine's potency was not sensitive to the trp 7.35→ala mutation. taken together, the diverging sensitivity of pilocarpine to the trp 7.35→ala mutation between the m3 and the m2 receptor suggests that the role of this epitope for receptor function may differ between even-and odd-numbered muscarinic acetylcholine receptors. in vivo experiments for inhalation toxicity are time and animal consuming. thus several in vitro methods aim to replace or reduce and refine the in vivo experiments. human 3dtissue models are commercially available reconstructed from different donors (normal, smokers, chronic obstructive pulmonary diseases), which show a normal human bronchiole tissue that reveals a pseudostratified epithelial structure, numerous microvilli and cilia on the apical surface of the cultures. the presence of tight junctions and mucus secretion has also been confirmed comparable to the in vivo situation. these 3d-models are cultured on a porous membrane as air-liquid interface. test substances can be applied apically, either as solution or with an aerosol-inducer. in our in house validation to test the strengths, handling and reproducibility of such 3dmodel systems as well as determining the correlation between in vivo inhalation data, we have assessed the epiairway tm model from mattek, usa. a set of 20 substances were selected with known in vivo toxicity data and mode of action. the substances were tested in the epiairway model an in parallel, in 3t3 and a549 cell lines to assess putative unspecific cytotoxic effects of the test substances. a comparison of toxicity data from the 3d-model and the in vivo data revealed, that the model is only predictive of respiratory toxicity in vivo for a subset of substances with specific modes of action. the epiairway tm model has proven to be robust, showing high reproducibility between pre-and main-tests as well as in the concurrent controls but it will need a strict definition of its applicability domain or further development of the test protocol to achieve a wider applicability. remodeling of intracellular ca 2+ handling and cyclic amp-dependent signaling in atrial myocytes from patients with chronic atrial fibrillation. voigt n. 1 background: in atrial myocytes ca 2+ entry through l-type ca 2+ channels (ica,l) triggers a larger ca 2+ release (ca 2+ transient,cat) from the sarcoplasmic reticulum activating contractile myofilaments. reduced ica,l is a hallmark of atrial remodeling in chronic atrial fibrillation (caf) and is supposed to contribute to action potential shortening and contractile dysfunction. however, the coupling efficiency between ica,l and cat and its regulation by camp-dependent signaling in caf patients are unexplored. methods: ica,l (voltage-clamp) and cat (fluo-3) were measured simultaneously in rightatrial myocytes from sinus-rhythm (ctl) or caf patients. a saturating concentration of the non-selective β-adrenoceptor (ar) agonist isoprenaline (iso, 1µm) and the nonselective phosphodiasterase (pde) inhibitor 3-isobutyl-1-methylxanthine (ibmx, 10µm) were used to increase cellular camp content. camp content was assessed with immunoassay. results: in caf amplitudes of ica,l (3.3±0.3pa/pf, n=12/6 [myocytes/patients] vs 6.2±0.8 pa/pf, n=15/10, p<0.01) and cat (182.2±26.8nm vs 307.5±30.9nm, p<0.01) were lower than in ctl myocytes, whereas diastolic [ca 2+ ]i levels were unchanged (caf, 312.3±56.7nm; ctl, 305.3±43.6nm). the coupling efficiency between ica,l and cat was similar in ctl and caf. application of iso increased ica,l amplitude to 10.4±2.0pa/pf (n=7/4) in caf and to 14.3±1.7pa/pf (n=9/7) in ctl. the corresponding cats increased to 493.0±132.3nm in caf and to 545.0±79.0nm in ctl. although the amplitudes of ica,l and cat also increased after pde inhibition with ibmx, the magnitude of these increases was smaller than the iso-induced enhancements. both iso and ibmx had no effect on diastolic [ca 2+ ]i and coupling efficiency. however, the relative iso-induced increases in ica,l (caf, +202.3±47.3% vs ctl, +98.4±16.1%, p<0.05) and cat (caf, +215.4±57.8% vs ctl, +101.9±20.3%, p=0.06) were significantly higher in caf compared to ctl myocytes and a similar tendency was found for ibmx. basal camp levels were higher in caf compared to ctl (caf, 9.9±1.5pmol/mg, n=6 vs ctl, 5.0±0.6pmol/mg, n=7, p<0.05), pointing to an increased camp-dependent signaling in caf patients. conclusions: these data point to remodeling of camp-dependent signaling in caf patients which likely contributes to the stronger relative increases of ica,l and cat amplitudes after β-ar stimulation and pde inhibition. remodeling of camp-dependent signaling might be a novel contributor to af pathophysiology. direct visualisation of g-protein-coupled receptors and heterotrimeric g-proteins using single-molecule microscopy wagner j. 1 g-protein-coupled receptors (gpcrs) form the largest family of membrane-bound receptors and mediate the effects of several extracellular stimuli. although the basic mechanisms of gpcr signalling have been extensively studied, a full characterization of the involved protein-protein interaction is still missing, largely due to technical limitations. in this study, we developed new methods for labelling gpcrs and g-protein subunits based on snap-and clip-tags and visualise them with single-molecule sensitivity. the snap-tag is a mutant of the dna repair protein o 6 -alkylguanine-dna alkyltransferase that reacts with fluorescent benzylguanine derivatives, whereas the clip-tag is reacting specifically with o 2 -benzylcytosine derivatives. these tags allow labelling proteins directly in living cells with very high specificity and low background. snap/clip-tagged receptors and g-proteins were covalently labelled with small organic fluorophores and visualised by total internal reflection fluorescence microscopy, which allows to selectively illuminate only fluorescent molecules located on or immediately underneath the cell surface. particles were automatically analysed with previously published as well as newly developed algorithms. the results indicated that both receptors and gproteins, although diffusing with high speed on the cell surface (diffusion coefficients: receptors ~ 0.05 mm 2 /s, g-proteins ~ 0.1mm 2 /s), can be visualised and correctly tracked. a variable fraction of receptors and g-proteins are immobile or show hop movements, possibly suggesting their interaction with cytoskeletal or other membranebound proteins. our data also suggest the feasibility of performing two-colour analyses with snap-and clip-tagged proteins aimed at directly visualizing transient interactions between receptors and g-proteins or among g-protein subunits. in-vitro screening systems are particularly well suited to preclinical toxicology testing at an early stage of drug development as they have the advantage of being fast and requiring only a small amount of test substance. the demands for in-vitro screening assays for systemic toxicity are multiple and include the need of organ specific cell systems, the use of optimal cell numbers, cell passages and incubation times. even minimal changes in the conditions of the test system may lead to significant changes of the biological system. therefore a reliable normalization compensating biological variability is crucial prior to any interpretation of results generated from a biological system. basf has developed an in-vitro metabolite profiling assay and a subsequently tuned normalization strategy allowing the prediction of specific organ toxicity. the in-vitro assay consist of exposing cells lines to test substances and to determine the metabolite profile using chromatography coupled to mass spectrometry systems. herein, we compare five different normalization strategies referring to their suitability in the application to in-vitro metabolite profiling data. the strategies comprise statistical approaches, approaches referring to reference values from each individual sample or samples generated in dependent batches. best results were achieved by an individual strategy using a new reference value correlating well over a large range of cell counts previously used for generating corresponding cell extracts. statistical analysis revealed the normalization based on the new reference value greatly improved the quality of the results compared to non-normalized samples as well as to all remaining strategies. generation and application of this new reference value and the corresponding normalization strategy will be presented the first time. validation will be featured on the basis of extracts of the human hepatocellular carcinoma cell line hep g2. molecular mechanisms of the inhibitory function of rhoh in phospholipase cmediated signalling walliser c., löschmann y., ziegler v., kühne e., schilling p., rasonabe z., bühler a., vatter p., gierschik p. universitätsklinikum ulm institut für pharmakologie und toxikologie, albert-einstein-allee 11, 89081 ulm, germany rho gtpases are a subfamily of ras gtpases regulating diverse signalling pathways, for example those regulating the reorganisation of the actin cytoskeleton. among them, rac2 and rhoh show an expression restricted to the hematopoietic lineage. rhoh is constitutively active, because it carries mutations in two positions (s13 and n62) known to be important for gtp hydrolysis. hence, rhoh is controlled on the level of protein expression and, possibly, by tyrosine phosphorylation. rhoh has been implicated in human malignancies, since the gene is subject to somatic hypermutation in its noncoding regions and to translocation to the gene encoding laz3/bcl6 or to other genes in human b-cell lymphomas. furthermore, rhoh is overexpressed in primary human chronic lymphocytic leukemia (cll) cells. these findings suggested that rhoh is involved in the initiation and/or progression of cll. we previously showed that rhoh acts as a potent inhibitor of both rac2-mediated phospholipase c-β 2 (plcβ2) and plcγ2 activation in intact cells. the aim of this study was to elucidate the molecular mechanisms of the inhibitory effect of rhoh on plc activity. the results showed that rhoh directly inhibited the activity of constitutively active variants of plcγ2, plcβ2, and plcδ1, but that it had little or no effect on the activity of plcγ1 and plcε. the amino acid residues s13 and n62, likely to be the cause for the gtpase-deficiency of rhoh, are not required for the inhibitory function of rhoh. furthermore, the switch-i and switch-ii regions of rhoh are not necessary for the inhibitory effect of rhoh, since rhoh mutants carrying switch-i or switch-ii regions of rac2 caused inhibitory effects on rac2-mediated plcβ2 and plcγ2 stimulation indistinguishable from wild-type. interestingly, rhoh seems to interact with regions of plcγ2 distinct from those which are necessary for rac2 interaction, as the split pleckstrin homology domain of plcγ2, which is essential for its interaction with activated rac2, is dispensable for the inhibitory effect of rhoh. in summary, our results indicate, that rhoh acts as a plc-isozyme-specific negative regulator of the activity of plcβ2 and plcγ2, both of which are specifically expressed in hematopoietic cells. these findings suggest a novel mechanism of plc isozyme regulation by rhoh. the results also suggest that rhoh plays an important role in b cell maturation, function, and leukemogenesis by modulating b-cell-receptor-mediated plcγ2 activation. effect of rac1 inhibition on doxorubicin mediated cell response wartlick f., fritz g. heinrich-heine-universität düsseldorf institut für toxikologie, universitätsstrasse 1, 40225 düsseldorf, germany background: the small gtpase rac1 is a well characterized member of the rashomologous (rho) family. rac1 is not only a key regulator of the actin cytoskeleton but also regulates the activity of nadph oxidase, stress kinases and transcription factors (e.g. nf-κb, ap1). furthermore, rac1 can translocate into the nucleus and interacts with topoisomerase type ii (topo ii). yet the general nuclear function of rac1 is still unclear. here, we address the question how rac1 influences the genotoxicity of the topo ii poisons doxorubicin and etoposide. methods: to study the function of rac1, human hepatoma cells were pretreated with the rac1-inhibitor eht 1864 before they were exposed to doxorubicin, etoposide or, for control, ionizing radiation (ir). to check the influence of rac1 inhibition on the outcome of genotoxin treatments, cell viability and cellular stress response were analyzed by the wst-assay, western blot (wb), co-immunoprecipitation experiments, facs-analysis and the alkaline comet-assay. results: as compared to the control, cells that have been pretreated with the rac1 inhibitor showed a higher viability, less phosphorylation of h2ax (s139) and a reduced dna damage formation (measured by alkaline comet-assay) after treatment with doxorubicin and etoposide but not after treatment with ir. furthermore inhibition of rac1 resulted in a reduced phosphorylation of topo iiα (s1106) and an increased interaction of topo iiα with hsp90 in doxorubicin treated cells. the data indicate that inhibition of rac1 protects human hepatoma cells against topo ii poisons due to interference with topo iiα function. the presence of drugs or other potential toxic substances in milk has enormous toxicological and nutritional consequences for consumers of dairy products. the atpbinding cassette (abc) transport protein breast cancer resistance protein (bcrp; abcg2) is expressed in alveolar epithelial cells of the mammary gland in cows, sheeps and goats. bcrp is known to play a major role in the active secretion of a variety of xenobiotics into human milk. so far there is little information about the transport activity and substrate specificity of dairy bcrp. therefore we aimed to establish a mdck cell in vitro model expressing bcrp of dairy animals. bcrp mrna was isolated from bovine, caprine and ovine mammary gland. full-length clones were generated using race (rapid amplification of cdna ends) pcr. the final full-length bovine, ovine and caprine abcg2 cdna-clone sequences were submitted to the ncbi genebank (eu570105, gq141082 and gq241418). stable transfection of bcrp in mdck cells was performed and the subcellular localization of bcrp at the apical plasma membrane was identified by confocal laser scanning microscopy. bcrp-mediated transport of the substrate hoechst 33342 was measured and the selectivity was determined by the bcrp inhibitor ko143. inhibition studies using hoechst 33342 identified various drugs including the antibiotic enrofloxacin or anthelmintic agents like oxfendazole as substrates of bovine, caprine and ovine bcrp. to further characterize bcrp carrier activity, bidirectional transport studies were performed with transwell® filter inserts that allow studying drug transport between an apical and basolateral compartment. cell monolayer integrity was checked by measuring teer values as well as by measuring the paracellular flux marker atenolol by lc/ms. bidirectional transport studies with enrofloxacin were performed to characterize the bcrp transporter activity. our results may contribute to increase the understanding of carrier associated drug transport into the milk of dairy cattle and therefore enlarge consumer protection. acrolein and acrylamide: excretion of mercapturic acids after consumption of potato chips watzek n., scherbl d., berger f., feld j., eisenbrand g., richling e. technische universität kaiserslautern fachbereich chemie; fachrichtung lebensmittelchemie & toxikologie, erwin-schrödinger-str. 52, 67663 kaiserslautern, germany acrolein (ac) and acrylamide (aa) may be formed from food constituents during heating of food. ac is supposed to be generated via heat induced formation from glycerides/glycerol, aa is known to arise during the maillard reaction from asparagine and reducing carbohydrates. ac also has also been suggested to be formed by endogenous metabolism as a side product of carbohydrate and/or amino acid turnover or by oxidative desamination of polyamines [1] . as an α,b-unsaturated aldehyde, ac forms 1,4-michael-adducts with biomolecular nucleophiles, such as sulfhydryl and amino groups. in the organism, ac and aa are preferentially conjugated to glutathione and are excreted as mercapturic acids (ma), n-acetyl-s-(3-hydroxypropyl)-cysteine , n-acetyl-s-(carboxyethyl)-cysteine (cema), (n-acetyl-s-(2-carbamoylethyl)-cysteine (aama), and (n-acetyl-s-(2-hydroxy-2-carbamoylethyl)-cysteine (gama). data on human exposure to ac and its occurrence in the diet are scarce. in general, contents in heat treated foods are considered to be in the low ppb range (µg/kg) [2] . nevertheless, in a pilot study in humans urinary 3-hpma excretion of 14 non-smokers was reported to be about three fold higher, as compared to aama [3] . in the present human intervention study we monitored the excretion of mas in five healthy volunteers (male) after ingestion of commercially available potato chips (175 g), equivalent to an uptake of 44 µg aa (absolute amount), together with an as yet unknown amount of acrolein [4] . urinary ma contents were monitored by hplc-ms/ms following solid phase clean-up of urine for up to 24 h after test meal uptake. the results demonstrated kinetics of 3-hpma and cema excretion in human urine to be clearly related to ingestion of the potato chip meal. on the basis of auc values, total excretion of 3-hpma plus cema exceeded that of aama plus gama by a factor of about four. the results confirm earlier findings on urinary mas, suggesting markedly higher human exposure to dietary ac / potential ac precursors than to aa. it is an as yet unresolved question, whether and to what extent concomitant substantial ac exposure may influence toxicology of such dietary heat-induced toxicants. [1] stevens, j.f. and maier, c.s. (2008) molecular nutrition & food research 52; 7 [2] osorio, v. m. and de lourdes cardeal, z., (2011) journal of chromatography a 1218; 3332 [3] schettgen, t., musiol, a., and kraus, t., (2008) rapid communications in mass spectrometry 22; 2629 [4] ewert, a., granvogl, m., and schieberle, p., (2011) lebensmittelchemikertag 2011 450 protective effects of increased nad + levels in human peripheral blood mononuclear cells exposed to dna damaging agents weidele k., beneke s., bürkle a. university of konstanz molecular toxicology group, department of biology, jacob-burckhardt-str.31, 78457 konstanz, germany the dna damage-activated enzyme poly(adp-ribose) polymerase 1 (parp-1) acts as a nick sensor and modifies target proteins by covalent attachment of poly(adp-ribose) [par] using nad + as substrate. the intracellular levels of par and nad + are important parameters for biological responses to genotoxic stress and influence diverse cellular functions including dna repair or maintenance of genomic stability. notably, loss of genomic stability is a hallmark of both carcinogenesis and the ageing process. here we analysed the impact of elevated nad + levels in human blood peripheral mononuclear cells (pbmc) with regard to (i) poly(adp-ribose) formation, (ii) cell death, (iii) initial dna damage and subsequent repair, as well as the influence on (iv) genomic stability under genotoxic stress. after ex vivo supplementation of pbmc with low concentration of nad + precursor nicotinic acid (na) intracellular nad + level significantly increased up to 2 fold in unstimulated [1] and 1.5 fold in mitogen-stimulated cells. after dna damage infliction, parp activity was dramatically increased in supplemented cells, necrotic cell death was reduced and dna strand break repair was significantly affected. furthermore the frequency of micronuclei decreased significantly after irradiation damage, emphasizing the fundamental role of adequate nad + levels in maintaining genomic integrity. the cyclic purine nucleotides adenosine 3':5' monophosphate (camp) and guanosine 3':5' monophosphate (cgmp) are well-examined second messengers with many proven biological functions. in a recent study, using a highly sensitive and specific mass spectrometry method, we have shown that cyclic 3':5' cytidine monophosphate (ccmp), a pyrimidine nucleotide, is naturally occurring in several mammalian cells [1] . ccmp activates both camp-and cgmp-dependent protein kinases with low potency [2] but the physiological function of ccmp is still very poorly understood. in an effort to delineate the function of ccmp, we analyzed expression of the early response gene egr1. we chose this gene because it is regulated by numerous stimuli including camp [3] . in our first study, we showed that dibutyryl-ccmp and ccmp failed to increase egr1 gene expression levels after stimulation of kb cells under various experimental conditions using real-time pcr (taqman®). we have now changed the experimental set-up using hela cells and the new ccmp analogue, ccmp-acetoxymethyl ester (ccmp-am), still focusing on gene expression of egr1. esterification of the negatively charged cyclic phosphate of ccmp allows better transport of the nucleotide across the cell membrane, thus augmenting possible intracellular effects. hela cells were stimulated in cell culture medium with extracellularly applied ccmp-am (3, 10, 33, 100 µm) over 15 to 240 min 24 h after seeding. analysis of real time pcr (taqman®) experiments, using β-actin as a housekeeping gene, showed a significant increase of egr1 expression in a time and concentration dependent manner. these effects were specific for stimulation with ccmp-am but not the control phosphate trisacetoxymethyl ester. hela cells were also cultured in serum free resting medium (mcdb 153, sigma) that induces growth arrest, one to eight hours prior to stimulation. here, even higher egr1 expression levels through ccmp-am stimulation could be seen. these results suggest that ccmp could function as a second messenger just as camp and cgmp do. studies are in progress to further examine the mechanisms of the ccmp-am effects on egr1 expression in hela cells. methyl-cpg-binding protein 2 (mecp2) recognizes methylated dna, it is involved in chromatin remodeling and it acts as a transcriptional repressor or activator. we have previously shown that expression of mecp2 is diminished in murine and human heart failure. prevention of mecp2 downregulation in transgenic mouse models aggravated cardiac hallmarks of heart failure. in patients with rett syndrome, which is caused by mutations in the mecp2 gene, mitochondrial function was found to be altered in the central nervous system. as the impact of mecp2 on mitochondrial function in the heart is unknown, the aim of the present study was to characterize the significance of mecp2 of cardiac mitochondria in mouse models with cardiac myocyte-specific expression or ablation of mecp2. in order to investigate the cardiac function of mecp2, two genetically modified mouse models were previously generated, including mice with inducible transgenic expression of mecp2 in cardiac myocytes under control of the tetracycline-system (mecp2-tg) and mice with targeted ablation of mecp2 in myocytes (mecp2 mlccre ). these mice were analyzed under basal conditions and after chronic transverse aortic constriction (tac). at baseline, cardiac-specific overexpression of mecp2 did not cause any difference in cardiac function as compared to control mice using millar catheterization. isolated interfibrillar mitochondria showed a decrease in citrate synthase activity. after chronic pressure overload, the decrease in cardiac mecp2 expression could be completely prevented by the mecp2 transgene. cardiac contractility and relaxation were significantly decreased in mecp2-tg animals. upon electron microscopical investigation, transgenic mecp2 expression was associated with a significant reduction of interfibrillar mitochondria, clustering of mitochondria in the perinuclear region and smaller mitochondrial cross sections as compared with control specimens. in contrast, cardiac myocyte-specific ablation of mecp2 caused a rightward shift in the size distribution of mitochondria as compared with mecp2-tg hearts. epigenetic processes, including the recognition of dna methylation by mecp2, may play an important role in the control of mitochondrial gene expression, structure, subcellular localization and function in the heart. thus, precise control of mecp2 expression and function is essential to prevent deterioration of metabolic function during chronic heart failure. normalisation of blood pressure does not prevent angiotensin ii-induced dna damage in kidney and heart of ren2 rats weissenberger s., hey v., lau d., schupp n. universität würzburg institut für pharmakologie und toxikologie, versbacher strass 9, 97078 würzburg, germany increased activity of the renin angiotensin system (ras) with enhanced levels of angiotensin ii (angii) leads to oxidative stress with endothelial dysfunction, hypertension and atherosclerosis. epidemiological studies revealed a higher cancer mortality and an increased kidney cancer incidence in hypertensive patients. we could show in vitro and in vivo that angii causes structural dna damage dose-dependently in kidney cells and in the kidney. elevated angii levels therefore might contribute to carcinogenesis of the kidney. in a model of high angii organ levels, the transgenic ren2 rat, carrying an additional renin gene, dna damage in the kidney was analysed in animals of 13 and 32 weeks. untreated ren2 rats exhibit increased blood pressure from the age of 8 weeks on. therefore, the line is kept on angiotensin i converting enzyme inhibitor therapy, which normalizes blood pressure and kidney function to values of control sprague dawley rats. despite this normalized blood pressure of the ren2 animals, a significant higher superoxide production could be observed in kidneys already in 13 week old animals. also a higher frequency of structural dna damage and double strand breaks could be detected in the comet assay and with an antibody against the double strand break marker γ-h2ax in kidneys. further, fittingly, an increased dna repair activity exists in kidneys of ren2 rats compared to control rats. as another organ affected by hypertension the heart of the ren2 animals was analysed for oxidative dna damage. although only a marginal increase of superoxide production could be found, also in the heart a significant higher frequency of dna double strand breaks and cells positive for dna repair activity could be observed. our data let us conclude that normalization of blood pressure in a state of activated ras is not sufficient to prevent angii-induced genotoxicity. this further implies that also patients with treated hypertension still might suffer from endorgan-damaging effects of elevated angii levels. the d541a pore mutation leads to complete inactivation of trpv6 channels in epididymis weißgerber p. 1 , kriebs u. replacement of aspartate residue 541 by alanine (d541a) in the pore of trpv6 channels in mice disrupts ca 2+ absorption by the epididymal epithelium resulting in abnormally high ca 2+ concentrations in epididymal luminal fluid and in a dramatic but incomplete loss of sperm motility and fertilisation capacity raising the possibility of residual activity of channels formed by trpv6 d541a proteins (sci signal 4, ra27, 2011) . it is known from other cation channels that introducing pore mutations even if they largely affect their conductivity and permeability can evoke considerably different phenotypes compared to the deletion of the corresponding protein. to gain insights whether the trpv6 d541a pore mutant still contributes to residual channel activity and/or channelindependent functions in vivo, we compared important fertility-parameters between trpv6 -/and trpv6 d541a/d541a mice: the fertilization rate observed in permanent matings, the in vivo fertilization rate as judged by the rate of embryos isolated from plug positive females of matings with males homozygous for either trpv6 mutation as well as the motility, in vitro fertilization capacity and viability of sperm were reduced to the same extent in both genotypes. also, no differences were observed in copulatory behavior between trpv6 -/and trpv6 d541a/d541a males. the profound reduction in ca 2+ uptake by the epididymal epithelium was identical in trpv6 -/and trpv6 d541a/d541a males. this direct comparison of these parameters indicate that deleting trpv6 does not further aggravate the phenotype observed in trpv6 d541a/d541a mice, and -in our opinion -allows the conclusion that the d541a pore mutant of the trpv6 protein leads to complete inactivation of the trpv6 channel activity or channel-independent scaffolding functions in epididymal epithelium. characterization of a naturally occurring c-terminal mutation (n996i) on herg channel function in hek293 cells sellmaier v. 1 , moretti a. long-qt-syndromes (lqts) are acquired or inherited disorders which predispose patients to cardiac arrhythmias and sudden death. in affected individuals, the electrocardiogram shows a prolongation in the qt interval, due to an unstable repolarization of the action potential. acquired forms of lqts are often the result of treatment with medications that block cardiac potassium channels, such as class iii antiarrhythmic drugs or antihistamines. inherited lqts are caused by mutations in cardiac ion channels. congenital long qt syndrome 2 (lqt2) is caused by loss-offunction mutations in the human ether-á-go-go-related gene herg (also known as kcnh2 or kv11.1). herg encodes the pore-forming α-subunit of the rapid delayed rectifier potassium current i kr, whose physiological role is to repolarize the late phase of cardiac action potentials. herg channel α-subunits exist as 2 isoforms (1a and 1b) that are identical except for structurally divergent n termini. native cardiac ikr channels are tetraheteromers containing 2 of each α-subunit types. a loss-of-function can be due to either defects in a) channel opening (gating), b) ion permeation or c) protein maturation and trafficking. we have identified a so far uncharacterized dominant missense mutation in the herg1 gene (n996i) in a patient with lqt. both herg1a and herg1b subunits were cloned from a human heart cdna library and the specific n996i mutation introduced by site directed mutagenesis. hek 293 cells were transiently transfected with equal amounts of mutated herg1a and wild type (wt) herg1b cdnas and the resulting potassium current compared to herg1a/b wt. whole-cell patch-clamp analysis showed similar current densities for wt versus mutated channels. also the voltage-dependence of activation was unchanged with a halfmaximal activation at -27 mv for wt and -29 mv for the mutated channel assembly. differences were found for the deactivation and inactivation kinetics. the deactivation was faster in the mutated channels with t fast = 62 ms and tslow = 480 ms versus tfast = 90 ms and tfast = 620 ms in wt channels, determined from the tail current at -40 mv after a 5-second pulse to + 50 mv. the half-maximal steady-state inactivation of the tail current was shifted by 20 mv to the depolarizing direction in the mutated channel compared to the wt. a defect in channel gating appears to be the most likely explanation. background: abcc2 is adjacent to p-glycoprotein the most important efflux transporter for various endogenous and exogenous compounds and is expressed at several compartment barriers. by increasing evidence it is shown that the abcc2 polymorphisms are of clinical significance. the aim of our study is to analyze the epigenetic regulation of distinct abcc2 haplotypes by the influence of micrornas. methods: abcc2 cdna clones containing five distinct haplotypes were generated by site-directed mutagenesis, cloned into pires-zsgreen expression vectors and transfected into different cell lines for further functional analysis. one modified vector set contained a short 3'utr sequence of abcc2 whereas the other contained the full length (fl) sequence. mirnas potentially interacting with abcc2 were identified form an mirna array after rifampicin stimulation of hepg2 cells. results: we could demonstrate that there is no difference in the basal protein expression level comparing the two vector types (fl 3'utr vs. mod. 3'utr) concerning the -24c/1249g/3972c (cgc) abcc2 wt in hepg2 cells. using the fl vector construct, the expression level of cac haplotype protein was increased (136,15% ± 20%). transfection assays with the mir-397, which was identified as a candidate mirna targeting abcc2 mrna, and the two different vector constructs harboring the cac or the cgc (wt) haplotype, confirmed that mir-379 was able to down regulate the abcc2 protein expression. there was no significance in downregulation for one abcc2 haplotype, respectively (modified 3'utr: cgc 22-34%; cac 20-40%, fl 3'utr: cgc 20-25%; cac 17-44% down regulation compared to mir-negative control). discussion: differences of abcc2 protein expression level are due to the epigenetic regulation of abcc2 haplotypes. to further characterize the effect of mir-379 on tcg, tgt and cgt abcc2 haplotypes, transfection assays are currently performed using cell cultures as well human primary leukocyte cultures. sex differences affect the pathophysiology and pharmacology of leukotriene biosynthesis werz o. friedrich-schiller-university jena, institute of pharmacy, philosophenweg 14, 07743 jena, germany inflammatory diseases affect more females than males. thus, women suffer more often from asthma, rheumatoide arthritis, alzheimer´s disease, and many autoimmune diseases than men. of interest, sex differences also exist in drug responses, with respect to both pharmacodynamics and pharmacokinetics. we have recently discovered a sex bias in the biosynthesis of pro-inflammatory leukotrienes (lts) due to testosterone, which may represent a molecular basis for gender differences in inflammation and asthma. interestingly, testosterone downregulates lt biosynthesis and also causes a sex bias in the efficiency of lt synthesis inhibitors, which demands for the clinical evaluation of a gender-tailored therapy with anti-lts. we found that certain inhibitors of lt biosynthesis were more efficiently in females than in males, and that androgens are responsible for these gender differences. in fact, the flap inhibitor mk886 effectively reduced ltb 4 pleural levels in female but not in male rats treated with carrageenan, and mk886 increased survival only of female mice in the lt-related disease model of paf-induced lethal shock. administration of testosterone to female mice abolished the protective effects of mk886. in view of the current active development of lt synthesis inhibitors as therapeutics in respiratory and cardiovascular diseases, our data prompt for consideration of gender issues in the development and use of such drugs, in order to optimize medical therapy both for men and women. considering the complexity of deposition and kinetics of air-borne nanomaterials in the lung, potential pulmonary toxicity of biopersistent nanomaterials should be evaluated by inhalation studies. those studies demand special equipment and large quantities of test material. intratracheal instillation appears as a simple and low substance-consuming alternative, although bolus dosing and the more central distribution of the particles in the lung are a well known trade-off. we compared the inflammatory response of the lung to amorphous silica (as) after instillation and inhalation. for inhalation the established short-term protocol for nanomaterials was employed (ma-hock et al. inhalation toxicology, 21:102, 2009 ): male wistar rats were exposed to the test items for 6 h/day on five consecutive days. the lungs were evaluated by analysis of bronchoalveolar lavage fluid (balf) and by histopathology three days and three weeks after the end of the exposure. in a parallel study, rats were intratracheally instilled and equally evaluated three days after instillation. assuming a deposition rate of 10%, the instilled dose corresponded to the aerosol concentration of 10 mg/m 3 used for inhalation. results show that inhalation and instillation of nominally equal mounts of amourphous silica elicit different results in the lung with inhalative treatment being less harmfull. this difference may be due to the bolus effect inevitable linked to instillation. instillation stuldies with amorphous silica may, therefore, be of limited value with respect to doseresponse assessment. sunscreen products containing uv filters protect consumers from the harmful effects of uv exposure. pigmentary grades of metal oxides like zno result in an opaque whiteness as a result of scattering visible light, whereasnanoparticles result in transparent products for better consumer acceptance and thus improved protection of human skin against uv-induced damage. in addition scatter uv light is most efficiently reflected at a nanosize of 60-120 nm. in the last 2 years the toxicological properties of nanozno in comparison with pigmentary zno were examined, the results of these comprehensive studies are presented. all tests were performed according to oecd guidelines, which were modified, especially in regard of substance preparation where appropriate. nanosized zno showed no acute toxicity after dermal application, in the bcop assay as well as in the epiderm assay it showed no corrosion / irritation potential. nanosized zno does not penetrate the intact as well as the sunburned skin. a dermal absorption test in rats (oecd 427) with 65 zn-labelled test item as well as penetration tests in weanling pigs after uv radiation did not show a penetration of the zno nanoparticles through the skin. genotoxicity was tested in vitro in the ames test, in the chromosomal aberration test in v79 cells, both showing negative results whereas the mouse lymphoma mutation test / l5178y/tk+/-cells was positive. in vivo no mutagenic effect was detected in two mouse micronucleus tests, on with intraperitoneally application and another after repeated inhalation. nanosized zno was tested in 5-days, 14-days and 90-days inhalation studies, in all studies the predominant effects were reversible local inflammatory changes in the nasal cavity and lungs, with a noaec of 1.5 mg/m3 in the 90-day study. in a prenatal developmental toxicity study according to oecd tg 414, with repeated inhalation exposure to female wistar rats from gestation day 6 through 19, maternal toxicity was observed by increase lung weights and inflammations in the lungs. but no substance-related effects on reproductive parameters (conception rate, corpora lutea, implantation sites, preimplantation loss, postimplantation loss, resorptions, dead fetuses) and no increase in external and soft tissue malformations and variations could be detected. the overall result of all the toxicological studies with nanosized and pigmentary zno is that the toxicological profile of both is very similar. studies were sponsored partly by cefci lri and partly by basf se. use of reach registration data for improving thresholds of toxicological concern (ttc) wieneke n., dorn s., jakupoglu c., schäfer c., sica m., wiegand c., mostert v. dr. knoell consult gmbh, marie-curie-str. 8, 51377 leverkusen, germany the threshold of toxicological concern (ttc) concept is utilised to identify human exposures that are so low that in-depth toxicological investigations are expendable. this is called "exposure-based waiving". exposure-based waiving serves to focus available resources on substances with relevant human exposure potential. important work into establishing ttc values has been published by munro et al. (1996) . the initial report used a database of 613 organic substances compiled from publicly available sources. in total, 2941 noels were collected in this fashion. the munro concept used the cramer classification to categorise substances according to their hazard potential. we broadened the ttc database by including noaels published on the echa website as per 3 november 2011, containing data for more than 3900 registrations. only nongaseous mono-constituent substances with oral noaels were included in the ttc database. organophosphates and genotoxic substances were excluded from the database as well as noaels obtained for surrogate substances. noaels for all systemic endpoints (general toxicity, developmental toxicity, fertility, neoplasia) were taken into account. where appropriate, default assessment factors of up to 6 were used to establish chronic noaels for each substance. for every eligible substance, we collected the published clp category for acute oral toxicity as a potential predictor of overall hazard potential. this gives rise to five categories of acute oral toxicity. a ttc is calculated from the 5 th percentile of noaels in each of these categories using the reach rules for establishing dnels for workers and the general population. this poster presents the preliminary results for more than 1000 substances. the results indicate that the ttc concept becomes more robust when using the very broad echa database. it also suggests that acute oral toxicity categories can be used as a predictor for the overall hazard potential of a substance. comparison of different in-vitro models for inhalation toxicology with respect to the effects of cigarette smoke total particulate matter wiese j. 1 , schumann b. b-and l-moc can be cultivated up to 70 days without loss of viability, as determined by resazurin-assay. viability of cell cultures was determined by mtt-assay after incubation with increasing doses of tpm. exposure of h322 to tpm (30 mg/l) reduced viability to 95% or 83% after 24 or 72h, respectively. in a549 viability was 67% after 72h with tpm (30 mg/l). the same dose of tpm lead to a decrease in viability to 82% (24h) or 25% (72h) in nhbec and to 74% (24h) or 28% (72h) in plc. as a marker of oxidative stress the level of intra-cellular glutathione (gsh) was determined by hplc. in both the tumor cell lines analysed gsh-level was increased by tpm (5 mg/l). in h322 the induction was 1.6 and 1.2 fold after 24 or 72h, respectively. while in a549 it was 1.3 (24h) and 1.4 fold (72h). in nhec and plc tpm (5 mg/l) did not have a significant effect on gsh-levels after 72h. in n-moc tpm (5 mg/l) also did not modulate gsh after 24h, but it diminished gsh-level by 0.5 fold upon prolonged contact (72h). in b-moc gsh concentrations also decreased to 0.6 or 0.8 fold the level of controls after 24 or 72h incubation. the results presented show that in-vitro models of varying complexity and origin within the respiratory tract clearly differ in their response to tpm, which was used a model inhalative toxicant. the tumor cell lines used seem to be better adapted to chemical stress, while the models closer to the in-vivo situation are more vulnerable. the nongenomic effects of the mineralocorticoid receptor in transgenic mouse heart winter s. 1 , schreier b. within the renin-angiotensin-aldosterone system (raas), the mineralocorticoid receptor (mr) is important for the regulation of fluid and electrolyte balance in the kidney, salivary glands, sweat glands and colon. however, survival of patients with severe heart failure is increased when mr blockage is combined with standard therapy suggesting aldosterone, the mr ligand, as a key factor in the development of cardiovascular diseases, but the mechanism is not yet fully understood. in recent years, evidence accumulated that besides its function as a hormone-activated transcription factor the mr also functions via nongenomic pathways. to investigate the function of the nongenomic effects of the mr in cardiovascular dysfunction, we generated a transgenic (tg) mouse model expressing a truncated human mr lacking the dna-binding site (hmr def ) under control of the cardiac specific α myosin heavy chain promoter (αmhc), a model for nongenomic effects of the mr in the heart. in this mouse model no enhanced mortality could be observed. body weight (bw), heart weight and relative heart weight were not different compared to wild type (wt) while left atrial weight/bw was increased by 20 % (wt 0,25 ± 0,01 mg/g vs. tg 0,30 ± 0,02 mg/g, p<0.05, n=12). compared to wt mice neither surface electrocardiographic experiments nor echocardiographic experiments revealed modified parameters for tg mice under basal (i.e. unstimulated) conditons as well as under β-adrenergic stimulation by isoproterenol (iso, 100 µl 1 mm iso intraperitoneally applied). to uncover the role of aldosterone in the development of cardiovascular diseases treatment with aldosterone and high-salt diet (1%) was performed. after 28 days cardiac function and heart dimensions were analyzed, surface electrocardiographie uncovered increased p duration (16 ± 0.5 ms vs. 14 ± 0.7 ms, p<0.05) and qtc interval (61 ± 2 ms vs. 50 ± 3 ms, p<0.05) in tg (n=7) compared to wt (n=5) animals. these findings probably indicate more sensitive conduction pathways to aldosterone in tg mice. oligomerization is important for regulation of phospholamban activity wittmann t., lohse m. j., schmitt j. p. institut für pharmakologie und toxikologie, versbacher straße 9, 97078 würzburg, germany phospholamban (pln) is a heart specific protein located in the membrane of the sarcoplasmic reticulum. it inhibits the ca 2+ -atpase serca2a, thereby decelerating cytosolic ca 2+ clearance during diastole of the cardiac cycle. upon phosphorylation the inhibitory activity of pln on myocyte ca 2+ transport is attenuated. further, it is believed that phosphorylation favors the formation of (rather inactive) pentamers and that pln pentamers itselves were an inferior substrate for phosphorylation compared to monomers. this would suggest an important role of pln oligomerization in the regulation of pln activity. to prove this hypothesis, we are investigating the patterns and kinetics of pln phosphorylation in the context of alterations in pln structure. the introduction of specific point mutations into the transmembrane region of pln yielded mutants that are purely monomeric (l37a, i40a and c41f) or favor pentamer formation (i45a, v49a). transfected hek293 cells expressing these mutants or wildtype pln were stimulated with forskolin to induce pln phosphorylation before lysis of cells and western blot analysis using antibodies directed against phosphorylated pln. surprisingly, phosphorylation was increased for both monomeric and pentameric pln after stimulation with 0.25µm forskolin for less than one minute. at increasing forskolin concentrations phosphorylation signals increased in parallel for monomers and pentamers. for measurement of phosphorylation kinetics stimulation of cells with 2.5µm forskolin was stopped at different time points. we found phosphorylation of both pln monomers and pentamers within seconds of stimulation. differences in phosphorylation patterns became more pronounced when assays were performed at low temperature (14°c). intriguingly, preliminary analyses suggest that pka dependent phosphorylation occurs first in pentamers and that phosphorylation of monomers may catch up only after pentamer phosphorylation is almost complete. our data suggest that both pln pentamers and monomers are suitable substrates for pka dependent pln phosphorylation. unlike the prevalent assumption, kinetics of pentamer phosphorylation seem to be at least as fast as that of pln monomers in transfected hek293 cells suggesting an important role of pln pentamers in the regulation of pln activity. regulation of cardiac contractility by nucleoside diphosphate kinases in zebrafish wolf n. m. 1, 2 , abu-taha i. in the heart, nucleoside diphosphate kinases (ndpks) can interact with heterotrimeric g proteins, thus regulating camp synthesis in a receptor independent manner and thereby influencing contractility in cardiomyocytes. we further investigated the interaction of ndpk isoforms with heterotrimeric g proteins in the heart in vivo and in vitro using zebrafish embryos and embryonic fibroblast from ndpk a/b double knockout mice (ndpk a/b ko mefs). in zebrafish the morpholino-mediated knockdown of ndpk a did not lead to an obvious phenotype, although the total ndpk activity was reduced. depletion of ndpk b caused a cardiac phenotype characterized by severely impaired atrial and ventricular contractility and insufficient blood flow. the depletion of ndpk b was associated with a significant decrease of protein levels of the heterotrimeric g protein subunits gβγ, gα s and gαi. the knockdown of ndpk c led to a more restricted cardiac phenotype with markedly reduced pumping function of the ventricle, while the atrium was unaffected. in accordance to the reduced cardiac pumping function, camp levels were significantly diminished in the ndpk b and ndpk c morphants. similar findings were obtained in ndpk a/b ko mefs. the absence of ndpk a and b resulted in a decrease of the plasma membrane content of gβ and gαs and a significant reduction in camp synthesis. the protein expression of the isoform ndpk c was also significantly reduced in the ndpk a/b ko mefs. the re-expression of ndpk b but not ndpk a rescued the basal camp production and the membrane content of g proteins. interestingly, the overexpression of ndpk c led to a 5-fold enhancement of the camp level and a significant increase of the membrane content of gβ and gα, and thus rescued the knockout phenotype. our data indicate, that the ndpk isoforms b and c are essential for cardiac contractility, most likely by forming a signaling complex at the plasma membrane including ndpk b, ndpk c and heterotrimeric g proteins. the isoform ndpk c, with its n-terminal hydrophobic region, might serve as a membrane anchor for the ndpk/g protein complex. induction of apoptosis via pka-dependent and pka-independent pathways by cyclic purine and pyrimidine nucleotides in mouse lymphoma cell lines wolter s. 1 camp is a second messenger that plays an important role in intracellular signal transduction of various hormones and neurotransmitters. a major function of camp in eukaryotes is the activation of camp-dependent protein kinase a (pka). pka is involved in the control of a variety of cellular processes. pka exists as an inactive tetramer of a dimeric regulatory (r2) and two catalytic (c) subunits that releases the active c-subunits upon binding of camp. stimulation of the mouse t-lymphoma cell line s49 wild-type (wt) with dibutyryl (db)-camp induces apoptosis by an intrinsic, mitochondria-dependent mechanism. apoptosis induced by db-camp occurs via a pka-dependent mechanism, since s49 kincells lacking the catalytic subunit of pka are resistant to db-camp-mediated cell death. db-camp is cleaved by esterases into the biologically active compound n 6 -mb-camp and into 2'-o-mb-camp. other cyclic nucleotides (cnmps) in addition to camp, like ccmp and cump can also function as second messengers and activate pka and cgmp-dependent protein kinase (pkg) 1 . therefore, we investigated the effects of a series of membrane-permeable analogues of camp, cgmp, ccmp and cump in s49 wt und s49 kincells on apoptosis. stimulation with db-ccmp or db-cgmp induced neither apoptosis in s49 wt nor in s49 kincells. interestingly, we observed apoptosis in s49 wt and s49 kin cells after incubation with membrane-permeable nucleotide acetoxymethyl ester (am)-analogues of cgmp, ccmp, cump and also camp. induction of apoptosis occurs via pkadependent and also pka-independent pathways. a potential role of pkg and of the exchange protein activated by camp (epac) in the induction of apoptosis is unsolved and will be explored by specific pkg-and epac-activators in this system. investigations are on the way to identify the targets, the involved signal transduction pathways and the mechanisms of pro-apoptotic actions mediated by cnmp-ams. (1) wolter s, golombek m, seifert r (2011) differential activation of camp-and cgmpdependent protein kinases by cyclic purine and pyrimidine nucleotides. biochem biophys res commun. in press apoptosis in s49 cells : induction of apoptosis in s49 wt and in s49 cells lacking the catalytic subunit of pka (s49 kin-) with a) db-cnmps and b) cnmp-ams for 72h. nebivolol reduces vascular inflammation in spontaneously hypertensive rats wu z., xia n., förstermann u., li h. institut für pharmakologie, obere zahlbacher str. 67, 55131 mainz, germany nebivolol is a third generation β1 receptor blocker with additional effects on endothelial nitric oxide production. the aim of the present study is to investigate the antiinflammatory effects of nebivolol in vivo. 48 spontaneously hypertensive rats (shr) were divided into two groups: control or nebivolol treatment group. nebivolol treatment (5 mg/kg/day for 10 days) significantly reduced the blood pressure and the heart rate in shr. the drug had no effect on coagulation. aorta from nebivolol-treated rats showed significantly improved endothelial function. nebivolol did not change the expression levels of aortic nf-kb, but significantly reduced its dna binding activity. furthermore, nebivolol decreased the expression of adhesion molecules (e.g. icam-1, vcam-1) and pro-inflammatory cytokines (e.g. il-6). in conclusion, nebivolol reduces vascular inflammation in experimental hypertension. it inhibits nf-kb activity, decreases the expression of adhesion molecules and pro-inflammatory cytokine, and improves endothelial function. characterization of the cellular activity of pde4 inhibitors using two novel pde4 reporter cell lines wunder f., quednau r., barg m., tersteegen a. bayer pharma ag lead discovery wuppertal, aprather weg 18a, 42096 wuppertal, germany cyclic nucleotide-specific phosphodiesterases (pdes) play an essential role in cellular signal transduction by regulating the intracellular levels of camp and cgmp and, therefore, are important pharmacological targets. we report here the generation and pharmacological characterization of two novel pde4 reporter cell lines. plasmid constructs encoding human pde4b1 or pde4d3 were stably co-transfected with the beta1-adrenoceptor in a parental camp reporter cell line expressing a cyclic nucleotide-gated (cng) cation channel, acting as the biosensor for intracellular camp. in this reporter cell line, camp levels can be monitored in real-time via aequorin luminescence stimulated by calcium influx through the cng channel. by using different pde4 and non-pde4 inhibitors, we could show that our novel pde4b1 and pde4d3 reporter cell lines specifically monitor pde4 inhibition with high sensitivity. pde4-selective inhibitors alone did not increase basal luminescence levels in this experimental setting. however, these inhibitors induced concentration-dependent luminescence signals in combination with the adrenoceptor agonist isoproterenol. in contrast, in a stable beta1-adrenoceptor reporter cell line with no recombinant pde4 expression, pde4 inhibitors had no effect on isoproterenol-stimulated luminescence signals. we compared the cellular activity of different pde4 inhibitors with the in vitro inhibition of full-length and truncated (catalytic domain) pde4d3 from cell lysates. two different groups of pde4 inhibitors could be identified. the first group, including the allosteric inhibitors pmnpq and d159153, showed high cellular activity and much better inhibition of full-length versus truncated pde4d3. the second inhibitor group, including classical competitive inhibitors like roflumilast, cilomilast and piclamilast, showed comparably lower cellular activity and similar inhibitory activity on full-length and truncated pde4d3. the results imply that these novel pde4 reporter cell lines are well-suited for the characterization of the cellular activity of pde4 inhibitors and may also support a better understanding of the complex pde4 pharmacology. plexin-b2 is required for kidney regeneration after acute renal failure xia j. 1 , gröne h acute renal failure is a common clinical problem with unsatisfactory therapeutic options and high mortality in humans. therefore, unraveling the mechanisms that promote kidney regeneration and repair may provide new therapeutic strategies for acute renal injury. plexin-b2 belongs to a family of transmembrane receptors which mediate the cellular effects of semaphorins. while plexins have first been described in the context of axon guidance, several recent studies have established them as key regulators of organogenesis, the immune system and cancer. we have recently found that plexin-b2 is highly expressed in the adult kidney, particularly in tubular epithelial cells which are most sensitive to acute ischemic injury. to study the role of plexin-b2 during kidney regeneration we generated mice lacking plexin-b2 specifically in tubular epithelial cells. under physiological conditions, these mice displayed normal kidney morphology and function. in contrast, following ischemia/reperfusion injury, plexin-b2 conditional knockout mice exhibited severely impaired kidney regeneration. while the renal function of control mice fully recovered within 3 weeks after injury, plexin-b2 knockout mice had strongly elevated serum creatinine and urea levels associated with increased morbidity and mortality. this was accompanied by hyperproliferation of tubular epithelial cells and obstruction of tubular lumina. we conclude that plexin-b2 is required for regeneration after acute ischemic renal injury and that pharmacological interventions activating plexin-b2 might represent a new therapeutic strategy in acute renal failure. the nadph oxidase enzyme complex consists of two membrane-bound catalytic subunits (a nox protein and p22phox) and several cytosolic regulatory components including p47phox, p67phox, p40phox and the small gtpase rac1. we have previously shown that treatment of apolipoprotein e knockout mice with resveratrol led to a downregulation of nox2 and nox4 in the heart. our recent data demonstrated that resveratrol also reduced the enzymatic activity of cardiac nadph oxidase. because activation of nadph oxidase enzyme complex is induced by translocation of the regulatory subunits, we studied whether the reduced enzymatic activity is due to an inhibition of such a translocation. indeed, resveratrol treatment prevented rac1 membrane translocation from cytosol. resveratrol is known as an activator and expression enhancer of the longevity gene sirtuin 1 (sirt1). we then wanted to find out whether the effect of resveratrol on rac1 was mediated by sirt1. sirt1 is a histone/protein deacetylase. in vitro incubation of rac1 with sirt1 led to a reduction of lysine acetylation. deacetylation of rac1 on lysine 166 could be identified by mass spectrometry analyses. the lysine 166 lies within the p67phox-binding region of rac1. consistently, in vitro incubation of rac1 with sirt1 markedly reduced its binding activity to p67phox. in conclusion, we provide evidence that rac1 is a direct target molecule of sirt1. sirt1 deacetylates rac1 on lysine 166 and thereby inhibits its interaction with p67phox. this is a novel mechanism of nadph oxidase inhibition by sirt1/resveratrol. mutational analysis of the effects of the cardioprotective drug dexrazoxane on topoisomerase ii beta in vitro yan t., deng s., frensch i., gödtel-armbrust u., wojnowski l. universitätsmedizin der johannes gutenberg-universität mainz institut für pharmakologie, obere zahlbacher straße 67, 55101 mainz, germany dexrazoxane (drz, icrf-187) is the only approved drug shown to protect against anthracycline-induced heart failure. the protection is usually ascribed to the ironchelation by drz, which is thought to reduce anthracycline-induced oxidative stress. however, similarly to anthracyclines, drz is also an inhibitor of the anthracycline target topoisomerase ii (top2). we hypothesized that the cardioprotective effects of drz are mediated by the prevention of the anthracycline-induced dna damage mediated by top2b, the dominant cardiac top2 isoform. this was investigated using top2b mutants resistant to drz, which were expressed in cells depleted of wild-type top2 isoforms. top2b-mediated double-strand dna breaks were assessed as γ-h2ax. the levesl of dsb generated by the top2b mutants in response to the anthracycline doxorubicine (dox) was indistinguishable from that mediated by a wild-type top2b. preincubation with drz depleted wild-type top2b and this was accompanied by a decrease in the dna damage following a subsequent exposure to dox. in contrast, neither top2b depletion nor the reduction of dsb by drz was seen in drz-resistant top2b mutants. furthermore, the cardially ineffective drz analog icrf-161, capable of iron chelation but not of top2 binding, affected neither the stability of top2b, nor the dox-induced dna damage mediated by this enzyme. these results indicate that drz may exert its cardioprotective effects by reducing the dna damage mediated by doxpoisoned top2b rather than by iron chelation. they also suggest a cardioprotective function of top2b, which is currently under investigation using cardiomyocyte-specific top2b mouse knockouts. aminoglycosides are important antibiotics in the treatment of life-threatening infections, especially those caused by gram-negative bacteria. their nephrotoxic and ototoxic potential is well-known, but little is known about the effects of aminoglycosides on the male reproductive system. we studied the effects of four aminoglycosides on sertolicells in vitro. rat sertoli-cells from the cell line serw3 were cultivated for 3, 6, and 9 days in dmem supplemented with three different concentrations of amikacin, streptomycin (30 mg/l, 100 mg/l, 300 mg/l), gentamicin or tobramycin (10 mg/l, 30 mg/l, 100 mg/l). we determined the expression of two junctional proteins (connexin 43, ncadherin) and one protein of the cytoskeleton (vimentin) by western blot. cells were solubilized in lysis buffer. lysates were separated by sds-page and electroblotted on a pvdf-membrane. after incubation with primary antibodies overnight and horseradish peroxidase-conjugated secondary antibody the visualization was achieved by a chemiluminescence-detection system. in addition, proteins were detected by immunohistochemistry. after three days in culture amikacin caused the most pronounced effect. at the lowest concentration tested (30 mg/l) connexin 43 and n-cadherin were reduced to 55±19% and 92±10% of the controls (n=6). no change was recognized for vimentin (102±16%). effects obtained with streptomycin were less pronounced for these these proteins (68±16%, 96±7%, and 102±13%, respectively). similar, but less pronounced effects were observed with gentamicin and tobramycin at a concentration of 10 mg/l (connexin 43: 88±15% and 81±15%; n-cadherin: 95±18% and 103±11%; vimentin: 81±12% and 102±19%) and 30 mg/l (connexin-43: 84±19% and 78±18%; n-cadherin: 98±19% and 103±13%; vimentin: 102±8% and 115±15%). after incubation for 6 and 9 days the effects occurred in the same range. the substances showed no influence on the viability of serw3 sertoli-cells up to 300 mg/l in the mtt assay. by immunohistochemistry we showed that the localisation of the proteins -connexin 43 and n-cadherin at the cell membrane and vimentin in the cytoplasm -was not influenced by the aminoglycosides. large conductance calcium-and voltage-gated potassium (bk) channels play an important role in controlling membrane potential and calcium influx, and are strongly modulated by protein kinases at multiple sites. the stress-regulated exon (strex) adds to the bk channel c terminus a cysteine-rich insert of 59 amino acids that inverts the channel regulation by protein kinase a (pka) from excitatory to inhibitory. here we investigated the mechanisms by which the strex insert influences bk channel regulation by protein kinase c (pkc). activity of bk channels without strex insert (bk-zero), transiently expressed in hek cells, was inhibited by pkc in inside-out membrane patches (~ 50% inhibition). bk channels with strex insert (bk-strex), however, were insensitive to pkc. phosphomimetic mutation of a pkc phosphorylation site (s700e) in bk-strex, resulted in a ~50% reduction of basal channel activity, whereas the s700a mutant retained normal activity. to examine whether palmitoylation, and thus association of the strex domain with the plasma membrane, prevents pkc inhibition of bk channel gating, palmitoylation was abolished by either site-directed mutagenesis (c12:13a) or by pharmacological inhibition of palmitoyl transferases with 2-bromopalmitate (2-bp). both, mutation and pretreatment with 2-bp resulted in the expression of bk-strex channels which were sensitive to pkc (~ 50% inhibition of channel activity). no inhibitory pkc effect was observed in patches of the bk-strex s700a channel mutant pretreated with 2-bp. in a clonal rat somatomammotroph pituitary cell line (gh3b6), in which pcr products without (zero) and with the 174 bp strex exon could be identified, the pkc activator pma blocked channel activity by ~25 %. this inhibition was increased to over 50% when gh3b6 cells were pretreated with 2-bp, indicating that both channel isoforms were functionally active. in summary, the present study demonstrates that palmitoylation of strex prevents bk channel regulation by pkc, which is mediated by phosphorylation of ser700, probably by steric hindrance. our results provide further evidence for a cross-talk between palmitoylation and phosphorylation as a crucial mechanism underlying the dynamic regulation of ion channels. human pleural mesothelial met-5a cells are a limited in vitro model system in determining potential asbestos-like genotoxic effects of multiwall carbon nanotubes ziemann c. 1 , reamon-büttner s. multiwall carbon nanotubes (mwcnt) are nanomaterials with important technological impact. depending on their diameter, length, and biopersistence, however, some mwcnt seem to exhibit a fiber-like cytotoxic and genotoxic potential, similar to asbestos. thus, a project funded by the german federal ministry of education and research (bmbf contract no. 03x0109a) focuses on potential adverse biological effects of different types of mwcnt to enlarge the knowledge base about toxicity determining parameters. this project comprises both in vitro (rat) and in vivo endpoints with long amosite asbestos as a positive control. as mesothelial cells are target cells for adverse effects of asbestos, in particular mesothelioma development, the human sv40transformed, non-malignant pleural mesothelial cell line met-5a was chosen as the main in vitro model in this project. in the present study part, met-5a cells were characterized concerning their usefulness as an in vitro model to study potential asbestos-like cytotoxic and genotoxic effects of different mwcnt varieties. using an mwcnt-optimized lactate dehydrogenase liberation assay and proliferation parameters derived from cell counts, concentration-dependent cytotoxicity of long amosite asbestos (2, 10, and 20 µg/cm 2 ) was demonstrated in met-5a cells. cells also showed asbestosinduced increase in dna-strand breaks and oxidative dna-damage in the hogg1modified comet assay. thus, met-5a cells were responsive to asbestos treatment. owing to asbestos potential to induce aneugenic effects and spindle fiber damage, micronucleus induction, determination of numerical chromosome aberration, and altered meta-, ana-, and telophase morphology were planned as in vitro endpoints. met-5a cells were thus initially characterized in this regard and were found to exhibit highly variable chromosome numbers with lower than 10% cells exhibiting a normal diploid chromosome set, an up to twentyfold higher spontaneous micronucleus frequency, as compared to polychromatic bone marrow erythrocytes in rodents, and a profound number of aberrant meta-, ana-and telophases with bridges, lagging chromosomes and multipolar divisions. in conclusion, met-5a cells are of only limited value as an in vitro model system to study potential asbestos-like effects of mwcnt and also biopersistent fibers. the cells are indeed responsive to asbestos, but unfortunately demonstrate marked genomic instability and thus limited significance concerning genotoxic effects. waixenicin a inhibits cell proliferation through magnesium-dependent block of trpm7 channels zierler s. 1 transient receptor potential melastatin 7 (trpm7) channels represent the major magnesium-uptake mechanism in mammalian cells and are key regulators of cell growth and proliferation. they are abundantly expressed in a variety of human carcinoma cells controlling survival, growth and migration. these characteristics are the basis for recent interest in the channel as a target for cancer therapeutics. we screened a chemical library of marine organism-derived extracts and identified waixenicin a from the soft coral sarcothelia edmondsoni as a strong inhibitor of overexpressed and native trpm7. waixenicin a activity was cytosolic and potentiated by intracellular free magnesium (mg 2+ ) concentration. mutating a mg 2+ -sensitive site on the trpm7 kinase domain reduced the potency of the compound, whereas kinase deletion enhanced its efficacy independent of mg 2+ . waixenicin a failed to inhibit the closely homologous trpm6 channel and did not significantly affect trpm2, trpm4, and crac (calcium release activated calcium) channels. therefore, waixenicin a represents the first potent and relatively specific inhibitor of trpm7 ion channels. consistent with trpm7 inhibition, the compound blocked cell proliferation in human jurkat t-cells and rat basophilic leukemia cells. based on the compound's ability to inhibit cell proliferation through mg 2+ -dependent block of trpm7, waixenicin a or structural analogs may have cancer-specific therapeutic potential, particularly since certain cancers accumulate cytosolic mg 2+ . release of metals from different sections of domestic drinking water installations zietz b. p., richter k., laß j., suchenwirth r., huppmann r. governmental institute of public health of lower saxony division of environmental medicine and environmental epidemiology, roesebeckstraße 4-6, 30449 hanover, germany different metals were used as important piping materials in the drinking water supply for a long time. due to corrosion metals can leach into the tap water. of special importance is the toxic element lead. however other heavy metals in drinking water such as copper, nickel and cadmium can also give reason for health concerns. in this study it was investigated in which amount relevant metals were released from different parts of domestic installations into the cold water. for the spatial allocation of the emission sources a sequential water sampling protocol was used after three hours of stagnation time representing the first 5 litre of the water column. after stagnation ten sample volumes were collected in series. existing facilities of domestic installations constructed with different plumbing materials were examined predominantly from residential buildings. the elements al, as, cd, cr, cu, fe, mg, mn, ni, pb, sb, se, u and zn were detected by means of icp-ms. in total 16 water pipe strands of 11 domestic installation systems were examined. they comprised 379 single water samples and 5306 single parameters. depending upon the type of plumbing different courses and concentration ranges of the elements could be measured in the tap water samples. terminal taps or installation parts were frequently responsible for a release of nickel and in several cases of cadmium. the concentration courses of the element zinc proved as a good indicator for the allocation of the emission source to a brass containing section of the installation (zinc as an alloy component of brass). one can conclude that an investigation by means of a sequential water sampling protocol and multi-element detection can be a valuable non-destructive method for drinking water-hygienic investigations of domestic installations. novel interaction partners of the murine trpc4 protein zimmermann j., beck a., flockerzi v. universität des saarlandes experimentelle und klinische pharmakologie und toxikologie, kirrberger str. 1, 66421 homburg, germany in this work novel interaction partners of the murine protein transient receptor potential canonical 4 (mtrpc4) were identified. the trpc4 protein is the major subunit of a cation channel, residing in the plasma membrane. it comprises six trans-membrane domains and cytosolic amino and carboxyl termini. two major splice variants of the trpc4 gene exist, trpc4a (974 aa) and trpc4b (890 aa), trpc4b lacks aa 781 to 864 of the trpc4a variant. both trpc4 variants are co-expressed in endothelial cells, intestinal smooth muscle and brain. to identify trpc4-interacting proteins a yeast two-hybrid system, cytotrap®, which allows identification of protein-protein interactions within the cytosol was used. a premade mouse brain cdna library was screened by the cytosolic amino and carboxyl terminal parts of mouse trpc4a (aa 1 to 324; aa 622 to 974). for the carboxyl terminal part fourteen proteins were identified. to independently prove the interaction, the fulllength cdnas of all fourteen proteins were cloned, fused to a flag-tag and coexpressed with trpc4 in hek 293 cells. co-immunoprecipitations were performed for all candidates using both the anti-flag-antibody and the antibody for trpc4. in addition, changes of cytosolic calcium were monitored and trpc4 currents were recorded in hek 293 cells expressing the candidate cdnas and stably expressing the trpc4a or trpc4b and the muscarinic receptor type 2 cdnas. the tarbp2 protein, one of the candidates shown to interact with trpc4, changed calcium influx when coexpressed with trpc4. in order to identify the domains of trpc4 responsible for its interaction with the tarbp2 protein, six trpc4-gst-fusion proteins covering the carboxyl terminal 353 aa of trpc4a were expressed in e. coli and used for pull-down experiments. by this approach two domains of trpc4 could be identified to interact with tarbp2. one of these domains is well conserved within the trpc5 protein, corresponding to the result, that trpc5 and tarbp2 effectively co-immunoprecipitate, too. the tarbp2 protein has been shown to be a component of the risc loading complex, also known as the micro-rna loading complex which is composed of dicer1, eif2c2/ago2 and tarbp2 (chendrimada et al. 2005) . by its interaction it may link trpc4 to pre-microrna processing. increased levels of angiotensin ii provoke dna damage and have influence on dna repair in mouse kidneys zimnol a., brand s., schupp n. universität würzburg institut für toxikologie, versbacherstr. 9, 97078 würzburg, germany the renin-angiotensin system (ras) plays a crucial role concerning the blood pressure, electrolyte balance and cardiovascular homeostasis. angiotensin ii (ang ii), the active hormone of the ras, in higher concentrations leads to vasoconstriction, oxidative stress and hypertension. hypertensive patients have an increased risk to develop cancer, especially kidney cancer. we have shown in vitro and in vivo, that ang ii is capable to cause an elevation of blood pressure as well as dna damage dose-dependently. to investigate whether the high blood pressure or the enhanced levels of ang ii are responsible for dna damage, male c57bl/6-mice were equipped with osmotic minipumps, delivering ang ii in a concentration of 600ng/kg · min during 28 days. additionally they were treated with ramipril, an angiotensin-converting-enzyme blocker, with the ang ii receptor antagonist candesartan, the vasodilator hydralazine, and the antioxidant tempol. dna damage was analysed with the comet assay. we measured the base excision repair (ber)-related dna repair in the kidney with a comet-based in vitro repair assay. furthermore, the distribution and expression of the ang ii-type 1 (at1) receptor in the kidney was analyzed by immunohistochemistry. treatment with ang ii led to a significant increase of blood pressure, whereas the medication with candesartan decreased the systolic blood pressure. the intervention with hydralazine lowered the blood pressure only for a short time. the other substances had no effect at all on the blood pressure. genomic damage, quantified with the comet assay, was augmented by ang ii and improved by all interventions, particularly by candesartan and tempol. beyond that, ang ii showed a tendency to reduce dna repair. treatment with candesartan, hydralazine and tempol increased the repair capacity. furthermore, ang ii tended to result in a downregulation of the at1 receptor in kidney tubule cells. candesartan and ramipril, especially were able to augment the expression of the at1 receptor, whereas hydralazine achieved the opposite. these results demonstrate that ang ii leads to dna damage in the kidney independent of blood pressure. apparently elevated levels of ang ii affect dna repair and expression of at1 receptor. to confirm these findings we are going to examine more precisely the manifestation of other enzymes, which are implicated in dna repair. regulation of hcn channel activity by cyclic cytidine 3´, 5´-monophosphate zong x., krause s., chen c. -c., gruner c., cao-ehlker x., fenske s., wahl-schott c., biel m. lmu münchen, department pharmazie, pharmakologie für naturwissenschaften center for integrated protein science munich (cipsm), butenandtstr. [5] [6] [7] [8] [9] [10] [11] [12] [13] 81377 münchen, germany hyperpolarization-activated cyclic nucleotide-gated (hcn) channels play a key role in controlling cardiac pacemaker activity and are essential for normal function of neuronal circuits. hcn channels are principally gated by voltage but are coactivated by the cyclic nucleotides camp and cgmp which directly bind to a c-terminal binding domain. recently, cyclic cmp (ccmp) was shown to be present in various cell lines and tissues at concentrations that are comparable to cellular cgmp levels. moreover, there is recent evidence that ccmp can activate camp-and cgmp-dependent protein kinases in vivo. here, we examined whether ccmp exerts effects on hcn channels. to this end, we recorded hcn channel-mediated currents (i h) in hek 293 cells that stably express hcn1, hcn2, hcn3 or hcn4, respectively. currents were measured either with a standard patch-clamp setup or by employing the planar patch-clamp technology. in hcn2 and hcn4 channels, ccmp shifted the membrane potential for half maximal activation (v0.5) to more positive values. in addition, ccmp accelerated activation while it slowed down deactivation kinetics. the ec50 for ccmp was 30 µm which is about 5 times higher than the ec50 of cgmp. cyclic cmp is a partial agonist of hcn channels since it activates only 65 % of the maximal current obtained with camp or cgmp. to identify in vivo effects of ccmp we recorded ih of murine sinoatrial node (san) cells in the presence and absence of 1 mm ccmp. like for heterologously expressed hcn channels, ccmp shifted v0.5 of ih by about +5 mv. importantly, the steepness of the diastolic depolarization of san pacemaker potentials which is mainly determined by the amplitude of ih was profoundly increased by ccmp, compared to control conditions. as a consequence of the upregulation of ih the frequency of san pacemaker potentials was increased by about 20 % in the presence of ccmp. our results suggest that ccmp is a physiological regulator of hcn channel activity. a 3d actinic keratosis like construct for assessment of innovative tumour therapeutics zoschke c. 1 the incidence of actinic keratosis (ak) has increased dramatically in the last decades and it is considered the most frequent carcinoma in situ today. especially immunosuppressed patients are at high risk to develop invasive squamous cell carcinoma (scc) [1] which asks for most efficient and well-tolerated ak therapy. yet, current measures do not fit with these demands. nucleotide analogues, recently identified by molecular modelling [2, 3] , outperformed the current standard for the therapy of actinic keratosis, 5-fluoruracil, when tested in the tumour cell line scc25, in normal human keratinocytes and fibroblasts [4] . as next step in pre-clinical drug assessment, we aimed to characterise the effect of the most selective nucleotide analogue oxbu in reconstructed human tumour skin. based on the 3d construct of scc developed by höller and co-workers [5] for start we introduced several adaptations with respect to keratinocyte, fibroblast and scc12 seeding to grow an aklike construct with scc12 cells forming nests in particular in the epidermis. in addition, first experiments with the oxbu on the ak like constructs showed promising results for an efficient treatment of actinic keratosis. efficacy was derived from immunohistology (marker for proliferation: ki-67, marker for scc: cytokeratin-10, axl, marker for invasion: mmp2, marker for apoptosis: caspase-7, nuclei were stained with dapi) as well as the effects on the secretion of cytokeratin-18 and its caspase-induced cleavage product into the culture medium following drug exposure for up to 7 days. efficacy of a 0.05% oxbu solution proved close to or even better when compared to both a 0.1% 5fluorouracil and 0.025% aphidicolin solution. the former being the gold standard of current ak therapy, the latter is a frequently used inhibitor of human polymerase alpha and delta, however, failed to be introduced into clinical use. -in fact, in monolayer cultures aphidicolin proved most toxic for normal human keratinocytes which was not true with oxbu [4] . -therefore, these 3d tumour constructs offer a new approach to pre-clinical drug assessment and may be added to other 3d models of skin diseases currently gaining increased interest as test platforms. ima910, a multi-peptide cancer vaccine for advanced colorectal cancer, induces multiple cd8+ and cd4+ t-cell responses associated with improved survival walter s. 1 , kuttruff s. ima910 is a novel peptide-based vaccine consisting of 10 hla-a*02 and 3 hla-dr binding synthetic peptides that were identified based on natural presentation on human colorectal cancer (crc) samples. ima910 was characterized in a phase i/ii trial in advanced/metastatic crc patients being at least clinically stable after 12 weeks of first-line oxaliplatin-based therapy. all patients received a single application of low-dose cyclophosphamide for immunomodulation. this was followed by repeated ima910 vaccinations in combination with low-dose gm-csf (cohort 1; n=66) or ima910 / gm-csf plus topically applied imiquimod (cohort 2; n=26). before and post vaccination, patients were analyzed by hla-multimer assay and intracellular cytokine (ics) assay for cd8 + t-cell responses and by ics assay for cd4 + tcell responses. as immune status biomarkers, 6 phenotypically defined myeloid derived suppressor cell populations (mdsc1-6) were analyzed prior to immunotherapy. tumor status of patients was monitored repeatedly by ct/mri according to recist and corresponding tumor scans were reviewed centrally. clinical assessment included disease control rate (dcr), time to progression (ttp), progression-free survival (pfs) and overall survival (os). ima910 overall was immunogenic in 73/81 (90%) evaluable patients, with 43% and 65% of patients mounting multiple cd8 + and cd4 + t-cell responses, respectively. patients that received the immunomodulator imiquimod presented with significantly more multiple cd8 + cell responses as detected by ics (p=0.016). multiple cd8 + and multiple cd4 + responses were individually associated with significantly better clinical outcome. the association was most pronounced for patients with both multiple cd8 + and multiple cd4 + responses. these patients had significantly higher dcr at 6 months (p=0.002), improved ttp (p=0.006) and improved pfs (p=0.009) than other patients. most importantly, a trend for prolonged os was also observed in these patients (p=0.088, hazard ratio 0.53). in the study population, levels of 5 different mdsc phenotypes were significantly increased as compared to age/gender matched controls. high mdsc levels were associated with fewer immune responses and for mdsc4 and mdsc5 high frequencies were associated with shorter os (p=0.007 and p=0.019, respectively). to summarize, both hla-a*02 and hla-dr restricted peptides in ima910 were immunogenic. a significantly better clinical outcome of multi-tumap responders in comparison to patients with one/no tumap response strongly indicates clinical activity of ima910. acrylamide (aa), a genotoxic carcinogen (iarc class 2a) is formed in food by thermal treatment from different precursors. after oral ingestion, aa is metabolically epoxidized in the liver by cyp450 2e1 into glycidamide (ga). ga binds to dna, forming covalent adducts, primarily at n7 of guanine (n7-ga-gua). both, aa and ga undergo conjugation to glutathione (gsh) to be excreted via urine as mercapturic acids (ma), namely nacetyl-s-(2-carbamoylethyl)-cysteine (aama), and n-acetyl-s-(2-hydroxy-2carbamoylethyl)-cysteine (gama). in a dose response study, encompassing the dosage range from human dietary exposure levels up to 10 mg/kg bw, female sprague dawley (sd) rats on a diet devoid of detectable aa content were gavaged with single doses of aa. formation of urinary mas and of n7-ga-gua dna adducts in liver, kidney and lung was measured 16 h after application, a time point where cmax of n7-ga-gua was reached. the untreated control group was found to excrete about 0.8 nmol (aama plus gama) in the urine (16 h), indicating a background of endogenous aa formation. compared to untreated control, the lowest dosage of 0.1 µg aa/kg bw neither resulted in significantly enhanced ma excretion, nor in a detectable n7-ga-gua adduct levels in any organ tested (limit of detection, lod, 0.2 adducts/10 8 nucleotides). at the tenfold higher dose (1 µg/kg bw), adducts were found in kidney (about 1 adduct/10 8 nucleotides) and lung (< 1 adduct/10 8 nucleotides), but not in liver. at 10 and 100 µg/kg bw, adducts were found in all three organs, at levels not significantly different to those found at 1 µg aa/kg bw (about 1-2 adducts/10 8 nucleotides). the results of this in vivo study and of further recent research on aa toxicology will be discussed with respect to risk assessment. exposure of rats to single doses of aa in the range of human dietary exposure (0.1-10 µg/kg bw ) leads to n7-ga-gua adduct levels in the tissues monitored obviously not exceeding the range of steady state background dna lesions associated with endogenous/exogenous exposure to various genotoxic electrophiles. thus, the question of significant impact on human background dna damage resulting from exposure to a given genotoxic carcinogen, and on potentially ensuing biological consequences may become a highly relevant issue in risk assessment. pharmaco-economic impact of price, volume and demographic development böcking w., kirch w. institut für klinische pharmakologie, medizinische fakultät der tu dresden, fiedlerstr. 27, 01307 dresden health insurance costs in germany have grown by 3% p.a. over the last ten years and amount to approx. 280 bn eur in 2009. while costs for stationary treatment as the largest cost category have been intensely analyzed over the past years, pharmaceutical expenses have been analyzed in less detail, mostly focusing on the statutory health insurance side, even though pharmaceutical expenses have grown almost twice as much as costs for ambulant treatments. therefore, the question was asked how pharmaceutical expenses in a large german private health insurance company are allocated with respect to age and indication groups, and how those have developed from 2007 to 2011. the data of a private health insurance company with more than 600.000 customers was split into price and volume effects per age group to understand if price or volume drives the cost development. additionally, the two largest indication groups are analyzed in detail. as a result, both price and volume effects drive an overall cost increase. these effects are even stronger in older age groups. this cost increase is not sustainable for the german health insurance system over a longer period of time and will even further increase due to the ageing of the german population. a novel animal replacement system for the detection of endocrine disruptive capabilities in sexual development scheider j. 1, 2 , winter p. alternatives to animal testing for prediction of local toxicity and genotoxicity have been recently established. however, currently these methods are not suitable for measuring endocrine effects in developing organs such as e.g. embryonic gonads. here we present a phenotypic anchoring of a comprehensive study on sex-specific gene expression analysis accompanied by histological analysis of endocrine disruption in chicken embryo gonads, having the potential for an animal replacing system for endocrine disruptive toxicologic and ecotoxicologic examinations of chemicals. chicken embryos were inoculated with different amounts of tributyltin (tbt) and bisphenol-a (bpa). embryos were incubated and their gonads analyzed histologically 2 d prior to hatching. from identically treated embryos right and left testes and ovaries were separated and genome-wide transcription profiles generated using supertag digital gene expression (st-dge, supersage) profiling. male and female gonadal tissues both revealed histological aberrations in response to tbt and bpa. female gonads became masculinized in response to tbt and, viceversa, bpa-treated male gonads underwent feminization whereas in female gonads clearly visible structural aberrations occurred. in both chemicals mortality increased especially in the most affected sex (tbt: females, bpa: males). the expression profiles of more than 60 million mrnas revealed massive effects of both chemicals, tbt and bpa, on important cellular signaling pathways. gene expression differences were most pronounced in the phenotypically most affected sex. our results demonstrate that endocrine disruptive chemicals exert their effects on several levels including but not restricted to known hormone-based pathways. together with an ongoing study of gene expression differences in very young life stages and different chemicals these data will form the base for a blow-by-blow analysis of sexspecific gene expression of embryonic development. the project builds on already existing and further to generate data with the aim of the development of an in vitro method for testing chemicals at chicken eggs for 1) replacement of tests on juveniles and (sub-) adult rodents, 2) stages with impossibility of sensation of pain in the individuals, 3) highly sensitive prospects of modes of action of chemicals, which 4) might show consequences in the next generations. 3 channels are critical for oscillatory burst discharges in the reticular thalamus and absence epilepsy differential distribution of three members of a gene family encoding low voltage-activated (t-type) calcium channels hippocampal seizure resistance and reduced neuronal excitotoxicity in mice lacking the cav 2.3 e/r-type voltage-gated calcium channel transcriptional upregulation of cav3.2 mediates epileptogenesis in the pilocarpine model of epilepsy structure and functional expression of a member of the low voltage-activated calcium channel family a molecular determinant of nickel inhibition in cav3.2 t-type calcium channels histidine residues in the is3-is4 loop are critical for nickel-sensitive inhibition of the cav2.3 calcium channel substrate recognition and translocation by polyspecific organic cation transporters proton pump inhibitors inhibit metformin uptake by organic cation transporters (octs) structural determinants of inhibitor interaction with the human organic cation transporter oct2 (slc22a2) functional characterization of the human organic cation transporter 2 variant p.270ala>ser extra-adrenal glucocorticoid synthesis in the intestinal epithelium: more than a drop in the ocean? local glucocorticoid production in the mouse lung is induced by immune cell stimulation biomimetic materials in tissue engineering biomaterials offer cancer research the third dimension synthetic biomaterials as instructive extracellular microenvironments for morphogenesis in tissue engineering injectable self-assembling peptide nanofibers create intramyocardial microenvironments for endothelial cells directed growth of fibroblasts into three dimensional micropatterned geometries via selfassembling scaffolds novel pcl-based honeycomb scaffolds as drug delivery systems for rhbmp-2 tissue engineering spatio-temporal vegf and pdgf delivery patterns blood vessel formation and maturation presentation of rgds epitopes on self-assembled nanofibers of branched peptide amphiphiles controlling mammalian cell interactions on patterned polyelectrolyte multilayer surfaces langmuir avintegrins as receptors for tumor targeting by circulating ligands heparin binding nanostructures to promote growth of blood vessels tirrell endothelial cell adhesion to the fibronectin cs5 domain in artificial extracellular matrix proteins design and bioproduction of a recombinant multi(bio)functional elastin-like protein polymer containing cell adhesion sequences for tissue engineering purposes stimuli-responsive thin coatings using elastin-like polymers for epac as a novel effector of airway smooth muscle relaxation ) camp inhibits airway smooth muscle phenotype modulation functional roles of epac and pka in human airway smooth muscle phenotype plasticity assessment of the sensitizing and irritative potential of preservatives by loose-fit coculture-based sensitization assay (lcsa) sonnenburg a nsc-631570 (ukrain) in the palliative treatment of pancreatic cancer. results of a phase ii trial association of funding and conclusions in randomized drug trials: a reflection of treatment effect or adverse events a general method for the covalent labeling of fusion proteins with small molecules in vivo robust single-particle tracking in live-cell time-lapse sequences correlation of structural class with no-observed-effect levels: a proposal for establishing a threshold of concern trbp recruits the dicer complex to ago2 for microrna processing and gene silencing index a 009, 019, 035 011, 014 003, 044 objective: hypertension and arterial stiffness is influenced by environmental and genetic factors. high plasma sodium concentration leads to mechanical stiffening of endothelial cells resulting in endothelial dysfunction and elevated blood pressure. here we investigated whether endothelial cell stiffness of ex vivo preparations of human arteries is linked to plasma sodium concentrations and functional genetic variants of the mineralocorticoid receptor (nr3c2), rs2070951 modulating blood pressure, renin, and aldosterone levels, and rs5534, which alters a mirna binding site. design and methods: twenty patients were enrolled after a vein stripping procedure and collateral arterial blood vessels were prepared for atomic force microscopy (afm). plasma sodium concentration was routinely determined and dna for genotyping was extracted from edta blood samples. sodium levels >140 mmol/l were defined as 'high'. after application of 5 µm amiloride, a specific blocker of the endothelial sodium channel (enac) changes in endothelial cell stiffness, were defined as 'weak' (≤10%), or 'strong' (>10%). statistical analyzes were performed by anova. results: in ex vivo artery preparations of patients with high sodium levels (n=12), mechanical stiffness of endothelial cells was tend to increase (∆ amiloride) (p=0.06). both nr3c2 variants were associated with a change >10% in endothelial stiffness after amiloride treatment. the rs2070951 c allele was significantly associated with a strong amiloride response (p=0.024), while the rs5534 a allele only showed a trend towards stronger amiloride effects (p=0.06). conclusion: our findings indicate that high plasma sodium concentration results in an increased endothelial amiloride response and thus influencing mechanical stiffness, modulated by functional nr3c2 variants. our novel approach linking patients' sodium levels and genetic status to endothelial stiffness by afm will be further evaluated in larger clinical settings. protein expression changes in bap-exposed human bladder cancer cells from spliceosome activation towards redistribution of the cytoskeleton after long-term exposure to subacute concentration schmitz-spanke s., pink m., jeske e., stempelmann k., rehn s., verma n., rettenmeier a. w. universitätsklinikum essen institut für hygiene und arbeitsmedizin, hufelandstr. 55, 45122 essen, germany deregulation of the β-catenin signaling pathway plays an important role in the development of hepatocellular tumors. activating mutations in ctnnb1 (encoding β-catenin) are frequently observed in murine and human liver tumors (e.g. human hepatoblastomas). activation of β-catenin signaling induces an overexpression of several cytochrome p450 (cyp) enzymes, including cyp2e1. cytotoxicity of acetaminophen (aap) is based on its cyp2e1-catalyzed metabolism to the electrophilic compound n-acetyl-p-benzo-quinone imine, which forms covalent adducts with cellular macromolecules if depletion of glutathione occurs. treatment with aap should therefore lead to a selective damage of cyp2e1-overexpressing ctnnb1mutated hepatoma cells. mice were injected with a single dose of the liver carcinogen n-nitrosodiethylamine (den) and subsequently treated with the tumor promoter phenobarbital to select for ctnnb1-mutated tumors. administration of a single dose of aap (300 mg/kg of body weight) followed the tumor promotion protocol. two days after treatment immunohistological analysis of the livers showed about 90% necrotic tissue in the larger tumors which were positive for glutamine synthetase (gs), a marker for ctnnb1-mutated tumor cells. by contrast, gs-negative tumors remained unaffected. at later time points we observed regeneration processes with infiltration of the necrotic tissue by inflammatory cells followed by fibrotic cells. proliferation of normal hepatocytes surrounding the damaged areas could also be observed. however, repopulation of parts of the former tumor areas by remaining gs-positive tumor cells was also detected. these results suggest that treatment with aap might serve as a future therapeutic possibility to selectively poison cyp2e1-overexpressing hepatoma. release of 5,6-epoxyeicosatrienoic acid (5,6-eet) upon neuronal activity induces trpa1-dependent mechanical pain hypersensitivity sisignano m. 1 , epoxyeicosatrienoic acids (eets) are cyp-epoxygenase (cyp450) derived metabolites of arachidonic acid (aa) which act as endogenous signaling molecules in multiple biological systems. we investigated the specific contribution of 5,6-eet to transient-receptor potential-(trp)-channel activation in nociceptor neurons, and its consequence for nociceptive processing. we found that during capsaicin-induced nociception 5,6-eet-levels increased in the drg and it is released from activated sensory neurons in vitro. 5,6-eet potently induced a calcium flux [10 nm] in cultured drg-neurons which was completely abolished when trpa1 was deleted or inhibited. in spinal cord slices 5,6-eet dose-dependently enhanced the frequency, but not the amplitude of spontaneous excitatory postsynaptic currents (sepsc) in lamina ii neurons that also respond to mustard oil (aitc), indicating a presynaptic mechanism. furthermore, 5,6-eet-induced enhancement of sepsc frequency was abolished in trpa1 null mice, suggesting that 5,6-eet pre-synaptically facilitates spinal cord synaptic transmission via trpa1. finally, intrathecal injection of 5,6-eet caused mechanical hyperagesia in wild type but not trpa1 null mice. we conclude that 5,6-eet is synthesized upon acute activation of nociceptors and leads to mechanical hypersensitivity via trpa1 at central afferent terminals in the spinal cord. sisnaiske j. 1 , hardelauf h. introduction: neurite outgrowth and plasticity of neuronal networks are essential processes e.g. during brain development and learning. thus, morphological readouts of neuronal connectivity are thought to be important endpoints to assess neurotoxic effects of environmental chemicals as well as when discovering new drugs. to analyze neurite outgrowth and connectivity level rapidly and easily in vitro we developed the network formation assay (nfa) (pct/ep2010/002811). this platform requires a spatially standardized hexagonal array for culturing neuronal networks with no need to fix or stain the cells to visualize neuritic processes. methods: to demonstrate the feasibility of the nfa we performed experiments in which we disrupted mature neurite networks or inhibited generating networks of human sh-sy5y cells with different concentrations of acrylamide (acr). we also observed the counteracting effects of brain-derived neurotrophic factor (bdnf) and calpeptin in these systems. to create the hexagonal array we used a poly(dimethylsulfoxide) bilayer stencil comprising through holes for adhesion spots and interconnecting tracks. plasma stencilling a peg-coated glass substrate produces adhesive nodes for the neurons and micron-scale-tracks for guiding neurite outgrowth and connectivity. results: in both systems, the developing and mature network, we found not only a concentration dependant effect of acr and bdnf but also a time dependant effect with a limited capability of the developing system to regenerate, even in the presence of acr. the co-treatment of the cells showed that inhibition of calpains by calpeptin might reduce the effect of intracellular elevated ca2+, a known neurotoxic mechanism of acr. moreover, the neurothrophin bdnf acts via trkb receptors on pathways stimulating neurite outgrowth and thereby counteracting the adverse effect of acr. conclusion: with the nfa we provide a rapid and simple way to analyze neurite outgrowth and connection formation in real time. by spatially standardizing the array we provide assay coordinates to streamline the analysis process and bring it towards high throughput testing. furthermore preliminary data showed that modification of the surface with biomolecules allows cell adhesion of other neuronal celltypes (e.g. primary mouse neurons) and extracellular matrix proteins (e.g. laminin) stimulate neurite outgrowth via integrins. transcriptional regulation of nox4 by histone deacetylases siuda d. 1, 2 , zechner u. 3 , prawitt d. 4 nox4 is a member of the nadph oxidase family, which represents a major source of reactive oxygen species (ros) in the vascular wall. nox4-mediated ros production mainly depends on the expression levels of the enzyme. the present study is aimed to investigate the regulation mechanisms of nox4 transcription by histone deacetylase (hdac). in cultured human ea.hy 926 endothelial cells, treatment with the pan-hdac inhibitors (scriptaid, trichostatin a, tsa, and suberoylanilide hydroxamic acid, saha) leads to a drastic decrease in nox4 mrna expression. a similar down-regulation of nox4 mrna expression can be achieved with sirna-mediated knockdown of hdac3. hdac inhibition in endothelial cells is associated with enhanced histone acetylation in the human nox4 promoter region, with no significant changes in dna methylation. consistently, scriptaid-treated cells show increased chromatin accessibility in nox4 promoter. in addition, we provide evidence that c-jun plays an important role in controlling nox4 transcription. knockdown of c-jun with sirna leads to a marked downregulation of nox4 mrna expression. in response to scriptaid treatment, the binding to c-jun to the nox4 promoter region is reduced despite the open chromatin structure. in parallel, the binding of polymerase iia to the nox4 promoter is significantly inhibited as well, which may explain the reduction in nox4 transcription. in conclusion, hdac inhibition decreases nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the nox4 promoter. this is very likely because of a hyperacetylation-mediated steric inhibition. cyclopentenone prostaglandins induce oxidative dna-damage in hamster lung fibroblast v79 cells solecki g. m. 1 key: cord-015021-pol2qm74 authors: nan title: third international congress on the immune consequences of trauma, shock and sepsis —mechanisms and therapeutic approaches date: 1994 journal: intensive care med doi: 10.1007/bf02258437 sha: doc_id: 15021 cord_uid: pol2qm74 nan this issue of the journal contains the abstracts for the third international congress on the immune consequences of trauma, shock and sepsis -mechanisms and therapeutic approaches. we hope that the information contained in this special issue will stimulate you to participate in the congress, to contribute to the knowledge being developed in this field and to use this information to help you in providing better care for your patients. we thank the editors and the editorial board and publishers of the journal for their interest and support in preparation of this special issue. we also, on behalf of the scientific committee, welcome you to the third international congress in munich on 2-5 march 1994. when, in the mid-1980s, we thought of having a worldwide congress, we hoped to bring together investigators to discuss this theme. the explosion of knowledge occurring around that time provided an excellent background against which the first conference in 1988 provided stateof-the-art information and consensus on factors involved in injury and sepsis. in 1991, the second congress was held at the time of another resurgence of research, study and information on injured and operated patients. it seemed then that there would be a lull in the development of new information and therapy, and that another state-of-the art conference might not be necessary until 1995 or 1996. however, the explosion in molecular biology has continued. the wonderful world of cytokines has gone from ill to il-6 to il-11, il-12 and il-13 and beyond. the vast amount of information about mediators and their importance in disease is impressive. this has all suggested a magic bullet that might be used to alter or block inflammatory responses. this has not happened, however, and the question is "why not"? our science is powerful, but our therapy is still weak. what are the issues, then, in 1994, to be dealt with at this symposium and congress? (1) proposals for new terminology. there have been a number of proposals for new terminology and new classifications of injury, sepsis, inflammation and various other problems related to human illness. the question is whether this is the way to go. will this contribute to better clinical trials, information basis and better research? the pros and cons of this development will be reviewed by those making the proposals and those questioning the need for and wisdom of this effort. (2) magic bullets: the prospect of a magic bullet to deal with inflammation in injury and infection seemed highly promising earlier. many preclinical trials and a lot of animal research suggested the possibility of a great breakthrough in clinical care. what has become, then, of all the expensive and extensive multi-institution randomized, placebo-controlled, double-blind clinical trials of agents that block mediators and endotoxin. many such studies have yielded equivocal, marginal or negative results. the reasons for this and the future of clinical research will be the subject of presentations and discussions to set the stage for further work. (3) should future clinical trials be based on new classifications of illness such as mods, sirs, apache iii, sap ii, mrm, etc., or should trials be dedicated to specific diseases -urinary tract infections, pneumonia, trauma patients, cardiac surgery and other specific problems, rather than generalized problems of sepsis, the sepsis syndrome and other classifications? in other words, should we now begin to have clinical trials on specific diseases with causes that are known and can be attacked? the causality of disease becomes an important consideration in this regard. (4) a multitude of potential therapeutic agents has been proposed on the basis of animal studies. how should we decide which of them should be brought to clinical trial? the possibilities are endless as we develop new clinical information about the mechanisms and pathogenesis of human disease. (5) information on the pathogenic mechanism of disease states and of injury continued to emerge in an explosive fashion, and in light of our gathering knowledge we can look forward to working out a cohesive system of response to injury. (6) additional information will be provided in plenary sections, many symposia and free communication sessions and posters, which will update the participants on a variety of relevant topics presented by many of the leading in-iv vestigators in these fields. topics will range from molecular mechanisms, such as signal transduction, through the explosive growth of information on the role of cytokines and pathophysiology, to practical considerations in the design of immunomodulatory therapeutic regimens. these merely touch on a few areas, from the basic to the clinical, which will be the subjects of those symposia. all this information will fit into the jigsaw of this exciting area and its stimulus to further research study. this promises to provide an exciting, educational programme with experts and participants from all over the world. we hope it will set the stage for many years to come and will increase our understanding of trauma, shock and sepsis and help us to provide better therapy for those of our patients who are affected by such problems. a. the clinical syndrome of mods versus mof will be reviewed in detail by those who have made these proposals. b. an extensive review of the design and interpretation of clinical trials in patients with shock and injury will be provided. the reasons why so many clinical studies in the recent past have been negative will be reviewed. the therapeutic strategies that are being developed for the treatment or prevention of mods or mof will be the subject of another panel discussion by experts who have been involved in and contributed to this area. a consensus conference or controversy conference will be presented about various aspects of mods or mof, including the benefits of supernormal oxygen delivery, bacterial translocation, parenteral nutrition, the immune response and other aspects. the successes and failures of completed clinical trials will be presented by those who are involved in these clinical trials, with a refreshing review of the problems related to that injury. there will be late news about studies just being completed at present or after the beginning of 1994 and where they stand. c. the mechanisms and biochemical profiles of specific organ dysfunction or failure will be reviewed. what are the definitions? what are the mechanisms? how can organ dysfunction and/or failure be defined? an extensive review of the biological mechanisms involved in production of injury by mediators will be presented. a session will be devoted to how future ongoing trials might be better designed and what can be done about the studies recently completed, many of which are negative. d. the immunological or inflammatory pathways resulting in organ injury will be reviewed in detail in presentations and a panel discussion. we look forward to welcoming you to an exciting and rewarding conference, which undoubtedly possesses the potential to become a landmark event and major reference point for any scientific discussion about the complex of host defense dysfunctions following trauma, shock and sepsis. studies over the past 25 years have established that the contact system, which forms bradykin/~, is gax important mediator in hypotensive septicemia. in addition to hradyk{nln, another product of the contact system, kailikrein, can mediate inflammation by virtue of its chemotaetic mad neutrophj/activating properties. using functional and immunochemical tech~2ques, we have demonstrated activation of the contact system in the adult respiratory distress syndrome in typhoid fever and clin/cal sepsis. we have also been able to inhibit the hypotension but not the disseminated intravaseular coagulation in a model of primate sepsis by the use of a monoclonal antibody directed agsi~st factor xii, the initiating protein of the contact system, in volunteers given e. coil endotoxin, who did not develop hypotension, we were also able to demonstrate activation of the contact system with a rise of alpha-2macrogiebulin-kalllkrein complex. we have also examined, j~ an i~tensive care situation, patients with sirs. we found that serial measuremezzts of the contact system were useful in eva~u~ting prognosis+ these studies suggest that inhibition of kalllkrein a~d l e r bradykinin actions might be useful i~ obviating many .of the features seen in sepsis and septic shock. dextran sulfate (dxs) activates the contact system and, in vivo, produces transient hypotension. in order to better define the mechanisms underlying the dxs-induced hypotension, we investigated the effects of either the plasma kallikrein inhibitor, des-pro2-iarg]5]aprotinin (bay 4620) or the b2 kinin antagonist, hoe 140 on the hypotensive response to dxs. in the first study, anesthetized miniature pigs (5 pigs/group, randomly assigned) were given one of the following treatment protocols: 1) dxs (5 mg/kg), 2-5) dxs plus bay 4620 (45, 90, 180, or 360 rag), or 6) saline. dxs alone produced a profound but transient systemic arterial hypotension with a corresponding reduction in plasma kinin-containing kininogen. circulating kinin levels, complement fragment c3adesarg and fibrin mom)mer were all increased. bay 4620 produced a dose-dependent delay or attenuation in these effects with the highest dose completely blocking dxs-induced hypotension and elevations of kinin, c3adesarg and fibrin monomer levels. thus, the effects of dxs are solely dependent on contact system activation and this activation is sensitive to bay 4620. llowev~:r, contact system activation is known to produce changes in a variety of vasoactive mediators, all of which can affect blood pressure. in a second study, two groups of pigs (3/group) were given either dxs alone (2 mg/kg) or dxs 10 minutes after a bolus injection of hoe 140 (30 #g/kg). dxs alone produced transient hypotenmon. this response was completely blocked by hoe 140 pretreatment. both groups had identical reductions in kinin-containing kininogen. we conclude that dxs-induced hypotension is produced by activation of the contact system which results in the production of bradykinin. liberation of bradykinin is both necessary and sufficient to produce all of the hemodynamic changes observed. dr. matthias siebeck, department of surgery, university of munich, klinikum lnnenstadt, nussbaumstrasse 20, d-80336 munich, germany in experimental animals exposed to i.v. injection of endotoxin accumulation of leukocytes in various organs as lungs and the liver is a prominent feature. as a part of these morphological changes damages of endothelial ceils are regularly seen. this process, which is a part of endothelial-cellular interaction, leeds to exposure of the sub-endothelial basement membran. the basement membran is known f6r its capacity to activate the contact system of plasma. during this cascade activation, coagulation factor xii is converted to the active factor xii. this activation might produce increased plasma kallikrein activities and thereby give release of the vasoactive substance bradykinin. using a porcine model we have noticed that endotoxin infusion (0,01 mg/kg) induces elevated plasma kailikrein activities within two hours after the start of the infusion. this enzyme activity remained increased during the next hours and reached value of up to 50 u/1. in patients with sepsis we also have observed elevated plasma kallikrein activities with enzyme activities up to 200 u/1. in order to further elucidate the significance of these elevated enzyme activities, we prepared human plasma kallikrein and injected it intravenously in anaesthetized pigs (1). when very small plasma kailikrein activities (0,3 u/kg bodyweight) were given intravenously a 50% decrease in arterial blood pressure was seen in the animals. in the patients with sepsis also decreases in prekallikrein values and functional plasma kallikrein inhibition are frequently seen. furthermore, degradation of high molecular weight kioinogen is found in these patients indicating formation of bradykinin. these experimental and clinical studies underline that contact activation in sepsis might results in the release of very powerful mediator substances which can be of pathophysiological importance in this disease. a number of pathological disorders as reperfusion injury, bone marrow transplantation, polytrauma and septic shock are associated with capillary leakage. as the activation of the complement system and the contact phase play a major role in these diseases we investigated whether cl-lnhibitor (c1-inh), which inactivates cl-esterase, kallikrein and clotting factors xii and xl, could abolish vascular leakage. a capillary leakage was induced in rats by the administration of interleukin-2 (5 x 106 iu/kg). the increased vascular permeability was monitored for one hour as the extravasation of fitc marked rat serum albumin from a mesenterial vessel by a video-image processing system. ci-inh (berinert®, behringwerke) given as a single i.v. bolus in concentrations of 100, 250 or 500 u/kg dose-dependently prevented the capillary leakage. carrageenaninduced inflammation in the rat leads to vascutar leakage and to edematous swelling of the paw. ci-inh in this model leads to a dose-dependent decrease in paw edema formation. finally, we investigated the effect of ci-inh (infusion (100-125 u/kg x h) on a lps-induced shock in the rat by combination therapy with the antithrombotlc agents antithrombin ill (kybernin®) or rec. hirudin (both substances from behringwerke). in this animal model mortality was 90 % in the untreated control. both antithrombotic agents decreased mortality rates by inhibiting formation of dic; a further significant improvement of survival was achieved by the treatment with ci-inh. thus+ it could be concluded that c1-inh has a beneficial effect in diseases associated with a vascular leakage. iclb and laboratory for experimental and clinical immunology, university of amsterdam, the netherlands; 2thrombosis research center, temple university, penn., usa; 3oklahoma medical research foundation,. ok. city, usa. to evaluate the contribution of the contact system to activation of other mediator systems in an experimental model of sepsis, we investigated the effect of mab c6b7 which inhibits activation of factor xli, on activation of complement and fibrinolytic cascades and activation of neutrophils in baboons suffering from a lethal sepsis. activation of the complement system was assessed by measuring circulating levels of c3b/c and c4b/c, and a significant reduction was observed in 5 animals that had received a lethal dose of e. coli together with mab c687 (treatment group), compared to 4 animals that had received a lethal dose of e. coil only (control group). activation of the fibrinolytic system as reflected by circulating plasmin-=2antiplasmin complexes and tissue plasminogen activator, and activation of neutrophils, assessed by measuring circulating elastase-=l-antitrypsin complexes, was also significantly less in the treatment group. we conclude that activation of the contact system protein factor xll during the inflammatory response to a lethal dose of e. coil in this baboon model, modulates directly or indirectly activation of the complement and fibrinolytic systems and that of neutrophils. in a prospective study, plasma levels of c3a, c3, and c5a were measured in 30 patients from an internal intensive care unit. 20 patients were clinically septic defined by the criteria of bone et al.(l) . the remaining 10 patients were critically ill but didn't fulfill the clinical criteria of sepsis. from both groups of patients blood samples were taken over a l0 days period. during the first 3 days blood samples were drawn every 8 h, on day 4-6 every 12 h and the last 4 days once daily. mean plasma concentrations of c3a within the first 32 h after clinical onset of sepsis were 1019 + 618 pg/ml, whereas non-septic-patients exhibited mean values of only 595 +_ 312 p_g/m/. c3 levels were lower for septic-patients (840 + 480 lag/ml) than for non-septic-patients (1340 _+ 770 lag/ml). the most profound difference between both groups was found, when the c3a/c3 ratio was compared (1.84 + 2.28 for septic-patients and 0.4 _+_ 0.18 for the control group). no significant differences between both patient groups were observed in c5a plasma levels (5.9 + 2.1 ng/ml in septic-patients vs. 5.6 _+ 1.5 ng/ml in control patients). in 13 of 20 cases of clinically defined sepsis causative organisms like bacteria, protozoa or fungi could be cultured from blood, bronchoalveolar lavages and/or section materials. application of the complement parameters to survivors (n=9) and non-survivors (n=l 1) within the septic-group revealed, that the c3a/c3 ratio could also be used as a prognostic parameter for clinical outcome. the possibility of rapid and easy measurement of c3a and c3 in only 20-25 minutes (2) and the significant difference of the c3ajc3 ratio between the septic and non-septic group renders this parameter a good candidate for early diagnosis of sepsis in the intensive care unit. hirudin, a single polypeptide chain composed of 65 amino acids with 6 cysteine residues (mr 7000 daitons), is the most potent and specific thrombin inhibitor, which is now available as a genetically engineered product (rec. hirudin -hbw 023, behringwerke; marburg). the aim of our study was to establish a rabbit model of tissue factor (tf) induced activation of the extrinsic pathway of coagulation and to evaluate the therapeutic efficacy of rec. hirudin. coagulation was induced in female nzw rabbits by infusion of 0.5 p.g/kgxh thromboplastin for 7 hours. development of disseminated clotting was manifested by a decrease of fibrinogen and platelets to 27.0 % and 33,0 % respectively, and by an increase of fibrin monomers from 13.2 to > 85.0 ~tg/ml. we administered rec. hirudin to rabbits in 3 different concentrations (0.5, 1.0 and 2.0 mg/kg); treatment started simultaneously with the infusion as an i.v. bolus. rec. hirudin significantly prevented the decrease of fibrinogen, platelets and the increase of fibrin monomers. this effect was dose dependent and long lasting, even 7 hours after the administration of rec. hirudin, clotting was still significantly reduced. as could be drawn from the plasma levels, rec. hirudin had been cleared from plasma at this time. in a post-treatment study we administered rec. hirudin (0.5, 1.0 and 2.0 mg/kg i.v. bolus) as late as 2 hours after the start of tf infusion. at this time there was already a prominent activation of coagulation. even in this post-treatment regimen rec. hirudin significantly prevented disseminated clotting. hence, it was concluded, that rec. hirudin by inkihiting thrombin could be effective in the prevention of coagulation disorders including disseminated intravascular clotting (dic) induced by a septic disease. research laboratories of behringwerke ag, 35001 marburg, germany $3 novel protease inhibitory activities of the second domain of urinary trypsin inhibitor (r-020) and its effect on sepns-lnduced organ injury in rat atsuo murata 1, hitoshi toda 1, ken'ichi uda 1, hidewaki nakagawa 1 , takesada mori 1, hideaki morishita 2, tom yamakawa 2, jiro hirese 2, atsushi ni~ 2, nariaki matsuura 3 1osaka university medical school, osaka, 2mochida pharmaceutical co. ltd. tokyo, 3wakayama medical schoof, wakayama, japan inhibitory-activities of the second kuntz-type inhibitor domain of human urinary trypsin inhibitor (uti) and its effect on sepsis-induced organ injury in rat were investigated by using the recombinant protein. uti is a glycoprotein with a structure in which 2 kunitz-type inhibitor domains are linked in a row. we isolated the gene encoding the second kunitz-type inhibitor domain of uti, and then constructed expression plasmids by ligating it to the e. coli phoa signal peptide gene. these plasmids expressed the second domain in e. coil strain je5505 which lacks the membrane lipoprotein. the recombinant second domain (r-020) innb[ted trypsin, plasmin, neutrophil elastase and chymotrypsin. in addition it inhibited blood coagulation factor xa and plasma kallikrein in a concentration dependent and competitive manner. the in vivo effect of the recombinant r-020 was investigated in a rat model of septic shock induced by cecal ligation and puncture. the administration of r-020 significantly improved the survival rate of the rats and attenuated the pathological changes of lung and iiver. we found out the novel protease inhibitory activities of the second domain of uti and its protective effects on sepsis-induced organ injury. macrophages are known to secrete lysosomal proteinases,mainly cathepsin b and cathepsin l, and also ~-proteinase inhibitor (pi),related to acute phase proteins.disturbances of proteinases/ proteinase inhibitors correlates with inflammatory process,leading sometimes to noncontrol "pathglogical" proteolysis (jochum et ai.,1990) . the cathepsin l-like and cathepsin b-like activity were measured in serum of 42 patients with chronic bronchitis (14 -with obstructive, 28 -with nonobstructive bronchitis),acute bronchitis (15) and 35 healthy persons.simultaneously the level of~pi was determined in the same groups.cysteine proteinases were measured with help of fluorogenic substrates,as was presented earlier (korolenko et ai.,1991) , ~pi with help of immune enzyme method. it was shown increase of cathepsin l-like and cathepsin b-like activities during aggravation of chronic bronchitis comparatively to the controls (2-4 fold) .after treatment there was a tendency to normalization of indices,but the increase was about 30-40% more than the control values.~pi level in this group was also increased (two-fold),in patients with acute bronchitis -2-4-times more comparatively to the control.it is possible to conclude that chronic bronchitis induced increased secretion both cysteine proteinases and d{pi into blood. some peculiarities of ratio were noted in patients with emphysema. endotoxins are microbial products derived from the outer cell membrane of gram negative bacteria. the active component of endotoxin is lipopolysaccharide (lps), a complex macromolecule consisting of polysaccharide covalently bound to a unique lipid, termed lipid a. now recognized to embody the endotoxic principle of lps, lipid a consists of a/31-6 diglucosamine backbone, both ester and amide linked fatty acids, some of which are acyloxyacylated, and charged constituents such as phosphate, phosphorylethanolamine and 4 amino arbinose lps, exerts its biological effects in vivo by noncytotoxic interactions with a variety of host inflammatory mediator cells, primarily the mononuclear phagocyte and the endothelial cell, although other host cells also participate. these interactions are modulated by lps-specific binding proteins found in plasma, including lps-binding protein (lbp) scd14 and perhaps other proteins as well. specific receptors for lps have been identified on mammalian cells which mediate signal transduction via multiple pathways. lps-activated host cells are stimulated to secrete or express multiple proinflammatory mediators, including tnf-a, illa, il-1/3, ifn-a, il-6, il-8, il-10, paf, pge, ltb 4 and procoagulant activity. the overproduction of these proinfiammatory mediators results in the manifestations of endotoxemia, observed experimentally as fever, hypotension, disseminated intravascular coagulation and death. modulation of activity of these mediators protects animals against lethality. similar pathways are thought to be operative in gram negative sepsis, and control studies with human volunteers support such conclusions. immunotherapeutic approaches in clinical gram negative sepsis have, to date, been less successful. in vitro experiments and studies in animal models have recently shown that several proteinaceous bacterial exotoxins can evoke cytotoxic effects that ultimately lead to cardiovascular collapse and shock. since the possible relevance of bacterial exotoxins in the pathogenesis of septic shock has received very little attention in the past, an attempt will be made here to provide a brief overview of this generaily neglected topic. protein toxins act intracellularly or they dz~nage the integrity and function of the plasma membrane. major representatives of the former group are the adenosine diphosphate (adp)-ribosylating toxins, e.g. cholera and cholera-like toxins, diphtheria toxin), and the neurotoxins. most medically relevant toxins of this category have been studied in great detail. although often responsible for severe and sometimes fatal disease, their association with septic shock is rare. in contrast, experimental evidence is accumulating for a role of membrane<lamaging toxins in the pathogenesis of inflammatory lesions. formation of transmembrane pores is probably the most widespread mechanism via which such physical membrane perturbation can occur (1,2), the present discussion will be restricted to this category of toxins. the author shall first discuss basic properties of pore-forming proteins, and consider the factors that direct their attack to specific targets. thereafter, attention will be drawn to diverse functional consequences that may follow membrane damage so that a general concept of how pore-forming toxins may contribute towards the development of shock will evolve. i. bhakdi, s. and tranum-jensen. j. 1987 gram positive bacteria produce exotoxins that, at least in part, exhibit the unusual properties of superantigens. superantigens (sag) activate v/3 selectively t cells to produce lymphokines including i1-2 and tnf/lymphotoxin. systemic application of the sag staphylococcal enterotoxin b (seb) to d-galactosamine sensitized mice causes lethal shock within hours. seb reactive v/38 + t cells accumulate within 90-120 min mrna for the ii-2, i1-4, tnfai~ and ifn-3,. parallel in time high concentrations of bloodborne i1-2 and tnf are noted. anti tnf t~//3 neutralizing mab protect mice against seb induced t cell dependent lethal shock thus indicating a key role of tnf in effecting lethal toxicity. within 12-18 h distinct levels of tolerance to seb become discernable on ligand reactive v/38 t ceils, dependent of the dose of seb used. these levels include anergy, t cell receptor downregulation, loss of cd2, cd4, cd8 accessory molecules and programmed cell death. m.freudenberg, y.yaegashi, p.nielsen, c.galanos. the sensitivity towards endotoxin (lps) is genetically determined, however it may be increased under different experimental conditions. these include treatment with hepatotoxic agents such as d-galactosamine, and with live (infection) or killed bacteria. it is well established now that d-galactosamine sensitizes to lps by increasing the susceptibility of the host to toxic activity of lps-induced tnf~. sensitization by bacteria, especially by gram-negative once is of especial interest because it implies that infecting microorganisms not only produce lps, but als0 sensitize the host to its lethal activity. sensitization to lps by bacteria proceeds in all lps-sensitive (lps n) mouse strains that have been investigated so far. it also proceeds in lps-resistant (lps d) c3h/hej mice. the absence of sensitization to lps in lps d c57bl/10sccr mice was identified as being due to their inability to produce interferon-7 (ifn?). administration of exogenous ifn? to these mice conferred sensitivity to lps. conversely, administration of anti-ifn? to lpsn; mice inhibited the development of sensitization'by bacteria. it may be concluded therefore that ifn7 is the mediator of sensitization to lps by bacterial infection. the ifn? defect of c57bl/10sccr mice concerns only the ifn7 response to bacteria, since the response to the t-cell mitogen con a and to cd3 monoclonal antibodies was not impaired. the ifn?producing cells themselves were shown to be intact. on closer investigation the impaired ifn? response was identified as the result of a defective accessory function of macrophages. macrophages of c57bl/10sccr mice are incapable of producing interferon-~ (ifn~) which we could show to be obligatory for the production of ifn? in response to bacteria. although antibiotics are required to clear bacteremia, they do not reverse evolving shock, because endotoxin free in the bloodstream or exposed on the surface of circulating bacteria continues to activate the production of inflammatory mediators, furthermore, antibiotics have been shown to induce release of endotoxin from gram-negative bacteria during the treatment of severe infection. in an in-vitro study, using live escherichia coil, we have observed the release of endotoxin upon treatment with several antibiotics like cefuroxim, imipenem, ceftazidime and aztreonam. incubation with imipenem or ceftazidime induced an early lyeis and a mild rise in endotoxin levels, whereas incubation with cefuroxime or aztreonam resulted in the formation of filaments with a late but dramatic rise in endotoxin levels. in a second study we studied the influence of different antibiotics on the tnf-= production of e.coli stimulated human monocytes. we found again great differences in the production of this important cytokine among the different antibiotics according to their capacity to induce liberation of endotoxin. these differences in the amount of endotoxin release are probably caused by penicillin-binding-proteins (pbp) specificities of the antibiotics with resultant differential morphological changes, in a rat sepsis model treatment with imipenem or ceftazidime resulted in a transient increase in il-6 levels, whereas treatment with aztreonam resulted in progressively increasing levels of il-6 and a significant increase in mortality. the increased mortality in pbp-3 specific aztreonam treated rats might have been the result of filament formation in the abdominal cavity of the septic rats. the endotoxin sequestered at local sites may account for the precipitation of gram-negative shock. finally, we've also demonstrated that in patients under treatment for septic shock endotoxin release can be related to the administration of antibiotics. carbapenem-and cephalosporin-lnduced release of lps from smooth and rough pseudomonas aeruginosa: in-vivo relevance j. jackson and h. kropp, merck research laboratories, rahway, nj b-lactam (cell-wall active) antibiotics are generally deemed the class of antibiotics most responsible for the release of free endotoxin (lps) from gram-negative bacteria due to their cell lytic actions although all antibiotic classes studied thus far induce release of endotoxin. we have recently shown in-vitro that antibiotics within the b-lactam class (i.e. imipenem , meropenem and ceftazidime , with equivalent mics) differ in their propensities to release excessive amounts of lps from both smooth-(s-) and rough-(r-) lps laboratory and clinical and antibiotic-sensitive and -resistant p. aeruginosa isolates and that lps release is independent of cell lysis. additionally, variations in the induction of endotoxin release is antibiotic concentration dependant and correlate with antibiotic penicillin-binding protein (pbp) affinities which result in morphological changes. these studies also show that lps differences measured via chromogenic lps (c-lal) assay correlate with lps lethality in lps-sensitive mice. release of lps was compared to changes in cfus, cell mass, morphology and antibiotic pbp-specificity. corresponding in-vivo studies in cd1 mice against s-lps p. aeruginosa isolates show that, despite equivalent in-vitro protection, antibiotic efficacy in mice differ as much as 100-to lo00-fold. to establish a possible in-vivo role for antibiotic-induced release of free lps we compared antibiotic efficacy results in mice challenged with virulent parent s-lps and its relatively avirulent r-lps mutant (lacking only its 0 side chain) p. aeruginosa isolates. results show the relevance of quantity and physiochemical composition (bioreactivity) of free lps to virulence and in-vivo antibiotic efficacy. in other in-vivo studies chemical agents that destroy cells (m~) which mediate lps lethality in-vivo were used to pretreat mice prior to antibiotic therapy and bacterial challenge with wild type pseudomonas. we conclude that circumstances in which antibiotic-induced filamentation occurs are also conditions that yield excessive lps release, the in-vivo effects of which are shown through the requirement of larger amounts of antibiotic to afford equivalent protection. bacterial endotoxins have been reported to play a significant role in the pathogenesis of gram negative sepsis by their interaction with, and stimulation of, host inflammatory mediator cells to produce cytokines, biologically active lipid intermediates, and procoagulant activity (tfa). although both microbe-associated and free endotoxin are capable of stimulating mediator production, studies from our laboratory suggest that free endotoxin may be the more potent stimulus for tnf and tfa. further, our studies suggest that the majority of the proinflammatory activity released from antibiotictreated e. coli coisolates with lps by two different fractionation techniques. to assess whether actions of endotoxin directly contribute to lethality in gram negative sepsis, an experimental model was developed in which mice were made hypersusceptihle to the lethal effects of endotoxin by treatment with d-galactosamine (dgaln). challenge of cf-1 dgaln-treated mice with e. coli olll:b4 resulted in an lds0 of approximately 2.5 x 104 cfu. resolution of the peritonitis and bacteremia by treatment with cell wall-active antibiotics resulted in an increase in the ldso. ceftazidime and imipenem, which differ in their mechanism of action and in their capacity to effect endotoxin release in vitro, also differed in their in vivo capacity to provide chemotherapeutic protection (3 fold vs 8 fold respectively, p< .01). parallel studies with endotoxin hyporesponsive c3h/hej mice indicate significantly greater levels of protection with imipenem (> 80 fold vs saline controls). collectively these data suggest that endotoxin may contribute directly to the pathogenesis of experimental gram negative sepsis. bacterial lipopolysaccharides (lps) are the endotoxins of gram-negative bacteria and represent their major surface antigens. lps is made up of three chemically, biologically and genetically disctinct regions, i.e, the o-chain, the core region and the lipid a moiety whereby the latter represents the endotoxic center. it is our current understanding that lps is responsible for many of the pathophysiological events observed during gramnegative infections and that one of the major mechanisms leading to shock and death is the lps-induced activation of macrophages resulting in the production and release of lipid and peptide mediators, among which tumor necrosis factor seems to be the most important. therefore, in the fight against the lethal outcome of gram-negative infections, modern strategies, in addition to antibiotic treatment, aim at i) the neutralization of tumor necrosis factor, ii) the inhibition of the production of tumor necrosis factor or iii) the neutralization of the activation potential of lps for macrophages by monoclonal, preferably human antibodies. the latter approach, to be effective against a broad spectrum of gram-negatives, must be directed against common structures of lps (lipid a and core region). the molecular basis of this approach and the controversy in this field will be discussed. passive immunotherapy has been used since 1893, when von behring described the administration of immune horse serum to treat a patient with diphteria infection. even if this therapy was sometimes successful in bacterial infections, it has been largely replaced by antibiotics. however, antibiotics have their limitations, especially in critically-ill patients. to improve outcome, adjunctive therapies such as immunotherapy with polyclonal and monoclonal antibodies particularly against endotoxin are again considered. the role of humoral immunity in host defenses against bacterial infections is weu known. for instance, tile importance of antibodies in the defense against gramnegative infections has been established clinically by studies relating the outcome of patients with gram-negative bacteremia to tilers of antibodies directed at the offending pathogens at the onset ofbacteremia (mccabe 1972; pollack 1979) . ever since we know the role of endotoxins in the pathophysiology of sepsis, antibodies against the s-and r-lps have also been detected in sepsis patients. the aim of the administration of iv/g to the sepsis patient is as follows: 1 ) enhancing of opsonization and phagocytosis(antibactericidai activity) 2) synergistic effects with [3-1actam antibiotics 3) neutralization of endotoxin, the main pathogenic mediator of gram-negative sepsis 4) modulation and/or inhibition of cytokine release the enhancement of opsonic-and phagocytic-activity especially with igg via fc and c3 receptors has been well documented. monoclonal antiendotoxin antibodies, proven in clinical studies, do not appear to neutralize endotoxin in vitro and are not reproducibly protective in animal models of sepsis. also they can not suppress endotoxin-induced tnf-~, il-6 release in mice (baumgartner 1990, corriveau and danner 1993) . in conlrast, recent studies of a polyclonal immunoglobulin preparation, containing high levels of antibodies against gram-negative bacteria and their o-antigen of lps in igg, igm and iga classes (pentaglobin®) provide evidence to neutralize endotoxin. this effect is demonstrated in vitro (berger 1990 (berger ,1993 , in animal models (stephan 1985 , berger 1993 and also in prospective, randomized, controlled clinical trials (schedel 1991 , poynton 1992 , behre 1992 . furthermore mortali b' was reduced statistically in patients with septic shock and endotoxemia by using this preparation, as has been demonstrated by sehedel. anti-core lps monoolonal antibodies: binding specificity and biological properties f.e. di padova, r. barclay, e.th. rietschel. bacterial lps and cytokines are responsible for the pathological processes of gram-sepsis and are suitable targets for therapeutic interventions. chemical characterization and structural analysis of different lps have revealed common features. the inner core region of lps shows a high degree of similarity among e. coli, salmonella and shigella. among a large number of broadly cross-reactive murine anti-core lps mab one of these igg2ak) has been selected and chimerized into a human igglk (sdz 219-800). in elisa and in immunoblots on purified lps both sdz 219-800 and wni 222-5 show a strong reactivity with all smooth lps from e. coli and salmonella. reactivity with all the known complete core structures from e. coli and salmonella (ra) is evident. reactivity with re structures or free lipid a is not observed. this mab cressreacts with all clinical e. coli isolates from blood, urine and feces and with other enterobacteriaceae. sdz 219-800 and wni 222-5 have biological activity as they inhibit the lal assay and the secretion of monokines (il-6 and tnf) by mouse and human macrophages. moreover, sdz 219-800 and wni 222-5 inhibit the release of il-6 and tnf in vivo. in vivo sdz 219-800 as well as wni 222-5 neutralize the pyrogenic activity of e. coli lps and protect mice from lethality in d-gain-sensitized mice. the possibility to use wni 222-5 as a capture antibodies in the immunolimulus assay opens the possibility to differentiate the origin of the lps in patients with endotoxemia. franco di padova, sandoz pharma ag, ch4002 basel, $chweiz $7 presentation of lps to cd14 by lps binding protein peter s. tobias, julie gegner, katrin soldau, lois kline, loren hatlen, douglas mintz, and richard j. ulevitch. the activation of myeloid cells by lipopolysaccharides (lps) has been shown to require the serum glycoprotein lps binding protein (lbp) and binding of lps to membrane bound cd14 (mcd14). other cells such as human umbilical vein endothelial cells (huvec), smooth muscle cells, and some epithelial cells, which do not express mcd14 but nevertheless respond to lps in the presence of serum, have receptors for complexes of lps with the soluble form of cd14 (scd14). these complexes of lps with scd14 are only formed efficiently in the presence of lbp. we have begun to characterise the mechanisms by which lbp enables lps to bind to cd14, either soluble or membrane bound. with the use of fluorophore and radiolabelled reagents we have developed procedures for quantitative measurement of the association of lps with lbp and of lps-lbp complexes with cd14. these results show that the delivery of lps to scd14 is catalysed by lbp, i.e., lbp is not included with the lps-scd14 complex. in contrast, on the surface of cells, lbp does not dissociate from the cells after lps binds to mcd14. the kinetics, equilibria and stoichiometry of these reactions will be discussed in the context of models for cellular activation by lps and cellular uptake of lps. supported by nltt grants gm37696, ai32021, ai15136, gm08172, and assistance from the pharmaceutical research institute of johnson and johnson. the scripps research institute, imm-12, 10666 n. torrey pines rd. la jolla, ca usa 92037. modulation of endotoxin-induced cytokine production by lps partial structures h.-d. flad, h. loppnow, t. mattern, and a.j. ulmer department of immunology and cell biology, forschungsinstitut borstel, d-23845 borstel lipid a constitutes the active moiety of endotoxin (lps) of gramnegative bacteria. it activates mononuclear phagocytes to produce cytokines, such as tnf, i1_-1, and il-6, which are the major mediators of the endotoxic effect of lps in vivo. lipid a precursor la (synthetic compound 406) does not induce cytokines, but is able to specifically antagonize lps-or lipid a-induced mediator production in human mononuclear cells, vascular endothelial cells, and smooth muscle cells. furthermore, we present evidence for the first time that t-lymphocytes proliferate in response to lps and express mrna for interleukin-2 and interferon-~ and that these responses are also antagonized by synthetic lipid a precursor la. when comparing the agonistic and antagonistic activity of lipid a and different partial structures at the functional and binding level, the number and length of the fatty acids and the number of phosphoryl groups were pound to be of crucial importance. unexpectedly, lipid a precursor la, although biologically inactive, turned out to be both the most potent antagonist and competitor in inhibiting the binding of lps. taken together, our results provide evidence for a model in which lipid a partial structures compete with lps for specific cell surface receptor(s). in this sense, biologically inactive lipid a analogues may be good candidates as therapeutic agents for the prevention of gram-negative septic shock. two mammalian lipid a-binding proteins have been identified that are believed to have important roles in mediating the host response to endotoxin: lipopolysaccharide-binding protein (lbp) and bactericidal/ permeability-increasing protein (bpi). human lbp shares a 45% amino acid sequence identity with human bpi. despite the sequence homology, the two lipid a-binding proteins have very different functional activities. lbp is an acute phase serum protein that markedly potentiates the proinfiammatory host response to gram-negative infection by a mechanism which involves binding of the lbp-lps complex to cd14 receptors on monocytes, neutrophils and endothelial cells. in contrast, bpi is a neutrophil granule protein with potent bactericidal and lps-neutralizing activities. the divergent functional properties of these two lps-bindlng proteins can be explained by the inability of bpi-lps complexes to bind to cell-surface cd14 receptors. a recombinant protein (rbpi23), corresponding to the amino terminal 23kd fragment of human bpi, has been shown to retain the potent biological activities of the hdlo protein and may represent a novel therapeutic agent for the treatment of gram-negative infections, sepsis and endotoxemia. for therapeutic effectiveness in many clinical situations, rbpi23 will have to successfully compete with relatively high serum levels of lbp (5-60 ~g/mi) for binding to endotoxin and gram-negative bacteria. to evaluate this issue, experiments were conducted to compare the relative binding affinities of rbpi23 and human recombinant lbp (rlbp) for lipid a. the binding of both proteins to iipid a was specific and saturable with apparent kd's of 2.6 nm for rbpi23 and 58 nm for rlbp. in a competition assay format rbpi23 was approximately 75-fold more potent than rlbp in inhibiting the binding of nsi-rlbp to lipid a. these results demonstrate that rbpi23 has a significantly higher affinity for endotoxin than does rlbp and may explain the potent inhibitory activity of low concentrations of rbpi23 in a variety of in vitro functional assays for lps activation of cells despite the presence of high lbp levels. for example, rbpi23 at 0.2 ~tg/mi was able to totally inhibit lps-induced tnf release from monocytes despite a 100-fold weight excess of rlbp over rbpi23. and for heparin binding. three separate domains which inhibit the lal reaction to lps and bind to heparin were identified in amino acid regions 17-45, 65-99 and 142-169 . a single synthetic peptide (85-99) was bactericidal. these results suggest that rbpi23 contains three separate functional domains which may contribute to its high affmity interaction with gram-negative bacteria and heparin. the individual activity of each domain and the cooperative interaction among domains provide the basis for developing rbpi23 analogues with increased biologic efficacy. a considerable body of experimental data has accumulated implicating tumour necrosis factor (tnf) as a principal mediator of the pathophysiological features of septic shock. these data prompted the development of clinical strategies designed to limit excess (inappropriate) tnf production. monoclonoal antibodies (mobs) were developed and a phase ii dose escalation trial in 80 patients confirmed that the mab was safe, and suggested that it was having a beneficial effect on certain parameters. preliminary results of a large phase iii study indicated that (a) the mob was safe; (b) that it was of no discernible benefit in non-shocked patients; (c) that it reduced mortality in shocked patients, especially during the first 3 days. an alternative strategy was to take advantage of the high binding affinity of soluble receptors for tnf (stnfr). stnfr-iggfc constructs were made for both the p55 and p75 receptors. both were effective in animal models of lps challenge, but when a clinical trial was done with the p75 stnfr-fc there was unexpected mortality in the treated arm. using an animal model of live e.coli sepsis, we have shown that this may have been due to the release of bound tnf from the construct. plasma enhances while bpi inhibits lps-induced cytokine production from peripheral blood mononuclear cells (pbmc). pseudomonas species produce cytokine-inducing substances which are different from lps as indicated by the fact that polymyxin b blocks only 40% of the cytokine-inducing activity of these pyrogens. we now tested the effect of plasma and bpi on the il-1[3-inducing activity of pseudomonas maltophilia -derived pyrogens (pmp). bacteria were cultured to the log phase and filtered (300kd) to obtain prop. dilutions of pmp or lps were added to pbmc alone or to pbmc in 5% plasma +/-bpi (500 ng/ml). pbmc were incubated for 24 hours at 37°c and total il-i~ was measured by ria. results: il-i[~ in ng/ml (n=7, mear~+sem, *p<0.05 vs control). 0 control 0.1_+0 + bpi 0.1+0 5% plas. 0.2_+0 + bpi 0.2_+0 pmp (ng/ml) lps (ng/ml) 60 600 3000 1 10 3.2_+1 8.0_+1 16.3_+3 1.2_+. 2 5.2+.6 0.2_+.1 3.3_+.9" 13_+3" 0.1_+0" 0.1_+0" 2.2_+. 5 5.7+1 14_+2 5_+.6* 14_+2" 0.6+. 4" 5.3+1 15-+2 0.1-+0" 0.5_+.3" cba, c57bl/6, balb/c, akr, dba, swiss mice, guinea pigs, rabbits have been used in research work. the toxicity, immunogenicity, mitogenic and immunomodulating activity of lps have been studied. the possibility of reduction of the toxic activity of lps on macroorganism by bioglycansimmunomodulators obtained from sea invertebrates anymals (crenomytilus grayanus, stromhus gigas) have been investigated too. lps has been shown to induce specific antibody response of laboratory animals. cba mice are high responsive to lps. lps stimulates humoral immune response of mice to tdependent and t-independent antigens and suppresses intensity of the delayed hypersensitivity. the small doses of lps stimulate functional activity of macrophages, the large doses of lps -decrease one and show the cytotoxic effect. the bioglycans enhance the resistance of mice to the lethal effect of lads and provide protection 59-75% of mice. one opens possibility to use of bioglicans for reduction of toxinemia in generalizated forms of pseudotuberculosis. thus, lps from y.pseudotuberculosis is immunogen and immunomodulator wich has influence on humoral and cellular factors of immunity and plays the important role in immunopathogenesis of infection. endotoxaemia is implicated in the pathophysiology of obstructive jaundice. the lirnulus lysate (lal) assay is the gold standard method for measuring endotoxin concentrations, but inherent biochemical and technical problems limit the usefulness of this assay. the endocab elisa is a novel assay which measures endogenous antibody (igg) to the inner core region of circulating endotoxins (acga). objectives we evaluated the significance of endotoxaemia in biliary obstruction using the endocab assay and subsequently the specificity of the humoral response to endotoxin compared with an exogenous antigenic challenge [tetamls toxoid (tt) ]. materials and methods in experiment i three groups of male wistar rats (250-300g) were studied [no operation (n=39) , sham operation (n=40), and bile duct ligation for 21 days (bdl)(n=50)]. plasma was collected and assayed for bilirubin, endntoxin(lal) and acga(endocab). in experiment ii 30 rats were actively immunised with tetanus toxoid ('it) and then randomised to have no op(n=8), sham op(n=8) or bdl(n=i4). blood was taken at this time (to) and 21 days later(t20 at sacrifice for acga concentrationslendocab] and igg produced to tt(ttab) [elisa] . antibody concentrations are expressed as % increase from control values.results in bdl rats, acga concentrations were significantly increased compared with controlslp<0.001, mann-whitney]. endotoxin concentrations were sporadically elevated in the jaundiced rats but the rise was not significant. in experiment [i there was no difference between the acga or ttab concentrations in the fllree groups at to, bdl rats had a significant rise in acga concentrations by t21 [p<0,001,paired t-test] and humoral response to tt was significantly impaired in bdl rats compared with control groupslp<0.05, paired ttest data plasma endotoxin was measured by means of an endotoxinspecific endospecy test after pretreatment of the plasma with a new perchloric acid method that we developed. the normal value of plasma endotoxin is less than 9.8 pglml. polymyxin b was administered at a dose of 12,500 u every 6 hours. plasma endotoxin rapidly decreased to the normal range in 40 of the 42 patients. body temperature fall significantly. apache ii scores were also significantly improved. tumor necrosis factor-o~ and interleukin 6 decreased in survivors, while in high values tended to persist in patients died. no side effects were observed in any of the patients. in conclusion, intramuscular injection of minute of polymyxin b was useful in the treatment of endotoxemia. 19-1 uchimaru, morioka 020, japan. l e v a n t g r a m n e g a t i v organisms. m e t h o d s : u n d e r general anesthesia, 12 n o r w e g i a n b r e d landrace pigs (17-30 kg) of either sex, 6 pr group, u n d e r w e n t t r a c h e o s t o m y a n d w e r e v e n t i l a t e d on a 50/50 air a n d o x y g e n m i x t u r e a i m e d at m a i n t a i n i n g a n o r m a l p h a n d a isocapnic level. ventilation w a s not readjusted d u r i n g the observation period. the anesthesia w a s k e t a m i n e 0.6 m g / k g h a n d d i a z e p a m 2.4 m g / k g h i n t r a v e n o u s l y . h e m o d y n a m i c m o n i t o r i n g of m e a n aorta, p u l m o n a r y artery, central v e n o u s a n d p u l m o n a r y capillary w e d g e pressures w a s p e r f o r m e d w i t h a 5f s w a n -g a n z catheter a n d an aorta catheter. a continous infusion of r i n g e r ' s acetate ( 1 0 m l / k g h ) w a s g i v e n intravenously. w h e n stabilised, the a n i m a l s w e r e g i v e n 2.5 x l09 cfu of e colt intraperitoneally as a bolus in 100ml saline, the a n t i b o d y g r o u p received in a d d i t i o n 5 m g / k g e5 a n t i e n d o t o x i n i n t r a v e n o u s l y over 1 h o u r via a n infusion p u m p at the start of the observation period. the a n i m a l s w e r e observed for 6 hours. results : a t 5 a n d 6 hours, the o x y g e n c o n s u m p t i o n increased by 30 % in the a n t i b o d y treated g r o u p w h e r e a s there w a s a significant fall of 30 % in the sepsis group. in the a n t i b o d y group, the arterial p h a n d the cardiac index were also significantly h i g h e r at the s a m e p o i n t s in time. there w a s no significant difference in arterial po2. in severe bacterial infections it would be beneficial to neutralize the plasma endotoxin content with complex forming compounds. the phenothiazines are able to form complexes with endoto×in and the existence of these complexes were already shown in differential speetrophotometry and animal experiments, however, the mechanism of partial neutralization was not clarified. therefore some representative phenothiazines and structurally related compounds were tested for anti-endotoxin activity. the endotoxin neutralizinb effects of several benzophenothiazines were investigated in differential speotrophotemetry, tnf induction and in the conventional limulus test. in animal experiments some beneficial effect of complex forming compounds was found. the benzophenothiazines were not able to inactivate the biological effect of endotoxin in the limulus test. the recent findings indicates that a multifocal effect can be responsible for "anti-endotoxin action in vivo". effects of tnf inducing effect of endotoxin in leukocytes and bypotensiv action in experimental animals were reduced by some phenothiazine derivatives. monophosphoril lipid a was without effect. of microbiology, albert szemt-gydrbyi medical university, odm t~r lo, h-6720 szeged~ hunbary 46 involvement of streptococcus pyogenes erythrogenic toxins in the induction oflstreptococcal toxic shock syndrome 3 heide mgller-alou~* , joseph e. alouf , die [er gerlach , ~atherine fitting., and jean-marc ca~aillon . unit6 des toxines microbiennes and "unit6 d'immuno-allergie, institut pasteur, 28, rue du docteur roux -75015 paris (france) ; institut f~r experimentelle mikrobiologie, jena (germany). superantigen erythrogenic toxin a (eta) is thought to be involved in toxic shock syndrome in humans by inducing massive release of cytokines by patient immune cells. the cytokineinducing capacity of eta w~:s £:ompa~ed to that of lps, a gram-negative bacterial cell wall component. eta elicited weak production of il-1 d and ~, tnf ~ and il-6 in purified human monocytes whereas lps stimulated the production of high amounts of these cytokines. in the presence of t cells, eta elicited the production of significant amounts of il-i~, il-i~, il-6 and il-8. however, the most preponderant cytokine was tnf~, which peaked at i0 ng/ml after stimulation with i0 ~g eta. comparable amounts of tnfd (ca 4 ng) were induced by 0.i ~g eta and 0.i ~g lp$. in contrast to lps, eta was a strong inducer of tnf~ which was produced only in marginal amounts by lps. these results suggest that the septic shock induced by gramnegative bacteria (lps) and by gram-positive bacteria {extracellular superantigens) follows different pathogenic pathways. lps-induced shock is mainly mediated by monocytes and monocyte-produced cytokines (il-i and tnf). the eta-induced shock is mediated by t-cells or depends on t cell help for the production of monocyte-liberated cytokines. production of t cell cytokines such as tnf~ and interferon 7 in addition to the other cytokines contribute very likely to the severity of the toxic-shock resulting from s. auzeus and s. pyogenes infections in humans. the present study was utidertakc~l to cvalu~tlc the effect of soluble chemically modified giucan during septic shock. carboxylnethyl-b-i,3-glucan (ram 120000) was injected twice 48 and 24 h before the shock i.v. in a dose of 50 ing/kg. shock was induced in u~?esthetizcd (sodikm~. l)mntobarbital) rats by i.v. injection of endotoxin of escherichia colli 0127 bs, 5 mg/kg. aiiofcmg pretreated ruts survived during first 24haher ¢ndotoxine, while in controi shock group the lethality was 90%. the concentration of ~col)terin in serum was significantly elevated 24 hafterthc second cmginjection (appare~tly 80 % if compare with the control rats), but didu't chartged 60 rain and 1 s0 rain after endotoxin injectjom cardiac output in cmogroup was higher a* the i20 and 180 min after endotoxine onset ( 8i % trod 72~, respectively of initial level) than in the control shock group (64 % and 52% at the same time). pretreatment of rals with soh~ble giucan w~ts associated with beneficial effects o~ the hepatic c~ergy $ia[tls after 3 h after challenge of endotoxiae: the tissue level of lactale was ahnost twice lower than in the control ruts, me~mthne the tissue atf in cmg pretreated group was higher at 40 %. twice injected macrophage stimuhttor soluble glucan can prevent the endotoxic shock, and extremely ir~creased survival rate after endotoxine injection. the national committee of surgical infections of the spanish association of surgeons have produced a computer program for the collection and analysis of information on surgical infections. the program is suitable for ibm compatible hard disk personal computers and works through the ms-dos system. the main menu is called up on the screen when the operating disk has been installed; it reads as follows: i. new record; 2. modify records; 3. erase records; 4. searches; 5. reports; 6. configure; o. ouit. if you ask fdr a new record the screen will prompt you to enter the number of case, record number, hospital, age and sex. the next screen will come up and the words "topographic diagnosis" will flash. a menu of 34 areas or organs will be displayed. then, the words "type of pathology" (inflammatory, neoplastic, traumatic and other). days of postoperative period. type of surgery (programmed and emergency). type of operation (clean, clean contaminated, contaminated and dirty). duration of surgery. this is followed by "order of operation" and the "type of anaesthesia (general, regional or local). you are then required to supply the "diagnostic code of who" (icd9) and the "procedure code of who. analytic and concurrent illnesses (total proteins, albumin, haemoglobin, haematocrit, leucocytes, red corpuscles, glucose and bilirubin). the next screen asks for "risk factors" (obesity, uraemia, neoplasia, malnutrition, urinary catheter, distant infection, artificial valve, immunosuppressive drugs, over 65 years and anergy. this is followed by a screen headed "postoperative complications". "evolution" (the questions asked are drainage, systemic antibiotics, and on each ocasion a choice of 31 antibiotics is displayed), local antiseptics, reoperation, etc. under "microhiology" is a choice of 37 organisms and the chance of identifyin 3 organisms. finally, "sepsis score". our recent work had shown that renshen-fuzi-chaihu mixture could increase the survival rate in experimented study. the purpose of this study was to determine the effect of combined administration of renshen-fuzi-chaihu mixtuer and antibitics (sa) in patients with septic shock. the result showed that, in sa group (40 cases), the total effective rate was 87,5%, in the contral group (combined administration of gentamycin and dexamethasone, 25 cases) the total effective rate was 84%. however the obviously effective rate in sa group 70% was significantly higher than in contral group 44% (p<o.os). sa group appeared to be more obvious than the contral group in raising arterial pressure, recovering consciousness and pulse, improving cold lilmbs and reducing fever (p<o.o5). it was found that sa could inhibit the pathogeneses changes of septic shock such as the decreases of superotide dismutase (sod) and glutathione peroxidase (gsh-px) in erythrocytes and the increases of plasma free hemoglobin (phb), plasma malondialdehyde (mda) and 13-glucuronidase (13-gc) occured in septic shock. sa had stronger effect than gd in inhibiting the changes mentioned above. hence, these results suggest that sa has beneficial effect in the treatment of septic shock. sa could diminish the decrease of the antioxdase activities and inhibit lipid peroxidization and stabilize cellmembrance. this may be one of the principal mechanisms of the beneficial effect of sa in the treatment of septic shock. donna l. lehman, heather a. childers, irshad h. chaudry and alfred ayala. the thymus is thought to be a privileged sight for tcell generation. here the t-lymphocytes are either selected for their potential to recognize and react competently to foreign antigens or de-selected, due to their potential to react to auto-antigens, de-selected. tcells with this latter capacity are thought to be deselected through a process referred to as programmed cell death or apoptosis. while it is known that thymic apoptosis is elevated by a variety of stressers associated with infection and inflammation, little or no information is available concerning its presence in polymicrobial sepsis. it was, therefore, the aim of this study to determine whether thymocytes exhibit increased apoptosis during sepsis. to assess this, thymocytes were harvested from c3h/hen mice at i and 24 h following cecal ligation and puncture (clp; to induce sepsis) or sham-clp (sham). thymic yields were assessed and the extent of apetosis determined by staining the cells with propidium iodide (a dna intercalating dye) for facs analysis or by examining the extracted dna electrophoretically for the endogenous endonuclease activity the results (n=6/grp) indicate that at i h post-clp there was no marked changes in any of these parameters. however, at 24 h the thymic cell yield from clp mice was significantly decreased (sham:3.6!0.2 x l06 vs clp:0.3i0.1 x 106* cells; *,p<0.05), the % of cells apoptotic by facs analysis increased from 1.7i0.1% in sham to 8.7±3.2%* in clp, and the septic mouse genomic dna exhibited dna degradation these results indicate that clp, but not simple laparotomy, induces marked thymic apetosis, which may contribute to the lymphocytic immune deficiency seen in these mice the purpose of this retrospective study was to evaluate effectiveness of irrigation to treat septic knee joints from longterm results. from 1982 to 1992 31 patients with septic knee joints have been treated. treatment in acute cases started with asp#at[on of the effusion to evaluate for cultures. without awaiting the result treatment was continued with immediate joint irrigation under arthroscopic control. three redon tubes ( ch 16 ) were introduced for continuous irrigation and for intermittent distention of the joint cavity. in case of chronic joint infection additionally all foreign and synthetic material was removed. systemic antibiotics were given. patients could be re -examined with a mean follow-up of 6 years. re-examination showed that immediate arthroscopic lavage started at the onset of septic sings had best results (less signs of osteoarthr[tis, less synovitis, less pain while loading and less range of motion loss ). 21 patients (80%) out of 26 patients with s tre_=.tsd acute se.~tic knee joint had excellent functional recovery, whereas all 5 patients with chronic septic knee joints showed poor results. additionally, in 3 of these 5 patients with chronic septic knee joint after irrigation a further procedure was necessary due to recurrent septic knee joint. the concept that bacteria can translocate across the intestinal mucosa under a variety of circumstances is well established. however, due to the inherent limitations of in vivo studies, the cellular mechanisms underlying the process of bacterial translocation (bt) across the epithelial barrier are poorly understood. this is especially true in those circumstances in which there is no merphologic evidence of mucosal injury. consequently, we have utilized an in vitro cell culture model system to investigate the cellular events involved in the translocation process. in this model system, a polarized monolayer of intestinal cells (caco-2 cell line) is grown on a 3 micron filter in a two compartment system. after tight junction integrity is established, bacteria are placed in the apical surface chamber and bt across the caco-2 monolayer is measured by culturing the basal chamber. the results of our studies utilizing the caco-2 system indicates that; l) several strains of non-pathogenic bacteria will translocate across the caco-2 monolayer in a time-dependent and dose-dependent fashion. however, the incidence and magnitude of bt are highest for those strains of bacteria (gram-negative enterics) that are most commonly cultured in vivo. 2) caco-2 cells are actively involved in the transcellular passage of bacteria across the caco-2 monolayer, since inhibition of caco-2 microtubule or microfilament function decreases the magnitude of st. 3) lectin but not integrin receptors are involved in bacterial translocation of e. col~ across the caco-2 cell monolayer. 4) caco-2 cells have the ability to ingest and kill certain strains of bacteria. the mechanisms involved in caco-2 bactericidal activity appear similar to that of pmns to the extent that both are oxidant but not nitric oxide mediated. these in vitro studies suggest that caco-2 cells can function as "nonprofessional" phagocytes and that both bacteria and enterocytes are involved in the translecation phenomenon. bacterial translocation due to gut barrier dysfunction is considered to be a major cause of septic complications and multiple organ failure following trauma, burn injury, hypoxia, shock and shock-like states. the invasion of bacteria into the circulation from the gastrointestinal tract or even from other sources, however, should not necessarily result in systemic infection or organ cohinisation, as long as the humoral and cellular defense mechanisms are not impaired. thus, a reduced res clearance capacity and hepatic clearance function of bacteria and toxins can be observed under the same conditions which enable bacterial translocation from the gut. in order to evaluate the influence of circulatory, metabolic and humorul disorders on the elimination of bacteria out of the blood circulation, a series of experiments were performed in anesthetized and ventilated rabbits which received defined numbers (1.3 x 108 colony-furming units) of escherichia coli as a bolus injection. in unaffected control animals the intravenously injected bacteria are eliminated about 99.9% within the first 15 minutes and are totaiy cleared within 90 minutes. 57% offue total cfu counted in the cultures of the organ homogenates were found in liver and spleen, whereas only very low numbers could be detected in the lungs and kidneys. systemic injury of the animals by hemorrhagic shock, endotoxinemia, hypoxia, coagulation disorder, systemic vasoconstriction by norepinephrine and other types of injury reduced the elimination rate of bacteria and changed the pattern and degree of their dissemination and tissue distribution. the bacterial clearance was delayed mostly in rabbits subjected to hypoxemic hypoxia in which about 100 cfu of viable e.eoli per ml blood could still be counted 3 hours following their injection. the number of viable bacteria was some ten powers higher in organs of hypoxic rabbits in comparison to control ardmais, and an excessive colonisation of the lungs and kidneys with 25% respectively 11% of the total cfu of the cultared organ homoganates was observed. unilateral iscbemia of the kidney prior to the intravenous injection of e.coli only moderately delayed the bacterial clearance, bowever, caused an intense eohinisation of the affected reperfused organ. in contrast to this, inhalation injury did not promote the accumulation of bacteria in the lungs. conclusion: shock, systemic and local affections reduce the clearance of bacteria from the blood circulation and enable their dissemination and accumulation in vital organs. the degree of this reduction and the organ pattern of the bacterial distribution varies with the type of injury. thus, these factors seem to be essentially involved whether or not bacterial translocation and multiple organ failure will follow the invasion of bacteria into the circulation as in the ease of gut barrier dysfunction. movement of enteric baaterla from gut to extralumeaal sites (bacterial tra~sloeation) m~y play a role ~. nraltipl~ orga~ failure. traumatic stress (shock, hffectiort, and im~lammation) and severe illness are common alateeedents. tnt~st~aal lleus is e common proximme causal factor. we have exanlined the hypothesis that bacterial ttanslecation from the gut in expe~me~tal paaerestitis is due to bacterial overgrowth as a cuas~ucnee of a deere~ae in intestinal transit. ne~rotit_.~g pancreatitls fbliowlng bile duct iigation in the opossum is associated with colonization of the pa,se ~re, aa by gut derived organisms or salmonella of biljary origin in infected animals (previously reported). to ti~rther define the mechanism of transloemdon in panereati* inflammation, pancteatitis was induced hy iigatlon of t~e lower bile duet distal to the pancreatic duets m the rat. intestinal transit, enteric bacteriology, and tromsloeatien of bacteria to mesanterie lymph nodes, blood stream and splanelmie organs wen deletmhaed at 24 (n~ 7), 4g (n ~ 10), and 96 (n~ 10) hours with the fallowing results: ly~testinai,..transfi". geometric mean of fluorescent marker -25 % at 24 hour~ with return to 51% of gut leugth (similar to ~m) at 96 hour~. bacterial overgrowt_hff -increase in total bsctetia ~ad gram negative rod.~ at 2,; hours with return to sham control at 96 hours (9 log cfu/g veraus 6 log cfu/g ia ileum). translocation to mesenteric nodes -71, 90, and 30 percent at 24, 48, and 9fi hours compared to 49b in sham controls (n=l of 26). conclusion -bacterial translocation following panetoatieobiliary duct ljgation induced panereatitls ha the rat is tighlly linked to intestinal trausit and bacterial overgrowth within the gut lumen. speclrjlation -the ilens in in/~arm~ation may be related to activation of eytokiaes and mediated by nitric oxide. preliminary evidence for this notion will be discussed. in a series of studies we investigated: a) the time course of bacterial translocation (bt), b) the kinetics of lps and cytokine appearance (tnf, il-6, soluble tnf receptor [stnf-r] ) in the circulation, and c) the role of lps and/or tnf with regard to haemorrhagic shock (hs) related pathophysiologie alterations and mortality in baboons and/or rats. method: baboons were subjected to femur fracture, soft tissue trauma (acute experiment), and hs (40 mmhg mean arterial pressure: map for 2-4 h terminated at an accumulated oxygen debt of 180-200 ml/kg followed by resuscitation). a chronic model of hs was set up by administration of zymosan activated plasma (zap) before and after hs but without trauma. hs was induced in rats by bleeding the animals to map of 30-35 mmhg 90-180 rain followed by resuscitation. results and conclusion: in rats, lps increased in the systemic circulation, plasma tnf levels were found to be significantly elevated at 90 min and were still markedly increased at 150 rain postshock. plasma tnf, il-6, and stnf-r levels increased already during the shock period in baboons, while tnf was not detectable. our data suggest that haemorrhagic shock may lead to early bt in the intestinal wall (at 30 min following resuscitation in rats subjected to hs for 90 min, similarly at 3 h after the end of oxygen debt period in baboons ) and transient access of gut-derived lps into the circulation (levels became significant at 120 min, while in baboons higher levels up to 300 pg/ml were found at the end of shock and 1 h after reinfusion, partially accompanied by bacteremia). in addition, since the treatment of rats with anti lps measures or anti tnf therapy significantly improved the 48-hour survival rate it can be assumed that endogenous lps and lps-induced mediator(s), in particular tnf, may lead to organ injury and mortality following haemorrhagic shock. alteration of the mesenteric blood flow and the consequent ischemia / reperfusion injury to the intestine appear to be one of the earliest events in the systemic inflammatory response following severe thermal injuries. the causative relationship between the subsequent disruption of the intestinal integrity and the potentiation of the translocation of indigenous bacteria and/or endotoxins to remote body organs and the systemic circulation has been deeumented in several studies. among other effects, endotoxemia solely or acting synergistically with thermal injuries has been shown to promote bacterial translocation. as these sequences continue without adequate intervention, multiple organ failure and eventually death may become inevitable. hence, the crucial need of treatment modalities with the capacity of modulating the intestinal blood flow and preventing the manifestations of these pathological incidents. although the underlying mechanisms of this process have not yet been fully elucidated, there is accumulating evidence that various interacting vasomediators are involved. the actions of these mediators can probably be modulated by either nonspecific or selective agents. among the nonspecific agents, the composition, volume and timing of the resuscitation fluids appear to play an important role in this process. nitropmsside, a nonspecific vasodilator, has been shown to prevent the decrease of the postbum mesentefic blood flow when selectively infused in the mesenteric artery. the nonspecificity of various vasodialators with their possible undesirable systemic effects emphasizes the importance of the identification and modulation of selective vasomniiators. thromboxane a2 (txa2) and angiotensin ii (a-ii) appear to be the primary mediators of the postburn vasoconstriction. both mediators were found to be elevated following thermal injuries. in a previous study, pretreatment with oky-046, a thromboxane synthetese inhibitor was found to attenuate the postbarn mesenteric vasoconstriction. in a bum/sepsis porcine model the efficacy of dup753, an a-11 receptor antagonist as a posthum treatment modality was evaluated. treatment with dup753 prevented the early posthum and the late posteodotoxin elevation in the mesenteric vascular resistance and subsequently abrogated the fall in the mesenteric blood flow. the incidence of burn & endotoxin induced bacterial translocation was significantly reduced by dup753 from 86% to 29% (p<0.05, fisher's exact test). the indigenous flora of the gastrointestinal tract exerts a potent influence on the developmental maturation of normal systemic immunologic responsiveness. moreover, both oral and intraportal administration of experimental antigen is associated with a systemic state of immunologic hyporesponsiveness, known as oral tolerance. since trauma and critical illness are associated with changes in the flora of the gut and with altered systemic immune reactivity, we have hypothesized that the interactions of an altered gut flora with immune tissues in the gut and liver play a role in the pathogenesis of the immunologic derangements of critical illness. prolonged feeding of two clinically relevant killed organisms-pseudomonas and candida-to a rat results in significant impairment of cutaneous delayed hypersensitivity (dh) reactivity. conversely, measures directed against gram negative bacterial overgrowth in experimental peritonitis result in partial restoration of impaired dh reactivity. to ascertain the role of the liver in the pathogenesis of these alterations, we studied the differential immunologic sequelae of portal versus systemic (ivc) infusion of killed bacteria. experimental infusion of live or killed gram negative organisms into the portal vein produces dh suppression in rive, and profound suppression of mitogen-driven lymphocyte proliferation in vitro; systemic administration of the same organism has no effect on these phenomena. suppression is tgf3-dependent, and mediated by a soluble factor released by fixed tissue macrophages of the lung and spleen. the suppressive influence appears to be mediated at the level of interactions between il-2 and its high affinity receptor. release of this factor can be induced by a second circulating factor present in the plasma of portally-, but not systemically-infused animals two hours after bacterial administration. these studies suggest that the release of inducible immunoregulatory factors from the liver, and possibly from the gut, may play a role in the pathogenesis of the profound derangements of systemic immune homeostasis that accompany trauma and critical illness. acad.hospital st. radboud,postbus 9101,6500 lib nijmegen introduction. the central question being tested in this study was whether liposome encapsulated dichloromethylene-diphosphonate (ci2mdp) mediated elimination of liver and splenic maorophages would influence zymosan induced systemic toxicity (st) and bacterial lranslocation (bt). materia|s and methods. 90 male swiss (icr) mice were used weighing 21-25 g elimination of liver and spleen macrophages was accomplished by intravenous injection of 200/11 suspension of ci2mdp-liposome suspension. control mice received an i.v. injection with 200 pt phosphate-buffered saline (pbs) . two days later all mice were challenged with an i.p. injection of zymosan suspended in paraffin (z) at a dosage of either 01 mg/g, 0.5 mg/g or 1.0 mg/g body weight (n=8 in each group), signs of st and bacterial translocation bt were measured 24 hours later the st was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a four point scale. bt to the mesenteric lymph nodes (mln) and systemic organs (liver, spleen, lung and blood) was tested by culturing on blood and macconkey's agar at the time of sacrifice sections of the distal ileum were taken for histology. an additional two control groups (n=6 in each group) were tested to verify that neither paraffin nor pbs or ci2mdpqiposome suspension alone induced bt or st. furthermore survival over a 12-day period was assessed in a control and macrophage-depleted group with a dose of 1d mg/g z. results. neither the paraffin nor ci2mdp-tiposome suspension induced bt or st the incidence of bacterial translocation to the mln was similar between macrophage depleted and control groups. however, macrophage depletion was associated with a significantly higher incidence of systemic spread of bacteria. data expressed as positive tissues/total tissues tested: 11/32 vs. 1/32 at 0.1 mg/g zymosan (p~o.01); 19132 vs. 10132 at 05 mg/g zymosan (p~q05); 23/32 vs. 11/32 st 1.0 mg/g zymosan (p~o.o1).the magnitude of bt however did not show any differences. although systemic bt was greater in the macrophage depleted groups, the systemic toxic response was significantly less at doses of 0.5 mg/g zymosan (3.14 -+ 0.69 vs. 7.13 -+ 3, p~o.01) and 1.0 mg/g zymosan (4.38 -+2.50 vs. 11.25 -+ 2.5, p<_.o.01) . the 12 day mortality after 1.0 mg/g zymosan was 27% in the control group and 0% in the macrophage depleted group (p=o.05). histology did not show any differences between the control and macrophage depleted groups. conclusion. the lethal and toxic effects of zymosan in this model appear to be more related to the excessive activation of macrophages than to the systemic spread of bacteria. bacterial translocation (bt) in hemorrhagic shock was studied using a porcine model. in this study three groups were randomized: one control (n=3) and two experimental (n=6 each). a portal vein catheter was placed and remained in situ 48 hrs. a femoral artery and vein catheter were in place 12 hrs. the arterial eaanula was used for monitoring mean arterial pressure (map) in all animals and to bleed the experimental animals and the venous cannula to repelfuse with ringer's lactate (rl) or autologous blood (ab). baseline samples and values were taken in this period. control animals were observed for a sham shock period of 6.5 hrs., given 2 more hours of anesthesia, and studied for further 48 hrs. 40% of the estimated blood volume was withdrawn from each experimental animal over 30 min. these animals were kept in shock for 6.5 hrs. at the end of the shock one group was perfused with rl and the other with ab. two hours later they were extubated and further studied for 48 hrs. samples from portal blood for cultures and measurement of leukotriene b4 (ltb4), prostaglandin e2 (pge2), and nitric oxide (no) metabolites were taken from all animals at intervals beyond baseline up to 48 hrs. after 48 hrs. under general anesthesia a second laparotomy was performed and samples for culture from mesenteric lymph nodes (min), liver and spleen were taken; as well as samples for histologic study with light and scanning electron microscopy were taken from jejunum, ileum, and colon. animals were then euthanized. results: significant shock was induced as indicated by map and arterial blood gases. significant bt to min was observed in all groups. no significant bt was observed in cultures from liver and spleen in any of the groups. increased values for ltb4, pge2 and no metabolites were found during the period of shock. these results suggest that the isehemic phenomenon triggers a significant inflammatory response, and that bt to min may be an epiphenomenon without apparent significant influence on the inflammatory response. objective: to study the potential effects and mechanisms of action of systemic administration of monoclonal antibodies anti-endotoxin (ha-1a) in a animal model of gut-origin sepsis. materials and methods: exoeriment a. balb/c mice were transfused with allogeneic blood (from c3hb-iej mice). five days post-transfusion the animals were gavaged with lxl09 viable escherichia coil and randomized into 3 groups (n=22/group) to receive a sham burn (sb group) or a 20% thermal injury immediately followed by the systemic administration, via a tail vein, of monoclonal antibodies (6 mg/kg) (ha-1a group) or a 20% burn injury and administration of equal volume of sterile saline (c group). all 3 groups were resuscitated by intraperitoneal injection of 0.3 ml of saline. the animal survival rate was observed for 10 days post-burn. experiment b, transfusion and burn procedures were identical to experiment a but the mice (n=8/group) were gavaged with 109 viable e.coli labeled with mmndium oxine. four hours after burn the mesenteric lymph nodes (mln), liver, lungs and blood were harvested to determine plasma endotoxin levels and the magnitude of transtocation of labeled bacteria as measured by the residual radioactivity (dpm) in the organs. circulating endotoxin levels were determined by limulus colorimetric assay. diamine 0xidae(dao) is an enzyme existing in the mucosa of small intestines, but its activity is low in the plasma. it is evoked in the blood with the intravenous injection of much heparin. it is well known that da0 declines under the chronic mueosal injury when the small intestines are cut off or the mucosae become atrophied. aim of this paper is to confirm that da0 goes up to the measureable level by the intravenous injection of a small amounts of low molecular heparin(lmh objectives: the aims of this study is to investigate the inhibitor" effect of the prolease inhibitor, urinastatin, on endotoxin induced bacterial transleeation in mice. materials and methods:lcr mice were divided in six groups as the fotlows, group i: control mice receiving intraperitoneal injections (ip) of saline 24.5 and 24 hr prior to sacrifice, group ih treated mice receiving ip of utinastatin 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group ilk sham4reated mice receiving ip of saline 24.5 hr and endotoxin (5mg/kg) 24hr prior to sacrifice,group iv: saline control mice receiving ip twice during 30 rain intervals,group v: mice received ip of utinasmtin, and 30 min later they were received ip of endotoxin (5mg/kg), group vi: mice received ip of saline,and 30 min later they received ip of endotoxin (5mg/kg). in group l,ii and ill ,the mice were killed by cervical dislocation.using stetile techniques,a middle-line incision was made and the mesenteric lymph nodes were harvested in order to culture on selective agars for quantitation of bacterial trauslocation. in group iv, v and vi, survival was recorded for 3 days. unnastatin pretreatment could rednce mortality significantly. first surgey, shiga university of medical science, seta ,ohtsu 520-21,japan increased gut permeability correlates with inflammatory response in multiple trauma aptients. pape, h.-c. dwenger, a. grotz, m. regel, g. purpose: disturbances of intestinal permeability have been advocated as a pathogenetic factor of posttranmatic multisystem organ failure (mof). a close relationship with impairment of the stationary reticulocndothelial system (res) and a systemic inflammatory response (sir) was discussed. these tactors were investigated prospectively in patients with multiple trauma showing posttraumatie complications (multiple organ failure, mof). methods: 31 polytranmatized patients were studied prospectively. according to a mof-score (goris) those with posttrattmatic complications were selected (>7 points at 2 days), others were excluded. every second day gut permeability according to the ratio of urine concentrations of lactulose and mannitol (l/m) was evaluated (enteral application). at parallel time points res clearance capacity (k-value, invasion constant, normal range 0.6 -0.8 mind) was studied after i.v. injection of 100 mbq 99rotehuman albumin. liver perfusion was calculated from these data, total serum bilirubin (/zmol/l) was documented. serum elastase (#g/l) levels were determined enzymatieally. results 86.0 148+ 239+ liver perfusion did not ehangu, bilirubin showed progressive worsening indicating mof. a positive correlation was present between l/m and k (r=0.88) and between l/m and ela (r=0.71). conclusions: there is a positive correlation between the time pattern of intestinal permeability dysfunction and res hyperactivity as well as between intestinal permeability and the systemic intlammatory response (elastase levels). the results speak in favor of an interaction between intestinal and extraintestinal inflammatory systems, which in eombiuation are likely to be responsible for post~anmafic complications. endotoxemia, il-6 release and consecutive acute phase reaction are observed as a host response to surgical trauma. as well vasodilative prostaglandins (pg) and thromboxane (tx) are released after abdominal meaenteric traction (mt). the following hypotension and acute hypoxeraja are duo to prostacyelin (pgiz) arm can be avoided by perioperative cyclooxygenase inhibition. we therefore focused on the effect of pg and tx liberated following mt on the induction of endotoxemia. methods: in a prospective, randomized double-blinded protocol 50 patients, who were scheduled for major abdominal surgery (pancreatic or infrarenal abdominal surgery), were studied. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. mt was applied in a uniform fashion. baseline values were obtained before induction of anesthesia. further measurements followed before the incision of the peri[onenm (tl) and 5, 30, 90, 180 min, . the plasma concentrations (,pc) of 6-keto-pgft,, txb: and-ki-12-pgf ~ (stable metabolites of pgi2, txa2 and pge~) were determined by ria. we measured endotoxin pc by limulus-amoebocyte-lysate test and il-6 levels by elisa. data are given as mean+sem (* p< 0.05 placebo vs. [ibuprofen] ). results: endotoxin plasma levels increased before incision of the peritoneum tl both in the ibuprofen pretreated and in the placebo group. peak pc were observed 180 minutes after mt. endotoxin pc were significantly higher in the ibuprufen treated group (t5 0.16+0.02e[0.32+0.06] eu/ml). il-6 pc demonstrated an increase continuously from t4 to t7 (t7 115+12 [91 + 15] ng/l) in both groups. after intentional abdominal mesenteric traction we observed a marked increase of 6-keto-pgf~,, pc up to 6h after mt in untreated patients with a peak of 2450*[60] ng/1 at tl. also txb: and kh2pge 2 pc showed a considerabe increase up to 6h after mt in the placebo group. in ibuprofen pretreated patients the pg and tx pc remained within the normal range. discussion: our data clearly indicate a significant endotoxemia and il-6 release following major surgical trauma which is not initiated either by prostaglandin or thromboxane release. moreover endotoxemia is accentuated by ibuprofen pretreatment. therefore we hypothesize that in major abdominal surgery prostacyclin release-after mt may play a crucial physiological role in maintaining splanclmic microcirculation and thus preserving gut mucosal barrier function. objectives of the study it has been shown recently that parenteral and certain euteral diets promote the translocation of gut flora to the mesenteric lymph nodes (mln) and systemic organs, a process termed bacterial translocation (bt). in chow fed rats bt usually does not occur without further promoting factors. the goals of the present study were to determine whether the provision of defined amounts of standard lab chow during iv-tpn administration wotfld redane the incidence of bt, materials und methods male spf spragnle-dawley rats were divided into 6 groups. group 1 received standard laboratory chow feeding ad lib. in group 2 a central venous catheter was placed, ligated and secured by a spring coil tether attached to a swivel allowing free movement in the housing cage and chow was fed ad lib. in group 3 50% of the calculated daily required calory intake (drci) (307/kcal/kg) was given by iv-tpn (28% glucose, 4,25% amino acids) and 50% by limited chow administration. groups 4 and 5 received 70% and 90% of the drci by i.v. tpn and 30% and 10% respectively by chow feeding. group 6 received iv-tpn only. after 7 days the rats were sacrificed and the mln, liver, spleen and cecum removed aseptically, homogenized and cultured for bt samples of distal ileum were taken for light microscopy. the group with the least amount of chow shown to be protective against bt received the amount of non-fermentable fiber of that chow regimen during iv-tpn feeding and bt was studied. 4 5,5+0,9 4 12 23,9 -2,3 16 ,7 2/12 63+11 125~1 "7,7-+0,7 6,7-+1 5 12 24~6 -3,5 67 8/12 241+~243 170+163 8_+0,7 7+1,1 6 18 25, 5 -4 88,8 16118 2061-+3224 2211+4015 8,4~0,7 8,1-+1~1 conclusions: the administration of 30% of drci by chow feeding during iv-tpn significantly reduced the incidence of bt and maintained gut barrier function. the addition of the respective amount of dietary fiber of this group did not prevent iv-tpn-indueed bt. dr. med. m naruhn., dep. of general surgery, eberhard-karls-university, hoppe-seyler-str. 3 previous experimental studies have suggested that a disturbed ecology of the enteric bacterial population might contribute to the development of bacterial translocation from the gut in acute liver failure (alf). in the present study, the effect of oral administration of lactobacillus reuteri r2lc and oat fiber on bacterial overgrowth and translocation was investigated in rats with acute liver failure induced by subtotal (90 %) liver resection. the oatmeal soup base was anaerobically inoculated with lactobacillae and fermented for 15 hours, after which the animals were fed with either fermented or unfermented oatmeal or saline daily for 6 days prior to the operation. bacterial translocation to mesenteric lymph nodes (mln) and the systemic circulation was determined, as well as the intestinal bacterial flora and enterocyte protein content. the incidence of bacterial translocstion to the systemic circulation was nit in rats subjected to sham operation and saline treatment and 17 % in animals subjected to 90 % bepatectomy and lreatment with fermented oatmeal, while 80-90 % and 34-50 %, respectively, in rats subjected to hepatectomy and treatment with either saline or unfermented oatmeal. only one rat with fermented oatmeal demonstrated bacterial growth in mln (p < 0.05 vs hepatectomy and treatment with saline or unfermented oatmeal). the enterocyte protein content significantly decreased (p < 0.01) in salinetreated animals following 90 % hepatectomy, while there was no significant difference between bepatectomized animals with oral administration of fermented or unfermented oatmeal. the number of anaerobic bacteria, gram-negative anaerobes and lactobacillus significantly decreased and the number of e.cnli increased in the distal small intestine and colon in hepatectomized animals with enteral saline or unfermented oatmeal as compared with animals subjected to sham operation or bepatectomy with fermented oatmeal. our results thus show that the occurrence of bacterial translocatiou from the gut in 90 % hepatectomy-induced alf could be prevented by enteral administration of fermented oatmeal, maybe partly due to a positive effect on the enteric bacterial ecology. 190_+20" 16+_3" 11.7" data=mean_+sd, * stats anova p<0.05 vs control. l+air and lap groups, both exposed to exogenous i.ps shnwm:t m significant increase (p<.05) in lps gut translocation compared to control and l+co2. this correlated with a significant increase in peritoneal inflammatory responses (o2-,tnf) above that of the control and l+co2 groups, while mac-1 and cr3 opsonized phagocytosis were significantly impaired. the absence of significant differences between l+air and lap groups indicates that lps rather than wound factors is the principle mediator. thus, lps plays a significant role in regulating peritoneal responses in the early post-operative period dept of surgery, rcsi, beaumont hospital, dublin 9, ireland brlke e, berger d, staneseu a, buttenschsn k, vasilescu c, seidelmann m, beger hg in 32 patients undergoing a colonoscopy, endotoxin, endotoxin neutralizing capacity (enc), thromboxane b o (stabile metabolite of tbmomboxane ~), 6-keto-prostaglansin, leueotriene c4, interleukin 6 and the incidence of bacteremia were determined before and then every five minutes during the procedure. twenty-one of 32 patients showed a significant increase of endotoxin plasma levels during colonoscopy (p=0.003), whereas only one patient had a positive blood culture with bacteria obviously derived from the gastro-intestinal tract. the enc decreased significantly five minutes after the beginning of eolonoscopy and was diminished further thereafter. the baseline values were reached after 24 hours. ~hromboxane b o levels also increased after five min. from 89 to 376 pgyml peaking at 30 min. with 1332 pg/ml. 6-keto-prostaglandin,leucotriene c 4, ii-6 and crp remained unchanged. a control group of i0 volunteers who were not subjected to endoscopy, were prepared for eolonoscopy by orthograde lavage. the blood sampling procedure remained identical. no differences were seen in all described parameters for the controls. these data show that the gut barrier can be compromised by mininml invasive procedures, at least, concerning bacterial products. living bacteria, on the contrary, do not pass the gastro-intestinal wall. endotoxin, when determined by enc, is more sensitive than the conventional limulus-amebocyte-lysate test. no acute-phase reaction was induceri by the observed endotoxin translocation. it can be speculated from the dramatically enhanced thromboxane b 2 levels, together with its hemodynamie effects, that the thromboxane release may support translocation of bacterial products. sepsis is common after hemorrhagic shock. this study aims to demonstrate that hemorrhagic shock alone can promote translocation of gut bacteria from intestinal tract to its regional nodes and subsequently to blood. one hundred twenty mice, divided into 4 groups were subjected to 30, 60 and 120 minutes of 20%, 30% and 40% of hemorrhagic shock. on the specified time, blood cultures were taken and mice were sacrificed. the intestinal tract were histologically examined for any changes which allows translocation and its regional nodes were quantitatively cultured for translocated bacteria. there was a direct relationship between duration and degree of hemorrhagic shock and incidence of translocation (p 0.05). there was a high incidence of gut bacterial translocation to the mesenteric and mesocolic nodes in all degrees of shock (p 0.05). bacterial growth in the regional intestinal nodes increased and blood cultures were positive in direct proportion to degree and duration of shock. histologic evaluation of segments of git showed submucosal congestion to allow bacteria normally contained within the gut to cause systemic infections. translocation of gut bacteria in untreated hemorrhagic shock is clearly shown in this study on animal models. in this study, guotobiotic rats with 5 known species of bacteria were subjected to total parenteral nutrition(tpn) and subsequent hemorrhagic shock. the purpose of the study was to observe the impairment of gut barrier function following tpn and hemorrhagic shock and to study the mechanism of enterogenic infection induced by tpn and shock.the results were as follows: 1.long term(8-12 days) tpn induced impairment of gut barrier function, evidenced by atrophy of intestinal mucosa, significant decrease in diamine oxidase activity of intestinal mucosa and blood, and marked microecologic imbalance of the intestinal mucosa flora with dorminant growth of aerobes and relative decrease in anaerobes. the degree of mucosal damage were proportional to the duration of tpn. 2.in tpn+shock groups, failure of gut barrier function was found. ri,~ere were further damage in the mucosa, with a large number of gramnegative organisms invading mucosa and submucosa and a significant decrease in dao activity as compared with each relative tpn groups. these changes were significantly correlated with enhanced bacterial translocation, elevation of lps and mda levels in the plasma. these findings suggested that long term standard tpn impaired the gut barrier function, precipitating posttraumatic gut barrier failure. thus infec. fion following shock might be oi'iginated from the gut and it was obviously related to the impaired gut defence resulted from antecedent tpn. the determination of plasma dao activity might provide a valuable tool for the ear. ly diagnosis of gut injut;y during tpn and after trauma. in our earlier studies we have investigated the dynamics of granuloayte infiltration of the ischemic/reperfused s~all intestine (g. illy~s, j. hamar int. j. exp. 9athol. 73. 161. 1992.) . there was a increasing infiltration of the mucosa c111m~nating at the 3d to 4th hours of reperfusion. in the present series we have studied sc~e of the conseqn/ences and the possible role of this cellular reaction. 45 ~in isehemia was followed by a 4 hour reperfusion in the anesthetized rat. arterial ~/ad mesenteric venous blood samples were collected at 5 m_in, i, 2~ 3, and 4 hours of reperfusion. elastase and lactate concentrations were determined and hamoculture was carried out from the blood samples, and tissue pieces from the heart, lung, liver and kidney were collected for histological analyses at the above mentioned times of reperfusion. all blood samples were free of cell bacteria. staphylococci appeared only occasionally at the 4th hour in the arterial blood .and at the 3d and 4th hours in the venous blood, respectively. arterial and venous elastase activities were high throughout the reperfusion, venous concentrations being higher at all times. lactate concentrations of the arterial and mesenteric venous blood samples increased during shock. ~ranuloeyte infiltration of all organs studied appeared during the 2d hour and it increased at later times of reperfusion. it is concluded that heavy infiltration of the intestinal mucosa can block bacterial translocation in most of the cases during reperfusion. granulocytes activated either by the reperfused area or by the released cytokines infiltrate other organs contributing by this way to the mesenteric shock s!rndrc~e. intestinal motility plays an important role for maintaining nutrient transport and absorption and for balancing the enteric bacterial population. disturbances of intestinal motility may be one of the earliest notable changes in intestinal function. in the present study, we aimed at determining early alterations in intestinal transit time following ischemia-reperfusion injury induced by occlusion of the superior mesenteric artery in the rat. intestinal ischemia was induced for 20 and 30 minutes by applying a microvascular clip on the superior mesenteric artery followed by reperfusion 2, 4 and 6 hours after clip removal. intestinal transit time was measured by the propulsion of a radiolabelled solution (cr51). light microscopy was performed on intestinal samples. macroscopical pathological changes were not observed. however, microscopically, mucosal epithelial oedema, degeneration or slight ulceration occurred in rats 6 hours after reperfusion in ischemia-20 rain group and 4 and 6 hours after reperfusion in the ischemia-40 rain group. delayed small intestinal transit time was seen from 2 hours and on after intestinal ischemia for both 20 and 40 rain ischemia followed by reperfusion. the distribution of radioactivity demonstrated that most radioactivity was accumulated in the first two segments following intestinal ischemia and reperfusion, significantly differing from what was seen in animals subjected to sham operation (p < 0.01). the distribution of radioactivity in segments 4 and 5 in the group with repeffusion 6 hours after intestinal iscbemia for 20 rain was significantly higher than that noted in the group with repeffusion 6 hours after intestinal ischemia for 40 min (p < 0.01). q'he results indicate that a delayed intestinal transit time may be one of the earliest pathophysiological alterations noted, associated with duration of gut ischemia, and a potential factor for the development of bacterial overgrowth, gut barrier failure and bacterial translocation, in hypovolemic conditions. bacterial infections still constitute a major cause of morbidity and mortality in patients with acute liver failure. the present study aimed at evaluating the effect of ethylhydroxyethyl cellulose (ehec) on bacterial translocation following surgically induced acute liver failure. acute liver failure was induced by subtotal hepatectomy (90 %) in the rat. water-soluble ehec was administered orally 1 and 12 hours prior to hepatectomy. the incidence of bacterial translocation from the gut to mesenteric lymph nodes (mlns) and systemic and portal circulation was evaluated and the number of isolated bacteria from these samples and from intestinal content were determined. intestinal transit time, bacterial adherence onto the intestinal surface, intestinal mucosal mass, bacterial growth and dna synthesis, bacterial surface characteristics (hydrobiology: hydrophobicity, hydrophilicity and neutrality; surface charges: positive, negative and neutral) were also determined. hepatectomized animals showed a 80-100 % translocation rate to mlns or blood 2 and 4 hours after operation, while only 0-1% of rats subjected to sham operation or animals with 90 % hepatectomy and pre-treatment with ehec (p < 0.01). bacterial overgrowth, increased bacterial adherence onto the intestinal surface as well as decreased intestinal mucosal masses were observed in animals with subtotal liver resection alone, alterations that were prevented by enteral ehec treatment. a delay in intestinal 2-hour transit time occurred in both groups with subtotal liver resection, with or without enteral ehec. ehec inhibited bacterial growth and dna synthesis, and altered bacterial surface properties following 1hour incubation with bacteria. in conclusion, the findings in the present study imply that ehec alters enterobacterial capacities for metabolism, proliferation and invasion by effects on e.g. bacterial surface characteristics. furthermore, ehec seems to possess a trophic action on the intestine, rather than exerting its effect by enhancing intestinal motility. department of surgery, lund university hospital, s-221 85 lund, sweden disturbances in intracellular calcium signalling can potentially result in impairments of cellular responses vital to the functional integrity of both immune and non-immune cells, and thus contribute to a decrease in host resistance against infection and to multiple organ system failure during sepsis. studies in our laboratory have focused on assessments of intracellular ca 2÷ regulation and ca~+-depended cellular responses in the liver, skeletal muscle and splenic tlymphocytes harvested from rats subjected to gram-negative intraabdominal sepsis. cytosolic ca 2+ concentration, [ca2*]i, and ca 2+ fluxes were measured by the use of fluorescent ca 2+ chelating dyes (fura-2 or indo-1) and 45 ca respectively. to assess sepsis-related changes in ca 2+ dependent cellular responses, we measured the acute phase protein response in the liver, the regulation of protein and sugar metabolism in the skeletal muscle, and the proliferation response in the splenic tlymphocytes. altered ca2+ i signalling with sepsis was correlated with an exaggerated inappropriate acute phase protein response (100% ¢) in the liver, and a blunted insulin mediated sugar utilization (60% 4) and increased proteolysis (50% ~) in the skeletal muscle. in t-lymphocytes, a decrease in mitogen induced elevation of [ca2+]i by 35-40% was correlated with a significant depression in their proliferative capacity. these studies clearly suggest that altered calcium signalling is correlated with disturbances in cellular responses in both immune and non-immune cells during sepsis. the altered cellular responses adversely effect the outcome of the septic injury. (supported by nih grant gm 32288). alfred ayala, ping wang and irshad h. chaudry. changes in macrophage capacity to respond to foreign pathogens are thought to be central to the developing immunosuppression associated with traumatic injury. in this respect, the suppression seen in m~ functions following hen (a common component of traumatic injury) may be mediated by the direct or indirect inhibition of their capacity to perceive external stimuli (e.g., opsonized & non-opsonized bacteria, and their cellular components, etc.} due to the breakdown of the receptormediated signal transduction system. results of a number of studies by our laboratory and others indicate that this inability to respond to external stimuli is in part due to the loss of cell surface receptors. decreases have been documented for not only la antigen, but also c3b, fc, and tnf receptors following hem in mice. furthermore, studies which have examined second messenger generation in these cells indicate that m~ derived from the peritoneum and spleen exhibit a decreased capacity to mobilize ca +2 from intracellular stores. this protein kinase dependent process of [ca+2] i mobilization appears to be linked to the inability to synthesize inositol triphosphate. of interest, the depression in ca +2 signal generation appears to be inversely related to presence of elevated levels of camp in m~ from hen mice. we have reported that m~ priming agents, such as ifn-7 (which exhibits salutary effects on m~ function following hem), appear to restore cell signal transductive capacity while reducing the levels of camp. nonetheless, the extent to which depressed receptor signal transduction in hem, is due to receptor loss~dysfunction or elevated antagonistic second messenger levels remains to be determined. conclusions: significant impairment of calcium signaling occurs at all time-points prior to and following pha stimulation in trauma patients. tcell activation failure can, in part, be explained by the inadequacy of this essential intracellular second messenger system. restoration of immunocompetence following trauma will have to address strategies to better assess and restore this vital step in the activation sequence leading to proliferation during the antigen recognition process. patrick a. bseuerle institute 01 biochemistry, albert-ludwigs-university, hermann-herder-str. 7, d-79104 freiburg, germany the active form of the transcriptional activator nf-~b is a heteredimer composed of a 50 and 65 kda polypeptide. in this form, nf-'<b can bind with high affinity to decameric sequence motifs (consensus: 5"-gggpunnf'ypycc-3") found in enhancers and promoters of many genes activated upon a wide variety of pathogenic conditions, including viral and bactedai infections, acute phase response, oxidative stress and uv and gamma irradation. clinically important target genes for nf-k:b encode inflammatory cytokines (il-1b, tnf, [l-6, il-8, gm-csf etc), cell adhesion molecules (icam-1, elam-1, vcam-1), acute phase proteins and immunoregulatory ceil surface receptors. in most cell types, nf-~:b is sequestered in the cytoplasm and, therefore, unable to initiate transcription of these genes. the cytoplasmic form of nf-~:b contains one additionai subunit, referred to as h,;:b. i~b can reversibly suppress dna binding and nuclear uptake of nf-~b by virtue of its association with p65. upon stimulation of cells, ikb is proteolytically destroyed, allowing nf-kb to enter the nucleus and activate genes upon binding to transcriptional enhancers. we have demonstrated that various antioxidative compounds inhibit the activation of nf-~b in response to many inducers. moreover, nf-~:b was shown to be activated upon treatment of cell cultures with p.m-amounts of hydrogen peroxide. these data suggested that nf-~b prinicipaliy is an oxidative stress-responsive transcnption factor. the notion is supported by numerous reports on the induction of oxidative stress by many nf-nb-inducing agents. nf-~b activition can likewise be suppressed by protease inhibitors. these drugs apparently inhibit a yet unidentified protease responsible for the inducible decay of inb. we propose to use nf-nb inhibifors as novel drugs with a very broad application under acute phases el inflammation, injury and infection. northern blot experiments revealed that expression of the recently discovered lipopolysaccharide binding protein (lbp), a 58/60 kd glycoprotein, in the liver rises substantially during the acute phase response. experiments with an in-vivo acute phase model using new-zealand-white rabbits and also in-vitro experiments employing stimulated hep-g2 cells showed that lbp transcripts could be induced 10 to 100 ford within 24 hours after induction with cytokines. by using a newly established nonradioactive nuclear run-on technique we show that induction of lbp during the acute phase is transcriptionally regulated. furthermore we screened a human genomic library and were able to clone the lbp gone, containing the promoter 1.5 kb upstream. several liver-specific and "acute-phase-typical" potential binding sites were found fn the promoter region and reporter gone assays were set up. several caat-boxes, the il-6 responsive elements hstf-hsp 70.5 and h-apf-1 rs as well as the transcription factor hnf-5 and the glucocrticoid-responsive elements oct-2-d and gcre were found, which correlates with the induction pattern of lbp mrna in hep-g2 cells by il-6, il-1 and dexamethasone. pcr-based analysis of the exon-intron pattern of the lbp gene revealed similarities with the genes of two other lps binding proteins, namely bpi and cetp. further analysis of this novel acute phase protein, that also has an important role in endotoxin recognition and immunomodulation will help in understanding the complex acute phase mechanism and potentially lead the way to new therapeutic strategies. lps activation of nucle?/r fa-ctor:~b " (nf:~bj"in cd14.transfected fi'broblasts does not appear to require tyrosine kinase activity d. t. golenbock, r. delude, r. savedra, s. vogel and m. j. fenton lipopolysaccharide (lps) is the major constituent of the outer leaflet of gram-negative bacteria. during the course of serious infection, shock may occur as a result of the interaction of lps with macrophage lps receptors. cd14, a 55 kda glycosyl phosphatidylinositol (gpi)-linked protein, functions as an lps receptor. a critical issue in lps activation concerns the mechanism by which cd14, which has no transmernbrane domain, transduces a sig,nal after lps binding. evidence exists which suggests that cd14 may signal cells after lps binding by interacting with an lps signal transducer with tyrosine kinase (tk) activity. wild-type chinese hamster ovary fibroblasts (cho) normally do not respond to lps, but can be activated following transfection with cd14 (cho/cd14). stimulation of cho/cdi4 with as little as 1 ng of lps/ml resulted in the release of 3h-arachidonic acid (3h-20:4) into the culture supernatant. in addition to 3h-20:4 release, we examined lps-imiuced activation (transl0cation) of the transcription factor nf-~b. similar to lps-induced 3h-20:4 release, these responses were observed only in cho cells lransfected with cd14 closely resembling the same response in the marine macrophage cell line raw 264.7. maximum nf-~cb expression occurred at 30 minutes. in contrast to lps-induced 3h-20:4 release, dexamethasone, cycloheximide, and actinomycin d had no effect on activation of nf-r,.b in cho/cd14, suggesting that nf-r,b activation begins early after lps binding to cd14 and does not require transcription or translation. we then examined cells for lps-induced tk activity by western blotting cellular ly~ates with anti-phcsphot-'rosin:~ anl',bodie~" although tk activity was seen in lps-stimulated raw cells and phorbol ester stimulated cho/cd14, lps-induced tk activity in cho/cd14 was not observed. although the tk inhibitor herbimycin inhibited the release of 3h-20:4 in cho/cd14 and raw cells, neither herbimycin nor genestein effectively blocked nf-r,b activation in either cell type. these results suggest that tk activity is involved in a portion of the signaling pathway that is distal to initial signal transduction. the absence of observable lps-induced phosphotyrosine residues in cho/cd14 cells as well as the failure of tk antagonists to inhibit lps-induced nf-~cb activation suggest that the proposed cd14-related lps signal transducer in cho/cd14 is not a tymsine ldnase. dimished cardiac inotropic function and response to 13-adrenergic receptor (13-ar) stimulation are frequently seen in septic patients, these cardiac defects are also seen in animal models of entoxemia. to determine the characteristics of the decreased transmembrane signal associated with the decreased beta adrenergic response we measured the force and rate of contraction of atria harvested from sprague-dawley rats exposed to endotoxin (0:2 gm/kg). to stimulate various components of the transmembrane signal cascade of 15-ar was isoproterenol (i3-ar), acetylcholine (m2ach receptor), naf(gs and gi proteins), forskolin (adenylate cyclase), and calcium (intracellular contracttile signal) were used. to eliminate the transmembrane control of calcium and test the intrinsic contractile response, hypothermia (22°c) was used the results showed alterations in the inotropic function with no significant difference in chronotropic response. the inotropic reponse for the endotoxin exposed atria and controls is shown below these results show that there is a transmembrane signal defect both for the i~-ar signal and for ca +. these data indicate that the level of the defect appears to be at the g proteins. ischemia-reperfusion of the rat intestine produces an injmry both to the intestine and to distal organs, notably the lungs and liver. prior studies have shown that 1 h of intestinal ischemia leads to a nemtrophil independent reperfusion injury of the gut. thus, rats rendered neutropenic by pl~l antiserum have been shown to express a severe local gut injury when subjected to ischemia and reperfusion. this gut injury is postulated to he mediated at least in part by the complement system. the soluble (s) cri receptor which binds to c3b and cdb was given to rats at a dose of 12 mg/kg by intravenous bolus, 15 min before reperfusion of the ischemic gut. a control group was given pbs buffer. at 4 h of rmperfmsion the animals were sacrificed. pbs treatment led to a significant activation of both classical and alternate complement pathways while scri inhibited both pathways (fall to zero of chbo ). intestinal mucosal injury score, assessed by histological grading was reduced by scri (2.49±0.22 to 1.73_+0.17,p<0.05) . intestinal neutrophil sequestration estimated by assay of myeleperoxidase was also reduced (0.05 ± 0.01 to 0.02 ± 0.01 u/g). c3 was detected in the gut hy i~unohistochemistry. remote organ injury was modified that is lung permeability (ration of concentration of radiolabelled albumin in broncho-alveolar lavage fluid to that in blood) was reduced (11.3±1.4 x i0 3 to 3.9±0.8 x 103, p<o.05). however, neutrophil sequestration by the lungs was unaffected. this remote protection is thougth to be related to prevention of ic3b deposition on imng andotheli~ which activates adherent neutrophils. these findings support the hypothesis of complement mediated local and remote injury. we postulate that complement fragments may be deposited either as a result of the ischemia induced loss of membrane bound complement regulatory proteins, such as decay accelerating factor or membrane cofactor protein, which allow complement fragments to accumulate on the endothelial cell surface. alternatively, increased expression of p-selectin on the endothelial cell surface may, by means of its complement binding domains, act as a lattice for complement deposition. indeed, p-selectin has been detected on the surface of endothelial cells ans platelet-neutrophil aggregates recovered from the intestine of these animals. ischemia (4hr) followed by reperfusion (dhr) of rat hind limbs resuits in local (muscle) injury as well as in remote (lung) injury, which is characterized by increased vascular permeability (leakage of j2sl labeled albumin) and neutrophil accumulation (tissue content of myeloperoxidase, mpo). within 60 minutes of reperfusion following ischemia, tumor necrosis factor-o~ (tnfc 0, interleukin-i (1l-i) and il-6 plasma levels increased significantly, reaching maximum levels after 2 hours ofreperfusion. polyclonal antibodies to tnfet and 1l-i provided significant protection against muscle and lung injury. muscle and lung vascular permeability decreased in anti-tnfct treated rats by 41% and 74% respectively and mpo was reduced by 72% and 83%. antml-1 yielded a protection in muscle and lung vascular permeability of 24.7°,4 and 52.4% respectively whereas muscle mpo was reduced by 46.4% and lung mpo by 25.9°,4. these results were confirmed by the use of soluble tnf~ receptor and il-i receptor antagonist (il-ira). when icam-i expression in vivo was examined using 125mabeled antmcam-i, constititive expression of icam-1 was evident only in lung but not in muscle. during reperfusion icam-1 expression increased by 98.2%. treatment with anti-tnfcx or il-ira reduced icam-i upregulation by 73.9°,4 and 74.8% respectively. frozen sections of normal rat lung revealed no evidence of e-selectin expression, whereas lung sections obtained after ischemia and reperfusion immunhistochemically demonstrated expression of e-sdectin. lungs from rats pretreated with anti-tnfct were negative for e-selectin staining. our data indicate a role for cytokines in the upregulation of adhesion molecules in ischemia/reperfusion injury. adherence of neutrophils to the coronary endothelium is associated with significant myocardial injury following 90 min of ischemia (mi) and 4.5 h of reperfusion (r). p-selectin is expressed by activated endothelial ceils and platelets within 10 min and tethers polymorphonuclear leukocytes (pmns) to the endothelium. we examined the cardioprotective effects of a monoclonal antibody (mab) designated pb1.3 to p-selectin in a feline model of mi+r. pb 1.3 (1 mg/kg) administered 80 rain post-occlusion (10 min prior to reperfusion) significantly attenuated myocardial necrosis compared to a non-blocking mab (nbp1.6) for p-selectin (15 + 3 vs 33 + 5% of area-at-risk, p<0.01). endothelial dependent relaxation to acetylcholine was also significantly preserved in ischemic-reperfused coronary arteries isolated from cats treated with pb 1.3 compared to nbp1.6 (67 + 6 vs 11 + 3, p<0.01). furthermore, pmn adherence to ischemic-reperfused coronary endothelium was significantly attenuated in pb1.3 treated cats (64 + 7 vs 136 + 12 pmns/mm z, p<0.01). also, normal coronary endothelium activated with thrombin (2 u/mt) had increased pmn adherence under conditions of shear stress, an effect which was significantly (p<0.01) blocked by pb1.3, but not by nbp1.6. furthermore, p-selectin is markedly upregulated 10-20 min post-reperfusion in coronary arterial and venous endothelinm. similar results were obtained in a rat model of mesenteric ischemia/reperfusion, including upregulation of p-selectin on mesenteric vascular endothelium. these results demonstrate that tethering of neutrophils to endethelium by p-selectin is an important early consequence of reperfusien injury, and a specific monoclonal antibody to p-selectin exerts significant endothelial preservation and cardioprotection in ischemia and reperfusion states. as recent interest has increased in the role of extracellular matrix proteins in inflammation, we examined the role and relationship of laminin and fibronectin to the processes of cell infiltration in a rat model of heart isografts. changes occurring in this system are on a nonimmunological basis and may be an effect of the initial surgical manipulation and the warm and cold ischemic times of transplantation. lewis (lew) donor hearts were transplanted into lew recipients, naive lew acted as naive controls (nc). grafts were harvested 2, 4, 8, 16, 24, 48, 72, 120 and 168h after transplantation (n=6/time point) . snap frozen sections of organs were stained for cd-4 (w3/25), cd-8 (ox8), t-lymphecytes (tl) (ox-19), macrophages (ed1, ed2), neutrophils (pmn) (mom), icam-i (iap29), laminin and fibrinogen. results were evaluated visually by counting cells/field of view (cf) for w3/25, ox8, ox19, ed1, ed2 and mom, staining for icam1, lain and fib was assessed visually using a scale (ve=0-10). as early as 2h after transplantation, fibrinogen and laminin expression increased in i, peaking 8h after transplantation (ve= 4.5; nc: ve=i.5) . at 16 and 24h in i, on both vessels and interstitial cells laminin and fibrinogen expression had declined to baseline (ve=i.5) but rose again at 48h, peaking at 72h (ve=4) and remaining elevated thereafter (ve=3). the high expression at 8h correlated well with the maximal pmn infiltration at that time (cf=6) in i returning to baseline at 24h (cf=0.5) and thereafter. the second peak at 72h correlated with the onset of macrophage and t-lymphocyte infiltration. thus, laminin and fibrinogen seem to guide leucocyte infiltration following graft ischemia during the time of reperfusion. background. reperfusion of ischemic kidneys with xenogeneic blood leads to activation of endothelial cells and circulating leucocytes with the final consequence of microvascular thrombosis. in concordant systems, the mechanisms of rejection can be studied due to cross-reactivity of mediators with anti-human monoclona~ antibodies. aim of the study. to obtain information about the kinetics of proinflammatory cytokines and production of soluble adhesion molecules in the acute phase of reperfusion, eight kidneys from rhesus monkeys were perfused ex-vivo with human blood (group b/o) for one hour in a closed system. blood levels of il-lb, il-6, tnf-a, soluble icam and e-selectin were measured in elisa-technique under steady-state conditions. results. cytokine levels rose significantly within the 60minute interval (il-lb: 6,1__+2,6 to 161,1__+98,5 pg/ml; il-6:30.24-7,7 to 274,2__+75,8 pg/ml; tnf-a: 544,2__+363,6 to 1651,0+25,7 pg/ml; p<0.05). immediately after the beginning of reperfusion, soluble icam-1 and selectin levels were abnormally high and constantly rising throughout the observation period, reaching significance at 60 minutes. discussion. high levels of proinflammatory cytokines may lead to an induction of adhesion molecules, thus upregulating the leukocyte-endothelial interaction in an complement-independent mechanism. specific pretreatment with monoclonal antibodies against icam-1, lfa-1 or other soluble mediators may be useful in downregulating hyperacute rejection in transspecies transplantation. prolonged ischemia has been associated with later dysfunction of solid organs as it may cause upregulation of adhesion molecules, production of cytokines on vascular endothelium and migration of host cells into the graft. these early events may lead to irreversible organ injury. this study evaluates the effects of global ischemia on early immunohistological changes in cardiac grafts transplanted in rats. isografted hearts (lewis->lewis) were were divided into ischemic and non-ischemic groups (n=30/group). all donor hearts were flushed immediately with cold saline. non-ischemic hearts were then transplanted within 30 rain, ischemic hearts were stored in cold ringer's solution for 3 hours before revascularization. representative grafts were removed after 12. 24, 72hrs, 10 and 100 days, and evaluated immunohistologically (cells/field of view=c/f). restitution of ventricular activity was significantly delayed in ischemic grafts (5 vs 1 rain). after 12 hrs, all ischemic grafts exhibited an extensive interstitial edema, declining slowly thereafter. at the same time, numbers of pmn peaked (3 vs 1 c/f in non-ischemic grafts), whereas edl+macrophages (9 vs 2 c/f) and tnfe expression peaked by 24hrs. by 72 hrs t-lymphocytes began to enter ischemic myocardium and icam-1 was moderately increased. after 10 days cellular infiltration had returned to baseline, and no differences were seen among both groups after 100 days. global myocardial ischemia inhibits initial graft function, and engenders a brisk inflammatory reponse, primarily pmn and macrophages, with increased mhc class ii and cytokine expression. leukocyte -endothelial interactions are the result of endothelial activation, leukocyte activation or combination of both, which are accompanied by nee-expression, upregulation or shedding of adhesion molecules (selectins, inlegrins). such interactions differ with regard to the stimulus (e.g. thrombin or histamine for p-selectin, endotoxin or tnf/il-1 for e-selectin), the time course of response (minutes versus hours) and the localisation in different organs. recently assays are available for circulating soluble fragments of the cell bound adhesion molecules e.g. se-seleetin was found to be increased in plasma concurrent with high circulating endoloxin and cytokine levels. the importance of adhesion molecules for the sepsis event is evident, while effectiveness of anti-adhesion inolecu]e therapy is controversial e.g. beneficial anti-e-selectin therapy in baboon bacleremia but deleterious effects of amti-cd18 treatment in the same model. in other species similar controversial results with anti-cd18 therapy in sepsis were reported. steven l. kunkel,theodore standiford* and robert m. stricter. the migration of leukocytes to the lung during endotoxemia is dependent upon the coordinated expression of lung vascular adhesion molecules and the subsequent production of appropriate leukocyte chemotactic proteins. in experimental animals, neutrophils accumulate within the lung soon after the administration of endotoxin, while mononuclear cell infiltration occurs in a more distal manner. a kinetic analysis of lung leukocyte levels revealed a 3-fold increase in neutrophil numbers associated with dispersed lullg tissues 2 hours after lps treatment, while macrophage levels increased by 4-fold at the 24 hour time point. thus, the recruitment of different leukocyte populations to the lungs during endotoxemia is likely directed by different mechanisms. recent studies have identified a supergene family of small inducible chemotactic cytokines (chemukines) which possesses chemotactic and activating properties for neutrophils. the prototype of this family is interleukin-8 (il-8). interestingly, a related supergene family has been identified which possesses activity for recruiting mononuclear cells. examples of this group of inflammatory chemukines are monocyte chemotactic protein-i (mcp-i) and macrophage inflammatory protein-i alpha (mip-i). in initial in viva studies we examined whether mip-i was expressed systemically or in a compartmentalized fashion post lps challenge. assessment of plasma cytokine levels revealed maximal tnf levels occurred i hour post lps administration, returning to baseline by 4 hours, while mip-i levels were maximal at 2 hours (2,5 ng/ml), with a second peak at 24 hours after lps challenge. interestingly, aqueous extracts of liver homogenates from lps treated animals demonstrated no mip-i levels, while aqueous extracts of lung revealed a 4-fold increase in mip-i levels over control lungs. immunohistochemical analysis of the lungs from 24 hour lps treated animals demonstrated the alveolar macrophage was a rich source of mip-i protein. cell-associated mip-i was also expressed by blood monocytes adherent to the pulmonary vascular endotheliun, however the expression of monocyte-mip-i was observed by 2 hours post lps administration. immunohistochemical analysis also demonstrated that mip-i antigen is associated with the extracellular matrix on the interstitial side of the endothelium. this suggests that the extracellular matrix, which is produced during inflammation, can bind mip-i and this may serve as a depot for the prolonged presence of nip-1. in additional studies we have demonstrated that the intratracheal instillation of rmui [ip-l(loong) activation of polymorphonuclear leukocytes by inflammatory stimuli may contribute to the development of multiple organ failure in septic patients. thereby pmnl are proposed to avidly adhere to vascular endothelium causing damage by the subsequent release of toxic agents. as cellular adhesion is primarily mediated by 62-integrins and lselectins, the present study compares the expression of these adhesionmolecules on pmnl in septic patients and healthy volunteers. methods: expression of 62-integrins and l-selectins on pmnl was measured in whole blood by flow cytometry using the monoclonal antibodies ib4 and dreg 200, baseline values were determined immediatley after drawing blood. in addition cells were incubated 45 min at 37°c to allow for spontaneous regulation of adhesion molecules. 55 blood specimens from 8 septic patients were obtained during the course of their illness. control values were determined in 12 healthy volunteers. results: baseline expression of 62-integrins and l-selectins was not signifcantly different in septic and in healthy subjects. in contrast, there was a significant upregulation of g2-integrins and shedding of l-selectins of pmnl in septic patients (sp) compared to healthy volunteers (hv). the local or systemic production of inflammatory cytokines, such as tumor necrosis factor alpha (tnfc~), can serve to modulate multiple aspects of neutrophil function. the ability of neutrophils to leave the circulation and migrate to areas of infection is one essential component of host defense. l-selectin, a leucocyte-associated adhesion molecule, is responsible for the initial reversible contact between neutrophils and endothelium and the subsequent roiling action of neutrophils along the vessel wall. in contrast to other adhesion molecules, l-selectin expression is rapidly down-regulated after neutrophil activation. the loss of l-seleclin may thus be a critical determinant of how neutrophils become unbound from their endothelial attachments and enabled to proceed towards an underlying extravascular area of infection. we hypothesize that the shedding of l-selectin is a strictly controlled process, occurring primarily at localized sites of inflammation, which may be modulated by tnf~, a flow cytometric method of staining neutrophhs by monoclonal antibodies in whole blood is described whereby the kinetics of l-selectin shedding may be followed in real time. the dose response and time course of in-vitro l-selectin shedding by neutrophils from normal human subjects was assayed after exposure to n-formyl-methionylleucyl-phenylalanine (fmlp) and tnfc~. either singly or in combination, our results show that l-selectin shedding can be reliably followed over time. a significant percentage of cells shed l-selectin after exposure to 2000 pg/ml tnfc~ or 1000nm fmlp (but not at 400pg/ml tnfc~ or 200nm fmlp). greater numbers of cells were able to shed their l-selectin when fmlp and tnf~x were presented in combination rather than alone. high levels of tnfc~ did not appear to alter the threshold concentration of fmlp required to induce shedding, we conclude that the extent and rapidity of l-selectin shedding may be modified by different combinations of ligands and that shedding, by vidue of the high concentrations of cytokines or chemotactic factors required, is a process localized to sites of infection or inflammation. we prospectively studied 23 patients with severe sepsis syndrome; group a : septic shock with or without adult respiratory distress syndrome lards) (n =7, bacteremia = 6); group b : sepsis syndrome without septic shock (n = 16, bacteremia = 9). serial plasma samples obtained on day 0, 1, 3, 6, 10 and 14, were assayed using elisas method (british biotechnology), normal control levels of soluble icam-1 and e-selectin, obtained from 11 healthy volunteers, were respectively 205 ± 19.4 ng/ml and 45 ± 5.7 ng/ml (mean _+ se), acute lung injury was quantified dally on a tour-point score system (murray, am rev respir dis,1989) . compared to control mean values, initial levels of groups a and b were significantly higher for icam-1 (p < 10 -4) and e-selectin (p < 10-3). comparisons of group a and [] (* = p<0.05; ** = p<0.00t) soluble icam-1 levels of group a enhanced significantly (p<0.05) during the first 24 hours, and a sustained high levels was of bad prognosis (25% of survivors at day 6). the evolution of soluble icam-1 and e-selectin levels were significantly correlated with murray's score (spearman test : p <0.001). conclusion: these results suggest that endothelial adhesion molecules are released into the plasma of patients with severe sepsis syndrome. soluble icam-1 and e-selectin are correlated with endothelial lung damage, and loam-1 seems to be a better indicator of the severity of endothelial injury. introductory remarks to anti-adhesion molecule strategies as a therapeutic modality ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the development of antimicrobial therapy represented a major breakthrough in the struggle against disease. it strengthened the notion that disease could be overcome by eliminating foreign invaders threatening the host. this paradigm has proven to be very successful, the threat of many infectious diseases has significantly changed, some have even been eradicated. nevertheless, sepsis has remained a severe condition, increasing in incidence while mortality remained very high. more recently, it has become increasingly clear that besides the nature and treatment of an exogenous agent, the reaction of the host defense itself plays a pivotal role in the outcome of the event. endogenous mediators, such as tnf, il-i, il-6 and il-8, govem many of the actions of the host defense system. while the expression of these cytokines more often than not benefit the host, (over)-expression can cause severe damage. based on this hypothesis,anticytokine strategies, such as those targeted against tnf or il-1, have been evaluated for the treatment of sepsis. results of these early studies have not yet indicated success in improving the outcome of the disease. it has been difficult to define a patient population where a benefit could be reproducibly shown. furthermore, it has been documented that synergy between cytokines occurs, but detailed knowledge of the cytokine network is not yet available. it is conceivable, that neutralization of one cytokine prompts the induction of another which will evoke the intended response in the host. recent data obtained in human endotoxemic volunteer models seem to confirm this. if this turns out to be the case, neutralizing a single cytokine may not be a successful approach. cytokines in tum, induce various adhesion molecules, such as icam-1. such molecules regulate for instance the neutrophil-endothelial cell interactions, which are thought to play an important role in the pathogenesis of systemic organ injury. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries. thus, altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by inappropriate behavior of host defense cells. targeting cellular surface interactions has been added to the efforts to change the outcome of disease. modulation oftheseprocesses seems very promising, but may temporarily leave the host without effective defense mechanism. great care therefore, must be exerted when studying this powerful two-edged sword in a clinical setting. our knowledge of the role of adhesion molecules in the intlammatory response has increased rapidly due to the availability of new reagents and mice geneticly deficient in adhesion molecules. these molecules are important in interactions of leukocytes with endothelial cells, other leukocytes, platelets, and epithelial cells. when these molecules are engaged, they can also play a role in activating leukocytes and their effector functions. in the venules of the systemic circulation, adhesion often occurs through a series of sequential interactions. initial interactions are mediated by members of the selectin family to loosely associate the leukocytes with the endothelium and are followed by firm adhesion requiring members of the integrin and immunoglobulin family. later interactions with endothelium may require pecam. adhesion molecules are usually required for leukocyte emigration in response to extravascular stimuli and for neutrophil-mediated endothelial cell injury. they are critical for host response in many diseases including infections. however, when the inflammatory response results in damage to host tissues, patients may benefit from blocking the leukocyte response. anti-adhesion molecule agents are an important potential antiinflammatory therapy. the focus of anti-adhesion therapy may be at any step of the sequence. diseases where anti-adhesion molecule therapy may benefit patients include ischemia/reperfusion injury in many organs, ards and mof, and transplantation, both to protect the donor organ from ischemia/reperfusion injury and to inhibit graft vs host disease. many strategies have been considered and include: 1) blocking the ability of adhesion molecules to recognize their ligand using antibodies that have been humanized or soluble receptors linked to igg to prolong their circulating halflife, 2) blocking the ligands for adhesion molecules using soluble adhesion molecules, peptide analogues, or oligosaccharides, and 3) blocking the production of the adhesion molecule using anti-sense oligonucleotides. because the synthesis of adhesion molecules is usually regulated by cytokines, inhibiting the action of cytokines is another potential site for interrupting the adhesion process. although important issues of safety must be evaluated, the potential for modulating the inflammatory response make this an exciting area of improvement in health care delivery. claire m. doerschuk, m.d.; riley hospital for children, room 2600; 702 barnhill drive; indianapolis, in 46202 usa. modulation of neutrophil-endothelial cell adhesion with anti-cdl i/cd18 monoclonal antibodies as a therapeutic modality. ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the central role of inflammatory cells in the pathogenesis of lung and systemic organ injury is well recognized. binding of neutrophils to endothelial cells and migration into the parenchyma are largely regulated by complementary adhesion molecules. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)18 chain covalently linked to one of three different alpha chains (cdlla, cdllb, cdilc) and exist on the cell surface as three distinct heterodimers. cdlla/cd18 is expressed on all leukocytes, whereas cd 11 b/cd 18 and cd 11 c/cd 1.8 are restricted to cells of myeloid origin. cd i 1/ cd 18 interacts with intracellular adhesion molecule-1 (icam-i), its ligand on endothelial cells. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in a large number of experimental models. protective effects with anti-cd18 antibodies have been observed in a wide variety of inflammatory, immune, and isehemia-reperfusion injuries, such as arthritis, burns, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degenemrion, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. blockage of cd18, however, would affect all leukocytes, as would antibodies to cdlla/cdi8. targeting cdllb/cd18 would affect cells of the myeloid lineage only, which could prove to be beneficial. cd11b/cd18 is not only involved in transendothelial migration, but is also implicated in adherencedependent formation of reactive oxygen species. blocking cd1 lb/cd18 may therefore not only reduce the numbe r of leukocytes accumulating in the tissue, but also attenuate the oxidant stress of infiltrated neutrophils. anti-cd 11 b treatment has been used effectively to reduce tissue injury initiated by ischemia-reperfusion, complement activation and endotoxemia. altering the host response by modulating the function of adhesion molecules may attenuate the inadvertent injury caused by leukocytes, but may also temporarily leave the host without effective defense machinery. overall, animal studies suggest that it may be safe to inhibit neutrophil adhesion for a limited period of rime. these observations will have to be confirmed in carefully designed clinical trials. c, arbobydrams are ubiquizom constir~uts of cell sv.rfaees, and possess many c~xssfies ttm~ m~,e ~em ide~. canaidates for r~ognifioa mole~ule& in m~y systems whe,~ cer udhesioa ~lays a critical ro~ car~hydram l:~dtag ~otegas have been shown to b~ad tocell surfa~ earbohydzaxes ~nd pzrl~pate in cell-ceil lumtaefion& such sys,.ems include ~rti~za~io=, deveaopmeat, l~thoge~-hcet reeog--ition ~d i~zmmadon_ in particular, tb.z recent di%~ve~ of lhe selec~ and th~ impo.~a~c~ in teukccy~udo~lelium adh~ion has -~f~m av.c~on ok l~in m~ted cell adhe~on. s~vere/poten~s/cs.rbohydr~ l~ga~s hrve ~e~l ~u~ilied for ~he s~lcc~ins. the,~ c~u be broadly di,,sded la~o ~wo m'oups -sibyl l~wis x m~ mh~.~l oligo~chadd~s, ~d sf/~ ca~ohydmma, all ~:~ ~l~dns bind m siflyl l~wis x (sie$ o!igos~ccb.e.rkms, zlthou~ w~ differing avi~re~. 'we have i~¢n~ed the functional g~oups 0a s~ex ~n~ med/a~ ~he b~u ~di~g of ~h~ c~b0hydmm = e-se/sedm we have used ~hat iv.formation to sya~esize sle ~ '~mt0gs r.he, t focus on replacing slslic ~sd ~nd fuc0s¢ wi~ simpler, more stable strunt~es. a[~ou~a ~ proeer~ is ongoing, we hve been ~ucee,.~ful a~ rep~aein8 t.ke si~ic a~id. residue wi~ std.fzte. ~ce~ or la~c amd groupa we t'we ex2aninad &e ten, bunion of ezed~ hydroxyl group of the fizeose residue ~ billding of e-, l-~nd p-selees..u. we have also found m~fi~fio~ of the reducing end ~¢.cha'i~ ~z increase mtagovsst activity. the, m¢ond. group of figs,rids a.r eontzin su~a~ 0u a ea.rbohydr~t¢ support,, und seem to bi~.d to t~e sele~ti~s wi~ dlf:ferem characteristics c0.an does sle:, s=h compounds are m2ogniz~d by l-selects. md p-selectia, bur., in genera/, not e, selecti~ these dam may mdicam r.hat l-and p-s~ ¢at~ h~d via o, second ~te thaz operates lu~.ead of, or in conjunction with ~tc sle" b~ding ~iite. dam rela~&~g to ±e, se two types of ,ml~ liga~ds have beam t~ed to desig~ potential the2~peutics for i~fi~anmat0ry disease. lr:rng maimai models of acute lung lu3ury we can demo~trate that eompmmds that inhibit seleetiu birding ~ ~i~o hzve ber~ficial effects when uc~d in rive. progressive microvascular damage in the tissue adjacent to a cutaneous burn injury results in extension of burn size. the role of leukocytes in the pathogenesis of microvascular injury was investigated by inhibition of their adherence to the microvascular endothelium using monoclonal antibodies directed to leukocyte cdi 8 or its endothelial ligaud, intercellular adhesion molecule-1 (icam-1, cd54). a model of thermal injury was developed using new zealand white rabbits. two sets of three full-thickness burns separated by two 5 x 30-mm zones were produced by applying brass probes heated to 100°c to the animals' backs for 30 sec. cutaneous blood flow determinations carried out with a laser doppler blood flowmeter were obtained for 72 hours. there were five experimental groups: controls given saline alone; animals given monoelonal antibody to the cd 18 r 15.7 prior to burn injury (pre-r 15.7); animals given r 15.7 30 min after burn injury (post-r 15.7); animals given a monoclonal antibody to icam-i, r 6.5 prior to burn (pre-r 6.5); and animals given the r 6.5 30 min postburn injury (post-r 6.5). blood flow in the marginal "zone of stasis" between burn contact sites was significantly higher in the antibody-treated animals. administration of the antibodies 30 min after injury was as effective as preburn administration in preserving blood flow. at 72 hr post-burn all antibody -treated animals had blood flow in the areas at risk for progression (i.e., the zone of stasis) at or above baseline levels while the control animals had levels equal to 34.7 _+ 12% of baseline (p < 0.05 by analysis of variance and mann-whitney u test). these results indicate that leukocytes play an important role in the pathogenesis of burn wound progression, and that this progression can be attenuated by moduiating adherence to endothelial cells. a wealth of information now supports the hypothesis that inhibition of cell adhesive mechanisms will nter the course of immunologicand inflammatory processes. what remains unclear is whether inhibition of specific mechanisms wfl[ be of therapeutic benefit in any specific human disease. current data derived from animal models are not inconsistent with the hope of therapeutic benefit, but techniques for inhibition (e.g., antibodies, antisense oligonucleotides, inhibitory peptides, inhibitory carbohydrates, smaii synthetic inhibitors, etc), tissue and species differences in the relative contributions of adhesion molecules to the inflammatory process, and the cascade model of adhesive interactions are all confounding issues, making predictions of therapeutic benefit in any specific human disease process very difficult. additional concerns involve the potential roles of adhesive mechanisms in host resistance to infection. as human therapeutictdals are initiated, more exact information on the roles qf specific adhesion molecules in human disease should emerge. inhibition of leukocyte adherence to endothelial cells can represent a novel therapeutic approach to septic shock. we performed a pilot study to evaluate the safety and tolerability to cy-1787, a monoclonal antibody against human e-selectin, in patients with septic shock. septic shock was defined by clinical signs of sepsis, a documented source of infection, and fluid-resistant hypotension requiring the use of vasopressors. eleven patients entered the study, but 2 patients who died during the first 24 hours were excluded, as this was part of the protocol. cy-1787 was administered as a single intravenous bolus of 0.1 mg/kg (n=3), 0.33 mg/kg (n=3) or i mg/kg (n=3) mg/kg. the antibody was well tolerated. none of the 9 patients died during the 28 day follow-up period. organ failure was assessed for 5 organs (cns, lungs, liver, kidneys and coagulation). the mean number of organs failing, which was initially 2.7 ± 1.2, decreased to 0.2 ± 0.4 at the end of the study (p<o.o01). these results are therefore encouraging. administration of an antibody to e-selectin in patients with septic shock requires further study. the importance of cytotoxic t lymphocytes (ctl) in the host defense mechanism against viral as well as parasitic and bacterial diseases is becoming increasingly appreciated. the design of vaccines to generate cytotoxic t ceils from nonviable or subunit constructs, however, poses challenges due to the unique characteristics of antigen processing and presentation by class i major histocompatibility complex (mhc) molecules. since the final antigen recognized by ctl are short peptides capable of high affinity binding to class i mhc molecules, we have designed strategies: a) to define the structural characteristics of peptides that are required for their high affinity binding to mhc; and b) to formulate these peptides into immunogenic vaccines that are capable of generating cytotoxic t lymphocytes. the strategy and results to accomplish these two goals will be discussed. amnon altman and erich gulbins ras proteins represent essential intermediates in receptor-mediated growth and differentiation pathways. they couple signals initiated by receptor or nonreceptor protein tyrosine kinases (ptks) at the cell surface to cytoplasmic targets which coordinate signal transduciion to the nucleus. ras also participates in t lymphocyte activation initiated by the antigen-specific t cell receptor (tcr)/cd3 complex, which leads to lymphokine production and proliferation. receptor ligation causes activation of associated ptks of the src and syk families as the earliest identifiable event in this pathway. the importance of ras in t cell activation is indicated by the findings that an oncogenic ras protein activates the interleukin-2 (il-2) gene promoter; conversely, a transdominant inhibitory ras mutant inhibits this event and the activation of map kinases. the rate-limiting step in ras activation is the exchange of bound gdp for gtp, which is catalysed by guanine nucleotide exchange factors (gefs). we identified the sh2/sh3 domain-containing vav protein, which is selectively expressed in hematopoietic cells, as a ras-specific gef coupled to several hematopoietic receptors. vav can be activated by two pathways, i.e., by ptk-mediated phosphorylation or by binding of diglyceride second messengers to a cysteine-rich, protein kinase c (pkc)homologous vav domain. these pathways are independent as demonstrated by structure/function analysis and the use of pharmacological inhibitors. the two activation pathways may enable vav to integrate signals from distinct hematopoietic cell receptors, and couple them to ras. t cells express another, ubiquitous ras gef, i.e., sos, which is known to be coupled to activated tpk receptors. t cell activation leads to membrane association of a signaling complex consisting of sos and two other signaling proteins, grb-2 and shc, via direct binding of the shc sh2 domain to the tyrosine-phosphorylated chain of the tcr/cd3 complex. thus, multiple receptors, ptks and ras activators participate in signaling pathways that lead to hematopoieitc cell growth and differentiation. the interactions and function of these various signal transducers will be reviewed. phagocytes. immunity is mediated by specific t lymphocytes, cytokines produced by these specific t cells activate antimicrobial capacities in mononuclear phagocytes, thus contributing to protection. other immune mechanisms also participate in the acquisition of resistance. on the other hand, t cells are also crucial for many pathologic sequelae of intracellular bacterial infections. although ifn-y is central to macrophage activation, synergistic and antagonistic effects with other cytokines occur. the cd/3 t cells, representing the major t cell population in experimental mice, are the main mediators of antibacterial immunity; an auxiliary role for y/~ t cells appears likely. the c~//3 t cell population further segregates into cd4 t cells which recognize microbial peptides in the context of gene products of the major histocompatibility complex (mhc) and cd8 t cells which see their peptide in the context of mhc class i. the -y/~ t cells are generally double-negative. knock-out mice in which the genes for various t cell receptor chains, for mhc class i or mhc class ii molecules, for cytokines or cytokine receptors had been deleted, have provided extremely helpful tools for analyzing the role of t cell subsets and cytokines in antimicrobial immunity. using these mutant mice, the role of distinct t cell subsets and cytokines in bacterial elimination and granuloma formation was analyzed in detail. these studies underline the central role of a//3 t cells and ifn-y in antibacterial immunity and, furthermore, show a distinct function of 7/$ t cells. moreover, these studies show that the relative contribution of cd8 or cd4 t cells to antimicrobial immunity varies in response to different pathogens. apoptosis is an active form of cell death in which the cell participates in its own demise, providing the biochemical pathways that trigger and mediate the complex events of the process. although this phenomenon has been studied for many years, it is only recently that significant progress has been made towards the elucidation of the molecular mechanisms that control apoptosis. once such mechanism came as something of a surprise: in a number of eases, apoptosis can be promoted by the action of the c-myc protein, a protooncogene product that had previously been associated only with cell proliferation (and presumably, survival) . expression of c.myc in cells can cause them to enter an apoptotic pathway or proliferate, mad this "decision" depends upon additional signals, such as growth factors that inhibit apoptosis and allow the ceils to proliferate. thus, c-myc directs cells out of rest and the choice of proliferation or death depends upon the presence or absence of survival signals. other survival signals are generated by oncogene products that can cooperate with my¢, including bcl-2 and abl there are implications of this interplay between apoptotie and anti-apoptorlc signals in cell iransformation and the resistance of some tumor ceils to therapeutic agents capable of inducing apoptosis. oncogene interactions therefore control the life or death of transformed ceils but also have important roles in normal, development processes. in the immune system, developing t ceils are "screened" for potential self-reactivity by a process of negative selection, in which activation via the t cell receptor (tcr) induces apoptosis. this process can be mimicked in t cell hybridomas, and appears to depend upon the expression of the c-mye protein. evidence that this represents a requirement for the myc/max heterodimer will be presented. as above, these cells are presented with the "choice" to proliferate or die and this depends not only upon myc but also other signals. thus, expression of functional v-an kinase in these ceils can render them resistant to the activation of apoptosis via tcr ligation, surprisingly, bcl-2 does not appear to be capable of blocking this form of apoptosis, and the reasons for this will provide new insights into the development of the immune system and the process of apoptosis. although the oncogene products discussed here probably do not directly participate in the biochemical process of apoptosis, they represent molecular controls on this complex mechanism. as such, they gave us new ways to look at the life and death of a cell recent attention has focused on macrophage release of cytokines, such as il-1, il-6 and tnf as important mediators of septic shock. however, serum cytokine levels have correlated poorly with outcome of septic shock. the purpose of this study was to compare the functional status of macrophages to serum cytokine levels in early sepsis by an analysis of splenic macrophage protein synthesis during abdominal sepsis. macrophage total protein distribution and biosynthetic activity was assessed by large format 2-d page, autoradiography of 35s-labeled proteins and computer assisted densitometric analysis. sepsis was induced in sprague dawley mts by cecal ligation and puncture (clp). untreated and sham operated rats served as controls. splenic macrophages were harvested at 3 and 24 hrs. following clp. serum il-6 levels were determined at 3,12, and 24 hours by the 7 tdi bioassay. by 12 hrs. 100% of the clp mls exhibited multi-microbiaj bacteremia. control or sham operated rats did not show bacteremia. clp resulted in a significant 286% elevation (p < 0.05) in serum il-6 levels at 12 hrs. there was no difference in il-6 at 3 and 24 hrs. when compared to the control groups. 2d page studies examined splenic macrophage total protein distribution and biosynthetic activity at 3 and 24 hrs. post-clp. at 3 hrs. macrophages from clp rats showed a substantial decrease in total protein pattern (353 protein spots vs 503 protein spots) when compared to non-septic control macrophages. a decrease in the number of lower molecular weight proteins (< 40 kd) was observed. at 24 hrs. post-clp, splenic macrophages from clp rats showed a substantial increase in the synthesis of low molecular weight (< 40 kd) proteins, when compared to non-septic controls. we conclude that: l) fulminate abdominal sepsis induces an early (3 hrs.) staining with cd14 and cd16 mabs will identify two monocyte subpopulations in human blood: a major population of regular monocytes, which strongly expresses the cd14 antigen (cd14++) and a minor population with weak expression of cd14 and expression of the cd16 antigen lcd14+/ cd16+ cells). in healthy control donors(n=35) the latter cells account for 45+22 cells/mm 3 and 9%+5% of the monocytes. in sepsis patients, the cd]4+/cd16+ cells can become a major population with more than 50% of all monocytes in 3 of 18 patients and with more than 500 cells/mm 3 in 4 of 18 cases. there was no correlation of cd14+/ cd16+ cells to any clinical parameter except for cd14+/cd16+ percentage and body temperature (p= 0.013). the cd14++ monocytes showed a substanttial decrease in cd14 antigen density in 9 of 11 patients. three-color-if shows that the cd14+/ cd16+ cells in sepsis patients when compared with the cd14++ monocytes exhibit a higher level of class ii antigen and a lower level of cdiib and cd33 antigens, consistent with a more mature nature of the cd14+/cd16+ cells. levels of il-6 were increased in sepsis patients: 3 of 5 patients with high numbers of cdi4+/cd16+ cells ( 200/mm 3) had high levels of il-6 (250u/ml). these data suggest that septicemia may lead to substantial changes in blood monocyte composition and this may be related to elevated levels of cytokines such as il-6. human peritoneal macrophages were exposed to increasing doses of lipopolysaccharide (lps) or a synthetic lipid a analogue (mrl 953) and production of the cytokines il-lb, il-6, tnf-a and g-csf was assessed at the protein-and mrnalevel. cells were also stimulated with low doses of lps and mrl 953 to study their "adaptation" to a secondary challenge with high doses of lps. tnf-a production after stimulation with lps could be almost completely relieved by preincubation of cells with low doses of lps and similarly, il-6 production was suppressed. surprisingly, however, "adapted" cells were able to synthesize even larger quantities of of g-csf and il-lb when exposed to a secondary lps challenge. the changes in tnf-a, il-6 and g-csf protein synthesis could a/so be detected on the mrna level, whereas transcript levels for il-lb remained unaltered as assessed by northern blot analysis. high doses of the synthetic lipid a analogue mrl 953 were also able to "adapt" macrophages for a secondary lps challenge by downregulating tnfa-and il-6-production, while priming secretion of g-csfand il-lb as well. taken together, here we describe for the first time the discordant adaptation of human peritoneal macrophages to a secondary lps stimulus in vitro. these findings appear to bear ramifications for the in-vivo endotoxin response during inflammation and gramnegative septicemia. shock states with inadequate cellular oxygen delivery may contribute to macrophage (mo) activation or dysregulation. in this study we compared the effect of transient hypoxia and endotoxin pretreatment (lps 1) on mo mediator release with a second endotoxin stimulus (lps2). methods: in vitro cultures of marine peritoneal exudate mo were exposed to 2 hr. of hypoxic or normoxic conditions, then incubated 22 hr under identical normoxic conditions _+ 10 ng/ml of lps 1 pretreatment. during the final 24 hours all m~l were exposed to a range of lps 2 concentrations. m~i supernatants were assayed for tnf, il-1, il-6, pge2, nitric oxide (no), and superoxide release. results: lps 1 markedly inhibited mo tnf release by lps 2 but hypoxia had no effect on lps 2-triggered tnf release. hypoxia increased mo il-6 production in the absence of lps 1, but inhibited lps 1 augmentation seen under normoxic conditions. pretreatment with lps 1 increased no production from lps 2 under normoxic conditions, but bypoxiainhibited this effect. pretreatment with lps1 signifcantly augmented mo release of il-i and pge 2, but hypoxia had no significant effect (data not shown) superoxide production was increased by lps 1 under normoxic conditions, but hypoxia significantly inhibited superoxide release (not shown a. lepape, c. docile, f. arpin, m. c. gutowski, v. banssillon and j. bienvenu. i aim of the study. increased levels of tnf are inconsistently correlated with the severity of sepsis. one possible explanation is a decrease ability of ceils to produce eytokines. the objectives of this study were m compare at different levels of of clinical severity the responsiveness of cytokine producing cells. this was evaluated by the response to a stimulation by lps as well as to the inhibitory effect of ptx. the technic used is a whole blood ex-vivo model, as described by desch et al. (1) . h materials and methods. different groups of icu patients, postoperative (n=12), sepsis (n=12), septick shock (n=13), and healthy control (n=10), were studied. blood was drawn in endotoxio free heparinized robes. stimulation was achieved by addition nf75 pl oflps (10ng/ml) to 675 id of whole blood in presence of various concentrations of ptx (0, 10 "5, 10 -4, 10 "3, i0"2m). after 5 hrs incubation at 37°c, the tubes were centrifuged and supematants frozen at -80°c. tnfa and/l6 were measured by elisa (medgenix r and immunotech r respectively). monocytes were quantified on a technicon h6000. the results are expressed as median values. non parametric tests are used for testing differences between groups. hi results, tnftx and 11,6, corrected for monocytes count, are given as % of their initial value, the baseline before stimulation or inhibition being 100%, 1) stimulation by lps. the control group is much more stimulated (>21000% for il6, >8000% for tnfa). blood samples taken postoperatively and in patients with simple sepsis are significantly less stimulated (>1500% for il6, >600% for tnfa ). the lowest stimulation was observed in patients with septic shock (median = 168%), some patients being not stimulated at all. 2)effects of ptx.the inhibitory effect of ptx on tnftx production is effective in all groups at 10 -3 m (reduction to less than '¼ of the median values), and is almost complete at 10" 2 m. the septic shock group has a decreased sensitivity to ptx. il6 production exhibits a lesser reduction at 10 -3 m (~ 'a to ½ of the median values), further increased at 10 -2 m. the septic shock group is again less sensitive to ptx. iv conclusion: the reduced ability of circulating monocytes to produce cytokines during severe infections is confirmed here. ptx is able to reduce significantly tnfc~ at 10 -3 m and the inhibition is nearly complete at 10 -2 m. surprisingly, there is a lesser, but significant suppressive action of ptx on il6, not found in experiments using purified monocytes. one possible explination could be the interplay between cytokines production. (1) lymphokine research (1989) cdna sequencing constitutes a powerful method of measuring steady-state mrna levels for all genes transcribed in a given cell or tissue at a particular stage of differentiation. by comparing transcript abundance both prior to and following differentiation, individual genes can be identified whose transcription is regulated both positively and negatively. in order to examine monocyte activation, the human monocyte line thp-1 was induced with phorbol ester (48 h) and activated for 4 h with lipopolysaccharide (lps) after which polya + rna was purified. the rna from control and lps-treated cells were each used to construct a cdna library under identical conditions, and all resulting clones were selected for cdna sequence analysis. each clone sequence was evaluated by matching with both genbank and our own gene databases. very different patterns of gene expression were seen in the two libraries, the latter reflecting very high levels of known inflammatory mediators such as il-1 and tnf. a second set of libraries were made from umbilical vein endothelial cells (huvec), both with and without lps stimulation, and were analyzed in a similar fashion. the effects of lps induction on specific gene transcription in both cell types will be discussed. t. tadros, md, th wobbes, me) phd, rja goris, md phd to investigate whether the preactivation of regional macrophuges by liposomes containing muramyl tripeptide (mtp-pe) can counteract the detrimental effect of blood transfusions on both anastomotic repair and host susceptibility to infections. methods eighty lewis rats received lmg/kg of either empty or mtp-pe encapsulated liposomes, intraperitoneally (ip). twenty-four hours thereafter, the animals underwent resection and anastomosis of both ileum and colon, and received 3 ml of either saline or blood from brown norway donors,iv. the animals were killed 3 or 7 days after surgery and examined for septic complications and anastomotic repair. the average anastomotic strength, as assessed by bursting pressure (+sd), was significantly diminished in the transfused animals, as compared to the non-transfused animals (ileum;day 3; 44-+8 vs 99+7, p<0.001). transfused animals pretreated with mtp-pe encapsulated liposomes showed a significant improvement of their anastomotic bursting pressure (93+9, p<0.001 vs transfusion). pretreatment with mtp-pe encapsulated liposomes decreased significantly the incidence of anastomotic abscesses in transfused animals ( from 90% in ileum on day 3 to 20%, p<0.001). conclusions preactivation of regional macrophges by intraperitoneal administration of mtp-pe encapsulated liposomes prevents the detrimental effects of transfusions on anastomotic repair and reduces the incidence of intraabdominal sepsis. academic hospital nijmegen, dept of general surgery, pb 910 i, 6500 hb nijmegen, the netherlands. leukemia cell line, teip-1. robin s. wa, gner*, perry v. halushka "~, and james a. cook*, departments of physiology , pharmacology "l" and medicine "t, medical university of south carolina, charleston, s.c. 29425. adherence of monoeytes to endothelium and extracella/ar matrix proteins is essential for accumulation at sites of inflammation. txa2, an arachidonic acid metabolite, inhibits human monocyte chemotactic responses suggesting that txa 2 may alter monocyte adhesiveness. we selected the thp-1 cell line, a human monocytic leukemia cell line to further investigate the effect of txa 2 on adhesion. we tested the hypothesis that txa 2 alters lpsinduced adhesion of thp-1 cells and that txa 2 exerts its effect on adhesion via a camp dependent mechanism. thp-i cells were exposed to s. enteritidis endotoxin (lp.g/ml) _+ the cyelooxygenase inhibitor lndomethacin (in), the txa 2 mimetic i-bop (0.11.tm,) or txa 2 receptor antagonists bms180291 and l657925 (10~m). cells were allowed to adhere for 24 hours and adherent protein/well was determined. lps-induced a significant (p<0.05;n=7) increase in adherence of thp-1 cells (basal, 2.4+0.85gg protein/well; lps, 20.4+_6.0p.g protein/well). the amino acid glutamine is an essential compound for synthesis of purine and pyrimidine basis and therefore necessary for rna-and dna synthesis. in human plasma the concentration of glutamine is between 0.5 -0.6 mm, and is reduced in septic patients up to 80 % (0.1 -0.2 mm). monocytes play a central part in the inunune system and it was of interest, whether glutamine is involved in the modulation of cell surface markers and phagocytosis of these cells. human peripheral blood mononuclear ceils were obtained from 500 ml heparinized blood of apparently healthy donors by ficoll-paque density gradient and isolated by counterflow elutriation. the puritiy was more than 90 %. subsequently cells were cultured in phenolred-free rpmi 1640 medium with various concentrations of glutamine (0.05, 0.1, 0.2, 0.3, 0.6, 1, 2 mm) in teflon-fluorinated ethylene propylene bottles to exclude cell adhesion and possible cell activation. aider seven days culture, cell viabilty was determined by trepan blue exclusion and varied between 70 and 90 %, independent of glutamine concentrations. cell surface markers were detected by flow cytometry, noaspecifie phagoeytosis was measured with latex beads and specific phagocytosis with opsonizied e.eoli using a facscan. lower concentrations of glutamine decreased the expression of hla-dr and icam-1/cd54 on monocytes in a dose-dependent manner. the receptor for fc'/rucd64 as well as the receptors for complement cr3/cdllb and cr4/cdllc were down-regulated. cr1/cd35 which is only slightly expressed on monocytes was not influenced. furthermore, no effects on the expression of cdi4, the receptor for transferrin cd71 and fc'friii/cd16 were seen. our data indicate, that lower concentrations of glutamme influence the phenotype of monocytes. we are now interested to study whether glutmnine influences non-specific phagocytosis, or whether specific phagocytosis correlates with the decreased expression of fc'/r and complement receptors. we investigated immunologically more than 300 patients who were admitted to icu because septic syndrom during the last four years. patients were immunologically followed up 2-3 times per week until release from icu. the expression of hla-dr antigen on monocytes turned out to be the best prognostic parameter. the persistence (> 5 days) of low hla-dr expression (< 30%) predicts fatal outcome (mortality > 85%). the altered phenotype was associated with a functional deactivation of monocytes (diminished apc, ros formation, cytokine secretion). we called this phenomenon "immunoparalysis". ifn-gamma and gm-csf were able to restore the altered phenotype and function in vitro. however, addition of autologous plasma from septic patients with "immunoparalysis" to these cultures prevented the cytokine-induced restitution. the inhibitory activity could not be removed by dialysis. therefore, we started a study to prove the therapeutic efficacy of plasmapheresis. indeed, [7 of 33 patients recovered from "immunoparalysis" following repeated plasmapheres; 16 of them survived (46 %). 7 patients recovered temporarely and 9 patients did not respond (all 16 died). the survival rate in the control group of septic patients with persistent "immunoparalysis" was 4 of 38 (11%; p<0,01). in summary, plasmapheresis in association with immune monitoring may be an alternative strategy to improve survival rate in severe sepsis. taurolidine, a synthetic taurine-formaldehyde derivative has antiadherent, bactericidal and anti-lps properties functioning primarily through binding of the lipid a region of the lps molecule. the active derivative of taurolidine, taurine, modulates calcium channel activity, critical to the initiation of a number of immunostimulatory pathways. we hypothesised that taurolidine may have direct immunostimulatory activity. the aim of this study was to investigate the immune effects of taurolidine on peritoneal macrophage (pmo) function and then determine the role of taurine in this response. study 1: in vivo stimulation:cd-1 mice (n=35) were randomized to receive taurolidine (200mg/kg bw/i.p.) or saline cor~trol. peritoneai cells were harvested after 24 hours and were assessed for pm0 function [superoxide anion generation (o2-), nitric oxide (no), tumor necrosis factor (tnf), fc/cr3-mediated phagocytic function (phago) study 2: control pm0 were harvested and cultured in vitro with taurine (1.0mg/ml for 10 hrs), after which time they were assayed for 0 2-and tnf release. in vivo stimulation with taurolidine taurolidine has specific immunological effects on m0. release of the inflammatory mediators 0 2-and tnf, and fc/cr3-mediated phagocytosis were significantly increased, while release of the endothelial relaxing factor no was significantly reduced. in addition, the amino acid taurine, which is released as a byproduct of taurolidines breakdown has an immunostimulatory effect on pmo and may be the active moeity of the compound tanrolidine. in sepsis, a number of mediators which affect vasomotor tone and cardiovascular function are produced. inasmuch as sepsis causes decrease in systemic vascular resistance (svr), attention is usually focussed on vasodilators such as lactate, tumor necrosis factor, interleukin-i & 2, and nitric oxide. but injury and inflammation als0 cause production of several vasoconstrictors whose effect may not be evident in changed svr, but may significantly affect organ blood flow or function in the paracrine environment. endothelin (et) is a 21 amino acid peptide vasoconstrictor produced by ischemic or injured endothelial cells (ec's). et is also a potent constrictor for renal mesangial and coronary vessels, an endocrine regulator, and a negative cardiac inotrope. systemic et levels increase significantly in hypoperfusion and ischemia. while et is principally produced by ec's, we asked if human monocytes might also produce et and thereby regulate vasomotor tone in areas of inflammation. monocytes from healthy donors were separated on ficoll, resuspended in rpmi 1640 +5% fetal calf serum and stimulated with i0 ug/ml endotoxin (lps). et was measured by radioimmunoassay. lps-stimulated monocytes produced 87 ! 6 fm of et/106 cells (vs. unstimulated controls of <25). this calculates to 15-30% of the amount of et observed in patients with low cardiac output, sepsis or ischemia. we conclude that et is a cytokine produced by both ec's and monocytes with potent effects on numerous cells and organs in the critically ill. wuppertal 5, germany we and other authors showed that fatal outcome in septic disease is associated with a decreased capacity of peripheral blood monocytes for the in vitro production of proinflammatory cytokines, especially tnf-alpha. we found that this monocytic deactivation is completed by a persistent and marked decrease of hla-dr expression on monocytes (< 30 % hla-dr+ monocytes) and a diminished antigen presenting activity whereas the capacity to form the antiinflammatory il-1 receptor antagonist remains high. in order to evaluate the in vivo situation and to determine at which level tnfproduction/secretion is altered we assessed the tnf-alpha mrna expression in freshly isolated peripheral blood mononuclear cells (pbmnc) from septic patients. tnf-mrna was onty rarely detected by semiqaantitative polymerase chain reaction in pbmnc's from septic patients with monocyte deactivation. meanwhile, it was found in almost all pbmncs from septic patients without monocytic deactivation. we wondered, whether il-i0, which ,is known to depress monocytic proinflammatoly response and mhc class ii expression, could be one of the mediators in fatal sepsis. in fact, we found that il-10 message in pbmncs of septic patients peaked in the beginning phase of monocytic deactivation. in further investigations we found that tnf-administration can induce monocytic deactivation in a murine model/n vivo and provoke il-10 message in human pbmncs in vitro. these results support our hypothesis that an excessive delivery of proinflammatory cytokines in a first phase can induce an overwheiming inhibitory feedback, mediated by immuninhibitory mediators like il-l0, which leads to often fatal monocytic deactivation in a second phase. interferon-gamma which is known to counteract il-10 production and the effects of il-10 on monocytes restores the function and phenotype of monocytes from septic patients with monoq, te deactivation in vitro and could be a possible therapeutic agent in otherwise fatal sepsis. our laboratory previously reported that lps dependent macrophagederived tnf-a production can be enhanced by pretreatment with lps at substimulatory lps priming doses coincident with a suppression of lps dependent nitric oxide (no) production (zhang and morrison, j. exp. med 17:511, 1993) . in order to extend the characterization of these lps priming effects in mouse macrophages, we examined the capacity of substimulatory lps to modify lps dependent il-6 production. macrophages were obtained from peritoneal exudate of thioglycollate treated c3heb/fej mice and cultured in rpmi 1640 medium containing 10% fetal bovine serum. macrophages were pretreated with various subthreshold stimulatory concentrations of lps (olll:b4) for 6 hours, washed three times, and then stimulated with the effective stimulatory concentration of lps for 18 hours. the amount of il-6 in the supernatant was measured by il-6 dependent cell line (b9 and 7td1) proliferation assay. il-6 was produced by macrophages at lower threshold doses of lps than those required for tnf-o~ or no production. subthreshold doses of lps modulated il-6 production in a biphasic manner characterized by an initial suppression and then potentiation. higher doses resulted in secretion of il-6 during the initial incubation with lps and subsequent desensitization. il-6, like tnf-~ and no, is, therefore, also affected by lps pretreatment. moreover, tnf-a and il-6 shared the similar potentiational pathway, but differed by the fact that only il-6 was inhibited. (supported by r37 ai23447 and po1 a54474.) department of microbiology, molecular genetics and immunology and the cancer center, 1000 wahl east, university of kansas medical center, kansas city, ks 66160-7832. korolenko t.,urazgaliev k.,and arkhipov s. the role of macrophage (mph) stimulation in mechanism of protective effect of new immunomodulators yeast polysaccharides -heteropolysaccharide cryelan and homopolysaccharide mannan rhodexman (both produced by petersburg chem.-pharm. inst.) was studied. in vitro according to nst test incubation of murine peritoneal mphs with cryelan or rhodexman, 150 ~g/ml,30 min was followed by increase of potencial microbicidic activity of mphs. in vivo mph stimulation by immunomodulators studied included increase rate of carbon particles phagocytosis during single i.v. or i.p. mode of administration to mice 1-14 days after (peak at 2nd day for i.v. and 5th day for i.p. mode of administration of the same dose of 5 mg/100 g b.w.).the preliminary injection of cryelan (5 mg/100 g, 24 or 48 h before) to mice with acute cold stress (-10 ° c, 1 h) revealed protective effect restorating the value of depressed phagocytosis up to the normal level;the positive effect on ultrastructure of hepatocytes was noted also.there was no changes of plasma corticosterone level between group with acute cold stress and mice with cryelan + acute cold stress (several fold increase comparatively to the control mice).as was suggested, the mechanism of protection can include mph stimulation and secretion of some acute phase proteins responsible for positive effect of immunomodulators. new yeast polysaccharides cryelan and rhodexman can be used for macrophage stimulation,especially in pathological states. immunomodulators were shown to increase production and secretion of lysosomal enzymes (like zymosan). secreted enzymes,especially cysteine proteinasescathepsins b and l -involve in the process of inflammation;however, excessive release of these enzymes may lead to noncontrolled proteolysis followed by tissue degradation (assfalg-machleidt et al.,1990) .the effect of zymosan,bcg and new immunomodulator carboxymethylglucan (cmg), second fraction on secretion of lysosomal enzymes by murine peritoneal macrophages was studied. zymosan increased the secretion of n-acetyl-~-d-glucosaminidase and ~-galactosidase into the culture medium (3-4 fold); bcg possessed similar effect.cmg in the same concentrations (50 /~g/ml) increased release of these enzymes only saightly (1.6 times).it's known that zymosan-induced secretion reflects the enzyme release from formed lysosomes (warren,1989) .it was suggested that cmg activated macrophages via interaction with scavenger-receptors,followed by weak secretion of lysosomal enzymes and as a result decrease of tissue damage. in vivo zymosan induced stimulation of mononuclear system of phagocytes followed by increase of cysteine proteinases activity in liver at the 5th day. in the same time in blood n-acetyl-~-d-glucosaminidase and n-acetyl-~-d-galactosidase activity increased 2-5 fold. it was concluded that in drug design it's possible to select such immunomodulators,e.g. cmg,which can activate mononuclear system of phagocytes and do not damage tissue. endothelin-i (et-i) is produced by injured/ ischemic endothelium, mobilizes intracellular ca ++ and is a potent vasoconstrictor. it is also a ca ++ agonist for anterior pituitary or renal mesangial cells and monocytes. et-i causes monocytes to produce interleukin-l, 6, 8, prostaglandin e2, and substances which trigger neutrophil superoxide production. et-i levels increase in shock and et may play a role in activating leukocytes post shock causing reperfusion injury. but blood flow experiments suggest splanchnic circulation changes more profoundly in shock than peripheral circulation. we therefore asked if et-4 (or vic), the et which predominates in splanchnic vessels, had any effect on monocyte cytokine production. human monocytes from health~ blood donors were separated on ficoll. 0.5 x 10ucells/ ml in rpmi 1640 + 10% fcs were incubated i0 min., 6 & 24 hrs. with 10 -8 m et-i, 10 -8 m vic or i0 ug/ml of lps. supernatants were assayed by elisa. we have shown that low dose endotoxin pretreatment (lps 1 ) for 24 hrs markedly inhibits the macrophage (mo) release of tumor necrosis factor (tnf) and increases interleukin-1 (il-i) in response to a subsequent endotoxin stimulus (lps2). in this study we examined the kinetics of lps 1 inhibition of tnf and augmentation ofil-1. methods: murine peritoneal exudate mo from balbc mice were exposed in vitro to medium or 10 ng/ml of lps1 for intervals of 0 to 24 hours. culture medium was then replaced with 0, 10 or 1000 ng/ml of lps2 for 24 hrs. tnf and il-1 in mo supernatants were measured by specific bioassays. during sepsis endotoxin (lps) activates macrophages (mo) to release mediators such as tumor necrosis factor (tnf), interleukin-6 (il-6), interleukin-i (il-i) and prostaglandin e2 (pge2). we showed that preexposure to lps (lps 1) alters the response of murine m~i to subsequent lps stimulation (lps2). we hypothesized that in vitro cytokine release by lps2 in human monocytes (mo) is also be altered by preexposure to lpsi. methods: human peripheral blood mo were obtained from healthy volunteers (n=8), cultured in vitro 24 hrs, then pretreated 24 hr _+ lps 1-cultures were then stimulated with lps 2 and mediators in mo supernatant measured: tnf, il-i, and il-6 by specific bioassays, pge2 by immunoassay kit. serum cytokine levels (specific elisa kits) were compared to in vitro supernatant levels. data is expressed as % control_+sem, lps2= 1000 ng/mh the table shows that all mediators were increased, in the absence of lps 1. pretreatment with lps 1 resulted in complete inhibition of lps2-triggered tnf release. in contrast, lps 1 significantly increased mo secretion of il-1, il-6 and pge 2 (data not shown). serum cytokine levels were as follows: tnf 407_+21, il-i 241+208, and il-6 8.0-+2.5 ng/ml. these serum levels were low, showed an extremely wide variation, and did not correlate with in vitro lps2-triggered mediator production. conclusion: human monoeyte mediator production is differentially regulated by preexposure to lps 1. provocative in vitro testing of monocytes may ultimately be clinically useful to identify prior in vivo lps exposure or mo macrophages release numerous secretory products involved in host defense and inflammation. activated macrophages with cytokines produced have been implicated in tissue damage in sepsis and multiple organ dysfunction. aimed to elucidate the organ-association phenomena,this study is to compare peritoneal macrophage(pm),alveolar macrophage(am), and kupffer cells(kc) during sepsis in terms of cellular protein contents as symbol of activation by flow cytometry analysis. sepsis were produced by cecal ligatien and perforation (clp) in wistar rats weighing 210-290g.pm were obtained by peritoneal lavage,am by bronchial lavage and kc by incubating the collegenase digested liver with pronase-e. leukocytes have been implicated as a mediator of the microvascular dysfunction associated with reperfasion of ischemic tissues. a role for ieukocytes is largely based on observations that rendering animals anutropenic with anti-neutrophil serum or preventing leukocyte adhesion with monoclonal antibodies attenuates the increased fluid and protein leakage from the vaseulature that is normally observed in postischemic tissues. we have recently undertaken studies designed to determine the relationship between leukocyte-endothelial cell adhesion and albumin leakage ia rat mesenterlc venules exposed ~o ischemia-reperfusion (i/r). leukocyte adherence and emigration as well as albumin extravasafion were monitored in single postcapillary venules using iatravital fluorescence microscopy, lschemia was induced by complete occ!usion of the superior mesenteric artery and ~dl parameters were monitored at various intervals following reperfusion. the magnitude of the leukocyte adherence and emigration, and albumin leakage elicited by i/r was positively con-elated with the duration of ischemia. the albumin leakage response was also highly correlated with the number of adherent and emigrated leukocytes. monoclonal antibodies against the adhesion glycoproteins cd18, cdllb, icam-1 and l-selectin, but not p-or e-selecdn, reduced i/r-induced leukocyte adherence and emigration as well as albumin leakage. phauoidln, an f-aetin stabilizer, largely prevented the emigration (but not adherence) of leukocytes and greatly reduced, the raicrovascular protein leakage. plateletleukocyte aggregates were formed in postischemic vemdes; the number of aggregates was reduced by antibodies against p-selecdh, cdilb, cd18, and icam-1, but not e-selectin or lselectin. a significant fraction of the mast ceils surrounding the posteapillary venules degranulated in response to ischemia/repeffusion, but mast cell stabilizers did not afford protection against the albumin leakage elicited by i/r. these results indicate that reperfusloninduced albumin leakage is tightly coupled to the adherence and emigration of leukocytes in posteapillary venules. this adhesiomdependent injury response is primarily mediated by cdllb/cdi8 on activated neutrophils and icam-1 on venular endothellum, and appears to require l-selecda dependent leukocyte rolling. mast cell degranulation does not appear to conwibate to the vascular pathology associated with i/r. m.d. rod=iek, boston, ma, usa the polymorphonuclear neutrophil (pmn) has long been known to pa~tlcipats in the inflammatory rebpons~ as a phagocyte and killer of invading organisms, but little attention has been given to its potential as a participant in the in~une interaction of lymphocytes and macrophages. we and others have shown that the pmn may have i~m~/nomcdulatory effects both in vitro and in vlvo. more recently it has been proven that the pmn can make mrna for and secrete the proinflammatory oytokines illa, il-ib, tnfs, il-6 and il-8 as does the other major circulating phagocyte, the monocyte/macrophags. furthermore it has been shown to make the potentially autoregulatory oytokines gcsf and gmcsf. these functional capabilities suggest that the pmn is not an wend cell ~, but one which has a potential role in regulation cf ~he immune response and that this potential ~cle should no longer be ignored when considering the immune abnormalities existing in patients following majo~ injury or surgery. we have investigated the proinflaznmatory oytokine secretion patter~ by pmn in patients following major ~hermal or tra~matic injury and in volunteers fellowinq endotoxemia. ?ollowing major injury there is variable pmn secretion of these cytokines when stimulated in vlero. following endotoxemia in a group of human volunteers pmn showed a hypo=esponsivenesa to lps 4 hrs following endotoxin infusion followed at 24 hre by an overshoot. pretreatment with steroids modulated this overshoot phenomenon, suggesting that receptors for steroids are involved in the regulation of cytokin® secretlon by fmn. these results sugges~ that the pmn, the most numerous cell in the circulation and the first to respond to an ins~l~ may be a so~rce of the prolnflammatory cytokine cascade following injury that has been recognized as significant in the process which often leads to multiple o;gan failure, the immunosuppresslon which occurs following major thermal injury may predispose these individuals to infection and sepsis, which remain a significant cause of morbidity and mortality. included among the many immune aheratlons are the p2 integrln (cdlla, b,c/cd18) dependent activities of adhesion, chemotaxls, diapodesls, and phagocytosls. our investigations indicate that, following major thermal injuries, the expression of the [~2 integrlns, but not cd14, is significantly decreased on neutrophlls (pmns). it remains unclear if pmns from thermally injured patients respond normally to lps, the effects of treatment in vitro with lps and f-met-leu-phe (fmlp) on the expression of cdtlb was examlned on pmns from the peripheral blood of healthy volunteers and non-septic burn patients (>30~; total body surface area, >ls~ full thickness), the pmns were incubated with lps (]ng-10p.g/ml) or f'mlp (10 "1 1 to 10"6m) et 37oc for 30 mln, in 1~; human ab serum, the expression of the 132 ]ntegrins was detected using monoclonat antibodies and flow cytometry. lps and f'mlp resulted in a slight increase (3 fold) in the expression of cd11b on pmns from burned patients compared to an 8 and 14 fold increase, respectively, on pmns from healthy individuals. this inability of lps or fmlp to increase cd11 b expression was not due to the amount of lps bound to the two cell populations. because the same defect is seen after either lps or fmlp stimulation, it is speculated that the defect must be in the amount of preformed cd1 ] b or its transport to the plasma membrane. platelet-activating factor (paf) and neutrophils have been implicated in the patbophysiology of ischemia-repeffusion injury, in addition, paf stimulates neutrophi[ (pmn) oxidative metabolism in vitro. the present study examined the potential role of paf in repeffusion injury in an in viva rabbit model. eight anesthetized rabbi~s underwent retroperitoneal exposure of the infrarenal abdominal aorta after percutaneous insertion of a catheter through the jugular vein into the infrahepatic inferior vena cava. doppler flow probes were placed around the abdominal aorta and the right common femoral artery to assess flow through these vessels. an occlusive ligature was placed around the abdominal aorta (superior to the flow probe) at t = 0 and total occlusion of blood flow to the lower extremities was maintained for g0 mins., after which the ligature was released allowing for reperfusion of the ischemic lower limbs. effluent blood from the ischemic hind-limbs was collected through the ivc catheter at the times indicated below and assayed for paf by a direct radioimmunoassay. in addition, neutrophil h202 production was determined by a previously described 2'7'-dichlorofluorescein flowcytametric assay. 185 _+ 53 amean _+ s.e.m, pg/ml blood; brelative fluoresenee (% of baseline); caortic and femoral artery flow (% of baseline); *p < 0.01 vs. baseline; 1"p < 0.05 vs. baseline. a significant elevation of paf was observed in ischemic hind-limb effluent blood at 15 min. after release of the aortic ligature during the repeffusion phase, as compared to baseline levels. in addition, pmn h20 2 production was increased by 2.3-fold above baseline values by 1 hour after ligature release during the reperfusion phase. both of these elevations were transient and returned toward baseline by 2 hours post-isehemia. tatar occlusion of hind-limb flow was achieved as evidenced by the absence of aortic or femorat flow at 90 rain. post-ischemia, however after release 01 the ligature a significant reactive hyperemia was observed by 15 mln. into the rapeffusion phase. histolog[c examination of reper[used gastrocnemius muscle revealed moderate pmn infiltration into the interstitium. in conclusion, these data indicate that paf is released into the circulation during repeffusion, and is likely involved as a mediator in the observed pmn oxidative burst activity, thereby contributing to reperfusien injury. following thermal injury and infection granulocyte function ts abnormal. to elucidate the mechanism by which thermal injury and infection affect the granulocyte's ability to polymerize and depolymedze actin, we serially measured f-actin levels in granulocytes from 14 burned patients (mean age 42,1 +_ 17.7 years, mean burn size 39.8% _+ 22.2%) during the first s weeks post injury. six of the patients had infections during the course of the study, (septicemia, wound invasion and pneumonia). actin levels in granulocytes from eleven healthy volunteers (mean age 34 years) were measured repeatedly and served as controls. lysecl white blood cell preparations were brought to 370c and incubated with n-formyl-met-leu-phe (stim) or with dulbecco's phosphate unbuffered sellne (unstim). the cells were concomitantly stained and fixed with formaldehyde, lysoleclthln and fiuoresceln phafioidin. actin depolymedzation (depol) was measured by incubating stimulated cells at 0°c before the stain-fixative was added. baseline (base) f-actln levels were assessed by adding stsln-fixatlve to icecold unstimulated cells. fluorescence was estimated in a facscan and expressed as ilnesr mean channel fluorescence_+ sem (mcf). figure 1 displays granulecyle fectln levels in infected and uninfected patients as compared to controls. f-actln levels were consistently lower in control cells than in those from burned or burn-infected patients under all measured conditions. granulocytes from infected burned patients demonstrated a significant decrement in their ability to depofymerlze f.actin compared to both uninfected burned patients and controls, while there were no significant differences between infected and 1,~ uninfected patients in the baseline, unstlmuleted and stimulated conditions. those results indicate la5 that grsnulocytas from burned and bum-infected patients contain higher levels of polymerized actln than ~ ,2s control cells. in order to study tumor necrosis factor (tnf) receptor sensitivity in septic critically ill patients we investigated 20 blood samples of such people in reaction of leucocyte migration inhibition. migration of their polymorphonuclear leucocytes (pmns) was studied with stimulation with human recombinant tnf in concentration of 166.7 u/ml (recommended by manufacturer is the range of 10-200 o/ml) and without such. ten healthy blood donors formed control group. the results obtained showed diminished pmn reactivity to tnf in patients (migration inhibition was absent) oscaring with significantly increased migration ability of their pmns (107.2% of that in control group). at the same time normal pmns in control group did show migration changes upon tnf stimulation. considering all the above we come to a conclusion that externally added tnf fails to activate pmns in critically ill patients more than they are by their endogenous tnf. moreover, this tnf no longer serves a positive chemotactic factor for such pmns. these findings may suggest that in critically ill septic patients reactivity of pmns to tnf is deeply altered. tnf receptors of pmns are either exhausted as such by excessive stimulation with endogenous tnf or further transmission of their message is impossible due to "fatigue" of the cell's activation mechanisms. we express our gratitude to reanal factory of laboratory chemicals for generously providing us with a tnf com~rcial sample. ~-sanguis medical, ekaterineburg russia; s-urals med.lnst. activated neutrophils infiltrating the local site of inflammation following trauma release high amounts of destructive lysosomal enzymes into the extracellular space. cytokines were discussed to be involved in regulation of this early process. the task of this investigation was to evaluate the possible regulatory role of interleukin-6 (il-6) and its potential immunosupressive opponent, the transforming growth factor-&, in regulation of neutrophil degranulation. we analysed the concentration of the al-proteinase-inhibitor complex of the lysosomal elastase as marker for the degranulation of neutrophils as well as the levels of il-6 and tgf-61 in the plasma probes of patients undergoing multiple trauma and severe surgeries. the time courses of il-6 and elastase were found to be highly correlated, wheras the concentrations of the cytokine tgf-e~ were found to be not significantly altered in comparison to the control group. this close temporal correlationship was confirmed by investigation of fluids derived from sites of inflammation. interstingly, the inhibitory potential (~zcproteinase inhibitor, antithrombin iii) was dramatically reduced in the early inflammatory phase. to prove this in vivo findings, the effects of il-6 and tgf-i~ 1 on the degranulation of isolated human neutrophils of healthy donors was investigated in vitro. pathological high concentrations of rhll-6 up to 104u/ml (as detected in fluids derived from local inflammatory site) were found to be capable to induce a significant release of lysosomal elastase in a concentration-dependent manner, whereas the degranulation of neutrophils was uneffected by tgf-61. in conclusion, these data suggest a contribution of il-6 in regulation of neutrophil activation at sides of inflammation. the immunosuppressive cytokine tgf-i&~ seems to have no direct regulatory effect beside its described chemotactic function on neutrephils. postirradiation chan~es of adhesive properties arid supercoiled nucleoid dna structure of blood leukocytes were studied in macaca nemestrina andrats. the dynamics of membrane chan~es after nonlethal irradiation of rats demonstrated the temporary increase of the leukocyte adherence at 24 h followed by return of this parameter to normal levels at 48 h. after lethal irradiation of both animal species the increase in adhesive leukooytes fraction was detected as early as at 3 h. this hi~her index persisted until the end of experiments (5 days). the early (3-5h) temporary loosin~ of supercoiled dna structure was demonstrated in the leukocytes of nonlethally irradiated animals. this phenomenon seems to be connected with the lymphocyte fraction chan~es. this process was not dependent on altered adhesive properties of leukocyte membranes. the membrane chan~es of leukocytes preceded decondensation of supercoiled dna after lethal irradiation of animals, in this case loosin~ of supercoiled dna pro-~ressively increased at 24 h and at the later terms of postirradiation period. the systemic inflammatory response syndrome (sirs) involves many inanunological reactions of the host including acfivatinn of inflammatory mediator cascades and depression of cellular reactivity in t-lymphecytes (1). there are reports of nentrophil dysfunction in inflammatory disorders of the skin (2), are there dysfunctions concerning the unspecific host defense in sirs, as well? in this study, we examined the reactivity of neutrophil granolocytes from patients suffering from sirs. twenty-one patients (apache ii-score 21±5) with diagnosis of sirs entered the study. granulocytes were prepared as reported previously (2) . in parallel, granulocytes from 20 healthy individuals were tested. two granulocyte functians were studied in vitro: 1. migration of the ceils in a boyden chamber through a filter matrix following stimulation with 5 different receptor dependent stimuli (c5a, intefleukin-8, platelet-activating-factor, leukotrien b4, fmlp). 2. release of 13glucuronidase following stimulation with the aforementioned activators. the results demonstrate, that the release of 13-glucuronidase in patients suffering from sirs was comparable to the enzyme release of granulocytes prepared from healthy individuals. each stimulant induced release of p-glucuronidase in a characteristic dose dependent fashion. all granulocyte preparations from the healthy donors showed a positive chemotaxis response in the migration-assay. in contrast, only ten out of twenty-one patients had granulocytes migrating after stimulation. the two groups of patients displaying reactive or non-reactive granulocytes differed clinically: the nonreactive group consisted of patients with multiple organ failure (10/11) and nonsurvivors (8/11), whereas 8/10 patients in the reactive group survived. thus, the in vitro chemotaxis of granulocytes is impaired in a subgroup of patients with sirs. this defect of the non-specific host defense may contribute to poor prognosis and outcome of these patients. dermatol. 85: 194-198, 1985 klinik ffir an~isthesiologie und operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiel, germany. objectives of the study: major emphasis has been given to the analysis of interactions of antibiotics with microorganisms. effects of antibiotics on cells of primary host defense mechanisms, such as the neutrophils, are less well known. therefore, attention has been focused on clindamycin, a member of the lincoseamide family. materials and methods: the effect of clindamycin (i -i00 ~g/ml) on 8 granulocyte functions (healthy volunteers) such as random migration, chemotaxis (agarose method), ingestion (radiometric assay), superoxide (cytochrom c reduction) and hydrogen peroxide production (phenol red oxidation), lucigenin-and luminol-amplified chemiluminescence (luminometry) and degranulation (turbidometry with micrococcus lysodeicticus) were investigated in vitro. results: motility and degranulation were inhibited, ingestion of saccharomyces cerevisiae, zymosan-induced lucigenin-and luminol-amplified chemiluminescence, superoxide and hydrogen peroxide production were stimulated in a dose dependent fashion. conclusion: clindamycin has granulocyte function modulating properties. recognition of immunomodulating effects of antibiotics may have therapeutic significance, especially in patients with long-term antibiotic therapy or immune deficiencies. the intense muscle activity (ea) of rats resulted in increase of neutrophil influx in muscles during the recovery. we investigated neutrophil proteinases involvement in neutral proteinases balance of skeletal muscles by na. the rats were submited to swim with the load (8% of body mass) till exhaustion. immediately after na the neutrophil antiserum was injected i.p. to rats of experimental group. saline was injected to control animals° injections were repeated in 12 h of the recovery and cytosol proteolytic activity (ph 7.4; fitc-casein) was determined. isolated soleus muscles were incubated also in vitro and proteolytic activity of incubation media was measured. it was found that there was 2-3 -fold proteinases activity increase in cytosols of all investigated muscles (soleus, white and red portions of quadriceps) of control animals by 6 h of the recovery (the comparison was done with the sedentary rats). in 12 h cytosol proteolytic activity decreased and then increased again by 24 h of the fast. antiserum injections resulted in relible decrease of the proteolytic activities at every investigated time. when incubating m. soleus in vitro the activities of proteinases in incubation media turned out reliably less if soleus muscles were isolated from the animals to which antiserum was injected. the conclusion is that neutrophil proteinases can be involved in the balance of rat skeletal muscle neutral proteinases after ~a. a lot and new clinical problems complicating the outcome of polytrauma, burn and septic patients in surgical intensive care units, have arisen as the care improvement prolonged the patient's survival: a progressive degradation of organ and system functions often develops, usually making its first clinical appearance by ards, followed by the other organ failure (mof) and sepsis symptoms. the clinical picture is polymorphic, the end result of a complex systemic pathophysiological reaction trigg~ed off by trauma consequences (tissues disruption, hypo~xygenatiun and necrosis). nowadays there is not a preventi~ or specific therapy to lower the mortality rate (70-80%) and-'mdy-a~ early, aggressive surgical approach .-evacuating haematomas, stopping bleeding, toileting all septic, necrotic foci and restoring anatomic continuity-, seems to be of some help this complex clinical entity has not an univocal denomination yet. the proper labelling of an illness should come from the full understanding of its pathopysiology and suggest the proper treatment choice. clinical and experimental studies demonstrated that pathophysiologic mechanisms involved in the past-traumatic illness, share the same anatomo-pathological elemem: the interstitial edema, due to a generalised endothelial micro circulatory injury. this alteration, as constantly seen in polytrauma patients, develops in a few hours after trauma as a consequence of the deregulation of the homoeostatic and immune mechanisms. in fact the overproduced oxygen free radicals and r~ombinam cytokines (il1,tnf), together with the complement degradation fragments, the proteolytic enzymes and many other mediators are all strongly h~l ~ ,_he e,,j,yheha! ceils. our~osect, atim~,-bnsed on examination of autopsical specimens from 85 polytraanm patients, showed that such endothelial damage, supporting the interstitial edema, is widely and simultaneensly distributed, ensues shortly arer trauma and shows its effects in different organs at different times, only because each apparatus has different fimctienal reserves: the lung is the first organ to fail just because its ah, celocapillary membrane is one of the most delicate bodily structure, and its function is irroplace~le. we think it will be of a great help, in planning a preventive therapy, to chose a denomination focusing the physician's attention on the earl)" generalized endothelial injury and its effects, as in trauma patients it is present -even if latenflysince the first few hours. we would like to see the generalised endothelial microcircolatory injury properly highlighted when considering the best definition and the optimal nomenclature for the post-traumatic s3mdrome. the presence of interleukin (il)-8 in bronchoalveolar lavage fluid of critically ill patients correlates clinically with the development of the adult respiratory distress syndrome lards), and inhibition of il-8 in animal models can attenuate lung injury. collectively, evidence to date suggests that il-8 attracts and activates neutrophiis (pmn), which are then responsible for the capillary leak of ards. however, an alternative explanation is that il-8 is directly toxic to the endothelial cell (ec). in this study, we have hypothesized that il-8 can disrupt endothelial integrity independent of pmn. meth ods: human umbilical vein (huv) ec monolayers were cultured to confluency on collagen-coated micropore filters. to assess ec integrity, 1251.albumi n leak was quantitated by measuring the 1251 counts which crossed the monolayer, using a gamma counter. il-8 (lpg/ml) was incubated in the culture medium with 1251.albumi n for 21 hrs. the il-8 dose was not cytotoxic. to determine the involvement of protein synthesis in this process, selected monolayers were pretreated with cycloheximide (ch) prior to 11.-8 addition. statistical analysis was performed using anovmfisher plsd. we have previously shown that platelet activating factor (paf) enhances cdt8 expression and primes pmn's for subsequent 02generation. both are important steps in pmn mediated injury and are assumed to occur in concert. following major trauma non-specific pmn inflammation is activated, however, unbridled systemic pmn activity needs to be minimized. since circulating catecholamines are high early post-injury, we hypothesised that they downregu/ate cd 18 expression and pmn priming via the [3 adrenergic signal transduction pathway. methods: normal human pmns were primed with paf (10ng/ml for 5 min) or pre-treated with 10-5m of isoproterenol (i) or forskoklin (f) for 5 rain and then primed with paf. cd18 expression was measured by flow cytometry (fig.l) and 02-generation in response to 10-rm fmlp was determined as sod inhibitable reduction of cytochrome c ( fig.2 holler** and georg w. bornkamm* lymphocyte-endothelial interactions are crucial for various immune responses, including cytokine driven inflammatory processes. protein kinase c (pkc)-inhibitors on the other hand are discussed as potential cytokine antagonists. in the present study we investigated the influence of the pkc-inhibitor gf109203x on cytokine-and endotoxin induced expression of intercellular adhesion molecule 1 (icam-1) and on adhesion of lymphocytes to cytokine activated endothelial cells. we found that tumor necrosis factor alpha (tnfo0-and lipopolysaccharide (lps)-induced icam-1 expression on human endothelioma celts (eahy926) were unaffected by the pkc-inhibitor and thus appeared to be independent of pkc activation. in contrast, gf109203x significantly reduced icam-1 expression induced by interferon-y (ifn-?) and interleukin-1 (il-1). the functional relevance of these findings was evaluated in an adhesion assay using human umbilical vene endothelial cells (huvec) and peripheral blood mononuclear cells (pbmc). in fact, the ifn-? and il-1 induced adhesion of pbmc to cytokine treated huvec could be downregulated by the pkc-inhibitor, whereas tnfc~-and lps-mediated adhesion was not influenced. additionally, the il-1 driven icam-1 expression on eahy926 cells as well as the il-1 induced adhesion of pbmc to huvec was found to be tnf-dependent, since both effects could be inhibited by an anti-tnf monoclonal antibody (195f) . these in vitro data further support the idea of examining pkc-inhibitors, such as gf109203x, for their biological relevance in cytokine related dysregulations. seiffge, d., bissinger, t., laux, v., during inflammation there are some key processes, which occur in the microcirculation: the release of mediators from various cell types, the migration of inflammatory cells towards a chemotactic stimulus in the tissue, the expression of adhesion molecules on different cells, and the extravasation of plasma proteins. the aim of the present study was to elucidate the mediator induced interaction of leukocyte adhesion and plasma leakage in postcapillary venules. using an analogous video-image analysing system we have studied the effect of different mediators on leukocyte adhesion and macromolecular permeability in the mesentery of the rat. the increase in permeability was measured as changes in optical density. we found that topical administration of leneotriene b 4 (ltb4, 5x10 "6 tool/l) or intravenous injection of interleuldn-2 (il-2, 5-106 iu/kg b.w.) and lipopolysaccharide (lps, 15 mg/kg b.w.) resulted in a significant extravasation of fitc-labelled rat serum albumin (fitc-rsa) in venules but not in arterioles. we could correlate the changes in vascular permeability with a locally increased number of rolling and sticking leukocytes in venules. both effects were dose dependently inhibited by different drugs. pentoxlfylline inhibits lps-indueed fitc-rsa extravasation and leukocyte adhesion at a dose of 3 mg/kg b.w., superoxid-dismutase (sod, 10.000 iu/kg b.w.) was able to decrease the ltb 4 effect, and the immuumodulating drug leflunomide (hwa 486) exerted inhibitory effects on il-2-induced permeability at a dose of 3 mg/kg b.w.i.v. the obtained results demonstrate that lps, ltb 4 or il-2 induced extravasation of fitc-rsa is mediated by activated leukocytes and can be deminished following administration of different drugs. platelet-endothelial cell adhesion molecule-i (pecam-i), a member of the immunoglobulin superfamily, is constitutively expressed at high levels on the endothelial cell surface. in vitro data have suggested that pecam-i functions as a vascular adhesion molecule, specifically in neutrophil transmigration across the endothelium. this current work is the first demonstrating the in vivo role of pecam-1 in neutrophil migration. blocking antibodies to human pecam-1, in which the antibodies are crossreactive with rat pecam-1, were able to block the movement of neutrophils into the rat lungs after igg immune complex deposition. furthermore, when human foreskin was transplanted into mice with severe combined immunodeficiency and the site injected with tnf-alpha, anti-pecam-i blocked neutrophil emigration into the dermal interstitium. it has already been established that neutrophil recruitment is dependent upon selectin mediated rolling, followed by firm adherence that is icam-1/ integrin mediated. these data suggest, for the first time, that a third endothelial adhesion molecule (pecam-i) is involved in the coordinated recruitment of neutrophils in vivo. to test whether trauma causes generalized activation or priming of pmns, cdi8 adherence receptors were measured with iinmunomonitoring in whole blood after lps stimulation ex vivo. anesthetized (fentanyl) mongrel pigs (15-25 kg) were subjected to 40% arterial hemorrhage + soft tissue injury and after liar, resuscitated with all the shed blood + supplemental fluid. blood was collected at 24hr intervals from unanesthetized animals with indwelling catheters, pmns were counted, and lps was added (0,1,5,10 i.tg/ml) ex vivo. after 3hr incubation at 22-24°c, %cd18 (+) pmns were determined with fitc-ib4 and flow cytometry from mean channel fluorescence histograms. 49±8 # p<0.05 vs baseline * p<0.05 vs sham $p<0.05 vs no anesthesia these observations provide direct evidence for time-dependent changes in pmn priming following major injury because cd18 expression was depressed for at ]east 24hr after trauma relative to sham but by 72hr, was enhanced, relative to sham, and because fentanyl anesthesia at 72hr had a greater effect on cd18 expression in trauma vs sham. neutrophil (pmn) adhesion to vascular endothelial cells (•c) is a key element in the inflammatory response and tissue injury. inflammatory mediators such as lps (exogenous) and tnf (endogenous) can promote pmn-ec interaction which is believed to be responsible for capillary leakage and subsequent organ injury. however, the mechanism of this injury remains unclear.we hypothesised that the mechanism of tissue injury is due to ec necrosis with release of toxic products and that activated pmn are responsible. human pmn were obtained from healthy donors, separated by density gradient, and activated with lps (50ng/ml), tnf(10ng/ml), and lps/tnf(25ng/10ng/ml). cultures of the human ec tine(ecv-304) were used as surrogates of the microvasculature, were exposed to either lps, tnf, lps/tnf and pmn activated with lps, tnf, lps/tnf and incubated for 6, 12, 18, and 24hrs. ec necrosis was assessed by a 51cr release cytotoxicity assay. pmn activation was assessed by cd1 lb receptor expression and respiratory burst activity 6hr 0_+0 1.7-+ 1 0-+0 3.2_+ 1 0_+0 4.4_+ 1 0_+0 2.3_+0.9 12hr 0+0 3.24_3 0_+0 9.3_+3 0_+0 11_+3" 0+_0 12+--2.1" lghr 04-0 0.9_+2 0+_0 284-12" o:fo 25,12" 0~0 30+-9.5* 24hr 0_+0 4.14-4 0-+0 33+_3* 0_+0 30_+9* 0_+0 32_+10" data = ec % necrosis mean_+sd stats: student's t-test with significance (*) set at p<0.05 vs control. ( our previous studies have indicated that despite the increased cardiac output and maintenance of tissue perfusion, hepatoceliular dysfunction occurs during early sepsis. nonetheless, it remains unknown whether vascular endothelial cell function (i.e., the release of endothelium-derived relaxing factor/nitric oxide) is depressed under such conditions and, if so, whether endothelial cell dysfunction also occurs at the microcirculatory level. to determine this, rats were subjected to sepsis by cecal ligation and puncture (clp), following which these and corresponding shams received 3 ml/100g bw normal saline. at 5 hr after clp (hyperdynamic sepsis) or sham operation, the thoracic aorta was isolated, cut into rings, and placed in organ chambers. norepinephrine (ne, 2xi0 -7 m) was used to achieve near-maximal contraction. responses for an endothelium-dependeut vasodilator, acetylcholine (ach, via nitric oxide), were determined. in additional studies, the small gut was isolated at 5 hr post-clp. after pre-contraction of blood vessels in the isolated gut with 5xl04 m ne, vascular responses to ach (5x10 6 m) and an endotheliumindependent vasodiiator, nitroglycerine (ntg, 5xl0 -7 m), were determined. total vascular resistance (tvr, mmhg/mi/min/100g) was then calculated as pressure/ perfusinn rate. ach-induced relaxation (%, n=6/group) in the aortic rings were: ach lxl0 i~s, st-in ~ ~ significantly at 5 hr post-clp (i.e., increased *p(o 05 vs. sham; n-4 per group. tvr) in the absence of any changes in ntginduced relaxation (fig. a) . thus, the vascular endothelial cell dysfunction observed in the aorta in early sepsis also occurs at the microcirculatory level. introduction: the cytokine-mediated adherence of leulcooytes to vascular endothelium is considered as an early step in the cascade of pathologic reactions culminating in the "systemic inflammatory response syndrome" (sirs); the purpose of this study was to evaluate the influence of interleakin-1 on leukooyteendothelial cell-interactions and microoirculation in the liver after hemorrhagic shock by means of intravital microscopy. methods: in anesthetized female sprdrats co.w. 200-230g) shook was induced by fractionated withdrawl of arterial blood within 5 rain and maintained for 1 h (map at 40 mm hg, cardiac output 50 % of baseline). rats were adequately resuscitated with 60% of shed blood and twice the volume in ringer's solution additionally. following 5 h of reperfusinn (map >100 mm hg, co >120% of baseline) the microcirculation in liver lobules was examined by intravital fluorescence microscopy after labelling of leukocytes. continuous administration of il-lra (synergen, boulder, colorado, 5mg/kg/h) was started at different time points in a randomized and blinded manner. the animals in group p (n=6) received the il-lra as pretreatment beginning 5 min prior to shock induction. in the group t (n=6) the application of il-lm started at the beginning of the reperfusion period with a bolus injection of 5 mg/kg and was followed by continuons administration of 5 mg/kg/h. the control group c (n=4) received equal volumes in nac10,9%, the sham-operated group s (n=4) was not exposed to shock. results: macrohemodynamics were comparable in all shook groups. the increased percentage of permanendy adherent leukocytes after hemorrhagic shook (s: 9,1% +1,1%; c: 42,1% _+5,4%) was significantly reduced by pretreatment or treatment with il-lra (p: 16,9% -+1,9%; p<0.001, t: 28,8% -+3,6%, p<0.01, anova). temporary adhesion of leukocytes was unaffected by application of il-lra. liver microcirculation measured by volumetric blood flow in liver sinusoids and sinusoidal diameters was impaired after hemorrhagic shock in all groups and was not affected (c: 31993iam3/s +40971um3/s, p: 32768llm3/s +4445}am3/s, t: 326211ams/s -+2998 lam3/s, s: 390201am3/s -+39041am3/s). di.seu~sinn: the results demonstrate that permanent adherence of leukocytes to endothelium is in part regulated by il-1. pathological adherence could be reduced by application of illra, even given at die time of resuscitation. the effect of ll-lm on permanent adhesion is a specific event and might be caused by reduced expression of specific receptors on sinusoidal endothelial cens and leukocytes. objectives of the study. the adhesion of activated neutrophils (pmn) to endothelial ceils (ec) and the concomitant production of reactive oxygen metabolites (rom) initiates organ damage after trauma, sepsis, shock and organ reperfusion. aien of this study was to investigate the effect on adhesion and rom production of the highly water-soluble, membrane-permeable and physiological ascorbic acid (asc). materials and methods. adhesion of pmn to nylon fiber (cell count) and simultaneous rom production (chemiluminescence-cl-response) were measured up to 6 retool/1 asc as well as adhesion, rom production and ec damage (lllln-release from labeled ec) of endotoxin-activated pmn to cultered ec moanlayers. in an in vivo animal model (sheep with lung lymph fistulas) the effect of asc (1 g/kg bw bolus, followed by 0.2 g/ kg-h infusion) on the endotoxin-induced (0.5 ixg/kg bw) neutropenia (cell count), lung capillary permeability damage (lung lymph protein clearance) and rom production of neutrophils (zymosan-induced cl response) was measured. results. asc scavenged rom dose-dependently during adhesion of pmn to nylon fiber (p<0.0005 at 6 mmol/l asc), adhesion itself was unchanged. during the activated pmn/ec interaction asc scavenged rom (p<0.025 at 6 mmol/l asc) and reduced the adhesion dose-dependently (p<0.0005 at 6 mmol/l asc); ec damage was also reduced (p<0.0005 at 6 retool/1 asc). in the in rive model asc increased the endotoxin-induced blood pmn decrease (p<0.05), decreased the protein clearance (p<0.05) as well as the zymosan-induced rom production (p<0.03), indicating the asc-mediated reduction of adhesion, rom production and lung tissue damage processes. conclusions. by in vitro and in rive experiments ascorbic acid reduced the adhesion-and rom production-initiated tissue damage. therefore, i.v. administration of ascorbic acid is recommended for oxidative stress-associated states after trauma, sepsis, shock and organ reperfusion. for neut rophi l-accumulat ion and activation. we investigated the influence of or to the activation and the expression of lecam-i and cdiib,cdi8 on neutrophils and lymphocytes. methods: from blood samples (n=5) all white blood cells (wbc) and neutrophils (nc) were isolated and cultured. or were produced via the xanthine oxidase/hypoxanthine system. after 0,5, 15,30,60 and 120 minutes a giemsa-staining to determine the granulation of neutrophils (n: normal, r : reduced ) and a facs-analysis with monoclonal antibodies detecting cdiib,cdi8 and lecam-i was performed. results: under the influence of or a degranulation of neutrophils starting at 15min was observed in wbc-cultures (n/r: 0min 91/9, 15min 76/24, 30min 68/32, 60min 52/48, 120min 49/51). these data were confirmed in the dot-plots of facs-analysis. only in wbc-cultures or induced a significant increase of lecam-i expression on neutrophils up to 30min followed by a decrease to normal values at 60min. lecam-i on lymphocytes disappeared totally during the observed period. cdllb,cdl8-expression was not altered. conclusion:increased lecam-i expression on neutrophils due to or could enhance the 'rolling' of neutrophils along the endothelium which is a prerequisite for neutrophil sticking and migration. further or are able to activate neutrophils without endothelium. these changes seem to be mediated by other wbc. introduction. multiple organ failure (mof) has been hypothesized to be the result of an excessive uncontrolled autedestructive inflammatory response. since the complement system is an important mediator and initiator of the inflammatory response, interruption of this cascade could theoretically lead to an attenuation of mof. in order to test this hypothesis we evaluated the response of c5-delicient mice in a model of zymesan indt~ed mof. materials and methods. 25 c5-deficient b2d10/oid and 25 c5-sufficient b2d10/new mice were used in this study. on day 0 all mice received an intraperitoneal injection with zymosan suspended in paraffin in a dose of 1 mg/g body weight. between day 0 and 12, biological parameters (temperature, body weight and clinical condition) were measured daily and mortality was monitored. clinical condition was assessed by blindly grading the degree of lethargy, conjunctivitis, diarrhea, and ruffled fur of each mouse on a two point scale (maximum score=4). on day 12 all surviving mice were sacrificed and relative organ weights of lungs, liver, spleen and kidneys (relative organ weight= (organ weight/body weight)x100) wore calculated. earlier experiments with our model have shown a good correlation between histological organ damage and relative organ weights. statistical analysis of biological parameter was performed using the koziol curve analysis. analysis was divided in an acute phase (day 0-4) and a late phase (day 8-12). relative organ weights were analyzed using wilcoxon's test and mortality rate using fischor's exact test. results. all zymosan injected mice showed a typical triphesic illness. deterioration of the clinical condition as indicated by the symptom score and the decrease in temperature and body weight in the acute phase were all significantly lass severe in c5 deficient mice (all p<0.005). in the late phase no differences could be noticed in the courses of biological parameters. overall mortality was 2/25 (8%) in c5 deficient mice and 8/25 (32%) jn c5 sufficient mice (p=0.049), a difference mainly due to a difference in the acute phase. organ damage assessed as the relative organ weights did not show any statistical differences for any organ between both strains. conclusion. complement factor c5 appears to play an important role in the acute hyperdynamic septic response in this model but deficiency of c5 could not prevent organ damage in the late mof phase. this suggests that other factors could be more important in the development of the inflammatory response leading to mof. proinflammatory cytokines are thought to play a critical role in the pathophysiology of multiple organ failure (mof). in mice, zymosan-lnduced generalized inflammation (ztgi) leads to mof. therefore we performed a sequential study into plasma levels of, and macrophage production capacity for, four cytokines during the development of mof in the zigi model. male young-adult c57bl/6 mice received zymosan (1 mg/g body weight) intraperitoneally. groups of 14 animals were killed after 3, 6, 8, 18 and 24h and subsequently at each day until day 12. plasma was collected and peritoneal macrophages were isolated and cultured overnight with or without lipopolysaccharide (lps). interleukin 1-ct, and -6 (il-lc~,~,), and tumour necrosis factor-o~ (tnf-c 0 were measured in plasma and culture fluid by means of a ria (detection limit 0.1 ng/ml). interleukin-6 (il~) levels were assayed using the b9 hybddoma cell proliferation assay. zymosan induces a three-phase disease in mice. after an acute phase the animals recover. around day 7, they start to develop clinical signs which resemble mof. plasma tnf-~ peaked within 3h after zymosan injection and disappeared within 8h. from day 8 onwards, tnf levels started to rise again. plasma il-6 behaved almost similarly in the acute phase, but in the mof phase plasma il-6 remained low. no circulating il-16 could be detected at any time point. macrophage lps-stimulated production of il-lcq il-11~ and tnf--c~ was suppressed immediately after zymosan injection. production of il-16 and tnf-~ was normalized within 6h, while production of il-lc~ remained lower than that in macrophages from untreated control mice. only at day 6 did production of il-i~ reach control values. il-16 production was higher than control values from day 3 onwards. il6 production was similar to that of ili-il the production of tnf-ct was strongly elevated between days 1 and 6 and again during days 10 to 12. the development of mof-like symptoms during zlgi in mice is accompanied by increased plasma levels of tnf-ct without enhanced il-16 or il-6. also, the ability of macrophages to produce excessive amounts of il-16 and tnf--~, as well as the suppressed capacity to produce il-lcq could be important mechanisms in the pathophysiology of mof. when conjugated to an asialoglycoprotein, dna and oligonucleotides are specifically taken up by the hepatocytes via the asialoglyccprotein receptor which is unique to the liver. human asialoglycoprotein (~1-acid, asgp) was derivatized with low molecular weight poly(l)lysine(pll) and complexed with 5 antisense dna's (as) complementary to the 5' region of the il-6 gpl30 receptor. the antisense were 5'-agtttagggatgagg-3' (asl), 5'-atcttcatcttctgaat-3' (as2), 5'-aagtgaatgattaaaacact-3' (as3), 5'-aaacctttataggcg-3' (as4), and 5'-cgttctacaactgcaacgt-3' (as5). using hepg2, the biological effects of these antisense complexes on the high affinity il-6 receptor were evaluated by scatchard analysis, cellular proliferation, and acute phase protein expression by radioimmunoprecipitation and two dimensional gel electrophoresis. scatchard analysis demonstrated that high affinity receptor expression was inhibited by incubation of cells with asgp-pll-asi for 24 h. underivatized asl was less effective and the complex, asgp-pll-as2, had minimal effects on high affinity binding. when the cells were treated with the conjugates and stimulated with il-6 (i00 units) asgp-pll-asi alone showed a dose dependent (0.i-0.4 ~m) inhibition of 3ss fibrinogen synthesis. two dimensional gel electrophoresis showed that expression of other acute phase proteins was also blocked. these results indicate that the targeted delivery of antisense molecules via conjugates recognized by the asialoglycoprotein receptor can block the cytokine stimulated acute phase protein response in hepatocytes, this approach may be relevant to the therapeutic management of patients with severe injury and sepsis. it has been established that immune cells are able to express neuropeptide genes and to release products that were considered to be of neuroendocrine origin. we have shown that proenkephalin (penk), a neuropeptide encoding gene, is expressed in lymphoid cells in culture. to study the physiological significance of these observations we have used the model of experimental endotoxemia. in this model, a disease state is induced by bacterial lipopolysaccharide (lps), that activates the immune system, the adrenocortical axis and the nervous system. we found that the expression of penkmrna is markedly enhanced in vivo immediately after lps injection both in the adrenal glands and in the lymph nodes. in situ hybridization analysis combined with immunohisto-chemistry indicated that the induced penk expression is confined to macrephages within the lymph nodes and chromaffin cells in the adrenal medulla. furthermore, this expression in lymph nodes is modulated by ligands of the adrenergic system. our results strongly support the notion that immune derived opioids participate in the bidirectional communication between the nervous and immune systems. of neurology hadassah university hospital, jerusalem 91120, israel. objectives of the study: multiple-organ-failure is recognized as the most severe, and often lethal, complication after multiple trauma. however there is no adeqate animal model available. our goal was to develop an animal model, in which reproducable irreversible failure of parenchymal organs is achieved in the late phase after insults in the early phase (trauma). materials and methods: l0 female merino-sheep were included (mean weight: 30kg). day 0: hemorrhagic shock (mean arterial pressure (map) 50mmhg for 2 hrs.), closed femoral nailing (ao-technique), day 1-5: bolusinjection of endotoxin (et) (0,75~tg/kgbw) und zymosan-activated plasma (zap) (20ml) every 12hrs., day 6-10: observation. bronchoalveolar lavage (bal): day 1, 6, 10. the course of representative parameters of organ function was documented: cocardiac output (i/min), svr -systemic vascular resistence (dyn ~ s cm-5), pap -putm.art.pressure (mmhg), pap2 -arterial oxygen pressure (mmhg), bill -bilirubin (;xmov1), crci -creatinin clearence (ml/min) statistics: data as means+sem, *significant from baseline (wileoxon test; p<0005) results: baseline day 4 day 6 day 8 day 10 heart: co 4,96_+0,32 5,33_+0,36 5,4_+0,2 7,30_+0,77* 7,98_+0,66* svr1520_+134 1395+141 1403_+89 1119+_122" 1089+-91" lung: pap 17,0_-20,7 19,6_+1,6" 22,0+-1,2"25,8+2,0" 28,8+-1,7' pap2 103,1+1,5101,5+-5,7 97,3_+4,7 91,9+-5,6 89,8+_3,9* liver: bill 2, 94_+0, 344, 82_+1, 34' 5, 13_+0, 68'6, 13_+0, 7" 7, 19_+0, 91" kidney:crcl 86, 5+_30, 9 109, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] 4 74, 7_+10, 868, 8+14, 1 53, 1+17, 6 histologic specimens showed all signs of fulminant mof. combination of hemorrhagic shock, femoral nailing, et und zap (insults in the early phase) lead to an irreversible organ failure in the late phase. prostaglandin e (pge) levels are elevated by trauma, shock or sepsis and can profoundly affect the immune response. pge2 is produced by many cell types including fibroblasts, macrophages, monoeytes, follicular dendritic cells, and epithelial cells and is induced by il-i, bacterial lps, components of the complement cascade, tnf, il-6 and crosslinking of surface fc receptors for igg, iga and ige. our research has shown that pge inhibits b cell activation (specifically enlargement, class ii ~c and fc~ rii expression), proliferation, igm and igg3 responses, t cell proliferation, and il-2 synthesis in the mouse model. in contrast, pge greatly promotes class switching to ige,the isotype responsible for type i allergic hypersensitivity. thus, our model mirrors th~ general immunosuppression and elevated ige titers of the trauma or sepsis patient. pge increases the number of cells secreting ige and iggl, acts on surface igm positive b cells, synergizes with il-4 and lp$ to induce preswitch germline transcripts, and induces more rapid expression of mature vdj~ mp~a than in eontro~ pge intracellular signalling occurs through cyclic adenosine monophosphate (camp) levels and can be mimicked by camp-inducing agents and blocl~ed by an inhibitor of campdependent protein kinase a. pge action requires de novo protein synthesis and candidate pge-inducible regulatory proteins have been identified by 2d gel eleetrophoresis. thus, pge inhibits a number of immune mechanisms while promoting ige production. a deeper understanding of pge immune regulation may lead to more effective treatment of immune perturbations as sequelae of trauma, shock or sepsis. during infrarenai aortic surgery mesetueric traction (re.t.) results in prostacyclin (pgi:) release and consecutively in hemodynamic disturbances (decreased systemic vascular resisteace, mean arterial pressure; increased cardiac output, heartrate). these symptomes are bypassed by cyclooxygenase inhibition. hemodynamic symptoms vanish after 30-45 rain even without cyclooxygenase inhibition although pgi2 levels remain elevated. to study the endocrine vasopressor system in a prospective double blinded protocol, we investigated 26 patients undergoing major abdominal surgery as compared to 26 ibuprofen (400 rag, i.v.) pretreated (ibu) patients. the surgeon applied m.t. in a uniform fashion. we chose a general anesthesia combined with a supplemental thoracic epidural anesthesia. at the points in time 5, 15, 30, 45, 90, 180, 360 , 1440 rain after and before (to) mesentzrie traction we determined the plasma concentrations (pc) of 6-keto-pgf~o~pr~, epinephrine, norepinephrine, dopamine, renin, aldosterone, adh and cortisol. pc of 6-k-pgf~,tp~, peaked 5 minutes after m.t. (2274_+284, ibu: 102_+12, to: 60+i ng/l) and declined monotonously over 6 h (203 +_42, ibu: 84_+ 12 ng/1). catecholamine pc "s did not exceed the reference range during the observation period. reninpc peaked after 45 rain (66_+23, ibu: 18+3, to: 16-+2/~u/ml); aldosteronc also presented a maximum after 45 rain (110 + 12, ibu: 66-+ 15, to: 56 +-7 pg/ml), whereas cortisol demonstrated irrespectively of circadian rhythms a maximum 6 h after m.t. (26+_1, ibu: 24-+3, to: 10+_1 ~g/1). adh pc peaked 5 min after m.t. (71+7, ibu: 20-+6, to: 5+_1 pg/ml) and showed analogously to 6-k-pgft~j~ pc a monotone decline over the observation period. our data demonstrate a counteractive reaction to pgiz mediated vasodilation via adh secretion. the second regulative is the renin-angiotensin-aldosterone system (raas), which is activated 45 min after m.t., the aldosterone pc does not paratlel the cortisol pc, which peaked post operafionem in both groups, probably due to the end of anaesthesia. a regulative release of catecholamines could not be documented. the activation of adh and raas after mt is not a hormonal response primaryly related to surgical trauma and/or stress but a counterregulation to systemic vasoditafion induced by prostacyclin. although adh and raas support systemic circulation, angiotensin and vasopressin may compromise local organ blood flow (e.g. splancimic vascular bed). insfitut f. klin. chemic, anaesthesiologie ~, chirurgie l*, univ. ulm, 89070 elm, expression of c-fos protein in rat brain following occlusion of superior mesenterie artery. takanobu there is general agreement that neurologic abnormalities are seen in sepsis. the aim of this study is to examine what effect does the brain receive in case of sma occlusion by immunohistochemistry using antibody to c-fos, an immediate early gene, which is recently recognized as a genetic marker of activated neurons. moreover, we investigated the correlation between c-fos induction in the brain and plasma endotoxiu level. 20 rats of them received sma clipping and others wee used as control. control and treated rats at 2, 4, 6, 8 hours were perfused and fixed. the brain were sectioned at 20 pm and stained by abc method using c-fos antibody. plasma endotoxin level of 5 rats were measured at 0, 2, 4, 6, 8 hours after the treatment by chromogenic limulus method. immunohistochemical study showed scarcely no immunoreactivity in control rat brain. in treated rat brain, the significant expression of c-los was detected in specific nuclei including the habenula, some hypothalamie nuclei, amygdala, locus ceruleus and nucleus tractus solitarii. such immunoreactivities were increased in time curse, which well corresponded plasma endotoxin levels. the mean plasma endotoxin level of 0, 2,4, 6, 8 hours after the treatment were 6.48 ± 5.57, 15.0 _-4-4.37, 10.0 _+ 4.18, 14.4 ± 3.52 and 58.4 ± 28 .6 pg/ml, respectively. the results indicate that limbic and hypothalamic-brainstem systems are involved in sma occlusion, and suggest that such neuronal actival.jon may precede the elevation of plasma endotoxin icy.el. systemic vascular resistance and increased cardiac output accompanied presumingly by a increased pulmonary shunt (qs/qt). this response is induced by prostacyclin (pgi2). we examined oxygen transport after traction on the mesentery root and the transpulmonary prostacyclin levels in a prospective placebo controlled study with intravenous ibuprofen. methods: with approval of the human [nvestigadon review board we studied 52 patients in a prospective, randomized double-blinded protocol who were scheduled for major abdominal surgery. ibuprofen (400 mg i.v.) or a placebo equivalent was administered 15 minutes before skin incision. pulmonary artery thermodilution and radial artery catheters were placed after induction of anesthesia. mt was applied in a uniform fashion. baseline values preceded the incision of the peritoneum (to). fulther assessments followed 5, 15, 30, 45, . tile plasma concentrations (pc) of 6-keto-pgft, (stable metabolite of pgi2) were determined in arterial and mixed venous blood by radioimmunoassay. at all points in time we measured arterial and mixed venous blood gases. qs/qt was calculated by standard formula. data are given as median (p < 0.05 placebo vs. [ibuprofen] [117] mmhg (*p< 0.0i). these changes were accompanied by a marked increase of 6-keto-pgf~ pc up to 180 rain after mt in arterial and mixed venous blood of untreated patients with a peak of 2450*[60] ng/l tl (*p< 0.00ol). there was no difference between arterial and mixed venous pc. ibuprofen pretreated patients (n=zr) demonstrated stabile qs/qt and pao2 while 6-keto-pgf~ pc remained within the normal range. discussion: our data clearly indicate that mesenteric traction response includes a critical rise in qs/qt followed by significant decrease of paov stable oxygen transport determinants following cyclooxygenase inhibition signify an action mediated by prostacyclin. an indicative transpulmonary gradient for 6-keto-pgft~ was not detectable. a splanchnic vascular source for pgi2 release seems to be likely, but could not be proved by our current data. department of anesthesiology, cliu. chemistry * and surgery*; university clinics uim, prittwitzstral]e 43, 89075 ulm, germany it is unclear whether injuries like bums, in general, directly result in alterations of cell-mediated immunity that, in turn, promote endotoxic and bacterial translocation or, alternatively, whether these conditions allow increased bacterial invasion that, in turn, inhibits cmi. aim: to determine whether infectious challenge, as clp alone or combined with ti causes further immune abnormalities in the days following clp. study plan: on day 1, two groups of n=36 8 week old aj mice were subjected to either a 20% scold burn (ti), or were untreated (c) n=18. on day 10, 18 mice (ti+clp) and 24 mice (clp) were subjected to clp. the two other groups (ti and c) were untreated. at days 11, 12 and 13 after thermal injury splenocytes (sp) were harvested and cultured with cona for an assay of il-2 and adherent splenocytes (as) were cultured with lps for il-1, tnf, il-6 and pge2. results: either ti + clp or clp alone result in significantly decreased secretion of all cytokines tested. in the ti group almost every cytokine production determined was elevated in comparison to ti + clp and prosmcyclin (pgi2) has been implicated in the pathophysiology of septic shock. however, pgi~'s role in the inflammatory response to sepsis is not well-defined. the purpose of this study was to identify which acute septic events are mediated by pgi 2 during graded bacteremia. methods: eleven ~nesrhetized, hemodynamically monitored adult swine were infused iv with aeromonas h. (109/ml) at rates increased incrementally from 0.2 to 4.0 mi/kg/hr over 4 hours. animals were studied in two groups: septic control (sc), graded bacteremia only (n=6); pga (n=5), graded bacteremta plus anti-pgiz antibody, 50 ml/hr iv, beginning at 2 hours. mean systemic (map) and pulmonary arterial (pap) pressures and arterial po2, mmhg, cardiac index (ci), l/min/m2, oxygen delivery index (do2i) and consumption index (vozi), ml/min/m2, and oxygen extraction (er), %, )latelet aggregometry (plt), %max., plasma pg6-keto f1 alpha ; in the first instance~ peak values of lt ~4 after i~ hrs post infarction were 6 times higher than in the controls and excess leucocyte infiltration was noted at the infarction zone. in second instance two levels of lt b4 led to weak infiltration of the infarction zone by leucocytes. a. mo~e~o, in~.~p~siolo~,d~t.e~.cardiolo~,bogotsolets4, ~ev252024, ukrmne systemic lesion$of erythron in traumatic disease and possibilities of their regulation by opioid peptides. redkin y. v., fominih s. g. using clinical (121 patients) and experimental material(128 rats and 74 dogs) we revealed general regularities of erythron lesions after hard mechanical trauma of various genesis as well as some mechanisms of development of posttraumatic anemia and possibilities of its correction with preparations of opioid peptides. the condition of central and peripheral compartments of erythron was studied with unified morphologic, immunogematological, biochemical and radiological methods. it was revealed that irrespective of the experimental animal species (dogs, rats) or in clinical experiments (patients) and irrespective of the injuring factor type (skeletal trauma, craniocerebral trauma, loss of blood) in erythron can be observed one-directed unspecific reaction realized by the considerable lowering of hemoglobin concentration, erythrocytes number and hematocrit. in the initial period (1-7 days) in the system of erythron prevail processes of distraction and elimination of er~zthrocytes relatively to the general production of stimulated erythropoiesis. the primary alterating factor is the prolonged intensification of peroxydation of membrane iipids of erythrocytes with simultaneous lowering of reserves of reduced glutathione. the distraction of erythrocytes is supported by the developing phenomena of autoallergization of organism that becomes apparent by the appearance of sensitized t cells and antierythrocyte antibodies. the intensified production of erythropoietin rules to the realization of he program of fetal and terminal (reserved) erythropoiesis. failure of erythropoiesis function is supported by disturbances of the processes of the injuring of cell metabolic apparatus. using of dalargin (15 microgram per kilogram of body mass intrap'eritoneally within 5 days after the trauma) showed the precise pharmacotherapeutic effect revealed by the diminishing of anemia of experimental rats, more . fiberbronohoscopic procedures are known to produce "peep-like" effects and to increase pulmonary artery (pa) resistance [2] . peep can affect rv function by reducing preload and ejection fraction (ef) [3] . since changes of rv function during bronchoscopy in septic patients are not reported, we measured rv parameters before, during and after fiberoptic bronchoalveolar lavage (bal). method: this 34-year-old patient (apache-ii:24) developed a hyperdynanlic septic state due to staphylococcus aureus (blood culture). we inserted a "fast response" thermistor pa-catheter (baxter-edwards) to evaluate rv performance [4] . the therapeutic procedure included volume replacement, vasopressors (dopamine 5, dobutamine 3 gg/kg/min. iv) and analgosedatior/. before bronchoscopy (olympus bf-30, od=6 mm) the patient received pancmonium for muscle relaxation. ventilation was not changed during the procedure (endotracheal tube: id=8 ram, bennett 7200a, pressure controlled mode, pm~x=25 mbar, peep=8 mbar, i:e=i:i, fio2=1.0). we measured rv enddiastolic volume (edv), stroke volume (sv), ef, heart rate (hr), cardiac index (ci) and mean pa pressure (mpap gerlach h, gerlach m, clauss m, falke kj renal hypoxia and/or ischemia initiates the development of a deteriorated medullary perfusion based on fibrin deposition in the peritubular capillaries, vasoconstriction, and perivascular edema, which is followed by a swelling of the tubular epithelial ceils, intraluminal tubular obstruction, and a backleak of fluid through the injured tubules into the renal interstitium, finally leading to an acute tubular necrosis (atn) [1 ], clinically diagnosed as acute renal failure (arf). one important pathway for induction of enhanced vascular procoagulant activity and permeability is based on the synthesis and expression of macrophage-derived cytokines, which bind to specific endothelial cell surface receptors. we recently described the identification and purification .of a new 55,000 dalton polypeptide, which is synthesized and expressed by murine macrophages after stimulation with lipopolysaccharide, and exerts procoagulant activity on cultured endothelial cells [2] . in the presented study, we demonstrate that the new polypeptid is also synthesized by macrophages under hypoxic conditions. the protein binds to specific receptors, which are expressed by endothelial cells dependent on the environmental oxygen tension. animal studies were performed after approval by the local committee for animal safety; the animals were anesthetized, treated and supervised in accordance with the guidelines of this committee. in contrast to other authors, who performed long-term hypoxia experiments in awake animals, we preferred to implement the studies under anesthesia for ethical reasons, although regulatory functions for ventilation might be influenced. animal studies demonstrated that the intravenous injection of the polypeptide initiates fibrin formation in the peritubular vessels. keeping the animals under hypoxic conditions induces similar effects, which are reduced by a rabbit-antiserum against the new protein. in conclusion, the new polypeptide obviously contributes to the pathogenesis of acute renal failure by tubular necrosis during and after hypoxic events. the use of verapamil as cardioprotective agents for management of patients with acute ischemic/reperfused heart is based on the assumption that the increased intracellular ca+ level is a key factor in causing cell death. our in vitro study was designed to focus on effects of verapamil on the metabolic potential of cardiac slices after reversible ischemia in rats. the material consisted of two main groups : group a (non ischemia/reperfusion group) and group b (ischemia/reperfusion group), each is subdivided into two subgroups (a and b). each subgroup included 10 rat hearts. group aa is the control group, group ab is verapami] added group. group ba is ischemia group without verapamil. group bb is verapamil added group. ischemic cardiac slices were obtained from rats subjected to 15 min. haemorrhage to induce reversible global ischemia. both nonischemic and ischemic cardiac slices were placed in well oxygenated krebs ringer phosphate buffer containing 150 mg% glucose & 5 gm% bovine albumin and incubated in dubnoff shaking water bath for 60min at 37°c the results revealed that there was an enhancement in release of free fatty acids (ffa) (240%) and lactate (163%) and in glucose uptake (160%) in group ba as compared with group aa. these metabolic alternations produced by ischemic cardiac slices were reversed by verapamil addition (200 ml%) but in group ab verpamil did not alter the release of ffa & lactate from non-ischemic cardiac slices, whereas it inhibited glucose uptake from these slices by 67%. the improvement of the metabolic intervention of ischemic myocardium indicates that verapamil may be of importance in reducing the extent and severity of acute myocardial ischemic injury in acute haemorrhage. severe endothelial dysfunction occurs following injury to carotid arteries which is characterized by a decreased ability of these arteries to dilate when challenged with ach or a23187, but not with a direct vasodilator (nano2). this failure to relax to ach and a23187 reflects an inability of endothelium to generate edrf, but relaxation recovers gradually to control values by 2 weeks. exogenous no donors (e.g., c87-3754 or spm-5185), accelerate the recovery of the injured endothelium in rat carotid arteries. intravenous infusion of an no donor (30 p.g/day) with an implanted osmotic pump significantly accelerated the recovery of regenerated endothelium to produce edrf at 7 days. rat carotid artery rings relaxed only 17 + 5% and 15 + 5% to 10 gm ach in vehicle treated rats and in inactive no donor treated rats respectively 7 days following injury compared with 67 + 6% in no donor rats (p<0.001). relaxation to 100 gm nan02 was normal in all groups indicating that the differences in relaxation were not the result of damage to vascular smooth muscle. contraction to l-name (1 mm) was markedly reduced by injury, but was protected by no donors (p<0.01). thus, exogenous no donors enhance the ability of the endothelium to regenerate and to release edrf in response to endothelium-dependent vasodilators. this may be due to an anti-proliferative and anti-mitogenic effect of no on vascular smooth muscle cells, allowing the endothelium to regenerate without intimal thickening. no also has been shown to inhibit platelet aggregation, and to attenuate neutrophil adherence and activation. the superoxide scavenging effect of no is not the basis for these effects since hsod is inactive in preserving endothelial function in injured arteries. thus, no exerts a variety of cytoprotective effects which may be of importance in protecting against vascular injury. much evidence has now accumulated to show that the excess production of the vasodilator nitric oxide (no) in sepsis is an important contributor to the hypotension and multiorgan failure characteristic of this condition. various cytokines play an important role in this process through their ability to induce the production of one of the enzymes responsible for no synthesis, the inducible no synthase (inos). we have studied the effects of cytokines on the induction of this enzyme both in vitro using vascular smooth muscle cells, and in a murine model of gram-negative sepsis. tn smooth muscle ceils, the cytokines il-1, ifnq', and tnf-oc show strong synergy with one another in the production of inos. in order to define the molecular basis for this synergic effect, we have linked the promoter of the inos gene to a "reporter" gene, chloramphenicol acetyl transferase (cat), and transfected these constructs into vascular smooth muscle cells. assays of cat activity reflect the activity of the promoter in this system, and by generating sets of deletion mutants of the promoter sequence we have been able to define the area within the promoter which mediates the synergic effect of these cytokines. in addition to stimufatory effects on inos production, certain cytokines are able to down-regulate the production of inos in vascular smooth muscle cells, and the effects of these counterregulatory cytokines will be discussed. the interaction of these cytokine effects in the whole organism has been studied in a murine model of gramnegative sepsis. widespread induction of inos occurs in this model as assayed by enzyme activity and through use of specific antisera to inos. neutralizing antibodies to tnf-~ and tfn-y are both able to prevent death in this model, but it is only the anti-ifn-y which attenuates the induction of inos assayed in the liver. clearly there is some redundancy in the effects of cytokines on the production of inos in sepsis, and greater understanding of the most important factors in inos production is required in order to target anti-cytokine therapy most appropriately. effects of nitric oxide on hepatocyte metabolism in inflammation. j. stadler, department of surgery, tu mqnchen, frg hepatocellular nitric oxide (no) synthesis is induced by proinflammatory mediators such as tumor necrosis factor, interleukin-1 and interferon gamma or by bacterial toxins such as lipopolysaccharide. stimulation of the hepatocytes (hc) with a combination of these agents leads to an output of no in quantities which are not seen in any other celltype. it has been demonstrated by various investigators that important effects of these cytokines and bacterial toxins on hc metabolism can be attributed to the action of no. in contrast to other celltypes hc seem to be relatively resistant to suppression of basic metabolic functions such as energy metabolism by no. therefore, cell damage has not been described to a significant extent following exposure to no. however, no does inhibit total protein synthesis. the exact biochemical mechanism of this phenomenon has not been uncovered yet, but it has been demonstrated for some specific proteins that their production is inhibited at a posttransscriptional level. as in many other celltypes cgmp generation is elevated in hc by no through activation of the soluble guanylate cyclase. cyclic gmp may possibly exert a plethora of metabolic functions, but it is interesting to note that most of the cgmp seems to be transported out of the cell. some very specific effects of no on hc metabolism include the inhibition of the glyceraldehyde-3-phosphate dehydrogenase (gapdh) and the cytochrome p450 (cyp) enzymes. inhibition of gapdh activity is mediated through nitrosylation of critical domains of the enzymes by no which enhances auto-adpribosylation. this effect on gapdh activity might be responsible for the inhibition of gluconeogenesis by no, which has been described recently. finally, no-mediated inhibition of cyps may help to explain the suppression of hiotransformation processes which is a characteristic featur,'~ r ~ "~flamed liver. nitric oxide (no) is an endogenous inhibitor of polymorphonuclear leukocyte (pmn) adhesion which limits pmn-endothelial cell interactions under normal conditions. we have previously demonstrated that following ischemia, no production by the vascular endothelinm is dramatically reduced. accordingly, we investigated the effects of no-donors on pmn accumulation and tissue injury following hemorrhagic shock and ischemia. hemorrhagic shock was induced in anesthetized rats by bleeding to 35 mmhg for 3 hours followed by reperfusion. segments of superior mesenteric artery (sma) were isolated and suspended in organ baths. in rats receiving saline sma relaxation to acetylcholine (ach, 100 nm) was reduced by 80% compared to control sma segments (p<0.01) while relaxation to sodium nitrite (100 gm) was unaffected. in addition, mesenteric tissue pmn accumulation as determined by myeloperoxidase (mpo) activity was significantly elevated compared to controls (p<0.0l). interestingly, treatment with the no-donating agent, s-nitroso-n-acetylpenicillamine (snap) significantly preserved sma relaxation (p<0.01), attenuated mesenteric mpo (p<0.05) activity, and significantly improved survival compared to saline vehicle. in anesthetized, open-chest dogs we investigated the cardioprotective actions of a novel no-donor, spm-5185 (schwarz pharma), following regional myocardial ischemia (1 hour) and reperfusion (4.5 hours) . treatment with spm-5185 (500 rim) significantly reduced myocardial necrosis by 70% (p<0.01) compared to an no-deficient analog of spm-5185, spm-5267. furthermore, mpo activity within the ischemic-reperfused zone was also significantly (p<0.01) reduced following treatment with spm-5185 compared to spm-5267 (1.6 + 0.5 vs. 3.8 + 0.3 u/100 mg tissue). these data strongly suggest that no is a potent inhibitor of pmn-mediated tissue injury following hemorrhagic shock as well as in acute myocardial ischemia-reperfusion injury. overproduction of nitdc oxide (no') may contribute to sepsis-induced hypotension. during septic shock, excess no" is produced by an isoform of nitric oxide synthase (nos) which is induced by inflammatory mediators. nonselective nos inhibitors have been proposed as a new therapeutic approach to treating hypotension in septic shock. we studied the differential hemodynamic effects of n~-methyi-l-arginine (l-nma), a nos inhibitor, in normal canines versus those challenged with endotexin (lps) and compared the activity of this drug across the venous, pulmonary and systemic vascular beds. awake canines were challenged with lps (0 mg/kg, n=15:8 mg/kg, n=21 ; or 16 mg/kg, n=30) and treated with l-nma (0, 1,2, 4,10 mg/kg/hr) for 22 hours following a 0, 10, or40 mg/kg loading dose. animals were resuscitated with iv ringers solution (10 ml/kg/hr). hemodynamic data were collected at 0, 2, 6, 10, 14, and 22 hours using intravascular catheters and radionuclide heart scans and analyzed by anova. in both normal and endotoxemic animals, l-nma at all doses studied similarly increased mean arterial pressure (p=0.07), and systemic vascular resistance index (p=0.ol) and decreased cardiac index (p=0.05) and oxygen delivery index (p=0.01). in contrast, the effect of l-nma on mean pulmonary artery pressure, central venous pressure, pulmonary capillary wedge pressure, and pulmonary vascular resistance index was greater in lps-challenged canines compared to normal animals (p<0.05), but this differential effect on the venous and pulmonary circulation occurred, >6 hours after lps challenge. l-nma did not significantly increase survival rates or times at any of the doses studied (1, 2, 4, or 10 mg/kg/h) in either the low (8 mg/kg) or high dose (16 mg/kg) lps-challenge groups. a nonsignificant (p>0.2) trend toward a beneficial effect on survival ol low dose l-nma (1 mg/kg/h) in animals given the 8 mg/kg lps-cha[lenge was not enhanced by increasing the lethality of the model or by administering higher l-nma doses. at the highest l-nma dose used in this study (10 mg/kg/h), survival time decreased significantly for both the low and high dose lps-challenge animals (p<0.05). this increased mortality was not explained by changes in plasma concentrations of either lps or tnfc~. thus, l-nma did not have a greater effect on the systemic arterial circulation in endotoxemic compared to normal canines. however, in the venous and pulmonary vascular beds, the effect of l-nma increased with time after endotoxin-challenge these data suggest the induction of nos activity by endotoxin in canines may be relatively greater in venous and pulmonary vessels compared to systernic arteries. l-nma, a nonselective nos inhibitor, did not decrease mortality in endoloxemic canines and the highest dose studied was harmful. pulmonary hypertension (ph) and arterial hypoxemia are characteristic features of the adult respiratory distress syndrome (ards). reducing pulmonary vascular pressures may promote the resolution of pulmonary edema. intravenously infused vasodilators lower ph in ards, but, as a result of their general vasodilatatory effects, systemic mean arterial pressure may also decrease. furthermore, blood flow may be increased to non-ventilated or poorly ventilated lung areas resulting in a rise of intrapulmonary shunt, thus causing a further fall in pad 2. recently, short term inhalation of low concentrations of the gas nitric oxide (no), an endogenous endothelium derived relaxing factor, which is rapidly inactivated in blood by hemoglobin, was reported to decrease ph without causing systemic vasodilation in sheep [1]. similar changes have been observed in patients with severe ards during repeated short term inhalation of no (18 and 36 ppm), which rapidly and selectively decreased the mean pulmonary artery pressure (pap) and, in contrast to intravenously infused prostacyclin, induced a remarkable increase of pad2 [2] . this improvement in oxygenation was caused by a redistribution in blood flow away from intrapulmonary shunt areas to normal ventilated lung regions. continuous no inhalation (5-20 ppm) consistently lowered the pap and augmented the pao2/f.o 2 for up to 53 days. no negative side effects were observed during the whole time span examined. in particular methemoglobin levels always remained below 1.5%. following these investigations, it could be shown that these effects may also occur using concentrations in the parts per billion range [3] , which may reduce possible toxic side effects. however, in the same study it was demonstrated that the dose-response curves for pa02 and pap have different patterns. whereas pap presented a continuous dose-dependent downward tendency with an eds o of approximately 2-3 ppm, the improvement of oxygenation had a maximum at 10 ppm and, at higher doses, drifted back towards the baseline data. the ed~o was estimated at approximately 100 ppb, i.e. more than ten times lower than for the reduction of pap. in conclusion, inhalation of no by patients with severe ards may result in persistent and reproducible decreases in pap associated with an evident improvement in pad2, thus allowing reduction of the f.o 2. no inhalation should be performed using low concentrations which are less toxic, although any possible risks still have to be considered carefully. dose-response studies for the individual patients are recommended urgently. finally, controlled randomized studies are required to demonstrate that additional no inhalation is able to reduce mortality of ards. inhibition of the activity of glyceraldehyd-3-phosphate dehydrogenase (gapdh), an enzyme of the glycolysis/gluconeogenetic pathway, through adp-ribosylation is promoted by nitric oxide (no). since no is produced in the septic liver and hypoglycemia is a major problem of late sepsis, it was investigated whether no interferes with gluconeogenesis of hepatocytes. hepatocytes (hc) were isolated from sprague-dawley rats using a collagenase perfusion technique and differential centrifugation. exogenous no was applied by incubation with the no-donors s-nitrosyl-acetylpenicillamine and sodium-nitroprusside. endogenous no synthesis was induced by incubation with cytokines (tnfcq il-1, ifnj and lipopolysacchafide (lps). 24 hrs later the incubation medium was changed to a solution containing lactate, ornithine, lysine, ammoniumchloride and glucagon for optimal conditions of gluconeogenesis. after 3 more hrs glucose and nitrite levels were determined spectrophotometrically. gapdh activity was measured by the nadh-dependent conversion of 1,3-diphosphoglycerate to glyceraldehyde-3-phosphate. incubation of hc with no-donors led to a concentrationdependent inhibition of gluconeogenesis and gapdh activity. however, gapdh activity was about 5 times more sensitive to the inhibitory effect of exogenous no. incubation of hc with cytokines and lps induced nq synthesis as measured by an increase in nitrite concentrations. endogenously produced no suppressed gluconeogenesis by 49_+3%. in contrast to exogenously applied no, the effect of endogenous no synthesis was less on gapdh activity resulting in an inhibition of only 32_+12%. in conclusion, exogenous and endogenous no inhibited gluconeogenesis as well as gapdh activity. however, there was no correlation between the extent of inhibition of these two parameters of hepatocellular glucose metabolism. we have shown that inhibition of hepatocyte (hep) synthesis of nitric oxide (no) potentiates cell injury in a model of acetaminopheninduced oxidative stress and the extent of damage was paralleled by depletion of reduced glutathione (gsh) stores. to clarify the role of no in modulating the redox state of hep, we studied the effect of inhibition of cytokine-mediated no production on hep gsh stores, in a system of isolated rat hep in primary culture, no synthesis was induced (stim) by exposure to il-1, tnf, ifn, and lps for 16 hours. 20, 60, 120 and 200 ~m of n-monomethyi-l-arginine (nmma), a specific inhibitor of no synthesis, was added. cells incubated in media alone served as controls (cont). the no metabolite (no2); aspartate aminotransferase (ast), an indicator of cell injury; and gsh were assayed. (data presented as mean + sem; n=4.) gsh (nmovma orotein) ..~ (nmol/ma orotein) cont 14.1 + 0.3 32 + 4.0# stim 13.4 + 0.6 139 + 12 stim+2o tzm nmma 12.7 + 1.1 88 + 5.0# stim+60 ~m nmma 9.9_..+ 0.4* 68 + 4.5# stim+120 pm nmma 7.6 + 0.6* 57 + 3.0# stim+200 )lm nmma 5.0 + 0.5* 37 + 2.5# anova 0,0002 0.0001 (* p < 0.05 versus stim, # p < 0.01 versus stim; anova with neuman-keuls) gsh in cont+120 i~m l-nmma was equivalent to that of cont (13.3 vs.14.1). ast release was equivalent in all treatment groups. these data show that inhibition of hep synthesis of no depletes intracellular stores of reduced gsh. we conclude that hepatocyte no production modulates cellular gsh homeostasis and as a result, may be hepatoprotective in oxidative injury. nitric oxide (no) is a modulator of immune response and may be involved in the changes in immune reactivity after major trauma and operations. we investigated no-generation in rat and mice spleen cells (sc) after partial hepatectomy (ph). c57bl/6 mice and lew rats underwent a 50% and 70% ph, respectively. sc were prepared 1-6 days after ph and plated at 1 to 2 x 10ecells per well. after 20 h incubation at 37°c, no-production was measured as nitrite levels (griess reagent). normal mouse sc did not produce no, neither basal nor in response to lps or con a starting at the second day after ph, we found a substantial production of no. in rats, also sc from control animals were able to generate no; both basal and stimulated no-generation were further enhanced after ph (table, values expressed as mean --se). after shame operation, there was only a modest elevation of noproduction in rat and mouse sc. in first experiments we could demonstrate no-production also in phagocytes from a patient 3 days aider liver partial resection (1.4 nmol nitrite/106 cells) enhanced no-production in macrophages may contribute to the changes of immune reactivity after partial hepatectomy. nitric oxide (no) is recognized as an important mediator in endotoxemia and sepsis. increased synthesis of no has been demonstrated in septic humans and animals, and no inhibitors have been used in the treatment of septic shock. recent reports have, however, suggested that this form of therapy may cause serious organ damage. in the present investigation circulatory and metabolic changes in the liver were studied during treatment with the no-synthase inhibitor n-nitro-l-arginine-methyl ester (l-name) in endotoxemia. methods: juvenile pigs were randomized to one of the following treatment groups: 1) encletoxin and l-name, 2) endotoxin, 3) naci and l-name, 4) nach preliminary results from groups 1 (n=7) and 2 (n=8) are presented. catheters for pressure measurement were introduced into the aorta, hepatic and portal veins and ultrasonic transit time flow probes were placed on the hepatic artery and portal vein. a catheter was introduced into the pulmonary artery. endotoxin (1.7 gg/kg/h) was given as a continous portal infusion over the entire observation period of 8 hrs. l-name (25 mg/kg) was given as a bolus after 3 hrs. of endotoxemia. results: endotoxin transiently reduced portal vein flow (pvf) by 25%* and hepatic artery flow (hal e) by 45%*, while l-name caused a further and lasting reduction in flow (pvf 78%, haf 90%)*. transhepatic (portal-hepatic vein) vascular resistance increased to 3 times baseline value during endotoxemia while l-name caused a further marked increase in resistance to 12 times initial value. portal oxygen saturation (so2) decreased by 60%* during endotoxemia. l-name caused a reduction in portal so2 by 87%*. arterial so2 was unchanged in both groups. hepatic oxygen uptake was not changed by endotoxin, but was markedly reduced after addition of l-name. endotoxin caused a 27% reduction in cardiac output (co). the addition of l-name reduced co by a total of 71%*. *: p < 0.05. conclusion: is the present model of endotoxemia treatment with the nitric oxide synthase inhibitor l-name markedly reduced liver perfusion and portal oxygen supply. this might explain the increased liver damage reported in previous studies using no-inhibitors. the increase in transhepatic resistance found after l-name treatment will tend to cause pooling of blood in the splanchnic veins, resulting in reduced filling of the heart and thus contribute to the observed reduction in cardiac output. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. we have investigated the role of tumour necrosis factor (tnf) and interleukin-i (il-i) in the induction of nitric oxide synthase (nos) by bacterial endotoxin (lipopolysaccharide; lps; 2 mg kg -i i.v.) in vivo. in anaesthetized rats, pretreatment with a monoclonal antibody for tnf (tnfab; 20 mg kg -i s.c., at 16 h prior to lps) or with an il-i receptor antagonist (il-ira; 16 mg/kg bolus and 2.4 mg/kg/h infusion) ameliorated the fall in mean arterial blood pressure (map) at 90-180 min after lps. for instance, endotoxaemia for 180 min resulted in a fall in map from 114-+6 (control) to 79-+4 mmhg (p<0.01; n=8). in contrast, animals pretreated with tnfab or il-ira prior to lps injection maintained significantly higher map at 180 min when compared to lps-control: 118-+3 mmeg (n=5) and 103-+7 mmhg (n=5), respectively (p<0.01). three hours of endotoxaemia significantly reduced the contractile effects of noradrenaline (na) in the thoracic aorta ex vivo. the hyporeactivity to na was partially restored by in vitro treatment of the vessels with ng-nitro-l-arginine methyl ester (l-name, 20 min, 3x10 -4 m). pretreatment of rats with tnfab or il-ira significantly (p<0.05) prevented the lps-induced hyporeactivity of rat aortic rings ex vivo. l-name did not alter or only slightly enhanced the contractions of aortic rings obtained from tnfab or il-ira treated lps-rats, respectively. at 180 min after lps there was an induction of calcium-independent nos activity in the lung (5.14-+0.57 pmol citrulline/mg/min, n=8), which was attenuated by tnfab and !l-ira by 37-+6% and 46-+6%, respectively (n=5; p<0.05). thus, the production of both tnf and il-i contributes to the induction of nos by lps in vivo. the protective effect of agents which inhibit the release or action of tnf or il-i in shock may be, in part, due to inhibition of nos induction. neal garrison, md objective: sepsis is often accompanied by organ dysfunction, in part due to impaired microvascular perfusion. recently, nitric oxide (no) has been described as an important mediator of the hemodynamic changes of sepsis, and no synthase (no-s) inhibitors have been advocated for treatment of septic shock, but their visceral microcirculatory effects are inadequately characterized. we postulated that no-s inhibition would exacerbate the impaired organ perfusion of sepsis. methods: six groups ofdecerebrate rats were studied. bacteremia was induced with 109 live e. coli, which consistently increased cardiac output 15-20% above baseline (bl). the no-s inhibitor nm-nitro-larginine methyl ester (l-name,1 mg/kg iv), prevented this increase and elevated map by 25-30%. in the first 3 groups, total hepatic blood flow (thbf, ml/min by time transit flowmetry) and microvascular perfusion (mi-ibf, ¼ bl by laser doppler flux) were measured. in the other 3 groups, in vivo videomicroscopy was used to observe renal microvascular responses (ila=interlobular artery, aff=afferent arteriole, eff=efferent arteriole; % bl for all). results: data are 60 rains after e. cob. n=5-7/group. * p<0.05 vs bl by remanova and § p<0.05 vs e. coli alone by anova. ec+l-name 11-+2 -57_+10" § -45_+4* § -89_+3* § -33+5* -20+5* § conclusions: l-name administration in controls decreased renal blood flow, indicating no contributes to basal renal tone. bacteremia decreased mtlbf but not thbf, and mi-ibf was further impaired by no-s inhibition. e. coli caused renal preglomemlar, but not postglomerular constriction and reduced flow. l-name exacerbated these e. coli-induced alterations and caused eff constriction. these data indicate that no-s inhibition exacerbates bacteremia-induced impairment of renal and hepatic blood flow, suggesting that no is an importam compensatory dilator mechanism in these organs during sepsis. irf (iron responsive factor) is the central regulatory protein of intracellular iron metabolism able to bind to responsive rna elements (ires) present atthe 5'untranslated region (utr) of ferritin mrna and 3'utr of transferrin receptor mrna. binding of irf to ires results in repression of ferritin mrna translation and increased stability of transferrin receptor mrna leading to enhancement of transferrin receptor translation. we describe here that either tetrahydrobiopterin dependent stimulation as well as cytokine (ifn-~)/lipopolysaccharidemediated induction of nitric oxide synthase activates irf, which is due to direct interaction of nitric oxide with the iron-sulphur-cluster of irf. this was shown by gene expression studies using a plasmid containing a ferritin ire and a cat indicator box which was transfected into k562 myelomonocytic cells, which were shown to have a constitutive form of nitric oxide synthase (nos). furthermore, the increased binding of 1re to irf due to irf activation of irf by nitric oxide was demonstrated by gel shift assays. irf activity was much more increased in cellular extracts from murine macrophages (j774) where a cytokine inducible form of nos has been characterized earlier as compared with irf activity in k562 cells, where nos was stimulated by increasing the availability of the essential nos cofactor 5,6,7,8-tetrahydrobiopterin. we then demonstrated that activation of irf by nitric oxide is accompanied by alterations in ferritin translation as checked by metabolic labeling and immunoprecipitation. these results suggest a reasonable mechanism for the regulation of iron disturbances under chronic inflammatory disorders, characterized by increased concentration of immune activation parameters like ifn-5or neopterin and low serum iron and hemoglobin concentrations. taken nitric oxide, no, the putative endothelial derived relaxant factor, edrf, has been shown to be a potent inhibitor ofplatelet aggregation in vitro. in vivo evidence however, is scarce. accumulation of platelets in the lungs has been shown to occur during extracorporeal circulation. the aim of the present study was to investigate the effect of inhaled no on this reaction. materials and methods: the animals were divided into two groups, each consisting of 7 pigs. platelets were selectively labelled with luln-oxine. dialysis was instituted via catheters in the femoral vessels. in group 1, no, 50 ppm, was added to the inhaled gas from the start of dialysis. in group 2 no was not given. the activity over the lungs was followed dynamically with a gamma camera. central hemodynamics was monitored via a swan -ganz catheter. results: the activity was significantly lower in group 1, from 2 minutes after start of dialysis and onwards, indicating diminished accumulation of platelets in the lungs. parallel to this the hemodynamic response in terms of increased pulmonary artery pressure and pulmonary vascular resistance was blunted in this group conclusion: inhaled no in this model seems to affect pulmonary platelet sequestration. an associated attenuation of the changes in central hemodynamics was also seen. previous studies from our laboratory have demonstrated that vascular contractility decreased in endothelium-intact blood vessel rings in early and late stages of sepsis. although endothelium removal in early sepsis restored vascular contraction, the depressed smooth muscle contractility observed in late sepsis was not restored by endothelium removal. this indicates that impairment of smooth muscleper se may be responsible for such dysfunction in late sepsis. the aim of this study, therefore, was to determine whether or not smooth muscle-derived nitric oxide (no) plays a role in producing vascular smooth muscle dysfunction during late stages of sepsis. to study this, rats (250-300g, n=4-5/group) were subjected to sepsis by cecal ligation and puncture (clp). septic and shamoperated rats then received 3 rrd/100g bw normal saline. the animals were killed at 10, 20, or 35 h post-clp (10 h post-clp=early sepsis; 20-35 h post-clp=late sepsis), and thoracic aortic rings were prepared for contraction studies using organ chambers. the complete removal of endothelial cells was tested by the absence of any significant acetylcholine-induced vascular relaxation. contractile responses to norepinephrine (ne, 10 9 to 10 -5 m) were determined in the aortic rings without intact endothelium. ng-monomethyl-l-arginine (l-nmma, 300/~m, an inhibitor of no synthase) was then added to the organ chamber and ne-induced peak contraction was determined before and after the addition of l-nmma. the peak contraction (rag/rag tissue, mean_+sem) is shown below: the results indicate that the addition of l-nmma did not significantly affect ne-lnduced peak contraction in endothelium-denuded vessel rings at 10 and 20 h after clp. in contrast, l-nmma administration produces an 18% increase (p<0.05) in peak contraction during late sepsis. therefore, the vascular smooth muscle contractile dysfunction observed at 35 h post-clp is partially due to smooth muscle-derived no over-production. thus, unlike macrophages in which inducible nitric oxide synthase (inos) is observed in early sepsis, the inos in vascular smooth muscle appears prominent only in the late stages of sepsis. in three cases of human septic shock in which ng-monomethyi-l-arginine, (l-nmma) a nitric-oxide-synthase-inhibitor was applied, we isolated three completely different types of pathogens: candida, pseudomonas aeruginose and multiresistant coagulase-negative staphylococci. this observation suggests that endotoxin alone is not the main factor triggering hypotension in septic shock by the nitric oxide pathway. in a 68-years-old woman in severe septic shock due to a candida and pseudomonas aeruginosa infection complicated by adult-respiratorydistress-syndrome conditions deteriorated despite adequate conventional therapy. in this trial, effects of l-nmma on cytokin-levels were investigated. the study-protocol was approved by the ethical committee of the department of surgery. after two boll of 200 mg of l-nmma, a continuous infusion was installed (0.05 mg/minute and kg body weight l-nmma). as expected mean arterial blood pressure rose (62 to 134 mmhg}, heart rate stayed stable (124 + 4 b/rain), systemic vascular resistance increased (225 to 354 dyne.sec/cm5), cardiac output decreased (17 to 15.2 l/rain), and cardiac index declined (9.94 to 8.63 l/min/m2}. before and after 90 minutes while the infusion of l-nmma, blood samples for immunological measurements were taken and processed together. pulmonary-shunt-volume was observed before the application of l-nmma, after one hour and after 140 matutes. neopterine increased from 16.51 to 32.55 ng/ml, tumour-necrosis-factor-a increased from 24.16 to 36.61 pg/ml and intedeukin-6 increased from 61.9 to 98.2 pg/ml. immunoglobulines a, g, and m (3.2 to 2.9, 8.1 to 7.6, 1.1 to 0.9 g/i), complement factor c-3c and c-4 (0.54 to 0.50, 0.22 to 0.23 g/i), alpha-l-antitrypsine (3.86 to 3.83 g/i), c-reactive-protein (111.1 to 105.6 rag/i), interleukin-1 (0 pg/ml) and soluble interleukin-2 (2128 to 1983 units/ml) did not change significantly. pulmonary-shuntvolume decreased from 54.1% to 35.4% within one hour and to 36.6% after 140 minutes. in septic shock blocking nitric oxide as an intervention at the end of a not ~,et ful!y understood cascade might have important influences on pulmonary-shunt-volume and inter-cell-communication. department of surgery, pharmacy* and immunology**, university hospital of zurich, r~imistrasse 100, 8091 zurich, switzerland we previously reported that hypoferremic cba mice had an increased resistance to salmonella infection, and that injection of ammonium ferric citrate (afc) to these mice led to enhanced infection (ganthier et at. 1986. microbiol.immuno130:425) . because nitric oxide (no) is involved in the antimicrobial activity of routine macmphages towards various inttacellular pathogens, we investigated the influence of iron on the bactericidal activity of cba mouse macrophages towards s.typhimurium and on the production and activity of reactive nitrogen intermediates (rni). peritoneal macrophages hum cba mice were cultured in the presence (or not) of afc 50 ,um, ifn-,/250 u/ml, lps 1 fig/m/, ngmonomethyl-l--arginine (mmla) 2ram. nitrite (no2-) content of the supematants was determined by a standard griess reaction, and h202 release was measured by the peroxidese dependant oxidation of phenol red. for intracellular killing, macrophages monolayers were infected, and, at various intervals, lysed by triton x-100, and surviving bacteria enumerated by colony counting on agar. for in vivo experiments, mice were infected ip with 0.5 ml of a suspension of 5.10 ~" s.typhimurium, strain c5, and injected with aminoguanidine (ag) 1 mg/ml in saline. our results show that the rn[ inhibitor ag strongly accelerates the mortality of infected mice, the survival rate decreasing from 60% in the control group to 20% in the treated group, 10 days after challenge. correlatively the rni inhibitor mmla induces in vitro a decrease in the rate of bacterial killing, fxom 78% to 57%, in macrophages triggered with ifn-? + lps. the cultivation of macrophages in the presence of afc leads to a decreased no 2-accumulation, 4.7 nmole/well v.s. 21 nmole/well. conversely h202 production is enhanced from 09 nmole/well up to 2,33 nmole/well. nevertheless, macrophages cultivated in the presence of afc exhibit an increased tale of intracellular killing, 77% in iron exposed macrophages v.s, 68% in control macrophages. when triggered with ifn-~, alone, macrophages have a reduced antibacterial activity (57% v.s. 68%) whereas the addition of afc to these macrophagas restores an elevated (72%) rate of killing. in conclusion, the results show that bactericidal activity of cba macrophages towards s.typhimurium depends on the production of no by these macrophages ; but they also demonstrate that no is not the only reactive species involved in the intracellular kil/ing of s.thyphimurium ; indeed afc which strongly inhibits rni production, stimulates h20 2 release by these macrophages and increase their bactericidal activity in vitro. nevertheless afc may promote bacterial growth in vivo. crssa. unit4 de microbiologie. bp 87. 38702 la tronche cedex france. henning jahr, ulrike noack, karin braun the large amounts of no produced by the inducible no synthase in rat macrophages have direct antimicrobial effects, but inhibit the activation of the lymphocyte-dependent host defense system. the aim of this study was to investigate if complement activation influences no-generation. spleen cells from lew rats were incubated at 37°in tcm-199/5% fcs, with or without additional rat serum. after 20 h, nitrite (end product from no metabolism) was measured by oriess reagent. in rat spleen cell preparations, most of the no is produced by macrophages. complement activation in vivo was carried out by i.v. injections of 240 u cobra venom factor/kg b.w. at days 0 and 2. significantly higher (p<o.o1) lps-stimulated no-generation was found in spleen cells prepared at day 4 (13.0 4-1.3 nmol nitrite/106 cells, n=6) compared to spleen cells from non-treated animals (3.6 ± 1.3 nmol, n=6) complement activation was effective also in vitro. when incubated in 25% zymosan-activated rat serum, both basal and lps-stimulated noproduction were enhanced in spleen cells (table, values increased production of nitric oxide (no) is associated with sepsis, however, the effect of no inhibition on survival during sepsis is unclear. we examined the effect of no inhibition on host's ability to eliminate bacteria and survival in mice sepsis model. sixty female balb/c mice were injected with e.coli (10qbody) into peritoneal cavity. the treated group received n-ultro-l-arginine-methyl-ester (l-name), an inhibitor of nos, one hour before bacterial challenge. thirty mice were observed for survival after e.coli challenge and the other mice were sacrificed at 4 hours. blood was obtained by cardiac puncture and peritoneal lavage fluid (plf), liver and lung were harvested. the number of peritoneal exudative cell (pec) was counted and colony forming units (cfld of bacteria in the tissues, blood, and plf were also determined. in addition, pec was cultured in vitro with or without lipopolysaecharide (lps). tnf levels in the blood, plf, and supematants of cultured pec were measured by l929 bioassay. survival rate (%) qrql/,p n 8 hr 24 hr 4 dav control (normal saline 0.1ml/body i.p.) 10 100 50 40 n10 (l-name 10mg/kg i. administration of l-name before e.coli injection increased the number of bacteria in plf and tnf level in plasma, the result suggests that nos inhibitor may depress the host's ability to kill the injected bacteria and that it may induce greater systemic tnf production. we conclude that nos inhibitor is detrimental to animals in sepsis. the hypothesis that sod prevents reperfusion injury to the liver by protecting the endogenous endothelium derived vascular relaxing factor (no) from being inactivated by oxygen free radicals should be investigated. male wistar rats were subjected to 60 min of partial liver ischemia (70%) and 30 min of consecutive reperfusion in situ. some rats were pretreated with the no synthase inhibitor nitro-l-arginin (nla: i0 mg/kg). sod, when used, was given as mn-containing preparation at a dose of 6000 u. i.v. i0 min prior to ischemia. results (sod/ nla+sod/ ni~,, respectively): bile flow (~i/g/min) : 0.93±0.11"#/ 0.52±0.08/ 0.48±0.05. alt (u/2) • 171±40~#/ 477±173/ 506±182. gldh (u/l): 5.2±1.2e#/ 10.7±4.2#/ 24.3±9.1. in absence of nla, sod enhanced postischemic bile flow and reduced leakage of alt and gldh into the plasma, while the effects of sod disappeared, when endogenous 140~synthesis was inhibited by nla. nla had no ~ffect on untreated animals submitted to the same protocol. these results may suggest, that oxygen free radicals interfe~:e with the endogenous vasodilator no and that the benefit of sod can be attribuated in large part to a protection of no during reperfusion. nitric oxide (no) reduces pulmonary vascular resistance and increases oxygenation by inhalation in ards patients [1] . animal studies on the endogeneous no production proved that no is exhaled after synthesis in the respiratory tract [2] . with approval by the hospital ethics committee, we studied healthy volunteers and ventilated patients by measurement of inand exhaled no by no/no2-chemiluminescence in segments of the upper respiratory tract. we found that no is synthesized predominantly in the nose and in the upper nasopharynx, leading to inspiratory tracheal no concentrations of 0.07-0.13 parts per million in non-smoking volunteers; data during mask ventilation per nose were as follows: non 0.089 0.0422 0.0721 0.0311,2 parts per million (ppm) no, mean, n = 10; ~ p < 0.05 (t-test) between smokers and non-smokers; 2 p < 0.05 (t-test) between in-and expiration. about 50% of no is resorbe.d by the lung, and the exhaled no -in contrast to former presumptions -is the remaining part of the autoinhaled no. intubated and ventilated patients are deprived of no autoinhalation, and the exhaled no in the trachea decreases more than 95% {o.043 ppm before intubation vs. 0.003 ppm after intubation, ~, < 0.01), whereas the nasal no concentration increases by cumulation. hence, low-dose no inhalation in ards patients might be considered as a no replacement, especially since the tracheal no concentrations we measured in volunteers are similar to those which could be demonstrated to be effective for ards in former studies [3] . the data demonstrate that no is synthesized in the nose, and inhaled and resorbed by the lung. this phenomenon of no autoinhalation, which is reduced in smokers and in ventilated patients, does possibly contribute to the regulation of the ventilation-perfusion ratio in the lung. kikuchi 1, yoshihiro inoue 1, masao yoshida 2 1 critical care and emergency center, and 2department of bacteriology, iwate medical university. nitric oxide (no) is produced by macrophages and vascular endothelial cells. it is thought to be the true form of endotheliumderived relaxing factor, and to be involved in the fall of blood pressure during septic shock. we have reported previously that endotoxin (lps), tnf-c~ and il-2 contribute to the occurrence of septic shock (circ shock 38:264; . the present study investigated the in vitro effects of lps, tnf-o~, il-2 and ifn-'i on the production of no by macrophages. peritoneal macrophages were cultured with lps, il-2, tnf-~ and ifn-¥. in culture with lps, no was produced in a dose-dependent manner, and the production was suppressed by a no production inhibitor, l-nmma. ifn-y, il-2 and tnf-c~ used alone did not promote the production of no, but helped lps to facilitate no production. a combination of il-2 and tnf-c( enhanced lps-facilitated no production to a greater extent than il-2 or tnf-c~ alone. the above results suggest that these cytokines are involved in endotoxin shock through the mechanisms that facilitate no production. 19-1 uchimaru, morioka 020. japan. t. yolk 1,2, i. ioannidis 1, w.j. kox 2 and h. de groot 1 objectives: nitric oxide (no.) is known to play an important role in neurotransmission, vasoregulation, and bactericidal as well as tumoricidal cellular defense systems. by means of no-donating agents we studied the mechanism of no-mediated cytotoxicity against rat liver microvascular endothelial cells. materials and methods: 3-morphoiinosydnonimine-n-ethylcarbamide (sin-l) and sodium nitroprusside (snp) were used as no. donators, ldh release and trypan blue exclusion were applied to indicate cell viability. results: sin-1 (5mm), which releases both no. and o2-., caused a time-dependent cytoreduction of 60% and 80% after 3 and 6 hrs, respectively. this effect was not inhibited by superoxide dismutase (sod), which removes 02--to form h202 and 02. in contrast, the addition of catalase (cat), which removes h202 to form h20 and 02, diminished the cytotoxic effect ot sin-1 to 30%, equivalent to high concentrations of snp (20mm), which solely releases no.. in addition, the amount of h202 formed by 5mm sin-1 equaled the amount ot enzymaticany produced h202 by 5mu/l glucose oxidase (god). whereas god in this concentration and snp in low concentration (5mm) alone were not toxic to endothelial cells, the combination caused an 80% reduction of viability, comparable to the effect observed with sin-1 (5mm) alone. moreover, toxicity mediated by high concentrations of snp (20mm and 50ram) was reduced by 20% under hypoxic conditions indicating synergism with no-and 02. conclusions: we conclude, that no.-induced toxicity against endothelial cells is enhanced slightly in the presence of oxygen and is not due to an interaction with 02-. however, toxicity increases dramatically in the presence of h202. this may possibly occur either by a chemical formation of more potent reactive hydroxyl radicals or by independent actions on different cellular targets. although it is becoming increasingly clear that nitric oxide (no) is associated with otxan dysfunctions in septic patients, little is known as to die kinetics of no production in these patients to clarify these kinetics, we have investegated serum arid monocyte associated no in septic patients. five patients who had undergone gastrointestinal surgeries mid were complicated postoperatively with severe sepsis served as the subjects in this study (sgroup), using twelve healthy volunteers as a control group (c group). serum no2 and no3 levels were measul*d sliectrophotometricallymonocyte cultures were done for 24 itrs with or without lps+ifn-t stimulation mid no2 and no3 levels in the supernat,'utts were also measured spectrophotometrically. furthermore,using an no selective electrode,time course of the lps+ifn-t induced no production by monocytes was studied. l) serum no2 levels in s group were slightly lfigher than dmse in c group,bnt there were no significoatt differences between the two. 2)both no2 levels in the supematmlts of monocytes cultured with lps+ifn-t mid no2, no3 levels ill lhe supernatants of monocytes cultured without lps+ifn-y were significantly higher in s group. further,the time required for the no production by enltnred monoeytes was siglfificantly shorter also in s groap. conclusions l)in severe septic patients, monocyte no productions wei~ not only primed, but also activated concomitantly, suggesting that these monocyte activations m'e strongly associated with organ dysfunctions in sepsis. 2)based on the restdts of serum no nteasuremeuts, it can be deduced that no p~'oduced by monocytes in septic patients affects tissues and org~s ioeoregiomflly. objectives of the study: nitric oxide (no) is an important messenger which accounts for a wide spectrum of physiological and pathophysiological processes, e.g. mediating vasodilation in endoloxin shock investigating the signal lransducfion pathways involved in the regulation of the inducible nitric oxide synthase (inos), we report the involvement of phosphatidylcholinespecific phospholipase c (pc-plc) and proteinkinase c (pkc). materials and methods: no-production was induced in the murine macrophage cell line j774 with lipopolysaccharide (lps) [lljg/ml] and interferon ;f (ifny) [200u/ml]. after 18 hours of incubation no-release was determined as nitrite (no2) by using a colorimetric assay based on the griess-reaetion. results: preincubation of cells with the pkc-inhibitors staurosporine (stp), chelerythrine, bisindolylmaleimide (bim) and d609, that recently has been identified as a selective inhibitor of pc-plc, diminished significantly lpsand ]fnt-induced no2-production (p<o,001). although we observed no toxic effect on cell viability, no2-production was related to protein concentrations to exclude any inhibitory effect on celt growth. celts preincubated with stp [lo0nm] released 20% less no 2 in response to lps and ifn,[ compared to control cells cultured without inhibitor. chelerythrine [30}jm] and bim [ioijm] reduced the no2-formation by 50% and 85%, respectively. interestingly, the most potent inhibitor of no-production turned out to be the xanthate d609 cells pretreated with d609 [301~g/mt] released 96% less no 2 than stimulated controls the inhibitory effect on no 2production decreased the later the inhibitore were added after stimulation; e.g. bim added 6 and 12 hours after stimulation reduced no2-production only by 50% and 12%, respectively. accordingly, inqs activity present in stimulated cell lysates supplemented with l-arginine and co-factors was not suppressed by the inhibitors tested. conclusions: our findings suggest, that the induction of inos but not its activity is a pkc and particularly pc-plc dependent process. dr. k tschaikowsky, institut for anaesthesiologie, universital erlangen--nqrnberg, krankenhausstr 12, 91054 erlangen interleukin-1 (il-1) affects nearly every cell type by increasing the expression of a variety of genes associated with the promotion of inflammatory processes. these include upregulation of cyclooxygenase and nitric acid synthases. the il-1ri transmits a signal(s) through the cytoplasmic domain which is structurally related to the fruit fly toll protein. we have recently cloned the promotor most proximal to the 5' utr of the human il-1ri gene. the genomic sequences reveal a lack of tata and caat boxes but rather a considerable (75%) gc rich region. the translation of the type i il-1r appears under a significant suppression and may limit the biological activities of il-1 by low level of expression. on the other hand, the type ii il-1r, lacking a significant cytoplasmic domain, does not transmit a signal and acts as a decoy receptor preventing the binding to the type i receptor. there are several approaches to reducing il-1 activity; inhibition of il-1 converting enzyme which cleaves pro-il-l[3, anti-il-1ri, the il-i receptor antagonist (il-1ra) and soluble (extracellular) il-1ri and il-1rii. il-1ra is structurally related to il-1 and binds at 4°c to the il-1ri with near equal affinity as that of bona fide il-i; however, il-1ra does not transduce a signal. il-1ra has been given to humans in pharmacologic levels (mean plasma levels of 30gg/ml) without evidence of agonist activity. in addition, there has been no affect on normal homeostatic parameters, suggesting that il-i does not play a role in health. in healthy humans given intravenous endotoxin, il-ira reduced endotoxin-associated neutrophilia by 50% and completely reversed endoloxin-iuduced immunosuppression, il-tra is produced naturally and is elevated in the circulation in several diseases. in a variety of diseases, plasma levels of il-1ra are in 100-fold molar excess to those of il-i [3. it is unclear whether these endogenous levels of il-1ra are sufficient to block il-1 activity in disease. a single injection of ]l-6 or ifna in humans does not induce il-1 but rather high levels of il-1ra (10ng/ml). prof.dr.l.a. aarden interleukin 6 (il6) is a multifunctionai cytokine with a central role in host defense mechanisms and consequently in inflammation. il6 is thought to be involved in the pathogenesis of various diseases. therefore, specific inhibitors could have therapeutic value. a human-mouse chimeric monoclonal antibody to il6 has been applied in a phasei clinical trial in patients with multiple myeloma and in primate models of sepsis. no toxicity was observed and the most remarkable outcome of this study was the build up in the circulation of il6-anti-il6 complexes, suggesting that plasma il6 measurements represent a gross underestimation of il6 production in the patient. until now no natural antagonists of il6 have been described. the biological activities of il6 results from binding to a lowaffinity il6-receptor (il6r). the il6/il6r complex induces disulfide-linked homodimerization of the signal transducing receptor component, gp130. studies on the structure-function relation of il6 revealed that disruption of the gpl30-interaction site on il6 gives rise to a mutant il6 that, by virtue of its intact binding to the il6r, acts as a potent antagonist on human hepatocytes and on human b cells. in vivo studies using this molecule will be initiated in the near future. the evolution of infla~ation involves a dynamic series of cellular events controlled by specific signals which are important to the initiation, maintenance, and final resolution of the inflammatory response. the various phases of inflammation are influenced via the expression and subsequent regulation of a number of key mediators which play a predominant role during each particular stage of the developing lesion. for example, the initial elicitation of blood born leukocytes to on inflamed area is a complex system that is dependent upon the expression of early response cytokineso these proximal mediators, including both interleukin-i (il-i) and tumor necroisis factor (tnf), are important in the initiation phase by activating both immune and non-immune cells and establishing a series of cytokine networks, thus, the above proximal mediators can stimulate endothelial cells, epithelial cells, smooth muscle cells, and fibroblasts to generate more distal cytokines. these latter cytokines include interleukin-8 (il-8), macrophage inflammatory protein-i (mip-i), and monocyte cbemoattractant protein-i (mcp-i). the importance of the above chemukines can be found in the role they play in leukocyte elicitation, as well as wound repair. numerous investigations have shown the diverse cellular sources of il-8 and the ability of il-8 to elicit neutrophils in vitro at pm to nm concentrations, however, recent studies have sho%~ an additional role for il-8 during the resolution phase of the inflammatory process. using corneal pocket animal models of angiogenesis/neovascularization, il-8 was found to be a potent angiogenic factor at concentrations ranging from 2-40ng/ml. sequential fluorescein angiograms demonstrated that il-8 induced neovascularization by 14 days, which regressed by 6 weeks. in further analyses, both conditioned media recovered from either inflamed human heumatoid synovial tissue macrophages or lps-stimulated peripheral blood monocytes were found to possess potent angiogenic activity, which was effectively blocked by neutralizing antibodies directed against human il-8. in addition, an il-8 antisense nligomunclentide blocked the expression of a functionally active angiogenic factor from lps treated monocytes. the above data demonstrate that il-8 has multifumctional activities during the initiation, maintenance, and resolution of a local inflammatory response. inhibits specific t-cell responses to soluble protein antigens and alloantigens. the proliferative responses of th0, thl and th2 cd4 ÷ t-cell clones and cytokine production by these t-cell subsets are equally well inhibited. the inhibitory effects of il-10 are indirect and related to inhibition of antigen-presenting capacity and accessory function of the antigen-presenting cells (apc). however, il-10 also directly inhibits t-cell proli/eration induced by cross]inked anti-cd3 or anti-cd2 mabs (e.g. in the absence of professional apc), by specifically inhibiting il-2 gene transcription and il-2 protein production. il-10 also inhibits the production of the pro-inflammatory cytokines il-l-a, ~, il-6, and tnf-a and the chemokines il-8 and mip-i-u, which are strong chemo attractants for neutrophils and macrophage, respectively. in contrast, il-10 enhances the production of the il-1 receptor antagonist, a cytokine with anti-inflammatory activity. il-l0 produced by monocytes downregulates class ii mhc expression and il-10 production by these cells in an autoregulatory fashion. collectively, these in vitro data indicate that il-10 is a powerful suppressor factor for immune-and inflammatory responses and therefore may have potential clinical utility as an anti-inflammatory agent. this notion is supported by recent studies which indicated that il-10 also prevents lethal lps-induced toxic shock in mice. five of the chamekines (hip-i~,era~tes. ip-io and il 8) also chemoattraet t lymphocytes, while others act on mast cells. we have studied the effaces of these chemokines on t cell subsets. il 8 preferentially chemoattracts uns~imulated t cells. raises ~ttracte resting as well as activated cd4 and cd8 memory t cells. ip-10 chemoattracts only preactivated (not resting) cd4 or cd8 t cells. hip-l~ preferentially chemoattracts gd8 t calla, while hip-18 more readily attracts the cd4 t call subset. the 8 chemoklnes and ip-io were also sho~rn to promote the adherence of ~he same subsets of anti-cd3 activated t cells to ili activated human umbilical vein endothelial cells (hitvec). this was not associated with a~y i~crease in the number of lymphocyte adhesion molecules, bu~ could be inhibited by neutralizing monoclonal antibodies re lfa-i and vla-4. these chemokines also rapidly increased ~ha adhesion of resting t l~phoeytes to plates coated wi~h 8 integrlns (i-cam or v-cam) or matrix proteins such as f~bronectln. this induction of adhesion began wiehln 30', peaked by 1 hr and was completed in 3 hr. this suggests that chemoklnee rapldly increase the binding affinity of adhesion prote~ns on t cells. we also recently observed ~hat mcaf/mcpi and rantes chemoattract unatimulated mast calls. furthermore, igs actlvared mast calls were chemoattraeted by mcaf, ra~tes, pf4 and mip.i~. however. ~he chemoklnee did not induce mast cells ~o release histamines. presumably, chemokines as regulators of the adhesion, migration and homing of all kinds of leukocytes participate in many types of inflammatory diseases. natural killer cell stimulatory factor or interleukin-12 (il-12) is an heterodimerie cytokine formed by two covalently linked glycosylated chains of 35kd and 40 kd. il-12 was originally purified from the supermatant fluids of ebv-transformed human b ly~phobiastoid cell lines. however, in addition to b cells, phagocytic cells, i.e. monocytes, macrophages, and neutrophils, have been identified as the major physiological producers of il-12. phagocytic cells produce il-12 in response to bacteria, bacterial products such as lps, and intracellular parasites. the cell types on which il-12 exert biological activity are t and nk cells. the major direct biological function of il-12 on these cell types are the induction of cytokine production, primarily ifn-y, the enhancement of a generation of cytotoxic cells, and a mitogenic effect on antlgen-activated lymphocytes. in part thromgh its ability to induce ifn-y production, il-12, produced in response to infections, represents an important functional link between effector cells of innate resistance, phagocytic cells and nk cells, and effector cells of adaptiye resistance, t and b lymphocytes. the ability of il-12 to induce ifn-y production from circulating. nk cells and at least a proportion of t cells determines a rapid, antigen-independet production of ifn-y during infections. ifn-y acts as a potent enhancer of phagocytic and bacteriocidal activity of phagocytic cells, participating early in infection to activate some of the inflammatory mechanisms that represent the first innate resistance against infection. this antigem-independet activation of phagocytic cells that involves, in addition to il-12, other monocyte derived cytokines such as tnf and il-l, has been shown to be important in experimental animals for the resistance against infection by microorganisms such as limteria monocytogens and toxoplasma gondii. in addition to this role, early in infection in the antigen-independet phagocytic cell activation, il-12 has a major effect in the ensuing antigenspecific admptive immune response by facilitating the generation of t helper type 1 (th-i) response and suppressing a th-2 response. the presence of il-12 during antigen stimulation in vitro using human peripheral blood lymphocytes, or both in vivo and in vitro in experimental animals, induces generation of th-i cells producing ifn-y and il-2, and inhibits the generation of th-2 cells producing il-4. il-12 induces a direct differentiative effect on th-cells, priming them for high ifn-y production. ~is priming effect is stable and maintained even when t cell clones are cultured for extended periods in the absence of il-12, however, il-12 needs to be present during the first few days after stimulation with antigen or mitogen and established th clones are resistant to the priming effect of ifn-y. unlike the generation of high ifn-y producing clones, the il-12-mediated inhibition of il-4 producing cells in probably due to selective mechanisms, which may include a selective mitegenic effect of il-12 for thl cells and an inhibitory effect of ifn-y on th2 cell proliferation. in several experimental systems, it has been shown that the enhancing effect of il-12 on th-i responses is dependent on production of if~-y and on the presence of nk cells, possibly needed for the early production of ifn-y. the importance of phagocytic cells and nk cells in determining the characteristics of the th response during antigenic stimulation indicated that the mechanisms of innate resistance have a determining influence on the subsequent antigen-specific immune response: il-12 appears to have a key role in mediating the interaction between phagocytic cells. nk cells, and t lympocytes in these proccessem. trinchieri, g. 1993, interleukin-12 and its role in the generation of t e 1 cells, imm~ol. on peripheral blood monocyte/macrophages, il-13 behaves like il-4 in a variety of assays, and shows both similar and contrasting, activties with either il-10 or ifn-y. il-13 reduces inflammatory monokine and il-10 production. it reduces the pyrogen-induced expression of tissue factor (herbert et el, 1993, febs lett. 328, 268) . it mpregulates manmose receptor and ~c class ii expression, while reducing cdi4 expression. il-13 produces morphological changes (extensive cell processes, aggregation, giant cell formation) which have previously been observed with il-4. it inhibits niv-i production (montaner et el, 1993, i. exp. mad. 178, 743) , and interferes with hiv-induced cell fusion. the activities of il-13 and il-4 differ on t-ly•ocyte and large granular lymphocyte population. in particular il-13 does not exhibit the suppressive effects of il-4 on thl-type-helper functions (ifny production) and cytotoxic functions (perforin production). data will be presented showing that il-4 and il-l3 share a common receptor on a nu~er of cell types, but not on t-lympocytes (see also zmrawski st el. 1993, embo j.7. 2663-2670), explaining both thai common and divergent activities. the levels of il-13~a in activated peripheral blood lymphocytes are greater than or equivalent to those of il-4 mp~ja and the two ml{nas are expressed by different t lympocyte population: in particular, t8 cytotoxic lymphocytes express markedly higher levels of il-13~rna. in vivo, il-13 may thus be contributing, significantly to some of the activites previously ascribed to il-4 il-10. recently, we cloned the human cdna encoding interleukin-13 (il-13). human il-13 is a non-glycosylated protein of 132 aa with a molecular mass (mr) of 10 kd and has biological activities on monocytes and b cells, il-13, like il-4, strongly enhanced the expression of class ii mhc antigens on monocytes and induced expression of cd23 (fc~rii). furthermore, il-13, like il-4 and il~10, inhibited the production of pro-inflammatory cytokines il-1, ]l-6, tnfo~ and chemokines miplc~ and tl-8 by lps activated monocytes. both il-13 and il-4 enhanced the production of il-lra by monocytes. in contrast, il-13 lacked the t cell stimulating activities of il-4. the responses of monocytes to il-13 and il-4 were not additive or synergistic, supporting the hypothesis that il-13 and il-4 receptors are complex and share a common component which is important for signal transduction, taken together, these data indicate that il-13, il~4 and il-10 have important anti-inflammatory activities on human monocytes. in addition, these results indicate that il-13 is unique in that it can modify inflammatory reactions without stimulating (as does il-4) or inhibiting (as does il-10) t cell responses. transforming growth factor beta i (tgf-fll) belongs to a family of proteins that have potent immunoregulatory properties ranging ftom effects on the growth and dfflerentiafion of primitive stem cells to the differentiated functions of immune system effector cells. tgf-i~i is synthesized as a large secretory precursor polypeptide of 390 amino acids that is inactive until processed to a disulfide linked homodimeric molecule of 1t2 amino acids each. recently it has been found that tgf-[~i can have autocrine as well as paracrine effects on the immune system indicating that immune cells can activate the inactive secreted form of tgi~-~i. in addition, tgf-i~i has differential intraceuular effects on cell surface receptor modulation, tyrosine phosphorylation and cytokine gene transcription as well as cell mediated cytotoxicity. the systemic administration of tgf-131 has proven beneficial in a variety of animal models such as septic shock, allograft rejection and experimental forms of autoimmunity including collagen induced arthritis and experimental autoimmune encephalomyelitis. moreover the increased levels of tgf-ill and other tgf-13 isoforms found in several disease states associated with immunosuppression such as different forms of malignancy, chronic degenerative diseases and aids implicate the involvement of tgt~-i~ in the pathogenesis of some diseases. hopefully, well designed clinical trials will define the potential roles of the tgf-[3 family of proteins in the treatment of diseases of man. patient development of systemic inflammatory response syndrome (sis) after severe trauma is accompanied by a wide range of immune aberrations and altered cytokine production. in particular, t lymphocyte proliferation and ifn, t production is suppressed while monocyte (mo) tnfc~, il-6, pge2 and tgf~ production is massively increased concomitant to decreased antigen presenting capacity. recently, two new cytokines, il-10 and il-12, have been characterized as critical in regulation of mo tnfa and il-6, as well as in induction of t lymphocyte proliferation and ifn 7 production. in this study, peripheral blood leukocytes (pbl) from severe trauma patients (injury severity score >35) were analyzed for their il-10 levels, their in vitro proliferation to mitogen (pha) and their response after il-12 addition. since il-10 produced either by mo or by t lymphocytes can depress m~ antigen presenting capacity, inhibit t cell ifn,/production and directly diminish t cell proliferation, it might be suggested that immunosuppressed patients' mo and/or t lymphocytes would have increased il-10 levels. increased patient il-10 production might also be resulting from the high levels of tnfa a known stimulator of il-10. conversely, since il-12 augments mo antigenpresenting capacity, thl induction and proliferation, post-trauma leukocytes might be il-12 deficient. pbl of trauma patients were compared to normals' pbl, either unstimulated or ptta induced, and their levels of il-10 found to be dramatically and significantly reduced. patients' isolated m~, either stimulated with the bacterial cell wall analogue, mdp, or unstimulated, also had depressed il-10 production concomitant to elevated tnfa production when compared to normals' mo. mechanisms for the depressed patients' mo il-10 were explored. increases in tgf[3 may have partially contributed to the patients' depressed il-10 level, but elevated pge2 had no effect. addition of il-12 to patients' pbl significantly increased their mitogen responses. these data imply that sis is characterized by disruption in the interactions between mci and t lymphocytes so that patients' m~i produce excesses of some mediators (tnfa, il-6, pge2) and a dearth of other monokines (il-12, il-io). t lymphocytes are not activated and, therefore, unable to function in both immune defense and monocyte regulation. it is known that lge receptor-mediated or ca-ionophore-induced activation of mouse bone marrow-derived mast cells (13mmc) may result in the production of different cytokines including the interleukins (il) 3, 4, 5, and 6 as well as gm-csf and tnf-a. in the present study we analyzed the effects of exogeneously applied pro-inflammatory cytokines (il-1, 1l-6, tnf-c 0 as well as various mast cell growth factors (il-3, il-4, il-9, il-10, ngf, kl (kit ligand)) on cytokine production in primary mouse bmmc using a standard activation protocol (lxl06 bmmc/ml; ll.um ionomycin; 24-48h). the actixdties of bmmc supernatants were assessed in specific biological (il-3, il-4 il-6, 1l-9) and/or elisa assays (il-5, il-9). here we show that homogeneous populations of bmmc (>97%alcian blue+/safranln-; in vitro age: 4 weeks) generated in the presence of recombinant (r) rail-3 from normal balb/c mice produced modest amounts of 1l-6 and low or undetectable levels of il-3, -5, and -9 after induction with lp.m ionomycin only. however, a dramatic increase (5-to 10-fold) of these cytokine activities was noted, when in addition to ionomycin also human (11) rll-la was provided during the induction period. this il-1 effect was dose dependent with a maximgm at 2-10 u/ml hrll-la and specific, as pre-incubation (lh) of bmmc with 20 ng/ml hrll-1 receptor antagonist abolished the action of 2 u/ml hrll-lcc similar effects were noted with hrll-lg or rurll-lb (lng/ml, respectively), but not with rhll-6 or rmtnf-~. both mrll-10 and hrll-10 substantially enhanced ionomycin-induced 1l-6 production of bmmc in the absence or presence of il-1. il-10 significantly enhanced il-6 and il-9 production while decreasing il-3 activities to abont 50-90% of control levels, when il-i0 was provided in the presence of il-l/ionomycin. a monoclonal anti-nfil-t0 antibody (ascites 1:200) abrogated the effects of mrll-10. other mast cell-active cy~okines (]1,-3, il-4, 1l-9, ngf, or kl) added to ionomycia-or 1l-1/ionomycin-treated bmmc had no major effects on cytokine production. il-1 and il-i0 did not induce significant cytokine release in the absence of ionomycin suggesting tlmt cadependent signalling was required. at doses of 10"6m, dexamethasone, corticosterone, or hydrocortisone almost completely abolished ionomycin/il-1/ll-10induced cytokine production. the inducer cocktails per se did not interfere with the cytokine bio-assays. in case of il-9 inducibility of this cytokine in bmmc was confirmed at the mrna level by northern blot analysis. hence our data show that activated mast cells are a source of il-9 previously recognized as a product of th2type lymphocytes only. moreover, our study reveals novel functional roles for i-l-i, il-10, and ghicecorticoids in the regulation of cytoldne production in mast ceils. accumulating data suggests that cytokines, peptides involved in regulation of both physiological and pathological immunological responses, predominantly are produced at the local site of antigen stimulation. a new method was used to detect cytokine-producing cells in haman tissue at the protein level. single-cell production of 17 different httman cytokines, ilia, ill [3, illra, il2, il3, il4, il5, il6, ils, ill0, gm-csf, tnfa, ifn 7 and tgf[31.3, was identified by indirect immunohistochemical staining procedures and use of carefully selected cytokine-specific mab's. frozen sections were fixed with 4 % paraformaldehyde and permeabilized by 0.1% saponin treatment, eluting cholesterol from the membranes. the intracellular presence of all cytokines except ill, illra (late) and tfg[31_3, could be demonstrated by a characteristic perinuclear configuration in producer cells. in addition, the immunoreactivity extended over a large extracellular area encompassing the producer cell. a localization of the cytokine to the golgi-organelle was established by use of two culour staining including a haman golgi complex specific mab. this staining pattern was only evident in producer cells because injection of recombinant human cytgkines into the tissue caused a membraneous and extracellular staining pattern. both the extra-and the intracellular types of staining reaction could, however, be blocked by preincubating the cytokine specific mab with pure human interleukins. oxygen radicals (or) directly induce lipid peroxidation, indirectly they trigger adhesion and activation of pmn leukocytes. we investigated whether or also lead to a release of acute-phase response cytokins such as tnf-alpha, il-i beta or il-6 in whole blood cultures to maintain the induced inflammatory reaction. methods: blood samples from healthy volunteers (n=10) were incubated at 37°c. or were produced by the xanthine oxidase (xo)/ hypoxanthine (hx) system. after 0,30,60,120,240 and 360 minutes plasma levels of tnf-alpha, il-i beta and il-6 were determined with elisa kits. results: under the influence of or tnf-alpha plasma levels increased from 0,5 pg/ml at 0min to 14pg/ml, 236pg/ml, 343pg/ml after 60, 120 and 240 min. il-ibeta (0,3pg/ml, 0,6pg/ml, 1,5pg/ml, 81pg/ml and 169pg/ml after 0,60,120,240 and 360min) and il-6 (0,2pg/ml, l,lpg/ml, 4,1pg/ml, 58pg/ml and 72,9pg/ml after 0,60,120,240 and 360min) plasma levels were increased 60min later than tnf-alpha. summary: these data suggest that or do not only play an important role in initial accumulation and activation of pmn leukocytes but also lead to a stimulation of monocytes to produce the acute phase reaction cytokins tnf-alpha, il-i beta and il-6 to maintain and strengthen the inflammatory reaction. department of general surgery, steinhsvelstr.9, 89070 ulm, germany jan k. horn md, greg a. hamon md, robert h. mulloy md, greg chen bs, rebecca chow bs, and christof birkenmaier md. transforming growth factor-i~l (tgf-131) is released from inflammatory ceils following injury and in sepsis. in vitro experiments have confirmed that low concentrations of tgf-131 (0.1-1.0 ng/ml) are chemoattractive for monocytes, whereas higher levels of tgf-131 (>5.0 ng/ml) potentiate production of the immunedepressive prostaglandin e 2. other investigators have shown that tgf-]31 can cause the appearance of cd16 (fc immunoglobulin receptor) on monocytes exposed to 10 ng/ml of tgf-[~i for 24 hours. monocytes also express on their surface a glycoprotein that binds complexes of lipopolysaceharide (lps) and lpsbinding protein (lbp). such binding is associated with generation of proinflammatory cytokines such as tumor necrosis factor alpha. we have shown that cd14 is depressed in septic patients and therefore we hypothesized that tgf-131 could account for the down-regulation of cd14 observed in these individuals. we incubated normal human monocytes with platelet-derived tgf-[31 for 2 and 24 hours at 37°c and examined ceils for cd14 and cd16 expression using flow cytometry after immunnfluoreseent staining with appropriate monoclonal antibodies. monocytes were selected on the by usual criteria for size and granularity. non-viable ceils were excluded with the use of propidium iodide. two populations of monocytes could be found afcer incubation at 37°c alone. one displaying high density of cd14 had increased fluorescence over the homogeneous expression of cd14 in cells maintained at 4°c (baseline). the other population displayed decreased cd14 expression relative to the baseline cells. tgf-i~i (10-50 ng/ml) caused a shift of ceils from the high density into the low density cd14 population. this trend was observed within 2 hours of incubation and was complete by 24 hours. we observed a net decrease in cd14 expression 0f32% for all subjects studied (p<0.05 vs controls). phorbol myristate acetate (100 ng/ml) also caused down-regulation of cd14 to a similar degree as tfg-i~i. we also confirmed that monocytes could be induced to express cd16 after incubation with tgf-131 (10 ng/ml) for 24 hours. these studies demonstrate that monocytes incubated with immunodepressive levels of regulation of cd14 by tgf-131 deplete their surface expression of cd14 while generating cd16. this down-regulation of cd14 by tgf-131 correlates with our clinical observations of lower cd14 expression on monocytes obtained from septic patients. for over 25 years, activated t lymphocytes have been considered to be the cellular source of mif. we recently isolated and cloned the murine homolog of mif after identifying the specific secretion of this protein by lpsstimulated pituitary cells in vitro and in vivo. however, further experiments showed that mif protein is detectable both in t-cell deficient (nude) and hypophyseetomized mice, suggesting that yet additional cell types may produce mif in vivo. since monocytes/macrophages are a major source of the cytokines that appear in response to lps administration, we examined the possibility that mif also is expressed in cells of the monocyte/macrophage lineage. we found that mif is expressed constitutively in the murine macrophage-line raw264.7 and in thioglycollate-elicited peritoneal macrophages. significant amounts of mif mrna (rt-pcr) and protein (western blotting) were observed in cell lysates. in raw 264.7 cells, mif secretion was induced by as little as 10 pg/ml of lps (e.coli 011 l:b4), peaked at 1 ng/ml, but was not detectable at lps concentrations >1 txg/ml. similar data were obtained with elicited macrophages, but higher lps concentrations were required, unless the cells had been preincubated with ifn 7. production of mif by lps-stimulated (l ng/ml) macrophages peaked at 12 hr. expression ofmif mrna and tnf mrna by lps-stimulated raw 264.7 macrophages was investigated by rt-pcr. as expected tnf mrna expression increased over the range of lps concentrations (1 pg/ml to 1 p_g/ml). in contrast, levels of mif mrna correlated inversely with lps concentration. by competitive pcr, mif mrna was observed to increase approximately 2-fold after lps induction (100 pg/ml). mif secretion also was induced by tnfoc (1 ng/ml) and ifn? (10 iu/ml), but not by il-113 and il-6 (up to 10 ng/ml). lps and ifn 7 had additive effects in inducing mif secretion. in separate experiments, macrophages stimulated with recombinant mouse mif (1 gg/ml) were found to secrete bioactive tnf~ (>750 pg/ml by l929 cytotoxicity). we conclude that the macrophage is an important albeit overlooked cellular source of mif in vivo. mif secretion is induced by lps, tnfc~ and ifn?. mif also stimulates macrophages to secrete tnf. taken together with previous observations that anti-mif antibody protects against lethal endotoxemia, these data implicate mif as a critical mediator of inflammation and septic shock. inflammation is characterized by an exacerbation of proinflammatory cytokine production. cytokines such as il-4, il-10, and tgf8, have been identified as anti-inflammatory mediators thanks to their ability to down regulate the production of il-1, il-6, il-8, tnfc~ by activated monocytes / macrophages. however, other cells, including polymorphonuclear cells (pmn) do contribute to the release of pro-inflammatory cytokines. we investigated the capacity of the so-called anti-inflammatory cytokines to control the release of il-8 by activated neutrophils. human pmn were purified following glucose-dextran sedimentation and ficoli-hypaque centrifugation. the cells were cultured at 37°c for 24h in the absence or presence of lipopolysaccharide (lps) or tnfa. il-8 release was measured in the supernatants using a specific elisa. among tested cytokines, il-10 was the most efficient inhibitor of il-8 production by lps-activated pmn. il-4 was also active, whereas no down regulation was noticed with tgfp~i. when tnfa was used as a triggering agent, none of the cytokine could prevent il-8 production. northern analysis are under investigation to precise the level of the il-4-and il-10-induced inhibition of il-8 production by pmn. our data illustrate that il-10 and il-4 possess the capacity to down regulate the production of il-8 by both monocytes and pmn, whereas tgfb has a more limited inhibitory activity. ciliary neurotrophic factor (cntf), a member of the il-6 superfamily, has recently been shown to promote axonal growth and neuronal healing. cntf production is also increased during neuronal and muscle damage, associated with soft tissue injury or trauma. we postulated that production of cntf may explain the loss of skeletal muscm protein that occurs in inflammation. 40 female, wistar (175-200 gm) rats received either 250 or 25 pg/kg bw s.c. injections of recombinant rat cntf for seven days, or received sham injections and were freely-fed. additional animals were pretreated with 10 mg/kg ibuprofen lp prior to 250 pg/kg bw cntf. rats treated with 250 ,ug/kg bw cntf lost 6.8_+0.4 gms bw as compared to freely-fed controls which gained 17.0_+3.1 gms (p<o.001 by anova and lsd), and ibuprofen treatment had no effect on cntf-induced weight loss (-5.8_+0.7). animals pair fed to the high dose cntf gained body weight (10.2-+3.5), although weight gain was less than seen in freely-fed controls. rats treated with 25 pg/kg bw had reduced body weight gain (+7.8+_4.4 gin). food intake was also reduced by 29% in 250 pg/kg cntf (from 23.7+_0.7 gm/day to 16.8_+0.6 gm/day; p<o.05) and was also unaffected by prior ibuprofen treatment (18.4+-0.6 gm/day). despite the loss in body weight noted in the high dose cntf animals, there was no appreciable hepatic acute phase response, since liver weight (8.6+0,1 gms vs. 9.4_+0.3 gins), total protein (54.5+-2.9 gms/l vs 44.8+-3.6 gins/l) and albumin (29.0+1.5 gms/l vs. 26.0+_1.6 gms/l) were not different between cntf and freely fed animals. we conclude that cntf causes anorexia, and increases body weight loss in excess of the associated anorexia. unlike the cachexia associated with il-1 and tnf infusions, which is associated with an acute phase response, cntf acts predominantly on food intake and carcass weight through a prostaglandin-independent mechanism and has only minimal acute phase inducing properties, n. joseph espat, md. university of florida, department of surgery, j-box 100286, gainesville, fl. 32610. of cytokines by neurotrophic factors, the neuroendocrine system and phenotiazines. cytokines, including tnf and il-1, have a pathogenetic role in various diseases related to infection and inflammation. the action and/or production of cytokines can be regulated by drugs or endogenous mechanisms that involve the cetral nervous system (cns) we found that the antipsychotic chlorpromazine (cpz) potently inhibits tnf production and protects mice against endotoxic shock. cpz also inhibits brain tnf production in mice intracerebrally injected with endotoxin, whereas cpz analogs that do not cross the blood-brain barrier inhibit only perypheral, not brain, tnf. tnf production and susceptibility to the toxicity of endotoxin, tnf and il-1 are increased in adrenalectomized mice, indicating that endogenous corticosterone (cs) controls cytokine production and toxicity, in a feedback fashion since endotoxin and cytokines increase serum cs. increased tnf production was also observed in mice where the hypothalamus-pituitary-adrenal axis was blocked by chronic administration of dexamethasone, following a two-day washout. administration of cytokines acting through the gp130 signal transd,uction protein, including il-6 and ciliary neurotrophic factor, cntf potentiated il-l-induced elevation of cs. il-6 and cntf also induced hepatic acute-phase proteins. thus il-6 and cntf might have antiinflammatory activity, by potentiating the feedback responses of the neuroendocrine axis. these data indicate how complex can be the interregulatory relationships between the brain and the immune system, how they can be used to pharmacomodulate cytokine action and how their disregulation might exacerbate il-1-and tnf-mediated pathologies. lipopolysaccharide (lps) constitutes a well established activator of monocytes/ macrophages (mizi) and murine b lymphocytes. the hydrophobic part of lps, termed lipid a, represents the endotoxic principle of lps. we now demonstrate that lps is also capable of inducing proliferation of human t cells at a dose of 10 to 1000 pg/ml. the specificity of this stimulation was analysed by the following experiments: i) the synthetic hexaacyl escherichia coiltype lipid a (compound 506) also stimulated human t cells; ii) the synthetic tetraacyl lipid a precursor (compound 406) or the 1phosphono-oxyethyl analogue pe-4, structures known to antagonize lps-induced monokine production in human mz, also inhibited lpsinduced t-cell proliferation. the t-cell proliferation expressed the following features: i) cd4 ÷-as welt as cd8 ÷ t cells proliferate in response to lps, ii) limiting dilution analyses reveal that the frequency of responding cells was between 1/500 to 1/1000 cells; iii) purified t cells alone cannot be stimulated by lps, but need the help of monocytes. this help cannot be replaced by b cells or fixed monocytes. furthermore, for activation a direct cell-to-cell contact between t cells and monocyte is neccessary; iv) t cells stimulated with lps express mrna for il-2 and ifnf, but not for il-4 or il-5 (determined by pcr). in supernatants, ifnf was detectable (elisa); v) lps-activated cd4 ÷ as well as cd8 + t cells express cd25, the high affinity il-2 receptor. our results indicate that in contrast to previous reports human t cells respond to lps with il-2 receptor expression, lymphokine production, and proliferation. the reactive cells appear to belong to the t,1 cell type, the finding that human t cells can be activated by lps is of important relevance for the understanding of the pathomechanisms of infections by gramnegative bacteria. this may lead to new strategies for the therapy of sepsis. we have recently shown that human eosinophils produce mrna for interleukin (il)-8 when stimulated with calcium ionophore (eur. j. immunol. 23 (1993), 956). we now present evidence that human eosinophils contain preformed il-8 protein although they do not express mrna for il-8 as measured by rt-pcr. il-8 protein expression was determined using specific anti-human il-8 monoclonal antibodies by western blotting, facs analysis and immunohistochemistry. moreover, preformed il-8 was re/eased into supernatant by 99% pure eosinophils after priming with gm-csf or il-5 and subsequent 25-min stimulation with paf, rantes, ionomycin or pma, however not with il-1 or il-2, as measured by elisa. this observation implies that activation of protein kinase c and/or intracellular calcium mobilization are necessary events for il-8 release in human eosinophils. the determined amounts of preformed il-8 protein was higher in patients with asthma compared to normal individuals suggesting a role for eosinonhi! derived il-8 in asthma. since the eosinopnit is the ,,r~:=z~mmant cell in the asthmatic airways, we determined il-8 concentrations in bronchoaiveolar lavage (bal. ~ids from normal !i~.~ijvtduals and asthmatic patients. indeed, il-8 concentrations in bals from both groups were similar to those seen in the supernatants released by activated eosinophils. the role for il-8 in asthma remains to be determined. based on a well established rat model named activity-induced ulceration (asu) or activity based anorexia (aba), we have developed a rat model for chronic stress. male rats were exposed to a feeding schedule in order to reduce body weight by 20-30% within 14 days. while one group of rats was kept sedentary, the other had access to a running wheel. food restricted rats allowed to exercise developed highly increased activity as compared to ad libitum fed rats in wheels (ca. 15 versus 2 kin/day). they developed elevated plasma corticosterone levels (30_+5gg/dl) at their circadian peak, increased adrenal weight and decreased thymus and spleen weight as compared to control rats and weight matched sedentary rats, establishing this paradigm as a model of chronic stress. no differences in plasma acth were seen among the various groups, suggesting increased sensitivity of the adrenal glands to acth. the effect of chronic stress accompanied by hypercorticism on baseline and endotoxin-induced cytokine levels was studied in this model. under baseline conditions,splenic il-1 a was suppressed in both food-restricted groups. this was more pronounced in rats with access to a running wheel. il-6 and tnf activity in plasma was not affected. after administration of 1001.tg/kg lipopolysaccharide (lps), tnf activity in plasma was suppressed by food restriction but there were no differences between sedentary or exercising rats. in addition, no differences were found among groups for corticosterone, spleen il-lc~ and plasma il-6 activity. we conclude, that the food restriction-induced hyperactive rat model is a useful model for studies on the effects of chronic stress. in this model, under basal conditions il-le~ in tissues may be subject to downregulation by glucocorticoids, whereas after lps-stimulation of cytokine production only tnf activity is regulated by fast acting feedback loop between cytokines and the hypothalamo-pituitary-adrenal axis (hpa-axis). this is supported by strong correlations between plasma tnf activity and corticosterone in stressed rats. cytokine antagonists modulate cytokine actions and it appears that a balance between production of cytokines and cytokine antagonists is important to control of host responses. recent observations show that during myocardial infarction there is an increase in circulating cytokines such as tumor necrosis factor (tnf) and interleukin 6 (il-6). we tested the idea that compensatory increases in cytokine antagonists likewise occur and investigated changes in plasma concentrations of tnf and interleukin i (il-1) and those of their specific antagonists interleukin-1 receptor antagonist (il-1 ra) and soluble tumor necrosis factor receptor type i (s tnf r-i). we also measured plasma concentrations of ~-melanocyte-stimulating hormone (~-msh), an endogenous neuropeptide that occurs in pituitary, brain, skin, gut and other tissues. when administered to laboratory animals, this peptide has potent antiinflammatory and antipyretic activity, perhaps by mimicking an endogenous modulatory influence on cytokine actions. samples were obtained from 20 patients with acute myocardial infarction at presentation in the coronary unit, every three hours for 24 hours, and daily thereafter until discharge. we found elevations in the plasma cytokines il-1 and tnf only in a proportion of subjects whereas cytokine antagonists c~-msh, il-lra, and stnfr were elevated in all patients, o~-msh was elevated at presentation but it decreased progressively in successive tests, reaching normal values within 24 hr whereas stnfr and 1l-lra peaked at 3-6 hr or later. there were significant correlations between plasma concentrations of cytokine antagonists and enzymes released from injured myocardium, including creatine kinase (ck), aspartate serum transferase (ast), and lactate dehydrogenase (ldh). the data show that cytokine antagonists increase in the circulation of patients with acute myocardial infarction likely to modulate prninflammatory effects of cytokines. objects of the study: interleukin-6 (il-6) is a multifunctionai cytokine known to be involved in important homeostatic processes. il-6 levels are increased in many pathological situations, and correlates with disease activity and patient outcome. to exert its effect il-6 binds to its receptor on the membrane of its target cell. this receptor is also present in a soluble form in plasma and in urine. in this study we quantitatively measured the availability of soluble il-6 receptor (sll-6r) in biological fluids in healthy volunteers. materials and methods: blood, urine, cerebrospinal fluid (csf), and synovial fluid (sf) are obtained from healthy volunteers, sil-6r and il-6 are measured using elisa's. both elisa's are tested for interference of sil-6r mid il-6. the effect of sll-6r in the il-6 bio-assay (b9) is tested. results: baseline levels are (mean + sd): 76.6 -+ 19.3 ng/ml in serum, 3.7 -+ 1.3 ng/ml in urine, 1.64 _+ 0.4 ng/ml in csf and 11.6 + 3.3 ng/ml in sf. there is no interference of sll-6r in the il-6 elisa or bio-assay and no interference of 1l-6 in the sil-6r elisa. conclusions: in all investigated biological fluids sll-6r can be found. these data provide baseline values for investigations concerning pathological situations. both elisa's described are useful tools to do these investigations. trauma-associated immune aberrations coincide with augmented release of immunoregulatory and proinflammatory cytokines. the present study examines the effect of thermal trauma on the capacity for specific (receptormediated) response of lymphoid cells to interleukin 1 (il 1) and to turnout necrosis factor ~x (tnfc0. methods. bum patients (n=24, 25->75 % total body surface area) were studied weekly up to 42 days post-injury. the limulus amoebocyte lysate (lal) test was used to measure plasma endotoxin levels. the percentage of il 1~-and tnfcz-binding t(cd 5) lymphocytes was assessed by flow cytometry analysis. levels of il 1 receptor antagonist (il lra) in patients' plasma and cultures of peripheral blood ceils (pbc) were determined by immunoassay. results. plasma endotoxin concentrations were significantly (p<0.05) increased up to 3 weeks post-bum (means 3.5+1 in non-surviving and 2.6+ 0.6 u/ml in surviving patients vs < 1u/ml in the control). within 2 weeks of bum, the percentage oft ceils expressing receptors for tnfa and il 1[~ constitutively was elevated (by 10-15 fold). in contrast, the capacity for de novo receptor expression by activated pbc was reduced. serum levels of il ira were significantly increased (range 2.5-40x10 j pg/ml vs <0.2x10 j pg/ml in the control). in all patients, high concentrations of il lm were released spontaneously in unstimulated cultures of adherent ceils (range 20-30x10 -3 pg/ml vs 4-8x10j pg/ml in the control). however, its secretion was decreased in lps-stimulated parallel preparations. conclusions. in the bum patient, susceptibility to the immunoregulatory effect of tnfcz and tl 1~ may be modulated by infection-related products. alterations in the capacity for receptor expression and secretion of 1l lra may affect il 1-regulated biological responses including specific immune reactions. while studies suggest that il-10 is an important lymphokine involved in cell-mediated immunity, little is known about this mediator's role in hem-induced immunesuppression. our aims, therefore, were to determine: i) if il-10 contributes to depressed t-cell responses seen following hem; and 2) how other agents, known to play a role in hem, effect il-10 release. to study this, c3h/hen mice were bled to and maintained at a map of 35 mmhg for 1 h and then adequately resuscitated. mice were killed 2 h post-hem to obtain splenic t-cells (nylon-wool purified). il-10's immunosuppressant role was demonstrated by the ability of monoclenal antibody (mab) to il-10 to markedly improve the t-cell proliferative response [2.5 #g the marked increase in capacity of t-cells from hem mice to produce il-10 was significantly reduced by treatment with either ibu or mabs. since ibu, tgf-~, as well as il-6 are all reported to directly/indirectly influence prostanoid synthesis, this implies that eicosanoids play a major role in inducing il-10 release by t-cells following hem which depresses t-cell function. the mechanisms underlying immunosuppression induced by thermal injury and alcohol ingestion are in part due to cytokine dysregulatinn. il-10 down-regulates production of eytokines by maerophages and may be an important regulator of the initiation of the immune response. il-10 has also been demonstrated to inhibit the production of no by macrophages. this study examined the alterations in eytokine production and effect of inhibition of no production on immunologic function in a routine thermal injury model. methods: balb/c mice (n=40) were randomized to 4 groups: saline-sham(ns-sham), alcohol-sham(etoh-sham), ns-bum, etoh-bum. animals received 20% etoh or ns daily for 14 days by gavage. a 30% full thickness bum was induced 4 hrs after the last dose of etoh or ns. animals were resuscitated, then sacrificed 4 days post bum. splenic lymphocytes were cultured for 5 days with lps, and lps with two concentrations of n-monomethyl-l-arginine, a nitric oxide inhibitor (l-nmma 2.5ug/ml, 10ug/ml). splenocyte production of il-10, interferon-gamma, il-2, pge2 were measured, and lymphocyte proliferative response examined. results: il-10 production was significantly suppressed in thermal injury. exogenous l-nmma normalized the suppression of 11.-10 in a dose-dependent manner, indicating nitric oxide may modulate il-10 and interferon-gamma production in thermal injury. il-10 production is normal in etoh-burn animals. conclusion: il-10 and interferon-gamma production is altered in this murine thermal injury model, and may contribute to this injury-induced immunosuppression. inhibition of no synthesis normalizes il-10 production and should be investigated further as an immanomodalator in thermal injury. surgery, infection and inflammation results in the production of pro-inflammatory cytokines which mediate metabolic and immunologic host responses. the aim of this study was to characterise the elaboration of cytokine release following a variety of surgical procedures. twenty one patients undergoing elective intermediate, hip, knee and major gastrointestinal surgery were studied. levels of interleukin-1 (i1-1), interleukin-6 (i1-6), the interleukin-1 receptor antagonist (i1-1ra) and the acute phase c-reactive protein (crp) were measured in bloods drawn 0, 1, 2, 4, 6, 12, 24 and 48 hours following operation. a portion of the results are shown (mean -+ sem). 135+37 1738-+843 145_+24 one and two factor anova; *p<0.0002, #p<0.0001, §p<0.0009, ¶p<0.0014, for differences between groups i1-1 was not detected at any time point. both ii-ira and i1-6 increased after surgery. maximum responses occurred following major git and hip surgery, minimal responses were seen after intermediate and knee surgery. ii-ira levels increased within two hours and remained elevated for 24 hours; the b-ira increase was a thousand fold greater than the rise in i1-6 levels. i1-6 levels increased up to 24 hours after surgery. crp levels reflected maximum ii-ira and i1-6 levels (r2=.676, p<0.0001 and r2=.282, p<0.04 respectively). high ii-1ra and i1-6 levels reflect major surgery, however the ii-ira response is more rapid and of greater magnitude. the strong i1-1ra correlation with crp may indicate that this regulatory cytokine is itself a mediator of host responses to surgery. dept. of surgery, meath/adelaide hospitals, heytesbury st., dublin 8, ireland. change of il-6 and soluble il-6 receptor levels after surgery s. hisano, k. sakamoto, s. mita, t. ishiko, m. ogawa [objectives] under surgical stress, il-6 plays a main role in producing acute phase proteins and contributes to host defense mechanism. soluble il-6 receptor (sll-6r) is considered to be agonistic to il-6, unlike other soluble type receptors of cytokines. here we measured il-6 and sll-6r levels in the serum and drain fluid from surgical field in order to investigate the changes of il-6 and sll-6r after surgery and their origins. [materials and methods] serum and drain fluid samples from 21 cases (6 of esophagectomy and 15 of gastrectomy ) were serially collected before and after surgery. il-6 and sll-6r levels were measured by elisa. [results] (1) serum il-6 : all cases reached the maximum level on pod-l, more precisely 3-6 hours after operation. (2) il-6 in the drain : maximal il-6 levels in the drain were recognized 3-6 hours after operation, at almost the same time as serum il-6. furthermore the il-6 values in the drain were much higher, about 100 times, than those in serum. (3) sll-6r in the serum : all cases reached minimum levels 3-12 hours after operation and recovered to the preoperative levels a few days later (decrease ratio : 43.9+5.6~,, range : 9-84~'). (4) sll-6r in the drain : sll-6r levels in the drain showed almost the same value and change as serum sll-6r. [conclusions] (1) il-6 is produced from the cells gathering around operative fields whereas sll-6r is considered to be produced in the cells which do not gather around the operative fields. (2) there may be a mechanism that down-regulates sll-6r in the early stage of surgery. [objectives] il-6 plays an important role in host defense in the early stage after surgery. in the present study, we examined changes in il-6 concentration after major thoracoabdominal surgery and elucidated the effect of surgical trauma and factors influencing postoperative elevation of serum il-6. [materials and methods] thirty-eight patients undergoing elective surgery of the thoracoabdomen were classified into 6 groups according to the location of the operation. bloods and drain fluids were serially obtained and samples were frozen until measured, keukocytes were simultaneously collected for northern blot analysis. concentration of il-6 was measured by elisa and il-6 mrna was detected by northern blotting after total rna was extracted by the acid guanidium phenol chloroform method. [results] (1) serum il-6 levels reached the maximum concentration on the 1st postoperative day in all patients. (2) the il-6 peak was significantly correlated with surgical trauma as defined by the operation length and the volume of blood loss during operation (r=0.554, p<0.01, r=0.427, p<0.01, respectively). (3) the peak concentration of serum il-6 in patients undergoing esophagectomy was significantly higher than in those undergoing pancreaticoduodenectomy (p<0.05), despite a similar degree of surgical trauma. (4) peak 1l-6 concentration observed in a patient who underwent esophagectomy was about 100 fold greater in the drain fluid of thorax than in the peripheral blood. (5) il-6 mrna was demonstrated in leukocytes from thoracic and abdominal exudate at 6, 24 and 48 hours after surgery. in contrast, il-6 mrna could not be detected in leukocytes from the peripheral blood. [conclusion] il-6 is mainly produced in the operative field and subsequently enter the peripheral blood to induce cytokinemia. the operation length, volume of blood loss and thoracotomy are factors influencing the concentration of cytokine in the blood. zaragoza spain age may be an important factor influencing the function of immunocompeteut cells releasing cytokines after both accidental and surgical trauma the aim of the present paper is to ascertain if patients (pts) over 60 years old show a different serum level cytokine pattern than pts under 60 after a standard surgical procedure considered as a "medium strength trauma". patients and methods: 33 pts(22 females 11 males)with gallstone disease were perspectively studied, pts were allotted in two groups: gr.a:17 pts under 60 years(mean age:49.5+-7)gr.b:16 pts over 60 years(mean age:65.5_+5). all pts underwent cholecystectomy and cholangiography. 6 pts in gr.a and 7 pts in gr. b underwent common duct exploration. 1 spbintercctomy was performed in each group. on the day of surgery (pre) and on the 1st and 7th postoperative day(leo, 7po) : percentages of cd19, cd5, cd4, cd8 and cd16 cells we measured by means of flow cytometry using moab. and levels of il-1, il-4, il-6 and tnf "in vivo" by elisa using moab. results: ere: cd16% was 5.3_+3 in gr.a and 7. 5 objectives of the study. after surgery for esophageal cancer multiple organ damage has been reported to be caused by polymorphonuclear leukocyte (pmn)-mediated injury. we measured serum granulocyte colony-stimulating factor (g-csf) and interleukin 8 (il-8) levels to determine a role of g-csf and il-8 in pmn function after surgery for esophageal cancer. materials and methods. peripheral pmn counts, peripheral pmn chemiluminescence, serum g-csf levels, and serum il-8 levels were measured before and after surgery in 12 patients with esophageal cancer (ec), and 12 patients of gastric cancer (gc). esophagectomy with thoracotomy and laparotomy were performed for patients with ec, while subtotal gastrectomy with laparotomy were performed for patients with gc. results. peripheral pmn counts (p<0.01) and peripheral pmn chemiluminescence (p<0.05) of patients with ec were significantly decreased compared to those of patients with gc at 4 and 8 hours after surgery. serum g-csf levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4 and 8 hours after surgery. serum il-8 levels of patients with ec were significantly (p<0.01) increased compared to those of patients with gc at 4, 8 and 24 hours after surgery. significant inverse correlations (p<0.0l) between peripheral pmn count and serum g-csf and il-8 levels were seen at 4 hours after surgery. conclusion. these results suggest that many circulating pmns, which are excessively activated by g-csf and il-8, may adhere to the endotherial cells and then migrate into the tissues, and cause multiple organ damage after surgery for esophageal cancer. immunnogical changes in patients with severe brain trauma receive increasing attention since morbidity and mortality ere still high. interleukin-6 (il-6) was previously detected in the cerebrospinal fluid (csf) during different pathologies of the nervous system (1, 2, 3). in our study we monitored il-6 and nerve growth factor (ngf) production in the csf after human brain trauma. since astrocytes within the brain constitute one of the major cell type contributing to the inflammatory response through the release of cytokines and other factors after injury, we investigated the functional relationship of il-6 and ngf on a single cell niveau using cultured astrocytes. methods csf was obtained from patients with severe brain injury (glasgow coma score (gcs) <8 and ct abnormatities or gcs <8 over 6 hours) after implantation of intraventricular icp monitoring device for therapeutic purpose and collected over 24 hours csf and serum. il-6 and ngf were assayed by elisa. astrocytes were isolated from neonatal mouse brain as described (4) . ngf production by cultured astrocytes was measured by elisa in the presence of csf, il-6 and il-6 antibody. astrocyte migration was tested in a chemstaxis chamber. results 17 head trauma patients were included in this study (approved by the university hospital medical ethics board) and the csf was obtained through intraventricular catheters. high levels of il-6 were detected in the csf of these patients when compared to serum during the first days after brain trauma. furthermore ngf could be found inside the intracerebral compartment. csf containing high levels of il-6 could stimulate ngf production in cultured astrocytes. this effect could be [nhibited partially by il-6 antibodies, purified il-6 exposed to cultured astrocytes in vitro, stimulated the migratory activity of these cells in a dose response fashion. il-6 was found in the csf of brain injured patients, suggesting a role for this cytokine in the pathophysiology of brain injury. since astrocytes are involved in maintaining the homeostasis of the brain, we further investigated the possible role o1 il-6 on astrocyte functions, il-6 promoted ngf production in vivo and in vitro, thus contributing to neuronal cell survival and regeneration. furthermore il-6 stimulated astrocyte migration in a dose response fashion, potentially contributing to astrocytosis following brain injury and inflammation, these results show that il-6 represents a key cytokine in traumatic human brain injury with possible systemic effects, which are at preserlt under investigation. we studied a) the role of tnf and b) the therapeutic effect of a mab to tnf with regard to haemorrhagic shock (hs) related ,pathophysiologic alterations and mortality in rats. method: a prolonged hs was induced by bleeding to a blood pressure of 30-35 mmhg for 180 pin followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. 15 animals received a bolus dose (20 mg/kg) of tnf mab (celltech, berkshire, uk) at 15 min after resuscitation (tn3). the control group (n = 15) was treated similar to the tn3 group but received ringer's lactate (con). results: at 180 min the prolonged hs resulted in a metabolic acidosis indicated by a significant decrease of blood ph (7.16 + 0.04), hco3-(16.3 ___ 2.0 mm), and base excess (-12.5 + 1.9 ram) values with pco2 (47.9 + 7.7 mmhg) and po2 (136.2 + 20.1 mmhg) in the tn3 with no difference to the con group. immediately after resuscitation (230 min) plasma endotoxin levels were found to be increased in both groups (48.8 + 73.3 in tn3 vs 30.1 _ 25.7 pg/ml in con group) . prior to the treatment with tnf mab (230 min) there was also no difference between plasma tnf levels of the two groups (42.1 + 60.7 in tn3 vs 60 + 141.8 pg/ml in con group). treatment with the tnf mab at 65 rain post-hs improved the 48hour survival rate to 73.3 % as compared to 26.7 % in the control group. macropathologic evaluations revealed frequency of intestinal bleeding in oniy 2 animals in the tn3 vs 11 in the con group. no bleeding in the kidneys was found in the tn3 but in 4 rats in the con group. the significant increase in lung wet weight observed in non-survivors in the con (n = 11) was prevented in animals which died in the tn3 (n = 4) group ((9.3 +_ 2.4 vs 6.3 +_ 1.0 g/kg). conclusion: our data suggest that tnf formation induced by hs in rats is an important mediator for pathophysiologic alterations leading to multi organ failure and lethality. antibodies to tnf might be a useful agent in the treatment of haemorrhagic shock related disorders. 55-+8 n=ll*$ 15-+5 n=7 78_+22 n=5* * p<0.05 vs baseline :~p<0.05 no anesthesia vs anesthesia thus 1) tnf production increased 2-3 fold by 48-72 hrs following trauma in unstimulated blood, but was reduced or not changed after lps stimulation, so circulating leukocytes are probably not an important source of tnf post trauma; 2) anticd18 had no obvious effect on tnf production in unstimulated or lps stimulated blood, relative to vehicle, which suggests that the protective mechanism of anticd18 does not involve tnf suppression; 3) fentanyl anesthesia at 72 hrs following trauma unexpectedly decreased lps-evoked tnf production, which suggests that anesthesia alone can influence an inflammatory response. proinflamrnato~ cytokines have been shown to play a signific~t role in the pathogenesis of sepsis, which is a very common occurrence in born injury. tnfa is infrequently detected in the blood of burned patients, the ability to detect the shed receptors of stnfg has not been determined. serial serum mmples from burn patients were collected from the time of admission until death from septic shock. these samples were analyzed using an enzyme-linked immunosorbent assay (elisa) for stnfr, l-ira, tnf-a, and il-ib. the patients ranged in age from 26 to 72 yeas of age. the percentages of bum ranged from 40% -90%. cytokine concenlrntions vmled from patient to padent irrespective of bum size. tnfa levels were consistentiy in the range of30pgjml -150pg/ml. peaks in the tnfa values were above 90pg/ml and were also associated with a peak in the stnfr levels. these levels began at <10,000pghnl within the in,st 6 ins of injury and gradually increased with time. clinically. ti~ appearance of eytoklnes was independent of positive wound, blood, or respiratory cultures however peak values in tnfa and stnfr were ~ialed with a fluid requirnmenl levels of il-i ra were also elevated independent of clinical findings as well as extent of injury. in pl5 there is a significant corresponding peak in il-trn (>40~8/ml) at the same time as t/~:a and stnfr levels. we aimed to characterise the pattern of secretion of interleukin-1 beta 0l-ii3), intefleukin-6 (il-6) and tumour necrosis factor alpha (tnfa) in multiply injured patients and to relate these results to their clinical condition and outcome. two hourly blood samples were taken from ten patients from the time of injury until 48 hours. cytokine levels were measured using sandwich enzyme-linked immunosorbent assays (elisas). injury severity scores (iss) were calculated and haemorrhage was assessed from the blood transfusion requirement over the 48 hours. patients' ages ranged from 17 to 86 years. iss varied from 9 to 50 and transfusion requirement from 0 to 14 units. five patients died after the study period. ]1,-6 was raised in 9/10 patients (max level 12,500 pg/ml) but was unrelated to condition or outcome. 5/8 showed a rise in il-1b (max level 90 pg/ml) which was negatively correlated to iss (i=-0.789, p<0.02). tnfa was raised in 2/10 (max level 95 pg/ml). peak tnfc~ was positively correlated with iss (1=0.676, p<0.05) and haemorrhage (i=0.602 but p<0.5). il-ib and tnfa production was mutually exclusive. there was no common cytokine profile for these patients. unlike elective surgery there was no correlation between peak 11,-6 and severity of injury: tissue damage may not be the stimulus for the cytokine response to multiple injury. periods of ischemia or hypoxia produce endothelial damage in peripheral organs. tumor necrosis factor-alpha (tnf) plays a central role for regulation of endothelial physiology during septic events, taking influence on vascular permeability and coagulant activity [1] . animal experiments demonstrated a synergism between hypoxia and septic shock on letality, leading to the hypothesis that low oxygen tension leads to enhanced sensitivity of target cells for tnf [2] . radioligand binding studies with ~261odid-tnf on cultured human endothelial cells were performed after incubation in several environmental oxygen tensions (pc2) for 12 hours. data were achieved by nonlinear regression of an idealized saturation curve according to the equation: b = n " k./(1 + k,); b = totally bound tnf; k,: association constant (concentration for half-maximal binding); n: number of binding sites per cell. p_o0o (mm h¢i): _k, (nm}: n (molecules/cell): 130 -150 1.83 ± 0.15 2600 _+ 500 55 -70 1.27 ± 0.15 3600 + 500 25 -35 0,41 ± 0.13 4800 -+ 500 10-15 0.18 + 0.07 5900 -+ 300 presented are calculated values on the idealized curve + 95 % percentiles. hypoxia induces enhanced binding of tnf to specific receptors on the endothelial cell surface in a time-and dose-dependent manner by a mechanism, which is not dependent on oxygen radicals, as shown by additional protocols with radical-scavenging drugs. with respect to former findings about a correlation between growth and tnf receptor affinity [3] , these data lead to the hypothesis that enhanced tnf binding during hypoxia is due to a biochemical conversion of the receptor protein from the low affinity to the high affinity state, possibly by posttranslational phosphorylation of the binding protein by intracel)ular kinases. the proposed involvement of tnf-dependent pathways in pathogenesis of organ dysfunction and multiple organ failure after hypoxia/ischemia may provide a basis for understanding the initiation of hypoxic vascular injury, as manifested by increased permeability and prothrombotic tendency, and, thus, merits further attention. the levels of activity of circulating cytokines (ill, il-6 and tnf-alpha) which are believed to play important regulatory role in response to trauma are determined (by hioassays and respective anti-cytokine antibodies) in mice and rats subjected to scald injury ion c,10 see, °0v bsa, ld 50) and (80 c, 30 see, 20 ~ b ~^)~ , respectively. biphasic increase of cytokine activity was noted in mice: initial increase of il-i and il-6, 1-3 hr following injury and of try activity 6hr after scald, followed by elevated levels of il-i and il-6 at 12hr, with tendency of decrease of activity at later time points. increased activity of tnf was noted 24 hr following injury, in rats, initial, short-lived increase of il-i and tnf activity was detected lhr following injury, folowed by increase on days i and 3 postburn. il-6 increase peaked 3-12 hr after scalding and levels remained elevated 3-5 days following injury. similar kinetics of appearance of proinflammatory cytokines (il-i and tnf-alpha) both in lethal and ncnlethal injury concomitant with differential profile of circulating il-6 activity (early,short-lived increase and later slow decrease of activity in lethal burn injury) with late persistent high levels of activity in nonlethai injury demonstrated in the present study highlight the need for investigation the relationship of these cytokines in burn-injury induced inflammation. zikica jovicic,lnstitute for medical research, mma,crnotravska 17,11002 belgrade~yu. asadullah k (1), woiciechowsky c (2), liebenthai c (1), doecke wd (1), volk hd (1), vogel s (2), v. baehr r (1); depts. of med. immunology (1) and neurosurgery (2) , medical school (char#d), humboldt university berlin, frg in patients after polytrauma or major abdominal surgery a hyperinflammatory phase seems to be followed by the development of a phase of monocyte inactivation. the latter is charaeterised by a decrease of monocytic hla-dr expression and a shift to anti-inflammatory cytokine production. as shown, by us and others, this phenomenon indicates severe immunodepression with a high risk of infection. however, the mechanisms leading to monocyte inactivation in the above mentioned syndromes may be multiple. to elucidate the influence of a selective, sterile trauma to the central nervous system (cns) on immune reactivity the neurosurgieal patient is an interesting model. initially, 30 patients who developed a systemic inflammatory response syndrome following neurosurgery were analysed. in all of them a marked decrease of monocytic hla-dr expression was observed soon after the operation. these results suggest that neurosurgery alone can induce immunodepression and lead us to conduct a prospective study, in which we closely monitored l0 patients undergoing neurosurgery from the first preoperative day until at least day 6 after the operation. hla-dr expression was decreased hi all patients to various extent only hours after surgery. in one patient only we found a persistently reduced hla-dr expression and this was the only patient to develop sepsis syndrome. this suggests that a prolonged, postoperatively decreased hla-dr expression is predictive of infection following cns trauma. in order to assess, whether a decrease of hla-dr expression was associated with a preceding inflammatory response, local cytokine release in the cns was compared with systemic cytokine release. for this purpose, paired samples of earebrospinal fluid (csf) from a vantricle drainage and peripheral blood plasma were obtained. in the csf extremely elevated futerleakin (il)-6 levels, peaking already a few hours after the operation were found. in plasma, by eontrast, il-6( and tnf-alpha) was detectable not until days later and only if infection was present. the antiinflammatory ili-ra, on the other hand, was also present in csf but peaked after il-6 and was detectable in peripheral plasma too. we believe there is an association between the inflammatory response in the cns and the following depression of hla-dr expression on peripheral blood monocytes. our results suggest that even a sterile cns-trauma by itself may contribute to general immunodepressinn leading to septic complications. the aim of this study was to evaluate the effect of haemorrhagic shock (hs) a) on total capacity of the host, and b) the circulating blood cells to produce tnf immediately after bleeding. in vivo studies: baboons were subjected to a limited oxygen deficit (180-200 ml/kg) hypotension phase (mean arterial pressure = map of 35-40 mmhg for 2-4 hours followed by adequate resuscitation). rats subjected to hs (map of 30-35 mmhg for 90 rain followed by reinfusion of shed blood and fluid resuscitation) were challenged with endotoxin (1 ~g/kg i.v.) at the end of shock (rhs group). the control group (rco) received the same dose of endotoxin as rhs group but without prior bleeding. in vitro studies: whole blood (wb) obtained from both baboons and rats before and at the end of hs were incubated with endotoxin (100 ng/ml) for 2 hrs at 37 °c. results: at 90 min post-lps challenge we found significantly higher plasma tnf levels in rats that were subjected to hs prior to the endotoxin challenge as compared to the control group (956 _+ 585 vs 276 + 199 pg/ml) . after hs the tpc was significantly decreased in in vitro stimulated cbc of both rats (12 + 4 post-hs vs 164 + 89 ng tnf/ml pre-hs) and baboons (8 ± 16 post-hs vs 233 ± 188 pg tnf/ml pre-hs). in contrast, the il-6 productive capacity was increased in baboons cbc (not yet analysed in rats) stimulated at the end of hs (224 ± 106 pre-vs 1545 ±_ 1254 pg il-6/ml post-hs). conclusion: from our data we suggest that despite of down regulation of the cbc to produce tnf the overall tpc is enhanced at the early stage of i-is. with regard to the related literature (chaudry's group) it can be assumed that among the macrophage/monocyte populations, as the main source only the kupffer cells (kc) exhibit enhanced tnf production capacity following haemorrhage. the mechanisms of down/up regulation of cytokine response of cbc and/or kc following hs remain to be examined. d. eg~er, s. geuenich °, c. dertzlin~er °, e. schmitt*, r. mailhammer, h ehrenreich #, p. drrmer, and l. h01mer gsf-instimt fox experimentelle h~znatologie, °medizinische kliulk iii, klinikum groghadern, munich, *institut for immunologic, johannes gutenberg universit/it, malnz, and #psychiatrische k/in& der georg-aagust-universi~t, grttingen, germany. it has been shown previously (ehranreich et al., 1992, new biol. 4: 147 ) that mouse bone marrow-derived mast cells (bmmc) synthesize and secrete endothelin-1 (et-i) and express eta-type endothelin receptors (eta). so far, however, no functions of et-1/et a in bmmc have been described. in the present study we investigated the effect of exogeneously administered et-1 on the release of histamine, serotonin, and leukotriene c 4 (ltc4) by primary mouse bmmc (in vitro age: 4 weeks) caltured with different recombinant mttrine cytokines (interleukin 3 (il-3) and/or kit ligand (kl) in the presence or absence of il4) for two weeks prior to activation. et-1 (5x10 -8 to lxl0 -6 m) induced an extremely rapid (_<lmin) and dose-dependent release of histamine and serotonin (up to 20% and 16% of total mediator content, respectively, comparable to ige/ag-indueed mediator release) from bmmc cultured with il-3 and il-4. only when cultured in the additional presence of il-4 rather than in medium containing kl or kl plus il-3 alone, bmmc released histamine and serotoaln upon challenge with et-1.furthermore, et-1 stimulated ltc 4 biosynthesis up to 4-fold in bmmc cultured in the presence of il-4. in contrast, the peptide was without major effect on ltc4 biosynthesis in bmmc culturd without il-4. the release of histamine and sereotonin was not altered if bmmc were preincubated for i h with il-4 prior to activation with et-i, suggesting that a differentiation process rather than a functional priming effect had been initiated by ]l-4. et-1 (10"6m) -induced histamine and serotouln release from bmmc was inhibited by an eta-specific antagonist (cyclic id-asp-pro-d-val-leu-d-tel ) in a dose-dependent manner, with a complete inhibition observed at an antagonist concentration of 10 -8 m. crude peritoneal cells (containing 2-3% serosal mast cells) obtained from normal balb/c-mice released 87% histamine upon challenge with et-1 (10 -6 m; 20 rain). taken together, these resu/ts demonstrate that et-1 can directly function as a histamine and serotohin secretagogue and as a stimulator of ltc 4 production in mast cells. il-4 appears to be involved in the differentiation of immature mast cell prec~trsors towards an et-l-reactive functional phenotype. when inflammatory reactions are advanced, type ii phospholipase a2 (type ii pla2) is produced in high levels by various cells at the site of inflammation. cytokines such as tnf-a and il-i activate type ii pla2 and promote the production of eicosanoid, which is one of the mechanisms by which they enhance the inflammatory reaction. in the present study, the blood levels of type ii pea2, endotoxin, tnf-c~, il-6 and il-8 were determined in patients with sepsis to examine the relationship between these levels and the patients' pathological conditions. the levels of type ii pla2 and tnf-a in these patients were 218.3 ± 179.9 ng/ml and 98.5 ± 92.1 pg/ml, respectively, the two parameters being significantly correlated (r = 0.6460, p = 0.0001 ). there were also a significant correlation between the level of type ii pla2 an il-6 (r = 0.4262, p = 0.0188). there was no significant correlation between the level of type ii pla2 and il-8. these in vivo findings suggest that tnf-a may be involved in the production of type ii pla2. a. filzmaver, g. reisbach, e. schmitt °, r. mailhammer, and l. hiittner. gsf-iustitut ff~r experimentelle h~imatologie, munich, and °iustitut fttr immunelone , johannes gutenberg universit~t, mainz, germany the product of c-kit, a transmembrane tyrosin ldnase receptor, and a corresponding kit ligand (kl) are critically involved in the control of different hemopoietic cell lineages including the proliferation, maturation, migration, and function of mast cells. it has been reported previously that interleukin (il)-4 down-regulates kit receptors in human primary myeloid leukemic cells and in a human mast celt line (sinaber et al. 1991, j. immunol. 147: 4224) . transforming growth factor (tgf) gl, was also found to down-modulate c-kit mrna and kit receptor proteins in human aml blasts, a mechanism probabely contributing to the anti-proliferative effect of tgfih on kl-stimulated aml ceils in vitro (de vos et at. 1993, cancer res. 53: 3638) . in porcine endothelial cells transfected with a retroviral construct carrying the human (hu) e-kit gena, exogenous hu kl transiently down-regulated kit receptors (blume-jeusen et al. 1991, embo j 10: 4121) . in the light of these previous findings we analyzed the effects of these exogenously applied cytokines (il-4, tgfgl, kl) on kit receptor expression and kl-mediated proliferation and chemotactic migration of primary mouse bone marrow-derived mast cells (bmmc) (in vitro age: 4 weeks). our results show that in bmmc cultures initiated with recombinant (r) marine (mu) il-3 the additional presence of rmull-4 for 3 or 14 days did not change the expression of kit protein (assessed by facs analysis with the anti-mouse c-kit antibody ack-2) nor kl-mediated proliferation or chemotaxis. in contrast, il-4 syeergistically increased kl-stimulated growth of bmmc in a short-term proliferation assay (mtt-tes0. pre-incubation of bmmc with rhutgfl~ 1 (5.0, 1.0, or 0.1 ng/ml) for 24h had no effect on kit receptor expression, although tgffil at concentrations of 0.5 -5.0 ng/ml completely suppressed the proliferative action of kl on these ceils. similar anti-proliferative effects of tgfg 1 were observed in various factor-dependent mast cell lines stimulated with different mast cell growth factors (il-3, 1i,-4, il-9, and/or il-10). moreover, pre-incuhation (24h) of bmmc with tgf-gl (>500 pg/ml) significantly enhanced spontaneous undirected cell movement (chemokinesis) and synergistically increased il-3-or kl-induced chemetaxis. when bmmc were preancuhated with rmukl (200 ng/ml) for 1, 3. or 5 days, a transient down-modulation of kit receptors with a maximum effect on day 1 was demonstrated by facs analysis and correlated well with a decreased chemotactic response of these cells. in conclusion our results show that neither il-4 nor tgfi31 affect expression of kit receptors in primary murine bmmc. it is reasonable to suggest that c-kit expression is controlled in a cell type-specific manner.interestingly, tgfgl is obviously able to dissect the proliferative from the migrational signal transducted by kl in these cells. objectives of the study: antisense strategies using dna-otigonucleofides (odn) to modulate the cytokine response are presently under investigation. odn are thought to act very specifically with little or no relevant negative side effects. we now report that odn unspeeifically protect wehi 164 cells from tnf-mediated cytolysis. material and methods: wehi 164 subclone 13 ceils (5 x 105), that are highly sensitive to the cytolytic activity of tnf, were grown on 96-well culture plates in rpm11640 medium. after 24 hours, phosphorothioate(ps)and partially ps-modified-odn as well as phesphodiester-odn (20-29bp) were added (0.1, 1 and 10 pm). four hours after incubation with odn, ce(i lysis was induced by recombinant murina tnf. after 18 hours the plates were washed and stained with crystal violet cell lysis was determined by reading the absorbance (abs) at 590 nm. results: wehi 164 ceils incubated with tnf (1-8ng/ml) were completely lysed after 18 hours (0% abs). interestingly, wehi cells incubated with tnf and odn resisted complete lysis, eg cells incubated with 2.5ng/ml tnf and 11jm odn showed still 30% of the absorbance observed in control ceils without tnf (100% abs). the protective effect of odn started at 0.1pm, reached a maximum at 1,um, and diminished at 101jm. with increasing amounts of tnf the protective effect of qdn decreased and no protection was detectable at 8ng tnf per ml conclusions: dna-oligonucleotides were found to unspecifically inhibit tnf-induced cytolysis. we hypothesize, that this protective effect of qdn results from an inhibition of the binding of tnf to its receptor, or from interference of odn with the subsequent signal transduction mechanisms. as a consequence, to discriminate the specific effect of odn in biologic systems, several control odn should be used. secondly, whether dna released by degradation of tumor cells or leukocytes can significantly impair tumor-and immune-defense mechanisms merits further investigation dr. med. michael meisner, institut for anaesthesiologie der universitat erlangen-nqmberg, krankenhausstral~e 12, d-91054 erlangen. in this study we investigated the involvement of serine protease and free radical generation in the systemic release of tumor necrosis factor-alpha (tnf) and interieukin i(il-1), in the sepsis model of lipopolysaccharide (lps, 5mg/kg i.p.) induced hepatitis in galactosamine (gain, 18rag/mouse, i.p.) sensitized mice. treatment of gain-sensitized mice with lps (gain/lps) led to dramatic increase in serum cytokine (tnf and il-i) ievels and transaminase activity at 3 hr and 8 hr respectively. pretreatment of serine protease inhibitor, c~jantitrypsin (a j-at, 50mg/kg i.p.), 30 rains prior to gain/lps treatment, fully protected the animals against the hepatotoxic challenge with significantly reduced serum tnf and il-1 levels. in order to block and scavenge superoxide generation, the mice were pretreated with xanthine oxidase inhibitor, allopurinol (al, 2 x 100mg/kg i.p.) and pyran polymer-conjugated superoxide dismutase (sod, 2 x 200 unit/mouse i.v) r6spectively. pretreatment with al and sod (24 and 1 hr prior to gain/lps) prevented gain/lps hepatitis and blocked lps induced released of tnf and il-1 into serum of the mice. the protective agents like cq-at or al/sod did not protect the mice against th~ hpp~totoxi£ ch~llpn-e indllee4 b'~ th~ recombinant mmlse tnf-o' (0.3 ~/rno~e j.p.) ~d oi~lps 1~ caln-.~dlfa%aed mlce. it-l cett~aged la tnf (x/gain treated mjde was not detectable in animals pretreated with oq-at or al/sod. our study suggests that a serine protease sensitive to cq-antitrypsin is responsible in regulating tnf release, possibly by proteolytic cleavage of a tnf-precursor or membrane bound tnf. in addition our evidence suggest that the balance of extracellular protease/antiprotease activity may be regulated by free radical generation, possible superoxide anion, resulting in inactivation of the antiprotease. il-1 release may be subsequent to tnf release. objective: during sepsis one can observe a dramatically impaired production of proinflammatory cytokines like the tumor necrosis factor alpha (tnf-a), interleukin i-alpha (il-la), intedeukin i-beta (il-i&) and interferon gamma (if~) upon in vitro stimulation of circulating cells. however there is also evidence of a decreased ability to produce cytokines in other immuno-deficient states. in this study we compared the capacity to secrete proinflammatory cytokines upon in vitro stimulation of patients in severe sepsis and patients with malignant tumors. methods: heparinized blood samples of ten patients (62+ 14 years) in severe sepsis (sepsis score > 15 according to e}ebute and stoner) were drawn at onset of disease, from fifteen patients with solid growing carcinoma (59+19 years) blood was drawn at diagnosis prior to any therapy. controls were obtained from fifteen healthy volunteers. 50 pl of whole blood were incubated either with 450/4 of a standard medium or with 400 pl of a standard medium and 50 pl of phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours plasma concentrations of tnf-a, il-la, il-16 and if-~ were determined by elisa. comments: our results suggest that down-regulation of cytokine secretion or of cell responsiveness to non-specific mitogens during sepsis has occurred. we observe a similar phenomenon for the group of carcinoma patients vs control significant for stimulated tnf-a and stimulated if-t. sustained immunological interactions between tumorcells and cytokine producing cells could effect responsiveness of the latter, a general increased immuno-tolerant state in patients with carcinoma has to be discussed. however we found significant differences between sepsis and cancer concerning the in vitro capacity of responsable cells to produce il-la and il-i#. the dramatically decrease of the ability to produce il-i upon in vitro stimulation could be more sensitive for a septic state than stimulated tnf-a or if-3,. objective: tumor necrosis factor alpha (tnf-a) has been implicated as a central mediator of sepsis and its sequelae. increased systemic levels of this cytoklne seem to be correlated with severity of sepsis and outcome. however mechanism of action and metabolism of tnf-g are not fully understood. in most studies blood samples for tnf-a determinations are obtained either by peripheral venipuncture, a central venous catheter or by an indwelling arterial catheter. very often blood samples are taken in different manners within the same study. in this study we measured circulating tnf-a and the amount of tnf-a released upon in vitro stimulation in arterial and central venous blood. methods: heparlnized arterial and central venous blood samples of ten patients (6 males, 4 females, mean age 62+_14) with severe sepsis (sepsis score > 15, elebute and stoner} were drawn on day 1,3,6,8, and 14 of disease. blood was immediately placed on ice and processed within 1 hour. 50 pl of whole blood were incubated with 450 pl rpmi-medium supplemented with antibiotics and l-glutamlne or with 400 pl of rpmi-medium and 50 pl phytohemagglutinin (pha) a potent mitogen. after an incubation period of 24 hours samples were centrifuged and plasma was harvested and stored at -30 ° celsius before assessment of tnf-a concentration by elisa. statistical analysis was performed with the paired student-t-test. results: we found a significant difference (p < 0,05) for circulating mean arterial tnf-a concentration (237 pg/ml _+ 34 sem} and central venous tnf-a (203 pg/ml +_ 29 sem). upon in vitro stimulation there was also a significant difference (p < 0,002) between released arterial tnf-~' {746 pg/ml _+ 88 sem) and venous tnf-a (637 pg/ml +_ 76 semi. conclusions: these results are difficult to interprete but could reflect the influence of pao 2 and sao 2 on tnf a release. it could also be the result of different concentrations of tnf-o release influencing factors like for example endotoxin, interferon-f or prostaglandin. a possible pulmonary and/or a hepatic metabolism of tnf-n and tnf-a producing cells cannot be ruled out. however for better interpretations of tnf-a release in septic states it is necessary to use either arterial or venous blood samples. early inflammatory processes following trauma and/or infections were found to be associated with the secretion of high amounts of proinflammatory cytokines. besides intedeukin-t (il-1), tumor necrosis factor-a (tnf-c 0 and interleukin-8 (il-8) the multifunctional cytokine intedeukin-6 (il-6) was described to be a central regulatory element of the primary cellular and humeral defence reaction. the previously described close temporal correlation of pathologically elevated il-6-concentrations and the extracellulary release of lysosomal enzymes from activated pelymorphnuclear neutrophils suggests, that il-6 may be a potential substrate of these preteases. the serine preteases elastase (ec 3.4.21.37 ) and cathepsin g (ec 3.4.21.20) derived from the azurophilic granules were assumed to be mainly involved in unspecific proteolysis at sites of inflammation by cleavage of structural as well as soluble proteins at random sites, if the inhibitory potential is decreased. the possible proteolytic activity of elastase and cathepsin g toward the proinflammatory cytokine interleukin-6 (il-6) was investigated. the addition of purified neutrephil elastase and cathepsin g to recombinant human il-6 leads to a rapid sequential degradation in vitro. at least two intermediate products could be detected by silver staining and western blotting following protein separation under reducing conditions. the serine protease inhibitor g-anitrypsin was shown to prevent the proteolytical degradation of intedeukin-6. furthermore the loss of the biological activity of both, recombinant and natural human il-6, was demonstrated by determination of the capacity of protease-treated il-6 to stimulate hybddoma growth (7td1 bioassay). these data suggest a possible downregulation of pathologically elevated il-6 levels by proteolytic activity of extracellulary released enzymes at sites of inflammation. the aim of the study was to compare circulating levels of three cytokines -il-1, il-6, 11_-8 -between critically ill subjects who developed gram-negative sepsis and who did not. materials and methods: the patient population consisted of 39 patients admitted to an intensive cars unit, with different underlying diseases. sepsis diagnosis was given according to pre-estabilished cdteda. nineteen cases were enrolled in sepsis group, twenty in control group. serum sampling was collected in sterile tubes at study entry and every three days until study dismissal. serum concentrations of il-1, 11_-6 and il-8 were measured using commercially available test kits, based on the dual immunometric sandwich principle. results: the causative patogens of sepsis were: pseudomonas aeruginosa, acinetobacter, eseherichia co~i, serratia marceseens, proteus mirobilis and citrobacter freundl the time of observation was equal to 12 days, for a total of four tests performed (to, tl, t2, t3). i1.-1 was not detected in any samples. the serological profiles of the two cytokines 11.-6 and 11_-8 were similar; augmented levels were found at study entry and throughout the observation period, peaking at t3 and decreasing at t4. however, in patients with sepsis, il-6 and 11_-8 concentrations were significantly higher in respect to control group. conclusion: our observations shown that in icu patients increased il-6 and il-8 release may be induced by cdtical illness; however, in subjects in which sepsis occurred, il-6 and il-8 production appears more significantly elevated, suggesting a role of il-6 and 11_-8 in the pathophysiology of sepsis. the fact that ii. objective: to check whether continuous veno-venous haemofiltration (cvvh) could remove the cytokines, namely tumour necrosis factor alpha (tnfc 0 and interleukin 6 (il-6) from the circulation of critically ill patients with sepsis ad multiple organ failure (mof). setting: the intensive therapy unit of the medical school teaching hospital. patients: nine critically ill patients with sepsis and mof treated with cvvh. methods: blood samples were collected before the cvvh had been started. then, blood and ultrafiltrate samples were collected simultaneously after 4 hours and every 24 hour. tnfct and il-6 levels were measured using the bioassays with cell lines wehi-164 ci13 and 7td1, respectively. other data were recorded from the patient notes and intensive therapy unit charts. results: no measurable concentrations of tnfct were detected in either blood or ultrafiltrate samples. il-6 was found in all the patients' plasma samples and five patients' (55.5%) ultrafiltrate samples. the il-6 blood level ranged from 17.4 to 3061.7 u/ml (mean 518.2, sd 686.8). the il-6 level in positive ultrafiltrate samples ranged from 21.0 to 1150.2 u/ml (mean 252.2, sd 401.8). conclusions: our preliminary results suggest that il-6 is present in bloodstream of septic patients. we assume we could not detect tnfa in any sample because we usually started observations when septic state had developed. cvvh could extract cytokines from the circulating blood. it remains under discussion, whether that extraction may be beneficial to patients with mof. the pattern of some significant cytokines tnf, il-1 and il-6 and their pharmacomodulation were evaluated in an experimental model of polimicrobial sepsis induced in cd-1 mice by cecal ligation and puncture (clp) in order to understand their roles. this model of sepsis, which resembles the clinical situation of bowel perforation, was also compared with that induced by administration of pure endotoxin (lps). tnf was detectable in serum and tissues during the first 4h with a peak 2h after clp at a significantly lower level than after lps. il-1 was measurable in serum only after 24h, significantly increased in spleen and liver after 4 and 8h and in mesenteric lymphonodes from 8 to 24h after clp compared with shammice. il-6 was significantly increased in serum throughout the first 16h after clp. pretreatment with dexamethasone (dex), ibuprofen (ibu) and nitro-l-arginine (n-arg) significantly reduced the survival time while chlorpromazine (cpz) and tnf did not affect it. only the antibiotics and pentoxifylline (ptx) significantly increased the survival in clp. however cpz and dex protected from lps-mor~ality. in conclusion, by inhibiting tnf with dex, cpz, ptx a reduced, unchanged and increased survival time was observed and by increasing tnf with ibu and tnf administration the survival was decreased or unchanged respectively suggesting that the modulation of this cytokine does not seem to play a significant role in clp unlike lps_ moreover the negative effects of ibu and n-arg suggest an important and protective role by prostaglandins and no in clp. to gain more insigths on the contribution of tnf~, il-i~ and if 7 to lps toxicity, we explored the time-course of the cytokine production in ealb/c mice given different doses, from the lethal (= 3 ld50) to the sublethal (= 1/3 ld50) of three different lps (e.coli oiii:b4 and 026:b6; p.aeruginosa r261) endowed with different degree of toxicity cytokines were measured in serum and organs with specific elisas up to i0 h after lps administration. results demonstrate that i) circulating and organ levels of tnf~ do not reflect lps toxicity. in fact, the lethal dose of lps 026:b6 induced as much tnf~ as the sublethal dose of lps 0111:b4; furthermore, lps r261, whose cytokine inducing capability is far lower than that of lps from e.coli, induced higher tnf~ levels at the sublethal than at the lethal dose. in addition, policlonal anti tnf ab, that were able to protect mice from e.coli lps induced mortality, failed in mice treated with lps r261 2) circulating il-i~ levels are generally low and increase significantly only in muribond animals. on the contrary, in spleen and lung very high levels of il-i~ are persistent from i to 8 h post lps administration moreover, the treatment with 6 mgr of neutralizing policlonal anti il-i~ ab, did not modify survival in lps challenged mice. 3) circulating and organ levels of if 7 are proportional to the dose and degree of toxicity of all the administered lps even if lps r261 was again a less efficient cytokine inducer than lps from e.coli. csa is an immunos~ppressive drug, able to inhibit gene expression for many cytokines, including if 7. to study the effect of cytokines modulation on lps toxicity, csa was administered to mice twice at the oral dose of i00 mg/kg before the challenge with lps. mice were monitored in terms of mortality and tnf~, il-i~ and if 7 production. together with the total ablation of if7, the strong reduction of tnfu and unmodified il-i~ levels, a significant increase of lps toxicity was also observed. these results suggest the hypothesis that the numerous factors that jointly mediate lps toxic effects, can also be protective, the final outcome depending on their relative ratio rather than on the absolute amount interleukin-1 (il-1) mediates the septic shock syndrome and affects intestinal secretion in vitro. we studied the intestinal production of il-t and its effects on diarrhea during endotoxic shock. cd-1 mice were randomized to 15 mg/kg e.coli 0111:b4 lps or saline infusion (i.p. or i.v.). diarrhea invariably occurred following lps infusion. mice were sacrificed at 0, 30', lh, 2.5h, 4h, 6h, 12h, and 24h (3 mice/group/time-point). the small bowel was compressed and the intestinal contents were weighed and expressed per g sb weight. the small (sb) and large bowels (lb) were eventually frozen, weighed, and homogenized for either cytosolic protein or total rna. il-i~ (cell-associated agonist) was measured with a radioimmunoassay specific for mouse il-l~ (detection limit 100 pg/ml) and expressed as ng/g weight + sem (lowest detectable amount 1 ng/gwt). northern analysis of total rna and in sfu hybridization of paraformaldehyde-fixed frozen tissue were done with [0~-32p]-iabeled mouse il-lc~ cdna probes. only sb had il-i~ constitutively present (6.2 + 1.6 ng/gwt). lps i.p. or i.v. induced elevation of il-lc¢ in both organs in a biphasic pattern; lps i.v. induced 3-fold more il-i~ than lps i.p. following lps i.p., il-i~ in sb was 19.2 + 0.6 ng/gwt at lh, reached maximal levels at 2.5h (31.8 -+ 0.5 ng/gw-i) and returned to baseline at 24h. saline controls maintained their constitutive il-i~ levels. sb had 2fold more il-1 ¢ than lb and identical kinetics, but lb showed a clearer doseresponse. northern analysis of sb-total rna showed induction of il-i~ mrna by lps in correlation with il-lc¢ kinetics. il-i~ mrna producing cells were mononuclear cells in the lamina propda and epithelial cells at the bottom of the crypts of ueberkuhn. mucus and fluid were increased in the small bowel post-lps in correlation with intestinal il-lc~ kinetics (r2=0.9). separate mice were pretreated with saline i.p. orthe il-1 receptor antagonist (irap, 30 mg/kg bolus i.p.) and were challenged 20 rain later with 1.5 mg/kg lps i.p. or saline i.p. specific blockade of il-1 by irap decreased intestinal secretion at 4h and 6h post-lps challenge (p<_. 0.05, student's-t-test). these data indicate that local (intrinsic) intestinal il-i~ mediates sepsis-induced intestinal changes. inflammatory cytokines initiate the host response to endotoxemia, causing severe physiological and hemodynamic changes which may lead to septic shock. among the regulatory systems that play an important rote in controlling host inflammatory responses is the pituitary. it has been known for many years for example, that hypophysectomized animals are extremely sensitive to lps lethality. while investigating the possibility that protective, pituitary mediators might explain this phenomenon, we identified the cytoldne mif to be a specific secretory product produced by pituitary cells in vitro and in vivo after lps challenge. analysis of serum mif levels in control, t-cell deficient (nude), and hypophysectomized mice revealed that pituitary-derived mif contributes significantly to the rise in serum mif that occurs after lps administration. of note, pituitary mif content (0.05% of total pituitary protein) and peak serum mif levels (80-340 ng/ml) were determined to be within the range observed for other pituitary hormones that are released after pituitary stimulation. to investigate a possible beneficial role for mif in septic shock, we co-injected mice with purified, recombinant murine mif (rmif) together with lps (15 mg/kg). surprisingly, rmif markedly potentiated lps lethality compared to control mice that were injected with lps alone (85% vs. 35%, p = 0.003). to confirm these results, mice were treated with anti-rmif antibody prior to injection of a high dose of lps (17.5 mg/kg). anti-rmif antibody fully protected mice against lps lethality, increasing survival from 50% to 100% (p = 0.0004). serum levels of tnf,~, the first cytokinc that appears in the circulation after lps challenge, were reduced by 38.0 _+ 9.5% in anti-rmif-treated mice. we conclude that pituitary derived mif contributes significantly to circulating mif in the post-acute response in endotoxemia and may act in concert with other pituitary mediators to regulate both pro-and antiinflammatory effects. moreover, mif may play a critical regulatory role in the systemic host response in septic shock. our results suggest that anti-rmif antibody might be of potential therapeutic use in the treatment of septic shock. although anti-interleukin-1 (il-1) antibodies and il-1 receptor antagonist have been shown to improve survival in animal models of endotoxemia and abrogate the lethal effects of tnf, the presence of il-1 in the serum does not correlate well with outcome. we hypothesized that this may be because il-1 acts mainly in a paracrine fashion and is metabolized before it diffuses into the circulation. methods: we measured the il-i~ mrna expression with the differential reverse transcription polymerase chain reaction (rt-pcr) using g-actin as internal standard in the peritoneal macrophages and lung tissue in normal controls and mice after cecal ligation and puncture (clp). clp resembles human intra-abdominal sepsis in that it is characterized by very slight elevations of serum il-1 levels. results: il-lg mrna levels after clp are expressed as % of normal (mean+sem, n=5 in several experimental models of infection exacerbation of disease was observed, when infected animals were depleted of tuajor necrosis factor (tnf). after sublethal cecal ligation and puncture (clp) leading to peritonitis and sepsis the survival of mice also critically depends on tnf as demonstrated in earlier studies, when clp-treated mice injected with anti-tnf antibody died, whereas mice injected with a control antibody survived after clp (echtenacher et al. 1990, j. inununol. 145: 3762) . from a panel of different cell types (macrophages, neutrophils, t lymphocytes, natural killer cells, mast cells) able to produce tnf upon activation~ the mast cell is apparantly the only one capable of storing in cytoplasmic granules preformed tnf-ct which is rapidly released following challenge. in the present study-we analyzed serum tnf after lps injections as well as the outcome of clp in severely mast cell deficient mutant mice (wav v) as compared to syngeaeic wild-type littermates (+/+). we proposed that concentrations and/or kinetics of serum tnf should be different between wavv mutants and wild-type mice, if mast cell-derived tnf significantly contributes to the rise in serum tnf levels following systemic stimulation with endotoxin. although similar levels of increased tnf were detected in the sera of both genotypes after 1 and 2 hours of lps injection (100 btg/ 0.5 ml / mouse i. p.), mast ceil-deficient mice indeed showed decreased serum tnf levels 30 iron after injection amounting to only 12 to 25% of the concentrations observed in the corresponding sera of normal wildtype mice. in the clp model of septic peritonitis we found that mast celldeficient mutant mice were dramatically more sensitive to clp than syngeneic normal mice resulting in 92% mortality in w/w v versus 8% mortality in +/+ mice 2.5 days after initiation of clp. further experiments with w/w v mutants selectively reconstituted with cultured bone marrow-derived mast cells from normal syngeneic wild-type mice and the use of an antibody specifically blocking the action of tnf tn vivo should clarify a potential protective function of mast cells in this model of septic peritonitis. interleukin-10 (il-10) inhibits cytokine production, including tumor necrosis factor (tnf), by lipopolysaccharide (lps)-aetivated maerophages. we recently observed that lps injection (e.coli 055:b5, 100 gg ip) into balb/c mice induces the rapid release of circulating il-10 (17±9 u/ml at 90 min). blocking endogenous il-10 using monocional antibody (jes5-2a5, 2 mg, 2h before lps) resulted in a massive increase in tnf production (107±47 in lps+anti-il-10 treated mice vs 6±2 ng/ml in lps alone, p<0.05, n=4 to 5 mice per group) and an enhanced lps-induccd lethality (53% vs 7% in anti-il-10+lps or lps alone respectively, p=0.01, n=15 mice per group). irrelevant igg1 rat monoclonal antibody (lo-dnp) did not influence neither tnf production nor lethality associated with endotoxin shock. this led us to study the production of il-10 during human septicemia. plasma samples were obtained from 65 patients with gramnegative (gns, n=22) or gram-positive septicemia (gps, n=43) and from 20 healthy volunteers. among these patients, 17 suffered from septic shock at the time of sampling. il-10 levels were measured by elisa (detection limit: i 1 pghrd). we found that 35 patients (54%) had increased il-10 plasma levels (range 12 to 2740 pg/nd). patients with gps had il-10 levels similar to the ones observed in gns (median: 12 vs 17.5 pg/m, respectively). patients with septic shock had higher il-10 values (median: 58 pg/ml) than septicemic patients without shock (11 pg/ml, p=0.002). no il-10 was detected in plasma from healthy volunteers. we conclude that il-10 is produced daring human septicemia. our experimental data suggest that il-10 might be involved in the control of the inflammatory response induced by bacterial products. dr arnand marchant, immunology department, hopital erasme, 808 route de lennik, 1070 brussels, belgium. to provide information about the role of tnf in sepsis and mods we measured tnf and stnfr-i levels in septic patients and investigated if there is a relation between plasma concentration of these molecules and the severity of sepsis evaluated by two scores (apache 1i and sss). patients and melhods: 20 septic patients fullfilling sepsis criteria of american college of chest physician and society of critical care medicine were studied. tnf-cc and stnfr-i (60kda) were measured by enzyme immuneassays (norms1 values = 13+8 pg/ml and 1.06_+0a ng/ml respectively). results: the mean tnf and stnfr-i values for each patient (mean+sd) were 93+64 pg/ml and 11.1+5.5 ng/ml respectively. these values are approximately seven and ten times greater than those observed in normal healthy volunteers (p<0.001). mean tnf concentrations for each patient were significantly greater in non survivors (112+52 vs 64_+74 pg/ml p<0.05); stnfr-i levels also were greater in this group, but the difference was not statistically significant (12.6+5.2 vs 8.8_+5.5 ng/ml). plasma tnf and stnfr-i concentrations were significantly correlated (r = 0.65 p<0.005). mean tnf levels were significantly correlated with apache ii (r = 0.49 p<0.05) and sss (r = 0.54 p<o.01); stnfr-i levels were likewise correlated with both scores (r = 0.80 p<0.001 and r = 0.76 p<0.001 respectively). tnf and stnfqgi levels increase with the number of dysfuctional organs; in particular tnf and stnfr-i increase when hemodynamics deteriorate and kidney failure ensues. conclusions: the correlations between tnf and stnfr-i levels and sepsis severity scores suggests that tnf is involved in sepsis and may influence the occurrence of mods and finally clinical outcome. tnf and stnfr-i are in fact especially increased when mods occurs. in particular their concentrations are elevated when cardiovascular system and kidney failure ensue, suggesting that tnf can have a direct role in hemodynamic derangements caused by sepsis and that kidneys are involved in tnf and stnfr-i excretion. lif is a pleiotropic cytokine that has been implicated in the pathogenesis of sepsis in animal models. we conducted a prospective study in 33 septic patients to correlate lif plasma levels with patient's clinical and biological data (among them il-6). plasma was drawn daily from day one to .ten and lif presence was determined with a specific elisa and with the dai,a bioassay (sensitivity of 100 pg/ml and 1 ngml respectively). risk factor associated wftt~ with elevated lif plasma level was determined by an univariate analysis. a multivariate stepwise logistic regression analysis was subsequently performed with a bmdp statistical software. lif was detected with the elisa and with the bioassay in 11 and 2 out of the 33 patients, respectively. lif peak plasma levels were statistically associated with high creatmin levels, temperature and il-6. multivariate regression analysis showed that high serum creatinin level was the major independent risk factor associated with elevated lifplasma levels. this correlation was also found for il-6. peak plasma levels of both lif and il-6 correlated inversely with patients survival duration. cox's analysis for plasma levels of lif >480 pg/ml yelded a hazard ratio of [exp (1.86)=6.44]. our study indicates that lif levels were associated with clinical and biological parameters of illness severity and significantly increased (cut-off value 480 pg/mi) in patients with fatal outcome. current consensus exists about the central role of tumor necrosis factor (tnf) alpha in initiating the systemic inflammatory response syndrome (sirs). a correlation with sirs has inconsistently been found. tnf effects its pleiotropic reactions upon two distinct cellular receptors. soluble extracel]ular fragments of the human 55 kda tnf receptor (stnfri) and the 75 kda receptor (stnfrii) are detectable in the circulation. the kinetics of these endogenously produced tnf-inhibitors were measured to evaluate their role in patients with sirs. fourteen patients of an operative icu were included with the diagnossis of sirs (mean apache ii score: 20 points). serial blood samples were obtained within 12 h after diagnosis of sirs, every 6 hrs for the first 48 hrs and every 12 hrs thereafter until patients died or recovered. soluble tnfri and stnfrii were assayed by an enzymed-linked immunological binding assay. soluble tnfri and ii could be detected in all samples with a significantly higher level (p<o,o01) compared to healthy controls (normal value: tnfrh 1,5 ng/ml; tnfrii: 2,3 ng/ml). the serum concentration of tnfri (14,1 ng/ml) as well as tnfrii (18,2 ng/ml) were significantly higher in nonsurvivors (n=6) compared to survivors (8,6 and 9,5 ng/ml; n=8) at the onset of sirs and during the course of the inflammation response (p<0,05). a positive correlation exists between both receptors (r-0,57; p < 0,01 ). increasing levels and maximal values of stnfri (59 ng/ml) and i1 (82 ng/mi) were detected only in patients who died. the serum concentrations of patients who recovered did not change and are remaining at the initial level. tnf alpha bioactivity was detected in 10 out of 14 patients. no significant correlation exists between the concentration of both receptors and tnf alpha as well as the apache ii score at the onset of sirs. elevated serum concentration of stnfri and 11 are found in patients with sirs. these naturally occuring antagonists reflect the inflammatory process. the measurement of soluble tnf receptor levels may be useful in monitorina sirs and in the prediction of outcome. 11-2 is given to patients with advanced melanoma or renal carcinoma. however, this therapy is accompanied by severe side effects, including the vascular leak syndrome (vls) that closely resembles septic shock. to investigate the involvement of cytokines and phospholipase a z in the pathogenesis of the vls we collected serial plasma samples from patients during the first 24 hours after administration of il-2. in these patients we monitored temperature, blood pressure and heart rate and assessed levels of the cytokines tnf, il-6 and il-8 using immuno assays. in these plasma samples we also measured phospholipase a 2 activity using an enzyme activity assay, since this enzyme is beleived to play a major role in the generation of lipid mediators. we demonstrate that during il-2 therapy the cytokines tnf, il-6, and il-8 are produced and that the release of these cytokines is followed by an induction of phospholipase az activity in the plasma of these patients, we further discuss the role of these mediators in the development of the vascular leak syndrome observed in patients receiving il-2. objectives of the study: sepsis caused by candida albicans (ca) is no longer a clinical rarity but a common consequence of immunosuppression and surgery, and is associated with high mortality. tumor necrosis faetor-c¢ (tnf-<~), interlcukin-lg (il-lg) and interleukin-6 (il-6) are thought to play an important role in the pathogenesis of the sepsis syndrome. we therefore studied the in vitro production of tnf-<x, il-ib and il-6 by human peripheral blood mononuclear cells (pbmc) stimulated by ca compared to stimulation by bacterial lipopolysaccharide (lps). materials and methods: pbmc were isolated from venous blood of 5 healthy volunteers and cultured at a concentration of 2.5 • 10 ~ eells/ml in culture medium with a dose-range of either heat-killed ca or lps. supernatants were harvested after 24 hours. using elisa, tnf-c~, il-ib and il-6 concentrations were measured. ca was isolated from a patient with ca sepsis and cultured under sterile conditions. results: ca and lps stimulate the production of tnf-~t, il-11] and il-6 by human pbmc. we observed a dose-dependent synthesis of these three cytokines in human pbmc. tnf-c¢, il-ib and il-6 in ng/ml as mean _+ standard error. ca/ml 0.0 1. (2) we constructed a single-chain fv-fragment (sc-fv) from the variable domains of a neutralizing antibody to human tumornecrosisfactor-alpha (tnf) because sc-fv should have some advantages in vivo. the sc-fv has a similar affinity (3,8x10-9m) as the fab-fragment (2,7x10-9m) but bind the antigen with a eightfold lower avidity compared to the parental mab (4,8x10-10). the parental mab as well as its fragments can compete the binding of rhutnf to both tnf-receptors. this was shown by competitive binding to the cell line hl-60 and to immobilized rtnf-receptors. in the nutralization assay on the cell line, l 929, the sc-fv can neutralize tnf but in comparison to the mab or its fabfragment, the capacity was three-fold lower as expected from the molar ratio of the dissociation constants. the in rive-neutralizing capacity was tested in a mice receiving toxic dose of rhutnf. the sc-fv showed a 15-fold lower neutralizing capacity in comparison with the fab-fragment. this could be explained in different manners: i) partial instability of scfv at 370c, ii) shorter half-life because sc-fv but not fab-fragments are eliminated via kidney, iii) elimination of tnf-immunocomplexes may be improved by opsonization mechanisms which requires some structures of the constant region (as in fab or mab). in summary, despite the encouraging in vitro experiments, the in vivo data suggest that sc-fv are not the right strategy for neutralizing tnf in vivo. tnf production in endotoxin non-responder mice, effect of a glucocorticoid antagonist g. iazgtr jr, e. duda, j. ol~th, e. husztik, g. iazar glucocorticoids inhibit lipopolysaccharide (lps)-induced tnf production and tnf can stimulate pituitary adrenocorticotropin secretion, resulting in the release of corticosterone. earlier studies (1) reveales that the glucocorticoid antagonist ru 38486, nufepristone, sensitizes both endotoxin responder and nonresponder, c3h/hej, mice to endotoxin lethality. we have also shown that the glucocorticoid antagonist ru 38486 sensitizes endotoxin-tolerant (endotoxin-pretreated) and normal mice to the lethal effect of septic and endotoxin shock. on this basis, we set out to study the influence of ru 38486 on tnf production induced by endotoxin in endotoxin responder and non-responder mice. one and 2 hrs following lps injection, ru 38486 significantly increased the tnf concentration in the serum, liver and spleen of treated animals as compared with the control and methylprednisolonetreated groups. in tissue cultures, ru 38486 induced the tnf synthesis of myeloid cells and increased the tnf production of genetically modified hela cells, which synthesize tnf constitutively. normal and tumor cell cultures exhibited an increased sensitivity toward tnf in the presence of ru 38486. however, the same dose of lps did not induce tnf production in the serum, liver and spleen of endotoxin non-responder mice and this was not influenced by concomitant ru 38486 treatment. these studies suggest that ru 38486 aggravates lps lethality via factors other than tnf in endotoxin non-responder, c3h/hej mice. the cytokine response to a novel exnacellular mitogenic factor, mf, produced by the group a streptococci (gas), and to the streptococcal pyrogenic exotoxins (spe) a and b, was determined by in vitro stimulation of peripheral blood mononuclear cells (pbmc) obtained from healthy blood donors. the induction and kinetics of 14 different cytokines was studied at single-cell level using cytokine specific monoclonal antibodies and intracellular immunofluorescent staining. all three mitogens induced a massive ifny and tnfi3 response in 10-16% of the pbmc after 48-96 hours of cell stimulation. in contrast, il2 and tnfc~ containing cells was only detected in 1-3% of the pbmc. the induction ofil3, il4, il5, and ill0, was weak or completely absent. thus, the response indicates activation of th1 cells. however, illc~, illl3, illra and il8, were consistently found and gradually produced, peaking at 24 hems in approximately 5-8% of the pbmc. mf induced an equally extensive cytokine as well as proliferative inducing capacity, as did spea and speb, which suggests that also mf is a superantigen. a marked inter-individual variation could be noted between lymphocytes isolated from different individuals, both in the toxin induced proliferation and cytokine induction. this may be one explanation for the varying clinical severity noted in patients after infection with clonal gas strains. introduction -tumour necrosis factor (tnf) is released by mononuclear cells in response to inflammation and sepsis. it has been implicated as a possible mediator in inflammatory bowel disease (ibd) but its pathogenic role remains uncertain. this study assessed the relationship between tnfct and disease activity by measuring plasma and faecal tnf concelnmtions in an experimcntai model of ibd. methodologo' -colitis was induced in male wistar rats by intmcolonic instillation of 30rag 2,4,6-trinitrobenzenesulphunic acid in 0.5ml 50% ethanol (n=12). control animals received an isovohlmetric instillation of normal saline (n=12). faecal pellets were collected before induction of colitis (day 0) and throughout the experiment. after 8 days the colon was excised, ueighed and the colon macroscopic score (cms) assessed plasma tnf (ptnf) samples were assayed by bioassay and elisa. faecal samples were vortcxed in water and the supernatant removed for estimation of protein and faecal tnf (itnf) content (elisa). there is a significant increase in itnf on day 4 -#p=0 02 alld the total ftnf measured during the study correlatcs with the cms oll day 8 -p=0.04 there is no correlation between ftnf and ptnf. conclusions -therc is evidence of coloific tnf excretion by macroscopically normal animals and following induction of colitis there is increased faecal tnf which correlates with disease actmty. there is no significant elevation in bioactive or imnmnoreactive plasma tnf in colitie animals this data would suggest that faecal tnf is a marker of colonic inflamrnatiort and serial tnf assessment inay be useful as an indicator &severity in ibd. to study the effect of in-vivo hypoxia/reoxygenation on cytokine elaboration by routine peritoneal macrophages. materials and methods: adult male cba mice (20-25g) were primed intraperitoneauy with either lipopolysaccharide (lps) 10ug]anlmal or saline. five days later, the animals were subjected to hypoxia (8%02) for 160, followed by 20 of normoxia. harvested peritoneal macrophages were plated in-vitro, culture supernatants were collected at 20, 40, and 60 and assayed for tumor necrosis factor (tnf), prostaglandin e2 (pge2) and nitric oxide (no). control animals underwent the same procedures, but were maintained in normoxia (21%o2 disease 5, berlin, germany the precise mechanisms of reactivation of latent human cytomegalovirus (cmv) infection are still unknown. apart from the permissive effect of diminished cellular immunity there is evidence of a cytokine mediated cmv reactivation as it is known for other systemic virus infections (e.g. hiv). we found that tumor necrosis factor-alpha (tnf) -in contrast to all other cytokines tested -can stimulate the activity of the cmv-ie-enhancer/promoter region in the human monocytic cell line, hl 60. we wondered whether our in vitro results were actually reflected by the incidence of cmv reactivation in diseases which go together with high tnfalpha levels. cellular immunodeficiency as well as at least temporarily increased tnf levels are known to occur in septic disease. we tested for the presence of cmv infection in peripheral blood mononuclear cells (pbmnc) by means of 24h centrifugation culture, immunocytological detection of different cmv antigens (apaap-technique), and pcr technique (cmv-ie dna). in striking contrast to healthy controls almost all examined septic patients were positive, irrespective of the method for cmv-ddtection applied, including the less sensitive immunocytology. follow-up studies showed that cmv-antigen positive pbmnc ceased to be detectable in patients with good outcome once septicaemia had been overcome. to further back up our hypothesis, we looked for a coincidence of enhanced tnf-alpha serum levels and cmv antigenaemia (immunocytology) in some other diseases which are known to have either enhanced tnf serum levels or increased incidence of active cmv infection and found the majority of patients with b-cell chronic lymphocytic leukemia, liver cirrhosis, and common variable immunodeficiency to be positive for these two parameters. we now wondered whether, apart from cmv reactivation, tnf-alpha could also cause cellular immunodeficiency this way promoting cmv replication and spreading and eventually causing cmv disease. in summary, we were able to show that a diminished cellular immune response results from/n vitro or in vivo methylprednisolone (mps) is used to suppress both inflammatory and immune responses. il-1 receptor antagonist (il-lra) and tnf binding protein (tnfbp) are naturally occuring anti-cytokine proteins, possibly modulating inflammatory and immunological responses. to define mps modulation of cytokine and anticytokine responses, we examined effects of mps on il-lra production and tnfbp generation as well as il-i~ and tnfa production by human peripheral blood mononuclear cells (pbmc) stimulated with lipopolysaccharide (lps). pbmc were purified from the blood of healthy volunteers, then incubated with lps (10ng/ml) supplemented with different concentrations of mps (0, 500pg/ml, 50mg/ml). after 24hrs incubation, the supernatants were collected for rias of secreted cytokines. the cell lysates obtained by 3 cycles of freeze-thaw, were also collected for r ias of cell-associated cytokines. both the supernatant for secreted cytokines and the cell lysates for the cell-associated cytokines were subjected to rias for il-i~, tnfa, il-1 ra, and tnfbp-i (tnfsrp55). il-la, il-1 ra, and tnfbp were produced mainly as cell-associated forms in response to lps, whereas most tnfa was secreted into the supernatant of lps-stimulated pbmc. mps supplement resulted in reduction of il-la, il-lra, and tnfa production. il-la production was reduced up to 5.4+1.6%(50mg/ml) by mps in a dose dependent fashion, whereas about 50% decrease in tnfc, and il-lra production was observed at both high and low dose mps supplement. however, total tnfbp generation was not reduced significantly by mps supplement. furthermore, tnfbp secretion was increased 12 times more in the supernatant of lps-stimulated pbmc with 500gg/ml of mps. these data suggest that mps could effectively block tnf activity by both reducing its production and up-regulating tnfbp generation and that mps works as an anti-inflammatory drug as well as an immunosuppressant by modulating cytokine and anticytokine responses. in severe gram-negative sepsis, excess of proinflammatory cytokines is associated with increased morbidity and the mortality. we have found that immunocytes possess functional 13-adrenergic receptors (~-ar) which, when activated, down regulate the lipopolysaccharide (lps) induced gene expression and subsequent secretion of tnf-~ in vitro. in the present study, we used a lethal endotoxic model in mice to test the hypothesis that 13-ar stimulation will down regulate the gene expression thus decreasing the circulatory levels of proinftammatory cytokines and improve the survivals in severe endotoxicosis. the animals (iso group) were injected 3 times with !3-ar agohist isoproterenol (0.5 mg/kg i.m. and 0.5 mg/kg s.c.) 4, 2 and 0 hours prior to a lethal dose of lps injection (25 mg/kg i.p.). control group (ctl) received same volume of saline at the same times before the lps insult. at the appropriate time points after lps injection animals were sacrificed and venous blood was collected from the inferior vena cava. the plasma levels of tnf-cc and il-loc were determined by elisa. in the mortality study, each grou the data showed that isoproterenol significantly decreased the mortality and the circulatory levels of tnf-c~ and il-lcc (fig. 1) . these data suggest that 13-ar activation of immunocytes down regulates cytokine gene expression, decreasing circulatory eytokine levels thus reducing endotoxicosis mortality. these results provide a new pharmacological approach to reduce the excess of proinflammatory cytokines and mortality in sepsis. introduction: cytokines released from monocytic cells are thought to play an important role in the pathophysiology of sepsis. during the last few years several investigations have been performed aimed at modulating this mediator response. in the present investigation using a full blood model we have studied the effect of a standard immune globulin and an antitipopolysaccharide (lps) immune globulin preparation and a monoclonal anti-lps antibody in modulating the endotoxin induced cytokine response. methods: citrated blood from healthy human donors was incubated at 37 °c in rotating polystyrene tubes. samples were taken before addition of 1 x 103 ng/l e. col± endotoxin and after 2 hours. plasma was assayed for the cytokines minor necrosis factor alpha (tnf-~) and interleukin-6 (il-6) using elisa methods. the eodotoxin concentration was assayed by lal and end-point chromogenic substrate technique. the blood was preincubated with standard immune globuline (human igg from pooled plasma, gammagardr, baxter) or with an anti-lps immune glubuline (human anti-lps igg obtained by screening of blood donors, novo nordisk) in the concentrations of 4mg/ml or 8 mg/ml or with the monoclonal lipid a igm antibody ha-1a for ten minutes before the endotoxin was added. results: two hours after endotoxin addition markedly elevated tnf-~ and il-6 values were found.in the plasma. cytokine values at 2 hours: tnf-ec 1689 pg/ml (mean) and 1l-6 1123 pg/ml (mean). preincubalion with 8 mg/ml standard immune globulins reduced il-6 values by 49 % (mean) while there were no effects on tnf-c~ values at 2 hours. a slight reduction in il-6 values was also found with the lower concentration, 4mg/ml, but this was not statistically significant. preincubation with anti-lps immune globulins had no effect on il-6 nor on tnf-cc values. preincubation with the monoclottal lipid a igm antibody ha-1a in variable concentrations had no effect on cytokine release in this model. in this full blood in vitro model standard immune globuline reduced endotoxin induced 1l-6 release while tnf-cc values were unchanged.the anti-lps immune glubuline preparation and the monoclonal lipid a igm antibody ha-1a had no effect on the il-6 or tnf-ct release. the effect of anti-tnf treatment in severe haemorrhagic/traumatic shock was investigated in a conscious rabbit model. the jugular vein (for administration of substances and blood withdrawal) and right femoral artery (for measurement of bp) were cannulated and an aortic thermistor probe inserted into the left; femoral artery for the measurement of cardiac output (co). the following day, upto 40% of the estimated blood volume was withdrawn over 80-40 rain, to reduce bp to 40-50mmttg and co by 2/8 mad withheld for 60 rain. shed blood (60%) was then reinfused followed by lactate solution to give a total potential dreulat%ag fluid volume of 120%. zymoeen activated plasma (0,36gg/ml) was given 10 rain prior to haemorrhage and during blood r~n%eion, all ~,~ir, ak were killed at 72h and o~gane removed for histology. rabbita received either monoclonal antirabbit tnf (r6, rat igg1, 8mgkg i.e. n=7) or emine (nffi6), 60 rain prior to haemorrhage. the cardiovascular and biochemical changes during the haemorrhage and hypotensive phase (is. haemorrhagic stimulus) were similar in both groups. upon reinfusion, haematccrit and white cell count remained sigutfia~utly (p<0.05) higher in saline compared to r6-treated animals, indicating a relative haemoconcentration (i,e. reduced circulating volume) in saline animals. the lettkocytosis persisted in the saline animals upto 72h. plasma glucose, lactate and asparteto .m~-o transferase all rose similarly in both gorups but plasma ¢rea~!~+-e levels were markedly (p<0.05) higher in ealine ve r6 animals at 48 and 72h indicative of kidney damage, using a macro and micro. pathological scoring system, major organ damage was seen in the lung, liver and kidney which was significantly (p<0.05) attenuated in r6 treated animals. in the hmg and liver, this was primarily due to a reduced bleeding and pmn infiltrate and to an additional maintenance of tubular integri~:y in the kidney. hence, in tlais model anti-tnf treatment markedly reduced the biochemical and histological indices of severe ,, ~gan damage during liaemorrhage and ranerfn~ion. thermal injuries and systemic bacterial sepsis produce a wasting diathesis with anorexia and marked loss of lean tissue and body fat. endogenously produced cytokines, including interleukin-1 (il-1 ), are thought to mediate many beneficial as well as pathologic host changes that occur during burn injury and infection. to evaluate whether bluckade of il-1 in vivo would affect survival and attenuate the anorexia and cachex[a associated with a thermal injury and chronic fulminant gram negative infection, 24 sprague-dawley rats were studied. anesthetized rats were divided in 3 groups, implanted with alzet r minipumps and randomized to receive the following treatment for 14 days:i: 25 gg/kgbw/min of il-lcc; ii: 25 ~tg/kgbw/min of il-1 receptor antagonist (il-ira); iii: buffered vehicle. the animals were subjected to a 30% bsa, full thickness scald burn and the wounds then infected with 10 + cfu/ml pseudomonas aeruginosa. eight additional rats were anesthetized, but were not burned, and served as healthy controls. . il-hx reduced food intake transiently but was otherwise without effect however, il-lra significantly attenuated weight loss over the first 8 days and improved food intake between days 5 and 8. we conclude that blockade of il-1 after a thermal injury with superimposed infection does not adversly affect the progression of the disease, but does attenuate the anorexia and weight loss associated with this model. these data indicate that il-i receptor blockade may offer a means to minimize the morbidity associated with catabolic illness. tumor necrosis factor (tnf) and its downstream induced mediators have pivotal roles in the pathogenesis of sepsis and septic shock. the suppressive effect of pentoxyfillin (ptx) on tnf production may be of clinical importance. when peripheral white blood ceils were stimulated with either lps or staphylococcus aureus, pentoxyfillin inhibited tnf production. in contrast, no inhibitory effect was observed on the induction of il-6. ptx inhibited not only the tnf production of monocytes but also the tnf secretion of both granulocytes, and unseparated whole blood. the facs analysis revealed that the expression of cd14 antigen on monocytes was not influenced by pentoxyfillin. the in vitro tnf and il-6 producing capacity in septic patients were higher than in the control group, but decreased on subsequent days especially in fatal cases. administration of ptx (200 mg/day) in septic patients resulted in tnf and il-6 productions similar to those found in control donors. it also subsequently led to an improvement of the clinical status. a further improvement in apache score could be achieved by administration of pentaglobin ° (biotest)( 5ml/kg/day ). the prevention of lpsinduced in vitro tnf and il-6 production by pentaglobin ° could be demonstrated in in vitro experiments involving the use of whole blood rather than purified lymphocytes, very probably because of the presence of lps binding protein in the plasma. the level of soluble icam-1 in sera of septic patients was significantly higher (800-1200 ng/ml) than in normal individuals (50-150 ng/ml), but it decreased following the ptx and pentaglobin ° therapy. it is presumed that ptx and pentaglobin ° can antagonize eytokine production at different levels, resulting synergistic action being beneficial in the treatment of sepsis. albert szent-gy6rgyi medical university, institute of microbiology, szeged, hungary h-6720 ddm t. 10. e~i~im~/tii, septic sh0~ '. tnf alfa/pge, and somatostatln lands ji., alvarez j., gram m., llanos k., alcalde j., sanchez ja., balibr~d jl. taurine, a semi-essential sulphur-containing t:~-amian acid, promotes neutrophil phagocytic activit3' against yeasts and enhances intracellular killing of e.coli. paradoxically it protects against hypochlorous acidmediated biomembmne oxidatio~ and abrogates the pyrogenic effects of intracerebral endotoxin. it is therefore possible that taurine mediates some of these effects by modulating the cytokine cascade. aim to assess the cytekine responses in a rat model of intraperitoneal sepsis, pretreated with supplenmntary taurine, by measuring tnf and il-6 concentrations. methodology female wistar rats (wt 160-200g) (n-6 per group) were prefed with 2% tanrine, 3.33% glyciuc ( in tap water) or tap water (tw) for 15 days prior to caecal ligation aud puncture (clp). normothermia was maintained intraoperatively and the animals received 0.9% saline subcutaneously (3 mls/100g) post-procedure. animals were venosected by direct cardiac puncture at 6 or 18 hours post-operation and the blood placed in endotoxin-free tubes. centrifilgation and storage of plasma at -70°c was completed within 2 hours. tnf and il-6 were measured using wehi and b9 bioassays respectively. results (means ± sem) bioactivc tnf concentrations were not significantly different between clp or sham groups. however plasma il-6 estimation revealed a statistically significant reduction at 6 hours in tanrine-treated animals compared to glycino and tw controls ( objective: to evaluate the effects of allogeneic blood transfusion, thermal injury and bacterial garage on interteukin 4 (il-4), tumor necrosis factor alpha (tnf) production and host mortality and to study if the administration of thymopentth (thy) could affect these events. materials and methods: balb/c mice were transfused with allogeneic blood (from c3h/hej mice) and treated daily with thy (i mg/kg)(t+thy) . control animals were transfused but not treated (t). after 5 days systemic blood was harvested to determine the circulating levels of il4 and tnf. a second group was treated with thy for 5 days and then received a gavage wide lxl09 escherichia coli and a 20% burn injury (bg+thy). one hour post-bum, blood was collected to measure cytokins. control animals received the same treatment but thy (bg). a third group was transfused, treated with thy for 5 days and then received gavage and burn (tbg+thy). one hour post-burn, blood was collected to measure cytokins. control animals received the same treatment but thy (tbg). all treated and control groups had 8 mice/group. in separate groups (n=20/group), receiving the same treatments, mortality was observed for 10 days post-burn or transfusion. the production and release of oxygen radicals by phagocytic leukomytee is one of the most relevant and important functions of these substances in biology. when neutrophilic granulocytes or certain macrophages phagocytize they produce super oxide and hydrogen peroxide and form secondary products of these activated species. all of these substances are released in to the phagosome. it has been appreciated since 1977 that the amount of oxygen consumed by phagocytes in producing free radicals is prodigious. what has not been totally appreciated is the significant contribution this 02 consumption contributes to total body oxygen consumption. our understanding of increased oxygen consumption following sepsis is compounded by a paradox of increased cardiac index and a narrow av02 difference. it was mclean in a study reported in 1967 that showed an increase in cardiac index and a narrowing of the avo z difference in humans following sepsis. other studies documented similar changes in humans and it has recently been speculated that increasing o~ delivery might have a favorable impact on outcome. at least three different explanations have been put forward as to why there are possible alterations in energy metabolism during sepsis. these include decreases in oxygen supply, peripheral shunts and direct action o~ cellular mediators on o~ delivery, a provocative review by hodgkiss and nark in 1992 shewed that in experimental animals sepsis did not cause any evidence of bioenergatic failure in muscle i liver, heart or brain tissue. they further showed that the increase in serum lactate is mot necessarily due to cellular hypoxia. they conclude that cellular hypoxia and defects in energy producing metabolites are not the cause of metabolic abnormalities in sepsis. we set out on a series of experiments to try to explain the paradox in cellular metabolism and whether or not white cells might contribute to total body oxygen consumption during sepsis. the first set of experiments involved growing rat cardiac myocytes on tissue culture. pyruvate was added to the medium as substrata and physiologic levels of peroxide were also added simultaneously, carbon 14 labeled co~ production was measured as was nucleotides and degradation products from the cell. the peroxide induced a significant inhibition of pyruvate dehydrogenase. in addition, there was a loss of cellular atp and a corresponding rise in the release of inosine and adenine from the cell. we concluded from this first sst of experiments that physiologic levels of peroxide are a potent inhibitor of pyruvate dehydrogenase in isolated cardiac mitochendria as well as the cultured myocyte. we further showed that pyruvate dehydrogenase inhibition blocks the aerobic oxidation of glucose and leads te adenine nucleotide metabolism with adenosine and inosine release from the cell. other enzymes within the tca cycle did not appear to be specifically blocked by peroxide. this would explain the increase in lactate and the ability of the cell to continue to make high energy phosphate by entry of metabolites in to other entry points of the tca cycle, the second set of experimbnts was designed to estimate the magnitude of whole body reactive oxygen generation via nadph oxidase following granuloeyte activation invivo. using a guinea pig model baseline oxygen consumption was determined. ~iaphenyl iodinium (dpi) was used as a specific inhibitor of granulocyte nadph oxidase. animals were divided into two groups; those that received dpi and those that served as control, all animals were then injected with phorbcl myristate acetate (pma) intravenously. pma is a potent stimulus for granulocyte activation and subsequent reactive oxygen generation. whole body oxygen consumption measurements were then repeated after the p~la injection. in addition, arterial blood gas amalysis was performed, circulating neutrophils were collected and assayed for their ability to generate hydrogen peroxide and the r~ght lung was removed for wet and dry weight measurement. total body oxygen consumption increased by 25 percent after pma injection. arterial oxygen tension fell significantly in the control animal. neutrophil hydrogen peroxide generation rate doubled and lung water in the untreated animals increased by 25 percent. in this animal model we could show that granulocyte activation substantially increases whole body oxygen consumption to approximately 120 percent of control values. the nadph oxidase inhibitor, dpi, decreases granulocyte hydrogen peroxide generation invivo and prevents the pulmonary injury induced by pma. using this animal modal it is possible to transpose this to the human septic state. if we assume there are 5,000 neutrophils per cubic millimeter of blood, a 70kg man would have ~1.75xi0 u circulating neutrophils. when activated ixl0 ~ neutrophils generate one nanemole of hydrogen peroxide per minute. t~erefore, the circulating neutrophil pool could generate -1.75xi0 nanomoles of hydrogen peroxide per minute. this corresponds to an oxygen consumption of -4.2ml/ojmin by the circulating neutrophil pool alone. this does not take into account the production of oxygen by macrophages or the amount of oxygen consumed to produce nitric oxide. manipulation of oxygen delivery in human sepsis may or may not be beneficial. irshad h. chaudry, ping wang, and alfred ayala life threatening blood loss, due to soft tissue and bony injuries, is a frequent complication in trauma victims. although numerous organs and cells are adversely affected by hemorrhage, the focus here will be to discuss whether or not there are any differential alterations in splenic and hepatic immune functions. in particular, m~ antigen presentation (ap) and associated processes will be described since one of the important functions of the m6 is to activate resting t-cell lymphocytes by processing and presenting foreign antigens in a mhc class ii-restricted fashion. studies have shown that splenic m6 and kupffer cell (kc) ap was significantly depressed following hemorrhagic shock and remained so for at least 96 h alter resuscitation. the presentation of antigenic fragments associated with mhc class ii antigen itself was not decreased following hemorrhage, but the catabolism of antigens in antigen-presenting cells was decreased. this is likely due to a significant loss of cellular atp. although m6 ap was depressed in splenic, as well as kc, only kc showed an enhanced capacity to produce inflammatory cytokines, il-1, il-6, and tnf. this difference in response between kc and splenic md may be due to differences in the microenvironment in which these cell populations are found, as well as potential differences in the effects that hemorrhage has on the environment from which these cells are obtained. splenic m6 from hemorrhage-resuscitated mice exhibited a significantly reduced cytotoxic response, whereas kc showed an enhanced cytotuxic capacity. the increased kc cytotoxicity is probably mediated through the increased expression of cell-associated tnf and the release of reactive nitrogen. the enhanced production of reactive nitrogen may play an important role in the clearance of microbes translocated from the gut following hemorrhage. however, excessive release of reactive nitrogen by kc may have deleterious effects on the adjacent hepatncytes. such an effect could, in part, explain the reduction in active hepatocellular function, which is seen following hemorrhage-resuscitation. thus, hemorrhage induces differential effects on splenic and kc m6 populations; it decreases splenic m6 but increases kc capacity to mount a cytotoxic response. the depressed splenic md and kc ap was, however, significantly improved by posttreatment of animals with atp-mgcia, chloroquine, diltiazem or interferon-'/. these agents also decreased the susceptibility to sepsis following hemorrhage. thus, the use of atp-mgci~, dfltiazem, chloroquine or interferon-7 offers new therapeutic modalities in the treatment of immunosuppression and for decreasing the susceptibility to sepsis, which is observed following severe blood loss. the production of macrophages from bone marrow precursor cells is a dynamic and highly regulated process. the differentiation of myeloid lineage-restricted progenitor cells is initially controlled by interleukin-3, which drives the progenitors along a maturation pathway that leads to their response to specific lineagerestricted colony-stimulating factors(csfs) we have previously hypothesized that thermal injury can initiate a self perpetuating cycle of production of defective immune cells: thermal injury can initially affect circulating immune cells. then these cells, by unbalanced production of colony stimulating factors and other mediators, can cause a derangement of hematopoiesis resulting in an abnormal production profile of tnf, il-i, and il-6 and cytotoxic activity of bone marrow derived cells. the results of our studies so far have supported parts of this hypothesis. il-3 and il-3+m-csf treatments increased the production of tnf in burn 4 day macrophages and progenitor cells compared to normal cells. il-3+m-csf+il-10 increased the production of il-6 by burn progenitor cells compared to normal macrophagee. preliminary results for the ratios of tnf/~ actin mrna indicate that il-3+m-csf increased the mrna for tnf in the 4-day macrophages derived from burned animals compared to macrophages derived from normal animals. ratios of il-6/~ actin mrna indicated that il-3, il-3+m-csf, and m-csf increased the il-6 ~rna in progenitor cells from burn animals compared to progenitor cells from normal animals. these preliminary results support our hypothesis that bone marrow derived macrophages and progenitor cells from burned animals act differently compared to cells from unburned animals regarding the production of inflanunatory cytokines. more specifically, the results suggest that the different cell types are regulated in different ways by cytokines, and cells from burned animals are regulated differently than cells from unburned animals. it i,~ to ~ssume that the translneatinn is due to a ixx~h,v functioning ~astroiutestlnal surface pro|ection system, which leads to an unacceptably high load ¢ln the imrsunc dcfcncc system, partlcul~ly the local system in the gastrointestinal wall, leading tu exh~ustiou of ihi~ system. tlae g&~lrointesliual i]'ac( can be regal'deal a.s a t~2servojf of appr 200 m ~ separating 10 i~ eucayotic ceils of the hosl ttom l0 ~4 bacterial culls. in the human body the the myriad of cndngan¢nts micrix)rganistns, exogenous itl,~ttdittg micro~ganisms altd taxies m-'e sepia"areal li'otll lhe rest of the body by a simple epithclhd layer. the barrier function is heavily tlcpcnding tm =~ proleclive layer cmtsisting ie. surl~:emts, nluclts, probiotic bacteria, and ,~ub~tances with strong antioxid~lt, antipfotease al~d other i~rotective eft'ecru, the surfaet~ml htycr consists at tile besl if1 i(.) bimolecular layers, fl is thus very iltil~, the epilhelial layer is renewed every 24 hours , and tile tanuwer of the mucus is ~so fa,~t, all attdphy of die epithelial layei induced by g&sttointestinal st~'vation does also include the mu~usprndocing cells, gnhlet cells, which leaduhg lu slmnage aad luwer quality of gi mucus, altered viscoelas,jc properties and reduced protection, ]~'obioiic bactcrla keeps the ppm's low, produce nutrients for the intestinal mllcosa, eliminates tuxia~ and stimulates the local immune system, the gl surface is conslanlly under hc wv wearing by ingested bacteria and their enzymes, ingested substaucas, chefftie,'ds at~d toxins. 'll~e n~tective flora is reduced in disease, by stress and by cm'clcss antibiotic therapy. wc have made attempts tn recondition the i,,.astroiniesli!l~d. tnucosa by rcstlpply of surfactants or sttrfactanfliko glyc~lipids, gels with pseudomuein [until.oil, and probiotl¢ laetobaeilli. by supply of glycolipids we have bt:cn able m prevent and heal intlammatory and tlleerallve injuries (and io p)'cveut microbe transhteatkln) in uppe~' and lower (.31 u'acl, this c~al be fu~'ther augmentented by shnultaueos supply el' a p.~uedomucin,likc gel like pectin. ()at contain~ one hundred times more ot membrane lipid$ th~a =nay other food, much of fiber, ~spceially watcrsnlnhlc betaglucaal~, at~d has an close to ideal amino acid cornpo~ition, <')at, termented by species-specific lactobacilli, has a strol~ ability to prevent local inflammatory and ulcerative chunges,transh)cadnn and sepsis in experimeut~d attimals mid to pl~ven~ revert mof in ba,nan.~.lt seems us, that it the mucos~lnucus/probiotic bacterial filnclinns cat'= 1%' restated by mucosa reconditioning, even in severely sick iudividuals) the hx:~d iuuuuue ~y,~teme will be capable tu pr~)tec! the ix~y ag~lls~ sepsis ~nd mof. the impaired immune response associated with multiple system organ failure (msof) has been most widely studied following surgery for inflammatory disease and trauma. the majority of late deaths following trauma are due to infection and msof. not all patients with multiple organ failure have concomitant infection present, yet most have been septic at some time in their hospital course. their generalized inflammatory condition often persists whether or not organisms have been recovered. immune failure probably precedes msof with or without overt infection and indeed may perpetuate such widespread sepsis. macrophage tumor necrosis factor-alpha (tnf) production is thought to represent an important pathogenic mechanism by which this is mediated. cecal ligation and puncture (clp) is a clinically relevant murine model of intra-abdominal infection and sepsis. serum tnf and endotoxin levels, and peritoneal macrophage tnf production are only slightly elevated in this model even in endotoxin sensitive animals. mortality in this model, therefore, does not appear to be attributed to overwhelming endotoxemia and/or tnf production. it has therefore been postulated that tnf and other cytokines may act in a paracrine fashion to cause local injury. tnf messenger rna (mrna) expression was therefore measured and found to be increased in the lung and liver following clp. a different pattern of expression was seen after endotoxin administration, characterized by a more rapid rise and fall, and enhanced tnf mrna expression in the spleen as well. route of spread of infection as well as the type of stimulus may determine the organ specific cytokine response. these data support the concept of locally produced tnf as a contributing factor in tissue damage and multiple organ failure during sepsis. the estimation of histamine's role in sirs changed over the years based on increased knowledge in shock pathogenesis, evidence later found to be faulty, and the discovery of other, more fashionable mediators. over the decades, the prevailing view has been that histamine is a noxious mediator contributing to the fatal outcome of shock. the analysis of experimental data on exogenously administered, endogenously released and/or newly formed histamine in septic/endotoxic shock suggests that histamine contributes to the early cardiovascular changes via hi-receptor vasoconstriction thus excerbating (directly and indirectly) the effects of catecholamines. in the later phase of septic shock (low out-put, high resistance states), however, it can be assumed that histamine exerts strong vasodilator and positive inotropic effects via h2-receptors. these effects are considered beneficial in counteracting or attenuating the effects of sympathetic responses of catecholamines and other mediators. thus, a blockade of the early reaction with hi-antagonists and a stimulation of the h2-receptors by h2-agonists are worthwhile therapeutic approaches. shock produc~s ~ij alterations, an encrgy crisis and eventual c~ll damage, however, shock in itse|f do~ not lead freqoenfly to r~mote organ damage. in animal ~xpcrhnents and clinical expariez:ces in korea and vietnam, shock i~r s¢ was not associated with lung damage, renal failure or ards. the same is true with g.l bleeding. however, when shock is due to trauma or associated with tlssus injmy, then organ malfunction m~d damage is frequent. shock is bclleved to increase the frequency of infection, but clinical evidcnc~ for this is scant. hemonhsgio shock akme is an unusual o~orrenee dinica[ly. immune dysfolletion dons occur witl~ experhnental hypovolemic shock and in pailcnts with hypotcnsion or after receiving blood ~ansfosions. tbc most significant factor affecting reoorrcnce of resented ca,lorectal liver mclastasis was hypotensivc episodes during operation. blood transfosions depress immune function, improve transplant ~raft survival and increase ttll'rmr rec~r'tcnos ill co[eli and head and nc~k cancers. in experimental hemorrhagic shock, we found decreased response of [ymphocytes to mitogens, el:clad mixed lymphocyte reactilln and i[,-2 decreased. othe~.x hsvc found decreased macrophage and t and b ¢¢lt function, deci~ased re,ease of/l~i, increased poe v depressed macrophagc antigen presanlatiota, increased ien y, decreased il-2, 3 and 5, and shared mscrophags funclion with loss nr f and c 3b r~ccpto~s. this is inhibited by chlornqoine, ibuprofen, dihiazem, and atp. shock may activate ncutrophils contributing to ilappitlg in cspillarics, no refluw, increased adhesion, cytotoxicity and organ damage. this may be related to decreased clearance of bacteria after shock. t~lls shock, hypotcnsion and decreased microcirculatory blued flow initiate or set the stage for immune dysfmtction. they, along with tissue injury trigger inflammatory cascades. this also contributes to immunologic dysfonctitm, which is associated with increased lufoction. thus altered bh,od flow and mediator cascades are bolh involved, the question now is can wc rapidly improve microcirculatory blood flow, and dg'masa inflsrmnatory fosponss, which is also necessary for survival the effe~ of ~nterferon ~amma @n wour~ healing was ewaluated ~n a routine mo~el. ~mma ~n~erferon we8 g~ven in doses of ~37.5 to ~2,500 u~%$ synchronous with ereatio~ of a pa~splnal woumd then daily for is ~lays. ~mteet~on ~amma impaired tensile strength a% all ~oses relative to control. m0zphology suggested ~reas~ ¢oll~gen deposition and r~du~-~ neovag~ulaeit¥ ~n t~eat~4 animals, interferon ~amma was a2so ~valuated in a murine mo~el of cecal llgatlon and punneure. one hundred to 22,s00 uni~ vere ~iv.n following ~nju~ an~ than daily. interferon qamma was associated with inco:ea~ed mortalit~ and earlier death a~/a~n ~n a dose dependant manner (p< 0,05). afmlnistra~ion the proinfiammatory cytokines tumor necrosis factor-c~ (tnf-c0, interleukin-16 (il-ii3), interleukin-6 (il-6), and interleukin-8 (il-8) have been implicated in the pathogenesis of the multiple organ dysfunction syndrome (mods) following shock and trauma. these mediators which are released in high concentrations by activated macrophages, endothelial cells, and connective tissue cells cause a systemic inflammatory response syndrome (sirs), thus leading to a shock-like state and tissue destruction. recently, a number of proteins have been characterized in in vitro and in vivo studies demonstrating potent anti-inflammatory properties. these latter cytokines include interleukin-4 (il-4), interleukin-10 (il=10), transforming growth factor-6 (tgf-j3), and the newly sequenced interleukin-13 (il-i3). they are considered antiinflammatory, since in vitro studies using purified macrophage cultures or whole blood assays revealed an inhibitory effect of these antmnfiammatory cytokines on ~he synthesis and release of tnf-cl and il-u3. in addition, they reduced the severity of disease and reduce plasma levels of tnf-c~ and il-ii3 when administered to animals with infection or inflammation. furthermore, naturally occurring substances have been described that specifically neutralize the bioactivity of il-1b and tnf-a. the il-1 receptor antagonist (il-i ra) is related structurally to il-i and binds to the same cell-surface receptors, but does not stimulate the cell. in animal models ore. cob-induced shock, inflammatory bowel disease, and peritonitis, injection of il-lra significantly reduced the severity of disease. the cytotoxicity of tnf-c~ can be inhibited through two types of soluble tnf receptors (stnfr) type i (p55) and type ii (p75). these stnfrs are proteolytic cleavage products of the cell-bound tnfreceptors. when given to baboons to combat e. coil-induced shock, soluble receptm's to the tnf type [ receptor decreased the severi~ of the shock-like state. it has been well accepted that shock and severe injury result in an increased release of proinflammatory cytokines with a high incidence of sirs and mods. our studies investigating plasma levels of anti-inflammatory mediators (il-4, il-lra, stnfrs) in patients after severe injury further suggest that shock and injury lead to an activation of anti-inflammatory processes with altered plasma levels of these mediators. however, while plasma levels of il-4 and 1l-lra were significantly increased in trauma patients compared to healthy volunteers, stnfrs did not differ from control samples. thus, shock and severe injury result in different activation patterns of anti-inflammatory processes. m,l. rodrick, boston, b~usa following severe thermal injury significant suppression of the immune response has been demonstrsfed. these changes are associated with increased susceptibility not only to co,on human pathogens, but to organisms not normally pathogenic in the uncompromlsed host. early observations included prolonged graft rejection and skin test anergy in patients following burn injury. work from our laboratory and that of numerous others has shown that the~e is a profound £mmunoeuppresslon following severe thermal injury. i~mune deficiency has been identified in these patients hyz ii lack cf t nell responses to mitogens, 2) early decreases in total t and helper t cells in the peripheral bloo~ 3} lack of response to tetanus toxoid i~uniza~io~ in spite of increased non-speoifi~ i~k~unoglobulin praduction and 4) severe and prolongei deficiency of interleukin-2 (il-2) prcductlon by peripheral blood mononuclear cells (pbmo). this il-2 deficiency could be an underlyin~ cause of all the afo~ementloned immune defects by failing to activate both helper and suppressor t cell functions. mo~a recent work has focused on the molecular mechanisms of this il-2 deficiency end shown that the defect lies post-transcrlptionally since mrna for il-2 is also depressed following thermal injury. we have also investigated the potential of a defect in ii-2 receptor status on peripheral blood mononuclear uolls from patients fqllowing burn injury and in a murine model. in both oases no decrease in il-2r was found either by phenotypic markers or by funational assays. another hallmark of thermal injury is hyperao~ivatlon of proinflammatory ~ytok~ne secretion, which may play a significant role in multiple organ failure following burn injury. therapy o~ major burn injury may reasonably be aimed, therefore, at up-regulating t cell ~unction and down-regulating hyperactive secretion of proieflaam~ntory cytokines. the majority of patients dying of bums succumb to sepsis which coincides with dysfunction in the specific and non-specific host defences. generally, concepts relating to the mechanism(s) of specific immune failure in thermal trauma imply that inhibition of t (cd4+) cell responses is imposed by the injury or infection-related factors. however, the same factors elicit strong biological reactions within the lymphoid compartment. recent evidence indicates that systemic activation is inherent in the burn patient. to establish the relation of molecular and cellular events to the development of post-bum anergy we examined the state of and the capacity for t cell activation with regard to: 1) occurrence of activation-induced cell death (aicd), 2) activation-related phenotypic and functional_ diversity in the pool of peripheral cd4+ t cells. initially, (up to 7 days post-bum), peripheral blood lympliocytes from severely injured (> 35% total body surface area) patients exhibited high levels of constitutive expression of surface receptor for ]l 2 (cd 25) and spontaneous blastogenesis. the presence of activation-related t cellproducts in bum plasma was also apparent. subsequent impairment of the t cell receptor (tcr)-regulated t cell responses in vitro was accompanied by significantly increased dna fragmentation that is associated with cell death by the mode of apoptosis. using molecular markers we established that flesh peripheral blood ceils from immunosuppressed patients also contain large numbers of apoptotic cells. fluctuations in the number of viable (pi-) peripheral blood lymphocytes involved primarily cd4+/cd45ro+ (memory) subset of t ceils. the above observations suggest that thermal trauma-associated t cell anergy develops through aicd, a phenomenon commonly associated with the tolerogenic activity of bacterial superantigens. persistence of staphylococcal infections in the burn patient may support this assumption. response following trauma jane shelby, ph.d. the immune system is integrated with other physiologic systems, and is exquisitely sensitive to changes in nervous and endocrine systems changes following traumatic stress challenge. the immune, nervous and endocrine systems interact via both direct and indirect pathways which utilize neuro and endocrine hormones, neurotransmitters, neurepeptides and immune cell products. it is now known that the immune system may be affected by all of the neuroendocrine products produced during a stress response, with evidence for innervation of iymphoid organs, lymphoid cell receptors for neuroendocdne products, and leukocyte production of chemicals which are virtually identical to certain neuroendocdne peptides (acth, endorphins). trauma induced alterations in the equilibrium of various neuropeptides and neuroendocdne hormones have a significant impact on immune response potential, affecting control of proliferation, differentiation and function of immune cells. for example, the neurohormone melatonin is thought to be a natural antagonist to counteract glucocorticeid associated immunosuppression resulting from stressful challenges, such as surgery and trauma, plasma melatonin levels are known to be significantly reduced in burn patients. the administration of exogenous me[atonin improved cellular immune response following burn injury in an animal model. melatonin was also shown to have in vivo cytokine regulatory activity, increasing the potential for il-2 secretion and downregulating excessive il-6 and ifn~ in burn injured, stress susceptible mice. the regulatory interactions between the immune, nervous and endocrine systems provide mechanistic pathways for trauma associated immune dysfunction. increased knowledge of these interactions will enhance the potential for the design of novei clinical interventions to improve immune response and decrease the risk for infection in trauma and surgical patients. . animals receiving e were given a single dose daily of either 2.4 g/kg of e in a 20% solution by garage (ge), or 1.25 g/kg of sterile ive in saline. four hours following the last dose, bum animals were subjected to a 30% body surface area bum injury to their dorsum. twentyfour hours following injury, the animals were sacrificed and spleen cells were harvested for assessment of lymphocyte function. splenocytes were prepared by mincing the spleen, followed by incubation on glass petri dishes to remove adherent macrophages. non-adherent cells were then tested for proliferative response to t-cell mitogen concanavalin a (con a) and b-cell mitogen lipopolysaccharide (lps). data were analyzed by anova. results: chronic alcohol exposure and burn injury independently inhibit lymphocyte response to con a but not to lps. the combination of e plus bum injury, however, pmfouedly decreases this response to both con a and lps as outlined in the this data clearly identifies the synergistic impairment of immune function produced by ethanol and bum injury. it is furthermore apparent that ibis effect is gut mediated and that gastrointestinal exposure to alcohol is necessary to produce this effect. further studies will work to identify cellular and subcellular mechanisms to explain this effect. in experimental animal studies and investigations on human volunteers endotoxin infusion is mgulary accompanied by the release of the cytokine tumor necrosis factor a (tnf-~) determined by elisa technique. in patients with menigococcal sepsis also elevated tnf-a values have been found using a functional assay. we have studied the role of tnf-et in surgical icu patients with sepsis. using functional technique, we were not able to detect tnf-~ activities in the patient plasmas. when this cytokine, however, was determined by immunochemicai technique (el1sa) elevated tnf-e~ values where frequently oberserved. in order to further elucidate these observations, we studied shedding of tnf receptors in the patients. in these studies, we noticed that shedding of tnf receptors oecured regulary in the patients. at the time of diagnosis, soluble tnf receptor p55 and p75 were both 2-3 fold higher than values found in plasma samples obtained prior to die diagnosis of sepsis. we also observed that the sepsis patients revealed higher maximum values of p55 and p75 during the icu stay compared to values found in surgical icu patients without sepsis. these observations indicate that soluble tnf receptors are available in sufficient amounts to bind tnf-ot which is released in surgical patients developing sepsis. this mechanism may explain why functional tnf-c~ was not detected in the patients. institute for surgical research, rikshospitalet, the national hospital, university of oslo, 0027 oslo, norway. decker, d., sch6ndorf, m., bidlingrnaier, f., hirner, a., yon rfcker, a. the advantage oflaparoscopic cholecystectomy over conventional open surgical approaches in the treatment of symptomatic cholelithiasis has been shown convincingly by clinical studies. in order to facilitate comparisons of different surgical approaches, we evaluated the cell biological characteristics of tissue trauma by measuring changes in various cell surface markers on leukocytes and eytokines in plasma as a possible means to assess tissue trauma in choleeystectomy. patients recruited into our study had experienced at least one typical bifiary colic, had ultrasound-proven cholelithiasis (stages 0-ii according to me sherry), were 35-55 years old, and presented for elective choleeysteetomy. patients could choose between laparoscopic and conventional eholeeystectomy after being informed about the advantages and disadvantages of each procedure. cell surface markers on leukoeytes were determined using whole blood techniques with the help of commercially available fluorescent monocloml antibodies and flow cytometry. shed cell surface markers in plasma and cytoldnes were measured with the help of sandwich-elisa kits. blood samples were drawn 24 h before surgery, immediately before incision (after anaesthesia), 2 h and 24 h after incision. seventeen cell surface markers were examined on different cell populations and cellular subsets in 18 laparoscopic and 15 open-surgery patients. three soluble cell surface markers and six cytokines were monitored. by statistical analyses (multivariate regression analysis, student's t test, wilcoxommann-whituey's rank sum test) the six markers/cytekines that best distinguished open surgical from laparoscopic procedurea were determined. these were 1. the interleuldn-2 receptor and im soluble form (cd25/scd25); 2. the activation antigen fd-1 and its soluble form (cd30/scd30), a member of the nerve-growth-factor receptor family; 3. the cd45ro epitope which characterizes t memory ceils; 4. the trausferrin receptor cd71; 5. the soluble adhesion molecule icam-1; and 6. the cytokines interieukin-6 and interleuldn-8. on the basis of these results, a tissue trauma activation (tta) index was calculated by combining the marker/cytoldne measurements by simple multiplication. anaesthesia and pre-ineision maneuvers did not significantly change cell marker or cytokine levels in either surgical approach as compared to 24 h before surgery. 2 h after incision the tra index in open cholecystectomy showed a distinct 22-37 fold increase, whereas in laparoseopic surgery a mere 2-3 fold increase was noted. 24 h after incision, the tra-index returned to near pre-surgery levels. in conclusion, our results demonstrate that changes in cell surface markers and cytokines can help evaluate the magnitude of tissue trauma in diffei'ent surgical approaches. the relationship between lymphocyte subpopulation changes after thermal injury and the increased susceptibility of burned patients to infection is unclear. in this study, we have attempted to correlate such subpopulation changes with the presence of infection in burned patients. peripberal blood from 19 patients was monitored for lymphocyte subpopulation changes three times weekly for three weeks postburn and weekly thereafter for three additional weeks. mean bum size was 44.7% (range 24%-84%) of total body surface and mean age was 38 years. infection was diagnosed by carefully defined clinical and laboratory criteria and its presence or absence noted each time blood was drawn. samples taken when patients had wound infection, bacteremia, or pneumonia were compared with samples taken in the absence of systemic infection. whole blood samples were stained with four monoclonal antibodies, the red blood cells lysed and the leukocytes fixed and analyzed by flow cytometry. for each patient sample, the proportion of lymphocytes falling within the light scatter gates was determined as the percentage of cells negative for cd14 and most strongly positive for cd45. this percentage was used to correct each sample for the presence of debris or nonlymphocytic cells. the proportion of cd4 and cd8 positive cells was slightly greatc~ in the samples from infected patients, while the proportion of b cells (cd20+) was unchanged and nk (cd16+) cells were decreased by ahnos[ 50% compared to sampie~ li'om uuiuleclcd patients. the percentage of cells positive for cdilb (c~ integrin) decreased sharply and cd4ro (memory cells) decreased slightly in samples from infected patients while the expression of the lymphocyte homing receptor and cd29 were unchanged. cd25 (il2 receptor) and cd69 (early activation marker) were significantly increased in the samples from the infected patients while hladr was unchanged. these changes in lymphocyte phenotype correlate with the presence of infection. if they closely precede or occur during the early development of infection they may be valuable clues to the mechanism of susceptibility following thermal injury. trauma patients are subjected to an immediate massive impact on their host defense integrity due to the combined effect of tissue trauma, shock and endotoxemia. cytoldnes are playing a crucial role within the course of an impaired cell mediated immune response (cmi) resulting from a disruption of intact m%/tcell interaction. the current study was undertaken to further elucidate the mechanisms of dysfimctional cmi following major burn and mechanical trauma -via comparative analysis of mrna expression and protein release. the major regulatory levels for different cytokines were determined in mitogen, respectively lps stimulated peripheral blood mononuclear cell (pbmc) cultures of trauma patients on consecutive days (13) t, 3, 5, 7 and 10 post injury. we analyzed the cumulative data for interleukin-1 beta (il-i[3), il-2, il-8 as well as tumor necrosis factor alpha (tnf-~) and saw a considerable impairment of the protein release in the stimulated pbmc cultures until d5 post-trauma and recovery thereafter. *p < 0.05, ** p < 0.01 vs control comparing the autoradiographies of the specific cytokine mrna expression with the protein release in the supernatants, we saw a good correlation between mrna signal intensity and protein synthesis for il-8 and 11,-2, suggesting that for these cytokines the main regulatory mechanisms are located at the pre-/transcriptional level. for the other cytokines investigated one has to suppose posttranseriptional mechanisms. the analysis of our data clearly indicates a severe impairment of forward regulatory immune mechanisms following trauma. most likely the regulatory mechanisms, that are involved are greatly different among the cytokines investigated. it may be concluded, that depressed cmi responses post-trauma are partly due to an impaired pro-inflammatory cytokine production. the severity of the injury (iss) correlated with the development at multiple organ failure (mof-score; r=0.612). the levels of mediators and markers of the inflammatory response were generally higher in the more severely injured group (iss>30, n=16). i1-6, 11-8, g-csf, fpa, and c3a -levels differed significantly (p<0.005) between the iss-groups (>-< iss 30) at the time of admission, whereas on day 3 tnfa, c3a, 11-6, and ealpi showed significant differences. beyond the first week, major differences were restricted to pge 2 and c3a. the formation of two groups with respect to later multiple organ failure (mof < 3; mof > 2 n= 14) yielded similar results. leukocyte-facs analysis revealed significant differences mainly in the cd14 (monocytes), cd3/cd25 (i1-2r + t-cells), and cd29/cd4 (th 2calls) populations. summarizing our findings we were able to detect some alterations in the surface antigens of immunocompetent cells. the inflammato d response, however, seemed to be more pronounced and correlates wi~ the further clinical course. using an experimental bum model in rodents, we have demonstrated that administration of a full thickness, scald burn involving 20% or more of the total body surface area (tbsa) elicits systemic responses which are characterized by numerous alterations in t-ceu function (i.e., lymphokine production and contact hypersensitivity (ch) responses) plus an enhanced susceptibility to bacterial infection. in the present study we questioned whether the apparent systemic effects mediated by large burns would be elicited as site-specific alterations in immune function following administration of small area burn trauma (5% tbsa). following a 20% tbsa burn, ch responses to contact sensitizing antigens were found to be altered. the depression in ch responses could be induced independent of the site used for topical skin sensitization. following a 5% tbsa thermal injury, development of ch responses were affected in a site-specific manner. immunization of 5% tbsa thermally injured mice in a site near the position of the burn resulted in depressed responsiveness, whereas immunization through a contralateral site resulted in responses that displayed both the intensity and kinetics of a ch response equivalent to sham-bumed mice. similar systemic and site-limited changes in lymphokine production were observed with 20% and 5% tbsa thermal injuries, respectively. a 20% tbsa injury affected the lymphokine producing potential of all cells regardless of which lymphoid tissue the cells were isolated from. the effect of a 5% tbsa burn was significant but site-specific. thus, ceils from lymph nodes receiving drainage from thermally injured tissue were specifically affected, whereas lymphokine production by cells from lymphoid organs receiving drainage from unaffected skin was normal. it was concluded that modulation of lymphokine production and cellular immune responses may be a normal consequence of burntrauma regardless of the size of the burn. changes in immune competence can be mediated either regionally or systemically in direct proportion to the area of skin exposed to the burn injury. this work is supported by phs grant gm46899 and the office of navy research n00014-92-j-1612. division of cell biology and immunology, department of pathology, university of utah school of medicine, salt lake city, ut 84132. post spleneetomy septic sequelae may be fatal, but the mechanisms remain unclear. the objectives ef this study were to assess the mortality from concomitant splen-'etomy and ]~eritoneal bacterial challenge and to elucidate the local cetkdar responses. 50 cd-1 mice were randomised to receive laparotomy and sham splenectomy (l) or splenectomy (s) with simultaneous ca'-cal ligation and "):mcture and the survival patterns assessed. subsequently, 150 cd-1 mice were randomised into control (c), l or s groups and peritoneal cells studied at 24 hours for bacterial phagocytosis and killi:~g, superoxide (02-) and tumour necrosis factor (tnf) production and macrophage activation vsing mac-i(cd-11b) receptor in~.ensity expressed es mean channel of fluorescence (mcf). these resides indicate that sf!enectomy predisposes to nrortal~ty from bacterial sepsis ia the early pos~ operative period compared to sham operated animals. failure ~f p'.acrophages to kill bacteria in the splenectomv group '~:cured in t?~e absence of impairment of oxygen freeradical or tnf pred:~ctien. the macrovh~ge ac!ivotion marker mac-1 was significantly reduced in both l and s groups and impaired phagocytosis of bacteria oceured in both operative groups compared to controls. laparotomy a!one reduces macrophage activity in terms of surface re:eptor mac-1 expression and !ingestive capacity. splenectomy however s~gnificantiy ~mpairs r-acrophage-wediated l~,acterial killing and this qefect rttav co~tribut~ sig~ifjcav'ly to th-~ dissemination of local infection and to n':ortalit). depts of haem~ tology & surgery, beaumont hosoital, dub!in 9,eire. introduction: loss of cell membrane integrity appears to be a common pathway of injury to tissues subjected to high-voltage electrical shock. the cell membrane is the most heat labile structure in the cell, and is also the most vulnerable to externally-imposed electrical forces. skeletal muscle and nerve cells are particularly susceptible to electroporation by clinically relevant electric fields. restoration of membrane integrity is essential for cell survival in victims of electrical shock. we have studied the effect of non-ionic triblock copolymers ( poloxamer class) on the transport properties of isolated rat skeletal muscle cells following electroporation-induced membrane disruption. 1-3 mm long adult skeletal muscle fibers were isolated by enzymatic digestion from the rat flexor digitorium brevus and maintained under standard culture conditions. they were loaded with the calcein-am dye and placed in a 37,c chamber for recording by real-time video confocal microscopy. the cells were subjected to 4 msec, 150 v/era, 1 a field pulses with a low duty cycle to allow thermal relaxation. peak temperature rise was 0,2.c. the uye content of the cell was monitored in real time. experiments were carried out in calcium-free phosphate buffered saline, with 1 mm mg%. experiments were repeated with 4 mm neutral dextran ( the aim of the present paper is to ascertain if thuracotomy induces a different pattern of variations of cytokines, immunocompetent cells and antibodies from laparotomy in the early postoperative period. 60 patients (34 males 26 females,mean age: 59.5_+5) 30 with gallstone disease and 30 with non neoplastic pulmonary disease were studied. none of these patients received blood transfusion, biological response modifiers, radiotherapy or surgery for at least 6 months before being included in our study. anaesthetic procedures were similar in all patients and none were matnourished. on the day of surgery and on the 1st and 7th postoperative days (pre, lpo, 7po) percentages of cd19, cd5, cd4, cds, cdi6 were measured by means of flow cytometry using moab., and levels of ig a, lgg, igm, ige. by nephelometry cytokine levels in peripheral blood(il-1, il-2, il-4, il-6, tnf) were measured in 10 pts. of each group by means of elisa using moab. _r. esults:variations of il-2 and il-4 were not s.s.. il-6 increased but differences between groups were not statistically significant (s.s). il-i decreased on 1po and increased on 7po in both groups but were only s.s. in the th.g., and therefore, the differences between groups were s.s (p<0.05).tnf decreased in the l.g. and increased in the th.g. on the 1po, the difference was s.s(p<0.05); on 7po, tnf decreased in the l.g. and decreased in the th.g. but these variations were not s.s. cell percentages decreased an lpo and increased on 7po, except for %cd19 cell that increased on lpo and decreased on 7po ,in both groups of pts. differences were not s.s. ig a, igm decreased and ige increased in both groups (p< 0.0i), but differences between them were not s.s. in contrast, igg decreased on 1po (p<0.01) and increased on 7po in both groups, but the decrease iu the th.g. was greater than in the l.g. twenty male children,aged from six months to 3 years,admitted for elective inguinal operation were studied. the operations were performed under balanced combined anaesthesia (fentanyl,thiopemtone,vecuronium, 70% nitrous oxide in oxygen) and blood samples were collected before flunitrazepam premedication,after anaesthesia,4 and 24 hours after anaesthesia. cells from the wound were collected with cellstick sponge which was removed from the wound 4 or 24 hours after anaesthesia. the study was approved by the local ethical committee. the percentage of neutrophils was increased and that of lymphocytes was decreased in perpheral blood after the operation.the values in the wound were close to the values found in peripheral blood. the percentage of t-lymphocytes (cd3) and helper-t-cells (cd4) decreased in peripheral blood being lower in the wound than in peripheral blood after the operation. the percentage of t-eytotoxic cells (cd8) also decreased in peripheral blood and was similar to that in the wound. b-lymphocyte (cd19) percentage was increased in pe~pheral blood after the operation and was higher than in the wound. the percentage of activated t-cells (cd3+hla-dr-positive cells) in peripheral blood increased while that of natural killer cells (cd3+cd16+leu19-pos) was increased just after anaesthesia being decreased at g and 24 hours after the operation. spontaneous lymphocyte proliferative responses didn't change while phytohemagglutinin a and concavalin a induced responses were decreased in peripheral blood samples 4 hours after the operation with recovery at 24 hours.pokeweed mitogen induced lymphocyte proliferative responses were decreased at 4 hours (p<o.01) and 24 hours after the operation we previously reported that plasma ige increased after traumatic injury. in this study, we measured plasma ige ievels in 40 trauma patients on hospital admission and 3x weekly in the intensive care unit to evaluate ige kinetics. patient characteristics were: mean age 36-+ 16 years (33 m, 9 f), mean injury severity score (iss) 26+ 10, sepsis (19 patients), sepsis syndrome (9 patients), ards (4 patients), renal failure (5 patients). ige increased in 28 patients (70 %). the mean increase was 527 ng/ml (range 0 to 7362 ng/ml; 95% ci was 160 to 895 ng/ml). increase in plasma ige was significantly greater in patients with sepsis syndrome (p=0.021) and renal failure (p<0.001). although mean plasma ige was higher with ards, pneumonia, hepatic dysfunction, and shock, these differences were not significant (p>0.05). plasma ige increase was not related to severity of injury by iss score (p =0.42). the mean day to highest ige was 9.1-+6.5. the day sepsis was first observed preceded the day of highest ige by 4.2+6.6 days. there was a significant association between the day of sepsis onset and the day of highest ige (p=0.018). eight of nine patients with sepsis syndrome had > 100% increase in plasma ige from admission. one patient's ige levels were normal (20-100 ng/ml) for 24 days and then increased to 7300 ng/ml over the next 12 days, after onset of sepsis syndrome. changes in ige plasma levels may reflect the action of cytokines, such as il-4, which concurrently regulate production of ige and il-1 receptor antagonist in a response to sepsis. sepsis remains a leading cause of late mortality in trauma and hs. although hs-induced bacterial translocation is supposed to be the major cause of sepsis and mof, depression of the res increases susceptibility to infection after injury. the purposes of this study were: a) to evaluate the res in the lung, spleen and liver after hs and subsequent hypertonic saline (hsl) treatment, and b) to document the patterns of phagocytic activity in these organs during 24 hrs. adult male wistar rats (250+_23 gin) were submitted to hs (sbp 40 tort) and after t hr (shock i hr) and 24 hrs (shock 24 hrs) hsl (nac17.5%, 3.5 ml/kg) treatment, 1131 e. coli (i 131) was injected into the portal vein 10 ~tci (n_>12). twenty minutes later, the lungs, spleen and liver were harvested and scintilographic counts obtained. data is depicted as mean_%+sem * p<0.05, ~" p<0.001 and statistical analysis was performed by analysis of variance and wilcoxon tests. one hr after treatment, lung 1131 uptake was increased and liver and spleen 1131 uptake were reduced compared to sham. twenty four hrs after treatment, all organs, except lung uptake, returned to normal values. radioautographic histological analysis revealed radiolabeled particles inside phagocytic cells of all organs. we conclude that pulmonary phagocytic activity increases after 1 hr of hs hsl reatment, diminishing by 24 hrs although still above normal values. in contrast, res suppression occurs in liver and spleen after 1 hr hs hsl treatment, returning to normal values by 24 hrs. these results may explain lung complications and immunosuppression after trauma. infusion of endotoxin as well as major surgery is followed by lymphopenia in peripheral blood. the purpose of this study was to investigate to which tissues the lymphocytes are redistributed in response to endotoxaemia and major surgery. in addition changes in lymphocyte subpopulations and expression of mecii was measured. lymphocytes were isolated from peripheral blood of 30 rabbits, labelled with 111indium-tropolene and reinjected intravenously into the rabbits, i0 rabbits received an infusion of escherichia coli endotoxin 2 ~g/kg, while i0 rabbits were subjected to a major sham operation and i0 rabbits served as a control group. the redistribution of lymphocytes were imaged with af gamma camera, and calculated with an interfaces computer before, and 2, 4 and 6 hours after major surgery or infusion of endotoxin or saline. interleukin-l~ and serum cortisol were measured. in addition we followed cd2, cd3, cdlla/b, cdis, cd20, cd44, mhcii and cd4/cd8 ratio. following endotoxaemia interleukin-lf~ increased significantly, following endotoxaemia as well as major surgery serum cortisol increased significantly. following major surgery as well as endotoxaemia there was significant lomphocytepenia in peripheral blood with a decreased cd4/cd8 ratio while the cd3 positive subpopulation increased. in addition there was a decrease in the expression of mhcii on the lymphocytes peripheral blood. the radioactivity of the lymphatic tissue in and around the intestine increased to 129% of initial values following endotoxaemia and to 125% following major surgery. the results indicate that endotoxaemia as well as major surgery induces redistribution of lymphocytes from peripheral blood to lymphatic tissue. among the lymphocytes staying in peripheral blood there was a decreased expression of mhcii and a relative decrease in cd4 cells compared to cd8 positive lymphocytes. in order to analyze the effects of immune suppressive substances on expression of mrna of interleukin-2(il-2) and interleukin-2 reeeptor(il-2r), this study was carried out. twenty male rabbits with comminuted fracture were used in the study. ten ml blood were taken at 0, i, 2, 4, 6 days after injury. the sera were tested for the effects on lymphocyte blastogenesis and induction of il-2 stimulated by concanavalin a(con a): the sera from the rabbits 2 days after injury were analyzed with sds-page gel eleetrophoresis, and divided into three groups by ultrafiltration (ufpi ttk, 30kd,milipore; centricon-10,10kd,amicon), that are less than 10kd, between i0 and 30kd, and more than 30kd. each group of the substances also was tested for the expression of il-2 and il-2r by the dot blot hybridization. the results showed that: i) all sera from the rabbits after injury had significant suppression on lymphocyte proliferation and secretion of il-2 by the con a-stimulated splenocyte in mice; 2) the sera from the rabbits 2 days after injury had more profound suppression than other injured sera; 3) there was a marked band at about 10kd in sera from the rabbits 2 days after injury, but nothing at the same position in normal sera analyzed with electrophoresis; 4) the substance with molecular weight of about iokd had more obvious suppressive action on expression of mrna of il-2 and il-2r than other groups substances, of which molecular weights are more than 10kd. it is concluded that: i) the sera from the injured rabbits can reduce immune response; 2) there is kind of substance, of which molecular weight is about 10kd, it is probable the main factor involved in the pathogenesie of postinjury suppression immune; 3} the substance can depress the expression of mrna of both il-2 and il-2r. research institute of surgery daping, chongqing, 630042 p. r. china acute ethanol uptake prior to injury modulates monocyte tnfo~, production and mononuclear cell apoptosis. g. szabo, b. verma, p. mandrekar, d. catalano monocytes (mo) have been shown to contribute to immunosuppression after both major injury and alcohol consumption. we reported that acute ethanol exposure of m(3 results in decreased antigen presentation, induces tgf-13 and pge2 while inhibiting inflammatory monokine production. we also showed that post-trauma immunosuppression is mediated by hyper-elevated mo tnfc~ and il-6. consequently, here we investigated rnonokine production in trauma patients (n=10) who had elevated (>o.lmg/dl) or had no blood alcohol level (n=t0) at the time of emergency room admission. none of the patients had chronic alcohol use history. met tnfc~ production from trauma patients with prior alcohol uptake was undetectable during days 0-3 post-injury in contrast to patients without alcohol exposure. furthermore, decreased tnf~x levels were found in alcoholic patients' mci after mdp or ifny + mdp induction. however, mcl tnfc~ levels during the 4-8 days post injury period became higher in alcoholic trauma patients. furthermore, over 9 days post-injury, alcoholic trauma patients showed significantly elevated mci tnfo~ production after adherence isolation, mdp, or ifn+mdp stimulation compared to patients without alcohol. these results suggest that acute ethanol uptake prior to injury decreases tnf(x inducibility in the early post-trauma period, but these patients' mo produce hyper-elevated tnfa levels later post-injury, thereby prolonging their cytokine shock risk. tnf ng/ml 0-3 days post-injury 9 days post injury stimulus ale. pt. pt 9.3 11.9 5.6 4.0 immunosuppression might also be increased by the elevated apoptotic activity found in trauma patients' mononuclear ceils, which was even greater in alcoholic trauma patients' cells. in non-alcoholic trauma patients' preactivated mo, in vitro acute ethanol (25-150mm) exposure resulted in a significant down-regulation of tnfc~ (p<0.001) and il-6 (p<0.01) production. in contrast, in vitro ethanol exposure increased the production of inhibitory monokine, tgfi]. these results provide both in vivo and in vitro evidence for the effect of acute ethanol exposure increasing immunosuppression and cytokine shock. the 'systemic inflammatory response syndrome' (sirs) with consecutive septic multi-organ dysfunction represents the major cause of late death following major mechanical and burn trauma. systemic hyperinflammation and concurrent depression of cell mediated immune response (cmi) render the traumatized host anergic, resulting in profound susceptibility to opportunistic infection. monooytes/macrophages (mo) play a central role within the host defense system in developing and manifesting states of injury, shock and sepsis. the mechanistic scrutiny of the synthesis patterns of crucial cccytokines appears to be a helpful tool to further analyse mo behaviour in the compromised individual. the objective of this study was to further dissect the characteristics of cytokine regulation in pbmc under stressful conditions, via analysis of the expression of cd14+ receptor, the proinflammatory mediator il-6, the macrophage activating factor ifn-5,, and neopterin (npt) a metabolite of activated mo. we investigated pbmc's on consecutive days 1, 3, 5, 7 and 10 after mechanical trauma of 9 and after bum trauma of 5 patients (mean age 38~17 years; mean iss 34±2 pts). in trauma patients we saw a massive increase of pha induced neopterin synthesis compared to controls. however, when discriminating the npt levels in the supernatants for the amount of mo stimulated, the npt output of the individual cell was lower compared to mo of nontraumatized individuals. interestingly there was a contrary coarse in the cumulative protein release patterns of il-6 and ifn-7 in mechanical versus burn trauma patients. wheras in burn patients ifn-y was decreased significantly (16+5 u/ml) compared to controls (58+6 u/ml) as well as mechanical trauma (59+12 u/ml). il-6 showed a significant suppression following mechanical trauma (662+58 u/ml) vs control (1266+91 u/ml) and bum patients. the rt~,na signal intensity for beth eytokines was in concurrence with the protein release in more than 85% of the individual patients investigated. from these data we can conclude that the inadequate low npt synthesis predominantly in bum patients appears to be a sign of cellular immaturity and is probably partly due to low t-cell ifno t signals. in addition we could state that the quality of trauma is apparently responsible for the different synthesis patterns of ]l-6 and ifn-q,. it has been postulated that bacterial invasion or endotoxemia are necessary for cytokine production following burn injury. we studied the organ distribution and kinetics pattern of il-fc~ (cell-associated il-1 agonist) in eutrophic rats subjected to either 20% tbsa cutaneous scald injury (bi), muscle scald injury of equivalent 20% tbsa (mbi), sham muscle bum (resection of skin only, up to 20% tbsa) (smbi), and sham cutaneous burn (sbi), followed by saline resuscitation (15 mukg i.p.). separate rats were infused with 15 mg/kg e.coli 0111:b4 lps or saline lv. unmanipulated rats were baseline normal controls. liver, lung, spleen, ileum, thymus, kidney, skin, and plasma were harvested at various time-points within the first 24h. tissues were frozen, weighed, homogenized, the homogenates centrifuged and the supernates assayed with a radioimmunoassay specific for rat il-l(z (detection limit 150 pg/rnl). il-lc~ was expressed as ng/g weight + sem (lowest detectable amount 1.5 ng/gwt). il-lo~ was constitutively present only in the skin (102 + 16.4 ng/gwt). cutaneous burn and sham cutaneous bum induced biphasic elevations of il-lcc in the liver and lung only, with maximal levels at 2.5h (in the liver, bi = 16.5 _+ 6.2 ng/gwt, sbi = 1.7 + 0.1 ng/gwt, p _< 0.05; in the lung, bi = 10.3 + 1.3 ng/gwt, sbi = 1.9 + 0.8 ng/gwt, p -< 0.002). of note, both bi and sbi rats had detectable il-i~ in the liver at timepoint 0 already (2 min real-time). these levels increased in parallel until 30 min and became eventually different by 1 log at 1 -2.5h. all other organs as well as plasma were below detection limits. muscle burn injury and sham muscle burn (skin resection) induced similar elevations of il-10~ in the liver at lh, indistinguishable from each other and from cutaneous burn. in contrast, lps challenge induced dramatic elevation of il-t~ in all organs tested except for the kidney; the spleen was the most responsive organ to lps-induced il-lo~ production. these data indicate that thermal or mechanical injuries induce very early and organ specific production of il-1 c~ in vivo by mechanisms other than endotoxemia. injury-induced complement and platelet activation may be involved as well as the neuro-endocrine axis, which may explain the low levels of il-lo~ induction observed in all rats at the very early time-points. trauma services, massachusetts general hospital, and department of surgery, harvard medical school. fruit, st, boston, ma 02114. j. f. schmand *#, a. ayala* and i. h. chaudry* studies indicate that i.v. infusion of the colloid hes in normal animals does not adversely affect non-specific immunity. it remains unknown, however, if lies affects cell mediated, specific immune functions after trauma and hemorrhage (hem). to study this, non-heparinized c3h/hen mice underwent midline laparotomy to induce trauma and were then bled to and maintained at a bp of 40 mmi-ig for 60 rain. the animals were then resuscitated with either 4 times (x) the shed blood vohune as lactated ringer's solution (lrs) or 2x lrs + lx 6% lies. sham mice were neither hemorrhaged nor resuscitated. at 2 or 24 hours post hem serum, peritoneal (pm~) and splenic macrophages (sm~) were obtained. bioassayes were employed to assess the levels of ii-l, il-6 ( alternatively pmqb showed no differences in il-1 release between all groups at 2 and 24h, while sm~ from the lrs + hen group showed a depression at 24h. tnf production by pm~ was depressed in all groups at 2h and remained so in the lrs + hes group at 24h. sm~b showed decreased tnf release values in both hem groups at 2 and 24h. in summary, the levels of inflammatory cytokines (particularly the values of circulating il-6) after trauma/hem are positively influenced by the administration of hes. this might be due to a protective effect on pmqb and sm~, but also on other cytokine producing cells, e.g. kupffer ceils. we conclude that hes is not only a safe, but also beneficial agent in the resuscitation of patients atler trauma/bemorrhagic shock. this study investigated endotoxemia and consecutlve immune response in 39 patients with multiple trauma (median injury severity score = 20,5). blood samples.were collected shortly after injury and after 0,5, 1, 3, s and l0 days. endotoxin was measured with limulus-amebocyte lysate test and the specific antibody content (sac) against endotoxins of the classes igg, igm and lga by elisa-technique. five antigens were used: lipopolysaccaride (lps) of e.coli (ec), lipid a of e.coli (la), lps of pseudomonas aerog. (pa), lps of vibrin cholerae (vc) and cx-hemolysin of staphylococcus anreus (oth). a nephelometer indicated the total concentrations of igg, igm and iga. differences were checked with wilcoxon-test and p<0,0s was considered significant. cross-reactivity was calculated with rank correlation coefficients. results: endotoxemia peaked shortly after injury (0-3h) at 0,425 eki/ml (median), decreased thereafter to 0,04 eh/ml at day s and remained on this level. sac oflgmclass increased to all endotoxins and peaked at day 3 revealing the lfighest level to la followed by pa (= 80% of la-sac), ec (= 65% of la-sac) and vc (= 40% of la-sac). lga antibodies increased as well but only slightly and not significant (exception: sac to la was elevated significantly at day 5). igg antibodies increased similar to iga class only slightly and again only sac to la was significantly higher at day 5 and 10. however sac to (xh of all ig-classes remained continuously on the same level troughout the observation time. correlation analysis revealed strong cross-reactivity (r>0,5; p<0,01) most often between antibodies of igm-elass (73%) followed by igaclass (40%) and lgg class (2%]. conclusions: multiple trauma is associated with temporary endotoxemia. endotoxins probably translocated from the gut cause specific increase of anti endotoxin antibodies in blood of the igm-class. endotoxins cause no increase of antibodies to gramposilave bacteria. igm antibodies are most unspecific. during cardio-pulmonary bypass, as well as postoperatively, high levels of endotoxin, interleukin-6 (ii-6) and c-reactive protein (crp) were measured in 30 patients. i0 female and 20 male, ageing from 30 to 73 with a median age of 59. blood sampling was done preoperatively, immediately after induction of anaesthesia, after thoracotomy, after cannulation of the aorta and right atrium after the first half of the reperfusion phase, after closure of the thorax, 2 and 4 hours after the operation and then every morning until the 5th postoperative day. blood was drawn into heparinized tubes (i0 iu/ml) which were free of endotoxin. crp levels were determined through the use of the behring nephelometer. 11-6 levels were measured by using commercially-available elisa test. the endotoxin level was determined by a chromogenic modification of the limulus amebocyte test. the statistical analysis was done using the wilcoxon ranks test and correlation analysis. a significant increase {p 0.01) in endotoxin plasma occurred during surgery, culminating in a peak (median value of 0.795 eu/m!) during reperfusicn. plasma levels of endotoxin continued to be slightly raised till the 5th day after surgery, whereas those of interleukin-6 rose at the end of the operation and were at their highest 4 hours later (median value of 217.5 pg/ml). crp levels were also high postoperatively with a median value of 114 mg/l, and were markedly raised on day 2 (191 mg/l). a definite, statistically significant correlation between the plasma levels of endotoxin and 11-6 during the operation was establisthed (p 0.05), leading us to conclude that the endotoxin liberated during cardiac surgery acts as the main trigger in the releasing of 11-6, and thus induces the postoperative acute phase reaction. there was no evidence of a correlation between crp and endotoxin or 11-6 plasma levels. impaired immune function is well described following trauma and hemorrhagic shock (hs). prior studies have utilized peripheral blood or spleen cells to index immune function following hs. however, changes in mucosal immunity are not weii characterized in this setting. gut origin sepsis is thought to be an important cause of organ failure and death following trauma. a rodent model was utilized to allow comparison of mucosal-associated immune function vs, systemic compartments after hs. fischer rates underwent hs (map 35±5mm hg) for 60 minutes followed by resuscitation with shed blood and lr. sham animals were instrumented only. rat tears were collected at 24 and 72 hours following hs for quantitation of slga by ria. animals were sacrificed at 24 hours and spleen (spl), peripheral lymph nodes (pln), and mesenteric lymph nodes (mln) harvested for cell population analysis using flow cytometry and mitogen stimulation analysis. cell marker expression analysis revealed no changes in t or b ceil populations following hs. mitogen mucosal immune function appears relatively spared following hs. the mechanism(s) for this variability in immune function requires further investigation. we have found that transplantation of bone marrow in a hind-limb graft to syngeneic lethally irradiated recipient is followed not only by rapid repopulafion but also overpopulation of bone marrow cavities. the question arises whether this unexpected phenomenon could be the result of stimulation of stem cells by factors (cytokines) released from surgical wound at the site of anastomosis of graft with recipient. aim of the study was to investigate which tissues damaged during the procedure of limb transplantation may be a potential source of humoral factors accelerating in vivo bone marrow proliferation. methods. experiments were carried out on lew rats in 5 groups. in group i, the hind limb was transplanted orthotopically to a syngeneic recipient; in group ii, sham operation was performed; in group iii, a four-cm long cutaneous wound was made on the dorsum; in group iv, limb skin was harvested, fragmented and implanted into peritoneal cavity; in group v, bm from femur and tibia was implanted intraperitoneally. bm, lymphoid tissues and blood were sampled 10 and 30 days later for cell concentration and phenotype evaluation. results. the yield of nucleated cells from tibia was on day 10 in the control 29.3 + 3.2, in group 1 58.1 +2.0, in group ii 47.1 + 1.6, in group iii 50.8+2.0, in group iv 48.5_+2.0, in group v 37.4_+4.9x10(6). the evident increase in bmc yield in all groups continued until day 30. increase in weight and total cell count of spleen and mesenteric lymph nodes in all but group iii was also found. no differences in percentage of maturing erythroid cells, but higher of mature myeloid cells and lower of lymphocytes were observed. conclusions. trauma of skin, muscles, and bone brought about an increase in bone marrow cellularity and acceleration of maturation of myeloid lineage. transplantation of bm ceils alone did not produce this effect. transplantation of bm in limb graft is a good model for studies of natural factors reaulatin~ bm hemormesis. this study sought to determine a relationship, if any, between the degree of hypochclesterolemia upon trauma patients' admission and their subsequent outcome. all blunt and penetrating trauma patients admitted to a level i facility from 1987 through 1991, and who had serum cholesterol assayed during the first 24 hrs were retrospectively studied for development of death or significant organ dysfunction. the mantel-kaenzel chisquared test was used to determine significance of data at the p< 0.05 level. results: 2909 trauma patients were admitted during the four-year period who had serum cholesterol assays performed in the first 24 hrs. 24 patients had cholesterol levels less than 50 mg/dl; 8 of these (33.3%) died, 3 (12.5%) developed ards, 5 (20.8%) developed acute renal failure, and 8 (33.3%) developed multisystem organ dysfunction; hypocholesterolemia in these patients was not due to liver injury or massive fluid administration. the risk of death was 5 times greater and risk of multi-organ failure 3 times greater in this group than in those with a normal serum cholesterol (>if0 mg/dl; 1820 patients; p<0.05). conclusions: admission serum cholesterol level in the trauma patient serves as a powerful marker for those at risk of subsequent organ failure or death. hypocholesterolemia in this setting may result from organ hypoperfusion and humeral mediator release. lung tissue contains many immunocompetent cells. resection, therefore, is expected to activate extensively inflammatory mediators such as pmn-elastase, pmstanoids and pteridines. in a prospective clinical study we compared 35 patients (pts) undergoing either thomcotomy with or without lung tissue msectioh and tboracoscopic lung resection concerning activation of inflammatory response. material & methods: group a pts (n=8) had thoraantomy but no lung tissue injury; group b pts (n=ls) had thoracotomy and lung tissue resection due to benign diseases; group c (n=9) represents group b tissue resection but using a thomcoscopic procedure. the following parameters were determined pre-, peri-, and postoperatively: elastase and crp as indicators of activation of pmn-leukocytes and injury severity; prostacyclin (pgi 2) and thromboxane (txa~) as parameters of lung endothelial response; prostaglandin f2~ (pgf~) and pgm representing pulmonaly metabolic activity; pge a and neopterin as proof of macmphage activation. statistics were performed using analysis of variance for repeated measures. results: group b pts revealed postoperatively an increase in crp (p<0.001) indicating a higher injury severity in comparison to the thoracoscopic procedure (c). both, controls (a) and group c pts did not show pmn-activation, whereas group b demonstrated a reversible increase in elastase. surgical trauma caused in all groups a release of pgi z and txa 2 which was more pronounced in c (p<0.05) and most in b (p<0.01). similar results were found for pge~ and pgf2=. there was no activation of maerophages since neopterin did not increase. apparently, metabolic lung function was not impaired because there was no marked rise in pgm except in b (p<0.05 vs. c). discussion: our results demonstrate that lung tissue injury aggravates the mediator release induced by thoracic traum. these mediators among others are able to increase capillary pressure and hence lung edema formation. impairment of lung function, however, seems dependent on the extent of the liberation. therefore, the maximal release reactions occured in group b and c after lung tissue resection, whereas the controls showed the highest levels immediately after the incision. we conclude that thoracoscopic procedures are superior in reducing the resection trauma per se and hence might prevent severe mediamr-induced (pulmonary/systemic) sequelae. in a prospective study we investigated 45 patients using radiochemical method according to sch~dlich (s) and photometric method according to hoffmann (h). serum of severly traumatized patients was withdrawn directly after admission at our emergency room and in narrow time intervals during first 24 hours after trauma. follow up control samples were taken daily until day ten. whereas no elevated pla-ca was found during first 24 hours, a peak was regularly observed around day four. there was high correlation between pla-ca and iss (r=0.71, p<o.o001) except patients suffering from thoracic trauma, thoracic trauma {tt) provoked similar gons-score-and pla-ca-values compared to multiple trauma (mt) despite significantly lower iss: pla-ca (h) mt 32.9 + 6.4 tt 28,7 +_ 9,5 n.s. pla-ca(s) mt 11.7 +_ 2.6 tt8.3 _+ 2.6 n.s. goris mt 3.5 4-0.7 tt3.6 _+ 1.3 n.s. iss mt 35 4-3 tt21 4-3 p<0.01 we found delayed elevation of pla-ca after severe trauma, however detection of delayed pla-ca in the serum of traumatized patients cannot give reliable information about the exact role of pla in the inflammatory reaction after trauma as latest results show that pla is secreted early but bound at lipophilic sites of vascular membranes. hern~mdez m j, amiguet ja, monreal ji, vid~n jr, jim6nez f j, l6pez-vivanco g acute phase reactant proteins increment in serum of patients with traumatisms and inflammation has been previously reported. alpha-1 antitrypsin (aat), alpha-2 macroglobulin (amg) and ceruloplasmin (cp) are evaluated in the following of 33 patients, 27 males and 6 females (mean age: 43 years), with compound fractures. 57 % of them were located in tibia and fibula. intercurrent infection developed in 4 patients and internal fixation rejection in one. measurements were made by radial immunodiffusion on days 0, 3, 10, 17, 24, 31 and 38. a control group of 30 healthy volunteers was also evaluated. aat and cp showed increased values from day 3 which kept elevated untill the end of the study. amg elevated from day 24. when infection developed, aat and cp values were even higher whereas amg values decreased. fixation rejection caused aat, amg and cp increment. aat and cp increment in non complicated fractures might be due to accompanying inflamation, by means of cytokines release and aprps hepatic synthesis induction. therefore, increment is higher when infection or rejection develop. amg behaviour at early stages of evolution is secondary to hiperconsumption, considering mean life shortening after proteases ligation. amg modifications in fixation rejection remain obscure. aprps increment modulates inflammation and bony callus formation by means their antyprotease and antioxidant properties. clinically, aprps might be useful parameters in determining complications onset in fractures evolution. hemorrhagic shock (hs) is a common complication of trauma, as well as some major surgical procedures. the alteration of the immune system by hs is thought to contribute to the increased septic morbidity and mortality seen in these patients. multiple mediators are released following hs. il-1, il-6, tnf, pge 2 and tgf-b have all been implicated in these immune system changes. in vitro, pge 2 causes many of the immune system changes following hs, yet the time course of pge 2 release has not been well deliniated. the aim of this study was to determine the time course of pge 2 release by peritoneal and splenic macrophages following hs. c3h/hen mice were bled to a mean bp of 35+5 mm hg for 60 minutes. the animals were resuscitated with the shed blood and normal saline. control animals were cannalated, but blood was not withdrawn. the animals were sacrificed at 2, 12, 24, 48, and 72 hours following hs. peritoneal and splenic macrophages were harvested from each animal and cultured with lps for 24 hours. the supernatants were collected and frozen until assayed. pge 2 was assayed using an elisa (cayman chemical). results are shown below for the peritoneal macrophages: pge 2 release by peritoneal macrophages was significantly elevated at 24, 48 and 72 hours over control. it was maximal at 48 hours, but by 72 hours it had started to decline. splenic macrophage pge2 release was much more variable. (data not shown.) there was less consistency between time points and no clear trend was seen. in conclusion, pge 2 is significantly elevated at 24 hours following hs, and reaches a peak at 48 hours. pge 2 release may be responsible for the immune system changes seen in the first few days following hs. splenic and peritoneal macrophages respond differently in their release of pge 2 following hs; which may be due to differences in their milieu. there is an obvious association between the altered immunocompetence of patients following traumatic injury of various types and the increased riskto develop complications. the present investigation was undertaken in order to analyze the value of both neopterin (n) and c-reactive protein (crp) in monitoring disease course in patients with multiple trauma. we were interested to compare nconcentrations as marker of t-cell/macrophage activation with crp levels as indicator of the inflammatory acute-phase response daily serum concentrations(n and crp) were measured in 12 patients (9 male, 3 female, mean age 31.42), admitted to our icu following serious trauma (mean iss=34) and in 10 control subjects. the clinical course of each patient was evaluated with apacheii score according to knaus. mean stay in icu was 9.25 (range 5-13). 5 patients didn't develop organ failure(nof-group), 4 patients survived, developing mof (s,mof-group), 3 patients died with mof (ns, mofgroup). three patients(one s and two ns) showed signs of septicemia and septic shock. the serum samples were measured for neopterin (immu-test-ria, henning-berlin) and for crp (immunoturbidimetric method) -at the university hospital for internal medicine, innsbruck, austria. the highest crp levels were measured 48h after trauma, but they were not accompanied by significant increase in n-values. we found significantly elevated n-concentrations from the days 5-6. n and crp levels showed a parallel release peaks on the days 6-7 and the values discriminated significantly among the three outcome groups(the second crp peak is lower in comparison to the first posttrauma peak). by the ns-group we found constant and parallel increasing crp and n levels, reaching extremly high values 24-48h and preceding clinical signs of mof and sepsis. the values increased dramatically before death. the serum levels by all 10 control subjects were upper the normal limit for n and crp.. the parallel occured peak changes in n and crp concentrations showed a significant correlation with clinical status, using a disease activity score (apac h eli). our findings suggest that both-cellular and humoral mediated immune mechanisms are activated after multiple trauma and that simultaneous determination of n-crp may be of advantage as diagnostic and prognostic parameter in polytrauma-patient monitoring panel. it is apparent, that measurement of very high levels may be of value as a srong indicator in developing of mof or septic complications and thus offer the possibility for earlier therapeutic intervention. the modification of t-cell/macrophage and acute-phase responses may be potentially useful in the treatment of icu patients, resuscitated after severe trauma, and suggest that n-crp may be relevant indicator for testing experimental immunomodulating therapies. although several in vitro studies suggested the catalytic action of iron in oxygen free radical generation, few data are available concerning iron-metabolism following ischemia-reperfusion injury and hemorrhage in vivo. aim of the present studies was to evaluate iron metabolism following mild and severe hemorrhage in the presence and absence of intraperituneal hematoma. methods. male lewis rats (300 g) divided in 4 groups (observation time 120 hrs): a: mild hemorrhage by sequential blood sampling (0.9 ml each)(0, 2, 4, 6, 24, 48, 72, 96, 120 hrs). b: a with hemoperitoneum (hp)(2 ml/100g), c: hemorrhagic shock: blood withdrawal of 3 ml/100g over 30 min and resuscitation with ringer's lactate (2 x shed blood) and isogenic donor blood; d: c with hp. parameters: serum iron ([g/dl], ferrozin-method); 59fe labeling of erythrocytas (application of 1 ci 59fe i.a. into a donor rat, 5 days later bleeding of the rat and injection intraperitoneally in experimental rat; plasma transferrin ([g/ml], ria). t-tests and mann whitney. data are expressed as mean + sem. results. compared to baseline, mild and severe hemorrhage caused a significant increase in serum iron at 24 and 48 hrs, respectively (212+30 vs. 248+35; 185+_28 vs. 255+_22). hp reduced this increase (209+_17 vs. 177+21; 194+25 vs. 197+-48, p<.05). i-ip significantly increased hemoglobin between 24 to 96 hrs in mild (12.1+_.8 vs. 14.2+.7) and severe hemorrhage (10.3+.6 vs. 13.1+.6). in mild and severe hemorrhage comparable maximal resorption (59fe labeled erythrocytes in circulating blood) of intraperitoneal hematoma occured at 24 hrs (mild: 318+_71 vs. severe: 364+-114 counts/rain). hp increased survival-rate following hemorrhagic shock (no hp: 10/17; hp: 10/13). in mild hemorrhage no alterations of plasma transferrin concentrations. in shock group without hp, there was at 96 hrs a significant higher transferrin level than with hp alone (3.2+_.5 vs. 2.23 + .2). in conclusion, hemoperitoneum with hemorrhage prevents increase in plasma iron levels by increasing hemoglobin through resorption of erythrocytes and thereby suppressing iron mobilization from endogenous iron resources, e.g. liver. this suggests that intraperitoneal hematoma not necessarily promotes iron-mediated oxygen free radical production, especially since survival in these groups was improved. increased transfarrin levels in group c at the end of the experiment suggest increased iron turnover which was not observed in shock groups associated with hemoperitoneum. primary immune response to thymus-dependent (srbc) and thymus-independent (vi-polysaccharide) antigens was determined during 30 days after cct. antigenic challenge was made on day i, 5, 15 or 30. number of spleen plaque forming cells (pfc) and serum haemagglutinin (ha) titre were studied on day 5 after immunization. cct stimulated immune response to t-dependent, but not to t-independent antigen. enhanced response was noted when srbs were given in posttraumatic day i, it was higher than preceding one after injection on day 5 and reached a maximum when antigen was presented on day 15 after trauma (pfc number 2.4 times exceeded the level of immunized, but nontraumatized rats). the rise of pfc number in spleen correlated with increased ha level. thymectomy had been performed 30 days before cct completely abolished posttraumatio stimulation of immune response to t-dependent antigen. standard thoracotomy also enhanced humoral response when srbc were administered on day 15 after the operation. thymus trauma modeled by pincett squeezing of its right lobe and performed at the same time with thoracotomy fully abrogated as well as thymectomy stimulation of immune reaction. thus, different experimental chest traumas are able to increase antibody production via thymus-dependent mechanisms. endothelin is a 21 amino acid peptide produced by ischemic or injured endothelial cells. in vitro and in animal studies, et is also a potent constrictor for renal mesangial and coronary vessels, a negative cardiac inotrope and an endocrine regulator. our lab has shown that et is an agonist for monocyte production of interleukins i, 6 & 8, prostaglandin e 2 (pge2) , and substances which trigger superoxide production. systemic et levels increase in hypoperfused and septic patients, and et may be yet another important cytokine in critical illness. but while et has been shown to be a potent vasoconstrictor in healthy dogs, systemic vascular resistance does not increase in critically ill patients with high et levels. (we hypothesize that this might be due to pgez-mediated vasodilation predominating over et-mediated vasoconstriction.) we next asked what happened to systemic et levels in patients with major burns (>20%.) ten hemodynamically stable patients resuscitated by a modified parkland formula to a urine output >30 cc's per hour had et levels drawn on admission, at i, 6 12, 24, and 48 hrs. et levels were measured by radioimmunoassay. mean levels were elevated at 205±28 pg/ml at all time points versus levels in healthy controls of 39±9. in summary, systemic et levels increase significantly in patients with major burns. et may be yet another cytokine playing a significant role in the immune, inflammatory and multiorgan dysfunction observed with major burns. restoration processes in an organism after ischemic damage are realized through ~n~lammatory mechanisms~ the intensity of which is significantly defined by blood levels of neuropeptides. myocardial infarction (mi) was chosen for studyin 9 these processes since it eradicates the influence of infectious factc~rs. 32 dogs~ in whom mi underwent different forms o¢ healer, g; bhn~ed ~h~t during the acute phase of the disease there was a characteristic rise of ne!~ropeptides in the blood. these neuropeptides had nociceptive and antinociceptive effects. particularly substance p and -endorphins triggered off the development of compensatory and adaptive mechanisms and defined the intensity of inflammatory reaction at the zone of ischem~t: damage-notable fall in substance p levels after an ~nitial increase, while the ~-endorphins stayed high was an important condition for non complicated healing of mi. on the other hand high levels of substance p with low ~-endorphin concentrations lead to increased infiltration o~ neutrophils into the infarction zone and weakened the activity of synthetic processes~ thereby leading to left ventricular aneurysm. at the same time low intitial levels of substance p slowed down the development of necrotic processes which lead to delay in refunctioning of the heart and complicated the healing process. thus, regulation of the levels of neuropeptides in the blood in trauma forms a perspective method of its treatment. of laparascopic versus open choleocystectomy c. schinkel, s. zimmer, v. lange, d. fuchs, e. faist the impairment of immune function due to surgical trauma may be followed by deleterious septic sequelae. compared to open abdominal surgical procedures (lap), laparaseopic surgery (lsc) is associated with a decrease in hospital stay and in accelerated patient recover. the aim of the study was to evaluate the sensitivity of the immune sermn parameters of il-6, saa and neopterin, the percentage of cd14+ cells, the in-vitro il-2 synthesis after mitogen stimulation and lymphocyte proliferation, in order to purposefully discriminate differences in the severity of trauma. we investigated the blood of 27 patients with cholecystolithiasis undergoing either laparascopic (16) or open (i 1) cholecystectomy on consecutive perioperative days -1, 1, 2 and 3. there was no significant difference between the two groups concerning age and sex. patients with clinical signs of acute cholecystitis were excluded from the study. operation time and hospital stay were obviously longer in lap patients (67 versus 145 minutes, 5 versus 20 days) compared to the lsc group. concerning the unspecific acute phase reaction we could show no difference in the increment of senun amyoid a (saa) synthesis in the lsc group (d-i 40 + 1 lng/ml, d1 362 + 43ng/ml) versus lap group (d-1 61 + 19ng/ml, d1 457 + 45ng/ml), while in serum il-6 levels we saw a less steep increment in the lsc group (7-fold from d-1 to d 1) compared to the lap group (10-fold from d-1 to d 1). the analysis of cd14+ receptor expression and serum neopterin did not reveal any difference between the groups. lymphocyte function showed an impairment of proliferation to antigen stimulation in lap (d -1:24.000 + 3.330 cpm, d 1:17.300 + 3.000 cpm) compared to the lsc group (d -h 22.500 + 4.200 cpm, d h 26.000 + 2.900 cpm). in both groups il-2 synthesis was decreased post-operatively. our data indicate that laparascopic cholecystectomy reusults in a less distinct unspecific acute phase reaction post-trauma compared to that following lap. neopterin serum levels and cd14 receptor expression show that these parameters apparently are less useful markers to detect differences of surgical trauma severity while it appears that the impact of lap is reflected most impressively on the lymphocyte compartment. trauma alters the host resistance of organism and is accompained by appearence of excgenic and endogenic proteins in the body. to understand the molecular mechanisms of host resistans disorders in trauma, as a first step, the genetic regulatory mechanisms of immune response after antigen injection has been studed. the appearence of specific protein factors (13-19 and 25 kda), in the nucleus of rat splenic and brain cells, accordingly, was shown after immunization with sheep erythrocytes. the stimulatory effect of these factors on the il-2 mrna and il-2 production was detected. the nucleotide sequences of the human il-2 gene regulatory region bounding by the splenic nuclear proteins were determined between +39-141 b.p. the il-2 trans-factors shows the affinity to splenic and thymic lymphocytes in vitro. thus, the antigen causes the appearence of specific protein factors in the cells,which act on the gene level,stimulate il-2 production and the host resistance. these results cause the next step of experiments using the same model, but after trauma. these investigations will let us verify the hypothesis that the protein il-2 gene trans-factors may play a definite role in the decrease of the cell immune responce after trauma. confronted with the routine procedure of prophylactic treatment of candidates for surgery in a rural african hospital, we initiated studies on the fre'quency of post-surgical malaria. in tanzania 195 non-pregnant patients from rural areas were followed. of preoperative patients 10% had a parasitaemia and those maintaining it showed no increase or complaints. nine percent of patients without detectable parasitaemia before surgery came down afterwards and one-third had malaria-like complaints. spinal and general anaesthesia were equally applied in these last patients. in burkina faso we studied 120 patients of which 16% had a parasitaemia on admission and 6% had postoperative malaria. half of the surgical patients came from rural areas, whilst only 14% of those with malaria lived in the city (with much less exposure and immunity). 57% underwent major surgery and 43% minor. bloodtransfusions (4% with parasites) never evoked a parasitaemia in recipients. post-surgical malaria is thus a reality in about 10% of the adult cases, both in east and west africa. surgery evokes a cascade of factors, varying from cortison to interleukines and acute phase proteins; immune responses may temporarily be suppressed. clinical attacks of malaria in otherwise immunes could be evoked by one of these factors. though malaria can easily be cured, the differential diagnosis is difficult because of post-surgery fevers; we found that 16% was treated without justified indication. the involvement of "student-doctors" a. this study examines glucose uptake and hexose monophosphate (i~ip) shunt activity in normal human peripheral lymphocytes and polymorphonuclear leukocytes (pmn). glucose uptake was determined by measurir,g the uptake of tritiated deoxyglucose, a non-metabolized glucose analogue. adsorption of co2 derived from [i-14c] glucose was used to determine knp shunt activity. in vitro assays were carried out in hormone concentrations approximating normal and elevated trauma blood levels. (normal -cortisol 0.12 ~g/ml, glucagon 80 #g/m1, epinephrine 20 ~g/ml, insulin 18 t~u/ml; traumaeortisol 0.40 ~g/ml, glucagon 500 /*g/ml, epinephrine 420~g/ml, insulin 36 ~ij/ml. analysis of twenty subjects showed a reduction of 207° ~mp shunt activity by lymphoeytes and a ]3% reduction in glucose uptake by p~n in normal vs. trauma hontc,nes p < 0.05. lymphocyte glucose uptake was also reduced by trauma hormones p~0.05. it ha~ be.ea~ suggested thgt idiopatno pulmonary fibrous (y.pf) [s a consequence of severe alveolar epithelial injury and is associated with an nveolar irnammamry reactio~ and the presence 0f.neutr0phils. there~bre, neutr0pk~ chemoattra~ant~ are probably important in the genegs oft.he infial lesions of ipf. the obse,"wson that stimulated macrophages are or~n histologically promin~t in fibmfio [-~gs ~.nd am capable of p~oducmg a v~dery 0f flbrogenic pep'ides also a~gues for their role ~n the pathogenic prc~e~ oflpf. the observation that stimume~ maerophages ere often histologica[iy prominent in fibrotio lungs and ~re ~pable of producing a varie~, offibroge.~e peptide~ also argues for tkek role in the pathogenic process, therefore, we ha-~e tested the potentn for iater!eukln-8 (i1..-8) and mo~tocyte chemotacde pop, de 1 (x¢cp-1) to induce neutro~hil ~d mononuclear phagocyte accumuhdon in lungs of pafient~ with pulmonary .~r~idosis and i~f. brenet~o.alveolar lavabo (bal) fluids from 12 ipf and 15 sar~qidosis patient were conexntratea by reversed-phase chromatography, ~d ii.4 arid mcp-i asso.~ed by ells& ehemotaxis mad enzyme-reieasing ~ssas's on msnocyte~ and neatrophiis. elisa revealed significenfly elevated b al-eoneentrations o£mcp-i (20.1 ng]mg aibumm) in purisms with p~monary sarcoidodis artd in ipf (41.8 ng!mg) in comparises to 1.1 normal individuals (4.24 ng/mg) and to 15 patients w~th obreic bronentis (cb) (~, 16 rig/rag). similarly, chemota*dc ac~a~' for monocles (mcp-1 e.qu/va]ent) was strongly increased in sareoidosis (86.03 ngjmg) as well as ~n 12f pag,nts (54.47 ng/mg). norra.al indlvidu~s and cb patiants hzd a 7. or 3-fold lower ~cn%i~y, re~peefively. patients with ipf and sarcoidosi~ also h~l eievated il-8 ievei~ (15.5 and 26.0 rig/rag, respe~veiy; nomzls: 2.14 rig/rag; cb: 4.23 ng/mg) mad nvatropmi ohemotax~ (60.25 ~'~d 49.68 nnmg, res!z~ztiveiy; aormals: 0.25 ng,'mg; cb: 1l06 ngmg). these data suggest that increased ievels of born mcp.1 ~d il-8 may be oharacted~tie for ~arcoidosis or ipf_ it appears iikely that both ehernoattraetants ~ontribute to the influx ofmonocytes and neutrophils into the pulmonary alveoius and interstit~um in these dlsea~es. we have recently shown that the combined administration of noninjurious doses of lps and paf in the rat produce ards-like lung injury characterized by neutrophil adhesion to lung capillary venules, neutrophil accumulation in lung parenchyma, pulmonary edema, and increased protein and neutrophil count in bal fluid. this new paradigm of lung injury was associated with elevated serum tnfc~ and pretreatment with anti tnfa mab dose-dependently prevented these responses. also, the combined administration of lps and paf induced lung mrna levels of tnfe~ (5 fold vs. lps or paf alone), ll-lg (80 fold), kc (60 fold) and il-6. taken together, these data suggest that this new paradigm of lung injury is cytokinemediated and that lps/paf in vivo can functionally couple to the activation of gone expression of a multi-cytokine network system, all of which may be involved in the pathogenesis of ards. materials and methods. the sheep model included hemorrhagic shock and closed femoral nailing at day 0, 12hourly injections of e. coli endotoxin and zymosan-activated autologous plasma at clays 1-5 and further observation and measurements at days 6-10. from venous blood and bronchoalveolar lavage(bal)fluid of ten merino sheep (mean weight 30 kg) neutrophil counts (10e6 pmn/ml blood or epithelial lining fluid-elf-), the elf/ plasma ratio of albumin (r), and the zymosan-induced (stim) and non-induced (spont) chemiluminescence response (cl) of blood (10e6 cpm/250,000 pmn), and of blood-and bal-isolated pmn (10e6 cpm/25,000 pmn) were measured. for statistical calculations the wilcoxon test was used. data of the changes in polymorphonucleur leukocyte (pivinl) metabolism have been suggested to play a pivotal part in the post-traumatic systemic inflammatory response syndrome. the underlying cellular mechanisms which control this response are not yet completely understood. since the 'ca 2+ second messenger'-system has been shown to be involved in regulation of pmnl-'respiratory burst', we investigated changes in pmnl-ca z÷ regulation in relation to oxygen free radical mediated injury. methods. in 12 polytranmatized patients (mean injury severity score = 32) arterial and venous blood samples during 15 days. daily evaluation of horowitz-quotiant (po2/fio2), plasma lactate (mg/dl) and body temperature ( results. body temperature peaked at day 6 and 7 (day 0: 36+.4; day 7: 39.8+.4). plasma lactate was significantly increased at day l (36+2) and day 7 (16.2+2). hurowitz-quotient (day 0: 344+43) was low at day 4 (199+19) and day 7 to 10 (178+25)(p<.05). at day 7 a substantial rise in venous pmnl-superoxide production (day 5: 1.9+_.45, day 7: 3.3+.7, day 9: 1.72+_.8), oecured with significant increase in plasma lipid peroxidation (day 1:0.6+0.2 ; day 7: 1.3+0.2). pivin~-myeloperoxidase activity was high at day 2 (2.3+--.5) and then continuously declined (day 7: 1.3+.3). plasma antiexidant activity (glutathione pemxidase) was reduced by 34% at day 7 (day 1: 5.3+.3; day 4: 4.4+_.2; day 7: 3.5+.2). whereas basal ca 2+ concentration remained unchanged (day 4: 68+_17, day 7: 68+_15), fmlp-stimulated cytosolic ca 2+ mobilization increased at day 7 (day 4: 457+89, day 7: 11084410, day 9: 670+262). conclusion. the present study in polytraumatized patients shows, that seven days after injury the agonist-induced pmnl ca 2+ mobilization is significantly enhanced. at the same time, pmnl-oxygen free radical release and phagocytotic activity, systemic fever response and lactate concentrations were maximal. these observations were accompanied by post-tranmatic respiratory failure and in some patients by clinical signs of multiple organ failure. preliminary data from an ongoing study using hes-and dextran-infusions in these patients show attenuation of this inflammatory response. stefan rose, m.d., trauma surgery, univ. of saarland, 66421 homburg/saar 1donnelly sc, 1haslett c, 1dransfield i, 2robertson ce, 3grant is, 4carter c, 4ross ja, 5tedder tf. 1dept's of respiratory medicine, 2accident & emergency, 3intensive care, 4surgery, university of edinburgh, scotland and 5dept. tumor immunology, dana farber cancer institute, boston. the selectins are a family of adhesion molecules (l-selectin, e-selectin, pselectin), all of whom are implicated in inflammatory cell transendothelial migration. they, as a family can be proteolytieally cleaved from their parent cell and exist in a soluble form within the circulation. ards is a disease state in whic neutrophils and neutrophil transendotheliat migration have been implicated. in this study we wished to investigate whether the levels of these circulating soluble receptors from patients at-risk of ards at initial hospital presentation, correlated with subsequent ards progression. eighty-two patients were enrolled (pancreatitis (n=19), perforated bowel (n=12), and multiple trauma (n=51)), of whom 14 progressed to ards. assays for soluble l,p & e-selectin were performed on collected plasma samples via a sandwich elisa. (ns = not significant, **** = p<o.0001) we have found a highly significant inverse correlation between a low circulating soluble l-selectin (and not soluble e & p-selectin) and subsequent ards. these results further our understanding of neutrophilendothelial interactions in the early stages of ards pathogenesis and offer the promise of sl-selectin in combination with other markers of inflammatory events being used to identify individuals at particularly high risk of ards progression on initial hospital presentation. it has been suggested that polymorphonuclear leukocytes aggregate in the lung capillaries of patients developing the adult respiratory distress syndrome (ards) and account for the endothelial damage while releasing toxic metabo-iites such as o.a. free oxygen radicals. among many antioxidants which have been investigated in in vitro systems and in animal models, n-acetylcysteine (nac) has been established to be useful as a free radical scavenger in vivo. to assess the influence of n-acetylcysteine (nac) on the activation of neutrophils in patients undergoing cardiopulmonary bypass (cpb) surgery with extracorporeal circulation (ecc), we evaluated the plasma levels of elastase and myeloperoxidase (mpo) throughout the operation and up to 24 hours after surgery. four hours after surgery also a bronchoalveolar lavage was performed. a spectrophotometric method was used for the detection of plasma elastase activity and mpo levels were measured using a radioimmunoassay. we found that pretreatment with intravenous nac prevented the increase in elastase activity (632 _+ 231 pmol/i vs 1666 _+ 187; p -0.027), but not the release of neutrophil related mediators (2.20 _+ 0.56 ng/i vs 1.80 _+ 0.17; p=0.63). besides, nac had the capability to prevent the enhancement of neutrophil numbers in lavage fluid as is normally seen during cpb (1.4 + 0.8 % vs 25.0 _+ 14.3; p =0.04). although not significant, nac prevented also the increase in elastase levels in lavage fluid (19.0 + 18.5 pmol/i vs 172.7 + 101.4; p = 0.12). it is concluded that nac is able to protect against the inactivation of a 1-proteinase inhibitor, the main inhibitor of elastase activity, and that nac interferes with adhesion and migration of neutrophils. therefore nac may be useful in the prevention of tissue damage. this study is supported by inpharzam belgium. sodium nitroprnsside (snp) has previously been shown to attenuate the neutrophil induced lung injury associated with limb ischaemia and repeffusion. glyceryl trinitrate (gtn), already widely used in aortic surgery, also increases nitric oxide but has a less marked splanchnic "steal" effect. we examined the effect of gtn on lung injury, neutrophil infiltration and activation in a model of aortic occlusion (30 rain) and repeffusion (120 min). sprague dawley rats were randomized into: control (n=i 1); ischaemia repeffusion (ir) (n=12); and ir treated with gtn (2ug/kg/min) during repeffusion (n=10). myeloperoxidase activity (mpo) measured pulmonary neutrophil influx. pulmonary endothelial permeability was measured by wet to dry weight ratio (w/d), broncboalveolar lavage protein (bal) and neutrophil counts (bal pmn). neutrophil superoxide release (mean channel fluorescence mcf) was measured by flow cytometry in a further ir v gtn experiment (n=6 per group). control 573_+t02"* 992_+125 579_+54** bal pmn/mm 3 36l+-126 3"219+1087# 820_~221 *p<0.05, #p<0.02 v control/gtn;**p<0.01 v ir. students t test the significant increase in mpo activity produced by ir was attenuated by gtn. the increase in pulmonary microvascular leakage after reperfusion was also prevented by gtn. neutrophil superoxide release increased significantly from 9.4+0.76 to 13.9_+1.92 mcf after 40 minutes repeffusion (p<.01). this increase in superoxide was prevented in the gtn treated group. these results indicate that gtn inhibits neutropbil superoxide release during limb reperfusion, thus preventing pulmonary vascular leakage and neutrophil infiltration. these data suggest that the beneficial effects of gtn in aortic surgery may be due in part to a protective effect onpulmonary microvasculature. department of surgery, beaumont hospital, dublin 9, ireland. lipton adult respiratory distress syndrome (ards) is marked by increased vascular permeability of the lung to white blood ceils (wbc). indeed, influx of wbc into the lung is believed to be the central mechanism in acute lung injury of ards. evidence is developing that the neuropeptide c~-melanocyte stimulating hormone (c~-msh), a proopiomelanocortin derivative, inhibits inflammatory reactions in animai models of inflammatory responses in humans. to test the influence of a-msh on experimental ards, the peptide was administered to rats after intratraeheal infusion of endotoxin (s. typhosa, 500 #g in 0.25 ml saline). three groups (n=10 each) received: intratracheai infusion of endotoxin plus ip injections of o~-msh (100 ~tg in 0.2 ml saline) at 0, 2, and 4 hr post-endotoxin treatment; intratracheal endotoxin infusion plus saline injections; control intratracheal saline infusion plus saline injections. the bal data showed overall differences among groups (f2,29=6.2, p<.003), o~-msh treatment inhibited endotoxin-induced wbc migration into the pulmonary tree (p<.05). circulating wbc counts were markedly lower in both groups given endoroxin than in the control group (p<.01), but there was no difference in wbc count between these two endotoxin-treated groups. the results indicate that c~-msh can reduce wbc migration in experimental lung injury. in further experiments we tested the hypothesis that c~-msh, administered alone or in combination with an inhibitor of gram-negative bacterial growth, increases survival in a model of peritonitis/endotoxemia/septie shock. cecal ligation and puncture were performed under sodium pemobarbital anesthesia (75 mg/kg, i.p.) in female balb/c mice (16-20g) that were then randomly assigned (15-17 mice per group) to receive at time 0 and 3 hr later: control saline thjectioas (i.p.), injections of ~-msh (100 ~tg), gentamicin sulfate (i0 mg/kg), or both ~-msh and gentamicin. one ml of normal saline was given s.c. to each animal for fluid replacement. both ix-msh and gentamicin treatments administered singly improved survival; when given in combination survival was even greater these findings suggest that the anticytokine ot -msh molecule might be useful for treatment of systemic inflammation, perhaps particularly when used in combination with agents that inhibit bacterial growth. previous studies showed a close relationship between xanthine oxidase activation, membrane damage and postischemic cardio-respiratory failure following aortic clamping and reperfusion. aim of the present study was to evaluate alterations of polymorphonuclear leukocyte (pmnl)-metabolism and signal lransduction systems during ischemia/reperfusion induced by aortic clamping in man. methods, in 14 patients (3 female, 59+4 years) with elective reconstruction of infrarenal aortic anearysms, arterial (a) and venous (v) blood samples were obtained before clamping and declamping, and 60 min after declamping. arterial pla 2 concentrations were higher than venous (113+32 vs. 71+30). while cytosolic basal pmnl ca 2+ concenlxation was not altered at the end of ischemia (v: 52.3+8; a: 42_+6) and during reperfusion (v: 60+8; a: 69+12), fmlp-stimulated cytosolic ca 2+ mobilization showed a significant arterial increase as compared to venous (1652_+601 vs. 638+85, p < .05). these presented data indicate that the reperfusion syndrome after infrarenal aortic clamping is characterized by 1) pulmonary membrane damage possibly due to pmnl-degranulation, 2) activation of pmnl-superoxide radical production with release of activated pmnl in arterial circulation, and 3) a significant arterial increase of pmnl cytosolic ca 2+ mobilization after fmlp-stimulation parallel to pmnl-activation. in conclusion, ischemia/reperfusion in man induces pulmonary damage and activation of pmnl superoxide production possibly due to an altered pmnl-ca 2+ messenger system by inflammatory mediators (e,g. ii-8, tnf, paf). the septic lung diseases (i.e. ards) generally show distinct alveolar damage and surfactant disturbances. it is thought that alveolar macrophages are involved in specific release products like h202 and cytokines like tnf. in this pathway the type ii alveolar epithelial cell (p2 cell) is not only an important target, but as recently shown it is also capable of producing h202. the aim of this study was to investigate the influence of endotoxin on h202 release of p2 cells. we obtained p2 cells from lungs of female wistar rats and investigated the effect of lipopolysaccharid (lps) on h202 release of fresh isolated cells and on cells cultured for 2 days in a fibronectin matrix. furthermore we conditioned isolated alveolar macrophages for 16 hours with 1 p.g/ml lps and tested the conditioned medium (mcm) on cultured p2 cells. p2 cells 5 h ex vivo were _> 70 % pure, _> 95 % vital and had an basal h202 release of 64.5 _+ 1.8 nmol h202 per hour and million cells. adding 1 p.g/ml lps to the incubation medium the h202 release decreases slightly but significantly to 54.2 _+ 2.3 nmol. adding 0.3 p.g/ml phorbol myristate acetate (pma) to the basal incubation medium the h202 release increased 3-fold to 148.3_+ 26 nmol. pma induced h202 release decreased to 128.3 + 41.5 nmol after addition of 1 p.g/ml lps. after 2 culture days the p2 cells were _> 98 % pure and showed a pma inducible h202 release of 174.8 _+ 9.9 nmol addition of 1 p.g/ml lps had the inverse effect as on freshly isolated cells as it increased the h202 release up to 216.2 _+ 2.4 nmol. addition of mcm to cultured p2 cells increases pma-stimulated h202 release to 223.3 +_ 10.7 nmol. the release decreased to 191.9 _+ 2.4 nmol when an murine anti-tnf-alpha antibody was added. vitality of cultured cells was > 96 % in all experiments. the results show that lps has an direct effect on p2 cells cultured on fibronectin. we conclude that the observed additional stimulatory effects of mcm seems to depend on tnf-alpha. the induction of h202 release of p2 cells could be important for generating internal oxidative stress in p2 cells before external oxygen radicals exceed. the produced h202 did not necessarily damage p2 ceils, but it can effect surfactant metabolism, especially when extracellular h202 release of alveolar macrophages following an immune response is increasing. introduction: primary stabilization of femoral shaft fractures in patients with multiple trauma is beneficial. however, in patients with associated lung contusion we have found an increased incidence of ards, apparently associated with primary reamed femnral nailing (rfn). previous animal studies revealed, that perioperative disturbances of lung ftmetion appear to be related to the reaming procedure, ix~ssibly due to pulmonary embolizafion of bone marrow fat. in a prospective clinical analysis we compared effects of intrameduuary nailing with and withont reaming on parameters known to be related to ards-pathoganesis. in order to gain further insight into the role of endotoxin and cytokines in the pathogenesis of the adult respiratory distress syndrome (ards), we enrolled 23 patients with severe lung injury after sepsis (17) or polytrauma (6) and obtained multiple blood samples (14 days) for endotoxin, tumor necrosis factor e (tnfa), interleukin 18 (il-18) and interleukin 6 (il-6) determination. to evaluate the cytokine releasing capacity of the blood, plasma concentrations of tnfe, il-l8 and il-6 were also determined after the "in vitro" stimulation of the whole blood samples with lipopolysaccharide (lps, 0.1 ng/ml) for 2 hours at 370 c (stimulated values). the difference among stimulated cytokines levels and the basal plasma concentrations were defined as "delta values", an expression of the cytokine releasing capacity of the blood. the pao2/fiao2 quotient was used as an index of the severity of lung injury (sli). the endotoxin plasma level was significantly higher in patients with sli < 200 (0.288 ± 0.038 eu/ml, mean values ± sem) versus the patients with a sli > 200 (0.175 ± 0.023 eu/ml, p<o.01). the basal plasma concentration of ll-6 also correlates with the sli index (p<o.o01). the delta tnfe and delta il-6 values were s~gnificantly decreased in patients with a severe lung injury. compared with the survivors (n:11), the deceased patients (n=12) showed higher endotoxin level, a reduced tnfa and il-6 releasing capacity of the blood and higher basal il-6 levels. we can conclude that endotoxemia, high il-6 plasma level and a decreased capacity of the blood to release tnfa an il-6 under endotoxin stimulation associates with the severity of lung injury. [objectives] experimental and clinical findings implicate the involvement of inflammatory cytokines in the pathogenesis of the decreased oxygenation in the lung after major surgery, though the mechanism remains unclear. in the present study, we elucidated the involvement of il-8 in the pathogenesis of lung injury defined by deterioration of pulmonary oxygenation after esophagectomy. [materials and methods] seventeen patients underwent subtotal esophagectomy through a right thoracotomy. in all patients, tbe alveolar-arterial oxygen tension difference (aado2) was measured everyday and bronchoalveolar lavage fluid (balf) samples were obtained from a single segment ($4 or $5) of the right and left lung through a bronchoscope on days l, 3, and 5 days after surgery. they were then examined for cell analysis, measurement of cytokines by elisa, and measurement of neutrnphil elastase (ne) by elisa. [results] aado2 unexceptionally deteriorated postoperatively and the mean aado2 in 17 patients reached the maximum (mean ± sem; 372 _+ 23 mmhg) on the 3rd postoperative day. the mean concentrations of il-8, ne and neutrophil count in balf also increased postoperatively and reached their maximum level (2137 + 618 pg/ml, 1603 -+ 493 gg/l, and 1561 ± 477 x 103 cells/ml, respectively) on the 3rd postoperative day. these increases in balf were recognized in both the right and left lung. a highly significant correlation was observed between il-8 and ne levels in balf (r=0.82, p=0.0001). also, significant correlations between aado2 and the neutropbil count, and the concentration of ne or 1l-8 were observed. in contrast to il-8, neither il-6, il-113 nor tnfa was detected in balf. [conclusion] major surgical trauma such as esophagectomy may induce recruitment of neutrophils to the lung and cause lung injury. il-8 increased in the lung is an important mediator of neutrophil recruitment and activation in the lung. phorbol myristate acetate (pma) is commonly used to cause edema and other tissue damages in the lung. lung injuries induced by the administration of pma has been shown to be mediated mainly by neutrophils. neutrophils recruited to the lower respiratory tract may damage lung tissues by releasing reactive oxygen species, neutral proteases and lysosomal enzymes. the present study was conducted to investigate whether c~tocopherol, entrapped in dipalmitoylphosphatidylcholine liposomes and delivered directly to the lung, could counteract some of the pma-induced lung injuries. plain liposomes or c~tocopherol-containing liposomes (8 mg c~-tocopherol/kgbody wt.) were instilled into the lungs of rats 24 h prior to pma exposure (25 pg/kg, intratracheally) and treated rats were killed 3 h later. lungs of control animals exposed to pma developed increases in lung weight and lipid peroxidation as well as decreases in lung angiotensin converting enzyme (ace) and alkaline phosphatase (akp) activities. pma treatment also caused an increase in myeloperoxidase (mpo) activity in the lung, suggestive of neutrophi] infiltration. pretreatment of pma-treated rats with plain liposomes had no effect on pma-induced injuries. in contrast, pretreatment of rats with liposomal c~-tocopherol, 24 h prior to pma administration, resulted in a significant elevation of pulmonary a-tocopherol concentration, accompanied by a concomitant reduction in mpo activity and reversal of pmainduced changes in lung edema, lipid peroxidation, ace and akp activities. these results appear to demonstrate that the intratracheal administration of a liposome-associated lipophilic antioxidant, such as a-tocopherol, can significantly ameliorate the toxic effects of reactive oxygen species, released from pmastimulated pulmonary target cells and infiltrating neutrophils. jk wojcik, g palasiewicz, w roszkowski immunology department,institute of tuberculosis and lung diseases~ warszawa, poland, m~larhospital, eskilstuna, sweden. introduction:the critical point in neutrophil migration to the alveolar space in the course of ards is the attachment between giyeoproteins of neotrophil membranes(sialyl lewis x carbohydrate epitope containing u-l-fucose) and lectin domains of endothelial p and e selectins (gmp-140,elam-i). a potential beneficial therapeutic strategy may be designep to suppress neutrophil recruitment to the lungs by preventing their rolling and attachment to the pulmonary endothelial cells. objeclive:to investigate if tz-l-fueose prevent experiments pulmonary hypertension in ards as effective as metylpredniselone. methods:only healthy rabbits (n-24) weighing 5.d-5.5 kg oaselinepao>11 kpa and mean pulmonary arterial pressure (mpap)<i.5 kpa were included in this study. anaesthesia was induced with thiopental 20-25 mg/kg bw and "blind" jntubation was performed. a 5f swan ganz catheter was introduced via the left jugular vein into the pulmonary artery. pma, as was used in our experiments activates neutrophils and produces acute respiratory failure with pulmonary hypertension and pulmonary edema. eight rabbits serving as control animals received pma 25 ug/kg bw iv only. sixteen other animals were divided into two groups receiving either ~-l-fucose 100 mg iv or metylprednisolone 150 mg iv 15 min before pma admininstration. results:after pma administration notable physiological changes including marked increase in mpap(2.0-+o.~6 kpe3 ..lere found. the increase of npap was observed to be completely blocked in both groups of animals which received ~-l-fucose or metylprednisolone. conclusion:in aur rabbit model ards cz-l-fucose pretreatment prevents pulmonary hypertension after pma administration. the effect is comparable with high dose metylprednisolone. the possible explanation is that g-l-fueose molecules block iectin domains of gnp-140 or elam-i preventing the adhesion of the neutrophils to the pulmonary endotheliom. bartens c, elmadfa i, steltzer h, fischer m, druml w introduction: various micronutrients and vitamins are particulary effective in modulating immune functions and host defense. the nutritive ant±oxidants vitamin c, e and b-carotene, as well as selenium lower the burden of reactive free radicals and protect the structural integrity of cells and tissues of the immune system, as well as other systems in the body. certain cells of the immune system like polymorphonuclear leucocytes (pmn's) use free radicals as a weapon to destroy invading pathogens. these reactive molecules, when released into the surrounding area, mediate lipid peroxidation and tissue injury. there is growing evidence that pmn's are activated in vivo by complement or immune complexes in disorders such as ards. the respiratory host defense mechanisms of patients with adult respiratory distress syndrome (ards) may be defective. the aim of this study was ta evaluate the status of the free radical scavengers and their activity in ards and to investigate the respiratory burst of ards patients. methods: seven ards patients (3 sepsis, 4 trauma) with an fie 2 of 0.59+0.(36, a peep of 8.50±0.99, and a murray score of 2.83 ±0.18 were included in this study. 15 healthy adults served as controls. superoxide anion production in granulocytes was measured photometrically, and the hydrogen peroxides by fluorimetric assay. vitamin a, e, and g-carotene concentrations were assessed by hplc, and plasma vitamin c photometrically. plasma selenium was determined by dectrothermal aas. plasma lipid peroxidation pruducts, expressed as malondialdehyde (mda), were determined by thiobarbituric acid assay and hplc. results: mda-plasma (/xmol/l) 0.74~: 0.46* 0.37 ± 0.16 *= <0.01, **=p<0.001 condusion: ards patients showed significantly decreased plasma concentrations of vitamin c, e and g-carotene, as well as selenium. these nutritive ant±oxidants are consumed during increased oxidative stress. mda, a final product of lipid peroxidation, is increased. however, a difference in rates of release of hydrogen peroxides and superoxide-anion of pmn could not be detected. therefore, oxygen radical accumulation is more likely a result of excessive inspiratory oxygen concentrations (f.io2), pro-oxidant drugs, and a high settlement of pmn in the lungs, and not an increased release of free radicals of the single pmn. kd glycoprotein secreted by the alveolar type ii cells. recent study showed that sp-a exists in the sera from normal healthy adults and that the significantly elevated serum sp-a levels were observed in the patients with interstitial pneumonia and pulmonary alveolar proteinosis. these findings means there may be a mechanism that the sp-a produced in the alveoli escapes into the bloodstream. here we examined the serum sp-a levels in the patients with critical illness including sepsis and ards. ]clinical subjects & methods] a total of 99 serum samples were collected from 39 patients hospitalized in the division of critical care medicine(icu), sappom medical college hospital. serum sp-a levels were measured by an enzyme-linked immunosorbent assay using monoclonal antibodies to human sp-a. ]results] there was no significant difference between the septic (n=12) and non-septic (n=27) patients. patients with ards (n=10, 52.0+4.85ng/ml) and with respiratory disease other than ards (n=12, 49.6+-5.35ng/ml) showed significantly higher levels of serum sp-a than those with non-respiratory disease (n=17, 27.4_+4.86ng/ml). as to the ards patients, it was likely that the serum sp-a levels were getting higher in their clinical courses. however, there was no significant correlation between the serum sp-a levels and the pa%/fio 2 ratio. ]discussion] at the present time, the exact mechanism of the escape of sp-a into the bloodstream remain to be unclear. our results may suggested that there was some relation between this leakage of sp-a and the alveolar-capillary permeability because of the higher sp-a levels observed in the ards patients. however it is also likely that higher serum sp-a level represents the proliferation of alveolar type ii cells as the regeneration of damaged lung. further studies are now being done. neutrophils contain a number of enzymes within intracellular granules,potentially damagingto lung tissue.release of these enzymes into the lung tissue from the sequestred activated neutrophils is felt to play significant role in lung injury in the course of aros. using 6-hours acute animal model of aros the activity of cathepsins,beta-glucuronidase and acid phosphatase was determined in the lung tissue. results of this research were compared with neutrophil proteases activity in serum and broncho-alveolar lavage.also hemodynamic,respiratory and biochemical investigations were performed before beginning of experiment,and in a two-hours time intervals of its duration.after 6 hours was determined total and free activity of cathepsins,beta-glucuronidase and acid phosphatase in full homogenate of the lung tissue,mitochondriallysosomal fraction and the final supernatant. in the course of our experiment we observed an increase (30.i-49.6%) both total and free activity of neutrophil proteases in all fractions of the lung tissue.generally accepted current pharmacological treatment of aros only attenuated this increase but never caused reversion to the level before beginning of our investigations. the presence of aros was histologically proved.an activity of acid phosphatase in serum and the lung tissue fractions was compared with histochemical pictures of the lung. results of our research indicate that neutrophil proteases are a very important mediators in aros and they are a cause of the lung injury. in addition neutrophil-derived proteases can amplify the inflamatory process by enzymatitally activating of many other mediators and they also may to increase an oxidant induced injury by imparing of antioxidant defenses. oepartment of general surgery, sk~odowskiej 24a 15-276 ~ia~ystok, poland, tel/fax +48 85 619742. in vitro studies demonstrated that paf activates polymorplionuclear leukocyte (pmn) 5-1ipoxygenase, but the generation of leukotrienes (lts) is critically dependent on exogenous arachidunic acid (aa) disposal (f. grimminger et at., mol. pharmacol. 41:757, 1992) . we investigated the features of paf-evoked lt synthesis in a model of pulmonary microvascular leukostasis, in the presence and absence of intravascular n-3-and n-6 -prescursor fatty acid supply. human neutrophils were infused into isolated, ventilated and perfused rabbit lungs to achieve microvascular sequestration of ligand-responsive leukecytes. leukotrienes (lt) and hydroxyeicosatetra(penta)enoic acids (het(p)e) were quantified by itplc techniques. intravascular application of paf (5 um) or arachidonic acid (aa;ioum) provoked the generation of limited quantities of 4-series lts and 5-hete (total sum of 5-1ipoxygenade products rd. 7 and rd. 27 pmol/ml.; both in pmn-preloaded and non-preloaded lungs). combined administration of pat r and aa caused amplification of 5-1ipoxygenase product formation with predominance of cysteinyl-lt synthesis both in lungs without (total sum rd. 67 pmol/ml) and, much more strikingly, with pro-application of neutrophils (total sum rd. 308 pmofml). eicosapentaeooic acid (10 um) elicited exclusive generation of 5-series lts and 5-hepe (total sum rd. 82 pmol/mi). dual stimulation with paf and epa provoked amplification of epa-derived 5-1ipoxygenase product formation, again with predominance of cysteinyl-lts, in lungs without (total sum rd. 224 pmul/ml) and in particular in those with preceding microvascular pmn entrapment (total sum rd. 545 pmol/ml). we conclude that the paf-evoked 5-1ipoxygenase product formation in the neutrophil-harbouring lung capillary bed is critically dependent on intravascular precursor fatty acid supply, with epa representing the preferred substrate as compared to aa. pmn-related transcellular eicosanoid synthesis is suggested to underly the predominant generation of cysteinyl-lts. these findings may be relevant for lipid mediator profiles provoked by infusion of n-3-versus n-6enriched lipid emulsions in diseases with pmn-related inflammators events. adult respiratory distress syndrome (ards) involves damage to the alveolar epithelium and microvascular endothelium. products released from neutrophils (pmn) are thought to be among the chief causes of this damage, while the role of mononuclear cells (mnc) is uncertain. we studied patients at risk of or with acute ards. we measured the concentration of myeloperoxidase (mpo) and elastase (el) within circulating pmn as an index of pmn activation, elastin degradation products (edp) in the plasma as an index of tissue damage, as well as the cytotoxici~y of pmn and mnc to endothelial cells (ec) in vitro. cells and plasma were prepared from venous blood of healthy controls; or systemic arterial blood of patients on ventilators in intensive care but not at risk of ards; at risk of ards because of sepsis; at risk because of multiple transfusion/major trauma; or with acute ards. lung injury, multi-organ failure and apache h scores were recorded. to measure cytotoxicity, human umbilical vein ec were labelled with cr-51, incubated with either pmn or mnc for 18 hours, and the supernatant counted. the adherence of the pmn and the mnc to the ec was also measured. standard assays were used to measure mpo and el in the pmn, mad edp in the plasma. the mnc cytotoxicity did not differ between the groups. the pmn cytotoxicity was higher in the transfusion/trauma patients compared to each other group, which did not differ from each other. there was no correlation between cytotoxicity and adherence for any group. the mpo and the el concentrations were significantly decreased in the pmn from each of the patient groups, including the patient controls, compared to healthy controls. the edp were only increased in patients at risk of or with ards. thus pmn degrannlation occurred in patients not at risk of ards, but tissue damage as measured by increased edp only occurred in patients with or at risk of ards, suggesting that factors additional to pmn activation and degranulation are required for the progression to ards. there was no correlation between the parameters measured and the indices of disease activity, or outcome. these measures of pmn and mnc activation and function are not of value as prognostic indicators in patients with or at risk of ards. the liver is made up of several different cell types which subserve distinct functions in vivo. in addition to the different functions of the distinct cell types, it is now evident that even within the population of hepatocytes substantial functional heterogeneity exists. this heterogeneity occurs in a very organized fashion in the normal liver based upon the position of the hepatocyte in the acinus. although it has been proposed that the acinar heterogeneity of hepatocytes arises from migration of immature hepatocytes from the periportal region to the perivenous region where they become senescent and die, many of the heterogeneous responses can be accutely reversed by reversal of the oxygen gradient via retrograde perfusion. under normal conditions in which the acinus is exposed to unidirectional flow, the periportal hepatocytes are exposed to relatively high levels of oxygen, substrates and hormones. as these substances are extracted along the aeinus their concentration decreases leaving relatively low concentrations for the perivenous hepatocytes. in the normal liver, the heterogeneity of response is attenuated by extensive communication via gap junctions. the major liver gap junctions are made up of hexamers of connexin 32 (cx32) which forms channels between cells capable of pas.~ing any molecule smaller than 1000 d. coupling is sufficient to allow diffusion of molecules such as atp or camp from one hepatocyte into > 20 adjacent hepatocytes within 60 seconds. during stress conditions such as endotoxemia or zymozan inflammation, expression of cx32 is markedly decreased while the secondary gap junction protein cx26 is either unchanged or even increased. while cx 26 readily effects electrical coupling, molecules > 350 d pass only very slowly. this would result in restriciton of transmission of moecules the size of atp or camp. since inhibition of gap junctions also attentuates metabolic response to hormone or nerve stimulation, it is evident that modulation of hepatocyte hetereogeneity by gap junctions must be considered in determining the mechanisms of metabolic alterations during stress. already minor haemorrhage decreases portal venous blood supply to the fiver and the reduction in portal blood flow becomes more pronounced with more profound btood loss. severe hacmorrhagic hypovolemia also reduces hepatic arterial blood supply which, however, is maintained over a vide range of haemorthage. the net effect of blood loss is a reduction in liver oxygee supply and this reduction is in proportion to the vulume iossed. however, oxygen supply to the liver exceeds the demands of the normal liver and this is the ca~ stilt following reduction of 30 % of blood volume. the situation in sepsis is more complicated. po~l venous supply to the liver is redur.~i fairly early following normovolemic sepsis while hepatic arterial blood supply is maintained at le,~t initialiy, oxygen saturation might be maintained in arterial blood but may also be slightly reduced during sepsis, oxygen saturation of portal venous blood is significantly reduced during sepsis due to increased extraction of the intestines. therefore oxygea delivery to the liver during sepsis becomes sigalfkzntly reduced. at the s,~ne time and for mai.v.ly unknown reasons the need for oxygen becomes significantly increased in the ~-~ptic liver. as a consequence liver oxygen consumption becomes flow dependent and the liver is likely to suffer from ischemia during septic conditions. $94 although liver failure is well recognized in sepsis, it is generally thought to be a late complication following pulmonary and renal failure. jaundice, hypoglycemia, encephalopathy and bleeding secondary to low levels of liver-synthesizing clotting factors are, however, signs of rather severe end-stage hepatic failure. furthermore, elevated liver enzymes (sgot and sgpt) represent hepatucyte damage and not hepatocellular dysfunction. in view of this, a more sensitive indicator of hepatic function is desirable in order to detect early hepatic abnormality. in this respect, indocyanine green (icg) is a tricarbocyanine dye that possesses several properties which makes it particularly valuable inthe assessment ofhepatic function. this dye is bound m albumin and is cleared exclusively by the liver through an energydependent membrane transport process and is nontoxic at lower doses. we propose that maximal velocity (vm~,) of icg clearance is a valuable measure of active hepatocellular function, since the total concentration of functioning receptors is directly proportional to vm~. we have utilized a fiber optic catheter and an in vivo hemoreflectometar to continuously measure the administered icg in vivo and consequently determine its clearance without the need of blood sampling. using this technique, we have found that in the early stages of sepsis (i.e., 2 and 5 h following cecal ligation and puncture), the vm~ and kinetic constant (k=) of icg clearance was significantly depressed. it should be noted that at this stage of sepsis, there was no elevation in serum enzyme levels. furthermore, hepatic blood flow and cardiac output increased at the above mentioned time points. thus, the extremely early depression in active hepatocellular function in sepsis, despite the increased hepatic blood flow and cardiac output, may form the basis for cellular dysfunctions leading to multiple organ failure during sepsis. additional studies indicated that following hemorrhage, active hepatocellular function was markedly depressed. this returned to prehemorrhage levels after ringers lactate resuscitation, however, this function was not maintained and decreased significantly after fluid resuscitation. nevertheless, the depressed active hepatocelinlar function following hemorrhage was markedly improved by post-treatment of animals with either atp-mgci2, peutoxifylline or diltiazem. thus, the use of icg clearance provides an early sensitive indicator of hepatic abnormality during sepsis and following hemorrhage and this method should be used, not only experimentally, but also in the clinical arena for the early detection of hepatocellular abnormality. although multiple organ dysfunction syndrome (mods) remains a major cause of mortality and morbidity in intensive care units, very little is known about the mechanisms that precipitate its development. since an episode of inadequate tissue oxygenation is considered to be the trigger for mods, we have proposed that a primary localized injury such as ischemia/reperfusion may be sufficient to cause a change of gene expression of remote and apparently unaffected organs. such modulation of remote organ gene expression may decrease the organ's tolerance to a subsequent stress contributing to the development of mofs. to test this hypothesis, rats were subjected to hepatic regional ischemia by clamping the blood flow (hepatic artery and portal venous inflow) of the left and median liver lobes. intestinal congestion was prevented by allowing flow through the smaller right and caudate lobes. after 60 minutes of ischemia, the clamp was removed and the blood flow restored. the animals were allowed to recover for 1, 4 and 24 hours. kidneys were removed, total rna was isolated and poly(a) ÷ selected by affinity chromatography on oligo(dt) columns. message was in vitro translated using rabbit reticulocyte iysates in the presence of radioactive amino acids. the gene products (radiolabeled polypeptides) were fractionated by two dimensional gel electrophoresis, and visualized by fluorography. analyses of the two dimensional fluorograms indicate that there is a dramatic change in the electrophoretic pattern of in vitro translated products in samples corresponding to kidneys obtained after 60 minutes of hepatic ischemia and 24 hours of reperfusion with respect to kidney samples obtained after sham operation or from control rats. the latter were not subjected to any surgical manipulation. these studies suggest that the gene expression of the kidneys is specifically modified after a remote organ injury (hepatic ischemia/reperfusion). we speculate that this change of gene expression in kidneys after an indirect injury may be part of the early events leading to the development of mods. a priming event, e.g. local ischemia, in combination with a second insult, e.g. sepsis, may amplify a host's response and lead to multiple organ failure. to better understand the mechanisms involved in the pathophysiology, male fischer rats were subjected to 20 min of hepatic ischemia followed by reperfusion (rp) and injection of 0.5 mg/kg salmonella enteritidis endotoxin (et) at 30 min of rp. et injection potentiated the postischemic liver injury as indicated by histopathology and an increase of plasma alt activities from 870+122 u/l (i/rp only) to 3900+470 u/l at 4h rp. inhibition of kupffer cells (kc) with gadolinium chloride (7 mg/kg) attenuated liver injury in this model by 70%, however, monoclonal antibodies (cl26, wt3) directed against adhesion molecules (132 integrins, cd18) on neutrophils had no effect on the injury despite the substantial accumulation of neutrophils in the liver at that time (467+52 pmns/50 hpf; baseline: 13+3). isolation of kc and neutrophils from the postischemic liver indicated a 10-fold increase of the spontaneous superoxide formation only in the kc fractions [3.85+0.68 nmol o2-/h/10%elts (kc2); 6.82_+0.92 (kca) ] at 4h rp compared to control cells. in addition, stimulation with phorbol ester or opsonized zymosan revealed a substantial priming of kc for reactive oxygen formation. in contrast to the short-term experiments (4h), the antibody wt3 (4 mg/kg) attenuated liver injury by 90% at 24 h of rp and improved survival. conclusion: liver injury during the early rp phase is mediated mainly by kc generating excessive amounts of reactive oxygen while neutrophils are primarily responsible for organ damage during the later rp period. (es-06091 and gm-42957) tumor necrosis factors (tnf) are cytokines which are cytotoxic towards some tumors in vivo and certain tumor lines in vitro. moreover, these polypeptides are powerful immunomodulators and have been found to be distal mediators in several models of septic shock and septic organ failure. one of the best-characterized experimental systems is the hepatitis caused by lps or tnf in galactosamine (galn)-sensitized mice. here we describe a cell culture system, in which the direct toxicity of tnf towards mouse hepatocytes was examined. the toxicity of tnf, as determined by ldh-release or formazan-formation, was dose-and time-dependent. the threshold of toxicity was 10 ng/ml, which corresponds to serum concentrations found in mice after lpsinjection. toxicity was only observed in hepatocytes sensitized with transcriptional inhibiters such as galn, actinomycin d (actd) or cxamanitin. sensitization was neither observed with different translational inhibitors nor with various other metabolic inlaibitors or toxins. inhibitors of protein synthesis or protein processing such as cycloheximide, puromycin, tunicamycin and ricin protected actdsensitized hepatocytes from tnf-induced cytotoxicity. tnf induced apoptotic changes and dna-fragmentation in sensitized hepatocytes which is in line with the above findings that cell death is dependent on protein synthesis. thus tnf may be a trigger of programmed cell death during inflammatory organ damage. with the purpose of studying the role of complement activation in tissue injury after ischaemia and reperfusion we blocked the complement cascade in a model of rat liver isehaemia and reperfusion, either by administration of soluble human complement receptor type 1 (scri), 25 mg/kg iv after vascular occlusion (n=8) or by depleting the complement system using cobra venom factor (cvf), 0.5 mg im, 24 and 6 hours before ischaemia (n=10). non-ischaemic rats (n=8) and ischaemic non-treated rats (n=15) were used as controls. the experimental procedure consists of the temporary interruption of arterial and portal blood flow to the left lateral and medial lobes of the liver during 45 minutes, followed by reperfusion, recording the liver blood flow and haemoglobin saturation with a laser doppler flowmeter and photometer during one hour after declamping; alt levels were assayed and immunoperoxidase stainings for c3 and c9 were performed. there were statistically significant differences between the experimental ~roups and the untreated ischaemic control group in terms of post-isehaemic blood flow (p<0.001) and haemoglobin saturation (p<0.001). c3 and c9 were present in the endothelium of the ischaemic control group. no deposits of c3 or c9 were found in the cvf group. few c3 and no c9 were found in scri treated rats. these results show that the effect of reperfusion injury in the rat liver is ameliorated either by depleting complement with cvf or by regulating complement activation with scri. hepatic dysfunction, a major cause of mortality following hemorrhagic shock, has not yet been well characterized. the present study was designed to assess the effects of liver blood flow and cytokine levels on hepatic function following resuscitation from severe hemorrhagic shock in normal and cin-hotic rats. methods: aftor pentobarbltal anesthesia, 23 control and 38 cirrhotic sprague-dawley rats were subjected to severe hemorrhage to reduce their systolic blood pressure to 50 + 5 mm hg. this level of hypotension was maintained until the skeletal muscle transmembrane potential (era) depolarized by 25%.; the animals were then resuscitated with ringer's lactate solution in three times the volume of the shed blood. serial blood samples for tumor necrosis factor (tnf) determination (a modified flow-cytomeuic wehi cell bioassay) were obtained at baseline, during hemorrhage and following resuscitation. liver blood flow measurements by low dose galactose clearance (glc) and functional bepatocyte mass (fhm; defared as galactose elimination capacity [gec] from the zero order portion of the plasma disappearance curve following an intravenous galactose bolus [500 mg/kg], divided by liver weight) were measured before shock and after resuscitation. results: higher survival rates (p < .05) were observed in control as compared with cirrhotic rats. shock produced a significant reduction in gec (to < 0.01); fhm (19 < .01); and liver blood flow (p < 0.01) in normal and cirrhotic rats. decreases in gec and fi-im were greater (p < 0.05) in cirrhotic rots. tnf levels were higher (p < 0.05) in cirrhotic rats at baseline and during induction of shock. pre gap junctions provide pathways for metabolic signals between cells. in the liver, the majority of gap junctions are composed of connexin 32 (cx32) polypeptide subunits, and are regulated by gluconeogenic hormones. since sepsis and other inflammatory states alter hepatic glucoregulatory control, we have evaluated the contribution of gap junctional conductance to the metabolic dysregulation in the liver. an acute inflammation was induced in rats by injection with e. coli endotoxin (lps lmg/kg). northern blot/hybridization analysis of total rna isolated from livers after endotoxin injection show a decrease in the steady state transcript levels of cx32 to 18% of sham controls. immunostaining of liver sections using anti-cx32 revealed punctate fluorescent staining on the plasma membrane at regions of call-cell contact in saline injected animals, whereas, staining was only observed in cytoplasmic vesicles 6 hrs after animals were treated with lps, suggesting the internalization of cx32 without replacement on the cell surface. the staining was quantitated and expressed as % of pixels above threshold. at 6hr post injection 3.6% ofpixels exceeded threshold, compared to 16.2% in sham controls. functional gap junctional communication was assessed by dye coupling using lucifer yellow in an isolated perfused liver under intravital fluorescence microscopy. dye diffusion was markedly decreased 6hr after endotoxin injection. this suggests that decreased metabolic coupling after lps injection results from decreased gap junction abundance. the present data suggest that metabolic dysregulation during sepsis may arise in part from changes in intercellular communication caused by a decrease in gap junctional expression and communication. given the marked metabolic heterogeneity of hepatocytes with respect to acinar location, metabolic signaling via gap junctions most likely serves to moderate this heterogeneity, contributing to a coordinated metabolic response. altered cellular ca 2÷ regulation might be a critical step in organ dysfunction during sepsis and ischemia/reperfusion events. the aim of the present study was to evaluate hepato-ceuular ca 2÷ regulation in isehemiah'eperfusion after hemorrhage and to assess effectiveness of tnfc~-monoclonal antibody (tnfo~-moab). methods. male sprague-dawley rats (250g, n>_6/group; pentobarbital 50 mg/kg) with hemorrhage for 60 rain at 40 mm hg. reperfusion by ringer's lactate (2 x maximal bleed out/60 min) and 60% of citrated shed blood. tnfcz-moab (tn3, ceutech, 20 mg/kg in 0.9% nac1) infused during flrst 5 min of reperfusion. at baseline, end of ischemia and 60 min of reperfusion, hepatecyte isolation by liver collagenase perfusion. " 45 hepatocyte incubation (30 mg w.w./ml) with caci 2 (0.05 2+ 2+ 2+ mbq/ml) for 60 rain (ca influx [slope, 1/mini; ca uptake [nmol ca /mg protein]) w/ and w/o epinephrine (epi, 100 nm). hepatecyte resuspension in radioisotope-free medium and farther incubation (exchangeable ca 2+ (ca2+ex) [nmol ca2+/mg protein]; ca 2+ membrane flux [nmol ca2+/mg protein'min]). during incubation, aliquots (100 ~tl) were centrifuged through oil/lanthanum gradient and acivity measured by scintillation counting. statistics: anova. mean + sem. results. hepatocyte ca2+ex and membrane ca 2+ flux were significantly increased at both, the end of ischemia (14.3+.9; 0.41+.05) and reperfusion (16.4+.9; 0.50+.2), as compared to sham-operated animals (7.6+_.5; 0.09+.02)(19<.05 ). tnfc~-moab treatment significantly prevented reperfusion-induced increase of ca2+ex (10+.8) and membrane ca 2+ flux (0.17+.03)(p<.01). fast ca 2+ influx was significantly increased by epinephrine in hepatecytes from sham-operated rats (0.23+.03 vs. epi: 0.52+.1, p< .05). this hormone effect was not observed in isehemia (0.47+.05, epi: 0.6!-_.2) or reperfusion (untreated: 0.13+.06, epi: 0.07+.01; tnft~-moab: 0.26_+.04, epi: 0.33+.07). conclusion. the present study clearly demonstrated hepato-cellular ca 2+ overload in ischemia and reperfusion as a result of hemorrhagic shock. analysis of membrane ca 2+ fluxes and hormone ca 2+ mobilization suggests disturbances of membrane ca 2+ transport mechanisms, e.g. through ca2+-atpases. reperfusion-induced oxygen free radical generation which affect exchange kinetics of cellular ca 2+ buffering compartments might also be operative. prevention by tnfct-moab indicates the pivotal role of tnf as an early inflammatory mediator of hepatocellular alterations in signal transduetion mechanisms and cellular homeostasis. although the precise mechanism has not yet been elucidated, bacterial translocation and endotoxin absorption have been frequently shown after burn, and have been postulated to be one of the underlying processes of sepsis. the purpose of the current study is to define the hemodynamic response of the liver to endotoxin release in burns, in correlation to bacterial translocation. twelve female minipigs, weighing 20-28 kg, underwent a laparotomy & transition time ultrasonic flow probes were positioned on the portal vein, the common hepatic artery, and the superior mesenteric artery. 6.5 fr catheters were inserted in the superior mesenteric vein and the left hepatic vein. a jejunostomy was also performed. after five days all animals were anaesthetized and randomized to receive 40% of tbs a third degree burn. eighteen hours after burn. 100 gg/kg e. coli lps was intravenously administered over 30 rain. ali animals were studied for additional 24 hours and then sacrificed. several recent data suggest that in severe injuries, such as shock state, the gradual activation of kupffer cells and the excessive release of destructive and immunosuppresive products from macrophages may contribute to the development of "multiple organ failure". in in vivo experiments in mice, the effect of kupffer cell phagocytosis blockade on the correlation between the tissue distribution of lps, endotoxin sensitivity and lps-induced tnf production was investigated. to depress the activity of the kupffer cells, gadolinium chloride (gdc13) or carrageenan was used. th~e studies indicate the dissociation of tissue localisation of cr jllabelled endotoxin and endotoxin lethalithy. both gdc13 and carrageenan depressed kupffer cell activity, but endotoxin sensitivity was enhanced only by carragenan treatment. however, there was a close correlation between the sensitivity to lps and lps-induced tnf production as measured in the serum, since lpsinduced tnf production was enhanced only by carrageenan treatment. on the other hand, gdc13 pretreatment significantly increased tnf production in the spleen. these results support our earlier findings that gdc13-indueed kupffer cell phagocytosis blockade leads to activation of the spleen, and may explain some of the immunological effects of gdc13. inositol(l, 4, 5) triphosphate (ip3) has been proposed as a second messenger for calcium mobilization. the addition of ip3 at low concentration has been shown to cause calcium release from intracellular microsomal store in rat hepatocytes. the effects of sepsis on the ip3 binding from microsomal fraction of rat hepatocytes during sepsis were investigated. sepsis was induced by cecal ligation & puncture (clp). control rats were sham-operated. three microsomal fractions (rough, intermediate and smooth) were isolated from rat liver. study of ip3 receptor binding was performed with tridium label ip3. the results shewed that the ip3 binding was significantly depressed by 40-47% (p<0.05) during late sepsis (18 hrs after clp), but not in early sepsis (9 hrs after clp). the ip3 binding depression during late sepsis was most significant on rough and intermediate endoplasmic reticulum (p<0.05), but not on smooth subfraction. since ip3 binding plays an important role in the regulation of intracellular calcium homeostasis in hepatocytes, an impairment in the calcium release due to depressed ip3 binding on smooth and intermediate endoplasmic reticulum during late sepsis may have a pathophysiological significance in contributing to the development of altered hepatic metabolism during septic shock. septic organ failure is currently recognized as an overactivation of the nonspecific immune system by bacterial stimuli giving rise to proinflammatory mediators. little is known about the mechanisms of the resulting cellular injury. here, a synergism is described between tnf as a major mediator of septic organ injury released by macrophages and hydrogen peroxide (h202) as a representative of reactive oxygen species as formed by e.g. neutrophils. rat hepatocytes are only slightly sensitive to either agent alone. when treated with a conbination of tnf and h#09 a stronq synergistic toxicity was found, especially w~e~ tnf-treatment preceeded challenge with h~o~. we have recently described a coculture model bfzrat liver macrophaqes and hepatocytes where lps induces hepatocyte cell death partially mediated by macrophage tnf release. when h202 was also employed in fhis more complex cellular system a similar synergism was found: the ecc~ of lps was 62 consecutive patients with liver cirrhosis admitted to the department of surgery over a 1 year period from january to december 1992 were studied for their complement profiles in relation to other parameters of liver function, the aim of the study was to determine if a direct correlation existed between low complement levels and end stage liver cirrhosis. cirrhotic patients were divided into child's a, b and c categories using child's classification. complement levels (c3, c4) were measured and functional assay for complement (ch50) were performed in each of these groupings in addition to normal blood donor controls. these results show that the qualitative c3, c4 and the functional chs0 complement assays have good predictive values in assessing deteriorating liver function• in particular, the functional assay for complement (ch50) showed marked impairment in child's c patients (p<0.000001) confirming the impaired immunological status of these patients. sera from this group of patients (child's c) were titrated with pig red blood cells (rbcs) in a haemolytic assay. the results showed that there were significantly less haemolysis of pig rbcs in these patients (p=0.0177) as compared to the controls. this findings strongly support an impaired immunological status in child's c liver cirrhosis and may explain the high incidence of sepsis as a terminal event in these patients. aim:kupffer cells(kc) have an importamt play to cause hepatocellular injury in sepsis, because these cells release many kinds of substances. we reported that oxygem radicals released by kcs stimulated by lipopolysaccharide (lps) caused hepatocellular injury. aim of this study is to investigate the relationship between imtracellmlar calcium(ca) concentration of cultured rat kcs stimulated by lps and release of oxygen radicals, and effect of prostaglandin e~ (pge~) on imtracellular ca concentration. production of acute phase proteins (c-reactive protein, crp, transferrin, tf) and £erritin (f) in rat hepatocytes (hps) and its dependence on extracellular matrix components were studied. hps isolated from the liver by collagenase perfusion were cultured at ~o5 per 0.5 ml medium fi2+dmem (1:1) with 10% fetal calf serum for 2 days on uncoated or type i collagen coated plastic surface or in the presence of dextrane sulphate in the medium. hps were stimulated by conditioned medium (gm) from i~ia-p or e. coli lps preineubated human blood mononuclear cells. production of crp, tf and f by hps was detected by elisa. it was found that both cms decreased tf synthesis in hps by 15-50% (p<o.os). the level of f synthesis didn't change or only slightly decreased. the addition of cms led to the enhancement of crp synthesis more than 2.5 times. the most reproducible results were obtained when plastics had been coated with collagen. dextran sulphate markedly increased crp synthesis. thus using this model of an acute phase reaction in vitro we found the enhancement of crp synthesis and the decrease of tf synthesis. our data correspond with nublished materials on the acute phase in vivo and point to relevance of the proposed model. metabolism of hepatocytes under traumatic illness has not been sufficiently investigated. therefore we have applied absorption and fluorescent cytophotometry techniques to study the content of rna, glycogen and its fractions in hepatocytes of patients with severe mechanical trauma, both with and without endointoxication at various stages. for these purposes slides were prepared from the repeated aspiration biopsy material according to special techniques (kudryavtseva et al., 1983) . slides were stained by gallocyanin-chromalum to measure rna or underwent fluorescent pas-reaction to measure glycogen and its fractions (kudryavtseva et al., 1970 (kudryavtseva et al., ,1974 the ability of 02 metabolites derived from the xanthine-xanthine oxidase system to inhibit mitochondrial function was examined using freshly isolated rat liver mitochondria. under uncoupled conditions, mitochondria exposed to free radicals exhibited a significant decrease in 02 consumption supported by nad+-iinked substrates, but showed almost no change in 02 consumption in the presence of succinate and ascorbate. the activity of electron transfer complex i (nadh oxidase and nadh-cytochrome c oxidoreductase) and the energy-dependent reduction of nad+ by succinate were unaltered by oxidative stress. exposure to free radicals also had an uncoupling effect at all three coupling sites. the degree of mitochondrial swelling was closely correlated with the inhibition of state 3 oxidation of site i substrates and with the increase in state 4 oxidation of succinate. the immunosuppressive agent cyclosporin a (cya) completely prevented the mitochondrial damage induced by oxygen free radicals (swelling, ca2+ release, sucrose trapping, uncoupling, and selective inhibition of the mitochondrial respiration of site i substrates). the same protective effect was found when ca2+ cycling was prevented, either by chelating ca 2+ with egta or by inhibiting ca2+ re-uptake with ruthenium red. these findings suggest that the deleterious effect of free radicals on mitochondria in the present experimental system was triggered by the cya-sensitive and ca2÷-dependent membrane transition, and not by direct impairment of the mitochondrial inner membrane enzymes. correspondence should be addressed to: naoshi takeyama the aim of this study was to clarify the critical role of reduced glutathione (gsh) on the progression of lipopolysaccharide (lps)induced liver damage of mouse. the conditions of gsh-depletion and -richness were produced by intraperitoneal administration of buthionine sulfoximine (bso), a specific inhibitor of gsh synthetase, and of gsh-monoethyl ester (gsh-me), respectively. gsh-me, which was esterified the caboxyl group of the glycine residue, is readily transported into cells and is hydrolyzed intracellularly; this leads to greatly increased the cytosolic and mitochondrial levels of gsh. bso and gsh-me were administered intraperitoneally 3 mmol/kg and 10 mmollkg, respectively, and lps (2 mg/kg) given 1 h later. hepatocytes were isolated by in situ perfusion of the liver with collagenase 6 h after lps injection. the degree of hydrogen peroxide (h202) synthesis and mitochondrial membrane potential were measured by flow cytometry using fluorescence probe dichrolofluorescein diacetate and tetrametyl rhodamine ethyl ester, respectively. the degree of mitochondria membrane permeability transition (mmp) was assessed by [014]sucrose entrapment. we found that lps treatment results in a decrease in hepatic gsh level and mitochondrial membrane potential, and an increase in h202 synthesis and mmp. lps administration to bsq-pretreated mouse worsened at[ these parameters. in contrast, gsh-me pretreatment ameliorates. all together, gsh may acts an important role in cellular defence against lps-mediated liver damage, and mmp may result in the decrease in mitochondrial membrane potential. it has been shown, up to now, that disturbances of enzymatic processes in the cell organella are the basic factor of the changes taking place during endotoxin shock it has been observed that lysosomes are biochemically changed as result of the effect of lipopotysacharides of gram negative endotoxic bacteria. the purpose of the experiment was: 1.evaluation nfthe course of the ees due to the biochemic changes 2.determination of the influence of the ees on the activity of lysosomal enzymes in liver cell 3.deterrmnatian of the influence of h and d on the activity of some lysosomal enzymes in liver cell during ees material and method: examinations ware carried out on 30 dogs. the shock was evoked by i.v. administration of endotoxin e.coli. the dogs were divided into 5 groups: icontrols, i[ -dogs with untreated shock, iii-dogs in shock treated with h, ivdogs in shock treated with d, v -dogs in shock treated with h and d. the following parameters were determined: 1.activity of acid phosphatase in the venous blood serum. 2-6.total and free activity of acid phosphatase and catepsins in the full liver cell homogenate, in mitochondrial-and-lysosomal fraction, and in the superuatunt (all after 4 hours of the experiment). conclusions 1. essential increase of activity of lysosomal enzymes was observed in the ees. 2. increased activity of lysosomal hydrolases in the liver cell homogenate corresponds, as a result of the endotoxin effect, with increased enzymatic activity in the peripherial blood. extensive investigations from our laboratory of the pathophysiology of hepatic no-flow ischemia (45 min) -reperfusion injury demonstrated substantial kupffer cell activation with reactive oxygen formation during the first few hours of reperfusion as indicated by increases in plasma glutathione disulfide (gssg) levels in vivo (am. j. physiol. 260: g355, 1991) and enhanced superoxide formation by kupffer cells (kc) isolated from the postischemic livers (free radic. res. commun. 15:277, 1991) . the early kc-induced injury initiated the later, primarily neutrophilmediated reperfusion injury (faseb j. 4: 3355, 1990 ). to test whether or not similar mechanisms are operative during hepatic ischemia induced by hemorrhagic shock, male fischer rats (230-250 g) were subjected to 60 rain of hemorrhage (mean arterial pressure 40 mm hg) followed by resuscitation (rs) (reinfusion of shed blood). hepatic atp levels declined from 2.3+0.2 txmol/g to 0.24_+0.08 (60 min shock) indicating severe ischemia, and recovered to 1.04--0.2 at 90 min rs. to test for potential oxidant stress during rs, plasma gssg conc. were measured. although gssg levels increased by 120% compared to baseline levels (0.69!-_0.31 i.tm), there was no significant difference to shamoperated controls. spontaneous superoxide formation of isolated kc was 0.32+0.05 nmol o2-/min/10ecells (kc2) and 0.51+0.11 (kc3) in controls and did not change significantly after shock and 90 rain rs. addition of phorbol ester or opsonized zymosan to isolated kc did not indicate a priming of these cells. conclusion: in striking contrast to our previous findings with hepatic no-flow ischemia, there is no evidence for a significant kc activation after 60 min of hemorrhagic shock and up to 90 rain of resuscitation. objectives-this study investigates morphometric changes in kc nltrastmcture which might account for this diminution in phagacytosis. materials and methods -three groups of wistar rats were studied: control, sham-operated [sham] and bile duct ligated [edl] . after 3 weeks animals were sacrificed and livers were "perfusion fixed" with 3% glntaraldehyde and processed for transmission electron microscopy and image analysis. results -in the the bdl group kupffer cell numbers were greatly increased. biliary ductular proliferation was evident and sinusoidal spaces were compromised. eleven nuclear and cytoplasmic parameters were measured and statistically significant results are summarised in the results: ascorbic acid levels were slightly elevated after the hs and returned to basal values after 120 rain. glutathione concentrations were increased significantly after the hs and the gsh levels kept rising continuously to as much as 4-fold of basal levels at the end of 120 min. mda showed no significant variation before and after the hemorrhagic shock. plasma concentrations of ratine, teucine, isoleucine, phenylalanine, proline, threonine and serine showed significant increase over basal levels during the whole ischemic period. glutamic acid was slightly elevated at the onset of hs and remained the same for the rest of ischemic period. hydroxyproline was significantly below the basal value at the mid-point of ischemic period. alanine, glycine, histidine and aspartate did not change significantly throughout experimental time course. conclusions: (1) oxidative stress might not be severe in the systemic hemorrhagic shock for pigs since no significant variations were observed for ascorbic acid and mda (2) the increase of gsh might be that it was released from hepatocytes when the animal was under systemic ischemia. (3) celzain amino acids might be acting as transmitters in the event of ischemia. however, fm"lher investigations are needed to clarify the detailed mechanism in regard with antioxidants and amino acids. dr. fang-ku p'eng taichung veterans general hospital talchung, taiwan 407, r.o.c. jia shi yong, huang zhi oiang and men xian jun. in patients underi~ent major hepatic surgery,obstructive jaundice has been implicated with fnoreased susceptibility to sepsis and liver failure. jaundice was said to he associated with derangement of iamune function and result in expression of pr,:.inflar...aatory cytoki~es that cause hepatooellular damage. this study is ~.o investigate the effects ,.,t lip,~polysaccharide(lps)rats. jaundice were produced in healthy ~istar rats of either sex weighing 180-220 m~ by common hi l~ duet l igation(bl)l).seven days post l igation, rats were given lps(236.gg.10)0.7rag/kg intraperitoneally. at the end ,:,f l,gand 5 hours after lp$ ehanllenge, liver were removed and prepared for forzen sections immediately. i)emonstration of inf in liver parenchyna were performed by method of abu i~munohistochemistry using r~onoolonal antibody against tnf, leve of mrna for tnf ~ere measured by in-situ hybridization using biotin-labeled edna probe. results sho'~ed that tnf stained granules appeared in kup~'fer eell~ and polymorphonuclear leukocytes(pnnl,but not in hepatoeytes nor endothelial eel is. they ~'ere located within cytoplasms and peaked at 3-5 hours after lps ohal lenge. there was vocomitant tnfmrna expression but peaked earlier 1 hour post lps challenge. tnfmrna vcere detected notably in necrotic area and barely in bdl rats ~ithout lps challenge. it is therefore postulated that production of tnf by inflammatory effeetor cells-kupffer cell and phn in site, in the obstructive jaundice rats during sepsis may atribute to the hepatocellular damage. it also provide evidence for the beneficial effects of early release of biliary obstruction. in the present study, an animal model of a0c in wlstar rats was developed by tigatlng the common bite duct and injecting d.3mt solution of e. goli % b, (g×10' efm/mt) into the bite duct. the mortality rate was 40~ at 48h after aoc. at 6, 12, 24, 48h after aoc, kcs were isotated and cultured atone or cocuttured with hepatocytes to evaluate the modulation effect of kgs on hepatoeyte protein synthesis. the results showed that tactodehydrogenase leakage was increased progressively as the injured k~ function and structure developed. tnf and il-i tn the supernatant were detected with the peak values at 12h after aoc. at 48h after aoc, ~-teueine incorporation was obviousty decreased in the normal hepatocytes eocuttured with stimulated kcs compared to the uormai hepatocyte cultured group (p<8.01), but it was slgnificantiy increased in the group cocultured with normal kcs. in the a0c group, 'h-teuclne ineorporatien was decreased progressively in the injured hepatoeytes cocuttured with stimulatedkgs and it was markedly elevated when cocuttured wlthnormat kcs at 48h after a0c (p<0. og). it is concluded that the impaired hepatocyte proterin synthesis was directly mediated hy the stimulated kc function during aoc. such a mechanism possibly may be involved in the patho0ensis of hepatic dysfunction and m0f following h06. " the project supported by nsfc. in the past few years it has become evident that cytokines are implicated in various pathophysiological mechanisms. therefore measurement of cytokines and their potential inhibitors in biological fluids may be helpful in diagnosing and monitoring of diseases. however, dependent on the type of assay used investigations performed in different laboratories have often yielded conflicting results. in order to assess reliability and reproducibility of cytokine measurements two comparative studies for the determination of cytokines in biological fluids were launched by several investigators interested in this field: one national austrian ~tudy and one european study in the context of the european workshop for rheumatnlogy research. serum and synovial fluid samples were distributed by the organisers, and participants had to measure one or several of the following cytokines in all samples: il-1, il-2, 1l-6, i58, tnf-alpha, ifn-gamma, gm-csf, and the soluble receptors for il-2 and tnf; both commercially available immunoassays and home-made assays were allowed. so far, the main conclusions emerging from these preliminary studies are: (1) the same immurzoassay (elisa) yielded comparable results in different laboratories; (2) immunoassays from different manufactureres usually measured the highest concentrations in the same samples, yielded similar relative values but disparate absolute values; (3) assays for soluble receptors yielded less discrepant results; (4) some assays seem to be more suitable for measuring cytokines in biological fluids than others. the mean retrieval of exogenous recombinant human cytokines was approximately 50% for most cytokines tested. however, it must be borne in mind that natural and recombinant cytokines may behave differently in immunoassays. this raises the question as to the necessity of setting up international standards for all cytokines. possible interferences by serum components such as (lipo)proteins, complement, rheumatoid factors, etc., which may either decrease assay sensitivity or yield false positive results, must also be considered. in addition, natural cytokine binding proteins (e.g. soluble receptors) might interfere with cytokine determination in immunoassays. the problem of immunoassays versus bioassays has not yet been approached, but is without any doubt worth indepth investigation. thus, these studies are a promising first approach to deal with the difficulties of cytokine analysis in biological samples, and it is hoped that they will help to overcome some of the major methodological problems in the near future. tumor necrosis factor (tnf) is a pleiotropic cytokine which plays a key role in a number of immunologically mediated disorders like septicemia or cachexia. tnf receptors are found on the surface of a variety of cells, where they provide molecular signals for the cytokine effect. there exist two types of soluble tnf receptors (stnf-rs), tnf-r p55 and tnf-r p75. these stnf-rs can compete with the cell surface receptors and block their activity. the expression on and shedding from the cell surface of stnf-rs is enhanced by interferon gamma. increased concentrations of stnf-rs can be found in patients with infectious as well as malignant disorders. in patients undergoing immune stimulatory therapy with interleukin-2 and interferon alpha a simultaneous increase of tnf and its soluble receptors can be seen. in patients with hiv infection a strong correlation exists between the levels of stnf-rs and markers of immune activation like neopterin or beta-2-microglobulin. likewise patients with hematological disorders show elevated stnf-r levels. patients with weight loss have higher concentrations than patients with stable weight. the final value of stnf-rs within the complex network of cytokines is not yet clear. however, there exist striking parallels between infectious and malignant disorders pointing to a key role of endogenous immune activation. biochemicatly, d-erythro-neopterin derives from guanosine triphosphate. in vitro studies show that large amounts of neopterin are produced by human monocytes/macrophages stimulated with interferon-j (ifn-j). neopterin is biologically stable, and its halflife in the blood seems to solely depend on renal excretion. specimen for neopterin determination can be stored without special precautions. increased concentrations of neopterin have been described in body fluids of patients suffering from diseases which are apparently associated with an activated cellular immune response and with enhanced endogenous formation of ifn-~', e.g,, 1 ) increasing neopterin levels predict rejection episodes in allograft recipients 2) virus infections induce increased neopterin concentrations, and the degree of neopterin elevation predicts prognosis in these patients which is particularly true in hiv-1 infection 3) in autoimmune disorders such as systemic lupus erythematosus or rheumatoid arthritis neopterin levels reflect activity of the disease 4) increased neopterin concentrations in patients suffering from certain types of cancer indicate poor prognosis. significant associations between changes of neopterin and decrease of hemoglobin as well as the development of weight loss and cachexia, e.g., in cancer patients and in patients with hiv-1 infection, support the concept that endogenously formed cytokines such as ifn-~" and tumor necrosis factor-~( are involved in the pathogenesis of such symptoms. in conclusion, neopterin monitoring is a sensitive tool to detect the activation status of the cell-mediated immune system in vivo. repair and healing or perpetuation of acute inflammation is characterized by a complex network of interacting cellular and humoral defence mechanisms. of the numerous inflammatory mediators/effectors investigated hitherto, the proteolydc lysosomal enzyme pmn elastase has turned out to be highly destructive when released extracellularly thus contributing considm:ably to organ dysfunctions in severe posttraumatic and postoperative courses. besides various cytokines, elastase in complex with its main antagonist c¢ 1-proteinase inhibitor (a 1pi) is also supposed to induce the shedding of intercellular adhesion molecules from inflammatory cells (pmns, monocytes/macrophages, endothelial cells). these soluble adhesion molecules may modulate the inflammatory process in many different ways, e.g. via acting as chemotaxins, blocking neutrophil activation or competing with the membrane-bound forms in cell-cell adhesion. thus, measuring circulating or local levels of elastase-a 1pi and soluble adhesion molecules (icam, e-selectin,-pselectin, l-selectin) may be a helpful tool in judging the severity of an acute inflammatory process. in several clinical studies on surgical patients suffering from multiple trauma and/or sepsis we could demonstrate that the measurement of these parameters in consecutive plasma samples and local body fluids was highly valuable for early diagnosis and prognosis of multiple organ failure. prof. dr. m./ochum, institut fdr klinischechemie und biochemie, lmu miinchen, nufibaumstrage 20, 80336 m0nchen, germany procalaitonin (proct) a 116 aa peptide is dramatically increased in the blood of patients with various sepsis and infections. the increase begins as soon as 3 hours after the andoto:edn release and readied maximal level with a 200 to 80b-fold increase 24 h after li~ edrmrdstration. prcc, alcitorfin response was temporally different from these of tnf~ and ii-6, that reached peak levels at 2 h and 3 h respectively. at the opposite of eytokine (tnfcz, ild, j, is), proct is a very stable molecule. the half iife of proct in ~he blood is surprising if we consider the half llfe (about nine mirtute) of mature ¢alcitonin (co. we have found that it is due to the binding of proct with a protein on the n-termktal part. thin complex of about 80 000 daltons is unable to cross the kidney and the c_n9 barriers and the half life is at least elghteen hours. this long half llfe is very useful in the evaluation of a sepsis. a very sensitive and specific sandwich in'ununoassay has been developped. the results cart be delivered in two hours. at time, we know that in the healthy adults, the values are less thau 0.1ng/ml. it is also clear that proct is not increased ~n patients wlth inflammatory dleeasea without lnfectlon. for instance, proct is not signlffcat3vely increased in purpura rhumatom, arthritis, crohn's disease, rectocolitis, also in various cancer patients with inflammatory components. am other interesting finding, is the absence of increase or a very low increase in the viral infections. at the opp0site, in all the patients with bacterial sepsis or in the malaria pat/ants, progt was dramatically increased, from i00 to 10 000 fold. p~oct can be useful to the follow up of patients with sepsis. actually, we don't know the origin of this proct production, in the united states, will also be discussed. the septest portion of this study will eventually enroll over 500 patients tentatively diagnosed as septic. the results of septest will be compared with commonly accepted symptoms and criteria associated with sepsis in order to determine the clinical utility of this endotoxin assay. preclinical results of patients and normals be presented as well as data on the time course of endotoxemia and clinical outcome of selected septic patients. preparation of dna libraries clifford w. schweinfest, ph.d. a fundamental tool of the molecular biologist today is the ability to work with purified dnas representing genes of interest with respect to the investigator's field of study. originally applied almost 20 years ago to the genetic dissection of simple organisms such as viruses and bacteria, molecfllar cloning was rapidly applied to study the more complex genetics of the mammalian cell. today, virtually any study of cell physiology which depends upon specific gene expression is approachable using the tools of the molecular biologist. therefore, stress induced gene expression (such as the heat shock gene, hsp70) or the regulation of the nitric oxide synthetase gene or the induction of cytokines and growth factors as a result of trauma or injury lend themselves to investigation at the level of the gene. during these workshops, we will discuss the means by which cdnas and genes are isolated, amplified and identified. we will discuss the strategies and methods for cloning cdnas and genomic dnas, for organizing such cloned libraries, for finding specific genes and for characterizing those genes. this speaker will focus on the techniques and considerations involved in the synthesis of cdna libraries. topics include rna isolation, mrna quality, reverse transcription, choice of cloning vectors and library quality. genomic dna library synthesis will also be discussed with respect to the vector/host systems available and applications which are specific to genomic dna. finally, polymerase chain reaction (pcr) will be discussed briefly with regard to its impact on dna cloning. the identification of the genes or gene products that have a role in cellular processes is fundamental to our understanding of genetic control and methodologies to achieve this aim are currently available. all levels of the various signal transduction pathways can b~ molecularly analyzed to define the mechanism by which critical steps in each pathway are achieved. the questions that are unresolved in each area of investigation serve to direct the scientist towards analyses of gene products or gene regulation of the gene itself. thus, selection of the type of library (genomic or cdna) to be used is dependent upon the specific goals of each investigator. to facilitate resolution of this question, an overview of the uses for the different types of libraries will be presented. at least four methodologies are currently available for screening a library, which are dependent upon specific interaction between nucleic acids, nucleic acids and proteins, antibodies and antigens or between different proteins. the use and advantages of each of these methodologies will be discussed along with a description of probe preparation. sequence methodologies required to begin characterization of gene clones will be presented. hopefully, the participants in the workshop will help define areas of emphasis and allow time for directed interaction to discuss specific applications based upon their own experimental systems. alterations of physiological conditions, such as those occurring after shock and sepsis, induce changes in gene expression of cells composing the major organ systems. the challenge is to detect and characterize these changes in geae expression and relate them to the injury. the development of cloning techniques has emerged as the methodology of choice. changes in gene expression are usually characterized by variations in the concentration of cellular messages because synthesis of the final product "the polypoptide" is usually proportional to the concentration of mrna. due to the rapid regulation of gene expression the cellular concentration of messages changes very quickly. this is commonly referred to as steady-state levels, a balance between transcription and degradation. steady-state levels of mrna can be detected in a single cell (in situ hybridization) or in total rna isolated from cells or organs and separated in agarose gels (northern blots). although analysis of mrna steady-state levels are very useful, they do not provide information about the mechanisms that regulate gene expression. changes in gene expression primarily occur at the level of transcription; characterization of the rna species being synthesized at a given time is performed by the nuclear run-off technique. when there is no a priori information about the gene that may be regulated after a particular situation such as sepsis, the total pool of messages present in cells at given time can be characterized by a combination of in vitro translation of the cellular messages and fractionation of the in vitro translated products by two dimensional gel electrophoresis. such approach permits the visualization of the general pattern of gene expression, a "finger print", of the physiologic state. in summary, researchers now have access to a great variety of techniques to identify and characterize genes expressed in response to a physiological condition that may be utilized as "molecular tools" to study the organism's responses to shock and sepsis. the acute phase proteins are a set of plasma proteins with greatly increased plasma concentrations during acute inflammations and after tissue trauma, e.g. after major surgery. the proteins counteract the source of injury, facilitate clearance of infectious particles by phagocytes, assist immune responses and initiate wound healing. the corresponding genes are expressed in liver hepatocytes and regulated by the inflammatory mediators interleukin 1 and interleukin 6 (ill, il6). class1 acute phase genes are mainly controlled by ill and by combinations of ill plus il6; class2 genes mainly by ii,6. the human c-reactive protein(crp), serum amyloid a(saa) and complement c3 genes will be discussed as examples of class1 genes, and rat c~2 macroglobulin as a prototypical class2 gene. both classes of genes use different types of ¢ytokine response elements in their promoter-proximal transcription control regions; class1 genes use the transcription factor nf-il6 or c/ebp as the key factor to mediate cytokine responsiveness; class2 genes use a different, so far unidentified factor to achieve responsiveness to il6. ongoing efforts to purify this factor, called the il6-response factor (il6-rf), from rat liver nuclear protein extracts will be described. the il6-rf apparently mediates the effects of il6 in a broad range of cell types, including b lymphocytes and other hematopoietic cells. it is therefore likely to be of equal general importance for gene regulation by il6 as nf-il6. the importance of transcription factors as potential targets for novel bioactive substances and possibly therapeutic agents will be discussed with the help of several recent examples. new methods for altering the germ lines of mice provide powerful tools for understanding the roles of individual genes in normal and pathological processes, and for reproducing specific genetic mutations that result in congenital disease. three approaches will be discussed. conventional transgeulc mice are frequently constructed using artificial transgenes that express genes from a promoter other than the normal gene promoter. this paradigm is used to produce very high levels of a gene product, for example, with a viral promoter, or to attempt to regulate gene expression using an inducible promoter. alternatively, normal promoter sequences can be hooked to a gene whose expression is lethal to the cell (e.g., diptheria toxin a chain) to ablate all cells expressing that gene. another approach uses an intact gene with its normal regulatory sequences. here, an elevated level of the gene product is delivered from the correct cells in the correct tissues at the correct developmental stages or in response to natural signals. this approach has been refered to as "genetic pharmacology," and provides a precise delivery of the gene product to the target. however, to be most effective, the transgene should contain arl regulatory sequences as welt as the entire genomic segment containing gene coding regions. the introduction of conventional transgenes is limited to roughly 50 kilobases, while genes containing all regulatory sequences frequently stretch over dozens or even hundreds of kilobases. to overcome this problem, procedures have been developed recently to introduce yeast artificial chromosomes (yacs) into mouse embryonic stem (es) cells. yacs can include over 2 mb of human dna, large enough to encompass the largest known human genes. es cells are also useful for targeted gene deletions and mutations. homologous recombination can be targeted to any known gene in es cells. when these modified cells are injected into a mouse blastocyst, they can be incorporated into all tissues of the resulting mouse, including germ cells, so subsequent generations will pass the genetic modification as an inherited trait. assessment of the effects of a null allele on development can provide valuable insights into its normal role. ultimately, these technologies may be adapted to human cells -not for embryonic intervention, but to provide modified stem cells for various tissues (e.g., gut, eye, hematopoietic system) which could then be applied to somatic therapies. with major illness/injury that respirswy faihne fi'equently followed sepsis, tilney el al nse, d the term "soqueutial syste~ failure" for patients with renal failure after solmir of ruptured abdominal aortic ~aeurysms, i then reviewed our experiences after inju~ and oporstion and suggested that multiple, progsesslve or sequential systems or organ failure was a syndrome to be reckoned with in the, 70's. eiseman et al then wrote about "multiple organ failure", especially with liver failure. polk el m found that renmte orgau failure often signalled occult anita-abdominal infection. fry et al ampbasized the role of uncontrolled infection in organ failure, border et ai described metabolic alterations with mof and used the term multiplesystems organ feilmo (msof). other early conltibutots to our knowledge of mof include faist, trunkey and miller, marshall and dimic&, cassone, catlleo, meakins and marshall, (}otis et at., mater and many others. mof or msof were used eommoifly. cerra coined the terms "post-trsumatic septic sfndromc" and "hyper metabolism otgau failure complex", and border et al, used lhe ex3~ression "gut origin seplic states" to illdioate important eurrem aspects of abe gt.'nerlc problem of organ failure and death, other terms include acute organ-system failure, multiple organ injury syndrome, post-traumatic multi-system organ failure nod post-~aumatic organ system infection s~dmmc (fi~sis), i bdieve the latin "maltiple organ failure" is clean, neat and says it all, now there is a proposal for a new set of torminolugios -mods and sirs. will they be helpful in the study and esro of patients? our speakers in this session will review thcsa proposals and the evidence as we strlvo for eoasensns. it has been well demonstrated that the administration of pro-inflammatory cytokines, and especially tumor necrosis factor (tnf) can result in a picture similar to septic shock with secondary multiple organ failure. it is also clear that tissue hypoxia can lead to organ damage. we believe that it is the interaction between the two phenomenons that can lead to mof. on the one hand, the inflammatory response is amplified in the presence of hypoxia. on the other hand, the release of the mediators of sepsis can increase tbe oxygen demand ef the tissues, alter their oxygen extraction and decrease myocardial contractility, and the combination of these elements accounts for the development of septic shock. this hypothesis is supported by two lines of evidence. first, mof is usually preceded by an episode of acute circulatory failure (even though frank circulatory shock may not be evident). second, recent experimental and clinical studies indicated that immunotherapy (especially antibodies to tnf) is particularly effective in the most severe forms of sepsis, associated with circulatory shock. hence, an effective way to prevent mof may be a combination of aggressive cardiovascular management and immunotherapy. development of the multiple organ dysfunction syndrome (mods) in the critically ill follows an heterogeneous group of stimuli including infection, sterile inflammmion, extensive tissue injury, ischemia, intoxication with a variety of substances, and immune system activation. whether the resulting physiologic abnormalities reflect a common pathologic process, or are simply a non-specific manifestation of tissue injury is unknown. moreover, the lack of clearly-defined functional criteria for the characterization of the syndrome on the one hand, and of reliable biochemical markers of its evolution on the other, has made attempts to define its epidemiology and natural history difficult. using a numeric scoring system to quanfimte the clinical severity of mods, we demonstrated that, although mods is more common in patients who have significant infection at the time of icu admission, a much stronger correlation is evident between the severity of mods and the subsequent development of nosocomial icu-acquired infection. furthermore the severity of mods correlates strongly with the severity of the clinical septic response, and with the degree of immunologic compromise evident on delayed type hypersensitivity skin testing. to determine the relative importance of the initial stimulus, and the host response to that stimulus, to the subsequent emergence of mods, we undertook a matched cohort study of 109 critically ill patients admitted to the icu with infection, matched by age, sex, and apache ii score to 109 uninfected controls. organ dysfunction scores were higher in patients admitted with infection, but were also significantly associated with the expression of a septic response at the time of icu admission, independent of the presence of infection. by stepwise regression analysis, the magnitude of the septic response was the most powerful predictor of the severity of mods in both infected and uninfected patients. these data suggest a model of mods in which a stimulus (eg infection) and the host response to that stimulus (eg the septic response) result in a state of altered systemic homeostasis (manifested in this example by immunologic dysfunction). moreover they suggest that the principle determinant of the emergence of mods is the response of the host, rather than the nature of the stimulus, and are consistent with the hypothesis that a stereotypie systemic response to an heterogeneous group of injurious stimuli underlies the pathogenesis of mods. arthur e. baue, m.d, ever since the attorapt 10 build the tower ef babel, as desclibcd in the old testament of the biblc, it has been difficult to got agreement o;x common definitions or language, although mof has served well, there will always he now classifications proposed with good reasons for them, thus mods and sirs are not the final oxpressinns in the lexico~aphic sweepstakes. attcmpts to develop standard animal shock and scpsis models (hemorrhage, endotoxin, foes[ pellets, cecal ligation and puncture, e. colt hlf0sioi0 to evaluate therapy have net been suoca.ssf~. clinical syndromes of mof, sepsis, sepsis syndrome and athers have not been aeacpte.d by all. our problem is that we arc tryittg tu d¢ffino a non-emily. mof or mods or sirs is net a disease or oven a syndrome. it is a series of complications of injury, disease and intensiva care which loads to death for many patients in intensive care units, it is not a fixed entity, but an ewilving picture. we have lumped (ugethet for therapy and definition a number of diseases and problems according to clinical manifestatieos rather than the eause of ihe problem, the search for unified mechanisms has not bean successful. will we benefit oleo from a more precise classification and definition of a non-outjty, a hodgu-podge of human disorders which have charsmeristic.s and manifestations of tissue injury, inflammation and infactian? re.hi clinical trials of potential "magle ballets" suggest that we should go in a different direction--instead of lumpors, we should be splitters, to seek effective therapy, we must fucus on specific disorders such as trauma, specific infections, inflammatory disease and chronic or acute organ damage (copd -renal failure, etc.), i will say more aboet this later ha the me.ethag. thcre is cue potentially important pert of the mods proposal, and that would be do~mcntation of organ dysfunction before fsiiure occurs and before tile need for external supporl dovelope. this could lead to hatter methods of preventing organ failure. preveution is ultimately the only answer to organ failure, m mof, mods and sirs. have wo reached a consensus?--only that we arc dealing wilh a diffl~x:lt probtolr looking for solutlons. acute lung injury (ali) and its most severe form, acute respiratory distress syndrome (ards) characterized by severe hypoxemia, diffuse infiltration of both lungs, a reduction of compliance and a wedgepresure lower than 18 mm hare related to direct or indirect injury. when aliresults from direct injury (toxic agents, lung contusion, pneumonia), the lesions of epithelial barrier and the activation of lung macrophages are the first events leading to the release of inflammatory mediators wh:ch are responsible for alteration of the membranar permeability and for invasion of the alveoli by fluids, proteins and cells. an inflammatory reaction develops, with injury to endothelial cells, adhesion of polymorpbonuclear leucocytes (pmn) and the release in blood stream of intlammatory mediators dispersing the inflammatory reaction to other organs and tissues. the failure of lung function also results into hypoxia in other organs, leading to tissular ischemia, triggering an inflammatory reaction leading to organ dysfunction. frequent complications of direct lung injury are septic pneumonia with endotoxin release, phagocytes activation and cytokine production disseminating inflammation. by this way, direct lung injury can be at the origin of the multiple organ dysfunction syndrome (mods), the frequence of which having been estimated to 40% and is increased when all has occured in patients alreadypresenting an organ dysfunction (immunodepresslon, cancer...). when ali results from redirect or extrathoraclc injury (trauma, infections, hypovolemic shock, acute pancreatitis...), inflammatory mediators and micro-aggregates released in particular organs or tissues reach the lung via the blood and lymph streams, are filtered in the organ or trapped in the lung capillaries, and are responsible for a local inflammatory reactaon (endothelial cell activation, pmn trapping and adhesion, platelet aggregation, release of mediators). sepsis acts by the way of endotoxiu and sequential eytokines release (tnf, ill,...) while trauma with important blood loss acts especially by the way of hypoperfusionreperfusion phenomena leading to a disseminatedinflammatory reaction and to a possible bacterial transloeation when intestinal hypopeffnsion is present. in these conditions of indirect injury, all (or ards) is a iocal early (often the first) manifestation of a general phenomenon of altered membrane permeability and inflammatory reaction, easily and rapidly recognized by its clin~cai signs. the iung is one of the numerous organs participating to the mods, in these conditions, the mortality rate is high, reaching more than 80% when sepsis is present or when at least 3 organs are implicated in mods. by direct or redirect injury, lung is so a target organ for mods and injured lung can be the cause of mods or simply a participant to this syndrome. jorge cervts-navarro main determinants for brain dysfunction are breakdown of the blood-brainbarrier and raise of intracranial pressure (icp), in second place decrease of systemic blood pressure, disorders of blood coagulation and respiratory function. the blood-brain-barrier of cerebral blood vessels can be opened by stroke, cerebral trauma, inflammation, convulsions, acute hypertension and changes in the blood osmolarity. the consequence is brain oedema, increase of lop and decrease of the perfusion pressure. a close correlation between intraventricular pressure and the fate of patients with severe brain injuries has been established. regional cbf values of 16 to 18 ml/100g/min are reflected in isoelectric eeg, and values about t5ml/100g/min, result in loss of somatosensory evoked potentials. for ionic pump failure the threshold has been determined by values of about 10 ml/100g/min and is reflected in massive release of intracellular potassium. in stroke and in brain trauma when the oedematogenous condition is initially localized the tissue pressure variance within and between the hemispheres lead to tentorial and falciform herniations are the common cause of death even before the critical threshold of intracranial pressure is reached. the increase of the icp over the level of the perfusion pressure leads to the arrest of the cbf. if this persists {or periods exceeding 6 seconds of global brain ischemia loss of conciousness and developing seizures appear. if it exceeds 10 min irreversible injuries of the brain appear. following cerebral ischemia and after severe head injury a tendency to increased coagulation can be detected. vascular occlusion may develop either as a result of local thrombus formation or from emboli caused by circulating platelet aggregates. the formation of microthrombi is triggered by the plateletactivating factor a mediator in central nervous injury also responsible for aggravation of posttraumatic brain edema. acute hemorrhagic leucoencephalitis and brain purpura are known to arise as complications of acute viral infections and following gram-negative septicaemia. institute of neuropathology, free university of berlin, hindenburgdamm 30, 12200 berlin, germany undoubtedly, attempts to quantify as objectively as possible the degree of dysfunction of the various organs is very valuable. however, cardiovascular failure has a peculiar place in the context of sepsis. the development of acute circulatory failure (shock) is of paramount importance as a cause rather than as a consequence of organ failure. it is therefore essential to clearly separate patients with and without circulatory failure. once this has been done, attempts to list the cardiovascular system among the other systems have been unconvincing. in a number of studies, cardiovascular complications are assimilated to a low systemic vascular resistance (svr), but since svr is basically the ratio of blood pressure over cardiac output, and since hypotension is already included in the definition of shock, then a low svr is basically equivalent to a high cardiac output, which is a characteristic rather than an ominous complication of severe sepsis. development of a low cardiac output unresponsive to fluids is usually a terminal event. it is not advisable to list other problems such as severe arrhythmias, myocardial infarction or tamponade as complications of severe sepsis. host defense dysfunction following trauma, shock and sepsis e. faist, g. f. babcock * major accidental, burn and operative injury often precipitates a profound multicentric immunologic depression that is believed to predispose patients to become anergic and thus to develop towards a higb probability of infection. anergy or a lack of immunologic reactivity stands for a total elimination of skin test reactivity and recall antigen responses in the inununocompromised patient. anergy following overwhelming trauma or major septic conditions results from a massive impairment of cell mediated immunity (cmi) together with a whole body malignant inflammationiike state. we and others have demonstrated clearly that the skeration of cmi following trauma is mainly due to the disruption of intact monocyte (moo) / t-cell interaction. within this phenomenon we see a shift of the cell ratio in the compartment of peripheral blood mononuclear cells (pbmc) with a considerable increase of prostaglandin e 2 synthesizing m~ and a simultaneous decrease of functionally competent cd3+ and cd4+ lymphocytes. t-cell dysfunction in states of profound stress is characterized by impaired synthesis of two crucial lymphokines -interleukin-2 (il-2) and gamma-interferon (y-ifn). the inability to produce adequate amounts of il-2, most likely attributable to alterations in the transmission of signals from the cell membrane to the nucleus, results in incomplete proliferative t-cell responses to antigenic stimuli, while a lack of ?-ifn results in inadequate m~ antigen processing and presentation. the role of the two functionally distinct t-helper subsets, t-helper-1 (thl) secreting principally il-2 and ?-ifn and t-helper-2 (th2) acting principally in inducing antibody formation with a secretion of il-4, il-5, il-10 and tnf-[3 has still to be established within the context of major trauma. antibody formation to common protein antigens is impaired, the predominant finding is a shift from igm to igg synthesis. although excessive secretion of prninflammatory cytokines (il-u3, tnf-c~, il-6, il-8) has been considered to be deleterious for the host in states of major inflammation and infection, in-vitro and whole blood investigation of me function has clearly revealed that there is initially a marked suppression of moo from septic patients to synthesize and secrete proinflammatory cytokines to endotoxin. this probably will result in immunodeficiency of traumatized as well as septic patients, since these cytokines in low concentrations are involved in the upregulation of the essential humoral and cellular immune functions. abnormalities of pmn function noted after serious injury have been interpreted by some investigators as signs of unresponsiveness of this cell population, others have demonstrated signs of pmn byperactivation. latest findings revealed that there is a clear pmn dysfunction in circulating blood: 70% of all pmn's in this compartment show downregulated or non existent ~-integrins within 72 hours posttrauma -these cells do not phagocytose, have a reduced respiratory burst and appear to be completely degranulated. restoration of these cell functions within 7 days post-trauma is significantly correlated to survival of traumatized patients. other reports however stress increased expression of cr3+ receptors (cdll/cd18 complex) on circulating pmn's, increasing the ability of these cells to adhere to intercellular adhesion molecules, thus contributing to play a major role in inducing the pulmonary capillary leakage. our knowledge about the perturbation of host defenses associated with serious injury and sepsis is continuously increasing and represents the precondition for the design of effective therapeutic measures to prevent and counteract the resistance to invading microorganisms. dept. of surgery, klinikum grosshadern, ludwig-maximilians-university, munich, germany. * dept. of surgery, university of cincinnati, cincinnati, ohio, usa lg thijs~ ce n~ck sepsis is the most common cause of acute disseminated intravascular coagulation (dic) and coagulation disorders as well as abnormalities of fibrinolysis are common in septic patients. some clinical and experimental studies indicate that dic is a pathogenetic factor for the development of multiple organ failure. endotoxin and various mediators (tnf, il-i) enhance tissue factor and decrease expression of thrombomodulin on endothelial cells in vitro. also, tpa activity is suppressed and release of pai-i is stimulated. in such a way the endothelial surface becomes procoagulant whereas fibrinolysis is inhibited. following administration of endotoxin to normal volunteers levels of prothrombin fragments (fi+2) and thrombin-antithrombin (tat) complexes increase, indicating thrombin generation. also the fibrinolytic system is activated as evidenced by a rise of levels of tpa and plasmin-antiplasmin (pap) complexes but this is counteracted by a subsequent release of pai-i. in severely ill septic patients levels of tat complexes are increased and concentrations of the natural inhibitors of coagulation (at iii, protein c) are usually decreased. also, tpa levels snd pap complexes as well as concentrations of pai-i are elevated. the severity of many of these alterations is related to mortality. thus, both coagulation and fibrinolysis are activated in sepsis, but not in a balanced way, thereby promoting coagulation. one of the hallmsxk pathological alterations associated with sepsis is the development of microvascular thrombosis, an entity ~lmre~erizod by microvas~tlar plugbing with leukoeyies (predominately nentrophils) and fibrin deposits. preranably, ischemia remlt~g fibm ve~-'ttlac ow,.luwion contributes sisni.ficantly to end orgen darnaje and consequent dysfatlctlon. p, er~t srldies haw focused pmdominately on the role of adhesion molecules in the pathogenesis of neu~ophil sequestration during infection and s~sis. this preparation will review the mechanisms underlying intravak-'v.ler i~brin deposition and its contribution to organ injury. the normal endothelial cell sure is generally conddered 1o be anticos~am. this state is maintained by two major anticoag~lam molecules on the cell so,ace: hepax'an su,lfale and thmmbomodulin. studies performed over the past decade have do~mentod a general shii~ towards a procoa~qtlant staw during gram negative hffeodon, aft e~| mediated mainly by changes in the endothalial cell surface. antt-tf antibodies have been shown to be pro~ective during l~'ml endotexemia. in summary, ~erova~'~ler fibrin deposition, in ~rt mediated by ~rcgulation of cellular tf, is a consistemt patholo~cal flndin~ during sepsis and contributes to organ injury. strategies designed to abrogate this event may n~nimjze the magnitude of erg~ dysftm~on. n, v, christou md phd, department of surgery, megill university, montreal, quebec, canada interactions between immune calls and the endothelium vie adhesion molecules serve to modulate immune cell traffic between the blood strum and the extrmvascular space, these families of molecules acting in a cascade and closely integrated with the oytoklna network, blood coagulation and the complement system, are responsible for the homing of leukocytes to areas of inflammation and the realrculatlon of tymphocytes. there are three types of immune ceils that must exit the blood stream into the extravascular space: lymphocytes ; polymorphonuclear leukocytes; and non-lymphocyta mononuclaar cells (monooytes, basophlls, aoslncphils). b lymphocytes exit the blood stream and recircu}ate mostly via the high endothelial venule (hev) in lymphatic tissue, rarely, in chronic }nfectlon=, they may recirqulate vie hev in non-lymphoi:t tissue. t-lymphocyte recircutation on the other hand can occur via hev in lymphoid tissue, as well as, the endothalium of the skin, returning to peripheral lymph nodes via the lymphatic channels. lymphocyte recirculation is the mechanism which allows ¢ontaot between the immune repertoire and pathogens, and homing delivers these cells to the appropriate environment for activation and for effeotor function, pmns exit the blood stream through endotbelium in close proximity to the site of inflammation after appropriate endothelial membrane receptor expression. they must reach the site of pathogen tnveslon quickly in order to be effective. their exudation to the inflammatory site can be modulated by their intravascular pdmlng, which results from appropriate receptor expression. because endotoxin leak from the gut has been postulated as an important trigger mechanism for the systemic inflammatory response syndrome seen after serious injury, the immunologic and metabolic effects of bacterial endotoxin infusion into normal volunteers can be expected to shed considerable light on the validity of this hypothesis. we and ethers have used tbis model to show that endotoxin causes a temporary paralysis in the ability of circulating monocytes to prudce the proinflammatory cytokines interleukin-i (il-i) and tnf-a. endotoxin, however, causes increased production of pge2 by the same cell population. endotoxin infusion causes significant suppression of circulating t-cell numbers and a profound suppression in the ability of the circulating t-cell population to proliferate and to produce interle~in-2 (il-2) upon in vitro stimulation. after endotoxin circulating t-cells are also more sensitive to the inhibitory action of exogenous pge2. administration of cyclooxygenase inhibitors at the time of endotoxin infusion largely abrogates the effect of endetexin on t-cell proliferation and il-2 production. endotoxin infusion also causes the release of increased levels of circulating catabolic hormones including cortisol. administration of cortisol alone or cortisol along with endetowin to volunteers has allowed dissection of cortisol effects from those of endotoxin itself. cortisol infusion alone causes a diminution in the circulating t-cell population but the remaining ceils continue to produce il-2 after appropriate stimulation. cortisol infused along with endotoxin blocks the overshoot in monocyte proinflammatory cytokine production seen 24 -48 hours after endetoxin infusion. at present, one may conclude that administration of sublethal doses of bacterial endetoxin to human volusteers produces alterations in cytokime production and tlymphocyte activation which are both similar and dissimilar to those seen following serious injury. hemodynamic support is based first on intravenous fluids and second on vasoactive agents. colloids are usually preferred to crystalloids, for their more rapid hemodynamic effects and their lesser passage in the interstitial space. in the presence of hypotension, the pharmacological profile of dopamine makes it the first agent of choice. it is only when high doses of dopamine are insufficient to restore a sufficient tissue perfusion pressure that norepinephrine should be added. even when cardiac output is not decreased, a dobutamine infusion is often indicated to counteract the myocardial depression, prevent vasoconstriction, and increase oxygen delivery to the tissues. the benefit derived from the administration of "renal" doses of dopamine is doubtful, but stimulation of dopaminergic receptors may increase blood flow also to the gut. in any case, none of these catecholamines has been shown to significantly improve the oxygen extraction capabilities of the tissues. agents with antiinflammatory properties but also with some vasodilating properties, such as n-acetylcysteine, prostaglandin el or pentoxifylline represent more attractive options to increase oxygen extraction. cardiovascular support should aim not only at the restoration of a sufficient arterial pressure but also at the maintenance of an optimal oxygen delivery to the cells. it should thus be guided also by measurements of cardiac output, mixed venous oxygen saturation and oxygen extraction, and indexes of cellular hypoxia, such as blood lactate levels, gastric intramucosal ph or vanearterial pc02 gradients. supranorma[ 02 delivery contributes to the prevention of tissue hypoxia tissue hypoxia is considered to be the common final pathway in the pathogenesis of multipte systems organ failure. it may directly contribute to cellular injury and organ dysfunction as well as enhance further mediator activation and release by a positive feed back mechanism. prevention of tissue hypoxia, is therefore, one of the most important aims in the supportive therapy of critically ill patients, especially in those with sepsis and septic shock. mismatch of cellular o= supply and demand in these patients may resuit from the impairment of the cardiocircu[atory system on all levels. 1. myocardial depression with a drop in 02 delivery (do~) 2. redistribution of blood flow between the different organ systems 3. inadequate nutritive blood flow due to tissue edema and endothelial damage (gic, leucocyte swelling etc.) these pathological changes may go along with increased metabolic needs. cellular and organ 02 supply may therefore be inadequately matched with cellular 02 needs in patients with normal and even supral normal doz. hyperdynamic circulation is often present in adequately volume resusucitated patients. it is most likely that one of the bodys main compensatory mechanisms would maintain cellular o= supply in the face of increased metabolic needs and impaired oz extraction capabillities. this pathophysiology may explain the findings in several clinical investigations that patients with normal or even supranormal doz can have signs of inadequate regional tissue oxygenation. it is also consistent with the results of different studies which demonstrated that the achievement of supranormal doz levels, either spontanuously or due to adequate supportive therapy, resuits in better patient outcome. any attempt to prevent or treat tissue hypoxia in these patients, however, has to take into account the effects of therapy not only on global dgz but especially on regional and microcirculatory blood flow. r. rossaint, d. pappert, k. lewandowski, h. gedach, k. slama, k.j. falke klinik for anaesthesiologie und operative intensivmedizin, ukrv, freie universit §t berlin, germany important contributory factors to the high mortality of severe acute respiratory distress syndrome lards) are the aggressive therapies required, such as mechanical ventilation with high inspiratory oxygen concentrations and airway pressures. as specific therapies for reducing or preventing the general inflammatory reaction of the lung resulting in severe hypoxemia is unknown, today's therapy is limited to procedures which predominantly support or maintain pulmonary function, i.e. pressure limited ventilation with peep and permissive hypercapnea, avoiding or treatment of fluid overloading, and positional maneuvers. extracorporeal lung assist combined with low frequency positive pressure ventilation has been recommended, when conventional therapy fails. today, extracorporeal gas exchange may be carried out using a system internally coated with covalently bound heparin. this allows for a lower level of systemic heparinzation reducing the risk of severe hemorrhagic complications of extracorporeal respiratory support. from april 1989 to august 1993 89 patients, aged from 15 months to 60 years, were transferred to our intensive care unit (icu) for treatment of severe ards. all fulfilled at least the slow entry criteria of the u.s. national ecmo study. due to hypoxemia 13 patients required veno-venous extracorporeal membrane oxygenation (ecmo) immediately after transfer to our icu. the other 76 patients were first treated with pressure controlled mechanical ventilation, permissive hypercapnea, prone position, and dehydration, if necessary. in 23 out of these 76 patients, in which the gas exchange did not improve in the following days, veno-venous ecmo with heparin-coated systems was additionally performed. heparin was given to achieve a partial thrombin time between 40 -55 s and an activated clotting time between 120 -150 s. if surgery was performed during ecmo, heparin infusion was interrupted. no severe bleeding complications related to anticoagulation for the extracorporeal bypass could be observed, however, in some patients, after three weeks of ecmo thrombi in the drainage cannula were observed. a problem of of membrane leakage shortened the life span of the membrane lungs to a mean of about four 4 days. in the conventionally treated group 87% survived and in the additionally with ecmo treated group 58% survived, representing a 75% survival rate in patients whose risk of death is considered more than 50%. on the basis of these experiences, we conclude that the described strategy, including veno-venous ecmo with heparin-coeted systems, may improve the survival in patients suffering from severe ards. abnormally high skeletal muscle po 2 in septic patients and its normalization during recovery: evidence against tissue hypoxia but for impaired oxygen metabolism of skeletal muscle? p. boekstegers, k. werdan department of medicine i, gro6hadern university hospital, munich, germany a pathological oxygen uptake supply dependence was suggested to indicate tissue hypoxia in sepsis-a hypothesis which is discussed controversially. because the detection of reduced oxygen transport to tissue and &tissue hypoxia would have important implications for therapy, skeletal muscle oxygenation was studied in patients with sepsis. intermittent and continuous determination of skeletal muscle po 2 by polarographic needle or catheter probes provided no evidence for skeletal muscle hypoxia even in severe stage of sepsis (boekstegers et al., crit care med 1994) . in contrast, mean skeletal muscle po 2 was found to be abnormally high in patients with sepsis (48 _+ 10,4 mmhg, n=39) compared to patients with limited infection (28, 4 + 4, 7 mmhg, p<0.001, n=16) , to patients with cardiogenic shock (22,3 + 6,2 mmhg, p<0.001, n=ls) and to healthy subjects (33,3 _+ 8,1 mmhg, p<0.001, n=10). furthermore, serial measurements in patients with sepsis showed that skeletal muscle po 2 was higher (44,1 _+ 10,3 mmhg) on days with severe sepsis than on days with intermediate state (30,1 _+ 8 mmhg) and than on days without sepsis (26, 8 _+ 5, i mmhg) . thus, a decrease of abnormally high skeletal muscle po 2 was related to improvement of sepsis. this was also demonstrated by comparing the decrease of abnormally high skeletal muscle po 2 with the decrease of apache ii score or elebute score as well as interleukin-6 serum levels in a separate study (boekstegers et el., shock, in press). in summary, our data provide no evidence for skeletal muscle hypoxia in patients with sepsis but support the assumption &impaired oxygen metabolism in severe sepsis. neutropnlic emigration from the vascular space into the extracellular matrix is important for the response to tissue injury or contamination by providing a large recruitable pool of tissue-based phagocytes. the consequences of neutrophil exposure to intravaseular chemoattractants, likely relevant for il-8 and c5a in sepsis, trauma, and in burn injury, are suppression of neutrophil attachment to endothelial cells and matrix proteins. it is probable that proteoglycan-bound attractants are of considerable biologic relevance, and may result in enhancement of responses to subsequently encountered cytokines. in the presence of connective tissue proteins expressing integrin-specific binding ligands, neutrophils respond to tnf-cq gm-csf and other cytokines by producing large amounts of hydrogen peroxide. this response is likely important in the digestion of contaminated or injured tissue by facilitating activity of neutrnphil granular proteases and inactivating plasma anti-proteases. this matrix-dependent oxidative response is important in defining potential novel targets for therapeutic intervention. the response appears to involve signalling by integrin-linked tyrosine kinases. the net effect of matrix protein injury through this mechanism is enhancement of the fibrotic response which underlies much of the prolonged ventilator dependence of patients with the adult respiratory distress syndrome. this adherence dependent response has also been implicated in direct hepatocyte injury, and may represent an early component of the syndrome of multisystem organ failure. study purpose: in this patient (pat.) population with a considerable sepsis morbidity and mortality, parameters proposed for sepsis assessment (elebute sepsis score [ele], sirs) were investigated with regard to their use in the early classification of pop. pat. at risk for developing sepsis. patients and methods: in a previous observational study on 110 consecutive pat. after elective open-heart surgery, we had determined ele daily for at least 3 pop. days (in pat. with a longer stay: until icu discharge). after publication of the newly proposed sirs definition, these criteria were retrospectively calculated and compared to ele. results: on the total of n =515 patient-days, both ele and sirs showed significant (p<0.0001) correlations with parameters reflecting the general and sepsis-related severity of the pop. clinical course. compared to sirs, the ele correlations were better (icu mortality: 0.59 vs. 0.32; icu treatment days: 0,51 vs. 0.32; days on mechanical ventilation: 0,54 vs. 0.32; days with vasopressor requirement: 0.59 vs. 0.33; number of microbiological findings: 0.44 vs. 0.29), the optimal classification criteria for the mainly sepsis-related outcome gave maximal youden indices of 0.87 for ele (ele >_12 on >_2 days, accuracy: 94%) compared to 0.42 for sirs (sirs present on >3 days, accuracy: 67%). accordingly, ele roc curve areas for both overall (0.92) as well as sepsis-related prognostic evaluation (0.99) were significantly (p<0,05) larger compared to sirs (0.79 and 0.86, resp.), this higher overall accuracy of the ele criterion was primary due to a more valid assessment already on the first and second pop. day, where sirs still had a high false positive classification rate (46% and 53%, compared to 7% and 1%, resp.). conclusion: in the early postoperative course after cardiac surgery, the sirs definition displayed a high false-positive classification rate (low specificity) for subsequent sepsis-related mortality compared to better classification results obtained by the elebute sepsis score. from the departments of medicine i and of "cardiac surgery, grosshadern university hospital, marchioninistr. 15, d-81377 munich, frg. correlation between physiological and immunological parameters in critically ill septic patients. ma rogy, h oldenburg, r trousdale, s coyle, l moldawer, sf lowry a relationship between physiological parameters of severe sepsis and immunological function has not been established. in an effort to assess such a relationship we prospectively evaluated nine severely ill septic patients. physiological risk was assessed by the apache iii score 1, while one component of immunologic function was evaluated by peripheral blood mononuclear cells (pbmc) eytokine production after in vitro lps stimulation 2. four of the nine patients died. apache iii scores at 72 h were lower in survivors (s) than in non-survivors (ns), (51-+13 vs 111-+1 5 p<0.05), while apache iii scores at admission were not significant different between s and ns (82-+13 vs 95-+13). down regulation of cytokine production by pbmc upon lps stimulation was a transient event in s. while s demonstrated an 3 fold increase of tnf-a bioactivity with[r~ 72 hours, ns did not demonstrate any increase at all. a similar pattern was demonstrated for il-1[3 and il-6 immunoactivity. tnf was measured by wehi bioactivity, il-1[~ and il-6 immunoactivity were determined by elisa. the sensitivity was 35 pg/ml for tnf, 30 pg/ml for il-ll3 and 35 pg/ml for il-6, respectively. in conclusion, both physiological as well as immunological functions of severe critically ill septic patients demonstrate predictive value for ultimate survival. while patients biological status seems to be more predictable by apache iii at day 3, p<0.05, the pattern of cytokine production by pbmc upon lps stimulation over the first 72h might be a reliable predictor as well. introduction: therapy of sepsis and its sequelae depends largely on its early recognition. many studies have investigated the change of certain mediators during sepsis and their potential to predict multiple organ failure and outcome. it was the objective of this study to investigate whether the onset of sepsis can be predicted by alterations of levels of interleukin-6 (il-6), tumour-necrosis-factor (tnf), pmn-elastase and c-reactive protein (crp). materials and methods: over a one year period, 40 polytraumatized patients were prospectively studied (mean age 42y, 71% male, iss 28). serum and edta-plasma samples were taken in 12h intervalls until the patient left the icu. il-6, tnf, elastase, and crp were determined immunologically. sepsis was defined according to the criteria of 'systemic sepsis' (veterans" administration study, 1987) with at least 4 of 7 clinical signs: (1) tachycar-dia> 100/min, (2) temperature >38,9°c, (3) blood pressure < 100mmhg, (4) mechanical ventilation, (5) leukocytosis > 15.000 /ml, (6) thrombocytopenia < 100.000/ml and (7) presence of an obvious septic focus. clinical parameters, sepsis severity and serum levels were documented on a daily basis, beginning on day 2 after trauma. results: 21 of 40 patients developed a systemic sepsis (52.5%), and 5 died. all mediator levels were elevated under septic conditions. the clinical severity of sepsis correlated well with the respective levels of mediators. in patients, who developed a sepsis the following day, il-6 (510 vs. 195 ng/l; p=0.029), crp (160 vs. 121 mg/l; p=0.035) and tnf (32 vs. 15 ng/l; p=0.045) were significantly increased as compared to those patients who remained non-septic. elastase levels were considerably elevated but did not reach the level of significance. we conclude that il-6, tnf and crp appear to be sensitive markers for prediction of septic complications in polytraumatized patients. objectives of the study: the assessment of liver function in polytraumatized patients who are at risk of developing mof is too inaccurate and late by using conventional biochemical parameters. methats: the injury severity of the patients (n=26) was determined by the injury severity score (iss). lidocaine is given at a dose of 1 mg/kgbw over 2 rain. i.v. and is metabolized in the liver by a cytochrome p-450 mechanism to monoethylglycinexylidide (megx). the metabolite is measured by a fluorescence polarization immunoassay. serial determinations of the test were performed between the 1 ~t and the 11 ~ day after trauma and were compared with other liver function tests (bilimbin, gldh, alt, ast). the systemic inflammatory response syndrome (sirs) is still a challenge concerning early diagnosis, therapy and prognosis. therefore, evaluation of inflammatory and disease activity becomes more important. c-reactive protein (crp) is a well established acute phase protein in chronic inflammatory diseases. recent reports suggest an induction of crp by interteukin-6 (il-6), a cytokine involved in the mediator cascade of sirs. on the other hand, tumornecmsisfactor alpha (tnfcx) is a very early released mediator in sirs removed very rapidly from circulation. in addition, soluble tnf receptors (stnfr~5, stnfr75) are released into circulation in the acute phase response. this study examines the kinetics of five acute phase proteins (crp, il-6, tnfot, stnfr55 , stnfr75 ) in patients suffering from sirs. eighteen patients entered the study after diagnosis of sirs. blood samples were drawn every six hours during the first two days and every twelve hours thereafter. crp was measured in an routine turbimetric assay. il-6 was detected in an biological assay using the/l-6 dependent 13-cell line 1313/29. detection of tnfc~ was performed in an elisa system using a monoclonal antibody" for tnfo~. soluble tnf receptors were also measured by elisa. crp levels were elevated (>10 mg/l) in all patients and at all time points. crp values did neither differ significantly in patients with (143±33 mg/l) or without (129a:39) multiple organ failure (mof) nor in survivors (133±41) or non-survivors (147:t:44). in contrast, 1l-6 was elevated in patients wilh mof (mean 740 pg/ml, range 0-8900 pg/ml). il-6 levels correlated especially with lung dysfunction. tnf(x levels were consistently elevated in patients with mof. crp, il-6 and tnfoc did not correlate with each other. in contrast, levels for both stnfr showed a positive correlation (r=0.7). patients could be divided into two groups by values for stnfr~5 and stnfr75: the group with higher soluble tnf receptor levels showed increasing values combined with a poor prognosis. the group with lower levels of soluble tnf receptor consisted of patients surviving mof or without mof. in conclusion, crp does not monitor the course of sirs adequately. in contrast, il-6 correlates with mof and episodes of high disease activity. high stnfr levels may indicate poor prognosis. klinik f0r an/isthesiologie and operative intensivmedizin der cau kiel, schwanenweg 21, 24105 kiei, germany. ch. waydhas, md; d. nast-kolb, ivid; m. jochum, phi); l. schweiberer, mi) objective: to evaluate the irfflarranatory response after different types of orthopedic operations and compare them with the systemic effects of accidental trauma of varying severity. patients: in 135 consecutive patients with multiple injuries (iss 40.6) the inflammatory response to trauma was prospectively studied. the patients were divided into 4 groups according to their iss points. additionally, the alterations after 79 secondary operations (>24 hr) were determined (msteosynthesis of the femur (n=28), pelvic girdle (n=ll) and spine (n=8), facial reconstruction (n=13), smaller osteosynthesis (n=13) and others (n=6)). methods: specific and unspecific parameters of the inflammatory response were determined in the trauma patients every 6 h, beginning on admission of the patient to the emergency room for a period of 48 hr, and in the operative patients on the morning of the operation, at the end of the procedure and every 6 hr during the first two days. results: lactate, neutrophil elastase, heart rate, po2/fio2-ratio, and other parameters discriminated significantly between the 4 injury severity groups during the first 48 hr (kruskal-wallis-test, p<.05). the degree of postoperative changes differed significantly (kmskal-wallis-test, p<.05) between the 5 types of operations for lactate, heart rate, po2/fio2-ratio, nitrogen excretion and showed a strong discriminating tendency for neutrophil elastase and c-reactive protein. the extent of changes were highest after operations of the pelvic girdle, followed by procedures on the femur, spine, smaller bones, and the facial region. the postoperative changes after osteosynthesis of the femur or pelvis were comparable to the alterations noticed after smaller (iss 1 to 24) or moderate (iss 25 to 40) accidental trauma for neutrophil elastuse, heart rate, po2/fio2-ratio and parameters of the coagulation system. conclusions: there is a considerable inflammatory response to operative procedures that varies with the type of surgery. large operations cause changes in the body homeostasis that resemble those after multiple injuries. it remains to be established whether the inflammatory sequelae of surgical trauma are additive to the changes caused by accidental trauma. objective of the study: we retrospectively compared characteristics of elderly patients (~65 years) and yeunger patients admitted to a surgical {sicu) and a medical intensive care unit (micu). we further studied the relations between advancing age, chronic disease, sepsis, organ system failure (osf) and mortality in the elderly group. material and methods: during a 1-year period, 538 patients were consecutively admitted into the 8icu; and during a 2-year period, 487 patients were consecutively admitted to t~mich. criteria for chronic disease, sepsis, osfsi.e. cardiovascular (cf), pulmonary (pf), renal (rf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature. results: patients from the sicu and~cu were similar in age, number of osf, and length of stay. however, when compared to sicu patients, micu patients had more cf (p<o.o1), sepsis (p<o.o01), and mortality rate (27% vs. ii~, p<o.ool). in contrast, sicu patients had more hp and nf, and prior chronic disease (all, p<o.o01). of the total group of 1,025 patients, there were 406 (40%) patients 65 years of age or older. elderly and younger patients had comparable length of stay in the icj, but elderly patients had significantly more chronic disease, sepsis, and number of osf (all, p<o.o02). all osfs, except hf and nf, were more ~o~mon i~ mdeljl, eompard to you-ger patients. ~he mortality is also higher in elderly patients (29% vs. 11% in younger patients). after multiple logistic regression, only number of osf increased mortality by a factor of 2.48 (95% confidence limits, 1.97-3.12), age did not increased [odds ratio 1.05 (i.01i.i0)], while admission to the sicu reduced risk of death by a factor of 0.15 (0.08-0.30). conclusions: based on these results, we conclude that age does not have any impact on icu outcome in the elderly. the only prognostic factor is the extent of acute disease, as measured by the number of osf. hence, prevention of oaf may influence outcome in elderly patients with critical illness. the lower risk of death in sicupatients may be explained by the large proportion of postoperative elective patients with relative mild chronic disease, and the lower incidence of sepsis. department of surgery, free university hospital, de boelelaan 1117, 1081 ~v amsterdam, the netherlands. septic shock and low output syndrome h.miyakawa, t.noguchi, s.hattori, a. mizutani, m. mori and n.honda objectives: cytckines are thought to cause the acute phase reactions under several critical conditions. we had investigated the relationships between cytokines network which included il-6,1l-8,acth and cortisol,and the outcome of the patients with septic shock or low output syndrome (los). materials and methods: in the septic group and los group,nine and four patients were enrolled respectively. furthermore, the patients in the septic group were divided into two groups,survival (n=5) and non-survival group (n=4).tn these patients,ll-6,1l-8,acth and cortisol had been measured every day after diagnosis of septic shock or los.the total number of measurements were 12 points in survival septic group, l l points in non-survival septic group and 10 points in los group.statistical analysis was used student's t-test to compare the mean of each group,and the changes were reported as clinicat significant if p<0.05. results: il-8 levels in non-survival septic group were higher than in both survival septic and los group.ll-6 levels in non-survival septic group were more significant than in both survival and los group.otherwise,there were no differences with respect to acth and cortisol among three groups. conclusions: we assumed that los would make several organs dysfunction as well as septic shock. but our results showed septic shock, especially non-survival,had different cytokins network mechanism for organs failure from los. lasson ,~, g/3ransson j, jijnsson k. eady diagnosis of complications after trauma is imperative to decrease morbidity and mortality. for this purpose an easy, cheap and fast diagnostic method is needed. the aim of this study was to analyse if complications after surgical trauma of various extent could be diagnosed earlier using preoperative or daily postoperative measurements of various plasma proteins than by using climcal signs of complications. material and methods: two acute phase proteins (c-reactive protein and antichymotrypsin), lbur main plasma protease inhibitors (alpha-2-maeroglobulin, c 1estemse inhibitor, antithrombin ill and alpha-2-antiplasmin) and one collagen precursor (procollagen iii peptide) were measured preoperatively and then once dally for 7 days postoperatively. for the plasma protease inhibitors immunorcactive as well as functional levels were measured. haemoglbin, albumin and the white blood cell count were also measured. measurements were done preoperatively in 256 patients and continued postoperatively in 78 patients, 70 % of the patients were operated on for malignancy. resulls: preoperative plasma c-reactive protein levels were higher in patients developing postoperative complications. this discrimination increased when plasma was sequentially analysed postoperatively, when a remaining high level ora second increase in plasma levels depicted a complication 1-3 days earlier than clinical signs. high levels were seen both in patients developing local as well as in patients developing general complications. there was no clear correlation between plasma protease inhibitory levels and complications, neither pre-nor postoperatively. procollagen iii paptide levels were slightly (but not significantly) higher postoperatively in patients developing complications. contusions: the acute phase response, especially concerning c-reactive protein, predicts postoperative complications better than both plasma protease inhibitors or collagen precursors. preoperative plasma c-reactive protein levels indicate the risk of postoperative complications, while daily postoperative measurements more readily depicts a complication, usually preceeding the clinical signs of complication by 1-3 days. dept. of surgery m'alm6 general hospital, s-2 i4 01 malmr, sweden. mof is a major threat to the extensive burned patients and associated with a high mortality rate. the role of endotoxemia in the pathogenesis of mof in burned patients remains controversial. in this study, we examined the relationship of plasma endotoxin levels to development of mof, and outcome in patients with thermal injury. methods: a prospective cohort study of 17 patients admitted with burns covering more than 70 per cent of body surface area was undertaken. circulating endotoxin concentrations were measured by modified limulus amebocyte lysate assay in serial samples of plasma. results: 7 out of 17 burned patients developed mof according to multiple criteria. the plasma endotoxin concentrations of patients with mof were 0.512--1.127 eu/ml, significantly higher than that of 10 patients without mof (0.216-0.553 eu/ml) on 3, 14, 21 and 28 days postburn (p< 0.05--0.01). significantly more incidence of positive endotoxin test (>_ 0.120 eu/ml) was found in patients who developed mof as compared to that of non-mof during the observation period (p<0.05). as the mean endotoxin levels increased, the prevalence of mof and death also increased (see table below), persistent endotoxemia carried a poor prognosis. conclusions: the present investigation provide further evidence that endotoxemia in severely burned patients commonly occur. cimulating endotoxin has also been found to be strongly associated with development of mof and mortality following major burn injury. multiple hemostatic changes occur in sepsis mad multiple organ failure (mof). to evaluate the role of platelcts in patients with sepsis and mof, we examined changes in surface glyeoproteins on circulating platelets of t4 patients with suspected sepsis and mof. the severity of sepsis and mof was assessed by eiebute and apache 1i scoring system, respectively.using flow cytometric techniques and platelets specific monoclonal antibodies, platelet surface expression of fibrinogen receptor on gpiib-iiia, ofvon willebrand receptor gpib, and of granula glycoproteins (thrombospondin, gmp-140, and gp53) was measured. receptor density of gpiib-illa mad gpib on circulating platelets was not affected by sepsis or mof. in septic patients surface expression of activated fibrinogen receptor (libs1 expression) was significantly elevated (p<0.05) and correlated well with severity of disease (f0.597). no significant change in surface expression ofthrombospondin, gmp-140 or gp53 was noted in septic patients. in contrast, degranulation ofgraanle glycoproteins was significantly elevated in mof (!0<0.05) that correlated well with severity of mof (gmp-140, r=0.611; thrombospondin, r=0.643).we speculate, that platelets in sepsis circulate in a hyperaggregable (fibrinogen receptor activation ) but still reversible state that results in increased risk of microthrombotic events. in the course of the disease, irreversible platelet degranulation might occur and may play an important role in development of mof. abdominal sepsis is still associated with high morbidity and mortality. the present study aimed at evaluating patients with abdominal sepsis treated at our surgical intensive care unit during a 10-year period with the aim of identifying potential prognostic factors, bacteriological cultures, diagnostic procedures, treatment and outcome. during the period 1983-1992 i66 patients with abdominal sepsis were treated at the icu at our university hospital. 60 patients were women and 106 men with a mean age of 64 (17-101) years. in 74 cases, the abdominal sepsis occurred as a postoperative complication. the patients were scored according to apache ii and bacteriological cultures and the occurrence of organ failure were noted. the patients were hospitalized in median for 40 (-273) days out of which 7 (-178) in the intensive care unit. 46 out of 166 patients (28 %) died in median after 18 (1-194) days. the primary cause of mortality was multiple organ failure (38/46; 83 %). apache ii scoring could not predict a fatal outcome. abdominal bacterial cultures were dominated by bacteria of enteric origin (83 %) and in 60 % cultures grew multiple bacteria. 134 patients bad organ failure and 64 multiple organ failure. 29/166 patients (17 %) had abdominal sepsis due to diffuse peritonitis despite a morphologically intact gastrointestinal tract and the absence of localized abscess formation. mortality in this group was significantly higher as was the percentage of positive blood cultures and the occurrence of multiple organ failure. abdominal sepsis is still associated with a high mortality, predominantly caused by multiple organ failure. abdominal culture findings are dominated by bacteria of enteric origin. in about 1/5 of patients with severe abdominal sepsis a diffuse peritonitis with intact gastrointestinal tract without localized abscess formation was found. in this group the mortality was increased as well as the risk of developing multiple organ failure. during the period from january 1985 to september 1993 56 patients, mean age 24+4 years were referred to our department of resuscitologywith the diagnosis of eclampsia. all the patients were delivered by cesarian section and were mechanically ventilated for 7.3_+5.7 days. diagnosis of sepsis was confirmed in 6 cases by clinical and microbiological methods. patients were divided in two groups: lnon septic patients, 2-patients with sepsis, the control group consisted of 20 patients after cesarian section without symptoms of eclampsia or infection. we determined plasma concentrations of immunoglobulins a,g,m(a,g,m), complement factors (c3,c4), alphal-antitrypsin (aat), trausferrin (trf) and albumin (alb) using beckman (usa) analyzer,protein concentration, using kone (finland) analyzer. a(mg/dl) g(mg/dl) m(mg/dl) c3(mg/dl) c4(mg/dl) k 203+-31 971+47 205_+21 134+12 30+-3 1 154-+29" 568-+48* 142_+24" 81-+14' 17_+4" 2 102+_21'* 420-+30** 96-+21"* 67-+10"* 11_+3" in a prospective study we investigated serum of 12 severly traumatized patients withdrawn directly after admission at our hospital (tr i). follow up controls were taken daily until day ten after trauma (tr ii). two control groups were performed: serum of healthy volunteers (co, n =24) was investigated as. well as serum of patients undergoing elective herniotomy (n=12) 24 hours before (op i) and 24 hours after operation (op ii). serum bactericidal index (sbi) was determined using a hemolytic e.coli strain 08:k87:h19. 200/11 suspension with a final concentration of 250 -400 cfu were incubated with l oopl serum. after overnight incubation sbi was calculated according a special formula. results: co 74.8 _+ 6.3 opi 77.8 _+ 6.4 opii 68.2 _+ 7.7* tri 38.4 _+ 9.1"* trii 57.6 + 8.9** (*:p< 0.05; **:p<o.01) sbi represents a simple and reproducible method to detect immunobiological alterations after trauma and elective surgery. patients undergoing elective surgery showed slight but significant diminuation of sbi 24h after operation. deterioration of sbi was observed in serum of traumatized patients directly withdrawn after admission at our hospital. ten days after trauma significant improvement of sbi was found. further investigations have to be done to proof sensitivity and prospectivity of this simple but reliable test. a sepsis like syndrome (sls) occurs in i0 % of our patients following cardiac surgery. sls is characterized by a severe decrease in systemic vascular resistance requiring the use of vasopressors to maintain adequate hemodynamics in the presence of negative blood cultures. in order to determine whether preoperative factors predict the occurrence sls we retrospectively studied 62 patients with sls (group i: age 61.1 ~ 10.8 years); they were compared to 52 control patients (group 2: age 58 ± 14.3 years). treatment of sls consisted of aggressive fluid replacement and norepinephrine infusion. rydrocortisone (200 mg/24 hrs) was given on the first day. antibiotics were switched prophylactic to cover gramnegative bacteria in 48 pts. preoperatively pts in group 1 revealed a significantly lower cardiac index (2.5 ± 1 i/min/m 2) compared to group 2 (3.1 ± 1.2 i/min/m2; p < .02) higher left atrial pressure (group i: 17.8 ~ 8.1 mmhg; group 2: 13.1 ~ 6.1 mmhg; p < .005) and lower ejection fraction (group i: 57.9 z 16.3 %; group 2 65.2 ± 10.9 %; p < .03). bypass time, minimum and mean blood pressure on bypass and aortic clamp time were not different between the groups. immediately postoperatively (pop), the white blood count was elevated (group i: 15,700 ± 8,200/ui; group 2: 10,200 ~ 4,400/ui; p < .0005) and core temperature 12 hours pop was higher (group i: 38.3 7.6 °c; group 2: 37.6 ~ 8.4 °c; p < .004). serum lactate was already elevated on arrival on the icu pop (group i: 5,0 z 1.9 mmol/l, group 2: 2.2 ~ 0.9 mmol/l) but dropped within 24 hours (p < .0005). icu stay (group i: 3.7 ! 4.3; group 2: 1.6 ± 1.9 days; p < 0.001) and mortality over 3 months (group i: 19.1 %; group 2: 3.1 %; p < .005) were significant higher in group i. sls is observed more frequently in patients with preoperative cardiac congestion and results in longer icu stay and higher mortality. although intraoperative hemodynamics are similar in both groups, the white blood count and serum lactate levels suggest an intraoperative cause for sls. further investigations will focus on possible intraoperative causes of sls. the objectives of the study were to estimate the problem of stress ulcers in critically ill patients and to compare ranitidine and omeprazole to determine their efficacy in the prophylaxis of stress ulcers. 33 patients who were admitted to the intensive care units between january and july '92 with polytrauma, sepsis or those requiring ventilatory support for more than 48 hours were selected for the study. the patients were randomized into 3 groups to receive ranitidine, omeprazole or a placebo. the changes in gastric ph were monitored 4 hourly and the patients underwent an upper gi endoscopy 48 hours after the start of the study to look for stress ulcers and hemorrhage. the drugs were withdrawn one week after the start of the study. of the 33 patients selected for the study, 14 had undergone cardiac surgery, 6 had polytrauma and 13 had sepsis. the change in average pre and post drug gastric ph was +1.1 in the omeprazole group, +0.5 in the ranitidine group and -).9 in the placebo group (p < 0.05). endoscopic evidence of stress ulcers was seen in 62% of patients not on prophylaxis, 18.1% of those on omeprazole and 54.5% of those on ranitidine. while all the patients in the placebo group who had stress ulcers showed endoscopic evidence of hemorrhage, 82% of the patients with ulcers in the ranitidine group and none of the patients with ulcers in the omeprazole group showed hemorrhage (p < 0.01). in critically ill patients the incidence of stress ulceration is high. an alkaline gastric ph is protective against stress ulcers. omeprazole when used for prophylaxis against stress ulcers in critically ill patients or those on short term ventilation will reduce the incidence of formation and hemorrhage from stress ulcers. the early and accurate diagnosis of sepsis is of key importance for critically ill patients survival. many studies have shown that tumor necrosis factor ~x (tnf ~) and interleukin-6 (il-6) are mediators of the inflammatory response in sepsis. bacterial antigens interact also with antigen specific t cell receptors and the activation of t ceils takes place. activated t cells release soluble interteuldn-2 receptors (sil-2r). the aim of this study was to evaluate: the significance oftnf c~, il-6 and sll-2r in the diagnosis of bacterial sepsis and to evaluate the correlation of clinical and laboratory parameters with serum levels of tnf ~x, il-6 and sll-2r. we examined 19 patients with clinical signs of sepsis treated in the intensive care units of university medical centre ljubljana. venous blood was drawn from each patient within 24 hours after the first clinical signs of sepsis. clinical data (body temperature, blood pressure), laboratory dam (peripheral white blood cell count with differential, platelet count, c-reactive protein), and quantitative blood cultures were recorded. serum levels of tnf c~, 11,-6 and sil-2r were measured in 19 septic patients and in 20 adult healthy controls. tnf ct, il-6 and sil-2r serum levels were measured by commercially available ria and elisa (amersharn and eurogenetics) kits. the differences in serttm levels of tnf ~, il-6 and sil-2r between healthy control and patients and correlation between clinical and laboratory parameters were calculated. we found that only sll-2r serum levels were significantly (p<0.005, student t test) increased in patients in comparison with healthy controls. of all indicators of sepsis that we compared, only hypotension was significantly correlated with tnf ~x levels and leukocytosis with ]1,-6 levels. we conclude that only the increased serum sil-2r levels were predictive of bacterial sepsis within 24 hours after the first septic signs. tnf c~ and il-6 levels were not significantly increased at that time in lifts small group of patients. in order to create high precision prognostic system for patients with sepsis and septic shock we have made prospective and retrospective analis of 200 cases of sepsis. patients were included in this analis according bone's criterions of sepsis. comparing with apache-h, we have excluded such phisiologic variables as:temperature,na+ ,k+ ,}it, ph,which had a little influence on prognos of illness. we have added the other, more important variables: biilirubin, plateles,pti. we took into account: the seat arrangement, methods of respiratory support, comorbid conditions as: caneer, diabets,obersity. in general new score has 5 headings: -physiologic variables, -age, -seat arrangement, -chronic comorbid conditions, -methods of respiratory support. roc -analis have showed a greater precision of our score, compared with apache-ii score. there is significant difference between the roc-curve of apache-ii scare. the k.p.tit~v.,i.e.gurmanchuk. objectives of the study. sepsis is a severe complication of the local infection, th e fever and the systemic inflammatory response syndrome may take place in such a case as manifistations of the inflammation induced by bacteria. the systemic fever observed in infection is known to develop due to the action of certain cytoldnes. other systemic manffistation of inflammation are the production of acute phase reactants, acute phase proteins, the activation of the complement system. ivratertals and methods. we observed 35 children with light and 45 children with severe course(l-3 class of serionuess) of the sepsis. the ftmetional activity of the complement system (ch50, level of c 1-c5, factors b told d, c3a), level of c-reactive protein (crp} and tumor necrosis factor (tnf) were investigated, results. the level of crp was increased in the children with more severe class of the septic process in both group (60%-1 st and ioo%-2 nd}( with light and hard eours of disese) of patients. parameters of the complement system -the activity of cl, c4, c3 components, activity of b and d factors-were increased in the seram of children with light course of sepsis. in children with severe course of sepsis these parameters were signffieantty decreased, especially in the group of chiidrellwith higer level of crp. we found the negative correlation of the tnp-alfa concentration with the functional activity of classical pathway components (cl~3) and the positive onewith the funfional activity of the alternative pathway factors b-and d of the complement system. conclusions. thus, in children with different course of sepsis certain changes in levels and functional activity of an acute phase o[ inflammation proteins -crp and the complement system ones -&ire characteristic. the relationship between the tnf-alfa level and the activity of classical and alternative pathways proteins indicates on its role in the complement proteins genes expression. schr6der j, braun j, rennekampff ha, schr6der dw various alterations of the immune response are known in trauma, but the course will not be complicated by multiple organ dysfunction syndrome (mods) in all patients. phenotyping of cells offers a rapid system of evaluation certain aspects of the immune response. different subsets of lymphocytes and neutrophils were determined to evaluate their prognostic role in developement of mods. in 16 trauma patients with an injury severity score (iss) > 20 (mean iss = 32; mean age 41 years) lymphocyte and neutrophil phenotypes cd 3 (t-cells), cd 4 (t-helper cells), cd 8 (t-suppressor cells), ratio cd 4/cd 8, cd 11b (receptor for cr 3) and cd 16 (fcriii) were measured on day 1,2,3,5,7 and 10 post trauma. the expression of class ii histocompatibility antigen (hladr) on monocytes (hladr+ cd 14) and il 2-receptors on t-helper cells (cd 25/cd 41 were determined as well. the percentage of cells was monitored by immunofluorescence using monoclonal antibodies and three color cytometry. the percentage of hladr+ cd 14 were significantly lower an day 2,5,7 and 10 in patients who developed mods (p<0,05) compared to patients without mods and a healthy control (p<o,o01). the expression of il 2receptors on cd 4 was significantly different only on day 2. no differences could be measured in lymphocyte phenotypes cd 3, 4, 8 and cd 4/cd 8-ratio as well as in expression of cd 11b and cd 16 on neutrophils. class ii histocompatibility antigen (hladr) on monocytes offers the best ability to reflect cellular immunosuppression in trauma. this parameter allows the best differentiation of patients with and without mods. we retrospectively studied the relative importance of age, prior chronic disease, sepsis and organ system failure (osf) to determine outcome in critically ill patients with acute renal failure (arf). arf was defined as serum creatinine > 280/zmol/i, a twofold creatinine rise in prior renal insufficiency or the need of acute renal replacement therapy. definitions for prior chronic disease and other osfs -i.e. cardiovascular (cf), pulmonary (pf), neurological (nf), haematological (hf), hepatic (lf), and gastrointestinal failure (gf)-were derived from the literature and described previously. of the 1025 consecutively admitted patients to a surgical and a medical intensive care unit during 1 -ye0r period, 136 (13%) had arf. arf mortality was 52%. ninety-eight percent had other osf. overall, cf, pf, gf, and nf was significantly more common in nonsurvivors than in survivors (all, p<o,02). although both the number of osf before and after arf was higher in nonsurvivors than in survivors (p<0.02), the number of osf before was higher compared to after arf in both groups of patients (p<0.001). furthermore, age, cf and pf before the ontset of arf, nf thereafter, and renal replacement therapy were related to mortality (all, p<0.03). however, prior chronic disease was not related to mortality and chronic renal insufficiency was inversely related to outcome (p<0.002). after multiple logistic regression, advancing age, cardiopulrnonary failure and sepsis before arf, and nf after arf remained significant. these results show the high prevalence of arf in critically ill patients and that arf rarely occurs alone. the prognosis of arf in critically ill patients is poor, especially if associated with advancing age, additional osf, particularly cardiopulmonary failure and sepsis before arf, and nf after arf. hence, prevention of cardiopulmonary and sepsis before the outset of arf may influence outcome in arf in patients with critical illness. the search cominnes for a magic bullet ta help critically ill petienta, bat recent elini ',.ml 1rials of agents that showed ixomise in animal studies raise questions about such trials. expcrlmemai uvidan~ :in anlmats and human volanteet~ for concepts, mechanisms and treatment of inju~ ur illness can be substentlal, l~rauasive and exciting but the positive effects of potential therapy suggested by such animal and ctinieal research may be difficult to prove in sick patients. efficacy of a new txeatment can be difficult to prove became elinie.al situations, hi spi~e of important biologic phenomena, are v~iable and conrplex. there is redundancy and overlap of mediators ~d problems of timing of therapy. a majt~r cause of this dilemtns is not with the trials or hypotheses, but rather the promem of the cause or the causability of the human dise~.se that is trebled. this is/hu "causa nets" or '*specific cause" as described by stehbens, when prevention or tre.atmcnt is based em the sauna nero, extinetiem of the disease is ix~ssible as with saul/ pox. only elimination of the cause wl12 cure the disease. careful animal studit~s evaluate single pcrturbmiuns for survival or other changes. an ldso preparation may be infiuancad aignificantly by a ~mall clangs which may be insignific~lt ht pstiants, most human experiments ate carried out in normal volunleers with a single porlurbation, such as a low-level, non-lethal dose of a substance. such studies are huportant in defining mechanisms attd mediators of disease, in our world of injm'ed, operated, infected and inflslned patients, there are many diseases, mof is not one of lhem. it isn't even a syndrume, it is the final pathway for a number of diseaaes~ each of which has a cause. mecormock believes thal a "multi-causal" etiology is a euphemism for ignoranc# or a synonym fo~ unknown etiology. admission iv sn icu, a high apache seer% the sepsis syndrome, mof, muds, or sirs and having predictors indicating potential mortality, are not diseases. the inmpors are getting negative results in trials of agents on eiek or septic icu patients and aru now becoming splitters, dafining subgaoups at higher risk for death. such an approach may produce positive results if the diseases are identified and the treatmeul is specific for them, a major challenge in deterraintng the efficacy of new therapies for sepsis nnd shock is identifying patients whose e#temtc inflammatory response is appropriate from those in whom the mediator| are contributing to end organ system damage end death, traditional and recently revised categorical patient descriptions such as sepsis syndrome, sepsis syndrome with shock, multiple organ system failure, or the recently proposed systemic inflammatory response syndrome (sirs) may not be sufilcient since they identify individuals at varying levels of risk for short term mortality, the efficacy of new lmmunotherapy may be directly related to the patient's severity end risk of mortality at entry to the study. we recently reported (jama 1993;270:1z33-1~41) a comprehensive risk assessment approach for patients with sepsis syndrome, a common eategarlcal description used as an entry eriterlon for many trials. by screening s database of 58,737 recent admissions to 107 hospitals in the united states and western europe, we identified 1,195 patients who met sepsis syndrome criteria but were not enrolled tn clinical trinh, we then developed a survival model end severlty assessment method to estimate their 2g.day mortality risk. k..ente physiologic abnormalities were the most important prognostic factors (82% of total chl square) influencing outcome. the specific disease resulting in ice admission and the days the patient spent in the hospital and icu before qualification were independent risks, as were age and a clinical history of cirrhosis. the predictive model was cross-validated within the original 1,195 patients with a receiver d_perator characteristics (roc) area of 0.76 and in two independent databases, the first independent database contained 295 hospitalized patients with gram-negative infection meeting criteria for sepsis syndrome (roc=0.80); in the second, the 302 placebo patients withtn the recently completed phase ill e~aluatlan of il-1ra (rocffi0.76). in all databases the risk model was able to accurately risk stratify patients throughout the range of risks that varled from a very low 1% to a very hlglt 99%. by drawing on the ready avatlabillty of large clinically aeuurate, current databases and using the power of modern statistical techniques to model the bodfs response to sepsis, we are able to produce very aeettrate pre-treatment risk estimates, these prospectively defined and continuous risk estimates reduce reliance on multiple subgroups and data dredging that can compromise a stady's integrity. they cart provide a nnlform method for patient description across clinical trials of different attempts at immmlotherapy. risk calculations can also be useful in developing entry criteria for phase ii evaluations in order to initially test efficacy on a limited number of high risk patients, fonowing sueeessinl completion of a definitive trial, the risk estimates can be used to develop specific |ndlcattons for s drug's use and can monitor the impact of both new and multiple compounds on patient outrome, when dealing with therapeutic trials for the treatment of sepsis, there are 2 ways of checking the comparability of the groups (placebo and treated groups) -to use several description items such as age, sex, preexisting disease, number and nature of organ failures, physiology score, such as saps il (1) -to use a model for risk of death either from a score (apache ii, lii, (2) saps ii) or directly (mpm ii system (3)) before using the model for risk of death in septic patients, is it mandatory to validate it (by calibration, i.e. goodness of fit, and discrin'dnation, i.e., roc curves) first on a data base using the same definition for sepsis as in the trial and secondly on the placebo group of the trial ? or is it better to use a specific model for each trial ? the general models usually do uot fit for septic patients. there are 2 methods to make them fit : either to add variables to the original score (model for sepsis using the apache iii system (4)) or to customize the model. for saps ii the customization is applied to the equation regression, using the same saps ii score. for mpm 1i each component of the model can be customized. several remarks : only one model is published to be applied at the icu entry ( mpm ii 0) before any therapy. 2 -most of the published scores are calculated from the lsl icu day. applying these scores to the let day of sepsis could have a very different significance. 3 -is the accp classification of sepsis useful at the bedside to select oatients ? ( the pathophysiology of sepsis involves a dynamic interaction between the host and the infecting microorganism(s). a multitude of biochemical and hemodynamic interactions occur which function in concert to determine the ultimate outcome of the septic episode. the complexities of the septic process preclude outcome analysis by in vitro systems or computer models, animal studies have become indispensable in evaluating new therapeutic agents in the treatment of sepsis. these studies provide valuable information on safety, pharmacokinetics, relative efficacy and dosing strategies for potential therapeutic agents in the treatment of sepsis. there are three basic types of animal models: (1) toxicity models with injection of bacterial endotoxin or exotoxins; (2) live or killed bacterial challenge models; (3) actual infection models. all models employ some form of external manipulation under controlled conditions to induce sepsis and to analyze potential therapeutic efficacy. these experimental requirements may limit the ability of animal models to accurately predict the clinical value of new agents in human sepsis. four major end points are analyzed in the various animal models: (1) hemodynamic parameters; (2) modulation of host-derived inflammatory mediators; (3) resolution of end organ injury; or (4) lethality. each animal model has certain advantages and limitations. species-specific differences in physiologic and immunologic features make it difficult to directly extrapolate the results of animal experiments into clinical medicine. while no ideal animal model exists, consistent benefit of a new immunotherapeutic agent in multiple animal systems provides the most compelling preclinical evidence of its therapeutic potential in septic patients. consensus-assisted development of a study protocol on sepsis: an important difference from previous randomized trials there have been several, and perhaps too many metaanalyses of cliuical trials on surgical infections, but their results have only rarely been incorporated into the design of new randomized trials in sepsis. metaanalysis is retrospective. it seems more important to analyse and to criticize the design of tria/s before they are ongoing. incorporation of that into development of a study protocol is the aim of the following procedure: the time-schedule of about two years should include further cell culture and animal experiments after the first successful studies showing effectiveness. exhaustive evidence for a dose-dependent cost-banefit and near complete explanations of the pathomechanism should not be awaited for development of the protocol of the clinical study. however, a discussion forum of experts should be performed in a recently published, almost perfect trial in order to see the frontiers of the present research in cell and molecular biology, clinical pharmacology, statistics and decision making. this should be followed by a metaanalysis of at least 10 study designs on sepsis with methods of formalized medical decision making (decision tree). clinical algorithms -developed by objective methods including nominal group processes -should be developed to standardize any concomitant treatment in the centers considered for a multinentre trial. the latter is usually necessary in the field of sepsis. finally, a pilot study should be performed to demonstrate not only feasability, but to learn about all possible types of interventions which modify endpoints as response variables. especially mortality is not free from such effects. the time for the individual phases of this consensus-assisted process of protocol development have to be shortened by their temporal hiterloeldug. due to the fast discovery of new chemical factors in sirs it wilt otherwise be impossible to come up with results of randomized trials which are fascinating enough to be incorporated in daily routine treatment. severity scoring systems are intended to provide a population-based estimate for the risk of an unwanted outcome resulting from some disease process. in the area of infections and the "septic" response, this has routinely been defined as death. for anti-infective trials, there has been a reasonable correlation of severity (apache ll) with outcome measured as persistent or recurrent infection, but this is not as robust as the association with mortality. it is expected that non-lethal failure would not immediately correlate with disturbed physiology: the basis for antiinfective failure depends on a variety of factors not measured in severity scoring, such as type of infecting organisms, density and susceptibility to administered antimicrobia[s, and anatomic features of infection (e.g., pancreatic). severity scoring is important in differentiating these "categorical" variables from continuous (physiologic) variables. there are, additionally, a series of problems related to the cause of death in relation to severity scoring. in those tdals in which cause of death is analyzed, high severity scores did not routinely predict death as an immediate sequelae of sepsis/septic shock. many of the predicted deaths occurred remotely of progressive background diseases. other important problems with severity scoring have to do with lead-time bias: hew long patients were ill before entry into the study. statistical techniques for condensing various categorical and continuous variables, as well as factoring in information requiring the pharmacodynamics of agents which affect outcome, are being developed to further refine deviation from predictive equations as an outcome measure. stepwise logistic regression analysis has identified the presence of shock, intm-abdominal or unknown source of infection, hospital acquired infection, age greater than 70 years, neutropenia and inappropriate antimicmbial therapy as independent variables associated with increased mortality in sepsis. maldistribution or" these variables is a concern in targe randomized clinical studies. it should be noted that only the selection of antimicrobiai therapy could be addre.~'sed and altered at the time of enrollment in a sepsis clinics] trial. inappropriate antimicrobial therapy has been noted in 41%-66% of septic patients. well designed, randomized sepsis trials do not assure the absence of imbalance between treatment and control groups with regard to inappropriate antibiotic therapy, especially with subgroup analysis. statistical manipulation to correct for any imbalance is appropriate but avoiding imbalance is ideal. selection of antibiotics in septic patients requires such considerations as clinical setting (history, physical examination and underlying disease), appropriate laboratory tests (such as gram stain), identification of source and associated bacterial flora, antibiotic susceptibility patterns for that hospital and likelihood of resistant organisms. determination of inappropriate antibiotic therapy is made retrospectively after culture results are available. in some circunmtances the organism and antibiotic sensitivities cannot be predicted, while in others it can. leibovici et al identified hospital acquired infection, antibiotic treatment before a bacteremic episode, endotracheal intubetion and thermal trauma as independent variables which predicted isolation of a multiresistant organism. the same authors developed a predictive index for resistant organisms, which when compared to prescriptions of attending physicians, improved antibiotic selection in critically ill patients. including a standard antibiotic regimen for all septic patients in a clinical trial is impractical. there is too much interhopsital variability in flora and antibiotic susceptibility. in addition, the aggressive broad spectrum coverage necessary for some patients is inappropriate for others. a protocol which guides a prescribing physician through a logical chain of reasoning might improve antibiotic selection in critically ill patients and could be included in the design of a clinical trial in sepsis. although this approach might minimize potential for maldistribution of inappropriate antimierobial therapy, it would likely create multiple problem areas. will the patient's physicians agree to the protocol choice? what is the availability of speciftc antibiotics at a participating hospital? what is the aplpiieability of study results in typical clinical situations? if there is agreement that thin is an appropriate antibiotic protocol, should it not be used in all seriously ill septic patients in the hospital? in other conditions such as ards, the success of protocol therapy is dependent upon measurable goals such as pao 2 and highest plateau pressure, while measurable goals are not as clear in choosing antibiotics. based on the above confounding issues, a complex and extensive algorithm covering many potential possibilities of error and specific antibiotics seems impractical; however, an algorithm focusing on three to four major common errors in antibiotic selection and dealing with classes of antibiotics is plausible for inclusion in sepsis clinical trials. in this session we will attempt to outline the methodology of such a future study, emphasizing the immense difficulties involved in its proper conduction including: design, selection of patients, surgical considerations, supportive treatment, the "human factor" and ~nd points. only a close cooperation between a few dedicated surgeons, obsessively studying a large number of well selected and stratified patients over years, could make such a study possible. algorithmis have frequently been mistaken for the graphic format in which they are presented. however, an algorithm is neither a flow chart nor a list of instructions; it is a step-by-step approach to solving a problem. likewise, a clinical algorithm is a step-by-step approach to solving a clinical problem. cas are deterministic, which does not mean that they are rigid. they are practical rather than mathematical algorithms, that tan be used only with clinical judgment, and must be tailored to each patient. there are a number of types of, or uses for, cas that can improve sepsis management, including: diagnostic or severity scorm cas, a management ca for managing sepsis constructed according to recently published standards for use in teaching research protocol algorithms that must be used to collect data or guide implementation of a clinical trial aimed at validating one or more dicisions in the management ca; finally, protocol-style cas drawn from the management ca and should be validated using matching outcome studies. organisational problems peculiar to multicentre trials the organisation of any randomised clinical trial is fraught with difficulties, and that of a multicentre trial particularly so. there are problems associated not only with the principles on which the investigation is based but also with the detailed organisation and analysis. are all the participants truly unbiased about the relative merits of the regimens being studied? is like being compared with like -are all the end points and the standards for measuring them clearly defined so that they cannot be misunderstood? are all eligible patients being studied, and their results reported? is truly informed consent being sought that will satisfy not only the patient's right to choose what happens to him, but also the law of the land? and, finally, how can the participants be sure that the trial will be stopped at the right time, to ensure that no treatment is given to any patient that might be harmful? these questions may seem insoluble, but until we tackle them we shall never be certain. dr.m. ev~s, scar~mu~ hospital, scarborough, nonhyo~s~ y0126ql, u.k. increasing knowledge of the pathogenesis of sepsis and septic shock has led to the development of many new therapies and technologies capable of preventing, blocking or even reversing the deleterious cycle of events. recent results of subsequent multicenter trials testing some novel therapeutic drugs, however, did not confirm the promising sometimes overwhelming effects obtained experimentally. it is postulated that this is largely due to inappropriate design and conduct of multicenter trials as currently performed. one of the shortcomings emphasized in this paper is the "treatment mix" problem. multicenter trials in sepsis are performed because no single center is able to recruit the necessary number of eligible subjects within a reasonable time. each single center (icu) has its own individual policy which cannot be standardized for the trial (treatment mix). even if we ascribe that each icu did the "best" for their patients, it is highly probable that the outcome differs between the icus. this translates to the effect of the new therapy under investigation. ]t is therefore worthwhile to consider that icus must first demonstrate a certain standard based on the patients' outcome (audit) before becoming accepted as a participating center. this prerequisite ensures comparable quality between participating centers, led to the reduction of "noise level" and increases the probability of positive study results. the riyadh-icu program based on standard mortality ratios is recommended as an audit instrument. during the last decade most clinical trials of potential therapeutic agents in systemic sepsis have failed to demonstrate benefit from the drug under study. although in many instances it is entirely possible that the agent in question does not have clinical efficacy, an equally plausible explanation for the multitude of negative results is that the studies were improperly designed and thus could not show therapeutic benefit, if it existed. problems which have been identified in these various trials include inappropriate or unrealistic definitions of response to the drug; inadequate sample size, with attendant insufficient statistical power to demonstrate a difference, if it existed; insufficient standardization of customary therapeutic maneuvers in septic patients; imprecise criteria for patient entry into the study, which creates unacceptable inhomogeneity between control and treatment groups and invites unjustified post-study subgroup analysis; the lack of a formal sequential monitoring procedure for interim data analysis, which could potentially affect patient safety; and a primary analysis of study results that excludes protocol deviants and is not according to intent-to-treat. these and other problems have impacted upon the validity of the various trials conducted to date and may well have prevented the resolution of controversies surrounding the use of certain agents in the treatment of septic patients. the complexity of bacterially-induced septic shock involves a cascade of events that evolve over time, beginning with the proliferation of offending organisms and followed by the release of toxic mediators from the bacteria. endotoxins [e.g. lipopolysaccharides) from gram-negative bacteria initiate septic shock by precipitating a systemic inflammatory response syndrome (sirs) through release of a broad spectrum of hostderived mediators. over the last century, hundreds of these endogenous mediators have been proposed to serve as pathophysiological substances in this "alphabet soup" of cytokines, eieosanoids, biogenic amines, kinins, peptides, ions and hormones. an evaluation of the literature on septic shock leaves the investigator with a sense that, although many pieces of the septic shock puzzle have been presented, no clue was provided to indicate how these pieces fit together, in an attempt to assess objectively the causal role of individual mediators, we recently completed a collective meta-analysis of 24 mediators that were individually reviewed and subjected to koch-dale analysis of causality by distinguished authorities in the field (1). in summary, our attempt analyze available evidence to support a cause-and-effect relationship for putative mediators of septic shock revealed that only eight of the 24 mediators analyzed could be strongly inferred as playing a causal role in septic shock. evidence supported the involvement of endotoxln, endorphins, complement, interleukins, tumor necrosis factor, eieosanoids, platelet activating factor and calcium. other mediators either lacked adequate supportive evidence or were not conclusively involved. posttraumatic outcome may be reduced by an amplified stress response mediated by neural and humoral stimuli. the classical hormonal catabolic response is predominantly mediated directly or indirectly by neural stimuli, while most changes in inflammatory responses such as leukocytosis, acute phase proteins and changes immunofunction are mediated by humoral mediators. clinical experience from controlled studies suggest afferent neural blockade and modification of stress response to improve organ function and postoperative outcome, and limited experimental studies also suggest neural blockade to be of beneficial value in shock states. of special value may be the improved gastro-intestinal motility after neural (visceral) blockade. no outcome studies are available from patients with major truma, multiple organ failure or sepsis, conditions where humorai mediators may be more important to modify than neurally mediated responses. future studies should investigate the clinical outcome effect of combined neural and humoral mediated blockade, as well as the potential role of an initial neural blockade in elective trauma (first hit) to improve the metabolic scene and outcome if a postoperative complication (second hit) should occur. neutrophil elastase is the predominant protease of the human neutrophil. this enzyme, and a variety of other enzymes, including neutrophil collagenase, are released by activated neutrophils in the setting of high concentrations of reactive oxygen metabolites. in addition, in this extracellular environment, the normal antiprotease defense mechanisms of the organism may have been severely inactivated oxidatively especially along capillary endothelial cells. accordingly, release of neutrophil elastase which mediates inflammation and degrades most extracellular matrix proteins, including elastin, fibronectin and many forms of collagen, may contribute to tissue injury and organ failure in a variety of diseases ranging in severity and acuity from pulmonary emphysema to the adult respiratory distress syndrome (ards). as a result, significant efforts by the pharmaceutical industry have been directed not only toward the development of human neutrophil elastase inhibitors, but also toward their characterization in a variety of animal models of neutrophil mediated diseases, and their evaluation as potential therapeutics for the treatment of a variety of human clinical conditions. data from animal models of organ injury and failure along with data collected from a series of patients at risk for ards and with ards will be presented and discussed as a rationale for inhibition of neutrophil elastase. parameters that need to be monitored to obtain success in future clinical studies will also be presented in this context. have turned out to initiate and maintain organ dysfunctions in severe posttraumatic and postoperative courses. such proteolysis-induced disorders are especially rendered possible by the concurrently arising consumption of inhibitory regulators (e.g. a vproteinase inhibitor=a 1pi, antithrombin iii=at iii) via cofnplex formation and/of proteolytic/oxidative inactivation. besides the well-known destructive potency against protein substrates, proteinases such as elastase or thrombin (active or in complex with the serpin inhibitors ~ 1pi or at iii) are also supposed to increase the inflammatory reaction by inducing cytokine release or shedding of soiuble adhesion molecules from various inflammatory cells (pmns, monocytes/macrophages, endothelial cells). those cell-derived mediators may provoke further cell activation through an autocrine/paracrine loop thus increasing significantly the proteinase burden at the inflammatory focus. therefore, this noxious circle might be interrupted by a convenient proteinase inhibitor therapy to ameliorate the inflammatory process. to verify this hypothesis, high dosis of at iii (mean plasma levels up to 140 %) were applied in first controlled randomized studies on surgical patients suffering from multiple trauma or severe sepsis. in control patients we could clearly demonstrate the sequential appearance of proteinase/serpin-complexes (fa 1pi, tat), cytokines (il-8, il-6), and soluble intercellular adh6sion molecules (icam, elan, p-selectin) in the circulation. these factors turned out to be a helpful tool for early diagnosis and prognosis of forthcoming organ dysfunctions. interestingly, m at iii-treated patients a significantly reduced release/production of inflammatory mediators became obvious concomitant with the improved clinical course in comparision to an increasing deterioration in control patients. soluble mediators of inflammation are cytokines (e.g. il-1, il-6 and tnfe) as well as breakdown products of complement proteins (e.g. c5a and terminal complement complex). therefore, attenuation of both, release and generation of these synergistically functioning mediators together with stimulation of release of antagonists including soluble forms of receptors are potentially beneficial in all kind of inflammatory diseases. intravenous immunoglobulins (ivigs) are known to have an anti-inflammatory effect in humans (nih consensus conference on wig, 1990) . the mechanism of action becomes more and more clear. in single cells stimulated by anti-cd3 mab wig was able to down-regulate production of il-2, il-10, ifn% and tnfj~ significantly. igg coated solid-phase stimulates release from monocytes of interleukin-1 receptor antagonist (il-lra) but not of il-1; this observation is very much in contrast to the effect of endotoxin in the same in vitro system. furthermore, naturally occurring anti-cytokine antibodies can be demonstrated in apparently healthy individuals and in wig prepared from plasma pools. some of these antibodies can be detected by antigen/antibody reactions in fluid phase (anti-ll-le, anti-il-6), some only by solid-phase detection systems (anti-ll-8, anti-ifn% anti-tnfc 0. infusion of wig to patients suffering from inflammatory disorders have resulted in a decrease in il-6 in circulation. during infusion wig might induce an acute but transient rise of tnfo~, il-6, and il-8. self limitiation of this process might be due to demonstrable instant release of il-1 ra and soluble tnfo~ receptors. in vitro ivig has also been shown to attenuate complement activation via the classical pathway. furthermore, acid haemolysis of erythrocytes in paroxysmal nocturnal haemoglobinuria could partially be prevented by ivig. ivig was able to attenuate forssman shock in guinea pigs. we have seen a patient with congenitally elevated turnover of alternative pathway of complement who repeatedly showed an increase of haemolytic titres after administration of ivig. thus, wigs apparently have multiple potencies including attenuation of soluble mediators of inflammation and might therefore be of benefit in trauma, shock, and sepsis. significance of sinusoidal liuing cells and of humoral factors on hepatic function following sepsis and major surgery hirata k, katsuramaki t, yamaguchi h, mukaiya m, yamashiro k. regeneration of the liver after hepatic necrosis or partial removal has been extensively studied, but uncontrolled mechanisms to irreversible hepatic failure following sepsis or major surgery of liver or with hepatic dysfunction have so far not been well documented. we suggested the importance of the intrasinusoidal aggregation between sinusoidal lining cells and intrasinusoidal running ceils in the mechanisms of hepatocyte dysfunction. thus, the purposes of this study was to investigate the changes in each sinusoidal lining cell during this process and the effect of cytokine on hepatocyte under various oxygenation. [ materials and methods ] ( 1 ) clinical study : thirty two patients with histologically proved malignant tumor underwent major hepatectomy were devided in two groups, i_e. the cirrhotic liver group and the non-cirrhotic [aver group. most of the former were the patients with hepatocellular carcinoma and most of the latter were the patients with metastatic carcinoma of colorectal cancer. five patients were complicated septic conditions with hepatic failure. following operation in each patient, a verous blood sample was taken for measurement of serum il-1, il-6, tnf and c-reactive protein concentration. and hepatic biopsies were sometimes undergone in patients with anomalous hepatic function. (2) experimental study : kupffer ceils, sinsoidal endothelial cells and hepatocytes were separated from mouse ( c3hhen ) liver. and also polymorphonuclear cells and platelets were purified from blood and peritoneal macrophages were from peritoneal cavity of mouse. co-cultures between or among these cells with iipopolysaccharide were performed. cytokine concentrations in media and hepatocytic function were compared. [ results and conclusion ] our findings in above experiments demonstrated the cleanly different concept for the mechanism of hepatocyte dysfunction following sepsis or major surgery. namely, the effect of hepatoeytie function by cytokines or superioxide were significantly affected under low oxygenation, but not significant under good oxygenation. hirata m.d.,nishi 17,hokkaido,japan $120 470 immune complexes as .mediators of sepsis and immune dyafumcffon laa k horn, chxistof birkenmaier, and greg h2mon departmen~ of surgery, san frandsco general hospital, urdversr 3, of calif(~rrda, san frandsco. immune complexes (ic) a;:e commonly found circulating in parians during infection and sepsis; and following traumm, bums, and massive l~ensfusion. clearance of 1c occurs by phago~'tmsis of esll-bou_nd or opsonized p~tlcles. monocyte activation du~jng phmgocytos/s could lead ~o release of cytokilies ieletexiot~ to the hosh to examine tb/s phenomenon, we formed insoluble ic by precipitating iow-endotoxin bovine serun~ albumin (bsa) with rabbit ant/-bsa igg. we have ~town that these ic are ingested by monocytes and concomitantly stimv/ate re~pizatory bu~t activity measuxed by assay of in%racellul~ peroxldaffon. we have shown that ic ingesffon produces signlqcant el,era,lore in the monoey~e phenotype. spedacally, cdi4 is down-regu/ated, along wl~ cd 32 and cd64. thus responsiveness to lipopolysacchmdde (128) a~xd i=m~unoglobulin fe st~mu/mtlon is reduced. in cert,+rest, the complement rote.p tot cdc42 is u~-regulamd, indicaling mcremsed re~ponalx=~ness to opsonized pat,rotes, we examined monocyte superna~n~s for production of hzmor necrosis factor alpha (tnfg) following ic stimulation and showed that ic are capable of provoking ~elaase of hrmaunl)reac~ve tni~. j[c also increase mono=yte produchon of prnstaglandin e2. in summary, ~/~ese results demonstrate lhmt ic are capable of indudng production of ¢ytokines and the imrau-nosuppressive prostaglandin pge2. ic also allez ~he surface expression of important receptors involved in actlvaffon by lp~ and phagocyiosis. thus, ic aze competent for indud/on of the mepl/e s!0otlse. by examining m0n0cyte phenotypms, it may be possible to d~mrmkne extent ohn'u~u~e complex activation and predict immune depression iu criffcally ill pa~ents. a great deal of experimental work has focused on preventing and/or treating sepsis and organ failure following injury. many of these strategies have tried to attack mediator substances released during the systemic inflammatory response following injury. sugermann has demonstrated the improvement of pulmonary function, but not eappillary leak using [buprofen in the porcine model. in a clinical trial, faist demonstrated the effectiveness of indomethacine in amelierating the post trauma of cellular amenity using in vitro parameters. morbidity and mortality, however, were not different between control and treatment groups. baue und chaudry have suggested that atp magnesium chloride may have protective effect in renal failure um kupfer cell dysfunction seen after shock. pentoxiphyllin has been found to be effective in improving tissue oxygenation and oxygen consumption following hemorrhage, and there may be promise for this drug in the ischemia reperfusion injury. monoclonal antibodies against endothxin and inflammatory mediators seemed promising at first glance; however, preliminiary clinical data of the treatment of sepsis with either ha-ia or e5 monoelonoal antibodies against bacterial cell wall have been disappointing. initial studies with il-i receptor antagonist also appeared encouraging, but no difference was seen in the most recent trial. currently studies are ongoing with monoclonal antibodies to tumor necrosis factor which may have more efficacy given the fact that it is a macrophage mediator released by endotoxin. although we seem closer to understanding and preventing the problems of sepsis and organg failure after trauma, we must continue to appreciate the complex interrelationships and delicate balances inherent in the host defense system. overzealous attempts at restoring immunity in the absence of understanding pathophysiology may be just as dangerous at post-traumatic immlmospremsion. severe acute pancreatitis was caused by different etiologic factors, but once pancreatic tissues were infected, patients show a similar pattern of aggravation and develop organ failure. remarkable elevation of serum il-6 was unexceptionally observed in patients with severe acute pancreatitis. the serum levels were more than 500 pg/ml (normal: less than 4 pg/ml) during the first one or two days after onset. there was a significant correlation between the peak serum il-6 level and the number of organs showing failure (r=0.883). tnf-a production by isolated peritoneal macrophages following lipopolysaccharide (lps) stimulation was significantly increased in cerulein-induced pancreatitis rats. serum tnf-a activity was elevated following intraperitoneal administration of lps as the septic challenge both in pancreatitis rats and in control rats, being significantly higher in the former (p<0.05). histological findings and liver function tests revealed that lps induced more severe liver damage in pancreatitis rats than in control rats. these results indicate that increased tnf-a production by peritoneal macrophages in acute pancreatitis augmented lps-induced liver injury. organ failure in severe acute pancreatitis could be caused, at least in part, by increased cytokine production. it is clear now, that in mods, organ injury is not directly due to exogenous factors, such as bacteria or toxins, but instead is largely a consequence of the host's own endogenously produced mediators. there is an extensive body of literature on the inhibition of mediator production, release and action in different cascade systems by glucocorticoids. they can serve as a host protection against overactive defense reactions if used adequately. a new understanding of the regulation of cytokine synthesis, especially tnf, may lead to an insight of the conflicting findings between the benefits of glucocorticoid therapy in animals and its current failure in recent clinical trials. glucocorticoid shares its role with other novel therapeutic approaches also shown to be successful only in experimental settings. a mete-analysis of steroids in sepsis, however, revealed a beneficial effect in the subgroup of patients with gram-negative infections. there is now a series of arguments to reevaluate the indication for steroid thera-py and/or prophylaxis in mods and sepsis. honjo i-1-1, kumamoto 860, japan in septic shock j. briegel, m. halter, h. forst, w. kellermaun, m. bittl, k. peter imrodnetlea and methods: hydrocortisone (hc) at an infusion rate similar to the maximal secretory rate of cortisol in man improves hemodynamics in human septic shock (1). we hypothesized that the hc-induced hypercortisolemia prevents a harmful overshoot of immune reactions. to test this hypothesis we prospectively investigated 12 patients admitted to the icu in refractory septic shock. after resuscitation (mechanical ventilation, fluid resuscitation, titration of vasopressors, and initiation of antimicrobial therapy) hc was started with a loading dose of 100 mg/ 30 rain, followed by a continuous infusion (10 mggh). with hemodynamic improvement (dopamine < 2 gg/kg/min) the infusion rate nfhc was tapered over a period of 3 days. phospbolipase a 2 (pla2), c-reactive protein (crp), and elastase-proteinase inhibitor complex (e-pi) were deierminated daily. results: according to our previous results hemodynamics improved during hc infusien. after 4 days dopamine requirements decreased to less than 2 gg&g/min in all patients. this corresponded to an attenuation of the systemic inflammatory response syndrome (sirs) indicated by the abrogation of fever and the decrease in heart rate and inflammatory mediators. pla 2 activity and e-pi concentrations decreased to near normal values and crp concentrations decreased as well. after cessation of hc infusion (between day 5 and 7) a reinforcement nfthe sirs was observed despite cortisol levels within the normal range. this was first indicated by e-pi, followed by fever, increases in heart rate, crp, pla2, and finally by the relapse of septic shock in four patients on days 7,8 9, and 1o. a second infusion of hc again resulted in shock reversal and in a decrease in pla2, crp, and e-pi in the patients under study. discussion: there is growing evidence that the endogenous steroid hc and proinflammatory cytokines integrate a complex immunoregulatory feec~ack circuit. the elaboration of tnf, il-16, il-6, and il-8 is attenuated by-100 rag hc prior to endotoxin challenge in humans (2, 3) . cytokines like tnf, il-16, il-6, and il-8 are potent proinflammatory signals for acute reactants (--, clip), neutrophil degranulation (--, e-pi), and pla 2 synthesis and secretion. our data provide evidence that hc at an infusion rate similar to the maximal secretory rate of cortisol in man attenuates the systemic inflammatory response in human septic shock. to evaluate the feasibility of a nigh-dose regimen of antithrombin iii (at iii) treatment in patients with multiple injuries regarding blood levels of antithrombin iii and undesired side effcts and to analyse effects of the initibitortherapy on the systemic inflammatory response. patients and methods: 20 patients with an iss nf more than 28 points who could be treated with at iii administration within 180 rain after trauma were randomized to receive either at iii or placebo. at iii was given to reach an antithrombin iii inhibitory capacity in plasma of 140% by using the following calculation: at iii to administer = (140% -at iii measured) x body weight. at iii was given every six hours for a period nf48 hours and on the 4th day. patients were followed up throughout their stay in the icu but at least 14 days. in this abstract the data of the first 10 patients are included. results: the patients' median injury severity score was 41 points with a median age of 42 years. the patients were equally distributed between verum and placebo groups with respact to age , iss, prehospital treatment, blood pressure, hemoglobin and at nmevel on admission, and time from the accident until the test solution was given. the patients of the at iii group received 18,100 units of the inhibitor on average during the first 4 days. with our regimen, the at iii levels could be kept between 125% and 140% of normal, whereas control patients an at hi concentration of 40% to 60% was observed. patients with at ni treatment required less red blood cells bur received the same amount of ffp and fluids. there were no differences with respect to platelet count, partial thromboplastin time, l~rothrombin time and prothrombim. we did not observe bleeding disorders or any other side effect with peak concetrations of up to 206% of normal. white blood count, pmn--elastase, interleukin 6 and 8 and c-reactive protein tended to be lower in the at iii group. there were no differences with respect to renal and hepatic function parameters but the duration od mechanical ventilation was shorter in the at ii1 group (8.5 vs. 15.8 days) conclusions: we conclude that our treatment protocol (a) was able to restore at iil levels to the desired range of 140%, (b) did not lead to coagulation disorders, (c) did not increase transfusion requirements, (d) did not reveal other undesired side effects, and (e) showed a tendency towards reduced posttranmatic inflammation and mechanical ventilation requirements department of trauma surgery, 45122 essen, germany antithrombin-lll (at-ill) acts as an inhibitor of thrombin, further proteases and the alternative pathway of the complement system. inhibiting thrombin, at-ill works as a functional antagonist of paf~ therefore, in state of hypevolemic-traumatic shock, continuous supranormal serum -and tissue -concentrations of at-ill may be efficient to reduce the local posttraumatic reactions resulting from complement and pmn-activation. 20 patients with severe multiple trauma (7 treatment [at-ill] --13 controls [c]) were investigated. study cdteda were age > 16 and < 65 years, injury severity (iss) > 30 points and glasgow-coma-scale > 8 points; randomization and treatment has to be started within 6 hours after trauma. permission for the clinical study was given by the local ethic committee. bradykinin (bk) and related kinins are potent inflammatory peptides which possess the ability to induce, vasodilation, increased vascular permeability and hyperalgesia. cp-0127, a novel homodimer bk antagonist has previously been shown to increase survival in rat and rabbit models of lethal endotoxin shock and is now in clinical trials for sepsis. we have now evaluated the effect of cp-0127 in other models of inflammation. male rats were precannulated with a catheter in the carotid artery. 24h later bk was injected ia and the pain score ranked from 0 (no responses) to 5 (vocalization). cp-0127 at 3.6 umoles/kg completely inhibited the pain responses for a period of 2.5 -3h. cp-0127 at 3.6 umoles/kg s.c. was also found to inhibit the increase in paw volume and hyperalgesia induced in rats over a 3-4h period by an intraplantar injection of 0.1% carrageenan. the abdominal constriction response 1o an intraperitoneal injection of kaolin was inhibited in a dose-dependent manner by cp-0127. when 50 ul of 0.5% formalin was injected into the paw of a mouse a characteristic licking response was observed which was biphasic in nature. cp-0127 significantly inhibited both the first (0-5min) and second (15-30 min) phase responses. ]n a rat burn model, where the hind paw is immersed in water at 55°c for 15 sec the increase in paw volume was significantly reduced by pretreatment with cp-0127, 3.6 umoles/kg s.c. finally cerebrai edema was induced in rats by applying cold (-78°c for 10 sec) to the dural surface following a craniectomy. cp-0127 at 3.6 umoles/kg s.c. produced a significant reduction in the amount of edema compared with sham controls 24 h later. these data suggest that bk is an important mediator of inflammation and hyperalgesia and that the bradykinin antagonist, cp-0127, may be useful in the treatment of such inflammatory, hyperalgesic disorders. partial hepatectomy in humans is associated with a considerable morbidity due to hemodynamic and metabolic derangements, which increase the risk for organ failure and mortality. we hypothesized that endotoxemia may play a pivotal role in these complications. we therefore, investigated whether peri-operative infusion of rbpi23, a recombinant protein of the human neutrophil bpi with bactericidal and endotoxin-binding capacity, could prevent postoperative derangements following partial hepatectomy. male wistar rats (230-250 g.) received a 70% liver resection (phx) or a sham operation (sh), and a continuous intravenous infusion of either 0.2 mg/kg/hr rbpi23 (phx-bpi, n=8; sh-bpi, n=7) or the (iso-electric, iso-kd) control protein thaumatin (phx-con, n=8; sh-con, n-8). various parameters were measured 4 h after the resection or sham operation. mean arterial pressure, cardiac output and heart rate were significantly decreased in phx-con rats compared with sh rats, which effects were not observed in phx rats treated with rbpi23. blood ph was significantly decreased in the phx-eon group, whereas the leucocyte count, hematocrite and il-6 levels were significantly increased compared to sham levels. in the phx-bpi group, these parameters were restored to near sham levels. in vitro experiments with rat plasma and human mononuclear cells (mncs) revealed that plasma of phx-con rats is highly capable of activating mncs, accompanied by the release of cytokines. this activation is attenuated with phx-bpi plasma. in vitro added acd14 or polymyxin b was able to reduce the activation by phx-con rat plasma to the levels of phx-bpi rats thus, these data suggest that systemic endctoxemia, possibly of gut origin, is a major cause of postoperative hemodynamic and metabolic derangements following phx and that rbpizz can prevent these changes. more recently we reported a transient appearance of both endotoxin and tnf in the circulation of rats subjected to the haemorrhagic shock (hs) already at 90 -120 rain. similar to bpi, recombinant bpi was found to bind lps and inhibit tnf formation in vitro. the aim of this study was to investigate the effects of rbpi21 (kindly provided by xoma corporation, berkeley, ca) against haemorrhage related endotoxemia and mortality in rats. method: a prolonged hs was induced by blood withdrawal to a mean arterial pressure of 30-35 mmhg for 180 rain followed by reinfusion of shed blood (sb) and resuscitation with two times of sb volume of ringer's lactate over 50 rain. rbplg. 1 was administered at a total dose of 5 mg/kg i.v. (2.0 mg/kg at the16-eginning followed by two doses of 1.5 mg/kg each at end of shock and the end of resuscitation). the control group was treated similar to the bpi group but received thaumatin as a protein control preparation at the same dose as rbpi21. results: imrffe?diately after resuscitation (230 min) the detected plasma endotoxin levels in the control group (mean = 61, range = 0-120 pg/ml) were almost neutralized by rbpi21 treatment (mean = 13, range = 0-53 pg/ml) . plasma tnf levyis were not significantly influenced by rbpi treatment at the two time points 230 and 320 min of experiment (means: 65 and 51 in bpi vs 49, 65 pg/ml in the control group). the 48-hour survival rate was improved from 6/16 (37.5 %) in the control to 11/16 (69 %). conclusion: these data suggest that haemorrhagic shock may lead to bacterial translocation and/or transient endotoxemia with concomitant cytokine formation that may play an important role in the pathogenesis after shock and trauma, rbpi21 might be a useful therapeutic agent against endogenous bacterfal/endotoxin related disorders in hemorrhagic shock. morbidity and mortality after hypoxia of the vital organs had been correlated to the production of oxygen radicle which is mediated by xanthine oxidase activity, in this study we have evaluated the survival rate after allopurinol. 42 rabbits weighed 900 + 200 grams divided into two groups. group i included 22 tabbits were treated with allopurinol 80 mg/kg for seven days before induction of haemorrhage. group ii as a control included 20 rabbits. all rabbits were subjected to 25% arterial blood loss through the central ear artery for one hour then resusciatation was done by the heparinized withdrawn blood through a marginal ear vein. during the experiment blood pressure and heart rate were monitored through the central ear artery. also uric acid, lactic acid, glutathione activity were estimated. animal survival was followed for 10 days. postmortem vital organ histochemistry and histopathology examinations were done. in group i the survival after three days was 10 out of 22 while in group ii it was two out of 20. our conc|usion, allopurinol had increased the survival in aiiopurinol pretreated rabbits which may indicate the value of allopurinol premedication for patient prepared for elective bloody surgical intervention . h2 receptor antagonists are commonly used for stress ulcer prophylaxis, but their actions on the septic response are largely unknown, in an experimental model, 26 pigs were first anesthetized, then injured with 100 joules of energy to the posterior thigh, then hemorrhaged 30-40% of their blood volume. after i hr of shock, all the shed blood plus 2x the hemorrhage volume as lactated ringers was infused. following resuscitation, ranitidine (1.5 mg/kg iv twice daily) or saline placebo was begun. the treatment group was randomly assigned in a blinded fashion. after 72 hrs, a septic challenge was administered (15 bg/kg of e. coil endotoxin (lps)). serial gastroscopy, gastric ph, hemodynamics, abg's, physiologic dead space ventilation, leukocyte counts, and tumor necrosis factor (tnf) levels were recorded for 180 min. baseline values and units were cardiac index 102 _+ 9 ml/min/kg (ci), arterial po2 228 + 11 mmhg(pao2), base excess 6.5 -+ 1 meq (be), physiologic dead space fraction 23 +_ 4% (pds), and tnf 0.4 + 0.1 units/ml. baseline gastric ph was 3.9 -+ 0.4 and 4.8 _+ 0.5 in the placebo and ranitidine groups, respectively. the gastritis following hemorrhage was marginally attenuated in the ranitidine group. following lps infusion the following were obtained: ci pao2* be* gastric* pds* peak* 120 rain 60 rain 120 rain ph 120 min tnf ranitidine 167_+17 118_+15 -4.4±1. bum injury results in hypermetabolism, fever and nitrogen wasting. endotoxin (lps) has been proposed to mediate these effects, either directly or via activation of macrophages to produce cytokines such as interleukin-6 (ii-6). this study was designed to clarify the role of lps and 11-6 in the metabolic response to bum injury. twenty-five burn patients (49 -+ 17%; 16 + 15% ft bsa burn; 32 _+ 16 years old) were studied serially for three weeks post bum. patients underwent partitional calorimetry to assess metabolic rate and compartmented heat loss. nitrogen was assayed using chemiluminescence. lps and i1-6 were measured with limulus amebocyte lysate assay and elisa. patients were excluded if they suffered smoke inhalation, showed any sign of sepsis or failed to rapidly meet their nutritional needs via the enteral route. ten patients received intravenous polymixin b (5,000 u/kg/day to bind lps). these patients did not differ for the remainder. all patients were hypermetabolic and febrile in proportion to the size of their bum wound but were not endotoxemic (3.1 +_ 5.6 pg/ml; normal <40 pg/ml). i1-6 did demonstrate a significant correlation with cole temperature (tr~ = 37.6 + 0.21ogi1-6, p=0.04) and with nitrogen excretion (nou t = -5.2 4-0.091ogi1-6 + 0.14tr, p=0.01). administration of polymixin b had no effect on metabolic rate, temperature or i1-6 levels but did reduce nitrogen excretion resulting in more positive nitrogen balance (0.t grn/day vs. -0.1 gm/day, p=0.04). although bum injury does not produce an obligatory endotoxemia, i1-6 does appear to play a role in the fever and nitrogen wasting seen with such injuries. the effect ofpolymixin b on nitrogen excretion suggests that lps may play a role either locally or in the portal system. introduction: there is substantial evidence that release of inflammatory mediators by activated kupffer cells contribute to the course of a systemic inflammatory process, e.g. after shock or lrauma. besides the systemic effects of mediators such as tnf, paf or interleukines, local actions on hepatic microvasculature and hepatic inflammatory response have to be considered. our aim was to assess the role of tnf and paf by blocking their effects using anti-tnf monoclonal antibody, pentoxifylline and a paf antagonist. methnds: in anesthetized sprd-rats, hemorrhagic shock was induced by withdrawl of arterial blood within 5 rain and shock state was hold for 1 h at a map of 40 mm hg (cardiac output of 50 %). following adequate resuscitation with 60% of shed blood and twice of this volume as ringer's solntion, animals recovered to map >1130 mm hg and co >120%. hepatic microcirculation and sinusoidal leukocyte-endothelium interactions were examined by intravital epi-fluorescence microscopy at 1, 3, or 5 hours after resuscitation. in a blinded fashion, a rat-specific monoclonal anti-tnf antibody [2 mg/kg, celltech, uk) , pentoxffylline (ptx, 50 mg/kg, hoechst, d), and a paf antagonist (web 2086, boehringer, ingh., d) were given either as pretreatment or at the time of resuscitation (n=6-8 group bolla. k*., duchateau, j., hajos, gy., mbzes, t., hern~di, f. prevention of temporary/secondary immune deficiencies or reduction of their severity and/or duration as well as the reduction of the perifocal inflammatory processes belong to the rational targets of posttraumatic/pedsurgical medication. such a targeted medication can result in less frequently occurring nosocomial infections, and in reducing the duration of the intensive care and convalescence period. the results of in vitro studies performed with the amino acid sequence 32-35 of thymopoietin, i.e., with thymocartin in whole blood and peripheral mono-nuclear celi(pbnc) cultures clearly show some characteristic effects of this immunomodulator. preincubation with the tetrapeptide significantly (p<o.ol) and concentration-dependent reduced the endotoxin(lps)-induced tnfalfa production from 95,7 to about 20 pglml, but did not abolish it. the inhibition of the lps(0.1 ug/ml)-induced tnf production occured already in presence of 0,1 pg peptide/ml and peaked at the concentration of 0,01 ng/ml. higher concentrations did not increase this effect. in contrast to the other two small thymic peptides (tp-3 and tp-5), thymocartin did not enhanced the pwm-induced pge 2 production in pbmc cultures up to a concentration of 1 ng/ml, inclusive. the selection of thymocartin (tp-4) for perisurgical medication and/or for treatment of trauma-induced temporary immune depression has been strongly supported also by targeted animal experiments. thus, mortality of about 90% registered in mice infected with pseudomonas aeruginosa after bum injury could be reduced with the tetrapeptide treatment to a level which did not differ significantly from that of the infected non-burned controls. moreover, observations gathered first of all with the inflammatory model of air poach/croton oil grenuloma (selye) clearly showed, that a very definite, antiexudative effect can be achieved with this thymic peptide. as to this effect, thymocartin has been confirmed, e.g., to be equipotent with locally administered fluocinolone acetonide and/or s.c. injected prednisolone. the interim results of two explorative double-blind clinical studies (involving more than 260 patients after polytrauma and hip-fracture untill now, respectively) correspond to the expectations. the treatment significantly reduced the occurence of nosocomial infections, the days spend in intensive care and on antibiotics as well as it shortened the bed-out time and reduced the additional medication. objective of this prospective randomized study was to further delineate the mechanisms leading to these alterations and to investigate the effects of immunomodulatory intervention. patients and methods: 20 patients (pts) formed control group a. 20 group b pts received 3x50 mg indomethacin (indo) from the first (dl) to the fourth postoperative day (d5) intravenously to block prostaglandin e2induced suppression of cmi response as well as 50 mg thymopentin (tp-5) subcutaneously preop., on d2 and d4 to augment cmi reactivity. in vitro investigations preoperatively, on dl and d7 included measurements of the percentage of cd4+ t-helper (th) cells, pbytohemagglutinin (pha)induced synthesis of interleukin (il)-2 as one cytokine primarily produced by thl-cells, and pha-induced synthesis of il-6 as one cytokine primarily produced by th2-cells. delayed type hypersensitivity (dth) skin response to an antigen skin test battery and specific antibody (ab) production following tetanus vaccination preoperatively and on d7 were used as in vivo tests for thl-and th2-induced immune response respectively. results: postoperatively, group a pts showed a persistent significant reduction of cd4+ th cells, il-2/il-6 ratio, and dth skin response as compared to baseline values (bl), while ab production increased (p<0.05 vs.bl). in group b pts no change in cd4+ th cells, il-2/il-6 ratio, or dth skin response was observed (p<0.05 vs.group a), and postoperative ab production increased significantly (n.s. vs. group a). conclusions: 1.these results clearly indicate a significant suppression of th1 induced cmi response, while th2 induced response remains normal following ohs. 2.these alterations may gain clinical significance whenever a postoperative infection requires a th1 response and may explain the susceptibility to opportunistic microorganisms observed in pts after ohs. 3 the aim of this study was to find out whether the establishment of administration of thymostimulin (tp-i) in jaundiced and anergic rats would return the rats to the state of immunocompetence. material and methods. male wistar rats weighing 250-300 g were injected with haemocyanin (klh). all rats with a positive response were included in the study and we evalu ated the t cell subpopulations and il-2. they underwent laparotomy and the common duct was ligated and divided. those rats whic become jaundiced and anergic one week after the operation, were divided in two groups: control group ( objective of study. the goal of this study was to prove the necessity of thymus derived immunomodulators treatment in acute period of infantine sepsis, materials and methods. immune status was investigated in 11o infants. clinical conditions of children were defined by syndrome of multiple polyorgan]c insufficiency. the heaviness nf condition was estimated as the sum of points according to efforts needed to restore of basic living functions -respiration, hemodynamjcs, hemocoagulatlon, metabolism, functions of liver and kidneys. it was expressed in form of clinic condition classes from 1 to 4. results.it was found that in 100% of infants with 3-4 classes of heaviness immunode[iciency took place simultaneously with ii and iii stages of hemocoagulattve syndrome. amounts of t cells and t helpers were decreased more then 2 times, ratio th/ts was reduced to 1.5 or lower. concentration of lgg and igawas reduced almost a hull in comparison to control. phagocytic and metabolic activities nf neutrophils were depressed. complement system state was changed: the functional activity of the alternative pathway factors b, d and the level or c3a subcomponent were increased but the level of c2, c3,, c4 and c5 components of the classic pathway were decreased. during the development of coagnlopathy the direct correlation between chso, levels of plasminogen and antithrombine iii was found. in greater extent the function of immune system was disodered in accordance with 2 point metabolic violations and hemocoagulative disturbances considered as disseminated inmtravaseular clotting syndrome on ii or ili stages. conclusions. the rehabilitation of the immune system functions was promoted by taetivine and thymaline in doses of i-3 mcg/kg per day and 2 mg/kg per day accordingly during a week in children with 3 -4 classes el heaviness. aiter 11-14 days tactivine had advantage. when eoagulopathy took place thynaaljne promoted restoration of (1-3)-~-d-glucan (bgc) is a major cell wall compornent of pathogenic fungi and shows many immunopharmacological activities on experimental animals. lps is also derived from gram-negative bacteria and have in~ttnomodulating activities on immune systems positively or negatively. because of difficulty to cure contaminating lps from bgc preparations, it is very hard to evaluate the exact activities of bgc. in this study, we compared the immunological activities of lps ans highly purified bgc on murine system. materials and methods: c3h/hen or c3h/hej mice, 6 to 12 wkolds of both sexes were used through experiments. highly purified (1-3 )-{5-d-glucan (gurdran; bgc)was courteous gift from seikagaku kogyo co. tokyo, japan and resolved in endotoxin-free ultra-pure water. escherichia cell lps was purchased from sigma chemicals, usa. mitogenic activities of bgc or lps on splenic cells were measured by mit dye assay. in vitro activation of peritoneal exudate macrophage (pen) have moniotored by phagocytosis and intraphagocytic killing of pseudomonas aeruginosa (z~b-139. induction of cytokine productions from pem or spleen cells induced by bgc or lps were detected by cytotoxic assay. results and (bnclnsions: i ) in vitro cultures of pem with bgc have induced enhanced phagocytic and bactecidal activities in c3h/hen and c3h/hej mice, whereas lps only induced such activities in c3h/hen mice. most effective dose of bgc was 10 ug/ml. 2) bgc-dependent proliferative response of c3h/hen splenic cells was not detected under the culture condition used. 3) induction of tnf~ prodction was apparently observed in bgc-stimulated c3h/hej pd4 cultures, but not in lps stimulated one. these findings indicate that activation mechanisms of pr94 and splenic cells by bgc are different from that by lps. to investigate the bsc-induced p~4 activation, intracellular signaling pathways of p~4 by bgc-stimulation are under consideration. yamagami k, uedono y, kawakami k, kitazawa y and tanaka t according to the latest reports of use of il-2 in a burned-sepsis model, the dose was too high to adjust for human therapy. adoptive transfer of l~ymphokine-activated killer (lak) cells has been demonstrated as a means of enhancing therapy in malignant tumors. we therefore attempt to use lak therapy on burned-infected mice instead of il-2. under anesthesia, 8-weekold male c3h-hej mice were subjected to a 20% scald or sham burn and then resuscitated. sham burned mice served as controls. scald burn mice were subjected to peritonitis by intraperitoneal innoculation of 105 organisms of p. aeruginosa 24 hr after burn. burned-infected-mice were divided into two groups. one group had no further treatment, and the other group received lak cells (2x105) intraperitonelly after septic challenge. the mice were scored for survival daily. on day 7 after burn, using moabs dual-color flow cytometry, we studied lymphocyte expression in mice with use of blood and spleen. simultaneousely we studied the alteration of il-1 and il-6 in their serum using immunoassy kits. sham burned animals had no mortality. the mortality of the lak-treated mice was significantly reduced when compared with that of the burned-infected mice. the most consistent changes observed were depressions in percentages of thyl, 2 positive cells and a remarkable depression of nk cells in spleen. after lak cell treatment, those two percentages of cells significantly increased. all the data of assay of ill and [l 6 were not detected on sham burn mice serum. due to burn and septic challenge the levels of illand il 6 (n=6) were raised to 45.9_+17.4 and 3866_+1045 pg/ml, while the levels in lak treated mice were reduced to 4.23_+2.37 and 437_+110 pg/ml, respectively. the results reported here demonstrate that lak therapy is able to improve cell-mediated immunity in burned-infected mice. this is associated with a significantly improved survival rate, since ill has been demonstrated to mediate immune and inflammatory responses. also a cause effect relationship between elevated endetoxin levels and increases of [l6 has been recently established. the aim of our present study was to investigate the effect of parenteral indomethacin on the immune system of patients under major operations. our study included 20 operated patients, 10 of which received 100mg indomethacin before and 1,2 and 3 days after surgery (group a) and 10 patients received no antiinflammatory therapy (group b). we measured total t-cells helper (cd4), suppressor (cd8), nk-cells and interleukin-1 and tnf production by pha or endodoxin (lps) stimulated peripheral blood mononuclear cells at day 0 and 1, 3 and 7 days after operation. in group a we detected a significant (p<0.05) increase in cd4/cd8 ratio on day 3 while no differences were ~oted in nk-cells or cytokine production in both groups (table 1) . it seems, therefore, that parenterai indomethacin may have a benefitial effect on the immune system of patients under major operations by increasing the t-helper /t-suppressor cell ratio while having no effect on the production of cytokines by peripheral blood mononuclear cells. this study was undertaken td dete~nnine the role of prostaglandin synthesis inhibitor on survival post traulna sepsis. analysis of the effects of prostaglandin synthesis inhibitor on survival of four groups of 15 mice each were made after laparatomy and intravenous administration of live e.coli. post trauma sepsis, group a received no treatment; group b received antibiotics im; group c received prostaglandin synthesis inhibitor im and group d received both antibiotics and prostaglandin synthesis inhibitor im. survival time was longest in group treated with prostaglandin synthesis inhibitor and antibiotics post trauma sepsis. the singular use of either antibiotic or prostaglandin synthesis inhibitor did not alter the outcome on survival. mortality rate was lowest in the group treated with combined prostaglandin synthesis inhibitor and antibiotics. use of this combination showed a reduction in bacterial growth in peritoneal and blood samples, mild morphologic changes secondary to sepsis in the heart, lungs, liver, kidney and stomach and marked enhancement of mean level of activity. the study indicates that addition of prostaglandin synthesis inhibitor in treatment of trauma-sepsis has clear beneficial effects. interferon-? (ifn-y) is a potent immunostimulant which has been shown to be detrimental when administered during septic shock. blockade of this cytokine however is beneficial during the acute septic response. the aim of this study was to evaluate the cellular effects of this cytokine and its blocking monoclonal antibody (moab) in lethal sepsis following injury. 100 female cd-1 mice were randomized into two groups: septic=lps vs injury=hind limb amputation (hla vs igg. nstats= anova=p<0.05 vs lgg ifn-y was detrimental when given to control septic animals. however its blocking moab was protective (p<0.05). conversely ifn-y was proteetive in injured septic animals compared with anti-ifn-? (p<0.05). these findings demonstrate that the immune stimulant ifn-? has therapeutic potential in the immunosuppressed injured host predisposed to sepsis, while blockade of this cytokine is required for protection in the septic host with a normal immune response. dept of surgery, rcsi, beaumont hospital, dublin 9, ireland. oxidants play a role in physiology. the potential noxious oxidant biochemistry is restrained by chemically distinct antioxidants. these antioxidants, which may reside in different cellular compartments, can act synergistically. in normal physiology an oxidant/antioxidant bai&nce exists. unbalance in favour of the oxidants is called oxidative stress. oxidative stress has been implicated in many pathologies including lung diseases(e.g, doxorubicin induced cardiotoxicity), ischemia/reperfnsion damage and central nervous system trauma. transition metals such as iron are involved in the early events leading to oxidative damage in these pathologies. this has been underlined by the finding that postischemic cns pathology closely resembles that caused by a chemical oxidative insult (e.g. iron microinjection). moreover, a protective effect of oxygen-radical scavening agents or compounds that inhibit lipid peroxidation (antioxidants) in brain ischemia/trauma is apparent. some known drugs (e.g. anti-arrhythmics or histamine h2-antagonists may act as non-specific antioxidants. howe~er, recently, interesting new membrancespecific antioxidants (e.g. 21-aminosterdids) have been designed. subt&e effects on iron redox chemistry contributes to the potent antioxidant activity of these 21aminosteroids. since a few years, one area that greeted enthusimastlcally in clinical medicine is that of reoxygenatlon injury or ischemia-repeffusion, a phenomenon accepted to make a majo:" contribution to organ dysfunction in trauma, shock and sepsis through the formation o( oxygenated free radical species. however, thei detection in vivo always remains a difficult challenge. efforts have been made recently to directly establish the formation of free radicals in biological samples as complex as blood, plasma or tissue with the use of electron spin resonance (esr) spectroscopy. using spin trapping agents, the presence of reactive carbon-and oxygen-centered radicals has been demonstrated in animals blood submitted to heart, renal and intestinal ischemia-repeffusion or to liver transplantation. recently, the in vivo formation of free radicals in the cerebra; extracellnlar fluid during cerebral isehemia-repeffusion has been assessed by the spin trapping technique coupled to brain microdialysis. esr investigations have also been conducted to detect endotoxin-induced free radical generation in rat or baboon liver and oxygen free radicals (ofr), produced both at intra-and extra-cellular levels, seem to play a major role in the pathophysiology of tissue injury and organ dysfunction in sepsis, through induction of lipid pemxidation in cell membranes. oxidative damage can result both to an ofr-overpmduction or to a depletion of endogenous antioxidant defenses, which are represented by scavenger enzymes (e.g. sod), hydrosoluble and liposoluble antioxidants (e.g. ascorbate, vitamin e), and other mechanisms such as metal-ion sequestration. in animal models, oxidative damage has been proved by detection of increased levels of lipoperoxidative products, and a depletion of antioxidant defenses was found both in plasma and tissues. in the few clinical studies a similar results have been reported, and oxidative damage appears to correlate with dic, with organ dysfunction and with red blood cell deformability. in a group of critically ill septic patients we found increased levels of conjugated dienes (cd) (bioproducts of lipoperoxidation) and decreased plasma levels of alpha-tocopherol and coenzyme qlo (coqi0)(endogenous liposoluble antioxidants). a relationship between cd and uric acid (p<0.004) was found, may be related to xantine-dehydmgenase to xantine-oxidase conversion. antioxidant depletion is due both to overconsumption and to a nutritional deficiency, even if a reduced synthesis can also be present. in fact one more relationship between coqi0 and plasma cholesterol (p<0.0005) demonstrates the commun hepatic biosynthetic defect. is there any role for antioxidative therapy in sepsis? can it achives a significant improvement in the septic course, organ dysfunction, and final outcome? several antioxidant drugs, have been evaluated in experimental and clinical sepsis: scavenger enzymes, natural antioxidants, alloporinol, 21-aminosteroids or lazaroids, have been proved to reduce lipopemxidative damage, to protect organ dysfunction, and in some cases to improve survival. several questions must be addressed in evaluating safety and utility of antioxidative therapy. more specific and standardized methods must be applied to detect and quantify the lipoperoxidative damage. antioxidant drags must act selectively on ofr-production, without interference with other than that are beneficial for the organism. antioxidants must be able to achive in adequate amount the tissue or cellular compartment where their action is required. many antioxidants have also a pro-oxidative effect which can he detrimental in some conditions. the timing of administration, the dosage used and the association wih others antioxidant drugs must be defined. nutrition plays an important role as antioxidative therapy, since it supplies all compounds needed to maintain adequately levels of endogenous antioxidant or to support their synthesis. thiobarbituric acid reactive substances (tbars) concentration in serum has been determined in a large population of healthy subjects and in patients suffering acute hepatitis and chronic cases of hepatitis c, as well as several other processes. the correlation with different physiological and clinical parameters in these populations is also presented. treatment with interferon of the chronic active hepatitis c patients, 5x10 u three times a week during two months, led in those patients whose sgpt activity normalized in serum, to a concomitant decrease in serum tbars content. the possible theoretical involvement of peroxidation and antioxidants in this beneficial effect of i~terferon in hepatitis c patients is discussed. the results presented confirm the value of tbars as laboratory test in the management of disease and as a useful tool for the study of pathogenic and/or therapeutic mechanisms. 4-hydroxyalkenals are among the different substances measured by the tbars assay. these compounds show several biological effects that have been demonstrated mainly in different experimental models. we have studied the capacity of different tissues to conjugate these compounds patients surviving the initial subarachnoid hemorrhage (sah) from a ruptured in~raeranial aneurysm are prone to develop cerebra] ischemia from vasospasm which is associated with significant morbidity and mortality. among the many treatments that have been prol~osed to treat this condition, none has been shown to be satisfactorily effective. recently, a new drug, namely the 21-aminosteroid tirilazadmesylats, has been studied in a large, prospective, multicentor trial for its effectiveness to improve the outcome of patients with aneurysma! said. the study was conducted in europe and australia in 41 neurosurgical centers and included 1023 patients. all patients received presently accepted standard treatment, including nimodipine. patients were randomized to four different groups: placebo and 8 groups of tirilazad in escalating dosage (0.6 -6 mg/kg/day). the clinical outcome was compared for these 4 groups and cerebral a~giography was performed in 75 % of patients on day 7 -11 follovang sah, to study the effect 0f'the dflf~ off cerebral vasospasm. the study reached its planned endpoint 6 months ago. preliminary results ~how a significant improvement with regardto mortality in those patients receiving the highest tirilazad dose as compared to placebo and the two lower dose groups. additional beneficial effects were seen in the 6 rag/day groups with regard to the occurence of symptomatic vasospasm and good clinical outcome. although the final analysis of all data is still pending, these results suggest that tirilazad-mesylate could represent a major improvement for the treatment of patients with sail. experimentally lipidperoxidation products (lpo) are increased in acute pancreatitis (ap) due to oxygen radicals (or). the purpose of this clinical study was to determine the antioxidant status and lpo in patients suffering from ap and to evaluate the effect of antioxidant treatment with sodium selenite (se) thus improving the function of the se dependant glutathione peroxidase which is the major detoxifying system for or. materials and methods: in z0 patients sufferin s from severe ap (c-reactlve protein >120 me/l) we determined on day 1 and day 8 after admission the lpo ma!ondialdehyd (mda), conjugated dishes (cd), reduced (gsr) and oxidized (gssg) glutathione, the vitamins a,c,e and se. moreover the patients were evaluated clinically using the ranson and the apache ii score. i0 patients were randomly treated with 600 ug/day of se for 8 days. results: all patients suffered from a severe depletion of antioxidants,especially a low concentration of se (only 1/3 of normal). thereby the increase in lpo correlated with the clinical course. during se treatment lpo decreased and the levels of antioxidant vitamins improved. se had no influence on leth-slity the lenl or the chan in rs or ap ii. background: since reperfusion injury occurs when oxygen is reintroduced into ischemic tissue, the ideal timing for administration of therapeutic compounds aimed at ameliorating oxygen radical mediated injury is at the time of initial fluid resuscitation. currently used colloid or crystalloid preparations do not provide optimal, or even significant, anti-oxidant protection. systemic iron chelation affords protection against the iron catalyzed components of oxygen and lipid radical mediated tissue injury. the conjugate resulting from chemical attachment of the clinically approved iron chelator, deferoxamine (dfo, desferal ®, ciba), to hydroxyethyl starch (hes) represents a novel approach to colloid based fluid resuscitation. hes-dfo contains 85% hes and 15% chemically bound dfo. the polymer-drug conjugate has a lower molecular weight than that of hes in order to allow more rapid excretion. results: preclinical and initial clinical trials indicate that hes-dfo is well tolerated, even at high doses. in animal studies, fluid resuscitation with hes-dfo does not significantly improve central hemodynamic recovery beyond that observed with hes, but hes-dfo seems to afford better protection of microcirculation in organs at risk (lung, liver and gut), possibly by decreasing neutrophil sequestration. in a burn model, total fluid requirements are lower and oxygen utilization higher in hes-dfo treated animals compared to hes controls, suggesting decreased vascular leak and improved tissue perfusion. conclusion: hes-dfo represents a means by which potent antioxidant protection can be administered at resuscitation. iron has been suggested to play a pivotal role in oxygen flee radical mediated tissue injury. in vitro experiments indicated its critical role as a katalyst in hydroxyl free radical generation 0fenton-reaction). since iron chelator deferoxamine administered in shock alone demonstrated severe side effects, a hydroxyethylstarch (hes)daferoxamine (dfo)-conjugute was used to modulate oxygen free radical injury during the ischemia/reperfi~ion syndrome induced by hemorrhagic shock. methods. female lewis rats (190-215 g, n>6; pentobarbital anesthesia 50 mgjkg), in hemorrhagic shock ( the aim of the study was to elucidate (1) whether the generation of or would affect lung and kidneys as primary shock organs in the very early phase of sepsis and (2) whether dfo-hes could prevent this tissue damage. methods: in 67 rats sepsis was induced by cecal ligation puncture (clp) peritonitis. the animals were randomly assessed to 2 groups: one group was treated with 3 ml dfo-hes (100 mg/kg iv), the other rats received solely 3 ml of the carrier starch solution. 30, 60, 120, and 240 min after induction of sepsis respectively, the animals were sacrificed, the organs collected, and tissue contents of glutathione (gsh), malondialdehyde (mda), myeloperoxidase (mpo) and conjugated dienes (cd) determined. plasma samples were obtained for analyses of endotoxin (chromogenic lal test). blood pressure (map) was measured via a carotid artery catheter. results: clp caused sepsis with high (> 1.84 eu/ml) endotoxin levels. map in both groups decreased slightly but significantly during sepsis regardless any treatment. in the lungs mpo concentration was increased (p<0.05) in the lies group already 30 min after sepsis induction. concomitantly, tissue gsh level decreased and lipid peroxidation was pronounced as shown by elevated mda and cd levels. dfo-hes diminished tissue pmn accumulation and mpo concentration. moreover, at each time point lung mda and cd levels were lower (p<0.02). histomorphological examination showed marked micro-atelectases, destruction of the alveolar septa, and splicing of the basal membranes in the lies group. in contrast, in dfo-hes treated rats the alveoli remained well-ventiiated and only some enlarged reticular fibers without splicing were observed. almost similar results were found for the kidneys. mpo levels differed neither within nor between both groups. the slight decrease in gsh levels seen after 60 min in the dfo-hes group seems to demonstrate an oxidative stress to a lesser degree. the most impressive effect of iron chelation, however, was revealed by the lipid peroxidation products. at each time point, mda and cd levels were lower (p<0.02) compared to the hes group. light and electron microscopic examination disclosed tubulotoxic and mitochondriat damages while dfo-hes lxeatment prevented that alterations. conclusion: both the biochemical and histological results of this study reveal an early and remarkable generation of or in peritonitis-induced sepsis. thereby, these or obviously cause pulmonary and renal tissue damages, intravenous application of dfo-hes may, however, benefit by preventing early lipid peroxidation of the tissue. the proteolytic irreversible conversion of xanthine dehydrogenase (xd) to xanthine oxidase (xo) is triggered by calcium flux. the aim of our study is to clarify ~he link between intracellular ca2+ levels and xo activity determined by uric acid release, and to evaluate the efficacy of verapamil, on the generation of hydrogen peroxide associated with reperfusion by assaying lactate & pyruva~e release and the levels of cytosolic free nad /nadh ratio. experimental protocol consisted of :(a) non ischemic/reperfused experiment in which normal cardiac slices of rats were perfusated with oxygenated kreb's ringer phosphate buffer containing glucose (150mg%) and bovine albumine (5gm%) for 60min at 37°c.it composed of 3 groups, group aa (control group), and groups ab & ac (perfusate supplemented with verapamil in the dose of loo&200 mi% respectively). (b) ischemic reperfused experiment in which ischemic cardiac slices were obtained from rats subjected to 15 min ~aemorrhage.lt was also divided into two groups; group ba and bb (verapam~/ 200mi% added to perfusate}. verapamil stimulated uric acid release from normal rat cardiac slices were 267% in group ab and 767% in group ac(dose related). rates of uric acid release is enhanced by verapamil in group bb. moreover, rates of uric acid release in groups ac & bb are insignificant. in verapmil added groups (group ab, ac & bb), increase uric acid release is associated with an enhancement in pyrurate release and with increase levels of cytosolic free nad+/nadh ratio, although it is not evident ~ ischemic group (group ba).it is concluded that the conversion of xd to xo is calcium independent. eicosanoids like thromboxane a2, leukotriene b4 and leukotriene c4 are known as promoters of initial inflammatory reactions. we investigated whether oxygen radicals (or) are able to induce a release of these eicosanoids in whole blood. blood from healthy volunteers was incubated with xanthine oxidase/hypoxanthine to generate oxygen radicals. after 0,5,15,30 and 60 minutes plasma levels of thromboxane b2 (txb2), leukotriene b4 (ltb4) and leukotriene c4 (ltc4) were determined via elisa technique. another volunteer had taken 1000mg aspirin one day before taking the blood sample (no7). results: txb2 plasma levels increased from 14pg/ml at 0min to 144pg/ml, 350pg/ml, 370pg/ml and 660pg/ml at 5,15,30 and 60min (p<0,01) . ltb4 and ltc4 plasma levels showed an increase during the first few minutes (ltb4: 0min: llpg/ml,5min: 87pg/ml; ltc4: 0min: 23pg/ml, 5min: 97pg/ml (p<0,05)) followed by a decrease to normal values at 15min. in the sample no7 the cyclooxigenase-pathway was completely inhibited, the txb2 plasma-levels did not alter at all, whereas ltb4 and ltc4-plasma levels weren't affected. opallogeneic blood transfusion jane shelby, ph.d., and edward w, nelson, m.d, there have been numerous investigations dudng the last two decades examining the effect of surgery, anesthesia, blood loss and transfusion on vadous immune parameters in humans and animal models. there appears to be concurrence among several well controlled studies that transfusion of whole blood (containing leukocytes), has regulatory effects on immune ceil function which include decreased cell mediated immune response, and inhibition of il-2 secretion. these effects occur following transfusion alone and in con.cart with the distinct immune effects of surgery, trauma and anesthesla, the clinical consequences of this immune modulation by transfusion include decreased allogeneic response to transplanted organs, which has been exploited clinicelly in renal transplant patients. additionally, there is evidence for a strong association with increased risk for infection in transfused patients following surgical procedures. aiiogeneio blood transfusions have been shown to inhibit cellular anti.bacterial mechanisms, causing increased susceptibility to bacterial pathogens, in humans and in animal models. there is also concern that allog~neic transfusion may adversely affect cancer patients, resulting in decreased disease-free survival. several stategies have been proposed to minimize the adverse effects of blood transfusion. there is evidence that the risk of immune mediated infectious complications associated with transfusion may be greatly minimized wlth the use of autologous blood and leukocyte free allogeneic blood.products in surgical and trauma patients, it also appears that the inhibition of cellular immune response and il-2 productiorl following atlogeneic blood transfusion may be mediated by increased prostaglandin e2 secretion, and that immune response may be preserved in allogeneio whole blood transfused subjects receiving c3lc~oxygenase inhibitors such as ibuprofen. among these are various alterations in immune function. efforts have therefore been made to utilize alternatives to homologous transfusions. these include the use of autologous predonation, supplemental iron therapy, and recombinant human erythropoietin. although initially considered innocuous, these therapies are now recognized to have potential deliterious immune sequelae. erythropoietin, by its ability to lower serum iron levels, can impair both lymphocyte and nk cell activity. autologous donation impairs nk cell function. finally, supplemental iron therapy can stimulate bacterial growth and increase the rate of infectious complications. this talk will present a discussion of these factors as well as a weighting of their importance. r.l rutan, rn;bsn, shriners burns institute and the university of texas medical branch, galveston tx, usa the serious sequelae of homologous blood transfusions have resulted in vigorous efforts at identifying alternate therapies for correcting red blood cell (rbc) deficits. erythropoietin (epo) was hypothesized to exist in the early 20th century, however the protein was not isolaled until 1977. the human gene was identified and cloned in 1983, which permitted the production of epo through recombinant techniques. the earliest clinical trials were performed in anemic end-stage renal failure palients on hemodialysis. treated patients experienced increases in erythropoiesis with normalization of hematocrit and hemoglobin levels, cessation of lrans-fusion requirements and improvement in general wellbeing. these studies, however, identified side effects of epo treatment such as hypertension, seizures and ee deficiency. volunteer trials have established that the hypertension is not a direct pressor effect but rather the result of abnormally rapid increases in red cell mass in the face of the incompetent volume-controlling mechanisms of the end stage renal failure patient. lower doses of epo and the subsequent gradual increases in red cell mass are associated with significantly lower incidences of hypertensive complications of epo therapy. likewise, seizure activity is not the result of a direct epileptogenie effect but parallels the incidence of hyper-tensive-related sequelae during high.dose epo treatment. in cross-over designed studies, pre-existing iron deficiency has been demonstrated to decrease or negate stimulated erythropoiesis but effective-hess can be restored with appropriate fe supplementation. exogenous epo is effective whether given by iv or sq routes and dose response curves do not vary with route of administration. increases in rbc mass are directly related to the dose of epo, both in amount and frequency of administration although there is a 3-5 day time lag between the first epo dose and laboratory indications of its action (i.e. increase in the number of reticulceytes in peripheral wood). epo is currently labelled for use in the treatment of anemias associated with end-stage renal disease and aids. however, its use in the surgical population has been explored because of its unique direct dose-response, epo has been used to effectively increase the blood harvest amounls in autologous pre-donation, significantly increase hematocrils in children following thermal trauma and successfully increase red blood cell mass following essential surgical procedures in patients with religious aversion to transfusion. by blood transfusion in colorectal cancer surgery mm heiss md, ch delanoff md, r stets md, j hofinann, e faist md, kw jauch md, fw schildberg md allogeneic blood transfusions are associated with an increased risk for postoperative infections in colorectal surgery when compared with autologous blood transfusions. attribution of this effect to immunomodulation was suspected in our previous study (lancet 1993; 342:1328-33) . task of the recent investigations was to analyze which specific effector systems were affected in-vivo by this transfusion-associated modulation. for global in-viva assessment of cell-mediated immunity (cmi) multiple recall skin-reactions were applied prior and post-operative. the specific humoral immune mechanisms were investigated by applying tetanus-toxoid one day preoperatively and deterimnating the quantitative igg-response. for indication of macrophage stimulation in-vivo tnf-levels were determinated by bioassay. dth-responses were significantly suppressed (p<0.05) in patients receiving allogeneic blood (n=36) or operated without blood transfusions (n=17). dthresponses were not suppressed and tendentiously increased in patients with autologous blood transfusions (n=24). in contrast, specific igg-levels increased sigmficantly (p<0.05) in patients receiving allogeneie blood (from 1.6 + 4.4 to 9.7 _+ 18.4 ie/ml) whereas in patients receiving autologous blood a smaller increase (from 1.9 + 4.1 to 4.0 + 6.7; p=0.068) was observed. tnflevels demonstrated a similar pattern with a higher increase in patients receiving allogeneic transfusions (l 1.4 + 12.0 to 28.5 + 11.4 u/ml) compared to those patients with autologous blood (8.7 + 12.5 to 17.1 + 23.0). in conclusion these data indicate that allogeneic blood transfusions lead to a remarkable macrophage/rhs stimulation. this is corroborated by the boostered humoral igg-response which was initiated before onset of surgical trauma and blood transfusion. concerning cmi this caused a substancial suppression probably due to a stimulated secretion of immunosuppressive monokines. objective: firstly, to analyse the concentrations of the cytokines tumor necrosis factor (tnc), interleukin-1 (il-i), interleukin-6 (il-6) and coagulatioo/fibrinolysis parameters in postoperatively retrieved blood from a surgical area, secondly to characterize the correspanding cytokine patters in the patients and thirdly to study cytokine concentrations in the initial portion of drainage blood from a surgical area. materials and methods: blood retrieval was performed in a closed-loop system without anticoagulant during 4-6 hours after surgery in 10 patients undergoing arthroplasty (9 hips and 1 knee). 7kf, il-1, it-6, thrembin-antithrombin complexes (tac) and antithrombin (at) ~ere determined in shed blood. patient plasma tn v, il-i and il-6 concentrations ~ere analysed at the beginnlqg and end of the 4-6 hour blood retrieval period. in a separate study (5 hip arthroplasties) f~f, il-i and il-6 ~ere determined in the initial portion of drainage blood. cytekine analyses ~re performed usiog ipmuooassays. an omidolytic method was used for at determinaf.ion and tac was analysed by elisa. n~n-poram~tric tests was used for the statistical comparison. results: the patient plasma il-6 coocemtratiems rose from a median value of 0 to 116 pg/ml, p<o.01, during the blood ret.rieval period. in c was detectable in 4 patients, range:15-50 pg/ml. il-i was not detectable in the patients. in retrieved blood tag was >200 mg/ml in all samples (ref:<4.1 mg/ml) and at was 0. 16-0.51 units/ml (ref:o.80-1.20) . the il-6 concentrations in retrieved blood was >1000 pg/ml in all samples. tn v or il-i was not detectable. in the separate study, (n=5), characterlzing eytokine content in the initial portiere of drainage blood, in= (range: 11-277 pg/ml) and il-i (range:5-125 pg/ml) ~re present in all samples but ii-6 (range:o-25 pg/ml) was detectable in o.qly one semple. conclusion: theses findings indicate that hypereoagulability and hic~ ccrcentratioos are present in retrieved blood. the cytokine pattern in the initial portion of blood from a surgical area differed from these observed in retrieved blood and in the systemic circulation. to identify the role of both autologous and homologous blood on postoperative infections in elective cancer surgery. materials and methods: 159 patients with colo-rectal cancer submitted to curative elective surgery were prospectively studied. on hospital admission the following nutritional measurements were assessed: serum level of albumin, cholinesterase, delayed hypersensivity response , total lymphocyte count and weight loss, as were age and sex, duration of operation , operative blood loss, amount and type of blood given, pathological dukes' stage of the disease and the attending surgeon were also recorded. results : eighty-four patients (52.8%) were perioperatively transfused. thirty-six (42.8%) patients were given autologous blood , while 48 (57.2%) received homologous blood. no patients received both autologous and homologous blood. twenty eight (17.9%) patients developed postoperative infections. non transfused patients had a 9.3 % infection rate , those receiving autologous blood had a 13.9 % infection rate, whi]e in the homologous blood group the infection rate was 33.3% (p < 0.001). univariate analysis showed that infections were significantly related to operative blood loss (p<0.01), length of operation (p<0.05) blood transfusion (p<0.05) and attending surgeon (p<0.05) . multivariate analysis identified homologous blood transfusion as the only variable related to the occurrence of postoperative infections , while the other variables failed to reach statistical significance. blood transfusion (bt) remains an essential life-saving treatment for surgical patients. however, besides the beneficial short-term impacts, negative longer-term effects are observed, which include various alterations in the immune responsiveness. in surgical patients these alterations may contribute to the increased risk for infections and cancer recurrence. since relatively few data demonstrate immunologic changes occurring in other lymphoid compartments than blood after bt, we studied the effect of et on the frequency and responsiveness of immune cells in bone marrow (bm), spleen (spl) and blood (b) in a rat model. normovalemic, 2 month old rats were transfused intravenously with syngeneic heparinized venous blood (3x2ml, every other day), and 3, 7 and 14 days after the last transfusion bm cells ( leh is an experimental oxygen-carrying resuscitation fluid. since leh is cleared from the circulation primarily by the mps, its effect on the development of sepsis and the nature of its relationship with the mps remain a major concern. preliminary in vivo data from our laboratory failed to show any leh effect on the hemodynamic and hematologic responses to endotoxin lipopolysaccharide (lps) in the rat. in contrast, leh exacerbated the lps-induced tnfa production and early mortality. the exacerbation of early mortality by leh was attenuated by pretreatment with the tnfu synthesis inhibitor rolipram. ex vivo, peritoneal macrophages from rats treated with leh and lps have shown increased il-lg mrna signal as compared to lps alone. also, leh increased tnftx production by peritoneal macrophages in response to lps stimulation in vitro. additionally, recent pilot studies indicate that leh attenuates pma-induced superoxide production from rat peritoneal macrophages and that leh augments fmlp-induced migration of human monocytes. taken together, these data strongly support possible interactions of leh with the mps and therefore the nature of such interactions should be further explored. over the last decade, we have developed liposome encapsulated hemoglobin (leh) as an artificial oxygen carrying fluid, or blood substitute. our efforts have focused on studies to define the safety and efficacy of this resuscitative solutions. leh consists of distearoyl phosphatidylcholine, cholesterol, dimyristoyl phosphatidylglyeerol, and alpha tocopherol in a 10:9:0.9:0.1 mole ratio and can encapsulate hemoglobins of different origin (bovine, human, recombinant human). leh is fabricated using hydrodynamic shear to create an average particle size of 0.3 microns. leh can be lyophilized using disaccharides and stabilized in the dry state and easily reconstituted before administration. histopathology and clinical chemistries indicate that leh rapidly accumulates in tissue resident macrophages in small animals injected in the tail vein, principai1y in the liver and spleen. the consequences of accumulation in the reticuloendothelial system are manifest by transient increases in liver transaminases (ast, alt), bilirubin, and bun over 24-48 hours with no change in biliary function (ggt, ap) . clearance through the liver and spleen is observed over the course of 1-2-weeks. more recent attention has been focused on secondary consequences of leh administration especially with regard to inflammatory eytokines. leh does not elicit expression of tumor necrosis factor in vivo and in isolated macrophage cultures, but does result in a transient increase in serum il-6. we have also examined the interaction of leh with lps in vitro macrophage culture to further understand how this blood substitute may effect the immune system. we have labeled leh with technetium-99m (99mtc) to study the biodistribution of leh non-invasively in anesthetized rabbits. rabbits were infused with a 25% topload of leh (200 mg of phospholipid, 2.5 g of hemoglobin per kg of body weight) and imaged continuously with a gamma camera. at 20 hours, images were again acquired. animals were then sacrificed and tissue counts obtained, images revealed an initial rapid uptake bythe liver, 8% at 30 minutes and 15% by 2 hours. the spleen accumulated activity at a slower rate, 3% at 30 minutes and 7% at 2 hours. at 20 hours, autopsy biodistribution studies revealed that approximately 42.6% of the dose is in the blood pool, 15.4% in liver, 18.1% in spleen, 3.2% in lungs, 2.4% in muscle and 1.6% in urine, with trace levels in kidney, brain and heart (<1 °/o). in a hypovolemic model, rats were 10% or 50% exchange transfused with 99mtc-leh. in the 10% exchange model, 99mtc-leh was rapidly taken up by the liver and spleen with minimal activity in the circulation at 20 hours. with the 50% exchange, 50% of the leh was in circulation at 20 hours. the interaction of leh with platelets labeled with indium-111 was also studied. after infusion of leh, the labeled platelets rapidly moved from the circulation to the lungs and liver. over the next 30 minutes, the platelets gradually returned to circulation. this effect was not seen with iiposomes of the same lipid composition but containing no hemoglobin. non-invasive imaging is proving to be a very useful tool for the investigation of leh. the need for a safe, efficacious and commercially viable blood substitute is unequivocal. of the several strategies pursued to invent an adequate blood substitute, liposome entrapped hemoglobin (leh) has been already established as a leading possibility. major advances in liposome technology have already resulted in liposome preparations compatible with clinical use for drug delivery. recent technological advances made by the u.s. naval research laboratories resulted in the capacity to entrap hemoglobin into liposomes in a way which secludes hemoglobin from interacting freely with biological systems. the leh produced has already been tested in in vivo systems and was foun.d to be well tolerated. moreover, the leh originally produced as a solution can be transformed into a lyophilized form which can be reconstituted and delivered as a fresh solution. while important milestones in leh development for a practical blood substitute have been achieved, several issues remain to be explored. most notably, the long term consequences of leh on host defense mechanisms and, in particular, immune cell function. in addition, it is important to understand more fully the metabolic fate and repercussions of leh delivered at clinically relevant dose/schedule regimens. finally, while leh is a highly promising strategy for a blood substitute, the present formulations consist of human hemoglobin derived from human blood, to improve the safety profile, a recombinant preparation for liposome entrapment will be much desired, aa-ginine, a semi-essendai dietary amino acid, possesses several unique and potentially pharmacologic properties. argirdun is a potent secretagogue for pituitary growth hormone and prolacfin and for pancreatic insulin and glueagon; it modulates host protein metabolism by increasing nkmgen retention and enhancing wound collagen synthesis. it also is a potent t call function regulator. ait of these effects coupled with its relative lack of toxicity and safety make it an a~antive nulritionai pharmacologic agem (t). rodents fed supplemeutal arginine exhibit increased thymsc weight which is due to increased numbers of thymic lymphocytes present in the gland. thymic lymphocytes from animals fed supplemental ar~e demonstrate increased blastogenesis in response to coma. and pha (2) . peripheral blood lymphocytes from humans given supplemental arginine also have heightened mitogunic responses to mitogen or antigens (3) . in postsurgery padents supplemental arginine abrogates or diminishes the deleterious effects of trauma on lymphocyte responsiveness and restores peripheral blood lymphocyte responses much faster than observed in controls. overall host immunity is also enhanced by arginine. allograft rejection is enhanced and septic animals survive longer when given supplemental arginine (4) . tumor bearing urginine-supplemented animals have decreased tumor growth and enhanced survival (i). lastly, asgmine can induce t cell maturation and t cell mediated responses in athyrnic nude mice. arginine also has remarkable effects on host nitrogen metabolism post-injury. in increases nitrogen retention in healthy human volunteers and in surgical patients. this beneficial effect on overall nitrogen metabolism is accompanied by a unique effect on the healing wound. supp]emental arginine increases wound collagen synthesis which also translates into increased wound breaking strength (5) . arginine has no effect ou epithelialization. douglas w. wilmom, m.d. boston, ma gintamine is the most abundant amino acid in the body, but it has long been considered a nonessential amino aeid because it is synthesized in many tissues. fohov~g st,~'vation~ injury or infection, skeletal muscle pmteln inoresses its net tale of degradation and releases amino acids into the blunds~mm at an aocelerared rate. app~o)~mately one-third of the amino nitmgea is ghitamine, which is metabolized by the kidney where it parth:~pates in acid-base homeostasis, is the primly ~ for lymphocytes, mac~optmgcs and untexocyms, and contm'butcs to the synthesis of giumth~une. olmamine degrades slowly while in ~olu~ou, especially at usual room teml~mtums. because giulamine was considered nonessential, it has beer absent r'om nil intravenous and most gluts.mine should be considered a cendittona]ly essential nutrient for individuals with serious ilinesses, uspccially those confoanded by infcctinn and inflammation. over the uc~:t 5-7 years, glutamine will be incorgorated into most feeding formulas designed for patients with critical illness. 526 o]~ga-3 pufa there continues to much interest in the application of the 0mega-3 pufa in clinical nutrition. the basic principle has been that the 0mega-3 pufa will displace arachidunic acid and result in a decrease in eic0san0id production. in addition these changes in pufa will after the physical characteristics of the membrane including flujdity, receptor function and transmembrane signals. animal studies have shown that there is omega-3 incorporation with continuou~ enteral feeding both in control and endotoxic animals within 3 days. this includes the liver, spleen, circulating and alveolar marc0phages and the lung. this incorporation resuls in significant changes in the eicosan0id production including pgf 2 and ket0-pgflalpha. there is improvement in the cardio-vascular reep0nse of these animals with ~ecreamed lactic acidosis and improved cardiac contractility. as well there is improved immune function with improved t cell response to mit0gens. the ~ of a mumber of pharmacological agents blocking cicosanoid production can enhance the cell effects of 0mega-3 pufa. clinical studies using short term entsral nutrition with 0mega-3 either alone or with other enteral supplements in a number of clinical settings have shown significant 0mesa-3 incorporation and decreased eicosan0id production. these positive results must be discussed with the additional evidence that long term omega-3 supplementation decrease eic0san0id production but als0 induce a state of immune suppression that is capable of increasing transplant sunvival. these 10ng te~ inune effects may benefit clinical conditions including rheumatoid arthritis and cr0hn' disease early enteral nutrition instituted i~mediately afte~ injury will decrease the entry of bacteria into the intestinal wall and decrease the number of bacteria that translocate into the portal blood. these reductions are associated with & decreased catabolic response, decreased plasma cortisnl levels, end decreased vma excretion in the urine and prevention of mueosal atrophy. sdecific nutrients also affect the transloeation process. addition of arginlne to the diet significantly improves the ability to kill translocated organisms. however. translooetion across the gastrointestinal barrier is not affected. in contrast, glutamine diminishes the rate of translooation across the imtestinal barrier and also improves killing of the beetarla that do translooate. the omega 3 fatty acids in the form of fish oil slightly decrease the rate of translocation but more significantly increase the ability of the animal to kill translo~ated organisms, all three dietary additives, i.e. argini~e, glu=amine and fish nil. significantly improve survival, hut adding glyoine or medium chain triglyeeridem do not, combinations of srginine and glutamlns, glutamine and fish oil, and fish ell end arginine each improve survival, and to a greater degree than a combination of all three. these studies add further evidence that translocation is an important determinant of survival after injury, early feeding with immunonutrlent enriched dices will improve survival and dsarease transloeation to varying degrees, depending upon the nutrients provided. objectives: we studied effects of supplementing a commercial enteral diet, impact r (imp, sander nutr lnc), with fiber (imp/fib) or alanyl-glutamine (imp/ag, exogenous glutamine (gln) 14 gms/l) on influencing the incidence of bt to mesenteric lymph nodes (mln) in burned mice. fiber has been shown to improve gi integrity under certain stress/treatment conditions. the dipeptide ag is a water-stable source of gln, which is a specific fuel for many cells including enterocytes. traumacal (trcal), a high-protein, high-fat enteral diet (mead johnson iuc), was also studied, as well as rodent chow (harlan teklad inc), which contains very high protein & fiber. methods: anesthetized cf-1 mice aged 8-12 wks received 32% tbsa fullthickness dorsal burns & were resuscitated with 2 cc ip saline. diets were allowed ad lib; caloric intakes were comparable in all gps except fasted gp (fast 24 hrs, chow 24 hrs). at 48 hrs postburn mln were sterily removed, homogenized and plated on heart brain infusion agar; cfu/g mln tissue were determined. bt was analyzed by fishers exact test, cfu/g by anova-bonferroni. * p<0.01, ** p<0.05 compared to imp and burn-fast gps. background. infectious complications following trauma, major operation, or critical illness adversely affect hospital cost and length of stay (los). some key nutrients have been shown to possess immune enhancing properties. this multicenter trial was conducted to determine if early administration of an enteral formula supplemented with arginine, dietary nucleotides and fish oil can decrease los and infectious complications in icu patients. methods. this was a prospective, randomized, double-blind study of 326 adult icu patients who required enteral feeding for > 7 days. patients entered the study within 48 hr of the event, were stratified by age and disease, and were randomized to receive either the supplemented formula (impact®) or the conventional formula (osmolite ® hn). feedings were initiated at full strength and advanced to at least 60 ml/hr by 96 hr after event. results. both groups tolerated administration of formula well. for patients fed > 7 days, the median los was 25% shorter (p=o.ol) for the--supplemented group (21 days) compared to the conventional group (28 days). the incidence of most infectious complications was lower in the supplemented group, but this difference reached significance only for urinary tract infections (p=o.o05). the supplemented group had a significantly shorter los from onset of infectious complication until discharge for patients with pneumonia (19 vs. 27 days) and skin/soft tissue infection (19 vs. 37 days). conclusions. administration of the supplemented formula was safe and well tolerated. when fed > 7 days, it reduced the incidence of most infectious complications, and significantly reduced los. materials and methods: twenty-seven patients were randomised into 3 groups ( n= 9 each) to receive either a standard enteral formula, the same formula enriched with arginine, rna and omega 3 fatty acids (enriched group) or isonitrogen, isocaloric parenteral nutrition. early enteral nutrition was started within 12 hours following surgery (10 ml/hour). it was progressively increased reaching a full regimen on day 4. on hospital admission and on post-operative day 1 and 8, the following parameters were assessed: serum level of transferrin , albumin , prealbumin, retiool binding protein (rbp), cholinesterase. delayed hypersensitivity response, igg, igm, iga, lymphocyte subsets and monocyte phagocytosis ability were evaluated on admission and on post-operative day 1, 4, 8. the three groups were comparable for sex, age, cancer stage, type and duration of surgery, intra-operative blood loss and amount of blood transfused . in all groups a significant drop in all the nutritional and immunological parameters was observed on postoperative day 1. comparing post-operative day 1 versus day 8 a significant increase of prealbumin (p<0.02) and rbp (p<0.005) was found only in the enriched group. with respect to immunological variables an increased phagocytosis ability (p<0.001) and a significant recovery in delayed hypersensitivity response (p< 0.005) was observed only in the enriched group. conclusions : these data are suggestive for a more effective post-operative recovery of both. nutritional and immunological status in cancer patients fed with enriched enteral formula. gastrointestinal intolerance was equivalent (18% in each group) and laboratory screening confirmed that both diets were safe. when analyzing clinical outcome for all patients, there were no significant differences in septic complications (immun-aid = 41% vs vivonex ten = 35%), mean mof score (immun-aid = l.b vs vivonex ten = 3.6), or mortality (immun-aid 0% vs vivonex ten = 6%) . kowever, when analyzing the subgroup of patients with severe injury (iss or ati _> 25), patients receiving immun-aid appeared to have fewer septic complications (33% vs 50%) and their mean mof was significantly lower (1.8 _+ 0.7 vs 5.8 + 1.7, p = 0.05, student's t-test) . these preliminary data indicate that immun-aid is tolerated well when aggressively delivered immediately postinjury. the ultimate affect on clinical outcome appears ~avorable for immun-aid, but needs to be confirmed in larger patient groups. kemp?n, m., neumann, h.a., he i[michh b: as both increased, normal and reduced phagocytic capabilities of polymorphonuclear leukocytes (pmn) and monocytes in acute batterial infections have been reported, the role of phagocytes in patients with severe sepsis is less clear.we examined pmn and monocytes from 10 patients in septic shock and 17 heailhy votunteers for phagocytic function. phagocytosis was determined by flow cytometry (facscan) and was measured by the ability of pmn and monocytes to phagocytose e.coli marked with fluorescent antibodies. a septic shock was defined by the presence of a ~ource of i, nfoctiqn with a known bacteriology, distinct signs of a systemic response and defined minimum scores in 3 icu scoring systems indicating the presence of a multiple organ failure. additionally we examined how phagocytosis is influenced when a new enteral diet formulation containing substrates suggested to improve immune function or arginine, one of its major compononts, is added in vitro in defined concentrations and incubated for 10 minutes. pmn (p{o,05) and monocytes (p<o,01) of the septic patients showed a significantly enhanced phagocytosis compared tolhe phagocytes of the healthy group. the incubation with the enteral diet in vitro was followed by a higher dose-related rate of phagocytosis, especially in the healthy group, that was not statistically significant. after addition of l-argintne we atso observed an iqcrease in phagocytosis, that was lower than in the group with the complete diet. using a modern immunefluorescence-cytometric essay we founfl an improved phagocytic function in septic shosk. there are signs for an influence of nutrient substrates on phagocytosis which seems to be complex and should be further investigated. arg and gln were lower in the 49%-bcaa vs 16%-bcaa group; arg was related directly to arg dose and inversely to bcaa dose (p<o.o01 for all). clearances of bcaa increased with increasing bcaa dose (p<o.o01); arg and gln clearances were directly related to the bcaa clearances (rz=o.65, p<o.o0i). relationships between arg, gln and other aa seem to be ruled by patterns involving specific intracellular aa transport systems. regulation of arg and gln clearances through bcaa administration may be related to an increased flux of aa into synthetic processes. intestinal segments from rats immunized by infection with the nematode trichinella spiruus undergo intestinal anaphylaxis or type i hypersensitivity when challenged in vitro with parasite antigen. immunized rats exposed to t-radiation (7gy) and sustained on rat chow,fail to fully express type i hypersensitivity up to 14 days post irradiation (dpi). we hypothesized that enteral nutritional support immediately following irradiation may be effective in preserving mucosal immune responsiveness or in reducing the recovery time after radiation-induced injury. rats were infected with 3x103 t.spiralis larvae. thirty to fifty days later rats received 7gy t-radiation from a co source as total abdominal irradiation (tai). immediately following tai, rats were fed an elemental amino acid diet (eaad) and at various dpi sacrificed and tested in ussing chambers for local anaphylaxis, expressed as antigen-induced ci secretion. the normal ci secretory response to antigenic challenge which is suppressed after irradiation,was restored by 3 dpi when rats were placed on the eaad. antigen-induced c[ secretion is dependent on the interaction o~ antigen with specific ige antibodies on the surface o5 mucosal mast cells and their subsequent degranulation. mast cell-derived 5-ht,histamine and pg, together effect ci secretion.in irradiated rats on rat chow,the failure to respond to antigenic challenge appears to be associated with the destruction of mast cells. they are undetectable with standard staining procedures and mucosal histamine levels are drastically reduced. also,el secretion can be induced by direct stimulation of epithelium with cr secrctagogues and circulating ige levels are normal. despite a more rapid recovery of immune responsiveness in irradiated rats receiving eaad,mast cells were not restored. we conclude that the eaad contributes to an adaptation that supports the expression of local anaphylaxis in the absence of mast cells. total parenteral nutrition (pn) and bowel rest may influence the host response to infection. this study examined the different host response to infection in parenterally and enterally fed animals. forty-three rats were divided into pn group and total enteral nutrition (en) group. they received identically constituted isocaloric isonitxogenous diets for seven days. animals were then challenged intraperitoneauy with 3xl(y escherichia coli and survival were observed. in other experiments, they were sacrificed before and two hours after bacterial challenge. peritoneal cavity was lavaged with 10ml saline and peritoneal exudative cells (pec) were harvested and cultured in vitro for 24 hours with and without lipopolysaccharide (lps). colony f(m, ning units of bacteria (cfu-b) in the peritoncal lavaged fluid (plf) were determined and tnf in the plf, serum and pec culture supernal,ants were measured by l929 bioassay. twenty-fonr hour survival rate in en group was significantly greater than that in pn group (60% vs 0%). the data suggest that local immune responses of pn-treated animals may be depressed with consequent disturbance in the clearance of bacteria. this may lead to a greater systemic cytokine response and resulting in a poor survival after bacterial challenge in pn group. the effects of intragastfic tube feeding of diets enriched with m-6 polyunsaturated fatty acids (pufa) from safflower oil (so) or (o-3 pufa from menhaden oil (mo) on eicosanoid production, and onthe membrane phospholipid fatty acid composition were investigated in a severe acute septic shock model. the percent calories from fat maintained at 30% in the diets was adjusted to provide 10% each of saturated, monounsaturated, and pufa using olive oil and palm oil as filler fats. after feeding for 5 days, lipopolysaccharide (lps, 9mg/100g) was injected through the tail vein. the percent of animals surviving in the mo group was 67% (14/21), and did not differ significantly compared to 57% (12/21) in the so group. the plasma concentrations of tnf ct for the groups of animals fed so (1.2+3.0 ng/ml) and those fed mo (1.8+1.1) did not differ significantly. the levels of prostaglandin(pg)e2, 6-keto-pgflc~ and tumor necrosis factor-ct (tnf-c 0 were determined 90 min after lps injection. the fatty composition (relative mole%) of the liver was determined before (lps-) and 3h after the lps administration (lps+). the data (mean_+sd; n=6) are as follows. group these data indicate that a marked reduction in the plasma levels of poe2 and 6-keto-pgflct for rats fed mo diet was associated with a reduction in aa which was displaced by epa in the membrane phospholipids. further, in these rats, we observed that there was a 2.8% reduction in the percent of epa following lps challenge compared to the basal levels. however, for the group of rats maintained on so diet, the liver membrane phospholipid fatty acid composition before and after lps administration was unaltered. these finding suggest that although a reduction in aa was associated with a decrease in the production of pro inflammatory eicosanoids after 5 days of continuous mo feeding, there was no marked effect on the survival following lps challenge. animal models have been used to examine the effects of various nutrients and nutrient combinations upon the immune system. we studied the effects of standard and specialized enteral formulations on the response of mice to infectious challenge with listeria monocytogenes, influenza a or candida albicans in order to test the efficacy of specialized ingredients. cf-1 outhred female mice (12-15 g) were fed purina #5002 chow, commercially available osmolite hn ®, perative ® or impact*. mortality and tissue burden of pathogen were examined during the 21 d period following induced infection. the results indicate that there were no differences between the groups fed the liquid formulas with regards to mean survival time or % survivors in these models of infection. examination of spleens in the groups challenged with l. monocytogenes and kidneys in the groups challenged with c. albicans revealed no differences in tissue burden of pathogenic organisms. alterations in the length of pre-feeding from 0 to 8 days prior to infection did not affect these results. these data demonstrate that, contrary to previous reports, the use of specialized nutrients did not improve resistance to disease in mice challenged with l. monocytogenese, influenza a or c. albicans as compared with mice fed osmolite hn. animals fed standard chow tended to be more resistant to infectious disease than those fed the liquid formulas perhaps due to the form of diet or complexity of ingredients. the results indicate that these animal models of infectious challenge may be of limited use to distinguish effects of modified nutrient composition of enteral formulas. laboratories the beneficial effects of sesame oil (ses) have been attributed to the presence ofsesamin, a non-fat constituent which is claimed to iultibit a-5 desaturase activity. we investigated the effects of ad libidum feeding of ses enriched diet on fatty acid composition of phospholipids from plasma, liver, on eicosanoid production, and on the protective effects in mice after cecal ligation and puncture (clp) and compared with safflower oil (so) diets. olive and palm otis were added as filler fats to the so diet to maintain the ratios of saturated, and unsaturated fatty acids similar to ses diet. thus, while the total fat remained at 15wt%, the only difference between the two diets is the presence ofsesamin in ses diet. the animals were fed for 3 weeks. the incorporation of dihomo-y-linolealc acid (dgla: 20:3 o)-6) in the phospholipids from plasma and from the liver for the group of mice fed so diet ranged between 1-3% compared to the undetectable levels for those fed so diet. there were no significant differences in the incorporation of other fatty acids either in plasma or in the cell membranes between the two groups. following an intraperitoneal administration oflps (10p.g/kg body wt), the plasma levels of both pge2, and txb2, were significantly decreased (p<0.02) by nearly 50% for the group of animals maintained on ses diet compared to those maintained on so diet. these findings suggest that sesamin inhibited the release of arachidonic acid (aa) which upon accumulation is retroconverted to dgla which could reflect a modest increase in the content of dgla. failure to decrease the formation of aa could mean that the effects of sesamin on chain elongation process may be after aa is formed, and that sesamin may not inhibit a-5 desaturase activity. on the other hand, our data suggest that sesamin present in sesame oil inhibited the activity of cyclooxygenase which converts aa to its metabolites), or alternatively could block the activity of phosphalipase a 2 (pla2) (which releases aa from membrane phospholipids) as is evident from a marked decrease in eicosanoid production. the percentage of animals surviving after cecal ligation and puncture in rite group of mice fed ses diet was 65% (13/20) which was markedly higher (p<0.01) compared to only 20% (4/20) for the so group. increase in survival in the group of mice fed ses diet was associated with nearly 50% reduction in the production of tnf in response to lps i.p. challenge, for ses group compared to so fed animals. our data suggest that sesamin, present in sesame oil, decreased the production tnf~, inhibited the activity ofpla 2 and consequently offered a significant protection against clp. thus sesamin or sesame oil appear to be novel antiinflammatory agents which are present naturally in many edible products. [dept. surgery, ~e. deasoness hosp.,harvard medical school, boston, usa] stress causes alterations in the homeostasis of an organism with major changes in various organs, lifespans of cells, protein turnovers, metabolic pathways of activation or stimulation, energy mobilization, etc. punctual changes have been described in several organs and various cell types of the nervous, endocrine and immune systems. many stored or newly synthesized proteins are released to the bloodstream to be transported to target organs or to be disposed of. as stress alters protein synthesis and requirements in the targets, the bloodstream is a useful system for monitoring the relevant changes. we present here the pattern of newly synthesized or released serum proteins in c57bl/6 mice that have been stressed by immobilization. the proteins have been labelled with 3ssmethionine in vivo and the study was performed by two dimensional gel electrophoresis based on the method originally described by o'farrel. the electrophoretic run was carried out in an isodalt apparatus. a molecular dynamics laser scanner and the kepler image analysis system (lsb, rockville, usa) were used for modelling the radiofluorographic images. the radiofluorographic images of the gels from serum samples obtained from mice injected with i mci of 3ss-methionine reveal about 440 protein spots, from which a small fraction -about 20are altered in both qualitative and quantitative manner. the major changes are in the range of 25 to 60.000 d. the analysis displays a highly reproducible pattern, where clear differences between stressed and non-stressed conditions are shown. differences between patterns attributed to chronic vs. acute stress will be presented. patients with severe trauma or major surgery are known to acquire alterations in neutrophil functions which contribute to their enhanced susceptibility towards microbial infections. we analyzed neutrophil functions from fourty patients admitted for major surgery 6 days and 1 days preoperatively and on the ist and 6th postoperative days (study-days i, 2, 3, and 4) in a randomized placebo-controlled double-blind study. patients were divided into two groups, one ~eceived 6 days preoperatively an enteral nutrition enriched with omega-3 fatty acids, arginiee, and rna (impact), the other an isocaloric, not enriched control nutrition (placebo). meutrophils were isolated from the peripheral blood and stimulated with the ca-ionophor a23187 (7.3 pm). the capacity of the respective neutrophils to generate leukotrienes was analyzed by rp-hplc. neutrophils from impact group generated significantly higher amounts of ltb5 (mean values 6.6 ng/l x i~ cells) compared to the placebo group (2.6 ng) at study-day 2 [p, 0.01). in contrast, the capacity of neutrophils to produce ltb4 was not significantly different in both patients populations at study-days 2 and 3. the omega-oxidation of ltb4 to its biologically less active metabolites oh-ltb4 and cooh-ltb4 was not significantly different in both groups. however, neutrophils from group f patients generate more ltc4 at study-day 2 (p(0/5). the capacity of the patients'neutrophils to generate reactive oxygen radicals upon stimulation with fmlp, pma or the caionopbore a23187 exhibited patient dependent differences but no correlations to the repective nutritional supplementation as was measured by luminol dependent ohemiluminescense= these data indicate that an enteral nutrition enriched in omega-3 fatty acids lead to alterations in distinct parameters of neutrophil functions. clinical patient characteristics and mean caloric intake were similar between the two groups. both formula were tolerated well and the incidence of diarrhea was low. the number of infectious and wound complications as well as the apache ii score was evaluated for the two study groups. although the number of complications in the group supplemented with arginine, rna and omega-3 fatty acids was lower no significant difference in the occurance of infectious and wound complications has been observed between the two groups. however, the apache ii score indicated a significantly improved clinical outcome in patients with supplemented diet. in conclusion, early enteral nutrition with an arginine, omega-3 fatty acids and rna supplemented diet helps to recover more rapidly after surgical total parenteral nutrition (tpn} is associated with intestinal atrophy and dysfunction attributed to the absence of the non-essential amino acid glutamine in current parenteral nutrition solutions. in this animal study with 18 male wistar-rats we tested the hypothesis that glutamine-dipeptide supplementation during tpn for 10 days would restore small bowel atrophy and tpn induced dysfunction. a conventional tpn solution (250 kcai/kg bw) was compared to an isocaloric and isonitrogenous tpn (0,5 g n/animal/d) supplemented with alanin-glutamin (ala-gin) dipeptide (0,15 g n derived from ala-gin =30% gin-n). an enteral fed cqntrol group was included. mucosal thickness, villous height and architecture, absorption of water and glucose as well as dissacharidase activity of maltase and alkaline phosphatase were evaluated. total parenteral nutrition in rats causes villous atrophy, a significantly reduced absorption rate and a decreased activity of villous enzymes compared to an enteral fed control (p<o,01). addition of ala-gin to parenteral nutrition solutions prevents intestinal atrophy and restores functional alterations with a significantly increased rate of water and glucose absorption as well as a higher dissacharidase activity compared to the standard tpn group (p<0,01). there was no significant difference found between the enteral fed control and the dipeptide supplemented animals concerning intestinal structure and absorption, the enzyme activity was significantly decreased in the ala-gin supplemented group compared to the enteral fed control (p<0,01). in this rat model supplementation of parentera] nutrition solutions with ala-gin dipeptide restores tpn induced intestinal atrophy and dysfunction. boston. ~ usa injury, infection and major o1~'ations s~nulate a variety of factors whi~ initiate the body's response to th~ st~..sses. 'i"hese reactions are m~liated by a cbmage ia the neta'al hormonal, eac.ranmemt, by the elaboration of eytokines and tl~ougb the stimu/ation of other inflammatory mediators. this eatabolio setting zesulis in body protein loss, which g-taduai1y erodes both fimetianal and stm~ tissue. wiu3_b the normally nom'ished individual can withslzod "d~ response for a brief period of time, ircolonged eatabollsm l~ associated with ~,nmunosuppmssion, poor wound healing, and proioagcd convalescence. surgeons have rej2ed on intraveaous or eater~ feedings to ~everse this process. a va~ety of data now indicates that it is extremely di~edt to replete protein-containing tissue d~g the eataholie state of illness; the umutt response to hypercaloise feedings in this setting is the incxeased accretion of body fat md water. administration of gxow~ ho~moae ~cesults i.a n shift of the body's oxidative machinery to fat rather than eazbohydmte utiq~afic~; ¢oncomitaatiy, protein synthesis is stimulated. this metabolic state may have clinical utility in p~e.n.ts who need to heal large wotmds, in those who need to gain strength in order to be w~ed 5ore veatilatory support, or those patients who ~ve mulfiorgan failure and nee~ to ealaaace protein syatsesis to in.suze recovery. 235als are now in progress to dcterm~e the benefit of growth hormone in these and other disease state.s, rn the fature, the increased use of growth hormone, will occur;, this and other growth factors will serve to su~2po~ the host and shorten convalesceace following catabolic diseases. our objective was to study effects of pre-trauma growth hormone (gh) treatment on liver and skeletal muscle metabolism in a trauma model in piglets. twenty-four piglets were randomized to three treatment groups; one group was pretreated with gh 24 iu daily three days before the experiment (gh-3), another group treated with gh 24 ie at the start of the surgical procedure (gh-1) and one group served as untreated controls (con). they underwent a standardized surgical procedure to place sampling cathethers in the portal, hepatic and femoral veins and doppler flow probes around the portal vein, the hepatic and the femoral arteries. all pigs recieved a primed constant infusion of u-14c-glutamine. substrate fluxes were measured one (lh) and five (5b) hours after termination of the surgery. nitrogen excretion was significantly decreased in the gh treated animals (gh-3:30.8+4.2 mg/kg, gh-i: 37.6+_.5.8 mg/kg, con: 55.4+-3.1 mg/kg, p=0.004). in the hindieg, there was reduced net glutamine efflux due to decreased absolute glutamine release (gh-3:0.9 + 0.8gmol/min at lh and -2.4+1.4 in.tool/rain at 5h, gh-i: -3.4+1.5 i.tmol/min at lh and -1.4+1.2 pmol/min at 5h, con: -8.7+_2.8g mol/min at lh and -11.6+3.4/amol/min at 5h, p=0.005, p=0.006), whereas the absolute uptake of glutamine in hindleg was unchanged (p=0.84). glucose uptake in the hindleg was decreased (p=0.0006). net glutamine uptake in liver was attenuated (p=0.03 at 5h), whereas net glutamate release from liver was increased (p=0.005). thus, pre-trauma treatment with gh improved nitrogen economy, attenuated net glutamine loss from hindieg due to decreased absolute release, and attenuated hindleg glucose uptake. liver net glutanaine uptake was decerased and net glutamate release increased. patients with major abdominal surgery exhibit a considerable catabolic response with negative nitrogen balance and protein breakdown, which may have detrimental effects on outcome. we investigated the effects recombinant human growth hormone on substrate metabolism in parenterally fed patients. 48 metabolic healthy patients were randomized to receive either placebo or hgh in a dosage of 0,075; 0,15 or 0,3 i.u. per kg bw. substrate oxidation rate, energy expenditure as well as short life proteins were measured daily. six patients were excluded from analysis as drop outs, due to surgical complications. we could find a dose-dependend effect of growth hormone on resting energy expenditure, carbohydrate oxidation, fat and proteine oxidation. with increasing growth hormone resting energy expenditure increased from 20.9 calories per kg body weight to 26.6 calories. this was mainly due to an increased carbohydrate and fat oxidation, while proteine oxidation decreased. this was in accordance with a decreased urea.production rate and an improvement in cumulative nitrogene balance, which increased from minus 12.7 to plus 8.5 g n / 7 days. in consequence short life proteins were also increased. the only negative side effect was an increased blood glucose concentration in some of the patients, making insuline administration mandatory in 3 patients. our results are in accordance with other studies showing improvement of nitrogen balance, maintainance of lean body mass and muscular strength. if growth hormone administration may have any impact on morbidity, mortality and length of hospital stay in these patients it should be evaluated in a larger number of patients. in 30 patient after liver transplantation (oltx) the effect of recombinant human growth hormone (rhgh) on protein metabolism and hormonal changes was studied. patients and methods: the underlying disease in all patients was end stage liver cirrhosis, non of the patients included in the study was suffering from malignancies. the patients were randomly allocated to receive either 81u of rhgh bid (sc) or no alternative medication. the study period lasted 14 days. from daily 24 hrs udne collection urinary nitrogen loss and daily loss of urea were determined. body compostion analysis (bca, bioimpedance, akern-ltaly) was performed preoperativly and at the postoperative days 7 and 14 to evaluate changes in body water and body cell mass (bcm). the effect on resting energy expenditure (ree) was analysed using indirekt calorimetry wrhin the same intervalls (metabolic monitor, datex). blood levels of gh, igf1 and igf2, igfbp3 were measured daily during the study period, on day 3 a 24 hour profile of gh was.performed. samples were collected each 20 minutes. results: cumulative udnary nitrogen and urea loss were not signifcantly different for the whole study period but for the first 4 study days. bia indicated for the rhgh-group a loss of bcm to a lesser extent than for the controls (n.s.), changes in bodywater where similar for both groups. ree changes (kcal/kg bcm) were not significantly different for the two groups. blood levels of gh and igf1 differed significantly between the groups for the whole study period, but not igf2 and igfbp-3. the circadian rhythm of endogenous gh-excretion had not been altered by gh, but absolute levels of gh were increased. conclusion: during the first 4 days after oltx we could proof the protein sparing effect of gh treatment. although the differences between the two groups were not significantly different for the whole study period the results showed on all days a less catabolic situation for the rhgh treated patients. to possibly improve protein balance after major abdominal surgery we studied the effects of gh on protein metabolism after ltx. excluding malignant diseases, 29 candidates for ltx were randomized to either postoperatively receive the usual treatment (co=control group, n=14, age range 25-59 yr, bmi 23.9+5.7 kg/m 2) or for 14 days additional gh (2x8 i.u.s.c.) (gh group, n=14, 21-59 yr, 21.8+2.2 kg/m2). metabolic studies (calorimetry and lsc-leucine infusion) were performed 5 (test a) and 11 days (test b) after surgery. tracer analysis was done by gas chromatography-mass spectrometry. during the 2-week study clinical parameters did not differ significantly between groups; however, there was a tendency for increased glucose levels, insulin need, and energy expenditure at the time of test b in the gh group. leucine oxidation (x+se) during test a;b was 117+10; 111+15 (co) and 110+7; 100±12 (gh) pmol/kg ffm/h (n.s.). protein breakdown was 134±16; 131+30 (co) and 156+14; 178+30 (gh) pmol/kg ffm/h (n.s.). nonoxidative-leuine disposal was 119±9; 127+16 (co) and 139±11; 177+20 (gh) pmol/kg ffm/h (p<0.05 for the rise in the gh group). tracer data were supported by cumulative urea nitrogen excretion (days 1-4: gh 52+3 vs co 66±7 g; p<0.05). we conclude that after major abdominal surgery gh lowers nitrogen excretion by increasing total body protein synthesis. it is not known which organs contribute to this effect. in several investigations it has been shown that the protein saving effect of recombinant human growth hormone (rhgh) in the postoperative state is accompanied by raised igf-1 levels in plasma. it was concluded that the anabolic effects of rhgh were probably, at least in part, mediated by igf-1. this study was aimed at detecting the efficacy of igf-i on modulation of the protein metabolism in the catabolic state. patients and methodes: 38 patients (both sexes, age: 40-75 years, bmi 17-30 kg/m 2) with major upper gastrointestinal surgery (gastrectomy or partial gastrectomy) received 80 ug rhlgf-1/kg body weight/twice daily or placebo during 5 treatment days beginning on the first postoperative day in a double-blind, randomized study. all patients received hypocaloric and hyponitrogenous total parenteral nutrition (3g chng, o.6g aa/kg) troughout the whole study period. cumulated nitrogen balance, 3-methyl-histidin exretion in urine as well as serum-igf-1 levels have been determined. the two tailed wilcoxon test was used for statistical analysis results: first results will be presented previous studies have suggested that growth hormone (gh) potentiates various activities of leukecytes. however, it is not clear whether gh can improve survival of animals with gram-negative sepsis. we investigated the effects of gh on host response to infection in septic mice model. methods balb]c mice were randomized to 3 groups (n=10/gronp): gh-high (4+8mg/kg/day), gh-iow (0a8mg/kg/day) or control (saline). following subcutaneous treatment (every 8 hours for 6 days) with gh or saline, mice were challenged i.p. with e.coli (lxl08/body). survival rate was recorded every 2 hours. in the next experiment, gh-high and control animals were sacrificed 4 hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative cells (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. pec were cultured in vitro for 24 hours with lipopolymccharide (10p.g/ml in normotensive adults growth hormone (gh) rises when clonidine challenge is performed. recently evidence was shown for gh to accelerate wound healing. while gh secretion patterns are inconstant the gh-dependant insulin like growth factor (igf-1) and the insulin like growth factor binding protein (igfbp-3) reflect the integrated gh secretion over days. since we administer clonidine to patients with acute alcohol abuse we determined plasma levels of igf-1 and igfbp-3. materials and methods: 21 patients sheduled for esophagus resection were investigated once they had given written informed consent. 8 patients showed acute alcohol abuse and were treated with clonidine postoperatively• 13 patients with no history of acute alcohol abuse served as controls. besides liver enzymes igf-1 and igfbp-3 were determined. results: both groups had a comparable hepatic capacity of synthesis with decreased cholinesterase levels as to be expected after a major operation. regression analysis of igf-1 and igfbp-3 levels showed no differences between the groups. discussion: igf-1 and igfbp-3 plasma levels are not increased in normotensive patients who are treated with clonidine for prevention of postoperative alcohol withdrawl syndrom. further studies are required to discriminate whether our results are due to impaired postoperative liver function or if clonidine has not any impact on the gh-igf-axis in patients with alcohol abuse. recent studies have revealed that igf-i has several immanoregulatory roles. however, little is known about its effects on septic animals. we tested whether igf-i could increase the survival o f mice challenged intraperitoneally (i.p.) with e. coli. m~thqd$ balb/c mice received either high dose igf-i (n=10, 24mg/kg/day), low dose igf-i (n=10, 2.4mg/kg/day) or saline (11=10) every eight hours subcutaneously for six days. animals were then challenged i.p. with lx108 e. coli. survival was observed every two hours. in the other experiment, mice that received high dose igf-i or saline were sacrificed four hours after bacterial challenge. peritoneal cavity was lavaged with 3ml saline and the peritoneal exudative ceils (pec) were harvested and counted. colony forming units (cfu) of bacteria in the peritoneal lavaged fluid (plf), liver, lung and blood were determined. in addition, pec were cultured/n vitro for 24 hours with lipopolysaccharide (10).tg/ml). tumor necrosis factor (tnf) in the supernatants of pec culture, plf and serum were measured by l929 bioassay. results igf-i improved the survival rate at a dose of 24mg/kg/day. severe chronic neutropenia (scn) is a disorder characterized by severe neutropenia secondary to either a maturational arrest of myelopoiesis at the level of promyelocytes (kostmann-syndrome; scn) or regular cyclic fluctuations in the number of blood neutrophils with a median absolute neutrophil count (anc) of less than 500/~tl (cyclic neutropenia). patients suffering from scn have an impaired acute inflammatory response to injury and an increased susceptibility to infections, i.e. bacterial or fungal infections, resulting in chronic oral inflammation, severe pneumonia, abscess formation and bacteraemia. we have treated 36 patients (32 scn, 4 cyclic neutropenia) with r-methug-csf (filgrastim). thirty of 32 patients with scn and all 4 cyclic neutropenia patients responded to this treatment with an increase of the median anc to greater than 1000/~tl. the dose of r-methug-csf needed to achieve and maintain the response varied between 0.8 and 120 ~tg/kg/d. long-term treatment did not exhaust myelopoiesis: the anc remained stable after up to 6 years of treatment and antibodies to r-methug-csf were not detected. the increase of anc was associated with dramatic clinical responses: significant reduction of severe bacterial infections, reduction of intravenous antibiotic treatment, and reduction of hospitalizations. no severe bacterial infections occurred in any of the r-rnethug-csf responders during longterm treatment. severe adverse events, most likely due to the underlying disease, included the development of mds/leukemia in two patients, and osteopenia/osteoporosis in 12 patients. these results demonstrate the beneficial effects of r-methug-csf treatment in severe chronic neutropenia patients. the newborn is predisposed to mortality during infections due in large part to developmentally immature host defenses. recently cytokines have been identified that regulate the development of these defenses. one such cytokine is specific for neutrophils and is termed granulocyte colony stimulating factor (g-csf). in the studies to be discussed, we have attempted to impact hematopoiesis in the fetus using exogenous rhg-csf delivered through the pregnant dam. our rationale was that an enhanced myeloid system may result from such in utero treatment leading to, perhaps, enhanced protection to microbial insult. rhg-csf crossed the placenta and reached peak fetal serum concentrations 4 hours after administration. these levels were 1000-fold lower than the levels detected in the adult dams serum. white blood cell counts were elevated approximately 2-4-fold over control newborn blood. this increase was due to circulating numbers of neutrophils. the marrows from these pups were hyperplastic consisting of predominately immature and mature neutrophilic cells. no changes were seen in the thymus or livers. pups born to mothers treated with rhg-csf since day 15 of gestation (parturition in rodents occurs on approximately day 21) survived a lethal challenge of group b-13 hemolytic streptococcus. in contrast their untreated yet infected counterparts did not survive. recent clinical developments have led to trials of rhg-csf in certain neutropenic states including cyclic and chronic neutropenia. we have identified several of these patients who were pregnant during treatment with rhg-csf. biologically and anfigenically identifiable rhg-csf was detected at increased levels in the cord serum of these neonates. we will discuss our findings with these patients in light of our animal model of neonatal myeloid development. we have previously demonstrated decreased g-csf production and g-csf mrna expression from activated mononuclear cells from newboms compared to adults (cairo, pediatr res 31:574, 1992) . we have also recently observed that administration of a single dose of rhg-cse to one-day-old newborn rats induced significant neatrophilia, while administration for 7 days induced neulrophilia and increased the bone marrow (bm) neutrophil storage and progenitor pools (cairo, blood 76:1788 , 1990 cairo, pediatr res 27:612, 1990) . we embarked on a phase i trial exploring the safety, pharmacokinetics, and biological efficacy of g-csf in newborns with presumed sepsis. 42 infants <72 hrs old with a diagnosis of presumed sepsis were stratified into three groups: very preterm (26-30 wk), preterm (31-35 wk), and term (>36 wk) and randomized to receive either a placebo or 1, 5, and 10 gg/kg/qd or 5 and 10 p.g/kg/bid of rhg-csf infused by pump over 1 hour for 3 consecutive days. cbcs were obtained at 2, 6, 24, 48, 72 and 96 hrs. tibial bone marrow aspirations were performed 72 hrs after study entry and differential counts and cfu-gm pools were determined. c3bi expression was determined at 0 and 24 hrs after rhg-csf, and g-csf pharmacokinetics were performed after the first dose of rhg-csf utilizing a sandwich elisa. a significant increase in the anc was observed at 48, 72 and 96 hrs following administration of both 5 and 10 ~tg/kg/d of rhg-csf. the maximum increase in the anc occurred 72 hrs after 5 and 10 ~tg/kg/d (397 -116%) (p<0.01) and (621% -+ 200%) (p<0.001), respectively. there was a significant dose-dapendeat increase in the bm neutrophil storage pool (18 _+ 4% vs. 62 + 6%) (p<0.01) (placebo vs. 10 ~tg/kg/d). there was no significant difference in the nantrophil proliferative pool. an increase in cfu-gm and cfu-gemm was seen at all doses tested, compared to placebo (53.5 _+ 8.6 vs. 87 -+ 15) (colonies/l(p cells/plate). c3bi expression was significantly increased 24 hrs after 10 bg/kg/d of rhg-csf (233 + 81% vs. 83 +-26%) (p<0.012). peak serum g-csf levels occurred at 2 hrs and were dosedependent. the half-life of rhg-cse was 4.44 + 0.4 hrs. most importantly, there was no observed toxicity from g-csf in all patients studied. 31 of 42 patients were on ventilators prior to administration of rhg-csf and there was no increase in pulmonary toxicity. these preliminary data suggest that rhg-csf is well tolerated at all gestational ages in newborns with presumed sepsis. a multi-center phase ii/iii randomized double-blindad placebo controlled trial is required to determine the efficacy of rhg-csf in this clinical setting. we investigated the effects of recombinant canine granulocyte-colony stimulating factor (g-csf) on survival, cardiopulmonary function, serum endotoxin levels and tumor necrosis factor (tnf) levels in a canine model of lethal bacterial septic shock (clinical research.41:240, 1993) . methods: awake 2 ylo beagles had serial cardiopulmonary and laboratory studies before and for up to 10 days after intraperitoneal placement of an e. celi infected clot. nine days before and daily until 3 days after clot placement, animals received high (n=17) or low dose (n=17) g-csf or protein control (n=20) subcutaneously. results: survival in high dose g-csf animals (14/17) was significantly improved compared to low dose (10117) and controls (12120) (p<0.04 wilcoxon). high dose g-csf also improved cardiovascular function evidenced by a higher mean left ventricular ejection fraction (day 1 after clot, p<0.001) and mean arterial pressure (day 2, p<0,02) compared to low dose and controls. high dose rcg-csf increased (p<0.001) peripheral neutrophil numbers both before and after clot implantation (2 hours to 4 days) compared to low dose and controls. in addition, high dose rcg-csf produced a more rapid (p<0.0001) rise (day 2) and fall (day 4) in alveolar neutrophils determined by bronchoalveolar lavage compared to low dose and controls. lastly, high dose rcg-csf decreased serum endotoxin (2 to 8h, p<0.002) and tumor necrosis factor (tnf, 2h, p<0.02) levels compared to low dose and controls. discussion: these data suggest that therapy with g-csf sufficient to increase peripheral neutrophil numbers during peritonitis and septic shock may augment host defense and endotoxin clearance, reduce cytokine levels (tnf) and improve cardiovascular function and survival. the use of g-csf in sepsis prophylaxis in neutropenic patients is well established and has been ascribed to accelerated recovery in granulccyte counts. here, an additional sepsis-prophylactic property could be demonstrated in healthy volunteers: eleven volunteers were employed in a sinqle-btind, controlled study and were given 480 uq g-csf or saline placebo via subcutaneous injection. blood was withdrawn immediately before and 20 or 44 hours later. lps-inducible tnf, il-1, stnf-r p75 and il-lra were assessed in the supernatant of whole blood incubations stimulated with 10 ug/ml lps from salmonella abortus equi. similarly to previous animal studies, lps-inducible tnf was attenuated by about 50% 20 hrs. after treatment. the same was true of il-lb. in contrast, lps-inducible stnf-r p75 which was indetectable in blood incubations from untreated donors increased dramatically 44 hrs. after g-csf treatment. il-lra found after lps challenge was increased tenfold by g-csf treatment. it is concluded that g-csf treatment switches peripheral leukocytes to an antiinflammatery state characterized by an attenuation of il-i and tnf releasing capacity and an augmentation of the release of cytokine antagonists. this findinq minht offer a novel concept in septic shock prophylaxis. objective.the aim of the study was to investigate the effect of recombinant human g-csf (rhg-csf) on survival, bone marrow neutrophil myelopoiesis, neutrophil counts, levels of bacteria and some important sepsis mediators in a model of rat abdominal sepsis. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. rhg-csf was administered as 10 /.tg/kg iv every 12 h, first dose at sepsis induction. bone marrow neutrophi] progenitors were determined as blast colonies, cfu-gm and cfu-g. neutrophils and bacteria were determined in peripheral blood and peritoneal fluid. lps, tnf, endothelin 21 and lactate were measured in blood from femoral vein. mortality rates were registered with g-csf treatment starting either 1 or 7 days before or 4 hours after cp. results. mortality was reduced from 90% to about 50% with rhg-csf intervention and there was no difference between the pretreatment and treatment groups. bone marrow blast colonies were not influenced while neutrophil myelopoiesis was augmented at the stages of cfu-gm and cfu-g. neutrophils in blood and peritoneal cavity were enhanced and numbers of bacteria in the same compartments were substantially reduced. circulating lps, tnf, endothelin 21 and lactate were attenuated the first 24 hours after cp. neutrophil myelopoiesis is augmented with increased number of neutrophils in blood and peritoneal cavity, resulting in enhanced clearance of pathogens. lps, tnf, endothelin 21 and lactate are suppressed the first 24 hours during sepsis course. a. wendel, j. barsig, g. tiegs gm-csf stimulates the proliferation and differentiation of granulocytic and monocytic progenitor cells. in addition the hemopoietic cytokine activates the inflammatory response in mature leukocytes. the priming effect of gm-csf towards lipopolysaccharide (lps)-induced cytokine production in vitro has been described, but little is known about proinflammatory gm-csf effects in vivo. we detected gm-csf in plasma of lps-challenged mice with kinetics similar to tnf, reaching peak levels 2h after lps administration. gm-csf pretreatment (50 ~tg/kg i.v.) enhanced mortality in mice challenged by a sublethal dose of lps. plasma levels of tumor necrosis factor (tnf) and interleukin-6 (il-6) were significantly enhanced. a monoclonal antibody, which neutralizes gm-csf bioactivity, rendered mice less sensitive towards lethal lps-challenge. tnf-and il-6-tevels were reduced in these mice compared to control animals without antibody treatment. in addition, severalfold potentiation of lps-induced cytokine release by gm-csf was observed in vitro in murine bone marrow cell cultures. these data demonstrate the proinflammatory capacity of gm-csf and suggest that the hemopoietic cytokine plays also a role as an endogenous modulator of lps toxicity. immune dysfunction, developing in the wake of multiple trauma, overwhelming infection and other forms of critical surgical illnes% is associated with increased infections, morbidity and mortality. the mechanisms responsible for alterations in immune regulation are incompletely understood but monocyte appear to play a central role. polymorphonuclear leukocytes (pmn) are known to play a central role in the inflammatory response of the host toward invading microrganisms. reports of defects in all the aspeots of pmn function have been accumulated in recent years. the possible role of gm-csf in modifing the state of immuno suppression detected in severe intraabdominal infected pt~. inspite of surgical appropriate procedures and in reducing the expected mortality is investigated. the safety of rh-gm-csf administration in sepsis is also evaluated. a double blind randomized study is proposed. this study include 60 icu patients who do not exhibit signs of shock and/or ards, with clinical signs and symptoms of abdominal infection. immunodepressed patients-aids, chronic chemotherapy or chronic steroid administration do not partecipate to the study. patients will receive rgm-csf (l~g/kg/day) or placebo in 24 hs. continuous infusion for 15 days. safetyandefyieacy will be assessed till to day 30. the apache ii score is adopted for risk stratification of patients because it is reliable and validated, objective and composed of information that is indipendent of diagnostic criteria. patient's entry criteria is apache ii > 16 (score 16 corresponds to expected mortality rate of 38%).in this protocol the surgeons report the judgement of the efficacy of surgical procedure to remove or not the focus of infection. objectives: infections and subsequent septic responses remain the leading cause of death among surgical intensive care (sicu) patients despite tmprovetaunts in supportive care and brond-epectrum antibiotics. usually invading bacteria are efficiently cleared by neutrophil granulocytes. however, during sepsis various neatrophil dysfunctions have been demonstrated, leading to impaired host defense. granulocyte colony-stimulating factor (g-csf) induces a sustained increase in circulating neutrophils and enhances various noutrophil functions. it was the purpose of the present study, to evaluate the safety and efficacy of g-csf (filgrastim) in sicu patients at risk of sepsis. materiel a.d methods: the study was designed as an open-label phase-ll study of filgrastim. ten consecutive slcu patients, with a therapeutic interveotion score greater than 30, were included in the study. filgrastim was given by daily continuous intravenous infusion for 7 days or discharge from the sicu. apache ll-score, multiple-organ-failure (mof) score, definitions of infections, sepsis, systemic inflammatory response syndrome (sirs), and acute respiratory failure were applied daily. a response to filgrastinl th_erapy was defined as an improvement in disease severity quantified by a decrease of > 4 apache 1i score points on day 4 after onset of treatment. results: none of the 10 patients developed a sepsis or mof later on and no patient died during hospitalization. specific postoperative complications occured in one patient ~jth a leekage of the oesophagou-gastric anastomosis after oesophageus resection. at study entry the leucocytes amounted to 10.110 + 2.000/~tl (mean + sem) and reached a level of 29.280 +_ 5.810/tal at day 6 after onset offilgrastim therapy. the apache ii score initally was 14 + 1.57 (mean + sem) and as an indicator of filgrastim response a decrease of 5 points ~dthin 4 days oceured in 7 out ot 10 patients. filgrastim was well tolerated, side effects were not noted. growth of solid tumors might be modulated by the activity of inflammatory and/or immune effector cells of undefined specificity. in this study patients undergoing surgical treatment for gastric (n= 41) or colorectal (n= 47) cancers were evaluated for endogenous serum levels of granulocyte colony-stimulatingfactor (g-csf) during a pre-and postoperative time period. from the same blood specimens mononuelcar cells (mnc) were prepared. the release of ifn-%, and il-2, which are secreted by thl cells, were stimulated in vitro by pha during a cell culture period up to 48 hours. the patients were further classified for their immunreactivity by responses in dth skin testing to seven different antigens (e.g. tetanus toxoid, ppd, diphtheria toxin, trichophyton, streptococcus, candida and proteus antigens). dth testing has been repeated in each patient two remarkable results were obtained. the serum levels of endogenous g-cse showed a biphasic increase with maximum values of 900 pg/ml (preoperative < 70 pg/ml) on day 1 and day 3 to 5 after surgical treatment. similar patterns of g-csf production were found in both groups of patients with gastric or colorectal cancers. high serum levels of g-csf were significantly (p < 0,01) correlated with infectious complications in patients whh gastric cancer (n= 17/22). secondly patients could be arranged into two groups according to an anergic (n= 25) or normergi¢ (n = 63) responsiveness in dth testing. the frequency of anergi¢ responsiveness was similar in both patients with gastric (n= 11/41) or colorectal (n= 14/47) cancers. interestingly we found a significant correlation (p < 0,01) between low serum levels of g-csf and anergy during the postoperative period in both groups. stimulation of mncs from anergic patients (n= 30) within the pre-and postoperative period resulted in reduced mean values (about 50%) for ifn-ff release (preoperative means llo0 pg/nfl), if compared to patients with normergic dth (n= 63, preoperative means 1960 pg/ml). similar, but less significant results were obtained for il-2 secretion. our results confirm a correlation between infectious complications and g-csf in the postoperative period, however elevated levels were also found in some patients without any signs of infections. more interestingly there might be an association between cytokine (c~csf, ifn-% and il-2) release and dth, which is known to be mediated by activated thl calls. to recognize anergic dth as a possible higher risk in the postoperative outcome of cancer patients extended periods of observation are needed. objectives of the study effects of recombinant huraan granulocyte colony-stimulating factor(rhc-csf)a galnst severe septic infections were investigated by its single use or by its corn b{nation with cephera antibiotlcs.we examined its effects on the mortality,and circulating blood neutrophyis counts and functlons,such as phagocytic activity and h 2 02 production using the rat severe septic model. rats were subcutaneously administsrd rhc~csf(s0orl0o ~ g/k~ body wt)after on set of peritonitis brought about by cecal ]igation and one puncture withe2-gaug e needle once a day for three days.in addjtlon,cefmetazol na(cmz)(50m$/k9 bo dy wt)was injected intrarnustularly to the rats tv~ce a day for three days. cirehlatlng blood neutrophyls counts were determoned electronically with a hem ocytometer,and blood smears stained with may~runwaldm.qlemsa~taln. neutrophyls functions in vltro,such as phagocytic activity and h 20 2 producti on using the rat severe septic model was analyzvd by automated flow cytometri c single cell-analysis methods. the reortallty rate after 2 weeks was significantly decreased by administratlon of rh~-csf(p<0,01).ln addjtion,a combination therapy of rhg-csf wlte cephern ant~biotics(cmz)showed a significantly survive] advantage and the rate had b een reached 57.1%. nextly,treatn%ent wlth rhg-csf(s0 ~ $/k9 body wt)increased the nuzaber of the peripheral blood neutrophjls slgn[fieantly(p<0.01). iv~oreover,functions of neutrophlis which were phagocytic activity and h2 02 p roduction were remarkably enhanced by admlnlstratlon of rhg-cs~(50 ~ 9/ks b ody wt) (p<0.(15). these findings suggest that combination therapy of rhcrcsf with cephern antib iotlcs(cmz)is an efficient regime against severe infectlons.and the increased ne utrophils counts and enhanced neutrophiis functions were played a important ro le about the survival advantage. granulocyte macrophage colony-stimulating factor (gm-csf) is a haematopoietic growth factor active on neutrophils and macrophages. leukopenia often occurs following renal transplantation and can be associated with infection and/or the myelosuppressive effect of azathioprine. aim: we report the use of gm-csf in 17 renal allograft recipients with leukopenia. nonglycosylated recombinant gm-csf was obtained from e. coli transvected by human gm-csf gene. m~terial ~,nd methods : written informed consent was obtained from all patients. patients were suffering from toxic neutropenia (neutrophils < 500/mm 3) with medullar hypocellularity on bone marrow aspiration, or leukopenia (neutrophils < 1000/ram 3) with cytomegalovirus infection requiring ganciclovir administtation. gm-csf was given subcutaneously at a dally dose of 2 to 5 mcg/kg/day, according to renal function. results : in all cases, neutrophil counts returned to normal levels within 1 to 4 days. in most of them, spectacular correction was observed within 24 hours, with a single injection. adverse events due to gm-csf at this dose were mild and easily managed (2 cases of bone pain treated with paracetamol). one acute rejection episode was observed after correction of leukopenia. conclusion : on the basis of this study, it appears that gm-csf at a dose below 5 mcg/kg/day is an effective treatment for renal transplant recipients with leukopenia associated with cmv infection or toxic neutropenia. department of nephrology, 161, rue de s~vres, hopital necker, 75743 paris, france. changes in serum g-csf and il-6 after surgical intervention hitoshi toda 1, atsuo murata 1, hidewaki nakagawa 1 , takesada mori 1, nariaki matsuura 2 1osaka university medical school, osaka, 2wakayama medical school, wakayama, japan we measured serum immunoreactive interleukin 6 (il-6) and granulocyte colony-stimulating factor (g-csf) levels of the patients undergoing major thoraco-abdominal surgery for esophageal cancer. serum samples were collected from eight patients on the day before surgery, at the time of operation, and thereafter at suitable intervals for one week. il-6 and g-csf were measured by means of enzyme linked immunoassay. the normal range of serum ]l-6 was less than 2 pg/ml and g-csf less than 4 pg/ml. values between groups were compared with linear regression analysis. both serum g-csf and il-6 levels reached their maximal levels at the first postoperative day and decreased thereafter. the correlation between g-csf (y) and il-6 (x) was y=3.28x+6.16 (r=0.786, n=86, p<0.01 ), showing a significant correlation. in the case who suffered from aspiration pneumonia and ards at the second postoperative day, the peak level of il-6 was 50 pg/ml and g-csf 69 pg/ml respectively. the estimated value of g-csf was 171 pg/mi by the regression equation. this means the real g-cse level was less than half of the estimated value. it suggests that low responsiveness of g-csf is one of the reason of immunodeficient state after the major surgery, neutrophils from injured patients ingest and kill bacteria less efficiently as compared to those of healthy individuals, probably reflecting the suppression in respiratoly burst which occurs after severe trauma. one of the main mechanisms of killing bacteria by neutrophil granulocytes is production of oxygen radicals (respiratory burst). granulocyte colony-stimulating factor (g-csf), a 20kilodalton cytokine, leads to a sustained, dose-dependent increase in circulating neutrophils. thus, it was investigated whether filgrastim (recombinant human granulocyte colony-stimulating factor, rhg-csf) therapy fits for prophylaxis of sepsis in postoperative/posttraumatic patients, and whether, besides an expected increase in neutrophil count, filgrastim would also augment neutrophil function. material and methods: this study was designed as an open label, prospective phase ii study of filgrastim and performed in a surgical intensive care unit (sicu) (university hospital). 10 postoperative/post-traumatic patients with a therapeutic intervention scoring system (tiss) score greater than 30 were treated with filgrastim (0.5 -1 l.tg/kg/day) for prophylaxis of sepsis on 7 days or until discharge from the sicu. production of oxygen radicals can be quantified by analysis of fmlp-and zymosan-induced chemiluminescence. neutrophil oxygen radical production was tested by fmlp-and zymosan-induced chemiluminescence by the polymorphonuclear cells (pmn) of these patients in multiple blood samples over a period of up to 14 days. results: none of the patients treated with filgrastim for prophylaxis of sepsis developed sepsis. in vitro fmlp-induced (10 -7 reel/l) neutrophil oxygen radical production was significantly increased under therapy with filgrastim by a maximum of 797% +-570% (217% -1826%) compared to pretreatment values of 100%. tapering of filgrastim resulted in a reduction of fmlp-induced neutrophil oxygen radical production within 48 hours. in contrast, zymosan-induced neutrophil oxygen radical production was not affected by filgrastim treatment. conclusions: besides its quantitative effect on neutrophil counts enhanced neutrophil function, documented here as increased fmlp-induced oxygen radical production, may account for the beneficial effect of filgrastim for prophylaxis of sepsis in posttraumatic/post-operative patients. granulocyte colony stimulating factor (g-csf) and granulocytemacrophage colony stimulating factor (gm-csf) have been recently introduced in the treatment of chemotherapy-induced neutropenia. effects of these csfs on cellular immune system were evaluated in 38 neutropenic gynecological cancer patients during chemotherapy. g-csf and gm-csf were equally able to induce a rapid recovery of white cell count within one or two days. g-csf treatment resulted in a significantly higher concentration of leukocytes measured in the peripheral blood although by gm-csf a sufficient effect was achieved (p<0.05). before initiation of csf treatment urinary neopterin was similar in both groups of patients (283+/-38 and 267+/-40 lamol/mol creatinine for gm-csf and g-csf respectively expressed as mean +/-one sd). in g-csf treated patient only a marginal induction of neopterin was observed. on day 4 the mean value was about 40% above the basal level (p<0.05). on the other hand gm-csf treated patients were characterized by a pronounced increase in urinary neopterin levels. in comparison with the basal level a more than 2 fold induction was noted and the difference between g-csf and gm-csf was highly significant (p<0.01). this effect was confirmed in vitro by investigating the effects of these csfs on interferon-gamma mediated pathways in thp-1 human myelomonocytic cells. results suggest activation of immune effector cells by gm-csf which may help the organism to overcome infections. however, activated macrophages produce several growth factors which may increase malignant proliferation, and augmented neopterin production as sign of macrophage activation has also been associated with poor prognosis m several malignancies. more data are therefore necessary to clarify whether csf mediated immune activation is beneficial or deleterious for cancer patients but considering our results caution in applying csfs in oncology seems advised. from a historical perspective, the development of humoral immunity to bacterial endotoxin has assumed a prominent position in the spectrum of therapeutic approaches which have been explored for the treatment of gram negative septic shock. predicated upon the fact that rough strains of bacteria manifest lps containing exclusively conserved structural features common to lps from all gram negatives, specific antibodies were elicited which conveyed cross protective immunity in experimental models of bacteremia and endotoxemia. such studies culminated in a well-conducted, randomized, double-blind placebo-controlled clinical trial using passively administered human polyclonal antiserum to treat patients with suspected gram negative sepsis. the efficacy of treatment established in that trial spurred efforts to develop monoclonai reagents which, to date, have not been uniformly successful in reproducing those earlier studies with polyclonai antibodies. nevertheless, the numerous successes which have been documented in experimental models of endotoxemia continue to foster promise for this immunotherapeutie approach. several recent studies with human polyclonalimrnunoglobulin preparations containing antibodies reactive with lps and lipid a have yielded promising results in treatment of patients with sepsis. in addition, the recent development of an antiidiotypic monoclonal antibody which reflects an internal image of a kdo specific monoclonal antibody has provided an alternative experimental approach to generate anti-lps antibody. immunization of mice with the antiidiotype provides significant protection against subsequent lps lethality consistent with the development of circulating immunoglobulin specific for lps. thus, the use of polyclonal immunoglobulins contrives to provide an alternative and potentially cost effective method for the treatment of endotoxin shock. supported by r37 a123447 and pot ca54474. john holaday, anne fortier, shawn green, glenn swartz, john madsen, carol naey, and jan dijkstra entremed, inc.. rockville, md, 20850. at the time of diagnosis, the signs and symptoms of septic shock are an indication that the systemic inflammatory response is well underway; thus, it has been argued that the endotoxin "cat is out of the bag", and that subsequent passive immunization may be too late to achieve therapeutic benefit. our approach has been to evaluate active immunization as a prophylax~s against sepsis. mice were inoculated twice (two weeks apart) with liposomes containing dmpc[i.8], dmpg[0.2], cholesterol [1.5] , and monophosphoryl lipid a [20-200 gg/txmole phospholipid] by several routes (i.p., i.m.), and serum was collected 10-11 days after each inoculation. after a single injection, highest tilers of ab were produced in mice inoculated i.p., but mice inoculated by all routes produced anti-lipid a ab. following the second injection. ab levels were roughly equivalent in mice inoculated by all routes, regardless of lipid a concentration. mice vaccinated i.p. with liposomes containing 0, 20 or 200 gg lipid a were treated with cyclophosphamide to produce neutroperda and then challenged with e. cole in an infection model of gram negative sepsis. the lds0 for control (liposomes with no lipid a) mice was 3x10 bacteria; ld50 for mice vaccinated with 20p.g was 24x103 (8-fold increase in resistance) and with 200~tg was 48x103 bacteria (16-laid increase in resistance). mice vaccinated as before were also treated with actinomyein d to increase sensitivity to lps (salmonella minnesota) challenge in an endotoxemia model of grain negative sepsis. the ld50 for control (liposomes with no lipid a) mice was 30 ng lps; the ld50 for 20gg lipid a was 70rig lps (2-fold increase in resistance) and for 2001xg was 570ng lps (19-fold increase in resistance). mice were similarly vaccinated and challenged with an aggressive gram negative pathogen, francfsella tularensis. the ld50 of franciseua in normal mice or mice inoculated with liposomes without lipid a was 20-40 bacteria. in contrast, mice vaccinated with liposomal lipid a (200ggl survived challenges as high as 30,000 bacteria, (3 logs of protection). the impressive protective capacity of this vaccine did not correlate with ab liter in any of the sepsis models, nor did it correlate with classic nonspeeific events, such as macrophage activation. maerophages harvested from the peritoneum of mice vaccinated and protected against sequelae of gram negative infections did not spontaneously kill the bacteria in vitro, but could be activated by ifn-y for antimicrobial activity equivalent to that of macrophages from unt#eated mice. research is underway to defme the protective mechanism(s) activated by this liposomal-lipid a vaccine. intervention by monophosphoryl lipid a in septic shock jon a. rudbach, ribi immunochem research, inc., hamilton, montana, usa monophosphoryl lipid a (mla), the clinical form of which is called mpl®-immunostimulant, has been tested extensively as an intervenient material in septic shock. mla is protective when given to experimental animals prior to a live microbial challenge or challenge with lethal doses of microbial products or certain cytokines. this is shown with gram negative and gram positive bacteria, gram negative bacterial endotoxins, and gram positive bacterial exotoxins. furthermore, animals treated with a regimen of mla which results in a refractory state to a lethal dose of gram negative bacterial endotoxin concomitantly display increased resistance to a live bacterial challenge. thus, both endotoxin tolerance and nonspeciflc resistance to infection can be manifested simultaneously. also, prophylactic doses of mla do not interfere with other therapies given subsequently; an additive or a synergistic protective effect can be demonstrated with certain combinatorial treatment regimens, such as mla followed by antiendotoxin monoclonal antibodies. the preclinical studies were extended to human trials wherein the safety of agonistic doses of mla was verified. furthermore, when mla was administered to human volunteers 24 hr before challenge with a pharmacologically active dose of reference endotoxin, febrile, cardiac, tnf, il-6, and il-8 responses were all decreased significantly as compared with the responses of subjects pretreated with a control solution and challenged with endotoxin. human trials with mla are being extended into patient cohorts which have high probabilities of developing septic shock; this will expand the safety base and establish clinical efficacy for mpl®-immunostimulant. a considerable body of in vitro evidence supports the concept that the effects of lps on cells of the immune/inflammatory systems are controlled by interactions of lps with cd14. to evaluate if blocking lps-cd14 interactions has potential as a therapeutic in septic shock we have evaluated the effect of anti-cdi4 monoclonal antibody (mab) on lps-induced cytokine production and physiologic changes in an experimental model of endotoxin shock performed in cynomolgus monkeys. a novel model has been established where animals were treated with interferongamma for three days prior to infusion of highly purified lps over an eight hour period. in this model lps challenge resulted in marked release of eytokines in the blood, substantial hemodynamic changes, release of liver enzymes and alteration in lung permeability observed over a 24 hour period. to evaluate the effect of treatment with anti-cd14 mab, animals were given either nothing, an isotype control or anti-cd14 mab (5mg/kg) 30 rains, prior to the beginning of the lps infusion. evaluation of physiologic changes including mean arterial blood pressure and cardiac output, quantitative analysis of eytoldne levels including tnfct, il-113,1i,-6, il-8 and il-10, and liver enzymes during a 24 hour period revealed that treatment with anti-cd14 mab markedly attenuated all parameters of injury including decreased mean arterial blood pressure, increased cytnkine levels and the release of liver enzymes observed in animals given the isotype control mab or those not treated. administration of anti-cd14 mab to interferon-gamma treated animals not challenged with lps did not induce any detectable physiologic changes or increases in cytoldnes. these studies suggest that strategies to block lps-cd14 interactions will have utility in diseases such as septic shock or ards where lps plays a central role in initiating injury. preclinical studies with recombinant bactericidal/permeability increasing proteins (rbpi and rbpi23). p.w. "frown, dept. of preclinical science, xoma corporation, berkeley, california, usa. bactericidal/permeability increasing protein (bpi), from neutrophils, binds to and neutralizes lipopolysaccharide (lps); it also specifically kills gram-negative bacteria (gnb). these properties, which reside in the n-terminal half of the molecule, indicate potential therapeutic application in the treatment of gram-negative sepsis. the gene for human bpi has been cloned and recombinant holoprotein (rbpi) and a 23 kd n-terminal fragment (rbpi;3) have been produced in sufficient quantities for preclinical studies. both rbpi and rbpi23 bind to lipid a and neutralize the biological activities of lps derived from a variety of organisms, rbpi23 has equivalent antibacterial activity to bpi against rough gnb but is up to 30x more potent than bpi vs. serum-resistant and smooth gnb. rbpi and rbpi23 compete with lps-binding protein (lbp) for binding to lps under physiological conditions. consequently, both rbpi and rbpi23 block the cd14-dependent lpsinduced synthesis of the cytokines tnf, il-1, el-6 and il-8 in vitro. rbpi23 has also been shown to inhibit the lps-induced synthesis of reactive 02 metabolites, endothelial adhesion molecules and the procoagulant molecule tissue factor. in animals, rbpi has been reported to increase survival of endotoxin-challenged rats and mice, to inhibit the dermal schwartzman reaction in rabbits and to increase survival of neutropenic rats with pseudomonas bacteremia, rbpi23 increases survival and decreases cytokine production in endotoxin challenged mice and rats. it normalizes lps-induced changes in hemodynamic, pulmonary and/or metabolic parameters in lps-induced rats, rabbits and pigs. treatment with rbpi23 also increases survival and decreases cytokine production in bacterial challenge models in rats and mice. rbpi23 was not toxic to rats after 10 daily consecutive i.v. doses of 10 mg/kg. this combination of properties indicate that recombinant bpi may be useful in the treatment of sepsis. phase i/ii clinical trials of rbpi23 have begun. the discovery of lps binding protein (lbp) and subsequent identification of cd14 as a receptor for lps or lps-lbp complexes has resulted in a new understanding o£ how lps responsive ceils are stimulated. cd14 is found either as a glycosylphosphatidyl-inositol (gpi)-anehored membrane glycoprotein (mcd14) of myeloid cells or as a soluble serum protein (scd14) lacking the gpi-anchor. binding of lps to mcd 14 triggers cell activation while binding of lps-scd14 complexes to cells such as endothelial or epithelial cells that normally do not express mcd14 activates these cells. these pathways are shown in schematic form below. 4 ~di mcd14 plays a crucial role in presentation of lps to additional membrane components that make up a functional lps receptor. an immediate consequence of engagement of this functional receptor is protein tyrosine phosphorylation. the molecular mechanisms leading to these events will be discussed. understanding of these pathways will lead to the development of new therapeutic approaches to controlling host responses to lps. pretreatmen t posttreatment (before or after tnf peak) d) with different antibody dosages: 15 mg/kg ---0.1 mg/kg pretreatment with anti-tnfab prevented death in most model situations (except peritonitis), but also posttreatment up to 4h after sepsis induction was successful in the few studies performed. there is additional evidence that low-dose tnfab is partially effective. especially baboon anti-tnfab studies provided many insights into the pathophysiological sequences of sepsis induction, due to crossreactivity with human reagents. those events include the cytokine sequence with tnf-dependent il-i, il-6, or il-8, but also il-lra or stnf receptor release. granulocyte as well as endothelial cell activation were shown to be partly tnf related, and the procoagulatory response was influenced by anti-tnf treatment. from many animal studies the concept that tnf plays a pivotal role in sepsis is clearly evident and therefore anti-tnf therapy is a major candidate tbr clinical studies. the beneficial or harmful effects of tnf-mediated inflammatory responses depend on the clinical context. decreasing exaggerated tnf-mediated inflammatory responses may be useful in some patients with organ failure. tnfr:fc (immunex, seattle, wa) is a recombinant human protein composed of two identical extracellular p80 tnf receptors linked by the fc region of iggl. it neutralizes tnf with an affinity for tnf<z of 10-10 m and has a t1/2 of • 63h in normal humans. methods: 18 normal volunteers were randomized to receive either tnfr:fc vehicle (n=6), or low dose (ld) tnfr:fc (10 mg/m 2 iv, n=6), or high dose (hd) tnfr:fc (60 mg/m 2 iv, n=6) infused over 30 rain prior to endotoxin (e) (escherichia coil 0:113; 4 ng/kg iv) administration. plasma cytokines were measured using elisa and tnf bioactiv'dy. systemic hemodynamics were evaluated with indwelling arterial and pulmonary artery catheters and radionuclide heart scans and compared before and after fluid loading in subjects given vehicle with hd tnfr:fc. results: endotoxin-related symptoms were unchanged after ld or hd. peak temperature occurred at a later time point (4-5h) in subjects given tnfr:fc compared to vehicle (3h) (p=.004). at 3h, 5h (post fluid load ), and 8h, subjects given vehicle developed increased cardiac index and heart rate and decreased systemic vascular resistance index (all p=.0001 ). hd tnfr:fc did not alter this hyperdynamic cardiovascular response. ld and hd inhibited the e-induced teukopenta at i h post endotoxin (p<.03) and later leukocytosis (p<0.05). peak tnf bioactivity in subjects given vehicle was 154 + 44 pg/ml and <10 pg/ml in the ld or hd tnfr:fc groups (p<.001). two immunoassays for tnf were performed because the detection of receptor bound tnf varies with elisa. tnf (biosource) was decreased in ld vs vehicle or hd (p=.0001) whereas tnf (r&d systems) was increased after hd or ld vs vehicle (p<.001). decreased levels of il-8 (p=.00t), granulocyte colony stimulating factor (p=.03), and il-1 receptor antagonist (p=.001) were found after ld or hd vs vehicle. il-6 levels were lower after ld vs vehicle or hd (p=.006). il-10 levels were similar among subjects given vehicle, ld, and hd tnfr:fc. conclusions: ld and hd tnfr:fc delayed the febrile response to e but did not alter the early hyperdynamic cardiac response. this suggests that mediators other than tnf play an important role in the initial cardiovascular response to e in humans. tnfr:fc attenuated the release of some e and tnf-induced cytokines. ti~e antiinflammatory responses of tnfr:fc may be useful in some patients with disease processes resulting from excessive tnf-mediated inflammatory responses. the biosynthesis of tnf within macrophages is regulated both at the transcriptional and the translational levels. the 5' flanking region of the tnf gene contains several transcriptional control elements, including two kb enhancers similar to those of immunoglobulin genes and a "y box" similar to that of the mhc class ii promoter. these elements are critical for the enhancement of transcription noted after stimulation of macrophages with lps. despite these and other elements, transcriptional regulation alone accounts neither for the absence of tnf protein during normal homeostasis nor the profound increasein tnf production following stimulation with lps or other secretagogues. the translation of tnf mrna into protein is also tightly regulated via mechanisms involving the octameric "ttatttat" motif present in the 3'untranslated region of tnf, human inducible no synthase, and several other cytokine genes. this region not only destabilizes mrna, but also confers a translational blockade so that tnf protein is not normally synthesized despite leaky basal transcription. lps stimulation releases translational blockade and, together with enhanced trancriptional rate, accounts for significantly increased tnf secretion. opportunities for inhibition of tnf production are available at each of these two levels. agents which increase intracellular camp (pentoxif¥1ine, amrinone) inhibit accumulation of tnf mrna; in contrast, corticosteroids act primarily at the level of translation, inhibiting lps induced translational activation. understanding the regulation of tnf biosynthesis may assist in clinical strategies designed to regulate tnf secretion. the serine protease thrombin is formed by enzymatic conversion from its proenzyme form after coagulation activation and may enhance the inflammatory response in various ways. in the last step of the coagulation cascade, thrombin cleaves its substrate fibrinogen, thus forming fibrin. the fibrin clot may prevent phagocytosis of bacteria and promote local bacterial growth. thrombin can also stimulate a specific th.rombin receptor that exists on a variety of cells. for example, minute amounts of thrombin ma), stimulate platelets to develop tissue factor and boost the activation of more thrombin, thus initiating a positive feedback loop. other cellular effects of thrombin receptor stimulation include increase in intracellular calcium of platelets and monocytes, contraction of smooth muscle cells, increase in endothelial permeability, thromboxane generation by neutrophils and lymphocytes, pulmonary vasoconstriction, and enhanced cytokine production. animal experiments have been conducted with a variety of thrombin inhibitors including heparin, antithrombin ili, hiradin, and hirudin-like peptides. an alternative approach is the inhibition of the coagulation cascade at an earlier point in the cascades, or administration of the anticoagulant enzyme protein c work from our own group in a porcine model of lipopolysaccharide-(lps)-induced shock with disseminated intravascular coagulation has shown that prelreatment with recombinant desulfatohirudin (r-h) or with a preformed complex of human donor antithrombin iii with heparin or post treatment with r-h are able to block the degradation of fibrinogen and the formation of fibrin monomer. in addition, after pretreatment with r-h it was found that lps-induced lung injury was reduced. since both natural hirudin as well as r-h were well tolerated by healthy volunteers, adminislration of r-h may be a promising therapeutic approach in patients with sepsis and sirs. corticosteroids have been used in the management of various shock syndroms including sepsis. the data concerning the effects of this therapy is, however, conflicting and in large prospective studies, the use of early administration of high doses of corticosteroids have been found to give no beneficial effects in patients with sepsis. we have particularly been engaged in studies on possible effects of corticosteroids on the plasma cascade systems and on the mediator release from leukocytes. in in vitro studies, high doses of methylprednisolone were found to facilitate endotoxin induced generation of plasma kallikrein. furthermore, plasma prekallikrein and kallikrein inhibitor values decreased together with formation of kallikrein-c1 inhibitor complexes (1). in this in vitro plasma model we also found that methylpredoisolone alone in doses of 1 mg/ml induced contact activation with the formation of kallikrein-c1 inhibitor complexes. methylprednisolone was also found to have an additive effect on endotoxine induced decreases in kallikrein inhibitor functions. in the same experimental model on endotoxemia methylprednisoione was found to give an additive effect on activation of the initial part of the complement cascade (2) . activation of the terminal part of complement, however, was inhibited by the same dose of methylprednisolone. addition of methylprednisolone alone to plasma was also found to activate the initial part of complement. using a full blood model, we studied the effect of methylpredoisolone in modulating the endotoxin induced cytokine response. when 103 ng/l e. coli endotoxin was added to citrated blood from healthy human donors preinkubation with 20 gg/ml methylprednisolone significantly reduced the release of tumor necrosis factor et (79 %) and interleukin-6 (63 % ) at two hours after exposure to endotoxin. in conclusion, these studies show that corticosteroids might facilitate contact activation of plasma and reduce the function of major protease inhibitors. furthermore, we also observed that this drag in in vitro conditions facilitated activation of complement. on the other hand, corticosteroids were found to reduce endotoxin induced cytokine release from leukocytes in whole blood. these studies also showed a dose dependence of the effects of corticosteroids on endotoxin induced cellular and cascade system activation. different predisposing conditions like extensive trauma, infection or pancreatitis result in a remarkably similar inflammatory response. although thls inflammatory response primarily aims at the removal of devitalized tissues, destruction of bacteria and reestablishment of the integrity of host tissues, it may potentially become unregulated and generalized and thereby producing deleterious effects to vital organs or the body" as a whole. cytokines play an important role in the development of the systemic inflammatory response. most of their biological effects, however, are not directly mediated but exerted through other mediator systems. when the inflammatory response results in the formation of capillary leak syndrome and refractory hypotension, the unregulated activation of complement and contact systems appears to be the cause. both systems are almost exclusively regulated by c1-1nhibitor. am unbalanced degradation of the inhibitor protein was found to be associated with the onset of capillary leakage in various conditions such as bone marrow transplantation, severe burns, and septic shock. these observations suggest a relative deficiency of biologically active c3-1nhibitor in these septic shock like syndromes. therapeutic intervention studies were initiated using high doses of c1-1nhibitor concentrate. first results are promising, as in most patients the administration of the drug was followed by a quick and marked improvement of the patient's hemodynamics. whether the administration of c1-1nhibitor alone or in combination with other drugs is also sufficient to improve long-time survival has to be investigated in double-blind, placebocontrolled studies. salutary effect of human soluble complement receptor type-1 in models of adult respiratory distress syndrome g. feuerstein, m. dimartino, and *r. rabinovici, smithkline beecham pharmaceuticals, king of prussia, pa, usa, and *jefferson medical college, philadelphia, pa, usa adult respiratory distress syndrome (ards) is a severe pulmonary insufficiency syndrome associated with diverse diseases and injuries. ards is believed to be the consequence of a massive acute inflammatory reaction consisting of activated neutrophils infiltration into the lung. neutrophil activation is likely the result of multiple factors of which complement is believed to play a major role. a potent complement regulatory system in the form of the complement receptor type-1 (cr-1) exists on many cells where protection against complement injury is provided by inhibition of both the alternative and classical pathways for c3/c5 convertase formation. we have recently purified the human recombinant cr-1 in a truncated form (brl55730, tp-io, scr-1) and tested this soluble protein for protective effects in several models of ards. specifically, we have used the following rat models: 1) endotoxin-induced ards in paf primed animals; 2) 11.-2induced ards; 3) thermal injury (30% body surface)-induced ards; 4) acid aspiration-induced ards. ards-like features included edema (increase in lung wet weight and water content), neutrophil accumulation (histological inspection and myeloperoxidase assay) and increase in cells and protein in the bronchoalveolar lavage (bad. scr-1, 1-25 mg/kg, i,v., given as prophylactic (and in some cases, therapeutic) treatment significantly inhibited edema formation and leukocyte infiltration and, in some cases (e.g., endotoxin/paf or il-2), virtually completely. these comprehensive studies strongly support the therapeutic potential of scr-1 in ards due to diverse injuries. the xanthine derivative pentoxifylline (ptx) and several analogs of ptx have been evaluated for their protective effect in various animal models of septic shock. data from these in vivo studies demonstrate that (1) ptx suppresses lps-induced tnf release from activated macrophages; (2) ptx prevents tnf-induced pmn activation; (3) ptx attenuates lps-or tnfinduced acute lung injury as evidenced by prevention of increased pulmonary vascular permeability and detoriation of gas exchange; (4) ptx exerts its .protective effects even when administered following initiation of sepsis. the sum of these actions suggest that administration of ptx in sepsis may have therapeutic potential. hans hoffmann md, dept. of surgery, klinikum grosshadern, ludwig-maximilians-universit~.t, marchioninjstrasse 15, d-81377 m0nchen, germany. pentoxifylline [pof] and related xanthins are drugs of known haemorrheologieal activity and widely used clinically. recently, new pharmacological aspects of these established drugs could be demonstrated. it was found that pof selectively inhibits the formation of tnf but not il-6 most likely by causing accumulation of intraceliular camp via inhibiting phosphodiestersse activity, and, secondly, by inducing prostacyclin in different cell types. furthermore, pof is able to counteract tnf-mediated adherence of neutrophils to vascular endothelium and to inhibit fmlp-indueed respiratory burst in neutrophils. we and others have, therefore, studied its effect in different animal models. it was found that pof protected from increased pulmonary vascular permeability and sequestration of neutrophils into the lung in different animal models of acute lung injury. moreover, in pigs with induced faecal peritonitis the drug improved haemodyrmmle variables as well as pulmonary compliance and reduced the number of pulmonary neutrophila and lymphocytes. consequently, as a general outcome, pof could be found to improve survival in different models of haemorrhagie and endotoxie shock. the protective effect of pof was dose-dependent and observed even when pof was given up to 4 hours after endotoxin. in order to evaluate these pharmacological effects of pof in humans, we studied pof under controlled conditions of endotoxlnemia in volunteers. we found that pof selectively inhibits the lps-indueed endogenous formation of tnf without affecting il-6 and il-8. moreover, pof counteracts the lps-indueed initial leukocytopenia by interfering with the adherence of neutrophils in the microvasculature. meanwhile the inhibition of endogenous formation of tnf by pof could be demonstrated under different clinical conditions of acute and chronic cytokine release syndromes, {okt3 first-dose reaction, cancer-or tuberculosis-induced cachexla}. it appears worthwile and promising to investigate wether pof exhibits beneficial effects also in septic patients. objectives: to determine the specific role of paf in both lethal primate bacteremia and nonlethal human endotoxemia. materials and methods: two double-blinded placebo controlled studies were performed. first, ten baboons (14.5 +.7 kg), were randomized to a control group receiving 101° cfu/kg of intravenously administered e. co/i (n=5) and a treatment group (n =5), pretreated with .1 mg/kg of paf receptor antagonist ro24-4736 two hours prior to e. co/i infusion. in a second study, ten healthy male volunteers were randomized to a control group receiving 4 ng/kg of intravenously administered endotoxin (lps) (n=5) and a treatment group (n=5) pretreated with 10 mg of ro24-4736 eighteen hours prior to lps administration. physiologic parameters and cytokine levels were measured continuously in both studies for 24 hours. results: baboons pretreated with the paf antagonist had improved oxygenation (96 + 11 mm hg v 59 -+ 6 in controls, p < 0.05) and creatinine clearance 12 hours post e.coli (39.8 + 23.4 ml/min v 0.1 _+ 0.1 in controls, p < 0.05). similarly, volunteers pretreated with the paf antagonist experienced fewer symptoms, including rigors and myalgias at 1 hour post lps. this was in concert with a diminished peak cortisol (668 +_ 107 mmot/l v 959 +-159 mmol/l in controls, p < 0.05), epinephrine secretion (1057 + 165 nmol/l v 2029 + 431 nmol/l in controls, p < 0.05). hemodynamics and circulating tnfa levels (data not shown) were unaffected in either study by prior paf antagonism. concluslons: paf influences some components of the multi-organ failure syndrome in lethal gram negative sepsis and the counter-regulatory hormonal system in human endotoxemia through tnf-independent mechanisms. paf antagonists may serve as adjuncts to cytokine blockade in the treatment of gram negative sepsis. the mediator role of platelet-activating factor in gramnegative septic shock pierre g. braquet, david hosford and matyas koltai institut henri beaufour, le plessis robinson, france considerable evidence has been accumulated to support the concept that a paf/cytokine autogenerated feedback network is responsible for priming and amplification of inflammatory mediator release in septic shock. cytokines, such as tnf~x, il-1 and other interleukins interact with paf which then amplifies cytokine production, therefore, paf may be the principle mediator in endotoxin shock. a single factor identified as tnf is suggested to play a pivotal role in the fatal phase of septic shock. presumably excess tnf release is the result of amplification process primarily triggered by paf. one may assume that the high tnf concentration in the blood of 'non survivors' is the consequence rather than the cause of cell death. the dubious results of clinical trials with anti-tnf antibodies and il-1ra, a naturally occurring il-1 antagonist protein, have risen debate around the exceptional role of cytokines in the pathomechanism of septic shock or systemic inflammatory response syndrome (sirs) induced by gramnegative microorganisms. there are similar problems with the interpretation of the results obtained in clinical trials of monoclonal igm antibodies against e. coli endotoxin, ha-1a and e5. future studies will answer the question as to the effectivity of inefficiency of this treatment. perhaps when the plasma concentrations of lps and cytokines exceed a critical level, treatment fails to save the patients life. with regard the causal role of lps in septic shock, the putative role of bacterial porins may have to be taken into consideration. the marked release of paf induced by endotoxin leading to excess txa2 production may be responsible for the microvascular failure and death in septic shock. antagonists of paf markedly protect against the release of txa2, and may interrupt the vicious circle of amplification process. no produced by the inducible no synthase plays a pivotal role in causing vascular paralysis in lps-induced sirs. to explore the relationship of paf to no synthesis will provide better understanding of the pathomechanism of septic shock. several paf antagonists, including bn 52021, have undergone phase ii and phase iii clinical trials. the strategy in the treatment of sirs, however, remains multifactorial, since little is known about the sirs caused by microorganisms other than gram-negative bacteria. evidence has begun to accumulate which suggests that in sepsis excess production of lps and cytokines can lead to uncontrolled production of inos and that this might in part explain the severe unresponsive hypotension seen in some septic patients. a number of strategies have been explored in an attempt to limit excess no production. a favourite target has been competitive inhibition of nos by arginine analogues such as l-nmma. in animals, some investigators have shown that the haemodynamic effects of lps challenge can be attenuated by l-nmma, but there is a narrow toxic-therapeutic ratio. in a model of live e. cell sepsis, we have been unable to reproduce these findings. localisation of no production by immunohistochemistry suggests that no production is compartmentalised, and more subtle methods of regulation may be required if this approach is to have clinical valuc. anti-adhesion molecule strategies ch wortel, repligen corporation, one kendall square, building 700, cambridge, ma 02139, usa. the neutrophil has been implicated as a pivotal player in the pathogenesis of multiple organ dysfunction. an important first step in the process of neutrophilmediated organ injury involves the binding of neutrophils to endothelial ceils. this process is largely regulated by complementary adhesion molecules, some of which are present constitutively on the cell surface and others that can be upregulated in response to chemotactic and pro-inflammatory stimuli. several different adhesion molecules have been described. l-selec tin is expressed on the plasma membrane of neutrophils and is shed from the surface early in the inflammatory process. it is therefore thought to mediate the initial interactions with endothelial cells. e-selectin and p-selectin are endothelial adhesion molecules. e-selectin is not constitutively expressed but is upregulated by inflammatory mediators, particularly il-1 and tnf. p-selectin is present in the weibel-palade bodies within the endothelial cytoplasm and can be upregulated rapidly in response to thrombin, histamine, and oxidants. all selectins recognize oligosaccharides, particularly sialylated lewis x antigen. this carbohydrate moiety is expressed on many proteins, including the selectins themselves. the leukocyte integrins are glycoproteins expressed on the neutrophil surface and in the cytoplasmic granules. integrins consist of a common beta2 or cluster differentiation (cd)i 8 chain covalently linked to one of three different alpha chains (cd11 a, cd1 lb, cd1 lc) and exist on the cell surface as three distinct heterodimers. cd1 l a/cdi 8 is expressed on all leukocytes, whereas cd1 l b/ cd18 and cd1 lc/cd18 are restricted to cells of myeloid origin. cd1 i/cd18 interacts with intracellular adhesion molecule-1 (icam-1), its ligand on endothelial cells. icam-1 is a member of the immunoglobulin family of adhesion molecules. it is constitutively expressed on endothelial cells and can be upregulated by cytokines. the potential for monoclonal antibodies to adhesion proteins to reduce vascular and tissue damage has been studied in alarge number of experimental models. protective effects have been observed in a wide variety of inflammatory, immune, and ischemia-reperfusion injuries such as arthritis, bums, endotoxic shock, bacterial meningitis, autoimmune diabetes, nerve degeneration, allograft rejection, allergic asthma, acute lung inflammation, skin lesions, and ischemia-reperfusion models of the intestine, myocardium, lung, skeletal muscle, and central nervous system. protective effects have also been observed in animals resuscitated following hemorrhagic shock. since modulating the function of adhesion molecules may temporarily leave the host without effective defense machinery, safety evaluations are of utmost importance in evaluating this therapeutic approach. treatment of gram-negative septic shock with an immunoglobulin preparation: a prospective, randomized clinical trial ingolf schedel, ursula dreikhausen; birgit nentwig; marion hockenschnteder, dirk rauthmann, salim balikcioglu, rolf coldewey, helmuth deicher, md endotoxin may play a major role in the pathogenesls of muitiorgan failure in the context of the toxic process in septicemia induced by gram-negative pathogens. the hypothesis which has led to a prospective clinical study consisted in the idea of inhibiting the biological effects ofendotoxin during the early phase of septic process, and thereby attampting to attain a positive effect on the clinical course of the disease. -objecave: to evaluate the effectiveness ofa polyclonal immunoglobulln (ig) preparation containing igg, lgm, and iga as an adjunctive therapy for septic shock. -desigru prospective, randomized clinical trial. patients: fifty-five patients w~th septic shock were randomly allocated to two groups according to criteria of septic shock. -intervention: one group of patients (n = 27) received a commercially available immunoglobulin preparation (containing high titers of antibodies specific for determinants to bacteria1 endotoxin) during the first 3 days after inciosion in the study. the other randomized group (n = 28) did not receive any immunogiobulin preparation. -measuremems and main resmts: during the period of <6 wks after the beginning of clinically apparent septic shock, death related to the septic process occurred in one (4%) of 27 patients who received immunoglobulln. by comparison, nine (32%) of 28 control group patients died during this period (p <. 01 ). within the first 48 hrs after onset of the clinically apparent septie process, significaptly increased activity of circulating endotoxin and simultaneously decreased specifie igg serum titers to lipid a were detected in the group of nonsurvivors. the rationale for the use of polyvalent intravenous immunogiobulins (ivig) to treat severe infections stems from in vitro and animal studies documenting that high-dose igg enhance opsonization and phagocytosis of pathogenic bacteria. moreover, recent clinical studies have shown that in septic patients the phagocytic function of circulating polymorphonuclear neutrophils (pmn) is defective; the degree of such impairment correlates to the severity of the septic state, the beneficial effect of administering high dose igo in septic patients can be two-fold: i) ivig provides large quantities of antibodies against invading pathogens; the igg bound to the pathogens can opsonize their phagocytosis; 2) the opsonization of phagocytosis by exogenously administred polyvalent igo would be of special importance in those patients who present a significant defect of their phagocytic function, when they are challanged by a large bacterial invasion. a prospective, randomized, double-blind study was carried out comparing the administration of ivig (sandoglobulin@) vs, paeebo (human albumin) in severely septic surgical patients, only patients with gepis score >_20 (meaning a mortality risk >70%) were accepted into this protocol. patients were randomized to receive 0.4 g/kg of ivig or placebo on day 0 (when they reached sepsis score>20), repeated on day +1 and +5. at the beginning of icu treatment, the two groups of patients were similar for severity of sepsis, age, concomitant disease, type of surgical procedures, antra and perioperative procedures, antibiotic administration. the results of the study indicated a significantly reduced mortality in patients with severe surgical sepsis treated with ivig as compared to placebo control patients (mortality: 38% vs, 67% respectively; p< 0,05). in conclusion, the results of our study in patients with severe surgical sepsis were the following: 1) ivig plus multimodal treatment of sepsis, including antibiotics, reduce mortality significantly', 2) the reduction of mortality seems to be due to a decreased incidence of lethal septic shock. despite substantial clinical research, the avallable data regarding the effectiveness of supplemental immunoglobulin (ig) treatment in sepsis in adult patients do not yet allow definitive conclusions. in view of the persistently high sepsis mortality there is a need to continue clinical investxqations regarding supplemental sepsis treatmen~ in general, as well as concerning ig administration in particular. we present and discuss the protocol of the ongoing ,,score-based-immuneglobulin therapy of sepsis (sbits)" study. the protocol (theoret surg 8(1993)61-83) of this multicenter, randomized, prospective and double-blind trfal relies on the results of an observational trial on i.v. igg treatment in 163 patients with sepsis and septic shock (infection 19~1991)216-227), carried out as a prerequisite for the present trial. using microcomputer-based bedside routine score monitoring, we regard quantitative measures of severity of disease and sepsis: only patients with a certain degree of both severity of disease (apache ii score 20-35) and severity of sepsis (elebute sepsis score 12-27) will be included. by observing these previously validated inclusion criteria, this trial snould iqentify a priori and include patients with potentially optimal response to therapy, consisting o~ either placebo (0.i % albumin) or polyglobin n" -12 ml (0.6g)/kg on day 0 and 6ml (0.3 g)/kg on day i. with an anticipatedpopulation size of 800 patients the study should comply with the statlstical requirements (estimated mortality: 30%, with a 33 % reduction in 28-day mortality in the treatment groupl to prove or disprove the question of igg effectiveness in sepsis in terms of improved prognosis. up to november 1993, more than 460 patients had been included; patient enrollment will be finished in 1994. previous studies have demonstrated rhll-i ra, a naturally occurring antagonist of il-1, increases survival in animal models of andotoxemia and eschehchia coli bacteremia and attenuates the decrease in mean arterial pressure resulting from challenge with both gram-negative and gram-positive bacteria. previously, in 99 patients, rhll-lra was demonstrated to increase survival in patients with sepsis syndrome and septic shock in a dose-dependent manner. methods: a randomized, double-blind, placebo-controlled, malticenter, clinical trial enrolled 893 patients at 63 academic medical centers in europe aad north america. eligible patients received either placebo (vehicle) or rhil-lra (anakinra) 1.0 or 2.0 mg/kg/hr by continuous intravenous infusion for 72 hours. the presence of organ dysfunction (i.e., ards, dic, renal, and hepatic) at study entry was determined prospectively by a clinical evaluation committee using definitions which were developed a-priori. survival time was evaluated over 28 days utilizing a linear dose-response model, assuming a log-normal distribution. results: 563 patients had one or more sepsis-induced organ dysfunction(s) at study entry. a dose-related increase in survival time was observed with rhll-lra compared to placebo in patients with ards, dic, and renal dysfunction (p --< 0.04 endotoxin infusion releases platelet-activating factor (paf), a potent phospholipid mediator which leads to an autocatalytic amplification of cytokine release. bn 52021 (ginkgolide b), a natural paf receptor antagonist, has provided significant protection against sepsis in different animal models• a randomized, placebo-controlled, double blind, multicenter trial on efficacy (mortality at d28) and tolerance of bn 52021 (2 iv infusion of 120 mg x 2/day over 4 days) in severe sepsis has enrolled 262 pts. the 28day mortality rate was 51% for the placebo group and 42% for the bn 52021 group (p = .17). the efficacy of bn 52021 was greater in pts with gram-negative sepsis: the 28-day mortality rate was 57% for the placebo group and 33% for the bn 52021 group (p = .01). bn 52021 also reduced mortality among pts with gram-negative septic shock (mortality was 65% for placebo vs 37% for bn 52021; p = .01). using statistical adjusments for pronostic factors, the relative risk of death of the bn 52021 group was 0.61 (0.34 -1.08, 95% confidence interval; p = .09). this risk corresponds to an adjusted reduction in mortality of 39% for pts receiving bn 52021. no differences in mortality rates were found between the placebo and the bn 52021 groups in the absence of gram-negative sepsis• there were no differences in adverse events between the placebo and the bn 52021 groups. bn 52021 is a safe and promising treatment for patients with severe gram-negative sepsis. a confirming study, focused on gram negative sepsis, is in progress. v~3lliam a. kanus m.d. and the rhll-lra it has been traditional within the field of infection and sepsis to think in terms of specific indications for drugs based on the type of infecting organisms, advances in antibiotic therapy now control or ltnflt the growth of bacteria. the majority of deaths are now caused by either an initial overwhelming response to infection or subsequent multiple organ system failure attributed, in part, to the effects of intrinsic biologic responses of the host. type of organism, therefore, may not be as critical as determining the exact severity of the host's severity or risk of death from infection. we also know that both the relative benefit of a new treatment across groups and its absolute benefit for an individual patient will vary with their risk in a predictable fashion. we recently iuve~iguted the relationship between one measure of host response, the acute risk of death as prospectively estimated by u comprehensive risk mode[ for 28-day mortality (jamb. 1993; 270:12,33-1241) , by its retrospective application to the results from the phase in evaluation of recombinant human intcrlenkin-1 receptor antagonist (rhll. ira). we found that there was a significant interaction between the patient's predicted risk of mortality at the time of entry to the study and the ability of rhil-lra to prolong survival time (x2 = 7.6, p [] 0.02, log.normal) for all 893 patients in the trial• survival benefit began st approximately 24% baseline risk of 28-day mortality. for the $80 patients with a predicted risk > 24%, there was a 22% reduction (p=0,00$ log normal). when we examined the variation in patients above and below the 24% risk level with hazard functions, i.e., their daily risk of death during the study period, we found that placebo patients with < 24% risk had lltile acute daffy risk during the hlltial two days follawh~g study entry and this risk was little affected by rhil-lra, in contrast, patients with > 24% risk had high daily mortality risks during the tuttlal two days that high dose rhtl-lro substantially reduced. these results are compatible with our current understanding of outcome from sepsis and the proposed mechanism of action o£ immunotherapy, the earliest deaths from sop sis are secondary to an immediate inflammatory response followed closely by deaths secondary to multiple organ system failure, later deaths (after 14 days) are not as closely related to the acute effeete of the inflammatory cascade. because of the timing and action of most proposed tmmunotherapy, they may be capable of preventing mortality primarily in these initial two phases. in this study, an independent predicted risk of mortality reflected this mortality pattern ned illustrated the potential benefit of immtmotherapy. use of a predicted risk of mortality in the design and analysis of clinical trials could improve our understanding of the clinical benefit of these new therapeutic approaches. the systemic inflammatory response syndrome (sirs) is a term recently proposed to describe patients with systemic inflammatory responses to insults such as infections (sepsis), trauma, burns, pancreatitis, and other initiating events. patients with sirs may have similar activation of inflammatory mediators and similar outcomes independent of the initiating event. these outcomes include organ dysfunction and failure, shock, and death. challenges to the successful conduct of clinical trials in sirs include the complexity of illness in these patients and the important--but limited--clinical benefits of novel compounds that may be limited to selected patient subsets. addressing these challenges will require new tools and approaches. these will include more sensitive and appropriate endpoints, and the use of methods such as baseline risk adjustment, to allow detection of drug risk interactions not captured adequately by categorical definitions, such as sepsis syndrome. on the basis of supportive preclinical and phase i safety studies, we have initiated phase ii clinical trials of a novel bradykinin antagonist, cp-0127, in four sirs subcategofies: sepsis, multiple trauma, burns, and pancreatitis. each of these studies is designed to measure the effect of cp-0127 on mortality, organ dysfunction and failure, and activation of mediators. in addition to investigating rates of organ failure using standard definitions--a new endpoint--a continuous summary measure of organ dysfunction (the acute physiology score of apache tm iii) is being used to quantify the degree of organ dysfunction and the speed and pattern of recovery of physiologic stability. in the sepsis study, another new approach--a study specific risk model based on the apache ill database--has been developed which will be used to assign a pre-treatment baseline risk to each patient enrolled. the primary outcome variable will be risk adjusted survival time to 28 days. this type of risk-adjusted analysis may allow for more efficient and powerful trials and more accurate and useful indications for use. study purpose: in post-cardiac surgical patients (pat.) at risk for sepsis, the efficacy of early i.v. immunoglobulin (ig) treatment was compared to a matching historical control (con.) population. postoperative risk assessment: using apache ii scores lap) (first postoperative [pop.] day) in a pilot study phase, we were able to differentiate between the large population (95.2%) of pop. low-risk pat. (ap< 19; mortality: 1%) and the small groups of pop. pat. at risk lap= 19-23) and high risk lap_24) with a significantly higher mortality (14% and 76%, mainly due to sepsis). subsequently, among 1341 consecutive pop. pat. we prospectively identified and treated these pat. iq treatment reqimens: first study period (n =41): (gg (psomaglobin n a, tropon biologische pr~parate, cologne, frg, day 1:8 ml/kg, day 2:4 ml/kg). second study period (n=25): iggma (pentaglobin r, biotest, dreieich, frg, 5 ml/kg on days 1 to 3). results: ig pat. and con. were comparable in demographic data, operation characteristics and baseline disease severity lap and elebute sepsis scores). in contrast to con. (risk: n=21, high-risk: n-21), the ig pat. showed a marked improvement in disease severity (fall in ap), especially in the high-risk group (igg, n=26: p<o.05; iggma, n=13: p: 0.08). in this group, ig led to higher (p<0.05) response rates, defined as an ap score decrease ->7 within four days (igg: 54%, iggma: 62%; con.: 19%), and reduction in mortality (igg: 46%, iggma: 46%; con.: 76%), statistically significant (p<0.05) for ig treatment as a whole (igg and iggma). conclusion: given the good comparability of the study groups, our results indicate, despite the non-randomized design, that early supplemental ig treatment can improve disease severity and may improve prognosis in prospectively apache ii score-identified high-risk patients after cardiac surgery. objective. elevated plasma levels of endothelin (et) have been demonstrated in both experimental and human sepsis. et has been proposed as a sepsis mediator leading to vasoconstriction with tissue hypoperfusion and organ failure. the aim of the study was to determine the effects of sepsis treatment with volume resuscitation, antibiotics and the anti-lps monoclonal antibody es® on big et and active, 21 aminoacids et (et 21) in rat abdominal sepsis. methods. lethal peritonitis was induced with a 4 mm coecal perforation (cp) in male wistar rats. plasma levels of big et and et 21 were determined with amersham tm endothelin rias 4, 8 and 12 h after sepsis induction. experimental groups: 1. cp control, 2. volume replacement (vr); 0,9% saline 10 ml/kg/h continous iv infusion started after 2 h, 3. antibiotic; imipenem 40 mg/kg iv after 2 h, 4. e5®; 10 mg/kg iv after 2 h, 5. vr + imipenem + es® after 2 h. results. high concentrations of both big et and et 21 could be demonstrated after 2 h and lasting for 12 h after cp. neither volume replacement nor imipenem did influence the elevated plasma et. e5® significantly reduced et 21 both 4, 8 and 12 h after sepsis induction, but did not reduce big et. when es® was combined with vr and imipenem, reduction of et 21 was the same as for e5® alone. these results strongly suggest that bacteria and hypovolemia per se are not decisive stimuli for et production during sepsis. e5® reduces circulating lps and tnf which is the probable mechanism of the suppressed et 21 synthesis. the unaltered big et fraction after e5® treatment indicates conversion of big et to et 21 as the site of action responsible for reduced et 21. conclusion. lethal peritonitis in the rat is followed by elevated plasma levels of big et and et 21. e5® anti-lps antibody significantly reduces plasma et 21 while volume resuscitation and antibiotics failed to do the same. es® did not reduce plasma big et. pmx treatment on severe endotoxemia with multiple organ failure was safety and effect in prognosis, and sepsis related parameters. it was certified that reduction of plasma endotoxin was effective in severe endotoxemia. a. lechleuthner,s. aymaz, g. grass, c. stosch, s. dimmeler, m. nagelschmidt, e. neugebauer. ii. dept. surgery, university of cologne, germany. introduction: the cardiovascular therapy of hypodynarnic shock states is a challenging problem. in clinical as well as experimental studies beneficial functions of a new hg-agonist bu-e-75 in congestive heart failure has been demonstrated 03aumann, 1989). therefore, we investigated the effect of bu-e-75 in hypodynamic shock in pigs. materials and methods: pigs (deutsches hausschwein, pitrain, [20] [21] [22] [23] [24] [25] were anesthesized with fentanyl/dormicum, ventilated (n20:o 2 = 2:1) and cardiovascular parameters were monitored with a complete icu-eqnipment. the hypodynamic model was established in a pilot study (4 animals) to evaluate the effective concentration of bue-75 in healthy and endotoxin (lps)-treated animals. endotoxic shock was induced by continous infusion of 10 ~g lps/kgkg/h (055:b5, fa. difco). the hypodynamic state was defined as a decrease of cardiac output by 30 % of steady state levels. a wedge pressure of 10-12 mmhg was kept constant by volume resucitation during the experiment. in a subsequent randomized controlled trial (rtc) 3 groups with 6 animals per group were studied. the groups were treated as follows: group i, lps and 0,9 % nac1; group ii, lps and bu-e-75 (100 #g/kgkg/h); group iii, famotidine (h2-blocker) pretreatment (1 mg/kgkg), lps and bu-e-75. results: the pilot study in healthy pigs revealed, that bu-e-75 had positive inotropic effects. these effects were inhibited by the h 2antagonist famotidin. bu-e-75 however had no beneficial effects in the hypodynamic phase of endotoxic shock in the rct. cardiac index (ci) and the oxygen delivery (do2) were not significantly influenced by bu-e-75 application (group i versus group ii). bu-e-75 did not ameliorate the negative inotropic effect measuring left ventricular stroke work (lvsw) in hypodynamic shock phases. on the contrary, bu-e-75 led to a further significant decrease of lvsw (p < 0,05). famotidin pretreatment did not affect the response (group iii versus group ii). conclusion: in hypodynamic shock states the h2-agonism seemed to have no beneficial effect under these experimental conditions. receptor down regulation or changes of signal transduction under septic conditions may be responsible. cellular studies may help to identify these mechanisms. objectives. antithrombin iii inactivation of proccagulant proteases is so far the only inhibitory therapeutic approach to disseminated intravascutar coagulation (dic). we therefore set out to investigate whether cll substitution reduces coagulation activation in an endotoxin induced rabbit dic model. materials and methods. 29 male rabbits chbb:hm(spf) were randomty assigned to one of the following groups. group k : naci 0.9% (control without endotoxin, n=10). group e : endotoxin 120 tjg kg "1 bolus i.v. + naci 0.9% (control with endotoxin, n=10). group c : endotoxin 120 pg kg -1 bolus i.v. + cll 400 u kg -1 bolus + 400 u kg "1 4h "~ i,v. (treatment group, n=9). all animals were anesthetized and mechanically ventilated. blood samples were drawn prior to endotoxin administration (m1) and after 120 (m2) and 240 rain. (m3). thereafter, lung and liver tissue samples were taken intravitatly in a standardized fashion for h&e microscopic fibrin quantification using a triple score (fibs). from all blood samples the prothrombin time (pt), activated partial thromboplastin time (aptt), fibrin monomers (fm), and d-dimers (dd) were measured. for statistical significance of differences between the groups anovas and the wilcoxon test (fibs) were performed. results. fibs for lung/liver were significantly different (p<0.05) between group e (lung 180, liver 159) and c (lung 89, liver 0) (group k : lung 11, liver 0). , a synthetic serine proteinase inhibitor, has an anticoagulant activity in the absence of" antithrobim iii. gabexate has been reported to be useful in the treatment of disseminated intravascular coaguiation due to neoplastic diseases. in this study, we investigated gabexate therapy for the treatment of dic due to sepsis in the postoperative critical patients. materials and methods: from july 1992 to june 1993, 15 patients in the surgical intensive care unit met the criteria of dic or pre-dic. eleven were male and four were female with the mean age of 66.7 years. all these patients suffered from some complication of operations which led to the development of sepsis. foy was administered at the rate of 2mg/kg/hr untii the coagulation profile retumed to normal or the patient died. the coagulation parameters were monitored before and on the 1st, 3rd, 5th and 7th day. results: fourteen of these fifteen patients died despite transient improvement of the coagulation parameters in five patients. these patients suffered from sepsis resulting from surgical complications which could not be well controlled. the only survival was a case of recurrent intrahepatic duct stone with biliary tract infection complicated with sepsis and dic. after choledocholithotomy and the use of foy, the patient recovered gradually. conclusion: dic is a late manifestation of sepsis in the critical surgical patients. the most important thing is to eradicate the cause of sepsis. if the underlying septic focus cannot be controlled, dic will persist despite the use of gabexate mesilate. emergency surgery, taipei veterans general hospital, taipei, taiwan. there are 2 main types of bradykinin (bk) receptor, namely bk~ and bk z. the bk 2 receptor is constitutive. the bk 1 receptor is also constitutive but in the majority of cases is inducible and involved in chronic inflammatory syndromes such as sepsis, hyperalgesia and airways hyperreactivty in animals. the mechanism(s) involved in the upregulation of the bk 1 receptor is unclear, however a variety of agents including lps, e coil and ill are particularly efficacious in vitro and in vivo. ill and bradykinin acting at their respective receptors are believed to be involved in sirs/sepsis. we have investigated the effect of antagonists at ill (antril), bk 2 (bradycor [cp-0127]),bk~ (cp-0298) and bkz/bk 1 (cp-0364) receptors on the de novo generation of bk~ receptors (reflected by hypotensive responses to a bk 1 agonist) in the lps-treated (10ug iv) rabbit. in lps treated rabbits hypotensive responses to bk~ but not bk 2 agonists increased with time and at time 210min appeared maximally induced. constant iv infusions of cp-0127 blocked bk 2 but not bk~ and cp-0298 bk~ but not bk 2 responses. cp-0364,cp-0127+cp-0298 and antril+cp-0364 blocked both bk 2 and bk~ responses. antril alone had no effect on bk 2 or bk~ responses. within 30-60 min after stopping the infusions of antagonists the responses to bk~ and bk z agonists were the same as those in nonantagonist infused rabbits. these results indicate, at least in the lps-treated rabbit, that neither bk2,bk ~ or ill receptors alone or in combination, are involved in the de novo generation of bk 1 receptors. in vitro studies demonstrated that beth bradycor and cp-0364 (but not antril) were antagonists at both bk z and bk~ receptors. if both bk z and bk 1 receptors are significantly involved in chronic inflammatory situations in man such as sirs/sepsis then the rationale for the use of compounds such as bradycor or cp-0364 is clear. infection is a major cause of or contributor for morbidity and mortality in liver transplant recipients. effectiveness of prophylactic and therapeutic protocols is important for the success of liver transplantation ( olt ). sdd is used as prophylaxis for reduction of infection caused by gram negative or fungal microorganisms. between september 1988 and july 1993 400 olt's in 368 patients were performed at our department. the actuarial 1-year patient survival is 90 %. infection prophylaxis is started with sdd and ciprofloxacin once the patient is accepted as an olt candidate. perioperatively metronidazol, tobramycin and cefotaxim, postoperatively cotrimoxazol are prescribed additionally. the table shows pneumonia, peritonitis, major wound and urinary tract infection are common nosocomial infections following severe injury. in a series of severely injured patients from the university of louisville hospital, pneumonia was the most common infection followed by peritonitis, intra-abdominal abscess formation and burn wound infection. pneumonia is actually the leading cause of death from nosocomial infection. these are defined as occurring from 48 to 72 hours after hospital admission. this definition has important implications for antibiotic therapy because the likely pathogens and their respective sensitivities are different for community acquired pneumonia. the diagnosis of nosocomial pneumonia is difficult following major injury as many patients will have pre-existing fever, leukocytosis, tachypnea, and chest x-ray changes. reliance on sputum gram stain and culture is important and best obtained by a bronchoalveolar lavage or protected specimen brush during bronchoscopy. predisposing risk factors include severe head injury, emergent intubation and shock, and such patients have been shown to benefit by early tracheostomy. staph aureus has been the most common pathogen isolated from the sputum and the remainder gram-negative organisms with pseudomonas aeruginosa, and klebsiella pneumonia predominating. bacteria recovered by site as well as by intensive care unit is published in the six month antibiogram which also includes recent antibiotic sensitivities. this aids in empiric antibiotic selection against such nosocomial organisms. in a series of severely injured patients (iss -30), mean temp. was 101.4f, leukocytosis was 16k, pan2 was 87, fin2 was .47, and peep was 5.1 at the time of diagnosis (ards excluded). there was marked reduction in class ii histocompatibility antigen (hla-dr) density on peripheral and bal monocyte/macrophages which recovered over time with resolution of pneumonia. immune suppression occurred prior to development of pneumonia, was especially localized to the infected tissue, but recovered with clinical improvement. specific immune modulation targeted to pulmonary white cells may hasten clinical recovery and minimize pulmonary dysfunction. -clinical experience j. tnllemar amphntericin b remains the drug of choice for many systemic fungal infections. its advantages include a broad spectrum of activity and intravenous administration. the major disadvantages of amphoterlcin b is its severe side-effects, especially the nephrotoxicity. to decrease the toxic side..cffccts various liposomal amphoteficin b formulations have been produced. it was found that these liposemal formulations were as effective as amphotericin b but in contrast had a low incidence of toxicity. at present there are three ~different variations of lipid formulations under assessment: amphotericin b lipid complex (ablc), amphotericin b coloidal dispersion (abcd) or true liposomes. the ablc has a ribbon like structure. it has been shown to have a reduced toxicity and an efficacy ranging from being as effective to four times less effective that conventional amphotericin b. regarding abcd the particles have a disk-like structure with a diameter of around t00 am and a thickness of 4nm. the ami-fungal efficacy is 2-4 times less than that of conventional amphotedcin b. both ablc and abcd are presently investigated in phase ii/iii studies in the us. ambiseme is currently the only commefieally available true lipesome. ambiseme is a spherical small unilamellar lipesome with a diameter less than 100 nm with a mutina ld50 of > 175 mg/kg. it has been used in dosages up to 6 mg/kg/day in compassionate based studies with good tolerability. the mycological efficacy range from a 83% response rate for invasive candida infections to 41% response rate for aspergillosis. ambisomc have been evaluated as anti-fungal prophylaxis in randomized trials in bone marrow (bmt) and liver transplant (ltx) recipients. it was well tolerated. in bmt recipients the incidence of proven fungal infections was 8% among placebo treated patients compared to 3% for the ambisome treated patients (ns). in ltx recipients ambisome prophylaxis was effective, significantly reducing the incidence of deep fungal infections from 16% to 0% ill placebo and ambisome treated patients respectively (p<0.01). prospective randomized trials comparing these various amphotericin b preparations with conventional amphotericin b is needed to determine their future place in the therapeutical arsenal. two patlentgroups ere particularly at risk to develop serious cmv disease: cmv seronegative transplant recipients of seroposltlva donors and those patlants treated for rejection with anti t-ceil preparations, we have evaluated the value of prophylactic anti-cmv immunoglobulin (cytotect", biotest pbarma gmbh, dreieich, frg) administration in high risk heart and kidney transplant recipients, in a double blind placebo controlled study 40 kidney transplant recipients, treated for biopsy proved re)action with rabbit atg, received globullntplacebo infusions. the preparatlons were given i,v, in a dose of 100 mg/kg at day 0,7,14,35,56 and 77 after the initiation of anti = rejection therapy, passive immunization completely prevented cmv related death, although it did not reduce th~ incidence of cmv isolation, viraemia or disease, this effect was mainly observed in cmv saronegativa recipients of a serop0sitive donorktdney. seroposltive recipients did not benefit from treatment and seronegatlve recipients of a seronegetlye donor were not et risk for cmv infection at e!l. in a open study the incidence of cmv infection and disease was evaluated in 143 consecutive i~eart sllograft recipients. sixty-five patients were cmv seronagatlve and they all received passive immunlzation according to the dosage schedule used in the kidney patients, but starting on the day of transplantation, this scheme resulted in median snti-cmv igg titers of 1200 elisa units during 3 months. cmv infection occurred in 21/65 ~eronegetlve and in 40/81 seropositive recipients (n,s,), in ssronegetive donor-recipients pairs the incidence was significantly lower (3/34] , the passively immunized seronegstive recipients of e seroposltlve donorheart showed comparable incidence of cmv infection f18t29) vs the seropositive recipients. primary infection more often resulted in disease than secondary infection (11121 v8 10/40), but no difference in incidence of disease (11165 vs 10/81 ) or severity in symptoms was noted between the immunoglobulln treated serone(]ative patients and the seropositiva recipients. apparently passive immunization induces anti-cmv immunity which crossly resembles naturally acquired resistance. abdulkadirov k.,chebotkevich v., moiseev s. the incidence of infection is still high in patients underwent bmt. this complication is the major cause of mortality if it is not recognized and treated promptly and properly. our data showed that from 21 patients with different types of leucemia after autologous and allogenzc bmt 19 had the episodes of fever. in the ma i ority of these episodes the bacterial etiolog$ gram negative bacflli and gram positive cocci) can be proved. on the other hand, in 62% of the fever cases we detected also viral respiratory (corona-, adeno-, rs-and other) infection. our previous investigations showed that even in healthy persons the viral infection has influence on antibacterial immunity, in the cases of model experimental reaction in volunteers we found the decrease of delayed hypersensitivity 10-14 days after intranasal inoculation of influenza virus a (h3n2-3) to bacterial (staphylococcal, streptococcal and pneumococcal) and ~iycoplasma pneumoniae antigens in the leucocyte migration inhibition test. these results showed that respiratory viruses may be the important pathogenic factor in the development of bacterial infection in posttransplanted period. we consider the constant control of latent and visual respiratory viral infection in bmt patients to be very important. ficcb the ~ter£~li of the nation~l institute of trad/~atoloqy in budapest 1.00 consecutive cases of revision hip grafting were carried out arthroplasties wlth hemoloquous bone between the years 1990 and 1993. in the same period of time 732 pri~ total hlp replacen~nts were performed under i4entieal technical conditions. the average septic rate for the 'total hip althroplasties was less than 1%. in the selected i00 cases the septic rate was 4% indicating the role of bone grafting° homografts were prepared by deep freezing~ it .is recognized that the cells of the hl~grafts become destroyed by the ium~unological, response of the host~ and the patients develop ~ti-hl~, ar~tib'o~ies. the dead ~trix, however, has a bone-inducing capacity that stimulates host osteoblasts to recolonize the *i~/trix which serves as scaffolding. the sequence of events favours the infections. for this reason, beside preventive perioperative systemic ant/biotic treatment, local ~ntibioties were also applied in the form of antibiotic-//npregnated cement. the role of age and the .immune status of the patients .is discussed.. the purpose of this study is to evaluate the rate of toxemia in patients with acute panereatitis and to find this coudition to the activation of cascade systems that are encountered in the subsequent complications of the disease. we studied a series of 45 patients with acute pancreatitis, the severeness of which was evaluated by the ranson's criteria and the apach-ii scoring system. all of them were considered to have severe acute puncreatitis. the determination of toxemia was made using the limulus test (lal test). we also determined the levels of the third (c3) and fourth (c4) complement components as weu as the coagulation factors, iibrinolysis faeters and kimns by serial measurements. the severity of the disease was serially determined by the apach-ii scoring system. it was found that complement activation ( which was also assessed using a graphically illustrated method by a aggregometer ) was followed by an increase of morbitity and mortality .we also detected that toxemia (positive lal-test) was closely correlated with complement activation and more of the ranson's criteria. a clear relation existed between the number of ranson's signs and the enmplieations' rate (1"= -0.766, p < 0.001). the documentation of toxemia and the complement activation cannot predict the kind and the severity of complications. the study of coagulation, fibrinolysis and kinms systems didn't reveal any results with statistical significance. necrotizing pancreatitis still represents a life-threatenthg disease. infectious complications dominate among the causes of death. differences in the individual immune response could possibly explain different clinical courses even in patients with comparable pancreatic morphology. to explore the inflammatory response in acute pancreatitis, the following investigation was performed. methods: peripheral-venous blood was withdrawn on admission and furthermore twice weekly in as yet 36 patients with acute pancreatitis and tested for the parameters mentioned below. in parallel, polymorphounciear granaiocytes were isolated using density gradient centrifugation and assessed for superoxide anion and hydroxyl radical producing capacity using electron spin resonance techniques. results: total leukocyte cotmt and total lymphocyte count did neither reflect the clinical course nor predict complications. this comes tree also for serum igg, igm, iga, c3, c4, crp, alpha-l-antitrypsin and neopterth as well as for plasma il-la, il-ib, il-1ra, il-2, il-2r, il-6r, tnf-ct, tnf-~r (p75) and icam-1. in contrast, pmn-elastase, il-6 and il-8 closely correlated to the clinical course. isolated pmn's in vitro capacity to produce oxygen radicals depended on the respective radical species and was slightly elevated (superoxide anions) or decreased (hydroxyl radicals), respectively. patients with a cd4+/cd8+ ratio below i were seen at risk of developing septic complications. in contrast, a percentage of monocytes of 30 % or more among total mononuclear cells indicated an uncomplicated course, in general. conclusions: the immune status of the individual patient may significantly influence the course of acute pancreatitis. the cytokine pattern in peripheral blood is very complex and most parameters are of little use for the clinician. the pmn-elastase, il-6 and il-8, however, closely correlate to the clinical course and may prove valuable for follow-up. the cd4+/cd8+ ratio was found the best predictor of septic complications, but it failed in non-septic patients. a percentage of 30 % or more of monocytes among total mononuclear ceils indicated a rather mild course. the reduced ability of the pmns to produce hydroxyl radicals may help to explain the frequent development of septic complications in severe necmtizing pancreatitis. peroxidation of membrane lipids contributes to ceil injury in pancreatitis. overwhelming release of toxic metabolites by infiltrating neutrophils is regarded a major pathogenetic factor, too. as yet little is known about the mechanisms by which oxidative stress and leukocytes damage pancreatic cells. the present study examines (i) the susceptibility of pancreatic acinar cells to attacks by oxidants and leukocytes and (2) the potential of antioxidants to prevent such damage in order to better understand the cellular mechanisms of pancreatic injury in inflammatory states. methods: freshly isolated rat pancreatic acinar ceils were exposed to a model system of oxidative stress consisting of 20 mu/ml xanthine oxidase (xod), 500 mm hypoxanthine (hx), 50 mm fec13 and 75 mm edta. in a second set of experiments, acinar cells were exposed to excess autologous neutrophils or neutrophils obtained from patients with acute pancreatitis. neutrophils were stimulated by zymosan a, pma, and il-8. cell viability was assessed by both cellular uptake of trypan blue (tb) and by release of ldh. results: the xod/hx system caused a time-dependent acinar cell injury. this injury was effectively prevented by catalase (cat) and gfutathione peroxidase (gpx). in comrast, superoxide dismutase (sod) enhanced cell injury. addition of both sod and cat abolished the damage seen with sod alone. the non-enzymatic scavengers mannitol, dmso, dmtu and the iron chelator deferoxamine were not protective and at a higher concentration even accelerated cell decline. the newly developed antioxidants of the lazaroid type effectively prevented oxidative acinar cell damage. stimulated neutrophils, both autologous and heterologous, did not damage healthy acinar cells but had even protective effects. conclusion: pancreatic acinar ceils are very susceptible to oxidative injury. a combination of catalase and sod prevented cell damage effectively. sod when given alone may rather damage than protect aelnar cells when h202 is generated in concentrations overwhelming the capacity of endogenous catalase. therapeutic approaches to pancreatic disease using antioxidants should, therefore, include combinations of protective substances. the lazaroids seem to be candidates for clinical use as antioxidants in pancreatitis. the results argue against direct toxic effects of stimulated neutrophils to pancreatic acinar cells. are ch~act~z~ by the presence of a polymicrobial flora, the pmtotyi~ cffthese inf~ons is secend~,y bacterial pedtonitlw, whereby a pathololoeal process in the ~trointesfimd tract r~ful~ in tim disrup~on ofi~ inteffrlty and ¢ollseqtlent sptl]nge of inte~.i,o~.l gontents into the peritoneal c~iry. the ensuing infection invariably contains a mixtm~ of gt~m negative enteric bacilli, gram positive b~eria and anaerobe& experimental and clinical =t~ies have de~ed the eantrlbution of each of th¢~ components to ti~ ovemu virulence of these in~ons, gram negative enteri~ such as f.veher~chla coil ere endowed with a virulent l~l~x~lyse~haride ptill~ly t~sponsible for lethality, by contrast, bacteroldes sl~cles, which rarely c~se death, prornot~ abscess fonllation, a uniqm~ capsul~ polyseccluu'ide, particularly on b.j~ogiljs slrai~, oontributes to tjtis erect, several mecltanims have bccn pml~ed whereby or~ microorganism mi~t interact with its microbial ~net to augment the overall virulence of a r~xed im~edan. these include: l) provision of nutrients by one apexes which stimulates the growth of its ~opathoge& 2) inhibition of host deletes by one of the migroorganisms so that the other microbes might persist and exert their virulence, 3) the trant~ of vim.©n~e traits between ~renr~a.,dsms and 4) the ~.mizatian d the mi~oe~vironmental con~tion$ by one d the baetez'isl pa#, so that the other might persist. exampl~ for each of these m~banisms imv~ been provided by experimental ttudies i~stigating e.co!l-b.p~flls synergistic in~ra~ons. byproducts ofg.coli metabolim l~¢ovide essential short ebath fatty acids £~ optimal b,frosili~ ga'owth. fm-ther, oxygen ¢ons~tmption by kcelt lowers oxygen tension end redox potantial to levels eomlucive to b#a#lts gro~h. coawr~ely, b,~agtlis rolea~s proteases and fatty acids wl~¢h impair pl'tsgocy~¢ ~lt rmctlon tnd permit f-..¢oli proliferation and expression of its intrinsic virulent. in summaxy, interactions among the separate microbial cemponents of mixed infections heighten the overall virttienee of these lafectiot~, this knowledge provides ~r rationale for targetting of antibiotic therapy against the knowa eantributors of these synergistic pro~¢sses, intraabdominal abscess formation and the macrophage william g. cheadle, m.d., department of surgery, university of louisville school of medicine, louisville, ky 40292 inflammation of the peritoneal cavity following bacterial contamination has been classified into primary, secondary and tertiary, the last two relating to bacteria originating from the gastrointestinal lumen. the natural history of such infection is either resolution without clinical sequelae, which is uncommon, abscess formation, or generalized peritonitis, which occurs as a result of failure of peritoneal host defenses. early clearance of microorganisms by peritoneal fluid circulation and filtration througti subdiaphragmatic lymphatics into the thoracic duct and systemic circulation occurs as well. simultaneously peritoneal macrophages and the omentum approach the area of inflammation and lead to neutrophil influx and abscess formation adjacent to the affected viscus. we have found a shift in peritoneal macrophage function from antigen presentation to proinflarnmatory cytokine production that occurs early after experimental peritonitis produced by cecal ligation and puncture. this is also reflected by reduced class ii histocompatibility antigen expression on peripheral blood mononuclear cells and peritoneal macrophages. this is accempauied by an influx of both neutrophils and macrophages into the peritoneum and subsequent abscess formation. interestingly, there is little serum endotoxin or tnf seen in this model despite tnf mrna expression in peritoneal macrophages. we believe this model is more clinically relevant than other models of endotoxemia or bacteremia in which different patterns of cytokine expression are seen. newer agents aimed at reduction of systemic manifestations of sepsis originating from intra-abdominal infection such as monoclonal antibodies against cytokines or il-1 receptor antagonists may need to be directed against remote organ macrophage populations while preserving peritoneal macrophage function. inflammation is a complex process involving microcirculatory changes, extravasation of fluid and a cellular influx in the affected body area. in our communication, we will only consider the regulation of the cellular infiltrate which plays a major role in the defense of the peritoneum against microbial invasion. until recently, it was thought that the influx of leukocytes in the abdomen was induced by bacterial products, local humeral factors and secretions of resident macrophages. there is now increasing evidence that this view is too simplistic. many other cell types present in the abdominal cavity or composing the peritoneal membrane (mast-cells, mesothelial cells, fibroblasts) are able to release or secrete vasoactive or chemotactic substances such as histamine, prostagtandines, or cytokines. they are most likely to play a role in the regulation of intraperitoneal inflammatory reactions. the emigration of leukocytes towards the abdominal cavity is also modulated by a previous contact with gram negative bacteria. in the rat, this intriguing phenomenon is long lasting, cannot be transferred by serum and seems independent from t lymphocytes. the clinical relevance of these various regulating mechanisms has still to be determined. kinnaert paul, h6pital erasme, route de lennik 808, 1070 bruxelles belgium generalized response in secondary peritonitis the clinical course of an intraabdominal infection may depend on a variety of variables including the capacity of host defense mechanisms and the degree of the inflammatory response. if local defense mechanisms fail to restrict the inflammation to the abdominal cavity a generalized inflammatory reponse will result. in a first stage generalized signs of a local inflammation become detectable whereas the second stage comprises the overwhelming systemic inflammatory response. the extent of this systemic response determines the outcome. sometimes it may appear to be unrelated to the severity of the intraperitoneal findings. the activation of plasma systems and cellular elements leads to a fast release of cytokines, inflammatory mediators and other substances. these parameters precisely reflect the degree of the generalized response. inflammation of the peritoneum causes significant morbidity. objektives: to test the hypothesis that peritoneal mesothelial cells play a role in regulating inflammatory responses within the peritoneal cavity, we examined neutrophil-chemotactic activity (interleukin 8) and monocyte-chemotactic cytokine (mcp) release by sytokine-etimulated mesothelial cells. confluent human peritoneal mesothelial cells were exposed to varying concentrations of phorbolmyristate-acetate (pma) and the cytokines tumorneerosis factor a (tnf a) and interleukin i~ (il-i~). the supernatant was examined for il-8 by elisa and for mcp by investigating the ehemotactic activity for isolated human monocytes. mesothelial cells express low levels of il 8 and monocyte chemotactic activity when cultured. these activies were significantly increased (2-fold) after stimulation with either tnf a or il-i~. additionally macrophage inflammatory protein was detected. these observations provide a probably important mechanism whereby peritoneal mesothelial cells respond to imflammatory stimuli released during peritonitis and how leucocyte recruitment by liberation of chemotactic cytokines is regulated. the perioperative course of lps, tnfa and il-6 in 19 patients with bacteriologic proven abdominal infection (intraabdominal abscess 10, diffuse peritonitis 4, pancreatic necrosis 2, pancreatic abscess 3) was followed prospectively and evaluated for possible correlation with septic state and organ function. methods: patients were studied in a 6 to 10 hours period during their first surgical intervention because of intraabdominal infection. all were monitored for their cardiovascular, respiratory, hepatic and renal function. plasma samples for lps. tnfa and il-6 determination were drawn preoperatively, intraoperatively, and until 2h postoperatively in regular intervals (min 7/pat), results: preoperative apache ii was 12 in median (rain 4, max 25). 10 patients fulfilled the criteria of sirs. 4 of them were in septic shock.there was a significant correlation between preoperative tnfa and apache ii (p=0,000 i, spearman coefficient). preoperative cardiovascular (systol. rr<90 mmhg) and respiratory (pao2<75mm hg) dysfunction were associated with significantly elevated tnfa (cardial: p=0,000 i, wilcoxon; pulmonal: p=0,0003) and il-6 (cardial: p=0,2; pulmonal: p=0.0001) overall, lps, tnfa and il-6 values varied considerably during the observation period. however, tnfa was markedly higher in patients with sirs and septic shock (group a: n= i 0, mean 182 pg/ml) than in those who did not fulfill these criteria (group b; n=9, mean 24 pg/ml; p=0,00 i, wilcoxon). il-6 was significantly higher in group a (mean 2169 pg/ml) than in group b (mean 191 pg/ml; p=0,0o i wilcoxon). conclusion: perioperative tnfa and il-6 were shown to correlate significantly with preoperative organ function, apache ii and the severity of sepsis. these results could help to define patients that might benefit from further therapeutic strategies, e.g. antibody administration. department of surgery, university vienna, akh wien, wahringer gurtel 18-20, 1090 wien. aim of the study: the purpose of this pilot study was to establish and to prove a standardized reproducible animal model of intraperitoneal sepsis induced by e.coli-endotoxinaemia in lew.lw-rats in order to investigate early immunoserological responses to find a mediator based evaluating system of peritonitis sepsis. materials and methods: in 30 lew. lw-rats, diffuse peritonitis was induced by intraperitoneal injection of a mixture of e.coli (khu +) and autogenous haemoglobin solution. in the control animal group (n= 12) an intraperitoneally injection of physiological saline solution was done. blood samples were obtained by heart puncture after 6 hours. stastistieal calculations were performed on a personal computer with the spss programm vers. 4.01 (correlation with pearson's r, mann-whitney-u-test, descriptives statistics, discriminant analysis). results: in contrast to the sham treated rats, the peritonitis animals showed significant differences in the concentrations of endotoxin, interferon-gamma (wn-y), the pteridin derivate biopterin and serum pla2-activities [endotoxin range from 0.064 eu/i, sd=0.19 to 102.38 eu/1, sd-145.4 (p < 0 0001), ifn-¥ levels, range from 147.9 pg/ml, sd-141.6, to 4591 pg/ml, sd=6541 (p < 0.0001), circulating pla2-activities range from 116.2, sd=45.4 to 185.4 u/1, sd=105.4 (p < 0.01) and biopterin range from 54.4 nmol/l sd=17.2 to 127.8 nmol/l, sd=76.8 (p < 0.001)]. for the peritonitis group we found strong correlations between the degree of endotoxinaemia to elevated levels of ifn-'~ (rp = 0.72, p < 0.0001) and bioptefin synthesis (rv= 0.82, p < 0.0001). the increase of ifn-t levels was correlated to the regulatory synthesis of biopterin (r = 0 73 p < . .. p • , . 0.0001) and to the pla2-actwtues (rp = 0.50, p < 0.005). the biopterin synthes~s correlates slightly with the pla2-actn,ities (rp= :0.38; p < 0.05). using the para, meters of endotoxin, ifn-y levels, biopterin and the pla~ -activities only, the statistical procedure of the linear discriminant analysis makes it possible, to distinguish between non-septic animals and septic animals correctly at a rate of 87 %. anaerobes were found in 53.4%, anaerobes were isolated in 22.8%. there were aerobic and anaerobic associations in 12.1% and microflora was not found in 11.6% of the cases. express method of anaerobes discovering let to receive information on 1 -3 days early than in generally accepted nethods. intraaotal transfusion of oxygenate blood and laser irradiation of blood reduces the duration of anaerobic sow, disminishes intoxication and accelerate the patients recovery. patients with abdominal sepsis are subject to long periods of hospitalization and high associated morbidity and mortality rates. this category of patients is thus consuming extensive facilities and costs. as the age-related outcome of abdominal sepsis is not fully known, the aim of the present study was to investigate abdominal sepsis in the elderly. out of 166 patients with abdominal sepsis treated at the surgical intensive care unit during a 10-year period, 72 (43 %) had an age of 70 years or more. 28 were women and 44 were men, a sex distribution not differing with patients younger than 70 years. the patients were scored according to apache ii and septic severity score (sss) upon arrival to the intensive care unit. bacterial cultures, the occurrence of organ failure, hospitalization and outcome was noted. in median two operations were performed for both "younger and elderly" patients. the median time of hospitalization in the elderly was 29 (-183) days including in median 10 days in the icu. figures in patients less than 70 years of age were comparable (34 (-293) days out of which in median 10 days in the icu). apache ii and sss-scores did not significantly differ (14.2 vs 13 and 23.3 vs 26.5, respectively), between the groups. neither did the incidence of organ failure differ (57/73 vs 77/94). however, the incidence of multiple organ failure was significantly lower in elderly patients (21/72 vs 42/94 (p < 0.001)). the mortality rate, however, did not differ between the groups (21/72 vs 26/94). in conclusion, severe abdominal sepsis in the elderly was not associated with an increase in mortality, incidence of organ failure or hospital stay. with the help of light transmissional scanning electron microscopy morphology of erythrosytes of peripheric blood was studied in patients with different stages of diffuse peritonitis before and after intravascu!ar irradiation of blood with heliun-neon laser. peritoneal morphology was investigated in patients who died from peritonitis, it was established that in all phases of peritonitis occured stomatocytoric and echinocytoric transformation of erythrocytes which progressed simultaneously with increase of intoxication. it combined with strongly pronounced vessels variability of microcirculatory peritoneal bed which displaied by erythrocytes aggregation, stasis and microtrombogenesis. in intravascular laser irradiation of blood number of erythrocytes which underwent to stomatocytoric and echinooytorie transformation was lower than in patients without laser irradiation. it indicated that the intravascular irradiation of blood with helium-neon laser can prevent development of severe alterations of rheological property of blood and consequently variability of microcirlatory peritoneal bed in patients with diffuse peritonitis. abdominal sepsis is still associated with high morbidity and mortality rates, frequenfly caused by multiple organ failure. it has been reported that changes in capillary permeability play a role in the pathogenesis of multiple organ failure. the present study aimed at evaluating the influence of intraabdominal sepsis induced by cekal ligation and puncture on capillary permeability in multiple organs and tissues. 96 adult male sprague-dawley rats were subjected to laparotomy with separation of the cekum (sham operation) or induction of intraabdominal sepsis by cekal ligation and puneatre (n--48 in each group). at 3, 6, 12, 24, 48 and 72 hours (n=6/timepoint), the animals were evaluated concerning mortality and capillary permeability as determined by the passage of :25i-labelled albumin from capillaries to the peritoneum, the proximal and distal small intestine, cekum, colon, spleen, kidneys, lungs. the mortality rate in rats with intraabdominal sepsis was 33 % both at 48 and 72 hours. capillary permeability in the peritoneum, cekum, colon and kidneys significantly increased from 6 hours and on in rats with intraabdominal sepsis. in septic animals, capillary permeability in the lungs and spleen increased from 12 hours and on and in the proximal and distal small intestine from 24 hours and on. different types of alterations in capillary permeability seem to appear: 1) a temporary short increase e.g. in the proximal small intestine and spleen; 2) a temporary longer increase e.g. in the colon and kidneys; 3) a persisting increase e.g. in the peritoneum, cekum, distal small intestine and lungs. we conclude that experimentally induced intraabdominal sepsis induces early alterations in capillary permeability in multiple organs and tissues. such changes may contribute to explain the development of sepsis-induced multiple organ failure. despite a number of significant advances in the care of burn and non-burn traumatic injury, infection and sepsis remain major causes of morbidity and mortality. the severe immunosuppresslon often seen in patients with severe trauma or large burns may predispose these patients to life threatening infections. included among the many immune alterations are changes in the functional capabilities of neutrophlls (pmns). we have examined the expression of the p 2 integrins (cd1 l a, b,c/cd18), and the fc'?r (cd16, cd32, and cd64), as well as several functional parameters, on pmns from thermal and non-thermal traumatic injury, pmns were obtained from patients sustaining severe trauma (initial apache ii score >10) or thermal injury (>30~ total body surface area, 15% full thickness), and healthy controls. the expression of cd11 b and c and to a lesser degree cdi 1 a was significantly reduced on pmns. the expression of cd16 and cd32 but not cd64 was also significantly reduced. pmns displaying this reduction in receptor expression have a significantly reduced ability to phagocytose bacteria and undergo the oxidative metabolic burst response. thermal and traumatic injury result in global reduction in the expression of 132 integrins and for which may lead to decreased functional capabilities, these abnormalities may in turn account at least in part for the increased rate of infection in these patlems, institute, dept. of surgery, 2~1 8ethesda ave, cincinnalt, oh, usa, 45267-0s5b, antibiotic-phagocytic cell interactions: their effect on endotoxin release. c g c-emmet1, dep[baeteriolog.z, univer_sitv of glasgow, scotlan~_d increasingly it is recognised that pathogenic bacteria are capable of surviving intracellularly within phagocytic cells in addition to their capacity to produce disease whilst in the extracellular milieu. as well as providing protection from certain antibiotics which fail to penetrate the phagocyte, such intraceltular bacteria may be transported from the initial site of infection to a distant more vulnerable body site wherein they may proliferate. it is also known that some antibiotics are capable of becoming concentrated within phagocytic cells mid displaying bioactivity therein. such bioactivity might be responsible for the release of endotoxia #orn gram-negative bacteria which when liberated from the celt could ~gger the cytokine cascade. anfib,.'otic-induced damage to the ultrastructure of bacteria can also occur when the target bacteria are exposed to low (sub-mic) concentrations of certain drugs. such bacteria may present quite altered surface components m host-defense cells as well as releasing biologically active ceil wall components such as endotoxin. the nature of these interactions at the cellular level as well as the consequences for the host will be discussed. new jersey medical school: umd, newark, nj 07107 a technique of physiologic state classification has been developed based on the m~itlvariable analysis of patient derived data sets of seventeen physiologic variables. these multivariable data sets obtained from critically ill patients requiring intensive care, were aormallsed by the mean and the standard deviation of recoverin~ trauma patients who were not critically ill, the resulting normalized seventeen variable sets were then clustered. seven independent data groupings were developed. the normal stress response hyperdynamic state seen post-trauma and in compensated sepsis (a stets)/ metabolic insufficiency seen in septic decompsnsation (b stste}; early (c,) and late (e2) respiratory insufficiency associated with ards; cardlogenlc dscompensation (n state); post-trauma hyvolemla without shock (r stats). the stats closest to a new patient's values allows patient classifi0atlon with regard to his previous physiologic state. classifying observations f~om patients who lived or died who fell into these physiologic states enables a probability of death (p death) to be obtalned. utilizing this criteria for the staging of severity in 44 recent trauma patients the physiologic states accurately and significantly predicted the likelihood that the patient had an increased circulating level of the eytoklnes tnf and il-1. the probability of death (p death) as well as the cytoklne levels appear to be a function of the physiologic b state with the highest levels being seen in the b state of metabolic insufficiency and the c~ state of oombined respiratory and metabolic insqffioienoy characteristic of septlc ards. the increase in the magnltude of metabolic abnormalities associated with the transition from non-sepsls to septic a, septic b, or septic c z states was associated with an increasing probability of death (p denth)(mean a state =.28, mean b state = .57, mean ~ state = .61). the accuraay of this estimate was prospectively analyzed in this group of 44 m~itlple patients of whom 72% had sepsis and 36% had ssptlo ards. the 22 survivors had a mean p death of 0.39 and the 22 deaths had a mean p death of 0.63. the severity of post-trauma sepsis can be quantified by probability analysis and stra~ifie~ by physiologic state. serologic tests have not been extensively tes'~ed in surgical patients but seem to be of limited value. we use nystatin as the main form of chemoprophyhxis. patients "~'ith signs of infection who do not rapidly improve with antibacterial therapy are candidates for anti-funsal therapy, amphoteradn b remains the first llne of therapy although combination therapy '~'ith flueonazole is use;l with increasing freque~;c)', the recovery of c~dida from an antra-abdominal site represents a challenging problem, anti~ngal therapy in such patients depends on the underlying disease, the nature of the infected material and overall patient risk. role of neural stimuli and pain principles and practice of anesthesiology effect of combined prednisolone, epidural analgesia and indomethacin on the systemic response after colonic surgery arginine: biochemistry, physiology and therapeutic irnplications immunosfimulatory effects of arginine in normal and injured rats arginine stimulates lymphocyte immune response in heahhy humans rote of arginine in trauma, sepsis and immunity arginine enhances wound healing in humans if labrecque t, gv campion t, and the rhll-lra phase i//sepsis syndrome study group the cleveland clinic foundation a murine-anti-human tnf-monoclonal antibody known as cb6 was the first anti-tnf mab which was studied in a phase ii multinational trial in the treatment of patients with severe sepsis.this was an open-label, dose-escalation trial consisting of 80 patients who were enrolled into one of four treatment groups: (1) 0.1 mg/kg of anti-tnf mab, (2) 1.0 mg/kg, (3) 10 mg/kg or (4) 1.0 mg/kg at study entry and the second dose 24 hours later. the small sample size in each group (n=20) precludes detailed statistical inference in this study. nonetheless, a considerable amount of useful information was obtained from this investigation. irst, this study demonstrated the clinical feasibility of specific anticytoldne therapy in septic patients. second, the measurement systemic levels of tnf proved to be an elusive target; interleukin-6 may prove to be a more useful indicator of cytokine activation. third, immunologic reactions including tnf: anti-tnf mab immune complexes and human anti-routine antibodies were frequently found in these patients. despite their apparent lack of overt toxicity in this study, these immunologic reactions may complicate this form of anticytokine therapy. additionally, the potential benefits of anti-tnf mab therapy occur within the first 48 hours of therapeutic administration in these septic patients. infecting organisms differ in their potential to induce tnf in vitro and these differences correlate with circulating tnf levels observed in septic patients. rapid methods to define those patients most likely to respond to anticytokine therapy are needed to determine the ultimate therapeutic potential of these agents in clinical medicine. wherry, j., abraham e., wunderink r., silverman h., perl t., nasraway s., levy h., bone r., wenzel r., balk r., allred r., pennington j. and the tnfa mab sepsis study group.tnfa mab (bay x1351) is a murine monoclonal antibody raised against human tumor necrosis factor. tnf~ mab has been shown to reduce morbidity and mortality in animal models of septic shock and has been safely administered to septic and non septic patients.to evaluate the efficacy and safety of tnf~ mab in patients with sepsis syndrome, a prospective, multicentered, double-blind, placebo-controlled trial was conducted in 31 hospitals in north america. patients were prospectively stratified into shock or nonshock groups and then randomized to receive a single intravenous infusion either of 15 mg/kg tnf~ mab, 7.5 mg/kg tnf~ mab or placebo (0.25% human albumin).patients received standard aggressive medical/surgical care during the 28 day post dosing period.the three treatment arms were well balanced with respect to demographics, apache ii score and other parameters. for all infused sepsis syndrome patients, those who received tnf~ mab had slightly reduced 28 day all cause mortality compared to placebo. among shock patients there was a more pronounced trend towards efficacy at day 28 post dosing with lower mortality rates in both active treatment arms. among nonshock patients tn~ mab did not appear beneficial. the initial clinical experience with a chimeric anti-tnf monoclonal antibody, ca2, was undertaken in septic patients. the objectives of the study were to determine the safety, pharmacokinetics and effects on cytokine levels of ca2. as a single infusion or in combination with ha-1a in septic patients. the study was conducted with the intent to progress to an efficacy trial based on the information collected.the trial was conducted in three stages. stage 1 was an open label trial in which 4 groups of 5 patients each with the clinical diagnosis of sepsis received ascending doses of ca2 (0.1, 1, 5, 10 mg/kg). stage 2 was a randomized, double blind study in which patients received a single dose of ha-1a (100 mg) and placebo or one of 3 doses of ca2 (1,5,10 mg/kg). stage 3 was a randomized, double blind study in which patients received a single dose of placebo or one of 3 doses of ca2 (0.1, 1, 10 mg/kg). in addition to usual laboratory tests, the following assays were performed: chimeric anti-tnf concentration, anti-chimeric antibody, endotoxin, tnf, il-1, and il-6 levels.a total of 141 patients were enrolled from 13 clinical sites (20 in stage 1, 60 in stage 2 and 61 in stage 3). primary analyses were performed on patients in stage 1 and 3. there were 65 patients who received ca2 exclusively and 16 patients received placebo. administration of ca2 was well tolerated at doses up to 10 mg/kg. no patient discontinued treatment due to adverse events. human anti-chimeric antibody responses were positive in 61% (30/49) of evaluated patients. mean cma × and auc increased proportionally with increasing doses of ca2. the mean half-life was -70 hrs (58-96 hrs). a dose related decrease in tnf concentration was observed 1 hr post infusion of ca2. tnf is considered to be one of the central endogenous mediators for the inili'ation of the pathophysiological changes in patients with sepsis and septic shock. high tnf levels were demonstrated to correlate with patient outcome. blocking or neutralising tnf with specific antibodies was effective in preventing death in some animal modets of sepsis. in a placebo controlled prospective randomized study we tested the mur~ne derived antibody mak 195f. it is a f(ab')2 fragment. the fragment rather the complete antibody was selected in order to reduce the potential immunogenicity and to facilitate tissue penetration. 122 patients with severe sepsis or septic shdck were enrolied in the study, three different doses of mak 195f or placebo were administered (0,1; 0,3 and i mg/kg) over a perid of 72 hours in random order. the patients were evaluated for side effects, hemodynamics, organ dysfunction, cytokines (il 6, il 8 and tnf), and outcome. at this time only an interim analysis of 60 patients is available i indicating that mak 195f in all 3 dosage groups resulted in a decrease in il 6. this contrasted to a further in crease of il 6 in the placebo patients. no serious side effects have been reported so far. a more detailed analysis on all 122 patients in the study will be presented and discussed.$152 s154 staubach,k.h., otto, v., kooistra,a,, rosenfeid,j.a., bruch, h.p., univ. lfibeek, germany once endotoxinemia occurs in sepsis a vieieus cycle with translocation of et can be established. increasing the clearance capacity for et would therapeutically be the ulimate aim. we developed a new et on-line adsorption (ad) system in whole blood by means of polymyxin b (pb) coupled eovalently to a matrix (acrylic particles) via a 13 atom-chain spacer. the detoxification capacity was 150 ug[et/ml column material. the biocompatbility resulted in 90~ platelet recovery. the column contained 7 ml of admaterial and was sterilized by high steam autoclave, anticoagulation was achieved by heparine 1.000 iu/h in the inflowline after bolus injection of 5.000 iu. hp was performed on 8 pigs at a rate of 50 ml/min by means of a roller-pump until the animals succumbed (h). 8 animals served as controls (c). serum et levels rose from 3.40 pg/ml to 74,9 pg/ml after 5 hours in the c and from 2.77 pg/ml only to 28 pg/ml in the h group after 7 hours whieh was highly significant. survival time could be extrended from 216 to 313 min. results are listed in the following l. blinzler, p. zaar, m. leier, r. b(2rger, d. heuser clinic of anaesthesiology , city hospital nuremberg, germany sepsis and multiple organ failure (mof) are still related with poor prognosis inspire of pharmacological and technical progress. impressed by revealing reports about blood purification the continuous veno-venous hemofiltration (cvvh) was used as supporting treatment beside the critical cam basic therapy of mof. from 1989 to 1991 78 consecutive patients were treated by cwh. mof was caused by hemolrhagic-traumatic noxa in 21°,4 and by septic-toxic event in 79%. all patients required mechanical ventilation (fio2>0, 5) . 86°4 showed hyperdynamic shock. 85% had renal and 53% hepatic failure. medium appache ii score amounted to 29,8 points. cvvh was performed in postdilution mode with a polyamide membrane (fh 66) and high volume exchange (50 l/die). anticoagulation was done with heparin. hemofiltration in mof was installed, when critical cam basic therapy including adequate respiratory and hemodynamic management, pamnteral nutrition, antibiotic treatment, etc., failed to stabilize organ functions. during consequent application of cvvh most of these patients showed improvement of their clinical course. pulmonary stabilization was seen in 71%, hemodynamic in 68% and renal in 66% of the cases. 42% of the patients survived and were discharged from hospital. 10 of 45 non-survivors (58%) died because of fatal mof within 24h after admission to icu. patients with early application of cvvh in mof showed a better survival rate.mediators of mof, i.e. products of the complement cascade measured in blood and nitrafiltrate by elisa, were partially removed by cvvh. the testing ultrafiltrate by hplc demonstrated decreasing spikes ofpolypeptides during hemofiltration. mof seems to be generated by cascade-activation of immune competent cells and plasmatic mediators (e.g. bmdykinin, eicosanoides, cytokines, anaphylatoxins, etc.). therapeutic approaches aim to inactivate or eliminate single substances. cwh with high-flux membranes in combination with high-volume exchange allows elimination of many mediators with different molecular weight and therefore may contribute to improve the prognosis of mof. other significant advantages of this teqalnique like adequate nutrition, optimized fluid balance and control of body temperature should not be negicctod. introductioni pseudomonas (p) aeruginosa has to be considered an important pathogen of nosocomial pneumonia and septic organ failure. the lung seems to be the predominant target organ for the pore-forming p. aeruginosa cytotoxin, thus inducing microvascular injury. with respect to therapeutical consequences, the potential protective effects of paf-antagonist (web 2086), cyelooxygenase inhibitor (diclofenac) and specific and unspecific antibodies on cytotoxin-induced pulmonary vascular reaction and mediator release were studied in the isolated perfused rabbit lung. methods: cytotoxin (6p_g/ml) was administered into the perfusion fluid in all groups, either in the absence of inhibitors (n=6), or after pretreatment with web 2086 (5xl0-gm, n=6), or diclofenac (10#g/ml, n-6). furthermore, the application of specific antitoxin (mg/ml, n=6) was tested in comparison with the unspecific immunoglobulins (venimmun®, behring, 7.5 mg/ml) (n=6) and the combination of immunogiobulins, web 2086 and diclofenac (n=6). six experiments without toxin served as controls. the arterial pressure mad the weight gain as an indicator of edema formation were continuously monitored during the three hour peffusion phase. arachidonic-ucid metabolites, as well as lactate dehydrogenase (ldh) and k + concentrations were determined at 30 rain intervals. results: cytotoxin caused a gradual increase in pulmonary arterial pressure, reaching a maximum value of 2.5 times higher than the control, starting after 1 min and a delayed onset of edema formation resulting in a mean weight gain of 20 g after 120 min. this was paralleled by a significant increase in prostacyclin generation and a continuous release of k + and ldh. thromboxane synthesis exceeded about 10 times that of controls in the toxin treated lungs. pretreatment with web 2086 or diclofenac significantly attenuated the pressure response and edema formation evoked by cytotoxin. the addition of the unspecific immunognbulin preparation alone induced a transient pressure increase within the first minutes, but mean values remained below those of the cytotoxin group in the continuing observation period. mmost complete inhibition of the pressure reaction, the edema formation and the metabolic alterations was achieved mainly by the combination of immunoglobulin, web 2086 and diclofenac and to lesser extend by the specific toxin antibody. conclusion: the current results point towards the crucial role of paf and aa-metabolites as mediators of cytotoxin induced microvascular injury. the systemic or local application of cytotoxin antibodies or even unspecific immunoglobolins in combination with paf-antagonist and diclofenac appears to be a promising therapeutic approach in the case of infection with cytotoxin-preducing strains. cytokines have long been shown to be of particular importance in the metabolic derangements occurring in lps-induced shock. recent studies strongly imply the involvement of platelet aggregating factor (paf) in the pathogenesis of gram-negative bacterial sepsis. an autocatalytic feedback network has been postulated to exist between paf and tumor necrosis factor (tnf), a key cytokine involved in septic metabolic cascade, leading to an uncontrolled amplification of inflammatory mediator release. we have previously shown that st 899 (4-n,n,n trimethylammonium-(r)-3-isovaleroyloxy-butanoic acid z-3-(5-chlorphtalidiliden) ethyl ester bromide) was quite effective in inhibiting the "in vitro" binding of 3h-paf (ki=7.5x10 -8 m) to rabbit platelets. the present study shows that pretreatment of c57bl/6 mice with st 899, administered by different routes, dose-dependently and significantly reduces the lethality induced by endotoxin (e.coli 055:b5 injected at 30 mg/kg intraperitoneally). very interestingly, st 899 administered at the same doses as above (i.e. 1.25, 2.5, and 5 mg/kg body weight) results to be significantly effective in reducing the endotoxin-induced release of serum tnf. the reported dual activity of st 899 (i.e. paf antagonism and decreased circulating tnf levels) may turn out to be greatly beneficial, in combination with current therapies, in the treatment of diseases that involve overproduction of tnf and paf such as septic shock. introduction: recently, we reported that prophylactic whole body hyperthermia (42.5°c) induces heat shock protein ('asp) 72 and increases smvival 2-4 fold in a mouse endotoxin model (am. j. physiol. in press). other investigators reported that prophylactic pharmacologic induction of hsp-72 by sodium arsenite improves survival in a rat sepsis model (abstract a95 am. rev. resp. dis. vol. 147, 1993) . the effects of heat are complex and in addition to formation of lisp-72 include release of cytokines, changes in cellular ph etc. thus, the protective mechanisms of heat may differ from those due to pharmacologically induced . the purpose of this study was to compare the protection of heat vs the protection of pharmacologically induced hsp-72 in a mouse endotoxin model to determine if different protective mechanisms were likely to be involved.. i%'lethods: both sodium arsenite (10 mg/kg) and ethanol (400 ~1 of 39% ethanol) caused marked induction of hsp-72 in lung, gut, kidney, and liver, which was comparable to heat-induced hsp-72. female nd4 mice weighing 22-25 gms were pretreated with arsenite or alcohol 8 hours prior to challenge with escherichia coli endotoxin (-ld 80) and survival was compared to control mice. results: survival at 24 hrs. for arsenite treated and alcohol treated mice was 96% and 88% respectively and was statistically different from the 40% survival for control mice. (p<0.01) (n=25 mice per group). however, at 7 days post endotoxin, there were no differences in survival in the 3 groups, i.e., ~ 4% survival for all 3 groups. in contrast, the protective effect of hyperthermia remains present at 7 days, i.e., ~ 70% survival vs 20% survival control. conclusion: the protective effect of heat is probably due to other factors such as the effect of hyperthermia to release il-lc~ and is not due solely to hsp-72 formation. it was the aim of the study to examine whether bacteria play a causative role in the pathogenesis of anastomotic insufficiency following gastrectomy in man.the study was carried out in form of a prospective, randemised, double-blind, multicenter trial. primary endpoints were the rate of anastomotic insufficiencies, infectious-and uncomplicated postoperative courses. all pat. received a periop, i.v. prophylaxis with cefotaxim. identical numbered vial either contained placebo or polymyxin b, tobramycin, vancomycin and amphotericin b . the vials were administered 4x per day from the day be ~ fore the operation until the 7th postop, day. insufficiencies were detected by gastrographin swallow and recorded by x-ray on day 7 postop.. evaluation was carried out on an "intention to treat'basis. statistical analysis was done with the pearson's chi square and fisher's exact tests~ results: interim analysis was carried out in 3/93 after 137 pat. had been recruited. along with a significant reduction of s.aureus and enterobacteria there was a reduction in the rate of anastomotic insufficiency of the esophago-jejunostomy from 10.6 % in the placebo-group to 3.5 % in the treatment group. the difference was not yet significant. the rate of nosocomial infections (e.g. respiratory tract infection and uti) were significantly reduced from 33.3 % in the placebo-group to 16.4 % in the treatment-group (p ~ 0.0281;fisher's exact test). in march 1994 final results with more than 200 patients will be presented for the first time. (= po2 <80 mm hg, b s-creatinin >2 mg%). respiratory insufficiency was the most frequent systemic complication followed by sepsis and respiratory insufficiency. etiology of pancreatitis and initial serum increase of pancreatic enzymes predicted neither complications nor outcome. only 2 of 14 deaths occurred during the 1st week, all other deaths occurred late (after 4-12 weeks), generally as the consequence of septic complications and multi-organ failure. high levels of crp were correlated with a compliacted course and a fatal outcome. although same cytokines (e.g. 11--6) were found increased in severe disease, the predictive value of these markers was not better than the combination of ctinical scores (ranson, imrie, apache ii) with gt or crp. conclusions: intensive care medicine can often control the inital shock situation in severe pancreatitis. thus. only 15% of deaths today occur eady in the course of the disease, whereas this percentage varied between 40-70% just 10 years ago. nowadays, most deaths are caused by late septic complications and multi-organ failure. ranson-and ct-scores as well as serum crp predict a course with systemic complications; they are less helpful for prediction of sepsis and late mortality. it is doubtful whether measurements of cytokines will help to better predict the late outcome. as yet, only careful and continuous monitoring of patients (e.g. by apache scores) may help to early identify those who develop septic complications and multi-organ failure. the classic description of severe acute pancreatitis has hinged upon the release of large volumes of activated enzymes into the peritoneal cavity and thertce the lymphatics and blood stream. these activated enzymes escape from the pancreas due to disruption of cells with associated ischaemia and occasional infarction of tissue. for 20 to 25 years it has been postulated that the bocly's defence system to activated pancreatic enzymes required supplementation iu the form of anti-protease support either in the vascular space or in the peritoneal cavity. all controlled studies have shown that this is either impracftcal or unnecessary.hore recently release of a large number of cytokines from monocytes, macrophages and neutrophils have been considered to be harmful to the body and various agent~ which oppose the action of tnf alpha, paf and similar cytokines are being examined in experimental anim~is and certain clinical trials, it has clearly been shown that higher levels of cytokines are released in the patients with objectively graded severe acute pancreatitis than in those with milder disease. we now seem to be moving into an exciting phase of potentially beneficial therapy in acute pancreatitis which has had no specific effective therapy through studies utilising aprotinin, gabexate mesilate and fresh frozen plasma. inflammation cascades may play a role in the pathogenesis of acute pancreatitis. to evaluate the status of the cellular immune system we examined serum concentrations of immune activation markers in 24 patients with acute pancreatitis (14 males, 10 females; median age: 56 years, range: 32-81 years). concentrations of neopterin, serum soluble tumor necrosis factor receptor (stnf-r) and serum soluble intercellular adhesion molecule type 1 (slcam-1) were determined using immunoassays (henning, bender, t cell sciences). 13/14 had increased concentrations of stnf-r compared to the 75th percentile obtained in healthy controls (>4.2 ng/ml), and 9/24 patients had increased neopterin (> 8.7 nmol/i), 9/24 presented with elevated slcam-1 (>440 u/i). all patients with increased neopterin also had increased stnf-r, 4 patients had concentrations of all three markers outside the normal range. there existed a significant correlation between neopterin and stnf-r (rs = 0.728, p < 0.001 ). weak associations between age and stnf-r (rs=0.435, p=o.05) or neopterin (rs=0.456, p = 0.044) were also found. our results demonstrate activation of the cell-mediated immune system taking place in a sub-group of patients with acute pancreatitis. the finding of increased neopterin and stnf-r levels implies that activated monocytes/macrophages are involved in the pathogenesis of the disease. further data are necessary to evaluate potential associations between changes of marker concent-rations and the course of the disease. pancreatic injury after heart surgery was reported as soon as 1970 ( 1,2) and characterized by increased serum or urine amylase levels (in about 33% of patients) in the 5 fi~t postoperafi.'ve days. this pancreatic injury, which sometimes led to acute pancreatitis, was atreaay at~buted to inappropriate perfusion of this organ. in the 198ffs, 3 studies were published dealing with pancreatic suffering alter heart surgery, in large series of patients, concluding ~n~at panc~a~c injury (with a low incidence of pancreatifis) is more common than previously recognized and is a potential source of complication after camliac surgery (3, 4, 5) . in a recent study (6), evidence of pancreatic cellular injury was found in 80 out of 300 patients undergoing cardiac surgery, with 22 out of these 80 patients presenting abdominal signs or symptoms and 3 developing severe pancreafitis. this injury was associated w~th preoperative renal insufficiency, valve surgery, ~..stoperalive hytxxension, calcium administered periopuratively and length of bypass. we studied 300 patients submitted to cardiopulmunary bypass (cpb) for heart surgery and used the measurement of un:~sin, pancreatic iso-amylase and lipase in plasma for biochemical characterization of pancreatic cellular injury. blood samples were obtained before surgery, directly aller surgery (return to inte~ve care unit), 8 hours alter surgery and in the folfowing days alter surgery (days 1, 2, 3, 4 and 8). computed tomography scan of pancreas was performed in patients presenting hi~ levels of amylase on day 1. we measured abnormal levels of trypsin and pancteatic iso-amylase in 30% of patients and observed 2 simultaneous releases of these 2 enzymes, the fi,'st one in the 24 hours after surgery and the second more intense from day 4 and pa~icularly on day 8 after smgery. this second release was concomitant with abnormal levels of llpase. these biochemical observations were accompanied by radiological and clinical signs of pancreatic injury in about 2% of our patients : pancrealic abnormalities were revealed by scan in 7 patients and acute pancreatitis in i patient. more pronounced pancreatic suffering was observed in patients undergoing valve replacement than in patients undergoing coronam-anrtic bypass grafm~g. analysis of trypsin and pare're, tic 1so-amylase are sw.cific of pancreatic cellular injury and their simultaneous ir~rease in plasma alter cpb in our padents confirms the presence of an exocrine pancreatic injury. the presence of a simultaneous peak of lipase mcaezse~ the specificity of overt pancreatic injtu 7 diagnosis. the precise cause of th/s injury could he related to hypoperfnsion leading to ischemic injury of foe splancbnic area, pancreas being largely sensible to hypoperfnsion (7). this hypoperfosion could he responsible for the ftmt release of pancrealac enzymes observed in our patients and would contribute to the deterioration of other organs leading to an inflammatory reaction developing in the following days and responsible for the second release of pancreatic enzymes observed in our patients. patients with necrotizing pancreatitis show a heigh rate of pulmonary, renal and septic complications, whereas the course in acute interstitial pancreatitis is generally very mild. we have prospectively analysed the value of endotoxin, interleukin-6(il-6) and transferrin in compare with c-reactive protein(crp) for the early assessment of the severity of acute pancreatitis. patients aud methods: the values of endotoxin(measured by limulus-lysate-test), ii-6(elisa), transferrin and crp (nephelometry) were analysed daily along the first i0 days of hospitalisation by 28 patients with acute pancreatitis admitted to our hospital from 4/1991 to 4/1993 . it was judged whether the patients have either interstitial (aip) (n=9) or necrotizing (anp) (n=lg) pancreatitis. 5 patients with anp have died during the course of pancreatitis (mortality=17.5%). results: 1-severity o~ pancreatitis: signifcant differences (p<o.05, mann-whitney test) could be calculated between aip and anp for endotoxin, il-6, transferrin and crp during the i0 days of monitoring. 2-mortality: except of il-6 on days 2, 3, 4 and 5 after ad-mission there were no significant differences between the values of analysed parameters in patients who survived and those values in patients who died. 3-organ failure: the patients with pulmonary, renal or multiorgan failure have significantly heigher values of endotoxin, il-6 and crp along the i0 days of monitoring in compare with those patients without an organ failure. conclusions: endotoxin, il-6 and transferrin, an important endotoxin carrier, are promising parameters to differentiate between the severe and mild form of pancreatitis comparable with crp. this parameters may be helpfull in the early clinical assessment of patients with acute panereatitis. the determination of cytokines may elucidate the pathophysiologic mechanisms that produce early systemic complications in acute interstitial (i) or necrotizing (n) pancreatitis (ap). the appearence of respiratory insufficiency (ri) is used to appraise the early systemic complications and is compared to the results of cytokine blood levels. in a prospective clinical trial from 10/92 -8/93, 23 patients with ap were recruited and blood samples taken for cytokine determination with commercial elisa-kits. the differentiation between i (n=12) and n (n=l 1) ap was made through ct-scan in all and laparotomy for drainage and necrosectomy in 13 patients. the etiology of ap was gallstones in 9, alcohol abuse in 7, hyperlipidemia in 2 and other or unknown causes in 5 patients.five of 11 patients with nap died early (n=l) or of late septic complications. none died of iap.the peak of cytnkine level in the first three days of hospitalisation was used for calculation. ri was diagnosed in the first week of hospitalisation, if oxygenmask or iutubation for at least 3 days was nescessary to treat arterial hypoxemia. for statistical analysis u-test of wilcoxon, mann and whitney was applicated.the il-6 concentration in blood reached up to 2600pg/ml in the first days depending on the severity of ap and dropped to almost zero in the next days, independently of the clinical course. the differentiation of i-versus nap, using a cutoff-line of 600 pg/ml was correct in 20 patients (87%, sensitivity (se): 82%= specificity (sp): 91%, (z:0,001). high levels of il-6 over 500 pg/ml correlated well with the prognosis ifri in the first week (accuracy: 87%, se: 80%, sp: 100%, c~: 0,001). the blood levels of il-8 reached a maximum of 1381 pg/ml in the first days, depending on the severity of ap, and showed a correlation to the clinical course in the following days. the peak of 1l,-8 blood levels indicated correctly the severity of ap in 18 out of 23 patients using a cut offtevel of 200 pglml (accuracy: 78%, se: 82%, sp: 75%, ~: 0,01). there was no correlation between cytokine blood levels and mortality, also there seemed to be no correlation between the levels of il-6 and il-8. in the bloodsamples of five patients with i-or nap no c~-tnf was detectable.the blood levels of il-6, and to a lesser extent of [l-8, can predict the severity and early systemic complications of ap in men. the excessive rise of cytokines can be explained by the stimulation of immunological cells ( macrophages, lymphocytes and endothelial cells) in the course of ap. objectives: in an opossum model, ligation of the sphincter of oddi or separate ligation of the bile and the pancreatic duct together leads to severe haemorrhagic pancreatffis while single ligation of the pancreatic duct causes only an exocdne atrophy. since bo has been discussed to be a contdbutor t¢, res dysfunction, this may depdve the organism of a potent defensive mechanism thus promoting the course of pancreatitis. the present study wa,,; carded out tc develop a new method for measuring dynamic liver res func.. tion and to test the hypothesis that bo causes a res dysfunction. material & methods: 18 opossums were randomly placed in three groups. all received a central venous line. after midline laparotomy, a specially designe0 tourniquet was placed around the common hepatic duct above its junction with the pancreatic duct (group a, n=6), around the pancreatic duct (group b, n=6) or around both ducts (group c, n=6). after one week, the tourniquets were closed. using a scintillation camera, the liver uptake of nanocoll, a 99mtcalbumin colloid with a diameter of 25 nm, was measured before, 2,5 and 6 days after the obstruction. the curves were analysed by a computer and two parameters (r, s) were calculated using natural logarithmic regression. as a common measure for res function, the corrected granulopexic index (a) wa,,; determined, after day 6, the pancreas of each opossum was searched for necrosis by morphometdc methods. results: all animals survived till day 6. the opossums in groups a & 8 had a macro-and microscopic normal pancreas, while those in group c showed a severe hemorrhagic pancreatitis. the granuinpexic index showed a significan~ change to the worse starting at day 5 in group a & b (p<o,05) and at day 2 it~ group c (p<0,ol). at day 6, res function in group c was significantly worse than that in group a & b (p<0,01). the regression parameter r showed no significant change in groups a & b, but a highly significant decrease in group c starting at day 2 (p<0.005), thus showing a dysfunction of the hepatic res. conclusions: obstruction of the hepatic or pancreatic duct alone does no{ result in immediate dysfunction of the hepatic res, while obstruction of beth ducts does. because only ligation of both ducts is followed by a severe hereof.. rhagic pancreatitis, res dysfunction could be an important factor in the pathogenesis of pancreatitis and resulting organ failure.logarithmic regressinrl has proven to be a valuable tool to analyse dynamic hepatic res function. (b6) in lewis rats weighing 220-250g. there were five groups: group a (n=14) were untreated controls; group b (n=7) were given isotonic saline intraperitoneally and observed for 48 hours; group c (n=7) 2 ml x 108 e. coli intraperitoneally and observed for 48 hours; group d (n=7) were given 4 ml x 108 e. coli and observed for 48 hours; group e (n=7) 4 ml x i08 e. coli and observed for 24 hours, the rats were than killed, the spleens were removed and heparinised blood taken for immunological tests. total t-ceils, helper t-ceils, suppressor t-cells, monocytes, and the interleukin 2 receptor were determined by monoclonal antibodies, indirect immunfluorescence, and flow cytometry (facs analyser 11). statistical analysis was by the t test on rank transformed data.in group b (isotonic saline) the total t-ceils increased in the blood (p=0,003) and spleen (p=0,0001 )com pared with the untreated rats (group a), in the groups with peritonitis (c, d, and e) we found highly significant rises in suppressor t-cells in the blood and spleen (both p<0,0001 ) compared with the non-infected control groups a and b. this increase was significantly higher in group d than in groups c and e. as well as other alterations, we found a uniform increase in the number of t suppressor ceils in our model of acute peritonitis that was both time and dose dependent, department of surgery, university vienna, akh wien, w~hringer gurtel 18-20, 1090 wien. d~'na~'aics of som~ l~mnunologic indices in children with abdo;~tinal shock v.z.i~ioskalenko, s.f.vesiol$,t.v.p~bina serum (iga,:~i,g, circulating imaune co!~plexes, co:apleaentary serum activity, antimesothelialvisceral and parietal antibodies) and cellular (i-l~q~hocytes, ~s/th,b-lyaphoeytes,i nlaunoleukosis to aesothelial allergens; spontaneous blast cell transformation to phytohemaaglutinin and .nesoth<-~lial ~,togen; phagooftic neutrophil activity) im:~une indices v.'ere studied in 60 children operated v:ith symi~to'as of abdo~ainal shock. ,'~!-~ of them had appendicnlar(26-1ocalized jo-diffuse, 9-~eneralized) and 5-hemoperitoni-~is (dull abdominal trau;na with injayy of 'the spleen-4, the spleen and liver-q), flo patients tjere operated on for co~iplete iieus (~-intussnsc.sption, 5-com:lissural ileus). 2he follo~'~ing serum factors: cireula:;ing im~aune complexes and ~).esothelial antibodies are the aost i~portant ~ar~ers of the abdominal shock course. :yith the progress of the process the indices increase irresponsive of t.l~e cha±.aeter of pathology. in case of ileus regress and hemoperitonotis an abrupt drop in ~he titer of anti~aesothelial antibodies is noted. in bacterial peritonitis a high ~iter of anti:aesothelial parietal antibodies in great dilutions ("+++","++++"-q ; 720) persisted even in the period of clinical recovery. cellular factors cham'acterize dfnaaics of the abdominal shock coulees less specifically llast cell transfor~aation and phagocyue neut~ophil activit 2 can inderectly characterize transition of shock f~orn reversible into irreversible phases. the past so years have brought a number of major advances in burn care. the initial advance was the discovery that burn victims required large volumes of resuscitation fluid in the initial 24 hours to maintain hsmodynamic stability, this was followed hy the dsvelopmsnt of topical antimicrohlal agents to prevent end treat infections. next, was the reporting that early excision of burn wounds, with autografting of the excised wounds was possible.finally, was the identification of the importance of aggrsssive nutritional support for the hypermstabolic burn patient.these advances have markedly increaesd survival rates for given burn mimes, s~ch that burns which would previously have been considered to be aniformly fetal, are now readily survivable.thsre remain two unsolved problems in burn care, which are limiting survivability cf burn patients.the first ie ths treatment of inhalation injury. ths sscond is how to obtain coverage of ths maaaivsly burned patient, who has limited skin graft donor sites available.this ssssion will focus on the new treatments being developed to obtain permanent coverage of burn wounds.these include the use of various tcplcal growth £aotore which can speed the rate of reepithelialization of wounds, both the beneficial and dstrimental effects of using cultured karatinooyte preparations, to obtain in excess of one squats meter of a pseudoskin from a single fifteen square centimeter biopsy of unburned skin, will be discussed, finally, the used for artificlal dermal preparations will be discussed. integumentary wound healing consists of simultaneous epithelializalion and contraction. not long ago, it was thought that healing proceeds at an optimum fixed rate and could only be delayed. the recent explosion in molecular biology and recombinant gene techniques have produced information on the biological activity of compounds previously believed to be biologically inert. many current attempts to modulate the rate of wound healing center on the topical application of various growth factors produced by integumentary cells or agents designed to modulate growth factor expression or response. epidermal growth factor (egf), plateiet derived growth factor (pdgf), transforming growth factor (tgf) and fibroblast growth factor (fgf) have been demonstrated, both in vitro and in vivo, to stimulate the proliferation and diferentiation of their designated cells types. egf and pdgf have been clearly demonstraled to increase the rate of wound closure in partial and full thickness wounds. pdgf also has demonstrated efficacy in the closure of recalcitrant pressure sores, whereas tgf increases the tensile strength of incisiona[ wounds and fgf stimulates angiogenesis. it appears that epidermal keratinocyte migration during wound healing depends on integrin-mediated interactions with compounds and ceils extracellular matrix. additionally, the activities of extracellular glycoproteins such as fibronectin, fibrinogen, laminin and thrombospondin are coordinated by multiple integrins with specific distributions and binding affinities. currently, agents which incorporate specific protein sequences essential for the interaction of the glycoproteins with the cells of the extracellular matrix are being investigated. it is has been clearly established that the rate and nature of wound healing can be influenced by the application of various agents and that sufficient quantities of these agents can be manufactured. the future challenge is to develop techniques to now the objective evaluation of the clinical efficacy of these various agents and to employ the appropriate agents in a cost-effective manner. transplantation; and 4) the immune status/manipulation of the host.multiple interactive variables will determine the effect of employing allogeneic cells and tissue in wound coverage design and a batter understanding of the dynamics of the wound-graft microenvironment will facilitate the dove opment of clinicauy beneficial graft composites. cadaver allograft skin (cas) is the "gold standard" for wound coverage, but has limitations including varied availability & quality, immunogenicity leading to rejection, & potential for disease transmission. our development of a temporary living skin replacement (tlsr) addresses these issues; the tlsr consists of highly screened human neonatal fibroblasts (hf) cultured in biobrane a, a bilayer dressing consisting of nylon mesh laminated to a thin silicone membrane which controls water-vapor transmission. studies in our lab indicated that "fresw ~ tlsr grafts reproducibly close full-thickness wounds in mice & perform better than bb controls. we studied wound adherence & structural/molecular properties of fresh & cryopreserved (cryo) tlsr. methods: tlsrs were prepared by seeding 4000/co 2 hf in bb nylon mesh in dmem. hf quickly attached to the nylon mesh & were allowed to proliferate 3-6 wks. fibroblast mitochondrial activity was assayed by mtt assay. matrix proteins were identified by immunostaining, mrnas for specific growth factors were identified by reverse-transcription polymerase chain reaction amplification, & quantitated by gel scans. bb structure was studied by scanning electron microscopy & stretching measurements pro/post cryo. 80 grafts were cryo'd in 10% dmso, quick-thawed & sutured onto 2x2 cm excised full-thickness dorsal wounds on athymic mice. 40 "fresh" grafts were similarly placed. controls grafts were cas and aeellular bb. adherence was quantitated by a device which peeled grafts from wounds at intervals following placement, using a serve motor and a strain gauge. results: postcryo, storage and subsequent thawing tile tlsrs maintained >60% cell viability by the mtt assay, indicating continued mitochondrial activity, and bb structure & stretchability were maintained. multiple matrix proteins secreted and deposited in the bb nylon mesh (types l/iii collagen, decorin, fibroneetin) were identified by specific immunostaining. growth factor mrnas in the tlsrs (afgf, bfgf, kgf, tgf~,p~,) were present in 10-10,000x higher levels in fresh/cryo tlsrs than in adult hcs. grafts adhered to wounds on mice through 40 days of followup. histologic exams on days 6-40 showed excellent vascular ingrowth and minimal inflammation. adherence of tlsrs to wounds was >cas adherence. burn wound coverage in the massively burned patient remains a difficult problem. although cultured keratinocytes have been utilized for burn wound coverage, their impact on the patient with burns greater than 80% total body surface area has not been spectacular, with poor graft take and unstable epithelium.current investigations have been directed toward dermal replacement beneath either very thin split-thickness autografts (stag) or utilizing cultured keratinocytes. current products include: collagen dermal replacement with thin stag (burke, et al). collagen dermal replacement with cultured keratinocytes and fibroblasts (boyce, et ai). allograft dermis with cultured keratinocytes (cnno, et al). allograft dermis with thin stag (life cell). polyglactin acid mesh and neonatal human fibroblasts with thin stag (hansbrnngh, et al).investigations regarding culture media, use of growth factors, topical nutrients and antibiotics, and melanocytes for pigmentation as well as safety and efficacy are needed before any of the current products become viable options for coverage of the massively burned patient. the~ is a growing world-wide problem with the ujc of cadaver tissues and ocgans bae, au~ of the tren~m~s~km of dilemma such a; cmutzfeldt.jukob disease and iiiv as we1] as ready availability of urdform lis~ue~. on dec~mt~r 14, 1993, the fda assumed control of as1 tissue bar~s in the uldtod st=tea in an attempt to bflng ~s difficult problem of dise~s~ transmission under ¢onlrol. in europe, ~om¢ of the governments are consldofll~ a c~mplcte bat) on the use of cadaverlc fissu~s such as ddn, 'this |ncroam in regulation of cadavefle ~s,quct will incmar¢ the difficulty of obtain~g and dlslflbulmg them. however, thc nc~ for these tissues contlnue~ m incrcaso, we will discuss ~'l¢ solulion to this important pmbl~n: tissue engineering. tlssu~ engineering is an in~rdisdpllnary field that applies pdnclplc~ of angin~edng and die life sclcnce~ reward the development of ~olok~¢al sub~dtute,~ ih= mslom, maintain, or improve tissue function, "13ssuc ongln~cdng can provide ~ho nccassary tlssuoa for wound repair ~d ibe assuranoe fl'~t the lissuos are d.ls¢~¢ free. in addition, a ds~uo-cng~ne~n~l wound covering will bo u~lvemally acceptable and evntlublc as "off g~o shell", consis~t products, them are several approaches to restating thls function in a large wound, 'l'nosc i~elud~ tmmcdiete long term coverage, short t=nn coverage, uandtl~el coverage and compost= dssu¢ coverage, "flssuo onglncrcd wound coverings that meet those vaflous ne,.cds will he r~vlowod.cllni~:sl and experimental d~la in venous ulcer, dlabctl¢ ulcers, prossur~ ulcers and bum wounds wgj be mvlcw~, a~ welt as new approacl~s u~ csrtilag¢, bone, liver and bone marrow it~suos. c oomplon, k nadirs, w press, g wetland, j fallen iv, shrtners burns institute and massachusetts general hospital, boston, ma~schusetts, usa the clinical "take" rate o? cultured epithelial autografts (cea) has been observed to increase with transplantation to allodermls, but the reasons for the improved clinical performance have not yet been defined. the aim of this study was to determine the biological impact of normal human dermis on cea differentiation and maturation, biopsies of cea transplanted to engrafted and de-opldermlzed human homograft dermis have been compared to nopsles of cea transplanted to granulation tissue in tullthickness burn wound beds on the same patient, each patient serving as hls or her own control. paired test and control biopstes from six patients have acquired from as early as one week postgrafting to as late as 3 years postgrafting (one patient) and analyzed histopathologlcally, ultrastructurally and immunoh[stochemloally, results demonstrate more rapid normalization of differentiation markers (e,g., involucfln, fllaggrln, cytokeratln profiles) in the cea transplanted to allodermls compared to their corresponding controls by in all patients, the proliferation rate within the basal layer ot the epidermis as determined by ki-67 (proliferation-associated antigen) is seen to norh~altze more quickly in the cea transplanted to allodermls in every case, persistence of allodermal matrix can be dooumented in all patients by elastic tlssue-trichrome stain, allowing visualization of the dermal elastin network. the popu;atlon densities ot intraepldarmal langerhans cells are conslstently and signlflcantly higher in cea transplanted to ,allodermls, possibly reflectlng an immunologlcal reaction to the underlying allogenlc tissue. overall, these preliminary results indicate that transplantation to a normal human dermal matrix accelerates the maturation of cea-deflved epidermis, wound closure continues to be a major problem in patients who have sustained a major thermal injury, cultured epidermal autografts (cea) have been utilized extensively since 1984 when galllco et el reported theh'use in two brothers with greater than 90% total body surface area burn. unfortunately, cea take rate varies widely and the resultant skin coverage is often fragile and the cosmetic results are less than optimal however the overall take rate and durability of the coverase can be markedly improved by using nn allodermls base as the recipient bed. a review of cea applications performed by physicians using cultured outologens epithelium obtained from blusurfaoe teclmology, inc. shows a marked discrepancy in the results obtained utilizing different methods of wound bed preparation. tgf-b is an important modulator coordinating complex physiological events associated with growth and development. it is assumed that tgf-b is also involved in the well-coordinated process of cutaneous wound healing by regulating proliferation, differentiation, chemotaxis and matrix deposition. the purpose of our study was to analyze the spatial and temporal pattern of tgf-b expression during granulation tissue formation in patients with accidanutl surgical trauma (monotraumata mid polytraumata) and bum wounds. after debridement (day 0), the full thickness wounds were covered with epigard, a synthetic dressing until day 10. after this time the granulated wounds were closed by transplantation of mesh graft. biopsies of the wound center were taken from 30 patients at the beginning of surgical treatment (day 0) and after 3, 6, and 10 days. cryosections were stained with antibodies against tgf-fi s using the apaap technique and -for standard histology -with hematoxylin-eosin. for identification of the cell type expressing tgf-6, double staining immunofluorescence experiments were conducted using antibodies specific for monocytes/macrophages, polymorphoanclear neutropkils and fibroblasts. the results showed a characteristic pattern of tgf-t~ distribution during wound development. tgf-fi appearence was mainly cell-associated znd the absolute and relative number of cells that were positive increased with lime. infiltrating cells and developing blood vessels were most prominently stained; epithelial and t-cells showed no immuno-reactivity. a delay of emergence for tgf-b during the time course could be seen in one patient group. this might reflect various regulation patterns depending on the type and severity of injury.(1) pharmatec gmbh, frankfurt (2) institut fiir immonologie and serologic, heidelberg ( immune cells extravasating specifically in skin recognize and eliminate the invading antigens (bacteria, viruses, etc.) either in situ or transport them to regional lymph nodes. they also participate in the process of skin wound healing. cells which traffic through the skin can be harvested from efferent lymph drained from a given area of skin. the type of migrating cells changes after trauma, heating and infection. we have developed a method for collection of human afferent lymph in lower limbs. the method allows obtaining immune cells from normal and injured skin and their characterization. aim of the study was to characterize skin immune cells in situ and in skin lymph with use of immunohistological methods (staining, facs). results. group 1, cells migrating through skin: 75+4% t lymphocytes (cd3), 6+3% langerhans and dendritic cells (cdla, hla dr, s100), 41+9% cd4, 18+6% cd8, no b cells (cd22, 23), 74% cd45r0 (memory cells), 6+3% il2r. approximately 90% cells possessed cdlla and 18 antigens. cd1 lc was expressed only on large cells. the frequency of all phenotypes was different from the blood populations. group 2, cells in skin: langerhans cells were found only in epidermis, cd3,4 and 8, cd45r0, rb, ila/18 cells around venules, cd68 (macrophages) uniformly dispersed, no il2r and b cells. hla dr positive were endothelial and some dispersed mononuclear cells. group 3, one, three and thirty days after surgical wound (simple varicous vein extirpation): high density of epidermal langerhans cells, hla dr positive keratinocytes and all endothelial ceils, few il2r cells, perivenular infiltrates of cd68, 45r0 but less cd3 cells, high density of cdlla/18 cells. classic staining of isolated and in situ located ccl!s with mgg or he did not allow to follow kinetics of changes. conclusions. this study presents the first in the literature quantitative data of immune cell traffic through normal and injured human skin. in the controlled release of biological response modifiers for soft tissue regeneration. alan s. rudolph, helmut speilberg, mariam monshipouri, and florence rollwagen, and barry j. spargo. we have employed lipid microstructures as controlled release vehicles for the delivery of growth factors in wound repair. traditional liposomes as well as novel lipid based microcylinders have been examined for their in vitro kinetics of the release of transforming growth factor beta (tgf-b). in vitro reiease has been examined by setting up models with examine the physical release of iodinated tgf-b as well as a cell based bioassay (based on the ht3 bioassay). the hollow lipid microcylinders (50 microns in length and i micron in diameter) show an initial burst (5-10 ng) followed be zero order kinetics which result in the release of approximately i ng tgf/day. this release behavior can be modified by temperature based on the phase behavior of the lipid bilayer which comprises the microcylinder.we have also examined the cellular response to lipid microcylinders applied in vivo. the lipid microcylinders are mixed in agarose and implanted as a composite hydrogel block under the flank of a mouse. the blocks are removed 1, 3, and 5 days following implant and the cells analyzed by facs sorter analysis. the observed pattern of ceil recruitment to the blocks mimics that seen in a local inflammatory response. cell surface phenotype studies included the determination of cd3 and cd4, mac-l, and ig bearing cells. we have also begun to examine the change in cell surface phenotype and kinetics of recruitment following the inclusion of tgf-beta in the lipid microcylinders.center for biomolecular science and engineering, code 6900, naval research laboratory, washington, dc. 20375-5348. expression pattern of heat shock proteins in acute, good healing and chronic human wound tissue. abstract: wound healing is a complex biologic process that is well characterized at the histological level, but its molecular regulation is poorly understood. after clot formation, inflammatory cells are rapidly drawn into the wound, followed by migration of fibroblasts and epithelial cells that divide and repopulate the wound area. during the last decade peptide growth factors and cytokine are thought to play a key role in initiating and sustaining the phase of tissue repair. these factors which are released from different cells appear to initiate the cascade of events that lead to healing. different studys described the rapid activation of a family of proteins,named heat shock proteins (hsp) in differnt tissue that were exposed to various forms of stress (heat, toxic agents, mechanical). in this context hsp's have the ability to regulate protein folding and assembly, to transport proteins across cytoplasm and membranes, to disrupt protein complexes, to stabilize, degrade and regulate the synthesis of proteins and to take part in dna replication and repair. we now attempted to find out if hsp-gene activation is also involved in injury and wound healing, which likewise resemble a stress situation for cells. therefore we collected tissue samples during operation and single biopsies from chronic wounds (decubitus for example) and granulation tissue. after rna preparation from these samples we used rna-pcr and nothern analysis to study the expression of objectives of the study chronic, non-healing cutaneous tflcers are a challenging clinical and socioeconomic problem. several animal studies have shown that cytukines (e.g. egf, pdgf, fgf, tgfb) accelerate the healing process and tissue repair in general. results from first clinical trials indicate a promising value of cytokines in the treatment of chronic non-healing diabetic and venous ulcers. recent reports in the literature indicate that the biological activity of the solution of platlet derived wound healing formula (pdwt~) released from c~-granules (mainly pdgf & tgfi~) is greater than the activity of the recombiant single factors like e.g. pdgf-bb (robson, lancet 1992) . the aim of our study was to determine whether a correlation exits between the concentration of tgfi~ & pdgf and the time course of wound healing. materials and methods pdwhf was prepared from 200ml of auto]ogous patient blood and diluted with a special buffer to a final concentration of 30 ng/ml g-thromboglobulin. the concentrations of pdgf and tgfg were determined by elisa-tests developed in our laboratory. 22 patients with chronic non-healing ulcers have been evaluated alter treatment by topical application of pdwhf. pdfg and tgff~ concentrations of the topical solution were measured and two patient groups formed for analysis the time course of wound healing was regularly and meticulously documented and evaluated by photography and casting. the time from initiation of treatment instil o wound volume reduction to 50 go of the origional size (t50%) was noted• results: healing of extensive burn wounds can be accelerated by grafting cultured autologous or allogeneic keratinocytes. the stimulation of granulation tissue formation and reepithelialization is presumably based on growth factors and cytokines released by keratinocytes. we wanted to prove this hypothesis by investigating the bfgf expression during wound development, bfgf is mainly described as an angiogenic protein with mitogenic activity on various mesodermal and ectodermal cell types pointing to its stimulating potential in wound heating. in the present study we compared the pattern of human bfgf m-rna expression and the localization of bfgf protein during the first days of wound healing. biopsies were taken from 5 juvenile human bum patients, immediately after wound debridemerit mad on day 8 after transplantation of cultured allografts. biopsies were snap frozen and cryosected. the pattern of bfgf expression was assessed by in situ hybridization of the bfgf m-rna with a digoxigenin-labelled antisense-rna and the parallel detection of the mature protein with an anfi-bfgf monoclonal antibody. our study revealed typical patterns of bfgf-m-rna-expression and intense bfgfprotein deposition during granulation tissue formation and reepithelialjzation of healing bum wounds. 'it, is known that major thermal injuries cause early impairment of wound healing followed by decreased influx of granuiocytes st. the site of injury. the role of granuiocytes in the process of wound healing is not ~"~ "" elucidated, it is now assumed that they are not merely phagocytic cells but active participants in ~n~*'1~.,.,a+~o~: processes secreting_ a number of various cvt-;kines, in order to investigate the effect of there is accumulating evidence that neuropeptides could be involved in the pathogenesis of several inflammatory reactions. vasocactive intestinal polypeptide (vip) and substance p (sp) have been detected by immunohistochemistry in normal as well as inflammed skin mostly in perivascular and periglandular location. both vip and sp are involved in vasodilatation, mast cell degranulation and irnmunomodulation.we determined the influence of sp and vip on the proliferation of lymphocytes in patients with psoriasis and healthy individuals. peripheral blood t-lymphocytes of 6 psoriatics and 6 healthy controls were isolated by density gradient centrifugation and passage over nylon wool. cell enrichment was controlled by facs analysis, lx105 t-lymphocytes were then incubated alone or in coculture with 2x104 irradiated autologous lymphocytes in culture medium containing 10 -7 mol/i sp or vip. cell proliferation was measured semiquanfitatively by tdr uptake in a betacounter. significance was tested by the wilcoxon signed-rank test.our results show that sp and vip exert only an effect on unstirnulated t-cells. in healthy individuals but not in patients with psoriasis sp increases significantly proliferation of t-cells. vip, however stimulates significantly the blastogenesis of t-lymphocytes only in psoriatics.our results confirm the psychoneuroimmunologic component in inflammatory reactions and vip and sp could be partially implicated in their pathogenetic mechanisms. moreover psoriatic lymphocytes show an altered reaction to sp and vip. this might be due to a preexisting (genetic?) or more likely to an epiphenomenal receptor defect. the adhesive interactions between endothelial cells and circulating ~enkocytes in shock and innammatory vondltions is mediated by several distinct families of ce11-surface determinants. of particular importance are the leukocyte integrins cdib / cdlla-c. in this study monoclonal antibodies to two of the u chains (cdlla & cdiib) and the common [~ chain (cdib) have been used to investigate leukocyte-dependent and leukocyte-independent plasma leakage in tee skin of rabbite. plasma leakage was measured as the local accumulation of t2si-hsa over a 30 rain period, the chemotac~c peptide imlp (0.1. 30ng) and bradykinin were used to induce cell.dependent and cell-5ndependent leakage respectively, the antibodies used were 6.5e (cdis), nri85 (cdlla) and antibody 198 (cdllb). ]ntradermal in~ections of bradyklnin and ~dlp both caused a dose dependent increase in plasma extravasatien (15.~. ffi 2.1p.l to 54.4 z b.bttl and 16.4 ,-1.8~ to 59.8 z 6.71d respectively. 6.5e (0.0072-2.4mf,/k~ iv) caused a dose dependent inhibition of imlp-induced but not bradyldnin.inducecl plasma exudation. at 0.24mk/kg, the plasma leakage was completely inhibited, antibody nr185 produced similar results, treatment with antibody 198 did not cause inhibition o£ plasma leakage due to either tnedi~tor. in vitro, the irmnune system ex~nination in persons with bone, chest and abdominal traumatic injury (i group . 22 patients without infectious coz~lications and 2 group -53 patients with wound infections development) was carried out. to restore found immunity disorders and host defense to infection 23 patients of the 2 group were treated with thymalin-the biologically active peptides prepared from bovine thymus. the examination on t~e i-3 days after injury revealed a considerable decrease of lymphocytes, ed3",$d4 ~ and cd8 cells amo~it in the blood, cd4 /cd8 ratio and indexes of let~ocyte migration inhibition test in both groups of patients. the imm~lity disorders recovered to norm on the 7-10 days in pateents of+the i group. but stable ~eple$ion of cd3 and cd4 cells amount, lower cd4 /cd8 ratio and indexes of leukocyte migration inhibition test in patients of the 2 group were observed~ besides that, these persons showed higher cd19cells amount and ig level in the blood. after thymalin therapy valid ii~rovement of inun~e status was discovered. also good clinical effect of immunotherapy and best wo~id healing observed in 80% of cases. these results allow us to propose that the thymus involution and the reduction of cell-mediated immunity responsiveness with disturbances of immu_uoregulatio~ on the level of restriction of activated cd4 tho cells play the most important role in the pathogenesis of wound infections development in persons with traumatic injury.dept. of immunology, military-nedical academy, lebedeva str. 6, 194175, st.petersburg, russia a severe impairment of neutrophil (pmn) function often occurs following severe thermal or non-thermal traumatic injury. our laboratory has previously reported that following severe burn or non-burn traumatic injury the expression of the p2 integrlns (cd11a,b,c/cd18) and the fw receptors (cd16, and cd32) were significantly decreased on pmns, in this study, the effects of gm and g-csf on the expression of the f~ r and the ~2 integrln family on pmns were examined, pmns were obtained from severe trauma (initial apache ii score ;z 10) or thermal injury (>30~; total body surface area, >15~ full thickness) and incubated /n v/tro with gm or g-csf. the j32 integrins or fcyr were detected with monoclonal antibodies and flow cytometry. gm end g-csf induced a sllght increase in the percentage of pmns expressing cd1 lb, cd16, and cd18 while gm bur not c-csf induced an increase in the percentage expressing cdi 1 a, cd1 lc, and cd32, gm-csf and to a lesser extent g-csf induced an increase in the density (1,5 fold) of the ~2 integrlns on pmns from normal, burn, and trauma patients, these data suggest that cytoklne modulation with csfs could have a role clinically in certain situations. institute, dept. of surgery, 231 bethesda ave, cincinnati, oh, usa, 45267-0558. funl~al infections after solid organ transplantatlon(sot) lewis flint, md and ed,~-afd e. etheredge, me) dept. of surgery tullrte univ. school of medicine new orleans. louisiana infections contribute to increased gra5 loss and mortaliw following sot. pr~isposing facton include diabetes, hepatitis, leukopenia, cc.¢xistem infection, and intense, especially triple drug, immunosuppression. funga] infections occur ~s isolated conditions in 6% and in association with bacterial infection(l 1%), viral infection(3*/.), and combined infections(it%), candida sp. is the most common fungus recovered but aspecgillus, coccidiodies, cryptococcus, histoplasma, mueor~ ghizopus, tinea, and toruiop~is s?. also are pathogens. clinical syndromes vary among orga.aizms or may be variable with a single p~tthogen, for ~ample, with aggressive immunosuppression, candlda my be localized esophagitis or cystitis or systemically iavaslve with an associated high mortality. aspergilius presents ~ a diffuse pneumonia while cryptococcus causes pulmonary and centrad nervons sy'stem infection, clinical examination, ct scanning and aggressive sampling for c'ultures a.s wall as serologic tests contribute to diagnosis. empiric the~py is ind',cated where there is a high level of suspicion. preventlon of ca.adlda izfection is ~ci~itated by early remov-a.1 of central }ants, ca~hetess and stents as well as by the use of oral nystatin. amphotericin ]~ remains the drug of choice for treatment of in.save fungd infection, surgical resection of infectious loci in the lung and brain is indicated in selected patients. the main problems of diagnosis in lower respirator-), tract infection are the differentation of infection from colonization or contamination, and the isolation of a reliable and true pathogen. expectorated sputum may be unreliable in pneumonia, because of contamination by oropharyngeal flora. although blood cultures may be negative, they provide a precise diagnosis and should be obtained in all pneumonias. other more invasive procedures are transtracheal needle aspiration, fibrobronchoscopic techniques including protected specimen brush and bronchoalveolar lavage with quantitative culturing and cytological analysis, transthoracic needle aspiration, thoracoscopy -guided biopsy and open lung biopsy. recently m. e1-ebiary, a. torres et al, 1993 reported quantitative cultures of endotracheal aspirates for the diagnosis of ventilator-associated pneumonia offering reliable results in these patients and should be further investigated. any invasive procedure in a severely ill patient should be carefully directed weighing the risks as well as the benefits, whilst taking the underlying diseases and expected survival into consideration. -current therapeutic approach is based mainly on monotherapy with broad spectrum antibiotics. combination therapy is apparently indicated only in p. aeruginosa infections and severe s. aureus pneumonia. graft infection can lead to fulminant graft failure or rapid progressive cirrhosis. for prevention of graft infection immunoprophylaxis, i,e. administration of human polyclonal anti hbs hypedmmunoglobutin (hig), starting in the anhepatic phase during operation, has proved to be at least partially succesful when performed on a long term basis.from a total of 415 olt in adult patients 100 olt were performed for hbsag positive liver disease (cirrhosis n=86, fulminant liver failure n=8, retransplantation n=6) in 94 pat. all pat. received 10.000 u hig in the anhepatic phase and 2.000 u/per day for the first week. a small group of 9 pat. received hig only for i week (short term immunoprophylaxis), in all other pat. hig is administered on a long term basis to keep anti hbs serum levels above 100 uii or until graft infection occurs (long term immunoprophylaxis);one-year survival rates are 100% in pat. who were transplanted for fulminant hepatitis, 96% in pat. with cirrhosis and long term prophylaxis, and 78% ir~ pat. with short term prophylaxis. all fatalities were related to hbv graft infection. the total rate of graft infection was 100% under short term prophylaxis and was independent from preoperative hbv dna status, under long term prophylaxis graft infection occurad in 28% in pat, negative for hbv dna. in hbv dna positive pat. infection rate was 56%, the total rate of reinfection for all pat. with long term prophylaxis was 36%the results of liver transplantation in hbsag positive pat. are comparable to other indications, graft infection with hepatitis b virus ist the major risk factor for these patients. under long term therapy with hig the rate of graft infection can be significantly reduced. the crucial cellular element for mods-mof: monocyi'f_./m acrophaoe ronald v. meier, m,d., f.a,c,s. the severely :injured or crldcally ill surgical patient is at high risk for immune dysfunction. a major consequence of this immune dysfunction is multiple organ dysfunction and failure leading to death, the underlying etiology is now recognized to be an uncontrolled, unfocused, disseminated activation of the host normally protective inflammatory. ,, cascades.. the resultant "mahgnant' systemic" inflan'a'natlon produces d~ffuso multiple organ bystander injury !eading to progressive organ dysfunction and failure. systemic malignant inflammation involves diffuse actlvatton of all components of the humoral and cellular inflammatory host response. of these various components, the macropha~e is the crucial central cellular element. the tissue fixed macrophage is ideally located diffusely throughout the various organs injured to orchestrate the inflammatory process. the macrophage is long-lived and highly metabolic, the macrophage regulates both the extent and the dissemination of the inflammatory processes. the macrophage is an exu'emely active c¢11 capable of producing and releasing not only directly eytotoxlc agents, s irnil~, to the neutrophil, including oxidants and numerous proteases out also the multitude of other cytokines and initiators of the interacting inflammatory cascades. the macrophage is the central source for ehemotactic agents (il-8, ltb4, c5a) for neutrophils and other inflammatory cells, production of vasoaetive arachidonie acid metabolites (tx, pgi2, poe, lt's), complement components (c3a, csa), thrombotic agents (pca, tx), metabolic and physiologic modulators (il,1, il-6 or tnf), and immunosuppressivc agents (poe2, il-10). these products of the macrophage are highly effective in enhancing and augmenting the inflammatory response. disseminated activation otthe macrophage is critical to the induction of the long-term diffuse activation of inflammation necessary to induce multiple organ injury and failure. our ability to elucidate the molecular mechanisms that control the macrophage will lead to our ability to conu'ol the maerophage response and prevent mods-mof.flarborview medical center, 325-9th ave za-16, seattle, wa 98104 usa key: cord-330549-ppuqvafd authors: christophi, george p.; panos, michael; hudson, chad a.; christophi, rebecca l.; gruber, ross c.; mersich, akos t.; blystone, scott d.; jubelt, burk; massa, paul t. title: macrophages of multiple sclerosis patients display deficient shp-1 expression and enhanced inflammatory phenotype date: 2009-04-27 journal: lab invest doi: 10.1038/labinvest.2009.32 sha: doc_id: 330549 cord_uid: ppuqvafd recent studies in mice have demonstrated that the protein tyrosine phosphatase shp-1 is a crucial negative regulator of pro-inflammatory cytokine signaling, tlr signaling, and inflammatory gene expression. furthermore, mice genetically lacking shp-1 (me/me) display a profound susceptibility to inflammatory cns demyelination relative to wild type mice. in particular, shp-1 deficiency may act predominantly in inflammatory macrophages to increase cns demyelination as shp-1-deficient macrophages display co-expression of inflammatory effector molecules and increased demyelinating activity in me/me mice. recently, we reported that pbmcs of multiple sclerosis (ms) patients have a deficiency in shp-1 expression relative to normal control subjects indicating that shp-1 deficiency may play a similar role in ms as to that seen in mice. therefore, it became essential to examine the specific expression and function of shp-1 in macrophages from ms patients. herein, we document that macrophages of ms patients have deficient shp-1 protein and mrna expression relative to those of normal control subjects. to examine functional consequences of the lower shp-1, the activation of stat6, stat1, and nf-κb was quantified and macrophages of ms patients showed increased activation of these transcription factors. in accordance with this observation, several stat6-, stat1-, and nf-κb-responsive genes that mediate inflammatory demyelination were increased in macrophages of ms patients following cytokine and tlr agonist stimulation. supporting a direct role of shp-1 deficiency in altered macrophage function, experimental depletion of shp-1 in normal subject macrophages resulted in an increased stat/nf-κb activation and increased inflammatory gene expression to levels seen in macrophages of ms patients. in conclusion, macrophages of ms patients display a deficiency of shp-1 expression, heightened activation of stat6, stat1, and nf-κb and a corresponding inflammatory profile that may be important in controlling macrophage-mediated demyelination in ms. multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system (cns) that remains a major cause of disability (1) . several studies demonstrated that ms lesions contain multiple leukocyte cell types including lymphocytes, macrophages, and dendritic cells all of which are believed to contribute to lesion formation by various distinct and interacting mechanisms (2, 3) . among these leukocyte subsets, infiltrating macrophages have been identified as major effectors of demyelination in both ms and animal models for ms (4) (5) (6) (7) (8) (9) (10) (11) . in accord with these studies, it was recently described that a predominant mechanism of demyelination in ms is macrophage-mediated (12) . these findings have stimulated intense interest on the various effector functions of macrophages in lesion formation including signaling events that draw these cells into the cns white matter and trigger the effector mechanisms by which these cells destroy myelin. for instance, macrophages have been identified as the major responders to cns chemokines, producers of a number of pro-inflammatory cytokines, chemokines, and toxic molecules known to promote demyelination (7, (13) (14) (15) (16) (17) (18) , and the major cells involved in phagocytosis/degradation of myelin sheathes. interestingly, both leukocytes and cns plaques of ms patients contain activated transcription factors like nf-κb (19, 20) , stat1 (20) (21) (22) and stat6 (23) (24) (25) , which can lead to enhanced expression of these inflammatory effector functions in macrophages. based on our previous work, we propose that modulation of inflammatory signaling via these multiple transcriptional pathways may be deficient in leukocytes, including inflammatory macrophages of ms patients, and that this deficiency is responsible for susceptibility to inflammatory demyelinating processes within the cns. shp-1 is a protein tyrosine phosphatase with two sh2 domains which acts as a negative regulator of both innate and acquired immune cytokine signaling via nf-κb (26) (27) (28) , stat1 (29, 30) , and stat6 (31) (32) (33) (34) . mice genetically lacking shp-1 (motheaten mice) display myelin deficiency, which may be mediated by increased innate inflammatory mediators in the cns (35, 36). furthermore, motheaten mice are highly susceptible to experimentally induced demyelinating disease (37-39). taken together, these studies indicate that shp-1 is a key regulator of inflammation in the cns that may be relevant to the pathogenesis of ms. indeed, others and we reported that shp-1 expression and function is deficient in leukocytes of ms patients compared to normal human subjects (22, 25) . we have shown that shp-1-deficient mice uniquely display an unusually rapid cns demyelination associated with extensive white matter cellular infiltration and clinical paralysis within the first week of theiler's murine encephalomyelitis virus (tmev) infection compared to their wild type littermates (37). recently we have shown that shp-1deficient mice show a profound and predominant infiltration of blood-derived macrophages into the cns following tmev infection and that these macrophages are concentrated to areas of demyelination (9) . importantly, these macrophages possessed an inflammatory profile in vivo with an unusual co-activation of stat6, stat1, and nf-κb and corresponding responsive pro-inflammatory genes. because of the rapidity of demyelination in this model, we proposed that shp-1-deficient macrophages possess a particularly pronounced demyelinating phenotype. accordingly, macrophage/monocyte depletion with clodronate liposomes resulted in a significant delay in the onset of clinical symptoms and decreased inflammation and demyelination in the spinal cords of shp-1-deficient mice. those studies suggested that shp-1 is an important regulator of cns inflammatory demyelination acting predominantly to control recruitment of macrophages into the cns and a broad modulation of macrophage inflammatory effector functions. in the light of recent studies demonstrating the importance of macrophages in mediating demyelination in ms (12) and our recent studies in shp-1-deficient mice cited above (9) , it became imperative to characterize the inflammatory profile of macrophages of ms patients compared to normal subjects. the present study demonstrates that the expression of shp-1 is deficient in macrophages of ms patients compared to normal subjects. corresponding with this deficiency, we show that phosphorylation of stat6 and stat1 and activation of nf-κb are higher in macrophages of ms patients compared to those of normal subjects. in accordance with this activation, several stat6-, stat1-, and nf-κb-responsive genes that are important for inflammatory demyelination were increased in macrophages of ms patients following cytokine stimulation. supporting a direct role of shp-1 deficiency in altered macrophage function, experimental depletion of shp-1 in macrophages of normal human subjects using sirna resulted in an increased activation of the above transcription factors and increased inflammatory gene expression to levels seen in macrophages of ms patients. taken together, we propose the potential involvement of shp-1 in mediating the augmented inflammatory profile in macrophages seen in ms patients and contributing to the pathogenesis of ms. patients were clinically diagnosed for multiple sclerosis in the suny upstate medical university ms clinic by established criteria (40). patients were selected that had not received any disease modifying treatment like ifn-β, glatiramir acetate, steroids or other immunosuppressive agents at least three months prior to donating blood. in this study, macrophage cultures were obtained from 27 patients with relapsing remitting (rr) ms (mean edss score of 3.6+1.6) and 24 normal control subjects. the patients and control normal subjects were matched in both age and gender, and the biometric data of the subjects used in this study are shown in table 1 . the institutional review board of suny upstate university approved all studies and both patients and control subjects granted informed consent before providing blood. separation medium (cellgro, herndon, va). after centrifugation, the 10 ml of the interface containing the pbmcs was collected and washed twice with hbss. adherent monocytes were cultured for a week in rpmi media, 15% fetal bovine serum, and 50ng/ml gm-csf as previously described (41, 42). medium was replenished every 3 days and non-adherent cells were removed at the second feeding (at 6 days). for experiments, cells were cultured for an additional day in the absence of gm-csf, washed twice with pbs and adherent cells were lysed in stat-60 (tel-test, friendswood, tx) for rna analysis or ripa buffer (43) for protein analysis. for flow cytometry cells were detached by incubated them in 5.0mm edta for 10 minutes at 37 degrees c. the method consistently yielded more than 95% pure macrophages assessed by both morphological criteria as described (44) and expression of the monocyte lineage marker cd14 as determined by flow cytometric analysis. macrophages of ms patients and normal subjects were cultured for 1 week then gm-csf was removed from the media. macrophages were then treated with either 10ng/ml tnf-α, 10 ng/ml of il-4, 100u/ml ifn-γ (r&d systems, minneapolis, mn), 5µg/ml of lps (salmonella minnesota re595 lps; sigma-aldrich, st. louis, mo, usa), 5µg/ml of dsrna (polyinosinic:polycytidylic acid; amersham pharmacia, piscataway, nj) or received medium alone for 18hrs. anti-shp-1 sirna was used to deplete shp-1 from macrophages of normal subjects. macrophages were transfected with sirna against human shp-1 or scramble sirna (dharmacon, chicago, il) at a concentration of 1µg/10 6 cells. the transfection reagent (dharmafect 4, dharmacon, chicago, il) was used as specified by the manufacturer. cells were incubated in the transfection medium for 24 hours, after which the medium was replaced with complete growth medium for another 48 hr before cytokine treatment. the effectiveness of the shp-1 sirna to lower shp-1 expression was evaluated by western immunoblot. total rna was isolated using rna stat-60. rna was quantified spectrophotometrically and 0.5 µg of total rna was converted into cdna. briefly, total rna and random primers (invitrogen, carlsbad, ca) were incubated at 72 degrees for 10 minutes. reverse transcription was performed using the superscript ii rt enzyme (invitrogen, carlsbad, ca) and followed the specification of the manufacturer. cdna was diluted to 200µl with water and 4µl was used for quantitative real-time pcr using sybr green kit (abgene, epson, uk). the pcr parameters were 15 minutes for 95 degrees and 35 cycles of 95 degrees for 15 seconds and 60 degrees for 1 minute in abi prism 700 (applied biosystems, foster city, ca). the primers were used at 10 nm. serial dilutions of cdna containing a known copy number of each gene were used in each quantitative pcr run to generate a standard curve relating copy number with threshold amplification cycle (45). gene expression levels were calculated during the logarithmic amplification phase by determining the initial mrna copy number using the standard curve. amplification of each gene specific fragment was confirmed both by examination of melting peaks and by agarose gel electrophoresis. the following primer pairs were used in this study: shp-1 (bc002523) forward-tggcgtggcaggagaacag and reverse-gcagttggtcacagagtagggc, shp-1 promoter (i) (nm080548) forward-tggcttccccctccctacag and reverse-ccctggttcttgcgactgg, shp-1 promoter (ii) (nm002831) forward-atctgaggcttagtccctgagc and reverse-ctgaggtctcggtgaaaccac, ccl17 (nm002987) forward-cgagggaccaatgtgggc and reverse-gggtgaggaggcttcaagacc, ccl11 (nm002986) forward-cacttctgtggctgctgctc and reverse-gctttctggggacatttgc, ccr8 (nm005201) forward-cagtgtgacaacagtgaccgac and reverse-gcaataaaagacagcaaggagc, adam8 (nm001109) forward-atcccgagagacccgctac and reverse-tgattcaccacctccagcac, arginase i (nm000045) forward-gacctgccctttgctgacatc and reverse-ttgacttctgccaccttgcc, mcp-1 (nm002982) forward-gctcatagcagccaccttc and reverse-gcttctttgggacacttgc, caspase i (nm033294) forward-catcctcaggctcagaagg and reverse-tgtgcggcttgacttgtc, cox-2 (nm000954) forward-gcatctacggtttgctgtg and reverse-actgctcatcaccccattc, gapdh (nm002046) forward-accaccatggagaaggc and reverse-ggcatggactgtggtcatga. whole cell extracts were prepared as previously described (26, 30) . briefly, macrophages were rinsed with pbs, and then lysed with ripa buffer. 10 µg of protein per lane was electrophoresed through a 12.5 % polyacrylamide resolving gel and electroblotted to a polyvinylidene difluoride (pvdf) membrane (millipore corporation, burlington, ma). membranes were blocked with 5% nonfat dry milk for 1 hour, and then incubated with anti-shp-1 (upstate, lake placid, ny) antibodies followed by horseradish peroxidase conjugated rabbit igg antibody. (dako corporation, carpinteria, ca) or anti-arginase i (bd biosciences, san diego, ca) followed by horseradish peroxidase conjugated mouse igg antibody. enhanced chemiluminescence (amersham life sciences, inc., cleveland, oh) was used to visualize reactive protein bands on x-ray film. western blot protein bands were quantified based on pixel density using the scion image software. for analysis of stat6 and stat1 activation, macrophages were treated with either 10ng/ml il-4, 100 u/ml of ifn-γ, or 10 ng/ml tnf-α, respectively for 1 hour. cells were then washed twice with pbs and incubated in 5.0 mm edta for 10 minutes at 37 degrees c. cells were detached with gentle pipeting and received 100µl of 16% stock formaldehyde per ml for fixation and then incubated in 90% methanol at 4°c for half an hour to permeabilize cells for intracellular staining. cells were washed twice with the staining media containing 0.5% bsa and 0.02% sodium azide in pbs. cells were resuspended in a 100µl of staining media and incubated with 20µl of either phosphostat6 (py-641) with alexa-488 (612600), phosphostat1 (py-701) with alexa-488 (612596), or mouse igg with allexa-488 isotype control (557721) (becton dickinson, mountain view, ca). in addition, the levels of total (phosphorylated plus unphosphorylated) stat6 and stat1 were concurrently analyzed. fixed and permeabilized cells were incubated overnight at 4°c with either 1µg of rabbit anti-total stat6 (santa cruz, ca), rabbit anti-total stat1 (upstate biotechnology, ny), or rabbit polyclonal igg for isotype control. the cells were incubated for 3 hours in 1µg of goat anti-rabbit secondary antibody conjugated to pe (invitrogen, carlsbad, ca). furthermore, fixed and permeabilized cells were incubated overnight at 4°c with either 1µg of rabbit anti-shp-1 (upstate, lake placid, ny) or rabbit polyclonal iggs for isotype control. the cells were incubated for 3 hours in 1µg of goat anti-rabbit secondary antibody conjugated to pe (invitrogen, carlsbad, ca). in addition several extracellular markers were quantified by flow cytometry. 5×10 5 cells were incubated in 15µl of cd14-apc, cd11b-fitc, cd49d-pe, or ccr2-apc (becton dickinson, mountain view, ca) for 1 hour at room temperature. cells were analyzed on an lsrii analyzer (becton dickinson, mountain view, ca) and the mean florescence intensity (mfi) was recorded. nf-κb dna-binding activity was analyzed using the transamnf-κb p65 transcription factor assay kit (active motif, carlsbad, ca) following the manufacturer's instructions and as previously described (46-48). briefly, nuclear extracts were prepared (26) from macrophages of normal subjects and ms patients that were treated with media alone or 20 ng of tnf-α or 5µg/ml of lps for 1 hour. protein levels of the nuclear extracts were quantified with the bradford assay (pierce chemicals, rockford, il) and 10 µg were incubated in a 96-well plate coated with oligonucleotide containing the nf-κb consensusbinding sequence 5'-gggactttcc-3'. bound nf-κb was then detected by a p65-specific primary antibody. an hrp-conjugated secondary antibody was then applied to detect the bound primary antibody and provided the basis for colorimetric quantification. the enzymatic product was measured at 450 nm with a reference wavelength of 650 nm by a microplate reader. to quantify the amount of nf-κb, serial dilutions of purified p65 recombinant protein (20ng -0.16ng) were measured to provide a calibration curve between p65 binding and absorbance. the specificity of the assay was further tested by the addition of wild type or mutated nf-κb consensus oligonucleotide in the competitive or mutated competitive control wells before the addition of nuclear extracts. the addition of the wildtype nf-κb consensus oligonucleotide completely abolished nf-κb binding. the levels of the cytokines tnf-α and il-6 were measured using r&d systems duoset elisa kits (r&d systems, minneapolis, mn) following the manufacturer's protocol. the amount of no produced was measured in amounts of nitrite in macrophage supernatants. macrophages from normal subjects and ms patients were treated with either media alone, tnf-α, il-4, ifn-γ, or lps for 18hrs and supernatants were mixed with the greiss reagent at a 1:1 ratio (sigma, st. louis, mo). the reaction was allowed to proceed for 15 min, at which time the absorbance at 540nm was quantified with an elisa plate reader. a calibration curve was generated using sodium nitrite (1-100µm) and used to calculate the amount of nitrite present in macrophage supernatants. arginase enzymatic activity was measured as previously described (31, 49) . briefly, cultured macrophages were lysed in ripa buffer and incubated in 10 mm mncl 2 and 0.5 m l-arginine at 37°c for 75 min. after the assay was stopped by addition of h 3 po 4 , 1phenyl-1,2-propanedione-2-oxime (sigma, st louis, mo) was added and the samples were incubated at 100°c for 60 min. urea production by arginase was measured by optical density at 540 nm. histograms contain statistical means with the standard error values. unless otherwise specified, 27 samples from ms patients and 24 samples from normal subjects were used. in the shp-1 sirna experiments samples of macrophages from 15 normal human subjects were used. the p-values were generated using the unpaired student's t-test and p-values between groups are displayed on histograms. a p-value of less than 0.05 was chosen to indicate statistical significance between two sample means. the expression levels of multiple macrophage markers were quantified by flow cytometry in cultured macrophages of normal subjects and ms patients ( figure 1 ). more than 95% of the cells stained for cd14 and no significant differences in levels of expression were observed between the subject groups ( figure 1a ). furthermore, we examined the expression of ccr2 (cd192), which is the receptor for the chemokine mcp-1 and a marker for inflammatory monocytes (50). importantly, macrophages of shp-1 deficient mice display increased levels of ccr2 (51, 52). macrophages of ms patients had significantly higher levels of ccr2 compared to normal subjects indicating that ms-derived macrophages have a relatively heightened inflammatory state. as such, we characterized two other potential activation markers on these cells. we analyzed the antigenic determinants of cd11b (α m β 2 integrin) and vla4 (α 4 β 1 integrin), which have been shown to be important in the trafficking, adhesion, phagocytosis, and migration of macrophages at inflammatory sites (53) (54) (55) (56) . integrin α m β 2 expression was significantly higher in the macrophages of ms patients compared to normal subjects. in contrast, the expression of integrin α 4 β 1 was not significantly different between macrophages of ms patients and normal subjects. we have previously described that pbmcs of ms patients have decreased expression of shp-1 compared to normal subjects (25) . cultured macrophages of ms patients had significantly lower levels of shp-1 protein than those of normal subjects measured by intracellular flow cytometry (figure 2a & 2b) . to corroborate these findings, the levels of shp-1 protein in macrophages were measured by western immunoblotting ( figure 2c ). on average, shp-1 levels in macrophages of ms patients were half the levels of shp-1 in cells of normal subjects. to examine whether macrophages of ms patients displayed a deficiency in shp-1 at the transcriptional level, the levels of shp-1 mrna were quantified using primers flanking the coding region of the gene common to both transcripts ( figure 3 ). shp-1 mrna levels in macrophages were significantly lower in ms patients compared to normal subjects ( figure 3a) . furthermore, analysis of shp-1 mrna expression in individual subject groups did not show any statistically significant correlation between either age or gender and shp-1 levels (data not shown). in order to determine the individual contribution of each of two known transcripts on the expression levels of shp-1 in macrophages, promoter i and ii transcript copy numbers were measured using promoter-specific rt-pcr primers. constitutive levels of shp-1 promoter i transcripts were not significantly lower in macrophages in ms patients compared to controls ( figure 3c ). on the other hand, constitutive levels of shp-1 promoter ii transcripts were significantly lower in macrophages of ms patients compared to controls ( figure 3d ). taken together, we concluded that macrophages of ms patients have constitutively lower levels of shp-1 protein and mrna compared to normal subjects and furthermore that a lack in promoter ii transcripts is primarily responsible for this deficiency. shp-1 regulates phosphorylation of several stats by enzymatic removal of the phosphate group from tyrosine either on activated cytokine receptors or downstream signaling molecules. thus, shp-1 modulates stat activation, translocation into the nucleus, and transcriptional activity on responsive genes. because several studies showed that shp-1 stringently modulates il-4rα/stat6 signaling molecules (31, 32, 34, 57) , and stat6 was shown to be elevated in ms (23) (24) (25) , we examined stat6 activation in macrophages from ms patients (figure 4 ). for analysis, we measured the level of total stat6 (unphosphorylated plus phosphorylated) and tyrosine phosphorylated stat6 (pstat6) in macrophages using intracellular flow cytometry. as expected, macrophages of ms patients had significantly elevated constitutive phosphorylation of stat6 compared to macrophages of normal subjects ( figure 4b ). in contrast, there were no differences in the expression levels of total stat6 protein ( figure 4a ), verifying that the differences observed in pstat6 levels are attributed to the relative phosphorylation state of stat6. staining the same cells for shp-1 revealed a reciprocal expression pattern of shp-1 and pstat6, such that lower constitutive levels of shp-1 in ms patients correlated with higher pstat6 levels compared to macrophages of normal subjects (data 18 not shown). furthermore, macrophages were treated for 1 hour with il-4, which induces phosphorylation of stat6, to determine possible differences in stat6 activation following engagement of the il-4 receptor. treatment with il-4 caused higher phosphorylation of stat6 in macrophages of ms patients compared to normal subjects ( figure 4b ). similar results were obtained when macrophages were treated with il-13, another cytokine that mediates stat6 activation via the il-4rα (data not shown). apart from stat6, there are several other transcription factors that are elevated in ms patients and are controlled by shp-1. in particular, tyrosine phosphorylated stat1 (py-stat1) that mediates interferon signaling was found to be elevated in ms patients (20, 21) and is controlled by shp-1 (29, 30, 58, 59) . therefore, we characterized stat1 activation by measuring the level of total stat1 (unphosphorylated plus phosphorylated, stat1) and tyrosine phosphorylated stat1 (pstat1) in macrophages using intracellular flow cytometry. no differences were observed in total stat1 protein ( figure 4c ). constitutive pstat1, although higher, was not significantly elevated in macrophages of ms patients compared to normal subjects ( figure 4d ). however, 1 hour treatment with ifn-γ resulted in higher phosphorylation of stat1 in macrophages of ms patients compared to normal subjects ( figure 4d ). taken together this data suggest that both stat6 and stat1 show enhanced cytokine-mediated activation in macrophages of ms patients, in accordance with shp-1 deficiency relative to normal subject macrophages. another important transcription factor involved in the pathogenesis of ms is nf-κb (19, 60) . notably, nf-κb activation was observed in inflammatory macrophages in ms cns lesions (61) . several reports have documented the negative regulation of nf-κb by shp-1 (26, 27, 59, 62, 63) . therefore, it was important to examine nf-κb activation in macrophages of ms patients ( figure 5a ). macrophages from normal subjects and ms patients were treated with medium alone, tnf-α or lps and then nuclear extracts were isolated and allowed to bind an nf-κb consensus oligonucleotide sequence. bound nf-κb was then detected by a p65 (rela)-specific antibody and quantified based on a calibration curve generated by using a purified p65 recombinant protein. constitutively, nf-κb binding was slightly, but significantly elevated in macrophages of ms patients compared to normal subjects ( figure 5a ). furthermore, treatment with either t n f-α or lps dramatically increased nf-κb binding and importantly nuclear extracts from macrophages of ms patients showed significantly increased binding compared to macrophages of normal subjects. in addition, we quantified the secretion of the nf-κb-responsive pro-inflammatory cytokines tnf-α and il-6 from macrophages of ms patients compared to normal subjects, since it was previously shown that a deficiency in shp-1 leads to higher expression of these cytokines (9, 27, 62, (64) (65) (66) . macrophages were cultured for 18 hours with either media alone, tlr ligands or tnf-α and the supernatants were analyzed for the presence of tnf-α or il-6 ( figure 5b & 5c) . constitutively, the levels of tnf-α and il-6 were slightly but significantly elevated in supernatants of macrophages from ms patients compared normal subjects. furthermore, stimulation of macrophages with lps or dsrna resulted in a substantial induction of tnf-α and il-6 in both subject groups but the induction was significantly higher in macrophages of ms patients compared to normal subjects. in all, these data demonstrate that macrophages of ms patients display an enhanced expression of nf-κb-responsive genes both constitutively and after stimulation with tlr ligands and cytokines that induce nf-κb. in order to determine whether increased activation of stat6, stat1, and nf-κb in macrophages of ms patients relative to normal subjects corresponded to heightened expression of stat6-, stat1-, and nf-κb responsive genes, the mrna levels of several genes were quantified following 18-hour treatment with several cytokines (figure 6 ). the expression levels of stat6-responsive chemokines were of particular interest because these mediate trafficking, maturation, and attraction of lymphocytes and macrophages to areas of cns inflammation (14) . ccl17/tarc (67, 68) and ccl11/eotaxin i (69) were substantially induced in macrophages following il-4 treatment and importantly macrophages of ms patients showed significantly higher induction compared to macrophages of normal subjects ( figure 6a & 6b) . similar results were obtained when macrophages were treated with il-13 (data not shown). similarly, the mrna of adam8, a stat6-responsive disintegrin matrix metalloproteinase (70, 71) that may be involved in demyelination (72) , was induced at higher levels in macrophages of ms patients compared to macrophages of normal subjects following il-4 treatment (data not shown). taken together, in agreement with depressed shp-1 levels, macrophages of ms patients display increased stat6 activation and increased expression of stat6-responsive gene expression. furthermore, because shp-1 controls stat1 activation (29, 30, 58, 73) , we examined the expression of stat1-responsive genes following cytokine stimulation ( figure 6 ). the chemokine ip-10, a stat1-inducible chemokine that was previously shown to be elevated in ms (74) (75) (76) (77) (78) , was highly induced by ifn-γ and moderately induced by lps in ms macrophages. moreover, ip-10 was induced to significantly higher levels in ms macrophages compared to normal subject macrophages ( figure 6c) . similarly, caspase 1, a stat1-inducible enzyme involved in il-1β-mediated inflammation (79) (80) (81) , was significantly induced to higher levels by ifn-γ in ms macrophages compared to macrophages of normal subjects ( figure 6d ). these data suggest that stat1 phosphorylation and stat1-responsive genes show enhanced activation in macrophages of ms patients compared to macrophages of normal subjects. moreover, since nf-κb binding was elevated in macrophages of ms patients, we wanted to examine whether nf-κb-responsive genes were elevated in macrophages of ms patients. therefore, we quantified the expression of the nf-κb inducible chemokine mcp-1 and cyclooxygenase-2 (cox-2) (82, 83) with real time rt-pcr ( figure 6e & 6f) . mcp-1 and cox-2 genes showed substantially higher induction in macrophages of ms patients compared to normal subjects following treatment with either tnf-α or lps. these data further demonstrate that nf-κb activation and nf-κb-responsive genes are elevated in macrophages of ms patients compared to normal subjects. several reports in mouse models point to the importance of different macrophage activation profiles in specific types of inflammatory disease (84) . recently characterized macrophage profiles are the classically activated (or m1 macrophages) that display increased stat1/nf-κb-responsive genes including inducible nitric oxide synthase (inos) and alternatively activated (or m2 macrophages) that display increased stat6-responsive genes including arginase i. interestingly, there is usually a profound antagonism of differentiation towards either m1 or m2 macrophage activation profile such that dual expression of inos and arginase i is seldomly observed (85) (86) (87) (88) . using this paradigm, we determined whether ms macrophages might be preferentially skewed toward either the m1 or m2 profile relative to those of normal subjects. examination of constitutive inos mrna expression and nitric oxide (no) production showed no differences between ms and normal subject macrophages (figure 7) . tnf-α and ifn-γ moderately and lps substantially induced inos mrna expression and no production in macrophages ( figure 7a & 7b) . importantly, macrophages from ms patients showed a significantly higher inos mrna expression and no production following cytokine stimulation compared to macrophages from normal subjects. with respect to constitutive arginase i expression, we found higher levels of both arginase i protein, mrna, and enzymatic activity in ms compared to normal subject macrophages ( figure 6c-e) . moreover, when macrophages were exposed to m2-inducing cytokine il-4, ms macrophages expressed more arginase i than normal subject macrophages. these data indicated that ms macrophages may be constitutively skewed towards m2 activation profile in vitro and this skewing may be further reinforced by il-4. nonetheless, ms macrophages also display m1-skewedness relative to normal subject macrophage following exposure to m1-inducing cytokines such as ifn-γ and tnf-α. the latter indicates the extreme plasticity of ms macrophages relative to normal subject macrophages and the potential importance of the cytokine milieu in determining the ultimate balance between m1 and m2 phenotype of these cells. to examine whether decreased shp-1 levels are directly responsible for transcription factor activation and inflammatory gene expression in macrophages, macrophages of normal subjects were treated with sirna to acutely deplete shp-1 ( figure 8 ). first, we observed that shp-1 protein was substantially depleted by more than 5-fold in the macrophages treated with the shp-1 sirna compared to the macrophages treated with control sirna of scrambled sequence ( figure 8a ). accordingly, shp-1 depletion resulted in a considerable increase of the stat6-responsive arginase i protein ( figure 8a ) and mrna (data not shown). further, we investigated whether lowered shp-1 levels in normal human macrophages treated with sirna led to enhanced activation of stat6 and stat1. shp-1 depletion resulted in a significant increase in constitutive stat6 phosphorylation and to a significantly higher induction of pstat6 in response to 1-hour treatment with il-4 ( figure 8b ). similar results were obtained when macrophages were treated with il-13, another cytokine that mediates stat6 activation via the il-4rα (data not shown). in addition, although shp-1 depletion did not result in a significant increase of constitutive stat1 phosphorylation, ifn-γ treatment for 1 hour induced significantly higher pstat1 levels in shp-1 depleted macrophages compared to control macrophages treated with scrambled sirna ( figure 8c ). to further validate that shp-1 controls stat6 and stat1 activation, we examined the expression of stat6-and stat1-responsive genes in macrophages of normal subjects that were treated with either scramble sirna or shp-1 sirna. the stat6-inducible genes ccl17 ( figure 8d ), arginase i, ccl11, and adam8 (data not shown) were significantly induced to higher levels in shp-1-depleted macrophages following an 18-hour il-4 stimulation. similarly, the stat1-inducible genes ip-10 ( figure 8e ) and caspase 1 (data not shown) were induced at significantly higher levels in shp-1 depleted macrophages following ifn-γ treatment. taken together, these results point to the important role of shp-1 in down-modulating pro-inflammatory stat6 and stat1 activities in human macrophages. furthermore, we examined the role of shp-1 in controlling nf-κb-responsive genes in human macrophages. first mrna levels of the nf-κb-inducible chemokine mcp-1 ( figure 8f ) and cox-2 (data not shown) were induced at significantly higher levels in shp-1depleted macrophages following tnf-α or lps treatment. furthermore, the secretion of tnf-α and il-6 in shp-1 depleted macrophages was significantly higher following lps or dsrna stimulation compared to macrophages treated with scramble sirna (figure 8g & 8h). additionally, tnf-α or lps treatment led to a significantly higher production of no in shp-1-depleted macrophages compared to macrophages treated with scramble sirna ( figure 8i ). taken together, we concluded that depressed levels o f s h p-1 in human macrophages led to a unique co-activation of stat6/stat1/nf-κb-responsive proinflammatory genes relative to normal subject macrophages. with the observation of heightened ccr2 (figure 1 ), we propose that ms macrophages may be inherently inflammatory in nature. in multiple sclerosis and animal models for ms, intense macrophage infiltration is present in active demyelinating lesions and both their nmbers and differentiation/activation correlates with disease severity (2, 4, 5, 89, 90) . macrophages can mediate myelin phagocytosis, degradation, oligodendrogliopathy, and axonal loss both through cell-mediated processes and the secretion of inflammatory mediators (1, 7, 90, 91) . based on our recent report of increase demyelinating activity of macrophages in me/me mice (9), a key question was whether macrophages of ms patients display similar defects in modulation of proinflammatory cytokine signaling that may contribute to activated inflammatory profile and demyelinating activity. therefore, we analyzed the expression and function of shp-1 as a possible cause of enhanced macrophage inflammatory activity in ms. examining monocyte-derived macrophages from ms patients and normal subjects revealed that shp-1 was deficient in macrophages of ms patients. interestingly, this shp-1 deficiency corresponded to a diminished expression of shp-1 promoter ii transcripts suggesting a specific alteration in promoter ii function in ms. accordingly, macrophages from ms patients displayed enhanced stat6, stat1, and nf-κb activity, transcription factors that were previously shown to be modulated by shp-1 in various cell types including mouse macrophages (9) . in turn, several stat6-, stat1-, and nf-κb-inducible genes that have been shown to play role in the mechanism of inflammatory demyelination were shown to be both higher in macrophages of ms patients and regulated by shp-1. the relevance of the present findings to the pathogenetic mechanisms of ms is supported by previous reports on activation of signaling molecules in the cns of ms subjects that are regulated by shp-1. for instance, phosphorylated stat6 was found to be highly expressed in ms lesions (24) , normal appearing white matter of ms patients (23) and leukocytes of ms patients (25) . furthermore, macrophages of mice that lack shp-1 show increased constitutive activation of stat6 and heightened levels of stat6-inducible genes (25) . moreover, the lack of shp-1 leads to severe macrophage-mediated cns demyelination following tmev infection (9, 31, 37) . thus, the present study suggests that stat6 activation and expression of several genes with confirmed stat6-responsive elements in macrophages may have functional significance to the pathophysiology of ms. for example, the chemokines ccl17/tarc and ccl11/eotaxin, which attract inflammatory cells to demyelinating lesions, were elevated in macrophages of ms patients and the expression of these chemokines is controlled by shp-1 (92) (93) (94) . analysis of other stat6-responsive genes showed that the matrix metalloproteinase adam8 is elevated in macrophages of ms patients and shp-1 controls its expression. proteinases may be involved in the pathogenesis of ms, since these can mediate the proteolysis of extracellular matrix molecules, disrupt the blood brain barrier, and promote leukocyte entry into the brain parenchyma (95) . adam8 which is highly expressed in macrophages was shown to hydrolyze myelin basic protein (72) and can therefore directly contribute to demyelination. in addition, arginase i is a stat6-responsive gene that has been shown to be constitutively elevated in the cns of shp-1-deficient mice and plays an important role in virus-induced demyelinating disease (31) . also, arginase i inhibition results in attenuation of eae onset and progression (73) . interestingly, the increased levels of arginase i in ms macrophages might make these cells more susceptible to viral infections relevant to ms pathogenesis (31) . although the mechanism by which arginase i activity may promote inflammatory demyelination still remains to be elucidated, its role in ms in light of the present findings should be further investigated. apart from stat6, there are several other transcription factors that were previously shown to be elevated in ms patients and are controlled by shp-1. in particular, tyrosine phosphorylated stat1 (py-stat1) that mediates interferon signaling was found to be elevated in ms patients and controlled by shp-1 (20-22, 30, 58) . in agreement with those studies, we found that stat1 activation is enhanced in macrophages of ms patients following ifn-γ stimulation. furthermore, stat1-inducible genes like the chemokine ip-10 and caspase 1 are elevated in macrophages of ms patients consistent with a role for stat1 in expression of these genes (74) (75) (76) (77) (78) (79) 96) . thus, lower levels of shp-1 seen in macrophages of ms patients may lead to activation of stat1 and stat1-inducible genes that play a role the pathogenesis of ms. furthermore, the transcription factor nf-κb mediates pro-inflammatory cytokine signaling and is elevated in ms pbmcs (19) and macrophages in ms lesions (60, 61) . thus, it may be important that mice genetically lacking shp-1 show elevated nf-κb activity and increased nf-κb inducible genes in both cns glia and hematopoietic cells (9, 26, 27, 59, 94) . therefore it was imperative to investigate whether nf-κb-responsive genes were elevated in macrophages of ms patients. both the chemokine mcp-1 and cox-2 showed significantly elevated induction in macrophages from ms patients compared to normal subjects reflecting the increased nf-κb binding activity observed in ms macrophages. in addition, the inflammatory cytokines tnf-α and il-6, which are regulated by nf-κb, were secreted at higher levels in macrophages of ms patients compared to macrophages of normal subjects following lps or dsrna stimulation which signal via tlr4 and tlr3 respectively to activate nf-κb. considering that shp-1-depleted macrophages of normal subjects display a very similar inflammatory profile to macrophages of ms patients, it is likely that the deficiency of shp-1 seen in macrophages of ms patients is directly responsible for the increased inflammatory profile. as such, the classification of macrophage phenotypic markers may be an important consideration in ms. several reports in mouse models point to the importance of different macrophage activation profiles in specific types of inflammatory disease (84) . for instance, so-called classically activated or m1 macrophages display increased stat1-and nf-κbresponsive genes, which have been shown to be critical for eradicating various intracellular microbes and causing some types of autoaggressive tissue inflammation such as rheumatoid arthritis. conversely, so-called "alternatively activated" or m2 macrophages display increased stat6-responsive genes including arginase i and may be more important for eradicating helminthes and promoting allergic-type inflammatory diseases. importantly, there is usually a profound antagonism between differentiation towards either m1 or m2 macrophage activation profiles such that dual expression of m1 and m2 genes are rarely seen. in possible distinction to these prior classifications, the present study indicates that macrophages of ms patients may exhibit abnormally high activation of either stat6, stat1 or nf-κb, depending on the nature of cytokine stimulation. as such, a deficiency in shp-1 may allow an aberrant co-activation of stat6/stat1/nf-κb responsive genes in ms macrophages especially in vivo where these cells may be simultaneously exposed to both m1-and m2-skewing cytokines. if so, the inflammatory nature of these macrophages may be unique and perhaps possess a corresponding enhanced demyelinating capacity. such a concurrent activation of normally opposed pathways in ms macrophages is supported by observations of patterns of gene expression in leukocytes including macrophages in ms, where increased co-expression of stat6/stat1/nf-κb-responsive genes is observed (20, 21, 24, 25) . adding to the promotion of broad activation phenotype of macrophages in ms are reports showing that high levels of both th1 and th2 cytokines are co-expressed in ms. for instance, cultured myeloid dendritic cells from rr ms patients polarize naïve t-cells to both th1 and th2 and induce increased levels of ifn-γ, il-4/il13, and tnf-α (97, 98) . in accord with these findings, several studies demonstrate that plasma, leukocytes, and csf of patients with active ms show a concurrent increase of ifn-γ, il-4/ il13, and tnf-α levels compared to normal subjects (99) (100) (101) (102) (103) (104) (105) (106) (107) . the high levels of plasma cytokines certainly contributes to the enhanced transcription factor activation previously observed in freshly isolated peripheral blood cells of ms patients (19, 21, 22) . in distinction to these prior studies, the present study exclusively employs cultured macrophages of ms patients and normal subjects, which offers the advantage of allowing stimulation of the cells with equal amounts of the cytokines ifn-γ, il-4, and tnf-α and therefore examining intrinsic differences in cytokine signaling. therefore, apart from the elevated cytokine levels that are observed in ms patients, this study further documents that macrophages of ms patients display enhanced signaling in response to both th1 and th2 pro-inflammatory cytokines and therefore the lack of shp-1 may be responsible for a broad inflammatory profile of these cells. another question that arises is how the shp-1 deficiency observed in peripheral macrophages of ms patients might only be manifested as cns inflammatory demyelination in ms patients. one possibility is that persistent cns viruses might attract macrophages into the cns and the lower levels of shp-1 in ms macrophages enhance migration to and demyelinating activity with the cns. the latter is supported by studies showing that tmev infection in shp-1-deficient mice results in severe cns demyelination that is exclusively macrophage-mediated (108) . furthermore, deficiency of shp-1 in macrophages enhances responsiveness to chemokines including mcp-1 that play an important role in macrophagemediated demyelination (9, 51) . another possibility is that macrophages are attracted into the cns through myelin-specific autoimmune events. once macrophages enter the cns, the shp-1-deficiency can contribute to enhanced fc-gamma receptor mediated myelin phagocytosis in the presence of antibodies and complement (12, (109) (110) (111) . these hypotheses indicate a two-step process in which shp-1-deficiency in macrophages is a precondition to tissue specific inflammation triggered by a cns specific inflammatory signal. the latter is consistent with a recent mouse study in which shp-1 deficiency alone does result in inflammatory disease but rather establishes a permissive state in which microbial infection triggers inflammation and autoimmunity (28) . two distinct promoters drive the expression of two different shp-1 transcripts from the shp-1 gene (112) (113) (114) . here, we demonstrated that promoter ii transcripts, which are preferentially expressed in hematopoetic cells, are selectively lower in macrophages of ms patients compared to normal subjects. the question thus arises as to the potential genetic basis for deficient shp-1 promoter ii activity in ms leukocytes. several studies have examined the contribution of genetics in ms (115) (116) (117) (118) and with exception to some weak association in some populations, no study has shown an absolute linkage to the shp-1 locus, chromosome 12p12 (119) that would explain a wide-spread deficiency in promoter ii expression in ms patients. as such, alternative mechanisms need to be considered including epigenetic alterations in promoter activity. indeed, numerous reports document the downregulation of shp-1 promoter ii transcripts in leukemia/lymphoma cell lines and attribute shp-1 deficiency to epigenetic methylation of particular cpg sites of shp-1 promoter ii (120, 121) . interestingly, in the context of ms, either viral infections (122) (123) (124) (125) (126) (127) or inflammation (128) (129) (130) (131) can cause de novo methylation or hypermethylate cpg promoter sequences resulting in decreased gene expression. furthermore, a recent studies report that htlv-1, which has been associated with the cns demyelinating disease known as ham/tsp (htlv-1 associated myelopathy/tropical spastic paparparesis) (132), profoundly and specifically suppresses shp-1 expression through methylation of the shp-1 promoter ii (133, 134) . in accordance with these reports, preliminary data indicate that treatment of pmbcs of ms patients with the demethylating agent 2'-deoxy-5-azacytidine increase shp-1 promoter ii transcripts to normal levels (personal unpublished observations). whether a stable epigenetic modification of promoter ii or another mechanism results in a deficiency in shp-1 expression in macrophages from ms patients requires further investigation. stat6 and stat1 activation in macrophages of ms patients and normal subjects. total stat6, tyrosine phosphorylated stat6 (pstat6), total stat1, and tyrosine phosphorylated stat1 (pstat1) levels in macrophages of ms patients compared to normal subjects. macrophages of ms patients and normal subjects were fixed, permeabilized, and stained with antibodies against either against total stat6, psta6, stat1, or pstat1. the histogram overlays are labeled as follows: shaded histogram for isotype control, solid line for normal subject, and dashed line for ms patient. a & b. the levels of stat6 and pstat6 were quantified based on the relative mfi in macrophages of ms patients and normal subjects before and after 1-hour treatment with il-4. c & d. the levels of stat1 and pstat1 were quantified based on the relative mfi in macrophages of ms patients and normal subjects before and after 1-hour treatment with ifn-γ. nf-κb activation in macrophages of ms patients and normal subjects. a. macrophages from normal subjects and ms patients were treated with media alone, tnf-α or lps for 1 hour and nuclear extracts were isolated and allowed to bind an nf-κb consensus-binding sequence. bound nf-κb was then detected by a p65 specific antibody and quantified based on a calibration curve generated by using a purified p65 recombinant protein. the nf-κb dna binding activity is reported as ng of bound p65 protein per 10µg of macrophage nuclear extracts. b. & c. tnf-α and il-6 secretion was quantified in macrophages of ms patients and normal subjects. macrophages were cultured in media alone or in the presence of the tlr ligands, lps and dsrna for 18 hours and supernatants were analyzed for tnfα or il-6 by elisa. gene profile of macrophages of ms patients and normal subjects. a number of genes that are stat6-, stat1-, or nf-κb-inducible were quantified in macrophages of normal subjects and ms patients before and after 18-hour treatment with tnf-α, il-4, ifn-γ, and lps. real time rt-pcr was used to measure the mrna levels of ccl17/tarc, ccl11/ eotaxin, cxcl10/ip-10, caspase i, ccl2/mcp-1, and cyclooxygenase ii (cox-2). the actual transcript copy number of each gene is reported in relation to the untreated normal subjects, which is designated as 1. arginase i and inos expression and activity in macrophages of ms patients and normal subjects. a. inducible nitric oxide synthase (inos) mrna expression was quantified in macrophages of normal subjects and ms patients before and after 18-hour treatment with tnf-α, il-4, ifn-γ, and lps by real-time rt-pcr. b. similarly, the levels of nitrite, as an indicator of nitric oxide (no) production, were quantified in the supernatants of macrophages from ms patients and normal subjects that were treated with tnf-α, il-4, ifn-γ, and lps. c. arginase i and actin protein expression were measured in macrophages of ms patients compared to normal subjects by western blot analysis. d. the enzymatic activity of arginase was quantified in macrophages of ms patients and normal subjects. e. arginase i mrna expression was quantified in macrophages of normal subjects and ms patients before and after 18-hour treatment with tnf-α, il-4, ifn-γ, and lps by real-time rt-pcr. shp-1 was depleted in macrophages of normal subjects using sirna and the inflammatory profile is the context of shp-1 deficiency was characterized. a. macrophages of normal subjects were treated with shp-1 sirna or control sirna (scramble sequences) and the levels of shp-1, arginase i, and actin protein levels were quantified by western immonoblot analysis. in the same panel the amount shp-1 and arginase protein measured by western immunoblot were quantified by measuring the pixel density. results were normalized to macrophages treated with scramble sirna represented as 1. b. & c. phosphorylated stat6 multiple sclerosis heterogeneity of multiple sclerosis lesions: implications for the pathogenesis of demyelination neuropathology of multiple sclerosis-new concepts macrophages in multiple sclerosis ccr1+/ccr5+ mononuclear phagocytes accumulate in the central nervous system of patients with multiple sclerosis theiler's virus persistence in the central nervous system of mice is associated with continuous viral replication and a difference in outcome of infection of infiltrating macrophages versus oligodendrocytes macrophages and neurodegeneration role of macrophages/microglia in multiple sclerosis and experimental allergic encephalomyelitis modulation of macrophage infiltration and inflammatory activity by the phosphatase shp-1 in virus-induced demyelinating disease maturation and localization of macrophages and microglia during infection with a neurotropic murine coronavirus microglial/macrophage accumulation during cuprizone-induced demyelination in c57bl/6 mice homogeneity of active demyelinating lesions in established multiple sclerosis chemokines and chemokine receptors in autoimmune encephalomyelitis as a model for central nervous system inflammatory disease regulation the expression and function of chemokines involved in cns inflammation effectors of demyelination and remyelination in the cns: implications for multiple sclerosis kallikreins are associated with secondary progressive multiple sclerosis and promote neurodegeneration activity of a newly identified serine protease in cns demyelination anti-ccl2 treatment inhibits theiler's murine encephalomyelitis virus-induced demyelinating disease changes in the activation level of nf-kappa b in lymphocytes of ms patients during glucocorticoid pulse therapy upregulation of transcription factors controlling mhc expression in multiple sclerosis lesions pstat1, pstat3, and t-bet expression in peripheral blood mononuclear cells from relapsing-remitting multiple sclerosis patients correlates with disease activity low expression of interferonstimulated genes in active multiple sclerosis is linked to subnormal phosphorylation of stat1 normal-appearing white matter in multiple sclerosis is in a subtle balance between inflammation and neuroprotection multiple sclerosis: cytokine receptors on oligodendrocytes predict innate regulation shp-1 deficiency and increased inflammatory gene expression in pbmcs of multiple sclerosis patients increased inducible activation of nf-kappab and responsive genes in astrocytes deficient in the protein tyrosine phosphatase shp-1 functional consequences of the shp-1 defect in motheaten viable mice: role of nf-kappa b inflammation and autoimmunity caused by a shp1 mutation depend on il-1, myd88, and a microbial trigger differential regulation of the alpha/beta interferon-stimulated jak/stat pathway by the sh2 domain-containing tyrosine phosphatase shptp1 the role of protein tyrosine phosphatase shp-1 in the regulation of ifn-gamma signaling in neural cells inverse regulation of inducible nitric oxide synthase (inos) and arginase i by the protein tyrosine phosphatase shp-1 in cns glia regulation of the dephosphorylation of stat6. participation of tyr-713 in the interleukin-4 receptor alpha, the tyrosine phosphatase shp-1, and the proteasome shp-1 regulates stat6 phosphorylation and il-4-mediated function in a cell type-specific manner regulation of the leishmaniainduced innate inflammatory response by the protein tyrosine phosphatase shp-1 leukocyte cd11/cd18 integrins: biological and clinical relevance the adhesion molecules used by monocytes for migration across endothelium include cd11a/cd18, cd11b/cd18, and vla-4 on monocytes and icam-1, vcam-1, and other ligands on endothelium increased expression of cd11b/cd18 on phagocytes in ischaemic disease: a bridge between inflammation and coagulation anti-alpha4 integrin therapy for multiple sclerosis: mechanisms and rationale protein-tyrosine phosphatase shp-1 is a negative regulator of il-4-and il-13-dependent signal transduction association of the interferondependent tyrosine kinase tyk-2 with the hematopoietic cell phosphatase receptor activator of nf-kappa b ligand stimulates recruitment of shp-1 to the complex containing tnfr-associated factor 6 that regulates osteoclastogenesis activation of nf-kappab and c-jun transcription factors in multiple sclerosis lesions. implications for oligodendrocyte pathology transcription factor nf-kappab and inhibitor i kappabalpha are localized in macrophages in active multiple sclerosis lesions lipopolysaccharide-activated shp-1-deficient motheaten microglia release increased nitric oxide, tnf-alpha, and il-1beta regulation of inhibitory protein-kappab and monocyte chemoattractant protein-1 by angiotensin ii type 2 receptor-activated src homology protein tyrosine phosphatase-1 in fetal vascular smooth muscle cells role of tumor necrosis factor-alpha in the spontaneous development of pulmonary fibrosis in viable motheaten mutant mice phosphatase shp-1 promotes tlr-and rig-i-activated production of type i interferon by inhibiting the kinase irak1 regulation of avoidant behaviors and pain by the anti-inflammatory tyrosine phosphatase shp-1 il-4 induces expression of tarc/ccl17 via two stat6 binding sites csf chemokine levels in relapsing neuromyelitis optica and multiple sclerosis interleukin-13 upregulates eotaxin expression in airway epithelial cells by a stat6-dependent mechanism the detection of adam8 protein on cells of the human immune system and the demonstration of its expression on peripheral blood b cells, dendritic cells and monocyte subsets expression and regulation of a disintegrin and metalloproteinase (adam) 8 in experimental asthma the enzymatic activity of adam8 and adam9 is not regulated by timps arginase and autoimmune inflammation in the central nervous system expression of specific chemokines and chemokine receptors in the central nervous system of multiple sclerosis patients expression of the interferongammainducible chemokines ip-10 and mig and their receptor, cxcr3, in multiple sclerosis lesions expression and modulation of ifn-gamma-inducible chemokines (ip-10, mig, and i-tac) in human brain endothelium and astrocytes: possible relevance for the immune invasion of the central nervous system and the pathogenesis of multiple sclerosis chemokine expression by astrocytes plays a role in microglia/macrophage activation and subsequent neurodegeneration in secondary progressive multiple sclerosis cxc chemokine receptors on human oligodendrocytes: implications for multiple sclerosis increased expression of caspase-1 and interleukin-18 in peripheral blood mononuclear cells in patients with multiple sclerosis activation of the stat signaling pathway can cause expression of caspase 1 and apoptosis caspase-1 regulates the inflammatory process leading to autoimmune demyelination transcription factor nf-kappab: a sensor for smoke and stress signals role of astrocytes and chemokine systems in acute tnfalpha induced demyelinating syndrome: ccr2-dependent signals promote astrocyte activation and survival via nf-kappab and akt macrophage polarization in bacterial infections il-4-activated stat-6 inhibits ifn-gamma-induced cd40 gene expression in macrophages/microglia regulation of macrophage gene expression by proand anti-inflammatory cytokines il-4 pretreatment selectively enhances cytokine and chemokine production in lipopolysaccharide-stimulated mouse peritoneal macrophages differential regulation of nitric oxide synthase-2 and arginase-1 by type 1/type 2 cytokines in vivo: granulomatous pathology is shaped by the pattern of l-arginine metabolism monocyte/ macrophage differentiation in early multiple sclerosis lesions determination of the sequential degradation of myelin proteins by macrophages brain microglia and blood-derived macrophages: molecular profiles and functional roles in multiple sclerosis and animal models of autoimmune demyelinating disease the role of the th2 cc chemokine ligand ccl17 in pulmonary fibrosis role of the chemokine receptor ccr4 and its ligand thymus-and activation-regulated chemokine/ccl17 for lymphocyte recruitment in cutaneous lupus erythematosus eotaxin (ccl11) and eotaxin-2 (ccl24) induce recruitment of eosinophils, basophils, neutrophils, and macrophages as well as features of early-and late-phase allergic reactions following cutaneous injection in human atopic and nonatopic volunteers the multiple sclerosis degradome: enzymatic cascades in development and progression of central nervous system inflammatory disease caspase-1 expression in multiple sclerosis plaques and cultured glial cells innate immunity in multiple sclerosis: myeloid dendritic cells in secondary progressive multiple sclerosis are activated and drive a pro-inflammatory immune response multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines increased transforming growth factor-beta, interleukin-4, and interferon-gamma in multiple sclerosis the cytokine storm in multiple sclerosis cytokine profile of myelin basic protein-reactive t cells in multiple sclerosis and healthy individuals cytokine secretion profile of myelin basic proteinspecific t cells in multiple sclerosis increased il-13 but not il-5 production by cd4-positive t cells and cd8-positive t cells in multiple sclerosis during relapse phase multiple sclerosis is associated with an imbalance between tumour necrosis factor-alpha (tnf-alpha)-and il-10-secreting blood cells that is corrected by interferon-beta (ifn-beta) treatment elevated serum levels of ifn-gamma, il-4 and tnfalpha/ unelevated serum levels of il-10 in patients with demyelinating diseases during the acute stage intrathecal activation of the il-17/il-8 axis in opticospinal multiple sclerosis intracellular cytokine profile in t-cell subsets of multiple sclerosis patients: different features in primary progressive disease modulation of macrophage infiltration and inflammatory activity by the phosphatase shp-1 in virus-induced demyelinating disease shp-1 regulates fcgamma receptormediated phagocytosis and the activation of rac cd47-signal regulatory protein alpha (sirpalpha) regulates fcgamma and complement receptor-mediated phagocytosis the effect of phosphatases shp-1 and ship-1 on signaling by the itim-and itam-containing fcgamma receptors fcgammariib and fcgammariia human protein tyrosine phosphatase 1c (ptpn6) gene structure: alternate promoter usage and exon skipping generate multiple transcripts molecular mechanisms underlying shp-1 gene expression transcriptional activity of the shp-1 gene in mcf7 cells is differentially regulated by binding of nf-y factor to two distinct ccaatelements multiple sclerosis genetics: leaving no stone unturned risk alleles for multiple sclerosis identified by a genomewide study multiple sclerosis: a polygenic disease involving epistatic interactions, germline rearrangements and environmental effects the gene encoding hematopoietic cell phosphatase (shp-1) is structurally and transcriptionally intact in polycythemia vera protein tyrosine phosphatase containing sh2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13 lack of phosphotyrosine phosphatase shp-1 expression in malignant t-cell lymphoma cells results from methylation of the shp-1 promoter reduction of hematopoietic cell-specific tyrosine phosphatase shp-1 gene expression in natural killer cell lymphoma and various types of lymphomas/leukemias : combination analysis with cdna expression array and tissue microarray reduced expression and promoter methylation of p16 gene in epstein-barr virus-associated gastric carcinoma foreign dna integration. genome-wide perturbations of methylation and transcription in the recipient genomes the short arm of chromosome 11 is a "hot spot" for hypermethylation in human neoplasia infection with human immunodeficiency virus type 1 upregulates dna methyltransferase, resulting in de novo methylation of the gamma interferon (ifn-gamma) promoter and subsequent downregulation of ifn-gamma production presence of simian virus 40 dna sequences in diffuse large b-cell lymphomas in tunisia correlates with aberrant promoter hypermethylation of multiple tumor suppressor genes a specific viral cause of multiple sclerosis: one virus, one disease inflammation-mediated cytosine damage: a mechanistic link between inflammation and the epigenetic alterations in human cancers endogenous cytosine damage products alter the site selectivity of human dna maintenance methyltransferase dnmt1 regulation of the dna methyltransferase by the ras-ap-1 signaling pathway methylation-dependent gene silencing induced by interleukin 1 beta via nitric oxide production the htlv-1 neurological complex negative regulation of the sh2-homology containing protein-tyrosine phosphatase-1 (shp-1) p2 promoter by the htlv-1 tax oncoprotein mechanisms of shp-1 p2 promoter regulation in hematopoietic cells and its silencing in htlv-1-transformed t cells macrophages of normal subjects were pre-treated with shp-1 sirna or control sirna and then treated with il-4, lps, ifn-γ, or tnf-α for 18hrs. the mrna levels of ccl17/tarc, cxcl10/ip-10, and ccl2/mcp-1 were quantified by real-time rt-pcr. g. & h. tnf-α and il-6 secretion was quantified in macrophages of normal subjects that were pre-treated with shp-1 sirna or control sirna. macrophages were cultured in media alone or in the presence of the tlr ligands, lps and dsrna for 18 hours and supernatants were analyzed for tnf-α or il-6 by elisa. i. the levels of nitrite, as a indicator of nitric oxide (no) production we would like to thank dr. isobel scarisbrick at mayo clinic for providing cdna plamids used in performing real time rt-pcr. we thank carol ozark for collection of normal subject blood samples. we would also like to thank dr. nick j. gonchoroff, for his expertise in flow cytometry/cell sorting performed in this study. table 1 biometric data of ms patients and normal subjects used in the studythe data are shown in mean value ± sd. for the gender f stands for females and m stands for males. the age at onset of clinically diagnosed ms and disease duration are shown in years. the clinical symptoms were measured on kurtzuke's expanded disability status scale (edss) at the time the blood was drawn. key: cord-005814-ak5pq312 authors: nan title: 8th european congress of intensive care medicine athens greece, october 18–22, 1995 abstracts date: 1995 journal: intensive care med doi: 10.1007/bf02426401 sha: doc_id: 5814 cord_uid: ak5pq312 nan objectives: evaluate the levels of tnf, il-6 and pai-i in different moments of the ards and the possible relationships among them. methods: 23 septic patients with ards were studied. also significant differences for: tnf, pai-i and il-6 in septic patients and both evaluations of ards with control gropup; pai-1 between septics and 2nd evaluation in ards, and between the ist and 2nd evaluation in ards; il-6 between septics and both evaluations in ards; and il-~ in both evaluations in ards patients in relation to mortality. conclusions: i) elevations of tnf, pai-i and il-6, with clinical signs, are suggestive of infection; 2) the persistent and progressive elevation of pai-i with any clinical criteria may suggest evolution to ards; 3) due to its own kynetics, il-6 takes part later in the acute phase, its levels being related to the magnitude of the injury in the tissues. objectives: the influence of long-term volume therapy with different solutions on plasma levels of circulating adhesion molecules was studied. methods: according to a randomized sequence, 30 patients with sepsis secondary to major surgery exclusively received either hydroxyethylstarch solution (10% hes, mean molecular weight (mw) 200,000 daltons, degree of substitution (ds) 0.5) or human albumin 20% (ha) for volume therapy for 5 days. plasma levels of circulating (soluble) adhesion molecules (endothelial leukocyte adhesion melecule-1 [selam -i] , intercellular adhesion molecule-1 [sicam -i] , vascular cell adhesion molecule-1 [svcam -i] , and p-selectin ) were serially measured on the day of admission to the intensive care unit (='baseline ' value) and during the next 5 days. results: selam-i, sicam-i, and svcam-i plasma levels were markedly higher than normal at baseline in both groups. in the hes-patients, selam-j decreased to normal range, whereas it further increased in the ha-group (from 89• to 106• during the study period, sicam-i and svcam-i plasma levels remained unchanged in the hes-patients, but further increased in the ha-group (from 626• to 1,329• sgmp-140 increased significatly only in the ha-group (483• to 683• only pao2/fio 2 was significantly correlated to plasma levels of adhesion molecules. conclusions: sepsis is associated with markedly elevated plasma levels of adhesion molecules indicating endothelial activation or damage. by long-term volume therapy with hes, these levels remained unchanged or even decreased, whereas volume therapy with human albumin did not have any beneficial effects on soluble adhesion. central venous catheters are frequently used in the care of the critically ill patient. the incidence of catheter related sepsis varies in the literature. we investigated the occurrence of contamination and sepsis compared to results of the epic study as part of quality assesment in our intensive care unit. from january until august 1994 all removed central venous catheters were examined for microbiological culture. the patients who showed signs of sepsis were also registered. the results of the contaminated catheters and septic patients were compared with results from the epic study. during the 8 month period ,2059 patients were hospitalized on our intensive care unit. 230 central venous catheters were examined for microbiological culture. 118 specimens appeared to be possitive (51%). 13 patients showed clinical signs of sepsis. the incidence of sepsis due to contaminated central venous catheters was 13/118 (11%). the incidence of sepsis due to the presence of all central venous lines was 13/230 (6%). the microorganisms responsible for the sepsis syndrom were : stapylococcus aureus (n=5), escherichia colt (n=7), others (n=6). in the epic study the percentage for sepsis on the icu was 17.6% for the netherlands and 17.8% for europe. despite a high number of positive culture from removed intravascular lines, a low percentage of sepsis was seen compared to results of the epic study. we recommend routine bacteriological culture of all removed central venous lines and recommend to look at colonization and sepsis due to intravascular lines as a measure of quality control in the intensive care unit. objectives: prognostic assessment of simplified acute physiology score (saps) in granulocytopenie patients with septic shock (ss). methods: the medical records of 59 admissions to an intensive care unit (icu) of granuloeytopenic patients with ss are reviewed. fiftytwo patients had haematological malignancies. seven patients had aplastie anaemia. patients were categorised as survivors (discharged from icl 0 and non-survivors (died in the icu). saps index was calculated for patients daily during their stay in icu. all patients were severe granulocytopenic (total white cell count less than 0,5 ]09]1). results: five patients (8,5%) were discharged from icu. fifty-four patients died in icu. non-survivors had saps on admission higher than survivors ( 20.9+4.6 and 16.5+3.0, respectively, p<0,01, mann-whitney u test). no patient with a saps greater than 20 survived. mortality among the 27 patients with saps from 9 to 20 was 81,5%o. the evolution of ss was rapid. the mean stay in icu among non-survivors was only 56 hours. an analysis of the saps index on admission of non-survivors showed an inverse correlation with the duration of their stay in icu (r=-0,52, p=0.001). all survivors recovered from granulocytopenia. they had normal white cell counts at the time of discharge from icu. there was inverse correlation in survivors between saps and white cell counts, when these parameters were evaluated daily. however, the saps index alone cannot be considered to be on individual predictor factor of mortality. patients who had failure of the malignancy to respond to chemotherapy and who had persistent granuloeytopenia died in icu despite saps index on admission and recovery from ss. conclusion: saps index greater than 20, failure of the malignancy to respond to chemotherapy and persistent leueopenia all point to a poor outcome of granulocytopenie patients with ss. introduction: antipyretics sometimes are used for fever control in febrile neutropenic patients with hematological malignancies(hm). we observed a dramatic fall of blood pressure(bp) and development of septic shock(ss) in some of the patients who received antipyretics. aim: to clarify can antipyretics provoke ss in neutropenic patients with infection. methods: retrospective review of medicat records of 52 neutropenic(wbc <0,5 109/1)patients with hm, admitted to the intensive care unit for ss, was performed. there was selected group of 8 patients receiving antipyretics shortly before a fall of bp. results: there was a definite causal relationship between receiving antipyretics and fall of bp in 4 from 8 patients. all patients had fever due to infection and had normal level of bp before receiving antipyretics. hypotension developed within 40 minutes up to 1,5 hours after administration of antipyretics. three patients received 0,5 g of metamisol and one 0,5 g ofparacetamol per os. in all cases we observed dramatic diaphoresis and the temperature fall to subnormal level (35.4+0.4~ accompanied'by hypotension. but in 8-12 hours the fever was coming back without blood pressure elevation. the fluid replacement was controlled by central venous or wedge pressures. there were required 1200+350 ml colloid and cristalloid solutions for volume loading. in spite of fluid administration the hypotension persisted and all patients required inotropic therapy. only one patient survived and is alive now. conclusion: it seems to us that our data offer to state that antipyretics administration can initiate ss in febrile neutropeuic patients with infection. objectives: to assess the agreement between cardiac output (co) measured by odm t and by 3 other methods used in icu patients. methods: we prospectively studied 12 adu t patients requiring hemodynamic monitoring with a pulmonary artery catheter. an esophageal doppler monitor provided measurements of co (odm), stroke volume and flow time (ft) used as an indirect evaluation of patient's volume status. patient hemodynamic status was evaluated by a modified fast response pulmonary artery catheter (baxter health care corporation, santa ana, ca), allowing co measurements by thermodilution 0"d) and an evaluation of right ventricular ejection fraction and end diastolic volume (rvef and rv-edv). in the last six patients co was measured by transthoracic echocardiography (echo) and oxygen consumption was measured by a deltatrack ii metabolic monitor (datex) allowing co calculation according to the fick formula (fick). the agreement between methods measuring co and their reproducibility, were evaluated by bland and altman analysis. results: agreement between co measurements is expressed as bias (d) and 95% limits of agreement (l of a = d_+2sd .15 td-fick -2.36 -8.06 to 3.34 fick-echo 0.60 -5.92 to 7.12 there was no correlation between ft and rv-edv. conclusions: although co measurements by odmil had the best reproducibility, the limits of agreement between the four methods tested were unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of co measurement in the icu. phd, a. paltzev, v.bajbikov, b.dobryakov d.sc., a.ostanin phd, o.leplifia phd, h.chernykh phd munieip. hosp. n l, n 12; inst. of clin. immunol., novosibirsk, russia objectivies: efficiency of native cytokines used in the treatment of patients with severe surgical infections has been studied. methods: for two years 120 patients were treated with cytokine mixture (ssp) obtained by arterio-venous perfusion of swine spleen and contained the following cytokines: il-1, il-2, il-3, tnfa, ifny, gm-csf. results: ssp intravenous infusions were shown to accompany with mortality decrease from 23.4% to 12.5% in patients with abscessed pneumonia and lung abscesses and from 50% to 13% if disease course was complicated with sepsis. in patients with purulent peritonitis and sepsis efficiency of ssp was decreased due to endotoxieosis. thus, we used adoptive immunotherapy with mnc activated in vitro with ssp or recombinant il-2. intravenous infusions of such cells resulted in transformation of a pathologic process from destructive into productive one. moreover, clinical manifestations of sepsis were controlled in 81% and mortality was decreased from 46% to 19%. conclusions: the use of eytokines themselves as well as cytokine-treated lymphoeytes permits to control the disease and leads to the mortnlity decrease owing to stimulation of host defence mechanisms. background: although red blood cell transfusions (rbct) are used to increase oxygen availability in septic patients, several lines of evidence suggest that rbct may actually worsen tissue hypoxia. thus, rbct may negatively influence outcome of septic patients. objectives: to determine the association of 1) rbct ; 2) number of units transfused; and 3) mean age of the units transfused on the first day of transfusion with mortality of critically ill septic patients. methods: we prospectively identified patients who met strict criteria for sepsis syndrome (ss) seen in the icu of st. paul's hospital from 1992 to 1994 and excluded patients who died in the first 5 days after the onset of sepsis. we recorded clinical characteristics, multiple system organ failure score, and apache ii at onset of sepsis. then, we retrospectively recorded the total number and age of rbc units transfused during the first 5 days after onset of sepsis. overall 30-day mortality was 22%. results: the main results are shown in the table. the mortality of patients who received rbct was nearly double the mortality of those who did not receive rbct even after adjusting for severity of illness using apache ii. objectives: gastric mucosal acidosis is frequently observed in patients with sepsis. the aim of this study was to determine whether volume infusion using pentaspan| decreases abnormal gastric mucosal pco2 (pico2) in patients who have sepsis syndrome (ss) who have already been resuscitated using clinical endpoints. methods: we prospectively identified 5 patients who met strict criteria for ss, had a pulmonary artery catheter and a gastric tonometer in place, and pico2 > 50 mmhg. pentaspan| (500 ml) was infused in 30 rain. measurements of hemodynamics, hemoglobin, arterial lactate, blood gas analysis, and pico2 were performed before and repeated 30 miff and 2 hr after pentaspun| infusion. we calculated the pico2 -arterial pco'2 difference (pico2-paco2) and phi (using henderson-hasselbach equation). anova was used to assess statistical significance. results: all patients werereceiving adrenergie drugs. map was 73 :1:13 mmhg and lactate 1.2:1:0.6 mmol/l. pentaspan| increased ci by 22% (p<0.05) but did not change pico2 ( and increase m oxygen o* wery were simimny achieved in both groups. nevertheless, epinephrine was associated with a lactic acidosis and increased laetate/pyruvatemia ratio (l/p) that evoke a dysoxia rather than a metabolic effect. an higher gastric mucosal pco2 in the ep group compared to nor-rob suggests the hypothesis of an anaerobic production of co2 in favor of a splanchnic hypoxia. in both group, arterial ketone body ratio that reflects hepatic mitochondrial redox state, compared to a control group without shock was decreased but increased between 12 and 24 hours after restoration of arterial pressure. the association norepinephrine-dobutamine seems to be better for splanehnic circulation than epinephrine and should be used for dopamine resistant septic shock. moreover, the increase in arterial pressure with nor-dob improved gastric mueosal ph and hepatic mitochondrial redox state and argue to reconsider arterial pressure as a significant goal for resuscitation in septic shock. conclusion: significantly higher malondialdehyde and ghitathione levels and glutathione-peroxidase activity in group ns at the end of icu stay were related to mortality these findings indicate an increased generation of free oxygen radicals together with increased anfioxidant activity in this group and sapport the employment of antioxidant interventions in critically ill patients. oblecfives: to determine the role of nitric oxide (no) in the mechanism of septic shock induced by isolated limb perfuslen with recombinant tnfcr methods: we have measured tnfr~ and metebo~ites of no in 5 patients with signs ot septic shock following treatment with isolated limb perfusion for nonresectable soft tissue tumors and melanomas of a limb. perfuslen was carried out with melphalan (burroughs wellcome) and recombinant tnfcr (boehringer). tnfc~ was determined by specific radiometric assay (medgenix diagnostics), nitrate and nitrite were measured with a modification of the guess reaction ~. results: results are shown in the table. conclusions: during isolated limb pedusion with recombinant tnf~ very high levels of tnfcr were measured in arterial blood in 5 patients. they all showed signs of severe sepsis syndrome with shock from vasodilafion, probably due to leak of recombinant tnft~ from the peduslen circuit to the systemic circulation. tnfc~-induced vasodilation was not accompanied by a rise in serum no-metsbolites. our findings do not confirm the widely accepted theory, mainly based on animal experiments, that genera• of no is the key pathogenefic mechanism in septic vasodilafion 2, nor that tnfrt invariably induces forreafion of no. the precise mechanism of shock in these patients remains to be elucidated. references: 1. moshage h, kok b, huizenga jr, jansen plm nitrite and nitrate determinaiions in plasma: a critical evaluation. clin chem 1995: 41/6. 2. moncada s, higgs a. the l-argioine-nitrio oxide pathway. n engl j med 1993; 329: 2002 -2012 ec is a commonly used for prolonged, stable animal anesthesia. noting that the hypotension after iv lps was attenuated by ec, we hypothesized ec also protects against lps toxicity. sprague-dawley rats received ip saline (s), thiobutabarbita180 mg/kg (tb), or varied doses of ec, followed 2 hours later by bolus 30 mg/kg iv lps. 7-day survival is shown below: group: s tb ec(0.1gmikgi ec(0.sgm/kg) ec(i.2gm/kg) alive (n) t 0 0 3 9 ~ total (n) 10 s s 7 10 "signiflcant;y different from all other groups, p<0.0s 5/5 rats given lps followed 2 hours later by ec (1.2 gm/kg) also died. additional rats were treated with s (n=10) or 1 2 gm/kg ec (n=10) followed by 30 mg/kg lps, then sacrificed at 4 hours. blood glucose (bg, mg/dl),.hematocrit (hct), leukocyte count (wsc/mm~ platelet count (pltxl0~/mm3), bicarbonate (hco, mg/dl), gross bowel hemorrhage (bh, 0-4 scale) and lung myeioperoxidase activity (mpo, ~vmirvgm wet lung) are shown below ( we conclude that ec reduces the lethality and multiple organ toxit;~ty of lps. its diverse effects suggest asite of activity upstream from the cytokine cascade. these results are important for studies of lps which may use ec anesthesia and may have potential in the therapy of septic shock. [zo = 0 hz impedance (z; {dyn.sec.cm "5 }); zl = first harmonic z; zc = characteristic z; z1 ph. = t'trst harmonic phase angle {radians}; f, #, * at least p < 0.05 between fio2 0.4 and 0.12, fio2 0.4 and fio2 0. 4&no -0.46_+0.1 -0.26_+0.1 # -0.24+0.1 m -0.85+0.1 * -0.85+0.1 * -0.74+0.1 * -0.88_+0.1 * in hyperoxia, compared to dogs at the same q, minipigs had a higher ppa (26+1 rnmhg versus 16+1 mmhg; p < 0.01). hypoxia increased (ppa-ppao) at all levels of q by an average of 13 mmi-ig in minipigs and 2 mmhg in dogs. inhaled no inhibited hypoxia-induced (ppao-ppa)/q changes in both species. conclusions: we conclude 1 ~ that the minipig is an animal model of elevated pulmonary vascular resistance and impedance, and 2 ~ that hypoxia-induced alterations in pvz spectrum are due to changes of resistance in small arteries. objectives: 1) to determine the toxicity of ng-monomethyi-larginine (nma) administered by intravenous bolus to patients with refractory septic shock. 2) to investigate the biologic activity of nitric oxide synthase inhibitors in septic shock. methods: from august 1993 to january 1995, thirteen patients with vasopressor refractory septic shock received nma intravenously in escalating doses from 1 to 40 mg/kg. results: no hepatic, renal, gastrointestinal, or hematologic toxicity was observed at doses of nma as high as 40 mg/kg. significant biological activity was observed at all dose levels consisting of increased blood pressure (systolic blood pressure from 70.9 mm hg + 3.4 to 109.0 _+ 4.3 s.e.m., p=0.0004, systemic vascular resistance (430 + 57 to766 + 93 dyne.sec/ cm s, p=.002), and a decrease in vasopressor requirements. the magnitude and duration of these effect were dose dependent. decreased cardiac output (8.1 _+ 0.8 to 7.0 _+ 0.9 i/min p=.003) and increased pulmonary artery pressure (35.6 _+ 2.1 to 43.5 _+ 2.0 mm hg; p=.004) were also observed. no significant effects on heart rate, pulmonary capillary wedge pressure, or central venous pressure were observed. four of 13 patients survived for more than 28 days, 4 patients died of cancer complications (all 5 patients had maintained blood pressure for 24 h on nma) and 4 patients died of complication attributable to septic shock (mods, ards, dic, refractory hypotension), and 1 patient was unevaluable. conclusions: no adverse clinical effects have been observed in patients receiving bolus doses of nma as high as 40 mg/kg. the increased pulmonary artery pressures observed in septic shock patients is further augmented by nma and may limit the dose which can be administered by intravenous bolus. other schedules of drug dosing may attenuate this effect. glucose-insulin-potassium (gik) solutions have been shown to improve cardiac contractility and increase oxygen availability in experimental and clinical settings of septic shock. several mechanisms have been proposed to explain these effects including a direct improvemeut of the energy balance by glucose, a direct influence of insulin on cardiac performance or an increase in intravascular volume due to the hyperosmolarity of the solution. to explore the role of hyperosmolapity, we compared the effects of gik to those of a isoosmolar hypertonic saliue solutiou in endotoxin shock in dogs. methods : the study included 18 mongrel dogs (25• pentobarbitalanesthetized aud mechanically ventilated with air. thirty minutes after the intravenotls administration of 3 mg/kg of e. coli endotoxin, the dogs were randomized to receive a 2ml/kg infusion in 30 rain of a hypertonic (2895 mosm]l) solution iucludiug either a mixture of glucose 50 % with 750 u insulin and 2000 meq kcl/l (glk-group 1) or hydroxyethyl starch 7.5 % in naci 8.4 % (hes-group 2). in each dog, a 0.9 % saline infi~sion was continued to maintain the puhnonary arlery occluded pressure at baseline level. hemodynamic, blood gas aualysis and laboratory data were collecled at baseline and 30 miu, 60 rain, 120 rain, and 240 nunutes later.. results : eudotoxin administration was followed by a fall in mean arterial pressure (map) aud cardiac index (ci) and a rise in blood lactate levels. resuscitation with either gik or hes hypertoaic solutions resulted in similm increases in map, ci, oxygen delivery and left ventricular stroke index (table 1) . we conclude that during resuscitation from endotoxic shock the use of gik solutions is not superior to hypertouic hes solutions. the higher blood lactate levels observed in the dogs receiving gik can be attributed to the glucose metabolism. 1, 75 for group 2, 105 for group 3) were drawn and immediately analysed at 37~ using the abl500 radiometer for po2, pco2 and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the ps0 at ph=7.40, pco2=40 mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood, as was the ps0"in vivo" (i.e. the ps0 at the patient's value of ph, pcoz and temperature), using siggaard-andersen's algorithm. the data were compared by the one-way anova test and by the t-test for paired and unpaired samples. results: the mean resulting values (in mmhg) with the statistical differences are shown in table i. in addition, the time series analysis shows the mean ps~st values as statistically below the psin vivo" in the septic patients while the opposite is shown for the cardiac patients. no differences in the time analysis are demonstrated for the second group. a possible clinical significance may be drawn from these different behaviours. objectives:toxemia degree and humoral immunity condition have been studied in 36 patients aged from 19 to 72 with progressive course of sepsis and polyorganic insufficience. methods: such toxemia and humoral immunity findings as lencositlcindex of toxication (lii), level of oligopeptides of the middle molecular mass registered at the wave length of 25nm(mmi) & 280nm (mm2), distribution index (id), immunoglobulins a,m,g, concentration of circulating immunocomplexes (cici & cic2) and also some clinical and biochemical findings on the 1,3,5 day after the operation serve as criteria for treatment effect. results: it was founded that in intensive therapy and detoxication, level of lii is successively decreased from 12.6~1.4 to 2.6+.5 on the 5-th day after the operation. true decrease of the level mm2 from .704~.09 to .402+.08 un & optimal density and increase of distribution index from .96 to 1.09 are argued. conclusions: in studlng the dynamics of the immunoglobulin's spectrum and the true increase of immunoglobulin g level from 8.4+.6g/i to i0.8+.7g/i on the 5-th day after the operation simultaneously with the decrease of cic2 from 806.9~60 to 624.7~54.8(p .05) were founded. some stages of the investigation true increase of lymphocytes from 9.0+2.6% to 15.0+1.8% was noted and it appeared to be a favourable prognosis finding for disease outcome. high correlation dependence between bacillus-and segmentonuclear neutrophils and immunoglobullns g & m (r=.5-.7 in p<.05) was discovered and it also showed positive dynamics of the course of the disease. a 34 year old male patient was admitted to the icu with severe paraquat poisoning. treatment consisted of gastic lavage and oral administration of fullers earth. because of very high plasma levels hemodialysis together with charcoal hemoperfusion was started within one hour after admission. this treatment was further continued by continuous veno-venous hemofiltration in order to remove the circulating paraquat and also circulating cytokines. nevertheless patient s condition worsened necessitating artificial. ventilation and hemodynamic support. patient died 24 hours after admission of acute multiple organ failure due to paraquat poisoning. serum levels of paraquat were determined by colorimetric method (table) . levels of interleukin 6 (il 6) and 8 (il 8), tumor necrosis factor (tnf-alpha), interleukin i receptor antagonist (il 1 ra) were determined both in plasma and ultrafiltrate ( q~!ectives : evaluate in critically ill patients the effects of tow-dose dopamine on gastric mucosal blood flow (gmbf) using laser-doppler flowmetry, a continuous non invasive method of assessing microcirculation. methods : 6 patients requiring both mechanical ventilation and pulmonary artery catheterization for multiple trauma (n=3), ards (n=2) and pancreatitis (n=l) were included. in each patient, the laser-doppler (ld) probe was inserted through a naso-gastric tube. the ld signal is proportional to the number of red blood cells moving in the measuring volume and the mean velocity of these cells. when the ld signal was satisfactory, an aspiration was created into a catheter which was fixed in parallel to the ld probe, to maintain the tip of the probe against the gastric wall at the site of measurement. data (systemic hemodynamic parameters and gmbf) were obtained at the end of a 30 rain resting period (baseline), then 30 min after dopamine (2 mcg/kg/min) infusion, and finally 30 rain after the end of dopamine infusion (recovery gmbf 185 _+ 23 (perfusion units) gmbf 0 ~a% vs baseline) * p < 0.05 vs "baseline" and "recovery". conclusions : 1) despite a slight increase in co (+13%), the dramatical increase in gmbf (+87%) with dopamine, strongly suggests a selective vasodilator effect of low-dose dopamine on gasaic mucosal perfusion. 2) laser-doppler flowmetry appears a promising method to assess gastric microcircalation in critically ill patients. increasing evidence suggests that the activation of inos is the final common pathway for vasodilation in human sepsis associated with endotoxic shock. activation of the cellular immune system induces the excessive release of the pteridines neopterin (n) and 7,8-dihydroneopterin (nh2) by human macrophages/monocytes. besides the well established diagnostic value of pteridines in several inflammatory diseases, it is speculated that these substances per se exhibit biochemical functions. thus we hypothesize that pteridines can modulate inos gene expression in vascular smooth muscle cells (vsmc) in vilro. cdtured rat aortic vsmc from female wistar kyoto rats were incubated with n (20 pm), nh2 (20 ilm), lipopolysaccharide (lps, 5 ~g/ml), and interferone-~/(ifn-~/, 100 u/ml) for 9 h, respectively, inos gene expression was measured by competitive reverse transcription polymerase chain reaction. the results are summarized in the table. the present study demonstxates a neopterin induced increase in inos mrna expression at the transcriptional level in vsmc. while coincuhation of cells with n + lps resulted in an additive effect on inos gene expression, n + ifn-7 seem to have a more than additive effect nh2 did not alter inos mrna synthesis, but it suppresses the lps as well as the ifn-yinduced augmentation of inos gene expression. we speculate that this pteridine-mediated modulation of inos gene expression is involved in the regulation of the vascular tone in endotoxic septic shock. the relationship of sepsis and coagulation abnormalities is well known, mainly in severe sepsis and septic shock. still farther, the extreme expression of hemostasis abnormalities (disseminated intravascular coagulation) in sepsis, has been extensively described. we studied the changes in several coagulation and fibrinolysis markers in septic patients, trying to correlate them with the evolution of the sepsis phenomenon, with an emphasis in its early stages, where therapeutic intervention might be more drastic. in 64 patients, 30 with sepsis, 22 with severe sepsis and 12 with septic shock, as well as in 14 healthy volunteers (control group) we measured : platelet (ptl), coagulation markers [fxii, fvii, fviii, fvw, fibrinogen (fibr) we conclude that all parts of the coagulation system are gradually changed during the evolution of sepsis phenomenon , even in the earliest stage of sepsis. the expression of an inducible nitric oxide (no) synthase (inos) plays a major role in the pathophysiology of septic shock (ss). inhibition of inos could therefore be of therapeutic value. however, such an inhibition has been shown to be detrimental, increasing tissue anoxia (and end-organ damage), possibly through the simultaneous blockade of constitutive nos (cnos). thus, selective inhibition of inos might be more suitable. we evaluated the effects of l-canavanine (can), a more potent inhibitor of inos than cnos, in an animal model of ss. method: in 30 anesthetized rats, catheters were placed in the femoral vein and artery. 23 rats were given an iv bolus of lipopolysaccharide (lps, 5 mg/kg), at baseline (to). after 1 h (t1), rats received at random an infusion of either can (20 mg/kg/h; can group, n=l 1) or an equivalent volume of 0.9% naci (2cc/kg/h; nac1 group, n=12), giyen over 4 h (t1-t5). a third group (sham group, n=7) received 0.9% nac1 in place of lps, and then was treated like the nac1 group. mean blood pressure (mbp), blood lactate and nitrates (no3) were measured each h. glucose, creatinine and asat were also measured in 18 rats (n=6 in each group). the can 98_+9* 304+52"t 3.2+1.1"~ 3.6+_1.4"t 138+46" 48+10" *p<0.05 can vs naci ?p<0.05 vs sham can suppressed the hypotension, reduced the hypoglycemia and hyperlactatemia, and attenuated the biological signs of renal and hepatic dysfunction induced by endotoxemia. these effects were associated with a lesser elevation of blood no3, confirming a partial inhibition of inos. conclusion: l-canavanine attenuates the hemodynamic and metabolic consequences of endotoxemia in the rat. these effects may be related to a partial inhibition of inos. they contrast with the deleterious effects described with non selective inhibitors of nos. l-canavanine could become a new tool for the treatment of septic shock. rocalc1tonin :marker of sepsis, ii~flammaiiur% t~ boifi .cheval*~ jf.timsit*, m.assicot**, b.misset*,/.carlet*, c.bohuon** saint joseph heap, paris**biochemistry institut g roussy, villejuif, ce bi~)l~i~ttectives_: high serum levels of procalcitoaln (proct) have been shown to be ~ss-ocinted with bacterial infection. however, few data exist about the ability of proct to differenciate septic shock and shock from other origin in which an activation of intlmmamtory mediators has been also demonstrated. methods: thirteen patients with bacterial septic shock (ss), 11 patients with non septic shock (nss), 14 patients with bacterial infection without shock (1nf) and 8 icu patients without shock and without infection (control) were compared for proct levels at dayl, 2, 3, 7, 14 . patients were classified blindly and independently fi'om proct results. twelve patients were excluded because any classification was impossible due to mixed pathology. proct was measured with ebemoluminescenee (brahms diagnostica-berlin). results: dayl, proct levels are significantly different between the four groups. dayl proct levels are correlated with saps (p=0.0002), infection (3.7+_3 vs 61_+25,p=0.0007), shock (13_+10 vs 60+.-27,p=0 .002), death at day28 (12_+7 vs 96_+44,p=0.003). when shock and infection are introduced in multifactor &nov& only infection remains correlated with day 1 proct levels (19=0.003) in patients with shock, dayl proct levels are correlated with saps, infection and death at day28, but not with arterial lactate levels (p=0.37), white blood calls (p=0.2) or fever (p=0.1). proct levels remain higher i~i septic shock patients at day 1, 2 and 3( figure) . i c0edpsion: procalcitonin levels in the first three days of shock are differen[" between septic and non septic shock patients. in patients with diseases known to induce acute an inflammatory process, procaldtonin seems to be a marker o~ infection. obiectives-to evaluate the effect of endotoxic shock on the distribution of blood flow between the mucosal and the muscular layer of the intestinal wall. methods: in 10 fasted pigs, mean aortic pressure (map, mm hg), cardiac output (co, ml/min-kg),superior mesenteric artery flow (q sma, ml/min.kg), and phi, where measured before (control) and after i.v. endotoxin (10 gg/kg). the blood flow to the mucosal and the muscular layer was measured in 3 regions (proximal jejunum (pj), mid-small intestine (mi) and terminal ileum (ti)) by colored microspheres, using 8 adjacent samples in each region. the muscular layer was separated from the mucosa by blunt dissection, and the flow determined independently in each layer. results: endotoxin with fluid resuscitation induced the expected decrease in map (95.8_+3.0 vs 53.9-+2.4, p<0.01), and phi (7.29!-_0.02 vs 7.13_+0.01, p<0.01), with a constant co (133_+4 vs 144_+8, p=0.28) and qst, aa (21.5_+0.8 vs 22.8_+0.9, p=0.09). the results of regional pertusion are presented in the table. (flow in ml/rain 9 100g of tissue; mean _+ sem ; * p<0.001 vs control by two-way anova) conclusions-these data indicate that the mucosal flow increased during septic shock. they suggest that a decrease in phi may be due to hypoper~usion of the muscular layer or to metabolic alterations within the mucosa, despite a 50% increase in flow. acute increase in wbc count (from a mean of lo.oo01mm a to 42 o00/mm~), between the 3rd and the 7th day of therapy. there was a decline of the wbc count to an average of about 25.0001mm a after decreasing the daily dose of the medication to 200 mcg there was no increase in tile absolute number of the eosinophils during the whole course of the medication. there was a slight decrease in the c3 complement between 0.26 to 0.29 g/i. normal values 0.5 to 0.9 g/i there was no change in c4 values. conclusions : an early increase in wbc count was observed (3rd day) without subsequent increase in the number of immature types from bone marrow, probably due to the mobilization of wbc from the periphery and this increase was dose dependent. there was a slight decrease in c3 fraction of complement, probably due to the consumption of this fraction in the process of opsonization. no adverse effects of the medication were observed, during the treatment with the above dose. these data sugest that cm csf may be a useful complement to tile main antimlcrobial treat,nent ~ of septic [cu patients. objectives: as part of a large multicentric, placebo-controlled, randomized clinical trial investigating the effects of interleukin-1 receptor antagonist (ii-lra) in the treatment of severe sepsis and septic shock, this substudy evaluated in dem.il the acute hemodynamic effects of ii-lra in patients who were invasively monitored. methods: in a total of 71 evaluable patients in whom vasoactive support was little altered, hemodynamic measurements were performed at baseline (twice), and i hour, 2 h, 3 h, 4 h, 8 h, and 12 h after the administration of 1 mg/kg (n=20) or 2 mg/kg (n=22) of i1-1 ra or the corresponding placebo (n =29). 58/71 patients (82 %) were treated with adrenergie agents and 66/71 (93 %) with mechanical ventilation. data were analyzed by a kruskal-wallis test. results: during the study, there was no significant difference with time or between groups in arterial pressure, cardiac filling pressures, cardiac index or left ventricular stroke work (figure). burmester, "~ man8 and h. djonlagic medical university (internal medicine, "cardiology, *'microbiology) and "**southern city hospital, lfibeck, germany obiectives: evaluation of the incidence of bacteremia and sepsis in patients with nontyphoidal salmonella (s.) infections, specification of risk factors, need of icu treatment, clinical course, and mortality in the group of the patients who developed septic complications. methods: data of all patients with microbiologically proven s. infections hospitalized in the medical university of lobeck and in the southern city hospital of l0beck from 1992 to 1994. results: within the observation period s. was isolated from the stool cultures of 748 patients. in 13 patients (g m, 4 f, median age 52 yrs) s. could be detected in blood cultures (9 s. enteritidis, 4 s. typhimurium). in addition, in 10 of these patients s. was also isolated from other specimens (urine, liquor, and tissue fluids derived from abscess punctures). in all 13 patients with positive blood cultures the clinical course of s, infection was complicated: ? patients developed mof (acute renal failure, ards, hemodynamic instability, dic) and required icu treatment for at least 4 up to 62 days, 4 of the 13 patients died. the predisposing disorders in the patients with s. bacteremia were (n=): aids (3), immunosuppressive drugs (2), chronic alcoholism (2), malignancies (2), none (4). septic complications in patients with nontyphoidal s, infections are relatively rare (in this study < 2 % of all hospitalized patients with microbiologically proven salmonellosis) but severe (mortality of approx. 30 %). patients at risk for a complicated clinical course are predominantly those with predisposing disorders but occasionally also patients without evidence for an underlying disease. age (yr) 65 + 9 58 + 14 death (n) 22 18 duration of shock (h) 27 + 37 54 + 44 noradrenaline (rag/h) 5,9 _+ 6 2 + 5 temperature (~ 38,2 + 2 38,1 + 1 pvr (dynxsecxcm -5) 1195 + 408 572 + 418 co (ljmin) 4,2 _+ 1,6 9,9 + 3,6 lactate (mmol/l) 7 + 5,9 9,2 + 9 interleukin-6 (pg/ml) 829 _+ 798 1331 + 888 interleukin-1 (pg/ml) 9,3 _+ 9,4 9,8 + 7,3 tnf-alpha (pg/ml) 23,5 + 31,7 166 + 209 neopterin (nmol/l) 43,2 + 43,7 218 + 193 crp (rag/l) 131 _+ 97 233 +_ 138 pro-ct (ng/ml) 22,6 + 50,5 77,9 + 160 there was no positive correlation between serum lactate levels, degree of shock, hypoxemia and pro-ct positivity. pts with septic shock of bacterial origin entirely developed hyperprocalcitoninemia, whereas pts with cardiogenic shock, who expired within 24 h did not. however, in late cardiogenic shock (>24h) all pts developed fever of unknown origin and consecutive hyperprocalcitoninemia. these data suggest bacterial inflammation and/or mucosal translocation of bacterial products in pts with prolonged cardiogenic shock. the use of a loading dose of quinine (16.7 mg/kg base in 4 h) is recommended in previously untreated patients (pts) with sfm, particularly in multi-drug resistance areas. this protocol is difficult to validate, since the viability of microorganisms is not assessed routinely in parasitology laboratories. objectives: to examine the evolution of parasite viability during the early phase of therapy of sfm. methods: from 02/1993 to 12/94, pts with sfm (who 1990) treated with iv quinine for less than 6 h were included prospectively. blood samples were collected at o, 6, 12, 18, 24, 36 and 48 h viability was assessed by culturing parasitized red blood cells in the presence of 3h-hypoxanthine, and radioactivity was determined at 42 h by scintillation counting. viability was expressed as the percentage of radioactivity compared to the initial sample. plasma quinine was determined by liquid chromatography. tile ratio plasma quinine (pmol/1)xlo00/icso for quinine (nmo]/]) was called the parasiticida/ index. results:5 pts were included, 42• saps1 18.6-+4.9. the initial parasitemia was 2t.4+7.2%. complications of malaria were coma (4 pts), shock (3 pts), renal failure (2 pts) and acute lung injury (2 pts). all strains were sensitive to quinine (icso 174--67 nmol/1). in 2 pts who were not given a loading dose, parasite viability increased by 63 and 157%, with concomitantly low quinine levels (22 and 19 #mow] at 12 h); 1 pt died. in 3 pts that received a loading dose (serum quinine at 12 h = 33.1--2.0 ~mol/]) a marked decrease of parasite viability (by 73+_10% at 12 h) was shown. viability was inversely correlated with plasma quinine (r=.677, p-.o11) and parasiticidal index (r=.678, p-.o1). conclusions: even with fully sensitive strains, the use of a loading dose of quinine seems warranted in severe falciparum malaria in order to reach rapidly adequate plasma quinine ]evels, necessary to inhibit significantly parasite viability. l nkka, e ruokonell j takala. critical care research program, department of intensive care, kuopio univ hospital, finland objective: to determine the incidence of positive blood cultures, their microbial subgroups and to evaluate the outcome of icu patients with different bacleremias. material and methods: we analysed all positive blood cultures in 3077 consecutive admission to a university hospital icu in 1992 -93 and the icu and hospital survival of the bacteremia patients. during these years 73 patients had 176 positive blood cultures that were considered as clinically relevant, excluding colonizations or contanfinations. results: patients with positive blood cultures had an icu survival of 65.8 % (vs. 92,7 % in all icu patients) and six month survival of 50.7 % (vs. 85.8 % in all icu patients). the most common bacteria were enterobacteriaceae (27,3 %), staphylococcus aureus ( 18,8 %) , coagulase negative staphylococci ( 14.2 %), pseudomonas ( 14.8 %) and slieptococci (9.1%). obiectives: to evaluate prognostic factors and mortality in consecutive patients (pts) with hiv infection and septic shock. methods: from 03-1991 to 12-1993 , records of consecutivepts with septic shock (crit care med 1992, 20: 864-74) admitted to the icu were reviewed retrospectively. results: among 76 pts with septic shock admitted during the study period, 28 had hiv infection-26 of whom had aids-(gr. i) and 46 were hiv-negative (gr. ill. ten gr. ii pts (21%) were irnmunosuppressed because of neoplastic or immune dlsease. mechanica] ventilation was required in 89% gr. i and 83% gr. ii pts in 8 gr . i pts (29%) a multivariate analysis demonstrated that hiv infection and sap5 i were independently predictive of death in pts with septic shock. ~onclusions: evidence of increased mortality, number of organ failures and higher severity scores (saps i does not take into account immunosuppression) is demonstrated in hi v-positive pts, infection with hiv appears to be an independent prognostic factor in pts with septic shock. the frequency of opportunistic infections (often responsible for delayed diagnosis and treatment) may contribute to the poor prognosis in this population. obiectives: to determine interleukin (il)-i 0 levels in plasma of patients with sepsis and septic shock. to analyze the relationship between plasma il-10 and the proinflammatory mediators, tumor necrosis factor-aifa (tnf) and il-6, the underlying severity of the disease and the evolution of patients with sepsis. methods: we studied 94 critically ill patients (63 men, 31 women; 18-86 years old) in three diferents groups. group i: 23 patients without evidence of infection, group i1:34 patients with sepsis and 37 with septic shock (group iii). we measured plasma il-lo, tnf and il-6 levels in the first 12 hours of diagnosis. severity of illness was estimated with the acute physiology and chronic health evaluation (apache ii) scoring sytem. results: plasma levels of il-10 were higher in group iii (median, 51 pg/ml; range, 5-6000 pg/ml) than in group ii (median, 10 pg/ml; range, 2-970 pg/ml; p <.001) and group i (median, 5 pg/ml; range, 2-133 pg/ml; p <.001). median il-10 concentrations did not differ among patients who survived (median 7 pg/ml; range, 2-6000 pg/ml) and those who died during the overall follow-up period (28 days) (median, 15; range, 5-5400 pg/ml); but patients who died in short-term (< 24 hours) with catecholamine-refractory hypotension showed the highest concentrations of il-io (median, 1200 pg/ml; range, 51-5400 pg/ml). in patients with bacteriemia (34%), levels of il-10 were higher (median, 51 pg/ml; range, 2-6000 pg/ml) than in those with negative blood culture (median, 8,5 pg/ml; range 2-5.400 pg/ml; p< .001). there was a good correlation between plasma il-io concentration and levels of tnf (r= .59; p < .001) and il-6 (r= .60; p < .001). the correlation between levels of il-10 and the apache ii score was significant only in the septic shock group (r= 0.48; p <.005). conclusions: in septic shock, il-io and proinflammatory citokines are released in high concentrations. the significant correlation observed in patients with septic shock between il-10 levels and apache ii, short-term death and bacteriemia can possibly be explained by the massive inflammatory response in septic shock with fulminant course. intensive care department -calmette hospital -59037 lille -france. in septic shock, inadequate splanchnic blood flow may play a prominent role in the pathogenesis of multiple organ failure. measurement of gastric phi has been propose to evaluate tissue oxygenation in splanchnic organs. objectives: to compare gastric phi values with hepatic icg clearance, an index of liver blood flow and function ; to determine if one of these two methods could be proposed to assess the entire splanctmic peffusion in septic shock. methods : 6 patients (age : 65• years ; saps ii : 46• were prospectively investigated (septic shock : bone criteria). following parameters were collected during 12 hours : systemic hemodynamic parameters (swan ganz catheter 93a434h -ref1 computer -baxter lab.), calculated systemic oxygen transport (do2), oxygen consumption (vo2) by indirect calorimetry (deltatrac datex lab.), gastric intramucosal pco 2 (pco2ss) and phi (trip -ngs catheter -tonometrics lab.) and plasma disappearance rate of icg (pdr dye) (femoral artery fiberoptic/thermistor catheter 2024, cold z021 computer -pulsian medizintechnik, germany). correlations were performed using a linear regression. elevated in all days with the highest value in second and third days of treatment. nonsurvivors had higher values of these parameters than survivors but differences did not reach statistical significance. another trend of changes were observed in selectin p (gmp-140) concentration. in all patients concentrations measured were elevated but in survivors after not significant decrease this parameter in second day another one had simmilar values. in patients who died we noted significant decrease in third day (p < 0.05) whereafter prominent increase, significant after seventh day, in comparison to third day value and value in survivors group. icam-1 concentrations in all patients reached high levels and in nonsurvivors after four day of treatment significant increase in comparison to survivors we found. conclusions: multiple trauma complicated with sepsis induce rapid elevation of concentrations of il-6, il-8 and increased expressior of adhession molecules (selectin e, p, icam-1) measure of icam-1 and selectin p concentration determine lung injury severity and prognosis as to health and life. (clp) .pathophysiology of cip is unclear, but changes in regional bloodflow may be a ~ignificant factor. nerve blood flow (nbf)is reduced in rat models of hemorrhagic shock (g),but no information is available in sepsis. we studied the comparative effect of acute endotoxemic shock {etx)& h on perfusion of rat sciatic nerve. methods: 20 male sprague-dawley rats were anesthetized with pentobarbital (ip), instrumented with a tracheostomy, carotid arterial & venous catheters and mechanically ventilated (fi02=0.5). the left sciatic nerve was surgically exposed. monitored variables included: a) mean arterial pressure (map,mmhg) ,b) nbf (ml/1o0 g/min) by laser doppler flow meter,c) nerve internal arterial diameter (id ~ m) by video image shearing and splitting method. after stable baseline measurements were obtained, acute hypotension was induced by randomly assigning the rats to etx (0.25 b6, difco) in saline at 1 mg/kg or h. both interventions produced 50% reduction in map within 3 min., which recovered to baseline values spontaneously in etx group, & by reinfusion of heparinized withdrawn blood in m. data were analyzed by linear regression, two-way repeated measures analysis of variance followed by bonferroni-t method. experimental stages were:(1)baseline, (2) mid-point of map reduction; (3) nadir of hypotension, (4)midpoint of map recovery, & (5) after stable recovery of map. both etx & h induced shock result in similar reduction in nbf consistent with lack of autoregulation in peripheral nerve vessels independent of etiology. since cip is primarily associated with sepsis, it is not likely that acute reduction in nbf alone causes cip. direct & indirect neurotoxic effects of mediators of sepsis need to be evaluated. .':_.~::::o4o:oc4., objectives : evaluate the relationship between il-10, a cytokine which inhibits tnf, production and protects mice from endotoxin toxicity, and the other proinflammatory cylokines, tnf~, il 6 and ils in severe sepsis and septic shock. methods : twenty-eight icu patients (19 m, 9 f, mean age 54 + 17 y) were studied as soon as they developped a severe sepsis (n = 16) or a septic shock episode (n= 12) as defined by a conference consensus in 1992 (1). tnf~, il 6, il s and il-10 plasma levels were measured by immuno-radiometrie assays from medgenix (fleurus, belgium). lc mean and range. results : the comparisons between cytokine levels in severe sepsis versus septic shock were made using the logarithm of the value in order to normalize the distribution of data, and student test. il-10 plasma levels were higher in patients with septic shock than in patients in severe sepsis. there was a significant correlation (p < 0.05) between il-10 and tnf a (r= 0.6), il-10 and il~ (r = 0.73) and il-10 and il s (r = 0.65) as well as between il-10 and apache n score (r= 0.52). patients who died (n = 13) had il-10 levels higher than patients who survived but this difference was not statistically significant (114 pg/ml vs 34.5 pg/ml; p>0.05). conclusions : during severe sepsis and sepsis shock, il-10 seems at least to follow the same evolution (increase in plasmatic level) with the severity of sepsis as the other cytokines. reference : (1) crit care med 1992;20:864-74. objectives: to evaluate the effects of steroids on hemodynamics and mortality in septic patients with konwn levels of cortisol concentration. methods: retrospectively we analyzed data ofpatients with documented septic shock who received steroids after assessment of adrenal function. in all patients hemodynamic parameters as well as the necessary vasoactive medication were assessed, before and 24 hours after corticosteroid medication. immediately before administration of corticosteroids adrenal function was evaluated with cortisol levels before and after synthetic corticotropin (0.250 mg). finally we studied mortality. we defined a positive respons on corticosteroids as an elevation of map of at least 30 mmhg and/or a decrease in the necessary vasoactive medication of at least 50% within 24 hours. adrenal insufficiency was defined as a cortisol level after stimulation of less than 500 nmol/l. results: 15 of 23 patients were found to respond to steroid medication, 8 did not. mean cortisol levels before and after corticotropin were 534 • 366 and 737 • 396 nmol/l in the responder group (rg) and 583 • and 907 • 301 nmol/l in the non responder group (nrg). in the rg 9 out of 15 (60%) were found to have an adrenal insufficiency, in the nrg 3 out of 8 (37%). in the rg 2-weeks mortality was 6.7% (l out of 15), the overall mortality 33% (5 out of 15). mortality in the nrg was 62% (5 out of 8) (p <0.01) and 75% (6 out of 8) (p <0.005) respectively. conclusions: in patients in septic shock there is a beneficial effect of steroids in case of adrenal insufficiency, but also in a subgroup with normal adrenal f{unction. obiectives: intercellular adhesion is a critical step in the accumulation of leukocytes. postischemic cardiac lymph has the capacity to stimulate icam-i. in the coronary microcirculation neutrophils can be trapped and in many cases obstruct capillaries, previously we found that troponin t (s-tnt) a marker for myocardial iechemia, was increased in septic patients. the aim of the study was to follow slcam-1 and s-tnt levels continuously starting at the beginning of sepsis. methods: 19 patients were ingluded in this institutionally approved study after relatives had given their informed consent. all patients were included within 24 hrs following the beginning of sepsis. blood was drawn every 4 hrs in the first ;~4 hrs, after 48 hrs, followed once per day for 7 days. s-tnt, icam-1, elam (elisa's, boehringer mannheim inc, r&d systems ltd.) arterial and venous blood gases were determined, an ecg and a complete hemedynamir measurement including cardiac output were obtained. all patients received adequate volume and catecholamine therapy (norepinephrine, dopamine, dobutamine; median (range) 0.6 (0.0-1.66), 3.12 (2.4-12), 6.29 (0.0-15.3) pg/kg/min, respectively). statistical analysis: wileoxon signed rank-sum test. .45 (0.06-7.6) 0.0003 13 patients had s-tnt levels >0.2pg/l. 11 of these died, whereas only 2 of 6 patients died with s-tnt values <0.2 pg/l (p=0.0296). all patients that died had elevated sjcam-1 levels (232 ilg/l:cut-off ) whereas in the survivor group only 50% had elevated icam-1 levels (p=0,043). conclusions: increased slcam-1 and s-tnt levels were found during early sepsis in the majority of patients, a high sicam-1 and s-tnt value was associated with a higher mortality. the research of the noninvasive haemodynamic monitoring accelerated recently all over the world. the aim of our study was to test whether the changes of the haemodynamk parameters measured by impedance cardiography (icg) were corresponded to clinical changes in septic patients. investigations were performed on 20 critically ill postoperative septic patients (their multiple organ failure score was 8-9/with icg monitor. in 9 cases the investigation~ were performed in septic shock. the measured parameters were: heart rate (hr), mean arterial pressure (map), cardiac output (co), peripherial resistance (svr),preejection period (pep), and ventricular ejection time (vet). these parameters were measured during 3-72 hours in every 30 minutes, depending on the patients cl~tnical condition. results: at the septic patients the hr and the co ]~reased. in septic shock the co was significantly higher the svr lower than in the septic group. in the hr there was no difference between the two groups. in septic shock noradrenalin influenced more effectively the measured parameters than dobutamin. conclusion: the trend of the measured icg parameters correlated with the clinical changes of septic patient's state. the noninvasive haemodynamic monitoring by impedance cardiography helps the planning and leading the adequate intensive therapy of these critically ill septic patients. to evaluate the development of sirs, sepsis and septic shock in hospitalized patients with fever, a prospective study was performed on 300 patients using previously defined criteria. methods: 300 normotensive patients with fever (temperature >38.0 ~ axillary), admitted to the department of internal medicine were evaluated for the existence of sirs during the first three days of the study and sepsis at inclusion. during a follow-up period of 7 days the patients were daily evaluated for the development of sepsis or septic shock. results: most patients (69%) had or developed sirs within the first three days, 16 patients (5%) did not. sepsis was present in 25% at inclusion. in patients with sirs, 76% did not progress to sepsis or septic shock, 24% progressed to sepsis (mean interval 2.55 • 1.97 days), and 1 patient (<1%) directly progressed from sirs to septic shock. in patients with sepsis, 17% progressed to septic shock (mean interval 2.08 • 1.56 days). sepsis was preceded by sirs in 40%. septic shock was preceded by sepsis in 92% and by sirs in 8%. conclusions: 94% of patients with fever in an internal medicine department develop sirs, or sepsis. furthermore, progression from sirs to sepsis or septic shock is poorly predicted by fever or sirs. nevertheless, all patients with septic shock were preceded bysirs or sepsis. taken together, this may indicate a severity hierarchy of the syndromes. however, fever, sirs and sepsis are relatively poor indicators of development of septic shock. this supports further research on additional predictors of septic shock. b. m.manuylov, v.b.skobelsky (moscow) in recent years sodium hypochlorite (sh) has been successfully used to eliminate pyo-septic complications. moreover, the mechanism of the sh effect on the immune system has not been sufficiently studied. the aim of the present investigation was to study the mechanism of sh effect in inflammatory pulmonary diseases. 20 patients with double pneumonia were subjected to the evaluation. sh in the concentration of 600 mg/l in the volume of 400-800 m1/24 hours was administered by drop infusion into the central vein. to evaluate one of the defence systems the leukocytes activity by the chemoluminescence technique was studied. in all the patients baseline secondary immunodeficiency which was indicated by the decrease in the luminescence level was established. even 1 hour after the sh administration the leukocytes activation exp-ressed by the enhancement of their chemoluminescence 0.5-5 times was observed. this supports the available findings that accumulation and liberation of the oxygen active forms (ol'oh, '02, h202) are accompanied by the increased phagocytosis, i,e. the signs of "the oxydation explosion" testify to the favourable sh effect on the course of inflammation processes. the use of sh permitted to decrease the percentage of lethality in double pneumonia by 15% in the intensive care unit over the year. at the same time, excessive activation of free radical oxygen may be a damaging factor. therefore, precise individual control over the choice of concentration, dosage and the preparation administration rate is required. prospective, double-blind, placebo-controlled, trial of atiii substitution in sepsis r. a. balk objective: pilot study to evaluate the efficacy and safety of atiii substimtion therapy in patients with sepsis. efficacy assessed using change in mortality or organ failure/dysfunction. adult patients meeting a definition of sepsis and cared for in a tertiary care academic medical center in chicago were identified and prospectively randomied to receive either atiii (kybernin p) or placebo in a double-blind treatment protocol. all other therapy and patient management were under the direction of the patient's attending physician. all patient's were followed for 28 days and the organ dysfunction/failure were scored using published scoring systems (jordan et al crit. care med. 1987 , goris et al arch. surg. 1985 , kuaus et al ann. surg. 1985 colldusions:wha~ we met the shomaeker objectiv% the mortality and the pro~os[s were i~ttc*. those criteria were obtained with file tradititmal t~ctor likr doht~mme, hut c.~vh ~,as ca1 in~aertam measure. they ac~s smxergically in the optimizatic~l of the fell vmtrictdar work index, tad fimdameatally cavh seox~s to have an impo.aat role in the better respiratory ev-altmtioa, leaving yet the possibility to coltrol the flui& r althou~l eomproved it's not aec~pt~xl file importmlce h* the diminution, of the sepsis modiat~lrs llke fnt and il-6 with h~wmotiltrafi(al, stopphlg the evolution to nmltiorganic failure mid de~easethe mortality. with ours clhlicals results, we could saythat cavii in multiol~atlie disfut~oa septic patieats, se~r~ to be an c4xilna] supoa or troatmeat maesure. of anaesthesia and intensive therapy, medical university of prcs, p~csf hungary. objectives: since some biological effects of bacterial endotoxin require an interaction between the lps molecule and a serum factor(s), we hypothesized that lps-induced no production and cgmp accumulation in vascular smooth muscle cells (vsmc), a mechanism ~thought to underlie cardiovascular collapse associated with septic shock, is modulated by serum factor(s). methods: cultured vsmc from rat aorta were challenged with e. coli lps for 4-6 hours either in the presence or absence of fetal calf serum (fbs), and no production was monitored by radioimmunoassay determination of cgmp content of hci extracts. results: in the absence of serum, 1o00 ng/ml lps was required to increase cgmp levels, whereas the presence of 10 % fbs shifted the lps concentration curve i00 times to the left. similarly to fbs, human serum also potentiated lps-induced cgmp accumulation. in contrast to lps, serum had no effect on cgmp accumulation elicited by sodium nitroprusside, a no releasing agent, suggesting that the sensitivity of vsmc to generate cgmp in response to exogenous no is not modulated by serum. heat inactivation (>80 ~ 30 min) but not removal of small molecules (<10,000 d) from the serum by dialysis, reduced the potentiation of cgmp accumulation by serum. time course studied indicated that serum is required within the first 120 min of lps exposure to increase cgmp levels. to investigate whether the effect of serum is specific for lps, we treated the cells with increasing concentration of interleukin 1-~ (il-i). 10% fbs shifted the il-iinduced cgmp responses five times to the left. conclusions: our study suggests that lower concentrations of e. cell lps and il-i require a heat labile macromolecule in the serum in order to elicit no production. this factor is present in the human serum and it may play a potentially important role during no synthesis induction in vsmc. objective: to evaluate the factors of acquisition and the outcome of methicillin resistant staphylococcus aureus (mrsa) bacteremia in an intensive care unit (icu). methods: all patients in which bacterermia due to staphylococcus aureus developed >72 hours following admission to our icu, during a 10 year period ( january 83 through january 94) were reviewed. 30 patients (pts) were included, mean age 68,1y (sd 13,1), saps 2 35,9 (sd 11,1), mac cabe (1 and 2) 53%, mortality directly due to sepsis 30%. 16 pts had mrsa bacteremia and 14 methicillin susceptible staph. aureus (mssa) . both groups were compared using the chi square (with correction of yates), fisher's exact, student's t or wilcoxon test. results: there was no statistically significant difference between mrssa and mssa regarding at age (71,8+ 4,8 vs 63,9+ 1,8) , saps2 (33,6+6,6 vs 38,2+14,1), use of vancomycin (94% vs 71%), mechanical ventilation (94% vs 100%), number of days (d) before the drawing of the first positive blood culture (median 20 d, range 7-150 d vs median 30 d, range 7-120 d). more mrsa than mssa pts had previous use of nonsteroidal anti-inflammatory drugs (nsaid) (25 % vs 0% p<0,001), central venous catheter infection due to staph.aureus (62,5% vs 14% p<0,01), but previous use of antibiotics was not significantly different (37,5% vs 21%). the outcome of the bacteremic pts was not statistically different: saps 2 at the first day of bacteremia (33,6+_.7,2 vs 40,7+14,5), severe sepsis and septic shock (31% vs 28%), persistence of the bacteremia (43% vs 78%), mortality directly due to bacteremia (25% vs 45%). conclusion: previous use of nsaid, infection of venous central catheter are more frequently associated with mrsa bacteremia. thus, similar to others studies (hershow infect control hosp epidemio11992; 13:587-593) , these results do not indicate that mrsa is associated with increased virulence. objectives: to closer definition of mosf formation mechanismes in nosocomial sepsis (ns) the complex clinicobiochemical, microbiological, immunological, functional exaroination of 62 cases with ns had been done. methods: examination of cellular and humoral immunity, nonspecific immunologic reactivity, systemic and hepatic circulation, microbiological examination of blood,electro-and echocardiography, sonography and computer tomography of chest and abdomen organs were obligatory. autopsy findings of 5 dead cases had been analized. results: in 45 cases (72,6%) opportunistic pathogen microscopic flora ( staphylococcus anreus,staphylococcus epidermidis, staphylococcus saprophyticus) had been found out in blood inoculations. in 36 cases (58%) side by side with destructive process in lungs the bacterial endo-and myocarditis with blood circulation failure had been determined.in 21 cases (34%) simultanious lesion of three organs (heart,lungs,liver) had been found. morphologic examinations of 5 dead cases (8%) internal revealed involvement of them in mosf-syndrome.hyperplasia of adenohypophysis;sclerosis of adrenal glands cortical layer;perivascular brain oedema,paralysis of brain capillaries and plasmorrhagia, cerebral thrombosis and cerebral abscess,necrobiosis of epithelium tubules of the kidney,pletora of hepar, fatty and granular degeneration of hepatocytes had been found.atrophy of white pulp and hyperplasia of red pulp, supress of lymphoid tissue, plethora and formation of infarctious had been found in spleen. mentioned changes in spleen were indispensable in ns. conclusion: in ns spleen can not secure it functions to support and appropriate detoxication potencial of organism,elimination of microbes,toxines,antoallergenes. insolvency of immunological link of antimicrobic defence is the starting mechanism of mosf developmentin ns. %neviere, jl. chagnon, b. vallet, d. mathieu, n lebleu, f. wattel ] ept of intensive care, hop calmette, lille, france ~everal studies have described tiypoperfusion of intestine during sepsis. 4owever, it is unknow whether the mesenteric blood flow is associated with nucosal hypoperfusion. additionally, the effects of resuscitation on the ntestinal microcirculation remain controversial. 3bjectives : to describe the effects of endotoxin in a porcine model during ~hock and resuscitation. ~ethods : ten pigs (30 kg) were anesthetized and instrumented for "neasurement of cardiovascular variables. gastric and gut oxygenation vere assessed by intra-mucosal ph and microvascular laser doppler lowmetry. after baseline data collection, a 30 minute intravenous infusion )f escherichia colt (serotype 34h4113, sigma, st. louis, mo) was begun ~t a rate of 150 pg/kg. an infusion of either saline at 1.7 ml/kg/min (group ; n=5) or saline and dobutamine at a rate of 5 pg/kg/min (group ii; n=5) vas begun 30 mn after the end of the endotoxin infusion. tesults : to td0 1120 t180 ~ fl0w fluid ioadin,q alone sfyras d, k perreas, e douzinas, k spanou, m pitaridis and c roussos critical care dpt, evangelismos hosp., athens univ, school of medicine. obiectives: much controversy exists concerning the beneficial effects of cvvh on sepsis. we studied the effects of cvvh application on septic patients with reference to the following parameters: i) survival rate ii) cytokines' removal and iii) timing of cwh onset. methods: 20 patients with sepsis (criteria according to accp/sccm, 1992) underwent cvvh as soon as they developed renal failure or dysfunction (urinary output<250 ml/8h, cr>2.5 mg/dl and bun>60 mgd'dl ). specimens were collected: blood samples before cvvh and therafter both blood and ultrafiltrate (uf) samples on 24, 48 and 72 hours. cytokines tnfa, i1-1 and ii-6 were measured by the immunoassay method in all specimens (uf and plasma -p) and sieving coefficient ([uf]/[p]) and 24 h solute mass transfer of tnf and i1-6 were calculated (v24 h x [uf] ). the apache ii score before cvvh onset, the duration of icu stay and the timing of cwh application related to the sepsis onset in days (ta) were recorded.with respect the mortality two groups were formed, i.e. group a (survivors) and group b (non-survivors) . the morbidity period in days of those septic patients who died in the past year and were not subjected to cwh (group c) was compared to that of group b. results: group a included 8 pts and group b 12 pts with mean+sd age (65 _+19 vs 64_+9, ns) and apache scores(24_+2 vs 24-+2.2, ns). the mean ta-+ sd was 3.6+2 vs 10-+6, p<0.05. the mean_+se morbidity period of group b vs group c was 20_+4 vs 8_+0.8 p<0.05 . the mean values of cytokines are presented in the following figures. the sieving coefficient for tnf was 0.2 and for i1-6 was 0.25. the solute mass tranfer was 6-fold the actual plasma content at a given time. . o conclusions: i) early application of cvvh seems to favourably affect the outcome of septic patients, ii) cytokine plasma levels do not decrease although cytokine removal is substantial, iii) it seems that cwh application in sepsis of any stage helps to buy time for further treatment. the most commonly monitored variables in shock stages idclude : arterial pressure, heart rate, central venous pressure, pulmonary artery wedge pressure and cardiac index. with vigorous therapy it is possible to bring these values back into the normal range in both survivors and nonsurvivors. therapeutic goal in septic shock stages is to maximize the values of cardiac index, 02 delivery (do2) and 02 consumption (c02). objectives: the main purpose of this article is to determine the relationship betwee~ 02 delivery an 02 consumption as a sign of hypoxia. fifteen patitents with septic shock were treated with intention to maximize the value of ci,d02 and v02. we compared the levels of these parameters between the survivors and nonsurvivors and found no significant differences after 24 hours. high levels of do2 and v02 may not guarantee against tissue hypoxia in early stage of septic shock. zjar~iic, dj janjic, lj. gvozdenovic, a.komareevic. t.petrovic, &marjanovic, institute of surgery, novi sad, yugoslavia objectives: evaluation and mutual comparison of clinical signs, laboratory data and microbiological monitoring in the patients with burn sepsis. method: retrospective analysis of the recorded data of all burn patients treated in our department between january 1989 and december 1994. specially attentions were given to data considering wound infection, positive haemocultures, positive urinocultures and characteristics of septic state. results: out of 372 patient there were 324 (87,09~) adults and 48 (12,9(3~) children. almost two thirds of the patients (238 -63,97~) were males. the predominantly cause (68,759~) of children's burns was scalding b~y hot liquids and flame burns ~63,97~) in adult patients. the most frequdntly species isolated from surface swat~ were pseudomonas aeruginosa (64"1796 in adult patients) and staphyloccocus epidermidis (78,57% in children). in only five patients (1,349~ 1 the haenmcultures were positive -pseudomonas aeruginosa was isolated in three and staphyloccocus aureus in two patients. urine infection was diagnosed in 6,72% of all patients. the treatment protocol included use of imipenem and polyvalent pseudomonas vaccine again~ pseudomonas aeruginosa and vancomycin and aminoglycosides against staphylococcus aureus. total mortality rate in this group of burned patients was 6,98~, but the mortality rate caused of sepsis was low (i 34%) . conclusions: early detection of any signs of wound infection and symptoms of septic state is a foundation for prevention and treatment of burn sepsis. the burn sepsis could be reliable detected by continuously monitoring the patient's status and by systematic microbacteriological monitoring of the burned patients. hyperdynamic vasoplegic septic shock p.f. laterre, p. goffette, j. roeseler, j.p, fauville, a. poncelet, p. lonneux, m.s. l~eynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. splanchnic ischemia is described as a common feature of septic shock and could determine the development of msof. therapy such as noradrenaline (na) aiming at improving blood pressure is expected to worsen splanchnic ischemia by its vasoconstrictive effect and subsequent reduction in intestinal blood flow. ob[ective: evaluate the effect of na on splanchnic blood flow. material and method : in a patient admitted for variceal bleeding, ards and sepsis with positive blood culture, a fiberoptie catheter was positionned in the portal vein after recanalisation of its portosystemic stent shunt. blood pressure (bp-mmhg) , ci, svr, do 2 (vigilance ~ baxter), v02 (indirect colorimetry), arterial, mixed venous and portal vein blood gases, phi were determined before (to) and during (t1) na infusion (0, 1 to 0, 19 hcg/kg/min.) . changes in splanchnic flow were assessed by changes in portal oxygen saturation (sp02) and arterio-portal oxygen saturation gradient (sao, -spoe laterre, ,lp. pedgrim, th. dugernier, v. delrue, ph. hantson, p. mahieu, m.s. reynaert. dept. of intensive care, st. luc univ. hospital, brussels, belgium. aim of the study : prospective determination of plasma levels of in patients with ss and their correlation with the type of microorganism and outcome. material and methods : in 19 patients (pts) with ss and severe sepsis, plasma levels of tnfti, ill-b, il6 and il8 were determined every 8 hours for 3 days and on day 7 after fulfilling the criteria of ss and severe sepsis. results : in 9 pts, sepsis was caused by a gram (-) microorganism, in 6 pts by a gram (+) and in 4 pts no microorganism was identified. there were 12 survivors (63%) (s) and 7 non-survivors (37%) (ns) . cytokines profiles and levels were not different between gram (+) and gram (-) sepsis. ill-b levels were seldom elevated whatever the group studied. tnfot and il-6 were significantly higher in ns than in s ( objective: to evaluate the effects on the nitric oxide synthase inhibitor l-n~ hcl (546c88) on myocardial performance in human septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70mmhg) or the requirement for a noradrenaline (na) infusion >_ 0.i ]tg/kg/min with a map _< 90mmhg. cardiovascular support was limited to na _+ dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t = 1); and at the end of treatment (t = 8) with 546c88. conclusions: 546c88 can restore systemic vascular tone in patients with septic shock enabling na therapy to be reduced and/or removed. the ci tends to fall whilst lv performance is sustained over time. 546c88 is a novel vasoacfive agent for the treatment of septic shock, which is undergoing further clinical evaluation. laterre, f. thys, e. danse, j.p. pelgrim, e. florence, z roeseler, m.s. r eynaert. dept, of intensive care, st. luc univ, hospital, brussels, belgium. therapy aiming at improving blood pressure and cardiac index in septic shock (ss) might have deleterious effects on regional blood flow. objectives : compare the influence of volume loading (vl), dobutamine (dobu) and noradrenaline (na) on sushepatic oxygen saturation (shoe) and svoe-sho, gradient in treated ss. material and methods : in patients with ss, ci (thermodilution) , doe, svo,. sho,, svoe-sho e gradient and lactate (l) were determined before (to) and after (t1); vl, dobu and na. results: in 5 patients with treated ss, 16 tests were performed (vl n=8; dobu n=4; na n=4 method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map<70 mmhg) or the requirement for a noradrenaline (na) infusion ~> 0.1 ~g/kg/min with a map _< 90mmhg. cardiovascular support was limited to na + dobutamine (db), 546c88 was administered for up to 8 h at a fixed dose-rate of either i, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at 1 h from the start of treatment (t = 1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 is a novel vasoactive agent that can sustain map in patients with septic shock, enabling na support to he reduced and/or removed. there is a tendency for the ci to fall during treatment, which may be reflex in response to the increase in systemic vascular tone. 546c88 is a promising new therapy for septic shock, which will now be evaluated in a randomised, placebo-controlled safety and efficacy study. k. guntupalli objective: to evaluate the acute effects of the nitric oxide synthase inhibitor l-n~ hc1 (546c88) on selected indices of organ function in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 [xg/kg/ min with a map _< 90 mmirlg. cardiovascular support was limited to na + dobutamine. 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. indices of organ function were assessed at baseline (t = 0); at the end of treatment (t = 8); and 12 h after treatment (t = 20) with 546c88. results. -median values (* assessment made at 8 h or when 546c88 discontinued). conclusions: there was no appareut dose-dependent adverse effect on these indices of organ function either during or after exposure to 546c88. the plmelet count tended to fall whilst creadnine appeared to increase over time in all dose cohorts. this novel and promising therapy for septic shock will now be evaluated in a randomised, placebo-controlled safety and efficacy sludy. pharmacokinetics of 546c88 in patients with septic shock preliminary results z. hussein, b. jordan, c. fook-sheung, k. guntupalli objective: to evaluate the pharmacokinetics of the nitric oxide synthase inhibitor l-n~ hc1 (546cg8) given by continuous infusion for 8 h in patients with septic shock. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion --> 0.1 ~tg/kg/min with a map _< 90 mmhg. cardiovascular support was limited to na • dobutamine. 546c88 was administered for up to 8 h at a fixed dose-rate of either 1, 2.5, 5,10 or 20 mg/kg/h iv. plasma was collected from each patient over a 24 h period and analysed for 546c88. pharmacokinetic parameters were derived from plasma concentration-time profiles using non-compartmental pharmacokinetic analysis. results: the (cm~ -maximum plasma concentration; auc -area under curve; cl -plasma clearance; v,, s -steady state volume of distribution; t'/2 -plasma elimination halflife). conclusion: the pharmacokinetics of 546c88 in patients with septic shock are dose-independent at infusion rates up to 2.5 mg/kg/h. at higher rates, clearance of 546c88 decreases without any marked change in volume of distribution. 546c88 metabolism may be partially saturable at dose-rates above 2.5 mg/kg/h. obiectives: investigate the effect of the no synthase inhibitor, l-nt-methylarginine hc1 (546c88) on the haemodynamics and survival rate in a conscious mouse model of endotoxin shock. methods: female cd-1 mice (25-35 g) were instrumented under gaseous anaesthesia (isofluorane, 2%) and connected to a swivel tether system for continuous monitoring of blood pressure and drug administration. results: after 24 h recovery, endotoxin administration (e. col• 026:b6, 6-12.5 mgkg -1 i.v.) elevated the plasma concentration of nitrite/nitrate (nox) and caused a progressive fall in mean arterial pressure (map) from 101 + 5 to 59 + 4 mmhg (n=5, p<0.05) at 12 h, with a survival rate at 24 h, 48 h and 72 h of 80%, 40% and 20% respectively. 546c88 administered as a 24 h continuous infusion (3 mgkg-th -t i.v., n=5), 4 h after endotoxin, inhibited the elevation of plasma nox and attenuated the fall in map from 105 + 2 to 70 + 3 mmhg (n=5) at 12 h, with an improved survival rate at 24 h, 48 h and 72 h of 100%, 100% and 60% respectively. conclusions: this study suggests that overproduction of no is involved in the hypotension and mortality characteristic of septic shock. inhibition of no synthase using 546c88 represents a novel and promising treatment for septic shock. cultures of e.coli (19,5%) and candida(8, 3%) were olso received from autopsy material of children;p.aeruginosa,unspored anaerobes,proteus sp.,s.aureus,b.pneumonia were found in the few cases. in adults the spectrum of bacterioflora was mo~ re limited speaking about the number of species and cultures. in generalized forms of bacterial pyo-septic pathology a wider specific spectrum of causative agents was revealed usua fly with associations. e.coli and k.pneumonia played the leading role in children as well as in adults. in general,k.pneumonia (23,7%cultures) and common e.coli(18,6%)prevailed according to the date of microbiological investigations of authopsy material in pyo-septfc pathology in 1994. objectives: .in spite of all clinical exertion sepsis is still the reason for high clinica! lethality. this study is characterizing the group of patients which survived a septi~ shock. methods: during a period of 12 months all surgical patients on icu were registrated prospectively, more than 270 parameters for each of them were documented'daily in a paradox file. results (see table 1): 20 of 286 patients fulfilled the criterion of a septic shock (r. bone, 1991 ) , 11 of them died at the 2 lth day, while the surviving group of patients stayed almost 51 days at icu. obiectives: to compare the effects of 6 and 10% pentastarch solutions to a human albumin solution on oxygen delivery (do2) in septic patients. methods: this stud}, included 41 septic patients with fever (t > 38~ tachycardia flqr > 90/rain), tachypnea (rr > 20/min) or mechanical ventilation, leukocytosis (wbc>12000/mm 3) or leukopcnla (wbc<400()/mm 3) and a clinical source of infection, who required a fluid challenge. in each patient the pulmonary arterial occlusion pressure (paop) was < 12 mmhg. patients were randomized to receive 400 ml of 4% albunun (n:i4), hydroxyethyl starch (hes -mw200/d.s. 0.5) 6% (n:14) or t0% (n=i3); 33 patients were also treated with adrenergic agents. results cardiac index (c1) increased significantly only in 10% lies (table) hemoglobin (hb) decreased significantly at 40 min in the same group. there was not significant change in oxygen delivery ( do2 ). baseline ci alb 3.7::1.3 (l'min/m 2) hes 6% 3.9=0.9 hes 10% 3. polyneuropathy of the critically ill (pci ) is a well recognized complication, acquired in the course of severe illness. we undertook a prospective study, to estimate the severity, extension and time of onset of pci in a selected group of 25 patient with established septic shock ( bone's criteria ). all patients received inotropic circulatory support and were mechanically ventilated. none received relaxants or aminoglycosides. pci was diagnose<by clinical investigation and by emg, and repeated weekly ( axonal degeneration ). after 3 days ( day 1 = onset of septic shock ) in 68% of the patients pci was demonstrable. after 14 days 80% of the patients had pci. there was a strong correlation between pci and other mof and between pci and encephalopathy ( eeg ). we conclude that i. pci is a common complication after septic shock. 2. pci develops early in the course of illness ( 85% after 5 days ). 3. pci can be considered as a part of mof, suggesting a common pathogenesis. objectives: we addressed two questions: 1) can serial measurements of vapco2 and avph also reflect the onset of tissue hypoxia during endotoxemia? 2) are whole body changes in vapco2 and avph associated with parallel changes in regional circulation? methods: in 12 anesthetized, mechanically ventilated dogs exhaled gases were sampled for determination of oxygen consumption (vo2). a catheter was positioned into the pericardial cavity to induce cardiac tamponade. ultrasonic flow probes were placed around superior mesenteric, left renal and left femoral arteries, and the corresponding veins were cannulated. group l served as control (n=6), group 2 (n--6) received 2 mg/kg of endotoxin. thirty min later, tamponade was induced to gradually reduce do2. results: systemic do2crit was higher (12.1_+2.2 vs 7.9+2.6 ml/kg.min, p <0.05) and critical oxygen extraction ratio (o2ererit) was lower (45.1+_9.7 vs 74.1+_9.1%, p<0.05) in the endotoxie than in the control group. meslenteric and femoral do2crit were higher in the endotoxic than in the control group (8.2_+2.5 vs 4.1_+0.5 mlll00 g tissue.min and 8.3_+2.3 vs 4.6_+0.9 ml/min, respectively, both p<0.05). regional o2ercrit were lower in we endotoxic than in the control group (mesenteric: 37.1_+15.4 vs 71.i_+7.4 %, renal: 30.7+ 24.6 vs 53.9-+28.7 % and femorah 48.1-+9.2 vs 75.3_+6.9 %, all p<0.05). vapco2 and avph followed a similar pattern in both groups, increasing slightly before do2crit was reached, but dramatically wh,en do2 fell below do2crit. in the presence like in the absence of endotoxin, systemic and regional do2crit calculated from vo2, from vapco2, or from avph were similar; do2crit was also significantly higher in the mesenteric and the femoral beds of the endotoxic than the control dogs. systemic and regional do2crit could be calculated from the blood lactate levels only in the control group. conclusions: during an acute reduction in blood flow i) vapco2 and avph can reflect hypoxic threshold in the presence like in the absence of endotoxemia. 2) alterations in organ oxygen extraction capabilities induced by endotoxin can be reflected by regional changes in vapco2 and avph. objectives: enhanced nitric oxide (no) release has been incriminated in the vasodilation and myocardial depression characterizing septic shock, but the administration of no blockers has yielded equivocal results. the present study explored the systemic and regional effects of a no donor linsidomine (sin-i) during endotoxic shock. methods: 14 anesthetized dogs received endotoxin (2 mg/kg iv), followed 30 min later by a saline infusion to keep cardiac filling pressures constant. oxygen uptake (vo2) was derived from the expired gas analysis. regional blood flow was measured by an ultrasonic flowmeter (transonic). results: the control endotoxie group (n=7) maintained low arterial pressure and systemic vascular resistance. the 7 dogs receiving a continuous infusion of 1,2,4 ~ug/kg.min of sin-1 starting 30 min following initial fluid resuscitation (each dose for 1 h), had a higher cardiac index (0.23-+0.03 vs 0.16_+0.08 l/min.kg, p<0.05) and lower systemic and pulmonary vascular resistance than the control group (1006-+170 vs 1460-+445 and 114-+38 vs 244-+84 dyne.sec/cm5, respectively, both p<0.05). arterial pressure and pulmonary artery pressure were not influenced. sin-1 significantly increased the mesenteric blood flow from 63-+17 to 148+49 % of baseline levels and renal blood flow from 67-+25 to 96-+24 % (both p<0.05) but did not influence femoral blood flow. the relative blood flow was increased in the mesenteric bed (at all doses), and reduced in the femoral bed (at highest dose). systemic oxygen delivery, vo2, oxygen extraction and blood lactate levels had similar course in the two groups. conclusions: in fluid-resuscitated endotoxic shock dogs, the no donor sin-1 increased cardiac index without deleterious effects on arterial pressure, and increased splanchnic blood fl0w. objectives: sepsis syndrome is associated with the release of a variety of inflammatory mediators, such as interleukins, tumor necrosis factor and platelet-activating factor (paf). administration of paf, a naturally occuring phospolipid, to laboratory animals has been demonstrated to resullt in pathological changes that closely resemble those found in sepsis. in addition, paf antagonists of various origins have been demonstrated to attenuate cardiovascular collaps and to protect against lethality in endotoxemia. in the present study the efficacy and safety of the paf-antagonist tcv-30g was investigated in sepsis syndrome patients. in addition, the putative inflammatory parameters tumor necrosis factor (tnf), interleukin il) -6 il-8, and soluble (s) e-selectin were measured in both tcv-309 and placebo treated patients. metho.~a randomized, double-biind, multi-center study was undertaken to compare the safety and efficacy of intravenously administered tcv 309 versus placebo (takeda chemical industries ltd., osaka, japan). tcv-309 was used as add-on to conventional therapy in the treatment of patients with severe sepsis and septic shock (1.0 mg/kg body weight intravenously over 2 hrs, twice a day for one week). day 28 mortality was registered, as well as vital signs, laboratory parameters, hemodynamic parameters, apache ii score and mof score. plasma samples for the determination of inflammatory parameters were collected twice a day for one week. the study was approved by each institutional review board and written informed consent was obtained for each patient. results: a total of 29 patients were included in the study. one patient had to be excluded as a result of protocol violation of the remaining 28 patients 12 patients were randomised to receive tcv-309 and 16 to be treated with placebo. there were no significant differences between the treatment group with respect to age. gender, and apache i~ score on admission. however, admission levels of il-6 and il-8, considered to reflect severity of disease, tended to be higher in tcv treated patients: 24.59. vs 2.37 ng/ml for il-6 (p.06) and 0.34 vs 0.04 ng/ml for il-8 (p.07) in tcv-309 and placebo treated patients, respectively. a remarkable difference in survival, however not significant, was observed at day 7 i.e. the end of tcv treatment (mortality: 2 out of 12 in the tcv group and 7 out of 16 patients in the placebo group). the inc;dence ui ~-,~verse events, 'n both treatment groups were comparable. plasma il-6 and plasma il-8 levels tended to decrease more rapidly in tcv treated patients compared to controls (p=.17 and p=.09, for il-6 and /l-8 respectively) conclusions: tcv-309 is safe as add-on treatment in sepsis syndrome patients. the data presented indicate that tcv-309 may enhance survival of sepsis syndrome which will be assessed in a phase ill study. objective: oxidation of lipids by free oxygen radicals playes an important role in sepsis. an important source of oxygen radicals is the respiratory burst of phagocytic leukocytes, especially~ polymorphonuclear leukocytes. clinically the hallmark of sepsis is the capillary leak, which is mediated in part by oxyradicals. the adult respiratory distress syndrom (ands} represents the most studied capillary leak phenomenon in sepsis. methods: in ii patients (df,~m, aged 20-81y.) with verified septicemia had been included in this study. beginning from day 1 oxidisable lipidautoantibodies (club), ~opterin and crp were determined in the patients serum and compared against the apache ii score during the days 1-7. 4 patients, 2f and 2m died @u= ring their stay at the icu, 3 of them in less than 48h~urs: after admission. at the begin surviving patients showed olab values between 75-119%, non-survivors between 68-104%, com= pared to neopterin values of 38-75 m~ol/l in survivors and 20-250 ~mol/l in non-survivors. apache ii score didn t show any difference. during the following days olab sbowed an in= crease in survivors, while in non-survivors olab stayed equal or showed a decrease. no difference between both groups was found in neopterin, crp and apache ii score. as result we conclude, tbat lipidperoxidation playes an important role during septicemia. olab as a marker of this process are predictive for the outcome of patients. obiectives : to evaluate the influence of cardiopulmonary bypass ongastrointestinal transit. meth~xls; gastrocoecal transit time (gtt) in 10 consecutive children on day 1 after uncomplicated open heart surgery was compared to gtt in 10 children after closed heart thoracic surgery and to healthy controls. gtt was measured by hydrogen breath testing, using lactulose (o,4g/kg in a 10% aequous solution) as nonabsorbable marker. exspiratory hydrogen content was measured using a electrochemical system (stimotron). patients in both groups had continuous infusion of morphine and midazolam and were comparable in respect to catecholamine dose. children with a history of toddler's diarrhoea and children with elevated central venous pressure were excluded. results: gastrocoecal transit was delayed in all patients after cpb, no transit was achieved within 480 minutes in 7/10. after closed heart surgery gtt was delayed 138(+-80) min vs. 56(+-20) compared to controls, but transit was achieved in all aptients within 285 rain. mean dopamine dose was 2,7mcg/kgmin after cpb and 4,8meg after closed heart surgery.. conclusions: after uncomplicated cpb gastmcoecal transit is extremely delayed. the degree of delay is not explained neither by the use of analgetic and sedative drugs neither by the use of catecholamines. factors directly related to cpb (e.g. low flow perfusion, hypothe,mia) may be responsible. these findings are of limited clinical relevance in ancomplicated cases. in critically ill patients delayed enteral feeding may contribute to infectious complications. studies of therapentic measures to accelerate gastrointestinal transit therefore are warranted. neonatal ecmo -czech experience. nekvasil r., penkova z, vasek l., plier p., bobula a novotna e., pavlicek v., hnilicka m. nicu and ecmo center brno and dept. pediatric surge13', children's hospital brno, czech republic. introduction. in spite of the fact that neonatal ecmo has been gradually considered a standard method of solving uncontrollable respiratol t failure in newborns in most countries of the western europe, the introduction of this sophisticated method in countries of the former communist block meets a lot of problem. material methods. in the course of two years, only 16 newborns, b.w. 2000 -4300 g, were reffered to neonatal ecmo because of uncontrollable respiratow failure. at the admission the average values of oxygenation index were 51 +12, and aado2 79,6 -+7,6 kpa. results. 4 newborns (25%) died shortly alter" admission or during transport without possibity to further is 5 newborns (31,3%) were treated using high-frequency ventilation (hfo or hfjv), one for conspicuous airway resistance paradoxically with low-frequency ippb. all ventilated newborns survived. ecmo was applied in 6 newborns (37,9%) and 3 of them survived the mortality rate of group mentioned was 44%. conclusion. the fact that in the czech republic (10 mil. population with natality rate of about 100.000 newborns/year) only small part of patients was reffered to neonatal ecmo has a number of causes. the most important are: unacquaintance of prognostic and indication criteria at forwarding workplaces and the aversion of neonatologist to all new.therapeutic interventions. last but not least, a negative role is also played by current change of health service systems when, under the influence of health-service insurance companies a newborn in serious condition given only a standard intensive care in primary and secondary nicu is literally "the golden calf", and therefore these workplaces either do not send him or send him too iate ~o an ecmo center. stndy aim: 8urfactant, which is being applied into the lungs for treatment of respiratory distress syndrome, can reach the circulatory blood system by absorption. it refinances local fimctiun of macrophages and lymphocytes and increases the accumulation of nentrophils into the hmgs this eunld influence the local acute phase reactiml in the hmgs after surfactant application. "ilia aim of the stud), was to investigate the influence of oatural and artificial surfactaat onto the acute phase reaction and the blood elottiog system. material :and methods: a) measurement of tnf-a, 11-6, ltb 4 and thromboxane b 2release in whole blood: each of 1.5 ml hepariaised blood of healthy adult volunteers was iucubated lbr 2 hears with eillier ill ug/ml lps, 1 mg/ml b&,ine smfactant (alveulhct/themae), 1 rag/nil artificial surfactant (exosurf/wellcome), 10 og lps + i mg/ml bovine surfactant or i0 ug lps + i mghal artificial sorfactant, tleparinised bhx-,d without additions was used for controls. '111e concentration of e)lokines and eicosanoids ".van measured semiquantilatively by eijsa or eia (dimmva/hamburg) after ceatrifagalion, b) measurement of platelet aggregation: each of 1.5 ml haparinised hltxxl of healthy adult vohmteers was inculmled lbr 2 hours with eilher 10 aghnl i,ps, i mg/ml bovine surfactant (alveafact/thomae), i mg/ml artificial snrlhctant (exosurf/wellcotne), it} ug lps + i mghnl bovine snrfactant or 10 ug lps + i mg/ml artificial surfaetant, tlepminised blood withont additions was nsed for controls. platelet aggregation in whole blood was measured by impedance in the aggregometer (g~nstaedt). c) measurement of the dotting activity: citrated bleed of healthy adult w~lunlccrs was mixed with difl'ereul couccalrations of imvine or artificial snrfactant fad then prolhrombiu tiaras and imrlial thrmnlx~plaslia filues were ineasnrcd. for measurement of the prokoagulatioo acfivily the reagent in the qnick test was replaced by surfaclant. results: dependmlt on the dose both surfactants stimulated the release of 11-6 but not or tnf-a io wlmle blood. "illere was no suppression of the lps-indaeed eytokine-release. both surfaclants did not directly influence the extrinsic blood coagulation system, while both activated the inniasie activity dependant on the dose. platelat aggregatiun was slightly stimulated by bovine snrfactunt and strongly inhibited by by artificial surthctant. in comhinafion with 1.1~ both factors showed no difft.'renca to lps ahme.'fhe reltu.tse of eiconasnid~." was stimolated more by artificial surfactant then by bovine surfilctant with regard to the cyehmxygelmse (thromboxsne b2) and the lipooxygenase (lencotrieae b4) pathways. conclusion: we cooclude: 1) i:lovine as well as artificial surfaelant stimulate the cellular inmnn]e response. 2) bovine as well as artificial surfnctant activate the intrinsic ctmgnlation cascade. 3) bovine surlilctant stimulates platelet aggregation, artificial sar-fijctant inhibits platelet aggregation.. introduction: although the use of modem noninvasive methods like measurement of tope2, tcpco 2 or san 2 leeds to an carly infonnation about pathologic alterations of the gas exdmage of ventilated patients, these methods cannot replace invasive bloodgas analysis. a new noninvasive method to monitor the capillary gas exchange is the reflection spectrophotometry. to record reflection spectra with a ldgh sensitivity on the surface of tissue we eonstnmted a special mierolightgnide photospectronmter (empito). this spectrometer enables the investigation of local helerogenities of llbo 2 and 1 lb content, capillary flow rate and oxygen uptake rate. aim of the study: 1 ) to compare the sousitivity and specifity ofthe empilo with other non-invasive method~ of monitoring the gas exchange in neonatal intensive care patients. 2) to investigate the possibility of obtainiug infonnations about the local 02nptake and the venous as well as arterial 02 satnration. patients and methods:we investigaled 16 non-selected patients of oar pediatric intensive care unit. for coulinnons monitoring we conslracted a special sappert to fix file end nf the microlightguide+ during the investigation tope2, tcpco 2 and san 2 were measured by standard methods. "fo investigate the local tisslm oxygen uptake we recorded 1000 spectres within 2 minutes by moving the lightgnide over an described area of the skin of the patient. 'lhe reselting i(gx) l ibo 2 vahms were sorted by a software package. on the assumption that the low values correspond to the tibo 2 value of the veaons ends of the capillaries and the high values to those of the arterial ends we 2) it is possible to gel infonnations about the local o2-uptake and arterial 02 saturation ia the peripheral tissue by photoslg'ctroscopy. 3) in combination with noninvasive pl i-measorement it seems possible to reduce withdrawal of blood for invasivn bhmd gas analysis. objectives:improve ventilation,tissue oxygenation and acid base balance among extremly low birth weightand premature infants. methods: fourteen cases were s~udied,their gestational age ranged from(27-32)ws.continnous positive air way pressure was applied to six cases at peep level from (3-6)cm h2o through nasal pronge,(group i),the other 8 cases were managed as routine,(group ii).blood gases, tcpo2,tcco2,resp.rate,depth and pattern were monitored for assessment of tissue oxygenation and ventilation, results: our rasults showed that early application of cpap improve ventilation among (83.3%)of cases,while (16.7%)of cases need imv.the cases of group ii need imv among (75%)of the studied cases during the second or the third day of life. conclusions:-this preliminary study showed the import-............ ance of early application of cpap before the onset of respiratory distress syndrom to improve spontaneous ve ventilation,tissue oxygenation and to gecrease the risk of intubation. comparative assessment of pediatric intensive care; a national multicenfre siudy. reinoud j.b.j. gemkc, gouke j. bonsel 2, the dutch picasso (pediatric intensive care assessment of outcome) study group. twilbelmina children's hospital and utrecht university, utrecht 2department of clinical epidemiology, academic medical center, : amsterdam, the netherlands the performance of all (10) pediatric intensive care units (icus) in the netherlands (6 tertairy and 4 non-tertiary) was analyzed in an open prospective muiticenter study. effectiveness of care was defined as the ratio of observed to prism-score derived expected mortality 0aenee, standardized mortality rate). efficiency was determined by 2 objective criteria (mortality risk > 1% or administration of at least 1 icu-dependent therapy)'. 1063 consecutive admissions aged < 18 years old were included. overall, observed and expected mortality were in good agreement (p > 0.5). between hospitals, crude mortality showed wide variations (mean 7.1%, range 1-10%). however, in each center, observed and expected mortality were similar (mean ratio 0.99, range 0.8-1.5). in tertiary care centres, severity of illness corrected mortality in high-risk patients was less than in non-tertiary care centres; paradoxically, in low-risk patients the opposite was found. probably the large proportion of low-risk tertiary care patients suffering from severe, incurable chronic disease, explains the higher mortality in this group. this indicates that simultaneous assessment of circumstances of dying and of long term morbidity in similar future studies is imperative. the average proportion of efficient icu days was 72%, however large variations between units were found (range: 22-95 %). in conclusion differences in mortality rates among pediatric icus were explained by differences in severity of illness. high efficiency rates in combination with adequate effectiveness, found in several centres suggest that admission and discharge decisions might be improved by a better selection of high risk patients requiring icu-dependent therapies, especially in less efficient centres. objectives: previously published studies showed that serum lactate levels correlated with outcome of severe ill adult, 'we hypothesized that critically ill newborns are often incurred hypopeffusion manifested by elevated lactate levels. these initial blood lactate levels should be related to nicu outcome. design: prospective study with ethical comfnittee approval. setting: the 14-bed neonatal intensive care unit of a university hospital material and method: a total of 209 consecutive outbem newborns admitted to nlod from 01,10.1991 to 31.,19.1992 were enrolled to the study. babies who died or were discharged from the unit within 48 hours of treatment were excluded from the study, mean birth weight was 2040g (+/-820r), mean gestatational age was 35 weeks (+/-3.5 wks), mean age at the admission was 50 h (+/-110hi. multiple (~_2j organ system failure occurred jn 38.3% of babies at the admission./~tertal lactates were measure/at the admission, among 22 -26 hour and 46 -50 hour of n[c'lj therapy. outcome was defined as a mortality and length of nicu stay. results" survival rate was 68.4%, mean length of nicu stay for survivors was 17.8 days (+/-15.1 day). we found high lactate levels at the admission in 82.8% babies (~9.2% with levels above 5.0 retool/i). the mean arterial lactate concentrations for nonsurvivors were signiftcahtly higher than for survivors durin~ consecutive da~ as follows: objectives: the purpose of our research was to analyze the frequency of bronchial asthma (b.a.) exacerbations in pregnant women and health status of infants. methods: the research was based on the epidemiological investigation and prolonged observation of 23 pregnant women with b.a. during the gestation period. remission of b.a. before the pregnancy in excess of 5 years was recorded in 9 patients (39.1%), 6 patients (26.1%) reported a 2-3 year remission and 8 patients (34.8%) had a remission lasting less than 12 months before they became pregnant. results: seven patients (30.4%) developed medium attacks in the second half of pregnancy, four patients (17.4%) experienced light attacks of b.a. asthma attacks were most frequently caused by acute respiratory diseases and stress factors. in two cases with grave manifestation of b.a., the pregnancy ended in abortion within the first 16-18 weeks due to the frequent and heavy choking attacks. to fight b.a. attacks, five patients used 132adrenomimetics (salbutamol, becotid) in sprays, six women were administered theophyllinum and salbutamol in the form of tablets during 1-2 weeks. a significant portion of pregnant women with b.a. (78%) exhibited frequent complications during pregnancy (toxemia, late gestosis, threat of miscarriage). our findings prove that babies born from women with b.a. of domestic and pollen origin had a low body weight (2800-2500 gr), functional immaturity and chronic antenatal and intranatal hypoxia twice as often as the infants born from healthy women without allergic background. conclusions: preventive treatment of women with b.a. prior to pregnancy is required to maintain a stable remission of the disease, which is a key to having healthy children delivered by mothers suffering from b.a. introduction. intracerebral hemorrhage (ich) is a common event in human prematudty, affecting about 25% of newborns weighing below 1500 g who are born before 32 weeks of gestation, however, little is known about the pathogenesis of ich with exception of the prematurity of the brain itself, (birth) trauma, and asphyxia. the postischemic production of oxygen free radicals (ofr) dudng reoxygenation as a cause of brain damage has been demonstrated in 9 animal research. since almost all preventive antioxidant activity of plasma is associated with ceruloplasmin and transferdn we investigated the association of such iron-oxidizing resp. iron-binding proteins and ich. we could demonstrate significantly reduced levels of both, iron-oxidizing and iron-binding proteins, in premature asphyxiated newboms pdor to development of ich. an increase of suparoxide after hypoxia in the presence of iron ions facilitates the formation ofthe highly reactive hydroxyl radicals. our data support the theory that ich may be caused by ofr, which can damage any sensitive tissue including growing endothelial cells. the estimation of transferrin-saturation and measurement of ceruleplesmin levels might help to identify an infant at dsk before the onset of ich. with the new medos | hia-vad | cardiac assist system the missing tool in the armamentarium of cardiac surgeons is available in two pediatric sizes: i0-ml and 25-ml pump volume. the right sided pumps are 10 % smaller for biventricular use. between february 1994 and may 1995 we implanted this assist system in 6 children. the indications and demographics are indicated in the following table (left ventricular assist device-lvad, right vad-rvad univentricular vad-uvad, post cardiotomy cardiac failure-pcf, dilated cardiomyopathy-cmr bland white garland syndrome-bwg, tetralogy of fallot-tof, hypoplastic left heart syndrome-hlhs). objectives: evaluate tile effeci'of inhaled nitric oxide (no) as puhnona] t vasodilating agent ill tile posloperalivc period after correclion of congenital heart defects in 3 infant. patient n.l: 4 kg, 7 lnonlhs, down syndrome undenvcnl rep~fir of atrioventricular septal defect (avsd). after surgery the puhnonary arlcry pressure (pap) slowly rose to tile syslemic dcspilc tnaximal eonvcnlional fllerapy (fentanyl 4 mcg/kg/h, hypocapnia of 27 mmhg and metabolic alcalinization). no was delivered into tile inspiratory branch of!be breathing circuit at 10 ppm, and the gas aoalyser for no and no2 (polylron dmger) were situated at the espiratory branch, a rapid dccrcasc of pap io i/3 of systemic was obtained with a dramalic improvement. no was continued at 5 ppm for six days and the baby was exlnbated if! days after surgery and discharged from the icu 5 days after. patient n.2 : 4.5 kg, 6 monlhs, onderwen! repair of avsd. the day after surgery the systemic oxygen salnralion was 76% wilh a pap at 75% of systemic. two hours of c01wenlional therapy failed 1o improve ihc patient and no administration was slarled at 10 ppm. so2 dramatically incrcased to 95%, but the pap dropped only to 50% of syslemic. nevertheless ihe clinical conditions improved and the no administration could be reduced at 5 ppm in the following 6 days. she was extubaled 8 days after surgery and discharged from the icu 20 days after. patient n.3:12 kg, 3 3'ears. underwen| hearl tral~splantalion for congenital heart disease with moderate hypoplasia of pulmonary arlcrics. at the end of cardiopulmonary bypass the transpnlnlonary al~erio-venoas gradient yeas higher than 7 mnfflg and we speculaled !hat w'ls due to a degree of puhnonary vasocostrictiont. the nsnal dose of no was otilised, however no significant modilicalion of pulmonary pressure or systemic oxygen saluralion was noled, and after 1 h no was discontinned. tile palienl was carried io the icu with maximal inotropic support, extubated after 4 d;b's and disclmrged from the icu after 15 days. in all patient no major adverse effect relaled to no admilfistration ",','as holed. conclusion: in our experience no ms a pulmonary vasodilaling agent is effective and easily adjustable to tile palienls requiemenls, however its use remains limited ill those palienl ill whoin tile alnonll! of fixed inlllllojliify vascular resistance is predominanl. we report the use of ecmo support in two unusual cases of severe tracheal disruption in which it had become impossible to achieve adequate ventilation. case 1: severe tracheal laceration due to aspiration of a share forelan bodv: a previously healthy 13 month old toddler was referred for ecmo following aspiration of a porcelain foreign body (with razor sharp edges) which had become embedded in the right mainstem bronchus with massive extrusion of air. this was removed on veno-arteda[ ecmo support, as the patient was unventilatable prior to bronchoscopy due to ongoing airieak. ecmg was continued after bronchoscopy to permit airway healing without the presence of an endotracheal tube. unfortunately, an extensive pulmonary haemorrhage on day 4 of ecmo necessited re-exploration of the airway. this revealed a posterior tracheal tear from the cricoid to the middle of the right lower lobe. following repair the patient was left on ecmo support together with high frequency oscillation ventilation (hfov), the latter being used to minimise potential aideak and maximise alveoli recruitment. ecmo was weaned after 17 days (420 hours) -the patient was extubated 7 weeks later. case 2: tracheal wound dehiscence due to seosls -tracheal transelant on ecmo: a 4 month old infant with a c[inically significant congenital long segment tracheal stenosis and left pulmonary artery sling underwent resection of the stenosis, followed by primary reanastomosis. this was complicated, 5 days later, by severe mediastinitis and complete dehiscence of the anastomosis. an autologous pericardial patch was used to repair this, however, the tracheal wound again dehisced 4 days later making mechanical ventilation impossible. in view of ongoing sepsis and a severely disrupted trachea ecmo was the only possible form of support. following resolution of the local sepsis (4 days) a definitive procedure in the form of a tracheal homograft (transplant) was undertaken on ecmo. the patient was managed on ecmo and hfov for a further 3 days, the hfov being used to optimize rapid lung inflation. unfortunately this patient died 9 months after weaning from ecmo due to complete disintegration of the homograft, which was not deemed reparable. conclusions: 1) ecmo can be used in the acute management of oxygenation when there is major airway disruption making mechanical ventilation impossible. 2) hfov was a useful adjunct in aiding recruitment of lung volume on ecmo in these two patients. backoreund: persistent pulmonary hypertension of the newborn (pphn) consists of a heterogenous group of diseases ranging from transient reversibte pulmonary hypertension to fixed primary malformations of the lung (primary pulmonary dyspfasia-ppd). inhaled nitric oxide (ino), a selective pulmonary vasodilator, has been proposed as a treatment for severe pphn. obiective and methods: ino was administered to 23 near term neonates with severe persistent pphn, oxygenation index > 25 and echocardiogrephic evidence of pulmonary hypertension, in order to further determine the clinical role of ino in the treatment of pphn. the response to ino was also analysed retrospectively to examine whether this could be of diagnostic value in differentiating at an early stage patients with reversible from fixed causes of pphn results: twenty one of the 23 patients studied responded to the initial trial of 1no (20ppm x 20 minutes), as defined by a greater than 20 percent improvement in pad2 as well as a fall in the el to < 40. these 21 patients were continued on ino therapy, with 3 patterns of response emerging: pattern 1 babies (n=8) continued to show a sustained response to ino and were successfully weaned from it within 5 days -all survived. pattern 2 babies (n=9) failed to sustain their response to ino over 24 hours, as definded by a rise in the el > 40. six survived, five with ecmo. pattern 3 babies (n=3) had a sustained dependence on ino for 3 -6 weeks. all three died and lung histology revealed severe primary pulmonary dysplasia (ppd). patients with ppd (pattern 3) not only required ino for longer periods of time than did the sustained responders (pattern 1), but also required significantly higher doses of ino we report on the air transport of 32 paediatric intensive care patients. these transports fall into three categories: 1) retrieval of critically ill neonates and paediatdc patients referred for either ecmo or inhaled nitric oxide (ino) (n = 12). one patient was transferred on ind. mean transfer time 2.2 hours (se +0.6hrs). 2) long distance international transport using chartered aircraft (n = 11). the indications for these transfers included both urgent retrievals for cardiac surgery and semi-elective transfer of stable patients back to their referring unit following treatment in tertiary centres. mean transfer time 4.4 hours (se +0.4hrs) 3) long distance international transport using commercial aircraft (n = 9). indications for transfer were either semi-elective retrieval for tertiary treatment or the return of stable chronically ventilated patients to their referring hospitals. mean transfer time 14 hours (se _+1 .fhrs, longest 24 hrs). the transport team consisted of a paediatric intensive care doctor of at least registrar grade and a registered sick chidrens nurse with intensive care experience. the administrative components of the transfer (ambulances, airlines, customs) were managed in collaboration with companies specializing in air ambulance transfers. outcome: all the patients were safely transported to their destination without mortality or morbidity. complications durino transfer ir~lv~; 1) patient complications -semielective endotracheal tube change and central access needed in the only patient brought to the commercial aircraft by the referring hospital (all others retrieved directly from referral hospital), seizure in patient with known encephalopathy, severe cyanotic spells in patient with fallots tetralogy who was retrieved for urgent surgery for this indication 2) mechanical compfications -ventilator failure, incubator battery failure, oxygen regulator failure -all occurred with equipment sent from referral hospital, this was unfamiliar and unchecked by our transport team -it was not the decision of the transfer team to use this equipment on this single occassion. 3) administrative complications -confiscation of incubator battery by airport security police, excessive delay by custom officials (2 hours) in the airport. the incidence of such problems were felt to be low and unpredictable. in conclusion: mechanically ventilated paediatric patients can be safely transported on both chartered and commercial airlines. these transports are best accomplished by trained intensive care medical and nursing staff with the backing of an air ambulance organization competent in arranging the necessary administrative details. it is essential to use your own equipment and to retrieve the patient _directly from the referrin(] hospital to minimise ootential complications. our experience with anaesthesia for paediatric electromyography _w_._pla_ti_k_a_n_o_v, r.eousseff, k.pavlova, d.marinova dpts. of anaesthesiology and int. care and clinika] neurophysiology, med. university, pleven, bulgaria ~)_b_j#~ti_v~. to t~st a " heavv sedation " regimen of anaest-es~a for the purpose of paediatric electromyography d#s~gil~ non-randomized,non-blinded human trial in the seting of an uriiversity hospetal. _m_a_t_eri_a_is_a_nd_ m_e_th_od_s_.110 children,asa i-if,median age 6 years,range 9-13 who undervent eleetrcmyography required anaesthesia. they recieved low-dose ketamine + i~iazepam or midazolam via musculary route( 25 children,age 0 -3 yrs,ketamine 2,5 mg/kg, diazepam 3-6 mg total dose ) or per os ( 85 children,ketamine 5-7 mg/kg,diazepam 0,3 mg/kg or midazclam 0,4 -0,5 mg/kg ) _resu_l_t_s. 20 -25 minutes after medication a state of heavy sedation with weak spontaneos and stimuli-provoked movements was achieved in all children, that lasted 30 -60 minutes and allowed adequate needle emg and nerve conduction investigation. 11 children recieved additional 0,6 -1,0 vol.% halothane during the placement of the needle. non -invasive blood pressure , breath and heart sounds and hb sad2 by pulse oxymetry were monitored.none of the older children disclosed memories of pain when asked after they regained adequate verbal contact.no complicationes were observed. antenatal maternal steroids reduce the risk of periventricular-intraventricular hemorrhage in very premature neonates treated with natural surfactants. i.apostolidou, c.papagaroufalis, g.touloumi, m.xanthou, n.kalpoyannis a' and b" neonatal icu "ag. sophia" children" s hosp. athens, greece. dept of hygiene and epidemiology, athens university, greece. obiectives: the aim of the study was to evaluate the association of periventricular-intraventricular hemorrhage (p-ivh) in surfactanl treated premature neonates with pre-and postnatal variables. methods: the population of the study was 88 neonates admitted during the years 1990 to 1992, with gestational age _< 32 weeks and severe respiratory distress syndrome (rds) (mechanical ventilation and arterialalveolar oxygen tension ratio (ajapo2) <0.22), who received rescue therapy of at least two doses of natural surfactants (alveofact or curosurf) and examined with ultrasound and/or autopsy for the presence of p-ivh (papile's classification). the examined factors in each neonate were the following: gestational age, birth weight, sex, multiple pregnancy, antenatal maternal steroids (complete and incomplete course of betamethasone), a/apo 2 before the administration of the 1st dose of surfeclant, delivery, apgar score at 5min, type of surfactant, pneumothorax and patent ductus arteriosus. the statistical methods used were x 2 and one-way analyses of variance followed by logistic regression medels, results: the incidence ot p-ivh was 31.8%. three factors were found to have an independent relation to p-ivh (final logistic regression model): gestalional age, a/apo 2 before surfactant administration, and antenatal administration of maternal steroids (complete and incomplete courses). for every 2 weeks of lower gestational age the neonates had an almost doubled associated risk of p-ivh (or: 1.92, 95% c1:1.14, 3.22). for every 0.02 on average decrease of a/apo 2 before surfactant administration the risk of p-ivh in the neonates was 1.27 times higher (95% ci: 1.02, 1.58). the neonates whose mothers received antenatally steroids had only one tenth of the risk of p-ivh of the neonates whose mothers had not (or: 0.10, 95% ci: 0.01, 0.82). conclusions: our results suggest that the antenatal administration of maternal steroids, even less than 24 hours before delivery, reduce the risk of pqvh in very premature neonates treated with natural surfactants, whereas the small gestational age and the lung immaturity still remain the main risk factors tor the development of p-ivh. we analysed retrospectively the management of 103 (51 boys, 52 girls) accidental ingestions of foreign bodies in children (mean age : 2.8 years, range : 7 months-10 years). no child had ingested more than 1 foreign object. the majority of the ingested foreign bodies were : coins (n : 44), toy parts (n : 11 ), jewellery (n : 3), batteries (n : 16), "sharp" materials such as needles and pins (n : 21), "large" amounts of food (n : 8). impaction of food occurs more frequently in children after oesophageal reconstruction in cases of oesophageal atresia. although according to literature "coca-cola" is reported to be effective, this was not seen in our experience. 28/103 patients had minor transient symptoms at the moment of ingestion, such as retrosternal pain. only 4 children experienced severe manifestations (cyanosis, dysphagia). in these children, endoscopy revealed oesophageal and gastric erosions. children were seen at the emergency ward within a few hours after the accident ( mean : 3 hours, range 20 min. -28 hours). chest and/or abdominal x-ray was performed as first-line investigation ( 93/103 objects were radio-opaque), and revealed an (unexpected) oeeophageal impaction in 6 children. in 87/103 the foreign body was in the stomach. batteries, sharp objects and objects trapped in the oesophagus were removed, either by endoscopy or by magnet-extraction whenever possible. the outcome of the patients was excellent. no complications were observed. extraction is recommended in symptomatic patients, and whenever the foreign body is trapped in the oesophagus, or if the foreign object is "sharp" or a battery. objectives: two strategies were used for management of malignant diphtheria in children aged from 0.5 to 13 years. methods: protocol n1 consisted of intravenous administration of diphtheria antitoxic serum, prednisolone (2 mg/kg bw/day), plasmapheresis and supportive care. protocol n2 included the use of antitoxic serum against the background of high-dose dexasone (2-3 mg/kg bw/day), hemocarioperfusion and a preventive use (before the clinical manifestation of myocardial damage) of inotropic medications, inhibitors of angiotensin-converting enzyme and pentoxyphylline. each of protocols included the monitoring of serum toxin (diphtherin) levels. results: the group of patients treated according to the protocol n1 consisted of 17 children with malignant diphtheria, 11 of them with severe malignant diphtheria (grade 2 and 3). all patients exhibited the circulation of toxin during at least three days after the start of treatment. all 11 patients with severe grade of disease demonstrated heavy cardiovascular disturbances associated with malignant diphtheria. of the 11 children in the group died seven. the children of the second group were treated according to the protocol n2. out of total of 22 patients of this group. 11 patients had severe malignant diphtheria. in all children a significant reduction in serum toxin level was revealed after hemocarboperfusion. in all but one case the satisfactory control of cardiovascular function on was achieved. of 22 children admitted to the trial 21 survived, one child with malignant diphtheria of grade 3 and congenital filbroelastosys of the left ventriculum died. the severity of neurological complications was similar in each of groups. conclusions: the use of hemocarboperfusion, high-dose dexasone and early prevention of heart failure as a adjunct to the standart treatment has been shown to be of benefit in the management of malignant diphtheria. t. schaible, i. reiss, j. m611er, l. gortner med. university of lqbeck, children's hospital, kahlhorststr. 31-35, 23538 l~beck, germany surfactant therapy seems a promising approach for the treatment of the biochemical and biophysical abnormalities of the pulmonary surfactant system in severe ards. patients and methods: over a 18 months period 10 non-neonatal pediatric ards patients (age 1-38 months) in a "pre-ecmo"-situation (oi 40 over 4 h) were treated with bovine surfactant (alveofact| the underlying conditions-of ards were pneumonia (5), sepsis (2), immunosuppression (1), near drowning (1), neurogenous ards (1). a total of 20-120 mg/kg b.w. was applied in several fractions. before surfactant therapy, we first tried different ventilation (best peep-finding, inversed i/e-ratio, hfo-ventilation) while monitoring the pulmonary mechanics. for hemodynamic stabilisation both norepinephrine and epoprostenol were used to optimize pulmonary perfusion for max. 4 hrs. if there was no improvement of the oi by at least 10, further treatment with surfactant was initiated. in addition to surfactant all patients received a treatment with dexamethasone of 1 mg/kg in 2 doses. patients with no benefit (oi remained unchanged or increased within the max. 2-4 hrs) were taken on ecmo. results: nine patients improved within 4 hours after surfactant therapy: the oi decreased from a level of 41 (mean, range 22-100) before our treatment to a level of 16 (mean, range 6-60) thereafter. in 6 patients we were able to continue the positive effects of our treatment and they could be weaned of the respirator within 3-10 days. the other 3 patients got worse despite respiratory improvement, they suffered of multiorgan failure of more than 3 organ systems. the last patient did not benefit from surfactant, he had to be put on ecmo, but died because of a complication (hemopericard)after 10 days. the autopsy of the ecmo-patient showed a pulmonary fibrosis, but the other 3 death were not due to pulmonary failure. conclusion: a different sequential ards treatment integrating surfactant therapy can reduce the number of patients requiring ecmo. but ecmo as a therapeutic tool should be available in centers involved in ards treatment. l.blindl, t.p.le, h.weinzheimer, centre for paediatrics, university of bonn, germany selective reduction of elevated pulmonary vascular resistance by inhaled prostacycliu (pgi) has been reported in adults with acute lung injury, neonates with persistent pulmonary hypertension and in one infant with idiopathic pulmonary hypertension. we report on the effect of aerosolized prostacyclin in two children with secondary pulmonary hypertension. patient 1: in a boy with down's syndrome an avsd had been surgically corrected at 11 month of age. at 5,6 yr of age a catheter examination revealed a pulmonary vascular resistance of 70 % of systemic vascular resistance in room air and at an fin2 of 1.0. prostacyclin (0.5 mcg/ml) was administered with a jet nebulizer at an fin2 of 0.21. pvr declined to 0.4 systemic vascular resistance and returned to baseline after stopping pgi-inhalation. subsequent intravenous infusion (5 ng/kg rain) had to be stopped after 5 minutes because of systemic arterial hypotension. patient 2: a 8 month old male infant with bronchopulmonary dysplasia developed suprasystemic right ventricular pressure inspire of therapy with oxygen and nifedipin. while he was spontaneously breathing 60 % oxygen via face mask pao2 was 37 mmhg, arterial ph was 7.35. systolic arterial pressure was 85 mmhg, a rv-ra gradient of 100 mmhg was measured by cw-doppler. while fio2 was maintained aerosolized prostacyclin was administered over 30 minutes. rv-ra gradient was 70 mmhg, systemic blood pressure 75 mmhg, pao2 58 mmhg. two hours later nitric oxide (20 ppm) was inhaled at an fio2 of (3,6. rv-ra gradient declined from 100 to 65 mmhg, systemic systolic blood pressure remained stable at 105 mlnhg. discussion: sporadic experience shows that aerosolized prostacyclin selectively reduces elevated pulmonary vascular resistance in some patients. in patient 2 the poor response to inhaled pgi1 compared to inhaled nitric oxide may be explained by the fact that the action of pgi is not independent from endothelial function, limiting it's effect in severe vascular disease. during the last two years (1993-94), 51 infants weighing less than 1000gr. admitted to our referral unit. thirty four of them (67%) survived, (28% of infants weighing 500-700g and 78% of infants weighing 701-1000gr survived) for the years 1986-87-88 the survival of these infants was 53% and for the years 1976-77-78, 14% (p<0.01). we analyzed the perinatal and neonatal factors influencing the outcome of these infants. the comparison among neonatal survivors (1) to neonatal deaths (2) shows: gestational age: 27.6 w (1) to 26.4 w (2) (s). birth weight: 923.5g (1) to 724.7 (2) (s). apgar score: 7,90 (1) to 7.56 (2) (ns). presentation and mode of delivery: breech presentation is associated with higher incidence of neonatal deaths. i.v.h. (at the age of 8 weeks): no one of the survival infants had evidence of i.v.h. respiratory problems: intubation, at the admittance of the infants 32.3",,(1) to 95% (2) (s) use of surfactant: 70% (1) to 95% (2). bpd observed in 62% of the babies and only one was dependent on oxygen at home. antenatal betamethasone was given in 20% of the mothers. in conclusion: 1) a great improvement in the survival rate observed in these infants the last 20 years in our unit. 2) factors with positive effect are increasing gestational age and birth weight, the absence of i.v.h. and the use of surfactant. the breech presentation and the severe respiratory problems increase the incidence of death. animal experiments demonstrated, that brain temperature determines the amount of neuronal damage caused by hypoxia and that mild hypothermia may have a protective effect. until now there is no method described and evaluated to measure brain temperature in neonatal intensive care units. we non-invasively measured brain temperature analogues, nasopharyngeal (tnasoph) and zero-heat-flux temperature (zht) at the temple whereby under zero heat flux surface temperature represents deep head and thus brain temperature. the aim of our study was to investigate the practicability of the method, the relationship of the two brain temperature analogues to rectal temperature (trect) and their dependence on insulation, thermal environment, body activity and time course. we investigated 19 healthy preterms less then 2 weeks postnatal age (gestational age 315 +_ 2.1 wks; x + sd, weight 1653 +_ 370 g) in an incubator. tnasoph was measured by a thermistor within a feeding tube, advanced to the nasopharynx, zht temple by a thermistor and a heat flux transducers both covered by an insulating pad, and trect thermal environment was characterised by operant temperature (tair .0.4 + twall 0.6). body activity was video taped. measurements were performed during the following interventions: i/ insulation increased by turning the temple with sensors onto the mattress (15rain). ii) insulation increased by a cap (30 min), iii) 30 min after its removal, iiii) increased operant temperature by 1.6 + 0.5~ (60min). results: seven children with ea had a gasless abdomen, the endoscopic procedure excluded (6) or diagnosticated an upper pouch fistula (1). in patients who suspected "h" fistula (2) broncoscopy has strong advocated method to make diagnosis and established cervical approach. from july 1992 14 newborns with ea and lower pouch tef received a selective transtracheal incannulation. we were not able to proceed just in 1 case with congenital subglottie stenosis. in these patients we provided gastric drainage by radiopaque and flexible 3-4 french catheter. the knowledge of the precise anatomic position of tef consent to adjust the tip of the endotracheal tube in order to achieve best ventilation. the presence of the catheter through the fistula helps the surgeon to identify, it quickly. no complications were correlated to the procedure and no babies had early pneumonia. alimentary continuity was achieved in all patients (30 primary anastomosis, 2 resections of tef, 6 oesophagocoloplasty and 1 died with gastrooesofagostomy). the late mortality 7.7% (3) was only directly related to the severity of associated malformations. conclusion: the advantages of this technical approach are unquestionable for the anaesthesiologist and the surgeon. in our experienc e the procedure improves perioperative management of babies and appears to be safe. relation between cytokines, prethrombotic markers and endotelial injury markers in children with septic shock objectives: to establish the relationship between cytokines (tnf, il-1, il-6) prethrombotic markers (d.d., pcam) and endothelial injury markers (tm, uwf) in pediatric patients with sepsis and bacteriemia without shock, and patients with septic shock. design and methods: prospective study, 18 children (9 months-16 years) were admitted in our picu in 1994 with the following diagnosis: bacteriemia (4) sepsis (4) and septic shock (10) according to jacob's r f criteria. measurements: il-1, il-6, tnf, tm, vnf, d.d. pcam and routine laboratory data on admision, 12, 24, 48 hours and on discharge. the prism (pediatric risk of mortality score) was also recorded. results and conclusions: two patients in the septic shock group died. significant differences were found between non-shock and septic shock patients in relation to tm, dd, pcam, il-2, il-6 and tne high levels of tnf and il-6 are closely associated with the severity of septic shock with purpura in children. low levels of pcam on admission were associated with severe shock. who underwent open hea~nt surgery, hypervotaemia with or without oliguria was the most frequent reason to start pd (77%). in 10 patients pd lasted less then one week and there were no complications; in 3 patients it lasted 13 -29 days (one child had a peritonitis). instillation of dialysis fluid into the peritoneal cavity was associated with a significant increase in central venous pressure. there were no significant changes in cardiac output or arterial oxygeu saturation. in all patients pd dhnjnished fluid overload or improved the metabolic status. 5 patients (38%) survived the postoperative course and all had complete reintegration of renal function. conclusion: pd is a useful method to treat the fluid overload and acute renal failure in paediatric patients following open heart surgery with file effects of little importance on the cardiovascular fimction. obieetives: with the marketing of computerised systems for lung function testing in newborns, there has been an increasing interest in clinical approaches. percentile curves of pulmonary parameters permit an appropriate and clinically useful interpretation. however, the manual evaluation of the results using different curves is an impractical technique. therefoi'e a computer programme was developed. methods: the percentiles (5%, 10%, 50~ 90%, 95%) of the most important pulmonary parameters were determined non-parametrically in 6 weight-classes. for the calculation we have taken results of our own as well as other laboratories using a meta-analysis of reference studies. in all, individual data of 300-600 healthy newborns ageing between 1-28 days were collated. using these percentiles, for every parameter in relation to the body-weight the cumulative distribution was calculated approximately using piecewise linear and exponential functions. as shown in the figure the results of computing are represented numerically as well as graphically and can be included in the patient report. conelusions: clinic~d experiences with the programme have shown that representation of all measured parameters on standardised 100% scales allows an easy interpretation at first sight and improves the detection of pathologic patterns in the parameters. ")supported by bmft, fp "risikoneugeborene" prism (pediatric risk of mortality) score is a well known, already validated scoring system that quantifies severity of illness based on 14 routinely clinical and laboratory variables measuring physiological instability. once computed the score by summing up the weights corresponding to the most abnormal value recorded during the first 24 hours, the overall risk of mortality can be predicted by using the coefficients estimated by a logistic regression where prism score is the main independent variable. (pollack mm et al, -pediatric risk of mortality (prism) score. crit. care med. 1988; 16:1110 -1116 . to assess the applicability and validity of prism in the italian setting we launched out a prospective data collection in a sample of 33 pediatric icus. measures of calibration (goodness of fit statistics) and discrimination (receiver operating characteristics and area under the roc curve) are planned to be adopted in the cohort of patients recruited during 1 year period. as the validation study started on july 94, data collection is still on going and validation analyses will be carried out on july 95. up to now 23 centers recruited 1116 cases. at present, characteristics of the sample recruited are the following: most of the patients were male (62%); the mean age is 3 years with 30% of patiens having less than 30 days; more than half were medical cases (59%) admitted from emergency room or from hospital floor (51%); 52% cases were admitted with an organ failure while 48% to be intensively monitored. icu-mortality was l 1%. the paper will present final results of calibration and discrimination analyses that will be carried out in the whole sample and across subgroups known to differ in terms of clinical relevance and prognosis. if calibration and discrimination assessment will produce not satisfactoty findings, a customization of the current coefficients will be made allowing a formal comparision of previous and new parameters. jf riera-faneao, m wells, j lipman. baragwanath intensive care unit, university of the witwatarsrand, south africa. [background the prism score is designed to assess the likelihood of death in ipaediatdc icu patients, using only acute physiological disturbances, age and [operative status to predict mortality. there is no evaluation of chronic health status, [including malnutrition. this may significantly affect its ability to accurately predict outcome in a population where malnutdtion is common. aim to determine the influence of nutritional insufficiency, as indicated by a low weight-for-age on outcome prediction by prism. patients & methods we analysed prism, weight and demographic data co ected prospectively from 1528 consecutive paediatdc icu admissions over a 6 year pedod. a proportional weight (pwt) was calculated as a percentage from the 50th centile of the who weight-for-age growth charts. the pwt was compared for survivors and nonsurvivors, and mortality compared for pwt categodes 0nho wellcome classification). multivariate statistical techniques were used to identity associations with non-survival and to develop a modified logistic regression equation including a measure of i nutdtional status. receiver operating characteristic (roc) analysis was performed including and excluding patients with low pwt for the odginal and modified equations. results non-survivors had a lower weight than survivors (6.8kg and 8.3kg medians p = 0 63) a lower pwt (85% and 90% medians p = 0.6"0. the incidence of malnutdtion , in our icu population was 33%. the mortality of manoudshed patients was' significantly increased (p = 0.001), with a good correlation with the degree of malnutrition. the accuracy of prism was significantly improved when malnourished patients were excluded from the analysis (roc value increased from 0.72 to 0.79). ! logistic regression and discriminant analysis identified a significant association between prism, pwt and outcome; age and operative status were not significantly related to mortality. the use of a modified equation including the raw prism score, pwt category and age can significantly improve the discriminatory power (az dm/elopmental sample 0.82, az validation sample 0.81). the modified formula is: legit = -2.864 +0.134*prism score -0.006*age + 0.463*weight category, where the probability of mortality is exp(iog/t)/1 + exp(iogio. discussion although we can improve the prediction of mortality by a modified or recelibrated formula, this still does not compare with the reference prism population. the need for validation of the score itself, in the association with outcome of the acute physiological variables themselves, is thus apparent. we conclude that while the odginal prism formula can be improved significantly, a modification of the basic variables in this and other third wodd populations may be essential. a high incidence of malnutrition is an independent risk factor of mortality, and an important cause of the poor discriminatory performance of prism. in order to improve the accuracy of prism, nutritional status should be taken into account. objectives: to assess the value of inhaled no to differentiate between pulmonary vascular constriction or fixed anatomical obstruction. methods: we assessed the response to 40 ppm inhaled no in 12 patients(9 m, 3 f, median age 4.5 months, range 1day to 17years) with signs of increased pulmonary vascular resistance, there were 5 pre and 7 postoperative patients. patients were divided into responders(+) or non-responders(-). a positive response was defined as a 20% reduction in pulmonary arterial pressure and pulmonary vascular resistance(pvr) or in the presence of a left to right shunt, a fall in pvr accompanied by increasing pulmonary blood flow. left atrioventricular valve atresia + 12 mustard pat: pulmonary atresia vsd: ventricular septal defect asd: atrial septal defect pda: patent ductus arteriosus tapvc: total anomalous pulmonary venous connection the responders(7/12) were characterised by left to right shunts or pulmonary venous hypertension(4/7). patient#11 was weaned from ecmo with inhaled no. patient#2, without congenital heart disease, underwent a lung biopsy which confirmed reversible pulmonary vascular changes. patient#1 had a pulmonary hypertensive crisis which responded to no. all non-responders(5/12) had evidence of anatomic obstruction to pulmonary blood flow (#7,10,12)or a low pvr(#8) on subsequent cardiac catheterisation. in patient #3, lung biopsy confirmed severe obliterative vascular disease. conclusions: inhaled no appears to be an effective pulmonary vasodilator. a failed response may be evidence of either irreversible pulmonary vascular disease or a residual anatomical obstruction which may be surgically remediable in the postoperative cardiac patient. therefore, inhalation of no may be a useful diagnostic test to differentiate between fixed anatomical obstruction and reversible vasoconstriction. results: during these 18 years, the incidence of sdra was 1.3% of the total of admissions. the most common etiology was meningococcic septic shock. since 1989, there is a decrease of its incidence. (from 24% to 11%) and an increase of pneumonia and immtmodeficiencies. mean age of our patients was 2,7 years (61% males, 39% females), total mortality by sdra was 59% and there is an increase up to 72% since 1989 mean time of stay of the dead was 4,3 days and 12,4 days those who survived. although during the late years we offer in the picu a better attendance quality to the patients with sdra and the mean stay is longer, both for those who die and for those who survive, mortality of patients with sdra have increased. the incidence of sdra secondary to the septic shock of a meningococcic etiology have decreased. on the contrary, the sdra secondary to infections by opportunistic germs in patients with congenital inmmunodeficiencies or acquired immuodeficiencies have a tendency to increase. in our series, this change of aetiology is the responsible for the increase in mortality. hospital infantil unlversitario "virgen de1 roclo". sevilla. espalqa aims:to assess the incidence, etiology, clinical course, sequelae and mortality of the patients admitted to a paedfiatic intensive care unit with the diagnosis of severe traumatism. material and method: 60 cases of severe traumatism in children admitted to our icu in the period from january 1990 to june 1990 were reviewed. age of patient ranged from 4 months to 9 years, 65% were males. in our series, 53% of cases suffered traumatism due to a traffic collision and 38% had a fall from a considerable height. only in one case was traumatism due to violence to the child. we assessed the first assistance received in 76% of cases: where was it performed, interval of time since the accident, and steps taken. these data were also studied in relation to the latter evolution. results: 75% of our patients suffered cranioencephalic traumadsm (ct); in 53 % it was an isolated picture and in 22 % of cases was associated to other lesions. there was participation of thoracic and/or abdominal organs in 16 % of cases. 10% of cases presented important maxillofacial involvement. only one case presented serious cervical medullar lesion. mortality in our series was 3.3%. in 8.3% important sequelae remained. all of these patients presented tepas on admission equal or lower than 4. 16% of those with traumatises had slight sequelae. 71.6% of the total evolve towards healing. a polytraumatized child is a patient that benefits considerably of it admission in a paedriatic !cu. the rapidity in receiving first aid and its quality are essential to avoid sequelae and to make mortality decrease. after unilateral lungtransplantation 20% of the patients develop a lung failure with decrease of perfusion and increase of pulmonary blood pressure in the transplantated lung. the improvement of perfusion is an importent task in the postoperative period. case report: a 14 year old girl with idiopathic pulmonary fibrosis received a left sided single lung transplantation. during the early postoperative period occured a higtter demand of oxygen and an increasment of the pulmonary vascular resistence in the left lung. the pulmonary ventilation and perfusion scintigraphy indicated in comparison with the right lung a reduced perfusion of only 30% in spite of a ventilation of 70% of the transplanted lung. to improve the perfusion of the transplant we administrated per inhalation prostacyclin in a maximal dose of 20 ng/kg/min. the arterial blood pressure decreased but the perfusion continued nearly at the same level. during the following administration of 10 ppm no in the respiratory air we achieved a significant reduction of the respiration pressure f~m 40 to 32 nun h20 and of the pulmonary arterial pressure. the perfusion in the transplanted lung increased to 70ca/of the total pulmonary perfusion. after 3 days of administration with no we were able to withdraw the axtifical respiration without any following complications. conclusions: the perfusion of transplanted lungs is a major proble_r~ in the postoperative period. this case demonstrated the advantage of no towards the inhalativ application of prostacyclin. no showed a significant improvement of perfusion in the transplanted lung of a 14 year old girl. results: a total of 11 children with ards were treated with bovine surfactant (alveofact| 9 cases were evalable. the median age was 1.3 years (range 2 weeks to 4,9 years). in six cases ards was associated with pneumonia, in two cases with lung hemorrhage; in one case isolated ards followed hemihepatectomy. the first surfactant application was performed with a median latency of 22 clays (range 3-68 days) after first symptoms of ards witha median doseof 84 mg/ kg (range 42-133mg/kg). in 9 patients 28 doses of surfactant were applied. during the hour before therapy, the median pao2 / fio2 -ratio was 70-65. within 30 min. after application of exogenous surfactant the pao2 / fio2 -ratio increased to 90 with successive decrease over a period of 8 hours to 75. accordingly, an increase in pao2 and oxygen saturation and (less significant) a decrease in ventilation parameters could be observed. analysis of broncho-alveolar lavage before surfactant application in children receiving repeated doses revealed in most examined cases either clear surfactant deficiency or pathological function. 2 of 9 treated patients survived (3 of the 11, respectively). 13 of the 28 surfactant doses were applied in the 2 surviving patients.conclusions: the application of exogenous surfactant in children with ards caused a significant increase in oxygenation, which declined over a period of 8-12 hours. the effect often could repeatedly reproduced, in one case after 11 applications. the increase in oxygenation often allowed the reduction of fio2 and/or the inspiratory pressure. no side effects were observed after exogenous surfactant application.in many cases the application of surfactant wag too late after first symptoms of disease (median latency 22 days). ards mostly due to pneumonia seemed to respond to surfactant therapy less well or not at all. permanent junctional reciprocating tachycardia (pjrt) is the most common incesant supraventricular tachycardia (svt) in children. it is usually drug resistant and its onset in early life has been associated with dilated eardiomyopathy. we report our clinical experience with 3 patients detected antenatally and another diagnosed at 2 months of age. method.diagnosis: negative p waves were detected in leads ii,iii and f, p'r > rp" and there was not warm-up at tachycardia onset.clinical records, ekg,x-rays, echo and holter were reviewed. ep studies were undertaken only with therapeutic purposes. results. in a 10 year period 13 patients under 14 y of age fullfilled diagnostic criteria; 3 were detected prenatally (25-34 weeks) and one was diagnosed at age 2 mo. the 3 fetuses had intermitent svt during gestation. all 3 of them had pjrt in the first month of life at rates between 180 and 240 bpm. they were admitted to the icu but did not develop signs of heart failure. they were controlled with digoxine (d); d and quinidine; d and propafenone in 2 to 20 days. one was in sinus rhytm until age 4y; he then showed persistent pjrt over 70% of the day on repeated holters and underwent successful radiofrecuency catheter ablation (rfca).the other two patients showed initially a lowering of tachycardia rate followed by sinus rhytm for over 90% of the day (follow-up 2ran and 4 y). the 2 mo. old infant was admitted to the icu in severe cardiac failure. echocardiogram showed marked systolic dysfunction (shortening fraction 15%) treatment with digoxine, amiodarone and propafenone were unsuccessful despite lowering heart rate to 185; rfca was performed at 3 m. of age with restoration of sinus rhytm and rapid recovery of contractility. all patients were given atp at admission with transient (15 to 35 see) recovery of sinus rhytm. ff,s162 clinical course of pjrt is variable. atp is useful only as a diagnostic tool. initial treatment with digoxine + amiodarone or propafenone is adviced. rfca is a very useful therapeutic modality and can also be performed in young infants twelve patients (5%) died. these were 4 meningitis, 2 head injury, 2 sub-arachnoid bleeds, 1 status epileptieus, 1 leukaemie, 1 drowning, and 1 multiple trauma. calculated from the a 2 admission day p edialric risk of mortality score (prism), the probability of death (p) ranged from 0-100%. of the 12 deaths, i 1 were predicted by prism analysis except for the leukaemie patient (p i%) who died from haematological complications following chemotherapy. two children predicted to die (p 43% & 73%) survived. the median length of stay was 2 days (range 1-34 days). 98 patlents(50%) received ventilatn~ support and 10 patienta(4%) were transferred to specialist units (5 neurosciences, 3 liver, 1 cardiac, 1 bums). this data supports the view that many paediatric patients are being adequately treated in a dgh icu. meningitis and other neurological illness caused the majority of deaths and respiratory problems caused most admissions. most deaths (9 of 12) occurred within a few hours of admission. ectopic junctional tachycardia (ejt) is one of the most dangerous arrhythmias in the postoperative setting of congenital heart defects since it does not respond to antiarrhythmics or defibrilation. the object of this presentation is to report on two patients who presented f_jt in the early postoperative period and developed intense congestive heart failure which could be controlled after treatment with moderate topical hypothermia. two patients, 8m and 2y, diagnosed of atdoventficular septal defect and tetralogy of fallot developed intense heart failure in the early postoperative period. taehyeardia rate was 215 and 225 bpm. medical drug therapy included weaning from vasoactive drugs, iv digitalization and iv amiodarone treatment. there was not response. they were both surfaced cooled by placing plastic bags filled with cold water over the patient's chest and abdomen. temperature was monitored to obtain a central temperature of 34~ there was a gradual decrease in heart rate in the following hours (130-150bpm) paralel to the degree of surface cooling and clinical course estabilized.both recovered normal sinus rhytm in 48 to 72 hours. there were not significant arrhytmias after the procedure and postop, was uneventful. conclusions. moderate hypothermia is a very useful manuever for the treatment of drug resistant ejt. since it lacks side effects of other antiarrthymics we beleave it should be the treatment of choice for the treatment of ejt in the postoperative patient. present understanding of the pathogenesis of sepsis, based on the theory of systemic inflammatory reaction, has risen new interest in the more invasive methods of treatment, like plasmapheresis, leucapheresis and exchange transfusion (et). obiectives: evaluate the effect of et in the treatment of neonatal sepsis. material and methods: from september 1 to december 31, 1994 a prospective study was carried out, where the severest cases of bacteriologically proven neonatal sepsis (n=9) were treated with et. in total 15 newborns were treated for culture positive sepsis in the intensive care unit during this study period. diagnosis of sepsis was based on the clinical criteria of suspected neonatal sepsis, used by mc harris et al., laboratory data and positive blood culture. newborns with severe congenital malformations were excluded. et was carried out with fresh (less than 24 hours old) adsol-conserved erythrocytes, from which buffy coat had been removed, and same donors plasma, using a slow continuous two-site technique. the mean volume of et was 164.3 ml/kg. the effect of et was assessed as a change in the score for acute neonatal physiology (snap), general treatment results were compared with a historical control group of 26 newborns, treated for culture-positive sepsis in the same icu during the first eight months in 1994. students ttest and chi-square test were used in statistical analysis of the data. results: with the use of el a significant decrease in mortality was achieved: 1 death of 15 cases during the study period, compared to 9 deaths among the 26 controls (p<0.05). no baby, receiving et, died. the incidence of severe complications did not differ in the two groups. the snap-score showed quick improvement by the first post-transfusion day (p<o.05) and the effect persisted during the five following days. conclusions: we conclude, that the use of et in the treatment of critical cases may help to lower the mortality of neonatal sepsis. it provides a quick improvement in the clinical condition of septic neonates. the granulocyte-colony stimulating factor (g-csf) is largely employed in granulocytopenic patients. experimental studies have' demonstrated the effectiveness and safety of the therapeutic use, nevertheless the human employ is reported almost exclusively in neoplastic patients submitted to chemotherapy and in patients with fungal or pseudomona~ spp infections. the authors report the data concerning three non granulocytopenic children: in the first, o girl 7 years old, affected by pseudomomm aerug/n~a pyeionephritis, the antibiotic therapy was not tolerated in consequence of renal and hepatic failure. the second patient, an infant 6 months old, has been treated with salbactam-ampiciilin, ceftriaxone and chioramphenicol because of haemepldlw iafluem~ meningitis; a late neurological aggravation required the antibiotic therapy recommencement the last, a 17 years old insulin dependent diabetic boy, was affected by cared/do spp systemic infection; the antifungnl therapy w~ not practicable in consequence of renal and hepatic involvement in all the g-csf was administred subcutaneously at the'dose of $ u for 7 days. a significant increase of granulocytes was ever noted already after the second administration until 24 hours after the last dose. clinical signs, fever and biochemical values are promptly influenced positively and all patients are restored to health. no side affects were noted. the granulocyte colony stimulating factor, at indicated doses, can prove of great help in critical patients, in who the antibiotic or antifangal therapies can result toxye or cannot be tolerated. the ~reptococcas p~, among the gram pmltlve~ represents the main causative organism of bacterial meningitis in children, with the majority of deaths occurring within hours of admission, the third generation cepohlosporim were indicated as antibiotic of first choice, and some literature reports have demonstrated no significant dlffereuces in dinical course of patients treated with ce~rlaxoae and of those treated with ampieibin and chlornmphenicol. the authors report the ease of on infant, male, 18 months old, submitted to a neurosurgical interventiol after 2 days the infant became febrile and lethargic and a bacterial meningitis by ~trepmcecctz ~ was diagnosed, in spite of he was given the eoftriaxone therapy.. an amelioration was found only when a systemic treatment with vaucomyein was added. after 2 days an intrathecal administration of vancomyein 40 m~day was started: the norbmlization of the spinal fluid profile was uoted two days later and the infant receve~d progressively. the follow-up after eight months doem't evidentiate motosensorini sequelae-the individuation' of ~cscc~ pmmmsm/ae strains heta-loctam antibiotics resistant requires alternative therapeutic regimens: the k~terity quead vitam and quoad valetudinem of these infections justifies an intrathecal therapy, especially failing o prompt answer to the systemic treatment objectives: to evaluate the incidence of the burnout (bo) of the staff in a picu in a university hospital. the bo is a syndrome charactedzed by many factors like emotional stress, lack of personality and reduced work ability. this syndrome represents a kind of abnormal response in the working habitat, above all, where psychological and personal characters are mostly involved. the symptoms of bo are represented by demoralisation, demotivation, incapability, boredom, isolation up to organic disturbances, like migraine, insomnia, dizziness and gastrointestinal disturbances. the major incidence of this syndrome was found in the hospital staff, especially among the nurses, working in an atmospher of high emotional dsk, just like in an intensive care unit. methods: from january '95 till april '95, all the staff of the picu of the a. gemelli hospital of rome, underwent a test to evaluate the bo. the test used was taken from the book "bumout: from tedium to personal growth", by pines and aronson. twenty seven people were studied (19 females, 8 males)i out of which 7 doctors, 7 residents and 13 nurses. it was considered sign!ficant as mild bo, a score between 3-4 and as severe, the score of bo >. 4. results: 10 subjects (37%) resulted positive for bo, out of which 8 were females (80%) and 2 were males (20%). the subjects with mild bo were 7/10:1 was a doctor, 3 residents and 3 nurses. the subjects with severe bo were 3/10, out of which 1 resident and 2 nurses. conclusion: the results obtained show that bo is a condition well represented in the staff of our picu. the category most at dsk seem to be the nurses (5 subjects), as well as residents (4 subjects), as in literature, which shows a major incidence of the syndrome in younger subjects and having a limited partecipation of functional decision. the results obtained obliged us to start a programme of serial controls so that the subjects most exposed can have a necessary psychological support to react adequately to this condition. the term systemic inflammatory response syndrome (sirs) was adopted by the consensus conference to denote a type of systemic response to severe infection or otherinsults in critically ill patients. when sirs occurs from infection it is called sepsis. sepsis occurs more frequently in persons with perexisting illness or severe trauma. there has been tremendous advances in prophylaxis, diagnosis, and treatment of sepsis. a comprehensive model of the disease progression from sirs to mods should be developed giving priority to severity of illness scoring system and other predictive methods. some recommendations for future clinical trials include: trials should not start with humans. before proceeding to human trials, animal studies should indicate an acceptable risk/benefit ratio. appropriate patient populations must be defined and treatment protocols should be standardized. full and rapid reporting of all results should be mandatory and a central repository of published and unpublished study results could be helpful. accrual at each center should be of sufficient size, and should include the number of patients accrued, mortality rates, and patient characteristics. pivotal trial should be preceded by sufficient pilot or phase ii studies. correct drug dosage and usage should be delineated in pilot studies. large, multicenter, trials should be used to enhance the unversality of trial results. analyses should be planned a priori. definitions for the target population should be explicit, reproducible, and include illness severity scores. outcomes should be relevant reproducible and include both measures of benefit and harm. mods and its reversal should be considered as an endpoint. quality of life should also be considered as an endpoint. the estimators of overall treatment effects should be controlled for base-line prognostic factors and subgroup anaiysis should only be used for hypothesis generation and not to modify the conclusoin of the trial. economic analysis should be included as part of clinical design. evaluatin of source control should be a critical component of any study. standardized clinical mediator assays should be pursued. placebo patients in clinical trials should be studied for a better understanding of the pathogenesis and epidemiology of sirs, evidence based medicine should be used to evaluate the validity of clinical. introduction: use of inhaled nitric oxide (no) as a modulator for optimizing ventilation-perfusion or lowering pulmonary artery pressure is becoming increasingly common. no is a free radical but little toxicological research has been published. clearance of nebulized 99mtc-dtpa is known to be, a sensitive indicator for early function impaimaent of the alveolocapillary barrier. we investigated whether exposure to no increased clearance of 9~tc-dtpa from the lung. methods: three groups of 5 white sealand rabbits (bw 3.5 kg) were anesthetized, tracheotomized and paralyzed. 2 groups were ventilated for six hours at pressure regulated volume control, set to deliver 10 ml/kg with a frequency of 30/rain, i/e ratio = 1:2 and peep = 3 cm hzo using a modified servo 300 ventilator (siemens, solna, sweden) with computerized no delivery system. gas mixture per group was either 0/25 or 20/25 [no (ppm) / fioz]. after six hours of ventilation in these 2 groups and immediately after anesthesia in group 3 (control), 99~tc-dtpa was nebulized into the inspiratory line of the breathing circuit and administered as a fine aerosol. gamma counting was measured for 20 minutes, monoexponential curves were fitted to the data and the clearance half-time (t 89 was calculated. the t~/2 mean • sd of the different groups were: t~a (mean 4-sd) h"e,i witl~ arf 1:14 di.ff:erent kinds, aged .q-ore 2 mon't.hes to [4 gears o11:1 (bodi weight .~rom 4.,5 to 45 kg), is presen .... "ed ( i,,~u::trl:e i:ibstraclive d:lse~se...14~ 2.ards'-8; :~,;,,arf o~ ::entral genes:i s .-3,0,~ :inc lud ing men ingeenceph 1 it :is-3~ reye ' s ~yrtdro~e-..#~,bri~:ln pes~.re~nimatior~ disease.."5). int:lrl~]. pa-. "iiulle'i,~s ariel regymes o+ l;mv,l;i"t"v were cle'l'.ermllled by ba'i~ier was. about 4. tuber,, dopamin tiara-:. t.io; was ~.".,,'.r:~r~led. cmv,cppv d~.!"~tion raniled -~rom f to 25 dayns.,~ <6.-:in 351, 6"t2-irl lo;and>12 davs'-in 6'l~atierr~{s i'i"ai3s:ltiol~ o4; patterers to imv, simv modee was per.r:)rmed, ~herl pif:' decrease.d to 16-17 ml~ar, fi02 ~ecreased to 0,4. lind less with 5a02=901/,,. i:lesq.lts:{ in pat:i.ents e{ group :l, who were tre,~d.ed w&th 2f'f'v, teoph :i. :1. l:i.r~ (is-24.mg/kg/day), g lucecdr t icostei~oids (2 .... :~;mg/kg/day), when r exceeded in 2,2-.];,4 times normal va i tea the e aqes/,'!:l"oln 2~j,, ite :i.~;::.!;, 8 ~ml"lrj), it was possible 't'(' ce 'e~ e aad]t:..~rom !1.20 2'.' 26i', 8 to !..';51,0-106, 1 mml-lg in ~}.. :~.[~ houi,!; ~d'l(:i to ru:}l",g'd!~l:i.2e i::h,:~e,'~c['el';i.stil obieetives : this chapter will describe what is knovca of the psychlogical responses of infant and children to hospiuiisation and attendant procedures. the factors which may modify these responses will he discussed and important considemtiorts will be outlined for optimal anaesthetic management and postoperative period of infants and children which will minimised the rise of emotional upset. methods : in this paper the autors will discttssed the probl 9 of: 1. health children (asa i, ii) facing single uncomplicated surgical elective procedures 2. various abnormal situations including neurotic children, children facing repeted operations, chronically ill, buaaes and tsaumatically impired ones 3. unfortunate young patient facing and often expoclting fatal outcome from le "ul'ukaemia, tumors, cystic fibroses or otheq" disease. : management of each child must vary greatly, ifi general the phases of emotional conditioning include home and preadmissiun preparation, admitiun preoperated and operative care and postoperative period. the authors would be happy if the child passes all stages without any trauma which could be prolonged in the future life. introduction ino is used to selectively reduce pulmonary vascular resistan(~e. we applied ino in the postoperative intensive care of patients with pulmonary hypertension and the risk of right ventricular failure after surgical correction of a congenital cardiac defect. methods 2-50 ppm no were added to the ventilatory gas mixture using a specially designed equipment (messer-griesheim, germany/austria). indications for application included pulmonary artery pressure >50% systemic pressure, critically depressed right, ventricular function or an oxygenation index >10. assessment of n oefficiacy consisted of on-off-on measurements according to the clinical stability of the patient including hemodynamic parameters, pulmonary gas exchange, continuous monitoring of ventitatory function and transesophageal echocardiography of the right heart. results in 29 situations (19 patients, age 3 days-71,1 years), ino was applied 0-628 h postoperatively. oxygenation was improved in 13 situations from 114_+51 to 171+53 mmhg pc2; pulmonary pressure was reduced in 17 situations from 70-*22% to 34_+17% of systemic pressure. in 7 situations, no reduction of pulmonary pressure was present, but measurement of cardiac output or echocardiographic analysis indicated an improvement of right ventricular function (right ventricular stroke volume +39-*12%, cardiac output +20-*11%). in 8 situations (immediately postoperativ with suprasystemic pulmonary artery pressures [n=4], multi-organ-failure [n=4]), no response to ino could be determined. conclusions for a special group of patients, the selective reduction of pulmonary vascular resistance by ino has become an important part of postoperative therapy. using this selective afterload reduction, postoperatively depressed right ventricular function can be improved. this effect of ino seems to be the most important one in the postoperative period. thus, ino appears justified to be appfleo when impaired right ventdcular function could be improved even when pulmonary artery pressure is not raised or remains unchanged. obiectives : premature infant are exposed to danger of apaea due to anaesthesia during their tirst months of life. it is yet unknown whether prematurity is corelated to any other kind of reslgratory disorder due to anaesthesia within the tirst year of life. methods : we theretbre researched retrospectively for respiratory disorders in all infants under 12 months of life belonging to asa group 1. they all had been anaesthetised in 1985.95 in our clinic for the following surgical reasons: ingvinal haemia, umbilical haemia, hydrocelae testis and phymosis. results : in 2350 cases we tbund: lafingospasm during induction in anaesthesia (0,5%), bronchospasm during induction in anaesthesia (0,22%), impaired intubation (0,1~ postanaesthetic laringospasm (0,1%), supposed aspiration (0,04%),postanaesthetic inspiratory stridor (0,05%), postinductional inngoedema (0,03%), death after 4 months in consequative of infection pneumonie (0,12%), none of these disorders was correlated the prematurity, 3 infants suffered of post anaesthetic apnea, 2 of them had premature medical history. concludions : prematurity does not enhance the risk of respiratory disorders due to anaesthesia within the first year of life, except the danger of postanaesthetic almea needs spetial cosideration. it could be demonstrated that aepgi 2 lowers pulmonary vascular resistance and indirectly improves cardiac function. this effect seemed to be selective, and was comparable to ino in the doses we have examined. therefore, aepgi 2 could represent a clinically useful alternate to inc. however, further research is necessary to work up the benefits of either therapeutic strategy. objectives: heat and moisture exchange filtem (hme) are used as artificial noses for intubated patients to prevent tracheo-bronchial or pulmonary damage resulting from dry and cold inspired gases. furthermore they are used for the prevention of bacterial contamination of the anesthetic apparatus by the patient's exspired air. so they are considered as a time-and money-saving device in anesthesia. filters are mounted directly on the tracheal tube, where they collect a large fraction of the heat and moisture of the exspired air, adding this to the subsequent inspired breath. the effective performance depends on the water-and bacteria-retention capacity of the filter. this study evaluates the efficiency of four different filters under clinical conditions. methods: four different types of filters ( dar hygrobac, gibeck humidvent, medisize hygrevent and pall bb 100 ) were investigated dudng mechanical ventilation over a pedod of 24 hours. 20 minipigs with hemorrhagic shock were intubated and ventilated for 5 days in an animal intensive care unit (icu). after 24 hours of mechanical ventilation the filter was randomly replaced maintaining the individual ventilatory conditions. the weight of the filter was determined before use and after removal after 24 hours. the airway pressure was monitored online to record changes during use. tracheal secretions and both sides of the filter were microbiolologically tested to see whether bacteria of the animal's respiratory system could be found on the patient's side of the filter or if they even would have penetrated the barrier. results and discussion: over a pedod of 24 hours 3 of 4 types of filters showed an increase in weight of 10 + 6% and airway pressure. bactedal celonisation 0ccured in nearly all fillers (93 of 100) on the patient's side, whereas only three of four types of filters showed identical bacterial colonisation on both sides. the only filter that did not show bacterial penetration, increase in weight or airway pressure was the pall-hme, a condensation humidifier without hygroscopic salts for moisture retention. with respect to our data one should use a condensation humidifier if airway conditions should remain stable dudng mechanical ventilation and desinfection of the anesthetic apparatus should be avoided after each patient. aim: to assess the clinical uses of, and experiences with, the hayek oscillator. this is a non-invasive device capable ef delivering not only continuous negative pressure (cnp) but also external oscillatory ventilation around a negative baseline (eov-nb) using an external cuirass. this type of ventilation avoids the need for intubation and intermittent positive pressure ventilation (ippv) and facilitates weaning in ventilator dependent patients. patients and methods: 21 patients in respiratory failure, age range 3 weeks to 15 years in a total of 29 patient episodes were treated using either cnp or eov-nb mode. duration of treatment varied from 4 hours to 8 days. indications for use ef the device were: 1) to facilitate weaning from ippv 2) prevent reintubation of patients following unsuccessful extubation, and 3) avoid intubation and ippv altogether using the hayek oscillator as the on[y means of respiratory support. results: there was an increase in pao2:fio2 ratio after cnp and eov-nb (p <0.0001, and p=0.01 respectively, wilcoxon signed rank test). patients who were in respiratory failure with hypercapnia showed a statistically significant reduction in paco2 both with eov-nb and cnp (p=0.02 and p=0.01 respectively) but the magnitude of change was individually greater in the patients who were treated with eov-nb. all patients, however, showed a fall in respiratory rate (p<0.0001) after the application of the cuirass in cnp mode. there was no physiological deterioration related to the application of external extrathoracic negative pressure in either cnp or eov-nb modes. conclusion: the improvement in pao2:fio2, the fall in paco2 and respiratory rate were indicators of an improvement in ventilation. the proposed mechanisms include improvement in frc, recruitment of additional alveolar units, and improvement in secretion clearance resulting in reduction in the work of breathing. meek to ~ month of the lifo,the bemodyuanicfacls were defined uitb the help of tetropolar reography method!. the excretion of !he catbocholauines fcfi] mith the urine gas detertend by taylor ll,laoorsy ~ iacg/dayl. hsaltl in the hypercuagulation stage of bic we deflorteeed the acliuutiun of the tbrubio and plasiin syaet~ mitb the increase of the inhihitnrs, in this case we registered in full uahe dot this process coabined uitb the dayl~ excreliou with lho urine epinopbr ne e], nor~pinopbr no tel and dophanine io], lbat shod the inlensificatiou of the s~nthosis prnoe-s~es and the release of ea in blood fron hissue deport the actffat on of the svnpathadrenui systen ]sfisl assisted to furl the b?perd~nanical rosins of the eircuidion and increase the ,icrocirculatinn, the klinicai sings of the insufissieutly of the circulalion have not defined,that has been associated the conpensatury character uf the ehan~es of ~ and heludy~enic status, t~e uun~u|p-lion ceugulupatby bus been donoustraled in the hypocougulatien stage ~bat man xauifosted b 7 the exhaust of lhe confulalion nod oessel-platel heuostasis, the consuxptton of cnnpononts tbronbln ,plnstin, kallek~eiu-kinln s~slots and the forniration eat in fell canoe clot uas accoqaued bs docrea,e of fl,nfl,o, the products of the xotabolisx of c~ and the activation of xonoaninoxydasu. the decrease of the extoll'on g and the exhaust deport co indicahd about t!e ]ou fund/anal reserve of ~fl~. it was one of the lain reason of ~bo heiod~uanic disbroed iheat insnfissient]~] and the uicrncireulaflion lintestinal codeme with the low effectife periferal flow] and nul[iplay organ failure,the distrued deport of sos mitb throubocytupenin no; be one of the nechanisn the dislrood of uessej-plalol heioshasis, the correlation bolueeo changes of boiostosis c~ and circulation ore reguired aduinistration nedidns, thai reslore the love s of c~ in the blood, prevent uulliplay organ failure and hetorrnge in children with sepsis, ~b~ectives: multi-measured correlative analysis of the most number of non-invasive indices of the cardiorespiratory system function was made to determine the structure of their interrelation and the ways of their adequate and effective correction. hethods: spiremetry, capno~raphy, oxygenography, indirect fick method at recurrent respiration, plethysmography, integral rheography -in all 52 indices were used. the received data were processed on a computer by a standard package of statistical bmdp programs. results: 70 women with ~h-gestosis (i group) and 48 somatically healthy pregnant women (ii group) were studied. cluster analysis has shown that the rate of the mean correlation connection between ventilation indices was 94% in the ist group and 90% in the iind group; gaseous metabolism -91% and 86%, respectively; central hemodynamics was 87~ in both groups. conclusion: cluster interpretation allowed to suggest that an increase of the rate of the mean correlation connection between the indices was characteristic of effective adaptation as the system was multi-component and well-regulated. on the contrary, the increase of the rate of strong correlation connection between the indices reveals the rigidity of the system and the tensity of adaptation mschaniams, i.e. the proximity to decompensation. it follows from this that in cases of eph-gestgsis, the reliability of regulating ventilation and gaseous metabolism decreases. seve/e hypoxemia in non intubated patients represents a major contraindicafion to fiberoptic bronehoscopy (fob) and bronehoalveolar levage (bal), but these procedures are often required for a correct diagnosis of the causative agent of pneumonia. aim of this investigation was to veaify the safety and efficacy of bronehoseopic procedures during pressure support ventilation administered through facial mask (fm-psv). five intensive care patients, all immunoeompromised, (3 males and 2 females; mean age 41.6• were enrolled in the study. all patients presented criteria for pneumonia with pao2/fio2 ratio ~ 100 and were responders to fm-psv. fob and bal were performed afte~ topical anesthesia with fm-psv ( ps = 16 em h20; peep = 5 emh20; trigger = -lemh20) continuously admires" tered ( 10' before fob fio2 = .7; during fob, fio2 =1 and for 90' alter fob, fio2 = 0.7). pao2/fio2 ratio as well as 02 saturation (sat) did not show signifteative changes during the procodure (fig.l) . no complication was observed and hemodynamic conditions were stable for all patients. cmv, pnenmoeystiis (2), legionella and mycobaetermm tuberculosis were identified from bal allowmg a prompt and targeted therapy. we concluded that mask psv can represent an excellea~ technique to pexform fob and bal in severely hypoxemic patients without deterioration of gas exchanges and avoiding endotraoheal intubation. intensive care unit, hospital general of albacete, albacet~ spain. objective: to analyze the current incidence and epidemiology of total parenteral nutrition (tpn) among critically ill patients placed on mechanical ventilation. design: prospective observational study. setting: medical intensive care unit in a tertiary hospital. patients: a total of 113 consecutive l'ritically ill patients with non-coronary related disease needing mechanical ventilation admitted in our icu during a 12 months period. measurements: data of sex, age, diagnosis, and outcome were recorded. severity of illness and therapeutic effort in the first 24 hours were measured using acute physiology score and chronic health evaluation (apache ii) and therapeutic intervention scoring system (ties). r~ults: 113 mechanically ventilated patients, 76 male and 37 female, were studied. only ten patients needed tpn and their main diagnoses were: five cases of multiple organ failure secondary to pneumonia (2), ards (2) and septic shock (1); two eases of acute panereatitis; and one mesenteric throngmsis, one status epilepticas, and one ,prolonged cholinergic crisis b~ suicidal organophnsphate insecticide subcutaneous injection. no statistically significant differences between both tpn and non-tpn groups were found: objectives: evaluate the efficacy of prone position in ards and determine its importance in the therapeutic algorithm. methods: 43 consecutive patients with severe ards (murray-score > 2,5; pao2/ fit 2 < 160 mmhg; 29 male, 14 female, mean age 62 years) were conventionally ventilated (pcv, peep 6-16 mbar, i:e=i:i, ppeak < 30 mbar). if after 24 hours pulmonary function did not improve patients were placed in prone position. change from prone to supine position was done every 12 hours. beside ultimate survival, parameters investigated were aado2, pao2/fio2, and venous admixture (qs/qt). results: during the first 12 hours in prone position 39 of 43 patients showed a significant decrease in qs/qt (25.3% vs. 17.8%) and aado 2 (235 vs. 187 mmhg), and an increase in pao2/fio2 (151 vs. 201 mmttg). changes were most pronounced in patients with high qs/qt, and in patients with an onset of ards less than 48 hours before first application of prone position. after an average of 6 position changes (2 to 16) 28 of 43 patients could be weaned from the ventilator. 22 patient could leave tile hospital. i11 the later course letality was primarily determined by additional organ failures and by the severity of the underlying disease. negative side effects were minor, including slight cardio-vascular depression and increase in p~co2, and never posed a limitation to continuation of prone position. especially in patients with septic shock skin lesions in exposed areas could not always be prevented, prone position could easily be combined with all ventilation modes and with all intensive care interventions. also immediately after major surgery and in patients with open packing prone position was possible. conclusions: in this investigation prone position proved to be an efficient and safe method in the treatment of severe ards. patients with a pronounced ventilation/ perfusion mismatch and patients in the early stages of ards appear to profit most from prone position. though the immediate effect on oxygenation is striking, still more the 40% of all patients die from multi organ failure and underlying diseases. a proposed therapeutic algorithm for ards is as follows: if under conservative ventilation (pcv, peep < 20 mbar, ppeak < 30 mbar) pulmonary function does not improve within 12 -24 hours prone position should be applied. when after 2 -3 position changes no lasting effect can be achieved further ventilation modes (e.g. pc-irv, aprv, no, etc.) should be used in addition to prone position. standard intensive care principles, such as fluid restriction and optimization of circulation, apply also to patients in prone position. objectives: nitric oxide reacts with superoxide to form peroxynitrite, an extremely reactive and toxic species. we quantified the presence nitrotyrosine, the stable product of the interaction ' of peroxynitrite with tyrosine residues in the lungs of pediatric patients that died with respiratory distress syndrome (rds). methods: paraffin embedded lung sections, obtained at autopsy, were incubated with a polyclonal antibody raised against nitretyrosine, followed by a secondary fluorescent antibody. alveolar structure-associated fluorescence was quantified using existing methods. results: tissue sections from patients who died with rds exhibited significant specific immunostaining which was uniformly distributed across the blood-gas barrier. in contrast only background levels of fluorescence were seen in the lungs of patients who died from non-pulmonary causes. intense staining was also seen in the lungs of rats that breathed 100% 02 for 60 h, a condition known to result in rds-type illness; no immunostaining was observed in air-breathing rats. conclusions: significant levels of peroxynitrite may be formed in the lungs of patients with acute lung injury. peroxynitrite may be contributing to the pathology of rds by damaging key components of the alveolar epithelium including the pulmonary surfactant system. mechanical ventilation time was prolonged 16,g • 10 days in patients with ardsvs 1,7 _+ l,4 days in control . mean staylcuwas lg _+ 10,g days in the ards group vs 4,9 • 2,7 days in control group postoperative mortality rate was 53% in ards patients vs 5,7% in those without respiratory failure. 1-ards incidence in liver transplantation is low (11,2% in our sene) but it causes high mortality (53%) page, gas ventilation of the perfluorocarbon-f'dled lung, supports gas exchange and circulation in small animals (<15kg) with lung disease. we hypothesized that large animals could be supported by page without adverse effects on bemodynamics. we first elucidated the determinants of gas exchange in normal sheep, and applied them to a model of adult respkatory distress syndrome (ards). methods: using the ventilator settings determined to be optimal in our pilot study (fio2 of 0.6, peep of 5 cm h20, imv of 6 bpm, it of 50%, and tv of 16 ml/kg), sheep weighing 58.9 ~ 8.3) kg had lung injury induced by instilling 2 ml/kg of 0.05n hc1 into the trachea. ten minutes after injury, sheep with pao2<100 ton" were randomized to continue gas ventilation (control, n=9) or to institute page (n=9). page was instituted by instilling 1.6 l of unoxygenated pefflubron into the trachea and resuming gas ventilation at the previous settings. abg's were drawn at baseline, 10 minutes after injury, 30 minutes after injury, and then every 30 minutes for 4 hours. objectives: inhaled nitric oxide (no) can improve oxygenation and decrease mean pulmonary artery pressure (papm) in hypoxemic patients with ards. in severe hypoxemic copd patients, it is not known whether inhaled no can exert a similar effect on hemodynamics and gas exchange. therefore, we investigated die response of inhaled no in hypoxemic copd patients and the results compared with those obtained in a group of ards patients. methods: ten copd patients (age 71_+2y;fev~ 0.98_+0.12l) and 11 ards patients (age 57_+5; lis 2.8_+0.1) mechanically ventilated were studied. hemodynamic parameters were measured using a swan ganz catheter. arterial and mixed venous blood gas determinations, sao2, svo2, hb and methb were measured (abl 500,osm3). mean intratracheal concentrations of no and no2 were continuously monitored using a chemiluminescence analyzer (nox 2000) . during the study the ventilatory pattern and fioz were kept constant. the protocol was for ards group: basalt, no loppm, basal~; copd group: basalz, no lo ppm, no 20 ppm, no 30 ppm and basal2 . after a steady state of 20 rain hemodynamic and gas exchange measurements were performed. a positive noresponse was defined as a 20% increment in pao 2. results: papm was similar in both groups and decreased significantly after no (ards, basal 33.6_+9.7 mmhg, no 29.7 +6.7 mmhg, p <0.01) (copd, basal 27.8_+6.3 mmhg, no-10 24.4_+5.3 nrmhg, p<0.01). all other hemodynamic variables remained unchanged after no. basal oxygenation was higher in copd group (paojfio 2 189_+53 mmhg) vs ards group (paojfio 2 100_+40 mmhg)(p<0.01). after no-10, pao2 increased (69_+20 mmhg to 97_+40 mmhg, p<0.01) and qs/qt decreased (37+11% to 31_+10%, p<0.01) only in ards group. in both groups, significant correlations between basal papm and inhaled no-induced decrease in papm were found. inhaled no-induced increase in pao2/fio2 was not correlated with basal paoflfio2. no responders were 8/11 (73 %) in ards group and 2/10 (20%) in copd group (p<0.05). conclusions. in hypoxemic ards and copd patients, inhaled no decreased mean pulmonary artery pressure. however, oxygenation only ameliorated in ards group because die number of responders to inhaled no were higher in ards group and this effect seems not to be related to the basal hypoxemia. these results might be explained by the v/q abnormalities present in copd patients. grant fis 95/1390. objectives: it has been recently reported that expired con slope as a function of time is modulated by total respiratory system resistance (rrs) in critically ill patients (chest 1994; 105:219-223) . in this study, we analyze the relative contribution of disease (dis), endotracheal tube resistance (rtube), airway resistance (rmin), additional resistance (~rrs), autopeep (peepi) and dylmmic/static elastance (ed/es) to the co2 elimination in different clinical conditions. methods: we have studied 37 adult patients (8 controls, 11 acute respiratory failure, 9 severe ards and 9 copd) mechalfically ventilated (servo 300 and 900c, siemens) without peep. we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. objectives: alveolar ejection volume (van) can be defined as the fraction of tidal volume (vt) with minimal dead space (vd) contamination. according to the classical paradigm: limvd_~ [vco2/vt] =facoz, vco2 vs vt relationship tends asyntotically to a constant slope when approaches end-tidal volume. we have defined van as the volume that defines this relationship until a limit of 5% variation. methods: six subjects with normal respiratory mechanics were studied during anesthesia for minor surgery. two subjects, otherwise normals but having high values of total resistance and dynamic compliance, were also studied. capnograms were recorded in steady-state at 3 levels of vt (0.3, 0.5 and 0.8 l) and four levels of peep (0, 5, 10 and 15 cmh20 objectives: patients with ards presented lung abnormalities which originate an increase in airway resistance (rmin), in additional resistance (~rrs) and in static elastance (ers). application of peep further increases ~rrs. capnographic indexes reflect lung ventilation]per fusion inhomogeneities. in these conditions, the effects of peep on lung mechanics could be better understood by simultaneous measurement of capnographic indexes. methods: we studied 3 groups of subjects. n: 8 normal subjects scheduled for minor surgery; arf: 9 critically ill patients with mild acute respiratory failure; ards: 8 patients with early ards (< 72 h). we recorded tracheal pressure, airflow and capnograms. signals were analogic to digital converted for posterior data analysis. respiratory system mechanics was assessed by constant end-inspiratory and end-expiratory occlusions technique. at equal tidal volmne (0.5l) a peep level of 0,5,10 and 15 cmh20 was applied in all patients. we calculated ers (cmh20/l), rmin, c~rrs (cmh20/l/s) and autopeep. capnographic indexes were alveolar ejection volume (vae)/vt ratio and expired co2 slope beyond vae (sipco2 in contrast to synthetic surfactant natural suffactants (alveofact|174 are able to inhibit pmn-activation. after incubation of activated neutrophils with surfactant, l-selectin expression is decreased. these effects depends on which preparation is used. we conclude, that natural surfactant (aveofact| can perhaps influence early recruitment (,,rolling") of pmn in patients with respiratory failure like ards. with ards hormann cb, baum m, putensen c, knapp r, lingnau w, putz g . clinic for anesthesia and general lntensiv care medicine, university of lnnsbruck, anichstrabe 35, 6020 innsbruck objectives: in thoracic ct scans of patients with severe ards atelectasis and pleural effusion can be found in the dependent lung regions. by rotating these patients from left lateral position to right lateral position a redistribution of the ct densities, a recruitment of atelectasis and therefore an improvement of gasexchange is possible within a few days (1, 2). the objective of this study was to find out the mechanism of alveolar recruitment during lateral positioning by ct scanning in left and right lateral position. methodes: after approvel by the local institutional reviewboard we investigated 7 ventilated patients with severe ards (entry criterias: murray score > 2,5) in the ct scann of the university hospital. after a stabilisation period of 30 minutes in supine position a thoracic ct scan slice 1 cm above diaphragm was taken. then two different positions of the patients were studied in a randomized order: a) 60 degree of left lateral position, b) 60 degree of right lateral position. each lateral position was held for 20 minutes. at the end of each of these periods a thoracic ct scan slice 1 cm above diaphragm was taken. quantitative analysis of ct scan data was based on the frequency distribution of the ct numbers. to quantify the alveolar recruitment during lateral positioning by means of ct scan we defined 3 compartments within the lungs: a) normaly inflated lung, b) poorly inflated lung, c) noninflated lung ( = atelectases) (3). results: independant of the side of lateral positioning (l) in the non-dependent upper lung a significant increase of the normaly inflated compartment (s: 45%; l: 65%) as well as a significant decrease of the noninflated compartment (s: 34%, l: 12%) was observed in comparison to supine position (s). in the dependant lower lung the normaly inflated compartment decreased significantly (s: 45%, l: 26%) whereas the noninflated compartment increased significantly (s: 34%, l: 51%). throughout the whole studyperiode we did not observe any significant change regarding gasexchange and hemodynamic parameters. conclusions: in lateral position the non-dependent upper lung is decompressed. therefore a significant recruitment of atelectases is observed in the upper lung within 20 minutes. on the other hand the dependent lung is compressed by the weight of the upper lung and the mediastinum. a great amount of the alveoli of the dependant lung collapse in this short time intervall. therefore the net effect of recruitment of one positioning maneuver is very small. when positioning patients one should be aware, that the patient is kept in each lateral position long enough to clean up the atelectases in the non-dependant lung and short enough to compress less lung tissue in the dependant lung. objective: to analyze effects of low-dose no inhalation ia patients with severe aeut~ respiratory distress syndrome (ards) over five days. methods: we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who required no inhalation with a dose of 5 ppm for at least 5 days. entry criteria for no injaalafioa were murray score >i 2.5 aud pat/fie 2 < 125 nun hg with peep >~ 8 em i~o for at least 24 hours. all patients were sedated, intubated and mechanicauy vantil~ed with volume assist-control ventilation, and had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) to measure cardiac output (by thermodilufion) and relevant intravaseular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and endotraeheal tube and flow was adjusted to obtain 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endouacheal tube by the chemiluminiscence method (nox 4000, see-seres, france). metahemoglobinemia levels were mesured daily. no inhalation was manteined if paojfio ~ improved at least 20 % and was stopped when the change in pao2/fio ~ was below 20% or when the patient presented a paojf02 > 150 mm hg a~er 30 minutes without no inhalation. every day we made an on-off test to determine if no inhalation improved pao2/fio ~. statistics: analysis of vmiance. data: mean + standard deviation. results: the mean age was 60.1 +_ 10.2 years and mean lung injury score was 3.3 • 0.2. mortality was 60 % (6/10), metahemoglobinemia 1.1 • 0.2 %, and no2 concentrations zero. paojf~o 2 always improved significantly al~er 5 ppm no inhalation (see :~ conclusions: reintubation in salf-extubated patients strongly depends on the type of meehamcal venfilatory support: the probability of needing a reintabation ff ese occurs during fult vontilatory support is higher than ff ese occurs during weaning. these data suggest that some patients may remain under weaning from mechanical ventilation for unnecessarily prolonged periods of time. objective: the aim of this study was to evaluate the acute effects on gas exehonge and hemodynamics due to positional changes from supine (sp) to prone (pp) in patients with severe acute respiratory distress syndrome (ards). methods: nine intubated, sedated, paralyzed and mechanically ventilated patients with severe ards were prospectively studied. all had a murray score > 2.5, and a pao2/f~o 2 < 100 with peep ~8 cm h20 for at least 24 h. all patients had indwelling arterial catheters in the pulmonary artery as well as in the radial or femoral artery in order to measure cardiac output (by thermodilution) mad relevont pressures, and to withdraw blood samples. arterial blood gases and hemodynamie parameters were measured first in sp, and then in pp after 60 minutes of stabilization. vontilatoly parameters remaing unchanged during all the study. statistical analysis was done by the non parametric wdeoxon test. data are expressed as mean ~= sd. results: there were 6 men and 3 women with a mean age of 54.2 years (21-71) and mortality was 55 % (5/9). main results are shown below: objective: to describe and compare a new method for obtaining p-v loops (p-vcv) by using a two-way collins valve (twv) with thosu obtained by the supersyringe method (p-vss). methodology: we prospectively studied 14 patients who had an aeute lung injury and were intubated, sedated and paralyzed, and mechanieany ventilated. we performed the p-vev loops and p-vss loops in random order, and the static inflation pressure was limited to 35 emh20 with both methods. pressure (p) was measured at the airway opening by means of a differential p transducer, and volume was obtained from flow (measured with a pneumotacograph) integration. the p-vse method has already been described (h~trf a,et al.bepr 1975; 11:709-28) . the p-vev method consists in the following: the inlet of a twv is connected to the ventilator's y-piece, and both outlets are couneeted to the endotraeheal tube by means of an additional y-piece; one of this outlets has a one-way rudolph valve in order to allow inspiration but not expiration during the inflation maneuver. changing the twv tap position allows basal ventilation or progressiveinflation of the respiratory system. this maneuver is as follows: during an end-expiratory occlusion, the ventilatory settings are adjusted to deliver a 100 ml v r with a respiratory rate of 20/min and i/e ratio 1:4; at the same time the twv tap is ehonged in order to divert flow through the one-way valve. inflation then begins alter releasing the expiratory oonlusion. pressure and flow signals were digitized and acquired by a computer for subsequent data analysis. we analyzed the following parameters: inflation compllonee ( objective: to analyze the variables which eventually may differentiate ards patients who do and do not respond to low doses of inhaled no. we prospectively studied 10 patients (9 men, 1 woman) with severe ards admitted to our icu between may 1994 and may 1995 who were treated with no (5 ppm). the onta'y criteria for no inhalation were murray score >/2.5 and paojfo z < 125 mm fig and peep >/8 cm i~o for at least 24 hours. all patients were sedated, intubated and mechanically ventilated with volume assist-control ventilation. tidal volume was between 6 and 10 ml&g, with constant inspiratory flow, respiratory rate was 15-25/rain, and i/e ratio between 1:2 to 1:3. all patients had indwelling arterial catheters (pulmonary artery, and radial or femoral artery) in order to measure cardiac output (by thermodiintion) and relevant intravascular pressures, and to calculate derived parameters. no was administered between y piece of the ventilator and ondotracheal tube, and flow was adjusted to obi~a 5 ppm no in the inhaled gas. the no, no 2 and no x concentrations were continuously measured at the distal end of the endotracheal tube by the chemilumiinscenee method (nox 4000, see-seres, france). metahemogtobinemia levels were measured daily. we considered a response to no inhalation when an improvement in paoz/fo 2 above 20 % was observed after the inhalation of 5 ppm no (group r) . when the cha~age in paojfi0 z was below 20 % it was considered a lack of response (group non-r small airways functional abnormalities have been recognized as a common feature of lung pathology. however peripheral airways contribute relatively little (~ 10%) resistance to flow and there disturbances can not be adequately estimated by conventional measurements of respiratory mechanics. the purpose of the study was to evaluate the relationship between raw and small airways conductance following weaning from ventilator methods. 37 patients (age:24-62 years; 22 males) with no serious complications al~er mitral or multiple valves replacements and with more than 15 hrs on mechanical ventilation have been enrolled in this study. the modified flow interrupter technique (ptg "gould" with fleish head #2; differential pressure transducer pm-131-tc "statham" w amplifier "kistler 7251") and flow-volume recording of forced expiration (fleish head #4) have been applied before surgery and following operation on mechanical ventilation (my), after extubation (t:xtijb), on 2 (2 nay) and 3 (3 day) days. airways specific conductance (sg aw) has been calculated as a mean of 7-10 consequent measurements in each patient at each stage. the sac was estimated by max expiratory flow at 50 and 25% of vc on 3-4 f-v curves (mef .~0, mef 25) all the data were statistically analyzed with t-test introduction : noninvasive ventilation (niv) reduces the need for endotracheal intubation, the length of stay in icu and the mortality rate in acute exacerbation of copd. however, some patients failed to be ventilated with niv. .objectives...; to further delineate patients who failed to be ventilated with niv and to obtain predicted factors of failure. patients : a cohort of 51 patients (72 • 10 years) presenting with acute exacerbation of copd (fevi: 610 • 396 ml, paco2:62 • 17, ph: 7.33 • 0.08) and nonmvasively ventilated (pressure support through a full-face mask) between april 1990 and may 1994 twenty-seven (53%) were successfully ventilated with niv (discharged alive without the need for endotracheal intubation) while 24 (47%) failed, requiring endotracheal intubation. .methods : patients successfully ventilated and those who failed were compared according to 35 respiratory and nonrespiratory variables univariate analysis (wilcoxon rank-sum test and fisher-exact test) was performed to select variables included in a multivariate analysis by stepwise logistic regression. results : underlying disease assessed by the simplified acute physiologic score (15 • 3 vs 11 • 3, p = 0.0003), creatinine serum concentration (122 • 45 vs 86 • 25 gm/l, p = 0.005), blood urea nitrogen (bun : 12 • 6 vs 8 9 3 mm/l, p = 0.009), age (75 • 9 vs 69 • 10, p = 0.01) were higher and encephalopathy (71 vs 30%, p = 0.005) more frequent in patients who failed. multivariate analysis showed that encephalopathic patients (or (odd ratio) = 4, p = 0.001) older than 65 years (or = 4, p = 0.04) and presenting with bun >_ 10 mmyl (or = 3, p = 0.01) failed to be ventilated with niv. variables related to the respiratory" status (i.e. paco2, pao2, fev1) were unable to predict tile failure of niv. conclusion : copd patients older than 65 years, presenting with acute exacerbation, encephalopathy and bun > 10 ram/l, should be carefully monitored because of high probability of failure with niv. methods:from february to december 1994 we studied 30 pa_ timnts,25 males and 5 females(mean age 68+/-5);18 of the se had emphysema,lo chronic bronchitis,2 dilatative car diomyopatia,with tracheostomy and emphysema.mean pac02 at admission in icu was 95+/-8mmhg,while when weaningbegan, 60+/-5.mean autopeep was 8 cmh20(4-12).all patients were ventilated in crpv as long as four hours to calculate st8 tic and dynamic cmpliance and autopeep.then the ventila tion was continued with psv+cpap(peep 7cmh20 objectives: analysis of the incidence of neurogenic pulmonary edema (npe) in a population of headtrauma patients with acute respiratory failure (arf). npe can occur after a central nervous system insult. differential diagnosis: cardiogenic pulmonary edema and other forms of non eardiogenic pulmonary edema. true incidence and pathophysiohigy remain poorly defined, however the role of catecholamines seems undeniable. early onset npe (within 12 h after trauma) is characterised by hypoxemia, transient pulmonary hypertension and bilateral central fluffy infiltrates on chestx-ray. characteristics of cardiogenic edema or pneumonia are absent. late onset npe, (beyond 12 hours after trauma), is more insidious. the clinical and radiographic picture has to clear within 24 to 48 hours. (1) methods: all headtrauma patients admitted from january 1 to december 31, 1993 in a nearotrauma icu setting were retrospectively analyzed for arf with as sole criterinm a pao2-fio2 ratio < 250. results: 151 neurotrauma patients were admitted during 1993.94 patients (63%) presented with severe head injury (gcs<8), 42 patients (27.8%) with moderate (gcs 8-12) and 15 patients (9.9%) with minor head injury (gcs 12-15). overall mortulity was 19.2% early (within 12 h. after trauma) and delayed onset respiratory incidents were distinguished, counting for 29 (19.2%), respectively 27 patients (17.8%), 7 patients (4.6%) had early and late respiratory complications. early respiratory insufficiency was caused in 9 patients (25.0%) by aspiration, in 11 patients (30.1%) by lung contusion, in 1 patient (2.7%) by fat embolism and in 15 patients (41%) by npe. in the late onset group 31 patients (91.2%) presented with pneumonia, 1 (3.0%) with fat embolism and 2 (5.8%) with npe. the npe group, 17 patients, presented as follows: 15 patients (88.2%) developed early npe, and 2 (11.8%) delayed onset npe. 9 patients (53%) died within the first days after admission, showing high mortality. gcs was less than 8 in 16 patients (94.1%), indicating severity of head injuries. conclusions: high incidence of arf with various etiology (41,7~ was found in this population. in about 10% of all admitted hcadtrauma patients (26,9% of arf) npe was causing attetial hypoxemia. occurrence of npe seems to be related to the severity of the brain injury and thus to outcome. these data call for extreme vigilance in respect of the insidious occurrence of npe. were included if recovering from respiratory failure and if in the opinion of the primary physician were ready for extubation. patients were excluded if undergoing compassionate withdrawal of support or had tracheostomies. the attending physicians were blinded to the measurements. included patients were placed on pressure support (ps) of 0 em h20 with demand-flow continuous positive airway pressure (cpap) 5 cm h20. after a minimum of 30 minutes on the above sehiogs: gastric intramucosai pc'o2, abg, and a p0.1 were measured. the padents were then disconnected from the ventilator for a period of one minute and the patients" respiratory rate and minute ventilation were measured using a wrights respirometer to calculate the frequency to tidal volume ratio (f/vt). patients were then extubated. extubafion failure was defined as the inability to maintain spontaneous ventilation for 24 hours for any reason. results: twenty patients met criteria and were studied over one month period in october 1994. six of the twenty patients (30%) failed weaning. the mean and standard deviation is outlined in failure 7.01+/-0.10 6.8+/-3.9 74.3+/-43.3 87.5+/-43.6 comparison between roc areas shows phi and p0.1 to each show a statistically significant difference from an area of 0.5 (p<o.05). the area at which the test has no discriminative value. this is not the case for either the integrative index or the f/vt ratio. the differences between areas for each curve is not statistically significant. the area's for each roc curve is shown in table #'2. results. 9 of the 21 newborns referred to us for ecmo-therapy developed barotrauma, 6 of the 9 required ecmo and one of the three other patients who did not recieve ecmo-therapy died. to determine the correlation between the risk factor barotrauma and the severity of the pulmonary situation, we determined the oxygenation index of each patient referred to us for ecmo-therapy . the table demonstrates 36, d-35392 giessen, frg. inhalation of nitric oxide (no) and aerosulizatiun of prostaglandin (pg) i: have been suggested to achieve selective pulmonary vasodilation and improvement of arterial oxygenation in patients with acute respiratory distress syndrome (ards). we directly compared these two modes of transbronchial vasodilator therapy in 16 ards patients mechanically ventilated for 24 -96 h (mean murray lung injury score [1] 2.75 + 0.05). patients were randomized to receive either first no and then pgi2 (n=8), or vice versa (n=8), with an intermediate baseline period. each drug was titrated individually to find the lowest effective dose for maximum improvement of arterial oxygenation. gas exchange variables, including data from the multiple inert gas elimination technique (miget), and hemodymmaics under application of no/pgi2 were compared to pro-and past-challenge values. no (mean dose 17.8 + 2.7 ppm) increased the mean oxygenation index (pao2/fio2) from 115 + 12 to 144 + 15 mmi-ig (p < 0.01) and reduced the shunt-flow from 33.1 + 3.6 to 26.6 5:4.5 % (p < 0.05). aernselized pgi2 (mean dose 7.5 + 2.5" ng/kg rain) augmented pao2/fio2 from 114 + 12 to 135 + 12 mmhg (p < 0.01), and decreased shunt from 33.5 + 3.8 to 26.0 + 3.9 % (p < 0.05). the miget analysis showed predominant redistribution of blood-flow from shunt areas to regions with normal ventilation-perfusion ratios in response to both agents. in 10 patients, both no and pgi2 caused an increase in pao2/fio2 by at least 10 rnmhg. two further patients displayed a corresponding improvement of arterial oxygenation in response to either no or pgiz. the mean pulmonary artery pressure decreased from 34.8 5:2.2 to 33.0 5:1.8 mmhg in response to no, and from 35.0 5:2.2 to 31.9 5:1.7 mmhg (p < 0.05) in response to pgi2. cardiac output, systemic arterial pressure, and right and left heart filling pressures were not affected by either agent. we r that individually titrated doses of inhaled no and aerosolized pgi~ effect selective pulmonary vasodilation and redistribute blood-flow from shunt-areas to weu-ventilated regions with nearly identical efficacy profiles. obieetives: the use of extraeorporeal bypass systems for oxygenation and co2-removal is performed in patients with acute respiratory distress syndrome (ards) to reduce mortality and to improve restitution of pulmonary functions. high-dose heparin -commonly used in such patients to prevent thrombotic complications -might cause incurable bleadings as a major risk. this lead to the development of heparinized bypass systems; results of animal and first clinical studies proposed that such systems might run with low-dose heparin or even without any systemic anticoagulation. methods: since 1989, 47 patients with severe ards were treated with extracorporeal lung assist (ela) using heparin-coated systems (type maxima, medtronic, carmeda coating) in our icu. they received a moderate systemic heparinization. standard clotting assays (act, aptt, tt, ptt, at-ill, fbg, fsp) as well as special tests (tat, r~-tg, pf4, pal-l, d-dimers, protein c, cl-inhibitor) were performed. results: covalent heparin-eoating of the ela-systems combined with lowdose heparinization could not prevent major changes in coagulation: 1) in mean, platelets dropped from 305/nl to 76/nl within 60 h after onset of ela. 2) tat levels were already elevated before ela start (55 ug/]) and demonstrated an additional peak 2 h after onset of ela (up to 240 ug/[). 3) in parallel, there was a transient peak of pai-i levels (from 5 to 75 au/ml} 2 h after onset of ela. 4) in mean, fibrinogen levels dropped from 470 to 310 mg/100ml within 24 h after onset of ela. 5) in mean, cl-inhibitor levels dropped significantly from 132% to 115% after onset of ela and reached the initial levels within 48 h. 6) after membrane change, there were significant changes for c1-inhibitor, fibrin-d-dimers and tt. 7) global clotting tests such as aptt, ptt and tt were within normal range. conclusions: patients with severe ards develop a prethrombotic state already before they get connected to the ela bypass system. after onset of ela, they experience additional involvement of procoagulant, anticoagulant, fibrinolytic, and complement systems. in parallel to laboratory findings, clinical data suggest that the use of heparin-coated systems with accompanying systemic low-dose heparinization does not avoid thrombembolic and hemorrhagic complications in tong-term ela patients. thus, at the present, higher dosed individually adjusted heparin treatment has to be recommended. thereby, standard clotting monitoring is not sufficient. objective: poor muscle performance contributes to prolonged dependence on mechanical ventilation. longitudinal data on muscle function in icu patients is scarce. we evaluated changes in inspiratory and peripheral muscle performance during prolonged intensive care in patients with acute respiratory failure. methods: 22 patients requiring intensive care for more than one week were studied. maximum inspiratory pressure (pimax) was measured twice a week for a maximum of three weeks. handgrip power (dynamometer) and subjective fatigue (keldet score) were obtained in survivors only. one measurement was done on the date of extubation. pimax pressure was obtained by occluding the airway for 20 seconds or at least five inspiratory breath attempts. obiectives -measurement of peep-induced changes in lung volume helps to assess respiratory mechanics in acute lung injury (ali). we evaluated the accuracy and stability of a new respiratory inductive plethysmograph (rip), in the measurement of peep induced changes in end-expiratory lung volume (eelv) and tidal volume (vt) against volume reference, pneumotachograph (pnt), methods -two hours periods of basal ventilation and stepwise increases and reductions of peep (5-10-15-10-5 cm h20) were recorded in patients with all. in addition, the new rip device (respitrace plus tm) was compared to an older rip (respigraph tm, nims, fl, usa) to determine its thermal stability using cold and warm devices and a ventilated lung model. results -the difference between the new rip and pnt in the measurement of vt was -30 _+ 3 mi (x _+ se) or a -5.2 _+ 0.6 % error. increasing peep from 5 to 15 cm h20 induced a 511 + 93 ml change in eelv. a difference of 18 + 12 ml (3.5 % of max. eelv change) (ns) was found between rip and pnt eelv readings. during controlled ventilation with constant settings the mean change in eelv was 187 _+ 69 ml/120 min (fig.i) . validation of calibration showed a mean error of 1 -+ 1.78 % after 120 rain. the new rip had a higher drift when cold (cold 28 + 10 ml/30 min vs warm 3 _+ 1 ml/30 min), whereas the old rip was thermally stable (cold -3 + 3 ml/30 rain vs warm i + 1 ml/30 min). trauma icu, hospital traumatologia y rehabilitacidn. vall d'rebron. barcelona. spain. recent data suggest that the proportion of patients with relevant discrepancies between two jugular bulb (jb) oxygen saturation (sjo 2) values is higher than suspected. objectives: determine the incidence, the significance and the pro9nosis value of those differences,if they exist,in severe head injured-patients. material and methods: 35 severe head-injured patients, coma glasgow scale (gcs) 8, with diffuse brain injury according to marshall criteria in the first ct scan, icp recorded, with an interval from accident-double jb monitoring < than 12 hours, were studied. data from bilateral sjo 2 were obtained simultaneously, every 6 hours. discrepancies were considered significant, when differences in sjo 2 were > 10%. no chan9es in treatment protocol (hyperventilation, man• etc) were carried out due to this study. results: 30 men and 5 women were studied, aged 32• yrs. at arrival at hospital, gcs were < 5 in 20 and ) 5 in to. the incidence of high icp() 20 mmhg) were 7sz at the entry. the mean therapy index level required to control lop was 4~l all patients required vasopressor therapy to maintain upp over ds mmhg. in 20 patients a s.s f swan-ganz fiberoptic catheter was used to obtain a continuous recording of sjo 2. in the others 15, sj02 were intermittently controhed.the mean time of monitoring were d.8• days. ten patients died within this period. a total of 1.240 blood samples were analized. at arrival, sjo 2 discrepancies were found in 22 patients, b2%. at 48 hours, the incidence were lower, 18/35, 51.4%. at 4th day, were h/29, 38z and at day 7, when the catheters were retired, ii[25, 44z showed discrepancies. the ct showed new injuries in g4z of patients with differences > 10~ in sd02 values throughout treatment period. none of those were considered for neurosurgical treatment. no correlation was found between iop and sjo 2 values and sjo 2 differences. conclusions: the incidence of discrepancies between sjo 2 was higher than expected in severe head-injured patients. these situation could reflect disturbances between 02 demands. when differences are known, and those lend to change, the ct scan, nearly always, will show new injuries. platelet-activating factor (paf) is an inflamatory mediator implicated in the pathogenesis of bronchial asthma and acute respiratory distress syndrome (ards). its inhalation in healthy subjects produces transient bronchoconstriction and mild ventilation-perfusion mismatch, together with peripheral leukopenia as a result of intrapulmonary neutrophil (pmn) sequestration. likewise our group has shown in healthy subjects and asthmatic patients that aaibutamol (s) inhibits both pulmonary and systemic effects of paf, suggesting that s may inhibit paf-induced venoconstriction in pulmonary microoirculation. the aim of the present study was to investigate if s inhalation decreases pmn by lung sequestration induced by paf. we studied 8 healthy, non-atop• nonsmoking subjects (6m/2f, 24+4 yr), which were pre-treated with s (300,ug) or placebo, with a randomized, double-blind, crossover, design, before paf (24,ug) inhalation. we measured the respiratory system resistance (rrs) by forced oscillation, arterial btood gases and both total white cell and pmn count every 4 min over a 30 min. period. simultaneously, we recorded continuously the lung dynamics of inm-neutrophil and tc99m-erythrocytes activity, with a gammacamara. after placebo, paf inhalation decreased white cells (from 5410 2 1125 to 33022934x109/l), and pmn(from 29752693to 1222 _+ 767 x109/l), and increased aapo 2(from 2.1 _+9.5 to 14.7+ 12.2 mmhg, p<o.05) and rrs(from 3.0 2 0.6 to 3.7 2 0.7 cmh20.s.i q ) (p < 0.05 each). the fall in total white cell count correlated with the intrapulmonary pmn retention (r =0.83, p <0.05). by contrast, after s, paf did not induce any change in white ceil count (from 596321766 to 6147+1721 x109/i), pmn count (from 354321628 to 2953 2 1793 x109/i), aapo 2 (from 7.3 2 10 to 5.6 + 8.6 mmhg), and rrs (from 2.9 2 0.5 to 2.9 2 0.7 cmh20.s.i 1). compared with placebo, after s there was a lower ~lln-pmn lung activity (2.020.4 vs 1.320.7 cts/mci/pixel, p<o.01 ), lower intrapulmonary pmn retention (15.627.7 vs 11.1 24.8 %, p=o,09), and a faster washout (0.1420.08 vs 0.1720.007 s ~, p=0,4). our results indicate that s inhibits paf-induced intrapulmonary pmn sequestration, being consistent with the hypothesis that s may offset the venoconstriction of pulmonary microcirculadon caused by this mediator. supported inpiratory muscle fatigue with failure of force generation as def'med by a tensiontime index (tti)>0.15-0.20 has been shown to occur in normal volunteers and in stable copd patients with a specific imposed breathing pattern. its role, however, in hypercapnic respiratory failure is less certain. we studied 10 failed weaning trials in 5 copd patients in which breathing pattern, tension-time index (tti) of inspimtory muscles, dynamic peepi, dynamic lung elastance, lung resistance, and arterial paco2 and ph were measured at the beginning and end of a t-piece weaning trial. in addition, the change in esophageal pressure during a mueller maneuver (apes max) was measured. a weaning trail has been prospectively defined to have failed if one of the following criteria was met: a rise in pco2 >20mmhg from baseline accompanied by a fall in ph<7.35; a respiratory frequency (f) >30/min; excessive accessory inspiratory muscle recruitment; and a marked increase in dyspnea. values are expressed as mean • se. weaning failure was characterized by a more rapid, shallow breathing pattern, worsened mechanics, hypercapnia and respiratory acidemia despite an unchanged tri and pes max. we conclude that in this setting hypercapnic respiratory failure is not a consequence of inspiratory muscle fatigue. rather the adopted breathing strategy and resultant hypercapnia may represent an adaptation to forestall the onset of muscle fatigue. concerning the investigated elf-par~eters, no stadstically signhqcant differences were detected between the pgi2 and the control group. histopathologlcal changes occured in both groups and consisted in rare focal flaaaning 0f tracheal epithelium with loss of cilia and slight inflammatory cell infiltration, as well as slight swelling of alveolar typo4 pneumoeytes. sections of generation 5, 10 and 15 from bronchial tree were free of pathological changes. conclusion: alter 8h inhalation of p~ji2 no signs of respiratory-lract tissue damage caused by the aerosol could be detected. the minor pathological findings in the trachea are most likely due to mechanical irritation by bronchoscopy, changes of the alveolar epithelium are known for long-term mechanical ventilation 3. objectives: the aim of this study was to evaluate of efficiacy of ganglion stetlate blockade in patients with respiratory failure. methods: two groups of patients were investigated: group i (n = 15) trauma patients with acute lung injury (ali), group if (n = 15) patients with asthmatic status. in all cases continuous mandatory ventilation (cmv) was used with bennett 7200 ae. in both groups bilateral ganglion stellate blockade with antero-lateral approach was performed, using 0.375 % marcain. the following parameters were analysed: pao2, sao2, paco~, pip and c~t~t. results: in trauma patients with aij after bilateral ganglion stellate blockade short -lived and slight improvement of pao 2 and sao2, decrease of pacoz and pir and increase of static compliance of respiratory system were found. in second group bilateral ganglion stellate blockade interrupted the asthmatic status and significant statistical improvement of parameters of oxygenation, ventilation and respiratory system mechanics were observed. conclusions: we suggest that the bilateral ganglion stellate blockade is a very useful method in treatment of patients with obstructive respiratory insufficiency. the aim of the study was to analyse whether there exists serum and urine electrolyte disorder in patients(pts.) with acute respiratory insufficiency(ari). the study included t8 pts. with ari (pao2:8,24@1,49 kpa. paco2: 5,01i-0,77kpa, ph:7 42~:0,59, hco3: 26,3:~8,10 mmol/1, sao2 : 90,4~-7,42%) who were hospitally treated due to pneumonia(9 pts.),emboly of the pulmonary artery(3 pts.) and severe attack of bronchial asthma (6 pts). among tham there were 12(66,7%) males and 6(33,3%) females, average age 51,5~:16,1 years, otherwise previously healthy. electrolyte concentracions were measured at the onset of the disease in serum and urine collected during 24 hours (sodium-na,potassium-k, chlorine-c1, calcium-ca,magnesium-mgand phosphorus-p). the measured serum and urine electrolyte concentrations were compared with respective referent values (rv). by serum electrolyte analysis, the following average velues were obtained: na:l 4o,94 the object of our investigation was a group of 21 pts with massive pneumonias, 14 males (66.6%), 7 females (33.3%),mean age 55 yrs.thirteen (62%) of them were smokers,8(38%) nonsmokers. only 1 pt (4.7%) had pre-existing chronic respiratory disease, and 20 (95.2%) were admitted for the first lime,with no previous respiratory anamnesis. diagnose was based on anamnestic data of productive cough in 15 pts(71.4%),physicaly ~onchial breathing in 19 i~s (90.4%),white cell count onder 10 x 109 /l in 18 pts(85.7%). radiographicly, bilateral massive homogeneous shadows were found in 7 pts (33.3%), onilateral in 12 pts(57.1%),pleural effusion in 2 pts (9.52%). abnormal renal function was found in 14 pts (66.6%). sputum culture was positive in 8 pts (38%): slr.pneumoniae, str.pyogenes, pse'udomonas aerug, in 4, 2, 2 cases respectively. all patients had remarcable hypoxernia (pao2 range from 4,75 to 8,1 kpa) without hypercalmea. all patients needed oxygenotherapy together with antibiotics and other .symptomatic therapy. nineteen pts had anaelioration of general condition and normalization of blood gas analyses, while 2 pts with the lowest hypoxcmia died.in conclusion, massive pneumonias are frequently followed by respiratory insufficiency which is one of the markers of pneumonia severity. as existing hypoxemia complicates the course of the disease,prolonges the recovery, makes therapy more complexe and may be cause of death , frequent blood gas measurement is recomanded. we studied the effects of bosentan (bos), an eta and etb receptor antagonist, to examine if endogenous et mediates pulmonary hypertension in anesthetized and ventilated dogs with acute lung injury due to oleic acid (oa). the gradient between pulmonary artery pressure (ppa) and occluded ppa (ppao), and gas exchange (evaluated by arterial blood gases and sf6 intrapulmonary shunt) were measured at controlled flow. in 8 dogs (treatment), data were collected at baseline, during long injury (obtained 90 rain after intravenous administration of oa 0.06 ml/kg), and again after bos (10 mg/kg intravenously). in 5 dogs (pretreatment), data were obtained at baseline, after bos and then after oa. in treated dogs, oa increased (ppa-ppao, mmhg, table, means + sem, * p < 0.05 vs base) and deteriorated gas exchange. after oa, bos did not affect pulmonary vascular tone nor gas exchange. in pretreated dogs, bos had no effect on baseline pulmonary vascular tone but prevented the increase in (ppa-ppao) after oa. the deterioration in gas exchange after oa was not influenced by bos pretreatment. objectives: the alveolar 02 tension is measured by the application of the alveolar air equation in which the arterial pco 2 is used or by the simplified form of this equation in which the respiratory exchange ratio is taken at the value of 0.8. the purpose of this study was to estimate the effective alveolar 02 tension (pao2eff) during spontaneous breathing with a new bedside technique which is simple non-invasive in 14 normal subjects and 27 patients with chronic bronchitis-emphysema. we also compared these values with the ideal alveolar po 2 (pao2(i)), measured from the alveolar air equation in which paco 2 was substituted by the effective alveolar pco 2 (paco2eff) and with the alveolar po 2 measured from the simplified alveolar air equation (pa02). this study is complemantary to previous work for the estimation of paco2eff. methods: the subjects breathed quietly through the equipment assembly (mouthpiece monitoring ring, fleisch transducer head) connected to a pneumotachograph and a fast response 02 and co 2 analyzer. the method is a computerised calculation of the effective alveolar po2quite similar to that of paco2eff, obtained from the simultaneously recorded at the mouth expiratory flow, 02 and co 2 concentration versus time curves. results: the results showed a mean difference (pao2eff-pa02(i)) of -0.061 kpa in normal subjects and -0,711 in patients. the mean of the difference (pao2eff-paq 2) and (pad2(i]-pao z) was much greater than 0.281 in all subjects. the limits of agreement for the difference (paozeff-pa02(i))were -0.691 to 0.568 kpa in normal subjects and -2.040 to 0.596 in patients, while those for the differences (pao2eff-pad 2) and (pao2(i)-pad 2) were very large ( > -1.5 to > 1.7) in all subjects. conclusions: the effective alveolar po 2 is very close to the ideal one in normal subjects, tn patients pao2eff may excessively deviate from pa02(i) due to the observed significant difference between the alveolar/tidal volume ratio for o 2 and that for co 2. the alveolar po 2 measured from the simplified alveolar air equation (pao 2) differed substantially from pao2eff and pad2(i) in all subjects. the essential role of glucoprotein hormone erythropoietin is to control red cell production. hypoxemia, reduced blood 02-carrying capacity and increased affinity of hemoglobin for 02 are the primary stimuli for erythropoietin production. both anemia and hypoxemia induce rapidly erythropoietin secretion. kidney erythropoietin rna levels correlate inversely with hematocrit and directly with plasma erythropoietin level. similarly, hypoxemia increases kidney erythropoietin rna and plasma erythropoietin. the effect of hyperoxemia (pa02>lo0 mmhg) on erythropoietin secretion isn't very well understood. the purpose of this study was first to evaluate the erythropoietin secretion in patients with acute respiratory failure and second to determine the effect of hyperoxemia on erythropoietin secretion in patients with and without anemia. sixteen patients with acute or acute on chronic respiratory failure needed mechanical ventilation were included in this study. these patient were divided in two groups. the patient who developed anemia were included in group i and the patients without anemia in group i1. erythropoietin was estimated in venous blood in three stages. the first sample was taken during hypoxemia, the second during hyperoxemia and third during normoxemia. all the patients had high erythropoietin level during the hypoxemia period (mean value 98• mu/ml). during hyperoxemia etythropoietin levels were reduced in both groups ( mean value 21.6+15.2 mu/ml in group i, 36.8• mu/ml in group ii). in normoxemia stage, erythropoietin increased again in anemic patients, and decreased more in the patients of group i1. we conclude that hyperroxemia inhibit erythropoietin secretion in spite of anemia and tow arterial oxygen content. hyperoxemia may be a factor of the insisted anemia in with oxygen treated icu patients. the purpose of this study was to determine the relationship between clinical features of acute lung injury (all) and parameters like total proteins, total and individual phospholipids, the presence of paf, and acetylhydrolase activity in bal of mechanically ventillated patients. acetylhydrolase catalyses the cleavage of acetyl-group from the second position of the glycerylether backbone of paf, leading to its inactivation. mechanically ventillated patients were divided to three groups. group i includes patients without all; group ii, comprisespatients with moderate degree all, (1.0<g-i1<2.5); and group iii comprises 5 patients with severe ards (all score > 2.5). broncoalveolar lavage (bal) was obtained after infusion of normal saline at 37~ to intubated patients and cooled immediately. cells were removed after mild centrifugation (350 x g, 30 min, 4oc). aliquots from the supernatant were used for total protein, phospholipid and paf analysis and determination. acetylhydrolase activity was assessed after incubation of bal with 3h-paf labelled on the acetyl group. released label was measured by liquid scintillation counter in the supernatant after trichloroacetic acid precipitation of the non-reacted substrate. kinetic characteristics of the enzymes were also studied. total phospholipids appear reduced in bal of patients with all, while total proteins increase. these factors appear to correlate with the severity of all. paf was not present in bal samples pretreatad with equal volume of 20% acetic acid to denaturate acetylhydrolase. detection limit for paf under our experimental conditions: 60 pg paf/ml bal. instead, acetylhydrolase activity was detected in amounts increasing with the total protein content. background: intubated patients without lung injury or impaired breathing control normally display an inspiratory peak flow of below 1l/s. the aim of our study was to investigate the inspiratory peak flow generated by patients with acute respiratory insufficiency (ari). we had to take into account that both an inspiratory pressure support (ips) and the resistance of the endotracheal tube considerably influence the flow pattern generated by the patient. patients and methods: to investigate the non-influenced flow pattern we developed a new ventilatory mode which automatically compensates for the flow-dependent resistance of the endotracheal tube (automatic tube compensation, atc). furthermore, the mode maintains a constant tracheal pressure in inspiration and expiratio n . consequently, the measured flow pattern exactly corresponds to the flow pattern generated by the patient except that the ventilator modified for this mode (evita, driiger liibeck, germany) was not able to deliver a gas flow of more than 2l]s. we have investigated 10 patients with ari arising from different reasons. results: the inspiratory peak flow measured in the atc-mode was 1.7l/s _+0.3l/s. the maximal deliverable flow of 2l/s was obtained in 3 of 10 patients. the figure shows the flow pattern under atc and ips in [~s] oi:) one of these patients. conclusions: patients with ari display a highly increased inspiratory peak flow. ventilators used for spontaneous breathing should therefore be able to deliver a gas flow of more than 2l/s. an overproduction of no and reactive oxygen species (ros) has been demonstratred in septic shock. ros and nitric oxide (.no) are free radicals which are known to react together leading to peroxynitrite anions that can decompose to form nitrogen dioxide (no2) and hydroxyl radical (oh~ thus, no has been reported to have a dual effect on lipid peroxidation (prooxydant via the peroxinitrite or antioxidant via the chelation of ros). in the present study we have investigated in different models the in vitro and in vivo action of no on lipid peroxidation. copper-induced ldl oxidation was used as an in vitro model of lipid peroxidation. ldl (100 ~g apob/ml) was incubated with cu 2+ (2,5 ~tm) in presence or absence of no donor (sodium nitroprussiate or glutathione-no) from 10 to 500 ~m. oxidation of ldl was monitored continuously with conjugated diene formation (234 nm) and 4 hydroxy nonenal accumulation (hne). exogenous no prevents in a dose dependent maner the progress of copperinduced oxidation. ischaemia-reperfusion injury (i/r), characterized by an overproduction of ros, is used as an in vivo model. anaesthetized rats were submitted to 1 hour renal isehaemia following by 2 hours of reperfusion. sham operated rats (sop) were used as control. lipid peroxidation was evaluated by measuring the hne accumulated in rat kidneys in presence or absence of l-arginine or d-arginine infusion. l-arginine, but not darginine, enhances hne accumulation in i/r but not in sop (<0.05 nmol/g tissue in sop versus 0.6 nmol/g tissue in i/r), showing that in this experimental conditions, no produced from l-arginine, enhances the toxicity of ros. this study shows that the pro-or antioxydant effects of no are different in vivo and in vitro and could be driven by environemental conditions such as ph, relative concentration of no and ros, ferryl species...these conditions are impaired in circulatory shock. methods:" the diagnostic and therapeutic approach was standardized so that data collected over a 10-year period were comparable. a progressive deterioration of clinical conditions and/or pulmonary gas exchanges was considered as indication for my. variables potentially predicting the need for hv were derived from clinical and arterial gas data, extrapulmonary diseases, use of drugs, chest x-ray and ecg abnormalities. results: rv, performed with external and/or internal ventilators, was necessary in 130 patients (22%). at the hospital admission, pac02 was higher and ph was lower in patients requiring rv ( pneumomediastinum, pneumothorax, ateleetasis and myocardial infarction are rarely seen in bronchial asthma. these complications occur as a result of the severe asthma.the aim of our retrospective study was to analyse the complications seen in acute asthma attacks. during the years 1990 through 1994, 244 patients were admitted to hospital in acute asthma episode. there were 11 (4,5%) pts with complications; mean age of 27 yrs; 6 females (54%). clinical history, ecg and chest radiogr~hs were analysed. the mean duration of bronchial asthma was 14 yrs (range from 2 months to 17 yrs), all patients were atopics. there were four ex-smokem and one smoker. the worsening of asthma symptoms begun two days before the admission (range from 1 to 7 days). on ecg all patients had tschycardia. rightward shift of the qrs axis and st-t changes indicative of right ventrieutur strain were found in three pts. these were the transient fmdings that improved after curing the acute asthma attack. non-q myocardial infarction oeeured in one patlent and resulted from the hypoxaemia of asthma. hyperinfl~ion was the usual finding on the chest radiograpk pneumomediastinum and subcutaneous emphysema were apparent in five pts and required no additional treatment unilateral pneumothoraccs were present in two pts and needed eontimous intrapleural drainage; one of these patienst died in eardiorespiratory insufficiency. ateleetasis of right upper lobe was present in one patient. it oceured due to inspissated secretions and needed no additional treatment all these patients, except one who died, improved on lreaanent with oxygcr~ steroids, beta-two agonists, theophylline and antibiotics. in conclusion, complications occur in acute asthma episodes as a result of the severe asthma mediastir,*l emphysema and atelectasis are not serious complications. pneumothorax and myocardial infarction are very serious life-treatening complications and always have to i:m considered in taati~ts with sev~ asthma. acute bronchial asthmatic episodes represent one of the most common respiratory mnergendes, its maximmum expression "status asthmatiens" is one entity of low incidence, still it is a risk to the physical integrity of the patient. during 1993 a total of 52 patients with diagnosis of status asthmabcas were hospitalized. out of these palients six had a near-fatsl asthma and they were subjected to a complex examination. near-fatal asthma was defined as either respiratory arrest or acute asttuua with paco2 greater than 6,7 kpa and/or an altered state of consciousness. mean age was 56,2-d:16,2 yrs, four male and two female sex. at presentation two patients suffered from coma, others were confused. they exh'bited severe dystmoes, diffieul~ speaking, used accessory muscles of respiration, increased whee~tg while two cases had silent chest on auscultation. cyanosis indicated a very severe asthma attack in all six patients. mean respiratory rate was 28~4/min and puts rate 118.d: 12 bts/imn. arterial blood gases revealed a pao2 of 6,95~1,33 kpa, paco2 of 7,87• kpa and ph of 7,274-+-0,132. area-careful evaluation they received conventional therapy (immediately continuous oxygen, impelled nebulization with high doses of betatwo agonists and ipmtropium bromide, intmvanous st~oids and theophylline). in two eases signs and symptoms of deteriorating airflow and respiratory muscle fatigue determined the need for mechanical ventilation. out of six near-fatal attacks aggressive lrealanent was suscessfull in four patients and fatal in two eases. one patient admittcxl in coma died in severe hypoxae~a upon one hour and one mechanicaly ventilated died from cardiac arrhythmia. life-threatening attacks in asthmatics in our group developed gradual worsening despite neatment which r symptoms in most other patients. one patient had "brittle asthma", other long-standing acute episodes ireated with systemic steroids. conclusions: idantitiechon of fatality prone subjects may lead to fttrther muetion of seveze episodes. respiratory affest and coma upon admission, severe dyspnoca with silent chest on ausouhation, oyanusis and use of accessory muscles of respiration constitute the basic cfinieal picture. hypoxasmia must be immediately eon'ected.the patients and physicians should be able to assess the severity of asthma, a major factor in near-fatal and fatal asthma attacks. objectives :our purpose was to asses if the evolution of patients with a adult respiratory distress syndrome (ards) ,shows any relation to the pulmonary or systemic origin of the disease and whether or not there were differences in the frequency of the syndrome in both groups. methods : randomized prospective study in multidisciplinary icu. one hundred and sixteen patients with a high risk developing ards were distributed into two groups. one was named systemic origin group(so) and the other pulmonary origth group (po).ai1 patients only showed one cause (pulmonary or systemic) with potential risk of ards.the patient's hemodynamic and respiratory status was evaluated every 6 hours the first day and every 12 hours the second and third day. at the end of 72 hours the patients were diagnosed as ards or non-ards. measurements and main results : of the total 116 patients, 57 were finally included in the so group and 59 in the po group.patients in so group and po group had comparable ages (p<.01).peep in both groups was comparable (=.06) at the mmnent of admission to the study. there were no statistically significant differences for cardiac index and systemic vascular resistances. the pulmonary vascular resistances (pvr) showed significant differences at 48 h.(p<.05) and 72 h. (p<.03).the oxygen comsumption (vo) in patients of the so group showed statistically significant differences at 48 h. (p<.05) with respect to initial values.fifteen cases of ards (26.3%) in the so group and twenty five cases (42.3%) in the po group were identified. the time of onset of ards was 35_+ 14 hours in the so group and 11 + 4 b hours in the po group.the final outcome was very similar th both groups : mortality of 36% in the so group versus 37% in the pc group. conclusions : the pathogenesis of ards depends on whether the lesion is originated at or outside the lung. the po group showed a sborter thne of onset of ards, a faster and more severe increase of pulmonary shunt and a higher percentage of patients developing ards compared with patients of the so group.the so group showed a higher and faster increase in puhnonary resitances tbat po group and a decrease th oxygen comsumption earlier and more severe than in the po group. these data thus seem to show that there could be two mechanisms involved in the genesis of ards depending on the cause. the fact that the ards genesis is shorter in the cases of pulmonary etiology with faster impairment of pulmonary shunt, and a slower increase in pulmonary resistances in this pulmonary group, would indicate that the underlying mechanisms responsible for the hypoxemia are different to those which thitiate the increase in pulmonary resistances. finally, the exclusive inapairinent of oxygen consumption, which appears earlier than the onset of ards in the systemic origth group, could show the generalized character of the process in this group. perfusion of prostacyclin (pgi2) to treat pulmonary hypertension in adult respiratory distress syndrome (ards) worse pulmonary gas exchange due to a marked impairement of ventilation/perfusion mismatch. recently has been shown that if prostacyclin is given by aerosol instead of intravenous the net effect is an improvement of arterial oxigenation due to a redistribution of blood flow to well ventilated areas. objectives: to asses the effects of inhaled proatacyclin on pulmonary haemodynamics and gas exchange in patients with severe ards. methods : two patients with severe ards (murray score >3) recived inhaled pgi 2 at 15-20 ng.kg.min "1 using an ultrasonic nebulizer. haemodynamic measurements, arterial and mixed venous blood gas analysis were performed before and after 30 rain of pgi inhalation. results: short-terro p~i 2 inhalation improved pulmonary g-~ e-'~hange in both patients. arterial oxygen partial pressure (pao2) increased from 101 to 166 mmhg in patient 1 and from 87 to 108 in patient 2, the ratio pao to the fraction of inspired oxygen increased from 1262 to 207 (patient 1) and from 124 to 154 (patient 2). venous admixture decreased from 36% to 29% and from 34% to 27% in patient 1 and 2 respectively. mean pulmonary artery pressure decreased slightly from 25 to 23 mmhg in patient 1 and from 41 to 37 mmhg in patient 2. no effects on systemic haemodynamics were observed in any patient. conclusions: pgi 2 inhalation improves gas exchange and produces selective pulmonary vaaodilation, thus can be an alternative therapy for the treatment of pulmonary hypertension and hypexemia in patients with severe respiratory falllure. methods: we treated 67 ards-patients (age 41 yr (16-75) mean, range) during 1991-94. the lowest pao2/fio2-ratio was 74 (29-140), the worst murray score 3.0 (2.3-4.0), icu-stay 41 (1-121) days and hospital mortality 40%. the costs of intensive care were calculated according to intensivity of patient care as assessed by tiss-scoring (therapeutic intervention scoring system). the more intensive the care, the higher are the costs. costs per year of life saved (=life-year" in us $) were compaired by other medical treatments (1-4). it is assumed that the mean expected length of remaining life in ards-survivors after intensive care is 25 years. treatment life-year ($) ' bone marrow transplantation (acute leukemia) 65 000 lowering cholesterol using iovastatin 51 000 treating hypertension using nifedipine 32 900 heart transplantation 28 000 intensive care of ards-patients 3300 conclusions: intensive care of patients with severe ards is highly more cost-effective as compared with many other routinely used medical treatment strategies, the usually good recovery and the reasonable quality of life in survivors justifies investments to care of these patients (5). there is a close correlation between these two methods of measuring evlw. however there is an underestimation of 38.5 % in this kind of pulmonary edema ( oleie acid induced ) with the double dilution method. although the size of the sample is small, in normal lungs there appear not to be this underestimation. the effect of peep on evlw has been studied with contradictory results, probably as a consequence oft differences in methods of measuring evlw, variations in the type and severity of lung injury, and different timings of peep application. objective= 1) to analyse the effect of different levels of peep (0, 10 and 20omh20) on evlw during hpe; 2) to establish whether increases in intrathoracic pressure due to high peep levels can obstruct lymphatic drainage. material and methodet hpe was provoked in 3 groups of dogs by inflating a foley catheter in left auricular to a pressure of 24-26 r~uhg. peep levels of 0, i0 or 20 m~hg were applied. resultst objective: to assess the effect on extravascular lung water (evlw) of the application of peep and the reduction of vt in an oleic acid pulmonary edema model in pigs, using three ventila~ary strategies. material and methods: twelve adolescent pigs (weighing over 30 kg) were randomly divided in three gmups immediately alter infusing via a central vein 0.1 ml/kg of oleic acid to produce a permeability pulmonary edema. the ventilatory parameters for each group were as follows: group i (n=4) : vt: 10-15 ml/kg; zeep. group 2:(n=4) : vt: 10-15 ml/kg; peep: 10 cm h20. group 3:(n=4) : vt: 5-10 ml/kg; peep: 10 emil20. (resulting in permissive hypereapnla) after a four-hour period of ventilation the animals were killed and the lungs excised to calculate gravimetrically the extravascular lung water using a standardized procedure ( hemoglobin content method ). ill evlw (ml/kg) group obiective: in the postoperative period, maintenance of adeguate arterial oxygen tension is a major problem in morbidly obese patients probably because of a large reduction in functional residual capacity (frc). the aim of this study was to evaluate the effects of peep on respiratory mechamcs and gas exchange in this kind of patients. methods: in nine postoperative mechanically ventilated morbidly obese patients (bmi>40 kg/m 2) we partitioned the total respiratory system mechanics into its lung (1) and chest wall (w) components using the airway occlusion technique associated with the esophageal balloon, during constant flow inflation (jap 1989; 67: 2556) . at three different levels of peep (0, 5, 10 cmh20 ) we measured: compliance (cst), airway (rim) and "additional" (dr) resistance, frc and gas exchange. obiectives. to describe the use of prone position in our icu we analyzed the clinical records of all patients admitted in 1993-94, selecting adult patients with arf defined as: intubation and pao2/fio2<250 mmhg plus an fio2>0.5 or peep>5 cm i120. results. 146 patients met the arf criteria: 40 of them (27.4%) underwent prone positioning (p+). prone position use began in the early phase of arf (3.5• days from the beginning, range 1-32, median 2).25 out of 40 p+ pts were treated with controlled ventilation (cppv or pcv), while 14 were on assisted ventilation (simv+ps) and 1 on spontaneous breathing (cpap). only 2 pts were awake when turned prone, while 11 pts required adjuncts of sedation to tolerate the change of position. the duration of prone positioning was variable (average lenght 4.7• h, range 0.5-12 h). only minor side effects were observed (eyelids and facial edema, chest and facial pressure bruises). we consider responders (r+) those patients presenting at least 12.5 mmhg increase in pao2/fio2:35/40 patients (87.5 %.) were responders when first pruned. the pao2/fio 2 changes induced by prone position are reported in the figure. pao2/fio 2 increased when patients were pruned (*p<0.001) and remained higher than baseline values when returning supine(*p<0.001). paco 2 remained unchanged. prone positioning was used at least twice in 21/40 ( conclusions. this retrospective analysis confirms that prone positioning improves oxtgenation in the majorib' of arf patients. altough we have no available criteria to discriminate in advance r+ from r-pts, we now routinely consider the use of prone position in the treatment of severe arf. palo a, otivei m*, galbusera c, veronesi r, sala gallini g, zanierato m, iotti g, braschi a.servizio anest. e rianim. i, *laboratorio biotecnologie e tecnologie biomediche irccs s. matteo, pavia, italy inhaled no can improve arterial oxygenation and reduce pulmonary hypertension in ards patients; little information is, however, available about the dose-response curves. methods seven ards patients (lis 2.7+.5) submitted to mechanical ventilation randomly received 8 inhaled no doses in increasing or decreasing sequence: 0.5, 1, 5, 10, 20, 50 and 100 ppm. reference measurements were obtained before and after the entire period of no inhalation. hemodynamic parameters and blood gases were measured after 25 min in each condition. cmv was administered under sedation and paralysis, with constant ventilation, peep (lol-_2 cmh20) and fit2 (.56+.14). the changes in vt and fit2 due to the no (1000 ppm in n2) injection in the ventilator external circuit were compensated for. results .34 the dose of 0.5 ppm, ineffective on papm, significantly improved oxygenation. the increase of pat2 and the decrease of q'va/q' and papm were nearly maximal at 5-10 ppm. no deterioration of arterial oxygenation was observed at no doses as high as 100 ppm. co2 exchange was not influenced by no inhalation. systemic hemodynamic variables did not change throughout the study. these results suggest that a concentration around 10 ppm is adequate for obtaining maximum effects on hypoxemia and pulmonary hypertension in patients with ards. low-dose inhaled nitric oxide (no) induces redistribution of pulmonary perfusion in patients with severe ards and causes improvement of oxygenation [1] . however, addition of exogenous lowdose no in the inspiratory gas mixture might be only a replacement of missing atmospheric no (2-130 ppb) in hospital central-supplied medical air. [2] we have realised nitric oxide measurements in ten healthy volunteers, (4 smokers and 6 non-smokers) breathing with a mouthpiece and occluded nostrils through a ventilator circuit, with separation of inhaled and exhaled gases by a valve. no concentration was measured with a double-chamber chemiluminometer (environnement sa, france) and with charcoal/silicate purified compressed air. there was no nitric oxide detectable in the inspirat0ry limb of the ventilator. unfiltered central supply medical air contained :20 -50 ppb of no and 10 -30 ppb of no2, whereas central supplied oxygen was no/no 2 free. samples were taken after equilibration periods of 5 minutes, with increasing fit2 levels of 0.21, 0.50 and 1.0 for subsequent 5 minutes periods; paired values were recorded every 30 s. the mean no value was 4.57 ppb (sd 2.51) and n o significant differences were found for different fit2 levels both in smokers and non-smokers. these data suggest that the no concentration of pulmonary origin in the exhaled air of' healthy volunteers is probably lower than that reported by other authors [2] and that, previously reported, differences between smokers and non-smokers are not always striking [3] . we suggest the use of activated charcoal/silicate filters for clinical trials in order to achieve standard conditions. [ objective: to compare efficacy and safety of two doses of salbutamol. methods: sixteen adults who had severe acute a~hma were randomly assigned to receive either 10rag (n=9) or 5rag (n=7) of nebulized sulbutamol. both groups were similar with respect to age, duration of a~hma, duration of attack before arrival at the hospital and severity of a~hma according to baseline measurements (table) . evaluation was performed 30, 60, and 120 rain after the start of nebulization. results: compared with 10mg regimen, 5mg regimen resulted in the same improvement in peak-flow and fischl index (figure). the changes in heart rate, respiratory rate and pace2 did not differ significantly between both groups. the incidence of side effects, which included tremor, palpitations, cardiac arrythmlas and other symptoms, was not sj~ificanfly different in the two populations. conclusion:the results of this study suggest that nebulization of 10ng of salbutamol is not more effective than 5rag in the initial treatment of acute severe asthma in adult patients. the prognostic factors of neutropenic patients admitted to the icu remain poorly known. the aim of this study was to determine the respective weight of underlying malignancy and organ system failures on the outcome of these patients. patients and methods: the charts of 107 neutropenic patients (wbc < 1000/mm 3 and/or pmn < 500/ram3), admitted to the icu between 1986 and 1990, were retrospectively reviewed. the characteristics of the neoplastic disease (h~emopathy or solid tumor, tumoral evolution, duration of cancer disease and of neutropenia), the mac cabe's score, the organ system (respiratory, hemodynamic, renal, neurologic, hepatic) failures and the severity scores (saps, saps ii ,osf) were registred within the 1 st day in the icu. when discharged from the icu, the patients were classified as alive or dead. results: fifty-seven patients (53.3%) had a h~ematologic malignancy, and 50 (46.7%) a solid tumor. fifty-nine of the 107 patients died (55.1%); the mortality rate did not differ between both groups (61.4 and 48% respectively, p = 0.16). with univariate analysis, none of the tumoral features is linked to the prognosis; only the respiratory (p < 10 -4) and cardiovascular (p < 10 -3) failures, and the number of organ system failures (p < 10 -4) are associated to the risk of death. the saps (p < 10 -3) and saps ii scores (p < 10 -4) were higher in patients who died. with multivariate analysis (logistic regression), only the respiratory failure is correlated to the risk of death (p = 10-4); neither the features of the underlying malignancy (p > 0.8), nor the duration of neutropenia before admission in icu (p = 0.83), nor the severity scores figs ii: p = 0.068) are linked to the outcome. conclusions: the tumoral characteristics do not modify the prognosis after admission to the icu. they should not influence the decision to admit or refuse a cancer patient in the icu. respiratory failure at icu admission has the predominent weight on the risk of death in the icu. patients with respiratory acidosis due to asthma occasionally require levels of mechanical ventilation that place them at risk for barotrauma. a few case reports have described the use of an extra-corporeal membrane oxygenator(ecmo) circuit as an alternative means of co 9 removal. generally, this has been used for short periods of time (<24h) without serious complications and with low blood flows through the extra-corporeal circuit. we report a case of refractory asthma who could not tolerate even small-volume breaths from a mechanical ventilator due to severe bilateral airleak. ecmo therapy was initiated at the referring hospital prior to helicoptor transport. high blood flows were used (70% of the patient's cardiac output), sufficient to achieve both co 2 removal and oxygenation. satisfactory gas-exchanged was accomplished (pco2=50-60 mmhg) with nearly total lung rest for a prolonged period (60h). however, the long ecmo duration was associated with two severe complica-ti0ns:1) bilateral hemothoraces due to anticoagu!ation in the extra-corporeal circuit, and 2) prolonged weakness as a result of neuromuscular blockade for six days. the patient was discharged from the hospital in good condition. we present the respiratory and hemodynamic features of this case aw well as the potential complications of ecmo therapy in asthma. objectives: parameters derived from tidal expiratory flow ~e) and volume (vt) can be used to detect airflow obstruction in copd patients who might be unable to perform forced spirometry (e.g., icu). however, indices such as ave/v t and at/re are highly variable (thorax, 1981: 36; 135) . methods: we investigated whether the standardized for v m effective time (teff~) of a tidal breath, which is derived by asimple mathematical procedure (teff,= j'vdt/vt2), is a more reproducible and sensitive detector of airways obstruction, we studied nine normal subjects (5 male, 31 -+5yr) and 13 copd patients (4 male, 61 -+10yr) in the seated position, with a noseclip on. they breathed quietly, through a pneumotashograph to measure flow (v). volume was obtained by numerical integration of thellow signal. each subject had an initial 10-15 min trial run, in order to become accustomed to the apparatus and procedure. when regular breathing had been achieved, all breaths over a5 min time interval were recorded. the mean value of six consecutive breaths (ers criteria) for each subject was used for analysis under the condition that within session variation of tidal volume (vt) was <10%. lung function tests were: in normals (mean-sd), fevl%pred = 100• fevl/fvc%=81-+3% , and in copd patients, fev~%pred=53__.20 and fevi/fvc%=51 --.12%. results: values are shown as mean-..+-sd in the following a su~ve~ os literature sources p~oves that t~aditlona], i.e. medicinal medication and physiothe~apeutic methods os t~eatment often p~ove to be insufficientl~ effective both currently and in the ~emote future. the goal of this study was to investigate the efficacy os t~eatment of b~onchial asti~ma patients by means os speleo-and artificial sp~ay therapy. speleotherapy t~eatment was conducted in the conditions os mic~oclimate os salt mine in solotvino hospital. a~tis sp~ay the-~apy was conducted by means os a self-made device. ou~ method is based on the p~inci-~ le os using the majo~ facto~ of speleo-he~apy -highly dispe~sed sp~ay 0s sodium chloride. the obtained ~esults ~e~e analyzed in five g~adations. at the end os the speleothe~apy improvement and considerable improvement was observed in 75,0~ os patients; inconsiderable improvement -in 15,9~ os patients. having evaluated the e~s os t~eatment using a~tis sp~ay therapy the indices a~e 68,7h and 20,5~ ~espectively. remote ~esults of t~eatment a~e an important index os t~eatment, the ~esult os ~hich ~e~e studied by means 0s a ~uestionnaive-method. patients ~ho had been t~eated by speleothe~apy mo~e f~eguently ~e-po~ted a ~elapse in disease 3ust afte~ the course o~ t~eatment (29,3h). ho~eve~, in a ]ate~ phase the ~emission ~ould last ]on-~e~ (s 6 months in 84,5~ os patients, till one yea~ in 69~9~). in 12,5~ os patients who passed the co~se os a~tificial sp~ay therapy a ~elapse was ~egiste~ed immediately as the co~se os t~eatment. then thei~ condition stabilized ~hile in 73,5~ os patients a period os ~emission lasted s ha]s a yea~. 42,9~ of patients dida't ~epo~t a ~elapse of the disease du~in~ one yea~. evangelismos hospital, critical care department, athens, greece method#: 19 mechanically ventilated patients (5 copd, 6 ards, 8 other pulmonary diseases) were studied in two phases: 1) during the acute phase of respiratory failure; 2) during recovery 5-73 days later. we measured mip and monitored the pattern of breathing while the patients were breathing spontaneously through the respirator (pressure support mode with 3-8 cmh20) until either the point they were unable to sustain spontaneous breathing (sb) any longer (phase 1) or for two hours when they could sustain sb indefinitely (phase 2). subsequently the patients were sedated, paralyzed and mechanically ventilated. then we simulated the pattern of sb at the end of the sb trial by manipulating the variables of the ventilator and assessed respiratory mechanics b y the end-inspiratory and end-expiratory occlusion technique. 1. during recovery, a combination of reduced inspiratory load and increased venfilatory capability makes a patient previously unable to sustain sb to breathe spontaneously. 2. inspiratory load is reduced during recovery, mainly because both intrinsic peep and breathing frequency are diminished. obiectives: although elevated concentrations of a few cytokines have been shown to be present in the bronchoalveolar lavage (bal) fluid (balf) of patients with the adult (acute) respiratory distress syndrome (ards), the pethogenesis of ards is largely unknown. leukemia inhibitory factor (lif), a growth factor recently recognised as a polyfunctional cytokine integrated in cytokine networks was measured in unconcentrated balf of patients from different patient groups. methods: lif was measured in balf by means of a specific and sensitive elisa (detection limit 10 pg/ml)in balf (lavage of 3 x 50 ml in the right middle lobe). results: lif was not detected in the balf of 13 healthy control patients and in only one (34 pg/ml) out of 26 patients at risk for ards (after cadiopulmonary bypass surgery) who underwent bal 4 h after the end of the extracorporeal circulation. high and detectable levels were found in the unconcentrated balf of 10 out of 12 patients with full-blown ards (196 + 80, mean + sem, range 10-985 pg/ml). there was a good correlation between the level of lif in the balf and a number of markers of inflammation: neutrophils/ml (r:0.70, p= 0.01), albumin ( r:0.75, p=0.008) and protein level (r:0.74, p=0.006). conclusions:the biological role of lif in these balfs is not readily explained by its currently known actions and it is unkwon whether lif contributes to or is a response to local tissue damage. our results indicate that this cytokine with lots of interesting _functions is a pert of the inflammatory cytokine cascade in ards. background and obiective : we recently demonstrated that cisapride -a new prokinetic drug -enhanced enteral feeding in a heter0genoas group of ventilated icu patients by significantly accelerating their gastric clearance (crit care meal, 1995 ; 23 : 481-485) . it remains unknown, however, whether certain subgroups of patients might benefit more from adding cisapfide to their enteral nutrition regimen than others. patients with chronic obstructive pulmonary disease (copd) might represent such a subgroup since their illness and its specific treatment put them at risk for gastric emptying disorders. design and setting : prospective, consecutive sample study in an adult medical intensive care unit in a university hospital. patients : 10 mechanically ventilated and hemodynamically stable copd patients. interventions : gastric emptying was evaluated by bedside scintigraphy and expressed as the time at which 50% of a tcg~-labelled test meal was eliminated from the stomach (t 1/2). baseline data (do) were recorded after enteral nutrition reached 1500 to 2000 ml daily. scintigraphic measurements were repeated 4 days after cisapride (10 ml orally, q.i.d) had been added to this regimen (d4). patients were considered cisapride responders when gastric clearance improved by more than 50% from baseline. results : normal values for the test meal and for scintigraphic acquisitions obtained in the supine position were found to be 31 + 15 min. in healthy volunteers (crit care med, 1995 ; 23 : 481-485) . five patients responded to cisapride (t 1/2 : 81 + 31 rain vs. 26 + 10 min at do and d4, respectively) and five did not (t 1/2 : 36 + 18 min vs. 33 _+ 11 rain at do and d4, respectively). in contrast with non-responders, all five responders had clinically significant maldigestion at baseline (excessive (> 150 ml) gastric residues, vomiting (> 3 times/day and abdominal distension) which disappeared in 4 of them after the administration of cisapride. conclusion : copd patients who tolerate enteral nutrition well have basal gastric emptying times which are comparable with those of healthy volunteers and are not influenced by cisapride. however, cisapride treatment provides both scintigraphic and clinical improvement in those copd patients who exhibit clinically obvious gastric emptying disorders. cernv v., dostal p., zivny p., zabka l. dept. of anesth. and critical care, charles university, faculty hospital, i-irade~ kralove 500 36, czech republic objective: the aim of the study was to evaluate the effect of early entera nutrition started within 24 hours of injury on the incidence of multiple orgar failure (mof) in trauma patients requiring vantilatory support. methods: after institutional approval 25 patients were enrolled in the study enteral feeding was begun within 24 hours of injury in 14 trauma patients (en group) admitted to icu. nasuenteric tube was placed as soon as possible after admission into the distal duodenum under endoscopy. additional parenteral nutrition was used to meet patients energy and protein requirements. the control group (pn) consisted of 11 patients fed during this period paretuerally. severity score apache ii, trauma score, cumulative balance of nitrogen (g), incidence of mof (three and more organs) and length of ventilatury support (days) were calculated. values are expressed as mean + sd. results: tab introduction : parenteral nutrition (pn) is an important aspect in the optimal treatment of patients on gastroenterology or intensive care. the aim of this bi-center study in 38 patients has been to assess tolerence and efficacy of a new protein-lipid mixture for pn from a simple preparation. patients and m~hods : patients were selected in two hospitals (tenon and saint-lazare, paris) and were divided into two groups : group a (gastroenterology~ 1 l short bowel syndrome) and group b (intensive care, 27 surgical patients). all patients likely to require pig for a period of 10 days (group a) or 7 days (group b) were studied. the pn regimens administered were the following : combination with 50 g of mct/lct fat emulsion end 9,6 g of nitrogen, in 1 liter end glucose requirements were met by imfizsion of l liter of glucose 20-30 % via a "y " connection. lipid thus provided 3040 % of the non introgen calories. total daily calorie intake was 1540 to ] 940 kced. this study monitored, before and at the end of infusions, the sennn albumin (alb), preaiburtun (prealb), triglycendes (tg), cholesterol (cs), and the serum ammotransferases (sgot and sgpt) end alkaline phosphatase (alp) activities. statistical significances were calculated using the wilcoxon-tost. introduction: many 1cu patients present a catabolic illness in response to inflammation and infection, characterized by a rapid loss in skeletal-muscle mass despite optimal nutritional support. growth hormone (gh) is responsible for a rise of lipolysis, enhancing the energetic balance, and of protein synthesis. recombinant human gh (rhgh) is nowaday available for clinical use, but its cost is very high. therefore, rhgh should only be prescribed to icu patients when its efficacy can reasonably be anticipated (ie. when the patients are catabolic or stressed, but in order to avoid overprescription for unstressed patients and for those who are overly catabolic). hence, we, as others, recently demonstrated that rhgh had no favorable effect in highly stressed icu patients. objective: to detect on a clinical basis, low (ls), mild (ms) and severe stress (ss) states in icu patients and validate this clinical judgement by objective metabolic mesurements, in order to select early those icu patients potentially able to benefit from rhgh therapy. methods: 36 consecutive icu patients were prospectively stratified as ls, ms and ss by two experienced icu senior consultants (temperature; agitation; heart rate; arterial blood pressure; presence of an infection; respiratory rate; exogenous catecholamines). anabolic (insulin, igf-1, gh) and catabolic (cortisol, ghicagon) hormones, and nitrogen balance were determined for each patient within 8 hours after admission in the icu. metabolic and clinical data were then compared. the clinical stress states determined by icu physicians correlate with an objective metabolic assessment. therefore, the patients who will more likely benefit from adjuvant rhgh therapy can be detected simply and early. a prospective study on rhgh therapy in ms icu patients is in progress. berger mm md 1, chiolero r md 1, pannatier a phd 2, berger l 2, cayeux c 1, voirol p 2, hurni m md 3. 1 surgical icu, 2 pharmacy, and 3 cardiac surgery, chu vaudois, ch-iotl lausanne, switzerland objective. nutrition of the compromised cardiac surgical patient is challenging. numerous factors influence the gastrointestinal (gi) absorption function, among which gut perfusion, which depends largely on the systemic hemodynamic status. patients in hemodynamic failure are prone to organ failure, and may benefit from an early jejunal feeding. the study was designed to assess the absorption function after cardiac surgery in patients with adequate and altered hemodynamic status, using paracetamol as tracer of gi absorption. methods. after cardiac surgery, 24 patients, aged 63_+8 years (mean_+sd) were assigned to 2 groups (anaesthesia: fentanyl 20 gg/kg + midazolam): group 1 (n=10): reference group, with normal hemodynamic status, easy recovery. group 2 ('n=14): patients in low output syndrome, cardiac index < 2.5 i/m2 on day 1 (d1) after surgery, requiring prolonged intensive care, mechanical ventilation + nutritional support. paracetamol 1 g, was given intragastrically on d1 + d3: plasma levels measured (h.p.l.c), at administration (to), t30-60-90-120-180-240 and 480 rain. hemodynamic status assessed with pulmonary artery catheter. 5 healthy subjects served as controls. results. compared to healthy controls, absorption was strongly reduced on d1 in all patients (no difference between groups). on d3, peak paracetamol level was significantly lower in group 2 (low cardiac output): in group 2 the area under the curve on d1 and d3 were similar. there was a large inter-patient variability, reflecting the hemodynamic status. conclusion. gi absorption was decreased on d1 in all patients, and reverted to normal between d2 and d3 in case of normal cardiac function, but not in case of low output syndrome. the decrease on d1 can be attributed to fentanyl, known to slow down the gi transit. in patients with cardiac failure, correction of altered absorption was correlated with the hemodynamic status, suggesting that gi absorption is dependent on adequate splanchnic perfusion. the aim of the work was to define specific significance and evaluate efficiency of enteral component of infusion therapy in the intensive care of gastroenterotogic patients of surgical profile with pyo-septic complecations. there were used the methods of radial diagnostics and polyelectrography; the laboratory control on oxygen-transporting function, volumetric and hemodynamic state, changes in metabolic, hormonal and immunologic status was conducted. from january, [992 till november, 1994 there was carried out the randomized study of 155 patients with general purulent peritonitis; among them 70 persons constituted the control group and 85 -the main one. in the main g~oup the intestinal lavage, enterosorption, enteral introduction of nutrient solutions with gradual turn to enteral nutrition by equalized mixture "ovolaet" were started from the first hours after operation. the data obtained allowed to define the specifity of the program of artificial medical nutrition in the group of examined patients, based on necessity of individual selection of media for enteral introduction depending on the stages of intestinal insufficiency syndrome. it was shown that inclusion of enteral component into the program of infusion therapy during early periods stabilized circulation in the regime of moderate hyperdynamia, considerably decreases the deficiency of circulating blood volume, normalizes the values of oxygen transport, consumption an}d extraction, provides the optimal level of mycardial adaptive possibilities without tension of its compensatory functions and pulmonary circulation overload. due to combined application of parenteral and enteral nutrition the metabolic processes are shifted towards anabolism. this is supported by decrease to normal values in the contents of blood aggresive hormones (acth,hydrocortisone) and increase in somatotrophic hormone. the complete parenteral-andenteral nutrition influences positively on restoration of cellular and tumoral immunity, activates the factors of organism nonspecific protection and recovery from immunodepression, prevents the development of immunodeficiency. impact tm vs control. s atkinson, n maynard, r grover, e sieffert, r mason, m smithies, d bihari departments of surgery and intensive care, guy's hospital, london, u.k objectives: comparison of the effect of an immunonutrient enteral feed versus a control on the outcome of a mixed intensive care unit (icu) population. methods: admissions to this multidisciplinary adu)t icu thought likely to stay more than three days and with tube access to the gi tract ~r randomised to receive either impact tm, a feed with supplemental arginine, dietary nucleotides and omega-3 fatty acids, or an isocaloric and isonitrogenous control feed. study end points included mortality and icu stay. approval was obtained from the hospital ethics committee. rosults: 390 patients were entered into the trial. the two groups were well matched for age, sex, and admission apache ii with an overall mean admission risk of death of 30.8 (std. dev. -+ 22.9). on an intention to treat basis, there was a no significant difference in icu mortality, icu stay or standardised mortality ratio (s.m.r.) between the two groups (see table) . similarly, there were no differences after stratification for patients receiving 5 or more litres of feed. conclusion: there is no evidence of an effect of impact@, an enteral immunonutrient feed, on pre-determined end-points (icu mortality, icu stay or standardised mortality ratio) in a mixed intensive care unit population over that of an isocaloric, isonitrogenous control feed. objeeflves: evaluate changes of blood laatate levels according to patient medical status after cvvhd initj,~ion using dialysate solution containing lactate. method: review of medioal records of 20 consecutive patients ~eated by cvvhd (dialysate solution hmnosol lg2, hospal,uk, lactate concentration 40 retool/l). date obtained 1 hr before and 4 -6 hrs at~er cvvhd initiation were analysed. results: all data are presented as mean + sem. in one patient, pre end post filter lactate levds were measured during standard cvvhd setting (blood flow 100ml/mlu, dialysate solution flow i 1/hr), and approximate daily lactate flux into the patient was calculated to be as high as 920 mmol/d. lactate leveh measured after cvvhd initiation increased significenfly compared to baseline levels (3.80+0.67 axtd 2.88+0.68,respectively; p<0.01,paired t-test). when patiente with increased basal lactete (~-7) were compared to paliente with normal basal values (n=13), no difference in laotete increase was fmmd (p=0.22, manova). patiente with severe liver dysfunction (2 points in mop scomlg, n=9) had higher basal laotate levels than patiente with normal or slightly abnormal liver teste (0 or 1 point in mof scoring, n=ll), rite values being 4.04 + 1.17 and 1.84 + 0.23, respectively (p<0.05, student t-test). increase in blood lactate did not differ between these two groups after cvvhd was stetted (p=0.86, manova). in 11 pafiente with invasive hemedynamio mo~, no oorrelation batween changes in lactate levels and eitlm" changes in oxygen ddivery (t2=o.ol; p--o.81) or oxygen consumption (reversed fie, k) (r2-q).o7;p--0.66) were found after cvvhd initiation. conclusion: blood lactate increases on cvvhd with dialysate soh~on rich in lactate. this increase is predominantly caused by influx of lactate into the blood via the filter end does not seem to depend on the liver fimotion and/or oxygen metabolism changes. objectives: the study was designed in order to determine the effect on plasmatic proteins, of two types of aminoacids solutions of parenteral nutrition (pn) adapted to stress, having different concentration of branched chain aminoacids (bcaa), when applying to politraumatized critical patients. methods: a prospective study was performed using a randomized double blind design of 20 polytraumafized patients, split in two groups of ten patients each, with mean ages of 35 _+ 17 an 45 -+ 20 years. due to their condition, all patients required p.n. for at least 9 days. both groups were subjected to isocalorie and isonitrogenous solutions (45 ci/kg/ day and 0.24 g of nitrogen/ks/day), varying only in the concentration of bcaa; solution a having a 10 % concentration and solution b 23 %. blood samples determinations during days 0, 3, 6, 9 after the beginning of treatment with p.n. were total proteins., albumin, trandferrine, protein binding retinol; prealbumine and fibronectine. the anova test (one and two way) was used to compare the values between the two groups. results: the administration of solution a, showed statistically significant increases in the determinations of the values of protein binding retino] (p < 0.05) and prealbumin (p < 0.05). no significant increases were observed in the values of total protein, albumin, transferrine and fibronectin. solution b produced statistically significant increases only in the values of total proteins (p < 0.05). the remaining proteins did not changed from their control values during the whole period of pn administration. comparing both groups, no statistically significant differences were observed related to the type of diet. nevertheless, differences were found in total proteins, albumin, protein binding retinoi, fibronectin (p<0.05) and prealbumin (p < 0.005) in relation to the time course of pn therapy. only the albumin values showed significant differences (p < 0.01) when considering the interaction of both the type of diet and the time course of pn. conclusions: 1. solutions of pn adapted to stress, can maintain the control values of slow turnover proteins and improve the values of rapid turnover proteins. 2. no significant differences on plasma proteins were found between the two solutions having 10 % or 23 % concentration of branched chain aminoaeids. &determination of rapid turnover proteins does not seems useful for discriminating different solutions of bcaa during pn. obiectives; the hormonal changes in the post-traumatic situation often leads to an elevated blood glucose and a negative nitrogen balance. to reduce the elevated glucose production by aminoacids the apprication of xylitol may be an alternative energy source. in a double-blind randomized study we investigated the effects of a xylitol/glucose solution (group a: aminoacids 50 g/i; glucose/xylito180 g/40 g/l) on metabolism and particularly on pancreatic and liver enzymes compared to a glucose based nutrition solution regimen (group b: aminoacids 50 g/i; glucose 120 g/i). methods: the clinical trial was carried out after the approval by the local ethical committee on 31 patients with severe brain injury. there was no difference in body mass index bmi (group a: 25.9 +/-2.7 kg/m 2 and group b: 25.1 +/-2.4 kg/m=), age, and sex. daily individual energy expenditure was measured by indirect calorimetry (deltetrac "~). nutrition was started 24 -48 hours after trauma or surgery with carbohydrates and aminoacids. fat was added 24 h after nutrition had started. to analyze the effects on pancreatic and liver enzymes we investigated the following parameters for 4 days: blood gtucose, serum lipase, serum amylase, asat, alat, ~gt, ap, and serum cholinesterase (che). results: due to the daily indirect calorimetric measurements energy requirements were satisfied. there was no difference in blood glucose concentration and cumulative nitrogen balance between the two groups. neither were there any significant changes in asat, alat, ap, and che for 4 days in both groups. serum tipase steadily rose to 202 lull in group a and 320.2 lull in group b, respectively. conclusions: there was no measurable influence of either nutrition solution on liver enzymes. the xylitol/glucose nutrition regimen does not have any advantage over the glucose based nutrition solution concerning blood glucose level or nitrogen balance. the elevation of serum lipase to a 2-fold level in either group needs further investigation on trauma patients. the effects of fat emulsions in lung function, particularly in lungdamaged patients, have been attributed to alterations in pulmonary vascular tone caused by eicosanoid production modificatione. as the eicosanoid production may depend on the fatty acid profiles of the intravenous fat emulsion, haemodynamic, pulmonary gas exchange and plasma levels of prostanoids were investigated in acute respiratory distress syndrome (ards) patients, during different intravenous lipid emulsions (providing different prostanoid precursors). we studied in a randomized double-blind design 3 groups (n=7 each) with ards. group i (lct) received a fat emulsion with long chain triglycerids (lct-20%), group ii (mct) an emulsion containing a mixture of medium and long chain triglycerids (mct/lct 50/50-20%) and group iii placebo (control), during 12 h (2 mg/kg/min each). we measured before, at the end of 12 h infusion, and 12 h after the end of the infusion: lipaemia, arterial and venous blood gases, pulmonary and systemic haemodynamics, and plasmatic levels (arterial and in mixed venous sample) of eicosanoids (txb=, 6-keto pgf~,, and ltb4). at the end of the fat emulsion, groups (i and il) to 1,10• to 0,51 • mmol/i), the paoz/fio z remained unchanged in the three groups; no changes in intrapulmonary shunt (qs/qt) were shown; neither in the mean pulmonary artery pressure. in contrast, only in the lct group: cardiac output and oxygen consumption increased significantly (12.5% and 19%) (p<0.05). eicosanoids were increased at baseline compared to reference values (p<0,05). a decrease (p<o,05l in the arterio-venous difference of the vasodilatador prostanoid (6-keto pgf~=) was shown in lct group. our results indicate that fat emulsions in ards patient do not influence pulmonary gas exchange, provided that the perfusion rate were keept at 2 mg/kg/min. the present study was designed to evaluate the metabolic changes of a carbohydrate-based diet versus a fat-based diet on oxidation rate of substrates and energy muscle metabolism in patients with an acute exacerbation of chronic obstructive lung disease. patients were mechanically ventilated with a volume-cycled respirator. after an overnight fast, patients were randomnly treated with a continuous enteral feeding over a nasogastrie tube with a carbohydrate/fat ratio of 2/1 in group i (n=6) , and a carbohydrate/fat ratio of 1/2 in group i] (n = 6) during 8 consecutive days. indirect calorimetry was set at baseline and 24 hours later the enteral feeding was started. muscular quadriceps biopsy was performed on baseline and on day 8 of enteral feeding. urine was collected during 24 hours and was used to measure 24h urea nitrogen production. the caloric intake was set according to the measured resting energy expenditure. respiratory gas exchange rate were measured using a computerized open-circuit indirect calorimeter. quadficeps femoral muscle samples were obtained by muscular biopsy after local anesthesia. analyses of glycogen, glucose 6-phosphate, lactate, phosphocreatine and adenosine triphosphate (atp) and enzymes (glycogen synthase and glycogen phosphorylase) were determined. after 24 h of nutrition patients in group i showed an increase in carbohydrate oxidation (from 17.8% to 55.4%, p=0.06), and in group ii a decrease in protein oxidation (from 27% to 21%, p=0.03). after 8 days of enteral feeding a tendency to increase in glycogen concentration (group i, p=0 .06; and group ii,p=0.1) was observed. while patients in group i showed a tendency (p=0.06) to decrease glycogen phosphorylase, patients in group ii maintained these enzymatic levels. the increased in glycogen was correlated in group li with degradation enzyme (p =0.01 ). in group i, the pao2/fio 2 ratio increased after 8 days of treatment (from 235.9 to 284.5; p=0.03), but no changes in days of mechanically ventilation, length of stay in icu, or mortality rate was evidenced between groups. it is concluded that the oxidation rate of substrates used for energy production varies according to the caloric sources administered. with nutritional therapy muscle glycogen concentration increases in both diets, with different enzymatic influence. more studies are warranted to further assess the clinical implications of the different pathophysiologic behavior of the two diets. under certain experimental and human conditions (radiation and/or thermal injury) the gi mucosa is unable to perform this protective function and could play an important role in the pathophysiology of the syndrome of multiple organ failure (smof). the objective of the present multicenter study was to know the gi mucosal permeability in critically ill patients and the relationship with their outcome. the method used to assess the integrity of the gi mucosal barrier measured mucosal permeability to various hydrophilic compounds. specifically, we measured the urinary excretion ratio (uer) of two sugars (lactulose and mannitol) that had been previously administered through a nasogastric tube. urinary excretion rate was measured in 10 healthy humans (control) and in 30 patients admitted to the intensive care unit (icu) at days 1, 3 and 5. the apache ii of the patients studied was 15_+4. there was an increase of uer in the critically ill patients compared with controls (o.26_+0.1 vs 0.036_+0.01) (p<0.001). in contrast, no changes in uer between days 1, 3 and 5 were observed (0.26 -+ 0. . significant changes were also established in the different groups: major and minor surgery, general and epidural anaesthesia , transfused and non transfused patients except for iron and ferritin plasma levels which didn't change significantly in patients who had received blood. a positive significant correlation was established between crp and ferritin (r=0.89) for patients under general anaesthesia, but not on epidural, regardless of the type of surgery. conclusions: iron and transferrin plasma levels fell acutely in surgical patients while ferritin and crp rose. transfusion implied an acute iron load.the established correlation between cpr and ferritin in patients on general anaesthesia suggests that ferritin behave as an acute phase reactant in that setting, while epidural anaesthesia may ameliorate the inflammatory response. objectives: to analize the effect of gastrointestinal complicacions (gic) related to the enteral nutrition (en) in the real volume of diet administered to icu adult patients. a prospective multicenter study was designed (comgine study) in wich en application, gic definition and his management were defined by collaborative consensus.patients treated with en in one month pedod were included. in each case, daily volume of diet prescribed (pv) and administered (av) was recorded and the volume ratio calculated (vr=pvxl00/av). patients were divided for days of en according to the presence (group 1) or absence (group 2) of gic (abdominal distension, increase of gastdc residuals,vomits or regurgitation, diarrhea,constipation and bronchoaspiration). differences between groups were analized by anova and mann-whitney u test with p<0.05 for statistical significance. (vr%) 53 61 58 89 67 63 65 72 62 60 64 59 67 7461 group2 (vr%) 85 90 90 94 94 93 92 90 92 91 92 94 90 92 tolerance to the enteral nutrition (en) has been related to the severity of illness in icu patients. the aim of this study was to evaluate this relationship. methods. a prospective, multicenter, study was performed (comgine study) in wich en application and related gastrointestinal complications (gic) were defined by collaborative consensus. adult icu patients treated with en in one month pedod were included. gic were classified as: 1) abdominal distension (abd), 2) increase of gastdc residuals (igr), 3) vomits (vom), 4) diet regurgitation (reg), 5) diarrhea (dia) and 6) constipation (con). patients were divided in two groups: group a: patients with gic dudng their evolution. group b: patients without gic. differences between groups were analized for apache ii admission score or evolutive variables ( carbon dioxide production is a major determent of work of breathing. increase in co2 production during nutritional support may precipitate ventilatory failure and difficulty in weaning from mechanical ventilation. in the present study, co2 output was calculated while passive mechanically ventilated patient were fed with different amount of calories and different proportion of protein, carbohydrate and fat. four clinical stable and mechanically ventilated patients were studied. carbon dioxide was calculated while patients were paralyzed, sedated and mechanically ventilated. six nutritional regimens were used: a) no calories, b) t00gr glucose/24h, c) calories equal to rest energy expenditure in special formula with low carbohydrates and high fat (pulmocare) d) calories equal to rest energy expenditure in standard formula (nutdson) e) calories double to rest energy expenditure in standard formula (nutrison) and f) calories equal to rest energy expenditure parenterel (tpn). carbon dioxide output was low while no calories and/or only 100 gr/24h glucose was provided, the mean values of vco2 output were 134_+_25 and 140!-_26 ml/min respectively. there was no difference in vco2 output between with low carbohydrates and high fat regimen and standard regimen, mean values were 16t .5-+27 and 163_+26 respectively.the high calories regimen and tpn resulted in higher vco2 output, mean values 184_+25 and 211• respectively. we conclude that the special with low carbohydrates and high fat regimen did not reduce vco2 output, in the contrary, high calories intake as well as tpn increased vco2 output under tested conditions. klouche k., cristol j.p., vela c., canaud b., b6raud j.j. m6tabolic intensive care unit and nephrology department. lapeyronie hospital. 34059 montpeuier france. predictive factors for rhabdomyolysis (rh) -induced acute renal failure (arf) were studied in a retrospective study from january 92 to january 95. 33 patients (pts) (male:23, female:lo; mean age: 57+4 y.o.) were admitted with rh defined as cpk>1000 iu/1. etiologies were: traumatic and ischaemic 24, infectious 4, toxic 4, excess activity 1. factors studied were: simplified acute physiologic score (saps: 10.3+1.1), organ systemic failure (osf: 1.02_-!-0.2), diagnosis delay (d: 33+_5h), clinical parameters (sepsis, dehydration), blood chemistry data (cpk, bun, creatinine, potassium, phosphorus, calcium, proteins, hematocrit) and urinary ph. severity of rh was estimated by ward score determined according to phosphorus, albumin, potassium, cpk, dehydration and sepsis. urea appearance rate (uar) and creatinine index (ci*) were determined over a 24 hours period. arf was observed in 25 pts. in non-arf and arf groups respectively, saps (5.5_+0.5 vs 11.8+1.3), deshydratation (0 vs 11), sepsis (0 vs 12), phosphorus (1.03+0.16 vs 2.21-+0.21), calcium (2.1+0.07 vs 1.8_+0.07), ward score (4_+0.65 vs 11.8+0.8) were significantly different. however, no significance was observed in uar (310-+89 vs 210-+35) and ci (26_+7 vs 34_+3). 16 patients required hemodialysis (hd) (85:2 sessions) and 9 remained dialysis free. only osf (1.1_+0.1 vs 1.9-+0.23), ward score (9.2_-/-0.95 vs 13.25_+0.92) and ci (29+_3 vs 41-+3) appeared significantly higher in pts requiring hd. 5 pts died from associated disease. all patients suffering from arf recovered a normal renal function. we confwmed that an elevated ward score (over 7) is a good predictive index of arf. in addition we found that ci is a severity factor for arf requiring hd. thus, patients suffering for rh with elevated ward score and ci, have a fair chance of dialysis and should be treated more intensively. * ci (expressed in mg/kg) = (car + feces creatinine) / weight. where car: creatinine appearance rate; feces cr~t..= mean plasmatic creatinine x 0.043. tr~er k., cetin t.e., tugtekin i., georgieff m., ensinger h. universit~tsklinik flir an~sthesiologie, 89070 uim, germany introduction: endogenous as well as exogenous adrenergic agonists have a profound effect on carbohydrate metabolism in human critical illness. in this study the effects of noradrenaline (nor) and dobutamine (dob) on carbohydrate metabolism during a 4 hr infusion were investigated. methods: after approval by the local ethic committee 14 healthy volunteers were studied. hepatic glucose production (hgp [mg/kg/min]), using 6,6-d2glucose as stable isotope tracer, as well as plasma concentrations of glucose (glc [mmol/i]) and lactate (lac [mmol/i]) were measured prior and during infusion of nor (0.14 pg/kg/min) and dob (6 pg/kg/min). blood samples were drawn before and during the agonist infusion. results: no major changes in insulin and gtucagon plasma concentrations could be found during the study period. ::i:::: :iiiii~ 8~ i ::i: ~:: : :: i:ii. mean-+sd are shown. # p<0.01, anova for repeated measurments. conclusions: the effect of nor on hgp and glc were smaller as compared to adrenaline (i) with a similar time course. in contrast to the effects of adrenaline and nor, dob had a different effect on carbohydrate metabolism: a decrease in hcp and glc, which is uncommon for a /3-adrenoceptor agonist. since hgp is an energy consuming process that might deteriorate hepatic oxygen balance in critical illness, the differential effects of adrenergic agonists may be of importance and need further clarification. the nutritional insufficiency often accompanies post-operative hypercaloric states, inanition, serious infections and weakening chronic illnesses. that is why the early nutritional support, sufficient and appropriate for each individual base, is a fundamental component of intensive care unit as an indispensable factor for recovery. per this reason, our unit, developed a software for the implementation and nutritional control of t~e assisted patients. this software is incorporated is an expert system called ~i~su, designed and developed by the computational division of our unit. this system arrives to inferred diagnoses such as : respiratory, hepatic, renal(with and without dialysis) dysfunctions, pancreatitis, ards, decrease of consciousness, diabetes. according to these data objectives: to compare the effect of short term enteral feeding versus parenteral nutrition, when a isonitrogenous and isocaloric feeding solution is administered by either mute. methods: in a prospective controlled clinical trial 30 patients were studied; all exhibited moderate degree of malnutrition, normal liver and kidneys, and a functi6ning gastrointestinal tract. the patients were randomized to receive a free amino acid and small peptide diet (15 patients) or an isonitrogenous isocaloric parenteral support (tpn) (15 patients) (total energy: 2880 kcal, nitrogen: 14.5 g, carbohydrates: 380 g, fat: 112 g, n/non protein calories: 1/175) at least for 10 days. results: there were no significant changes in anthropometric parameters within either group. nitrogen equilibrium was aqhieved by day 3 in the tpn group and by day 5 in the enteral group (66.6% of the enterally fed patients and 80% of the tpn patients maintained in positive balance the day 10 of the study). there were no significant changes in serum albumin within either group. serum level of transferrin reached a significant increase in both groups (p=0.003). thyroxine-binding prealbnmin rose significantly in both groups as well (p=0.019 and 0.004 respectively). statistically significant rises in lymphocyte counts (p=0.003 and 0.001 respectively), in levels of c 3 (p=0.009 and 0.01)1 respectively), iga (p=0.002), igg (p=0.004 and 0.003 respectively) and igm (p=0.004) occurred in either treatment group. there was a high incidence of negative skin tests at the start of the study in the enteral group (73.3%) and the tpn group (60%). by the end of the study the incidence of negative responsiveness was 40.0% and 26.6% respectively. despite maintenance of similar glucose levels in both groups, tpn led to significantly higher serum insulin levels. the serum insulin increased almost linearly over the study period and eventually prevented fat mobilization and lipolysis, so that free fatty acid levels had fallen significantly. a significant elevation of the liver enzymes over the study period occurred in 73.3% of the tpn group, but not in the enterany fed patients. conclusions: the present findings provide no evidence that enteral diets containing free amino acids and small peptides, as their nitrogen sources, are in any way inferior to isonitrogenous isoealoric regimes parenterally given. aim: the aim of this study is to describe and explore the expectations of the functions of the critical care nurse to enable the formulation of guidelines for the scope of practice for the critical care nurse with a south african context, methods: phase i was to determine the expectations of the critical care nurse, the nursing service managers and the doctors with regard to the functions of the critical care nurse. a focus group interview was held with a group of experts in the field of critical care. the results were used to compile a questionnaire. this questionnaire was sent to the critical care nurses, the nursing service managers and the doctors in south africa for completion. from these results the functions of the critical care nurse were determined. phase ii was to formulate guidelines for the scope of practice for the critical care nurse within a south african context. through usage of the date (phase i) the scope of practice was formulated. guidelines were formulated for the practise, education and research regarding the limitations of the professional-ethical authoration and the implementation of the scope of practice for the critical care nurse. objectives : high output gastric aspirates arc occasionally observed during fasting in critically ill paticnts, preventing any attempt of feeding via the enteral route. although these patients are often said to suffer from "gastroparesia", the motor correlates of this condition arc lurgcly unknown. in this stud?', wc recorded the gastrointestinal motility of critically ill patients with abundant (>250 ml/24 hours) fasting gastric aspirates. methods : antral (4 sites separated each other from 1.5 cm), duodenal (1 site) and jejunal (1 site) contractions were recorded simultaneously by ~eans of a multihimen tube assembly positioned trader fluoroscopic control (perfused catheter technique). tracings from prolonged recordings were obtained on a multichannel recorder (7758a recorder, hewlett-packard) then anal)7,ed visually, with a special attention for the following abnormalities which are characteristic of intcstinal pseudoobstmctiou: l) absence or aberrant propagation of the migrating motor complex (mmc), 2) presence of bursts (> 2min) of nonpropagated phasic pressure and 3) presence of sustained (>30 min) uncnardinate pressure activity. 11 patients with a volume of gastric aspirates of 731 • 506 (sd) [median 5001 ml/24 hrs were investigated for 538 4-271 [median 4551 minutes. results : only one patient had no detectable motor abnormality. mmcs were either absent (n=4) or migrated abnormally (retrograde propagation : n=4; retrograde and stationnary : n=2) in 10 pts. bursts of nonpropagated phasic pressure activity were present in the duodenum in 9 pts and sustained uncoordinate pressure activity was found in 2 pts. additional abnormalities included episodes of prominent pyloric activity. (n=l) and sustained antral pressure activity (n=2}. conclusion : critically ill patients with large volume of gastric aspirates have manometric evidence of intestinal pseudoobstruction. prokinetic therapy in these patients should thus focus not only on enhancing gastric motility, but also on restoring a normal propagative contractile activity in the intestine. this prospective, open-label, randomized placebo-controlled study included 20 patients with hypokalemia in whom rapid potassium replacement (20 meq kci in 1 h) was performed: 14 patients received mg sulfate (6 g in 3 hours) and 6 patients received a corresponding saline infusion. measurements were made at time 0, +1, +3 and +6 hours results: k levels increased more in mg treated patients than in the patients who received saline infusion at time 1 and 3 h (p < 0.05 -students-newman-keuls). (table 1 ). introduction. dual lumen uaso-gastrojcjunal tubes are a major ads'ance in nutritional therapy of mechanically ventilated critically ill patients since the3" authorizc jejunal feeding with concurrent gastric decompression, there,, reducing the risk for aspiration. unfortunately, placcmem of these tubes in the jejunum regularly dictates to resort to endoscopy in order to facilitate pyloric intubation. recently, the remarkable gastrokinetic properties of the well known macrolide antibiotic er}lhromycin have been demonstrated in gastroparetic critically ill patients 1. aim. in the presem stu~,, we evaluated the feasibility of placing dual lumen naso-gastrojcjunal feeding tubes at the bedside without endoscopy, using edthromycin to help iranspy'loric migration of the tube under fluoroscopic control. methnd each patient admitted in our icu during a 2 months period and requiring artificial ventilation and enteral nutrition for a period of at least 3 days was included in the study.. after inserting the tube (stayput| sandoz, usa) in the gastric anmnn, e.rythromycin (200 rag) was aduunistored intravenously, to help fluoroscopic positioning of the tube into the jejunum. the total duration of the procedure (from nasal intabatiun to jejunal placement), as well as the duration of ftuoroscopy were recorded in each patient. results. 15 patients (male/female : 13/2: mean age : 56.9 + 22.2 years; mean apacbell score : 23.t • 7.0) wore enrolled into the study.the procedure was performed within the 2 dab,s following institution of mechanical ventilation. jejunal access was obtained in all 15 patients without resort to enduscopy in 10,81 • 7.31 min.(total duration of the procedure). mean duration of fluoroscopy was 3.54 + 2.97 rain. conclusion. we conclude that placement of dual lmnen naso-gastrojejunal tubes can be obtained in mechanically ventilated critically ill patients without resort to endoscopy., provided that e rythromycin is used as gastrokinetic agent to help pyloric intubation. the following ad and dis parameters were considered in all patients: -mid arm circumference, triceps skinfold thickness, serum transferrin, albumine and lymphoeites and urinary creatinine/height index. patients whose results were bellow 80% of normal values in 3 or more of the 6 above criteria were considered undernourished (und).statistical analysis was performed using %2 analysis.statistical significance was established at p<o.o5. results: a total of 129 patients(47 female, 82 male) -with a mortality rate (~) of 31,8% -were studied, aged 56-+8 years and a median ]enght of stay of 22 days. 1 -nourished (nou) at ad and at dis -22; ~ 0%; 2 -nou at ad and und at dis -17; ) 41,2% ; 3 -und at ad and und at dis -73; ~ 45.2%; 4 -und at ad and nou at dis -17; ~ 5,9%. we observed a p< 0.05 when we compared groups 1 and 2 (p=0.0012), and groups 1 and 3 (p=0.00026). variation in nutritional status and longht of stay: 1-nou at ad and nou at dis = > median lenght of stay 18 days; 2 und at ad and und at dis = > median lengbt of stay 22days; nutritional status and age at admission: 1-age > = 60 years : nou (13) , und (53) 2-age < 60 years: nou (26), und (37) nutritional status and age at discharge: 3-age > = 60 years : nou (12) , und (54) 4-age < 60 years: nou (36), und (27) we observed a p<o.05 when we compared groups 1 and 2 (p=o.01) and groups 3 and 4 (p =0.004) conclusion: such study permits to conclude that most of our patients (69.8%), staying in icu for more than 6 days, were und, namely the elder. we observed that this kind ef patient had a longer median length of stay in icu, associated with higher mortality rate. in conclusion, the nutritional management is an essential element in suportive care of critical)y ill patients. ohiectives: sirs is associated with hypercataholisnl and resistance to nutritional support, despite raised circu&ting levels of growth hormone and insulin. serum igf-1 levels however are low; thercti~re rhigf-i administration might produce nsefnl anabolic effects. the pharmacokinetics of exogenously administration rhlge-i in such patients are likely to differ from that seen in the less seriously ill due to changes in circulating blood volume, increased capillary permeability, poor nutritional stares, and alterations in binding proteins. the ol2jective of this study was to determine the clearance, apparent vohnne (if distribution (vd), time to peak concentration (tmax) and elimination half-like (tia) of a single subctkaneous il~ieetion of rhlgf-i in patients with sirs on the intensive care unit (icu). methods: 9 icu patients with sirs were entered into the study, which had been approved by the ethics comjnittee. in each patient 6 baseline hlood samples for circulating 1gf-i were obtained over a 24 hour period, prior to il!iection of 40 p.g/kg of rhlgf-1 subcutaneously. a further l4 samples were taken fi'om each subject over the next 48 hours. circulating ige-i was measured in serum at each time point by radioimmtme assay, and the area under the curve tbllowing administration of rhlgf-i was derived tbr individual patients fixm~ baseline a~!justed igf-i levels. clearance values were caietdated by dividing the administered dose of rh[ge-[ by the total area under the etnve. vd and tv2 were calculated by regression analysis of the terminal part nf the log sertnn concentration time profiles. results: results are expressed as median (range). age 35(29-73) years, apache ii score 18(12-34), and length of icu stay 26(10-92) days. baseiine circulating igf-1 levels were low 28( < 20-166) ng/ml reaching a peak of only 84( < 20-246) ng/ml. 2 of the 9 patients had basal levels predmnhaantly below the inwel-limit of detection of the assay, and showed no appreciable rise in serum igf-[ levels tbllowing rhigf-i injection. parmacokinetic variables could not therefi)re be determined in these two patients. following rhlgf-i injection in the remaining 7 patients tmax was at 3(2-6) hours, clearance was 89(31-452)0 ml/min, vd was 0.29(0.12-0.73) l/kg, and tia 9.2(4.2-30.1) honrs. conchlsions: there was marked patient wu-iability in response to rhlgf-i administration. vd was similar to that seen in poshq~erative maior surgery patients (0.29 vs 0.33 l/kg), but clearance was greater (89 vs 25 ml/min), tmax earlier (3 vs 5 hours) and t~a shorter (9.2 vs i0.8 homts). if riaigp-i is to be administered to dtically ill patients the dose should he nlodified m the light of these findings. baekgronnd: acute bacterial endoearditis continues to be a condition with high morbidity and mortality. the majority of patients are treated with high dose antibiotics, but a patient group requires surgical treatment. methods and results: from january 1991 to march 1994, 65 patients underwent surgery for acute bacterial endocarditis: 36 had native valve endocarditis and 29 had prosthetic valve endocarditis. there were 48 men and 17 women. streptococcus viridans was the predominant infecting agent in native ~alve endocarditis and staphylococcus epidermidis in prosthetic valve endocarditis. progressive congestive failure, uncontrolled sepsis and peripheral emboli were the most common indication for surgery. although the mitral valve is more commonly involved in acute bacterial endocarditis, the aortic valve most often requires surgical inlervetion. the postoperative bleeding in the no-aprotinin group was 1902+1450cc and in the aprotinin was 850:t:80 (p < 0,0001). the mortality in icu was 10,7%, the staphyloccocical endoearditis mortality was 18,2% and the no-staphyloccocical endocarditis mortality was 2,6% (p<0,05). conclusions: the early surgery is indicated if endocarditis has no response with medical treatment. the mortality of staphyloceocical endoearditis involving left valves was higher than caused by other infecting agents. aprotinin treatment improved prosta#andin metabolism and preserved pletelet function during open heart surgery and could be useful in this type of interventions. urgent surgery had a high mortality (24%). selective digestive decontamination (sdd) has increasingly become the subject of critical discussion sine~ the development of multiresistant organisms has become an issue. moreover, a selection of gram-positive pathogens must also be taken into account when using the sdd regimen, which is primarily directed at the gramnegative bacterial specumn, with the methicillin-resistant staphylococcus aureus strains (mrsa) being of particular importance. the objective of our study was to determine the extent to which colonization with mrsa strains is promoted by sdd. method: 65 patients (ventilation period > 5 days) were randomized and allocated to the sdd group (n=32) or the control group (n=33). in their general intensive care theraw, there were no differences between the groups. the sdd regimen consisted of the four times daily administration of 50 rag polymi~ 80 mg tobramycin and 500 mg amphotericin b in the nesc, mnoth and stomach. systemic prophylactic ~dmini~/rution of antibiotics was not part of the sdd regimen. smears were taken from the nose and the rectum twice wceldy and from the pharynx and trachea once wceldy, and tested for mrsa. further samples were taken as clinically reqnircr results: 625 smears were examined in the sdd group. mrsa strains were detected in 66 samples (10.5%) from 7 patients, and in 5 patients they were detected for a period of up to 4 weeks. the positive smears were districted as follows: tracheal 11/117 (9.4%), nasal 28/199 (14.0%), pharyngeal 15/111 (13.5%) and rectal 121198 (6.1%). severe mrsa-induced infections were observed in 2 patients (infection rate 28.6% of the colonized sdd patients). 560 smears were examined in the control group. ivlrsa swains were r in 15 samples (2.6%) from 6 patients, but only repeatedly over a period of up to 10 days in 3 patients. the po~tive snmars were distributed as follows: traclmal 1/114 (0.8%), nasal 8/174 (4.6%), pharyngeal 4/98 (4.0%) and rectal 2/174 (1.1%). there were no mrsa infections in the control group. conclusion: the data collected support the view that the use of sdd promotes a selection and persistence of mrsa strains. longer-term colonization with mrsa and sovere systemic inf~ons were only found in the sdd group. although the clinical and epidemiological impact of resistance develol~ng when sdd is applied ~maine unclear, this question should be given close scrutiny. tazobactam/piperacillin (taz/p1p) is a new broad spectrum antibiotic, in which the acylaminopenicillin piperaeillin is protected by the betatactamase inhibitor tazobactam from hydrolization by bacterial enzymes. taz/pip has shown to possess a high antibacterial activity against almost all clinically relevant bacteria and is a registered drug in germany. obiectives: purpose of this investigation was to evaluate, whether faz/pip 4.5g is suited for efficient antibacterial monotherapy of severe infections and what influence dosage frequency reveals on clinical efficacy. methods: 2151 hospitalized patients have been documented in this multicenter trial during a 2 year period. as this investigation should reflect the usual clinical treatment, the only criteria for enrolment were the typical signs of infection as e.g. temperature > 38~ leucocytosis or an isolated pathogen. exclusion criteria did not exist and the patients were treated in accordance to the severeness of infection, underlying diseases, risk factors etc. with taz/pip 4.5g t.i.d, or b.i.d. results: patients suffered in most cases from infections of the lower respiratory tract (n=926), followed by intraabdominal (n=765) and skin and soft tissue infections (n=460). 61% of the 926 lrtis wvre nosocomial acquired and in 75% the treatment was conducted as monotherapy. in 53% the lrti was treated with taz/pip b.i.d, and in 45% t.i.d. pseudomonas spp. (n=138) and staph..aureus (n=134) were the most isolated pathogens pretrcatment. the clinical response rates (cured/improved) after treatment with taz/pip 4.5g b.i.d, and t.i.d, were 89% and 81% respectively. results for intraabdominal-and skin and soft tissue infections will be presented. conclusions: in hospitalized patients with severe infections successful treatment with taz/pip in monotherapy is possible. in this population a reduction of the dosage frequency to 4.5g b.i.d, revealed equivalent clinical response rates. objectives. retrospective evaluation of 9 cases of severe generalized tetanus (sgt), treated in our icu the last 7 years. we review 9 cases of sgt (6m, 3f), mean age 66.7 years. in 5 eases the entry site of c.tetanus was a skin laceration, in 1 case it proved to be the external genitalia, while in the rest no portal of entry could be determined. in the first 6 cases incubation period was short (3-11days) and so was the period of onset (1-6 days). all patients needed mechanical ventilation (range 15-58 days), initally through an orotracheal tube,and later through a tracheostomy, performed 6• days after admission. clinical manifestations of sgt included muscle rigidity and i generalized spasms, persisting for up to 6 weeks in the most severe cases. significant autonomic nervous system dysfunction was present in 3 cases occurring 5-12 days after the admission and following the time course of generalized spasm. besides general supportive measures, specific treatment included passive +active immunization, penicillin g, magnesium sulphate and sedation in a variety of regimens. neuromuscular blockade was required in 5 cases. nosocomial infections occurred in 7 eases, with sepsis and mof in one. average stay in the icu was 18-62 days. one patient died with severe septic complications and one was discharged with severe 9 disability due to anoxaemie ancephalopathy, after a cardiac arrest on admission. ~ disinfectant in suspension test, without presence of organic load, 12 disinfectants showed efficacy on lm. in the carrier test, in the presence of organic load, 6 out of 14 examined disinfectants did not exposed efficacy on lm. the results of examinations clearly showed that evaluation of disinfectant's efficacy partly depend on the used test method. antun basi6, intensive care unit, kb firule split spin~ideva 1! jugoslavia bacteremia and sepsis are frequent complications encouuntered in severe icu patients.microorganism identification with hemoculture presents the basis for adequate and successful antibiotic treatment.in many patients damage and vulnerability of the peripheral veins presents an obstacle for obtaining the blood culture from the central venous (cv) catheter sample could be also used. material and methods blood cultures were perfomed in lo4 patients on blood samples simultaneously obtained from the peripheral vein and cv catheter three times in a 24-hour period.criteria for the suspected bacteremia were body temperature above 38 c and leucocytosis above ioooo leucocytes/dl. the site for venipuncture and the cv catheter stopcock port were cleansed with povidon iodine.after the initial 5 ml of blood were discarded,lo ml were used for the blood culture.standard laboratory technique for blood cultures was used. results and discussion in 76 (73%) patients hemocultures was negative at both sites,whereas in the remaining 28 (27%) they were positive.for twentyone (26~176 of the positive patients the same results were obtained at both sites (peripheral vein and cv catheter),whereas in 7 (6.7%) patients the blood culture were positive only for the cv catheter samples.the cv catheters were in place for less than 4 days in 81 patients and for more than 7 days in 23 patients.from 7 patients with positive blood culture from the cv catheter,one patient had the catheter for three days,whereas the other 6 had the catheter from 6-1o days. we neither found significant differences in hemodynamic dates : objectives: 1, to count and evaluate bacteria isolated from endotracheal (et) suctiori samples (with and without saline). 2. to establish the exogenous source(s) of pathogens isolated from carer's hands and the equipment involved in sampling in order to reduce the incidence of contamination and infection. method~: this prospective study included 20 consecutive ventilated patients (15 male and 5 female, 56_ + 16 yr; apache ii score 19-+7) over a period of 3 months. et aspirated samples with and without saline were taken daily from day 0 of intubation until pathogen~ were presented in counts of _> 105 per ml. at the same time, samples from both carer's hands were taken before and after et suction and a swab from the ventilator tube. results: the overall length of intubation varied between 3 to 65 days. bacterial transfer between staff and patients was noted in 80% of patients until day 5 of intubation. there was no significant correlation between severity score and appearance of colonization. the incidence of pneumonia in studied patients was 45% with an overall mortality rate of 30%. acinetobacter anitratas (no 15), staphylococcus aureus (no.15), klebsiella pna~moniae (no.9) and pscudomonas aeruginosa (no.4) isolates predominated in all our specimens. we noticed increased resistance to most antibiotics with the exception of imipenem for gram (-) bacteria and vancornycin for gram (+) bacteria. conclusions: i. tracheobronchial colonization appears directly in the maiority of intubated patients. 2. there is a close relationship between the microflora of personnel, patients and equipment. 3. bacteria transfer was noted both to and from patients. 4. strict hand disinfection policy remains an important measure for the proper care of mechanically ventilated patients to reduce respiratory infections. nnseeomial pneumonia is the most common nnsocomiai infection in the icu-settiag, reported in up to 20% of patients admitted to the icu following surgery. it is associated with significant mortality that ranges from 30~ to 70%. enteric gram-negative bacilli have been implicated in 75% to 85% of ventilntor-associated pneumonias and pseudomonas aeruginosa accounts for 27% to 40% of these pneumonias. importantly, epidemics of/3-1actamnse-pruducing enterobacter spp or klebsiella spp that are resistant to extended spectrum cephalosporins or penicillins, pose serious obstacles to effective antibiotic choices. carbapenems provide in ~tro activity against a wide range of enterobacteriaceaeand other gramnegative aerobic bacteria, except steaotrophomonns maltophilia. in vitro meropcnem is more active against pseudomonas spp than imipanem (especially p. aeruginosa and p. cepacia), imipenem and meropenem are effective against more than 95% of strains responsible for nnsocomial infections. all major pathogens associated with lrti are usually covered by the carbapenems, exceptions are pathogens involved in so-called atypical pneuomouia like mycoplasma, chlamydia and legionella. carbapenems are highly stable in the presence of most chromsomal and plasmid-mediated blactumases and usually offer a postantibiotie effect lasting for three hours against most of the enterubacteriaceae. reeent studies comparing imipenem/cilastatin with other ~-lactams and fluoroquinolones in severe lrti in icu patients resulted in favourable clinical cure rates and good tolerance, but development of resistance in p. aeruginosa and 5;. aureus during treatment were of some concern. meropenem offers the advantage of greater stability against enzymatic degradation, so no concomitant administration of an enzyme inhibitor is necessary, and meropenem appears to be associated with a lower risk of seizures, particularly when used at high doses. results from studies with meropenem in lrti, especially in critically ill patients with acute exacerbations of chronic bronchitis, demonstrated excellent cure rates and better gastrointestinal tolerance of this new carbapenem. both earbapenems are effective candidates for use as empiric monotherapy in nosucominl infections of critically ill patients. qbl~ctives a favourable effect of iv immunoglobulins in septic surgical patients has been reported, but not sufficiently validated. we conducted this study on trauma patients to: i) investigate the effect of ivig on septic complications and il) quantify this effect by means of serum bactericidai activity (sba) assessment and iii) to explore the effect of temperature increase (from 37 to 40 ~ c) on the sba methods: twenty trauma patierits matched on admission for age, sex, inju~ severity score and glasgow coma scale, were allocated to receive either wig (ivig group; i0 patients) or equal volumes of human albumin 20% (control group; 10 patients). wig (sandoglobulin) was administered in a total dose of 1 g/kg divided in a four time regimen on days 1, 2, 3 and 6 post-admission. three blood collections were performe& before the first dose (day 0) and 24 hours after the third and the fourth dose (days 4 and 7 respectively). complement, lgg fractions, the sba at 37 ~ and at 40 o c and clinical parameters were recorded. results-similar lgg and igg] serum levels were found in groups ivig and control on day 0 (743+_130 vs 898• ns and 394+103 vs 472+101, ns), whereas they were significantly higher (p<0 05) in the 1v1g group on days 4 (1700_+_274 vs 799+197, p<0 05) and 7 (1740_+227 vs 864+i64, p<0.05). the various complement-fractions increased in both groups without inter-group differences the mean (• sbas (37 ~ c) at 30 rain in ivig group vs control group were: -53_+32 vs -56• ns for day 0, 9_+46 vs -54_+46 p<005 for day 4 and 7_+34 vs -54+47 p<005 for day 7. the mean (+sd) sbas (40 ~ c) at 30 rain presented a significant improvement over those of 37 ~ c but for the control group remained negative a~d were respectively as following: -~8• vs -26+33, ns for day 0, 22+_39 vs -29_+35, p<0.05 for day 4 and 24_+31 vs -27_+36, p<0.05 for day 7. the increase of temperature induced a 3-fold improvement of sba in iv1g group and 2-fold ofcontrol-~oup positive blood cultures, and the product of the infectious episodes number multiplied by days of occurence, were significantly lower (p<0 05) in the ivig group than in the control (2 vs 7, and 440 vs 1900, respectively). conclusions: our study shows a significantly favourable effect of ivig administration on septic complications and on sba of trauma patients. the increase of temperature results in a significant improvement of sba of patients that received ivig, which theoretically means a farther prevention of infection in the febrile state. pharmaceutical microbiology, university of bonn, meckanheimer aune 168, d-53115 bonn, germany infectious diseases in intensive care patients are common in comparison to patients on other wards and out-patients. the main difference is that intensive care patients are much more sensitive even to less virulent bacteria. thus, the spectrum of infecting organisms is different. strains often regarded as pathogens with low virulence cause serious infections in these patients. strains such as serratia, however, have intrinsic resistance to most commonly used agents such as 3rd generation eephalosporins. furthermore, the common pathogens like staphylococci, psoudomonas aeruginosu, enterocneei and gram-negative bacteria, enterobacteriaeceae as well as the non-fermenters are less sensitive if isolated from intensive care patients. it is difficult to generalize on intensive care units as different patient groups are in different icus aud there are great changes from one hospital to another and from one country to another. if we take s. aurens strains from one study from 1990 the'overall resistance in intensive care units towards oftoxacin was 22 %, whereas in other hospital wards the percentage of resistance was 5.3 %, in out-patients, however, only 2.$ %. the same trend was true for entercnecus faecnlis, coagulase-negntive staphylococci, and other bacteria as well as other drugs. one most striking difference was found with klebsialla pneumoniae and gantamycin resistance, which was $ times higher in intensive care units as compared with outpatients, whereas in the same species no difference was to be seen with the resistance towards carbapenems. however, differences between countries seem to be even more striking, as example gantamycin resistance and staph. anrens is given. the extreme difference is more than 60 fold. thus, it is evident that there is a general trend towards higher resistance in intensive care units, but no generalizatiouis possible. therefore, surveillance studies in intensive care units are needed and the antibiotic policy has to be adapted to the specific needs of the unit. in the icu setting the most potent antimicrobial agents are required to address problem organisms including those resistant to penicillins, cephalosporins and aminoglycosides. carbapanems would appear to present a useful option in this setting. objectives of this study was the evaluation of systemic candid•177 in postoperative cardiac surgery patients (pts) with prolonged icu stay. methods: out of 2617 postoperative adults pts of mean age 61.1+8.9 years old, with a mean icu stay of 1.6_+0.6 days, following an open heart surgery from july 1993 to april 1995, 54 pts (2%) remained in icu for more than 10 days because of severe perioperative complications. patients were included in the protocol if they had clinical signs of infection or sepsis, and fungi isolated in blood culture or in culture from at least three different sites. the patients who developed systemic candidiasis received iv fluconazole (800 mg/day) (10 patients) or amphotericin-b for at least four weeks, and then they were closely monitored. results: out of 54 postoperative pts with prolonged jcu stay, 11 pts (20.3%) developed systemic candid•177 usually after the 20th postoperative day. they were 8 males and 3 females of mean age 64+_7.4 years old. this group of pts had prolonged bypass and aortic cross-clamp time compared to control group (119 min vs 84, and 64 vs 49 min). all these pts received inotropes per• (mean value=2.3). during their icu stay, 9 pts developed sepsis of bacterial origin, while the other two severe infection, and received antibiotic regimens for prolonged period. the patients were submitted to mechanical ventilation for a median period of 50 days. the median icu and hospital stay was 58 and 60 days respectively. all pts have been improved and finally negative cultures were obtained. conclusions: 1. a significant percentage of patients who remained in the postoperative icu for more than 10 days developed systemic candidiasis. 2. all patients who developed systemic candidiasis had received antibiotics because of sepsis or severe infection, for prolonged period. 3. fluconazole seems to be a very good alternative to amphotericin-b. 4. fluconazole is a safe antifungal agent with few side effects. botulism is the most severe and an odd food poisoning. although it is more commonly related to preserved meat derivatives, preserved fish and vegetables are also responsible for a number of cases. obiectives: to evaluate four familiar outbreaks of botulism . methods: we study the patients that were admitted in our hospital because of botulism from may 1982 to february 1995. results: the thirteen pacients involved had a previous history of home preserved beans ingestion. after a 24-hours incubation period, gastrointestinal symptoms (abdominal pain, vomits, constipation) appeared and lead them to hospital consultation in the 4th to 7th day after ingestion. two patients died (acute respiratory failure before admission), seven were admitted in icu, two in ward and two of them were discharged from emergency room. clinical symptoms and the previous history of the ingestion established the diagnosis, that was emg confirmed. in all cases, symptoms were consistent with b-toxin botulism. b-toxin was isolated in serum and food proceeding from the third outbreak, and the serum was negative in the other ones. neurological symptoms were predominant: midriasis (100%), dry mouth (100 %), dysfagia (100 %), asthenia (55 %), palpebral ptosis (55 %), accomodation paralisis (66%) and urinary retention (55%). muscle weakness lead to acute respiratory failure in three patients (one of them required mechanical ventilation). four patiens developed infections (respiratory, urinary and phlebitis). both died patients and one another presented severe hypertension. all admitted patients were treated with polivalent anti-toxin. the two patients who underwent a more severe muscle weakness received also guanidine hydrochloride, with no answer in one case and provoquing a cholinergic crisis in the other one. icu length of stay was 10 days. at hospital discharge, patients continued symptomatic, mainly with dry mouth, disfagia and impaired vision. conclusions: although botulism is a serious illness, the pronostic seems favorable if treatment and support measures are avaible. usually neurological symptoms we predominant and at discharge some of them could still persist. the arrow "hands-off" (aho) thermodilution catheter (tc) is completely shielded during balloon testing, preparation, and the insertion procedure. in order to assess the value of the aho thermodilution catheter in the prevention of systemic infections associated with pulmonary artery catheterization (siapa), we conducted a randomized prospective study over an 18-month period. methods : the patients (pts) were randomly assigned to two groups : group i for a standard tc customarily used in the department, versus group 2 for the aho thermodilution catheter. the diagnosis of siapa was determined on the basis of a positive culture of tc and bacteremia with the same organism, with out any other nearby focus, in association with regression or disappearance of the clinical signs of infection after removal of the thermodilution catheter. results ( objectives: the mortality rate (mr) of tb requiring mechanical ventilation (mv) is high (70-100%). the aim of the study was to evaluate mr, associated factors, and prognostic significance of mv and hemodynamic disorders from tb in icu in 35 patients with tb. methods: clinical parameters on admission, and complications in icu were related by univariate analysis to icu, hospital, and 6 month outcome. 18 patients required mv; 10 were immunocompromised (ic) including 8 hiv. tb was pleuropulmonary in 24, disseminated in 9 and meningeal in 2. results: mr was 31% in icu, 34% in hospital and 47% at 6 month. 12/16 (75%) <0.001 mortality was associated with a high saps score, initial shock, mv and nosocomial septicemia. the mr dramatically increased when ards occurred during illness, despite the lack of correlation between mr and initial po2/fio2 ratio or initial murray score. the site of infection did not influence the mr. surprisingly, the mean therapy delay was shorter for non survivors. mr was not related to ic status, nor hivstatus, but was only related to previous steroid therapy. conclusion: mr of tb requiring icu is high (47% at 6 month). need for mv increased mortality (72% vs 18%). general severity and respiratory dysfunction seem to be major prognostic factors in icu rather than tb per se or than therapy delay. in spite of the improvement in the prognosis of pneumococcal meningitis (pm) with third generation cephalosporins (tgc), this infection still presents a great mortality which could be increased with the appearance of antibiotic resistant streptococcus pneumoniae. objectives: to asses intensive care mortality and morbidity of pm and to define patients (pts) at risk of complicated evolution. patients and methods: a retrospective evaluation of pm cases (all diagnosed by csf culture) admitted in our icu from january 1985 tit march 1995. in all pts we analized: demographic data, underlying disease, apache ii score, clinical symtomps, treatment, complications and outcome. statistical analysis was done using bmdp sofware package. results:a total 0f42 pts were studied, 26 males; mean age 55,8 _+ 16 (16-81); apache ii score 16,6 + 7,9; glasgow coma scale (gcs) at admission 12,5 _+ 1,7; 17 (40%) pts suffer from cronic pathology; 5 (12%) pts diabetes mellitus (dm), 4 (9,5 %) pts had had a previous cranial traumatism. in 22 cases the source of infection was otic and also in 22 (52 %) episodes of pm there were bacteriemia. in 21 out of 26 (80%) pts that ct was performed no radiologic abnormalities were shown, 3 of them presented cerebral oedema and 1 pts a cerebral abscess. twenty-eight percent presented seixures, 14% hemiparesia, 46,3% respiratory failure, 17,5% shock, i5% renal failure, 5,1% multiple organ failure (mof). as for treatment refers 5,5% pts recieved only penicillin, 69,4% pts only tcg, 11,1% pts tcg followed by penicillin and 8,3% pts tcg+vancomycin. seventy-five percelat of pts recieved corticosteroids and 25,6% vasoaetive drugs. the mean icu stay was 7,5 5:6 days (1-28). twelve (28,5 %) pts died, two of them presented pm relapse (resistant streptococcus pneumoniae) and another two pts developed neurological sequelae. factors associated statistically with bad prognosis were dm, the use of vasoactive drugs, shock, mof, the apache ii score at admission, the gcs at the 48 and 72 hours from admission in the icu but not the gcs at admission. didn't resulted statistiealy signifcative age, previous eronie pathology, seizures, baeteriemia, renal failure and coagulation disorders. conclusions: mortality was high and associated to apache ii score at admission, to gcs at 48 and 72 hours after admission, shock, vasoaetive drugs and mof. objectives:the aim of the study was to analyse some of significant immunologycai changes in surgical patients,requiring intensive health care,and to determinate the possibility for evaluation,dynamical examination and importance of immunologycal problems for treatment. methodes:the study concerns a number of 30 patients with expanded surgical intervention or serious postoperative complications.the results has been carried out with fiowcytometryc analyses of lymphocytic suhpopulations and routins methods for investigation of humeral immunity.the"panel" for evaluation of 2(} immunologycal parameters has been offered:t-calls total/cd3+/;t-helper/cd4+/;t-supressor/cd8+/ th/ts ratio;b-cells/cd19+/;naturai kilier/nk/cells;skin test for cellular immune function;phagocytic and oxidative activity;serum levels of immunogiobulins-g ,a,m;protease inhibitors;c-reactive protein.all patients have been studied during suffering and after surgical procedures dynamicaly. results:there have been estimated significant changes in immunologycal parameters especially:decrease of t-cells: cd3+mean=37.62%/14.3%-47.9%/and cd4+mean=22.11%/9% -28.8%/;inverted th/ts ratio ,mean=o.72/0.37-0,90/;reduced or negative skin teste;reduced phagocytic and oxidative activity before septic complications. conclusions:dynamical examination of immunologycal parameters shows,that the prolonged t-total,t-helper lymphocytopenia with functional deficience of ceils-mediated immunity correlates with the stage of clinical condition of the patients and has prognostic importance.it's clear,that immunologycal monitoring gives a possibility for immunecorrection. patients (pts) with the human tmunodeficiency virus (hiv) infection have a decreased immune response and are particularly susceptible to infectious endocarditis (ie). the aim of our study was to analyze the prevalence of ie, its clinical and therapeutic implications in a hiv population 9 we prospectively studied 245 pts, 9.4% (23/245-group ie+) with ie during the clinical course of this disease. we analyzed the following parameters: age, gender, race, type of hiv, cdc classification, number of t4 and t8 type cell population and its ratio, therapeutic with azt, type and number of opportunist infections (inf, mycobacteriosis (mb), neoplasm's (nee) 9 the echocardiographic parameters were lv internal diastolic and systolic diameters, lv percentage of fractional shortening, interventricular and posterior wall thickness, the degree of valvular regurgitations and the presence of pericardial effusion. el was located at the mv in 2.7%, tv in 6.0%, av in 2% and pv in 0.9~ and was multiple in 2.0%. hiv el+ pts had larger lv diameters and more frequent significant valvular regurgitations (39% tr, pe 33%, mortality 32%). these two groups differed significantly in the following clinical parameters: the typical symptoms were watery diarrhea, high fever, tachycardia,luekocytopenia and oligouria within 7th postoperative days. the patients with mrsa enterocolitis had positive mrsa culture from the many materials except feces.mesa strains frequently had coagulase type 2,enterotoxin a and toxic shock syndrome toxin-1 .eight of 1 6 patients had postoperative organ failure.most of the mrsa strains in japan were similar in coagulase type to our hospital and our department.all of mesa strains were susceptible to vancomycin and arbekacin,tbough most of them showed resistant to many other antibiotics.we have employed guidelines for therapies such as oral or enteral administration of vancomycin and correction of the hemodynamics for dehydration and circulatory failure due to diarrhea from 1992.futhermore we have placed colonized or infected patients in private room,worn gown and mask,and carefully washed our hands from 1992. these countermeasures for prevention of nosocomial infections after 1992 significantly reduced the incidence of mrsa enterocolitis. conclusions:earlier diagnosis and treatment, and distric prophylactic measureres against mrsa infections are very important. -cdo ivda leptespiresls affects all the organs with widespread hemorrhage that is more prominent in skin, mucosa, skeletat muscles, liver and kidneys. lung involvement is usually mild and less common. suli, it is very uncommon acute respiratory failure to be the pr6sontirlg symptom. a case with leptosplrosl..,s which was presenting with acute respiratory failure is described. a 36 year-old man admitted to icu becauso of fever, myaigla, aevere c~, hemopty~s. his blood gases showed: pao2:46mmhg with fio2:1.0, pco2:27 mmhg, ph:7.4, hco3:20mecl chest x-ray film demonstrated diffuse bilateral alveolar pattern occupying beth lung4/4). trarmamlnase, bllllrubln, ~ and esr were elevated, wbc was 8.7001mm8, platelet: 40.0001ram3, hematesrlt:30%, hemoglobin: .sgrldl=. there was no clinical or ecttlographlc evidence of left heart failure.patient fulfilled the criteria for diagnosis ards he was found to have an ~lutinatlon tlter for leptoq~lral antigens(indirect he~lutlnatlon atomy, ilia} very high (1/800, negative<ill00) and iglm antibodies also very high (elisa, a=1.492, negative<0.150) strongly suggesting leptospirosls. treatment with tetracycline and non-tnvaslve mechanical ventilation by nasal mask were started. pre~jre support mode and cpap was usod. the following days the clinical picture,the oxygenation and the chest x-ray of the patients were improved, and patient dl~herged from the icu. the control of leptosplremlc phase as well as his good cooperation during nipsv contributed to rapid recovery and excellent outcome in hiv infections, pneumonias (pnm), mainly due to pne~ mocystis carinii (pc) have an outstanding place in pu ! monary complications. the aim of this work was to compare two group> of patients admitted with pnm in our icu during the same period (1990-94): group a, 19 patients hiv+, and group b, 152 patients hiv-. apache ii was identical in the 2 groups (p=ns). group a required more often mechanical ventilation (p=0,o07), had a higher p(a-a)o2 (p=0,004) and metabolic acidosis was more frequent (p=0,001). regarding laboratorial parameters group a had a lower no. of linfocytes (p=0,02), a higher ldh (p=0,04) and a more marked hypoalbuminemia (p=o,03). mortality was higer in group a (52,6%) than in group b (29,6%), (p=0,04). analysing the a group patients, we found no significant differences between alive and deceased patients, with exception for albuminemia, which was lower in the deceased patients (p=0,02). in conclusion, the hiv+ patient's pnm have a more agres sive behavior when compared with community acquired hiv-patient's pnm. the prognosis was not influenced by the apache ii. perhaps other parameters such as p(a-a)o2, metabolic acidosis, linfocytes, ldh and albumin shoud be more evaluated as possible predictive indices. some prognostic factors, usually accepted as predictive in the analysis of hiv+ patients do not seem to be worth in the late stages of aids, mainly when they reqquire intensive care. intensive care unit, onassis cardiac surgery center, athens, greece. objectives of this study was the comparison of two different antibiotic regimens as prophylaxis in cardiac surgery patients. methods: in a prospective randomised comparative study, two different forms of antibiotic regimens were investigated : a single dose of cefuroxime (zinacef, 3 gr) (group a) given during the induction of anaesthesia, versus a four days combination of amoxiculine (amoxil, 2 gr tid) plus netilmicin (netromycin, 150 mg bid) (group b). a total of 926 patients (pts) (767 males and 159 females, of mean age 60.6+8.7 years old) were included in the study over a period of one year; 424 in group a and 502 in the group b. patients were checked for the occurrence of infection during the first postoperative month. results: the total rate of infection in cardiac surgery pts was 5.8%; 5.4% in group a and 6.1% in group b (p=ns). 34 pts (4.7%) developed infection following cabg, 17 pts (7.9%) following valve replacement and 6 pts (17.6%) after other cardiac surgery. they were 43 males (5.6%) and 11 females (6.9%). endocarditis has occurred 0.4 % in group a and 0.2 % in group b. severe wound infection was recorded in 0.4% in group a and in 0.8% in group b. one case of sepsis (0.2%) in group a and in group b (0.2%). respiratory infection occurred in 11 pts of group a (2.6%) and in 11 pts of group b (2.2%). two cases of urinary tract infection was in group a and one in group b. catheterrelated infection was occurred in 5 (1.1%) in group a and 6 (1.1%) pts in group b. 3 pts (0.6%) had fever of unclear aetiology in group b. conclusions: there was no statistically significant difference regarding the rate of infection in both groups. a single dose administration of cefuroxime is accordingly just as effective as a four days regimen of amoxicilline plus netiimicin. legionella pneumophila is a common bacteria of the environment, and it is an agent responsible for severe community acquired pneumonia (cap). we analyzed the 8 patients with lpp admitted in our icu during the last 8 years (1986) (1987) (1988) (1989) (1990) (1991) (1992) (1993) (1994) . they represented 4.6% of cap. seven patients were males and 1 female, with mean age 46.7+12.1 years. tiss was 24.1+10.9 and apache ii 21.0+5.2. all, but 1 patient, were under mechanical yen tilation (mv) during a mean period of 11.7• (min-l, max-44) days. two pneumonias occurred beyond the season, while 4 patients had an epidemiological history. only 1 patient had no risk factor. in all the others tobacco smoking and alcohol abuse was quite frequent. diagnosis was based on serologic test and culture or direct fluorescent antibody staining of bronchial secretions. seven patients had a multisystemic disease with hepatic dysfunction in 5, renal failure in 4 (due to rhabdomy~ lysis in 3). one patient had a prosthetic valve endocarditis and another developped ards. nosocomial septicaemie occurred in 3 patients. mortality rate was 50%. deceased patients had initially higher apache ii, (a-a) 02, and lower natriemia. comparing lpp with the other cap (n=84), both submitted to mv, mortality rate was similar (57,1% versus 54.7%). in conclusion lpp can occur all over the year. there was a high incidence of severe complications and outcome was similar to the other cap when requiring mv. prospective specimen brash (psb) with culture > 10 cfu 10 3 cfu/ml. broncho-alv~lat lavage (bal) ~= 104 c'fu/rnl or positive blood culture. 10 were excluded for rapture of treatment ; 63 were analysed (shift with oral antibiotic8 : 3 ; prohibited antibiotics associations : 5 ; resistant germ : 2). clinical data : age 60,6 • 18,7 ; saps 12 • 2,86 ; mac cabe i : 76,2% -ii : 22,2 % -iii : 1,6. 63,5% of the patients were intubated and under mechanical ventilation. the pneumoaiae were : primitive in 35 (55,6%), copd 9 (14,3%), aspiration pneumonia 19 (30,2%). 75 germs were isolated (psb 67, bal 1, blood culture 7) : s. pneumoniac 28 (37,3%), h. influeazae 14 (18,7%), sttep~:occns 10 (13,3%), saar6ns 10 (13,3%), enterobaetdrindr 5 (6,7%), mosexella catarrhalis 2 (2,7%), othem 6. 71/75 (94,7%) were sensitive to freatment. the ltentment was 100 mg/kg/d of ampiclllin and 50 mg/kg/d of sulbactam in continuous iv adminisu'ation during at least 10 days. clinical eff~ienev : success 46 (73%), failures 17 (27%) with superinfeetion 7, worsening or relapse 3, dead 5, side effects 2. there was no difference between etiologies : primiti~;e~ 74,3%, copd 77,8%, aspiration pneamoniae 68,4%. the bacteriological effieieacy was evaluated only for 41 patients with eradication 30 (73,2%), eradication but super~ection 6 (14,6%) : with pseadomoaas a&ogiuosa 2, eater~ac~ 3 ; beeteriological failure 5 (12,2%). in conclusion, the aasor ampicillin -sulbactam is effective for the i~eatment of severe acquired community pneumonise. objectives : to assess the efficacy of chlorhexidine (cl) gel or suspension applied in the nose and in the op for the prevention of the tmcheobronchial colonization. methods : thirty-seven patients expected to be intubated for > 48h were randomized to received topical application oga cl suspension (2%) qshrs, a cl gel (1%) q6hrs or a placebo. in addition all vpts received a nasal and a op spray (2%) of either cl or placebo administrated according to the same schedule. semi-quantitative cultures of the anterior nares, the oropharynx (op) and the trachea were obtained on admission and once a day until extubation (just before the next application). the results were assessed according to the following criteria: success = no acquisition of gnb in the trachea ; failure = acquisition of gnb in the trachea. acquisition was defined by a follow-up culture positive for a gnb not present in the trachea on admission. results : success failure nosocomialpneumonia overall morality clsusp. placebo clgel placebo n=8 n=10 n=9 n=10 5/8 6/10 7/9* 3/10 3/8 4/10 2/9* 7/10 1/8 2/10 3/9 4/10 0/8 1/10 2/9 2/10 i *p = 0,03byfisher'sexacttest conclusions : these results suggest that topical cl gel administered q6hrs may prevent tracheal colonization by gnb. f. daumal*, m. daumal**, c. plot**, v. vurmmen ~ e.colpurt**, b. manonry** * hygiene hospitali&e, ** service de r6enmmtion, * service des admissiens-urgeuces centre hospitalier g-6ndral -02 321 saint-quentin -france obiectives: evaluate the nosocemial risk due to peripheral venous inserted short catheters, and the quality of care. patients-methods: the intensive tare unit (i.c.u.) is a 9 beds unit. the prospective study includes all the patients comn~ in from 01/01/1993 to 30/03/1995. the recruitemont uses an evaluation schedule of local clinical signs. the nurses aimed to create this evaluation data which includes the place of entry site, the duration of catheterization and the cause ot withdrawal. only patients staying longer than 2 days in the i.c.u. are accounted for. the diagnosis of uosoenmial infection is assured by the physician taking care of the patient and by the hospital epidemiologist on the next signs: evident pus at the catheter entry site, positive culture of the strain, with or without the same pathogen in the blood sla'uam,the patient having no other distant source of infection. analyses were performed on epi/nfo. results: the occurrence of 3 nosoeomjal inthrtions: i abcess and 2 bacteremia during the first part of the study lent the medical staff to modify the protocol of insertion end survey of the device. so we analysed 2 different periods: period 1( 1/01/93 to 31/10/93 ) and period 2 (01/11/93 to 30/03/95 ) for all 1 .e peripheral catheters inserted in the i.c.u. period 1 44,9 % 46,2 % en infection due to peripheral venous device is a daily threat. the severity of some clinical situations requiring admission in icu proves it. the motivation of nurses for rigid adherence to established protocol, the daily survey of the entry site, the withdrawal of the peripheral catheter every 72 hours aimed to reduce significantly the local signs of inflammation end infection of peripheral catheters inserted inside the i.c.u. objectives: to investigate the use of a new metabolic monitoring device for different ips levels by comparing oxygen consumption (vo2) to measurements of the mechanical work of breathing (web) and p0.1. methods: the study was approved by the institutiotml ethics committee. eight patients were investigated during weaning after prolonged mechanical ventilation (6-75 days) for various diagnoses when the clinical physician judged the patient to be ready fur weainag. ips was setto 15, 10, 5, 0 mbar far 20 rain periods each. all patients had a peep between 5-8 mbar.. respiratory frequency (f), tidal volume (tv), minute ventilation (ve) were read from the ventilator display (7200ae, puritan bennett, carlsbad, usa). flow and airway pressure were measured at the endotracheal tube site. esophageal pressure was measured using an esophageal balloon catheter (fa. ruesch, frg). web was determined as the area subtended by the pleural-pressure-vohime curve. p0.1 was determined by using standard occlusion technique and graphical analysis of the airway pressure tracing. vo2 and vco2 were measured using the pb 7250 metabolic monitor (puritan bennett, carlsbad, usa) connected to the pb 7200ae ventilator. all data are given as mean• deviation for each ips level. comparison between the different ips levels was performed using anova for repeated measurements. significance was considered at p<0.05, compared to ips 0 mbar. results: the values for breathing pattern, web, p0.1, vo2 and vco2 are given in the table for the different ips levels; significance is indicated by ~. objectives: fluidized beds are often used in the management of critically ill mechanically ventilated patients. critically ill patients are increasingly colonized with resistent pathogens [ie: p. aeruginosa, methicillinresistent s. aureus (mrsa), extended spectrum i~-iactamase producing enterobacteriaceae ] that can ultimately cause nosocomial infection. methods: we prospectively monitored bacterial colonization of mechanically ventilated patients and of the fluidized bed (clinitron) inwhich they were treated. multiple samples for quantitative bacterial cultures were taken from oropharynx, trachea, feces and bedsores. samples of ceramic beads from the bed were also taken both during and after patient stay (after bed operation in the absence of patient). re,~ults: 13 episodes in 12 consecutive patients (mean age: 57.6 years) were analyzed. all had bedsores and/or urinary catheters and fecal incontinence, 7 patients had nosocomial pneumonia, 6 had urinary tract infection [2 with extended spectrum imactamase producing k/ebsie//a pneumoniae (ki~lse)], one had positive blood cultures with mrsa, and one patient had a ki~lse found in high concentrations (103 -10 s cfu/ml) in 2 occasions in feces. patients were heavily colonized: the , samples from ceramic beads showed no growth or became sterile without any sterilisation procedure (even in one case of presence of kf~lse) during the patient stay. conclusions: fluidized beds do not put patients at high risk of acquiring nosocomin pathogens, and cross-contamination between patients seems unlikely, even when multiple resistent organisms were initially present. the recommandation from some manufacturers to undergo extensive sterilization of fluidized beds after use does not seem warranted, at least with the bed used in this study. ant. koutsoukou, a, tahmitzi, p. kithreotis, m. koutonlidou, k. stavrakaki, kainis e, g. vlahogiorgos and e. eliopoulos icu-centre for respiratory failure -chest diseases hospital of athens. the cost-effectiveness issue is becoming vital in modern medicine and may lead to moral dilemmas since sometimes certain groups of patients may not have access to highly specialised modalifies. objective: our study compared the mean daily cost for antimicrobial medication in copd patients treated in icu versus all other patients in the context of relevant epidemiological, prognostic and outcome data. methods: age, sex apache ii score, length of icu stay (los) and in -icu fatality were retrieved from the files of all icu admissions over 1994. mean daily cost for antimicrobial therapy per patient (dcat) was estimated. these variables were statistically compared between copd and non-copd patients. significance was assumed at p<0.05 results: 140 of the total 178 admissions were fully evaluable. 38 of them (27%) were copd patients. data (m---sd) results for statistical test are given in table i . copd patients were significantly older spent more time in the icu and presented with significantly higher apache ii scores. outcome and dcat were comparable in the two groups. objectives: the use of heat and moisture exchangers (hmes) during long term mechanical ventilation (mv) is increasing. in icu patients, they are routinely changed every day, according to the recommendations of the manufacturers, but the clinical basis for such a daily practice is lacking. we therefore prospectively assessed whether changing hmes (dar hygrobac, spa, mirandola, italy) every 48h only would affect their clinical and bacteriological efficiency. methods: two consecutive groups of patients requiring mv for >48h were compared: group1= hme replaced every day, n= 61 episodes of mv in 61 patients; group 2 = hme changed every 48h, n=68 episodes in 64 patients. tubings were not changed in the same patient during the whole length of ventilatory support. diagnosis of nosocomial pneumonia (np) was based on a positive quantitative culture (~103 cfu/ml) of a protected specimen brush in patients with clinical signs of pneumonia. quantitative cultures of pharynx, trachea and y-cannector were performed every 48h. results: the groups were similar in terms of age, indication for and overall duration of mv (10+_8.6 vs 10+_9days, p=0.9), and severity of illness (saps: 16---4.9 vs 16.4+_5.5, p=0.6). the maximal values for peak airway pressure were identical in both groups (33.4-+7.8 vs 33.7• cmh20, p=0.9). obstruction of the tracheal tube was observed in only one instance in a group 1 patient who had tracheal bleeding. circuit colonization was very rare, and of low grade in both groups. the level of patient colonization and the type of organisms were identical in both groups. more importantly, the incidence of np was the same (6/61 vs 8/68, p=0.7), as was duration of mv before the occurence of pneumonia (9• vs 10.5+_4.7, p=0.6) and overall mortality rate (17161 vs 17168, p=0.7). conclusions: the clinical efficiency of this hme does not seem altered after 2 days of use. indeed, replacing this hme every 48h only neither affect circuit and patient bacterial colonization nor the incidence of np. therefore, substantial savings could be obtained changing hmes every other day only. obiectives: to evaluate the usefulness of different paraclinical investigations for the diagnosis and prognosis of acute viral encephalitis in icu patients. methods: we reviewed 13 patients (pts) admitted to our icu from july 1989 to december 1993 with the diagnosis of acute viral encephalitis. all were in coma and were initially treated as presumed herpes simplex virus (hsv) encephalitis. the causative agents were: hsv (2 cases), herpes zoster varicellae (1), measle (1), rabies (1), unidentified (8). eleven pts survived and three presented neurologic sequelae. twelve pts were investigated by mri, and eleven also by spect and multi-modality eps. including brainstem auditory eps (baeps). these investigations were obtained as soon as possible following admission and were repeated during icu stay when possible. the clinical outcome was noted. results: six pts (6/12) had an abnormal mri. among them, 2 pts made a complete recovery, in comparison with 5/6 pts with a normal mri. in one hsv infected patient, mri remained normal despite clinical deterioration and bad outcome. when repeated, mri became abnormal in 3 cases (with poor outcome in one) and was improved in one. spect was found abnormal in 10/11 pts (among them, 4 pts had thus a normal mr/). the correlation regarding the topography of brain lesions was poor between mri and spect. the findings of spect could not be correlated with a poor outcome. the baeps confmned in 56% of the pts the clinical diagnosis of brainstem involvement. changes in visual and somatosensory eps were mild in all the pts and were not helpful for the prognosis. eps were otherwise interesting for the follow-up of the coma in these sedated and ventilated pts. conclusions: the value of mri and eps for the diagnosis of acute viral encephalitis is of limited interest. spect seems to show early modifications, even in pts with a normal mri, but this test is poorly specific and does not correlate with mri changes when present. concerning the prognosis, larger studies should probably confmn that a normal mri could usually result in a good outcome. this serie illustrates also that hsv encephalitis could be demonstrated only in a small number of cases and that the prognosis of non hsv encephalitis is not easily assessed. objectives: to study the influence of gram (-) bacterial lung infections on liver function i~ mv icu pts. pts and methods: we studied 102 pts, 68 # (66,7%), 34 (33,3%). hean age:48,3• years (16-82). mean stay in icu:13,3• days (8-75). they were divided in 2 groups: a(44 pts) who did not suffer from pneumonia and b (58 pts) who developed a gram(-) bacterial pneumonia. both groups were consisted of pts with same age, sex and disease distribution and same systemic failures. we measured sgot, sgpt, total bilirubin(tb), direct bilirubin (db), alk.phosphatase (al.ph.), v-gt and albumin (alb.) 3 times: on days o, 4 and 7 of the pneumonia for group b and respectively for g~oup a. conclusions: 1) in elderly intubated pts of an icu, kp is isolated more frequently than in icu pts<65 years (p<o,01). 2) the frequency of isolation of kp in icu pts during 1985-92 was 25-30%, but now (1992-95) kp becomes more and more rare (12-15%) and is isolated especially in elderly pts. 3) its sensibility to antibiotics remains always the same. 4) the prognosis of np in our study was independent from age (p<o,1), sex, presence of bacteremia (p<0,1) and type of invading microorganism. gram neg bacteria and renal failure (rf) from aminoglycoside toxicity are common and serious complications in critically ill patients. the aim of this prospective, pilot study was to compare the safety and adequancy of the endotrecheal (et) vs the intravenous (iv) administration of aminoglycosides. were measured in the plasma and the bronchial secretions at 1 and 4 hours after the iv (bolus) or the et (nebulized) administration of 80 mg of gm in seven criticcally ill patients (3 with established renal failure). safety was defined as peak and trough plasma gm levels _< than 8 and 2 ijg/ml respectively and adequancy as gm levels in bronchial secretions _> 0,5 ijg/ml. results: gentamicin was administered by the et and iv routes in 18 and 7 separate sessions respectively. a total of 107 samples were assayed, 69 in bronchial secretions (bs) and 38 in serum. the et route resulted in higher gm levels in the bronchial secretions compared to the iv route (3,26 + 2,86 vs 2,1 _+ 2,1 pg/ml respectively, p = ns ). adequate bronchial gm levels were achieved in 100% of patients after et administration, compared to 66% after iv aaministretion. the blood levels of gm were significahtly lower after the et vs the iv route (1,56 + 1,95 vs 5,56 • 1,96 pg/ml respectively, p _< 0.01). the et administration resulted in toxic bronchia~ gm levels in 47% of the specimens. 66% of these samples were from patients with renal failure, however toxic blood levels were reached in only 12% of these. gentamicin seems to be a safe and adequate alternative route of treatment for the lrti. however, in patients with renal failure the et administration of the aminoglycosides should also be modified and continuously monitored. in order to evaluate the pathogenic role of anaerobes in nosocomial pneumonia (np), we investigated the systemic humoral response in patients who developed a np with anaerobic bacteria, especially prevotella species. methods: blood samples from 4 groups of patients were tested. group i: 13 patients with a np in which prevotella spp. was isolated from protected specimen brush (psb), group ih a control group of 30 patients with a np without anaerobic bacteria, group ill: a control group of 27 patients with dental stumps but without pulmonary infection, group iv: a control group of 30 healthy voluntary people with prevotella spp. isolated from the dental plaque. an elisa was used to evaluate the total antibodies level against a mixture of four prevotella strains and a western-blot method was done to identify the antigenic proteins. results: data are expressed as means .+ sd. the antibody levels in patients of group i (63• was statistically higher (p=o.o05) than in the 3 control groups (respectively: 29+25, 32_+25, 31_+23). using western-blot method, the intensity of the response was roughly superposable to levels obtained by elisa and the profiles were different according to the prevotella species. the occurence of a np with anaerobic bacteria (prevotella species) isolated from psb leads to an antibody response which seems specific of the prevotella species isolated. fever is common in the intensive care unit, but is not always related to an infection. we sought to define the epidemiology of febrile patients in a general medical/surgical icu. methods: we prospectively analysed the source of fever (t >38.2 ~ c) in all adult patients admitted for >-48 hours in the icu during a two month period. these patients were studied for 14 consecutive days. and werc classified in 3 groups according to the evidence of infection (center for disease control criteria) after complete evaluation: documented infection: cdc criteria + isolation of pathogen (d); possible infectron: cdc criteria without isolation of pathogen (p); unlikely infection: patients who did nol meet the cdc criteria (u). results: of a total of 208 patients studied, 74 dec'eloped fever (35 6%). including (after complete evaluation) 39 d, 15 p and 20 u palients. both the highest temperature in tile first day of fever and the maximal temperature were higher in d than in u (38.7•176 versus 38.5•176 and 39.2-~0.9~ versus 38.64-0.4, respectively p= 0.05 and p= 0.003). most common sources of infection in d were the lungs in 25 patients (64%) and urina .ry tract in 4 (10%). 14 of these patients had positive blood cultures (36%). the overall mortality was 27% (23% in d, 40% in p and 25% in u. differences ns). antibiotics were given in 100% of d, 73% of p and 15% of u (3 patients). in p there was a non significant lower mortality." in patients who received antibiotics (3/11 (27%) versus 3/4 (75%) patients, respectively). conclusions: in febrile icu patients both the highest first day" temperaturc and maximal temperature are significantly higher in infected than in non infected patients, but the differences are too small to be useful clinicall). mortality rate is not significantly influenced either by the presence of an infection or by the administration of antibiotics, obiective: retrospective study to determine the influence of candida infection on icu outcome. methods: 126 patieet with a stay of more than 7 days in inteaasive care were screened for candida infection. 70 patients were treated with antifungal therapy due to either an increased antigen titre of -> 1:8 or clinical evidence of candida colonization. serological candida-antigens (ramco, pastorex) and antibody titres (hemagglutination, lgg-, igm-elisa) were examined routinely. seroconversion was defined as a threefold increase of antibody titre or a titre of 1:640 or higher. results: the median length of stay was 37 (ranging from 8 to 132) days, the mean apache ii score on admission was 18 (+_ 5.8 sd) points. of 126 patients 31 patients died (24.6%). in the group treated with antifungnls (71 patients) 19 patients died (26.7 %). although of the 126 patients only 51 (40.4 %) developed a candida infection as defined above the mortality in the group that showed signs of infection was significantly higher (37.2% vs. 14.6%, p < 0.05 [chi-square-test]). in 34 patients an antigen concentration-> 1:16 was measured. seroconversion was found in 41 patients. the most common fungus was candida albicans (66.4 %). furtberm0re, candida glabrata was found in 21.1%. most of the patients were treated with 2 x 200 mg fluconazole (66 patients). in 38 patients therapy was changed to amphotericin b/flucytosine. in 5 patients therapy was started with amphotericine b and flucytosine. in 40 patients a threefold decrease of candida antigen titre was found. 27 patients showed a decrease of candida antibody titre. conclusions: meticulous screening for eandida infection seems to be necessary since the number of patients with fatal outcome is significantly higher in the group with signs of fungal infections and thus requires immediate antifungal treatment. objective: early diagnosis of patients with ventilator-associated pneumonia (vap), and subsequent identification of causative microorganism, and selection of the appropriate therapy are critical important points that affect morbidity and mortality. the results of the quantitative bacterial cultures are not available for at least 24 hours, while a two hours period, since the specimen are obtained is enough to know the gram stain results. the aim of this study is to determine the usefulness of gram stain in specimens obtained by bronchoaiveelar lavage (bal), through the bronchoscope. material and methods: we studied 47 patients (36 males and 11 females, age 49 + 23) with suspected ventilator-associated pneumonia. the bal gram stain was considered positive when the specimen after a centrifugation at 1500 rpm for 15 min revealed: i) more than 20 leukocytes per optic field, ii) squamous epithelial cell less than 1 percent and iii) one or more microorganisms per optic field on 1000 magnification. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 8 patients had vap and 39 patients did not. in 5 cases the bal specimens (quantitative bacterial cultures) established the diagnosis of vap in the remaining three patients the vap diagnosis was established by other procedures (blood or pleural fluid culture, clinical outcome, autopsy). apache fl score in patients with vap was 15,7 -+ 5,5, while in patients without vap was 17,9 + 6,4. there was a significantly higher incidence of vap in patients who had i) coma (gcs <8) and ii) been receiving neuromuscular blockade (p<0.05) . the sensitivity of the gram stain for vap diagnosis was 75%, the specificity 89,5%, the positive predictive value 60%, and the negative predictive value 94,6%. conclusion: our data indicate that the gram stain of bal specimens is useful for the early diagnosis of vap and the subsequent administration of the appropriate treatment. the role of anaerobes in mechanically ventilated patients with pneumonia (mvp) have been poorly investigated aim of the study : analyse the prevalence of anaerobic isolation in mvp. methods : between october 1992 and february 1995 all suspected mvp were investigated using protected specimen brush (psb) technique. brushes were rapidly transported in shaedler broth to laboratory. a special care was tooken for anaerobic isolation. results : among the 153 psb performed for suspected mvp (132 nosocomial and 21 community-acquired pneumonia), 81 yielded at least one micro-organism (positive psb : 53%). 63 of positive psb demonstrated only aerobic bacteria and 18 (23%) yielded with anaerobes. in 14 out 18 patients, anaerobes were associated with aerobic bacteria. anaerobes were mostly isolated in nosocomial pneumonia (17/76 positive psb). 27 strains of anaerobes were isolated. prevotella species represent 19 out these 27 strains (70%) the most frequent anaerobic species were prevotella oralis (6) p. intermedia (5) and p. buccae (4). comments:using adequate methods, anaerobic bacteria are frequently isolated in mvp. it could be off importance to take in account anaerobes in the choice of empirical antibiotic therapy in mvp. objectives: the majority of patients with multiple trauma are considered immunocompromised. the aim of this study was to identify risk factors of pneumonia in mechanically ventilated patients with multiple trauma or after surgery. methods: in this prospective study we studied 64 multi-trauma patients (mean age 58 + 19 years, apache ii 16.5 + 6), admitted to a general intensive care unit (icu). all patients were intubated and mechanically ventilated. we were considered that a patient had ventilator associated pneumonia (vap) when the specimens of bronchoalveolar lavage (bal) or protected specimen brush (psi?,), ebb'ned through the bronchoscope, had one or more microorganisms in concentrations greater than 105 and 103 cfu/ml respectively. all patients had been receiving antibiotics, with no change during the last 3 days, prior to bronchoscopy. results: 14 patients had vap, and 50 patients didn't. in the bivariate analysis, the glasgow coma scale (gcs)<8 (x2=4.15, p<0.05), the administration of neuromuscular blockade (x2=7.9, p<0.05), the duration of mechanical ventilation to be greater than 5 days (x2=5.5, p<0.05), the flail chest (x2=4.1, p<0.05), the parenteral nutrition (x2=5.6, p<0.05), the ards (x2=3.9, p<0.05), the abbreviated injury scale (ais) of more than 4 for thorax (:,:2=5.9, p<0.05), the pneumothorax (x2=5.1, p<0.05) were statistically significant related to development of vap. in multivariate regression analysis, using the stepwise technique, three of the seventeen studied factors showed to have an indepantent association with the development of vap:the administration of neuromuscular blockade (f: 4.8, p<0.001), flail chest (f:3.0, p=0.003), and gcs (< 8) (f:2.1, p= 0.039). conclusions: in patients admitted to icu for multiple trauma or major surgery, the administration of neuromuscular blockade, the flail chest, and the gcs (<8), in the population under study, were the indepedent risk factors for vap. mof is a sereous complication of differem states: infection, sterile inflamation, extensive fissure injure, intoxication, ets. there is close correlation between extension of mof and death, developement of nasocomial infection. immunologic disfunction. in order to prgnose probability of risk of mof development among the patients with sepsis and septic shock, we achived an eqation, allowing to recive a coeficient, closely connected with this probabiliti. we have used retrospective analisis of 160 cases of sepsis. diagnosis of sepsis was based according to bone's criterions of sepsis. mof was assessed as disfunction of 2 or more systems according to bone's classification of mof. having used correlation analisis we have estimated factors which have had high correlation coeficient with the probability of development of mof. there were: apache-ii score points, evidenceof septic shock, endocrinopathy. with the help of multyple regression analisis we acheved next equation: y= 0,2042 + 0,0243x~ +0,2931x 2 +0,1504x3 , were x i-apache-ii score points, x2-evidence of septic shock, x3-endocrinopathy. the explanatory power of this quation was evidenced by roc of 0.88, se (v 0 -0.033 introduction: the presence of liver dysfunction in the process of multiple organ failure is associated with an adverse outcome, particularly when it becomes progressive to liver failure. disturbances of liver function may occur early and their detection may be of significant importance for the further development of organ failure. routinely used liver function tests appear to be inconsistent indicators of hepatic damage. in this study, we used p_lasma disappearance rate (pdr) of indocyanin-green dye (icg) as an early estimate of liver function. methods: we serially evaluated pdr and routine liver function tests (serum bilirubin, sgot, sgpt), as well as acute phase and non-acute phase proteins (crp, transferrin) in 26 patients during the first week after trauma or the onset of sepsis. patients: group 1: (n = 11) multiple trauma iss > 30, group 2: (n = 15): abdominal sepsis, acute necrotizing pancreatitis (anp) grade iii. patients were selected on the basis of clin4cal estimates that these patients would require continued icu observation. pdr was determined by means of a fiberoptic catheter and a computerized system (cold z-021, pulsion), which permits repeated bedside measurements. the initial values of pdr, serum bilirubin and transaminases were not significantly different in trauma, sepsis and anp. in trauma patients pdr improved during the first week. in patients with sepsis and anp pdr remained low and worsened with time. the decrease in pdr preceeded an increase in biochemical liver function tests in these patients. 1+4.4 110&-_11 7(4-9) discussion: routinely available blood tests of liver function are usually altered several days after injury. however, they are generally non-specific indicators and they are influenced by extrahepatic factors. pdr seems to be useful to evaluate impaired liver function early after the onset of sepsis and trauma. objectives: to study frequency of organ system failure (osf) and it's influence on outcome in granulocytopenic patients with hematological malignancies and septic shock(ss). materials and method: retrospective review of medical records of 52 granulocytopenie(wbc<0,5xl09) patients with hematological malignancies and ss, who were admitted to the intensive care unit (icu). frequency of osf before and after ss was analysed. the patisnts were categorised on survival and non-survival. results: signs of osf were observed in 28.8% of patients before ss and in all patients after ss. only 3 patients presented with hypotension refractory to inotropic therapy. nevertheless there was a significant increase of frequency of acute respiratory failure (arf), acute renal failure (arenf) and liver injury (li) after ss occurred(showed on the figure). only 5 frequency of organ failure before and after objectives: statusmetria allows to define the effective level of oxygen status and accordance to it means of carbon dioxide and elec-trolyte8 in critical care. the conception of syndrome int~ive care (sic) is exhausted itself and invariable outcomes of sic of multiergan system failure (mosf) confirms that. therefore, an alternative to sic should be advanced. methods: efficlenoy of treatment has been asscsaed in 257 patients with mosf using value of metabolic rate and ability of an organism to cover it by oxygen and substrate supply. oxygen pulse (op) and index of efficacy of oxygen transport (ieto2) was monitored. ~lt~.lntenaive care is considered to be homeostasis-securing therapy (hst) if energostructure deficit is eliminated and necessary for recovery regeneration rate is .restored. op in patients with mosf was 0.8 mt-m "2, and le,~ and ie'i~ w~ 2.4 units in sic. we managed to maintain op of 1.0-1.7 ml.m "2 and ieto2 of 1.9-2.6 units in hst. 41 patients from 135 with mosf survived in sic and 96 patients from 122 survived in hst. efficiency of hst appeared to be two times as much as efficiency of sic. cr of homeostasia-se-'uring therapy is advancing. the conception provides restoration of regeneration rate due to effective then in sic elimination of en=gostructure deficit. the conception may be a basis of new technology for treatment of mosf. helen f goode phd, nigel r webster phd. anaesthesia & intensive care, university of aberdeen, ab9 2zd, uk. objectives: xanthine dehydmgenase is converted under conditions of ischemia, reperfusion and endothelial damage to xanthine oxidase, with superoxide anion as a co-product of its catalytic activity. multiorgan dysfunction syndrome is associated with splanchnic vasoconstriction resulting in significant and prolonged gut ischaemia. aggressive volume resuscitation with prompt restoration of blood flow results in reperfusion of the tissue and is likely to cause xanthine oxidase-mediated release of oxygen-derived radicals. this study investigates xanthine oxidase activation and oxygen-derived free radical-mediated damage in such patients. methods: fourteen consecutive patients on itu who met established criteria for septic shock and secondary organ dysfunction were studied. serum xanthine oxidase activity was measured using oxidation of a chromagen in a dual enzyme system and plasma malondialdehyde was measured using a specific spectrephctometdc assay. apache ii scores, blood pressure, svr, cardiac output and 28 day survival were also recorded. biochemical data were compared with results from 20 healthy subjects. results: xanthine oxidase activity was 6.30 + 1.59 units/i in patients (mean :t: sem) and 0.74 + 0.12 units/i in controls (p<o.01, mann whitney u test), and was highest in the 6 patients who survived (p<0.05). malondialdehyde concentrations were elevated (0.81 • 0.11 prnol/i compared with 0.29 • 0.05 pmol/i in controls, p<0.001). xanthine oxidase activity did not relate to apache score or any of the cardiovascular parameters recorded, and was not influenced by the degree of organ dysfunction. conclusions: patients with sepsis and secondary organ dysfunction have xanthine oxidase activation and evidence of free radical damage. the finding that activity was highest in those patients who survived suggests that reperfusion was incomplete in patients who died, with decreased tissue xanthine oxidase ~tash out' into the circulation. influence objective : evaluation of the feasibility of measuring the intragastrie partial pr~sure of carbon dioxide (pco2) using a chemilumin~nt optode and fibre~ptics originally developed for intrav~cular blood gas monitorlng(p7) methods the pco 2 electrode w~ calibrated i~-vitro according to the m~ufaeturers instructions the sensor w~ then stripped of its outer c~ing and threaded down the pile tube of a g~ c enema e (gt) so ha he ens ng portion sat well within the balloon. the modified gt was inserted n~og~trically after induction of anesthesia in ten patients undergoing c~diopuimonary byp~s throughout results: according to the datas of integral rheography 70% of patients were revealed to have hypodynamic type of blood circulation (cardiac output was decreased on 35-40% and general peripheral resistance increased on 80-100%). it was effective to use complamin (xantinoli nicotinas) in the complex therapy of su~h patients. the dosage was 10-15 mg/kg during 4-6 days. as a result of such therapy was also the oliguric stage shortening (ac n 1464083).in 9,6% of the cases unsatisfactory datas of central hemodynamic,combined with high (more than 15 cm h20) venous pressure,lung oedema.then nanipruss (0,5-1,5mkg/kg/min) was succesfutly used in complex with routine therapy.in the patients with low cardiac output and low general peripheral resistance mesaton (0,25-0,5 mg/kg) and dopamine (5-10 mkg/kg/min) were effective.change for the worse in the hemodynamics datas, inspire of therapy during 5-7 days,was prognostically unfavourable. conclusions:we consider early diagnostic of hemodynamic disorders and adequate correction of them is one of the most important factors, determined the outcome and prognosis in arf patients. obiective: to evaluate the results of renal replacement therapy (rrt) in surgical icu patients with acute renal failure (arf). arf in icu patients is associated with high mortality rates. we investigated the relation between mortality and organ failure in 4 different categories of surgical icu patients receiving renal replacement therapy (rrt) for arf. methods: the records of 76 surgical icu patients with arf requiring rri-(haemodialysis or cavhd) were examined. five different patient categories were defined; 1 .ruptured aneurysm of the abdominal aorta (raaa) n =20 2.elective vascular surgery (evs) n =20 3.abdominal sepsis n -17 4.trauma n = 8 5.others ( e.g. malignancy, obstetric emergencies) n-11 seven organ systems (cns,circulatory,respiratory,hepatic,renal,digestive,haematologic) were scored for possible failure using the mof score. results: mortality was as follows: all patients 72% , group 1 75% , group 2 70% , group 3 88% , group 4 38 % , group 5 75 % . mortality increased with the number of failing organ systems; mortality for all patients with > 4 failing organsysterns was 80% the only exception being the subgroup of trauma patients where mortality under these circumstances was 5o% conclusions: mortality in surgical icu patients receiving rrt for arf is high. no significant difference in mortality is found between raaa and evs. mortality increases with the number of failing organ systems. the subgroup trauma patients shows a lower mortality compared to the group as a whole, even with > 4 failing organ systems. to look for the most accurate scoring system to measure the severity of the complications occuring in the early phase ( first 30 day) of kidney transplantation and to asses their prognostic value. methods: in our retrospective study we applied the apache li and the goris scoring system for the kidney recipients who developed multiple organ failure (mof) as a consequence of their pulmonary and. cardiovascular complications following kidney transplantation. we evaluated the recipients the distribution of the women and men ( 60% ~ 40 % ) was the same as in the kidney recipients. applying the apache ii system most of the patients had their score between 10 and 19, and the function of 2,3 or 4 organs were affected at the time of the onset of mof. the apache ii system gave adequeate information about the disturbance of the function of other organs beside the kidney failure even at the time of the transplantation. the scores and the number of the affected organs correlated with the condition of the patients in the goris scoring system but not as sensitively as in the apache ii scoring system. conclusions: both the goris and the apache ii scoring system can be applied to measure the severity of the multiple organ failure occuring during the early phase of kidney transplantation. however the apache ii system is more suitable to follow not only the stateof the patients at the time of the admission but also the changes occuring in their condition during the complication. v.v.erofeev, v.v.ivleva scientific research institute for general reanimatulogy russian amsci, moscow, russia objectives: the analysis of ssc and results of their treatment in patients following critical states showed the necessity of developing a combined antibacterial therapy. methods: according to the protocol 97 patients (18-60 years old) with combined trauma and massive hemorrhagy following vast aml traumatic operations were examined. microflora's composition and resistence to up-to-date antibiotics was studied using the anaiyser iems reader by "labsisteme"(finland). general clinical, bacteriological, immunological indices, as weil as the duration of the treatment and recovering rate served as criteria of the combined antibacterial therapy effectiveness. results: it was proved expedient to administer antibiotics in staphylococcus infection in the following combinations: riphampizin with fluoroquinolones; i-ii degeneration, cephalosporins with aminoglycosides; cephalosporins with fluoroquinolones. in case of singling out the exciters of the euterobacteriaceae family, including the pseudomonas aereginosa, -fluoroquinolones combined with modern amynoglycosides; fluuroquinolones with ureidopenicillines; ureidopenicillines with amynoglycosides; amynoglycosides with the ii-iii generation cephalosporins; cephalosporins with fluoroquinolones. in severe ssc caused by combined infection (including anaerobes) clindamicin with modern amynoglycosides was prescribed. conclusion: the combined antibacterial therapy allows: 1) to increase the effect on microbic agents and the efficacy of treatment in combined infections; 2) to lessen the possibility of the exciters'resistence to antibiotics; 3) to prevent the development of superinfection: 4) to decrease the doses of medicine and its toxic effect. objectives: two methods of blood volume measurement in a group of critically ill patients were compared to investigate the practical possibilities of a new easy to use method based on carbon monoxide (co) uptake. methods: all patients had multi-organ failure and haemodynamic monitoring with a swan-ganz catheter. mean apache ii score was 19 (10-25). when indicated, 9 patients had blood volume measurements simultaneously based on the techniques of, i) dilution of 5~cr labelled red cells, and ii) inhalation of carbon monoxide gas with measurement of the rise of carboxyhaemoglobin produced. the co was administered via a newly designed, ventilator driven, fully closed circle system ensuring co retention and co2 removal with automatic addition of oxygen to m}ttch patient uptake. a portable computer performed all necessary calculations. results: volumes obtained by co uptake were compared with the "gold standard" radiolabelling method. mean blood volume determined by the co method was 6310ml (4710-7959ml) compared with 4690ml(3755-5778ml) with slcr labelled red cells (r=0.9). regression analysis produced an intercept at 769ml. the slope of the regression line was 0.62 (0.33-0.9, 95 % confidence limits). discussion: the co method produces volumes in excess of the radiolabelling method. there appears to be a systematic error, and one possible explanation is co binding to substances other than haemoglobin. conclusion: the co method is easier to use than radiolabelling and of the lower cost, since cohb measurement only is required. aceuraey is sufficient for clinical use and our preliminary findings suggest this system will meet the requirements. objectives: this study was conducted to determine the role of nitric oxide (no) in the pathophysiologic alterations and multiple organ damage, and the possible effects of " " " (l-n -monomethyl-l-arglnlne nmma) on hemodynamics and mortality in rats caused by a prolonged hypovolemic insult. methods: a prolonged hemorrhagic shock (30-35 mmhg for 180 rain) was induced in anesthetized rats followed by adequate resuscitation. l-nmma was administered intravenously at doses of 2.0 mg/kg or 20.0 mg/kg at the end of resuscitation. results: infusion of 2.0 mg/kg l-nmma diminished the fall in mean arterial pressure, significantly increased the cardiac index (ci) and stroke volume (sv), together with remarkable protection from multiple organ damage compared to the controls. the 48 h survival rate was significantly improved from 26.7% in the control group to 68.8% in the treatment group (p<0.05). in contrast, the high dose of 20.0 mg/kg l-nmma resulted in a strong blood pressure response but a marked reduction in ci and sv concomitant with an increased total peripheral resistance index within the observation period, and caused severe damage to various organs at 2 h after treatment. in addition, marked elevation in both endotoxin and tnf levels were observed in animals subjected to shock insult. conclusions: these results suggest that no induced by hemorrhagic shock in rats is an important mediator for pathophysiologic alterations associating with cardiovascular abnormalities, multiple organ dysfunction, and even lethality. thus, regulation of no generation and use of no inhibitors might provide new aspects in the treatment of hemorrhage related disorders, and the use of l-nmma would be either deleterious or salutary in a dose dependent manner. (hebert, chest-1993) . the purpose of this study was to assess the risk factors for hepatic dysfunction in mosf. methods: 733 patients have been hospitalized in our icu from january 1992 to may 1. 994, 198 (27%) with mosf. among mosf pati~ts, 57 (29%) have had hepatic dysfunction defined according to hebert (bilirubin ~ 60 ttmop1, chest 1993). thirty six of these 57 patients acquired hepatic dysfunction after admission in the icu. these 36 patients were compared with 36 mosf patients without hepatic dysfunction selected blindly. chrorfic diseases, severity scores, eanse of admission, clinico-biologieal and hemodyunrrfic parameters, use of vesopressors, use of hepaiotoxic drugs, use of nutritional support and mortality were compared for hepatic failare and non hepatic failure groups.twenty nine patients had postmortem hepatic histologic examination, results: univaciate analysis: only parameters with p _< 0.05 are pre~nted. including these paramet~'rs in a multivariate analysis, anly c~hosis and vascular surgery remain independent risk factors for hepatic dysfunction. in particular, pao2/fio2, arterial lactate, do2 were not different between the two groups, some de~'ee of histological abnormalities was found in all liver samples, despite a normal bilirubin level in 15 % of the cases conclusions: in our patients, conu'ary to previous studies, hypoxic and hemody~anfic parameters were not independent risk factors for hepatic dysfantion. this might be due to the inadequacy of the usual biologic definition of hepatic dysfunction as well as to the poor sensitivity of general hamodynamic parameters. critical states of various origin are complicated with the mldtiorgan farm (moi~ oceuzr~ce. due to their and functional features the lungs become the primmy damage target in various critical.states. ard8 that occurs in such states is associated with pulmonary edema development because of capillary permeability increase mediated by humeral and cenular responses to 0amag/~5 factors exposure. r nmst be emphasized that mediators and effecto~rs of this respo~e affect not only puknonary capillaries, but other organs capiu~es as wellenhancing their permeability. orsans edema is a conmm~ finding at the autopsy of patients died from mof.clinical and radiolosial findings allow to have a diagnosis of pulmonmy edema before ~mi!ar lesions in other organs occm. additionally, there are some techniques that permit quantitative assessment of pulmonary edema flv.id (evlw) volume. in conclusion, we suggest that evlw changes in .dyn~rmcs in patients with mof are considered as a critical state severity measure which reflects indirectly the edema in other organs. objectives: we compared three different dialysis membranes to find out whether or not there were differences between their clearance characteristics on substances such as inuline, creatinine, urea, and phosphate to be eliminated in acute renal failure (arf). moreover, if a loss of clearance did occur we were interested in whether this was due to heparinization and a high production of the thrombine-anti-thrombine-complex (tat). methods: we carried out a randomized controlled study on 13 consecutive critically ill patients presenting with arf, most of them in association with multi-organ failure, to be treated by continuous pump-driven arterio-venous renal replacement therapy on continuous low-dose heparinization. three different types of high-flux filter membranes (f 60 tm [fresenius] , ct 190 tm [baxter] , and filtra116 tm [hospal]) were assessed. each filter was changed intentionally after a 24 hours" use. together the data of 54 filters were evaluated, each at three different times (immediately after its onset [0 hi, after 8 h, and after 24 h). the clearances of creatinine, urea, phosphate, and inuline were measured. results: there were some significant differences in clearance characteristics of inuline, creatinine, urea and phosphate between the filters (p<0,05) showing the f 60 tm membrane excelling filtra116mand ct 190 tm the more. the loss of inuline clearance (3 mi/min/m 2) after 24 h, however, was insignificant for all 3 filter types. a continuous low-dose heparinization scheme was applied without any relevant prolongation of the aptt. even lower losses were noted for the clearances of creatinine, urea, and phosphate. we found the tat-producfion increased after 8 h (p<0,05), but it did not rise any further. conclusions: as we could demonstrate in our study the clearance data of different types of filter membranes applied during continuous renal replacement therapy do show significant differences. on the other side, no relevant loss of clearance occurs during a 24 hours" period indicating a high efficiency over time. to consider commercial aspects as well it shows that inexpensive conventional filter membranes can successfully be applied even for a longer renal replacement period, if needed. a retrospective study was performed on 100 patients with acute renal failure (arf). we analysed survival in continuous (cd) and intermittent dialysis (hi)). mean age of the patients was 60 years (y), 57 patients (57 %0) were <65 y, 43 patients (43%) were >= 65 y. the incidence of dialysed arf in our mixed intensive care departement is 3%/admission/y. statistics: fischer's exact test, mann-whitney-u test. efioloev: the contribution sepsis, cardiac failure and aminnglycosidcs was respectively 70%, 44 % and 35 %. treatment: cavh (cd) or cvvh (cd) was used in 40 patients (40%), hemedialysis (hd) was used in 60 patients (60 %). data: mean apache 2 scores were the same for cd and hd (27 for both groups), patients treated with continuous dialysis techniques had significantly (p<o,05) more need for ventilation (100 % vs 70 %), elevated bilirubin (82 % vs 57 %) and a higher vasopresser need (89 % vs 46 %@ than patients treated with hemedialysis. there was a trend towards more sepsis (83 % vs 62 %; p=0.06) mad coaguiation disorders (50 % vs 27 %; p=0.07) in cd. taere were sign/ficantly more younger patients (<65 y) treated with cd (70 % vs 30 %, p<0.os). mean apache 2 score was significantly lower in patiants<65 y than as patienta>=65 y (26 vs 30; p<0.05). patients<65 y had significantly (i}<0.05) more coagulation disorders (53 % vs 17 %) and elevated bilirabin (81% vs 52 %). there was no significant difference in vasopressur need and ventihatio~ between age groups. outcome:. hi) had a better sr compared to cd (43 % vs 15~ p<0.05). patiants>=65 y had a comparable sr vs patients<65 y (3") */e vs 28 %; p----a.s.). tha global survival rate (sr) was 32 % (32 patients). conclusions : diaiysed arf has a well known lowsurvival rate (32 %): hc~raedialysed patients had a better survival rate than patients treated with continuous dialysis. this can be explained by the fact that the latter were in a worse condition considering organ failure (more vantilatian, elevated bflirubin and need for vasepressurs), apache 2 score couldn't illustrate that. patient~65 y with arf have the same survival rate as patients<65 y: although patients >=-65 y have a higher apache 2 score they have less organ faille. the avacbe 2 score is not a good oredictor of survival in p with organ failure. departments of surgery and intensive care, guy's hospital, london, u.g-obiectives: a randomised controlled trial of a management protocol utilising the regular measurement of gastric intramucosal ph (phim) to control the administration of dopexamine. methods: patients admitted to a multidisciplinary teaching hospital intensive care unit (icu) undergoing insertion of a pulmonary artery catheter were managed according to a resuscitation protocol. randomisation was to either the protocol alone or to insertion of a nasogastric tonometer and subsequent management guided by phim. phim < 7.32 initiated volume and inotrope resuscitation and, if unsuccessful in elevating phim, dopexamine was commenced. approval was obtained from the hospital ethics committee. results: 94 patients were considered for analysis and the two groups were well matched for age and sex. overall, there was a high hospital mortality of 64.9%. there was no difference in icu or hospital mortality between the two groups (see table) . objectives: to compare cardiac output (co) measurements between continuous termodilution (cco) by thermal wire on pulmonary artery catheter (cco/svo2 vigilance. baxter critical care), and co measurement using a trans-esophageal doppler (dco) ultrasound system (odm ii, abbott laboratories), in the immediate postoperative period of cardiac surgery. methods: 15 patients undergoing myocardial revascularization were monitored with cco by a swan-ganz catheter and an intra-esophageal dco probe, after induction of anesthesia. exclusion criteria were: aortic valve disfunction, previous valvular surgery esophageal disease, absense of sinus cardiac rhythm, and need of ventricular or intraaortic assistance. hemodynamic parameters, co by both cco and dco, svo2. sao2, diuresis, pha, and hemoglobin were repeatedly registered during the first 6 hours after surgery, as the patients were kept under sedation and mechanical ventilation. results were compared using the method described by bland and altman. results: 176 measurements of co were obtained, ranging 2. objectives: a decreased tissue oxygen delivery is responsible for a higher morbi-mortality rate among surgical patients; this diminished oxygen delivery/consumption rate (dojvo2) may origin the lactic acidosis observed in the gastrointestinal tract, reported in patients undergoing hypothermic cardiopulmonary extra corporeal surgery, and can be registered by tonometry as result of the gastric mucose ph. the purpose of this study is to evaluate the reliability of the intramucosal ph (phi) measurement by a nasogastric catheter as indicator of the do2/vo > its co> relation to other parameters of do2/vo 2 disturbance, and with postoperative complications and clinical course. methods: 20 patients (16 male, 4 female) undergoing cardiac surgical procedures were included (16 myocardiai revascularizations, 3 valvular substitutions, 1 constrictive pericarditis). mean age was 63 + 12 years, mean weight 70 _+ 10 kg. a nasogastric probe (trie tonometrics) was placed after anesthesia induction; phi values were registered in the postoperative period (90', 120', 240", 360' and 18 h after surgery end). the corresponding hemodynamic parameters, venous oxygen saturation (svo2), diuresis and arterial ph (pha) were also recorded. results: phi values ranged 7.20 to 7.65 (mean 7.40 (0.8); the mean values of clinical evolution were: extubation time, 20 _+ 12 hr.; discharge from postoperative care unit, 88 4-50 hr.; and hospital total postoperative time, 10 _+2.2 days. complications registered were: 2 perioperative acute myocardial infarctions, 2 cases of respiratory insufficiency, 1 occlusion of coronary bypass, an 1 ease of hyperamilasemia. all patients with severe complications needing specific treatment showed either a low phi value, or a considerable descent in comparison with the initial register. statistic correlation between low phi and presence of complications was found; the low significance (p > 0.05) degree may be due to the low population size. conclusions: phi measurement in cardiac surgery patients is a non invasive, uncomplicated method for prediction of doz/vo 2 disturbances, thus reflecting risk of increased major complications, and may precede changes in other usual indicators (svo2, pha, cardiac output, ...). work-in-progress with a greater population size may offer more significant results. references: (1) gutidrrez g: lancet 1992; 339:195-202. (2) landow i: acta anaesthesiol scand 1994; 38: 629-639. the haemoglobin-level (hb) is besides the arterial oxygen saturation and the cardiac index one of the relevant parameters of oxygen supply to the tissue. in contrast to otherwise healthy patients, there is no agreement on tile so-called transfusion-trigger in critically ill patients. in i?ont of this background the question arises, whether and to what extent blood transfusion in critically ill patients improves oxygen supply io tile tissue. this study was performed in 34 critically ill/septic patients in the postoperative period alier an inlcclive/scptie revision operation of the hip or knee joint. on cardiac/seplic reasons monitoring consisted beside other measures of a pulmonary arlery catheter and of an indwelling arterial line li~r measurering/calculating standard haem~dynamic as well as systentic oxygen parameters. the indication for blood transfusion was given by hb together with the cliuical slatus of thc patienl (asa-scorc and multiple organ dysfunction (moi))). statistical analysis w~ks performed by mann-whitney-u-test. by fisher's exact-test and by wii.coxon-test: statistical significance was set with p<0.05. according tu the pretransfusion value of hb and of lactate (lac) palicnts ;,,'ere divided into groups as follows: a: hb<8 and b: >sg/dl: i: 1ac<2.8 and ii: >2.smm. in either group blood transfusion results in zt significant increase in hb (a: 7.5_+0.4 to 9.4+0.8 g/dl; b: 9.(~0.8 tt, 10.5+0.09 g/dl; i: 8.0-+1.0 to 10.2-+0.6 jdl; i1:8.4-+0.9 to 10.1+0.7 g/dl). wlailc, however, haemodynamic parameters do not difl)r significantly from each other before and alter blood transfusion, oxygen delivery (do, -ml/min x m-') increases significantly hi either group studied (a: 467-+86 to 581-+158; b: 521+125 to 577+137; 1:512 -+1 13 to 599-+141; i1:516-+123 to 632-+214), in contrast oxygen consumption (vo~ -ml/min x m e) does not change significantly in either group (a: i68-+148 to 158-+38; b: 180-+162 to 175-+52; i: 171-+38 tu 179-+35; 11: 199-+60 to 219+_71); oxygen exlraction ratio decreases. this study in critically ill/septic patients demonstrates, that in this group of patients studied blood transfusion at a base-line-value of >7.5-+0.4 g/dl expectedly rises do~, however, it does not improve vo=; even not in septic patients with elevated lac-values. paclitaxel in a new anticancer agent, extract from the bark of the yew tree (taxus brevifolia), employed against breast and ovarian cancers resistant to chemotherapy. it promotes the polymerization of tubuline, and disrupts the normal microtubule dynamics. hematologic toxicity, hypersensitivity reactions (bronchospasm, urticaria and hypotension), and peripheral neuropathy are the main reported toxic effects. cardiac side effects are rare: atrioventricular blocks of higher degree are reported in 0.1% of patients; congestive cardiotoxicity was discussed only in one trial in patients treated with paclitaxel and doxorubicin. we describe the history of a 48-years-old worn an with a breast cancer, diagnosed in 1989, initial staging t3nim0, treated with mastectomy, axillary lymphadenectomy, andchemotherapy with a cumulative dose of anthracyclines of 678 mg/m2 until august 1994. the patient complained of dyspnea and severe hypotension immediately after an intravenous infusion of 100 mg paclitaxel, given over 1 hour for the treatment of bilateral, malignant pleural effusion. at echocardiography die left ventricular ejection fraction was reduced to 20%. she died 20 days later because of a severe cardiac low output with hepatic and renal failure; an impressive hepatic cytolysis was observed. the post mortem examination confirmed the dilatation of the cardiac cavities, especially of the right ventricle, bilateral pleural fluid, and ascites. the histology was suggestive for a cardiomyopathy secondary to anthracyclines. the electron microscopy revealed a deposition of an unusual pathological pigment in the myocytes; subsarcolemmal deposition or membranous were absent. we hypothesize that paclitaxel was the cause of a major hypersensitivity reaction with shock and severe hepatic cytolysis, worsening the myocardial damage induced by anthracyclines. the possibility that a low doge of paclitaxel could directly increase anthracyclines cardiotoxicity -as decribed in the medical literature -will be discussed. objectives: activated endothelial cells release soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selectin (selam-1). sicam-1, svcam-1, selam-1, and inflammatory cytokines were determined. methods: sicam-1, svcam-1, and selam-1 were determined by elisa. tnf-a, il-6, and il-8 were also measured by elisa. endotoxin was measured by an endotoxin-specific endospecy test after pretreatment of new pea method. results: the sicam-1 and s vcam-i levels were significantly higher in the septic multiple organ failure (mof) and sepsis groups than in the non-septic mof group. the selam-1 level was slightly higher in the septic mof group than in the sepsis withut mof group and non-septic mof group. the increases of soluble adhesion molecules were not in agreement with changes of plasma endotoxin level. levels of soluble adhesion molecules were correlated with the levels of plasma tnf-a and il-8, but the level of il-6. discussion and conclusion: the slcam-1 and svcam-1 levels in septic patients closely reflected the severity of the pathophysiological conditon. it was possible that the release of sluble adhesion molecules were not stimulated by plasma endotoxin, but endotoxin in the local infectious region. tnf-c~ and il-8 also were suggested to be involved in the release of these soluble adhesion molecules. obiectives: cardiopulmonary bypass (cpb) surgery is associated with a systemic inflammatory response attributable to the release of various inflammatory mediators and the activation of complement or coagulofibrinolytic system. in addition, adhesion molecules, such as icam-1, elam-1, and vcam-1, appear to be of central importance in the inflammatory process following cpb surgery. we previously reported the effects of a synthetic protease inhibitor, fut-175, reduced release of inflammatory cytokines (tnf, il-lg, il-6), activation of complement (c3a, c4a) or coagulofibrinolytic system (tat, pic, fpa) and protected platelet function (gpib, gpiib/llla) following cpb surgery. methods: in this study, we analyzed fut-175 on soluble adhesion molecules following cpb surgery. 20 patients undergoing cpb surgery were divided into two groups, group a consisted of 10 patients who received 1omg of fut-175 in priming solution, followed by a continuous infusion at 2mg/kg/hr during cpb in addition to initial heparin dose of 3mg/kg. group b, a control group, included 10 patients who were injected with heparin only. the plasma slcam-1, selam-1, and svcam-1 concentration was measured by elisa. results: every soluble adhesion molecules decreased during cpb in both groups, and rose after cpb. selam-1 and slcam-1 reached their peaks on 3 hours after cpb and on pod 1 respectively in both groups, but they remained lower in group a (selam-i: 32.1+11.5 vs. 38.6• ng/ml, p<0.05, slcam-i: 327• vs. 483• ng/ml, p<0.05), svcam-1, in both groups, remained lower than preoperative levels, but did much lower in group a. conclusions: fut-175 reduced adhesion molecules and suggested to be the effect on postoperative organ dysfunction. in the last few :,'ears the conditions of treatment in continuous hemofiltration/hemodiafiltration were discussed controversially. a significant removal of tnf-alpha and il-i could be demonstrated in cvvhd. the aim of our study was to investigate the elimination of tnf-alpha, 1l-2, il-6, il-8, s-cd-14 and ifn-gamma in cvvh by measurement in plasma and hemofiltrate of 10 critically ill patients with an acute renal failure. the patients of our study were treated with a continuous veno-venous-hemofiltration (polysulfone-filter, blood flow: 100-130 ml/h, filtration rate 500 ml/h). the samples, hemofiltrate and plasma, were taken one hour after the start of treatment. the patients suffered from septic shock (4), the so called hepatorenal s~aldrome (3) and a severe pancreatitis (3). the cytokine concentrations were measured with elisa-method. in contrast to elevated concentrations in plasma for tnf-alpha (5 cases), scd 14 (9 cases), il-2 (l case) and il-6 (4 cases), hemofiltrates contained no activities. only il-8 was removed in significant amounts with even higher levels in hemofiltrate than in plasma. this phenomenon was described so far for tnf-alpha and il-1 and may be due to the absence of metabolic properties (possibily enz~natic) in hemofiltrate. it can be shown, that tnfalpha, il-2, il-6 could not be eliminated in cvvh with a filtration rate to 500 ml/h. in contrast to findings of other investigators with a higher filtration rate (> 1000 ml/h), we found no significant concentrations of tnf-alpha and il 6 in hemofiltrate. we conclude, that for a significant removal of important cytokines higher filtration rates (>1000 ml/h) are necessary. objectives: multiple organ dysfunction syndrome including liver and renal impairment is a fatal complication in patients with the diagnosis of sever sepsis. this study focused to the effects of removing toxic substances from inflamnatory tissue by hemodiafiltration. ~4ethods: eleven patients were admitted to the icu in emergency center and met the criteria of systemic inflammatory response syndrome in association with infection. all patients developed liver and renal dysfunction and were treated by hemodiafiltration with high flux membranes (fb-u:nipro). the hemodiafiltration were performed 19 times using nafamostat mesilate as an anticoagulant in 5 hours with 12 l of substitution fluid (hf-b:fuso). the serdm levels of endotoxin, cytokines, endothelin-i (et-]), human neutrophil elastase ~1-proteinase inhibitor complex (hne-pi), fibronectin (fn), lactate, and amino acids were measured before and after the hemodiafiltration. the hemodiafiltration would be effective to renal dysfunction by reducing endothelin and beneficial to tissue metabolism represented in fisher's ratio, but might be harmful to respiratory function by activating neutropila in patients of severe sepsss. background : intermittent hd may be poorly tolerated in the early phase of arf in hemodynamically unstable patients (pts). this technic may fail to achieve steady state urea low levels in hypercatabolic pts. method : nt = 25 consecutive pts treated with hd; n 2 = 25 consecutive pts treated with cvvhf. hemodynamic unstability is defined by arterial hypotension and requirement of inotropie support despite adequate filling. rate of change in urea (u), ereatinin (cr), k + , ph were computed from a linear regression .analysis of data vs time in each treatment group during the 5 first days of application of the two technics (anova). dally worst values were recorded. results : hd-group : apach% score = 22 _+ 7; mean number of organ system failure (osf) = 1.5 -+ 1; mean blood pressure (mbp) = 75 • 22 mmhg (first day of application of hd). cvvhf-group : apachen score : 25 + 6; osf = 3 -+ 1; mbp = 57 + 20 mmhg (first day of application of cwhf discussion : during the 5 first days of application of hd/cvvhf, u and cr decreased much more rapidly in the cwhf-group. k* and ph were maintained within normal range in the two groups. initial mbp which was much lower in the cwhf-group significantly improved during the application of cvvhf while mbp remained unchanged in the hd-group. conclusion : despite higher severity of disease in cvvhf group (apachen score, osf, lower initial mbp), we obtained a better performanco with cvvhf regarding the decrease of u and cr and the improvement of mbp. in relation to the different and continuous renal replacement techniques, the continuous venovenous one is the alternative method to continuous arteriovenous for critical patients with acute renal failure (arf). we present you our experience with cvvh in patients with mof. in our intensive care unit (icu) 20 patients with mof were treated with cvvh in the period between january in 1992 to march in 1995. the mean (• age of our patient population was 52,1• years, being 65% male and 35% female the whole patient population was with mof iust at the moment the technique was accomplished; 75% was in mechanical ventilation, 90% needed vasopressor support and 65% required both of them (mechanical ventilation and vasopressor support) apache ii score mean of the patient population was 17,84~:6,97 (range 5-28) and ati of them were with arf oligoanudc. technique: cvvh was accomplished using a single-d~al iumen catheter, ptaced in either a temoral or subclavian vein by the stand ard seld{nger technique. pol{sultone hemofitiers were also used, and the extracerporeal circuit used standard arterial-venous blcod tubing. blood flow and hence oltrafiltration pressure, within the circuit was generated by a roller blood pump. the modulus has a roller pump, a pressure transducer connected in an arterious and venous line, such as an air-transducer which is adapted to a drip-chamber in the return way. the replacement used was a peritoneal dialysis solution. medicine 1, st. george's hospital medical school, london. england. hepatic sinusoidal endothelium shows a major inflammatory response in porcine sepsis that can be attenuated by the administration of dopexamine hydrochloride. dopexamine is a beta 2 and dopaminergic receptor agonist. the specific beta 2 adrenoceptor antagonist ici 118551 has been shown to reduce the protective effects of dopexamine. we investigated the effect of this antagonist on hepatic ultrastructure in porcine sepsis. six pigs (25-30kg) divided into 2 groups were anaesthetised and intubated. cardiac output and portal blood flow were measured using standard techniques. the 2 groups were; placebo, (peritonitis induced); blocker, (peritonitis induced and 200 pg/kg ici 118551 bolus infused then given hourly). caecal content was aspirated and peritonitis induced. colloid was infused to maintain pawp at 10-12mm hg for eight hours the animals culled, hepatic tissue removed and prepared for electron microscopy. in the placebo group hepatic endothelium was swollen and the sinusoids occluded by wbc. but in the ici 118551 blocker group, much of the sinusoidal endothelium was absent and there where large extra sinusoidal spaces among the hepatocytes. an assessment of the two groups showed worse hepatic architecture in the blocker group. the b2 antagonist blocked any protective effect of endogenous beta 2 adrenoceptor agonist (adrenaline) on hepatic endothelium in porcine sepsis. george's hospital medical school, london. england. dopexamine hydr0chloride, a beta 2 and dopaminergic receptor agonist reduces hepatic damage in porcine sepsis. we tested dopexamine's effect on cerebral oedema. the beta 2 adrenoceptor antagonist ici 118551 was infused to block any protective effect of dopexamine. nine anaesthetised pigs (25-30kg) were randomised into 3 groups; placebo, (peritonitis induced); dopexamine, (peritonitis induced and 5 ~tg/kgdar of dopexamine infused); blocker, (as in dopexamine group but in addition 200 pg/kg ici 118527 bolus given then infused at that rate hourly). caecal peritoneum was induced and colloid infused to maintain pawp at 10-12mmhg for eight hours when the animals were culled, cerebral tissue removed, prepared for electron microscopy and digitisation. digitisation of the area of oedema surrounding the blood vessel and expressed as a percentage of the micrograph. 30.5_+4.1, dopexamine 13.5+2.9", blocker 31.5+3.7. data expressed as mean + sd. significance p<0.05. * dopexamine compared to placebo and blocker. in the dopexamine group the area of tissue oedema was significantly lower than either the placebo or blocker groups. there were no significant differences between the placebo or blocker groups. the 62 antagonist completely blocked the protective effect of the drug on cerebral oedema in porcine sepsis. beta 2 adrenoceptor stimulation is protective of cerebral oedema in porcine sepsis. objectives: the hemodynamie~ of hepatic circulation during multiple organ failure (mof) have not been suffleienly studied. we investigated liver hemodynamics in two subgroups of patients with mof, those with either liver or lungs as the main organ of involvement. methods: three groups of patients were created: i) mof-hepatic involvement (mof-hi) (7 patients) with bilirubin >3.5 mg/dl and lung injury score <1.8, it) mof-ards (9 patients) with respective values <2.0 and >2, iii) 5 patients with head injury with respective values <2 and <1, served as group control. all patients were in haemodynamieally stable state with an oxygen delivery index >300 ml/min/m2 prior to measurements. two swan-ganz catheters 'were inserted, one in the hepatic veins and one in pulmonary artery and the following measurements were determined: the hepatic vein free pressure (hvfp), the hepatic vein wedge pressure (hvwp), cvp, paop and co. the gradient of hvwp-hvfp represents liver perfusion pressures. by injecting contrast media at dose of iml/lokg with the balloon inflated to achieve sinusoidai image, the hepatic blood flow (hbf) was concluded by the time in seconds of media removal after balloon deflation. results: the co, cwp and cvp were comparable to all three groups. namely, for mof-hi, mof-ards and control groups the mean (+sd) value of co was 7.2_+0.8 vs 6.9_+0.3 (ns) and 6.3_+0.6 respectively, of the paop was 8.7+_1.6 vs 10+:3 (ns) and 8.2+3.1 respectively and of the cvp was 12.+.2.3 vs 11.6+2.3 (ns) and 5.8 respectively. in contrast the two mof groups were different after the cut-offinclusion criteria ie the mean (+sd) value for bilirubin was 6.8+9.5 vs 1.20+0.7 (13<0.05) and 0.8_+0.2 respectively and lung injury score was 1. objectives: oxygen delivery (do2) and oxygen consumption (vo2) are increasingly monitored parameters in the icu. there still remain controversies about an oxygen supply dependency in critical illness particularly with respect to vo 2 determination by either indirect calorimetry (vo2m) or tick calculation (vo2c). the purpose of this study was to investigate the changes in vo2m and vo2c following do 2 increase. methods: the relatives of 24 critically ill patients (mean age 63 years, mean apache ii 24, mean mof-score 9) gave their written informed consent to participate in this institutionally approved, prospective study. do 2 was increased by fluid loading (hydroxyethylstarch 10%: mean volmne 750 ml, mean duration of infusion 80 min) and catecholamine support (dobutamine: mean dose 14,3 ~g/kg/min). changes in vo2m and v02c were recorded sinmltaneously before, during and following interventions. calorimetry was obtained with the metabolic monitor 7250 integrated in the ventilator 7200 (puritan bennett, carlsbad, ca adaptive endocrine response of organism to septic shock consisting in activation of the production of adrenal hormons, renin -angiotensin -aldosterone system (raas) and other hormonal systems has an influence over microvascular changes in these states and for development of multiple organ failure (mof). in 25 patients with peritonitis of different origins (18 nonsurvivors and 7 survivors) were followed the changes in cortisol level and raas by radioimmunological methods and many variables for evaluation of respiratory, renal, hepatic function, coagulation etc. as a signs of mof. it was observed significant increase of the level of cortisol (1099 +_ 4,83 nmol/ i), aldosterone (0,895 • 0,687 nmol/i). by factorial statistical analysis we found significantly high correlations between hormonal changes and respiratory function (for example r=-0,539, p < 0,02 between cortisol and pao2; r = 0,817, p < 0,001 between cortisol and d (a-v) 02; olso renin -cao 2 r=-0,824, p < 0,001, renin d ~,vl o2 r = 0,626, p < 0,001). such significant correlations was found and for raas with respiratory, renal function, byproducts of arachidonic acid thromboxan b 2 and p6fla, soluble fibrine degradation products etc. these correlations between the degree of endocrine changes and multiple organ failure in patients with septic shock produced by peritonitis suggest that their effects upon peripheral vascular resistance and constriction of the splanchnic, splenic, renal and other organ vasculatures are not always with physiologic expediency and there are perhaps the possibilities of therapeutic influence. intredu~on : dopexamlne has previously been shown to control hyperkalaemia ia patients with acdto renal failure (arf), however effects on the subsequent course of art are undomunente~ ob_iectlv~ : to evaluate clinical progress in patients with acute renal failure (arf) in an intensive care unit (icu) with regard to biochemical control, need for -and time to -dialysis, and outcome in patients receiving dopexamine. m~ods : 14 consecutive patients meeting standard criteria for diagnosis of arf were included in the study. full cardiovas~dar, biechemical and intervention/outcome details were recorded. dopex.~min~ was infilsed at a dose of 2 pg/kg/min in conjunction with a regimen of inotropir support and blood volume optimization. resn]~ : following the intzoduetion of dopc',~mine ilrinr vohlmes increased slightly over the next 24 hrs fzom 516 + 140 ml/24 hrs to 817 + 229 ml/24 hrs (ns). data expres,uxl as mean + sem. three patients (21%) became polyuric with urine output >100 ml/hr within 3 days and did not need dialysis. in the remaining patients the time to dialysis (to correct acid-base deficits or volume overload) was 5.09 + 0.84 days. serum potassium levels were well controlled. day 5 or immediate pre-dialysis levels were 4.48 + 0.19 mmol/l compared with pre-lreatment 4.67 + 0.2 mmol/l overall mortality in this series was 4/14 (28%). duration of acute dialysis in survivors with renal recovery was 16.8 +_ 1.82 days. 2 patients (14%) progressed into chronic renal failure and needed continuing renal replacement therapy. no adverse cardiovascular altects were seen at this low dopoxami~ dose although its competitive inhibition to adrenergic reuptake mechanisms meant that doses of pressor agents could often be reduced. : dopcx:~minr nsed in conjunction with inotropic support and blood volume oplimitntion, can safely postpone, or even avoid, the necessity for acute haemodialysis in icu patients. no evidence of tachyphylaxis to the effect on serum potassium levels was seen over the duration of the study. hen'era m., suarez g., dagn d., varela a., ramos j., garoia jm, aragdm c, jurado l, medina a. icu. hospital regional. malaga. spain. objective: to evaluate the haemodinamic tolerance to the veno-venous continuous hemefiltration (vvchf) system in patients with systemic inflammatory response sindrome (sirs), and the possible beneficial effect of this technique on the haemodinamics in these patients. material: 13 patient admitted to the icu, with diagnosis of sirs and monitored with a pulmonary artery catheter at the beginning of wchf. we performed a complete haemodinamic study to all these patients (cardiac output, vascular resistanoss, ph and co2 in arterial and mixed venous blood samples, saturation of pulmonary mixed venous blood, do2 and vo2 calculations and temperature) and determined the respiratory mechanics (compliance and pao2 /fie2 relatinship) before starting the procedure, after 10 minutes operating with the ultraflltrate branch closed (without filtered fluid production), afler 30 and 60 minutes of zero fluid balance bemofiltration and after 120 minutes of filtration with negative balanos adjusted to the patients conditions. for the statistical analisis we have performed the anova test over the mentioned variables. results: we have not detected statisticaly significant differences of the analyzed variables before the beginning after operating the pun'@ for 10 minutes without filtered fluid production and after 60 minutes of zero fluid balance hf. only temperature shows a meaningful decrease in time. objectives: among many organs, playing the important role in pathogenesis of multiple organ failure, the particular place is taken by the intestine. ~ethods: the study was carried out in 5 dogs !~n"~h pi was modelled by severe operative trauma (ot). the dcm was estimated by the indices values of work time (wt), contraction frequency (cf), mean amplitude of contractions (~ac) and motility index (mi) measured by method of tensography. "sl", created on the basis of sorbit and sodium lactate (1800 mosm/l), was injected in the dose of 10.o ml/ kg into v. cephalica antebrachii after 24 hrs of ot. the results of the present study are the evidence of "sl" stimulative action on dcm and are experimental ground for "sl" using in complex therapy of pi in clinic. with splanchnic venous blood pc02 p.f. laterre p. goffette, j.p. fauville, a. poncelet, p. loneux, m.s. reynaert. intensive care unit, st. luc univ. hospital, brussels, belgium. determination of gastric intramucosal ph (phi) by gastric tonometry using the henderson-hasselback equation is expected to allow the detection of splanchnic ischemia in critically ill patients. because of bicarbonate concentration and acidbase balance influences on the calculation of phi, it has been proposed to use arterio-gastric pco,_ gradient [p(gast-a)co,] to assess splanchnic perfusion. htpothesis : pcoz in the gastric mucosa is in equilibrium with intraluminal co z and with co, in the blood leaving the stomach (mesenteric and portal blood). objective: mesure pco; and ph in portal vein blood and compare its value with pco 2 and phi obtained simultaneously by gastric tonometry. material and method : in a patient (55 y.), a fiberoptic catheter (baxter r) was positionned in the portal vein after transhepatic stent shunt repermeabilisation. hemodynamic parameters, do, (vigilance n baxter), gastric co 2 and phi (tonometrics baxter) and portal blood gas were determined at regular intervals. results : 19 sets of data were obtained and are expressed in mean + sd. gastric pco z was 46,5+18 compared to 40,4+3.5 mmhg for portal pco 2. phi was 7.32+._0,17 vs 7.34+._o,04 for portal ph. no correlation was found for these 2 parameters. p (gast-a) c02 was 6.4+15 mm hg vs 2+1.6 mm hg for p (portal-a) coz (no correlation). there was a good correlation between do e and p (portal-a) co z (r = 0,61) [figure] but no correlation with p (gast-a) c02. obiectives: desaturation is a common finding during haemodialysis (hd). pulmonary oedema might be one cause for impaired gas exchange (1). the aim of this study was to quantitate the amount of extravascular lung water (evlw) and gasexchange in chronic renal failure patients during and after a regular hemodialysis session. methods: 10 chronic renal failure patients without symptoms or diagnosis of cardiac or respiratory disease were studied at the start (i), at the end (ii) and two hours after (iii) a regular bicarbonate hemodialysis session. the double-indicator 2 dilution method, with indocyanine green and the stable isotope h20 as tracers, was used to measure evlw (2). arterial bloodgases and endtidal co2 were registered. evlw data was compared to a group of 18 renal healthy patients (0). dcp n evlw, ml -pao2, mmhg h~o +, nmol/l control group 0 18 243 -2--61 l 10 332 _+ 91"* 13 -+ 3 38 _+4 crfgroup ii 10 269 -+ 84~ 11 +-2ns 34-+2"(" iii 10 283 +-78t 11 _+3ns 34-+2t ** p < 0.01 dcp i from dcp 0, t p < 0.01 dcp li or i1 from dcp i, :~ p < 0.001 dcp ii from dcp i the evlw at the start of dialysis was larger in the crf group than in the control group. the evlw decreased significantly to a level not different from the control group in response to the reduction in weight after hd. pao~ was normal at the start of hd and showed a nun-signficant reduction after hd. paco2 (5.3+0.6 kpa) and etco2 (5.2+0.8 kpa) were unchanged while h3 o+ decreased and bicarbonate increased significantly. conclusions: the elevated level of evlw at the start of hd did not impair gasexchange. the decrease in evlw did not inhibit the decrease in pao2. the reduction in h30 + followed by a fall in alveolar vantilation is the most plausible cause for the decrease in pao2 in bicarbonate dialysis. 1. prezant lung 1990; 168: 1-14.2. wallin j appl physio11994; 76: 1868-75. a. dona~ d. battis& l col~ r danieli, d. achill~ l viglienz;~ c. giov-anaini, p. piaropao~ oblectives: to verify if intraoperative modifications of mtramucosal gastric ph (phi) below the normal lowest value 7.32, can be predictive for important complications, as perforation, sepsis, mof or death. methocls: we have considered 20 patients who andenvent major abdominal surgery. all patients received the same drugs in pre-anaesthasia, the same type of anaesthesia (balanced anaesthesia) and the same treatment with h2-bloekers. after the induction of anaesthesia a gastric tonometer was positioned and a catheter was positioned in the radial artery. during the operation, every 30 minutes, the following parameters were measured at the same time: phi, arterial ph (pha), blood lactate, mean arterial pressure. in follow up we considered death and complications happened during the hospital stay, in relation to intraoperative phi falls below 7.32. results: among the 20 patients, 9 had a drop of phi below 7.32 during surgery. in three of them this fall was a single episode and happened within the first hour after the begiluting of the operation. after that phi rose to nomml values until the end of the operation these patients had a normal post-operative period, without complications, the other 6 patients had a fall of phi during the demolitive manoeuvres. two paticots of them died. the first had a lowest phi=7.25 and the second 6.97. the first one ~zs operated on for hepatic istiecitoma, suffered a complete del'dseenco of the surgical wound on the 20th day after operation and died on the 25th day, the second one was operated on for a hepatic carcinoma had an intraoperative haemorrhage and died ~vo hours after the end of the operation. the other 4 patients with a fall of phi had a lowest phi=7.24. 7.18. 7.26. 7.28 respectively.the first patient,operated onfor sigmoid carcinoma, underwent on a second operation for a transmural necrosis of the colic segment on the 25th day; the second one, operated for carcinoma of the right colon, had a cardiac ischelnia on the 5th pest-operative day and a dehiscence of the surgical wound on the 8th day: the third one, operated on for a sigmoid carcinoma, had melena in 41h post~ operative da b, and finally the fonrth patient, operated on for carcinoma of the tight colon, suffered a fistula of the surgical enteral anastomosis.all these 4 patients were discharged alive from the hospital. the other 11 patients, who had not reductions of phi ditring the operation, had a normal pest-operative period, without complications. conclusion: phi was able to predict the arising of some complications, probably due to intraoperative ischemic events. we can say that gastric tenometry, for its low invasivi.ty, can be included among the intraoperative monitoring in patients that tmdenvent on major abdominal surgery. (ttd),t"ea~rrerj.~ of 3 hours duraticn. all l:atients nm.'-~ms_(~lly va~2ated in eantrol wcde ard_ la':'ad a a,~m--ganz catheter, with optic fibers for contirums mmsuremmt of svo mic studies were performed, c~e before the hegir~ of hd, c~e 15 rain after the ~, ~ne at the middle, ~ne 15 rain before lhe erd ard one 30 rain after the erd of hd. paired t test ~as used far slatistical eval~ti~n. results: daring i~d there was a significant'reductton (p<o.~) of: c~tr~ venc~s eres~.re (0ve)emm 9 to 7.5 ~ ~, 2) ~arn'~w capil~ ~f~e pressure (i<~p) fr~n 13 to 11 ~mg hg, 3) i~09 from 33 to 30.5 mmg hg. ~here was also a less pmnameed bat also-slatistics/ly significant r~ucticn (p<o.05) of: i) s~o~ from 67 to patients undergoing surgical reconstruction of the aorta due to anenrysms of its abdominal part may develop transient and sometimes sustained episodes of dysoxia despite the conventional appeoreneas of being adequately resuscitated. indirect masurement of gastric mussel ph (phi) is being widely evaluated as a minimally invasive and sensitive means of assessing the adequaey of tissue oxygenation in these circumstances. the duration end degree of gastric intramucosal acidosis are related to the risk developing injured gastrointestinal tract end its putative consequences, i.e. translceation, cytokine release, organ dysfunction end failure, sepsis end death. measurement of phi is obtained indirectly by measuring pc02 in the lumen of the stomach with a silicone balloon tonometer (tonometrics, inc., worcester, ma, usa) and the bicarbonate concentration in arlz,,rial blood, end substituting these two values in the henderson-hasselbalch equation. objectives: to evaluate whether perioperative phi changes are predictive of complications end outcome end how they compare to standard haemodyuamic and oxygen trensport parameters. methods: 16 patients (15 males and 1 female) urtderwent surgical replacement of abdominal aortic anonrysm with the aortic simple or bifurcated grail. 14 patients were operated on eleetively, 1 had an emergency surgery for raptured enentysm. haemodynamic, arterial ph and bicarbonate concentration measurements were taken before and after induction of anaesthesia, after cross-clamping and declamping of the aorta end at admission to itu. phi calculations ware done at the sime time except before induction, as tonometers were inserted after patient had been asleep. we used pulmonary utilizing a computer billing survey as well as hospital service transfer data from a 67 month period (1/89 -7/94), all obstetric patients that were transferred to the med/surg icu were identified, as well as their diagnoses and procedures. twenty-eight obstetric patients were transferred to the med/surg icu, compared to 12,489 deliveries occurring during that time period for a .22% rate. the following procedural indications for icu transfer occured: 4 insertions of endotracheal tubes, 2 arterial catheterizations, 1 temporary tracheostumy, and 1 venous catheter. the following medical diagnoses were found: 5 hypertensive/eclamptic episodes, 2 hypotensive episodes, 1 pulmonary embolism, 1 mitral stenosis, and 1 coagulation deficiency. there were four episodes of respiratory failure (14%). the historical rates of respiratory failure from the medical literature seen at a university hospital ore higher than those at our community hospital despite comparable icu transfer rates for critically ill ob patients. objectives: to evaluate the usefulness of several isss used in order to predict risk of death (rd). design: prospective data collection for 7 months in a multidisciplinary 18-bed icu. methods: data from 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995 were studied. all the necessary informations were stored in a computer using special software for every day computation of three isss (saps, saps ii, apache ii) and of the rd predicted by saps ii, mpm0, mpm24 and odin. data from 2453 days of hospitalisation were used for comparative evaluation of isss and rd, while sensitivity and specificity in death prediction (cut off point of rd = 90%) were evaluated from data collected during the day of admission (saps ii, rpm0, odin) or after 24 hrs (mpm24). agreement between rd predictions was evaluated by bland and altman analysis. results:our results were the following: objectives: a) to create and validate software specially designed for the collection of demographic data and estimation of illness severity and predicted rd and b) to provide data necessary for the evaluation of the effectiveness and the efficiency of our icu. design: prospective data collection in a multidisciplinary 18-bed icu. methods: prospective data on specific physiological variables, selected demographic characteristics, comorbidity and the reason for icu admission were recorded every day for 270 consecutive patients admitted to the icu from 1/10/1994 to 1/5/1995. data were entered into a portable computer using specially designed software allowing the calculation of usual illness severity scoring systems (isss) and the corresponding predicted rd. results: we studied 176 men and 94 women, with a mean age of 55 years. we conclude that, although observed mortality was higher than predicted rd, this difference was not statistically significant. the fact that the sd of predicted rd was too large, limits the usefulness of isss predictions for a comparative evaluation of different icus. objectives :the importance of objective evaluation of patients prognosis at their admission to an emergency department is clear. the aim of this study was to appreciate the prognosis of patients with the simplified acute physiology score ii (saps ii), and correlate this score to the prognosis and orientations of patients. methods : the score which is a reduced form of saps ii was calculated from 6 clinical variables and 6 laboratory items for 200 patients. sensibili}y (se), specificity (sp) and accuracy (ac) were calculated to evaluate the performances of saps i1. thresholds were calculated with the youden's test (se+sp-1). results : 23 patients were admitted in the intensive care unit (icu)and 177 patients in medical units; 23 patients died. the saps ii was higher in patients which died than the survivors (31,0+11,4 vs. 1,9+9,3, p<0,0001 ; respectively), the best threshold was 24, se was de 86%, sp was 92%, the ac was 920/0. thus for patients admitted in icu than in medical unit (27,0:1:13,5 vs. 12,5+9,9 , p<0,0001; respectively); the best threshold was 21, se was 69%, the sp was 78%, the ac was 77%. conclusions : these preliminary results suggest that saps ii can be used to determine the short term prognosis and the destination of patients seen in an emergency department. design: data was collected on all admissions over a six month period. all patients had a screening hiv elisa test. confirmatory ifa tests, western blot and flow cytometry were performed on hiv positive patients. the institutional ethics committee considered the major clinical impact of the study sufficient cause to waive the patient's right to informed consent, icu staff and patients were blinded to hiv results. following discharge patients were given the option of being advised of their hiv status. setting: the study was conducted in a 16 bedded surgical icu at a tertiary care teaching hospital. patients, participants: all patients admitted during the period in question were included. interventions: "all patients were treated according to standard icu protocols. there were no interventions. results: most patients were admitted following trauma. there were no patients with aids, 52 hiv positive and 350 hiv negative patients. with respect to the latter two groups, the mean age and sex distribution was similar, there was a significant difference in organ failure (71% versus 49%, p < 0.05) and septic shock (39% versus 15%, p , 0:05) but no difference in icu/hosp[tal mortality or duration of icu/hospital stay. flow cytometry changes in hiv positive patients were consistent with the quiescent phase of hiv infection. conclusion: while morbidity is higher in hiv positive patients, there is no difference in mortality. hiv status should, therefore, not be an exclusion criterion for icu admission in this patient population. 1, n= 112) and january-march 1994 (period 2, n= 127) were studied. interventions: a resident micu team led by a trained intensivist took over the medical care from the primary physicians when the patients were admitted to the micu. the intensivist also vetted micu admissions and decided on micu discharge. in addition, there was a resident respiratory therapist to attend to vendlatory care during office hours. after office hours, the care of the micu was delegated to the on-call team on a rotational basis among the medical departments. results: the patients in the two groups were similar with respect to age, sex, race, source of admission and apache 11 scores. the icu and hospital mortalities decreased from 33.8% to 27.6% (p=ns) and 38.2% and 34.2% (p=ns) respectively in period 2. the mean icu and hospital stay was also decreased from 4.6 __+ 7.1 to 3.6 + 4.0 days (p=ns) and 19.3 __+ 357 to 17.2 • 22.2 days (p=ns) respectively. the improved micu length of stay for survivors in period 2 was statistically significant (4.2 • 4.2 days vs 2.8 • 2.8 days = 0.015). there was no significant differences in the vse of peritoneal dialysis (5.4% vs 6.3%) and mechanical ventilation (55.4% vs 49.6%). however, utilisation of intra-arterial lines and pulmonary artery catheters increased from 0% in both to 23.6% and 5.5% respectively in period 2. conclusion: the duration of critical illness was reduced in period 2. hence, we believe that the closed |cu which was staffed by an intensivist and had the services of a respiratory therapist was beneficial for the care of the critically ill. the intensivist, was also more likely to utilise invasive monitoring. in the absence of liver transplantation, intensive care for patients with acute hepatic decompensation arising from alcoholic liver disease (ald) is indicated only for those who are likely to benefit from conventional means of organ support. we have attempted to elucidate potential risk factors and the efficacy of organ support in relation both to hospital outcome and icu costs so as to allow the earlier identification of relatively futile intensive care in this group of patients. methods: over a period of one year, 59 ald patients (37 males; 22 females; median age 46 years, median apache ii score 19; hospital mortality 68%)with severe complications (30 [50%] with bleeding oesophageal varices, 16 [27%] with hepatorenel failure, 5 [9%] with respiratory failure, 3 [5%] with septic shock, and 5 [9%] others) were studied in our liver icu. organ system failure, daily therapeutic interventions and icu costs were assessed using a patient management system (riyadh intensive care program). results: whilst survivors had a significantly lower admission apache ii score compared with non survivors (medians 12 and 20, p<0.0003), 2 or more organ systems in failure in the first 24 hours of icu admission was associated with a 91% (21123) mortality. mortality was also related to child's grading and the numbers of organs supported. of the 29 patients who received more than one form of organ support only one survived. organs supported mortality a (n=2) 0% none 3/13; 23% b (n=6) 33.3% one 8/17; 47% c (n=51) 74.5% two or more 28/29; 97% the icu costs for the 59 patients were only s 440 but s 687 (75%, 216 patient days) was utilised by the 40 patients who died, giving an effective cost per survivor (ecps) of s 496. although the cost of treating the 23 patients with 2 or more organ systems in failure on the day of admission was s 510 (36% of the budget) the ecps was s 755. conclusion patients with ald who have 2 or more organ systems in failure early in their icu course have a poor prognosis and in the absence of liver transplantation, traditional strategies of organ support are ineffective. this begs the question whether such costly care is appropriate as although the ecps in this sub group is comparable with other patient groups with multiple organ failure, their outcome is considerably worse. objectives: to evaluate the economic impact to the healthcare sector of using dopexamine hydrochloride infusions to deliberately increase perioperative oxygen delivery in high-risk patients. methods: effectiveness data were obtained from a controlled clinical trial in which 107 surgical patients were randomly assigned to either a control group (n = 549 to receive best standard optimal fluid resuscitation perioperatively, or a protocol group (n = 53) to receive, in addition, an infusion of dopexamine hydrochloride to increase the perioperative oxygen delivery index to greater than 600 ml/min per square metre. resources consumed by patients during treatment were recorded prospectively and costs of resources at 1993/94 prices were obtained retrospectively. cost-effectiveness ratios were estimated by dividing the total cost for each treatment group by the total number of patients in each group and by the clinical outcome of mortality 28 days postoperatively. results: in the protocol group, there was a 75 % reduction in mortality 28 days postoperatively (p = 0.015), a significant reduction in the number of complications (p = 0.008) and a reduction in length of stay in the icu and surgical ward. the average cost per protocol patient to achieve a 94.3 % survival rate 28 days postoperatively was s 9,894, resulting in an average cost of s 10,488 per surviving protocol patient. the average cost of a control patient was s 11,331 to achieve a 77.8 % survival rate 28 days postoperatively, resulting in an average cost of s 14,568 per surviving control patient. the use of dopexamine to deliberately increase oxygen delivery perioperatively generated a cost saving to the nhs of s 1,436 per patient together with a 75 % reduction in mortality at 28 days postoperatively. hence, the cost per surviving protocol patient was s 4079.8 less than the cost of a surviving control patient. conclusion: the additional cost of dopexamine in protocol patients was offset by a lower incidence of complications and a shorter stay in the icu and on the surgical ward. hence, increasing oxygen delivery perioperatively in high-risk surgical patients saves both money and lives. objective: although king's college criteria are widely used, indication and timing for liver transplantation in acute liver failure is still far from certain. long-latency sensory evoked potentials -a proven measure of metabolic brain dysfunction (grimm et al. lancet 1988; 2: 1392-94; madl et al. lancet 1993; 341: 855-58 ) -are markedly affected in patients with acute liver failure. serial sensory evoked potential recording should provide very useful information for the management of patients with acute liver failure. methods: 90 recordings of standard bilateral sensory evoked potentials were performed in 25 patients with acute liver failure. findings were focused on cortical n70 peak -a stable negative deflection occuring 70 • 9 ms after median nerve stimulation in healthy subjects, which can be recorded by skull electrodes over the sensory cortex. results: nine patients recovered spontaneously, 8 patients were referred to emergency liver transplantation, and 8 patients died. in all 9 patients who recovered spontaneously, n70 peak was detectable between 74 and 162 ms. all 8 patients who were referred to liver transplantation and 7 of 8 patients who died, developed a loss of a prior detectable n70 peak during the course of acute liver failure. objective: to determine high risk arid low risk patient groups in a medical intensive care unit regarding short term and long term outcome. methods: data on all admissions of the year 1993 were collected prospectively. according to their mode of admission patients were classified as admissions by the physician staffed ambulance service (as), admissions through the emergency department (ed)i referrals from non-intensive care wards (ni), and secondary referrals from other hospitals (oth). outcome was assessed in terms of in-unit mortality, in-hospital mortality, and long time mortality. patient groups were compared using the \2-test. life table analysis were per{ormed by kaplan-meier method comparing patient groups by log-rank test. the software spss for windows 6.0.1 was used for statistical calculations. results: in-unit mortality: 184/1853 patients (9.9%) --oth 20.0% > as 10.6%> ni 9.1% > ed 6.8%; p = 0.01. in-hospital mortality: 365/1853 patients (19.7%) --oth 30.9% > ni 20.4% > as 19.6% > ed 16.2 %; p = 0,05. mean survival time in days after discharge: as 620 < ni 682 < oth 708 < ed 777; p = 0.66. conclusions: despite an excess in-unit mortality of secondary referrals from other hospitals the iongtime course of this special patient group is not different to others. solsuam, j, marrugat*, g, mirs, j, nolla, a, vazqu~z-sanchez, l alvamz, ~ioio s xndioina i~siw. ir~itate l(~icipal da l~sti~isn l~di~*, ~ospits dal objective: to study the influence of modifiable variables (complications derived from therapeutic activities) on the prognosis of ~atients admitted to the icu indapemently on thn severity of illnsss. patients am methods: between january 1990 asd ]lay 1993 data from 1,425 patients over 14 years of aqe who retained in the icu for mare than 24 hours ~ere pr~pectively regiatered. a cohort st~ly with follo~-~ nf patients durin~ ~eir stey in the hospital was deni~.el in all patients, reasons for a~issien, principal diagnosis sad severity of illn~s moasared by the saps scare vare recorded. fastens affecting patients' outcome that my be proventsd or modified included technical :omplisafioss, heapital-acqnired infections and in~pro~riate therapeutic decisions. a logistic regression model was used to assess the relative risk (l~} for in-heapital mortality adjusted for each variable. results: ic~ mortality ~s 17.2% and in-hospitul mortality 22.7%. patients who died showed a higher spas score then survivors (15,3 ~ i0,i). after adjusting hy severity of illness, co~;licetices that statistically increased the risk of in-hospital death were septic shock secomery to hoapitul-acqdired infection (1~ 7.18; 95% el, 1.9 to 27.1), pmo~othor~x related to mocasnical ventilation (@ 6.28; 95% cl, 1.7 to 22.3) and delay in the insertion of a fln~-quidod catheter (ii~ 5.49; 95% ic, i.i to 26.9). col~lusien: registration of complicaticas derived from therapeutic activities is a valuable tool far quality central in the icu. g, ~i~5, j.l mle~ma, j, ~amqat*, j..~lla, a, vazquez-saltemz, f, alvamz , servioia de nndicina l~siu. i~stitutu ~icipal de ln~sti~acidn ~4i:a*, hospital dsl objective: to dstsr~ine the incidence of self-extebatien and its effect on ~ortality. patients and ]~etheds: betveen january 1990 and april 1994, all i~tiente in whom selfextubatien w~s registered were inclnded in a prospective study. patients were divided into @nee who needed r~intabatinn within 24 hoers and those who did not. in all patients, dsmoqraphie and ciinical data were recorded as well as icii mortality, in-hoapital mrtality and severity of illness according to saps score. 0eta were analyzed usi~ the cbj-square test for cathgorical verinbls, the analysis of varianc~ (anva) for aontinuc~ ~ria~les and a leqi3tic regression anal~is to estimate the relative risk (iiii) for mortality as result of celt-nxtt~ation after adjusting for severity of illness. results: a total of 815 intnmtsd patients amre stndied. self-extu~atien occurred in 54 (6.6%) patients and 25.6% required reintuhot~pn. when a co,arise was made between patients who did not required reint@atinn and patien~.s who did, statistically significant differences in eqe (52.1 v_s 60.4 years, p = 0.~02), ~verity of illness (11.4 ~ 13.1 spas score, p = 0.02), dia~isstia category (4s.6% v_s 66.7% of patients with res~iratury conditiono, p =0,002} and mean length of stay (10,9 ~ 20,7 days~ p = 0.05) were fo~m, a~ter ad~sti~ for severity, patients with self-ext@atinn who did not reqnired reintalatien showed a 0.4 iir for mortality (95% ci, 0.i to 0.9) as co~arod with patients in when self-ext@ation did mot occur. conclnsien: self-~extamtice that does not require reint@ation is associated with a isamr in-hospital natality probably dt~ to a prolonged period of weaming. patients' admissions to ices am often delayed doe to the shortage of beds available. @ile amaltieq icu admission, these patients are treated in observation nits of @e emergency services which bare ,either tile structure nor the trained ~reomenl that are available in leb~. objective: to daterdno the effect on the patient's proqusis of a delay in tile admission to the icu when criteria for icij admission are fulfilled. ~terials and methods: between jme am l?ece~ber 1993 all patients who fulfilled criteria to be almittod to the ic0 who for waste~r reason retained in tile observation unit for more than 24 hours were included in a prospective stedy. in all patients, des~raphic end clinical dabs amre recorded as well as severity of illness aencrdi~j to saps score. a cesucontrol dasi~ was eend with a total ss~ln of 1,425 patients who suffered no delay is admission to icii over a period of 3 years. data wen analyzed using the chl.-squ~re test (to aeons the association hetwenn in-patienty mortality end categorical vari~lns) and a maltipln logistic reqression model to sstimta odds ratio for) for in-hospital mortality as result of delay in icy admission as compared with early ad~issi| after adjusting for severity of illness end use of assisted mchenical ventilation. ~9&ults: a total of 50 patients remained in the observation nit for more than 24 hours with a del w in igd admission of 55.8 _+ 25.1 hoers. assisted mechanical ventilation was requited in 22% of patients and only monitericatien in 46%. itsse patients were cspared with 112 ntients from the tet~l sample ratchod by age, sp~ score and rennoss of admission. in-hospital mortality for cases warn 16% as compared with 17.5% for controls (p = s). after adjamtilg fen spas, age and mobamioal ventihtien, no statistically significant differences between both ~renpa were foam, altho~b there was a tendency towards a higher mortality amen@ patients with delay in icu admission (or = 0.779; 95% ci, 0,2 to 2,5). conclnnien: ~se findings suggest that prognosis of critically-ill patients is no worse as a result of admission to the loll being deln~d for 24 borers. all data appropriate for the calculation of the apache ii score (aps) together wi'th other specific cardiac details relevant to these .patients were collected daily, verified and enter~ into a computer database. results: 150 patients were studied. six patients died and five of thee underwent cardiac surgery. the mean aps was 9 for survivors and t6 for non-survivors (p < 0.001). the mortality ratio was 1.1 and the major markers of mortality were apache ![ score, presence of chronic ill health, mean duration of ventiiation, mean length of icu stay and need for emergen~ surgery. sixteen percent (233) of icu bed days were occupied by 4% of patients (non-sarvivors) which resulted in cancellation of 60 cardiac sot#cat sessions in 6 momhs. conclusions: this study concludes that apache 1t could be used as an audit tool in a cardiac surgical icu and demonstrates the severe compromis~don of cardiac surgical throughput by a few non-survivors, organ to determine the number of organ failure free days (offd) in a cohort of survivors and non-survivors with sepsis syndrome followed over a 30 day period. 2) to determine sample size requirements for clinical trials utilizing a increase in the number of organ failure free days as the primary outcome as opposed to mortality. methods: beginning december 1990 through to april 1992, patients who met inclusion criteria of the "cardiopulmonary effects of ibuprofen in sepsis syndrome" and who did not have hiv/aids. brain death or moribund state were prospectively identified. presence or absence of failure of 7 organ systems (pulmonary, cvs, renal, hepatic, gi, hematologic, & cns) was recorded daily until death or until 30 days. a score of one was assigned to each organ system free of organ failure in patients still alive, ie, maximum daily off score=7, maximum 30 day off scorn=210, sample size estimations were performed for variable detectable differences in off scores (delta). alpha was set at 0.05 (two-sided), with n/group = 2[(z a +z b ) o conclusions: a clinically relevant increase in off days may be detected with as small a sample size as 30 to 50 patients per group. this represents a significantly smaller sample size than needed to detect a change in mortality from 40% to 30% (25% relative risk reduction) where the n/group=356. scoring patients in this manner prevents a lethal inte~entien from providing an improved organ failure score. in addition, an intervention that prolongs survival must also provide greater organ failure free days in order to be counted by this scoring method. survival as an outcome provides no information about the quality of that survival. off days provides a measurement of burden of illness. interventions which lessens this burden may be just as valuable as those that decrease mortality by providing a measure of the quality of survival and by decreasing costs of care. they may also prove to be an accurate surrogate marker of mortality. the advantage of this approach is that the event rote is much higher and sample size requirements are subsequently smaller. this would mean that clinical trials can be completed faster and at lower cost. outcomes such as mortality could then be assessed at a later date utilizing recta-analysis. we suggest that the use of off days is a valid outcome measure that may be utilized in clihieal trials of sepsis syndrome. the icu is perceived by many as being a stressful environment for both patients and staff. stress has been defined in three ways: a stimulus producing a particular response; the physiological and psychological response to a stimulus; an interaction butwom an individual and their environment. stress is currently thought to be a dynamic system of stimulus and. response which takes into account the individual's perception of the stimulus and their ability to respond effectively. stress may, therefore, be positive and allow personal development but an individual unable to respond effectively to a stimulus will experience negative effects or strain. critical illness is an intense stimulus to which the body needs to respond effectively. physiological responses are vital and most of intensive care involves supporting these. alternatively, blocking them, for instance with atom(date, increases mortality. psyehological responses are also vital but often poorly appreciated because of communication problems. many of the problems patients experience in an icu are evidence of psychological strain. this can be exhibited in various ways, for instance, anxiety, depression, passivity and confusion. dealing with critically ill patients is perceived as stressful. we recently studied occupational stress in our icu. most aspects of intensive care were not generally perceived as stressful indicating a self-selectien of icu staff. the most stressful aspects of icu work for nursing staff were the structure of the organization and career opportunities. medical and nursing staff had different stressors and different coping strategies. support for occupational stress, therefore, should focus on the individual and concentrate on information and communication. atmosphere, and especially at intensive care units, we face up to daily decision making. in most cases these are taken on the basis of personal opinion and the processing of a very limited amount of information. rising need to optimize the results of medical attendance becomes necessary to set structured system of d@cision making in which ethical basis have a sp@dial significance in view of next considerations: -we live into a pluralist society in which the importance of values is different. -most persons consider health as the first value only in the event of illness. -medical resources available are limited, whereas medical, attendance demand from population increases in a way many people consider it unlimited. in consequence, it becomes necessary to set up priorities in patients treatment. ehtical basis that rule decision making are essentially these ones: i. beneficence: to provide the patient that is being treated the highest profit. 2. non maleficence: it is our first duty to avoid hurting or damaging the patient."primum non nocere" 3. autonomy: in every particular medical attendance, the patient has ability to decide by himself. 4. justice: as equity: to provide the same treatment for those who have the same pathology, ignoring another factors such as age, sex or race. severe application of these principles can cause difficulty, which resolution requires a systematization of decision making. (1-84) . the lenght of stay between survivors and non survivors didn "t show statistical significance (p =0.51 ). the mean aiii score when considering all admissions was 59,9 (8-153) . the initial score between survivors and non survivors showed ststistical difference (48.6 vs 92.5) respectively (p < 0.0001). univariate logistic regresion analysis demostrated a 90% increment in death probability for every 10 points augmentation in the aiii score with a sensitlbity of 94.9% and specificity of 62.7%, the roc curve showed that the best cut off point for death prediction was 75 points with a sensitivity of 75.6% and specificity of 79.9%. if a patient is classified as high risk (> 75) the bayesian analysis showed a 52.8 probability of death and for one class(fed as low risk (<75) a death probability < 10%. conclusions: the first day aiii score in this population showed to be a good discriminator between survivors and non survivors, and the risk of death augments as the aiii does. in this population an aiii score > 75 points is asociated with a greater risk of death. using the aiii score in conjuntion with the clinical judgement will help clinicians reducing uncertainty in the every day decision making and better predict outcome, the results from this study should been taken with caution because the data were obtained from a small sample. objective: the quality of life has been considered a "uniquely personal perception" resulting from a mixture of health related factors and social circumstances [t. m. gill, jama 1994, 272: 619] . the aim of this study was to evaluate two measures of pqol in intensive care unit (icu) admitted patients. patients and methods: during icu stay and six-months after hospital discharge, 160 co-operative icu admitted patients were directly interviewed about their pqol. we administered ftrstly the uniscale (pqolu) [sage et al crit. care med. 1986, 14: 777-782] and then a 5 step verbal scale (pqolv): best, good, fair, poor, worst. of the 160 studied patients, at the first interview, 116 were able to use both scales, but 44 (27.5%) understood only the verbal one. at the second interview, 8 patients were not able to answer, 113 used both scales and 39 only pqolv. statistical analysis was performed using wilcoxon signed ranks, spearman rank correlation, student's t and chi square tests. results: of all cardiac surgery pts, 42 pts (1.6%) died in icu. they were 33 males (1.5%) and 9 females (1.6%). their mean age was 66 (+7) years and mean ef was 0.38 (+0.1). nineteen pts (45%) had low (<0.35) preoperative ef. mortality was 0.9% in the coronary artery bypass grafting (cabg) group (n=2014) and 2.8% in the valve replacement (vr) group (n=359). in the cabg +vr group, mortality was 8.4% (n=95), and 3.3% in the remaining pts (n=149). cardiogenic shock was the sole cause of death in 24 pts (57%), septic shock in 6 pts, whereas sepsis in combination with ards in 4 pts, sepsis and stroke in two pts. in addition, 6 pts died from cerebrovascular accidents, one from ards and one from pulmonary embolism. the pts who died in the icu had a significantly longer bypass and aortic cross clamp time and received more blood transfusions (p<0.001) than a matched control group that survived to icu discharge. the duration of mechanical ventilation and length of icu stay were greater in the pts who died in the icu than in the control group. conclusions: 1. although cardiogenic shock is the main cause of death (57%)in cardiac surgery pts, sepsis and cerebrovascular accident are relatively frequent causes. 2. patients who died in the icu had longer bypass and aortic cross clamp time and received more transfusions, compared with the control group. 3. although renal or hepatic failure contributed to death in some pts, they were not the primary cause of death in any patient. objectives: evaluate the acute and follow-up outcome of 27 patients (pts) treated with primary ptca (without prior thrombolysis) in acute myocardial infarction (ami) after 12 and up to 24 hours after onset of typical thoracic pain ("late" primary-ptca). methods and patients characteristics: from 12/89 to 4/95 364 consecutive pts with ami were treated by primary ptca in the wuppertal heart center 9 27 pts (7,4%) were admitted to our hospital > 12 hours and < 24 hours after symptom onset with ongoing chest pain and typical ecg-changes.mean age was 62 years (49-78). 23 pts were male, four female. 37% had an anterior wall myocardial infarction, 63% suffered an inferior/postero-lateral wall myocardial infarction.two pts were in cardiogenic shock at admission. singlevessel-disease was documented in 70.4%, multi-vessel-disease in 29.6%. average time of onset of pain to recanalisation was 929 min (720-1440). angiography revealed timi-flow 0 in 85.2% of the pts, timi-flow i in 11.1%, timi-flow ii in 3.7%. average follow-up (fu) period was 12 months (4-28 months). timi iii lv-ef ~ 30-day major late re-late flow p.i.* aeute/fu mortality bleeds infarction mortality 92.6% 58%/63% 7.4% 7.4% 3.7% 0% early mortality occured in the two pts, who were in cardiogenic shock at admission 9 no pt required emergency coronary artery bypass grafting.restenosis >50% was seen in 37% of the pts. conclusions: "late" primary ptca achieves a favourable high recanalisation rate of about 90% (timi ill-flow) in our study group. additionally, there seems to be a trend for lv-ef improvement in follow-up. early high mortality is influenced by the patients admitted in cardiogenic shock. there might be a trend for increased major bleeding complications. objective: to assess the validity of saps ii (new simplified acute physiology score), comparing it with the previous version, (saps), in a sample of patients recruited by giviti, a network of 128 icu's representative of the italian icu system 9 methods: measures of calibration (goodness-of-fit statistics) and discrimination (receiver operating characteristics curve and area under the curve) were adopted in the whole sample and across subgroups differing in relevant prognostic characteristics. of the 3004 patients recruited during one month period, a total of 1813 patients were included in this study. for the purpose of the comparison of the two scores, patients with less than 18 years, or having cardiac surgery or staying in the icu less than 24 hours were excluded. vital status at icu discharge in the whole sample and at hospital discharge in half cases wher adopted as outcome measure. re$01~: saps ii fits the data equally well compared to the older version (goodness-of-fit p=0.29 and 0 9 in the new and old versions, respectively) but its performance is somewhat better in terms of capability to distinguish patients who live from patients who die (areas under the curve 0.81 and 0.73, respectively). furthermore, saps ii is better in terms of uniformity of fit across relevant subgroups, although substantial over prediction of mortality was observed in trauma patients and in patients admitted without organ failure to be intensively monitored. saps ii performed very wet] also in the subsample where hospital mortality was the dependent variable.satisfactory measures of calibration (goodness-of-fit p--0.47) and discrimination (receiver operating characteristics area=0.80) were observed. c0nr saps ii, a multipurpose scoring system developed in an international study, retains its validity in this independent sample of patients recruited in a large network of italian icus. although it has shown a good performance when adopted to predict icu and hospital mortality in the entire sample, further investigations are warranted. the observed over prediction of mortality in a few subgroups indeed call for a through assessment of the impact of confounders and biases on model performance when saps ii is adopted in samples that do not reflect the "average" icu patient. objectives: 1) assess the effectiveness in a group of intensive care units by means of a quality performance index (qpi); 2) assess the efficiency by means of a resource use index (rui); 3) evaluate the performance of individual icus with respect to both indices (clinical and economical) while controlling for severity of illness. 1270 critical from 17 ucis in catalonia patients alearic islands have been included in the study. inhospital mortality and weighted hospital lenght-of-stay (los) have been considered the outcome variables. severity of illness has been measured with the mpm ii at admission. in each icu, expected mortality has been obtained adding the probabilities of dying for its patients. expected los has been estimated adjusting a second order polynomial to the severity of illness. performance indices have been obtained by dividing the observed by the expected outcomes. re~ult~: the overall qpi was 1.15 and it ranged from 0.58 to 2.05 in the 17 icus. the overall rui was 1 and it ranged l~ont 0.61 to 1.51. there was not a trade-offpattern between clinical performance and resource use. objectives: teaching hospitals often provide [cu care across a variety of specialized services. overall, this approach appears to result in the best risk adjusted survival rates, but at the highest cost (critical care medicine 1993;21:1432-42): recently, there has been increasing focus on markers of overall hospital performance. however, in large teaching institutions, such markers may fail to detect intra-institntional variation at a large tertiary care medical center. methods: first intensive care unit (icu) day, acute physiology and chronic health evaluation iii (apache iii) and active therapeutic intervention scoring system (tiss) data were collected on 1621 random admissions to 8 specialty icus with 90 beds (range 8-14) between february i and december 3 l, 1994. post-operative solid organ transplant recipients were excluded. units included 2 general medical, 2 general surgical, and trauma, neurosurgery, cardio-thoracic surgery, and coronary care units. data were analyzed for risk adjusted outcomes: icu and hospital mortality and length ef stay (los); risk of requiring active 1cu treatment; and icu readmissinn using apache iii risk prediction models. results: the study icus cared for a diverse group of patients. mean apache iii scores ranged from 36.9-55.5; predicted risk of hospital death ranged from 8.5-21.1%. standardized mortality ratios ranged from 0.40 to 1.24 with 4 icus performing significantly better and 1 performing worse than predicted (p<0,05). los ratios and icu readmission rates ranged from 0.95 to 1.09 (ns) and 2.1 to 13.2% respectively. patients predicted at low risk of requiring active icu treatment ranged from 6,6 to 45.8% conclusions: there was wide variation in the mean level of patient severity between icus. after controlling for this severity, outcomes also varied widely. no clear pattern of overall institutional performance was evident. these data suggest that efforts to assess performance, improve quality, and maximize efficiency must be focused within individual units. programmatic evaluation of outcome allows for focused review of the processes of care contributing to good outcome (best practices) and where to focus ongoing quality improvement and cost reduction activities. background and method : we compared icu mortality in different age groups presenting with the same severity of disease. we assessed severity of illness by the physiological day 1 -apache~ (physio-aa) score (thus excluding the age related points). for each of the following physio-a n score intervals (0-5; 6-10; 11-15; 16-20; > 20) , we compared tcu mortality within 6 age intervals (< 40; 41-50; 51-60; 61-70; 71-80; > 80 years -10, 11-15, 16-20) . in these groups mortality may be twice higher in the > 60 years patients than in the _< 60 years. mortality does not vary with age in low (physio a n = 0-5) and high (physio a n = > 20) risk groups. in the low risk group, mortality is low in all the age intervals because of the begninity of illness. in the high risk group, extreme severity of disease probably blunts the impact of age and leads to high mortality rates in all age intervals. introduction: to access the actual social/clinical outcome of the patients who undenvent intensive care therapy oct) is rather difficult, quality of lilr is not easih.' defined and ohserver subjectivity is a prime factor in the evaluation. mortality ratio after discharge must be established and its causes understood. obieetives: the propose of this stud)-is to look into the mortality ratio that occurred on a series of patients that undorwent ict at our unit from of the ~iew point of severity of the original illness and the diagnostic groups. material and methods: during the period of one )-ear (1994), 216 patients were treated at the unit, 45 of them died, and 16 ~ere not matched in our series because os incumpletc records. thirteen patients died in hospital after their reference to other departments, twelve patients were lost after discharge. thus. at the end. only 142 patients were evaluated on the fu. the, were classified into the follov4ng three groups: acute medical, elective surge d 9 and acute and emergency postoperative. the patients were seen at 3, 6 and 12 months after discharge. the, were evaluated in accordance to their abili~, to being self supported in their daily life and capecity to fully return and hold to their pre~4ous jobs. apache 11 scores were evaluated for each of the three groups and correlated to the icu dead, hospital dead, and mortality after hospital discharge, spss package was used for statistical analysis. remlts/conclasions: data shows that 19/142 patients died after discharge from the hospital, of ~itch nine died in the first three months. seventy-eight per cent of the patients were fully self supported in their daily life and 20% showed some kind of handicap. fosty-nine per cent of the patients wore on retirement either due to age or some form of chronic disease, when admilled to our unit. thirty-two peg cent had not been able to return to work, because the" were incapacitated on discharge. only 7% had return to their fully jobs but the period of the stu~, is not enough for all of them to be fully physically recovered. preliminmy statistical analysis shows us significant differences among groups. the aim of the present study is to compare the prognostic performance of five general severity indices ou coronary patienta and to find out if a proper ntatistical hundling of these indices could provide better results in these patients. methods: saps ii, mpm ii (mpm ii0 i mpmp ii24), apach ii end gaprik were evaluated o~ 456 patients with acute myocardial infurction admitted to 17 intensive care units from catulunye. calibration and discrimination were calculated for each index. calibration was calculated by th bosmer-lemeshow test. discrimination was evaluated by the area under the relative operating characteristic (roc)curve. if a model did not show a good performance it was customized using multiple logistic regression. finally, tworeduced models were developed, one fro~ the mpm series (mpm ii24cor) and one from the group apache-saps (sapsiicor).their performances were again evaluated. results: discrimination was high enough for all models. neverthelees, oelibration of apache ii, saps ii and mpm was not satisfactory. thus,mpm ii24, saps ii and gaprik were customized for coronary patients using the logits of both models, and obtaining good calibrations. mpm ii24, and apache-saps were adapted and reduced to 5 (mpm ii24cor) end to 4 variables (sapsiicor), respectively . both models showed better oalibrutions end discriminations than the original models. conolusion| models developed for multidisciplinary patients show a good discrimination when applied on aoronar i patients, but some needed customization in order to improve calibration. the number of variables of the principal model can be reduced (even to 5 or 4 variables) without loosing prognostic accuracy. objective: to compare the ability of two methods to predict outcome for intensive care patients. methods: we included 343 consecutive intensive therapy unit (itu) admissions with an itu stay>24 hrs in a 18 month prospective study (exclusion criteria: burn injury and age <16 yrs). data were couectsd applying the criteria described by the developers [1, 2] . the definition of coma (mpm24ii) was modified and the best assessment within 24 in's, rather than the admission score, was used. statistical analysis included classification tables and receiver operaung characteristics (roc) curves to assess discriminative power, and lemeshaw-hosmer statistics and calibration curves to test accuracy of prediction. results~ average abe was 58 yrs (ranse:16-92) with a male:female ratio of 1.6:1. the actual hospital mortality was 26.8%, mean predicted death rates were 22.8% (mpmz4ii) and 15.2% (ap hi). non-survivors had siguitlcanfly higher predicted risks than survivors applying both methods (p<0.000l, t-test). the total correct classification rates (tccr) for apache iii were bett~r for all decision criteria applied (tccr, decision criterion 50%: apache ]/i 77.1%, mpm24ii 71.4%). the area under the roc curve was 0.75 (ap iii) and 0.66 (mpm24ii) confirming the better discrimination of apache ill. accuracy of risk prediction was similar for both models (ap nl ~2-59, mpm24b ;(2-52, lemeslmw-hosmer). showing some fluctuation, calibration curves lay close to the ideal line for predicted risks -<50% with increasing deviation for higher risk groups (s. figure) . apache iii underestimated the risks of hospital death for almost all risk groups (curve above diagonal), whereas considerable overestimation for predicted risks >40%0 ceenred with mpm~ii. objective: to assess the goodness-of-fit of the apache iii model for british itu patients. methods: we prospectively studied a cohort of 715 adult patients consecutively admitted to a medical-surgical itu over a period of 18 months. patients with burn injury, age < 16 yrs and itu stay < 4 hrs were excluded. using a eomputerlsed database, we routinely recorded 24 hrs apache ill scores. predicted risks of hospital death were computed by critical audit ltd, london. accuracy of risk prediefion was assessed by hosmer-lemeshaw chi square (;(2) statistics and calibration curves [1 ]. discrimination was tested employing classification tables and receiver operating characteristics curves (roc). restths: the mean age of the 453 male and 262 female patients was 59 yrs (range: 16-92 yrs). of these patients, 64% were medical admissions, 17% were admired after emergency and 21% after elective surgery. the observed hospital mortality was 25.4%, the overall mean predicted death rate was 16.8%. mean predicted risks were siguifieanfiy greater for nonsurvivors (38.0 %o) than for survivors (9.6%, p<0.00l, t-test). apache iii showed good calibration (z2-~59, lemeshaw-hosmer). however, the calibration curve lay above the diagonal for almost all risk groups reflecting the tendency to underestimate actual mortality (s. figure) . the best total correct classification rate (tccr) was 89.3% (decision criterion: 50%). the area under the roc curve was 87.6% confirming the good discriminative ability of the model. objectives: the aim of this study is to point out the discrepancies between needs and actual treatment of less severely ili patients admitted in italian intensive cam units (icus) requiring only intensive monitoring, and verify the substantial likelihood of data comparing those collected from a national short term study with a regional long ternl use. ~: less severely ill patients ("observed patients") were only monitored; they did not require intubation, even if for a short period (less than 24 houm) or major cardioeiranlatory supports, and were neurologically normal. epidemiologieal national data were obtained from giviti group (gruppo italiano valutazione interventi in terapia intensiva); this cohort study, collected 5092 patients, in two months in summer in 1992 all over italy. regional data were echieved in a three years entlection (1990-i992) in lombardia' icus from archidia group (arehivio diagnostieo), including 10065 patients. mortality, severity score, diagnostic category and some typical intensive procedures were analysed and compared in both studies. patients' disgunstie categories were defined as surgical, medical and trauma, according to the main diagnosis and the presence/absence of surgical procedures. rr162 observed patients account for 23.2% and 22% of all icu's patients respectively in national and regional data. very tow mortality rate was found in national data (2.3%) and extremely low mortality in regional data (0.6%). in both studies mortality, s.a.p.s. and length of stay were much lowor in "observed patients" than in general icu's population (mortality: 25.7% and 22.3%; 8.a.p.s. score: 10.6 and 13; iength of stay: %7 and 9). homologous distribution of patients in the two studies was noted for what concern their diagnostic category, aside from a slight prevalence of tranmatised patients in the giviti study. in the two groups the surgical patients were respectively 47% vs. 57%, medical patients were 34% vs. 31% and traumatised were 19% vs. 13%. 92% of "observed patients" in national study and 93% in the regional did not received any intensive procedure. only a minority of these patients availed haemodynamie eonu'ol with swan-ganz or renal haemofiltration. conclusions: these results underline that about one fourth patients admitted in italian icus benefit an oversized slructure i, relation to the real needs of their pathology. in hot more than 90% did non received any advanced treatment and mortality and s.a.p.s. score were substantially lower respect to general population. the results obtained from these two studies are similar, suggesting an uniform distribution of the case mix in italy, even if a different recruitment period and a different gengraphieal distribution were used. some discrepancies in the two studies were found in the diagnostic categories moreover regarding the tranmatised patients (19% vs. 13%); this can be explained from the seasonal (summer) characteristic of the national study. mutuality, yet very low, is different in the two groups, but these data do not allow any definite explanation. finally these epidemiologieal survey suggest need of further studies settling more strict criteria of admission in icu. this study aims to evaluate patients outcome, quality of care and effectivity of therapy in our intensive care unit. the main goal was to indentify factors that the most influence that outcome. during 1994. the authors collected data of patients outcome and predictor variables. overall mortality rate was 39,3%. the most common causes of death were infection. the diagnosis of sistemic inflammatory response syndrome (sirs) and multiple organ dysfunction syndrome (muds) significantly correlate with death (90%). average length of stay was 6.6 days ~. 55% patients died in the first ten hosiptal days and only 18% after 30 days. age was directly correlated with death 50% of dead were older then sixty years. an analysis of physiological variables showed that serum levels of gl~cose (55%) and natrium (71%) were in optimal physiological values. serum proteins (72%) and haemoglobin (50%) levels were inversely related to death. multivariate showed that alveolo-arterio difference in 02 content was the most informative of all mortality predictors (mean value 22,4 mmhg in 90% patients io>mrnhg). factor that most influence the patients outcome was infection (sepsis) and muds. use of predictive indicators of outcome in critically ill patients may help to assess treatment regimens and to compare patient groups. acute physiology and chronic health evaluation (apache if) score (crit. care had. 1985; 13: 818-29) and the sepsis score of elebute and stoner (br. h surg. 1983; 70: 29-31) have been used, objectives: to compare sepsis score and apache ii score in predicting outcome of critically ill patients. methods: overall survival during the past 8 years for patients in our icu was calculated = 62% (prior probability). the outcome of 230 patients who were admitted to our icu for > 72 hours was observed. apache ii score on admission, patient predicted risk of death (apache ii risk) and the sepsis score on the first day of antibiotic course were prospectively recorded. discriminant function analysis of the scores in relation to outcome was performed. results: apache ii and sepsis scores in the survivors were significantly lower than in those who died (21.6 i 7.2 v~s 25.6 • 6.5 and 10.9 • 5 v's 15.2 • 5.9 respectively p < 0.001). correct prediction of outcome by each score is shown in discussion and conclusions: although both scores have been previously evaluated in predicting outcome of icu patients, studies of the sepsis score were conducted in small numbers of patients or involved additional measurements not routinely available. this study demonstrates that the sepsis score alone or in combination with apache ii score is more effective than apache ii score in predicting outcome. objective to test the hypothesis that resuscitation titrated against gastric intramucosal ph (phi) improves survival in critically ill patients as suggested by gutierrez et al~. method emergency admissions to the intensive care unit were randomized into control and intervention groups. in the control group phi was measured at 0, 12 and 24 h while in the intervention group phi measurements were made 4 hourly for 24 h. both groups were managed according to the same guidelines to achieve the following targets: mean arterial pressure >70 mmhg, systolic arterial pressure >90 mmhg, urine output >0.5/ml/kg, haemoglobin >8 g/dl, blood glucose < 12 mmol/1, arterial oxygen saturation >94% and correction of uncompensated respiratory acidosis. if the phi was < 7.35 after achieving these targets, or after maximal therapy to achieve the targets, patients in the intervention group were given fluid to ensure an adequate cardiac preload and then dobutamine at 5 then 10 mcg/kg/h, titrated against phi. this additional therapy was continued until 24 h after entry into the study. in each year patients were subdivided in two series with random selection, so that the 1st series contained abeat 2/3 and the 2nd 1/3 of the patients. the 1st series of all the years constituted the devdoping data set and the 2nd series the validation data set. with data of the 1st series (642 patients), we created the predictive model, using stepwise logistic regression (bmdp, usa). each patient has been evaluated in die 1st, 5th, 10th and 15th day, calculating for each lime the apache ii score (for a total of 1444 records), independent variables were, besides time and apache ii of the time ( michaloudia g,, melissaki a., alexias g., gogafi c., kolotoura a., krimpeni g., pamouktaoglou f, filias n. objectives: to determine the medical staff's attitude towards various ethical issues methods : between january 1994 and february 1995,185 anonymous questionnaires were sent to 20 intensive care units, all over greece. results : 107 questionnaires (57,7%) were replied and returned back. of them 58,9% were answered by male and 41,1% by female. the doctors replied in the following rate :81,2% aged up to 34,80% aged between 3544 and 94,4% aged over 45. 36 questions were answered and were divided into 3 main topics, as following: 1. admission criteria: limited bed availability was the main cause for refusing admission in 54,5% of icu's. 54,5% evaluated each case's viability and only 10,3% used some prognostic score system. 21,5% of icu's accepted all cases and a significant percentage (64%) gave in to pressure coming from their colleagues (72,7% female and 58,7% male). 2. informing the patient/relatives: only 6,5% was willing to tell the whole truth, while 39,2% had given selective information.. in the case of iatrogenic incident, 58,9% withheld it, because either they feared legal implications (34,6%), or lost of trust (46,7%). doctors are asking consent from the patient and/or his family, in order to include him/her in research protocols, in a rate of 82,3%, while only 55,1% found informed consent necessary for the proposed treatment procedure. 3. withdrawal of therapy/dnr orders/organ donation: 80,4% were willing to withdraw complex treatment in patients with short life expectancy, except of administi'ating intravenous fluids, feeding and analgesics. in 34,6% such a decis~n was unanimous, while the percentage of those carrying it out was 69,1% (72,2% female, 63,9% male). in case of brain stem death 87,8% (67,3% female, 85,7% male) withdrew any life support. 67,3% would like therapy withdrawal to be legally established, while only 12,1% would perform euthanasia, if there was substantial legal cover. for these cases, relatives' consent was considered to be necessary from a percentage of only 11,2%. 83,2% considered organ donation to be a necessary proposal, while 10,3% refused to ask the patients' relatives for an organ donation, either because they didn't have the psychological strength for it (3,7%), or because they doubted the procedures' objectivity (4,7%). note: in greece, icu beds are less than 1% from the total number of hospital beds available. only a percentage of 35-50% of these admissions comes from the same hospital, with a potentially direct evaluation. usually an icu doctor has to be informed through the telephone. finally, employment conditions in greece are such that any changes of the medical and nursing staffare limited. conclusions: the mathematical model we found has been validated also in the second series and the discrimination capability increases with time. using this model we can evaluate the probability of survive at every, time. its application at different times permits a better evaluation of haemodinamically instable patient trend. introduction: the feasibility to assess pulmonary capillary pressure (pcap) offers the opportunity to determine the longitudinal distribution of pulmonary vascular resistance (pvr). the purpose of this study was to measure pcap and to calculate pvr to determine whether relevant shifts in the distribution of pvr could be expected after routine cardiac surgery. methods: the study population consisted of 25 consecutively admitted patients after cardiac surgery. surgical procedures included coronary artery bypass graft (cabg) (n=14) and mitral valve replacement (mvr) (n=t 1). pcap was estimated by analysis of the pressure decay tracing after pulmonary artery occlusion. after estimation of pcap precapillary (ra) and postcapillary resistance (rv) was calculated. a complete set of hemodynamic variables was obtained at 1 hour and at 6 hours after operation. results: there were no significant hemodynamic changes during the first 6 hours after surgery. the mvr group maintained pulmonary hypertension and higher levels of pcap. ra/rv, reflecting the longitudinal distribution of resistances, remained unchanged. however, rv predominated ra during the postoperative period in both groups. objectives: evaluation of the influence of long-term continuous i.v. administration of the ace-inhibitor enalaprilat on regulators of circulatory homeostasis. methods: t9 trauma and 26 sepsis patients randomly received either 0.25 mg/h (group i, n=15) or 0.5 mg/h (group 2, n=15) of enalaprilat i.v. or saline solution (control, n=15) as placebo for 5 days. plasma levels of endothelin-1 (et), atrial natriuretic peptide (anp), renin, vasopressin, angiotensin-ii, and catecholamines were measured before injection of enalaprilat (='baseline' values) and during the next 5 days. results: except for et, plasma levels of all vasoactive substances exceeded normal range at baseline. angiotensin-ii significantly decreased during enalaprilat infusion (0.25mg/h: from 53.1• to 22.1• pg/ml; 0.50mg/h: 62.1• to 17.9• whereas it remained significantly elevated in the untreated control patients. vasopressin increased only in the control group (p<0.01) and decreased after 0.50mg/h of enalaprilat. et remained almostunchanged in group 2, whereas et increased significantly in the control patients (from 4.9• to 10.t• on the 5th day). catecholamine plasma levels (epinephrine, norepinephrine) markedly increased in the control group (p<0.01), but they did not change significantly throughout the study period in both enalaprilat groups. conclusions: continuous i.v. administration of the angiotensin-converting enzyme inhibitor enalaprilat beneficially influenced systemic and local vasoactive regulators of the circulation, which are normally increased in the critically ill. thus patients at risk of (micro-) circulatory abnormalities may profit from enalaprilat infusion. objectives: to determine the time taken for hemodynamic and gas exchange variables to a reach stady-state after a change from supine to trendelenburg position (trp). methods: we prospectively studied 8 adult patients with severe sepsis or septic shock requiring hemodynamic monitoring. usual cardiorespiratory parameters were measured at baseline, 15 min after the patient was placed in a trp and again 15 min after the return to a supine position. a fiberoptic pulmonary artery catheter (svo~ oximetrix, abbott) allowing continuous svo 2 monitoring wa~used. during the protocol we also continuously measured sao~ by pulse oximetry and vco~ and vo 2 by monitoring partial concentration of o2and co 2 ir~ inspiratory and expiratory gases (deltatrac metabolic monitor, datex). therefore, we were able to monitor cardiac output variations by dividing vo~ with arteriovenous difference according to the fick equation (co-fick). results: no significant difference in hemodynamic status was observed 15 min after the patients were placed in trp. despite the fact that no significant change was observed in co and vo~ estimated by thermodilution, co-fick had a tendency to dedrease continuously in trp and then to return to its initial value when patients regained supine position. respiratory gas analysis showed a small but persistent continuous increase in vco 2 without a similar trend in vo 2 values. conclusions: we conclude that no significant hemodynamic effect was detected in our patients after 15 min in trp. evaluation of vo 2 from respiratory gases analysis after a change in body's position should be interpreted with caution, since the patient may not yet have reached a stady-state after 15 rain. since vo 2 did not change, vco~ increase was probably due to position related changes in-pulmonary gas exchange and not to a change in patient's metabolic status. objectives: to determine whether changes in svo 2 and/or other hemodynamic parameters during weaning trials could be used to predict successful weaning. methods: we prospectively studied 10 adult patients with a history or clinical evidence of cardiovascular dysfunction, who were unable to tolerate spontaneous breathing (sb) for 3 hours. for all these patients right heart catheterisation was considered necessary in order to detect hemodynamic alterations during weaning. a fiberoptic pulmonary artery catheter (svo 20ximetrix, abbott) allowing continuous svo 2 monitoring was 5sod. hemodynamic status was evaluated ~t baseline and after one hour of spontaneous breathing through a t-piece. patients were assigned to one of two groups depending on whether they tolerated sb for 3 hours. data were analysed by analysis of variance and unpaired student's t-test we also used multiple linear regression analysis to determine which hemodynamic variables were correlated with the magnitude of svo~ change and multiple discriminant analysis to determine if asy of the above variables were associated with toleration of sb for 3 hours and/or successful weaning (s-w). (j physiol 1995; 78." 696-701) . we tested the hypothesis that the ventilatory stimulation by dead space (vd) loading and 3% co2 inhalation is accompanied by a proportionate cardiovascular change. methods: six healthy subjects, mean age, 25 year, performed three incremental exercise tests in a randomized order: 1) inspiring air without vd (air control, ac); 2) inspiring air with vd of 920 ml (avd); 3) inspiring 3% co2; 21% oxygen, balance nitrogen. the ventilatory responses were examined at matched heart rate (hr) equivalent to 90% peak hr. results: ventilation (vi) was significantly greater (p<0.0001) during the avd and co2 tests than during the ac test at the same work rates. end-tidal co2 (petco2) and estimated arterial co2 (paco2) were significantly greater (p<0.01) at 150 w and 200 w. oxygen saturation was significantly lower (p<0.05) during the avd test than during the ac and 3% co2 exerdse. at matched hrequivalent to 90% peak hr, vi was significantly greater (p<0.01) during the avd and 3% co2 tests than during the ac exerdse (123 l, 121 l, and 91/). conclusion: we conclude that the increase in xri and petco2 due to vd loading and 3% co2 inhalation is not associated with an acceleration in hr. sup.ported by mrc (canada). objeetlve: the production of large amounts of oxygen radicals from the onset of ~en may be responsible, st least in part, for peroxidative damage to myocardial tissue. the aim of this study was to evaluate the time dependence of plasma tbars in patients with am] receiving thrombolytie therapy (tt). patients and m~hods: filiy eight patients admitted in icu (46 men and 12 women; mean age 50.6 4-16.02 years) rec~ving systemic tt for possible am] were ~died. all patients received recorabinant haman tissue-type plasminogen activator (r-tpa). the mean time fi'om the onset of symptoms and the be~nning of tt was 3.01 4-2.13 hours. peripheral veao~s blood samples were obtained fi'om each patient before and serially after tt (0, 3, 6 and 9 hours). tbars levels woe determined by using a spectrophotometrie technique. rq~r fusion was identified by the timing of ereatine phosphate kkmse (cpk) peak (<15 hours). table i list the variation of plasma eoneenlrations of tbars (mean 4-sd) in groups (a,b, and c) as a function of time from the beginning of tr. co,arisen oftbe 0 time cuncentzatiens reveal a difference p<o.01 between group ami (a and b) versus group c. the between-groups (a-c) comparison showed significant differences (p<~).05) at 3,6 and 9 hours fi-om de begin~g of'it. comparison ofthe 3,6 and 9 hours eonocnlratiens did not reveafl any difference becween n~ (group b) and angor (group c). condmien: we have observed a maintenance of high level of tbars in reperfused patients enly. these results suggest that the increase of tbars levels may be due to phospholipid derangament of reperfused myocytes. objective: to detemline if tbars levels may be an early index reflecting peroxidative damage in ami patients. patients and methods: by using a spectrophotometric teclmiqtm, based on a modificatien oftbe buege and aust method, tbars levels ware determined in 51 healthy subjects (group i) , 43 patients with several risk factors of ischaemic cardiopathy (group if) and 99 icu patients with possible aim (87 were confirmed ami (group ma) and 12 were angor (group iiib)). peripheral blood was obtained at the time of the icu admission in the group i11. electrecardiographic data and eehocardiographic wall motion were used to identify ami location (anterior, inferoposterinr and indeterminate). statistical analysis was made with a standard statistical package (rsigma, horus hardware). , h 4, s, , h statistically significative increase of ' ix~e/ea.ee x~* tbars levels (p< 0.001) only in pstients with con/tuned ami (group ilia) . no differences were found between the rest of the groups. the figure shows the relationship between the tbars values and the time from the onset of the aim's symptoms. conclusion: we have found that in am/patients the tbars levels can be used as an early index reflecting peroxidative damage occurring to ischemie tissues. perioperative risk in patients with ischemic heart disease. the protective role of diltiazem. lg. hatziantoniou, p. tassiopoulos, c. fakiolas, ! g. foussas~ m. lycourinas perioperatlve morbidity and mortality are mainly of cardiac origin, particularly among elderly patients (pts) ~ith ischemic heart disease. we performed a randomized trial, in order to investigate the possible protective activity of diltiazem (d). methods: 58 pts (aged zo• with a history of coronary insufficiency were subjected to major urological operations. they were randomized to two groups: group i (29 pts) -p:ithout d and group ii (29 pts) -d ~as administered 0.15 mg/kg iv bolus 12 hours prior to anesthesia and 0.15 mg/kg/h upon the end of operation and for at least 12 hours post-operatively. iv was substituted by oval administration of 2x120 mg starting the next day and for the rest of hospital stay. all pts underwent daily clinical, electrocardiographic and laboratory (ck-mb) control. results: are shown in the following interactions between the heart-lung unit and a cardiac assist device are the crucial issues for successful outcome of patients under mechanical circulatory support. several factors have been indicated and we can diffrentiate between anatomical and hemodynamic interactions. especially right heart failure and pulmonary hypertension have been proven to limit left ventricutar assist device (lvad) performance. in order to study the effects of right heart failure (rhf) and pulmonary hypertension (pht) on mechanical circulatory support, we designed an experimental study in dogs and combined in six groups left heart failure (lhf) and rhf and pht, respectively. in the control group we induced lhf with subsequent occlusions of lad and cx followed by reperfusion: in the next group we supported with an lvad during 90 minutes starting 5 minutes after cx occlusion (group if). in the next group we added right ventricular ischemia by occluding the rca simultaneously to the cx (group iii). these both groups we repeated only with pre-existing acute pht induced by the injection of 1o0 fm glass beads (groups vi and v). a last group was similar to group v (lhf and rhf and pht), but instead of a lvad, we used bvad support. in i and v no animal survived the second perfusion period (4 hours). in ii and iii (no pht), all dogs survived in stable conditions. in iv (lvf, pht), 60 % survived, the remainder died of incomplete recovery. in vi with bvad, only 40 % survived. in iii and v, during occlusion of the rca, the right ventricle stopped contracting and right atrial pressure and mean pulmonary pressure equalized. this lead to a significant drop in inflow on the left heart side, which still provided sufficient lvad performance in case of absent pht (iii) and lead to death due to "low lvad output" syndrome in v. this "low lvad output" snydrome could be partly overcome by bvad support. we conclude that heart-lung interactions play a major role in lvad performance. by optimizing the perioperative management, they all can be overcome, except the occurrence of alveolar leakage syndrome. obiectives : we propose a new approach of transmural pulmtmary artery occlusion pressure (paoptm), in presence ef peep, from the folluwing hypothesis : if respiratory swings of paop are compared to those of alveolar pressure (apalv = plateau pressure -total peep), an index of transmission of airway pressures to pulmonary vessels is obtained (i t = apaop/apalv). the value of the product of it by peep can be substracted from end-expiratery paop (paopee) to obtain a calculated paoptm (paoptrnc). thus paoftmc = paopee -(apaop/apalv x peep). methods : we tested this hypothesis in 67 patients by comparing paopee, paoptmc, and paopnadir obtained within the first 3 seconds after disconnection from the ventilator, value of reference of paoptm in absence of intrinsic peep (peepi). at zeep, peepi was absent in 38 patients (group a) but present in 29 others (group b). patients were studied under the peep level chosen by the attending physician-(gr a : 11 +_ 2 ; gr b : 11 _+ 3 cmh20 ). results : l. gr a : paopee (12 _+ 6 mmhg) differed (p < 10 -3) from paopnadir (8 • 6 mmhg) and paoptmc (8 _+ 6 mrahg). gr b : paopee, paopnadir, paoptmc differed between themselves (14 "2-6, 12 + 6, 10 + 6 mmhg respectively). 2. good correlatkm (r (i.99 ) and agreement between paopnadir and paoptm were found in gr a, while there was no agreement in gr b (bland and altman analysis). 3. lung compliance (v t/bpah,) correlated well with i t in both groups (r -0.92), suggesting that our hypt)thesis was strtmg even in patients with peepi. conclusions : paoptm under peep ventilation can be obtained, even in patients with peepi, from parameters easily measured at the bedside. introduction: cardiopulmonary bypass (cpb) may cause ischemia in several organ systems like the heart, brain and kidneys. reactive oxygen species (ros) are formed by subsequent reperfusion and stimulation of neutrophils by cpb. ros are harmful to biological systems e.g. cellular membranes, enzymes~ dna. many natural antioxidant systems protect against the effect of ros. in patients undergoing cabg we evaluated the production of ros by total plasma antioxidant status (tas) and lipid peroxidation measured by lipofuscin flip). methods: tas (randox, uk) and concentrations ofllp (tsuchida et al.) were measured in 16 consecutive patients undergoing cabg. all the patients had a normal serum creatinine clearance (>50 ml/min). serum samples were obtained a) before operation, b) after removal of the aortic crossclamp, c) at admission to the icu, d) 4 hours after operation, e) 22 hours after operation. results: tas was significantly decreased after removal of the aortic crosselamp ( b, c and d lower than a), followed by a subsequent significant increase of lip ( c and d higher than b). the levels of tas and lip returned to baseline 22 hours after operation. methods: 10 patients with preoperative lvef<40% undergoing coronary artery bypass grafting were studied. after surgery, a 3f femoral artery catheter was inserted and connoted to a fiberoptic monitoring system (cold z-02t; pulsion medizintechnik, germany); this allows, with a double-indicator dilution technique, the calculation of cardiac index (ci,l/min/m2), intrathoracic bood volume (itbv,ml/m2), pulmonary blood volume (pbv,ml/m2) and extravascular lung water (evlw,ml/kg). with a 7f pulmonary artery catheter, wedge (w,nunhg) and central venous pressure (cvp,mmhg) were measured, while 02 extraction ratio (o2exr,%) and oxygen delivery (do2,ml/min/m2) was calculed. peak inspiratory pressure (pawp,cmh20) and mean airway pressure (mawp,cmh20) were measured with a varflex flow transducer (bicore,sensormedics,us). the patients were studied after 60 minutes (to) of volume controlled standard ratio ventilation (vc), and after 60 minutes (ti) of stabilisation period of pcirv (67% inspiratory time, 0 % pause). vt,ve and total peep were held constant in every mode of ventilation. 1+_0.4" *'p < 0,05 versus to conclusions: these data show that pcirv 2:1 is a safe ventilatory support also in cardiac patients with impaired ventricular function, and monitoring of itbv is more reliable to measure and optimise circulatory volume status, than w and cvp. c.ledeki-,g.rldisis,s.karotzai,c.micheilidis,m.agioutantb, g.beltapaulos. objeolivee:to evaluate the influence of lvswl on the well known correlation of sr and svo2. paw eight patients (12 melee end 16 females) were included in this study regerdlen of the icu ~h"niseion couse. all paints were ,'~theta~ with e fiboroptir pulmonary artery catheter connected with an oxymetfir (r)~ so2/co abbot computer.for any pulmonary artery catheter insertion, two pain= of sr and svo2 were obtained, one dudng inserlion and one during taking the catheter out. for any pair obtained, we eleo collected the deta concemig with the pedient's hemodynamir and oxygenation end we calculated the lvswi. were significantly (p<o.05) higher in group i. conclusion: breathing room air, the less hypoxemic patients had a rather satidactory do2 and tissue oxygenation (pro2) because they had properly adapted their ci. the absence of this mechanism (right heart impairement ?) in the hypoxemic patients, in spite of higher blood ne and e, was responsible for the poor do2 and pro2. objectives: acute massive pulmonary embolism (pe) increases pulmonary artery pressure (pap) and dght ventdcular aftedoad, which may lead to dght ventdcular failure. inhaled nitdc oxide (no) selectively dilates pulmonary vessels. thus, inhaled no may be of potential therapeutic value in the management of the acute phase of pe. methods: following institutional approval, ten piglets (body weight 17+2 kg) were anaesthetised (midazolam/piritramide) and ventilated using a modified servo ventilator (siemens elema, sweden; fio 2 0.3). the following variables were obtained: systolic pap (spap), mean pap (mpap), diastolic pap (dpap), mean artedal pressure (map), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), cardiac output (co), and artedal (pao2) and mixed venous (pvo2) oxygen saturation. pulmonary vascular resistance (pvr) was calculated. to induce pe, 300 pm microspheres (sephadex g50 coarse, pharmacia biotech, freiburg, germany) were injected in an amount sufficient to increase mpap to 45 mmhg initially. 45 rain after embolisation when haemodynamics were in a steady state (control 1), inhaled no was administered. further measurements were taken 5 min after 40 ppm no (no 40), 5 min after 80 ppm no (no 80), and 10 min after discontinuation of no administration (control 2). the administration of inhaled no resulted in a significant decrease in spap, mpap, and pvr. the cessation of no inhalation led to a complete return to pretreatment control values (table) . co, map, cvp, pcwp, pao?, and pv~ remained unchan no administration. objectivss:evsluate the yield of recombinant tissue plasminogen activater(rt-pa, actiiyse ~ 9oehringer ingelheim) as a thrombolytic agent ie pulmonary embolism ie .kr~auma patients. methods: in 1994 five patients with diagnosed pulmonary embolism(blood gases, chest x-ray, echocardiography, perlesion lung scans) were given 100rag of actij.yse as ~ 2hrs infusion, followed by heparin~single 5000j. ihjaction snd iefusion ie doses 300-400j./kg/day).the effectiveness of rt-pg thrembo].ytic sctier~ was svaluated im 3.24. hrs(blood gases) amd on the third dayafter t~eat-,ment(~chocardiography and lung scan). results: three hrs after actilyse was given we observed significant clinical imprevemeet,confirmed by elevation of pao 2 and sao 2 in dlood.echocardiegr~m on the third day showed regression of pulmonary hypertension and lung scans showed 45% improvement in total lung perfusien. except slight bleeding from the site of injection nc~ more thromboiytic complicatioos were observed. conclusiens: actilyse used in five hrauma patients with pulmonary embol.ism was a very effsctive and safe thrombolytic agerlt, inducing rapid and evident clinical improvement. intermittent positive pressure ventilation (1ppv) by increasing pleural pressure, decreases the pressure gradients for systemic venous return and left ventricular (lv) ejection. we have previously shown that when inspiration is synchronized with systole using synchfjv in patients with severe cardiomyopathy, that cardiac output (co) was increased relative to both ippv and non-synchronized hfjv (1). however, it is not known if similar effects could be realized in ventilatordependent patients with lesser degrees of lv dysfunction and fluid overload. we thus compared synci-ifjv to ippv in stable patients following coronary artery bypas grafting. methods: 13 normothermic who were stable (< 20 % variance/h.) were studied following admission to the sicu. heart rate (hr), mean arterial (pa), pulmonary artery occlusion pressure(ppao), co by continuous thermodilutiun (vigilance, baxter, usa), mixed venous saturation (svoz) and arterial blood gases (abg) were measured after 30 rain. of each step. ippv was simv adjusted for a rate, tidal volume and fit2 to insure both normal abg and a peak airway pressure < 35 cm h,o. syncfifjv was delivered by a hfjv ventilator (acutronic) during systole with ventilator parameters adjusted to optimize abg. protocol: patients received three ippv runs (ippvi.3,5) with synchfjv runs interposed (hfjvz4). statistics: anova for repeat measures and paired t-test were used to compareruns and demonstrate variability. results:no significant difference in any of the measured hemodynamic variables were seen among the ippv and synchfjv runs (table) . post hoc separation of patients by preoperative ejection fraction (ef; ef > 40% ; n=10 and < 40%; n=4) did not alter these results. back~ound: in man, vascular endothelium-bound ace is expressed in concentrations greater than 50x that in serum and is believed to be the site of synthesis of circulating angioteusin il it is unclear whether ace inlubitors interact similarly with ace in different vascular beds. coronary vessels possess all the components of the renin-angiotensin system, including ace which may be involved in normalcardiac homeostasis, as well as in the pathogenesis of various cardiomyopathies. obiecfive: to develop a method for assaying the interaction of ace inkibitors with coronary endothelium-bunnd ace in man, methods: ace a~aty was meas~ed in five patients undergoing cabg surgery, from the transeuronary hydrolysis of the synthetic ace substrate 3h-bpap. trace mnou~ of ~fi-bpap (4gci) were injec~d as a bolus in the root of the aorta and simultaneously blood was withdrawn from a coronary sinus catheter into a syringe containing protease inhibitors which prevented the convession of umeaet~ ai-i-bpap by blood ace. the sample was later centrifuged to separate cells from plasma and the radioactivities due to formed product (~rl-bphe) and total sh were astimated in a [b-counter. two additional such determinations of ace activity were perform~ the second in the presence of 1.5pg/kg e (coinjected with ~-i-bpap) and the third ten minutes after e. results: all subjects were hemodynamically stable throughout the course of the there were no noticeable hemodynamic effects of e. control transcorunary metabolism of~-bpap averaged g0-a:3%, in agreement with previously reported data. in the presence of e, % metabolism of ~-bpap was reduced to 21• reflecting a 85• inhibition of normal ace activity. ten minutes after e, ~ri-bfap metabolism had partially recovered to 52:l:10%, representing a 50-a:15% inhibition of control ace activity. from this data, the dissociation constant of e for coronary ace in vivo was estimated as 6.8x10 "4 sec "l. conclusions: we have demonstrated the feasibility of repeated, reproducible measures of coronary endothelium-bound ace activity and of its inhibition by e. this procedure is safe and can be used to study the role of ace in normal cardiac function and in card pathologies. objectives. primary pulmonary hypertension (pph) is a progressive fatal disease of unlmown origin, with median life expectancy of less than three years after diagnosis. the responsiveness of pulmonary hypertension to a variety of vasodilator agents led to the speculation that, concomitant with vascular renmdelling processes, persistent vasoconstriction is an important feature of the disease. long term use of ca-channel blockers and intravenous pgiz may improve mortality in certain populations of pph patients, but both of these treatments lack selectivity for tire lung vasculature. the aim of this study was to test the efficacy of aerosolised prostacyclin and its stable analogue, [loprost for selective pulmonary vasodilatation in pph. methods: in three patients with pph, we compared aerosolisation of prostaglandin iz (pgi2) and iloprost to a battery of vasodilatory agents (diltiazem, nifedipin, inhaled nitric oxide, intravenous pgiz). results: nebulisation of pgi2 and iloprost tumed out to be most favourable for achieving effective and selective pulmonary vasodilatation. pulmonary vascular resistance decreased from 1664 + 75 to 1054 -+ 93dyn*s*cm 4 (p<0.001) and pulmonary artery pressure from 63.3 + 3.1 to 528 + 3.4 mmhg (p < 0.05), cardiac output increased from 2.66 + 0.11 to 3.57 _+ 0.16 i/rain (p < 0.001), mixed venous oxygen saturation from 49.6 _+ 2.2 to 63.3 + 2.8 % (p < 0.001) and arterial oxygen saturation from 87.9 + 2.6 to 93.6 _+ 2.2 % (mean _+ sem of 7 trials in 3 patients). 5-month iloprost nebulisation in one patient (100 gg/day in six aerosol doses) demonstrated sustained efficacy of the vasodilator r~men. conclusion: aerosolation of pgi2 or its stable analogue may offer as new strategy for selective pulmonary vasodilatation in pph. endothelial adhesion molecules may play an important role in the pathogenesis of myocardial cell damage, and may contribute to the progression of heart failure. we measured the plasma soluble intercellular adhesion molecule-1 (sicam-1), vascular cell adhesion molecule-1 (svcam-1), and e-selecfin (selam-1) levels in 27 patients with acute myocardial infarction admitted within 6 hours after onset. peripheral venous plasma-samples were collected at the time of admission, 12, 24, 36, 48, and 72 hours after onset. plasma soluble adhesion molecule concentrations were determined by elisa. patients were divided into 3 groups as follows: group 1; killip's class (k) 1 and 2 without thrombolytie therapy, group 2; k 1 and 2 with thrombolytic therapy and group 3; k 3 and 4. both plasma sicam-1 and svcam-1 concentrations in group 2 and 3 were elevated rapidly and significantly and maintained at a high level during the first 3 days. plasma selam-1 level did not change in any of the groups. these results suggest that the adhesion molecules icam-1 and vcam-1 may play a role in the pathogenesis of myocardial reperfusion injury and may indicate its severity in myocardial infarction. objectives: nitric oxide (no) is known to exert cytotoxic and negative inotropic effects on cardiomyocytes. no synthase activity has been reported to be increased in infarcted area in animal model of myocardial infarction. these findings suggest that no may be an important regulator for myocardial damage and cardiac function after myocardial infarction. we measured plasma no27no3-(nox) levels and estimated serial changes in acute phase of myocardial infarction. methods: subjects were 15 patients admitted within 3 hours after onset. venous blood samples were collected at 3-hour intervals on the first day, 6-bour intervals on the 2nd day and 12-hour intervals on the 3rd day and 4 th days after onset. plasma nox concentrations were determined by griess method. results: the time course of the plasma nox levels (mea~+sem) displayed a tendency to gradually increase and to make a biphasic pattern with two peaks about 24 hours and 2-3 days after onset (basal level; 32.8_+4.9, first peak; 42.0!-_7.0, second peak;47.0+7.6 ram/l). plasma nox concentration was not influenced by the thrombolytic therapy, and nox values at the time of 60 hours after onset were significantly correlated with maximal plasma creatine kinase level (r=0.83, p<0.01). the levels of plasma nox in the early stage of myocardial infarction (from admission to the 4th day after onset) did not correlate significantly with the hemodynamic parameters (left ventricular ejection fraction, pulmonary capillary wedge pressure). conclusion: the early and late increase in no production after myocardial infarction may be implicated in the deterioration of myocardial contractility and induction of myocardial damage in the early phase of myocardial infarction. range 36-85) fullfilling the high risk criteria of shoemaker (colectomy 13, gastrectomy 10, pancreaticoduodenectomy 4, others 6). patients were admitted to the icu preoperatively. arterial and pulmonary artery catheters were inserted and hemodynamics and oxygen transport were measured at admission and after stabilization to predetermined physiological end points. patients were considered stable when ci >2.5 l/min/m 2, pcwp >10 mmhg, hb >100 g/l, sat2 >.94. objectives: evaluate the acute effects of 0,08 mg ipratropium bromide and 0,2 mg fenoterol (ibf) inhaled dose on pulmonary function in 17 nonsmocers (nb:m) and 14 smocers (s) with sever (new york heart association class ii-iii), stabile congestive heart failure(chf) and 12 healthy subjects. methods: pulmonary function tests were performed < 3h postprandial. the tests consisted el arterial blood gas aspiration followed by routine spirometry and pletismography, and single-breath gas analysis. after performance of these maneuvers, the patients was administred 4 puffs-ipratropium bromide (0,08rag) and fenoterol (0,2 rag). for 0,5 h, spirometry was repeated. results: in resting, pulmonary abnormalities observer in the s group were more severe then abnormalities observere in the nsm group. after treatment with ibf the improvement in pulmonary function was even more marked in patients who had smoked. the mean changes by forced expiratory volume in 1 second(eevt) was 8,1% (p<0,00t) improvement and 3,9% (p<0,ob), forced expiratory flow betwen 25% and 75% of the forced vital capacity (fef25.75) was 30,8% (p<0,001) and 23,6% (p<0,001) and maxamal voluntary ventilation (mw) was 21,7% (p<0,05) and 16,2% (p<o,05) improvement. the mean changes in normal subjects were mild (fev 1 increased by 3,4%,fef 25-75 by 14,5% and mvv by 6,3%). we observed no adverse heamodinamie conseevence and no arrthytmogenicity. conclusion." in patients with chf the airway response to ibf is highly significant. further study is needed to determine whether therapy with ibf will load to improvement quality of life in patients with chf with dearly documented venti/atory defects. compared with unilateral ima grafts, usage of bilateral ima grafts in cabg patients causes greater thoracic trauma and further reduces blood supply to the sternum and intercostal muscles. these effects may be associated with increased postoperative respiratory complications obiectives: to study the effects of bilateral ima grafts on postoperative respiratory morbidity in cabg patients. methods: two groups of patients undergoing cabg were prospectively studied: group 1 (n=13) received both imas as grafts and group 2 (n=12) received only the left ima. the two groups were matched for age, smoking habits, total number of grafts and preoperative ejection fraction. pao2/fio2 ratio was measured in all patients in the icu, at 30,120,240 minutes on mechanical ventilation (mv), at 30, 120, 240 minutes after extubation (ex) and on postoperative day (pod) 5. in addition, radiografic scores of atelectasis and pleural effusion were assessed postoperatively. results: group 1 had significantly longer bypass (101_+23 vs 70+15. p<0,001) and aortic cross clamp time (45+-19 vs 28+7, p=0,009). pao2/fi02 ratio was significantly higher in group 1 only at 30 minutes after extubation (table) . there was no difference in the incidence or severity of atelectasis or pleural effusion between the two groups. the duration of mv was longer in group 2 (13+_3 vs 9_+2, p=0,004), but the length of icu stay and hospitalization were similar. there was one case of pneumothorax in group 2 and one case of pulmonary infection in each group. after extubation 30 m[n 120 min 240min 30min* 120rain 240min pod 5 1' 342_+87 302_+92 369+87 278-+67 2664-79 294-+102 376_+76 2 319_+105 324_+89 320_+76 223-+52 257_+94 249_+82 400-+43 mean_+sd, *p=o, o03 conclusions: compared with unilateral ima grafts, the usage of bilateral ima grafts in cabg patients is not associated with increased postoperative respiratory morbidity. the diagnosis of rv ischaemia is difficult in the critical care setting. the purpose of this study was to determine the ability of rv endomyocardial and interstitial ph electrodes to detect rv ischaemia in an animal model of rv infarction produced by fight coronary artery ligation. we hypothesized that changes in transmural and interstitial ph would accompany the induced tissue hypoperfusion. rv interstitial (phint) and transmural endomyocardial ph (phendo) were measured in 10 adult anaesthetized pigs before and serially after coronary ligation. phendo and phint fell progressively and significantly following coronary occlusion. this was accompanied by ischaemic ekg changes. the absolute and relative rates of change were greater for phendo compared to phint, particularly after 4 minutes of ischaemia, ph was unchanged in control experiments when the electrode was placed within the fight atrial or ventricular chambers, as well as when coronary ligation was not performed. these data suggest that rv myocardial ph measurements reflect coronary ligation induced rv ischaemia, ph recorded from an electrode placed against the rv endomyocardial wall via a central vein was as useful as an interstitial measurement that required a thoracotomy. this newly described technique may be helpful in clinical settings where differentiation between ischaemic and nonisehaemic causes of rv dysfunction is important. centre. depts of cardiac surgery and anesthesiology, university of bari, italy. lung injury secondary to cardiopulmonary bypass (cpb) is well recognized. nevertheless, mechanical respiratory changes after cpb have been poorly investigated. aim of the study was to evaluate the effects of cpb on respiratory mechanics. elastance of the lung (est l), chest wall (est cw), and total respiratory system (est rs) were measured in 8 patients without previous pulmonary disease undergoing elective cardiac surgery. there were 6 males and 2 females; no pleurotomy was done. cpb was carried out with membrane oxygenator. mean cpb and cross clamping times were 69_.27 and 39• min. flow (pneumothacograph), airway opening and esophageal pressures (pressure transducers and esophageal balloon) were measured on patients sedated and paralysed. measurements were obtained before sternotomy, at the end of cpb after chest closure, four and seven hours later. est rs, est l and est cw, were measured by occluding the airways and the end of a tidal breath. before end cpb-4 h after 7 h after stemotomy closed chest cpb cpb estrs (cmh20/l) 17.7+4.9 19.94-5.6" 22.5_+5.8* 22.64-4.3* est cw " 6.7_+4.1 6.3• 6.61+4.09 6.5-+4.1 est l " 10.9--4.3 13.54-5.4" 15.9-_4.9" 16.1_+5.1" x + sd. * p < 0.05: anova vs "before sternotomy". our data show that cpb induces increase in est l, whereas est cw remains unchanged despite sternotomy. impairment in est l after cpb evolves within the first 4 hours and stabilizes 7 hours later. during the clinical course of human imunodeficiency virus infection (hiv), patients (lots) can develop congestive and dilated cardiomyopathy syndrome (dcm). the aim of our study was to analyze the prevalence of dcm among an hiv population, its clinical and echocardiographic changes under a long term follow-up and therapy with an ace-inhibitor agent (captopril). we prospectively studied 220 pts, 23% (50/220) developed dcm during the follow-up study. among this group 26% (13/50 pts) were in class i and ii and 64% (37/50 pts) in class iii and iv of the nyha classification. we divided this population in a acei+ (22 pts) and acei-(28 pts), according to the administration of captopril. we analyzed the following parameters: age, gender, race, ivda, type of hiv, cdc classification, number of t4 and t8 type cell population and its t4/t8 ratio, therapeutic with acei, type and number of opportunist infections, mycobacteriosis and neoplasm's. we analyzed several echocardiographic parameters, which included the initial (i), final (f) and variation (a) of the lv internal diastolic (lvdd/mm) and systolic (lvsdimm) diameters, lv % of fractional shortening (lv%fs/%), ivs and posterior wall thickness and lv mass (lvm/g). the two groups differed significantly in the following parameters: left ventricular functional indices of patients (pts) with iscjhemic dilated cardiomyopathy (idcm) have a critical value in terms of clinical prognosis of this disease. three-dimensional echocardiography (3-de) is a recently developed method that gives a better evaluation of lv morphology and function. the purpose of our prospective study was to assess the clinical applicability of this new 3-de method in a population with ischemic dcm diagnosis and calculate its lv indices of global and regional function. we studied a group of 16 idcm pts, mean age 58_+11 yrs, 70% male gender, using a combined 3-de tee approach. all pts were in class ill/iv of the nyha classification. first, ekg gated images were acquired through a mechanical pullback of the tee probe, and each set of data was transferred to a conventional ibm-pc system using a dedicated 3-de software. computer processing and analysis of the tee images led to a 3-de reconstruction matrix and off-line calculation of several lv global and regional indices. in all idcm lots the lv cavity was clearly reconstructed and end-systole and diastole in several orthogonat projections, out in multiple planes and its function quantitated using 3-de derived indices. mean values of 3-de lvesv=149-+20 ml, lvedv=197_+32 ml, lvstv=48_+25 ml and lvef=24-+5% were obtained. for all these 3-de parameters, good correlations were observed with 2-de lv measurements obtained through biplane tee analysis of the lv cavity (r>.70; p<.01) as well as regional analysis of sequential 3-de cut planes. conclusion: in our group of patients with the diagnosis of ischemic dilated cardiomyopathy, this new 3-de method could be applied. our results show that this method allows a better assessment of the lv morphology and spatial geometry, with the calculation of global and regional indices with critical clinical and prognostic value in this particular cardiovascular pathology. simultaneous left atrial (la) and left ventricle (lv) inflow analysis assessed by pulsed doppler tee illustrate the loading conditions and reflect the hemodynamics of the left heart. we performed a prospective tee pulsed doppler study with recordings of the transmitral lv filling and pulmonary venous (pv) flow drainage in a group of patients with dilated cardiomyopathy (dcm). a group of 23 dcm patients, mean age 57_+11 yrs, 74% male were studied. this population was divided according to tee severe lv dysfunction (group slvd+ 62% pts; group slvd-38% pts) in each pt we measured the peak velocities (vel/m/sec) and time velocity integrals (vti/m) of the transmitral early (e) and late (a) filing waves, the vel and vti of the pv systolic (s), diastolic (d) and atrial contraction (c) reversal flows. 3-de tee evaluation of the lved, lves, lvst volumes and lvef were obtained. we calculated other parameters, such as e/a, s/d and a/c ratios and the sum of c+a vel, that refelect la systolic function and lv compliance. 19+5 305-_8 0.02 simultaneous and quantitative analytical approach of the pulmonary venous and transmitral flows and ventricular volumes improve the non invasive assessment and understanding of left ventricular diastolic function and cardiac performance in dilated cardiomyopathy patients. objectives : to assess the hemodynamic effects of fluid loading (fl) in acute circulatory failure (acf) due to acute massive pulmonary embolism. methods : hemodynamic measurements (fast-response thermistor pulmonary artery catheter) were performed at baseline (baseline) and after a rapid fluid loading with 250 (fl250) and 500 (fl 500) ml of dextl'an 40 (rhemacrodex| in 12 patients free of previous cardiopulmonary disease (66 • 3 yrs) with acf (ci < 2.5 l/rain/m2) due to angiographicalty proven mpe (miller score > 21) . results : are expressed as mean _+ sem and compared by anova. a significant negative correlation (r = 0.83) was observed between baseline rvedv[ and the effects of fl on ci. such correlation was not observed between baseline rap and the fl induced increased in ci. conclusion : fusibmificantly increases ci in acf due to mpe. however, the simultaneous decrease of arterial 02 content due to hemodilution, limits the benefits expected from improved ci on peripheral oxygenation. obiective: to examine the hemodynamic effects of external positive endexpiratory pressure (peep) on right ventricular (rv) function in acute respiratory failure (arf) patients. methods: incremental levels of peep (0-5-10-15 cmh20) were applied and rv hemodynamics were studied by a swan-ganz catheter with a fast response thermistor for right ventrieular ejection fraction (rvef) measurement in 20 mechanically ventilated arf patients (lis = 2.6 ~-0.45 sd). according to the response to peep 15, two groups of patients were defined: group a (9 pts.) with unchanged or increased rv end diastolic volume index (rvedvi) and group b (h pts) with decreased rvedvi. results: in the whole sample cardiac index (ci) and stroke index (sj) decreased at all levels of peep, while rvedvi , rv end systolic volume index (rvesvi) and rvef remained anchange d. at zeep the hemodynamic parameters of the two groups did not differ. in group a, ci decreased at peep5, rvef decreased at peep10 (~0.8%)~ rvesvi increased only at peep15 (+21.5%) and rvedv[ reded unchanged. in group b, ci and rvedvi started to decrease at peep5, 'rvesvi decreased only at peep15 (-21.4%), anf rvef was unchanged. individual behaviors of the hemodynamic parameters at the 4 levels& peep were studied. rvedvi and ci were significantly correlated in 10 out of:l 1 patients in group b, and in no patient of group a. on the contrary, mpap and rvesvi were significantly correlated in 5 out of 9 patients in group a, and in no patient of group b. the slope of the relationship between rvedvi and rv stroke work index (rvswi) expresses rv myocardial performance. this relationship was significant (no change in rv contractitity)in 8 patients of group b and in 2 patients of group a. in some patients of group a, increments of peep shifted the rvswi/rvedvi ratio rightward inthe plot (rv function decrease). conclusions: in arf patients peep causes more often a preload decrease with unclmnged rv conctraetility. on the contrary, the finding of increased rv volumes during the application of peep is related to a decrease in rv myocardial performance. thus, these data suggest that application of peep might be considered as a stress test to assess rv function. right introduction: after heart transplant (ht), the right ventricle can be subject to an acute pressure overload, especially in cases where there is a preexisting severe pulmonary hypertension. this provokes right ventricular failure and, occasionally, circulatory collapse in intensive care unit. desire the advances that have been made in systems for preserving the donor heart and in post-surgical management, we have failed in our attempts to totally avoid this problem. the right ventricular function, although it usually remains within tolerable limits in these patients during the post surgery period, represents a factor which limits the results achievable in clinical transplant programmes. objectives: to determine the maximum tolerance of the right ventricle (mxtrv) when faced with acute pressure overload. to study the function of both ventricles of the healthy heart (donor) when faced with different degrees of pulmonary hypertension. to detect possible interactions between the ventricles in the absence of the pericardium to approximate the experimental model to the clinical model of ht. materials and methods: the pulmonary artery is progressively constrained in an experimental model until biventricniar failure is detected. this experiment is performed in two diffferent situations: with and without pericardial integrity. results: when pericardial integrity is maintained the mxtrv faced with a pressure overload is 73.2 + 8.56 nun hg. when this pressure is exceeded there is a circulatory collapse with a sharp fall in the cardiac output and in the aortic pressure. however, when pericardectomy is performed (model similar to ht), only 52 • 6.71 nun hg is tolerated (p < 0.01). conclusions: with the pericardium open, as in heart transplant, the maximum pressure that the right ventricle can support is significantly less than with the pericardium closed. the pericardium has a positive effect in protecting the systolic ventricular interaction. it is, therefore, advisable to close the pericardium after heart transplant. jb prrez-bernal, a ordrfiez, a. heroandez, jm borrego, map camacho, c cruz, mac s~nchez, j monterrubio, c garcia, e. gonz~lez. hospital uulversitario " virgen del rocio ". sevilla. espaiqa. introduction: nowadays cardiomyoplasty isused incases of cardiac insufficiency as an alternative to cardiac transplant. after surgery the patients show a noteable improvement with the aid of this "biological circulatory assistance". some researchers suspect that the improvement could also be due to the formation of new blood vessels from the muscle that wraps the heart, nourishing the ischemic myocardium. objectives: our cardiovascular research group has proposed as an objective, the detection of any possible myocardial neovascularization through the muscle used for cardiomyoplasty. in the case that there are new blood vessels to the diseased myocardium through the wide dorsal muscle in which it is wrapped and which aids it mechanically, it would be possible to confirm the worldng hypothesis that cardiomyoplasty not only improves the cardiocirculatory funcfinn mechanically but also by facilitating a better blood flow to the ischemic myocardium. materials and methods: the cardiomyoplasty technique is described using an experimental model of myocardial ischemia. the vascular cast is achieved by injecting methacrylate simulataneously into both the coronary tree and the wide dorsal muscle, in five experiments the connections between the coronary vascular system and the vascular structure of the wide dorsal muscle are demonstrated, conclusions: we have demonstrated that cardiomyoplasty, as well as improving ventricular function, favours the revascularization of the myocardium. cardiomyoplasty could be indicated for cases of ischemic cardiopathy in patients in whom it is not possible to perform direct revacularization using conventional methods. a the therapeutic cardiological manouevres necessary in cases of ischeima reperfusion have increased considerably: fibrinolysis, transluminal angioplasty, coronary revascnlarization surgery and cardiac transplant. the appearance of a specific pathology ht acute reperfusion has been related to free oxygen radicals (for) generated by oxidative damage. objectives: to evaluate the appearance of for during a conti-olled process of ischemia-reperfusion in an experimental biological model and compare it with that in clinical cases. materials and methods: transitory cardiac ischemia was performed in five rabbits by reversible surgical ligation of the descending anterior coronary artery. after 15 minutes coronary reperfusion was performed. blood samples were taken in the basal situation, at the end of ischemia and at 5, 10 and 15 minutes after the start of reperfusion. malondialdehyde (mda) was measured to evaluate the degree of lipid peroxidation (oxidative damage to the membrane). in ten patients undergoing conventional cardiac surgery the production of for was measured after aortic clamping. results: we observed that after 5 minutes of reperfusion there was a highly significant increase (p < 0.001) in the mda values (mean =2.00 /zmols/l). these returned to basal levels after 10 and 15 minutes of reperfusion. conclusions: an "explosion" of oxygen free radicals was detected very quicldy, just a few minutes after post-ischemia reperfusion. thus, if antioxidant agents are to be used to reduce the toxic effects of the for, these will ordy have a therapeutic effect if they are administered in the early phases of reperfusion. introduction: aortic connterpulsation is a ventricular assistance widely used in intensive care units in patients with cardiogenic shock as a provisional ventricular assistance. paraaortic or external aortic counterpnlsation is been investigated as a definitive veutricular assistance in those cases of terminal congestive heart failure and when heart transplantation is counterindicated. aims: to assess the haemodynamic effects of an aortomyoplasty in a biological model of congestive heart failure. material and method: as specimens, we used 10 "large white" pigs. mean weight was 22 kg. after the administration of conventional anaesthesia, dissection of the ladssimns dorsi muscle was performed on the samples at the laboratory of experimental surgery of our hospital. then we performed a thoracotomy at the level of the fourth intercostal space to reach the thoracic aorta. the aorta is dissecated 7 centimetres from the exit of the subclavia and it is wrapped by the dissecated muscle. a cardiomyostimulator is provided in order to allow the synchronization between the diastole and the muscle contraction. the model of heart failure was provoked using verapamil plus propanolol i.v.. results: a significant increase of the aortic diastolic pressures and a significant decrease of the left ventricle telediastolic pressures were observed. this improvement in the parameters (dpti/tti) implies an increase of the coronary perfusion in a model of heart failure. conclusions: using the external aortic counterpulsation, the aortomyoplasty improves the coronary perfnsion and the heart efficiency in patients with heart failure in whom no conventional therapeutic action is possible. the permanent character of the paraaortic counterpulsation is it main advantage. the appearance of specific pathologies as a resuk of myocardial reperfasion has been related to the oxidative damage secondary to the release of oxygen derived free radicals (ofr). during the myocardial ischemia induced during heart surgery with extraeorporeal circulation, severalsubproducts of the oxygen are produced that shall cause toxic effects after the reperfusion which could be counteracted by the physiological antioxidant systems and/or provided by the medication. aims: to asses the ofr during heart surgery. to check whether an antioxidant treatment administered in the preoperative period make decrease the levels of ofr before and after the myocardial reperfusion and to verify whether its administration have any beneficial effect on the intra and extraoperative management. material and method: the study comprehends 20 patients studied as two groups of 10 individuals each (a and b). all patients underwent conventional heart surgery of valvniar substitmion or myocardial revaseularization. group a patients were administered 400 rag/8 hours of vitamin e (tocopherol acetate) 72 hours prior to the intervention as antioxidant treatment. group b patient were not administered vitamin e. we assessed the quantity of malondialdehido (mda) to assess the degree of lipidic peroxidation or oxidative damage of the membrane during the myocardial ischemia and 15 nm after the reperfusion. conclusion: patients who underwent heart surgery and were treated with tecopherol acetate in the preoperative period presented levels of rlo significantly lower than those who were not administered the drug, both during the intraoperative period and after myocardial reperfusion. we detected in these patients a need for antiarrhythmicals and pharmacoiogical support with catecholaminas, although not significant, both in the introaperative period and the immediate postoperative period. recommendations for the treatment of pulmonary embolism (pe) in the presence of right atrial thrombus (at) are conflicting. because of a significantly higher mortality rate due to fulminam or recurrent pe, there is a necessity to treat patients (pts) with mobile type a thrombi compared to pts with adherent type b thrombi. therapeutic strategies include anticoagulation, thrombolysis (t) or surgical thrombembolectomy. combination thrombolysis (cot), predominantly used for the treatment of acute myocardial infarction proved to prevent reocclusion of the infarct related artery at a comparable rate of hemorrhagia. benefit has been related to the alteration of hemostatic proteins by non-fibrinspecific thrombolytic s. administration of cot in pe has been performed sporadically. in the present case, a 55-year old male with no history of prior cardiovascular disease developed acute dyspnea which was related to pe in the presence of deep vein thrombosis of the left femoral vein. therapeutic anticoagulation was installed for a couple of days until there were several bouts of deterioration. biplane transesophageal echocardiography (tee) was performed and revealed a large, wormlike, hypermobile thrombus within the right atrium. computer tomography (ct) of the chest detected a saddle embolus in the bifurcation of the pulmonary tmnk almost occluding the entire left pulmonary artery (pa) and parts of the right pat consisted of 100 mg frontloaded rt-pa and the subsequent continuous administration of urokinase in a dosis of 200.000 u/hr for 24 hrs followed by therapeutic anticoagulation. symptoms, blood gases and ecg improved steadily during infusion, no adverse effects, i.e. minor or major hemorragia were registered. follow-up ct promptly after termination of t showed almost complete resolution of the saddle embelus, whereas tee showed complete dissolution of the at. ' finally, the patient was switched to oral anticoagulants and had an uneventful clinical course until he was discharged. conclusion: in the present case, cot was effective for the treatment of a complicated pe without any adverse effect. introduction: nowadays we can assist hearts with problems of insufficiency by techniques other than transplant. many researchers believe that the best way of assisting insufficient heart muscle is with another muscle from the patient. this technique of ventficular assistance is known as cardiomyoplasty. we describe the surgical technique of cardiomyoplasty using a biological model. the transformed skeletal muscle is transferred to the thoracic cavity where it wraps the heart and assists it. the choice and preparation of this muscle is currently under investigation. our group has focussed on the development of protocols for electrical stimulation to transform a skeletal muscle into a muscle which resists fatigue and which is functionally similar to the myocardium. we detect the optimum time at which this muscle has been transformed, by studying the transmembrane action potentials using intracellular electrodes. when the action potential of the trained muscle behaves like cardiac muscle we consider it ready for cardiomyoplasty. conclusions: cardiomyoplasty is an alternative surgical technique to cardiac transplant, which has a great future in the treatment of patients with advanced cardiac insufficiency. we describe methodology which, by intracellular techniques, allows selection of the optimum moment of transformation of a skeletal muscle trained to perform,like cardiac muscle, without suffering fatigue. purulent pericarditis is a rare disease. its treatment associate systemic antibiotics and drainage of the pericardium. we report a ease of purulent constrictive pericarditis in which intraperieardial fibrinolysis was use. a 38 years old patient admitted in our icu for a constrictive pericarditis as a complication of a purulent pericarditis diagnosed seventeen days before. he had also an aehalasia and the o'esogastric endoscopy had found an oesophageal neoplasm. a fistula was not seen, indeed pericardial of flora was the same that oropharyngeal. hemodynamie and echographic study had confirmed a constrictive pericarditis. because of the poor state of the patient an intraperieardial fibrinolysis was prescribed (250.000 ui of streptokinase on days 23, 25, 27, 29). fluid drainage was improved and cardiac output was also improved (day 23 : 2.53 1.min "i, day 27 : 3.58 l.min'l). no change ofhemostasis was noted. a pericardeetomy and an oesophagectomy were performed after 37 days of evolution. eighteen months latter the patient was still alive. intraperieardial fibrinolysis seems an interesting therapeutic way if rapidly prescribed in the purulent pericarditis course. the decrease in the systolic pressure following a mechanical breath, termed ddown (delta down), has been shown to be a sensitive indicator of preload (1,2) . however, the clinical use of this method necessitates the introduction of a short apnea. we have therefore developed a respiratory systolic variation test (rsvt) which obviates the need for apnea. the test is based on the delivery of 4 successive breaths of increasing magnitude (5, 10, 15, and 20 ml/kg). a line of best fit is drawn between the 4 minimal systolic values (one after each breath) and the downslope calculated as the decrease in blond pressure for each increase in airway pressure ( mmhg / cmh20). in 14 mechanically ventilated patients the rsvt was performed during controlled mechanical ventilation under sedation. the test was repeated after the administration of 7 ml/kg of plasma expander. the initial mean downslope of the rsvt was -.40 + .40 mmhg/cmh20. following volume loading the downslope decreased to -.23 + .44 (ns). at the same time, cardiac output (co) increased by .96 + 1.2 l/min (p<.02), end-diastolic area (determined by tee) increased from 18.5 + 6.9 to 20.3 + 7.1 cm2 (ns), and paop increased from 12 + 8 to 17 + 9 mmhg ( p < .001). the preinfusion downslope value of the rsvt correlated significantly with the increase in the co (r = .81) and the eda (r = .70). methods: an expert system has been constructed running on a multimedia computer with the two objectives in mind, viz training of inexperienced staff, and protocol guidance with treatment regimes for all staff. the system is based on experience gained from two previous systems, the one for dealing with acid-base and electrolyte problems in icu patients; the second for stabilisation of patients with heart rate and blood pressure abnormalities. the training section takes the form of a stage-by-stage account of the insertion of the pac and displays of correct waveforms, coupled with indications of possible incorrect placements, and guidance when failing to achieve the perfect positioning. the treatment protocol section extends an existing protocol for correcting abnormalities in heart-rate and blood-pressure, and now takes account of all the indices as measured by the pac. the system will suggest treatment to correct such things as abnormal wedge pressures concomitant with parameter values throughout the rest of the cardiovascular system. the type of patient eg post-operative cardiothoracic or i. c. u. trauma, will be taken into account when recognising abnormal parameter values and when prescribing treatment. results: a working system which will be improved by the finetuning being carried out. the results and lessons learnt will be presented at the conference. method: septic shock was defined as severe sepsis with either persistent hypotension (mean arterial pressure; map < 70 mmhg) or the requirement for a noradrenaline (na) infusion ~ 0.1 9g/kg/ rain with a map --< 90 mmhg. cardiovascular support was limited to na + dobutamine (db). 546c88 was given for up to 8 h at a fixed dose-rate of either 1, 2.5, 5, 10 or 20 mg/kg/h iv. during 546c88 infusion, na was to be reduced and if possible withdrawn, whilst maintaining map above 70 mmhg and the cardiac index (ci) as clinically appropriate. assessments were made at baseline (t = 0); at i h from the start of treatment (t -1); and at the end of treatment (t -8) with 546c88. conclusions: 546c88 does not appear to increase mpap or worsen pulmonary gas exchange in patients with septic shock, when given by infusion for up to 8 h. 546c88 is a novel vasoactive agent for the treatment of septic shock which will now he evaluated in a randomised, placebo-controlled safety and efficacy study. objectives : to compare cardiac output (q) data obtained for thermal indicators in pulmonary artery (qtpa) and aorta (qtao) and for the stable isotope 2hzo in aorta (q2v~2o) with indocyanine green (icg) in aorta (qicg) as reference. methods : an indicator solution of ice cold h20 (9.4 ml), 2h20 (0.6 ml) and icg (10 mg) was injected as bolus via the injection port of a swan-ganz catheter. qlco and qzmo was measured using a dual optical system (penn lab instruments, philadephia, pa, usa). qtpa and qtao was measured using a in contrast to the recoveries of thermal indicator in pa and 2h20 in aorta the :~covery of thermal indicator in aorta was significantly increased in group ii (n= 18 boluses) over group i (n=18 boluses) (1.3<-0.3 vs. 1.0+-0.1, p=0.04). conclusions: the "overrecovery" of thermal indicator in aorta is in agreement with " biscks deconvolution study (i) and results in erroneous values for q. the most pausible explanation is the distortion of the thermal curve caused by the slow response time of the thermal detection instrument as shown by ganz (2) objectives: to compare data obtained with the double indicator dilution method using indocyanine green (icg) and the stable isotope 2h20 for the estimation of extravascular lung water (evlw2hzo) to gravimetriu lungwater data (evlwg~). methods: an indicator solution oflcg (10 rag) and 2h20 (0.6 ml) was injected as bolus via the injection port of a swan-ganz catheter. dilution curves for icg and zh20 was registered in aorta with a dual optical system (penn lab instruments, philadephia, pa, usa). cardiac output and mean tranist time was measured for both tracers (qico, tlco, q2n2o, t202o) (1). data analysis: evlwg~av was reference for evlwzhzo calculated as q2hzo times the difference in mean transit time between t2nzo and rico (atm2n). as reference for atzn2o evlwg~,v was divided by q~cg to obtain atg~,. a reference distribution volume for 2h20 was calculated as the sum of central blood volume and evlwg=v. 54 boluses were administrated in a group (i) of 6 anaesthetized pulmonary healthy sheep while q was altered. another 18 boluses were administrated in a group (ii) of 6 anaesthetized sheep with stable oleic acid induced pulmonary oedema. evlwg~v measurement was performed postmortem. 2 results: for 72 boluses h20 parameters were not significantly different from their respective reference parameter: at2vao 5.3+_3.5 s vs. atg~, 5.4+3.2 s, evlwzh2o 332-+195 ml vs. evlwg~,~ 338+169 ml. in group i the ratio between 2hzo parameters and respective reference parameters (n=54) were independent of qlco from 1.4 to 7.1 l/min. obiectives: to assess the thermo dye method using indocyanine green (icg) and thermal indicator for the estimation of lung water (evlwt). methods: ice cold indicator solution of icg (10 mg) in water (10 ml the aim of the study was to assess left and right ventricular function in the early postoperative period after orthotopic heart transplantation to elaborate therapeutic approaches of heart function abnormalities correction. mathefial and methods. haemodynamic monitoring data of twenty one patients ( 19 men, 2 women ) age from 18 to 56 were studied. cardiac output, pulmonary artery, right atrium and pulmonary wedged pressure were measured with swan-gans catheter. central haemodynamic indices were calculated with the help of computer-based monitoring system. relations of ventricular stroke work index to it's end-diastolic pressure were used for ventficular function assessment. results. in most cases right ventricular disfunction was the main problem. isolated fight ventficular failure with high pulmonary vascular resistance (pvr) was observed in 24% (5pts), without high pvr-in 43% opts) and with left ventricular failure-in 33% (7pts). one of the most important reasons for fight ventricular failure was the time of heart ischemia more than 90min, which is of great importance in the ease of distance harvesting. the most effective treatment for cardiac failure was combination of dobutamine with i8oprotherenol, atrial pacing and vasodilatators in case of right ventfieular disfunction. all cases with isolated right ventricular failure were treated sucsessfully. biventricular heart failure was a sighn of bad prognosis and the reason of death in 2 cases. conclusion. right ventfieular disfunetion is the main problem during transplanted heart adaptation in the early postoperative period. optimal therapeutic management of cardiac disfunction includes infusion of dobutamine in combination with isoprotherenol, atrial pacing and vasodilatators. cardiology-department of clinical centre-kragujevac institution for occupational health "zastava"-kragujevac, sr yugoslavia the aim of the investigate is analisis five years survives patients with a.i.m.in dependence of locality and risk-factors. we ana~sed-39~-pat~e~ts (273 males and 118 woman), average 59,8 years. for statistic evaluation we used life-table slstem in oder to estimate prognostic determinants. patients with respkatory muscle paralysis may benefit from respiratory assistance by abdomino-diaphragmatie pneumatic belt. we used a non invasive technique, m-mode sonography, to assess the effect of this device on diaphragmatic excursion. we measured the amplitude of right diaphragm motion in seven patients with duehenne muscular dysl~ophy in supine position with various thoracic posture (0 ~ 45 ~ 75~ without and during pneumatic belt respiratory assistance. without respiratory assistance, the thoracic posture had no significant consequence on the amplitude of diapttragm motion, either in quiet or deep breathing. the pneumatic belt increased the diaphragm motion amplitude from 7.1 +__ 3.6 mm to 17.71 +_ 5.5 ram (p = 0.009) at 45 ~ tilt angle, and from 8.4 + 3.8 mm to 19.3 + 5.8 mm (p = 0.009) at 75" tilt angle. the tidal volume increased from 211 + 78 to 373 + 99 rut a145* tilt angle, and from 229 + 78 to 447 + 143 ml at 75* tilt angle (p = 0.009). two patients could not bear the horizontal position (0' tilt). in the five other patients, the pneumatic belt increased but not significantly the amplitude of diaphragm motion (9.2 + 4.9 mm to 15.5 + 7.3 ram). after an overnight respiratory assistance, pao2 increased from 66.4 +_. 8.7 to 73 + 10.1 mmhg (17 = 0.015), sao2 increased from 91.1 + 2.5 % to 91.9 +_. 3 % (p = 0.015), and paco2 decreased from 52 + 6.4 to 46.4 +_. 4 mmhg (p = 0.015) according to the ventilatory pattern result, m-mode sonography allows to measure non invasively the improvement of diaphragm kinetics obtained by pneumatic belt respiratory assistance, and may be helpful for its adjustment. objective: to study the effect of flow triggering (flow sensitivity 1 and 5 l/min) vs pressure triggering (-lcmh20) on inspiratory effort during pressure support ventilation (psv) and assited/controlled mode (a/c) in 8 stable copd patients non-invasively ventilated with a full face mask. methods: the patients were studied during randomized 15 min. runs using a bird 8400 st ventilator at zero peep (zeep). trigger values for pressure (-lcmh20) and flow (1 l/rain) were the lowest allowed by this ventilator. the transdiaphragmatic pressure time product per breath (ptpdi), dynamic intrinsic peep (peepi,dyn), maximal airway pressure drop during inspiration (apaw) andl ventilatory variables (ti,te,ttot,rr,vt and minute ventilation) were measured. results: no major problems due to airleaks or to auto-triggeriffg phenomena were observed in the patients, so that all of them were able to perform all the protocol runs. minute ventilation and respiratory pattern were not different using the two triggering systems. the ptpdi was significantly higher during both psv (10.6+6.8 cmh:0 x sec) and a/c (10.1+2.5) with pressure triggering, as respect to psv (8.9+7.5, p<0.02) and a/c (5.8+4.4, p<0.001) with flow triggering (1 l!m). no differences were observed between 1 and 5 l/min flow triggers. apaw was also significantly larger during pressure triggering; peepi,dyn was reduced during flow triggering being 5.5+1.5 cmh20 (psv flow trigger) vs 8.1+1.5 (psv pressure trigger) and 4.4+_1.3 (a/c flow trigger) vs'f~5+l (atc pressure trigger). conclusions: in stable copd patients non-invasively ventilated, flow triggering reduces the respiratory effort during both psv and aic mode as compared to pressure triggering. this may be partly due to a decrease in peepi,dyn using a flow-by system. objective. cardiac output is higher during alternating ventilation (av) (i.e. differential ventilation of the lungs with a phase shift of half a ventilatory cycle) than during synchronous ventilation (sv) of both lungs 1 . we verified the hypothesis that the higher cardiac output depended on a lower central venous pressure and intrathoracic pressure, due to a lower mean lung volume, which we attributed to part of the expansion of the inflated lung at the expense of the expiring, opposite lung 2. we studied this interaction between the lungs during one-sided inflation, which we called cross-talk. method. in 6 anaesthetized and paralyzed piglets we applied short periods (30 s) of one-sided ventilation (10 breaths per rain, bpm), while the other lung was open to the ambient air. the air flow into the non-ventilated lung during expiration of the ventilated lung was integrated to volume. we studied 1-to-r and r-to-i cross-talk at ventilatory rates of 10, 15 and 20 bpm. the amount of cross-talk was the volume displacement in the non-ventilated lung. results. during 10 bpm the r-to-i crosstalk was 23 _+ 4.7 % (mean +__ sd) of the tidal volume to the right lung and the 1-to-r crosstalk 31 _ 6.3 % of the left tidal volume. both values increased at 20 bpm to 30 _ 4.1% (p < 0.05) and 39 _ 7.7 % (p < 0.01) respectively. the values at 15 bpm were in between., conclusion. we concluded that the lower mean lung volume and lower thoracic expansion during av compared to sv depends on partial expansion of the inflated lung into the non-inflated lung, resulting in a lower mean intrathoracic pressure as the main reason for the higher cardiac output during av. obiective: natural surfactant given for rds in premature infants leads to a rapid improvement in oxygenation, but lung compliance did not improve in most studies. however, acute effects on lung mechanics during and immediately after surfactant administration have not been studied before. methods: a total of 13 administrations of bovine surfactant in recommended doses was given via a small catheter into the distal endotracheal tube either as a bolus (n = 8) or as a slow infusion (n = 5) in 10 infants with established rds. static compliance (c), resistance (r) and time constant (tc = cxr) of the lung were measured every 3 minutes with a lung function cart (sensormedics 2600) without interrupting ventilation. 3 infants receiving synthetic surfactant were studied as controls. results: after surfactant as a bolus or during infusion c first decreased but then increased, whereas r increased immediately with great fluctuations but did not return to baseline. this pattern was more pronounced in infusion than in bolus administration. change of c and r varied greatly in the individual case, maximum c was > 400 %, maximum r > 700 % of baseline value. retreatment was followed by an increase in r in all 3 patients, but c increased only in the one who was responder. patients receiving synthetic surfactant had no change of c or r and were non-responders. ob~i31ctives= acute lung injury (ali} sometimes induces severe hypoxernla which may be refractory to conventional modes of mechanical ventilation (mv). the elm of this study was to observe some cardio-pulmonary effects of an alternative method of ventilatory management of severe ali. five patients with severe ali (murray scores >3) requiring mv were studied. protocol inclusion was considered when a control-mode of mv (with a pzo~=l.0 and a peep level <15 cme=o} was not able to get either a p.ojf=o= ratio >55 or a s.o= >85%. patients were sedated, paralyzed, and a ventilator (serve 900c) was used for pressuz'e-control ventilation (pcv). fio= was maintained at 1.0 and peep removed. continuous gas flow (250• ml/kg] was humidified and jet delivered through a tube (7 ram id, 18 ml capacity, 0.09 ml/cm h=o compllancel ended in a nozzle (0.8 mm is) attached to the endotracheal tube connector. a thermodilution flcw-dlrected catheter was inserted in pulmonary artery. following variables were recorded 15 minutes before and after protocol started: tidal volume (vt), minute ventilation (vz), intratracheal pressures (p~w), wedge pulmonary artery pressure (wp), central venous pressure (cvp), mean arterial pressure (map), cardiac index (ci), arterial and mixed venous oxyhemoglobin saturation (sao=, svoa) , oxygen delivery (do~) , oxygen consumption (vo2) , intrapulmonary shunting (q./qt) , and oxygen extraction ratio (ero). this observation suggests that hfpv could allow to ventilate at lower fin2 and improve blood oxygenation during the acute phase after inhalation injury reducing toxicity risk related to high fin2. further studies are necessary to confima these results and evaluate the possible implications on mortality alter smoke inhalation and for other icu pts. objectives: to design a system for volume controlled high frequency ventilation (hfv) and to estimate the dependence of the tidal volume (vt) on frequency (f) in normocapnic ventilation in rats at frequencies 2 -25 hz. methods: a new system for volume controlled hfv was devised consisting of the generator of the constant flow during inspirium and the constant pressure during expirium. the ventilator allows ventilation at frequencies 2 -25 hz with the relative inspiratory time (ti) 0.2 -0.8. the airway pressure was measured at the proximal port of tracheostomic cannula , at the same site inspiratory and expiratory flow was measured using modified lilly-type of pressure-differential flow sensor. non-linearity of flow sensor was compensated on line by derived equation based on calibration at static and dynamic conditions. flow and pressure data were evaluated on line using original software. value of the positive end expiratory pressure (peep) was serve-regulated by analogous feed-back. in animal experiments white wistar rats (400-430 g) narcotized with ketamine/xylazine with cannulated carotid and femoral arteries were kept at the rectal temperature 37~ the arterial pressure was monitored. after traeheotomy the metal cannula (2mm [.d.) was inserted, animals were curarized and ventilated at the following condition: peep = 0.1 kpa, ti = 0.5. the dead space of ventilator including canula was 0.45 ml. the initial frequency was 2hz and 10 rain after each change of the ventitatory regimen the blood gases analysis was performed. the frequency was changed according to the following schedule : 2 hz-->4 hz-->8 hz-->4 hz-->16 hz-->4 hz--~25 hz-->4 hz. vt for each frequency was regulated to maintain normocapnie ventilation with arterial pco2 = 40 + 2 mm hg. the arterial po2 was always above 70 mm hg. results: for normocapnie ventilation in rats the following tidal volumes vt [ ml/kg] were found : vt1 = 5.91 --+ 0.30 ml/kg for ft = 2 hz, vt 2 = 4.31 +0.12 mukg for fz =4 hz, vt3 =3.30 +_0.27 ml/kg forf3 = 8 hz, vm4=2.61+0.08ml/kg forf4=16hz andvmt= 2.18 + 0.13 mukg for fs = 25 hz (presented as mean values _+ s.d., n = 6 ). the regression analysis using the mean values resulted in the equation for normocapnic vt in rats in our experiments : vtn = 37. 5 * f-e.30 . conclusions: the described system allowing ventilation in a wide frequency range 2 -25 hz with accurate measurements of airway pressures and vt might be useful for optimisation of artificial ventilation in new-barns with different lung pathologies. supported by grants iga mz cr nr 1448-3 and gacr nr 305. s122 intensive care unit. university. hospital of south manchester, uk. methods: measurements were conducted on 6 ventilated patients (puritan bennett 7200ac with metabolic monitor pb 7250 set to measure end tidal co2). all measurements were repeated with the patient stabilised at 5cm. 10cm and 15cm peep. inclusion criteria were: 1) haemedynamic stab(l( .ty for 1 hr; 2) pulmonad" anon" flotation catheter in situ: 3) volume control ventilation with plateau of 0.5s: 4) fio2~ > 11.6 to maintain pao~. > 10 kpa with 5em peep: 5) qs/ot > 20%; 6) pao2/fio2 ratio <150. measured variab!es included: r162 minute volume: plateau ainvay pressure: applied and intrinsic peep: fractional end tidal co2; arterial and mixed venous blood gases and hacmod).ttamic variables. results: statistical analysis was performed using repeated measures anova. significant decreases in cardiac index (ch p<0.01), compliance (p<t).05) and o,'gcn deliver, index (do2, p<0.02) occurred with increasing peep (table 1) . no other variable showed any significant change. methods : all consecutive patients orally intubated in the micu between january to april 1995 were studied. etts were positioned according to the reference marks mentioned above (measurements from upper incisors). the position of the ett tip in relation to the carina was measured on the chest radiograph done with the patient's head in neutral position. results : a total of 59 men and 46 women were studied (n= 105). the mean distance of ett tip to carina was 4.1 cm. using the reference markings, 29 cases (27.6%) had the ett tip < 3 cm from the carina and 5 cases (4.8%) > 7 cm. one case resulted in an endobronchial intubation. the mean height of all patients were 167 cm (153-187) for males and 155 cm (140-170) for females. of the patients with ett tip < 3 cm from carina, the mean height was 163 cm and 151 cm respectively. ~2onclusion : adopting the above quoted reference marks did not result in ideal positioning of the ett in a significant proportion of cases (32.4%). we postulate that [s because our asian population is generally shorter than those in previous studies. objectives: to measure the changes of pulmonary mechanics before and after tracheostomy in patients with prolonged mechanical ventilation and to determine factors that predict the outcome of liberation from mechanical ventilation. design: prospective. setting: respiratory intensive care unit (ricu) in a tertiary hospital. patients: twenty patients with chronic lung disease requiring long-term mechanical ventilation. tracheostomy is indicated for further care. intervention: tracheostomy. measurements and results: pulmonary mechanics including respiratory rate (rr), tidal volume (vt), peak inspiratory pressure (pip), intrinsic positive end ex~ piratory pressure (peepi), lung compliance (cld), mean airway resistance (rawm), work of breathing (wob), pressure time product (ptp) by bicore cp-100 pulmonary monitor were recorded 24 hours before and after tracheotomy. ventilator setting parameters remained the same during surgical intervention and were also recorded for comparison. generally, the mechanics including pir wob, raw~x and ptp showed improvment after tracheostomy. but only pip was significantly reduced (pre 33.4 _+ 11.8 to post 28.6 _+ 9.2, p < 0.05). changes of wobp showed significant correlation with pre-operation rr, minute volume (mv), wobp, and peep(. changes of raw m were also significantly correlated with pre-operation peep, vt, and raw m. the patients were divided into two groups according to their outcome after two week follow-up. group 1 included eight patients who were completely weaned from ventilator; group 2 included twelve patients who still remained ventilator-dependent or were mortality. there was no difference in age, duration of mechanical ventilation, pro, post or changes of several lung mechanics between the groups of patients. pre-tracheostomy peep i and cld showed significant difference between these two groups (1.1 _+ 1.6 vs 2.7 + 1.4 in peepi; 47.3 _+ 36.9 vs 28.8 _+ 16.5 in cld, p < 0.05). pre-tracheostomy ventilator setting in mode of assist/control also showed significant higher percentage in group 2 (3%5 % in group 1 vs 66.6 % in group 2). conclusion: in prolonged mechanical ventilation patients with chronic lung disease, tracheostomy will significantly improve pip and slightly reduce wobp, raw m and ptr patients who used pressure support mode before tracheostomy had better underlying lung conditions (lower lung compliance and auto-peep) will have better chance to wean from mechanical ventilation. forty-eight infants with congenital diaphragmatic hernia presenting within the first 6 hours of life, who underwent surgical rapair,were analysed prospectively in order to produce a reliable inde x of severity of disease that would reliably predict eventual outcome. there were 25 survivors and 23 deaths in this series (mortality 48%).using arterialpco 2 values measured 2 hours after surgical repairand correlating them with an index of mechanical ventilation,we have been able to clearly define two groups of diaphragmatic hernia based on their response to hyperventilation. the first group, with co 2 retention and severe preductal shunting,was unresponsive to hyperventilation with high rates and pressures the mortality was 90%. the second group responded well to hyperventilation and demonstrated reversable ductal shunting only. survival in this group was 97%. arterial co 2 accurately reflects the degree of lung development in this disease and separates those patients with severe pulmonary hypoplasia where the outcome is invariably fatal, from those with a well developed contralateral lung where there is excellent potential for survival. respiratory failure unit, dpt medicine, univ. thessaloniki, thessaloniki, greece the variability of arterial blood gases (po2, pc02) and the ph (abg) was examined in 20 stable icu patients, few hours before a successful weaning from the ventilator. all patients were lightly sedated and the ventgatory conti~ons were pressure support (ps) for 9 and ps plus intermitted mantatory ventilation in ii. [n each patient, 6 speciments of abg were measured at 10 min intervals during a 1-11 study period. at the same time with abg the arterial blood pressure (bp), the heart rate (cf), the tidal volume (tv) and the respiratory rate (n r were measured. for all the patients, the mean coefficient of variation (c) was 3.45 percent for po2, 3.53 percent for pco2 and 2.27 percent for hco3. the average sd for ph was 0.008, the corresponding c for systolic bp, diastolic bp, cf, tv, rf were 4.35, 5.21, 3.68, 2.51, 4.18 percent. we conclude that the spontaneous variability of arterial blood gases in icu patients is not substantial ~hen they have stable the heamodynamic and the ventilatory parameters. deptx?fa'aaesthesioiogy and reanimation, rhe sechenov medical academy, moscow, russia objective: ~he prevention and treatment of hypoxia in the critical patiems. methods: i~fusions of perphtoran -a blood substitute with gas-transporting fimclion based on perphtorhydrocarbon -in 496 patients with acute hypovolemia, microcirculatory distnrbance~ tissue gas exchange and metabolism; pulmonary iavage in 104; iongterm extrapulmonary oxigenation with tleoroearboa oxygenator in combination whb ~trafiltra!ion, hemosorption and hemodialysis -in 73 patients. results: pe~htoran increases blood volume, co,sv, decreases svr, improves capillary blood flow, increases the blood oxygen capacity, tissue oxygen tension, 102 del, vo2 by improving the rheologic properties of blood and plasma, normalizes 02 ext., prevents and eliminates fat embolisation and ards. decreases the need for blood transfusions and infusions of plasma expanders by 1.3-1.4 limes. alveolar venti!ation-perfusion ratio remains unchanged with its increased effective utilization. there was no surfactant destruction during lavage. extrapulmonary oxygenation of small volumes of venous blood eliminates venous destruction and then arterial hypoxia and increases pulmonary oxygenation. the use of lluorocarbon cxygenators during hemosorption and hcmodialysis provides the atraumatic and iongterm oxygenation of arterial blood and increases elimination of co2 which prevents the development of hypoxic complications. conclusions: perphtoran and fluorocarb~n oxygenators are effective in the correction of hypoxia in the criticat patients. objeqtives: to determine if there are differences in oxygen consumption (vo2) during weaning from mechanical ventilation (during total ventilatory support and spontaneous ventilation with cpap), and to compare different predictive parameters of weaning in predicting success of weaning. methods; prospective study in 20 critically ill patients treated with mechanical ventilation for at least 48h, who fulfilled at least 3 of 4 standard weaning criteria (vt>5ml/kg; respiratory frecuency (f) <35; pimax > 20 cm h20; pao2/fio2 > 150). baseline measurements: t, vt, p0.1, pimax, f/vt, p0.1*(f/vt), p0.1/pimax. study protocol: measurement of vo2, vco 2 (medgraphics), vt, f, ve, and arterial blood gases during total ventilatory support (cmv), and after 30 and 120 minutes of spontaneous ventilation with cpap 5 cm h20. the weaning trial was stopped, failure to wean diagnosed, and mv resumed it a patient presented significant tachypnea, tachycardia, bradycardia, cardiac rythm disturbances, hypertension, hypotension, hypoxemia or hypercapnia. results: four patients did not complete the weaning trial, 16 were extubatad, and 2 of them had to be reintubated before 48h, being considered also weaning failures. during cmv, vo2/kg was 4.07 + 0.2 ml/kg/min, and 5.09 _+ 0.4 mlo-2/kg/min after 30' on cpap 5 cm h20 (p < 0,01 ). 14 of 15 patients (93%) with 4 standard criteria were extubated, while only 2 of 5 (40%) with 3 criteria (p<0,01). next objectives: compare the extent and distribution of lung injury in dogs preinjured with oleic acid (oa) and ventilated with high tpp and adequate peep in the prone and supine position. methods: lung injury was induced with oa (0.06-0.09 ml/kg) in anesthetized, paralyzed, and intubated dogs (n=10) during volume controlled ventilation: rate=12/min, peep=5 cmh20, ti/ttot=0.3, fio2=0.6, vt=15 ml/kg. animals were rotated during the oa infusion and the following 90 minute stabilization period to assure uniform injury. in the supine position, peep was set 1-2 cmh20 above the lower inflection point (as determined by the pressure-volume curve), and vt was set to obtain a tpp of 35 cmh20: animals were ventilated in either the prone (n=5) or supine (n=5) position for four hours. pulmonary artery occlusion pressure was maintained constant (4-6 mmhg) with saline infusion. at the end of the protocol the lungs were removed and divided by template into dependent (d) and nondependent (nd) sections for wet weight/dry weight (v~n/dw) and grading of nstologic lung injury (hli; scale 0-3). oseillatron | is a pneumatic device that generates high frequency, oscillation by means of a reciprocating system in the form of a membrane. it generates sinusoidai wave form at 2(10 to 10(10 cycles/rain. the system does not deliver gas but must be adapted to the proximal respiratory, circuit of a conventional ventilator, resulting in ci-ifo. it was developed to enhance intrapnlmona~ diffusion during mechanical ventilation and to mobilise endebronchial secretions. methods. we measured arterial blood gases and haemedynamics during a first period of conventional ventilation (cppv) followed by. two 30 rain periods of chfo (sequences : 6(10 and 901) c/rain : group l, n = 1 l: 900 and 600 c/rain : group 2, n = 8). measurements were made at the end of each period. cardiac output was measured using thermedilution method: flu2 and peep were kept unchanged throughout the study. intrinsic peep was also evaluated by, means of an occlusive valve. results. pa02 is not significantly modified during chfo at 600 or 900 c/rain. paco2 is slightly decreased at 600 c/rain (p = 0.(16). however, intrinsic peep remains unchanged. there is no sequential effect (gr. l vs gr. 2). there is no more effect of chfo for patieets who are at a flu2 higher than 0.50 (n = 9). no changes in haemodynurmcs are observed except a slight increase in central venous pressure (cvp) during ci-ifo (p < 0.ol). obiectives: to examine the effects of inspiratory muscles unloading on neuromuscular output at controlled levels of chemical stimuli. methods: the ventilatory response to co 2 was examined in ten normal subjects using rebreathing method. ventilation ~) and respiratory muscle pressure output (pmus) at the same end-tidal partial pressure of co 2 (petco~) were compared with and without combined flow and volumeproportional pressure assist in two protocols (a and b). protocol a (n = 10): two levels of assist were studied; flow assist (fa) of 2 cmh20/i/sec and volume assist (va) of 2 cmh20/i (assist 1), and fa of 2 cmh20/i/sec and va of 4 cmh20/i (assist 2). all conditions were applied randomly. v~, tidal volume (vt) and breathing frequency (f) were measured breath by breath and plotted as a function of petco~. protocol b: in 5 subjects, in addition to above measurements, esophageal (pes) and gastric (pg) pressures were measured and the time courses of transdiaphragmatic pressure (pdi) and pmus were calculated. one level of assist (assist 2) was studied in this protocol. results: in both protocols inspiratory muscle unloading did not change the f response to c%. compared to control, with assist v t response was displaced upwards; at petco2 of 55 mmhg v t was increased significantly by 0.4+0.1 i and 0.7+0.2 i in protocol a with assist 1 end 2, respectively, and by 0.5_+0.1 i in protocol b with assist 2 (p<0.05). ~/~ responses showed similar changes as vtresponses. in both protocols the slope of v~ response (s did not change significantly with unloading. at low petco~ (50 mmhg), pdi and pmus waveforms did not differ with and without assist. with unloading, at high petco2 (59 mmhg), pdi and pmus at the end of neural inspiration decreased by 18.8-+8.3% and 11.7+15.7%, respectively, from control values. neither change was significant (p>0.05). by theoretical analysis we estimated the expected changes in vt and ~/~ when the levels of assist used in both protocols were applied in the absence of : any change in neural output response to co z. the predicted response was similar to that observed, indicating that the small difference in pdi and pmus between control and unloading runs was due to intrinsic properties of respiratory muscles end respiratory system. conclusions: these results suggest that when chemical stimulus is controlled, respiratory motor output is not downregulated with unloading. the determinants of the response of the respiratory output to inspiratory flow rates (v~) were examined in awake normal subjects. subjects were connected to a volume-cycle ventilator in the assist/control mode and v~ was increased in steps from 30 to 90 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. in six subjects the effects of breathing route (nasal or mouth) and temperature and volume of inspired gas (protocol a) and in 8 subjects the effects of airway anesthesia (upper and lower airways, protocol b) on the response of respiratory output to varying v~ were studied. in protocol b, in order to calculate muscle pressure during inspiration (pmus), respiratory system mechanics were measured using the interrupter method at end-inspiration. independent of conditions studied breathing frequency increased . significantly and end-tidal concentration of c% decreased as v~ increased. the response was graded and reversible and not affected by breathing route, temperature and volume of inspired gas and airway anesthesia. with and without airway anesthesia (protocol 8) neural inspiratory and expiratory time and neural duty cycle, estimated from pmus waveform, decreased significantly as v~ increased. at all conditions studied the rate of change in airway pressure prior to triggering the ventilator tended to increase as v~ increased. the changes in timing and drive were nearly complete within the first two breaths after transition with no evidence of adaptation during a given ~/~ period. we conclude that v~ exerts an excitatory effect on respiratory output which is independent of breathing route, temperature and volume of inspirate and airway anesthesia. the response most likely is neu~'al in origin, mediated through receptors not accessible to anesthesia such as those located in chest wall or below the airway mucosa. it has been shown, in mechanically ventilated awake normal humans, that increasing inspiratory flow rate (~/~) exerts an excitatory effect on respiratory output. it is not known if this effect persists during sleep. to test this seven normal adults were studied during wakefulness and nrem sleep. subjects were connected through a nose-mask to a volume-cycled ventilator in the assist/control mode and ~/t was increased in steps (3-4 breaths each) from 30 to 70 i/min and then back to 30 i/min. v~ pattern was square, and all breaths were subject-triggered. forty-one trials during nrem sleep and 10 during wakefulness were analyzed. both during sleep and wakefulness minute ventilation increased and total breath duration (ttot) decreased significantly in a graded and reversible manner as ~' increased. these changes were complete in the first breath after v{ transition. the response was significantly less during sleep than during wakefulness (p<0.05); at 30 i/min ttot, expressed as % of that at 70 i/rain, was 110.2+_1.3% during sleep and 127.8+_3.9% during wakefulness. during wakefulness, at 30 i/min, the rate of change in airway pressure prior to triggering the ventilator, an index of respiratory drive, was 60% of that at 70 i/min (p<0.05). the corresponding value during sleep, was 86% (p>0.05). in four sleeping subjects the increase in v~ was sustained for 1.5-2 min. there was no evidence for adaptation of the response; tro t, averaged over the last three breaths, did not differ from that obtained when vj was sustained for only 3-4 breaths. we conclude that 1) vt exerts an excitatory effect on respiratory output, mediated by a reflex neural mechanism and 2) the gain of this reflex is attenuated by sleep. chest radiographs is a common complementary technique for patients in critical care units, with a low cost and easily available. however, it has certain well-known limits in diagnosis, the most important derived from the low quality of some pictures. in this paper we make a general review of some new technical approaches developed for improving the quality of the images, and so incrensing the diagnostic value of conventional radiology. we begin deaeng with the correct positioning of the patient, trough the filtering techniques, the synchronization of radiology and ventilation, and we make reference to the new computerized systems for digital image processing. conclusions: the portable radiographic system is a device that probably with maintain for many years in critical care units as a basic non-invasive diagnostic tool. but we need an increase in the efficiency of it, applying means as simple as a correct positioning of the patient, or the use of fitlers or synchronizers. thus we should improve the general standards of portable radiography. "are circular circuits safe? quantifying undelivered tidal volume in pediatrics patients". objectives: to evaluate the overall influence of internal compliance of circular circuits on delivered tidad volume (vt). methods: we studied prospectively 14 asa i pediatrics patients (2 to 10 yr. old) scheduled for elective general surgery. mechanical ventilation was supplied by an ohmeda excel 210 (circular circuit). the internal compliance of the circuit (cc)-anesthesia machine plus external circuit-was determined by the supersyringe method: corrugated dar tubes of 10 mm. id and 1.5 m. long (children < 30 kg), and a corrugated dar set of 15 mm. id and 1.5 m. long (children > 30 kg) were respectively used for ccl an cc2 values of 9.3 and 9.5 ml/cm h20. a vtof 10 mlg/kg and respiratory frequency was adjusted for an end-tidal co2 (etpco2) between 30 35 mmhg. tidal volumes (measured by spirometry) and airway pressure (paw) data were recorded every ten minutes. volumes and thorax-lung compliances were calculated as follows: (vt delivered = vtadjusted-vol compressible, being vol. compressible = co x ppeak (aw). apparent compliance (ca) = vt adjusted/pplateau(aw), and true compliance (ct) = =vt delivered/pplatean(aw)). comparative statistics were separately designed between calculated compliance data and tidal volumes on a paired sample ~test basis. results: calculated values for volumes and thorax-lung compliances were: conclusions: due to the elevated internal compliance of the circular circuit there is a remarkable dilference between adjusted and delivered vt: mean undelivered vt was 38.8 % and reached as high as 64.1%. teere is also a significative error in calculating true thorax-lung compliance: its overestimation can be as high as 69.6 %. circular circuits are considered safe and cost-saving for anesthetical practice. nevertheless we conclude that anesthetists should bearin mind vt losses when using circular circuits, due to compressible volume. tracheal stenosis is one of the most serious complications of patients submitted to prolonged endotracheal intubation, in which the decrease in inner diameter of upper airway makes it very difficult to achieve a correct ventilation. objectives: compare the results of applying high frequency jet ventilation (hfjv) to some of these patients with conventional controlled ventilation (cmv). methods: we used a prototype of high frequency jet ventilator (santiago-2) developed in our university, and we developed a tracheal tube in wich we modified the distal tip (conic tip). we applied this system to two patients which were initially ventilated in the operating room with usuai controlled mecanical ventilation (cmv) following the standards of our department, and then intubated with the special endotracheal tube and ventilated with hfjv. results: we could verify a proper ventilation of both patients with cmv and hfjv. during hfjv, the airway pressures were lower than those recorded during cmv. a lower airway pressure prevents lesions due to high pressures. conclusions: hfjv is a good method of ventilation for patients with significative stenosis of the trachea, not only during surgical procedures, but also during ventilation for long periods in critically 111 patients. the ventilatory setting is pressure support mode. the pressure level and fit2 were kept constant during h/d. arterial blood gas, wbc count, and mean bp was checked according to the schedule: 0'(immediately before h/d), 15', 30', 60', 120', 180', 240'. respiratory drive (represented by poa), tidal volume(ti) and minute ventilation(ve) were continuously recorded by pulmonary mechanics monitor (bicore cp-100). the mean value of the breaths 5 minutes before blood sampling were used to represent the ventilatory status of that period. anova test is used for comparison between groups. for poa, hierarchical cluster method is applied to divide the cases into two groups of similar change. conclusions: our data suggest that pl is very useful, non invasive and low-expensive emergenc e support for arf, expecially in the elderly with severe chronic pulmonary disease and relative controindications to eti. pl seems to be an effective alternative when it is not immediatly possible to perform etl. the multiple inert gas elimination technique (miget) can be used to assess the effects of any given mode of mechanical ventilation on the pulmonary and systemic factors determining arterial po2 and pco> however, a potential problem in mechanically ventilated patients is that the 10 l mixing box (mb-10l) placed in series in the expiratory side of the circuit of the ventilator to sample mixed expired gas may provoke substantial discrepancies between the tidal votume set in the ventilator and the effective tidal volume delivered to the patient, due to the increase in the compression volume (vc) of the circuit. the effects of the mb-10l on the v c were compared with those produced by a new 1 l mixing box (mb-1 l) specifically designed to produce adequate gas mixing and to prevent loss of the two most soluble gases (ether and acetone) used in the miget. at any given peak cycling pressure (p~ak, cm h~o), the v c (ml) provoked by the mb-10l was substantially higher (vc= 7.4*ppeak) than that provoked by the new mb-1l (vc= 1.4*ppeak). at a ppeak = 50 cm h20 ~ the v c were 377 ml (mb-10l) and 67 m{ (mb-1l), respectively (p< 0.001). in a group of 6 subjects (4m/2f, 57_+6 years), for each of six the gases used in the miget, the regression line between the mixed expired partial pressures simultaneously obtained from mb-1 l and mb-10l fell on the identity line. it is concluded that the new mb-1l allows adequate assessment of the effect of different modalities of mechanical ventilatory support on pulmonary gas exchange, with less potential for gas compression and thus hypoventilation. objectives evaluate the influence of different pressure support ventilation (psv) levels on cardiovascular and respiratory funcion in icu polytrauma patients. metbed&we studied 15 polytrauma icu patients , who were in weaning process , after long term mechanical ventilation for acute respiratory failure . mean age 52 (37-71) yrs . they all were connected to servo ventilators siemens 900c , and all were in stable condition , without sedation , inotropes or diuretics. the hemodynamic studies were done with continuous svo2, swan ganz catheter (oximetrix, abbott). they all were in spontanuous mode (spent) with 5 cm h20 cpap for at least one hour. we turned them to psv with 0 inspiratory assistance (psv 0 cm h20) and after 60 rain we applied psv 10 cm h20, and after 60 min psv 20 cm h20 . hemodynamlo and respiratory measurements were done before and after the application of insiratory assistance. the results were statistically analyzed with anova. resets . respiratory variables . no significant changes in minute volume (ve). tidal volume (vt) and mean airway pressure (mpaw) increased statistically significant (p< 0.001 ) . respiratory rate (rr) decreased significantly (p<0.01) . blood gase showed no difference . cardiovascular variables. cardiac output (co) decreased ns , heart rate (hr) had no change , central venous pressure (cvp) , mean pulmonary artery pressure (mpap) , pulmonary capillary wedge pressure (pcwp) , increased ns , oxygen delivery (do2) decreased ns, oxygen consumption (vo2) decreased ns. conclusions. psv is a very useful respiratory mode helping patients to be weaned from long term mechanical ventilation . it has beneficial effects on respiratory function and oxygen consumption without affecting seriously the hemodynamic parameters, possibly due to a decrease of the work of breathing. a. michalopoulos, a. anthi, k. rellos, j. kriaras, s. geroulanos intensive care unit, onassis cardiac center, athens. objectives of this study was to examine the effect of different levels of peep on postoperative svo2 and pvo2 values in a group of patients, following open heart surgery. methods: upon transfer to icu, 67 patients (54 males and 13 females) of mean age 63_-+6 years, were randomly assigned to receive 0 (n=22), 5 (n=24), or 10 cm of peep (n=21). there were no statistically significant differences in demographic data or preoperative respiratory status among the three groups. all patients were ventilated on the assist control mode with a tidal volume of 10 ml/kg. the fraction of inspired oxygen (fio2) was adjusted to keep a pao2 around 100 mmhg. mixed venous po2 and svo2 were measured at 30 min, 4 and 8 hours after application of mechanical ventilation in the icu, just before extubation (be), half hour after extubation (ae), and at 4 hours post-extubation. differences at each study time were analysed by anova. results: mean svo2 and pvo2 values among the three groups, for all study intervals, are presented in the table. conclusion: we found no differences (p=ns) in tissue oxygenation (expressed by svo2 and pvo2) among the three groups, at any study interval, in the early postoperative course of patients following open heart surgery. intrinsic peep (peepi), and high elastance and resistance increase inspiratory work load in copd. cpap reduces work of breathing by counterbalancing peepi. pav provides flow (fa) and volume (va) assistance proportionally to patient resistance and elastance and inspiratory effort. we studied the effects of partitioned support (cpap-fa-va) on breathing pattern and inspiratory effort in five copd patients on pav compared to spontaneous ventilation (sv) and full support (fs: cpap+fa+va). flow, volume, minute ventilation (ve) respiratory rate (rr), inspiratory swing in esophageal pressure (apes), and its integral per breath (pti/b) and per minute (pti/m) were measured. objectives: to evaluate airway pressure fluctuation (apf) during spontaneous breathing in a high compliance cpap system. methods: the cpap system consisted of two 7l weighted balloons in a wedge shaped holder. ventilating gas flowed from one balloon through a low resistance one way valve into a tracheal tube (ett) provided with a pycor co 2 sensor to monitor rebreathing. the ett was connected to a piston drive mechanical lung. expired gas flowed through a low resistance valve into a second weighted balloon, from where it was exhausted through a peep valve connected in parallel with the second weighted balloon. we evaluated system performance at v r from 70 to 500ml, at rr from 10 to 120 bpm, while closely monitoring cpap airway pressure swings. at v v of 400 and 500ml the rr was limited to 60 bpm. for comparison we explored aps of a one 16l balloon cpap system, the cpap mode of the puritan bennett 7200, and siemens 300 ventilators, when connected to a healthy adult volunteer breathing through an ett. results: the compliance (cpl.) of one 7l balloon system was linear over a range from 1.0 to 3.3l, with a cpl. of 4.0 l/em h20.the cpl. of the 16 l balloon (0.5 l/em h20) was linear between a volume of 13 and 14.5 l. apf of the weighted balloon system was under 1 em h20 at all v r (except at a v r of 500ml aps was 1.5em h20), while the apf in the 16l balloon was up to 3 em h20. apf witli human volunteers with the two commercially available ventilators in the cpap mode was about 7 cm h20; while under identical conditions apf in the 16l balloon system was 1.5 emhzo; and in the two 7l balloon system was below lcm h20. conelusions: cpap using the two balloon system exhibits lower airway pressure fluctuations than a single balloon system; and is substantially lower than found in the two commercially available ventilators when used in the cpap mode. objective: to perform independent lung ventilation (ilv) with individual tidal volume (vt) set at a value generating a plateau airway pressure (pplat) < 25 crnh~o and to evaluate the usefulness of the continuous monitoring of endtidal co2 (etco2) as a guide to titrate individual lung vt during ilv and for the weaning from ilv. methods: in seven patients, ilv was performed with ttvo ventilators set with the same fio: and respiratory rate. each lung was ventilated with a vt that developed a pplat < 25 cmh~o. this setting led to a lower vt on pathological lung (pl). vt was increased in pl following etco~ and paco2-etco2 variations. ilv was discontinuated when etco~., vt and statical compliance (cst) were similar in both lungs. results: one hour after starting ilv (ti), pl mean vt was significantly lower than in normal lungs (nl) (224 + 46 ml vs 377 + 766 ml, p<0 001) two individual behaviours were observed on tl in pl: four patients presented low etco: (range 18 -31 mmhg)and normal pacoz (range 38 -42 mmhg), while three patients had normal etco2 (range 35 -45 mmhg) with high pac02 (range 44 -61 mmhg). one hour before stopping ilv (t2), vt, etc02 and paco2 were the same in each lung. the pao2/fio: ratio improved in all patients from the beginning ofllv cst of pl was 526 + 30 % of the normal lungs' cst on ti and improved to 97.6 + 27 % ofnl's cst on t2 (p<0.005 vs conclusions: setting vt of pl to a value not overcoming a pplat threshold does not impair oxygenation and is helpful in avoiding barotraumatism. measurements of differential etco2 and of the differential paco2-etco2 gradient can be used to titrate vt allocation during ilv and as a guide for the weaning from ilv. total respiratory resistance in mechanically ventilated patients exceeds values obtained in normal subjects, due to the added and highly flow dependent resistance of the endotracheal tube (rett). this can adversely effect the efficacy of pressure regulated modes of assisted ventilation, such as pressure support (psv) and proportional assist ventilation (pav). recent work demonstrates that the influence of rett during psv can be overcome by using tracheal (ptr) rather than airway opening (pao) pressure to regulate the pressure applied (intensive care med 20:$41, 1994) . the purpose of this study was to see if this approach would also be effective during pav. flow, volume, pao, ptr, and transdiaphragmatic pressure (pdi) were measured in 5 intubated patients in which either pao or ptt were used to regulate the pressure applied during pav where volume assistance was varied from 20 to 80% of respiratory elastance. representative results (mean + se) are shown below. compared to spontaneous breathing (pav 0%), pav increased tidal volume (vt) while reducing respiratory rate (rr) so that minute ventilation ('~e) also rose. this was associated with a reduction in inspiratory effort, as reflected by a decrease in the pressure-time integral ( [ p) of pes and pdi both per minute and per liter ~re. the effects on breathing pattern were similar for pao and ptr regulated pav. in contrast, the reduction in inspiratory effort was always greater for ptr regulated pav. in conclusion, the volume assistance provided by pav is more effective when ptr rather than pao is used to regulate the pressure applied. pav methods: retrospective data analysis of 596 adult patients with normal pulmonary function before operation and uneventful course following coronary artery bypass graft surgery over an 18 month period. we compared assist/controlled mandatory ventilation (s-cmv, 123 patients), synchronized intermittent mandatory ventilation with inspiratory pressure support (s-imv/psv, 431 patients) and biphasic positive airway pressure ventilation (bipap, 42 patients). results: patients ventilated with bipap had a significantly shorter mean duration of intubation (10.1 h, p< 0.05) than patients treated with s-imv/-psv (14.7 h) and s-cmv (13.2 hi. with s-cmv 39.9% of the patients required single or multiple doses of midazolam but only 13.5% in the s-imv-/psv group and 9.5% in the btpap group. the mean total amount of midazolam of these patients was significantly higher in the s-cmv group (8.8 mg) than in the s-imv/psv group (6.6 mg, p<0.05) and in the bipap group (4.3 mg, p<0.05). the consumption of pethidine and piritramide did not differ between s-cmv and s-imv/psv but was significantly lower during bipap (p<0.05). after extubation the paco2 patients was highest in the s-cmv group. conclusion: ventilatory support with bipap reduces the consumption of analgesics and sedatives and duration of intubation. unrestricted spontaneous breathing as well as fully ventilatory support allow adequate adaptation to the patients requirements. bipap seems to be an alternative to s-cmv and sqmv/psv ventilation not only in patients with severe ards but also in short term ventilated patients. _objectitives: after end-inspiratory airway occlusion we examined the ensuing gradual decrease in tracheal pressure (ptr) with the following equations proposed by bates et al. and hildebrandt: pv = p'v e'~cccl~2 +pst, rs (bates) [1] where p'tr is tracheal pressure immediately after occlusion, to= is occlusion time, "r 2 is viscoelastic time constant of respiratory system, and p t is static elastic recoil pressure of respiratory system. p~(t) = h 1 -h 2 log t (hildebrandt) [2] where h~ and h 2 are parameters depending on lung volume, and initial time is 1 s for analytical reasons. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecuronium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pressure was measured in the trachea. flow was measured with a pneumotachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a frequency of 200 hz and processed on a pc. the influence of the cardiac artifacts during the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean (+ sd) coefficient of correlation using eq. 1 was 0,912 -+ 0.168, and using eq. 2 was 0.884 + 0.045. the values ofz~ (eq. 1), however, decreased with increasing the tidal volume (vt) according to the following equation: "~2 = 1.52 -0.65 v t, similary, the values of h~ and h 2 increased with increasing v t according to the following functions: h~ = 4.4 + 13 v i and h 2 = 1.15 + 1.88 v t. conclusions: the behaviour of "% of eq. 1 suggests that the linear viscoelastic model is not sufficient to further describe the mechanical properties of the respiratory system over the vt range (6-14 ml/kg) in ventilated patients. infect this model predicts that "c 2 is constant and independent of tidal volume. on the other hand the plastoelastic model is not sufficient to further describe the mechanical properties of the respiratory system. in fact "r 2 obtained by fitting an exponential for data of eq. 2, is determined by the time of endinspiratory airway occlusion. obiectives: according to the viscoelastic model, the viscoelastic pressure of the respiratory system pv=rs during lung inflation with constant flow e~ is t/ r 1 2 1 wh t lsms ira tlmeand r given by:pv~c.~ = 2d~( -'e-~ )[ ] ere " ' p" tory " 2 and "r 2 are resistance and time constant of viscoelastic unit. in the past, the viscoaletic constants were determinated by performing a series of occlusions at different lung volumes, or a sedes of occlusions at a fixed lung volume achieved with various inflation flows. in the present study we have developed a new method for determining "c 2 and r 2 which requires a single constant flow inflation. our method is based on determination of pv~r, during a single breath constant flow inflation, and of z 2 during the ensuing end-inspiratory airway occiusion. dudng the occlusion the tracheal pressure p~, declines according the following function: ptr = p'lr e " too= " z2 + e~t.r= [2] where p'~r is tracheal pressure immediately after occlusion, toc c is occlusion time, p,i.rs is static elastic recoil pressure of respiratory system, and ~ is viscoelastic time constant. we first determinated "~2 by analyzing the time-course of ptr according to eq 2 and next determining r 2 according to eq. 1, using the expedmental values of p,i=~, ~ and ti, as well as "~2 obtained with eq. 2. materials & methods: we studied 8 healthy patients intubated, anestethized with propofol, paralyzed with vecurenium, and mechanically ventilated with constant flow (0.5 i/s) at zeep for minor surgery. pres-sure was measured in the trachea. flow was measured with a pneumniachograph and volume was obtained by numerical integration. the rapid occlusions were produced by an external valve. the signals were sampled at a fi'equency of 200 hz and processed on a pc. the influence of the cardiac artifacts dudng the occlusion time (4 s) was reduced by a software low-pass filter kaiser finite duration impulse response of elevated order. results: the mean coefficient of correlation with eq. 2 was 0.912. with v t of 7 ml/kg, the mean values (+ sd) of ':2 and r 2 of the 8 subjects amounted to 1.128 • 0.100 s and 3.990 • 0.890 cmh20 i "~ s. with the traditional multi breath method the corresponding values were 0.711 + 0.257 s and 4.445 _+ 1.474 cmh20 i "1 s, respectively. with the t-test the difference between new and traditional "~2 was statistically significant, between new and traditional r2 was not significant. conclusions: with the single breath method it is possible to compute ':2 and r 2. the mean values of r 2 with v t of 7 nd/kg, however, was slighuy different than those obtained with the traditional multi breath method. the application of modem principles of respiratory care and mechanical ventilation in icus has resulted in increased survival of critically ill individuals with neuromuscular, skeletal and irrevers~le pulmonary diseases. in these chronically ill individunts mechanical ventilation, long term 02 therapy (ltot) and continuous home care is considered a chronic life supporltng technique that can not be withdrawn after their discharge from an icu. the aim of this study was to present the results of a rehabilitation programme and home care that runs in our ward. twenw three patients were referred to our clinic f~om icus during 1993-94. a specific rehabilitation programme designed according to individual's needs was performed. patients that benefitted from this programme were grouped into the following disorders. 1) post tb respiratow failure 6(26%) 2) neuromuscular diseases, 3(21%) 3} undiagnosed sas 3{13%) 4) cope) 9(39%) (3 patients had a overlap syndrom). the programme consists of : 1) assessment and mechanical support ff needed of the respiratonj system with non invasive methods (nasal or via tracheostomy). 2) group and individual respiratory therapy 3) mobilization 4) nutritional support 5) educational classes for the members of the family. three from the patients passed away (during the year), 11 are under nippv during night with or without 02 supply, 13 pts recieve ltot. conclusion: the development of a programme for chronically ill individuals in especially designed wards in hospitals and the overall care at home is considered necessary at least in hospitals with icus. a rehabilitation programme and home care permits the fast but safe discharge of these patients from units of acute medicine that the cost of treatment is high and besides permits beds that are invaluable. we considered that the rehabilitation prod'amine and home care in our ward is the first performed in greek chronically ill pts and even though there is no special administxative support we think that the results are quite saltsfactory. objective: we postulated that the product of the respiratory frequency (f) and the ratio of inspiratory pressure (ip) to maximal inspiratory pressure (mip) would predict the weaning outcome in deeompensated copd patients better than either variable alone or other indices previously proposed. methods: in 28 decompensated copd patients with difficult weaning, we measured, daily, respiratory mechanics data both during mechanical ventilation and after ten minutes of spontaneous breathing. then we calculated weaning indices reported in literature and some new integrated indices. according to the results of the discriminant analysis, we considered the integrative index crop (acronym of compliance, rate, oxygenation and pressure), the rapid shallow breathing index f/vt, the load/capacity ratio ip/mip, and the following new index: f x ip/mip. we used receiver-operatingcharacteristic (roc) analysis by calculating the area under the curve considered as the overall probability of correct classification. results: main results are reported in the following objective: to evaluate the reliability of some indices of endurance in predicting the weaning outcome of decompensated copd patients. methods: in 28 decompensated copd patients with difficult weaning from mechanical ventilation (mv) we measured, daily, blood gas analysis, ventilatory and airway pressure pattern during mv, breathing pattern (frequency (f) and tidal, volume (v~)), inspiratory pressure (ip), and maximal ip (mip) during spontaneous breathing (sb). thereafter we calculated the following weaning indices: crop (compliance * mip * (pao2/pao2) / f), flvt, ip/mip. data obtained the day at which the patient was considered ready for a trial of sb on clinical grounds but weaning failed (wf) and those obtained the day of the successful weaning (ws) were compared statistically through the wilcoxon rank-sum pair analysis. in order to quantify the predictive accuracy for each index with respect to successful weaning we calculated sensitivity, specificity, and diagnostic accuracy according with the standard formulas. methods : five patients (64 + 6 yrs) suffering from ards (lung injury score > 2.5) for 48 hours or less entered into the study. irv (volume controlled, decelerating flow, 20 % inspiratory pause, lie = 2/1) was compared to conventional ventilation (cv) (volume controlled, constant flow, no inspiratory pause, iie= 1/2). these two modes were applied for 6 hours in a randomized order, with the same levels of total peep (peept = peep + peepi), tidal volume (8.0 • 0.7 ml/kg), respiratory rate (20 • 0"bpm) mad fit2 (63 • 2 %). measurements (respiratory mechanics, hemodynamics, arterial and mixed venous blood gases) were performed after 1, 2, 4 and 6 hours of application of each mode. rvsuils : are expressed as mean + sem and compared by anova. backeround and methods: periodic breathing (pb) is characterized by repetitive cyclic variation in minute ventilation. pb is considewxl to be provoked by an instability in the respiratory control. inintubated, spontaneously breathing patients conventional modes of pressure support ventilation, i.e., triggered inspiratory pressure support 0ps), do not allow patients to breathe with theirinherent breathing pattern. therefore, pb, if existing, will appear mainiy after extubation. since our new mode of pressure support ventilation" automatic tube compensation" (atc) continuonsly corrects for the flow-dependent tube resistance during insnmdon and expiration ("electronic" extubatim), it pemaits patients to maintain their own inherent breathing pattern. then, ff necessary, tracheal pressure can be additionally supported by volume-proportioead and/or by flow-proportional pressure support (proportional assist ventilation, pav). (~as~: we report the case of a 70-year-old male patient who was intubated due to acute respiratory insufficiency after acute myocardial infarction with left ventricular dysfunction. during ips of 10 mbar the patient showed a regular breathing pattem which became periodic during atc. in addition, proportional assist ventilation of 10 mbar/l increased periodic breathing in such a way that the typical cheyne-stokes breathing pattem occurred (see figure) . baqkground: the hering-breuer reflex (hbr) is characterized by an inhibition of inspiration during lung inflation. this response has been recognized as an important vagally mediated mechanism for regulating the rate and depth of respiration in newborn mammals. in adult man the hbr is considered to be active only at lung volumes well above functional residual capacity, i.e., at tidal volumes above 1000 ml. assessment of the hbr requires specialized methods such as single breath or multiple occlusion technique. methods; in the presence of desynchronization between ventilator and patient, which frequently occurs during triggered inspiratory pressure support ventilation (ips)(see figure) , prolongation of the interval between inspiratory efforts (indicated by negative deflection of the esophageal pressure) due to lung inflation exposes an active hbr. we examined the occurrence of hbr in intubated critically ill patients. strength of hbr was assessed by the formula: prolongation [%] = ((inspiratory interval of interest -preceding inspiratory interval)/preceding inspiratory interval) * 1(30. rr162 18 of 50 patients examined showed moderate to severe desynchronization. in 17 of these 18 patients a (re)activation of the hbr was found. the strength of hbr amounted to 134 + 51%. there was a significant correlation between tidal volume and strength of hbr. in contrast to previous reports, an active hbr was shown during lung inflation well below 1000 ml. b pck~round: triggered inspiratory pressure support ventilation (ips) is commonly used to support inspiration in intubated spontaneously breathing patients. despite its usefulness ips shows some disadvantages which can be deleterious in crificauy ill patients: -additional work of breathing to be performed by the patient due to the flow-dependent tube resistance -desynchronization between patient and ventilator due to inherent triggering failures of the ips mode suppression of the patient's inherent breathing pattern -inability to predict successful extubation in difficult-to-wean patients methods: based on the known flow-dependent tube resistance our new mode "automatic tube compensation" (atc) compensates for the pressure drop across the endotracheal tube ("electronic" extubation). then, if necessary, tracheal pressure can be supported by volume-proportional pressure support (vpps) and/or by flow-proportional pressure support (fpps). results: hitherto, we have examined 20 patients after open-heart surgery and 50 patients with acute respiratory insufficiency (ari) or ards using atc with/without vpps/fpps. preliminary results suggest that the new mode avoids additional work of breathing due to accurate compensation of the pressure drop across the endotracheal tube during in-/expiration prevents desynchronization between patient and ventilator allows patients to breathe with their inherent breathing pattern accurately predicts the outcome of extubation even in difficult-to-wean patients due to "electronic" extubation conclusions: the new mode atc with/without vpps/fpps allows to support ventilation in a more physiologic manner and overcomes the disadvantages of conventional modes of pressure support in intubated patients. backgound: cheyne-stokes respiration (cs) is characterized by regula]; recurring periods of hyperpnea and apnea. in normal subjects, cs may occur after hyperventilation, after arrival in high altitude, or during sleep. it has also been observed in patients with prolonged circulation time due to congestive heart failure, as well as in some neurological patients. there is no report about the influence of sedative drugs on periodic breathing (pb) and cs. methods: in intubated patients conventional modes of pressure support do not allow patients to breathe with their inherent breathing pattem. therefore, periodic breathing and cs are rarely seen. since our new mode of pressure support ventilation "automatic tube compensation" (atc) continuously corrects for the flow-dependent tube resistance during inspiration and expiration ("electronic" extubation) it permits patients to maintain their own inherent breathing pattem even if pathological, e.g., periodic. results: using this new mode of pressure support ventilation, periodic breathing was unmasked in 13 of 37 intubated patients, 6 of which showed cs. in 4 of these 6 patients the occurrence of cs was linked to impaired left ventricular function with increased circulation time. normal left ventricular and neurologic function was found in the remaining 2 patients. in 1 of these 2 patients cs disappeared after intravenous administration of the benzo-diazepine antagonist flumazenil (figure). consequently, in this patient cs was induced by benzodiazepine sedation. objecti',~s: in contrast to conventional rhodes for pressure supported spontaneous breathing, our newly developed ventilatow mode ,,automatic tube compensation" (atc) completely compensates for the flow-depandant pressure drop tlpm-r across endotracheal ttlbe (ett). in the atc mode, the ventilator supplies a flow v' in order to maintain a constant tracheal pressure p~,,~. to this end, pk,,= has to be oontinuousiy determined. since continued measurement of p,,~ by introducing a catheter via the ett is not reliable, we opted for its continuous calculation socordng to the following equation: p~ = p,,, -aperr, pw being the continuously measured airway pressure. this also requires the continual measurement .of flow v' to calculata apm-r using the non-fineer approximation: aport = kvv' + k2.w. the constant tube coefficients k~ and k2 are mathematically determined by mesns of a least-squares-fit procadum based on laboratory investigations. tracheal secretions, however, reduca the omss-saction of the ett. consequently, ~ values of ki end k2 are changed rendering the p~,ch calculations inaccurate. therefore, k1 and ~ have to be pedodcally updated to ensure an a~urete monitoring of pn,~ and a complete tube compensation under atc at any time. background: one of the first steps in weaning patients from controlled mechanical ventilation is to stop muscle relaxation and to reduce sedation. it can take several hours, however, until the patient is able to trigger the ventilator and to breathe spontaneously. during this period, many patients display a sudden increase in peak airway pressure of up to 30%. patients and methods: to investigate the reason for this potentially dangerous effect, we continuously measured lung and chest wall mechanics in post-operatively ventilated patients. lung mechanics (airway resistance and lung compliance) was measured using the esophageal balloon technique as described in [1] . chest wall mechanics (tissue resistance and chest wall compliance) was calculated from lung mechanics and total respiratory system mechanics as described in [2] . results: we found a decrease of chest wall compliance (cw) to be the main reason for episodes of sudden airway pressure increase while lung compliance (cl) remained unchanged. the decrease of c w can be intergil cano a, san pedro jm ~, sandar d, herntndez .1, carrizosa f, , herrero a. emergency and intensive care department, hospital of jerez, spain objective: 1) to determine the incidence of hypoteasion (h) associated with emergency intabatian of mechanical ventilation, and 2) to establish its relauonship with respiratory mechanics (rm) and arterial blood gases. mechanical ventilation performed in the emergency room, in a prospective eans~eative manner, were evaluated. data collected included patient demographics, diagnoses, blood pressure and arterial blood gas levels before and at~er intabatian, and p_m, including calculated pulmonary end-inspiratory volume above functional residual capacity (veic) and calculated dynamic hypetinflatien (dhc). all patients received midazolen and awaanrinm to facilitate tracheal intubatien and rm measurement. hypotension was defined as a decrease in systolic pressure higher than 40 mmhg or an absolute decrease in systolic blood pressure below to 90 mhg within 1 hour of intabatian. 14 patients were excluded because met at least one of the following exclusion criteria: preexisting shock or h (8), cardiac arrest (5) .1 there weren't any association between peepi or other airway pressures (paw) and h, but calculated pulmonary volitmes had tendency to be larger in patients with h (p < 0.1). high paco 2 before lrasheal intubatian (87.4 4-9 mmhg) with a quickly decrease alter starting mechanical ventilation was a usual finding (p < 0.01) in patients who developed h. paw. 3) thexe was a good relatienship between h and high arterial paco 2 before traqueal intahatian and its fast "washing" with mechanical ventilation. 4) because cao patients had the highest incidence of h, controned mechanicel hypoventilatien driven by paco 2 changes and pulmonary volumes monitoring instead paw, should be attempted in these patients to avoid this cemplication after tracheal intubatiert. introduction: the endotracheal tube (ett) and demand valve devices cause an added work of breathing (wobadd), which is the work necessary to overcome the resistive load of the ett and the breathing circuit (1). application of ips has been shown to partly compensate this added work (1). since tbe amount of wobadd is flow dependent, a fixed ips is not adequate to completly compensate the wobadd (2). therefore, atc has been developed as a new form of assisted spontaneous breathing (3), which provides a flow-dependent pressure support. thereby, it theoretically should compensate all the wobadd due to the tube. the purpose of this study was to evaluate the reduction of wobadd with ips and atc for different ett. methods: a mechanical lung model (ls 4000, dr*alger, liibeck, frg) was used to generate a constant spontaneous breathing pattern. the ls 4000 was connected to an artificial trachea (at, 10 cm long, 22 mm id). the at was intubated with three different tubes of 7.0, 8.0, 9.0 mm id and connected to an evita 2 ventilator modified to provide atc as an option (dfager, liibeck, frg). flow and airway pressure were measured between the y-piece and the ett for four different modes of ventilation: cpap, ips of 5 and 10 cm i420 and atc all with a peep of 10 cm h20. the tracheal pressure (ptrach) was measured in the at. total wobadd was calculated as the area subtended by the ptrach-volume curve below peep. results: the results for total wobadd in nd/1 are shown in the figure for the three different ett: breath/mln, s=success, f=failur% *~p<.05, **-p<01, ns = non significant, f versus s neveltheless, in 5/26 patients, invasive ventilation was necessary in mean 12.6_+12 hours after beginning of fmpsv. there was no significant difference between the two groups (success, failure) in following parameters : sex, age, previous histoly, medical treatment, saps1 & 2, clinical signs (rr, spo2, heart rate, blood pressure, glasgow score...), radiological and echocardiographic findings and standard biological parameters. only two parameters were related with failure : 1.a low value of pac02 on admission until the patients were intubated. 2. an increased level of cpk in relation with an acute myocardial infarction (4/5 cases in the failure group, vs 3/21 cases in the success group, x~(with continuity correction) : p<.05). conclusion : fmpsv is a noninvasive, safe, rapidly effective method of treatment in acpe, which may avoid tracheal intubation. further studies are necessary to precise if association of arf and low paco2 (<35mmhg) and/er acute myocardial infarction represents an indication of immediate invasive ventilation. introduction: since the added work of breathing (wobadd) imposed by the endotracheal tube (ets and the breathing circuit is regarded as an important contribution to the total work of breathing, considerable effort has been tmdettaken to compensate for this added work. ips has been fotmd to decrease the wobadd imposed by different ventilators (1, 2). because of the flow dependent pressure drop across the etf the tracheal pressure (ptr) should be measured to estimate the total imposed wobadd (wobtut) (3, 4). the aim of this study was to assess the circuit imposed work (wobcirc) and wobtot (including ett) for different demand valve ventilators during cpap and/ps. methods: a mechanical lung model (ls 4000, driiger, lfibeck, frg) generated a constant spontaneuus breathing pattern. the ls 4000 was connected to an artificial trachea (at), intubated with an 8.0 nun et]', end connected to one of four ventilators (servo 900c and servo 300, siemens,-elema, sweden; evita2, driiges, liibeck, frg; pb 7200ae, puritan bennett, carlsbad, usa). three different modes of ventilator settings were tested (cpap, ips 5 and 10 mbar; trigger set at maximal sensitivity, peep always 10 mbar). flow and airway pressure (paw) were measured between the y-piece and the etr; tracheal pressure (ptr) was measured in the at. wobtot was calculated as the area under the ptr-volume curve below peep, wobcirc was calculated as the area under the paw-volume curve below peep. results: in the foti g., patroniti n., cereda m., sparacino me., giacemini m., pesenti a. inst.of anesth.and intensive care-univ.of milan -sgh monza i aim of the study was to assess cpl,rs measurement obtained by the airway occlusion method during psv. we therefore studied 31 paralyzed cppv ventilated ali patients (lung injury score =2.25• that were weaned to psv. we performed end inspiratory and end expiratory airway occlusions using the hold function of the ventilator (siemens serve 900c), first during cppv and then within the 24th psv hour. airway pressure and flow signals were recorded (cpi00 bicore) for subsequent analysis. an airway pressure plateau was defined as a 0 flow tracing in which airway pressure was stable for at least 0.25 sec. end inspiratory (pel,rsi) and end expiratory (pel,rse) recoil pressures were then measured as the mean airway pressure during plateaus. cpl,rs was computed as tv/ (pel,rsi-pel,rse i) cpl,rs can be adequately estimated during psv using the airway occlusion method; 2) during psv inspiratory plateaus are longer than the expiratory ones; 3) the length of plateaus is negatively affected by the respiratory drive. foti g., de marchi l., *tagliabue m., gilardi p., giacomini m., sparacino me., pesenti a. inst.of anesth.and intensive care,-univ.of milan *dept.of radiology-sgh monza i we retrospectively compared ct scan and gas exchange findings between a group of patients successfully weaned from vcv to psv (group s = ii patients) and a group who failed the weaning (group f = 6 patients). we selected 17 ali patients (lis=2.5• in vcv mode who had available a chest ct scan performed within 4 days from the weaning trial. a psv trial was began as soon as the patient reached hemodynamic stability and a pao2 >80 mmhg, irrespective of fie2 (peep <15 cmh20). maximum psv level was < (pel,rs-peep) measured during vcv, where pel,rs was the respiratory system elastic recoil pressure at end inspiration. psv ventilation was considered successful if a respiratory rate <40 bpm, an increase in fie2 lower than 0.2 compared to vcv, a pace2 increase <20% of vcv value and hemodynamic stability were maintained during the next 48 hours of psv. if any of these conditions was not met the trial was declared a failure. interdisciplinary critical care unit, regional hospital lugano-ch *surgical critical care unit, university hospital, geneva-ch objective: to assess the degree of correlation of cardiac output measured by thoracic electrical bioimpedance and thermodilution in mechanically ventilated patients with different levels of positive end-expiratory pressure (peep). methods: prospective study with 10 ventilated patients, 7 after head injury and 3 with postoperative sepsis, with normal cardiac output: simultaneous determination of cardiac output by thermodilution and thoracic electrical bioimpedance performed with different levels of peep (0-5-15 cm h20). results: cardiac output measured by thermodilution during sequential increment of peep did not vary: 7.3 + 2.5 for peep 0, 7.4 + 2.7 for peep 5 and 6.9 + 1.7 l/rain for peep 15. simultaneously the bioimpedance device recorded a significant increase in cardiac output from 4.4 + 1.3 for peep 0 to 6.0 + 1.9 l/mi for peep 15. (p < 0,05). conclusion: cardiac output measured by bioimpedance cannot replace the invasive thermodilution methods of cardiac measurement output during mechanical ventilation with peep. we also isolated a subset (h) of 12 patients who had been hypercapnic (paco2>50mmhg) for at least 3 days (range 3 to 60 days) before the end of cv. the psv trial was started as soon as pao2 was > 80 mmhg, irrespective of fie2 and with peep < 15 cmh20 and the psv level had to be < (pplateau-peep) as measured during cv. pace2, pha, base excess (be) were collected before discontinuation of cv and on the ist day of psv: 05) . 2) weaning is more difficult in pts with head injury(p<o,05) and iss>30 (p<o,ol), in elderly(p<o,05) and in pts who need fi02>0,6 (p<o,05) and peep>io cm h20 (p<o,05). 3) pts with iss>30 need longer duration of mv (p<o,ol). 4) the mortality rate is higher in pts with iss>30 (p<o,o01), with head injury (p<o,ol) and in elderly (p<o,05). 5) paradoxally, compliance was found to be higher in pts>60 years than in pts<60 years (p<o,05). s. crotti, p.pelosi, d.mascheroni, m.cerisara, a.lissoni, l.gattinoni. istituto di anestesia e rianimazione -irccs, milano, italia. obiectives. we investigated by ct scan the effects of extrinsic peep (peepe) on lung inflation, tidal volume distribution and regional compliance in sedated, paralyzed chronic obstructive pulmonary disease (copd) patients with dynamic hyperinflation (peep• methods. two ct section (end expiration, end inspiration) were obtained at lung base level in 4 patients (individual analysis for each of the eight lungs) at 4 peepe levels: peepe = 0 cmh20 (zeep) and peepe amounting to 50%, 100% and 150% of the peep• at zeep (9.5 • 4 cmh20). tidal volume was kept constant (8 • 1.4 ml/kg). each lung section was divided into 3 zones equally spaced along the vertical axis: upper (the most ventral), middle and lower (the most dorsal) zone. for each zone we computed: gas volume at end expiration (gase) and at end inspiration (gas• tidal volume (dgas=gasi-gase) and compliance (cpl=dgas/dp;.dp=plateau pressureactual peep• we also computed the lung inflation as the entire ct section gas volume at end expiration (total gase). results. at each peep level both gase and gas• were higher in the upper than in the lower zone ( we cone!ude that in copd patients with dynamic hyperinflation only the application of peepe higher than peepi produces a further increase of lung inflation, decreasing cpl of the upper zone (the more expanded one) probably by stretching phenomena, and causing dgas redistribution from the non dependent to the dependent lung zones. i. ravagnan, p. vicardi, f. valenza, d. tubiolo. p. pelosi l. gattinoni. 1st . anestesia e rianimazione, universita' di milano, ospedale maggiore -irccs, italy objectives: to investigate the effects of peep and ps on respiratory. pattern and inspiratory effort during ps ventilation in patients with acute lung injury or acute exacerbation of chronic lung disease. methods: 4 pts with acute (a) (paco2= 39• mmhg, pao2/pao2= 0.4• and 4 pts with chronic (c)(paco2= 51• mmhg, pao2/pao2= 0.3:50.1) lung disease were studied in ps ventilation during the weaning phase. measuring airway pressure, esophageal pressure and gas flow we computed respiratory rate (ril bpm), tidal volume (tv, ml) and pressure time product (ptp, cmh20*s/min), which is an index of oxygen muscle consumption. measurements were collected, after 15 rain of stabilization, at 5 and 15 cmh20 ps and at two different peep levels (0 and 8 cndd20 conclusions: during psv: 1) breathing pattern and ptp are different between a and c; 2) in both groups peep does not modify breathing pattern; 3) peep reduces ptp in c but not in a; 4) ps modifies breathing pattern and reduces ptp in c but not in a. to test the performance of a new heat and moisture exchanger (hme): twistair, baxter. this is a hydrophobic-hydrophilic hme without hygroscopic salts. method: we evaluated the hme performance with a previously described method (i); it consists of a "lung simulator" connected to a mechanical ventilator and a flow divider, with a dry and a wet thermal probe inserted into inspiratory and expiratory limbs. on average ps level changes were associate( with opposed changes in both p0.1 and rr. changes in p0.1 were remarkable, homogeneous and highly significant (p<.002), while changes in rr were less pronounced, inhomogeneous and less significant (p<.02). it seems therefore that p0.1 is a better index than rr for estimating a change in load for the respiratory muscles. recently, the lsf method has been described as an interesitng alternative to conventional tecniques used to measure total respiratory mechanics, providing, over these latter, advantages such as no need for hold maneuvers and no need for particular flow patterns. data on the reliability of the lsf method, however, are still few. methods. 9 sets of measurements were obtained in each of 7 ards patients, paralyzed and ventilated in cmv with constant inspiratory flow and an end-inspiratory pause equal to 15% of ttot. the lsf method provided data for total compliance (ctotlsf) and total resistance (rtotlsf) by multiple linear regression analysis of airway pressure, flow and volume change, applied over the entire breath. the lsf measurements were automatic and continuous, and each value used for analysis was the average of 160 consecutive cycles. conventional measurements of dynamic.compliance (cdyn), quasistatic compliance (cqs), minimum inspiratory resistance (rmin) and maximum inspiratory resistance (rmax) were obtained by the constant flow, end-inspiratory occlusion method, each value being the average of 3 measuremts. ctotlsf was compared with cdyn and cqs, while rtotlsf was compared with rmin and rmax. results. ctotlsf showed a high correlation both with cdyn (cdyn=.87.ctotlsf+l.14, r2=.982), and with cqs (cqs =.94.ctotlsf +1.19, r2=.984). the average difference between ctotlsf and cqs, yet, was much lower (+.36+1.79 ml/cmh20) than the one between ctotlsf and cdyn (+2.43+2.40 ml/cmh20). rtotlsf correlated much better with rmax (rmax=.88*rtotlso+.98, r2=.914) than with rmin (rmin =.58.rtotlsf +. 1.71, r2=.804). the average difference between rtotlsf and rmin was +4.48+3.48 cmh20/1/s, while the one between rtotlsf and rmax was +.81+2.03, confirming the better agreement between rtotlsf and rrnax. conclusion.the agreement between rtotlsf and rmax suggests that rtotlsf takes into account both the airway and the tissue components of resistance, unlike rmin which only expresses airway resistance. the agreement between ctotlsf and cqs indicates that ctotlsf expresses the pure elastic components of the respiratory system. in the paralyzed patient, the expiratory time constant (rce) of the unit represented by total respiratory system and ventilator can be calculated from the slope of the expiratory flow-volume curve. recently it has been suggested that an estimate of this slope is provided by the ve/v'epeak ratio, ve being the exaled tidal volume and v'epeak the peak expiratory flow.the reliability of this method is still little known. methods. 9 sets of measurements were obtained in 7 mechanically ventilated ards patients during paralysis and cmv. measurements of ventilator expiratory resistance (rext) and of total respiratory mechanics were performed in order to calculate these 3 reference measurements for ve/v'epeak: rcdyn=(rmin+rext)ocdyn, rcstat=(rmax+rext)ocqs, rclsf=(rlsf+rext)~ cdyn (dynamic compliance), cqs (quasistatic compliance), rmin (minimum inspiratory resistance), rmax (maximum inspiratory resistance) were obtained by the constant flow, end-inspiratory occlusion method. clsf and rlsf respectively corresponded to measurements of total respiratory system compliance and resistance by the least squares fitting method. ve/v'epeak showed a high correlation with all 3 reference measurements, rcdyn (ve/v'epeak= 1.07orcdyn-.18, r2=.939), rcstat (ve/v'epeak = .97 9 rcstat -. 12, r2= .929), and especially rclsf (see fig the static pressure volume curve of the respiratory system (p/vst) provides valuable information, but the extensive data analysis prevents its use in clinical routine. dynamic p/vcurves are simpler to record. the objective of this study was to develop an automated method to obtain quasi-static p/v-curves (p/vqs) during low flow inflation as well as to measure resistance. preliminary tests were done in normal subjects and subjects with varying degree of lung disease. methods: a computer/ventilator interface (cvl) was constructed for control of a servo ventilator 900c during an automatic sequence: a) a normal breath, b) a prolonged expiration at zero peep, c) a low flow inflation of a predetermined volume, d) analysis of ventilator pressure and flow. pressure was corrected for tube resistance. airway resistance was calculated from a) and used to derive p/vqs from c). as reference, p/vst was obtained by the occlusion method. results: in non-obstructive diseases the method performed well at volume and pressure controlled ventilation. static and quasistatic compliance were virtually identical. p/vqs clearly reflected the lower inflection point and, when present, the upper inflection point (uip). in some patients uip was more evident in p/vqs, especially at an increased inflation flow rate. in obstructive diseases, the method for subtraction of resistive pressure was inadequate. conclusions: the system for automated computer controlled studies of lung mechanics based on a standard ventilator provides comprehensive information. differences between p/vst and p/vqs imply that the latter reflects also other factors than p/vst that contribute to impedance at hyperdistension. the clinical significance of the two curves remains to be shown. obiective: to evaluate the effect of combined high frequency jet ventilation (chfjv) in patients with severe respiratory insufficiency (sri). methods: in a retrospective study of the period 1985-1995 we analyzed patients suffering from sri who were refractory to conventional mechanical ventilation and were treated with combined high-frequency jet ventilation (chfjv). refractory to conventional mechanical ventilation was defined as a pao2/fio 2 < 100, despite maximal ventilator settings: peep > 8 cm hz0 , fit 2 > 0.6. a total of 43 patients matched these criteria, 27 males and 16 females with a median age of 44 (17-72) years. seventeen suffered from severe trauma. chfjv was started following a median period of 3 (1-22) days of conventional mechanical ventilation. prior to chfjv ventilation parameters expressed as median were the following: fit 2 0.8, pao2/fio 2 78, peep 12 cm h20 peak airway pressure (pap) 48 cm h20. chfjv consisted of high frequency jet ventilation with a frequency of 100 to 300 breaths/minute, driving pressure of 1.8 to 3.5 arm, and inspiration time of 20 to 30 percent, superimposed on the whole cycle of conventional mechanical ventilation with a frequency of l0 to 20 breaths/minute and tidal volumes of 100 to 400 ml. results: following two days of chfjv 31 of 43 patients showed an improvement of ventilatory parameters; peep could be reduced to < 8 cm h20 in 14 patients, the pap was decreased with > 5 cm h:o in 20 patients, fio 2 could be reduced to < 0.6 in 27 patients and finally the median pao2/fio 2 ratio changed from 78 to 133. during chfjv 23 patients died, 4 of respiratory failure and 19 due to multiple organ failure, 6 died within two days of chfjv. the median duration of chfjv in survivors and nonsurvivors was 6 days in both groups. conclusions: our data show that with chfjv in the majority of patients with sri who are refractory to conventional mechanical ventilatior" the ventilatory parameters can be improved. backeround and obiectives: although ventilation with peep above the inflection point (pinf) has been shown to reduce lung injury by recruiting previously closed alveolar regions, it carries the risk of hyperinflating the lungs. in the present study we set out to develop a new strategy to recruit the lung during ventilation with small vt, while maintaining peep levels as low as possible. we hypothesized that if the lung was recruited with a sustained inflation (si) to total lung capacity, recruitment would be maintained as long as the peep level was higher than the critical closing pressure of the lung, as observed on the deflation limb of the pv curve (ajrccm 1995; 151(4) :a432). the purpose of this study was to examine the hypothesis that a strategy using si and a peep<pinf would induce less lung injury during small tidal ventilation than ventilation at the same low peep level without using a recmitment strategy in a model of respiratory distress syndrome. methods: we used a randomized protocol with 4 groups to ventilate 40 nonperfused, lavaged rat lungs with small vt (5 to 6 ml/kg) for 2 hours and studied the effect on compliance and lung injury. group 1: peep>ping group 2: peep<pin f with an si, inflation to 30 on h20 over 30 sec, to recruit volume; group 3: peep<pinf without si; group 4: control group, lungs were inflated at peep<pin f, but not ventilated. results: in group 2 and group 4 static compliance did not change after ventilation (table) . in group 3 static compliance fell significantly. group 1 showed significant changes in the pv curve, which were less severe than in group 3. results from morphological examination are pending but preliminary results showed less lung injury in group 2, compared to group 3. conclusions: small tidal ventilation following a recruitment manoeuvre in a model of respiratory distress syndrome allows ventilation on the deflation limb of the pv curve at a peep below pinf. this strategy (1) minimizes lung injury as well as, or better than using peep above pint" and (2) ensures a lower peep to minimize the detrimental consequences of high lung volume ventilation. i~peep>pin~ 2 _objectives-this report is presenting the results of the clinical study for using eeg examination as a method of the evaluation of patients ability for weaning. methods: the study inclljqles 42 eeg examinations with fourier spectral analysis' of 37 patients ~vith respiratory insufficiency and prolonged control mechanical ventilation (cmv). all patients have had a-rhythm of eeg before weaning. we have followed respiratory rate, tidal volume, respiratory pa{tern, end-tidal co2 and blood gases during weaning. results: 13 patients had invariable eeg activity or short 13-waves period (till one hour). the weaning of this patients was fast arid sucsessful. other 24 patients have had a decreasing of a-activity, an appearence of 13-waves for an hour and more, a short episodes of a-and e-activity. after that this patients had gas exchange and respiratory disorders with regression of the weaning right up to cmv. conclusion: eeg could be used as a method of the evaluation of patients ability for weaning from cmv. some eeg signs shows the overstrain of compensatory systems before the change to the worse of gas exchange and respiratory pattern. s. elatrous, p. aslanian, d. touchard, d. corsi, h. lorino, l. brochard. medical intensive care unit, inserm u 296, hopital henri mender, cr~teil, france. in vitro comparison of flow triggering (ft) systems demonstrated advantages compared to pressure triggering (pt) systems for some ventilators (puritan bennett 7 200) but not others (siemens serve 300). we studied the two types of systems in two groups of 8 patients mechanically assisted with pressure support ventilation (15 + 6 cmh20). in the first group (pb 7 200) the effort of breathing, assessed by the esophageal pressure time index, was significantly lower with the ft than with the pt (139 + 40 cmh20.s/min -1 vs 158 + 32, p< 0.05). by contrast no significant difference appeared in the second group (serve 300), as predicted by the bench study despite marked interindividual differences (134 + 55 cmh20.s/min -1 vs 160 + 61, p = 0.1). we conclude that 1) rigorously performed bench studies can predict in vivo effects, 2) mild advantages can be found for the new triggering systems on some ventilators. objectives: pressore-volume curves (pv) of the respiratory system is of interest for the determination static compliance (cs0, lower (lip) and upper (uip) inflection points which indicate zones of airway recruitment and overdistension. this study aimed to compare an "automated low flow inflation" method (alfi) to the reference occlusion (oc) method. the ability of the former method to identify cst, lip and uip was tested in icu patients. me,otis: 16 (8 arf and 8 ards) sedated paralysed patients were studied using a serve 900c ventilator linked to a computer which automatically forced the ventilator to insufflate at a low constant flow a velum up to 1500-2000 ml or a maximum paw of 50 cm h20 (alfi). the quasistatic elastic pressure (pel,qs0 was obtained by subtraction of the resistive pressure of tubing and patient and related to volume for calculation of compliance cqst. for oc tidal volumes (v0 from 50 up to 1500-2000 ml were followed by a 3 s post-inspiratury pause for determination of static pal (pel,st) in relation to volume. compliance was defined from the linear part of the p/v curves. lip and uip were defined from the consistent deviation of p/v data from extrapolated the linear part. ~,~111i~: in ards, mean cst was 27.9 + 3.5 and cqst 29.7 + 3.9 ml/cm h20 (us), lipst 5.2 + 5.0 and lipqst 7.0 + 4.6 cm h20 (us), uipst 23.1 + 10.8 and uipqst 26.0 + 5~4 cm h20 (us). nosocomial pneumonias (np) are frequent and often unsuspected during ards (bell, !983). in the present study, we evaluated prospectively the onset of np during severe ards (group b of the european study). patients and methods: the charts of 15 patients with severe ards have been prospectively recorded. a plugged telescopic catheter (ptc) specimen has been systematically performed every 48 hours, for quantitative bacteriological analysis. the diagnosis of np was defined by a number > 103 colony forming units / ml. results: for the 15 patients studied, the mean saps score (+ sd) was 16+_2, the initial pao2/fio2 ratio was 100-&-_35, the duration of mechanical ventilation (mv) was 19+9 days. the mean delay before the onset of the first np was 8.6+5.6 days (5-12), and the mean pao2/fio2 ratio was 110+-28. respiratory symptoms (purulent aspirates, new pulmonary infiltrates, or gazometric changes) were present in 80% of the patients studied. alteration of gas exchange was present in 8 of the 15 patients (7 np) . a new pulmonary infiltrate was present in only 1 np (10%). an increase of fever was noted in 6 patients, an increase of leukocytosis > 20% in 8 patients, an increase of volume and purulence of sputum in 3 of the 10 patients with np. the degree ofgazometric worsening (pao2/fio2 before np minus pao2/fio2 during np) during the first episode of np was 44+17 mmhg. excluding the bacteriological criteria of np, the number of criterias of np present was 1 in 1/10 patients, 2 (5/10), 3 (2/10) or 4 (2/10). two patients only had a pulmonary colonization (ptc: < 1102 cfu / ml) before the first episode of np. the incidence of np is high (53%) during severe ards. the first episode occurs in average:at the 9 th day, and is the cause of a severe hypoxemia (pao2/fio2 110) . the onset of a np may contribute to the high mortality rate observed in our patients (93%). each worsening of hypoxemia during severe ards should induce to suspect a np. respiratory system during mechanical ventilation. the me~hod quantifies the dissipative energy consumption of the respiratory system in terms of energy loss aek, inefficiency ~k~ and respiratory dissipative resistance rk~ over a given partition of the tidal volume. the method can be applied in intensive care units with no interference to ventilatory support. it allows for monitoring the combined effects of inhomogeneities, non-linearities and visco-elastic effects, that are subject to change in the respiratory system. the method is studied on pigs~ in the presence of a log-dose response curve of methacholine (mch) induced disease. in healthy pigs~ we find a mean value of energy loss, ae, of 0.27 • j/l, a mean value of inefflency, ~ of 0.25 ~=0.05 and a mean value of resistance, 7~, of 4.40 • cm h20 s/1. the respiratory resistance, rk, shows a variation over the partition of tidal volume with armax ----3.90 • 0.66 cm h20 s/l. during methacholine provocation~ ae rises more than five-fold up to 1.48 • j/l~ doubles to 0.54 • and t~ increases to a maximum of 22 • cm h20 s/l, with armax : 15.1 • 7.0 cm h20 s/1. the variation in rk becomes more pronounced with higher doses of methacholine. methods: 10 ards patients were prospectively studied. initially they were ventilated in the amv (assist mechanical ventilation) mode with the settings prescribed by their primary physician. after stabilization, ventilatory gas exchange and hemodynamic variables were determined. patients were then ventilated in the mrv (mandatory rate ventilation) mode with 20 breaths as the target rate. in mrv the target rate is set and the ventilator autoregulates the pressure support level delivered ~o achieve this rate. after stabilization, the measurements done on amv were repeated. finally, patients were sedated and paralyzed and ventilated in cmv (control mechanical ventilation) with the ventilatory variables they had during mrv. measurements done in amv and mrv were repeated and respiratory mechanics were assessed with the constant flow end inspiratory occlusion method. results: two groups were recognized based on their response to mrv. tn group 1 patients responded to mrv by decreasing their v and increasing the t/t t ratio. ve, vo 2, and aado 2 decreased while paco 2 increased and tda vo ume and co remained unchanged. on the contrary, in group 2 v, vr and ve increased; ppeak and trr t remained unchanged, paco~ decreased while vo 2 and aado 2 increased with constant co, the pressure support level needed to achieve the target rate was much lower in group 1 than in group 2 (19,8-+1.3 vs 29.4_+2.0). obiectives : in the newly developed mode of ventilatory support ,,automatic tube compensation" (atc) the ventilator compensates for the flow-dependent pressure drop across the endetracheat tube (ett) thus allowing ,,e]ectronic extubation". the aim of the study is to investigate whether healthy subjects perceive atc in inspiration (atc-in) and in expiration (atc-in-ex) and whether atc provides an increase in subjective comfort compared with the conventional assisted spontaneous breathing mode (asb). methods : healthy volunteers (no preceding lung disease, non-smokers, male, 20-40 years)breathed spontaneously through an uncut ett of 7.5 mm id via a mouthpiece. the ett was connected with a prototype ventilator evita 2 modified by the manufacturer (drfiger, lebeck) for atc. flow and airway pressure were measured at the outer end of the ett. three ventilatory modes, (1) asb (10 mbarover 5 mbar peep), (2) atcin, (3) atc-in-ex were selected in random order. immediately following the transition from one mode to another the volunteers answered by hand sign how they perceived the new mode compared with the preceding mode: ,,better" (+1), ,,equal" (0) or ,,worse" (-1). inspiration and expiration were investigated separately by presenting 120 mode transitions (in total; including ,,placebo" transitions). results : the difference between atc and conventional asb is perceived in inspiration and in expiration. atc is positively judged; asb is nega ively judged. the diagrams show mean values _+ sd of five volunteers investigated up to now. the new mode atc is perceived as an increase in subjective comfort. our explanation is that atc preserves the natural breathing pattern better than conventional asb. objectives: to determine the role of cerebral vasoconstriction in the delayed hypoperfusion phase in comatose patients after cardiac arrest. to correlate the results with indices of cerebral oxygenation and the levels of several vasoactive hormones in the jugular bulb. methods: in comatose patients after cardiac arrest we measured the pulsatility index (pi) of the medial cerebral artery by transcranial doppler sonography. the pi is a reliable indicator of cerebral vascular resistance. we also sampled blood from the jugular bulb and measured cerebral oxygen extraction ratio and jugular bulb levels of endothelin, nitrate and cgmp. the first measurement was done within 4 hours after cardiac arrest and repeated 3, 6, 9, 12, 18 and 24 hours later. results: we studied 10 patients, 6 females, mean age 64,1+13,7 years. the pi decreased s!gnificantly between th~ first and the last measurement from 1.86 _+ 1.02 to 1.05 + 0.22 (p = 0.03). cerebral oxygen extraction ratio decreased also from 0.39+ 0.13 to 0.24 + 0.11 (.p = 0.015). endothelin levels were high, but didn't change during the studied period. nitrate levels varied in a wide range, but didn't change significantly. however, cgmp levels increased significantly from very low levels in the first measurement to very high levels 24 hours later, rasp. 2.95 pmol/ml (median; 25th 2.48-75th 5.43) and 7.5 pmol/ml (median; 25th 6.2-75th 14.00) (p = 0.02). eighteen and 24 hours after the first measurement we found a strong correlation between pi and cerebral oxygen extraction ratio ( r = 0.64, p = 0.05 and r = 0.76, p = 0.01). we.also found 12 hours after the first measurement a significant correlation between pi and cgmp levels ( r = 0.69, p = 0.03). we found no correlation between pi and endothelin or nitrate levels. conclusion.~; our results show a high cerebral vascular resistance in the first few hours after cardiac arrest, gradually decreasing during the next 24 hours. this is accompanied by an initially high cerebral oxygen extraction ratio and low cgmp levels, suggesting that the cerebral vascular resistance is induced by active vasoconstriction because of insufficient cgmp levels, leading to a decrease in cerebral blood flow and a compensatory ~ncrease in cerebral oxygen extraction. objectives: sudden cardiac arrest is a major cause of mortality in western countries accounting for over half of all cardiovascular deaths. in most cases the mechanism of death is prolonged cardio-circulatory arrest due to ver:tricular fibrillation (vf) preceding final asystole. recurrent syncopes due to idiopathic vf with good neurological prognosis have been reported in patients with and without cardiac etiology (1,2). in the past measurements of cerebral hemodynamics have been repeatedly done in humans during cpr, but until today no studies of cerebral blood flow velocity (cbfv) have been reported during controlled cardiac arrest in humans not under-going cpr. it was the purpose of our study to evaluate the acute hemodynamic effects of untreated vf on cbfv. methods: after approval by the local university ethics comittee, five male patients aged 34-48 years without evidence of cerebral disease were investigated during vf while undergoing implantation of a pacer cardioverter defibrillator system (model 7219d; medtronic| a standard anaesthetic regimen was used (propofol, fentanyl). after implantation of the automated cardiac defibrillator vf was induced by electrical countershock to test effective sensing, pacing, and defibrillation. to measure cerebral blood flow velocities (cbfvmca) the doppler probe was placed above the zygomatic arch between the lateral margin of the orbit and the ear and directed towards the m1 segment of the middle cerebral artery (mca). results: a total of 12 phases of vf were investigated. duration of vf ranged from 6 to 26 seconds, with cbfvmc a (mean_+sd, cm sec -1) flow pattern changing from pulsatile to laminar flow immediately after onset of vf. conclusions: the underlying mechanism of the laminar cerebral blood flow observed during vf in our patients is uncertain, but it may provide insight into the prognosis of patients with idiopathic vf. theoretically, the laminar cerebral blood flow observed in our pulseless patients may provide a substantial amount of cerebral perfusion even during clinical cardiocirculatory arrest objective: to investigate whether the intensive care nursing staff can inflate more accurately a specific air volume with the laerdal resuscitation bag when they receive feedback after each inflation about the delivered volume compared to no feedback. method: 42 icu nurses were asked to inflate a testlung model 10 times with a specific air volume (600 ml, 800,ml or 1000 ml) under three different conditions (normal, decreased compliance and increased resistance) without and with feedback. we measured the mean absolute difference from the specific airvolume after each ten inflations. results: the largest absolute difference was found when icu nurses inflated 600 ml (250 ml). the mean inflated volume for this group was 843 ml. when the icu nurses had to inflate 800 ml the mean absolute volume difference was 181 ml with a mean inflated volume of 913 ml. inflating 1000 ml produced an absolute volume difference of 131 ml with an mean inflated volume of 1042 ml. the absolute volume difference decreased when the compliance of the testlung was decreased and even more when the resistance of the used endotracheal tube was increased. when the icu nursing staff received volume feedback after each inflation the mean absolute volume difference was reduced between the 42 ml and 66 ml for all specific air volumes. 42% of the last 5 inflations with feedback were significantly smaller than 50 ml from the specific air volume (p < 0.05). conclusion: the majority of nurses overinflated the specific air volumes. the largest over inflation occurred when 600 ml and the smallest when inflating 1000 ml. when nurses were provided with volume feedback the performed significantly better. we concluded that icu nurses are not able to inflate a specific air volume with the laerdal resuscitation bag without receiving volume feedback. feedback is desirable in order to reduce the volume trauma. objectives: a pro_found impairment in systolic and diastolic myocardial function following successful cardiopulmonary resuscitation (cpr) has been demonstrated by using langerdorff method in rats. in the present study we have investigated post resuscitation myocardial dysfunction in a porcine model of cpr. methods: ventricular fibrillation (vf) was electrically induced by alternating current applied to the ep{cardium of the right ventricle in 11 domestic pigs. following 4 rain of untreated vf, precordial compression and mechanical ventilation was initiated and maintained for 8 min. electrical defibrillation was then attempted and 6 of 11 animals were successfully resuscitated. results: following successful cardiac resuscitation, stroke volume index (svi) decreased from prearrest value of 1.13 ml/kg to 0.74 ml/kg (p<0.05), and left ventricular stroke work index (lvswi) from 1.57 to 0.77 mmhg,ml/kg (p<0.05). both svi and lvswi remained depressed for another 3 hours. these decreases were associated with increases in heart rate from 145 bpm to 185 bpm (p<0.05). no significant changes from baseline in mean arterial pressure, mean pulmonary pressure, right atrial pressure and pulmonary artery wedge pressure were observed. prehospital resuscitation efforts c. k6ppel. g. fahron, h. lufft, a. kruger, c. th(jrk, f. bertschat, f. martens dept, of nephrology add medical intensive care, virchow-klinikum, humboldt-universit~t, d-13353 bedin, germany obiective: the success rate of prehospital resuscitation in patients with cardiocirculatory arrest in an emergency medical system (ems) may reach 30 -40% depending on the time of calling the ems, the distance to cover by the emergency ambulance and the training of the emergency physician and his staff. in the berlin ems, which is associated with the berlin fire brigade, the time between alarm and arrival at the scene ranges from 2 -31 min, mean 8 min. resuscftation is based on the advanced cardiac life support (acls) according to the guidelines of the american heart association. if resuscitation efforts fail to restore circulation, they are terminated after 30 -60 min, depending on duration of cardiocirculatory arrest, pre-existing disease, age, absence of an even transient response to cpr. however, there is a lack of practical criteria for termination of cpr in individual decision making. patients: we report 5 cases of prehospital cpr with primary asystolia terminated after 45 -60 rain of frustraneous cpr efforts including highdose epinephrine and dopamine. results: after termination of cpr, the ecg monitor remained connected and showed permanent asystolia in all patients while the emergency physician completed his records. spontaneous resumption of respiration and circulation was observed in these patients after 2 -5 min and cpr efforts were immediately resumed, nevertheless, 3 of the patients died at the scene, while 2 could be hospitalized with stable circulation. one of them died 3 hours after admission to the icu, the other survived for 3 weeks in a vegetative state. spontaneous resumption of circulation and respiration is most likely due to the development of extreme hypercapnia and acidosis, which -at least in some patients -seems to be a stronger stimulant of the circulatory and respiratory brainstem centers than cpr with high-dose catecholamines, conclusion: because of the legal and ethical implications of this rare phenomenon, emergency physicians should continue ecg monitoring for at least 5 rain. after termination of cpr efforts. pulmonary artery catheterezation is used for patient's monitoring [1]. we reported our results on such monitoring in 1969 [f.coaobbeb,r.fe6enb~-kap~monorm~,1969,n7,p.28-39] .however not all of the received criteria assessments meet demands that are necessary for early diagnosis of critical states. here we report the data on po2,pco2 (mm rg),so2,ph levels in femoral [af) and pulmonary (ap) arteries blood, as well as on summary gas pressure (sgp) calculated from pe=(po2+pco2) in mm hg in ap blood. these data were derived from:i)86 subjects free of cardiovascular pathology according to catheterization data during their spontaneous air breathing (n group in ap blood appears to be a measure of adequacy ratio between pc2 and sgp in ap blood during air breathing; partly its characteristics and variations ranges are presented earlier [2j. in control group it is equal to 1,91• mm hg. tests on sgp neither exclude nor substitute conventional (pc2 and pco2) tests, but rather include them as a part choosing only additive characteristic -pressure. they appear to be a part of general system of human metabolism regulation by pressure (arterial,venous,intracardiac, tissue,liquor,onco-osmotic,etc ietraabdeminal pressure produces perturbations of cardiac, pulmonary, and renal physiology. this most often occurs fonowing eeliotomy for peritonitis or intestinal obstruction; bowel edema and distention prevent wound closure without unacceptable compromise of blood pressure or pulmonary compliance. a variety of temporizing measures have been reported for managing wounds that cannot be closed: 1) using towel clips to reapproximate skin only, 2)i sewing silastic, marlex or other prosthetic grafts to the fascia to "enlarge" the peritoneal cavity, 3) using loosely tied retention sutures for partial closure, 4) simply packing the wound without attempts at c~osure. these techniques either traumatize the abdominal wall (complicating definitive closure), expose the bowel to damage, or allow excessive loss of fluid and heat. since 1989 we have evolved a suturelees technique which permits the abdomen to be partially closed in a quick, safe, sterile, sealed, atraumatic fashion -while providin! decompression of unphysiologic intraabdominal pressure. methods: whenever possible omentum is interposed between bowel and the open incision. viscera are covered by a layer of sterile, non-reactive plastic, placed deep to the fascia and extending we~t beneath the edges. sump tubes are placed above the plastic and covered in turn by two layers of an adhesive plastic drape which sticks to the skin and seals the wound in all directions, the patients remain intubated and paralyzed. results: we have used this technique in a total of 27 patients, four of whom suffered from compartment syndrome. all of the latter were males and ranged in age from 19 to 51. all four showed immediate physiologic improvement. all four incisions were eventually closed without complication. one compartment syndrome patient died 4t days later of multiple organ failure. there were no complications related to the closure technique in any of the 27 patients. conclusions; 1. selected patients with abdominal compartment syndrome will benefit from decompression using this temporary sutureless technique. the technique a) is quick, safe, sterile, sealed, and atraumatic, b) minimizes loss of fluid and heat, c) facilitates eventual definitive abdomina| closure. although m. brunner m. mitllncr objectives: to determine incidence and predisposing factors for cardiac arrest occurring during the first 24 hours after open heart surgery. methods: the study included patients who, following open heart surgery, had adequate cardiac function and in whom cardiac arrest was not anticipated. all data were prospectively recorded and analyzed. results: from 12/1993 through 3/1995, 2140 pts underwent open heart surgery at our hospital. of th~se, 23 pts (1%) (age 65_+9 yrs) had a cardiac arrest during the first 24 hours after transfer to icu. they were operated on for coronary artery bypass grafting (cabg) (17 pts), valve replacement (vr) (3 pts), cabg and vr (2 pts) and aortic aneurysm (1 pt). the preoperative ejection fraction was 44_+12% whereas bypass and aortic cross-clamp time were 127+70 and 72+42 rain, respectively. prior to arrest, they had a cardiac index of 2.23_+ 0.5 l/min/m 2 and were receiving 1.3+1 inotropes. arrythmias leading to cardiac arrest were ventricular tachycardia/fibrilation (10pts) and bradyarrythmia (9 pts). closed-chest cpr was initially performed on all pts and was followed by open-chest cpr in 12 pts. eighteen pts (78%) survived to icu discharge. causes of arrest included perioperative myocardial infarct (t2 pts, 52%), tamponade (3 pts, 13%), rupture of the proximal vein gra& anastomosis (1 pt, 4%), graft occlusion (2 pts, 9%); no cause was found in 5 pts (21%). conclusions: postoperative cardiac arrest in stable cardiac surgery pts is relatively infrequent (-1% incidence) and is associated with a high survival rate following successful cpr. perioperative myocardial infarct is the most common predisposing factor. group ~deptof anaesthesia and intensive care, semmelweis univ. medical school, 2 buda military hospital intensive care unit, budapest background: when a cardiac arrest occurs in-hospital, the outcome can be improved by a higher quality of basic life support provided by the witnessing health care workers until the code team arrives. this basic life ~pport (bls) should include the best available method for airway management as well. since not all medical staff are ready for carrying out endatracheal intnbation, we investigated the effieacy of the use of different airway management methods during bls. methods: we have investigated the efficacy of airway management of 25 doctors and 25 nurses from different hospital wards: internal medicine, department of surgery, trauma, urology and gynaecolagy. comparing the bag-valve-mask, laryngeal mask and the endotracheal intubafion, we have measured the following parameters: time needs for correct application (sec.), number of incorrect applications (out of ten trial), efficacy of artificial ventilation provided by the device. we used a computerised als trainer manikin for the evaluation of the performance. total performance score was created after the measurement between 0-10. after the first screening we held a 2 x 2 hours training. 8 doctors and 8 nurses were trained for the endotracheal intubation (group it1, 1t2) , 8 doctors and 9 nurses were trained to use the laryngeal mask (group lm1, lm2) . all respondent were trained to use the bag-valve-mask device. 1 day, 1 month and 3 month after the training we have carried out retention study using the same method. results: we have found that the efficacy of the artificial ventilation using the above mentioned devices were poor before the training. the average after-training performance scores of the groups are presented in the table below. (bls) should be initiated by the witnessing health care professional. the cpr study introduced a multi level code system, which means bls included sophisticated airway management, early defibrillation and early epinephrine administration provided before the code team arrives. our previous studies confirmed a poor level of cpr performance and a high demand for cpr training among health care professionals. method: we established a cpr training course centre, where doctors and nurses are being trained for in-huspital basic and advanced life support. 3 x 6 hours of training were held. after the theoretical introduction a step-by-step training method ws used for trainees to be familiar with all sequences of basic and advanced life support. then we synthetised all separated sequences. afterwards, a r01e play of rescue groups was taken in simulated situations. we also trained the multi level alarm system fur the in-hospital resuscitations. after the training all respondents had to sit for examination. the quality of performance was scored and compared to our previous results. semi-structured interviews were carried out before and aider the training among all respondents to collect information about the course. results: we have found a remarkably high interest among doctors and nurses in our cpr training courses. it was very important to use proper equipment for the training: audio-visual training facilities, computerised als trainer manikin, manual and automatic defibrillator units. the evaluation of the examination held immediately a~er the training course showed a significant higher quality of performance than before the training. the self.-eonfidence of the trainees for initiating and carrying out resuscitation had increased. their overall feeling about the course was positive and 100% responded the course "very useful". 73.6% of doctors and 79.4% of nurses claimed fur regular training facilities with als trainers, conclusion: the cpr training for health care werkers is mandatory including the training of sophisticated airway management and use of elad~l~ills~tt~r wlaa ~en ~r a~ti~atir ~nel r rm~a'*h*nr m~thnd for training will improve the efficacy, the satisfaction of trainees, therefore their compliance for further co-operation will also increase. s 144 objectives: the effect of reinfusion in emergency surgery and gynecology. methods: we had an experience of autologous blood transfusion in 22 patients whom was produce t an emergency surgical or gynecological interventions in occasion with break tubal pregnancies (45.5 %), penetrating abdominal wounds with injuries of mesenterial vessels (22.8 %), injuries of the liver (9.1%), blunt abdominal trauma with lien ruption (22.8 %). in 27.3 % patients had the previous somatic pathology. blood loss volume was 1500-4500 ml, & the reihfuside blood volume was 500-2000 ml, consisting 30-70 % of blood loss. it was needn't to fransuse donor blood in 18.2 % in further but 300-2500 ml of contanined erythrocytes were frasfused for supporting of hb concentration on the 80 g/l (8 g/dl) rate at the other patients with isovolemie hemodiluttion. results: the arterial blood pressure fast stabilisation on the perfusion level had noted after reinfusion, excluding the case, when the volume of reinfused blood had conisted just 40 % of blood loss at the patient with massive blood loss. complications have noted in two cases. one patient with slash wound, injury of arteria gastrica dextra and total blood loss of 4500 ml, has an episode of asystoly, dic (disseminated intravascular coagulation) syndrome, acute renal failure, and acute pancreatitis that we haven't connected to reinfusion. all the complications were successfully corrected and at thirty first day patient with subcapsular wound of the lien that has happened 14 days before complicated with external rupture of the capsull & massive intraabdominal bleeding, has the hemolytical shock, dic syndrome, acute renal failure developed after reinfusion. he was died. all another have no complications. posthemorrhagic anemia had corrected rapidly than in case when hemorrange corrected exclusively by donor blood. conclusions: we consider that simplicity, accessibility, high effectiveness, quite well further results of blood reinfusion, except the case of blood reinfusing that was for time-expired out of blood vessels (more than 10 days in our case) will promote to the wide spreading of this method, especially in emergency surgery, in massive injuries, & in disarters, all the cases of insufficiently of time for selection of lot of donor blood. objectives: study of a reaction of the oardioreepiratory system of pregnant women to i/v microperfusion of clophelinum which is known to eliminate hemodynsmic and endocrine nociceptive reactions and can be used for treating hypertensive syndrome in pregnancy and labor. methods: the following non-invasive methods were used: capnography, spirometry, oxygenography, indirect fick principle based on the circle breathing, plethysmography and integral rheography~ 52 functional indices of cardiorespiratory function were evaluated. results: 74 pregnant women with ~h-gestosis were examined before and after i/v infusion of i00 ml of 0.0001% clophelin solution, 0.005 mg/kg/hour. before the treatment intensification of carbohydrate metabolism, hyperventilation with moderate hypooapnia and complete respiratory compensation of metabolic acidosis~ increased alveolar ventilation, decreased alveolar volume, predomination of perfusion over ventilation, hypokinetio type of circulation with dominated load by peripheral vascular resistance to the blood flow was observed in this group of patients. microperfusion of clophelin imp~-oved the ventilation/perfusion ratio, ventilatory and gaseous exchange efficiency, resulted in a decrease of congestion in the pulmonary circulation, possibly owing to a decrease of peripheral vascular resistance by 17%, of the heart rate by io.5%, of the oardial output index by 9.5%. conclusionm: the resulted type of circulation with a decreased load on the heart both by resistance and volume allowed to improve the cardioreepiratory system function in pregnant patients. objectives: the injury severity score is a measure of severity of anatomic injuries. iss is a sum of squares of the highest degrees of the abbreviated injury scale (ais) for each of three most severity injured regions. the purpose of the study is to establish correlation between the iss values and mortality rate in older, polytraumatized patients. methods and results: iss was determined for 214 patients. the mean iss value was 27.65 + 17.36 while the median value was 21. minor injuries were present in 90 (42 %) patients with iss less than 21, while 124 (58 %) patients with iss more than 22 had severe injuries. increased mortality of the older patients was noted in the range 21-30. all patients older than 50 died while 20 % of patients below 50 yrs of age survived, indicationg correlation between iss and mortality rate in polytraumatized patients above 50 yrs of age. conclusions: this mode of evaluating severity of injuries may help in triage, determining appropriate level of care and as an indicator of future outcome of polytraumatized patients. objectives : tissue hypoxia is a non exclusive cause of hyperlactatemia. other serious medical situations induce hyperlactatemia. therefore, lactatemia could be a non specific indicator of severity in patients admitted in emergency unit. the aims of this study were to examine the correlations between lactatemia with the short term survival course prognosis and the unit of hospitalisation; intensive care unit (icu) or medicine unit, in patients admitted in our emergency department. methods -lactatemia was measured as soon as the admittance, in arterial blood sample of patients which needed arterial blond gas. sixty-one patients were included during 4 months. to assess the statistical performances of lactatemia, sensitivity (se), specificity (sp) and accuracy (ac) were calculated for the threshold determined by the youden's test (se+sp-1). results : fifteen patients were admitted in icu and 46 in a medical unit. fifteen patients died. a group of 35 patients had a lactatemia up to 2 mmol.l" 1. in this group of patients, 3 had acidocetosis, 3 had asthma, 3 had cerebral vascular ischemia, 3 had neoplasia, 2 had cardiogenic shock, 1 was epileptic, 8 had congestive heart failure, 6 had acute respiratory failure, 2 had septicaemia, 2 had hyperosmolar status finally 3 had medicinal intoxication. lactatemia was significantly higher in non survivor than survivor ( 5.5• vs. 2.3+1.0, p<o.oool, respectively), the cut point of lactatemia was 3.7 mmoll" 1 se was 60%, sp was 91% and the accuracy was 83%. on the other hand lactatemia was higher in patients admitted in icu than in medical unit (4.5+3.6 vs. 2.0• p<o.01, respectively). conclusions : this preliminary results suggest that lactatemia is a good indicator of the short term survival course in patients admitted in an emergency unit. furthermore, hyperlaotemia is present in several pathology seen in emergency. objective: to determine the pattern of injury, total peripheral white cell, neutrophil, and lymphocyte responses, and the outcome of trauma patients who are leucopaenic on admission to the icu. design: prospective, descriptive study of consecutive admissions over4 months. setting: a 16-bed surgical intensive care in a teaching hospital. patients: thirty consecutive adults admitted to the icu following trauma. leucopaenia was defined as a total peripheral white cell count of < 4 x 109, neulropaenia < 2 x 109, and lymphopaenia < 1.5 x 10 ~ cells per litre. measurement and results: the total peripheral white cell count was documented daily and the neurtophil and lymphocyte counts and differential percentages on days 0, 5, and 10. the incidence of leucopaenia was significanfiy higher in patients with gunshot wounds (p < 0.05) and hollow visceral intra-abdominal injury (p < 0.001). eight (27%) patients died. no significant differences were found in the initial mean total white cell, neutrophil, or lymphocyte counts nor in the differential percentages between survivors and non-survivors. the total peripheral white cell count increased significantly in survivors compared to those who died (p < 0.001) and significant differences were found in absolute neutrophil counts (p < 0.02) and differential percentages (p < 0.01) on days 5 and 10. no significant differences were found in lymphocyte counts or differential percentages. conclusions: there is a signficant association between leucopaenia, neutrepaenia, and intra-abdominal hollow visceral injury and peritoneal contamination. survival is related significantly to resolution of these white cell deficiencies. these findings suggest a possible role of granulocyte colony stimulating factor in the trauma patient with intra-abdominal infection and persistent neutropaenia. amelioration of organization emergency care team in order to improve caring for urgent cases. objectives: emergency care team (ect), as integral part of health, respectively as activity of primary health protection care all acute difficult conditions which vitaly danger patients and distressed. the aim of our investigations was to indicate on the possibilities of amelioration of organization ect in order to improve caring for urgent cases. methods: in our investigations we used epidemiologicai and statistical metods all informations and matherials from terrain ects. results: the number of traumatized in traffic accidents etc. have a epidemic character. it is well known that 58.1% traumatized died in first two hours. ect isn't enough qualified to care a traumatized on the place of accident and during transport. because that medical personnel must have an education with solid qualities. conclusions: 1. besides a physician specialized (vii --+ vii2 -~ viii gradus) in urgent medicine, it's necessary to introduce two (2) medical technicians instead of one as is existing now (1 ~ 2). 2.the medical technicians can drive cars and one of them always, drives the ambulance car ("b" category). 3.the technicians have been specially educated for urgent cases (iv ~ v ~ vi). 4. such a new team structure should be more capable in professional sense. 5.we can see an importance of team-work in ects. 6. such teams should also be more economic in comparison with existing one. 7. we think, that on the general medical studies need to include a new discipline-urgent medicine! objectives: to detoxicate an organism since 1983 we have used biological sorbents: isolated from liver and spleen of human or animal cells or tissue fragments. methods: cellular dialysis (164 dialyses) in patients with an acute hepatic insufficiency poisoning by hepatotropic poisons: cc14, dichloethane, mushrooms -were conducted using "artificial kidney" apparatus, by means of arteriovenous shunt, formed at forearm with disposable dialyzers. hepatocytes suspension was poured into dializing circuit of dialyzer at 5-10 mg of cells per i kg of patients weight. dialysis was performed during 1-2 hrs. 168 patients with purulent-septic states were treated as for hemoperfusion through prepared sorptional column: 40.0 ml of hemosorbent and 30.0 ml of fragmented spleen or hepatocytes suspension with the help of roller pump with the rate of 80-120 ml/min, during 90-120 min. average volume of hemoperfusion made 3.51. conclusions: in all cases we used both native and cryopreserved biomaterial. analysis of the dynamics of clinical, biochemical, scintigraphic and us-investigastions allows to conclude that application of cellular-sorptional method of detoxication enable to prolong life and reduce lethal outcome in some severe category of patients with endotoxicosis. after infusion of diazepam (0.2mg/kg),thiopentone (4mg/ kg) and vecuronium (lmg/kg),patient was intubated and mechanical ventilation was adjusted to mantain normoca= pnia. ct scan showed left parieto-occipital hypodensity with brain swelling. after admission in neurological icu,dexamethasone (24mg/ kg/die),mannitol (0.smg/kg) and diphenylydantoin (20mg/ kg/die) were administred. two hours later neurological status of the patien~ was normal and she was extubated. the risks of stereotactie radiosnkgety are related to: hemorrhgge during the latency interval;transient radia= tion effects;permanent radiatio~einduced complications; radiation induced neoplasia. no epilepsy is reported in pts with avm and no seizure before radiosurgery. in c0nclusion, epilepsy can be a posmib~e delayed eompli c~t~n, ~fe~r rgdi0surgery for cerebral avm. serum potassium deficiency is a frequently reported finding in the time course of acute myocardial infarction (ami). if this is also correct for patients with ami undergoing primary percutaneous transluminal coronary angioplasty (ptca) is unknown.for that reason we analysed in 20 patients (14 m.,6 f.,61 + 10 y.) serum potassium concentrations on ad ,mission and directly after ptca.the reference intervall for potassium in our clinical laboratory is 3,5 -5,0 mmol/l. although more than 90 % of the patients with ami undergoing ptca showed normal potassium serum concentrations on admission a measurable decrease of potassium could be found in 65 % of patients (13120) after ptca.therefore for the majority of patients with ami potassium supplementation prior to ptca can be justified to avoid electrolyte disturbances as a possible trigger factor for cardiac arrhythmias in the setting of ami and acute reperfusion due to primary ptca. objectives: diagnostic procedures are very important in the management of abdominal injures in trauma. the aim of this paper is to present our experience with peritoneal lavage as diagnostic procedure in abdominal injury. methods: we analyzed patients to whom peritoneal lavage was applied in the surgical emergency from 1st of january 1991 till 31st of december 1994. results; in this four years period there were 250 abdominal injures. there were 110 blunt injures. among them in 43 patients peritoneal lavage was applied. results were positive in 28 patients and negative in 11 patients. false positive finding were in 2 patients and false negative in two patients. in patients with positive lavage there were more than 90,000/mm3 red blood cells, more than 1,000/mm3 white blood cells and the level of amylase was up to 8 u/i. the gall, urine and stool were positive as well. in patients with negative lavage there were less than 40,000/mm3 red blood cells, less than 500/mm3 white blood ceils and amylase were negative. conclusion: peritoneal lavage is useful and important diagnostic procedure in the management of abdominal injures in trauma. our experience with urgent surgical management of the acute gastric and duodenal bleeding s.se6en md, z.milo~evi6 md, *s.srdi6 md, k.sar6ev md. clinic for abdominal and endocrine surgery, institute for surgery; *clinic for cardiology, institute for cardiovascular diseases; school for medicine novi sad, university of novi sad, yugoslavia objectives: acute gastric and duodenal bleeding are very common complication of gastric and duodenal ulcer. the aim of this paper is to present efficiency of our surgical management of these acute conditions. methods: in this paper results of surgical management of patients with acute gastric and duodenal bleeding, treated in our clinic from the 1st of january 1991 till 31st of december 1994, are presented. results: among 92 operated patients there were 31 females (33.70%) and 61 males (66.30%). there were 52 patients (56.53%) with gastric bleeding and 40 patients (43.47%) with duodenal bleeding. bleeding from gastric ulcer were treated with the following methods: excision of ulcer with suture in 23 patients (44.24%), ulcer suture in 12 patients (23.07%), billroth i in 9 patients (17.31%) and billroth ii in 8 patients (15.38%). heinecke-mikulicz-weinberger pyloroplastica was the most frequently used in the treatment of the duodenal ulcer bleeding. bilateral truncal vagotomy was done in 34 patients (85%), duodenotomy with ulcer suture was done in three patients (7.5%) and three patients underwent billroth ii operation. we had three complicationsduodenal dehiscence in pyloroplasty. there were no lethal complications. conclusions: based on our own experience we conclude that acute bleeding from gastric and duodenal ulcer can be safely managed with urgent and modern operative techniques. dept. of anaesthesiology. emergency center of serbia, belgrade, srj 0b0ectives and methods: cardiac contusion was evaluated by serial determination of cpk-mb fraction and co using non-invasive doppler technique in 60 patients with blunt trauma of the chest. results: miooardial contusion was diagnosed in 34(57%) patients, while in 26 (43%) it appeared unaffected. cpk-mb of 3% and more in comparison to total cpkwas 6.4+4,2% in 34 patients, and 1,9+0,6% in 26 pts (p<0.01).c0 of 5.8+1.17 l/min in patients with cardiac contusion, measured 16 hrs after admission at the latest, was significantly lower than in group of 26 patients with co of 4.8+0.86 l/min (p<0.01). conclusions: co values were significantly lower in patients with cardiac contusion and correlated with pathological values of cpk-mb fraction. objectives: extracorporal plasmophoresis (pph) was implemented by non-stop method in 28 patients with obstructive jaundice. methods: changes of serotonin (s) and histamine (n) in arterial (ab) and venous (vb) blood were investigated before and after one-time session of pph. s and h in blood serum were determined with fluoremetric method. results: before pph the average s contents in ab and vb did not differ. after one-time pph session s in the blood outflowing lungs was 23 % lower than in the blood inflowing. 24 hours after extracorporal detoxication implemented the indices kept the same level. similar dynamics after pph was observed in h. before the session h contens was equal both in the blood inflowing and in the blood outflowing lunge. after pph h contents in ab was 18 % lower compared to the vb and did not change for 24 hours. conclusions: dynamics of changes of biogene amines shows that under the influence of pph there takes place not only mechanical removal of their surpluses together with plasm, but there sharply grows inactivating role of lungs towards ba substances, which is confirmed by reduction of their concentration in the blood outflowing lungs. this effect is probably connected with the "deplasmation" of the lung cellular elements. simultaneously lungs' cells are freed not only from plasm but from toxic adsorbents on their surface. as a result the lung cells activate absorption of the surplus number of amines from the vessel channel. it is not excluded that s inactivation is due to growing activity of monoaminooxidase resulting in deminishing of intoxication. acute pulmonary embolism: thrombolytic therapy or pulmonary embolectomy? i.lodiena md, phd, s.shevchenko md, pphd., medical university, kharkov, ukraine objectives: pulmonary embolism (pe) remains a challenging problem for diagnosis and management for the emergency physician. the aim of this work is to identify the best treatment for massive pulmonary thromboembolism. methods: during recent 5 years 150 patients with symptomatic acute serious pe were reffered to our observations. selective pulmonary arteriography confirmed this diagnosis in all cases. 4 (2.7 %) underwent urgent surgery, of them 2 (1.33 %) with cardiogenic shock and 2 (1.33 %) with massive pulmonary embolism. 146 patients (97.3 %) underwent thrombolytic therapy. results: in the patients who received thrombolytic therapy successful results were achieved in 133 cases (91.1%), in 13 (8.9 %) patients subsequent pulmonary embolectomy was performed. mortality rate was 2 (1.36 %) in the patients who underwent pulmonary embolectomy after unsuccessful thrombolytic therapy and 4 (100 %) in the patients with cardiogenic shock and massive pe. lower extremity deep venous thrombosis caused pe in 89 % patients. pe mortality rate is repoted to be 4 %, but all patients with cardiogenic shock and massive pe died. this very high mortality rate related to the clinical status of the patients whose condition became worse despite intensive treatment. conclusions: the results of this study show that in most cases thrombolytic therapy is an effective rapid method of treating pe. however, surgical treatment is preferable when the patients reveal adverse effects to drug therapy, when medical therapy is unsuccessful or when the patients are seriously ill with reccurent cardiac arrest. high-quality pulmonary angiography remains the most accurate and reliable means of diagnosing pe. this prospective study was conducted to investigate the serum thyroid hormone levels in traumatized critical ill patients. serum thyroid stimulating hormene(tsh),total thyroxine(tr4),free thyroxine(ft4),total tri-iodothyronine(tr3),free tri-iodothyronine (ff3) levels were measured within 24 hr.of intensive care unit admission(first day) in 17 multiple tratmmtized male patients(injury severity score higher than 17). in fifth day the same hormone levels were repeated. tsh,et4,ft4,tr3 and ft3 levels were determined by radioimmueoassay(ria). the levels of 'i~sh,xt4,fr4,et3 and ft3 in survivors and nonsurvivors were compared with first and fifth days (table) . in conclusion, we are convinced that there was a high correlation between low ser~n thyroid hormone levels and mortality, serum thyroid hormone levels are prognostic value in traumatized critical ill patients. objectives: glucagon improves myokardium contractility, intensifies kidney blood flow, stops bronchospasm (1), these were the reasons of investigating its effect on hemodynamics and metabolism in hypovolemic shock expermentally and in clinic. methods : in experiments on white rats with traumatic shock glucagon (novo nordisk) was injected intraperitoneally in dose 20 rocg/kg. patients with hypovolemic shock (trauma, hemorrage) were injected glucagon intravenously in dose 1 mg on the background of infusion therapy. results : glucagon hightened recovery indexes of glucose in rats and patients with shock, whereas in groups without glucagon hyperglycemia developed. rats with glucagon injection had double times urea content than the norm ( 10,5+1,5 mmol& norm -4,9+0,3 mmol/i ), whereas rats without glucagon had urea content 30,3 +1,8 mmol/1 ( p < 0,001 ). osmolarity of blood plasma in rats having glucagon injections was 301,5 + 2,9 mosm/kg h20, without the drug -317,0 + 3,5 mosm/kg h20 ( p < 0,01), ldh activity -27,7 + 2.9 and 51,0 + 9,6 ( p < 0,01) mcmol cec-1 accordingly. lethality in the group of rats without glucagon was 70% /n=50/, with glucagon -0 (n = 70 ). in patients with hypovolemic shock in 15 minutes after glucagon administration stroke volume increased to 71% (sv), miocardium contractility -to 70% (mc), peripheral resistance of vessels ( vr ) decreased to 10%. in 1-1,5 hrs sv increased to 100%, mc-to 126%, pvr -decreased to 116%. conclusion : the study provides the evidence that glucagon has an anti-shock effect, improves hemodynamics and metabolism indexes in cases of hypovolemic shock. reference : picazo j. glucagon in acute medicine : pharmacological, clinical and therapeutic implications (norwell, mass:kluwer publishing,1993 ), 172 p. n, vasina (t) ,n.sebbota hph (2). dept; of acute endotoxicosis, n.v. s~lifosovslay scientific research institute of ermergency ltealth care, boscow, lk'ussia (i}. institute for problems of cryobiology and cryouledicine of the national academy of sciences of the f/maine, kl'k3r-key, [7maine (2), objectives: the investigation of isolated hepatocytes using's efficacy in patients with acute hepatic fai lln-e. methods: native and crioconsrved allo-and xeno-!mmatocztes fixed on semi;ermeable ~mbr~s were used. results: the artificial supporting system with isolated hepatocttes as metabolic ~its was. ap-plied in 85 patients. during tl~ treatmen~ with this cytotherapz an improvement of clinic state and biochemical rates were observed even inthe first 8 we~s. for instance, the kncreasing of glutamin acid's level in blood up to normal value and decreasing of ammontes -concentration from i9~26 mmol/l down to 89#15 mol/l were discovered. ~ae diminution of ence,%halo;athy was noted too. general lethality was' equal 37 z, there of 2i z concerned acute hepatic failure (/k~). conclusions: the using of sum~rting hepatic system is able to cope the ~nenomenon of ~i~. objectives: fat embolism (ee) is an important and insufficiently studied problem of the intensive care medicine. fe is clinically diagnosed in 25% and morphologically in 80-90% of the victims with severe multiple skeletal injury that is accompanied by the shock. methods: during the last 5 years 27 patients with the cerebropulmonary form of fe were treated in the icu. the prognosis of the probability of fe development was done using the computer system "cite-prognosis" in which the triss-method, dynamic and statistical prognosis of the injury outcome~ and the values of the most severe injury in points were realized. the treatment consisted of the surgical stabilization of the fractures with the authors' design of the rod apparatus in the acute period, long-term mechanical ventilation with peep via tracheostomy, complex fluid therapy using perfluorochemical emulsions (pe), and electrochemically active solution of na hypochlorite (snh) via the central vein. results: the treatment of 27 patients with fe gave a good result that was manifested by the complete regression of psychoneurologic and respiratory disorders. conclusions~taking into account the prognostic data~ it is necessary to provide early complex of intensive care to the polytraumatized patients. it includes the surgical stabilization of fractures, fluid therapy using pe and snh~ early long-term mechanical ventilation. dr. alexandr e. poladko, station of medical aid. kiev ukraine. objectives: the reason of investigation is to prove the necessity of beginning of the intravenous infusion of nitroglycerin before the hospitalisation. methods: from january i to june 30 1994 protocols of treatment myocardial infarction patients in kiev medical aid station. results: the results of treatment are better if infusion began early or prolonged during hospitalisation. conclusions: there were 30 patients with acute myocardial infarction treated before hospital by the cardiological groups. 100 patients were treated with the early infusion of nitroglycerin and 200 without. the results of treatment were: in the group with infusion: mortality in the first hours 5 % the full disappear of pain 90 % in the group without infusions mortality in the first hours 8 % the full disappear of pain 82 % that's why our opinion is that infusion of n. is necessary in the treatment of the patients with myocardial infarction from the first minutes of illness. dr. gennady d. kyrzhner, station of medical aid, kiev, ukraine. object: looking for safe medication which is good for the treatment of arterial hypertension and is simple for use. we inspected the effect of prazosin in the conditions of the cail during the year. method: we used 1 mg prazosin tablets sub lingua and measured blood pressure 4 ones during 2 hours. the parameters were registrated in protocol. result." one hundred patients took part in investigation. the middle age of patients was 69. the reason of hypertension was not taken into consideration. in two hours after beginning of the treatment we caught the reliable lowering of blood pressure in 90 % of patients. the general condition of patients became better in 10-15 min. it was only one accident of complication during the treatment -the orthostatic hypoter~sion, conclusion: prazosin hydrochloride in the dose 1 mg sub lingua is safe and simple for use in the urgent situations. objectives: hypomagnesemia is frequently observed in severely burned patients during the early period after injury. increased urinary excretion is insufficient to explain the magnitude of the magnesium (mg) depletion. the possibility of exudative cutaneous mg losses has been proposed, but not yet demonstrated. the study aimed at measuring the mg content of the cutaneous exudates and urine during the first week after major burns. methods: 16 patients, aged 34_+9 years (mean _+ sd), and burnt 37_+11% of body surface, were studied from day 1 (d1) to d20. serum samples were collected at do, serum and urine from d1 to d7, and on dio, d15, d20. cutaneous exudates were extracted from the textiles surrounding the patients, collected over 24h periods from d1 to d7, using bidistilled acidified water. mg supplements (8.2 to 123 mmol/24h) were prescribed from d1 to dio according to the severity of hypomg (serum mg < 0.7 mmol/i). results: mg serum levels decreased below reference ranges in 12 patients between d1 and d4, and normalised thereafter during supplementation. urinary mg excretion was increased in 11 patients, mean excretion being 4.8_+1.3 mmol/24h until d20. mean daily mg cutaneous losses, were 16 mmol/24h, i.e. 112 mmo; in 7 days. large inter-& intra-patient variability: the daily exudative losses ranged from 0 to 83 mmol/24h. conclusions: the mean daily mg cutaneous losses after burns were larger than the urinary losses, and equivalent to the recommended intakes for mg; the addition of the exudative and urinary losses largely explained the increased mg requirements after burns. complex immunologic alterations occur following thermal injury. the activation and consumption of the complement and dotting systems are suggested to play an important role in the development of the capillary leak syndrome, tn burned patients, a generalized inflammatory reaction as well as the impairment of the host defense are considered to be the major reasons for complications, such as sepsis and multiple organ failure. in a pig model we investigated a possible protective effect of cl-inhibitor (berinert, behring). before conducting the animal experiments the human cl-inhibitor concentrate was analyzed for its inhibitory capacity on purified pig cls and its pharmacoldnetic behaviour in pigs (t1/2,id50). pigs received anesthesia and analgesia and arterial, venous and pulmonary (swan ganz) katheters were placed into the carotis and jugular veins. the pigs were scalded for 25 seconds with 75~ hot water to achieve a 30% tbs partial-thickness burn. blood samples were taken prior and after surgery as well as 15, 45 minutes, 4, 8, 12 and every further 12 hours after thermal trauma. two control groups (each n=6) included animals which were either scalded or not scalded and treated only by resuscitation of ringers lactat solution. besides various parameters blood samples were analyzed for complement. the pigs (n=4) received c 1-inhibitor concentrate at an initial dose of 100 u/kg body weight either immediately or 12 hours after thermal trauma (n-2), followed by three further applications (of reduced amounts) every 12 hours. the clinical outcome as indicated by vital parameters and as well as demonstrated by reduced edema and inflammatory tissue destruction suggested a protective effect of cl-inhibitor. complement analysis revealed a faster recovery of the alternative and classical pathway activities in cl-inhibitor treated pigs as compared to the control group but only if the regulator was given immediately after thermal trauma. from these results a protective function of c 1 -inhibitor on thermal trauma can be assumed. objectives: the aim of study was to characterise the pattern of secretion of interleukin-6 (il-6) and tumor necrosis factor alpha (tnf alpha) in multiply injured or not injured critically ill patients and to relate these results to their outcome. methods: blood samples of 12 multiply injured ( 238 (73) 148 (46) 154 (57) il-6 (nis) 156 (81) 88 (54) 65 (39) il-6 (nin) 117 (68) 150 (76) 157 (04) conclusions: this data suggest tnf alpha is a better prognostic marker in patients with multiple injury, than in critically ill patients without injury. il-6 is not significantly higher in nonsurvivors of both groups. sepsis is associated with an increased production of nitric oxide (no), as reflected by a rise in plasma levels of nitrites (no2) and nitrates (no3). severe bum injury is associated with similar symptoms of inflammation like hypotension, high cardiac output low vascular resistance and increased vascular permeability so that an increased no production may be involved. the present study included 16 patients admitted to the intensive care unit of the burn center of brussels (age 35:1-18 years; burned surfaee area 374-19 %), and 6 control (non-septic) patients hospitalized in the neurological rehabilitation unit of the erasme hospital (age 64 4-18 years). the patients in both groups were fed enterally with 30 to 40 kcal/kg/day of a standard solution. in the burn group, daring the study period, 6 patients were mechanically ventilated, 4 required vasopressor (dopamine) therapy, and 5 received antibiotics; 13 patients were discharged alive from the intensive care unit, and no patient died during the study period. plasma was sampled for no2/no3 determinations (griess reaction) daily from day 1 to day 5 (n=ll) or to discharge (day 3, n=2; day 4, n=3) in the burn group, and once the control group. in the burn group, no2/no3 levels were elevated at day 1 (70_+ 12 #moles/i), reached a maximal value on day 2 (874-26 p.moles/l) and progressively decreased to day 5 (fig). these values were higher than in the control group (5.6:t:7.6 #moles/l, all differences p<0.01). however, no correlation was found between the plasma no2/no3 levels and the age of the patient or the total burned surface area; burned patients who became infected tended to have higher plasma no2/no3 levels than those who did not (177 + 131 vs 83 4-48 #moles/i, ns). the present study indicates that burn 120 noa/noa (llmol**/l) injury is associated with increasedloo] [__ ] ~ no2/no3 plasma levels, which are ao so consistent with an enhanced no pro-4o duction, ao obiectives:urapidil has been recommanded for therapy of hypotension in neurosurgical patients, because it was found not to increase intracranial pressure in patients with compromised intracranial dynamics (1). in contrast several authors reported an increase of icp after urapidil administration in patients with head injury (2). our study was designed to determin the influence of urapidil on mean lumbar cerebrospinal fluid pressure (csfp) in awake humans with uncompromised intracranial compliance. methods: after approval by the local university ethics comittee, six volunteers (asa 1, 4 male, 2 female) were studied in the lateral decubitusposition with the vertebral column positioned horizontally. a 25 gauge needle was inserted into the lumbar subarachnoid space at level l3/l4 and connected to a trancducer (mx860| medex inc.) for csfp measurements. the volunteers were advised to keep respiratory rate and etco2 constant. after a resting period of 10 minutes basline measurements were performed including csfp, cvp, map, hr, etco2 (cardiocap 2| datex). then the volunteers were given urapidil 0.2mg/kg over a period of 1 minute. 5 minutes later measurements were repeated. results: statistics: wilcoxon signed rank test, p<0.05 significant; values: mean_+sd, si = mmhg. 10-2_1" 74_+5* 64_+5* -3_+1" 35_+2 csfp: cerebrospinal fluid pressure, cpp: mean cerebral perfusion pressure. conclusions: during normoventilation urapidil leads to a decrease in map parallel by an increase in csfp and therefore in icp, most likley mediated by an autoregulatory dilatation of cerebral vessels. if this cerebrovascular response is suppressed by hyperventilation, no increase in icp is found despite a uredipil induced drop in map (2). after longterm hyperventilation cerebralbloodflow is normalized. this might be the reason why the drop in bloodpressure following urapidil administration again led to an increase in icp ( background and objectives: the intestine is one of the most sensitive tissues to ischemia-reperfusion injury. reperfusion of the intestine is often associated with increase in mucosal permeability and ulceration. d(-)-iactate is produced by indigenous bacteria found in the gastrointestinal tract and mammals do not possess the enzyme systems to rapidly metabolize it. the present study was designed to determine the kinetics of plasma d(-)-iactate alteration in rats paused by acute intestinal ischemia-reperfusion injury. methods: following the anesthesia, the superior mesentcric artery (sma) was exposed and it was occluded by a micro-bulldog clamp applied at its origin from the aorta. after 75 min of occlusion, the arterial clamp was removed and the supply area of the sma was allowed to be reperfused ( 6 h). plasma d(-)lactate levels were measured by an enzymatic spectrophotometric assay. the endotoxin content of plasma sample was assayed by the limulus amebocyte lysate test with a kinetic modification of the test kit procedure. results: intestinal ischemia for 75 min resulted in a significant increase in d(-)-lactate levels within the portal vein as compared to sham-operated animals. plasma d(-)-lactate levels had a tendency to further increase after reperfusion up to 6 h. it was showed that significant elevation of endotoxin concentrations in portal vein was alread~r detected at the end of 75-min ischemia, reaching peak at 0.5 h post-reperfusion and decreasing gradually throughout the study period. at the end of ischemia, marked mncosai damaged such as edema, hemorrhage and necrosis was observed. there was evidence of injury to ti, e muscuiaris propria together with diffuse mucosal damage and destruction following reperfusion, particular at 6 h postreperfusion. in addition, penetration of bacteria was commonly found in the intestinal wall at various time points following ischemia/reperfusion injury. conclusions: these data suggest that acute intestinal ischemia is associated with permeability change of mucosal barrier resulting in increased plasma d(-)qactate, which is also remarkably influenced by subsequent reperfusion. plasma d(-)-lactate may be a useful marker of intestinal injury following both ischemia and reperfusion insult. objective: to assess the clinical usefulness of two methods of intracranial pressatre (icp) monitoring: intraparonquimatous recording (icpc) and epidural recording (icpe), versus intraventricular icp (icpv) as a gold standard. we prospectively studied 13 patients with severe head injury (glasgow coma score < 8) admitted to our icu between fobmaly 1990 and december 1993. icpv was monitored in all pativrats by means of a multipzffolated catheter connected to a water coinnm. six of 13 patients were additionally icpc monitored by a fiber optic transducer (comma 420r~), and seven of 13 patients were icpe monitored by a couplvd fluid system (plastimedr). icpc was placed homolatetal to the focal, lesion m two cases and contralateral in three; icpe was placed homolateral to the focal lesiou in two cases and contralateral in three. all three systems were calibrated at the ear level. stali~cs: correlation coefficient and lineal regression were done between icpe and icpv, and between icpe and icpv with the hourly p~rs of values obtained dttting assistontial period. results: of the six icpc -icpv studied pa~onts, we observed a correlation coef~cieut r = 0.84 (p < 0.001) with a regres~on line y = 0.75 + 0.93x. four of ~hx lcpc -icpv patients had r > 0.85 when correlaliou eoet~dent was obtained indixddually. of the seven icpe -]cpv studied patients, we observed a cortelafiau ooeffioiont r = 0.47 (p < 0.001) with a regression line y = 7.2 + 0.43x. corralalmu eoetfieiont was inwer than 0.5 in all seven patients. corrdation eoelfieients for levals of icpv > 20 man hg, > 25 mm hg and > 30 tuna hg with icpe showed r = 0.89, r = 0.91 and r = 0.97 respectively; and with icpe r = 0.25, r = 0.13 and r = 0.09. the obtained values did not change during the study. conclusdns: in our study icpe was considered a good type of icp monitoring. /cpe signiticantly infravalorates icp values. we observed a good correlatinn between icpc and icpv values in patients with high inttacramal presanre. objective: midazolam is a benzodiazepine agonist widely used for sedation in emergency medicine. few studies in animals and humans point to a direct analgesic effect of midazolam probably mediated by spinal antinociceptive receptors and/or peripheral benzodiazepine receptors (1,2). in our experience in the berlin emergency medical system (unpublished results) with anecdotal cases of extreme chest pain due to binge drinking but no evidence of acute myocardial infarction or extreme abdominal pain due to peritonitis, acute intermittent porphyria, peutz-jeghers syndrome or testicular torsion, we found that small doses of midazolam (2 -5 mg i.v.) were much more effective in relieving pain than repeated administration of high doses of buprenorphine or morphine, which may be associated with a considerable respiratory depressant effect. the dose of midazolam required for pain relief in these patients is non-narcotic and allowed further communication on the character and localization of' the residual pain, which might be very important for the further diagnostic procedure. patients: ten patients with abdominal pain due to acute gastrointestinal bleeding, suspected pancreatitis, suspected acute porphyria, and chest pain with no evidence of acute myocardial infarction received first-line midazolam i.v. at an initial dose of 1 mg and were asked how it affected the intensity and character of pain. results: at the chosen dose of midazolam (2-8 mg), all patients were responsive to detailed questioning on basic orientation, the character, intensity and localization of the pain, and medical history. none of the patients required an additional opiate. all patients stated that the pain was tolerable after midazolam alone. conclusion: our preliminary clinical observations suggest that low-dose midazolam might be an alternative to opiates in extreme pain of presumably visceral odgin. objectives: it is known that severe head injury in elderly patients is associated with higher mortality than in younger patients. it remains however to be clarified whether the preinjury pathology which is frequent among these patients, affects the outcome. methods: in an attempt to investigate this hypothesis, 79 patients aged over 60 years suffering from head injury, with glasgow coma scale (gcs) of 8 or less, were studied retrospectively. twenty-six patients (32.9%) had preinjury pathology i.e. diabetes mellitus, arterial hypertension, heart failure, alcoholism, parkinson's disease etc. (group a) and fifty-three (67.1%) did not (group b). the following data were recorded: mortality in the i.c.u., duration of hospitalisation, incidence of infective complications and neurologic status at discharge. results: groups were comparable in terms of mean gcs (6.57 vs. 6.56) and median age (67.5 vs. 67). the incidence of brain pathology in the two groups was the following: epidural haematoma 7.69% vs. 11.32%, acute subdural! haematoma 30.7% vs. 30.19%, intracerebral haematoma 19.23% vs. 5.66%, subarachnoid haemorrhage 38.46% vs. 39.62%, diffuse haemorrhage 11.54% vs. 13.21%, contusion 26.92% vs. 49.06% and non-visible pathology (normal ct) 3.85% vs. 1.89%. unilateral pupilary dilatation was found to be 15.38% in group a and 18,87% in group b. the mortality during hospitalisation in the i.c.u. was almost the same: 50% iu group a and 47.2% in group b patients. however, group a patients had significantly more infective complications, required longer hospitalisation and had lower gcs at discharge. conclusions: the results show that the existence of preinjury pathology does not seem to affect the short-term outcome of elderly patients with severe head injury. it has however an impact on morbidity and perhaps long-term survival of these patients. the assessment of clinical development in intensive care patients with severe head injury still remains a problem. to optimize the monitoring of intracraniel prassure (icp) we rautlr~dly implant an eplduml measuring device in our hospital. the aim of this study was to prove the correlation of the icp-values with ct findings and clinical development. during a 12 month period (1993 -9r the icp was monitored in 23 p~,tients (14 male, 9 female) with severe head injury by an eplclural measuring device (epldyn~/$plegelberg| the mean age was 36.9 years (4 -83). the glasgow coma scale at admission was 6.9 (3 -15). in all cases the device was placed wfihln the first 10 hours after admission. the tcp was compared with physical examination, radioidglcal or intraoperatlve findings and cunlca! outcome. the average time of measuring was 7. 2 days (1 -19) . the traatment depended on the !cp values recorded. rising icp-valuea ~ed to radlologlcal c0ntra!s by ct-scan. in 1 case an intracranlai hemorrhage was detected and drained. the overall survival rate was 78.3 %. 113 showed a complete resolutl0n, in other 33.3 % psychological residuals like decreased mentatlon, in 17.4 % sensomotorlc residuals like cerebral nerve dysfunction and aphasia, and 11.1% of the injured remained in a comatous status. in 87 % of our cases the measured values correlated with clinical course and management. in 2 cases (8.6 %) we observed a displacement of the icp-pevice. there was no icp induced infecllon. istituto di anestesiologia e rianimazione, universit& ,,la sapienza", rome, italy * istituto superiore di sanit& -servizio di epidemiologia e biostatistica, rome, italy objectives: acute renal failure (arf) can be a severe complication of trauma. the current incidence of post-traumatic arf is associated with high mortality 1. identification of risk factors and prevention of this complication could improve the outcome of trauma patients. methods: one hundred fifty three consecutive trauma patients (age 37.6 _+ 19.6, injury severity score 28.3 +10.9) admitted to icu were studied. incidence of arf was 31.4 % (48/153). arf was defined as persisteat plasma creatinine >2mg/dl with or without oligoanuria 2. arf was defined as early when occurring within the first 96 hours (earf) and late when the onset was after the first four days (larf). results: earf occurred in 31 patients while larf developed in 17 patients. age, iss, and incidence of rhabdomyolysis and acute respiratory failure were not different in the two groups. an higher incidence of multiple organ failure (mof) and sepsis (76.6% for both) were observed in larf group, when compared to earf (25% and 23% respectively). abdominal trauma was more frequent in earf group (32% vs 18%). the gs for earf and larf were respectively 8_+4.4 and 9_+4.15 while in the group who not developed arf (narf) the gs was 10.5• conclusions: gs score difference seems suggestive and can be that an abnormal cerebral activity (hipofisary hormones?) may play a crucial role on onset of arf in these patients. moreover the frequency of acute respiratory failure in the group of arf was higher (91.7 versus 64.5) than narf group. the early ipoxia in the early phase of trauma, then, may be another crucial point for development organ failure. these are preliminary data. a more exact statistical analysis must be perform to have definitive conclusions. to compare the active compression-decompression cardiopulmonary resuscitation (acd-cpr) with the standard cardiopulmonary resuscitation (s-cpr) in out of hospital cardiac arrest patients. is a controlled, randomized study. two groups of patients with cardiac arrest out of the hospitalwere formed. group i, (acd-cpr) and group ii (s-cpr). for the acd-cpr groupweusedthecardiopumpdeviceofambulnternational. asfortherest, the erc (1992) algorithms for acls were followed. the utstein style (for out of hospitat cardiac errest) was used for listing and evaluating all cases of the study. the cpr was contucted by the crew and the doctors of our mobile intensive care units (micu). we studied 146 consequitive patients (75 in group i) and (71 in .group ii). demographics pre-cpr characteristics (e.g. ecg form of cardiac arrest) and procedures (eg bystanders or second tiers crew cpr, defibrillation, drugs) were quite similar for both groups. the mean arrival time of micu was 9min. in group i we recorded r.o.s.c. (return of spontaneous circulation) 17,5%, death 73%, continuation of cpr efforts 9,5%. while in group ii, 21%, 69%, and 9,9% respectively (recorded percentage until the admission to the hospital). no significant difference was found in anyofthe short term outcome parameters. no complications related to the acd-cpr technique, were noted. not any significant difference between the two methods was proven (from this small evaluated sample). the results of previous clinical studies are controversial (i) . more sophisticated studies proved the superiority, in a certain number of parameters (e.g pressures, flow, etc) of the new technique although there are many difficulties for establishing clinical results. in the pre-hospital setting that is related to many parameters (speed of the intervention, effectiveness of bystanders cpr, education ofparamedics, etc.)the evaluation is even harder. the superiority ofthe acd-cpr can be proven when it is performed in almost 0 times increased number of studied patients as w~ll as improvement of the technique could lead us to more established results. objectives; infectious morbidity is the major cause of mortality after burn injury, and is due to multiple factors. trace elements (te), which are involved in both humeral and cellular immunity, exhibit severely altered status after burns. te supplementation has been shown to be associated with increased leukocyte counts and shortened hospital stay. the trial aimed at studying the immune responses in severely burnt patients receiving normal te supplies or early large supplements. methods: 12 patients, aged 40_+16 yrs (mean_+sd), with burns covering 49+18 % of body surface were studied from day 1 (d1) to d30 post-injury, were randomised in 2 groups (g): g1-control receiving recommended te supplies + placebo; g2 -receiving in addition large supplements of cu, se and zn from d1 to d8. enteral nutrition was started within 12 hours of injury in all patients. immunological parameters: peripheral leukocyte counts, proliferation of mononuclear cells to mitogens, cell surface molecule expression, and neutrophil chemotaxis at d10 and d20. infectious episodes and micro-organisms were monitored until d30. results: the patients' characteristics were similar g1 & g2. the total leukocyte counts were higher in g2 between d10 and d20, due to increased neutrophils (significant from d13 to d15). total cd3+ and cdlg+ cells did not differ, whereas cd14+ (monocytes) were significantly increased at d20. proliferation to mitogens was significantly depressed in all patients. chimiotactism was not altered. the number of infectious episodes was significantly decreased in g2 with a mean of 2.0_+ 0.9 infections during the first 30 days versus 3.3_+ 0.8 in the control group (p < 0.03). conclusions: the large te supplements for 8 days was associated with a significant decrease of the number of infectious episodes. supplementation was associated with increases in total leukocyte, monoeyte and neutrophit numbers. further studies are required to determine the precise mechanism underlying the improved immune defences. objectives: evaluate the efficiency of local adsorption (la) with the use of carbon adsorbents in case of severe burns in expertment and clinic. methods: experimental studies on la were performed on a model of 20% body surface area iiib-iv burn in 335 rats. a burn eschar was excised on the 3rd day after burn, the wounds were dressed with the gauze bandages (control) or with adsorptive dressings (la), dressings were regularly changed. clinical investigations were carried out in the course treatment of 78 patients with severe thermal and radiation ilia-iv burn. in the dynamics of bum disease some indices of proteometabolism and intoyacation criteria were evaluated. results: the experiments have demonstrated that the application of la after early excision of a burn eschar exerts a pronounced normalizing effect on a protein electrophoregram and the activity of proteases and their inhibitors in burned tissues preserving vitality. thus, by the 14th day after burn infliction the activity of cathepsin d in injm'ed muscles is 6 times lower under an adsorptive dressing than under a gauze bandage (control) (p<0,05), the activity of trypsin-like proteases is 1.5 -3.4 times lower and the antitryptie activity does not differ significantly from the normal level. the cytotoxicity of extracts of burned tissues after the adsorptive dressing application fn vivo and adsorption in vitro is 25-35% and 7-20%, respectively, of the toxicity of control extracts. a similar normalizing effect of la is ok~rved for an intact muscular tissue and blood serum. the dectron-spin-resonance studies have demonstrated that la allows to normalize antitoxic activity of liver and functional activity of kidneys. the application of la in the treatment of patients with severe burns have been shown to localize a region of irreversible tissue changes, accelerate rejection of a burn eschar, attenuate an endogenous intoxication level and, as a result, shorten the time for grafting of a burn wound and accelerate wound heating. conclusions: proceeding from the obtained results, we can consider la as an effective method of localization of a region of irreversible tissue changes as well as of correction of local and general metabolism failures and overcoming burn autointoxication during burn disease. c de deyne, t vandekerckhove*, j. decruyenaere, b. vaganee, v vandewalle*, f colardyn depts of intensive care and neurosurgery*-university hospital gent-belgium. jugular bulb oximetry is the first bedside available cerebral monitoring technique providing an estimation of the adequacy of cerebral perfusion. its routine use in all patients suffering from severe head injury admitted to our ic unit enabled an extensive analysis of all very early cerebral perfusion data in order to evaluate the incidence of abnormal sjo~ data (and their possible causes) in this very eady period after traumatic insult and to search for possible implications as to the emergency management. these very early data were defined as the first 6 hours icu data and icu admission had to occur within 12h of traumatic insult. over the last 2 years, 150 pts with severe head injury (gcs<8) were monitored by jugular bulb oximetry, starting immediately after their arrival at the icu (mean of 4.8h after trauma, range between 2-9h). in a total of 85 pts (=56.6%), jugular bulb desaturatiens (<55%) were noticed during this early 6h period. in 24 pts (=16%), jugular bulb saturations higher than 75% were observed, whereas 41 pts (=27.4%) revealed no abnormal sjo 2 data (55-75%) during these first 6h. concerning the periods with too low jugular bulb saturations (n:85), we found the following correlation ; in 49 pts (=57.6%) cerebral perfusion pressure (cpp) was below 70 mmng, in 36 pts (=42.3%) paco~ was below 30 mmhg and finally in 6 pts (=7%) we found primary intracranial hypertension. for the high jugular saturations (n:24) we found a primary intracraniaf hypertension in f0 pts (=41%), and a pace 2 level above 40 mmhg in 6 pts (=25%). in all patients we could restore jugular bulb saturation within normal range (55-75%) with the correct!on of the presumed causative factor. we can conclude that ultra early jugular bulb saturation data revealed a high incidence of abnormal values, with a predominance of jugular bulb desaturations, confirming once again the high incidence of disturbed and too low cerebral perfusion within the first hours after severe head injury. these jugular bulb desaturations were especially correlated to systemic causes, as a too low cpp (caused in the vast majority by primary map insufficiency, and not by intracranial hypertension) and hyperventilation were the 2 major causes of the desaturation periods. as jugular bulb desaturatione are known to be significantly correlated to a worse neurological outcome after severe head injury, one might improve outcome by an emergency management avoiding these possible causes of jugular desaturation. therefore, extreme attention should be paid to the maintenance of an adequate mean arterial blood pressure (above 90 mmhg?) even duhng the few time spent at the emergency department. one should be as attentive to the maintenance of normoventilation during this very early period of admission and hyperventilation without any knowledge of icp or sjo2 should be abandonned. recently, indomethacine has been proposed for the treatment of therapy refractory intracranial hypertension in pts suffedng from severe head injury (1). indomethacine, a cyclo-oxygenase inhibitor, gives rise to a significant fall in cerebral blood flow by inducing cerebral vasoconstriction. therefore, its use could result in a drastic lowering of the intraeranial pressure (;cp) in pts suffering from intracranial hypertension secondary to cerebral hyperaemia and in whom the use of other cerebral vasoconstrictive drugs (barbiturates or hyperventilation) appears insufficient to control icp. for the last 18 months, we included the use of indomethacine in our therapeutic flow chart for severe head injury management. pts revealing intracranial hypertension (icp>20 mmhg) and cerebral hyperaemia (sjo~>75%) and in whom icp was not efficiently controlled by the combined use of hyperventilation and barbiturates were given indomethacine in a trial to control icp. a total of 98 head injured pts received treatment for intracranial hypertension over the last 18 months. six of them met the criteria set for the administration of indomethacine. in 2 pts, no decrease in icp or in sjo 2 was observed and both pts died due to therapy refractory intracranial hypertension. in the other 4 pts, a significant fall in icp and in sjo 2 was observed shortly after indomethacine administration. in 2 pts we observed a catastrophic fall of sjo= even below 55%, indicating an extreme cerebral vasoconstriction with the possible risk of inducing cerebral ischaemia. in one of the 4 pts, icp remained under control without further administration of indomethadne, but he died 3 days later in multiple organ failure. the other 3 pts, needed multiple indomethacine administrations (for 1 pt even during 4 consecutive days) to finally control icp. in all 3 pts, icp was finally controlled, but only 1 pt survived. both other pts died from systemic causes (multiple organ failure in 1 pt, massive gut infarction in the other tat, possibly due to the systemic vasoconsttictive effects of the indomethacine administration). in conclusion, indornethacine might have a role in the treatment of intraoranial hypertension, especially when caused by cerebral hyperaemia. we observed however a poor final outcome and a threatening high incidence of systemic events (multiple organ failure, gut infarction) in those pts receiving indomethacine for icp control. therefore, indomethacine in the treatment of intracranial hypertension should be reevaluated in controlled study settings, before its routine use can be considered. untill recently, intracranial hypertension (ich) in pts suffering from severe head injury was managed in a staircase approach, with csf drainage as first therapeutic step, mannitol as second step, hyperventilation as third step, and finally, barbiturates as the last rescue step for therapy refractory ich. this staircase approach for the treatment of tch was only guided by the intracraniat pressure, and not by other parameters such as e.g. the actual state of cerebral perfusion of the concerned pt. jugular bulb oximetry provides us with the first, bedside and continuous available, estimation of cerebral perfueion. its implementation in a rigourous flow chart, based on as well icp-as jugular bulb oximetry-data might result in an altered strategy for ich management. we adopted a '~ugular bulb saturation (sjo~)-guided approach" for ich management in 86 consecutive pts, suffering from severe head injury (gcs<8). we maintained csf drainage as first therapeutic step, but the decision for the second step was guided by sjo2 information. pts revealing ich and sjo=values above 75%, were treated with hyperventilation, and did not receive mannitol. if ich persisted, barbiturates were added as a third step. on the other hand, pts with ich and sjo= vales less than 75%, received mannitol administration as second step. hyperventilation and/or barbiturates were only added if ich persisted and if no cerebral hypoperfusion was discerned (sjo=>55%). our objectives were to prospectively analyze this new therapeuticstrategy, as compared to the formerly used staircase approach of ich. we managed 86 pts with ich, with an overall mortality of 13.7% due to therapy refractory ich. all pts received standard primary care with head elevation, full sedation and normovenfilation. fer 16 pts, csf drainage alone was sufficient to control ice of the remaining 70 pts, 38 pts received mannitol and 32 pts were hyperventilated as second approach. in the third line, 14 pts were managed with barbiturates, 12 with mannitol and 10 pts with hyperventilation. finally, barbiturates were used as the final rescue in 14 pts. these results reveal a less frequent use of mannitol as only 50 pts received mannitol, compared to the 70 pts that would have received mannitol using the former staircase approach. hyperventilalien was used much earlier in the treatment course, as 32 lots were already hyperventilated in the second line approach, were this was formerly exclusively reserved for the third line approach. finally, also barbiturates were used much eadier (14 pts received barbiturates as third approach). we may therefore conclude to a important change in the management of ich, induced by a sjo2-guided flowchart. however, future studies will have to elucidate if this new strategy for the intensive care management of severe head injury will also result in an improved outcome. obsectives: in a first series of experimental brain injury we investigated the course of brain po2, icp and cerebral blood flow after traumatic brain injury (tbi), whilst accordingly there are very few data available and the mechanisms leading to secondary brain damage are poorly understood. methods: in 6 piglets (14 days old, 3,3-5 kg) of either sex we produced a moderate brain injury (1,5 arm., 20 msec.) using a lateral fluid percussion {fp) device. complete measurements were made before and 5 min. after brain trauma and after 3, 5 and 24 hours including blood gases, cardiac output (htermodilution), heart rate, eeg, laser doppler flow probe (ldf} and icp values (camino), brain temp., po 2 by a clake type oxygen electrode (licox) and coloured microspheres for regional blood flow. results: immediately after the trauma a typical "cushing"response to the icp peak up to 130 mm hg being highly significant (before mean i0 mm hg, range 4-12 mm hg) could be observed: mean arterial blood pressure rose from appr. 85 mm hg to ii0 mm hg for 3-5 min. in two animals this was followed by an ischemic period lasting 15 min. accordingly icp values gradually returned to starting measures within 3 hours; in the ischemic animals they remained at a level of about 30 mm hg.-no secondary increase of icp could be observed, once icp dropped to starting values within 24 hours. cerebral blood flow (ldf) fell from mean values being i00 before trauma to appr. zero and recovered to around 50. brain po 2 started at mean values of 20 mm hg (range 15-30 mm hg) and fell to around zero depending upon the severity of the ischemic reaction. on average values of 15 mm hg were reached over the time course. conclusions: with our fp trauma model we can reproduce the well known "cushing"-response after brain injury; secondary icp elevations cannot be achieved, although local edema is observed. direct brain po 2 measurement seems to be a very sensitive variable for detection of cerebral ischemia and anticipates eventually following icp elevations by far. pulmonary aspiration s,traoaras. v. sgountzos, p. agouridakis, m eforakopoulou, e. ioannidou. intensive care unit (tcu) of "kat" hospital, athens, greece ob!e=ives: the reported mortality rate after pulmonary aspiration is variable in several series. the purpose of this study was to find out the influence of preexisting disease or situation on morbidity and mortality of intensive care unit (icu) patients with pulmonary aspiration. methods: patients who were treated in icu and had pulmonary aspiration, were studied, entrance's criteria in the study, all of them obliged, were: 1) suction of gastric contents from trachea during intubation, 2) presense of a predisposing factor, e.g. coma. 8) recent hypoxaemia or new infiltrates in xray. preexisting disease was recorded and correlated with complications and outcome. patients with glasgow coma scale 3, because of cerebral injury, and patients who died within 3 days from cause other than aspiration, were excluded from the study. method of statistical analysis: chi-square test, results: one hundred forty five patients were studied. the trauma patients were 96 and the non trauma patients 49. from the trauma patients, 77 had cerebral injury and 19 were polytreumatized without cerebral damage. from the non trauma patients, 13 had malignant neoplasms, 14 neurological diseases in terminal stage, 7 old age, 10 drug overdose, and 5 several diseases. eighty seven from 96 trauma patients (91%) and 45 from 49 non trauma patients (92%) manifested several complications (pneumonia, ards, etc), so there was no statistical difference in complications' frequency between the 2 groups (p>0,1). the severity of complications was also proportional in the 2 groups. eighteen deaths were recorded in the trauma patients (mortality 19%). only 7 deaths correlated directly or indirectly with the aspiration (7%). in non trauma patients, 32 deaths were recorded (71%). twelve deaths were recorded in 13 patients with neoplasms, 12 deaths in 14 patients with neurological diseases, 6 deaths in 7 aged patients, 1 death in 10 drug overdose patients, and 1 death in 5 patients with several diseases, the mortality difference in trauma and non trauma patients was statistically significant (p< 0,001). in patients with drug overdose the mortality was significantly lower from the other non trauma patients and the difference was statistically significant (p<0,001). conclusion: the preexisting disease or situation plays a major role in the outcome of the patients with pulmonary aspiration. the mortality of patients with aspiration seems to be caused by severe preexisting situations rather, that lead to death, than from the pulmonary aspiration per se, which may be a final happening in a predetermined course. obiectives; the purpose of this study was to compare fluconazole and amfotericin-b in the treatment of fungal infections in severe trauma patients. methods: thirty five severe trauma patients who were treated in intensive care unit (icu), were studied prospectively. they all developed fungal infections, prooved with blood positive cultures and at least one of the following: fever, positive urine or bronchial secretions cultures, infiltrates in xrays. the patients were separated randomly in 2 groups. the patients of group a (15 patients) received fluconazole 200 rag/day for 15 days. and the patients of group 8 (20 patients) amfotericin-b 50 rag/day for also 15 days. compaiison's criteria were the clinical responce to treatment (fever etc), the fungal elimination (blood and other cultures), the relapses of the disease, the side effects of drug, and the outcome of the patients. as method of statistical analysis was used the chi-square test. results: nine patients from 15 of the group a (60%), and 18 from 20 of the group b (90%), presented remission of fever (patients of group b had better clinical responce than patients of group a, and the difference was statistically significant, p<0,05). all the patients before treatment had positive for fungi blood cultures. after 10 days of treatment, 3 patients of group a and none of group b had positive cultures. eight patients (from 13 who had positive cultures of bronchial secretions before treatment) of group a. and 5 (from 17) of group 8. had positive cuttures of bronchial secretions after 10 days of treatment, so positive bronchial secretions were fewer in group b than in group a, but this difference wasn't statistically significant, (p<0,1 and p>0,05): ten patients (from 12) of group a and 7 patients (from 16) of group b had positive urine cultures, after 10 days of treatment (positive urine cultures were fewer in group b than in group a and this difference was statistically significant. (p<0,05). two patients of group a and none of group b had a relapse of fungal disease. in group a, no side effects were obsepced, while in group b were observed only minor side effects (small increase of serum creatinine in 2 patients, chills and fever during infusion in 3 patients, and hypokalemia in 12 patients). three patients of group a and 1 patient of group b died, because of sepsis. conclusion: amfotericin-b (even i~ short regimen of 15 days), is superior to fluconazole in the clinical and laboratory responce and also in the relapse of fungal disease, fluconazole is superior to amfotericin-b as it has no side effects. ob!ectives: flail chest after thoracic trauma is a serious injury. it is controversial if flail chest by itself orthe concomitant intrathoracic injuries e.g. pulmonary contusion, is the cause of the reported significant morbidity and mortality. in this study we searched the influence of concomitant thoracic injuries in the course and outcome of patients with flail chest. methods: eighty five patients with flail chest after isolated chest injuries were studied, for the purpose of analysis, we separated the patients into 4 groups, patients with isolated flail chest were included in group a, patients with flail chest and hemo-pneumothorax in group b, patients with flail chest and pulmonary contusion in group c, and patients with flail chest and hemo-pneumothorax and pulmonary contusion in group d. complications from the chest, duration of mechanical ventilation and mortality were compared in the 4 groups. statistical comparison of results belween groups was made using chi-square and t-studend tests. results: the patients were 85. all patients received mechanical ventilation, twenty eight patients were ihcluded in group a, 19 in group b, 20 in group c. and 18 in group d. seventy three patients manifested complications from the chest, especially pulmonary infections. there was no statistical difference among the 4 groups as to number of complications ( twenty four patients had chest complications in group a, 16in group b, 17 in group c, and 16 in group d. p>0,1}. the duration of mechanical ventilation was not statistically different among the 4 groups (the mean duration was 15,9 days in group a, 16,8 in group b, 16,5 in group c, and 17,5 in group d, p>0,1). there was also no statistical difference in mortality among the groups (six patients died in group a. 4 in group b, 4 in group c, and 5 in group d, p>0,1 ). conclusion: flail chest by itself is a serious thoracic damage with many complications, regardless of the presense of other thoracic injuries, which don't contribute to greater morbidity and mortality. the present study investigated the correlation between blood lactate mortality and organ failure in 129 trauma patients admitting between december 1, 1992 and july 31,1993 in the icu. road traffic accidents were the most common cause of trauma in this studded population. brain damage was the main cause of mortality .nevertheless, 29 of patients died from sepsis and multiple organ failure without significant brain damage and these deaths were potentially preventable. respiratory failure was the most common complication and was developed in 44 (44%) of survivors and in 26 (86%) of non survivors .we noted low fncidence 5 of renal failure may be do to the early and aggressive ittv'asive hemodynamic monitoring and cardiopulmonary support. as part of our routine case protocol serial blood lactate levels were measured in each patient at least 3 times a day until the valses returned within the normal range or until death. we analysed the blood lactate levels on admission, the highest value and the number of days until the first normal value (<l.smeq/i). initial lactate and highest lactate levels were significantly higher in patients with than without organ failure.(3.4 vs 2.4, 4.1 vs 2.8meq/l, p<0.01)the duration of hyperlactemia also was higher in the patients with organ complications. (2.2 vs 1.0 days, p<0.01). both initial lactate and highest lactate levels were higher in the non survivors than in the survivors.(4.0 vs 2.8,4.6 vs 3.4meq/l, p<0.01) the present study demonstrated that not only the degree but also the duration of hyperlactemia can be related to the development of posttraumatic complications. serial blood lactate measurements are reliable indicators of morbidity and mortality following trauma. s155 current evidence suggest that peep only affects intracrenisl pressure (zcp)when it interferes with systemic arterial pressure, although the effects of peep over cerebral oxygenation remains unknown. objective: determine the effects of peep over icp and cerebral metabolism measured by sj02, kjdo 2 and ceo 2 in severe head injured patients. meterial ~ methgds: patients with glasgow coma scale l 8 at arrival, with difusse brain injury in the first gt, according to marshall criteria,lop and 8jo 2 monitored, under analgesia and sedation, normoventilated with zeep, without mannitol treatment in the previous 4 hours, normal x ray chest and within the first 48 hours from injury, were considered candidates. peep value where increased fro~ zero to 15 cmh20, in three different steps, each one with 20 min of duration. all date were analized on line, pmax p'p ..... p.._, from the ventilator, systemlc haemodynem]c data, cerebra perfuslon pressure (cpp), icp and sjo 2, in the case of hemodynamic deterioration, during the study, ppc 6ommhg, extra-volume were administered. if persistence, dopamine were begun or increased dosage. if no good response were obtained, patients were retired from the study. results. 40 patients were candidates, 15 of them were exc;uded due to haemodynamic instability, k total of 25 completed all steps and were e~amined, 22 men and 3 women, age 32!7.9 yrs. at hospite; arrival, glasgow were < 5 in 18 patients, and >5 in the rest 7. 15 patients 20 mmhg at the beginning. zeep ob/ectives. critically ill patients are transpoded to an intensive care unit(icu), under conditions, which have not been systematically evaluated. therefore, we set suite investigate transportation and admission condition of these patients to our department. methods. we studied 36 patients(16 females), aged (mean-..+-sd) 56.2_ 17.3yrs, which were consecutively (from august 1994 to march 1995) admitted to the icu, through the greek national emergency transporta~on service. apache ii severity score upon admission was 17.4-+6.8 (range 4-31). the following data were evaluated: 1) number of medical departments, where health care was provided until final admission to the icu, 2) ambulance transportation conditions, 3) catheters and tubes inserted before admission, 4) vital signs upon admission 5) information provided by referring physician (scored on a 1 to 5 scale: history, electrocardiogram, chest x-ray, laboratory data, drug therapy already administered), 6) comparison of the state of the patient described by referring physicians, to the actual state u pen admission. resu/ts. one to four medical departments had provided health care before the palient was admitted the icu (1:22.2%, 2:47.2%, 3:27.7%, 4:4%). thirty/36 (83.4%) patients were escorted by a physician. twenty-six/36 (72.2%) were transported on oxyge n, fio2(mean__.sd): 46-+3%, pao2: 78.6-+35.2mmhg. five of the remaining 10, for whom no oxygen was provided, had pao2: 46.2-+7mmhg. twelve/36 (33.3%) were intubated and ventilated during transportation. thirtyfour/36 had a peripheral venous line, 5/36 had an arterial line, 13/36 had a nasogastdc tube, 20/36 had a urinary catheter. eleven/36 were sedated and 2/36 were paralysed. three/36 were on inotropes. vital signs upon admission were: arterial blood pressure, systolic 100.6-+44mmhg, diastolic 57-+23mmhg, heart rate 104-+22 bpm, temperature 36.3 -+2cc. patient information score was 217--. 1.7. the actual state upon admission was found substantially different, as compared to the description of the referring physician, in 28/36(77.7%) patients. conclusions. we conclude that several aspects of the greek national emergency transportation service to an icu should be reevaluated and further improved, i. e. ventilatory support, adequacy of information provided and accuracy of prior description of the patient's state. a new perspective must be applied for critically ill patients transportation since 78.8% of the patients were evaluated and treated in more than one, medical departments, mostly primary care, before they were finally admitted to our icu. dclhb is a human derived hemoglobin molecule that has been cross-linked to stabilize and permit heat pasteurization to remove residual proteins and inactivate viruses. dclhb is mixed with a lactated electrolyte solution to yield a total hemoglobin concentration of log/dl objective: to present an overview of four recently completed clinical safety studies of dclhb in the u.s. and europe, and to discuss the properties, actions and potential indications for dclhb. method: patient populations in the four studies included males and females ranging in age from 18 to 84 years. dosing ranged from 25mglkg to 300mg/kg. the controlled randomized safety studies were conducted in chronic renal failure patients, surgical patients undergoing total hip replacement or abdominal aorta repair and in hemorrhagic hypovolemic shock patients. these very diverse patient populations allowed safety evaluation of the product in patients who were generally elderly, often hypertensive with some degree of cardiovascular disease, and receiving medications for treatment of other conditions. results: over 150 patients received dclhb in the four:studies. no product related sarious adverse events occurred during the clinical trials. conclusion: results from phase itll safety studies of dclhb in patients undergoing chronic renal dialysis, abdominal aorta repair, or total hip replacement and in patients in hemorrhagic hypovolemic shock, indicate that the product was well tolerated in these distinct populations. although these studies were designed to evaluate safety, the data suggest clinical benefit. follow-up efficacy trials are indicated. prehospital emergency services represent the extension of emergency care into the community and constitutes the manpower, communications, transportations and facilities used to provide care for patients outside hospital. one of the main points of the system is how to decide the hospitalization of patients and what kind of facilities to provide : emergency medical service, fire brigade, locat general praclitionner or ambulance officers. objectives : to realize guidelines for using the prehospital emergency medical service in case of patient'calls outside hospital. methods : from 1st june 1994 to 14 july 1994, all the calls for emergency care were analysed using a questionnaire of 114 items (origin of the call, responses to the questions of an emergency practitionner, kind of emergency service provided and the issue of the patient). after taking account of the appropriatness of the decision, statistical method used was a logistic regression. results : 996 calls were analysed. the criteria, for prehospital emergency medical service using, given by the logistic regression were as following : existence of a call for emergency, thoracic pain, dyspnea, seizures, cyanosis, drug intoxication, fall of the patient, fracture, age, the state of consciousness and the neurologic reactivity. the minimal and maximal predictive values of the model given by the logistic regression are respectively 2% and 100%. the performance of the model is 88 %. conclusion : it seems possible to help medical decision of emergency medicine by using only some easy criteria and a predictive model. (italy) objective: to evaluate the incidence of blunt carotideal injury (bci) in patients admitted to our icu after head injury. methods: we reviewed the medical records of all patients diagnosed to have a bci. at admission, the severity of trauma was assessed either with glasgow coma scale (gcs) and with ct scan. bci was demostrated by doppler ultrasography (us) and by angiography (ang). results:since may 1991 to april 1995, 4 patients were admitted to our icu with bci (2m,2f, age 29+ 1 3). a history of direct trauma was present in 2 patients. admission gcs was 15 in all patients, and was associated with hemiparesis in 3 of them; the last became paretic 48 hours thereafter. two patients had concomitant injuries (a homoiateral clavicular and a controlateral zygomatic fracture, respectively). the initial ct scan was negative in every patient, and showed signs of ischemia after a variable timespan (2-4 days) after the onset of the symptoms. the bci was diagnosed with us and ang, which demonstrated a thrombosis of the internal carotid artery (ic). in two patients, an intimai dissection was also present. three patients were treated with heparin associated with antiaggregating agents and were discharged alive. the last patient was referred to our icu after the development of a massive hemispheric infarction, and died three days after the admission. at necropsy, the ic thrombosis was associated to an extensive homolateral extra and intracranial venous thrombosis. conclusions:the presence of focal neurological signs despite a negative ct scan should address the diagnosis toward a bci, thus implementing the diagnostic workup with us and/or ang. tab i: distribution of l~tients (%) in the 3 groups the outcome were monitorett results were sabmitted to statistical analysis using a continence table 3x2 in z2 test. res.cl~s: of 43 patients 34 were submitted to thrombolysts and 3 died. the higher incidence of bracb, ar~lhmias (ii degree gg p t39e 1 and 2 av block. 11i degree av block. avsb 6.141 <ii.05 sinus arrest) that requited the insertion of avsc 11.121 <cl005 temporar~ pace-maker was recorded in group a b vs c 0.004 ns in 64% of the cas6~. the rapid recognition of life tab li:;~ test. threatening conditions suggests the monitoring of v4r-lead r b' in the course of inferior ami. the authors report their own expirience in application of ringer lact.(rl)and 7,5%nacl/6%dextran 70. methods:in 40 polytraumatized patients with developed hae morrhagic shock,injures of the chest(qg)and abdomen(q2) prevalid.replacement of the volume loss 5egan in institutions of general medicine,frequently by rl and 69dextran 70 or haemaccel.after receiving necessary medical aid,polytraumatized patients wer transported to mma by helicopter,continuing replacing of circulation volume by the same solution. results:average quantities of the solution 8dministered ~o the injured wer~2375-2550ml.a group of the inoured which,after the admission to mma was resuscitated by adml nistration of the 7,5%nacl/6%dextran 7@(groupii)(4-sml/kg observed to the final surgical management,had statistically higher map(increase of 58% in relation to admission), cvp(average increase of 8,25cmh~o),diuresis(averagely 35omi after 30min.),better gas ~nalyses and acid-base status in relation to the group which was administered only rl(groupi)(increase of the map of q8%,average increase c~ of cvp 2,5cmh20 and average diuresis 20oml after 3omin). total quantity of the administered solutions was significantly smaller in the groupii(2800ml:4925ml).the only ob served complication was hyperhatr~f~a::and~moderat~hyper osmolerity of plasma which disappeared within 32hours. the quantity of replaced blodd was considerably smaller in the groupii(qooofnl:20ooml).in the group i interstitial pulmonary oedema was observed in two patients(qo%),while: in the group ii no complication was observed.coagulation i disorder and hypotension were not recorded because of the fast infusion of hypertonic/hyperoncotic solution in the groupil as the infusion of 7,5%nacl/6%dextran 70 lasted 5 minutes. w.scherzer(l~,k.lechner(1),r.simon(2). ll)dept.anaesthesiology,trauma hospital,vienna-meidlin 9 !(2)neurotraum.rehabilitation center,vienna-meidling both austrian workers'compensation board,vienna,austria what we usually call"unconcious" means only that the patient is not able to interact in any kind of way.experiences in intensive care medicine (robinson,1975) ,in general anaesthesia (bennet,1985) and with evoked potentials show,that unconcious patients are not automatically unhble to process and recall information.this implicates a challenge to start the efforts to restore the traumatica-!ly desintegrated brain function as early as possible in the acute phase ia (zieger,1992) .we present a method to ~repare the patient for the rehabilitation goals of the postacute phase ib,the phase of stabilisation ii and of rehabilitation iii within one concept. to achieve sensory stimulation in phase la we use: attempt at some kind of dialogue by speaking to and tou-ching the patient,to make him experience his body limits, ~acio-oral therapy by ice application,tast and smell pro4 vocations and trials of feeding,early adaption to circadian rhythm and guiding the patient in every-day-life-ac-tivities e.g.:in the brushing of teeth,washing etc. to achieve motor stimulation and orientation in terms of ~-ace,time and gravity in phase la we use: avoidance of perceptual impairment as produced by air-:sack-beds or habituation to supine position,using method~ according to affolter(1980) ,basal stimulation (bienstein, 1991) , bobath-therapy(1966) and facilitation of a physiological moving pattern(pnf)according to knott(1968) . conclusions: by integrating all these measures in the ~herapy and nursing even in the acute phase la one prei ares the comatose patient for multisensory stimulation nd motor training and all other rehabilitation activities in the following phases of therapy. s157 objectives." measurement of cardiac output (co) and stroke volume (sv) requires insertion of a central venous catheter and is associated with a considerable risk for complications. non-invasive methods for determination of hemodynamic parameters have been introduced recently. one of these methods is the aortic modelflow program, which provides co and sv continously using a three-element windkessel model. the aim of the study was to evaluate the accuracy of the aortic modelflow program in critically ill patients. methods: 21 patients (mean age: 69) admitted to the emergency department after cardiopuimonary resuscitation (n=ll) and for myocardial infaction (n=5), acute congestive heart failure (n=4) and anaphylactic shock (n=l) were included to the study protocol. after stabilization all patients received a swan-ganz catheter (baxter~; edwards swan-g-anz) via a central vein. co and sv were measured by the use of a cardiac output catheter and injection of 10rrd ice-cold glucose 5% non-invasive cardiac output measurement was done by using the continuous cardiac output software package modelflow (tno; portapres| results: overall, 159 paired measurements were used for the evaluation of the accuracy of non-invasive obstained sv and co. mean values, standard deviation and range of co and sv evaluated by invasive and non-invasive methods are summarized in the aortic modelflow provides reliable hemodynamic data in critically ill patients. it may be a useful alternative due to its non-invasive approach and continous measurement. objectives: to determine the feasability and accuracy of a rapid urine screening test (triage tm, merck-diagnostika) in an emergency department. methods: prospective analysis of the triage tm testkit (tt) in patients admitted because of suspected drug abuse during an observational period of 6 months. the tt is a eompetitve immunoassay which gives qualitative results within i0 minutes for methadone, benzodiazepines, cocaine, amphetamine, opiates, barbiturates and cannabis. urine samples were analysed with the tt and compared to the results of a enzyme-lihked tmmuno-assay (el.a) in our labratory (gold standard). results: during the study 25 patients were enrolled, in 19 of these patients substance abuse was proven by tt and verified by eia. we recorded no false positive or false negative results for benzodiazepines, barbiturates, opiates, cocaine and cannabis. two results were false positive and one false negative for methadone; one result was false negative for amphetamines. the triage-testldt had an overall sensitivity of 0.94 and specificity of 0.94. conclusions: the triage tm testkit seems to be a a useful rapid test device in addition to quantitative tests for drugs of abuse in an emergency department. objectives: the purpose of this study was to evaluate the influence of several factors of the renin-angiotensin-aldosterone system on blood pressure response in patients with hypertensive crises. methods: patients with systolic blood pressure >210 rorohg and diastolic blood pressure > 110 nunllg were included into the study. prior to treatment blood samples for determination of plasma renin activity (pra), angiotensin converting enzyme (ace), angiotensin ii (ang ii) and aldosterone (aldo) were collected. all patients received 5 rog enalaprilat intravenously. success of treatroent was defined as a reduction of systolic blood pressure below 180 mmi-ig and diastolic blood pressure below 95 mmi-ig within 75 minutes after start of treatment. results: 35 patients were included in our study, 20 (57%) patients responded successfully to treatment. mean arterial pressure decreased in responders by 36.5 mmhg and in non-respenders by 12.7 mmhg (p<0.001). responders and non-respenders differed signii'icantly concerning pra (p=0.001), ace (p=0.003) and ang ii (p=0.04). 0.003 0.04 the extent of blood pressure reduction correlated positively with the pretreatment pra and ang ii concentrations (correlation coefficient for pra: r=0.43; ang ii: r=0.66). conclusion: our data confirm that in patients with hypertensive crises blood pressure response to ace inhibition is mainly determined by circulatory pra, ace and ang ii. as the extent of blood pressure reduction correlates with pra, ace-inhibitors in patients with suspected high renin status cannot be recommended, as excessive blood pressure reduction, which carries a considerable risk for further organ damage, may occur. f. staikowsky, n. grillon, f.pevirieri, c.jedrecy, c. zanker, f. michard, a. haft medical emergency department. hospital bichat, paris epidemiology of acute intentional self medications-poisoning (smp) in france is especially known by data of poison control centei,s and intensive care units (icu). the purpose of this study is pro~,ided characteristics of this problem in a med for adults. method: july 1992 to june 1993, files of patients consulting to the ed for smp have been retrospectively analyzed. results: 727 patients, 482 women and 245 men, 33.3 + 12 years old (range 15-92) have been admitted for 804 episodes of smp (4% of all consultations) whose 77 relapses during the period of study. psychiatric disorders, drug addiction or hiv patients was found for respectively 42.6%, 9.1% and 2,9% of patients. the interval of time between the ingestion and emergency consultation was noted for 43% of smp (332 + 532 min, ranges 15-4320). the involved products name was known in totality in 89% of cases with an average number by episode of 1.7 + 1 drugs (ranges 1-8). the most often, 1 (52%) or 2 (21%) different products were interfered. the nonbarbiturate psychotropic drugs accounted for 76.7% of the products (benzodiazepines 67%, antidepressants 9.5%, neuroleptics 8%, carbamates 5.8%, imidazopyridines 5.1%, cyclqpyrrol0nes 2.7%). analgesics and nonsteroidal antiinflammatories represented 6.8% of all drugs, anticonvulsants 3.4%, cardiovascular drugs 2%, antiinfective agents 1.9%, drugs against cough 0.86%, muscle relaxants 0.86% and antihistamines h1 0.5%. the benzodiaz6pines were present in 531 episodes, alone in 316 episodes. in 36.5% of cases, there was a simultaneous intoxication with alcohol. the processing consisted of gastric lavage in 32.5% of cases, activated charcoal in 16.7% of cases, flumazenil in 16.9% of cases, naloxone and acetylcysteine in 3.4% of cases; orotracheal intubation was performed in 12 patients. admission in hospital was effective for 280 patients, in medical ward (n = 156), psychiatry (n = 63) or icu (n = 62); no fatal case was recorded. conelusion: smp to ed are often benign. the benzodiaz6pines are the most often incriminated but the new anxiolytics and hypnotics (imidazopyridines and cyclopyrrolones) take a growing place. the latsion burn center of athens. its planning constructive and functional refinements j. ioannovich, a. petalas-vourekus, d~ serbetis, h. carsin a 18 bed burns unit is under construction following a donation to the general hospital of athens. the plan of the unit, covering a surface of approximately 3.500 m2 is based on the principle of three identical 6 bed satelites which may function totally independent from each other. in the center of the unit the common facilities are installed, like operation theatres, storage rooms etc. this new modification in the plan of a burn unit is presented in this paper. the advantages from the fucntional, administrative and medical point of view are discussed. tiffs anisotropic conduodon could favour the ocenrence of a circular movement of the impulse that leads to tachyeardias by reentry. purposes of this work were to study, with the help of epicardial mapping, the influence of a trieyclie antidepressant, clomipramine (c), on the conduction velocity longitudinal (vl) and transverse (vt) to myocardial fiber orientation and on anisotropy (a = ratio vl/vt), and their modificutions by the sodium bicarbonate (13). method: a plaque of 64 electrodes, positioned on the left anterior ventricular wall of 9 anesthetized dogs, allowed to deliver, thanks to central electrodes, programmed electrical stimulations inducing vcuttienlar complexes, and to collect them. each entailed unipolar dectrogram was processed by a computer system that drew the isochrones and a map of activation allowing the calculation of v. the c was infused (0.5 mg/kg/min iv) during 75 rain; at t60, dogs received the b until the retuni of qrs to its initial value fro). a lengthening of qrs of at least 30% of its value at to was demanded before the administration of b. results: 1 dog was excluded because of an.~nsufficient prolongation of qrs before the administration of b. all values (map : mean arterial pressure, i-ir : heart rate, qrs andqt intervals, v) differed significatively (13<0.05) compared to values control fro)except qrs at t65. the b (7 + 6 ml/kg; ranges 2.8 and 20.5 ml/kg) modified no studied dements outside of the ( }rs. to ti5 t30 t45 t60 t65 t75 a 2,1 + 0,6 2,1 + 0,5 2,1 + 0,4 2,1 + 0,4 2,1 + 0,7 2,1 + 0,7 2,1 +-0,~ conclusion : the c slowed v l and v t without modify the anisotropy. the b did not modify the v of~conduction while the qrs prolongation was corrected. the c acts as a class i antiarrythmie drug on the inward sodium current during the phase 0 of action potential; the gap junctions have shown to be important in the conduction and an action on the gap junctions such as a modulation of the junctional resistivity, can not be rule out. is the doctor a heroe ? p. t.schies~.he, t. bauer, m. seyr dept. of anaesthesiology and intensive care, aokh krems, austria objectives: helicopter emergency services (hes) are getting popular more and more. the results concerning outcome are encouraging. however, some recent accidents with dead or badly wounded hescrew-members have shown the relatively high risk for the crews. therefore we were interested to eval0ate the motivation of physicians to participate in a hes. this survey was designed to investigate current concerns about safety and motivation of doctors on emergency call. methods: a questionnaire was sent to 205 doctors of the austrian emergency system. the survey consisted of multiple choice questions and subjective scoring tables from 1 (--full agreement) to 5 (=disagreement). overall, 64"/. of the active emergency physicians participated in the survey. results: 61.1% of the doctors assume the system is basically safe, experienced doctors tended to have less trust in safety. only 13% would not hesitate to go into action by dark. 14.8 % stdctly refuse night flights to accidents outdoors. although defibrillations are assumed to be safe dudng flight, only 29% would do it. 52.8% of the doctors would rather stop flying. the most common reasons for 9,uitting were wish of family and fear of an accident. 47.2% conclusioq: short transportation times help to avoid trauma related stress, pain and shock-induced organ complications. therefore the physiologic and economic advantages of hes are undebatable. however, the survey data indicate a considerable concern about safety of the medical personal in a hes. 14 crash landings within less than 10 years with 15 deadcases and 17 badly wounded crew members in a small country like austda make desire for safe flying conditions understandable. obiectives: to evaluate the clinical usefulness of trachlight. methods: trachlight is a new device facilitating endotracheal intubation. a stylet with a lightprobe is inserted into the endotracheal tube. intubation is guided by the light glowing through the neck tissues, thus rendering direct laryngoscopy unnecessary. intubation using trachlight was studied in 37 patients (age 21-68 years). the indication for intubation was elective surgery in 21 patients (asa i-ii) and emergency intubation in 16 patients. in the elective patients, anaesthesia was induced with thiopentone supplemented with fentanyl, and intubation was facilitated with vecuronium. the cause for intubation in the 16 emergency patients was dyspnea in 8, cardiac arrest in 2, trauma in,2 and unconsciousness due to drug overdose or seizures in 4 patients. intubation was facilitated with medication in 12 patients. results: of the elective 21 patients, 19 (91%) were successfully intubated. six patients (29 %) needed two attempts before successful intubation. the duration of intubation exceeded 30 seconds in 8 patients (38 %). of the emergency patients, 14 (88%) were successfully intubated. six patients (38%) needed two attempts, and the duration of intubation was more than 30 seconds in 9 patients (56 %). in 54 % of all 37 patients, intubation was assessed as easy. no or insufficient glow, prolonging intubation or necessitating two attempts, was noted in 11 patients (30 %). oesophageal intubation occurred in 2 patients. conclusions: trachlight may be a valuable adjunct for intubation in varoius settings provided that adequate training is provided. a learning curve was found to exist. objectives: to compare enoxaparin and standard heparin in cavhd and calculate the value of laboratory controls in the treaanent. patients and methods: twenty patients needing dialysis for acute renal failure participated in the study. the main exclusion criteria were massive bleeding or a thrombocyte level < 50 x e9/i. in each treatment the same type (av-400, fresenius ag, germany) of a polysulfone capillary haemofilter was used. the study scheme consisted of two consecutive four-day cavhd treatments, one course for each type of heparin. the order of heparin administration was counterbalanced between patients. the standard heparin was given as a continuous infusion aiming at an activated coagulation time between 200 and 250 s. the initial enoxaparin dose was 80 rag every 8:th hour intravenously, but was modified by any signs of coagulation in the dialysis blood lines or bleeding complications. results: the dialysis treatment was adequate in both treatment modes, with mean blood urea levels 24.3 and 25.2 mmol/l respectively (ns). the bleeding complications were moderate and similar in both treatment modes. the mean life-span of haemofilter using enoxaparin as an anticoagulant was some longer than using heparin (35.7 +31.6 h versus 22.3+26 h, ns). the mean aptt-levcl during heparin treatment was 79s and during enoxaparin treatment 54s (ref. 24 -34s). the mean daily dose of heparin was 422 nag, that of enoxaparin lg7mg. the mean anti-xa activities were 0.40 u/mi and 0.47 u/mi, respectively, reflecting a better bioavallability of enoxaparin. conclusions: both anticoagniation modes were equally effective and well tolerated. the amount of enoxaparin needed for a proper anticoagulation was, however, less than half of that of standard heparin. the changes in aptt level were too slight to make its use possible in controliing the dose of enoxaparin. the use of enoxaparin seems to be rather safe in cavhd even without laboratory controls. the adv~ucea in the management of computerized data of an intensive care unit have been petalled to the clinical advauces and the increasing sophistication of methods of diagnosis fop the clinical application an therapy. this has led our unit to design and develop a computational system called timbu which is used to help physicians assist patients. among its various uses, this system has a software for the hemodynsmic control of a critic patient. this program was carried out to get as fast as possible the hemodynamic data of the patients in an intensive care unit. as an example, we can mention that when we load 17 data obtained through direct measurement from the monitors and the lab, the program calculates 18 parameters that guide, intelligently, to the diagnosis and therapeutic behaviour of the hemodynamic problem through screen messages. the validation of this program in the unit of intensive care has demonstrated that its use allows a more efficient handling of the patient with serious hemodynamics and respiratory disorders. ohieetlve: traema is a heterogeneotm 'disease' that ecatr~ a~"o~s all age ~oupe with v~ying degrees of severity. this imerogeneity has made the di~e, trmma, diflkaflt to r the ehn of this stady wa~ to assr the fitaen of saps in ibis popeleties. methode: in order to compute the ~ probability, a model derived from logistic regression w~ developed. meam'e8 of calibration (goodaess-of-fit stetislj.r and di~'riminafion (roc ou~e) were adopted in developmm~ and validetlon set randomly taken from a database of 10065 pts eeeseemivety admitted in icu (arohidia). ~ witho= salm, p~ yom~ am is yam, with los ~horter thma 24 hotam wore exr fa'om thi~ mmly~ir thi~ model v~s then evahmed on the ~per ~mbgro~ (i.e., trmma pts). if'it did t~t fit the data well ~, new model wm developed rer the logit only on trm=~apm. reims: data were availabte for 8059 pts during aperiod of three .y~m , treama pts were 1156 04.3%), teats of calibration iadioaled probability model did mot provide m adequate refle~on of the mortality ezperieace in pm with ireutae, being the observed mortality lower flma the expected (figm'o). a aew model was then variable. this oastomized model fit~ the de~t of trmara pts very well (g 2=-8a7 p>0.25; roc = 0,94 ). the di:lferencea between the two modele were evident. conclusion: this ltudy shows that mortality in iramna pts is over wcfe~d when ~se~ed by menm of saps. however the r mode! meets high standmcd in terms of calibration mid dil~'iminat'~o~ ']"he advaatage of ~imd models meaas the colleotion of the ~ set of variables for all pm admitted in icu e~einat the ase of diasma specific ~oring syatex~. ("sl"): effects on cardiovascular and hemostasis systems (cvs, hss) a.oborin~ph, ~.~yndiuk~ph, b.kondratsky ~pt. of'""su~gery and transfusiology, research institute of hematology, lvov, ukraine objectives: great interest has been shown recently in the use of hoss for the initial resuscitation of hypovolemic shock. methods: the study was carried out in 6 dogs 1-~h hs was induced by jet momentary hemorrhage (h) from a. femoralls (the bloodloss volume made 29.8+1.9 ml/kg). the treatment was begun after 7.u+o.2 hrs of h. "sl", created on the basis of-sorblt and natrium lactate (1800 mosm/l) was injected into v. femofalls at the dose of io.0 ml/kg. results: it is established that before treatmen-~rterial blood and central venous pressures (abp, cvp) diminished to 30.0 mm hg and -0.6+0.2 cm h20 (p4.o01), while heart rate (hr)-increased to 190.0+9.6 per min (p<.o01). by this the indices of ~latelet counts (pic) and plasma fibrinogen (pf) lowered by 42.2% (p<.i) and 6.4% (p~.05), while fibrin degradation products (fdp) enlarged by 215.6% (p~ .001). after 30-40 min of treatment termination abp and cvp increased to 98.3+6.1 mmhg and 4.1+o.2 cm h20 (p<.o01), and ~[r diminished to t80.0+6.3 per min (p>.5). at the same time the indtces of pic and pf enlarged by 36.4% and 2.6% (p>.i), while fdp diminished by 8.2% (p>.i). one of 6 dogs survived. life duration of the other 5 dogs was 3.9+1.2 hrs. conclusions: the obtained data are ~he evidence of normalizing influence of "sl" on cvs and hss, and allow to recommend it as a mean of initial resuscitation of hs in clinic. oblectives: we prospectively studied 64 icu patients with severe head injury (hi), which cerebral lesions monitorized with sjo2 through opljcal fiber and the cerebral flux with tcd. methods: since january 1990 until june 1994, we collected 152 ht admitted to the icu, and 64 of them monitorized with optical fiber in the right jugular bulb and tcd. all patients needed mechanical ventilation related to gcs <__ 8, with ct in admission (classifing lesions according to marshall and al.) . we related the final results to the evolution of sjo 2 and tcd, with other monitorizing methods like gcs, ct and icp. ~sults: conclusions: in patients with gcs _< 8, sjo2 is useful to evaluate the evolution towards vegetative state, still more in cases with ct type ii in admission and higher apache ill. elevation of icp implies an evolutive nsk to brain death and data of tcd is a good indicator of brain death, the complete monitorization of these patients can improve the therapeutic control of this neurologic problem, , (16m,6f) , (m. age: 39+4 years), divided in two groups (a and b) under specific criteria(tremor and/or fever during admission in i.c.u., or not). the injury severity score was >25 in all studied patients. tbe group a (9 m, 41") had no tremor and/or fever on admisskm, while em group b (tin, 211 the above criteria were ix)sitive. bhx~d samplings were taken 2-9 hours after accident and 10-25 rain. after admisskm in i.c.u. micro-eli~ method was used for measuring cytokinc-levcls. statistic analysis was performed by studcnt-t test. as control group, 25 healthy people were examined. _resu!_ts-il-lct, il-ii~, il-2 and tnf-tt levels were similar to control group levels in both groups a and b. i!,-6 and g-csf levels were found increased in both groups (p<0jxjl), while il-6 levels were statistically significant comparing to group a. in con_tin_skin, during immediate post4raumatic period,proinflamatory cylokines il-i~, il-i~ and tnf.-ct, produced in an earlier stage than 11,.6, cannot be detected,whereas 11.-6 was increased significantly, especially in group b. g-csf was fimnd in increawal levels in both gr(mps, without statistically significant difference between gnmps a and i|. objectives-l~valantc proteolitic activity, disorders in" eariy, period after combined trauma and p(~.ssibilit, i' of their correction by injection of proteo[ysis inhibitors contrycal and s-fto~:nracil in combination with driving an isotonic snlu~ion of sodlum chloride and polig[ucine. methods: biochemicai studies of proteolitic activity in dogs with limited deep burn and acute bloodloss, . result:s: in case of deep 5% burn, cornplicated by bloodshed the of blood grows at 6-7 times. it; is the restdt of the pancreas glandischemi demage, caused by the centralised circulation of blood and intensifies the deviations of haemodiaamics and albumin exchange. the degree of endogene intoxication by mean mofecular peptides which are the products of albumin decay reses to 30%, and 77% in 3 hours. in 3 hours after the trauma the-process is accompanied b3! 59,6% lower inhibitory activity of blood, where as at the peak of the trauma it was 14,5~ higher. that proves the nnfavuurahle process of the shock in case a combined trauma. conclusion: the vein injection of 'proteolysis inhihitotz cnntrycal and 5-fforuraei[ in cumbination with driving an isotonic solution of sodium chloride and p.dligh]cine to refill lhe loss of blood helps to lower at 2 times the profeolitic activity of blood. but it still remains above the initial level. the degree of endogene intoxication lowers at 2 times; [15emodinamics aml albumin exchange stahilised. objectives: nimodipine, a known calcium antagonist, has been shown to dispose a beneficial effect on patients with subarachnoid hemorrhage, but its efficacy on traumatic or spontaneous intracerebral hematoma has not been justified. therefore, we studied the effect of nimodipine on the histopathological changes following an experimental intracerebral haematoma in rabbits. methods: twenty-three new zealand albin rabbits of both sexes, weighing 2-3,5 kgr and at age of 4-6 months were anesthetized and a small burr hold in the left parietal aerea was carried out under aseptic conditions. the dura was opened and 0.1 ml (this volume assuring a normal incranial pressure after kaufman 1985) of autologous blood was injected into a depth of 3 mm via a needle of 0.36 mm bore. the wound was closed and the animals were left to recover. nimodipine, of 2,1 mg/kgr of by weight per day was given via a nasogastric tube to fifteen animals for a period of time of fifteen days (group b). six rabbits were given water and served as control (group a). both groups of animals weie sacrified on the fifteenth day, their brains were removed and immersed into 10% formalin solution. tissue sections of 5 ~ were embedded into paraphin and stained with haematoxyline and eosin, mason and gfap stain for gliac cells. results: two animals died after the surgical procedure, because they developed large intracerebral bematoma. no animal developed neurological deficit except one of group a which manifested a right side hemiparesis. the results of the bistopathological changes are the following: i) the mean -+ sd diameter of the lesions in the group a was 260 --. 26 ~t while that of group b was 76 + 12 ~t (p<0,01) ii) secondary ischaemic neural tissue changes, characterized by the extravasatlon of red cells, the presence of haemosiderin-containing macrophages and signs of low grade inflammation zpredominated in the specimens of group a and were totaly absent from those of group b. iii) a ring of gliac hyperplasia and a low grade local fibrosis was found, encircling the lesions in the specimens of group a in contrast to those of group b. conclusions: nimodipine when administered in rabbits following the development of a non increasing the icp experimental intracerebral haematoma, prevents the extention and the severity of the lesion. objectives: to study the efficacy and side effects of adding intramuscular clonidine (clophelinum) to analgesic regimen in early management of patients with serious burn injury. methods: 20 pts with 20-40% bsa second to third degree flame burns (respiratory tact injury excluded) 19 to 61 yrs of age were randomised to study (n=10) and control (n=10) groups. burn shock was treated with hypertonic saline -bicarbonate solutions (250 mmol/l na +) 2ml/kg/%bsa for the first 24 hours and 1 ml/kg/%bsa for second day. analgesia in control group for the first 48 hours was provided by regular 6 hourly intramuscular administration of 20 mg of morphine sulphate and 500 mg of analgesic -antipyretic analgin with 10 mg of diphenhydramine (dimedrol). from the 3rd day regular administration of morphine was finished. in the study group 100 ixg of clonidine was added 8-hourly for 72 hours and dose of morphine halved. vas, verbal rating scale for sedation (vrs, 1 -5), sleeping time, spo2, hr, bp, diuresis, vomiting and other complications were comparatively evaluated during patients' stay in icu. results: addition of 300 ~g of intramuscular clonidine daily allowed to achieve better analgesia and sedation with halved consumption of morphine. mean vrs in study group for the first 3 days was 3.1 -3.3 vs 1.3 -1.7 in control group with twice longer sleeping time. there was significantly less tachycardia in study group; dynamics of bp for the first 24 hours did not differ considerably; later, there, was tendency for hypotension in study group without adverse effects on diuresis or other indices of tissue perfusion. because of high incidence of chronic ethanol abuse among study population 7 pts of control group suffered from psychomotor agitation or delirium, probably as a sign of alcohol withdrawal syndrome (aws). this made regular evaluation of vas impossible. in the study group only 1 pt showed sign of aws. mean vas score was in 2.4 -2.9 range for first 3 postburn days. 7 pts appeared excessively drowsy due to clonidine, but it had no adverse effect on their overall clinical course. mean spo 2 values in study group were in 95 -98 % range, among controls 90 -95 %; vomiting was absent in. cionidine group vs 4 cases among controls conclusions: clonidine could be a valuable addition to analgesic -sedative regimen in burns, especially for prevention of aws and deserves further study in this regard. hemodialysis -hemoflltration modifications and/or intratracheal gas insuflation have been recently used for blood gas exchange in several models of respiratory failure. objectives: evaluate the combination of cavh-m and igi for respiratory support in experimental acute lung injury. methods: five mongrel dogs (22-+4kgr) were mechanically ventilated inroom air, paralysed, heparinized, connected with a cavh-m system (diafilter-30 polysulphone membrane) and remained stable for one hour (pao~= 98.8• peco2=34-+8mmhg, ph=7.37-+0.07, bp=137-+12mmhg and pap=15-+2mmhg). all was induced two hours after oleic acid infusion (0.07ml/kgr) into the pulmonary artery (poo~=46.6_+6 -p<0.001, paco~-50.2_+10 -p<0.05, ph=7.10-+0.10 -p<0.01, bp=158-+25 -p=ns, and pap=29_+5 -p<0.01). fio 2 70% for the next 30 minutes did not significantly altered the b3ood gas abnormalities. afterwards, pure oxygen applied simultaneously a) through the inlet of the filtrate's compartment of the hemofilter (2l/min) while filtrate and gas were removed from the outlet port (bypass flow 220 ml/min) b) through a thin intratracheal catheter positioned 2cm above the carina (4l/min). the fio 2 given through the ventilator readjusted to 21%. results replacement fluids/filtrate during the next four hours were not exceed 0.7l/hour, whilst the blood gases and pressures were improved as follow: cavh-inlet:pao.=88. objective. to compare the changes in humoral immunity in trauma patients following massive transfusion of autologous and homologous blood. methods. we studied 3 randomised clinical groups of patients each containing 24 patients with trauma and operation of large arterial vessels. the amount of autologous or homologous blood transfused to the patients was exceeding 1 500 ml, while the patients in the control group did not recieve blood or blood products. results. we recorded most pronounced and characteristic changes on the 1-st and on the 7-th day in the group of patients recieving homologous blood transfusion, i.e. decreased amount of igg,iga,igm,c5 and c4 fractions of the complement system, haptoglobin and significant and sustained rise of circulating immune complexes up to the end of the study period. in the control group of patients the decrease was weaker and lasted only during the 1-st post-operative day; the dynamics of the circulating immune complexes level were almost the same as in the first group of patients. in the group of patients recieving autologous blood transfusion, the parameter values did not change significantly from preexisting levels after the 1-st day, while on the 7-th and on the 30-th day showed a tendency towards aslight rise. conclusions. autologous blood has a favourable effect upon humoral immunity and should be the transfusion medium of choice in cases where autologous blood reinfusion is technically possible. ivan petkov, m.d., rumen farashev, m.d. and dimitar terziiski, m. d. medicine, military medical academy, 3 g. sofiiski str.,1606 sofia, bulgaria objective. the amount of blood lost during trauma and operation could hardly be forseen and donor blood supplies are not always available in sufficient amounts. rare blood group types and/or unexpected haemorrhage pose a great challenge to the transfusion therapy and the methods of intraoperative autologous blood transfusion. methods. we report a case of a 18-year old male patient with extremely massive intraabdominal haemorrhage ( 7 300 m( blood loss ) during an abdominal aorta reconstruction following a traumatic injury of the abdominal aorta. we achieved a successful reinfusion of 6 600 ml of autologous blood using an original autotransfusion system developed by us ( pat. no 95311/ 11.10.1991) . results and conclusions. the autotogous blood in the case reported here was the only and the most suitable transfusion medium for the rapid intraoperative compensation of the acute haemorrhage and the favourable outcome of the patient. the post-operative period was smooth and no significant disorders in the clinical course as well as in the laboratory tests ( morphological,biochemical,coagulation and immunological) were recorded. there were no complications during the postoperative period despite the fact that the amount of blood reinfused to the patient was slightly exceeding his own volume of circulating blood. objective. the haemoglobin concentration and the perfusion pressure value could not be the only criteria for the early signs of tissue and organ dysfunction. because of this, we employed the extensive monitoring of oxygen transport during severe trauma in order to. achieve dynamic evaluation of physiologic compensatory mechanisms and to assess the efficacy of intensive care management. methods. we conducted a prospective controlled trial on the blood oxygenation, oxygen transport and tissue perfusion during the first 3 days after the trauma in 20 patients with polytrauma. we used a swan -ganz pulmonary artery catheter (beckton -dickinson, u.s.a.), deseret 1000 cardiac output computer (medical inc., u.s.a.) and hewlett -packard monitor (hewlett -packard, germany) to measure and calculate all the parameter values. the severity of the injury was assessed using the apache ii score system. all the patients had scores over 18. results. the results show a significant decrease in the arterial blood oxygen content and in the arterio-venous difference, as well as an increase in alveolo-arterial oxygen difference and in the transpulmonary right-to-left shunt. the tissue oxygen supply and the tissue oxygen consumption reveal a tendency towards a decrease below the physiologic minimum of adeqate values. the erythrocyte current velocity and the ratio between oxygen transport and erythrocyte current velocity also decrease inspite of the optimal blood rheology. conclusions. the dynamics in the parameters values are most pronounced between the 2-nd and the 18-th hr after trauma, which predisposes patients to the risk of developing stable hypoxemia and characterizes this period as the most critical for tissue metabolism and organ dysfunction. posttraumatic changes in immune mechanisms in lung compartment in trauma were analyzed in ao and da inbred strains of rats which differ in their immunological reactivity: the former being low responder and lat-~er hiperresponsive. methods: the levels of tnf-alpha activity in the 24 supernatants of cultured lung lobes and dynamics of cells migration from tissue explants in 6h lung cultures were assessed in ao and da rats subject ted to severe burn trauma. results: increased levels of tnf activity (160+3 pg/ml compared to 50+4.9 pg/ml in control) were found od day 3 following trauma in lung sups of ao rats while no changes in the levels of activity of this cytokine were found in lung-sups od da rats more pronounced extent and dynamics of cell emigration were noted in da rats, while almost unchanged in ao rats sharp rise in pmn percentages 3h following trauma (60-70% compared to rare pmns in control), followed by increase in lymphocyte numbers at later time points among lung cell emigrants was detected in ao rats. slower but persistent increase (25%, 3h following trauma and 60% and 50% on days 1 and 3 after trauma infliction, respectively) in pmn numbers among da lung cell emigrants was detected, which appeared to be activated, as judged by their nbt reduction capacity. increased percentages of peripheral blood pmns and increased state of leukocyte aggregation/adhesion were detected in both strains, but different levels of plasma tnf: increased levels in ao rats on days 1 and 3 following trauma, and initially but persistently high levels of plasma tnf alpha in da rats (4-5 fold higher compared to initial levels in ao rats). conclusions:different patterns of local (lung) and systemic changes in cell numbers and cytokine levels implicate differential posttraumatic migratory capacity of pmns vs. lymphocytes in lungs in ao and da rats. early diagnosis of acute intestinal ischemia by color doppler sonography e. danse, b.van beers, p.goffette, f.hammer,aav.dardenne, f.thys, p-f.laterre, m,s. reynaert, .lpringot dept of radiology (profb.maldague) and dept of intensive care ( prof m,s.reynaert), st.luc univ.hospital, brussels, belgium ob emergeny medical squad service is the most important segment in the process of saving the people, in the cases of mass accidents, like industrial accidents caused by the: explosion, fire, chemical poisoning, traffic accident, elemental catastrophes and the war. because of that, each emergency medical squad service needs to have in its motor-pool vehicle for the mass accidents/ for provoding at least 100 people, wounded as well as the people became ill/. objectives: presentation of such special vehicle, produced by "zastava-kamioni" and it's medical-technical equipment. methods: descriptive and comparative analysis of the medical and technical characteristics, based on the actual norms/din, 75080, iso 9001, yus.../ results: on the base of doctrinaired requirements of the emergency medical squad in the case of mass accidents, our researches resulted in the following medical and technical characteristics -the vehicles for mass accidents are gvw/with a payload off cca 5-8t, with the fixed, closed body, type: universal van, -technical equipment aggregates, stretches, anti-fire device, equipment for pitching the tent and for maintaing technical conditions of the work -medical equipment: linen bags with complete sets of bandage material, means for the reanimation and immobilization, for the infusion, medical instruments and remedies as well as the tent for lodging at least 50 wounded and sik people. in federal republic yugoslavia, it was proposed 24 such vehicles for the emergency medical squad needs. conclusion: we suggest to introduce this vehicle in the production range of the ambulance vehicles for saving, especially in the circles where can occur serious accidents. introduction : carbon monoxide (co) poisoning commonly generates central nervous system abnormalities though an important cardiac morbidity and mortality must be considered. long-term exposure to co with cohb levels < 30% may be more dangerous than short-term levels of 45-50%. we report a case of an adolescent who after prolonged exposure to co developed a severe reversible cardiac dysfunction with low levels of bloed cohe c a.ase history : a 15 year old boy was found comatose at home. his mother in the neighbouring bathroom died severn hours earlier of what was later proven to be a co intoxication. on arrival the gcs was 8/15 and the patient was breathing spontaneously. a postictal status with eventual postanoxic encephalopathy was suspected. a coh'b level of 10% was objectivated. the cardiorespiratory situation quickly deteriorated requiring mechanical ventilation. chest x-ray showed diffuse bilateral patchy infiltrates. ecg revealed signs of ischemia. severe left ventricular dysfunction was evidenced by pulmonary artery catheterisation and echecardiography and later by isotopic angiography (lvef 20%). treatment was intensified with inotropic support, intta-aortic balloon counterpulsation and oxygen therapy. the clinical course was further complicated by a crush syndrome and renal failure. the patient's condition gradually improved and he fully recovered without any residual lesions (lwf 80 %) conclusion : even after prolonged exposure cohb levels can be misleadingly low. high tissue levels of accumulated co can be associated with coma and fulminant cardiorespiratory failure requiring advanced life support facilities. introduction : both neuroleptics (nlp) and tricyclic antidepressive agents (tca) can induce arrhythmias, prolongation of the qt segment and the pr interval and hypotension. we report a case illustrating that combined overdose of these agents increases the toxicity of each compound and the risk for adverse cardiac events. .c, gse history : a 44 year old male ingested 2500 mg doxepin (sinequanr), a tca and 3500 mg prothipendyl (dominalr), a potent nlp in an attempted suicide. upon arrival in the emergency department the patient was unconscious (gcs 6/15), breathing superficially, and presenting signs of recent vomiting. physical examination revealed a taehycardia of 140 b.p.m., an arterial blood pressure of 90/70 mmh4g. ecg showed a brood qrs complex tachycardia. a chest x-ray revealed the presence of an aspiration pneumonia. laboratory investigation demonstrated increased levels of crcatine phosphokinase, lactate dehydrogenase and aspartate transaminase ; hyperglycemia and leucocytosis were present. the plasma concentrations of doxepin and prothipendyl were respectively 410 gg/l (toxic level 5130 #g/l) and 3900 i.tg/l (no reference). treatment consisted of mechanical ventilation, gaslric lavage and administration of activated charcoal and iv fluids and antibiotics. a hemodynamically well tolerated veatricular tachycardia developed 1 1/2 h later. nahco3 (250 meq/24 h) was administrated inducing an ectopic atrial tachycardia with a normal qrs complex and prolonged qt. 8 h after admission a normal sinus rhythm was present; the prolongation of the qt segment persisted for 2 days. the patient fully recovered. conclusion : the treatment with nahco~, alkalizing the blood and thus increasing the protein binding of the tricyclic antidepressant molecule, can readily correct the potentially life-threatening cardiac arrhythmias and therefore should be part of the routine treatment of combined tca-nlp overdose. ob/ectives: the development of diabetes insipidus (di) in patients with brain injury is a known negative prognostic sign. the aim of this study was to investigate whether this is also a reliable early prognostic sign of brain death. methods: this is a retrospective study of 85 patients treated" during a two year period (1-3-1992 to 1-3-1995) in our i.c.u who meeted the following criteria: (1) coma score _< 8 gcs within the first 24 hours, (2) positive brain ct scan on admission classified according to marshall's diagnostic classification (classes 1-6), (3) normal renal function during the entire icu stay. for the definition of di were used the usual di criteria plus hypematriaemia (serum na" >_ 155 meq/l). survival was defined up to the 30th postadmission day. conclusions: according to the findings of this study, the development of diabetes insipidus in brain injured patients seems to be a highly specific index for brain death (positive predictive value = 0.95). however, further prospective studies are needed for the definitive evaluation of these findings in such patients. emergency care in italy, despite all efforts, is still lacking a nationwide organized prehospital care system and, until today, there are only different regional solutions. the majority of these realities imply rather simple ambulance first-aid services without attending emergency physicians and without resuscitation equipment. the emergency medical service (ems) system in falconara m., italy, was implemented in august 1994 by a collaboration between the school of anesthesiology and intensive care of the university of ancona and the, already existing, volunteer rescuer organisation "yellow cross". according to the guidelines pubblished in 1992 [1] the pre-existing equipment of the volunteers was completed with type a ambulances and 1 special equiped motorcar (patient monitor, defibrillator) for ambulance indipendent physician transpur[. a special data collecting schedule was created to memorise every emergency intervention in a computerised data-base. the intraining members of the school of anesthesiology and intensive care provide 24 hour ready intervention. in this report the authors describe their experience concerning primary firstaid medical interventions. for a preliminary evaluation we considered, retrospectively, 300 consecutive emergency interventions in the time period from novembre 1, 1994 to april 30, 1995. the emergency physicians treated 131 male (44 %) and 169 female (56%) patients, 15 patients died before hospital admission and 75 patients (25%) were treated at home by the ambulance indipendent physician and did not need any further medical treatment. in the same time period 1 year earlier (november 1993 to april 1994) without attending physician the volunteer rescuers transferred all 257 first-aid interventions to near-by hospitals. we conclude that the presence of an attending, iudipendently motorised physician in emergency interventions is essential for the establishment of precise priorities and may be helpful to reduce hospital admissions by ambulance intervention, though reducing primary" health care costs. we have developed the method of liquor filtration which allows to purify the cerebrospinal liquor from blood and its decay products in the subarachnoid bloodstroke. the hemipermeable dialysis membrane was used as a filter, which lets only in water, electrolytes and substances with small molecular weight. the liquor filtration was used for the treatment of 19 patients with the subarachnoid bloodstrokes of different etiology. the perfusion of liquor was performed at the rate 3 ml/min in the recirculatory mode. its duration was 180 -240 min depending on the bloodstroke intensity. the filtration makes possible the most completely purifying of the hemorragic liquor, the reducing of the content of blood ceils and its decay products 80 -300 times as less. the monitoring of the patient's state during the perfusion didn't revealed the departure from the norm of the main vital part. the liquor filtration technique compares favo-~ rsbly with the routine method of cleaning by the absence of toxical effect of heterogenous solutions on the central nervous system. the filtrstion of the cerebrospinal liquor in the subarachnoid bloodstroke sllows to provide the the early cleaning of liqour, the regression of meningeal syndrome and to improve the patient's state of health. e3tabli~mczr 3bd ~ of rei~idnal medical first-aid zhoulittoing, ed., tan zi, m.d. dept. of sargery, the first teaching t[ospitat, 29 yejin-l)a-l)ao, wuhan 430080 fltlna objectives: the medical first-aid is the most important task of the public hc atth department. in general, single hospital model couldn't fatty, effective ly rescue mony severe patients who need mergant treatment in the scene. bub establishing the medical first-aid network, the severe patients can be given the most timely und the most scientific emergent treatment. so that, the suc cessfut rate of the saving wilt be greatly increased. methods..; our hospital is a general big hospital. through developing and cons tructlng for more than ten years, the medical first-aid network distributed art over the area under our jurisdiction has been set up. it consists of thr ee units: the medical first-aid unib center comartd and mnagment unit, co m~nlcation and tiaison unit. the principle of the network operation is with oat having to 90 far to mergoncy, specialized emergency and the best merge acy. results: the results of the network operation were notable. cmpari~ the to tat successful rate of the saving (91.6~), the successful rate of saving tra ma (93.~), the suscessfut rate of saving shock (98.~) and the successful rate of cardioputmonary resuscitation (52.4~) daring the three years after t he network operated with these before (86.6~), (91]. 4~), (92. ~) and (4ft. 5~), the successful rates after operating were remrk~iy higher ( p<i).o5 ). conctusions~ the above results showed estabiishmnt and mena0emont of region at medical first-aid network had very si~nificont action during mergentty s avin9 the severe patients. moreover, the regi~at medical first-aid network atso take an important part in prevention and health protection of the mass. objectives: se related mortality is due to the occurrence of both rv and lv dysfunction evoking cardiogenic shock and pulmonary edema (f. abroug, intensive care med 1995; s. nouira. chest 1995) . prospective evaluation of scorpion antivenon (sav), the only available specific treatment, is lacking. the aim of this study is to assess the effects of sav on the basis of a case-control study. methods:among 600 consecutive victims of se presenting to the emergency department, 135 were managed using sav (group sav+). these patients were matched to 135 envenomated patients who did not receive sav (group savchest injuries are the cause of death in 25% of trauma fatalities and a major contributing factor in an additional 50%. pneumothorax is a major complication of chest injuries and can be initially overlooked bringing to additional morbidity and mortality especially in patients who require mechanical ventilation. the real incidence of pneumothorax in severe blunt chest trauma hasn't yet been established, as many pneumothorax can be missed on the initial chest x-ray (cxr) and computed tomography (ct) has not been routinely used. to evaluate the incidence of pneumothorax in patients with severe blunt chest trauma. methods: a prospective study. from january 1 1993 to december 31 1994, all trauma patients with one of the followings conditions: fractures of four or more ribs, signs of lung contusion on the initial cxr or presenting a severe hypoxemia on admission (pao2/fio2 < 200) in the absence of pre-existing pathologies, were evaluated by initial cxr and subsequently submitted routinely to a ct. results: 52 severe trauma patients were considered. 8 of them, presenting a severe hypoxemia on admission, had no evidence of major chest trauma (ais<3). hypoxemia was the consequence of severe trauma in other districts; these patients were therefore excluded. 44 patients with severe thoracic trauma (ais>=3) were admitted into the study. the mean iss was 36.2 (16-75). thirty-six patients required artificial ventilation for at least 24 hours during the icu slay. three of them, who had a tension pneumothorax, were submitted to an emergency thoracic decompression on the field by the emergency helicopter team. in 7 cases pneumothorax was diagnosed an the initial cxr 14 more patients had a pnx which was identified only on the ct. in 4 cases a large pnx with lung collapse was missed on the cxr. in our group of severe blunt trauma patients, 54% (24/44) presented a pnx that required the insertion of a thoracic drainage. only one third (7/21) of the pneumothorax could be recognised on the initial cxr, while other 3 were decompressed before performing the cxr. as many as 58% of the cases of clinically significant pnx were missed on the cxr, and a ct performed soon after admission allowed an early diagnosis bringing to changes in the treatment. (as the patients were mechanically ventilated a chest tube was inserted in all these cases). in 4 cases, the initial cxr overlooked a huge tended pnx which was the cause of hemodynamie instability. conclusion: in patients with severe blunt chest trauma even large pnx can be missed on the initial cxr. moreover due to the non compliant compressible lung, a 20% pneumothorax which can be recegnised only on a ct, can bring to high intrapleural pressure altering eardiopulmonary function. n. andoeli6, 0.~osid, m.zesevid, m.risovid, d.stepi6, d.djokid b~rga~yc~qterclinicalcaqterafserbia, belgrade cb~ctives:~lis study ~ the use of ~rq]ofol earbired with k~t~ine (aq a~sjgh~ic s@~qt widn inirjrsic armlgesic pro~mities) or with fsqtmtyl,with psrtial azgmsis an hgenxlyn-a~ic ~ durirg ~ ~ re:~ver~ f~m ~ in hxh ~ of ~ti~. ~: yali~mial and ~bod:30 a~it p~tie~ts a~ i-ii were included in ibis shxly. patients were rsrd]nly dieided in two ~ns. all d~tie~ts ~me given 5-5 prcpofol bolus doses (o,5 ~gkg) for ird~iqn of ~. ~ia ~s m~sjn~ with an infusion 6 ~ ~ropafol. as sdflitianal were given fan-i~l (o,2 n]g) ~tely before ~ anj trad~e~ irfojoation followad by feasted bolus of o,i mg in ~ro4o l.patients in gr~4o 2 received i~ (an initial bolus dose of 35 rg slowly intcavax~ 8rd 25 mg as infusion over ~0 rain) .infusions of pro~fol or imcpofol with kg~mine ~ stopfsj 10-15 rain ]:~o~ extuhation.arterial blood ~ (sistolic arterial blood preassu-re~zap,mean ~rterial blood pr~,d~lic arterial preassure-[zp a~ h~art rate-~) ~ m~ before induction of a~ io, 30 snd 60 rain aftem ~ intutation. results: arterial blood preasstre ~s decreases duri~ irn~ction of sn~wd~sia in hy~ ~n~s,tnt mare in th~ ~ who r~eived fsqtanyl.~ere w~s statisticslly sifnific~ntly difemerme dmir~ m~ of an~ia. arterial blood r~easatre and heart rate were stable in the t-..e~min 9-~a4~. all th~,fl-e keta'nire grcqo hsd e~rly :~e~y time. ctrmlusi~s: ~e ombiretion of protxfol wilh keta/ne for irduorion a~d ~ of sn~sd~esis w~s yell accept~ by p~tierfcs anj coald he ~ as an alterrstive ~o ccnva~icrsl a~es -d~sia. objectives : assess the relation between cytokine or endotoxin release and indices of splanchnic malperfasion after hemorragic shock in multiple trauma patients. ]~r study was approved by the local ethical committee. trauma patients admitted to the emergency room who met the entrance criteria of more than 1 hour map < 60 mmhg or use of vasoactive agents or blood lactates > 5 mmol/1 were selected for study. a nasogastric tonometer (tonometrics, inc, plastimed, france) and a swan ganz catheter were placed on admission. phi, lactates, hemodynamics, plasma cytokine and endotoxin concentrations were measured on admission and at 3.6, 12, 24, 48 hrs. an immunoradiometric assay was used to determine plasma concentrations of il6 (n<0.03ng/ml) and tnfc~ (n<5pg/ml). plasma endotoxin concentrations were measured using a chromogenic limulus assay (n<0.1eu/ml)(1 endotoxine unit= 100pg). results : 9 severe multiple trauma patients (age = 42_+18 yrs, iss = 40-!-_15, saps = 19+'~, mean-+sd) were studied. they received 15+6 packed red cells during the first 24h. mean duration of collapsus before inclusion was 5.1_+2.9 hrs. death occm'red in ~tients. ~ pglml, *: ng/ml, etox : endotoxin(eu/ml), lact: lactate (retool/l) a significant correlation between initial il6 level and saps was observed. in the early post-injury period phi, sao2, svo2, vo2 were significantly associated with ;il6 release (p<0.05 at ho, h3, h6). later a significant correlation existed between lactates and ii6 (h6, h24). a peak of tnf was detected at 24 and 48 hrs. it was associated with low phi and low arterial ph of the early post-injury period (p<0.05 iat ho, h3, h6 ,h12, h24) and with high lactate levels of later period (_>h12). only the late release of endotoxins (i{48) was correlated significantly with initial !oxygea-delivered parameters. iconclusion : there was a marked increase in il6 in the early phase of trauma . i16 and tnf release after major trauma iwith hemorragic shock is associated with splanchnic malperfusion, as assess by the ivery low values of phi. lactates seem to be a later indice. toxic effects are a well-known complication of an overdosage of prescription theophylline. what is less known is that over-the-counter (otc) asthma medications contain theophylline, and that in some cases this might cause toxic effects. a case seen by us involved toxic effects from theophylline in an otc medication and to date is the only published case in the english literaturet the rationale for this study was to delineate the otc products containing theophylline from whatever data sources available. hyperthermia frequently occurs in intensive care treated patients and intentional application of whole body hyperthermia together with chemotherapy is a therapeutical access to treatment of malignant disorders. anaesthetic support is required in either condition. due to the marked decrease in systemic vascular resistance seen in hyperthermia an additional vasodilatory effect of the anaesthetic is unwanted. the vascular effects of anaesthetics in hypertherm organisms is not known in detail. therefore, we performed an experimental study to detect the effects of inhalational anaesthetics in whole body hyperthermia. in 30 sprague-dawley-rats katheters were inserted into trachea, jugular vein, and carotid artery. for continuous monitoring of cardiac output a flow probe was placed around the aortic arch. the rats were mechanically ventilated with different concentrations of inhalational agents in oxygen. we compared the effects of enflurane, isoflurane, and halothane in stepwise increased body temperature by submerging in a temperature controlled water bath. results: isoflurane lowers arterial pressure more than halothane or enflurane. the inhalational anaesthetics lower the cardiac output similarily and independently of temperature. isoflurane decreases systemic vascular resistance independently of core temperature and the decreasing effect of halothane on the resistance is completely abolished in hyperthermia. conclusions: the influence of hyperthermia on the systemic vascular resistance is dangerous. this allows no additional effect of the anaesthetic management. in spite of the vasodilating effect of inhalational agents in normotherm subjects, this effect is abolished in hypertherms using halothane. the condition of management of analgosedation in hyperthermia is different from normothermia. objectives: to evaluate a bedside computer processed cerebral function monitor for assessment of brain wave activity when clinical/visual clues are not present. methods: ten icu patients undergoing neuromuscular blockade monitored with the aspect 1000 brain wave monitor from january 1 to june 1, 1995. results: time to onset and depth of sedation were readily apparent to icu physicians not specifically trained in eeg reading. objectives: to determine whether non-depolarising neuromuscular blockade reduces oxygen consumption (vo2) in sedated, apnoeic patients. methods: haemedynamic. metabolic and oxygen transport variables were determined in 5 sedated, apnoeic patients with severe acute lung injury. all patients were ventilated using a puritan-bennett 7200ae ventilator with integrated 7250 metabolic monitor. inclusion criteria were; 1) stable cardiorespirator s" status; 2) systemic and pulmonary artery catheters already in situ; 3) inspired oxygen < 80%. patients were sedated with midazolam or propofol to abolish response to verbal stimuli, and sufficient morphine or alfentanil to abolish all spontaneous respiratory efforts. following baseline measurements, neuromuscular blockade was induced with intravenous vecuronium, 150 ug/kg, followed by an infusion of 80 ug/kg/h to maintain the train-of-four ratio at 0. a further four sets of measured and calculated variables were obtained at 20 min intervals. results: statistical analysis was by repeated measures anova. there were no significant changes in any variable over time. the changes in calculated oxygen consumption (vo2fick) , and measured oxygen consumption (vo2gas), and in energy expenditure (ee), are shown in the table. objetive: to study the effects on coronary hemodyrtamics and myocardiai metabolism of administering propofol during postoperation sedation of patients with normal coronary circulation and good ventricular function undergoing cardiac surgery. patients and methods: 18 patients (12 women and 6 men) undergoing aortic and/or mi~-a/ valvular cardiac surgery were selected, with an ejection fraction greater than 0.5 and normal coronary circulation. for postoperation sedation propofol was administered in 0.5 mg/kg i.v. bolus, followed by a 2.2 mg/kgth perfusion. all data were registered before administering propofol and after 20 minutes, the patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -~ systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabofic variables were measured. results: the patients studied were about 56 years old, and the average period of aortic cross-clamp was 77.50 min. the adminstering of propofol caused a decrease in the coronary blood flow (-9%), great curonary vein flow (-23%), myocardial oxygen consumption (-14%), regional myocardial oxygen constanption (-11%), myocardial oxygen extraction (-6%), regional myocardial ooxygen extraction (-10%), while coronary vascular resistances and global coronary vascular resistances did not change. oxygen saturation increased in the coronary sinus (+16%) as well as in the great cardiac vein (+32%). in no patient were significant changes suggestive of myocardial ischemia objectified. there was also found a decrease in systolic (-23%), diastolic (-20%) and mean (-25%) arterial pressure, systemic vascular resistance (-20%), and cardiac output (-8%). conclusions: in accordance with the clinical conditions of this study, the administering of propofol is not likely to cause changes in coronary autoregulation, oxygenation and myocardial metabolism. obietive: analyse the effects of 0.4% "end tidal" isoflurane (sedative dosage) on the metabolism and coronary hemodynamics during the postoperation period of patients undergoing cardiac surgery. patients and methods: 16 patients (12 women and 4 men) undergoing aortic and/or mitral valvular cardiac surgery, with an ejection fraction greater than 0.5 and normal coronary anatomy, were selected. after the surgical operation, 0.4 "end tidal" isoflurane was administered for postoperadon sedation. the determination of variables to be studied was carried out before and 20 minutes after administering isoflurane, die patients being hemodynamically stable and a rectal temperature of 34 _+ 0.5 -+c. systemic and pulmonary hemodynamics, and global, as well as regional myocardial blood flow, and metabolic variables were measured. results: the average age of the patients studied was 57 83 -+ 8.87 years. during surgical operation the period of aortic cross-clamp was 78.56 _+ 32.09 rain. the administering of isoflurane was followed by a statistically significant drop in coronary perfusion pressure (-26%), coronary vascular resistance (-29%), regional coronary vascular resistance (-29 %), regional myocardial oxygen consumption (-7%), regional myocardial oxygen extraction (-6%) and accompanied by a significant rise in oxygen saturation in the coronary sinus (+16%) and in the great cardiac vein (+32%). myocardial oxygen consumption, myocardial exu'action of lactate and regional myocardial lactate extraction did not change. in no patient were enzyme or electrocardiograph changes objectified. systolic (-23%), diastolic (-25 %), mean (-25 % ) arterial pressure, and systemic vascular resistances (-28%) decreased, while cardiac output did not. discussion: the administering of 0.4% "end ddal" isoflurane, in the clinical conditions of this study, produced a decrease in systemic arterial pressure due to a reduction of systemic vascular resistance without deteriorate cardiac output. at coronary circulation level, has and effect on coronary autoregulation but had no effect on oxygenation and myocardial metabolism. the idea of tiva implies the realisation of major anesthesia components (los of consciousness, neurovegetative inhibition, analgesia, myorelaxatiou, providing the adequate gas-exchange) through i.v. introduction of drugs exclasively. aim: providing for the main tiva components with minimal side effects of the drugs used, taking into consideration the patients characteristics and the surgery specific character. methods: 78 anaesthesias have been conducted in patients aged 15 75 years (28 females, 50 males), undergoing planned and urgent operations with the pathology of lower, extremities, perinaeum, small pelvis, hypogastrium and with reserved spontaneus respiration against a background of 100 % 02 insnffladon through mask. operations lasted from 0.5-1.5 h. anaesthesia adequacy was assested by constant monitoring: "cardiocap" (nibr hr, rr, sao2, t), through glykhaemia level and mimicry reactions. standart premedicatioo of m-cholinolytics (0.01 mg/kg) and h2-blockers (0.3 mg/kg) on the operational table was sumplemented by administration of 0.5-1.0 mg/kg of lidocaine, 1.50.0 mkg/kg of clonidine, 0.5-1.0 mg/kg of pentamidine by the tachifilaxia method. the premedication adequacy was assessed through haemodynamics characteristics. sedation: 0.05-0.1 mg/kg of droperidoi, 0.l-0.15 mglkg of diazepam and analgesia: 2-3 mkg/kg of phentanyl, 1.0--1.5 mg/kg of ketamine were introduced fractionally according to indications. infusion rate of ringer-lactat solution was 5-15 ml/kg/h and depended on the intraoperational blood loss volume and on the patients preoperational condition. the duration of postoperative analgesia was registered. results: clinical assessment of analgesia according to this techniques allowed to decrease the anaigetics dosage to the subauaesthetic levels. smooth stabilisation of haemodynamics (bp) at proper age norms in patients with the initial hypertension by the 30-th min. of anaesthesia as well as the absence of its increase in response to the additional introduction of anaesthetic have been achieved. (hr) had no abrupt changes and remained in the range of 70-80 per rain. adequate external breathing: decrease (rr) by 2-3 per rain., with sao2 increase from 9446 % to 98-100 %. hypoventilation was avoided by respirate ventilator. according to unauthentic data the glykhaemia level had been lowered by 10-t5 % to the end of the operation with the initial moderate hyperglykhaemia of up to 10 mmol/l the cutaneous covering grew warm and got pink colouring. no mimicry reactions. in the postoperative period patients were in the superficial sleep state (3-48) and analgesia lasted 6-8 b. there were no complications due to anaesthesia. conclusion: combined using of bz, opiates, neuroleptics potentiate the i.v. anaesthetics effects allowing lowering of each tiva component dosage and, as a consequence avoiding their negative influence on respiratory and heart vascular systems. complex application of adrenergetics (therapeutic doses of cionidine and pentamini with using of taehfilaxy effects) permitted to provide for analgetic and neurovegetative components of general anaesthesia under subanacsthetic doses of tiva main components, and manifestation of hyperdynamic reactions of haemodynamics decreased while using of lidocaine -the economicai activity of heart-vascular system. good level of muscle relaxation was achieved allowing for widening of surgical intervention extent without respirator ventilators and inhalation anaesthetics application. anaesthesia is easily controlled due to fractional introduction of drugs with quick recovery of cns functions after anaesthesia. postanaesthetic analgesia is increased while concurrent opiates doses are decreased. absence of marced haemodynamic, endocrine and metabolic reactions during the operation and after it resulted in shortening the period of patients staying in hospital. a 64 yo white man was admitted to hospital for dyspnea and a productive cough. he had cabg in past, but no recent cardiac ischemia. physical exam: decreased breath sounds over right lung. chest xray: consolidation of right lung. admission medications included diltiazem, furosemide (both were continued) and trazodone (which was discontinued). admission ecg: sinus rhythm, qt 0.44/qtc 0.49 sec, with st and t wave abnormalities similar to prior tracings. he required intubation and mechanical ventilation for progressive hypoventilation and hypoxemia. between icu days 8 and 16 he received haloperidol, 10-44 mg/d (cumulative dose 209 rag) for agitation and delirium. icu day 11: qt 0.46/qtc 0.57 sec. icu day 12: for better control of delirium, trazodone " 50 mg q hs was added. icu day 15: he developed frequent nonsustained ventdcular ectopy. icu day 16: qt 0.70/qtc 0.74 sec, pha 7.48, paco2 50 mm hg, pao2 72 mm hg, k 4.9 meq/l, mg 2.0 meq/l. later in icu day 16 the patient had 3 brief episodes of torsades de pointes, each responding to precordial thump, and finally rhythm stabilized with i.v. lidocaine and magnesium. haloperidor and trazodone were discontinued. ecg was unchanged and myocardial infarction was ruled out. next day, icu day 17: qt 0.42/qtc 0.53 sec. torsades de pointes, a form of ventricular tachycardia characterized by a twisting qrs axis, is commonly associated with qt prolongation. haloperidol is used frequently in icu for control of agitation and delirium, with reported doses up to 1000 mg/day. over past decade, 9 cases of torsades de pointes with prolonged qt related to haloperidol have been reported. trazodone may also prolong qt and cause ventricular arrhythmias, especially in patients with pre-existing cardiac disease. in this patient, trazodone likely exacerbated qt prolongation from halopeddol leading to torsades de pointes. critical care physicians must be aware of this interaction. it is imperative to follow the qt interval for patients receiving halopeddol, especially when another drug also known to prolong qt is added. one must consider discontinuing the drug when qt/qtc becomes prolonged. objectives: analgesics and intravenous anesthetic drugs are routinely used in critically fll patients, who often suffer from a secondary impairment of the immune system. previous in vitro studies have demonstrated inhibitory effects of these drugs on polymorpho nuclear cells (pmn). the potentially important role of endothelial cells (ec), however, was not investigated, since suitable test systems were not available until recently. therefore a physiologically more relevant in vitro migration assay through cultured human endothelial cell monolayers (ecm) we established. using this assay system, the comparative effects of fenlanyl, sufentanil, propofol and the known pmn inhibitor thiopontal were tested. methods: human umbilical vein endothelial cells (huvec) were isolated and cultured on microporous membranes (cyclopererm) until an ecm was grown. pmn from male and female volunteers were separated by standard procedures. ecm and pmn were preincubated with clinically relevant concentratious of thiopental (104 m), propofol (4p_g/ml), the solvent of propoful (intralipid), fentanyl (30ng/ml) and sufentanil (sng/ml). after preincubatiun (ecm 30 minutes, pmn 15 minutes) with the reslx~tive drug, leukocyte migration towards the chemoatfractant fmlp (1o 7 m) was measured in a two chamber 24 well system for 3 hours. the migration rate of untreated (untr.) and treated (treat.) pmn through untreated and treated ecm were determined. as a control untreated pmn and untreated ecm were used. results are given as means from 5 independent duplicate determinations and expressed as a percentage of control (table) . statistical analysis was done with student's t-test. results: clinical concentrations of fentanyl, sufentanil and prupofol showed similar inhibitor~ effects as the known pivin inhibitor thit e 1 ). 77% conclusions: for the first time we could show that analgesics and anesthetics exert their inhibitory effects not only on pmn, but mainly on the interaction of pmn with endothelial cells. moreover, we could shmv a significant suppressive effect of the opinids fentanyl and sufentanil on both ec and pmn. the known inhibitory effect of thiopental obtained in ec-free test systems were also confirmed in our physiologically more relevant assay system. objectives: to investigate when and how sedation is used in a consecutive cohort of patients admitted in a large sample of italian intensive care units (icus), gathered in a network named giviti, representative of the italian icus system. methods; the study called for a recruitment period of one month, from january 10 to february 8, 1994, data collection included age and other demographic variables, acute diagnostic broad profiles, severity of illness scores, treatments, lenght of stay and vital status at icu discharge. as concerned sedation, each patient was observed until discharge or for a maximum period of seven days. information on all the drugs used for analgesia/sedation, the route and modalities of administration, the timing, dosages and purpose of the administration have been recorded. results: the study involved the cooperation of 138 icus, 128 of which enrolled at least one case. the total sample included 2932 patients. overall, 60.7% of patients analyzed (t780/2932) received at least one prescription of sedative during their stay. globally, at least one sedative drug was prescribed to these 1780 patients in 5014 days in icu. although over 38 drugs were reported to be used, 10 pharmacological principles accounted alone for 89% of all prescriptions. opioids were actually used in 33% of prescriptions; propofol in 24% and benzodiazepine in 18.3%. as regards the way of administration, intravenous administration was applied in 74% of cases and, followed by intramuscular in 17.3%. moreover, non-steroidal anti-inflammatory drugs (nsald) were used in 19% of patients and neuromuscular blockade agents (nmba) in 23%. detailed analysis on certain subgroups (surgical, trauma, ventilated patients etc.) have been also carried out in order to describe the practice of sedation in these peculiar subgroups. findings will be widely discussed during the presentation. conclusions: these results should be interpreted keeping in mind how peculiar is the intensive care setting compared to many other less complex settings of hospital care. in conclusion we thought it was important to present the data currently available in the most neutral form, to start moving in a direction which will enable us -by means of more specific and detailed studies, and with the cooperation and involvement of all those participating in the project -to shed light on one of the many aspects of medical practice in the field of intensive care which deserve closer attention. introduction: the aged run perilously high risks in cardiac surgery: among others, of haemodynamic fluctuations, respiratory depresskm and organ failure. response to anaesthetics is a crucial determinant for post<)perative complications, none the less being reintubation due to mechanical ventilation difficulties which increase morbidity, mortality and intensive cdre unit (icu) stay. objective: we wanted to assess our a,aesthesia window (selection, and a view of the induction -extubation period) for predicting safe and swift awaking, thus: icu dismissal for the aged. methods: in 1994, 162 selected patients (pts) (>70y, 62f) followed a regular elective cardiac surgery protocol (propofol given at precisely designated time intervals). upon 1cu arrival, they were subjected to an admission protocol. our predictive criteria for early extubation at 8h included: a) alertness and ready response to commands; b) adequate gag reflex and sufficient protection for respirak)ry tract; c) pao2 >75 mmhg with flu 2 <0.4; d) stable ph>7.35 with spontaneous respiration; d) stable haemodynamics without dysrhythmias; e) adequate perfusion and diuresis (>1.(i ml/kg/h); f) mediastinal bfeeding<100ml/h for at least 2h; g) normothermia (core temp>36~ and no shivering). subsequent reintubation was for: 1) rr>35/min; 2) spontancx)us ventilation for 30 rain with paco2>50 mmhg; 3) pao2<50 mmhg with fio2>0.4; 4) ph>7.45; 5) heart rate>]20 bm; and/or 6) non mental alertness; and 7) other medical disorders, after which adequate weaning therapy was necessary. then, successful weaning after 24h was considered: 1) spontaneous breathing without any forrn of mechanical assistance; 2) stability in haemodynamics; and 3) elimination of fever threat. results: 122 pts (75%) were extubated at 8h without complication; 29 other pts (18%) at 8h but had to be reintubated because they were hypoxic and began weaning therapy; finally, they were all re-extubated by 48h. only 11 pts (7%) proved problematic. conclusion: a,aesthesia wimhlw options (selectkm, extubation, reintubation and weaning) predicted quick (times propofol administration) and safe (rigid criteria) extubation (75%=8h and 18%=24h), exempting pts with developed post-operative complications (7%=extubation<72h) unrelated to al~aesthesia window or icu protocol. dismissal and recovery then became an abbreviated question of time. fifisetll p, domeneg~i ~, sforzini i., veronesi i~, maconi a.g. *, breg~ massone p.p 30h [] ic+pca request conclusions:using e~aprenorphine, a synthetic,long-acting, ago-antagemist opinid drug as analgesic, in the major surgery we obtained the best clinic results with association of conttheus infusion of haft dose drug with bohts of pca in the first 15-20 hours and just pca in the secmad day after surgery when the patient is less sleepy. in this way we dent have a great sav~g of suppled drug but the major well-belng of patient without ~erious side-effects and quick mobilization; the dosage used don't compromise a good awake of patient: all patients are sleepy but ready for answer, no allueinatian, bradipnea but not less than 10 b/m without ipoxia. also the patient proffered this kind of truit meut than the traditional at demand. the ward staff feel it useful] and rehabl~ the negative feed-back technology of the electronic infuser system makes possible to use it safe in the ward with high drug's concentration too. the infusion rate of low dose of drug assure a continuative analgesic covering ~n the first postoperative periad; the pca mode involves the patient him-self in the managemenl of therapy and enables him to choose the best way to confront the dll~icuity of postoperative period without call medical stall using pca-device we have had no probicm~ no accident. analgesia during extracorporeal shook wave lithot ripsy a .levit, b.grinbezg regional hospital, ekaterinbu~g, russia 0b~ectives: our task was to compare ~he analgetic effect of norphin and tramel. methods: study was made of two groups of uro-li~patients aged 25-61. group a (23 patients) received baprenorphine hydrochloride (norphin) at dosages of #.52• mg/kg. group b (30 patients) received tramadel hydrochloride (t~aasl) st dosages of 1.50z0.38 mg/kg. before the procedure diazepam was administrated i.v. (0.24!0.03 mg/kg). blood saturation (spoz), hemodynamics incides (bp, hr,sv,co,sap,svr) were examined and the patients' subjective assessments of snsesthesis quality were analyzed. the hospital ethics committee approved the investigation. results: when using norphin hr increased by 17.7% on the onset of the procedure while sap and sv decreased by 8.%% and 9.6%, respectively (p<0.05). however, there were no reliable co chsnges. spoz ~educed by @.2% (p<0.05) and remained lower than the initial one after the procedure was oyez. when administrating tramsl 50 min. after ste~ting the procedure sap and svr increased by ~1.2% and 7.3% respectively. sv and co decreased insignificantly. nine patients in group b saffeting some dlscomfo~t needed additional tm~msl in~ection. in the course of the whole p~oced~e spo, was constant and was highez than that in ~he case of nozphin (p<o.05). conclusion: respiratory suppression by no~phin can limit its use in case of lithotzip@y while the advantages of tremsl a~e: lack of dyspnoe and good contact with the patti-b_ the role of uncommon agents of antinociception, placed epidurally, in the control of pain transmission in the spinal cord is well documented. somatostatin as an inhibitory agent is found in the dorsal horn of the gray matter of spinal cord. the effects of somatostatin is similar to the opiates if injected into the epidural space (ref. i, 2, 3, 4) . the aim of the present study is to present the clinical effect of above mentioned palliative therapy in the case of cancer and non cancer pain. informed consent had been obtained from every patient before treatment was initiated under the legalisation of the local ethics committee. using the permanent epidural catheter and continuous infusion pump, we administer somatostatin in the dose of 5 up to 20 microgram per hour for one up to three days. the patients paln feeling was evaluated using a vas (visual analogue scale). we found that the opiate resistant pain level decreased by 50%. the rest pain could be controlled by pereral opiate analgetics or by combination of somatostatin and an opiate plus a local anaesthetic agent epidurally. theoretically, it seems that epidural administration of somatostatin and local anaesthetic agent is useful in the control of acute pancreatic pain and systemic effect of somatostatin is beneficial in the treatment of acute pancreatitis. few studies have been performed in the critically ill (ci) and because of the complex pharmacodynamic and pharmacokinetic changes that occur, it is difficult to predict accurately drug responses and interactions in individual patients. midazolam (m) is a watersoluble benzodiazepine with a rapid onset and short duration of action in normal individuals; however its metabolism is decreased in the ci. critical care physicians every day experience suggest that among the ci there is a different pattern of response to m; here it is hypothized that such a response could be due to different metabolic behaviours of ci. m (i.v.infusion rate 1-7 rag/h) and antipyrine (1000rag p.o.) were infused to ci with (10 patients -group 1) and without (10 patientsgroup 2) endotracheal intubation. blood samples were collected at fixed times during and after m infusion. total urine nitrogen and antipyrine elimination half-life demonstrated different metabolic characteristics in group 1 chypermetabolizer") as compared to group 2 chypometabolizer"). results follow. the results of our study are preliminary and more kinetic data in critically ill patients are needed to statistically confirm our approach of "hypometabolizer" and "hypermetabolizer". objectives: some clinieai and experimental studies suggest that propofol decreases myocardial contractility in coronary artery bypass grafting (cabg) patients. the intrinsic negative inotropic action of the drug may be independent from changes of preload and afterload, whereas the myocardial depression induced by propofol may depend on changes in ca ++ ious availability. aim of this study was to verify the hemodynamic effects of propofoi in a group of cabg patients with uneompromised contractile function and to minimize myocardial depression, during induction of anesthesia, choosing to associate calcium chloride (cac12) in an other group and to define its role in producing this effect. methods: fourty patients, randomly divided in two groups (20 pts a and 20 pts b), undergone elective cabg, were enrolled in this study. anesthesia was induced by senior anesthetist in both groups by means of fentanyl 7 mg• kg < in 60", pancuronium 0.1 mg x kg -1 and propofol 2 mg • kg -1 in 60". a blind investigator administered via another venous way at same speed (60") saline in patients of group a and 10 mg x kg -1 cac12 in patients of group b. hemodynamic data (heart rate hr, mean arterial pressure map, mean pulmonary artery pressure pap, pulmonary capillary wedge pressure pcwp, central venous pressure cvp, cardiac index ci, stroke volume index svi, systemic and pulmonary vascular resistances indexed svri and pvri) were obtained at baseline (to), 2' after anesthesia induction (t1) and 2" after tracheal intubation (t2). results: hr decreased moderately in both groups (p = ns). map decreased as well, more markeatly in group a at ti and t2. this trend was statistical significant (p < 0.05) in both groups. pap, pcwp and cvp unchanged following induction in any of two groups. ci decreased in group a (p < 0.05) dramatically dropping at t2, while in group b it showed a negligible decrease at t1 (p = ns) and value similar to baseline at t2. svri and pvri did not exhibit statistically significant changes. conclusions: the use of propofol as a starter of anesthesia in cabg patients allows to reduce total dose of fentanyi, dramatically reducing post-operative intubation time. propofol-fentanyl association prevented the hyperdynamic response to stress (i. e. laryngoscopy, intubation) but severely depressed ci and svi. cac12 simoultaneous administration effectively minimized the negative inotropic effect of propofol. moreover no unwanted side-effects due to cac12 were observed. diseoordinated labor activity (dla) is one of most serious in obstetric pathology.medication sleep effect to a woman in birth is of limitation of pathologic impulsation from the central nervous system, but it does not influence processes resulting in and supporting dla on the organ level. constant discoordinated uterine contractions during medication sleep (ms) imerease disturbances of uterine blood flow, organ hepoxemia connected with hyperergia of vascular wall and myometrium to catecholamines, that reduces efficiency of anesthesiologic protection. we applied hexoprenalin in complex with ms to 20 primaparas. efficiency control was being accomlished through hysterograms, grade scale of analgetic effect where hemodynamics reaction to pain, significance of emotional reactions and their vegetative manifestations before and during ms were registered, and also through hydrocortisone level in plasma before and during ms. the examined women's average duration of ms was 2 hours more than in control and made 4 hours, though their average labor duration and in control was the same and made 12 hours. 98 % of the examined women experienced independant labor, as for the control group -only 68 %. supplementary methods of anesthesia were required in 73 % in control,that increased medicamentous depression of the fetus, but for the examined group -only in 16 %. the examinees's newborns experienced favourable terminations, in control 3 newborns were in state of light rate asphixia. analgetic effect was complete in control just in i5 %, and of examinees -in 82 %. on the women's hysterogrnms before ms discoordinated contractions were registered on the background of increased tonus. during ms in control similar uterine contractions were constantly registered, but for the examinees they were not marked at all or had the proper character. the examinees' hydrocortisone level was reduced for 42 % and in control only for 24 %. the study has allowed to make a conclusion that hexoprenalin applications in complex with ms in case of dla make anesthesiologic care safer and more effective, promote simultaneous removal of patholgic processes in the central nervous system and uterus, which lead to dla development. objectives: the aim of report is to present a clinical study results for the evaluation of anesthesia for a patients in unconscious states by eeg examination: methods: the study was conducted on 17 patients after a severe abdominal operations on stomach and intestine and on one patient after traumaticat exarticulation of upper extremity. all patients have different unconscious state levels from somnolence to coma ]. also thay hav~ had respiratory insufficiency and control mandatory ventilation have been used. we were used intrave,~ous injections of morphine hydrochloride from initial dose of 20 mg. than we were increased the dose to 80 mg i.v. by drops with an accompanying eeg monitoring and fourier spectral analysis. initially all patients have had 13-rhythm with slow 0and a-activity. an c~-activity index have been lower than 5%. results: after anesthesia the o-and a-activity have disappeared. the or-activity index have increased to 35%. also we have noted decreasing of the unconscious state levels to a possibility of a verbal contacts (13-14 points gcs). conclusion: the quality of anesthesia could be evaluated by eeg monitoring. a regaining consciousness afte~ the injections of high doses of morphine, probably, may be a result of an opiate deficiency (full or partial). anesthesia with the following drugs: clonidine, l-arginine, l-n-arginine-methyl-ester (l-name), and their combinations. tail-flick latency (tfl) was determined at time zero and 15 min after each treatment. clonidine (i-4 ~g/mouse) prolonged tfl in a dose-dependent manner. at a clonidine concentration of 4 gg/mouse, tfl increased 2.37-fold compared to time zero. l-arginine increased tfl at a high concentration (i00 gg/mouse). l-name suppressed tfl 1.2-fold compared to time zero at a concentration of 50 gg/mouse. conclusion: we have found that no is involved in clonidine spinal analgesia. studies are currently being undertaken to assess the effect of combination treatment of the above drugs on clonidine supraspinal analgesia. objectives: hemodynamic changes ussualy accompany laryngoscopy and tracheal intubation.the aim of this prospective study vas tu present the effectivness sufentanil in preventing reflex circulatory respone of laryngoscopy and tracheal intubation and provides stable hemodynamio condition for induction of balanced anaesthesia. methods: 30 adult asa i-ii patients sheduled for elective surgery.they were allocated randomly and divided into two groups:fifteen patients received single bolus of placebo prior to laryngoscopy.and tracheal intubation. anaesthesia induction was then performed with thiopental (smg/kg) and atracurium (0,5 mg/kg), followed by neurolept anaesthesia. mean arterial preassure (map) and heart rate (hr) were measured before, during and 5 min after intubation. althought ecg was recorded at the same time intervals. results: the groups were compared with regard to the changes in arterial blood pressure,heart rate and electrocardiogram. mean arterial preassure and heart rate were incresed significantly during and after laryngoscopy and tracheal intubation in control group but remained unohange the baselin values in the group who received sufentanil. control group ecg suggested more changes than sufentanil group. conclusions: the results indicated that 1,2 mg/kg sufentanil for anaesthesia induction reducing the pressor response to laryngoscopy and tracheal intubation. obiective: the aim of this experimental study was to evaluate morphologically the influence of anesthesia in liver as well as in isolated hepatocytes (hc). methods: a total of 16 female swine (20-22 kg) were used as liver donors and were randomly assigned to one of two groups regarding anesthesia protocol: group a (n=8): induction with intravenous sodium thiopental in a dose of 5 mg/kg, group b (n=8): propophol in a dose of 2 mg/kg for induction'and 10 mg/kg/h for maintainance of general anesthesia. both groups were under the same preanesthetic regimen (diazepam, ketalar and atropin) and received standarised doses of fentanyl and pancuronium during anesthesia. in beth groups total hepatectomy was performed and the liver was perfused with 4-5 it of cold (4oc) university of wisconsin solution for a period of 5 hours before hepatocyte isolation. liver tissue samples were fixed in formalin for the morphological study and stained with hematoxylin-eosin and pas. for hc isolation collagenase solution (type v, sigma, c-9263,1.3mg/ml) was infused via portal vein and the liver was incubated at 37oc for 45 rain. cells were filtered and then washed in cold hanks' solution. isolated hc were fixed and prepared for staining or simply cryopreserved (-20oc). results: histology was completed in 8/16 experiments (4 in group a and 4 in group b). mononuclear infiltration (halothane type injury) was found in all specimens (8/8). focal zonal necrosis and acidophilic bodies were found in specimens from group a (2/4 and 1/4 respectively) while portal inflammation with piece meal necrosis was found in one specimen from group b (1/4). smears of hepatocytes -beth formalin fixed and cryopreserved at -20oc -were stained with hematoxylin-eosin and showed morphologically intact hepatocytes. conclusion: pre[imineq~' study of our material reveals mote frequent livet injury in the thiopental group, but this finding has yet to be established by increasing the number of observations. objectives: in this paper we present our experience and point out the importance of sedation of those patients who suffered severe head injuries/contusic cerebri with signs of decelebration/and who were put on ventilatory machine to obtain better mechanical ventilation in neurosurgical intensive care unit of emergency center in belgrade. methods: 256 patients were treated from jan. 1994 till dec. 1994 results: in 157 patients we successed to reduce agitation and decelebration. conclusions: the major demand of sedation in neurosurgical patients are that the patient should be calm, comfortable and painless. references we compared three analogous groups of 20 patients in each, with orthopedic operations on lower extremities. in each group we applied different kind of analgesia: first group rece-ned local anesthetic (4 ml 0.5~ bupivacaine) when analgesia was first demanded; second group received 2 ml fentanyl via peridurai catheter when analgesia was first demanded, and third group received 2 m] fentanyi intravenously on demand for analgesia. we used visual analog scale (vas) for estimation of pain intensity and success of applied therapy. statistical data analysis was performed using student's t-test. tha best vas had group in which local anesthetic was applied periduraly. second was the group with periduraly applied fentanyl, and third was the group with intravenously applied fentanyl the longest duration of the effect of analgesia ~/as in the second group. there were no adverse effects and complications, apart from mild sedation in the third group. for orthopedic operations on lower extremities, application of peridural catheter for anesthesia and successful postoperative analgesia with local anesthetic, or opioid analgesics is acceptable. background and obiective : tympanic temperature measurement (ttm) is a relatively new procedure which provides accurate estimates of core temperature in stable postoperative patients and in patients admitted to the emergency ward. however, experience with this technique in hemodynamically unstable adult icu patients is limited. design : prospective study with patients serving as their own control. se:ttijlg : adult medico-surgical icu in a tertiary care hospital. patients : 51 consecutively enrolled patients with thermodilution catheters in place and without contraindieation for rectal temperature measurement and without hypothermia (core temperature <35~ interventions : tympanic temperature, measured by a commercialized tympanic thermometer (genius 3000 a first temp) was compared with pulmonary artery (pat) and rectal temperature (rt). within 10 minutes, core temperature was assessed in each patient by the same trained individual using the three techniques in random order. measurements and main results : mean bias (ix~ and its variability (sdr of method comparison pairs were calculated using the methods comparison analysis as described by bland and alunan (lancet, 1986 ; i : 307-310 objective: to measure the prevalence of pressure sores on the intensive care unit and the effect of risk calculation on pressure score prevalence and pressure score severity. method:in 1993 during a period of 5 months a series of prevalence studies was carried out on the sicu to investigate how many patient days with pressure sores were taken care for by the icu nursing staff, what kind of preventive interventions were done and what the patient risk was on developing a pressure score. results: on average there was a prevalence of pressure sores on the buttocks of 18.5% on the short stay unit and 42.8% on the long stay unit; preventive interventions increased when the pressure score was visible for the nurse and the majority of patients had a high risk or extra high risk for developing pressure sores according the pressure score risk calculator. as a result of these findings the nursing management decided in 1994 that nurses had to complete daily on every patient the pressure score risk assessment in order to be able to take adequate preventive interventions. when the results of the two studies were compared, the most important results were that: 1) there is no indication of a reduction in patient days with pressure sores (short stay unit: 14%, long stay unit: 55%); 2) there is a reduction in severity of pressure sores (grade iv decreased from 2.4 % to 0.7 %) because adequate preventive measurements are initiated by icu nurses in an earlier stage. possible explanations for the increase of patient days with pressure sores on the long stay unit are that the average pressure score risk score was increased from 17.8 to 19.8 and the mean frequency of nursing patients on alternate sides decreased from 1.4 to 0.6 times each day. conclusion: daily assessment of the individual patient risk on developing pressure sores does not decrease development but reduces the severity of the developed pressure sores. method: the conventional woundcare method of patients with an open abdomen is to change frequently the saline soaked ribbon gauze pads (4-6 times each day). this method is labour intensive for the itu nursing staff and rather uncomfortable for the patient. problems that often occur are: insufficient hygiene, frequent nursing interventions, a progressive risk for deterioration of the skin and underlying tissues, difficult to measure abdominal output and in case of bowel fistulas enteral feeding is hardly possible. in using the new method, stomahesive is applied on the skin surrounding the wound. a large size of surgical film dressing, opsite | is applied over the abdomen. two or more foley ~ catheters ch 22 are inserted through an opening in the surgical filmdressing. the application of several lateral openings in the catheters is advised. the drains are held firmly in place between two layers of sterile opsite | "flexigrid ~'' 6x7 with the so called bookend method and are connected to a low vacuum suction system (1-2 kph) results: we studied the frequency of dressings changes on 12 patients in the itu. in total they had 363 days with an open abdomen. during this period there were 63 dressing changes. this means one dressing change every 6 days. further advantages of the new method are; more hygiene in patient care, no maceration and irritation of the skin, nursing on alternate sides is possible, output can be measured, in case of bowl fistula, enteral feeding is possible and an abdominal lavage is possible on the sicu by opening the opsite | conclusion: this new method of woundcare management is more patient friendly, prevents several nursing complications and decreases the work load for the nursing staff. objective: to investigate the interaction between the ventilator and the closed suction system related to possible induced negative pressure by this sytem. method: we studied in a testlung model what the effect was of the different ventilators (evita: dr~iger; servo 900 c: siemens), ventilation modes 0ppv, simv) and ventilator settings (tv, trigger level, frequency, peep level) on the induced negative pressure by the closed suction system during suctioning. results: when using the evita the magnitude of the negative pressure increased when the ventilation frequency and tv decreased.. the largest negative pressure (-76 cm h20) was produced with a tv of 600 ml and a frequency of 6 /min in the ippv mode with trigger level on -3 cm h20 and a peep level of + 10 cm h20. the servo 900 c showed a complete different pattern. the largest negative pressures were measured when no trigger level was set (27 cm h20), the induced negative pressure was not depended on the tv or ventilation frequency but more depending on the ventilator mode. the simv mode produced the smallest negative pressures ( 1-4 cm h20 ) and the ippv mode the largest (16-27 cm h20 ). conclusion: the effectiveness of the closed suction system is very much depending on the kind of ventilator, ventilator mode and ventilator setting that is used. if the interaction with the used ventilator is not known by the icu nurse this piece of equipment can cause damage to the patient by inducing large negative pressure in the comprimised lung and there fore creating the possibility of alveolar collaps and atelectasis or oedema. introduction : ethical problems are daily and disturbing preoccupations for the icu staff. objectives : improving ethical management in icu by creation of an intelprofessional group of ethical reflexion ( iger ). methods : existing was evaluated by a preliminary formulated series of questions sended to the whole staff (q1, n = 43, 13 items) after two training sessions (laws, deontology, professional values, culture, norms of quality) followed by 38 persons (88 %) and directed by an external chairman, satisfaction and needs of the staff were evaluated by a second series of questions (q 2, n = 38, 12 items). at the issue, iger was created. results : q1 : 30 responders (70%). law's misinformation : 33 to 70 %, disagreement for limitation of intensive cares : 40 %, hope to improve collective decisions and creation of iger : 88% q 2 : 29 responders (76%) : satisfied by the training sessions : 65%, non satisfied : 31%, want to continue the project : 68 %, refuse : 10 %. iger was constitute by physicians, nurses, secretary, dietetic (n = 15). the first working theme was {< therapeutic's withholding and withdrawing decisions in icu ~>. four subgroups of iger's members (having access to an ethical library) worked independautly and submitted their reflexions in a tdmestrial plenary session of iger in the presence of an external chairman, allowing a synthesis. at the issue a report was writted to be used as a reference for bedside and individual decisions. conclusions : constitution of iger seems to improve ethical management in icu. the first result of iger is that it is now possible to began collectively a reflexion concerning therapeutic's withholding and withdrawing in icu. the work is going on and further subjects will be studied. objectives: 1) to compare the value of heat-moisture exchangers with bacterial filters (hmef) and without bacterial filters (hme) in the prevention of colonization of ventilator tubing and ventilator-associated respiratory infections. 2) to asses the temperature and relative humidity of inspired all using both types of heat-moisture exchangers. methods: 48 mechanically ventilated patients were randomized, to either hmef or hme. endotraeheal aspirates, pharyngeal swabs and samples from tubing were collected for bacterial cultures on the 1st, 2nd day mechanically ventilation and weekly thereafter. temperature and relative humidity were measured in 23 patients (13 hmef and 10 hme) 3 h and 24 h after placing the hme or the hmef. results: both groups were comparable as regards age, mechanical ventilation period, severity score (saps ii), leukocyte count, and number of patients with prior antibiotic treatment. from the hmef group, 10 (42%) ventilator tubing yielded microorganisms in, at least, one sample as compared to 7 (29%) of the hme group; p=ns. the incidence of respiratory infection was similar in both groups (25% vs 17%, p:ns, for hmef and hme respectively). among the 16 bacterial species isolated from ventilator tubing in the hmef group, 7 (44%) were not isolated from pharyngeal swabs. a similar ratio was shown in the hme group (6/15, 40%). both heat-moisture exchangers were efficacious in keeping a good relative humidity of inspired air (97% • vs 96% • 3.%; p=ns, for hmef and hme respectively). relative humidity was significantly higher after 3h of mechanical ventilation in the hme group as compared to hme group (28.5% • vs 26.5% • 2%; p=0.03). conclusions: both types of heat-moisture exchangers have the same effect on the prevention of colonization of ventilator tubing. similar relative humidities are achieved when using either type of heat-moisture exchanger. results: tumor and nontumer enhrgements of the thyroidea were present in 85~ of the operated, surgicel adrenal disease in io!, hyperplssle or persthyroid gland tumor in 2~ end endocrine pancreatic tumors in 3%. in the intensive oere unit, these patients wore screened by noninwsive monitoring in 85~ of cases: and invasive monitoring was applied in 15% of ceses.the basic noninvesive methods included: electrocardiogram with standard end precerdial leeds, percutaneous eutomotlc measurement of systolic, diastolic and mean arterial pressure, measurement of hourly diuresis and body temperature, frequency, hearing capacity and rhythm of one s own breathbng bs well as pulse oxymetry. a special plece in monitoring and control of vital parameters in postoperative period belonged to the nurse, thoroughly trained for enelysis end interpretation of the observed parameters which would be discussed in the paper. it has been believed that the leader sits at the pinnacle of power. over the years, this has proven to produce frustruation and anguish instead of the expected results. leaders have not been able to produce the changes they know are essential to their organization's survival with this command-and-control paradigm. through literature reviews and evaluating leadership styles, one can clearly see the most effective form is that of empowering people to a new level of performance -not ordering it. changing the leadership paradigm to a manner/style that has been shown to be effective and one of people empowerment shifts the focus to personal responsibility for performance. removing obstae}es~ stimulating self-directed actions, and determining focus and direction are just a few elements used to create the successful environment of empowerment. with increasing pressure in the health care arena, it becomes critical that a leader's job is to get the people to be responsible for their own performance. developing ownership, creating an environment where people want to be responsible, being a mentor or coach, and learning faster while encouraging others to do so demonstrates the commitment to effective leadership. this presentation will illustrate the critical components that are achieved when every person in the institution is empowered to perform at a level that is directed toward positive, effective results. herrera m. (md) . icu. hospital regional. malaga. spain. the systems of veno-vanous continuous haemofiltration (wchf) have a high cost and a limited life span. in an attempt of lengthening their mean life it has been proposed to accomplish programmed washes of the ~-stems. this practice supposes an increase in nursing workload. in order to evaluate the real efficiency of this practice we have accomplished this study. material: prospective randomized study of all the filters of vvchf used during the last year in our icu. we have determined two groups of filters, in the first (group a) we accomplished washed in a programmed way, and in the other (group b) only when the alarms of the system suggested a clotting of the filter. for the statistical analysis we used the kaplan-meier test for survival analysis. results: we have studied a total of 24 patient submitted to wchf during the last year. we used a total of 32 filters with this results. objectives. sounding out the nurses about the need to inform patients" relatives and the rigth kind of such information, like a preliminary approach to an information cuality assessment, methods: we inquired all the nurses of the intensive care unit of an regional hospital by an semiestructurated questionary which included personal data: age, sex, contractual relation, professional experience.., and opinion data: do you think to inform relatives is a nurse task?. which of the next informafions do you think is more important?, please, write others topics about information you think are relevant. we process the data on epi-info estatistical program and use x 2 test to compare the results. results" from 80 nurses of staff 5 refused to flu the quetionary, and 8 were not available. of the 67 remaining, 71%were v~men and 29% men. the mean age were 31.51% had an svable contract and 499( eventual, the mean professional experience were of 10 years and 44% worked in the unit since more than 6 years. the 88% answered that offer information to relatives is part of the nurse activities. we did not find differences with nurses who answered negatively comparing by sex, age, contractual relation or proffesional experience. the three information topics found out like more important were: 1) to inform about patient mood. 2) to inform about happenings from the last visit. 3) to inform about dressing instrument required by the patient, nurses who answered negatively think that to inform is a doctors task or that nurses are not competent. conclusion~ intensive care unit teams (nurses, doctors and auxiliar personnel) should get accord on who and how to inform relatives, we consider the nurses' role on information as unquestionable. objective: investigate the respiratory and cardiovascular response after discontinuing oxygen therapy durir~ intr~/]o~pital transport. desiqn: fifty-one patients (29 male and 22 female, aged 69+2,5 and 73,912,4 years respectively, ~+sym) being on 02 therapy were studied prospectively in two consecutive intrahospital transports. oxygen therapy was continued in the first transport while the second one was performed as usually, i,e, without 02. during transport each patient was monitored by pulse oxymeter and holter whereas arterlal blood gases were tested just before a~xl aft~-trar~portation. results: compared to daseline, pa02 and sa02 were signif~canthy decreased in the case of oxygen discontinuation (p<0,00i). paco2 was significantly inur~ds~i only in the subgroup of patients with obstructive lun[ disease (p<0,01) . heart rate increased in all phases of the transport when 02 administratlon was discontinued. blood pressure remained stable in either case. the percentage of supraventricu!ar extrasysto!es, ectopic v~r[hicui~r contractions and st-s6~ment depression was progressively increasing and became very high at the end of transport in the case of 02 therapy discontinuation. other arrhythmias did not change significantly. conclusion: discontinuation of oxygen therapy during intrahospital transport causes severe drop of pao2 and sa02, increases the heart rate and contributes to the appearance of arrhythmias which were not present before. methods:for evaluation of the functional state of brain the complex of methods was used,whieh included electro encephalngraphy ( brain mapping ), rheoencephalography, tetrapolar transtorax rheography. for the estimation of humoral status the level of histamine and serotonine, products of free-radical oxidation,enzimatic markers of ishemic damage of brain and of endogenous intoxication was investigated. results:92 patients with encephalopathies after resuscitation were observed.asystolia was as a result of:shock, trauma, asphyxia,poisonings,appiication of drugs, eclamp sia,injury of the heart,diseases of fhe cardiac vessels. all patients with postasystolic syndrome entranced in comafose condition.in the 1 group (reconvalescents) the depth of coma by glasgo~ pittsburg"s scale was 23,3+-1,78. the duration of coma was from 30 rain. to 48 hour,average 11,9+-4,sh.ln the 2 group (the deads) the depth of come was 13,8+-0,86.the artificial lung ventilation was used in all patients:in the 1 group 2,64+-0,92 days,in the 2~ 6,1 +-1,1 days.apallish syndrome developed in 5 cases,in 5 patients diagnozed <<brain death~. the 4th degree encephalopathy (coma) was found to be associated with disorganized eeg-pattern with predomi nant alpha-( 1 group) and slow (2 group) activity. the relative power of delta-and theta-ranges was about 77-87 per cent.the patients with apallich syndrome demonstrated greatly elevated theta-range power, that of beta -range was essentially decreased. the degree of disturbances of circulation in the both groups was different.cardiac index (ci) and stroke index (si) in the 1 group decreased on 19,5% and 44,3z, in the 2 -on 45,9% and (;3,3%.general peripheral resistance (gpr) increased in the 1 group on 40,5%,in the 2 -on (;9,4%.in the 1 group brain blood flow was increased, in the 2 -decreased on 37,6n.in the 1 group there was the normotonic type of reg-curve,in the 2 -atonic or hypotonic type (without reaction on pharmacologic influ ence).disturbances of permeability of cellular membranes (enhanced free-radical oxidation with an increase in di enic conjugates on 107 and 121n) and vascular ones (an increase of serotonin concentration on 90 and 110%, hi stamine content on 116 and 180%) resulted in hyperfermentia (an increase of concentration of creaune phosphoki nase in the 1 group on 59%,in the 2 -69%.the level of middle molecules (mm) increased in the 1 group on 79,2%, in the 2 -on 120,8%,of polyamines (p) (spermidin,spermin,putrescin).coefficient of correlation between mm and p was 0,891. immediate correction of neurocyte metabolism is impossible due to marked brain oedemaswelling,severe dis ordes of cerebral circulation, disturbances of membrane permeability.thus,the following treatment algorythm might be advisable: 1. protective inhibition of brain and reduction of its energy requirements. 2. recovery of cell and vascular membrane fuoctions.3.recovery of blood circulatiom4.oxygenation of nervous tissue and drainage of brain disintegration products.in 22 patientshbo carried out.5.active methods of detoxication (homo-and enterosorption).6.correction of cerebral metabolism.7. dehydrative therapy must be very cautious. conclusion: the activation of energetic metabolism in neurocytes must be carred out only under neurophysiolngical control and after definite preparation. (67). the clinical estimation of acute cerebral failure carried out by olasgo-pittsburg"s classification of coma. we studied such groups of patients: poisoning co (36), asystolia (8), eclampsia (5), acute renal failure (5), control group (13). methods: electroeneephalngraphy (brain mapping) was fulfilled by means of encephalograph <,medicor ~ in 8 monopolar channels. the estimation of eeg patterns by zhirmunskaya method (1984) was carried out. there were analyzed and changes of eegb by results of rapid transformation furie after rhythmical photostimulation(phts) 2,6,10,20 hz. we studied also the figures of absolute and relative power in such diapason : delta (1-4 hz), theta (5-7 hz), alpha (8-12 hz), betal(13-25hz), beta2(25 hz and more). results: all eeo changes were divided into following types: 1 -organized (3,4 groups); iii -asynehronic (8 group); iv -disorganized with prevalence of alpha-waves (12,13,14 groups); v -disorganized with prevalence of delta-and theta activity (16,17,18;19,20 groups) akkording to our model of hormonal-metabolic disbalance in pregnancy , one of the main reason of destosis is hypoergos , reesults in endotoxemia and neuro-endokrine disregulation. so, stady of central nervous system function give us the information about adaptation processes. the aim of our work is to study the degree of neurologikal deficit in destosis when olifen and lipin were used. there were studied 20 pregnants with different severe forms of gestosis. the degree of neurologikal deficit was valued by datas of neurologikal status . eeg with ,~brain mappinng~, , electrikal resistance of the skin. the komplex inteensive therapy with applikation of olifen and lipin allowed to improve the patient,s condition , decrease the degree of neurolodikal deficit and mortality . kostenko v.phd,kabanko t.,phd,galalu s.md,beliavtseva n. ,shramenko k. phd intensive care department,donetsk medical university, donetsk, ukraine plasmapheresis (pph),as other extracorporal methods of detoxicatin,has a great importance in contemporary medicine. the role of pph is special,because of its simplicity, effectiveness, atraumatic.we incalcated pph in obstetrical clinic. there are rough changes in agregate condition of blood in more of pregnants.these changes lead to disturbances of central,organ and peripheral hemodynamic.the therapeutic effect of pph is due to influence on agregate condition of blood. we considered that application of pph is very effective in pregnants with:bronchial asthma, severe forms of diabet, psorias, gestosis, allergic diseases. we used the membrane pph in pregnants: apparatus-,,hemos-pf>,, plasmofllter pmf-800,with effective area-800 cm,the volume of extracorporal contour-60 ml.such pph has no the ~ agressive effect,,, as in cases of application another extracorporal methods. this method was incalcated in our practice recently, so results will be reported in further publications. (2). post-operative cerebral neoplasm (1), post-operative subdural hematoma (1). icp was monitored via a catheter inserted in the lateral ventricle and values were continuously digitally recorded by means of a bedside computer data acquisition system (maclab). the fiberoptic tracheobroucosenpe, which guided the procedure, was passed between the nasotracheal tube and the trachea in order to avoid hypoventilalion. the patients had stable baseline hemodynaimcs. propofol infusion and fentanyl boli were administered to mantain stable mean arterial pressure values. peak (mean(sd)) icp duping the 30 minutes pre-ciaglia procedure (baseline values) were compared with values during ciaglia procedure, and the 30 minutes p0st-ciaglia procedure. data were compared with repeated measures anova. results: ciaglia procedure duration was (mean(sd)) 30 (14) objectives: transient global amnesia (tga) is a syndrome caracterized by impairment of short-term memory, inability to form new memories, retrograde amnesia and repetitive queries, without other neurological signs and symptoms. the pathophysiology of tga is unknown; thromboembolic, epileptic, migrainous and metabolic mechanisms have been suggested. to address some of these issues, we undertook a study of 25 cases of tga in whom we examined clinical, laboratory data, electroencephalogram, ct of the head, ultrasonography ecodoppler. methods: 25 patients were included in this study: 9 men and 16 women. the mean age was 64 years. all cases underwent a standard clinical examination, electrocardiogram, routinary humoral tests and x-ray, electroencephalogram (eeg), ct scan of the head, ultrasonography ecodoppler. results': the mean duration of amnesia was 5 h. 32 m. +/-7 h. 10 m. hypertension was found in 19 patients (76 %), ischemic heart disease in 4 patients (16 %), hypercholesterolemia in 10 patients (40 %), hypertrigliceridemia in 3 patients (12 %), smoking in 2 patients (8 %), atrial fibrillation in 1 patient (4 %), history of epilepsy in 1 patient (4 %), migraine history was not recorded. ct scans of the head showed multiple small deep infarcts in 4 patients (16 %), a single hypodense lesion in 4 patients (16 %). in 11 patients electroencephalogram was normal (44 %), in 8 patients there were widespread nonspecific electrical changes (32 %), in 6 patients there were focal nonspecific eeg abnormalities (24 %). conclusion: in our study tga was more common in women (64 %). we showed a prevalence of hypertension, hypercholesterolemia and cerebral infarcts compared to normal controls. we have demonstrated a higher incidence of nonspecific electrical changes in tga of lower length, while ischemic lesions in ct of the head were more frequent in tga of greater length. these data seem to be in agreement with the hypothesis that tga is a heterogeneous clinical syndrome, consisting of pure, epileptic, and ischemic types. however we did not find any correlation useful in discriminating pure from associated tga forms. from our study it is tempting to speculate that pure tga is a rare event, underlying still unknown mechanisms wich differ from ischemic, epileptic, migraineous causes. objectives: aneurysmal subarachnoid haemorrhage (sah) is special condition increasing intracranial pressure (icp) in various ways. at the other hand cerebral vasospasm and related delayed ischaemic deficit (did) could answer for the poor outcome. triple h therapy seems today a basic option to prevent did, but it may increase the icp worsening the altered intracranial pressure condition and thereby the cerebral perfusion pressure (cpp). is there any way to individualise the triple h therapy when it is necessary? methods: between sept. 94 march 95 thirty-seven patients with intracranial aneurysms were operated on within 48 hours following sah. five patients were in hunt-hess iv at admission. all patients received triple h therapy in a preventive fashion following surgery and were monitored by daily transcranial doppler ultrasonography (tcd). icp and cpp was measured in twenty-four cases. twenty-two of them received lumbar liquor drainage (lld) and nineteen were administered induced hypertension. the other group was treated by basic triple h therapy. results: in group with monitored icp the outcome was twenty-one excellent, one poor, two died (one of them died from extracranial decease). in the other group four had excellent, six moderate, two poor outcome, and one died. conclusion: according to our recent observation the patients can be divided into two groups of therapy. in group i, the patients with elevated tcd values and either low or high icp reacted to lld. we are concerned that haemodilution and slight hypervolaemia should dominate in the triple h therapy. in group ii patients having high icp with tcd and/or symptomatic vasospasm should be managed by the induced hypertensionhypervolaemia dominated therapy focusing on cpp (icp) and focal neurological signs. air emboli were detected in lo% (n=12) of natients undergoing coronary srtery bypass craftin~ (cabg). central nervous system ~ysfunction occured in 23~$ of the nstients with air embnli and in none of those ~ithhout air embo!i. hvtothermia is the classic form of oro-tect~on used dur~nc ~"~" " ~ ~ ca~.,~modu] :r, on~_,_.7 bj/oass. the surf~eon sho,;,ed thorough!~: evecnnte air from the heart, but the onesthesio!o[[ist can signifieamt!y influence the outcome by emt!oyin2~ methods to detect and treat air emboli. the changes in head rate are primarily due to alterations of autonomic tone. the heart rate variability (hrv), that express the degree of heart rate fluctuation around the mean heart rate, reflects somehow the condition of central nervous system. hrv may be measured by a number of techniques. short-term time-domain variables of hrv are reflect generally the vegal activity. in this study the changes in hrv variables of patients with brain damage, and in addition the changes in hrv measurements in comparison with the clinical evolution were evaluated. eight patient with brain damage and six normal individuals as control group were studied. a elecrocardiographer with availability of computation the sequence of beat-to-beat intervals for one minute was used. the following variables of hrv were measured: 1) standard deviation (sd) of beat to beat r-r interval differences that reflects the respiratory control, 2)the maximum/minimum (max/rain) interval that reflect variability related to baroreflex and thermoregulation and 3) the coel~cient of variation (cv), the results are shown in the in the patients with brain death and in vegetate state there were virtually no hrv. increased hrv pattern was found with clinical improvement, the changes of hrv precede of the changes of gcs, we conclude that time-domain hrv could reflects the degree of brain damage, it is good prognostic index of the brain damage and may change earlier than the gcs. objectives: cerebral co 2 vasoreactivity is an important determinant of cerebral blood flow (cbf) and has been shown to be of prognostic value in head trauma (acta anaesthesiol. scand. 1991; 35:113-122) . we wondered whether co 2 vasoreactivity could be selectively altered in one hemisphere in comatose patients. methods: 6 patients (5m/1f, age 32-65yrs, glasgow 4-8) in coma due an acute brain lesion (trauma, hemorrhage, or infection) were studied. cbf was measured bilaterally using jugular thermodilution at paco 2 25, 30, 35, and 40 mmhg by increasing pico 2 with mechanical ventilation kept constant. normal co 2 vasoreactivity was defined as an increase in cbf of at least i ml/min.100 g per mmhg paco 2. results: 2 patients had normal co 2 vasoreactivity bilaterally, 2 patients had altered co 2 vasoreactivity at both sides, and 2 patients had a normal response at one side (left or right) with an altered response on the other side (dght or left). for the 6 patients left cbf was in mean !7 ml/min.100g lower than right cbf (figure methods: following institutional approval 4 piglets (body weight 25:tl .5) were anaesthetized by 2% fluothane. a catheter was placed in the right femoral artery for blood pressure monitoring and a fiberoptic catheter (oxymetncs-3 abbott) was advanced via the right internal jugular vein to the jugular bulb for sjo 2 determinations. another catheter with a balloon on the tip was advanced in the right atrium via the right femoral vein. a mean arterial pressure (bp) at 25 mmhg was achieved by appropriate balloon inflation for 10 rain and two groups were cleated: i) the hypoxemic group by respirator disconnection (*) and it) the hyperoxemic group by fio2=l on respirator (o). samples were obtained at 0 time (1), 10' min at hypoperfusion (2) arid at reperfijsion at 1' (3), 3' (4) and 10' (5). pao2, pjo 2 and oxidative brain stress evaluation was performed from jugular bulb blood. the latter included: i) no synthase (nos) and xanthine oxidase (xo) activities by a method based on the oxidation of scopoletin detected fluorometrically, it) no levels estimated as onoo-by luminol enhanced chemiluminescence in the presence of 500~tm hydrogen peroxide (h202). resul'~s: the mean pao 2 was 34 mmt-ig for group i and methods: we retrospectively reviewed all 411 upper gi-endoscopies, performed in the period january 1992-july 1994 in 301 patients (199 men and 102 women) admitted at the 4 icu's of our hospital. results: it concerned 129 surgical, 103 medical, 50 eardiological and 19 neurological patients with a mean age of 57.9 yrs (range: 14-91). in 86%, the endoscopy was performed at the icu and in 14% at the endoscopy department. in 56% of the cases, the endoscopy was primarily diagnostic, of which 70% was performed for localization of upper gi blood loss. in 44 % the endoscopy was primarily thempentic, of which 89 % was performed for placement of a duodenal feeding canula. location of the upper gi bleeding was: variees (31%), duodenal ulcer (20%), oesophagitis (13%), gastric ulcer (11%), others (13%) and none (10%). as coincidental findings were noted: cesophagitis (37%), gastritis (16%), gastric deer (14%), duodenal ulcer (9%), duodenitis (8%), oesophageal ulcer (7%) and others (8%). conclusions: there were marked differences in indications and findings of endoscopy at the different icu's. these differences reflect an admission bias and differences in populations and treatment preferences. compared with cardiological and neurological icu's, substantially more endoscopies were performed at surgical and medical icu's. in a considerable number of cases, no source of upper gi blood loss could be found endoscopicaiiy. when upper gi blood loss was the icu admission diagnosis, the main cause was needing varices, which could be controlled endoscopically in the vast majority of cases. when upper gi blood loss was ndt the icu admission diagnosis, peigie ulcer and oesophagifis were the main causes of bleeding. because of the considerable number of coincidental almom~adities found at endoscopy, there is still room for debate whether antacid medication and/or motility stimulating agents should be given prophylactically at icu's. many studies have shown that blood lactate levels in survivors and nonsmvivors of traumatic and septic shock are significantly different. the degree of multiple organ failure is related to the duration of lactic acidosis (1). the aim of this study was to evaluate blood lactate level as a prognostic marker of high risk postoperative patients who may benefit from invasive hemodynamic monitoring and aggressive fluids administration and early inotropic support based on oxygen transport parameters. methods: 32 patients undergoing elective long term vascular and abdominal surgery (asa i-bi) were studied. blood lactate levels were measured after icu admission. in the case of blood lactate level above 2 mmoltl, measurement was repeated every 4 hours for 12 hours or until normaiisation (blood lactate level less than 2 mmol/1). type of surgery, length of surgery, amount of fluids delivered intraoperatively and postoperatively, hemoglobin levels, hemodynamic variables, diuresis, postoperative complications, length of icu stay and clinical outcome were recorded. because no attempts were made to randomisr therapy or change our standard therapy protocol institutional approval was not required. rebuts: the frequency of postoperative complications was 12,5 % and mortafity was 5,5 % in a group of patients with blood lactate level less than 2,5 mmol/l (n = 18). frequency of complications (62,5 %) was significantly increased in a group of patients with blood lactate levels 2,5-4 mmol/l (n = 8), mortality was 12,5 %. mortality (60 %) and frequency of complications (80 %) were significantly increased in a group of patients with blood lactate levels above 4 mmol/l (n = 5). conclusion: blood lactate levels can serve as early marker of high risk postoperalivr patients and may predict increased risk of postoperative complications mad ~e death. objective.~: investigated practicability and clinical value of the routine measurement of hepatic venous oxygen saturation (shvo2) after major liver surgery, as shvo 2 is considered an indirect parameter for splanchthc and hepatic blood flow. methods: 30 consecutive patients were included in this study after liver resections for primary or secondary liver tumors. 5 patients suffered from liver cirrhosis (childs a). immediately after post-operative admission on the icu a pa-catheter ,was inserted under fluoroscopy via the right jugular internal vein into the hepatic vein contralateral to the resection area. hepatic venous and arterial blood samples were drawn every two hours. shvo 2 was correlated to the clinical course, macro hemedynamics, abgs aug other established lab parameters. results: in 26 out of 30 attempts the catheter could be placed correctly. in four cases after right hemihepatectomy the left hepatic vein could not be intubated due to a dorso-lateral tilting of the left liver. this is also reflected in a significantly longer time of fluoroscopy for catheterization of the left hepatic vein (12.9 _+ %5 rain vs. 3.7 + 2.5 rain; p < 0.001). the procedure requires a total of between 45 and 75 minutes. relevant clinical complications were not observed except for short term supraventricular arrhythmias during passage of the catheter through the right atrium. hemodynamics and pulmonary function could be considered normal in all individuals at time of measurement. shvo 2 showed a span from 27.4% to 90.0% with a mean of 67.0% -+ 10.8%. the following statistically significant findings could be obtained: (a) patients with liver cirrhosis showed a significantly lower shvq than patients without (53.4% • 5.3% vs. 68.7% • 10.1%; p < 0.001). (b) a negative correlation between shvo 2 immediately after operation and the duration of intraoperative hepatic vascular occlusion could be observed (r = -0.58; p < 0.05). this correlation could also be seen for the first 12 post-operative hours (r = -0.42; p < 0.01). (c) a negative correlation between shvo2 and the difference between arterial and hepatic venous lactate levels was found (r = -0.39; p < 0.02). conclusions: the routine measurement of shvo 2 appears to be a promising extension of post-operative monitoring after major liver surgery. it is a safe method easily feasible on any major surgical icu though relatively time consuming. a further validation of this method is necessary in larger studies. therapeutic recommendations on the basis of shvo 2 findings cannot be given yet. methods: in 5 cases after major liver resection, in which abnormally low readings of shvo 2 suggested an impaired hepatic blood flow, pgi 2 was applied at a dose rate of 5 ng/kg/min. as shvo 2 can be considered an indirect parameter for hepatic blood flow, the effect of pgi 2 infusion on shvo 2 was measured. moreover, the changes of macro hemodynamics and pulmonary function were monitored. results: before the application of pgi z mean shvo 2 for all 5 patients .was 54.1% (47-9 -47-3). in three cases without major structural alteration of the remaining liver tissue the continuous intravenous administration of pgi 2 lead to a sustained increase of shvo z to an average of 67.1% (65.6 -69,1 ). the postoperative course in these three cases was uneventful. in two cases with compensated liver cirrhosis after hepatitis c no change in shvoz under pgi 2 infusion could be observed. both patients died 32 and 45 days respectively after operation in protracted liver failure. side effects of pgi 2 included a slight decrease of systemic and pulmonary vascular resistances. consequently map decreased by up to 10% as did intrapuimonary right-left shunt increase. in none of the observed patients did these side effects posed a limitation of continuous application of pgi z. conclusions: in patients without structural alteration of the liver the systemic application of prostacyclin at a dose rate of 5 ng/kg/min could significantly increase an abnormally low hepatic venous oxygen saturation after major liver resections, tn two cases of severe liver cirrhosis a similar increase could not be observed. after first clinical investigations and with the results of recent studies in animal further controlled clinical studies of prostacyclin in the postoperative management after liver surgery appear justified. any delay in gastric emptying can promote micro-aspiration and give rise to ventilator associated nosoarnnial pneumonia. h2-receptor antagonists have been suspected of promoting pneumonia by changing the gastric ph. in a few tri',ds on humans ranitidine was noted to delay gastric emptying. the aim of this prospective, randomised, blinded study was to evaluate in a ventilated icu population if there was a difference between cimetidine (c) and ranitidine (r) on the gastric filling index (gfi conclusion: in this population there was no difference in gfi between c and r; however the age and creatinine were significantly different and could have favoured the c group. also the very long t/2 could have hidden smaller differences between c and r as has been described in volunteers. between april 22, 1990 and april 19, 1993 , 102 patients with severe acute pancreatitis were admitted to 16 participating hospitals. patients were entered into the study if severe acute pancreatitis was indicated, on admission, by multiple laboratory criteria (imrie score >_ 3) and/or computed tomography criteria (balthazar grade d or e). patients were randomly assigned to receive standard treatment (control group) or standard treatment plus selective decontamination (norfloxacin, colistin, amphotericin; selective decontamination group). all patients received furl supportive treatment, and surveillance cultures were taken in both groups. results: fifty patients were assigned to the selective decontamination group and 52 were assigned to the control group. there were 18 deaths in the control group (35%), compared with 11 deaths (22%) in the selective decontamination group. (adjusted for imrie score and balthazar grade: p = 0.048). this difference was mainly caused by a reduction of late mortality (> 2 weeks) due to significant reduction of gram-negative panreatic infection (p = 0.003). the average number of laparotomies per patient was reduced in patients treated with selective decontamination (p < 0.05). failure of selective decontamination to prevent secondary gram-negative pancreatic infection with subsequent death was seen in only three patients (6%) and transient gramnegative pancreatic infection was seen in one (2%). in both groups of patients, all gram-negative aerobic pancreatic infection was preceded by colonization of the digestive tract by the same bacteria. reduction of gram-negative colonization of the digestive tract, preventing subsequent pancreatic infection by means of selective decontamination, significantly reduces morbidity and mortality in patients with severe acute necrotizing pancreatitis. ieco by sodium hypochlorite (nacio) infusion is considered to be a model of microsomal oxidation in liver on cytochrome p-450. active c10 provides oxidation of toxic metabolic products in the blood and exfused during plasmapheresis plasma, and also hydrophobic to hydrofilic transformation of substanses. sterile nacio in necessery concentrations was obtained by electrolysis of saline (0,85-0,9% naci solution) in electrochemical set e~io-4 (russin,moscow). methods: 1. the nacio in concentration 600 ragfl (400-800 ml/24h ) was administred into central veins in patients with extensive peritonitis and endotoxicosis 2-3/t. erytrocytes resistance to nacio, circulating blood volume glycemia and hemostasis were initially estimated. 2. after plasmapheresis exfused toxic plasma was mixed with nacio conccantration of i000 mg/t in 10:1 ratio in sterile "hemacons".the effectiveness of plasma detoxication and possibility of its reinfusion were evaluated by determination of albumin effective concentration (eca 35 g/l), the concanlration of medium molecular oligopeptides (mm 0,2) and other biochemical tests (bilimbin, creatinine, carbomide and so on). results: 1. the intravenous administration of nac10 excels detoxicative effect of hemosortion by 17-20% provides effictive presentation of protein components and blood cells and improves the transport function of albumin by 37%. 2. the return of exfused plasma after its purification ieco was 70-80%. only the remaning 20-30% of deficient plasma were compensated by fresh cryoplasma and albumin solutions. ischemic hepatitis (ih) is a severe complication in critically ill patients. acute circulatory failure of multiple etiology can lead to splachnic hypoperfusion and cause acute and reversible anoxic damage. over a period of 26 mos 12 pts, 8 m and 4 f, mean age 64+6.6 yrs developed liver disease compatible with ih. eight pts had a documented hypotensive episode (six pts with septic shock and two hypovolemic shock), while cardiogenic pulmonary edema in the absence of hypotension was responsible for ih in the remaining four pts. all the pts had a rapid striking elevation of ast, &lt and ldh with equally rapid resolution of these parameters to near normal wimin 9 days (mean 6.25). the mean peak level of ast, alt and ldh was 4340 iu/l (range 2105 to 7500), 3453 iu/l (range 1685 to 5150) and 2868 iu/l (range 1440 to 6960) respectively. serum total bilirubin levels rose transiently with a moan t:eak level of 1.95 mg/dl (range 1.1 to 2.7), while altered coagulation paran-,ete's (pt> 1.5 times normal) was observed in four pts and clinically significant coagulopathy with fibrin degradation products occurred in one pt (8.3%). renal impairment (cr>2.0 mg/dl) was manifest in all pts; six pts developed non-oliguric renal failure (50%) while two pts required hemodialysis. ten lots required vasoconstrictor inotropes [dobutamine (range 3-10pg/kg/min) and dopamine (range 7-25 pg/kg/min), while replacement of circulatory blood volume was performed in two pts with hypovolemic shock. eight lots expired (66.6%), but none died as a direct result of hepatic damage. the mortality rate was higher among pts with concurrent renal failure (75%). it is concluded that: 1) ih is not uncommon complication in the icu with the prognosis depending on the underlying disease. 2) clinically significant coagulopathy is uncommon complication of ih. 3) titration of inotropes is required to obtain optimal cardiac output support and subsequently liver blood flow. it is difficult to ascertain the perfusion of free flaps such as jejunal loops after surgery. objectives: to assess ischaemia as evidenced by intramural ph of jejunal free flaps used for reconstructive surgery following total pharyngolaryngectomy. methods: the sigmoid ph tonometer ( tonometrics inc.,usa ) was used to monitor intramural ph of the jejunal free microvascular flaps ( phig ) in 15 patients who underwent total pharyngolaryngectomy. a standard general anaesthetic was given and all patients were admitted to the icu for controlled ventilation and monitoring. all had similar postoperative care. phig was measured pre, post-revascularization of the flap and on icu admission, 4, 12 and 24 hours postrevascularization. objectives: to classificate the wide spectrum of itc of anp into distinct pathophysiological patterns according to presentation and course. patients (pts) and methods: 52 pts, 34 ~(65,4%), 18 (34,6%) were admitted in the icu because of anp and acute respiratory failure(arf), ilean age:54,3• years. hean stay in icu:29,2• days. 38 pts were operated, 15 of them twice. hean value of ranson's scale:4,4• (2-7). we analyzed hemodynamic measurements,arterial blood gases(abg), x-ray findings(xrf), ct-scans and operative records. results: 5 patterns of pleuropulmonary complications were identified: a)early hypoxia without xrf -33 pts. b)early ards with typical xrf -5 pts(1 died), c)early arf with xrf(atelectasis,infiltrates)-15 pts(9 died). d)late ards with typical xrf-32 pts(31 died), e)pleural effusions in various combinations with the above patterns -38 pts. overall mortality rate: 41/52 = 78,8%. conclusions: l)frequent x-rays and abg are important for the classification of itc of anp. 2)even though patterns of classification in anp are not clearly distinguishable,they facilitate an anticipatory management. 3)deterioration of abg and xrf indicates that preventive measures for arf must be intensified and agressive surgical therapy is required. 4)delay of surgical therapy is related to worse prognosis(p<o,05) and prolonged hospitalisation time(p<o,ol). in the contrary, the early timing of the operation before infection and extrapancreatic necrosis is essential for a better prognosis (p<o,05). objectives: the development of cholestasis during intensive care treatment is allied with an inferior prognosis. this study investigates the sensivity, specification and also the positive and negative forecast of patients survival -exemplary for bilirubine. methods: during a period of 12 months all surgical patients on icu were registrated prospectively (n = 1572). for documentation of disease development a standard foldcrform based on a computer-data-system was used. the results of this kind of documentation showed aapprax. 5,2 m!ll. of single patient informatioas. results: 1300 of 1572 patients didn't suffer from liver disease or have any operation on liver or bile tract. 1201 of them have had a normal scale of bilimbine in the postoperative period, a higher scale of bilirabine was regisu'atcd by 99 patients. at a ,,cut-off" of 4rag% (total-bilirubine) the sensitivity was 0,7, specivity 0,6, positive predicive level 0,8, negative predictive level 0,5 for survival criteria of a patient. the max. reached level of bilirubine, also the daily increase turned out almost the same results as they were found for other parameters nf cholcstasis like ap or gamma-gt. objective: the aim of this experimental protocol was to evaluate the morphological and functional parameters of isolated pig liver grafts, perfused in an extracorporeal liver support system. methods: the system consists of the graft liver, a membrane oxygenator (oxygenation surface 1.2 cm z, 02 flow=3 itlmin), a heater, a centrifuge pump and a fluid reservoir. twenty pig livers, mean weight 790 gr (min 610, max 870 gr) were obtained and after a mean cold ischemia time of 35 min were perfused fo} a mean of 7.5 h (min 4.5, max 9 h). perfusion solution consisted of r/l and hemacell. inflow to the graft liver was performed through the portal vein at a mean pressure of 16 (12-25) mmhg at 38~ while outflow was secured through the suprahepatic inferior vena cava. during perfusion the following parameters were evaluated through pre-and posthepatic hourly (to-t8) sampling: po2, 4" + +4" pco2, ph, hco3-, na , k , ca , osmolality, glucose, lactate, ast, alt, alp, u bilirubin and coagulation factors i, v and viii. biopsies were obtained periodicallty. b_p_~: results were as follows: mean ph fell from 7.45 at t o to 7.168 at t 8 while mean output po 2 fell from 527 at t o to 10 at t 8. mean output ast values increased from 361 at t o to >2500 at t 8 while mean output alp values increased from 3.66 at t o to 197 at t 8. mean output k + values increased from 3.93 at t o to >8 at t 8. histology revealed lesions of ischemic necrosis, more prominent after t 6. conclusion: results show that the isolated liver graft presents satisfactory function and morphology at least for a five hour perfusion period in the described extracorporeal circuit. correction of ph contributed to an increase in bile flow. between 1982 and 1993 the practice of transplantation has changed drasticaily in switzerland -besides kidneys also hearts, heart and lung, lung, iiver and pancreas transplantation has started in several centers. major information efforts have been made, organ exchange rules were set up and a national coordination center was initiated. the aim of this retrospective single center study was to assess the influence of transplantation on organ donation. in the past eleven years 205 organs were donated from 458 potential donors i139 single, 66 multi organ donations) analysis of refusal was evaluated categorized into medical and/or familiar reasons. the number of potential donors increased from 28 (1982) ,to 61 (1992) with a concomitant drastic reduction of donations from 64% in 1982 to 26% in 1992; amounting to a net unchanged number of donations over the last 10 years (1982 = 18; 1992 = 17) . the import and export of donor organs was balanced since the introduction of the national coordination center. in contrast multi organ donation increased from 0% in 1986 to 90% in 1993 despite of the more stringeant selection criteria, in conc]usion the introduction of a full range of transplantation procedures at several new university programs and the increase of multi organ donation has not had the forecasted impact on organ donation despite a sustained informative and promotional campaign, objective: monitoring hepatic venous oxygen saturation (svho2) provides online information about hepatic-splanchnic oxygen supply-demand ratio [1]. previously, x~ reported hepatic venous catheterization in patients undergoing orthotopic liver traru~lantation (olt) [2] . in the present study, we assessed the effects of nitroglycerin (ng), a vasudilator that affects the venous capacitance vessels more than arterial vessels and prostaeyclin (pgi2, flolan r~, wellcome, uk), an arterial and splanchnic vasodilator on hemodynamies and hepatic venous oxygen saturation (svho2) in human liver transplantation. methods: with institutional approval and informed consent, 14 consecutive patients, mean age 50-2-_10 years, were studied following olt. postoperatively, fiberoptic pulmonary artery catheter was inserted into the right hepatic vein. timed infusions of ng at a rate of 0.1 gg/kg/min and pgi2 at 5 ng/kg/min were initiated for a 45 rain period. each sequence was followed by baseline therapy for 45 rain. results are expressed as mean=tsd. statistical analysis was performed using friedman's-two-way-anova-test, significance was accepted at p<0,05. results: ng at 0.1 gg/kg/min induced a decrease of mean arterial pressure (map) (84_49 [baseline] vs. 75+9 mmhg) and pulmonary artery wedge pressure (pcwp) (8j:2 [baseline] vs. 65:1 mmhg). cardiac index (ci) (5-41 vs. 4+1 l/rain/m2), oxygen delivery index (do2i) (655-+108 vs. 618+123 mgnfin) and svho2 (74_~12 vs. 69-l-_19%) were decreased (p<0.05). pgi2 at 5 ng/kg/min induced a reduction in map (73• nm~. _g) and pcwp (6+1 mmhg). ci (6_+1 l/rain/m2), do2i (7555:135 ml/min) and svhoz (81+6%) were increased (!o<0.05). 9 vasedilatation induced by ng decreased systemic oxygen supply and impaired splanclmie oxygenation. 9 pgi2 increased systemic oxygen delivery in parallel with svho2, suggesting a corresponding improvement of hepatic-splanchnic okygenation. 9 thus, if vasedilator therapy is indicated in th6 15orient receiving liver grafting, pgi2 appears to be advantageous. however, due to its platelct aggregation inhibiting properties, the usefulness and safety of pgi2 in olt patients has still to be determined. objectives: to analyze the effect of steroid treatment given to donor on the early function of transplanted kidney. methods: from january, 1994 until now 56 donors were involved into this prospective study. every other donor was treated with 30 mg/kg solu-medrol one hour before organ retrieval. according to the steroid treatment of the donor the recipients were divided into two groups: group 1 -steroid pretreatment goup (y~=35), and group 2 -control group (n=37). the donors and the recipients were treated using the same kidney transplantation protocol onl~r the adults, and the first cadaver kidney transplanted patients were involved into the study. the daily routine parameters were analyzed pre-and intraoperafive, and on the 0-5th, 14th and 30th postoperative days. results: we could not show any clinically important differences between the two groups in respect of donor parameters. preoperative, the patients in group 2 had slightly lower ereatinin level (819 -+ 244 g.,non vs.923 -+ 254gmol/1) which persisted into the early postoperative phase. the values of the other examined pre-and intmoperativc parameters were almost the same. during the first 5 postoperative days the patients in group i needed less diuretics (furosemide and renal dose of dopamine) and their sodium excretion was closer to the physiological range than in group 2. the other parameters did not differ significantly. the less furosemide need in group ! pe~isted to the end of the first month. conclusions: according to our data the steroid treatment of the donors improves the early function of the transplanted kidney in some respects. to prove the real benefit of the donor steroid treatment needs more data and further analysis. objectives: severe infections may compromize the outcome of liver transplantation..determination of new parameters may increase the knowledge of pathophysiologic mechanisms and may lead to changes in postoperative therapeutic management of patients at risk. methods: between august 1993 and september 1994, 81 patients with 85 transplants were monitored for cytokines and extracellular matrix pammeters on a daily basis. serious infections (n=10) included microbiologic evidence and more than 2 secondary organ failures. patients with cholangitis (n=ll) or uneventful postoperative course (n=37) referred as control groups. results: 1-year patient survival was 88.9% (72/81): 5 patients died due to serious infections, while 4 died for other reasons. mean bilimbin, stnf-rii-, ifn-7-, il-4-, il-8-, il-10-, laminin-and neopterin levels were significantly elevated in patients with serious infections compared with patients experiencing mild cholangitis or with an uneventful postoperative course. a further increase of all parameters was observed in patients who subsequently died; tnf-ri/: 28310_+788 pg/ml vs 20452• pg/ml; ifn-7:466_+57 pg/ml vs 4.4-+1.8 pg/ml; il-4:214-+35 pg/ml vs 148-+29 pg/ml; il-8:667-+48 pg/ml vs 251_+26 pg/ml; il-10:149_+52 pg/ml vs 52• pg/ml; laminin: 3010-+312 ng/ml vs 1263-+ 117 ng/ml; neopterin: 247_+37 nmol/1 vs 96_+19 nmolb for non surviving vs-surviving patients. a significant decrease of sialic acid yeas observed in patients with serious infections; and a further decrease occurred in patients who subsequently died: 455-+31 mg/l vs 685• mg/1. conclusions: the increase or decrease of various cytokines and extracellular matrix parameters may be indicative for severity of infectiolx routine monitoring of these parameters may improve current diagnostic tools and poss~ly lead to changes in therapeutic management of patients at ~k. objectives: evaluation of the cytokine network after liver transplantation may give some insight in pathophysiologic mechanisms of rejection and may lead to detection of patients at high risk. methods: 81 patients with 85 transplants were monitored for various cytokines on a daily basis between august 1993 and september 1994. rejection was assessed by histology in combination with clinical signs of rejection and laboratory investigations. results: during the first postoperative month, 28 patients (34.6%) developed rejection; 14 patients were successfully treated with methylprednisolone (steroid-sensible rejection), while further 14 patients required additional treatment with fk506 or okt3 (steroid-resistant rejection). 4 patients subsequently developed chronic rejection. mean levels of various cytokines and extracellular matrix parameters including tnf-rii, ifn-7, il-ib, il-2r, il-4, il-6, il-8, hyaluronic acid and neopterin were significantly higher in patients with steroid-resistant than in patients with steroid-sensible rejection. a further increase of some parameters was observed in patients who subsequently developed chronic rejection; bilirubin: 18.2-+4.1 mg/dl vs 11.2-+1.7 rag/all; tnf-rii: 23374-+798 pg/ml vs 18246_+679 pg/ml; il-8:1024+-192 pg/ml vs 275-+67 pg/ml; neopterin 148_+37 nmol/1 vs 49-+21 nmol/1; hyaluronic acid: 290_+63 ~tg/l vs 223_+28 ~tg/l for patients with chronic versus patients with acute steroid-resistant ~ejection. sialic acid levels decreased in patients with acute steroidresistant rejection; and a further decrease was observed in patients who tieveloped chronic rejection: 437_+34 mg/l vs 671_+55 mg/1. ~onclusions: various cytokines and extraeeuular matrix parameters were indicative of severity of rejction. the extensive increase of bilirubin, tnf-ii, il-8, hyaluronic acid and neopterin may indicate subsequent chronic ection. monitoring of these parameters may, therefore, lead to changes in immunologic management after liver transplantation. background : combined kidney and pancreatic transplantation is being performed with increasing frequency in patients with diabetes mellitus and renal failure, as it offers more chances of success and better results than kidney transplantation alone. mycotic arterial aneurysm constitutes a devastating complication following pancreatic transplantation. all cases of mycotic arterial aneurysms have been however reported with exocrine pancreatic drainage into the gastrointestinal tract. intervention : we describe a series of 8 consecutive whole kidney-pancreas transplantation performed at the university of geneva hospitals (1500 beds) between december 1992 and may 1994. exocrine pancreatic drainage into the bladder (epdb) was performed to improve early detection of rejection episodes. epdb was hypothesized to reduce the risk of contamination from the gastrointestinal tract and the subsequent possible occurrence of potentially fatal infectious complication. in all patients the dual transplantation was performed through a median incision according to the procedure described by nghiem. results : two out of the 8 patients who received kidney-pancreatic transplant developed arterial mycotic aneurysms 15 and 35 days following surgery. aneurysms developed at the site of the arterial anastomosis used to rearterialize the homograft. both patients had peritonitis caused by candida albicans requiring surgical drainage and intravenous antifungal therapy. rupture with hemorragic shock occured in both patients leading to graft removal in one patient, and three episodes of lffetreateniug hemorragic shock followed by graft failure and removal 32 days after transplantation in the other. conclusion : arterial mycotic aneurysm constitutes an early, lifetreatening complication of kidney-pancreatic transplantation; it mandates graft removal. although exocrine pancreatic drainage into the bladder consitutes a definitive advantage for caller diagnosis of graft rejection, it does not eliminate the risk for retrograde colonization and subsequent severe infection in our experience. s. bocharov, i. teterina, regional clinical hospital, irkutsk, russia acute profound loss of blood can result from the very different injuries and hepato-pancreato-duodenai operations enter such a rank. ill-timed and inadeguate correction of operation hemorrage is one of the reasons for postoperation complications, including polyorganic insufficiency. the pathogenesis seems to be very complex. in early stages of bleeding the liquid enters the vessel bed, followed by hypoproteinosis and hematocrit fall. however, as decompensation develops, the fluid leaves the vessel system in the result of increasing postcapillary resistance and lowering col-ioidnooncotic blood pressure (cop). the resulting hypovolemia causes primarily acute disturbance of central hemodynamics and then of microcirculations and transcapillary exchange. central hemodynamic failure after acute loss of blood manifests itself through cardiac output lowering and capillary blood flow deceleration. taking into consideration, that 35 % is critical value for cpv loss and for cev it is 65 %, we consider arising the level of cop to the immediate task. cop raising allows to normalize transcapillary exchange, which we assess through cop and mcp (mean capilary pressure) gradient. the next task is to make up for globular volume till homeostasis providing level. considerable attention is given to catabolism inhibition and maximum possible enegry provision. control over high proteolitic activity of blood and callicreinkinin system activity implies direct proteases inhibitors. reologic, membrane stabilizing, antihypoxanthine and anticoagulant therapies are obligatory. virehow clinic, dept. of surgery, humboldt university berlin, germany regarding a high mortality up to 85 % of fulminant hepatic failure orthotopic liver transplantation seems to be the only promising therapeutic approach in many cases. this study shows experiences from a transplantation center. between june 1991 and april 1995 39 patients suffering fulminant hepatic failure were admitted to our surgical intensive care unit all patients showed severe liver dysfunction with grade ii to iv encephalopathy. after a period of diagnostics and conservative treatment ranging from few hours to 10 days (mean 2.4 days) we reported 22 of these patients as possible organ recipients to eurotransplant. all of these 22 patients were transplanted within 48 hours, 16 (73 %) of them even within 24 hours. the principal aetiologies were hepatitis b (7), hepatitis c (1), nanb hepatitis (5), mushroom poisoning (amanita phalloides 1). after transplantation 2 patients suffered from initial-non-function and underwent re-transplantation. the one-year-survival rate was 82 %, 5 patients died within 3 months after transplantation due to various reasons. 17 patients were not referred for liver transplantation. 10 of them never met transplantation criteria, improved by conventional therapy and could finally be discharged from hospital. the known reasons for liver failure in this group were mushroom poisoning (4), paracetamol intoxication (4) and fulminant hepatitis a (1). 7 patients suffering from fulminant hepatitis (6) or intoxication (1) were excluded from emergency liver transplantation for various contraindications. 6 of these 7 patients (86 %) died despite conventional intensive care. we don't know if some of the patients in the transplantation group would have survived without transplantation, because whenever we decided on transplantation we could perform the operation within 48 hours. but 9 the good survival rate in the transplantation group (82 %) 9 the 100 % recovery rate in the group, where there was no transplant-indication in our opinion 9 and the fatal outcome (86 % mortality) in patients with contraindications are an encouraging proof of a successful therapeutic strategy in acute liver failure. these results are based on a close cooperation between experienced transplant surgeons, hepatologists and intensive care doctors, using sophisticated laboratory and imaging techniques in a specialized center. introduction: during brain death patients suffer from multiple endocrinologic disturbances. one of the most important are those related with thyroidal axis. it is well described the euthyroid sick syndrome whose more frequent pattern consist of decreased triiodothyronine (t3), increased reverse t3 (rt3) with normal levels of tetraiodothyronine (2"4) and tsh, this lacking in "1"3 levels lead to a change from aerobic to anaerobic metabolism which results in tissular damage. objective: 1.to study thyroidal pattern in brain death patients potential organ donors. 2.to avoid organ impairment by administration of t3. 3.to study the hemodynamic and hormonal changes after the administration of t3 in these patients. material and methods:population: 22 brain death patients of any etiology potential organ donors admitted to the intensive care unit. patients were classified in hemodynamically stable (group 1) and unstable (group 2). group 2 received a bolus of 0.25p.gr/kg. and a perfusion at a dose of 2-3.5 p.gr]h of t3. hormonal assays: total t3 (tt3), total 2"4 (tt4), tsh. fxee t3 (ft3), free 1"4 (ft4) and rt3 were determine at the moment of clinical brain death (0 hrs) and in group two these assays were repeted at hours 4, 8 and 12. results: 22 patients (17 male) with a mean age of 33 years (range 17 to 71yrs.) were studied. the clinical brain death was confirm later with other explorations (eeg, doppler). there were 15 patients in group 1 (68,1%) and 7 patients in group 2 (31,8%). hormonal pattern: at the moment of brain death tt3 was normal in 21 cases (95,4%) and decreased in i (4,6%); tt4 was normal in 9 patients (40,9%) and decreased in 13 (59,1%); ft3 was normal in 3 cases (i3,6%), decreased in 19 (86,4%); fl'4 was normal in 19 patients (86,4%) , decreased in 3 (13,6%) .rt3 was normal in 17 cases (77,2%) and increased in 5 cases (22,8%). there were no statistically significant differences in hormonal pattern between the two groups. only t3 levels at hours 0, 4 and 8 were significant in group 2. in the 19 cases with ft3 decreased, the tt3 was normal in 18 (84%) and decreased in 1 (16%), tt4 was decreased in 11 (57,8%) and normal in 8 (42,1%), tsh was decreased in 1i (57,8%), normal in 7 (36,8%) and increased in i(5,2%) and ft4 decreased in 3 (15,7%) and normal in 16 (84,2%) and rt3 was normal in 14 (73,68%) and increased in 5 (26,3%). there were no statistically significant differences in cardiac index, vascular resistances and pulmonary shunt before and after the administration ef t3. conclusions: 1. the hormonal pattern most often find in brain death patients was: normal tt3, decreased tt4, normal tsh, decreased ft3, normal fr4 and normal rt3. 2 . there were discrepancies in the values of ft3 and tt3 3. there were no statistically significant differences in hemodynamic and pulmonary parameters. objectives: magnetic resonance angiographie (mra), a non-invasive procedure, provides flow-related information additionly to the anatomy of the vascular system. measurement of signal intensity and edge detection of vessel structures permits to calculate blood flow velocity and vascular diameters. we examined whether cerebral hemodynamic changes by altering the arterial pressure of carbon dioxid (pace2) could be detected by mra. methods: following institutional approval and informed consent, 10 mechanically ventilated patients without elevated intracraltial pressure underwent mra with defined periods of hyper-, hypo-and normoventilation (pace2: 30, 50, 40 mmhg; arterial blood gas probes; avl). mra was performed with a 1.5 tesla magnetom (vision, siemens). two different mra techniques were used: a conventional time-of-flight-3d-angiography (tr: 39 ms; te: 7 ms; fl: 20 deg; slab: 56 mm) for vessel diameter detection and a flash-2d-gradient-echo-sequence (tr: 28 ms; te: 5 ms; fl: 30 dog) for measurements of blood flow velocity. an axial view parallel to the ac-pc-iine (anteriorposterior-commissur-line) was used for repeated imaging of identical regions of interest 0toi) of the proximal part of the internal carotid (ica) and middle cerebral artery (mca) as well as of peripheral branches of the mca and the posterior cerebral artery (pca). results: changes of pace2 correlated with changing signal intensities, whereby under hyperventilation a decrease of 23,7% (p 0.01) and under hypoventilation an increase of 28.4% (p 0.01) was observed compared with normoventilation. blood pressures were stable throughout the whole study period, pace2 dependent changes in vessel diameters were more pronounced in peripheral branches of mca and pca. a change from normo-to hyperventilation produced a decrease in proximal vessel diameter of -3.5% (p _< 0.01) and in peripheral diameter of -22.2% (p _< 0,001). a change from normo-to hypoventilation produced an increase in proximal diameter of +6.1% (p < 0.05) and of +21.3% (p -< 0.001) in peripheral diameter. conclusions: pace2 related changes of cerebral vessel diameter can be easily detected by mra without injecting a contrast agent. the results confirm that co2-reactivity is more pronounced in peripheral cerebral vessels, which are subjected to greater changes in diameter than major basal arteries. hyperventilation leads to a decrease and hypoventilation to an increase in signal intensity thus reflecting the corresponding changes in blood flow velocity, intensive care unit (icu) of "kat" hospital, athens, greece, ob!ective$; the value of bronchoscopy in pulmonary atelectasis of icu patients is under question the presence of an air bronchogram sign in xrays, which is considered as evidence of central bronchus patency, is referred in several studies as a negative criterion for bronchoscopy, whereas its absence as a positive one. it is also referred that air bronchogram sign correlates with delayed resolution of atelectasis, probably because of obstruction of many periferal airways (not central). the purpose of this prospective study was the evaluation of the air bronchogram sign on frontal chest film as a negative criterion for bronchoscopy and as criterion of delayed resolution of atetectasis, methods: icu patients with atelectasis were studied prospectively. they underwent bronchoscopy, bronchoscopic findings, presense of air bronchogram sign, and outcome of atelectasis were recorded, correlations were made, between: 1) bronchoscopic potency of airways and air bronchogram sign 2} resolution time of atelectasis and broncoscopic potency of airways. 3) resolution time'of atelectasis and air bronchogram sign, methods of statistical analysis were the t-student test and the chi square test, results:the patients were 23, men 19 women 4, seventeen patients had atelectasis of whole lung, 3 of upper lobe, and 3 of lower lobe. ten patients had atelectasis in right and 13 in left lung. eight from 28 patients had air bronchogram sign in x-ray, there was no statistical correlation between air bronchogram sign and bronchoscopic potency of airways [6 from 8 patients with air bronchogram sign (75%) and 11 from 15 without air bronchogram sign (73%), had bronchoscopic potency of airways, p>0.1], resolution time of atelectasis didn't correlate statistically with bronchoscopic potency of airways (mean resolution time in patients with bronchoscopic potency 2,29 days and in bronchoscopically closed bronchi 2,33 days, p>0,1). there was also not a statistical correlation between resolution time of atelectasis and air bronchogram sign (mean resolution time in patients with air bronchogram sign 2,25 days, and without air bronchogram sign 2,33 days. p>0). conclusion~i; the presense of an air bronchogram sign in x-ray of icu patients with atelectasis, does not coexist obligatorily with bronchoscopic patency of airways and cannot be used as a negative criterion for bronchoscopy, neither as a criterion of delayed resolution of atelectasis. th. wertgen chest sonography (cs) is routinely used in our department to examine icu patients with clinical symptoms of pulmonary embolism, pneumonia, pleural effusion or unclear chest pain. we perform cs with a sector transducer (4.0 mhz) and a linear transducer (7.0 mhz) using acuson 128xp/10 c. the sonographic signs of pulmonary embolism and infarction are most well demarcated, mainly wedge shaped and triangular pleural based lesions, more roughly structured, observed with a hyperechoic reflex in the center corresponding to the bronchitic (fig. 1) . pneumonia is characterized by homogenously hypoechoic, wedge shaped parenchymal lesions, containing air or fluid bronchograms; they move with respiration (fig. 2) . pleural effusions are spaces of various echogenicities, from anechoic to homogeneously echogenic, which may contain floating strands or complex septa, located between visceral and parietal pleuras (fig. 3) . from march 1994 to april 1995 we did 55 examinations by cs in 34 icu patients (20 male, 14 female; age from 29-87). patients examinations pulmonary embolism 10 16 pneumonia 7 16 pleural effusion 13 19 us-guided thoracic punctions were performed in 7 patients. in two patients we found pneumonia or pleural effusion caused by a lung carcinoma. another two patients showed a normal cs (diagnosis: inflammation of the gall bladder, inflammation of the myocardium). conclusion: cs is a very useful method for icu patients with chest diseases. it takes less time and is less expensive than ctand sometimes of a higher diagnostic value than x-ray. last but not least cs is invaluable for the icu patient, because the examination is done save and quickly at bed side and the results of cs are very helpful in diagnoses and treatment. results : inter-observer reliability was evaluated as an 83 % concordance. results of the tee classification were : class 0 : n = 21 (34 %) ; class 00 : n = 13 (21%) ; class 1: n = 7 (12 %) ; class 2 : n = 8 (13 %) class 3 : n = 12 (20 %). therapeutic implications of tee in class 3 patients were : cardiac surgery in 5 patients (two cases of acute mitral regurgitation, two valvular abscesses and one hematoma compressing the left atrium), discontinuation of peep in one ventilated patient with an atrial septal defect, weaning of mechanical ventilation in one patient with an atrial septal defect, prescription of antimicrobial therapy in 8 patients with endocarditis and prescription of anticoagulant therapy in 2 patients with left atrial thrombus. the only noteworthy complication was a case of spontaneously resolving supraventrieular tachycardia. conclusion : tee is safe and well tolerated, and is useful in the management of icu patients with shock, unexplained and severe hypoxemia or suspected endecarditis. the aim of this study was to determine whether ultrasound guidance can help interns to improve the results of jugular vein access in icu. methods : in a prospective and randomized study, we compared, in 79 patients admitted to the icu, an ultrasound-guided method (ultrasound group : 37 patients) with an external landmark guided technique (control group : 42 patients). all jugular vein accesses were performed by young interns with an experience of < 5 procedures. results : internal jugular cannulatian vein was aci~ieved in all patients in the ultrasound group and in 10 patients (24 p.cent) in the control group (p < 0.01). average access time was longer in the control group (235 • 408 sec. vs 95 • 174 see. ; p = 0.06) and puncture of the carotid artery occurred in 5 patients in each group (p = 0.83). 32 patients (86 p.cent) in the ultrasound group and 23 patients (55 p.cent) ia the control group (p < 0.05) were cannulated in 3 rain. or less. the cannula was therefore unabie to be inserted within 3 minutes in 19 patients in the control group, with failure of eannulation in 10 of these patients (53 p.cent). failure was due to thrombosis (n = 1), small calibre of the internal jugular vein (< 4 ram) (n = 5), abnormal vascular relations (n = 3) or cervical irridation (n = 1). among the 10 primary failures of cannulation, an internal jugular vein catheter was able to be inserted in 4 cases by an experienced physician on the side initially selected and with ultrasound guidance in 2 cases. the catheter was inserted into the contralateral internal jugular vein under ultrasound guidance in the remaining 4 cases. jugular cannulation was obtained at the first attempt in 26 p.cent in the control group and 43 p.cent in the ultrasound group. conclusion : ultrasound guidance improved the success rate of jugular vein cannulation by inexperienced operators in icu patients. when the internal jugular vein has not been successfully eannulated within 3 minutes by the external landmark guided technique, the authors recommend the use of the ultrasound guidance. in the majority of cases right atrial or ventricular thrombi represent pulmonary emboli in transit. these may be fatal in patients (pts) treated conservatively with anticoagulation only. in literature the incidence of right heart thrombi in pts with proven pulmonary embolism (pe) is said to be in the range of 3-4%. extremely mobile, long, worm-shaped masses in the right heart cavities carry an especially high early thrombus-related mortality rate which ranges from 40-50%. current therapeutic strategies favour fibrinolytic therapy with consecutive anticoagulation. we report five cases (4 male, i female, 55-74 years) of right heart and pulmonary thromboembolism. in these pts diagnosis and regression of thromboemboli following systemic intravenous lysis therapy with recombinant tissue-type plasminogen activator (rt-pa) was documented by transesophageal echocardiography (tee). a submassive pe occured in 3 pts, a massive pe in 2 pts. one patient (pt) had a cardiac arrest. in all 5 cases tee clearly identified the extensive thrombns formation in the right-sided cavities of the heart and in the central pulmonary artery in 2 cases. all pts were treated with 100 mg rt-pa, 3 pts in a front-loaded regimen over 90 minutes, 1 pt over 120 minutes, and, due to the life threatening situation, in one case a bolus injection as ultima ratio was performed with no intracerebral bleeding complication. regression of thromboembolic masses after fibrinolytic therapy was demonstrated by transthoracic and transesophageal echocardingraphy after 1 to 15 hours. all pts survived and were put on coumadine, 1 pt developed an intracerebral bleeding with persistent hemiplegia. conclusions: the use of thrombolytic therapy is highly efficacious for the therapy of pts with pe and concomitant right or ventricular thrombus formation. transthoracic and especially transesophageal echocardiography are powerful bed-side diagnostic tools for the immediate diagnosis and follow-up of successful treatment in this life-threatening condition. although widely used, catheterisation of the femoral vein in the groin using "landmark" technique is frequently complicated by accidental arterial puncture. suboptimal hygiene and patient discomfort are also associated with this technique. with regard to these last two factors cannulation of the femoral vein 10-20 cm below the inguinal ligament would seem an attractive alternative. as "landmark" technique is not possible for the cannulation of the femoral vein in this part of the thigh, ultrasound was used to locate the vessel and the results of this technique were evaluated. methods: a portable compact ultrasound device (site rite,dymax corp.) featuring a 7.5 mhz transducer (ultrasound depth 4-5 cm) fitted with a needle guide and a 6 cm screen was used by residents with no previous experience in ultrasound guided cannulation. patients consisted of a surgical icu population. results: in 46 patients 55 catheters were introduced.in 6 cases more than one (2-4) attempt was made and in 3 patients the procedure was unsuccesfull due to the fact that the vessel was situated out of reach of the ultrasound (vessel depth > 4-5 cm), during the 55 procedures one accidental arterial punction was registered. the catheters remained in situ for a mean of 9 days (range 1-22) and were used for volume suppletion, medication, parenteral nutrition and haemodialysis.co-ionisation rates compared to those of subclavian catheters in our icu. in the first 20 patients 3 cases of asymptomatic thrombosis of the femoral vein were seer on ct-scans performed for other indications, in the following 26 patients duplex scanning performed after removal of the catheter yielded another 3 cases of asymptomatic femoral vein thrombosis. conclusions: ultrasound guided femoral vein catheterisation 10-20 cm below the inguinal ligament is a safe and simple technique that can easily be performed by residents without prior experience. the incidence and impact of thrombo-embolic complications associated with this technique are still subject to further investigation. objectives: to estimate the cost of antibiotherapy (ab-cost) in a multidisciplinary 8-bed greek icu and to correlate ab-cost with total cost of drugs and consumables and with patient's outcome, severity of illness and type of admission. methods: prospective data from 137 consecutive patients admitted to the icu from 1/10/1994 to 30/3/1995 were studied. a tick chart was designed to record all drugs, materials and consumables regularly used for icu patients, but did not include low price drugs and consumables, which are provided from hospital's pharmacy as stock and were included in a fixed icu cost calculated for a 12 month period. the chart also contained demographic details and data necessary for the calculation of several illness severity scoring systems. obiectives: over 3 years evaluate the necessary efforts and expenses to implement a cis in the routine of a 16-bed stcu. methods: in june 1992 a commercially available, unix-based cis was installed on a 16-bed surgical icu. the goal was a paperless documentation at the bedside. after more than 2 years clinical experience two aspects were investigated: what effort is necessary to install and support a cis, and what is the benefit for patients and personnel on the icu? results: the installation and support of a full-fledged cis requires a considerable effort: (a) the conceptual framework for the cis has to be defined. this includes the definition of documentation standards, as well as nursing and therapeutic standards, which is the essential basis for the configuration of any cis. (b) configuring a cis, i.e. "fine-tuning" it to the user's specific needs, is always a laborious task. moreover, constant maintenance is necessary. these tasks require the following personnel: experienced health care professionals for defining the conceptual framework, 1-3 trained health care professionals for configuration, 1 system administrator. on a single icu (12-20 beds) these are not considered full-time jobs. (c) training is best done employing the "train-the-trainers" approach. (d) beside the necessary amount of man power and money to install and purchase a cis, administrative and mis support is needed, especially when interfaces to the hospital and laboratory information systems have to be set up. in general, a cis needs the commitment of all people involved. without a really professional approach with a longterm goal any major cis can turn into an unnecessary but inevitable night mare. after 3 years clinical use and a thorough implementation of a cis on a major sicu it can be said that full-fledged cis offers an opportunity to dramatically improve the working environment on an icu. moreover, it adds to patient safety, quality of care and cost efficiency in one of the most advanced and expensive areas of medicine. conclusion: a major investment in man power and money is necessary to install and maintain a full-fledged cis. a sincere professional commitment to the goals of a cis is necessary. in exchange, a well configured and well maintained cis dramatically improves the quality of therapy and care on the icu. even return of investment and financial profitability of a cis seem feasible todayl from the clinical perspective it appears that the users themselves are the central determinant whether a cis makes a dream come tree or turns into a night mare. objectives: to establish a relationship between the activities of the staff and the occurrence of auditory alarms on the i. c.u. ard to evaluate confusion between auditory alarms. methods: laboratory based studies which investigated aspects of confusion between alarms in current use on the i. c. u. the observational studies were conducted over an 18 month period and examined the frequency and duration of alarms together with the concurrent activites being undertaken by staff on the unit. the laboratory based studies showed that there were enduring confusions between the alarms on various items of medical equipment, for example a ventilator alarm and an e. c. g. monitor alarm. the results of the observation studies demonstrated that alarms are activated when specific activities are being undertaken by staff. sounds could be used in future recommendations for alarms on medical equipment. suggestions are also discussed for improving and rationalising auditory warnings in the i. c. u. obiectives: we investigated inferior petrosal sinus (ips), the lowest affluent to jugular bulb (jb), as a possible source of contamination of samples in jb for monitoring oxyhemogiobin saturation (sjbo2). pulling back the catheter the oxyhemoglobin saturation usually rises indicating extracerebral contamination (jakobs en met al: j cereb blood flow metab 1989;9:717). methods: the study was carried out on patients undergoing ips sampling to differentiate cushing disease from ectopic acth syndrome and to lateralize any resulting pituitary lesion. we studied the value of oxyhemogiobkn saturation high in jb (sjbo2), at ips (sipso2) and at mid jugular vein (5th cervical vertebra) (smj 02) bilaterally. results: we found significant differences between right sjbo 2 and both right sipso 2 (p= 0.007) and right smjo 2 ( p= 0,017) and between left sjbo 2 and both left sipso 2 (p= 0.01) and left smjo 2 (p= 0.017) we did not fred any difference bilaterally. objectives: we studied various methods of receiving and editing of clinical datas in critically ill patients (different ethiology). 163 patients were investigated in regional intensive care center. methods : the following datas were studied : anamnesis, status praesens objectivus ( organs and systems ) ,. clinical and biochemical markers of critical condition , datas of eeg ,rheography . the medical information complex contained : 8channel electroencephalograph, 4-channel roencephalograph, ad-converter (16 analog inputs, 12 bit resolution, 60 k hz), ibm 486 dx2, software includes set of routines for spectral eeg analysis, eeg-mapping, correlative analysis, and brain bloodstream reg-monitoring (written in turbo pascal 4.0), expert programs for estimation objective and humoral patient status (written in clipper 5.0) and statistics. there were used following programme-language instruments : borland c++ 3.0, nantucket clipper 5.01, ca-clipper tools ii. as the methods of statistical processing of dates were used: t-students criterion , fisher criterion, methods of correlation analisis, calculation of the regression levels, dispersion analysis, results : there was created the optimal structure of hard and sofware complex of search steady objective regularity in dynamic of critically ill patients condition. conclusion : the created system allowed to value effectiveness of intensive care and give us new opportunities in study pathogenesis of systems disorders in critical condition . over a five year period a patient data management system has been installed which allows individualised patient data to be accurately collected. using this data a costing system has been developed which ascribes costs thus: 1. direct costs -drugs, fluids, consumables, interventions. these are ascribed to individual patients, according to data collected from the pdms. 2. indirect costs -energy, depreciation, admm costs, maintenance etc. these are summed for the year and ascribed as an overhead per patient day. n.b staffcusts contain art element of both cost types the aim is to make as many costs as possibie 'direct', hence 'activity costs' have been calculated winch comprise staff time, drugs and consumables -these are direct costs. these costs of patient care are then searnlessly integrated into the financial and budget management of the icu environment. it was found that by calculating costs in this manner 50% of the total cost of icu are captured within the 'direct' element, and so are able to be ascribed to individual patients. this is much more accurate than simply dividing the total costs of ~cu by the number of patient days. temporal costs (variations during patient stay) and cross sectional costs (cost differences between admitting specialities) were also noted with interest. results of the initial analysis of data captured by the system will be presented. little is known about the resource costs (not simply cash costs) of icu. even less is known about individual patient costs, with previous estimates of these costs varying widely. however, if cost effectiveness studies are to be undertaken accurate calculation of individual, group and total icu cost is an essential, prerequisite, which, via this system of costing, is now achievable. information about intensive care of cancer patients is limited in the literature, despite the increasing use of such facilities in oncology over the two last decades. in order to determine if and how critical care facilities can be used specifically for these patients, we performed a world-wide inquiry in anticancer centers selecting the hospitals by using the international directory of cancer institutes and organizations. we mailed a questionnaire to 146 centers and we received 84 responses (57.5 %). there was at least one uncological (i.e. with > 50 % of cancer patients) icu in 59 (% % an 18-year old woman with graves disease presents with sore throat, vomiting, diarrhea, sinus tachycardia at 155/minute and a temperature of 40~ several weeks before, treatment with propylthiouraeil had been stopped (rash and fever) and replaced by methimazole and ledide prior to a minor surgery. however, both drugs were discontinued by the patient two weeks before admission. shortly after arrival in hospital, patient's condition progressed to respiratory failure (upper airway edema), delirium and shock requiring icu admission, intubation and resuscitation with fluids and vasopressors. white blood count was 1300/mm ~ with 0 neutrophils. patient's hemodynamic data showed initial hyperdynamic profile followed by low output state with decreased sv02 (59%) (n 70-80%) and cardiac index (2,37) (n 2,5-3). echocardiogram confirmed cardiac chambers dilation as previously described in thyroid storm. lithium carbonate, corticosteroids, antibiotics and beta-blocker perfusion were given. plasmapheresis was started. free t& (n=9,2-21pmo/l) went from 143,6 to 16,6 after the first two pheresis. after a remarkable clinical recovery, sub-total thyroideetomy was done i0 days after admission. in life-threatening thyroid storm, plasmapheresis is a very effective therapy when anti-thyroid drugs are counterindicated. purpose: to compare the reliability of prognostic indexes in crhically iu patients admitted in an intesive care unit (icu) who had acute renal failure (arfi and were treated with different dialytic techniques. material and methods: 1087 patients were included in a prospective study from june 92 to november 94. 220 patients presented arf defined by creatinin serum leve(s greater than 150 pmol/l and previous normal levels. patients were divided in three groups. group i (control) : 156 patients with arf who did not receive substitutive techniques. group ih 21 patients under intermittent hemodialysis (hd) or peritoneal dialysis (pd). group ii1:43 patients under continuous hemodiafiltrstion (hf). the statistical analysis was chi-square test and analysis of variance. results: the table shows the results we obtained, we did not find any significant difference betwen the two groups of patients undergoing dialysis. d(fferences were observed only between group i and the other groups as shown below. we did not find any significant association between the theoretical mortality predicted and the observed mortality according to saps in the three groups. due to exposure to a wide variety of unpleasant stimuli, for example, tracheal suctioning, venipuneture and physiotherapy, most pataents admitted to the icu will require some form of sedation. this review will describe the suggested properties of an ideal sedative agent for use in the icu and review the current limitations of some of the available agents from this perspactive. methods used to quantify the level of sedation, such as the ramsay score, glasgow coma score, newcastle sedation score and visual analogue scores, and their deficiencies will be examined. consideration will be given to defining the optimal level of sedation and the circumstances under which sedation might be varied over the icu course will be discussed. preliminary results from an ongoing study examining the role of light versus heavy sedation and ischaemia in a cardiac surgical icu population will be presented. the pharmacceconomics of icu sedation will be briefly addressed. finally, the role that sedation may play in increasing morbidity, pastieuiarly nosocomial pneumonia, in the icu will be discussed. objectives : therapy cost(tc) in icu patients is a substantial component of total hospital care cost. estimation of tc during this year, partitioning to various groups of drugs used and attempt to minimise it, were considered practically useful. methods : in collaboration with the hospital pharmacy we were able to have a complete report of au drugs used for icu patients (including enteral and parenteral nutrition). mean apache ii severity score upon admission was 19.7 and mean length of tcu stay was 6.7 days. price per drug unit and cost per group of drugs were also available drugs were divided into two groups: antibiotics (1) cardiovascular drugs (2), gastrointestinal system drugs (3), enteral and parenteral nutrition (4), respiratory system drugs (5), sedative, analgesics and paralysing agents (6), parenteral solutions with electrolytes, vitamins and trace elements (7), anti-inflammatory agents (8), protein substitutes and immunomodulation agents (9), anticoagulative agents (10). antibiotics were further subdivided into those "freely" prescribed (a) and those whose prescription and administration requires filling of a relevant form (b). results : !) tc for icu patients/day was 30.530 drs ($122). total tc/patient was 295.195 drs ($1.108.7). ii) partitioning total tc per group of drugs reveals : (1) 43%, (2) 2.7%, (3) 2.7%, (4) 9.2%, (5) 0.3%, (6) 8.6%, (7) 9.3%, (8) 1.3%, (9) 15.8%, (10) 2.5%. t11) concerning antibiotics which consist the major cost component, group a and group b contributed by 29.1% and 13.9% to the total icu tc respectively. group b were administered to 13.9% of all icu patients. conclusions : i) for the above studied patient population antibiotics consist almost half of total tc followed by protein substitutes and immunomodulation agents. ii) if tc control could be attempted in the icu, prescription of beth groups must be reviewed. appropriate treatment should be prescribed and readily provided to any patient. clinical significance of routine protein substitution, currently controversial, should be re-evaluated. new antibiotics (third & fourth generation cephalosporins, quinolones, carbaponems) should be prescribed on the basis of strict diagnostic procedures using modern technology available. rationalisetion of antibiotic therapy will lead to cost control, redistribution of icu expenses and substantial contribution to infection policy in our country. objectives: i -to investigate the clinic efficiency of the monitoring of the rso2 cerebral, in relationship to the stroke prevention, in patient undergoing carotid surgery. 2-to determinate the variations of the rso2 during the different surgical and anesthetic procedures in these patients methods: ten patients undergoing carotid endarterectomy. precise neurological exploration previously to the surgery and in the immediate postoperative period. angiography evaluation to the extend of carotid artery disease. invasive blood pressure, ecg, pulse-oximetry ( pso2 ) and rso2 were collected previousty to the induction of anesthesia. the premedication was administered intravenously -midazolam (50 mcgr/kg) and fentanyl (i rncgr/kg) -. thiopental (4 mg/kg),fentanyl (3 mcgr/kg) and atracnrium (0,5 mg/kg) have been used for induction of anesthesia. co2te is monitoring al~er the orotraqueal intubation ! the anesthetic maintenance is accomplished with lsofluorane (0,5 -1,5%) and bolus of atracurium and fentanyh the surgical procedure is standard (without arterial shunt during the carotid cross-clamping). we register each 5 minutes: blood pressure, cardiac frequency, pso2, co2te and rso2. the rso2 cerebral variate in relation with: the anesthetic induction, blood ~ressure, co2te, cross-ulampping carotid and with the modifications of the head position. the maximum decrease of rso2 cerebral was in relation with the :ross-clampping carotid ( minimal value: 52 ). no patient had neurologic complications and postoperative stroke after carotid endarterectomy were not observed. objectives: there are more than 8000 anesthesia in chelyabinsk emergency hospital every year. to 80% patients of it emergency anesthesia is applied. more than 900 patients have ishemie heart disease (ihd), hypertansion (hp) and previos miocardial infarction (pmi). more than 5% of all patients are old patients (op). the resalts deep noninvasive bioimpedance monitoring (nbm) in surgical patients have been studied by us. methods: our nbm system "kentavr" includes 21 parameters of cardiac and vessels function. it is realised by monitors in operation theatres and computer network. moreover we are able to examine surgery patients before anesthesia and perioperatively by using special computers system for cardiovascular reflex control by fast fourie transform (fft) of 12 parameters simultaneously. results: 187 pathients extremly needed peryoperative monitoring of hemodinamics. from these 187 patients more 40% had stroke volume (sv) less than 30 ml, 18n -co less than 2.1/mim/m2, 25% -ejection fraction (ef) less than 65n and 32% -puls bioimpedans microvessels (pbm) less than 10 morn. 100 patient had intensive care in special department. 42 out of 187 died. comparing with survived with these patients before operation hr was larger, sv, co,ef, pbm and puls bioimpedance aortha was smaller. much more of these patients were with ihd, pmi, hd, op. even with survived patients these parameters decreased the towards the end of operation. surgery patients had different variability of 12 basic hemodinamical parameters with common tendency to increase power amplitude in low frequency by fft. conclusions: using of bioimpedanee noninvasive parameters allows to have criteria for corrections (infusies, vasodilatators, inotrops and others) and then us the final goal, to have more sucssesful surgery. with survived patients was perioperatively and postoperatively care more intensive. obiectives: the aim of the study was to compare the phi with the hemodynamically derived tissue oxygenation indexes as: oxygen delivery (do2), oxygen consumption (vo2), cardiac index (el), and arteriovenous difference in oxygen [(a-v)do2]. methods: 18 patients (15 males and 3 females) with major trauma or major abdominal surgery were studied. on admission, a nasogastric tube allowing phi measurement was introduced and a pulmonary artery catheter was inserted for optimal hemodynamic management. each phi measurement was accompanied with a complete hemodynamic study comprising systemic and pulmonary artery pressures, blood gases, and cardiac output measurements with the thermodilution method. derived parameters vo2, do2, ci, (a-v)do2 were measured according to the standard formula. hemodynamic parameters were opt• as soon as possible with fluids, inotrepes, and vasopressors according to repetitive hemodynamic measurements. all patients were under mechanical ventilation. after hemodynamic stabilisation phi and hemodynamic measurements were repeated every eight hours, during a 24-hour study period. a total number of 52 measurements were obtained and compared. statistics: results are presented as means + sd, correlations were performed between phi and the hemodynamically derived oxygenation parameters. a p<0.05 value was considered as significant. results: mean values were phi=7.19+0.1, do2=984+313, vo2=181+71, c.1= 3.7+ 1.2, (a-v)do2 = 4.47+1.2. no correlation was found between phi and do2, phi and vo2, phi and c.i, phi and (a-v)do2. on the contrary in 14 patients phi remained below 7.30 for more than 24 hours despite adequate hemodynamically derived tissue oxygenation parameters. mortality in this group of patients was very high (85%). conclusion: no correlation was found between phi and the hemodynamically derived tissue oxygenation parameters our data suggest that phi is a better oxygenation indicator than the hemodynamically derived tissue oxygenation parameters, because it is closely related to the patient's outcome. objectives: the pathogenesis of septic shock and multiorgan failure is believed to be related to tissue hypoxia of the gastrointestinal tract. therefore new monitoring techniques, preferably organ specific, are required to establish the adequacy of tissue oxygenation. peep is used to reduce pulmonary shunt volume and improve blood oxygenation, but is accused to impair splanchnic perfusion. we studied mucosal oxygenation and perfusion on the capillary level in the stomach and the duodenum. methods: we used the erlangen microlightguide spectrophotometer (empho ll) together with a specifically designed fibre probe (bodenseewerk ger~tetechnik, 0berlingen) in combination with a standard gastroscope. measurements were performed on 9 ventilated, traumatized patients (ages 17 -75 years), with no evidence of shock or severe infection, after informed consent was obtained from the relatives. all patients were hemodynamically stable without inotropic support. an area of 9 cm 2 was analysed in the gastric corpus, the antrum and in the duodenum. in three patients we simultaneously measured the muc0sal blood flow using a laser doppler flowmeter ( objectives: to investigate the influence of hb-o 2 affinity in the monitoring of svo~ during improvement of cardiac index (ci) in cardiogenic shock. design: to state whether changes in svo: were associated in changes in actual pso (p~0) and standard p~0 (ps0st) 22 consecutive measurements of artero-venous bga, before an.d after therapy-induced changes in ci, were evaluated in 11 patients (mean age 73-*7 y) suffering from cardiogenie shock, all under mechanical ventilation in psv modality. methods: together the hemodynamic measures, m~xed venous samples were analysed at 37 ~ c using the abl500 radiometer for po2, pco: and ph, and the osm3 radiometer for hbo2%, hbco% and methb%. psost (i.e. the p~0 at ph=7.40, pco:=40mmhg and temperature at 37 ~ c) was calculated automatically by the instruments on mixed venous blood as was the ps0"in vivo" (i.e. the pso at the patient's value of ph, pco2 and temperature), using siggaard-andersen's computerizated algorithm. mean time between paired measurements was 6.1 -* 1.2 houm. the data were compared by anova test for linear regression and t-test for paired samples. results: a dose linear relationship was found between svo2 and oxygen extraction ratio (oer), r=0.94,p=0.00000. the improvement of ci (1.41 -* 0.47 to 2.55 + 0.5 l/min/m 2, p<0.0000001) induced a significant increase in svo~ (0.495 -* 0.131 to 0.636 • 0.060 %, p<.0001). a significant decrease in p50 (32.5 • 6.7 to 27.9 • 2.5 mmhg, p<0.05) without any significant change in p~0st (29.1 • 2.2 to 28.7 • 2.3 mmhg, p=ns) was also found. these data show that either oer or the shift to the left of the oxygen dissociation curve account for increase in svo2 occurring with restoration of systemic blood flow. the program is intended to help the intensive care unit interne providing him with a practical tool when making decisions concerning patients in a critical condition. in his daily practice in intensive care unit, in this case the interne of the unit, uses this program for each patient as follows: on the first stage of data collection he should complete the following modules: (1)personal data (2)patient's pathology (3) laboratory and~ monitor lug data (4)drugs prescribed or toxic elements ingested. in this way, the system allows optionally the consult with a computerized data base about the drugs prescribed, standardized parameters and techinques performed by the central laboratory. (5)reference to an antibiotics guide regarding becterian sensitivety in our unit, whitch ee checked every six month (6) access to de questionnaired apache ii to load up new data. (7) statistcs about patient's admission and discharge. results: once all data collection is finished the system performs the followin duties: (1)detailed drugs interactions, including toxic elements (2)diagnosis starting from the clinical, laboratory and monitoring data. in some cases, it also establishes therapeutic strategies, e.g. a coagulopathy (3) give the l~narmacological incompatibilities between the drugs p~escribed and %he diagnosis established, and (4)perform dosage adjustments based upon the personal and pathological data. objeatve: to assess the power of diseri~,~ion ofa multiperpose severity score (sai~) when applied to subgroups ofpatieals (pta) according to their lemg~ of ~ay (los) in icu. design: in order to compute the saps probability, a model derived fi~m legible regression was developed. meaumree of calibration (goodmem..of.fit statistics) end discrimination (roc cm've and relative area under the cm've) were adopted in develotammtul asd validation set. the whole databue was ~ati~ed in five gronps reeked on los as follows: los = 2 days, los = 3-4 days, los = 5-7 da~, los = 8-14 days, los >15 day~. area under the carve (auc) was ud~ninted for each 8ro~. s~ing: 25 imlimlcus. patents: of 10~65 pts comec~ively admired ~ a period of three yeet~ (1990) (1991) (1992) , a total of 8059 was i~leded in this study. pts without saps, p~ yolmger them 18 yearn, p~ with los shorter ~ 24 hom'~ were excluded from this maly~is. iaterventinns: nose mema'onm~ end result: the logistic model developed gave good remits in terns of calibration md discrimin~on, both in developmental set (do.s g2: 9.24, p > 0.25; auc = 0.79i-0.01) and in validation ~t (g.o.g g2: 8.95, p > 0.50 ; auc = 0.78..+0.01). auc of each grottp showed a loss in di~zimination (i.e., prediaton) closely related with los, being 0.90i-0.01 in pts with los = 2 days el 0.59~.02 ia tm with los > 15 da~ (figure). following the present guidelines of integral management, in order to achieve optimization of sanitary resources and better use of facilities, we feel that the setting up of objetives is a key factor in the continuous process of improvement of quality care. postsurgical intensive care services maintain an interdepent relationship with other hospital services. within the general plan of the hospital it's of the utmost importance to delegate autonomy to the various depertments and service units in determining and achieving objetives. it's also necessary to establish mechanism for coordination of the activities in order to assure the succes of the program. the objetives cannot be improvised, they must be carried out in a specific manner in the following stages: 1.-analysis of the present situation (starting point). where are we?. defining objetives and making explicit the activities and methods to achieve them is to anticipate the future; it is of the utmost importance to comunicate said plans to all whom affect by encouraging them to attain the desired results. in the present paper we intend to show the guidelines to follow in carrying out a course of objetives. introduction:we presents results related to the quality of life (qol)of critical patients, from paeec project data. material and methods: the paeec project is a multicentre study define the type of patients cared for in spanish icus, and the therapeutic activity provided. ninety-five icus from spain are taking part. this study analyzes the qol of critical patients prior to their icu admission.for the evaluation of qol a questionnaire designed by our team for critical patients was used, with 15 items grouped in 3 sub-scales: physiological functions (4 items); functional capacity (8 items) and subjective aspects (3 items). qol is classified in 4 levels: normality (0 points); slight deterioration (1-4 points);moderate deterioration (5-9 points); significant deterioration (>i0 points). the we present results related to therapeutic activity in critical patients and their age, from the paeec project. material and methods: the paeec project is a multicentre study to define the type of patients in spanish icus, and the therapeutic activity provided. ninetyfive icus from spain are participating. this study analyzes therapeutic activity in the first 24 hours as evaluated by tiss, and related factors. results: the sample was 9,291 patients, sge 57.91~17.46 years. severity by apache ii system was 15.59• points. the tiss score was 19.87• points, distributed as follows: i (<i0 points): 14%; ii (i0 -19 points): 44%; iii (20 -39 points): 36%; iv (>39 points):5%.there is a positive correlation between the level of therapeutic activity and severity by apache ii (r = 0.46, p < 0.001), and a very weak but negative correlation between tiss and age (r = -0.048, p < 0.001), so that an increase in age corresponds to a lower level of therapeutic activity.patients the multivariate analysis of the relationship between tiss and age took into account: severity, existence of previous history, need for mechanical ventilation, size of hospital, diagnosis and mortality. it indicated that there continued to be a relationship between therapeutic activity and age, so that as age increased, therapeutic activity diminished. conclusions: therapeutic activity performed on critical patients is less in the oldest patients, in whom excessively aggressive procedures are limited. a relational data base management system in the icu. c. kotsavassiloglou*, d.matamis, g. dadoudis, j. kioumis, d. riggos. icu dep., g. papanicolaou gen. hosp., exohl, thessaloniki, and * a' neurological clinic of aristotelian university, thessaloniki, greece. objectives: the introduction of the information technology in the i. c. u seems to be unavoidable because of the large amount of produced data and the need for their systematic analysis. such an information system should be a) easy to use, b) friendly to the user, c) powerful and d) modular. on that basis, we created a patient data management system (pdms) according to the expectations of the medical staff of an eighteen bed multidisciplinary icu. methods: we selected paradox for windows v4.5 for the implementation of a relational data base because this program meets the above mentioned criteria. informations regarding the patients include a) demographic data, b) previous medical history, c)diseases upon admission, d)complications during hospitalization and e) outcome data. the diseases' registration consists of 421 items classified in 15 categories upon the principal system affected. specific informations about the need and duration of mechanical ventilation, nutrition, renal replacement, right heart catheterization and icp monitoring are also available. an extension was added concerning icu infections and related informations about antibiotic-resistant pathogens. all icu pathogens can be matched to their resistance or sensitivity and cost of antibiotics. the program can perform queries and various statistical analyses based on complex criteria. new modules can be added later according to the future needs and remarks of the users. results: the program was well accepted by the medical staff and 300 patients were registered as a test. the first analysis of the data related a) observed mortality versus the apache ii predicted mortality, b) mortality according to the age, gender, pathology aud duration of icu stay and c) pathology upon admission and icu related complications. conclusions: the long term use of this pdms can be an efficacious research tool. it can be used in retrospective or prospective studies by addition of necessary modules. the first data analysis revealed the iack of an international diseases' classification system. the development of a worldwide common classification system is essential for the compatibility of the data analysis among various icus. this will allow the realization of multicenter trials on a large scale. s. nanas= n. sphiris, a. precates, a. lymberis, m. pirounaki, and ch. roussos dept. of critical care, university of athens, athens, greece the complexity of the cases submitted to an icu, the variety of underline disease, tbe severity, as well as the large number of substances administered to each patient constitute obvious the need of support with an easy available dss. this system will assure the safety of the administered treatment will help to adjust the dose according to the situation of each patient and it will screen for possible interaction and incompatibilities between the administered drugs. the goal of the present effort is the design and development of a software system acting as a decision support tool to physicians of icu. the application is organised around a relation database management system (rdbms) that consist of: a) all available substances (1.850), b) all generic names of medications available in our country for each substance, c) incompatibilities (2.300 cases) and d) interactions with other substances (50.000 cases). the following figure shows the structure of the rdbms. y ta~ortato~ [ c~rs using the stored parameters for each patient the dose and the rate of administration of selected substances will be possible to calculate. the continuous monitoring of the treatment for each patient supports the medical staff to make the necessary changes of the prescriptions. the application is currently developing in wireless pen based computer systems which place patients at the centre of "islands of information" located throughout icu. in conclusion this dss is a powerful and useful tool for icu staff because it provides without additionai work to the routine of daily practice, the currently available information for each order concerning drug interaction and incompatibilities as well as treatment monitoring is to obsea~ among 113 critically ill pfdieats, stdjdivided following the diagn~s at the adn~ssio~ the diffmeax:es in the ~ and oxyplx~efic l~mmems bawe~ strvwors [s] and non sumvors ins] and to test the pc~'bih'ty to have soar survival criteria, as earliest as tx~able. method~ :we made a ~ study on 113 consexa~e ~ilically ill paliffas, subdivided in 3 series following the diastases at the admission: medical pafiea~ (29 s and 23 ns), surgical patients (19 s and 22 ns), a~d poliwauntas ( 14 s and 6 ns). follow up was done at 20 d,.ays from the admission in ice. all the patienls were ramitored with a ~ c~eter and 18 laeno:lymmi. "c and o .x.xyphorefic txuamaers va:~e couected at 7 fin~es (t): at fiae ~draission (t0), at 121x~ars from t0 (t1), at 24 (f2), 48 (y3), 72 (t4), % (t5) and 120 horus from t0 cf6). in~,h ~ies, for ~y ~ a all the lin'~ n~an and sandaid d~viation was ~ tx~h for s and for ns. th~ betw~ s and ns tl~ roeaas of ~h porarneter ~e ccmpared tt~ng t-lest and p < 0.05 w~ considered ska~ significant in each series in the t wheae the mast significative diffemx:as ~goeamd bet~en s and ns, we made a txedictive criterion, asamting as predictive indices for stnvival the i:r values, higher or lower than flae treans of the ~rar~ers of au flae patients, axx)rdhlg to those ones t~iatistically diff~'e~ betw~m s and ns. fhmlly xse co:weatxt onaong the 3 series the nrametees of the st~rs with the analysis of variance, to daserve the lxjsable differealt irea~ of sty1 hflices, following the diagn~s of admission: :nedkal, angical patient or poll~tam results: we c~ld not find ~ predictive criterion for politraonaas, perhaps ixx:ause of the few ntanber of l~fients. for high ri~ saw~cal patieras the following criterion at t2 has a sensitivi .ly of 100~ ,and a ~ecificity of 27.8%: sv1>32.89 nffmin/n~, map>92 mmhg, pmap<27 nmalqg cvp<i0 nunhg will4 nmah& lvswi>34 g m/m 2, sxo2>67~ do21>515 mlhnin/m2, o2er<31%. for lx~dical l~tienls at t5 the following criteric~a has a ser~tivi.ty of 100% and a ~zificity of 36.8~ cvp<7.5 mn~g, sao2>97%, s,g)2>74~ vo2i<133 ml/nfin/m 2, o2er<25%, shunt<19% survlvops' data of the 3 series ~ signitic~atly differenl~ both for the t~mody~nic a~ for fl~e ox3rphomfic lxlmn~s; moreover we ~ that the vatt~ of hemodynamic mad ox.~ho~tic indices were higher in politrautms. conclus'ions: acx~ording to the fftffe~mt patho!o~es, the ~ rnelabo~c needs are diffeten~ so that it is juslified to mash ~ the~alceutic goals, following the type oflmthology. 2hen~ we foru~d for high ~k mrgical pmka~ and for medical patier~s assme, ff mllslied, a good prognosis while, if n0[ ntljsfled~ the plinsliclioil ofdl~tth is no[ g(ioct finally, ab~ high iis~ supgical palieaats, according to what other atmhors say, txatws sh0~'n~ers ' therapeutic goalsvvould seem inadeqt~te, bec~jse they need a gear physiologic and themtx~ic elth~ in rdation to the rretabolic needs. figure 1) . thus, the smaller european nations had a greater participation than ~e larger ones, with the exception of norway. a similar result was evidenced for contributions to intensive care medicine (figure 2 ). these findings can be explained by different submission policies and language banners. however, there was no significant correlation with the gross national product of each country. conclusion: we conclude that the smaller european countries generally contribute more to international intensive care journals than the larger ones. objectives: to evaluate the agreement between a new and three old methods measuring ctp and to assess their reproducibility. methods: we studied 20 patients ventilated with a siemens 900c respirator. we measured ctp by dividing the tidal volume with the increase in airway pressure (paw), either with the respirator setting used (ca) or with a fixed setting (cf). by modifing the inspiratory time (ti) without changing inspiratory flow, we were able to deliver two series of 10 inflations (100, 200,... 1000 ml) before and after curarisation of the patient. the same volumes were also inflated in paralysed patients with a super syringe. at the end of each inflation a plateau of 3 sec was performed and paw was recorded. the above three sets of pressure-volume (pv) points were used to reconstruct the corresponding pv-curves ((31, c2, c3 the new method for ctp measurement without a super-syringe had the best reproducibility in paralysed patients and gave similar results without curarisation in the majority of them. however, agreement between the methods tested was unacceptable for clinical purposes. further investigation is required in order to improve the accuracy of ctp measurement in icu patients. m kunert, r.sorgenicht, l.scheuble, k.emmerich, h.g01ker med.clinic b (dept.of cardiology) i heart center of wuppertal/university witten-herdecke,germany objective to determine the accuracy of activated partial thromboplastin time (apl-l) and activated clotting time (act) studies when samples are drawn through heparinized central venous catheters (cvc). methods a total sample of 80 paired act/p't-/" values was analysed in 40 patients (28 m.,12 f.,66 + 12 y.) for monitoring heparin therapy.all patients had a cvc (certofix trio,braun,frg) in the internal jugular vein receiving a continous infusion of 20.000 u heparin via the central catheter.act (hr-act, hemotec,usa) and ap'i-f (neothromtin, behring,frg) samples were drawn from the cvc using the double syringe technique (removing and discarding 5 ml blood before drawing the sample). these blood samples were compared to act/ap'cf blood samples obtained by venipuncture (v.fem.) at the same time, act values were analysed directly in the intensive care unit (icu),api-i samples were measured in the hospital laboratory within 30 minutes. results ac-i -~ pi-f~ cact/~pi7 r = 0,62) cvc samples 132 88 +34 +22 . v.femoralis samples 1"28 84 +29 +24 p-value n.s. n.s. conclusion there is no difference in heparin anticoagulation studies drawn from heparinized central venous catheters compared to those obtained by femoral venipuncture,withdrawing 5 ml blood prior to obtaining the blood specimen is a safe way for eliminating heparin contamination.not only the aptt test but also the act test is a useful method for heparin anticoagulation assessment in the icu. objectives: evaluation of the delicate balance between filter-coagulation and patient-hemorrhage using heparin as anticoagulant in continuous renal replacement procedures. methods: from january 1991 through august 1994, we studied filter surviva[ and hemorrhagic complications during 240 filter periods in 78 critically d[ patients, treated with continuous arterio-venous hemo(dia)filtration, with special emphasis on the heparin dose, concurrent use of coumarins, systemic activated partial thromboplastin tirne(aptr), platelet count, mean arterial bloodpressure and the type of filter used. results: 141 filters (59%) were disconnected because of coagulation. mean survival of multiflow an69 filters was twofold shorter compared to survival of fh66 gambm filters. a total of 48 hemorrhagic complications occurred of which three patients died at aptt values of respectively 39, 48 and 56 seconds. after adjustment for mean arterial bloodpressure, platelet count and the type of the filter, the risk for filter-coagulation decreased 25% (relative risk 0.76, 95%c1 0.68-0.85) for each ten seconds increase in aptt. the risk for patient-hemorrhage increased 50% (relative risk 1.50, 95%ci 1.38-1.64) at an aptt-increase of ten seconds. the occurrence of filter-coagulation and patienthemorrhage was not correlated with the administered dose of heparin. concurrent use of cournarines had a positive effect on filter-survival, without increasing the overall incidence rate of patient-hemorrhage. conclusions: the systemic apt]" is a good predictor of the risk for filtercoagulation and patient-hemorrhage. heparine therapy seems optimal at an aptt between 35 and 45 seconds, although one should realize that fatal hemorrhagic complications still can occur. objectives: the alterations in vascular tone which are primarily regulated by adreno-sympathetic tone(ast) are compensatory responses in hemorrhagic patients. this study was designed to evaluate the correlation between vascular tone and ast in patients with hemorrhage, methods: the vascular tone was expressed by volume elastic modulus (ev) that is defined as; ev = ap/(av/v) (ap; the arterial pulse pressure, av/v; the volume change ratio). ev was measured using a non-invasive transmittance infrared photoelectric plethysmography (tipp) and a volume oscillometric sphygmomanometer . we prospectively studied 6 patients with hemorrhage. the initial ev measurement was performed on arrival and repeated for a 48hours duration. as a parameters of ast, serum concentrations of adrenalin (ad), noradrenalin (nor), plasma renin activity(pra) were measured simultaneously. we analyzed the correlation of ev and conventional parameters to ast by multivariate statistical analysis. results: ev values at transmural pressure 40mmhg on admission and 48hours later were respectively 864.2 + 249.5mmhg, 270.0 +_ 92.0 mmhg (mean + sd). systolic pressure(pas) and serum hormones on arrival and 48hours later were respectively, pas; 96.5_+20.4, 152+18.7mmhg, ad; 1.21_+1.02, 0.07_+0.04 ng/ml, nor; 1.60_+1.48, 0.65+0.39 ng/ml, pra; 26.6_+37.8, 2.5_+2.9ng/ml/hr. the ev values correlated significantly with ad (r=0.47, p=0.006, n=33), nor (r=0.47, p=0.005, n=33), pra (r=0.38, p=0.032, n=33). by multivariate statistical analysis, ev correlated more significantly with ad and nor and pra (p=0.00079) than the conventional parameters such as pas, heart rate and pulse pressure. conclusions: the alterations of ev correlates closely with ast. the compensatory mechanism in hemorrhagic patients can be detected noninvasively by ev monitoring. obiectives and method: autologous oxygenator blood was processed at the end of cardiopulmonary bypass (cpb) by either hemofiltration (hf 60, 1,2 m 2, fresenius) or by cell washing with a onntinous autologous transfusion system (cats, fresenius). prospectively the blood of 10 patients for each group was processed and then retransfused intravenously to the patient. besides, volume and time requirements, standard hematologic chemistry, coagulation and complement activation were measured. results (mean values for oxygenator blood at the end of cpb, and results of concentrate after processing by filtration or washing): both processing techniques show excellent hemoconcentration of the diluted cpb blood with a good transfusion effect for the patient. filtration retains all plasma proteins and large molecular weight plasma bound waste products. in contrast, cell washing with cats significantly depletes plasma proteins and waste products. the newely developped cats machine gives eonsisinnt laboratory result in a fully automatic continuous processing mode. in conclusion, both filtration and washing are effective for processing cpb blood. filtra tion yields a highly concentrated whole blood, whereas cats washing produces a high quality autologous erythrocyte concentrate. soluble fibrin has during the last years gained interest as a marker for the activation of the coagulation in connection with various clinical conditions, e.g. disseminated intravascular coagulation, deep venous thrombosis and myocardial infarction. elevated levels of soluble fibrin in plasma can be detected by the chromogenic assay coaset fibrin monomer, relying on the ability of fibrin to enhance the tpa-catalyzed conversion of plasminogen to ,plasmin. using this test, it has been shown that the level of soluble fibrin can be correlated to severeness of illness in critically ill intensive care unit patients. a revision of the coaset fibrin monomer kit has now been made and the new product, coatest soluble fibrin, is considerably more convenient to handle and gives higher resolution at low fibrin levels. the test is performed by the addition of a buffer dilution of the plasma sample to a microstrip well containing the colyophilized mixture of tpa, plasminogen and the plasmin specific cbromogenic substrate s-2403. the reaction is allowed to proceed at,. room temperature for 15 minutes before discontinuation. the absorbance at 405 nm, measured in a microplate reader, is proportional to the content of soluble fibrin in the sample. the assay is carefully standardized and calibration curves are provided in the kit. the convenient and rapid assay procedure makes the coatest soluble fibrin test well suited for single test analysis in acute situations. objectives : blood coagulation abnormalities have been reported in the systemic blood of patients with cerebral lesions. the physiopathology of such events is not yet completely understood. we compare the coagulation profile of blood from the right jugular bulb with systemic blood of patients with head injury. methods: we studied 4 patients, who were admitted to our neurosurgical intensive care unit between january and march 1995 with head injury and no other associated pathology (age 20-60 yrs), a glasgow coma score <= g, no abnormality in baseline coagulation profile and no history of coagulopaties. the patients did not undergo angiography. a one-way 16 gauge certofix catheter was inserted through the right internal jugular vein up to the jugular bulb. an identical catheter was inserted through a subclavian vein. blood was sampled from either catheter (a=atrial; j=jugular) 6-10 hours after trauma (t 1) and t 2 hours later (t2 the inddence dpontolx'rative thmmhi~e and haumord~gic complieatiom were assessed in padents treated with indobefen, heparin calcine caeca), low mollecolar weight heparin (lmwh) (f.nosheparin) and undergoing hemodiludun, blood predeposhing, intra mad postoperative blood saving. ]'he indolmfon tempota~.norks platelet aggregation through ,,elective inhibition of the cyclatygenasis and thus atacbldonicadd(1).tbe n'mimum effect occurs after 3 hours from the fast administration and is still present after 24 hours. ~-979 patients, mean age 62---11 yrs., weight 68---10 kg were studied. 321 (32.8%) were male and 658 (67.2%) female. 858 onderwent 713 hip prosthesis (7 previously plate and screw removal) 145 hip revim'un (10 stem, 33 cop and 102 stem + cop), 121 tutal knee prosthesis, in the 1st anaesthesidogy depl from 14-1992 to 30-6-1994. as for antithromboembolic ptephylam, apart from hemodihitiun 668 pts were with treated indobufen 0ndo), 199 with heparin ealdum caeca) and 112 with low mo!lecular weight hepam (lwr,91). as the slightest clinical and/or imtmmental suspidon of deep vein thrombosis (dv'i') or polmonary umbolism(pe), a phlebogram or sdndgram were respectively carried out. -the inddence of homologom transhisiom was significandy lower (p=0.000l) in the padeats treated with indobufen (4.256) compared 7.'ith heca (14.5%). the con~gency table shows statistical signifleance for the use of heca in patients with vein deficiency in the lower limbs, past dvr and/or pe, coronary heart disease (cdh'), while there is no correlation for renal, cardiac or liver defidency, obesity, systemic hypertemion, atrhythmy, diabetes, chronic bronchitis and rheumatoid arthritis. by comparing the postoperative cumplications with the risk factors, there ks a highly significant correlation (p=0.000l) between cdh and thrombotic and humord~agic complieatiom (pe, death, he~atoma, die use of hum_ologous blood). thee data show that hep~in, preferred in patients with c'dh, roost likely for leagal-tuedical reasons, did not have the de~'ed effect. conclusions -the stastisfical aar~ais shows ~nifieanfly different efflea~ (pro0.0001) between the therapies (see table) : it can be seen that in patients undergoing autotramfusiun and hemedihidon, indobufen produo~ a lower incidence of haemotrhagic complieatiens compared to heca and lmwh and is more effective in the prevention d ~c complications at clinical e~idence. the duration of i~toperadve hospital stay is signi~cantlylonger for patients transfused with homologous red ceils and treated with hec,7.a (13.7-+1.4 days) and lmwh (13.5+-1a days) compared with indo(ll.8_+1a days). one of the main causes for postoperative complications in major orthopaedic surgery is postopemtive bleeding with local effects in the operation site (hematomata, pain and delayed mobilization) and/or systemic and subsequent cardiodrculamry repercussions that are sometimes severe. the aim of this study is to assess the possibility to apply a new system of monitoring, control and saving postopemtive blood loss from the drainage. the bt797 recovery dideco (marandola, modena-italy) ~ used since it is the only apparatus capable of doing this. the apparatus consists of a pressure transducer, adjustable from -100 a +50 mmhg, which activates a peristaltic pump connected m drainage robes. the bt797 recovery display shows hourly bleeding in the first 8 hours, total bleeding, time passed since the start of monito~g and subsequent salvage and the aspimtioo pressure on the drainage robes; the latter is inserted at -10 mmhg and then modified according to bleeding/minute, g bt797 recovery also has an alarm that sounds automatically if.' blood loss is more than 300 ml/hour; air is in the circuit; the batteries are running low. materials and methods: 191 pts were studied (53 m and 138 ~), aged 63.5-+11.lyears, basal hemoglobin 13.1-+ 13(range 7.8-16.6)g/all, treated from 1st january, 1992 to mst december, 1994 in the 1st service of anesthesia and intensive care unit of our hospital. the patients underwent the following surgical treatment: total hip revision (132pts), cup revision (~ipts), stem revision (13 pts), total knee revision (2 pts). the average dumtion of the operations was 173-+58 min. intranpemtive monitoring and blood salvage was applied to all patients. genera! anesthesia was used on 57 pts. and integrated (epidural analgesia + light general) on the remaining t34. anttthromboembolic prophylaxis consisted of external pressure bandage, isovolemic hemodilution with iodobufen in 131(68.6%)pts., calalc heparin in 35(18.3%)pts., low molecular weight heparin in 25(13.1%)pts.; 1 pt did not give a predepoalt of blood, 4 gave 1 unit, 45 pts 2 units, 110 pts 2 units, 31 pts 4 units. the data obtained was statistically analysed using contingency tables and anova. results: average intmop salvage was 420-+345 ml, average postop salvage was 420-+265 mi the average intra+postop 909+-460 ml. average postop loss was 677-+359 ml. the global incidence of postop complications was: h~natomata 5.2%, dvt 1.1%, pulmonary thromboembolism 1,196, myocardiac ischemia 0.5%, acute myocardic infarction 0.5%, respiratory deflciecy 2.6%, arrhythmia 2%, cystitis 0.5% there were nn complications in 86.4% of pts. postop bleeding over 300 ml in under 60 minutes (with bleeding alarm activation) occurred in 30 pts (15.7%). this sta~tically correlates only with the type of operation performed (more frequently in total hip revision p=0.034) and with a significant decrease (p~0.003) in the pruthrombic activity detected about 8 hours after the operation. this bleeding, also made the alarm sound, calling the attention of staff who could act accordingly, by making the drainage pressure positive and incre~sthg the tension of the external pressure bandage. conclusions postop monitoring, control and blood loss salvage combined with predepoalting and intmop salvage has enabled allogenic transfusions in 16% of cases to be avoided in operations with high postop blood loss like hip or knee revision. the usefulness of the system can be seen by the fact that in the 30 patients with so much bleeding to set off the alarm, there was no significant difference in the incidence of allotransfusions and complications. references 1)borghi b., bassi a., de simone n., laguardia am., fonnaro g. an injury of the brain may result in various disorders of hemostasis caused by the release of • into the circulation through a damaged blood-brain bar tier. disseminated intravascular coagulation(dic) is one of these disorders. it is a freguent but relatively rare ly diagnosed complication of subaraohnoidal haemorrhage. the aim of this study was to evaluate some parameters of both blood coagulation and fibrynolisis in patients with sah.in addition one wanted to find out wh~ther potential changes correlated with the pa• condition in the acute phase of sah and whether they influenced the course of this disease. 60 patients with sah were studied. in 17 of them sah was due to closed eraniocerebral injury and in the rema ining 43 resulted from vascular malformation. the following parameters were evaluated:the prothrombine time,the activated partial thromboplastin time, the thrombine time,level of factor v,fibrinogen degrada tion products and fibrin monomers. the results let us show the presence of oic in 3 patients with closed craniocerebral injury and in 14 with vas. cular malformation despite the lack of clinical symptoms the tests in 5 posttraumatic patients and in 6 patients from second group showed incomplete dic.on admission patients with such changes in measured parameters were in poor condition.the course of the disease and the effe cts of treatment were also worse in these patients. the results showed ihal in patients with sah complex disorders of both coagulation and fibrynolisis occur, and they depend on clinical condition of the patient. they also influence the course of the disease. methods : charts of all patients admitted with d.i.c. over a ten year period (85-94) were reviewed. diagnosis of dic was based on the association of fibrinogen < 2 g/1 -platelets <150 109/1 -fpd > 40 ~tg/ml in the 24 hours of the admission. results : 40 patients -mean age 29+6 y -saps 8+_5 -gestanional age 35_+5weeks -the two first conditions associated with d.i.c. were placental abruption (35 %) and preeclampsia or eclampsia (22,5 %). bleeding episode was present in 22 pts (55 %) and surgical treatment has always been necessary. 26 pts (65 %) were given packed red ceils (12+10 u) and fresh frozen plasma (9+8 u). 6 patients were given platelets packs. heparin was never administered. 6 pts required mechanical ventilation and two patients hemodialysis. all the 40 patients survived. correction of prothrombin time (p.t.) and fibrinogen (f) was quick (p.t. at t24 h 69~2 % -f at t36 h 2,60+1,5 g/i). but platelets count remained low (plat. at t48 h 80 +42109/1) -no difference was observed in patients who received platelets. conclusion : prognosis of critically ill o.p. is good. blood loss is the main complication. correction of hypovolemia and anemia with concomitant surgical treatment are essential. the administration of coagulation factors or platelets is still under discussion. objectives: to evaluate the effects of antithrombin iii i at-iii) and a protease inhibitor, gabexate mesilate foy), on the coagulation and fibrinolysis in disseminated intravascular coagulation (dic). methods: after the approval of our institution and consent from patient's family, 40 patients with a dic score (1988, japan) more than 5 points (dic or having a risk for dic) entered this study. they were randomly divided into two groups, foy (i-2 mg/kg/h for 7 days or more) treated group and no foy group, each of 20 patients. platelet count (plt), fibrinogen (fen), at-iii fibrin degradation product (fdp), d-dimer (do), fibrin monomer (fm), thrombin-antithrombin complex (tat), plasmin-plasmin inhibitor complex (pic), and prothrombin time ratio (ptr) were measured before the start of treatment (at admission) and i, 3, 5 and 7 days after the admission. at-iii at 1500 units for 3 days was administered if the at-iii at admission was less than 70%. finally the patients were divided into four groups: group a, foy (+) and the at-iii ~ 70%; group b, foy (+) and the at-iii < 70%" group c, foy (-) and the at-iii 70%; group d, foy (~) anffthe at-iii <70%, each of 7 patients, to match the patients for backsrounds. all parameters, dic score and survival rate in a month following treatment were compared among the four groups. results: the at-iii and plt from day 3 to 7 were significantly higher in groups a and c than in groups b and d. the fdp, dd, tat, and pic after treatment decreased significantly from the baselines in groups a and c but not in groups b and d. the fgn and fm were not significantly different among the four groups. the ptr decreased in groups c and d but increased in group b. the dic score decreased significantly in groups a and c than in groups b and d. survival rates were 57%, 43%, 71% and 57% in groups a, b, c and d, respectively, although not significantly different. conclusions: in patients with dic or a risk for dic, foy had no expected effects but at-iii had suppressive effects on the coagulation and fibrinolysis mechanisms. a prognostic factor ? carbon monoxyde intoxication is a classical complication of inhalation injury. carbon monoxyda is also physiologically produced during the heme metabolism: heme is conversed to bi]irubin by the hemeoxygenase which is an intracellular stress protein. 20 icu patients (pts) were studied prospectively for apache ii score and carboxyhemnglobin (hbco) arterial level to assess if hbco level could be correlated with the severity of the pts. objective: to evaluate a new technique of non-surgical tracheotomy. patients: 55 adults, mean age 54 years and 11 children, mean age 18 months (2 me.-5 yrs). method: through a needle inserted in the trachea, a guide wire is retmgradely pushed out of the mouth and attached to a special device formed by a flexible plastic cone with pointed metal tip joined to an armoured tracheal cannula. this device is then pulled back through the oral cavity, larynx and trachea, and outwards across the neck wall by applying traction on the wire with one hand and counterpressure on the neck wall with the fingers of the operator's other hand. when the cone and 2/3 of the eannula have emerged, the cannula is cut off from the cone, straightened perpendicular to the skin, rotated and advanced caudally to its final position. results: endoscopic control facilitates and improves the safety of all manoeuvres. the pointed cone easily pierces the tissues, and the cannula is extracted without difficulty since it has the same outer diameter as the cone. tissue adherence around the cannula is absolute thus preventing local inflammation. the time in apnea required for dilation and cannula placement does not exceed 60 see., and it is well tolerated because within safety limits in patients hyperventilated with oxygen. only one case of bleeding occured in a patient on dialysis with severe coagulopathy. autoptic findings in subjects who died due to progression of primary disease showed a very regular stoma with an almost complete lack of hematic and flogistie infiltration in recent tracheotomies. .conclusions: translaryngeal tracheotomy (tlt), by virtue of its greater inherent safety and lower tissue trauma than percutaneous techniques, can also be carded out in infants and children, a severe test bench for any tracbeotomy technique. further specific indications are recently stemotomized patients, since tlt is associated with a low rate of infection, and short term tracheotomies after laryngeal surgery, to prevent obstructive complications. references: fantoni a., translaryngeal tracheotomy, apice, ed. gullo, trieste, 1993, 460 . background: inhalation of no has been shown to reverse hypoxic pulmonary vasoconstriction 1, to reduce pulmonary pressure in pulmonary hypertension of different origin and to improve gas exchange. in putmoflary embolism, pulmonary hypertension is caused by mechanical vascutar obstruction and by reactive vasoconstriction. the effects of inhaled no in putmonary embofism has been partiatly studied' the purpose of this study was to investigate and determine the effects of no inhalation on pulmonary hemodinamica and gas exchange in a hypoxic canine model of pulmonary embolism. methods: two groups of adult mongrel dogs were studied: group 1 (control} 5 dogs and group 2 (no inhaled) 6 dogs. both groups were anestesized with tiopental, mechanically normoventilated with an hypoxjc mixture of 02 and n~ (f[q2 0,16) and instrumented (swang-ganz catheter, femoral artery catheter) pulmonary embolism (pe) was induced by fisher's method s. no inhalation (80 ppm) in group 2 was started 15 rain. pdor to pe and kept constant throughout the experiment. no inhaled concentration was analyzecf by chemiluminiscence technique. pulmonary artery pressure (pap), central venous pressure and sistemic arterial pressure were continuosly recorded. cardiac output, artedat po~ (pan2) and mixed venous po~ were measured in both groups under hypo)dr conditions, before pe and 5, 15, 30 and 45 rain. after pe. pulmonary vascular resistance (pvr) and gas exchange (pao21fio:~ ratio), were calculate using standard formulas. data were process and analyzed with non pararnetdc test, and reported as mean -so and statistical significance was considered if p < 0,05. : no produced an increase in arterial oxigenation (pao2/fio~ ratio) and reduced pap before pe induction in group 2. after pe we found no significant difference with .respect to the time eour.se of pap, pvr and gas exchange between beth groups throughout the experiment. probably, the severe mechanical obstruction produced in pulmonary embolism masked the small effects of no inhaled. obiectives: blood volume measurement would be useful in critically ill patient management if it were easy to perform. this is not the ease and current methods are based on radiolabelled red cell dilution. inhalation and uptake of a known mass of carbon monoxide (co) gas and measurement of earboxyhaemoglobin increase can give results accurate enough for clinical use. this requires a rebreathing system providing oxygenation and carbon dioxide removal, yet complete retention of all carbon monoxide administer&l, and so most authors hand ventilate with a bag and waters soda-lime canister, adding oxygen as necessary. we aim to popularise this method by; i)design of an automatic co administration system driven by the itu ventilator and ii)writing of software for a portable computer to perform all necessary calculations 9 method: we show the computer is use estimating the co dose required and later estimating the blood volume. we also show the new gas administration system. this is a fully closed circle attached to a "bag in bottle", driven by the ventilator. the novel feature is the mechanism by winch driving gas (set to 100% 02) spills automatically into the circle, balancing o5 uptake by the patient, yet allowing no co loss. conclusions: this equipment is easy to use, reduces human error and allows optimum ventilator settings to remain. the operator merely administers the volume of co determined by the computer and takes blood on two occasions. carboxyhaemoglobin measurement is easy to perform, thus there is a cost saving also. with our modifications use of this technique may potentially become more widespread, the video demonstrates the method in use in our itu. -10 (25%) underwent conventional surgical therapeutics. 9" (90%) with resection of tracheal stenosis with end-to-end anastomosis(rts). i (10%) with broncoscopic dilatation. one patient died and the others still have stable patency(sp) without continued treatment. -29 (72,5%) have received endoscopic laser ablation with or without calibration tubes. 17 of them (58,6%) are receiving continued endotracheal treatment until now. 12 (41,4%) have sp wihout continued treatment. -i (2,5%) endoscopic laser therapeutic case turned to rts and is having sp. conclusion: conventional surgical aproach has been progressively replaced in our hospital by endoscopic laser ablation and silicone calibration tubes. this study suggests that these technics are effective and could be the elective treatment for iatrogenic stenosis. obiectives: hemorrhagic disorders due to thrombocytopenia and thrombocyiopathia remain one of the most serious complications during long-term extracorporeal membrane oxygenation (ecmo) in patients with severe acute respiratory distress ~drome (ards). in the presented study, nitric oxide (no), kwown as a potent endogenous platelet antiadhesive, disaggregating and antiaggregating compound, was evaluated for its possible antagonistic effect on platelet trapping when added to the gas compartment of membrane oxygenators (mo). meti~ods: two parallel separated extracorporeal circuits, consisting of heparin bonded hollow fiber oxygenators (minimax, medtronic, carmeda eioactive surface), tubing systems, low pressure reservoirs, and roller pumps were prepared. for each measurement, a pair of circuits was simultaneously filled blood from the same volunteer. low-heparinized fresh warm blood was obtained from four healthy volunteers, who had no drugs for at least two weeks. the gas inlets of both oxygenators received dry gas (21% oxxygen, 5 % carbon dioxide, 84 % nitrogen); gaseous no (20ppm) was added to the gas of one of the oxygenators (no-mo), whereas the other one (mo) was used as control. after 270 minutes no gas was switched off, so that the no-mo received no more no, and no was added to the gas inlet of the membrane, which had no no before_ to assure iutracircnit volume stability, drawn blood for measurements was replaced with saline, and platelet counts were corrected for dilution by hemoglobin values. the mean of four platelet counts (coulter counter) of each timepoint (start, 30, 90, 150, 210, 270, 330, 390 , and 450 minutes) was used for statistical analysis (paired sample t-test). results: in the no-mo platelets remained at 96 + 3,2 % (percentage of baseline value, mean -+ sd) until 270 min. in contrast, platelets of the mo continuously decreased after start and were significantly lower after 150 minutes ( 96,4 9 +3,5% vs90_+3,1%(p<0.05);210min. 95,9-+4,5%vs84,5_+2,2%(p< 0.05); 270 min. 82,7 _+ 2,5 % ( p < 0.05). after switching of no gas to the mo, further decrease of plateleta was stopped and platelets remained at 81,4 +_ 4,5 % until termination of circulation. platelets of the former no-mo decreased slightly after cessation of no gas to 92,6 _+ 5,4 %. conclusions: these data indicate that gaseous no significantly attenuates platelet trapping in hollow fiber oxygenators, when added to the gas compartment. this might be a new therapeutical approach for membrane oxygenator induced thrombocytopenia during long-term ecmd. objectives: nitric oxide (no) plays a pivotal role in regulation of vascular hemostasis. several studies elucidated the antiadhesive, antiaggregating, and disaggregating properties of endothelially synthesized no to platelets. additionally, agonist-induced no production in platelets by the l-arginine-no pathway was found as a negative feedback mechanism after platelet activation. although noplatelet interactions were intensively studied by several investigators, no data exist, about changes in platelet surface molecule expression in no-modulated platelets measured by flow cytometry using monoclonal antibodies (moabs). methods: p-selectin (alpha-granule-membrane protein, gmp-140, cd62p) and glycoproteiu 53 (gp53, lysosomal protein, cd63) are expressed only after platelet activation and degranulation. activation was quantified in thrombin (0.4 u/ml) and adp (0.1 ram) stimulated platelet rich plasma samples (prp). blood was obtained from healthy volunteers (n=7), who had no drugs for at least 14 days. for evahiation of no-modulated activation, the spontaneously noreleasing compound sin-i (0.1 mm) (3-morpholino-syndonimin-hydrochlorid) was added in parallel prepared samples prior to the addition of agonist. platelet surface molecule expression was evaluated with moabs directed against cd41a (gpilbliia, fibrinogen-receptor, phycoerythrin(pe)-conjugated), cd62p (fitcconjugated), and cd63 (fitc). only cd41a-positive signals were gated in sideangled light scatter, and assayed for activation marker expression (defined as percent of gated population). results: basal p-selectin expression was 1.6 + 0.7 %, and increased to 75.2 _+ 12.2 % after thrembin-activation, and to 26.7 + 5.3 % in adp-stimulated samples. addition of sin-1 attenuated p-selectin expression to 34.0 -4-193 % in thrombin (p<.001, two-tailed paired t-test), and 5.2 + 2.2 % (p<.001) in adpactivated platelets. basal gp53 expression was 1.7 _+ 0.5 % and increased to 63 .0 + 6.4 % in thrombin, and to 7.7 _+ 3.4 % in adp-stimulated samples. with sin-l, gp53 expression decreased to 346 _+ 10.4 % (p<.001) in thrombin, and 3.0 5:1.4 (p .001) in adp-stimulated samples. conclusions: these data implicate, that no leads to a significantly reduced activation of surface molecule expression in thrombin and adp-stimulated platelets. in addition, flow cytometry might be a useful tool for studying modulation of platelet activation by no or no-releasing compounds. introduction: acute cadmium poisoning is very rare. on initial presentation may mimic metal-fume fever, but acute inhalation cadmium toxicity may produce fatal chemical pneumonitis. case report: we present a case of acute fatal respiratory failure secondary to cadmium-fume irthalation. a 53 year old patient was trasferred from another hospital with acute respiratory failure presumably due to pneumonia. the last days before he had had commom cold symptoms. he had been cutting with a welder during one hour without any respiratory protective measure. three hours after exposure he developed progressive dispnea and was admitted to hospital. with presumtive diagnosis of respiratory infection, antibiotics were begun, however be failed to improve. all microbiological studies were negative. chest x-ray showed bilateral diffuse infiltrates. on seventh day he needed intubation and mechanical ventilation and on 10th he was admitted to our icu. antibiotics were stopped and new microbiological studies were performed including brochoalveolar lavage and virologic studies. all results were negative. he developed progressive hipoxemia and hipercapmia and finally, multiorganic disfunction syndrome. he died 19 days after exposure. the metal he had been working with was a 10% cadmium alleation. blood cadmilam concentration 15 days after exposure was 0.34 mcg cd/g cr, and urine cadmium concentration was 16.9 mcg/l. on postmortem examination, tissue cadmium concentrations were: blood 175 ng/ml, liver 823 ng/g, kidney 3571 ng/g and lung 1143 ng/g. these values confirm that cadmium was the cause of the fatal respiratory illness in this patient. conclusion: this case evidences the considerable hazard of acute poisoning after inhalation of eadmium-fume and stresses the need of appropiated safety measures against metal-fume poisoning. aim : lactic acidosis is considered the hallmark of cyanide poisonirig. however, the relationship between plasma lactate and blood cyanide levels has not been determined. the aim of this study was to determine the significance of plasma lactate concentration (plc) during the course of cyanide poisonings. methods : the patients were included according to the clinical suspicion of pure cyanide poisoning at the time of presentation. fire victims were excluded. serial blood samples were collected before and after intravenous hydroxocobalamin (hoco). blood cyanide concentration (bcc) was measured colorimetrically. plc was measured enzymatically. results : 8 patients were studied. on admission, plc ranged from 4.8 to 53 mmol/l, and bcc from 12.7 to 256 gmol/l. mean systolic blood pressure was 80 • 56 mm hg, mean arterial ph 7.34 • 0.14, mean anion gap was 29.7 + 7.7 mmol/l and mean pao 2 32.2 • 27.0 kpa. three patients died. before antidotal treatment, there was a significant correlation between plc and arterial ph (p = 0.008), anion gap (p = 0.008) and bcc (p = 0.016) but not with heart rate, pao2, paco 2 and blood glucose, or blood pressure. during the whole course of the poisoning, a plc _> 7 retool/1 was a sensitive and specific indicator of a blood cyanide concentration > 40 ~tmol/1. sustained catecholamine administration reduces the correlation coefficient. conclusion : baseline measurement of plc allows assessment of severity of acute cyanide poisoning. thereafter, plc may be used to assess the adequacy of antidotal treatment, more especially in patients not requiring sustained infusion of catecholamines. aim: the aim of this case report was [o study the correlation between the plasma lactate levels and several clinical, biological, and toxicological parameters serially measured during the course of a cyanide poisoning treated with a high dose of hydroxocobalamin. a 63-year-old male ingested potassium cyanide leading to cardiac arrest. cpr was performed prior to hospital arrival where the patient received 10 g hydroxocobalamin. sbp rapidly returned to normal allowing withdrawal of epinephrine. the patient remained comatose and died from brain injury 12 days after the ingestion. methods plasma lactate and blood cyanide levels were measured serially. blood cyanide levels were measured using a colorimetric method.~ plasma lactate levels were measured using an enzymatic method. for correlation spearman rank correlation test was used. results. initial plasma lactate and blood cyanide levels were 53 mmol/l and 256 gmol/l, respectively. there was no overall correlation between sbp and either blood cyanide or plasma lactate levels. similarly, there was no overall correlation between arterialvenous oxygen saturation difference with either blood cyanide or plasma lactate levels. in contrast there was a strong correlation between blood cyanide and plasma lactate levels (r=0.976, p<0.0001). the time-course of the blood cyanide concentrations was described by a mono-exponentiai decay (r2=0.968) with a blood half-life of 1.14 h. similarly, the time-course of plasma lactate levels was described by a mono-exponential decay (r2=0.986) with a blood half-life of 3.94 h. discussion. in this case of acute human poisoning, sbp was a much poorer indicator of continuing cyanide effect both before and after antidotal treatment, than was lactate production. this suggests a potential clinical role for following serial plasma lactate levels as a marker of the evolution of cyanide toxicity. aim : cyanide (cn) poisoning in fire victims is frequent and rapidly fatal. in a prospective study we tried to assess the clinical tolerance of a high dose of hydroxocobalamin (hoco) administered at the scene of the fire in fire victims suspected of cn poisoning. methods : inclusion criteria : soot in mouth or sputum ~ any degree of neurological impairment. exclusion criteria : children, pregnant women, burns of total surface body area > 20 %, multiple trauma. protocol desigrl following examination and the collection of a blood sample in dry heparin, a 5 g dose of hoco (10 g in case of cardiovascular collapse) was administered intravenously over 15 min. the systolic blood pressure was monitored before and after the administration of hoco, and one hour later. results : there were 28 females and 22 males. the mean blood cn concentration was 83 • 73 pmol/1. the mean blood carbon monoxide was 3.2• 2.1 mmol/1. nineteen fire victims eventually died. among the non-cn-intoxicated patients (blood cn < 40 ~mol/1), there was no significant change in arterial blood pressure. in the 33 cn-intoxicated patients (blood cn > 40 gmol/1) a significant increase in blood pressure was observed both immediately (p < 0.001) and 1 hour later (p < 0.001) after the admistration of hoco. no allergic reactions were observed. conclusions : in fire victims with cyanide poisoning, the administration of a high dose of hydroxocobalamin was associated with an improvement in systolic blood pressure. hydroxocobalamin is well tolerated in fire victims without cn poisoning. objectives: tricyclic antidepressant (tca) overdose can lead to serious complications including cardiac arrhythmias [ 1] . because of the known risk of early deterioration and the implication for management, emergent evaluation is essential. we determined the diagnostic usefulness of the electrocardiogram (ecg) in tca poisoning. methods: retrospective study of all patients with tca intoxication (pos. ,toxicology screening in urine and/or pos. history) in a 800-beduniversity hospital from 1989 through 1994. the severity was graded with mild= no symptoms or agitation; medium= disorientation, somnolence, tachycardia, or convulsions; and sever~ coma, significant arrhythmias or death. we analysed the first ecg after admission with a special emphasis on qrs-and qtc-intervals and the terminal 40ms frontal plane qrs-vector (tqrs), which, was reported to lie typically between +130 and 270* 798+310 915+453 5609• the best correlation with severity grade was found with qrs-and qtc-duration (p=0.0001), the tca-dose (p=0.0003) and hf (p= 0.027); tqrs did not correlate. 2 patients died (5.7%). conclusion: qrs-and qtc-prolongation in the admission ecg, and the reported dose of ingested drugs are useful predictors for severity of poisoning due to tricyclic antidepressants. we did not find additional benefit in determining the terminal 40ms frontal plane qrs-vector. objectives: since treatment of amphetamine poisoning is usually symptomatic and often associated with a fatal outcome, a search for specific drugs to help the amphetamine-intoxicated victim is sorely needed. methods: we report a case of a suicidal ingestion of large amounts of the amphetamine-derivative 3,4-methylenedioxy-ethamphetamine (mdea) and heroin (diacetylmorphine) and present the hypothesis that the two drugs produce opposing clinical effects. results: a 25 year old caucasian male was admitted to the emergency ward because of acute-onset confusion. at presentation, he was agitated and showed increased muscular rigidity. he had taken 40 tablets of "eve" (mdea, approx. 4 g) and 12 g of "smack" (heroin) by oral route approximately 2 h before admission. because of rapidly progressive tachypnea and exhaustion, the patient was intubated and ventilated. the serum concentration of "eve" on admission was 1400 ng/ml (lethal range 950-2000 ng/ml). trace amounts of cocaine and substantial amounts of heroin (115 ngtml; mean value in heroin-related deaths: 190 ng/ml) were also found in the serum. the patient was successfully weaned from the ventilator by day 4 and recovered without persistent neurobehavioral disturbance. despite high serum levels of both drugs, the patient did not present with the classic signs and symptoms normally seen during intoxication with these drugs. amphetamines in general, and mdea in particular, have opposite clinical effects to heroin or diacetylmorphine. none of these were however present in the case presented despite the high ingested doses and the serum levels in the lethal range. conclusions: the fascinating fact that, apart from the respiratory depression, none of the clinical signs reported after massive overdose with these two drugs were present, might be attributed to the opposite pharmacological effects of mdea and heroin. we believe that the patient unwittingly saved his own life by the oral coingestion of both mdea and heroin. our clinical data raise an interesting point about the pharmacological treatment of acute poisoning with amphetaminederivatives. introduction: the acute attack of aip still carries a significant risk of mortality of around 10 %. a succesful outcome depends on early diagnosis, removal of pricipitating factors and provision of intensive supportive therapy. objectives: twenty one patients (20 females, 1 male) with documented aip were seen over a 10-year period in the university hospital. 1 patient was in clinical remission and 20 were with the acute attack of aip, among them 4 with respiratory paralysis were required artificial lung ventilation and 4 -assistant ventilation with peee pathologic treatment during the attack was normosany, adenil, androgenes, glueosa, riboxin parenteral and enteral nutrition via nasogastric tube. symtomatic treatment -pethidine, propranoton, antibiotics, bronchoscopia. methods: intermittent phasmapheresis was performed on 15 patients. the following measurements were peformed: level of porphobilinogen (pbg) in the wire and delta-aminolevulinic acid in the blood. hematological and routine chemical evaluations, hepatic, hemodynamic and respiratory function. results: after plasmapheresis the median pbg excretion (normal range 1-2 mkg per/1 kgr creatinine) fill from 188 mkg on admission 140.8 mkg, then on 3-5 day raise to 193 mkg and then during treatment with normosong and prasmapheresis lowest level was 32.9 mgk. fatalities occured in two females during attacks with proforma cerebral involvement and 13 patients attained clinical remission. conclusion: after therapy with plasmapheresis normosong we found that there was consistently reduce the urinary excretion of pbg and shortening the duration of the acute attack. objectives: pigs has been reported to present with a higher pulmonary arterial pressure (ppa) and stronger pulmonary vascular reactivity than many other species, including man. aim of the present study was to compare pulmonary vascular impedance (pvz) before and after embolisation in weight-matched adult dogs and minipigs. methods: we investigated pvz spectra in 6 anaesthetized and ventilated (fio2 0.4) minipigs and 6 dogs. after baseline measurements the animals were embolised with autologous blood clots to reach a ppa above 35 mmhg. results: flow (03 and ppa matched pvz data (mean-+sem) are shown in the table. [zo = 0 hz impedance (z; {dyn.sec_em-5}); zl = first harmonic z; zc = characteristic z; z1 phase = first harmonic phase a@e {radians}; fmin = frequency of pvz the first m{n~mam; *, f p at least < 0.05 between dog and minipig, and before v~. after embolisation respectively]. before case report: a 53-yr-o]d woman affected by legs recurrent thmmbophlebitis, was admired in medmine department for tach.~pnea, chest pain, tachycardia and cyanosis. before starting two-dimensional transesophageal echocardiography (tee) to confirm the suspicion of pulmonary embolism, she suddenly had ventricular fibrillation. resuscitation and defibrillation were readily performed. when sinus rhythm was reinstituted she was in superficial coma with preserved corneal and light reflexes: right hemiplegia, poor perfusion and h~posphygrma of the left arm. tee showed dilation of rigth ventricle (rv), incomplete occlusion of pulmonary arter~ (pal at it~ hifurcation, severe tigth-to-left shunt through a patent foramen ovate, paradoxical embolism with incomplete occlusion of left subclavian artery mechanically ventilated with vt=800 ml, rr=12/mm, fio2=l, the patient had ph=7.28, pao2=57 mmhg and paco2=45.1 systemic bp was 130/80 mmhg and hr=80 b/min with low dose epinephrine (0.12 g/kg/min) a thrombolytic infusion (rtpa: 100mg/2h) through a peripheral vein was started tee imaging and clinical status 3 hours later were unmodified. a new rtpa infusion was performed through the pulmonary hole of a swan-ganz catheter with the tip close to the embolus. one hour later pa pressure decreased from 46/30 mmhg to 36/25 mmhg, etco2 increased from 26 to 30 mmhg and sao2 improved from 89% to 96% three days later the parietal, spontaneously breathing and with normalized tee scans of rv and pa, was transferred to rehabilitation service to perform physical therapy. conclusions: massive pulmonary embolism in a patient with patent foremen ovale, paradoxical embolism and refractory hypoxaemia was unaffected by systemic rtpa infusion, while intrapulmonary rtpa administration dramatically improved gas-exchange, hemodinamics and the general conditions of the patient. the presence of a large rigth-to-left _atrial shunt and the rapid rtpa metabolism could likely explain the effectiveness of its intrapulmonary administration in front of failure of systemic thrombolysis. introduction. cardiogenic shock during massive pulmonary embolism (blpe) is due to an acute increase of right ventricle (rv) afterload and possibly rv ischemia causing a failure of rv pump function. the rec~;mmended therapeutic strategies are: xoiume augmentation ~n ~rder m }ncrease rv pre-h~ad, adrenergic drugs to increase t'ontractillly and maybe coronary perfusion, fibrinolytic drugs to delermine clot lysis. there have been several reports of noradrenaline (na) as a useful drug in this setting for its sluing ~z, but also 1~, properties. case report.an obese 75 },ears old woman was transferred to our icu for tetanus. she was given the usual antibiotic and immunoglobuline therapy. l'wo thoracic epidural catheters were put in place at different levels and replenished with marcaine qid. a continous infusion of sedation (diazepam § was started together with mechanical ventilation. curarization ~,as given occasionally. fraxiparine 0.3/die was used for prophylaxis of thrombotic disease, on day 8th at 11.00 a.m. she started to be hypoxic (sa02 90%), tach3,tardic l1 l(i b/rain.), her blood pressure(rp) dropped frum norma~ values to 6r mm/hg, the central venous pressure (cvp) raised [rom lb to 27 mm/hg and the end tidal co2 was 7mm/hg lower than one hour before. the physical examination of the chest revealed a clear bilateral ventilation and the chest x-ray was normal apart from an elevation of the :tiaphragm as compared to the previous. an e.c.g. showed sinus tachycardia, right bundle branch block and a possible inferior necrosis (which was already present on admission). a trans-thoracic echozardiography was performed which showed "an acute overload of the right centricle wilh remarkable dilatation. tricuspidal regurgitation ++. paradoxical movement of septum. small left ventricle with normal wall kinetics". the cardiac enzymes were later shown to be normal. an acute massive pulmonary embolization was assumed m be present.. a bolus of streptokinase 750 x i(i 3 u. was given fonowed by a continous infusion . two liters of colloids were also given in a sh~rt time, two hours later the patient was still deeply hypotensive, hypoxemic and anurir(bp 54/32 mm/hg, cvs 23 mm/hg, spo2 90%) despite a cominnus infusion of dobutamine 20 fag/kg/min and adrenaline 0.5 ~tg/kg/min. at this stage a bolus of aoradrenaline 20 ,g was given followed by a cnntinous infusion of 0.05 !*g/kg/min. an immediate improvement of the hemodynamics was noticed and one hour later the bp was 149/77 mmhg, the cvp 24 mm/hg, the sao2 100% and a brisk diuresis started. the hemodynamics kept stable and weaning from vasoactive drugs was achieved within two days. one month iater the patient was discharged home in good conditions.. con c i u sio n.ne administration may help to restore rv coronary flow and ;~ump function during mpe. aeute putmonary t~omboembo~sm [ffe) cou16 be mamfeslated with either respiratory or cardiovascular syndromes or both. the arm of the study was to establish leading respn'atory symptoms, frequency and form of the roendganographic (rig) changes as well as blood gas disturbance degree in acute pte with dommam respiratory disease appearance. the study includes retrospeotive analysis of i 14 pte patients (pts), 63 males (average age 47,7 yrs) and .q females (average age 53,2 yrs). they were admitted at university, olinie" with suspection ofpleuropnlmonary disease, including pte. final diagnosis of pte was based o~ evident risk factors in 94,7% of the eases (deep venous thrombosis, surgery, trauma, imobilisation, malignancy ere), acceptable clinical, rtg, sdntigraphic and laboratory findings, as well as deep veins examination by dopple~-sonographie and radioisotopic -~enogmphy. respiratory symptoms appeared in all cases: sudden pleural pain (79%), dyspnea (64%), hemoptysis (49%), cough (39%) with association of two or more symptoms in 93%. chest xrays findings were abnormal in 92% with diaphragmal elevation (74,2~ lung opaeilies (69,5%), atelectasis (48,5%), plemal effusion (35,2%), main pulmonary brancah asimetry (22,8~ oligemia (19%), heart shadow changes (10,4%) and pulmonary arteries "cut off' (6,6%). the association of two or more abnormalities was found in 92,1% while normal chest x-rot was found in 8~ of the cases. hypoxemia with pao2<10,4 kpa was found in 64,4% followed with hypocapnia and respiratory alealosis in 34,6% in 27,8% of the gas exchage analysis were within normal limits. among cardiovascular symptoms short syn~cpa appeared in i0,5%, ecg changes-st q3t3 type in "~1,6%. results show high frequency of positive ~g findings in pte pts that is opposite to oppinion that chest x-ray in acute fie is the most ofran normal. leading symptoms are pleural pain and dyspnea, while hemoptysis were found in a half of the study group. blood gas changes were present in two thirds of the cases. kakkar, in his classic work ,clearly demonstrated the efficiency of low doses of heparin in prevention of deep vein thrombosis (lancet 2:669,1971) .after this first study the application of heparin prophylaxis became more and more diffused until to be considered a routine in many surgical departement.actually application of blood saving technique induces postoperative hemodilution effect. in that condition prophylaxis routinely applied seems a nonsense and can be at risk for postoperative hemorrhage. methods: to analize this problem we compared 100 patients arrived in our intensive care unit (i.c.u.) in.1980: (group a) with 100 arrived in 1994: (group b) .every patient was operated for major abdominal surgery.in each one we considered the hemoglobin (hb) value,hematocrit(hct), and coagulation pattern (c.p.) at the arrive in i.c.u. and 24 hours later. the patients was also divided in those receiving heparin prophylaxis (i) from not treated patients (ii) results:the application of blood saving technique clearly appears from the hb and hct level wich have a mean value of 11,4 +/-1,8 (hb) and 34 +/-2 (hct) in group a while in group b mean value are 9,7 4-/-1,2 (hb) and 29 +/-2(hct).patients of group a (ii) are the only one where a pathologycal c.p. with statistical significance has been demonstrated.in this goup we got four cases of evidence of venous thrombosis and one of pulmonary embolism.in patients of group b(i) we encontered the incidence of two cases of severe hemorrhage despite the absence of statistical significance in c.p.modifications. oxygen desaturation during broncho-alveolar lavage: role of oxygen saturation monitoring in prevention of acute respiratory insufficiency g. galluccio, b. valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the broncho-alveolar iavage is a diagnostic procedure employed in interstitial diseases of the lung. it requests the introduction through the working channel of a fiberoptic bronchoscope, after occlusion of a segmentary bronchus, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove cells and proteins from elf (endoalveolar lining fluid), which is related to interstitial medium. bronchoalveolar lavage induces deep effects on pulmonary function: -lowering of the alveolar surface of exchange; -shunt effect, depending on the perfusion of non-ventilated districts; -increased pulmonary arterial pressure, due to hypoxic vasoconstriction; -decrease of lung compliance. in this report the authors present the result of oxygen saturation monitoring in a group of patients with interstitial lung disease, who underwent diagnostic broncho-alveolar lavage. in most patients with severe interstitial involvement, the lavage performed without supplement of oxygen induced a severe fall in the oxygen saturation during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. in patients without thickening of interstitium, in whom the lavage was performed in order to obtain material for bacterial or cytologic examination, no modification of oxygen saturation was observed in standard procedure. as conclusion the authors strongly reccomend monitoring oxygen saturation in patients with radiologic evidence of interstitial involvement also in patients with no evidence of dyspnoea. g. galluccio, b.valeri, s.batzella, m. di lazzaro*, servizio di endoscopia toracica, ospedale forlanini, rome, italy * servizio die anestesia a rianimazione, osp. forlanini the treatment of choice in patients with alveolar proteinosis consists of pulmonary lavage. this procedure requests the introduction, through the working channel of a fiberoptic bronchoscope, segment by segment, of aliquots of saline solution at 37 c, subsequently gently reaspired, in order to remove the proteins deposited in the alveolar spaces. the method is very similar to that used in bronchoalveolar iavage, a diagnostic procedure used to obtain cells and substances from elf (endoalveolar lining fluid), which is related to interstitial medium. as known, bronchoalveolar lavage induces oxygen desaturation, because of shunt effect. understandably, one lung lavage has remarkably more deep effects on pulmonary function than bronchoalveolar lavage, for the amount of fluid introduced, the length of the procedure and the conditions of controlaterai lung. in this report the authors present the result of oxygen saturation monitoring in a patient who underwent pulmonary lavage for alveolar proteinosis. in the lavage performed without supplement of oxygen a severe fall in the oxygen saturation was observed during the late phase of the procedure. if supplementary oxygen was delivered during bronchoscopy, since its beginning, only slight modifications of the curve were detected. as conclusion the authors strongly reccomend the subministration of supplementary oxygen in pulmonary lavages, also in patients with excellent respiratory conditions. a. b. dublisky prof., m. r. isaakjan ass., v. a. zasukha, s. m. vinichuk prof., v. p. tserty ass. prof., chair of anaesthesiology, resuccitation and medicine of catastrophes, neurology of ukrainian state medical university, kiev, ukraine. objectives: detection of plasmophoresis's influence of results in treatment of ishemic insult. methods: we ve investigate 25 patients with ishemic insult, treated with reverse plasmopheresis in complex treatment. after primary infusive therapy we took 400 ml of patients' blood and separated it within 15 min with rotation frequensy of 2000/rain. after separation of erythrocytes from plasma, the latter has been returned to patients. we made 3-4 procedures during 3-4 days. hemoglobin, hematokrit, time of blood coagulation were determinated. the brain blood flow in internal carotid arteries, regional volum brain blood flow and total brain biood flow were evaluated with tetrapotar chest rheography and tetrapolar rheoencephalography. obtained date were comparised with control group after traditional treatment. results: it was found that after reverse plasmopheresis the hemoglobin and hematokrit levels decreased significantly in studied patients' plasma (from 140 + 3.2 g/l to 120 _+ 2.3 g/1 and from 44 + 2.1% to 35 _+ 1.8 % respectively). the time of blood coagulation by lee-white has increased by 2-2.5 times (up to 10-12 rain). the level of brain blood flow has been increased significantly after reverse plasmopheresis in comparison with control group. the following tests of brain blood flow have been increased: a) the total volume brain blood flow from 480.7 + 34.6 ml/min to 625.4 _+ 35.4 ml/min (p < 0.05); b) the regional brain blood flow from 52.2 _+ 2.8 ml/min to 87.1 + 6.2 ml/min (p < 0.01); c) the brain blood flow in internal carotid arteries from 166.1 _+ 12.2 ml/min to 206.3 + 14.6 ml/min (p < 0.05). conclusions: the use of reverse plasmopheresis in complex treatment of patients with ishemic insult aiiows to improve rheological blood patterns, helps to increase volume brain blood flow. it results in quicer reparation of neurological functions. objectives: a prospective evaluation of the efficacy of continuous infusion of verapamil in reducing the incidence of postoperative atrial fibrillation after pulmonary surgery. methods: a total of 199 consecutive patients, 100 on verapamil, 99 on placebo was included after lobectomy or pneumouectomy. a loading bolus of verapamil (10 mg over 2 minutes) was followed by a rapid loading infusion (0.375 mg/min) for 30 minutes and finally a maintenance infusion (0.125 rag/rain) for 72 hours. results: a mean plasma level of verapamil of 150 ng/ml was obtained only after more than 24 hours. atrial fibrillation occurred in five out of 78 patients who tolerated the verapamil infusion, and in 15 out of 99 patients on placebo (p = 0.08). verapamil infusion was not tolerated in 22 patients because of hypotension or a heart rate of less than 50/min, within 6 hours of the start of the therapy. when atrial fibrillation occurred, the ventricular response, mean _+ sd, was not significantly slower during verapamil infusion (132 + 22) compared to placebo (147 + 20). conclusions: because of its frequent side effects and the only modest efficacy verapamil should not be considered for prophylactic therapy of atrial fibrillation after pulmonary surgery, and is probably not a good first choice for slowing the heart rate in case of rapid ventricular response once atrial fibrillation has occurred in these patients. results: study of haemostasis in these patients has showed deep disturbances of blood coagulation. fibrogen level has reduced to 0.62 + 0.03 g/l, fibrinogen and/or fibrine degradation products concentration have enhanced to 0.40 _+ 0.04 g/l, monofibrin soluble complex concentration to 0.08-+ 0.04 g/l, blood plasmin level was enhanced to 34.0 + 0.2 mmol/1, plasminogen proactivator level was also enhanced to 153.0 + 0.60 ram, plateletes aggregation has decreased to 52 %. after plasmopheresis aggregation was decreased in 1.6 times. it has been connected with decrease of fibrin and/or fibrinogen degradation products level and level plasmin in 1.7 times, and plasminogtnt activator level in 4.6 times. at the same time we have observed increase in total antifibrinalitic activity of blood in 1.3 times. activity of activators plasmine and plasminogene proactivators has decreased in 1.2 times and in the same time activity of activation inhibitors and antiplasmines has increased in 7 times. conclusions: plasmapheresis leads to considerable improvement of a general condition and reduction of the haemorrhagic syndrom's sings (controlling of gastrointestinal haemorrage, reduction of intensity of subcutaneons haematoma). evaluation of continuous cardiac output (cc0) monitoring based on thermodilution technique in 35 critically ill patients. methods: cardiac output (co) was monitored continuously using a modified pulmonary artery (pa) catheter, on which a heating filament is located and by which energy is transmitted to the circulating blood. a microprocessor calculated co by a new algorithm. standard bolus thermodilution technique (10ml of ice-cold saline solution) was used to compare cc0 with intermittent bolus cardiac output (ic0) measurements. the following subgroups were prospectively studied: i. heart rate (hr) >120 beats/min, 2. cardiac output >10 i/min 3. cardiac output <4.5 i/min, 4. rectal temperature >39.0~ and 5. pa catheter was inserted for more than 4 days. results: a total of 404 pairs of ic0 and cc0 measurements were obtained from the 35 patients. bias (ico measurement minus cc0 measurement) of all measurements were 0.03• i/min and the 95% confidence limits (mean difference• were -1.01/1.06 i/min. also in the subgroups, cc0 measurement agreed closely with ico measurement (c0 >10 i/min: bias=0.16• i/min; co <4.5 i/min: bias=-0.17• i/mln). elevated temperature and prolonged lay-days of the pa catheter did influence agreement of cc0 measurement with ic0 measurement neither (>39~ bias=0.09• i/min). conclusions: monitoring of cc0 using a modified pulmonary artery catheter with a heated filament has proven to be accurate and precise also in the critically ill when compared with "standard" intermittent bolus thermodilution technique. this method enhances our armamentarium for more intensive monitoring of these patients under various circumstances. background: the number of patients who need coronary artery surgery was) grows every year. most of these surgical operations are with extrar eircuiation (ecc). since january 1994, this surgery is made without ecc in selected patients in our hospital. this technique is exceptional in spain. this type of surgery has proved useful in patients requiring revascularization of the left anterior descending, eireunflex or right coronary artery (not for grafting the pos~tefio~r descending branch}. blethods and results: since 1994, 30 patients aged 54 to 77 years (mean 66 years) underwent cas without ecc. the mortality in programmed surgery was 0%. no patient was reexplored for hemorrhage. the mean values of some clinics parameters v~ere: a) blood requeriments: 2 units per patient, b) need of mechanical ~entilation: i3,6 hours, c) postoperative bleeding: 900 cc, d) days at icui 2,5. we used the student % t test or fisber~s exact test to compare these results with the mean values of surgery with ecc: a) blood requeriments 4 per patient (p<0,0001), b) need of mechanical ventilation: 29 hours (p<0,0001), c) postoperative bleeding: 1300 cc (p<0,004), d) days at icu: 4 (p<0,001), e) programmed surgery mortality: 7% (p<0,05). conclusion: our limited experience shows that this surgery is an alternative in the treatment of coronary disease, especially for aged patients with associated pathology and in jehova's witness. the need of mechanical ventilation, days at icu, blood requeriments and morbi-mortality were fewer than surgery with ecc. to study the hemodynamic and antiarrhythmic influence of ace-inhibitor enalapril in acute myocardial infarction (mi). methods: holter ecg monitoring, heart rate variability analysis, echocardiography (3 and l0 days after beginning of the treatment), stress-echocardiography and stress ecg (8-10-th day after the onset of mi). enalapril was included into the treatment of 42 pts with mi (study group), with normal or increased blood pressure, from the 1-st day of the disease. the data were compared with 30 pts treated without enalapril (control group). results: silent ischemia during stress-test was registered in 6 pts of the study group and 8 of control group, the arrhythmia episodes during stress test -in 5 and 8 pts and episodes of silent nocturnal isehemia -in 7 and 12 pts correspondingly. enalapril importantly attenuated the hypertensi~re re~aetioh %0 stress test. in 10 pts of the study group the number of perifocal hypokinesis zones decreased; in the control group it didn't change. the quantity of ventricular extrasystoles in the patients of the study group decreased by 25%; the heart rate variability indices improved as well; in the control group the character of ventrieulir arrhythmias, heart rate and its va]~i~bili%y didn't change significantly. conclusions: the inclusion of enalapril into the treatment of mi is a useful t0ol to improve hemodynamie parameters and decrease the incidence of ventricular arrhythmias. objectives: to study left ventricular (lv) systolic function in the patients with acute myocardial infarction (ami) before and after peroral captopril test. methods: the original echocardiographic parameter of lv contractility, "coefficient of effective systolic function" (cesf), was proposed in the study. cesf is calculated from lv stroke volume (sv), obtained from doppler aortic flow in lv outflow tract and lv end-diastolic diameter (edd): cesf =sv/edd. the study included 60 patients with ami, who had local lv dyskinesia and global lv systolic dysfunction (ef<45%). besides cesf, the ejection fraction was calculated before and after administration of 25 mg eaptopril (on the fifth day of ami) by methods of bullet and simpson. results: the dynamics of these parameters, as well as heart rate (hr) and mean blood pressure (bp), is shown in the tabte. before cal~topril ef (bullet) 32.12 • 2.51 ef (simpson) 35 . introduction: the cold system is a monitoring system for measurement of right (copa) and left (coart) ventricular cardiac output, cardiac function index (cfi), fight ventricular ejection fraction crvef), fight ventricular cnddiastolic volume (rvedv), intrathoracic blood volume (!tbv), global enddiastolic volume (gedv), lung water (etv) and excretory liver function (pdr). patients and methods: 41 pts have been monitored by the cold system. above mentioned parameters are measured by thermal dye dilution and a fiheroptic femoral artery catheter. copa, rvef and rvedv measurements additionally were compared to measurements by the baxter explorer. :::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::::: ;;;k;;;;i cov (%) explorer ! 6 13 ! 1 [ gedv, itbv and pdr showed a significant decrease dufing the first 12-24h after the operation, cfi and rvef si~canfly improved after 48k wheras etv showed a i~ in the early postoperative phase and fell to normal ranges at 48h. comparison of cold/explorer m~ements sb0wed good correlations. discussion: concerning m0~toring of ri,ght ventric~ar function cold and explorer can he seen as equal. rvef gives an ar report about the performance of the right ventricle without use o f echocardiography. measuring itbv and gedv ~ improve ~gement and con~ol of th.e volume status, monitoring etv helps preventing lung edema. pdr shows good corre|ati0n to liver blood chemistry and is bedside avai|ab|e. thus the cold system offers additional parameters for comprehensive m~nitofing of pts. ~e~ ~c surgery. obiectives: to evaluate the influence of an a!'~ered cardiac function on the cardiovascular response to the increase in oxygen demand induced by an increase in core temperature. methods: this preliminary study included 12 adult critica!ly ill patients monitored by arterial and pulmonary artery catheters in whom thermodilution cardiac index {ci) and arteria! and mixed-vef)ous blood gases measurements could be obtained before and after an acute change in core temperature of at least 0.5~ (max 60 rain apartl the patients were separated in two groups according to their cardiac function: 4 patients had an impaired cardiac function as defined by a history of cardiac disease and an ejection fraction below 45% and 8 patients had normal cardiac function. results: individual data are shown in the figure. in contrast to the control group (continuous line) in which c! increased without changes in oxygen extraction (02er), the q2er in patients with impaired cardiac function (dottled line) increased without changes in ci. conclusions: the increase in oxygen demand associated with changes in temperature is met by an increase in c! in patients with unaltered cardiac function and in an increase in o2er in patients with altered cardiac function. temperature should be taken into account in the assessment of the adequacy of cardiac output in patients with impaired cardiac function. objectives: to define the hemedynamic and metabolic response to physical therapy(pt) in relation to the type/level of sedation and the cardiac status in icu patients. methods: we studied 34 mechanically ventilated icu patients (64• years) in stable hemodynamic status (no change in vasoactive treatment for at least 2 hours), separated in 4 groups: group 1 = deep sedation, cardiac dysfunction required dobutamine (n=5)r group 2= deep sedation (barbiturates), unaltered cardiac function (h=lo), group 3= moderate sedation, altered cardiac function (h=7) and group 4= moderate sedation, unaltered cardiac function (n=12). complete hemodynamic data, arterial and mixed venous blood gases, respiratory gas analysis (metabolic cart ccm, medgraphics) were obtained at baseline (2x) and twice (q.10 min) during leg mobilization. data were analyzed by anova. calcium channel blockers were used in complex preoperative preparation of 59 hypertensive surgical patients. patients were allotted to 3 groups based on their hemodynamic profile: hypokinetic: ejection fraction (ef)<0.6, 29 patients; eukinetic (ef>0.5),i6 patients and hyperkinetic (ef>0.6),i4 patients. the most noticable change in hemodynamics was in the hypokinetic group: ef and cardiac output (co) were significantly decreased (p<0.001) while systolic arterial pressure (sap) (p<0.05) and peripheral resistance (pr) (p<0.01) were elevated. the results showed that in hypokinetic patients on nifedipine ef (p<0.00t) stroke volume (sv) (p<0.0l) and co (p<0.001) were increased while pr(p<0.0t), sap(p<0.001) and diastolic arterial pressure(p<0.05) were decreased. eukinetic type patients also showed an increase in ef,albiet to a lesser extent,than in the hypokinetic group. increased sv and co(p<0.01) were observed in eukinetic patients though this was to a lesser extent than in the hyperkinetic group. in the hyperkinetic group of patients nifedipine had no effect on the aforementioned parameters except for a decrease in sap(p<0.0 i). nifedipine increased ef in all hypokinetic patients. comparative results show that isoptin was less effective than nifedipine in decreasing peripl~eral vascular resistance and had a depressive effect on the myocardium. it can be concluded that the action of calcium channel blockers normalizing the circulation in the hypertensive surgical patient depends on: the condition of myocardium, the patients hemodynamic profile and their pharmacological properties. they were most effective in the hypokinetic group. zalo/nthinos e., daniil z. zakynthinos s., armaganidis a., kotanidou a., nikolaou ch..,roussos ch. critical care department, university of.athens, evangelismos hospital, athens, greece. introduction : surgical is the optimal treatrnent for ioculated effusions and the preferable procedure when multiple bands are seen in the pericardial sac by echo. patients : 6 palients, 4 post cardiac surgery, 2 uremic (3men, 3 women) with large pericardial effusion and clinical or echocardiographic findings of tamponade or both. these particular patients displayed numerous linear echo-dense bands and s~'ands crossing the pericardial space (in one of them a ioculated effusion compressed the left ventricule). one had aptt increased, four were mechanically ventilated. technklue : a 8fr polyurethane catheter with end and multiple side holes over 18 ga needle was echo-guided to the ideal site (fluid abundant and closest to the transducer). the catheter was attached to a close system with a heimlich valve for continuous drainage (pneumothorax kit). subcostal entry was selected in one patient and chest wall in five. the patient's position was changed every hour at least. (we believe that the small changes in the position of the catheter and the mechanical breaking of the bands in relation with the movement of the heart assist the pericardial fluid to remove). results : in all cases only a small quantity of fluid was withdrawn in the first minutes(30-70ml) with some clinical and echo-findings improvement. the fluid was bloody or serosanuginous with high protein content (ht=15% ,protein 5,1gr/dl) in all cases. in first 24 hours the mean volume of fluid removed was 550ml (350to 720ml). in that period echo showed no residual fluid. the catheter remained within the pericardium 1 to 3 days .. no complications are mentioned. conclusion : cardiac tamponade due to hemorrhagic high protein pericardial effusion in uremic and postcardiac surgery patients,, as it is revealed by echo dense bands, can be faced by 2-d echo guided perieardiocentesis. a 8-fr polyurethane catheter with multiple side holes, attached to a heimlich valve was effective to evacuate the pericardial fluid. no catheter was occluded though heparin infusions were not used. multiple changes of the patient's position may be fundamental. this 2-d echo guided pericardiocentesis performed in in~nsive care unit seems to be useful , safe and quick technique. determining the best inotropic drug represents a very serious problems. the use of more selective and potential inotropic and vasodilatative drugs does not always lead to improvement of hemodynamic parameters in patients with low cardiac output syndrome. this paper presents patients with acbp who need an inotropie support after extracorporeal circulation in first 24 hours. the patients were divided into dobutamin et dopamine groups. the heart rate (hr). mean sistemic arterial pressure [map), central venous pressure (cvp). and termodilution cardiac index (ci) were measured. the measurements were without using inotropic drugs, and then using them after 5 rain, 30 min, and finally with one hour rate, within first 24 hours. the statistical analysis shows that both drugs lead to an increase in hr in the first hour of the application. the final effect of dobutamine is no change in hr, whereas the effect of dopanime is very significant increase in hr. thus. an absence of taehyeardie response selects the dobutamine as a better choice. backeround: pulmonary vascular eadothelium possesses major metabolic functions, which when altered contribute to the development of serious pathologies such as ards. one such function is the conversion of angiotensin i to angiotensin ii, catalyzed by angiotensin converting enzyme (ace), located on the luminal surface of the endothelial cells. ace activity has been extensively studied in animals in vivo, by means of indicator-dilution techniques, providing: i) under toxic conditions, an early index of lung injury, and it) under normal conditions, estimations of dynamically perfused capillary surface area (pcsa). objectives: to validate the use of these techniques in matt: i) for pulmonary endothelial function assessment, and it) for pcsa estimation. methods: ace activity was estimated in ten adult haman volunteers, with no pulmonary medical history and normal pulmonary artery pressures, undergoing cardiac catheterization for coronary artery disease assessment. single-pass traspulmonary hydrolysis of the specific ace substrate 3hbenzoyl-phe-ala-pro (bpap; 30p.ci) was measured by means of indicatordilution techniques, and expressed as %metabolism (%m) and v=-hi(1-m). bpap was injected as a bolus i) into a main pulmonary artery, and it) inside the right atrium, to assess ace activity in one and both lungs. we also calculated a,~,/i~, an index of pcsa. pulmonary plasma flow (fv) was determined by thermodilution. fp in one lung was estimated as 0.5xf v. results: similar values of %m (69.6+3.8 vs 68.9• and v (1.29• vs 1.25• were observed in both and one lung respectively. a~k~ decreased from 4185• ml/min (both ltmgs) to 2122:~175 (one lung). conclusions: i) pulmonary endothelial ace activity and thus pulmonary endothelial function may be assessed in humans by means of indicator-dilution techniques, it) our data denote homogeneous pulmonary capillary ace coneentratious and capillary transit times in both haman lungs, iii) the 50% reduction of a=~/k~ in one lung suggests that this procedure can be used to quantify pcsa in man. (supported by the fonds de la recherche en saute du quebec and the national health system of greece). objective: verify whether antioxidant activity is higher in reperfused than in no-reflow myocardium after i.v. thrombolysis for acute myocardial infarction (ami). methods: 37 patients with ami were included. blood for estimation of catalase (cat), glutathione peroxidase (gpx) and mn-superoxide dismutase (sod) was drawn before initiation of i. the mechanism of myocardial cell defence against free radicals is probably identical in both reperfusion and no-reflow phenomena. therefore, antioxidants cannot be used as reperfusion markers. objectives_ to evaluate the precipitating factors of hypothermic phrenic nerve injury following cabg with lima. methods: fifty two consecutive patients (8 females), with a mean age of 59+8 (mean +sd) years were studied. during the ischemic arrest time topical hypothermia was obtained in al~ patients wffh ice slush and no cardiac insulation pad was used. all patients received a lima graft, with or whithout additional vein grafts. supramaximai, bilateral phrenic nerve stimulation was performed percutaneously preoperatively and whithin 24 hours postoperatively. square wave stimuli of 0.1 msec duration were applied at the posterior border of the sternomastoid muscle. the compound muscle action potential of the diaphragm was recorded, using surface electrodes on the anterior chest wall. the time interval from the application of stimulus to the onset of diaphragmatic activity, phrenic nerve conduction time (pnct), was measured. values exceeding 9.75 msec were considered as abnormal. besults: preoperatively, all patients had normal (mean+sd) pnct, 7.69• msec for the left nerve and 7.98• mseo for the right nerve. on the first postoperative day, right pnct was normal in atl patients (7.93• msec) , whereas left pnct was normal in 45 patients (7.86• msec) and abnormal in 7 patients (incidence 13.5%). in 6 patients the left phrenic nerve was inexcitable and in 1 patient left pnct was prolonged (10.50 msec). comparing patients with normal and abnormal pnct there was no difference in age, gender, number of grafts used, aortic cross-clamp and bypass time. however, patients with abnormal pnct had a lower preoperative ejection fraction (45• vs 53• p=0.03). moreover, in all of them lima was dissected from its origin ligating all upper arterial branches, which provide the blood supply to the left phrenic nerve, whereas in those with normal pnct the small vessels originating from the upper 2 to 3 cm of lima were preserved (p=0.001). conclusiojel~ a hypoperfused left phrenic nerve seems to be more susceptible to hypothermic injury during cabg with a lima conduit. objectives: to test if necessary interventions on systemic vascular resistance (svr) along with preset pump flew (q) during cpb could adversely affect autoregulatory response and cause vo 2 shifts. methods: we studied 26 males (554-7 yrs) who underwent cpb for cardiac surgery. at o oesophageal temperature 27-28 c we set pump flow at 2.1 i.m~2.min -1. when map was higher than 85 mmhg we calculated vo 2 by using fick equation. then we infused sodium nitropruaside (sn) to control map at 55-65 mmhg for 10 min and we calculated vq 2. without changing the sn infusion rate we set q at 2.5 i.m'2.min "1. ten min later we measured vo 2. we took vo 2 changes into consideration if greater than 15%. statistical analysis using students-t-test for paired data and analysis of variance was used as appropriate. results: depending on the biphasic vo 2 response to sn infusion during low and high q we classified pts in four groups (table). i. vo 2 increases with sn and increases further during high q unmasking hypoperfusion and supply dependency. ii. vo 2 increases with sn but the addition of high q results in systemic shunt. iii. vo 2 increase during high q proves that vasodilatation can turn flow insufficient. iv. vo 2 does not change with any intervention. the small number of pts and the wide standard deviation did not allow any statistical significance. conclusions: cpb is an interesting model for the behavior of microcirculation. intervention on svr and q can improve or impair effective regional oxygen delivery, resulting in either better perfusion or systemic shunt. vo 2 monitoring seems necessary during cpb. preoperative cardiovascular optimization (opt) to ci > 2.8 l/min/m 2, 8 _< paop < 18 mm hg,and svri __< 30 mmhg/ll/min/m 2 decreases cardiac events (events) and mortality (mort) in peripheral vascular surgery patients (pvs). objectives: to determine if opt to the same endpeints decreases events in patients undergoing abdominal aortic aneurysm repair (aaar) and to study the r predictive value in pvs patients. methods: 44 aaar patients and 41 pvs patients were admitted to the s cu monitored with e pa and arterial catheters and treated to achieve opt. patients underwent surgery independent of success of opt data included demograph cs, incremental risk factors, laboratory and hemodynamic data pre, intra, a~nd postoperatively events, and mort. events included arrhythmias requiring treatment or prolonging the sicu stay > 24 hours, a st depression > !mm or t wave inversion, an acute mr defined by a new q wave > 0.03 sec or cpk-mb > 5%. results are presented as means _4-. sd. opt was achieved in 32 of 41 (78%) and in 36 of 44 (82%) in the pvs and aaar group, respectively. events did nat differ between groups 9 of 41 (21,9%) and 12 of 44 (27,2%) in the pvs and aaar group, respectively (p>o.05). mort was 0 of 41 (0%) and 1 of 44 (2.2%) in the pvs and aaar group, respectively (p > 0.05), while there was no difference in endpoints of opt between patients with and with.out events in the aaar group, there was a significant difference in ci between patients with and without events in the pvs group. of note, 8 of 9 (89%) patients who developed events in the pvs group had a ci < 2.8 in contrast to 4 of 12 (33%)in the aaar group. the positive and negative predictive value were 89% and 97% in the pvs and 50% and 75% in the aaar group. conciusione: f. the endpoints of opt used for pvs patients cannot be ~sed to reduce events in aaar patients; 2. pvs patients who have net achieved opt are at extraordinary risk of perioperative events; 3. preoperative card ovascu ar opt in aaar patients makes no difference in cardiac related events, background : comparison of the right and left filling pressures (cvp/pcwp ratio) is considered as a useful diagnostic clue : the normal ratio is _< 0.6; ratio >_ 0.8 may suggest right ventricul~ infarction while equalization of the cvp and pewp is a classic sign of tamponade (1). however after cardiac surgery, many conditions (diastolic dysfunction, pulmonary hypertension, positive pressure ventilation) are susceptible to modify the '*normal" cvp/pcwp ratio. material and method : we determined cvp/pewp ratio in 100 consecutive patients (pts) after uncomplicated cardiac surgery (78 coronary artery bypass grafts; 22 valvular replacements) measurements were made before and after tracheal axtubation. results :cardiac index : 3.2 _+ 0.7 1/minlm~; laotate: 144 + 68 rag/i; cvp range : 4-17 rnmhg; pewp range : 2-16 mmhg. mean cvp/pcwp ratio before extubation is 0.94 (95 % confidence imerval : 0.86-1.02) and after extubation, 0.93 (95 % confidence interval : 0.83 -.1.03), (ns, paired t-test). in 25 % of the pts, cvp was higher than pewp. there are no correlation between the cvp/pcwp ratio and c! before (r = -0.04) and after extubation (r = -0.09) nor between the cvp/pcwp ratio and mean pulmonary arterial pressure (mpap), before (r = 0.08) and after extubation (r = -0.13), discussion : cardiac performance is adequate according to ci and lactate. however the cvp/pcwp ratio is markedly higher than the "normal" (_< 0.6) ratio. this difference is not related to mechanical ventilation because the ratio is similar before and after extubation, nor to pulmonary hypetaension because of absence of any correlation with mpap, post-cpb diastolic dysfunction of the right ventricle could be an alternative explanation. in this group of pts, increased cvp/pewp is not associated with any impairment of cardiac performance (absence of correlation with ci), conclusions : cvp/pcwp ratio as high as 1 within a large range of cvp (4-17 mmhg) and pcwp (2-16 mmhg) may still be considered as normal after cardiac surgery. this emphasizes the limitations of the hemodynamic monitoring after cardiac surgery (in comparison with echographic technics). careful analysis of the morphology of the cvp and right ventricular pressure curves (x descent, y descent, dip-plateau) is mandatory rather than relying on the quantitative assessment alone. reference : (1) ntensive care.-university hospital -m~laga (spaink introduction. fibrinolitic treatment (ft) permits the treatment of acute myocardial infarction (ami) addressing the etiology, thereby eading to mproved ventncular function and a marked reduction m mortality. the main clinical oroblem is the reduced time of application. delay in hospitalization, which can be from 50 to 120 minutes, is potentially the most avoidable delay. method. to reduce delays in hospitalization, the following was carried out in two chases. 1 audit: analysis of the time lapse from onset of symptoms to start of ft. showed that during "(he period 1 june to 31 december 1994, 79 patients with chest paros were treated within a 0eriod varying from 30 minutes to 6 hours from onset of symtoms. ages ranged from 33 to 86 (average 64,4), oelng 49 males and 30 females. they were glved initial ecgs to determine st mcreases suggesting ami. median t~me for this orocedure was l0 m.. 204 potentia ami patients were then admitted to the coronary unit, 103 [)atients, under age 75 with no contraindications received ft the median time apse from admission to corona-y care and administration of ft was 15 minutes (12. 17), -he total median delay was 58 minutes ~40-i h.45min,~ delays n start of this procedure are grouped as follows: extra-hosdita delays (from onset of symtoms to arrival at hospital) diagnostic delays (from hospital arrival to ecg). treatment delays (from diagnosis to ft). 2 objectives: protocol of procedure to implement a fast-track method. a protoco was drawn up with the object of reducing diagnostic delays to 8-i 0 minutes and treatment delays to less than i 5 minutes results. following rmplementatlon of this protocol in january 1995, 30 fts were glven, with an over all average delay of 24 minutes. this fast-track method did not reveal any inappropnate ft or any increase m complications, conclusions: detailed study of the various times taken for diagnosis ane treatment of ami patients, showed up weaknesses in the system and improvements througn the protocol based on performence orocedures which led to a 59% reduction in the start of ft background: the importance of the early use of thrombo!ytic agents in acute myocardial infarction (ami) is based in the better remaining ventrictjlar function and smaller mortality rate because of the greater reperfusion and sma!ler infarction size, therefore, it is very impodant to apply this treatment to the maximum number of patients without thrombolytic contraindicati0n, and within the minimun period of time. the "thrombolytic fast track" implementation allows to optimize the time to administrate thrombelytic agents avoiding multiple delays~ methodology: we anal!ze the application of thromboly0c agents to 73 patients with suspect of ami from the begin!ng of september 1994 until the end of february 1995. in this time there are two different periods, during the first 4 months thrombolytic agent were admin!strated at intensive care unit (icu), and during the second period we carried out a protocol of quick detection and thrombolysis therapy in susceptible patients at the emergency room in order to reduce the time to treatment. ma!n results are shown in the faffewins de ay h=hours m=minutes the implementation of the fast track does not need supplementary personal or equipment but a protocelized approach and training of the personal involved the main problem detected was the usual attendance overload of the emergency department that makes difficult to follow many structurated actions. conclusions: pratocqlized changes in the management of ami can significantly reduce the detay in the administration ef thrombolytic agents. it is not necessary to eomplet the procedure iq the emergency department, as the use of bolus schedules allows to begin the treatment in this area and to transfer the patient to icu afterwards. elective cardiac surgery. b calvet, f ryckwaert, p trinh duc, p colson. anesthesia -reanimation, hopital arnaud de villeneuve, montpellier, france. obhectives: the study was aimed at analysing the incidence of renal dysfunction following cardiac surgery and its prognosis (acute renal failure, post-operative morbidity and mortality). methods: two hundred and thirty seven patients (aged from 28 to 90) were consecutively operated on for elective cardiac surgery and retrospectively included in the study. patients with preoperative infections and operated on in emergency were excluded. each patient had preoperative invasive cardiac investigation with angiography and calculated ejection fraction (ef). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest management were similar in all patients. general body temperature was reduced to 28 -30 ~ c. renal dysfunction was defined as a 20 % increase from baseline of serum creatinine. demographic data, asa, treatments, pre-operative creaunine level, cpb and clamping (axc) times, intra and postoperative use of inotrope, serum lactate level before surgery, at the end of cpb, at the time of admission in intensive care unit (icu) and on post operative day one and apache score were compared in patients with or without renal dysfunction using anova test for repeated mesures and x2 when appropriate. data are expressed as mean +__sd. p value less than 0.5 was considered statistically significant. results: thirtytwo patients (13,5 %) suffered from renal dysfunction. age, serum lactate level at the end of cpb, at admission in icu, at pod1 and apache level at admission in icu, intra-operative use of inotropes were statistically different in patients with or without renal dysfunction (p<0,05). mortality rate was statistically different in patients with or without renal dysfunction(~,2,5 % and 0 %, respectively, p=0,001). incidence of acute renal failure following renal dysfunction was 6,2 % (2 patients required hemodialysis). conclusions: although our cdteria for defining renal dysfunction were very sensitive, the incidence of renal dysfunction following elective cardiac surgery was lower than communly accepted in the litterature (1). however renal dysfunction appeared significantly associated with a poor prognosis. reference: 1-settergren g, ohqvist g current opinion in anaesthesiology 1994, 7:59-64 r 66; 159, 200 tzelepis, g. 112, 127, 142 late complications were observed in 14% of cannulations: local infection in 2 (i,7%), catheter displacement by the patient in 4 cases (3,5%), catheter displacement during nursing care in 5 (4,4%) and malfunction in 5 cases (4,4%). conclusions: central venous catheterizations are followed by immediate and late complications in almost the same percentage acute poisoning with amphetamines (mdea) and heroin: antagonistic effects between the two drugs methods: after institutional approval and informed consent, 33 selected patients (65_+9 years) undergoing peripheral vascular surgery (n=17) or carotid endarterectomy (n=16) were investigated. patients included had either documented cad (n=18) or two or more (n=15) dsk factors (age >65 years, smoking, diabetes meltitus, hypertension, hypercholesterolaemia >240 mg/dl). 12-lead ecg recordings were carded out preoperatively, on ardval in the postanaesthetic care unit, and 20 h, 48 h, 72 h, and 84 h postoperatively. ecg recordings were analysed by an independent blinded cardiologist for signs of pmi (new st segment depression >0.2 mv and/or new t inversion). in addition results: 22 of the patients investigated developed ecg-documented pmi, 86% occurdng in the immediate postoperative phase. troponin i levels >1.6 ng/ml were found in 19 of these 22 patients thus, comparing a cardiac troponin i cut-off level of 1 ng/ml with intermittent 12-lead ecg recordings, we found a sensitivity of 86% and a specificity of 91% methods: demographic, clinical and ecg data were analyzed. 53.8% of patients were male; 46.2% female. cad was the most common underlying cardiac disease (85.7%) and 58.4% underwent open heart surgery. 69% received proeainamide for supraventricular and 31% for ven~cular arrhythmias. 27% received a loading dose. maintenance was provided by iv route in 36.8% and by po in 63.2% (40.8%sr end 22.4% ir). 40.4% of patients were obese right ventricular function following cardiopulmonary bypass: is important the mode of myocardial protection we underwent this study in order to examine its safety and usefulness in pts with trustable coronary conditions (unstable angina ua the mean age for group a was 54 • 16 years, for group b 64 • 11 years, and for group c 59 • 13 years. a history of previous myocardial infarction was present in 6 pts of group a, in 3 of group b and in 54 of group c. three pts in group a, 5 in group b and 15 in group c had previous coronary artery bypass grafting. the median time between the onset of symptoms and a was 5 days (2 -19) for group a we used a continuous fixed intravenous a infusion at a dose of 140 the sn was 100% in groups a and b, 98% in c, and sp 100% for group a, (fixed defects included) and 50% for groups b and c. there was no difference of side effects among groups: chest pain (i pt -group a, 2 pts -group b, and 8 pts -group c), transient hypotension (1 pt -group c), headache (5 pts, group c), dyspnea (1 pt -group a), while st depression was seen in 2 pts of group b and in 2 pts in group c. the rate of a infusion was decreased to 70 7/kgr/min in one group b pt due to development of chest pain s228 five year follow up of humoral immunity in paced patients athens polyclinic hospital, department of cardiology athens, greece author index a abiad ch 133 bertschat, e 141 betbes6 75 blanch, l1 177 del nogal saez 93 e1-meneza 220 nolla, j. 98 nolla-salas 24 pilz~ u puig de la bellacasa e 38 scarpa, n. 77 217 van de wetering objectives: only 50% of patients suffering from acute guillain-barr@ syndrome (gbs) respond promptly to established therapies like plasma exchange or intravenous immunoglobulines. in contrast to serum, cerebrospinal fluid (csf) of gbs and ctdp patients contains enriched portions of antiexcitatory factors(i) and cytokines (2) able to induce pronounced conduction block (3). to reduce or remove such pathologic factors we introduced a technique with direct access to the subarachnoid space. methods: with informed consent we lumbally inserted 18 g catheters in 24 gbs-and 22 cidp -patients under sterile conditions. some of them had not responded very well to established therapies. 30-40 ml of csf were withdrawn and retransfused by a bidirectional pump (flofors) after passing newly developed filters (pall). daily filtrations with several cycles were performed (200 -300 ml) over one week. results: the 24 gbs patients improved after 19 days (median) for one grade (according to the gbs-scale from the gbs study group) . the ventilator dependent patients were weaned after 16 days (median). patients not at all treated before (16/24) responded better than patients that had been pretreated (8/24) with plasmaexchange or intravenous immunoglobulines. 18/22 cidp patients drew benefit from treatment, 10 stabilized iongterm. conclusions: csf-filtration is a relatively save and well tolerated additional procedure. the costs are considerably lower (1/3) than those for plasmaexchange or intravenous immunoglobulines. references:(1)wsrz aet al: csf and serum from patients with inflammatory polyradiculopathy have opposite effects on sodium channels. muscle nerve (1995) . (2) clinical observations were made in 24 patients admitted to the clinic. they were in coma associated with acute alcohol intoxication.standard evaluations (ecg-monitoring, electrocardiography, neuromonitoring, studies of acid-alkali condition, biochemical and toxicologic investigation of blood and urine) prior to and following the treatment conducted were undertaken in all the patients.to correct irreversible impairement of functions twofold laser blood irradiation by means of alok-01 apparatus, the exposure within 20 minutes, was carried out.the data obtained confirm more rapid coma withdrawal of the patients, reconstruction of the heart and central nervous system electrophysiologic indeces, reliable reduction in complications compared with the control group. objective: to know the actual incidence of the critical illness polyneuropathy(cip). setting: fourteen intensive/critical care unit beds, in 550 bed university hospital, covering 345.000 inhabitants (majority rural area). the icu patients are medical, surgical and coronary, excluded the neurotrauma and neurosurgical. design: a conseculive and prospective study. all the patients admitted during three months, from january lth to march 31th 1993, were eligible (patients with admittance diagnosis of polyneuropathy were excluded ). methods: patients with apache ii score >10, at the admission and six days after admissions were included into the study protocol. diagnosis of sepsis, mof, and all the drugs administered days before were recorded. a complete neurological exam, by a neurologist, in absence of ssdatives and muscles reliant (7th, 25~ and 60th days after icu admittance) was made. we evaluated the nerve and muscles function with and electromyography study in all patients, at same days. in some paeents with cip we performed a nerve biopsy. results: from 285 patients ( apache ii score: 12.82) admitted in the icu, 16 (5.6%) enter the study protocol. seven (2,45%) had an axonal polyneuropathy(cip), three very severe. only four of the patients with cip had pathologic clinical exam. apache ii score: cip vs non-cip was 22.6 vs 16.6. the incidence of cip by diagnosis (cip/diagnosis) was: sepsis, 5/9 and mof, 6/11. conclusions: 1. -we think that it is necessary to define the "critically ill" for some score, before designing a study to know the incidence of this syndrome. 2. -we think that the incidence of the cip is lower that the latest papers say. objectives:acute pancreatitis(ap)is becoming a more important problem among the elderly as the population ages. the increasing presence of gallstone disease,as well as the use of certain drugs,may also contribute to the occurrence of pancreatitis. methods:all patients(>60 years)admitted to our medical department over an eight year period were included.pancreatitis was confirmed by biochemical tests and imaging techniques.scores were developed using ranson's criteria and a multiple organ system failure(mosf)index . overall, 103 patients were evaluated; 21(23%)had pancreatitis of unknown etiology . results:(1)patients with pancreatitis of ~nlqnown etiology were sicker and had greater morbidity(48% vs 22%),mortality(24% vs 8%),and longer hospital stays than p~tierf~ with pancreatitis of known cause.(2)the best predicto~of severity and outcome was the mosf index and not ranson's criteria;the higher the score,the greater the associated disease,the worse the outcome.(3)curlously,no difference existed in associated medical conditions between patierts withknown and ur6~own causes of pancreatitis. conclusions:greater organ dysfunction exists in patients with pancreatitis of unknown etiology, even though age and associated medical conditions do not differ . the application of the total enteral nutrition in the burns disease has minimized the complication rate and consequently increased the survival rate of children and adults. time of initiation, composition, duration and way of administration are very important in obtaining the optimum beneficial effect from the treatment and diminishing the complication rate and side effects. the above features will be discussed in view of our experience in 240 cases. ta buckle?,, ra freebalm, c gomersall g joynt, r young. tg short. department of anaesthesia and intensive cm+e, prince of wales hospital. the chinese university of hong kong, shatin, hong kong introduction: gastric mucosal ph (phi) monitoring has been proposed as a relatively noninvasive index of the adequacy of aerobic metabolism in the gut. to examine the accuracy of gastric intramucosal pit measurements as a function of time and as a function of the catheter itself to determine whether the measurement error between catheters is clinically acceptable. patients with a gastric tonometer (trip tm, tonometrics, worcester. ma) insitu for >3 days were studied. following informed consent two new tonometers were inserted equidistantly & correct position was confirmed radiographically. measurements of intramucosal gastric ph were then performed over a 36 hr period. eight -ten measurements were made in each of ten critically ill patients.percent differences between the two new catheters were 1.6% ie at ph 7.3 _+0.12 (95% limits) and between old & new catheters were 2.2%, ie ph 7j3 _+0.16 (95% limits). conclusions: the results suggest that the function of the tonometer deteriorates over time and that the absolute values of phi m~ not ~ufficiently accurate. however as a trend monitor phi may be useful in the clinical setting. despite a continuous decline both in li'equency and severity of gastro-intestinal stress-lesion/-bleeding (gisb) due to both improvement in preclinical support and in intensive care medicine, patients with cerebral lesion are still considered at high risk for developing gis8. therefore the question arises, whether m> specific (}lsb-prophylaxis besides general and neurological intensive care, specific pharlnaeothcrapy or even the combination of two specific drugs reveals any protective efli~ct on frequency and severity of gisb.this pntspcclive randomized study has been perfornted in 173 patients snfrering t'rttna head-injury/cerebral lesion and with a glasgow-coma-scale on admission (gcs:,)of <9. according to randomization the patients have been grouped as tbllows: h analgesia/sedation (n=37); ih analgesiajsedation plus pirenzepine 60 mg/day (n=54); .[ih anatgcsia/sedalkm plus sncraltate 6 x [ g/day (n=47); iv: analgesidsedatkm plus pirenzcpine 60 mghlay plus sucralfate 6 x 1 e/day (n=35). slalislical analysis has been performed by chl:*tt~sl. rank correlatinn and unpaired t-test; statistical significance has been set with p <0.05. 28/173 patients (16.2 %) developed gisb. although the mean gcs~-value (x -+ sd) did not reach significance between patients with and without gisb (5.61 + 1.65 vs 6.12 -+ 1.65). a significant inverse correlation between gcs:, and the incidence of gtsb (rs~ = 0.89) has been shown. the frequency of gisb among the groups is as follows: h 18.9 %; lh 18.5 %; llh 17.0 %; iv: 8.6 % (ch1 -~ =1.94; not signilicant). no gisb-induced blood translusion or mortality, respectively, could be demonstrated. survival rate between the groups did not differ significantly (chi-" =5.86; p=0.1186) and reached an overall-value of 75.1%.drug-specific glsb-prophylaxis -administered either as monotherapy (pirenzepine, sueralfate) or in combination of these two specific-drugs -reveals no additional significant influence on the incidence of gisb in patients with cerebral lesion compared to no specific prophylaxis besides the general trauma-/disease-specific intensive care measures. critical care dpt, evangelismos hospital, athens university scho~" of medicine objectives: the correlation of longterm presence of nasogastric tube (ngt) to gastroesophageal reflux (ger) is still in question. in case of positive correlation, peg should represent an alternative to tube feeding in patients unable to be fed orally. therefore, we investigated: i) the correlation between ng and ger and ii) the effect of peg on ger. methods: a 24-h esophageal ph-metry was performed in 40 patients in recumbent position at 30 ~ who had a ngt for more than 10 days and were on sucralfate for gastric mucosal protection. the tip of the ph-probe was lied 5 cm over the esophagogasttie junction, confirmed by x-rays. patients who presented a percentage of ger-total (i.e. with a ph less or more than 4) (ger-t) more than 3%, underwent ~t peg. the presence of a creseent-notch on the esophagogastric junction persisting on inspiration and the grade os endoseopic and histologic esophagitis (scale=0-3) was noted. two ph-metrles repeated on 48 h and on 7 days post-peg were compared to the pre-peg one, with the followin~ parameters taken in consideration: i) % ger-t, ii) number of ger-total per hour (no/h ger-t) and iii) the duration that ph was less than 4 (tph<4). in case ot ger persistence at the ph-metry on ?th day post-peg (group ii) another endoscopy was performed, while patients with reduced ger (group i) were considered as esophagifis-free.results: 23 out of 40 patients presented a ger-t>3%. eleven out of 23 group i group (n=6) 1i ( objectives: the aim of the present study was to compare the performance of a specially modified version of a photo-and magnetoacoustic (pa/ma) gas analyzer (br~)el & kjaer, denmark) with a conventional quadrupole mass spectrometer (ms) (innovision, denmark) in inert gas rebreathing (rb) tests such as determination of functional residual capacity (frc), pulmonary capillary blood flow (pcbf) and lung tissue volume (vtc). methods : from simultaneous readings of inert gas concentrations with the ms and the pa/ma analyzer during rb experiments a comparison was made of the pcbf, vtc and frc values. the rb tests were performed during rest and exercise (0,50 and 100 w) in ten healthy subjects. results: the differences (mean +/-sd) between simultaneous estimates of rebreathing parameters were the following (pa/ma -ms) for pooled data, pcbf: 0.18 +/-0.38 i/min, vtc: -33 +/-108 ml and frc: 0.028 +/-0.048 liters. conclusions: smell but significant differences were found between the estimates of pcbf, vtc and frc using the ms and pa/ma, respectively. reference: p. clemensen, p. christensen, p. norsk, and j. gr~nlund. a modified photo-and magnetoacoustic multigas analyzer aplied in gas exchange measurements. j appl physiol 1994; 76: 2832-2839. objectives: because transcranial doppler (tcd) has been proposed to explore cerebral co 2 vasoreactivity in brain injury (stroke 1992;23:962-6), we compared this technique with the kety-schmidt reference method to assess cerebral vasoreactivity in comatose patients. methods: 17 mechanically ventilated patients (age 30-81 yrs, glasgow 3-10) in coma due to acute brain injury were investigated during stepwise changes in paco 2 (25, 30, 35, and 40 mmhg) by increasing inspired pco 2. middle cerebral artery velocity (vm) was measured by tcd. after insertion of a catheter in the ipsilateral jugular bulb, cerebral blood flow (cbf) was determined by the kety-schmidt method, using the inhalation of 10% n20 through the inspiratory line of the ventilator. for each patient a cerebral co~ vasoreactivity index was calculated as the slope of linear relationship between vm or cbf and paco2. objectives: after cardiac surgery the fluid shill, between interstitial and intravasal space may be marked. this is due either to the intraoperative volume loading by the extracorporeal circulation or the increased postoperative diuresis. therefore, infusion of a large amount &fluids is necessary during the first postoperative hours. it still remains unclear which of the substances at disposal is the best for this purpose. aim of the present study was to compare the different fluids with special regard to postoperative bleeding and rheological behaviour. methods: 93 patients undergoing cabg-surgery were investigated and randomizedly distributed to three different groups of postoperative volume replacement to stabilize the mean arterial pressure at 80 mm hg. 1. ringer's solution, 2. 3.5% gelatine solution, 3. 6% hydroxyaethylstarch (mean m.w. 70.000). we evaluated the following parameters within intervals of 30 min: arterial and central venous pressure, heart rate, postoperative bleeding, urinary output, volume replacement. results: there was no statistically significant difference between the groups with regard to urinary output and bleeding. in spite of larger amounts of fluids necessary in the ringer treated group patients of this group showed symptoms of hypovolemia. hematocrit was increased in the ringer patients. this was statistically significant. introduction: pulmonary wedge pressure (pcwp) and central venous pressure (cvp) are frequently used as parameters for cardiac preload, although it is known that both are poorly correlated to the cardiac index (ci). it has been claimed that intrathoracic blood volume (itbv) measured with the thermal dye dilution method reflects cardiac preload better than pcwp and cvp. we studied the correlation between itbv and ci in a mixed population of critically ill patients. methods: in 17 consecutive patients (6 sepsis/sirs, 2 acute heart failure, 3 ards, 6 transjugular intrahepatic portosystemic shunt) monitored with a pulmonary artery catheter, itbv was measured on regular intervals using the pulsion cold z-021 system (pulsion, munich, germany). ci, pcwp, and cvp were recorded simultaneously. results: a total of 1ol measurements was made. pcwp and cvp did not correlate to ci, nor did apcwp or acvp correlate to aci. itbv was correlated to ci in a non-linear fashion (f -139, df = 99, p < 0.001, (figure) ). aitbv was correlated to ac1 in a linear fashion (r = 0.76, f = 134, df = 99, p < 0.o01). a rapid and efficient circulatory support system may save a patient in cardiogenic shock. left heart bypass with percutaneous and transseptal placement of the aspiration canuia simplifies the circuit and avoids the need for an oxygenator. we assessed this preclinical set-up in 5 anaesthetized pigs using a centrifugal pump with a 14 f arterial catheter and a 16 f left atrial aspiration line. animals were supported for two hours at a mean flow of 3.1 liter (3'680 rpm), a mean hematocrit of 29% and low heparinisetion (act double baseline). hemodynamic and laboratory samples were taken at baseline (a), 10 minutes (b), one hour ( pulmonary hypertension (ph) usually involves obliteration and loss of functional pulmonary microvasculature. the microvaseular endothelium normally acts as a major metabolic organ, converting angiotensin i to angiotensin ii via the angiotensin-converting ectoenzyme (ace). it is unknown whether the loss of functional vasculature and altered pulmonary blood flow seen in ph will affect lung ace metabolic activity. we therefore estimated pulmonary vascular ace activity in 9 patients with ph of various causes: 2 primary; 1 post atrial septal defect closure (asd); 2 chronic thromboembolic (te); 1 anorexigen; 1 iv drugs; 2 collagen disease. single-pass transpulmonary hydrolysis of the specific ace substrate 3h-benzoyl-pbe-ala-pro (bpap) was measured and expressed as % metabolism (%me0. we also calculated an index of peffused functional capillary surface area (amax/km). all patients with ph had an abnormality of %met or amax/km, or both. as compared to 9 control humans (mean %met = 71.4% _+ 11.3% s.d.), the mean %met in ph patients was 54.3% _+ 14%. the %met in ph patients correlated inversely with cardiac output (r=0.74), possibly reflecting more complete bpap hydrolysis with longer pulmonary transit times. amax/km was markedly decreased in ph (1663 + 536 ml/min) as compared to controls (4225 _+ 1018 ml]min), consistent with a significant loss of functional capillary surface area. patients with collagen disease, asd and anorexigen-induced ph had the most marked abnormalities. in conclusion, patients with pulmonary hypertension have decreased pulmonary endothelial angiotensin converting enzyme activity, likely due to a loss of functional or perfused pulmonary microvaseulature. supported by the funds de la recherche en same du quebec and the national health system of greece. objective: to investigate adrenocortical function in patients with ruptured aneurysm of the abdominal aorta (raaa). studies investigating adrenocortical insufficiency in critically ill patients report an incidence ranging from 20% to less than 1%. this may in part be explained by difference in methods used (single cortisol measurement vs short acth stimulation test) and populations studied (heterogenous groups of patients with great individual variation in underlying disease as well as duration and severity of illness). methods: we investigated the adrenocortical function in 32 patients with (raaa).a short acth stimulation test (synacthen test; 250 ug 1-24 acth iv) was performed at 0800 hrs within 24 hrs of admission. plasma cortisol was measured before (cort basal) and after stimulation (cort stim). a plasma cortisol level > 0.55 umol\l before or after stimulation was considered normal, severity of illness was assessed using apache ii. results: of the 32 patients investigated 6 died and 26 survived. mean cort basal in nonsurvivors was significantly (p<0.o04) higher than in survivors; 1.03 (range 0.72-1.29) vs 0.69 (range 0.24-1,14). this difference between nonsurvivors and survivors was also present for cort stim but lacked significance; 1.30 (range 0.96-2.25) vs 1.00 (range 0.57-1.53). while 8 patients showed a cort basal < 0.55, no cort stim <0.55 was found. there was no significant difference in mean age or apache ii score between survivors and nonsurvivors; 70 vs75 and 19 vs 21. conclusions: single plasma cortisol levels were inadequate to assess the adrenocortical function in the patients studied, judged by a short acth stimulation test, our investigation in patients with raaa showed no adrenocortical insufficiency. mortality in raaa is associated with elevated plasma cortisol levels. obiectives: mortality in acute myocardial infarction (ami) prinicipally depends on hemedynamic impairment. thus, patients (pts) with elevated pulmonary wedge pressure (pwp) present high in-hospital mortality. however, the complete right heart catheterization is laborious, so the central venous pressure (cvp) alone is frequently used to assess the severity of ami. the accuracy of cvp in estimating pts with ami was tested in this retrospective study. methods: 131 pts. aged 68+14 years, admitted to our ccu from 1992 to 1994 with their first ami, were inctuded in this study. all had undergone right heart catheterization because of overt or suspected heart failure. swan-ganz catheters (7f, 85cm, abbott, il, usa) had been used, every treatment had been temporarily interrupted l h before the calheferization. based on ecg findings the pts were retrospectively divided into 3 groups. in group a we included 54 pts with anterior ami, in group b, 30 pts with inferior ami, and in group c, 47 pts with inferior and right ventricular ami. the initial values of cvp and pwp were considered for the linear regression of the pwp variable on cvp and p<0.05 was accepted as statistically significant.results: in g~oup a, the cvp and pwp vaiues were 8+3 mmhg and 14_+7 mmhg respectively. despite the signifanf correlation (p<0.01) between the two variables, it was not possible fo predict the exact value of pwp based on cvp value, 19 pts (35%) presented cvp>8 mrnhg and 16 of these (84%) had pwp_>15mmhg. in group 3, the cvp was 11_+8 mmhg and the pwp, 13_+6 mmhg. significant correlation (p<0.05) between the two variables also existed, however it was impossible to predict the pwp value. 6 pts (20%) had cvp>8 mmhg but only 2 of these (33%) had pwp>15 mmhg, similar was the relation between cvp and pwp in group c (p<0.01). cvp averaged 12+6 mmhg, and pwp, 17_+8 mmhg. 33 pts (70%) had cvp>8 mmhg and 25 from these (76%) presented pwp>15 mmhg,conclusions: a single measurement of cvp in ami does not ensure an accurate assessment of pwp. because every pt with ami needs optimal values of pwp in order to prevent pulmonary congestion or manifestations of low preload, the significance of complete right heart catheterization becomes apparent. in patients (pts) with advanced hf the need and the prognosis for heart transplantation (ht) can be predicted from vo= max. indirect measure of functional capacity with the six-minute walk test can also predict smvival in moderate hf. to predict vos max from indirect astinmtions of functional capadty such as 6-1~q~/, pulmonary and heart function tests, and to assess the prediddve value of the above parameters in hf pts survival. we evaluated 35 pts (age 48+12 yeats nyha class: 12 ii, 15 hi, 8 iv) with hf for pit. they underwent a pmgmmive exercise test on cycle ergometer for vo2 max determination, a 6-mw, a right heart catheterization and a spirometry and dlco estimation. introduction: brain death causes myocardial impairment by mechanisms that are not well understood yet. the aim of this work was to assess the echocardiographic features found in these patients from the clinical onset of brain death to somatic death, methods: seven brain dead patients were studied (patients" relatives refused to allow them to be used as donors). mean age was 23.5 (18-32) years old. four of the patients were female, none of the patients had any history of cardiac disease. transthoracic echocardiogram (echo) and electrocardiogram (ecg) were obtained at the onset of clinical brain death and were repeated every 24 hours until somatic death. we we detected severe diffuse hypokinesia (ef<50%) in 2 patients and mild hypokinesia in 3 others (ef 50-60%). systolic function was strictly normal in only 2 patients. corrected qt interval (qtc) in ecg was 54.6_+5.5 msec (normal range 38-43 msec) just before somatic death (b). conclusion: in patients with brain death we observed a significant increase of left ventricular mass due mainly to ivs "hypertrophy" without any important change in the dimensions of the left ventricle. to our knowledge, this finding has never been reported before and its importantance in heart transplantations may be of particular interest. predict right ventricular outcome. l. jacquet, r. dion, p. noirhomme. m. van dijck. m. goenen cardiothoracic intensive care unit, st-luc univ. hospital(ucl) we have registred: heart rate (hr), blood pressure (bp), pulmonary artery pressures (pap), central venous pressure (cvp), pulmonary capillary wedge pressure (pcwp), pulmonary and systemic vascular resistances (pvr, svr), right ventricle end-diastolic end end-systolic volume (redv, resv), right ejection fraction (ref), right sistolyc ventricular work (rsvw) and cardiac output (co) using a thermodilution thechnique and a microprocessor (model ref-1; baxter-edwards laboratory); duration of cpb and aortic clamping, and the requirements of haemodynamic support after cpb.results: in the c group an increase post-cpb of the fc (62 + 13 95.8 + 18.4, p < 0.01) was produced without significantly changes in the redv, resv, ref, rsvw neither co. in the w group, hr increased from 58.5 + 8.8 to 79.9 + 8.6 (p < 0.01); redv was reduced from 227.7 -+ 76 to 139.14 _+ 36.4 (p < 0.05); resv was reduced from 142 • 68.7 to 82 + 35.3 (p < 0.05). there were not changes in the other haemodynamyc parameters. there was a trend (no significantly) to an increase of ref in the w group (40.07 + 9.7|• 4.7) compared with the c"group (39 • 10.9($ 38.3 • 8.5) post-cpb. the need for haemodynamic support was similar in both groups.conclusions: the warm, continuous, anterograde-retrogade myocardial protection has obtained a decrease of preload, hr, and a trend to an increase in the ref, making an improvement in the right ventricular global performance when is compared with the classic form of cold myocardial protection. objective: to evaluate the effect of dobutamine on gastric mucosal ph (phi) after coronaly artery bypass surgery. design: prospective study in a university hospital intensive care unit (icu). subjects: 20 elective cardiac surgery patients. interventions: dobutamine was infused at 4 ug/kg/min for 3 hours immediately after admission to the icu. hemodynamics were measured every 30 minute periods until 3 hours and again 2 hours after stopping dobutamine. results: there were no significant differences in mean gastric phi between the groups but mean phi decreased in both groups during the study period. oxygen delivery and consumption both increased during dobutamine infusion but decreased to the control group level after stopping the dobutamine infusion. lactate levels did not change. baseline 90 objectives: the aim of the study was to evaluate the usefulness of a low dobutamine dose in conjunction with intraaortic balloon pumping and mechanical ventilation in cardiogenic shock. we studied 21 patients 58.9-+ t4.4 years of age suffered of post infarction cardiogenic shock characterized by a systolic arterial pressure< 80mmhg, urine output< 20 ml/h and mental confusion or purpueral signs of low output, non responded to dobutamine infusion up to 9 pg/kg/min. all patients underwent mechanical assistance by the intra-aortic balloon pump (iabp). five patients were additionally placed on mechanical ventilation due to blood gases disturbances. the end points in our study were: reversion of cardiogenic shock, improvement of patients survival or both on the 15th post infarction day and 6 months later. results: three patients refused iabp treatment and 0/3 survived on the 15th day. on the 15th day 3/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p <0.01). on the 6th month 2/13 supported by the iabp and 5/5 that underwent mechanical ventilation plus iabp were alive (p<0.01). conclusions: in conclusion, the combined use of mechanical ventilation and iabp assistance in severe cardiogenic shock might improve survival. obiectives: the study was aimed at analysing predictive factors of swan ganz pulmonary catheter (pc) requiremen t during elective cardiac surgery according to the need of sustained inotropic support after surgery. methods: three hundred patients (aged from 27 to 85; 89 females and 211 males)were consecutively operated on for elective coronary artery bypass surgery (cabg, n=179), valvular replacement (vr, n=98), combination of both (vr-cabg, n=15), or others (n=6) and retrospectively included in the study. each patient had preoperative invasive cardiac investigation with calculated ejection fraction (ee). anaesthesia, cardiopulmonary bypass (cpb) and cardiac arrest managements were similar in all patients. pc requirement was estimated from the need of either dobutamine, adrenaline, dopamine or enoximone use during the first 48 hours after cardiac surgery. demographic data, asa and nyha classifications, preoperative ef and treatments, type of surgery, cpb and aortic cross clamping (axc) times, and postoperative incidence of complications were compared in patients with or without inotropic support using either student's t test or x2 with continuity correction when appropriate. results: seventy4hree patients (24.5%) required inotropic support after surgery. axc .and cpb times, mean stay in icu were significantly longer in patients with inotropie support (p<0.001). type of surgery, preoperative ef, and nyha classification are the first 3 significant factors related to inotropic support (p<0.005). most patients operated on for double-vr or vr=cabg required inotropic support (57 and 60%, respectively). postoperative mortality was higher in patients receiving inotropic support (8,2% vs 0,9% 'overall mortality, p=0.003). conclusions: since pc insertion is most.often justified because inotropes are required, these results suggest that elective rather than routine systemic pc insertion could be helped by considering several but selected preoperative factors. background: cardiovascular depression due to anaesthesia, old age and major gastrointestinal surgery is becoming an increasingly frequent challenge .to the anaesthesia-surgory team. deliberate preoperative manipulation of haemodynamics and oxygen transport parametres towards prede~t~mined optimal values may prove to be effective "in reducing 12 morbidity ~nd mortality in high risk surgical patients,. a new concept of using conlimaous perioperative measurement of cardiac'output to obtain and maintain supranormal oxygen delivery (do2i) is presented. methods: continuous measurement of cardiac output is a relatively new form of on-line monitoring, in which trains of impulses are emitted from a thermal filament mounted on a pulmonary artery catheter. computer software recognizes patterns generated by minute changes in blood temperature and ealoalates cardiac output every 30-60 seconds. cardiac 3 output and mixed venous blood oxygen saturation are displayed graphically on line. in tins tm study cardiac output was measured continuously by vigilance cardiac outpu t compl/ter (baxter). preoperative haemodynamic optimization was performed with the goal of increa-2 1 sing do2i to at least 600 ml/min/m accordfing to shoemaker's algorithm . this was.done by infusing colloids (albumin or hydroxy ethyl starch (haes-steril| until the desired do21 was reached. infusion was stopped if cardiac output ceased to increase with infusion, if there were signs of pulmonary oedema or if wedge pressure reached 18 mmhg. vasoactive or inotropic drugs were infused if the desired do21 was not reached by infusion alone. anaesthetic technique included continuous thoracic epidural and isoflourane anaesthesia. expected mol:bidity and mortality rates were calculated by the "possum" score aasing preoperative clinical and paradinical estimates of organ function as well as surgery characteristics 4. materials: 15 asa group ill-iv patients with a mean age of 75 years (range 60-92) and a mean weight of 67 kg (range 36-93)) scheduled for major abdominal surgery were included. results: 2 patients were excluded because do2i could not be raised at all. mean do2i was increased from 488 ml/min/m 2 (range 384-610) to 688 ml/min/m 2 (range 490-967). mean volume of preoperativdy infused colloid was 954 ml (range 0-2000). during surgery 1230 ml (range 5002300) of colloid was infused. mean length of surgery was 150 minutes (range 60-300). mean blood loss was 920 ml (range 04800). expected mortality and morbidity rates ("possum") were 56% and 92%, respectively, whereas patient follow up upon discharge or at death revealed mortality and morbidity rates of 8 % and 54%, respectively. conclusion: based on experience from the present study, continuous measurement of cardiac output has proved to be a valuable tool for perioperative optimization of do21 in asa group ili and iv patients during major surgery. however further studies including a greater number of patients are necessary to confirm the promising preliminary findings. we studied the hemodyn~c effects of three different combinations of positiv inotropic .agents, vasodilators, diuretics and av-filtration (av) in 16 patients (pts) with severe left heart faille (left veutrieul0x filling pressure (lvfp) >25 mmhg) due to acute myocardial infarction. hemodynamic measurements (intravascular pressures (lvfp), thermodilution (cardiac index (ci)) were made before (control) and after each therapy. in 11 furosemide (f) + d0butamin (d) + nitroglycerin (ni) reduced lvfp and a small increase of ci occurred. in 6 of these pts :(group a) nitroprusside (hip) instead of ni increased ci significantly, in the other 5 pts adding of amrinone (a) resulted in a pronounced increase of ci. group c (n=5): the combination of ni and av reduced lvfp but did not increase ci which was achieved by av+d+ni. in order to optimize the treatment of acute heart failure a combination of inotropic agents, vasodilators, diuretics and av-filtration should he used guided by hemodynamic monitoring. arias jr, miragaya d, sandard, san pedro dm ~, herndndez d, valenzuela 12 . objectives: to evaluate the variation in nomdrenaline (na) plasma concentrations in patients with acute myocardial infarction (am1) after thrombolytic therapy with noniltvasive reperfusion criteria (clinical, electrocardiographic and enzymatic), in relation to infarct size and location.methods: 42 consecutive patiens with ami, from october 1, 1994 to february 28, 1995, admitted within 6 hours alter onset of symptoms, undergone successfull systemic thrombolysis. 21 of them were anterior (group a) and 21 inferior (group b) . noradrenaline plasma levels at 0 (na1), 60 (na2) and 240 (na3) minutes after admission were compared with ck-peak plasma levels by linear regression. differences were tested for significance by student-t-test for paired and unpaired values. na plasma concentration was measured by high-presssure liquid chromatography. p< ns 0.01 ns means 4-sem (normal limit for our laboratory: na < 37/0 pg/ml; ck < 170 u/i ) conclusions: 1. the na plasma levels at admission (nai) are more increased in anterior than inferior amis, probably in relation to infarct size. 2. the decrease in na is more evidence in amis with anterior location. 3. this decrease is probably due to the major efficacy of thrombolytic therapy in amis with anterior location. arias jd, miragaya (group b) , probably due to certain degree of t~cg'rfueion. 3. there is not significant variation in na in conventional treated ami (group c). v.suchanov, a.levit, p.trofimov, icu, regional hospital, ekaterinburg, russiaobjectives: our task was to improve the technique of preservation of platelet rich plasma. methods: 38 patients scheduled for multiple cardiac valve replacement in 1994 were divided into two groups: group i (10 patients) -without pp; group ii (28 patients) -pp was performed preoperatively. the first pp was made ten days and the second -3 days before the operation. prp was preserved by cryoconservation. our technique of cryoconservation is distinguished by the speed of freezing (17-18~ and absence of dmso. this made it possible to preserve 90 % functionally active platelets during 20 days. the prp was transfused back after heparin neutralization. the hospital ethics committee approved the investigation.results: the blood loss through the 1st p. o. d. was significantly greatest in the group i (725 _+ 97 ml) and all the patients required transfusion of the donor blood (480 + 112 ml) whereas the blood loss in group ii was 489 +_ 75 ml and olny 12 patients required the donor blood. the number of platelets on the 1st p.o.d, was 107 _+ 12. 109/l (group i) and 153 + 19. 109/l (group ii), p < 0.05.conclusions: our technique of prp cryoconservation makes it possible to avoid the crystallization phase during freezing of prr thus the infusion of prp may improve hemostasis after open heart surgery and limit the use of the donor blood. in-hospital outcome of women suffering an ami is generally considered worse than that of men, but it is still debated whether female sex is per sea negative prognostic factor or is merely associated with other negative determinants of prognosis. the purpose of the present study is to evaluate the independence of the association between female sex and mortality (in the 567 patients of the swiss centers) and in the 36381 patients randomized in the isis-3 trail mortality rate in women was 14.8% (1421/9600) compared to 9.1% (2417/26480) in men; in switzerland: in-hospital mortality for women was 15.5% (20/129), for men 7.1% (31/438).the table shows the results of isis-3 in terms of odds ratios and their 95% confidence intervals either after unadjusted analysis or after adjustment for age, known to be the major confounding variable when prognosis of women after myocardial infarction is considered, and for all the available clinical and epidemiological characteristics collected at trial entry: these observations suggest that there is a small but independent effect of female sex on short-term mortality after acute myocardial infarction. (27) and bubble (13) oxygenators a, ere used. anaesthesia was balanced and pts were extubated 12 to 18 hrs after cpb. pts were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that time qs/qt was calculate( according to )be standard shunt equation. after the sgc had been removed, an estimated shunt was calculated. measurements of qs/qt were performed: before induction of anaesthesia (1), after induction of anaesthesia (i[), 5 mins after cpb (iii) 2 (iv) and 6 (v) hrs afiter cpb, 30 rains after extubation (vi), 24 hrs after cpb (v[1) and on the 2nd, 3rd, 5th, 8th and 13tb postoperative day (pd) (viii, 1 x, x, xi, xi1, respectively). analysis of data was performed by two-way analysis of variance, p < 0.05 being regard as significant.results: the figure shows the values for qs/qt expressed as means + sd. there was a significant increase in qs/qt above b~setine throughoul the whole investigated period except on the 13th pd. qs/qt reached maximum at 30 rains after extubation (vi). objectives: many stndies have shown advantages of membrane oxygenalors over 9ubbie type oxygenators. the aim of this study was to evaluate the influence of 3x3'genator type on pulmonary shunt (as/at) after coronary surgery. methods: 40 patients (pts) gave their informed consent to the study which was approved by the university ttuman research committee. pts were divided into two groups: a1 (n = 27) with a membrane o~genator and a2 (n = 13) with a bubble oxygenalor used during cardiopulmonary bypass (cpb). ths were monitored with swan-ganz catheters (sgc) for 24 hrs after cpb. at that tfme os/ot was calculated according to the standard shunt equation. alter the sgc had been removed, an estimated shunt was calculated..measurements of os/qt were performed: betore induction of anaesthesia (i), 30 mins after extubation (11), 24 hrs alter cpb (111) and on the 2nd, 3rd, 5th, 8th and 13th postoperative day (iv, v, vi, vii> viii, respectively). analysis of data was performed by one-way analysis of variance, p < 0.05 being regarded as significant.results: the figure shows the values for qs/qt expressed as means _+ sd. os/qt was significantly greater at 30 rains after extubation (ii) in a2 group. the difl'ereuce between the two groups was no more significant from 24 hrs after cpb (iii) to the end of the investigated period. ! i * p < a.0s betw~n ~o~ conclusions: membrane ox3'genation during cpb is accomplished by reduction in blood cellular destruction and less alteration in blood. the results of our study show the influence of oxygenator type on value of qs/ot only after extubation (12 to 18 hrs after cpb). the difference in qs/qt disappeared 24 his after cpb and since that time the oxygenator type had no influence on qs/qt. it may be of particular importance in patients with severe forms of cardiopulmonary disease who are at risk of higher postoperative morbidity and mortality. objectives: hypomagnesemia has been reported with a variable prevalence (20 to 61% ) in icu patients. magnesium deficiency can induce a number of climcal symptoms (primarily cardiovascular and neuropsychiatric) but can also be clinically silent (10-65% are asymptomadc), methods: we measured whole blood ionized magnesium (lmg++) in 74 patients on admission to the icu, using a nova 8 electrolyte analyzer (nova biomedical), containing an img++ electrode. blood was collected in syringes with dry heparin (radiometer qs 50 ). normal range of img++ was found between 0.45-0.55 mmot/l (healthy volunteers). results: for the entire population, we found a 61% prevalence (45/74) of hypomagnesemia (figure 1) . among the surgical patients, the prevalence was highest after cardiac surgery (85%) and after thoracic surgery (80%) and was lowest after neurosurgery (8%). hypomagnesemia was also common in patients after liver transplantation (lvtx) or with hepatic failure (100% for both groups). conclusion: our findings confirm that hypomagnesemia is common in acutely ill patients, especially in those after cardiothoracic surgery or those with liver disease. nevertheless. it is difficult to define the associated factors with sufficient specificity, so that measurements of img++ are warranted to diagnose hypomagnesemia. hepariu influences platelet function and may lead to thrombocytopenia called heparin-associated thrombocytopenia (hat) regardless of the dose and route of administration. additinnal venous and/or arterial thrombosis may lead to life-threatening complications. the incidence of so-calied heparin-associated thrombocytopenia and thrombosis (hatt) ranges between i-5%. hatt is confirmed by a heparin induced platelet activation assay (hipa). results: from 11/93 to 11/94 1146 consecutive patients of our icu were reviewed retrospectively. all patients were treated with heparim the incidence of hatt was 1% (12). in all cases diagnosis was proven by a positive hipa. 2/12 patients died. in 3/12 hatt could be confirmed before severe thromboembolic complications occured. 4/12 patients developed a deep vein thrombosis (dvt), 2/12 dvt and pulmonary embolism (pe), 2/12 dvt, pe and arterial thrombosis (at) and 1/12 a dvt, pe~ at and a sinus thrombosis. conclusion: the incidence of hatt in a r series of 1146 pts. is 1%. presence of thrombocytopenia and thrombosis of the great 'vessels is associated with a significant mortality (2/12). computed tom0graphy (ct) and transthoracic/transesophageal echocardiography (tte/tee) are important tools in diagnosing and monitoring the extent of cenlrai venous and arterial thrombosis. a. cabral md, m. shahla md c. meneses-oliveira md and jl vincenl md.phd. department of intensive care. erasme university hospital, brussels, belgium objective: to determine extreme hemodynanuc patterns in cardiogenic shock. although ~.~xdiogenic shock is characterized by a low cardiac index (ci), high systemic w~,scular resistance index (svri), and high cardiac filling pressures, some patients may develop art atypical pattern. we reviewed the hemodyuamic pattern of 73 patients with cardiogenic shock, as defined by an initial ct below 2.5 l/rain/m: in the presence of myocardial dysfimction attributed to ischemic heart disease (n=26), heart failure (n=7), valvulopathy (n=2) or recent cardiac surgery (n=38). after exclusion of 10 patients with concurrently suspected/documented infection, this study included 63 patients, of whom 23 (36.5%) survived. treatment of shock included dopamine (n=43), dobutamine (n=56), norepinephrine (n=18) and epinephrine (n=23). 39 patients with arterial hypertension (ah) and initially law plasnla renin activity (pra) had been studied. in all patient changes of arterial pressure (ap) after single administration of enap was studied. nypotensive reaction wiht deereasin e of average ap about 20-25 mm hg ayter single drug administration observed only in 4 patients. ezap monotherapy accomplished during one week with 20 mg daily dose. hypotensive effect observed in 5 patients including ones which were susceptible to single enap administration. after that first stage of therapy all patints began to combinate enap with hypothyazid in dose of 25 mg per day~ after week of treatment such drugs combination lead to veritable ap lowering in 3 addition patients. in the remaining resistant to such drug combination patients was add corinfar in daily dose of 40 mg. this new drug combination permits to lower ap in 23 patients. subsequent discontinuation of enap administration to such patients aid not connected with increasing of again.therefore the most of the patients with ah and law pra(78,7%)did not susceptible to enap therapy and enap and hypothyazid combination. on the contrary-combination of corinfar with hipothyazid was effective in 59% patients with ah and low pra. methods: in 35 patients with cardiogenic shock due to ischemic heart disease (n=26), heart failure (n=7) and valvulopathy (n=2), hemod31aamic data including measures of intravascular pressures, cardiac output and mixed venous gases were collected at regular times intervals, at least 3 times a da?. all measurements were obtamed in a relative steady state and in the absence of severe anemia or hypoxemia. treatment of shock included dobutamine (n=30), dopamine (n=24), norepinephrine (n=i2) and epinephrine (n=7 objective: based on our previous studies of the function of isolated liver grafts, this experimental protocol aims at developing a novel extracorporeal liver support circuit, with an incorporated pig liver. methods:the graft liver was obtained from pigs weighing 15-20 kg. under general anesthesia the aqimals underwent total hepatectomy,following cannulation of the portal vein, the infrarenal aorta and the infrahapatic vena cava and peffusion wit h 4 it of heparinised r/l solution at 4~ the circuit consisted of the graft liver connected to a fluid reservoir and a centrifuge pump. ten healthy pigs weighing 30-35 kgr were connected to the circuit as follows: the rt carotid artery was connected to the portal vein of the graft and the rt jugular vein was connected to the fluid reservoir, through the centrifuge pump. the fluid reservoir collected the outflow from the graft's suprahepatic inferior vena cava. the cystic duct of the graft was ligated and the bile.duct cannulated for bile collection and measurement. bridges were adapted to the circuit to bypass the graft liver when necessary, in cases of by pass blood perfusing the graft was oxygenated through a bubble oxygenator. mean total priming volume of the circuit was 600 ml. temperature was maintained at 38~ and portal vein pressure at 16 (12-20) mmhg. the flow was 0.5-0.7 ml/gr of graft liver mass per minute. observation period was 8 hours (t8). results: results of the hemadynamic and metabolic monitoring of the recipients [map (t0=124mmhg , t8=118mmhg), hr (t0=177, t8=201), rap (t0=11mmhg , t8=19mmhg), pap (t0=26mmhg, t8=31mmhg), pcwp (t0=14mmhg, t8=15~mhg), svr (t0=1940dyn'sec/cm '5, t8=2190dyn'seclcm~ pvr (t0=206dyn.sec/cm o, t8= 354 dyn.sec/cm ,'~), co (t0=4.631t/min, t8=3.61t/min), do 2 (t0=662ml/min, t8=261.6 ml/min), vo 2 (t0=118ml/min, t8=111ml/min), o2er (t0=17.8%, t8=42.5% ), ph (to= 7.48, t8=7.39 ), po 2 (t0=292mmhg, t8=371mmhg), pco 2 (t0=28mmhg, t8=30 mmhg), pvo 2 (t0=47mmhg, t8=36mmhg), svo 2 (t0=84%, t8=66%), be, na, k, ca ++, lactate, osmolality, ast, alt, pt, aptt, revealed hemodynamic and metabolic stability of the animal. 02 consumption, co 2 production and tissue oxygenation of the graft were also studied. conclusion; the described circuit proved to be safe and well tolerated by healthy animals but its value for temporary liver support is currently being estimated, in a surgically induced experimental fulminant hepatic failure modal. introduction: prosthetic materials like silikone, dacron, teflon e.tc. produce auto immune responses and may even trigger clinical syndromes like scleroderma, sjogren, sle el.c. in our study we followed the evolution of humorial immunity parametrs for up to five years in a cohort of paced pts with implanted metallic and silicone materials. method: 24 paced pts (mean age 55+-13 yrs) without clinical or laboratory findings of malignancy or immune disorders were included. we measured the immunoglobulins, the complement, the auto antibodies and the proteins involved in inflammatory reactions every 6 months. the initial and final mean values are shown in the obiectives: hsp, a systemic leucocytoclastic vasculitis and anaphylactoid purpura can be accompanied by abdominal pain and life-threatening intestinal bleeding. recently we could disclose, that these patients develop severe fxiii-deficiency and immense haemorrhagic oedema of the intestinal wall. by the following case report we will demonstrate and discuss the importance of fxiiideficiency for pathogenesis, therapy and outcome in hsp. case report: a 41 year old man developed typical skin manifestations of hsp following an episode of severe (biliary ?) pancreatitis and percutaneous draining of a pancreatic pseudocyst. two days later he had a paralytic "ileus with immense hemorrhagic wall-oedema and massive dilatation of the small bowel. he got fever up to 39.5 ~ and developed severe gastrointestinal haemorrhage (blood transfusions necessary). the coagulation data disclosed a severe fxhi-deficiency (activity 34%), whereas quickvalues, platelet count and atiii-level were found to be within the normal range. elastase was markedly elevated. substitution of fxiii to normal levels leeds to the cessation of bleeding symptoms and abdominal pain, later resulting in a restitutio ad integrum. conclusions: hsp with intestinal involvement is a life-threatening vasculitis, in which careful and frequent examinations of the coagulation system, especially of fxiii are necessary. detailed analysis of the coagulation data suggest, that the severe fxiiideficiency is due to a specific degradation by proteolytic enzymes (like elastase) as well as consumption within the immense haemorrhagic oedema of the intestinal wall. knowing these facts, even most severe cases of hsp with intestinal involvement can be successfully treated by substitution of fxih. a 49-year-old woman presented a 3 year history of occasional self-limited episodes of weakness, generalized edema and o!!~aria. the immunologic testing showed no~nnai levels of complements, clq inhibitor, and serum chemistry values, between or during a attack, she was not treated. she was a~mitted to the hospital with symptoms including nausea, vomiting, weakness and ol!guria. on examination, the patient presented facial and g~neralized edema. the systolic blood pressure was 60 mm hg, pulse 140 beats/mir~ute, hematocrit 0.59, seln~n protein 46 9/i, and se~um albumin 23 q/l. an leg-kappa pa[apfotein was demostrated (7.82 g/l) and urine was neaative for puotein. c~'stalloid and colloid don't increased the blaod pressure but resulted in anasarca, with a total of ii lit[as of in~ravenous fluids. therapy wink flozen plasma, 1.000 units of clq inhibitor, cortlcosteroids, annihistwnines and antifibrinolytic agents was uns~iccessfull. the a~minist~ation of dopamine, norepineph~ne and epinephrine was inefective. the patient died at the 48 bores, only a few cases have been reported, all had igg paraprotein, the pathophysio!o~] is urd~no~n% but is possible that the paraprotein may be zesponsib!e for the increased capillary pe~leabilityo despite efforts to res~scinate the patients during an acute attack, the syndrome is often fatal. the variable course of systemic uapiliary leak syndrome and the unpredictability and self-limited nature of attacks cloud assessment of therapeutic inte~-vention. the purpose of the present work is to provide some information about the nursing care and results from our experience in continous arteriovenus hemofiltration (cavh).cavh is an extracorporeal technique, especially applicable in the critically ill patients, for disturbances, and for the control of azotemia.we used this method in 30 critically ill patients 16 men and 14 women ages from 32-74 who had sepsis -arf 10 congestive heart failure 8 postoperative multiple organ failure 8 and polytrauma 4.this method was applied to these patients from 24 to 168 hours. 20 % of the patients recovered completely their kidney function, 50 % improved their kidney function and 30 % died.we concluded therefore that this method was very effective for the critically ill patients to whom it was applied, but it requires excellent and continuous nursing care; under the above mentioned circumstances the method works effectivelly. an animal model with rats undergoing a dialysis procedure was designed to test the hypothesis that recovery from ischemic acute renal failure (airf) may be affected by the type of membrane used in hemodialysis. male sprague dawley rats were allocated to 2 groups: in group i, (n=48) airf was inducted by bilateral renal artery clamping for 60 rain. group h (n=48) rats underwent a sham procedure. in each group, rats were dialyzed twice (4th and 8th day) with either a cuprophan (cupro), a hemophan (hemo) or a pan (an69) minidialyscr or stayed nondialyzed (no hi)). renal function was monitored daily by measuring urea and creatinine values and by two single shot inulin clearances on the days following dialysis. additionally hemolytical activity of complement was determined. inulin clearance on day 5 was reduced significantly but there was no difference in the degree of decrement in glomular filtration rate (gfr) between dialyzed and undialyzed rats, nor between the dialyzed animals with different membranes (gfr: no hi): 0.78_+0.54; cupro: 0.84_+0.9; hemo: 0.82_+0.28; an69: 0.77_+0.31). the evaluation of renal function by day nine revealed significant recovery for all airf-groups compared to day 5 (p<0.001), irrespective of wether they underwent dialysis or not, or the type of dialysis membrane. complement activation could be detected in all dialyzed groups but no statistical differences between the animal groups dialyzed with different membranes were noticed. our findings refute the hypothesis that in airf exposure to complement-activating cellulosic membranes impairs the recovery of renal function in rats. changes patients: 150 patients who underwent first cadaver kidney transplantation in our unit between january and december in 1994 were involved. the recipients were divided into 3 groups: group i." non functioning graft (n=27); group ii: delayed graft function (n=59), group ili: good graft function (n=64). the grouping criteria were: a/haemodialysis in the fii~t 5 postoperative days, b/diuresis in the i st postoperative day, c,' scram crcatininc difference between the 1st postoperative day and the preoperative level. all of the parameters were involved into the exarainatio, which we measllre in our every, day practice. results: the preoperative haematocrit level differed significantly between group i. (0.36) and croup ii. and iii. (0.31 and 0.30, p< 0.05). intmo!0emtive significant differences were found between the different groups in systolic blood pressure (group i. 110 hgrmn, group ii. 140 hgnnn, group iii. 165 hgmm, p<0.05), mean arterial pressure (group i. 66 hgmm, vs. group ii. 83 hgnun p<0.05, vs. group iii. 116 hgmm p<0.001), and pulse-amplitude and rate-pressure product too. the second warm ishaemic time in group iii. was significantly shorter than in the other two groups (group iii. 39 inin. vs. group ii. 43 rain. p< 0.05, vs. group i. 49 rain. p< 0.00!). the rejection rate was higher in the first 5 days in the patients with non-functioning grafts (group i. 53% and group ii. 24% vs. group iii. 12 %) . the other examined parameters have not differed significantly. conclusion: according to our results the success of the kidney transplantation is mnitifactorial. the most important factors of this relationship are: the perioperative fluid-balance, the maintenance of adequate perfusion blood pressure during the operation, good surgical technique and immunological problems. key: cord-022888-dnsdg04n authors: nan title: poster sessions date: 2009-08-19 journal: eur j immunol doi: 10.1002/eji.200990224 sha: doc_id: 22888 cord_uid: dnsdg04n no abtract the humoral pattern recognition receptors of innate immunity include collectins, ficolins and pentraxins. ptx3, the prototype of long pentraxins, plays a nonredundant role in resistance against a. fumigatus lung infection. the model proposed suggests that upon binding, ptx3 facilitates recognition, phagocitosis and killing of a. fumigatus conidia by alveolar macrophages, dendritic cells and neutrophils and the subsequent development of a properly th1-oriented adaptive response. actually, ptx3-deficient mice are highly susceptible to aspergillosis and develop th2 skewed responses; moreover, ptx3-deficient resident macrophages and neutrophils show defective conidia phagocytosis. both in vitro and in vivo defects can be rescued by the administration of recombinant ptx3, which does not show direct activity on fungal cells. finally, ptx3 alone or in combination with antifungal agents, induces a curative response in mice with aspergillosis, even when given prophylactically. in the present study, we investigated the mechanisms underlying the ptx3-mediated opsonic activity and the involvement of complement, complement receptors and fcg receptors, by in vitro studies and genetic approaches in vivo. in vitro ptx3 amplified the complement-dependent effects on a. fumigatus conidia phagocytosis by human neutrophils, activated through the alternative pathway. accordingly, in the presence of ptx3-opsonised conidia, cd11b activation, internalization, recruitment to the phagocytic cup and cd11b-dependent phagocytosis were increased. as pentraxins interact with fcgreceptors, which in turn can control cd11b activation, the phagocytic assay was performed in the presence of fcgr blocking abs. data obtained strongly suggest that upon conidia opsonisation with ptx3, fcgriia/cd32 mediates inside-out activation of cd11b and consequently phagocytosis of c3b-opsonised conidia. in vivo phagocytosis experiments performed with c1q-and fc common gamma chain-deficient mice and complement inhibitors support in vitro data. these data confirm and extend the paradigm of cooperation among innate receptors, in particular among the humoral arm of innate immunity (complement, ptx3) and the cellular arm (fcgrs, cr3). moreover, they confirm previous studies on the interaction between pentraxins and fcgrs and support the idea that pentraxins behave as predecessors of antibodies. innate immunity is the first line of defence against pathogens and plays a key role in the initiation, activation and orientation of adaptive immunity. the humoral arm of the innate immunity includes soluble pattern-recognition receptors (prrs) such as collectins, ficolins, complement components and pentraxins. the prototypic long pentraxin ptx3 is rapidly produced and released by diverse cell types in response to proinflammatory signals. ptx3 binds selected microorganisms such as aspergillus fumigatus and restores protective immunity against this pathogen in ptx3-/-mice. neonates have an immature innate immune system and are more susceptible to bacterial infection than older children or adult. a beneficial effect of breast feeding on newborn health is highly demonstrated. this protective effect is mediated by nutrients, immunomodulatory mediators (ifn-g, tnfa, or tgf-b), innate immunity factors (soluble cd14, immunoglobulins, lactoferrin), and leukocytes contained in milk that can penetrate the newborn circulation. we thus hypothesized that milk may contain ptx3. we found high concentration of ptx3 in human colostrum (47.62 ± 13.5 ng/ml at day 1 post-delivery) compare to the one found in human serum ( x 2 ng/ml). the presence of ptx3 in human colostrum seems to be due to the secretion of ptx3 by human mammary gland since we report the production of ptx3 by these cells. this prr is also found in human milk cells (hmc), mainly in leukocytes, and penetrate into newborn tissus after suckling. furthermore, human colostrum upregulated the ptx3 production by adult and neonate immunocompetent cells and we demonstrate that neonate mice present a deficit in their ptx3 production after lps injection. collectively, these data demonstrate that newborn have three distinct ways of ptx3 supplying by breast feeding: (i) soluble ptx3 in colostrum (ii) hmc that can secrete ptx3 upon stimulation in the specific tissue, (iii) an increase of ptx3 production by immune cells in the presence of colostrum. thus, soluble or cell-derived ptx3 may participate to the beneficial role of breast feeding on the newborn health. a. m. baru 1 , j. stephani 2 , h. wagner 2 , t. sparwasser 1 1 twincore, institute for infection immunology, hannover, germany, 2 technical university of munich, institute for medical microbiology, immunology and hygiene, munich, germany toll-like receptors (tlrs) represent the best characterized pattern recognition receptor family in mammalian species. the family currently comprise of 10 receptors in humans (tlr 1-10) and 12 in mice (tlr 1-9, 11-13). as transmembrane receptors, tlrs are expressed on the cell surface (tlr 1, 2, 4, 5, 6, (10) (11) (12) (13) and at endosomal membranes (tlr 3, 7, 8 and 9) . toll-like receptors recognize specific patterns of microbial components and regulate the activation of both innate and adaptive immunity. bacterial dna has been shown to possess immunomodulatory activity about a decade prior to the identification of cpg motifs. about 5 years later to this, toll-like receptor 9 (tlr9) was identified and shown to be the receptor for unmethylated cpg dna which is present mainly in non-vertebrate genome. studies have defined potential role of tlr9 as adjuvant enhancing protective immune responses against tumours and infectious diseases in murine models. although promising results are obtained from a few human clinical trials, overall efficacy and safety could not yet be translated entirely from murine studies to human trials. one explanation for these discrepancies could be the fact that expression of human-tlr9 (hutlr9) is restricted to b-cells and plasmacytoid dendritic cells (pdcs) whereas murine-tlr9 (mutlr9) is also expressed on conventional dendritic cells (cdcs). consequently, tlr9 ligands induce distinct cytokine profiles in mice and human thereby probably regulating immune responses in a different manner. by employing bacterial artificial chromosome (bac) technology, we generated transgenic mice with hutlr9 (henceforth called as hut9 mouse) integration in their genome under human epigenetic control. to avoid effects seen due to overlapping ligand specificities, we crossed this mouse onto mutlr9 knock-out background. we expect that hut9-mutlr -/mice mimic the human specific expression pattern of tlr9, i. e. exclusively in b-cells and pdcs, allowing us to investigate detailed in vivo functions of hutlr9. by studying infection and tumour models as well as models for autoimmunity, allergy and transplantation we could then define appropriate and safe implications for employment of tlr9 ligands in human immunotherapy. the fractal analysis provides unique physical insights into the interactions between c1q and the prp protein. if one may take the liberty to extend this to cellular surfaces, where presumably these reactions are taking place, then one has access to a possible avenue by which one may control these reactions in desired directions. if this is true, then surely, this is worth exploring further. any effort, no matter how small that assists in help providing better insights into these debilitating and neurodegenrative disorders such as alzheimers is defintely worth the effort. interleukin-12 is a heterodimeric cytokine consisting of the two subunits p35 and p40. the main inducers of il-12p40 are microbial components activating toll-like receptors with the magnitude of il-12p40 induction depending on the specific tlr engaged. differential induction of il-12p40 upon tlr stimulation correlated with striking differences in the kinetics of nfkb activation. cpg-dna strongly induces il-12p40 due to its outstanding capacity (i) to induce nucleosomal remodelling in proximal il-12p40 promoter region and (ii) to stimulate prolonged rela activity. here we were interested in further changes in chromatin structure of the il-12p40 promoter upon tlr triggering. we did not observe a change in dna methylation, but using chormatin immunoprecipitation (chip) we were able to detect a strong increase in histone 3 and 4 acetylation in specific regions of the proximal promoter region. acetylation of h4 showed a specific distribution pattern and occured mainly in regulatory elements within the il-12p40 promoter, whereas acetylation of h3 took place over all regions analyzed. tlr tolerance has been reported to be associated with specific chromatin alterations. methylation status of lysine residue 4 on h3 turned out to be important for the inhibition of gene expression upon repeated stimulation. modifying the chromatin structure of gene promoter regions therefore seems to be a sensitive mechanism to modify cytokine expression to exogeneous stimuli in innate immune cells thereby allowing adaption of innate immune responses. a. d. koepruelue 1 , w. ellmeier 1 1 medical university of vienna, institute of immunology/division of immunobiology, vienna, austria macrophages are important in innate and acquired immunity. failures are associated with inflammatory and autoimmune diseases. understanding their stimulation is the basis for therapeutic targeting. members of the tec kinase family (bmx, btk, itk, rlk and tec), expressed in the haematopoietic system, constitute the second largest family of non-receptor tyrosine kinases. mutations in btk represent the source of human x-linked agammaglobulinemia (xla). a mutation in the murine btk gene accounts for a similar syndrome, x-linked immunodeficiency (xid). although the tec family members tec, btk and bmx are expressed in monocytes/macrophages, little is known about their function there. tec kinases become activated upon signaling via divers receptors including antigen receptors, receptor tyrosine kinases or tlrs. several studies in xla or xid macrophages and in monocyte/macrophage cell lines implicated roles for tec kinases in tlr signaling and as well as other macrophage effector functions like phagocytosis. inspired by these findings, we aim to determine the role of tec kinases in bone marrowderived macrophages (bmm), during macrophage activation and in other macrophage functions such as recruitment or phagocytosis. in a comprehensive functional analysis of tlr-mediated bmm activation from mice deficient for one or more of the tec family members in vitro, we reveal which of the kinases play a role in which tlr pathway. based on the results of this analysis, we set the goal to further study how tec kinases regulate the respective signaling cascades. our study will contribute insights into the role of tec kinases in this important cell population of the innate immune system. g. lunazzi 1 , m. buxadé 1 , j. minguillón 1 , r. berga 1 , j. aramburu 1 , c. lópez-rodríguez 1 1 universitat pompeu fabra, department of experimental and health sciences (dcexs), barcelona, spain nfat5 is a transcription factor that regulates the expression of cytokines such as tnfa and lymphotoxin b in response to osmotic stress. in addition, nfat5 participates in multiple processes not linked to the response to hypertonicity. in this regards, it has been recently reported that nfat5 is required as a novel host factor that supports hiv replication in macrophages. given the established connections between nfat5, the expression of certain inflammatory cytokines, and its role in the response to specific pathogens in macrophages, we aimed at studying whether nfat5 could be activated by receptors for pathogens expressed in macrophages. the activation of toll-like receptors (tlrs) is central to innate immunity. upon stimulation of tlrs, cells of the immune system induce signalling pathways that lead to the activation of different transcription factors. as a result of that, cells such as macrophages and dendritic cells induce the expression of genes that participate in the response to pathogens such as those encoding proinflammatory cytokines, antimicrobial products, survival factors or mediators of cellular migration. we have analyzed whether nfat5 is expressed in primary macrophages through the activation of different toll-like receptors. likewise, we have explored whether the activity of nfat5 is induced during the response to tlrs. in addition, we have studied whether the specific inhibition of different signalling pathways positioned downstream of tlrs could interfere with the expression of nfat5. our work indicates that nfat5 is a novel transcriptional regulator acting in response to the activation of tlrs. our work extends the knowledge about mechanisms that participate during the innate immune response to pathogens and offers a new regulatory pathway as a possible target to modulate this response. objectives: compelling evidence support a link between inflammation, cell survival, and cancer, with a central role played by nf-xb, a master switch of inflammation. recent studies implicate some tlrs in tumor development or regression, and immune escape. however, mechanisms leading to tumor growth or apoptosis induced by tlr stimulation are not fully understood. several studies strongly suggest that chronic inflammation in lungs induced by chronic bronchitis, chronic obstructive diseases, emphysema, asbestos or tobacco smoke, increases the risk of carcinogenesis. we hypothesized that some tlrs can contribute to lung inflammation and tumor development in vitro and in vivo. methods: tlr expression in lung cancer was assayed by immunohistochemistry or flow cytometry. nfxb activation was determined by western blot and nuclear translocation assay after tlr stimulation. clonogenicity of stimulated cells was analyzed by colony assay. transcriptomic analysis were performed by taqman lda technology. tumor growth in vivo was analyzed in nod/scid mice after subcutaneously engraftment of human lung tumor cell lines. we have observed that primary human lung tumors express tlr3, tlr4, tlr7 and tlr8 and that stimulation of these receptors in lung tumor cell lines by poly i:c, lps, loxoribine or poly u induces nfxb activation through atypical signaling pathway, with phosphorylation of ixba without its degradation and nuclear translocation of p50 and p65 nfxb subunits. interestingly, we observed that tlr3 stimulation induces apoptosis depending of the histological type of the tumor. on the contrary tlr4, tlr7 and tlr8 stimulation induces cell survival and increases clonogenicity. this is correlated with an up-regulation of bcl-2 expression. moreover, despite a common atypical activation of nfxb, our transcriptomic analysis revealed major differences in gene modulation after triggering of tlr3, tlr4, tlr7 and tlr8. finally, in vivo tlr7 stimulation of human lung tumor cells dramatically increases tumor size and metastasis. conclusions: altogether, these data emphasize that tlr4, tlr7 or tlr8 triggering can directly favor tumor development whereas tlr3 signaling can induce tumor cell death. these data suggest that anticancer immunotherapy using tlr adjuvants should take into account the expression of these tlrs in lung tumor cells. objective: dasatinib (bms-354825) is a small molecule src/abl tyrosine kinase inhibitor approved for the treatment of chronic myeloid leukaemia and philadelphia chromosome-positive acute lymphoblastic leukaemia. members of the src family of kinases are involved in normal physiological processes, and play a significant role in the induction and regulation of innate and adaptive immunity. the purpose of this study was to evaluate the inhibitory action of dasatinib on toll like receptor (tlr) signalling, natural killer (nk) cell cytotoxicity as well as antigen-specific cd8 + and cd4 + t cell function. methods: to analyse tlr signalling in vitro murine bone marrow derived (bmd) macrophages were stimulated with the tlr 4 ligand lipopolysaccaride (lps) in the presence of dasatinib and tumour necrosis factor a (tnf-a) in the culture medium was measured. the response to tlr stimulation was also tested in vivo, dasatinib-treated mice were challenged with lps and tnf-a in the serum was quantified. in addition, the clearance of the rma-s cells, a mhc class i deficient thymoma sensitive to nk cell lysis, was analysed in mice undergoing dasatinib treatment. to investigate the inhibitory effects of dasatinib on adaptive immune responses, transgenic cd4 + and cd8 + t cells specific for ovalbumin were utilised to measure antigen specific t cell proliferation. endogenenous cd4 + and cd8 + t cell responses were determined following immunisation of dasatinib-treated mice with a nonreplicating recombinant virus. results: we show that dasatinib impairs: 1. innate immune response; dasatinib treatment reduced the (a) production of tnf-a following tlr 4 stimulation of bmd macrophages in vitro, (b) production of tnf-a in vivo in response to lps and (c) ability of nk cells to eliminate mhc class i deficient cells in vivo . 2. adaptive immune response; dasatinib treatment inhibited (a) proliferation of antigen-specific murine transgenic t cells, (b) endogenous antigen-specific helper t cell recall-responses and (c) t cell-mediated cytotoxic effector function. conclusions: these findings suggest that dasatinib has the potential to modulate the host immune response and highlights scope for off target applications, for example therapeutic immunosuppression in the context of autoimmune pathogenesis, or in combination with other interventions for the treatment of endotoxic shock. i. zanoni 1 , r. oatuni 1 , m. collini 2 , m. caccia 2 , p. castagnoli 3 , g. chirico 2 , f. granucci 1 1 university of milano-bicocca, biotechnology and bioscience, milan, italy, 2 university of milano-bicocca, physics, milan, italy, 3 singapore immunology network (sign), biomedical sciences institutes, immunos, singapore, singapore the recognition of mamps by tlrs expressed on dendritic cells (dcs) plays an essential role for the regulation of the immune responses. by recruiting different combinations of adapter proteins, individual tlrs turn on signal transduction pathways leading to the activation of different transcription factors. interleukin-2 (il-2) is one of the molecules produced by dcs shortly after stimulation with different tlr agonists. based on this observation and by analogy with the events following t-cell receptor (tcr) engagement leading to il-2 production, we hypothesized that the stimulation of tlrs on dcs might lead to activation of the ca2+/ calcineurin and nfat pathway. we found that dc stimulation with lps induces extracellular ca2+ influxes, leading to calcineurin-dependent nfat activation. the activation of this pathway was independent of tlr4 engagement, depending instead exclusively on cd14. we also found that lps-induced nfat activation in dcs was necessary for the efficient synthesis of cyclooxygenase-2 (cox-2) that, by generating prostaglandins (pgs), such as pge2, regulates different dc functions including migration and polarization of t cell responses. our findings reveal novel aspects of the molecular signaling triggered by lps in dcs and define a new role for cd14. given the essential involvement of cd14 in many diseases, including sepsis and chronic heart failure, the discovery of signal transduction pathways activated exclusively via cd14 represents a major step towards the development of potential treatments with modes of action involving interference with cd14 functions. we have examined the interaction of cd55, a 80-kda glycosyl-phosphatidylinositol (gpi)-anchored membrane protein, with the monocyte signalling receptor, cd14. human monocytes were isolated from healthy adult donor's peripheral blood. this involved labelling molecules at saturation with different coloured fluorophores and determining their positions separately by dual wavelength imaging. the cells were labelled at saturation with anti-cd14 antibody coupled to biotin visualised by qd-525-streptavidin and anti-cd55 antibody coupled to allophycocyanin. the images are analysed to quantify the overlap of the particle images and hence determine the extent of co-localization of the labelled molecules. single particle fluorescence imaging (spfi) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labelled with small fluorescent particles. the images of particles are diffraction-limited spots that are analysed by fitting with a two-dimensional gaussian function providing the basis for determining the dynamic and associated behaviour of receptors on living cells. changes in the numbers of receptors, and in the proportion of receptors showing colocalisation, indicated that lps promotes the interaction of cd55 and cd14, suggesting a new functional role of cd55 as a member of a multimeric lps receptor complex. l. lundvall 1 , r.r. schumann 1 1 charité -universitätsmedizin berlin campus mitte, institute for microbiology and hygiene, berlin, germany meningitis is a life-threatening disease mainly caused by bacteria and viruses. bacterial components such as lipopolysaccharide (lps), lipoproteins or peptidoglycan breakdown products (i. e. mdp, mesodap) stimulate pattern recognition receptors (prrs), such as toll-like receptors (tlrs) and the intracellular nod-like receptors (nlrs) for an inflammatory response. we hypothesize that a synergistic effect of tlr-induced nf-xb activation and nlr-mediated caspase-1 induction leads to an increased release of mature il-1b during bacterial meningitis in brain-derived cells. a mouse meningitis model with s. pneumoniae (d39) was established for assessing il-1b induction during this disease. the murine raw 264.7 cell line, the human astroglial u-87 mg and the murine microglial cell-line bv-2 were stimulated with the tlr4 ligand lps, the tlr2 ligand pam 2 cys, the nod2 ligand mdp, or the nod1 ligand c12-ie-dap, and, as control, atp alone or in combination. we assessed il-1b by elisa and caspase-1 and pro-il-1b expression by western blot. furthermore, primary mouse astrocytes isolated from the cortices of mouse puppies were used for stimulation followed by sirna suppression of elements of the il-1b induction pathway. s. pneumoniae (d39) infected mice showed a significant increase in il-1b release after 24 hours. in vitro, an increase in il-1b levels after costimulation with lps or pam 2 cys, and mdp or c12-ie-dap was observed in a dose-dependent manner. a synergistic enhancement of il-1b by tlr-and nlr-ligands was observed in raw cells, bv-2 cells, u-87 cells and primary astrocytes. active caspase-1 (p10) was induced by mdp or c12-ie-dap, corresponding with high il-1b responses when lps or pam 2 cys was added. sirna experiments show that a knock-down of nod2 leads to a diminished il-1b release after lps-and mdp-stimulation. the precursor forms of il-1b and caspase-1 seem to be constitutively expressed in astrocytes and microglia. a synergistic enhancement between tlrs and nlrs in il-1b release in brain-derived cells was observed. so a two-step stimulation seems necessary for the release of high levels of mature il-1b by astrocytes and microglia. bacteria containing both, tlr-and nlr-ligands thus have the potential to induce high levels of il-1b which may contribute to disease pathology and may point to novel intervention strategies. j. rosenberg 1 1 toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i-like (rlrs ) are more well-characterized in their identity and expression as signaling markers which effect the ealry innate immune response and elicit adaptive immunity , . in the case of tlrs most sutides to date have delineated tlr expression and function on antigen presenting cells like dendritic cellof this research. extension of the profiling and presence of tlrs on cell characterized as adaptive immune cells such as t cells is the subject of this line of research. using a cd3 and cd28 activation model system -tlr4 presence on cd4+ cells is found in mouse t cells, human t cells and jurkat cell lines. following cd3/cd28 activation for 48 hours we have identified a small but distinct populationof tlr4+ cells. further characterization indicates these cells to be cd4+cd25+ cells. further characterization of the expression and functional acitvity of the tlr4+ t cells indicates co-expression of tlr4 with md-2 indicating a functional tlr4 receptor. in addition lps activiation did not lead to upregulation of tlr4 expression in t-cells. the data indicate that tcr activation leads to tlr4 expression. the expression appears to be associated with cd4+cd25+ cells and refelecting an activated t cell phenotype which will be further characterized as perhaps related to tregs or other tcell subsets. s. m. lehmann 1 , d. kaul 1 , c. krüger 1 , f. zipp 1 , r. nitsch 2 , s. lehnardt 1 1 charité-universitätsmedizin berlin, cecilie-vogt-clinic for neurology, berlin, germany, 2 charité-universitätsmedizin berlin, institute for cell biology and neurobiology, berlin, germany the innate immune system is the first line of defense against various pathogens and requires the expression of toll-like receptors (tlrs). in macrophages, tlr7 plays a crucial role in immune responses elicited by gu-rich ssrna (i. e. ssrna40) as well as synthetic antiviral chemicals, including imidazoquinoline components (i. e. imiquimod) and some guanine nucleotide analogs (i. e. loxoribine). these compounds were initially described to activate mouse tlr7 (and human tlr8) and are potent immune response modifiers leading to important antiviral and antitumor activities. microglia serve as the major innate immune cells in the central nervous system (cns). employing various techniques including pcr, in situ hybridization, and immunocytochemistry, we demonstrate that tlr7 is expressed in these cells. incubation of microglia with all three of the above mentioned tlr7 ligands leads to activation of these cells displaying an ameboid shape and releasing inflammatory cytokines such as tnf-a and il1-b in a dose-and time-dependent fashion. analysis of wild type (wt) and tlr7 knock out (ko) microglia by realbecause neutrophil apoptosis plays a key role in resolving inflammation, identification of proteins regulating neutrophil survival should provide new strategies to modulate inflammation. using a proteomic approach, coronin-1 was identified as a cytosolic protein cleaved during neutrophil apoptosis. coronin-1 is an actinbinding protein that can associate with phagosomes and nadph oxidase but its involvement in apoptosis was currently unknown. in coronin-1-transfected plb985 cells, coronin-1 overexpression did not modify the kinetics of granulocyte differentiation as assessed by cd11b labeling. concerning apoptosis, increased coronin-1 expression in dmf-differentiated plb985 significantly decreased gliotoxin-induced mitochondrial depolarization as compared with controls. likewise, coronin-1 significantly decreased trail-induced apoptosis with less mitochondrial depolarization, caspase-3 and caspase-9 activities, but not caspase-8 or bid truncation suggesting that coronin-1 interfered with mitochondria-related events. to validate the prosurvival role of coronin-1 in a pathophysiological condition involving neutrophil-dominated inflammation, neutrophils from cystic fibrosis (cf) patients were studied. circulating neutrophils from cf patients had more coronin-1 expression assessed by immunoblotting or proteomic analysis of cytosolic proteins. this was associated with a lower apoptosis rate than those from controls evidenced by delayed phosphatidylserine externalization and mitochondria depolarization. in addition, inflammatory neutrophils from cf patients lungs showed an intense coronin-1 immunolabeling. we concluded that coronin-1 could constitute a potential target in resolving inflammation. p.-n. hsu 1 1 national taiwan university, graduate institute of immunology, taipei, taiwan, republic of china human osteoclast formation from mononuclear phagocyte precursors involves interactions between tumor necrosis factor (tnf) ligand superfamily members and their receptors. many of the proinflammatory cytokines and growth factors implicated in inflammatory processes have also been demonstrated to impact osteoclast differentiation and function. recent evidence indicates that the tnf-related apoptosis-inducing ligand (trail) of the tnf ligand superfamily, which was initially thought to induce apoptosis in many transformed cell lines, can serve as an effector molecule in activated t cells. we show in this work that trail can induce osteoclast formation from human monocytes and murine raw264.7 macrophages. we demonstrated that both cell models differentiate into osteoclast-like cells in the presence of trail in a dose-dependent manner, as evaluated in terms of tartrate-resistant acid phosphatase (trap)-positive multinucleated cells and bone resorption activity. the trail-induced osteoclast differentiation is independent of caspase activation and apoptosis induction activity. however, trail-induced osteoclastogenesis is dependent on activation of nf-xb, erk, and p38 map kinase. the trail-induced osteoclastogenesis was significantly inhibited by treatment with traf-6 sirna and traf-6 decoy peptide, indicating this pathway is traf-6 dependent. thus, our data demonstrate that trail induces osteoclast differentiation via direct engagement with the trail death receptor through a signaling pathway distinct from apoptosis. our results indicate that in addition to triggering apoptosis, trail induces osteoclast differentiation. it provides a novel role for trail in regulating osteoclast differentiation and in osteoimmunology. microglia are considered to be the local antigen presenting cells (apcs) of the central nervous system (cns) which are thought to play a crucial role in local reactivation of autoreactive t cells during cns autoimmunity e. g. in multiple sclerosis (ms) and its animal model experimental autoimmune encephalomyelitis (eae) . in this study we investigated if the anti-inflammatory nuclear transcription factor peroxisome proliferator-activated receptor gamma (pparg) that has been described to negatively regulate macrophage activation has an influence on microglia immunogenicity. sustained activation of pparg both reduced microglial signalling via mhc molecules and costimulatory molecules and concomitantly increased signalling via the coinhibitory molecules b7-h1 and b7-dc. moreover, also production of pro-inflammatory cytokines like tnf-a and il-6 was profoundly reduced if microglia were pre-treated with the pparg-agonist pioglitazone (pio). in contrast to this, the lack of pparg in microglia resulted in increased expression of pro-inflammatory cytokines not only following an inflammatory stimulus but also in the steady-state indicating that pparg might play a cell-intrinsic role in controlling microglia immunogenicity. importantly, if pparg was activated in microglia, the capacity to prime ovalbumin-specific t cells was impaired. t cells primed by pio-treated microglia produced reduced amounts of il-2 and ifn-g which could not be overcome by restimulation with acd3. this indicates that t cells primed by pio-treated microglia did not undergo functional differentiation but were impaired in exhibiting effector functions. furthermore, microglia were able to induce antigen-specific differentiation of naive cd4 t cells into t helper 17 (th17) cells, which have been associated with autoimmune pathogenicity during eae. however, if pparg was activated, microglia were no longer able to induce th17 differentiation. in conclusion, activation of pparg impairs microglial apc function leading to reduced activation of antigen-specific t cells and, in addition, inhibits the induction of th17 cells. therefore, activation of pparg in microglia is a promising approach to limit local activation of autoreactive t cells in the cns in cns-autoimmune deseases. bacterial lipopolysaccharide (lps) triggers monocytes and macrophages to produce several inflammatory cytokines and mediators. however, once exposed to lps, they become hyporesponsive to a subsequent endotoxin challenge. this phenomenon is defined as lps desensitization or tolerance. previous studies have identified some components of the biochemical pathways involved in negative modulation of lps responses. in particular, it has been shown that the il-1 receptor-related protein st2 could be implicated in lps tolerance. the natural ligand of st2 was recently identified as interleukin-33 (il-33), a new member of the il-1 family. in this study, we investigated whether il-33 triggering of st2 was able to induce lps desensitization of mouse macrophages. we found that il-33 actually enhances the lps response of macrophages and does not induce lps desensitization. we demonstrate that this il-33 enhancing effect of lps response is mediated by the st2 receptor since it is not found in st2 ko mice. the biochemical consequences of il-33 pretreatment of mouse macrophages were investigated. our results show that il-33 increases the expression of the lps receptor components myeloid differentiation protein-2 (md2), cd14 and tlr-4 and the myeloid differentiation factor 88 (myd88) adaptor molecule. in addition, il-33 pretreatment of macrophages enhances the cytokine response to tlr-2 but not to tlr-3 ligands. thus, il-33 treatment preferentially affects the myd88-dependent pathway activated by the tlr. c. klotz 1 , b. lenz 1 , r. lucius 1 , s. hartmann 1 1 humboldt-university berlin, molecular parasitology, berlin, germany chronic helminth infections are shown to be negatively associated with allergic disorders in humans and animal models and parasite cysteine protease inhibitors (cystatins) have been identified as a major class of modulators from filarial parasites. recently we showed that recombinant parasite cystatin (avcystatin), derived from the model parasite acanthocheilanema viteae, effectively abolished ova-induced allergic airway responsiveness in a mouse model of asthma (schnoeller et al., 2008) . the cystatin effect was blocked by the application of anti-il-10 receptor antibodies and by depletion of macrophages using clodronate liposomes. we hypothesize that parasite cystatin induced regulatory macrophages characterized by secretion of immune suppressive interleukin-10 (il-10). the aim of the present study was to elucidate the molecular mechanisms by which avcystatin induces il-10 in primary macrophages. in vitro experiments with peritoneal macrophages from balb/c mice confirmed specific and concentration dependent il-10 production after avcystatin stimulation. application of specific inhibitors revealed that the il-10 induction was p38 and erk dependent, and inhibitor titration indicated a higher sensitivity towards p38. western blotting experiments confirmed the phosphorylation of p38 and erk in macrophages after avcystatin stimulation. in addition, by using specific inhibitor and western blotting, we showed that avcystatin induced il-10 is also regulated by the phosphatidylinositol-3-kinase (pi3k) -proteine kinase b (akt) pathway. further analysis indicated a hierarchical signalling pattern and cross regulation of the identified pathways. hence, we conclude that avcystatin renders macrophages into a regulatory state by addressing a broad range of signalling cascades that ultimately lead to the expression of il-10 and possibly other regulatory markers. in general, revealing fundamental knowledge about induction of regulatory macrophages by helminth immunomodulators will help to design new strategies for the treatment of inflammatory disorders. we screened approximately half the (putative) human kinome to identify novel candidates interfering with macrophage activation in response to endotoxin. this screen revealed the impact of several novel kinases as well as kinases with previously established function. one of the top candidates identified to block endotoxininduced tnf-a secretion was carkl, a gene with no previously described function. subsequent biochemical analyses unequivocally revealed that carkl is a phosphotransferase protein using sedoheptulose as a phosphate acceptor and atp as a donor. sedoheptulose is a monosaccharide consisting of seven carbon atoms and a functional ketone group. the product sedoheptulose-7-phosphate (s-7p) is also an intermediate metabolite of the pentose phosphate pathway (ppp) and so far was only known to be produced by condensation of ribose-5p and xylulose-5p via a transketolase reaction. to identify the molecular mechanism by which carkl modulates the immune response, we investigated its endogenous regulation and function in the course of macrophage activation. so far, our data favor a model where post-stimulatory downregulation, i. e. loss of carkl is essential for the activation of macrophages by various pro-inflammatory stimuli. disentangling the signaling pathways responsible for the rapid regulation of carkl unearthed nf-kb and p38/jnk but not erk as driving forces. counterbalancing endotoxin induced loss of carkl by over-expression led to an impaired cytokine response and a concomitant block of free radical production. comparison of wild type and catalyticinactive forms of carkl unveiled that most of the effects of carkl on the inflammatory response were due to its phosphotransferase activity. expression profiling using gene chip analysis further supported the concept that carkl may represent a new key modulator of inflammatory processes. taken together, detailed analyses to study the molecular function of carkl should ultimately lead to a more profound understanding of cellular metabolism and especially clarify new mechanisms involved in the regulation of inflammation. in addition, connecting the ppp and its impact on the cellular redox state with inflammatory disease models might reveal new therapeutic targets. in this context, the sedoheptulose kinase carkl and its product s-7p may provide a novel basis for interfering with adverse immune responses. t. bosschaerts 1 , y. morias 1 , p. de baetselier 1 , a. beschin 1 1 vib, cmim, vub brussels, brussels, belgium the development of classically activated monocytic cells (m1) is a prerequisite for effective elimination of parasites, including african trypanosomes. however, persistent m1 activation causes pathogenic damage including liver injury during infection, resulting in death of the host. we aim to identify mechanisms involved in regulation of m1 activity in order to dampen their pathogenicity and increase the resistance of the host to parasitic diseases. methods: we have scrutinized the phenotype and cellular origin of liver m1 in trypanosoma brucei infected by facs analysis and bone marrow transfer experiments. the contribution of different signaling pathways, including myd88, ifng, il-10, ccr2 and nf-kb to the development and/or recruitment of pathogenic m1 in the liver was investigated using knock-out mice or by delivering il-10 in infected mice. results: we established that cd11b+ly6c+cd11c+ tnf and inos producing dcs (tip-dcs) represent the major m1 liver subpopulation. tip-dcs differentiated in an ifng/myd88-dependent manner from cd11b+ly6c+ inflammatory monocytes in the liver of infected mice. ccr2 promoted the egression of inflammatory monocytes from bone marrow to blood but not their entry, differentiation and maturation to tip-dcs in the inflamed liver. as a consequence, ccr2 ko mice experienced reduced pathogenic symptoms. on the other hand, the absence of il-10 enhanced the recruitment of inflammatory monocytes as well as their differentiation and maturation to tip-dcs, resulting in exacerbated pathogenicity and early death of the host. in addition, the therapeutic liver-specific delivery of il-10 in t.brucei infected mice efficiently limited the differentiation and maturation to tip-dcs, hereby limiting disease-associated pathogenicity. finally, the absence of the nf-kb member p50 was associated with increased tissue injury associated with increased production of pathogenic tnf and no by inflammatory monocytes, but not by tip-dcs. conclusion: our data demonstrate that nf-kb p50 and il-10 play a role in preventing infection-associated pathogenicity in hosts confronted with a chronic inflammatory situation by limiting the activity of pathogenic m1, in particular tip-dcs. the inflammatory activity of liver m1 is controlled by il-10 and/or p50 nf-kb at different levels, including recruitment of inflammatory monocytes to the liver, their differentiation to pathogenic tip-dcs, or their production of tnf and no. a. popov 1 , j. driesen 1 , z. abdullah 2 , a. niño castro 1 , t. chakraborty 3 , m. krönke 4 , o. utermöhlen 4 , c. wickenhauser 5 , j.l. schultze 1 1 limes institute, laboratory for genomics and immunoregulation, university of bonn, bonn, germany, 2 institute of molecular medicine and experimental immunology, bonn, germany, 3 institute of medical microbiology, university of giessen, giessen, germany, 4 institute for medical microbiology, immunology and hygiene, university of cologne, cologne, germany, 5 institute for pathology, university clinic leipzig, leipzig, germany dendritic cells (dc) and macrophages play an important role in pathogen sensing and antimicrobial defense. here we report on a new role for the myeloid antigen presenting cells (apc) in granulomatous infections. infection of myeloid dc and macrophages with listeria monocytogenes results in a distinct regulatory phenotype characterized by expression of multiple inhibitory molecules, including indoleamine 2,3-dioxygenase, cyclooxygenase-2 and cd25 and production of prostaglandin e2 (pge 2 ) and interleukin 10. all these molecules are strictly dependent on autocrine tnf, released during infection, and are in concert suppressing t-cell responses; cd25, expressed by regulatory myeloid cells, acts as an il-2 scavenger. importantly, myeloid cells with regulatory phenotype are characterized by increased resistance to infection and demonstrate significantly improved bactericidal activity against intracellular bacteria. furthermore, infected cells can transfer the regulatory phenotype to the uninfected ones in a cell-cell contact independent manner, thereby extending the pool of infection-resistant myeloid cells. induction of regulatory and protective phenotype in macrophages and dc require at least two signals provided by tnf and either pge 2 or tlr ligands. transcriptional changes in human macrophages, infected by mycobacterium tuberculosis, resemble the ones induced in dc during infection with l.monocytogenes. in fact, granuloma in patients with tuberculosis and listeriosis are enriched for cd25 + ido + cox-2 + regulatory myeloid cells, whereas most effector cell populations, such as t cells, b cells and nk cells, are expelled from the granuloma. of note, in tuberculosis granuloma consist mostly of macrophages, whereas in listeriosis dendritic cells predominate. altogether, our studies provide strong evidence that intracellular pathogens such as m.tuberculosis and l.monocytogenes induce a specific polarization of myeloid dc and macrophages characterized by a functional preponderance of inhibitory mechanisms. we postulate that these regulatory myeloid cells play a dual role during life-threatening granulomatous infections. on one hand, they promote pathogen containment by efficiently killing intracellular bacteria; on the other hand, these myeloid cells inhibit granuloma-associated t cells and thereby might be involved in the retention of granuloma integrity protecting the host from granuloma break-down and pathogen dissemination. the interferon-gamma (ifn-g) component of the immune response plays an important and essential role in infectious and non-infectious diseases. induction of ifn-g secretion by human t and nk cells through synergistic co-stimulation with interleukin 12 (il-12) and il-18 in the adaptive immune responses against pathogens is well known, whereas a similar activity by macrophages is still controversial, largely due to criticisms based on the contamination of macrophages with nk or t cells in the relevant experiments. the possible contribution of macrophages to the interferon response is, however, an important factor relevant to the pathogenesis of many diseases. to resolve this issue, we have determined the production of ifn-g at a single cell level by inmunohistochemistry and by enzyme-linked immunosorbent spot (elispot) analysis and have unequivocally demonstrated that human macrophages derived from monocytes in vitro through the combined stimulation of il-12 and il-18 or with macrophage-colony stimulating factor (m-csf) were able to produce ifn-g when further stimulated with a combination of il-12 and il-18. in addition, naturally activated alveolar macrophages immediately secreted ifn-g upon treatment with il-12 and il-18. therefore, human macrophages in addition to lymphoid cells contribute to the ifn-g response, providing another link between the innate and acquired immune response. a. j. denzel 1 , m. rodriguez gomez 2 , m. niedermeier 2 , y. talke 2 , n. göbel 2 , k. schmidbauer 2 , m. mack 2 1 unversity hospital regensburg, internal medicine ii, regensburg, germany, 2 university hospital regensburg, regensburg, germany we have shown previously that basophils recognize and react to free antigen during a memory immune response in vivo and release large amounts of il-4 and il-6. activation of basophils is dependent on the presence of free antigen, antigen specific immunoglobulins and expression of immunoglobulin fc-receptors. we now have analysed in more detail the binding of antigen to basophils, the recruitment of basophils to lymphoid organs and the basophil dependent migration of other leukocytes during the first days of a memory immune response. following restimulation with soluble antigen only antigen specific basophils but not basophils from naïve mice migrate from bone marrow and spleen to the site of restimulation (e.g. the peritoneum) and the draining lymph nodes. peripheral blood basophils are markedly reduced during the first hours after restimulation. in the blood, spleen lymph nodes and bone marrow basophils can bind intact antigen on their surface for up to 24h, with basophils in the bone marrow binding the lowest amount of antigen. depletion of basophils also affects the recruitment of various other leukocyte subsets in immunized mice. our datas show that basophils are recruited to draining lymph nodes during a memory response. tnf-a is a pro-inflammatory cytokine that mediates inflammation in response to various pathogens, including mycobacterium tuberculosis. it is also a key factor in the pathogenesis of autoimmune diseases like rheumatoid arthritis. three tnf-a-blocking drugs have been approved to treat selected autoimmune diseases; two are monoclonal antibodies against tnf-a (adalimumab and infliximab); the other is a soluble tnf receptor/fc fusion protein (etanercept) . tnf-a-blockers have been shown to increase the risk of reactivation of latent tuberculosis and this risk appears to be higher in patients treated with the monoclonal antibodies. we studied the effects of tnf-a blockers on the maturation of mycobacteria-containing phagosomes in human macrophages. all three drugs had an inhibitory effect on ifn-g-induced phagosome maturation in pma-differentiated human thp-1 cells infected with m. bovis bcg, the avirulent m. tuberculosis h37ra strain and the virulent m. tuberculosis h37rv strain. adalimumab and infliximab, but not etanercept, suppressed phagosome maturation in primary human peripheral blood monocyte-derived macrophages (mdm) in the presence or absence of ifn-g. macrophages secreted tnf-a in response to infection with mycobacteria and this response was enhanced by activation of the cells with ifn-g. treatment of infected macrophages with tnf-a increased maturation of mycobacteria-containing phagosomes. these results suggest a role for tnf-a in activating phagosome maturation and highlight a novel mechanism through which tnf-a blockade can affect the host innate immune response to mycobacteria. z. g. dobreva 1 , l.d. miteva 1 , s.a. stanilova 1 1 trakia university, faculty of medicine, molecular biology, immunology and genetics, stara zagora, bulgaria il-23 is a heterodimeric cytokine composed of a p19 subunit associated with the il-12/23p40 subunit. like il-12, il-23 is expressed by the activated antigenpresenting cells and both cytokines induce ifn-gamma secretion by different t cell subsets. the proper balance between il-12p40-related cytokines controls the appearance of normal th 1 and pathological th 17 mediated immune responses. in this study, we examined the dynamics of inducible il-12p40 and il-23p19 mrna expression and protein production in purified human monocytes and how jnk and p38 mapks inhibitors influenced il-12p40 and il-23 production. the cytokines' quantity determination was performed by elisa. quantitative real-time polymerase chain reaction (qrt-pcr) was performed for mrna transcripts detection. results were calculated in fold increase compared with gene expression in nonstimulated monocytes. il-23p19 gene expression was higher than those observed for il-12p40 gene at all time-points. the level of il-23p19 mrna increased after 6 th h and reaching a maximum level at 9 th h (43.4 fold for c3bgp and 22.7 fold for lps). c3bgp and lps triggered il-12p40 gene transcription were almost equal at the 3 rd h (4.4 and 4.1 fold) and at 9 th h (7.8 and 7.9 fold, respectively) after stimulation. the higher level of il-12p40 gene expression was detected at 6 th h in lps compared to c3bgp stimulated monocytes (8.1 vs. 4.9 fold). however, il-12p40 and il-23 protein production was increased in the highest level after c3bgp stimulation. the inhibition of p38 led to the statistical significant augmentation of c3bgp stimulated il-12p40 production. the inhibition of the same map kinase enhanced lps stimulated il-12p40 production without significant difference. the inhibition of jnk and p38 mapks significantly decreased c3bgp stimulated il-23 production from human monocytic cells.in summary, the present study demonstrates the different time-course and ability of c3bgp and lps to induce the expression of il-12p40 and il-23p19 mrnas in purified human monocytes. we showed that inhibition of p38 mapk down regulated il-23 and upregulated il-12p40 production in stimulated monocytes. we concluded that in human monocytes p38 map kinase activation has an opposite effect on the il-12p40 and il-23p19 expression. neutrophils represent key components of the innate immune system with the ability not only to phagocytose and killing invading pathogens, but also to produce a variety of proteins, including cytokines and chemokines, with important consequences on the recruitment and activation of other immune cells, such as monocytes, dendritic cells, t and b cells. for instance, it has been shown that neutrophils can directly interact with, and induce functional maturation of, immature monocyte-derived dendritic cells (modc). indeed, upon interaction with neutrophils, modc up-regulate the expression of costimulatory molecules, such as cd83, cd86 and cd40, and secrete il-12, thus acquiring the ability to induce proliferation and th1 polarization of naï ve t cells. in order to extend these findings, the present study was designed to address whether human neutrophils interact with peripheral blood-derived dendritic cells and the pathological consequences that such interaction could eventually produce. in human peripheral blood, dendritic cells can be divided in plasmacytoid dendritic cells (pdc) and myeloid dendritic cells (mdc), the latter further divided in three different subsets based on the expression of cd1c, bdca-3, and cd16. by analyzing different chronic inflammatory pathologies, such as crohn's disease, psoriasis and sweet's syndrome, we found that neutrophils co-localize with a subtype of myeloid dendritic cells (mdc) with characteristics resembling the cd16+ subset of mdc. in order to characterize the interaction between the two cell types, autologous neutrophils, highly purified by an in-house built immunonegative selection protocol, and cd16+ dc were isolated from healthy donors and analyzed in a co-culture system under different stimulatory conditions. here we show that neutrophils modulate different effector functions of cd16+ dc, including their survival and their ability to produce il-12p70. besides providing the basis for a better understanding of the cellular interactions that occur in pathological conditions, our results further emphasize the importance of neutrophils in the modulation of the inflammatory response. chitin is a linear polymer of n-acetyl-d-glucosamine (glcnac) residues present in human pathogenic fungi or nematodes. chitotriosidases (cht) and acetic mammalian chitinase (amcase) have been identified as the only functional chitinases in mammalians. the expression of both chitinases appears to be strongly species dependent, indicating distinct physiological functions. amcase is considered as predominant chitinases in mice while cht is regarded as major chitinases in humans. interestingly, cht is constitutively expressed by human phagocytes at high levels while it is absent in mice phagocytes. although, amcase received increased attention as modulator of the innate immune response against chitin in mice, the physiological function of cht in humans is virtually unknown. to evaluate the physiological function of cht we have characterised the substrate specificity of human cht and the mode of substrate cleavage by analysing chtproduced fragments of chitosan, a close but water soluble derivate of chitin. degradation products of chitosan have been investigated by gel electrophoresis and maldi-tof mass spectroscopy. moreover, the application of a computer-based model of cht activity revealed the mode of substrate cleavage. we found that cht is a processive endo-cleaving chitinase resulting in the production of only small diffusible chitin/chitosan fragments. in further studies we could show that those cht-produced small chitin/chitosan fragments exhibit a strong ability to induce a pro-inflammatory response in human blood derived monocytes/macrophages as indicated by an increased release of the pro-inflammatory cytokines tnf-a, il-6, il-8 and mcp-1 involving the transcription factor nfxb. moreover, these stimulated monocytes/macrophages revealed an increase of cht expression indicating an autocrine positive feed-back regulation. our data suggest that human cht is involved in the early recognition of chitin/chitosan containing human pathogens due to the generation of immuno-stimulatory chitin/chitosan fragments. m. hasenberg 1 , s. wolke 2 , a. brakhage 2 , m. gunzer 1 1 otto-von-guericke universität, institut für molekulare und klinische immunologie, magdeburg, germany, 2 hans-knöll-institut, abteilung für molekulare und angewandte mikrobiologie, jena, germany since their discovery in 2004 nucleic extracellular traps (nets) released by certain cell types including neutrophil and eosinophil granulocytes were shown to play a crucial role in mediating innate immune responses towards different bacterial und fungal pathogens. recently it was found by us and others that neutrophil granulocytes release nets also upon contact to the filamentous fungus aspergillus fumigatus. in the present study we aimed to characterize this process in more detail focusing on the kinetics of net-formation as well as clarifying the responsible cell-biological mechanisms. by the use of several microscopic techniques (scanning electron microscopy, fluorescence widefield microscopy, confocal-and 2-photon microscopy) we initially demonstrated the generation of net like structures after coincubation of a. fumigatus germlings and freshly isolated murine or human pmn. the analysis of our time lapse video microscopy data allowed us to examine the exact time course from initial contact to the fungal surface to explosive release of nets up to 3 hours later. moreover, we investigated the dependency of this phenomenon on the induction of an oxidative burst. therefore we added the nadph-oxidase inhibitor dpi to the cell coincubation and found clearly reduced net formation. by fluorescence staining of reactive oxygen species we could demonstrate that ros are released prior to net detection. interestingly, our data currently suggest that in contrast to other pathogens investigated so far, nets are not directly toxic to fungal elements. whether and how nets control growth of a. fumigatus currently remains open. to summarize our data, we found rapid net formation as a commonly observed immune response of neutrophil granulocytes contacting a. fumigatus. consistent with studies on different pathogens this mechanism seems to be ros-dependent, however not toxic for the fungus. thus, in the future we will have to clarify whether net-formation really occurs in vivo and how nets can control the outgrowth of a. fumigatus at sites of infection. production of type i interferons (ifn-i, mainly ifn-a and ifn-b) is a hallmark of innate immune responses to all classes of pathogens. when viral infection spreads to lymphoid organs, the majority of systemic ifn-i is produced by a specialized 'interferon-producing cell' (ipc) that has been shown to belong to the lineage of plasmacytoid dendritic cells (pdc). it is unclear whether production of systemic ifn-i is generally attributable to pdc irrespective of the nature of the infecting pathogen. we have addressed this question by studying infections of mice with the intracellular bacterium listeria monocytogenes. protective innate immunity against this pathogen is weakened by ifn-i activity. in mice infected with l. monocytogenes systemic ifn-i was amplified via ifn-b, the ifn-i receptor (ifnar) and transcription factor interferon regulatory factor 7 (irf7), a molecular circuitry usually characterisitic of non-pdc producers. synthesis of serum ifn-i did not require tlr9. in contrast, in vitro differentiated pdc infected with l. monocytogenes needed tlr9 to transcribe ifn-i mrna. consistent with the assumption that pdc are not the producers of systemic ifn-i, conditional ablation of the ifn-i receptor in mice showed that most systemic ifn-i is produced by myeloid cells. furthermore, results obtained with facs-purified splenic cell populations from infected mice confirmed the assumption that a cell type with surface antigens characteristic of macrophages and not of pdc is responsible for bulk ifn-i synthesis. the amount of ifn-i produced in the investigated mouse lines was inversely correlated to the resistance to lethal infection. based on these data we propose that the engagement of pdc, the mode of ifn-i mobilization, as well as the shaping of the antimicrobial innate immune response by ifn-i differ between intracellular pathogens. t. naessens 1 , s. vander beken 1 , p. bogaert 1 , j. grooten 1 1 ghent university, biomedical molecular biology, zwijnaarde (ghent), belgium introduction: although the effector and modulator functions of activated macrophages in innate and adaptive immunity are well documented, their exact role in the initiation and propagation of immune pathologies is still not fully understood. recent insights in monocyte and macrophage heterogeneity render the picture even more complex. in addition, it is unclear to what extend resident and elicited macrophages differ functionally and hereby differentially contribute to immune pathologies. in this study we focused on the dynamics and function of resident alveolar macrophages (ram) during and after allergic bronchial inflammation. strategy: we used an ovalbumin (ova)-alum based mouse model of allergic asthma and an ova-complete freund's adjuvant (cfa) based mouse model of hypersensitivity pneumonitis, constituting a th1-driven immunological counterpart of the th2-driven experimental asthma. ram were distinguished by prior in situ labelling with fluorescent polystyrene microspheres. as complementary approach, ram and elicited alveolar macrophages (eam) were distinguished using cd45 bone marrow chimeric mice. combined with flow cytometry and fluorescence activated cell sorting, both approaches allowed us to trace resident and elicited am populations in the course of th1-and th2-driven allergic airway inflammation. results: during the acute phase of the allergic response, isolated ram and eam showed distinct gene expression signatures, reflecting a possible functional heterogeneity between these two macrophage subsets. in both types of allergic inflammation, microsphere-tagged cd45.1 + ram remained constant in cell number for the first 2 days of chronic ova-exposure and then dropped sharply, having nearly completely disappeared from the alveoli by day 4 of ova-exposure. as a consequence, following the clearance of inflammation, inflammation-experienced ram replaced the initial ram population. strikingly, in both types of allergic inflammation, this secondary ram population showed a markedly altered responsiveness to lps stimulation. this involved macrophage activation markers and nf-kb inducible inflammatory genes. however, especially genes induced by ifn-beta showed strongly increased expression in secondary ram as opposed to their near lack of induction in primary ram. this switch from an ifn-beta deficient to an ifn-beta adequate phenotype may increase the inflammatory sensitivity of allergic inflammationexperienced lungs as also observed in asthmatic patients, showing an increased sensitivity to bacterial infection. e. schlecker 1 , a. stojanovic 1 , a. cerwenka 1 1 german cancer research center, innate immunity, heidelberg, germany myeloid-derived suppressor cells (mdsc) are a heterogeneous population of cells that expand during cancer, inflammation and infection. these cells play a critical role in suppressing t cell responses. the exact nature and function of mdsc remain unclear. here we show that a subpopulation of mdsc (gr-1 + cd11b + f4/80 + ) isolated from rma-s tumor-bearing mice did not suppress but rather activated nk cells to produce ifn-g. additionally, nk cells eliminated this subpopulation both in vitro and in vivo. in order to identify molecules and pathways that might be involved in mdsc accumulation in tumor bearing mice and their suppressive/activatory function, gene expression profiling of mdsc subpopulations was performed using whole genome microarrays. understanding the reciprocal interaction of mdsc with nk cell could improve the efficiency of cancer immunotherapy. g. solinas 1 , f. marchesi 1 , m. fabbri 1 , s. schiarea 2 , c. chiabrando 2 , a. mantovani 1,3 , p. allavena 1 1 istituto clinico humanitas, rozzano, italy, 2 istituto mario negri, milano, italy, 3 università di milano, milano, italy experimental and clinical evidence has highlighted that tumor-associated macrophages (tam) represent the principal component of the leukocyte infiltrate and are usually associated with tumour growth, progression and metastasis. macrophage population is generally divided into two distinct subsets: m1 and m2. m1 macrophages act as a first line of defence against pathogens whereas m2 cells participate in wound repair and maintenance of tissue integrity. in the tumour micro-environment tam interactions with the extracellular matrix, neighboring cells, and soluble stimuli largely influence their gene expression and behavior. to investigate the role of the tumor micro-environment on macrophage differentiation, we cultured freshly isolated human monocytes with pancreatic cancer cell line supernatants, in the absence of exogenous cytokine addition.. in selected cultures, about 50 % of the monocytes differentiated after 5 days into macrophages. the phenotype analysis of tumor-conditioned macrophages (tc-macro) demonstrated high expression of the mannose receptor, cd16, cd68 and low levels of mhc class ii. tc-macro produced il-10, il-6, tnf but not il-12, even after lps stimulation. moreover, tc-macro produced a panel of chemokines including ccl2, cxcl8, ccl17 and cxcl10. the transcriptional profile of tc-macro revealed that several genes in line with an m2 polarization are highly expressed. the nature of the tumor-derived factors inducing macrophage differentiation is currently under investigation; biochemical analysis indicated that the biological activity is excluded from exosomes and have a high molecular weight ( g 100.000 kda). il-3 and il-6 were not detectable in tumor supernatants whereas m-csf was present at low levels. by mass spectrometric techniques, we surprisingly found that the tumor-derived m-csf had peculiar migration patterns which were different from those expected for the common human homodimeric glycosilated protein, suggesting an interesting structural differences for the tumor-secreted isoforms of this primary regulator of mononuclear phagocyte. the characterization of tumor-derived factors inducing macrophage differentiation could better clarify the intricate cross-talk between tumor cells and macrophages and thus might aid in the process of devising novel anti-tumor treatments. genomic effects of glucocorticoid hormone (gc) are exerted by glucocorticoid receptor (gr)-mediated changes of gene expression. this is relatively timeconsuming process, needing hours to develop. in contrast, non-genomic effects may occur within minutes. gcs are used for a long time for the therapy of anaphylactic reactions, where mast cells play crucial role. moreover, many cells and cell lines of haemopoetic origin are sensitive to gc-induced apoptosis. recent findings indicate, that non-genomic gc effects mediated by mitochondrial gr may have important function in generating pro-apoptotic signals. we aimed the investigation of non-genomic gc effects on in vitro cultured rbl-2h3 rat mast cell line. we demonstrate that gr nuclear translocation begins within 5 minutes and completes after 30 minutes in dx treated rbl-2h3 cells. since genomic effects occur in the nucleus through gene expression changes, we considered gc effects within 5 minutes as non-genomic. studying gc-caused apoptosis, rbl-2h3 cells proved to be gc-resistant and no mitochondrial gr translocation neither impaired mitochondrial function could be observed upon gc treatment. in further experiments we used rbl-2h3 cells sensitized with anti-dnp (dinitrophenyl) ige and dnp-conjugated bovine serum albumin was used for stimulation. 5 minutes of dx treatment inhibited ca 2+ -signaling in antigen stimulated rbl-2h3 cells in the concentration range of 100nm -10mm. moreover, 5 minutes of dx treatment altered the tyrosine phosphorylation pattern of rbl-2h3 cells. dx treatment alone caused slight increase in tyrosine phosphorylation, while dx treatment of activated cells caused also an increase in tyrosine phosphorylation compared to the solvent-treated controls. the tyrosine kinase syk plays indispensable role in regulating mast cell activation through the fc[epsilon] receptor i. our immunoprecipitation studies show, that dx treatment results in decreased syk phosphorylation in both resting and activated cells. this finding raises the possibility, that syk phosphorylation thus kinase activity may be directly or indirectly regulated by gcs via non-genomic pathway. taken together, our experiments along with the clinical experiences suggest that gcs rapidly influence mast cell activation via a non-genomic pathway, too. the elucidation of the exact signal transduction mechanisms behind rapid gc effects need further experiments. high mobility group box 1 (hmgb1) is a non-histone nuclear protein that binds chromatin and has transcriptional and architectural functions. notably, hmgb1 is highly mobile in the nucleus and is passively released by necrotic cells, while it is bound firmly to apoptotic chromatin (1) . extracellular hmgb1 can act as a cytokine and a chemoattractant, mediating inflammatory responses. interestingly, hmgb1 exerts antibacterial functions in human adenoid and testis (2) . recent investigations have revealed that neutrophils eliminate microbes not only by intracellular phagocytosis but also by trapping them in three-dimensional structures called neutrophil extracelluar traps (nets), made of dna fibers, nuclear proteins (histones) and granule proteins. it has been shown that histones on nets have an anti-microbial activity (3). we asked whether hmgb1 from neutrophils is a component of nets and whether it has a function in nets. we purified human primary neutrophils from peripheral blood of healthy volunteers on ficoll gradients. to induce net formation, we stimulated cells for 40 or 120 minutes with 25 nm phorbol ester (pma), 100 ng/ml interleukin 8 (il-8), or 100 ng/ml lps. the presence of nets was assessed by immunofluorescence using antibodies directed against the granule protein myeloperoxidase (mpo) and against a dna-histone h2a-histone h2b complex. dna was stained with hoechst. using a polyclonal antibody we found hmgb1 in the euchromatin of polylobulated nuclei of resting neutrophils and on the filamentous structure of nets induced by all stimuli. elisa assays revealed that hmgb1 is not present in the supernatants of activated neutrophils, confirming its binding to nets. in conclusion, we found that hmgb1 localizes on nets. we hypothesize that net-bound hmgb1 might exert a direct antimicrobial function, or that nets might concentrate hmgb1 locally to recruit macrophages to the site of infection. these receptors were present on the mast cell surface. incubation (37°c, 3 h) of hlmc with vegf-a, vegf-b, vegf-c, vegf-d and placental growth factor-1 induced concentration-dependent chemotaxis that was blocked by a combination of anti-vegfr-1 and anti-vegfr-2 antibodies. these data indicate that human mast cells represent both a source and a target of vegfs and therefore may play a role in inflammatory and neoplastic angiogenesis through the expression of proangiogenic factors and their receptors. macrophages are important effector cells in immunity to intracellular pathogens and at the same time are exploited as host cells by a number of microorganisms such as mycobacterium tuberculosis. a very important mechanism of intracellular killing is delivery of invading microbes to phagolysosomes. whilst mycobacteria can block phagosome maturation in resting macrophages, and hence survive and replicate inside the host cell, the ifn-g activated macrophage utilizes a diversity of defense mechanisms to eliminate the invader. these include putative killing by antibacterial peptides/proteins and overcoming phagosome maturation block, possibly by induction of autophagy, production of reactive nitrogen or oxygen intermediates and deprivation from nutrients such as iron. mycobacteria are not eliminated even upon onset of protective immunity rather leading to persistence. we hypothesize that the very early steps of pulmonary infection directs the outcome of disease. therefore, we investigate initially infected lung cells and their role in infection in the lung with respect to their anti-microbial mechanisms against mycobacteria in vitro as well as in vivo. preliminary data show that m. tuberculosis is able to persist in the alveolar space for several weeks and bacterial numbers do barely drop even after very low dose infection, indication that bacterial killing is inefficient from the very beginning. cells harboring mycobacteria are found during early and late stages of infection. both, autophagy and nitric oxide production seems to contribute to growth restriction of mycobacteria by macrophages. neutrophils, although recruited in vast numbers to infected lungs, are not able to reduce bacterial numbers in the absence of il-18. altogether, the initial response in the barrier organ lung executed by resident and immigrating cells restricted by the local environment can determine the outcome of infection. human cd1 molecules are dedicated to lipid presentation to t cells and are implicated in inflammatory and auto-immune responses. the cd1a protein is almost exclusively expressed at the cell surface of dendritic cells and is dedicated in surveying extracellular environment. our previous studies have demonstrated that ii associated with cd1a and cholesterol-dependent lipid rafts impact on cd1a surface expression and cd1a-restricted t cell response. bacterial infections can induce an increase in self glycolipid synthesis in dendritic cells and such activated dcs acquire the ability to stimulate cd1-restricted autoreactive t cells. this mechanism of self recognition induced by bacterial infection is believed to be involved in the development of auto-immune disorders. sulfatide, which is a major component of the myelin sheath, is also the only known self-antigen presented by cd1 group i molecules. the functional role of these molecules has not been investigated in auto-immune diseases and we propose that regulation of glycolipid presentation by cd1a molecules could impact in such pathologies. we have thus conducted a preliminary study to understand the implication of cd1 molecules in multiple sclerosis. we first analyzed cd1 expression on monocytes from 16 ms patients and the influence of sera and plasma from these patients on dendritic cell differentiation from healthy donors. results obtained in this preliminary study demonstrate that cd1a was not expressed on ms patient monocytes, while the other members of the cd1 family were expressed. moreover ms sera and plasma induced an earlier and more rapid dendritic cell differentiation than ab sera. these preliminary results confirm our hypothesis that cd1 molecule expression is modified in ms and also reveal that serum from patients with ms modifies lipid-antigen presenting cells. further studies should contribute to define precise mechanisms involved in lipid presentation by cd1 molecules in this context. c. ohnmacht 1 , d. voehringer 1 1 ludwig-maximilians-universität munich, institute for immunology, munich, germany basophils are effector cells of the innate immune system which are associated with allergic inflammation and infections with helminth parasites. however, their development and in vivo functions are largely unknown. here, we characterize basophil turnover, tissue localization and effector functions during infection with the gastrointestinal helminth nippostrongylus brasiliensis. for this purpose, brdu incorporation experiments and in situ fluorescence microscopy of il-4 reporter (4get) mice as well as in vivo depletion of basophils are used to uncover their role during type 2 immune responses. our results demonstrate that under homeostatic conditions basophils have a lifespan of about 60h. n. brasiliensis induced basophilia is caused by increased de novo production of basophils in the bone marrow. basophils are found near the marginal zone in the red pulp of the spleen, in the lamina propria of the small intestine and in the lung parenchyma. activated basophils promote systemic eosinophilia, were associated with differentiation of alternatively activated macrophages in the lung and contributed to efficient worm expulsion of n. brasiliensis in the absence of th2 cells. these results demonstrate that basophils play a crucial role as effector cells in type 2 immune responses which might hold great potential for the treatment of helminth infections and allergic diseases. during acute bacterial infections such as meningitis, neutrophils enter the tissue where they combat the infection before they undergo apoptosis and are taken up by macrophages. neutrophils show pro-inflammatory activity and may contribute to tissue damage. in pneumococcal meningitis, neuronal damage despite adequate chemotherapy is a frequent clinical finding. this damage may be due to excessive neutrophil activity. we here show that transgenic expression of bcl-2 in haematopoietic cells blocks the resolution of inflammation following antibiotic therapy in a mouse model of pneumococcal meningitis. the persistence of neutrophil brain infiltrates was accompanied by high levels of il-1beta and g-csf as well as reduced levels of anti-inflammatory tgf-beta. significantly, bcl-2-transgenic mice developed more severe disease that was dependent on neutrophils, characterized by pronounced vasogenic edema, vasculitis, brain haemorrhages and higher clinical scores. in vitro analysis of neutrophils demonstrated that apoptosis inhibition completely preserves neutrophil effector function and prevents internalization by macrophages. the inhibitor of cyclin-dependent kinases, roscovitine induced apoptosis in neutrophils in vitro and in vivo. in wild type mice treated with antibiotics, roscovitine significantly improved the resolution of the inflammation after pneumococcal infection and accelerated recovery. these results indicate that apoptosis is essential to turn off activated neutrophils and show that inflammatory activity and disease severity in a pyogenic infection can be modulated by targeting the apoptotic pathway in neutrophils. objectives: to investigate the existence of systemic inflammatory response to subchronic oral warfarin (wf) consumation in rats. methods: dark agouti (da) rats were treated with warfarin in drinking water (10 mg and 100 mg daily) for 30 days. oxidative activity (cytochemical nbt reduction) and myeloperoxidase (mpo) intracellular content of peripheral blood neutrophils, plasma levels of il-6 and tnf-a (elisa) and superoxide dismutase (sod) activity (red blood cell lysates) were analyzed as inflammatory parameters in rats following warfarin consumation. changes in prothrombin time (pt), as basic biological warfarin activity was determined as well. results: significantly increased pt was noted at the lower wf dose, with tremendous rise after the higher dose. increase of pma-stimulated neutrophil nbt reduction capacity (neutrophil priming) was noted at both wf doses, while increase in mpo intracellular content was noted at the higher wf dose solely. warfarin consumation resulted in no changes in plasma levels of il-6 and tnf-a. significant decrease in the sod activity was detected in red blood cell lysates at both wf doses, suggesting systemic oxidative activity. conclusion: increased neutrophil priming as well as prooxidant activity in peripheral blood of rats following subchronic warfarin consumation imply proinflammatory effects of oral warfarin administration. absence of the rise in inflammatory cytokines in circulation, suggest low-grade inflammation in these rats. this work is funded by serbian ministry of science and technological development (grant 143038). objectives: although many different macrophage receptors and serum proteins have been shown to play a role in phagocytosis of apoptotic cells, the unique microenvironment of an inflammatory site will have considerable influence upon the molecular pathways which are utilized in apoptotic cell removal. we have recently reported that immune complexes (ic) are able to specifically bind to the surface of human apoptotic neutrophils which may have profound implications for their physiological clearance. in disease situations where immune complexes are present neutrophils undergoing apoptosis would be predicted to become coated with ic. here we address the consequences of ic opsonisation of apoptotic cells upon phagocytosis and cytokine response by macrophages that would be expected to be present at the earliest stages of inflammatory responses (type-1 macrophages, mph1), and during resolution of inflammation (type-2 macrophages, mph2). methods: mph1 / 2 were generated by culturing cd14 + human monocytes for 6 days in the presence of gm-csf or m-csf, respectively. phagocytosis by mph1 / 2 of ic opsonised and unopsonised neutrophils was assessed by flow cytometry. after phagocytosis mph1 / 2 were stimulated with lps and secreted il-6, il-8, il-10, il-12p40 and tnf were quantified by elisa. results: mph2 are relatively efficient phagocytes for apoptotic neutrophils whereas mph1 are only poorly phagocytic. opsonisation with ic leads to enhanced neutrophil uptake by both mph1 and mph2 which is specifically inhibited in the presence of a blocking mab for macrophage fcyrii. uptake of ic opsonised neutrophils causes a shift towards an anti-inflammatory cytokine profile. in both macrophage subsets il-6, il-12 and tnf production is suppressed while il-10 secretion is increased. in contrast, engagement of macrophage fcyr with ic alone induces the release of pro-inflammatory cytokines. conclusion: our data demonstrate that ic opsonisation of apoptotic neutrophils increases the proportion of macrophages capable of phagocytosis and that apoptotic cell recognition interactions provide a dominant anti-inflammatory signal, suppressing macrophage responses, even in the presence of ic opsonisation. we suggest that ic present in the inflammatory milieu would opsonise apoptotic neutrophils, enhance macrophage phagocytosis and thereby facilitate the process of resolution of inflammation. excessive production of reactive oxygen species (ros) produced by neutrophils is known to be a factor accelerating ageing because of damaging effect on cells. on the other hand, intracellular heat shock proteins (hsp) are involved in protecting cells from the damaging effects, and provide cell resistance to stress. in this work, correlation analysis was applied to analyze relationship between ros production and intracellular hsp70 in neutrophils of elderly people. neutrophils were isolated from peripheral blood of donors of 90 years old and older (long-livers). intracellular ros and hsp70 levels were registered by flow cytofluorimetry upon labeling with 2',7'-dichlorofluorescin diacetate (invitrogen) and anti-hsp70 antibody (brm-22, sigma), respectively. intracellular level of hsp70 was also estimated in neutrophils after heat shock (hs) performed at 43°c for 10 min. extracellular ros production from zymosan-activated neutrophils was detected by luminol-dependent chemiluminescence. a positive correlation was determined for intracellular ros level and zymosan-mediated extracellular ros release although the dynamics of ros release at 1-15 min time range varied within the group. the correlation was unaffected by hs of neutrophils performed for 1 min at 42°c, although this short heat treatment decreased significantly ros release. there was no correlation between basal intracellular hsp70 (hsp70 basal ) and ros level, both intracellular and extracellular. at the same time increased hsp70 level immediately after hs (hsp70 (0 min)) correlated negatively with intracellular ros (initial and after hs). the hsp70 increase value (hsp70 (0 min) -hsp70 basal ) correlated negatively also with intracellular ros and extracellular ros release in response to zymosan; and the correlation with ros level became lower when hsp70 increase was registered in 60 min after hs (hsp70 (60 min) -hsp70 basal ). thus it was found that within this age group the alteration in hsp70 induced by hs in neutrophils but not basal hsp70 itself is the parameter associated negatively with both spontaneous ros level and ros production in response to activating action of zymosan. this work is supported by istc grant #3303. d. goyeneche-patiño 1 , z. orinska 1 , f. mirghomizadeh 1 , s. bulfone-paus 1 1 forschungszentrum borstel, borstel, germany several studies have shown different roles of mast cells (mc) in innate and adaptative immune responses. in fact, crosstalk between cd8 + t cells and mc has shown to induce multiple genes implicated in the signaling of specific programs such as type 1 ifn. two novel genes, receptor transporter protein (rtp4) and virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible (viperin) are ifn inducible and were found to be over-expressed in chip analysis. the aim of this study is to characterize the expression and protein production of rtp4 and viperin in mast cells after tlr ligand stimulation in mice lacking of ifnra and the adapter proteins myd88 and trif. bone marrow derived mast cells (bmmc) from wt, ifnra -/-, mydd8 -/and trif -/mice, were exposed to tlr ligands (lps, pic, cpg, p(da-dt) and new castle disease virus (ndv)) during 8 and 48 h. mrna and protein extraction were performed for further qrt-pcr and sds page analysis for rtp4 and viperin. intracellular stimulation of tlr was performed transfecting cells with nucleic acids using lipofectamine 2000. stimulation of wt cells with pic, pda-dt and ndv showed an increased expression of viperin and rtp4 in comparison to control cells (untreated). the same trend was observed for mc from the trif and myd88 knockout mice. in contrast, in the ifnra deficient mice, expression of genes and protein production was abrogated to the same levels of wt untreated cells. lipofectamine stimulation does not increase the expression/production of the genes. direct stimulation of the well recognized viral sensors tlr 3 and 9, as well as, infection of mast cells with ndv (rna virus) induce the expression of rtp4 and viperin. the findings suggest that activation of mc with the ulterior expression of genes is type i ifn dependent. in contrast, the adaptor proteins myd88 and trif and the pathways that they represent are not relevant in the expression of rtp4 and viperin. these findings provide bases for performing further studies focused to elucidate the functions of these proteins and show an alternative role of mc in innate immune responses. in recent years, it has been suggested that the phenomenon of "myc-dependent cell competition" described in drosophila melanogaster, could be a critical step when a cell initiates nascent tumour field. we have taken a step forward and applied the phenomenon of cellular competition to the human macrophage system: inflammatory macrophages theoretically have the ability to eradicate cancer due to their tumoricidal capability and, at the same time, acting as antigen-presenting cells (apcs) to activate lymphocytes; but inflammatory macrophages do not express c-myc and, within the tumour, they encounter two powerful rivals: tumoral cells and alternative tumour-associated macrophages which express c-myc. we studied some phenomenons suggested to be myc-dependent such as the ability to feed, the ability to survive in a competitive medium, the ability to proliferate and the ability to eliminate competitors and we observed that alternative macrophages have more resources to survive in a tumoral microenvironment and could be involved in tumour growth by collaborating with tumour cells in transforming inflammatory macrophages into anergic cells which enter into apoptosis and are then phagocyted. finally, using lentiviral vectors, we over-expressed exogenous c-myc in inflammatory macrophages in an attempt to increase their chances of survival in the tumour microenvironment, in vitro and in vivo, and to determine whether it can be utilized as a potential anti-tumoral cell therapy. g. germano 1 , e. erba 2 , r. frapolli 2 , m. d'incalci 2 , a. anselmo 1 , s. pesce 1 , p. allavena 1 , a. mantovani 1, 3 1 humanitas clinical institute, rozzano, italy, 2 mario negri institute, milan, italy, 3 institute of general pathology, university of milan, milan, italy several lines of evidence suggest a strong association between chronic inflammation and tumor progression; therefore the use of anti-inflammatory drugs may be beneficial in anti-tumor therapies. inflammatory mediators (e. g. cytokines, chemokines) are produced at the tumor site both by tumor-associated macrophages (tam) as well as tumor cells, and are attractive target of novel anti-tumor therapies. trabectedin (et-743) is a natural product derived from the marine tunicate ectenascidia turbinate, it binds the minor groove of dna, affects transcriptional factor activity and blocks cell cycle. this novel anti-tumor agent is currently used in phase ii studies in patients with sarcoma, ovarian and breast cancer, with clinical regressions. we previously demonstrated that trabectedin is selectively cytotoxic, in vitro, to monocytes/macrophages, being active at concentrations that spared lymphocytes suggesting a possible alternative target for the anti-tumoral role of this drug. we tested the effect of trabectedin on primary cultures and liposarcoma cell lines showing that at sub-cytotoxic concentrations the production of some inflammatory mediators were down-modulated. trabectedin significantly reduces ccl2, cxcl8 and the inflammatory protein pentraxin3 (ptx3) either at transcriptional and protein level, especially after tnfa/il1b stimulation. down-regulation of ccl2, cxcl8, ptx3 and also of il6 and vegf were confirmed in primary cultures of liposarcoma. according to the previous in vitro data we now show in a mouse model , using the fibrosarcoma mnmcai , that trabectedin treatment selectively affects monocytes in the blood and bone marrow. moreover trabectedin treatment strongly reduces the number of macrophages and of cd31+ vessels in the tumor microenvironment, in line with its selective activity on monocytes/macrophages. overall these results suggest a possible triple role of trabectedin. besides its direct cytotoxic effect on tumor cells, trabectedin also affects tumor associated macrophages and at low dose the transcriptional activity of inflammatory genes involved in the tumor-microenvironment cross-talk. as the local expression of inflammatory mediators may play a role in tumor progression, this newly recognized effect of trabectedin makes it an attractive candidate in inflammation-associated tumors. interleukin-4 (il-4) is a key cytokine of the t helper 2 cell response. il-4 has been found to be a major regulator of immunoglobulin class switching to ige and has important functions in the regulation of allergic diseases. here, the onset of the il-4 production after birth was investigated in equine neonates. the form of equine placentation does not support the transfer of cytokines or immunoglobulins in utero and maternal immunity is exclusively transferred to the neonate with the colostrum after birth. il-4 producing cells were measured in peripheral blood mononuclear cells (pbmc) of neonates and foals by flow cytometric analysis. at day 3-6 after birth, a small population of il-4 producing cells was observed in the absence of any stimuli. the il-4 + population was not detectable at 6 or 12 weeks of age. other cytokine producing cells (ifn-g, il-10) were not detected using these conditions. the stimulation of neonatal pbmc with pma and ionomycin did not alter the il-4 + cell population. phenotyping of the neonatal il-4 + cells showed that they were ige + /mhcii -/cd4cells. the occurrence of cd4 + il-4 producing cells after pma stimulation increased slowly with age and did not reach adult levels by 12 weeks after birth. magnetic cell sorting of the ige + /mhciicells identified them as basophils. previous work has shown that foals do not produce endogenous ige for at least six months of life. ige bound to the surface of neonatal basophils was found to be of maternal origin and transferred with the colostrum after birth. here, the stimulation of neonatal pbmc with anti-ige induced the secretion of il-4 at day 5 after birth. neonatal pbmc collected before colostrum uptake did not produce il-4 in response to anti-ige. in summary, equine neonates provide a model to investigate ige mediated il-4 responses after birth. the transfer of maternal ige from allergic individuals could potentially provide a direct mechanism for the early induction of an allergen-specific neonatal il-4 response mediated by the mare's accumulated acquired immunity to allergens. s. schmechel 1 , d. voehringer 1 1 ludwig-maximilians-university munich, institute for immunology, munich, germany macrophages display broad phenotypic heterogeneity depending on their microenvironment. the initial inflammatory response to th1 cytokines is predominantly mediated by classically activated macrophages whereas macrophages undergo alternative activation in a stat6-dependent manner when stimulated with the th2 cytokines il-4 or il-13. alternatively activated macrophages (aam) are implicated in diverse disease pathologies such as host response to parasitic infection and asthma. furthermore, it has been shown that aam suppress the proliferation of t cells by a yet to be determined mechanism. currently there is still very limited information about the phenotype, migration and function of aam. we began to elucidate whether macrophage turnover and recruitment to inflammatory sites is regulated in a stat6-dependent manner. to this end we generated mixed bone marrow chimeras with bone marrow from congenic wild-type and stat6-deficient mice and infected these chimeras with the helminth nippostrongylus brasiliensis to determine whether lack of stat6 in macrophages affects their turnover and recruitment to the lung side-by-side in the same animal. the highest turnover of macrophages was found in the peritoneum, irrespective of stat6 expression. no major differences in tissue distribution and turnover were observed between both populations suggesting that macrophage proliferation and recruitment during parasite infection is not dependent on stat6 expression in macrophages. we could further confirm that in vitro generated aam from wild-type but not from stat6-deficient mice have a strong inhibitory effect on t cell proliferation. we are now trying to identify the mechanism(s) by which t cell proliferation is inhibited. furthermore, we work on the cellular cross-talk between eosinophils and macrophages and try to determine the plasticity of macrophage differentiation. we have previously shown that aam can recruit eosinophils to inflammatory sites and we now try to clarify which chemotactic factor are involved in this process. to identify potential aam-derived eosinophil chemotactic factors we currently compare the gene expression profile of il-4 exposed macrophages from wild-type and stat6-deficient mice. candidate genes will be expressed using retroviral transfections of stat6-deficient macrophages and supernatants from these cells will then be used to induce eosinophil recruitment in transwell assays. macrophages are an essential component of leukocytes infiltration in the tumor. they are identified as tumor associated macrophages (tams). these cells are also present in pleural effusions which appear as a consequence of spreading of neoplasm in the pleural cavity. the aim of the study was to assess the influence of the pleural macrophages on cells from human malignant cell lines. we tested the dynamics of growth of the malignant cells, their apoptosis and expression of proteins regulating this process under the influence of conditioned media from cultures of macrophages isolated from pleural effusion. we have also attempted to interpret our results by assessing the expression of a variety of immune modulating factors, their receptors on the malignant cells surface as well as the transcription factors. in the study we used macrophages isolated from a total of 38 pleural effusions, including 15 malignant and 23 nonmalignant tumors. the following human malignant cell lines were tested: a549, ht29, hct116, sw60, mcf7, mda-mb231, jurkat and hl60. results: our results suggest that the conditioned media isolated from the cultures of pleural macrophages can up-regulate the proliferative activity of the human malignant cell lines. macrophages from pleural effusions can act as a factor promoting or inhibiting apoptosis of malignant cells. down-regulation of apoptosis may depend on modulation of expression and activity of proteins regulating this process. macrophages can affect the apoptosis regulatory proteins and their activity through the immune-modulatory molecules, e. g. cytokines, chemokines, and growth factors. the up-or down-regulation of transcription factors expression may control the expression of pro-and anti-apoptotic proteins. the results indicate that macrophages from malignant and non-malignant pleural effusions differ from each other insignificantly; however the macrophages isolated from the non-malignant tumors show a pattern comparable to m1, and the tams isolated from the malignant effusions similar to m2. among the alternative stimuli, glucocorticoids are the most effective stimulus up-regulating ms4a4a and ms4a6a: highest trascriptional level after 18h of stimulation with 10-6m dexamethasone. ms4a murine genes are differently expressed respect to the human counterpart and only the homologs of ms4a6a (ms4a6b, 6c and 6d) have a similar regulation. finally, egfp-tagged ms4a4a, ms4a6a, and ms4a7 expressed in cho cells showed that all molecules traffic to the cell membrane. though the biological functions of these ms4a proteins has not jet been defined, their membrane localization and the structural relationship with other better characterized ms4a members suggest a potential involvement in signal transduction, either as components of multimeric receptor complexes or as components of ligand-gated ion channels. during inflammatory reactions endogenously produced cytokines and chemokines act in a network and interact with hormones and neurotransmittors to regulate host immune responses. these signaling circuitries are even more interfaced during infections in which microbial agonists activate toll-like (tlr), rig-like (rlr) and nod-like (nlr) receptors. on the basis of the discovery of synergy between chemokines for neutrophil attraction, we here extended this phenomenon between the chemokine monocyte chemotactic protein-1 (mcp-1)/ccl2 and the gpcr ligand fmlp or the tlr4 agonist lipopolysaccharide (lps) on monocytes. in fact, the bacterial tripeptide fmlp, but not the cytokines il-1b or ifn-g, significantly and dose-dependently synergized with ccl2 in monocyte chemotaxis. furthermore, lps rapidly induced the expression of interleukin-8/cxcl8, but not of the ccl2 receptor ccr2 in monocytic cells. in turn, the induced cxcl8 synergized with ccl2 for mononuclear cell chemotaxis and the chemotactic effect was mediated by cxcr1/cxcr2, because cxcl8 receptor antagonists or antibodies were capable of blocking the synergy, while keeping the responsiveness to ccl2 intact. these data recapitulate in vitro the complexity of innate immune regulation, provide a novel mechanism of enhancing monocyte chemotaxis during bacterial infections with gram-negative bacteria and demonstrate the importance of local contexts in inflammatory and infectious insults. objectives: in recent years the existence and effects of cell-derived vesicles (e. g. exosomes, microparticles) have been revealed in several physiological functions, such as antigen presentation, hemostasis or receptor transfer to innocent cells. most data were collected on endothelial cells and on thrombocytes. however, there are only few data on vesicles derived of neutrophilic granulocytes (pmn), and most of these investigations applied only pharmacological agents. our aim is to investigate pmn-derived cell-free particles and their possible role in bacterial killing methods: preparation of pmn and investigation of bacterial killing by our semi-automatic method was described by rada et al. (blood, 2004) . cell-free vesicles were prepared after co-incubation of human pmns with different activating agents for 20 min at 37°c with gentle shaking, followed by spinning down of pmns for 5 min, at 4°c and 500g. the supernatant was sedimented at 15000g for 10 min, 4°c, and we used the sedimented fraction for our investigations. formation of particles was followed by fluorescent and electron microscopic assays. the amount of particles was estimated with flow cytometer and by their protein content. we observed that upon co-incubation of pmns with s. aureus, opsonized by mixed human serum, pmns produce a well detectable amount of vesicles. omitting opsonization or opsonizing with heat inactivated serum caused a minimal amount of particles. production of particles could be inhibited with diphenyl-iodonium (dpi), cytochalasin b (cb) or with azide treatment. treating pmns with dnase or withdrawing glucose during co-incubation had no effect on vesicle formation. in killing assays we detected remarkable antibacterial effect, which correlated well with the protein content of the used fraction. this antibacterial activity could be inhibited by dpi, cb, azide treatment or by withdrawing glucose from the medium during the killing assay. however, treatment of the microvesicles with dnase had no effect on their antibacterial capacity. for long, cd56 has been used for the detection and identification of natural killer (nk) cells. recently, the presence of a minor subset of cd56 low cd33+ blood monocytes (mo) in healthy individuals and the increase in cd16+cd56+ blood mo in patients with inflammation has been reported. the functional activity of human cd56+ blood mo has been studied in vitro but not tested ex vivo so far. healthy people living permanently in malaria endemic areas are exposed to plasmodium infection, and we hypothesized that blood mo of these individuals could be activated and display increased cd56 expression. we tested if this phenotypic expression was associated with detectable changes in the mo anti-parasitic activity. the mo phenotype of healthy malaria naï ve and malaria exposed individuals was determined by three-color flow cytometry. myeloid cell markers included cd33 and activation markers such as hla-dr and trem-1. percentages of blood mo involved in phagocytosis activity either with or without immune sera were then identified by flowcytometry, and the potential association between a given mo phenotype and phagocytosis activity was then looked for, using spss ® and statview ® softwares. our results showed that, compared with malaria naï ve individuals, there was a 12.3 fold increase (p x 0.0001) in the total number of circulating cd56 low mo present in the blood samples of healthy malaria exposed asian individuals living in thailand. according to the density of surface antigen determined by fluorescence intensity (fi), the decrease in cd33 and the concomitant increase in hla-dr expressions indicated that in this malaria endemic area, blood mo were mature and highly activated by comparison with surface markers of mo from malaria naï ve donors. the relative levels of cd56+ blood mo were associated with the percentages of membrane-bound ifn-g present at the mo surface. in conclusion, (i)-a subset of cd33+ blood mo expressed increased levels of cd56 on mo of healthy malaria exposed individuals; (ii)-blood mo with activated (hla-dr+) and mature (trem-1+) phenotypes were present in these healthy individuals; (iii)-increased expression of hla-dr and cd56 on cd14 high mo was associated with a high phagocytosis activity. introduction: adipokines, initially described for their function within metabolism, have been characterized to exert a regulatory role on the immune response. for instance the appetite-regulating hormone leptin has been identified to modulate the response of the innate as well as the acquired immune system. the present work focuses on the effects of leptin on the reactivity of m1-and m2-polarized human macrophages. methods: monocytes were isolated from the peripheral blood by magnetic cell sorting. polarization to m1 and m2 macrophages was induced by culture in the presence of mcsf or gmcsf respectively. polarized cells were characterized by flow cytometry, stimulated with lps and response assessed by characterization of cytokine profiles via cytometric bead array (cba). results: culture of monocytes in the presence of mcsf or gmcsf induced two different phenotypes. cells cultured in the presence of gmcsf represented the m1 type and were cd14 negative but cd80 and mhcii positive and produced high levels of il-8, tnfalpha and il-6 following lps stimulation. culture in the presence of mcsf resulted in induction of the m2 phenotype. these cells were cd14 positive with intermediate expression of cd80 and mhcii expression and produced high levels of il-10, il-6 and il-8 following lps stimulation. interestingly, already baseline il-8 production was high in these cells. stimulation with leptin alone increased cytokine production in both cell types as compared to cells cultured in medium alone. however, if leptin was present in cultures stimulated with lps, the induction of cytokine production was significantly reduced in both, m1-and m2-polarized cells as compared to cells stimulated with lps alone. summary: whereas presence of leptin enhances baseline cytokine production in polarized macrophages, it reduces the cytokine production in response to stimulation with the tlr4 ligand lps. thus, abundant leptin levels like present in obesity or in the hypertrophied fat as present in crohn's disease patients might exert modulating effects on macrophage response to bacterial antigens. methods: hl60 cell line was used as a model of leukemic myeloid cell differentiation cultured in suspension or on fibronectin matrix prior to pma (50 ng/ml) treatment for 48h. morphological evaluation was performed with conventional microscopy and electron microscopy. immunephenotype and phagocytic activity of the cells were determined by flow cytometry and immunocytochemistry. a colorimetric nitro-blue-tetrazolium reduction assay was performed to assess the production of reactive oxygen species (ros). results : besides their distinctive macrophage morphology and ultrastructure with spindle cell-like features and high granularity, the pma-treated fibronectinadherent hl60 cells expressed antigen receptors cd14, tlr2, tlr4 and cd68 , and displayed enhanced phagocytic activity and production of ros. expression of cd13, cd33 and cd15 was also maintained however the cells were hla-dr and cd1a negative. conclusion: we describe the enhanced ability of fibronectin-adherent hl60 cells to differentiate into macrophages in response to pma. hl60 may provide a functional model for macrophage differentiation. above all, this finding may stimulate further research on myeloid leukemia biology and potential adjuvant therapies. a. aporta 1 , n. ferrer 2 , a. gómez 2 , j. gonzalo 2 , a. arbués 2 , a. anel 1 , c. martín 2 , j. pardo 1 , apoptosis, immunity and cancer 1 university of zaragoza, molecular and cellular biochemistry and biology, zaragoza, spain, 2 university of zaragoza, mycobacterium genetics, zaragoza, spain mycobacterium tuberculosis is an intracellular pathogen that uses alveolar macrophages as its preferred habitat, being capable of produce both a progressive disease and an asymptomatic latent infection. it has been postulated that infected macrophage apoptosis may contribute to host defence against this intracellular infection by, firstly, eliminating supportive environment for bacterial growth and, secondly, by leading to the formation and release of apoptotic vesicles containing mycobacterial antigens. it has been proposed that m. tuberculosis inhibits host cell apoptosis thus interfering with the immune system response. however the biological relevance of this process is not clear. our group has generated so2, a m. tuberculosis phop mutant strain that was shown (perez et al 2001) to be more attenuated than the present attenuated vaccine strain bcg and conferred protective immunity against m. tuberculosis infection in mice and guinea pigs (martin et al 2006) . in the present study, we compare the time course and phenotype of cell death induced by so2, bcg and wild type m. tuberculosis on the murine macrophage cell line j774 and on bone marrow derived mouse macrophages. our results indicate that wild type m. tuberculosis induces macrophage cell death analysed by a clonogeneic assay much faster than the attenuated bacteria. of note cell death presented apoptotic features like caspase-3 activity and nuclear condensation. in order to analyse the consequences of this apoptotis-like cell death, it has been invetigated whether dead cells translocate phosphatydilserine to the outer part of the plasma membrane and if this traslocation is enough to promote phagocytosis by fresh macrophages. experiments are ongoing with macrophages derived from trl2x4 deficient mice and wt animals in order to study the role and implication of those receptor on the susceptibility to infection and death induced by the virulent and attenuated phop m. tuberculosis strain. objectives: vip is a potent anti-inflammatory peptide, mainly acting as endogenous macrophage deactivating factor. type 1 receptor for vip (vipr1) gene is highly conserved through species and, in humans, is highly polymorphic. vipr1 has been reported to be down-modulated in cells of the immune system after activation. an association of some snps with some autoimmune diseases has also been reported. in this study we have investigated the correlation between these snps and gene expression in monocytes exposed to lps. methods: monocytes from 53 blood donors were separated from pbmc and stimulated with lps. total rna was reverse transcribed and the level of vipr1 in untreated or lps-stimulated monocytes was measured by real-time rt-pcr and protein expression. protein level was measured by western blot and densitometric analysis. the kinetic of expression of vipr1 after 3, 6, 9 and 12 h of exposure to lps was firstly analysed in monocytes from five individuals. there were two kinetics: one in which a reasonable high levels ( g 50 %) of mrna was maintained trough time and a second one in which the decrease of mrna was pronounced. the experiments were repeated using monocytes from 53 donors that had been typed for the relevant vipr1 snps. the down-regulation of vipr1 correlates with the presence of a t at rs896 mapping in the 3'-end of the gene (p= 0.004). the vipr1 protein level was decreased about 40 % in monocytes of 3 subjects typed as t/t at rs896 whereas 3 subjects typed as c/c at rs896 maintained a high level of expression after 9h of lps treatment. the data show that different haplotypes of the vipr1 gene correlate with a different kinetics of gene expression in activated monocytes. a possible consequence of these data is that the anti-inflammatory properties of vip governed by the vipr1 vary in different individuals and can eventually contribute to the genetic predisposition to some autoimmune diseases. j. oujezdska 1 , t. vavrochova 1 , d. filipp 1 , immunobiology 1 institute of molecular genetics as cr, immunobiology, prague, czech republic phagocytes which appear in early mouse development (e7.5-13.5) represent a unique embryonic macrophage lineage that differs from adult macrophages phenotypically, biochemically and by their origin. recent studies suggested that there are at least three waves of macrophages populating an early embryo: a maternallyderived one and two waves of extraembryonal, ys-derived phagocytes. in addition, the occurence of early embryonic phagocytes of undetermined origin in the anterior head mesoderm in several invertebrate and vertebrate species is well documented. this origin-related heterogeneity among early embryonic phagocyte subpopulations coupled with the lack of their specific surface markers makes it difficult to distinguish them phenotypically and study their potentially distinct physiological roles in early development. the aim of this study is to identify a set of surface markers expressed on embryonal phagocytes suitable for phenotypic distinction among embryonic phagocyte subpopulations. here we report the temporal and spatial expression of toll-like receptors (tlrs) and cd14 in the early mouse embryo (me). facs analysis of cell suspension prepared from 10.5 day me showed that about 0.7-1 % of cells were positive for cd11b. these cells exclusively were also positive for cd14, tlr2, and cd45 antigens. using qpcr and flow cytometry we show that tlrs and other tir domain-containing signaling molecules are expressed in the embryo through embryonic days 7,5-13,5. reciprocal matings between wild type and mhcii-egfp knock-in mice revealed that while maternallyderived mhcii + macrophages are present in the embryo from early developmental stages (e7,5), embryo-derived mhcii + macrophages start to appear in the embryo around day 13. multicolor facs analysis of cd11b, cd45, cd14, f4/80, tlr2, tlr4, c-kit and mhcii surface markers revealed differential expression of tlr2 and c-kit on embryonal phagocyte subpopulations. moreover, the microarray analysis of cd11b + tlr2 + cells isolated from the e10,5 embryos has revealed significantly upregulated expression of several novel genes in comparison to their expression in murine peritoneal macrophages. these molecules are currently being tested for their use as embryonic phagocyte specific-lineage markers. these results are first to characterize the regulated expression of tlrs on early embryonal phagocytes and demonstate their potential to serve as novel markers for their detection and isolation. humans may be exposed to a variety of mycobacteria ranging from environmental or bcg vaccine to more pathogenic mycobacteria. only a minority of individuals exposed develop disease, this susceptibility may result in part from variability of host immune responses genes through simple (mendelien disease) and complex (polymorphisms with milder effect) inheritance mechanisms. interestingly, key elements of inflammatory pathways are particularly involved in this susceptibility to mycobacteria. il12/il23-dependent ifng pathway of macrophage activation plays a central role in inflammation and cell-mediated immune responses to mycobacteria. due to the high rate of consanguineous marriages in the north african countries, recessive genetic disorders including primary immunodeficiencies occur with a relatively high prevalence. in tunisia, among patients affected with primary immunodeficiencies 16 presented with disseminated bcg infection (bcg-osis). among them, five have an underlying well-defined primary immunodeficiency either a severe combined immunodeficiency or a chronic granulomatous disease and 11 have a mendelien susceptibility to mycobacterial disease. using a candidate gene strategy, we have identified in 9 out of these 11 patients mutations in several ifng pathway genes, other candidate genes are being investigated for the 2 other patients. in the general population, common polymorphisms with milder effect on the risk of tuberculosis have been identified including mhc and nramp1. we did focus on the study of 2 genes which are considered as important pathogen recognition receptors of the innate immune system: tlr2 is the principal mediator of macrophage activation in response to mycobacteria through nfkb pro-inflammatory signaling pathway and dc-sign is the major receptor of m. tuberculosis on human dendritic cells and in contrast induces anti-inflammatory il-10 cytokine. using a case/household-contact cohort we did investigate polymorphisms of these 2 genes in tunisian patients affected with active pulmonary tuberculosis and have shown specific patterns of snp and microsatellite polymorphisms associated with susceptibility/resistance to tuberculosis. host inflammatory responses play a major role in granuloma formation and control of the infection. unraveling these pathways might be crucial in order to identify new therapeutic targets and strategies including immunotherapy e. g. ifng therapy for tuberculosis, particularly in this era of emergence of multi-drug and extensively-drug resistant m. tuberculosis strains. francisella tularensis is a gram negative bacterium that is the causative agent of tularemia. research into francisella has expanded over recent years due to its designation as a potential biological warfare agent. several species of francisella exist and have varying degrees of pathogenicity. f. tularensis live vaccine strain (lvs) is an attenuated strain of the holarctica subspecies and has been shown to be an effective vaccine in humans. however, it is pathogenic in mice which can, therefore, act as a useful model of human tularemia. f. tularensis is an intracellular pathogen and is able to invade several different cell types, in particular macrophages, most commonly through phagocytosis. therefore, if phagocytosis could be disrupted via the addition of inhibitors, uptake of f. tularensis would decrease and antibiotic treatment may be more effective. a flow cytometric assay was developed to measure bacterial uptake. this method used a fitc labelled anti-f. tularensis antibody in conjunction with antibodies to cell surface markers to determine specific cell phenotypes that were positive for bacteria. a series of phagocytic inhibitors have been tested in vitro on an alveolar macrophage derived cell line (mhs) and on ex-vivo mouse lung tissue to determine whether uptake of f. tularensis lvs could be altered. the presented data shows that several inhibitors work efficiently to reduce lvs uptake by up to 70-80 % in both the in vitro and ex vivo assays. however, cytotoxicity of some of the inhibitors was high and, therefore, it was essential to concentrate on inhibitors with low cytotoxicity for further assessment. in addition, bacteriological data suggests that the combination of inhibitors with antibiotics may be a useful therapeutic against f. tularensis. it may also work against other intracellular pathogens that use phagocytic mechanisms to enter their optimal niche.ã crown copyright. dstl, 2009. hsp70 are intracellular proteins but it is known that these proteins can be expressed on cell surface and contained in extracellular medium, in particular in peripheral blood serum. it is also known that extracellular hsp70 have pronounced immunomodulatory properties. to study the pathways of the protein modulating action on immune system we investigated effect of exogenous and cell surface hsp70 on reactive oxygen species (ros) release from phagocytes, namely human neutrophils, during process of phagocytosis (respiratory burst). neutrophils were isolated from human peripheral blood by using a standard protocol. respiratory burst induced by opsonized zymosan was measured by method of luminol dependent chemiluminescence. for the experiments human recombinant hsp70 (low endotoxin) and paraformaldehyde fixed mouse thymocytes exposed surface hsp70 were used. exogenous hsp70 was used in concentration 1-10 ug/ml, fixed thymocytes were added to neutrophil samples in quantitative ratio 1:1 and 2:1 directly before the measuring. as the control we registered amplitude of oxidative burst in samples supplemented with pbs or live mouse thymocytes having no hsp70 on their surface. results demonstrating effect of exogenous hsp70 on phagocytosis-induced ros release from human peripheral blood neutrophils have been obtained. it was demonstrated marked dose-dependent inhibiting action of exogenous hsp70 on amplitude of respiratory burst. the cells expressing surface hsp70 impacted on ros production in this model similarly. the results of chemiluminescence analysis demonstrated that zymosan induced ros production was essentially decreased under action of fixed thymocytes, and was decreased slightly in presence of live thymocytes in the neutrophil samples. the effect was more pronounced for increased amount of thymocytes added to the samples. thus, immunomodulatory effects of exogenous hsp70 might be caused by influence of the protein on ros release from phagocytes. we suppose that the registered effects are connected with ability of hsp70 to inhibit activity of nadp-oxidase -the key enzyme for ros production during respiratory burst. results: we recruited 28 pts, with so far five complete pathological remission, five partial responses and five no responses. no substantial changes were detectable in the number of circulating monocytes. in contrast we observed a clear expansion of cd14/cd86 and cd14/cd163 double positive subsets. this event was transient; it abated at the later time point suggesting a causal relationship to the treatment. it correlated with sensitivity to the treatment. in fact we observed that in the responder patients the expansion of the cd14/86 subset was clear in the first weeks of treatment and decreased there after. in contrast in non-responder patients it was already expanded before the neo-adjuvant therapy. all the patients had an initial expansion of the cd14/163 subset. in the responder patients this population was still present at the time of surgery. the immunohistochemical study revealed a massive tumoral infiltration by macrophages that displayed clear features of alternative m2 polarization. conclusion: these data suggest that neo-adjuvant therapy modulates the cellular components of innate immune responses that could represent valuable predictive factors. m. dimitrijević 1 , i. pilipović 1 , s. stanojević 1 , k. mitić 1 , k. radojević 1 , v. pešić 2 , g. leposavić 1,2 1 institute of virology, vaccines and sera "torlak", immunology research centre "branislav janković", belgrade, serbia, 2 faculty of pharmacy, university of belgrade, department of physiology, belgrade, serbia the primary aim of our current study was to ascertain whether rat resident peritoneal macrophages synthesized catecholamines and to unmask putative effects of catecholamines on nitric oxide (no) and hydrogen peroxide (h 2 o 2 ) production and phagocytic activity of these cells. in addition, given that chronic administration of b-adrenoceptor antagonist increases the density of b-adrenoceptors on both non-immune and immune cells and thereby affects their sensitivity to catecholamine action, we hypothesized that such treatment could also affect macrophage responsiveness. to address our proposition, we determined adrenoceptor expression on peritoneal macrophages from rats subjected to 14-day-long propranolol treatment and measured both no and h 2 o 2 production and phagocytic activity of these cells. using both immunocytochemical and flow cytometric analyses of rat peritoneal exudate cells constitutive expression of tyrosine hydroxylase and both b 2 -and a 1 -adrenoceptors on macrophages was revealed. furthermore, according to the characteristic assemblage of tyrosine hydroxylase and adrenoceptor subtype expression different macrophage subsets were identified. in vitro treatment of macrophages with the non-selective a,b-adrenoceptor agonist arterenol and/or the b-adrenoceptor antagonist propranolol indicated that b-adrenoceptors potentiated no production and suggested a-adrenoceptor-mediated suppression of hydrogen peroxide h 2 o 2 production. an increase in h 2 o 2 production in the presence of the a 1 -adrenoceptor antagonist ebrantil provided support for this. chronic propranolol treatment in vivo led to increased no and h 2 o 2 production by peritoneal macrophages. furthermore, this treatment resulted in opposing effects on the expression of b 2 -and a 1 -adrenoceptors on peritoneal macrophages (a stimulatory effect on b 2 -adrenoceptors and a suppressive effect on a 1 -adrenoceptors). in conclusion, a subset of resident peritoneal macrophages synthesizes catecholamines, which may exert differential effects on h objectives: monocytes display great phenotypical and functional heterogeneity and are divided into two major subsets: cd14 ++ cd16 -('classical') and cd14 + cd16 + ('pro-inflammatory') monocytes. a central monocyte function is cytokine production in response to toll-like receptor (tlr) ligation. the cd14 + cd16 + monocytes display higher tlr2 and -4 expression, produce higher levels of pro-inflammatory cytokines and have increased potency for antigen presentation than the cd14 ++ cd16monocytes, suggesting that the two subsets could play different roles in antimicrobial responses. newborns are vulnerable to infections and an immaturity of both adaptive and innate immunity has been described. studies of neonatal monocyte antimicrobial responses show contrasting results and much remains to be learned, especially regarding monocyte subpopulations. thus we aimed to compare monocytes from newborns and adults, focusing on monocyte subpopulations and responses following tlr2 stimulation. methods: cord blood (n=8) and peripheral-blood (n=8) mononuclear cells were stimulated in vitro for 24hrs with peptidoglycan and subsequently analysed for cd14 and cd16 and intracellular il-12p70 and tnf expression. the mann-whitney u-test was used to evaluate differences between groups. results: a significantly higher percentage of neonatal monocytes were positive for il-12p70, both unstimulated and after peptidoglycan stimulation, as compared to adults. geomfi of il-12p70 was low and similar between groups, although significantly higher in newborns after stimulation. in both newborns and adults, il-12p70 (% positive cells and geomfi) was significantly higher for cd14 + cd16 + cells than for cd14 ++ cd16cells, unstimulated and stimulated. regarding tnf, neonatal and adult monocytes did not differ in unstimulated cultures, however geomfi of tnf was significantly higher in neonatal monocytes after stimulation. whereas the tnf response following stimulation was similar between the adult monocyte subsets, in newborns the cd14 ++ cd16cells were positive for tnf to a significantly higher extent than the cd14 + cd16 + cells. in particular the tnf response to tlr2 stimulation differed between newborns and adults, with neonatal monocytes having a higher per cell production of the cytokine. notably, in newborns the cd14 ++ cd16monocytes were positive to a higher extent for tnf following stimulation pointing towards a functional immaturity of neonatal monocyte subset responses. objective: chronic granulomatous disease (cgd) is an uncommon congenital phagocyte disorder characterized by recurrent life-threatening infections. cgd generally present with recurrent suppurative infections; however, intracranial fungal abscess complicating cgd may cause a diagnostic problem to anyone who is unfamiliar with its clinical and radiological features. we report a 16-year-old boy who admitted with complaints of seizures during the previous 2 months. there was a history of axillary and perianal suppurative skin infections and cavitary pneumonia. the family history was unremarkable, and the parents were unconsanguineous. physical examination was only remarkable for oral moniliasis and skin scars at axillary and perianal region. a large frontol mass with diffuse peripheral vasogenic edema was discovered on mri. subfalcine herniation was noted secondary to mass effect. cgd was suspected and the analysis with flow cytometric dihydrorhodamine assay (dhr assay), for functional analysis of neutrophils was compatible with the diagnosis of cgd and no bimodal histogram pattern spesific for x-cgd was found in the mother and sister. after the diagnosis of cgd, neurosurgical removal of the abscess cavity was performed due to peri-lesional edema and herniation risk. aspergillus fumigates grew from the culture; liposomal amphotericin b and voriconazole were started; which were found to be sensitive to the cultured species. in addition, interferon-g (50 mgr/m2/day, subcutaneously every other day) was started. after 2 months, control mri showed regression of the lesion, and the anti-fungal treatment was continued for 3 months. the screening of the other family members with dhr assay demonstrated that one of his sisters had also cgd and phenotype was autosomal recessive. mutaton analysis in "hot spot" in ncf1 gene concerns the well-known gt deletion in the second exon of ncf1 gene both at the patient and his sister. results: this was an atypical clinical presentation of cgd in an adolescent boy with cerebral aspergillosis, mimicking intra-cranial tumor. we documented a good response to the combination of ifn-g, liposomal amphotericin b and voriconazole after surgery. conclusion: cgd should be considered in the differential diagnosis for all children presenting with invasive fungal infections particularly, those involving the central nervous system. recent data suggest that ubiquitin has anti-inflammatory properties and therapeutic potential after severe trauma and brain injuries. therefore, we hypothesized that ubiquitin treatment can modulate the local inflammatory response triggered after brain injury. to test this hypothesis, a focal cortical contusion was induced using a controlled cortical impact (cci) model in sprague-dawley rats. animals (n = 45) subjected to moderate brain injury were randomized, and received either 1.5 mg/kg ubiquitin or vehicle (placebo) intravenously within 5 min after cci. levels of tnf-a, il-1b, il-6, il-10 and il-1 receptor antagonist were analyzed in brain tissue using real time rt-pcr at 4 and 72 hours after treatment. immune cell infiltration was studied by immunostaining for neutrophils and macrophages/ microglia at 24h and 7 days. data were analyzed with the mann-whitney u test and a two-tailed p x 0 .05 was considered significant. all cytokines were highly up-regulated 4 hours after cci but no differences between the groups were observed at this time point. three days after trauma the levels of il-10 were significantly lower in the ubiquitin treated animals, whereas the levels of il-6 and tnf-a were higher when compared to the placebo group. interestingly, macrophages/ activated microglia were significantly increased in the pericontusional cortex after ubiquitin treatment at day 7. the infiltration of neutropils was not affected by ubiquitin treatment. here, we could demonstrate for the first time that a single injection of ubiquitin immediately after brain trauma is able to modulate the inflammatory response triggered after brain injury at the cellular as well as at the cytokine level. macrophage activation and oxidative metabolic changes are commonly implicated in pulmonary tuberculosis (ptb) patients. efficient plasma antioxidant activities are needed to neutralize high free radical load in pulmonary tuberculosis (ptb) patients. there is limited information about the plasma levels of neopterin (a marker of macrophage activation) and oxidative stress indices such as total plasma peroxide (tpp), total antioxidant activity (taa), malondialdehyde (mda), and oxidative stress index (osi) in ptb patients during chemotherapy with or without micronutrient supplementation. the present study was designed to assess the levels of neopterin, tpp, taa, mda, and osi during chemotherapy with (c+m) or without (c-m) micronutrient supplementation using elisa and spectrophotometric methods. thirty-eight (38) newly diagnosed ptb patients and forty non-ptb apparently healthy subjects volunteered to participate in this study. twenty of the ptb patients were on anti-tuberculosis drugs supplemented with micronutrients (c+m) while 18 were treated with anti-tuberculosis drug alone (c-m) for a period of four weeks. the levels of neopterin (p=0.02), tpp (p=0.00), osi (p = 0.00), mda (p = 0.00) were significantly raised but taa (p = 0.01) was significantly reduced in ptb patients compared with controls. the levels of mda (p = 0.04), neopterin (p=0.00) and tpp (p=0.00) were significantly reduced in c+m after two weeks of treatment compared with baseline values before commencement of treatment. the levels of tpp (p=0.00), mda (p=0.00), neopterin (p=0.02), osi (p=0.00) were significantly reduced while taa (p=0.01) was significantly raised in c+m after 4 weeks of treatment compared with the baseline concentrations. in c-m, only mda showed significant decreased after 4 weeks of treatment when compared with the baseline values. plasma level of neopterin, tpp, osi and mda declined faster in c+m than c-m. therefore, micronutrient supplementation of ptb drugs with synthetic antioxidants or naturally occurring ones (fruits and vegetables) should be attempted. this will improve deranged macrophage activation and reduce oxidative stress indices in ptb patients. a. p. aguas 1 , e.m. cunha 1 , m.j. oliveira 1 1 icbas, university of porto, anatomy, porto, portugal the acute in vivo intake of mercury (hg) microparticles (20 nm in diameter) by neutrophils and macrophages was studied with the use of in situ detection of hg by scanning electron microscopy coupled with x-ray elemental microanalysis (sem-xem). the intracellular distribution of hg particles was compared, at high resolution, between macrophages and neutrophils, and between activated and non-activated phagocytes. balb/c mice were injected intraperitoneally (ip) or in a subcutaneous air-pouch with mercury chloride, and the animals were sacrificed up to 5 minutes after the injection. in some mice, before the hg injection, peritoneal phagocytes were activacted by ip injection of bsa. pre-injections with a selenium (se) salt were also performed in order to study the putative modulatory role of se on hg intake by phagocytes. peritoneal cells were collected by washing of the peritoneal or subcutaneous cavities with pbs, they were cytospinned, fixed with formaldehyde, and processed for observation by sem-xem. five min after the hg injection more than half of the mouse phagocytes were positive for hg. a higher percentage (70 %) of macrophages contained the metal particles than neutrophils (55 %). phagocyte activation enhanced the number of hg particles seen inside the phagocytes. pre-injection of the peritoneal cavity of mice with se resulted in finding that more than half of the hg intracellular particles were coupled with se. subcellular topography of hg particles showed that they were presented in individual small cytoplasmic vesicles. we conclude that hg microparticles are rapidly ingested by macrophages and neutrophils, a processed that is enhanced by cell activation. hg particles are ingested by pinocytosis and sorted in the cytoplasm of macrophages and neutrophils inside individual small vesicles. this study was supported by a grant from fct, portugal. mast cells play central roles in allergic inflammatory reactions and innate immunity. swap-70 is a rac-interacting protein expressed in several cells types of the hematopoietic system including mast cells. in b cells and mast cells swap-70 regulates f-actin cytoskeletal rearrangements, cell polarisation and cell migration. (pearce et al., 2006; sivalenka and jessberger, 2004) . swap-70-/-bone marrow derived mast cells (bmmc) are specifically impaired in fceri-mediated activation and degranulation and in c-kit-induced activation, migration and cell adhesion (gross et al., 2002; sivalenka and jessberger, 2004; sivalenka et al., 2008) . crucial regulators of these processes are members of the rho family of small gtpases such as rac1 and rhoa. swap-70 interacts with rac1 in vitro and preferentially binds the active gtp-bound rac1. swap-70 supports the increase of active rac1 in vitro by a yet to be defined mechanism (shinohara et al., 2002) . in this study, in vitro pull-down assays with purified recombinant proteins were employed to characterize the interaction between swap-70 and rac1. it was found that fulllength swap-70 preferentially binds to constitutively active rac1 (rac1q61l) but not to its dominant negative form (rac1t17n). binding assays with swap-70 truncated mutants showed interaction of swap-70's n-terminus with gtpgs rac1 or rac1 depleted of guanine nucleotide, whereas swap-70 central or c-terminal regions do not bind to any form of rac1. preliminary competitive-binding assays with overlapping 18mer peptides, spanning the entire swap-70 sequence, mapped the rac1 binding site near the n-terminus of swap-70. full-length swap-70 site-specific mutants will be generated to test the relevance of these interactions in mast cells in terms of adhesion, migration and activation of rho gtpases. elucidating the molecular interactions of swap-70 with rho gtpases and the relevance of these will shed light on the biology and biochemistry of mast cells and possibly other hematopoietic cells, which express swap-70. v. c. barbosa 1 , c. d. polli 1 , m.c. roque-barreira 1 , m.c. jamur 1 , c. oliver 1 , g. pereira-da-silva mast cells are essential cells in ige-associated immune responses. fceri crosslinking induces mast cell degranulation and release of proinflammatory mediators. we have previously shown that the lectin artinm induces mast cell activation but the mechanisms involved in this activity remain unknown. objective: the present study was undertaken to further characterize the ability of artinm to activate mast cells. methods: rbl-2h3 cells were sensitized with ige anti-tnp and stimulated with dnp 48 -hsa or artinm. artinm binding to rbl-2h3 cells was assessed by flow cytometry. mast cell degranulation was determined by measurements of released b-hexosaminidase activity. microplate binding assays were utilized to assess artinm binding to ige. to investigate fceri recognition by the lectin, western blots of cell lysates were stained with biotinylated artinm and be's antibodies specific for fceri b-subunit. intracellular protein phosphorylation was detected by specific antibodies and analyzed by confocal microscopy. mcp-1 and tgf-b levels released by mast cells were measured by elisa. results: artinm binding to the cell surface was dependent on sugar recognition and resulted in mast cell degranulation in the presence or absence of ige. the release of b-hexosaminidase doubled when cells were sensitized by the immunoglobulin and was abrogated in the presence of d-mannose, suggesting that mast cell degranulation induced by artinm might be the result of interactions between the lectin crds and glycosylated components on the cell surface, like fceri or ige. indeed, it was observed that the lectin bound to ige in a dose-dependent manner and recognized the fceri b subunit in western blot analysis. exposure to artinm resulted also in phosphorylation of intracellular proteins, mcp-1 release and tgf-b production. significant increases in these activities were observed upon sensitization with ige. conclusions: these results suggest that artinm may bind to glycans of the high affinity ige receptor and/or of the ige (bound to fceri) and that such interactions would be implicated in its ability to activate and degranulate mast cells. in view of the well-established significance of mast cells in allergic inflammation, the participation of sugars as binding receptors on mast cell surface opens new ways of controlling allergic disorders. the adhesion receptor l-selectin is a key player of the innate immune response in the process of leukocyte migration from the blood stream to inflamed tissue. it is expressed on leukocytes and promotes the initial contact to the endothelium resulting in steady rolling and eventually diapedesis. a distinct feature is the exclusive presentation of l-selectin on the tip of finger-like cell membrane protrusions called microvilli which cover the entire leukocyte surface. this topography was shown to facilitate the first transient interactions of the free flowing cell to the static counterreceptor particularly in the context of high dynamic shear. other adhesion molecules such as p-selectin glycoprotein ligand 1 (psgl-1), b1 and b7-integrins also share this special phenotype. taken together, prominent adhesion receptor positioning reflects a widespread biological principle contributing to inflammation as well as hematogenic tumor metastasis. despite the functional relevance and frequent occurrence, however, molecular mechanisms of cell surface receptor compartmentalization remain largely unknown. in this study we identified the highly conserved transmembrane domain of l-selectin to regulate microvillus receptor positioning and adhesion under flow. taking advantage of the inverse surface expression pattern of cd44 (cell body) compared to l-selectin (microvilli) in a myeloid cell line, we investigated domain swapped chimeric receptors regarding their substructural surface localization and their ability to initiate rolling under flow. transmission electron microscopy showed a crucial impact of the transmembrane domain to target the chimeric receptors to a certain cell surface compartment independent of the intracellular anchorage. in turn, the receptor shift from microvilli to the cell body goes along with a substantial decrease of rolling cells in an in vitro parallel flow chamber assay. thus, contrary to the common view of single membrane spanning domains to simply act as a mechanical anchor, our results attach an important functional component as well and might point out a new general principle for targeting receptors to specific membrane compartments. objectives: macrophages are one of the principal effector cells involved in the innate immunity response. they kill microbes through phagocytosis and upon activation, secrete pro-inflammatory cytokines such as il-1b, il-18 and tnf-a. herpes simplex virus 1 (hsv-1) is an enveloped dna virus that infects mostly oral mucosa and sensory neurons. innate immunity responses activated by hsv1 infection consist of: activation of macrophages; activation of the complement cascade, and production and secretion of a variety of cytokines and chemokines. il-18 and tnf-a are cytokines produced by macrophages that contain known anti-hsv properties. the objective of this study was to characterise the secretome of human primary macrophages infected with hsv1. methods: human monocytes were purified from the peripheral blood mononuclear cells of healthy blood donors and differentiated in vitro into macrophages. macrophages were left untreated or primed with poly(i:c) (10ug/ml), a mimetic of double-stranded rna, after which cells were left uninfected or infected with hsv-1 for 18 h. after this, cell culture supernatants were collected, concentrated and proteins purified. the secreted proteins were digested into peptides, identified and quantified using itraq (isotope tagged relative and absolute quantitation) -labelling of the peptides followed by peptide fractionation by cation exchange chromatography and analysis by nanolc-ms/ms. the raw ms/ms data was analysed using proteinpilot 2.0 software. results: in the first itraq experiment over 300 human proteins were identified in the hsv1 infected cell supernatants. from these proteins 119 had at least 3 fold increase after poly(i:c) + hsv1 infection compared to the uninfected cells. hsv1 infected cells had clearly more proteins in their cell supernatants after infection compared to the uninfected cells: itraq labelling showed a total of 2.7 fold increase in the protein amount in the poly(i:c) + hsv1 infected cell supernatant and a 2.6 fold increase in the hsv1 infected cell supernatant when compared to the uninfected cell supernatant. amongst the upregulated proteins there were known inflammatory proteins: chemokine (c-x-c motif) ligand 10, il-6, tnf-a induced protein 6, complement factor b, galectin-1 and mxa. at present, further experiments are on-going for more detailed analysis of the hsv1 infected macrophage secretome. h. p. prakash 1,2 1 german cancer research centre, translational immunology, heidelberg, germany, 2 max planck institute for infection biology, molecular biology, berlin, germany chlamydophila pneumoniae are the major etiological factors for worldwide pneumonia, chd and copd. chlamydia lives and multiplies inside their host epithelial cells where they confer resistance for apoptosis by inducing expression and stability of anti-apoptotic proteins called inhibitor of apoptosis proteins (iaps). the significance of cellular inhibitor of apoptosis protein-1 (ciap-1) and x-linked inhibitor of apoptosis proteins ( xiap) in chlamydia pneumoniae pulmonary infection and innate immune response of macrophages was investigated in ciap-1 and xiap knockout (ko) mice using a novel non-invasive intra-tracheal infection method. in contrast to wildtype, iap knockout mice failed to clear the infection from their lung. wildtype mice responded to infection with a strong inflammatory response in the lung. in contrast, the recruitment of monocytes and macrophages was reduced in iap ko mice compared to wildtype mice. the concentration of interferon gamma (ifn-g) was increased whereastumor necrosis factor (tnfa) was dysregulated in the lungs of infected iap ko mice compared to infected wildtype mice. ex vivo experiments on mouse peritoneal macrophages and splenocytes revealed that iaps are required for innate immune responses of these cells. our findings thus suggest a new immunoregulatory role of iaps in c.pneumonaie pulmaonry infections. methods: human monocytes were purified from venous blood of normal volunteers by ficoll density gradient centrifugation. hrgal-3 (25 mg/ml) binding to monocytes, in the presence or absence of 10mm lactose or sacarose, was assessed by flow cytometry and confocal microscopy. in transwell systems, assays were performed using hrgal3, laminin or fibronectin immobilized or not on the filters. these were added to wells containing soluble hrgal3 or rpmi and monocytes (1x10 5 ) were added into each insert. when necessary, hrgal3 was pre-incubated with 10mm lactose or sacarose. mcp-1 (100ng/ml) was used as positive control. we observed that hrgal-3 binds to the surface of human monocytes through its crd, since this interaction can be inhibited by lactose. we corroborated some data of literature that hrgal-3 is able to induce monocyte migration in a dose-dependent manner, resulting in a bell-shaped curve as seem with other known attractants. when we evaluated the participation of the ecm laminin and fibronectin in monocyte migration induced by hrgal-3, we observed that the association between these glycoproteins and hrgal-3 resulted in a 60 % increase in the number of migrating cells. both n-and c-terminal domains of hrgal-3 are involved in the association between laminin or fibronectin and hrgal-3, since the presence of lactose resulted in 50 % and 20 % inhibition of monocyte migration induced by the lectin, respectively conclusions: our results showed that hrgal-3 induces monocyte migration by haptotaxis, through the interactions established between both n-and c-terminal domains of the lectin and ecm glycoproteins, laminin and fibronectin. in a vertebrate embryo, macrophages develop in two sites (yolk sac and liver) and constitute the primary mechanism of host defense. their phagocytic function may be required during the earliest stages of development both for survival and for organogenesis. recent studies have shown that monocyte heterogeneity is conserved in humans and mice. the different monocyte subsets seem to reflect developmental stages with distinct physiological roles but nothing is known whether the macrophage diversity arises in early ontogeny. in order to study the ontogeny of the monocyte-macrophage lineage, we developed a new culture technique using human embryonic stem cells (hesc).culturing of embryoid bodies for 3 weeks in the presence of bmp4,vegf and a mixture of hematopoietic cytokines resulted in a generation of a significant cell population of cd14+cd45+ cells. the sorted cd14+cd45+ cells were further cultured for 7 -10 days in the presence of m-csf and gave rise to a homogenous population of adherent mature macrophages. embryonic stem cells derived macrophages were identified by several criteria including morphology and ultrastructural features observed by microscopy and by expression of nonspecific esterase and myeloperoxidase by histochemical staining. while virtually all embryonic-derived macrophages expressed the lps-receptor cd14, m-csf receptor cd115 and the scavenger-receptor cd36, we characterized two distinct subpopulations of macrophage based on their difference in size and density and the expression of the cd14 and cd16 (fcgammariii) : the cd14lowcd16-and cd14+ cd16+. trancscriptional, phenotypic and functional assays suggest the alternative (m2) polarization of cd14+cd16+ embryonic stem cell-derived macrophages.(anti-inflammatory cytokines secretion, active phagocytosis, m2 -related gene expression).the exact chemokine receptor expression pattern, phenotype and transcriptional activity of their foetal counterparts are currently under investigation. collectively, our data provide insight into alternative macrophage polarization in humans and and adds further data to the growing body of evidence that establishment of macrophage heterogeneity is related to early ontogeny. b.-s. choi 1 , p. kropf 1 1 imperial college london, immunology department, london, united kingdom the balance between t helper (th) 1 and th2 cell responses is a major determinant of the outcome of experimental leishmaniasis, but polarized th1 or th2 responses are not sufficient to account for healing or nonhealing. we have recently shown that arginase-induced l-arginine depletion results in local suppression of antigen-specific t cell responses in nonhealing leishmaniasis. healing, induced by chemotherapy, resulted in control of arginase activity and reversal of local immunosuppression. moreover, supplementation with l-arginine restored t cell effector functions and resulted in reduced lesions size and parasite load. however, despite the efficient production of ifn-g by cd4 + t cells at the site of infection and despite the reduced pathology, the mice did not heal. we hypothesised that arginase-expressing macrophages contribute to persistent disease and become refractory to ifn-g mediated signals. to test this hypothesis, we used a well-defined model of bone marrow derived macrophages and determined whether the differentiation state of parasitized arginase-expressing macrophages could be altered. in addition, we also tested whether alternatively activated macrophages can be induced to switch off arginase and upregulate inducible nitric oxide synthase (inos) to kill the intracellular parasites. vg9vd2 t lymphocyte are activated following recognition of non-peptidic phosphorylated metabolites. the phosphoantigen isopentenyl pyrophosphate (ipp) is overproduced by tumors following hyperactivation of the mevalonate pathway of isoprenoid synthesis. previous work has shown that a molecular complex homologous to mitochondrial atp synthase (ecto-f1-atpase) is expressed on many cell types and is a possible specific ligand for the vg9vd2 tcr. the present study aims at understanding the role of f1-atpase in antigen regognition. using video microscopy calcium imaging in single vg9vd2 t lymphocytes, we can now show that the t cell response to ipp requires contact with bystander cells of variable tissue origin but that this requirement is not fulfilled by a cell line deprived of surface f1-atpase. purified f1-atpase immobilized on polystyrene beads can partly replace the need for cell-cell contact. ipp in soluble form is highly sensitive to terminal phosphatases and addition of these enzymes in t cell activation assays clearly shows that it is not recognized as such on tumors. however, we could detect nucleotide derivatives of phosphoantigens which are resistant to terminal phosphatases in the cell lysates of stimulatory tumors. one of these, a derivative of ipp, is barely able to stimulate vg9vd2 cells in the absence of apcs, as opposed to the non-nucleotidic antigen ipp. however it can bind stably to f1-atpase. thus the f1-atpase complex acts as a presenting structure for nucleotide phosphoantigens. altogether, our data suggest that vg9vd2 t cells are dedicated to the recognition of phosphoantigens in the form of nucleotide derivatives, on the surface of tissue cells and that antigen recognition involves multiple antigen modification steps, in including final cleavage by a nucleotide pyrophosphatase activity. surface plasmon resonance was used to analyse the molecular interaction between tcr and f1-atpase. by using purified f1-atpase and peptides derived from vg9vd2 tcr sequences, interaction sites between f1-atpase and tcr were identified on both ligands. based on these findings a generalized model for vg9vd2 t cell activation is proposed. ligands for the cytotoxic lymphocyte activating receptor nkg2d are highly expressed on cells stressed by numerous agents including genotoxic damage, thereby contributing to the elimination of transformed cells by nkg2d(+) lymphocytes. a key question is whether this represents a primary inductive means of immune surveillance, or merely enhances responses initiated by dendritic cells and antigen-specific t cells. a second key issue is the scope and scale of events that follow nkg2d activation in vivo. by transiently overexpressing the nkg2d ligand rae-1-beta in the skin of transgenic mice, we showed that this alone provoked rapid, coincident and reversible changes in the organization, morphology and activation state of tissue-resident vgamma5vdelta1 gamma-delta t cells and langerhans cells (lc), that were swiftly followed by epithelial infiltration of unconventional alpha-beta t cells. these data indicate a novel primary immune surveillance pathway whereby epithelial upregulation of nkg2d ligands is sufficient to provoke a series of multicomponent immunological changes. the effects on lc, which lack nkg2d and presumably respond to changes initiated by local gamma-delta t cells, are particularly interesting. ongoing microarray and co-culture experiments are now providing a molecular definition of the immume surveillance response to nkg2d ligands in vivo. to assess the scope of this response, ovalbumin was applied to the skin concomitant with rae1 induction. the primary systemic th2 response is increased by concomitant responses to a stress antigen. we will now resolve whether this increased response contributes to the adaptive memory pool, or whether it is a primary, regulatory response that may limit adaptive responses to auto-antigens exposed during stress. in addition, the many ligands available to the nkg2d receptor suggest that different ones may play unique roles. a novel nkg2d-ligand, h60c, is uniquely expressed in mouse skin. when the expression of this was further increased in a novel transgenic system, there was again an overt alteration in the local immune compartment, but with features that are seemingly distinct from the action of rae-1 induction. such studies may help resolve a long-standing puzzle over the pleiotropy of nkg2d ligands, and dissect immune surveillance of changes in gene expression levels rather than absolute levels. a.-s. invariant natural killer t (inkt) cells are a distinct lineage of t lymphocytes that co-express a highly conserved ab t cell receptor (tcr) along with typical surface receptors for natural killer (nk) cells. these lymphocytes recognize glycolipid antigens presented by the non-classical class i molecule cd1d. inkt cells are characterized by their capacity to produce rapidly large amounts of both th1 (ifn-g, tnf) and th2 (il-4, il-13) cytokines, which enables them to play a role in the regulation of many different types of immune responses, ranging from self-tolerance to responses against pathogens and tumors. converging studies in mouse models suggest that inkt cells can prevent the development of type 1 diabetes. the frequency of inkt cells is lower in non-obese diabetic mice (nod mice). manipulation of inkt cells, either by increasing their frequency or by stimulating them with agonists such as a-galcer, inhibits diabetes onset in nod mice. recently, a new population of cd4 -nk1.1 -inkt cells producing high levels of the pro-inflammatory cytokine il-17 has been identified (inkt17 cells). given that this cytokine has been implicated in several pathologies including autoimmune diseases, we investigated the role of inkt17 cells in type 1 diabetes. interestingly, nod mice exhibit a higher frequency of inkt cells producing il-17 as compared to c57bl/6 mice. this increased frequency was observed in the thymus as well as in peripheral lymphoid tissues. as previously described in normal mice, inkt17 cells present in nod mice were mainly cd4 -nk1.1 -, express the ror-g transcription factor and il-23 receptor, both molecules being usually associated with th17 commitment. we are currently analyzing, using co-transfer experiments, whether these inkt17 cells play a beneficial, a deleterious, or any role in the development of type 1 diabetes in nod mice. j. s. dodd 1 , r. muir 1 , s.s. affendi 1 , p.j. openshaw 1 1 imperial college london, respiratory medicine, london, united kingdom natural killer t (nkt) cells are a heterogeneous population of innate t cells that have attracted interest because of their potential to regulate immune responses to a variety of pathogens. upon activation with their cognate glycolipid antigen presented by cd1d molecules, activated nkt cells produce copious and numerous cytokines which endow these cells with potent immunoregulatory properties. consequently, nkt cells have become the focus for the development of vaccine adjuvants, cancer immunotherapeutics and modulators for autoimmune and inflammatory conditions. respiratory syncytial virus (rsv) is a common cold virus of the family paramyxoviridae. it is the most frequent viral cause of serious lower respiratory tract infection in infants and children worldwide and a significant contributor to winter deaths in the elderly. despite its global impact, there is still no safe and effective vaccine and our understanding of the immunological mechanisms that regulate protection and pathology is incomplete. it is known that cd1d-deficient mice with poor nkt cell responses have inefficient induction of cd8 t cells and reduced clearance of rsv, perhaps because of ifn-g release by activated nkt cells. we now show that activation of lung nkt cells with intranasal agalcer during rsv infection of mice boosts th2 immunity (increasing il-5 and il-10), promoting pulmonary eosinophilia and ablating cd8 t cell recruitment. by contrast, intraperitonal injection of agalcer enhances nk cell recruitment and boosts pulmonary cd8 t cell activity (as measured by cd25 expression), increasing ifn-g production in the airway and lung and inhibiting viral replication. effects on illness (as measured by weight loss) were similarly distinct: intranasal agalcer induced early (d4) weight loss independent of conventional t cells, whereas intraperitonal agalcer enhanced late (d7) weight loss by a cd8 t cell dependent mechanism. therefore, nkt cells stimulated by agalcer administered via different routes induce distinct types of immune response to viral infection in the lung with the intraperitonal route leading to optimal viral clearance. in general, neonatal conventional t cells, especially cd4 + ab t cells, are regarded as immature or t h 2 biased. vg9 + vd2 + t cells are unconventional lymphocytes: they are mhc-unrestricted and can react rapidly upon activation with pyrophosphates (e. g. (e)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (hmb-pp)) or aminobisphosphonates (e. g. zoledronate) in adults. until now, little is known on the functional reactivity of neonatal vg9 + vd2 + t cells towards these activators. because il-23 is preferentially secreted by neonatal dendritic cells (dc) upon tlr stimulation, we investigated the potential costimulatory effect of this cytokine on hmb-pp and zoledronate-treated neonatal vg9 + vd2 + t cells. herein, we observed that zoledronate induced neonatal vg9 + vd2 + t cell proliferation and ifn-g production in cord blood mononuclear cells (cbmc) cultures. other t h 1-like cytokines like tnf-a and gm-csf were also produced upon this stimulation, but less than ifn-g, while t h 2-like cytokines such as il-4 and il-5 were not induced. addition of il-23 to zoledronate selectively costimulated ifn-g production from neonatal vg9 + vd2 + t cells. furthermore, zoledronate/il-23 treatment resulted in neonatal vg9 + vd2 + t cells expressing high levels of the cytotoxic mediators perforin and granzyme a. zoledronate induced the expression of the receptor for il-23 (il-23r) and the transcription factor t-bet, which is known to be important for the production of ifn-g in gd t cells. in addition, costimulation with il-23 resulted in a further increase of t-bet expression in neonatal vg9 + vd2 + t cells. these changes in the expression of il-23r and t-bet likely contribute to the observed selective ifn-g response towards zoledronate/il-23 treatment. of note, in contrast to adult peripheral blood vg9 + vd2 + t cells, hmb-pp had no or only a minor effect on the functional reactivity of neonatal vg9 + vd2 + t cells. altogether, these observations show that neonatal vg9 + vd2 + t cells are functionally active and that this t cell population might play a role in protective immune responses to infections with intracellular pathogens in early life, in particular when dc-derived il-23 is produced in response to microbial stimuli. the evasion of antigen presentation is a feature common to herpesviruses. one of the strategies employed to inhibit antigen presenting molecules is ubiquitination, internalisation and lysosomal breakdown by viral e3 ligases such as hhv8 encoded k3, k5 or mhv68 encoded mk3. these viral genes represent homologues of the march family of cellular genes whose function is the regulation of cell-surface antigen presentation and reduction of the lifetime of loaded antigen complexes. ubiquitination targets surface molecules to the lysosome via the multivesicular body (mvb), a structure which also has an important role in the budding of many viruses. we investigated the existence of alternative fates for antigen presenting molecules post-ubiquitination, and how viral e3 ligases manipulate them. we discovered that both the cellular march and viral e3 ligases ubiquitinate cd1 molecules. however, whereas viral molecules inhibit cd1-antigen presentation, the march molecules are essential for the recirculation and function of the long-lived and lysosome-resistant cd1 molecules. in contrast mhc class ii was only targeted by cellular and not by viral e3 ligases. furthermore cd1 molecules could be found in viral particles as a result of ubiquitination, presumably via the mvb. thus, virally expressed and cellular e3 ligases have opposite effects, despite their homology. how this is achieved is a matter of active investigation. gamma delta (gd) t cells recognize stress-induced auto-antigens and contribute to immunity against infections and cancer. our previous study revealed that vd2 negative ( neg ) gd t lymphocytes isolated from transplant recipients infected by cytomegalovirus (cmv) killed both cmv-infected cells and ht29 colon cancer cells in vitro. in order to investigate the anti-tumor effects of vd2 neg clones in vivo, we generated hypodermal ht29 tumors in immunodeficient mice. concomitant injections of vd2 neg clones, in contrast to vd2 + cells, prevented the development of ht29 tumors. vd2 neg clones expressed chemokine c-c motif receptor 3 (ccr3) and migrated in vitro in response to chemokines secreted by ht29 cells, among which were the ccr3 ligands macrophage inflammatory protein (mip)-1d and monocyte chemoattractant protein (mcp)-4. more importantly, a systemic intraperitoneal (i. p.) treatment with vd2 neg clones delayed the growth of ht29 subcutaneous (s. c.) tumors. the effect of in vivo gd t cell passive immunotherapy on tumor growth could be reverted by addition of a blocking anti-ccr3 antibody. gd t cell passive immunotherapy was dependent upon the cytotoxic activity of the gd effectors towards their targets since vd2 neg clones were not able to inhibit the growth of a431 hypodermal tumors. our findings suggest that cmv-specific vd2 neg cells could target in vivo cancer cells, making them an attractive candidate for anti-tumor immunotherapy. more recently, we generated ht29 cells expressing the luciferase and realized orthotopic injection of ht29-luc cells. progressive tumor development and regression following « gd treatment » will be observed in vivo using bioluminescent imaging. intraepithelial lymphocytes (iel) compose large, oligoclonal, tissue-associated repertoires of non-mhc-restricted t cells that play key roles in immunosurveillance. it is commonly considered that the characteristic iel repertoires are positively selected by thymic epithelial molecules that are also stress-induced in specific tissues, thereby activating iel function. however, no such molecules have been identified. here we characterise skint1, currently the only known determinant of a canonical iel compartment, that is selectively required for vg5vd1 + dendritic epidermal t cell (detc) development. we show that both peripheral and thymic skint1 expression is essential for full detc development. its effects are highly specific since even substantial and ubiquitous over-expression neither negatively selects detc, nor affects any other t cells. unexpectedly, however, skint genes are not expressed by cell lines and are downregulated rather than activated by carcinogenesis. mouse genetic models allow powerful insight into skint1 function; for example, we demonstrate that the constitutive expression of wild-type skint1 fully restores detc development in a skint1 mutant mouse, but does not rescue normal detc function. thus, skint1 provides a novel perspective into how epithelia regulate the development and function of specific tissue-associated t cell compartments, and how normal versus dysregulated tissues may be demarcated. marginal zone (mz) b cells are strategically localized in the mz of the spleen. since most of the blood reaching the spleen is passing through this region such localization favors contact with blood born antigens and pathogens. besides being able to rapidly secrete antibodies, mz b cells may also act as professional antigen presenting cells (apcs). they are known to express high levels of cd1d which is the presenting molecule for nkt cells which are also located in the mz. therefore we hypothesised that mz b cells may be efficient activators of nkt cells. to test this hypothesis, we used freshly sorted splenic mz b cells (cd19 + cd21 hi cd23 lo cd11c -) and splenic conventional dendritic cells (cdcs) (cd11c hi cd8a +/-cd11b +/-b220 -) from wt and cd1d -/mice as apcs for nkt cells from va14-ja18 transgenic or wt mice. the apcs were treated with agalactosylceramide (agalcer) or heat killed (hk) listeria monocytogenes or salmonella typhimurium. both mz b cells and cdcs proved to be highly efficient apcs for priming of nkt cells and induced robust proliferation. in contrast, other populations of b cells failed to activate nkt cells. we showed, using cd1d -/mice as well as blocking antibodies to icosl, that proliferation of nkt cells depends on tcr/cd1d and in case of mz b cells, also on icos/icosl interactions. importantly, apcs primed with hk bacteria were not able to induce nkt cell proliferation. interestingly, mz b cells exclusively induced production of il-4 by nkt cells. in contrast, cdcs mostly induced production of ifn-g and il-4 producing cells were scarce under these conditions. cytokine production by nkt cells proved to be independent of tcr signalling, but dependent on icos/icosl interactions when mz b cells were used as apcs, and gitr-dependent when cdcs were used. taken together, our data suggest that both mz b cells as well as cdc act as professional apcs for nkt cells. notably, the nature of apcs appears to be critical for polarization of the immune response: mz b-cell-primed nkt cells induce cytokine milieu fostering a t h 2 response, whereas cdc-primed nkt cells rather favor a t h 1 response. objectives: il-18 is an innate cytokine present in elevated levels in sera from patients suffering from autoimmunity (eg. sle and ra) and the allergic disease atopic eczema. in mice, injections of il-18 give rise to an early polyclonal isotype switched antibody response which is absent in inkt cell deficient (cd1d -/-) mice. we set out to investigate the activated b cells in il-18 injected mice and how these are regulated by inkt cells. methods: mice received daily i. p. injections of il-18 (2 mg) for 10 days and the antibody response in serum was monitored using elisa. the b cell activation in the spleen at day 14 was evaluated by flow cytometry and immunohistology. results: mice injected with il-18 developed self reactive (anti-pc and anti-dna) antibodies in the serum, in line with the autoreactive antibodies in patients with e. g. sle and atopic eczema. the antibody producing cells formed cd138 + cell clusters in the red pulp of the spleen, a typical feature of extrafollicular activation frequently associated with autoreactive responses. surprisingly, the antibody response induced by il-18 was increased in inkt cell deficient (cd1d -/-) mice, in contrast to published data. an increased response to il-18 was also observed in ja281 -/mice, which lack the a-chain of the tcr used by inkt cells, and thus our data suggest that inkt cells inhibit antibody producing cells in il-18 induced antibody responses. further characterization of the recruitment of b cells in il-18 injected mice revealed a marked expansion of the marginal zone b cell (mzb) population in the spleen, suggesting an important role for mzbs in the il-18 induced autoreactive antibody response. mzbs are innate-type b cells that express high levels of cd1d, are prone to autoantibody production and often involved in early immune responses. the il-18 induced antibody response in mzb deficient (cd19 -/-) mice was either decreased (igg) or delayed (ige), supporting the importance of mzbs in il-18 induced antibody responses. we conclude that the role for inkt cells in il-18 induced antibody responses is to inhibit the production of autoreactive antibodes from mzbs in extrafollicular foci. objectives: amoebiasis is a widespread human parasitic disease caused by the intestinal protozoan entamoeba histolytica. there are two major clinical manifestations of the disease, amoebic colitis and amoebic liver abscess (ala). interestingly, only a small proportion of e. histolytica-infected individuals develop invasive disease, whereas the majority harbors the parasite within the gut without clinical symptoms. so far, cells of the innate immune system have been described to constitute the main host defense mechanism for the control of amoebiasis, relying largely on the early production of interferon-g (ifn-g). however, information is lacking about the sources of early ifn-g production as well as the amoeba antigens involved in this activation process. methods: using a recently developed c57bl/6 mouse model for ala, the contribution of natural killer t (nkt) cells for protection against amoebic disease was investigated. applying nkt cells and dendritic cells as antigen-presenting cells from various ko-mice, the signaling pathways implicated in recognition of amoebic antigens and activation of cytokine-secretion by nkt cells was analysed. results: nkt cells were found to play a key role in the defense against ala. specific activation of nkt cells by a-galactosylceramide (a-galcer) induced significant protection, whereas jalpha 18-/-and cd1d-/-mice lacking inkt as well as dnkt cells suffered from more severe abscess formation. a lipopeptidophosphoglycan, which is present in large quantities on the surfcae of e. histolytica trophozoites (ehlppg), was identified as a major amoeba antigen that activates nkt cells resulting in the production of ifn-g, but not of il-4. moreover, ifn-g production required the presentation of ehlppg by cd1d and signaling through the tlr receptor cascade in combination with a simultaneous secretion of il-12. similar to a-galcer application, treatment of mice with purified ehlppg significantly reduced the severity of ala in amoeba-infected mice. our study provides a mechanism for the innate control of amoeba invasion that might explain why the majority of e. histolytica-infected individuals do not develop amoebic disease. a few years ago, we have observed a significant expansion of circulating effector gamma delta t cells following cytomegalovirus (cmv) infection in kidney transplant recipients (ktr). these unconventional t cells display tcr dependent cytotoxicity against both cmv-infected cells and carcinoma cells. in the present study, an extensive phenotyping of gamma-delta t cells allowed us to demonstrate an over-expression of cd16 in cmv-infected individuals. cd16 is the fcgammariiia, a natural killer cell marker usually absent on conventional t cells. we found that 71.9 ± 15.9 % of gamma-delta t cells from 21 cmv-infected ktr expressed cd16, when compared with only 19.8 ± 16.7% in 11 non cmv-infected ktr (p x 0.0005). similarly, 51.9 ± 25.7 % of gamma-delta t cells from 13 cmv-seropositive blood donors expressed cd16 compared to 27.1 ± 25.1 % in 15 cmv-seronegative donors (p x 0.01). cd16+ gamma-delta t cell lines generated from cmv-infected individuals were able to produce ifn-g (a potent anti-viral cytokine) in a cd16-dependent manner when activated by cmv/igg immune complexes. this production greatly increased in the presence of il-12 and ifn-alpha, two cytokines highly produced during cmv-infection. the supernatants of gamma-delta t cells activated with agonist anti-cd16 mab inhibited cmv replication in vitro and this effect was abrogated in the presence of a blocking anti-ifn-g antibody. cmv/igg immune complexes were also able to induce the expression of the cytotoxicity marker cd107a on cd16+ gamma-delta t cell lines. cd16 is well-known to mediate antibody-dependant cellular cytotoxicity (adcc), especially in natural killer cells. accordingly, we demonstrated that cd16+ gamma delta t cell lines could make adcc against the daudi lymphoma cell line and the a431 skin carcinoma cell line pre-incubated either with rituximab (anti-cd20) or cetuximab (anti-egfr), respectively. in contrast, no addc could be observed against cmv-infected fibroblasts pre-incubated with polyclonal anti-cmv igg (cytogam), probably because cytogam weakly stained infected cells. these data reveal a new cd16-dependent anti-cmv function of gamma-delta t cells through recognition of immune complexes and secretion of ifng. moreover, they demonstrate that these cells are able to kill through adcc lymphoma and skin carcinoma cells, two tumour types frequently encountered in ktr. dendritic epidermal t cells are a prototypic population of intraepithelial gd t cells in the mouse skin. found in the basal layer of epidermis and in close contact with langerhan's cells and keratinocytes detc facilitate vital immunological and physiological processes e. g. wound healing, homeostasis, tumor surveillance and regulation of inflammation. gd t cells respond rapidly to non-peptidic microbial and stress induced self antigens in a non-mhc restricted manner and are therefore proposed to bridge the gap between innate and adaptive immunity. by using gd t cell knock-out mice tcrd-/-, ovalbumin transgenic k5mova mice and a skin grafting model we aimed to elucidate the role of gd-detc in adaptive immune responses associated with elimination of foreign antigen presented in the skin.we show that in the absence of gd t cells in the skin there is a decrease in rejection of ovalbumin expressing skin grafts compared to wildtype mice. we show that optimal regimens of antigen delivered subcutaneously in conjunction with adjuvant elicits comparable responses in wildtype and knockout mice. however frequency of primed host animals is reduced in tcrd-/-mice when antigen is delivered epidermally via skin grafting; suggesting detc enhance cross presentation of classical mhc bound antigens in the skin. considering the incapability of gd t cells to recognize peptide antigens in the context of mhc we plan to dissect the relationship between detc and professional antigen presenting cells in the skin. understanding the underlying mechanisms of this relationship will expand our knowledge of enhancing professional apc function in skin by detc and potentially other epithelia by intraepithelial gd t cells and can be useful in designing therapies to epithelial infections and malignancies. we demonstrate a rapid and hmb-pp-dependent crosstalk between gd t cells and autologous monocytes that resulted in the production of inflammatory mediators including il-6, ifn-g, tnf-a, osm, ccl2, cxcl8, cxcl10, and trail. moreover, under these co-culture conditions monocytes showed enhanced survival and differentiated overnight into inflammatory dcs with antigen-presenting functions. these cells expressed cd40, cd86, hla-dr, and dc-sign, and lost cd14, ccr2, ccr5, and cxcr4. addition of further microbial stimuli (lps, peptidoglycan) induced ccr7 and enabled these inflammatory dcs to trigger antigenspecific cd4 + effector ab t cells expressing ifn-g and/or il-17. importantly, our in vitro model replicated the responsiveness to microbes of effluent cells from pd patients and translated directly to episodes of acute pd-associated bacterial peritonitis, where vg9/vd2 t cell numbers and soluble inflammatory mediators were elevated in patients infected with hmb-pp-producing pathogens. conclusion: our findings suggest a direct link between invading pathogens, microbe-responsive gd t cells, and monocytes in the inflammatory infiltrate, which plays a crucial role in the early response and the generation of microbe-specific immunity. the mechanism(s) responsible for their dichotomous behaviour are poorly understood, and the outcome of nkt cell manipulation remains unpredictable. there is growing evidence that the nkt cell pool is composed of functionally distinct subsets, but such a possibility has not yet been investigated in a model of nkt cellmediated immunosuppression. we examined the differential ability of nkt cell subsets from the thymus and liver to prevent type i diabetes when transferred into prediabetic nod mice. the transfer of abtcr+dn thymocytes (a population enriched for nkt cells) has previously provided robust protection against tid development; however it has not been formally shown that nkt cells are solely responsible for the protection. our study found that while the transfer of thymic dn nkt cells can prevent tid and severe insulitis in nod mice, not all nkt cell subsets show the same tolerogenic capabilities. these findings both formally demonstrate the disease-preventing effects of nkt cell transfer in nod mice and provide further evidence that nkt cells are a functionally heterogeneous population. objective: vg9/vd2 t cells constitute a minor t cell population in human blood that expands specifically and rapidly in response to the microbial metabolite hmb-pp. our previous microarray studies showed that vg9/vd2 t cells stimulated with hmb-pp in the presence of il-21 express markers associated with a possible follicular b cell helper function. we therefore investigated in more detail whether and how hmb-pp and il-21 regulate expression of the b cell attracting chemokine cxcl13/bca-1, its receptor cxcr5, and co-stimulatory molecules involved in b cell help. purified peripheral vg9/vd2 t cells were co-cultured with autologous monocytes or b cells (as feeder cells) for up to 4 days with and without hmb-pp, in the absence or presence of il-2 or il-21, or in medium alone. cells were analysed by flow cytometry and immunofluorescence microscopy. results: high levels of cxcl13 protein were detected in co-culture supernatants only when both il-21 and hmb-pp were provided, implying an il-21-dependent and tcr-dependent expression. vg9/vd2 t cells were confirmed as producers of cxcl13 by flow cytometry and immunofluorescence. under the same conditions, activated vg9/vd2 t cells expressed cd27, cd28, cd40l, cd70, icos and ox40. in contrast, neither cxcr5 nor ccr7 changed markedly by il-21 stimulation of peripheral vg9/vd2 t cells. conclusion: our findings confirm on the protein level that stimulation of vg9/vd2 t cells with hmb-pp and il-21 induces markers typically associated with follicular b helper t (t fh ) cells. these data suggest that gd t cells contribute to humoral immune responses and play a role in germinal centre formation and production of high-affinity antibodies in microbial infection. ongoing analyses of gd t cells in inflamed and non-inflamed lymphoid tissues (tonsils, appendices) aim at demonstrating the physiological relevance of our findings. y. emoto 1 , m. emoto 1 1 gunma university school of health sciences, department of laboratory sciences, maebashi, japan invariant (i) natural killer (nk)t cells become undetectable after stimulation with a-galactosylceramide (a-galcer) or interleukin (il)-12. although downmodulation of surface t cell receptor (tcr)/nkr-p1c (nk1.1) expression has been shown convincingly after a-galcer stimulation, it is unclear whether this holds true for il-12 stimulation. to determine whether failure to detect inkt cells after il-12 stimulation is caused by dissociation/internalization of tcr and/or nkr-p1c or by block of de-novo synthesis of these molecules, and to examine the role of il-12 in disappearance of inkt cells after a-galcer stimulation, surface (s)/ cytoplasmic (c) protein expression as well as mrna expression of tcr/nkr-p1c by inkt cells after stimulation with a-galcer or il-12, and influence of il-12 neutralization on down-modulation of stcr/snkr-p1c expression by inkt cells after a-galcer stimulation were examined. the s/ctcr + s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of a-galcer, which was partially prevented by il-12 neutralization. whereas s/cnkr-p1c + inkt cells became undetectable after in-vivo administration of il-12, s/ctcr + inkt cells were only marginally affected. mrna expression of tcr/nkr-p1c remained unaffected by a-galcer or il-12 treatment, despite the down-modulation of ctcr and/or cnkr-p1c protein expression. in contrast, ctcr + cnkr-p1c + stcr -snkr-p1c -inkt cells and cnkr-p1c + snkr-p1c -inkt cells were detectable after in-vitro stimulation with a-galcer and il-12, respectively. our results indicate that tcr and nkr-p1c expression by inkt cells is differentially regulated by signaling through tcr and il-12r. they also suggest that il-12 participates, in part, in the disappearance of inkt cells after a-galcer stimulation by down-modulating not only snkr-p1c but also stcr. the fetus and infant are highly susceptible to viral infections. a number of viruses, including human cytomegalovirus (cmv), cause more severe disease in early life compared to later life. it is generally accepted that this higher susceptibility to viral infections is due to the immaturity of the immune system. gd t cells are unconventional t cells that can react rapidly upon activation and show mhc-unrestricted activity. herein, we show that upon cmv infection in utero, fetal gd t cells expand and become differentiated. the response was restricted to vg9-gd t cells, irrespective of their vd chain expression. differentiated gd t cells expressed high levels of ifn-g, transcription factors t-bet and eomes, natural killer receptors and cytotoxic mediators including perforin and granzymes. in addition, congenital cmv-infection induced a highly restricted complementary-determining region 3 d1 (cdr3d1) and cdr3d2 repertoire, with a striking enrichment in a specific germline-encoded cdr3d1 sequence. differentiated gd t cells and the enriched cdr3d1 sequence were detected as early as after 21 weeks of gestation. our results indicate that functional fetal gd t cell responses can be generated during development in utero and suggest that this t cell subset could participate in anti-viral defense in early life. results: spectratyping showed only in-frame selection for vd1-jd1 and vgi-jg1.3/2.3 rearrangements in tcrgd thymocytes and to a lesser extent in tcrgd cb cells. in contrast, clear in-frame vd2-jd1 and vg9-jg1.2 selection was seen in pb tcrgd cells. detailed analysis of the cdr3 motifs revealed selection determinants in both vg9-jg1.2 (canonical length and cdr3 motif) and vd2-jd1 (minimal cdr3 length in combination with an invariant t nucleotide) rearrangements. upon evaluation of the replication history we found a clear increase in the number of cell divisions from naïve tcrgd thymocytes (˚4) and tcrgd cb cells (6-7) to tcrgd pb cells (˚10 or more). no increase was seen between cb and pb tcrgd t cells within the first year of life, suggesting that peripheral proliferation occurs later in life. our results indicate that the human peripheral tcrgd repertoire is shaped by (antigenic) selection and proliferation processes. moreover, the ontogenetic changes in the gd repertoire between the central and peripheral immune systems are clearly influenced by proliferation. background: natural killer (nk) t cells have been implied in the regulation of disease in the non obese diabetic (nod) mouse model of type 1 diabetes (t1d). we have previously shown that transgenic expression of a cd1d-restricted, va3.2-vb9 tcr in nod mice lead to an increase in cd1d-restricted type ii nkt cells (24abnkt cells), and prevention of the development of t1d in the transgenic mice. in this study we have investigated the requirements and underlying mechanism of disease protection by type ii nkt cells in a disease transfer model. to investigate the mode of regulation by 24abnkt cells, we explored a disease transfer model into nod.scid mice using transgenic diabetogenic bdc2.5 cd4+ t cells, in the presence or absence of selected cells from 24abnkt cell transgenic mice. results: in 24ab transgenic mice a high frequency of activated transgenic nkt cells was found in the pancreas of the protected mice. in this organ, 24abnkt cells expressed a high level of cxcr3 and a low level of ccr7 and cd62l, a pattern similar to that observed in t cells homing to inflammatory tissues. adoptive transfer of cd4+ bdc2.5 t cells into nod.scid recipients rapidly induced onset of diabetes. using this model, we found that co-transfer of spleen cells from 24ab transgenic mice with bdc2.5 cd4+ cells resulted in the prevention of diabetes development. the protection from disease required a minor cd4+ subset of 24ab+ nkt cells, but was independent of cd25+ t regulatory cells. analogs of alpha galactosylceramide (a-galcer) that may modulate the strong activation of inkt and at the same time prolong their effect upon in vivo administration are a long standing goal of research in this area due to their putative immunotherapeutical applications. a new class of non glycosidic analogues bearing an aminocyclitol ring as galactose surrogate have been synthesized and assayed in their capacity to be presented by cd1d and recognized by inkt. the structural novelty of these compounds resides in the presence of a cyclohexane that substitutes the sugar moeity and the substitution of the o glycosidic linkeage with the ceramide by a n. in this basic structure, substitutions in the cyclohexane ring with oh in different conformations mimicking different sugars, differences in the length of the sphingosine lipid and differences in the orientation of the n linkeage conform a series of analogs that have been analyzed in their capacity to stimulate inkt cells. proliferation assays in bulk splenocyte cultures and cytokine secretion determinations show that inkt cells are specifically stimulated by some of the analogs tested. in particular, the active compound hs44, induces in vitro inkt cell expansion and ifng and il-4 secretion in a similar fashion but less potently than a-galcer. dose response assays show a bias towards a th2 profile response after recognition by nkt cells, more similar to the response induced by och. the degree of structural similarity of the cyclitol ceramides with a-galcer parallels their cellular activities. these data open the way towards the development of a new class of a-galcer lipid analogues having charged amino substituted polar heads resistant to glycosidase degradation, thus enhancing their in vivo biodisponibility, and expands the range of potential inkt cell sphingolipid agonists that can modulate the immune response due nkt cell activation. objectives: invariant natural killer (ink) t cells represent an innate lymphocyte subset with important modulatory functions. in the presence of pathogens or tumors, inkt cells play an adjuvant function that boosts t cell immunity through cytokine secretion and dc maturation. in steady-state conditions, i.e. in the absence of pathogens, inkt cells acquire a regulatory function that promotes t cell tolerance and prevents autoimmune disease. our aim was to assess the mechanism of action of inkt cells in the steady state and, specifically, to test the hypothesis that inkt cells promote immune tolerance through modulation of dcs. methods: to assess the direct influence of regulatory inkt cells on dc maturation in resting conditions, we derived murine inkt cell lines in vitro and, after staining with agalcer-loaded cd1d tetramers and magnetic purification, we tested their capacity to modulate bone marrow-derived myeloid dcs in the absence of any other maturation signals. we analyze the transcriptional profile (microarray analysis) as well as maturation, cytokine expression profile and pro-tolerogenic antigen-presenting function of inkt cell-modulated dcs (inkt-dcs). the cell-cell interaction with inkt cells provoked dramatic phenotypical changes on immature dcs that acquired the cardinal features of tolerogenic dcs such as intermediate levels of mhc class ii and co-stimulatory molecules expression and high secretion of il-10 with no release of pro-inflammatory cytokines. most importantly, inkt-dcs acquired tolerogenic antigen-presenting function inducing the differentiation of regulatory tr1 cells and immune tolerance in vivo. dcs, simultaneously stimulated with inkt cells and through toll-like receptor (lps) completely lost the pro-tolerogenic phenotype and acquired a proinflammatory cytokine profile. conclusion: it is still mysterious how inkt cells can play a dual role and either boost t cell immunity or promote immune tolerance. our results suggest that the same mechanism could underlie both inkt cell functions. in the presence of pathogen-driven maturation signals, the inkt cell-modulation of dcs favors their acquisition of a pro-inflammatory phenotype and function. on the contrary, if inkt cells are activated in the absence of pathogens, e. g. during autoimmune conditions, their interaction with immature dcs promotes their tolerogenic maturation to maintain peripheral tolerance and counter-regulate autoimmune diseases. th17-type immune responses have been reported to fight extracellular bacterial infection, but as well to cause autoimmune diseases and allergy. the th17 immune response is characterized by the secretion of il-17a and il-17f. the il-17 locus encodes the highly conserved il-17a and il-17f cytokines that are syntenic in 44kb distance to each other. besides cd4 + th17 and nkt cells, approximately 50 % of the il17a producers are gd t-cells. like cd4 + th17 cells, il-17 producing gd tcells have recently been implicated to play a major role in the immune response to infections with extra-and intracellular bacteria. our findings show a difference between the il-17 production of gd t cells in the peripheral system and mucosal epithelia. mucosal gd t-cells generally do not produce th17 cytokines. in the periphery, we define novel subsets of gd t-cells that can produce either il-17 or ifn-g. combined with the well known classification of il-17 producing gd t-cells along the markers cd27 and cd122, our data point at specialized functions of the different gd t cell subsets depending on their location and origin. functional studies are currently carried out in order to address the role of the different gd t-cell subsets for th17-type immune responses in vivo. in this context, the potential redundancy of il-17a and il-17f may complicate the analysis. so far, most studies were carried out with il-17a single-deficient or il-17f single-deficient mice. to further clarify these issues, we will have to address the above mentioned findings in il-17a and il-17f double-deficient mice. several subsets of gd tregs have been described and intensively studied, but the potential regulatory role of innate t cells in controlling immune responses remains unclear. lymphocytes expressing gd tcr are involved in both innate and adaptive immune responses. vg9vgd2 t cells, which represent a major peripheral blood gd t-lymphocyte subpopulation in humans, display a broad reactivity against microbial agents and tumors.here we report that tgf-b1 and il-15 differentiate in vitro a subset of gd t lymphocytes with regulatory functions (vd2 tregs) in the presence of specific antigen stimulation. these cells express the forkhead/winged helix transcription factor (foxp3) and, similarly to ab tregs, suppress the proliferation of anti-cd3/anti-cd28 stimulated-pbmc. detailed knowledge about the phenotype and functionality of vd2 tregs will improve our understanding of the role of gd t cells in the pathogenesis and regulation of autoimmune, infectious and cancer diseases. a-galactosylceramide (a-galcer) has the potential to activate invariant (i) nkt cells, which in turn release a wide variety of cytokines that stimulate immunocompetent cells. although this rapid and vigorous cytokine release appears critical for regulation of various immune responses, it remains elusive whether protection against intracellular bacteria can be induced by a-galcer. here we show that treatment with a-galcer ameliorates murine listeriosis, and inhibits inflammation in the liver and spleen following listeria monocytogenes infection. liver infiltration of granulocytes and g/d t cells was accelerated by a-galcer treatment. granulocyte and g/d t cell depletion exacerbated listeriosis in a-galcer-treated mice, and this effect was more pronounced in granulocyte than in g/d t cell depletion. although secretion of gm-csf and il-17 was detected among the nkt cell population in the liver and bone marrow immediately after a-galcer treatment, infiltration of granulocytes into the liver was not prevented by neutralizing mab. yet, in parallel to the numerical increase of granulocytes expressing cd11b in the liver following a-galcer treatment, numbers of cells lacking cd11b diminished in the bone marrow. in addition, respiratory burst in granulocytes was enhanced by a-galcer treatment. our results indicate that a-galcer-induced antibacterial immunity is caused, in part, by accelerated infiltration of inflammatory cells, in particular granulocytes and to a lesser degree g/d t cells, into the liver. we also suggest that the infiltration of granulocytes is caused by an accelerated supply of granulocytes from the bone marrow, rather than by accelerated granulopoiesis. objectives: the aim of this work is to evaluate whether phenotypic and functional features of vgamma9/vdelta2 t cells are influenced by the activity of mevalonate pathway in tumor cells and contribute to determine disease aggressiveness in cll. methods: eighty seven previously untreated cll patients were evaluated for in vitro vgamma9/vdelta2 t cells expansion upon stimulation with zoledronic acid (za) and interleukin-2 (il-2). gammadelta t cells subset distribution and natural killer receptors profile were evaluated by multicolor flowcytometry. the mutational status of the tumor immunoglobulin heavy chain variable region (igvh) was analyzed by dna sequencing. the activity of the mev pathway was determined by 1) the bioinformatic analysis of gene expression profiling data 2) the quantification of mev pathway metabolites. results: proliferation of gammadelta t cells was observed in 43 patients (49 %) (responders, r), whereas 44 patients (51 %) were non-responders (nr). vgamma9/vdelta2 t-cell subset distribution was well balanced in r patients, whereas effectors subsets [i. e., effector memory (tem), and terminally differentiated effector memory (temra)] were largely predominant in nr patients. temra of nr patients mainly expressed the inhibitory receptor ilt2, whereas temra of r patients had an higher expression of the costimulatory molecule nkg2d. the proliferative response of vgamma9/vdelta2 t cells was significantly associated with igvh mutational status, which is a well known prognostic factor in cll. indeed, 82 % of r patients were m, whereas 77 % of um patients were nr (p x 0.001). given this association, we evaluated the activity of the mev pathway in tumor cells of m and um patients. the pathway was more active in tumor cells of um than m patients, suggesting that the former can more easily engage gammadelta t cells and drive their differentiation into functionally exhausted t emra . given the association between the r/nr status and the igvh mutational status we also analyzed the independent prognostic impact of r/nr status in multivariate cox analysis. nr patients had a significantly shorter time to first treatment thus pointing to r/nr status as an independent prognostic factor. conclusion: these data define a novel mechanism of immune escape which can contribute to determine disease aggressiveness in cll patients. the studies reported here were undertaken to ascertain and delineate the ability of kupffer cells to regulate the response of inkt cells to biliary obstruction. methods: c57bl/6 mice were not treated or rendered kupffer cell-depleted by intravenous inoculation of liposome-encapsulated dichloromethylene diphosphonate. to clarify the factors that elicit inkt cell activity, additional mice were administered anti-il-12 p40 (clone r2-10f6; atcc) or anti-cd-1d (clone 1b1) monoclonal antibody (mab) prior to surgery. midline laparotomies were performed; the common bile duct was ligated twice and divided. sham-operated animals served as controls. blood and liver samples were collected at periodic intervals post-surgery. the hepatic lymphoid population was purified and characterized by flow cytometry. the nkt cell population was increased significantly in the livers of control, but not kupffer cell-depleted, mice at 18 hours post-bdl. the response of inkt cells was diminished in mice pretreated with mab specific for il-12p40, a component of both il-12 and il-23; pretreatment with anti-cd1d mab had no effect. il-12rb-deficient mice also exhibited a marked increase in hepatic inkt cells following bdl suggesting that il-12 was not a critical factor. this suggestion is supported by the increased expression of il-23p19 and il-12p40 (but not il-12p35) mrnas by kupffer cells purified from the livers of bdl animals. these findings imply that il-23 production by kupffer cells promotes the response of hepatic inkt cells to biliary obstruction. objectives: p-glycoprotein (pgp or abcb1) is a member of the abc family of transporter proteins which are characterized by their ability to pump molecules across membranes in an atp-dependent manner. although pgp was first identified for its ability to confer resistance to chemotherapeutic agents in tumor cells, it has now also been described in cells of the immune system. our work primarily focuses on gd t cells that complement and regulate the activities of ab t cells, particularly in tissues. we have recently described functional subsets of gd cells based on cd27 expression. gd 27+ cells secrete interferon-g, while gd 27cells are capable of producing il-17. this study investigates the role of pgp in gd cells with specific reference to these recently-identified cd27-defined subsets. methods: pgp activity was measured based on the expulsion of rhodamine 123. cells were incubated with rho followed by a period in the presence or absence of the pgp inhibitor cyclosporine-a. cell populations were identified using monoclonal antibodies and flow cytometry. percentages of subpopulations were compared by anova, statistical results are shown as p values that were calculated using a newman-keuls multiple comparison post-hoc test. results: up to 40 % of intraepithelial lymphocytes (iels) from the small intestine are tcrgd + . of these, virtually all displayed pgp activity. indeed, pgp activity was generally higher in tcrgd + than tcrab + iels. in the thymus, pgp activity was observed in only˚2% of gd 27+ cells but not at all in gd 27cells. by contrast, in peripheral lymph nodes, mesenteric lymph nodes and peyer's patches, 40-60 % of gd 27+ cells were positive for pgp activity, although their gd 27counterparts remained largely negative (p x 0.01). conclusion: this study demonstrates that subsets of gd cells display different levels of pgp activity depending on their location in the body and their expression of the newly identified functional marker cd27. as pgp activity may play a role in cytokine release, cytotoxicity and protection from harmful toxins, it confirms our hypothesis that gd 27+ and gd 27cells have very different roles in immune responses and provides insight into the mechanism by which gd cells cope with diverse body locations. objectives: an effective immune response orchestrates different cellular activities of both innate and adaptive immune compartments. in this context, the vgamma9vdelta2 t cell biology presents some critical features for their ability to display a broad antimicrobial activity by directly killing infected cells and by inducing an effective adaptive immune response. the activation of vgamma9vdelta2 t cells by aminobisphosphonate drugs such as zoledronic acid (zol) results in a massive release of cytokines and chemokines that may induce a bystander activation of other immune cells such as dendritic cells (dcs) and b lymphocytes. the aim of this work was to evaluate the ability of activated vgamma9vdelta2 t lymphocytes to orchestrate granulocytes functions in terms of migration capability, phagocytic activity and alpha defensin release. methods: peripheral mononuclear cells (pbmc) and purified vgamma9vdelta2 t cells from healthy donors were stimulated with different compounds (zol, ipp) for 24 hours and supernatants from these cultures were tested for their ability to induce granulocytes activation. briefly, we analysed the migration activity, the phagocytic activity and the degranulation process by perforimg migration assays, flow cytometry and elisa tests. we showed that soluble factors released by zol-stimulated vgamma9vdelta2 t cells activate granulocytes by inducing their chemotaxis, phagocytosis, and alpha-defensins release. proteomic analysis allowed us to identify a number of cytokines and chemokines specifically released by activated vgamma9vdelta2 t cells. moreover, mcp-2 depletion by neutralizing ab revealed a critical role of this chemokine in induction of granulocyte alpha-defensins release. altogether, these data show a vgamma9vdelta2-mediated activation of granulocytes through a bystander mechanism, and confirm the wide ability of vgamma9vdelta2 tlymphocytes in orchestrating the immune response. conclusion: an immune modulating strategy targeting vgamma9vdelta2 t cells may represent a key switch to induce an effective and well-coordinated immune response, and can be proposed as a way to strengthen the immune competence during infectious diseases. objectives: the aim of this study was to analyse the activity of vg9vd2 t lymphocytes against glioma cells and to verify the possibility to target these innate cells in new immunotherapeutic approaches. human vg9vd2 t cells recognize and kill several cancer cells presenting a disregulation in mevalonate pathway. interestingly, drugs already in clinical use, such as zoledronic acid, are able to promptly activate vg9vd2t cells through an indirect mechanism involving the block of farnesyl pyrophosphate synthase of the mevalonate cycle. the vg9vd2 t cell activation by zoledronic acid results cytokines and chemokines synthesis and cytotoxic activity. glioma are tumors arising from glia in the central nervous system. unfortunately, the majority of glioma patients die in less then of a year from diagnosis and new treatment strategies are therefore hardly needed. methods: in order to analyse the activation of vg9vd2 t cells and their effects on the viability of glioma cells, we expanded in vitro vg9vd2t cells from pbmcs of healthy donors by using phosphoantigen stimulation and tested the ability of vg9vd2t cell lines to kill three different glioma cell lines (t70, u251, u373) by cytokinic/cytotoxic mechanism by flow cytometry. results: our results demonstrated that vg9vd2t cells lines are able to recognize glioma cells, to differentiate in effector memory cells, and to kill glioma cells by releasing perforin. moreover, we analysed whether zoledronic acid treatment could improve the susceptibility of glioma cells to vg9vd2 t lines. we showed that zoledronic acid is able to directly induce cell death on glioma cells and to strongly enhance the cytotoxic activity of vg9vd2 t lines. conclusions: altogether, our results suggest that the induction of a strong antitumor response in vitro of vg9vd2 t cells by using aminobisphosphonates could represent a new interesting immunotherapeutic approach for glioma treatment. viral-induced cancers, such as cervical cancer and liver cancer, contribute to approximately 15 % of all cancers and represent a failure of host immunity to control chronic viral infection. natural killer t (nkt) cells are a population of regulatory t lymphocytes that are pivotal to the outcome of host protection to a range of viral infections and cancers, but their role in controlling host defenses to oncogenic viruses in epithelial and cutaneous tissue is virtually unexplored. using a mouse model of chronic viral infection in the skin, in which human papillomavirus (hpv) oncoproteins are expressed as a transgene in epithelial cells, we investigated the role for nkt cells in abrogating protective immunity to viral antigens in cutaneous tissue. we show that local hpv-e7 protein expression in the skin attracts a large lymphocytic infiltrate, including a population of cd1d-restricted nkt cells. this nkt infiltrate is required to maintain local hpv-e7-induced immune suppression and results in graft survival when transplanted onto a naive, immunocompetent host. the local suppressive environment evident in e7-expressing transplanted skin is dependent on interactions between populations of cd1d-expressing cd11c+/f480+ myeloid cells and nkt cells. removal of either donor-resident or host-infiltrating nkt cells is sufficient to break immune suppression and allow e7 graft rejection. dissecting the suppressive properties of nkt cells in this novel model of chronic viral antigen presentation in the skin will provide valuable new insight into the potential for clinical manipulation of nkt cell populations to restore chronic anti-viral and anti-tumour immunity in epithelial tissues. nkt cells were expanded from total pbmcs from healthy donors by treatment with il-2 and a-galcer. expression of cd1a, cd1d and the costimulatory molecules cd86 and hla-dr, was established by flow cytometry. rna was quantified by real time-pcr. functional assays were performed by analysis of nkts cytokine production (ifn-g, il-4) and cytotoxicity against treated-idcs. results: idcs stimulated with olive pollen lipids up-regulated cd1d expression on the cell surface in comparison with control cells. in contrast cd1a expression was decreased. cd86 and hla-dr slightly increased, indicating certain grade of maturation. the amount of cd1d mrna was higher in treated cells than in control cells. by contrast, there was less transcription of cd1a, cd1b and cd1c genes than in control cells. nkt cells efficiently killed treated idcs as "in vitro" cytotoxic killing assays showed. ifn-g producing cells increased slightly in response to treated idcs compared to unstimulated cells, but the number of il-4 producing cells was not modified. similar results were obtained using monocytes as antigen-presenting cells. conclusions: idcs treated with lipidic extracts from olive pollen up-regulate the expression of cd1d on the cell surface. in addition, nkt cells are able to recognize idcs and monocytes treated with lipids from pollen, producing ifn-g and cytotoxicity. all these data suggest that nkt cells may play a role in the control of the immune response to allergens, such as the lipids present in pollen grains. outline: in humans, 0.5-10 % of circulating lymphocytes express a vg9vd2 t cell receptor, yet strikingly little is known about the function and properties of such unconventional t cells. we performed cdna microarrays to find vg9-enriched genes compared to conventional mhc-restricted cd8 + ab t cells, and found reciprocal enrichment of nectin-like adhesion molecules igsf4 & crtam in gd and ab t cells respectively. because igsf4 binds to crtam, the data fuel a hypothesis that this may be a novel axis of communication between the two cell types. interestingly, previous studies show that activated nk, nkt and cd8 + ab t cells express crtam, and that engagement of igsf4 on epithelial cells renders the latter targets for enhanced cytolytic and cytokine responses. our data extends this to the prospect of cytolytic immunoregulatory interactions between t cells mediated by igsf4/crtam. we therefore sought to answer: 1. what is the function of igsf4/crtam on gd t cells? 2. how is the igsf4-crtam axis regulated in t cells? results and conclusions: flow cytometry showed igsf4 enrichment on resting gd t cells, with expression also detected on˚10 % of ab t cells. the properties of those cells are being examined. however, igsf4 generally correlates with markers of activation/antigen experience such as cd45ro. thus, igsf4 cells may comprise activated-yet-resting/pseudo-memory unconventional t cells and memory-effector conventional t cells. stimulating vg9 + t cells in vitro led to rapid crtam induction, resulting in the majority of cells co-expressing both igsf4 and crtam within 48 hours. however, engagement of igsf4 by crtam or vice versa is not sufficient to induce cytotoxicity, as stable cho cell transfectants expressing either molecules were not specifically lysed by pbmc in vitro, compared to efficient and parallel targeting of mica + cells. instead, our current experiments address the possibility that crtam-igsf4 may regulate cytotoxic interactions promoted by other receptor-ligand interactions, such as mica-nkg2d. this may explain why cells can tolerate co-expression of both molecules, and would refute the hypothesis that crtam-igsf4 interactions are sufficient for cd8 t cells to kill gd t cells and/or vice versa. instead, crtam-igsf4 interactions may set the threshold for cytotoxic immune-surveillance responses. t cell receptor (tcr) is a multisubunit complex in which the invariant subunit cd3z is a 16 kda transmembrane protein indispensable for coupling antigen recognition by tcr to diverse signal transduction pathways. approximately 3-6 % of human peripheral blood lymphocytes express the gd tcr and the majority of these cells express the vd2 tcr variable segment associated with the vg9 segment, and recognize phosphorylated non-peptidic metabolites from microbial or self origin. these compounds trigger vg9vd2 t cells without antigen presentation. in vitro stimulated vg9vd2 t cells with antigens are able to produce ifn-g and tnf-a and exert a powerful cytotoxic activity against infected cells as hiv-infected cells. however, during hiv infection a marked decrease of vg9vd2 t cells was observed and the remaining cells are unable to respond to their non-peptidic ligands. aim of the present work was to study the mechanisms of vg9vd2 t cell anergy observed in hiv+ patients. to this aim, cd3z expression and ifn-g production by vg9vd2 t cells from hiv+ and hiv-subjects were analyzed. we show that vg9vd2 t cells from hiv-infected patients expressed lower level of cd3z compared with healthy donors. a direct correlation between cd3z expression and ifn-g production capability by vg9vd2 t cell was found. however, pkc activation by pma is able to restore cd3z expression and ifn-g production. our findings may contribute to clarify the molecular mechanisms of vg9vd2 t cell anergy found in hiv+ patients and have implication in the design of effective immune-based therapies. l. abeler-dörner 1 , m. swamy 1 , s.l. clarke 1 , a. hayday 1 1 king's college london, immunobiology, london, united kingdom gut intraepithelial lymphocytes (iel) constitute one of the largest t cell compartments in mice and in man. their functions and their interactions with surrounding epithelium are likely to be crucial to the fine-tuned balance between tolerance to harmless food antigens, immunity to gut-associated pathogens, and overall intestinal immune surveillance. intestinal iel comprise many unconventional t cells including tcrgd cells and tcrab cd8aa or cd8 -cd4cells, which have been assigned innate-like immune functions and key roles in surveillance of stressed tissue. unlike conventional t cells, iel might initiate an immune response rather than simply being late effector cells. it is therefore important to elucidate the "immunological information flow" in the gut. to this end, this project characterizes different subsets of iel and their interactions with epithelium in steady state and under immunostimulatory conditions in vitro and in vivo. in the past, it has been notoriously difficult to study iel ex vivo. to solve this problem, we developed a novel culture system that allows us to expand the cells ex vivo and study their responses for up to 15 days. the cells are initially activated by plate-coated acd3 antibody and a cytokine cocktail and maintained further in medium containing low levels of il-2. after a resting period, the cells can be restimulated in vitro. in this new system, we studied responses of different iel subsets to stimulation via tcr, nkg2d and cytokine receptors, either alone or in coculture with epithelial cells. as readouts we monitored proliferation, cytokine secretion (ifng, il-6) and expression of activating and costimulatory molecules. reactivation in response to various stimuli could already be observed after 6 hours. the in vitro data set forms the basis for analysing iel responses in vivo to stimulatory molecules ectopically expressed as transgenes in the gut. the characterization of iel responses opens new insights into the nature of gut immune responses and should provide a better understanding of the immunology of inflammatory bowel diseases which still remain a major problem in the clinic today. objective: behcet's disease (bd) is a multisystemic disorder with a possible underlying pathology of immune-mediated vasculitis. increased expression of cd94 in bd patients suggested that nk receptors may play a pathogenic or regulatory role in the pathogenesis. considering the regulatory functions of nkg2 molecules in heterodimer with cd94, we screened the presence of these receptors on t cell subsets in bd. the expression of nkg2 a/c/d molecules on gd and cd8+ t cells were analyzed in 17 active and 9 inactive patients with bd and 21 healthy controls. expression of nkg2 molecules was evaluated on cd8+, gd t and cd56+ nk cells by using flow-cytometry. results: gd t cells were increased in patients with bd compared to controls (4.4 vs. 2.8 %, p=0.001). in addition to the increase of gd t cells, increased expression of activating nkg2c molecules was also observed on gd t cells (20 % vs. 11 %, p= 0.01). nkg2a expression on gd t cells was found to be higher than nkg2c expression in patients and controls; but nkg2a expression on the t cells was not statistically different in both groups (33.5 vs. 40 %). nkg2d receptors were present on most of the gd t cells in both groups. however these activating molecules on cd8+ cells were decreased in patients with bd compared to controls ( revlimid is a therapeutic agent used to treat myelodysplastic syndrome (mds), a group of haematological disorders characterised by ineffective haematopoiesis. the mechanism of action for revlimid is poorly understood, but there has been increasing interest in the strong association reported between mds and defects within the immunoregulatory nkt cell compartment. indeed, some studies now suggest an important outcome of revlimid treatment is the restoration of normal cytokine production by nkt cell levels and an increase in their overall numbers. we have conducted the most thorough study to date of the nkt cell compartment of mds patients treated with revlimid/ancestim and can report that mds patients had normal nkt cell levels prior to treatment, and no significant increase as a result of revlimid/ancestim treatment. furthermore, nkt cells from mds patients produced high levels of th1 and th2 cytokines when stimulated with pma/ ionomycin and the proportion of nkt cells capable of cytokine production did not increase significantly after revlimid/ancestim treatment. these are highly significant findings given the recent emphasis on nkt cells as a potential therapeutic target for mds. our study provides an extensive analysis of the impact of revlimid/ ancestim treatment on the nkt cell compartment and sheds new light on the role of nkt cells in mds and the mechanism of revlimid immunomodulation. objectives: human gd t cells are potent killers of a variety of tumour cell lines, and mice lacking gd t cells suffer from high incidence of experimentally-induced tumours. however, the molecular mechanisms mediating tumour cell recognition by gd t lymphocytes remain largely unknown. we aim at identifying potential tumour antigens and co-stimulation molecules expressed in ex vivo tumours and in tumour cell lines that activate human gd t cells for tumour cytolysis. as immune evasion mechanisms that down-regulate tumour antigens may operate in vivo, we have identified candidates from human tumour cell lines of hematopoietic origin that constitute in vitro cytolysis targets for vg9/vd2+ lymphocytes. we have screened a panel of 26 lymphoma and leukaemia cell lines using a conventional in vitro killing assay using vg9/vd2+ cells, and selected two susceptible ("target") cell lines (over 60 % death in the assay) and two vg9/vd2+ resistant ("non-target") cell lines (under 20 % death) for cdna microarray analysis. we compared the differential expression in pairs of tumour cell lines of identical origin: the burkitt's lymphoma cell lines daudi (target) vs raji (non-target), and the pre-b cell leukemia cell lines rch-acv (target) vs 697 (non-target), and validated the results by rt-qpcr quantification. results: we identified 37 commonly up-regulated and 50 commonly down-regulated genes that encode cell membrane-associated proteins in susceptible tumours. ulbp1, ifitm1 and prame, for example, are up-regulated, whereas cd274 and clec2d are down-regulated in target cell lines. as these encode membrane-bound proteins with relevant functions in tumour immunity, they constitute potential ligands for gd lymphocyte recognition of tumour cells. the expression of these candidate genes was studied by rt-qpcr in a broader panel of cell lines and primary biopsies. we are currently testing, in functional assays based on rna interference and overexpression, these and other candidate genes in order to determine whether they provide activating or inhibitory signals to gd t cells. the comparison between the transcriptomes of vg9/vd2+ target versus non-target cell lines allowed the identification of candidate genes, whose individual function we are currently dissecting, that may be involved in tumour cell recognition by human gd t cells. mice and humans are the only species in which phenotype and function of inkt cells have been properly described. our aims are to directly identify this cell population and to investigate cd1d, in the rat. mice and rats have very similar cd1d and inkt tcr genes, with the exception of the va14 gene segment, which is a multimember gene family in the rat. novel monoclonal antibodies with nearly identical binding capacities to mouse and rat cd1d revealed a very similar pattern of cd1d distribution, and could inhibit cytokine production after agalcer stimulation of primary cells in both species. response to agalcer was studied in five different rat strains, showing big inter strain differences. notably, ifn-g and il-4 production was 10-100 fold lower in the best responder rat strain (f344) compared to mouse (c57/bl6). since nkrp1a (rat homologue of mouse nk1.1) and tcr are not appropriate markers for rat inkt, cd1d oligomers where tested for binding to inkt-tcr transduced cells. newly generated agalcer loaded rat cd1d dimers, recognized rat inkt tcr and, although less efficiently, bound to mouse inkt tcr. however, mouse cd1d agalcer dimers did not bind to rat inkt tcr. agalcer loaded rat cd1d dimers were then used to stain primary intrahepatic lymphocytes. but, although mouse inkt cells were stained to some extent, the identification of a discrete population in the rat was not possible. the reasons behind could be: that the avidity of the dimers for the tcr is not high enough to stain primary cells and/or that the frequencies are so low that the detection by facs analysis is difficult. in order to clarify these issues we currently produce and test rat cd1d tetramers. burkholderia pseudomallei is a highly virulent bacterium which causes the potentially fatal disease melioidosis in humans. this disease is endemic in tropical regions, especially thailand and northern australia, and has a serious outcome for many infected individuals. b. pseudomallei is an intracellular bacterium and many b. pseudomallei strains are resistant to antibiotics so antibiotic treatment is aggressive and relapse of the disease is frequent. in addition to this, no vaccine is currently available to prevent the disease. human g9d2 t cells are involved in the immune response to infection with a number of intracellular pathogens including brucella suis and mycobacterium tuberculosis. g9d2 t cells respond to non-peptidic phosphorylated molecules known as 'phosphoantigens' which are byproducts of essential metabolic pathways in both bacteria and mammals. phosphoantigens cause expansion and activation of g9d2 t cells during infection with intracellular pathogens including fransicella tularensis and m. tuberculosis. analogues of natural phosphoantigens have been developed to manipulate g9d2 t cell responses as a cancer therapeutic and are currently in clinical trials for the treatment of hepatitis c virus. we aimed to determine in vitro whether enhancing gd t cell responses in human blood using the synthetic phosphoantigen picostim could reduce growth of intracellular b. pseudomallei in the human monocytic cell line thp-1. a significant (p x 0.01) reduction in intracellular bacterial numbers was observed (n=8) in the presence of pbmcs cultured with picostim+il-2 in comparison with pbmcs cultured with il-2 or media alone. picostim+il-2 caused significant expansion and activation of gd t cells following culture of pbmcs for 10-14 days. purified gd t cells stimulated with picostim were able to reduce intracellular b. pseudomallei numbers 100-fold. this data demonstrates that pbmcs, stimulated with the synthetic phosphoantigen picostim+il-2, reduced growth of intracellular b. pseudomallei in a gd t cell-dependent manner. objectives: vgamma9/vdelta2 (gd) t cells play a major role in innate immunity against microbes, stressed and tumor cells. they represent less than 5 % of peripheral blood lymphocytes (pbl), but can be expanded in vitro by zoledronic acid (za)-treated monocytes or dendritic cells (dc).the purposes of this study are: 1) to determine whether dc generated from multiple myeloma (mm) patients are as effective as their normal counterparts in the ability to activate gd t cells; 2) to evaluate whether gd t cells can exert immunoadjuvant activity on dc generated from mm patients and primed with tumor-specific antigens (survivin-sv); 3) to establish whether the same issues could be solved using a simplified protocol of dc generation. 1) dc were generated from cd14 + cells of healthy donors/mm patients; immaturedc on day 6 were induced to fully mature by incubation for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za. after 7 days of co-culture dc:pbl, percentages and total counts of gd t cells were determined by flow cytometry; 2) idc generated from cd14 + cells of hla-a*0201 + healthy donors/patients were pulsed with sv-peptide and stimulated for 18 hours with tnfa + il-1b + pge2 in the presence or absence of 5 mm za; after 2 rounds of autologous t cells stimulation by dc, the frequency of sv-specific cd8 + t cells was determined by svpentamers staining; 3) the same experiments were performed both with dc generated following a standard protocol and a 48h protocol (dc fast objective: depletion of or deficiency in gd t cells aggravate colitis in different animal models. additionally, reconstitution of mice with syngeneic gd t cells ameliorated chemically-induced colitis indicating a suppressive or regulatory role for murine gd t cells in intestinal inflammation. therefore, we asked whether human gd t cells possess also suppressive or regulatory potential, which could be of therapeutical use in chronical inflammatory diseases such as ulcerative colitis or crohn's disease. hence, the proliferation, suppressive activity, and cytokine profile of human peripheral gd t cells were determined in vitro. methods: human gd t cells were isolated from whole blood of healthy donors by macs technology. the proliferation was determined by [6-3 h]-thymidine incorporation, while suppression of responder cell proliferation was measured by flow cytometry via cfse fluorescence intensity. the cytokine profile was determined by elisa from culture supernatants as well as by flow cytometry intracellularly. finally, the in vitro characteristics of gd t cells were compared to those of cd4 + cd25 + regulatory t cells (treg). human peripheral gd t cells show suppressive activity against responder cell proliferation, though being themselve anergic, that is, they produce negligible amounts of interleukin-2 on stimulation and proliferate poorly. while the proliferation of gd t cells and treg cells is comparable, the suppression of gd t cells on responder cell proliferation is even stronger than the suppression by treg cells though gd t cells being foxp3 negative. additionally, gd t cells are strong producers for tgf-b, particularly by the vd1 subset. conclusion: human peripheral gd t cells possess regulatory potential and could be of therapeutical use in treatment of chronical inflammatory diseases as they are anergic and act suppressive. their suppressive activity is even superior to treg cells and might be due to strong tgf-b secretion. for application of human gd t cells in therapy their expansion under maintenance of their regulatory properties should be elucidated. there are previous descriptions of gamma-delta t lymphocytes (gd) from behçet's disease patients (bd) but, in most of cases, they are incomplete or contradictory. it has been suggested that nkg2d on gd is involved in bd lesions through interaction with mica molecules. furthermore gdcd8+ have been recently proposed as a new regulatory t subset (treg). objectives: to study gd phenotype in bd active (bda) (n=8) and inactive (bdna) (n=20), versus healthy controls (hc) (n=30) and patients with recurrent oral ulcerations (ru) (n=14). to determine gd cytokine profile and surface markers treg-related in bd (n=9) and hc (n=11). methods: we obtained mononuclear cells from peripheral blood (pbmc). we determined by flow cytometry: -surface expression of: gd tcr, vdelta1, vdelta2, cd8alpha, cd8beta, nkg2d, nkg2a and cd103. -intracellular expression of ctla-4, and foxp3. -intracellular expression of il-2, il-4, ifngamma, il-10 and tgfbeta after pbmc polyclonal stimulation. we used two tailed test for means comparison (mann-whitney u or student's t test). -vdelta2+ cells were significantly increased in ru. vdelta1+ and gdcd8+ lymphocytes were significantly increased in bd versus ru and hc. -the mean fluorescence intensity of nkg2d was slightly increased in gd from bda. -nkg2a expression by gdcd8+ was not different in bd versus hc. -most of gdcd8+ presented cd8alpha-alpha homodimers in bd and hc and were negative for cd103, foxp3 and ctla-4. gdcd8+ and gdcd8-subsets were (in bd and hc): -high ifngamma-producers without differences. -low il-2-producers: il2+ cells were lower in gdcd8+ than in gdcd8-. -low il-10-producers: il10+ cells were lower in gdcd8+ than in gdcd8-. -low tgfbeta-producers: tgfbeta+ cells were lower in gdcd8+ than in gdcd8--very low producers of il4 in most of cases. the hallmark in bd was the increase of gdcd8/vdelta1+. this subpopulation has recently been described as immunosuppressive in infiltrates of human tumours and its function related to nkg2a in intraepithelial intestinal lymphocytes from celiac patients. we did not find a cytokine profile or a phenotype t-reg-related for gdcd8+, except a lower percentage of il-2+ cells than in the gdcd8-subset. gdcd8+ from bd did not show significant differences versus hc. natural killer t (nkt) cells comprise a highly heterogeneous subset of t lymphocytes that co-express a t cell receptor (tcr) and nk cells markers such as cd56 in humans. a subgroup, the invariant nkt cells (inkt), expresses the va24vb11 tcr rearrangement representing a minority subset in peripheral blood and virtually absent in the newborn. objectives: to establish a method to growth cord blood-derived nkt cells (cd3 + cd56 + ), in order to evaluate their phenotypic characteristics and the tcrvb repertoire. methods: mononuclear cells were isolated from 10 healthy umbilical cord blood samples and stimulated with ifn-g (50 ng/ml), anti-cd3 (25 ng/ml) and il-2 (500 ui/ml). these cells were cultured for 21 days and the expanded cd3 + cd56 + cells were isolated by immunomagnetic methods. surface markers were determined by flow cytometry. total rna was extracted from the purified cd3 + c56 + cell suspension using trizol ® reagent and mrna expression of twenty tcrvb gene families was measured by semiquantitative rt-pcr. statistical analyses were performed using mann-whitney u test and one-way anova, a p value of x 0,05 was considered significant. results: we could significantly expand cord blood cd3 + cd56 + nkt cells from 0,87±0,57 % to achieve an enrichment of 46,89±13,31 % (p=0,0002). table 1 shows the percentage (mean±sd,n=10) of phenotypic markers in cd3 + cd56 + cells at baseline (day 0) and after 21 days of culture. expression of mrna for the vb families studied was confirmed in each individual cell culture with a significant high expression of vb4 and vb8 families (p x 0,001). conclusion: our results show that cord blood-derived nkt cells are mainly cd8 + and cd94 + subsets, similar to peripheral blood nkt cell with a low percent of inkt cells. additionally, we confirm a diverse tcr vb repertoire with a significant expression of the vb4 and vb8 families in these cells. l. marischen 1 , d. wesch 1 , p. rosenstiel 2 , a. till 2 , d. kabelitz 1 1 institute of immunology, kiel, germany, 2 institute of clinical molecular biology, kiel, germany gd t cells account for a minority of t cells in human blood, but represent the majority of intraepithelial t cells in the intestinal tract. due to their ability to respond rapidly and in an mhc-independent fashion to particular antigens by cytokine production, gd t cells are considered as a link between innate and adaptive immunity. in addition, the expression of distinct pattern recognition receptors such as toll-like (tlr) and nod-like receptors (nlr) are characteristic for cells of the innate immune response. recent reports have demonstrated the tlr expression in human and murine gd t cells. here we provide evidence also for a gd t cell responsiveness to muramyl dipeptide (mdp), the putative ligand of the nlr family member nod2. peripheral blood mononuclear cells (pbmcs) containing gd t cells as well as freshly isolated gd t cells were stimulated via the gd t cell receptor in the absence or presence of mdp and analyzed for proliferation and ifng-production. while the proliferation of gd t cells within pbmcs was decreased, ifng-production was increased after costimulation with mdp compared to the stimulation with a non-activating dd-stereoisomer of the ligand (mdpi). the enhanced ifng production of pbmcs after costimulation was mediated mainly by gd t cells as shown by intracellular flow cytometric staining. with regard to the ifng-production after co-stimulation with mdp vs. mdpi, freshly isolated gd t cells from different healthy blood donors can be divided into responder and non-responder. responder gd t cells showed a significant increase of the ifng-production due to mdp-stimulation, whereas ifng-production was not influenced in non-responder gd t cells. in further experiments, as first approach to explain the different reactivity patterns of gd t cells, it is planned to analyze the polymorphisms of the nod2 gene in various donors. taken together, our preliminary data indicate that gd t cells are a major source of ifng-producing cells among pbmcs when challenged with specific antigens plus mdp, and support the role of gd t-cells as an important team player in the early immune response against bacteria. objectives & methods: an increasing of gamma-delta t cells during acute p. vivax infection and convalescent period has been reported. moreover, the activation of gamma-delta t cells leads to the inhibition of blood stage p. falciparum parasites in vitro. to determine the killing mechanisms of p. vivax parasites by gammadelta t cells comparing with what has been found in p. falciparum, the gamma-delta t cells were enriched by isopentenylpyrophosphate (ipp) from naïve pbmc. different number of gamma-delta t cells and normal pbmc were incubated with intact of p. vivax parasites and protein extract of p. vivax parasites, recombinant pvmsp1 19 and pvama1 proteins. gamma-delta t cells was daily determined the cytokine and granzyme intracellular releasing by flow cytometry until day 5 culturing. results: among the enriched gamma-delta t cells, the percentage of cells expressing cd69 + and cd25 + was elevated after co-culturing with intact and the proteins of p. vivax parasites. the overall gamma-delta t cells showed proliferation at day 3 after the co-cultivation. moreover, the gamma-delta t cells expressing ifngamma + and cd107a + (lysosomal associated membrane proteins: lamp-1) elevated from the first day of pbmc collection after co-culturing with the intact and p. vivax antigens. this level was correlated with the significantly decreasing number of parasites and the increasing percentage of parasite growth inhibition. our results showed the activation of gamma-delta t cells during p. vivax infection in vitro. this suggests that gamma-delta t cells could be stimulated by p. vivax parasites and these actively activated gamma-delta t cells could kill the parasites via mechanism of granzyme and cytokines at the early stage of cell activation. this study provides more understanding in activation of the innate immunity during acute malaria infection which may lead to the selection of appropriate malaria proteins as vaccine candidates in the future. objectives: several evidence suggest that invariant nkt cells (inkt) connect innate and acquired immune system. they are able to produce both th1 and th2 cytokines after stimulation. atopic dermatitis (ad) is a chronic inflammatory skin disease. th1-like and th2-like cytokines have been implicated in the pathogenesis of ad, but there are controversial data on their role in ad. the frequency and absolute number of inkt cells in mononuclear cells (pbmcs) of peripheral blood of patients with atopic dermatitis (ad) (n=43) and healthy controls (n=13) were determined by flow cytometry using anti-cd3 and monoclonal antibody specific for the cdr3 loop of the invariant tcr a chain of inkt cells (clone:6b11). furthermore, after pma/ionomycin stimulation for 4 hours, intracellular ifng and il-4 cytokines were detected in cd4+cd8-, cd4-cd8-(dn), cd4-cd8+ and cd4+cd8+ subsets of inkt cells by five colour flow cytometry in patients with ad (n=10) and healthy controls (n=10). results: both frequency and absolute number of inkt cells were significantly lower in patients with ad (p x 0.01) compared to healthy controls. the frequency of dn subpopulation was significantly lower in ad patient (p x 0.01). there was a positive correlation between the frequency of dn cells and inkt cells both in ad patients (r=0.726 and p x 0.001) and healthy controls (r=0.693 and p x 0.001). in the intracellular ifng level there were no significant difference in any of the inkt subsets of ad patients, however the intracellular il-4 level was significantly higher in dn subpopulation of inkt cells of ad patients compared to healthy controls (p x 0.05). the frequency, the number of inkt cells and the cytokine producing capacity of the cd4/cd8 inkt subsets are different in peripheral blood obtained from ad patients compared to healthy controls. our result suggest that the dn inkt cell subset can serve as a source of il-4 that promotes the th2 differentiation in ad patients and might play a role in the pathogenesis of this disease. introduction: intrahepatic immune cells (ihic) are known to play central roles in immunological responses mediated by the liver, and isolation and phenotypic characterization of these cells is therefore of considerable importance. aims: in the present investigation, we developed a simple procedure for the mechanical disruption of mouse liver that allows efficient isolation and phenotypic characterization of ihic. these cells are compared with the corresponding cells purified from the liver after enzymatic digestion with different concentrations of collagenase and dnase. results: the mechanical disruption yielded viable ihic in considerably greater numbers than those obtained following enzymatic digestion. the ihic isolated employing the mechanical disruption were heterogeneous in composition, consisting of both innate and adaptive immune cells, of which b, t, natural killer (nk), nk t cells, granulocytes and macrophages were the major populations (constituting 37.5%, 16.5%, 12.1%, 7.9%, 7.9% and 7.5% of the total number of cells recovered respectively). the ihic obtained following enzymatic digestion contained markedly lower numbers of nk t cells (1.8 %) . the b, t and nk t cells among ihic isolated employing mechanical disruption were found to be immunocompetent, i. e. they proliferated in vitro in response to their specific stimuli (lipopolysaccharide, concanavalin a and alpha-galactosylceramide respectively) and produced immunoglobulin m and interferon-gamma. conclusions: thus, the simple procedure for the mechanical disruption of mouse liver described here results in more efficient isolation of functionally competent ihic for various types of investigation. nature killer t cells (nkt) are a special t cell population with co-expresses nk and t cell surface markers. murine nkt cells include cd4 + nkt and cd4 -cd8 -nkt cells. nk1.1 + nkt cells may release large amounts of il-2, il-4, ifn-g and il-10 after they are activated. it has been reported that a-galactorsykeramide (a-galcer), a glycolipid, may induce proliferation of nkt cells with the role of immune regulation by stimulating mouse spleen cells. this study demonstrated that superantigen staphylococcal enterotoxin b (seb) , a kind of peptide, can activate the nkt cells with the function of immune tolerance. the response ability of seb-activating effect cells to cona, lps and il-2 had significantly decreased compared with that of normal lymphocytes. the effect cells exerted an inhibitory effect for the response of normal lymphocytes to cona and il-2. there was a significantly increase in the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cells identified from the seb-activated cells. based on the cell distribution detected in the upper part of the facs picture, expression of cd69 molecule existed in 68.95 % of the cells from large-scale selection. the percent of cd8 + nk1.1 + and tcrvb8 + nk1.1 + nkt cell subsets in the giant lymphocytes were enhanced to 84.0 and 38.9 folds, respectively. under a light microscope at x400 magnification, the seb-activating lymphocytes in size were larger than not only the cona-activated cells but also the adherent macrophages with an increase of 5 fold observed under a microscope. there were a few granules seen in cytoplasm. the value of cytoplasm vs nuclei was less than 1.0 and they are non-adherent cells. the differentiation pathway of the seb-activating cd8 + and tcrvb8 + nkt cells was not relative to a nk source. they were produced directly from t cell population and were considered as a subsets of t lymphocytes. our results suggest that the superantigen seb can act on the cd8 + nkt cell and tcrvb8 + nkt cells. and the two nkt cell subsets may play a critical role in seb mediated tolerance. gd t cells in the intestinal intraepithelial compartment (gd iiel) show an intrinsic activated phenotype. we hypothesised that their t cell receptor gd (tcrgd) is implicated in the activation of gd iiel. because the tcr gd ligands in mice are not well described, monoclonal antibodies (mab) directed against the gd tcr, like the clone gl3 which binds the d subunit of tcr gd, are important tools to specifically activate gd t cells. using cytometric indo-1am measurement, we could detect calcium flux of intestinal and peripheral gd t cells from tcrd-h2begfp reporter mice. stimulation with anti-gd clone gl3 or anti-cd3 clone 2c11 elicited activation of gd t cells suggesting that tcr gd and cd3 molecules in gd t cells are functional and signalling competent. next, using elisa and cytometric bead array, we found that iiel stimulated with plate bound gl3 in vitro produced ccl4, ifng and tnfa. therefore, we were interested whether the ccl4 production of gd iiel influenced the homing of ccr5 cells such as lamina propria (lp) cd4 + foxp3 + cells (tregs). to test this, wt mice were i. p. treated with gl3 mab and lp tregs were analysed by cytometry at various time points post inoculation. we found similar frequencies of lp tregs population but a slight decrease in ccr5 + tregs. however, when we compared wt and tcrd -/mice, we found both lower percentages of total lp tregs and of lp ccr5 + tregs in tcrd -/mice compared to wt mice. in conclusion, our data suggest that intraepithelial activation of gd t cell may directly or indirectly induce changes in the iiel and lamina propria (lp) lymphocyte compartment and influence the ccr5 expression and the homeostasis of lp treg. the ability of nkt cells to serve a variety of different immunoregulatory functions in vivo may reflect a diversity in function of different nkt cell subsets. diversity in cytokine production by nkt cell subsets has been observed in murine and human studies, although this analysis has largely been following in vitro restimulation. here, we investigated cytokine production by murine nkt cell subsets in vivo under conditions where minimal manipulation of the cells was required. to this end, we examined il-4 production in g4 reporter strains in which dna encoding green fluorescent protein (gfp) was inserted into the first exon of the il-4 gene. in the absence of any manipulation gfp was expressed from the il-4 locus in populations of immature thymic nkt cells (predominantly cd4+cd44lotcrhi cells on a balb/c background, and cd4+cd44lonk1.1-on a c57bl/6 background) and some splenic nkt cells, with overall numbers of gfp+ cells in both tissues decreasing with age. after i. v. administration of the nkt cell ligand a-galactosylceramide, il-4 production was induced predominantly in cd4+ nkt cell subsets of the liver and spleen, and after i. n. administration, in cd4+ nkt cells of the airways. spontaneous and a-galcer-induced expression from the il-4 locus occurred in the absence of stat6 signalling, and did not require initial exposure to il-4 protein from other sources in the host. diversification in cytokine expression by nkt cells subsets therefore occurs early in ontogeny, and is also a significant feature of responses to exogenous activating stimuli. interleukin-17 (il-17) plays an important role in neutrophil recruitment. herein, we investigated the role of il-17 receptor signaling in polymicrobial sepsis induced by cecal ligation and puncture (clp). methods: adult c57bl/6 (wt) and il-17 receptor gene-deficient (il-17r ko) mice were subjected to non severe (ns-clp) sepsis. intraperitoneal neutrophil migration, bacteremia, cytokine, chemokines and liver injury were evaluated 6 hours after surgery. the ability of il-17 mediate the neutrophil microbiocidal activity in vitro, as well the neutrophil migration in vivo and in vitro were also evaluated. the means of different treatments were compared by analysis of variance (anova), followed by bonferroni's t test and the survival rate by the mantel-cox log rank test. results: it was observed that il-17r ko mice, subjected to ns-clp sepsis, show reduced neutrophil recruitment into peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared to wt. as a consequence, the mice showed an increased mortality rate. moreover, il-17 induced neutrophil migration in vivo and in vitro. besides, we demonstrated that neutrophils harvested from il-17r ko mice already show reduced microbiocidal activity, compared with wt, suggesting a physiological role of il-17receptor signaling in the microbiocidal activity of neutrophils. furthermore, wt neutrophils treated with il-17 showed strongly enhancement of microbiocidal activity by a mechanism dependent of nitric oxide. conclusion: during ns-clp besides the importance in recruit neutrophils to focus of infection, il-17 also enhances the microbiocidal activity of neutrophils. therefore, our results demonstrated that il-17 receptor signalization plays a critical role on host protection during polymicrobial sepsis. objectives: members of the toll-interleukin-1 receptor (tir) family are important for host defense, inflammation, and immune regulation. their canonical signaling pathway involves adaptor proteins and il-1r associated kinases to activate nfxb and p38 mitogen-activated protein kinase. the il-33-induced signal transduction in mast cells is poorly understood. in this work we studied the signal transduction of il-33 in different mast cell subsets. methods: different mast cells subsets (hmc-1, human cbmcs and murine bmmcs) were stimulated with il-33. the resulting signal transduction was investigated by immunoblot for activated signaling molecules (pc-kit, perk1/2, pakt, pnfxb, p38 and pjnk). additionally, we studied the signal transduction of il-33 in il-33r transfected hek293t cells. results: we found, that a tir family member, il-33r, transactivates the receptor tyrosine kinase c-kit in mast cells and that il-33-induced cytokine production depends on c-kit transactivation. il-33r and il-1r accessory protein (il-1racp) form a physical complex with c-kit. thereby the complexation is dependent on the activity of c-kit. conclusion: these results show for the first time that the biological function of an il-1r family member is dependent on the presence of an activated receptor tyrosine kinase. furthermore, these results reveal that certain il-33-induced signaling pathways and effector functions are dependent on activated c-kit and could therefore explain the effects of il-33 in mast cells in absence of iger activation. (1) . we now provide a molecular mechanism underlying this pathogenic effect by which free heme sensitizes hepatocytes to undergo tnfmediated programmed cell death. independently of newly gene transcription and/or protein synthesis, free heme cytotoxicity is mediated by the unfettered generation of free radicals in response to tnf, presumably due to the participation in the fenton reaction of the fe atom present in the protoporphyrin ix ring. once exposed in vitro to free heme, a sustained c-jun n-terminal kinase (jnk) activation was observed in hepatocytes in response to tnf, an effect that promotes further free radicals production. pharmacologic or genetic (shrna) inhibition of jnk in hepatocytes avoids free radicals accumulation and caspase-3 activation, also mimicked by the anti-oxidants n-acetylcystein (nac) or butylated hydroxyanisole (bha). expression of the heme catabolyzing enzyme heme oxygnease-1 (ho-1) in hepatocytes affords protection against heme sensitization to tnf cytotoxicity. recombinant adenovirus mediated ho-1 expression in the liver suppresses tnfmediated hepatocyte apoptosis and prevents the lethal outcome of plasmodium infection in mice. in conclusion our data reveals a novel signal transduction pathway via which heme sensitizes hepatocytes to undergo tnf-mediated cytotoxic effect, critically involved in the outcome plasmodium infection. the multi-step leukocyte extravasation process is governed by adhesion molecules and chemotactic factors dynamically interplaying in the presence of shear forces. responsiveness to chemotactic ligands is mediated by g protein-coupled receptors (gpcrs) which are finely regulated by a family of cytosolic proteins, betaarrestin 1 and 2. recent evidence indicates that, in addition to playing a regulatory role in gpcr desensitization and internalization, beta-arrestins may contribute to gpcr signaling by functioning as scaffolds for the recruitment of signaling proteins into complexes with agonist-occupied receptors. on this basis, we investigated the physiological role of beta-arrestin 2 in chemokine-driven dynamics associated with leukocyte extravasation, with special interest to the activation of the rap1 small gtpase, recently emerged as pivotal regulator of integrin function. the analysis of kc the (keratinocyte-derived chemokine) rap1 activation profile in rbl (rat basophilic leukemia) cells expressing mcxcr2 shows a bimodal kinetic, with the first peak at 30''/1' and the second at 5' after stimulation. rna interference-mediated depletion of beta-arrestin 2 specifically inhibits the occurence of the second wave of rap1 activation, whilst it has no effect on the early pick, thereby suggesting that beta-arrrestin 2 is involved in rap1 activation and that the oscillations in the formation of rap1-gtp are regulated by different molecular mechanisms. in order to elucidate the gefs and gaps involved in the gtpase regulation we are at present down-regulating the expression of c3g (rap1gef) and spa1 (rap1gap): preliminary results suggest that spa-1 has probably a role in the early activation peak. since this oscillatory chemokine-induced rap1 activation is present on other myeloid cell lines (hl60, 32d) and fresh pmn's we are also translating our research to these more appropriated cells. interestingly betaarrestins amino acid sequence and three-dimensional structure reveal a unique and evolutionary conserved proline-rich sequence in beta-arrestin 2, localized in a solvent exposed loop which may serve as a docking site for migration-associated transducers/adaptors. in order to find sh3 containing proteins that interact with beta-arrestins, we have performed an overlay screening assay of 153 different sh3 domains that revealed over 20 putative beta-arrestins putative interactors, some of which isoform specific. granulocyte-macrophage colony-stimulating factor (gm-csf), interleukin (il)-3 and il-5 stimulate proliferation, differentiation, survival and functional activation of myeloid cells. the cell surface receptors for these cytokines consist of cytokine-specific a subunits and a common b-receptor (bc), required for the activation of intracellular signaling following cytokine engagement. aberrant signalling, stimulated by these cytokines, has been implicated in the pathogenesis of many diseases, including arthritis, asthma and leukemia. as a result, we have sought to define key molecular determinants of these receptor-cytokine interactions in order to gain a greater understanding of receptor activation. here we present novel insights into the role of the ig-like domain of the gm-csfra in gm-csf binding. deletion of the ig-like domain abolished direct gm-csf binding and we identified specific residues directly involved in ligand binding by site directed mutagenesis and binding studies. the results indicate a previously unrecognized role for the ig-like domain of gm-csfra. furthermore, we address a longstanding controversy in the field of gm-csf, il-3 and il-5 receptor biology, by performing a systematic study of the role of n-glycosylation upon on the bc, and related murine b il-3 , in ligand-binding and receptor activation. these data demonstrate definitively that n-glycosylation does not play a role in mediating ligand-binding or receptor activation. these findings clearly establish that the determined human bc structures lacking glycosylation at asn 328 are biologically relevant conformers of the human bc ectodomain. our results appear to suggest that the potency of receptor signalling can be influenced by the biophysical and structural properties of the extracellular receptorligand interactions and it also addresses important, poorly-understood aspects of mechanisms underlying ligand recognition and activation of the gm-csf: gm-csfra: hbc receptor complex. reference: (1) micrornas (mirnas) are endogenous small non-coding rna molecules acting as key regulators of immune cell differentiation and innate immune responses. mirna-146 expression is induced by activation of the toll-like/interleukin-1 receptor pathway (tirpathway), where it targets essential adaptor and signaling molecules, thus serving as a regulator preventing the cells from an exacerbated pro-inflammatory response. since tnfa also up-regulates the expression of mirna-146a, we decided to explore whether this mirna is involved in the regulation of apoptosis. to this end, we used the hela human epithelial cell line as a model system for tnfa signaling. following tnfa and cycloheximide (chx) treatment mirna-146a transfected cells showed significantly reduced levels of the active proapoptotic caspases 8 and 3 (casp8/3). in line with this, mirna-146a conferred enhanced protection against tnfa-induced dna fragmentation and mitochondrial potential drop-down. our results demonstrate that mirna-146a is a regulator of receptor-mediated apoptosis. similar to the tir-pathway, mirna-146a seems to be part of a negative feedback mechanism of the tnfa signaling cascade. ongoing research focuses on the identification of the specific pro-apoptotic molecules targeted by mirna-146a. furthermore, we are exploring the relevance of our observations for the mycobacterial infection of human macrophages, where the regulation of apoptosis is critical. objectives: the aim of this study was to evaluate the role of single nucleotide polymorphisms (snps) located in il-15, il-7r a-chain and il-15r a -chain genes in hiv disease progression. methods: we studied 70 antiretroviral treated patients (progressors) and 71 long term non progressors (ltnp). we analyzed 2 snps in the il-15 gene, 5 snps in the il-7r gene and 3 snps in the il-15r gene. in univariate analysis, we found an association between the presence of at least one mutated a allele in il-15r aa 182 and a higher possibility of being ltnp ( our study suggests that genetic polymorphisms located in il-7r and il-15r genes can influence the rate of disease progression in hiv+ patients, especially when a combination of aplotypes is present. mutations in the coding regions might compromise the binding of the cytokines or the intracellular signal transduction pathways, therefore leading to the alteration of cd4 and cd8 t cells homeostasis. aims: mono-adp-ribosyltransferases (arts) are gpi-anchored ectoenzymes that covalently modify cell-surface or soluble target proteins by transferring an adpribose moiety from extracellular nad+ to specific arginine residues of target proteins. in this study, we report that human tumor necrosis factor (tnf) is adpribosylated by art1, and that adp-ribosylation affects both the release of tnf from cells and its cytolytic action. methods: transcription of art1 in human leukocytes was analyzed by rt-pcr. adp-ribosylation of tnf was detected by monitoring the incorporation of adpribose from labeled nad. release of tnf from transfected hek cells was monitored by elisa. binding of tnf to tnf receptors was analyzed by biacore. tnf cytotoxicity was monitored by flow cytometry. the adp-ribosylation site on tnf was analyzed by lc/ms mass spectrometry. results: we identified art1 transcripts by rt-pcr analysis in human blood leukocytes. soluble art1, released from the surface of transfected cells by phosphatidylinositol-specific phospholipase c (pi-plc), adp-ribosylated recombinant human tnf in vitro. co-transfection of hek293 cells with art1 and tnf resulted in modification of tnf at at least 2 distinct sites, i. e. one within the tnf ectodomain, and one on the stalk that remains connected with the cell membrane after cleavage by tnfa converting enzyme (tace). analysis of modified recombinant tnf by mass spectrometry provided evidence that the tnf ectodomain is adp-ribosylated at r32, a site that has previously been implicated in binding to tnfr2. binding assays indicated that adp-ribosylation inhibited binding of tnf to its receptors. importantly, modified tnf was less potent at inducing cell death in the human t cell lymphoma line kit225 than wildtype tnf. furthermore, cell surface adp-ribosylation of hek cells co-transfected with tnf and art1 resulted in reduced release of tnf into the supernatant. conclusions: adp-ribosylation of tnf or other cell surface proteins interferes with the biology of tnf signals by at least two distinct mechanisms. adpribosylation of tnf blocks binding to its receptors, thereby inhibiting tnf-mediated cytotoxicity. additionally, adp-ribosylation of tnf or another protein on the surface of tnf-producing cells inhibits the proteolytic release of tnf. noninflammatory chronic pelvic pain syndrome : immunological study in ejaculate g. n. drannik 1 , t.v.poroshina 1 1 institut of urology amsci of ukraine, laboratory immunology, kyiv, ukraine chronic prostatitis (cp) is a disease which likely is associated with abnormalities in local immune responses. secretions of the urinary and reproductive tract mucosa contain various protective effector molecules, produced by mucosal cells, lymphocytes, macrophages and neutrophiles. the aim of this prospective study was to observe local immunophenotypic patterns in patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome for further description and as possible surrogate markers for diagnosis and treatment. methods: 23 patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome (cp/cpps) and 11 control men were assessed for slpi, tnf-alpha, il-8 and free tgf-b1 in ejaculate by elisas using stat-fax 303 plus. a 3 to 5 day sexual abstinence period was required from the subjects before semen collection. after liquefaction and centrifugation, seminal plasma samples were kept at -20 degrees c°until assayed. the materials were processed after the standard programmes for statistical analysis.the study was approved by the local ethics committee. the slpi concentration was elevated in all patients (5127.571 ± 971.731 pg/ml, p x 0.001) in seminal fluid, in comparison with the healthy control subjects. the tnf-alpha concentration was elevated in all patients in seminal fluid (125.49 ± 17.9 pg/ml; p x 0.05). the il-8 concentration was elevated in all patients (366.7± 49.5 pg/ml; p x 0.05) in seminal fluid. free tgf-b1 was present in normal seminal plasma in high concentrations (346.4 ± 29.2 pg/ml), while in ejaculates of patients with noninflammatory cp/cpps tgf-b1 concentrations were 36.2 ± 6.2 pg/ml. conclusion: ejaculate's slpi, tnf-alpha, il-8 and free tgf-b1 are possible surrogate markers for the diagnosis and treatment of patients with noninflammatory chronic prostatitis/chronic pelvic pain syndrome. m. r. marrakchi 1 , e. a. elgaaeid 1 1 faculté des sciences de tunis, biology, tunisie, tunisia ulcerative colitis (uc) and crohn's disease (cd), collectively referred to the inflammatory bowel disease (ibd), represent a group of multifactorial autoimmune disorders of the gastrointestinal tract sharing many clinical and pathological characteristics, however, differing in histological features and cytokine profiles. the excessive production of either th1 or th2 cytokines due to perturbed regulation of immune system activation results in chronic inflammatory processes and loss of immune homeostasis that may be implicated in the genesis of ibd. studies have identified a gene that encodes the nod2/card15 protein, which is involved in the immune system's response to bacterial infection and confirmed to influence susceptibility to cd. indead, it has been suggested that high rates of asca (saccharomyces cerevisiae ) in absence of panca (perinuclear anca: anti-neutrophil cytoplasmic) antibodies were associated with aggressive forms of cd and that the important rise of panca was more frequent at uc . in a sample of tunisian patients, we examined the contribution of nod2/card15 gene in cd. we performed a cases /controls study upon 75 cd patients and 90 healthy controls. this study suggests that in northen tunisian population, 3020insc mutation in nod2/card15 gene is a prevalent mutation leading to the typical crohn's disease including ileal location, stricturing and penetrating clinical types and asca expression. since conflicting results were obtained on il-10 polymorphisms as risk factor for ibd, the aim of our study was also to explore anti-inflammatory il-10 cytokine genetic profile in patients with ibd. we examined the contribution of il-10 gene promoter polymorphisms (-627 and -1117) to crohn's disease (cd) phenotype, and the possible genetic epistasis between these polymorphisms and card15/nod2 gene mutations in cd presentation and location. in tunisian population, the 3020insc insertion in nod2/card15 gene is a marker of susceptibility to cd, while the a allele at position -627 in the il-10 promoter increases the risk of cd ileal location and severe disease presentation. a genetic epistasis between il-10 gene polymorphisms and card15/nod2 gene mutation was suggested. in conclusion, genetic and serologic markers might be useful in defining patien gangliosides were shown to inhibit the il-2-dependent proliferation of t-cells, implying that gangliosides interfere with one or more of the il-2-driven events. it is known that the major mechanism of inhibition is the direct interaction between ganglioside and the cytokine and, as a result, the capture of il-2 molecule by ganglioside. but gangliosides apparently can also form complexes with il-2r; such complexes influence on the signal transduction through il-2r. this effect of gangliosides may lead to the failure of this pathway. unfortunately, the biological and structural aspects of this problem are poorly understood. in this study we propose possible modes of interactions between exogenous gangliosides and il-2r subunits. in our work we use il-2-dependent cytotoxic t-cell murine line ctll-2. two different approaches for study of possible interaction types between exogenous gangliosides and il-2r subunits were applied: antibody staining of il-2r subunits followed by flow cytometry analysis, and photoaffinity labeling of living cells with 125 i-dcp-gm1 followed by immunoprecipitation of il-2r subunits. the fluorescence intensity of the antibody-labeled il-2r a-subunit substantially decreases after the treatment of cells with gangliosides. it has been shown that the fluorescent labeled cell fraction decreases by 29.5 % after cells incubation with ganglioside gm1, and by 10.7 % after incubation with gm2. labeling of the cells with antibodies to the il-2r b-subunit results in a less significant fluorescence decrease after cells incubation either with gm1 (2.8 %) or with gm2 (5.9 %). to determine the mode of this impressive masking influence of ganglioside gm1, photoaffinity cross-linking has been used. in our modification this method could identify is interaction of gm1 with subunits of il-2r occur with or without incorporation exogenous ganglioside into plasma membrane. electrophoresis followed by immunoprecipitation with appropriate antibodies resulted in appearance of the radioactive band only for b-subunit of il-2r, but not for il-2r a-subunit. these results demonstrate that exogenous ganglioside gm1 can interact with a-and bsubunits of il-2r in different modes. interaction of il-2r b-subunit with ganglioside gm1 requires incorporation of the ganglioside into plasma membrane, but it is not the case for interaction with a-subunit. a. respa 1 , j. bukur 1 , s. purpose: loss of interferon (ifn)-g inducibility of hla class i antigens has been found in some tumor cell lines, but the underlying molecular mechanisms of such deficiencies have not yet been elucidated in detail. this kind of tumor escape mechanism might lead to an inefficient recognition of tumor cells by the immune system. methods: phospho-specific western blot analyses were performed to verify the functionality of the different ifn-g pathway components, intra-and extracellular flow cytometry experiments were employed to determine the expression of antigen processing components and hla class i cell surface antigens, quantitative real time-pcr experiments to confirm the absence of jak2 and presence of pathway relevant molecules as well as, genomic pcr and chromosome typing technique to prove the deletion of jak2. results: different ifn-responsive phenotypes were defined in human melanoma cell lines varying from loss to low, delayed as well as strong ifn-g inducibility. resistance to ifn-g treatment was associated in one melanoma cell line with the lack of jak2 expression due to a gene deletion on chromosome 9, whereas the expression and functionality of other ifn-g signal transduction components like stat1 and jak1 were not affected. jak2 blockade by two jak2-specific inhibitors resulted in reduced levels of hla class i surface antigens. conclusion: structural abnormalities of jak2 leading to a lack of jak2 expression are associated with loss of ifn-g inducibility as well as reduced constitutive hla class i surface expression. in addition the jak2 inhibition modulates the expression of the hla class i antigen processing components. of renal cell carcinomas (rccs) are associated with the size, grade, t, n, m, stage and death of rccs patients. the genotypes were compared with those of a random sample of 506 controls of the spanish population. methods: two il18 polymorphisms located on the il-18 promoter region, snps -607 a/c (rs1946518) and -137 g/c (rs187238) were genotyped using taqman snp genotyping assays. the functional il-18 gene polymorphisms studied do not influence the susceptibility to rccs in the spanish populations (il-18 -607 p=0,318. il18 -137 p=0,740) but may contribute to disease onset and aggressiveness: the genotype il-18 -607 cc genotype, which is associated with higher il-18 production, was significantly associated with higher tumor size (p=0,001), grade (p=0,030), t (p=0,001), m (p=0,012) and stage (p=0,002). the influence of the il-18 -103 gg genotype was less relevant, however was correlated with higher tumor size (p=0,036), grade (p=0,017), t (p=0,026) and stage (p=0,011). the multivariate analysis with cox proportional hazard model revealed that, in this serie, nuclear grade and stage grouping were independent prognostic factors, whereas il-18 polimorphism can not be considered as independent prognosis factors. our results suggest that the polymorphism variants from the il-18 gene (il-18 -607 and -137) may be associated with an worse prognosis of rcc. high levels of il-18 production can play an important role in grow, invasion and metastasis of renal cancer. interleukin-18 (il-18) is a pleiotropic cytokine that is involved in regulation of both the innate and acquired immune response. the most prominent biologic property of il-18 is its ability to induce the production of ifn-gamma in presence of il-12. moreover, it stimulates the expression of tnf-a and il-l, enhances the differentiation of t cells to the thl and impairs the synthesis of the anti-inflammatory cytokine il-l0. then it seems that il-18 has a crucial role in immunity against brucella infection. since the expression of il-18 can be affected by polymorphisms in its gene, we decided to investigate any probable relation between six different il-18 gene polymorph isms and brucellosis. methods: a total of 188 patients with brucellosis and 77 healthy farmer who consumed contaminated row milk and dairy products from animals with brucellosis, were included in this study. all individuals were genotyped for six il-18 polymorphisms at positions -656, -607, 137, +113, +127 and codon 35/3 using polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). the distribution of alleles for il-18 polymorphisms at position -137 g/+113t/+127c (correlated with high production of il-18), codon 35/3c and -656g/-607c (correlated with higher production of il-18) were significantly higher in the healthy controls than the patients (p=0.000022, p=0.00185 and p=0.0441, respectively). discussion: as data revealed genotypes that have correlation with higher production of il-18 are more frequent in the controls than the patients. then it might be deduced that individuals who inherited these genotypes/alleles are able to produce higher level of il-18 at the onset of infection and it leads to more ifngamma production and control brucella infection before appearing brucellosis. abstract withdrawn by author objectives: a dsyregulated cytokine response has been shown to play a role in inflammatory bowel disease (ibd) pathogenesis. to dissect the influence of these cytokines, specifically interferon gamma (ifng), on the inflammatory response and colitis we have created a cell specific ifng receptor 2 (ifngr2) mouse mutant. we have generated a mouse line carrying a conditional ifngr2 allele using the 2 loxp 2-flp recognition target (frt) approach. a targeting vector with 2 loxp sites flanking exons 4-6 and 2 frt sites flanking the neomycin resistance cassette was generated. after confirmation of homologous recombination the neomycin resistance cassette was deleted by crossing with flp deleter mice. cell specific deletion is being performed by crossing conditional 'flox/flox' mice with specific cre expressing mouse lines. functional inactivation of the receptor has been demonstrated by performing western blots to detect phosphorylated and total stat1 following ifng stimulation. results: successful deletion of the gene and conditional mutants without the presence of the neomycin resistant cassette have been generated. functional inactivation of the receptor with no stat1 activation following ifng stimulation has been demonstrated in the complete knock out mice. furthermore, flox/flox mice retain full responsiveness to ifng. breeding with lysm cre and cd4 cre mice will been completed to create a cell specific deletion of the ifng receptor in macrophages/granulocytes and t cells respectively. the specificity of this deletion will be confirmed through cell sorting and functional assays. in order to determine the influence of ifng on a specific cell type a conditional gene targeting approach has been utilised. this has allowed the generation of conditional mice mutants with deletion of ifngr2 on macrophages and t cells. this has generated a very powerful tool for dissecting the role of this cytokine in numerous disease models. moreover, the ability to cross the conditional mice with additional cre lines will enable the analysis of more cell types in the future. a. gonzalez 1 , r. carretero 2 , p. saenz-lopez 2 , j. cantón 2 , j. carretero 2 , f. ruiz-cabello 2 , l.m. torres 1 , cts-143 1 hospital universitario virgen de las nieves, departamento de ginecología, granada, spain, 2 hospital universitario virgen de las nieves, departamento de análisis clí nicos, granada, spain objetives: cervical cancer is almost associated with infection by human papillomavirus (hpv). however, only a subset of infected women will ever develop the malignancy. ifng dinucleotide (ca) repeat polymorphism is responsible for genetic differences in interferon-gamma production. our objective was to investigated the relationship between ifgn polymorphism and cervical intraepithelial neoplasia (cin) methods: we have studied nineteen women with cin and 130 normal women control. dna was extracted from blood samples, and was genotyped for functional microsatellite (ca) repeats in the first intron of ifn-gamma gene. results: heterozygosis in (ca)12 allele of ifgn is significant more frequent in healthy control than in cin patient (p=0,030) in contrast homozygosis for (ca)12 allele did not show significant differences between both population. analysis of relation between this polymorphism and the cin stage showed that both heterozygosis and homozygosis is correlated with advanced stage (p=0,030 and p=0,045 respectively). conclusions: our study suggest that ifng (ca)12 allele may influence in cin risk and progression. ifng is associated with hpv clearance, but it also plays a role as inflammatory cytokine, promoting cervical malignant progression. further studies of the role of ifng and other cytokines may contribute to the understanding of cin promotions and progression. introduction: macrophages play a fundamental role in controlling of brucella infection, mainly through the secretion of cytokines such ifn-y and tnf-a. interleukin-15 (il-15), a th1-related cytokine, triggers inflammatory cell recruitment and increases the expression of ifn-y. as the cytokine production is under the genetic control, we decided to investigate the association between il-15 single-nucleotide polymorphisms (snps) and susceptibility to brucellosis in iranian patients. methods: 190 patients with brucellosis and 78 healthy animal husband men who kept animals with brucellosis, were included in this study. all individuals were genotyped for il-15 (c267t, g367a, c13687a and a14035t) polymorph isms using pcr-rflp. results: at position 267, the distribution of c allele and cc genotype were significantly lower in the patients than the controls (p=0.025 and p=0.042, respectively). at position 13687, the distribution of c allele and aa genotype were significantly higher and lower in the patients than the controls, respectively (p=0.050, p=0.015). discussion: as shown the frequency of cc genotype and c allele at position 267 were higher in healthy controls than the patients. hence, our study provides evidence that presences of cc genotype and/or c allele are significantly associated with susceptibility to brucellosis. the frequency of aa genotype at position 13687 is lower in patients than the healthy controls and the frequency of c allele at position 13687 is higher in patients than the healthy controls. hence, our study provides evidence that presences of c allele and lack of aa genotype are significantly associated with susceptibility to brucellosis. objectives: increasing evidence is emerging regarding the ability of mammary epithelial cells to respond to various cytokines. our aim was to investigate the cytokine receptor phenotypes of two distinctive human mammary cell lines, mcf-7 and skbr-3, as well as their ability to respond to several cytokines and to the g-1 gpr30 agonist. the mcf-7 and skbr-3 human adenocarcinoma cell lines were investigated by immunocytochemistry and flow cytometry for the expression of the following cytokine receptors: il-2ra, il-2rg, il-3/il-5/gm-csfr, il-4/il-13r, il-5ra, il-6ra, il-6r (gp130), il-7ra, il-10r, tnfr i, tnfr ii, ifngra, cxcr1, cxcr2, cxcr3, cxcr4, cxcr5. cells were incubated with il-10, ifn-g, tnf-a and tnf-b (for which both cell lines displayed receptors) alone and with the gpr30 agonist. cytosolic ca 2+ concentrations [ca 2+ ] i were measured by the microfluorimetric technique. results: different cytokine receptors phenotypes emerged for the two cell lines. the less well differentiated skbr-3 cells were found positive for a larger number of receptors than the mcf-7 cells. both cell lines displayed an important heterogeneity for each of the investigated molecules, in terms of number of positive cells and expression intensity, with chemokine receptors percentages constantly higher than for the other receptors. pretreatment of mcf-7 cells with il-10 reduced the calcium response to g-1, while pretreatment with ifn-g and tnf-a potentiated the calcium response to g-1. tnf-b had no effect on mcf-7 g-1 stimulation. no direct effect on basal [ca 2+ ] i stimulation could be noticed when administering the cytokines alone. in skbr-3 cells, pretreatment with il-10 or tnf-b had no effect on basal [ca 2+ ] i and did not significantly alter the calcium response to g-1, while pretreatment with ifn-g induced calcium oscillations and potentiated the calcium response to g-1. pretreatment with tnf-a produced calcium oscillations and reduced the response to g-1. conclusions: mcf-7 and skbr-3 cell lines express distinctive cytokine receptor phenotypes. furthermore, their ability to respond to cytokines in terms of modulating the gpr30 stimulation proved to be different. the susceptibility towards various soluble factors of these cell lines can offer us some insights on the complexity and individuality of each primary tumor and thus the distinctive evolution of each particular patient. results: in initial analyses of the early infection phase we identified splenic pdcs expressing ifnb/yfp. furthermore, we show for the first time in vivo that these ifnb producing pdcs are not directly infected with mcmv and that not only cdcs but also cd8a -pdcs expressed gfp as a marker for mcmv infection. we observed that at early time points equal frequencies of cd8a -pdcs and cdcs were infected with mcmv, whereas after 24h p. i. the frequency of infected cdcs wins over that of the pdcs. conclusions: with this experimental system we are able to visualize the ifnb response vs. the infection status of mcmv in vivo on a single-cell level. from our initial results we can conclude that infected cells in the spleen induce ifnb production in noninfected pdcs which initiate the antiviral immune response in this organ. recently, we have developed live-attenuated arenavirus vaccine candidates based on lymphocytic choriomeningitis viruses (lcmv) carrying the vesicular stomatitis virus (vsv) envelope gene (rlcmv/vsv-g). since interferon (ifn) signaling is known to be crucial for adaptive immune responses against wildtype (wt) lcmv and control thereof, we wanted to assess the innate and adaptive immune requirements for containing rlcmv/vsv-g infection. mice lacking both, ifn type i and ii receptors generated potent virus-specific cd8 t cells and neutralizing antibody responses against rlcmv/vsv-g, even exceeding the respective responses in wild type animals. in further contrast to wt lcmv infection, ifn type i and ii signaling was dispensable for rlcmv/vsv-g control. rlcmv/vsv-g infection of rag1-deficient mice (lacking mature t and b cells) resulted in persistent levels of circulating viral rna that was solely detectable by qrt-pcr but not by classical measures of infectivity. overt viremia was only found in mice lacking both rag1 and ifn type i receptor (ar). thus, viral attenuation for vaccine use was found to considerably relax the ifn-dependence of adaptive immune responses and virus control. this redundancy of ifn and adaptive immune responses in rlcmv/vsv-g control provides a better understanding of the attenuation of this vector. it furthermore suggests that the virulence of a particular virus may influence the interferon-dependence of cd8 t cell and antibody responses, which may have implications for vaccine development. objectives: the class of type i interferons (ifns) consist of multiple ifnas and a single ifnb subtype. although being important for anti-viral defence they have been shown to be detrimental for the host during bacterial infections. while in general the first ifn to be produced is ifnb, the cell types responsible for its initial production remain unclear. to assess the cellular sources of ifnb and its role for the outcome in bacterial infections we use an ifnb reporter-knockin mouse model, in which yellow fluorescent protein (yfp) is expressed from a bicistronic mrna linked by an internal ribosomal entry site (ires) to the endogenous ifnb mrna. methods: to induce a type i interferon response we intravenously injected the tlr agonists poly(i:c) and cpg 1668, respectively, or infected reporter mice or bone marrow derived macrophages (bmdms) or dendritic cells (bmdcs) with the facultative intracellular pathogen listeria monocytogenes. the spatiotemporal tracking of the ifnb response was done using facs analysis and immunofluorescent microscopy. results: after poly(i:c) injection in vivo a small subpopulation of ifnb + cd8a + dendritic cells relocalize from the red pulp via the marginal zone to the t cell areas of the spleen whereas ifnb + activated pdcs were localized in the splenic white pulp at the t/b-cell interface after cpg administration. in vitro infection of bmdms, bmdcs and flt3-l derived dcs with listeria monocytogenes resulted in a low frequency of ifnb + cells depending on the listeria strain used and multiplicity of infection with bmdms being the most potent producers of ifnb. in vivo mostly activated f4/80 + macrophages were accountable for the ifnb expression. simultanous visualization of the cellular state of infection shows that ifnb + bmdms carry a higher bacterial load then ifnbcells. the cellular detection of ifnb expression reveals a remarkably low frequency of ifnb-producing cells in response to distinct pamps or the infection with intracellular bacteria. this hints at a superior role of few specialised cells for mounting a significant response against distinct stimuli or bacterial disease. additional data will be presented further resolving the timecourse of the ifnb response vs. the state of infection after bacterial challenge. the spleen is a secondary lymphoid organ that is characterized by highly specialized structures and plays a crucial role in the defense of blood-borne pathogens. we investigated the role of the spleen in the generation of antigen-specific cytotoxic cd8 t cell (ctl) responses in a systemic infection with recombinant adenovirus expressing ovalbumin (adova). although adenovirus mainly infects the liver, the ctl response requires the spleen, as splenectomized mice were incapable to mount an antigen-specific ctl response upon systemic challenge with adova. additionally, dendritic cells (dc), macrophages and an intact splenic structure were mandatory for the induction of an efficient ctl response. we detected adova specific ctl responses only in splenectomized mice that received splenic autotransplants but not after adoptive transfer of single cell splenocytes. furthermore, we asked how toll-like receptor (tlr) ligands influence adova-specific ctl responses. tlr ligands as "danger signals" are generally known to exert immune stimulatory effects. importantly, we observed that systemic administration of single-stranded rna (ssrna) prior to adova infection inhibited the generation of antigen-specific ctl responses in a tlr7 and type i interferon (ifn) dependent manner. ssrna injection induced the production of type i ifns as detected in sera and supernatants of splenocytes isolated from wild-type mice. experiments performed in ifnbeta reporter mice revealed that splenic plasmacytoid dcs represented the cellular source of type i ifns. additionally, splenic cd11c+ dcs purified from ssrna pretreated mice showed a reduced capacity to cross-prime ova-specific cd8 t cells upon adova challenge. in vivo pre-activation of endogenous ova-specific cd4 t cells as well as an adoptive transfer of in vitro activated transgenic ova-specific cd4 t cells prevented the ssrna mediated suppression of the ctl activity. we assume that dcs preactivated by systemic ssrna were impaired in their ability to activate naive cd8 t cells in response to an adova infection due to an impaired cd4 t cell help. taken together, we show that type i ifns cannot only stimulate, but also inhibit the induction of ctl responses in the spleen. within hours after infection many viruses induce early type i interferon (ifn) responses that can confer protection against lethal infection. modified vaccinia virus ankara (mva) is a highly attenuated vaccinia virus (vacv) strain generated by more than 500 passages in chicken embryo fibroblasts. mva stimulates systemic ifn responses, whereas vacv-induced cytokine milieus are dominated by il-12. to study the impact of virus-triggered ifn on the induction of t cell responses, we used recombinant mva-gp33 and vacv-gp33 expressing the gp33 epitope of lymphocytic choriomeningitis virus. for adoptive transfer experiments transgenic mice expressing the p14 t cell receptor recognizing the gp33 epitope were intercrossed with mice devoid of the ifn receptor (ifnar -/-) to obtain p14ifnar -/mice. upon adoptive co-transfer of p14ifnar -/and p14ifnar wt/wt t cells and subsequent challenge with mva-gp33, a massive expansion of p14ifnar wt/wt t cells was observed, whereas p14ifnar -/-t cells expanded less efficiently. in contrast, challenge with vacv-gp33 induced a rather similar expansion of ifnar competent and ifnar deficient p14 t cells. in the absence of ifnar-triggering t cell expansion was associated with reduced proliferation capacity and increased apoptosis. to study the impact of ifnar-signaling on the expansion of endogenous t cells, conditional mice with a t cell-specific ifnar-ablation were infected with mva. interestingly, in those mice the virus-specific t cells showed a reduced expansion compared to t cells of wt mice. additionally, we found that ifnar-triggering of dendritic cells but not of macrophages further supported specific t cell expansion. thus, upon virus infection virus-associated properties affected the overall cytokine milieu that influenced the quality and the quantity of expansion of virus-specific t cells. to delineate h5n1-specific signaling patterns we used a genome-wide comparative systems biology approach analyzing gene expression in endothelial cells infected with three different human and avian influenza strains of high and low pathogenicity. blocking of specific signaling pathways revealed that h5n1 induced an exceptionally nf-kb dependent antiviral response. irf3 is essential part of this interferon-response of human endothelia. furthermore, we identified hmga1 as a novel transcription factor specifically responsible for the overwhelming proinflammatory but not anti-viral response induced by h5n1. finally, nfatc4 was found to be a transcriptional regulator for specifically h5n1-induced genes. we therefore describe for the first time defined signaling patterns specifically activated by h5n1 which, in contrast to low pathogenic influenza viruses, are responsible for an imbalance of overwhelming proinflammatory and impaired anti-viral gene programs. objectives: to study the early events of immune antagonism by influenza virus in vivo and how this process impacts the timing at which adaptive immunity is generated. methods: to dissect the contribution of the unique viral antagonist ns1, delta-ns1 influenza virus (a recombinant influenza virus lacking the ns1 gene) was compared side by side with wild type influenza virus in vivo. mice infected with influenza virus were sacrificed at different time points after infection. to study the onset of inflammation during infection lung and blood samples were isolated with a selected panel of inflammatory and antiviral proteins that were measured by multiplex elisa and quantitative pcr (qpcr). to determine the bridging between innate lung inflammation and adaptive immunity, the kinetics of lung antigenbearing dendritic cell (dc) migration to the draining lymph nodes was quantitated in infected mice. further, functional in vitro and in vivo assays in infected animals with influenza-ova viruses were used to determine whether antigen-bearing migratory dcs were capable of priming transgenic t cells. to investigate the effect of ns1 during the onset of immunity in vivo, we systematically studied the early events occurring in the lungs and draining lymph nodes upon infection with influenza virus. strikingly, no sign of innate immunity was detected in the lungs for almost two days after infection when a sudden inflammatory burst including ifns, cytokines, and chemokines occurred. this burst preceded the robust dc migration and t cell activation in the lymph nodes. virus-loaded dcs appear in the lymph node starting 2 days post-infection, reached its maximum at day 4, and triggered t cell priming in vivo. a direct comparison of delta-ns1 virus with wild type virus infection demonstrates that virus can only trigger rapid inflammation in vivo when it lacks the ns1 protein. we demonstrate that the delay in the generation of immediate lung inflammation is mediated by the influenza ns1 protein. thus, we propose that the virally encoded ns1 protein establishes a time limited "stealth phase" where replicating influenza virus remains undetected thus preventing the immediate initiation of innate and adaptive immunity. keratinocytes represent the major cell population of human epidermis which provides a first line of defense barrier for the host. in addition, keratinocytes actively participate in immune response. viral double-stranded rna (dsrna) is the most important viral structure involved in activation of innate immune response. intracellular detection of dsrna triggers secretion of soluble signaling molecules, interferons, and activates pro-inflammatory response and programmed cell death, apoptosis. here we have used subcellular proteomics to characterise dsrna-induced human keratinocytes. cells were transfected with a mimetic of dsrna, poly(i:c), after which the cells were fractionated into cytoplasmic and mitochondrial fractions. these proteomes were analysed using two-dimensional electrophoresis in combination with mass spectrometry, immunoblotting and confocal microscopy. we show that several proteins involved in apoptosis, cytoskeleton reorganization and intracellular transport are up-regulated upon dsrna stimulation. in mitochondrial proteomes the expression of structural proteins, especially fragments of cytokeratins, is highly up-regulated. we show that cytokeratin 18 is cleaved during poly(i:c) stimulation and fragments are solely localized in mitochondria. similar degradation of cytokeratin 18 is seen in emcv-and vsv-infected keratinocytes. cytokeratin fragmentation after dsrna stimulation is dependent on caspase activation, which indicates a role for cytokeratins in the regulation of apoptosis during viral infection. in addition, we show that 14-3-3 proteins are upregulated in both mitochondrial cell fraction and cytoplasm after dsrna-stimulation and during viral infection. viral dsrna also induced transient phosphorylation of 14-3-3 target proteins. thus, these results suggest that 14-3-3 proteins have a regulatory role in host defence against viral infections. i. wessels 1 , d. fleischer 1 , l. rink 1 , p. uciechowski 1 1 institute of immunology, rwth aachen university hospital, aachen, germany objectives: the proinflammatory cytokine interleukin (il)-1b mediates the expression of a variety of proteins responsible for acute inflammation and chronic inflammatory diseases. however, the molecular regulation of il-1b expression is not elucidated, yet. it is known that the il-1b promoter is packaged into a nontranscribed but poised architecture in monocytes, rapidly producing il-1b when stimulated. b-cells which are il-1b non-producers reveal an inaccessible promoter structure. in this study the chromatin structure of the il-1b promoter and the impact of methylation on il-1b expression were examined in a cellular monocytic differentiation model. methods: promyeloid hl-60 cells were differentiated into monocytic cells after dihydroxyvitamine d 3 treatment. the monocytic phenotype was confirmed by flow cytometry. the il-1b promoter was analyzed using the chromatin accessibility by real time pcr (chart) assay. to test the influence of methylation, cells were treated with 5-aza-2-deoxycytodine (aza) and changes in il-1b expression were measured by pcr, elisa and western blot analyses. results: in contrast to undifferentiated hl-60 cells, differentiated cells displayed upregulation of cd14 antigen and acquired the ability to express il-1b. by comparing the accessibilities of il-1b promoter we detected that the il-1b promoter was not accessible in undifferentiated hl-60 cells but highly accessible in differentiated monocytic cells. the accessibilities of differentiated cells were comparable to that observed in primary monocytes. lps stimulation did not affect promoter accessibility in promyeloic and monocytic hl-60 cells, demonstrating that the chromatin remodeling of the il-1b promoter depends on differentiation but is independent of the transcriptional status of the cell. demethylation via aza led to the induction of il-1b expression in both undifferentiated and differentiated cells which could be increased after lps stimulation. conclusion: two independent mechanisms involved in the regulation of il-1b expression were found. our data indicate that the il-1b promoter is reorganized into an open conformation during monopoiesis and that the established poised structure is a privilege of mature monocytes but not of the entire myeloid lineage. as a second mechanism, il-1b expression is regulated by methylation acting independent of the developmental stage of myeloid cells. a. holweg 1 , g. wetzel 1 , h. arnold 1 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany the p65 family members of interferon (ifn) inducible gtpases also known as guanylate binding proteins (gbps) are among the most abundantly induced transcripts after stimulation with ifn-g. although the stimulatory capacities of the toll like receptor ligands lps and cpg, cytokines like ifn-b and il-1b and the intracellular pathogens listeria monocytogenes and toxoplasma gondii have been described to induce their expression, the function of gbps during bacterial infections is still ill defined. here we report for the first time the massive induction of murine gbps in two independent in vivo models of pneumonia (infection with streptococcus pneumoniae and pseudomonas aeruginosa). a strong and rapid induction of mgbp2, 3, 4, 5 and 6 mrna after intratracheal infection was detected by realtime pcr analysis of bronchoalveolar lavage (bal) cells. using newly generated antibodies in western blots and fluorescence microscopy, we identified macrophages as the main producers of mgbps in bal samples. although the signaling cascade involved in the upregulation of mgbps after ifn-g stimulation has been extensively studied, the mechanisms responsible for mgbp induction upon bacterial stimuli are unclear. in this study we could show that the induction of mgbps upon infection is abrogated in ifn type i/ type ii receptor double-deficient mice and thus absolutely dependent on endogenous interferon production. in contrast, the tlr adaptor molecule myd88 was found to be dispensable arguing for a trif-mediated interferon production subsequentially resulting in enhanced gbp expression. based on these findings our future experiments will address the functional role of gbps in innate and adaptive immune responses against extracellular bacteria. (2)). activation of resident microglia cells and infiltrating brain macrophages appeared to play a role in modulating virus replication shortly after infection but also appeared to be responsible for the inflammation in brains of infected mice. clearance of replicating virus from the cns required mcmv specific cd8 + t lymphocytes whose effectors functions still remain incompletely defined (j. immunol. 2008 aug 1;181 (3)). humoral immunity appeared to also play a role in the control of mcmv infection in developing brain. infected newborn mice treated with mcmv-specific antibodies had lower viral titers in the cns, significantly less cns inflammation and improved neuronal migration and increased cerebellar area as compared to control mice (j. virol. 2008 dec; 82 (24) ). conclusion: peripheral inoculation of the virus induces focal infection and inflammation in the developing mouse cns followed by strong innate and specific immune response that could also alter developmental programs required for normal development of mouse brain. passive immunization of infected newborn mice reduces mcmv-related pathology in infected brain suggesting that antiviral antibodies are an important component of immunological responses during cmv infection of developing cns. chronic inflammation is associated with the promotion and enhancement of malignancy and tumor growth. tumors enhance the accumulation of myeloid derived suppressor cells (mdsc), which contribute to tumor escape, immune tolerance and immune suppression. previously, we have shown that tumor-derived inflammatory cytokines, such as il-1b in the tumor microenvironment can induce a massive accumulation of mdsc in the spleen of tumor bearing mice and induce t cell suppression. in this work, we describe a novel polymorphnuclear mdsc subpopulation -inflammatory mdsc (infmdsc) which accumulates in the bm and spleen of mice bearing inflammatory 4t1 breast cancer cells over expressing il-1b (4t1/il-1b) tumor cells, but rarely in untransfected 4t1 cells. secretion of il-1b from tumor cells is crucial for infmdsc generation and accumulation. infmdsc have the ability to suppress nk cell activity via reduction of the nk activating receptor nkg2d in vivo, and in a cell-cell contact dependent manner in vitro. inflammation up-regulates il-4ra expression on the cell surface, which correlates with tumor growth and induction of suppression on nk cells. our data suggest that tumor derived inflammation enhances a specific mdsc subset which has the ability induce suppression of nk cells, and perhaps can serve as a new chemotherapy target. objectives: lps constitutes a main target of innate immune recognition of gram-negative bacteria and other lps carrying pathogens. cytokine production after in vitro stimulation of whole blood with lps is used as an expression of individual lps reactivity. we assess genome-wide data to analysed the association to lps induced cytokine response, and replicated the top findings in two independent cohorts. materials and methods: we used 130 healthy caucasian blood donors as discovery samples, and replicated snps from affymetrix -genome-wide human snp array 5.0 having p x 10 -4 in two independent cohorts each consisting of 200 blood donors using a customized chip from illumina and real-time pcr respectively. in all the three cohorts whole blood samples was stimulated for 24h and we measured the levels of il-6, il-8, il-10, tnf-alfa and il1-ra with r&d ® assays on luminex platform.. the association analysis was performed with wald statistical test assuming an additive model. the discovery sample statistical analysis revealed 150 snps with p x 1,0*10 -4 . to identify/replicate the association of cytokine production for these 150 we reanalysed these on a 200 cohort. a combined analysis revealed 10 snps with p x 9,1*10 -5 .these results are not genome wide significant. the 10 snps showing nominal association to lps cytokine response are being analysed in a replication cohort conclusion: we find 10 nominal associated snps between lps stimulated cytokines in blood samples of healthy caucasian blood donors. the importance of these snps are to be determined in a replication cohort. adipocytes, so far known for their lipid-storage capacity came into the focus of interest because of their immunoregulatory properties resembling those of innate immune cells. adipocyte-derived pro-inflammatory mediators contribute to the development of chronic inflammation, thereby promoting the progression of insulin-resistance/metabolic-syndrome and diabetes. the physiological signals inducing the secretion of inflammatory mediators by adipocytes are unknown. heat shock proteins, such as hsp60 have been identified as potent regulators of inflammatory innate immune cell-activities, whereas their influence on adipocyteactivities remained elusive. here, we investigated the regulatory effects of hsp60 on adipocytes. for the first time we could show a hsp60-stimulated release of the proinflammatory cytokines il-6, cxcl1 and mcp-1 in a time-and concentration-dependent manner from murine 3t3-l1 adipocytes. analyses of hsp60-signalling in these adipocytes revealed that members of the mapk-family (erk1/2, p38) and the transcription factor nfkb are involved in hsp60-mediated induction of the mediators il-6, cxcl1 and mcp-1. binding-studies with fluorescence-labelled hsp60 demonstrated that the interaction of hsp60 with adipocytes exhibits basic features of a receptor-mediated binding. hsp60-binding to adipocytes was saturable and reached its maximum at 3.5 mm. binding was inhibitable only by the unlabelled ligand (52 %), but not by unrelated proteins, thereby proving the specificity of hsp60-binding. further analyses to characterize hsp60-receptor structures on adipocytes revealed the presence of toll-like receptor (tlr)4 on adipocytes. tlr4 has been found to be expressed on macrophages and to interact with hsp60, therefore suggesting tlr4 as a potential receptor candidate for hsp60 on adipocytes. in order to identify the responsible binding-epitope of hsp60 we investigated the effect of specific antibodies directed against different epitopes of the hsp60-molecule. incubation with antibodies directed against the n-terminus of hsp60 (aa1-200; 5-25 mg/ml) were capable of inhibiting the hsp60-binding to adipocytes (47-80 %) indicating that the n-terminal region of hsp60 is involved in receptor binding. our experiments demonstrate that hsp60 stimulates the release of proinflammatory adipocyte mediators. the findings implicate that the hsp60-mediated induction of adipocyte mediators contributes to inflammatory processes observed in obesity-associated disorders and could serve as a target for the development of therapeutic strategies for patients suffering from diabetes or diabetes-related disorders. legionella pneumophila, a gram-negative facultative intracellular bacterium, is the causative agent of a severe pneumonia known as legionnaires' disease. classically, type i ifns (ifna/b) have been associated with antiviral immunity. ifna/b signal through the ifna/b receptor (ifnar) leading to the induction of hundreds of ifn-stimulated genes (isgs), many of which have anti-microbial activities. recently, it was demonstrated that ifna/b are also produced in host cells infected with (intracellular) bacteria or stimulated with cytosolic dna. here we show by rnai that l. pneumophila infected host cells produced ifnb dependent on irf3. we observed enhanced l. pneumophila replication in mouse macrophages lacking ifnar and human cells after irf3 knock-down, suggesting that endogenously produced ifnb activates a cell-autonomous defence against legionella. moreover, ifnb treatment restricts legionella replication in human and murine host cells. ifna/b impacts formation of large replication vacuoles, but appears not to influence the recruitment of er markers nor fusion of the legionella-containing vacuole with the lysosome. moreover, ifna/b-mediated cellautonomous defence was independent of autophagy and pyroptosis. we thus hypothesize the crucial involvement of antibacterially acting isgs. ongoing studies focus on the role of ifn-induced immunity-related gtpases (irgs). mesenchymal stem cells (mscs) are identified by their capacity to differentiate into connective tissue cell types. mesenchymal stem cells also show a high plasticity and account for a potential therapeutic efficacy. several authors have reported the expression of alfa-smooth muscle actin (a-sm actin) by msc. this protein has been considered a marker for the myofibroblast, has the capacity to polymerize into the cytoplasm and contribute to the cell contractility. this activity may be crucial for the changes of the cell shape, for cell-cell interactions and may therefore be relevant for the physiology of msc. in this work, we study the presence of alfa-sm actin in human msc by flow cytometry and immunoflurescence. we also study the contractility of these cells under the effect of different cytokines. human bone marrow samples were obtained from bone marrow aspirates. bone marrow mononuclear cells were isolated by density gradient centrifugation and cultured in opti-mem culture medium with 3 % of fetal calf serum at 37°c and 5 % co2. bone marrow nonadherent cells were removed after 24 h, and culture medium was refreshed twice per week thereafter. cells grew adherent, with a fibroblastic morphology and expressed cd10, cd29, cd73, cd21 and stro-1 and were negative for cd45. intracellular staining was performed for alfa-sm actin. cell contractility was measured with the collagen gel contraction assay. alfa-smooth muscle actin was detected in almost 100 % of msc. interleukin-2, ifn-gamma and tnf (th1 cytokines) increased msc contractility, whereas il-10 (a th2 cytokine) decreased msc contractility. by immunofluorescence, we observed that il-2, ifn-gamma and tnf increased the incorporation of alfa-sm actin into the stress fibers of msc, whereas il-10 decreased that incorporation. our results suggest that th1 and th2 cytokines regulate msc physiology by acting on their contractility. aims: thapsigargin (tg) is a sesquiterpene lactone (sl) of guaianolide type isolated from the mediterranean plant thapsia garganica l. it is widely recognized as an inhibitor of sarco-endoplasmic reticulum ca 2+ -atpase leading to elevation of intracellular calcium. this activity is shared by trilobolide (tb), a sl from laser trilobum (l.) borkh. tg has been shown to possess prospective immunotherapeutic properties. it kills slowly proliferating and non-proliferating cells, and inhibits replication of viruses. the aim of our work was to investigate possible immunostimulatory potential of tg and tb. methods: the effects of the agents were analyzed under conditions in vitro using rat and mouse resident peritoneal cells (pecs), and human peripheral blood mononuclear cells (hpbmcs). they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. supernatant levels of ifn-g were determined by elisa. production of no by animal pecs was assayed using griess reagent. possible contamination of the samples with lps was excluded using the chromogenic limulus amoebocyte assay. results: we have found that both tg and tb at as low doses as 40 nm and 400 nm induce ifn-g secretion in rat pecs and hpbmcs, respectively. the concentration of ifn-g produced by the highest dose of the agents (10 mm) at the 24-h culture interval reached the values of approximately 3 ng/ml and 2 ng/ml in rat pecs and hpbmcs, respectively. the increase was apparent within the interval of 2-6 h in rat pecs. the 24-h interval was optimal for accumulation of ifn-g in cultures of hpbmcs. only modestly enhanced secretion of ifn-g was observed in mouse pecs. production of ifn-g was found to depend on activation of nf-xb and map kinases p38 and erk1/2. it was not suppressed by the calcium chelating agents bapta-am and tmb-8. the tg-and tb-induced ifn-g production was associated with activation of the high-output production of no. conclusions: sesquiterpene lactones tg and tb are potent inducers of ifn-g in animal and human cells. the effect is independent of their serca inhibitory potential. underlying mechanism(s) of the immunostimulatory effects remain to be elucidated. acknowledgements: the work was supported by the grant gacr 305/07/0061. pin1 is a peptidil-prolil-cis-trans isomerase that specifically binds phosphorylated ser/thr-pro protein motifs and catalyzes the cis/trans isomerization of peptides. mitotic proteins, cytoskeleton, transcription factors and apoptotic proteins are pin1 substrates and targeting sites. recent data show that pin1 interacts with apo-bec3g (a3g). the pin1/a3g interaction results in a reduced a3g expression and a diminished a3g-mediated restriction of hiv. two single nucleotide polymorphisms (snps) in the promoter region of the pin1 gene (-842 g/c and -667 t/c) modulate pin1 expression; in particular, the -842 gc genotype or cc haplotype are associated with reduced protein levels (neurobiol. aging, 28; 69-74, 2007) . the -842 c/g and -667 t/c polymorphisms in the promoter of pin1 gene as well as pin1 protein levels were analyzed in 30 exposed seronegative individuals (esn), heterosexual partners of hiv-infected patients; 40 hiv-infected patients (hiv) and 40 healthy controls (hc). the genotype and allele distributions of the -842 snp was skewed in esn (genotype: p= 0.008; allele: p= 0.013). in particular esn showed a significantly lower frequency of the -842 gg genotype compared to hiv and hc (p=0.017 and p=0.019, respectively) and consequently a lower g allele frequency (p=0.026 and p=0.028, respectively). no significant differences were found for the -667 snp. these snps are in linkage disequilibrium and combine to form haplotypes. conclusions: our findings support the role of hiv viral replication as the most important promoter of immune activation and prove the importance of art in reducing immune activation and viral replication even in a sub-saharan african environment, where patients are exposed to an abundance of other infectious agents. our data further indicates that hiv replication rather than host genetics is the key determinator of circulating cytokine levels among the studied hiv infected participants. aims: acyclic nucleoside phosphonates (anps) are synthetic analogues of natural nucleoside monophosphates. they have proved to be outstanding antivirals inhibiting replication of both dna-viruses and retroviruses. tenofovir, 9-(r)[2-(phosphonomethoxy) propyl]adenine [(r)-pmpa] is broadly used for therapy of aids, adefovir, 9-[2-(phosphonomethoxy)ethyl]adenine (pmea) has been approved for the treatment of hepatitis b. the aim of our work was to investigate possible interactions of anps with production of cytokines and nitric oxide (no) implicated in antiviral defence mechanisms. the immunobiological effects of anps were screened in vitro using mouse resident peritoneal cells. they were cultured in density of 2 × 10 6 /ml in complete rpmi-1640 medium. secretion of cytokines was determined after the 5-h culture by elisa. production of no was assayed at the interval of 24 h using griess reagent. approximately 300 compounds belonging to several distinct anp groups were included in the study: a) pmea derivatives, b) pmedap i. e. 9-[2-(phosphonomethoxy)ethlyl]-2,6-diaminopurine derivatives, c) 9-[2-hydroxy-3-(phosphonomethoxy)propyl]-adenine (hpmpa), and hpmpdap derivatives, d) guanidino analogues of pmpdap, e) 9-heteroalkyl substituted 2-amino-6-guanidinopurines, and f) 2-amino-3-(purin-9-yl)propanoic acid derivatives. possible presence of lps in the stock solutions of the samples was checked and excluded using the chromogenic limulus amoebocyte assay. results: approximately 30 compounds were found to activate the secretion of anti-hiv effective chemokines rantes and mip-1a and cytokines tnf-a and il-10. although these anps did not stimulate biosynthesis of no on their own, they substantially augmented production of no triggered by ifn-g. no clear-cut relationship between the chemical structure and biological effects of anps was observed. however, the most effective proved to be the n 6 -cycloalkyl derivatives of pme-dap. the effects were produced in a dose-dependent fashion, exhibiting the immunostimulatory effects at as low concentrations as 2 to 5 mm. the remarkably enhanced secretion of chemokines was reached within 2-4 h of the cell culture. the effects were found to depend on activation of nf-kb. conclusions: it may be suggested that anps represent a new generation of antivirotics with combined antimetabolic and therapeutically prospective immunostimulatory properties. acknowledgements. the work was supported by the grant 1m0508. host related immune factors in childhood chronic hepatitis b and change of initial profile with ifn-a treatment needs to be clarified. sixteen patients were included, and 10 million units of ifn-a treatment three times a week for 6 months was initiated. before and after treatment: percentages of the il-2 and ifn-g in cd4+ t cells were assessed to determine intracellular t helper cell 1 (th1) type cytokine expression. similarly, percentages of intracellular il-2 and ifn-g were detected to verify cytotoxic t cell 1 (tc1) type cytokine expression in cd8+ t cells. percentage of th2 and tc2 type cytokine expression, (il-4 and il-13) were determined in cd4+ and cd8+ t cells, respectively. six (50 %) of these were evaluated as having no response and the other half with partial/complete response. all patients had higher percentages of th2 cells with respect to healthy controls before treatment. tc percentages, both tc1 and tc2, were significantly different between these groups, being higher in the patient group. when values of no responder group were compared with healthy controls, il-4 expression was higher and the percentages of th1 type cells were significantly low. il-13 expression in th and tc cells decreased after treatment in the unresponsive group. intracellular cytokine profiles of treatment responders and normal controls were not different. this has been the first study in children comparing baseline and post treatment intracellular cytokine profiles with healthy controls. the frequency (29,8 %) of high concentration of igg anti-oxldl antibodies in patients with hcv infection were significantly elevated in comparison to healthy subjects (6,6 % according to bibliographic data). the immune response was significant but it was not assosiated with the viral load. it is probable that humoral immunity plays a critical role and contributes in an immunoinflammatory reaction of hcv-infection. abstract withdrawn by author t. schwandt 1 , f. juengerkes 1 , b. schumak 1 , g. gielen 1 , j. kalff 2 , p. knolle 1 , b. holzmann 3 , a. limmer 1 1 university hospital bonn, institute of molecular medicine and experimental immunology, bonn, germany, 2 university hospital bonn, department of surgery, bonn, germany, 3 department of surgery, tu munich, munich, germany bacterial translocation is a possible risk of abdominal surgery and could be the cause of life-threatening consequences such as organ failure and septic shock. patients surviving septic shock often suffer from opportunistic infections as well as defects in adaptive immunity. here we investigated the influence of bacteremia and bacterial translocation on systemic adaptive immune responses using murine models. to mimic abdominal surgery, mice were subjected to intestinal manipulation (im). to study septic conditions, mice underwent colon ascendens stent peritonitis (casp) or received e.coli intravenously. we monitored the distribution of gut-derived bacteria by in vivo imaging (xenogen) and additional microbiological assays and determined antigen-specific ctl responses towards subsequent infection with recombinant adenoviruses (av). we detected comparable amounts of bacteria in lung, liver and spleen of mice that underwent casp or were injected i. v. with e.coli. in addition, mice infected systemically with av lacked an antigen-specific ctl response, whereas the ctl responses of locally av infected mice were not affected. in contrast, bacteria were detected in lung and liver but not in spleen of mice that were subjected to im or received e.coli by injection into the hepatic portal vein. here, the ctl response was not impaired. depletion experiments imply that kupffer cells as well as soluble mediators such as tumor necrosis factor alpha play an important role in trapping and clearance of translocated bacteria in liver and lung. our experiments demonstrated that translocation of bacteria did not cause immune suppression as long as they did not reach the spleen in high numbers. we suggest that liver and lung fulfill a filter function to prevent systemic distribution of gut-derived bacteria. failure of or bypassing these barriers might enable bacteria to access the spleen and thus cause systemic suppression of adaptive immunity, whereas induction of local immunity is not affected. objectives: varicella-zoster virus (vzv) is one of the most frequent pathogens for which a vaccine is available. tropism of vzv for skin is the most obvious manifestation of vzv infection, producing vesicular cutaneous lesions that are associated with varicella and herpes zoster. the striking difference in the clinical outcome of infection by rush inducing circulating virulent vzv strains and asymptomatic infection by the vaccine leads to the assumption that the virus interferes with cutaneous immunity. therefore, we comparatively investigated the impact of vzv clinical isolates and the vaccine on the reciprocal crosstalk of immature dendritic cells (idcs) and epithelial gd t cells. methods: skin punch biopsies of herpes zoster patients were analyzed by dual immunofluorescence microscopy. phenotypic changes of cutaneous dcs and monocyte-derived dcs after vzv infection were investigated by flow cytometry. the cytokine profile of vzv-infected dcs and epithelial gd t cells was determined through elisa. results: we observed that innate lymphocytes recognize dcs, which infiltrate herpes zoster lesions, after infection with vzv. strikingly, only the vaccine but not vzv clinical isolates could license the bidirectional crosstalk between idcs and gd t cells resulting in release of ifn-g and il-12, the signature cytokines of antiviral immune responses. this is the first demonstration that virulent virus strains disrupt dendritic cell instruction whereas the corresponding vaccine does the opposite. we describe a novel immune evasion strategy in the skin, which provides the opportunity for efficient and symptomatic virus replication. this result is also of practical importance: future vaccine design has to ensure that candidate vaccines do not impair dc instruction in order to allow stimulation of powerful antiviral immune responses. a. jafarzadeh 1 1 rafsanjan university of medical sciences, rafsanjan, iran, islamic republic of objective: it has been reported that the caga + h. pylori strains induce more severe gastric mucosal inflammation. the aim of this study was to investigate the association of the h. pylori virulence factor caga with serum levels of il-17 and il-23 in h. pylori-infected duodenal (du) patients and h. pylori-infected asymptomatic (as) carriers. methods: totally, 45 h. pylori-infected du patients (23 patients were positive for anti-caga antibody and 22 patients were negative for anti-caga antibody), 30 h. pylori-infected as carriers (15 subjects were positive for anti-caga antibody and 15 subjects were negative for anti-caga antibody) and 15 healthy uninfected subjects (as a control group) were enrolled to study. a blood sample was obtained from all participants and the serum levels of il-17 and il-23 was measured by elisa method. the mean serum levels of il-17 in total du patients (9.28 pg/ml ± 5.48) was significantly higher than those observed in total as subjects (5.19 pg/ml ± 3.75, p x 0.001) and healthy control group (3.55 pg/ml ± 3.76, p x 0.0001). in du group, it was found that the mean serum levels of il-17 in subjects with positive test for anti-caga (10.84 pg/ml ± 5.79) was significantly higher than those observed in subjects with negative test for anti-caga (7.65 pg/ml ± 4.74; p x 0.05). the mean serum levels of il-23 in du (8.66 pg/ml ± 8.41) and as groups (7.25 pg/ml ± 5.66) was significantly higher than those found in uninfected control group (3.64 pg/ml ± 3.36, p x 0.02 and p x 0.03, respectively). however, no significant difference was observed for mean serum levels of il-23 between du and as groups. moreover, in both du and as groups the mean serum levels of il-23 was not significantly differ in subjects with positive test for anti-caga and those were negative for anti-caga antibody. the results of the present study showed higher serum concentrations of il-17 and il-23 in h. pylori-infected subjects as compared with control group. in du group the expression of il-17 influenced by the bacterial caga factor. a. aral 1,2 , a. atak 1 1 gazi university faculty of medicine, department of immunology, ankara, turkey, 2 gazi university institution of health sciences, department of immunology, ankara, turkey objective: ebv is a human herpesvirus, which infects human b lymphocytes latently and immortalizes the cells due to transformation. ebv infection is asymptomatic in childhood while it may cause a self-limiting lymphoproliferative disorder called infectious mononucleosis (im) in adolescence. in immunodefective patients, the virus may lead to malignancies like burkitt's lymphoma, nasopharyngeal carcinoma and immunoblastoma. the virus can also cause latent infections. cytokine production in response to ebv infection differs according to the phase of the infection. neopterin, ifn-g and il-6 levels are elevated in acute ebv infection in vitro; but in chronic phase, particularly, il-6 elevation could not be detected. tnf-a enhances the effects of ifn-g on neopterin synthesis while neopterin enhances the secretion of tnf-a via various stimuli. ifn-g levels are elevated particularly in the acute phase of im. since the elevation of cytokine levels changes according to the phases of disease, it's thought that the association between host defense and viral replication depends on different parameters. ifn-g is the major stimulus for neopterin synthesis, which stimulates monocytes and macrophages primarily. increased production of neopterin in body fluids can be used to monitor the activation of cell mediated immunity. method: in order to analyze the effects of neopterin release and other cytokines, mononuclear cells have been isolated from peripheral blood samples of healthy donors and transformed via ebv. neopterin, ifn-g, tnf-a and il-6 levels have been measured with eia kits in culture supernatants. results: neopterin levels increased dependent on time and independent of ebv transformation. in ebv-transformated cell culture tnf-a levels increased beginning from the 48th hour and reached to maximum levels at the 1st week and decreased again at the 3rd week; however there were no significant differences between the levels among three weeks. likely tnf, ifn-g levels also increased at the 1st week and started to decrease at the 3rd week. il-6 reached to its peak at the 3rd week. conclusion: according to these results, neopterin levels, which increased dependent on time but independent of ebv transformation, may be a helpful marker for evaluating the acute response to viral infection but not for transformation. v. jurisic 1 , m. jurisic 2 1 university of kragujevac, school of medicine, kragujevac, serbia, 2 university of belgrade, school of dentistry, belgrade, serbia tnf-alpha is a pleiotropic cytokine that is considered as a primary modifier of inflammatory and immune reaction in response to various inflammatory diseases and tumour. we investigated tnf concentration in 43 radicular inflamed cysts and 15 odontogenic tumours obtained from patients undergoing surgery, under local anaesthesia, and after aspiration of cystic fluid from non-ruptured cysts. further, we estimated the role of cyst wall on production of tnf-alpha in respect of presence of inflammatory cells, degree of epithelial proliferation and degree of vascularization. the concentrations of tnf-alpha in the cystic fluids were measured by elisa, while proteins analyzed after separation by two-dimensional gel electrophoresis. the presence of pericystic inflammed cells were analyzed in thick section for routine histological examinations and by immunohistochemisty for cd3, cd20 and cd68. tnf-alpha is elevated in both cysts fluid, but higher values were found in radicular inflamed cysts in comparison to odontogenic tumours. higher concentration of tnf-a were associated with higher protein concentration, higher presence of inflammatory cells in peri cystic tissues, cysts wall thickness and higher degree of vascularisation (mann-whitney u-test, p x 0.05) in radicular cysts. no correlation was found, based on these parameters in odontogenic tumours, but all sumple have detectable concentrations of tnf-alpha. objectives: interactions between the neuroendocrine and immune system play an important role in maintaining and restoring homeostasis. in susceptible individuals a dysfunction of the neuroendocrine system may be one of the risk factors involved in the pathogenesis of septicemia and bacterial infection at all. we will study prolactin role in defensive reaction of immune system to bacterial infection. as a type of this bacterial infection we have chosen septic status, where we are expecting the most significant alterations of immune reaction, and specially septic statuses in blood malignancies, where we are expecting deficiency of immune system. our idea is that prolactin takes part in this defensive reaction by its cytokine effects, and it has contraregulative role against activation of adrenocortical system. the aims of this study are to extend our knowledge about the activation of peripheral prolactin expression and by this way to contribute elucidation of its role in periphery. 1) drawings blood from 20 patients and 20 healthy donors. blood of patients and healthy persons were sampled together with past histories after getting their acquainted approval. status of patient had to meet these conditions: a) the presence of systematic inflammatory response syndrome (sirs) according to standard clinical and laboratory criteria. b) positive hemoculture or determination of septic focus with demonstration of microbiologic source. 2) detection of the gene expression of prolactin and tlr-2 in cd14+ peripheral blood monocytes from patients with septic status and from healthy controls has been performed by rt-pcr at the level of mrna and flow cytometry at the level of intracellular protein results: we have found statistical significant differences (p p 0.05) in expression profile between patient and control groups. these differences were at both levels of expression, mrna and protein. conclusion: septic statuses change tlr-2 and peripheral prolactin expression in cd14+ monocytes. the function of interferon (ifn)-induced immunoproteasomes (i-proteasomes) is at present almost exclusively acknowledged in connection with improved processing of mhc-class i antigens. the experiments performed here challenge this existing paradigm of i-proteasome function. we demonstrate in vivo and in cellulo that the key role of i-proteasomes resides in the protection of cells against the formation of protein aggregates, which is ultimately crucial for preservation of cell viability under ifn-induced oxidative stress. ifns up-regulate the ubiquitylation machinery and enhance the formation of oxidant-damaged, nascent, poly-ubiquitylated proteins, which essentially require i-proteasomes for their efficient degradation. i-proteasome deficiency results in the formation of aggresome-like induced structures and increased sensitivity towards apoptosis. enhanced degradation of poly-ubiquitin conjugates designed to protect cells, will therefore also increase the peptide supply for antigen presentation. thus, only by executing their physiological function in the maintenance of protein homeostasis i-proteasome induction also provides a mechanism for cellular immune-adaptation. background: revived by the description of new functions, b cells are considered to be essential in the genesis of autoimmune diseases. in strong support of this interpretation, baff would play a key role in their physiology. however, the correlation between circulating baff levels and disease activity is not clearly established. conflicting results concerning levels of baff and b-cell repopulation after rituximab treatment have been reported. in fact, basal serum levels of baff reported in literature vary according to the antibodies (ab) used in the elisa and the mode of calculation. the aim of this study was to understand these variations. material and methods: different anti-baff abs were tested to verify whether they recognize glycosylated baff purified from u937 culture supernatant. sera from patients with autoimmune diseases were also tested. a western-blot analysis of sera was performed to evaluate anti-baff abs specificity and the best combination of anti-baff abs validated for elisa. then, different commercial baff elisa-quantification kits were compared to our "in-house" elisa. results: unexpectedly, the binding of some anti-baff ab was restricted to glycosylated baff. however, both glycosylated and non-glycosylated forms of baff were found in sera from patients with autoimmune diseases. most of the kits commercially designed to quantify baff suffer from some limitations. some sera are indeed positive with a kit and negative with another and vice versa. furthermore, there seems that rheumatoid factors (rf) interfere and correlate with the optical density of the anti-baff elisa. conflicting results concerning levels of baff and disease activity or auto-abs titers should be reconsidered in light of the glycosylation status of baff. (table-2 ). when tb household contacts and healthy controls were compared, cfp10 and esat6 seemed to be more useful than tst in tb contacts for displaying ltb (table-2) . although cfp10 spot numbers were much more than esat6 spots at the beginning and follow up period, statistically there was no difference in terms of median values(p: 0,069)( table-3 ). both esat6 and cfp10 spots were prone to decline during the follow up period. [ is some evidence to suggest that aspects of innate immunity (e. g. triggering of cytokine production by dendritic cells) may be impaired by hcv. to gain insight into some features of the innate immune response activated in vivo in the context of acute hcv replication, we analysed cytokine and chemokine levels in serum samples from chronic hcv patients undergoing liver transplantation. luminex multiplex assays and elisas were used to quantitate serum levels of g 20 analytes immediately prior to liver transplantation and at sequential time points up to 90 days post-transplantation. the increase in serum hcv rna levels associated with acute viral infection of the transplanted liver was found to be associated in most patients with elevations in serum levels of cytokines and chemokines including ifn-gamma, tnf-alpha, ip-10, il-6 and il-10. notably, the pattern and magnitude of systemic analyte elevations were very similar to those accompanying the acute burst of viral replication in primary hcv infection. high-magnitude elevations in systemic type 1 ifn levels were not observed in either setting, which may reflect an in vivo impairment of plasmacytoid dendritic cell functions by hcv similar to that observed in in vitro studies. we suggest that the liver transplant setting can be used as a model to study aspects of the innate immune response activated in acute hcv infection. to test the hypothesis that virus interactions with sialic acid receptors may play a role in innate antiviral immunity, we used recombinant viruses and differentiated cultures of human airway epithelial cells (hae). the hemagglutinin of the pandemic virus a/hong kong/1/68 (h3n2) (hk) differs from its putative avian precursor by 7 amino acid substitutions. we generated the complete recombinant virus rhk and its ha variants with amino acid reversions back to the ancestral avian sequence (rhk-5aa-i62r, n81d, k92n, s193n, g144a, human (2-6); rhk-r2-l226q, s228g and rhk-7aa-i62r, n81d, k92n, s193n, g144a, l226q, s228g, avian (2-3)). among these variants, the double mutant rhk-r2 and the seven mutant (rhk-7aa) had a typical avian-virus-like receptor-binding specificity owing to substitutions l226q and s228g.we infected hae cultures with the viruses and collected samples from the apical and basolateral sides of the cultures at different times post infection. virus titers were determined in mdck cells, and concentrations of about 50 pro-and anti-inflammatory mediators and chemokines were measured using a multiplex bead assay.concentrations of most cytokines progressively increased at the apical side of the cultures in the course of the infection. many cytokines, including t-cell-attracting chemokines such as ip-10 and rantes, were induced to similar levels by different viruses. however, some mediators were induced significantly stronger by avian-like viruses rhk-r2 and rhk-7aa as compared to rhk and rhk-5aa. in particular, avian-like viruses stimulated a higher release of potent chemo-attractants of innate immune cells, such as g-csf and il-8, shedded adhesion molecules (cd25, vcam-1, icam-1), and pro-apoptotic factors (trail). remarkably, the patterns of secreted cytokines differed between the apical and basolateral sides of the cultures. whereas avian-like viruses typically induced similar or higher levels of cytokines at the apical side than did rhk and rhk-5aa, the human-like viruses were stronger inducers of basolaterally secreted mediators. these data provide the first direct experimental evidence that receptor specificity of influenza viruses can significantly affect patterns of innate immune responses in human airway epithelium. further studies are warranted to determine the role of the observed effects in the host range and pathogenicity of influenza viruses in humans. a total of 98 newborn infants were enrolled in the study. forty-nine newborn infants (group i), who met the criteria of sepsis, had a routine sepsis evaluation as well as measurement of pct, il-10, and neutrophilic cd64 levels, procalcitonin and il-10 were measured by elisa technique while, neutrophilic cd64 by single colour flowcytometric technique. of these 49 "infected" infants, 16 had positive blood culture (subgroup ia: culture-positive sepsis), and 33 infants were diagnosed to have clinical sepsis with negative blood cultures (subgroup ib: culture-negative sepsis). another 49 newborn infants classified as control group (group ii) . results: sensitivity, specificity, positive predictive value, and negative predictive value for diagnosis of sepsis were analyzed for pct, il-10, cd64, and crp. il-10 had the highest sensitivity and specificity, 92 % and 84 %, respectively, using cutoff n 17.3 pg/ml. for pct, the highest sensitivity and specificity, 65 % and 60 %, respectively, were at a cutoff value of n 36.4 pg/ml. neutrophilic cd64 had maximal sensitivity and specificity of 92 % and 71 %, respectively, at cutoff value of 2.6 %. combinations of different markers may improve the sensitivity and specificity of biomarkers such as the tests used in this study. we found that the best combination was il-10 and neutrophilic cd64, which together provided sensitivity and specificity of 95 % and 83 %, respectively, and npv 86 %. the combination of il-10 and crp had high sensitivity and moderate specificity, 93 % and 79 %, respectively. conclusions: il-10 and neutrophilic cd64 levels determination can be used as good tests for early detection of neonatal sepsis. procalcitonin measurement might be used as an additive parameter to improve the diagnostic efficacy of the other markers in neonatal sepsis, but it is not helpful as a single test. objectives: the assembly and activation of inflammasomes are essential processes in the immune response to many inflammatory stimuli. inflammasomes are typically formed by at least one member of the cytosolic innate immune sensor family, the nod-like receptors (nlr). the nlr family members nalp3, naip5 or ipaf and the adaptor asc are involved in caspase-1 activation in response to bacterial infection, triggering the processing and secretion of il-1b and il-18. recent studies have demonstrated that tlr-dependent inflammasome activation in macrophages is modulated by autophagy, a homeostatic mechanism for the catabolism of cytosolic constituents. autophagosome biogenesis and maturation requires activation of the class iii pi3k, vps34 and can be inhibited with the pi3k inhibitors wortmannin and 3 methyladenine (3ma). in contrast, activation of akt, via class i pi3k, results in inhibition of autophagy. the aim of this study was to determine the nature of this inflammasome and whether autophagy influences il-1b secretion in dendritic cells. methods: murine bone marrow-derived dendritic cells (bmdm) were treated with tlr ligands in combination with 3ma, wortmannin or an akt inhibitor. supernatants were analysed for il-1b by elisa. results: 3ma enhanced il-1b secretion by bmdc treated with the tlr3 and tlr4 ligands poly(i:c) and lps, but not with any other tlr ligands tested. similar results were obtained using wortmannin. this increase in il-1b secretion was greatly reduced in bmdc from nalp3 -/mice compared to wild type c57/bl6 controls. treatment with the akt inhibitor had no effect on lps-induced il-1b secretion by bmdc. tlr-dependent secretion of il-1a was also enhanced by treatment with 3ma. conclusions: these data demonstrate that il-1b secretion by bmdc in response to treatment with pi3k inhibitors, in combination with lps or poly(i:c), is dependent on the nalp3 inflammasome. this response is limited to tlr3 and tlr4 agonists. inhibition of akt had no effect on lps-induced il-1b production, suggesting that the effect of wortmannin and 3ma on inflammasome activation is not dependent on the inhibition of akt activation by class i pi3k. objectives: in the last few years, several evidences have shown the modulation of toll-like receptors (tlrs) by g-protein coupled receptors, i. e. our group has recently demonstrated the attenuation of tlr2 signaling by the inflammatory lipid mediator sphingosine 1-phosphate (s1p) through receptors 1 and 2 in human monocytes-macrophages, which could explain some of the s1p anti-atherogenic effects. since adhesion of monocytes to endothelial cells is considered an important event in atherogenesis, our goal was to investigate the putative implication of tlr-s1p receptors crosstalk on the expression of adhesion molecules in endothelial cells. methods: for the study, in vitro cultured endothelial cells from arterial and venous origin were challenged with a combination of tlr ligands and s1p, and later analyzed by flow cytometry. a pharmacological analysis of the s1p receptor subtype involved in the response was also performed. results: data from flow cytometry experiments revealed that icam-1 expression was increased following lps and s1p concomitant stimulation in both venous and arterial cells, suggesting that tlr4 and s1p receptors cooperate in the expression of icam-1. conversely, no cooperation was observed when tlr2 ligands were used. in order to elucidate which s1p receptor subtype was involved in the increase of icam-1 expression, we used a pharmacological approach with s1p receptor inhibitors and pertussis toxin. results showed differences between arterial and venous cells. while in arterial cells elevated icam-1 after lps and s1p challenge was significantly reduced by blocking s1p receptor 3 and the effect was pertussis toxin-insensitive, in venous cells the response was pertussis toxinsensitive and not blocked with inhibitors of s1p receptors 2 and 3, which suggest that s1p receptor 1 might be involved in the effect. conclusions: altogether these data demonstrate that tlr4 and s1p receptors can interact to increase adhesion molecules such as icam-1 in human endothelial cells, and the s1p receptor subtype involved in the effect differs between arterial and venous cells. 15 with ssc without pah) and a pool of 12 sera of healthy controls (hc) were tested. results: in 1 dimension immunoblot, serum igg from ssc patients, patients with ipah and hc tested individually reacted with 7-10, 4-8 and 2-5 protein bands in a human vsmc protein extract with qualitative and quantitative differences between groups, respectively. in 2 dimension immunoblot, igg of pools of patients with ipah, igg of pools of patients with ssc with or without pah, and igg of a pool of hc recognized 145±48, 127±26, 130±25 and 150 protein spots respectively. twenty one protein spots were recognized by more than 80 % of igg of pools of sera in each group of patients and not by igg of hc. twenty seven protein spots were recognized by the great majority of igg of pools of patients with a higher intensity than igg of pools of hc. identified proteins were constituents of cytoskeleton, proteins involved in oxidative stress as stress-induced phosphoprotein 1 and peroxyredoxin 6 and proteins involved in regulation of cell energy metabolism as triosephosphate isomerise. we have identified anti-vsmc abs in the serum of patients with idiopathic and ssc-associated pah. these abs bind to cytoskeleton, oxidative stress and cell cycle antigens. objectives: this study aimed to verify that subcutaneous lymph node transplantation inducing lymphatic regeneration is possible in healthy adult rats, as obtained in other species. methods: this rat model was used to determine the effects of lymph node fragmentation as well as sheep erythrocytes and platelet-rich plasma injection on the regeneration of the transplanted lymph nodes. results: this rat model is adequate to study the regeneration of transplanted lymph nodes. lymph node fragmentation seems to affect transplant regeneration negatively. an immune challenge by injection of sheep erythrocytes in the drainage area of the transplanted lymph nodes does not improve fragment regeneration. however, injection of syngeneic platelet-rich plasma containing several growth factors resulted in an improvement in regeneration. conclusion: lymph node fragment regeneration, although still experimental, could be relevant for lymphedema prevention. acquired lymphedema has a high prevalence in developed countries as a consequence of the removal and/or radiotherapy of tumor-draining lymph nodes in cancer patients. this disease causes lifelong disability due to chronic swelling and increased risk of infections. it currently lacks an effective treatment. methods: 27 patients suffering from different diseases were enrolled in our study. 16 patients were suffering from bone diseases (osteomyelitis, necrosis, tumour) whereas 11 of them were suffering from inflammatoty diseases (soft tissue inflammation, diabetic ulceration). blood specimens were collected before hyperbaric oxygen treatment and the serum levels of icam-1 and vcam-1 were assesed by an enzyme immunoassay (elisa). results: 11 out of the 27 patients (40,7 %) had elevated levels of the intercellular adhesion molecule. 6 out of the 16 patients suffering from bone diseases (37,5%) had raised values (mean value 400 ng/ml) whereas 5 patients out of the 11 suffering from soft tissue diseases and diabetes (45,5 %) had raised values (mean value 735,5 ng/ml). reference value for icam-1 was 130-299 ng/ml. vascular cell adhesion molecule's assesment revealed no elevated levels in our patients. conclusions: our study revealed a high rate of patients (40 %) having increasd levels of icam-1. high icam-1 levels were more prevalent in patients suffering from soft tissue inflammatory diseases and diabetes (45,5 %) than in patients with bone diseases (37.5 %). mean values were found 735,5 ng/ml and 400 ng/ml accordingly. those findings verify the positive correlation between icam-1 and inflammatory diseases and tissue damage but not for vcam-1. colorectal cancer (crc) was the first solid tumour to be successfully targeted with anti-angiogenic therapy in the clinic. tumour angiogenesis is critical for cancer progression in that it permits expansion of the tumour mass and fosters malignant dissemination. angiogenesis is a multistep process involving endothelial cells as well as numerous stromal components within the tumour microenvironment that also represent potential therapeutic targets. inflammation dependent-angiogenesis is increasingly recognised as a central force in tumour growth and progression, while use of anti-inflammatory drugs has been found to reduce incidence of crc carcinoma potentially through repression of tumour angiogenesis. we investigated the link between inflammatory angiogenesis and colorectal cancer in archival tissues across a range of pathologies that represent diverse steps in the progression of crc: 16 cases of ulcerative colitis (urc), 16 adenocarcinomas developed from preexisting tubular or tubulo-villous adenomas, 33 tubular or tubulo-villous adenomas with low grade dysplasia, and 33 infiltrating adenocarcinomas. immunohistoobjectives: to determine the effect from the administration of preoperative pravastatina to therapeutic dose in the expression of cd18 in the leucocitaria adhesion to endotelio vascular in the isquémico-reperfundido miocardico weaveal endotelio vascular en el tejido miocardico isquémico-reperfundido by the circulation extracorpórea (cec). methods: they were included in way randomizada double blind 20 patients with intervened controlled hiperlipidemia of surgery coronary low circulation extracorpórea (cec). 40 mg of pravastatina oral 2 hours they were administered before the procedure (group study, n=10) or placebo (group placebo, n=10), and control (group control, n=10). samples of outlying veined blood were extracted to the 24 hours. the separation of leukocytes was made in peripheral blood, to determine the expression of cd18 in such. in all the samples one quantified the intensity of the expression pattern and the percentage of leucocitarias cells. results: 3 types of patterns were distinguished: cytoplasmic, of membrane and compound. the intensity of the expression was classified in 3 degrees: degree 0. without expression. degree 1. weak; degree 2. moderate; degree 3. intense. in the group control: most of the samples they presented/displayed a mixed pattern (cytoplasmic and of membrane) with an intensity degree 0-1. the placebo group: mixed pattern, degree 1-2. group study (40 mg. oral pravastatina): most of the cells they presented/displayed a predominance of membrane pattern: degree 2-3. the percentage of cells that expressed cd 18 was greater in the group study (40 mg. oral pravastatina). the preoperative oral pravastatina to therapeutic unique dose ours study produces a greater expression cd18 answer induced by the cec; it seems that these molecules located in the leukocytes participate in the adhesion to the activated endoteliales cells, necessary for the extrusion of the lymphocytes through endotelio towards the inflammatory center and in quimiotaxis of the leukocytes towards the inflammation sites. several surface molecules on endothelial and epithelial cells undergo regulated cleavage by the disintegrin and metalloproteinases adam10 and adam17. we recently identified transmembrane chemokines, junctional adhesion molecule-a (jam-a), and members of the proteoglycan family as novel substrates for these proteases. here we demonstrate that cell lines and primary cells of human endothelial or epithelial origin release considerable amounts of soluble jam-a and proteoglycan ectodomains. this release is enhanced by treatment with the proinflammatory cytokines ifng and tnfa. the enhanced release was not caused by an increased gene induction but rather associated with a reduction of the surface expressed molecules. both, constitutive and induced release required the presence of adam17 as demonstrated by specific inhibitors, lentiviral silencing experiments as well as treatment with the recombinant catalytic domain of adam17. these data suggest that the proinflammatory cytokines ifng and tnfa induce enhanced proteolytic shedding of cell surface molecules on endothelial and epithelial cells. to investigate the physiological relevance of this induced shedding, mice were treated systemically with ifng/tnfa leading to increased presence of soluble jam-a in the blood serum. both cytokines also stimulated jam-a release from excised murine aortas with was associated with enhanced adam17 activity in the tissue. in the presence of the adam17 inhibitor induction of jam-a release was suppressed. in cultured epithelial cell lines enhanced shedding of jam-a or proteoglycans was not associated with increased mrna or surface expression of adams but rather with increased activity of cellular adam17 as shown by means of a synthetic substrate assay. our study demonstrates that the proinflammatory cytokines ifng/ tnfa upregulate adam17-mediated shedding activity rendering the protease an important modulator of endothelial and epithelial surface molecules in inflammatory settings. rium, and the haplotype vegf-460/ vegf+405 is associated with rcc risk ( p= 0,017), metastases ( p=0,043), nuclear grade ( p=0,05), tumor stage ( p=0,029), and tumor size (p=0,04). on the other hand, the polymorphism vegf -2578 a/c is not associated with rcc risk and clinical parameters. our results shed a new light to the knowledge on the association between vegf polymorphism and rcc risk and development. these data could help to improve our understanding of the rcc pathogenesis and disease progression. pten is a lipid phosphatase, whose substrate is phosphatidylinositol 3,4,5-trisphosphate. therefore, pten is one of the main antagonists of the pi3-kinase, which plays a major role in many important cellular functions, such as proliferation, migration or response to inflammatory stimuli. here we investigated the role of pten in collagen induced arthritis. we show that conditional deletion of pten under the lysm promoter (lysmcrepten flox/-) leads to a significant reduction in clinical severity of collagen induced arthritis (cia). histological analysis of cia, lysmcrepten flox/mice displayed significantly reduced joint inflammation as well as erosive bone destruction. total anti-collagen antibodies, however, as well as anti-collagen iggs were identical in both groups. upon analysis of inflammatory cytokines in serum after immunisation we found a significant reduction of il-6 as well as il-8 levels. furthermore, pten deficient macrophages and dendritic cells showed reduced induction of il-6 as well as il-12 and il-23 mrna upon stimulation with various tlr-ligands. since these cytokines play an important role in the induction of pathogenic th-17 t cells, we measured th-17 cytokines in lymph nodes after immunisation with collagen. although dendritic cell and macrophage recruitment to the draining lymph node was comparable in both groups, there was a slight reduction of il-17 and a strong reduction of il-22 mrna in the draining lymph node of immunized lysmcrepten flox/compared to wild-type mice. one of the mechanisms through which il-10 exerts its anti-inflammatory effects consists in promoting the release of anti-inflammatory molecules. in this context, particularly important is the production of il-1ra, whose expression is induced by lps in human neutrophils and monocytes and significantly potentiated by the presence of il-10. based on our previous observation that support a direct role of il-10-activated stat3 in the enhancement of il-1ra transcription induced by lps, we plan to characterize the transcriptional activators recruited to the il-1ra promoter in vivo and responsible of the increased rate of transcriptional initiation upon exposure of lps-treated cells to il-10. quantitative chromatin immunoprecipitation (chip) studies were employed to examine the in vivo binding of transcriptional activators to the il-1ra promoter. crosslinked nuclear lysates were immunoprecipitated 30 and 60 min after il-10 addition with different antibodies and immunoprecipitated dna was analyzed by quantitative real-time pcr for the presence of target sequence located in the il-1ra promoter. chip assays showed that the pol ii recruitment to the il-1ra promoter induced by lps is significantly increased by il-10, further strengthening the concept that the rapid enhancement of lpsinduced il-1ra gene expression by il-10 initially occurs by targeting transcriptional events. as expected, real-time pcr of anti-stat3 immunoprecipitated dna showed statistically significant levels of stat3 binding to the il-1ra promoter only in cells stimulated with lps in the presence of il-10. surprisingly, anti-p65 and anti-p50 chip assays revealed enrichment of both p65 and p50 recruitment to the il-1ra promoter when il-10 was added to lps-stimulated cells, suggesting that il-10 enhances the recruitment of nf-kb to the il-1ra promoter. interestingly, when nf-kb is recruited to this promoter in lps + il-10-treated cells, the overall nf-kb nuclear translocation (analyzed by western blot) and dna binding activity (detected by emsa analysis) were not modified with respect to cells stimulated with lps alone. the enrichment of nf-kb at the il-1ra promoter site is dependent on il-10-activated stat3, since it is greatly reduced when stat3 activation by il-10 is impaired. the molecular mechanism through which il-10-activated stat3 promotes the recruitment of nf-kb to the il-1ra promoter is currently under investigation. major components of mast cell secretory granules are proteases. we could recently report that intracellular stored mast cell-produced cytokines regulate mc protease activities and provided evidence that il-15 acts as a specific negative transcriptional regulator of mouse mast cell protease-2 (mmcp-2). we examined the mechanisms underlying the repression of mmcp-2 gene expression. our data show that the "repressor" effects of il-15 on mmcp-2 promoter activity are still operating on the mmcp-2 591 bp long minimal promoter. moreover, il-15 deficiency in mast cells causes a specific dysregulated expression of the transcription factors c/ebpb and yy1. furthermore, chromatin immunoprecipitation revealed that il-15 promoted specific reciprocal recruitment of c/ebpb but not yy1 to the mmcp-2 promoter. finally, il-15 deficient mast cells display a predominantly non-cpg methylated pattern of the mmcp-2. thus, we proposed that the expression of mmcp-2 and possibly other immunoregulatory genes may be regulated by il-15 through epigenetic modification and by balancing the content and binding of c/ ebpb and yy1 in mast cells. i. nagy 1 , k. filkor 1 , a. vörös 1 , l. kemény 2 , a. szász 1 1 bzaka, baygen, szeged, hungary, 2 university of szeged, department of dermatology and allergology, szeged, hungary micrornas (mirnas) are evolutionary conserved small non-coding rnas that act as key regulators of gene expression at post-transcriptional level by targeting mrnas for translational repression and/or degradation. mirnas have been shown to have unique tissue-, developmental stage-and diseases-specific expression patterns. during the last years several studies highlighted that mirnas play critical role in the differentiation and function of the adaptive as well as innate immunity. little is known however, about the differential regulation of mirnas following the activation of pattern recognition receptors. in order to tackle this issue, we treated hacat keratinocytes with staphylococcus aureus-derived peptidoglycan (pgn) once or repeatedly, the latter mimicking persistent infection. after appropriate treatments we first analyzed the expression profile of mirnas mir-203, mir-146a and mir-155, which are known to participate in immune processes of the skin. repeated pgn-treatment significantly decreased mir-203 expression; in contrast, pgn re-stimulation had no further effect on mir-146a and mir-155 expression. next, we investigated the correlation between the expression of mir-203 and its two known direct targets: regulatory protein p63 and suppressor of cytokine signalling-3 (socs-3). although the gene-expression profile of neither p63 nor socs-3 changed, we found that the expression of mir-203 reversibly correlates with both p63 and socs-3 protein expression, a phenotype that we verified by two independent protein analysis methods (western blotting and immunofluorescent labeling). importantly, transfection of hacat cells with anti-mir-203 prior to pgn-treatment completely abolished both p63 and socs-3 down-regulation, revealing the involvement of mir-203 in pgn-induced transcriptional regulation. finally, methylation-specific pcr experiments unravelled the role of dnamethylation in regulating mir-203 expression upon pgn-treatment. taken together, our results strongly suggest that sets of mirnas may be differentially regulated during persistent infection. results: tgfb1+/-had a lower incidence and burden of benign papillomas when compared to tgfb1+/+ animals. however, more scc developed in the tgfb1+/-mice. after acute and chronic promotion, tgfb1+/-skin showed a reduced proliferative response with no increase in epidermal tgfb1 or nuclear p-smad2 compared to tgfb1+/+ mice. tpa-induced pkca activity as well as phosphorylation of specific pkc substrates in keratinocytes correlated with tgfb1 gene dosage. further, pharmacological inhibition of alk5 suppressed tpa-mediated pkca activation suggesting that physiological levels of tgfb1 are required for maximal activation of pkc-dependent mitogenic responses. even though the tpa-induced inflammatory response was greater in tgfb1+/-skin, tgfb1+/+ papillomas had more tumor infiltrating neutrophils. tpa-induced proinflammatory gene expression was sustained in tgfb1+/-skin and primary keratinocytes but it was elevated in v-ras ha -transduced tgfb1+/+ but not tgfb1+/-keratinocytes, indicating that tgfb1 switches from an anti-inflammatory cytokine in the skin to a proinflammatory factor in tumors dependending on an activated ras. despite this differential proliferative and inflammatory response to tpa and enhanced papilloma formation in the tgfb1 +/+ mice, there was no increase in conversion to scc in this genotype. conclusions: tgfb1 acts to promote benign tumors enhancing cell proliferation and inflammation through its ability to regulate pkc activation in skin, yet retains a suppressive function for malignant conversion. background: proto-oncogene survivin has been recently shown as a prognostic marker distinguishing patients with destructive rheumatoid arthritis (ra). in the present study we studied the relationship between survivin and urokinase (upa), a fibrinolytic serine protease being over expressed in the inflamed joints and exhibiting arthritogenic properties. material and methods: levels of survivin and upa were measured in the paired blood and synovial fluid samples of 132 patients with ra, using elisa and compared to controls with non-inflammatory joint diseases. the ability of upa to induce survivin and requirement of upa receptor (upar) was studied in primary synovial fibroblasts and pbmc of ra patients, human monocytic (thp-1) and fibroblast (mrc-5) cell lines employing antibodies against upar, sirna technique, and synthetic inhibitors of intracellular pathways. the ability to prevent urokinase-induced arthritis by interruption of survivin expression was evaluated in mouse model of arthritis. results: in the present material of 132 ra patients and 82 controls the levels of survivin correlated to urokinase (upa) (r=0.46), a plasminogen activator over expressed in inflamed joints and known to exhibit potent arthritogenic properties. we found that 30/132 ra patients had high circulating levels of survivin. these patients had erosive arthritis and were characterized by high levels of upa. in vitro studies showed that upa induced survivin in leukocytes and this process was dependent on signaling through upa receptor. in turn, survivin was required for expression of upar. additionally, survivin was essential for upa production in mrc-5 and synovial fibroblasts. down-regulation of survivin with sirna was followed by significantly reducion of upa synthesis. finally, treatment with downregulation of survivin by sirna in vivo efficiently abrogated upa-induced arthritis in mice model. these findings indicate that survivin is an essential mediator of arthritogenic properties of upa regulating its synthesis in synovial fibroblasts and upar expression in leukocytes. close correlation between survivin and upa in patients with ra supports the improtance of these interaction for the pathogenesis of arthritis. upon cell activation, ubiquitously expressed inositol 1,4,5-trisphosphate 3-kinase type b (itpkb) phosphorylates inositol (1, 4, 5) trisphosphate (ins(1,4,5)p 3 ), a calcium-mobilizing second messenger with pleiotropic effects. itpkb inactivation leads to severe t cell deficiency, altered thymo-independent b cell responses and neutrophil hyperactivation. we here report that itpkb-deficient (itpkb -/-) mice also display profound alterations in mast cell development and function. indeed, while mast cell number, c-kit and fcepsilonri expression were comparable in itpkb-deficient and proficient mice, itpkb -/mast cells were almost completely devoid of granules. this phenotype could be partially reversed upon treatment with sodium cromoglycate. nevertheless, fcepsilonri or c-kit activation on mast cells led to increased ca 2+ responses and to stronger erk phosphorylations. however, itpkb -/mice displayed an attenuated sensitivity to ige-mediated passive systemic anaphylaxis, correlated to the absence of fcepsilonri-dependant histamine release and to downregulation of h1 and h2 receptor expression due to high basal histamine concentrations. production of neosynthesized mediators remained normal. finally, itpkb deficiency also severely impaired scf-induced mast cell differentiation in vitro. taken together these results demonstrate that itpkb is a key regulator of mast cell activation. itpkb antagonists might thus be of therapeutic interest for programmed and progressive depletion of histamine stores. the large percentage of immune relevant genes that are alternatively spliced and the connections between splicing and disease, strongly indicate that alternative splicing plays a central role in the regulation and fine-tuning of physiological immune responses. il-1b is an important proinflammatory cytokine produced by activated macrophages and monocytes. il-1b is produced as an inactive cytoplasmic precursor that is proteolytic processed by the inflammatory caspase-1 to generate the mature secreted active form. caspase-1 is also synthesized as an inactive form that requires processing by the inflammasome to become active. we have used a subset of the trc lentiviral human library to generate loss-of-function phenotypes for most of the splicing factors and splicing regulators. we were able to silence the expression of 425 genes involved in splicing with an average 5-fold coverage. after the primary screen and several rounds of phenotypic validation, we have identified 30 genes that significantly affect the production of il-1b by thp-1 cells after a 24h challenge with pfa-fixed e. coli, as measured by elisa. knockdown levels were analyzed by qrt-pcr for the most significant candidates to validate the phenotypes observed. exon array analysis are being performed to identify possible targets of the most significant splicing factor candidates obtained by the shrna screening in order to dissect their mechanism of action in the regulation of the inflammasome and il-1b secretion. tissue transglutaminase (tg2) has a critical role in the pathogenesis of chronic inflammatory diseases such as celiac or neurodegenerative diseases. we have previously described the key role of tg2 in cystic fibrosis (cf), a genetic disease characterised by chronic lung infections and inflammation. in cf, mutation on the cftr gene results in an increased tg2 expression and activity leading to functional sequestration of the anti-inflammatory pparg and increase of the classic parameters of inflammation. here we tested whether in vivo inhibition of tg2 can reverse inflammation in chronic inflammatory diseases. to assess the importance of tg2 not just in cf but in chronic inflammatory diseases in general, we injected cystamine, a potent tg2 inhibitor, in a transgenic mouse model cf and in the taz10 transgenic mice that spontaneously develop autoimmune thyroiditis. intraperitoneal administration of cystamine had a significant impact on the lung epithelium in the cf model, where it decreased tg expression and activity. the treatment was also able to dampen all the classic inflammatory parameters as well as restoring normal cellular levels of functional pparg. interestingly, cystamine injections could also block inflammation in the taz10 tcr transgenic mouse model with chronic thyroiditis, highlighting the pivotal role of tg2 in generating inflammation in two very different pathologies. this work underlines the critical role of tg2 in inflammation and provides new opportunities to develop therapeutic strategies for sufferers of chronic inflammatory diseases. angiogenesis, the growth of new blood vessels, is a process that is essential during tissue repair, foetal development, and female reproductive cycle. angiogenesis is also a relevant process associated to many pathologic conditions including autoimmune diseases and tumors. we have shown that dendritic cells activated in the simultaneous presence of pro-and anti-inflammatory signals (alternatively activated dc, a-dc) display potent angiogenic activity in vivo which is mediated by the release of biologically active vegf-a. here, we investigates the molecular mechanisms leading to vegf-a secretion in lps+pge 2 stimulated a-dc. preliminary results indicate no accumulation of hif-1alpha in a-dc, therefore suggesting that vegf-a is induced by a non-classical, hif-1alpha independent pathway. in addition, we found that vegf-a secretion depends on the activation of mapk p38 but not erk1/2 or jnk. inhibitor studies, nascent transcript analysis and polimerase ii recruitment on the promoter show that the induction of vegf-a is largely due to new transcription and not to changes in mrna stability. chromatin immunoprecipitation studies aimed at the characterization of the modifications of vegf-a regulatory regions in a-dc and at the identification of transcription factors bound to vegf-a promoters are being performed. this will possibly allow the description of novel transcription factors involved in vegf-a activation in a-dc. wnt proteins are secreted palmitoylated glycoproteins with multiple functions in cell proliferation, migration as well as tissue organization. they are best known for their role in embryonic development and tissue homeostasis. deregulation of wnt signaling has been shown to promote carcinogenesis. recently we identified wnt signaling to be involved in the regulation of inflammatory processes: wnt5a is induced in human macrophages in response to mycobacteria and conserved bacterial structures and contributes to the regulation of the proinflammatory cytokines il-12 and ifn-gamma. to gain deeper insights into wnt mediated modulation of inflammatory processes we now used murine bone marrow derived macrophages and analyzed the effects of the addition of exogenous wnt homologs. we monitored wnt-mediated activation of primary macrophages by measuring the activation of signaling pathways and transcription factors, analyzed the expression of target genes by real-time pcr and measured the secretion of inflammatory cytokines by elisa. exogenous wnt5a -but not wnt3a -was able to induce cytokine expression in primary macrophages. in infection experiments wnt5a promoted the mycobacteria-induced macrophage activation and enhanced the expression of inflammatory mediators in murine macrophages. in contrast, addition of wnt3a reduced the expression of inflammatory mediators upon mycobacterial infection. these data corroborate our previous findings and further support the notion that tlr/nf-kappab and wnt signaling, both being evolutionary highly conserved pathways, are functionally interconnected infection of immunocompetent mammals with t. gondii induces a chronic infection of the brain. t. gondii cysts persist in neurons and escape elimination by the immune system. in immunodeficient individuals, the infection can be reactivated resulting in a lethal toxoplasma encephalitis (te). in te, the parasite is primarily controlled by infiltrating t and b cells. also brain resident cells may contribute to control of the disease and however, the mechanisms of brain resident cells leading to the protection of the vulnerable brain in chronic te are largely unknown. in a previous study, we could show that expression of gp130 on astrocytes in mice is critical for survival of te. in the present study, we analyzed the function of neuronal gp130 in te. after infection with t. gondii, mice lacking neuronal gp130 (synapsin-cre gp130 fl/fl ) died significantly earlier in the chronic phase of infection than control gp130 fl/fl mice. death of synapsin-cre cre gp130 fl/fl was due to a severe encephalitis with larger inflammatory lesions and higher numbers of inflammatory leukocytes. additionally, te of synapsin-cre gp130 fl/fl mice resulted in a substantial apoptosis of neurons both in the vicinity of inflammatory lesions and also in brain areas without inflammation. in vitro, apoptosis of gp130-deficient neurons was also significantly increased upon infection with t. gondii or stimulation with tnf as compared to gp130 expressing neurons. interestingly, the intracerebral parasitic burden was not increased in synapsin-cre gp130 fl/fl mice indicating that the immunoregulatory role of neurons is more important than their anti-parasitic function. t. objectives: persistent production of tnfa in many autoimmune diseases, including intraocular inflammation (uveitis), can lead to significant tissue damage. targeting tnfa with neutralising antibodies or tnf receptor fusion proteins is often, but not always, an effective therapy. high serum concentrations of tnfa, il-1b, il-6 and il-8 have been detected in a spectrum of autoimmune diseases; while in contrast, the levels of il-4, il-10 and tgfb are reduced. this suggests, indirectly, that failure to regulate an appropriate balance of inhibitory factors contributes to the pathogenesis or propagation of tissue inflammation in autoimmunity. thus, understanding the homeostatic control of tnfa by tgfb1 further may generate more effective therapies. as tnfa mrna 3' untranslated region (utr) contains an au-rich element (are), which targets mrnas for degradation, we wished to test whether tgfb1 suppresses tnfa protein production by upregulating the rnabinding protein fxr1, which can bind to tnfa mrna and inhibit translation. methods: using raw 264.7 cells and mouse bone-marrow derived macrophages stimulated with lps and tgfb1, we assessed mrna expression by q-pcr and tnfa protein expression by flow cytometry. the 3'utr of tnfa mrna was isolated and inserted into a luciferase reporter vector on a constitutive promoter. transfected raw cells were treated with lps and tgfb1 and luciferase expression was quantified. cells treated with lps and tgfb1 were also examined for fxr1 expression using pcr and western blot. following fxr1 knockdown using sirna, the influence of tgfb1 and lps on tnfa protein production was examined by flow cytometry. results: we find that while tnfa mrna expression remains constant, lps induced tnfa protein expression is suppressed by tgfb1. using the luciferase-tnf-3'utr vector we show that tgfb1 targets the 3'utr of tnfa. furthermore, tgfb1 and il-10 both upregulate fxr1 mrna and protein; and treatment with tgfb1 and lps can synergistically upregulate mrna expression, more than tgfb1 alone. following sirna inhibition of fxr1, tgfb1 can no longer inhibit lps-induced tnfa production. comtb up-regulated mmp-1 and mmp-3 secretion from saecs, nhbes and fibroblasts to a peak of 2.5 +/-0.5 ng/ml, at 72 hours. interleukin-17 augmented comtb-stimulated up-regulation of mmp-1 and mmp-3 secretion from saecs and fibroblasts in a synergistic manner. in contrast, interleukin-17 down-regulated mmp-9 secretion from saecs by 50 %. interleukin-22 up-regulated mmp-1 and mmp-3 secretion from fibroblasts but not from saecs. timp1 secretion from saecs was enhanced by interleukin-17 but there was no effect of interleukin-22. mmp up-regulation by interleukin-17 and comtb was inhibited by the pi3kinase inhibitor ly294002 and on western analysis akt (protein kinase b) was phosphorylated at 30 minutes. chemical inhibition of the p110d isoform of pi3kinase with ic87114 abrogated the il-17 and comtb driven secretion of mmp-3 from the small airway epithelial cells. chemical inhibition of the tumour suppressor phosphatase, pten (phosphatase and tensin analogue on chromosome 10) accentuated mmp-3 secretion. these inhibitory effects were confirmed with sirna. mmp-3 up-regulation was secondary to increased gene expression with promoter activity peaking 24h after stimulation. in summary, interleukin-17 and interleukin-22 drive transcription dependent mmp-1 and mmp-3 secretion from airway epithelial cells and fibroblasts. interleukin-17 also increases timp but down-regulates mmp-9 gene expression and secretion. this may contribute to the matrix degrading phenotype in tuberculosis. the pi3kinase pathway is central in interleukin-17 driven tissue destruction in the context of m. tuberculosis infection. v. delgado-maroto 1 , l.s. moreira 2 , e. gonzalez-rey 2 , m. delgado 1 1 institute of parasitology and biomedicine lopez-neyra , csic, granada, spain, 2 university of seville, sevilla, spain objectives: atherosclerosis is an inflammatory chronic disease characterized by the formation in the arteries of lesions that involve inflammation, lipid accumulation, cell death and fibrosis. over time, the rupture of these atherosclerotic plaques releases prothrombotic material to the blood and causes thrombotic occlusion at the site of disruption. atherosclerosis will probably become the most common cause of death within 15 years. one of the initial hallmarks of the disease is the uptake of oxldl particles by macrophages, which leads to intracellular cholesterol accumulation and the formation of foam cells. t cells undergo activation after interacting with foam cells, which process and present local antigens including oxldl, generating a t helper 1 response. cholesterol metabolism is regulated by factors such as pparg1 (proliferator activated receptor g), srb1 (a class b scavenger receptor), cd36 or abca1, that can induce cholesterol exit from the macrophage which may help to solve the lesions. expression of these factors depends on intracellular camp. adrenomedullin (am), urocortin (ucn) and vasoactive intestinal peptide (vip) are novel neuropeptides synthesized by immune cells that have various characteristics to be considered as possible therapeutic agents for atherosclerosis. they are potent anti-inflammatory agents, which downregulate a broad spectrum of pro-inflammatory mediators, and inhibit th1 immune response. their mechanism of action involves binding to gpcr and adenylate cyclase activation with subsequent increase of camp in the cell, which is recognized as an anti-inflammatory response. methods: we investigate am/ucn/vip effect on bone marrow-derived macrophages stimulated with oxldl. we determine the levels of pparg1, srb1, cd36, and abca1. we also analyze the cholesterol metabolism of oxldl-stimulated macrophages after neuropeptides incubation using oil red o staining of lipids drops and tritium labelled cholesterol. objective: endovascular aortic repair (evar) is considered a minimally invasive procedure, and the patients are expected to be discharged after a day or two. however up to 60 % develop a systemic inflammatory response syndrome (sirs), resulting in prolonged convalescence. as yet there is no satisfactory explanation to this severe response. previous studies have shown a high level of il-6 in the mural thrombus lining the aneurysm. the thrombus is manipulated during the procedure, but whether or not it is the source of circulating il-6 and/or other cytokines during and after the operation is unknown. methods: quantitative analysis of the pro-inflammatory cytokines il-6, tnf-a, il-1b, il-8 and il-12, and the anti-inflammatory cytokine il-10, in plasma from five patients, as of yet, was carried out by means of cytometric bead array, while analysis of plasma il-23 was performed using the luminex platform. the cytokine levels were compared to the clinical response, in terms of sirs. results: evar induced the production of il-6 and il-10, and in some cases, of il-23. the maximal plasma levels of il-6 and il-10 were found at 24 hours and of il-23 between 48-72 hours. modest plasma levels of il-8 were also observed, with maximal production at various time points (4-24 hours). by contrast, production of tnf-a, il-1b and il-12 did not occur to a significant extent, while production of il-17 occurred sporadically. although our preliminary data indicate that sirs is associated with enhanced cytokine responses, the production of il-6, il-8, il-23 and il-10 also took place in patients who did not develop sirs. conclusion: evar is associated with the sequential production of il-6, il-10, il-8 and il-23, i. e. a mixed pro-and anti-inflammatory response, even in the absence of sirs; but the production seems to be exaggerated in patients developing sirs. further studies involving 165 patients are in progress, and will clarify this. we hypothesize that sirs is elicited by il-6, activated by manipulation of the mural thrombus. to reveal whether this is the case, studies involving immunohistochemistry of the thrombus, will be performed. il-33 is a novel il-1 cytokine family member that is expressed as an intracellular precursor (pro-il-33) and is thought to be cleaved by caspase-1 to yield a mature bioactive form of the molecule (mat-il-33). to date however, evidence of cell-associated proteolytic processing and caspase-1 dependent secretion of mat-il-33 has not been reported. here we show that pro-il-33 but not mat-il-33 is released from uvb-irradiated keratinocytes. we demonstrate binding of pro-il-33 to the il-33r and also il-33r-dependent bioactivity of pro-il-33 on mast cells. we propose a previously unrecognized role for pro-il-33 as a pro-inflammatory mediator and suggest a direct link between uvb-mediated epithelial cell damage and cutaneous mast cell activation. we have previously shown that induction of er stress and tlr signalling synergistically enhance il-23 p19 mrna expression in myeloid cells, and markedly increase secretion of il-23, but not il-12, by dendritic cells. the aim of this study is to investigate the mechanism of this synergy. we examined the il-23 promoter for potential binding sites for er stress induced transcription factors and identified a putative site for chop10. chromatin immunoprecipitation (chip) assays using anti-chop10 and isotype control mab were performed using nuclear lysates from u937 cells and il-23 promoter dna measured by qpcr. chop10 binding on the il-23 promoter was detected following stimulation of u937 cells with lps or tp alone, but this was significantly enhanced when er stress and tlr stimuli were combined. il-23 promoter dna was not detectable following chip with the isotype control antibody. to confirm the role of chop10 in il-23 gene transcription, u937 cells expressing shrna's specific for chop10 or non-specific gene target were tested for their ability to express il-23 following tlr and er stress stimulation. u937 expressing three independent shrna targets for chop10 exhibited significant reductions in il-23p19 mrna (up to 87 % reduction of the response to lps+tp) compared to u937 expressing a control shrna. chop10 shrna expression did not affect the expression of other lpsresponsive genes, including il-1, il-8, ccl3 and sod2. to identify if er stress induction of il-23 mediated by chop10 expression plays a role in a more physiological setting, we examined the role of chop10 in the induction of il-23p19 gene expression following chlamydia trachomatis (ct) infection. infection of u937 cells with live but not g-irradiated ct induced expression of er stress response genes, including chop10. u937 infected with live ct exhibited increased il-23p19 mrna expression compared to u937 infected with nonviable bacteria. chop10 silencing significantly reduced the ability of live ct to induce il-23p19mrna, confirming the important role of chop10 in this response. these data suggest that er stress induction of chop10 could contribute significantly to the pathogenesis of diseases in which il-23 plays an major role, through induction of il-17 and il-22 producing cells. the clonal deletion of thymocytes by negative selection is an important process to ensure immunologic tolerance, even though the underlying molecular mechanisms are poorly understood. here, we show that gadd45b, a regulator of mitogen-activated protein kinases, is critically involved in selection processes in the thymus. gadd45b expression was inducible in different in vitro and in vivo models of negative selection. strikingly, only tcr-ligating peptides resulting in negative selection induced gadd45b expression, while positively selecting ligands or dexamethasone, a tcr-independent apoptosis agonist, failed to do so. expression of gadd45b maintained a sustained activation of p38 kinase and thereby promoted tcr-mediated apoptosis. in contrast, thymocytes from gadd45b-deficient mice showed only transient p38 activation and reduced caspase activation. interestingly, we observed a switch to positive selection in gadd45b-deficient mice since a higher percentage of single positive thymocytes was found. moreover, markers of positive selection as cd5 and cd69 were elevated on gadd45b-deficient thymocytes. thus, we provide evidence that gadd45b and a resulting persistent activation of p38 constitute a novel apoptotic pathway involved in negative selection. these results also provide evidence for the novel concept that not only the on-off switch of a signaling module but also its spatiotemporal regulation may crucially determine cell fate decisions. di santo 1 1 institut pasteur, paris, france, 2 monash university, victoria, australia > the thymus represents the ''cradle'' for t cell development, with distinct thymic stroma components providing multiple soluble and cellular membrane cues that foster in a step-wise fashion developing thymocytes. although il-7 is recognized as an essential factor for thymopoiesis, the nature of the thymic il-7 niche remains poorly characterized in vivo. > using a novel bacterial artificial chromosome transgenic mice in which yellow fluorescent protein (yfp) is under control of il-7 promoter, we identify a subset of thymic epithelial cells (tecs) that co-express yfp and high levels of il7 transcripts (il-7 hi cells). il-7 hi tecs arise during early fetal thymic development, persist throughout life, and co-express homeostatic chemokines (ccl19, ccl25, cxcl12) and cytokines (il15) that are critical for normal thymopoiesis. in the adult thymus, il-7 hi cells are found in cortico-medullary regions and display traits of both cortical or medullary immature tecs. interestingly, the frequency of il-7 hi cells decreases with age, suggesting a mechanism for the age-related thymic involution that is associated with declining il-7 levels. conversely, the frequency of il-7 hi cells is markedly increased under severe lymphopenia imposed by genetic mutations that cause an early and profound block in t cell development. this augment indicates that thymocyte-tec crosstalk may condition il-7-expression by tecs. > together, our temporal-spatial analysis of il-7-expressing cells in the thymus suggests that thymic il-7 levels are dynamically regulated under distinct physiological conditions. this novel il-7 reporter mouse provides a valuable tool to further dissect the molecular and cellular mechanisms that govern thymic il-7 expression in vivo. two lines of evidence have recently demonstrated that the pre-b cell receptor (pre-bcr) is associated with autoimmunity, through its surrogate-light-chain (slc) components l5 and vpreb. it has been shown that pre-bcrs are polyreactive for several self-antigens. the polyreactivity of the pre-bcr induces pre-bcr signaling and activation. furthermore, in human a self-reactive b cell subset was identified that co-expresses immunoglobulin light chain (ig lc) and the slc components. these vpreb + lc + b cells, found in healthy individuals, are potentially harmful as they express autoreactive antibodies associated with autoimmune diseases, like sle and ra. to elucidate the contribution of pre-bcr components to the development and activation of autoreactive b cells, we have recently generated a slc transgenic (slc-tg) mouse model in which all b cells express slc proteins. slc-tg mice exhibit spontaneous igm + plasma cell development. moreover, aging slc-tg mice have elevated anti-nucleosome igm levels, accompanied by immune complex deposition in the kidney, but do not display auto-immune pathology. nevertheless, vpreb + lc + b cells may induce pathology when self-tolerance mechanisms fail. to test this hypothesis, slc-tg mice were crossed on two autoimmune-prone genetic backgrounds: (i) em-bcl2-tg mice with b cell-specific overexpression of the anti-apoptotic protein bcl2 and (ii) fcgriib-deficient mice. both in young slc-tg;em-bcl2-tg double tg mice and in slc-tg;fcgriib -/mice spontaneous germinal center (gc) formation -which is associated with autoimmunity -was significantly enhanced, when compared with control em-bcl2-tg and fcgriib -/mice. in slc-tg;fcgriib -/mice, numbers of splenic igg2 plasma cells and serum igg2 levels were˚5-10 fold increased. importantly, serum from young slc-tg;em-bcl2-tg and slc-tg;fcgriib -/mice contained high titers of igg auto-antibodies in 2/3 and 6/6 cases, respectively. these values were increased when compared with control groups: 1/6 in fcgriib -/and 0/6 in bcl2-tg. finally, we found that the collagen induced arthritis (cia) was significantly enhanced in slc-tg;fcgriib -/mice, compared to fcgriib -/mice. taken together, these findings demonstrate the slc components have the capacity to induce auto-antibody formation in the mouse and and to enhance autoimmune pathology in ra. t cells are generated from progenitor cells that enter the thymus from bone marrow via blood. these progenitor cells once within the thymus have little selfrenewal capacity. differentiation from hematopoietic stem cells to early t lineage cells proceeds through a series of intermediate precursor populations. however, it is largely unknown to what extent these cell populations contribute to t cell development in the presence of other precursor populations and how the earliest intrathymic t cell progenitors are generated from extrathymic precursors. to assess the relative contribution of potential precursors to t lineage differentiation we developed a strategy based on the depletion of well-characterized precursor populations rather than their enrichment and subsequent adoptive transfer together with an equal amount of congenic non-depleted bone marrow. thus, the physiological ratio of extrathymic precursors remained largely intact and we were able to address the question whether there is only one physiological t cell precursor or many. we showed that, under such competitive conditions, t lineage progenitors are confined to the cd27 + cd135 + fraction of bone marrow cells. notably, t lineage reconstitution was not restricted to either cd117 hi cells, representing multipotent progenitors, or cd127 + cells, representing common lymphoid progenitors, both of which contributed to t lineage differentiation with different kinetics. in conclusion, our data suggest that multiple physiological extrathymic t cell precursors exist, which are able to compensate for the loss of depleted populations. thus, our findings may have implications for devising strategies for improved t lineage reconstitution after hematopoietic stem cell transplantation. background: previous results from our group have demonstrated ephb2 and ephb3 expression on both thymocytes and thymic epithelial cells (tecs). we used chimeric models to determine that those molecules govern in an autonomous and non autonomous manner thymocyte and tec development, and how they regulate interactions between both cell types. objectives: in order to better define the importance in thymus of eph-ephrin b interactions we have analyzed the effects of the lack of ephrin b1 and/or ephrin b2, the ligands of ephb2 and ephb3 receptors. this approach is specially interesting taking into account that eph-ephrin signaling is transmitted to both the two cells participating in the interaction and that the cell responses depend on the type of signals (reverse, forward or both), their direction and intensity. methods: for this purpose we have used cre-loxp recombination systems for deleting ephrinb1 or ephrinb2 genes specifically on either thymocytes or tecs. results: animals with ephrin b deficient thymocytes showed thymic hypocellularity and alterations on t-cell development whose severity depended on the background of the analyzed mice. in these mice only a few changes occurred in the cortical tec network. on the contrary, mice with conditioned deletions in tec, especially ephrinb1/b2 double mutants, showed a more severe phenotype that began early in the ontogeny and resulted in very small thymi exhibiting an extremely compact cortical and medullary network, decreased numbers of cd45+ cells in the cortex, increased proportion of k5+k8+ cells and high presence of cysts. in addition, t-cell development was partially blocked at the dn cell stage. conclusion: these data reveal an autonomous and non-autonomous role for ephrinb1 and ephrinb2 in the development of both t cells and tecs, confirming the importance of these molecules in the establishment of a crosstalk between the main two cell types of thymus. we discuss how eph-ephrin contacts modulate cellular homotypic and heterotypic interactions that take place during thymus organogenesis and in t cell differentiation. a. rolink 1 , d. vanhecke 1 1 university of basel, developmental and molecular immunology, basel, switzerland the importance of normal t lymphocyte development in the immune system is exemplified by the occurrence of inherited and acquired human immunodeficiencies where the development or functional maturation of t cells is defective. in order to identify molecules/genes and elucidate developmental processes that are essential for human t cell development we use a novel in vitro tool, the op9-dl1 cell culture system (1) . using this in vitro assay we obtain large numbers of human cycd3 + and cd4 + cd8 + double positive thymocytes starting from umbilical cord blood (ucb) derived cd34 + hsc. signals and molecules that are involved in t cell development are being addressed by using blocking antibodies and/or chemical inhibitors. similar as in mice we found an essential role for il-7 and notch mediated signaling in the development and survival of particular developmental stages of human thymocytes. among the molecules that are rapidly induced in cd34 + cells upon notch signaling is cd7 followed by cd127. t cell specification is accompanied by the induction of cd1a and loss of cd34 on cd7 + cd127 + cells. these cd34 -cd1a + cd7 + cells become dependent on continuous il7 and notch signaling for sustained survival and further differentiation into cd4 + cd8ab + dp thymocytes. we found that flt3l is not essential for the differentiation of cd4 + cd8 + human thymocytes but that addition of exogenous flt3l in the co-cultures increases the number of cd34 + precursors and consequently result in higher yields of developing cd4 + cd8ab + dp thymocytes. finally few mature tcrab + t lymphocytes develop from the cycd3 + cd4 + cd8ab + dp subset in this in vitro assay suggesting that op9 stromal cells lack the required selecting mhc-antigen complexes and/or costimulating molecules to induce and sustain positive selection of human thymocytes. this in vitro assay will allow us now, by using rna interference, to test additional genes for their role during human lymphoid development. from a clinical standpoint, a better understanding of the mechanisms controlling human t-cell development is a fundamental step towards the development of specific therapies for the treatment of primary and acquired immunodeficiencies as well as for the treatment of malignant t-cell disorders. brain derived neurotrophic factor (bdnf) promotes various neuronal functions such as survival, regeneration and synaptic plasticity. emerging evidence also indicates an essential role for bdnf in the immune system, e.g. in the b and t cell lineages. we therefore investigated the impact of bdnf on thymocyte development using bdnf knockout (ko) mice and conditional ko mice lacking bdnf specifically in t cells. in both models, we found reduced thymocyte numbers and a significant increase in double negative thymocytes. in contrast, the percentage of naturally occurring regulatory t cells and the expression of activation markers were unaltered. moreover, the lack of bdnf did not result in enhanced thymocyte apoptosis. the increase in double negative thymocytes was due to a partial block in the transition from the dn3 to dn4 stage, where bdnf and its receptor p75 are expressed as revealed by real-time pcr. the observed partial block in thymic maturation results in mild peripheral lymphopenia without affecting the activation status of peripheral t cells, their homeostatic proliferation and without compromising peripheral immune responses in general. in summary, our findings point to a critical role of t cell lineage derived bdnf in thymocyte development acting in an autocrine and/or paracrine manner. r. berga 1 , c. lópez-rodríguez 1 1 pompeu fabra university, barcelona, spain nfat5 is a transcription factor that belongs to the rel family (nf-kb and nfatc proteins). its expression in primary cells and organs is restricted to certain proliferative tissues, like activated t lymphocytes and thymocytes, where levels of nfat5 are relatively high and its subcellular distribution is predominantly nuclear. recent mouse models suggest that nfat5 participates in thymocyte development and also indicate its involvement in t cell proliferation and survival. nfat5 deficient mice present a t cell immunodeficiency consistent on lymphopenia, which is more accused for cd8 + lymphocytes. these observations are of substantial relevance as we and others have described that, in vivo, nfat5-null mice are unable to mount cd4+-and cd8 + -immune responses. data from our laboratory indicate that nfat5-null mice suffer from hyperosmolarity in plasma (hypernatremia) as a result of the incapacity to induce an osmoprotective gene expression program at a systemic level. to selectively analyze the t-cell autonomous effects derived from the lack of nfat5 during the development of t lymphocytes, we developed mouse models that delete nfat5 at early (lck-cre + /nfat5 flox/flox ) or late (cd4-cre + /nfat5 flox/flox ) stages of thymocyte maturation and that present isotonic plasma. our work indicates that nfat5 is expressed at all stages of t cell development. in addition, analysis of mouse models that lack nfat5 at different points of t cell development indicate that it participates at early stages of the ontogeny of t cells. objectives: apoptosis mediated by the tumor suppressor molecule p53, is regarded as a major player in tumor prevention but this may not be its only role. we have investigated this by creating a mouse (m ¿ pro) lacking residues 58-88 of the proline-rich domain of p53. methods: we compared the ability of various hemaptopoietic tissues from m ¿ pro mice and wild type mice to undergo apoptosis following irradiation or treatment with pro-apoptotic drugs. apoptosis was measured by staining with annexin v in vitro and by detection of caspase activation in vitro and in vivo. we also compared their ability to undergo cell cycle arrest using brdu staining. tumor development was monitored in cohorts of m ¿ pro, p53 null (p53-/-) and wild type (p53+/+) mice, with or without prior irradiation. results: apoptotic function was lacking in m ¿ pro mice, but they were able to arrest cell-cycle progression in hematopoietic tissues. m ¿ pro developed late-onset b-cell lymphoma, but not the thymic t-cell tumors found in p53-/-mice. interestingly, m ¿ pro lymphomas were comprised of incorrectly differentiated b-cells. bcell irregularities were also detected in m ¿ pro prior to tumor onset, in which aged mice showed an increased population of inappropriately differentiated b-cells in the bone marrow and spleen. we propose that the apoptotic function of p53 has an important role in b-cell homeostasis, which, in turn, is important for prevention of b-cell lymphomas moreover, our data suggest that the apoptotic function of p53 is not important for preventing thymic t-cell tumors. s. myrczek 1 , r. pardi 2 , a. gessner 1 1 microbiological institute-institute for clinical microbiology, immunology and hygiene, university hospital erlangen, erlangen, germany, 2 vita-salute san raffaele university school of medicine, milano, italy jab1 is the catalytic subunit of the highly conserved cop9 signalosome. this complex plays a central role in various cellular processes as proliferation and cell cycle control. jab1 regulates the neddylation of ubiquitin ligases and thus contributes to degradation of many proteins. furthermore jab1 regulates the activity of ap1 transciption factors. to date jab1 is thought to be essential for every cell type as jab1 knock out mice are embryonic lethal and t cell development is blocked by t cell selective absence of jab1. to investigate the function of jab1 in b cells we established a mouse strain deficient for jab1 selectively in b cells. mice with floxed alleles of jab1 kindly provided by r. pardi were crossed with a mouse strain expressing the cre recombinase under control of the mb1-locus (m. reth, freiburg). ablation of jab1 expression resulted in an almost complete block of b cell development at the pro b cell stage. the absence of peripheral mature b1 and b2 cells and serum immunoglobulins resulted in chronic arthritis with high pathogen burden after experimental infection with borrelia burgdorferi. the observed block in b cell development is rescued by over expression of the anti apoptotic protein bcl2 under the control of the m enhancer. facs analyses revealed that all b cell subtypes analyzed in the jab1-deficient, bcl2-transgenic mice are present albeit at reduced numbers compared to wild type animals. serum immunoglobulin titers are detectable and after borrelia infection specific antibodies are produced. we confirmed the absence of jab1 in sorted spleen b cells by immunoblot analysis. in summary, we show for the first time that cells are viable and functional without jab1 when apoptosis is prevented. t. nitta 1 , s. murata 2 , k. tanaka 3 , y. takahama 1 1 university of tokushima, tokushima, japan, 2 university of tokyo, tokyo, japan, 3 rinshoken, tokyo, japan how self-peptides are generated and displayed in the thymus to select a useful and self-protective repertoire of t cells is largely unknown, whereas the role of thymic self-peptides in eliminating self-reactive t cells and thereby preventing autoimmunity is well established. a recently identified form of proteasome, termed thymoproteasome, is specifically expressed by thymic cortical epithelial cells (ctec) and is required for the optimum generation of cd8 t cells. here we show that ctec display a thymoproteasome-specific spectrum of class i mhc-associated self-peptides, which is essential for positive selection of major and diverse repertoires of class i mhc-restricted t cells. indeed, cd8 t cells generated in the absence of thymoproteasomes display a markedly altered tcr repertoire that is defective in both allogeneic and antiviral responses. these results demonstrate that thymoproteasome-dependent self-peptides are required for positive selection of a diverse repertoire of immunocompetent cd8 t cells. defects in helper t cell number or function causes susceptibility to infections and in some cases autoimmunity or allergy. our understanding of the genetic control of helper t cell differentiation into specific functional subsets is still far from complete. here we present the results to date from a genome-wide enu mutation screen for mice with inherited deficits in specific helper cell subsets. these deficits were detected by multi-colour facs analysis of peripheral blood samples, and by antibody production following immunization with heat-killed b.pertussis and cgg coupled with the hapten arsonate (aba) in alum, which induce internally polarized th1 and th2 antibody responses, respectively. using this screen, a number of new mutant strains have been isolated with complete or partial loss of cd4+ t cells or functional deficits that selectively interfere with th1 or germinal centre responses. in this talk i will present data from some of the first 12 strains that have been identified including the first 5 strains where we have been able to identify the causative mutation. systematic genetic analysis of helper t cell differentiation in the resulting strains will illuminate how t cell help is correctly polarized for immunity and to avoid immunopathology. intrathymic t-cell development provides a unique model system to study cell fate determination because of the well-defined cellular stages and the confined microenvironment of this process. in order to highlight the differences and similarities between fetal and adult t-cell development at the molecular level we performed a microarray study. labelled rna from facs purified fetal and adult dn1 c-kit high (etp), dn2 and dn3 thymocyte populations was hybridised to affymetrix mouse 430a-2.0 genechips. the resulting data were grouped into four distinct gene clusters: cluster i contained genes over-expressed throughout adult development and included a large proportion of transcription factors (85 out of 623 genes), illustrating a significantly different transcriptional program acting during adult differentiation. conversely, cluster ii consisted of genes that were over-expressed in fetal progenitors and included 64 signal transducers (out of 590 genes) such as acvr1, bmpr1, fzd7, chemokine receptors cx3cr1, cxcr6 and integrins a2, a4, a9, ae and av, pointing to a difference in microenvironments. genes that showed uniform down-regulation during consecutive stages of fetal and adult development were restricted to cluster iii. amongst these were transcripts governing alternative developmental choices, therefore emphasising a common mechanism of lineage restriction during thymopoiesis. on the other hand, cluster iv was limited to genes that were homogeneously up-regulated during development. these included gata-3, tcf-1, notch-1, rag-1, rag-2 and pre-ta, which are indispensable for t cell development. interestingly, levels of expression of these genes were elevated in fetal progenitors, especially at the etp and dn2 stages, suggesting that the molecular program of t-cell development is more advanced in the early stages of fetal differentiation. discriminant analysis with the use of the support vector machine arrived at the same conclusion that demonstrated a nearby clustering of all fetal stages with the adult dn3 population, therefore implying a more committed state of fetal progenitors. finally, transcriptional signatures of each developmental stage were defined by "recursive feature elimination" with support vector machines. this approach can now be used to classify characterised and aberrant hematopoietic progenitors and thus construct an ontological scheme of hematopoietic development based upon transcriptional signatures of populations under normal and pathological conditions. tcrgd+ cells and tcrab+cd8aa+ intraepithelial lymphocytes (iels) of the gut are unconventional t cells that reside in tissues and provide innate-like immune responses to "stressed-self". as these cells share common functional properties in the periphery, we have hypothesised a common mechanism of development in the thymus; their progenitors diverging from the conventional t cell developmental pathway based on tcr signal strength at the dn stage. the pre-t-alpha chain (pta) that pairs with tcrb to generate the pre-tcr, has two isoforms; pta a and pta b . both can form a functional pre-tcr with tcrb. ligand-independent signalling by the pre-tcr is a result of spontaneous oligomerisation (followed by internalisation), that is mediated through charged residues on the pta chain. pta b lacks 3 out of 4 of these essential residues and therefore, we speculate results in higher surface expression and different signalling capabilities. we have hypothesised that pta a and pta b permit differential signal strength through the pre-tcr at the dn stage, facilitating the divergence of the conventional and unconventional lineages of tcrab+ t cells. preliminary semi-quantitative pcr data suggest that pta a and pta b are differentially expressed in wt thymocytes at different stages of ontogeny. retroviral transduction of pta -/-e14 thymocytes with either pta a or pta b alone, followed by fetal thymic organ culture, confirmed the rescue of abt cell development by both isoforms. however the two isoforms appear to differentially regulate the kinetics of thymocyte development by 7-10 days of culture; pta b expression generates a greater percentage of tcrab+ cells while pta a expression results in the accumulation of isps. these results suggest different roles for the two isoforms of pta in the thymic development of abt cells. in order to determine the mechanism by which pta a and pta b may generate qualitatively different signals, site directed mutagenesis was used to produce mutant chains of pta a and pta b that lack the "dimerisation residues" necessary for internalisation of the receptors. in addition, bac transgenic mice that express singly either pta a or pta b under the pta promoter are being generated to fully characterise their role in conventional vs. unconventional lineage commitment. erythroid, myeloid and lymphoid cells are initiated in parallel in appropriate cytokine environments so that specific number of erythrocytes, myeloid cells, natural killer cells, thymocytes and t cells, and precursor of b cells can be detected and counted at day 18 of culture. if needed for further functional analyses, long-term proliferating lines and clones of progenitor t and b cells can be established at this point of "in vitro" development. hence the "in vitro" differentiation of es cells to different hematopoietic cell lineages and their progenitors can be quantified. it allows for testing the efficiencies for hematopoietic development of genetically or epigenetically different es or ips cells. aim: increasing evidence includes wnt proteins inside the group of master-signalling pathways which govern immune and non immune differentiation systems. although their precise functions in bone marrow and thymus are still controversial, numerous studies show that wnt signalling is able to control the proliferation of hsc and thymic progenitors and might also affect both their cell-fate decisions and subsequent maturation. in the present work we analyse the effect of transient stimulation of canonical wnt pathway in the differentiation potential of lin -cd34 + cd1ahuman thymic progenitors, a multipotent and heterogeneous cell population which has the capacity to develop into t cells, nk cells, monocytes, conventional dendritic cells (cdc) and plasmacytoid dcs (pdcs). methods: human thymus samples from patients aged 1 month to 3 years undergoing corrective cardiac surgery were obtained and used according to the declaration of helsinki. transient b-catenin stabilization was triggered culturing purified thymic lin -cd34 + cd1precursors with recombinant wnt3a (100 ng/ml) or with licl (10 mm) for 12hr. active b-catenin, was detected by flow cytometry using anti-human active b-catenin mab (8e7) under conditions of phosphatase activity inhibition. wnt3a or licl pre-treated precursor were assayed in chimeric human-mouse ftoc, in il-15 and scf-supported cultures for generation of nk cells and in co-cultures with murine bone marrow stromal st2 cells suplemented with il-7 and flt3l. phenotype of recovered cells, apoptosis and cytokine receptors were analysed by flow cytometry. expression profile of transcription factors was analysed by real-time quantitative rt-pcr . our results demonstrate that giving a boost to canonical wnt signalling triggered by transient exposure of thymic progenitors to wnt3a or licl, change their differentiation capacity enhancing nk cell production. on the contrary, wnt3a or licl pre-treated thymic progenitors generate a significant lower number of myeloid lineage cells, monocytes and cdc, as well as reduce their capacity to differentiate into pdc lineage. as a possible mechanism for this effect we show that wnt pre-treated progenitors change their expresssion of receptors for cytokines pivotal for their expansion and differentiation, such are il-7 and flt3l and modify the transcription factor profiles of cd34 + cd1thymocytes mainly increasing hes-1 and id3 expression levels. human th17 clones and circulating th17 cells showed lower susceptibility to the anti-proliferative effect of tgf-beta than th1 and th2 clones or circulating th1oriented t cells, respectively. accordingly, human th17 cells exhibited lower expression of clusterin, and higher bcl-2 expression and reduced apoptosis in the presence of tgf-beta, in comparison with th1 cells. umbilical cord blood naï ve cd161(+)cd4(+) t cells, which contain the precursors of human th17 cells, differentiated into il-17a-producing cells only in response to il-1beta plus il-23, even in serum-free cultures. tgf-beta had no effect on constitutive rorgamma t expression by umbilical cord blood cd161(+) t cells but it increased the relative proportions of cd161(+) t cells differentiating into th17 cells in response to il-1beta plus il-23, whereas under the same conditions it inhibited both t-bet expression and th1 development. these data suggest that tgf-beta is not critical for the differentiation of human th17 cells, but indirectly favors their expansion because th17 cells are poorly susceptible to its suppressive effects. m. irla 1 , w. reith 1 1 university of medecine, pathology and immunology, geneva, switzerland objectives: medullary thymic epithelial cells (mtecs) are specialized for inducing central immunological tolerance to self-antigens. to accomplish this, mtecs must adopt a mature phenotype characterized by expression of the autoimmune regulator aire, which activates the transcription of numerous genes encoding tissue-restricted self-antigens. the mechanisms that control mature aire(+) mtec development in the postnatal thymus remain poorly understood. however, the generation of mutant mice exhibiting blocks in thymocyte differentiation at different stages, together with studies on embryonic development of the thymus, have demonstrated that reciprocal interactions between developing thymocytes and tec control not only t cell development but also the differentiation and organization of tec, a phenomenon designated as 'crosstalk'. the aim of the project outlined here is to elucidate the cellular and molecular mechanisms by which thymocytes control the numbers of mature mtec, key mediators of central tolerance. we have demonstrated by generating different transgenic mouse models, that although either cd4(+) or cd8(+) thymocytes are sufficient to sustain formation of a well-defined medulla, expansion of the mature mtec population requires autoantigen-specific interactions between positively selected cd4(+) thymocytes bearing autoreactive t cell receptor (tcr) and mtecs displaying cognate self-peptide-mhc class ii complexes. these interactions also involve the engagement of cd40 on mtecs by cd40l induced on the positively selected cd4(+) thymocytes. conclusion: this antigen-specific tcr-mhc class ii-mediated crosstalk between cd4(+) thymocytes and mtecs defines a unique checkpoint in thymic stromal development that is pivotal for generating a mature mtec population competent for ensuring central t cell tolerance. q. qiu 1 , i. ravens 1 , g. bernhardt 1 1 hannover medical school, institute of immunology, hannover, germany cd155 is originally identified as human poliovirus receptor (pvr) and as rodent tage4, which is overexpressed in rodent colon carcinoma. cd155 is also known as necl-5, a particular notable nectin-like molecule belonging to immunoglobulin superfamily, owning its unique expressing frofiles. cd155 expression is very low in most adult organs, but is abundant in the developing or regenerating liver. in addition, cd155 is overexpressed in transformed cells and promotes the cell cycle. thus, cd155 seems to be an oncofetal protein that functions in embryonic development and cancer progression. t-cell development is characterized by the progression through several phenotypically distinct stages, defined as double negative (dn), double positive (dp) and single positive (sp) based on expression of the co-receptors cd4 and cd8; the dn subset is further subdivided into four stages (dn1-4) by differential expression of cd44 and cd25. thymocytes at different stages of development occupy distinct spatially restricted domains in the adult thymus, indicating that differentiation occurs concomitantly with a highly ordered migration. during their final maturation in the medulla, semi-mature sp thymocytes down-regulate activation markers and subsequently exit into periphery. while semimature cd4+ sp are sensitive to negative selection, it remains elusive when negative selection occurs in the cd8 lineage. here we show that the frequency of terminally matured cd8+ sp cells but not that of cd4+ sp present in thymus varies depending on age. in mice lacking expression of the adhesion receptor cd155, a selective deficiency of mature cd8+ sp thymocytes was observed emerging first in adolescent animals at the age these cells start to accumulate in wild type thymus. evidence is provided that the mature cells emigrate prematurely when cd155 is absent thus cutting short their retention time in the medulla. moreover, in unmanipulated wild type mice semi-mature cd8+ sp thymocytes are subjected to negative selection as reflected by the diverging t cell receptor repertoires present on semi-mature and mature cd8+ t cells. in cd155 deficient animals, a shift in the tcr repertoire displayed by the pool of cd8+ sp cells was found demonstrating that cd155 is involved in negative selection. in the adult, steady-state, homeostatic conditions, lymphohematopoietic cell lineages display high rates of cell turnover. yet, the frequencies of simultaneously cycling cells are small, except in intermediate cellular stages of transit-amplifying precursor cell stages. the analysis of the molecular targets controlling these proliferation rates may provide relevant information to understand differentiation pathways along the ontogeny as well as mechanisms of leukemic transformation (passegué et al. j. exp. med. 2005 , 202, 1599 . during development, hematopoietic stem cells and their derived cell lineages need to expand to cope with continuously-increasing somatic demands. by using complementary, quantitative analyses (brdu labelings, hoescht 33342, propidium iodide), we are dissecting the proliferation rates of hematopoietic cell lineages and their differentiation stages along the whole mouse gestation from e9 (e, gestational day) on, in yolk sac, splanchnopleura/agm, blood, liver, spleen and bone marrow. we have observed that around half of cd71 + ter119 + erythroid and cd45 + cd11b + myeloid cells are simultaneously cycling (s/g2/m) in the post-gastrulation mouse embryo (e10-12). the peak of lymphohematopoietic cell proliferation occurs at e13 in a sort of wave-like pattern. these high-proliferation frequencies are present not only in immature, but also in mature cells, the latter thus displaying a different behaviour from the one present in the adult. later on, the proliferating cell subsets are restricted to fetal liver, whereas the equivalent cells become arrested in the periphery. interestingly, nucleated erythroid cells suddenly go into quiescence 24-48 hours before they enucleate, suggesting that this cell arrest is required for the enucleation process. we are also analysing the proliferation state of the first b and t lymphoid progenitors emerging at e11-12 that give rise to perinatal lymphocytes and, in some cases, to innate-like lymphocytes displaying self-renewal in the adult. we attempt to dissect the mechanisms regulating proliferation and death in the embryo versus those of adult lymphohematopoietic precursors, which may influence the functional activities of the mature cells. objectives: the role of cd40-cd154 interactions in t cell activation of antigen presenting cells and b cells is known, but a role for this receptor-ligand pair in hematopoiesis control has not been described. following an initial discovery that b lineage cells in the bone marrow (bm) as early as pro-b cells express cd40, we hypothesised a role for cd40-cd154 interactions in the control of b cell haematopoiesis. the objectives of this study were to investigate this hypothesis further. methods: flow cytometry was used to investigate cd40 expression by precursor b cells using b cell specific markers. reverse transcription of bm stromal cell rna and pcr were used to assess the presence of cd154 message and cell lineage specific mrna. irradiation and bone marrow transplantation (bmt) in both directions between cd154-/-and wt mice was used to assess potential functional contributions of stromal or haematopoietic cd154 on reconstitution of b cell numbers following depletion. we show that cd40 is expressed by pro-b cells, and these cells proliferate in response to cd40 signalling in vitro. pcr identified a source of cd154, negative for cd3eta, in the bm of wt mice showing this cd154 is not provided by activated re-circulating t cells. we have shown that when cd154 -/-mice are recipients, but not donors of bmt, b cell recovery after irradiation is significantly delayed regardless of the donor cell source. in the in vitro experiments we found that the pta gene is expressed from the dn1 (cd4 -/cd8 -/cd44 + /cd25 -) to the dp (cd4 + /cd8 + ) stage, whereas no yfp expression could be observed in the b lineage. the in vivo analysis of thymocytes confirms the appearance of yfp positive cells during t cell development from the dn1 stage on. in the bone marrow we found yfp + /b220 + and yfp + /b220populations. thus these pta expression analyses show closely similar pattern to those observed with hucd25 preta-reporter transgenic mice (gounari f. et al. 2002 , martin et al. 2003 . the bac pta reporter system can be used together with specific markers of other hematopoietic lineages and their progenitors to trace lymphopoiesis. gounari f et al., nat. immunol. 3, 489-496 (2002 ) martin c. h. et al., nat. immunol. 4, 866-873 (2003 . the individual functions and the reason for the tightly regulated expression of igm and igd during b cell development are poorly understood. our data show, that igd requires stronger stimuli than igm to induce b cell activation and that this silences autoreactive vdj recombination products when expressed as igd. in agreement with this, mhc and dhc, the respective heavy chains of igm and igd, differ dramatically in pre-bcr signaling, which represents the prototype of an autoreactive receptor. together with published data, our results reveal a novel role for igd and suggest that the differential expression of igm and igd is important to raise the activation threshold of mature b cells, thereby avoiding hypersensitivity and ensuring tolerance towards self-antigens. p. d. rymkiewicz 1 , g. klein 1 1 zmf (center for medical research), section for transplantation immunology and immunohematology, tübingen, germany thymic conduits which are exclusively found in the medullary region of the thymic lobules have been recently identified. the core of the conduits consist of fibrillar collagen bundles and is surrounded by a basement-membrane-like structure which contains the typical basement membrane components such as laminins, collagen type iv, nidogens and perlecan. a marker molecule for the conduits in the human thymus is the laminin isoform lm-332 which is synthesized by the medullary thymic epithelial cells (tecs) which tightly surround the conduits. functionally the conduits are too small to transport cells but they are able to transport small molecules x 70kda.mmp-19, a secreted member of the matrix metalloproteinase superfamily, is a protease capable of digesting lm-332. in the human thymus medullary, but not cortical thymic epithelial cells strongly express mmp-19. by western blotting the zymogen and the activated form of mmp-19 can be detected in whole thymus lysates, whereas in lysates from isolated tecs mainly the activated form is present. an in situ zymographic analysis revealed an increased proteolytic activity in the medullary region of the thymus. using confocal laser scanning microscopy double immunofluorescence staining showed that lm-332 and mmp-19 can be found in close neighbourhood, but they do not exactly co-localize. why activated mmp-19 which can be secreted by medullary tecs does obviously not destroy the surrounding basement membrane of the conduits has not been solved so far. two natural inhibitors of mmp-19, timp-2 and timp-3, are found in the thymic medulla, but they are not expressed and secreted by tecs. whether mmp-19 plays a role in processing medullary chemokines which are produced by the thymic epithelial cells is presently under investigation. to study the process of t cells differentiation in more detail, we intend to establish an inducible gene expression system (tet-on system) in primary t cells. the tet-on system comprises two retroviral vectors. the response vector contains an inducible modified minimal cmv promoter which per se is unable to induce expression of the gene of interest (goi). the second vector encodes a transactivator which is constitutively expressed and undergoes conformational changes upon binding of doxycycline. in this state, the transactivator enables the minimal cmv promoter to transcribe the gene of interest. therefore, co-transduction of both vectors is required to achieve transcription of the gene of interest. to date we have tested two tet-on systems (revtet system and retro-x tet-on advanced inducible expression system) that differ in the sequence of their inducible promoters. to monitor successful transfection in retrovirus-generating phoenix cells and transduction in t cells, respectively, we have cloned the reporter gene gfp under the control of a constitutive cmv promoter, into the response vector of the revtet system. this allowed identification of transduced gfp-positive cells via facs. however, when we used a red fluorescent protein, tomato, as a surrogate goi, we detected considerable leakiness of the promoter irrespective of the presence of the transactivator or doxycycline. in contrast, we found comparably low leakiness when using the retro-x tet-on advanced inducible expression system. here, co-transfection of phoenix cells with the transactivator and supplementation of doxycycline yielded an induction of 15-20 % compared to only 5 % basal rate. therefore, the retro-x tet-on advanced inducible expression system appears suitable for our studies. future experiments will aim at establishing this system in primary t cells. although a number of different experimental approaches has been used to elucidate impact of basal levels of adrenal gland-derived glucocorticoids (gcs) on t-cell development, and thereby t-cell-mediated immune response, their relevance for these processes is still far from being understood. the study was undertaken to explore relevance of basal levels of gcs for t-cell differentiation/maturation. eight days post-adrenalectomy in adult male rats thymocyte yield, apoptotic and proliferative rate and relationship among major thymocyte subsets defined by tcrab/cd4/cd8 expression were examined using flow cytometry analysis. it was found that adrenal gc deprivation affects: i) thymocyte apoptosis, producing thymic hypercellularity and ii) kinetics of t-cell differentiation/maturation leading to an overrepresentation of the cd4+cd8+ double positive (dp) tcrab low cells entering selection, and their cd4+cd8+ dp tcrab-immediate precedents followed by underrepresentation of the selected cd4+cd8+ dp tcrab high and the most mature cd4-cd8+ and, particularly, cd4+cd8-single positive (sp) tcrab high cells. the study suggests that withdrawal of adrenal gcs produces alteration in thymocyte selection processes that may affect diversity of functional t-cell repertoire and generation of potentially self-reactive cells as indicated by the reduced proportion and number of cd4-cd8-double negative tcrab high cells. in addition, it indicates that gcs influencing post-selection maturation of thymocytes play a regulatory role in controlling mature cd4+cd8-/cd4-cd8+ sp tcrab high cell ratio. in the thymus a specific subset of thymic stromal cells -medullary thymic epithelial cells (mtecs) -express a highly diverse set of tissue-restricted antigens (tras) representing essentially all tissues of the body, which is known as promiscuous gene expression (pge). this allows self-antigens, which otherwise are expressed in a spatially or temporally restricted manner to become continuously accessible to developing t cells. the scope of central tolerance is to a large extent dictated by the pool of promiscuously expressed genes. thus, even lack of a single tra can result in spontaneous organ-specific autoimmunity. promiscuously expressed gene which have no structural or functional commonality display two prominent features, they are highly clustered in the genome and show a preference for tras. for better understanding these features, we set out to precisely define the genomic organization of this gene pool. in particular, we probed to what extent and according to which rules predefined genomic clusters of tras are transcribed in mtecs. our analysis proceeded from the bioinformatic definition of tra clusters via gene expression analysis in mtecs using whole genome arrays to the in depth analysis of selected tra clusters by rt-pcr at the population and single cell level. patterns emerging from these studies will hopefully yield insight into evolutionary mechanisms responsible for selecting this gene pool. conceivably, positional cues in the genome and/or particular properties of self-antigens (e. g. immunogenicity) could have been driving forces during the co-evolution of pge and adaptive immunity. although catecholamines have been shown to influence thymocyte proliferation and differentiation, long-lasting b-adrenoceptor (ar) blockade failed to show any significant effects on thymic cellularity. bearing that in mind, the present study was undertaken to explore: i) a 1 -ar expression on thymic lymphoid and nonlymphoid cells and ii) putative role of a 1 -ar-mediated mechanisms in modulation of thymic cellularity and t-cell development. for this purpose a 1 -ar expression on thymic cells was assessed using both immunocytochemistry and flow cytometric analyses, while their putative modulatory role in thymopoiesis was estimated by analyses of thymocyte proliferation and apoptosis, as well as expression of major thymocyte differentiation antigens (cd4/ cd8/tcrab), in adult wistar rats subjected to 14-day-long treatment with a 1 -ar blocker urapidil (0.20mg/kg body weight/day s. c.). the a 1 -ar immunoreactivity was found in both thymocytes (mainly less mature cd3and cd3 low cells) and thymic nonlymphoid cells (thymic epithelial cells located mainly at cortico-medullary junction and cortical ed1-postive cells, which comprise macrophages and dendritic cells). chronic treatment with urapidil increased thymic weight and caused the organ hypercellularity. the thymic hypercellularity reflected, at least partly, increased frequency of proliferating thymocytes, which was followed by diminished thymocyte apoptosis. in addition, in these rats changes in distribution of major thymocyte subsets delineated by cd4/ cd8/tcrab expression were observed. these changes comprised of an increase in the percentage of cd4+8+ tcrabthymocytes, which was accompanied by the reduction in that of cd4+8+ tcrab low cells in urapidil-administered rats, and divergent changes in the percentage of the most mature single positive tcrab high thymocytes. compared with saline-administered controls, the percentage of cd4+8-tcrab high thymocytes in urapidil-administered rats was increased, while that of the cd4-8+ tcrab high was reduced. in addition, the percentage of cd4+ t regulatory and cd161+tcrab+ nkt cells was increased. collectively, this study clearly showed the expression of a 1 -ar on both lymphoid and nonlymphoid thymic cells, and indicated that a 1 -ar-mediated mechanisms may be implicated in modulation of multiple steps of t-cell development. we have recently shown that the thymus is a common target for mycobacterial infections. of notice, while bacterial growth is arrested in the spleen around 4 weeks post infection with mycobacterium avium, it takes several weeks longer within the thymus to reach a bacterial plateau. this observation suggests that a specific immune response occurs in the thymus, although this seems to be distinct from that occurring in the spleen. since t cell differentiation occurs, to a large extent, in the thymus, and depends, among other factors, on the antigens encountered within the thymus and on the cytokine milieu of the organ, we decided to characterize the pattern of the thymic immune response against mycobacteria and investigate possible consequences on the normal function of this organ. methods: c57bl/6 mice infected with m. avium (10 6 cfus, iv) were sacrificed at different time points after infection (5, 16 and 25 weeks). non-infected animals were used as controls. bacterial load was assessed in the spleen and thymus and cytokines (such as ifn-g, tnf, il-10) were quantified by rt-pcr in tissues (normalized for hprt expression) or by elisa in the supernatants of cultured thymocytes and splenocytes. statistical significances were determined by anova. we observe increased levels of ifn-g in infected thymi at 16 weeks post infection. this increased expression of ifn-g is concordant with the late bacterial growth arrest within the thymus. at 24 weeks post infection this difference is still present. throughout the course of infection no significant differences are found in the expression of tnf and il-10 in this organ. in the spleen, ifn-g reaches a peak of expression earlier (5 weeks post infection) and this is accompanied by increased tnf expression. conclusion: our cytokine analysis of the thymus and spleen confirm that an immune response against mycobacteria is mounted within the thymus, although different in timing and pattern from the one in the spleen. since the cytokine milieu influences t cell differentiation within the thymus, our observations raise the question on the consequences of such response on the normal function of this organ. such implications should next be investigated. precise regulation of eukaryotic gene expression requires interactions between distal cis-acting regulatory sequences with the looping out of the intervening dna, but how trans-acting regulatory proteins work to establish and maintain dna loops during gene activation remains largely unexplored. lps-induced transcription of the mouse igx gene in b lymphocytes utilizes three distal enhancers and requires the transcription factor nf-xb, whose family members include rela and c-rel. using chromosome conformation capture technology in combination with chromatin immunoprecipitation, here we demonstrate that lps-induced igx gene activation creates chromosomal loops by bridging together all three pair-wise interactions between the distal enhancers and rna polymerase ii, the apparent molecular tie for the bases of these loops. rela and actin polymerization are essential for triggering these processes, which do not require new transcription, protein synthesis or c-rel. we have thus identified both essential and non-essential events that establish higher-order chromatin reorganization during igx gene activation. this investigation was supported by grants gm29935 and ai067906 from nih and grant i-823 from the robert a. welch foundation to wtg, and by grants hl067256 and hl61897 from nih to lst. allelic exclusion of immunoglobulin (ig) genes supports burnet's clonal selection theory. the recognition that m-chain expression is sufficient for the maintenance of the silenced allele status by a process of feedback inhibition is yet not enough to explain the earlier monoallelic activation by the rag complex. attempts to prove the probabilistic or epigenetic nature of monoallelic v(d)j recombination were insightful and favor the epigenetic hypothesis, mainly by the observation that, like autosomal imprinted or x-chromosome inactive genes, ig genes are differentially marked at the chromatin level, and replicate asynchronously in virtually any cell of the mouse organism ever since the embryonic life (mostoslavsky, singh et al. 2001) . we are testing this hypothesis, i. e., that an epigenetic event has previously marked, on each b cell progenitor, which of the ig alleles is going to be activated for rearrangement and expression. for this, we are generating b cell clones from b cell progenitors of (c57bl/6 x balb/c)f1 mice, because the original strains have ig heavy chain (igh) alotypes distinguishable by monoclonal antibodies. we are analyzing if individual heterozygous clones show biased expression of a particular igh allele, and we expect to map the cell stage at which the (epigenetic) allelic marks are fixed. we have already shown, for the igh gene, a clear segregation of monoallelic expressors among b cell clonal lines that were generated from the common lymphoid precursor, but no allele bias was observed among multi-potent progenitor or hematopoietic stem cell b cell clones. this result, although suggesting epigenetic silencing starting at the common lymphoid precursor stage, does not favor the prevailing epigenetic hypothesis in its original formulation. we are currently exploring this result by the analysis of the igh silenced allele rearrangement status in sorted fractions of igm+ b cell clones. we are also testing the same epigenetic hypothesis for the ig kappa light chain gene (igk), using f1 mice in which the igk constant region from both alleles can be distinguished by antibodies. a. giniewski 1 , s. lang 1 , m. stein 1 , t. winkler 1 1 friedrich-alexander university, erlangen, germany vdj recombination is considered to be regulated by lineage and stage specific changes in accessibility of the loci to rag recombinase. accessibility is expected to correlate with certain histone modifications such as acetylation (e. g. h3ac) or methylation of h3 on lysine 4 (h3k4me2/3). previous studies in our lab revealed three regions in the intergenic part of the distal v h cluster (ivars), which are associated with high levels of active chromatin marks (h3ac and h3k4me2/3) only in pro b cells but not in pro t cells. it is also known that vdj h recombination is accompanied by sense and antisense transcription, however, little is known about the function and origin of the antisense transcripts. since one ivar (ivar #3) shows promoter activity in antisense orientation, it was analysed in more detail. we found three transcription start sites by 5'rlm race at the ivar #3 element. background: t-all is a malignancy of the lymphoblast committed to the t-cell lineage with translocations between tcr genes and oncogenes as a genetic hallmark. these translocations are thought to be driven by v(d)j-recombination mechanisms. we believe that these mechanisms only partly facilitate the occurrence of tcr translocations and that the accessibility of involved genes plays an intrinsic role in promoting these events. the lmo2 locus, is thought to be accessible only during the double-negative (dn) 1 and 2 thymocyte stages based on mrna expression, implying that translocations between lmo2 and tcr genes can only occur within these stages. gene expression as a readout for accessibility can not elucidate the involvement of other oncogenes such as tlx1 (hox11), which are not expressed in thymocytes, as being accessible for translocations to occur. objectives: we aimed 1) to evaluate lmo2 and tlx1 breakpoint-site accessibility during thymocyte development; 2) to determine in which stage of development there is an increased chance for lmo2 or tlx1 translocation to occur based on this accessibility. methods: dna of immunologically "healthy" sorted thymocytes was isolated using faire (formaldehyde-assisted isolation of regulatory-elements). this dna was used to quantitatively assess lmo2 accessibility during thymocyte development at both the transcription start site (tss) and negative regulatory element (nre), and within different in t-all documented breakpoint-sites of both the lmo2 and tlx1 loci. results: quantitative analysis on the tss showed a correlation with mrna expression, with the dn1 and dn2 development stages showing the highest accessibility. the nre, showed an inverse pattern of accessibility to the tss region. analysis of breakpoint-sites revealed the highest accessibility levels within the earliest stages of development, dn1, dn2, dn3 and immature single positive (isp) stages for both lmo2 and tlx1. conclusion: our findings show that both the lmo2 and tlx1 loci are accessible during thymic development irrespective of gene expression and that this accessibility is not restricted to dn1 and dn2 stages, suggesting that these loci are much more active than assumed, thus increasing the opportunity for translocations to occur. we addressed this issue, by building a model able to account for of v-ja gene rearrangements observed experimentally during thymus development of mice. we developed, based on experimental data, a numerical model on the whole tra/trd locus to estimate va and ja genes accessibility to rearrangements. the progressive opening of locus to v-j gene recombinations is modeled through windows of accessibility of different sizes and with different speeds of progression. furthermore, the possibility of successive secondary v-j rearrangements was introduced in the modeling. the model points out some unbalanced v-j associations resulting from a preferential access to gene rearrangements and from a non-uniform partition of the accessibility of the j genes, depending on their location in the locus. the model shows that 1 to 3 successive rearrangements are sufficient to explain the use of all the v and the j genes of the locus. finally, the model provides information on the kinetics of rearrangements and on the frequency of each v-j association. the model accounts for the essential features of the observed rearrangements on the tra/trd locus and may provide a reference for the repertoire of the v-j combinatorial diversity. the genetic programs of b-cell differentiation and the first dj h gene rearrangements appear in the post-gastrulation mouse embryo (e10-12), shortly after the first multipotential hematopoietic progenitors do emerge. these dj h joints represent the unselected baseline of the ig repertoires. we have undergone a systematic sequencing of embryo dj h joints obtained from normal balb/c embryos and heterozygous embryos obtained from rag2 -/mothers mated to balb/c males (to discard any mother-derived contribution), as well as newborn and adult control groups. the embryo dj h s displayed unexpected mechanisms of diversity, including short stretches of non-templated n nucleotides in one-third of the studied sequences (in the absence of tdt expression) and frequent dj h s with large nucleotide deletions, as a consequence of ligation to joint-distal microhomology sites. because the dna polymerase m (polm), a highly-homologous tdt member of the x dna polymerase family, showed an increased expression in the embryo, we analysed the dj h s of polm -/mouse embryos. we observed that polm was mainly responsible for introducing n nucleotides at the mouse embryo dj h joints. also, and based on its dna-dependent polymerization ability, polm filled-in small sequence gaps at the coding ends, and ligated highly-processed ends by pairing to internal microhomology sites, although at the cost of germline sequence losses and the generation of "useless" gene products. we think that, more than attempting to increase diversity, polm acts as a "connector" in the embryo, subsequently participating in the repair of rag-induced double-strand breaks, to preserve genomic stability and cellular homeostasis in cells with high proliferation rates. along the end of gestation, further selective pressures acting over these first v-dj h products will contribute to establish the differential neonatal ig repertoires. although mortality from infectious diseases peaks during infancy, many vaccines are ineffective in early life. most children with infantile bronchiolitis are under 6 months of age, and most cases are due to respiratory syncytial virus (rsv) infection, for which vaccines continue to be elusive. we now show that, compared to adults, the antibody response to rsv infection is very poor in neonatal mice and is unaffected by cd4 cell depletion. however, cd8 depletion in infancy led to a remarkable boosting of antibody responses during adult re-challenge. to test the possibility that poor antibody boosting is caused by rsv-specific cd8 t cells killing rsv-infected b cells, we sorted cells from the lungs of infected neonates. viral copy number was high in neonatal b cells, but viral load in surviving b cells was unaffected by cd8 cell depletion. in addition, fas ligand (fasl) deficient gld mice responded to rsv infection in the same way as normal mice, indicating that fasl is not required for the inhibition of antibody responses. this new mechanism of regulation of b cell responses by cd8 t cells has important implications for vaccine development against neonatal infections. (2008) showed that irf8 knockout mice had significantly reduced numbers of pre-pro-b cells in marrow and a phenotype similar to agammaglobulinemia. these results prompted us to consider icsbp/irf8 as a candidate gene in the pathogenesis of defective early b cell development. therefore we decide to undertake direct sequencing of the gene encoding icsbp/irf8 in a small cohort of patients with autosomal recessive agammaglobulinemia. methods: eight patients affected by agammaglobulinemia were included in this study. all patients were under regular ig replacement therapy. informed consent was obtained from all patients. genomic dna was extracted from whole blood and amplified with specific primers designed on the flanking regions of every exon. direct gene sequencing of the eight exons of icsbp/irf8 were obtained using abi prism sequencer. results: seven of the eight patients result wild type while only one patients present a synonymous snp in exon v, yet documented as rs17444416. conclusions: although recent findings indicated that irf8 function is essential for early b cell development, our data in a small cohort of patients affected with autosomal recessive agammaglobulinemia did not evidence any mutations in icsbp/irf8 that may be responsible for this disorder. the hh/ptch signaling system is known to control the development and neoplastic transformation of several cell types. however, the role of hh/ptch for the differentiation of b and t lymphocytes from hematopoietic stem cells (hsc) has not been assessed so far. to analyze the function of hh/ptch for lymphopoiesis in vivo, we have employed a genetically engineered mouse mutant in which the ptch gene can be conditionally inactivated by virtue of the cre/loxp recombination system. we show that targeted disruption of ptch in the adult organism results in a dramatic specification and differentiation defect of the lymphoid lineage leading to rapid disappearance of newly generated b and t lymphocytes from peripheral lymphoid organs. the developmental block occurs at the level of the common lymphoid progenitor cell (clp), which defines an early branching point of hsc differentiation and lineage commitment. in contrast to the lymphoid lineage, development of cell types of the myeloid lineage from common myeloid progenitors (cmp) appears normal. our data identify hh/ptch-induced signaling as a key regulator for proper development of immunocompetent lymphocytes. hence, the progression of tumors, which are initiated upon oncogenic hh/ptch mutations, may be further promoted due to impaired tumor surveillance by a compromised immune system. l. calderon dominguez 1 , t. boehm 1 1 max-planck institute for immunbiology, developmental immunology, freiburg, germany in many organ systems of animals and plants, specialized niche microenvironments maintain and specify stem and progenitor cells. the ability to modify or artificially create such niches in vivo and in vitro has many implications for stem cell research and therapy. by analysis of several mutant mouse strains and subsequent transgenesis in the mouse, we disentangle and individually modulate niche functions responsible for collection, maintenance and specification of multipotent thymocyte progenitors. we demonstrate how an epithelial niche, rendered functionally inactive by disruption of the foxn1 transcription factor, can be specifically rebuilt in a modular and combinatorial fashion to only attract, or attract and maintain, or attract, maintain and specify progenitor cells into the b and t cell lineages, respectively. the strategy of engineering niche functions in a modular fashion might be applicable to other progenitor cell systems. silencing of dc-sign using lentiviral rna interference revealed its critical function for pd-l1 expression on dcs after m. tuberculosis infection. as a counterpart to expression of its ligand, we showed that cd4 and cd8 t cells from tuberculosis patients highly express pd-1 when compared to healthy uninfected individuals. in addition, analysis of pd-1 expression in lung biopsies from tuberculosis patients revealed that pd-1 is expressed on cd4 and cd8 t cells confined to lung granulomatous lesions. finally, blocking of the pd-1/pd-l1 axis using monoclonal antibodies abrogated the down-modulation of t cell proliferation and ifn-g production induced by manlam, a mycobacterial cell wall glycolipid and ligand for dc-sign. taken together, our results suggest that the pd-1/pd-l1 pathway is involved in the exhaustion of t cell responses to m. tuberculosis. the inflammatory canonical nfkb pathway is critically involved in virtually all aspects of inflammation in general. yet, the role of the alternative, non-canonical nfkb pathway in inflammation and adaptive immunity remains largely elusive. the alternative pathway is primarily mediated through the nfkappa-b inducing kinase (nik) which in turn leads to the phosphorylation and the cleavage of p100 to p52. among the receptors engaging nik is the ltbr, which is also required to form the anlage for secondary lympoid tissues (slts). due to a point mutation within nik, alymphoplasia (aly) mice do not develop slts and are highly immunodeficient. however, while the immunodeficiency of aly mice is widely held to stem from their developmental malformation, it has been overlooked, that the mutation of nik itself could potentially lesion the development of immune responses. to verify this notion, we generated a series of bone marrow chimeric mice (bmc) in which the absence of slts was disconnected from the hematopoietic loss of nik function. we generated mice, which lack all slts, but are equipped with a normal systemic immune system (wt 1 aly), and conversely, mice with normal slts, but lacking nik in all leukocytes (aly 1 wt). surprisingly, we discovered that nik is vital for the development of autoimmune disease, while slts (ie. lns, spleen etc.) are essentially dispensable for cell-mediated immunity. we found that nik is required for the polarization of effector t cells and that th17 and th1 cells cannot be generated in the absence of nik. preliminary data implicate the involvement of nik in a discrete and novel pathway required for the formation of cell-mediated immune responses. the family of nfat (nuclear factor of activated t-cells) transcription factors is indispensable for t cells, for example playing an important role in cytokine gene regulation. in peripheral cd4 + t cells, nfatc1 and c2 are predominantly expressed. nfatc1 is synthesized in six isoforms which have partly opposing functions regarding activation and apoptosis. here we address the functional difference of the short isoform nfatc1/a, which is highly induced upon t cell activation, and the long constitutively expressed isoform nfatc1/c. as demonstrated by y2h screen and co-ips, nfatc1/c-specific c-terminus can be highly sumoylated. confocal microscopy studies revealed that upon sumoylation nfatc1/c -but not the unsumoylated nfatc1/a -translocates to promyelocytic leukemia-nuclear bodies (pml-nbs). this leads to interaction with hdacs followed by deacetylation of histones (co-ips), which in turn induces transcriptionally inactive chromatin (chip and confocal microscopy). as a consequence, multiple expression studies revealed sumoylation dependent suppression of the nfatc1 target gene interleukin-2. other lymphokines like ifng and il13 are reversely regulated. interestingly, ntreg cells which do not express il2 exerted only nfatc1/c, but no nfatc1/a expression (qrt-pcr). these findings demonstrate that the modification by sumo converts nfatc1 from an activator to a site-specific transcriptional repressor, revealing a novel regulatory mechanism for nfatc1 function. therefore, especially ntreg cells and anergized cd4 + t cells might be regulated by the long sumoylatable isoform nfatc1/c. lnk/sh2b3 and aps/sh2b2, two members of the lnk/sh2b family of adaptor proteins, play an important role as negative regulators in b cell lymphopoiesis. they possess several protein-protein interaction domains and motifs that allow their interaction with different signalling effectors. mice deficient for these proteins demonstrated that lnk inhibits expansion of pro/pre-b cells while aps controls mature b-1 cell population, suggesting specific roles for these adaptors during b cell development. however, the molecular mechanisms underlying their regulatory function in these cells, have not been identified. to address this question, we used primary and b cell lines at different stages of differentiation as our cellular system. analysis of lnk/aps expression pattern showed that lnk is expressed at all developmental stages, while aps is only detected in immature and mature cells. we then first examined the role of lnk in il-7 signalling in pre-b cells overexpression of lnk dramatically inhibits il-7-dependent growth demostrating that lnk negatively regulates il-7 pathways. furthermore, we showed that il-7 stimulation induces lnk phosphorylation and its subsequent association with important signalling effectors, notably the e3 ubiquitin ligase cbl. we next analyzed the role of aps in mature b cells by imaging and biochemical techniques. our results showed that aps colocalizes with the bcr complex after bcr triggering. interestingly, lnk is not recruted to the bcr signalosome in these cells, suggesting that interaction of the adaptors with the receptor complex regulates their function at different development stages. moreover, we showed, for the first time, that aps can associate, upon bcr stimulation, with the signalling molecules cbl and vav1. to address the functional implications of these interactions, we examined specific b cell responses, notably bcr trafficking and cytoskeleton remodelling. we demonstrated that overexpression of aps enhances ligand-induced endocytosis of bcr, possibly through interaction with cbl and affects the kinetics of bcr-induced cell spreading. our results therefore suggest a regulatory function of aps in bcr internalization and cytoskeleton dynamics. altogether, our findings demonstrate that lnk and aps display sequential specific regulatory roles during b cell development that are important for maintaining b cell homeostasis. signaling through the t-cell receptor (tcr)-cd3 complex is a critical event in adaptive immunity. it is still not clear how ligand binding to the tcr is communicated across the plasma membrane and leads to phosphorylation of the cytoplasmic domains of the cd3 complex. it is widely accepted that dimerization or multimerization of tcr is required for tcr triggering. in our model t-cell activation is initiated by recognition of monomeric mhc/peptide complexes on the surface of antigen presenting cells (apc). critical to tcr triggering is the movement of the t-cell across the apc. engagement of a mhc/peptide complex on the surface of the apc will change the mobility of the tcr leading to partitioning with lipids of lower mobility that are enriched in signaling molecules critical for t-cell activation. furthermore, the change in mobility will lead to dislocation of the itams from the plasma membrane so that they become accessible to tyrosine kinases. to test the hypothesis we established a new approach where we created a soluble bifunctional complex composed of a pmhc and a fab that recognizes an epitope tag that we express on the t-cell surface. binding of the fab to the expressed epitope tag will constrain the lateral mobility of the tcr that is engaged by the pmhc arm of the same complex. the bifunctional complexes induced activation and proliferation as well as ca influx and cytokine production in human cd4 + t-cell clones that displayed the epitope, but not in t-cells that did not display the epitope. activation required interaction of the fab with its epitope on the t-cell surface because no activation was observed when soluble epitope peptide, which acts as a competitor for the fab binding site, was added. these results demonstrate that a monomeric copy of a pmhc is sufficient to trigger tcr and that formation of a tcr dimer is not an obligatory step in t-cell activation. the bifunctional complex we generated may also have a great immunotherapeutic impact. exchanging the fab with a fab or cytokine directed to a surface molecule may allow an antigen specific stimulatory or inhibitory modulation of t-cell responses. adaptor proteins are crucial in signal transduction, cell cycle regulation, apoptosis and stress response. adaptor proteins containing characteristic sh2 or sh3 domains known to mediate protein-protein interactions are key players in these processes. sly2 (sh3 domain protein expressed in lymphocytes 2) was identified as a putative adaptor protein containing a sh3 and a sam domain as well as a bipartite nls. sly2 belongs to a family of three molecules: sly1, sly2 and sash1.in humans, the sly2 gene is located on chromosome 21, in mice on chromosome 16. sly2 is widely expressed for example in immune tissue as well as in hematopoietic cells, brain, lung and pancreas. subcellular fractionation showed that the sly2 protein is located in the cytoplasm and the nucleus and to a lesser extend in the plasma membrane.to elucidate the function of sly2 we searched for possible interaction partners by yeast two hybrid screening with a mouse t cell lymphoma library. this approach identified sin3-associated polypeptide p30 (sap30) as a putative interaction partner of sly2. sap30 is a conserved member of the sin3a-hdac corepressor complex that contains histone deacetylase 1 (hdac1) and histone deacetylase 2 (hdac2) and acts as a transcriptional repressor for a variety of genes. we confirmed this interaction by implementing coimmunoprecipitations with lysates from transiently transfected 293t cells. in addition, we could show a direct interaction between sly2 and hdac1. to investigate the functional impact of this molecular interaction, we performed hdac enzymatic activity assays. we were able to show that sly2 increases the activity of hdac1 in whole cell lysates and, more precisely, in nuclear extracts of 293t cells. the interaction of sly2 with sap30 and hdac1 indicates a transcriptional function of this protein. within the sin3a-hdac corepressor complex sly2 might act as a switch for the activity of hdac1. cd46-cyt1 and cyt2 are co-expressed in human t cells and undistinguishable from the cell surface. in order to determine their specific role in t cell activation, we have expressed chimeric proteins consisting of the extracellular domains of cd19 (b cell marker) fused to the transmembrane and intracellular domain of cd46-cyt1 or cyt2 in primary t cells. we show that these two isoforms differently control human t cell function. specific cyt1 coengagement controlled il-10 secretion, while cyt2 coligation inhibited ifng production. moreover, our preliminary data suggest that cd46-cyt2 inhibits the phosphorylation of several molecules known to be activated by cd46 stimulation. these data suggest that these two isoforms act as molecular switches for t cell activation, either promoting or turning off t cells. they demonstrate for the first time the distinct roles of cd46 cytoplasmic isoforms in primary human t cell activation. this also suggests that the modulation of their expression and/or activation might provide new therapeutic avenues. nck is a ubiquitously expressed adapter protein that is almost exclusively built of one sh2 domain and three sh3 domains. nck connects receptor and non-receptor tyrosine kinases to the machinery of actin reorganisation. in t cells, nck participates in different and interdependent signalling pathways linking t cell activation and effector function with actin remodelling proteins that in turn initiate changes in cell polarity and morphology. we previously showed that nck directs the death factor fasl to the cytotoxic immunological synapse when t cells encounter putative target cells. we now performed a systematic screening for interaction partners of the four individual interaction modules of nck in primary and leukemic t cells. we precipitated putative binding partners from untreated or pervanadate-treated pha blasts, jurkat and hut78 cells with gst fusion proteins containing full length nck, the three sh3 domains or the individual sh3 and sh2 domains. binding proteins were excised from gels after staining with coomassie, silver or flamingo pink and processed by tryptic in gel digestion for mass spectrometrical analysis. as expected, we observed major differences in nck binding proteins precipitated from resting versus activated t cells. we not only verified established interactions (e. g. with the tcr signalling components slp76 and cd3epsilon, the actin-regulatory proteins wasp and wip and the nuclear protein sam68) but also identified novel nck-interacting proteins. the interaction with the actin-binding protein hip55 once more underscores the fundamental role of nck in tcr-mediated actinreorganization. the identification of the nuclear proteins sfpq/nono points to novel, yet unknown functions of nck that might be associated with the recently reported nuclear translocation/localization of nck. accordingly, employing laser scanning microscopy, we clearly detected nck within the nucleus also in human t cells. the present data highlight that nck serves versatile functions in t cells, which include the different interdependent pathways of tcr-induced actin reorganization but also novel, yet poorly defined protein networks that are associated with a nuclear translocation of nck. cytotoxic t lymphocytes (ctl) mediated killing is tightly regulated according to the strength of t cell receptor signal. killing is regulated by the delivery of perforin-containing lytic granules moving along microtubules towards the centrosome, which polarizes and docks at the central supramolecular activation complex (csmac) within the immunological synapse. although much has been learnt about the mechanisms controlling the strength of tcr signal and the mechanisms required for release of the lytic granules, little is known about how the strength of the tcr is able to control the degree of ctl-mediated killing so finely. here we examine how the strength of tcr signal controls polarization of the secretory apparatus leading to ctl-mediated killing using tcr transgenic ot-i ctl. decreasing the tcr signal by reducing the concentration of ova peptide or using the weak agonist peptide, g4, results in a slight reduction in the number of ctl target cell conjugates formed, and the number of conjugates in which a csmac (visualized by a patch of lck-staining at the immunological synapse) was formed. tcr signals result in reduced or absent (in the case of g4) staining with psrc and perk antibodies in the immunological synapse and reduced or absent (g4) degranulation as measured by cd107a assays. the centrosome docks at the csmac of the immunological synapse even with relatively weak tcr signals, but the lytic granules require a certain threshold of signaling to successfully polarize to the immunological synapse. inhibitors support a role for pi3k in granule polarization. together these data demonstrate that the strength of tcr signal controls the level of ctl mediated killing at the single cell level by controlling, the number of conjugates formed, the formation of the csmac and the accumulation of psrc and perk at the synapse. the centrosome polarizes to the csmac even with relatively weak tcr signals, but granule recruitment requires a higher threshold of signaling. these findings reveal how ctl can fine tune the degree of killing in response to tcr signals at the single cell level. cytotoxic t cells play an essential role in the immune system, particularly in the elimination of tumor and virus-infected cells. cytolytic t-cell activity is mediated through the pore-forming molecule perforin allowing granzymes to enter the target cell and to initiate apoptosis. perforin and granzymes are stored in specialized secretory granules, called secretory lysosomes. they are capable of undergoing regulated secretion in response to a t cell receptor engagement which involves binding to a cognate mhc class i-peptide complex. the intracellular transport of lysosomal proteins from the golgi to the lysosomes is mediated by the cationindependent mannose-6-phosphate receptor which exhibits structural and functional similarity to the vps10p-receptor sortilin. sortilin was characterized predominantly in neuronal cells where its function in protein sorting was identified. in the secretory pathway, sortilin is putatively involved in trafficking of proteins in the constitutive and regulated pathway. to explore whether sortilin has a broader functional relevance, we asked if sortilin might act as an alternative receptor for the cation-independent mannose-6-phosphate receptor in cytotoxic t cells. first, we demonstrate that sortilin is expressed in t cells. to examine its function during an adaptive immune response, we analysed sortilin-deficient cytotoxic t cells derived from a knockout mouse strain. in strong contrast to the results reported from neuroendocrine cells, we obtained a reverse phenotype in sortilin-deficient cytotoxic t cells. whereas the regulated release of secretory lysosomes was enhanced, the constitutive release of interferon-g was found to be decreased. the enhanced release of cytotoxic molecules from sortilin-deficient cytotoxic t cells translated into an increased cytotoxicity in vitro. thus, the deletion of sortilin imposed a specific phenotype in cytotoxic t cells which could not be compensated for by other sorting receptors. our localisation studies of sortilin in t cells were consistent with the results previously described in neuronal cells which indicated that sortilin acts as a sorting receptor during the anterograde transport of lysosomal hydrolases from the trans-golgi-network to endosomes and lysosomes. taken together, we suggest that sortilin might play a modulatory role in the regulation of the adaptive immune response through the control of the constitutive and regulated secretory pathway. there is growing interest in the soluble splice variant of ctla-4 (sctla-4) as an immune inhibitor secreted by t cells, because genetically determined variation in its production is associated with susceptibility to autoimmune disease. however, little is known of the biology of sctla-4 in immune responses. using a specific anti-human soluble ctla-4 monoclonal antibody, jmw-3b3 that selectively binds the soluble isoform but not membrane bound ctla-4, or cleaved fragments of it, we demonstrate that sctla-4 plays a vital role in regulating antigen-specific immune responses. we used antibody blockade to show that antigen-specific t cell responses are strongly enhanced upon blockade of sctla-4, secreting increased amounts of cytokines including interferon-g, il-17 and tnf-a, but lower amounts of il-10. soluble ctla-4 was also prepared from sera for use in experiments by antibody based affinity purification techniques. addition of sctla-4 induced secretion of the immunoregulatory cytokine il-10 by human pbmcs both in an antigen-selective and dose-dependent manner, while antibody blockade abrogated that effect. the immunosuppressive indoleamine 2,3 dioxygenase enzyme cascade was also initiated by sctla-4. it is clear that the importance of this natural soluble molecule has been overlooked and like membrane-bound ctla-4 it is crucial to t cell inhibition. membrane-bound ctla-4 exists as a homo-dimer on t cells but sctla-4 is usually considered to be monomeric in form, implying its functional capacity is diminished because of an inability to cross-link b7 ligands on antigen presenting cells. a third important observation from this study is that sctla-4 exists both in serum and culture supernatants as a natural 46kda homo-dimer, and not as a monomer. this goes some way to explaining why this molecule has such potent immunoregulatory effects on antigen-specific immune responses. together, these results lead us to reappraise sctla-4, concluding it to be a mediator of negative feedback, secreted as a recall regulatory t cell response to antigenic stimulus, rather than a product of resting t cells. this work also raises the possibility that where il-10 dependent regulation is most critical, boosting sctla-4 secretion by regulatory t cells could be a novel therapy for immune mediated diseases. recently, we identified a new adaptor protein, swiprosin-1/efhd-2, in lipid rafts of b cell lines that undergo apoptosis after b cell receptor (bcr) stimulation. swiprosin-1/efhd2 is expressed in immature b cells of the bone marrow, in resting and activated splenic b cells, in t cells, macrophages, mast cells and some nonlymphoid tissues. ectopic expression of swiprosin-1/efhd-2 in the immature murine b cell line wehi231 enhanced spontaneous and bcr-induced apoptosis. in contrast, shrna-mediated down-regulation of swiprosin-1/efhd-2 impaired spontaneous and bcr-elicited apoptosis, but not bcr-induced g1 cell cycle arrest. to understand how swiprosin-1/efhd2 enhances pro-apoptotic bcr signals, we analyzed whether swiprosin-1/efhd2 is involved in proximal bcr signalling. in fact, ectopic expression of swiprosin-1/efhd2 enhanced bcr-induced calcium flux in wehi231 cells, whereas shrna-mediated down-regulation of swiprosin-1/ efhd2 impaired bcr-elicited calcium signals. concomitantly, gst-pulldown experiments revealed that swiprosin-1/efhd2 interacts with phospholipase cg2 (plcg2) and with the tyrosine kinase syk (splenic tyrosine kinase), both of which are important for bcr-induced calcium flux. the interaction of plcg2 and swiprosin-1/efhd2 was further established by co-immunoprecipitation. reconstitution of bcr-elicited calcium signals through complementation of swiprosin-1/efhd2 silenced wehi231 cells with swiprosin-1/efhd-2 was inhibited by the syk inhibitor bay 61-3606. in analogy, swiprosin-1/efhd2 regulated syk activity positively. moreover, swiprosin-1/efhd2 re-expression accelerated tyrosine phosphorylation of several proteins, specifically tyrosine phosphorylation of plcg2 and of syk tyrosine residue 352, which is involved in syk activation. finally, reconstitution of swiprosin-1/efhd2 knock-down cells with swiprosin-1/efhd2 mutants revealed that the n-terminal putative sh3-binding site, the first ef-hand, and to a lesser extent, the second ef-hand and the c-terminal coiled-coil domain, are important for bcr-induced calcium flux in wehi231 cells. interestingly, swiprosin-1/efhd2 re-expression in swiprosin-1/efhd2-silenced cells induced already in unstimulated cells raft partitioning of syk, plcg2 and the bcr, which was reversed after 2 min of bcr stimulation. in summary, swiprosin-1/efhd2 is an accelerator of proximal bcr signalling and acts through syk and plcg2 by assembling a syk-dependent calcium initiation complex in lipid rafts. this might be relevant for memory b cell signalling or central b cell tolerance. to test the biological relevance of cbl-b e3 ligase activity, these mice were analyzed for t cell proliferation, susceptibility to autoimmunity, in vivo t cell tolerance responses, and tc1 tumor rejection. results: when stimulated, t cells from rf mutant mice hyperproliferate compared to wild type t cells, even in the absence of cd28 co-stimulation. preliminary data also suggest that rf mutant mice are more susceptible to autoimmunity. in addition, rf/p14 mice die within hours after a second challenge with p33 peptide, indicating a severe defect in t cell tolerance induction. more importantly, cbl-b e3 ligase dead mice can spontaneously reject tc1 tumors. conclusion: cbl-b e3 ligase dead mutant mice phenocopy total body cbl-b knock out mice, thus indicating that cbl-b e3 ligase activity is indispensable for its regulatory in vivo functions. intriguingly, our data suggest that its inactivation could be sufficient to confer anti-tumor activity. to further elucidate the cellular mechanism of cbl-b mediated tumor rejection we have now generated the conditional cbl-b e3 ligase dead mutant mice to for the first time study the cbl-b ubiquitination function in a tissue specific and temporal fashion. our research is also currently focused on identifying the relevant in vivo cbl-b ubiquitination substrates. interferon alpha (ifn-a) has been broadly used in the treatment of specific malignancies and chronic viral diseases. for a long time it was thought that the direct inhibitory effects on malignant or virus infected cells were the major mechanisms involved in the response to ifn-a therapy. however, recent studies in mice have revealed that ifn-a/b also exerts effects on several host immune cells. ifn-a has been shown to enhance cd8 t cells (ctls) responses against soluble antigens in mice. this immunostimulatory activity of ifn-a results at least partly from its direct ability to induce maturation of dendritic cells. several studies have recently demonstrated that ifn-a/b also acts directly on murine ctls, inducing clonal expansion and differentiation into effector and memory cells. to date, little is known about the effects of ifn-a on human ctls. to approach this issue, magnetically sorted untouched human cd8 + cd45ro -t cells (mainly naï ve cells) were unstimulated or stimulated with human ifn-a and gene expression profiles were compared using an affymetrix human array. interestingly, ifn-a stimulation of highly purified human ctls without any other concomitant signals remarkably enhanced the expression of several molecules involved in death receptor signalling (trail) and chemotaxis (ip10 and itac). in a second genome-wide array analysis, we analyzed the effects of ifn-a on human ctls responding to antigen (signal 1) and co-stimulatory signals (signal 2), provided by beads coated with anti-cd3/cd28 antibodies. gene expression patterns were compared for cells stimulated with anti-cd3/cd28 beads alone or along with ifn-a. ifn-a regulates the expression of a number of genes that promote proliferation, activation and survival of ctls, tcr stabilization, chromatin remodelation, and, importantly, enhances the expression of genes involved in ctls effector functions (granzyme-b, ifn-g, trail, fasl) and chemotaxis (ip10, itac). the enhanced expression of granzyme-b, ifn-g, trail and ip10 were further confirmed at the protein levels by flow cytometry analysis and/or elisa. enhancement of granzyme-b-and trail-mediated cytolitic functions was also found by functional assays using anti-cd3-coated p815 cells and trail-sensitive caki-i cells as targets. our results show that ifn-a provides a strong signal-3 to human ctls leading to their differentiation into effector ctls. t cell activation is an important process of the adaptive immune system, which requires recognition of mhc-associated antigens by antigen presenting cells (apcs) via the t cell receptor (tcr). to induce a productive t cell response the interaction of t cells with apcs needs to be stabilized by adhesion molecules. junction adhesion molecules (jams) are a recently discovered group of immunoglobulin (ig) superfamily proteins, which are involved in the regulation of various inflammatory and vascular events. the third member of the jam protein family, jam-c, is highly expressed in platelets and endothelial cells, whereas expression in t cells is largely unknown. to investigate the regulation of jam-c in t lymphocytes, we determined jam-c gene expression in quiescent and activated human t cells. treatment with the polyclonal t cell activator phytohemagglutinin (pha) increased surface and total jam-c expression in t cells time-and dose-dependently, as determined by flow cytometry and immunoblot analysis. by contrast, no up-regulation of jam-a in activated t cells was detectable. the highest level of jam-c up-regulation by pha was observed in cd3 + foxp3 + and cd4 + cd25 high t cells. moreover, t cell receptor activation with combined anti-cd3 and anti-cd28 stimulation induced jam-c expression in t cells. jam-c induction occurred at the mrna level suggesting a transcriptional regulatory mechanism of jam-c expression. accordingly, we studied the regulation of the human jam-c gene promoter in transiently transfected t cells. luciferase activity of a jam-c promoter gene construct with three potential consensus sites for the transcription factor nfat was markedly induced in activated t cells. finally, pretreatment with two pharmacological inhibitors of calcineurin, cyclosporin a and fk-506, but not with mapk inhibitors, blocked jam-c induction in activated t cells. in summary, the present data indicate that jam-c is induced in activated human t lymphocytes via a transcriptional mechanism and suggests a major regulatory function of jam-c for the t cell response. hiv-1 infection leads to immune dysfunction owing to a successive loss of the cd4 + t cell compartment. the molecular mechanisms underlying this depletion are not well-understood but may involve the viral nef protein. nef is a multifunctional accessory protein that is required for full hiv-1 virulence and the maintenance of high viral loads. nef enhances viral infectivity and replication by downregulating cell surface receptors, e. g. cd4 and mhc class i, and modulating signal transduction pathways. the latter is thought to raise the cellular activation level and in this way may increase the infected cell's susceptibility to apoptosis. in this study we identify a signaling complex assembling at the n-terminus of nef, which contains the kinases lck and pkcv. formation of this complex, termed nakc for nef-associated kinase complex, led to activation of lck, as assessed by in-vitro kinase assay, and recruitment of pkcv to membrane rafts, as detected by discontinuous sucrose density gradient ultracentrifugation. recruitment of pkcv to membrane rafts is a hallmark of t cell activation and has been associated with activation of the nfxb transcription factor. however, contrary to our expectations, nef-mediated nakc formation did not activate nfxb. instead, it led to a strong induction of erk1/2. this correlated with a nakc-mediated increase in hiv transcription that was demonstrated by luciferase reporter assays suggesting that erk1/2 directly targets hiv transcription, possibly via induction of transcription factors. to our surprise, however, the effect of nakc on hiv transcription was found to be independent of ap-1, nfat and nfxb suggesting an alternative mechanism of nakc-mediated enhancement of hiv transcription. on the basis of our previous results we propose that nef enhances hiv transcription via removal of inhibitory factors and thus derepression of the hiv promoter. how erk1/2 is involved in this mechanism and whether nakc targets other cellular promoters, which may enhance the cellular activation level and thus sensitize the cell to apoptosis, remains to be determined. p. otahal 1 , t. brdicka 1 , v. horejsi 1 1 institute of molecular genetics as cr, praha, czech republic aims: c-terminal src kinase (csk) and cd45 are key regulators of src-family kinases in leukocytes. while cd45 is a transmembrane phosphatase, csk is localized mostly in cytosol. however, a fraction of csk is found at the cell membrane and in lipid rafts where it inhibits signaling by phosphorylating inhibitory tyrosine of src-family kinases. currently, it is accepted that sh2 domain of csk binds phosphotyrosine 317 of transmembrane adaptor protein pag and via this interaction is recruited to the cell membrane and lipid rafts. however, pag knock-out mice still have cell membrane-associated csk and do not show any apparent dysregulation of signaling which would be expected due to the low levels of membrane csk. thus, the mechanisms of membrane targeting of csk remain unclear. to analyze the role of membrane and lipid raft targeting of csk on lymphocyte signaling we targeted csk to different membrane compartments by fusing csk with transmembrane domains of lat, lax, cd25 and n-terminal part of src kinase. methods: csk chimeras containing n-terminal membrane targeting motif and c-terminal orange fluorescent protein were cloned into retroviral vector pmxs. jurkat t cells expressing individual constructs were subsequently prepared and analyzed for the inhibitory effect of these csk chimeras on t-cell receptor (tcr) signaling by measuring calcium flux and cd69 upregulation. the efficiency of inhibition depended on the membrane targeting motif, while lat-csk chimera completely inhibited tcr signaling and src-csk chimera inhibited the signaling only partially; lax-csk and cd25-csk chimeras showed almost no inhibition of tcr signaling despite efficient presence at the plasma membrane. conclusions: our data demonstrate that the function of csk strongly depends on its targeting to the specific areas of plasma membrane. it also strongly supports the idea that membrane compartmentalization is critical for regulation of t-cell signaling. peripheral cd8 t cell tolerance can be generated outside lymphatic tissue in the liver. however, the course of events leading to tolerogenic interaction of hepatic antigen presenting cells with circulating t cells is unclear. here, we demonstrate that systemically circulating antigen was preferentially taken-up by liver sinusoidal endothelial cells (lsec) and not by other antigen presenting cells in the liver or spleen. uptake and cross-presentation of circulating antigen was followed by rapid antigen-specific naï ve cd8 t cell-retention in the liver but again not in other organs. using bone-marrow chimeras and tie-2kb mice, we could show that antigen cross-presentation by lsec was both essential and sufficient to cause antigen-specific t cell-retention under non-inflammatory conditions, which was followed by cd8 t cell proliferation and expansion, but ultimately led to the development of t cell tolerance. our results show that cd8 t cell tolerance towards circulating systemic antigens is predominantly generated in the liver by lsec, which preferentially take-up and cross-present circulating proteins to cd8 t cells, leading to their rapid local antigen-specific retention and subsequent tolerisation. these insights broaden our understanding not only of physiological immune regulation towards circulating antigens but also of therapeutic manipulation of cd8 t cell responses. alphapix is a rho gtpase guanine nucleotide exchange factor domain-containing signaling protein that associates with other proteins involved in cytoskeletalmembrane complexes. it has been shown that pix proteins play roles in some immune cells, including neutrophils and t cells. in this study, we report the immune system phenotype of alphapix knockout mice. we extended alphapix expression experiments and found that whereas alphapix was specific to immune cells, its homolog betapix was expressed in a wider range of cells. mice lacking alphapix had reduced numbers of mature lymphocytes and defective immune responses. antigen receptor-directed proliferation of alphapix deficient t and b cells was also reduced, but basal migration was enhanced. accompanying these defects, formation of t-cell-b-cell conjugates and recruitment of pak and lfa-1 integrin to the immune synapse were impaired in the absence of alphapix. proximal antigen receptor signaling was largely unaffected, with the exception of reduced phosphorylation of pak and expression of git2 in both t cells and b cells. these results reveal specific roles for alphapix in the immune system and suggest that redundancy with betapix precludes a more severe immune phenotype. s. merluzzi 1 , s. parusso 1 , b. frossi 1 , g. gri 1 , c. pucillo 1 1 university of udine, dstb, udine, italy in this study, we investigated whether primary mcs could modulate the activation and proliferation of primary b cells. we performed co-culture assays using mouse splenic b cells and bone marrow-derived mcs. naï ve and activated b cells proliferation could be induced by nonsensitized mcs while an increase in b cell proliferation was observed when mcs are activated. moreover, b cell proliferation was partially abolished when mcs and b cells were separated by the transwell membranes suggesting that cell-cell contact is important in this event. using both il-6 -/-mcs and anti-il-6 receptor antibody, we demonstrated that in co-culture of primary b cells and mcs, il-6 derived from activated mcs is a key cytokine implicated in the b cell proliferation. moreover, we showed that activated mcs can influence the surface expression of costimulatory molecules as cd40 on naï ve b cells and the interaction of cd40 on b cell surface and cd40l on mcs is important for the further differentiation of b cells to plasmacells. indeed, we presented for the first time evidence that cytokines produced by activated mcs and interaction between cd40l e cd40 on mc and b cells respectively can contribute to differentiate mature b cells to iga secreting cells. in conclusion, in the present report, we showed a novel role of mcs as promoter of both the survival and activation of naive b cells and of the proliferation and further differentiation of activated b cells through soluble factors production and cell-cell contact, suggesting that mcs can contribute to the regulation of specific immune response. e. fourmentraux-neves 1 , n. bercovici 1 , a. caignard 1 1 inserm u567, paris, france inhibitory killer ig-like receptors (kir2dl1-2/3) which bind to hla-c molecules are expressed by human natural killer cells and effector memory cd8 + t cell subsets. these receptors suppress cd8+ t cell activation through recruitment of the src homology 2 domain-containing protein tyrosine phosphatase 1 (shp-1). to further analyse the yet largely unclear role of inhibitory kir receptors on cd4+t cells, kir2dl1 transfectants were obtained from a cd4 + t cell line and primary cells. the transfection of cd4 + t cells with kir2dl1 dramatically increased the t cell receptor (tcr)-induced production of il-2 independently of ligand binding, but inhibited tcr-induced activation after ligation. kir-mediated tcr activation requires intact itim motifs, involves kir2dl1-itim phosphorylation, shp-2 recruitment, zap-70 and pkc-v phosphorylation. synapses leading to activation were characterized by an increase in the recruitment of p-tyr, shp-2 and p-pkc-v but not of shp-1. in contrast, the kir2dl1/hla-cw4 interaction led to a strong synaptic accumulation of kir2dl1 and the recruitment of shp-1/2, inhibiting tcr-induced il-2 production. kir2dl1 may induce two opposite signaling outputs in cd4 + t cells, depending on whether the kir receptor is bound to its ligand. these data highlight unexpected aspects of the regulation of t cells by kir2dl1 receptors. b cell receptor (bcr) binding by antigen initiates activating signaling cascades and facilitates the exposure of specific b cells to powerful co-stimulatory signals, such as t cell help or toll-like receptor ligands. the role of bcr binding in modulating the access to these second signals is complex and varies between stimulatory conditions. by quantitative tracking of b cell responses in vitro we can measure which signals affect b cell proliferation or differentiation, or both, and thereby establish a novel understanding of how b cells respond appropriately to different combinations of stimuli. we utilised hel-specific bcr transgenic sw hel mice to assess the effect of a specific antigen signal on b cell responses to the t-independent mitogen lipopolysaccharide (lps). the presence of antigen renders a greater proportion of cells responsive to lps stimulation and profoundly influences effector cell differentiation. antibody secreting cell formation is dramatically inhibited by hel, but we found that isotype switching to igg1 is strongly upregulated. both of these alterations to differentiation outcomes occur independently to the proliferative effects induced by antigen. when b cells are exposed to antigen for a limited period of time, switching to igg1 still occurs but some capacity to differentiate to antibody secreting cells is recovered, leading to effective secretion of igg1 antibody during these conditions. the observed igg1 switching behaviour mimics that of b cells responding to lps and il-4, but is mediated by a different, stat6-independent pathway. these data are indicative of the important role specific antigen signals play in regulating b cell responses in stimulatory environments. a. quintana 1 , c. schwindling 1 , m. pasche 2 , c. junker 1 , c. kummerow 1 , u. becherer 2 , e.c. schwarz 1 , j. rettig 2 , m. hoth 1 1 saarland university, biophysics, homburg, germany, 2 saarland university, physiology, homburg, germany the adaptive immune response requires the interaction between antigen-presenting cells and t cells. this cell-cell interaction, called the immunological synapse (is), facilitates the activation of t cell receptor-mediated signalling cascades including a rise of cytosolic calcium through the activation of crac/orai1 channels. to allow sustained activity of crac/orai channels, the calcium-dependent inactivation of the channels through local calcium microdomains has to be prevented. objectives: the purpose of the study was to analyze local and global calcium signals in t cells and to test the hypothesis that the is controls these signals through mitochondrial positioning. methods: we used different microscopy techniques including very fast wide-field microscopy with subsequent deconvolution, total-internal reflection microscopy, and confocal microscopy in combination with electrophysiological techniques in primary human t helper cells and cell lines. to test the statistical significance of our data, we used two-sided student t-tests or non-parameterized tests. results: following is formation, we found that mitochondria translocated to the is in a calcium-dependent way. the distance between mitochondria and the plasma membrane at the is was lower than 150 nm. following accumulation at the is, mitochondria limited calcium entry to the orai channels localized right at the is by preventing their calcium-dependent inactivation. in contrast, no calcium influx was observed at sites where no mitochondria were accumulated as orai channels were inactivated at these sites. mitochondrial positioning at the is thus induced local calcium influx at the is without the necessity to enrich orai channels at the is. mitochondria took up calcium at the is distributing it further into the cytosol by releasing it at different sites, which kept the local domain at the is low enough to prevent calcium dependent orai inactivation and to prevent excessive calcium clearance by the calcium aptases in the plasma membrane, which could inhibit an efficient t cell activation. conclusion: mitochondria positioning at the is controls local calcium entry through orai channels. mitochondria prevent orai inactivation and excessive calcium clearance at the is to facilitate calcium-dependent t lymphocyte calcium signalling. we aimed to determine the functional correlates of cd4 + t cell tolerance and immunity in vivo. ovalbumin (ova)-specific transgenic cd4 + t cells were adoptively transferred into syngeneic mice immunized with soluble ova protein ± lipopolysaccharide (lps) by the i. v. route, and analyzed for a variety of immunological parameters over a period of 21 days. under tolerogenic conditions (ova alone), cd4 + t cells showed substantial early activation, but their activation profile differed markedly, both in magnitude and quality (icos, 4-1bb), from t cells activated by ova+lps. this difference in activation also translated into differing cd4 + t cell expansion and contraction kinetics in the early phase of the t cell response (days 1-6). in the late phase of the primary response (days 7-21), under immunizing conditions, the large majority of transgenic cd4 + t cells in the spleen developed into mature effectors with a prominent capacity to secrete il-2, ifn-g, and il-17a, and only few ova-specific foxp3 + regulatory t cells ( x 10 %) were observed. germinal centers were prominent and ova-specific ig of all isotypes were generated. in contrast, under tolerizing conditions, antigen-specific cd4 + t cells failed to migrate into the b cell follicles, but production of ova-specific igm was nevertheless observed. in these animals, the proportion of splenic ova-specific regulatory t cells (30 %) was substantially increased. on day 14, both groups of mice were re-challenged via the airways with ova+lps to functionally assess their immune status. in tolerized animals, the transgenic t cell population in the lung infiltrate was composed of ova-specific regulatory t cells (50 %) or t cells with a reduced capacity to secrete effector cytokines. in contrast, in immunized animals, this population almost exclusively consisted of cd4 + effector t cells with a pronounced inflammatory cytokine profile (ifn-g, il-17a). with this model we provide a comprehensive analysis of the many functional correlates of "immunity" versus "tolerance" to soluble protein antigen in vivo. we identify and characterize a number of the key players (cell surface molecules, cytokines, cell subsets) representing the decision between immunity and tolerance in the immune system. mast cells (mcs) are well-recognized as key effector cells in immunoglobulin e (ige) -associated immune responses and as prototypic regulators of innate immunity. the characteristics, importance, and molecular requirements for interactions between mast cells (mc) and cd8 t cells (tc) remain to be elucidated. using myelin/oligodendrocyte glycoprotein (mog), we demonstrated that mcs induce antigen-specific cd8 tc activation and proliferation. the antigen crosspresentation by mcs induces the secretion of interleukin-2, interferon-g and macrophage inflammatory protein-1 by cd8 tc. in vivo evidence that mcs modulate t cell responses has been obtained so far in the murine experimental autoimmune encephalomyelitis (eae), the standard animal model for multiple sclerosis, in which both cd8 tc and mcs are now recognized as key players. one of the main central nervous system (cns) antigens recognized by autoreactive tc in eae is the myelin oligodendrocyte glycoprotein (mog). to investigate the in vivo-relevance of the identified mc-cd8 tc interactions, we have employed the eae as a model of organ specific autoimmune disease in wild type mice and mc-deficient w/w sh mice. wt and w/w sh mice were immunized with the mog 35-55 protein. our results provide direct evidence that mc contribute to cd8-specific priming in eae and show that the tc proliferation failure is specific for cd8 tc from mog 35-55 -immunized w/w sh mice. the role of mc-cd8 tc interaction in induction of autoimmunity will be further investigated in eae. in summary, we provide the first evidence that mcs regulate antigen-specific responses of primary cd8 tc in vitro and in vivo. our study further supports the emerging concept that mcs, protagonists of innate immunity are also important regulators of adaptive immune responses and corresponding cd8 tc responses. this newly uncovered mc function might be of great biological relevance in situations where effector cd8 tc are critically involved, e. g. viral infections or infections with intracellular pathogens and/or autoimmune diseases such as multiple sclerosis. activation of resting t cells in vitro is triggered by combined t cell receptor (tcr) and cd28 engagement and can be modulated by simultaneous ligation of various other surface receptors. although the fasl is best known for its capacity to initiate cell death in fas-bearing cells, it has recently been implicated in the regulation of t cell activation. thus, a crosstalk between the tcr and fasl is likely, but far from being biochemically elucidated. we report that fasl engagement by immobilized but not soluble fasfc fusion protein and anti-fasl antibodies blocks the activation of primary human peripheral t cells even in the presence of cd28 costimulation at the level of an early signal initiation. inhibition is thus associated with a reduction of tyrosine phosphorylation of a number of key elements in tcr signal transduction and also with a lowered calcium response. the data presented stress the importance of the fas/fasl-system for signal initiation via the tcr/cd3complex and provide further arguments for a retrograde signaling capacity of fasl or a crucial role of fas as a costimulatory molecule. golgi network (tgn). moreover, trim specifically associates with the cytoplasmic tail of ctla-4, but not via any conventional motifs in this region. overexpression of trim augments ctla-4 surface expression, whereas down-regulation of trim expression by shrna results in disturbed ctla-4 localisation, mainly restricted to the tgn. ctla-4 vesicles and surface expression were significantly reduced but not abolished, suggesting that other factors are involved in ctla-4 trafficking. here, we identify additional transmembrane adapter protein (trap) family members as novel binding partners and regulators of ctla-4 expression. although there is some redundancy amongst traps, our results highlight the importance of this family of proteins in ctla-4 transport to the cell surface. it is imperative to reveal the mechanisms by which ctla-4 is transported to the cell surface, given that minor changes in expression can have major effects on t-cell function and in the development of autoimmunity. natural killer t (nkt) cells are found within the liver and are known to exhibit immune regulatory function. upon recognition of glycolipids presented on cd1 molecules, nkt cells are activated and release cytokines, including ifn-g, il-2 and il-4. nkt cells are efficiently recruited to the liver via cxcr6-dependent chemotaxis toward cxcl16 and constitute a large proportion of the liver-resident lymphocytes. we have previously shown, that liver sinusoidal endothelial cells (lsec) can scavenge circulating soluble antigens, and can cross-present these antigens to naive cd8 t cells. cross-presentation leads to initial t cell activation and expansion, but ultimately these cd8 t cells are rendered tolerant. as both naive t cells and nkt cells come into close contact with lsec in the hepatic sinusoids, we investigated whether nkt cells can modulate cd8 t cell tolerisation via interaction with lsec. to this end we analysed cd1d expression on lsec and their ability to activate nkt cells by presentation of the cd1d-binding glycolipid a-galactosylceramide (agalcer). we found that lsec express functional cd1d, as agalcerpresenting-lsec were capable to induce tnf-a, il-2, il-4 and ifn-g production in nkt cells in vitro. the interaction of agalcer-presenting-lsec with nkt cells led to the upregulation of cd54 and b7-h1 on lsec. as naï ve cd8 t cell tolerisation by lsec critically depends on b7-h1, we hypothesise that hepatic nkt cell activity may contribute to the immunological capabilities of the liver by regulating the tolerogenic function of lsec. improved antibody responses by class-switched memory b cells require enhanced signaling from their antigen receptor (bcr). however all bcr classes on naïve and antigen-experienced b cells utilize the canonical iga/igb subunit for signaling. we identified the signal amplification mechanism of the igg-and ige-bcr. for these isotypes tyrosine-based signaling is not confined to iga/igb but extends to a conserved tyrosine residue in the cytoplasmic segments of immunoglobulin heavy chains. the phosphorylated immunoglobulin tail tyrosine recruits the adaptor grb2 in order to sustain protein kinase activation and generation of second messengers causing robust cellular proliferation. hence membrane-bound igg and ige not only recognize antigen but also exert bcr-intrinsic costimulation to render memory b cells less dependent on t cell help for activation. objectives: the majority of circulating human gd t cells harbor tcr containing vg9, jg1.2, and vd2 gene products. they recognize nonpeptide antigens like (e)-4hydroxy-3-methylbut-2-enyl pyrophosphate derived from pathogenic microbes and isopentenyl pyrophosphate (ipp) in malignant cells. recently, we and others found out that gd t cells express a variety of costimulatory molecules including icos, and pd-1. one of the inhibitory receptors, pd-1, is a member of cd28/ctla-4 family and contains a single ig v-like domain in its extracellular region. pd-1 can bind to two b7 homologue molecules, pd-l1 and pd-l2. it has been reported that interaction of pd-1 with its ligands resulted in peripheral immune regulation and tolerance in ab t cells. in this study, we show that pd-1 is expressed on activated human gd t cells and regulates the effctor functions of gd t cells. methods: peripheral blood mononuclear cells were resuspended in yssel's medium and stimulated with 2-methyl-3-butenyl-1-pyrophosphate plus il-2 to obtain gd t cells. pd-l1+ and pd-l1-human tumor cell lines were established from cancer patients. in order to prepare anti-pd-l1 mabs, the pd-l1 extracellular domain was expressed in e.coli as inclusion bodies and refolded in the standard arginine-based buffer. mice were immunized with the refolded protein and mabs were established. to determine the function of pd-1 in gd t cells, we determined cytokine production and cell mediated tumor lysis by activated gd t cells in the presence of inhibitors of pd-1/pd-l1 interaction. results: gd t cells expressed pd-1 upon simulation with nonpeptide antigens and many tumor cell lines expressed pd-l1. we first examined whether or not the engagement of pd-1 receptor could modulate the cytotoxic activity of gd t cells. pd-l1-expressing tumor cells tempered cytotoxic activity of pd-1+ gd t cells, and cytokine production such as tnf-a was down-regulated by pd-1 engagement. in addition, inclusion of anti-pd-l1 mab reversed cytotoxic activity and cytokine production when pd-l1-expressing tumor cells were challenged by pd-1-expressing gd t cells. conclusion: pd-1 delivers inhibitory signals in gd t cells upon engagement with pd-l1. peripheral tolerance plays an important role in preventing t lymphocyte responses to self or harmless antigens. one of the mechanisms that contribute to this form of tolerance is anergy, which is characterized by a lack of proliferation and il-2 production by t cells in response to antigenic challenge. the acquisition of the anergic phenotype is an active process, with negative regulators of t cell signalling being induced. among these are the e3 ubiquitin-protein ligases which recognize target proteins for ubiquitination and catalyse the transfer of ubiquitin to them, directing them to the proteasome or to the endosome-lysosomal pathway, and hence downregulating their activity. the e3 ubiquitin-protein ligases cbl-b, itch and grail have been shown to be upregulated in anergy and to ubiquitinate and downregulate tcr signalling elements. our objectives were to determine the expression of cbl-b, itch and grail in antigen-specific cd4 + t cells in both the induction and maintenance phases of anergy, in vitro and in vivo, and to investigate their functional signalling role(s) in the maintenance of the tolerance phenotype. in order to accomplish these objectives we induced priming or tolerance of ovalbumin (ova 323-339 peptide)-specific t cells from do11.10 tcr transgenic mice in vitro or, following adoptive transfer of near physiologically relevant numbers of such cells into recipients, in vivo and correlated functional outcome (via proliferation and cytokine readout assays or antibody production) with e3 ubiquitin-protein ligases expression and the ubiquitination status of the tcr signalling machinery. cbl-b, itch, grail and target ubiquitination status, in terms of tissue, cellular and subcellular protein expression, modification and localisation, were assessed by a combination of immunoprecipitation and western blotting studies. moreover, we have performed quantitative analysis at the single cell level by tracking such antigen-specific cells in vitro and in vivo by using laser scanning cytometry. our current work focuses on the functional consequences of adenoviral transfection of such antigen-specific t cells by mutant e3 ligase-, signal target-and ubiquitin-constructs. collectively, these approaches have facilitated the dissection of the potential differential roles of ubiquitin signalling in priming and tolerance of antigen-specific t cells. s. j. keppler 1 , p. aichele 1 1 immh, university freiburg, immunology, freiburg, germany interleukin 12 (il-12) is produced by cells of the innate immune system during infection and plays an important role in controlling various pathogens. it was postulated recently that il-12 has a direct influence on cd 8 + t cells in vitro, enhancing expansion and the development of effector functions as a third signal, additionally to tcr engagement (signal 1) and costimulation (signal 2). we analysed direct il-12 signaling to cd8 t -12 signaling exhibited normal degranulation activity, cytolytic functions, ifn-g and tnf-a production. however, cd8 t cells lacking il-12 signaling failed to up-regulate klrg1 and to down-regulate cd127 in the context of listeria but not viral infections. thus direct il-12 signaling to cd8 t cells determines the cell fate decision between short-lived effector cells (slecs) and memory precursor effector cells (mpecs), dependent on the pathogen-determined local cytokine milieu. cd8 + t lymphocytes are required for effective host defense against pathogens but also for mediating effector responses against uncontrolled proliferating self tissues. we could now reveal that individual cd8 + t cells are tightly controlled in their effector functions by cd152 (ctla-4). we demonstrate that signals induced by cd152 reduce the frequency of interferon-gamma (ifn-g) and granzymeb expressing cd8 + t cells. for this novel function cd152 specifically represses the transcription factor eomes, but not t-bet. a cd152 mediated induction of the inhibitory transcription factor ckrox has been ruled out. ectopic expression of eomes reversed cd152-mediated inhibition of effector molecule production. additionally, enhanced cytotoxicity of individual cd8 + t cells differentiated in the absence of cd152 signaling could be demonstrated in vivo. the novel insights that cd152-mediated signal transduction in vivo indeed alters cd8 + t cell cytotoxicity qualitatively at the single cell level and not only quantitatively by enhancing expansion extend the understanding how to selectively modulate immune responses of cd8 + t cells. objectives: atp constitutes a damage associated molecular pattern (damp) and contributes together with pathogen associated molecular patterns (pamp) to the efficient priming of the innate immune system. atp is a ubiquitous extracellular messenger, which activates plasma membrane receptors for extracellular nucleotides termed p2 receptors. p2x 1-7 receptors open to non-selective ion channels, whereas p2y1, 2, 4, 6, 11-14 are g-protein coupled receptors, which bind preferentially adp, udp, utp or udp-glucose. as the role of p2 receptors in the control of b cell activation has been poorly investigated, aim of the present study is to understand better the mechanisms of intracellular atp production and release by human b cell subsets. methods: intracellular atp measurement has been performed using a bioluminescence assay while extracellular atp has been measured by hplc. storage and release of atp by b cells have been elucidated using confocal and tirf microscopy, to study vesicles distribution and dynamics near the plasma membrane. results: in both human naive and memory b cell we observed a prominent increase of atp synthesis upon tlr9 but not bcr stimulation. glycolytic pathway rather than oxidative phosphorilation was involved in atp synthesis. p2x7 antagonists inhibited both proliferation and differentiation to plasma cells of human b cells thus suggesting that atp is released in the pericellular space. labelling of resting and activated human memory b cells with quinacrine, a nucleotide binding component, revealed a typical vesicular pattern of atp, confirmed with subcellular fractionation on sucrose equilibrium gradients. tirf imaging showed a fluorescently labelled vescicle underwent fusion with the plasma membrane after stimulation with anti-ig and this event was ca(2+)-dependent. conclusion: these data provide evidence that atp is produced by b cell preferentially by glycolytic pathway and vesicular exocytosis is a key mediator of atp release in human b cells. atp released in the pericellular space might act as an autocrine and paracrine signalling molecule that regulates the functions of b cells. o. ballek 1 , a. brouckova 1 , d. filipp 1 1 institute of molecular genetics as cr, laboratory of immunobiology, prague, czech republic two src family tyrosine kinases lck and fyn are critical for the proximal t-cell signaling. we have previously demonstrated that induced lck activation outside lipid rafts (lr) results in lck translocation to lr. central in this sequence of events is the rapid translocation of kinase active lck to lr, yet the mechanism underpinning this process is unknown. the main aim of this study is the characterization of molecular mechanisms and its functional elements regulating the early recruitment of signaling molecules to lr and forming immunological synapse. we have recently characterized the c-terminal yqpqp sequence as a novel cis-acting component essential for partitioning of lck to lr. here we report that the expression of the c-terminal truncate of constitutively active lck ( ¿ fqpqp) in nih3t3 cells failed to phosphorylate several proteins detected in the presence of untruncated kinase active y505flck. comparative 2-d gel analyses followed by ms/maldi identified rack1 as a candidate protein for interaction with the c-terminal tail of lck. co-expression in nih3t3 cells of ha-tagged rack1 with either a wild type lck or constitutively active y505flck revealed a significantly enhanced complex formation between y505flck and rack1 compared to that of wtlck. ectopic expression of y505flck with its domain-inactivating mutations showed that lck-rack1 interaction depends on functional sh2, sh3 and the c-terminal tail sequence of lck. lck-rack1 interaction is readily detectable also in primary cd4 + lymph node t cells. upon their activation, only the pool of lck molecules associated with high molecular weight complexes can translocate to lipid rafts. co-purification of rack1 with these fractions further suggests that it plays a role in the translocation of lck to lr. in addition, lck and rack1 co-redistribute to both forming immunological synapse and to antibody-mediated capping clusters. moreover, the importance of interaction between activated lck and rack1 in the context of lck translocation to lr is further strengthen by the observation that rack1 is associated with elements of cytoskeleton. these results are the first to characterize rack1 as a candidate molecule involved in the regulation of lck translocation to lr through linking the c-terminal sequence of lck to cytoskeletal network. human b cells are currently not known to produce the pro-apoptotic protease granzyme b (grb) in physiological settings. we have discovered that b cell receptor stimulation with either viral antigens or activating antibodies in the context of the acute phase cytokine interleukin 21 (il-21) can induce secretion of substantial amounts of grb by human b cells. grb response to viral antigens was significantly stronger in b cells from subjects recently vaccinated against the corresponding virus as compared to unvaccinated subjects. both, naï ve and memory b cells differentiated into grb-secreting cells, which featured a homogeneous cd19+cd20+cd27-cd38-igd-phenotype, improved survival and enhanced expression of co-stimulatory, antigen-presenting and cell-adhesion molecules. b cellderived grb was enzymatically active and its induction required activation of similar signaling pathways as in cytotoxic t cells. our findings suggest grb-secreting b cells play a role in early anti-viral immune responses, thereby contributing to the elevated serum grb levels found in various viral diseases. further studies will elucidate whether b cell-derived grb induces cytotoxicity towards virus-infected cells or exhibits other functions. results: transfer of otii cells augments the wild type th2 response to alumova inducing large early germinal centres and massive plasma cell formation with more than 75 % of these switching to igg1. the plasma cells up-regulate cxcr4, but not cxcr3, a chemokine receptor that attracts plasma cells to inflammatory sites. oti cells respond to alumova by producing ifng, a th1-associated cytokine. when both oti and otii cells are transferred switching is diversified with plasma cells being igm (˚5%), igg2a (˚30 %), igg2b (˚30 %) or igg1 (˚30 %). in addition to cxcr4, some 70 % of these plasma cells strongly express cxcr3. the induction of cxcr3 in these plasma cells correlates with their increased expression of the transcription factor t-bet, which has been linked with igg2a switching during th1 responses. this is functionally significant for oti-dependent cxcr3 expression, as well as induction of switching to igg2a, are dependent on t-bet expression by the responding b cells, although t-bet-deficient b cells still switch to igg2b. t-bet is known to be induced in b cells exposed to ifng or tlr9 stimulation. these two hypothetical mechanisms are currently being tested in mice injected with blocking anti-ifng antibodies or mice deficient in myd88. objective: a successful immune response against malaria has to be tightly controlled. the early production of pro-inflammatory cytokines is required to control the growth of intraerythrocytic parasites but the same cytokines are also involved in the induction of severe malaria in both humans and mice. activation of t lymphocytes through tcr signalling takes place within the context of numerous other cell surface protein interactions. to prevent unnecessary activation of t cells the immune system has developed an intricate balance between positive and negative costimulatory signals. costimulatory signals determine whether antigen recognition by t lymphocytes leads to full activation or to anergy. in contrast negative costimulators expressed by t cells seem to mediate the regulation of immune responses and thus play a pivotal role in the maintenance of peripheral tolerance. recently, btla (b and t lymphocyte attenuator, cd272) was described as a novel negative costimulatory receptor. btla is predominantly expressed on t and b cells and dampens t cell activation. in this study, we analyzed the function of btla during experimental malaria infection. to study the function of btla we employed a mouse model of blood-stage malaria using p. yoelii nl infection of btla-deficient and hvem-deficient mice. p. yoelii provokes a high parasitemia in infected mice that is cleared within three weeks from time of infection. immunity in this model depends on cd4+ t cells and hence the role of negative costimulators that modulate t cell function can be studied using this model. results: peak parasitemia of p.yoelii-infected btla-deficient and hvem-deficient mice was much lower compared to wild type mice. the increased immunity of btla-deficient mice depends largely on cd4+ t cells. we found that btla::hvem interaction regulates the size and the cytokine-production of the responding t cell pool. however, in contrast to the ctla-4 pathway, the manipulation of btla::hvem interaction triggers no pathology during infection. the hvem::btla interaction dampens the protective immune response during experimental malaria and thus manipulation of this pathway is an attractive target for therapeutic interventions. . so far, the contribution of actin regulatory proteins to this process remained largely unknown. here we demonstrate that the actin-bundling protein l-plastin is indispensable for segregation of lfa-1 and cd2 in the psmac of untransformed human t-cells. in marked contrast, tcr/cd3 accumulation in the csmac is not dependent on l-plastin. the relocalization of l-plastin in the immune synapse occurs within seconds of t-cell/apc contact formation and relies on actin polymerization. importantly, binding of calmodulin to l-plastin is required for the maintenance of l-plastin in the immune synapse and inhibition of calmodulin prevents psmac formation. thus, receptor segregation in the immune synapse is a consequence of the combined activities of the actin-bundling protein l-plastin and calmodulin. protective t cell responses are based on expansion and persistence of clones with optimal affinity for antigen. presently, it is unknown which mechanisms guard the selection and expansion of the highest affinity clones from the very diverse naï ve pool. rapid cell division creates shifts in selective pressure, which is a basic biological prerequisite for elimination. therefore we hypothesized that apoptosis might play an important role during this phase of t-cell biology. here we show that the balance between the pro-apoptotic protein noxa and its antagonist mcl-1 regulates interclonal t cell competition during acute and chronic immune activation. we found p53-independent noxa gene induction and mcl-1 downregulation upon t cell activation. concomitant we observed the release of death-inducing factor bim from mcl-1, which was delayed in noxa -/cells. using ot-1 cells and altered peptide ligands we observed that the level of mcl-1 downregulation in activated t-cells depended on the antigen affinity of the t-cell receptor. since mcl-1 -/mice are embryonic lethal, noxa -/mice were used to study the functional implications of this mechanism in vivo. at a young age noxa -/mice have a normal lymphoid compartment, but accumulate effector t cells over time. upon acute influenza infection, normal levels of effector cells were generated. however, the quality of the antiviral (np366) response was impaired in these mice as many subdominant clones persisted in the effector t-cell population of noxa -/mice at the peak of infection. this increased diversity correlated with exacerbated pathology and a reduced rate of viral clearance. in a model of chronic immune activation, effector t cells rapidly accumulated in the noxa -/mice and infiltrated the peripheral organs, culminating in severe multi-organ pathology and premature death. these results establish a novel role for the noxa/mcl-1 axis during immune responses and suggest that the formation of a high-affinity effector population of restricted clonal diversity depends on a darwinian selection of t-cells during the expansion phase based on antigen affinity, with survival of only the fittest clones. cytotoxic t lymphocytes (ctls) are essential for immunosurveillance, a process that requires the presentation of virus-or tumor derived antigenic peptides in context of antigen presenting cells. insight into intracellular mechanisms facilitating lytic granule release and formation of the immunological synapse as a prerequisite for target cell destruction was primarily obtained from loss-of-function mutations in hereditary human diseases and gene-mutated mice. here, we refer to estrogen receptor-binding fragment-associated antigen 9 (ebag9) as a negative regulator of secretory lysosome release. in gene deleted mice we show that loss of ebag9 confers ctls with enhanced cytolytic capacity, in vitro and in vivo. here, we show that ebag9, which was previously identified as a snapin-interacting protein in neuronal cells, interacted with the adaptor molecule g2-adaptin in t cells. both interactions suggested an involvement of ebag9 in endosome-lysosome related organelle biogenesis and membrane fusion. efficiency of granzyme b sorting towards secretory lysosomes was improved, which was consistent with the enhanced kinetics of cathepsin d proteolytic processing. while the formation of the immunological synapse remained unaffected in ebag9-/-ctl, relative size distribution of lytic granules revealed a shift towards smaller granule diameters and volumes in ebag9-deficient ctls. these data imply a role for ebag9 in regulating the formation of mature ctl granules and identify ebag9 as a tunable inhibitor of ctl-mediated adaptive immune response functions. finally, ebag9 defines a novel negative regulator of secretory lysosome release in ctls. thus, the elucidation of the ebag9-related pathway might provide a fresh impuls on therapeutic approaches in the treatment of autoimmune disorders. the liver is known to induce tolerance, rather than immunity, through tolerogenic antigen presentation or elimination of effector t cells. recently, we could show that liver sinusoidal endothelial cells (lsec) inhibit activation of naive cd8 t cells by antigen-presenting dc. this regulatory effect of lsec on dc function was mhc-independent and not limited to soluble mediators, but required physical contact. interestingly, interaction with lsec led to reduced dc expression levels of cd80/86 and il-12. in addition to indirect inhibition of t cell activation by de-licensing of dc, we now detected another influence of lsec in form of direct inhibition of t cell priming. in the presence of lsec, stimulation with acd3/acd28 or pma/ionomycin could not significantly activate cd4 and cd8 t cells. thus, lsec did not only inhibit t cell priming triggered by tcr activation but also after elicitation of ca 2+ influx into the cytoplasm. furthermore, we found that ifn-g secreted by t cells in the early phase of activation is crucial for licensing the inhibitory function of lsec. taken together, these data indicate that the inhibitory effect of lsec is mediated by a machinery induced at the early phase of t cell activation, however, interferes with late events in the t cell activation cascade. we propose a model of "inducible inhibition", where on the one hand naive t cell priming is directly inhibited by lsec, and on the other hand tolerogenic priming by antigen-presenting lsec is still allowed. taken together, these results reveal a novel principle, operative in hepatic tolerance induction, in which lsec not only tolerize t cells themselves, but also inhibit the responsiveness to local activation stimuli. m. almena 1 , s. carrasco 1 , i. merida 1 1 centro nacional de biotecnología csic, inmunología y oncología, madrid, spain self-tolerance acquisition is essential for the immune system to control its own response. t cells achieve self-tolerance trough thymic selection and anergy, two processes where rasgrp1-ras-erk signal intensity is critical to determine the final cell outcome. rasgrp1 is a gef for ras that is activated in a diacylglycerol (dag)dependent manner. dag is generated by plcg after tcr stimulation and is consumed by diacylglycerol kinases (dgk). dag generation, as a result of the concerted regulation of these two enzymes, activates ras, providing a mechanism to translate the strength of the stimulus into a quantitative cell response. 1. analyze the impact that dag metabolism plays in t cell tolerance in vivo, using transgenic mice where dag generation is impaired. 2. develop a method to sense dag production and localization, in both thymocytes and peripheral t cells, and its correlation with the strength of the stimulus used. methods: we generated transgenic mice expressing a constitutively active dgk in t cell lineage. this protein was anchored to the plasma membrane, thus diminishing the lipid levels in this specific location after tcr stimulation. ot-i cd8 cells expressing gfp-c1 domains were used with peptide-pulsed apcs to study dag generation and dynamics by confocal microscopy. results: transgene expression was obtained in thymic and peripheral t cells. no major defects were observed in t cell subsets but analysis of peripheral t cells demonstrated important defects in t cell activation. we are currently studying thymic selection breeding our transgenic with h-y mice, in order to check if t cell populations are being properly selected. using t cell-apc conjugates with peptides with different tcr binding affinities we found a clear correlation between the strength of the stimulus, dag production and ras-mapk activation. conclusion: our data demonstrate that dag generation not only activates c1 domain containing proteins but regulates a mechanism by which t cells sense the magnitude of the stimulus received, translating it into the intensity of the generated response, a process essential in t tolerance. future experiments will help to define the exact contribution of the lipid to tcr signaling pathways and to t cell homeostasis. the inducible costimulator (icos, h4, cd278), a cd28-like costimulatory molecule, has an important role in the development of efficient t cell responses. early data showed that icos costimulation produced th2 biased responses and high production of the anti-inflammatory cytokine il-10, and was essential to the development of germinal centres. however, icos can also help in the il-21-dependent differentiation of inflammatory th17 cells. these different functions could be due to differences in the apcs bound by icos-expressing t cells and/or because of the intervention of distinct molecules binding the cytoplasmic domain of icos. icos shares with cd28 a yxxm cytoplasmic motif that can bind, upon tyr phosphorylation, the regulatory p85 subunit of class ia pi-3 kinases. these can complex with one of the three 110 kda catalytic subunits (p110a, p110b, and p110d) expressed by leukocytes that generate pip 3 affecting cell growth, cell cycle progression, survival, intracellular traffic, cytoskeletal changes and migration. there is also evidence that the regulatory and catalytic class ia pi3k isoforms fulfill specific functions in macrophages and lymphocytes. we have used proteomic and immunochemical approaches to identify molecules binding the phosphorylated or unphosphorylated cytoplasmic domain of icos, and particularly the presence of distinct pi3 kinase isoforms. then, the functional importance of these molecules has been analyzed by using pharmacological inhibitors specific for downstream mediators of icos activation. pull down of t cell lysates using phosphorylated or unphosphorylated synthetic peptides covering the cytoplasmic domain of icos was carried out. proteomic and immunoblot analysis of bound proteins showed that phosphorylated icos bound the different pi3-k regulatory (p85a, p85b, p53a) and catalytic (p110a, p110b, and p110d) pi3-kinase subunits expressed by leukocytes. these data were confirmed in icos immunoprecipitates from pervanadate-activated cells. icos bound regulatory and catalytic subunits in the order p85a g p53a g g p85b and p110a g p110d g g p110b, in agreement with quantitative rt-pcr and immunochemical estimation of subunit abundance in the t cells and t cell lines used. the use of specific pi3-kinase inhibitors has confirmed the relative importance of the catalytic isoforms in icos function, including reorganization of actin cytoskeleton induced by icos ligands, or costimulation of tcr/cd3-induced secretion of il-10 and il-4. during the process of antigen recognition between t-cell and antigen-presenting cell (apc), structural and spatial changes take place at the cell-cell contact, where the molecules involved in the formation of the immune synapse (is) reorganize, displaying a segregated localization. in this context, the translocation of the microtubule-organizing center (mtoc) is an early event that occurs during the formation of the is, bringing with it the golgi apparatus, thus providing the basis for a polarized secretion. however, the molecular mechanisms involve in the localization of the mtoc at the contact area between the t cell and the apc are not completely understood yet. we have studied the possible role of scaffolding protein akap450, a member of the a-kinase anchoring protein (akap) family that localizes at the centrosome and interacts with pka regulatory subunit and other signalling molecules, in mtoc polarization and immune synapse formation. either the overexpression of gfptagged c-terminal cg-nap/akap450 construct that acts as a dominant negative, or sirna knockdown of endogenous akap450 expression in t cells prevents the correct organization of cd3z and pkcv to the is and mtoc reorientation towards t cell-apc contact area in antigen and superantigen-dependent human models, resulting in a disorganized is; lfa-1 localization was also analyzed to assess p-smac architecture and, interestingly, confocal 3d reconstruction revealed that lfa-1 ring was not clear in the akap450-disrupted cells. moreover, akap450 was required for tcr signalling since the knock down with specific sirna and overexpression of c-terminal of akap450 decrease the phosphorylation of molecules such as lat, plcg1 and pkcv. these defective activation events as reflected in a reduction of il-2 production. together, our results underscore a key role for akap450 in the organization of the immune synapse and in the antigen-specific reorientation of the mtoc. the tcrbeta/ptalpha pre-tcr complex signals the expansion and differentiation of developing thymocytes. functional properties of the pre-tcr rely on its unique ptalpha chain, which suggests the participation of specific intracellular adaptors. in fact, we have recently identified cms, a member of the cin85/cms family of adaptors, as a ptalpha-binding protein that specifically interacted with the human ptalpha cytoplasmic domain via its sh3 domains, and to the actin cytoskeleton via its c-terminal region. we found that cms co-localized with polymerized actin in pre-tcr clusters at the pre-tcr activation site, and also in the ptalpha endocytic compartment. since actin polymerization plays a critical role in regulating signalling through the alpha/beta tcr in mature t cells, we decided to investigate the potential function of cms as a regulator of actin polymerization and pre-tcr signalling in pre-t cells. using pre-t cells expressing a mutant pre-tcr lacking the cms-binding motif in the ptalpha tail and short hairpin irna-based gene silencing, we demonstrate that binding of cms to ptalpha contributes to cytoskeleton dynamics and pre-tcr-mediated signalling in human pre-t cells. cms-deficient cells specifically showed defects in pre-tcr-induced ca2+ mobilization and cell activation involving the pi3k, nfat, plcg and erk signalling pathways, together with defects in actin polymerization and cell motility. cms therefore links cytoskeleton dynamics with the function of discrete pre-tcr signalling components, suggesting the functional implication of cms in human t-cell development in vivo. abstract withdrawn by author j objectives: most signaling pathways engaged after bcr activation have been described. however, several negative regulators of these pathways are unknown. the characterization of these regulators is important to understand the control of transduction pathways in adaptative immunity. carabin (tbc1d10c) has been recently described as a negative regulator of tcr signaling. it interacts with calcineurin and inhibits the formation of calcineurin/calmodulin complex, blocking nfat nuclear transport. moreover, carabin maintains ras protein under an inactive form, thus inhibiting ras-mapk cascade. expression of carabin is finely regulated following tcr signaling, and its knockdown (kd) enhances t cell activation. considering the important molecular similarities of antigen receptor signaling pathways in t and b cells, we studied the role of carabin in b cell. could carabin play a role of negative regulator of b cell function? methods: we studied by quantitative rt-pcr 1) the expression of carabin in different purified subsets of bone marrow and splenic mouse b cells, as well as 2) the kinetic of expression of carabin in bcr stimulated murine splenic mature b cells. 3) we then studied the phenotype of carabin kd (shrna expressing) a20 b cells after bcr stimulation. 1) the expression of carabin is significant in murine b cells, with an increase during b cell development, from bone marrow pro/preb to immature, to splenic t1 tot2 b cells and to follicular mature b cells. 2) the kinetic of expression of carabin in bcr stimulated murine mature b cells suggests a fine regulation of carabin expression. 3) bcr simulation, but not lps stimulation, of carabin kd a20 b cells shows an acceleration of ras target erk1/2 phosphorylation, without any for the phosphorylation of mapk jnk, which is not targeted by ras. conclusion: carabin is expressed in murine b cells in a developmental regulated manner, with the highest expression in mature compartment. bcr stimulation leads to a fine regulation of carabin expression in wild-type mature b cells, and to a faster activation of erk1/2 pathway in carabin kd b cells. altogether, these results strongly suggest a role of carabin as a negative regulator of b cell function toll like receptors are pattern recognition receptors, which recognize invariant pathogen associated molecular patterns. toll like receptor 3 (tlr3) binds doublestranded rna, a nucleic acid frequently associated with viral replication. we observed that freshly isolated human cd4 + t cells express tlr3 and respond to the well characterized synthetic tlr3 ligand polyinosinic-polycytidylic acid [poly(i:c)]. the expression of activation markers and cytokine production by cd4 + t cells upon t cell receptor (tcr) stimulation is enhanced in response to co-stimulation via tlr3. tlr3 stimulation on its own had no effect on expression of activation markers and cytokine production. to elicit the molecular basis of a potential cross-talk between tcr and poly(i:c) induced signaling, we used jurkat cells to perform luciferase assays. we observed that costimulation with poly(i:c) in comparison to tcr stimulation alone enhanced nf-kb but not nfat activation in jurkat cells. similarly to jurkat cells, tcr stimulation activated nf-kb in primary cd4 + t cells. this effect was further enhanced by additional poly(i:c) stimulation as shown by real-time-pcr and western blot analysis. on the other hand, we observed that poly(i:c) stimulation on its own activated the transcription factor interferon regulatory factor 3 (irf3) as revealed by realtime-rcr analysis of ifn b and irf7, whose transcription depends on the activity of irf3. combined tcr and poly(i:c) stimulation further enhanced the transcription of these two genes. these results indicate that tlr3 signaling modulates tcr-driven responses and vice versa both in jurkat cells and in freshly isolated cd4 + t cells. this study was supported by dfg spp 1110 "innate immunity" (ka 502/8-3). the initiation of protective t cell responses requires the recognition of mhc-bound peptides from pathogen or tumor antigens by the t cell receptor (tcr). how this signal is transmitted across the t cell membrane to the cytoplasmic signaling motifs is still unknown, and is the focus of this project. in textbooks, the cytoplasmic domains are depicted as flexible chains in the cytoplasm, but biochemical studies show that the cd3e cytoplasmic domain (cd3e cd ) binds to synthetic lipid vesicles that contain acidic phospholipids. this binding is predominantly due to electrostatic interactions between basic residues of cd3e cd and acidic phospholipids. in the cell, acidic phospholipids are enriched in the inner leaflet of the plasma membrane. phosphatidylserine in particular is concentrated on the inner leaflet of the plasma membrane due to active transport mechanisms, explaining how such charge-charge interactions are generated. to study the interaction of the cd3e cd with the membrane in live cells, we have developed a fluorescence resonance transfer (fret) assay which measures the proximity between a fluorescent protein (tfp) attached to the c-terminus of cd3e cd and a fluorescent membrane dye (r18). with this assay, we show that the cd3e cd is membrane-bound in resting cells and that binding is abrogated by introduction of mutations that disrupt lipid binding in the biochemical assay. additionally, in vitro analysis confirm functional domains for cd3e cd lipid binding and conformational change. finally, nmr spectroscopy analysis reveals key features in membrane binding dynamics of cd3e cd to lipid bicells. membrane binding by the cd3e cd could thus be subject to dynamic regulation during the engagement of the tcr and further activation of the t cell. m. xydia 1 , y. ge 1 , u. quitsch 1 , p. beckhove 1 1 german cancer research center (dkfz), heidelberg, germany in peripheral tissues and the factors affecting their proliferation. cd4+ t cell help is believed to contribute to optimal cd8+ memory expansion via cd40l on cd4+ t cells binding cd40 on dendritic cells. however, a few reports suggest that cd40l-cd40 engagement may mediate direct cell-cell contacts between cd4+ and cd8+ t cells. in this study, we investigated the importance of cd4-cd8 co-operation and cd40l-cd40 interactions for t em proliferation. methods: we isolated human cd4+ and cd8+ t em cells from peripheral blood of healthy donors by facs or macs sorting. separated or mixed cd4+ and cd8+ populations were activated in vitro using anti-cd3/cd28 beads. proliferation was measured by [ 3 h]-thymidine incorporation, in some experiments after irradiation of one t em subset and/or incubation with blocking mabs against cd40 or cd40l. furthermore, facs staining was used to assess cell-surface markers. statistical comparison was performed by student's t test. results: upon activation mixed t em populations showed a highly better proliferative response than separated cd4+ or cd8+ t em cells, demonstrating that optimal t em expansion requires direct cd4-cd8 interactions. surprisingly, not only cd8+ but also cd4+ t em cells proliferated much more in mixed populations compared to the separated ones, indicating that optimal cd4+ t em proliferation depends on signals from cd8+ t em cells. activation induced the expression of cd40 on both populations and cd40l on subsets of cd4+ and cd8+ t cells. blocking of cd40l on cd8+ t em cells impaired significantly cd4+ t em proliferation, which confirms that the improved expansive potential of cd4+ t em cells in mixed populations depends on cd40l co-stimulation by the cd8 t em subset. conclusions: our data demonstrate for the first time that activated cd8+ t em cells deliver help to the cd4+ t em subset via cd40l-cd40 signalling and may play an important role for cd4+ t em expansion upon stimulation. the t cell surface glycoprotein cd5, a member of the scavenger receptor cysteine-rich (srcr) family of proteins, targets to the immunological synapse upon t cell binding to antigen presenting cells (apc). however, it has not been established whether this translocation is due to the binding of a ligand expressed in the apc, or to intracellular interactions with signaling molecules or components of the cytoskeleton, that may control cd5 localization upon t cell:apc conjugation. we have questioned which domains of cd5 mediate the localization within the is, and for this we have expressed cd5 mutants as gfp fusion proteins in human t lymphocytes. we have also used jurkat cell lines expressing different cd5 mutants. t cells were incubated with superantigen-loaded raji b cells, and following the establishment of stable interactions between the cells, we analyzed the localization of cd5 by immunofluorescence and confocal microscopy. interestingly, our results show that the translocation of cd5 depends on sequences within the cytoplasmic domain, as a cd5 deletion mutant lacking most of the cytoplasmic tail, cd5.k384 stop , is randomly distributed through the whole cellular surface, even in sustained t-apc interactions. the cytoplasmic domain relevant to cd5 translocation was mapped within amino acids glu 418 and his 449 since the cd5.h449 stop mutant, just short of 22 aa is still able to translocate to the is, whereas cd5.e418 stop , that lacks 2 important tyrosine residues, is no longer transported to the is upon t cell: apc interactions. although these studies do not exclude a role for the extracellular domain binding to an elusive apc-expressed ligand, they suggest that a major mechanism of regulation of cd5 translocation is dependent on molecular association of a short stretch of its cytoplasmic region (glu 418 -his 449 ) to intracellular signaling effectors. however, in hodgkin's lymphoma (hl) ebna-2 is missing, but lmp-1 is still expressed. using a hl derived cell line, we have shown that the cytokine il-4 can induce lmp-1 expression in vitro and can replace ebna-2. we have investigated the molecular events for this mechanism. stat proteins bind to the palindromic ttc(n) x gaa sequence, where × is 2, 3 or 4. a high affinity stat6 binding site is spaced by 4 nucleotides. we found three potential stat binding sites in the lmp-1 promoter, which we named lrs, tr and edl1. they were spaced by 4, 3 and 2 nucleotides, respectively. electrophoretic mobility shift (emsa) experiments were performed with nuclear extracts prepared from il-4-treated or non-treated kmh2-ebv cells. dna binding activity was analyzed using a double stranded oligonucleotide corresponding to the germline (gl) epsilon promoter, which is known to contain a high affinity stat6 binding site, or lrs-stat6. a stat6 complex binding to the gl-epsilon promoter and lrs-stat6 was induced by il-4. the specificity of the stat6 complex was shown by supershift experiments with anti-stat6, but not anti-stat5 antibodies. when gl-epsilon or lrs-stat6 was used as cold competitors in a 100-fold excess, both unlabelled probes could compete out the labeled probe, providing evidence that the lrs-stat6 contains a functional stat6 binding site. oligonucleotides, corresponding to lrs in which the stat6 site had been mutated, could not compete for stat6 binding. interestingly, the unlabeled lrs-tr with 3 nucleotides as spacer could also function as competitor. however, when ttc/gaa palindrom was spaced by 2 nucleotides (lrs-edl1), it could not compete. thus, expression the transforming protein lmp-1 can be induced directly by the t cell derived cytokine il-4 in a stat6 dependent manner. it is likely that this mechanism operates in vivo as well and determines expression of the ebv encoded protein lmp-1 and thus the pathogenesis of ebv carrying hls. established knockout/ knockin mice with a fasl deletion mutant that lacks the intracellular portion (fasl ¿ intra). co-culture experiments confirmed that the truncated fasl protein is still capable of inducing apoptosis in fas-sensitive cells. preliminary immune histochemistry data suggest that, in contrast to published data, the absence of the intracellular fasl domain does not alter the intracellular fasl localization in activated t cells. we are currently investigating signalling and proliferative capacity of b-and t-cells derived from homozygous fasl ¿ intra mice. our data point to a rather inhibitory role of fasl reverse signaling during immune responses. during an immune response numerous receptor-mediated signals delivered to t cells direct their proliferation, survival and differentiation. we are using a quantitative model and in vitro methods to assess the "calculus", or decision-making algorithms t cells use to process these multiple signals. previous experiments with ot-i cd8 t cells revealed that tcr affinity regulated both the frequency of cells responding and the average time taken for cells to reach their first division (ji 2007 (ji . 179:2250 . furthermore, affinity was the sole regulator of the rate of cell death in subsequent divisions. here we examine the same question for cd4 t cells. again we find that lower affinity peptides stimulate t cells to divide rapidly, however, a high proportion of cells die within each division round, revealing an important potential mechanism for affinity maturation and selection of dominant clones over time. in contrast varying the number of dendritic cells used to stimulate cd4+ t cells primarily affect the proportion of cd4+ t cells going into division rather than affecting division time or cell death in subsequent divisions. currently we are using these quantitative methods to measure the effect of cytokines and co-stimulatory molecules cd40, cd80 and cd86 on parameters of cd4+ t cell proliferation to inform quantitative models of the immune response under different conditions. our goal is to develop quantitative models of t cell behaviour that can accommodate information at the molecular, cellular and population level. interaction between cd40, a member of the tumor necrosis factor receptor superfamily constitutively expressed on antigen-presenting cell as b cells, and cd40l, a member of the tumor necrosis factor family transiently expressed on activated t cells, are essential for the development of humoral adaptative immune response. various studies have shown that dual stimulation of b cell through antigen binding on bcr and cd40 leads to an enhancement of ig and cytokine production. the current dogma postulates that these 2 signals are necessary and sufficient to drive naive b cell proliferation and differentiation to ig secreting plasma cells. however, recent evidence suggests that the innate immune responses could regulate humoral adaptive immune response. indeed, b cells can be activated through engagement of a variety of innate immune receptors, including toll-like receptors (tlrs). soluble cd40l is unable to induce murine b cell proliferation. however, we and others have shown that recombinant mouse cd40l (rmcd40l) can increase proliferation induced by tlr3 (poly ic) and tlr4 (lps) agonists. by contrast, we never observed any synergy between rmcd40l and tlr1/2 (pam3csk4) or tlr2/6 (pam2csk4) agonists. to go further in the study of cd40l/tlr agonist synergetic effect, we have developed trimeric synthetic molecule to mimick cd40l, named mini-cd40ls, based on a c 3 -symmetry core holding cd40-binding motif lys-gly-tyr-tyr. in surface plasmon resonance experiments, mini-cd40ls bind to immobilized human cd40 and compete with the binding of cd40l homotrimers and diplayed effector functions that matched those of the much larger recombinant cd40l homotrimers as maturation of mouse dendritic cells and activation of in vivo immune response in a mouse model of trypanosoma cruzi infection. as soluble cd40l, mini-cd40ls synergize tlr4 (lps), tlr3 (poly ic) and tlr7/8 (r848) agonist-induced murine b cell proliferation but no synergy was observed between mini-cd40ls and tlr 1/2 (pam3csk4), tlr 2/6 (pam2csk4) and tlr9 (odn 2395) agonists. synergy between cd40l and tlr agonist provide the ground to use such a combination as adjuvant in vaccination strategy. however, to reach this goal, evaluation of cd40l/tlr combinations on murine and human b cell activation and differentiation in antibody producing cells are under investigation. interaction of naïve cd8 + t cells with immature dendritic cells (idc) expressing self-peptides can result in their abortive activation (aa), which leads to the induction of cd8 + t cell tolerance. we have defined a phenotypic profile for cd8 + t cells undergoing such aa. these cells undergo limited proliferation which is associated with lack of ifn-g production, low cell surface expression of cd25 and cd69, and high levels of expression of cd62l and ly6c. whereas, cd8 + t cells undergoing productive activation (pa), following encounter with mature dc, form effector ctl which is evidenced by extensive t cell proliferation, high levels of ifn-g, cd25 and cd69, and loss of cd62l and ly6c expression. ly6c is a gpi-anchored cell surface glycoprotein expressed on cells of hemopoietic origin: however, its role in peripheral tolerance induction is not understood. in this study, we show that mab-blocking of ly6c in vivo and in vitro results in pa rather than aa. we hypothesize that the interaction of ly6c, expressed on naïve cd8 + t cells, with its ligand on idcs, may be vital in controlling the induction of peripheral tolerance amongst self-reactive cd8 + t cells. objectives: organophosphorus compounds (opcs) are commonly used in the manufacture of insecticides and pesticides. exposure to opcs is associated with neurological toxicity but the effect on the immune system remains ill-defined. in this study, we used a subchronic exposure model to investigate the effect of the organophosphorus compound, paraoxon, on the murine immune system. methods: balb/c mice were injected i. p. daily with saline (control group) or paraoxon (experimental group) for 3 weeks. during the treatment, animals were weighed and blood was collected weekly for determination of acetylcholinesterase activity in red blood cells. at the end of treatment, mice were sacrificed and spleen cells analyzed by flow cytometry. spleen cells were also cultured in the presence or absence of mitogens and supernatants were analyzed for cytokine content by elisa. for in vivo survival studies, mice were treated as described above and then orally infected with a virulent strain of s. typhimurium. animal survival was followed for up to 60 days after infection. results: daily injection of paraoxon induced g 50 % reduction in acetylcholinesterase activity by the end of the first week of treatment, a level which was thereafter maintained during the remaining 2 weeks of treatment. mice exposed to paraoxon exhibited g 80 % reduction in the rate of body weight gain over the treatment period in comparison with control group. at the end of treatment, ex vivo analysis of spleen cellularity and function revealed no significant differences between control and experimental groups. to analyze the status of the immune system in vivo, mice were infected with a lethal dose of a pathogenic strain of s. typhimurium and followed for survival. unexpectedly, paraoxon-treated mice exhibited a significant degree of resistance with 80 % of mice surviving the infection compared to 20 % in control group. protection in paraoxon-treated group was dependent on the reduced acetylcholinesterase activity as it was abrogated by coadministration of a reactivator of cholinesterase. conclusion: our data demonstrate that a reduction in the level of acetyl cholinesterase rendered mice more resistant to a virulent infection. this suggests a hitherto novel function of the neurotransmitter acetycholine in modulating the immune response to infection. t cell-dependent (td) and t cell-independent (ti) igg autoantibodies have been described in the context of the autoimmune disease systemic lupus erythematosus (sle). however, their different roles in autoimmunity are unknown. here we show that ti antigens induce anti-inflammatory igg antibodies and protect from antigen-specific immune pathology. administration of antigen-specific anti-inflammatory igg antibodies was sufficient to mediate this effect independent of the igg inhibitory receptor fcgammariib. ti but not td igg autoantibodies were further associated with inhibition of pro-inflammatory th1 and th17 cells and disease in mice deficient for fcgammariib, a spontaneous model for sle. the data suggest a novel immune regulatory function for ti immune responses through the generation of anti-inflammatory igg antibodies. objective: class i phosphoinositide 3-kinases (pi3k) constitute a family of enzymes that generate 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (tyr) kinase-associated receptors or g protein-coupled receptors (gpcr). the class i pi3k are divided into two types: class ia p85/p110 heterodimers, which are activated by tyr kinases, and the class ib p110g (p110gamma) isoform, which is activated by gpcr. although the t cell receptor (tcr) is a tyr kinase-associated receptor, previous studies showed that p110g deletion affects tcr-induced t cell stimulation. mice lacking p110g show a partial defect in t cell differentiation, activation and survival. p110g participates in signaling pathways that regulate pre-tcr dependent differentiation and cd4+/cd8+ t cell lineage commitment. in the mrl/lpr mouse model of systemic lupus erythematosus, administration of a pi3kg-specific inhibitor causes a reduction in the number of cd4+ memory t cells that mediate renal injury. similarly, pi3kg deletion in p65 pi3k transgenic mice also reduces the numbers of cd4+ memory t cells. there is therefore evidence that pi3kg has an important function in tcr-mediated t cell activation, although the mechanism by which pi3kg regulates this process is not well understood. we studied the specific role of p110g in t cell activation. methods: we studied whether the tcr activates p110g and the consequences of interfering with p110g expression or function on t cell activation. results: we found that after tcr engagement, p110g interacts with and forms a complex with ga q/11 , lck and zap70. tcr stimulation activates p110g, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. we show that tcr-stimulated p110g controls rac1 activity, f-actin polarization, and the interaction between t cells and antigen-presenting cells (apc). we show that p110g deletion affects the activation of many pathways downstream of tcr crosslinking, as well as the interaction between t cells and apc; these findings could explain the defective activation of p110g-/-t cells. our observations clarify the activation mechanism and mode of action of p110g in the control of t cell activation, confirming a crucial role for p110g in tcr-induced t cell activation. we investigated mechanisms controlling central location of lytic granules and kinetics of their release within immune synapses formed by cytotoxic t lymphocytes (ctl). we show that cytolytic granules in ctl can be delivered to the secretory domain via two different pathways -"short" and "long". the choice between these pathways is regulated by the kinetics of early tcr signaling which depends on the strength of tcr/pmhc/co-receptor interactions. meanwhile, the molecular hardware used to deliver the granules remains the same. we conclude that the difference in temporal and spatial coordination of the two principal events, i. e., granule movement toward microtubule organizing center (mtoc) and the mtoc polarization, accounts for two different pathways of granule delivery to the secretory domain that influence efficiency of ctl cytolytic response. our findings reveal a mechanism of well-documented flexibility in t cell responsiveness that is derived from differential use of the similar set of immune receptors, signaling proteins and intracellular effector molecules. objectives: in addition to specific immune cytokines, lymphocyte activation and immune response are modulated by universal mediators like acetylcholine. nicotine was shown to suppress both cellular and humoral immune responses. previously we found that two nicotinic acetylcholine receptor (nachr) subtypes, a4b2 and a7, expressed in mouse b lymphocytes regulate their development within the bone marrow. the aim of the present study was to evaluate the roles of these two nachrs in b lymphocyte activation. methods: b lymphocytes were magnetically separated from the spleens of c57bl/6j mice. they were stained with fluorescently labeled igm-, cd40-or cd23specific antibodies in the presence/absence of unlabeled nachr subunit-specific antibodies to be examined by flow cytometry. b lymphocyte activation was studied by 3 h-thymidine incorporation upon stimulation with anti-cd40 and nachr-specific agonists or antagonists. the antibody response of mice immunized with cytochrome c with or without a7 nachr antagonist methyllicaconitine (mla) was studied by elisa. results: antibodies against a4 or b2 nachr subunits inhibited binding of igm-and cd23-specific antibodies but facilitated that of cd40-specific antibody. in contrast, antibody against a7 subunit prevented binding of anti-cd40 but not of anti-igm or anti-cd23 suggesting that a7 nachrs are located close to cd40, while a4b2 ones are close to bcr/cd23. consequently, anti-cd40-induced b lymphocyte proliferation was increased by mla much stronger than by a4b2-specific antagonist dihydro-b-erythroidine. it was also increased when cells were incubated with the inhibitor of acetylcholine synthesis hemicholine-3. in contrast, proliferation of b lymphocytes from mice consuming nicotine was significantly weaker than that of control mice. mice co-injected with cytochrome c and mla responded with igm antibodies faster than those injected with cytochrome c alone, while the secondary / igg responses were similar. the cd40-mediated b lymphocyte proliferation, but not the igm-igg switch or memory b cell activation, is negatively controlled by either endogenous acetylcholine or consumed nicotine through a7 nachrs. therefore, acetylcholine may be regarded as an auto/paracrine regulator of lymphocyte activation.this work was supported by philip morris usa inc. and philip morris international. binding of cd4 + t h -lymphocytes to antigen presenting cells or of cd8 + cytotoxic t-lymphocytes (ctl) to their target cells lead to a tight contact between these two cells, called immunological synapse (is). formation of the is induces calcium signaling, rearrangement of the actin cytoskeleton, and the recruitment of various molecules to the is, all of which are crucial for t-cell functions such as cytokine release or target cell killing. objectives: using primary human t-lymphocytes, none of the proteins involved in either calcium influx, cytokine release, actin cytoskeleton rearrangement nor in killing of target cells can be analyzed by knock-out strategies. for testing protein functions, down-regulation by rnai technology is thus an important tool. we used short interfering rnas (sirnas) to analyze the role of proteins involved in calcium influx and proliferation (stim1 and trpc3), and to analyze snare proteins which were shown to accumulate at the is and are good candidates to play a role in cytotoxic granule fusion and exocytosis to kill target cells. methods: to validate down-regulation of different mrnas quantitative rt-pcr was used. down-regulation of proteins was confirmed by immunocytochemistry, western blotting and various functional assays depending on the potential role of the protein of interest (calcium imaging, proliferation, cytokine release, killing assay). results: transfection efficiency of sirnas in t-lymphocytes was about 96 %. down-regulation of stim1 was confirmed by qrt-pcr and by calcium imaging, but only for early time points following activation of cd4 + t h -lymphocytes, probably because of stability problems. to increase stability of sirnas within t-lymphocytes we used modified sirnas published by mantei et al. (eji, 2008) . we show that these sirnas down-regulate various snare proteins in ctls more efficiently than non-modified sirnas. the optimal sirna concentrations for transfection in primary human t-lymphocytes was found to be 300-600 pmol, which is lower than the concentrations reported in other cell types. conclusions: following optimization, down-regulation of mrnas by sirna is a powerful tool to investigate the role of different proteins involved in the activation of t-lymphocytes in primary human cells. chemical modifications increase the lifetime and efficiency of the sirnas in primary human t-lymphocytes. stress-inducible heat shock protein 70 (hsp70) has gained plenty of attention because of its potent adjuvant capability to induce antigen-specific cd8 + cytotoxic t-lymphocyte (ctl) and cd4 + t-helper cell (th1) responses. in this study, we investigated the behavior of t-cell subsets stimulated with endotoxin-free recombinant hsp70 with respect to proliferation, cytokine expression, cytotoxicity against allogeneic b-lymphoblastoid cell line (b-lcl) and k562 cells as well as targetindependent cytotoxicity. cd4 + cells exhibited a strong increase in proliferation after stimulation with hsp70, with rates of up to 29 %. in the presence of target cells, a 35-fold up-regulation of granzyme b mrna was observed after stimulation of cd4 + t-helper cells with hsp70 in combination with il-7, -12 and -15. the target cell-independent secretion of granzyme b by cd4 + cells was greatly augmented after stimulation with hsp70 plus il-2 or il-7, -12 and -15. in this study, we have shown that hsp70 is capable of inducing a cytotoxic response of t-helper cells in the absence of lps or any other pamps. the granzyme b secretion and the cytolytic activity of cd4 + t cells is induced in a target-independent way, whereas the cytotoxic activity of cd3 + and cd8 + t cells can be further enhanced in the presence of the target cells. our data provide novel insights into the role of extracellular hsp70 on t-cell immune response concerning the induction of target-independent t-helper cell cytotoxicity. jun n-terminal kinases (jnk) have been shown to play controversial role in regulation of cell fate. cd40, which is responsible for germinal centre formation in lymph nodes, trigger jnk activation. the role of other b cell co-receptor molecules that may be involved in antigen-driven differentiation were not clarified. the aim of this study was to find out whether cd150 receptor contributes to jnk activation in mature human b cells. protein expression and phosphorylation were studied by western blot analysis. protein associations were evaluated by immunoprecipitation and gst-pull down assays. hpk1 overexpression in a model system was achieved by transfection. pjnk1/2 expression in primary hrs cells was assessed by immunohistochemistry. ligation of cd150 on resting (dense) and activated (buoyant) human tonsillar b cells lead to jnk2, but not jnk1 activation. cd40 ligation on primary tonsillar b cells also resulted in jnk2 activation. however, bcr crosslinking did not affect the level of jnk1/2 phosphorylation. cd150-mediated jnk2 activation was independent from sh2d1a/sap adaptor protein expression, and was demonstrated for all studied b-lymphoblastoid, burkitt's lymphoma and hodgkin's lymphoma (hl) cell lines of b cell origin. we were searching for serine/threonine kinase that could coprecipitate with cd150 and link this receptor with jnk pathway. using immunoprecipitation and gst-pull down assays we found that hematopoietic progenitor kinase 1 (hpk1) was associated with cd150 in primary b cells as well as in b cell lines. cd150-hpk1 association was independent from cd150 tyrosine phosphorylation and sh2d1a expression. overexpression of hpk1 in a model system significantly enhanced cd150mediated jnk2 phosphorylation. it is known that tnf family receptors such as cd30, cd40, rank trigger survival signals in hrs cells. we observed the expression of pjnk1/2 in hrs cells of primary classical hl. cd150 could be involved in sustained jnk2 activation in primary hrs cells, and this may reflect the role of cd150 receptor as well as other receptors in the regulation of hrs survival. overall, it was shown that jnk2 is activated via cd150 in primary b cells and in all studied cell lines of b cell origin. serine-threonine kinase hpk1 is involved in cd150-mediated jnk2 activation. objectives: cd5 has been shown to act as a negative regulator of tcr signaling during thymocyte development. however, the molecular mechanisms involved in this process remain elusive. one potential key molecule involved in the downmodulation of tcr signaling is c-cbl, a ubiquitin ligase that physically associates with cd5 upon tcr crosslinking in thymocytes. the objective of this study was to determine which sequences within the cytoplasmic tail of cd5 are involved in c-cbl phosphorylation and association. methods: el4 thymoma cell line was stably transfected with wild-type human cd5 or hcd5 cytoplasmic tail mutants: cd5.k384stop (maintaining only a pseudo itim); cd5.h449stop (lacking the distal s and y in the carboxy-terminal region); cd5. ¿ e418-l444stop (lacking the pseudo-itam, putative site for c-cbl association). phosphorylaton of y700 in c-cbl was analyzed, which is required for vav recruitment and c-cbl dependent degradation by the proteasome. stable clones were stimulated with anti-murine cd3 in combination or not with anti-human cd5 biotinylated antibodies and phosphorylation of c-cbl was detected by flow cytometry after intracellular staining anti-phospho c-cbl (py700) antibody. murine thymocytes were used as positive control. data was analyzed using flowjo software. unpaired two-tailed student t test was used to calculate statistical significance (p x 0.05). in murine thymocytes, co-crosslinking of cd3 with cd5 induces an increase in c-cbl phosphorylation compared to cd3 alone. analysis of the el-4 transfectants showed that mutants cd5.k384stop and cd5.h449stop lost the ability to costimulate cd3-mediated phosphorylation of c-cbl. in contrast, cd5. ¿ e418-l444stop mutant, was able to efficiently costimulate cd3-mediated c-cbl phosphorylation, similarly to the hcd5wt. our results indicate that the absence of the pseudo itam in cd5 does not interfere with c-cbl phosphorylation in response to cd3 plus cd5 crosslinkiing on the other hand, sequences present in the carboxy-terminal region of cd5 appear to be important for c-cbl phosphorylation. therefore, c-cbl phosphorylation might not require physical association with the cd5 cytosplasmic tail, but rather, may indirectly associate with cd5 through the interaction with other sh2-sh3 domain-containing molecules, that may be recruited to cd5 through its carboxy-terminal region. l. kolly 1 , s. narayan 1 , j. tschopp 2 , a. so 1 , n. busso 1 1 chuv, rheumatology, lausanne, switzerland, 2 unil, biochemistry, epalinges, switzerland apoptosis-associated speck-like protein containing a caspase recruitment domain (asc) is an adaptor protein that is essential for the recruitment of pro-capase-1 into inflammasomes and thus plays a key role in regulating capase-1-dependent il-1b and il-18 production. despite recent evidence implicating asc in adaptive immunity against infections, hyperresponsiveness and vaccination, the cellular and molecular basis for asc involvement in adaptive immune responses remains largely unexplored. to investigate the impact of asc on t cell activation and subsequent effector function. asc +/+ and asc -/-t cells or purified cd4 + and cd8 + t cells were activated in vitro through anti-cd3 stimulation and their proliferative potential and cytokine profiles characterized. proliferative responses by asc -/-t cells were significantly inhibited two-fold following tcr-cd3 ligation when compared to asc +/+ t cells. furthermore, cytokine analysis revealed that anti-cd3 activated asc -/-t cells predominantly displayed a more th 2 phenotype, producing more il-10 (199 vs. 692 pg/ml; asc +/+ vs. asc -/-t cells respectively; p=0.0074) and less ifn-g (15,831 vs. 6 ,921 pg/ml; asc +/+ vs. asc -/-t cells respectively; p = 0.0021). when asc +/+ and asc -/-t cells were purified into cd4 + and cd8 + t cell fractions and activated individually using anti-cd3, no inhibition in proliferation was observed amongst activated asc -/-cd4 + and cd8 + t cells. interestingly, the activated asc -/-cd4 + t cell fraction produced significantly more il-10 when compared to activated asc -/-cd8 + t cells and asc +/+ cd4 + and cd8 + t cells (asc -/-cd4 + t cells = 380 pg/ml il-10; asc -/-cd8 + t cells = undetectable il-10; asc +/+ cd4 + t cells = 11 pg/ml il-10; asc +/+ cd8 + t cells = undetectable il-10). cd4 + and cd8 + t cell mixing experiments revealed that asc -/-cd4 + t cells are able to inhibit the proliferative ability of asc -/-cd8 + t cells, asc +/+ cd4 + and cd8 + t cells in vitro and that this suppression appears to be mediated by a soluble factor secreted by activated asc -/-cd4 + t cells. collectively, these results demonstrate that the absence of asc drives cd4 + t cells towards a suppressor cell phenotype, suggesting that asc might play an important role in determining the fate of cd4 + t cells. various members of the eicosanoid family derived from arachidonic acid participate in inflammatory reactions and may act as potent regulators of the immune response. in particular, e-series prostaglandins, pge 1 and pge 2 suppress some t-cell functions including proliferation, activation and cytokine production. pge 2 signals through four types of gpcrs called the ep receptors. at low concentrations, pge 2 is believed to be necessary for t cell function, whereas at higher concentrations, pge 2 inhibits t cell proliferation. these effects are largely governed by various cell specific stimuli and tissue microenvironment. objectives: to delineate, compare and contrast the effects of pge 2 and ep receptor antagonists on t cell activation. methods: flow cytometry, proliferation assays, migration assays. we have observed that pge 2 diminishes expression of early, intermediate and late t cell activation markers. in contrast, pre-treatment of cd4 + t cells with ep receptor antagonists was found to impair cell surface expression of cd71, cd69, cd25 and ox40 but not cd44. suppression of t cell proliferation by pge 2 has already been widely studied. however, blocking ep receptors in cd4 + t cells by the use of ep antagonists prior to activation surprisingly caused a defect in t cell proliferation. migration of cd4 + t cells to the chemokine sdf-1b was also found to be reduced due to pre-treatment with ep antagonists. in order to study the physiological relevance of these findings we studied the trafficking of basal and activated t cells to regional lymph nodes during inflammation in the presence and absence of ep receptor antagonists. this model revealed that the use of ep antagonists causes a reduction in the amount of cd44 + cd4 + adoptively transferred t cells in the regional lymph node following the induction of a local inflammatory response. conclusions: in our study we show for the first time that ep receptors are required for expression of activation markers and activating proliferation in murine cd4 + t cells. our results also suggest that considering pge2-mediated camp signaling in cd4 + t cells, it will be absolutely necessary to distinguish between transient increases, which have potentiating effects, and sustained increases, which have inhibitory effects in t cell activation. objective: our objective was to investigate how ros affect the different stages of t cell activation. because activation is initiated by changes in intracellular calcium concentration, we addressed whether and how ros affect calcium signalling. the experimental results were obtained using a combination of fluorescence microscopy, patch-clamp, t-cell activation assays and molecular biology. results: we show by direct measurement of ros that t-cells are exposed to high concentrations of oxidants when they are in close vicinity of activated phagocytes. the effect of ros on calcium signalling in jurkat t-cells as well as in primary naï ve and effector cd4 + human t-cells was examined. oxidation affects several ca 2+ signalling pathways by altering the activity of ip 3 receptors, trp channels and store operated ca 2+ channels in a concentration dependent manner. interestingly, calcium signalling is differentially affected in naï ve and effector t cells. thiol reducing agents were able to significantly reduce the effects of oxidation implicating thiol oxidation as a major player in the regulation of ca 2+ signalling in t-lymphocytes. cysteins are the main carrier of thiol groups in proteins and we show that orai ion channels contain reactive cysteine groups that mediate ros effects on the calcium influx pathway. conclusion: ros regulate the calcium dependent t-cell activation in a complex way, affecting all three major calcium signalling pathways. by mutational analyses of the orai proteins, we are able to pinpoint molecular targets of regulation. the activation of t cells during an immune response is a crucial but tightly regulated event. to make the grade, the t cells upregulate costimulatory but also inhibitory receptors upon antigen recognition. this enables the t cell to be stimulated for proliferation to keep pace with pathogens infection, but also to become dampened upon successful defense against the pathogens via negative feedback mechanisms. in this study we present data of the signaling mechanisms underlying the potent t cell inhibitory receptor cd147 (emmprin, basigin) , a member of the ig-family. previous studies reported that lymphocytes from cd147 knockout mouse possess enhanced mixed lymphocyte reactions and cd147 monoclonal antibodies can interfere with t cell activation. these observations already pointed to a negative crosstalk of cd147 signals with the t cell antigen receptor or co-stimulatory signals. consistent with these studies, we found that rna interference (rnai) with cd147 in jurkat t cells augments the secretion of the t cell growth-factor interleukin-2 (il-2) upon t cell activation. this up-regulation is at least partially due to an increased activity of the nuclear factor of activated t cells (nfat), which resulted in an enhanced il-2 promoter activity. by reconstituting the rnai-mediated knockdown with various truncated rnai-resistant forms of cd147, we identified the immunomodulatory sub-domain of cd147. supported by the gen-au program of the austrian federal ministry of science and research. mirnas play a critical role in the control of hematopoiesis. the goal of this project is to determine whether mirnas function also during the antigen-induced activation of mature b lymphocytes. therefore, we determined mirna profiles in primary splenic b cells before and after polyclonal activation with either lps (simulates t cell-independent activation) or a combination of anti-igm, anti-cd40 and il4 (simulates t cell-dependent activation). microarray assays identified about 104 mirnas in unstimulated b cells. 35 of these were downregulated and one was upregulated upon stimulation. in silico analyses with various mirna target prediction programs revealed an interesting and promising set of transcripts whose translation/stability could be controlled by mirnas during the antigen-induced activation phase of mature b cells. among these targets are bcr signalling molecules and transcription factors that control proliferation, igh class switch as well as differentiation in antibody-secreting plasma cells. one of these transcripts codes for the interferon regulatory factor-4 (irf-4). the graded expression of this important transcription factor has been shown to coordinate isotype switching with plasma cell differentiation. first results indicate that the expression kinetic of irf-4 transcripts differs from that observed for irf-4 protein abundance after b cell stimulation. further analysis identified the irf-4 transcript as a target whose expression is obviously fine-tuned by a mirna upon antigen stimulation. we are in the process to biochemically verify potential targets for each of the differentially regulated mirnas and determine the effect of ectopic and retrovirally mediated expression of mirnas on b cell differentiation. the work was in part supported by the izkf erlangen, the dfg graduiertenkolleg gk592 and the dfg forschergruppe for832. objective: transforming growth factor-b (tgf-b) signals through type i (tgfbri) and type ii (tgfbrii) tgf-b receptors and receptor regulated smad proteins. tgf-b exerts predominantly anti-proliferative and pro-apoptotic effects which are frequently lost in cancer. the mechanisms of resistance against tgf-b have not been fully elucidated. our aim is to describe how b cell lymphoma cells respond to tgf-b compared to normal peripheral b cells, to create an overview of the different signaling pathways involved, and to characterize the mechanisms behind the loss of sensitivity to tgf-b. methods: proliferation assays were performed on 11 different b-cell lymphoma cell lines and normal peripheral b cells to screen for tgf-b-induced effects. western immunoblotting analysis was conducted to characterize protein expression and phosphorylation related to tgf-b signaling pathways. facs analysis was used to measure tgf-b receptor surface levels. cells were treated with demethylating agents to examine changes in gene expression levels. s. manthey 1 , f. hauck 2 , i. berberich 1 , f. berberich-siebelt 2 , gk 520 -immunomodulation 1 institute for virology and immunobiology, university of wuerzburg, würzburg, germany, 2 university of wuerzburg, department of molecular pathology, würzburg, germany the transcription factor ccaat/enhancer-binding protein b (c/ebpb) can not act only as a transcriptional activator but also as a transcriptional repressor. in murine cd4+ t lymphocytes, the transcription factor is predominantly expressed in t helper 2 (th2) compared to t helper 1 (th1) cells. in contrast, by binding to the c-myc promoter(s), c/ebpb represses c-myc expression thereby arresting t cells in the g1 phase of the cell cycle. both, transactivation and repression depend on the n-terminal transactivation domain of c/ebpb. blimp-1 encoded by prdm1 is a transcription factor necessary for terminal differentiation of b cells to plasma cells. furthermore, blimp-1 is expressed in differentiated effector t cells where it is higher in th2 than th1 cells. the regulation of the blimp1 expression is not fully understood. interestingly, we found that c/ebpb can bind to the prdm1 promoter and activates blimp-1 expression in t cells. as c/ebpb is also expressed in b cells, we hypothesize that this transcription factor might as well influence the expression of blimp-1 in b cells. so far, we were able to show a similar expression profile of c/ebpb and blimp-1 in b cells using cre recombinase. moreover we found a new putative blimp-1 isoform lacking exon 2. currently, we analyze the expression of c/ebpb and blimp-1 in primary b cells and b cell lines after various stimulations. to get more insights into the function of c/ebpb in b and t cells, we are generating mice carrying a b as well as a t cell-specific deletion of c/ebpb. engagement of antigen receptors on lymphocytes leads to rapid increases in intracellular free calcium concentrations via phosphorylation of phospholipase c gamma (plcy) and plays an important role in activation of cells. by screening 53 cvid patients with a flow cytometric assay we demonstrate that calcium flux is significantly reduced in b and t cells isolated from the peripheral blood of patients in the group ia of the freiburg classification as compared to non-ia patients and healthy donors (hd). ia patients are characterized by the expansion of an unusual cd21low b cell population in which calcium mobilization is strikingly lower than in other b cell subsets. common subpopulations like naï ve and mz-like b cells as well as cd4 + t cells but not transitional b cells or cd8 + t cells also revealed significantly decreased calcium peaks. the cytometric data correspond to a semiquantitative rt-pcr assay and functional data showing reduced induction of the calcium dependent macrophage inflammatory protein-1a (mip-1a), and abrogated activation and proliferation, respectively. preliminary data on b cell receptor (bcr) mediated phosphorylation of plcy2 revealed constitutively high background levels in cd21low b cells of ia patients. since phosphorylation in the other b cell populations as well as calcium flux upon ionomycin were the same for patients and healthy donors, we postulate an abrogated amplification or altered inhibitory pathway targeting the signalling events downstream of plcy and upstream of internal store release, thus resulting in defective calcium signalling. the underlying mechanism yet remains to be elucidated and is part of our work in the future. c. balas 1 , v. courtois 1 , k. de luca 1 , r. sodoyer 1 1 sanofi pasteur, marcy l'etoile, france the presence and relative abundance of cytokines at different stages of infection is relatively well documented, but their involvement in immune status, pathogenesis or disease progression is still unclear. a potential explanation to the difficult interpretation of the results obtained might be related to the intrinsic weakness of the analytical techniques. for instance monitoring of the expression level of cytokines, such as il-2, il-4 or il-6 could lead to misinterpretation if molecular isoforms are not detected by antibodies currently used to measure them. the analysis of the human transcriptome is a way to access the subset of genes involved in the immune response upon infection by various pathogens. such an analysis might be completed and enriched by the analysis of the relative expression of some cytokine splice variants. methods: genetic tools (primers and qpcr probes) capable of discriminating and quantifying alternatively spliced messenger rnas from il-2, il-4 and il-6. furthermore, the recognition by several commercial antibodies of the different cytokine isoforms (expressed as recombinant proteins) has been investigated. the genetic tools have been validated on in vitro models as well as on biological samples (please refer to the abstract no a-136-0033-01835). conclusion: implication of such kind of analysis in diagnostic application and disease progression survey will be discussed. in a different context, the same kind of analysis could be applied to the monitoring of the immune response upon vaccination or more generally for new antigens or adjuvant screening. parasitic helminths affect about one third of the world population. therefore the mechanisms, which are involved in the persistence or the expulsion of the parasite, are of special interest. from other parasitic infections it is known, that the regulatory receptor cytotoxic t lymphocyte antigen-4 (ctla-4) plays a crucial role during infections. here, we use the strongyloides ratti infection of mice as an experimental system to investigate the role of ctla-4 during nematode infections. we employed a quantitative real-time pcr (qtpcr) analysis to quantify the migrating larvae (il3) in the tissue and the released eggs and first stage larvae (l1) in the feaces. the cytokine response of lymphocytes, prepared from the spleen and the mesenteric lymphnodes (mln) upon stimulation with polyclonal a-cd3 and s. ratti antigen was determined. additionally the humoral response was analysed in the primary and the secondary infection. to investigate the role of ctla-4 during the infection, a neutralysing antibody (a-ctla-4; 4f10) was administered intraperitoneally (300 mg) two hours before subcutaneous infection with s. ratti il3. the in vivo neutralisation of ctla-4-signalling by applying a-ctla-4 during s. ratti infection led to an altered cytokine response, compared to infected mice treated with a control antibody. we detected an increase in th2 cytokines, such as il-4 and il-5 and a reduction of the proinflammatory cytokines ifn-g and il-17. the investigation of the humoral response showed a remarked increase of the igg1-titer in the serum during secondary infection in mice that had been treated with a-ctla-4 during primary infection. furthermore, the blockade of ctla-4 resulted in a diminished worm burden as indicated by reduced release of l1 in the faeces. these results suggest that the blockade of ctla-4 during s. ratti infection induces an activation of the appropriate effectors of the immune system that are beneficial for the host defence. in particular the transition of the t cell cytokine profile towards a th2 response supports this hypothesis and might be the reason for the reduced worm output in the primary infection. the strong increase of igg1 during secondary infection also reflects the induction of a potent th2 response. objectives: cd36 is a class b scavenger receptor, which has been shown to be involved in the pathogenesis of atherosclerosis as well as in the clearance of apoptotic cells by macrophages. this clearance is important in regulating the immune system to avoid autoimmune reactions, as seen in systemic lupus erythematosus (sle). it was recently described that cd36 is highly expressed also on the marginal zone b cell subtype. we therefore set out to investigate the role of cd36 on the regulation of b cell in the setting of apoptotic cell clearance and autoimmune activation. we used a mouse model for sle where apoptotic cells were injected repeatedly in order to study the auto-reactive antibody response that follows. elisa was used to measure antibody levels and flow cytometry to study cell activation as well as cd36 expression. cd36 knock-out (ko) and wild type mice were used. results: preliminary in vivo data show a tendency for a higher antibody response towards ds-dna and the common self-antigen pc in cd36 ko compared to heterozygous mice. since reduced levels of cd36 are expressed in heterozygous mice we are currently repeating this experiment using wild type mice as controls for comparison. in support of the in vivo findings, the immunosuppressive effect of injected apoptotic cells seen in wild type mice after in vitro stimulation of splenocytes with lps is gone in cd36 ko mice. after one injection of apoptotic cells, cd36 ko b cells are activated while wild type b cells are not. after four injections a break of tolerance is seen and apoptotic cells do no longer have an immunosuppressive effect and we show that cd36 on b-cells are involved in setting this threshold. conclusion: our data suggest that cd36 is involved in the early regulation of b cell response towards apoptotic cells and production of autoreactive antibodies. it does so by being involved in regulation of the tolerance effect exerted by apoptotic cells. successful t cell immunity requires lymphocytes to be at the right time at the right place. the co-receptor cd152 acts as a major check-point of immune responses, but the mechanism by which cd152 controls peripheral t cell responses is unknown. the consequences of cd152 signaling on murine th cell migration were analyzed using chemotaxis assays in vitro and radioactive cell tracking in vivo. the genetic and serological inactivation of cd152 in th1 cells reduced migration towards ccl4, cxcl12 and ccl19. crosslinking of cd152 together with cd3 and cd28 stimulation on activated th1 cells increased expression of the chemokine receptors ccr5 and ccr7, which in turn enhanced cell migration. sensitive liposome technology reveals that mature dendritic cells but not activated b cells were potent at inducing surface cd152 expression and cd152-mediated migration-enhancing signals. importantly, migration of cd152 positive th1 lymphocytes in in vivo experiments increased, as compared to cd152 negative counterparts, showing that indeed cd152 orchestrates specific migration of selected th1 cells to sites of inflammation and antigenic challenge in vivo. these data show that cd152 signaling does not just silence cells, but selects individual ones for migration. this novel activity of cd152 adds to the already significant role of cd152 in controlling peripheral immune responses by allowing t cells to localize correctly during infection. it also suggests that interference with cd152 signaling provides a tool for altering the cellular composition at sites of inflammation and antigenic challenge. here we analyzed the role of cd152 signaling on the longevity of cd28 null t cells. using a sensitive staining method for cd152, we show that human cd4 + cd28 null and cd8 + cd28 null t cells rapidly express surface cd152. serological inactivation of cd152 using specific fab fragments or blockade of cd152 ligands using ctla4ig in cd4 + cd28 null and cd8 + cd28 null t cells reduces the number of non-apoptotic cells in a fas/fasl-dependent manner. cd152 crosslinking on activated cd28 null cells prevents activation-induced cell death (aicd) as a result of reduced caspase activity. apoptosis protection conferred by cd152 is mediated by pi3'k dependent activation of the kinase akt resulting in enhanced phosphorylation and thereby inhibition of the pro-apoptotic molecule bad. we show that signals triggered by cd152 act directly on activated cd28 null t lymphocytes and, due to its exclusive expression as a receptor for cd80/cd86 on cd28 null t cells, prevention of cd152 mediated signaling is likely a major target mechanism taking place during therapy with ctla4ig. objectives: cd45 is a transmembrane protein tyrosine phosphatase (ptp) expressed in all nucleated leukocytes. it activates src family kinases (sfks) by dephosphorylating inhibitory tyrosines in their c-terminal tails. in cd45 -/mouse t cell signaling and development is severely impaired, while other leukocyte populations seem much less affected. at least in part, it is due to the activity of another transmembrane tyrosine phosphatase cd148 (ptprj, dep-1) which acts as a positive regulator of sfk in cd45 -/-b cells and macrophages and can compensate for cd45 deficiency in these cells. indeed, combined deficiency of cd45 and cd148 in mice results in defective macrophage and b cell signaling and development, a phenotype much more severe than the loss of either protein alone. naïve murine t cells do not express cd148 and its expression is increased only after activation. accordingly, no defects in t cell development and signaling in cd148 -/mice were reported so far. however, in human t cells the role of cd148 may be different since naive human t cells express cd148 at a level comparable to b cells. using cd45 -/-/cd148 -/human t cell line (jurkat-derived js-7 cells) we tested the ability of cd148 to complement cd45 deficiency in t cells. we used retroviral transduction to express human cd45 or cd148 in js-7 cells and tested their ability to reconstitute major signaling pathways. we also employed substrate trapping mutant of cd148 to identify direct substrates of this phosphatase. in agreement with previously published data, defective t cell receptor (tcr) signaling was observed in js-7 cells. expression of wild type cd45 or cd148 in js-7 cells resulted in more rapid calcium mobilization, enhanced tyrosine phosphorylation, and increased cd69 upregulation after tcr cross-linking. moreover, the carboxy-terminal tyrosine of lck, major t cell sfk, was hypophosphorylated in js-7 cells expressing cd148 when compared to control cells. finally, cd148 substrate trapping mutant expressed in js-7 cells interacted with lck in vivo suggesting that lck is a direct substrate of cd148 in js-7 cells. the results suggest a level of redundancy between cd45 and cd148 in human t cells not appreciated so far. during the past decades, great efforts have been made to get insights into the complex process of antigen-induced t cell activation and the underlying signal transduction pathways. the t cell antigen receptor signaling cascade is initiated by phosphorylation of itam-tyrosine residues through the t-cell specific src protein tyrosine kinase family member lck. during t cell activation, lck is supposed to undergo structural changes from a closed inactive to an open active conformation followed by phosphorylation of the itam-motifs. in order to resolve conformational changes of lck in living cells with high spatio-temporal resolution, we designed biochemically active conformational-sensitive förster resonance energy transfer (fret) biosensors using cyan and yellow fluorescent proteins inserted at special positions of the complete kinase backbone. for the live-fret imaging and biochemical assays we complemented lck-deficient jurkat t cells (jcam1.6) with the biosensors. by introducing point mutations affecting the two major regulatory tyrosines tyr 505 and tyr 394 we found a dramatic decrease and increase, respectively, of intramolecular fret efficiency compared to the wild type biosensor. these results correspond to unfolding of the biosensor to its active conformation on the one hand and condensation of the kinase structure to its inactive form on the other hand. thus, our biosensor is able to detect phosphorylation modifications of key residues. however, we could not detect any overall change in fret and thus conformation of membrane-associated lck molecules during t cell activation indicating that other mechanisms, presumably reorganization of localization, underlie lck regulation. furthermore, we observed a contribution of intermolecular fret, which indicated homophilic interaction of lck. indeed, by performing single molecule analysis and native 2d immunoblotting we found lck dimers and higher order oligomers. together, these advanced imaging studies in the live cell context provide a novel picture of the function and regulation of this key kinase in signaling via the t cell antigen receptor. it has been reported that mitochondria accumulate under the immunological synapse (is) in response to tcr (t cell receptor) stimulation. this process seems to be required to allow proper tcr-induced calcium influx in t cells in contact with antigen presenting cells (apcs), because mitochondria can sequester calcium and thus keep crac (ca2+ release-activated ca2+) channels open. however, antigen-induced calcium signaling is very fast, and clearly much faster than mitochondrial translocation toward the is. thus, we speculated that other signals are involved in recruiting the organelles to the contact region between t cells and apcs. we found that the adhesion molecule lfa-1 (leukocyte function-associated antigen 1) induces localization of mitochondria at the is. this process is antigenindependent and is enhanced by the presence of chemokines in the t cell environment. however, tcr triggering stabilizes mitochondria at the synapse and it is important to sustain their recruitment in time. our data suggest that, by recruiting mitochondria to the cell-cell contact region, lfa-1 prepares and facilitates tcr signaling. we are performing experiments to understand the signalling pathways involved in mitochondria translocation at the is. burkitt lymphoma (bl) is a high grade b cell malignancy (non-hodgkin lymphoma (nhl)) derived from germinal center b cells, that harbours a chromosomal translocation juxtaposing the protooncogene myc next to the regulatory elements of one of the immunoglobulin loci. however, the precise contribution of myc to the pathogenesis of this tumour is poorly understood. based on the definition of a distinguishing gene expression signature for the molecular burkitt lymphoma (mbl) with myc as one hallmarking signature gene (hummel et al.2006) we describe a non-viral vector based approach (vockerodt et al. 2008) to express myc in primary human gcb cells from pediatric tonsils. comparative whole genome gene expression profiling was performed in 9 independent preparations. our data reveal a global change in gene expression in lymphoma precursor cells by myc giving new insight into potential changes of the gene expression program of gcb cells on the accidental way to bl in addition as a first step the function of selected signature genes in bl is accomplished. in a representative cell line with a mbl signature and with a non-mbl signature rnai directed inhibition of elements of the cd40 signaling cascade was conducted. after activating this particular signaling cascade (cd40) we analysed respective gene expression profiles of ikks, trafs and mapk deficient cells. based on these different rnai-mediated ge-profiles a comparison between both lymphoma types is performed. first attempts are made to reconstruct the topology of the respective signaling pathway by using the nested effects bioinformatic model, which has been described recently (markowetz et al. 2005 ). a rat thymic epithelial cell (tec) line (r-tnc.1) was established from a long-term tec culture. this line was characterized as a type of rat cortical tec with nursing activity (tnc). very little is known about molecular mechanism of the tnc/thymocyes interaction. in our previous studies we investigated molecular mechanisms involved in the binding and emperiopolesis of resting thymocytes by r-tnc.1 cell line in vitro. it was found that a number of adhesion molecules, such as cd2, cd4, cd8, cd11a, cd18, cd54, cd90 was involved in these processes. objectives: a main goal of this study was to define the adhesion molecules involved in the interaction between r-tnc.1 line and activated thymocytes. methods: experiments was performed on inbred ao rats. monoclonal antibodies (mabs)-mediated modulation of thymocyte binding and emperiopolesis was tested by adhesion and engulfment assay, respectively, using a coculture of cona and il-2 activated syngeneic thymocytes and unstimulated or ifn-g stimulated r-tnc.1 cells. we found that both the adhesion (30 min and 3h) of activated thymoytes were partially blocked by mab to cd2 and cd8 molecules (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells). early adhesion was inhibited by mab to cd90, abtcr, mhc class i molecule (ifn-g stimulated r-tnc.1 cells) and cd4 molecule (ifn-g unstimulated r-tnc.1 cells). after prolonged incubation, significant inhibition was obtained using anti-mhc class i mab (ifn-g unstimulated r-tnc.1 cells). almost all mabs which were inhibitory in the binding assay were inhibitory in the engulfment assay (6h), namely mab to cd2, cd4, cd8, cd90 molecule (ifn-g unstimulated and ifn-g stimulated r-tnc.1 cells) and mhc calss i and mhc class ii molecule (ifn-g unstimulated r-tnc.1 cells). our results also suggest the involvement of cd11a/cd18 dependent -cd54 independent pathway in adhesion and cd11a/cd18 dependent -cd54 dependent pathway in emperiopolesis. the obtained results imply that adhesion, deadhesion and emperiopolesis of activated thymocytes by r-tnc.1 cell line are tightly regulated processes in which multiple adhesion molecules are involved. the crucial roles of cytokines in shaping t cell responses have been documented in both healthy and disease conditions. interleukin-27 (il-27), a recently described cytokine, has been shown to exhibit both pro-and anti-inflammatory properties. il-27 favours naï ve cd4 t cell differentiation into th1 cells to the detriment of th17 or th2 differentiation. the il-27 receptor (il-27r) is a heterodimer composed of tccr, which confers ligand specificity, and gp130, a signal transducing chain that is utilized by several other cytokines. il-27 has been demonstrated to promote cytotoxic lymphocyte functions of mouse cd8 t cells, but the potential impact of il-27 on human cd8 t cells has not been elucidated. our goal is to investigate the impact of il-27 on human cd8 t cell functions. we used peripheral blood mononuclear cells (pbmc) from healthy donors, either exvivo or after short term in vitro activation to perform our analyses. we first assessed whether the il-27r is detectable on ex-vivo t cells using flow cytometry. we observed a greater proportion of cd8 than cd4 t cells expressing the complete surface il-27r (gp130+tccr). however, we detected high amounts of intracellular tccr in both, cd4 and cd8 t cells, but only polyclonal activation (anti-cd3) of cd8 t cells led to an actual increase of il-27r surface expression. purified cd8 t cells from healthy donors were shortly stimulated in vitro and then analyzed using flow cytometry-based functional assays. il-27 activated stat1 and stat3 signalling with rapid kinetics in both cd8 and cd4 t cells, indicating the capacity of il-27 to signal through these molecules. addition of il-27 to anti-cd3 activated cd8 t cells led to a significant dose dependent increase of proliferation (as measured by cfse-based assay) and ifn-gamma and granzyme b production (determined by intracellular staining). these results demonstrate a pro-inflammatory impact of il-27 on human cd8 t cells. defects in immune regulation could result in the breakdown of immune tolerance leading to development of multiple sclerosis (ms). the pd-1/pd-l1 pathway is associated with production of the immunoregulatory cytokine il-10, the suppression of t lymphocytes proliferation by inhibition of akt phosphorylation (pakt), and the elicitation of apoptosis of antigen-specific cells; an impairment in this pathway could play a pathogenetic role in ms. we analysed by flow-cytometry the surface expression of pd-l1 and pd1, as well as myelin basic protein (mbp)-stimulated il-10 production, pakt inhibition, and apoptosis (annexin v), in 50 ms patients with relapsing-remitting disease. twenty-six patients were diagnosed as being affected by acute disease (ams); 24 had a diagnosis of stable disease (sms). results showed that: 1) pd-l1 -expressing cd14+ and cd19+ cells are reduced in ams compared to sms individuals (p=0.04); and 2) pd1 expression is increased in cd4+ t cells of sms individuals and is comparable on cd8+ t cells of ams and sms patients. this is associated with a significant decrease in il-10 production by mbp-stimulated cd14+ and cd19+ cells of ams patients (p=0.03). additionally, cd8+ anexin v+ (av+) cells were diminished and cd8+ pakt+ cells were higher in ams compared to sms patients, while similar percentages of cd4+av+ and cd4+ pakt+ were observed in both groups of individuals. data herein show that the impairments of the pd-l1/pd-1 pathway seen in ams patients result in a reduced mbp-specific il-10 production by cd14+ and cd19+ cells as well as in a reduced apoptosis (annexin v) and an augmented proliferation (pakt) of mbp-specific cd8+ t. the pd1/pdl1 pathway plays an important role in the pathogenesis of multiple sclerosis. monitoring of the expression of these proteins could be a novel diagnostic tool. anti-4-1bb in cd8 cells. this difference could be due to down regulation of cd28 by activated lymphocytes and possible preferential response of cd8 cells to anti-4-1bb costimulation. moreover, increase in ifn-g concentration in costimulated cultures also may enhance the suppressive function of mscs which again could explain the inability of costimulation in proliferation recovery. likewise, reducing tgf-ß by costimulation is not sufficient to abolish suppressive effect of mscs. in overall, these results suggest that lack of costimulation expression by mscs is not the mechanism of msc suppression and other mechanisms are involved. cytotoxic t lymphocytes (ctls) kill target cells by secretion of cytotoxic components contained in lytic granules at the contact zone between the target cell and the ctl, the immunological synapse (is). t cell receptor (tcr) enrichment at the is is one of the early and key events of is formation. objectives: soluble nsf attachment receptor (snare) proteins are required in almost all fusion events in cells. in the present study we tested if the snare protein syntaxin7 (stx7) is part of the is and whether it serves as a key player of is formation and/or the fusion process itself. methods: pcr-techniques, cell transfection, immunocytochemistry and different microscopic techniques like confocal microscopy and total internal reflection microscopy (tirf) were used on primary human ctls to test the function of stx7. rna interference technique was also used to down regulate stx7 expression in primary human ctls. results: we identified stx7 in ctls by pcr and immunocytochemistry. stx7 accumulates at the is after ctl/target cell contact. when stx7 function was blocked by overexpression of a dominant negative stx7 mutant (deletion of the transmembrane region), functional studies with tirf showed a reduced accumulation and fusion of lytic granules at the is. furthermore, confocal studies showed a loss of tcr accumulation at the ctl/target contact side. conclusion: these results imply that the snare protein stx7 is present at the is and moreover is required for is formation in ctls. the observed block of lytic granule release is probably caused by disturbing an upstream process such as vesicle transport, recycling or sorting. objectives: despite the 20 years history of mouse t h 1 and t h 2 subpopulations, relatively little is known about the differences in their signaling mechanisms and the membrane organization of critical receptors and signal transducing molecules. we have developed mouse t h hybridomas to study these differences between polarized t h cells. the in vitro established hybridomas were first characterized as t h 0, t h 1 or t h 2 phenotypes, based on their cytokine production (il-2, ifng or il-4). a comparative analysis of t-bet, ifng and il-4 mrna levels was also done on quiescent and activated t h hybridomas. in the present study, the ca 2+response, membrane raft expression/organization, k + -and ca 2+ -ion channel expression/function and sensitivity to apoptosis (aicd) were compared in these hybridomas. expressions and molecular localizations were investigated by flow cytometry and confocal microscopy, respectively. ion channnels were functionally analyzed by patch-clamp technique. apoptosis was analyzed using three markers (mitochondrial membrane potential, caspase activation, dna fragmentation) and flow cytometry. results: expression level of plasma membrane rafts/gangliosides (assessed by cholera toxin b-staining) showed the following rank: t h 1 g t h 0 g t h 2, although the membrane cholesterol level (detected with anti-cholesterol ab, ac8) was similar in the three cells. in connection, tcr displayed stronger colocalization with rafts and appeared more polarized in t h 1 cells upon activation than in t h 2 cells. t h 1 cells produced a more sustained calcium response with higher amplitude than t h 2 cells to the same tcr-mediated triggering signal. interestingly, this does not coincide with the expression of cav1.2 and kv1.3 ion channels, major functional determinants of the sustained calcium influx. t h 2 cells expressed the highest levels of these two ion channels. there were also marked differences in their sensitivity to activation induced apoptosis (aicd) as assessed by three different markers of apoptosis. the results suggest that a different membrane compartmentation/organization rather than the differential expressions of certain receptors, ion channels and/or other upstream signaling molecules of these t h hybridomas may be responsible for the observed differences in their functional characteristics. objectives: bone morphogenetic proteins (bmps) belong to the tgf-b superfamily, which plays a central role in controlling cellular processes like proliferation, differentiation, apoptosis and migration. whereas tgf-b is well established as one of the most potent negative regulators of hematopoietic cells, the role of bmps in b lymphoid cells remains more elusive. in this study we investigated the effects of bmps on mature human b-cells. methods: b cells were isolated from peripheral blood of healthy donors using cd19-dynabeads. cd19 + isolated cells were facs sorted into cd19 + cd27naïve b or cd19 + cd27 + memory b cells. dna synthesis was measured by 3h-thymidine incorporation, immunoglobulin (ig) levels in cell supernatants were measured by elisa and phospho-protein levels were measured by western immunoblotting analysis. results: all bmps significantly suppressed anti-igm-induced proliferation of cd19 + cd27naï ve b cells, of which bmp-6 and -7 were most efficient (40 % suppression). similarly, all bmps suppressed cpg-induced proliferation of cd19 + cd27 + memory b cells by 40 -50 %. to induce differentiation, both naï ve and memory b cells were stimulated with cd40l and il-21. this increased the production of igm, iga and igg 10 -100-fold compared to medium control, whereas addition of bmps inhibited the production of all ig classes. all bmps highly induced phosphorylation of smad1/5/8 in cd19 + b cells. the mechanisms for how bmps mediate their inhibitory effects are currently being explored in more detail. conclusion: bmps have prominent inhibitory effects on anti-igm-and cpg-induced proliferation of naive and memory human b cells, respectively. they also suppress cd40l/il-21-induced production of igs in mature human b cells. s. gutenberger 1 , k. warnatz 1 1 university medical centre freiburg, freiburg, germany background: signals through the b cell receptor (bcr) and co-receptors are essential for the survival, differentiation and effector function of b cells. the stimulation of the bcr initiates several independent but interrelated signaling pathways. one important pathway leads to the activation of mitogen activated protein kinases (mapk) and especially the phosphorylation of extracellular signal-regulated kinases 1 and 2 (erk 1/2). in a subgroup of patients with common variable immunodeficiency (cvid) we have previously demonstrated intrinsic defects in the activation of b cells revealed by the insufficient cd86 upregulation and proliferation after b cell receptor (bcr) stimulation. therefore we assessed signaling pathways downstream of the bcr in order to identify defects in the activation of b cells. methods: pbmc of 20 hd and 25 cvid patients were stimulated by anti-igm. different igm expressing b cell subsets were analyzed separately for erk1/2 phosphorylation by intracellular flow-cytometry using phospho-specific antibodies to erk1/2. to increase the signal intracellular phosphatases were inhibited by h2o2. as markers of activation and initiation of proliferation, cd86 and ki67 expression were measured after 2 days of in vitro stimulation. k. theil 1 , p. aichele 1 1 immh university freiburg, immunology, freiburg, germany type i interferons are homone-like molecules that are produced early after viral and bacterial infections. they signal via the type i interferon receptor (ifnar) and have pleiotropic effects on different cells of the immune system. their best known function is the antiviral activity. to test the direct effect of type i interferons on cd8 t cells in vivo we adoptively transferred lcmv glycoprotein specific tcr transgenic p14 cd8 t cells that are deficient in type i interferon receptor (ifnar-/-) into wild-type b6-recipient mice and compared their expansion with wild-type (wt) p14 t cells after viral infection. we could demonstrate a severe impairment in the capacity of p14 t cells lacking type i ifnr (ifnar-/-) to expand after lcmv infection. following infection of recipient mice with recombinant vaccinia virus, recombinant vsv (vesicular stomatitis virus) or recombinant listeria monocytogenes expressing lcmv glycoprotein, p14 t cells expansion was considerably less dependent on type i ifnr expression. therefore direct type i ifn signalling is essential for cd8 t cell expansion and survival only after lcmv infection. our experiments showed that the lcmv generated cytokine milieu is responsible for the failure of expansion of ifnar-/-t cells during lcmv infection. a suitable model for elucidating the impact of the lcmv generated cytokine milieu is the transfer of p14 t cells into h8 mice. h8 mice ubiquitously express the lcmv immunodominant glycoprotein-epitope gp33-41. therefore the antigen-presentation can be uncoupled from the lcmv induced cytokine milieu when the h8 mice are infected with lcmv8.7. this is a lcmv variant that has got a point mutation in gp33-41 and consequently cannot be recognized by the p14 t cells. s. frischbutter 1 , r. baumgrass 1 1 deutsches rheuma-forschungszentrum, signal transduction, berlin, germany antigen-specific stimulation of t helper cells induces activation of the main transcription factors nfat, nf-kb and ap1 which are important for expression of cytokines such as il-2, ifng and il17. it is known that the immunosuppressive drug cyclosporin a (csa) blocks the activity of the ser/thr phosphatase calcineurin and thereby the activation of the transcription factor nfat. however, we and others observed that this drug also inhibits the activation of nf-kb. to detect targets of calcineurin within the nf-kb pathway we analyzed phosphorylation and degradation levels of different nf-kb signaling proteins in the presence of csa and other calcineurin inhibitors. we found that phosphorylation of the signaling protein bcl-10 was prolonged in cells treated with inhibitors. our data do not indicate an enhanced bcl-10 phosphorylation but rather an inhibition of bcl-10 dephosphorylation. furthermore, calcineurin and bcl-10 co-precipitated with each other. interestingly, this interaction was observed only in t cell receptor-but not in tnfa-stimulated cells. in our proposed model, we hypothesize that calcineurin interacts with the carma/bcl-10/malt1 signaling complex and dephosphorylates bcl-10 and, thus, promotes nf-kb activation. therefore, calcineurin is not only a hub for nfat but also for nf-kb activation. a. t. fulop 1 , j. lamoureux 1 , c. fortin 1 1 université de sherbrooke, medicine, sherbrooke, canada objectives: aging is accompanied by a decrease in immune functions, called immunosenescence. the exact cause is still not known. changes in t cell subpopulations, thymic involution were invoked. we have demonstrated that the signal transduction is altered with aging. in the present work we studied the negative regulatory molecules in the t cell signaling to explain the altered activation of t cells with aging leading to decreased clonal expansion. methods: 25 healthy young and elderly subjects were studied. lymphocytes were separated by fycoll-hypaque. the molecules participating in the negative control loop of lck were studied by western blot and confocal microscopy. the surface expression of ctla-4 has been studied by facscan. the translocation of the molecules in the membrane lipid rafts (mlr) was also studied by western blot. the activity of phosphatases was also determined. results: we found that the phosphorylation of pag was altered with aging explaining the decreased release of csk from mlr and the decreased lck activation. the activation of fynt was also altered. the phosphatase activity studies showed an increase in their activities with aging. the ctla-4 expression was higher after stimulation in t cells of elderly. there was differences between cd4 and cd8 t cells with aging. conclusion: these results suggest that the negative regulation is preponderant in t cells with aging on the positive activation and as such explaining the defect in t cell functions with aging. this opens new therapeutical avenues in the future. in contrast to other members of the tumour necrosis factor superfamily, fas ligand (cd95l) contains a cytosolic proline-rich domain (prd) that enables interactions with sh3 and ww domain proteins. since fasl surface expression is regulated by adam10-mediated ectodomain shedding and fasl might be subsequently released into the cytosol by regulated intramembrane proteolysis (riping) through the secretase-like enzyme sppl2a, we are interested in defining interactions involving the generated intracellular fragment of fasl. employing a monoclonal antibody directed against the intracellular domain of fasl, we observed that previously described fasl-interacting proteins of the pch family selectively bind to the full length molecule but not to n-terminal fragments (ntfs). in order to identify other sh3 domain proteins that potentially interact with the riped fasl prd, we used a sh3 domain phage display library containing all 288 sh3 domains expressed in humans. the screen confirmed several previously identified interactions but also revealed numerous new and interesting candidate binding proteins includig non-receptor tyrosine kinases and adaptor proteins or enzymes implicated in membrane, organelle, and actin cytoskeleton dynamics. selected interactions were verified biochemically and by laserscanning microscopy in transfected cells. it could be demonstrated that tec kinases known to be involved in immune receptor-associated signal transduction as well as members of the snx9 family, which are crucial regulators of endocytic and endosomal dynamics and trafficking, join the list of known fasl-interacting proteins. of note, in contrast to pch proteins, the snxs bound both ntfs and unprocessed fasl, indicating that individual interactors might influence different facets of fasl biology. in conclusion, the present data provide substantial evidence for a selective binding of individual interaction partners of fasl to the full length protein or ntfs. this more detailed glance at the fasl interactome will facilitate focussed strategies to clarify unanswered questions regarding reverse signalling and functional conseoptimal t cell activation requires the engagement of the t cell receptor (tcr) by the specific mhc/antigen complex and costimulatory signals as the interaction of b7 family members on antigen-presenting cells with cd28 on t cells. remarkably, whereas classical glucocorticoids (gcs) effectively suppress solely tcrtriggered t cell activation in vitro, additional cd28 co-stimulation leads to gc-resistance. in this study, we compared the non-steroidal selective glucocorticoid receptor agonist (segra), compound12, with classical gcs regarding their suppressive effect on cd28-costimulated t cells. human primary t cell subpopulations and jurkat cells were stimulated in vitro with plate-bound anti-cd3 and anti-cd28, and proliferation, cytokine secretion as well as phenotypic activation parameters were determined. remarkably, a clearly improved inhibition of ifn-gamma secretion was observed in cd28-costimulated human memory/effector cd4+ t cells by compound12 than by classical gcs. interestingly, apoptosis and activation antigen expression were similarly regulated. improved inhibition of lymphokine secretion by compound12 was also seen after pma / ionomycin stimulation of human primary t cells and jurkat cells. when investigating the in vivo effects of compound12 and prednisolone in acute and subacute dnfb-induced contact hypersensitivity models in mice, we observed comparable efficacy for inhibition of t cell-dependent skin inflammation when treating before hapten challenge. in contrast, however, when treating around hapten sensitization markedly stronger effects were demonstrated for compound12 than prednisolone. when evaluating possible mechanism for the increased activity of compound12 in inhibition of t cell activation we got hints for a specific inhibition of the calcineurin pathway by compound12 which was not prevented by the partial gc receptor antagonist, ru-486, in vitro. moreover, in vivo we observed less induction of il-1beta and tnf-alpha by pre-treatment with compound12 than with prednisolone. our data indicate that the non-steroidal segra, compound12, may represent a promising drug candidate for the treatment of t cell-dependent inflammatory diseases where therapy with classical gcs is hampered by t cell resistance. influenza a infection of b6 mice elicits robust cd8+ t cell responses, with virus-specific cells showing a distinct pattern of cytokine production: tnfa+ cells always express ifng; and il-2+ cells are contained entirely in the ifng+tnfa+ subset. interestingly, the co-expression of ifng and tnfa varies for different epitope specificities. almost all ifng+ pa 224 -specific cells also express tnfa, but only about half of the ifng+ np 366 -specific cells co-express tnfa. this was originally linked to the avidity of the responding population for the specific peptide/mhc complex, with the ifng+tnfa+ phenotype representing cd8+ t cells with higher avidity and a more differentiated phenotype. however, the same cytokine pattern is seen in adoptively transferred cd8+ t cells expressing a clonal tcr, implying avidity alone cannot control development of cytokine profiles. co-expression of ifng and tnfa by adoptively transferred cfse-labelled ot-i cells following infection with influenza a virus expressing ova 257-264 peptide shows a close correlation with division in vivo. early after antigen encounter (0-2 divisions) the vast majority of cells express only tnfa. after 3-4 divisions cells begin to co-express ifng and tnfa. the emergence of an ifng+tnfa-phenotype increases with subsequent divisions (4-6 divisions), indicating cytokine profile is closely linked to cell cycling, as described previously for both b cells and cd4+ t cells. titration of adoptively transferred ot-i cells, which controls the level of expansion in vivo, reveals that more cd8+ t cells develop an ifng+tnfa-phenotype with increased expansion. thus we conclude that while tcr avidity and co-stimulation can impact the differentiation of cd8+ t cells, expansion plays a very important role in the regulation of cd8+ t cell effector function. in addition to its chemo-attractant function, sdf-1a (stromal-cell derived factor-1a, cxcl12) has been described to costimulate cd4 + t cell during tcr triggering. our objective is to clarify the mechanism regulating this costimulatory activity. tcr-driven proliferation of human cd4 + t cells was increased by immobilized sdf-1a to a level similar to that obtained with the costimulatory molecule cd28. as visualized by real time confocal microscopy, t cells entering in contact with sdf-1a formed a tether and displayed an active scanning activity. since sdf-1a induced a similar activity in t cells stimulated with a sub-optimal dose of anti-cd3 mabs, it is conceivable that the sdf-1a-driven scanning may favour productive tcr engagement. to test this hypothesis, we are studying the effect of sdf-1a on tcr internalization, calcium mobilization, mapk activation and actin cytoskeleton reorganization. we are also studying the role of sdf-1a in the context of cd4 + t activation by antigen-presenting cells secreting sdf-1a. this study should help us to better define how sdf-1a modulates cd4 + t cell activation beyond its chemo-attractant function. background: propolis, an ancient herbal medicine, is well known for the management of respiratory diseases. caffeic acid phenethyl ester (cape), an active component in propolis, is known to have anti-tumor, anti-inflammatory, and antioxidant properties. in this study, the effect of cape on the functions of t cells, which play the major role in chronic airway inflammation of asthma, was evaluated. method: cd4 + t cells isolated from human peripheral mononuclear cells by automacs were stimulated with anti-cd3 and anti-cd28 antibodies and cape for 2 days. cytokine levels were dertermined by elisa and lymphoproliferation was analyzed by 3h-thymidine incorporation method. signaling pathway of t cells was studied by western blot. result: it was found that cape significantly inhibited ifn-g and il-5 production and lymphoproliferation in cd4 + t cells stimulated by anti-cd3/cd28. cape could inhibit nuclear factor-kb (nf-kb) activation, but not mitogen-activated protein kinase (mapk) family phosphorylation in t cells. cape could also inhibit akt phosphorylation. conclusion: these results indicated that cape inhibits cytokine production and lymphoproliferation of t cells which might be related to the nf-kb and akt signaling pathway. this study also provided a new insight into the mechanism of cape in immunology and the rationale for propolis in the treatment of allergic disorders. objective: upon activation, cd4 t cells express a variety of molecules on their surface, such as mhc-class ii, cd80, cd86, cd70, whose ligands are constitutively expressed on resting t cells. whereas these molecules are physiologically expressed on antigen presenting cells, their function on t cells is not understood. we tested the hypothesis that activated cd4 t cells might induce t cell proliferation and differentiation from cd4 resting t cells through interaction of activationinduced surface molecules and their constitutively expressed ligands. methods: cd4 t cells from the peripheral blood of healthy donors were co-cultured with fixed activated t cells from the same donor. after 5 days of co-culture, the phenotype of the resulting cells was analyzed by assessing their surface molecules and production of cytokines. results: cd4 memory t cells but not naive t cells proliferated in response to contact with activated t cells. these cells showed a mild activated phenotype assessed by the expression of cd25, cd30, and cd69. analysis of the cytokine profile of these cells revealed the differentiation of il-10-and ifn-g-double-producing cells in response to contact with th1 effector cells, and il-4-producing cells in response to contact with th2 effector cells. the levels of produced cytokines were, however, significantly lower than those produced by activated cells in response to anti-cd3/cd28 stimulation. whereas neutralization of ifn-g or il-4 during culture did not diminish the frequency of the arising cytokine-producing cells, separation of the responder cell population from effector cells by a transwell system led to a significant decrease of cytokine secretion. blocking particular receptor/ligand interactions by neutralizing antibodies against hla-dr, cd70, cd80, and cd86 could not prevent cytokine production induced by t-t cell interaction. however, simultaneous addition of all antibodies significantly inhibited cytokine production to 64-85 %. conclusion: interaction of cd4 memory t cells with activated t cells resulted in the production of the cytokines il-4, il-10, and ifn-g. given the immunomodulatory capacity of il-4 and il-10, these findings might indicate a novel potential negative feedback mechanism to control t cell-driven immunity. a. nasir 1 , s. thompson 1 , j.j. murphy 1 1 king's college london, division of immunology infection and inflammatory disease, london, united kingdom the murine bcl1 leukaemia cell line can be induced to undergo plasmacytoid differentiation in vitro with cytokines il-2 and il-5 and this is characterised by a marked reduction in proliferation and production of large amounts of secreted igm. these cells were observed to express significant levels of the zinc fingercontaining protein zfp36l1 by western blot analysis. this protein is reported to act in post-transcriptional regulation of gene expression by binding to au rich elements (ares) of mrnas of certain genes and consequently promoting mrna degradation. at a cell functional level, zfp36l1 has been described to have roles in apoptosis, proliferation and differentiation in different cellular contexts. cytokine-induced bcl1 differentiation was observed to be associated with downregulation of zfp36l1 protein. in an attempt to determine whether zfp36l1 downregulation was directly linked to bcl1 differentiation, a zfp36l1 shrna expressing lentivirus (psicor) was employed to knockdown zfp36l1 expression. this reagent downregulated zfp36l1 expression very effectively . shrna infected cells proliferated less well than either control virus infected cells or wild-type cells with or without cytokines. zfp36l1 shrna infected cells also produced more secreted igm per cell than either control virus infected cells or wild-type cells in the presence or absence of cytokines. these results are consistent with a role for zfp36l1 downregulation in promoting bcl1 plasmacytoid differentiation. vidual lysates of peripheral blood lymphocytes (pbl) of 32 patients with igg multiple myeloma and healthy controls were investigated for the expression of sialic acid (sa), galactose (gal) and n-acetylglucosamine (glcnac), the sugars known to specify the glycoforms of human serum igg. the degree of glycosylation and signaling status of all 32 isolated myeloma igg bcrs were correlated and compared with the glycosylation of the igg paraproteins isolated from sera of the same patients. it was shown that bcr igg in myeloma is more heavily sialylated when compared with normal controls, that the increased sialylation of igg bcr is associated with higher levels of tyrosine phosphorylation (signaling activity) of the igg bcr supramolecular complex and that bcr igg and serum igg paraprotein from the same patient differed in all cases in the levels of terminal sugar expression. the results suggest that the development of the malignant clone in mm from postswitch b cells expressing igg bcr at their surfaces to plasma cells secreting igg paraprotein may be followed by permanent glycosylation changes in the igg molecules. caused by thapsigargin-induced release of calcium from the endoplasmic reticulum was insensitive to tpen. conclusion: the signal with fluorescent probes for the detection of calcium ions in response to thimerosal is entirely due to zinc release, and no indication for a calcium signal was detected. in light of these observations, zinc may also contribute to calcium signals caused by mercury containing compounds other than tms, and a potential involvement of zinc release in the immunomodulatory effects of these substances should be considered. although best known for its pro-apoptotic function, it seems clear now that cd95 (fas, apo-1) also exerts anti-apoptotic effects associated with costimulation and the induction of proliferation. we investigated effects of fas co-ligation during tcr/cd3/cd28-triggered activation of freshly isolated human t-lymphocytes. to this end, tcr-triggered cells were incubated in presence or absence of different ligand concentrations of anti-apo1 mab, faslfc or faslstrepfc fusion proteins, or leucin zipper (lz-)cd95l. interestingly for all ligands tested, we could clearly demonstrate a correlation between ligand concentration and t cell response: low doses drastically augmented proliferation in the sense of costimulation, whereas high doses completely blocked tcr-induced cell proliferation without inducing cell death. the positive costimulatory effect of fas at low concentrations is associated with elevated il-2 and ifng production, upregulation of activation markers, adhesion molecules and cell-cycle regulating cdks and cyclins. in addition, we observed an increased activation of important signalling molecules including mapk and caspases. using pharmacological inhibitors, we demonstrate that fas is internalized upon ligation. we also observed an increased tcr internalisation following fas co-incubation potentially resulting in the generation of larger signalling platforms that allow optimal t cell activation. in stark contrast, most fas ligands at high concentrations almost completely inhibited cell proliferation of tcr-triggered lymphocytes. in this context, crucial events associated with t cell activation, i. e. tyrosine and erk1/2 phosphorylation, the expression of various activation markers, the il-2 production and caspase activation were almost completely abrogated. these findings highlight that fas-triggering accelerates or blocks t cell activation, depending on the strength of the stimulus. in addition, we provide further evidence for an anti-apoptotic function of fasl during signal initiation in human t lymphocytes. sponsored by the dfg (sfb415) and the medical faculty kiel (to oj) it has been shown that glycosylation of cell surface proteins controls critical t cell processes, including homing, thymocytes maturation, activation, and cell death. plant lectins have been long used to study changes in cell surface carbohydrate structures, to identify leukocyte cell subsets, and as surrogates for authentic t cell activation stimulus. the galb1,3galnac-specific lectin from amaranthus leucocarpus (all) shows a differential binding pattern to murine thymocytes and peripheral cd4+ and cd8+ t cells. in addition, mitogenic stimulus increase 3-fold the all binding to cd4+ t cells. previous studies in human pbmc showed that all binds to human cd4+ t cells and all-binding increased after a mitogenic stimulus using total cell cultures as murine studies. these data suggest that all detects selectively activation-related changes in cd4+ t cell surface carbohydrate but none study has been performed to examine the all effect on human t cell activation. to examine the effect of all on human t cell activation, we analyzed the anti-cd3-dependent activation of purified cd4+ t cells from pbmc in presence or absent of all by measuring proliferation using cfda-se staining, expression of the surface activation marker cd25 and calcium influx by flow cytometry. results showed that all did not induce significantly t cell proliferation or cd25 expression, but enhanced the anti-cd3-dependent proliferation and cd25 expression of purified cd4+ t cells. analisis of calcium influx showed that all enhanced anti-cd3 dependent calcium influx. our findings indicated that all alone does not affect t cell activation but suggested that all induces a costimulatory effect on human cd4+ t cells by up-regulating t cell activation mediated by anti-cd3 stimulus, as further studies have to be performed to elucidate all-induced costimulatory effect. financed in part by papiit-unam (in214609) a. the adaptor protein lat (linker for activation of t cells) has a prominent role in the transduction of intracellular signals elicited by the tcr/cd3 complex. upon tcr engagement, lat becomes tyrosine-phosphorylated and thereby recruits to the membrane several proteins implicated in the activation of downstream signaling pathways, leading to tightly equilibrated programs of activation and survival or induced cell death. the balance between cell survival and cell death is critical for normal t cell development and activation, and is maintained by signals through lymphocyte antigen receptors and death receptors such as cd95 receptor. it has been previously demonstrated that cd95 ligation in t cells induces the proteolytic cleavage of several adaptor proteins, including gads, slp-76, slap-130 and lat. given the dual role of lat as a transducer of activation and negative signals in t cells, we have analyzed the role of the lat cleavage in t cell functions and studied the proteases responsible for this cleavage. objective: the study is designed to explore preliminarily the need of t cells for cytokines during the culture in vitro, which are associated with the activation, proliferation and apoptosis of t cells, and by detecting the expressions of il-rs, co-stimulatory molecules and apoptotic receptors/ligands onto human peripheral blood lymphocytes (hpbls). the results may lay a theoretic and experimental basis for developing the condition media qualified especially to t cell culture. methods: pbls were isolated , and cultured in different media. both immunocytochemistry staining and cell enzyme linked immunosorbent assay (celisa) were used to detect the expressions of il-1r, il-2r, il-3r, il-7r, il-12r, il-18r, cd27, cd28, cdw137( 4-1bb), cd 95(fas) and cd178 (fasl) on hpbls in different cultured time, i. e. 0d, 1d, 3d, 5d, 7d, 9d, 11d and 14d. using typan blue staining, the living cells, dead cells and total cells of each cultured group were counted, then their cell growth curves were drawn out. to evaluate the cellular activity, growth situation and cell cycle of t cells, both mtt and fcm analysis were also performed separately. 1. the expressions of several membrane immune molecules on the lymphocytes in different cultured conditions. 1) the expressions of membrane immune molecules before cultured. 2) expressions of the membrane molecules on hpbls during culturing. 5% fbs rpmi 1640 group (1640 group), il-2 group, pha group... (1) mtt assay. (2) proliferative times and growth curves of hpbls... 1. during cultured in vitro, there are expression changes of the il-1rs (il-1ra, il-2ra, il-2rg, il-3r, il-7r, il-12r, il-18r), co-stimulatory molecules (cd27, cd28, 4-1bb) and apoptosis associated molecules (fas/fasl) on hpbls in different time and cultured media. the expression patterns of the most molecules checked are similar in 1640 group, il-2 group and pha group, but the rests are different. 2. our data also suggest that the hpbls cultured in cd3mcab+cd28mcab+il2+il1a group has a great proliferative potential compared with the other groups. using this condition medium, may have a practical prospect to tumor therapy. 3. celisa will become probably an effective test to detect the expressions of membrane receptors or molecules quantitatively on a large scale. f. beceren-braun 1 , r. tauber 1 1 zentralinstitut für laboratoriumsmedizin und pathobiochemie, berlin, germany l-selectin is a leukocyte cell surface glycoprotein involved in carbohydrate-specific ligand binding which mediates tethering of leukocytes to the endothelial surface during inflammation. apart from its role in adhesion, l-selectin functions as a signal transduction molecule. crosslinking of l-selectin with antibodies or ligand binding to the receptor have been shown to elicit a wide range of cellular responses. in addition to process signals coming from outside of the cell, the intracellular part of l-selectin (lscyto) is also able to conduct intracellular signals, e. g. activates tyrosine kinase p56lck and the ras/rac2 signalling pathway (1) followed by mitogen-activated protein kinases (2) and c-jun n-terminal kinase (1), which leads to an enhanced binding of l-selectin to soluble ligands (3). in our previous work we described an association of lscyto with isozymes of the pkc family which phosphorylate the receptor on serine residues (4). here we show that the protein phosphatase 2a inhibitor phapii is a novel direct interacting partner of the lscyto. we propose a model in which the l-selectin mediated signalling is regulated by the interaction of pkc, pp2a and phapii: phapii binds to the unphosphorylated lscyto. upon l-selectin crosslinking lscyto is phosphorylated, pha-pii dissociates and inhibits the phosphatase pp2a. in addition we have started structural analysis to investigate ligand binding induced conformational changes of the cytoplasmatic domain of l-selectin. v. heissmeyer 1 , e. glasmacher 1 1 helmholtz center munich, molecular immunology, munich, germany during self-antigen recognition, roquin dependent posttranscriptional downregulation of icos prevents t cell help to b cells and autoantibody production. the molecular mechanism by which roquin interferes with icos translation remained unclear. we have identified two critical regions in roquin. the amino-terminus is required for rna binding and can be functionally replaced by conserved sequences from its paralog mnab. the carboxy-terminus mediates p body localization and has specialized in roquin for efficient repression of icos in t cells. using knockout cells of dicer or ago1-4 genes, we prove that roquin mediated repression of icos occurs in the absence of mirisc formation. instead, roquin function required intact p bodies, and was impaired after knockdown of lsm1 and rck or expression of dominant-negative gw182. interestingly, roquin activity is blocked through induced mirisc formation implicating the mutual regulation of different mechanisms of posttranscriptional gene silencing in immune responses. s objectives: upon encountering their antigens, naï ve t cells are activated and driven to clonal expansion and differentiation into armed effector cells. according to the two-signal hypothesis, the induction of an optimal cd4 + t-cell immune response requires both antigen-specific and co-stimulatory signals. in contrast, stimulating naïve cd8 + t cells with specific antigens and costimulatory signals is insufficient to induce optimal clonal expansion and effector functions. thus, cd8 + t cells require additional signals for full activation and further differentiation into effector cells. methods: in this study, we adopted an in vitro approach to dissect the cellular and molecular requirements for cd8 + t-cell activation and differentiation. naïve cd62l hi cd44 lo cd8 + t cells were sorted and stimulated by anti-cd3 and anti-cd28 antibodies. results: firstly, we show that the activation and differentiation of cd8 + t cells require il-2 provided by activated cd4 + t cells at the initial priming stage after stimulation. secondly, this critical il-2 signal is delivered through il2rbg of cd8 + cells and is independent of il-2ra. besides promoting cell proliferation, il-2 stimulation increases the amount of ifng and granzyme b produced by cd8 + t cells. conclusion: therefore, our studies demonstrate that a full cd8 + t-cell response is elicited by a critical temporal function of il-2 released from cd4 + t cells, providing mechanistic insights into the regulation of cd8 + t cell activation and differentiation. most antigenic peptides recognized by cd8 t lymphocytes are produced through degradation of intracellular proteins by the proteasome. however, some antigenic peptides are produced by a proteasome-independent pathway, which is poorly characterized. mage-a3168-176 is a tumor antigenic peptide presented by hla-a1 and widely used for vaccination of melanoma patients. we observed that proteasome and tppii inhibitors failed to block presentation of the antigen by tumor cells. however, processing of this peptide occurred in the cytosol because tap inhibition prevented its presentation. to characterize the cytosolic peptidase producing mage-a3168-176 we setup an in vitro digestion assay using a 20-mer precursor peptide encompassing the sequence of the antigenic peptide. we observed that only the cytosolic fraction was able to produce the antigenic peptide from this precursor. this production was abolished by treating the cytosolic fraction with o-phenanthroline, a broad-spectrum inhibitor of metallopeptidases. this inhibitor also blocked the presentation of mage-a3168-176 by tumor cells. by electroporating hla-a1 cells with a precursor peptide blocked at the c-and the n-terminus, we could exclude the involvement of exopeptidases in the processing of this peptide, and conclude to a major role of a cytosolic metalloendopeptidase. one such enzyme is insulin-degrading enzyme (ide). we observed that depletion of ide abolished the capacity of a cytosolic fraction to produce the antigenic peptide. furthermore, recombinant ide was able to produce the peptide in vitro from the precursor peptide. lastly, silencing of ide with sirna reduced presentation of the peptide by tumor cells. with tppii, ide is the second example of a proteasomealternative pathway in the production of class-i restricted peptides. antigen-specific t cell based tumor immunotherapy, though extensively studied, has only been of limited clinical success so far. immune escape, due to impairment of hla dependent tumor epitope presentation is believed to be one major reason for this failure. to identify novel mechanisms by which tumors can become refractory to immune elimination, human melanoma cells of different donors expressing the transmembrane mart-1/melan-a tumor antigen were exposed to two or three rounds of brief co-culture with mart-1/melan-a 26-35 specific cytotoxic t lymphocytes (ctls). immune selected melanoma cell clones, being resistant to lysis by mart-1/melan-a 26-35 ctls due to impaired epitope processing were further investigated. our results show that in addition to previously described immune evasion mechanisms like down regulation of mhc class i and mart-1 expression, the ifn-gamma independent endoplasmic reticulum associated degradation (erad) pathway is crucial for mart-1/melan-a 26-35 epitope generation. moreover, deregulation of several erad components is essentially responsible for the observed immune escape of the immune selected melanoma cells. in support, re-expression of down-regulated erad components in ctl-resistant melanoma cells completely restored immune recognition by mart-1/melan-a 26-35 ctls. thus, our studies demonstrate for the first time that erad not only plays a central role in the production of cd8 + t cell epitopes from membrane proteins but also contributes to tumor escape mechanisms by cancer immunoediting. studies of t cell responses to hen egg lysozyme suggest that several conformers of peptide-mhc class ii complexes can be generated for a single peptide epitope and that distinct cd4 t cell repertoires known as type a and type b recognise these different conformers (lovitch and unanue, immunol rev 207: 293-313, 2005) . type a t cells recognise peptide-mhc complexes generated from intact proteins after intracellular antigen processing under h2-m (dm) control, where as type b t cells respond to synthetic peptides in the absence of dm editing, but fail to respond to processed intact protein. type b t cells escape thymic deletion in mice (petersen et al., immunity 11: 453-462, 1999) , with implications for autoimmunity. so we studied whether type a and type b recognition patterns occur in t cell responses to autoantigens such as the rheumatoid arthritis (ra)-associated proteoglycan aggrecan, and whether naturally occurring extracellular ligands that activate type b t cells are found in inflamed joints. lymph node cells from aggrecan-immunised balb/c mice proliferated in response to intact aggrecan and to the immunodominant peptide 84-103, whereas peptide-immunised mice responded to peptide, with low or absent responses to intact aggrecan. t cell hybridomas generated from 84-103 peptide-immunised mice either recognised peptide only (the majority) or peptide and intact aggrecan (the minority), a pattern consistent with type a and type b t cell recognition. responses to staggered and alanine-substituted peptide sets showed that type a and b t cell hybridomas recognized the 84-103 epitope in the same register, consistent with this peptide epitope binding to mhc in distinct conformers. type b t cell hybridomas recognised aggrecan fragments in supernatants from cartilage degraded by stimulation with proinflammatory cytokines that induce raassociated aggrecanases. our data suggest that inflammation generates extracellular peptides that activate type b t cells. we are also characterising human type b t cell responses as well as searching for type b t cell ligands in synovial fluid from ra patients. we propose that extracellular cartilage degradation generates ligands that induce autoreactive type b t cell responses which participate in the pathogenesis of autoimmune arthritis. a to cope with mhc i antigen presentation hcmv encodes for several post-translational strategies which have been extensively studied in transfected cells. in this study we analysed the plc in naturally hcmv-infected cells and monitored the composition of the plc throughout hcmv replication. metabolic labeling experiments revealed the absence of tapasin incorporation into the plc. in contrast, western blot analysis demonstrated only a slow decline of tapasin steady state levels in infected cells, suggesting a blocked synthesis rather than degradation. tapasin mrna levels were found to be continuously downregulated during infection, however, the tapasin transcripts were stable and long-lived. taking advantage of a novel method, in which newly transcribed rna is selectively labeled and analysed (dölken et al, 2008), we found, after an initial induction at 8 hrs p. i., a strong inhibition of tapasin transcription at 24 hrs p. i. furthermore, also reduction of tap1 and tap2 transcription was observed contrasting to the elevated levels of erp57 and mhc i transcripts. importantly, ectopic expression of tapasin restored the incorporation of tapasin into the plc in hcmv-infected cells. the data indicate that hcmv controls mhc i antigen presentation also on a transcriptional level and show for the first time the regulation of tapasin transcription as a viral immune evasive function. most peptides presented by mhc class i molecules are produced by the proteasome during degradation of intracellullar proteins. two main proteasome types have been described, differing in their content of catalytic subunits. the standard proteasome comprises catalytic subunits ß1, ß2 and ß5, which are replaced by their ifng-inducible counterparts ß1i, ß2i and ß5i in the immunoproteasome. the thymoproteasome represents a third proteasome type, where catalytic subunit ß5i is replaced by a thymus-specific subunit ß5t. the standard proteasome is present in most tissues, the immunoproteasome in found in cells exposed to ifng and in dendritic cells, while the thymoproteasome is found exclusively in the thymus. we produced a panel of novel antibodies that recognize subunits ß1i, ß2i, ß5i and ß5 in their native form. using these antibodies for successive immuodepletions performed on tumor lysates, we identified two new proteasome types that are intermediate between the standard proteasome and the immunoproteasome, i. e. they contain only one or two the three catalytic subunits of the immunoproteasome. one comprises ß1,ß2 and ß5i (single intermediate proteasome), and the other comprises ß1i, ß2 and ß5i (double intermediate proteasome). we quantified these intermediate proteasomes in a series a tumor lines of various origins, and found that they represent 10-20 % of the total proteasome content of those tumor cells. they are also present in dendritic cells, where they represent about 50% of the proteasome content. we characterized the activity of these intermediate proteasomes, not only on fluorogenic substrates but also on actual antigenic peptides recognized by anti-tumor ctl. with respect to antigens known to be processed differently by the standard and the immunoproteasome, the intermediate proteasomes often behaved like the immunoproteasome. importantly, we identified two tumor antigens that are processed exclusively by either the single intermediate proteasome ( tapasin is a multi-functional protein dedicated to mhc-i biosynthesis; it serves as a structural component in the so called mhc-i peptide loading complex (plc), as a chaperone putatively acting as an active peptide editor and mhc-i quality control mechanism, as an er retention signal for immature mhc-i, and as a chaperone stabilizing tap expression and increasing tap-performance. furthermore, tapasin has been found outside the er, where it has been suggested to regulate retrograde transport of escaped immature mhc-i back to the er from the trans-golgi compartment. the role of tapasin as an active peptide-editor has been debated and we here set out to study the effect of tapasin on binding of peptides of both high-and low-affinity to a human mhc-i allele (hla-a*0201) using protein interaction-and peptide-competition assays. specifically we wanted to in detail compare the binding of two peptides of the same affinity. at high concentrations all of the tested hla-a*0201 binding peptides (tap-transported high-affinity peptide (ttp-ha), signal-peptide of high affinity (sp-ha), tap-transported mediumaffinity peptide (ttp-ma)) induced dissociation of hla-a*0201 from tapasin, but only ttp-ha dissociated hla-a*0201 from tapasin at lower concentrations. using peptide-competition assays against ttp-ma, a peptide of lower affinity, we could show that ttp-ha, one of the two peptides of equally high affinity was a significantly more efficient competitor than peptide sp-ha. however, analysis of mhc-i peptide loading in the tapasin-negative cell line lcl-721.220-a2 showed no competitive advantage of ttp-ha compared to sp-ha supporting a role for tapasin as a selective facilitator of mhc-i peptide binding. in conclusion, we here show that peptides of different affinities dissociate hla-a*0201 from tapasin in a dose-dependent manner, and that tapasin facilitates ttp-ha, but not sp-ha replacement of a lower-affinity peptide (ttp-ma). together these data strongly suggest a role for tapasin as a selective facilitator of peptide binding to mhc-i. importantly, this study implies that criteria in addition to peptide-affinity determines whether tapasin will promote peptide binding to hla-a*0201. m. basler 1,2 , c. lauer 2 , m. groettrup 1,2 1 biotechnology institute thurgau, kreuzlingen, switzerland, 2 university of constance, division of immunology, department of biology, konstanz, germany two lmp7-dependent antigens have been described that relied on the 'structural presence' of lmp7 in the proteasome but not on the activity of lmp7. here we have investigated processing of the h-2d b -restricted uty 246-254 epitope of the male minor antigen uty reported to be lmp7-dependent. using splenocytes from lmp7 -/-, lmp2 -/and mecl-1 -/mice we found that the uty 246-254 epitope requires lmp7 and lmp2 but not mecl-1. curiously, a selective lmp2 inhibitor did not interfere with uty 246-254 presentation. objective: we investigated why the deletion but not the inhibition of lmp2 interferes with uty 246-254 presentation. we hypothesized that the 'structural' requirement for lmp2 is based on replacement of the caspase-like activity of b1 in the proteasome. methods: it was determined if t1a mutants of lmp2 and/or b1 can rescue the uty 246-254 epitope. we used a b1-selective inhibitor to determine if the inhibition of the caspase-like activity of b1 preserves the epitope. finally we determined by mass spectrometry if the uty 246-254 epitope embedded within a 25mer precursor peptide is differentially cleaved by lmp2-deficient and proficient immunoproteasomes in vitro. results: we found that t1a mutants of lmp2 and b1 rescue presentation of uty [246] [247] [248] [249] [250] [251] [252] [253] [254] . also inhibition of cells with a b1-selective inhibitor preserves uty [246] [247] [248] [249] [250] [251] [252] [253] [254] presentation. an aspartate in position 7 of the uty 246-254 sequence wmhhnmdli is preferentially used as a cleavage site by lmp2-deficient but not half as frequently by lmp2-proficient immunoproteasomes. the generation of the uty 246-254 epitope relies on the replacement of the caspase-like activity of b1 by lmp2 because the b1 activity destroys the uty 246-254 epitope. this is the first example for the 'structural' requirement of lmp2 for generation of an epitope. eliminating the activity of their constitutively expressed homologous subunits may explain the requirement for immuno-subunits of the proteasome also for the generation of other antigens. thus we have discovered a so far unrecognized mechanism how lmp2 and perhaps also lmp7 and mecl-1 exert their function in antigen processing. a. linnemann 1 , a. musiol 2 , r. lindner 1 1 hannover medical school, cell biology, hannover, germany, 2 hannover veterinary school, graduate school for biomedical sciences, hannover, germany objectives: mhc i molecules are constitutively endocytosed and recycled to the cell surface. this process is required for the turnover of aged molecules and for some forms of cross-presentation of exogenous peptides on mhc i. in fibroblasts, mhc i is known to internalize via a clathrin-independent, arf6-regulated pathway that is highly sensitive towards the cholesterol-sequestering drug filipin. although this observation suggests that membrane rafts are involved in the internalization of mhc i, no evidence for an association of mhc i with membrane rafts has been found in this cell type. methods: a novel detergent extraction protocol was used to investigate the association of mhc i with membranes rafts. endocytosis of mhc i was measured with a biotinylation-based biochemical assay and with a cell biological assay employing confocal laser scanning fluorescence microscopy. for characterization of mhc i internalization pathways, dominant negative mutants of gtpases (dynamin and arf6) were overexpressed in 3t3 fibroblasts. we show that antibody-mediated oligomerization of mhc i in 3t3 fibroblasts shifted this molecule from soluble fractions to detergent-resistant membranes. this change in detergent resistance coincided with a switch to a novel internalization pathway: oligomerized mhc i internalized faster and more completely and arrived at different endocytic organelles. the two mhc i internalization pathways differed in their sensitivity towards dominant negative arf6: endocytosis of oligomerized mhc i was not affected, whereas non-oligomerized mhc i endocytosed more slowly and changed its subcellular distribution. unlike transferrin receptor internalization, none of the mhc i endocytosis pathways was affected by overexpression of dominant negative dynamin suggesting internalization mechanisms independent of clathrin, caveolin and rhoa. conclusion: we propose that mhc i switches from an arf6-regulated to a novel, arf6-independent internalization pathway in response to a change in membrane environment induced by oligomerization of mhc i. since mhc i is one of the cellular receptors for sv40 virus and since sv40 binding triggers mhc i oligomerization, this novel pathway may be involved in sv40 uptake. antigen cross-presentation in dendritic cells is a complex intracellular membrane transport process, but the underlying molecular mechanisms remain to be thoroughly investigated. in this study, we tested the effect of sirna-mediated knockdown of 57 rab gtpases, the key regulators of membrane trafficking, on antigen cross-presentation. twelve rab gtpases were identified to be associated with antigen cross-presentation, and among which rab3b, 3c were found to be colocalized with mhc class i molecules at perinuclear tubular structure. tracing with fluorescence protein tagged beta2-microglobulin demonstrated that the mhc class i molecules were internalized from plasma membrane to rab3b and rab3c postitive compartment. moreover, the recycling ligand transferrin was enriched in the rab3b or 3c positive vesicles. furthermore, the rab3b, 3c positive compartment were colocalizd with a fraction of rab27a at a juxtaposition of phagosomes. together these data demonstrate that rab3b and rab3c positive vesicles is involved in and may constitute the recycling compartment of exogenous antigen crosspresentation. introduction: while the proteasome is thought to generate most of the hla peptidome, other proteases were also proposed to be significant for this process. both t cell based assays and proteasome inhibitors were used in the past to follow presentation of specific model hla peptides. the hla-peptidomes presented at the cell surface depend on the rate of peptide generation within the cells, their transport from the cytoplasm and loading in the er, binding stability at the cell surface and retrograde uptake of the hla molecules back into the cytoplasm. objectives: the role of the proteasome in hla peptide presentation was evaluated using proteasome inhibition, while following the turnover rates of the entire hla peptidome. the peptidomes of both the authentic membranal and a recombinant soluble form of the hla molecules were collected for analysis at different time points after the inhibition of the proteasomes. the turnover rates of the hla-peptides were followed using pulse-chase analysis with stable-isotope labeled amino acids concurrently with epoxomicin treatment. the hla molecules were immunoaffinity purified and the peptides were analyzed by capillary chromatography and orbitrap tandem mass spectrometry. both the endogenous membranal and soluble mhc molecules were studied in parallel from the same cells. peptides were identified by their ms/ms fingerprints and the turnover rates were determined by the shift from the 'light' to the 'heavy' leucine of each peptide. results: a few thousands hla-peptides were identified, and for a large portion of them, the turnover rates could be defined. proteasome inhibition did not affect the complexity of the hla peptidomes or reduced significantly the amounts of membranal hla molecules. many peptides were labeled relatively rapidly with heavy leucine, indicating that the hla peptidome contains also the products of newly synthesized and rapidly degrading proteins. the source proteins of the hla peptides seemed to have similar biological functions and cellular origins in both the inhibited and untreated cells. the centrality of proteasomal degradation in hla-peptide presentation is put into doubt and the role of the proteasome in the generation of each peptide and each cleavage site can be defined. epstein-barr virus (ebv) is a ubiquitous y-herpesvirus, infecting over 90 % of adults worldwide. it can cause mononucleosis and several lymphomas and carcinomas, reflecting the tropism of the virus for b-lymphocytes and epithelial cells. ebv persists for life despite the presence of virus-specific adaptive immunity, indicating that it has evolved strategies to counter the host immune response. one such strategy is the persistence of the virus in the latent phase of its life cycle, where expression of viral proteins is minimized. however, for ebv replication and dissemination to occur, it must enter the lytic phase. here, over 80 viral proteins are expressed, creating many potential antigens for presentation to cytotoxic t -lymphocytes. ebv can circumvent possible eradication by cd8+ t lymphocytes during the lytic phase by interference with antigen processing and presentation through hla class i in the infected cell. the viral proteins bnlf2a and bglf5 have been shown to achieve this by impairing peptide-loading of hla class i and inducing the degradation of mrnas encoding hla molecules, respectively. a third ebv lytic phase protein, the g-protein coupled receptor (gpcr) bilf1, has now been found to down-regulate cell surface hla class i expression (zuo et al, plos pathogens 2009). this represents a novel function for a virally-encoded gpcr. bilf1 is expressed early in the ebv lytic cycle and is localized predominantly at the cell surface. there it can interact with hla class i molecules, resulting in their internalization and lysosomal degradation. this has a profound effect on the ability of cytotoxic t-lymphocytes to recognize cells displaying antigens derived from ebv proteins. interestingly, bilf1 displays a differential effect on distinct hla class i haplotypes. furthermore, we have shown that the intracellular c-terminal tail of bilf1 is required for its effect on hla class i expression. however, the ability of the gpcr to activate intracellular signaling pathways is dispensable in this regard. thus, by reducing the cell surface expression of hla class i molecules, ebv bilf1 can hinder the recognition of virally-infected cells by cytotoxic cd8+ t lymphocytes, thereby facilitating the evasion of adaptive immune mechanisms. t lymphocytes mature in the thymus, generating a non-dangerous t cell repertoire. for the adquisition of tolerance, thymocytes suffer positive and negative selection processes. during t cell maturation, tcrs contact with different mhc-peptide complexes on the surface of pressenting cells, allowing tolerization against self proteins. to obtain a non-self-reactive t cell repertoire, it is of most importance that pressenting cells in the thymus express a repertoire of mhc-peptides complexes representative of the proteins that t cells will found in periphery, including tissue restricted antigens (tras)-derived peptides. in the last decade, transcription of tras in thymus has been well-reported. furthermore, the expression of many genes codifying for tras are dependent.on the expression of the autoimmune regulator (aire). aire is mainly expressed in medullar thymic epithelial eells (mtecs), which are involved in negative selection. so far no systematic study have been made to describe the peptide repertoires associated to hla molecules in the thymus. in addition, although many data of tras transcription in thymus have been reported, much less work has been performed at biochemical level, and to our knowledge, no hla ligand arising from any tra have been reported in thymus. in this report we present the results of analyzing the hla-dr-associated peptide repertoire from whole tissue samples of different human thymi by mass spectrometry. we describe 131 natural ligands, including two peptides derived from semenogelin-1, a tissue restricted antigen expressed mainly in the prostate, and present in semen. using qpcr we demonstrate that semg1 is transcribed in thymus from both male and female individuals. finally, we detected the semg1 mrna expression in a fraction enriched in stromal cells, but not in the thymocyte fraction of the thymi. the proteasome is the major protease complex for non-lysosomal protein degradation in eukaryotic cells, which generates most peptides for mhc class i antigen presentation. vertebrates express two sets of catalytic subunits, constitutive (beta1, beta2, beta5) and immuno-subunits (beta1i, beta2i, beta5i). deficiency in beta5i results in profound reduction of mhc class i expression, demonstrating the significance of this subunit for efficient antigen presentation. currently, this is attributed to the specific proteolytic activity of the beta5i subunit, its role in the maturation of immunoproteasomes or both. however, re-expression of catalytically inactive beta5i subunits is capable to rescue antigen presentation suggesting that the proteolytic activity of this subunit is not limiting in this process. here, we show that following infection with listeria monocytogenes induction of beta5i expression increases the cellular proteasome content in the infected organs. our results indicate that this is due to the high chaperone activity of its propeptide which drives proteasome neosynthesis and thus enhances the overall proteasome quantity. further, mhc class i antigen presentation on beta5i-deficient cells could be restored by treatment with d3t, which increases the amount of proteasomes independent of beta5i via induction of mixed proteasomes containing beta1i, beta2i and beta5. consequently, not the lack of the specific proteolytic activity of beta5i or immunoproteasomes, but the reduced proteasome quantity in beta5i deficient cells is the major limiting factor for mhc class i cell surface expression. . we have previously shown that lc, in contrast to ddc, do not express cell surface tlr2, 4 and 5, which results in their inability to respond to both gram-positive and gram-negative extracellular bacteria in terms of maturation into immuno-stimulatory cells and production of inflammatory cytokines. therefore, the question remained what the role is of lc in class ii mhc-mediated activation of anti-bacterial t cells. we determined the capacity of ddc and lc to internalize and process whole bacteria and present bacterial antigens to cd4 + t cells. in vitro generated lcs and ddcs were cocultured with gfp-expressing bacteria and subsequently analysed by clsm and facs for their uptake capacities. furthermore we investigated their capacity to stimulate autologous bacteria-specific t cell lines as a measure for antigen presentation. results: we found that lc are principally able to internalize bacteria, but far less efficient than ddc. moreover, visualisation of bacterial uptake by em revealed different uptake mechanisms by lc and ddc. both in lc and ddc internalized bacteria were detected in the endosomal and lysosomal compartments of the mhcii processing route. nevertheless, presentation of bacterial antigens by lc on mhcii was inefficient compared to that of ddc, as indicated by a low capacity to activate autologous bacteria-specific cd4 + t cells. the presence of exogenous tlr3 and tlr8 ligands did not overcome the differences between lc and ddc, indicating that the impaired capacity to internalize and process bacteria and activate bacteria-specific t cells is not due to the lack of tlr signalling or insufficient expression of co-stimulatory molecules, but could be an intrinsic characteristic of lc. conclusion: we propose that the epidermis of the skin is an immune-privileged site where lc play a minor role in anti-bacterial immunity and may play a role in inducing tolerance to the bacterial skin flora by steady-state presentation of antigens from commensal skin bacteria. e. james 1 , i. bailey 1 , t. elliott 1 1 university of southampton, cancer sciences division, southampton, united kingdom regulatory t cells (tregs) play a pivotal role in the suppression of tumour specific t cell responses. depletion of tregs in balb/c mice results in a robust immunity to the normally poorly immunogenic ct26 colon carcinoma. this response is long lasting and mediated by both cd4 and cd8 t cells. importantly, the treg depleted ct26 specific immunity is cross-protective; capable of mediating rejection of tumour lines of different histological origins (a20, c26, bcl1, renca) implying a broader repertoire of response. we have characterised one of these cross-protective antigens, gsw11, which is h2-d d restricted. analysis of the generation of gsw11 in ct26 revealed that the peptide is susceptible to over-processing by the er-resident aminopeptidase eraap. inhibition of eraap in ct26 cells substantially increased the amount of gsw11 present, observed by increased t cell responses to the tumour in vitro and hplc analysis. this increase was in spite of an overall reduction of mhc class i molecules at the cell surface. to investigate whether the increase in immunogenicity following knockdown of eraap would protect mice, we generated stable eraap knockdown (kd) ct26 and immunised balb/c mice. greater than 80 % of mice injected with eraap kd ct26 were found to reject the tumour. analysis of t cell responses revealed the presence of gsw11-specific t cells, however, these responses were small (0.5-1 %). this compared to a much larger response to ct26 (˚5 %). preliminary results indicate that the majority of the t cell responses (non-gsw11-specific) in these mice are directed toward unstable peptide/mhc complexes, possibly indicating presentation of n-terminally extended peptide antigens. this highlights manipulation of the peptide repertoire as a potent tool for the generation of t cell responses in vivo. minor histocompatibility antigens play important roles in the outcome of stem cell and organ transplantation as they are involved in the development of graftversus-host-disease and in the graft-versus-tumor reactivity in hla-identical stem cell transplantation [1] . the di-allelic hla-a2 restricted minor histocompatibility antigen ha-1 locus codes for the highly immunogenic ha-1 his and the non-immunogenic ha-1 arg nonapeptides, differing in one amino acid. the only difference that could explain the absence of the ha-1 arg immunogenicity was the estimated numbers of cell surface presented copies i. e. 80/cell for ha-1 his and less than 5/ cell for ha-1 arg [2] . as ha-1 his/arg is hematopoietic system specific and shows additional expression on epithelial cancer cells while absent on the normal epithelial cell counterpart, the ha-1 his allele is currently used for boosting the graft-versus-tumor responses after hla matched ha-1 mismatched stem cell transplantation. to elucidate the mechanisms underlying the differential cell surface presentation of the ha-1 allelic peptides, we investigated the impact of the ha-1 his/arg polymorphism on molecular and cellular processes involved in the intracellular generation and stable cell surface presentation of hla class i-bound peptides. therefore, proteasome-mediated digestion experiments, tap translocation analyses, and hla-dissociation assays with ha-1 his and ha-1 arg peptides were performed. moreover, the crystal structures of hla-a2 in complex with either ha-1 his , ha-1 arg or a ha-1 variant with a citrulline residue at position 3 were determined in order to obtain atomic level insights into the conformation of the hla-a2/ha-1 peptide complexes. our results exclude a role for antigen processing in preventing ha-1 arg to be presented at the cell surface and both the structural and hla-dissociation data clearly show that the lack of cell surface expression essentially results from an increased instability of the ha-1 arg allele in the hla-a2 peptide binding groove [3] . they provide a rationale for the lack of ha-1 arg peptide immunogenicity essential for the choice of tumor peptides for stem cell based immunotherapeutical application. proteasomes play an important role in mhc class i antigen processing. exposure of cells to proinflammatory cytokines such as tnfa or ifng leads to the expression of three facultative catalytic proteasome subunits (i. e. immunosubunits) that replace the constitutively expressed subunits in the cellular proteasome population. immunoproteasomes generate many pathogen-derived cd8 t cell epitopes with high efficiency and thereby shape the specificity of the pathogen-specific cd8 t cell response. on the other hand, immunosubunit expression is not essential for development of cd8 t cell-mediated protective immunity, thus the physiological relevance of these cytokine-induced proteasome subunits remains unclear. we observed that mice that lack the immunosubunits lmp7 (ib5) and mecl-1 (ib2) develop a variety of autoimmune responses, including a latent form of t1d (or insulin dependent diabetes mellitus, iddm), following irradiation and bone marrow reconstitution. iddm development in these mice is characterized by inflammation of the islets of langerhans, glucose intolerance and increased water consumption, and is dependent on the presence of cd8 but not cd4 t cells. a cd8 t cell epitope, encoded by the islet beta cell-expressed "islet-specific glucose-6-phosphatase catalytic-subunit-related protein" (igrp) mrna, was identified as an important target of the cd8 t cell response. this epitope, like many other known diabetes-associated epitopes, binds its presenting mhc class i molecule with low affinity. as t cells specific for low affinity binders most likely can escape central and peripheral tolerance while t cells specific for high affine binders do not, we postulate that inflammation-induced immunoproteasome expression primarily functions to replace self-peptides that are derived from tissue-associated antigens and bind mhc class i molecules with low affinity, by a higher affine peptide species towards which t cell tolerance exists. thus, the inducible proteasome subunits may play an important role in immune regulation, by removing the targets of potential auto-immune cd8 t cells that enter inflamed tissues. endocrine epithelial cells, targets of the autoimmune response in thyroid and other organ-specific autoimmune diseases, express hla-ii molecules with compact conformation and are therefore expected to stably bind autologous peptides. the role of these molecules is not known but they could be involved in the maintenance and regulation of the in situ autoimmune response. to study in situ t cell responses without characterizing self-reactive t cells, we have identified natural hla-dr-associated peptides from autoimmune organs that will help finding peptide-specific t cells in situ. here we report the first analysis of hla-dr natural ligands from ex-vivo graves' disease-affected thyroid tissue. using mass spectrometry, 162 autologous peptides were identified from hla-dr-expressing cells, including thyroid follicular cells, some corresponding to predominant molecules of the thyroid colloid. most interestingly, eight of the peptides derived from a major thyroid autoantigen, thyroglobulin. cell-free in vitro binding assays were performed with the thyroglobulin peptides and some other thyroid-eluted peptides as controls, to identify to which hla alleles were these peptides associated in vivo. all but two of the thyroglobulin peptides showed low binding with the corresponding alleles. the two peptides with relatively high binding affinity were presented in the context of dr3 and dr4. analyzing the digestion patterns used for the generation of the thyroid peptides, a preferentially cleavage after a lys and arg was observed for all of them, independent of the restricting allele. our data demonstrate that although the hla-dr-associated peptide pool in autoimmune tissue mostly belong to abundant ubiquitous proteins, peptides from autoantigens are also associated to hla-dr in vivo and therefore may well be involved in the maintenance and the regulation of the autoimmune response. the t cell response generated following herpes simplex virus type 1 (hsv-1) infection is known to be crucial in the clearance of replicating virus and in limiting the severity of infection. despite this, the relative contributions of cd4 + and cd8 + t cells in hsv-1 immunity have yet to be clearly elucidated. to better understand the role of hsv-1-specific cd4 + t cells in immune control we have identified a 13 amino acid epitope derived from glycoprotein d of hsv-1. following flank infection, gd-specific cd4 + t cells were first detected in the draining brachial and axillary lymph nodes (ln) 5-days post-infection (pi), peaking at day 7 and declining thereafter. gd-specific cd4 + t cells were first recovered from the spleen, skin and dorsal root ganglia (drg) at day 6 pi and peaked at day 9. while hsv-specific t cells were first observed in the draining ln at day 5 pi, hybridoma assays showed ex vivo presentation of the gd epitope by brachial ln cells as early as 2 days pi, with peak activity 4 days pi before declining to background by day 7. however presentation of the gd epitope was much more prolonged in vivo as proliferation of transgenic gdspecific cd4 + t cells was observed up to 23 days post-infection in the brachial ln. ex vivo analyses suggest that only cd11c + cells were involved in gd antigen presentation at days 2, 5 and 15 post-infection. subdivision of dendritic cells (dcs) populations indicated that both skin-derived dcs and cd8a + dcs can present the gd antigen to cd4 + t cells at day 2 pi, whereas by day 5 pi the skin-derived dcs were the predominant population presenting the gd epitope. together these data show that following hsv-1 infection, antigen presentation is initiated rapidly and persists well after clearance of replicating virus. furthermore, we present evidence that different dc populations have distinct roles in the presentation of viral antigens and that they may vary during the course of infection. complementary zippers induced complete dimer formation, whereas identical zippers impaired stable interactions of the tagged peptidases. we also verified that the zippers did not influence the substrate "preferences" of the respective erap. our results from in vitro digestions suggest that the stabilised heterodimer is significantly more efficient in the production of a model epitope than the mix of monomeric erap1 and erap2 unable to form dimers. this observation is not due to mere thermodynamic stabilisation but involves positive cooperative effects in the heterodimers. conclusion: allosteric interaction of erap1/erap2 in heterodimeric complexes enhances the global efficiency of precursor peptide trimming in the human er. during the biogenesis of class i molecules, newly synthesized heavy chains fold and acquire disulfide bonds while interacting with the lectin-chaperone calnexin (cnx) and its associated thiol oxidoreductase erp57. upon assembly of the heavy chain with b 2 m, the class i molecule enters a peptide loading complex (plc) that consists of the tap transporter, tapasin, the calnexin homologue calreticulin (crt) plus associated erp57. both crt and erp57 are required for efficient assembly of peptide-loaded class i molecules and their subsequent expression at the cell surface. we examined functional sites on crt and erp57 to gain insights into their mechanisms of action in class i biogenesis. for crt, its lectin function is thought to be crucial for its association with class i molecules. however, when crt mutants lacking lectin function were expressed in crt-deficient cells, they completely complemented all class i biosynthetic defects. thus polypeptide-based contacts either mediated through erp57 or directly between crt and the heavy chain are sufficient to effect the chaperone and quality control functions of crt in class i biogenesis. we also tested the notion that erp57 must be recruited by cnx or crt to function on class i molecules. we found that the rates of heavy chain disulfide formation were normal in cells lacking cnx, crt or both chaperones. furthermore, an erp57 point mutant that fails to bind to cnx or crt was just as effective as wild type erp57 in normalizing rates of disulfide formation. we conclude that erp57 does not require recruitment by cnx or crt and likely acts directly on class i heavy chains to promote disulfide formation. furthermore, in cells expressing the erp57 point mutant, class i heavy chains, crt and the tapasin-erp57 disulfide conjugate were present at normal levels in the plc, indicating that the interaction between erp57 and crt is not required for plc assembly. finally, we show that mutations that destroy the enzymatic function of erp57 have no effect on plc stability or class i surface expression, suggesting that erp57 plays a structural as opposed to catalytic role in plc function. autoimmune pancreatitis (aip) underlies 5-11 % of cases of chronic pancreatitis and is characterized by prominent lymphocytic infiltration. a strong association of aip with the hla-drb1*0405/dqb1*0401 haplotype has been reported, but identification of the predisposing hla gene(s) has been precluded by strong linkage disequilibrium. here, we show that hla-dr*0405 transgenic ab0 nod mice suffer from aip and additional pneumonitis after sublethal irradiation and adoptive t cell transfer from syngenic donors, leading to complete pancreatic atrophy. pancreas histology is characterized by destructive infiltration of the exocrine tissue with cd4+ and cd8+ t cells, b cells and macrophages. mice with complete pancreatic atrophy have reduced serum lipase activity, develop fat stools and loose weight on regular chow. hla-dr*0405 transgenic mice (cd4+ t cell competent) develop aip even unprovoked, similar to ab0 nod mice (cd4+ t cell deficient), while hla-dr*0401, hla-dq8 or hla-dr*0405/dq8 (double-) transgenic controls all remain normal after same treatment. we conclude that hla-dr*0405 fails to protect from aip, likely due to defects in the induction of cd4+ regulatory t cells. our results identify hla-dr*0405 as a prominent risk factor for aip on the hla-drb1*0405/dqb1*0401 haplotype. this humanized mouse model should be useful to study mechanisms that underlie the hla association of autoimmune diseases, but also immunopathogenesis, diagnostic markers and therapy of human aip. s. khan 1 , c. britten 1 , h. overkleeft 2 , g. van der marel 2 , k. melief 1 , d. filippov 2 , f. ossendorp 1 1 leiden university medical center, section tumorimmunology, leiden, netherlands, 2 leiden university, biosynthesis group, leiden, netherlands objective: we have targeted peptide antigens to dendritic cells by the use of synthetic peptides chemically coupled to synthetic tlr ligands to study the impact on mhc class i and class ii antigen presentation. the potency of the vaccine was addressed by monitoring antigen presentation, priming of t-cells and tumor protection. results: our data show that this type of targeting of peptides greatly improves antigen presentation and t-cell priming compared to free peptide. vaccination of mice with the tlr-ligand peptide conjugates induced high numbers of functional cd8 and cd4 t-cells that could protect mice for aggressive melanoma. this potency relies on tlr signaling since peptide coupled to a non-functional tlr ligand was unable to support induction of specific t-cells. these data indicate that simultaneous encounter of antigen and a maturation signal are crucial for optimal t-cell activation by dendritic cells, and show the potency of tlr-l peptide conjugates as a vaccine modality. y. shi 1,2 , x. hu 1,2 , a. kawanatachikawa objectives: nef protein of human immunodeficiency virus (hiv) holds some important immunodominant ctl epitopes. two overlapping 8-mer and 10-mer epitopes (rypltfgwcf (nef138-10) and rypltfgw (nef138-8)) were found to be presented by hla-a*2402 and some immune escape mutants of these two epitopes have also been found in some patients, e. g. y2f, y2w, t5c, f6l, w8r, f10r, f10y etc. or their combinations. it's important to study the molecular basis of the peptide being displayed on the cell surface, through which we can analyze the mechanism of immune escape of hiv. methods: refolding method was used to attain the soluble protein pmhc. crystals are grown using hanging drop vapor diffusion method and x-ray diffraction technology is used to determine the structure. we have determined six peptide-mhc(pmhc) structures containing nef138-10 (wild type) and its four mostly common immune escape mutants (y2f, t5c, y2f&t5c, f6l), and also nef138-8 (wild type). we found that there was little difference between the nef138-10 (wild type) and nef138-10 (y2f) when they were displayed in the peptide-binding groove of mhc molecule, except water molecule distribution near the anchor residue y2 or f2. interestingly the central bulge region of the peptide was becoming very flexible for the nef138-10 (t5c) and nef138-10 (y2f&t5c), which may affect the binding of peptide and the recognition of t cell receptor. for nef138-10 (f6l), the side chain of l6 was more flexible compared to the nef138-10 (wild type). alignment of the nef138-10 and nef138-8 showed that the nef138-8 became flat and the side chain of f6 was not solvent-exposed due to shortening of the length of the peptide. conclusion: as the peptide nef138-10 was featured, while the peptide nef138-8 was featureless, so the different topology of these two epitopes indicates that they have different tcr repertoire diversity in hiv-specific responses. different immune escape mutants of nef138-10 was using different strategies to avoid the killing of host ctls, which indicates that the therapy strategy based on the cellular immune response should be diversity. for the in vivo or ex vivo activation of antigen-specific t cell responses long synthetic peptides are used to activate both cd8+ and cd4+ t cells. in this study we investigated the efficiency and mechanism of cross-presentation of these long synthetic peptides in mhc class i. we observed a large variation in the effectiveness of activation of specific t cells by the extended peptides corresponding to different epitopes, indicating a difference in the efficiency of processing and presentation of these peptides. for the hla-a2 restricted cmvpp65 derived nlv epitope specific t cells were most efficiently activated by n-terminally extended variants of the minimal epitope, while the use of c-terminally extended variants resulted in a 2-3 log reduction of activation efficiency. this pattern was seen for 5/9 epitopes tested in different hla restrictions. furthermore, for all epitopes tested, extending both the c-terminus and n-terminus led to 2-5 log less efficient activation of the specific t cells, compared to the minimal peptide. exchange of the c-terminal sequence of the c-terminal extended hla-b7 restricted cmvpp65 rph peptide with the c-terminal extended nlv peptide led to the enhancement of t cell activation by the exchanged nlv peptide, indicating a role of the extended peptide sequence in the efficacy of processing and presentation of the peptide. tap-deficient t2 cells loaded with extended nlv peptides efficiently activated nlv-specific t cells, indicating that the route of presentation was tap-independent. addition of lactacystin did not affect activation of specific t cells, illustrating that crosspre-sentation was proteasome-independent. primaquine reduced the activation of specific t cells by extended nlv peptides, but not by the minimal nlv 9-mer peptide, suggesting that cross-presentation was dependent on endosomal recycling. these data suggest that long synthetic peptides can be processed by peptidases in endocytic compartments and presented by recycling mhc class i molecules. not all immunogenic epitopes that have been selected in vivo for efficient processing and presentation by the classical pathway may be presented efficiently by cross-presentation. therefore, a rational design of peptides is crucial for efficient activation of cd8+ t cells in approaches of vaccination, adoptive transfer and immune monitoring. antigenic peptides presented by mhc class i molecules are fragments that are usually excised from intracellular proteins while these are degraded by the proteasome. recently, three antigenic peptides were found to result from the splicing of segments that are not contiguous in the parental protein. for two of these peptides, splicing was found to occur in the proteasome by a mechanism of transpeptidation resulting from the nucleophilic attack of an acyl-enzyme intermediate by a free peptide fragment. one of them is derived from melanocytic protein gp100 and requires excision of a four-amino acid intervening segment. the other peptide is derived from protein sp110, and requires splicing in the reverse order of two segments initially separated by six amino acids. the first spliced antigenic peptide described was derived from fibroblast growth factor-5 (fgf-5) and was recognized by human cytotoxic t lymphocytes directed against kidney cancer cells. it is made of two spliced fragments, which are initially separated by a long segment of 40 amino acids. the splicing mechanism of this peptide has not been worked out. the length of the intervening segment made the transpeptidation model more difficult to account for the splicing of this peptide. we therefore evaluated the role of the proteasome in the splicing of this peptide. we observed that the spliced fgf-5 peptide was produced in vitro after incubation of proteasomes with a 49-amino acid long precursor peptide. we evaluated the mechanism of the catalytic reaction by incubating proteasomes with several peptide precursors in a pair wise manner. the results confirmed the transpeptidation model of splicing. we further compared the production of the fgf-5 spliced peptide by cells transfected with mutant constructs encoding fgf-5 proteins where the intervening segment was shortened from 40 amino acids to 30, 20 or 8 residues. we observed an increase in the production of the spliced peptide that was proportional to the reduction in length of the intervening segment, as predicted by the transpeptidation model. finally, using the spliced gp100 peptide model, we observed that splicing did not occur at a significant level between fragments of two distinct proteins in the cell. the polymorphic residues within the peptide binding cleft of hla class i molecules not only diversify the range of peptides presented to cytotoxic t lymphocytes but also influence the pathway of antigen presentation. in order to acquire high affinity peptides, some class i allotypes, such as hla-b*4402, are heavily dependent upon tapasin and other molecules comprising the peptide loading complex (plc). other class i molecules, like hla-b*4405, appear to largely bypass this complex but are consequently loaded suboptimally with peptide. hla-b*4402 and b*4405 are naturally occurring allotypes that differ by only a single amino acid, making this difference in behaviour all the more remarkable. we have previously speculated that such tapasin-independent class i molecules may have been selected in response to viral inhibitors that target the plc, such as the human cytomegalovirus us3 protein. to address this hypothesis, us3 was stably coexpressed in b lymphoblastoid cell lines expressing hla-b*4402 or hla-b*4405. in the presence of us3, the surface expression of hla-b*4402 was substantially reduced whereas hla-b*4405 expression was relatively unaffected. although us3 was able to form complexes with both hla class i allotypes, only hla-b*4402 was retained intracellularly in an immature form whereas hla-b*4405 was transported to the cell surface. accordingly, in the presence of us3, hla-b*4405, but not hla-b*4402, constitutively presented a hla-b44 restricted alloantigen to reporter t cells, suggesting that us3 binds hla-b*4405 without interfering with peptide loading. us3 has been reported by others to bind the plc but surprisingly we have not detected such us3-plc complexes in our system. rather, in the presence of us3 we identified a pool of class i molecules distinct from the plc and only present in us3 expressing cells, implying that us3 may act independently of the plc. these findings demonstrate how hla class i polymorphism not only impacts upon the t cell repertoire and diversifies determinant selection, but also serves to evade the impact of viral inhibitors on antigen presentation. c. massa 1 , b. seliger 1 1 martin-luther-university halle-wittenberg, institute of medical immunology, halle, germany in the attempt to optimize vaccine dc, modifications have been proposed both in the antigen loading and in the maturation protocols. for dc loading "whole antigens" are now preferred to peptides. therefore, it is important to consider not only the costimulatory properties of the vaccine dc, but also their antigen processing abilities. this is even more important since there is the trend to stimulate dc with tlr ligands combined with ifn-y in order to induce dc not only able to correctly migrate, but also secreting the bioactive il12p70. since ifn-g is known to influence the expression of multiple proteases involved in antigen processing, aim of this study was to compare the various maturation cocktails for the consequences on the antigen processing capabilities of the dc in parallel to their costimulatory potential. for this purpose monocyte-derived dc were stimulated for 24h with the gold standard of maturation (tnfa, il1b, il6 and pge2) or a combination of ifn-g and different tlr ligands. the dc obtained exhibit a similar expression of costimulatory and adhesion molecules together with the ability to induce proliferation of allogeneic pbmc, but differ for the pattern of proteases expression as evaluated by real time pcr. with the exception of the downregulation of the tripeptidyl peptidase ii (tppii), no dramatic differences were observed for endo-and aminopeptidase between immature and "gold standard" mature dc. in response to the "ifn-gcontaining" cocktails there was a similar tppii downregulation, but also the induction of many other enzymes. the cytosolic leucine aminopeptidase-3 (lap3) had a more than 20-fold increase in transcription levels, whereas the mrna expression of the aminopeptidases of the endoplasmic reticulum erap1 and erap2 and of the immunoproteasome subunits lmp2 and lmp10 was enhanced between 2 and 5-fold under these culture conditions. with regard to the different tlr ligands used in combination with ifn-g, there was a reproducible higher mrna induction in the presence of the tlr4 ligand mpla in comparison to the tlr-3 and 7/8 ligand polyi:c and r848. these data suggest that the maturation cocktail of dc may alter the peptide repertoire presented by hla class i surface antigens. it has been suggested that mast cells might serve, under certain circumstances, as antigen presenting cells for t cells. however, whether cognate interactions between mast cells and class ii restricted cd4 + t cells actually occur, is still an open question. we addressed this question using peritoneal cell-derived mast cells (pcmc) as an antigen presenting cell model. our results show that in vitro treatment of pcmc with ifn-g and il-4 induced surface expression of mature mhc class ii molecules and cd86. when ifn-g/il-4 primed pcmc were used as antigen presenting cells for cd4 + t cells they induced activation of effector t cells but not of their naive counterparts as evidenced by cd69 up-regulation, induction of proliferation and cytokine production. confocal laser scanning microscopy showed that helper ot-ii t lymphocytes form with pcmc functional immunological synapses, characterized by pkcq enrichment and ifn-g polarized secretion towards the antigen-presenting mast cells. finally, upon cognate interaction with ot-ii t cells, mast cells lowered their threshold of activation via fceri. our results show that mast cells can establish cognate interactions with class ii restricted helper t cells, implying that they can actually serve as resident apc in inflamed tissues. h the vast majority of peptide ligands presented by mhc class i molecules is thought to be produced by cytosolic degradation of source proteins by the proteasome. although, next to cytosolic and nuclear proteins, proteins targeted to the endoplasmic reticulum (er) can also be degraded through this pathway following retrograde transport into the cytosol, antigen processing of er proteins remains little characterized. studying processing and presentation of er-targeted and cytosolic forms of proinsulin (pi), an autoantigen playing a pivotal role in triggering of cellular autoimmune responses in type 1-diabetes, we found that er-targeting of this model antigen has profound effects not only on how pi is degraded, but also on regulation of its synthesis. as expected, proteasome inhibition inhibited degradation of cytosolic pi as well as presentation of the epitope insulin b15-23 to specific cd8+ t cells. in contrast, prior exposure of cells to proteasome inhibitors strongly reduced production of er-targeted pi (pre-pi) through induction of er stress, both in cells infected with a recombinant vaccinia virus and in cells transfected with a tetracycline-regulated expression system. experiments using conditions permissive for pre-pi expression showed that er-targeting modified proteolytic processing of pi for mhc class i presentation. these experiments suggested that two proteolytic pathways contribute to degradation of er-targeted pi, with their relative contribution depending on the stability of the protein. while degradation of unmodified pre-pi was partially dependent on the proteasome, removal of one or several disulfide bridges increased the role of the proteasome in processing of pre-pi for presentation, while introduction of a site for n-glycosylation had the opposite effect. these findings imply that er-targeting together with structural features can have profound effects both on antigen production and on the pathway of proteolytic antigen degradation and presentation. cd8 + t cell immune response to exogenous antigens relies on cross presentation by dendritic cells (dcs) in secondary lymphoid organs. recently, in several infectious murine models, it has been shown that in addition to dc located in tissues, de novo differentiating dc participate in the protective th1 immune response. the role of de novo differentiating dc in cross presentation is however poorly documented, and difficulties of human immunology prevent the accurate identification of the apc subsets patrolling for exogenous ag. a prerequisite for cross presentation is a moderate ag degradation rate in the endocytic pathway, allowing the generation of antigenic epitopes and their binding to mhc molecules. this prerequisite is of special importance considering dc precursors (such as monocytes), which are not yet dcs and may take up antigen before differentiating into dcs. the objective of our in vitro study is to evaluate whether ex vivo purified human blood monocytes are able to cross present long antigenic peptides to cd8+ t cells and whether they are able to sustain this cross presentation while differentiating into dcs. we have previously shown the unique property of dendritic cells to maintain for several days the capacity to stimulate cd8+ tumor-specific t cell clones when pulsed with long antigenic peptides (that need to be processed before presentation to cd8+ t cell clones, faure, 2009, eur j immunol 39 (2): 380-90). in the present study, we address the question of the mechanisms of long peptide cross-presentation by blood monocytes along the course of their in vitro differentiation into dcs. we have shown that despite their high degradative capacity, ex vivo purified monocytes pulsed with long peptides are able to stimulate cd8+ t cells after their in vitro differentiation into dc, 6 days following their antigenic pulse. the delineation of apc subsets able to sustain ag cross-presentation and t cell stimulating potential might be of clinical relevance in immunotherapy using synthetic long peptides. viral genomes contain alternative reading frames (arfs) encoding for mhc-i restricted epitopes (arf-epitope). in the siv/macaque model, ctl responses directed against arf-epitopes participate in controlling viral replication. we previously described that hiv-1 genome contains arfs within gag, pol and env genes encoding for a panel of hla-b*07 restricted epitopes. qprsdthvf (q9vf/5d) is one such epitope but its parental epitope qprsnthvf (q9vf/5n) has a significant higher frequency among hiv-1 isolates. strikingly, q9vf/5d-or q9vf/5n-specific ctls recognize apcs infected with hiv strains encoding for q9vf/5d (e. g. hiv lai ). in contrast, hiv strains (e. g. hiv nl-ad8 ) encoding for q9vf/5n do not activate ctl responses raising the possibility that q9vf/5n epitope is not presented by infected cells. we asked whether introducing mutations within q9vf might be a mean for the virus to escape ctl responses directed against this arf-encoded epitopes. we dissected the mechanism responsible for the lack of q9vf/5n mhc-i presentation. we modified hiv lai to introduce a d to n mutation in q9vf. introducing this single amino-acid mutation abrogated ctl recognition indicating that this asparagine (n) alters q9vf mhc-i presentation. we performed in vitro proteasomal digestions of 28mer peptides encompassing q9vf/5d or q9vf/5n and cleaved polypeptides were analyzed by mass spectrometry. the asparagine (n) in q9vf/5n is a preferential proteasomal cleavage site. thus suggesting that proteasome cleavages within q9vf/5n might be responsible for its lack of mhc-i presentation. we then sought in hiv-infected patients for the presence of proviruses encoding for q9vf/5d or q9vf/5n, and ctls responses directed against these epitopes. far thus, two out of three donors tested recognized the q9vf/5d peptide. we cloned and sequenced hiv-1 genomes from the three donors. surprisingly, out of 20 hiv proviral genomes isolated from pbmcs of q9vf/5d reactive donors, we could not find any virus bearing the q9vf/5d sequence. the isolated hiv sequences either encoded for q9vf/5n or had a stop codon within the epitope. in contrast, viruses encoding for q9vf/5d were isolated from pbmcs of the q9vf/ 5d nonreactive patient. altogether, our data suggest that ctls exert a selection pressure on viral arfs. hiv-1 seems to escape immune surveillance by introducing mutations altering processing of arf-derived epitopes. i. e. flesch 1 , y. wang 1 , d.c. tscharke 1 1 the australian national university, biochemistry and molecular biology, canberra, australia vaccinia virus (vacv) was the live vaccine used to eradicate smallpox and some strains are now being used as vectors for recombinant vaccines. cd8 + t cells recognizing viral peptides in association with mhc class i molecules on infected cells play a crucial role in the defence of viruses. despite the large number of possible mhc class i-peptide combinations, cd8 + t cells only recognize a small number of epitopes, a phenomenon called immunodominance. using recently defined cd8 + t cell epitopes for vacv in mice, we have investigated how heterozygosity of mhc class i molecules influences immunodominance patterns in h-2 bxd f 1 mice compared with their inbred parent strains. we find that the immunogenicity of vacv peptides defined using inbred mice is variable in f 1 progeny, with some peptides being almost equally immunogenic in f 1 and inbred mice, while others elicit responses that are reduced by more than 90 % in f 1 mice. during acute infection as well as memory responses, the dominance hierarchy in inbred mice did not predict the epitopes that would be poorly immunogenic in f 1 mice. in line with these findings, a multiepitope construct expressed by a recombinant vacv was less immunogenic in f 1 mice than would be predicted from its performance in parent strains. in terms of mechanism, we find evidence of altered tcr repertoires including in the case of one epitope, the loss of many diverse tcr vb clones and outgrowth of cd8 + t cells with a restricted vb usage in f 1 mice. these data have implications for our interpretation of experimental vaccine work done in inbred mice and for our understanding of how mhc diversity can alter the range of epitopes that are immunogenic in outbred populations. objective: tlr ligands are being exploited as potential adjuvants, and have impact on the antigen processing and presentation by dendritic cells (dc). therefore we aimed to study the efficacy of a tlr2 agonist, s-[2,3-bispalmitoyiloxy-(2r)-propyl]-r-cysteinyl-amido-monomethoxyl polyethylene glycol (bppcysmpeg), a synthetic derivative of the mycoplasma macrophage activating lipopeptide (malp-2), as an adjuvant for cross-priming against cellular and soluble antigens. malp-2 has been characterized as an effective mucosal adjuvant and synthesis of bppcysmpeg further improved solubility and pharmacokinetic features of the adjuvant. methods: dc isolation, in vitro and in vivo t cell stimulation, intracellular cytokine staining, in vivo cytotoxicity assays. results: systemic administration of bppcysmpeg induced maturation of cd8 + and cd8 -dc in the spleen resulting in enhanced cross-presentation of intravenously co-administered soluble antigen in mice. in addition, administration of bppcysmpeg and cell-associated ova resulted in generation of an effective ctl response against ova in vivo in a t-helper cell-dependent manner, but independent of ifna. delivering antigenic peptides directly linked to bppcysmpeg led to superior ctl immunity as compared to giving antigens and adjuvants admixed. in contrast to other tlr ligands such as cpg, systemic activation of dc with bppcysmpeg did not result in shutdown of antigen presentation by splenic dc subsets, although cross-priming against subsequently encountered antigens was reduced. we provide evidence that bppcysmpeg stimulation of dc via tlr2/6 results in the generation of an effective ctl response and that delivering antigenic peptides linked to bppcysmpeg is a promising strategy for vaccination. while bppcysmpeg-matured dc retain their antigen uptake and presentation capabilities, cross-priming against subsequently encountered antigens is inhibited, indicating that mechanisms beyond down-regulation of macropinocytosis and phagocytosis contribute to shut-down of cross-priming after tlr-mediated dc maturation. altogether our study promotes synthetic lipopeptides as potential adjuvant for specific applications (e. g. viral infections, cancer) for the reason that they can be chemically engineered to carry specific antigenic peptides which allows targeting of antigens and simultaneous activation. tumor immunevasion. to verify whether the loss of erap1 expression could confer a survival advantage on tumor cells and enhance tumor progression, we stably knocked down expression of eraap (murine erap1) in a murine t lymphoma cell line, rma. we used a method that allows an efficient and continuous expression of mirnas that directly silence eraap and obtained several eraap-deficient rma clones with different levels of eraap expression (up to 90 % of reduction at the protein level). microsomal aminopeptidase activity and mhc class i surface expression were decreased in all clones proportionally to eraap expression. moreover, low expression of eraap affected the stability of mhc class i molecules as evaluated after acid and brefeldin a treatment. de-regulated er peptide trimming also drastically affected the tumor formation of rma cells and host survival. eraap-deficient rma clones with different levels of eraap, 50 and 10 % as compared to control rma cells, were injected s. c. in the flank of c57bl/6 syngenic mice, and analysed tumor growth. all mice injected with control rma cells developed a tumor but survived up to 45 days after injection. all mice injected with rma clone with a 50 % level of eraap expression developed a tumor and died within 23 days after injection. surprisingly, any animal injected with rma clone with a 10 % level of eraap died or showed a visible tumor. thus, knockdown of eraap expression appears differently to affect the immunogenicity of rma cells, depending on the eraap silencing level. hemophilia a is an x-chromosome-linked bleeding disorder caused by the absence or dysfunction of clotting factor viii (fviii). treatment consists of regular administration of fviii, but is complicated by the formation of inhibiting antibodies against fviii. both genetic and treatment-related factors play a role in the etiology of inhibitor development in patients with hemophilia a. the development of inhibitory antibodies in hemophilia a patients has been shown to be a cd4 + t-cell driven process. therefore, in order to better understand the process of inhibitor formation, we aim to identify the epitope specificity and phenotype of t cells against fviii in hemophilia a patients using mhc class ii tetramers. cd4+ t-cell responses of two monozygotic twins with severe hemophilia a were analyzed. one of these subjects developed a high titer inhibitor (207 bu/ml) following intensive factor viii (fviii) treatment. high dose immune tolerance therapy together with anti-cd20 therapy resulted in eradication of the inhibitor. in contrast, his twin brother developed a low titer inhibitor (2.5 bu/ml) which declined rapidly after tolerance induction. fundamental differences in the twins' antibody responses were further suggested by elevated and persistent igg4 levels in the subject with the high titer inhibitor. in order to gain a better understanding of processes leading to inhibitor formation versus tolerance, we investigated drb1*0701-restricted t-cell responses of the high titer inhibitor subject, using fluorescent mhc class ii tetramers loaded with 20-mer synthetic fviii peptides to stain epitope-specific cd4+ cells.cd4+ t-cells from the high-titre inhibitor subject recognized three peptides corresponding to the fviii a2 domain: fviii 405-424 , fviii 421-440 and fviii 653-672 , as well as the c1 domain peptide fviii 2093-2112 , but not any c2 domain peptides. the c1 domain peptide contains a sequence that was reported as a promiscuous t-cell epitope (jones td et al., j thromb haemost.85:123-33, 2005 ). analysis of t cells from the lower titer inhibitor subject is expected to reveal differences in the epitope specificity and phenotypes of t cells that may underlie the discordant immune responses of these twins to infused fviii. m. forloni 1 , s. albini 1 , m.z. limongi 1 , l. cifaldi 1 , d. fruci 1 1 ospedale pediatrico bambin gesù, rome, italy neuroblastoma (nb) is a pediatric tumor that derives from neural crest. the most aggressive forms are characterized by amplification of the mycn oncogene and severe reduction of hla class i expression. mycn has been claimed to hinder hla class i expression through affecting the expression of the transcription factor p50 nf-kb subunit. since in many human tumors the expression of hla class i molecules is positively co-ordinated with that of er aminopeptidases, erap1 and erap2, we wondered whether in nb cell lines mycn may impair expression of these aminopeptidases. to explore this possibility, nb cell lines that differ in mycn expression were quantified for expression of mycn, erap1, erap2 and hla class i heavy chains by western blotting and for surface hla class i expression by flow cytometry. we found that mycn negatively correlates with expression of hla class i, erap1 and erap2. this negative correlation was confirmed in a nb cell line expressing a tetracycline repressible mycn transgene. then, by the use of tnfa (a nf-kb nuclear translocation stimulator), sulfasazine and ikba mutant (two nf-kb nuclear translocation inhibitors) and knockdown of p65 nf-kb subunit, we demonstrated that nf-kb is involved in erap1 and erap2 expression in nb cell lines and that mycn does not affect nf-kb expression. furthermore, we showed that mycn and nf-kb are recruited to the promoter regions of erap1 and erap2 and that mycn affects the recruitment of nf-kb binding to these promoter regions. in conclusion, the present results indicate that an enhanced mycn level, linked or not to mycn amplification, represses erap1, erap2 and hla class i expression in nb cell lines by affecting the recruitments of nf-kb binding to their promoters. s. brosch 1 , s. tenzer 1 , h. schild 1 , e. von stebut-borschitz 1 1 uniklinik mainz, mainz, germany infection of inbred mouse strains with the intracellular protozoan parasite leishmania major either leads to self-healing cutaneous disease (resistant phenotype; e. g. c57bl/6 mice) or systemic disease (susceptible phenotype; balb/c mice) depending on the genetic background of an individual. healing of leishmania infections is based on th1 immunity, whereas ifng secretion of both cd4 + th1 and cd8 + tc1 cells is critically important for protection by inducing oxidative radicals in macrophages, which enables them to kill the parasite. stimulation of antigen-specific effector t cells is driven by l. major-infected dendritic cells (dc) in an il-12-dependent manner. proteasome/immunoproteasome-dependent antigen processing is necessary for clearance of viral or intracellular parasitic diseases to induce effective cd8 + t-cell responses via the mhc class i. here, we analysed the role of the ifng inducible immunoproteasome for the priming of cd8 + t cells in l. major infections. using an in vivo model, we show that the functional knock-out mouse in the chymotrypsin-like catalytic domain of the immunoproteasome lpm7 (lmp7 -/-) does not exhibit an altered course of infection (lesion development, parasite loads, cytokine profiles) in intradermal, low dose infections with l. major mimiking natural transmission of the parasite as compared to wild type c57bl/6 mice. in addition, ex vivo co-cultures with infected dc from either lmp7 -/or wild type mice together with antigen-specific t cells from infected wild types showed no differences in tc1 cell ifng secretion and the dc restimulatory capacity of cd8 + t cells. furthermore, significant differences in the proliferation of antigen-specifically restimulated (with soluble leishmania antigen; sla) cd8 + t cells, isolated from low dose infected c57bl/6 wildtype or lmp7 -/mice, were not detected. in summary, our data indicate that despite the fact that cd8 responses in l. major infections are important for disease outcome, processing of antigen and thus priming of cd8 + t cells against l. major is independent of the lmp7 subunit of the immunoproteasome. studies have defined an essential requirement for autoantigen-specific b cells as antigen presenting cells in rheumatoid arthritis. however, the cellular mechanisms involved in antigen processing and presentation of joint-derived autoantigens by b cells are unknown. in this study we have developed a system to investigate how antigen-specific b cells recognise and present the proteoglycan aggrecan, a major component and candidate autoantigen of joint cartilage. we have utilised these cells to characterise the mechanisms by which aggrecan-specific b cells could induce autoimmunity. we have constructed plasmids encoding an aggrecan-specific b cell receptor and have transfected them into the b cell line a20, generating b cell lines that specifically recognise and target aggrecan for presentation to t cells. in addition, we have established conditions for a panel of aggrecan-specific t cell hybridomas to recognise aggrecan pulsed b cells following fixation, to allow the kinetics and mechanisms of aggrecan processing to be studied. we used inhibitors of mhc class ii transport, endosomal ph and enzymes involved in aggrecan degradation. we found that aggrecan-specific b cell lines presented the major arthritogenic cd4 + t cell epitope (84-103) from the g1 domain of aggrecan 10,000 times more efficiently than non-specific b cells and over 100 times more efficiently than the macrophage line j774. however, despite this highly efficient aggrecan capture, processing and presentation of the 84-103 epitope took at least 5 hours, comparable to the time required for presentation of aggrecan by j774. treatment of aggrecan-specific b cells with ammonium chloride to raise endosomal ph or brefeldin-a to disrupt golgi transport inhibited presentation of the 84-103 epitope, suggesting a requirement for low endosomal ph and presentation by newly synthesised mhc class ii. interestingly, aggrecan presentation by antigen-specific b cells was also reduced by phenanthroline, an inhibitor of the aggrecan-degrading metallo-proteinases that are found in abundance in the arthritic synovium understanding the mechanisms of antigen processing and presentation by autoantigen-specific b cells may explain their role in the pathogenesis of diseases such as rheumatoid arthritis. tapasin is an mhc-dedicated chaperone that facilitates peptide loading and optimization of the peptide cargo of mhc class i molecules within the peptide loading complex (plc). class i molecules differ in their dependence on tapasin for efficient cell surface expression, dependence that is determined by the nature of amino acids at positions 114, 115 and 116 at the peptide binding groove. position 116 also determines the strength of tapasin binding and influences peptide specificity, but its precise effect is probably context dependent. the mhc class i antigen b27 is strongly associated to ankylosing spondylitis (as) and other spondyloarthropathies. hla-b27 subtypes differ in their dependence of tapasin for cell surface expression and incorporation into the plc. tapasin also modulates b27 folding but not maturation and although tapasin optimizes the constitutive peptide repertoire of b*2705, peptide loading is relatively independent of this chaperone. we analyzed the effect of b27 subtype polymorphism on tapasin binding and the correlation of this feature with the affinity of the peptide repertoires, the maturation kinetics and the folding efficiency of b27 subtypes. the association of b27 heavy chain with tapasin was analyzed in c1r cells transfected with hla-b27 subtypes and mutants by pulse-chase analysis and co-immunoprecipition with the monoclonal antibody pasta-1, which recognizes human tapasin. we also analyzed the global thermostability, as a measure of the stability of the peptide cargoes, and the optimization of the b27 peptide repertoire with thermostability assays, by pulse-chase analysis and immunoprecipitation with the me1 monoclonal antibody that recognizes b27properly folded b27/peptide complexes. the formation of fully assembled b27 molecules was analyzed by pulse-chase analysis and immunoprecipitation either with the monoclonal antibody hc10, which recognizes mhc class i free heavy chains (hc), or with me1. maturation was analyzed by pulse-chase analysis, immunoprecipitation with me1 and treatment with endoglycosidase h (endo h). hla-b27 polymorphic positions other than 116, both at the a and c/f pockets modulate tapasin binding and the optimization of the peptide cargo. the stability of the peptide repertoires critically influences the folding efficiency of b27 subtypes. from as early as the initial phases of infection, hiv is coated with complement (c) fragments and following seroconversion, the circulating virus forms immunecomplexes with igg and complement. recent in vitro experiments revealed differences with respect to productive infection of immature dendritic cells (idcs) with differentially opsonized hiv. the opsonization pattern of hiv may additionally have profound consequences for the outcomes of the antigen-presenting capacity of dcs and their ability to mount an adequate immune response. in this context, we compared the impact of differential hiv-opsonization on the antigen-presenting capacity of dcs and found that c-opsonized hiv triggered ctl responses, while igg-coated virus did not. these in vitro generated ctls showed an enhanced ifn-g secretion and recognized the help independent ctl epitope slyntvatl. c-generated ctls also degranulated upon stimulation with specific hiv peptides and were able to elicit antiviral activity against hiv-infected cd4 + t cells. our results indicate that c-opsonization of hiv drives the virus towards the mhc class i pathway in dcs, thereby promoting a more efficient stimulation of naïve cd8 + t cells. this ctl-stimulating property of c could be exploited when searching for a novel approach against hiv. igg isolated from patients with high titers of anti-ccp antibodies showed a cross-reactivity with hcit peptides. vaccination experiments supported a triggering role of hcit for the development of arthritis in mice model. conclusions: diamination process is significantly increased in patients with ra while carbamylation is suppressed. production of specific antibodies against diaminated residues in ra patients may have a modulating role for the development of autoimmune arthritis. the classical pathway of mhc class i antigen presentation involves cytosolic degradation of viral proteins by the proteasome. peptides generated entry the endoplasmic reticulum through the transporter associated with antigen processing (tap). previous reports have shown that viral epitopes are presented to ctl independently of tap in smaller viruses. we hypothesized that presentation of vacv by mhc class i might proceed by alternative pathways. the aim of this study was to characterize these alternative pathways in tap-deficient mice. our results show that ctl derived from c57bl/6 mice immunized with vacv, recognized tapdeficient dendritic cells infected with the virus. approximately 15 % of vacv global presentation in the context of h-2 b was independent of tap. in addition, vacv infection induced a virus-specific ctl response in mice deficient in tap. dendritic cells (dc) initiate robust ctl immunity via the presentation of antigen-derived peptides by surface major histocompatibility complex class i molecules (pmhc). two major dc subtypes have been described, cd8+ and cd8-dc, which differ in their mhci antigen presentation capacities. cd8+ dc are the major dc subset responsible for cross presentation (presentation of exogenous antigen by mhci), while cd8-dc display little cross presenting capacity. here, we examined the mhci antigen presentation pathway of cd8+ and cd8-dc in more detail. first, turnover (half-life) of total mhci at the cell surface of cd8+ and cd8-dc was determined. surprisingly, cd8+ dc exhibit rapid surface mhci turnover compared to cd8-dc (following culture in the presence of brefeldin a). following activation of dc with cpg, mhci levels at the surface of both cd8+ and cd8-dc were stabilized and no longer underwent rapid turnover. this suggests that cd8+ and cd8-dc differ in their regulation of surface mhci turnover and that this is subject to regulation by antigen-associated signals. second, we examined the ability of cd8+ and cd8-dc to generate pmhci complexes containing cross presented antigen. we utilized the model antigen ovalbumin (ova) and an antibody that can detect h-2kb loaded with the ova-derived peptide, siinfekl. cd8+ and cd8-dc isolated from ova-expressing mice (actin-ova transgenics) displayed abundant kb-siinfekl complexes at their cell surface. in contrast, in response to exogenous soluble ova protein, only the cd8+ dc, but not the cd8-dc, displayed kb-siinfekl complexes at the cell surface. similarly, when dc were pulsed with ova-coated splenocytes, kb-siinfekl complexes were only detected on the surface of cd8+, and not cd8-dc. this data further validates the role for cd8+ dc as the major cell type responsible for cross presentation and provides insight into the mechanisms that prevent other dc subsets from accessing the important cross presentation pathway. objectives: the action radius of matrix metalloproteinases or mmps is not restricted to massive extracellular matrix (ecm) degradation but extends to the proteolysis of secreted cytokines and membrane-bound receptors and adhesion molecules. although many instances exist in which cells disintegrate, often in conjunction with induction of mmps, the intracellular mmp substrate repertoire or degradome remains relatively unexplored. the aims of the present study were to identify novel intracellular mmp targets and to answer the question whether the proteolytic modification of intracellular proteins alters the immunogenicity of released intracellular contents. methods: multidimensional degradomics technology was developed by the integration of broadly available biotechniques and applied to thp-1 cytosol using gelatinase b/mmp-9 as a model enzyme. in the first dimension, ion exchange chromatography separated the thp-1 proteins by their net charge and/or isoelectric point (pi) followed by cleavage of the proteins by mmp-9. in the second dimension, potential substrates were separated by molecular weight on sds-page. to evaluate the effect of proteolysis by mmp-9 on the immunogenicity of the intracellular protein pool, mice were immunized twice with thp-1 cytosol in complete freund's adjuvant. lymph node t cells were isolated and stimulated with mmp-9-cleaved or intact thp-1 cytosol. proliferation was assessed by measuring incorporated 3 hthymidine. results: 100-200 mmp-9 candidate substrates were isolated, of which 69 were identified, revealing many novel mmp-9 (candidate) substrates from the intracellular matrix (icm), such as actin, tubulin, stathmin,... about 2/3 of the identified substrates were described as systemic autoantigens in one or multiple autoimmune conditions. remarkably, a significantly lower t cell proliferation was observed in the presence of cleaved vs. intact cytosol. conclusion: multidimensional degradomics technology is a valuable tool for high-throughput identification of novel mmp substrates. proteolysis by mmp-9 decreased the immunogenicity of the intracellular contents, suggesting that mmps may contribute to the dampening of inflammation by the clearance of toxic and immunogenic burdens of intracellular (matrix) proteins released after extensive necrosis and tissue injury. a preference for hla-a versus -b molecules; e3-19k did not detectably associate with hla-c molecules under identical conditions. this locus specificity may provide a functional advantage to ads by inactivating t-cell receptors, while avoiding activation of nk receptors. finally, we showed that residue 56 in hla-a11 and residue 93 in e3-19k are highly critical for association of both proteins. this defines a putative interaction surface between e3-19k and class i molecules. conclusions: our studies provide novel insights into the functional relationship between e3-19k and the class i antigen presentation pathway. moreover, because soluble e3-19k can differentiate between polymorphic gene products encoded in the mhc, our results may contribute to define paradigms for how class i substrate specificity is established for er retention. overall, our studies represent an important step towards a molecular understanding of the strategy evolved by ads to establish life-long persistence in host cells. objectives: the transporter associated with antigen processing (tap) belongs to the abc transporter superfamily and is a heterodimer consisting of the two subunits tap1 and tap2. tap transports peptides yielded by proteasomal degradation from the cytosol into the endoplasmic reticulum (er) and is thus a key element of the mhc class i antigen processing machinery (apm). methods: target-specific tap knock downs were generated by shrna technology. the resulting transfectants were subsequently analysed regarding mhc class i surface expression using flow cytometry, whereas mrna and protein expression levels of tap1 and tap2 were analysed by rt-pcr and western blots, respectively. furthermore, the protein stabilizing effect of tap1 on tap2 was investigated in the presence of two distinct proteasome inhibitors. results: previous findings obtained with rare tap1 mutants suggested that the lack of tap1 protein expression is associated with a strong reduction of tap2 protein levels, which could be restored by tap1 gene transfer, whereas no such regulation is found vice versa. to investigate this stabilizing effect of tap1 on tap2 different shrna plasmids specifically targeting tap1 or tap2, respectively, were stably transfected into constitutively tap1 and tap2 expressing hacat keratinocytes and colo794 melanoma cells. in both cell types the shrna-mediated tap1 and tap2 inhibition resulted in a significant downregulation of the respective transcript and protein expression levels. the knock down of tap1 caused not only an almost complete loss of tap1, but also a strong decrease of tap2 protein expression. in contrast, the tap2 knock down exhibited no influence on the tap1 expression. specific inhibition of the proteasome prevented the degradation of tap2 in the tap1 knock down variants. the results of our study emphasize that an unidirectional stabilisation of tap1 on tap2 protein expression is not restricted to rare tap1 mutants, but rather suggest a common regulatory mechanism for the tap complex. uv injury profoundly affects the skin immune homeostasis by promoting strong inflammation and cellular immuno-modulation. in this study, we characterized the inflammatory cell subsets that emigrate in the epidermis the days following uv exposure. therefore, the buttock skin of 27 healthy volunteers was exposed twice to 1.5 minimal erythema dose of uv. blister roofs were then collected before and 1, 4 and 10 days after uv-exposure from un-exposed and exposed skin and the resulting epidermal cells were analysed by flow cytometry. we demonstrated that, along with the rapid activation and migration of langerhans cells (lc), uv skin radiation exposure promotes the infiltration into the epidermis of a monocytic cd14 + cd36 + and of a macrophagic cd14 -cd36 + cell subsets that emerge 1 and 4 days post exposition, respectively. more importantly whereas classical cd1a hi cd207 + lc are the unique dendritic cell (dc) subset found in the epidermis of unexposed skin, we detected two new subsets of epidermal dc namely cd1a low cd207and cd1a low cd207 + that emerged 1 and 4 days post irradiation, respectively. these two distinct populations of epidermal dc (edc) differ from classical epidermal lc by their activation/maturation profile as assessed by the strong expression of cd86 and hladr. finally, 10 days post-exposure, we observed that lc represented almost the only haematopoietic cell population in the epidermis. these results suggesting that the uv-recruited edc and monocytic/macrophagic subsets participate to the progressive recovery of the epidermal immune cell network homeostasis. i. bailey 1 , e. reeves 1 , t. elliott 1 , e. james 1 1 university of southampton, school of medicine, cancer sciences division, southampton, united kingdom there is accumulating evidence that cd4+ cd25+ regulatory t cells (treg) play an important role in anti-tumour immunity by preventing effective t cell responses to tumour antigens. tregs have also been shown to inhibit development of organ-specific autoimmune diseases suggesting they inhibit immune responses to tissue-specific self-antigens. the depletion of tregs prior to challenge with the murine colorectal tumour, ct26, stimulates a robust, protective t cell response which is also protective to challenge with other tumours of different histological origins, such as b cell lymphomas and a renal cell carcinoma. this cross protection has not been seen with other tumour cell lines. we have identified a ct26-derived cross-protective antigen, gsw11, which was found to be encoded within the ectotropic murine leukaemia virus (emv-1) envelope protein, gp70. this protein has previously been shown to encode ct26-specific cd8 and cd4 antigens, implicating it as a 'hot-spot' for ct26 tumour antigens. interestingly, we have identified a truncated version of gp70 which may be responsible for generation of gsw11. expression studies have revealed increased gp70 expression in ct26 compared to other tumour cell lines, indicating the ability to cross-protect is related to the quantity of antigen (gsw11) generated. the current knowledge of hla class ii antigen presentation and peptide binding is mainly based on studies of hla-dr molecules. they contain a large hydrophobic p1 pocket, which can accommodate large hydrophobic amino acid residues and is the most important pocket in selecting and binding peptides, while p4, p6 and p9 tune the peptide repertoire that can be presented by individual hla-dr alleles. the same rules and requirements do not necessarily exists for peptide binding to hla-dp molecules, however. the present study adresses this issue. we have expressed and affinity purified soluble recombinant hla-dp2 molecules from drosophila melanogaster cells and studied its binding of a number of peptides known to bind hla-dr, -dq or dp molecules. unexpectedly, the immunodominant epitope in multiple sclerosis (ms), the myelin basic protein derived peptide, mbp85-99, bound to hla-dp2 with high affinity (10-30 nm). binding studies of mbp85-99 derived peptides containing single alanine substitution at each position revealed that only three of the peptides (f91a, f92a and k93a) were affected, and only by a 20-50 fold reduction in affinity for hla-dp2. the observation that none of the substitutions resulted in a complete loss of binding to hla-dp2 indicates that 1) hla-dp2 binding to peptides does not depend on a large hydrophobic residue accomodated in p1, or 2) mbp85-99 can bind in more than one register. we will present data addressing this issue. the hla-dp peptide binding capacity was increased at neutral as compared to acidic ph, and by the presence of n-butanol, a small organic mhc loading enhancer (mle). in summary, the hla-dp2 molecule binds the immunodominant epitope in ms, mbp85-99, possibly in more than one register. additional studies are required to resolve the hla-dp2 peptide binding properties, and to determine whether expression of hla-dp2 affects the disease course in ms patients. results: depletion of mncs for cd14+ cells abrogated the tg-induced cytokine production and proliferation of cd4+ t cells, indicating a primary role for monocytes as apcs. however, the encounter of t cell with antigens presumably occurs in b cell-rich compartments such as lymph nodes or lymphoid tissue in inflamed organs. to mimic the conditions prevailing there, we depleted pbmcs for g 98 % of the monocytes (without significant loss of t-cells), and compared the tgelicited t-helper cell responses in the presence and absence of b cells. the tg-induced cd4+ t cell proliferation was significantly reduced in cd14/cd19-depleted mnc cultures, as compared to cultures depleted for cd14+ monocytes alone. the same applied to the production of il-2, il-6 and tnf-a. production of ifn-g and il-10 was generally not observed. our data indicate that normal b cells are capable of inducing a pro-inflammatory cytokine response in mnc-cultures, where monocytes and monocyte-derived cells are not preponderant. studies addressing the relative contributions to this cytokine production, by b cells themselves and by t cells (following antigen-presentation by b cells), are in progress. j. kyosiimire-lugemwa 1,2 , p. pala 1 , g. miiro 3 , j. todd 3 , p. kaleebu 1,2 , n. imami 2 , f. gotch 2,3 1 mrc uganda, basic science, entebbe, uganda, 2 imperial college, london, united kingdom, 3 mrc uganda, entebbe, uganda background: hiv-1-specific t-cell responses are preserved in hiv-1 infected individuals with non-progressing hiv-1 disease. "long term non progressors" (ltnps) were defined as art naï ve individuals infected with hiv-1 for g 8 years, maintaining cd4+ t-cell counts g 500, and with minimal cd4+ decline over time. we tested the hypothesis that gag-specific t-cell responses are inversely correlated to disease progression whereas nef-specific t-cell responses are not. methods: 17 art naï ve hiv-1 infected patients from the entebbe cohort in uganda were recruited and stratified by cd4+ t-cell count, cd4+ decline slopes, and time of enrolment, into 2 groups -10 ltnp and 7 rapid progressors (rp). all patients were women reflecting the patient base at the entebbe cohort. we measured plasma viral load, current cd4 t-cell count, and ifn-g, il-2 and il-4 elispot responses to pools of 22 to 34 peptides (18-mers overlapping by 10aa). peptides were based on consensus sequences of gag and nef from hiv-1 clades a1, a2 and d. medians and inter-quartile ranges were calculated and comparisons between groups were performed using the mann-whitney u test. correlations were presented using spearmann's linear correlation coefficients. results: some gag-specific ifn-g and il-4 responses were significantly higher in the ltnp than in rp (p=0.02, ifn-g responses to gaga2 pool 1; p=0.04, il-4 responses to gaga1 pool 2). il-2 responses were low and not significantly different between ltnp and rp. there was a positive correlation between il-4 responses to gaga1 pool 2 and cd4 t-cell counts (r 2 =0.502, p=0.04), but no correlation between either il-4 or ifn-g responses and viral load. cytokine responses to nef peptides were not significantly different between the ltnp and rp. conclusion: overall, gag hiv-1 specific responses were higher in ltnps than in rps confirming previous results. non-specific il-4 responses were high possibly reflecting baseline th2 responses to helminths a common environmental exposure in the study population. objectives: in human and murine tumors and in in vitro oncogene-transformed cells defects in the expression of components of the hla class i antigen processing machinery (apm) have been described, which were associated with a reduced antigenicity of these cells. so far, the molecular mechanisms of such defects have not been elucidated in detail. to investigate whether impaired apm component expression was due to altered transcription and associated with cell growth properties murine her2/neuand her2/neu + fibroblasts were employed. methods: using tapasin as a model molecule its cell cycle-dependent expression was analysed in a time kinetics upon serum starvation followed by stimulation with complete culture medium over time. cells were harvested at different cell cycle phases and expression of tapasin was analyzed by qrt-pcr and western blot. flow cytometry was employed for determination of the distinct phases using 7-aad for dna analysis and specific antibodies directed against the proliferation marker ki-67, the m-phase specific phistone h3 as well as for the h-2l d surface antigens. in addition, chromatin immunoprecipitation (chip) experiments with an antibody directed against rna polymerase ii were performed to investigate the transcriptional levels of tapasin in her2/neuversus her2/neu + cells. results: serum starvation and subsequent stimulation with complete culture medium led to the enrichment of cells at the g 0 /g 1 -, s-and g 2 /m-phases of the cell cycle, which was associated with an altered tapasin transcription during the cell cycle. tapasin mrna level decreased during cell cycle progression, whereas an inverse protein expression was observed with low expression levels at the g 0 /g 1 -phase, which continuously raised and peaked within the s-phase. however, h-2l d surface antigen expression was not altered in her2/neucells during cell cycle progression. in contrast to her2/neufibroblasts the her2/neu + transfectants exhibit a decreased tapasin transcription, which was accompanied by an altered h-2l d surface expression. this was confirmed by a reduced promoter activity and decreased accessibility of the rna polymerase ii to the tapasin promoter. conclusion: these findings lead to an improved understanding of immune escape mechanisms demonstrating a cell cycle dependent and oncogene-mediated tapasin regulation that may provide novel targets for therapeutic intervention. recent studies suggest that dendritic cells (dcs) are key players in shaping the respiratory syncytial virus (rsv) specific immune response. before, dcs within the airway epithelium were characterized as langerhans cells. in this study, in vitro counterparts of langerhans cells expressing langerin (cd207) and ccr6 were cultured from cd34+ stem cells under the influence of tgf-b (tgf-b-dcs) and compared to cd34+ derived dcs, which passed through a monocytic stage . after infection with rsv, both types of dcs generated viral rna and viral-proteins. although tgf-b-dcs expressed higher levels of viral proteins as revealed by flow cytometry and fluorescence microscopy, more than hundredfold more viral particles were released by il-4-dcs. the increased expression of viral proteins is most likely responsible for the pronounced inhibition of t-cell functions by tgf-b-dcs. since there is evidence that langerhans cells are expressed in airway epithelium not before the age of one year, the results may indicate, that an inhibition of rsv replication is characteristic of a more mature answer against rsv. the occurrence of inhibitory antibodies against exogenous factor viii (fviii) remains the major concern of fviii replacement therapy in patients with hemophilia a. initiation of the immune response implies the endocytosis of fviii by professional antigen presenting cells (apcs): b lymphocytes, dendritic cells (dcs) and macrophages (mø). the organ where the anti-fviii immune response is initiated and the type of apcs involved in this process have not been investigated. we hypothesized that the spleen, which is the principal filter for blood-born antigens, is the principal organ where apcs interact with fviii to initiate the anti-fviii immune response. we first administered radiolabeled fviii at therapeutic doses to fviii-deficient mice. fviii was found to preferentially accumulate in the spleen and liver of the mice. levels of fviii in the spleen remained stable for up to 45 min following fviii administration, while they rapidly decreased in the liver. unlabelled fviii was then administered to fviii-deficient mice that had been splenectomized or sham operated and the anti-fviii humoral responses were compared. removal of the spleen resulted in significantly reduced levels of anti-fviii igg. using flow cytometry, fviii was found to preferentially accumulate with splenic mø than dcs and b cells. elimination of apcs by treatment of the mice with clodronate-containing liposomes prior to fviii administration resulted in a drastic reduction of the anti-fviii igg response, as compared to control mice treated with pbs-containing liposomes. taken together, our results suggest that the spleen is the principal organ in the initiation of the anti-fviii immune response and that splenic mø have an important part in this process. the interactions between antigen presenting cells (apc) and t-lymphocytes are a relevant current issue. the area of contact between an antigen presenting cell and a t-lymphocyte is termed immunological synapse (underhill et al, 1999) . the present work started, in experiments with leukocyte of healthy individuals from the finding that under certain experimental conditions, cell-cell association with closely contact between monocyte-derived macrophages and human autologous lymphocytes are produced when the cells are harvested from total leukocyte cell cultures. in this way, such cells selective forming rosettes with a central macrophage and adherent lymphocytes. objectives: as central hypothesis it was postulated that the phenomenon would be due to antigen presentation made (performed) by macrophages to lymphocytes and that would be t cells. methods: autologous total human leukocyte cultures, from samples of 30 healthy blood donors were harvested at various times and centrifuged and performed as previously reported (cabral and novak, 1992, 1999) . cytopreparations of each experiment were performed. statistical analysis: regression model. results: experimentally, it was found a) phagocytosis of autologous antigens by macrophages, stimulates the formation of rosettes, b) a linear relation between rosettes formation and culture-time occurs, (p x 0,0001), anova for regression, c) the cell-cell approximation is very important and was performed by centrifugation of the cells to form pellets, d) the forming rosettes lymphocytes are t-cells, cd4+, e) purified macrophages and lymphocytes produced few rosettes, however if antigens were added, the phenomenon was stimulated, f) if inhibitors of the antigen processing and antigen presentation, such as chloroquine or brefeldin a, were added, rosettes were not formed, g) monoclonal antibodies anti-human mhc ii precluded the formation of rosettes, h) gangliosides diminishes rosettes formation. conclusion: taken together, the findings suggest that the model of rosettes formation might be useful to study cell-cell interactions during antigen presentation in immunological synapses and other immunologic aspects on the cells involved, in short time assays. objective: to investigate if patients with multiple sclerosis (ms), without the typical increase of antibodies in csf, are less likely to develop neutralizing antibodies (nabs) against ifnb-treatment, and whether the absence of such an immunological response might reflect a difference in their antigen presenting ability due to a distinct genetic background. methods: overall, 2252 patients were obtained from the swedish multiple sclerosis registry and the swedish nab registry, and treatment information was available for 2207 of them. for 538 of these patients hla-drb1 data was available. results: a significant correlation between lack of antibodies in csf and nab-negativity was found (p=0.02). patients without csf antibodies were to a lesser extent nab-positive when treated with the ifnb-1a preparations, whereas no differences were shown for ifnb-1b. an association between hla-drb1*11 and nab-negativity was detected (p=0.028). the known associations between hla-drb1*15 and csf-positive ms and hla-drb1*04 and csf-negative ms were confirmed. conclusion: we show for the first time that patients without antibodies in csf have a different propensity to induce nabs compared to csf-positive patients, indicating an extended immunological difference between the two ms sub-groups. hla-drb1 potentially contributes to this, which indicates that it might have something to do with differences in antigen presentation. in csf-negative patients the reaction against ifnb-1a molecules, possibly through a t-cell dependent pathway, is lower than for csf-positive patients. however, reaction against ifnb-1b, which might also be activated through a t-cell independent pathway, shows no difference in seroprevalence between the groups. abstract withdrawn by author tyrosinase-derived epitope was confirmed by five independent assays: flow cytometry on multiple melanoma lines generated from patients, confocal microscopy immuno-staining of melanoma lines, frozen sections staining of authentic melanoma tissue from patients, cytotoxicity assays using tyrosinase-specific ctls, and finally mass spectrometry analysis of peptides isolated from a melanoma cell line. there was no correlation between the level of antigen presentation and mrna expression levels for the three antigens; however, our data suggest that tyrosinase protein stability may play a major role in the high level presentation of this antigen. measurement of the half lives of these proteins revealed a hierarchy in protein stability, with mart-1 and gp100 more stable than tyrosinase. by the use of the cofactor dopa, which stabilizes the tyrosinase protein, significant decrease of hla-tyr complexes presentation was achieved. in addition to the study of antigen presentation, these tcr-like antibodies can also actively participate in immunotherapy as targeting molecules, considering their high affinity and specificity. by generating a whole igg antibody, tumor cell lysis was achieved by antibody-dependent cell-mediated cytotoxicity (adcc). with the addition of point mutations in the fc fragment, which increased the affinity of the fc to the fc receptor, enhanced tumor cell lysis was achieved. g. schiavoni 1 , s. lorenzi 1 , f. mattei 1 , f. spadaro 1 , l. gabriele 1 1 istitituto superiore di sanità, cell biology and neurosciences, rome, italy cross-presentation is a crucial mechanism for generating cd8 t cell responses against exogenous antigens (ag), such as dead cell-derived ag, and is mainly fulfilled by dendritic cells (dc), particularly cd8a + dc. however, apoptotic cell death occurring in steady-state conditions is largely tolerogenic, thus hampering the onset of effector cd8 t cell responses. type i ifn are a family of cytokines induced upon infection and acting as danger signals by stimulating multiple arms of the immune response. in particular, type i ifn have been shown to promote the cross-priming of cd8 t cells against soluble or viral antigens, partly through the stimulation of dc. in this study we evaluate the role of type i ifn to affect dc capacity to capture and cross-present apoptotic cell-derived ag. by using uv-irradiated ova-expressing eg7 thymoma line, we show that type i ifn promote the ability of cd8a + dc to capture apoptotic eg7 cells and to undergo phenotypic activation, both in vitro and in vivo. remarkably, ifn-treatment prolongs the survival of ag-bearing cd8a + dc and the persistence of apoptotic eg7-cell ag within the phagosomal dc compartment, a process that is known to facilitate the recruitment of ag into the mhc-i presentation pathway. accordingly, type i ifn-treatment increases cross-presentation of apoptotic eg7-derived ova ag by dc, as revealed by higher expression levels of siinfekl peptide in association to mhc-i molecules on cell surface of phagocytic cd8a + dc. as a result, eg7-loaded dc become competent at inducing ot-i cd8 t cell proliferation and activation both in vitro and in vivo. our data indicate that type i ifn promote the cross-presentation of apoptotic cell-derived ag by cd8a + dc and suggest that these cytokines may act as a switch signal for cross-presenting dc, thus skewing the immune response from tolerogenic to immunogenic. (2). we have investigated the mechanisms of cross-presentation of soluble antigen in freshly purified splenic dc subsets. using biochemical methods, we show that only cd8+ dc efficiently transfer soluble antigen to their cytosol. the amount of antigen detected in the cytosol increased up to ten-fold after a short exposure to tlr ligands cpg, poly i:c or pam3csk4, and this correlated with enhanced cross-presentation. the increase in antigen accumulation within the cytosol was not due to increased uptake of antigen. measurement of the proteasome activity at different times after exposure to tlr ligands revealed that tlr signalling induced transient inhibition (maximum at two hours) of the proteasome in cd8+ dc but not cd8-dc, thus promoting accumulation of exogenous antigen in the cytosol of cross-presenting dc. this correlated with formation of aggresome-like structures only in cross-presenting dc exposed to tlr ligand. by limiting the degradation of transferred proteins during early activation, when endogenous proteins are being stored in aggresome-like structures, this mechanism could favour the loading of exogenous antigen peptides over endogenous peptides, promoting cross-presentation. to our knowledge this is the first report of a direct, immediate effect of tlr activation on proteasome activity. exosomes are nano-sized membrane vesicles of endosomal origin which can exert both immune stimulatory and tolerance inducing effects depending on their cellular origin. they are currently being investigated for use in vaccination and immune therapy strategies, but their physiological role has not been elucidated. here we explore whether exosomes of different origin can selectively target different immune cells. we compare the binding of exosomes from human breast milk, monocyte derived dendritic cells and b cells to peripheral blood mononuclear cells. flow cytometry, confocal laser scanning microscopy and multispectral imaging flow cytometry (imagestream) reveal that exosomes derived from human dendritic cells and human breast milk preferably associate with monocytes, whereas exosomes from an epstein-barr virus (ebv) transformed b cell line selectively target b cells. our data suggest a highly selective association between cells and exosomes which can be a way to direct their functional effects. one of the hallmarks of cancer cells is the resistance to cell death. it has been suggested that cancer cells also have the capacity to evade the surveillance by the immune system. the proteasome and macroautophagocytosis are attractive effector mechanisms for the immune system, because they can be used to degrade foreign substances, including pathogenic protein, within cells. here, we investigated that dm1, which is a saponin derivative isolated the stem bark of dracaena mannii induced autophagocytosis on a549 human lung cancer cell line. methods: dm1 induced cell cytotoxicity was measured by mtt assay and propidium iodide staining on a549 cells. we examined the morphological change using optical microscope. and gfp-lc3 punctation was measured using confocal. the protein expression was measured by western blot analysis and the mrna expression was measured using reverse transcription poly chain reaction (rt-pcr). results: dm1 was showed high cytotoxicity on various cancer cell line, especially a549 cells. dm1 treated a549 cells did not show regular dna fragmentation and caspases activation. we also analyzed protein expression of apoptotic marker, but protein level didn't change. as a result, we hypothesized that this non-apoptotic cell death is mediated autophagocytosis. we checked lc3 and beclin-1 protein and mrna expression and inhibitory effect of autophagocytosis inhibitor. the expression level of lc3 was increased concentration and time-dependently. beclin-1 also was increased. and then, we examined gfp-lc3 punctation on a549/ gfp-lc3 stable cells using confocal. a549 cells were formed gfp punctuation after dm 1 treatment. to confirm these data, we measured cell death ratio using 3-methyladenine (3-ma), an autophagocytosis inhibitor. after 3-ma treatment, dm1 induced cell death was decreased comparing with dm1 treatment. conclusion: dm1 did not induce apoptotic cell death. but dm1 showed several characteristics of autophagic cell death. taken together, dm1 induced autophagocytosis on a549 cells. these finding indicated that therapeutic potential of dm1 by triggering autophagic cell death. s. b. rasmussen 1 , s. r. paludan 1 1 university of aarhus, department of medical microbiology and immunology, aarhus, denmark during viral infections, different pattern recognition receptors detect specific pathogen associated molecular patterns (pamp)s. in the case of herpes simplex virus (hsv), detection is, among others, conducted by toll like receptor (tlr)9, which is a transmembrane receptor located in the endosomes where it detects unmethylated cpg rich dna of extracellular origin, e. g. viruses. upon binding to dna, tlr9 initiates downstream signalling cascades resulting in induction of antiviral cytokines, interferon (ifn)a and ifnb being some of the most essential ones. the exact route of hsv to the endosomes and thus tlr9 recognition is not clear-cut. the endocytosis pathway is believed to be a central mechanism in which viruses translocate to the endosome, but recently the role of autophagy in tlr mediated viral recognition has been drawn in to focus. we have found indications of an autophagy dependent ifn expression during hsv-1 infection. by use of an entry defect glycoprotein l and glycoprotein h deficient hsv-1, we found that tlr9 dependent ifn regulatory factor 3 activation was abrogated. in addition, inhibition by 3-methyladenine of phosphoinositol 3-kinase class iii, which is crucial in autophagosome formation, abrogates ifnb induction. these findings points to a role for autophagy in tlr9 dependent recognition. in the ongoing project we are examining how hsv-1 triggers formation of autophagosomes. especially the role of endoplasmatic reticulum stress and doublestranded rna-dependent protein kinase will receive attention. also the newly found involvement of ubiquitin and acetylation in autophagy execution and regulation will be investigated. j. zovko 1 , i. berberich 1 , gk 520 -immunomodulation 1 universität würzburg institut für virologie und immunbiologie, würzburg, germany members of the bcl-2 family control the integrity of mitochondria and thereby influence survival and death of cells. most bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their c-terminal portion. murine a1 and its human homologue bfl-1 are anti-apoptotic members of the bcl-2 family. a1 is expressed in small amounts in the bone marrow and immature b cells, but in high amounts in mature b cells. thus the protein seems to be important for b cell maturation. we analyzed the function of the c-terminus of a1. unless the c-terminal ends of other bcl-2 proteins the tail of a1 does not function as a strong membrane anchor. nevertheless, the last amino acids of a1 are important for the protein. in fact, the c-terminus of a1 serves a dual function by being required for the instability and the anti-apoptotic potential of the protein. we show that a1 undergoes proteosomal degradation controlled by its c-terminus. interestingly, binding to the proapoptotic bcl-2 factor bimel results in increased stability of a1. this is due to reduced ubiquitination of a1 after binding of bimel. we conclude that the cterminus of a1/bfl-1 serves as a docking site for e3 ubiquitin ligase(s) that control the stability of a1 by targeting the protein to the proteasomal pathway. very recently, we have identified a putative e3 ubiquitin ligase that interacted with a1 in a yeast two-hybrid screen. currently, we are trying to confirm this interaction in mammalian cells. suppressors of cytokine signaling (socs), initially identified as negative regulators of cytokine signal transduction, have recently emerged as multi-functional proteins regulating inflammation, survival, differentiation, and apoptosis of immune cells. here we describe novel function of socs-1 in the suppression of rosmediated apoptosis and associated mechanisms using tnf-alpha induced t cell apoptosis as a model system. both in jurkat t cells and primary splenocytes, socs-1 is induced under tnf alpha-induced apoptosis conditions. the tnf-induced apoptosis was mediated by generation of ros, and the over-expression of socs-1 significantly suppressed tnf-induced ros levels and the subsequent apoptosis. such anti-apoptotic function of socs-1 was manifest not only by the suppression of jaks acting upstream of p38 mapk, but also protection of ptps which down-regulate the tnf alpha -induced jak 1/2 activities. we further show that tnf-alphainduced ptp inactivation can be prevented by socs1 which up-regulates thioredoxin levels. finally we present data that there is a molecular interaction between thioredoxin and ptp which is formed in response to ros-generating stimuli and sustained in socs-1 overexpressing cells. the results strongly suggest that socs-1 acts as an anti-oxidant and anti-apoptotic factor to sustain t cell homeostasis and survival under oxidative stress imposed upon inflammatory cytokines during infection or other immune scenarios. aim: inflammation is a cardinal host response to injury, tissue ischemia, autoimmune responses or infectious agents. the effects of inflammatory mediators on the glial function are of particular interest since astrocytes contribute to the local inflammatory responses in the cns by producing cytokines, chemokines, and maintain local homeostasis clearing released neurotransmitters. cxcl12/sdf-1a not only regulates cell growth and migration of hematopoietic stem cells but may also play a central role in brain development. moreover, it has been described that tnf-a mediates in cxcl12 effects on primary astrocytes, and it is clear that tnf-a participates in the pathogenesis of many neurological conditions. methods: we used the astrocytoma cell line u-87, and sk-n-mc as neuroblastoma cells. detection of tnf-a mrna expression was carried out by rt-pcr. transcriptional activity was measured using luciferase reporter gene assays in transiently lipofectin transfected-cells. we performed cotransfection experiments of nfat promoter construct with a dominant negative version of nfat (dn-nfat). neuronal death was performed by mtt and tunel assays. nfat translocation was confirmed by western-blot. p53 and fas-l expression was carried out by western-blot. results: cxcl12 induced mrna-tnf-a and transcriptional activity of tnf-a promoter in human astrocytes. this cytokine by itself was not toxic when added directly to astrocytes, but when we investigated its effect on nb cultures, neuronal death increased in a direct and indirect way. surprisingly, tnf-a did not induce nf-kb activation in nb cells but it induced nfat activity. nfat translocation was confirmed by western-blot. neurotoxicity was absolutely reverted in the presence of ciclosporin. we discard p53 pathway as responsible for tnf-mediated toxicity since we did not find any alteration in p53-ser46 levels in stimulated cells. in addition, we found increased fasl expression in neuroblastoma cells after 24h of tnf-a treatment. conclusions: cxcl12 induced-tnf-a promotes nfat activation in neuroblastoma cells and this event leads to increased apoptosis through an increase of fasl expression without alter p53function/levels. s. y. demiroglu 1 , r. dressel 1 1 universitätsmedizin göttingen, zelluläre und molekulare immunologie, göttingen, germany objectives: intracellular hsp70 is part of the cellular stress response system and can inhibit specific apoptotic pathways. extracellular hsp70 on the other hand, is an immunological danger signal that can induce the antigen-specific activity of cytotoxic t lymphocytes (ctl). interestingly, hsp70 does not protect against cell death mediated by ctl. acute overexpression of hsp70 can even increase the susceptibility of target cells to ctl, which use the granule-exocytosis pathway to induce apoptosis (dressel et al. cancer res 63: 8212) . granzyme b is one of the main effector proteases of cytotoxic granules. therefore, we analyzed the effect of acute overexpression of hsp70 on granzyme b-induced apoptosis. methods: hsp70 was expressed in a human melanoma cell line under the control of a tetracycline-inducible promoter (ge-tet). the effect of hsp70 induction on granzyme b-mediated apoptosis was now analyzed after delivery of granzyme b into the cytosol of the target cells by the endosomolytic activity of adenovirus type 5. results: hsp70 did not protect the melanoma cells against granzyme b-mediated apoptosis when annexin v binding, loss of mitochondrial membrane potential, release of mitochondrial cytochrome c, and activation of caspase-3 were evaluated. instead, we observed a moderate but significant pro-apoptotic effect of hsp70 in ge-tet cells when late apoptosis was analyzed at the nuclear level by sub g1 peak measurements (p=0.01). in contrast, a partial protection of ge-tet cells was observed after acute hsp70 overexpression when apoptosis was induced by staurosporine. conclusion: acute overexpression of hsp70 does not seem to protect tumor cells from granzyme b-induced apoptosis; it appears even to accelerate the progression of apoptosis to dna fragmentation and nuclear condensation. it is conceivable that this hsp70 effect is mediated by chaperoning pro-apoptotic molecules and improving their function as it has been reported for the caspase-activated dnase (liu et al., blood 102:1788) . thus, granzyme b might be able to kill even those tumor cells that undergo an otherwise protective stress response. the work has been supported by the grants grk1034 and dr394/2-3. the authors thank prof. c. j. froelich (evanston, usa) for the granzyme b and dr. t. seidler (göttingen) for the adenoviruses. tumor necrosis factor (tnf) elicits its biological activities by stimulation of tnfr1 and tnfr2, both belonging to the tnf receptor superfamily. tnfr1-mediated signal transduction has been intensively studied and is well understood, especially with respect to activation of the classical nfkb pathway, cell death induction and map kinase signaling. in contrast tnfr2-associated signal transduction is poorly defined. earlier findings demonstrated that only membrane tnf, but not soluble tnf, properly activates tnfr2, resulting in depletion of traf2 from the cytoplasm. here we confirm that tnfr2 induced depletion of cytosolic traf2 by the use of tnfr1-and tnfr2-specific mutants of soluble and membrane tnf. corresponding with the known inhibitory role of traf2 in the alternative nfkb pathway, we show that tnfr2 induced activation of the alternative nfkb pathway. thus, we identified activation of the alternative nfkb pathway as a tnf signaling effect that can be specifically assigned to tnfr2 and membrane tnf. j. c. morales 1 , d. ruiz-magaña 1 , d. carranza 1 , c. ruiz-ruiz 1 1 universidad de granada, instituto de biopatologí a y medicina regenerativa. centro de investigación biomédica, armilla, spain different molecular mechanisms have been involved in the resistance of tumor cells to tumor necrosis factor-related apoptosis-inducing ligand (trail)-mediated apoptosis. epigenetic modifications commonly associated with tumor development, such as histone deacetylation, may influence the resistance of some tumor cells to trail by regulating gene transcription of trail receptors and other trail pathway related-genes. in the present study we have analyzed the effect of several histone deacetylase inhibitors (hdaci), belonging to different structural families, on trail-induced apoptosis in leukemic t cell lines. moreover, we have analyzed the activity of hdaci in normal t lymphocytes. we have found that, to a greater or lesser extent, all hdaci potentiate the induction of apoptosis in leukemic t cells by enhancing the signals triggered upon trail ligation, from the activation of the most apical caspase-8. in contrast, hdaci do not sensitize primary resting or activated t lymphocytes to trail-mediated apoptosis. the analysis of the expression of several pro-and anti-apoptotic proteins involved in the trail-signalling pathway indicates that most hdaci regulate the expression of trail-r2 and c-flip in leukemic t cells, but not in normal cells, which may explain their selective pro-apoptotic effect on leukemic cells. zfp36l1 is a zinc finger containing protein that is involved in post-transcriptional gene regulation. it can bind to mrnas containing adenine uridine rich (are) regions and subsequently mediate their degradation. we have previously reported a role for this protein in promotion of b cell apoptosis. one mechanism whereby zfp36l1 may mediate cell apoptosis could be by degradation of cell survival gene mrnas. the bcl-2 protein is an important cell survival protein at different stages of b cell development. bcl-2 mrna also contains are regions that could possibly be targeted by zfp36l1 protein. in the present study, we have tested the ability of zfp36l1 protein to bind to a bcl-2 mrna are probe and to degrade bcl-2 mrna. recombinant bacterially expressed zfp36l1 protein was shown to bind specifically to a bcl-2 are probe by rna electrophoretic shift assays (remsas). furthermore, remsas using cell lysates of ramos burkitt lymphoma b cells stimulated to express high levels of endogenous zfp36l1 also provided evidence that endogenous zfp36l1 in b cells could bind to the bcl-2 are. in order to examine whether zfp36l1 binding to bcl-2 are resulted in bcl-2 mrna degradation, actinomycin d rna degradation assays were carried out on murine embryonic fibroblast (mefs) cells from zfp36l1 knockout mice and wild-type mice using quantitative real-time pcr analysis. bcl-2 mrna was expressed in both wild-type and knockout mefs. the half-life of bcl-2 mrna was found to be extended in knockout mefs compared to wild-type mefs suggesting that zfp36l1 does play a role in degradation of bcl-2 mrna. overall, our data are consistent with a role for zfp36l1 in degradation of bcl-2 mrna which could be a mechanism for the reported role of this protein in induction of b cell apoptosis. the epstein-barr virus (ebv) is a common human herpes virus, which can predominantly infect two types of human cells: lymphoid cells and epithelial cells. its infection is associated with several human malignancies (hodgkin's lymphoma, burkitt's lymphoma, nasopharyngeal carcinoma), where it expresses limited subsets of latent proteins among which the latent membrane protein lmp1. since lmp1 is able to transform numerous cell types, it is considered as the main oncogenic protein of ebv. the principal mechanism of lmp1 function is based on mimicry of activated member of the tnf receptor super family (tnfr), by its ability to bind a similar sets of adapters and to activate overlapping signalling pathways like nfkb, c-fos-jnk, pi3-kinase...involved in the regulation of cellular processes. we previously generated two unique model, a monocytic (te1) and lymphocytic (nc5) immortalized by ebv and which expressing type ii latency program. here we developed original dominant negative (dn), by generating a fusion between gfp and tes1 or tes2 (transforming effectors site) derived from the c-terminal intracellular part of lmp1, in inducible vectors. then, we generated cell lines conditionally expressing these dns. we showed these dns not only inhibit survival processes resulting to the impairment of nfkb and akt pathway but increase apoptosis in this cell lines. we demonstrated that this pro-apoptotic effect is due to i) the depletion of lmp1's specific adapters and ii) the recruitment of theses adapters by dns interestingly allowed generation of apoptotic complex involved tradd, fadd and caspase-8. using this nc5 tumorigenic model in scid mice, we showed that induction of the dn lmp1-tes2 prevent development of tumours and mouse death. these dominant negative derived from lmp1 could be used to develop therapeutic approaches in malignant diseases associated with epstein-barr virus, but also in inflammatory pathologies. recent studies indicate that suppressors of cytokine signaling (socs) proteins play, in addition to their action as cytokine signaling inhibitors in the immuneinflammatory response, multiple roles in cell survival, differentiation, and apoptosis in diverse cell systems. since tumor cells often exhibit aberrant expression of socs genes, which may be involved in determining resistance to anti-tumor therapies, we have investigated the role of socs isoforms during dna damageinduced apoptotic response and cell cycle changes in various tumor cell types. by using tumor cell lines transduced for over-expression or knock-down of distinct socs isoforms, it is found that socs1 and socs3 differentially affect apoptosis and cell cycle changes induced by dna damaging agents in a cell type-specific manner. in t lymphocytic leukemia cell line jurkat, socs1 exhibited anti-apoptotic effect in response to ionizing radiation, hydrogen peroxide, and etoposide by inducing suppression of p38mapk activities, while socs3 promoted apoptosis with an increase in p38 activities. in contrast, both socs1 and socs3 display proapoptotic effect in rko colon cancer cell lines upon exposure to gamma radiation or ros-generating agents. notably, effects of socs proteins on cell cycle changes induced by dna damaging agents were rather similar in that over-expression of either socs1 or socs3 induced a slight decrease in g1 or s phase cells and a prominent increase in g2/m cells, regardless of their distinct effects on apoptosis. the analysis of cell cycle regulator proteins, however, revealed that different mechanisms are operating to regulate cell cycle via distinct cyclins and cdk inhibitors affecting g1/s transition and g2/m arrest induced by socs1 or socs3. socs1 promoted dna damage-induced p21 induction and g2/m cyclin b expression, while socs3 induced decrease in g1 cyclin e expression. the results suggest that socs isoforms potentially modulate growth of tumor cells exposed to dna damage via complex network involving apoptotic response and cell cycle regulation in a cell type-specific manner. the heat shock protein 90 (hsp90) is a highly conserved a widely expressed molecular chaperone. it is known to regulate the activity of several protein kinases or the proper folding of client proteins. hsp90 has also been identified as an important regulator of cellular survival. besides these intracellular functions, extracellular hsp90 can initiate cross presentation or immune responses. apoptotic cell death occurs permanently in multicellular organisms, without initiation of an immune response. however the mechanisms which prevent an inflammatory response to apoptotic cells are not understood to date. hsp90 is released during necrotic cell death and proinflammatory effects of extracellular hsp90 have been observed. thus, we asked whether apoptozing cells cleave hsp90 during apoptosis or how hsp90 is disposed by these cells. we induced apoptosis either in activated or resting primary human cells and analyzed the hsp90 protein content. we observed that hsp90 is degraded during apoptosis resulting in the formation of a fragment of about 50 kda. this fragment was to be observed exclusively in activated cells, while it was not detected if resting cells were induced to undergo apoptosis. analyzing the isoforms of hsp90 (hsp90 alpha and hsp90 beta) we could show that the 55 kda fragment is formed after degradation of the alpha isoform of hsp90. further, we were able to show, that hsp90 cleavage is dependent on caspase activity and most probably mediated by calpain. analyzing the cytokine response of monocyte derived phagocytes to apoptotic cells in presence or absence of exogenous hsp90 and caspase inhibitors. we observed a rather proinflammatory cytokine profile, if cleavage of hsp90 was inhibited or if exogenous hsp90 was added. these results demonstrate that cleavage of hsp90 represents a mechanism preventing the release of proinflammatory molecules from apoptozing cells. activity. mifc integrates the advantages of flow cytometry and fluorescence microscopy in one system, the imagestream. upon induction of autophagy, cytosolic lc3-i is processed to lc3-ii, which then remains associated with the autophagosome until its degradation upon fusion with the lysosome. an increase in steadystate levels of autophagosomes can be due to enhanced autophagy or decreased lysosomal activity. mcf-7 gfp-lc3 cells were therefore incubated in starvation medium for 3 hours, +/-bafilomycin, which potently inhibits lysosomal activity. classical gating strategies allowed the detection of cell populations of interest, which were further analyzed on single cell levels. we conclude that imagestream-based analysis provides an improved method in terms of objectivity, sensitivity and significance, to quantify autophagic activity. our results clearly show the need for discrimination between "steady-state" levels of autophagosomes and "current flux" of fully functional autophagy, i. e. quantification of autophagic flux. jnk seems to mediate the bcl-2/beclin-1 control of autophagy. recently, jnk was shown to be necessary for beclin-1 upregulation, and jnk-mediated phosphorylation of bcl-2 is associated with both, starvation-and ceramide-induced autophagy. the nfkappab pathway mediates critical survival signals during starvation, which have been linked to the inhibition of autophagy. we report here the novel findings that under conditions of starvation, pharmacological inhibition of nfkappab decreased the autophagic flux in mcf-7 cells, while jnk inhibition shows an enhancing effect on autophagy induction. ingenol 3-angelate (pep005), a novel activator of protein kinase c (pkc), has been shown to induce apoptosis in acute myeloid leukemia cells. we show here, that in contrast to leukemic cells, pep005 provides a strong survival signal to resting and activated t cells. this anti-apoptotic effect was dependent upon the activation of pkcv, a pkc isoform restricted to t cells and myocytes. expression of pkcv in the acute myeloid leukemia cell line nb4 turned their response to pep005 from an increased to decreased rate of apoptosis. furthermore, our data show that pep005 inhibited t cell apoptosis through the activation of nfxb downstream of pkcv, leading to increased expression of the anti-apoptotic proteins mcl-1 and bcl-xl. we conclude that pkcv expression determines whether pkc activation leads to an anti-or pro-apoptotic outcome in the cell types analyzed. this finding may be of considerable importance for the development of pkc -targeting antileukemic therapies. the neuronal growth factors, neurotrophins, and their receptors are widely expressed in a variety of non-neuronal tissues including the immune system. several reports indicate that survival and activation of normal b lymphocytes are regulated by nerve growth factor (ngf) and brain-derived neurotrophic factor (bdnf) autocrine circuits. however, the production and the role of neurotrophins were not evaluated in b lymphoma cells. diffuse large b-cell lymphoma (dlbcl) is a common and often fatal malignancy. despite major advance in the treatment (r-chop protocol) which improves the clinical outcome of patients, a subset of patients does not respond or relapses after the initial treatment; the exact mechanism of such resistance is not entirely clear. we hypothesized that autocrine neurotrophin survival circuits could contribute to the chemoresistance of dlbcl tumor cells. this hypothesis was investigated with dlbcl cell lines (su-dhl). thus, we evaluated the ability of su-dhl cells to produce neurotrophins (ngf, bdnf) and to express their receptors (p75, trka and trkb) in different cell culture conditions. our preliminary data show for the first time the production of neurotrophins by dlbcl tumoral cells whose level decreased in apoptotic conditions, in association with bad dephosphorylation suggesting its pro-apoptotic role. furthermore our results suggest that up-regulation of autocrine circuits (expression of trka known to be involved in survival signaling pathways) may contribute to cell survival and thus drug resistances of tumoral b cells. objectives: ataxia telangiectasia (a-t) is a rare disorder caused by mutations in the ataxia telangiectasia mutated (atm) gene. this gene encodes atm, a protein kinase which has a major role in dna double strand break response. a-t patients suffer from a variety of immune system defects including lymphopenia, immunoglobulin deficiencies and impaired class switch recombination, they also have an increased incidence of cancer especially leukaemia and lymphoma. the susceptibility to lymphoid tumours and immunodeficiency could be partly due to failure of extrinsic apoptotic processes involved in regulation of the immune system. although atm is known to have a central role in the induction of apoptosis in response to unrepaired dna double strand breaks its role in extrinsic apoptotic pathways is unclear. this study aimed to investigate if atm has a role in fas induced apoptosis. a bank of lymphoblastoid cell lines (lcls) derived from a-t and normal individuals and tumour samples with wildtype or mutant atm were used in the study. apoptosis was induced by incubating cells with the fas activating antibody ch11 and analysed by flow cytometry. expression of the caspase 8 inhibitor cflip which inhibits fas induced apoptosis was detected by western blot. results: there was no significant difference in the susceptibility to fas induced apoptosis or cflip protein expression between atm mutant and control groups. however cells expressing high levels of cflip protein do show greater resistance to fas induced apoptosis than those with lower expression. whilst the lcls expressed both long and short forms of cflip, the tumour cells expressed only the long form. conclusion: atm mutations do not affect susceptibility to fas induced apoptosis or alter cflip protein expression in lcls or tumour cells. cflip protein levels and fas susceptibility vary greatly between individuals but this is independent of atm status. high expression of cflip protein correlates with reduced apoptosis in response to ch11 treatment but there is no clear difference in cflip expression between atm wildtype and mutant cells. labdane diterpenoids have a broad spectrum of biological activities including antibacterial, antiviral, and anti-inflammatory properties. however, little is known about their possible role in the apoptotic cell death machinery. we report that labdane diterpenoids induce apoptosis in different tumor cell lines by activating caspase 8 in the extrinsic death receptor pathway, with subsequent participation of mitochondrial signaling. activation of caspase 8 by diterpenoids was followed by a decrease in mitochondrial membrane potential, the release of apoptotic factors from mitochondria to the cytosol, and subsequent activation of caspases 9 and 3. diterpenoids also led to time-dependent cleavage of bid. inhibition of caspase-8 abrogated these processes, suggesting that the death receptor pathway plays a critical role in the apoptotic events induced by labdane diterpenoids. in addition, pretreating cells with neutralizing antibodies to fas ligand, tumor necrosis factor receptor 1 (tnf-r1), and tumor necrosis factor (tnf)-a receptor 2 (trail) inhibited diterpenoid-induced apoptosis, revealing it to be dependent on these death receptors. diterpenoid treatment also induced a significant increase in the generation of reactive oxygen species (ros). however, increased ros production was not directly involved in diterpenoid-triggered apoptosis. these results demonstrate that labdane diterpenoids induce apoptosis through activation of the death receptor pathway. conclusion: cell proliferation and differentiation are tightly regulated networks and it is believed that in cell differentiation, even in cancer form, cells precluded from proliferation. whether these changes affect the level of differentiation or the change of survivin expression can affect the proliferation and differentiation pathways are the hypotheses that need further investigation. synthetic alkyl-lysophospholipids (alps) are a group of unnatural lipids with promising anticancer capability. a prototypic member is the ether lipid 1-o-octadecyl-2-o-methyl-rac-glycero-3-phosphocholine (et-18-och 3 ; edelfosine), which induces selective apoptosis in tumor cells through activation of fas/cd95 independent of its ligand fasl/cd95l. fas/cd95 is activated by edelfosine via its translocation in lipid rafts. in this study we showed that edelfosine promotes cell death in multiple myeloma and various solid tumor cell lines in a death receptor-independent manner. edelfosine-treated cells could not be protected against cell death after inhibition of caspases by zvad-fmk while fasl-stimulated cells stayed mostly alive. furthermore cells could not be rescued by addition of zvad-fmk in combination with necrostatin-1, an inhibitor of death receptor-induced necrosis. fas resistant solid tumor cells overexpressing members of the anti-apoptotic bcl2-family as well as cells overexpressing the cellular regulatory protein flip went in contrast to fas stimulation to apoptosis after treatment with edelfosine. therefore we suggest that edelfosine induces a death receptor-independent cell death pathway in a wide range of tumor cells. apoptosis represents a cellular "suicide" mechanism which allows the control of cell number from tissues and elimination of cells that present dna mutations or having an abberant cell cycle, those cells being predisposed to malignant transformation. thus, elucidating the mechanisms of programmed cell death process seems to be of great importance for malignant transformation, tumour evasion and therefore for anti-cancer therapy. many anti-cancer drugs act during physiological pathways of apoptosis, leading to tumour cell destruction. the present study focused on the potential influence of oncolitical treatment (5-fluorouracyl) associated with natural compounds (curcumin, genistein, quercitin) on the dynamics of the cell cycle and levels of apoptosis in colon cancer cell lines (e. g. colo 201, sw1116, lovo, caco-2, ht-29) . in addition, expression of antigens involved in tumour proliferation and apoptosis (ki-67, pcna, p53, bcl-2) was compared with gene expression in the presence or absence of stimuli treatment. percentages of apoptotic cells were detected by using annexin v/fitc and propidium iodide double staining, while progression through cell cycle phases was evaluated by using pi staining. correlation analyses between the individual profile of the stimuli modulated gene expression with the coded protein expression were performed by using data from rt-pcr with specific primers, and indirect immunofluorescence followed by flow cytometry, respectively. stimuli treatment of colon cancer cell lines differentially induced higher levels of apoptosis as compared to untreated tumour cells, while cell cycle distribution of dna changed. data obtained showed a various expression and functional behaviour of the markers under study associated to colon cancer cells, suggesting their possible involvement in regulating the interactions between tumour cells and host immune system. the results obtained might further lead to the establishment of an experimental pattern for the corroboration of cell and molecular mechanisms involved in the tumour progression and the treatment resistance of colon tumors using cell lines. the effect of modulatoy agents on proliferation and apoptosis could be used in clinical departments in order to elaborate new therapeutical approaches and act as useful instruments in elaboration of individualized treatment schemes. extensive tissue trauma and malnutrition results in disorders of programmed cell death influencing the patients susceptibility to infections. the purpose of our study was to assess the effect of pancreatic cancer surgery and immunonutrition on the apoptotic signaling pathways. the randomized studies were performed in 88 patients after pancreatic cancer resection with preoperative standard or enteral immunonutrition. lymphocytes expressions of bcl-2, bax, caspase 3, 6, 9, nfkb, parp1/89kda, tnfr1/cd120a and cd95/fas were assessed by western-blot and flow cytometry. results: before and after surgery the expression of bcl-2, bax, nfkb, parp1 was significantly lower and expression of caspases, tnfr1 as well as percentage of cd95 cells significantly higher as compared with control group. caspase 3 expression was significantly higher as compared with nfkb, parp1 and tnfr1. in comparison to the standard nutrition preoperative immunonutrition increased bcl-2 and nfkb expressions and decreased caspases and parp1 expressions. in addition, we found a significant down-regulation of bcl-2 expression after surgery, but insignificant in patients with preoperative immunonutrition. conclusion: preoperative enteral immunonutrition has an modulative effect on apoptotic signaling pathways after pancreas resection and possesses antiapoptotic properties. this modulatory effect of glutamine and omega-3 fatty acids has no influence on patients outcome. the capacity of medicinal herbs to modulate cellular and humoral immune response could have useful applications in some immune-mediated disorders, infections and cancers. in this study the immunomodulatory effects of salvia mirzayanii a native plant that is widely distributed to iran was investigated. s. mirzayanii is used for the treatment of infectious and inflammatory diseases and as a tonic in folk medicine. study of the effect of this plant on the activated human peripheral blood lymphocytes showed stimulatory effects at lower concentrations and inhibitory effects at higher ones (p x 0.01). in flow cytometry analysis, accumulation of apoptotic cells in the sub-g1 phase of cell cycle of the mitogen-treated lymphocytes exposed to the inhibitory doses of the extract was observed. dna fragmentation analysis of these cells showed a typical dna laddering. immunization of the extract-treated mice with the antigen decreased delayed hypersensitivity skin reaction as well as the antibody titer at higher concentrations (p x 0.007). these results indicated the presence of immunomodulatory compounds in the extract of s. mirzayanii and suggest that the induction of apoptosis in lymphocytes might be the mechanism responsible for the inhibitory effect of the extract observed at higher concentrations. a new randomized, double-blind, placebo-controlled clinical trial was conducted with 54 healthy volunteers receiving either la1 (10 9 cfu/day) or placebo, during 57 days prior to uv (2 × 1.5 med). blister roofs, liquid and skin biopsies were collected 1, 4 and 10 days after uv exposure from non-irradiated and irradiated skin areas and used for identification of cells involved in uv-induced immune response, quantification of inflammatory cytokines, a dna damage marker (p53). while a similar decrease of lc for both groups was observed on day 1 after uv exposure compared to placebo, la-1 group presented a faster increase of a new subset of epidermal dendritic cells (dc), namely early lc precursors (cd1a low cd207 -) associated with a minor recruitment of monocytes. concomitantly, inhibition of il-10 and a tendency to inhibit il-6 was observed in la-1 group compared to placebo. on day 4, la-1 group presented a greater recruitment of early lc precursors and a trend to increase cd1a low cd207 + lc precursors compared to placebo. additionally, a faster reduction of inflammatory and immunosuppressive cytokines (il-6, tnfa, il-8, and il-10) was observed in la1 group compared to placebo. we show that la1 limits uv-induced immune-suppression and skin inflammation. this contributes to the recovery of the skin immune homeostasis, confirming the previously observed benefits of la1 supplementation for photoprotection at a lower dose (1 log). the thymus is one of the primary lymphoid organs and plays a central role in the immune system. it provides the essential microenvironment for proper t cell development. in the thymus, the maturation of t cells depends on many interactions between t cells and different stromal cell types, mainly composed of epithelial cells (tecs). foxn1 is a winged-helix/forkhead transcription factor, which is crucially required for proper epithelial cell differentiation in the thymus. foxn1 appears to be expressed in all epithelial cells of the early thymic rudiment starting around e11.5. previously, we have used a lineage-tracing system to confirm the existence of a bi-potent epithelial progenitor cell. using the cre-loxp system, we showed that a single epithelial cell, when reverted to express foxn1 in a nude (foxn1-deficient) background, can give rise to a functionally competent thymic microenvironment. hence, we hypothesize that the epithelial progenitor cell expresses foxn1. if true, it should be possible to target this cell type by use of foxn1-promoter driven transgenes. conditional targeted cell ablation is a powerful method to elucidate the physiological function of cell populations and their regenerative capabilities. currently, we are using three different strategies of conditional targeted cell ablation in order to examine functional characteristics of epithelial bi-potent progenitor cells within the thymus. intracellular accumulation of poly glutamines is known to cause neurodegenerative disorders, such as huntington's disease. considering this, we are expressing transgenic egfp variants containing either 19 or 82 glutamine residues under the control of foxn1 promoter, leading to different degrees of tec degeneration. furthermore, also under the foxn1 promoter, we are using the transgenic expression of human diphtheria toxin receptor and the transgenic expression of the bacterial nitroreductase enzyme that converts the pro-drug metronidatole (mtz) into a cytotoxic cross-linking agent for conditional cell ablation. preliminary results describing the phenotypes of these mice will be presented. t. kamei 1,2 , y. toriumi 3 , k. kimura 4 1 european university viadrina, frankfurt (oder), germany, 2 shimane institute of health science, izumo, japan, 3 shimane university faculty of medicine, department of pediatrics, izumo, japan, 4 japan yoga niketan, yonago, japan as a method in relieving stress, yoga is popular today. many reports of physical changes describing how yoga improves respiratory, circulatory, endocrine, and metabolic functions by yogic practice have been reported until now. we examined changes of electroencephalograph (eeg) and cellular immunity before, during, and after yoga exercises, in an endeavor to detect the correlation between them. the subjects consisted of eight yoga instructors who had been practicing yoga for several years. a 10-minute-rest period, followed by a 15-minute yoga exercise called asana, a 15-minute respiratory exercise called pranayama (various specialized respiration methods continuously performed with the eyes closed), and a 20-minute meditation were performed. throughout rest and yoga, brain rhythms were continuously recorded via two disc electrodes placed on each forehead (fp2). blood samples were drawn before and after each exercise. nk activity and percentages of t-cell and b-cell subsets were measured. during the pranayama period, both a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in nk activity (r=0.83, p x 0.02), and a positive correlation between the change in abundance of the activated alpha waves and the ratio of changes in the number of t lymphocytes (r=0.77, p x 0.05) were observed. furthermore, a positive correlation was also observed between the change in amplitude of the activated alpha waves and the ratio of change in the number of cd4 (r=0.96, p=0.0001). these findings suggest that yoga creates a stress-free and mentally concentrative state which activates the functions of nk cells and t lymphocytes, mainly of cd4, within a short period of time. we conclude from these results that yogic respiratory exercise may be able to activate cellular immunity and to help recover the mental and physical harmony of human. yoga is considered to have an effect of some re-activation of a latent ability of harmonization in which humans naturally possess. t regulatory cells play a central role in the suppression of immune responses thus serving to induce tolerance and to control persistent immune responses that can lead to autoimmunity. several recent studies suggest also that diverse populations of regulatory t cell play an important role in regulating t-helper 2 response to allergens, maintaining functional tolerance and preventing allergy. here we demonstrate that cd4 + cd25 + t regulatory (t reg ) cells are critical in controlling the immediate hypersensitivity response of bone marrow mast cells (mcs) without affecting cytokine release. this effect is shown to require a cell-cell contact and depends on interaction between ox40l expressed on mcs and the constitutive expression of ox40 (members of the tumor necrosis factor [tnf] and tnf receptor family, respectively) on t reg cells. this interaction does not alter the activation of plc-g, syk and lat in ige/ag stimulated mcs upon co-incubation with t reg cells, whereas it induces a decrease in the phosphorylation levels of akt. moreover, we find that upon co-incubation with t reg cells, mcs show increased levels of camp, which is known to inhibit mcs function, as a result of ox40l signal. antagonism of camp in mcs reverses the inhibitory effects of t reg cells restoring normal ca 2+ responses and degranulation. the cross-talk between t reg cells and mcs through ox40-ox40l interaction defines a previously unrecognized mechanism controlling mcs degranulation. loss of this interaction may contribute to the severity of allergic responses or inflammatory disease. active regulation has emerged as a very essential mechanism for both inducing and maintaining peripheral tolerance to non-pathogenic environmental antigens. a healthy immune system responds to antigens with a combination of polarized th1 or th2 effector cells and the induction of antigen specific foxp3+ regulatory t cells (treg). it is believed that the dominant subset determines the quality of the eventual immune response. in allergic asthma there is a clear dominance of th2 cell responses to non-pathogenic environmental antigens. recently it was shown that the specific transcription factors that characterize the th2 and treg subset, gata3 and foxp3 respectively, counter regulate each other (mantel y et al., 2008) . we hypothesize that children with allergic asthma will respond to allergens with low induction of foxp3+ tregs and high gata3+ th2 cells. in order to prove this hypothesis pbmc of children with allergic asthma and non-sensitized healthy controls are stimulated with allergens, tetanus toxoid, and lps. almost 100 million allergic patients are sensitized to the major birch pollen allergen bet v 1, which cross-reacts with major allergens of fagales (e.g., alder, hazel, hornbeam, oak) pollen and plant food allergens. the epitopes of bet v 1 recognized by allergic patients' ige antibodies belong to the conformational type and therefore have not been characterized in detail. here we used antibodies raised against peptides spanning the bet v 1 molecule in ige competition experiments to search for sequences which are involved in ige recognition. the strongest inhibition (i.e., g 90 %) of patients' ige binding to bet v 1 was obtained with polyclonal and monoclonal antibodies specific for peptides comprising aa 30-59 (p2) and aa 74-104 (p6) of bet v 1. cross-reactive ige epitopes between bet v 1 and related pollen allergens and plant food allergens involved primarily p2. p2 and p6 are not adjacent peptides in the bet v 1 sequence but define a surface-exposed patch on the three-dimensional structure of bet v 1. as determined by surface plasmon resonance, monoclonal antibody mab2 specific for p2 and mab12 specific for p6 showed high affinity, i. e., dissociation constants, k(d) = 8.35e-11 m and k(d) = 1.05e-9 m, respectively. interestingly, peptide-specific mabs inhibited allergic patients' ige antibodies equally well as peptide-specific polyclonal rabbit antibodies but only the latter inhibited strongly allergen-induced basophil degranulation. this finding indicates that the surface patch defined with anti-p2 and anti-p6 antibodies contains several ige epitopes. in summary, we have defined a surface-exposed patch on the bet v 1 allergen which seems to harbor the majority of the ige epitopes and may be used for the rational design of active and passive immunotherapy strategies against birch pollen and related allergies. background: antigen-specific th1 cells as well as tc1 cells, induced by biolistic gene transfer using plasmid dna encoding the model allergen b-galactosidase (bgal) under control of the fascin promoter (pfascin-bgal), inhibited the elicitation of systemic th2 immune responses and suppressed ige production in an experimental mouse model. moreover, protective biolistic dna vaccination with pfascin-bgal prevented th2-mediated lung pathology (eosinophilia) in sensitized mice locally challenged with bgal protein, but led to the recruitment of th1/tc1 cells into the lung, associated with substantial neutrophilic infiltration and the induction of airway hyperresponsiveness (ahr). objective: to analyze the modalities of ahr induction in mice biolistically vaccinated with pfascin-bgal. methods: balb/c mice were immunized with pfascin-bgal using the gene gun. subsequently, mice were challenged by consecutive intranasal application of bgal protein. cd4 + and cd8 + t cells, respectively, were depleted before and during the provocation phase by intraperitoneal injection of anti-cd4 (gk1.5) or anti-cd8 (53.6.72) monoclonal antibodies. neutrophilic granulocytes were depleted by treatment of animals with either anti-gr-1 monoclonal antibody rb6-8c5 or monoclonal antibody nimp-r14. one day after the last challenge airway reactivity was assessed by whole body plethysmography, bronchoalveolar lavage (bal) was performed and the frequency of ifn-g-producing cd8 + effector t cells in the lung was determined. results: whereas neutrophilia in the lung of immunized and challenged mice was considerably alleviated by depletion of cd4 + t cells, ahr was not significantly affected, implicating that the elicitation of ahr by cd8 + t cells is dissociated from the activity of neutrophils. this notion was verified by elimination of neutrophils during the provocation phase, likewise leading to unaltered ahr. in contrast to cd8 + t cells, cd4 + t cells induced strong neutrophilic infiltration and ahr. transfer experiments with cd4 + or cd8 + t cell, separated from the airways of vaccinated and challenged mice, will probably reveal details of the effector mechanisms of th1 and tc1 cells operative in the elicitation of airway inflammation. conclusions: robust type 1 immune responses, although highly effective in the counter-regulation of local th2-mediated pathology, might as well trigger inflammatory reactions in the lung and provoke the induction of ahr. respiratory epithelial cells function as physical barrier and have shown to be active participants within the process of defense against pathogens and recognition of allergens. upon activation they release inflammatory mediators thereby creating a micro environment in which recruited immunocompetent cells induce a local immune response. house dust mite (hdm) extract as a source of allergens has been shown to induce a broad panel of genes upon stimulation of epithelial cell line nci-h292. the proteolytic activity of these hdm allergens has been proposed to be involved in the activation process. the aim of this study was to compare the influence of hdm extract on respiratory epithelial cells with grass pollen allergen-induced activation of these cells with regard to the mechanism of activation, gene expression level, and the level of induced cytokine and chemokine release. in contrast to the hdm major allergen der p 1, we were able to show that the major allergen of phleum pratense, phl p 1, although sharing molecular similarities with der p 1, does not display any enzymatic activity under physiological conditions. therefore, in this study respiratory epithelial cells were stimulated with grass pollen extract and purified phl p 1. chemokine and cytokine release was determined by multiplex enzyme-linked immunosorbent assay and mrna was used for cdna-microarray analysis. first data show that both, hdm extract and grass pollen allergens, induce the release of il-6 and il-8 from nci-h292 cells. furthermore, stimulation with hdm extract leads to the release of tnf-a, gm-csf and ifn-g. interestingly none of these mediators was induced after stimulation with grass pollen extract or purified phl p 1. in contrast to hdm extract grass pollen allergens induce the release of mcp-1 from respiratory epithelial cells, as well as moderate levels of il-12. detailed characterization of the response on gene expression level might give new insights into the pathophysiology of grass pollen allergy and a comparison with hdm induced expression profiles will be helpful towards understanding the allergic response in general. (supported by dfg sfb tr22) results: collectively, responses to blg, but not to bsa, were observed in all groups analyzed, included healthy controls. nevertheless, 3 distinct profiles of response were obtained: children with ige mediated cma had a significant increased level of proliferation (mean±sd of stimulation index(si): 7.2±5.7) and of il-13 (mean±sd: 1157±909 pg/ml), and reduced il-10 (mean±sd of il-10-spot forming units/2x10 5 cells (sfu): 912±510), compared to healthy subjects (3.4±2.7 si, p x 0.05; 355±396 pg/ml, p x 0.05; 1272±623 sfu, for proliferation, il-13 and il-10, respectively); children with non-ige mediated cma had a significant reduction of il-13 (192±362 pg/ml), compared to ige patients (p x 0.0004) and an increased, although not statistically significant, production of ifn-g (33.7±54.6 sfu) compared to control (9.5±9.5 sfu) and to ige-allergic patients (0.6±0.8 sfu). finally, tolerant patients showed reduced il-13 (636±1048 pg/ ml, p x 0.05) and proliferation (3.9±3.5 si), compared to acute ige-cma children. interestingly, the high level of il-10 observed in all groups might have a counter-regulatory effect, since its neutralization resulted in an increase of proliferation to blg; by contrast, il-4 was undetectable in all patients even blocking the il4-receptor. conclusion: blg-specific, immune responses can be recalled in peripheral blood of cma patients, as well as of normal and tolerant children. a th2-like response with il-13 and proliferation is dominant in ige-mediated cma patients; by contrast a th1-skewed response with ifn-g is present in non-ige-mediated allergic and in those children who outgrew ige-allergy. y. f. tang 1 , b. chua 1 , f.c. lew 1 , a. ho 1 , k. wong 1 , k.l. wong 1 , d. m. kemeny 1 1 national university of singapore, immunology programme, yong loo lin school of medicine, singapore, singapore allergic inflammation of the airways causes changes in the lung wall that can lead to chronic inflammatory disease such as asthma. using a mouse model, this response can be divided into an induction phase, in which cd4 th2 t cells specific for airborne allergens are produced, and an effector phase, during which they are recruited to the lung. in the lung, recruited th2 cells orchestrate the inflammatory response marked by eosinophilia, mucus hyper secretion and increased airway hyperresponsiveness (ahr). previously we, and others, have shown that transfer of cd8 t cells inhibits the induction of the th2 response. here we have investigated the effect of cd8 t cells on the effector phase of the inflammatory lung response. in vitro activated ot-i cd8 t cells were transferred to ovalbumin (ova)/ alum immunized mice one day before the first of 3 airway challenges with ova. eosinophil infiltration was inhibited by transfer of cd8 + t cells (36.7 %±4.1 % to 17.6 %±2.7 %). when ifn-gamma -/-ot-i cd8 t cells were transferred, we found that the inhibitory effect on eosinophilia was reduced (39.6 %±5.1 %), suggesting an important role for cd8 t cell ifn-gamma. cd11c + cd103 + cd11blung dcs from cd8 transferred mice secreted higher levels (500pg/ml) of il-12p70 following ex-vivo stimulation as compared with animals that were not given cd8 t cells. these data show that, in addition to regulating the induction of the allergic immune response, cd8 t cells can subsequently divert the local lung environment to one that favors th1 immunity. the chain terminator drug abacavir triggers a serious hypersensitivity reaction in 8 % of patients with hiv infection. this reaction is strongly associated with hla-b*5701 and appears to be mediated by cd8+ t cells producing inflammatory cytokines. we show that cd8+t cell responses can be primed in vitro, in normal blood donors who are hla-b*5701+, but not in non-b*5701+ donors. cd8 t cells, but not cd4 t cells, are expanded by abacavir pulsed autologous apc over a13-day culture, producing ifn-gamma and tnf-alpha upon re-stimulation with apc expressing hla-b*5701. similar responses were detected in abacavirhypersensitive hlab*5701+ patients. responses were not detected using mutant apc deficient in tap or tapasin, or when normal apc were aldehyde fixed before loading with abacavir, indicating a reliance on the conventional mhc-i ag presentation. responses were exquisitely restricted to hla-b*5701 since they were undetectable using apc expressing closely related hla-b57 or b58 allotypes. responses to apc expressing mutants of hla-b*5701 demonstrated a crucial role for residue 116. isolation of peptide fractions from abacavir-loaded cells has led to the identification of specific fractions recognised by an abacavir-specific t cell line. our data suggests that abacavir forms a conjugate with an endogenous peptide that is presented by hla-b*5701 triggering cd8 t cells. we speculate that this form of altered self is highly immunogenic, behaving like a form of allogeneic mhc-i, contributing to the responses observed in abacavir naï ve individuals. the molecular mechanisms underlying altered hla-b*5701 may be relevant to the role of other disease-associated class i allotypes such as hla-b27 and b51. a. jenckel 1 , s. bulfone-paus 1 , n. föger 1 1 research center borstel, immunobiology, borstel, germany mast cells play a crucial role in acute inflammatory and allergic reactions. upon activation, mast cells secrete a vast array of preformed and newly synthesized inflammatory mediators. recent work has begun to appreciate an important role of the actin cytoskeleton in mast cell activation. the actin-associated protein coro-nin1a (coro1a), a coronin family protein preferentially expressed in hematopoietic cells, is critically involved in various actin-mediated cellular functions of leukocytes. recent data of our group also indicate a regulatory role of coro1a in mast cell function. coronin proteins have been described to be differentially phosphorylated in vivo. however the molecular mechanisms by which coro1a is regulated in response to physiological stimuli are still poorly characterized. here we investigated the modalities of coro1a phosphorylation during the activation of mast cells. immunoprecipitation studies combined with phospho-specific western blotting techniques revealed a transient phosphorylation of coro1a on serine residues upon antigen-specific engagement of fc-epsilon-receptors. as the phosphorylation status of coro1a can influence its association with the actin cytoskeleton, we analyzed the subcellular localization of coro1a during mast cell activation. cell fractionation experiments demonstrated that the association of coro1a with the actin cytoskeleton significantly decreases in response to mast cell stimulation, concomitant with the increase in coro1a phosphorylation. a functional correlation between coro1a phosphorylation and its association with the actin cytoskeleton in mast cells was further indicated by structure function experiments employing specific phosphorylation mutants of coro1a. thus, coro1a is a downstream effector molecule of fc-epsilon-receptor signaling and likely is involved in the dynamic reorganization of the actin cytoskeleton during mast cell activation. allergen-specific t and b lymphocytes play an important role for the pathogenesis of asthma. t cells orchestrate the infiltration of the lung tissue with eosinophils and neutrophils and provide help for allergen-specific immunoglobulin production. recently, we have shown in a mouse model for allergic airway inflammation that b cells directly interact with t cells in the inflamed tissue and locally produce ige. to analyse t/b-interaction in the inflamed tissue in more detail, we developed a novel adoptive transfer system using ovalbumin-specific t cells and nitrophenolspecific b cells. recipient mice are then challenged intranasally with an np-ova conjugate. this system allows to track single allergen-specific t and b cells in all stages of the immune reaction using flow cytometry and immunohistology. in addition, cells can be re-isolated by flow-sorting for in-depth analysis. using this system we could define several phases of the inflammatory reaction. t and b cells first become activated in the lung-associated lymph nodes. granulocytes can be found very early in the lung tissue and also activated t cells very rapidly emigrate to the site of inflammation. however, clusters of allergen-specific t and b cells can only be found in later stages of the reaction. as another focus, we used our in vivo system to define the role of t cell costimulatory molecules for airway inflammation. costimulatory receptors are key regulators of t cell activation and differentiation and therefore promising targets for therapeutic intervention. of special interest is the t cell-specific icos molecule which is important for t/b cooperation as well as the regulation of chronic inflammatory reactions. using icos knock-out mice we were able to delineate the specific role of icos for the different stages of airway inflammation. in particular, we analysed the impact on t cell subset differentiation, cytokine production and allergen-specific immunoglobulin production. the integrity of the actin cytoskeletal network is critcal for a large variety of cellular functions. coronins constitute a family of evolutionary highly conserved wdrepeat containing proteins that have been implicated in the regulation of actin cytoskeletal dynamics. in mammalians seven coronin family members have been described. the high degree of sequence conservation amongst coronin family proteins suggests common features and functions. however, individual family members may also have developed additional selective and specific functions. our recent studies on coronin1a (coro1a) deficient mice have demonstrated that coro1a exhibits an inhibitory function on the cellular steady-state f-actin content, is required for chemokine-mediated functions in t cells and is involved in the maintenance of t cell homeostasis. coronin1b (coro1b) is a closely related homolog of coro1a and the two genes are co-expressed in hematopoietic cells. to address the question of functional redundancy in vivo, we have generated coro1b deficient mice and crossed them with coro1a deficient mice to obtain coro1a/coro1b double deficient mice. analysis of t lymphocytes from coro1a/coro1b double deficient mice revealed defective chemotactic responses and a severe peripheral t cell lymphopenia in double-deficient mice, which was significantly exacerbated as compared to the respective single knock-outs. an analysis of coronin deficient mast cells also revealed an involvement of coro1a/coro1b in the regulation of actin cytoskeletal dynamics and the function of mast cells. however, in contrast to the inhibitory effects of coro1a/1b deficiency on t cell function, mast cell degranulation and migration was enhanced in coro1a/1b double deficient mast cells. thus, depending on cell type specific requirements, coronin proteins can either exhibit positive or negative regulatory functions. additional studies will investigate molecular and regulatory mechanisms by which coronin proteins control actin cytoskeletal organization and function of immune cells. together, our studies here reinforce and expand our appreciation of the importance of actin-cytoskeleton regulatory proteins for immune cell function. initially found by serial analysis of gene expression, murine samsn1 (also known as hacs1 or sly2) is a putative adaptor and scaffold protein with a sterile-alphamotif (sam), a src homology 3 (sh3) domain and a predicted bipartite nuclear localization signal. the samsn1 gene is located on mouse chromosome 16 and encodes a well conserved protein with 364 amino acids, which is predominantly expressed in hematopoietic tissues. initial overexpression studies suggest a contribution of samsn1 in b cell activation and differentiation, however its physiological function is yet unknown. to investigate samsn1 expression in lymphatic and myeloid cell types in greater detail we employed the sensitive method of quantitative real-time pcr. our data revealed an expression of samsn1 in all tested hematopoietic cell types. the highest expression level of samsn1 mrna was seen in mast cells compared to lower levels in macrophages, dendritic cells, cd4+ and cd8+ t cells and b cells. the other two members of the sly family of adaptor proteins -namely sly1 (hacs2) and sly3 (sash1) -were expressed only at a very low level in mast cells. the high level of samsn1 mrna expression in mast cells, together with minimal expression of other sly family proteins in these cells, implicates an important role of this adaptor protein for mast cells. to address the potential role of samsn1 in mast cell differentiation and function we are analyzing bone marrow derived mast cells from samsn1 deficient mice. initial in vitro experiments indicate normal proliferation and differentiation of samsn1 deficient mast cells. in additional studies we are now investigating the effects of samsn1 deficiency on mast cell activation processes, such as degranulation, cytokine production and the signal transduction cascade. analyzing the role of samsn1 in mast cells will help to define the biological function of this novel class of adaptor proteins. introduction: we showed previously that the ability of murine igg1 antibodies to mediate anaphylactic reaction is directly dependent on the amount of sialic acid residues attached to the carbohydrate chain n-linked to the antibody fc region (silva et al; j.immunol., 2008). then, we hypothesize that differences in the glycan composition mainly the sialylation grade observed between the anaphylactic and non-anaphylactic igg1 abs may be resultant of the differential expression of the glycosyltransferase, essentially sialyltransferase, coding genes during its synthesis in b cells. objective and methods: to prove this hypothesis it was analyzed the expression of st8siai-v; st6galnac i-iv, st3gal ii -v genes quantitatively by real time-pcr in the hybridomas producer of these two types of igg1 abs. results: we observed that the expression of st3gal i, iii and v coding genes was similar in both hybridomas, while the st3gal ii and iv genes were less expressed in the hybridoma producer of non-anaphylactic igg1. in addiction, the expression levels of st8sia and st6galnac genes in the hybridoma producer of anaphylactic igg1 were significantly higher when compared to those observed in the hybridoma producing of non-anaphylactic igg1. conclusion: these data suggest a direct correlation between the sialylation grade observed in the carbohydrate chain attached to the igg1 abs and the expression of sialyltransferase enzymes in the hybridomas producer of these molecules. financial support: cnpq, capes, fapesp. basophils are innate immune cells endowed with important effector functions during allergic inflammation and parasite infection. their activation in terms of histamine and cytokine production is mediated through immunoglobulin-dependent and -independent mechanisms, raising the question whether stimulation of tolllike receptors (tlrs), which have been described in basophils, has a similar effect. we found that, in contrast to other tlr agonists tested, only the doublestranded rna poly(a:u) induced the typical t h 2 cytokine and histamine production in vitro. this compound was also fully active when administered in vivo since it activated basophils and promoted their recruitment to the periphery. we took advantage of a murine model of allergic asthma to establish the pathophysiological relevance of this finding. using both adoptive transfer and depletion of basophils, we established not only that these cells contribute directly to the severity of asthma symptoms, but also that a mimic of viral infection can aggravate the disease through their activation. this is the first evidence for a mechanism of exacerbation of allergic asthma induced by a mimic of viral infections, mediated through basophils. ishes the airway hyperresponsiveness and airway inflammation in experiment murine asthma models. to investigate the effect of activation of nkt cells at different allergic asthma progression, we administered balb/c mice with a-galactosylceramide (a-galcer), a stimulator for nkt cell activation, before or after ova immunization and measured the airway inflammation of that mice after 2 times of intranasal ova challenge. in our results, the total numbers of bronchoalveolar lavage (bal) cells were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. moreover, significant increased percentage and cell numbers of eosinophils in bal of mice administered with a-galcer before ova immunization was noted. il-5 and eotaxin are the most potent cytokine/chemokines for the recruitment of eosinophils. il-5 and eotaxin levels in the bal fluid were higher in mice administered with a-galcer before ova immunization compared to that of mice administered with a-galcer after ova immunization. these data demonstrate that activation of nkt cells at different allergic asthma progression dictates the different outcome of asthma. in addition, the activation of nkt cells in naïve mice induces airway inflammatory responses. the potential risks of treatment with nkt cell activation on human diseases should be considered. objective: bronchial asthma is a complex disease of the lung and is characterized by a variety of symptoms such as airway hyperresponsiveness, reversible airway obstruction, high serum levels of ige and inflammation. histologically, there are infiltrations of eosinophils, degranulated mast cells and hyperplasia of airway globlet cells in addition to lymphocytes. the transcription factor irf1 mediates the differentiation into th1 cells by activating multiple genes which are independently crucial for the development of naive t cells into th1 cells. because irf1 is expressed in many different tissues, it can be considered as a master switch factor for th1 cell differentiation. methods: here, we tested mice deficient in irf1 in the murine acute asthma model to evaluate its importance in this th2 cell-mediated disease. the protocol setup was the following: 3 sensitizations s. c. with ova, followed by 3 challenges via ova inhalation and adoptively transferred wildtype cd8+ t cells prior to initial sensitization. in our experiments, we could demonstrate that only after priming of irf1 deficient mice with the help of adoptively transferred cd8+ t cells, asthma symptoms in these mice were more severe than in wildtype controls. as an example, eosinophil infiltration into the lung was increased by 3.5 fold. likewise, ovaspecific antibodies and numbers of goblet cells (fig. 1) were also significantly higher in irf1 deficient mice. conclusion: interferon regulatory factor 1 plays a role in the severity of the development of asthma. in its absence, proinflammatory parameters in the lung are increased significantly. this effect is only visible in the presence of wildtype cd8+ t cells. mechanistically, a potential counterregulation of asthma by th1 cells is not available in irf1 knockout mice. together with our previous report that irf1 represents a susceptibility gene for allergy in the human, our data highlight irf1 as key in regulating the severity of asthma. the sensory neuropeptide substance p (sp) acts as an important stress mediator with its own stress axis in the skin modulating mast cell as well as antigenpresenting cell (apc) activity. here we postulate that stress-dependent communication between nerve fibers and immune-competent cells can also occur in spleen and affects the course of inflammatory disease. to address this question, atopic dermatitis-like allergic dermatitis (ad) was induced in c57bl/6 mice by intraperitoneal sensitization and intradermal challenge using chicken egg ovalbumin. animals were additionally exposed to noise stress for 24hrs prior to challenge. in this model, stress lead to a relative hyperinnervation of the immune-competent areas of the spleen. at the same time, an increased number of apc could be observed in these areas and contacts between nerve fibers and apc were found. under the same conditions, we were able to show increased nk1-receptor and ppt1 mrna levels. accordingly, sp had the capacity to raise the number of antigen presenting cells in spleen and altered the profile of cd11c expressing apc substets characterized by cd4 and cd8 expression in vitro. in vivo we found a stress dependent shift of cytokine mrna levels towards a th-1 cytokine profile and increased levels of il-2 mrna. further the number of cd25 + t-regulatory cells was increased in vitro. additional analysis of the quality and function of neuro-immune interactions in the spleen will reveal the role of the observed stress-induced alterations in allergic inflammation. proton pump inhibitors (ppis) that are the cornerstone of gastroesophageal reflux disease therapy have been reported to improve asthma and eosinophilic esophagitis (ee) in patients with associated symptoms. the most accepted explanation for these findings is based on the belief that pathologic acidic reflux can act as a triggering factor for these diseases through proximal extent and laryngopharyngeal reflux in asthma and impairment of the epithelial barrier in ee. under these considerations, acid suppression is believed that could prevent these pathogenic mechanisms. nonetheless, a number of evidences suggested the possibility that ppis could have a direct effect in molecular pathways involved in asthma and ee: 1) the inhibitory mechanism of ppis implies alkylation of cysteine residues in gastric atpase3, 2) asthma and ee are prototypic th2 diseases in which the cytokines il-4 and il-13 play a principal role through the activation of the transcription factor stat6, and 3) we have recently demonstrated that some chloromethyl ketones can downregulate stat6 by mechanisms involving cysteine alkylation. on the theoretical basis that cysteine reactivity of ppis may affect the regulation of stat6, we analyzed its effect in the activation of stat6 by il-4 and il-13. we found that treatment of cells with ppis inhibited the ability of il-4 and il-13 to signal stat6 activation in a dose-dependent manner in multiple cell types from different origin. given the important role of these mechanisms in asthma and related diseases, our findings show a novel mechanism to understand the effect of omeprazole in these diseases. in argentina more than three million people suffer from asthma, and the number is rising. asthma is defined as a disorder characterized by chronic airways inflammation that results in high mucus production and airways hyperresponsiveness. a th2 mediated immune response prevails in these patients. in the asthmatic exacerbation period, crisis (cr), triggered by viral infections or other factors, there is a high prevalence of bacterial overinfection. our objective is to compare immunological parameters and in vitro response of lymphocytes to bacterial antigens in the same patient, at that moment and at a time of stability between episodes (i). we studied 12 asthmatic patients both at cr and i. we evaluated eosinophils, basophils and ige expressing b lymphocytes; as well as t regulatory cells (by expression of cd4 and cd25 high (treg)), that might inhibit the development of a th2 response, together with gdt cells, which function in asthma is not completely understood, but could have a role in the increased airway responsiveness. to evaluate the t cell response, mononuclear cells were cultured for 48 hours in the presence of m. tuberculosis (m) or s. pneumoniae (spn), or absence of them (c). then the percentage of activated cells was determined (expression of cd69 or cd25 at 48 hours). all the parameters were evaluated in peripheral blood by flow cytometry. discussion: even though the pathophysiological characteristics of asthmatic patients in periods of cr and i are different, no significant differences were observed in the parameters (cell populations and cell response to bacterial antigens) evaluated when compared for the same patient at cr and i. we might be able to detect differences if we studied cells from the lungs, the target organ. we demonstrate that murine and human lc expressed the h4r. the level of intracellular ccl2 production in human lc was reduced after stimulation with h4r agonists and basal production could be restored when h4r was blocked with the specific antagonist jnj7777120. moreover histamine and a h4r specific agonist augmented the migration of lc from the epidermis as shown in ex-vivo migration experiments using human skin and in-vivo migration experiments in mice. in conclusion, the h4r is expressed on murine and human lc and influences the immunomodulatory function and migration of these cells. these findings underline the relevance of the h4r in allergic skin diseases and encourage further exploration of the h4r as a therapeutic target in allergic skin diseases. expression data of the non-coding rna gene, prins (psoriasis susceptibility-related rna gene induced by stress) identified and characterized by our workgroup, suggests a role for prins in psoriasis susceptibility and in cellular stress response. in order to asses the function of prins, we aimed to identify genes regulated by prins and intracellular molecules interacting with this stress-induced non-coding rna. to identify prins regulated genes, we carried out a cdna microarray chip experiment on hela cells where the expression of prins was silenced. this experiment identified g1p3, an interferon-inducible anti-apoptotic gene that was down-regulated by prins silencing. g1p3 was strongly expressed in proliferating keratinocytes and markedly upregulated in involved psoriatic epidermis compared to healthy epidermis. to detect prins interacting proteins we applied ribonucleoprotein (rnp) purification in hacat cells. with the help of matrix-assisted laser-desorption ionization time-of-flight (maldi-tof) method we identified nucleophosmin, a protein that physically interacts with prins rna. nucleophosmin is a ubiquitously expressed nucleolar phosphoprotein which shuttles continuously between the nucleus and the cytoplasm. immunohistochemical experiments revealed that the expression of nucleophosmin was significantly elevated in psoriatic involved epidermis, localized to the dividing cells of the basal layer. our data indicate that the non-coding prins rna forms a molecular complex with nucleophosmin that regulates stress-induced cellular processes. we suppose that the abnormal functioning of this complex may result in the altered regulation of genes among them the anti-apoptotic g1p3 which can contribute to the pathogenesis of psoriasis. atopic dermatitis (ad) is a chronic inflammatory skin disorder based on a genetic predisposition and triggered by environmental factors characterized by eczematous skin lesions, pruritus, and typical histopathological features. rituximab is a monoclonal anti-cd20 antibody therapy that targets pre-b cells and mature b cells, but not plasma cells. ad is generally considered as a biphasic, with switch to initial th2 to chronic th1-predominant disease, in which rituximab may have multiple effects. objectives: to report three patients with severe ad refractory to conventional treatments and to anti-ige monoclonal treatment (omalizumab). materials and methods: three patients with severe refractory ad with high levels of serum ige that received 4 weekly intravenous infusions of rituximab at a dose 375 mg/m 2 body surface each. subsets of lymphocytes were analyzed with multiparametric-flow cytometry (facscalibur, bd) at baseline and at specific intervals after treatment. serum immunoglobulins levels were quantified by nephelometry. results: at baseline, all patients had highly elevated levels of total ige ( g 5,000; g 6,000; g 19,000 mg/dl, respectively). all patients underwent prior treatment with omalizumab for 6 months, with only partial response. then, we started rituximab therapy, resulting in a clear and complete improvement of ad eczema area and severity of skin lesions in all patients. remission of pruritus was observed from the 2 nd week after initiation of rituximab therapy up to 1 year. whereas allergen-specific ige levels were not altered, we observed a large reduction in total serum ige concentrations after initiation of therapy with rituximab. in the first treated patient (follow-up 1 year), ige levels decreased from 5,512 to 1,500 mg/dl. the other two patients are in the 3 and 1-months of the follow-up period. importantly, during follow-up no other therapies were required for ad control. conclusions: treatment with an anti-cd20 antibody led to a dramatical improvement in our series of patients with severe refractory ad. this study support further evidence on the efficacy and safety of rituximab in severe ad. we have previously demonstrated that chronic topical exposure of mice to the contact allergen dncb or to the respiratory sensitiser trimellitic anhydride (tma) preferentially activates t helper (h) 1 and th2 cells, respectively. in addition, a single application results in divergent cutaneous cytokine production and the migration of langerhans' cells (lc) with different tempos. to explore events occurring after allergen application, balb/c strain mice were exposed to a single topical dose of either 1 %dncb, 25 %tma or to vehicle alone for 0.5-6h. measurement of cytokine production from skin exposed to the allergens was performed by cytokine bead array. exposure to dncb provoked rapid production of il-2 (mean=131pg/ml, n=12, p x 0.001), il-17 (mean=69pg/ml, n=12, p x 0.001) and il-1a (683pg/ml, n=12, p x 0.001) in skin compared with tma-or aoo-treated mice. in subsequent experiments, mice received an intradermal injection of 50ng/ear of murine recombinant il-2 or of the known regulator of lc migration; il-1b. interleukin-1b induced a significant loss of epidermal lc numbers, measured as a function of reduced frequency of mhc class ii positive cells within epidermal sheets, after 4 (32 %) and 16h (31 %) (n=6, p x 0.001). in contrast, il-2 or control injections were without effect. however, il-2 administration caused an increase in cutaneous il-17 production (347pg/ml, n=12, p x 0.001) compared with control injection and naï ve tissue (19 and 63 pg/ml, n=12, ns). in addition, systemic treatment with anti-il-2 antibody failed to impact on lc migration provoked by dncb (33 % reduction; n=6, p x 0.001). in parallel experiments dncb-induced lc migration was blocked by treatment with anti-tumour necrosis factor (tnf)-a antibody, another cytokine known to regulate lc migration. however, dncb-induced cutaneous il-1a (987pg/ml, n=10, p x 0.001) and il-17 (248pg/ml, n=6, p x 0.01) expression was reduced to baseline levels by anti-il-2 treatment. these data demonstrate that il-2 is not involved in the regulation of lc migration, unlike il-1b and tnf-a. however, il-2 is involved in the regulation of the production of other cutaneous cytokines provoked by dncb. therefore it is hypothesised that il-2 may influence lc and dermal dc maturation, via the expression of il-1a and il-17. allergic contact dermatitis (acd) caused by nickel ions (ni) represents the most common form of human contact hypersensitivity. along with other allergies its incidence is increasing in the us and worldwide (nhanes iii survey 2005), but the majority of molecular events underlying this kind of t-cell mediated disease are still widely unknown. to elucidate initial molecular mechanisms (sensitization phase) taking place at the primary allergen contact site in human skin a differential proteomic approach was chosen. by applying dige technology (differential gel electrophoresis), software analysis and mass spectrometric protein identification to cell lysates of allergen stimulated human keratinocytes, seventeen proteins were identified that are specifically regulated by metal allergen ni. in the attempt to further characterize the role of a certain down regulated p38-mapk-pathway related protein (p38prp) in acd, we analysed its regulation, differential distribution of phosphorylated isoforms as well as its subcellular localization. our results strongly support an involvement of p38 mapk pathway in allergenspecific signaling responses. it is expected that identification of differentially allergen-regulated proteins and detailed analysis of acd-associated signaling events in primary keratinocytes will lead to a better biomolecular understanding of the initiation of human contact hypersensitivity. (work supported by eu-project novel testing strategies for in-vitro-assessment of allergens, lshb-ct-2005 -018681, www.sens-it-iv.eu.) objectives: schnitzler's syndrome is a rare disease characterised by chronic urticaria, monoclonal gammopathy, fever, and arthralgia/arthritis with marked elevation of acute phase reactants. in the long term, 15 % of patients develop a lymphoproliferative disorder. schnitzler's pathogenesis is unclear; immunosuppressive treatment is ineffective and high dose steroids are usually required. the recent finding that treatment with il-1 receptor antagonist (il-1ra; kineret) is extremely effective has raised the issue of the role of the inflammatory cytokine il-1b and of il-1-like cytokines in the pathogenesis of the disease. methods: two patients with recently diagnosed schnitzler's syndrome were treated with kineret, obtaining rapid disappearance of fever and urticaria and normalisation of acute phase reactants in one month. blood samples were collected before and after initiation of therapy. serum cytokine levels were measured by elisa, and expression of il-1-related genes by real-time pcr on mrna from blood cd14+ monocytes. results: compared to normal controls, schniztler's monocytes had similar expression of il-18, il-18bp, and caspase-1, both before and after therapy with kineret. il-1b expression was similar to controls before therapy, and was decreased five-fold after therapy. at the serum level, neither inflammatory (il-1b, tnfa, il-12) nor anti-inflammatory cytokines (il-10, tgfb) were detected. as expected, il-1ra was only detectable after therapy. il-18 was detectable in schnitzler's sera at higher levels than in controls (23.0 vs. 10.3 pm) and decreased after therapy (13.2 pm). the circulating il-18 inhibitor il-18bp was lower than in controls and not affected by therapy. thus, free il-18 levels were increased in schnitzler's patients as compared to controls (17.6 pm vs. 7.2 pm in controls) and decreased after therapy (9.9 pm). conclusions: schnitzler's syndrome is not associated to enhanced expression of il-1-related cytokines (il-1b, il-18), nor of the il-1/il-18-converting enzyme caspase-1 in blood monocytes. however, the high circulating levels of il-18 suggest an increased activity of caspase-1, as in the case of autoinflammatory diseases. experiments are in progress to test this possibility. atopic dermatitis (ad) is a chronic relapsing allergic skin disease with a high and growing prevalence. currently around 20 % of the children in industrialized countries are affected. in most cases patients exhibit increased systemic ige-levels (so-called extrinsic form) accompanied by sensitization to allergens. while ad is frequently cleared until adulthood, many patients develop allergic rhinitis and asthma. most ad-patients show topical colonization with staphylococcus aureus indicating a defective innate immune response. as a class of pattern recognition receptors, toll-like receptors (tlrs) are essential for pathogen recognition and critical for the induction of an effective adaptive immunity. all known tlrs except tlr3 signal via myd88 to induce nfxb-dependent gene transcription. tlrs are also known to be involved in the pathogenesis of autoimmune diseases. as chronic ad also has an autoimmune component, the study of myd88 signaling in ad might provide new insights into the function of tlrs. to investigate the role of tlrs in the immunopathology of allergic reactions and skin infections, we induced ad-like symptoms in c57bl/6 myd88-deficient mice by repeatedly sensitizing the mice to ovalbumin (ova) after mechanical disruption of the skin barrier by tape stripping. first results show that myd88-/-mice display reduced inflammation of the treated skin area compared to wildtype mice. immunostainings of skin biopsies reveal reduced acanthosis and infiltration of inflammatory cells into the dermis compared to wildtype. skin-draining lymph nodes are less enlarged in the ova-treated knockout mice compared to wildtype and differ in cellular composition. serum antibody levels determined by elisa show reduced systemic total and ova-specific igg1-titers in ova-treated myd88-/-mice compared to wildtype mice, although the nacl-treated myd88-/-control group has higher total antibody titers than the wildtype nacl control group. total ige levels are increased in the knockout mice compared to wildtype mice under both conditions. to further investigate the role of staphylococcus aureus during ad development, we will include topical application of the superantigen staphylococcal enterotoxin b (seb) or living bacteria into our analyses. following this approach, we anticipate to obtain new insights into the role of the innate immune system in allergic reactions of the skin. introdution: the skin of vertebrates is the target for over 15,000 species of hematophagous arthropods. among these are ticks, which are long-term feeders and interact with host defenses for days to weeks. little is known about specialized strategies for eliminating ectoparasites, but ticks can induce immune responses in hosts. bovines present variable and heritable levels of resistance to the tick rhipicephalus microplus and are the only model in which distinct outcomes of infestation can be examined in the same species of host. in order to obtain some of the immune correlates of these outcomes, we examined expression of candidate genes and quantified populations of leukocytes and subpopulations of lymphocytes present in the inflammatory infiltrates elicited by tick bites in skin of genetically resistant and susceptible bovine breeds, respectively, nelore (bos taurus indicus) and holstein (b. t. taurus). methods: skin biopsies (6 mm punch) were taken at the feeding sites of ticks from susceptible and resistant cattle (each phenotype n = 4) or from non-infested contra-lateral sites. expression of mip-1a, igf-1, mcp-1 and ip-10 genes was quantified with realtime rt-pcr. sections of paraffin-embedded skin were stained with may grünwald-giemsa for differential cell counts. lymphocytes in sections of frozen skin were phenotyped with specific antibodies using immunoperoxidase technique; in infested skin, histological sections were limited to the area of the tick's cement cone. results: as expected, hosts recruit cutaneous inflammatory infiltrates around the tick's mouthparts. however the composition of infiltrates presented with significant differences that varied according to the phenotype of infestation. inflammation of nelores contained significantly more basophils, eosinophils and mononuclear cells expressing cd3, cd4, cd8, cd21, mhc class ii, and p46 than that of holsteins. lymphocytes expressing wc1 and cd21 antigens were significantly diminished in infested skin of holsteins when compared with control skin (p x 0.05). infested skin of nelores contained significantly more message for mip-1a, igf-1, mcp-1 and ip-10 than that of holsteins. conclusions: although ticks secrete molecules that inhibit cell adhesion and chemokines, resistance correlates with the capacity to recruit and maintain populations of leukocytes that generate effector immune responses. supported by cnpq, capes, fapesp, and icttd. in the last decade it has become clear that keratinocytes play an important role in the skin immune system. upon stimulation, keratinocytes produce high amounts of proinflammatory chemokines and cytokines and express receptors which are involved in immunoregulation. in a number of inflammatory skin diseases such as eczema or psoriasis infiltrating lymphocytes are found in close vicinity to keratinocytes, enabling interaction of these two cell types. it has been proposed (goodman et al.) that keratinocytes rather support a th2 response by interacting lymphocytes. we examined this hypothesis with autologous cultures of keratinocytes derived from the outer root sheet of the hair follicle co-cultured with cd3+ t cells from the same donor. during the coculture either seb or antigen were added. in all experimental approaches the addition of keratinocytes resulted in higher production of ifng by t cells. furthermore, we set up an experimental approach were autologous antigen-pulsed monocytes were also added. again, the induction of ifng by the presence of keratinocytes resulted in a marked and significant increase of ifng production by t cells. we were able to show that il-1 plays a crucial role in the induction of ifng in t cells keratinocyte interaction. in addition blocking of lfa-1 in the co-cultures resulted in significantly reduced ifng production by t-cells underlining that icam-1/lfa-1 binding is also crucially important for ifng induction. we conclude from our study that keratinocytes rather support a th1 than a th2 local response pattern by virtue of il-1 secretion and icam-1/lfa-1 interaction. this property of keratinocytes may account for the observed cytokine switch in allergic eczematous skin from a th2 like micromilieu in acute towards a th1 dominated milieu in chronic lesions. the genotyping of ccl4l gene in patients with psoriasis could allow describing subcategories of patients based in clinical parameters and disease severity. therefore, it could be also used as a clinical diagnostic tool, potentially modulating the efficacy of new treatments, or even to be used as a therapeutical target of psoriasis. this work was supported by project grants of merck-serono and instituto de salud carlos iii (pi07/0329). psoriasis is an inflammatory dermatosis with 2 % prevalence among caucasians. hla-cw6 allele is the gene that confers susceptibility to psoriasis and it is placed near to tnf loci with several snp in promoter region. the most common polymorphisms are two g to a transitions in -308 and -238 positions. strong association was found between polymorphisms in the -238 region with psoriasis. in several diseases, the association with hla and clinical manifestation is different between genders, for example in spondyloarthropathy and hla-b27, and this is a question of increasing interest. the objective of this study was to identify clinical and molecular differences between male and female in brazilian psoriatic patients. sixty-nine individuals assisted at the dermatology outpatient clinic of the teaching hospital, university of campinas, with diagnosis of psoriasis of early-onset (up to 40 years of age) were selected. hla-a -b -c -dr -dq alleles and tnf-238 and -308 snp were differentiated by pcr/ssp. analyzing the total group, 21 patients (30.5 %) were male, 48 (69.5 %) were female. in the male group, the mean age at disease onset was 16,3 years. severe forms were seen in this group (psoriatic arthritis in 4 cases and erythroderma in 2). seven patients (33,3 %) had a favorable evolution of the disease, but 14 (66,4 %) developed extensive psoriasis, covering over 30 % of body surface requiring systemic treatment. the main molecular risk factor for the disease, cw*06 allele was positive in 10 cases (47,62 %), tnf 238 g/a genotype was found in 5 (23,8 %) and tnf 308 g/a in 4 (19,1 %). in the female group, the mean age at disease onset was 14,6 years, one case of psoriatic arthritis and one of erythroderma. twenty-nine (60,4 %) had a favorable evolution of the disease and 19 (39,6 %) an unfavorable evolution. cw*06 allele was positive in 26 cases (54,2 %), tnf 238 g/a genotype was presented in 16 (33,3%) and tnf 308 g/a in 8 (16,6 %). severe disease was seen in male patients. there was no difference in frequency of cw*06 allele between male and female groups, but there was a tendency of significant difference in tnf 238 g/a genotype. we found that c57bl/6 mice were more susceptible than balb/c and dba/2 mice. higher susceptibility was reflected by higher footpad swelling and transient systemic dissemination. analysis of serum cytokine level revealed differences in production of proinflammatory cytokines, such as il-6 and mrp8/14, among different inbred strains of mice. furthermore, we identified the cells which are involved in this cytokine production. as expected, histopathological analysis showed that s. aureus infection induces an influx of monocytes and granulocytes. our study shows that not only bacteria-but also host-specific differences are associated with different courses of s. aureus skin infection. aims: to investigate the cause and to study the clinical symptoms and the laboratory findings of the anaphylactic reactions in the pediatric population of our country, considering that these are very often dangerous situations which demand direct treatment and increased alertness. methods: 136 cases, which were studied retrospectively, included children (84 boys and 52 girls), aged 3-14 years, who had an anaphylactic reaction, out of the 915 that were examined in total. the statistical analysis of the data was held with the spss program. the commonest causes were proved to be food (44 %-particularly sea food and dried fruit), drugs (25 %-usually antibiotics and non-steroidal antiinflammatory drugs), as well as insect bites (9 %-mainly caused by hymenoptera). the symptoms included mainly the presence of pruritic pomphus with erythema (76 %), and gastrointestinal symptoms (37 %), while there were quite many cases with dyspnea, nasal congestion, but also angioedema. total ige g 100 was found in 26 out of the 47 severest cases (55,4 %), in which the adequate control was held, while in their vast majority (45 out of 47) there was no previous anaphylactic reaction. on the other hand, it was proved in total, that in 53,7 % (73 cases) there was a hereditary family history of atopy, while in 52 children (38 %) there was also a personal history of asthma. finally, at a great percentage (69 %) eosinophilia was found, while a statistically significant seasonal distribution during spring and summer was registered. conclusions: it has, therefore, been shown that 1)the anaphylaxis is quite often in the pediatric population, with the commonest causes to be food and drugs, which are often thoughtlessly used. 2) in particular, in many cases it is proved that there is a personal but also a family history of atopy. 3) increased attention should be, thus, given in these cases -especially during spring and summer-for their early diagnosis as well as for their effective treatment (adrenaline, antihistamines and corticosteroids) particularly for the severest cases, where the hospitalization of the patient is also necessary. allergic contact dermatitis (acd) is an adaptive inflammatory response of the skin triggered upon exposures to certain chemicals or metal ions. as classical type iv delayed hypersensitivity reaction this response is mediated by t-cells. since many ingredients in consumer products might exert allergenic potency, there is a need for an appropriate screening and characterization of the chemicals used according to this toxicological endpoint. up to now the identification of potential allergens completely relies on animal testing, like buehler assay or guinea pig maximization test (gpmt). due to economical and ethical reasons, as well as driven by the enforcement of certain governmental regulations (i. e., cosmetics directive), the development of an in vitro test system for identification of potential sensitizers is mandatory. since dendritic cells (dcs) play a pivotal role in the initiation of contact dermatitis we chose dcs to characterize known sensitizers in their ability to activate these cells and subsequently examined the molecular interplay between dcs primed by allergens and t-cells in the test tube. the known allergens nickel, dinitrochlorobenzene (dncb) and cinnamic aldehyde were tested for their ability to alter the expression of several immunomodulating surface molecules on dcs derived from monocytes that display a langerhans cell (lc) type-similar phenotype. we used multicolour flow cytometry to detect differences in expression patterns of surface molecules that have been associated with maturation. in addition to the upregulation of cd86 we could observe dose dependent upregulation of programmed death ligand 1 (pdl-1) and downregulation of the dendritic cell immunoreceptor (dcir). furthermore we observed enhanced t-cell proliferation in mixed leukocyte reactions (mlrs) applying lcs stimulated with allergens ex ante. since changes in the expression of only single cell molecules are unlikely of being sufficient for reliable identification of possible contact allergens, we are aimed at analyzing a wide pattern of various surface molecules by multicolour facs and propose that this might be a reasonable approach to screen for contact sensitizing properties of chemicals. our findings are of particular interest for further development of new in vitro assays, using immune cells, to detect the sensitizing potential and quantify the sensitizing potency of chemicals. we want to present the case of a 60 year old iraqui patient with arabic ancestors who had been suffering from psoriatic arthritis since 35 years. in march 2006 a treatment with fumaric acid esters in combination with ibuprofen was introduced. this led to the complete healing of the skin lesions. for this reason the dose could be reduced to one tablet fumaric acid esters (120 mg) every second day. in april 2008 the patient presented himself in the consult with multiple livid papules with a diameter of 3 mm in the area of the auricle. the histological examination showed an hhv-8 positive kaposi sarcoma. the differential blood cell count demonstrated a lymphocytopenia. the hiv-serology was negative. the staging examinations (chest x-ray, gastroscopy, coloscopy, abdominal and lymph node sonography) showed no signs of visceral involvement. after the diagnosis the treatment with fumaric acid esters was discontinued. over the course the livid papules showed a spontaneous complete regression. a spontaneous regression is known from the iatrogenic ks caused by immunosuppressive therapy when the immunosuppression is terminated. as our patient also showed a spontaneous regression of the kaposi sarcoma after stopping the treatment with fumaric acid esters we propose a causative relation. sarcoidosis is a multisystemic granulomatous disease with unknown etiology. although the immunopathogenesis of sarcoidosis remains unknown there are some supportive evidence for the significant role of th1 type immune response. recently, suppressor of cytokine signaling (socs) proteins have been identified as regulators of cytokine signaling pathways. in this study we aimed to evaluate the roles of socs1, socs3 and foxp3 in the immünopathogenesis of sarcoidosis and their association with responsiveness to treatment. peripheral blood (pb) and broncholaveolar lavage (bal) mononuclear (m) cells from sarcoidosis patients in remission following treatment (responders, n:4), the patients who showed recurrence or progression after treatment (non-responders, n:4) and stage i/ii sarcoidosis cases which were followed up without any treatment (untreated, n:7) were evaluated for socs1, socs3 ve foxp3 mrna expressions by taqman pcr, and also flow cytometric analysis was performed for lymphocyte markers including cd3, cd4, cd25, foxp3, cd4 + cd25 high , cd4 + foxp3 + . expression of socs3 and foxp-3 mrna in pbmcs and balmcs from responders were found to be significantly higher in comparison to other two groups . socs1 was found significantly elevated in pbmcs of responders when compared with other two groups. it was also elevated in balmcs of responders when compared with with those of untreated cases. the proportions of cd25, foxp3, cd4+cd25 high , cd4 + foxp3 + cells in pbmcs and balmcs of responders were found to be increased in comparison to nonresponders and untreated cases. our data demonstrates that socs1, socs3 and t regulatory cells may have potential roles in the control of sarcoidosis. we think that if the roles of socs1 and socs3 molecules and t regulatory cells are well characterized, new therapeutic approaches targetting cytokine signal supressors, which can strenghten the regulatory responses, may be beneficial for the sarcoidosis cases resistant to conventional therapy. the inorganic dust, containing free crystalline silicon dioxide (fcs) is critical for the development of silicosis. several studies supported the view that fibrotic responses mainly depends on the regulation of the immune response to the fcs in affected individuals. the role of fcs in induction of a local and systemic inflammation and pulmonary fibrosis are still debates.we studied the changes of neopterin, as a marker for ifn-g dependent macrophage activation and circulating immune complexes (cic), as a marker of humoral immune response, in patients with silicosis and workers exposed to dust containing fcs.we survey a group of 62 silicosis patients, with mild (21), moderate (23) and severe (18) silicosis, 92 coal workers, exposed to inorganic dust containing fcs (exposed), and 43 healthy workers without exposure to dust aerosol (controls).the serum quantity of neopterin and cic, containing iga(igacic), igg(iggcic) and igm(igmcic) was detected by elisa. differences between investigated groups were detected by student's t-test and a p-value less than 0.05 was considered significant.neopterin level was significantly elevated in exposed (3,2±0,8ng/ml) compared to controls (1,8±1,3ng/ml; p=0,0001). moreover, the neopterin level in exposed was similar to silicosis patients (2,9±1,6ng/ml).the levels of iggcic was significantly elevated in the exposed compared to controls (81,9±22,7au vs 64,3±16,7au p=0,0001) and to silicosis patients (69,6±20,0au p=0,002). in contrast, igmcic was significantly elevated in silicosis than in exposed (70,2±18,8au vs 62,1±24,2au; p=0,03).in comparison with exposed, significantly higher igmcic was found only in mild, but no in moderate and severe silicosis. in contrast, the level of iggcic in mild and moderate silicosis was significantly lower compared to the exposed (p=0,001 and 0,02 respectively).the obtained results showed that activation of alveolar macrophages mainly depends on the presence of fcs in the respirable dust fraction and precedes the clinical data for pulmonary fibrosis. the dynamics of cic suggest the involvement of fc-receptors mediated regulation of the immune response in the progression of pulmonary fibrosis, and could be useful marker for exposure to inorganic dust containing fcs. described pathologic similarities between sarcoidosis (sa) and tuberculosis (tbc) suggest m. tuberculosis antigens as caustaive agentes. it seems that in the genetically different predisposed hosts, the same antigens may cause the development of sarcoid or tuberculous inmune response. so different hla haplotypes have been described as a predisposing factor to develop sarcoidosis (hla a*01, b*08, drb1*03 (these of good prognosis) *12/*13/*14/*15) or tbc (drb1*02/*05/*14/*16 and associations with dqa1*02/*03/*05) we describe two cases of two female patients from the same geographic region with mantoux and zhiel-neelsen negative tests and high levels of tnf diagnosed of tbc and sa respectively. both of them debuted with the same clinical manifestations: fever, abdominal pain, and asthenia and shared similarities in the images from the tc study (pulmonary nodules and mesenteric adenopaties). we found the same results for the flow citometry analysis of the non-caseificant granulomes as well as the same anatomopathologic characteristics. after being treated with anti-tbc drugs, the first one presented a good clinical improvement, so she was diagnosed as tbc. the second one did not improved, so she was treated with corticosteroid, with good results. therefore, she was diagnosed as sarcoidosis. after hla analysis, we noticed that the tbc patient was hla a*01, b*08 and drb1*03 (sarcoidosis good prognosis haplotype) and the patient diagnosed as sarcoidosis was hla a*01, drb1*14. as the results show, could there not be a direct relationship between the hla system and the development of sa or tbc, or in contrast, was the first patient missdiagnosed of tbc being a good prognosis sa? objectives: experimental mouse models for acute asthma are well established, yet models for chronic asthma have several shortcomings. for example, current chronic models show decreased inflammation over time and only marginal effects on airway remodelling. experimental models for chronic asthma are essential for development of new therapeutics and must include changes that closely resemble clinical conditions. ovalbumin (ova), house-dust-mite (hdm) and cockroach (cra) proteins are commonly used to trigger an asthma like response in mice and, for this reason, were used in the present study. the objectives of our work were to compare the most frequently used mouse models of chronic asthma and to develop a mouse model of chronic asthma that clearly displays pivotal features of severe human asthma. methods: for the induction of asthma, mice were initially sensitised by intraperitoneal injection of ova, hdm, cra or a combination of all three, followed by repeated challenge by intratracheal application of ova, hdm or cra. inflammation in lung was measured by analysis of cell influx into the bronchoalveolar lavage (bal) and by determination of chemokine and cytokine levels in bal and lung tissue using elisa and multiplex technology. additionally, serum levels of ige and igg antibodies were measured. airway remodelling was assessed by histological staining for mucus production, immune cell influx, smooth muscle thickening and fibrosis. results: significant differences were measured in cell influx, chemokine/cytokine and total ige levels. compared to hdm and cra, ova induced an higher cell influx in the bal, hdm showed an increase of chemokines in bal and increased ige levels in serum. using a combination of all three proteins resulted in the most severe form of asthma. conclusions: to our knowledge, this is the first study that directly compares the most commonly used mouse models in regard to their potential to display a pathology specific of severe asthma. the most sustained and severe form of asthma was induced by the combination model. this model offers particular advantages for evaluating existing and novel therapeutic agents. furthermore, this model could contribute to understanding of the mechanisms underlying chronic asthma. the present study focused on peri-smi connective tissue capsule formation, the most frequent post-operative local complication in patients receiving smi. we investigated the local immune processes via the phenotypic and functional characterization of lymphocytes within the fibrotic tissue. to this end, intracapsular lymphoid cells and peripheral blood mononuclear cells (pbmcs) from the same patients were isolated and analyzed via facs, concentrating on t-effector cells (teff) and t-regulatory cells (tregs: cd4 + , cd25 ++ , foxp3 + ), cytokine profiles, t-cell receptor (tcr) repertoire and reactivity against human heat shock protein 60 (hhsp60). intracapsular tregs were visualized by immunohistochemistry and functionally tested in suppression assays. the cellular composition of intracapsular mononuclear cells showed a preponderance of cd4 + t-helper cells and a significant subset of tcrg/d + cells, exceeding that observed in peripheral blood. il-17, il-6, il-8, tgf-b and ifn-g production prevailed, pointing to a th17/th1 weighted immune response. furthermore, intracapsular t-cells displayed a restricted tcr a/b repertoire (monoclonal/oligoclonal) as well as a preferential reaction with hhsp60. importantly, numbers of intracapsular tregs were inversely proportional to the degree of fibrosis and showed less suppressive capacity as compared to peripheral tregs. our results suggest that silicone triggers a specific local immune response via activated th17/th1 cells, promoting fibrosis due to the production of profibrotic cytokines. clonal restriction of the tcr repertoire is a further indication for a specific antigen driven immune response preceding capsular fibrosis. in this context, hsp60 might be a prominent candidate. taking into consideration that it is ubiquitously expressed, it might be the "missing link" between local and systemic side effects of smi. the inverse correlation between the degree of capsular fibrosis and the number of intracapsular tregs suggest that tregs may initially be able to inhibit the progress of capsular fibrosis. however, as numbers of tregs, as well as their suppressive capacity decreases over time, fibrosis develops. supported by the competence center medicine tyrol (kmt) and the lore-and-udo-saldow donation. objectives: recent findings have proven that silicone induces a local inflammatory response with subsequent fibrotic reactions. the present project deals with the standardization and further development of a modified elisa test system (silisa ® ) for the identification of patients with a risk for fibrotic side effects to silicone mammary implants (smis) based on the protein signature adhering on the surfaces of such devices (1) . the current silisa ® is a test system for the simple detection of the adhesion pattern of proteins from patients' sera to silicone. the optimization of the silisa ® comprised inter-and intra-assay standardization, robustness, specificity and sensitivity. the essay was further developed with antibodies against annotated proteins that were not yet tested in the past. all experiments were carried out in a 384 well plate format for high-throughput analysis. statistical analysis has been performed using spss. the extended essay has been successfully established in the system with antibodies against seven already tested proteins, including c-reactive protein, collagen-i, collagen-iii, fibronectin, igg, c3-complement, myeloid related protein 14 and two new proteins, integrin-ß4 and fibrinogen. data from more than 100 patients have been obtained and exploited so far. the intra-and inter-assay variability of the test was reduced to less than 10 % and 16 %, respectively. patients with fibrotic reactions to silicone were successfully identified using a pattern of protein deposition to silicone. conclusion: applying the silisa ® , sera from five different groups were tested: silicone patients with and without fibrotic reactions, female and male individuals without any contact to silicone and hospital's medical staff with potential silicone contact. the distribution pattern of eight proteins showed differences in patients developing strong fibrotic reactions to silicone compared to controls. muscular lesion is a frequent matter in sportive medicine and myodegenerative diseases. necrosis of the damaged tissue and activation of inflammatory response characterize the initial phase of muscle repair. this work aimed to analyze the tissue repair after induction of lesion in skeletal muscle from mouse lineages with distinct cytokine secretion patterns. it was included at least 3 mice per group with distinct cytokine pattern: th1 (c57bl/10, c57bl/6) and th2 (balb/c). muscular injury was performed by injection of bupivacaine. both th1-dominant strains presented more areas with regenerating myofibers and macrophages at 4 dpi. regional lymph nodes showed significant increase of cellularity and relative numbers of cd3 bupivacaine-inoculated balb/c mice compared to non-inoculated matched mice at 4 dpi. balb/c mice showed increased collagen expression and decrease of mmp-9 activity associated with more mrna for tgf-b1. this study shows that the immune background of the mouse may affect the remodelling processes in skeletal muscles that occur in response to bupivacaine injection promoting muscle regeneration (th1 cytokines) or myonecrosis and collagen deposition (th2 cytokines). the severe, life-threatening heart failure in some of the patients with dilated cardiomyopathy (dcm) is imputed to the stimulatory autoantibodies against the second extracellular loop (ec ii ) of the ß1-adrenergic receptor (anti-ß1ec ii ). to analyze their pathogenic impact as a single causal factor we used a human-analogous lewis rat model of dcm, where monthly subcutaneous immunization of the rats with the ß1ec ii peptide as a glutathione-s-transferase (gst) fusion protein induced production of anti-ß1ec ii and eventually dilated cardiomyopathy. in this model we isolated a ß1ec ii -specific rat monoclonal antibody (clone 13f6), and showed by elisa that it binds to the linearized ß1ec ii peptide. additionally, we confirmed with flow cytometry that 13f6 also binds the ß1ec ii in its native conformation, i. e. directly labeled circular ß1ec ii (dyl649-ß1ec ii ) peptide. moreover, we demonstrated activation of the ß1-adrenoreceptor by 13f6 using a fluorescence resonance energy transfer (fret) assay system in vitro. these data further corroborate the pathogenic role of anti-ß1ec ii antibodies in mediating dcm in this animal model, thus rendering them a potential therapeutic target. therefore, we investigated a novel anti-ß1ec ii -specific peptide-based therapy, by intravenously applying a circular ß1ec ii peptide in the dcm lewis rat model to neutralize the anti-ß1ec ii antibodies. while the peptide therapy strongly reduced the anti-ß1ec ii titers in the serum by up to 80 % and consecutively lead to clinical remission, elispot assays for the detection of ß1ec ii -specific antibody-secreting cells (asc) indicated no difference in the number of long-lived plasma cells in treated animals. in contrast, elispot and flow cytometrical analyses revealed a decrease in the number of ß1ec ii -specific memory b cells in the treated animals, indicating that this cellular compartment is most likely also targeted by the peptide therapy. our newly developed anti-ß1ec ii -specific therapy, thus, not only neutralized the pathogenic autoantibodies, but also depleted antigen-specific memory b cells involved in the generation of these autoantibodies. these results provide the rationale for further development of this therapeutic strategy for eventual application in patients with autoimmune dilated cardiomyopathy. cardiovascular diseases like myocarditis and subsequent dilated cardiomyopathy (dcm), are a frequent cause of mortality in humans with dcm being the most common reason for heart failure in young adults. infections with coxsackievirus b3 or cytomegalovirus can lead to an acute inflammation of the heart muscle that is followed by an autoimmune response directed autoantigens in the heart, such as the alpha isoform of cardiac myosin (myhca). immunization with the well-characterized myhca 614-629 epitope elicits autoreactive cd4 + t cell responses that have been shown to be the major mediators of autoimmune myocarditis in balb/c mice. it is known that professional antigen presenting cells (apcs) such as dendritic cells are crucial for initiating and maintaining t helper (th) cell responses affecting the heart muscle. however, the detailed analysis of the interaction between these cells in the context of autoimmune myocarditis has been hampered by the lack of appropriate analytical tools. we therefore generated a tcr transgenic mouse harboring t cells that specifically recognize the myhca 614-629 peptide. in a first step, hybridoma cells were generated by fusing bw5147 tcra -cd8lymphoma cells with myhca 614-629 -specific th cells. tcr expression and antigen specificity was assessed by facs analysis and elispot assay. following subcloning, the variable regions of the expressed tcr were characterized by pcr-sequencing. the rearranged v(d)j regions were subcloned into tcr cassette vectors and linearized constructs were injected into the pronuclei of fertilized oocytes. using this novel tcr tg mouse we plan to investigate in detail the activation of myhca 614-629 -specific t cells during the process of autoimmune myocarditis. furthermore, this new tool will help to generate a high resolution analysis of the contribution of different apcs in the activation and differentiation of autoreactive th cells during inflammatory heart disease. m. relle 1 , a. schwarting 1 , p.r. galle 1 1 university medical center of the johannes gutenberg university mainz, medical clinic i, mainz, germany several mouse or rat models have been established to explore the role of proteinase 3 (pr3), in anca-associated glomerulonephritis, vasculitis or pulmonary inflammation but these studies have demonstrated that anca alone are not sufficient to induce these diseases directly. therefore, we assessed the expression, mobilization and enzymatic activity of pr3 in mouse bone marrow, kidney, spleen and peripheral blood by immunohistochemistry and immunoblots, as well as the proportion of pr3-positive neutrophils in the peripheral blood of frequently used mouse strains. neutrophils were mobilized from the bone marrow by an intraperitoneal injection with human il-8. pr3-mrna from the murine cancer cell line wehi-274 was amplified by race-pcr and subsequently sequenced. sequence comparisons were done with dnasis software package and the blast tool of the ncbi. promoter analyses were performed with the genomatix software matinspector. we could demonstrate, that mouse bone marrow is a reservoir for functional neutrophils, which are rapidly mobilized after injection of furthermore, we identified an alternative pr3-promoter in the second intron of the mouse pr3 gene. this promoter is active in the bone marrow, in embyros and in cancer cell lines, indicating that its expression is not restricted to myeloid cells. fine structural analyses of this alternative promoter revealed differences not only between the rat and the mouse promoter but also between different mouse inbred strains. taken together, we have shown that the maturation processes of mouse neutrophils differ from those of human granulocytes. the identification of an alternative pr3 transcript and its promoter indicates that the murine pr3 may have additional, as yet not described, functions in hematopoiesis and cancerogenesis. objective: recent studies show that in vivo administration of och, a synthetic lipid that specifically activates natural killer t (nkt) cells, results in suppression of th1 mediated immune responses in autoimmune diseases. nkt cell activation depends on lipid presentation via the mhc-i like molecule cd1d on antigen presenting cells such as mature dendritic cells (mdcs) and upon activation by och nkt cells rapidly produce large amounts of th2 cytokines. the goal of this study was to investigate the effect of och and och-primed dendritic cells on atherogenesis. methods & results: ldl receptor deficient (ldlr -/-) mice were fed a western type diet and atherosclerosis was induced via collar placement around both carotid arteries. subsequently the mice were treated i. p. with och (n=13) or pbs (n=11) twice a week for seven weeks. the injections with och did not affect atherosclerotic lesion size. to improve the presentation of och to nkt cells in vivo, bone marrow-dendritic cells were maturated via tlr4 activation, in the presence/ absence of och. subsequently we transferred 1.5x10 6 mdcs (n=11) or och-primed mdcs (n=11) (3 times) to ldlr -/mice. afterwards the mice were put on a western type diet to induce atherosclerosis. vaccination with och-primed dcs resulted in a 70.6 % reduction in plaque size compared to mice treated with mdcs (p x 0.05). during the experiment no effect on serum cholesterol levels was observed, but at the end of the experiment there was a significant 23.7 % (p x 0.05) reduction in cholesterol levels in the mice treated with och-primed dcs. the number of nkt cells in blood and liver was monitored and a 2 to 3-fold increase in these cells was detected 3 days after the last treatment with och-primed dcs (p x 0.05). additionally, the nkt cells in the liver of mice treated with och-primed dcs produced more il-10. discussion: we conclude that immunotherapy using och-primed dendritic cells efficiently activates nkt cells, resulting in a th2 phenotype of the nkt cells and this leads to an efficient protection against atherosclerosis. these data indicate that immunotherapy based on ligand specific primed dcs may be a novel way to treat atherosclerosis. systemic lupus erythematosus (sle) is characterized by high serum titers of igg anti-nuclear antibodies secreted by plasma cells. however, the characteristics of the igg+ plasma cell antibody repertoire in sle has never been determined on a single cell level and little is known about the role of germinal center (gc) reactions for the development of sle autoantibodies. the igg inhibitory fcgriib knock-out mouse on the c57bl/6 background is a strain specific lupus autoimmune model that is characterized by the spontaneous development of autoantibodies to nuclear antigens such as dsdna and chromatin. to characterize the igg+ plasma cell compartment under normal circumstances and in autoimmunity we have cloned and expressed 350 igg antibodies from single isolated gc b cells and plasma cells derived from spleen, bone marrow and lymph nodes of wild-type c57bl/6 and fcgriib deficient mice. igh and igl chain gene sequence analyses revealed no major differences in the ig gene usage between wild-type and autoimmune mice, but gc b cells of fcgriib were enriched for antibodies with positively charged igh cdr3 regions and anti-nuclear specificity. the overall frequency of autoantibodies was similiar between wild-type and fcgriib deficient mice. however, strongly autoreactive antibodies to dsdna and murine igg2c were isolated only from fcgriib deficient mice, but not from c57bl/6 control mice and somatic mutations contributed to their generation. in summary, our data suggest that the gc reaction plays an important role for the development of self-reactive antibodies in fcgriib deficient mice. the finding that the frequency of autoreactive antibodies is higher in gc b cells than in spleen or bone marrow plasma cells may indicate that autoreactive gc b cells are partly regulated even in the absence of fcgriib. autoantibodies against double-stranded dna (dsdna) and nucleosomes (ncs) represent a hallmark of systemic lupus erythematosus (sle). however, the factors leading to the autoimmune response against these nuclear autoantigens are not fully identified. high mobility group box 1 protein (hmgb1), a nuclear dnabinding protein and an extracellular proinflammatory mediator gets tightly bound to modified chromatin during apoptosis. it is not released, since apoptotic cells are immediately engulfed by phagocytes. conversely, in conditions of clearance deficiency, which is observed in a subset of patients with sle, non-ingested apoptotic cells, may undergo secondary necrosis, thereby releasing ncs containing the "endogenous adjuvant" hmgb1. we investigated if hmgb1-containing ncs contribute to the breakdown of immunological tolerance against dsdna and ncs. we found that hmgb1 remains associated with ncs released from late apoptotic cells in vitro. hmgb1-ncs complexes were detected also in the blood of patients with sle. hmgb1 containing ncs from apoptotic cells induced secretion of il-b, il-6, il-10, and tnfa as well as expression of co-stimulatory molecules on human and murine macrophages and dendritic cells (dc), respectively. cytokine release from murine macrophages was dependent on myd88 and toll-like receptor 2. neither hmgb1-free ncs from living cells nor from apoptotic hmgb1-or hmgb1/2-deficient cells induced marked cytokine production or dc activation. specific inhibition of hmgb1 activity by the antagonistic a box domain significantly reduced capacity of "apoptotic "ncs to induce tnfa and il-10 release by macrophages. immunizations with hmgb1-containing ncs from apoptotic cells induced anti-dsdna and anti-histone igg responses in non-autoimmune mice in tlr2-dependent manner. in conclusion, hmgb1 in complex with ncs activate antigen presenting cells thereby contributing to the loss of immunological tolerance against ncs/dsdna and, hence, to the immunopathogenesis of sle. objective: apoptotic cells are considered to be a major source for autoantigens in autoimmune diseases such as systemic lupus erythematosus (sle). in agreement with this, defective clearance of apoptotic cells has been shown to increase disease susceptibility. still, little is known about how apoptotic cell-derived self-antigens activate autoreactive b cells and where this takes place. methods: injections of fluorescently labelled syngeneic apoptotic cells were traced using immunofluorescence microscopy. binding studies were performed using apoptotic cells and cho cells transfected with class a scavenger receptors (sr). repeated injections of syngeneic apoptotic cells in sr deficient and wild type mice were conducted and antibody production by autoreactive b cells was measured. autoreactivity against sr was followed in two sle prone mice strains over the development of disease and in a cohort of sle patients. an antibody against the sr was injected together with several antigens to directly evaluate the possible role of autoantibodies against the receptors. results: in this study, we find that apoptotic cells are taken up by specific scavenger receptors expressed on macrophages in the splenic marginal zone and that mice deficient in these receptors have a lower threshold for autoantibody responses. autoantibodies against sr are found before the onset of clinical symptoms in sle-prone mice, and they are also found in diagnosed sle patients. furthermore, injections of an antibody binding sr enhance the antibody production by b cells when co injected with either apoptotic cells or tnp-ficoll. conclusion: our findings describe a novel mechanism where autoantibodies toward scavenger receptors can alter the response to apoptotic cells, affect tolerance, and thus promote disease progression. because the autoantibodies can be detected before onset of disease in mice, they could have predictive value as early indicators of sle. e. glasmacher 1 , k.p. hoefig 1 , e. kremmer 1 , v. heissmeyer 1 stretches and helps in the selection of the correct splicing borders. a allele of (r61h) creates a strong binding site for a splicing enhancer protein srp40 according to bioinformatics. our findings indicate that, the putative branch point, r61h snp and the t stretch located downstream of exon two, plays a role in the alternative splicing of bank1. finally, we believe that bank1 delta 2 protein work as a dominant negative isoform in b cell activation and antobodies production, and may antagonize the effect of the full-length protein. these properties of the delta 2 protein may contribute to the observed reduction in sle susceptibility. s. beermann 1 , r. seifert 1 , d. neumann 1 1 hannover medical school, pharmacology, hannover, germany the biological function of histamine is mediated by four different receptors, namely histamine h 1 receptor (h 1 r), h 2 r, h 3 r, and h 4 r. during an immune reaction histamine acts as a local proinflammatory mediator and contributes to the polarisation of the adaptive immune reaction by modulating the activity of dendritic cells and t cells. in these cells, histamine may modulate the synthesis of characteristic t cell cytokines such as ifng, which plays a central role in a number of autoimmune diseases. the present study was initiated to analyze the involvement of histamine on the induced production of ifng by immune cells. mouse spleen cells were stimulated in vitro by either immobilized a-cd3 antibodies or cpg-oligonucleotides (cpg-odn) in the presence or absence of histamine or 4-methylhistamine, a h 4 r-selective agonist. ifng production was evaluated by analysis of cell culture supernatants by elisa. both, histamine and 4-methylhistamine concentration-dependently reduced ifng production in splenocytes obtained from control c57bl/6 mice induced by either a-cd3 antibodies or cpg-odn. this histamine effect was completely inhibited by the h 2 r-specific antagonist famotidine, while h 4 r-, h 1 r-, and h 3 /h 4 r-selective antagonists had no or only moderate effects. interestingly, the h 4 r-selective reagent jnj7777120, which serves as an antagonist on human cells, did not inhibit the histamine-mediated reduction of a-cd3 induced ifng synthesis, but in contrast it slightly enhanced the histamine effect. thus, at the murine h 4 r, jnj7777120 may be a partial agonist. we conclude that histamine modulates the induced production of ifng by t cells via mainly the h 2 r and, to a much lesser extend, the h 4 r. using this assay system, cells obtained from control c57bl/6 mice will be compared to those from sle-prone mrl lpr/lpr mice and the respective wild type strain mrl +/+ . i objectives: resolvins are products of omega-3 fatty acids and they exert potent anti-inflammatory properties. in this study we examined their effects on cytokine release in healthy subjects and autoimmune patients. to test the in vitro effects of 20 ng/ml resolvin e1(rve1) on the release of tgfb, il-6 and il-17 in the culture of peripheral mononuclear cells (5x10 6 /ml) stimulated by phorbol ester (pma) (1nm), and the combination of pma and ionomycine (3 mg/ml) for 72 hours. methods: mononuclear cells were prepared by ficoll-uromiro gradient centrifugation from 7 healthy subjects and from 10 patients each with sle and sjögren's syndrome (ss). level of cytokines was measured by elisa method. results: in the patients with sle (p = 0.010) and sjögren's (p=0.017) mononuclear cell stimulation by pma resulted in a reduced release of tgf b compared with controls. rve1 significantly reduced tgfb release from control mononuclear cells stimulated by either pma (p=0.041) or pma+ionomycin (p=0.021), however rve1 was ineffective at reducing tgfb release in the sle and ss patients. rve1 caused a non-significant decrease in il-6 release from control mononuclear cells, but was again ineffective in sle and ss patients. the production of il-17 was not significantly modified by rve1 in any of the groups tested. the release of tgfb by 20 ng/ml of rve1 can be significantly reduced in healthy control subjects but not in subjects with sle or ss. at the single dose of rve1 tested, il-6 and il-17 release were not significantly affected in healthy or autoimmune patients. omega-3 fatty acid derived rve1 may affect inflammation in healthy patients by reducing tgfb production but its effects on inflammation in sle and ss patients may be expected to be smaller or non-existent. in addition, the tgfb release in the pma activated mononuclear cells of sle and sjögren's patients is less than that of healthy subjects. g. e. fragoulis 1 , a.k. tsirogianni 1 , m. herrmann 2 , h. m. moutsopoulos 1 , m.n. manoussakis 1 1 university of athens, dpt pathophysiology, athens, greece, 2 university of erlangen-numberg, institute for clinical immunology, erlangen-numberg, germany objectives: altered phagocytic capacity has been shown to characterize systemic lupus erythematosus (sle) that is thought to lead to impaired clearance of apoptotic remnants. herein, we assessed comparatively the phagocytic capacity in the peripheral blood of ss and sle patients and investigated the phagocytosis of apoptotic/necrotic cells in the salivary glands of ss patients. methods: patients studied included 29 with primary ss (american-european criteria 2002) and 14 with sle (acr criteria 1997). age-and sex-matched healthy blood donors to the ss and sle groups (13 donors each) were also studied in all assays. the phagocytosis capacity (phagocytosis index) was assessed by flow cytometry, as previously (gaipl et al, j autoimmunity, 2007) using heparinized whole blood from individuals studied mixed with a commercially available preparation of fluorescent microbeads (mb-phagocytosis) or a preparation of propidium iodide-stained necrotic cell-derived material obtained from heat-treated normal pbmc (snec-phagocytosis). salivary gland biopsies of patients with ss with and without malt lymphoma (5 patients each) were also assessed by confocal microscopy for the presence of apoptotic/necrotic material (tunel assay) and the presence of macrophages (cd68-staining). results: in agreement to previous studies, mb-phagocytosis was found significantly decreased in granulocytes and monocytes of sle patients (both for p=0.0001). in ss patients, defective mb-phagocytosis involved only monocytes (p x 0.0001) and significantly correlated with the presence of extraglandular manifestations (p=0.02). compared to controls, snec-phagocytosis was significantly increased in the granulocytes of sle (p x 0.0001) and of ss (p=0.001). in the salivary gland biopsies of ss patients, the lymphoepithelial lesions and germinal center-like structures manifested significantly increased infiltrations by macrophages. these lesions were also characterized by notable accumulation of apoptotic/necrotic material that resided both inside and outside the phagocytes. these phenomena were significantly more intense in the salivary gland lesions that manifested malignant in-situ b-cell lymphoma. conclusion: in a manner similar to sle, ss patients appear to manifest altered phagocytic capacity. this may be associated with the observed accumulation of apoptotic/necrotic cells in the salivary glands that in turn, may participate in the chronic autoimmune reactions and/or the lymphoma-generating processes that characterize the disorder. the autoantibodies to various enzymes are often found out in sera of systemic lupus erythematosus (sle) patients, but clinical value of such antibodies often is not understood. the purpose of work was to study the of antibodies generation to the basic enzyme of purine metabolism -adenozine deaminase (ada) in sle and to reveal the relationship of studied antibodies with clinical and laboratory features of pathological process. methods: 30 healthy persons have been included in our study and 71 sle patients (66 women and 5 men) with various clinical signs (44 persons had 1 st degree of disease activity, 27 persons -2 nd degree of pathological process activity). 18 women had habitual noncarrying of pregnancy (hnp) in anamnesis. antibodies of igg class to ada (anti-ada) determined by technique of indirect elisa developed by us with the use of immobilized form of ada as an antigenic matrix. b 2glicoprotein-i-dependent antiphospholipids (aphl) of igg classes were determined using commercial "anti-phospholipid screen igg/igm" test set (orgentec diagnostica). results: at admission an anti-ada was revealed in 36,6 %, aphl of igg class -in 45,1 % sle patients. it has been noted that igg-aphl were found out in anti-adapositive patients more often and in higher antibody titer, than in anti-ada-negative sle patients (x 2 =6,4; p x 0,02). development of cytopenic syndrome was noted reliable more often in sle patients with associated presence of igg-aphl and an anti-ada in comparison with patients who has not the combinations of these antibodies in blood (x 2 =3,9; p x 0,05). the increased levels of anti-ada were revealed in 11 of 18 women with hnp, and the combination of anti-ada and aphl (9/18) was found out more often than isolated anti-ada (2/18, x 2 =6,5; p x 0,02) or isolated aphl (3/18, x 2 =4,5; p x 0,05). conclusion: taking into account the imbalance of immunoregulatory functions in sle, the further studying of autoantibodies to ada generation seems to be very promising. presence of hnp in anamnesis is the evidence of necessity of careful biochemical monitoring of aphl and anti-ada in women for the prevention of abortus fetus and administration of adequate therapy. objectives: sjögren's syndrome (ss) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterized by dryness of the mouth (xerostomia) and the eyes (keratoconjunctivitis sicca). dendritic cells (dc) are the most potent antigen-presenting cells that play a crucial role in initiating and maintaining primary immune responses. two main subsets of dc have so far been identified in human peripheral blood: myeloid dc (mdc), which can be further divided into mdc1 and mdc2, and plasmacytoid dc (pdc), also known as ifn-a/b producing cells. the pivotal role of dc in inducing and maintaining tolerance could be critical in ss as alterations among dc populations might contribute to autoimmunity. purpose of this study was to quantify mdc1, mdc2 and pdc in peripheral blood from primary ss patients by flow cytometry and compare the results with gender-and age-matched healthy controls. methods: blood samples from 31 pss patients fulfilling the american european consensus group criteria (aecc) and 28 gender-and age-matched healthy controls were collected in heparin tubes. dc populations were stained with the blood dc enumeration kit, miltenyi, according to the manufacturer's manual. cells were analyzed on a facs canto ii, bd, and data analysis was performed with flowjo software, tree star. for the statistical analysis, a two-tailed mann-whitney u test was performed using prism, graphpad. results: pss patients have significantly reduced amounts of pdc (p=0,0002) and mdc2 (p x 0,0001) in peripheral blood. conclusion: alterations in dc populations have been considered to play a role in autoimmune diseases such as systemic lupus erythematosus (sle) or diabetes. in ss patients, up-regulation of interferon-regulated genes has been shown previously. therefore, decreased pdc numbers in peripheral blood from pss patients might explain the fact that an increased ifn signature is found in salivary glands of pss patients, but no elevated levels of ifn-a are measured in serum. recently we reported that malignant cd5+ b cells from patients with b chronic lymphocytic leukemia (b-cll) produce granzyme b (grb) and are rapidly undergoing apoptosis in a granzyme b-dependent manner following interleukin 21 (il-21) stimulation. several autoimmune diseases have been linked to both elevated frequencies of cd5+ b cells and increased il-21 levels. we therefore hypothesized that il-21 may have similar biological effects on cd5+ b cells in autoimmune diseases. here we demonstrate that the amount of il-21 in the serum of systemic lupus erythematosus (sle) patients but not of healthy subjects highly correlated with serum levels of grb. in contrast to b cells from healthy individuals, where no baseline grb expression was found, we demonstrate that up to 14 % of cd5+ b cells in sle individuals expressed grb. in-vitro experiments revealed that il-21 was able to induce expression of grb in b cells from individuals with sle and other autoimmune diseases including psoriasis and rheumatoid arthritis. this effect was direct and was strongly enhanced by engagement of the b cell receptor or toll-like receptor 9. importantly, il-21 significantly decreased the cd5+/cd5-b cell ratio in both sle peripheral blood and healthy cord blood samples, suggesting a preferential induction of cd5+ b cell death. these results suggest that il-21-induced grb may play a regulatory role for cd5+ b cells similar to what we described earlier in b-cll cells. this is the first report uncovering an interrelation between il-21 and grb levels in sle and showing that il-21 reduces the cd5+/ cd5-b cell ratio in b cells from sle peripheral blood and healthy cord blood. endogenous il-21 may therefore play a disease-modifying role and may explain elevated grb serum levels in autoimmune diseases. further studies should evaluate the therapeutic potential of il-21 in sle and other autoimmune diseases. r. de palma 1 , e. d'aiuto 1 , s. vettori 1 , g. abbate 1 , g. valentini 1 1 second university of naples, clinical & experimental medicine, napoli, italy ssc is considered an autoimmune puzzling disease whose pathogenesis is unknown. in the last years, there have been increasing evidences that an interplay between activated t cells and fibroblasts could play a pivotal role in promoting matrix accumulation in systemic sclerosis (ssc). we have previously shown that peripheral t cells from ssc patients with early diffuse disease co-cultured with autologous fibroblasts expand the same t cell clonotypes found in the affected skin. here, using the same experimental approach, we found that the t cell clonotypes expanded in co-cultures are ab positive, hla-dr positive, and promote apoptosis of autologous ssc fibroblasts. we also found that, in these co-cultures, ssc fibroblasts up-regulated fas and underwent apoptosis that paired with the expression of fas ligand (fasl) on cd4+ t cells. finally, when we added a blocking anti-fas antibody to the co-cultures, we observed a marked reduction of fibroblast apoptosis, suggesting that engagement of fas/fasl had a critical role in mediating apoptosis in co-cultured fibroblasts. it has to be reminded that the absence of fasmediated apoptosis in vivo could be due to several reasons, as the increase of soluble fas in sera of patients affected by ssc. moreover, in the co-culture supernatants we found tgf-beta, il-1beta, il-6 and il-8, cytokines known to have a role in promoting fibrosis in systemic sclerosis. taken together, these data suggest that t cell response in ssc may represent an attempt of the immune system to kill fibroblasts, cells that are likely to be altered and expressing (auto)antigens. indeed, fibroblasts of ssc patients have been shown to display a persistently activated phenotype characterized by excessive production of collagen and other extracellular matrix proteins. however, the overall outcome of the t cell response triggered by fibroblasts in ssc, while unable to control the activity and the growth of fibroblasts, contribute to sustain inflammatory loops leading to fibrosis. these findings may lead to change our view about the pathogenesis of this disease and other autoimmune diseases. systemic lupus erythematosus (sle) is a chronic inflammatory autoimmune disease that is associated with a major breakdown in b cell self-tolerance as reflected by elevated serum igg levels of predominantly antinuclear antibodies (anas). serum antibody titers are maintained by antibody-secreting plasmablasts and longlived plasma cells, which reside in survival niches of the bone marrow. however, the antibody repertoire of bone marrow plasma cells, which may include cells expressing autoreactive and potentially pathogenic antibodies, has not been characterized in sle. to determine the frequency, specificity and immunoglobulin gene characteristics of autoantibodies in the long-lived plasma cell compartment in sle, we cloned and expressed 169 igg antibodies from single facs purified cd19+cd27+cd38+cd138+ bone marrow plasma cells of 3 patients with sle and tested the recombinant monoclonal antibodies for self-reactivity. our preliminary data on the ig gene repertoire and reactivity profile of human igg+ sle bone marrow plasma cells in comparison to healthy controls will be discussed. z. amirghofran 1 , e. moazemi godarzi 1 , e. kamali sarvestani 1 , e. aflaki 1 1 shiraz university of medical sciences, shiraz, iran, islamic republic of interleukin 6 (il-6) has been shown to be related to the pathogenesis of systemic lupus erythematosus (sle). two polymorphisms in the promoter region of il-6 gene at positions -572 g/c and -174 g/c have been described that are key regulators of il-6 gene. in the present study the relationship between these two polymorphic sites and disease susceptibility in a group of iranian patients with sle was investigated using polymerase chain reaction-restriction fragment length polymorphism method. the genotype distribution and allele frequencies of il-6 gene polymorphism at -174 position showed no significant difference between sle patients and controls. at this position the frequency of gg genotype as well as g allele was higher than c allele in both patients and control groups. in contrary, both allelic and genotypic frequencies at the -572 position significantly differed in sle patients and controls. at this position gg genotype was observed in 77.9 % of patients compared to 68.9 % in the control group (p x 0.014). the frequency of -572 g allele in patients (87.3 %) was also higher than in controls (83.2 %) (p=0.034). the haplotype study showed no significant difference between patients and healthy subjects. the relationship between these polymorphisms and clinical manifestations and laboratory parameters were investigated. -174 polymorphism was associated with the presence of antinuclear antibodies in all patients and rash and hematuria in male patients (p x 0.04). at -572 polymorphism, a significant difference with regard to photosensitivity in male patients (p=0.04) was found. in conclusion, results of this study showed that -572 polymorphism plays an important role in susceptibility to sle and that -174 polymorphism could influence the presence of antinuclear antibodies in the patients. the eukaryotic constitutive proteasome is the main protease expressed in most tissues. recently we have elucidated a functional importance of the second proteasome form, inducible immunoproteasomes, in regulating nf-kb activity during the intestinal inflammation. in comparison with healthy controls and patients with ulcerative colitis (uc), there was increased expression of immunoproteasomes in the inflamed mucosa of patients with crohn's disease (cd) at both mrna and protein levels. in our very recent work we have shown that the proteasome subunit pattern might be suitable for diagnostic differentiation between cd and uc patients. since ifn-g has been shown to be the main inducer of immunoproteasomes in various murine and human cell lines and the ifn-g levels are highly elevated in inflamed intestine of cd patients, induction of immunoproteasomes in cd might be mediated by this cytokine. our data with human leukemic t cell lines and primary macrophages show a significant increase in the nf-kb controlled production of proinflammatory cytokines after the ifn-g-mediated induction of immunoproteasomes in these cells. in the dss-induced colitis model we have observed a diminished colonic inflammation in the absence of the proteasomal immunosubunits. therefore we here suppose that immunoprteasome are involved in the complex inflammatory response during the chronic intestinal inflammation by increasing nf-kb activity in the epithelial and immune cells. however, it remains to be determined whether these results have an important implication for the treatment of chronic gut inflamation in humans. objectives: inflammatory bowel diseases (ibd) including crohn's disease (cd) and ulcerative colitis (uc) are characterized by unknown etiology and chronic intestinal inflammation. noninvasive serological tests to differentiate cd from uc have been searched for a long time. testing for panca together with ascas has good predictive values to identify patients with ibd.the aim of this study was to find evidences for diversity of ascas and anti-mycobacterium paratuberculosis antibodies (anti-mpt) by elisa method. in addition, to examine whether combination of these elisas is useful for distinguishing cd from uc. methods: the study population contained 161 patients with ibd (89 with cd, 41 with uc, 31 with gluten sensitive enteropathy, gse) and 33 healthy control subjects. serum asca igg, asca iga and anti-mpt antibody levels were measured by solid phase enzyme immunoassay. adsorption of 7 asca positive sera was performed by baker's yeast suspension. results: elevated level of asca igg, iga and anti-mpt was shown in cd and gse but not in uc compared to healthy controls. serum levels of asca igg, iga showed a significant positive correlation with anti-mpt antibody levels in cd. repeated adsorptions with yeast removed asca igg and iga from sera of patients, but did not change levels of anti-mpt. these results indicate the diversity of asca igg, iga and anti-mpt (accordingly their antigens) and suggest that combination of these elisa can have a role in the differential diagnostics of ibd. it is now well recognised that the majority of lymphocytes may be located within tissues, not in blood, and yet these tissue-resident lymphocytes are relatively understudied, especially in humans. we have extracted cells from human gut biopsies (both normal and inflamed gut) in order to characterise the immune cell populations that exist therein and which molecules may be of paramount importance to their function. we show that distinct populations of t cells exist within the gut and that the ratio of these populations changes down the length of the gut, with the so-called 'unconventional' double negative t cell population (ie tcrab+ve, cd4-ve cd8b-ve) predominating in the healthy colon whereas these cells are overwhelmed by infiltrating cd4(+) cells in inflammatory bowel disease (ibd). having previously shown in mice that gut-resident t cells express high levels of the regulator of g protein signalling-1 (rgs-1) protein, we have now found substantial over-expression (10-100 fold) of rgs-1 in human gut-derived t cells, particularly in this unconventional t cell population. furthermore, levels appear even higher in t cells derived from inflamed gut. transfection of rgs-1 decreases primary t cell responses to cxcl12 and ccl19, strongly implying that it may regulate t cell localisation. thus, rgs-1 may be a novel target for modulating t cells in ibd, consistent with which snps in rgs-1 have been associated with both coeliac disease and type 1 diabetes. mechanisms involved in the induction of oral tolerance (ot) or systemic immunization through the oral rout are still poorly understood. in our previous studies we have shown that when normal mice eat peanuts they become tolerant, with no gut alterations. conversely, if they are immunized with peanut proteins prior to a challenge diet (cd) containing peanuts they develop chronic inflammation of the gut. our aim is to evaluate the consequences of the introduction of a novel protein in the diet of animals presenting antigen specific gut inflammation. adult, female c57bl/6j mice were divided in control (c) and experimental (e) groups. c1-c3 received peanut protein immunization, animals of the control groups c4 were sham immunized, and control group c5 received ovalbumin (ova) immunization. the experimental group was immunized with peanut protein extract. before initial exposure to a 30 day peanut containing cd, the experimental group was divided into 5 groups (e1-e5). ova feeding began 7 days prior cd (e1) on day 0 (e2), 7 (e3), 14 (e4) and 21 (e5) during cd. our results show that oral exposure to a novel protein (ova) in the absence of gut inflammation (e1) leads to low levels of systemic antibody titers, comparable to tolerant animals. conversely, as off initial induction of inflammation, groups submitted to ova (ot) protocol develop increasingly higher systemic antibody (ab) titers similar to animals of the immune control group. in conclusion our protocol indicates that timing is more important than the antigenicity when a novel protein is offered, in the diet. nanoparticles of various types are increasingly used as constituents of food supplements and so called nanofood. since nanoparticles induce inflammatory reactions in the lung, there is an urgent need to also study the toxicological potential of nanoparticles in the intestine. therefore, we assessed the effect of particles on dendritic cells (dc) as key players in the manifestation of intestinal immunity.in in vitro studies we could show that ultrafine tio 2 as well as ultrafine silica led to a mature phenotype of the cells when particles were added to cultures of immature bone-marrow-derived dc. this effect appears to result from enhanced cell death in immature dc but also from direct stimulation of the cells.to analyse the mechanisms underlying this effect we looked for apoptosis as well as for induction of the inflammasome since it has been shown that crystalline silica leads to activation of caspase 1 and secretion of bioactive il1-b.in our hands certain nanoparticles induced apoptosis of immature dc, as well as enhanced secretion of active il1-b. we therefore hypothesize that particles can induce the inflammasome which leads to the activation of dc.to study the impact of nanoparticles on intestinal inflammatory processes in vivo, we induced colitis by applying 5 % destrane sulphate sodium (dss) in the drinking water for 8 days to wildtype mice. when ultrafine nanoparticles were administered on day 6 and 7 by gavage feeding, we observed an amelioration of disease symptoms when scoring the degree of epithelial disruption and inflammation. in future experiments we will also analyse the effect of different particles in the il10 -/model of colitis to assess the contribution of particles to the induction and pathogenesis of disease. m. schmohl 1 , n. schneiderhan-marra 1 , m. blum 2 , g. stein 2 , m. schmolz 2 , t. joos 1 1 nmi-natural and medical sciences institute at the university of tübingen, biochemistry, reutlingen, germany, 2 edi gmbh, reutlingen, germany the human immune system represents a highly complex system that protects the organism against diseases. there is an impressive network of immunoregulatory signals within the immune system as well as between the different healthy and diseased organs. epithelial layers function as a barrier against pathogens. as the gastrointestinal tract, which is occupied with a large variety of microorganisms, represents the outside of the body, the immune system has to establish and maintain a strong presence at the mucosal boundary. the ability to discriminate between pathogens while remaining relatively unresponsive to food antigens and the commensal microflora is achieved by a plethora of largely unknown regulatory mechanisms. this ability appears to be breaking down with chronic inflammatory bowel diseases (ibd) like crohn's disease and ulcerative colitis [1] . to date treatment options are restricted to controlling symptoms, putting and keeping the dis-eases in remission and preventing relapse. therefore, there is an urgent need for a more detailed understanding of the inflammatory events taking place during the disease. for this purpose a human organo-typic (hot) co-culture model is used, which allows analyzing the collaborative regulation between the immune system and the gut epithelial cells. the human caco-2 cell line, as a model for the gut-epithelium cells, are cultivated on the top side of special culture vessels, fitting as inserts into carrier wells of 24-well culture plates, containing whole-blood. this co-culture set up mimics the physiological barrier to perorally applied biologicals/drugs and allows measuring their effect on the immune system. as a read out miniaturized and parallelized sandwich immunoassays will be used to detect alterations in the intracellular mapk and rtk-signalling of the epithelial cells as well as in the extracellular communication via cytokines and chemokines at the interface of the two organs. this approach will provide new insight into the inter-and intracellular signalling of gut epithelium and the immune system, which will finally result in a better understanding of the etiology of inflammatory bowel diseases. inflammatory bowel disease (ibd), including crohn's disease (cd) and ulcerative colitis (uc), is characterized by an upregulation of pro-inflammatory cytokines that play an important role in pathogenesis. osteopontin (opn) is a cytokine implicated in several immunological diseases and, although expressed constitutively in normal intestine, is upregulated in intestinal mucosa and in the plasma of ibd patients. opn has been shown to be either pro-inflammatory or anti-inflammatory for experimental uc, indicating a controversy in this field, while its role in experimental cd remains unknown. in our study we investigated the role of opn in experimental colitis using two mouse models: trinitrobenzene sulphonic acid (tnbs) colitis, a t h 1-associated model that resembles cd, and dextran sulphate sodium (dss) colitis, a t h 2-like-associated model for uc. deficiency of opn (either by antibody-mediated neutralization or use of opn -/mice) resulted in suppression of disease phenotype in both colitis models, revealing that opn, and especially, the secreted isoform of opn (opn-s) is important for the initiation of acute intestinal inflammation. importantly, we discovered that opn drives il-17 production and t h 17 polarization and decreases recruitment of cd4 + cd25 + foxp3 + t regulatory (treg) cells in mesenteric lymph nodes (mlns) of mice with colitis. also, there was an effect of opn on recruitment of cd11c + dcs, which were significantly elevated in mlns of opn -/or anti-opn-treated, as compared to opn +/+ or ig-treated control mice. this finding implies that opn deficiency results in enhanced recruitment of regulatory cd11c + dcs which may mediate treg induction and protect from colitis. overall, our findings indicate that opn is proinflammatory in both types of colitis, by promoting pathogenic t h 17 and attenuating treg cell recruitment, implying also common mechanisms in the pathogenesis of cd and uc. c. shen 1,2 , g. van assche 3 , p. rutgeerts 3 , a. liston 1 , j. l. ceuppens 2 1 k.u. leuven, autoimmune & genetics lab, vib, leuven, belgium, 2 k. u. leuven, experimental immunology lab, leuven, belgium, 3 k. u. leuven, department of pathophysiology, gastroenterology section, leuven, belgium background: haptoglobin (hp) is one of the acute phase proteins synthesized during inflammation. hp-1 allele is associated with the disease behavior in crohn's disease but not in ulcerative colitis. however its role in inflammatory bowel disease has not been defined. aim: to determine whether hp modulates the immune responses in experimental colitis. methods: we induced 3 types of colitis dss (th1/th17), tnbs (th1) and oxazolone (th2) in hp ko mice. neutralizing anti-il-17 mab was injected into dss and tnbs hp ko mice. severity of colitis was evaluated by body weight, colon length and histology. th17/th1 cells were analyzed by flow cytometry. cytokines were measured by elisa or rt-pcr. 1) compared to the wt mice, hp ko mice developed much severer dss and oxa induced colitis. dss induced lethal colitis in hp ko but not in wt mice; 2) in dss but not in oxa colitis mice, il-17, ifn-g, tgf-b and il-6 were significantly increased (p x 0.01, dss vs control) in lamina propria and mesenteric lymph nodes (mln), and this is much evident in hp ko mice compared to those in the wt (p x 0.05, ko vs wt). in tnbs colitis, we found elevated il-12 and ifn-g (p x 0.01, tnbs vs control). although not significant, il-17 was also somewhat upregulated; 3) in dss colitis we observed that il-23 enhanced differentiated th17 cells in vitro, this effect could be abrogated by coculture with serum from wt but not hpko mice. furthermore, in vitro in the presence of tgf-b, il-6 and il-21, more mln-t cells from hpko mice differentiated into th17 cells; 4) anti-il-17 mab improved dss and tnbs colitis, and partially rescued hp ko mice from lethal dss colitis. in line with this, mice treated with anti-il-17 showed reduced il-6, il-17 and ifn-g in both mln and lp (p x 0.05, anti-il-17 vs control). our results reveal that hp has a protective role in the development of mucosal inflammation. in dss and tnbs colitis hp may exert its beneficial effect partially through inhibiting production of il-17, supporting further pre-clinical and clinical application of hp for treatment of crohn's disease. p. engelmann 1 , g. talabér 1 , g. süt" o 2 , p. németh 1 , t. berki 1 1 university of pécs, clinical center, department of immunology and biotechnology, pécs, hungary, 2 university of pécs, clinical center, department of immunology and rheumatology, pécs, hungary objectives: inflammatory bowel disease (ibd) resembles as an autoimmune-like disease. ibd is most common in developed countries: it is calculated that 2.2 million people in europe suffer from ibd. several hypotheses are raised in the pathogenesis of inflammatory bowel disease. one of the most favored is the dysregulation of the immune response due to failure of regulatory t cells. the most well known regulatory t cells are the cd4+cd25hi+ t (treg) cells. furthermore, other immune-regulatory cells are known such as invariant natural killer t (inkt) cells producing both th1 and th2 cytokines rapidly upon antigen (lipid) stimulation. methods: based on this hypothesis we aimed to investigate the role of various immune-regulatory t cells in human ibd. we attempt to measure the proportions of inkt cells, treg cells in peripheral blood of patients with crohn's disease (cd) and ulcerative colitis (uc) compared to normal controls. blood samples were collected from normal controls and ibd patients; then lymphocytes were labeled for inkt and treg markers with specific monoclonal antibodies and measured with flow cytometry. results: according to our results a decline in the total inkt cells of ibd patients was observed, interestingly the proportions of cd4+ and double negative (dn) inkt subgroups showed a characteristic shift among the study groups. percentages of dn and cd4+ inkt subpopulations were assessed after gating of total inkt populations. in controls we observed high percentage of dn inkt cells (74.5 ± 3.5 %, mean ± sem), while cd4+ inkt cells ratio was moderate (25.4 ± 3.5 %). in uc and cd patients we found a reduced proportion of dn inkt cells (uc: 38.0 ± 7.1 %; cd: 34.0 ± 5.2 %, mean ± sem), while the percentage of cd4+ inkt cells was elevated (uc: 62.0 ± 7.1 %; cd: 65.9 ± 5.2 %, mean ± sem) in both disease groups. proportions of foxp3+ treg cells also showed a decline in ibd patients comparing to normal controls. conclusion: this study can provide useful data about the pathogenesis of ibd and can lead to identify and characterize new cellular and molecular targets with possible therapeutic use in human autoimmune disorders. objectives: the aim of this project is to explore whether exosomes from tgf-b1 gene modified bone marrow-derived immature dendritic cells (md-imdc) have the function of systemic immune inhibition and protective effect on the development of inflammatory bowel disease (ibd) in mice, the underlying mechanism was also investigated. methods: exosomes were isolated from supernatant of md-imdc transfected with tgf-b1 adenovirus (tgf-b1-exo). the t cell inhibitory function of tgf-b1-exo was determined by mixed lymphocyte reaction (mlr) in vitro. to evaluate the protective effect of tgf-b1-exo in the development of ibd, dextran sulfate sodium(dss) induced murine ibd was established and mice were treated with tgf-b1-exo. the main symptoms of ibd were observed. the inflammatory degree of colon was also evaluated by histological examination. the relative cd4 + foxp3 + treg cell numbers from spleens and mesentery lymph nodes (mlns) were analyzed by facs. results: it was demonstrated that tgf-b1-exo could inhibit the proliferation of t cells in mlr in vitro. in murine ibd model, after treated with tgf-b1-exo, the main symptoms of ibd such as weight loss, diarrhea and grume sanguinopurulent stool were all alleviated and the inflammatory degree of colon was also reduced. analysis of cd4 + foxp3 + regulatory t cells (treg) revealed that the relative numbers of cd4 + foxp3 + treg increased in lymphocytes from mesentery lymph nodes (mlns) of inflammatory site but not from spleens. conclusions: these results demonstrate that immunosuppressive exosomes obtained from tgf-b1 gene modified md-imdc can delay the development of ibd. this protective effect is mediated by the induction of cd4 + foxp3 + treg. tgf-b1-exo might provide a novel strategy for the therapy of ibd. results: hcv-specific cytokine expression by cd8+ t-cells was similar in the four vaccinees as observed by ifng, il-2 production-profiles. however, the killing capacity of expanded cd8+ t-cells was distinct as observed by the competence to kill ns3-peptide presenting transfectants in vitro. as depicted in figure 1 , cd8+ t-cells cells from both vac1 (cleared ) and vac2 (chronic) produced il-2 and ifng after stimulation with ns3-peptide59. however, specific killing of the peptide loaded transfectants was only observed in vac 1, who was able to clear its hcv infection, and this was not observed not in any of the other chimpanzees, who became chronic carriers. [ figure 1 ] killing of ns3 peptide presenting cells was restricted to the vaccinee that was able to clear hcv infection. these results suggest that controlling hcv replication as initiated by this dna-prime mva-boost vaccine-protocol was partly mediated by antigen specific cd8+ t-cells. hence, the effector mechanisms induced were distinct between the animals and clearance of the infection was correlated with induction of killing competent cd8 t-cells. objectives: infection by hepatitis c virus (hcv) is characterized by its high tendency to chronicity, which is usually associated with a low or absent t-cell response against viral antigens. immune response specific for non-structural protein ns3 from hcv was associated with viral clearance. we have demonstrated that fusion of an antigen to the extra domain a from fibronectin (eda) targets the antigen to tlr4-expressing dendritic cells and improves its immunogenicity. thus, we tested if covalent linkage between eda and ns3 might constitute an alternative for vaccination against hcv infection. methods: recombinant plasmids expressing a secretable version of ns3 or eda-ns3 under the control of cmv promoter were prepared. recombinant ns3 and the fusion protein eda-ns3 were produced in e. coli. the recombinant proteins were tested in vitro on their capacity to activate maturation of bone marrow derived dendritic cells and to favour antigen presentation. hhd transgenic mice (expressing the human hla-a2 molecule) were immunized with the recombinant plasmids or with the recombinant proteins, in the absence or presence of poly(i:c) and anti-cd40 agonistic antibodies. elispot and chromium release assays were carried out to measure the immunogenicity of the different vaccination strategies. intrahepatic expression of hcv-ns3 rna was measured after a hydrodynamic injection with a plasmid encoding hcv ns3. results: immunization of mice with the plasmids expressing eda-ns3, but not ns3 alone, induced strong t cell responses against the main hla-a2 restricted cytotoxic t cell determinants from ns3. the recombinant eda-ns3 fusion protein, but not ns3, was able to activate in vitro maturation of bone marrow derived dendritic cells as well as the production of tnf-a by the thp-1 monocyte cell line. immunization of hhd mice with eda-ns3 fusion protein induced both cd4+ and cd8+ t cell responses against ns3 and, when immunized with poly(i:c) and anti-cd40 antibodies, was able to down-regulate the intrahepatic expression of hcv-ns3 rna. the recombinant eda-ns3 fusion protein may be considered for the development of prophylactic or therapeutic vaccines against hcv infection. vaccination is the most efficient strategy to prevent from microbial infections and to control epidemics but are still not available in the case of hiv infection even 25 years after virus detection. therein we propose the intra-dermal inoculation of dna vaccine that present a plasmid vector exploiting the binding capacity of the bovine papillomavirus e2 protein encoding an artificial multi-component hiv antigen. this inoculation is followed by electroporation in order to increase dna uptake. we used skin as site for vaccination because, being the first line in host defence, it is populated with various cells of immune system. among them, langerhans cells (cd207+cd1a+), located in the epidermis, are dendritic cell subset capable to elucidate specific cd8+ responses. the present work emphasizes molecular and cellular biodistribution of the dna vaccine in the skin after intra-dermal vaccination in macaques, as one of the most relevant animal models in hiv studies. technical approach considers an intra-dermal injection of dna followed by topical electroporation of the injection sites. skin and draining ln biopsies were collected at different time points. these biopsies were used for ihc fluorescent staining in order to establish biodistribution dna-encoded antigens and co-localisation with different cell types. kinetic of antigen expression was studied by bioluminescence in vivo imaging. t cell responses were measured by ifn-g elispot assays up to 3 years after dna vaccination. we show that a dna vaccine delivery method combining intra-dermal injection and electroporation dramatically increased the expression of the vaccine antigen selectively in the epidermis, increased the frequency of cd1a+ cells in the draining ln in association with the antigen expression, and increased the cellular response persistence, at high levels, for more than two years after the last vaccine boost. our data suggest that electroporation after intradermal injection of dna vaccine involves langerhans cells from the epidermis that elucidate qualitative anti-hiv immune responses. this new approach that comprise new dna vaccine followed by non-invasive electroporation, induce long-lasting cellular response that could be crucial in prophylactic / therapeutic vaccine design. presenting cells was developed. murine coronavirus-based virus-like particles encoding epitopes from the lymphocytic choriomeningitis virus glycoprotein or human melan-a, in combination with the immunostimulatory cytokine gm-csf, selective targeted dcs in vitro and in vivo resulting in vector-mediated antigen expression, and efficient maturation of dcs. in mice, a single application of only low doses elicited strong and long-lasting cytotoxic t-cell responses which provided protective antiviral and antitumor immunity. furthermore, the efficient activation of human tumor-specific cd8+ t cells by mature dcs transduced with melan-a-recombinant human coronavirus 229e indicates that this novel vaccine platform mediates the delivery of antigens and immunostimulatory cytokines to those cellular components of the immune system that initiate and maintain protective immunity. as the application of gm-csf already enhanced immunogenicity, we are now trying to further modulate the coronavirus vector-induced immune response with the reverse genetic setup of recombinant coronavirus-based vectors expressing different immunostimulatory cytokines. thereby cytokines will be acting on t cell and dc level. to enhance t cell response interleukin 2 (il2) and interleukin 15 (il15) will be involved, and fms-like tyrosin kinase 3 ligand (flt3l) will be expressed to modulate dendritic cells. il2 is known to enhance early t cell expansion and limits t cell overshoot, whereaes il15 guarantees survival of high affinity t cells during memory phase. on the other hand flt3l enhances dc proliferation and accumulation. with these approaches modulation of the immune response generated by this novel vaccine platform will be examined in viral and tumour models to get insight on the antigen specific ctl response, synergistic effects of the cytokines and protective as well as prophylactic vaccination approaches. f. demircik 1 , ag waisman 1 uniklinik mainz, 1. med, mainz, germany in murine cytomegalovirus (mcmv) infection, cytotoxic cd8 t cells and nk cells play a critical role. previously it was shown that mice deficient for b cells are more susceptible to mcmv-related disease, caused by virus reactivation. to better understand the role of b cells and antibodies in the response to mcmv, we made use of different mouse strains that lack b cells, secreted antibodies or il-10 production by the b cells. we found that for the initial t cell response to the virus b cells are important, but antibodies do not play an important role. this implicates b cells as potential important antigen presenting cells (apcs) in the activation of the virus-specific t cells. the reduced t cell response to the virus was observed whether the mice were b cell deficient from birth or if they were depleted later in life. six month after infection mice were tested for the memory cd8 t cell response. interestingly, we found that in mice that lack antibodies (mice that lack b cells all together and mice that have b cells but no secreted antibodies) maintain a rather high t cell response to viral peptides, in a level similar to the acute response 7 days after infection. we conclude that antibodies probably remove residual viruses from the body and therefore prevent the continuous activation of t cells. finally, we tested the role of il-10 produced by b cells by conditional deletion of the il-10 gene in these cells. we found that b cell secreted il-10 has a suppressive effect on the t cell response to mcmv, as this response is elevated in these mice. we conclude that b cells are important for an efficient acute response to mcmv and that antibodies play a role in eliminating residual viral particles, thus implicating a dual role for b cells in the efficient acute and memory response to mcmv. this work is supported by the deutsche forschungsgemeinschaft grant sfb490 to aw. objectives: ebv infection leads to life-long viral persistence. although ebv infection can result in chronic disease and malignant transformation most carriers remain disease-free due to an effective control of the virus by t cells. ebv-specific ifng-producing t cells could be demonstrated in acute and chronic infection by many researchers. recent studies in hiv and leishmania provide, however, evidence that assessing ifng alone is insufficient to assess the quantity and quality of a memory t cell response and support the crucial role of multifunctional t cells in disease control. in this study we therefore analyzed ebv-specific t cell responses in peripheral blood (pb) and bone marrow (bm). methods: paired pb and bm samples were obtained from 8 healthy virus carriers who underwent total hip arthroplasty. t cells were expanded for 10 days in the presence of il-2 and il-7 with exposure to overlapping peptide pools of latent ebna-1 and lytic bzlf-1 antigens. ebv-specific immune responses were assessed exvivo and after expansion by multiparameter flow cytometry staining for live/dead discrimination marker, cd3, cd4, cd8, ccr7, cd45ra, il-2, tnfa, ifng and cd107a. the majority of ex vivo ebv-reactive cd4+ t cells as well as ebna-1-reactive cd8+ t cells were il-2 and tnfa-producing memory cells, the later being more frequent in bone marrow (cd4+, median, ebna-1: bm 0.69 %;pb 0.12 %; bzlf-1: bm 0.37 %;pb 0.01 %, p=0.039). after in vitro expansion a major subset of ebv-specific cd4+ and cd8+t cells displayed a differentiated effector ifng/tnfa phenotype. a comparable number of ebv-specific cd4+ and cd8+ t cells retained, however, a tnfa single, tnfa/il-2 or triple producer phenotype resembling early differentiated or multifunctional memory t cells, respectively. interestingly, both cd4+ and cd8+ t cells generated from bm revealed significantly higher cytotoxic potential. sorting of ccr7/cd45ra differentiation subsets, revealed that ebv-specific t cells were predominantly expandable from the central memory compartment. conclusion: our data shows that multicolor assessment of ifng, tnfa and il-2 delineates various subsets of ebv-specific memory t cells, which reflect the profile of a protective immune response. human adenovirus (hadv) can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation (allosct). reconstitution of hadv-specific t cells has been reported to be associated with sustained protection from hadv disease, but epitope specificity of these responses has not been further characterized. furthermore, the relative contribution of hadv-specific cd4 + and cd8 + t cells in the protection from hadv disease after allosct remains to be elucidated. in this study, we demonstrate, by sensitive measurement using intracellular cytokine staining combined with cd154 or peptide-mhc tetramer staining, that clearance of hadv was associated with a combined hadv hexon specific cd4 + and cd8 + t cell response in both pediatric and adult allosct recipients. based on this observation, we developed a clinical grade method for the rapid generation of t cell lines with high and defined specificity for hadv hexon epitopes for adoptive immunotherapy. activation of hadv hexon-specific cd8 + and cd4 + t cells in peripheral blood with a hexon protein-spanning pool of synthetic 15-mer peptides followed by ifng-based isolation allowed rapid expansion of highly specific t cell lines from healthy adults, including donors without detectable frequencies of hadv hexon-specific t cells. the frequency of hadv-specific t cells was increased to 29-90 % in the t cell lines and the absolute numbers of both hexon-specific cd4+ and cd8+ t cells were 2 to 3 log increased compared to the starting material. detailed analysis showed that hadv-specific t cell lines recognized multiple mhc class i and ii restricted epitopes, including known and novel epitopes, and showed specific and efficient lysis of hadv infected target cells. this strategy may be used for adoptive transfer of donor-derived hadv hexon-specific cd8 + and cd4 + t cells for treatment of disseminated hadv infection after allosct. several studies showed that hbv persistance correlates with a failure of an efficient virus-specific t-cell response. induction of hbv-specific t cells by vaccination may be an innovative approach to overcome virus persistance. dna prime-recombinant adenovirus serotype 5 (ad5) boost strategy proved to be effective in stimulating t cell responses and control of viral infections. woodchuck hepatitis virus (whv) and its host the woodchuck are a useful peclinical model for investigating the new therapeutic approaches. the efficacy of plasmid dna and ad5 vaccine vectors expressing whv core protein was first examinated in c57bl6 mice. groups of mice were immunized with a dna prime-ad5 boost regimen or with dna and ad5 alone. ad5 was injected i. m. or s. c. t cell response was evaluated by intracellular ifng staining of splenocytes stimulated in vitro with whc-derived peptide pools. anti-whc antibodies were detected by elisa. we detected cd8+ t cell responses against peptide pools 1 and 3 in spleens of dna and dna-ad5 immunized mice. however, in prime-boost group the percentage of of detected ifng+ cd8+ t cells was lower in comparison to dna group. in splenocytes of animals vaccinated with ad5 very weak cd8+ t cell response was observed. in dna vaccinated animals we determined high level of anti-whc already after second immunization. after boosting with ad5 level of antibodies did not change. those antibodies were only igg2a subclass what indicates th1 t helper type of response. ad5-immunized mice had over 3-fold lower level of anti-whc: both igg2a and igg1 subtypes were detected. the weak response induced by ad5 may be due to the low expression of whcag. in ongoing expreriments we improved the protein expression level by insertion of an intron. we currenly investigate the new construct in mice. the new peptide construct containing four m2e-peptide sequences coupled to t helper epitopes from the plasmodium falciparum cs protein and the hepatitis b virus antigen was administered together with adjuvants intranasally and subcutaneously as described (mozdzanowska et al., virology journal 2007) into various mouse strains. in contrast to its predecessor peptide, we found that vaccination induced much higher anti-m2e serum ab titers against peptide and native m2e. this correlated with a large number of m2-specific ab-secreting cells in lungs and bone marrow. moreover, the serum of vaccinated mice was also crossreactive against the influenza virus subtype a/fm (h1n1), which contains a variant m2e-sequence different in 3 amino acid positions. importantly, this new peptide vaccine regimen showed significant protection against viral challenge with influenza a strains x31 (h3n2) and the highly pathogenic pr/8 (h1n1) with remarkably reduced viral titers in lungs and noses of mice. in conclusion, our studies show promising results towards the further development of vaccination with m2e as a potential "universal" influenza vaccine. this research is supported by a nih t32 fellowship ca09171-32, the nih grant ai 46457 and a grant from the commonwealth of pa. l. yu 1 1 zhejiang university, zhangzhou, china interleukin-18 (il-18) is a cytokine produced by stimulated mononuclear macrophage system. in this report, 18-day-old chicken embryos were vaccined with the plasmid dna (pci-chil-18) encoding chicken interleukin-18 and the copy numbers of chil-18 in peripheral blood, spleen and bursa of fabricius at different time points post-embryonic-vaccination were detected by real-time fluorescent quantitative pcr. the polyprotein of infectious bursal disease virus (ibdv) was prepared into dna vaccine, and the dna vaccine was co-administrated with pci-chil-18 in 18-day-old chicken embryos, then boosted after two weeks, and challenged with virulent ibdv four weeks later. the results indicated that allantoic cavity vaccinated with pci-chil-18 could accelerate high concentrations of chil-18 in nonage peripheral blood, accelerate high expression of chil-18 in nonage spleen and bursa of fabricius and promote the body early immune response capacity. embryo vaccination with chil-18 could significantly enhance the nonage proliferation responses of t lymphocytes from spleen and b lymphocytes from bursa. meanwhile, it could raise the nonage neutralization antibody level and inhance the protection against virulent ibdv induced by dna vaccine. the results indicated that the nonage immune responsing to ibdv dna vaccine was highly enhanced by embryonic coadministration with chil-18 (p x 0.05). due to the unique role of the hair follicle in percutaneous penetration, drug delivery systems, which target active compounds to the hair follicle, may result in a better penetration and a higher efficiency of hair and skin therapy ("follicular targeting"). applications in immunotherapy, e. g. transcutaneous vaccination, are of particular interest, because skin antigen-presenting cells (apcs) can be found at particularly high densities in hair follicle-bearing skin, where they are concentrated around the upper portion of the hair follicles. in in vitro studies on human skin explants, we demonstrated that nanoparticles, due to their ability to aggregate in the hair follicle openings and to penetrate along the follicular duct, are promising carrier systems for transfollicular drug delivery. transcutaneously applied nanoparticles in the size range of 40nm, were capable of penetrating the epithelium and entered into human epidermal lcs, suggesting that such particles may be used to transcutaneously deliver active vaccine compounds, via the hair follicle. the use of the skin as target organ for vaccine has been spurred by recent implication of epithelial dendritic cells (dc) in cd8 cell cross-priming and suggests that vaccination via the transcutaneous (tc) route may be relevant in the induction of cellular immune responses. advanced studies in vivo using functional vaccines are, however, essential to further assess the potential of particle-based vaccines in transcutaneous vaccination. for this purpose, we developed a standard operating procedure (sop) for transcutaneous vaccine delivery on human skin based on our current knowledge on follicular penetration. in a pilot study on 12 volunteers and a phase i study on 24 volunteers vaccinated with an influenza vaccine, we found that this newly developed sop is safe and efficient at inducing a significant increase in cellular immune responses mostly composed of antigen-specific cd8 cells. induction of t cell responses has become one of the major goals in therapeutic vaccination against viral diseases and cancer. this study proposes new perspectives for the development of vaccination strategies that triggers t cell immune responses in humans. objectives: all anti-hiv-1 neutralizing antibodies are directed toward the viral envelope glycoproteins (gp) 120 and the transmembrane protein gp41. two sites on gp120 and gp41 are attractive targets for vaccine design: the epitope in the third hypervariable region (v3) is recognized by the human monoclonal antibody 447-52d and the epitopes in membrane proximal external region (mper) were recognized by the human monoclonal antibodies 4e10 and 2f5. in order to elicit anti-hiv-1 neutralizing antibodies we have designed virus like particles (vlps) displaying either the gp120-v3 region or the gp41-mper. the vlps are based on the acyltransferase component (e2 chain) of the pyruvate dehydrogenase complex of geobacillus stearothermophilus. the e2 chain self-assembles into a 24 nm protein scaffold resembling a vlp and that contains 60 copies of e2. efficient display and refolding of the v3 and mper regions in e2 vlps are obtained by using engineered plasmid which allows insertion of exogenous oligonucleotides at the 5' of the gene coding for e2. the priming and boosting with a combination of vlps and specific hiv-1 envelope dna were used to immunize mice and rabbits. results: the v3-e2 and mper-e2 vlps were purified as stable 60mers from e. coli cells after refolding in vitro from inclusion bodies followed by gel filtration chromatography. binding of 447-52d, 4e10 and 2f5 antibodies to hiv-e2 monomers was confirmed by western blot. we obtained high titers of hiv-1 gp140-specific antibodies in mice immunized with a combination of vlps plus dna (hiv-1 sf162 gp160). these antibodies generated a low (20 %-27 %) level of neutralization. moreover immunizations were also performed in rabbits, a better model for induction of neutralizing antibodies. three doses of e2 vlps plus dna elicited a low titer of hiv-1 gp140 specific antibodies. additional rounds of immunizations in rabbits will be performed, in combination with gp160 plasmid dna, to enahance the responses to envelope and to induce neutralizing activity against these key epitopes. our results demonstrate that e2 vlps are able to display antigenic determinants of hiv and to induce high titers of hiv-1-specific antibodies. the e2 vlps represent a promising tool for a vaccine design. now a day we paid for vaccination of previous generations. as a result morbidity sharply increases, but we haven't well-tried scheme of immunity renewal yet. every clinic, every center do it in there own way, while vaccination is continued, even when it's not necessary, for example, grip, nobody know strain exactly. the most unpleasantly think is that most of physicians don't know what immunity mean specifically, general they think about vitamins, that isn't fit for forming immunity because of many reasons. we offer a way of immunity according to the world scientific theory and practice. the method is based on biochemical, electrophysiology, and biology way of correction physical status. at first we normalize and activate current settings that are going to the diseased organ, vascular system, gastrointestinal tract, spleen. all of it attends indemnity necessary microelements that were extracted from wild officinal herbals. we don't concentrate only on the one or two types of immunity, fist of all we take into account structure and dynamic of immunogeneration system. in our clinic we use this method; immunity is restored very quickly and kept during long time even if organism gets any complications, which can worsen the situation. that's why when we secure new physical statement in the cns program we forming new nearest and distant men health. we tell local state mechanism of disturbances from disturbances, that develop in blood, lymphatic system, tissues and hypothalamus, when pathological process exist long time. it's completely different disturbances of physician state, which should have different therapeutic approach. the threat of an influenza pandemic has become evident in recent years, emphasizing the requirement for influenza vaccines that are broadly cross-reactive against different subtypes with pandemic potential. we have previously shown that baxter's vero cell-derived h5n1 whole virus candidate vaccines are highly immunogenic both in animal models and in human clinical studies, and cross-protective in mice and ferrets. more recently, it was reported that cross-reactive heterosubtype immune responses against highly pathogenic h5n1 influenza virus could also be achieved by immunizing subjects with a trivalent seasonal influenza vaccine; however the induction of cross-subtype protection could not be addressed in this study with human subjects [1] . the study reported here evaluated whether the seasonal influenza vaccine, when used either as a monotherapy or in combination with a h5n1 whole virus wild-type vaccine, could induce an immune response and protect mice against h5n1 influenza virus infection. a trivalent seasonal influenza vaccine was shown to elicit anti-h5n1 antibody and t cell responses and partially protected mice against a lethal challenge with wild-type h5n1 virus. the protective efficacy of the trivalent vaccine derived mainly from the h1n1 component. moreover, passively transferred serum of mice immunised with seasonal influenza vaccine protected naïve mice from infection with h5n1 virus, suggesting that antibodies are the main contributor to protection. h1n1 specific serum did not inhibit neuraminidase activity of h5n1 virus suggesting that protection was not mediated by neuraminidase n1-specific antibodies. next, we investigated the combination of the trivalent seasonal influenza vaccine and the h5n1 whole virus wild-type vaccine. a prime with the seasonal influenza vaccine followed by immunisation with the h5n1 vaccine enhanced anti-h5n1 antibody response, cellular immunity and protection compared to a single immunization with an equivalent sub-optimal dose of the h5n1 vaccine. hence, hetero-subtype immunity can be achieved by immunization with a trivalent seasonal influenza vaccine, which can be further boosted with a h5n1 candidate vaccine. [1] gioia c et al. aims: to register the compliance of the population to the old and new vaccines of the national vaccination program for the children up to 6 years old, and to investigate the possible causes of the potential shortages, in order to approach even more successfully the further goal of this whole attempt, which undoubtedly is the future control of important generalized infections. methods: in the study we checked the vaccination history of 335 children in the first grade of primary school in the area of central and west macedonia. there were 234 greek and 101 foreign children. as fully vaccinated were considered those who had already undergone at least one dose of hib, meningococcus and pneumococcus, two doses of hav, as well as four doses of dtp-sabin, while in the cases of a lack of vaccination, the causes were investigated and the adequate recommendations and information were given. in all the cases, except for the nationality, the sex, and the educational and social level of the parents were registered. results: the percentages of the compliance found, are presented in the following 1) it should be underlined that, as shown in the table, the percentages of the obligatory-free of charge vaccines were close to 100%. 2) high percentages were noted also for meningococcus, either because it is an old vaccine (it has been available for seven years), or because the bacteria is considered quite dangerous (it has been emphasized through the media). 3) on the contrary, as far as the hepatitis a and the pneumococcus vaccines are concerned, low percentages were found, either because of the lack of adequate information-fact that was also shown in our study-or even because of their cost. 4) finally, a statistically significant difference was found relating the response to the vaccination coverage, between greeks and foreigners, but also between the greeks themselves, in relation to their educational and socioeconomic level. objective: over the past three decades, the incidence of type 1 diabetes has dramatically increased in europe and north america, inversely correlated to the decrease of infections. according to the hygiene hypothesis, pathogens may prevent the onset of the disease. om-85, a bacterial extract of both gram positive and gram negative bacteria already used as an immunomodulatory treatment in children, has been shown to protect non obese diabetic (nod) mice from diabetes development. we aimed here at understanding the mechanism underlying this protection. methods: nod mice and nod-cd28 -/mice, which are devoid of natural regulatory t cells (tregs), were treated with om-85. cytokine secretion, activation and proliferation of b cells and foxp3+ tregs were monitored. as toll-like receptors (tlr) recognise microbial molecules and trigger innate and adaptive immunological response, cells from mice deficient for tlr2, tlr4 or the myd88 adaptor protein were used to further address the mechanisms driving the immunomodulatory activity of om-85. two synthetic tlr4 agonists used as adjuvant in human (om-174-dp and om-197-mp-ac) were also tested for their capacity to protect nod mice from diabetes. the om-85-induced protection of diabetes required natural tregs, as nod-cd28 -/mice were not protected. remarkably, om-85 activated b cells and not t cells, promoting their proliferation and il-10 secretion, two phenomena that were tlr4-and myd88-dependent. om-174-dp and om-197-mp-ac two synthetic murine tlr4 agonists effectively prevented diabetes onset in nod mice, promoted the expansion of cd4 + cd25 + foxp3 + t cells and the proliferation of il-10 secreting b cells in a dose-dependent manner. conclusion: our results argue for the involvement of tlr4 signaling in the protective effect of om-85 on development of diabetes and show that two other tlr4 agonists induce proliferation of b cells and their secretion of il-10 as well as stimulation of regulatory cd4 + cd25 + foxp3 + t cells. activation of the innate immunity by tlr-stimulation using those products already used in clinics, may prevent the onset of diabetes in those at risk of developing the disease. d. de wit 1 , a. legat 1 , s. thomas 1 , m. van mechelen 2 , p. hermand 2 , m. goldman 1 1 institute for medical immunology/université libre de bruxelles, gosselies, belgium, 2 glaxosmithkline biologicals, rixensart, belgium aminoalkyl glucosaminide 4-phosphates (agp) are lipid a mimetics which are considered as interesting candidates for the development of synthetic vaccine adjuvants targeting toll-like receptor 4 (tlr4). since natural lipid a from bacterial lipopolysaccharide (lps) depends on membrane-bound or soluble cd14 (scd14) for its tlr4 ligand activity, we investigated the involvement of both forms of cd14 in the responses elicited by crx-527, a prototypical agp. first, we found that crx-527 efficiently induces nf-kb and irf3 activation in hek cells transfected with tlr4 and md-2 genes, whereas the responses to lps required co-transfection of the gene encoding membrane-bound cd14. likewise, crx-527 efficiently induces the synthesis of nf-kb and irf-3 dependent cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which a defect in membrane-bound cd14 prevents lps responses. we then observed that monocyte-derived dendritic cells (dc) which are devoid of membrane-bound cd14 respond to crx-527 but not to lps in serum-free medium. the addition of the soluble form of cd14 did not modify the levels of il12 and tnf produced by crx-527 stimulated dc but increased the levels of interferon-b (ifn-b). when scd14 was added to hek cells expressing tlr4/md-2, nf-kb activity was not modified but irf3 activity was increased in a dose-dependent manner in response to crx-527. we will further compare the responses induced by crx-527 in wild-type and cd14 deficient mice. we previously showed that the transcriptional transactivator (tat) of human immunodeficiency virus possesses the unusual ability to raise a humoral immune response in the absence of adjuvant. these observations prompted us to examine whether such a property can be used to boost the immune response raised against poorly immunogenic peptides. as we previously observed that the autoadjuvant property is controlled by a determinant located within the core-and cysteine-rich regions of the protein, we decided to investigate whether the grafting or the co-injection of a peptide partially containing this determinant (ptat) can raise a humoral immune response against two model peptides. these two peptides, which originate from diphtheria toxin (pdt) and from toxin alpha (pt), both contain an i-ad restricted t-cell epitope but are nonetheless non-immunogenic in balb/c mice of the h-2d haplotype when injected with alum. the ptat, pdt, pt, ptatpt and ptatpdt constructs were prepared by chemical synthesis, purified by reverse phase hplc and characterized by mass-spectrometry. pdt+ptat, pt+ptat, ptatpt and ptatpdt were respectively injected twice at two weeks interval in balb/c mice and animals were bled 14 and 28 days after the second immunisation. the sera were subsequently incubated in microtiter elisa plates previously coated with pt and pdt peptides respectively in order to assess the humoral immune response. we observed a lack of antibody response for the immunizations made with the mixture of peptides (pdt+ptat and pt+ptat) but an anti-pdt and anti-pt response for the immunizations made with the two hybrid constructs (ptatpt and ptatpdt). our results indicate that a humoral immune response can be raised towards non-immunogenic peptides using a determinant involved in the autoadjuvant property of tat, that the phenomenon requires the covalent coupling to the peptide antigen and that it is therefore not related to a bystander effect. interleukin-15 gene polymorph isms (c267t, g367a, c13687a and a14035t) and susceptibility to brucellosis in iranian patients russian federation some epidemiological and observational data suggest that farm and pets exposure [1] in early childhood may be conducive to reduced atopy. currently, there is a lack of consensus regarding underlying immunological mechanisms, especially in prenatal period. as we previously reported the decreasing of intracellular ifn-g production by cbmc statistical analysis was performed using the kruskal-wallis and mann-whitney tests. results: we revealed that newborns from rural mothers (n=14) have higher amount of both nonactivated (subtype infg+/cd69-, p=0.02) and activated (subtype infg+/cd69+, p=0.028) cbmc, producing ifn-g, as compared with newborns from urban mothers (n=79) exposure to pets and the risk of allergic symptoms during the first 2 years of life intracellular interferon-g production by cord blood mononuclear cells as predictor of atopic dermatitis forming in infants: a one-year prospective birth cohort study pc09/16 to what extent t-spot.tb could be used in the diagnosis of tuberculosis in children exposed to tb infection? s. a tb) in children, especially in bcg-vaccinated is difficult for diagnosis because of the low percentage of smear positivity (12-14 %) and clinical futures only in severe forms of disease. the purpose of the present study was to evaluate the diagnostic value of t-spot.tb (oxford immunotec, oxford, uk) compared to tuberculin skin test (tst) in children exposed to tb contact in the family. forty three children with a history for bcg vaccination/revaccination, treated in the university clinic for lung diseases in children sofia, bulgaria were enrolled in the study. the patients were divided according to age in the following groups: 5 months -3 years (n=22), 4 -7 years (n=15) and 8 -12 (n=6) tb has the highest diagnostic value in children n 4 years of age in early childhood the diagnostic value of t-spot.tb and tst does not differ cfp-10 antigen is more sensitive for detection of tb-specific t cells compared to esat-6 antigen. 4. in children with tst 5-14 mm t-spot.tb has a high diagnostic value objectives: the goal of this study is to determine the role of tlr2 and tlr4 in the development of spontaneous lupus disease by creating tlr2 or tlr4 deficient c57bl/6 lpr/lpr mice. methods: tlr2 and tlr4 deficient lupus prone mice have been generated by crossing c57/bl6-tlr2 -/-or c57/bl6-tlr4 -/-mice with c57/bl6 lpr/lpr mice which develop a moderate type of lupus related to fas deficiency. we analysed the phenotype of the disease, autoantibody production and renal injury. statistical comparisons were performed using the mann-whitney u-test. results: these mice developed a less severe disease and few immunological alterations. indeed, in tlr2 or tlr4 deficient lpr mice, glomerular igg deposits and mesangial cell proliferation were dramatically decreased and anti-nuclear, anti-dsdna and anti-cardiolipin autoantibody titers were significantly reduced. however, the response against nucleosome remained unaffected, indicating a role of tlr2 or tlr4 in the production of autoantibodies directed against certain slerelated autoantigens. analysis of b cell phenotype showed a significant reduction of mz b cells, particularly in tlr4 deficient mice suggesting an important role of tlr4 in the sustained activation of these cells likely involved in autoantibody production. interestingly, the lack of tlr4 also affected the production of cytokines involved in the development of lupus disease. conclusion: our data show that deficiency in tlr4 pc14/13 expression of full length mcl-1 and its splice variant in juvenile systemic lupus erythematosus (jsle) neutrophils: differential modulation by gm-csf granulocytemacrophage colony-stimulating factor (gm-csf) can prolong neutrophil survival by increasing mcl-1, an anti-apoptotic protein. a splice variant of mcl-1 arises by removal of exon 2 and induces cell death rather than preventing it. here we investigate the expression of both the full length mcl-1 (mcl-1l) and its splice variant (mcl-1s) in jsle neutrophils compared to controls and investigate whether the addition of gm-csf changes the expression of both isoforms of mcl-1. method: neutrophils were isolated from children (diagnosed x 17 years) with jsle (n=14) and non-inflammatory conditions (control, n=14) and incubated with control serum, jsle serum alone or with jsle serum plus 20pg/ml gm-csf. quantitative real time pcr was used to assess mcl-1l and mcl-1s mrna expression (mean ± sem) following incubation in the above conditions and immediately following neutrophil isolation the ratio of mcl-1s to mcl-1l was also higher in jsle patients compared to controls (p x 0.05). the addition of gm-csf to jsle serum was associated with an increase in mcl-1l (1.66 ± 0.31) and a decrease in mcl-1s (2.56 ± 1.1) mrna expression the addition of gm-csf to jsle serum can abrogate the increased neutrophil apoptosis. alternative splicing is recognised to play a significant role in the regulation of proteins involved in cell death. our results suggest that jsle neutrophils may be more apoptotic due to differential expression of mcl-1 compared to controls, with jsle neutrophils having greater expression of the pro-apoptotic isoform mcl-1s, and less anti-apoptotic full length mcl-1 cyld is a tumor suppressor gene known to play an important role in the nf-kb pathway. to analyze the function of cyld in vivo we used the cyld ex7/8 mouse strain, which is characterized by loss of the full-length transcript and overexpression of a short splice variant of the cyld gene (scyld) to further investigate the connection between scyld overexpression in t cells and colonic inflammation, we used an adoptive transfer model of colitis. therefore naive cd4 + cyld ex7/8 t cells were transferred into rag1 -/-mice which were analysed by mini-endoscopy weekly after cell transfer. here we could demonstrate that cyld ex7/8 cd4 + t cells exhibit less capacity to induce colitis compared to control cells. consequently we investigated if regulatory t cells (t regs ) of cyld ex7/8 mice are capable to control inflammatory responses. for this purpose cd4 + cd25 + cells were co-transferred with naïve wt cd4 + t cells into rag1 -/-recipients. interestingly, rag1 -/-recipients of cyld ex7/8 t regs displayed strong features of colitis compared to control recipients showing that these cells were unable to inhibit inflammatory responses. our findings demonstrate that overexpression of scyld leads to a hyperresponsive t cell phenotype and higher production of inflammatory cytokines by t cells pc17/16 the role of hla complex in inflammatory bowel disease: crohn's disease and ulcerative colitis de investigación biomédica en red de enfermedades hepáticas y digestivas (ciberehd). university hospital virgen arrixaca the allele frequencies of hla class i in cd and uc patients were not different to those observed in controls, although we found an increased frequency of a*03 in cd vs uc. haplotype frequencies of hla class i and ii in cd and uc were also not different to those observed in controls. however, we found increased frequencies of drb1*13, *01 and *0103 alleles, and a decreased allele frequency of drb1*15 in cd vs uc patients and controls. these data are in concordance with other previous studies suggesting that, in patients with isolated colonic cd, drb1*0103 is associated with the development of severe disease and positive association of cd with drb3*0301 and drb1*13. indeed, drb1*15 was negatively associated with cd. this allele appears to confer protection against all subgroups of cd, in all ethnic groups including japanese. however, hla-drb1*07 frequency allele, associated in unselected patients with cd in other studies, was not different in our cd and uc patients, and controls. additionally, an increased frequency in hla-drb1*04 in cd was not found in our patients in a different manner to other reported studies. on the other hand, in our uc patients, allele frequencies of drb1*15 were strongly increased with respect to cd and controls. however, the frequency of drb1*04 was decreased in uc with respect to cd and controls. in this sense, our data are agreed with other reports showing that hla-drb1*15 is associated with uc in european, north american, japanese and korean populations methods: a total of 176 children were studied, (92 boys and 84 girls), up to 17 years of age, with symptoms suspicious for epstein-barr virus infection. the elisa method was used to look for specific antibodies against the capsid of the virus vcaigg and against the nuclear antigen ebv-igm, while taking into consideration the possible increase of the vcaigg title between two serum samples. results: totally, 51 positive cases of children were found (29 %) with active infection : 28 boys (14 x 5 years of age, 12 5-10 years of age and 2 g 10 years of age) and 23 girls (10 x 5 years of age, 6 5-10 years of age and 7 g 10 years of age). pharyngitis was present in 47 children (92,2%), 39 had fever (76,5%) and 48 had lymphadenitis (94 %). the lab tests revealed leukocytosis up to 20.000 leukocytes in 29 cases (56,9 %) and leukocytosis g 20.000 in 9 cases (17,6 %). the most frequent complication documented was streptococcal superinfection in 13 children (25,5 %) and thrombocytopenia in 8 children (15,7 %). a past infection (negative ebv-igm values and positive vcaigg values) was virus infection is common among children and teenagers serum negative are mainly the children of little age and 3) there is no statistically important difference between the two sexes, while on the contrary there is a seasonal distribution of the infection, with winter and summer outbreaks general hospital of rethymno, rethymno, greece shows that the il-13ra2, previously believed to be a decoy for il-13 only, is able to transmit a signal via il-13. our results support this and may suggest that il-13/ il-13ra2 signalling causes disease in oxazolone-induced colitis. currently we are dissecting the role of single cell populations expressing il-4ra to establish which cells play a role in regulating the immune response to oxazolone-induced colitis. together this data can define a role for il-13 or il-13ra2 and identify specific cell populations methods: splenic apcs exposed to enteroantigen (eag) +/-probiotics were used to stimulate cultured cd4 + cd25 -t cells to which titrated numbers of tregs were added. neutralizing antibodies against il-6 and il-1b and elisa-based cytokine analyses were used to monitor the effect of cytokines secreted in the t cell cultures. results: exposure of apcs to eag and probiotics did not influence eag-specific cd4 + cd25 -t cell proliferation. however, exposure to three of the six probiotics tested (b. bifidum bi-98, l. acidophilus ncfm tm and b. bifidum bi-504) consistently reduced regulatory activity of tregs in a cell-dose dependent manner. the tregreducing activity of probiotics was analyzed using fractionated components of the b. bifidum bi-98 strain. data indicated that bacterial cell-wall components were responsible for reducing treg activity and not components of nucleus or cytoplasm. the probiotic-induced down-regulation of treg activity was not mediated by increased intra-culture secretion of inflammatory cytokines such as il-6 or il-1b. conclusion: we conclude that certain probiotic strains can modify apcs to cause reduced treg activity in an eag-specific t cell proliferation assay. this effect apparently depends on a direct apc-to-treg cell contact and not secreted cytokines. the apc/probiotics-mediated inhibitory effect on tregs may oppose antiinflammatory activities desired from probiotic therapy palmieri 1 1 'la sapienza dysregulated innate and adaptive immune responses against commensal flora lead to crohn disease (cd) and ulcerative colitis (uc), two different forms of inflammatory bowel disease (ibd), a lifelong inflammatory condition of the gastrointestinal tract methods: we analyzed 21 pediatric cd patients (13 active, 8 remission), 24 pediatric uc patients (17 active, 7 remission), and 37 age-matched non-ibd controls. nkg2d/ligand expression was evaluated by immunostaining and multiparametric facs analysis (on pbmc subsets), and by immunohistochemistry and twocolour immunofluorescence (on intestinal biopsies). differences between groups were analyzed with non-parametric and parametric tests; a level of p x 0.05 was considered significant. results: nkg2d expression is selectively upregulated on circulating "innate-like" t cell populations (g/d and cd3+cd56+ nkt cells), in active, but not in quiescent ibd patients; receptor upregulation correlates with disease type (observed in uc, but not in cd patients). in the same patient groups, the appearance of nkg2d ligands on circulating monocytes is also observed. the dramatic increase of nkg2d+ lymphocytes, and the strong upregulation of nkg2d ligands on both epithelial and immune components, are observed in active ibd lesions. conclusions: our observations document the dysregulated expression pattern of nkg2d/ligands on selected innate immunity populations in pediatric ibd patients, both at mucosal and systemic level pc17/26 peripheral and intestinal regulatory t cell dynamics in pediatric ibd patients is a chronic inflammatory condition of the gastrointestinal tract characterized by dysregulated innate and adaptive responses against commensal flora. regulatory t cells (t reg) represent an important mechanism to suppress uncontrolled immune responses to bacterial flora. aims: to evaluate the frequency of regulatory t cells in the peripheral blood, and in inflamed and non inflamed mucosae of pediatric ibd and non mucosal regulatory t cells were identified by immunohistochemistry; circulating regulatory t cells were analysed by immunofluorescence and facs analysis. differences were analyzed with parametric and non-parametric testsconsidered significant. results: foxp3+ t reg were significantly increased in the intestinal lesions of active ibd patients (cd or uc), and returned to normal levels in post-therapy remission phase. at variance, circulating cd4+ t reg frequency was elevated in patients affected by both forms of ibd, independently of disease activity, as it persisted in the remission phase. a selective imbalance in the frequency of t and nk subsets characterized the abundant inflammatory infiltrate present in active intestinal lesions, and the normal immunological profile was only partially restored in mucosal samples of quiescent ibd patients. conclusions: regulatory t cells dynamics are differently regulated in mucosal tissues and at the systemic level, during the distinct phases of disease; t reg dynamics in pediatric ibd patients only partially matches previous data obtained in the adults; quiescent ibd is characterized by the imbalance of selected lymphocyte subsets, both in the mucosa and systemically the increased expression of immunoproteasomes in the inflamed mucosa of ibd patients was shown to contribute to this pathology by enhancing nf-kb activation. due to the relation between nf-kb and the immunoproteasome we have investigated whether specific inhibition of immunoproteasomes is suitable for therapeutic intervention in ibd. lmp7 knock-out mice are deficient in the essential catalytic immunoproteasome-subunit ß5i and therefore are devoid of immunoproteasomes. to test our hypothesis, we employed the dss colitis model. in contrast to wild-type mice, colitis was attenuated in lmp7 knock-out mice characterized by reduced weight loss and less infiltration of lymphocytes in the mucosa confirmed by histology. in addition, lmp7 knock-out mice had lower levels of proinflammatory cytokines and chemokines compared to wild-type mice validated by rt-pcr and elisa. especially nf-kb regulated genes show enhanced induction in wild-type mice unlike lmp7 knock-out mice synaptic systems gmbh, braunschweig, germany objectives: although more than 200 million people worldwide are chronically infected with hepatitis c virus (hcv) no prophylactic or therapeutic vaccines do exist to prevent or cure hcv infections. our major objective is to develop a dendritic cell (dc)-based immunotherapy enhancing virus-specific cellular immune response for treatment of hcv infections based on this approach we aim at generating adec-205 antibodies conjugated with immunodominant hcv proteins to induce hcv-specific protective immunity. methods: recombinant hcv proteins are expressed using "expression-ready-clones" containing n-terminally his-tagged hcv-core (aa 1-191) or hcv-ns3 (aa 1027-1218) sequences. protein purification is performed by metal-affinity chromatography on ni-nta-agarose hcv-specific t cell responses are monitored at different time points after immunization by facs and in vitro t cell proliferation assays. results: to obtain high amounts of recombinant ns3 and core we successfully optimized culture and protein purification conditions. briefly, ns3 was purified natively using pbs-based buffers with ph-gradient. in contrast, purification of core was performed under denaturing conditions in presence of guhcl and urea and a ph-gradient elution. moreover, optimized conditions allowing conjugation of adec-205 to recombinant hcv proteins were established with respect to duration of conjugation and buffer requirements needed to avoid protein precipitation. efficient conjugation was verified by western blot analysis. after successful generation of adec-205/ hcv-protein conjugates we are currently establishing optimized vaccination conditions to induce hcv-specific immune responses pd01/2 mva-nef vaccination induces polyfunctional cd4 t-cells and increases the proliferative capacity of cd8 t-cells in hiv-1 infected individuals under haart several vaccination trials have made use of the modified vaccinia virus ankara (mva) as delivery vector. in a therapeutic vaccination trial, we demonstrated that mva expressing the hiv-1 protein nef (mva-nef) was safe in hiv-1 infected individuals under haart and immunogenic in regard to the elicitation of ifn-g mediated cd4 t-cell responses. recent advancements in polychromatic flow-cytometry technology revealed that the sole evaluation of ifn-g provides limited information on the quality of antigen-specific t-cell responses. the evaluation of several functions is essential, as simultaneous production of multiple cytokines by t-cells is associated with superior control of viral replication. methods: in a retrospective setting, we simultaneously assessed the production of ifn-g, il-2 and mip-1b, the expression of the activation marker cd154 and the differentiation marker cd45ra in nef-specific cd4 and cd8 t-cell populations during the course of the vaccination trial. furthermore we applied a multi-colour cfse based proliferation assay investigating the proliferative capacity and the simultaneous expression of ifn-g, il-2 and mip-1b. results: following mva-nef vaccination, we observed a significant increase of the total nef-specific cd4 t-cell response and a significant increase of polyfunctional nef-specific cd4 t-cells, simultaneously expressing ifn-g, il-2 and cd154. using the standard ics no increase of nef-specific cd8 t-cell responses was observed. however, by the cfse based proliferation assay, we could show a clear expansion and a generally enhanced proliferative capacity of nef-specific cd8 t-cells following mva-nef vaccination. notebly, we observed a correlation between the increase of ifn-g, il-2 and cd154 expressing cd4 t-cells and the increase of proliferating cd8 t-cells suggesting the possibility of a causal link between the two functions. conclusions: the mva-nef vaccine is able to change the quality and quantity of the nef-specific cd4 t-cell immune response and has the potential to increase the proliferative capacity of nef-specific cd8 t-cells in hiv-1 infected subjects under haart this preferential binding to the complex was evident in classical immunochemistry assays, as well as in surface plasmon resonance (spr) tests. this ab inhibited hiv-1 mediated membrane fusion and p24-detected replication. db81 was found to nicely recapitulate the characteristics of the unconventional, protective immune response, which is taking place in naturally resistant esn individuals. further characterization of the antibody and of its binding epitope is ongoing following intradermal vaccination with 25mg dna and electroporation of balb/c mice, splenocytes have been incubated with peptides representing class i and ii epitopes, and specific t cell-responses were examined by elispot-assays. the specific antibody responses have been measured by sandwich eli-sas, and neutralizing antibodies have been investigated by hi-assays. results: the vaccibody constructs have been found to be expressed and correctly folded in vitro. the in vivo experiments further demonstrate the presence of neutralizing antibodies as well as the strong induction of antigen specific cd4 + and cd8 + t cells. conclusion: antibody and cellular immune responses against influenza hemagglutinin are enhanced when targeted to apcs. methods: the hcv recombinant proteins rns4 (1677-1756 aa) and rns5a (2061-2302 aa) were conjugated with immunomax using the heterobifunctional reagent sulfo-smcc. balb/c and dba/2j mice were immunized intraperitoneally 2 times at a month interval with different doses (0.1 -2 mg/mouse) of the proteins without adjuvants, as conjugates with immunomax, or with complete freund's adjuvant (cfa) the other combinations were not immunogenic at given doses. it should be noted that only conjugates stimulated production of antibodies that bound not only to recombinant protein but also to peptides imitating epitopes of ns4 protein. immunization with rns5a-immunomax conjugate and rns5 in cfa (1.4 mg/mouse) induced a similar antibody activity, but a different t-cell responses. the conjugate induced splenic accumulation of t cells specifically reacting in vitro with ns5a recombinant proteins of various genotypes, with peptides and with phages by cell proliferation and/or cytokine secretion. immunization with rns5a in cfa induced cells proliferating in vitro after stimulation only with peptides; none of the antigens stimulated cytokine secretion. conclusion: covalent conjugates of hcv nonstructural proteins with immunomax effectively induce humoral and cell immune responses pd01/21 degree of cross-genotype reactivity of hcv-specific cd8 t cells directed against ns3 the existence of multiple hcv genotypes characterized by marked sequence differences is a challenge for immune control. the aim of this study was to compare the antiviral cd8 t cell response targeting hcv genotype 1 (gt1) and genotype 3 (gt3) as the most predominant genotypes in germany and to determine the extent of cross-genotype reactivity of specific t cells. we analyzed a cohort of patients with past or ongoing intravenous drug use (ivdu) hypothesizing that multiple exposures to different genotypes may occur. methods: 53 subjects (17 with gt1, 22 with gt3 and 14 anti-hcv-pos/hcv-rna-neg) were analyzed. hcv-specific t cells were expanded from pbmc in the presence of peptide pools covering ns3 from gt1 or gt3. individual reactive peptides and the degree of cross-reactivity between the gt1 and gt3 variants were determined by ics. complete ns3 is sequenced from all viremic patients pd01/22 anti-retroviral effects of type i interferon subtypes in vivo ifna subtypes 1, 4, 6 or 9 suppressed fvreplication in vitro, but differed greatly in their antiviral efficacy in vivo. treatment of fv-infected mice with the ifna subtypes 1, 4 or 9, but not 6 led to a significant reduction in viral loads. decreased splenic viral load after ifna1 treatment correlated with an expansion of activated fv-specific cd8 + t cells and nk cells in the spleen, whereas in ifna4-and ifna9-treated mice it exclusively correlated with the activation of nk cells. other ifna subtypes like ifna2, 5 and 11 are under investigation pd01/23 elimination of immunodominant epitopes from multispecific dna-based vaccines allows induction of cd8 t cells that have a striking anti-viral potential immunodominance limits the tcr diversity of specific, anti-viral cd8 t cell responses elicited by vaccination or infection. to prime multispecific t cell responses, we constructed dna vaccines that coexpress chimeric, multidomain antigens (with cd8 t cell-defined epitopes of the hepatitis b virus (hbv) surface (s), core (c) and polymerase (pol) proteins, and/or the ovalbumin (ova) antigen as stress protein-capturing fusion proteins. priming of mono-or multispecific, hla-a*0201-or k b -restricted cd8 t cell responses by these dna vaccines differed. k b /ova 257-264 -and k b /s 190-197 -specific cd8 t cell responses did although chronic infections remain asymptomatic in most cases, immunocompromised patients can suffer from severe and life-threatening ebv-associated diseases, such as posttransplant lymphoproliferative disorders (ptld). thus, immunotherapeutic strategies using adoptively transferred ebv-specific t cells are promising. one option is the generation and expansion of cd4 + and cd8 + t lymphocytes by using ebv-specific synthetic peptides for the stimulation of pre-existing memory t cells. aim of our study was to identify a set of mhc class-ii peptides for each antigen promiscuitive peptides with high syfpeithi scores were tested for immunogenicity using an ifn-g-elispot. pbmcs of at least 16 healthy, randomly chosen blood donors were cultured for12 days in the presence of each candidate peptide. functional and phenotypic analysis of t cells of several donors was performed by multicolor flow cytometry. 48 out of 72 tested peptides could be identified as t-cell epitopes. two of them were defined as immunodominant, as more than 50 % of tested blood donors showed peptide-specific t cell responses. so far, eight of the tested peptides could be identified as mhc class-ii epitopes. furthermore, a highly immunodominant class ii peptide mix consisting of 5 peptides was selected. in conclusion, we could identify several new ebv-specific mhc class-ii epitopes which can be used for united kingdom, 3 hospital de clínicas during persistent hbv infections, patients usually develop poor or no protective immune responses against viral antigens, which not only leads to the chronicity but also the unresponsiveness to conventional treatments.in order to overcome the unresponsiveness and to generate an effective therapeutic strategy for treatment for chronic hbv infections a chimeric tcr against hbsag, which aims to increase the percentage and quality of antigen-specific cd8 + t cells, was developed moreover, we pre-conditioned the liver microenvironment by injection of cpg oligodeoxynucleotides (odn) to optimize the recruitment of transferred cd8 + t cells to the liver and to overcome the tolerogenic microenvironment of the liver. we found that the il-12-exposed cd8 + t cells showed at least five-fold increase of survival rate in vivo than il-2-exposed cd8 + t cells did treatment of the recipients with cpg-odn could increase the percentage and also the total amount of transferred cd8 + t cells mainly in the liver. by in vivo brdu incorporation, we demonstrated that the higher in vivo survival rate of il-12-exposed cd8 + t cells and the effect of cpg-odn were due to the up-regulation of the proliferation of those cells. to sum up, the cocktail therapeutic strategy could not only increase the survival rate of transferred cells but also direct the antigen-specific cd8 + t cells to the liver to exhibit their effector functions. the detailed mechanisms responsible for the il-12 and cpg-odn effects on the regulation hyper igm (him) and wiskott-aldrich syndrome (was) than those of corresponding controls (p x 0.01) . there was a significant elevation of t ada and ada1 activities in iga deficient patients as compared to healthy individuals (p x 0.01) . our results hypothesized that altered ada activity may be associated with altered immunity. therefore, serum ada level could be used as an indicator along with other parameters pd01/61 hiv-1 sequence evolution after dendritic cell-based immune therapy in a phase i/ii clinical trial hiv rna was extracted from plasma samples collected before the startof haart and early after vaccination when haart was terminated. rna was amplified by rt-pcr and sequenced using standard protocols. sequences of the vaccine genes tat, rev and nef as well as control genes vif, vpr, vpu and parts of env were analyzed for variation between pre-and post vaccination time points. hiv sequences spanning known and predicted epitopes of the relevant hla alleles from each participant were analyzed in detail. results and conclusion: immune therapy was well-tolerated and no severe adverse effects occurred. after haart termination, plasma viral load became detectable in all patients after 2-6 weeks. follwing the viral rebound a set point was reached, that was lower than the viral load before start of haart. using various methods we evidenced newly induced or enhanced immunity after immune therapy (see abstract b. de keersmaecker et al.). for studying sequence evolution, complete sets of both pre-haart and post-vaccination sequences were obtained in 12 out of 17 patients. with one exception, variation in sequences of vaccine and control genes of pre-haart samples compared to samples taken early after vaccination was limited. this indicates that there was no significant impact of the immune response on virus evolution at this stage. more focussed analysis on viral sequences spanning specific hla islamic republic of newcastle disease (nd) is regarded throughout the world as one of the most important diseases of poultry, not only due to the serious and high flock mortality, but also through the economic impacts. the purpose of this study was to be informed from the possible influence of infectious bronchitis virus on immune response of chickens to nd live vaccine. one hundred and twenty, 10-day-old ross 308 broiler chickens divided randomly into 3 groups, 2 experimental and a group as control one. the first experimental group vaccinated by a monovalent nd live vaccine with cl/79 strain, and the second experimental group vaccinated by a bivalent newcastle disease and infectious bronchitis live vaccine with cl/79 and h120 strains, via the drinking water at 10 days of age at the same time, and the control group received no nd vaccine. the antibody response to vaccination was assessed using the hemagglutination inhibition (hi) test by taking blood samples three times, first the day before and the next, 7&14 days post vaccination. results indicated that, although the strain of studied nd live vaccines were the same united kingdom t cell-based ifng release assays from blood are an important advance for diagnosing tuberculosis infection but do not permit reliable treatment monitoring or distinction of active tb from successfully treated disease or latent infection. t-cell cytokine profiles vary with in vivo antigen load in viral infections cd4 t cells from 25 patients with active tb and 28 patients with successfully treated tb were analysed for simultaneous expression of ifng and il2 at the single cell level using multi-colour flow-cytometry after 6 hours stimulation with ppd. moreover, cells were stimulated with esat-6 and cfp-10 receiver operator characteristics analysis revealed that a percentage of ifng /il2 dual positive cells x 56 % served as an accurate marker for active tb patients (specificity 100 %, sensitivity 65 %), while frequencies g 56 % were observed in treated as well as active tb patients. in conclusion, quantitation of antigen-specific t cells based on the analysis of ifng only does not allow distinction of patients with active and successfully treated disease pd03/7 necessity of postpone bcg vaccination -lesson from primary immunodeficiencies v. thon methods and results: the czech national database of primary immunodeficiencies (pid) was established in 1993 and is connected with the european database of primary immunodeficiencies (esid). the prevalence of pid in the czech republic (approximately 10 100 000 inhabitants) is 5.33 to 100 000. among these patients there are children diagnosed with severe combined immunodeficiency (scid) and chronic granulomatous disease (cgd) too. according to the czech national database of pid, 12 out of 14 children with later proved scid were immunised with bcg vaccine in the first days of life. nine of them developed disseminated and generalized bcg infections. five children with scid died. moreover, reactivation of bcg was also seen in healthy children after admission of combined vaccines with hepatitis b given at the age of twelve weeks. on the other hand, this was not the case in thousands of children of hbsag positive mothers who were vaccinated against hepatitis b after delivery in the first place and later immunized with bcg vaccine. systematic vigilance against tuberculosis (tb) and vaccination significantly lower the prevalence and risks of tb. in the czech republic, the prevalence of tb is currently 6.12 to 100 000 inhabitants. unfortunately, temporary interruption of bcg vaccination in three large districts in the period of 1986 to 1993 led into higher incidence of tb and appearance of new cases of aviary mycobacteriosis. these complications were not observed in vaccinated children. conclusion: we recommend a change of current practice of bcg vaccination considering new immunization schedule with hexavalent vaccine pd03/8 novel analogues of thalidomide inhibit cd80 expression and production of tnf-a, il-12, ifn-g, cxcl-9 this work describes the synthesis and characterization novel thalidomide analogues, prepared in good yields using simple methodology. our results suggest that anti-inflammatory and immunomodulatory activity of these diamine compounds is potentially applicable in treating enl and other diseases. supported by: cnpq, fapemig and capes, brazil. of the b cell follicle. cta1-dd augmented gc-formations, specific antibody responses and cell-mediated immunity to the t cell-dependent antigen np-cgg, but failed to do so when used together with t cell independent antigens, such as np-ficoll or np-dextran. this effect required adp-ribosyltransferase activity, as mutant cta1r7k-dd failed to exert an adjuvant effect. the adjuvant function appeared to correlate with the fdc-localization and turned out to require complement and/or complement receptors (cr) chitosan formulations varying in molecular weight, counterion and structure (i. e. soluble v/s particulate) were used in assays to examine expression of maturation markers via flow cytometry and cytokine production by elisa. we found that, in contrast to alum, plg and ps particles, chitosan induced bmdc maturation on its own, as determined by the expression of cd80 and cd86. these effects were most prevalent with soluble chitosan chloride formulations but were also notable with soluble chitosan glutamate chitosan. the effect of chitosan on cytokine production was investigated using a panel of different tlr agonists in combination with chitosan particles. results show an increase in the secretion levels of il-1a and il-1b, while il-6 levels were not affected. finally we studied the role of inflammasome activation in the enhancement of il-1b production. using bmdc from nlrp3 -/-mice we examined il-1b production in response to different tlr and chitosan combinations. results show that the ability of chitosan to enhance il-1b production is dependent on nlrp3. collectively our data indicate that upregulation of maturation markers and enhancement in proinflammatory cytokine secretion mediated by chitosan severe sepsis, induced in mice by cecal ligation and puncture (clp), led to ho-1 expression in infiltrating peritoneal leukocytes, kidney and liver. mortality rate of clp increased from 20 % in wild type (hmox1 +/+ ) mice to 87 % in ho 1 deficient (hmox1 -/-) mice. hmox1 -/-but not hmox1 +/+ mice developed end-stage multiorgan failure. mortality of hmox1 -/-mice was associated with increased peritoneal leukocyte infiltration, but not with increased pro-inflammatory cytokine secretion or bacterial load in peritoneum, blood or organs. clp induced a significant increase in cell-free hemoglobin free heme was found to sensitize primary hepatocytes to tnf, anti-fas antibody, h 2 o 2 or peroxynitrite mediated apoptosis. this cell death was associated with outward nuclear translocation and extra-cellular accumulation of the late-stage pro-inflammatory cytokine hmgb1. similarly, circulating and cytoplasmic hmgb1 was increased in hmox1 -/-relative to hmox1 +/+ mice following clp. in conclusion, these data suggest that free hemoglobin and heme, released during severe sepsis, are important factors in the organ failure and death associated with severe and b-1,2-linked mannose residues elicit inhibition effect. it was found that inhibition activity of oligosaccharides increases with chain length. immunization with mannan-hsa conjugate allowed for the maturation of immune response generating specific antibodies with high avidity/affinity, whereas immunization with mannan alone elicited only low-affinity antibodies. in the future, an effective antifungal subcellular vaccine would be constructed using selected mannooligosaccharidic epitope and the appropriate carrier protein as inductor of immunological memory. acknowledgements: this work was supported by the grant agency of slovak academy of sciences all subjects received dtwp vaccine at 4-6 years of age (booster vaccination), following the national vaccination schedule of iran. blood samples were collected before and 2-4 weeks after the vaccination. immunogenicity of the vaccines was assessed by elisa using commercial kits. results: the geometric mean titers (gmt) of the antibodies induced against diphtheria and tetanus by dtwp-local were 7.7 and 9.4 iu/ml and those of dtwp-pasteur were 8.2 and 8.6 iu/ml, respectively. there was no significant difference between the immunogenicity of the two vaccines against diphtheria and tetanus. the gmts of antibodies produced against pertussis were 30.2 eu/ml for dtwp-local and 47.9 eu/ml for dtwp-pasteur vaccines, respectively (p x 0.001). no significant differences were observed in the antibody titers against diphtheria, tetanus and pertussis between the two vaccines before vaccination. conclusion: immunogenicity against diphtheria and tetanus was similar for the two vaccines pd05/18 united kingdom haemorrhagic septicaemia (hs) is an acute disease of cattle and buffaloes in tropical countries, caused by pasteurella multocida serotype b:2 , a gram-negative coccobacillus. jrmt12, an aroa mutant of pasteurella multocida, constructed previously in our laboratory, attenuated for virulence in the mouse and protects mice from challenge with the virulent strain. in this work, the immune response of calves was tested after intramuscular vaccination with single dose of 10 8 cfu of jrmt12. a possible contributory role of cellular immunity against hs was investigated in vaccinated and in control calves after challenge. a lymphocyte stimulation assay was used to assess the effects of a cell-free extract (cfe) of p. multocida on peripheral blood mononuclear cells (pbmcs) isolated from calves at different times after challenge. the results were indicative of a possible immunosuppressive effect of challenge with p. multocida b:2 on calf pbmcs. the suppressive effect was further investigated by in vitro experiments. calf pbmcs obtained from normal calves were treated with cfe for 1 h before adding concanavalin-a (cona) pd06 -vaccination and immunotherapy against parasitic diseases pd06/1 evaluation of simian adenoviral vector adch63 expressing msp-1 as a candidate blood-stage malaria vaccine this successful regime incorporated a human adenovirus serotype 5 (adhu5) prime, boosted eight weeks later with a modified vaccinia virus ankara (mva) vector. adenoviral vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. recent clinical trials have suggested that the simian adenoviral vector adch63 is a promising clinical candidate. we have developed vectors (of human and simian origin) and mva encoding a novel construct based on p. falciparum msp-1 and have undertaken comparative immunogenicity studies in mice. the antigen, termed 'pfm128 while asymptomatic per se, the heterozygous sickle cell trait confers a survival advantage against malaria, the disease caused by plasmointo carbon monoxide (co), iron and biliverdin. when infected by plasmodium hb sad mice are protected against experimental cerebral malaria (ecm), a lethal neuroinflammatory syndrome that in many aspects recapitulates human cerebral malaria. ho-1 expression and activity are strictly required to suppress ecm in hb sad mice, as demonstrated by functional deletion of the hmox1 locus or pharmacologic inhibition of its enzymatic activity. the protective effect of ho-1 is mediated by co, which inhibits the accumulation of protein-free heme in plasma following plasmodium infection conclusion: topical treatment of cutaneous leishmaniasis with gsno accelerated healing and reduced local parasitism in the mouse suggesting that it may be ben gp63 expression was confirmed by sds-page and elisa using monoclonal antibody against gp63. discussion: today researchers attempt to find a suitable vaccine for leishmaniasis. although some researchers have reported proper vaccines of interest, a5-180recp is recognized only by sera collected from resistant bovines infested with all stages of r. microplus, but not by sera from similarly infested, susceptible hosts. furthermore, this recognition was specific since sera from resistant non-infested bovines (naï ve animals) did not react with a5-180recp. our results show that reverse immunogenomics can be useful for discovery of new antigens for development of an anti-tick vaccine. supported by cnpq and fapesp. for the maintenance of ab-mediated vaccine-induced protection after re-challenge with the pathogen or the vaccine antigen. memory b-cell elispot together with ab titres might therefore prove useful as independent marker for duration of protection. objective: this study focused on establishing experimental conditions and optimizing the performance of the memory b-cell elispot assay by detection of specific memory b-cells against anti-tetanus vaccine and naturally acquired toxoplasma gondii infection as a model. methodology: twelve healthy subjects who had received the tetanus vaccine at least 6 month previously were enrolled. peripheral blood mononuclear cells (pbmcs) were isolated from each donor using cell preparation tubes (cpt). plasma was obtained after centrifugation of cpt and stored at -20°c until used for elisa. specific igg-secreting b-cells were determined by elispot assay, using tetanus toxoid (tt) and t. gondii surface antigen as model antigens. results: to optimize our assay, conditions were changed and compared to the previously established protocol. we detected low frequencies of total igg memory b-cells and tt-specific memory b-cells in all donors four seropositive and 2 seronegative donors had positive responses in elispot. no correlations were found with serum antibody titers and frequencies of memory b cells (r=0.216, p=0.641) or with t. gondii-specific b-cells conclusions: following optimization of several assay parameters, we demonstrated that the memory b cell elispot could be reliably used to determine low numbers of antigen-specific memory b-cells in individuals naturally exposed to infection or following vaccination our previous work demonstrated that il-17 also affects the cells of erythroid lineage, by stimulating development of early erythroid progenitors, bfu-e, but inhibiting the growth of late stage erythroid progenitors, cfu-e, from normal murine bone marrow. we also provided in vitro evidence that at least part of its effect on cfu-e is mediated by nitric oxide (no) generation. in the present study we demonstrated that the in vivo reducing effect of il-17 on bone marrow cfu-e was prevented by co-treatment with the no synthase (nos) inhibitor, l-name, implying that this effect is mediated through nos activation. the data obtained in cultured bone marrow cells showed the ability of il-17 to upregulate the expression of mrna for both the inducible (i)nos and the constitutive, endothelial (e)nos isoform. both the nos-inducing effect of il-17 and il-17-related inhibition of cfu-e growth were dependent on p38 mapk activity, since the p38 mapk inhibitor, sb203580, markedly downregulated il-17-induced activation of nos and reversed the growth inhibitory effects of il-17 on cfu-e. the in vivo stimulating effect of il-17 on bfu-e colony growth in the bone marrow was not affected by co-treatment with the nos inhibitor, pointing to different mechanisms for il-17 effects on bfu-e and cfu-e. however, the in vivo exposure of the mice to l-name, increased the number of various hematopoietic progenitor cells in the bone marrow, indicating that no itself is important regulator of hematopoietic progenitor cell activity. overall, the data presented gave an insight into the mechanisms by which il-17 acts on bone marrow cells and also revealed a link between the il-17, no and hematopoiesis. further studies on il-17-mediated induction of both inos and enos methods: a total of 1785 blood donor samples were tested for hbsag and anti-hbc with the immunoenzymic method elisa, while simultaneously, molecular blood test (nat) was applied. the positive samples for anti-hbc were also tested for anti-hbs and anti-hbc igm. results: a total of 68 samples (3,8 %) were found anti-hbc positive conclusions: it is proven, therefore, that in some cases the levels of hbsag, following an infection from the hepatitis b virus, are probable to remain low, so that it is not possible to detect them using elisa method. in these cases anti-hbc can be the only serological marker of the infection. consequently, patients with positive anti-hbc and levels of anti-hbs x 100 iu/l are possibly not immune enough, so that they can become blood donors. that was the reason why some blood donation centers in our country, until recent years when there was no capability for nat testing of blood donors, had 100 iu/l as a limit for anti-hbs levels. however, in present days that nat testing of blood donors is used in our country, it has offered great safety and it is possible that anti-hbc testing will not be necessary, despite the fact that many blood donor centers have preserved the safety limit of 10 iu/l anti-hbs in all the blood units, which also goes for our study pd14/20 neonatal allo immune thrombocytopenia and igg glycosylation patterns michaelsen 1,2 1 national institute of public health in milder cases it can cause petechia and in more severe cases it can cause intracranial hemorrhage and death. the reason behind the variation in clinical symptoms is not fully understood, but is probably not due to differences in immunoglobulin isotypes or antibody affinity. recently influence of glycosylation patterns of igg on the biological activity has been realized. variation in carbohydrate structures attached to asparagine 297 can cause differences in the interaction with fc-receptors, and hence a difference in thrombocyte elimination capacity of the igg molecule. patient sera from norway and the netherlands with different levels of antibody titres and severity of symptoms have been used to affinity isolate igg antibodies against the hpa-1a alloantigen and analyze the glycopeptides using mass spectrometry. the glycosylation patterns have been analyzed for a possible link between severity of symptoms and variation in the glycosylation patterns. so far patients with serious symptoms seem to have increased galactosylation and sialylation and a high level of non core-fucosylated n-glycans on their anti-hpa-1a iggs we monitored 12 children (9 boys and 3 girls) in ages from 3.2 to 17.2 years with average age of 8.9 years. in 11 of them all was diagnosed for the first time. 1 subject had the second relapse of all. one patient received maintenance chemotherapy, all the rest (11 subjects) induction chemotherapy. methods: leukocyte count and hemiluminescent analysis of whole blood were performed for all the patients during infectious complications twice: on neutropenia background and after the recovery of neutrophil number. hemiluminescent analysis for whole blood allows to estimate the functional activity of phagocytes, namely their bactericidal power and phagocytosis completeness. we valuated spontaneous and zymosan induced hemiluminescence. we used onsonised zymosan as the inductor of "respiratory paroxysm mice with a homozygous mutation in the rc3h1 gene, that encodes the zinc finger and ring finger containing protein roquin, develop severe autoimmune disease. the observed lupus-like phenotype involves follicular helper t cells, which express higher levels of icos. these cells provide inappropriate t cell help to b cells, leading to the production of autoantibodies (vinuesa et al. 2005, nature 435, 452-8). it has been shown that the half-life of icos mrna is shortened when roquin is over-expressed. such repression requires the 3'utr of icos, in which a 47bp sequence, containing a possible mir-101 binding site, was sufficient (yu et al. 2007, nature, 450, 299-303). mnab is the paralogue of roquin, and has been shown to bind to nucleic acids (siess et al. 2000, j biol chem 275, 33655-62). we demonstrate that in primary mouse t cells and embryonic fibroblasts roquin, but not mnab, inhibits translation of icos. we map critical domains in the roquin protein to icos repression using deletion-and point-mutants of roquin, as well as chimaeras that swap sequences from roquin to mnab and vice versa. addressing the mechanism of roquin mediated icos repression; we demonstrate binding of roquin to icos mrna in primary mouse t cells and in cotransfection experiments. our current work dissects the requirement of cellular rnai, the stress response pathway or p-body function by testing roquin repression of icos mrna in dicer-, tia-1-and ago2-deficient mef cells and in knockdown approaches. acknowledments: j m m-v is a recipient of a harvard real colegio complutense (rcch) grant. work in dr tsokos' lab is supported by grant phs nih r01 ai 42269. we have recently reported that 6-hydroxyl-1-methylindole-3-acetonitrile (6-hma) isolated from brassica rappa inhibit nuclear factor-kappa b (nf-xb) activity in raw 264.7 macrophages. in this report, we investigated the effect of 6-hma on dextran sulfate sodium (dss)-induced colitis model in mice. methods: we induced colitis with dss in mice and evaluated disease activity index (dai), including body weight, stool consistency and gross bleeding, and tissue myeloperoxidase (mpo) accumulation. through h&e staining, histological change was observed. the expression of inducible nitric oxide synthase (inos), inhibitory kappa b-a (ixba) and nf-xb were detected by western blot and immunohistochemical staining. in-vitro system, the expressions of interleukin-8 (il-8), monocyte chemotactic protein-1 (mcp-1) in ht-29 human colon epithelial cells were measured by rt-pcr. results: in dss colitis model, the dai score and detection of mpo accumulation brevealed 6-hma significantly inhibited loss of body weight, suppression of diarrhea and bleeding, and infiltration of macrophages, leukocytes. moreover, h&e staining also indicated 6-hma suppressed the thickness of muscle layer, edema, mucosal damages by dss. these results were related to the regulation of nf-xb activation. 6-hma attenuated the dss-induced phosphorylation and translocation of nf-xb subunit p65. in addition, this effect was accompanied with parallel blocking degradation of ixba. moreover, pretreatment of 6-hma significantly reduced the mrna levels of il-8 and mcp-1 stimulated by tumor necrosis factor-a (tnf-a) in the ht-29 cells. pretreatment of 6-hma also significantly blocked the ixba degradation and nf-xb p65 nuclear translocation stimulated by tnf-a in the ht-29 cells. these results were concurred with the effect on nf-xb activation in dssinduced colitis model. conclusions: these results for the first time demonstrated that alleviation of 6-hma mediated by regulation of nf-xb activation and suppression of chemokines in vitro and in vivo. therefore, 6-hma could be new potential therapeutic agent for inflammatory bowel disease.cd4 serves as receptor of hiv and is a self-antigen. we have previously characterized the anti-cd4 igg immune response in hiv-1-exposed, seronegative (esn) subjects and we know that there is a peculiar specificity of these antibodies for epitopes induced by gp120-binding and that there is an epitope specificity distinct from that seen in hiv-infected patients (second cd4 domain preferred). to generate antibodies able to inhibit the infection of hiv virus trying to learn from what happen in nature in esn we used a particular immunization procedure. we immunized mice with autologous cells expressing gp120, reacted with the 2 external domains of soluble human cd4, in the absence of the target cells expressing the co-receptor ccr5. the latter is the membrane molecule, which allows the complete reshuffling of the epitopic make-up of the cd4-gp120 complex and trigger the membrane fusion between effector (gp120 expressing) cells and target (ccr5 expressing) cells. thus, in the absence of ccr5 we specifically enriched our immunogens with "frozen" conformational intermediates, that are presumably transiently exposed on the cell membrane during hiv-1 infections. a conventional protocol for the generation of monoclonal antibodies was used. db-81 (igg1, x), one of the anti-cd4 antibodies obtained, recognized preferentially cd4 complexed to gp120, as compared to cd4 alone, not competed for the gp120 binding site on cd4 and was specific for the second extracellular domain of cd4. g. röder 1 , l. geironson 2 , a. darabi 3 , m. harndahl 1 , c. schafer-nielsen 4 , k. skjödt 5 , s. buus 1 , k. paulsson 2 1 copenhagen university, institute of international health, immunology and microbiology, department of experimental immunology, copenhagen, denmark, 2 lund university, immunology bmc d14, lund, sweden, 3 lund university, rausing laboratory, division of neurosurgery, department of clinical sciences, lund, sweden, 4 schafer-n, copenhagen, denmark, 5 department of immunology & microbiology, university of southern denmark, odense, denmarkcytotoxic t-lymphocytes become activated by binding to mhc-i molecules presenting antigenic peptides. the loading of peptides onto mhc-i takes place in the er and involves different chaperones and enzymes. tapasin binds mhc-i molecules, integrates them into peptide-loading complexes, and assures that only 'optimal peptides' are bound to surface exported mhc-i molecules. how tapasin exerts this quality control, and the criteria for being an optimal peptide, are still unknown. here, we have generated the first 87 n-terminal amino acids of human tapasin, tpn , and shown that this fragment of tapasin facilitates peptide dependent folding of hla-a*0201. to further investigate the properties of tpn and tapasin, we generated multiple mouse monoclonal antibodies towards tpn and wildtype human tapasin. one clone, atpn 1-87 /80 , was found to be specific for natural human tapasin and stained cellular er localized tapasin. using peptide chip technology, the epitope of atpn /80 was demonstrated to be located on tapasin [40] [41] [42] [43] [44] , which recently was shown to be a surface-exposed loop of the tapasin structure. together, these results demonstrate that, the first 87 n-terminal amino acids of tapasin are able to facilitate peptide-binding to mhc-i, and as well, this fragment can be recombinantly expressed in e.coli and fold into a structure, which at least partially, resembles that of wild-type human tapasin. we speculate that this region of tapasin might support empty, open and receptive mhc-i peptide-binding clefts effectively allowing an otherwise inherently unstable molecule to exchange peptide; i. e. this tapasin region might be essential for enabling peptide editing. a objectives: antigen processing and presentation through hla class i molecules is critical for an effective destruction of infected or transformed cells by cd8+ t lymphocytes. different intracellular routes governing the processing of endogenous and exogenous antigens have been described. we show here a strategy to introduce epitopes inside the cells for a productive cross-presentation to ctls. methods: to produce genetic in-frame tat fusion proteins, dna sequence encoding for the amino acid region 301-498 of the influenza a virus nucleoprotein (np) was inserted into the expression vector ptat-ha. starting from tatnpflu recombinant protein we produce hybrid proteins, in which the hla-b*2705-restricted np-flu epitope (aa 383-391) was replaced by hla-b27 or hla-a2-restricted epitopes of ebv and hcv, respectively. cross-presentation was evaluated according to the standard 51 cr release assay and through the ifn-g production. results: using hla-b27 or hla-a2 restricted viral epitopes we show that the two molecules cross-present the epitopes following two different pathways of processing: the hla-b27 molecules follow a proteasome-independent pathway which is active in different cell types, whereas the hla-a2 molecules present the epitopes in a classical proteasome-dependent pathway performed by dcs. furthermore, different hla-a2 restricted epitopes can be inserted in tandem and presented to the specific ctls without interfering each other. the data reported here offer new insights on how a same construct containing multiple epitopes from different viral or oncogenic proteins could be designed for vaccinal strategies. these findings also enlighten hla-b27 as a remarkable hla-class i molecule that, differently from hla-a2, can present peptides through additional, unconventional antigen presenting routes. this could concur to an imbalance of the immunological properties of the hla-b27 molecules leading to a more effective response towards viral as well as self -antigens. objectives: although cytotoxic t cells (ctl) in human immunodeficiency virus (hiv-1)-infected individuals can potentially target multiple virus epitopes, the same few are repeatedly recognized. ctl play a key role in limiting viral replication in infections caused by e. g. epstein-barr virus, cytomegalovirus, hepatitis c virus and hiv1. consistent patterns of immunodominant and subdominant ctl-responses have been found between individuals with the same hla-alleles in both acute and chronic infection. as the ctl-response frequency in a population closely correlates with its relative magnitude in an infected individual, the terms immunodominance/subdominance have been used in both contexts. however, the factors determining these ctl-response hierarchies are largely unknown. while structural differences between peptide-hla class i complexes may be important for tcr-repertoire selection and clonal expansion, it is less obvious how they impact ctl-response hierarchy formation and timing. other factors may also contribute, e. g. epitope abundance at the cell surface. methods: antigen processing efficiency of ctl epitopes from the p17-gag and p24 region was determined in vitro. 25mer peptides were digested with i20s and c20 proteasomes and the fragments identified by mass spectrometry. for epitope precursor peptides generated by the proteasome, we then determined tap affinity, trimming by eraap and hla-binding affinities and analyzed patient responses by elispot. results: we show that ctl-immunodominance in regions of hiv-1 p17-and p24-gag correlates with epitope abundance, which is influenced strongly by proteasomal digestion profiles, transporter-associated-with-antigen (tap) affinity and endoplasmatic reticulum aminopeptidase (eraap)-mediated trimming, and moderately by hla affinity. proteasomal cleavage-preferences were affected by flanking and intra-epitope ctl-escape mutations and could modulate the number and length of peptide-epitopes, thereby affecting t cell response avidity and clonality. conclusion: our analyses reveal that antigen processing plays a pivotal role in determining ctl-response hierarchies, that viral evolution may modify cleavage patterns, and suggest strategies for in vitro optimization of ctl-epitope-based vaccines. t. f. gregers 1 , g. koster 1 , o. landsverk 1 , f. skjeldal 1 , o. bakke 1 1 university of oslo, molecular biosciences, oslo, norway mhc ii is synthesized and assembles in the er together with invariant chain (ii). ii facilitates mhc ii assembly followed by transport to the mhc ii loading compartment (miic) where peptide loading occurs. miic is multivesicular late endosomal compartments resembling conventional multivesicular bodies (mvbs) found in all cells. it is not known whether the biogenesis of miics is regulated by the same mechanisms as formation of mvbs. expression of ii induces the formation of enlarged endosomes and we have previously shown that ii modulates antigen processing and presentation. we have suggested that ii itself can act as a tethering factor involved in fusion of ii containing endosomes, and our main question is whether ii can regulate the formation of an endosomal pathway dedicated for antigen processing and mhc ii loading.in order to investigate this we use cell lines expressing ii controlled by an inducible promoter, thus being able to control the ii expression level and thereby the endosomal size. live imaging and high through put microscopy of ii expressing cells treated with inhibitors and/or specific sirnas have revealed that ii induced endosomal fusion is independent on type iii pi3 kinases and thus ptdins(3)p. this is in contrast to conventional endosomal fusion and mvb formation. thus other factors might be important for miic biogenesis. by using small rnai libraries targeting proteins known to be involved in endosomal pathways and microscope based screening we aim to identify factors that are able to knock out the formation of enlarged endosomes in ii expressing cells, and thus potentially identify molecules defining an antigen presenting cell. m. bouvier 1 , l. visvabharathy 1 , j. fu 1 1 university of illinois at chicago, microbiology and immunology, chicago, united statesobjectives: adenoviruses (ads) cause persistent infections. the e3-19k protein from ad targets class i mhc molecules for retention in the endoplasmic reticulum (er), thereby preventing the cell-surface presentation of viral peptides. this escape from immune surveillance allows ads to freely replicate in host cells. the molecular mechanism of e3-19k-mediated class i retention is mostly undefined. it is clear that further characterization of this mechanism is important to understand the susceptibility of the class i antigen presentation pathway to immunomodulatory proteins and to elucidate the molecular basis of ad pathogenicity. we used biophysical and cell-based approaches to examine interaction between ad type 2 e3-19k and class i molecules.results: we showed that e3-19k associates with immature (peptide-deficient) and mature (peptide-filled) hla-a11 molecules, with the mature form being more tightly associated. we also provided evidence that e3-19k does not compete with the class i assembly proteins for binding onto class i molecules. importantly, immature class i molecules sequestered by e3-19k can still bind peptides. together, these results suggest that ads have evolved to interfere with the early and late stages of the class i antigen presentation pathway. evidence was also provided that e3-19k displays an allele-and locus-specificity towards class i molecules with high-density lipoprotein (hdl) reduces the risk for atherosclerotic cardiovascular disease by promotion of cholesterol efflux from macrophage foam cells and by antioxidative as well as anti-inflammatory properties. recent data indicate that qualitative changes of hdl including oxidative modifications and alterations of the protein cargo of hdl may alter its biological activity. here we analyzed the anti-inflammatory potential of hdl and compared it with hdl obtained from patients with end-stage renal disease (esrd), which are characterized by a proinflammatory state and an associated significantly increased cardiovascular mortality. we demonstrate that freshly isolated, but not oxidized hdl from healthy individuals exerts profound anti-inflammatory properties on professional antigenpresenting cells (apc) such as monocytes and dendritic cells, which are regarded as the most potent apc. production of typical proinflammatory cytokines (il-12, il-6, tnf-a) were significantly suppressed by hdl after stimulation of monocytes or dendritic cells with toll-receptor ligands 2 and 4, but also with the t-celldependent stimulus cd40l (cd154) indicating an immunomodulatory effect independent of agonist neutralization by hdl. moreover, surface expression of crucial activation and costimulatory molecules like cd40, cd83, and cd86 was inhibited by freshly isolated, but not oxidized hdl. the negative regulatory effect of hdl on cytokines and surface receptors occurred at the transcription level, while hdl did not modulate the activity of the major inflammatory transcription factor nf-kb or the map kinases p38 and erk-1/2. strikingly, hdl from esrd patients not only failed to block, but rather promoted proinflammatory cytokine production and apc activation. these data identify hdl as a novel potent anti-inflammatory regulator of professional apc, which may help to dampen excessive inflammatory responses of the innate immune system. conversely, qualitative changes of hdl leading to a loss of its anti-inflammatory function might contribute to a proinflammatory state that is linked with excessive cardiovascular mortality in esrd patients. objectives: cd4+ t cell abnormalities may play a role in the autoimmune pathogenesis of churg strauss syndrome (css). on one side, th2 (il-4+) cells may sustain autoantibody formation and eosinophilia, which are hallmarks of css. on the other, th1 (ifn-g+) cells could participate in vessel wall damage and granuloma formation. in order to define this th1 / th2 balance and to identify potential t cell target antigens (ags), we analyzed circulating cd4+ t cell responses to polyclonal stimuli and to myeloperoxidase (mpo) in css and healthy subjects. methods: ifn-g and il-4 expression in peripheral blood cd3+cd4+ lymphocytes were measured in 9 ccs patients and 7 healthy subjects (hs) upon polyclonal stimulation, both by intracellular staining and by elisa. mpo-driven il-4/ifn-g production was assessed by elispot on t cells co-coltured with autologous dendritic cells, stimulated either with heat-inactivated mpo, negative control protein or hexavalent vaccine (positive control recall ags). results: upon polyclonal stimulation, higher il-4 and lower ifn-g intracellular expression were detected in cd4+ t cells from css patients, as compared to hs (il-4: 1.3±0.3 % vs. 0.50±0.21 %, p x 0.05; ifn-g: 14.2±4.5 % vs. 27.0±4.8 %, p x 0.025). similar results were obtained by elisa (il-4: 0.39±0.16 vs. 0.30±0.07 pg/ml, p x 0.05; ifn-g: 31.0±14.3 vs. 79.0±15.0 pg/ml, p x 0.05). elispot counts of hexavalent vaccine-stimulated cd4+ cells were positive for il-4 in 4/5 (80 %) css patients and in 5/5 (100 %) hs, and for ifn-g in 2/9 (22 %) css patients and 7/7 (100 %) hs. mpo stimulation determined significant ifn-g release in 5/8 (62 %) css patients, but not in hs (0/5) no il-4 response to mpo in both groups was observed. conclusion: polyclonally or recall ag-stimulated cd4+ cells from css patients show a th2-polarized cytokine profile. mpo is here first identified as a css-related ag targeted by cd4+ t cells, and responses towards it are instead th1-polarized. these data unfold one molecular target and possible pathogenic mechanisms of cd4+ t cells in css. a. voigt 1 , e. opitz 1 , k. savvatis 2 , k. klingel 3 , k. stangl 1 , u. kuckelkorn 1 , p.-m. kloetzel 1 1 charité -universitätsmedizin berlin campus mitte, berlin, germany, 2 charite -campus benjamin franklin, berlin, germany, 3 universität tübingen, tübingen, germanymurine models of coxsackievirus b3 (cvb3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection. immunoproteasomes (ip) are crucial in the modulation of adaptive immune responses, in the maintenance of protein homeostasis and in the preservation of cell viability under stress conditions. our previous work has established that ip expression in the infected myocardium is linked to a strong enhancement of viral epitope generation.here, we investigated the impact of ip function in enterovirus myocarditis. mice, which are deficient in immunosubunit lmp2 of the stress-induced ip, were infected with 1x10e5 pfu cvb3 nancy strain. in concurrence to wt littermates, we observed a pronounced up-regulation of cardiac ip subunit lmp7 as early as day 4 p. i. in lmp2-deficient mice. however, lmp2-deficiency was linked to less severe myocarditis at day 8 p. i. (he stain of cardiac tissue sections: wt 1.95 ± 0.20 vs. lmp2-deficiency 0.71 ± 0.06 (grade of myocarditis; scale 0-4; p x 0.001). whereas the cardiac output (co) was reduced in wt littermates in enterovirusmyocarditis (p x 0.05), there was no difference in lmp2-deficient mice in comparison to sham-treated mice. maximal left ventricular pressure and dpdt max were impaired in acute myocarditis in wt littermates. in contrast, systolic function was not affected by cvb3 infection in lmp2-deficient mice. likewise, diastolic function was preserved in lmp2-deficient mice upon enterovirus infection. our findings of less severe myocarditis in lmp2-deficient mice were associated with tremendously reduced viral load in the myocardium of this strain.in conclusion, this study suggests an impact of lmp2-immunosubunit function in regulatory processes of viral replication. absence of lmp2 confers host protection in enterovirus myocarditis. h. w. liao 1 , j. xu 1 , j.q. huang 1 1 sun yat-sen university, guangzhou, chinathe characteristic of the dengue hemorrhagic fever/dengue shock syndrome (dhf/dss) is hematologic abnormality, which results from multiple factors including thrombocytopenia, coagulopathy and vasculopathy. the pathogenesis of endothelial dysfunction associted with vascular leakage syndrome however remains unknown. in this work, we showed that dengue virus serotype 2 (den-2) strain induced apoptosis in human umbilical vein endothelial cells (huvecs). additionally, fas expression was increased on infected huvecs. trailr1 and tnfr1-2 were constantly very low whereas trailr2-4 decreased after den-2 infection. fasl was expressed at similar levels on huvecs throughout den-2 infection. the apoptotic rates in huvecs were decreased upon addition of caspase family inhibitors and activated caspase 8, caspase 3 were also observed by western blot after by den-2 infection. there were no significant changes of no in our study. we thus proposed that the fas/fasl pathway might be involved in apoptosis induced by dengue virus in vascular endothelial cells in vitro. dermatolymphangioadenitis is a common complication of interruption of afferent lymphatics by cancer surgery combined with partial lymphadenectomy. it seems that skin microbes normally penetrating epidermis during hand work or walking are retained in the skin and subcutis because of lack of lymph drainage and evoke host reaction. aim. to study lymph node cellular reaction to bacterial antigens before and after ligation of afferent lymphatics. materials & methods. group i. s. epidermidis was injected daily for 7 days into wis rat paw web tissue in saline containing 7.5x10 7 cells. group ii. s.epidermis was injected as in group 1 after ligation of lymphatics below the popliteal lymph node. nodes were isolated on day 8.they were weighed, the cell number was counted and cells were stained with mabs for immunohistochemical analysis. immunohistochemical pictures were analyzed by microimage program. results. group i. skin contained some mhcii cells. the popliteal lymph nodes became enlarged on the bacteria injected side. there was an increase in lymph node weight and cell concentration per g of tissue, compared to controls by factors 2,23 and 3,91 respectively (p x 0.05). immunohistochemical pictures showed increase in percentage of ox62 (migrating dendritic cell), mhc ii, his48 (granulocytes), ox7 (stem cells) and cd54 (icam i) subsets in the subcapsular , follicle, paracortex and medullary areas. group ii. after ligation of afferent lymphatics the weight of nodes was not significantly increased. skin showed presence of multiple mhc ii, ed1 (macrophages) and ox62 cells. popliteal lymph nodes contained evidently less of ox62, his48 and mhcii cells than in group i (p x 0.05). summary & conclusions. afferent lymphatics transport microbial cells and/or microbes phagocytized by dendritic cells and macrophages to the regional node. local skin reaction is limited, whereas lymph nodes reveal acute reaction with mobilization of granulocytes from blood perfusing nodes. interruption of lymphatics saves nodes but skin reaction is strong and long-lasting. these observations seem to explain why damage to lymphatics during mastectomy or groin dissection is followed by recurrent attacks of skin inflammation. omega-3 fatty acids, and in particular docosapentaenoic acid (dha) and eicosapentaenoic acid (epa) from fish origin, have recently emerged as nutrients capable of modulating the expression of genes involved in inflammation and atherosclerosis and thus reduce the risk for cardiovascular events. our presentation focuses on the role of omega-3 fatty acids in the prevention and treatment of cardiovascular disease. it is based on reviewing and processing data obtained by search of scientific and medical databases. search terms used were: atheroma, atherosclerosis, cardiovascular disease (cad) ,coronary disease, antiinflammatory drugs, omega-3 fatty acids, epa, dha. we also searched epidemiological research web sites and screened the results of numerous controlled clinical trials which monitored the effects of omega -3 fatty acids consumption. the results indicate that omega-3 fatty acids supplementation is associated with a significant cardioprotection effect on both healthy individuals and patients with an established cardiovascular disease. omega-3 fatty acids appear to work by decreasing endothelial responsiveness to pro-inflammatory and pro-atherogenic stimuli, affecting molecular events not targeted by other drugs thus allowing their use as complementary treatments for the already implemented pharmacological treatments in inflammatory diseases. combined therapy with omega-3 fatty acids and statins shows a synergistic effect. methods: on ultracentrifugation of serum at density 1.24 and 202,000g, top 20 % layer contained lipoproteins only and 20-50 % layer contained lipoproteins as well as immunoglobulins. the bottom layer was shown to contain immune complexes (ic) by binding to coated anti apo(a) and detection with peroxidase labelled anti human immunoglobulins.both these forms of lp(a) were western blotted and probed with jacalin-hrp, anti-gal-hrp and anti apo(a)-hrp. anti-gal was prepared by affinity chromatography on guar galactomannan and complexed with lp(a) in vitro. ic formation by lp(a) was measured in terms of reduction in response in a new elisa for lp(a) involving addition of lp(a) sample to plate-coated jacalin, followed by anti-apo(a)-hrp detection. ic formation was also shown by migration of lp(a) from free lipid layer to ic layer below in ultracentrifugation. results: anti-gal and lp(a) could be liberated from precipitated ic using specific sugar. immune complexed lp(a) in serum was found to be more o-glycosylated, larger in size and binding more anti-gal than lp(a) in free form in western blots. while ic formations within homologous free anti-gal-free lp(a) pairs were few, those within heterologous pairs were more rampant. conclusions: lp(a) is a risk factor in vascular disorders including atherosclerosis, aneurysm, stroke and peripheral vascular diseases and is a component of atherosclerotic plaques, though mechanism of its uptake remains unclear. anti-gal comprising 1 % of serum igg is rich in igg capable of complement fixation and macrophage mobilisation. present results offer a viable mechanism of lp(a)-mediated immune injury to vessel walls leading to vascular damage. even though no receptors have been detected for lp(a), unlike for ldl, the present results may explain the internalization of lp(a) in the form of lp(a) immune complexes by macrophages since the latter can phagocytose ic. extended specificity of the a-galactoside-specific anti-gal for t-antigen in lp(a) is akin to that observed in jacalin, pea nut agglutinin and galectin-1. objective: the aim of this study was to determine if the combination of two genetic alterations, one affecting cell cycle regulation, such as the e2f2 mutation, and other affecting b cell apoptosis control, such as bcl-2 over-expression, can induce the development of ais. methods: mice: mice with both genetic abnormalities were generated in a non-susceptible c57bl/6 (b6) genetic background. e2f2-/-bcl-2tg were obtained backcrossing e2f2-/-and e2f2+/+hbcl-2tg mice. e2f2-/-bcl-2tg, e2f2-/-, e2f2+/+hbcl-2tg and control mice (e2f2+/+) were followed up to 18mo-old. serologic studies: serum samples obtained at 3, 9 and 15 month of age were test for igg and iga ana and anti-dsdna by elisa. histopathologic studies: kidney paraffin sections of 3, 9 and 15 mo-old mice were stained with hematoxylin-eosin (h&e) and masson's trichrome to identify histological changes. immunecomplex deposits were studied by direct immunofluorescence on kidney using fluoresceinated goat anti-mouse igg, igm and iga. to evaluate b cell homeostasis, absolute number of b cell in blood, primary and secondary lymph organs were assessed by flow cytometry. in vitro proliferation was measured with [h3]-thymidine and brdu was used to assess in vivo proliferation capacity of immature b cells. results: overexpression of hbcl-2tg in b lymphocytes of e2f2-/-mice induced the production of high titres of igg and iga ana and anti-dsdna, together with the development of a glomerulonephritis characterized by a moderated mesangial proliferation, mesangial immunecomplex deposits, mainly of the iga isotype, and the presence of tubular casts and lymphoid infiltrates with the presence of glomerular deposits. e2f2-/-bcl-2tg mice showed an altered b cell homeostasis as demonstrated in proliferation and apoptosis studies. e2f2-/-mice showed neither autoantibodies nor nephropathy. this study demonstrates that the isolated deficiency of e2f2 or the overexpression of a bcl-2 tg in the b6 genetic background do not induce an ais. when combined both genetic alterations, involving deregulation of cellular proliferation and survival affect lymphocyte homeostasis, induce a mild ais with overproduction of iga autoantibodies. an alteration in the b cell compartment, but not in the t cell compartment, seems to be underlying the syndrome described in the present work. in different mouse models of the autoimmune disease systemic lupus erythematosus (sle) loss of toll-like receptor 9 (tlr9) abolishes the generation of antinucleosome igg2a and igg2b autoantibodies but exacerbates lupus disease. however, the tlr9-dependent tolerance mechanism is unknown. here we show that loss of tlr9 in b cells of lupus prone mice prevents the generation of protective t cell-dependent self-reactive igm and thereby enhances the development of th1 and th17 t cells. transfer of a synthesized monoclonal polyreactive igm to tlr9 deficient lupus prone mice inhibits t cell activation and abolishes development of lupus disease. thus, these results document a protective tlr9-dependent tolerance mechanism in b cells that induces the generation of self-reactive igm to prevent autoimmunity. cloning and production of polyreactive or antigen-specific igm might therefore be a powerful tool to treat autoimmunity. objectives: to investigate cytokine and autoantibody levels in serum from patients with primary sjögren's syndrome (pss), and to determine possible associations with focal mononuclear cell infiltrates, lymphoid organization, and age at the time of biopsy. methods: minor salivary gland tissue was obtained from a group of patients fulfilling the revised eu-us criteria for pss (n=115) (vitali et al. 2002) . ninety-seven of 115 (84 %) patients had focal mononuclear cell infiltrates corresponding to focus score (fs) g 1 (fs+), while biopsies from 18/115 (16 %) patients lacked characteristic focal mononuclear cell infiltrates (fs-). germinal center (gc)-like lesions were determined in 27/115 (23 %) minor salivary gland biopsies. serum samples were used for cytokine and autoantibody evaluations. the mean level of unstimulated whole saliva was significantly lower in the fs+ patients compared with the fs-patients, and in the gc+ patients compared with the gc-patients (p x 0.05). interleukin (il) 17, il-1ra, il-1beta, il-12p40, il-15, macrophage inflammatory protein (mip) 1alpha, mip-1beta, eotaxin, interferon (ifn) alpha, and il-4 levels were significantly increased in the gc+ patients (n=27) compared with the gc-patients (n=70). in addition, minor differences in cytokine levels were found when comparing age groups. degenerative changes such as atrophy/fibrosis and fatty cell infiltration observed in the minor salivary glands of patients with pss may represent "burned out" inflammation. no significant differences were found in autoantibody levels in either of the groups, nor when comparing cytokine levels in the fs-and fs+ subgroups. the reduced salivary flow observed in gc+ patients may be influenced by the elevated levels of il-4 found in these patients (gao et al. 2006) . increased titers of th17-associated cytokines, il-17, il-1beta and the il-23 subunit il-12p40, may indicate a higher activity of these cells in gc+ patients (nguyen et al. 2008) . differences in cytokine levels may be utilized when sub-grouping the ss patients into disease phases and may consequently have implications for treatment. objectives: c-reactive protein (crp) is an acute phase protein, produced by hepatocytes in response to the pro-inflammatory cytokine il-6. the rapid increase of crp during inflammation makes it an excellent inflammatory marker, but for unknown reasons, blood levels of crp typically remain low in disease flares of systemic lupus erythematosus (sle), a systemic autoimmune disease. another feature of sle is the so called 'interferon (ifn) signature' which implies high levels of ifn-alpha and/or up-regulation of ifn-alpha related genes. ifn-alpha has a wide spectrum of immunomodulatory functions but is mainly known for its antiviral and anti-tumour effects. since high levels of ifn-alpha coexist with a muted crp response in sle disease flares and in viral infections, we hypothesized that ifnalpha inhibits crp synthesis. methods: crp promoter activity was studied in a crp-promoter and luciferase reporter transfected human hepatoma cell-line, hepg2. production of the acute phase protein serum amyloid a (saa) and the negative acute phase protein transferrin were analysed by elisa as reference. results: the crp-promoter activity was inhibited by all ifn-alpha subtypes. mixes of type i ifns that were induced by sle-like immune complexes or virus also inhibited the crp-promoter activity. virus-induced purified leukocyte ifn-alpha had the most prominent inhibitory effect ( g 50 %) on crp promoter activity. saa synthesis was inhibited by ifn-alpha in a similar fashion as for crp promoter activity, whereas transferrin was unaffected. conclusion: our data indicates that ifn-alpha is an inhibitor of crp-promoter activity. we suggest that this could explain the muted crp response seen in sle disease exacerbations. further, i may contribute to differences in crp response between viral and bacterial infections. background: b cell activating factor of the tnf family (baff) is an essential b cell survival and maturation cytokine. mice overexpressing baff (baff tg mice) develop lupuslike autoimmunity, b cell hyperplasia, and lymphomas. autoimmunity in these mice involves proinflammatory autoantibodies driving nephritis and sialadenitis, and was previously found to be t cell-independent (ti) and myd88-dependent. this suggested the involvement of transmembrane activator and caml interactor (taci), which is a receptor for baff that is essential for ti immune responses and is upregulated by myd88-dependent tlr activation. we assessed the role of taci in baff-driven ti autoimmunity. methods: we tested the importance of taci in ti autoimmunity by generating baff tg bone marrow (bm) chimeras reconstituted with taci -/or taci +/+ bm then comparing their disease severity by flow cytometry, autoantibody elisa, immunofluorescence microscopy for ig deposition. results: as expected, baff tg chimeras reconstituted with taci +/+ bm produced high levels of circulating proinflammatory autoantibody isotypes and rheumatoid factors (rhf), and ig deposition in the kidneys and salivary glands was observed. by contrast, baff tg chimeras reconstituted with taci -/-bm had greatly ameliorated levels of circulating proinflammatory autoantibodies, rhf, and ig deposition. b cell hyperplasia was greater in taci -/-1 baff tg chimeras. defects in the regulation of apoptosis contribute to the pathogenesis of human systemic lupus erythematodes (sle). autoantigens not being properly removed and thus exposed to the immune systeme might lead to the emergence of autoantibodies. physiologically apoptotic cells are removed without initiation of an inflammatory immune response and myeloid dendritic cells are believed to actively tolerize t-cells after phagocytosis of apoptotic material. these processes of silent apoptotic cell clearance seem to be disturbed in sle patients. a characteristic of apoptotic cell death is the shedding of membrane coated vesicles from the cellular surface (apoptotic cell blebbing). these microparticles have been recognized as mediators of intercellular communication. therefore, we were interested whether apoptotic cell derived microparticles can influence the function of monocyte-derived dendritic cells and whether those interactions might play a role in the pathogenesis of human sle. we observed an engulfment of microparticles by monocyte-derived dendritic cells. further, apoptotic cell-derived microparticles stimulated differentiation of immature dendritic towards a mature phenotype. however, microparticles caused a remarkable downregulation of mhc class ii molecules. further, we observed only a minor release of proinflammatory cytokines from monocyte-derived dendritic cells pulsed by membrane microparticles when compared to lps stimulated dendritic cells. finally, these dendritic cells pulsed by membrane microparticles did not cause a significant t-cell expansion. interestingly, dendritic cells obtained from sle patients showed significant variations in phenotype and cytokine secretion compared to normal healthy donor cells with absence of the mhc class ii downregulation and a higher constitutive secretion of il-8. objectives: increased levels of il-18, an innate and inflammatory cytokine of the il-1 family, can be detected in serum and organs of human autoimmune pathologies, as well as in autoimmune animal models. here, expression of il-18 and other genes of the il-1/il-1r families was examined in human systemic lupus erythematosus (sle) and in mrl lpr/lpr mice, which develop a chronic progressive lupus-like syndrome. methods: serum, urine, and monocytes were collected from 48 patients and 32 healthy controls. lymphoid (lymph-nodes, spleen, thymus, peyer's patches) and non-lymphoid organs (kidney, lung, liver, salivary and lacrimal glands) were collected from mrl +/+ and lpr/lpr mice of different ages. il-18 and il-18bp were measured by elisa. gene expression was assessed by real-time pcr and expressed relative to b-actin. results: in sle, serum and urine levels of total and free il-18 are higher than in controls. serum il-18 correlates with disease activity and decreases upon remission. monocyte expression of the receptor il-18rb is increased and correlates with disease severity, while expression of tir8/sigirr (a down-regulatory receptor of the il-1r/il-18r family) is reduced. in mrl lpr/lpr mice, expression of il-18, caspase-1 and il-18rb genes precedes disease onset in lymph-nodes. in other organs, changes in il-1-related genes (il-33 and tigirr-1 up-regulation, tir8/sigirr down-regulation) occur after disease onset. free il-18 levels are abnormally high in lpr/lpr lymph-nodes before disease onset, while in other organs the increase occurs with disease. conclusions: free il-18 levels correlate with autoimmune lupus both in mice and humans. free il-18 may be pathogenic in murine lymphadenopathy, while is a disease correlate in lpr/lpr and a severity correlate in sle. both in human and mouse syndromes, upregulation of il-18rb is a marker of pathology, suggesting increased il-18-dependent activation. both in mouse organs and human monocytes, tir8/sigirr expression decreases with disease, suggesting impaired control of il-18r activation. thus, il-18 may be involved in autoimmune lupus pathology, and il-18-related molecules can be both original diagnostic markers and novel therapeutic targets in autoimmunity. in this study we compared the epitope specificity of anti-topo i autoantibodies present in sera of dcssc, lcssc and sle patients. we have constructed an antigen fragment library displayed on bacteriophage lambda and screened this library with igg purified from patients' sera. regions of topo i selected from the library were expressed as recombinant fusion proteins and were tested with elisa and western blot. we unexpectedly found that antibodies against a fragment of topo i (fragment f4 (amino acid (aa) 451-593) could be detected in sera of healthy individuals and patients with inflammatory rheumatic diseases other than ssc and sle. using sera of dcssc, lcssc and sle patients we showed that the pattern of recognized epitopes is different between these patient groups. fragment f4 was recognized by all patients. fragment f1 (aa 5-30) was recognized by 9 of 34 dcssc patients. fragment f8 (aa 350-400) was recognized by 4 of 8 sle patients. analysis of clinical data revealed a significant difference between the f1 negative and f1 positive groups of ssc patients in age and in the duration of the disease. according to our results the newly identified fragments f1 and f8 could represent characteristic epitopes for dcssc and sle, respectively. background: previous studies demonstrated that depletion of regulatory t cells (tregs) results in autoimmunity in mice while their adoptive transfer prevents autoimmune diseases. studies performed by us and others showed that in human connective-tissue diseases a reduced number of tregs exists and this abnormality seems to be correlated with autoantibodies production and disease activity. objectives: based on these observations and the fact that rapamycin (rapa) has the ability to expand tregs and to induce anergy, we proposed to study the possibility to restore peripheral tolerance of cd4 + t cells isolated from systemic lupus erythemaosus (sle) patients by ex vivo expansion of tregs. methods: pbmcs or peripheral cd4 + t cells from sle patients were cultured in the presence of specific stimulation with or without rapa and ril-2. by facs the initial percent of tregs and after expansion protocol were determined. in order to verify the suppressive capacity of expanded tregs, cd4 + t cells enriched in tregs were co-cultured with activated cd4 + effector t cells (teff) stained with cfse, after one week teff cells proliferation was measured by facs. additionally, cytokine and igg release in cell culture media were analyzed by multiplex and elisa, respectively. expanded cd4 + t cells anergy was also evaluated based on cbl-b, grail and foxp3 mrna by realtime rt-pcr. results: in vitro expansion of tregs was more efficient when the starting cells were cd4 + t cells. the presence of rapa during expansion protocol significantly increased the number of tregs. sle tregs cells expanded in vitro in the presence of rapa had the capacity to suppress proliferation of both sle and hd teff cells. rapa inhibits igg secretion in the pbmcs culture, inhibition dependent on tregs level. rapa during tregs expansion protocol stimulated some type of cytokines while suppressed others. rapa had the capacity to re-establish sle cd4 + t cells anergy by induction of anergy genes, grail and cbl-b. conclusions: our data show that the above described protocol permits ex vivo tregs expansion and that suppressive capacity of the expanded tregs depends on the source of both tregs and teff cells. in this study, we look for a more specific approach to remove b-1 cells through targeting p110d by shrnas strategy. methods: we used the drugs, ly294002 and wortmannin, pan-specific inhibitors against pi3ks. then we designed shrnas carried by the lentiviral system and validated that several segments of them can sufficiently knock down the expression of p110d. we then introduced either pan-specific inhibitors against all pi3ks or p110d-targeting shrnas into an sle-prone animal model, nzb/w f1 mice, for therapeutic purposes. the results suggested that pi3ks are not only important for the development of b-1 cells but also remain essential to maintain their population after birth. shrnas carried on lentiviral systems were designed to knock down the expression of p110d. either pan-specific inhibitors against pi3ks or p110d-targeting shrnas were introduced into the sle-prone animal model, nzb/w f1 mice. one inhibitor, ly294002, and shrnas delivered by low dose of lentivirus exhibited certain potential to retard the rising of anti-dna auto-antibodies and prolonged the life span. conclusions: our findings are promising for developing treatments for sle. moreover, knowing pi3ks are critical for the maintenance of b-1 cell populations might shed light on future treating other diseases associated with b-1 cells, such as certain melanoma, lymphoma, or leukemia. a. m. zaghlool 1 , m. alarcón-riquelme 1 , s. kozyrev 1 1 institution of genetic and pathology, uppsala university, uppsala, swedenrecently, we discovered that the bank1 gene, which plays a role in b cells activation pathway, is associated with systemic lupus erythematosus through a nonsynonymous substitution g/a (rs10516487, r61h). we identified that bank 1 gene expresses two alternatively spliced isoforms, a full-length, and a shorter isoform that lacks exon 2 (delta 2). the two isoforms were detected differently in susceptible lupus patients depending on the presence of a risk haplotype. to address the question of how bank1 is spliced and what are the signals governing the expression of each isoform, minigenes with different genetic variants were constructed and the expression of the bank1 isoforms were tested in vitro. qpcr analysis revealed that, another t/c snp (rs17266594), which is in complete ld with r61h snp and located in the putative branch point, has a strong affect on the isoforms expression levels. deletion of a polypyrimidine (py) stretch downstream of the skipped exon produced a dramatic decrease in the full-length expression levels, probably due to the loss of the binding site for protein tia1, which bind to t objectives: cerebral ischemia is the most common presentation of antiphospholipid syndrome (aps), but several other neuropsychiatric features, including chronic headache, dementia, cognitive dysfunction, psychosis, depression, transverse myelitis, multiple sclerosis-like disease, chorea, and seizures have been associated with the presence of antiphospholipid antibodies (apl). we report the case of a subject with atypical movement disorder related to aps successfully treated with oral anticoagulation agents. case report: a 57-year-old woman with a previous history of recurrent foetal losses was admitted to our hospital due to cognitive dysfunction and headache. she presented involuntary movements that were characterized as mioclonic seizures and tonic spasms lasting from few minutes to several hours, followed by bilateral arrhythmic rapid purposeless jerks of the legs. mild executive dysfunction was observed. her deep tendon reflexes were symmetric and normal. pathological reflexes were absent. biochemical analysis, renal, hepatic and thyroid functions were preserved, prothrombin time and partial thromboplastin time were all normal. the immunoglobulin g (igg) isotope of anticardiolipin antibody (acl) was elevated, whereas igm isotype and anti-2gpi antibodies were undetectable. the lupus anticoagulant (la) was negative such as antinuclear antibodies (ana). no evidence of epilepsy was revealed from electroencephalogram or signs of denervation from electromyographic studies. brain magnetic resonance imaging (mri) showed multifocal encephalomalacia probably linked to previous cerebrovascular accidents. she was diagnosed as having an atypical neurologic manifestation probably linked to aps. she was thus discharged with a low-molecular-weight heparin therapy subsequently changed to mild oral anticoagulation . the therapy leads to a late, gradual improvement of symptoms that persisted at the last 1year follow-up evaluation. conclusions: antiphospholipid syndrome may constitute a rare but treatable cause of atypical neurologic manifestation such as myoclonic movements. due to the possibility of an effective treatment, it is important to rule out this diagnosis, moreover in women with other associated features of aps (foetal losses, livedo reticularis, thrombosis). a.-s. korganow 1 1 cnrs 9021, strasbourg, france b lymphocytes from patients with systemic lupus erythematosus are hyperactive and produce autoantibodies. several b cell phenotypic characteristics have been reported, as the expansion of activated populations, and of a newly investigated memory compartment. a few genes have been suggested to be implicated. one of the thing that makes these results difficult to interpret is the heterogeneity of the lupic disease, and sometimes the analysis all together of quiescent, paucisymptomatic and highly symptomatic patients, treated with immunosuppressors or untreated.we made the postulat that "intrinsic" abnormalities of b cells could be a common point in very quiescent patients. we choosed 18 patients, with minor clinical and/or biological manifestations of their disease, for at least 6 monthes. known of them received immunosuppressive drugs since this period. the mean sledai score was below 2. b cell surface markers expression was determined by flow cytometry. we analysed most of the already described and phenotypically distinctive b cell populations. we confirm the presence of activated b cells even in quiescent patients. we do not confirm the significant increase of a specific memory b cell compartment. above all, we described a decreased expression of the cd19 surface protein for all patients. this cd19 lower expression is associated with cd45 lower levels. it is not associated with an evident gene expression alteration and in vitro stimulation restores a control phenotype. these findings suggest some mechanisms in lupus genesis. objectives: tgf-beta is a pleiotropic cytokine with wide ranging effects in proliferation, differentiation, immune suppression and apoptosis. recent work from our group has shown that tgf-beta signalling in t-cells is protective in a mouse model of colitis associated cancer. smad ubiquitin regulating factors (smurf) are ubiquitin ligases that are involved in the regulation of tgf-beta signalling. the aim of this study was to determine the function of smurf2 expression in t-cells on the pathogenesis of experimental colitis associated colon cancer. methods: we could isolate a known splice variant of smurf2 lacking an exon in the c2-domain. to analyse whether this form has a regulatory role in colon associated cancer we generated a transgenic mouse strain that overexpresses smurf2 in t-cells. smurf2 expression were analysed by qpcr. wild type (wt) and transgenic (tg) mice were treated once with the mutagenic agent azoxymethan (aom) followed by three cycles dextran sodiumsulfate (dss). after each cycle, the inflammation of the gut and the tumor growth and size of every mouse were monitored by colonoscopy. results: smurf2 expression was upregulated by tgf-beta stimulation in t-cells and smurf2 was markedly upregulated in tumor infiltrating cd4+ lymphocytes in aom/dss treated mice. whereas wt mice suffered from severe colitis resulting in colon tumors beginning at day 42, smurf2 transgenic mice had less colitis and were significantly protected from tumor development. interestingly, t-lymphocytes overexpressing smurf2 showed an upregulation of the tgfbrii and an activation of smad2 and 3 as compared to wild-type t-lymphocytes, which were previously described as typical smurf2 targets for degradation. in addition the transfection of smurf2 and a caga-luc plasmid into cos-cells for smad3-promotor studies yielded the same effect as shown by upregulation of the smad3 activity. conclusion: although, wt-smurf has been described as a negative regulator of the tgf-beta signalling pathway, our data show surprisingly that a smurf2 splice variant upregulates the tgf-beta receptor expression and increases tgf-beta signalling effects. due to immunosuppressive effects on t-cells smurf2 has beneficial effects on mucosal inflammation and tumor development. smurf2 thus emerges as an attractive target for modulation of chronic intestinal inflammation and colitis associated carcinogenesis. the transcription factor stat3 has important functions in cytokine signalling in a variety of tissues. however, the role of stat3 in the intestinal epithelium is not well understood. we demonstrate that development of colonic inflammation is associated with the induction of stat3 activity in intestinal epithelial cells (iec) both in humans and in mice. studies in genetically engineered mice showed that epithelial stat3 activation in dss colitis is dependent on il-22 rather than il-6. il-22 was secreted by colonic cd11c+ cells in response to toll-like receptor stimulation. conditional knockout mice with an iec specific deletion of stat3 activity were highly susceptible to experimental colitis, indicating that epithelial stat3 regulates gut homeostasis. stat3 iec-ko mice, upon induction of colitis, showed a striking defect of epithelial restitution. gene chip analysis indicated that stat3 regulates the cellular stress response, apoptosis and pathways associated with wound healing in iec. consistently, il-22 and epithelial stat3 was found to be important in wound-healing experiments both in vivo and in cell culture experiments in vitro. in summary, our data suggest that intestinal epithelial stat3 activation regulates immune homeostasis in the gut by promoting il-22-dependent mucosal wound healing. stat3 seems dispensable for gut homeostasis under steady state conditions, but is activated upon challenge to drive tissue regeneration and protection in situations of increased demand, as during colitis and injury. map and ma infection induced an increase in both cd40 and tlr4 expression at day 3 and day 7 after infection. mycobacterial infection did not result in differential tlr2 expression as compared to uninfected cells. cd40 is involved in stimulating th1 pro-inflammatory responses, although map may interfere with cd40 signalling (1) . tlr4 signalling elicits anti-inflammatory responses, which can contribute to bacterial replication (2) .in conclusion, monocyte-derived macrophages from crohn's disease patients show an increase in cd40 and tlr4 receptor expression in response to both map and ma infection. as ma is a known human pathogen of immunocompromised hosts, this findings further support a role for map in the immunopathology of crohn's disease. objectives: for our understanding of the pathogenesis of human ibd, animal models of intestinal inflammation are indispensable. most of them are based on a compromised intestinal barrier, and a deregulated immune response against components of the flora is considered to be critically involved in the development of ibd. the occurrence of extraintestinal manifestations suggests that cross-reactions against hitherto undefined auto-antigens could be responsible for the activation of the adaptive immune system. to further dissect the pathophysiological mechanisms responsible for initiation and progression of ibd and associated extraintestinal manifestations, we established a new antigen-specific model, in which the local activation of cd8 t cells by exogenous antigen leads to colitis. methods: eight million naïve cd8 + ot-i cells, transgenic for a t-cell receptor specific for an ova-derived peptide (siinfekl) in the context of h2-kb, were transferred i. v. into b6 mice. at day 0 and 4, mice were treated intra-rectally (i. r.) with 50 % ethanol. thirty minutes later, ovalbumin (ova) or bovine serum albumine (bsa) were applicated i. r. proliferation of cfse-labelled cells was measured at day 2 after the injection of ot-i cells. the phenotype of effector cells was evaluated at day 5 by measuring ifng production and by in vivo cytotoxicity assay. based on histology and immunhistochemistry for cd8, the severity of colitis was scored. results: local application of the exogenous antigen ova but not of bsa led to antigen-specific activation and proliferation of adoptively transferred naïve ot-i cd8 + t cells. these cells differentiated into fully activated effector t cells with the capacity to secrete ifng upon re-stimulation ex vivo and possessed in vivo cytotoxicity to siinfekl-loaded target spleen cells. furthermore ova treated mice displayed an inflammatory infiltrate in the colonic lamina propria with strongly elevated numbers of cd8 + t cells. our study demonstrates that the local activation of antigen-specific cd8 t cells by exogenous antigen in the colon leads to fully activated effector t cells with the capability to promote local intestinal inflammation in non-immune-compromised b6 mice. aims: to determine the immune system response of the greek population against helicobacter pylori (hp), given the fact that hp infection is a frequent causal factor of gastroduodenal ulcer and gastritis, and to study the distribution by age and sex, as well as the possible correlation with anemia markers (hematocrit, hemoglobin, iron, ferritin etc). the results of express qualitative detection method for igg and iga antibodies were studied of 535 patients, (248 male and 287 female), with age average 69,7 years of age. patients who received antibiotics and excretory medicine in the last year were excluded. anemia laboratory tests were performed (hematocrit, hemoglobin, iron, ferritin), which were followed by statistical processing, using spss, x 2 and t-test programmes. results: in 372 patients (69,5 %,with age average 62,7 years of age) no antibodies were detected. on the contrary, in the remaining 163, 52 male and 111 female, (30,5 %, with age average 76,4 years of age), antibodies were detected. out of them, in 106 cases the results were strong positive (32 male and 74 female) and weak positive in 57 cases (20 male and 37 female). the statistical analysis that followed, showed no statistically important correlation with any of the anemia markers who were determined (hematocrit, hemoglobin, iron, ferritin, mcv and rdw). conclusions: it is proven, therefore, that: 1) helicobacter pylori infection is relatively common in the general population (30,5%).2) there is a statistically important correlation, as far as age (increased in elderly patients) and gender is concerned (clearly greater in women).3) there seems to be no correlation with anemia. it is evident, that the method is very useful, especially in elderly patients with dyspeptic complaints, (who frequently cannot undergo invasive procedures), and should not be neglected, given the fact that there is a great risk of helicobacter pylori infection in our country. abstract withdrawn by author m. durilova 1 , t. ulmannova 1 , k. stechova 1 , k. tesarova-flajsmanova 1 , v. stavikova 1 , j. nevoral 1 1 charles university, pediatrics, prague, czech republicobjective: was to analyze composition of cytokines in breast milk of mothers whose infants were diagnosed with allergic colitis and compare it to cytokine composition in breast milk of healthy controls. methods: breast milk of 20 mothers whose infants were diagnosed with allergic colitis and 20 mothers of healthy infants and no history of allergic disease was analyzed for presence of cytokines. breast milk samples were collected at the time of diagnosis of allergic colitis (2-27 weeks, average 16.8 weeks of infant's age) or at the age of 12 weeks in control group. concentrations of the following cytokines were analyzed using elisa method: il-4, il-6, il-10, il-17, il-23, ifn-gamma, tgf-beta1, egf and eotaxin. man-whitney u test was used for statistical analysis, p x 0.05 was considered statistically significant.results: il-10 as the only cytokine was not detected in any of the tested samples in both groups. significant difference was seen in concentration of ifn-gamma, which was higher (p x 0.001) in breast milk of mothers whose infants were suffering from allergic colitis (range 0-8.45 pg/ml, mean 2.1 pg/ml) than in control group (range 0-3.41 pg/ml, mean 0.35 pg/ml). higher concentrations of il-4 and lower concentration of tgf-beta1 were observed in breast milk received by infants with allergic colitis but the difference was not statistically significant. conclusion: immunologic factors including cytokines present in breast milk passively and actively influence the developing immune system of the newborn. although their role is not exactly known, they are important in regulation of immunologic reactions and might be responsible for protective effects of breast milk from many diseases. inter-individual differences in cytokine composition of breast milk were previously found in many studies and their presence is influenced by various factors. the results of our study indicate that there might be a risk cytokine pattern in breast milk of mothers whose infants are suffering from allergic colitis. supported by national project no. 8310-5. background: ulcerative colitis is associated with excessive neutrophil infiltration into the lamina propria and intestinal crypts leading to the formation of crypt abscesses. the chemokine il-8 (murine homologs kc and mip-2) and its receptor cxcr2 are involved in neutrophil recruitment, thus blocking this engagement offers a new therapeutic strategy for inflammatory bowel disease. this study aimed to develop and characterize a pre-clinical in vivo model to test potential therapeutics targeting neutrophil migration. methods: peritoneal exudate neutrophils from transgenic b-actin-luciferase mice were isolated 12 h post intraperitoneal injection of thioglycollate and phenotypically (facs analysis) and functionally characterized in an in vitro chemotaxis assay. four million exudate cells were injected intravenously into recipients with dextran sulphate sodium (dss) colitis followed by bioluminescence imaging of whole body and ex vivo organs at 2, 4, 16 and 22 h post-transfer. anti-kc antibody or its isotype control was administered at 20mg/mouse one hour before transfer followed by whole body and organ imaging 4 hours post-transfer. results: facs analysis revealed 80 % neutrophil purity, 35 % of which were cxcr2 + . in vitro, the cells migrated towards kc and this was inhibited by anti-kc. in the bioluminescent imaging model, trafficked neutrophils were evident in whole body and ex vivo organ images of dss recipients at all time points. neutrophil recruitment to the colon was detected only in dss recipients and was inhibited by anti-kc, 4 h post cell transfer. this study describes a novel in vivo model of neutrophil trafficking that can be used for pre-clinical studies to evaluate potential inhibitors of neutrophil recruitment. the human gut contains more than 10 15 bacteria (known as the commensal microbiota) that are essential for normal function of our digestive and intestinal immunologic systems. the barrier function of the mucosal epithelium is reinforced by innate defense mechanisms and by immune exclusion mediated by secretory (s)iga and sigm. sigs are generated via epithelial polymeric ig receptor (pigr)-mediated transfer of iga and igm from the lamina propria to the intestinal lumen. to assess the role of sigs in colitis development, we constructed pigr knockout (ko) mice and tested them in the dextran sodium sulfate (dss) colitis model (1.5 % dss in drinking water for 1 week, followed by pure drinking water for 1 week). pigr ko mice suffered increased morbidity and mortality compared with wild type mice, but colitis was cured by depletion of intestinal commensals suggesting that one role of sigs is to prevent pathology induced by commensal microbiota. in contrast, 2 % dss was lethal to all commensal-depleted mice, but these mice became anemic rather than suffering from bloody diarrhea. as previously documented by medzhitov and co-workers (rakoff-nahoum et al, cell 2004), treatment of commensal-depleted mice with the tlr4 ligand lps in drinking water protected against the lethality of 2 % dss. thus, the commensal microbiota serve two distinct roles in the dss colitis model. at dss concentration of 1.5 % they may become pathogenic and drive an intestinal inflammation. at 2 % dss commensals protect against the toxic effect of the chemical via their tlr ligands. in mice lacking sigs, due to deleted pigr, the severity of colitis induced by 1.5 % dss was greatly enhanced suggesting that one role of sigs is to prevent commensal microbiota from becoming pathogenic. ulcerative colitis (uc) is a human inflammatory bowel disease associated with chronic inflammation of the gastrointestinal tract. although uc is associated with a type 2 immune response, current treatment strategies use broad anti-inflammatory drugs which are aspecific for the disease. in a mouse model resembling uc, oxazolone induces il-13 production which is an important pathological factor. neutralizing il-4 or il-13 prevents or ameliorates disease significantly. as many aspects of the mechanisms involving these th2 cytokines in colitis remain undefined, we used mice deficient in il-4/il-13 or the key receptor through which they signal, il-4ra, to further dissect their role in oxazolone-induced colitis. disease was exacerbated in il-4ra -/mice with increased weight loss, mortality, inflammation and immunopathological symptoms. this was in contrast to il-4/il-13 double deficient mice which were protected from colitis. removing il-13 production from il-4ra -/mice, by using il-4ra/il-13 double deficient mice, reversed the susceptible phenotype to protection. together these data strongly suggest that il-13 mediates susceptibility in an il-4ra independent manor. recent evidence pc17/33 introduction: the activation of cd4 + t-cells in the lamina propria play an major role in the pathogenesis of inflammatory bowel disease (ibd). whereas cd is associated with increased production of th1-like cytokines, the cytokines profile in chronic uc is characterized by the increased production of several th1 cytokines, such as il-5,-6 and il-13. however, the functional role of t cell transcription factors such as nuclear factor of activated t cells (nfat) in ibd is poorly understood. the aim of this study was to further analyze the role of this signal transduction pathway and its pathogenic significance in uc. cryosesctions of uc and cd patients were analysed by immunohistochemically methods. a significantly higher expression of nfatc2 was found in uc and cd colonic tissue compared to control specimen. transmitted to the th2-mediated oxazolone-induced colitis model, nfatc2-production is significantly increased in both diseases, too. nfatc2 deficient mice were analyzed in colitis model and are significantly protected against the development of intestinal inflammation compared to control mice, documented by loss of weight, histological score and miniendoscopy. interestingly, cyrosections of inflamed colonic tissue displayed a higher apoptotic rate in nfatc2 deficient mice compared to control mice, which can be observed by tunel assays, caspase3 and annexin v staining, as well as in lamina propria t cells. contrary, anti-apoptotic proteins, like bcl-2 and bcl-xl were downregulated for induction of apoptosis. this observation was associated with a reduced production of il-6, ifn-gamma, il-13 and il-17 by mucosal t lymphocytes, tested by elisa assays. further studies with the oxazoloneinduced colitis model showed that nfatc2 regulates il-23/il-17 in an indirect way. last, administration of il-6 blocked the protective effects of the nfatc2 deficiency in experimental colitis, suggesting that nfatc through il-6 signal transduction plays a direct pathogenic role in vivo. conclusion: our data define a unique regulatory role of nfatc2 in colitis by controlling mucosal t cell activation in an il-6 dependent manner. the examination of this signal transduction pathway emerges as a potentially new therapeutic target for inflammatory bowel diseases. the pivotal role of micrornas in the regulation of gene expression, in particular genes involved in the immune response, indicates that they may play an important role in the pathogenesis of inflammatory bowel disease (ibd) as well. the study of the expression of micrornas in ibd will unravel their role in this disease. in addition, micrornas by their mechanism of action, are promising new therapeutic agents or targets. a possible therapeutic application of micrornas is the introduction of novel, artificial micrornas or microrna mimics to regulate specific genes. because ibd is a heterogeneous disease in human we decided to define microrna expression in a well defined model of experimental colitis. as a result of this study we found a number of micrornas involved in different phases of experimental colitis. to study the role of mirnas in experimental colitis in mice we have used a well defined colitis model that resembles human ibd. this colitis is mediated by cd4cd45rb high t cells that are injected i. p. in scid mice. in control mice in addition to the cd4cd45rb high t cells also regulatory cd4cd45rb low t cells are transferred and no colitis develops. to study mirna expression we collected colonic tissue from the mice at 3 different time points during colitis progression. after 3 weeks a chronic progressive colitis developed characterized by a progressing wasting disease that was terminated at 9 weeks. microrna was isolated from colons of mice in different stages of colitis progression (3, 6 and 9 weeks) and control mice that do not induce colitis (n=3 for each timepoint). from all mice we also processed a part of the colon for immunohistochemistry to determine disease progression at the various time point after induction of colitis.the rna isolation as well as the microarray analysis has been outsourced to miltenyi biotec gmbh, bergisch galdbach, germany. we used the mirxplore tm microarrays for microrna expression profiling. from 11 micrornas that demonstrated an induction during the development of disease we selected 4 micrornas for in situ hybridization and for a proof of principle of the efficacy in the cd4cd45rb high transfer model. objective: the purpose of this clinical trial (id: nct00287677 of www.clinicaltrials.gov) is to investigate whether the expansion of the thymus in adults can restore specific immune responses by administration of growth hormone (gh). methods: successfully highly active antiretroviral therapy (haart) treated hiv infected patients that failed to elicit a humoral response to tetanus toxoid (tt), or to hepatitis a (hva) or to hepatitis b (hvb) virus have been selected for the trial. growth hormone was given for 6 months with the hope that they will reactivate thymic input and restore their specific responses to these vaccine antigens. patients have been randomized in 3 groups: group a (n=8) receiving haart+ gh (for 6 months) + tt+hva/b vaccines (at month 6 post gh adminsitration); group b (n= 6) receiving haart+gh but not vaccines; and group c (hiv control group, n=7) with haart+vaccines (at month 6) but without gh. all patients are followed up 6 months further. results: preliminary results show that an increase in thymic size was observed in gh recipients and not in controls. furthernore after 24 weeks of administaring hormone the absolute numbers of cd4 incresase from 562 ± 93 to 704 ± 112 cells per mm 3 (mean and sem; p x 0.0025). in contrast, pacients who have not received the hormone but have been vaccinated showed a significant decay of the cd4 absolute numbers from 550±97 to 470±103 cells per mm 3 (p x 0.02). viral load remained undetectable in all patients. despite the increase in cd4 counts the percentage of recent thymic emigrants (as assessed by the expression of cd31) as well as the proportion of naï ve and memory cells remained constant throught the trial in all patients. finally, specific responses to hepatitis a virus seem to be restored in a major proportion of patients treated with gh (group a) than in the other groups. conclusions: although the clinical trial is ongoing, the preliminary results seem to indicate an increase in the thymic size and some immmune restoration in patients treated with growth hormone before vaccination. a major problem of current vaccines is the requirement for cold chains to maintain vaccine potency. in the course of the eradication of small-pox, freeze-dried vaccinia virus which proved to be extremely stable was used to overcome this limitation (dryvax ® ). before usage, dryvax has to be reconstituted before vaccination using a bifurcated needle reflecting another drawback of classical vaccination -transmission of blood-borne pathogens. an alarming report by the who has estimated that as many as one-third of immunization injections are unsafe in four of its six geographical regions. each year, an overwhelming number of infections with hiv (80,000-160,000), hepatitis c virus (hcv; 2.3-4.7 million) and hepatitis b virus (hbv; 8-16 million) are thought to originate from the reuse of needles and syringes by health-care providers. in this report, we took advantage of the stability of freeze-dried vaccinia virus mva and directly applied it into the nostrils of mice without prior reconstitution. this direct mucosal application induced systemic antibody and t cell responses comparable to those achieved by intramuscular administration. importantly, mucosal application of lyophilized mva conferred protective immunity against a lethal vaccinia virus challenge. additionally, recombinant mva expressing the model antigen ovalbumin induced long-term and protective immunity against listeria monocytogenes-ova challenge. the data clearly demonstrate the potency of a simple needle-free vaccination, combining the advantages of mucosal application with the stability and efficiency of lyophilized mva. methods and results: seventeen haart-treated asymptomatic hiv-1 infected patients with g 350 cd4 + t-cell counts and plasma hiv-rna levels of x 1.7 log 10 copies/ml were treated with a dc-based vaccine. the vaccine consisted of autologous mature dc electroporated seperately with either sig-tat-dc-lamp, sig-rev-dc-lamp or sig-nef-dc-lamp mrna and were each administered at a distinct anatomical site. after four monthly vaccinations haart treatment was interrupted. pbmc from 4 timepoints, before during and after vaccination, were analysed for ifn-g production (elispot), proliferation (cfse assay) and lytic capacity (fatt-ctl) in response to the antigens used in the vaccine. pbmc were screened upon ex vivo stimulation with pools of overlapping tat, rev, nef and gag peptides and ifn-g secretion was analysed using elispot ('peptide elispot'). elispot was also performed on re-stimulated t-cells with electroporated dc ('dc elispot') in vitro. responses were considered positive when the number of spot forming units per million t-cells was g 50 and when the responses were g 2 times the standard deviation above the mean of replicate negative controls (mock electroporated dc). 16/17 patients were screened with both peptide and dc elispot. an increase of responses to the vaccine-antigens after vaccination was found in both assays. based on the dc elispot data, we observed immune reactivity against tat in 4/16 patients before and 11/16 patients after vaccination. for rev, 7/16 patients showed a pre-existing rev specific response and 14/16 patients responded to rev after vaccination. nef was the most immunogenic antigen used in this vaccine with already 11/16 patients responding before and 16/16 patients responding after vaccination. for our control antigen gag, we observed an anti-gag response in 13 out of 16 patients before vaccination and 16/16 patients after vaccination. the results of the other assays correspond to the dc elispot results be it less pronounced. conclusion: therapeutic vaccination of hiv-infected patients with dc electroporated with tat, rev and nef induces and/or enhances antigen-specific t-cell responses, especially when monitored with the dc elispot. background: enterovirus 71 (ev71) is an etiologic agent responsible for seasonal epidemics of hand-foot-and-mouth disease and causes significant mortality among young children. no effective vaccine for ev71 is available yet. polysaccharide purified from ganoderma lucidum (ps-g) has been known to be a strong immunopotentiator, therefore, we studied the immune enhancing effect of ps-g as the possible adjuvant with ev71 inactivated virus. methods: mice were immunized intraperitoneally with 10 mg inactivated virus /mouse with one of the following samples: pbs, cfa/ifa, and ps-g. each mouse received the same dose of boosters after 0, 2, and 4 weeks. blood samples were collected at 0, 21, 35, and 45 days. the total serum anti-ev71 igg level was determine by elisa, and the neutralization assay was also done. to evaluate the cellular immune responses, spleens were harvested from all mice for splenocyte proliferation assay. cytokines assay regarding ifn-g and il-5 from splenocytes was also measured. results: immunization with ev71/ps-g showed that the anti-ev71 igg levels were significantly increased compared with ev71 alone or ev71/cfa/ifa in balb/c mice. neutralization assays demonstrated an effective protection of ev71/ps-g group compared to ev71 alone. the splenocyte proliferation test showed that production of ifn-g significantly increased in ev71/ps-g-immunized mice compared to those of ev71 or ev71/cfa/ifa-immunized mice, indicating a th1 cells response elicited by heat-inactivated ev71 vaccine with ps-g adjuvant. conclusions: vaccine design is important for the development of immune response for pathogen, and adjuvants play the very important role to enhance the effect of vaccine. the results here suggested that ps-g can be used as a novel, safe and natural vaccine adjuvant. objectives: the search for a vaccine against hepatitis c virus is hampered due to the lack of an animal model to study vaccine-efficacy other than chimpanzees. here we describe the differential modulation of cd8+ t-cell responses induced by a dna prime mva boost vaccine regimen in four individual chimpanzees and their association with viral clearance. methods: an ex vivo expansion protocol was used to map peptide specific cd8+ t-cell responses against hcv-ns3, studying induction of il-2, ifng and tnfa cytokine production as well as killing capacity. to assess the killing capacity and mhc restriction of the peptides, a non-radioactive killing assay was designed. peptides that induced both il-2 and ifng production by cd8+ t-cells were tested for their competence to induce killing of transfected target cells that expressed chimpanzee mhc class i molecules. introduction: high levels of hiv-1-recognizing cd8 + cytotoxic t lymphocytes (ctl) with a widespread specificity, especially against conserved epitopes, are considered to play an important role in the control of hiv-1 replication, and for the prolonged survival of infected individuals. a potential immunotherapeutic strategy would be the adoptive transfer of t cells, which are reprogrammed by introduction of an hiv-specific t cell receptor (tcr). up to now, such ctl were generated by retroviral transfer of tcr-encoding genes, which harbors several challenges (i. e., activation/inactivation of genes, life-long autoimmunity). methods: therefore, we investigated the transfer of tcr-rna into cd8 + t cells by electroporation, and chose tcrs which were able to recognize the hla-a2 restricted hivpol-peptide iv9, or the hivgag-peptide sl9. results: t cells, reprogrammed with these receptors, released the pro-inflammatory cytokines il-2, tnf, and ifng simultaneously, and showed up-regulation of the activation marker cd25, after stimulation with peptide-loaded target cells or target cells (i. e. ebv-transformed b cell and cd4 + t cells) presenting the naturally processed epitope. furthermore, these cells maintained their ability to proliferate after stimulation. more importantly, killing assays demonstrated that the tcrreprogrammed cd8 + t cells were capable to specifically lyse target cells (for at least three days) loaded with the cognate peptide, or presenting the naturally processed epitope. a comparison of our reprogrammed t cells with the parental ctl showed that the transfected t cells were only one order of magnitude lower in avidity than the parental ctl. also, the parental clone's recognition pattern of mutant peptides was preserved in tcr-rna-transfected t cells. the transfection of tcr-encoding rna into cd8 + t cells, may represent a simple, secure, and more flexible alternative to retroviral transduction, but has the benefit that a better evaluation of the transferred tcrs is possible. background: dendritic cells (dcs) are able to capture, internalize, and process antigens leading to potent activation of antigen-specific cellular immunity. the aim of this study was to investigate the capacity of splenic dcs that ingest antigen coated magnetic beads to induce hcv cellular immune responses. methods: splenocytes of flt3l pretreated balb/c mice were incubated for 3 hrs with hcv ns5-coated magnetic beads. the cells were harvested and cells that contained beads were purified by passage over a magnetic column. the isolated population contained g 80 % dcs and was used for immunization. dc expression of the maturation markers cd40, cd80 and cd86 was determined before and after ingestion of ns5-coated beads, showing a significant increase of all maturation markers induced by phagocytosis. cellular immunity was assessed using a conventional ctl assay, a cfse-t-cell proliferation assay, intracellular cytokine staining and tumor challenge (with stably ns5 expressing sp2/0 cells). results: in immunized animals, the ctl activity was increased 3-fold compared to mock immunized mice. accordingly, tumor challenge with ns5 expressing tumor cells showed a significant reduction in tumor growth. the number of cd4 + ifn-g + cells was increased g 3-fold and the number of cd4 + il-2 + increased g 5fold in the dc-ns5-bead immunization group. these results paralleled the proliferative response of splenocytes to ns5 protein obtained from immunized animals with the most significant response in the cd4+ population of dc-ns5-bead immunized animals. the use of ns5 coated beads combines three important aspects of dendritic cell based immunization in a single step: targeting of the antigen, enrichment and maturation of dendritic cells. the induction of a strong th1 biased t cell response makes this approach a promising new tool in therapeutic immunization in chronically hcv infected patients. the success of anti-dec-205 antibody as a stimulator of strong inflammatory immune responses depends on the coadministration of non-specific dendritic cell maturation factors. in their absence, anti-dec-205 induces antigen-specific tolerance rather than immunity. we hypothesize that regulatory t-cell epitopes contained in anti-dec-205 promote a tolerogenic reaction that is only overcome through the co-administration of non-specific immuno-stimulators. this hypothesis is based on our recent discovery of a set of natural regulatory t-cell epitopes derived from human immunoglobulins that induce tolerance by stimulating regulatory t cells. we have verified experimentally that these epitopes generate an expansion of regulatory t cells and suppress inflammatory immune responses. here, we embarked on a proof-of-principle demonstration that a pro-inflammatory and non-tolerogenic anti-dec-205 antibody can be developed. we screened the anti-dec-205 sequence computationally for putative hla dr4-restricted, regulatory t-cell epitopes as targets for mutations that will reduce epitope binding affinity for hla. amino acid substitutions predicted to interfere with hla binding were identified and are being incorporated into an array of anti-dec-205 antibody variants recombinantly fused to a test antigen, hiv gag. variant antibodies that do not interfere with dendritic-cell targeting will be evaluated for reduced tolerogenicity, as well as for enhanced gag immunogenicity, in terms of cellular and humoral responses. we predict that the modification of regulatory t-cell epitopes will significantly diminish tolerogenicity, enabling the use of modified anti-dec-205 as a hiv antigen-delivery system that obviates the dangers associated with non-specific activation of the immune system. supported by nih 1r21ai078800 a live oral vaccine based on human adenovirus (ad)4 has proved safe and effective in us military recruits for nearly 50 years. in these experiments, we have investigated whether replication-deficient ad4 can be an efficient potential vaccine carrier for oral vaccination. ad5 vectors were used throughout to provide a benchmark for efficacy. we generated novel ad4 and ad5 vector systems based on dna recombineering to facilitate manipulation of the vector backbone and high throughput transgene insertion (http://adz.cf.ac.uk). egfp was inserted with a hcmv ie promoter as a model transgene. preliminary in vitro studies on bloodderived human and murine cells demonstrated that primary lymphocytes are less susceptible to transduction with ad4 than ad5. ad5 routinely infected and provided transgene expression in˚10 % of human cd4+ and cd8+ t cells. stimulation of the hcmv ie promoter post infection increased detection of egfp to 25 -30 % of cd8+ t cells present, showing that ad5 infected a surprisingly large proportion of t cells. in comparison, ad4 provided egfp expression in x 2 % of either cell type, even after t cell activation. in contrast, infection rates and transgene expression in dendritic cells of both human and murine origin with ad4 and ad5 vectors were comparable. preferential infection of dcs is likely to be beneficial in the context of a vaccine. in vivo, we observed that following oral delivery both ad4 and ad5 induced restricted yet strong expression in the intestine. the vectors were rapidly taken up into the peyers patches, with optimal expression detected day 3 after dosing, and transgene expression being reduced below detectable levels by day 8. interestingly, when delivered together ad4 and ad5 vectors targeted the same subset of cells. together, these data show that ad4 is a viable alternative to ad5-based vaccines which may also avoid unwanted infection of activated t cells. aims: monoclonal antibodies (mabs) represent some of the most promising agents for anti-cancer and anti-viral immunotherapies (20 recently commercialized; g 300 in clinical trials). to date, their therapeutic antiviral efficiency has mainly been measured by their direct effects on viral spread. however, their indirect effects on long-term immune control of viral replication through their immunoregulatory properties upon interaction with other components of the immune system has hardly been assessed. as induction of long-term protective antiviral immune responses still remains a paramount challenge for treating chronic viral infections, we asked whether neutralizing mabs, in addition to blunt viral propagation, may also modulate the endogenous immune response. methods: we have developed a preclinical mouse model of fatal leukemia induced by the frcas e murine retrovirus. mice were infected with frcas e and treated, or not, with a neutralizing mab (the 667 mab). viral propagation, survival and development of immune responses were followed up for several months. results: using this model, we have shown that 667-treated/infected mice develop a long-lasting protective humoral immune response as well as a strong and sustained cellular immune response with high cytolytic activity which are not observed in leukemic non-treated/infected mice. these results show that neutralizing mabs act as immunomodulatory agents capable of inducing long-term protective immunity ( g 8 months) with both humoral and cellular contributions, despite the fact that they were administered over a short period of time (gros et al, 2005; gros et al, 2008; michaud et al. submitted) . although the initiation and maintenance of this long-term immunity is multi-factorial, we have demonstrated the crucial importance of the uptake of antibody-coated, infected cells by dendritic cells in the development of enhanced primary and memory antiviral t-cell responses. conclusions: our results show that infected-cells/antibody immune complexes play an important role in the induction and maintenance of protective antiviral immunity through enhancement of primary and memory antiviral t-cell responses. our observation might have important consequences on the design of antiviral mab-based immunotherapies. objectives: we have analyzed the potential of virus-like particles (vlps) from rabbit hemorrhagic disease virus (rhdv) as a delivery system for foreign t-cell epitopes. to accomplish this goal, we generated chimeric rhdv vlps incorporating a cd8 + t-cell epitope (siinfekl) derived from chicken ovalbumin (ova). the ova epitope was inserted in the capsid protein (vp60) of rhdv at two different locations: 1) the n-terminus, predicted to be facing to the inner core of the vlps (rhdv-vlp-2), and 2) a novel insertion site predicted to be located within an exposed loop (rhdv-vlp-306). both constructions correctly assembled into vlps and we analyzed the immunogenic potential of both chimeric rhdv vlps in vitro and in vivo. in vitro, dendritic cells (dcs) were able to process and present siinfekl peptide in the context of mhc class i from chimeric rhdv vlps for cd8 + specific recognition in a dose-and insert position-dependent manner. moreover, chimeric rhdv vlps activated dcs for tnf-alpha secretion.in vivo, mice immunized with the chimeric rhdv vlps without adjuvant were able to induce specific cellular responses mediated by cytotoxic (ctl) and memory t cells. although both chimeric rhdv vlps were able to induce specific ifn-g-producing cell priming, insertion of the siinfekl peptide at the amino-terminal position (rhdv-vlp-2) was more immunogenic than insertion at position 306 for induction of ctls and anti-viral immunity.more importantly, immunization of mice with chimeric rhdv vlps at the highest dose tested was able to control an infection by a recombinant vaccinia virus expressing ova in target organs. in addition, immunization with chimeric rhdv-vlp-2 at the highest dose tested was able to resolve vv-ova infection. conclusion: our data demonstrated that immunization with chimeric rhdv vlps was able to protect mice from a viral challenge, suggesting the potential suitability of these constructions for new vaccine development against animal and human viral infections. objectives: a major issue pertaining to use of dna vaccines is that despite successful proof of principle results in small animal models, low efficacies have been reported in human clinical trials and large doses are required to induce protective immune responses. in this study, we describe the targeting of antigen-encoded dna directly to dendritic cells (dcs) through a pathway that results in internalisation and transfection using a cationic lipopeptide containing arginine residues and the lipid dipalmitoyl-s-glyceryl cysteine, a known tlr-2 ligand. methods: agarose gel electrophoresis was used to confirm the electrostatic binding of lipopeptide to dna encoding for green fluorescent protein (gfp), influenza hemagglutinin (ha) or nucleoprotein (np). transfection efficiencies of dcs using these complexes were determined by flow cytometry using specific antibodies for each encoded protein. stimulation of t cells by np-transfected dcs was also investigated by measuring their ability to induce in vitro cytokine secretion by influenza virus-specific cd8+ t cells. subsequently, vaccine immunogenicity was ascertained by immunisation of mice via the intra-nasal route. results: electrostatic binding of the lipopeptide to dna plasmids was confirmed by gel electrophoresis where the positively charged amino acids of the lipopeptide were able to neutralise the negative charged phosphate groups within the dna backbone and retard its ability to migrate towards the anode. high levels of gfp, ha or np were detected in murine spleen-derived cultured dcs following transfection with these complexes concomitant with the upregulation of surface mhc class ii molecules compared to when dna alone was used. the ability of transfected dcs to stimulate cd8+ t cells from influenza virus-infected mice was also investigated. subsequently, vaccination by lipopeptide-dna complexes resulted in the induction of higher numbers of ifn-g producing np 147-155 specific cd8+ t cells in the spleen and lymph nodes of mice compared to those that received dna alone. conclusion: altogether these results demonstrate that the use of a tlr-2 targeting lipopeptide-based system that can facilitate the delivery of dna by directly targeting and concurrently activating antigen-presenting cells, such as the dc, could prove to be advantageous in enhancing cellular responses induced by dna vaccination. the level of interferon in blood serum of non-infected mice was determined by elisa kit and by the cytopathic test in the l929cell culture after aplication of ridostine and ridostine ointment. the effect of the preparation on phagocytic activity of macrophages was evaluated in the monolayer peritoneal cell culture. the statistical processing of the data was carried out by the student t-criterion. ridostine was administered to patients once or twice in the case of high temperature. the clinical signs were recorded (temperature, rhinitis, headache etc). for prophylactic and treatment purposes the ridostine ointment was intranasally applied twice per day during 7 days. the effectiveness of the preparation was evaluated by clinical sings and the level of cd2+, cd4+, cd8+, cd16+ t -lymphocytes. results: ridostine significantly decreased accumulation of the virus in lungs of infected mice at the initial stage of influenza. ridostine and ridostine ointment stimulated synthesis of interferon and phagocytic activity. ridostine in clinical practice decreased the duration of influenza, attenuated clinical signs and was more effective at an early stage of the infection. prophylactic intranasal application of ridostine ointment by healthy volunteers (nurses and doctors) resulted in a high degree of protection during the whole epidemic season and an activation of t-cell immunity. application of ointment at an early stage of disease markedly activated t-cell immunity, reduced the duration of the disease and the intoxication syndrome by 1,5-2-fold. conclusion: interferon inducer based on natural dsrna stimulates some reactions of innate immunity and resistance to influenza virus. the drugs based on dsrna show promise for treatment of influenza. objectives: the creation of gene engineering vaccines against hepatitis c virus (hcv) based on recombinant proteins is one of relevant approach. since the immunogenicity of these proteins is low as a rule, the choice of adjuvant is very important. the aim of this work was to evaluate immunogenicity of covalent conjugates between nonstructural ns4 and ns5a hcv proteins and immunomax ® , an acid peptidoglycan of plant origin, which displays immunomodulating activity. objectives: ifn-g takes part in the development of an anti-infectious reaction of the organism, which is connected with the peculiarities of its immunomodulating action. a/h5n1 influenza virus inhibits the ifn-g synthesis (mibayashi et al., 2007) and causes a decrease in cd4 + and cd8 + t-lymphocytes content in lung and lymphoid tissues associated with an impairment of this cytokine production (tumpley t. m. et al., 2000) . thus, ifn-g is a promising drug for prophylaxis and treatment of avian influenza under conditions of monotherapy or complex therapy. an essential defect of this cytokine is its fast degradation in blood. the goal of this work was to study immunomodulating properties of an ifn-g structural analog with increased proteolytic resistance when it was used alone or in combination with double-stranded ifn inducers. methods: a recombinant human ifn-g analog deltaferon is distinguished by a 10 amino acid deletion at the c-end of the molecule and substitutions of amino acids in positions 129-131 (tat'kov c. i. et al., 2000) . deltaferon was i. p. administered to male non-inbred mice in doses of 2-40*10 4 iu once or twice at an interval of 48 hours, alone or in combination with double-stranded yeast rna preparation (5 mg/kg). the content of ifn-a and ifn-g in mouse blood serum was determined by the immunoenzyme method, proliferative activity of splenocytes -by mtt-test (mosmann t., 1983) . results: when deltaferon was administered in doses of 2-20*10 4 iu alone it did not influence the content ifn-a in blood, but caused a transient increase in the level of ifn-g. the injection of the preparation in a dose of 2*10 4 iu led to a an increase in both spontaneous and conconavalin a-induced proliferation of splenocytes. the two-fold administration of deltaferon in the maximal dose increased a level of ifn-g and inhibited cell proliferative activity. the combined administration of deltaferon (2*10 4 iu) and dsrna markedly increased the level of ifn-a and enhanced splenocyte proliferation. the recombinant human ifn-g analog is able to enhance ifn-g synthesis, splenocyte proliferation and to modulate the effect of ifn inducer. these data suggest that the studies of the preparation as an antiviral agent during influenza are perspective. by using a combined approach of routes of immunization and vaccine delivery systems such as poly-lactic acid (pla) biodegradable nanoparticles, we have dissected the intensity and quality of both cellular and humoral immune responses in mice. we showed that the amplitude and quality of the immune response (humoral, cellular) at systemic and mucosal sites (blood, vagina) could be largely influenced by the choice of a pertinent cutaneous route of vaccination (intradermal (id), transcutaneous (tc), subcutaneous (sc)). while id and tc route remain mostly efficient for the induction of antigen-specific cd8 responses (tetramer+ hiv-1 gag p24 cells), the quality of humoral responses (igg, iga) remained distinct between the two routes. in addition, sc route is less efficient than id and tc routes for the induction of cd8 responses after pla-p24 immunization. we have also shown that a lower antigenic charge of pla particles was needed when pla-p24 were injected using id and tc routes of immunization. currently, we are dissecting innate cellular mechanisms that gave rise to distinct quality of immune responses. this unique possibility to modulate the quality of the immune response according to the skin route of immunization paves the way for new vaccine design strategies and highlights the capacity of nanoparticles-based vaccine delivery system. b. dí az-freitas 1 , c. prego 2 , s. vicente 2 , m.j. alonso 2 , a. gonzález-fernández 1 1 university of vigo. area of immunology, department of biochemistry, genetics and immunology, vigo, spain, 2 university of santiago de compostela, nanobiofar group, department of pharmacy and pharmaceutical technology, santiago de compostela, spainobjectives: the design of effective vaccine delivery nanovehicles is opening up new possibilities for making immunization more equitable, safe and efficient. in this work, we purpose polysaccharidic-based nanocarriers as delivery structures for virus-like particle antigens, using recombinant hepatitis b surface antigen (rhbsag) as a model. our aim was to evaluate in a murine model if these nanocarriers induce an immune response after intramuscular and intranasal administration. materials and methods: loaded chitosan-based nanocarriers were prepared by cross-linking the polysaccharide chitosan, in the presence of the stabilizer poloxamer 188, with a counter ion, tripolyphosphate, containing the free rhbsag. the immunogenicity of these polysaccharidic-based nanocarriers with 1 or 2 immunizations to balb/c female mice (4 weeks old) was assessed following intranasal or intramuscular immunizations. blood samples from the mouse maxillary vein were collected at different intervals (from day 15 to 260 post-primary immunization). specific igg antibodies levels directed against rhbsag in serum were measured by indirect elisa in miu/ml. results: chitosan-based nanoparticles with particle size in nanometric range, positive zeta potential and an important rhbsag loading were prepared. the results of the specific igg levels achieved following intramuscular administration of the antigen-loaded nanocarriers, and also of the alum-adsorbed vaccine showed the significant adjuvant effect of the nanocarriers. the response elicited was delayed respect to the alum based vaccine, and very interestingly, igg concentration was much higher using antigen-loaded nanocarriers than with the conventional vaccine. after intranasal administration, chitosan-based nanoparticles generated a lower immune response compared with the intramuscular route, but increasing over the time, showing an interesting slow release of the antigen. the igg titers elicited were enough to induce full seroprotection against the disease (100 miu/ml). polysaccharidic-based nanocarriers with interesting properties for improving vaccination with complex antigens were designed and in vivo behavior of these nanocarriers suggests their potential utility as controlled release vehicles for reducing the number of intramuscular doses administered. more studies are currently underway to fully validate the potential of these nanocarrier prototypes and to optimize alternative routes of immunization such as the intranasal route. the success of immunotherapeutical approaches strongly relies on specific antigen targeting to dendritic cells (dcs) in an environment that provides optimal immunostimulatory signals. in our research group a bio-safe coronavirus-based vaccine vector platform that delivers multiple antigens to professional antigenbackground: infection with human immunodeficiency virus type 1 (hiv-1) is characterized by dysfunction of hiv-1-specific t cells. to control the virus, antigenloaded dendritic cells (dcs) might be useful to boost and broaden hiv-specific t-cell responses. poly electrolyte microcapsules are potent protein delivery vehicles which can be tailored with ligands to stimulate maturation of dendritic cells. we investigated the immune stimulatory capacity of dendritic cells loaded with these microcapsules, containing both p24 antigen from hiv-1 and the tlr3 ligand poly i:c as a maturation factor. methods: monocyte-derived dc (mddc) from healthy subjects were cultivated with polyelectrolyte microcapsules containing, poly i:c. potential maturation of dc was evaluated by flow cytometry. mddc from hiv-infected patients under highly active anti-retroviral therapy (haart) were similarly pre-incubated with p24 microcapsules containing p24 and poly i:c. these antigen loaded mddc were used to directly stimulate autologous peripheral blood lymphocytes (pbl) in elis-pot. they were also co-cultivated for 10 days with autologous pbl to evaluate the immunogenic capacity. potential expansion of specific t cells was measured by comparing elispot responses of pbl before and after coculture, using a pool of overlapping p24 peptides. intracellular staining of interferon-gamma (ifn-g), interleukin-2 (il-2) and cd107a after p24 stimulation was also performed. results: mddc from hiv(-) subjects, incubated for 24 hour microcapsules alone did not induce maturation of dc, but when poly i:c was present the dc did mature. mddc from haart treated hiv-infected individuals, cultivated with p24 containing microcapsules with poly i:c, were able to efficiently expand and broaden autologous effector memory t cells which contain and secrete ifn-g and il-2, upon p24 peptide restimulation. objectives: we aimed at investigating whether and how the distance of a cytokine from the vlp surface impacts on its function and whether the relative distance towards a co-presented antigen is critical for its biological activity. methods: we inserted one, two or four ig-like domains of hcd16b between our model cytokine il-2 and the minimal gpi-anchor acceptor sequence. subsequently, we compared particle production by western blotting for p30gag, targeting of cytokines to lipid rafts and and vlp upon isopycnic membrane separation and biological activity in il-2 dependent proliferation assays of il-2 variants. results: murine il-2 attached to either of the four fusion partners was biologically active in vitro as shown by induction of proliferation of the il-2 dependent cell line ht-2. we found that the membrane anchors comprising one and four ig-like domains (il-2::1iggpi and il-2::4iggpi) resulted in severely reduced vlp production by 293 producer cells and despite of an increased targeting of il-2::4iggpi to vlp, a reduced stimulatory capacity of producer cell crude supernatant, when compared to il-2 fused to the minimal gpi-anchor acceptor sequence of hcd16b (il-2::gpi). il-2::2iggpi, however, showed comparable particle production and biological activity in vitro when compared to il-2::gpi. furthermore, il-2 fused to 2ig::gpi showed an increased capacity to co-stimulate primary p14 tcr transgenic t-cells specific for lcmv-gp 33-41 in the context of h2-d b . conclustions: besides the minimal gpi-anchor acceptor sequence we have identified one additional membrane anchor, which displays superior capacity to target cytokines to vlp. 2ig::gpi has a biological activity in vitro, which is comparable to the minimal gpi anchor. moreover, il-2::gpi displays increased co-stimulatory potential in the context of specific mhcp complexes. this work was supported by grants sfb f1816-b13 of the austrian science foundation, the austrian research promotion agency (forschungsförderungsgesellschaft) bridge grant 812079 & biomay ag, and the christian doppler laboratory for immunomodulation. a. roemhild 1 , interdisciplinary transplant laboratory 1 charite berlin, insitute of nephrology and medical immunology, berlin, germany immunosuppressive treatments, e. g. after transplantation are often followed by an impaired or dysfunctional immune system. missing viral immunity, particularly against ebv, is an essential key player in the development of severe infections and posttranplant lymphoproliferative disorders (ptld). ptld affects 2-25 % of solid organ transplant recipients, depending on the organ transplanted. healthy individuals control ebv infection by ebv-specific cytotoxic t lymphocytes (ctls), but some patients under immunosuppression are unable to do so. in these cases, immunotherapy is increasingly used as a new approach for re-establishing a functional immune response by retransferring in-vitro expanded autologous virusspecific t cells into the patient. currently these t cells are generated by repetitive stimulations with ebv-infected autologous lymphoblastoid cells (lcls). due to a generation time of 2-3 months, many patients suffering from missing viral immunity and subsequent severe viral disease are excluded from therapeutic benefit. therefore, shortening the generation time would be an important step to make adoptive immunotherapy available for more patients. t cell lines were generated with two different protocols. in the first protocol t cells are generated by repetitive stimulation with ebv-infected autologous lcls. the second protocol is based on stimulation with 6 different overlapping ebv peptide-pools and immunomagnetic cell isolation. expanded t cells were analysed using multicolour flow cytometry. cells were stained for diverse surface markers and intracellular cytokine production. cytotoxic capacity and specificity was determined by a calcein release assay. our group developed a new protocol for the production of ebv specific t cells, thereby shortening the generation time from 2,5 month to 16 days. t cell lines are composed of cd8 and cd4 cells with a mainly effector memory like phenotyp. after restimulation the cells produce more tnfa than ifng. depending on the generation protocol t cells specifically recognized and lysed autologous lcls alone or loaded with ebv-peptides. the detailed characterization of ebv-specific t cell lines should help to further improve the adoptive immunotherapy and its outcome. the novel, short time generation protocol did not affect phenotyp and cytokine production of the t cells. nevertheless their therapeutic potential in vivo has to be tested in further experiments. s. s. schmucker 1 , m. assenmacher 1 , a. richter 1 1 miltenyi biotec gmbh, r&d cell biology, bergisch gladbach, germanyadoptive transfer of virus-specific t cells provides a promising treatment of infection in immunocompromized patients. as expansion of virus-specific t cells from antigen-experienced donors is feasible, no reliable protocols for generation of antigen-specific t cells from naive hosts exist. in this study we established a cell culture system for priming of highly rare naive cmv pp65-specific cd4 + and cd8 + t cells from cmv-seronegative donors in vitro.magnetically isolated naïve (cd45ro -cd25 -) cd4 + and cd8 + t cells from pbmc of cmv-seronegative donors were co-cultured with autologous mature monocytederived dc loaded with cmv pp65 peptide pool and cd3-depleted autologous pbmc as feeder cells in the presence of il-2, il-7, and il-15. already 9-13 days after primary activation pp65 495-503 /a2-tetramer + cd8 + t cells were detectable for 3 hla-a2 + blood donors. to analyze cd8 + t cells having other specificities than for the peptide pp65 495-503 as well as probably primed cd4 + t cells, we looked for the production of cytokines after a second stimulation. we found ifn-g secretion in up to 3.9% of the cd8 + t cells and up to 3.8% of the cd4 + t cells after restimulation with pp65 peptide pool, but not with either irrelevant ie-1 peptide pool or without antigen, in each of eight donors tested. for generation of t cell lines, we magnetically enriched the primed t cells according to their ifn-g secretion. subsequent cultivation for 20 days led to a 10 -100 fold expansion of pp65-specific t cells, defined by their sustained capability to produce ifn-g. evaluation of the antigen-specificity of the expanded t cells also showed upregulation of the activation markers cd154 and cd137 only if restimulated with the pp65 peptide pool. further cytokine analysis of the cells revealed co-production of ifn-g, tnf-a, and il-2, indicating the functionality of the in vitro primed and expanded t cells.in conclusion, we established a cell culture system, which enables the in vitro priming and expansion of cmv-specific cd4 + and cd8 + t cells derived from the naive compartment. this should extend the application of adoptive t cell therapy to patients for whom immune donors are not available. a. i. wolf 1 , k. mozdzanowska 1 , l. otvos 2 , j. erikson 1 1 the wistar institute, philadelphia, united states, 2 temple university, philadelphia, united statesthe influenza virus a matrix protein 2 ectodomain (m2e) sequence has remained highly conserved among various human influenza a strains and is therefore a promising target for a protective vaccine. based on previous work using a synthetic m2e-based multi-antigenic peptide vaccine (mozdzanowska at al., vaccine 2003; virology journal 2007), we generated a novel peptide and investigated its efficacy in inducing an anti-m2e antibody (ab) response and its ability to confer protection against viral challenge.objectives: cytomegalovirus (cmv) causes significant morbidity and mortality in patients after haematopoietic stem cell transplantation (hsct). due to limitations of current antiviral therapies, alternative approaches, involving transfer of donor-derived cmv specific cd8 + t cells, have been considered. clinical data confirm a crucial role for antiviral cd8 + t cells inversely correlating with the incidence of cmv reactivation and disease. cmv specific cells have to reach protective levels in order to be effective. levels of such cells correlating with protection against cmv infection and disease have only been reported in patients expressing hla-a*0201 and hla-b*0702 previously. considering other frequent hla alleles cmv specific cd8 + t cells were monitored longitudinally in 30 hsct patients in this study to establish the cell number thresholds at which patients are protected from cmv reactivation. methods: we have correlated the pattern of different ex vivo cmv peptide specific cd8 + t cell responses (frequency analysis using tetramer staining and interferon gamma elispot analysis) with the cmv viral load (dnaemia) and clinical status in patients. different response groups were compared using the mann-whitney-u test.results: our results demonstrate that the presence of different cmv specific cd8 + t cells inversely correlates with the ability to detect of cmv reactivation in patients at different cell number thresholds. we show that the cell number thresholds for hla-a*2402/pp65 (341-349) (7.6x10 6 cells/l) and hla-b*3501/pp65 (123-131) (4.4x10 6 cells/l) specific cd8+ t cells are significantly lower than those for hla-a*0101/pp50 (245-253) (17.2x10 6 cells/l) and hla-a*0201/pp65 (495-503) (13.2x10 6 cells/l) specific cd8 + t cells in hsct recipients post transplant. this difference is also evident in healthy cmv seropositive volunteers. conclusion: these findings suggest a differing efficiency of the responses restricted by the two sets of alleles. the data merit further studies using larger patient cohorts and are important for considerations regarding the epitope restriction and quantities of ag specific t cells to be monitored after therapeutic strategies for cmv in hsct patients. (2,5 -50 mcg) . no adverse effects were indicated during trials (up to 25 month of observation).hiv-specific antibodies were induced by dose-dependent manner, the most prominent response was detected after 4th immunization with 50 mcg of vichrepol.no differences were detected in cd4+ and cd8+ t cell counts and cd4+/cd8+ ratio, so there was additional safety issue concerned to the possible sensitivity of vaccinees to hiv infection. the results of phase i clinical trials of vichrepol vaccine were approved by who authorized russian national control institution and transition to phase ii immunogenicity trials was recommended. objectives: to improve the vaccination efficiency of adenoviral vectors for anti-retroviral vaccination, we constructed adenoviral nanoparticles by fusion of the vaccine antigen to the adenovirus capsid protein pix. the adenoviral nanoparticle vaccine was evaluated in the friend virus (fv) mouse model and compared to conventional adenoviral vectors. methods: adenoviral nanoparticle vectors were constructed by deletion of pix from the adenoviral genome and insertion of the fusion protein encoding sequence as transgene. for vaccination against fv, that is a retrovirus complex of friend murine leukemia virus (f-mulv) and spleen focus forming virus, we constructed fusion proteins of pix and the f-mulv surface env protein gp70 or gag. to elucidate underlying mechanisms we produced displaying-only nanoparticles and plasmid dna encoding either pixgp70 or gp70 alone. conventional adenoviral vectors were used that express full-length f-mulv env and gag. the vaccines were tested in cb6f1 hybrid mice that are highly susceptible to fv infection and develop viremia and splenomegaly after fv infection. results: vaccination of cb6f1 mice with adenoviral nanoparticles expressing fusion proteins containing gp70 resulted in protection from viremia and splenic enlargement after fv challenge that was superior to vaccination with conventional vectors. immunological analyses showed that the adenoviral nanoparticle vaccine induced a significantly higher number of f-mulv env-specific cd4 + t cells and higher antibody titers than a conventional adenoviral vaccine expressing the vaccine antigen. we could show that for the beneficial effect it is necessary that the fusion protein is incorporated into the adenoviral particle and it also has to be expressed from the adenoviral vector in vivo. conclusion: adenoviral nanoparticles are a useful tool for the induction of antibody and cd4 + t cell responses that are superior to conventional adenoviral vectors. this new type of adenovirus-based vaccination vector combines genetic and protein vaccination and should make adenoviral vectors even more interesting for vaccination purposes. . antibody levels were monitored by elisa and hemagglutination inhibition assay, viral excretion in nasal washes was assessed by quantitative rt-pcr, and cellular production of ifn-gamma was measured via flow cytometry. results: we found that animals vaccinated with caf01 exhibited higher levels of serum igg and mucosal iga than the ones which received the vaccine alone, and that they excreted 90-99 % less virus. animals that received only vaxigrip were producing ifn-gamma after challenge, a sign of infection by low virulence influenza strains, whereas the animals that received also caf01 did not show any increase in their levels of ifn-gamma. conclusion: caf01 enhances the protection conferred by the commercial inactivated vaccine against strains matched by the vaccine. evaluation of the t-cell specific immune response is very important for global eradication of measles and rubella. peripheral blood lymphocytes (pbl) from 13 children aged 1-2 years old (6 boys and 7 girls) -group 1, and 11 children (6 boys and 5 girls) 6-7 years old -group 2 were isolated on a gradient of density before vaccination ( or revaccination) with priorix, 1 week, and 2 months after and incubated with cfse. then 2 million/ ml pbl were incubated in rpmi-1640 supplemented with 10 % fcs (the negative control), at presence of 5 mcg/ml pha (the positive control) or at presence of the measles or rubella viruses antigens in a humidified atmosphere containing 5 % cî 2 at 37°c within 7 day. intensity of a fluorescence estimated on fl1 by flow cytometr facscalibur (bd biosciences, usa). cytokines production was measured in the same cultures by bioplex technology (biorad, usa). in the negative control 90 % pbl in both groups did not enter mitosis. in the positive control 90 % of cells have passed one and more mitoses. in group 1 measles or rubella antigens did not induced lymphocytes to enter mitosis, like in negative control, before the vaccination and in a week, however in 2 months 15-25 % of lymphocytes demonstrated antigen-specific proliferation. in group 2, on the contrary, before the vaccination the most part of cells (75-80 %) has not entered division, but 20-25% of cells have passed 2 and more mitoses. in a week specific lymphocyte proliferation decreased and in 2 months it was increased up to 40-50 %. production of the interleukin (il) 6, ifn-g, tnf-a, il-4, il-1 was more informative than il-5, il-7, il-8, il-12. measles and rubella antigens induced cytokines production in pbl of immune children and did not influence on pbl of intact children. thus, it was shown, that both methods can be applied to revealing the specific cellular immune response to measles and rubella antigens. objectives: broadly neutralizing human monoclonal antibodies (mab) and patients' sera recognizing functionally conserved epitopes on hiv envelope (env), such as the gp120 cd4-binding site (cd4bs), appear to be uncommon. therefore, new approaches are needed to elicit the humoral response on these conserved epitopes. here we describe the generation of two anti-idiotype (ai) murine antibodies recognizing human anti-hiv-1 neutralizing polyclonal iggs directed against the cd4bs. the mabs were shown to react with an anti-cd4bs human neutralizing mab (b12), to elicit antibodies that recognize the gp120 molecule and an anti-hiv-1 neutralizing response in rabbits, confirming them as cd4bs mimotopes. these mabs were also used as probe to detect the expression of clonally distinct anti-gp120 antibodies in sera of hiv-infected individuals. methods: broadly neutralizing sera were collected from long-term non-progressor patients. anti-cd4bs iggs were purified and used to immunize mice for hybridoma generation. mabs reacting in elisa with the anti-cd4bs igg fraction were used to immunize rabbits. rabbit sera were then tested for anti-gp120 titer and hiv neutralizing activity by pseudovirus-based neutralization assay. sera from hiv-infected individuals at various clinical stage of infection were studied to validate an immunoenzymatic assay able to detect the reactivity to the ais. serial dilution of b12 in sera from healthy hiv-negative donors were used to determine elisa sensitivity. results: two clones (p1 and p2) reacted in elisa only with the cd4bs-directed igg fraction. the clones were also recognized in elisa by b12. p1 and p2-immunized rabbit sera showed a strong anti-gp120 titer. in the pseudovirus assay the ais-immunized rabbits showed a neutralization activity against virions bearing hxb2 strain glycoproteins. in particular, 3/5 rabbits in the p1 group and 1/5 in the p2 group showed an 80 % hiv neutralization at dilutions ranging from 1:20 to 1:150. the immunoenzymatic assay used, allowed to detect a p1 and p2 reactivity in hiv-positive sera and was able to detect a b12 concentration equal to 10 ng/ml. conclusions: these data demonstrate that immunogens designed on the idiotype of broadly neutralizing abs are feasible and could help in the design of effective anti-hiv vaccines or diagnostic assays. yellow fever vaccines (17d and 17dd) are well tolerated, with a very low rate of severe adverse events (yf-sae), such as serious allergic reactions, neurotropic (yf-and) and viscerotropic (yf-avd) diseases. viral and host factors have been postulated to explain the basis of yf-sae, especially those able to modify the host immune response to the yf vaccine. however, the mechanisms underlying the occurrence of yf-sae still remain unknown. in the present investigation, we present a detailed immunological analysis of a 23-year-old us citizen female patient, who developed yf-and characterized by encephalitis associated with pancreatitis and myositis following 17d-204 vaccination. our findings highlighted that yf-and exhibited decreased expression of fc-g-r in monocytes (cd16, cd32 and cd64) along with increased levels of nkt-cells (cd3 + cd16 +/-cd56 +/-/cd3 + ratio) and activated t-cells (cd4 + and cd8 + ) and b-lymphocytes. enhanced levels of plasmatic cytokines (il-6, il-17, il-4, il-5 and il-10) besides exacerbated ex vivo intracytoplasmic cytokine pattern, mainly observed within nk-cells (inf-g + , tnf-a + and il-4 + ), cd8 + t-cells (il-4 + and il-5 + ) and b-lymphocytes (tnf-a + , il-4 + and il-10 + ). the analysis of cd4 + t-cells revealed a complex profile with increased frequency of il-12 + and ifn-g + and decreased percentage of tnf-a + , il-4 + and il-5 + cells. depressed cytokine synthesis was observed in monocytes (tnf-a + ) following in vitro antigenic stimuli. these results support the hypothesis that a robust magnitude of the adaptive response and abnormalities in the innate immune system may be involved in the establishment of yf-sae. this is the first case report of yf-sae investigated by members of the international laboratory network for yellow fever vaccine associated adverse events. g. mester 1 , h.-g. rammensee 1 , s. stevanović 1 1 eberhard-karls-universität tübingen, department of immunology, tübingen, germany adenovirus (adv) is a widespread pathogen in humans and can persist in its hosts after infection. persistent virus is an important cause for severe disease in immunocompromised individuals, e. g. bmt recipients, with high rates of mortality. however, the cellular immune response against adv is poorly characterised, and very few t cell epitopes have been published up to now. thus, our aim was to detect dominantly immunogenic adenoviral cd8 t cell epitopes by analysing the responses of healthy blood donors who have overcome infection. we have predicted possible cd8 t cell epitopes for the frequent mhc class i alleles a*01, a*02, and a*24 from the proteins pii (hexon), pviii, and e1a of adv strains ad2 and ad5 by using the syfpeithi software developed by our group (www.syfpeithi.de). subsequently we performed a 12-day recall stimulation of pbmcs from at least 16 healthy donors with synthetic peptide followed by ifn-g elispot screenings to identify naturally occurring t cell responses and assess their frequency in the population. tetramer and intracellular cytokine stainings were also carried out to confirm the presence of specific cd8 t cells. we could identify 27 new peptides eliciting ifn-g responses, several of which were confirmed as novel cd8 t cell epitopes. amongst others we found at least one immunodominant epitope recognised by more than half of the healthy donors for each examined hla restriction as well as, to our knowledge, the first adenoviral epitope derived from a protein other than hexon. these findings will be helpful to identify frequently immunogenic and thus promising candidate peptides for in vitro t cell priming or expansion preceding adoptive transfer, which has been proven to be a valuable therapeutic approach in the treatment of persistent viruses in immunocompromised patients. methods: sle (5 ara criteria) was diagnosed in a 40-year-old african female patient with hiv-1 (clade c) infection. good initial response occurred on hydroxychloroquine and steroids followed by disease flare and drop of cd4 t-cell count x 200 cells/mm 3 . initiation of 500 mg mmf bid was associated with biological and clinical remission of sle and cd4 t-cell increase. no opportunistic infections or cancers were noted during a 3-year follow-up and the patient remained always naive to art. hiv-1-specific cd4 and cd8 t-cell responses were analyzed after 18 months of mmf by ifn-g elispot assay and polychromatic flow cytometry assessing ifn-g, tnf-a and il-2 production following stimulation with a panel of 192 hiv-1-derived optimal epitopes (9/10-mers) covering various hiv regions and a pool of 105 hiv-1-derived peptides (15-mers overlapping by 11 aminoacids) encompassing the entire gag protein. all peptides are derived from hiv-1 consensus strain iiib. results: highly polyfunctional hiv-1 specific cd4 and cd8 t-cell responses against gag were detected. 11 epitope-specific cd8 t-cell responses were identified: except for one response restricted by hla a*23 and another one by hla cw*07, all the others were restricted by hla-b alleles and mostly by b*58 (n = 5). seven out of 11 responses were strong enough to be further analysed with regard to their functional profile and shown to be highly polyfunctional (i. e. ifn-g+, tnf-a+ and il-2+) regardless of the viral region and hla restriction. conclusion: strong, broad and polyfunctional hiv-1 specific cd4 and cd8 t-cell responses known to be associated with nonprogressive infection were detected during mmf treatment.we therefore suggest that mmf use in the context of sle-hiv is not detrimental to the establishment or preservation of protective hiv-1 t-cell immunity. the rabies virus was propagated in the vero cell line. virus was titrated by focus fluorescent units. virus preparations having a titer of 10 6 dl50/ml were inactivated with b-propiolactone. aluminium hydroxide gel or squalene, at different concentrations were adsorbed to the inactivated rabies virus. male mice of the strain cf-1 of 12-16 g and no less than four weeks age, were distributed in six groups for intraperitoneal immunization, group a was immunized with virussqualene, group b with virus-aluminium hydroxide, group c with the antigen alone, group d with saline buffer-squalene, group e with saline buffer-aluminium hydroxide and group e was inoculated with mock-infected cell culture supernatant. mice were boosted at the 7 th day. all mice were properly bled to prepare preimmune sera and hyper-immune sera. at the end of the immunization protocol the igg raised against the rabies virus was tested by an indirect elisa. results: the highest titers of neutralizing antibodies were obtained with similar concentrations of either squalene-or aluminium hydroxide-based vaccine formultaions. there was a significant difference in the neutralizing antibody titers produced by mice immunized with the antigen (inactivated rabies virus) adsorbed to the adjuvant, as compared to those obtained from mice immunized with the antigen alone, as expected, no neutralizing antibodies were detected on mice inoculated with saline buffer or mock-infected vero cell supernatant. conclusions: the use of either squalene or aluminium hydroxide as adjuvant in the canine antirabic vaccine formulation increases immunogenicity, almost to the same extent. aluminum hydroxide adsorbed to the antigen seems to be a better option, since squalene is more expensive than aluminium hydroxide. supported by: concyt-46767, cofaa and cgpi-20090305. . state of vaccine-induced measles immunity was determined by means of elisa in 1-3, 4-6 and 7-9 years since revaccination with live measles vaccine (lmv) before and after tuberculosis chemoprophylaxis. statistic data were processed with t-, w-and u-criteria. results: during the first three years since lmv revaccination igg level was middle (children with negative and long-term positive mt) and high (children with conversion and hyperergic mt). in 4-6 years since lmv revaccination uninfected and long-term infected children showed a significantly decreased (p p 0.05) measles immunity and antibody level much lower (p p 0.05) than among children with mt conversion. in 7-9 years the comparison group kept decreased (p p 0.05) measles immunity, the majority (92±5.54 %) of persons had minimal protected igg level, but the observation groups were characterized by average immunity level, which was higher (p p 0.05) than in the comparison group. comparing measles immunity level before and after tuberculosis chemoprophylaxis demonstrated the following: measles igg level among long-time infected children on completion of chemoprophylaxis decreased (p p 0.05), the majority (83.3±4.1 %) of persons lost protected antibody level; among children with mt conversion in 1-3 years since lmv revaccination immunity state didn't change, but in further periods antibody level decreased (p p 0.05) to low values; among children with hyperergic mt igg level decreased (p p 0.05) and reached low (in 1-3 years), minimal protected (in 4-6 years) and lower than protected (in 7-9 years) values. -at the early stage of tubercular infection process measles immunity was higher compared to uninfected with mycobacterium tuberculosis persons, which fact is connected with immunomodulatory action of low-molecular peptide of bacterial cell wall -muromildipeptide.-in remote periods since lmv revaccination and on completing preventive tuberculosis treatment decreased measles immunity was observed.-in countries with high tuberculosis morbidity chemoprophylaxis level among children with latent infection is high, which can indirectly influence population measles immunity. objectives: to evaluate the balance of ifn-gamma and il-17 producing cells in lungs during the immunotherapy of tuberculosis with the dna vaccine encoding the heat-shock protein 65 (dnahsp65). methods: balb/c female mice were infected by intra-tracheal route with 10 5 h37rv mycobacterium tuberculosis. immunotherapy with endotoxin free dnahsp65 genetic vaccine was done at days 30, 40, 50 and 60 post-infection. each dose consisted of 100 micrograms of dna vaccine in the quadriceps. intracellular cytokine staining of cd4+, cd8+ and gamma-delta t cells from lungs were determined 10 and 50 days after the end of the therapy. bacilli loads, histopathological and morphometric analysis of lungs were also evaluated. differences of p x 0.05 were considered significant (t test). results: at day 10 after the end of the immunotherapy, dnahsp65 treated mice exhibit increased numbers of absolute cd8+ and gamma-delta t cells when compared to non-treated animals. the percentage of ifn-gamma and il-17 producing gamma-delta t cells were the same between treated and non-treated animals. in contrast, dnahsp65 treated mice showed more ifn-gamma producing cells in both cd8+ and cd4+ cell populations. at day 50 after the end of the therapy, the main observation in mice which received dnahsp65 treatment was the augment of all three populations producing ifn-gamma. although non-treated animals also increased the frequency of cd4+ and gamma-delta t cells positive for ifn-gamma, they did not increase the numbers of ifn-gamma cd8+ cells, together with a more frequency of gamma-delta t cells producing il-17. finally, the immunotherapeutic effects of dnahsp65 vaccination also included the diminution of bacilli loads in lungs, spleen and liver and the reduction of inflammation in lungs as determined by the histopathological and morphometric analysis. the results presented here indicates that cd8+ cells producing ifn-gamma and the reduction of the frequency of gamma-delta t cells secreting il-17, are the main effects of dnahsp65 immunotherapy of murine tuberculosis. furthermore, these results have important implications since they indicate the importance of an appropriate balance of il-17 and ifn-gamma levels for the combat of the bacilli and the reduction of the immunopathologic damage in lungs. the detection of quantitative changes in mrna expression levels are currently being performed using either genome-wide (microarray) or single gene (real-time pcr) screening methods. because these techniques are technically challenging and too costly to be applied on a routine basis in resource poor settings, we have developed a reverse-transcriptase multiplex ligation-dependent probe amplification (rt-mlpa) method. rt-mlpa is a reliable, robust, low cost and user friendly technique permitting rapid mrna expression profiling of as many as 60 loci in a single reaction. genes of interest can be selected on a tailor-made basis. the assay is highly reproducible, has an extensive dynamic range of 3-5 log depending on the genes of interest, and a pcr amplification step within the rt-mlpa ensures assay sensitivity, which is an essential prerequisite for the relative quantification of scarcely expressed genes. since this assay is relatively high throughput (96-well format), requires only 100 ng rna per sample, and allows mrna profiling in direct ex vivo whole blood samples (from e. g. pax-gene tubes), it is an exceptionally suitable technique for performing semi-large scale gene expression analyses in human cohort studies. to illustrate this, we have been able to successfully implement this assay in 5 different laboratories in sub-saharan africa. thus far we have applied rt-mlpa to characterize the human immune response to mycobacterium tuberculosis, with particular emphasis on the expression of genes associated with protective host cellular immunity and human disease susceptibility. a particularly useful application of the rt-mlpa is the identification and monitoring of host-biomarker profiles that predict (protection from) tuberculosis (tb) disease in latently infected household contacts or (in)adequate responsiveness to therapy in active tb patients. initial data sets already probe differences in immune reactivity in populations, yielding new candidate biomarkers associated with tb disease. these biomarkers may provide new and relevant information that can be applied in future tb studies for rapid, easy, semi-quantitative and reliable detection of host immune biomarker profiles. preclinical m. leprae infection is a major source for leprosy transmission. therefore, early detection of individuals infected with m. leprae is crucial. however, to date there are no diagnostic tests available that can identify preclinical leprosy. such tests will contribute to the prevention of leprosy disability and its further transmission by otherwise undiagnosed and untreated index cases.newly developed hla based bio-informatic tools combined with comparative genomics have created novel opportunities to help design improved tests for early detection of m. leprae infection.using this post genomic approach, we were able to identify candidate proteins and peptides unique to m. leprae containing predicted t cell epitopes restricted via several major hla-class i and ii alleles. since the selected genes were of unknown function, their expression in m. leprae bacilli was assessed.evaluation of the immunogenicity of these m. leprae proteins in pbmc from a brazilian population showed that 5 candidate antigens induced significant ifn-g levels in m. leprae infected individuals but not in healthy controls from an endemic area.importantly, among exposed healthy controls 71 % had no detectable igm antibodies to the m. leprae specific pgl-i, but instead responded to one or more m. leprae antigen(s). to further improve the diagnostic potential of these m. leprae sequences, synthetic peptides spanning all 5 m. leprae proteins were analyzed similarly. determination of cumulative t cell responses towards 4 of these peptides that activated pbmc of leprosy patients increased the sensitivity compared to single peptides to 100 % in pb, 75 % in rx and 93 % in hhc, without compromising specificity.since diagnostic tools should be applicable in several populations regardless of the genetic background, these m. leprae antigens are also tested in populations on the african (ethiopia) and asian (nepal) continent.in addition, we have applied these antigens in a new user-friendly ucp-lf assay to detect different cytokines. this assay proved to be more sensitive than elisa for detection of ifn-g and can be easily applied in field sites. tuberculosis, an infectious disease caused by mycobacterium tuberculosis (mtb), affects millions of people. m. bovis bcg is the vaccine against tuberculosis but its efficiency is variable for the pulmonary form of the disease. paratuberculosis, an enzootic bacterial disease in ruminants, due to mycobacterium avium subsp. paratuberculosis (map), has a significant economic impact on livestock production, and moreover, map infection may be one of the microbial triggers of crohn's disease in humans. map vaccines can delay apparition of clinical symptoms, but they do not prevent infection and they have a confounding effect in the skin-test based bovine tuberculosis control programs. cd40l, a co-stimulatory molecule preferentially expressed on activated cd4+ t cells, is the ligand of cd40. cd40-cd40l interaction induces the production of il-12 and the initiation of a th1-type immune response. several studies show that cd40l is required for the activation of macrophages and the maturation of dcs. moreover, cd40l enhances the capacity of cd8+ t cells to produce ifn-g and to lyse mtb-infected monocytes. in this study we attempt to improve existing tb and map vaccines with a recombinant bcg expressing cd40l. we have constructed the recombinant bcg strain expressing cd40l (rbcg2) by electroporation of bcg with pgfm11/signalsequenceag85b-cd40lec and an another recombinant strain with empty vector pgfm11 (rbcg1) as a control. the expression of cd40l has been evaluated by western blotting. balb/c mice were vaccinated with the recombinant bcg vaccines. bcg growth kinetics were compared by counting viable bacteria (cfu) in spleen and lungs. the immune response was evaluated by measurement of th1 type cytokine secretion of splenocytes after in vitro restimulation with immunodominant antigens and selected peptides. two months post vaccination, mice were challenged with mtb and map and protection was evaluated. preliminary results show normal persistence of the two recombinant bcgs. analysis of the immune response shows an effect of cd40l 2 weeks after vaccination but not at 4 and 8 weeks. rbcg2 seems to be more protective against paratuberculosis than rbcg1, but not against tuberculosis. another vaccination experiment is required to confirm these results. the effects of bcg-cd40l on cultured dcs in vitro will further be explored. objectives: tuberculosis is a major health problem globally and it is of critical importance to develop an effective vaccine to prevent further spread of the disease. iron is a key nutrient for both mycobacterial infection and for a successful protective immune response by the host. the regulation of iron availability within the host involves the intracellular iron-binding protein ferritin and it is proposed here that the regulation of ferritin is tightly controlled in the host immune response to tuberculosis. methods: using the guinea pig model of mycobacterium bovis bacillus calmette-guérin (bcg) vaccination, populations of immune cells were isolated and restimulated ex vivo over a time-course study using purified protein derivative (ppd) of mycobacterium tuberculosis or infected with bcg or m. tuberculosis. the expression of ferritin in co-ordination with key immuno-regulatory proteins, tnfa, ifng and il-1a, was examined using real-time pcr. to determine whether immuno-regulatory proteins are involved in the regulation of ferritin, cytokine cascades were inhibited in the ppd re-stimulation studies by the addition of guinea pig specific tnfa and ifng antibodies. results: a typical pro-inflammatory immune response was observed with significant up-regulation (p x 0.05) of tnfa, ifng and il-1a after re-stimulation with ppd and mycobacteria. of interest was a trend in ferritin down-regulation after re-stimulation with ppd and bcg and this was significant (p x 0.05) after restimulation with m. tuberculosis. the down-regulation of ferritin was also affected by the addition of tnfa antibody in the ppd re-stimulation study. conclusions: ferritin is important in the storage and management of intracellular iron and its regulation must be tightly controlled to restrict iron availability from invading mycobacteria from sequestering free iron. the data indicate that the regulation of ferritin is very subtle and is affected by cytokine cascades that involve tnfa. these results contribute to our understanding of the role of iron and intracellular ferritin in developing a protective immune response to mycobacteria in the guinea pig model of tuberculosis. this work is funded by health protection agency phd studentship award. methods: anti-cd40 mab and ag85a were chemically treated with sata and sulfo-smcc respectively, in order to produce a stable crosslinker between both proteins. crosslinking was confirmed by western blotting and cd40 binding on cd40 transfected l929 fibroblasts. the conjugates were tested in vivo in wild type and cd4 + cell-depleted mice for the induction of specific anti-ag85a serum antibodies. splenocytes were challenged ex vivo with ag85a and were examined for their ability to produce th1-related cytokines. elispot assays were performed to determine ifng production and flow cytometry was used to analyse intracellular cytokine staining for tnfa, ifng and il-2. we developed a method to successfully crosslink anti-cd40 mab to ag85a. serum antibodies against ag85a were detected after immunisation with this conjugate vaccine in both wild type and cd4 + cell-depleted mice. t cells derived from mice immunised with conjugate vaccine, and stimulated ex vivo, showed an increase in ifng production (elispot), when compared to mice vaccinated with ag85a alone. production of two other th1-related cytokines, tnfa and il2, was also increased in these t cells as shown by intracellular cytokine staining. conclusion: our results suggest that anti-cd40 conjugate vaccines could provide a new way to increase vaccine efficacy. this new conjugate vaccine may be able to by-pass the need for cd4 + t cell help in the production of specific antibodies, which would be a major benefit of any therapeutic vaccine to be used in immunocompromised patients. h. schäfer 1 , r. burger 2 1 robert koch-institute, cellular immunology, berlin, germany, 2 robert koch-institute, infectious diseases, berlin, germanyobjective: immunity against mycobacterial infections is mediated by both cd4-positive and cd8-positive t-lymphocytes. cd8-positve cells respond to peptides derived from cytosolic proteins and presented on mhc class i molecules of antigen presenting cells (apc) via the endogenous pathway. some apc however, are able to take up extracellular antigens and present peptides thereof on mhc class i molecules. this process has been termed cross-presentation and has been shown to be of importance in the immune response against intracellular bacteria. to define the contribution of cross-presentation to activation of cd8+ t cells in the response against mycobacterial antigens, we analyzed the secondary immune response in the guinea pig. methods: purified t lymphocytes from guinea pigs immunized with bcg or complete feund's adjuvant were labeled with the intracellular fluorescent dye cfse and incubated with ppd and/or apc for 5 days. surface phenotype and proliferation of t-lymphocytes were analyzed by flow cytometry.results: up to 30 % of lymph node t lymphocytes form immunized guinea pigs proliferated after in vitro restimulation with ppd-pulsed macrophages. no difference was observed between bcg-(living mycobacteria) and freund's adjuvant (heat killed mycobacteria)-immunized animals. the responses of both t cell subsets were equally strong, although the killed immunogen should primarily target the exogenous pathway of antigen presentation and therefore preferentially prime cd4+ t-lymphocytes in vivo. similarly, the cd4-positive subpopulation should primarily respond to soluble antigens presented on mhc class ii molecules. proliferation of both the cd4+ and the cd8+ subpopulation depended on the presence of apc. stimulation oft cd8+ cells as a consequence of direct loading of peptides onto mhc class i molecules was ruled out by using mhc-class i-positive fibroblast cells instead of professional apc, which did not lead to proliferation of primed t-lymphocytes. conclusion: cross-presentation of soluble antigens to cd8-positive t cells is a highly effective means to stimulate the response of cytotoxic t cells against mycobacterial antigens even without direct contact to infected cells. therefore cross-priming might represent an important mechanism for the induction of the cellular immune response against intracellular pathogens and should be useful for the rational design of vaccines against mycobacterial diseases. objectives: due to broad antigenic cross reactivity of purified protein derivatives (ppd) with bcg vaccine strains and environmental mycobacteria, results of currently used tuberculin skin test (tst) is not reliable to evaluate the specific anti-tuberculosis immune response. therefore, new tools are required to improve mycobacterim tuberculosis (tb) diagnosis and treatment, including enhanced ability to compare new treatment strategies. among different antigens early secretory antigenic target 6 (esat-6) protein is highly specific for tb complex and elicit strong t-cell response in human. in order to monitor the immune response against the pathogen, 83 iranian and afghan adults (38 patients with sputum smear and culture positive tuberculosis, 24 recovered patients during 6 months after full course of chemical treatment and 21 healthy individuals) were recruited to quantify the frequency of esat-6 and ppd specific t-cells in their peripheral blood by home made elispot ifn-gamma assay. results: considering cut off of 4 spot forming unit ( g 20 spots per million), we found detectable response to esat-6 in almost 80 % of patients with active disease. this frequency among treated patients after disease recovery was not significantly different and 77 % of these individuals had detectable esat-6 specific response even after six months completing treatment. neither of healthy individuals showed such response. t cell response against ppd was identified in 14 %, 57% and 62 % of healthy participants, active patients and healing individuals, respectively. conclusion: elispot ifn-gamma assay showed a sharp induction of th1 immune response, against esat-6, in tb patients which persists after successful treatment and full recovery. these results may show potential application of tuberculosis-specific elispot testing as a proxy measure of tb diagnosis and treatment. bcg is the only available vaccine today to fight tuberculosis, but it has been reported to be variably efficacious in the field. both environmental and genetic host factors as well bcg strain variability and virulence of the intruding m. tuberculosis strain have been suggested to affect the efficacy of bcg vaccination. in mouse and bovine models it has been shown that pre-exposure to mycobacterium spp. negatively affected protective efficacy of bcg. we use non-human primates (nhp) for the evaluation of new tb vaccine candidates and possible identification of immune mechanisms of protection. however, in naive rhesus macaques a variable efficacy of bcg reminiscent of the clinical situation was revealed. by meta-analysis we compared immune response parameters measured after vaccination using bcg strain danish 1331 in the context of protection measured by gross pathology evaluation after experimental infection with a constant m. tuberculsosis strain erdman. although numbers of animals used are relatively low, data suggest that both breeding origin as well as the immune status of monkeys impact on the efficacy of bcg. most remarkably, while bcg induced levels of ifng secretion did not correlate with protection, kinetics of secretion monitored after in vitro stimulation of peripheral blood lymphocytes did correlate. our findings would suggest that, in accordance with mouse and bovine experimental data and epidemiologic observations, possible pre-exposure to mycobacterial antigens beyond the current sensitivity of tb diagnostics for nhp, negatively affects the protective efficacy of bcg. together, these results are relevant for evaluation and interpretation of tb vaccine tests in nhp and support further research into the identification of (mechanisms of) protective immunity in the primate host. p. s. nagpal 1 , p.k. upadhyay 1 1 national institute of immunology, pdc-1, delhi, indiaobjective: study was aimed for the preparation of dry powder formulation containing live mycobacterium indicus pranii for pulmonary immunization against tuberculosis. pulmonary delivery evokes both systemic as well as mucosal immune response against the antigen. secondly, pulmonary delivery is a needle-free delivery system, long desired for vaccine delivery. dry powder aerosol one such method, in which vaccine can be directly delivered to lung without any kind of invasion and it has an edge over liquid formulation being feasibility of storage at room temperature, long term shelf stability and higher drug content per unit mass as compared to liquid one. method: sodium alginate solution with suspended mycobacterium indicus pranii was aerosolized using laboratory modified nebulizer assembly. aerosols so generated were entrapped in cacl 2 solution with poly-vinyl alcohol (pva) as surfactant. particle so formed collected by centrifugation and lyophilized for dry powder formulation. pva and alginate concentration varies the size, surface and shape of the particles. formulation so prepared was delivered to c57bl/6 mice directly into lung by endotracheal intubations of mice. proliferation index (pi) of spleenocytes of immunized mice was measured after in-vitro stimulation with mycobacterium tuberculosis antigen. result: 2.4 % alginate, 0.012 % pva concentration gives particles with size of 2-10 micrometer as confirmed by particle size analyzer and scanning electron microscopy. viability of the mycobacterium indicus pranii was best achieved with 5 % trehelose and 3.5 % pvp (poly vinyl pyrollidone). there was 6 fold increase in proliferation index of spleenocytes and releases 600 pico-gram of interferon gamma after 4 week of immunization. formulation also induces the activation of dendritic cells after their in-vitro incubation as shown by 10 % increase in cd86 and 10.5 % in ccr7 expression as compared to blank alginate formulation. bacterial exopolysaccharides (epss) are heterogeneous polymers containing a wide array of homo-or hetero-carbohydrates as well as organic and inorganic substituents. epss are produced by many bacteria and play a critical role in helping these microorganisms to cope with adverse environmental conditions. some epss contain sulphate groups as inorganic substituents. the presence of these groups contributes to the biological activity of epss, which have been shown to have anticoagulant, insulinotropic, antiviral, antitumoral and immunomodulatory properties, among others. b100 is a constitutively sulphated eps produced by halomonas maura, a recently discovered halophilic bacterium. in preliminary experiments we found that modification of its eps by adding sulphate groups to the native polymer (thus obtaining b100s) resulted in vigorous antiproliferative activity in several haematopoietic tumour cell lines. at the same time we found that other epss produced by closely related strains had only a very limited antiproliferative effect on these same tumour cells. it was therefore of interest to determine whether the antiproliferative activity of b100s was mediated by the induction of apoptosis, and if so, to dissect the pathway triggered by b100s. by cell cycle analysis we determined that b100s is able to induce apoptosis in up to 80 % of jurkat and molt-4 t cell leukemias. the examination of a large panel of haematopoietic and nonhaematopoietic cell lines revealed that apoptosis induced by b100s is restricted to cells of the haematopoietic lineage and that leukemic t cells are particularly sensitive to death induced by b100s, but that untransformed cells are not. a time-course of caspases activation indicated that caspase 9 is the first to be cleaved, followed by caspases 3 and 8, thus suggesting that b100s triggers apoptosis through the mitochondrial pathway. it is noteworthy that b100s also induces vigorous apoptosis in primary leukemic t cells obtained from the peripheral blood of patients. therefore, b100s may well provide a satisfactory therapeutic alternative to patients with acute t cell leukemias, since current antitumoral drugs are very inefficient in the treatment of these types of cancer. particulate antigen delivery tools have been shown to enhance the induction of immune responses by targeting dcs. polyelectrolyte microcapsules form a new class of microcapsules generated by the sequential adsorption of oppositely charged polyelectrolytes onto a sacrificial spherical template which is consequently dissolved, yielding a hollow microcapsule surrounded by a thin shell. this layer-by-layer approach allows an efficient incorporation of macromolecules under nondenaturing conditions. by using the biopolyelectrolytes dextran-sulphate and poly-l-arginine, biodegradable microcapsules can be obtained. in this study, we have chosen the lungs as a non-invasive route for vaccine delivery. as demonstrated by flow cytometry and confocal imaging, dextran-sulphate/poly-l-arginine microcapsules were readily taken up by local pulmonary apcs and transported to the mediastinal lymph nodes, making them excellent tools for antigen targeting towards apcs. microcapsule instillation also affected the pulmonary apc activation status, indicated by the emergence of an apc population expressing increased levels of mhcii, the co-stimulatory ligands cd40, cd80 and cd86, and of the inflammatory cytokines il-6, il-23 and mcp-1. using ovalbumin (ova) as a model antigen, we have analysed the adjuvant properties of these polyelectrolyte microcapsules. analysis of the alveolar infiltrate, cd4 t cell and antibody profiles revealed that polyelectrolyte microcapsules display different adjuvant properties than the standard th2 and th1/th17 skewing adjuvants alum and complete freund's adjuvant (cfa). in response to ova aerosol exposure, microcapsule based vaccination resulted in an alveolar infiltrate dominated by monocytes, while alum and cfa respectively induced typical eosinophilic and neutrophilic inflammations. striking differences were also observed on the level of cd4 t cell responses. microcapsule based vaccination resulted in a marked induction of il-17 secreting th17 cells, without inducing strong th1 (cfa) or th2 (alum) responses. these differences were also reflected on the level of the humoral immune response, with microcapsules being the sole adjuvant producing antibodies of all isotypes tested (igg1, igg2c and ige).in conclusion, polyelectrolyte microcapsules allow an efficient targeting of antigens to lung apcs, and possess immune stimulating activities distinct from alum and cfa. due to their capacity to generate th17 responses, polyelectrolyte microcapsules may become interesting tools to combat fungal and bacterial infections. pneumolysin is an important virulence factor produced by virtually all clinical isolates of streptococcus pneumoniae. the protein binds to cholesterol in cell membranes and creates transmembrane pores, leading to cell lysis. published findings have proposed that, at sublytic concentrations, the toxin causes a range of effects including activation of host complement, activation and chemotaxis of cd4 + t cells and increased production of pro-inflammatory cytokines in immune cells. in this study we investigated the interaction of pneumolysin with murine dendritic cells (dc). we found that pneumolysin induced the activation of dc, reflected in the enhanced expression of the costimulatory molecules cd80, cd86 and cd40 and mhc class ii molecules. the toxin alone was found to be a poor inducer of cytokine production by dc but it did enhance the secretion of tlr agonist-induced cytokines such as il-6 and tnf-a. previous published findings have shown that pneumolysin activates peritoneal macrophages in a tlr4-dependent manner. however, we found that pneumolysin was capable of activating dc from both wildtype and tlr4-defective c3h/hej mice, by inducing cell maturation and synergising with tlr agonists to enhance cytokine secretion. importantly, we also found that pneumolysin is a strong inducer of il-1b secretion by dc, through its effects on caspase-1 processing, which is also tlr4-independent. the results suggest that pneumolysin is a potent stimulus for dendritic cell activation and that this does not require tlr4 signalling. objectives: transmission of immune competence from mothers to newborns during pregnancy and lactation is crucial for education of neonate immune system in order to develop optimal protection against early life infections. the objective of the present study was to assess whether maternal supplementation with probiotics may enhance neonatal responses to measles immunization. methods: pregnant balb/c mice were supplemented with placebo (maltodextrin) or probiotics (lactobacillus paracasei ncc2461 (st11) or lactobacillus rhamnosus ncc4007 (lpr), each at 5x10 8 cfu/day), suspended in the drinking water, throughout the gestation period and up to the weaning of pups. at weaning, pups were immunized with live attenuated measles vaccine (mv-s, aventis-pateur). weight evolution of pups was followed from week 1 to week 7 of life. fresh feces were collected at 3, 5 and 7 weeks of life for determination of iga levels (assessed by elisa). pups were bled 2 and 4 weeks after immunization for determination of measles-specific igg1 and igg2a antibodies. analysis of microbiota composition (plating on semi-selective agar media) was performed on fresh feces collected one week after weaning. results: all newborns grew normally and no significant differences in the weight were observed between the groups all along the trial. fecal iga production increased progressively in all pups from weaning, reflecting a normal development status. nonetheless, feeding mothers during pregnancy and lactation did not significantly affect post-weaning s-iga production in pups. lpr supplementation of the mothers significantly potentiated post-weaning measles-specific antibody responses in pups in comparison to control group. interestingly, no significant effect was observed in the st11-fed group. finally, a modification of the microbiota composition was observed in pups of supplemented mothers. particularly, there was a significant increase in lactobacilli in pups from the lpr group as compared to controls. conclusion: this study supports the benefit of perinatal intervention with probiotics during pregnancy and lactation on immune maturation in the offsprings. moreover, these first results seem indicate that the effects are strain specific. chitosan, (1-4)-2-amino-2-deoxy-beta-d-glucan, is a deacetylated form of chitin, an abundant biodegradable, positively charged natural polysaccharide. chitosan (chi) is used for antigen delivery through mucosal barrier due to its ability to disrupt tight junctions. here we studied the ability of chitosan nanoparticles to form complexes with proteins of different size and charge. nanoparticles (chi-np) were prepared from 200 kda chitosan by ionotropic gel formation. bovine serum albumin (bsa) and myoglobin, human immunoglobulin g and superoxidedismutase (sod), and chicken lysozyme were fitc labeled. chi-np were preincubated with proteins at 3:1 ration and washed 3 times. after washing chi-np containing bound proteins were run by denaturating gel electrophoresis. all proteins were able to form complexes. most effective binding was shown for bsa, sod, and lysozyme. the stability of chi-np complexes with proteins was studied in vitro on macrophage cell line raw264.7 by confocal microscopy. for this chi was labeled with rhodamine and nanoparticles were coincubated with fitc labeled proteins before addition to the cells. we showed co-localization of chi and fitc for all proteins studied. these results demonstrate that chi-np form stable complexes which are internalized by macrophages. the family euphorbiaceae consists of a large group of plants whose compounds have been documented to possess anti-inflammatory activities, however, their effects as modulators of innate or acquired immunity has not been described yet. in the present study, different aspects of the immunomodulatory activity of 24 extracts from 10 euphorbiaceaes on peripheral blood mononuclear cells (pbmc) from healthy individuals were evaluated. the pbmc were exposed to the extracts w/o phytohaemagglutinin a (pha), cycloheximide (chx) or lipopolysaccharide (lps) as agents that induce proliferation, apoptosis and cytokine production in pbmc. the lymphoproliferative activity of pbmc was evaluated by thymidine incorporation and cfse dilution assay using flow cytometry. the mitochondrial membrane depolarization (as an early apoptosis indicator) was measured using dioc6/propidium iodide staining by flow cytometry and tnf-a secretion in the culture supernatans by elisa. we found that 15 up to 24 euphorbiaceae's extracts had the ability to modulate one or more of the immune parameters evaluated in this study. however, only the bark extract of croton spp. insoluble in hexane:diclorometane:methanol (hdm) and the latex extracts of euphorbia cotinifolia and euphorbia tirucalli induced strong proliferation, apoptosis and also tnf-a production in pbmc. these extracts were subfractioned by sephadex column chromatography obtaining three subfractions with enhanced activity in comparison to the crude extracts. additionally, we started with the characterization of the specific immune effects of these subfractions on pbmc. all three subfractions induced proliferation predominantly on cd3+ cells. these effect was also observed in isolated t cells indicating that accessory cells are not necessary for the subfractions'activity. the lymphoproliferative activity of these subfractions was also not inhibited by the carbohydrates d-(+) galactose or a-methyl-mannopyranoside. these results demonstrate the presence of immunomodulatory compounds in plants from the euphorbiaceae family and suggest an antigen-presenting cell-and carbohydrate-independent mechanism of the subfractions to exert their effects. we found significant increase on lymphocytes and eosinophils populations obtained from lps + p. acnes-treated group in relation to control group.on 35 th day, we detected a significant negative correlation between eosinophils absolute number and fec. both il-5 and ige serum levels were increased on animals from group i when compared to control.the enhancement on th2 immune response pattern induced by lps and p. acnes treatment diminished drastically parasitic load. conclusion: our findings support the idea of the use of immunostimulant as a helminthiasis control strategy in sheep, which stimulate non-specific mechanisms of resistance and therefore can act against nematodes infections. vaccines based on partially purify populations from the organism or recombinant subunits proteins have been recently developed and are often not sufficiently immunogenic by themselves due to the lack of innate immune stimuli. indeed, current influenza a vaccines do not generate significant immunity against serological influenza a virus subtypes and would thus be ineffective in the face of a pandemic novel variant. hence adjuvant usually needs to be added to those types of vaccines. here we show that wittycell compounds significantly augment cellular and humoral immune responses to commercial seasonal influenza vaccines. experiments performed in mice showed induction of specific cd8+ ctl cells against conserved proteins that were accompanied by the induction of ifn-g producing cd4+t cells, following single immunisation. in addition increased hi titres and higher levels of specific igg2a and igg2b antibodies were found even long periods after single vaccination with reduced doses of vaccines. consequently, protection from lethality was observed following challenge with homologous or heterogonous influenza viruses in vaccinated animals. this promising finding on the improvement of seasonal influenza vaccines by wittycell compounds in these preclinical studies strongly provides support for the careful evaluation in phase i clinical trials in humans. s. lindgren 1,2 , n. almqvist 2 , a. lönnqvist 1 , s. östman 1 , c. rask 1 , e. telemo 2 , a.e. wold 1 1 university of göteborg, department of clinical bacteriology, göteborg, sweden, 2 university of göteborg, department of rheumatology and inflammation research, göteborg, swedenobjective: dietary antigens normally evoke immunological tolerance. a prerequisite is their processing by intestinal epithelial cells, which leads to the appearance of a tolerogenic form in the serum of fed animals that confers antigen-specific tolerance when transferred to naï ve recipients. the gut microbiota may influence the handling of dietary antigens as atopic diseases have increased in western societies in parallel with reduced complexity of the infantile commensal microflora. we have observed that children neonatally colonized with s. aureus in the gut seem protected against development of food allergy. here we examine whether a s. aureus toxin affects tolerogenic processing by the intestinal epithelium. methods: mice were given s. aureus enterotoxin a (sea; 0.8 mg/ml) in the drinking water for 5 days and, 3 days later, 50 mg ovalbumin per os. one hour postfeeding, serum was transferred to naï ve recipients, whose tolerance to ovalbumin was tested in a model of allergic airway inflammation (sensitization followed by intranasal challenge with ovalbumin). results: recipients of serum from sea pretreated ovalbumin-fed donors exhibited increased tolerance compared to recipients of serum from ovalbumin-fed donors not pretreated with sea. this was demonstrated as reduced ovalbumin-induced airway inflammation with diminished influx of eosinophils into the lungs and reduced antigen-induced production of interleukin-5 and interleukin-13. examination of gut sections from sea treated donor mice revealed increased density of cd8a + intraepithelial lymphocytes. our results show that sea promotes oral tolerance induction, possibly by facilitating tolerogenic processing of soluble antigens by the absorptive intestinal epithelium via activation of intraepithelial lymphocytes. abstract withdrawn by author to develop an efficient vaccine against cp pneumoniae we cloned chlamydial genes encoding proteins of the outer membrane like ompa, omcb, and pmp21, proteins of the inclusion membrane like incc, secreted proteins like cpaf, and the heat shock protein groel. cpg-dna 1826, a highly stimulatory oligonucleotide for apcs, was used as adjuvans. subcutaneous co-injection of ompa, omcb, pmp21, or groel together with cpg-dna 1826 reduced the chlamdial burden in nasally infected mice. however, symptoms like substantial loss of body weight were not influenced. in contrast, a low dose infection with cp. pneumoniae almost completely prevented the loss of body weight upon challenge. to improve the efficacy of the vaccine we used poly-dl-lactide-co-glycolide microspheres loaded with the protein ompa or pmp21 together with cpg-dna 1826. the microsphere based vaccine offers the advantage that antigen and adjuvans are delivered to the same apc. intranasal but not subcutaneous vaccination of mice with ompa or pmp21 microspheres efficiently lowered chlamydial burden upon challenge and prevented loss of body weight. pmp21 microspheres induced protective ifng-secreting cd4 + t-cells and raised pulmonary pmp21-specific iga levels in vivo. also, pmp21 microspheres caused lower il-6 serum levels upon administration than the injection of pmp21 together with cpg-dna 1826, indicating fewer side effects. objectives: staphylococcus (s.) aureus superantigens are highly potent t cell mitogens and the causative agents of toxic shock syndrome (tss) and food poisoning. most s. aureus have superantigens and patterns are highly variable. to date, the role of superantigens in bacteraemia is not well defined.to analyse whether superantigens play a role in bacteraemia, we investigated s. aureus strains and anti-superantigen antibody responses in 44 cases of s. aureus bacteraemia in iv drug users and 44 cases in nonaddicts. a rise in neutralising antibody titers indicates that superantigens are produced during infection. the study comprised 44 iv drug users with positive s. aureus blood culture and an equal number of age-and sex-matched nonaddicts from the original fintrova and finlevo trials (ruotsalainen 2006).all s. aureus isolates were analysed by sequence-based genotyping (spa-typing), and multiplex-pcr was applied to determine the superantigen gene pattern. sera from patients were obtained at diagnosis (day 0) and four weeks thereafter (day 28). neutralising capacity of the sera was tested against the superantigen cocktail produced by the respective infecting strain as well as a panel of representative recombinant superantigens.results: genetic analysis confirmed our previous observation that most strains harboured superantigen genes, which were linked to staphylococcal lineages (holtfreter 2007) . there were no major differences in superantigen gene patterns in isolates from iv drug users and nonaddicts. interestingly, the staphylococcal lineage st59 (spa-type t172, agr 1, and sea, seb, sek and seq) was much more prevalent among bacteraemia strains from iv drug users than from nonaddicts (p=0.01).most iv drug users had neutralising antibodies against enterotoxins already at onset of bacteraemia, likely due to previous encounters with the infecting strain. we frequently observed a rise in antibody titers during infection. surprisingly patients with st59 strains did not show any elevations in neutralising antibody levels. conclusion: s. aureus bacteraemia induces an antibody formation against staphylococcal superantigens. this indicates that superantigens are produced during infection. however, the action of superantigens is frequently modulated by specific neutralising antibodies. this and the special behaviour of s. aureus st59 strains need further investigation. objectives: down syndrome (ds) is associated with recurrent infections, hematological malignancies and auto-immune diseases, suggesting immunological changes. to test for more severely disturbed specific antibody response we investigated the antibody response to the highly immunogenic protein antigen tetanus toxoid (tt), which is part of the dutch immunization program. methods: after booster vaccination at 4 and 9 years of age, quantitative (titer) and qualitative (avidity) tt responses were investigated in 15 and 7 ds children, respectively. samples were taken before and 3-4 weeks after vaccination. tt-specific igg and igg-subclass antibodies were measured in serum by quantitative enzyme linked immunosorbent assay (elisa), avidity of igg 1 -anti-tt by an avidity elisa. the results were compared with reference values from the laboratory. results: at 4 years, post-vaccination anti-tt-titers were decreased (geometric mean total igg, igg 1 , igg 2 and igg 4 ). at 9 years, ds children had lower postvaccination geometric mean igg 4 anti-tt-titers only. post-vaccination igg 1 -anti-tt avidity levels were decreased in 8/15 and 4/7 ds children at four years and nine years of age, respectively. the quantitative and qualitative anti-tt-responses in both ds groups are shifted downwards compared to the reference values. although the anti-ttresponse increases towards normal titers with increasing age, the avidity (qualitative response) is still abnormal at that age, showing that ds children have profound and lasting difficulties with specific anti-tt antibody formation. 2 kda; 30, 6 kda; 23, 9 kda; 19, [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] 6 kda and 18, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] 7 kda were expressed by a majority of examined strains independently of the associated diseases. we assume that these omps could be conservative proteins of h. pylori. conclusion: considering omps as potential targets in the search for disease-related biomarkers and potential vaccine antigens, the identification of h. pylori omps as well as the elucidation of their role in modifying the host immune responses seems to be very important research subjects. the increasing cases of severe diarrhoea and invasive lethal infections in children caused by salmonella typhimurium are a major public health problem in mexico. the rapid dissemination of multidrug-resistant s. typhimurium, and the lack of a licensed vaccine against non-typhoidal infections reduce the possibilities of an effective treatment. the objective of this study was to evaluate if the high incidence of non-typhoidal multidrug-resistant salmonella infections was associated with a reduced in anti-salmonella immunity. a cohort of 100 families, from a mexican agricultural community with a high incidence of endemic salmonella infections, was followed prospectively for an 8-month period. sera were obtained from healthy subjects from the same community (2 months to 88 years of age). the highest incidence of salmonella-associated diarrhea, 74/1000, occurred in children under 5 years of age. the lowest incidence, 10/1000, was observed in the population aged 10 to 59. whereas serum from individuals ranging 15-70 years of age showed maximum igg1, igg3 and iga anti-s. typhimurium titres, children less than 5 years-old did not show detectable igg1 and igg3 titres and had weak igg2, iga and igm antibody levels; only their igg4 levels were comparable to those detected in adults. moreover, the levels of igg2 and igg3 antibodies were lower in adults with a diarrheal-associated episode. interestingly, s. typhimurium yuhs 07-18, a commonly isolated human strain from this endemic area, resisted the complement-fixing activity of antibodies although it was sensitive to opsonisation and to fc-mediated phagocytosis by human monocytes. these data contributes to define the protective immune response involved in anti-s. typhimurium immunity. diseases caused by the yeast of candida genus are a serious clinical and social problem. despite this fact, there is no effective prevention against these opportunistic pathogens yet. although c. albicans is the major cause of the mycoses (67%), the number of the multiresistant non-albicans isolates increases. c. dubliniensis, which was described only recently as serious human pathogen, belongs to the group of these resistant isolates. the surface mannan of candida cells is component of the cell wall mannoprotein complex and participates in an initial contact with its host and subsequently with the host defense mechanisms. because of complexicity of this homopolymer it is necessary to identify a subunit of the mannan that is most effectively recognized by the immune system and thus influences the specificity of the induced antibody response (immunodominant epitope). in our study we prepared oligosaccharides from acid-stable part of mannan c. dubliniensis by conventional acetolysis. this procedure specifically cleaves the a-1,6linked mannopyranose units of mannan backbone and releases the side oligomannosyl branches. obtained oligosaccharides were used in inhibition elisa and spr (surface plasmon resonance) measurements. the reason of these measurements was to quantify interactions of these oligosaccharides with anti-mannan antibodies present in rabbit serum after 7-fold immunization with mannan-hsa conjugate injections in week intervals as well with inactivated c. dubliniensis whole z. neščáková 1 , s. bystrický 1 1 institute of chemistry, slovak academy of sciences, bratislava, slovakiaour newest approach to sub-cellular vaccine against gram-negative bacterial pathogens exploits detoxified lipopolysacharide (detoxified lps) as the target antigen. this is achieved by conjugation of carbohydrate to a protein carrier which secures the t cell dependent immune response. the goal of the immunization with this conjugate is to generate the effective production of memory b cells. here we prepared subcellular conjugate with the detoxified lps from vibrio cholerae strain o135 using polymer carrier and a protein. the cell immunity induced by the vaccination with the conjugate was evaluated in mice, namely their peripheral blood and the spleen. activation and differentiation of b-cell populations in the time-dependent manner was determined by flow cytometry analysis of these samples. a single-platform approach based on flow cytometry and defined number of fluorospheres was used to count b cells. however in our hands this method, previously used in humans, had to be adapted for mouse blood samples first. the protocol allows quantifying cells simultaneously with cytometric immunophenotyping without cell loss or other cell preparation steps. like pan-b cell marker cd19, expressed almost on all blood and tissue b cells, was used. here we investigate the characteristics and development of antibody (iso)types after secondary immunization with mencc or plain polysaccharide and the possible role of certain antibody responses in maintaining immunity after vaccination. methods: volunteers, age 18-55 years, were immunized with mencc or received a secondary immunization with mencc or plain menc ps. blood samples were obtained before and seven time-points after immunization. igg, iga, igm, igg1, igg2 and avidity were assessed by a multiplex immunoassay. functional antibodies were determined by a serum bactericidal assay. results: high levels of antibodies were still present 5 years after primary mencc immunization. secondary immunization resulted in increased igg and sba titers after 5 to 7 days. in primed individuals, igm was still present, and this only increased following a secondary immunization with plain ps. in addition, immunization with ps induced a higher igg2 response compared to mencc immunization. discussion: secondary immune responses are quiet slow. the composition of the ig (iso)type distribution is different between mencc and plain ps and might be of influence on functional titers. although this study indicates that immunological memory was previously induced by a single mencc vaccination, it highlights the importance to sustain protective antibody levels against a rapid invasive organism such as n. meningitidis. the immunological effectiveness of these two semi-synthetic immunogenic conjugates was established according to antigen-specific titers of igg, iga and igm isotypes and by phagocytic and respiratory metabolic activities of granulocytes. results: prime-boost immunization strategy resulted in enhanced production especially dlps-specific igg and igm isotype antibodies in both experimental groups (peak titers 1:3200). igm-igg isotype switch was more pronounced with o-sp. peak values of dlps specific iga isotype were signicantly lower than igg and igm ones (1:800 vs. 1:3200). flowcytometric simultaneous determination of phagocytosis and stimulated oxidative burst of granulocytes revealed conjugate induced enhancement, more evident with o-specific polysaccharide and ip final boost (stimulation index was 5. 85 fold of normal control). subcutaneous immunization gave a weaker stimulation: 1. 52 fold of normal control. the second de-oac conjugate exerted different pattern of stimulation, sc intervention was more effective. our results are indicative for immunological effectiveness of novel dlps derived glycoconjugates; thus promising further application in cholera subcellular vaccine. this work was supported by apvv 0032-06 and vega 2/7029/27 grants of the slovak grant agencies. background: the yeast candida albicans is an opportunistic pathogen that causes infections in immunocompromised individuals with a high morbidity and mortality levels. a long-acting, effective and safe vaccine that protects against medically important candida species should significantly reduce the incidence of various forms of candidasis by these etiologic agents. mannan, polysaccharide component exposed at the most external layer of the fungal cell wall, contains a backbone consisting of a-1,6-linked d-mannopyranose units and many branches composed of a-1,2 a-1,3 and/or b-1,2-linked mannopyranose units that are connected to the backbone. investigation of oligosaccharides immunomodulatory functions could be considered as an important part of their protective immunity against fungal diseases. objective: in this study, for mice immunization, synthetically prepared oligosaccharide (heptamannoside) conjugated to protein carrier (bovine serum albumin, bsa) was used. methods: in order to study the immunogenicity of heptamannoside -bsa conjugate as inducer of hummoral and cell-mediated responses, balb/c mice were subcutaneously immunized without adjuvant (6mg oligosaccharide per one conjugate dose) two times in 14 days intervals and then intraperitoneally or subcutaneously boosted. cell-mediated and humoral responses were analyzed on day 14 after injections by flow cytometric immunophenotyping of peripheral blood leukocytes and by measuring the levels of mannan specific antibodies presented in serum using elisa. results: prepared conjugate was immunogenic and re-injection elicited increase of mannan specific serum antibodies levels. intraperitoneal boost elicited significantly higher igg and igm levels than subcutaneous boost. immunization also induced changes in proportions of major lymphocyte subpopulations in peripheral blood. introduction: bulgarian immunomodulator respivax enhances the natural resistance of organisms and specific immunity towards the most frequent respiratory pathogens. it is composed of killed bacterial bodies and lysates of six microbial species (streptococcus pneumoniae, branhamella catarrhalis, streptococcus pyogenes, haemophilus influenzae, staphylococcus aureus, klebsiella pneumoniae). the immune response in respiratory tract includes not only systemic immunity in lungs, but also balt as a part of common mucosal immune system. most animals develop balt after antigen stimulation and this tissue plays a central role in antigen uptake and local immune response regulation. therefore, immunostimulation of balt may contribute to more efficient mucosal immunity in respiratory tract. aim: to study balt development in different terms after oral application of respivax in guinea pigs. methods: male guinea pigs (250g-350g) were treated orally with 50 mg respivax five consecutive days. after the last application, on days 3, 7, 10, 14, 28 and 42 six animals on each term were sacrificed and lungs were removed. morphological changes were evaluated on 4mm thick serial sections, stained with hemalauneosin. the populations of cd8, cd4 and b cells were identified on cryosections by using indirect peroxidase immunostaining. zio technique was used to detect intraepithelial dendritic cells (dc). results: balt was not identified in control animals. in the treated group on day 3 subepithelial lymphocyte infiltrates and diffuse lymphocytes in lamina propria were found. the following two terms were presented by hyperplasia of lymph epithelium with massive complexes of intraepithelial lymphocytes. they were composed mainly of cd8 positive cells, which number reached maximum at the end of the second week. on day 14 b cell lymphoid follicles with different size were found. lamina propria was presented by abundant lymphocyte infiltrates, composed of cd4 and cd8 positive cells. on days 28 and 42 the morphological reaction in the airways was reduced, characterized with small size lymphocyte accumulates. numerous intraepithelial dc's were detected in treated animals, comparing to controls in which only a few were identified. conclusion: oral administration of respivax in guinea pigs resulted in significant immunomorphological reaction in the airway mucosa presented by increased number of dc and balt development. g. gupta 1 , s. majumdar 1 1 bose institute, molecular medicine, kolkata, indiavisceral leishmaniasis (vl) caused by the protozoan parasite, leishmania donovani, is characterized by the loss of ability of the host to generate an effective immune response in the form of free radicals and proinflammatory cytokines. chemokines, particularly cc chemokines, have been shown to render protection against leishmania infection. there is no clear understanding about the immunoprotective role of cxc-chemokines in vl.in the present study, the comparative potential of cxc chemokines, interferon gamma inducible protein-10 (ip-10) and interleukin-8 (il-8) in restricting leishmania donovani infection via the release of nitric oxide (no) and proinflammatory cytokines was studied in an in vitro model. no, a crucial mediator for ip-10 mediated leishmanicidal activity, was found to be dependent on inducible nitric oxide synthase2 (inos2) expression and was linked to the mapk signaling pathway via antagonistic regulation of p38mapk and erk1/2. further, ip-10 was also able to abrogate the survival of leishmania in an in vivo model of vl by restoration of th1 cytokines and no. thus this study strongly demonstrates that ip-10, like cc chemokines, is involved in rendering a protective response in vl via upregulation of proinflammatory mediators. african trypanosomiaisis (at), known as sleeping sickness, is an orphan and extremely debilitating disease in human, cattle and domestic animals. at is caused by the protozoan trypanosoma brucei and at the present, there's no safe or efficient pharmacology intervention. the dna vaccines could be the answer for this disease by being able to induce production of igg antibodies and induce of th1/th2 cytokines mediated by cd8+ t cells and activating cd4+ t helper cells. in this study, we shows that balb-c mice immunized intramuscularly with a single dose of plasmids encoding three antigenic candidate genes from trypanosoma brucei, named invariant surface glycoprotein (isg), trans-sialidase (tsa), and fosfolipase c (plc) are able to produce igg antibodies anti-trypanosoma. this immunization process was able to control the mortality level when mice were submitted to challenger assay with trypanosoma brucei brucei parasites. in mice co-infected with s. ratti and l. major (nl) neither clearance of l. major nor strongyloides infection was changed. mice co-infected with s. ratti and p. yoelii (nl) showed the same course of parasitaemia as single infected mice. these results suggest a strictly compartmentalized and successful immune response in both murine co-infection models, s. ratti and l. major or p. yoelii. if this compartmentalization is also observed in the antigen specific cytokine response of ex vivo prepared lymphocytes will be the topic of further investigations. in the present study ,we evaluated tsa -encoded dna vaccine against l.major in balb/c mice. igg and ifn-g values were markedly increased in the immunized group ,which were significantly higher than in the control groups (p x 0.05) following immunization and after challenge with leishmania major. il-4 values were increased in all groups, but there was no statistical difference between the groups(p g 0.05) following immunization and after challenge with leishmania major. the immunized mice with the dna vaccine presented a considerable reduction in diameter of lesion comparing to the control mice and indicated a significant difference was observed between the immunized and the control groups (p x 0.05) in this regard . the survival time of the immunized mice with the vaccine was significantly higher than the control groups (p x 0.05) after the challenge with leishmania major. the immunized mice had significantly lower parasite load comparing to the control mice(p x 0.05). the findings of this study indicated that the tsa -encoded dna vaccine increased the cellular response and induced protection against infection with leishmania in the mice. the tsa -encoded dna vaccine may be an excellent candidate for future vaccine developments against leishmania. there is a lot of evidence showing that bcg vaccination at mucosal site via intranasal, intragastric and intrarectal routes are effective in conferring protection against virulent mycobacterium and several non mycobacterial infectious diseases. in this study the protective effect of autoclaved leishmania major (alm) vaccine in combination of either rectal or subcutaneous bcg on susceptible balb/c mice was evaluated.one month after bcg vaccination, balb/c mice were immunized subcutaneously twice with alm+alum at 3 week intervals. three weeks after booster injection, 5×10 5 stationary phase l. major promastigotes were inoculated subcutaneously in one footpad. immunological evaluation at before and post infectious challenge, showed strong proliferative responses in the spleen cells of the rectal immunized group after stimulating with parasite lysate. high level of interferon gamma was induced in the spleen and significant increase in the serum ratio of igg2a/igg1was observed only in rectal immunized group. rectal immunized mice showed comparable nitric oxide production and inos induction in peritoneal macrophages .the obtained results in rectal bcg vaccinated group showed no mortality but low parasite burden in the liver and spleen and suggested protective efficacy of intrarectal bcg immunization against leishmaniasis might be due to the long-lasting induction of type 1 immunity. methods: two groups of balb/c mice were infected by l. tropica. one group was infected subcutaneously into the left footpad and the other group intradermally into the left ear dermis. mice were challenged by l. major in the right footpad after establishment of l. tropica infection. the immune response was evaluated at two intervals: one week and one month after challenge. single cell suspensions were prepared from draining lymph nodes of mice. cells were stimulated by phorbol myristate acetate (pma). cell surface markers and cytokine production were determined by intracellular cytokine assay using flow cytometry. the following parameters were assayed in the two experimental groups: lesion development, delayed type hypersensitivity (dth) to l. major challenge, production of gamma interferon (ifn-g) and interleukin 10 (il-10), and cellular expression of cd4 and cd25. results: infection through subcutaneous route in comparison to the intradermal route induces significantly higher levels of dth and ifn-g, lower levels of cd4+ lymphocytes, and higher protection against l. major challenges. conclusion: intradermal infection of l. tropica, in comparison to subcutaneous infection, induces significantly more protective immunity in balb/c mice. therefore, we propose the route of infection as an important variable in this experimental model. this factor should be considered for development of an appropriate experimental model for human l. tropica infections. objectives: many mammals exhibit a periparturient relaxation of immunity (ppri) to gastrointestinal nematode parasites culminating in increased worm burdens. it has been suggested that the extent of ppri may have a nutritional basis as this effect on host resistance is considerably augmented when protein supply is scarce. subsequent studies have shown that increased dietary protein intake can ameliorate this phenomenon. however, this effect is often confounded with increased food intake and thus increased energy levels. here, we aim to dissect the effects of protein and energy nutrition on the immune status and resistance to gastrointestinal nematodes in the periparturient host. the nippostrongylus brasiliensis lactating re-infected rat model was utilised as a well established model for mammalian ppri. lactating rats, re-infected with 1,600 infective n. brasiliensis larvae on day 2 post parturition, were offered one of three levels of crude protein at one of two levels of metabolisable energy (me). parasite burdens were assessed by counting worms in the small intestine at day 6 post secondary infection. histological counting of intestinal inflammatory cells, assessment of antibody levels and measurement of cytokine mrna levels in the mesenteric lymph nodes were carried out to assess the host immune status. results: increasing cp supply, but not increased me supply, reduced worm burdens. whilst feeding treatment did not affect eosinophil and goblet cell numbers, increased cp supply increased mucosal mast cell numbers and levels of n. brasiliensis specific antibody (total igg, ige, igg1 and igg2a). this was independent of level of me supply. feeding regime did not affect levels of the type-2 cytokines il-4 and il-13. conclusion: this study effectively demonstrates that increasing protein supply per se can decrease periparturient parasite burdens. this anti-parasitic effect correlates strongly with an upregulation of immune effector mechanisms, namely accumulation of mast cells and production of antibody. this data emphasises the role of immunonutrition in combating infectious disease. protein supplementation of periparturient mammals has considerable potential as a non-chemotherapeutic method of controlling gastrointestinal nematode parasites. background: gp63 is the major surface glycoprotein of leishmania that exhibits protease activity and has an important role in the biology of the parasite. the aim of this study was cloning and expression of gp63 of l.major strain mrho/ir/75/er. methods: l.major promastigotes were grown in rpmi1640 supplemented with 10 % fcs. l.major rna extraction and cdna synthesis were carried out. gp63 gene segment was amplified by specific primers and cloned into ptz57r to construct ptz57r/gp63. the presence of gp63 into ptz57r was confirmed by pcr. then, ptz57r/gp63 was sent to determine the sequence of its nucleotides. after that the gp63 gene segment was sub-cloned into pet32 a (+) expression vector and transformed into e.coli bl21 (de3) plyss and gp63 protein was expressed in presence of 1mm iptg. objectives: the development of a vaccine against malaria caused by plasmodium falciparum is an urgent public health priority. influenza virosomes represent an innovative human-compatible antigen delivery system that has already proven its suitability for subunit vaccine design. at appropriate antigen doses, seroconversion rates of 100 % were achieved against two synthetic malaria peptide-mimetics in malaria naï ve volunteers (genton et al., plos one, 2007) . the aim of this clinical trial is to proof that virosomes are a suitable delivery system for malaria peptide-mimetics in malaria semi-immune subjects. objectives include demonstration of safety and tolerability of virosome formulated malaria peptide-mimetics and determination of the humoral and cellular immune responses against these malaria peptide-mimetics. particularly, boosting of pre-existing naturally acquired anti-malaria immunity will be investigated. the study design was a single centre, randomized, controlled, double-blind, age deescalating trial including 50 volunteers. 10 male volunteers (18 and 45 years) for the adult group, and 40 children of both sexes (5-9 years) were enrolled. subjects received virosomal formulations containing 50 mg of ama 49-c1 (pev301t), an apical membrane antigen-1 derived synthetic phospatidylethanolamine (pe)-peptide conjugate and 10 ug of uk39 (pev302t), a circumsporozoite protein derived synthetic pe-peptide conjugate. comparator groups received the influenza vaccine inflexal v. volunteers received two injections at study days 0, and 90. results: safety and tolerability defined as occurrence of local and systemic adverse events and incidence of clinically significant hematological and biochemical abnormalities are assessed. this vaccine showed a very good safety and tolerability profile in all study participants. curcumin dissolved in dmso when administered orally to p.berghei infected mice has been shown to have antimalarial activity, enabling 29 % of the treated mice to survive till 21 days after infection by which time all of the untreated mice had died. under such condition we found that bioavailability of curcumin was only 0.04 % of the amount fed and it remained in circulation in the blood only for 30 minutes post feeding. we therefore prepared curcumin bound to chitosan nano particle to improve it's delivery and found that oral feeding of such particles not only increased its bioavailability to 0.4 % ( of the amount fed but it's circulation was sustained till 6 hrs post feeding. under such conditions when 200mgm of curcumin bound to 300mgm of chitosan nano particles were fed one time daily for 10 days post infection to plasmodium yoelii infected mice 100 % of mice were cured and survived atleast for 100 days without any infection and were resistant to reinfection with the same parasite. curcumin under such condition accumulated preferentially in infected erythrocytes, the quantity increasing with increase in parasitemia and fluorescence microscopy revealed that it was bound to the parasite. like chloroquine, curcumin inhibited hemozoin formation in vivo and heme polymerization in vitro in a dose dependent manner. we believe that it is one of the ways by which curcumin may be killing the parasite. among immune cells, nk and gamma-delta t cells are suspected to play a critical role in the early control of plasmodium falciparum parasitaemia and to influence malaria adaptive immunity. gamma-delta vgamma9vdelta2 t cells, a non-conventional t cell subset specific of primates, are activated and expanded during primary p.falciparum infections in response to malaria non-peptidic phosphoantigens, and they are an important source of ifn gamma. furthermore these cells inhibit in vitro growth of p.falciparum blood stages by a granule exocytosis-dependent cytotoxic pathway and granulysin -an nk and t cell specific cytotoxic molecule has been incriminated. so far, the precise mechanism of the parasite inhibitory capacity of those cells, as well as the parasite blood stages involved remains unclear. to further investigate the anti-parasitic activity of gamma-delta t cells an rnai strategy based on a lentiviral vector approach was undertaken. we demonstrate that granulysin, but not perforin is essential for the anti-parasitic activity of gamma-delta t cells. concerning parasite blood stages, we show that both mature infected red blood cells and the free invasive form (merozoite) trigger gamma-delta degranulation and granulysin release, but noteworthy merozoites were the only stage affected by gamma-delta t cells. in addition, we also provide evidence that such a mechanism may occur in infected patients. altogether these data highlight a new mechanism by which gamma-delta t cells might directly contribute to malaria immunity opening new perspectives based on gamma-delta t cells to prevent or cure malaria. the immune system has a number of mechanisms to prevent self-destructive responses. amongst these, regulatory t cells (treg) have the ability to actively suppress effector responses. many questions surround the issue of antigen specificity of treg, since selective inhibition of only the pathogenic response, leaving the rest of the immune system intact, is the ideal therapeutic goal. the purpose of the project is to develop a model of robust, highly specific regulation operating in vivo that can be studied to understand the underlying mechanisms. such a model is provided by murine autoimmune hemolytic anemia (aiha) induced by immunisation with cross-reactive rat red blood cells (rbc). mice recover from disease due to the development of regulation with exquisite specificity, which suppresses only responses to self-epitopes whilst selectively allowing those to rat-specific determinants to be boosted. the re-establishment of tolerance is associated with the loss of t-cell proliferative responses, and emergence of il-10 responses, to epitopes on the dominant rbc autoantigen, anion exchanger-1 (ae-1, or band 3) protein, and protection can be transferred by injecting splenocytes from recovered mice into naï ve recipients. here we show that transfer of tolerance to naï ve recipients is dependent on ido mediated immunosuppression as mice receiving previously tolerised splenocytes under the cover of 1 methyl tryptophan, an inhibitor of ido, were refractory to tolerance and developed hemolytic disease. induction of ido is therefore an important process in antigen-specific tolerance, and initiators of ido activity, including ctla-4 + regulatory t cells or soluble forms of ctla-4, may also be crucial components of this regulatory pathway. consequently, this finding has important implications for our understanding of tolerance processes in autoimmune disease. objectives: it was shown alpha-fetoprotein (afp) induced immunosuppression of cell-mediated immunity in vivo. our previous work discovered afp-activated mice bone marrow hematopoietic stem cells (hscs) suppressed effector reactions of cell-mediated immunity in vitro. we investigated relationship existed between afp-induced hscs suppressor activity and immunosuppression of cell-mediated immunity during afp-produced teratocarcinoma development. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, methods of molecular biology and rna interference (rnai) were used in this work. results: as a result, there was a negative correlation (r medium =-0.81) between dynamics of hscs suppressor activity elevation in spleen and inhibition of nk cells, nkt cells and cd8 + t cells cytotoxic activities ex vivo during tumor growth. besides, the inhibition of spontaneous and induced cytokines productions such as ifng, tnf-a and tnf-b from these types of immunocompetent cells negatively correlated with increasing of suppressor factors expression such as tgf-b 1 (r medium =-0.74) and il-10 (r medium =-0.65) in isolated splenic hscs ex vivo. analysis of effector cd4 + t cells in spleen showed decrease of t h 1 cells quantity and simultaneous t h 2 cells number increase during teratocarcinoma development. moreover, it were found elevated numbers of cd4 + cd25 + ctla-4 + -and cd4 + cd25 -il-10 + il-4regulatory t cells in spleen as well as increasing suppressor activity of isolated regulatory t cells ex vivo. number boost kinetics of t h 1, t h 2 and regulatory t cells were correlated (r th1 =-0.74, r th2 =0.86 and r th3 =0.80 and r tr1 =0.68) with kinetics of hscs suppressor activity level. in addition, dynamics of regulatory t cells activity were linear (r th3 =0.84 and r tr1 =0.76) to hscs suppressor activity level in spleen during tumor growth. quantities of tgf-b1-and il-10-produced hscs in spleen were correlated (in some cases negatively but in other positively) with cell-mediated immunity effector reactions alteration during teratocarcinoma development also. however, inhibition of afp expression by rnai caused to inhibition as immunosuppression activity of hscs and their appearance in spleen as well as normalization of cell-mediated immunity effector reactions. conclusion: thus, hscs suppression activity is correlated with changes in cell-mediated immunity during endogenous afp productions by teratocarcinoma cells and may play a role in afp-mediated dysfunction of normal immunoregulation during afp-produced tumor development. syphacia obvelata, a murine pinworm gastrointestinal nematode, is common even in well-managed animal colonies. although often considered as irrelevant, pinworm infections were shown to alter hosts' immune responses and to interfere with the experimental settings. our studies showed that naturally aquired s.obvelata infection also influences the hosts' hematopoietic responses, inducing the increased production and release of the cells of granulocyte-macrophage, as well as of erythroid lineage from the bone marrow of the infected cba mice. while the enhanced myelopoiesis compensates the increased peripheral demand for a larger supply of tissue neutrophils and macrophages, the cause of stimulated erythropoiesis is less obvious, but as infection consequence clearly underscores the disturbed and altered hematopoiesis. beside cellular changes, we also evaluated the impact of the s.obvelata on mitogen-activated protein kinases (mapk) signaling in bone marrow cells and found that infection upregulated all three mapk families, p38, jnk and erk. additionally, s.obvelata enhanced the expression of mrna for the inducible nitric oxide synthase (inos). to evaluate how this pinworm infection modifies hematopoietic cells' reactivity, we also examined the influence of interleukin-17, t cell-derived cytokine implicated in the regulation of hematopoiesis and inflammation, on the bone marrow cells. bone marrow myeloid and erythroid progenitors from s.obvelata-infected mice displayed altered sensitivity to il-17, as compared to non-infected controls. the infection also altered the effect of il-17 on mapk activation by preventing its stimulating effect on p38 mapk. moreover, in s.obvelata-infected animals il-17 markedly down-regulated the expression of both inos and constitutive, endothelial (e)nos, not affecting the low basal nitrite production, which was opposite to the effect previously observed in noninfected mice, i. e. il-17 induced no production through the activation of both inos and enos. besides highlighting the importance of working under pinwormfree conditions when using experimental murine models for immunohematopoietic investigations, the data obtained pointed to the multiple layers of modulatory ability of this pinworm parasite and confirmed that the overall orchestration of the host response to the parasites is a complex process still being unraveled at both the cellular and molecular level. 2.-validation of the method: in order to determine linearity, analytical range, and reproducibility, three different sera with previously identified mc were serially diluted from 1 ⁄2 to 1/64 with a normal serum pool. 3.-implementation as a standard method for analysis of the mc in patients with paraproteinemia. the method showed good linearity: r 2 g 0.98. the analytical range was from 1 g/l to 70 g/l. the coefficient of variation (cv) was x 10 % for [mc] n 1 g/dl, and x 20 % for [mc] x 1 g/dl. this procedure was successfully implemented to quantify the mc in 1100 serum samples between march 2008 and february, 2009. among these samples, we have quantified light chains, heavy chains, igd and biclonal paraproteinemias. conclusions: 1. we have developed a simple, reproducible and low-cost method to quantify the mc using standard analyses of serum protein electrophoresis (spe), serum albumin, and densitometric quantification of mc and albumin regions. 2. the procedure allows monitorization of the mc in patients at diagnosis, after therapy, and evaluation of complete remission. objectives: direct influence of alpha-fetoprotein (afp) on immunosuppressor factors synthesis as well as immunosuppressor activity of bone marrow hematopoietic stem cells (hscs) was detected in our previous work in vitro. we investigated possible role of endogenous-produced afp in induction hscs immunosuppressor activity at tumor-bearing mice. methods: animal models, experimental oncology methods, immunomagnetic separation, cultural methods, cellular biology methods, flow cytometry, multiplex protein analysis, inhibition assay, colorimetric elisa, methods of molecular biology and rna interference (rnai) were used in this work. results: our results demonstrated afp endogenous synthesis adduced to elevation of immunosuppression activity of hscs in bone marrow. this afp effect becomes developed from 7 day and reached plateau level after 30 day of teratocarcinoma insertion. moreover, cd34 + cd38cells showed in spleen and main lymph nodes from 15 day and achieved plateau level after 60 day of teratocarcinoma growth. however, immunosuppression activities of purified hscs from spleen and lymph nodes discovered at 22 day and had a maximum pick at 60 day of teratocarcinoma inoculation. besides, immunosuppression activity of hscs from spleen and lymph nodes was more than 1,5 times lower than in bone marrow in the same period of tumor development. isolated hscs from bone marrow, spleen or lymph nodes produced similar spectrum of suppressor factors such as tgf-b 1 , il-10, pge 2 and no. inhibition of such suppressor factors lead to levelling of hscs immunosuppression activity ex vivo. kinetic of hscs quantity and activity had significant correlation (r cell number =0.81 and r activity =0.92) with afp level dynamics in blood serum. in addition, inhibition of afp expression by rnai caused to diminishing of hscs immunosuppression activity as well as hscs appearance in spleen and lymph nodes. conclusion: therefore, afp plays role not only specific inducer of hscs immunosuppression activity but also as a factor of activated hscs penetration into spleen and lymph nodes during afp-produced tumor development. cd40-ligand molecules -that are powerful immunomodulators -are strongly expressed by activated platelets; membrane-associated cd40l is cleaved to soluble (s)cd40l. we sought to examine the levels of scd40l in platelet concentrates (pcs) having led to an acute transfusion reactions (atr), and to test for its biological effect on b-lymphocytes. we recorded 5 atr episodes that could lead to investigation of residual platelets in container. two fractions of aliquots from each pc (and controls) were prepared, one for assay of individual supernatant fractions and one of corresponding lysates of platelets; scd40l -along with other products -were assayed by quantitative elisa. levels of il8, cd62p and pdgf-ab in pc supernatants and lysates from pcs associated with atr were similar to that in controls. supernatants of pcs associated with an atr contained higher scd40l levels compared to controls, and -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l . to examine if scd40l was biologically active, we stimulated purified b cells recovered from healthy blood donors and exposed those normal b cells to supernatants and cell lysates of pcs implicated in atr, or control material, and we measured il-6 secretion. the il-6 concentration was consistently below 5-10 pg/ml in pc supernatants and lysates, and unstimulated b cells did not secrete detectable levels of il-6. the addition of supernatant from atr-associated pc samples to purified b cells consistently resulted in sustained il-6 production over control (p x 0.05) at d2 after the onset of the culture, while -in a inversely correlative manner -corresponding platelet lysates contained lower levels of scd40l (p x 0.05). pre-incubation of b cells with cd40-blocking antibodies substantially abrogated il-6 secretion, unlike isotype-matched control. the partial blocking of cd40 binding on cd40 + b cells strongly suggests a potentially synergistic role in b cells for cytokines other than scd40l (under investigation) and indicates a sustained role for pc-derived scd40l. these data prompt us to investigate a larger series of events and controls to delineate on the one hand if certain factors can be responsible for an enhanced production of scd40l by collected/stored cpa. objectives: there is accumulating evidence for a role of natural killer (nk) cells in the antitumor response against hematological malignances. nk cells exert their action by means of a large panel of structurally distinct activating receptors that recognize their ligands on target cells. analysis of activating receptor pathways in nk cells has revealed a dominant role for natural cytotoxic receptors (ncrs) and dnax-accessory molecule-1 (dnam-1) in the lysis of acute myeloid leukemia (aml) blasts. here, we investigate the expression of these activating receptors on nk cells from aml patients stratified by age. methods: we analyses by flow cytometry peripheral blood mononuclear cells (pbmcs) from 30 aml patients before specific anti-leukemia therapy and 47 healthy donors. all results were analyzed statistically using spss version 15. results: aml patients under 65 years showed a significant reduction in the expression of dnam-1, nkp30 and nkp46 compared with age-matched controls. both healthy individuals and aml patients older than 65 years showed a reduction of these receptors compared with young donors. in contrast, we have found that nkp44 expression was increased in some patients of aml. on the other hand, the analysis of ligands for these activating receptors on leukemic cells showed a high variability that was not correlated with age or fab subtype. in addition, an inverse correlation in the expression of dnam-1 on nk cells and its ligand cd112 on aml blasts has been found in aml patients under 65 years. to analyze if leukemia cell were involved in the modulation of these receptors we have performed in vitro cocultures of leukemic blast and healthy nk cells. the initial recognition of aml cells by nk cells may represent a crucial process to prevent tumor development. here we described for the first time, a decrease of dnam-1 expression on aml patients and confirm previous reports showing a significant decrease of nkp30 and nkp46 on aml-nk cells. altogether, these alterations of the major receptors involved in nk cell-mediated cytotoxicity of leukemic cells represent an important mechanism of immunoescape that may correlate with disease progression and patient survival. a. stelmaszczyk-emmel 1 , e. gorska 1 , u. demkow 1 , m. wasik 1 1 medical university of warsaw, department of laboratory diagnostics and clinical immunology of developmental age, warsaw, polandglucocorticosteroids are often used in leukemia treatment. their therapeutic use is limited due to several side effects. one of them is multidrug resistance phenomenon, which causes lack of patients response for treatment. dexamethasone is used in schedule of children's all-b treatment and the response on glucocorticosteroids therapy is very important. the aim of this study was to examine whether dexamethasone changes multidrug resistance of lymphoblasts in all-b and their tendency to begin apoptosis. the study involved 10 children with all-b. bone marrow cells isolated by centrifugation on histopaque at the day of diagnosis were cultured for 2 or 48 hours with or without dexamethasone in concentration 10 -6 m. analysis of: p-gp surface expression, p-gp function (rhodamine 123 test), phi (carboxy-snarf) and apoptosis test (annexin-v and pi test) were performed with the use of flow cytometer coulter epics xl. for statistical analysis nonparametric wilcoxon test was used.the results showed that p-gp expression on lymphoblasts was 14,52 %±11,32. after 48 hours of lymphoblasts incubation with or without dexamethasone any statistically significant changes were observed. average percentage of lymphoblasts with rhodamine 123 efflux, which characterized p-gp activity, was 2,62 %±3,15. after 2 hours of cells incubation with dexamethasone there was seen significantly higher percentage of cells able to eliminate rhodamine 123 (4,31 %±4,03, p=0,015). average phi in lymphoblasts was 7,78±0,19. acidification of cells incubated 2 hours with dexamethasone was seen in 10-25 % percentage of cells 17) . rest of lymphoblasts showed alkalization (phi -8,00).the percentage of lymphoblasts in early stage of apoptosis after 48 hours incubation with dexamethasone (annexin-v test) was higher than in control cells (19,34 % vs 14,13 %; p=0,01). we concluded that dexamethasone does not influence surface p-gp expression on lymphoblasts of patients with all b but significantly increases activation of this protein. functional test should be performed to evaluate multidrug resistantace of leukemic cells, because surface expression of p-gp is not identical with its activity. moreover, dexamethasone alkalizes cytoplasm of lymphoblasts and induces early stage of their apoptosis. those effects may contribute to the treatment outcome. . however, small numbers of clonal pc can also be detected in the peripheral blood (pb) of the majority of patients and, in a minority of cases, mm is transformed into pc leukaemia (pcl). here, we describe that tumoral pc can express cd49d/cd29 integrin with high or, sometimes, low affinity states, which is associated with their retention in or release from the bm, respectively. objectives: to evaluate the activation state of cd29 on malignant pc from bm and pb, as well as its regulation. methods: pc and active integrin expression on these cells were detected with anti-cd38 and anti-cd29 (clon huts21) moab, respectively, by flow cytometry. to study the integrin activation with divalent cations and pc index proliferation (brdu+ cells), we used short-term pc cultures (18 hours). results: cd29 active form was expressed in the majority of normal and tumoral bm pc from healthy subjects (67.3±6.6, n=9) and mm patients in the early stages of the disease (32.6±7.5, n=17). in these cells, huts21 epitope was clearly upregulated by mn 2+ . in contrast, circulating pc were almost all huts21 negative, and levels did not significantly augment when these cells were exposed to mn 2+ . moreover, not only pb but also bm malignant cells from pcl patients were also huts21 negative and divalent cation refractory. it was also observed that pc from pcl patients showed an increased proliferative index (2.35±0.9 brdu+ cells, n=3) in comparison to pc from mm patients in the first stages of disease (1.3±0.2 brdu+ cells, n=5). these results suggest that the active form of cd29 must be expressed on pc to retain these cells within the bm environment. moreover, its downregulation is associated with increased numbers of circulating pc and disease progression. multipotent mesenchymal stem cells derived from (hucb) represent promising candidates for the development of future cellular therapy strategies. they are able to self-renew and they terminally differentiate into multiple lineages, including bone, cartilage, muscle, bone marrow, fat and other diverse connective tissues. in the first part of this study, we compared different protocols for the expansion of human mesenchymal stromal cells (hmsc) starting from diagnostic samples of bone marrow aspirates and the cord blood (cb). the protocols differed in the presence of either 10 % fetal bovine serum (fbs) with and without fgf2,or 10 % human platelet lysate (hpl), 5 %hpl, (10 % fbs + 10 % hpl), (10 % fbs + 5 % hpl). we obtained a significantly better expansion with hpl, compared to cells with a selected batch of fbs and in fewer days.in the second part of this study, we focused on proteins that were differentially expressed during osteogenic, adipogenic and vascular muscular differentiation by western blotting. we compared the quality and the quantity of protein expression before and after differentiation (day 18). two bm and two cb differentially expressed spots were observed between the two groups (before and after differentiation).we noted the low pourcentage of hmsc in cb samples: in ten samples, only two made msc colonies as in bm samples. we were also interested to the different coloration: osteogenic diffentiation was determined by alizarin red s staining, for adipogenic differentiation, the cells were stained with oil red o to visualize lipids droplets. background: inherited bone marrow failure syndromes (ibmfs) comprise a group of genetic disorders characterized by single or multiple cytopenias, as well as distinctive clinical features and varied molecular pathways. activation of p53 tumor suppressor pathway leads to cell cycle arrest and initiates apoptosis. we studied the presence of p53 dna (as a marker of cell cycle dysregulation); in bone marrow of children with fanconi anemia (fa) and those with acquired aplastic anemia (aaa). subjects and methods: this is a cross sectional study that involved: 1) ten cases with fa diagnosed on the basis of dna breakage analysis, 2) ten cases with aaa, and 3) ten normal control cases. the presence of p53 dna was measured in both bone marrow and peripheral blood samples using a real-time quantitative pcr by taqman assay. results: p53 dna was demonstrated in bone marrow of 90 % of children with fa, compared to 10 % in children with aaa (p x 0.001), while, no p53 dna was seen in normal control. a positive correlation between dna breakage and presence of p53 dna was seen in bone marrow from fa (p x 0.02, r0.81). the presence of p53 tumor suppressor gene by real time pcr in bone marrow of fa may represent an early indicator of significant dna genetic alteration in those patients. key: cord-015394-uj7fe5y6 authors: nan title: scientific abstracts date: 2008-12-23 journal: reprod sci doi: 10.1177/19337191080150020102 sha: doc_id: 15394 cord_uid: uj7fe5y6 nan by reduced placental oxygenation, hypoxia-induced oxidative stress is a predominant mechanism. we investigated the effect of hypoxic pregnancy, with and without antioxidant treatment, on placental and maternal circulatory indices of oxidative stress in rats. methods: on pregnancy day 6 , wistar rats were randomised into: normoxia (21% o 2 7 litters), hypoxia (14% o 2 8 litters) and hypoxia + vit c (14% o 2 + 500mg.100 ml -1 vit c in water, 6 litters). on day 20, dams were anaesthetised, maternal blood taken, pups measured and weighed, placentae weighed and frozen. only placentae from two male pups from any one litter were investigated. blood was processed for ascorbate, urate, l-cysteine and glutathione (gsh) measurement. placental protein was analysed for heat shock protein 70 (hsp70; western). results: hypoxia + vit c did not affect maternal food or water intake. vit c elevated maternal ascorbate by 70 % of baseline; a similar increment to human trials (poston et al. 2006) . hypoxia elevated placental hsp 70 and maternal plasma urate and l-cysteine, but decreased gsh. vit c in hypoxic pregnancies prevented all stimulated effects, but the reduction in gsh persisted. hypoxic pups had a reduced ponderal index and elevated head diameter: body weight ratio; effects also prevented by vit c. fetal oxygen uptake in normal and gdm pregnancies. emanuela taricco, tatjana radaelli, veronica cozzi, gabriele rossi, danila puglia, giorgio pardi, irene cetin. department of obstetrics gynecology "l. mangiagalli", irccs policlinico, mangiagalli e regina elena, milan, italy. background. diabetes in pregnancy has been associated with alterations of fetal growth probably due to increased nutrient availability and placental transport. fetal hypoxia and acidemia have been reported in pregestational diabetic pregnancies with poor glicemic control but this is still uncertain in well controlled patients. the role of placental function and the relationship between maternal and fetal circulation is crucial for efficient exchanges of oxygen and nutrients. since umbilical blood flow can be obtained by us in utero, we studied normal and gdm pregnancies in order to evaluate fetal oxygen uptake. methods. 21 normal (n) and 21 gdm pregnancies were studied at term, at the time of elective caesarean section. umbilical vein volume flow (qumb) was measured by us before caesarean section and blood samples from umbilical vein (uv) and artery (ua) were obtained. blood gases and acid-base balance were evaluated. results. average fetal weights were similar in both groups (n=3274±62; gdm=3381±111 g) while placental weights were significantly different (n=474±16; gdm=571±35 g). n and gdm pregnancies showed similar values of qumb and qumb/kg of fetal weight. (qumb: 245.3±19.0 in n and 242.2±18.5 ml/min in gdm; qumb/kg: 74.6±5.0 in n and 71.4±4.6 ml/min/kg in gdm). in fetuses from gdm pregnancies a significant reduction in o 2 sat, o 2 cont and po 2 and a significant increased lactate conc was found in both uv and ua compared to n (table 1) . o 2 umb uptake (n=0.78±0.08; gdm=0.56±0.08 mmo/l/min) and o 2 umb uptake/kg (n=0.24±0.02; gdm=0.16±0.02 mmo/l/ min/kg) were significantly lower in gdm compared to n fetuses. conclusions. our data indicate that fetuses from gdm pregnancies show a significant reduction in oxygen supply despite a normal blood flow/kg of fetal weight. these data may suggest that a good maternal metabolic control is not sufficient to ensure normal placental oxygen supply and/or utilization by the gdm fetus. background: synthetic glucocorticoids (sgc) are given to mothers at risk of preterm delivery to promote fetal lung maturation. evidence is emerging indicating long-term effects of such treatment on endocrine function and behavior in offspring. however, virtually nothing is known concerning potential transgenerational influences on growth, endocrine function and behaviour. we hypothesize that repeated treatment of grandmothers (f0) with sgc will alter hypothalamic-pituitary-adrenal (hpa) function in f2 offspring with no manipulation of the f1 pregnancy. methods: pregnant guinea pigs (f0) were subcutaneously injected with betamethasone (beta; 1mg/kg) or vehicle (veh) on gestational days 40/41, 50/51 60/61. adult f1 female offspring from each group were mated with control males. hpa function was assessed in adult f2 offspring by non-invasive measurement of salivary cortisol concentrations: 1) under basal conditions, 2) during and following exposure to psychological stress (high frequency strobe light) or psychological/physical stress (forced swim) and, 3) following dexamethasone suppression. results: there was no effect of beta (f0) on bodyweight from birth to adulthood in f2 offspring. basal salivary cortisol in the betaf2 females was lower than in the vehf2 group in the morning but not the afternoon; there were no differences in male f2 offspring. in contrast, both male and female betaf2 failed to mount adrenocortical responses to psychological stress compared to vehf2 offspring that mounted robust responses (p<0.02). swim stress induced robust adrenocortical responses in all groups (p<0.0001), however, the response was consistently lower in betaf2 offspring. beta exposure also led to a significant difference (p<0.003) in the cortisol response to dexamethasone suppression in female (f2) offspring, with a similar trend in males. conclusion: prenatal exposure to sgc (f0) causes transgenerational programming of adrenocortical function in adult f2 offspring. grand maternal exposure to beta results reduced basal adrenocortical activity in betaf2 female offspring, and caused stress hypo-responsiveness in both males and females. dexamethasone suppression tests indicate altered central glucocorticoid feedback. these findings have important ramifications for the management of human preterm labor. support: canadian institutes of health research. in the uterine and umbilical vasculatures, we hypothesized that their remodeling would also be blunted in pregnant enos -/-mice, leading to an elevated vascular resistance and decreased blood flow to the placenta contributing to fetal growth restriction. methods: utero-and umbilical-placental blood velocity waveforms and umbilical arterial diameters were measured using 40 mhz ultrasound biomicroscopy in control (c57bl/6j) and enos -/-mice at 17.5 days of pregnancy (n=6 mothers). spiral artery and fetal capillary morphologies, and uterine arterial diameters were evaluated from vascular corrosion casts. tissues were collected for hydroxyprobe-1 and actin immunohistochemistry to identify hypoxic and smooth muscle regions. we calculated resistance index ((s-d)/s) from systolic (s) and diastolic (d) velocities, and blood flow from mean velocity and vessel area. results: calculated uterine blood flow normalized to the weight of the uterus and its contents was 55% lower (p<0.05) in pregnant enos -/-mothers due to large reductions in uterine artery diameter (-33%, p<0.001) and mean velocity (-17%, p<0.05). uterine arterial resistance index was 52% higher (p<0.001), and the spiral arteries were less coiled and contained more smooth muscle actin in enos -/-mice than controls. more intense hypoxic immunoreactivity was detected in the spongiotrophoblast and trophoblast giant cell layers of the junctional zone of enos -/-placentas, whereas fainter staining was only detected in the spongiotrophoblast cell layer in controls. in the umbilical circulation, flow normalized to fetal weight was not significantly changed although the resistance index was slightly elevated (4%, p<0.05) and capillary lobule length was reduced by (-28%, p<0.05). fetal organs showed increased hypoxic immunoreactivity suggesting reduced organ oxygen delivery in enos -/-fetuses. conclusions: results suggest that enos plays an important role in uterine and spiral artery remodeling and in augmenting utero-placental blood flow during pregnancy. it also appears to enhance oxygen delivery to the placenta and fetus. enos may contribute to normal fetal growth by these mechanisms. integrin 5 methods: hpmcs isolated from term placentas were assessed for their phenotype markers, mutilineage capacity, and the expression of integrin molecules. the hpmcs were induced to endothelial cell differentiation in the presence of endothelial cell growth medium 2 with 2% of fcs and 50 ng/ml vegf for 14 to 21 days. the angiogenesis ability of these cells was demonstrated by using an in vitro angiogenesis kit and in vivo chick chorioallantoic membrane assay from ten-day-old embryos. blocking antibodies specific to integrin 4 , associated with gdm. this study was adequately powered to detect association in the caucasian and asian group. the absence of association suggests that gdm and t2dm may have more divergent molecular pathophysiology than previously suspected. background: uterine endometrium has a unique cycle of physiological angiogenesis. in mice, endothelial progenitor cells (epc) contribute to endometrial angiogenesis being incorporated after oestradiol administration. objective: to determine whether circulating © epc number and function vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: ten healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years) with regular menses (27-29 days) were studied. venous blood was collected during menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15, 20-22) . cepcs, serum oestradiol (e) and progesterone (p) were measured at each phase. cepcs were quantified by phenotype using flow cytometry (leukocytes co-expressing cd133, kdr and cd34) and function by the epc-colony forming unit (cfu) assay. epc-cfus were stained for endothelial markers including uptake of acetylated low-density lipoprotein, binding of lectin (ulex europaeus) and endoglin. results: luteal p was >30 nmol/l in all women. cepc numbers increased in the menstrual and follicular phases being 3-fold higher in the follicular compared to periovulatory phase (p<0.05). there was no significant variation in cepc function over the menstrual cycle. there was no correlation between serum e or p levels and cepc number or function. conclusion: cepcs number but not function (epc-cfu assay) vary during the menstrual cycle with numbers increasing during the menstrual and follicular phase and falling in the periovulatory and luteal phase of the menstrual cycle. this may represent mobilisation of cepcs from the bone marrow and subsequent incorporation into the endometrium. neither function nor cepc number correlate with serum p or e. our previous studies demonstrated that tissue factor (tf), which binds factor vii to act as a potent pro-coagulant and angiogenic factor, is over-expressed in endometriotic lesions. thus, we determined whether tf could serve as a target for the elimination of pre-established ectopic human endometrial growth in a mouse model. icon is composed of a mutated factor vii (fvii) domain targeting tf and an igg1 fc (fvii/igg1 fc) effector domain that activates antibody-dependent immune responses against tf bearing endothelial cells. methods: athymic, ovariectomized and estrogen-treated mice received intraperitoneal (i.p.) injections of 1.0 mg of proliferative phase human endometrial tissue derived from normal (disease-free) women. twelve days after inoculation to establish lesions, icon protein (10 ug) was delivered i.p. once a week for 4 weeks. after sacrifice, animals were subject to gross inspection. residual endometriotic tissue was formalin fixed, paraffin embedded and immunostained for von willebrand's factor (vwf) and tf. results: compared to control mice, treatment with 10ug of icon abolished all lesions in 7 of 10 mice and reduced both size (2.33 to 1.4mm) and number of lesions (1.9 to 1 per diseased mouse) in the remaining mice. moreover, residual lesions from icon treated mice were atrophic and displayed significant reductions in vessel areas of 87% +/-26% (p=0.002, mean +/-sem, n=3) as determined by vwf immunostaining. no hemorrhagic or thrombotic sequelae were observed in icon treated mice. conclusions: unlike other treatments that target developing angiogenesis, icon can target both developing and established human endometriotic lesions in athymic mice. the gross and microscopic vessel analysis suggests that icon directly or indirectly destroys endometriotic vessels. thus, icon presents a novel, non-toxic therapy for endometriosis. compared to the miscarriage and top groups. ihc for crisp3 demonstrated increased secretion of crisp3 in the glandular epithelium and expression in the leucocytes of the tubal uterine decidua. conclusions: there are differences in decidual gene expression in tubal compared to iu pregnancies. we believe that potential biomarkers of tubal pregnancy can be discovered by focusing on secreted proteins associated with uterine decidualization. one of these proteins, crisp3, is significantly increased in decidua of tubal ectopic pregnancies and we are currently investigating its expression pattern in sera from women with tubal compared to iu pregnancies. progesterone and hoxa 10 regulate gaba-a pi receptor expression, membrane translocation and activation. homayoun sadeghi, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale school of medicine, new haven, ct, usa. objective the expression of the gaba-a pi receptor has been previously described in the human endometrium in both luminal epithelium and stroma. it is upregulated during stromal decidualization in the rat and in the implantation window of human endometrium. the gaba receptor is modulated by progesterone metabolites, with the resultant opening of the receptor ion channels which allow the water flux necessary for trophoblast attachment. here we identified regulators of pi subunit receptor gene expression and activity. the well-differentiated human endometrial adenocarcinoma cell line (ishikawa) and human endometrial stromal cells (hesc) were transfected with hoxa10, treated with progesterone, or treated with vehicle or empty plasmid controls. gaba-a pi receptor mrna upregulation was evaluated by real time rt-pcr. protein expression was evaluated using immunohistochemistry. results gaba-a pi receptor mrna expression was increased with either progesterone treatment (31%, p=0.049) or hoxa10 transfection (11%, p=0.027). coadministration of progesterone along with increased hoxa10 transfection had no additive effect on the expression of gaba-a pi receptor mrna (p=0.98). gaba-a pi receptor protein expression was similarly increased by each treatment. either hoxa10 or progesterone independently caused translocation of the gaba receptor from the cytoplasm to the cell membrane in ishikawa cells. conclusion gaba-a pi receptor expression is increased in the human luminal epithelium and stroma in the window of implantation. activation of the pi subunit leads to opening of ion channels, likely allowing flux of water into the epithelial cells and out of the uterine lumen. progesterone and hoxa 10 each increase both pi subunit receptor expression and membrane translocation. the lack of additive effect suggests progesterone induced pi subunit receptor expression is likely mediated indirectly through progesterone's regulation of hoxa 10 expression. finally, after receptor expression and translocation, progesterone mediated gaba receptor ion channel activation mediates water resorption necessary for implantation. cancer. suzy davies, donghai dai, kimberly k leslie. obstetrics and gynecology, university of new mexico, albuquerque, nm, usa. objectives: endometrial cancer is the most frequent gynecologic cancer in women. it affects an estimated 40,000 women in the us every year, and long term outcomes for patients with advanced stage or recurrent disease are poor. targeted molecular therapy against the vascular endothelial growth factor (vegf) and its receptors constitute a new therapeutic option for patients that is now under study by the gynecologic oncology group in a phase 2 trial, gog 229e (now in second stage accrual). the goal of our work was to assess the potential effectiveness of vegf/vegfr blockade in preclinical endometrial cancer models. methods: these studies employed two agents, bevacizumab (avastin, a vegfa blocking antibody) and vandetanib (zactima, a tyrosine kinase inhibitor of egfr and vegfr2). ic 50 experiments were performed on endometrial cancer cells in culture using four established cell line models. xenografted athymic mice were also employed to test the ability of compounds to inhibit tumor growth in vivo. tumors were isolated from controls and treated animals, mrna was extracted, and affymetrix gene expression arrays were performed to determine the genes consistently modulated by treatment. results: compared to vandetanib with an ic 50 of 1.15 m, bevacizumab showed little activity on cell proliferation in vitro, and cell numbers were not reduced by 50% using concentrations up to 2.5 m. however, bevacizumab demonstrated robust activity in the athymic mouse model, resulting in a significant decrease in tumor formation and growth compared to vehicle treated animals when dosed bi-weekly in a concentration of 0.2 mg/mouse ip. tumors from this model demonstrated that eighteen genes were consistently up or down regulated in the presence of bevacizumab. among the regulated transcripts was microrna21, which was significantly down-regulated. microrna21 is an anti-apoptotic factor, and its inhibition by bevacizumab predicts for increased expression of the tumor suppressor pten, decreased cell proliferation, and a reduced capacity for metastasis. conclusions: these studies confirm that the vegf pathway is a good target for new therapies against endometrial cancer. blocking this pathway not only inhibits angiogenesis, but also results in changes in gene expression that enhance apoptosis and reduce cellular proliferation and tumor invasion. we collected fibroid and adjacent normal tissues following hysterectomy with patient consent and institutional irbs. to date, 840 data points for each gene from 420 arrays have been collected from 42 patients. the fluorescence readings were log-transformed and normalized with the robust quartile normalization method. quality control of the normalized data was performed to remove 13 arrays that deviated from twice the inter-quartile range calculated from the 420 array signals. results: the custom microarray profiling identified 38 genes that were differentially expressed between normal and fibroid tissues (p <0.01; fdr<0.13). among them, 12 genes encode receptor tks, 15 genes encode tk ligands, and 11 genes encode cell cycle and apoptosis proteins. clustering analysis of the mean log2 ratios of these 38 genes has led to the division of the 42 patients into two major groups. thirty four (81%) belong to a group characterized by the significant downregulation of the cyr61 (ccn1) gene in fibroid tissues. the cyr61-down group can be further divided into two sub-groups based on the expression of another tk ligand, efn4a. other receptor differentially expressed tk did not segregate with the three defined sub-groups. finally, there was no sub-group segregation based on age of the patient, menstrual phase, or weight of the fibroid. conclusion: our results have demonstrated that tyrosine kinases and their ligands are uniquely differentially expressed between normal and fibroid tissues. unique tyrosine kinase ligands in our population were cyr61 and efn4a, and these markers created three molecular classification groups based on their differential expression. these results support the hypothesis that tyrosine kinase ligands are involved in fibroid growth, and may offer targets for a strategy to avoid hysterectomy. microvascular perfusion sonographic imaging to detect early stage ovarian cancer. joanie mayer hope, 1 arthur c fleischer, 2 brian day, 1 stephanie v blank, 1 bhavana pothuri, 1 robert wallach, 1 john p curtin, 1 david a fishman. 1 1 gynecologic oncology, new york university school of medicine, new york, ny, usa; 2 radiology, vanderbilt university medical center, nashville, tn, usa. objective: epithelial ovarian cancer (eoc) is the 4th leading cause of death in us women due to the inability to detect early stage disease. recent sonographic developments involving harmonics, pulse inversion, and the use of contrast agents justify the hope that depiction of aberrant tumor microvascularity associated with early disease can occur. this study utilizes these new techniques to assess the unique microvascularity associated with early stage eoc. methods: we used pulse inversion harmonic microvascular imaging (mvi) technology to depict differences between benign and malignant ovarian lesions. contrast enhanced harmonic transvaginal (tv) sonography was performed using the philips iu22 scanner after intravenous injection of 1 g of definity (bristol-myer-squibb). split screen real-time images were acquired displaying conventional sonographic views adjacent to harmonic, low mechanical index images. morphologic features (thickened wall, papillary excrescence, calcifications) and aberrant vascularity were noted. q-lab quantification of wash-in, peak enhancement, and wash-out times as well as area-under-thecurve (corresponding to microvessel perfusion) were compared using student's t-tests. results: to date, contrast enhancement patterns of 93 ovaries have been analyzed, 78 benign and 15 malignant. of the malignancies (3 fallopian tube, 12 ovarian: 3 stage i, 12 stage iii), 12/15 women were correctly identified using conventional tv imaging and 3 others were identified using mvi 15/15. all benign lesions were correctly identified by mvi 78/78 while tv detected 81 normals (3 false negatives). the lesions detected as malignant by mvi were 2-stage i fallopian tube and 1-stage i eoc. contrast enhancement kinetics of malignant lesions demonstrated similar wash-in (26.1 ± 6.3 vs 24.9 ± 7.6, p = 0.7), greater peak enhancement (23.3 ± 2.8 vs 12.3 ± 3.9, p < 0.01 ), longer wash-out (139.9 ± 43.6 vs 46.3 ± 19.7 (p < 0.01), and greater perfusion (2012.9 ± 532.9 vs 523.8 ± 318, p < 0.01) when compared to benign lesions. conclusion: contrast enhancement patterns are significantly different in benign vs. malignant ovarian masses. this technique has clear potential in differentiating benign from malignant lesions and for detecting occult stage i disease. identification and characterization of mir-199a as regulator of ikk expression and its function in ovarian cancer cells. rui chen, ayesha b alvero, thomas rutherford, gil mor. department of obstetrics and gynecology, yale university school of medicine, new haven, ct, usa. introduction: the proinflammatory environment associated with tumor growth and chemoresistance is produced by both immune cells and cancer cells. the nf-b pathway plays a critical role mediating the capacity of cancer cells to produce pro-inflammatory cytokines. recently, we described a group of epithelial ovarian cancer (eoc) cells characterized by ikk expression as the main factor promoting nf-b activation and cytokine production. in this study we evaluated the regulation of ikk in eoc cells. we describe the identification and characterization of mir-199a as a regulator of ikk expression, thus indirectly of nf-b activity and function. materials and methods: 11 human eoc cell lines were established from malignant ovarian cancer ascites. protein expression were determined by western blotting. ikk mrna was measured by rt-pcr. cytokines were profiled by the luminex 200 system. ikk transfection was done with roche fugene6 transfection reagent. mirna microarray was done with invitrogen ncode multi-species mirna microarray kit. mir-199a qrt-pcr was performed with invitrogen ncode sybr greener mirna qrt-pcr analysis kit. mirna transfection was done with ambion siport neofx agent. the ikk 3'-utr luciferase reporter plasmid was established based on ambion pmir-report mirna expression reporter vector. results: ikk expression was associated with nf-b cyclic activity and the ability of type i eoc cells (but not type ii) to produce inflammatory cytokines. transfection of ikk into type ii eoc cells reversed their phenotype. mirna microarray identified 18 mirnas differentially expressed in type i versus type ii cells, one of which, mir-199a, had 3 putative binding sites in the 3'-utr of ikk mrna. mir-199a introduction into type i cells inhibited ikk expression, and direct inhibition through ikk 's 3'-utr was confirmed by luciferase assay. conclusion: we describe for the first time the identification of ikk as a potential key switch between chemo-resistant and chemo-sensitive phenotypes, by regulating nf-b activity in eoc cells. furthermore, we identified mir-199a as a direct regulator of ikk expression. ikk expression may represent an adaptational stage in tumor progression allowing cancer cells to create their own inflammatory environment. these findings may provide novel molecular targets and potential markers for individual therapy selection. regulation of cd55 in the rat uterus by nitric oxide and the involvement of p13 kinase pathway. uma yallampalli, rebakah elkins, pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cd55 is expressed in many cell types including uterine cells and has been shown to play an important role in protecting against compliment attack. we have previously shown in endometrial cell lines that cd55 levels were down regulated by nitric oxide (no) . in this study we extend our previous observations to determine if no down regulates cd55 in rat uterine tissues and assess its mechanisms of action. methods. non pregnant rats (200g; b wt) were bilaterally ovariectomized under ketamine anesthesia. groups of ovariectomized rats were implanted with alzet mini pumps to deliver nitro-l-arginine melthylester (l-name) at 10 or 50/mg/rat/day in saline or saline alone. uteri were obtained from these rats at 48 or 72 hours after infusion. in another set of experiments uteri were obtained from ovariectomized rats and cut in to small pieces and incubated in vitro with either l-name (3mm), diethylenetriamine-no 1mm) or worthmanin (0.1 m) in mem without phenol red for 24 hours. uterine tissues were homogenized in trizol and mrna levels for cd55 were measured using rt-pcr and expressed as a ratio to 18s. results. results show that in vivo treatment with l-name to ovariectomized rats caused elevations in uterine cd55 mrna levels in a time and dose dependent manner with maximal responses seen with 50mg l-name and at 72 hours. in vitro studies show that deta-no suppressed cd55 mrna levels in the rat uterus. both l-name and pi3 kinase inhibitor, worthmanin caused increases in cd55 levels in these tissues and the effects of worthmanin are reversed by deta-no. these results suggest that cd55 levels in the rat uterus are down regulated by no and are upregulated when no synthesis is inhibited by l-name. further, pi3 kinase appears to be involved in cd55 regulation in the rat uterus and no donor appears to modulate this response. lung. lakeitha r foster, 1 daniel b hardy, 2 carole r mendelson. 2 1 dept of pediatrics; 2 depts of biochemistry and ob/gyn, ut southwestern medical center, dallas, tx, usa. during 95% of human pregnancy, the maternal uterus is maintained in a state of almost complete quiescence by elevated circulating levels of progesterone (p 4 ). we previously observed that p 4 acting through the progesterone receptor (pr) serves an anti-inflammatory role and inhibits uterine contractility by antagonizing nuclear factor b activation and cyclooxygenase-2 (cox-2) expression (hardy et al., 2006) . our previous findings also suggest that the fetus provides an important signal for the initiation of labor near term through augmented secretion of the major lung surfactant protein, sp-a, into amniotic fluid (condon et al., 2004) . secreted sp-a, in turn, activates fetal macrophages which migrate to the maternal uterus where they release cytokines and promote an inflammatory response, leading to labor. in the present study, we tested the hypothesis that maternal p 4 also maintains uterine quiescence by inhibiting sp-a production by the fetal lung. age matched icr mice were injected s.c. once daily either with sesame oil (control) or p 4 (1 mg/ml) from 15 to 19 days post-coitum (dpc). as expected, treatment with p 4 delayed parturition by 24-48 h. this also was associated with a decrease in uterine cox-2 mrna expression, as compared to the vehicle-injected controls. interestingly, maternal p 4 treatment caused a marked decrease in the levels of immunoreactive sp-a protein secreted by the fetal lungs into in amniotic fluid at 19 dpc. furthermore, sp-a protein and mrna levels were reduced in the fetal lungs of p 4 -injected mothers, as compared to controls. this was associated with an inhibitory effect of p 4 treatment on cox-2 protein and mrna levels in the fetal lungs. these findings were of interest, since cox-2 expression is markedly upregulated during differentiation of human fetal lung (hfl) explants in culture (hardy et al., 2005) and endogenous and exogenous prostaglandins increase sp-a expression in hfl (acarregui et al., 1990) . collectively, these findings suggest that maternal p 4 treatment prevents increased uterine contractility, in part, by inhibiting inflammatory response pathways within the fetal lung. this, in turn, blocks the developmental induction of sp-a expression and its secretion into amniotic fluid. in this manner, maternal p 4 inhibits an important fetal signal leading to labor. supported by nih p01 hd011149; nih r37 hl050022. recent evidence suggests that leukocytes infiltrate uterine tissues at the time of parturition implicating inflammation as a key mechanism of human labor. ccl-2 is a pro-inflammatory cytokine that may contribute to the development of inflammatory reaction in the myometrium. previously we showed upregulation of rat ccl-2 gene expression in myometrium during term and ru486-induced preterm labor. also ccl-2 was elevated specifically in the gravid horn of unilaterally pregnant rats suggesting that mechanical strain imposed by the growing fetus controls its expression in the myometrium. the objective of this study was to investigate the role of mechanical stretch as a possible regulator of myometrial leukocyte infiltration and ccl-2 as a mediator of this stretch response. we also studied the effect of progesterone (p4) on the myometrial secretion of ccl-2. methods. we used primary culture of rat myometrium smooth muscle cells (smcs) to study in vitro ccl-2 gene and protein induction by static mechanical stretch. ccl-2 gene expression analysis was performed by real-time rt-pcr and immunoreactive (ir) protein content was measured by elisa assay. we used primary rat monocytes to access whether stretch-induced ccl-2 production by myometrial smcs resulted in enhanced monocyte chemotactic activity. results. myometrial cells were stretched for 2-24 hours and the supernatants collected. analysis of media conditioned by primary myometrial smcs revealed that static mechanical stretch (25% elongation for 24 hours) caused a significant accumulation in ir ccl-2 which was repressed by pretreatment with p4 (1 um). the rise in ccl-2 protein levels was preceded by a transient increase on ccl-2 mrna. the migration of primary rat monocytes in response to conditioned medium from stretched myometrial smcs was much greater than that of conditioned medium from control non-stretched cells. co-incubation with a neutralizing antibody to ccl-2 significantly reduced the chemotaxis of monocytes in response to the stretch-conditioned medium. conclusion: uterine smcs play an active role in uterine inflammation by producing chemokines and promoting the chemotaxis of immune cells into the myometrium. the blockade of this effect by p4 offers a potential explanation for the therapeutic actions of this hormone in the prevention of preterm birth. membranes during human labor. nardhy gomez-lopez, 1,2 guadalupe estrada-gutierrez, 1 lourdes vadillo-perez, 1 felipe vadillo-ortega. 1 1 direction of research, instituto nacional de perinatologia, mexico city, df, mexico; 2 escuela nacional de ciencias biologicas, ipn, mexico city, df, mexico. introduction. leukocytes arriving to the choriodecidua (chd) during labor are capable to secrete cytokines and matrix metalloproteinases that may play a role in the fetal membranes (fm) extracellular matrix degradation. objective. the aim of this work was to identify changes in the leukocyte subpopulations in the chd and fm during human labor. methods: fm were obtained from two groups of women: 1) term without labor (n=4) and 2) term with spontaneous labor (n=4). chd cells were isolated and analyzed by flow cytometry. explants of fm were embedded in paraffin and analyzed by confocal microscopy. in both techniques, cd45, cd56, cd14, cd19 and cd3 subpopulations of leukocytes were identified. intracellular mmp-9 was also identified in these cells. results: major changes in leukocytes subpopulations during labor involved a higher amount of cd3+, cd+14 and cd56+ cells, both in the chd and inside the fm. mmp-9 was associated to cd 56+ cells. cd3+ exhibited a more widespread localization in the fm and cd56+ cells were localized in the contact with the trophoblast layer during labor. conclusions: leukocyte populations changes both in the chd and fm during labor and are characterized by arrival and infiltration of specific subpopulation of lymphocytes and monocytes. nk cells are enriched in mmp-9, which may be related to a role in extracellular matrix degradation leading to the rupture of fetal membranes. women with a history of a pregnancy complicated by preeclampsia or intrauterine growth restriction (iugr) have an increased risk of future cardiovascular disease. excessive weight, particularly abdominal fat mass, is associated with cardiovascular morbidity and mortality. objectives: the aim of this study was to investigate differences in body composition and fat distribution between women with a history of preeclampsia or iugr and uncomplicated pregnancies. methods: from a genetically isolated population in the southwest of the netherlands, non-pregnant women with a history of preeclampsia (n=45), iugr (n=53) and uncomplicated pregnancies (n=106) were recruited at a mean follow up time of 10.8 years after pregnancy. body composition and fat distribution were assessed by dual energy-x-ray absorptiometry (dxa) and anthropometric measurements. results: women with a history of preeclampsia compared to controls had higher mean total-, fat-and lean mass (p <0.05) as well as higher mean indices of body mass, fat mass and lean mass (p< 0.05). no significant differences were found for these variables between women with a history of iugr and controls. women with a history of preeclampsia had higher waist circumferences and waistto-hip ratios (p<0.001) as well as excess of android fat mass and increased android-to-fat ratios (p<0.05). women after pregnancies complicated by iugr had higher waist-to-hip ratios (p <0.001). after controlling for body mass index, both women with a history of preeclampsia or iugr had higher waist circumferences (p <0.001) and waist-to-hip ratios (p <0.001) as well as smaller hip circumferences (p < 0.001). conclusion: despite differences in body mass index, both women with a history of preeclampsia and women after pregnancies complicated by iugr have a metabolically adverse fat distribution, marked by an excess of fat deposition in the abdominal region relatively to the hip region. these findings may explain, at least partly, their increased cardiovascular risk. women with a history of preeclampsia. marc ea spaanderman, 1 timo h ekhart, 2 robert aardenburg, 2 louis lh peeters. 2 1 obstetrics and gynecology, radboud university medical center, nijmegen, netherlands; 2 obstetrics and gynecology, university medical center maastricht, maastricht, netherlands. background: a history of preeclampsia is associated with persistent short-term alterations in circulatory function and remote cardiovascular disease. in this study we tested the hypothesis at least 20 years after preeclamptic pregnancy renal and central hemodynamic function is impaired as compared to women with uncomplicated pregnancy. methods: in 20 formerly preeclamptic women (pe) and 24 healthy parous controls (control) who were normotensive at 6 weeks post-partum follow up, we assessed at least 20 years after delivery blood pressure (mmhg), cardiac output (co, doppler ultrasonography, l/min) and effective renal plasma flow (erpf, pah clearance, ml/min/1.73m 2 ) after which we calculated total peripheral vascular resistance (.100 dyne.s/cm 5 ) and renal vascular resistance (.100 dyne.s/cm 5 ). data were analyzed parametrically (p<0.05). results: age and bmi were comparable between groups. blood pressure was higher in formerly pe. moreover, 40% of formerly pe women and 13% of control were hypertensive (p<0.05). although cardiac out was comparable between groups, total peripheral and renal vascular resistance were about 20% higher and erpf 15% lower in formerly pe women as compared to control. conclusion: at least 20 years after gestational hypertensive disease, women who were normotensive at direct follow up have impaired renal and central hemodynamic function and developed more often chronic hypertension. long-term follow up may also be warranted in apparently healthy formerly pe women who are normotensive at post-partum follow up. circulatory function in formerly preeclamptic women and healthy parous controls co erpf tpvr rvr formerly pe 5.0±0.6 397±61* 18±2* 120±29* control 4.7±0.8 469±80 15±2 97±25 * = p<0.05 101 pregnancy increases blood-brain barrier permeability coefficient (l p ) to lucifer yellow: role of estrogen. marchien j wiegman, 1,2 marilyn j cipolla. 1 1 neurology, ob/gyn and pharmacology, university of vermont, burlington, vt, usa; 2 ob/gyn, university medical center groningen, groningen, netherlands. background: eclampsia is similar to posterior reversible encephalopathy syndrome in which an acute rise in blood pressure causes breakthrough of autoregulation, blood-brain barrier (bbb) disruption, and cerebral edema formation. we previously showed that late-pregnant (lp) animals developed cerebral edema during breakthrough, a response that was absent in nonpregnant (np) animals. in the current study we hypothesized that pregnancy predisposes the brain to edema during acute hypertension by enhanced bbb permeability. we further hypothesized that the underlying effect of pregnancy on the bbb permeability is due to elevated estrogen levels. methods: permeability coefficients (l p ) to lucifer yellow (ly), a polar compound that does not pass through tight junctions, were compared in posterior cerebral arteries (pca) from 4 groups of sprague dawley rats: np (n=7), lp (d20; n=7), ovariectomized and implanted with 17 -estradiol (0.5mg, 21-day release) and estriol (5.0mg, 21-day release) pellets for 14 days (ovx+e; n=6), and ovariectomized and implanted with placebo pellets for 30 days (ovx; n=6). pcas were isolated, pressurized in an arteriograph, and perfused with 0.5mg/ml ly in saline. concentration changes of ly outside the vessel wall were determined at pressures from 60-200mmhg. the slope of the pressure vs. permeability curve is the rate of flux, or l p for ly. results: l p for ly was significantly increased in pcas from lp and ovx animals vs. np (p<0.05; figure 1 ). estrogen was protective of the bbb only in ovariectomized animals, decreasing l p 150% in ovx+e vs. ovx. however, pregnancy did not afford protection and had a l p that was 256% greater than np. conclusions: pregnancy significantly increases bbb permeability to ly, an effect that may predispose the brain to edema formation during acute hypertension. these data also show that estrogen modulates l p in ovariectomized animals differentially than pregnancy, suggesting that the increased bbb permeability in pregnancy is caused by a mechanism other than elevated estrogen levels. in both pre-and early pregnancy, the sympathoinhibitory response to volume expansion is blunted in formerly preeclamptic women with low plasma volume. ineke krabbendam, 1 marc ea spaanderman, 1 dorette a courtar, 2 robert aardenburg, 2 ben j janssen, 3 fred k lotgering, 1 louis lh peeters. 2 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 obstetrics and gynecology, university hospital maastricht, maastricht, netherlands; 3 pharmacology and toxicology, university of maastricht, maastricht, netherlands. background: the circulation of formerly preeclamptic women with a low plasma volume (lpv) is characterized by sympathetic dominance. these women respond to a new pregnancy with an aberrant rise in atrial natriuretic peptide (anp) and a 3 times higher chance to develop recurrent gestational hypertensive disease compared to their counterparts with normal plasma volume (npv). anp has sympathicomimetic capacity. we postulate that the sympathetic overdrive in lpv-women is associated with a reduced venous capacitance. to this end, we compared the response to volume expansion (ve) in women with lpv and npv, both before and in pregnancy. method: in 24 non-pregnant normotensive formerly preeclamptic women, we measured pv (hsa i 125 indicator dilution method) at least 6 months post partum. we intravenously infused 500 ml of iso-oncotic fluid over 30 minutes. during the infusion, we recorded changes in heart rate (hr, bpm), blood pressure (bp, mmhg), cardiac output (co, l/min), sympathetic activity (lfsys, mmhg 2 , low frequency component of spontaneous fluctuations in systolic bp, portapress) and anp (nmol/l). eight women became pregnant within 1 year and were evaluated at 12 weeks gestation. changes in circulatory and autonomic function between and within groups were analyzed non-parametrically (p<0.05). results: before pregnancy, ve leads to comparable changes in hr, bp and co in women with lpv (17/24) and npv (7/24). in npv, lfsys decreased 28%, but only 7% in lpv (p<0.05). anp remained unaltered in npv, but increased in lpv. in the pregnant group, 4 women had lpv and 4 had npv. in both groups, pregnancy did not alter the response to ve. conclusion: irrespective of pregnancy, the sympathoinhibitory response to ve is diminished in lpv. these data suggest that in these women ve leads to venous overfill, giving rise to anp-release and consequently sympathetic activation, flattening the normal baroreceptor-mediated sympathoinhibitory response. we speculate that this mechanism contribute to circulatory maladaptation to pregnancy, sympathetic dominance and subsequent gestational hypertensive disease. in both pe and ht, serum sflt-1 was increased, and plgf reduced at all gestations (p<0.0001). seng levels were also increased in pe. after 28 weeks (but not before) antihypertensive treatment was associated with a significant fall in serum sflt-1 and seng, in pe only. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). only sflt-1 was significantly reduced in the placenta in women who received antihypertensive therapy. conclusion in pe, antihypertensive therapy after 28 weeks' gestation is associated with a significant fall in serum sflt-1 and seng, and in placental sflt-1. these findings raise the possibility that these drugs may have an effect on the pathophysiology of pe other than their known antihypertensive action. the synergistic effect of soluble vegf receptor 1 pre-treatment and small doses of tnf-on endothelial cells. tereza cindrova-davies, 1 debbie a sanders, 2 olivera spasic-boskovic, 1 graham j burton, 1 d stephen charnock-jones. 2 1 dept of pdn, university of cambridge, united kingdom; 2 dept of obstetrics and gynaecology, university of cambridge, united kingdom. introduction: preeclampsia is marked by an enhanced endothelial inflammatory response manifested by maternal endothelial activation. soluble fms-like tyrosine kinase-1 (sflt-1, svegf-r1), a naturally occurring circulating antagonist of vegf-a and plgf, is one of the secreted factors implicated in the pathogenesis of preeclampsia. in women who develop preeclampsia, sflt rises sharply, preceding the onset of the clinical disease. the aim of this study was to examine the effect of a combined treatment with recombinant sflt-1 and tnf-on the activation of human umbilical cord endothelial cells (huvec) by examining leukocyte adhesion, and the expression of icam, vcam, endothelin and vwf. methods: huvec were seeded, grown overnight and pre-treated with recombinant sflt (50-250 ng/ml) in a basic 2% fbs-dmem medium for 24 hr. on day 3, low doses of tnf-(0.5 ng/ml) were applied to pre-treated cells for 6 hr. antagonism of the vegf-a action was mimicked at the protein level by pre-incubating huvec with anti-flt antibody (5-10 g/ml), anti-kdr antibody (1 g/ml), anti-vegf antibody (5 g/ml), or vegf receptor inhibitor su5614 (5 m). at the rna level, the effect of sflt was mimicked by sirna transfection of huvec with siflt or sikdr. at the end of each experiment cells were either harvested for western blotting, fixed in 2% pfa for immunofluorescence or incubated with labelled hl60 leukocyte cells, followed by fluorescent detection of adhesion. results: pre-incubation of huvec with sflt and subsequent treatment with low doses of tnf-increased the adhesion of hl60 leukocytes and increased icam-1, vcam-1, endothelin and vwf, compared to tnf-treatment alone. similar results were obtained when cells were pre-treated with su5614, anti-flt, anti-kdr or anti-vegf. transfection knock-down of flt or kdr gene also significantly increased leukocyte adhesion when small doses of tnfwere added. conclusions: pre-incubation with recombinant sflt, anti-flt, anti-kdr, anti-vegf, su5614 or knocking-down flt or kdr transcripts all antagonised the autocrine actions of vegf and/or plgf. this predisposes huvecs to be more sensitive to the effect of tnf-. our study shows that sflt and tnfcombine to induce an enhanced synergistic effect, activating endothelial cells. maternal obesity is associated with increased production of inflammatory cytokines and risk of poor perinatal outcome. inflammatory cytokines can stimulate production of reactive oxygen (ros) and nitrogen (rns) species, which can covalently modify protein function. placental oxidative and nitrative stress are increased in pathological pregnancies and associated with altered placental function. objectives: determine the effect of increasing body mass index (bmi) on placental nitrative stress, measured by the expression and localization of nitrated (nitrotyrosine) and oxidized proteins. methods: placental tissue was collected at term (37-41 wks) from lean kg/m 2 ), overweight (25-29.9 kg/m 2 ) and obese (30-40 kg/m 2 ) patients (n=5 or 6/group). tissue was sectioned for immunostaining with nitrotyrosine antibody. protein samples were either dot blotted onto nitrocellulose membrane, probed with nitrotyrosine ab, and nitrated proteins detected using ecl, or derivatized using 2,4-dinitrophenylhydrazine (dnph) (oxyblot® kit), separated on sds-page, probed with anti-dnph ab (1:150) and oxidized proteins detected using ecl. protein band intensity was measured by densitometry. oxidized proteins were selected for maldi mass spectrometry analysis. results: nitrotyrosine residues were immunolocalized primarily in the fetal capillary endothelial cells and the villous stroma, but were almost absent in the syncytiotrophoblast. by dot blot, nitrotyrosine expression differed across the three groups (p<0.003, anova) with expression in tissue from obese women being significantly increased compared to lean (p<0.005) and overweight (p<0.05, tukey test). several oxidized proteins were detected with significantly greater expression seen in the lean versus overweight groups (p<0.02, mann-whitney u). one oxidized protein was identified by maldi-ms with four peptide matches and 19.2% coverage as 3-beta hydroxysteroid dehydrogenase (3bhsd). discussion: with increasing bmi, an increase in nitrative stress appears to occur in parallel with a decrease in oxidative stress. oxidative stress is apparently reduced as ros are consumed by the interaction with rns to give nitrative stress. 3bhsd is involved in the biosynthesis of steroid hormones and glucocorticoids. its activity and hence steroid metabolism in the placenta may be regulated by oxidation with implications for fetal development. background teenagers are susceptible to delivering small-for-gestationalage (sga) infants. previous studies implicate continued maternal growth as a causal factor 1 . growing adolescent sheep have reduced fetal birthweight due to impaired placental development and nutrient transfer 2 . we hypothesized that placental function is impaired in human teenage pregnancy if there is maternal growth. methods placentas were collected from 29 teenagers (15-18 years) and 21 adults. activity of the amino acid transporter, system a, was quantified by the sodium-dependent uptake of 14 c-methylaminoisobutyric acid into placental fragments. teenagers were defined as growing (>2mm increase in kneeheight per 90 days) or non-growing. system a activity was analysed in relation to individualised birthweight centile and maternal growth. results placental system a activity was significantly lower in teenage compared to adult pregnancies (p<0.05). this was unrelated to birthweight; teenagers who delivered infants appropriate-for-gestational age (aga) had significantly lower placental system a activity compared to aga infants delivered to adults (p<0.01). growing teenagers did not deliver lower birthweight infants than non-growing teenagers (median birthweight 3550g and 3290g respectively). furthermore, system a activity in placentas from growing teenagers was significantly elevated compared to that in non-growing teenagers (p<0.01), and was similar to placental activity in adults. conclusions system a activity was reduced in placentas from teenagers compared to adults. this suggests that inherently lower placental function predisposes teenagers to sga, but that other factors (e.g. adequate nutrition) can compensate in those teenagers delivering aga infants. in contrast to our hypothesis, placental system a was elevated in growing teenagers and mimicked that of adults. this may be related to a hormonal-milieu in growing teenagers that is conducive to fetal growth, in part through stimulating placental transport. homocysteine inhibition of system a amino acid transport activity in human placenta. eleni tsitsiou, susan l greenwood, colin p sibley, stephen w d'souza, jocelyn d glazier. maternal and fetal health research group, st. mary's hospital, university of manchester, manchester, united kingdom. background: elevated plasma levels of the amino acid homocysteine (hcy) during pregnancy are associated with vascular-related complications and adverse neonatal outcomes including a reduced birthweight. fetal and maternal plasma hcy concentrations are positively correlated suggesting placental transport of hcy may be an important determinant of fetal plasma hcy. the mechanisms involved in placental hcy transport are uncharacterised. evidence that the system a amino acid transporter, which transports neutral amino acids in a na + -dependent manner, is important in promoting fetal growth and that a reduced system a activity is associated with intrauterine growth restriction (iugr), led to our hypothesis that system a provides one mechanism for placental hcy transport. this hypothesis was tested by measuring the ability of hcy to inhibit system a activity in isolated microvillous plasma membrane (mvm) vesicles and placental fragments. materials and methods: mvm vesicles and placental fragments were isolated from placentas of normal pregnancies at term. system a activity was measured at initial rate (30s and 20 min respectively) as na + -dependent 14 cmethylaminoisobutyric acid (meaib) uptake into mvm vesicles (0.165mm) or fragments (0.0085mm) in the absence (control) or presence of l-hcy and dl-hcy or model substrates. results: 20mm l-hcy (custom-synthesised) and dl-hcy (commercial source) significantly (p<0.05) inhibited na + -dependent 14 c-meaib uptake into mvm vesicles compared to control; comparable in magnitude to other model substrates (meaib, l-ala, l-ser, l-met; n=9, kruskal-wallis with dunn's multiple comparison test). l-hcy, l-met and meaib (0.05-20mm) caused a dose-dependent inhibition of na + -dependent 14 c-meaib uptake into mvm with ec50 values (in mm; mean ± se) of 0.53 ± 0.04, 0.27 ± 0.02, and 1.13 ± 0.12 respectively, n=6). na + -dependent 14 c-meaib uptake into fragments was reduced substantially in the presence of 10mm l-hcy or dl-hcy causing a 85 ± 13 and 88 ± 12% reduction (mean ± sd, n=2-3) of control respectively. conclusion: these observations suggest that hcy is a relatively high affinity substrate for system a in the human placenta. we speculate that inhibition of placental amino acid uptake by hcy could impact on fetal growth and development. supported by the mrc and action research endowment fund. 110 glucose regulates placental mtor activity and glucose deprivation down-regulates placental system l activity in an mtor-dependent manner. sara roos, 1 theresa l powell, 2 thomas jansson. 2 1 department of physiology, institute of neuroscience and physiology, gothenburg, sweden; 2 department of obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. placental amino acid transporters are down-regulated in intrauterine growth restriction (iugr). we have previously shown that mammalian target of rapamycin (mtor) regulates placental system l transporter activity and that placental mtor activity is decreased in iugr. however, the upstream regulators of placental mtor are unknown. in iugr, fetal hypoglycemia and reduced maternal glucose levels are common and the placenta may therefore be exposed to low glucose levels. hypothesis: we hypothesized that glucose availability regulates placental amino acid transporter activity mediated by changes in mtor signaling. methods: cytotrophoblast cells were isolated and cultured until syncytialization at 66 hours. cells were cultured for an additional 24 hours in culture media containing 0.5 mm, 4.5 mm, or 16 mm glucose (control), which corresponds to standard culture media. at 90 hrs, the activity of the mtor signaling pathway was assessed by measuring the protein expression of s6k1 phosphorylated at thr-389, the primary site of mtor phosphorylation. in another set of cells, system l activity was assessed by measuring the bchinhibitable uptake of 3h-leucine. results: as compared to control, phospho-thr-389-s6k1 expression was reduced by 24% in cells incubated in 0.5 mm results: fgr pregnancies were delivered earlier (39±0.2 v 32±0.6w; p<0.01), weighed less (3.5±0.2 v 1.2±0.1kg; p<0.01) and had higher s/d ratios (2.2±0.1 v 5.3±1.1; p<0.01). eaa concentrations were similar between groups, but there were differences (non-parametric testing; p<0.05) in transport rates between groups with his crossing considerably faster and ile crossing slower in fgr . the table shows fetal vein/maternal (fv/m) ratios standardized to the leu fv/m ratio for all 9 eaa for both groups. amino acids fell into 3 groups for fgr pregnancies: 1) his (ratio 1.38), 2) leu, phe, met (ratios 1), and 3) val, thr, ile, trp, lys with intermediate ratios ( 0.6 ns conclusion: this is the 1 st study to compare the relative rates of in vivo placental transport for all eaa between normal and fgr human pregnancies showing striking differences in the transport rates of two eaa. in the absence of eaa concentration differences between groups, the higher his transport rate in fgr suggests higher utilization by the placenta. vasculature of women who received exogenous estrogen compared to those who received placebo. the purpose of this investigation was to identify the gene expression in response to the estrogen, equilin, as the major component of conjugated equine estrogen and genistein, a phytoestrogen in human coronary artery endothelial cells. human coronary artery endothelial cells from a 43-year old female were used. cells were treated with estradiol [e2], equilin [eq] (0.1 nm) or genistein [gen] (5 micromolar) for 48 hours. focused oligomicroarrays for cardiovascular disease and endothelial cell function were used to study gene expression. rt-pcr was used to confirm the transcription of genes analyzed in oligoarrays. vascular adhesion molecules and genes involved in the inflammatory response were most affected with the three estrogen sources. significant reduction of integrin alphae was seen with all three 2.63, 2.06 and 16.74 for e2, eq and gen respectively. similarly nfkb1 was also reduced 2.62, 2.07 and 6.33 fold compared to controls. significant reduction of ccl2 was demonstrated with eq (3.11 fold) and gen (5.49 fold). tnfreceptor1a and 1b were only significantly decreased by gen. vcam-1 which is regulated by proatherogenic factors and upregulated mainly at atherosclerosis -prone sites, was significantly reduced (25.95-fold) with genistein, and a slight reduction was seen with e2 and no change was observed with eq. our data support the clinical findings that estrogen has favorable effects on the genes involved in atherosclerotic disease. while e2 has been shown to be slightly more effective than equilin in the modulation of gene expression, genistein was significantly more effective in the favorable expression of genes involved in particularly the inflammatory response. olga n lekontseva, sandra t davidge. physiology and obstetrics/gynecology, university of alberta, edmonton, ab, canada. introduction: the prevalence of cardiovascular disease in women dramatically rises in the postmenopausal period. although deficiency of estrogen has been implicated in the pathophysiology of systemic vascular dysfunction, the effects of estrogen on vasculature are complex and not completely understood. we have previously shown that estrogen exerts a beneficial effect on the aging vascular system by reducing circulating levels of the inflammatory cytokine tnf . tnf is a known regulator of matrix metalloproteinases (mmps), proteolytic enzymes that may modulate vascular tone through cleavage of vasoactive peptides such as big endothelin-1 (et-1). the role of estrogen in this pathway is unknown. we tested the hypothesis that in aging/estrogen deficiency, tnf -induced mmp activity mediates greater vasoconstriction, in part, through the et-1 pathway. we further hypothesized that estrogen replacement reduces vascular sensitivity to the constriction by preventing mmp activation. methods: aged (12 month old) female sprague dawley rats were ovariectomized and treated with either placebo [ovx] , 17 -estradiol [ovx+e 2 ], or the tnf inhibitor etanercept [ovx+etan] . after four weeks, resistance mesenteric arteries were isolated and studied on the pressure arteriograph. concentrationresponse to exogenous big in the absence or presence of mmp inhibitor (gm6001, 10μm) was assessed in the vessels. results: treatment of "menopausal" [ovx] rats with either estrogen or the tnf inhibitor reduced sensitivity of arteries to big et-1 (ec 50 =0.07±0.01μm in ovx versus 0.15±0.04μm in ovx+e 2 or 0.16±0.03μm in ovx+etan groups; p<0.02). although mmp inhibition attenuated maximal constriction in all of the arteries, there was a significantly greater (p<0.05) role of mmps in big et-1-induced vasoconstriction in the ovx+e 2 group (reduction in max constriction=70.3±11.8%) compared to ovx (40.7±9.1%) and ovx+etan groups (35.4±3.9%). conclusions: both estrogen and tnf inhibition reduced big et-1 vasoconstriction. however, contrary to our hypothesis, tnf is not contributing to mmp modulation of et-1 vasoconstriction. interestingly, our study demonstrates a novel role for estrogen to increase mmp contribution to big et-1 vasoactivity with the net effect being less vasoconstrictive. understanding this unique pathway of regulation by estrogen in the aged vasculature will allow for development of new therapeutic options for women. mo, usa. introduction: the etiology of pelvic organ prolapse is multifactorial, with both inherited and acquired components. the molecular mechanisms of prolapse have not been established yet. we have previously shown that lysyl oxidase (lox) expression is suppressed in uterosacral ligaments of women with pelvic organ prolapse. it has also been shown that lox is a tumor suppressor gene inactivated by methylation in human gastric cancers. hypothesis: the aim of this study was to analyze the dna sequence of the promoter region of the lysyl oxidase gene in tissues from women with pelvic organ prolapse and identify whether methylation is present. our hypothesis is that the promoters of the lox gene in women with pelvic organ prolapse have significantly more methylation sites than women without prolapse. materials and methods: genomic dna was isolated from the uterosacral ligaments of eight women with pelvic organ prolapse and 8 women without prolapse (controls). genomic dna samples were treated with ez dna methylation kit (zemo research, orange, ca). the lox gene promoter region of -246 to +74 was amplified by pcr and then cloned into pcr2.1-topo (invitrogen, carlsbad, ca) and transformed into an e. coli dh5a strain. amplified plasmid dna samples containing the lox gene promoter region from each woman were sequenced and methylated cpg islands were identified by sequence comparison. results: a total of 66 methylated cpg sites were found in the patient group with pelvic organ prolapse while only 1 methylated cpg site was found in the non-prolapse control group. conclusion: these findings suggest that methylation in the promoter region suppresses lox gene expression in women with pelvic organ prolapse. background: reports from case-control and cohort studies have suggested an inverse association between lactation and breast cancer risk, but findings have been inconsistent. methods: we conducted a prospective observational cohort study of 57,585 parous women participating in the nurses' health study ii from 1997 to 2005. our primary outcome was incident premenopausal breast cancer. results: during the study period, 621 cases of premenopausal breast cancer were diagnosed during 355,871 person-years of follow-up. women who had ever breastfed had a 24% lower incidence of premenopausal breast cancer (95% confidence interval [ci] 5-40%) compared with women who never breastfed, adjusting for parity, age at first birth, year of first birth, height, body mass index (bmi), bmi at age 18, family history, personal history of benign breast disease, participant birth weight, preterm birth, age at menarche, oral contraceptive use, physical activity, and alcohol consumption. no trend was observed with duration of lactation (p=0.52). the association between ever-breastfeeding and premenopausal breast cancer was modified by use of medication to suppress lactation (p=0.05); in analyses restricted to women who had never used suppressive medication, ever-breastfeeding was associated with a 46% (95%ci 23-63%) reduction in incident disease. the association between lactation and premenopausal breast cancer was further modified by family history of breast cancer (p=0.03). among women who had never used suppressive medication and reported a family history, those who had breastfed had a 67% lower covariate-adjusted risk of premenopausal breast cancer (95% ci 38-82%) than women who had never breastfed. among women without a family history, ever-breastfeeding was not associated with breast cancer incidence (hazard ratio 0.68, 95% ci 0.42-1.09). among women who had ever breastfed, we observed no association between breast cancer risk and duration of lactation amenorrhea or exclusive breastfeeding. conclusion: in a large, prospective cohort study, ever-breastfeeding was inversely associated with risk for premenopausal breast cancer. at the durations observed in our cohort, we observed no trend with duration of breastfeeding or lactation amenorrhea. the inverse association with ever-breastfeeding was stronger among women with a family history of breast cancer. regulatory and nkt cells at the maternal-fetal interface. thomas f mcelrath, 1 rachael a clark. 2 1 brigham women's hospital, boston, ma, usa; 2 harvard skin disease research center, brigham women's hospital, boston, ma, usa. introduction: pregnancy represents an immunologically challenging event requiring maternal tolerance of the fetal semi-allograft. an increase in decidual cd4 + cd25 + t cells has been documented but it is unclear if these represent foxp3 + t cells (tregs) . the possibility also exists that other cd3 + lineages with potential regulatory function exist within the decidua parientalis. we examined if cd4 + cd25 + cells are true foxp3 + tregs and evaluated the frequency of other t cell subsets with possible regulatory potential. methods: we extracted t cells from term deciduas of planned cesarean deliveries. cells were stained with directly conjugated monoclonal antibodies and were analyzed on a six color flow cytometer. results: we found that only a subset (median 33%) of cd25 + t cells were true foxp3 + regulatory t cells. from 65 donors, foxp3 + tregs accounted for a median of 7.1% of all cd4 + cells. these cells were memory cd45ro + t cells lacking ccr7, and l-selectin but expressing cd25, ctla-4, gitr, and hla-dr. additionally we found that a median 21% of cd3 + t cells expressed the nkt marker cd161. these cells were a mixture of cd4 and cd4 -cd8 -t cells, with variable numbers of cd8 t cells, suggesting they do not represent merely recently activated t cells. there was enrichment for t cells with nkt markers after culture on hela cells expressing cd1d, suggesting that these cell represent true nkt cells. nkt cells were of the non-classical type, with a diverse t cell repertoire (0.4% iv24jq) and also expressed cd69, hla class ii, cd45ro but were ccr7 and l-selectin low. comments: we find that only a minority of cd25-expressing t cell in the decidua are true foxp3 + tregs. because much of the work on treg in pregnancy has used cd25 as a treg marker, this suggests additional studies are needed to confirm the role of these cells in pregnancy. we find that a novel population of non-classical nkt cells exists in the human decidua. nkt cells can either promote or antagonize tolerance, depending on the immunologic context. the large number of these cells in the decidua suggests they may play a role equal or exceeding that of regulatory t cells. prolapse. marsha k guess, 1 kathleen a connell, 1 richard bercik, 1 lloyd g cantley. 2 1 obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 internal medicine/neprhology, yale university school of medicine, new haven, ct, usa. objectives: thy-1 is a cell surface glycoprotien expressed in human fibroblasts, neurons, hematopoetic stems cells and endothelial cells. thy-1 expression affects fibroblast proliferation and migration, cell-cell, as well as, cell-matrix interactions. moreover, thy-1 expression has been shown to play a critical role in fibroblast dedifferentiation into myofibroblasts, as well as in extracellular matrix (ecm) production and fibrosis. women with pelvic organ prolapse (pop) have alterations in vaginal ecm protein expression and metabolism, as well as decreases in smooth muscle fractional content. in the current study, we evaluated thy-1 as well as the smooth muscle markers alpha-smooth muscle actin (asma) and desmin expression in women with pelvic organ prolapse compared to women with normal pelvic support (controls). methods: anterior apical vaginal wall specimens from women with pop and controls were collected at the time of hysterectomy. messenger rna and protein expression of thy-1, asma and desmin were evaluated using semiquantitative rt-pcr, real-time pcr and western blot analysis. gapdh and beta actin were used as internal controls. results: rt-pcr demonstrated the presence of thy-1, asma and desmin in vaginal tissue from women with pop and controls. further, thy-1 mrna expression was downregulated 57% in women with pop compared to controls (p = 0.004). a parallel decrease in thy-1 protein was seen in women with pop compared to controls (p=0.02). although a 44% and a 37% decease were seen in asma and desmin mrna, these differences were not statistically significant. similarly, no differences were seen in asma and desmin protein expression. conclusion: we demonstrate that there is significantly less thy-1 expression in vaginal tissue from women with pop compared to controls. differential expression of thy-1 in prolapsed vaginal tissues suggests that thy-1 may have a functional role in mediating ecm metabolism in the female genitourinary tract. hur expression is altered in ectopic endometrium. s karipcin, t altun, ua kayisli, e seli. ob gyn, yale u., new haven, ct, usa. introduction: cytokines and growth factors contribute to cyclic turnover of the normal endomterium and to the pathogenesis of endometriosis. cytokine and growth factor messenger rnas (mrnas) undergo rapid turnover that is primarily mediated by au-rich elements (are) that consist of multiple stretches of adenylate and uridylate residues located in the 3' untranslated region (3'-utr) of their mrnas. hur is a ubiquitously expressed rna-binding protein that stabilizes are containing mrnas and prolongs their expression. we hypothesized that hur may play a role in the regulation of cytokine expression during normal menstrual cycle and in endometriosis. methods: tissue sections obtained from normal (n=14) and ectopic (n=7) endometrium were immunostained for hur. staining intensity was evaluated by hscore and grouped according to menstrual cycle phase. statistical analysis was done with one-way anova. cultured stromal cells isolated from normal endometrium were treated with vehicle, estradiol (e2; 10 -8 m), or progesterone (p, 10 -8 m) for 24 and 48h, and hur expression was determined using western analysis and normalized to ß-actin. results: hur immunostaining was nuclear in endometrial cells. hur immunoreactivity was significantly lower in the early proliferative and late secretory phases (157.5 ±11.08 and 190.0 ±15.2, respectively), compared to the mid-late proliferative (270.0±8.0) and early-mid secretory phases (256.6±20.2 )(p<0.01). moreover, hur expression was significantly lower in ectopic endometrial cells when compared to normal endometrium in midlate proliferative and early and mid-secretory phases (p<0.01). progesterone suppressed hur levels significantly in cultured endometrial stromal cells at both 24 and 48 h compared to control (p<0.05) while estrogen did not cause a significant change. discussion: decreased hur levels in the late secretory and early proliferative phases are likely to contribute to degradation of cytokines and result in lower cytokine levels observed mid-cycle. late secretory decrease in hur levels may be mediated by progesterone as suggested by in vitro findings. in ectopic endometrium, persistent low expression of hur compared to normal endometrium most probably results from elevated cytokine levels associated with endometriosis. the effect of lower hur expression in ectopic endometrium on other are-containing transcripts, and on the pathogenesis of endometriosis remains to be elucidated. tissue. hong zhao, joy innes, scott reierstad, mehmet bertan yilmaz, serdar e bulun. department of obstetrics and gynecology, northwestern university, chicago, il, usa. background: aromatase is the key enzyme for estrogen biosythesis, and is encoded by the cyp19a1 gene. thus far, only three unique untranslated first exons associated with distinct promoters in the mouse cyp19a1 gene were described (brain-, ovary and testis-specific exon i). however, it remains unknown whether aromatase is expressed in other mouse tissues via previously unknown tissue-specific promoters activating new exon is. methods: real-time pcr was used to examine the aromatase expression levels in various c57bl6 mouse tissues. 5'-rapid amplification of cdna ends (5'-race) was used to determine the transcriptional start sites of cyp19a1 transcripts. promoter activity was measured using serial deletion mutants of dna fused to the luciferase reporter gene. results: real-time pcr results showed that aromatase was expressed in male gonadal fat and the expression level is lower than that in testis. the adipose tissue-specific untranslated exon i of cyp19a1 transcript was isolated using 5'-race and this novel gonadal fat-specific exon i of cyp19a1 mrna did not show sequence similarities to previously reported ones. this new adiposespecific exon i was mapped to 75 kb upstream of the translation start site in the coding exon ii. the genomic region upstream of the adipose-specific exon i was cloned into luciferase plasmids. transfection of murine 3t3-l1 cells with these plasmids showed that promoter activity was conferred by the sequence located at -1 to -343 bp upstream of the transcriptional start site. dexamethasone significantly induced activity of the adipose-specific promoter region. conclusion: taken together, our results suggest that a novel cyp19a1 transcript is regulated by a tissue-specific promoter in male murine gonadal fat. these sacrificed; reproductive and selected other organs were removed, weighed and evaluated histologically by an observer blinded to treatment. results: the table below shows that tcc augmented effects of tp on weights of accessory sex organs. histological assessment revealed that tcc induced greater glandular distention with more secretions compared to the effects of tp alone; furthermore, mitotic figures were seen only in prostates from rats exposed to tp+tcc. conclusions: tcc is a newly identified endocrine disruptor with unique and novel actions resulting in potentiation of androgen effects on sex organs. these observations underscore possible environmental risks related to exposure to tcc. background: blastocyst implantation is dependent on the differentiation of human endometrial stromal cells (hesc) into decidual cells. transforming growth factor family members have well defined roles in cell differentiation and proliferation. activin a, a tgf superfamily member, enhances hesc decidualization and localizes to decidualized cells in human endometrium. other tgf superfamily members, including bmp2, bmp4, bmp7, gdf5, gdf8 (myostatin), gdf11 and nodal, may also be present in decidual cells and therefore may also play a role during this important process. this study aimed to determine whether activin is the major family member driving decidualization or whether other family members contribute to the process. methods: broad ranging activin inhibitors (activin-m108a and sb431542) that effect receptor-ligand interactions of other tgf superfamily members were used in hesc decidualization. protein localization was examined in secretory phase endometrium and first trimester decidua by immunohistochemistry and mrna expression was examined in an ex vivo model. the secretion of candidate proteins was measured during hesc decidualization and certain recombinant proteins added during decidualization to examine their effect. results: m108a (25nm) and sb431542 (10 m) significantly reduced decidualization (60% and 77% respectively) demonstrating that activin and possibly other tgf family members are involved in decidualization in vitro. bmp2, gdf5 and tgf 1 protein were detected in decidual cells of mid-late secretory endometrium and first trimester decidua whilst all ligands except nodal, were expressed by hesc. both bmp2 and tgf 1 secretion increased during hesc decidualization and administration of both these proteins significantly enhanced decidualization in vitro. conclusions: these data support a role for activin a, bmp2 and tgf 1 in hesc decidualization. this is important as the elucidation of factors involved during decidualization will aid in better understanding implantation and fertility. abnormal chromatin remodeling in diabetic murine oocytes. laura lawrence, ann ratchford, cybill esguerra, qiang wang, kelle moley. ob/ gyn, washington university, st. louis, mo, usa. background: diabetic women experience increased miscarriages and adverse pregnancy outcomes. previous studies suggest adverse diabetic outcomes may occur earlier than the preimplantation period, particularly during oogenesis. we hypothesize that diabetes affects chromatin remodeling and chromosomal condensation in murine oocytes. methods: mii oocytes from diabetic and control mice were fixed with 4% pfa, permeabilized with 0.5% triton x-100 for 15 min, and immunostained against a-tubulin and the nucleus for 1 hr at rt. images were obtained with laser confocal scanning microscope. protein expression levels of chromatin with dimethyl h3k9 modifications were measured in nondiabetic and diabetic denuded murine oocytes at 44 hours post pmsg (0 hour) and at six hours post hcg (6 hour) via western immunoblots. mature nondiabetic denuded oocytes were fixed in 4%pfa and permeabilized with 0.5% triton x-100. they were stained via immunohistochemistry against histone protein h3 at lysine 9 (h3k9me2) and heterochromatin. results: immunohistochemistry reveals that diabetic mii oocytes have aberrant spindle formations and metaphase chromosome alignment. approximately 10/50 (20%) diabetic oocytes examined had abnormal spindles and metaphase alignments compared with only about 1/70 normal oocytes (1.4%). western blot demonstrated 3 times higher expression of dimethylated chromatin in diabetic oocytes at time 0 compared with nondiabetic oocytes. at time 6 hours, diabetic oocytes had significantly fewer h3k9 modifications than the controls. when staining mature murine nondiabetic oocytes for dimethylation of h3k9me2 by immunohistochemistry, we demonstrated h3k9me2 expression in a condensed heterochromatin ring surrounding the nucleolus, consistent with transcriptional silencing. conclusions: diabetic mii oocytes have a significant increase in abnormal spindle formation and metaphase chromosome alignment. they also have increased dimethylation compared with normal oocytes at a time point when they should be transcriptionally active, storing maternal mrna in preparation for the silencing period. in addition, after hcg injection to trigger maturation and gene silencing, the diabetic oocytes had decreased dimethylated chromatin changes. our findings suggest that diabetic oocytes may be exiting the transcriptionally active period prematurely and may ultimately experience decreased, partial, and incomplete gene silencing. and xenopus epab genes and proteins were performed. expression of human epab and pabpc1 mrna was tested in ten different somatic tissues, testes, and ovaries by rt-pcr. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. epab and pabpc1 expression in human prophase i (pi) and metaphase ii (mii) oocytes, 8-cell embryos and blastocysts was evaluated using quantitative real time pcr. results: human epab is a 625 aa protein with 77% identity and 84% similarity to mouse epab and contains 4 rna recognition motifs and a pabp domain. human epab mrna is detected in ovaries and to a lesser extent in testes and several somatic tissues including kidney, liver, and muscle. similar to its mouse orthologue, human epab mrna is expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. pabpc1 mrna is ubiquitously present in all tissues as well as 8-cell, and blastocyst stage embryos. however, its levels are significantly lower than that of epab in oocytes. conclusions: in this study we report the identification of human epab. similar to that observed in xenopus and mouse, human epab is the predominant poly(a) binding protein in oocytes and it is replaced by pabpc1 following zga, which occurs at 4-to 8-cell stage in human. our findings suggest that the unique translational regulatory pathways that control gene expression during oogenesis and early embryo development may be common between model organisms and humans. objective: c-jun nh2-terminal kinase (jnk), a member of mitogen-activated protein (map) kinase family, is involved in cell proliferation, differentiation, and survival. fsh is required for granulosa cell proliferation and antral follicle growth but its mechanism of action in preantral stages is not well defined. we previously showed that pharmacological inhibition of jnk pathway halts invitro growth of murine preantral follicles in serum free media supplemented with fsh (100miu/ml). sigcs (spontaneously immortalized rat granulosa cell line) have characteristics similar to preantral granulosa cells and hence they were used in this study to determine whether the jnk pathway plays a key role in preantral granulosa cell proliferation. our specific aims were to determine whether: a) fsh activates jnk pathway in granulosa cells; b) the inhibition of jnk pathway blocks cell cycle progression. material and methods: sigcs were treated with 100miu/ml recombinant fsh in serum free media two days after serum starvation. activation of jnk pathway was analyzed with if and wb using phospho-jnk and phospho-cjun expression, respectively. fsh receptor expression (fshr) was analyzed with if. the inhibition of jnk pathway on cell cycle progression was analyzed by facs using a jnk inhibitor sp600125. results: fshr protein was expressed in sigc indicating that they can respond to fsh (fig 1a) . likewise by if, phospho-jnk expression was significantly increased in sigc 1 hour post fsh exposure (fig-1a) . similarly on wb, phospho-cjun expression increased as early as 30 min after fsh exposure and peaked at 4 hrs. cjun phosphorylation was abolished 1 hr after treatment with sp600125 (50mm) (fig 1b) . facs analysis showed that the inhibition of jnk by sp600125 resulted in cell cycle arrest at g2/m transition in a dose dependent fashion (fig 1c) . conclusion: these results strongly suggest that the proliferative effect of fsh on immature granulosa cells is mediated through the activation of jnk pathway. this is the first experimental observation implicating jnk signaling in granulosa cell cycle control. (nichd 043339-01). the induction of 17{alpha}-hydroxylase (cyp17) expression in granulosa cells. satin s patel, 1 victor e beshay, 1 william e rainey, 2 bruce r carr. 1 1 reproductive endocrinology and infertility, university of texas at southwestern medical center at dallas, dallas, tx, usa; 2 physiology, medical college of georgia, augusta, ga, usa. according to the traditional two-cell two-gonadotropin hypothesis of the ovary, androgen production arises exclusively from theca cells. the granulosa cells, in turn, utilize androstenedione, which is aromatized eventually to estradiol. studies involving immunohistochemical analysis of normal ovaries have shown that granulosa cells express significantly higher levels of the activator protein-1 (ap-1) transcription factor, cfos compared to theca cells, where cfos expression is virtually absent. we hypothesize that cfos functions to inhibit the expression of cyp17 in granulosa cells, thereby suppressing androgen production. hence, the inhibition of cfos activity might result in cyp17 expression in the granulosa cell. our objective was to define the role of cfos, in the regulation of cyp17 expression in granulosa cells. transformed human luteinized granulosa (hgl5) cells were utilized for all experiments. hgl5 cells were cultured in monolayer for 72 h. cells were treated for 48 h with and without pd98059 (pd), a mapkk inhibitor, which also blocks cfos expression. rna was isolated and real time rt-pcr was performed for cyp17. cfos rna interference experiments were carried out using rnaifect, cfos smartpool sirna and scrambled sirna for 48 h. rna was isolated and rt-pcr was also performed for cyp17. immunochistochemical studies were performed on normal ovaries, staining for cfos and cyp17. treatment of hgl5 cells with the mapkk inhibitor pd for 48 h, resulted in a 10-fold increase in cyp17 mrna expression compared to basal conditions. in cfos gene silenced cells, cyp17 mrna expression also increased by 10-fold compared to control sirna conditions. immunohistochemical staining for cfos and cyp17 showed significant staining of cfos in the granulosa cell layer, but absent staining for cyp17. conversely, the theca cell layer did not stain for cfos, but staining was evident for cyp17. these results suggest that the ap-1 transcription factor, cfos, may play a role in the inhibition of cyp17 expression in granulosa cells. this may provide an explanation for the lack of cyp17 expression in granulosa cells. the g2/m stages of the granulosa cell cycle. as clip-170 has been identified as an mtor substrate, we hypothesized that its function at the mitotic spindle would be positively regulated by mtor during the late g2 and m phases of the cell cycle in granulosa cells. during periods of stress (e.g., mtor inhibition), mtor would fail to phosphorylate clip-170, leading to spindle checkpoint failure and follicle undergrowth. objectives: the expression of clip-170 and mtor were evaluated. computational analysis of potential clip-170 phosphorylation sites and comparison with residues on known mtor targets were performed. clip-170 threonine 182 and serine 186 were chosen and evaluated as bona fide mtor phosphorylation sites. a preliminary assessment of the effects of mtor inhibition upon clip-170 function was performed. methods: for protein expression analyses, western blots, immunostaining of tissues and primary granulosa cells in culture were performed. computational analysis of potential clip-170 phosphorylation sites was followed by in vitro assessment of mtor kinase activity upon clip-170 and a peptide substrate. results: clip-170 was expressed in the ovarian stroma, blood vessels (including the endothelial cells of both arteries and veins), granulosa cells, and in the oocytes of primordial and growing follicles. overlapping expression was found between clip-170, mtor, and the mtor cofactors raptor and rictor in granulosa cells. this expression was conserved between the mouse and the human. evaluation of clip-170 phosphorylation supported thr 182 as a bona fide mtor target. conclusions: clip-170 was supported as an mtor substrate protein during granulosa cell mitosis. the mechanism of mtor action during granulosa cell growth and survival is likely to include the phosphorylation of clip-170 and subsequent positive regulation of mitotic spindle function. the effect of a selective oxytocin antagonist (barusiban) in threatened preterm labour: a randomised, double-blind, placebo-controlled trial. steven thornton, 1 thomas m goodwin, 2 gorm greisen, 3 morten hedegaard, 4 joan-carles arce. 5 1 warwick medical school, university of warwick, coventry, united kingdom; 2 maternal-fetal medicine, university of southern california, los angeles, usa; 3 dept of neonatology, rigshospitalet, copenhagen, denmark; 4 dept of obstetrics, rigshospitalet, copenhagen, denmark; 5 clinical research development, ferring pharmaceuticals, copenhagen, denmark. objective: a mixed oxytocin/vasopressin v 1a antagonist, atosiban, has been shown to reduce uterine contractions in placebo-controlled clinical trials and is useful in the management of preterm labour. the objective of this study was to determine the effect of a selective oxytocin antagonist, barusiban, in delaying delivery and reducing uterine contractions in women with threatened preterm labour at a late gestational age and relatively high risk of delivery. methods: this was a randomised, double-blind, placebo-controlled multicentre study in 6 countries. a total of 163 women between 34+0 and 35+6 weeks gestation, and with 6 uterine contractions of 30sec duration during 30min, cervical length 15mm, and cervical dilatation >1 and <4cm were randomised to receive a single intravenous bolus dose of either barusiban 0.3mg, 1mg, 3mg, 10mg or placebo. rescue tocolytics were prohibited. the primary end-point was percentage of women who did not deliver within 48h. uterine contractions were monitored by cardiotocography. obstetrical and neonatal outcomes were determined. results: there were no significant differences between the placebo and any barusiban group in percentage of women who did not deliver within 48h (72% in the placebo group and 65% to 88% in the barusiban groups). there was no dose-effect relationship nor an effect at 12 or 24h. none of the barusiban groups were associated with a significant reduction in number of uterine contractions compared to placebo at any time point up to 48h post-dosing. postpartum blood loss and time to established lactation were not significantly increased with barusiban. barusiban was well tolerated and was not associated with safety concerns for the women, fetus or neonates. conclusion: a single intravenous bolus of a selective oxytocin antagonist, barusiban (dose range 0.3-10mg), did not delay delivery or reduce uterine contractions compared to placebo in women with preterm labour at late gestational age and with short cervical length. the results contrast those of the mixed oxytocin/vasopressin v 1a antagonist, atosiban. prolonged delivery intervals in triplet gestations. tracy a manuck, heather l mertz, leah passmore, david c merrill. obstetrics and gynecology, wake forest university health sciences, winston-salem, nc, usa. objective: delayed interval delivery is one management strategy for previable preterm labor affecting multiple gestations. prior reports of asynchronous deliveries have examined twins and higher-order multiples as a group. this study was conducted to analyze the unique situation of asynchronous triplet deliveries. study design: cases of asynchronous triplet deliveries resulting in an ongoing twin gestation were ascertained through medline. data were abstracted and combined with two similar previously unpublished cases. patients were grouped by management with and without rescue cerclage. variables compared included use of tocolytics, antibiotic administration, gestational age at delivery of each fetus, interdelivery interval, delivery mode, birthweights, and short and long term outcomes. chi-square or t-test analyses were used where appropriate. results: fifty-one cases of asynchronous triplet deliveries met inclusion criteria and were analyzed. twenty-three patients (45.1%) underwent placement of a rescue cerclage following delivery of the first infant. these patients delivered the first fetus at a significantly earlier gestational age as compared to those patients without a cerclage (20.3 +/-3.4 weeks vs. 23.5 +/-3.5 weeks, p=0.0019). patients with a rescue cerclage had a significantly longer prolongation of the remaining twin gestation (59.4 +/-41.3 days vs. 25.9 +/-30.1 days, p=0.0016). no significant differences in use of tocolytics or antibiotics, gestational age at delivery of triplets "b" and "c," mode of delivery, short term outcome (alive at 24 hours), or long term outcome (alive at discharge) were noted, despite delivery of triplet "a" at a significantly younger gestational age. conclusion: rescue cerclage, particularly when placed following previable delivery of a first triplet, may significantly prolong the delivery interval for the remaining twin gestation. mean pregnancy prolongation (days). objective the aim of our study was to evaluate the role of amnioinfusion in pregnancies complicated by pprom. we studied 52 singleton pregnancies with pprom at <25 weeks gestation. all patients were managed conservatively with bed-rest, prophilactic antibiotics, tocolytics and steroids. only patients without vaginal bleeding and/or contractions were included: 11 patients showed an amniotic fluid pocket (afp) persistently 2 cm and did not undergo amnioinfusion (group b) whereas 41 had a maximum afp < 2 cm and were offered amnioinfusion to restore an adequate amount of amniotic fluid (group a). in 17 patients of group a amnioinfusion was successful (afp 2 cm for 48 hours following the procedure: group a1) whilst in 24 it was unsuccessful (afp<2 for 48 hours: group a2) and repeated. results were analized with the student t test for unpaired samples and with the 2 when appropriate. p values <0.05 were considered significant. results the group where amnioinfusion was not successful (group a2) showed the worst outcome (see table) . there were 6 intrauterine deaths, all in this group. pulmonary hypoplasia was present in 11/52 (22.5%) newborns (both survived and deceased) newborns, 10/11 in group a2. no maternal complications were recorded. conclusions our data confirm that a conservative-active management with amnioinfusion can be considered a reasonable option in women with pprom. in our series it was effective in preventing both neonatal death and pulmonary hypoplasia. group a1 (infusion successful) background. synthetic progestogens are effective in reducing the risk of spontaneous preterm birth in high risk singleton, but not multiple, pregnancy. we hypothesized that myometrial stretch may inhibit the response of human myometrium to progestogens. methods. myometrial strips obtained with written consent at the time of term planned cesarean section were studied using a modification of the method of young and zhang (jsgi 2004; 11:478-82) . strips were maintained in individual tubes in tissue culture media in an incubator for a period of three days. the effect of prolonged stretch was assessed by comparing strips connected to a 0.6g weight with those connected to a 2.4g weight. the effect of prolonged exposure to progestogen was studied by adding medroxyprogesterone acetate (mpa, 100nm or 1000nm). following the 3 day incubation, myometrial strips were transferred to an organ bath containing kreb's solution. all were placed under 2g tension and responses obtained to 50mm potassium then oxytocin. contractility was expressed as the ratio of the maximum response to potassium or oxytocin to the wet weight of the tissue (units = g.tension per gram), summarized as the mean (sem) and compared using student's paired t test. prolonged stretch increased the maximum response to both potassium (0.6g weight = 24.6 [5.3]; 2.4g weight = 43.1 [6.5], n=6, p=0.01) and oxytocin (0.6g weight = 30.4 [8.0]; 2.4g weight = 50.3 [7.6], n=6, p=0.01). in strips with a 0.6g weight, incubation in mpa for three days reduced the maximum response to potassium (vehicle = 30.1 [5.5]; mpa = 23.2 [5.2], n=5, p=0.02) and there was a trend towards a reduced maximum response to oxytocin (vehicle = 37.6 [8.2]; mpa = 31.5 [7.2], n=5, p=0.1). in strips with a 2.4g weight, incubation in mpa for three days had no effect on either the maximum response to potassium 1. prolonged stretch increases human myometrial contractility in vitro. 2. prolonged exposure to a progestogen inhibits the contractility of human myometrium but this effect is blocked by prolonged stretch. these properties of human myometrium may explain the failure of 17oh progesterone caproate to reduce the incidence of spontaneous preterm birth in multiple gestations. sangeeta jain, william l maner, janet l brandon, gary dv hankins, robert e garfield. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine if transabdominal uterine electromyography (emg) correlates with parturition factors such as measurement-to-delivery time (mtdt), cervical dilation (cd), cervical effacement (ce), and station (s) for preterm labor patients with and without tocolysis. materials and methods: 17 pregnant preterm labor women were included. uterine electromyography (emg) was measured for 30 minutes. cd, ce, and s were assessed at or near the time of uterine emg measurement. the power density spectrum peak frequency (pdspf) was calculated on emg. patients were grouped (g1: tocolysis, n= 4; g2: no tocolysis, n=13). pearson-product-moment test was used for correlation. significant differences were sought between groups using student-t test. p<0.05 significant. results: there was a significantly higher uterine emg activity (pdspf: 0.453 ± 0.051 vs. 0.379 ± 0.016), but no difference in cd (5.125 ± 1.727 vs. 3.714 ± 1.976), for patients delivering within 6 days of emg recording compared to those who delivered later, regardless of tocolysis. there was no apparent difference in uterine emg in tocolytic vs. non-tocolytic patients, regardless of mtdt (table 1) . conclusions: uterine emg activity is significantly greater in patients in preterm labor who delivered within 6 days of measurement, making it a viable alternative diagnostic parameter for assessing the state of parturition. tocolytics may not affect uterine emg, but this should be further verified with larger studies. supported by grant nih r01-hd037480. objective: calcium sensitizers are a novel class of drugs with unique molecular and phsiological actions. levosimendan, the best characterized of these compounds and is used in the treatment of acute and chronic heart failure. levosimendan can exert an inotropic effect via sensitization of myofilaments to calcium. it also exerts a relaxant effect on vascular smooth muscle through the opening of atp-dependent potassium channels and has been shown to be a potent inhibitor of human uterine contractions in vitro. for these reasons we investigated the effects of levosimendan on uterine contractions, both spontaneous and agonist induced, in the presence of glibenclamide, a k-atp channel blocker. method: biopsies of human myometrium were obtained at elective caesarean section (n=20). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to glibenclmide (100mmol) followed by cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. control experiments were performed simultaneously. results: levosimendan exerted an inhibitory effect on spontaneous and oxytocin induced contractions in human m yometrium in vitro, in comparison to control experiments. the effect of levosimendan was significantly antagonized by glibenclamide with the mean maximal inhibition seen due to levosimendan greatly reduced (n=6, p<0.05). conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on human uterine contractility in vitro. this action was antagonized by glibanclamide and this study demonstrates that the effect of levosimendan on uterine smooth muscle is mediated at least in part through the k-atp channel. introduction: the determination of the beginning and ending points for "bursts" of electrical activity occurring during uterine contractions is sometimes difficult. if bursts cannot be discerned, the preferred burst-by-burst analysis cannot be performed. one solution to is to analyze any given electrical recording in its entirety. but this approach has often lead to meaningless results when traditional analytic methods are applied. chaos analysis, using lyapunov exponents, may provide an answer. materials and methods: 46 term patients were included in the analysis: 27 were in labor (group 1), while19 were non-labor (group 2). 30 minute recordings were analyzed using "lyapunov exponent." for each pair of subsequent trajectories in phase space, only the most positive lyapunov exponent was calculated. the mean largest exponent was found by averaging over all of the trajectories in the recording. the lyapunov exponent is given in units of bits per data sample. thus a value of +1 means that the separation of nearby orbits doubles on the average in the time interval between data samples. the mean largest exponent was found for each patient recording. these values were compared using t-test (p < 0.05 considered significant). results: the mean and sd of the lyapunov exponent for all the patients was 0.0955 ± 0.0701. moreover, the lyapunov exponent calculated for each patient was positive. comparing lyapunov exponents of the two groups showed a statistically low value (low chaos) for the laboring group, compared to the non-laboring group (figure) . conclusions: there is a chaotic component associated with uterine emg traces, since small but non-negative lyapunov exponents were found in all the traces observed. the lyapunov exponent indicated significantly lower chaotic behavior in the whole emg traces of patients who were in labor than found in those who were not in labor, implying that this measure could be a good diagnostic parameter for labor, possibly eliminating the need for tedious burst-by-burst analysis. supported by grant nih r01-hd037480. comparing uterine emg to tocodynamometer for monitoring contractions. robert e garfield, lynette b mackay, sangeeta jain, william l maner. obstetrics and gynecology, the university of texas medical branch, galveston, tx, usa. objective: to determine whether uterine electromyography (emg) plots contractions similarly to tocodynamometer (toco). study design: 20 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. uterine emg signals were sampled at 100hz and band-pass filtered in the 0.34 to 1.00 hz range. root-mean-square (rms) function was calculated from the uterine emg signals in order to produce a "toco-like" trace from the original emg trace. emg-generated rms contraction plots were then compared to toco contraction plots using the following criteria: contractions were assigned a marker value of "1." in-between contraction periods were assigned a "0." from these marker values, contraction rates were established. correlation was found between the contraction rates of rms and toco. temporal overlap of contractions plotted by the two methods was used to find overall percent agreement (opa), positive percent agreement (ppa), and negative percent agreement (npa). these parameters were corrected for within-patient variation using a bootstrap method. results: uterine rms contraction plots were seen to correspond with toco contraction plots (fig. 1) . corrected opa, ppa, and npa were high at 90.68%, 84.52%, and 95.77%, respectively. there was a large, statistically significant correlation between uterine emg and toco contraction frequency (fig. 2) . conclusions: the similarity between toco and uterine emg contraction plots (specifically, using rms to convert) will allow emg to be used interchangeably with toco in the clinic. supported by grant nih r01-hd037480. introduction -this is a secondary analysis of women participating in a 14 center randomized placebo controlled trial (rct) evaluating the impact of 17-ohpc in preterm birth (ptb) prevention among women with twins. objective -to evaluate the relationship between plasma 17-ohpc concentrations and gestational age (ga) at delivery in women with twins receiving weekly injections of 17-ohpc. methods -women with twins were randomized between 16-21 weeks to receive weekly im injections of either 17-ohpc (250 mg) or placebo until 35 weeks. after a minimum of five consecutive injections had been administered to assure steady state concentrations a plasma sample was collected between 24-29 weeks. the sample drawn just prior to the next scheduled injection was analyzed for 17-ohpc by hplc-ms in a blinded manner. the lower limit of quantification of 17-ohpc was 1 ng/ml. we conducted univariate analyses to assess the association of 17-ohpc concentration and ga at delivery. we also conducted a proportional hazards model to evaluate the time from sample draw to spontaneous delivery (censoring indicated preterm deliveries), and a logistic regression to evaluate ptb<35 weeks; in both analyses we adjusted for bmi, race and ga at sample draw. results -96 women assigned to 17-ohpc were included; all received all of their scheduled injections. the concentration of 17-ohpc was significantly higher in women delivering < 35 weeks compared with those women delivering >35 weeks (p=0.002, table) . concentration of 17-ohpc was significantly correlated with ga at delivery (r = -0.38, p=0.0001). each unit increase of 17-ohpc was associated with a 30% increased odds of delivering < 35 weeks (odds ratio 1.3, 95% ci,1.14-1.50, p=0.001) and a 12% increase in hazard of spontaneous delivery (hazard ratio 1.12, 95% ci, 1.05-1.19, p=0.0004) after adjusting for confounders. gestational age at delivery mean (sd) -ng/ml < 35 weeks (n=34) 13.9 (5.9) >35 weeks (n=62) 9.4 (3.5) conclusion plasma 17-ohpc concentrations after weekly injections were inversely related to ga at delivery in women with twins. since 17-ohpc induces its own metabolism it is possible that higher concentrations during initial treatment are associated with lower plasma concentrations and reduced efficacy in later pregnancy . clearly more studies are needed. objective: in many non-human species, maternal circulating progesterone levels fall prior to delivery, leading to the theory that in humans progesterone withdrawal occurs on a local and/or functional level. our objective was to characterize maternal and fetal progesterone in human preterm and term labor. methods: women between 23.7 and 34.7 weeks' gestation (cases) or term controls (37-41 weeks) with either labor with intact membranes or premature rupture of the membranes prior to labor (prom) were enrolled in a prospective case-control study. progesterone was measured by immulite assay in maternal serum collected upon enrollment and again within 60 minutes after delivery and in umbilical cord serum obtained at delivery. maternal progesterone treatment was not used in any subjects. results: 20 cases and 20 controls were studied (see table for comparisons). controls p value ga at enrollment, weeks 29.4 ± 3.2 39.3 ± 1.1 <0.0001 interval to delivery, days (median, range) 2 (0 -25) 0 (0 -1.4) <0.01 maternal progesterone at enrollment, ng/ml 125 ± 87 185 ± 64 <0.02 maternal progesterone after delivery, ng/ml 55 ± 57 47 ± 27 0.6 cord progesterone, ng/ml 878 ± 602 762 ± 457 0.5 among cases, fetal but not maternal progesterone was significantly lower in preterm labor with intact membranes (475 ± 372 ng/ml, n = 8), as compared to prom (952 ± 417, n = 12), p<0.02. this difference increased further when cases of clinical chorioamnionitis were excluded. conclusions: serum progesterone in laboring patients prior to delivery is higher at term than in the preterm period, which may be attributable to increased placental mass in late pregnancy. this disparity disappears shortly after delivery of the fetus and placenta. fetal progesterone levels are several-fold higher than peripartum maternal levels. preterm labor with intact membranes is associated with diminished fetal progesterone, a phenomenon unrelated to clinical infection. these findings suggest the possibility of fetally regulated progesterone withdrawal as a mechanism underlying preterm labor with intact membranes. [snps] and ptb. however, many of these studies are inconclusive and non reproducible. the challenge of identifying robust associations between genetic variation and either susceptibility or protection from ptb is enormous. a systematic review of literature followed by metaanalysis was performed to understand true associations between snps and ptb. methods: for systematic review, articles were chosen based on medline and embase searches (1990 ( -april 2007 and relevant articles were chosen based on stringent inclusion criteria. primarily, studies reporting genetic associations between snps in maternal dna in singleton pregnancies and spontaneous ptb were included. other criteria included, but not limited to, provided genetic data in a complete enough format so that it could be evaluated in meta-analysis and defined the clinical outcome clearly. meta-analysis was performed wherever >3 replication data sets were available results: a total of 5422 abstracts were reviewed and 88 were selected for full text review. data were extracted from 50 articles. over 100 associations were reported between snps on various candidate genes and ptb; however only 32 had replication dataset. meta-analysis documented significant association between pon2a148g (odds ratio [or]=1.64 (95%ci 1.18-2.26), pon1(rs#662)(or=1.14; ci-1.007-1.34), tnfrsf6-670a/g (fas) (or=1.52; ci-1.11-2.08) and ptb. two snps pon2s311c (or=0.56; ci-0.36-0.85) and ifn gamma (rs2430561; or-0.62; ci-0.43-0.91) documented protective effect. conclusions: systematic review concludes significant heterogeneities leading to lack of reproducible data in genetic association studies of ptb. heterogeneities are contributed predominantly by lack of adequate power, poor phenotype selection, and population admixture. the functional relevance of the risk and protective alleles needs to be verified. jignesh parvadia, 1 mounira habli, 2 jeff livingstone, 2 william polzin, 3 foong lim, 1 timothy crombleholme. 1 1 pediatric and thoracic surgery, cincinnati children's hospital medical center, cincinnati, oh, usa; 2 obstetric and gynecology, university of cincinnati, cincinnati, oh, usa; 3 obstetric and gynecology, good samaritan hospital, cincinnati, oh, usa. objective little is known about the response of ttts to treatment either by amnioreduction or selective fetospopic laser in triplet pregnancy, particularly the survival of the bystander fetuses. in order to define the response of triplet pregnancies to treatment for ttts we reviewed our experience with higher order multifetal gestations complicated by ttts. study design retrospective chart review of patients diagnosed with in high order gestation from 2004-2007 was performed. results among 254 cases of ttts 11/254(4.3%) patients with high order gestations were identified (n=11) with a mean ga at diagnosis of 20.3±0.8 weeks. 9 pregnancies (81.8%) were dichorionic triamniotic and 2(18.1%) were monochorionic triamniotic. cincinnati modification of quinterro staging was utilized to characterize recipient cardiomyopathy as mild (stage iiia, n=3), moderate (stage iiib, n=1) and severe (stage iiic or iv, n=3) categories. 4/11 (36.3%) were treated with amnioreduction alone (ar), 2/11(18.1%) with selective fetoscopic laser photocoagulation (sflp) alone, 2/11(18.1%) with ar followed by sflp and 1/11(9.09%) with ar followed by intrafetal radio frequency ablation (rfa). 1/11(9.09%) patient had a cervical cerclage. 2/11(18.1%) patients were treated but remain undelivered. mean ga at delivery was 29.5±1.04 weeks. overall survival was 21/27(77.7%) with bystander survival was 9/9(100%), donor survival 6/9(66.6%), recipient survival was 6/9(66.6%). conclusion triplet pregnancies treated for ttts have 100% survival rate for bystander fetuses and have 66.6% survival rates for donor and recipients comparable to twins treated for ttts. ga at diagnosis 20.3±0.8weeks cincinnati modification of quinterro 1(i), 1(ii), 3(iii), 3(iiia), 1(iiib), 2(iv) ga at delivery 29.5±1.04 weeks live birth -donor 6/9(66.6%)* # -recipient 6/9(66.6%)*# -bystander 9/9(100%) # birth weight -donor to assess the effect of breast feeding (bf) on perinatal outcome in relation to maternal antenatal methadone dose. study design a retrospective chart review study of 170 methadone dependent mother and infant pairs. patients were categorized into 3 groups based on maternal dose at time of delivery: group 1: dose 50 mg, group 2: dose 51-100mg, group 3: dose >100 mg. the finnegan's scoring system was used to monitor neonatal abstinence syndrome(nas). treatment for nas was initiated if there were 2 scores of 8. neonatal outcome data included:% nicu admission, % of babies discharged(d/c) at time of maternal d/c, % nas, % treated for nas and total hospital stay. data were analyzed by t-test and fisher's exact test. maternal characteristics between the 3 groups were similar. regardless of maternal methadone dose, bf infants have shorter hospital stays and higher rates of d/c at time of maternal d/c, lower incidence of nas and fewer nicu admission (table) . in all three groups, breast feeding did not impact the severity of nas as reflected in finnegan's score(fs). (table) conclusion regardless of maternal methadone dose, breast feeding improves perinatal objective: to evaluate the effects of preventive collagen plugging of the fetoscopic access port at the time of balloon removal on pregnancy outcome in fetoscopic endoluminal tracheal occlusion pregnancies. study design: fifty-one pregnancies involving fetuses with severe congenital diaphragmatic hernia (cdh) were studied. all patients underwent feto between 26-29 weeks gestational age (ga) and fetoscopic balloon removal around 34 weeks ga. at the time of balloon removal, a purified dried collagen plug was inserted through the fetoscopic access port in 23 consecutive pregnancies but not in the first 28 pregnancies considered as controls. all patients underwent post-plugging ultrasound and magnetic resonance imaging studies to evaluate for membrane dehiscence, amniotic fluid volume and fetal well being. ga at delivery, incidence of premature rupture of the membranes (pprom), bleeding at port retrieval and adverse fetal effects were compared in both groups. results: mean (sd) ga at feto [28.2 (1.8) vs. 27.5 (1.5) weeks; p= ns] and balloon removal [33.9 (0.7) vs. 33.7 (1.1) weeks; p= ns] was similar in the treatment and in the control group. incidence of pprom following the second fetoscopy was 2/23 in the study group compared to 9/28 in the control group (p<0.05). mean (sd) ga at delivery was 37.0 (1.8) weeks in the study group, compared to 36.4 (2.0) in the control group (p=0.28). bleeding from the trocar insertion site occurred in 4 cases in both groups, but clinically significant bleeding occurred only in one of the controls. membrane dehiscence was noted in 4 patient in the treatment group compared to 6 in the control group (p=ns). conclusion: preventive collagen plugging of the fetal membrane defect created by the fetoscopic access resulted in a significant reduction in pprom rates and a trend towards increased ga at birth without adverse fetal effects in feto pregnancies. wider application of this technique should be considered, but needs evaluation in larger, randomized trials. hydrops fetalis is an uncommon fetal condition characterised by the abnormal accumulation of fluid in two or more body cavities, traditionally associated with a poor prognosis. the relative rarity of this presentation has meant that published case series have consisted of small numbers. a retrospective review of case notes of all cases managed at kemh between 1995 and 2005 was performed. in western australia, kemh is the only tertiary maternity hospital incorporating a maternal-fetal medicine unit. cases were obtained from the mfm database. in the period 1995 to 2005 there was a total of 60 pregnancies affected by hydrops (incidence 0.2 per 1000 births). the average maternal age was 29 years. in 5 cases a fetal abnormality had occurred in a previous pregnancy. the median gestational age at diagnosis was 18 weeks (range 11-36 weeks). in just over half (53%) of cases, the diagnosis was confirmed prior to delivery. a post-mortem was performed on all but 2 of the babies not born alive. edema was present in at least 3 cavities in over half of cases (n=36). chromosomal anomalies included trisomy 21, trisomy 18 and turners syndrome. in all cases of infection, parvovirus b19 was implicated. cardiac arrythmias included svt and atrial flutter. cases classified as other included alpha thalassemia and syndromic disorders. in 33 cases an interruption of pregnancy was performed at a mean gestational age of 19 weeks. of those who did not elect to terminate the pregnancy, there were 15 fetal deaths in utero, 11 live borns with 1 neonatal death. for the live borns, the median gestational at delivery was 36.3 weeks (range 33 to 40.7 weeks). the causes of hydrops in live birth cases included cardiac arrythmia (n=3), infection (n=2), chromosomal abnormality (n=1), unknown (n=4) and other (n=1 (dmv) have been noted to play a role in the development of hemorrhagic and periventricular leukomalacic lesions in premature babies. since deep vein drainage system is relatively more prominent in the developmental brain than adult brain, we investigated if dmv anomalies could be associated with clastic lesions in-utero. methods two senior neuroradiologists reviewed 900 fetal brain exam performed between 2001 and 2007, seeking for unequivocal anomalies in dmv, such as periventricular venular engorgement. all mr scanning is performed at 1.5 tesla, using surface abdominal coils and single-shot fast spin-echo t2-weighted 3-4 mm thick sections, with 1.1-1.25 mm in planar spatial resolution. we found 6 cases with dmv anomalies (tab.1). most of the dmv engorgement is located at frontal lobes level. from this limited preliminary series it appears that dmv involvement plays a role in the development of periventricular leukomalacia and periventricular hemorrhagic necrosis. the observation that these lesions are mostly located at frontal level may suggest that some of the term neonates carrying sequelae of atypically located leukomalacia (i.e. deep frontal lobe) might have developed these lesions in-utero. it is of interest to notice that most of our cases were related to heart failure. therefore, central venous hypertension affecting immature deep cerebral venous system has to be taken into account. in our center, patients with an estimated risk for chromosomal abnormalities at term greater that 1 in 220 after 1 st trimester combined screening test (ft) are offered non-directive genetic counseling. the aim of this study was to evaluate the responses of women younger than 35 attending this genetic counseling session. material and methods: data from patients referred for a positive ft from september 1, 2003 to july 1, 2007 was retrieved from our database. information concerning women younger than 35 years of age at the estimated date of delivery was extracted and tabulated. results: during the study period 591 women had genetic counseling for positive ft. thirteen patients were excluded from further analysis (8 had incomplete clinical documentation and 5 had spontaneous miscarriages prior to 16 weeks gestation). four hundred and twenty-five patients were older than 35 and 153 were younger than 35 at the estimated date of delivery. table 1 depicts summary statistics for studied variables in this younger group of women. conclusions: overall this data suggests that approximately 25% of this younger group of women opted for chorionic villous sampling (cvs), 40% for amniocentesis and more than 34% declined prenatal genetic testing. moreover, this data also suggests that: 1) these women opted for cvs when the ft risk (mean = 1 in 58) almost doubled the cvs procedure related risk quoted at 1% and, 2) when the ft risk is between 1 in 100 and 1 in 220 almost half (53 out of 115) declined not only the 1st trimester cvs but also the 2nd trimester amniocentesis. we believe that understanding our patient population is important to optimize both the efficiency and efficacy of the alternative prenatal screening programs. acceptance for prenatal genetic testing after a positive first trimester combined screening test in women of less than 35 to determine the optimal diagnostic test using prenatal ultrasonography and mri for predicting pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. methods: relevant papers were identified by searching medline (1966 medline ( -2007 , embase (1988) (1989) (1990) (1991) (1992) (1993) (1994) (1995) (1996) (1997) (1998) (1999) (2000) (2001) (2002) (2003) (2004) (2005) (2006) (2007) and the cochrane library (2004 issue 2). in addition, the specialist literature on the topic and reference lists were hand searched for relevant articles. studies were selected if they examined diagnostic tests for the prenatal prediction of pulmonary hypoplasia in fetuses with congenital diaphragmatic hernia. the primary outcome measure was perinatal survival. study selection, quality assessment and data abstraction were performed independently and in duplicate by separate observers. results: of a total number of 8581 articles (published studies), there were eighteen studies that fulfilled the entry criteria. six examined entirely unique heterogeneous parameters. of the remainder, all 12 examined the ultrasound measurement of lung to head ratios (lhr) at a 'cut-off of greater than or less than the thresholds of 0.6, 1.0, 1.4, 1.6. in addition, the presence of liver in the fetal thorax was included (if present) as a co-variable (liver "up"). a lhr >1.0 compared to <1.0 provided the strongest association with perinatal survival (peto or 5.73, 95% ci 3.38-9.71). the finding of "liver up"in the fetal thorax had a negative association with survival (peto or 0.25, 95% ci 0.16-0.39). only three studies provided data for lhr in conjunction with the presence of liver in the fetal thorax (peto or survival for lhr > 1.0 compared to <1.0 was or 7.65, 95% ci 3.42-17.10). data was also available for liver up and lhr > 1.4 which had a peto or of 2.2 (95% ci 0. 83-5.82 ). discussion: the data supports the view that lhr may be a useful prognostic indicator of perinatal survival in fetuses with congenital diaphragmatic hernia. however, heterogeneity still exists regarding the timing of ultrasound measurement and the use of mri. the majority of studies have small sample sizes. objective: to estimate, in fetal anaemia, the diagnostic value of fetal ultrasonography and doppler blood flow in the evaluation of fetal anaemia methods: literature from 2000 to 2007 was identified using medline and embase, the cochrane library and relevant specialist register of the cochrane collaboration, and by checking reference lists of known primary studies and review articles. studies were selected if the accuracy of the fetal ultrasound parameters or doppler studies of blood flow in the fetal vessels was estimated compared with a reference standard. diagnostic tests evaluated were ultrasound measurement of the fetal spleen and liver length and doppler studies from the umbilical vein and middle cerebral artery. data from the selected studies were abstracted as 2x2 tables comparing the diagnostic test result with the reference standard. results were pooled where appropriate. diagnostic accuracy was expressed as sensitivity and specificity. twenty-nine primary studies were identified containing suitable data. twentyone of these gave data on middle cerebral artery doppler peak systolic velocity (mca-psv) and 15 could be pooled in the meta-analysis giving a sensitivity of 0.75 (0.63-0.86) and a specificity of 0.89 (0.84-0.95) for 1078 cases in detecting severe anaemia. four studies gave data on spleen perimeter and it was possible to pool three of these giving a sensitivity of 0.20 (0.10-0.35) and a specificity of 0.28 (0.18-0.41) for 112 cases. three studies had data for liver length measurements and two were pooled. the sensitivity was 0.16 (0.05-0.36) and the specificity was 0.77 (0.46-0.95) but only 38 cases were used in the analysis. there were three studies on umbilical vein maximum velocity doppler studies and all were suitable for meta-analysis giving a sensitivity of 0.73 (0.61-0.83) and a specificity of 0.54 (0.33-0.73) with 97 cases analysed. two studies gave data on middle cerebral artery time-averaged mean velocity score giving 52 cases. the sensitivity was 0.95 (0.83-0.99) and specificity was 0.85 (0.55-0.98). discussion: middle cerebral artery peak systolic velocity dopplers remain the gold standard for non-invasive screening of fetal anaemia. middle cerebral artery time-averaged mean velocity scores require further investigation. other tests perform poorly when diagnostic accuracy is assessed. cochrane review of treatment in twin to twin transfusion syndrome. twin-twin transfusion syndrome is a condition affecting monochorionic twin pregnancies associated with a high risk of perinatal mortality and morbidity. the objective of this review was to evaluate the impact (maternal,fetal and pediatric)of treatment modalities in twin-twin transfusion syndrome. register and cochrane controlled trials register. we also searched conference proceedings and made personal contact with experts active in the area of the review. randomised and quasi-randomised studies of amnioreduction versus laser coagulation, septostomy versus laser coagulation or septostomy versus amnioreduction. eligibility was assessed by one reviewer. study authors were contacted for additional information. two studies were included. this review shows that laser coagulation of anastomotic vessels results in less fetal (rr 0.71; 95% ci 0.57, 0.89) and neonatal deaths (rr 0.30; 95% ci 0.17, 0.56) than amnioreduction ( figure 1 ). there is no difference in perinatal outcome between amnioreduction and septostomy. more babies in the laser arm are alive without neurological abnormality at six months of age (developmental delay at <2 years rr 0.70, 95% ci 0.57, 0.86)( figure 2 ). conclusions: endoscopic laser coagulation of anastomotic vessels should be considered in the treatment of all stages of twin twin transfusion syndrome to improve perinatal outcome. further research on the effect of treatment on milder forms of twin twin transfusion syndrome (quintero stage 1 and 2) are required. (produced in collaration with the cochrane collaboration, uk). (cvs) concluded that although the risks of pregnancy loss are relatively low, lack of adequate controls tends to underestimate the true added risk of prenatal invasive procedures (obstet gynecol.2007; 110 (3):687-94). the west midlands is a large region within the uk containing approximately 12% of the total uk population. the congenital anomaly register for this region is able to monitor pregnancy outcomes with accurate deminator data. results: there were 371 first trimester cvs performed, by ten operators, in the west midlands (uk) in 2005. this equates to 5.4 procedures per 1000 births. significantly higher rates were noted in areas of high socioeconomic status. the median number of procedures performed per operator was 35 (range 9-81). all operators were performing other invasive tests such as amniocentesis or cordocentesis,etc. the most common indication for cvs was: i) fetal anomaly on dating scan (24.25%) ii) abnormal (>1 in 300) risk on combined first trimester screening (22.4%) iii) molecular genetic diagnosis (18.1%) and iv) maternal request (35.25%). using a combination of first trimester scanning and cvs, 33% had abnormal karyotype/structural anomaly.the corrected loss rate (background and procedure-related) following cvs in normally formed, singleton pregnancies was 3.6% (95thci 1.1-6.0%) up to 7 days postnatal (perinatal loss) and 2.7% (95thci 0.60-4.8%) with 28 days of procedure. the proportion of cvs in which an adequate sample was not obtained was 1.1% (95th ci 0.0-2.1%). conclusions: this epidemiological study using accurate demoninator data provide interesting statistics relating to the uptake and prenatal risks of first trimester cvs. with the increasing prevalence of obesity in the last two decades, we have seen a tremendous increase in bariatric procedures in reproductive aged women. malnourishment and vitamin deficiency are common complications after gastric bypass which may impact on fetal development. we present the case of a 27 yo who underwent a duodenal switch procedure in 2005. she was discovered to be pregnant during evaluation for persistent malnutrition in 2006. multiple prenatal ultrasounds were performed; the first at 15 weeks gestation was unremarkable. the 19 week sono revealed a male fetus with shortened femurs and humeri bilaterally, nasal bridge hypoplasia, macroglossia, poorly defined hands, and possible clubbed feet. amniocentesis revealed a normal karyotype. 3d ultrasound redemonstrated the abnormal facial findings. a fetal echocardiogram was normal. the lagging long bone measurements continued to worsen, ultimately with femurs 6 weeks behind. the fetal thoracic circumference was two standard deviations below the mean, giving rise to concern for pulmonary sequelae. growth restriction was noted at the 33 wk sonogram. delivery by cesarean section was at 33 5/7 weeks secondary to nonreasuring fetal status. the birthweight was 1468gm; apgars were 1, 4, and 6. postnatal radiographs confirmed antenatal ultrasonographic findings and demonstrated evidence of epiphyseal stippling. the infant remained intubated until 12 weeks of life, after which it died secondary to respiratory complications associated with pulmonary hypoplasia. gene mapping studies have not found any point mutations on the recessive gene as an etiology of this disorder. rhizomelic chondrodysplasia punctata refers to a heterogeneous group of bone dysplasias with a familiar radiographic phenotype involving punctate calcifications and epiphyseal stippling. the etiology of this may be secondary to chromosomal abnormalities, mendelian gene disorders, or teratogens, notably warfarin. this case may be explained by vitamin k deficiency of the embryo due to maternal malabsorption after bariatric surgery. the maternal vit k level was <.03 ng/dl at time of delivery(normal > 0.10). the teratogenic effects of vitamin k deficiency in this instance highlight the need for strict counseling and screening for vitamin deficiency in those women undergoing bariatric surgery since previous obesity-related anovulation is reversed as patients lose weight, resulting in unexpected pregnancies and potentially preventable fetal abnormalities. term preeclampsia: any risk for the neonate? sindhu k srinivas, jamie bastek, christina m andrela, emmanuelle pare, michal a elovitz. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: preeclampsia continues to be a major contributor to maternal morbidity and mortality worldwide. preeclampsia at term is not associated with the same risk of neonatal morbidty and mortality as preterm preeclampsia . however, neonatal outcomes in term women with preeclampsia have not been adequately studied. we sought to compare short term neonatal outcomes in term infants born to women with and without preeclampsia. methods: this study was part of a large case control study. women with preeclampsia (n=228) and term controls (n=563) were prospectively identified. infants with congenital anomalies were excluded. hospital length of stay (los), admission or transfer to the nicu, and use of mechanical ventilation or cpap within first week of life were assessed. associations between neonatal outcomes and preeclampsia were evaluated using chi-square analysis and wilcoxon rank sum test. significant confounders were controlled for using multivariable logistic regression. results: discharge day of life was significantly different between neonates born to women with preeclampsia (median =2; mean =3.6) versus those born to women with uncomplicated term deliveries (median =2; mean =2.8, p<0.001). this difference persisted even when neonates with iugr and those born to diabetic mothers were excluded (p<0.001). term infants born to women with preeclampsia have a higher odds of being admitted or transferred to the nicu (aor=2. 16 [1.36-3.43 ], p=0.001) after controlling for iugr, delivery mode, race, and gestational age at delivery. these infants also have a higher odds of mechanical ventilation (aor=8 [1. 43-44.6 ], p=0.018) and cpap use (aor=2.6 [1. 23-5.32 ], p=0.012) after controlling for the same confounders. there was no difference in ivh or nec between the two groups. conclusion: neonates born to women with preeclampsia have differences in short-term morbidity when compared to neonates born to women without preeclampsia, despite being born at term. whether this increase in neonatal morbidity is attributable to medications used in preeclampsia, such as magnesium sulfate, is unclear. these findings may have implications for patient counseling as well as hospital resource allocation. further investigation correlating these findings with long-term morbidity is warranted. could confined placental mosaicism account for adverse perinatal outcomes in ivf pregnancies? benoit c jacod, 1 gh schuring-blom, 2 kd lichtenbelt, 2 jse laven, 3 d van opstal, 4 mjc eijkemans, 1 nick s macklon. 1 1 reproductive medicine gynaecology, university medical centre, utrecht, netherlands; 2 clinical genetics, university medical centre, utrecht, netherlands; 3 obstetrics gynaecology, erasmus medical centre, rotterdam, netherlands; 4 clinical genetics, erasmus medical centre, rotterdam, netherlands. background: ivf singletons have poorer perinatal outcomes than singletons from spontaneous conceptions. this may be due to the influence of ovarian stimulation on the chromosomal constitution of the embryos which could be translated into localized chromosomal anomalies in the placenta. aim: to compare the incidence of confined placental mosaicism (cpm) in ivf/icsi pregnancies and spontaneous conceptions. methods: multicentre retrospective analysis of karyotype results obtained by chorionic villus sampling (cvs) performed because of advanced maternal age ( 36 years at 18 weeks of gestation) in the netherlands between 1995 and 2005. results: from a total of 322246 pregnancies, 20885 cvs results were analysed: 235 in the ivf/icsi group and 20650 in the control group. the mean age of women in both groups was 38.4 years (mean difference -0.08, 95% ci -0.35 -0.18). foetal karyotype was missing in 152 cases of possible cpm, all in the control group. when taking into account missing data, the incidence of cpm was lower in the ivf-icsi group than in the control group, 1.3% vs. 2.2% (odds ratio 0.58, 95% ci 0.18 -1.81) whereas the incidence of foetal chromosomal anomalies was increased 4.3% vs. 2.4% (odds ratio 1.82, 95% ci 0.96 -3.46) although both differences are not significant. conclusions: the incidence of confined placental mosaicism is not increased in ivf/icsi pregnancies compared to spontaneous conceptions. cpm probably does not account for the adverse perinatal outcomes following ivf/icsi. fetal rh d, cc and ee genotyping using fetal dna from maternal blood is not impaired by the presence of maternal alloimmunization. chad a grotegut, 1 stacey l jeronis, 2 john p gaughan, 3 enrique hernandez, 2 ossie geifman-holtzman. 2 1 obstetrics and gynecology, duke university, durham, nc, usa; 2 obstetrics, gynecology and reproductive sciences, temple university, philadelphia, pa, usa; 3 biostatistics, temple university, philadelphia, pa, usa. this study was conducted to assess the impact of maternal alloimmunization on the accuracy of fetal rh d, cc and ee genotyping from maternal blood. we performed a literature search of english-written articles describing fetal rh d, cc and ee determination from maternal blood (am j obstet gynecol 2006; 195:1163-73) . using this database, we determined the accuracy of rh d, cc and ee genotyping in the presence of maternal alloimmunization. for each subgroup, 95% confidence intervals for a proportion were calculated and compared between groups. we found 37 english-written publications reporting non-invasive rhd genotyping and 4 reporting non-invasive rh ce genotyping from maternal blood. fourteen (37.8%) of the rh d articles and three (75%) of the rh ce articles provided accuracy results in the setting of alloimmunization. the accuracy results are reported as follows: 67 .7 % of the samples were determined correctly in the presence of alloimmunization.* this accuracy was significantly lower than the accuracy reported for all rh cc samples. when only studies utilizing free fetal dna for rh cc genotyping were used (vs fetal cells), fetal cc genotype was determined correctly in 11/11 (100%, 95% ci 70.0, 100), which was similar to the overall success rate for rh cc determination. overall, there were no differences in the success of rh d, cc or ee determination in the setting of alloimmunization compared to the overall accuracy seen when free fetal dna was used. the presence of maternal alloimmunization does not reduce the accuracy of fetal rh d, cc or ee non-invasive genotyping from maternal blood utilizing free fetal dna. further research into structure and rearrangements of the rh d, cc and ee genes may further improve diagnostic accuracy of free fetal dna from maternal blood. fetal dna, most likely of trophoblast origin, is present in both the plasma of pregnant women and provides a potential basis for non-invasive fetal diagnostic tests. however, fetal dna in maternal plasma is highly contaminated with maternal dna, and this contamination is the main technical challenge in trying to accomplish non-invasive detection of fetal chromosome abnormalities. existing methods for the selective amplification of fetal dna have generally relied on specific sequence differences between the mother and fetus. as an alternative, we have developed a method for selective amplification of fetal dna that makes use of observation that trophoblast dna is globally hypomethylated in comparison with dna from other sources. in this method, a dna mixture is first digested with a methylation sensitive restriction enzyme and then amplified by linker-mediated pcr. after an initial amplification, a second isothermal rolling-circle amplification is performed. this procedure results in the differential amplification of short, relatively hypomethylated fragments. after amplification, the resulting "representations" are comparatively hybridized to a microarray consisting of oligonucleotides that correspond to restriction fragments generated by the initial digest. copy number differences are then detected through statistical analysis of array addresses that show significant amplification. to test the feasibility of this method for detecting aneuploidy, we have prepared mixtures of peripheral blood dna and first trimester trophoblast dna from either normal or from samples with trisomy 18 and trisomy 21. we present data showing that aneuploidy can be detected even when 90% of the starting dna sample was derived from a euploid source and only 10% was from an aneuploid trophoblast sample. two different approaches to data analysis are presented. one relies on prior analyses of trophoblast methylation and the second is independent of any prior knowledge or analysis. both methods provide similar ability to detect aneuploidy. future work will focus on testing whether this approach can be used for non-invasive prenatal diagnosis. chromatin immunoprecipitation and emsa analysis of nf-b and c/ ebp synergism on the otr promoter. shirin khanjani, yun s lee, vasso terzidou, mark r johnson, phillip r bennett. institute of reproductive developmental biology, imperial college, london, united kingdom. we have shown, the transient transfections of the transcription factors nf-bp65 and c/ebp leads to a synergistic increase in otr promoter activity in human myocytes. this effect is mediated through a 20bp region of the promoter between -712 to -692 from tss. we now report that this sequence binds both nf-bp65 and c/ebp in vitro and chip studies show binding of both transcription factors to be increased by il-1 in vivo. materials and methods: emsa studies were performed using a 33 bp oligonocleotide sequence (-712 to -672) , containing the 20 bp region responsible for the synergistic activation of the otr promoter and nuclear extracts from primary human myocytes treated with il-1 1ng/ml for 6 hours. for chip analysis, dna protein complexes were crosslinked and antibodies recognizing p65, c/ebp and h4 (positive control) and igg (negative control) were used for immunoprecipitation. primers were designed to amplify the region -744 to -593, which includes the 20 bp response sequence. to further confirm the interaction between nf-bp65 with c/ebp a 6xnf-b consensus/luc reporter construct was cotransfected with an expression vector for either nf-bp65 or c/ebp . results: specific nf-bp65 and c/ebp binding was seen in the emsa study. preincubation with antibodies to nf-bp65 and c/ebp led, not to supershift, but to elimination of dna binding for both nf-kb p65 and c/ebp . chip analysis confirmed increased in vivo binding of nf-b p65 and c/ebp to this region of the otr promoter following stimulation with il-1 . transfection of the nf-b/luc reporter construct with an expression vector for c/ebp caused a significant reduction in the basal promoter activity suggesting that c/ebp is binding to nf-kb. this interaction was further confirmed using a tf-tf interaction array. conclusion: these data support the role of nf-b and c/ebp in regulation of otr. the 20 bp region contains a c/ebp but not a nf-b dna binding site suggesting that c/ebp primarily binds to this part of the otr promoter but also interacts with nf-b. the emsa data shows that the 20bp region binds both nf-b and c/ebp . the loss of the supershift observed in previous emsa studies suggests that the antibodies inhibit the interaction between c/ebp and nf-b, therefore inhibiting dna binding. chip analysis supports the concept that il-1 leads to binding of nf-b and c/ebp to the 20bp region. regulation of pro-labour genes by c/ebp, nf-b and ap-1 in human uterine myocytes. suren r sooranna, 1 shirin khanjani, 2 yun s lee, 2 phillip r bennett, 2 mark r johnson. 1 1 imperial college parturition research group, imperial college, london, united kingdom; 2 irdb, imperial college, london. introduction: the transcription factors c/ebp (lap), nf-b (p65) and ap-1(c-fos and c-jun) are implicated in inflammatory processes such as parturition. the promoter regions of the pro-labour genes il-8, pghs-2 and otr contain putative transcription factor-binding sites for these transcription factors. our aim was to determine the effect of transfecting these transcription factors either alone or in combination into uterine myocytes and to determine their effects on the expression of pro-labour genes including il-8, pghs-2, otr, connexin-43 and fp. methods: primary cultures of human uterine myocytes (n=6) were grown from myometrial samples obtained at the time of elective lscs. cells were cultured in 24 well plates to 80% confluence at which point expression constructs for c/ ebp (lap), nf-b(p65), ap-1 (c-fos and c-jun) were transfected either alone or in different combinations. the empty expression vector psg5 was used as a filler construct. cells were cultured for 24 and 72h after which culture medium was collected for elisa and cells frozen at -80°c prior to rna extraction. copy numbers of il-8, pghs-2, otr, fp, connexin-43 and gapdh were measured by qpcr using a rotor-genetm (corbett research). results: 24h post transfection with c/ebp (lap), nf-b (p65), c-fos and c-jun alone or in combination showed no significant changes in pghs -2, otr and connexin-43 expression. over expression of p65 alone or together with either c-fos or c-jun increased fp expression by 56, 76 and 73% respectively (p=0.028). nf-bp65 consistently increased il-8 expression either alone (by 1108%; p=0.018) or in combination with lap, c-fos or c-jun (by 1110, 1385 and 527% respectively; n=6; p=0,028). rel a, lap, c-fos and c-jun together also increased il-8 expression (by 502%; p=0.018) and a small but significant increase was seen with a combination c-fos and c-jun (by 137%; p=0.018). the changes observed in il-8 expression were reflected in the medium il-8 concentration at 72h post transfection. in the presence of rela and c-fos il-8 increased from 3.7 ± 0.2 to 71.0 ± 16.7pg/ml of culture medium (mean ± sem; n=4). conclusions: nf-b (p65) consistently increased fp and il-8 expression in human myometrium. the data suggest that pghs-2 activation has greater dependence upon other transcription factor(s) in addition to p65. true identity of myometrial pr-c: fact or fiction? yun s lee, suren r soorana, mark r johnson, jan brosens, phillip r bennett. imperial college parturition research group, hammersmith campus, london, united kingdom. progesterone is thought to be central to maintenance of pregnancy. multiple progesterone receptor (pr) isoforms underlie complex and diverse biological action of progestins. previously two human pr isoforms have been identified: pr-b and pr-a. pr-a is n-terminally truncated form of pr-b (initiation site methionine 165). in some cells pr-a inhibits pr-b. it has been proposed that increased expression of pr-a in myometrium underlies a 'functional progesterone withdrawal'. the breast cancer cell t47d contains a 60 kda progestin-specific binding protein that is not found in pr negative cells. it was proposed that there is a downstream methionine (met595) which serves as a translation initiation site for the generation of a pr isoform of 60 kda. based on such findings condon et al (mol endo 2006) have focused on the possible role of 60kda pr-c in human parturition. they found that expression of "pr-c" using pr antibody (sc-538 santa cruz, sc) is increased in upper segment myometrium with labour and that overexpression of this protein inhibits pr-b function. we cloned the same human pr cdna into psg5 expression vector. in vivo translation produced a protein of only 39kda. furthermore overexpression of pr-c595 did not significantly decrease pr-b activity in human myocytes. we examined other downstream initiation sites, which may produce a 60 kda protein. we constructed potential pr isoforms in psg5 vector with initiation sites at met 289 and 301. in vivo translation produced proteins of approximately 70 and 62 kda respectively. to determine the effect of pr isoforms on pr-b function, myocytes were co-transfected with pr-a, pr-c289, pr-c301 and pr-c595 with constant amounts of pr-b. the progesterone dependent pre/luc was used as reporter. unlike pr-c595 both pr-c289 and pr-c301 significantly inhibited ligand dependent pr-b mediated transcriptional activity. we found in western analysis that the antibodies pgr-312 (nc) and the sc-539 (sc) detected both endogenous and overexpressed pr-a and pr-b but none of the pr-c isoforms. the sc-538 antibody detected only pr-a and pr-b very poorly. our data suggests that the sc-538 antibody would not detect any pr-c protein and that none of the commercially available antibodies in the uk do so. great care needs to be taken when over-expressing pr isoforms to ensure that proteins are of the expected size. if pr-c does exist in vivo then the 60kda but not the 39kda isoform might inhibit pr-b function. tnf receptor 1 antibody (tnf ri ab), nf-b inhibitor (nf-b activation inhibitor) and erk inhibitor (u0126) (p<0.01), but not by tnf receptor 2 antibody (tnf rii ab), p38 mapk inhibitor (sb203580) and jnk inhibitor (sp600125). by western blot analysis, we found that the protein level of tnf receptor associate factor 2 (traf2) was higher than that of tnf receptor associate factor 1 (traf1) (traf2>traf1) in untreated ct cells. however, after tnf treatment for 4h to 24h, traf1 protein level was increased, but traf2 protein level was reduced (traf1>traf2). the increase of traf 1 and decrease of traf2 were blocked by tnf ri ab, but not by tnf rii ab. we also found that tnf rapidly (within 5-30 min) and significantly increased phosphorylation of ikk , erk1/2 and jnk1/2/3 and the phosphorylation of these protein kinases by tnf was reduced significantly by tnf ri ab, but not by tnf rii ab. moreover, we found that the changes of increased traf1 and decreased traf2 in ct cells (traf1>traf2) resulted in a dramatic deficiency in phosphorylation of the above protein kinases induced by tnf compared with the normal ct cells (traf2>traf1). nuclear localization of nf-b p65 in tnf treated cells was increased compared to untreated controls. conclusion: we have demonstrated for the first time that tnf stimulates mmp-9 production in ct cells through tnf ri-trafs-ikk /erk-nf-b signaling pathways, but not through the jnk/p38-ap-1 pathway. these studies reveal steps within this pathway as possible therapeutic targets to inhibit mmp-9 expression potentially attenuating tnf -induced degradation of extracellular matrix and pre-term rupture of the fetal membranes. objective: women with preterm birth are at elevated risk of cardiovascular disease, but mechanisms that might relate these conditions are not understood. we hypothesized that women with spontaneous preterm vs. term births may have early gestation evidence of activation of the fibrinolytic cascade, as measured by the thrombin-antithrombin iii (tat) complex. we also tested if this relation may be associated with inflammation. methods: tat was measured in plasma collected <21 weeks gestation (mean 10.2 weeks, sd 3.9) among women without chronic medical conditions, preeclampsia or growth restriction who delivered singleton liveborn infants. inflammation was assessed by c-reactive protein (crp) measured in serum from the same samples. women with spontaneous preterm birth (sptb) <34 weeks (n=32) and 34-<37 weeks (n=72) were compared to women with term births >=37 weeks (n=215). high tat was defined as >5.2 ng/ml (highest quartile among women with term births) and high crp was defined as >=8 ug/ml. multinomial logistic regression was utilized to relate elevated tat and inflammation to risk of sptb subtypes. results: women with sptb were more likely to have tat concentrations in the highest quartile compared to women with term births (<34 weeks, 53.1%; 34-<37 weeks, 30.7%; >=37 weeks 24.11%, p<0.01). women with high tat concentrations had a 3.3-fold (95% ci: 1.5-7.3) increased risk for sptb <34 weeks, after adjustment for body mass index, race, age and gestational age at sampling. there was no relation between high tat and sptb 34-<37 weeks (or 1.2, 95% ci 0.7-2.3). additional adjustment for elevated crp (>=8ug/ ml) did not effect the estimates associated with tat, and elevated crp was independently related to risk for both sptb subtypes (<34 weeks, or 2.9 [1.1-8.0]; 34-<37 weeks, or 2.7 [1.3-5.6]). thus, women with high tat and elevated crp appeared to be at particularly elevated risk of sptb <34 weeks (or 8.3, 95% ci 1.8-38.1). conclusions: the thrombin-antithrombin iii complex was elevated early in gestation among women with sptb <34 weeks, perhaps secondary to microvascular injury. this effect was independent of inflammation, suggesting that the elevated fibrinolytic cascade may function independently from inflammation among women with sptb. plasma cortico-releasing hormone and cortisol concentrations and psychological stress among pregnant women. katherine p himes, hyagriv n simhan. obstetrics and gynecology, university of pittsburgh medical center, magee womens hospital, pittsburgh, pa, usa. objective: many studies have found an association between psychological stress and preterm birth. we sought to determine if women with greater psychological stress during pregnancy had higher concentrations of plasma cortico-releasing hormone (crh) or cortisol. study design: this is a secondary analysis of a multicenter case-control study, nested within an observational cohort. of 3,073 participants, plasma crh and cortisol concentrations at 24 and 28 weeks gestation were available in 178 controls who delivered after 37 weeks and 159 cases who delivered before 37 weeks. the abbreviated scale for the assessment of psychosocial status in pregnancy (asaps) was available for all women. concentrations of crh and cortisol were compared between women above and below the lowest quartile score on the asaps among cases and controls. the same analysis was done for the portion of the scale related to psychological stress. concentrations of crh and cortisol and psychological stress were also compared between black and non-black cases and controls. univariate analysis was performed with kruskal wallis or chi-square. results: there was no difference in crh or cortisol concentrations at 24 or 28 weeks among women above or below the lowest quartile on the asaps (controls:p=0.17-0.66 cases:p=0.47-0.97). greater psychological stress was not associated with higher concentrations of crh or cortisol at 24 or 28 weeks(controls:p=0.33-0.63 cases:p=0.17-0.78). crh concentrations were not different between blacks and non-blacks. among both cases and controls, cortisol concentrations at 24 and 28 weeks were lower in black women than non-black women (controls:p<0.01 cases:p< 0.01). the median cortisol concentration among control black women was 11.0 g/ml at 24 weeks compared to 14.4 g/ml among non-black women and 14.4 g/ml compared to 18.6 g/ml at 28 weeks. black women reported less psychological stress than non-black women (p= 0.001) conclusion: we found no relationship between psychological stress and plasma crh or cortisol. furthermore, while stress is hypothesized to play a role in the racial disparity of preterm birth, black women reported less psychological stress and had lower cortisol concentrations than non-black women. improved assessments of psychological stress and additional biomarkers involved in the stress response may broaden our understanding of how stress contributes to preterm birth. expression, tissular traffic and activation of mmp-9 in human fetal membranes during labor. rodrigo vega-sanchez, arturo flores, marisol castillo, nardhy gomez, felipe vadillo-ortega. direction of research, instituto nacional de perinatologia, mexico city, df, mexico. introduction. rupture of the fetal membranes (fm) during human labor occurs as a consequence of extracellular matrix degradation. this process is controlled by increased secretion and activity of matrix metalloproteinases, particularly mmp-9.several evidences suggest that mmp-9 is mainly produced by infiltrating choriodecidual leukocytes that could arrive from placental circulation. characterization of the synthesis, transport and activation of mmp-9 within the fm is critical to understand the process of membrane rupture during human labor. objectives. expression and secretion of mmp-9 in placental leukocytes, trafficking of the enzyme through the fm and one possible mechanism for its activation were analyzed. methods. leukocytes were isolated from placental blood of women after active labor. maternal leukocytes were used as controls. cells were cultured for 96 h. relative expression of mmp-9 by rt-pcr and enzyme secretion by elisa and zymography were followed at different times. to analyze the traffic of mmp-9 through the fm, fluorescein-conjugated prommp-9 was added to the choriodecidual side of the fm in an in vitro system that allows the separation of amnion and chorion. labeled mmp-9 was localized at distinct times by confocal microscopy. the protease responsible for the activation of mmp-9 was identified using neutralizing antibodies and specific inhibitors. results. no difference in the relative expression of mmp-9 in leukocytes throughout the culture was found. however, secretion of the enzyme significantly increased since 24 h (p<0.05). experiments using labeled mmp-9, repeatedly showed that after 12 h in culture, the enzyme was mainly localized within the amniotic epithelium. specific inhibition of mmp-3 significantly decreased the activation of pro-mmp-9. conclusions. our results demonstrate that the increased secretion of mmp-9 by placental leukocytes is not associated to increased gene expression, suggesting that homing of a specific leukocyte subpopulation to the choriodecidua is occurring during labor. mmp-9 can be trafficked from the choriodecidua to the amnion, suggesting a transmembranal pathway that may regulate the tissular localization of the enzyme to the area of the fm with high content of connective tissue. once secreted by the placental leukocytes, activation of mmp-9 depends mainly on mmp-3, which seems to be derived from the same leukocytes. objective: preterm labour is a major problem in terms of perinatal morbidity and mortality. the histone-deacetylase inhibitor (hdaci), trichostatin a (tsa) has been shown to have an inhibitory effect on myometrial contractility. the aim of this study was to evaluate the effect of the hdaci's suberic bishydroxymate (sbha) and valproic acid (vpa) on human uterine contractions and hence their potential role as tocolytic agents. methods: biopsies of human myometrium were obtained at elective caesarean section (n=18). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of sbha in the concentration range of 1 nmol/l to 100 mmol/l and vpa (100nmol/l-1mmol/l). control experiments were run simultaneously. results: sbha and vpa exerted a potent and cumulative inhibitory effect on spontaneous and oxytocin-induced contractions, compared to control strips. the mean maximal inhibition (mmi) values for sbha were 65.35% for spontaneous contractions (n=6; p< 0.05), and 53.53% for oxytocin-induced contractions (n=6; p<0.05). the mmi values for vpa were 36.57% for spontaneous contractions (n=6; p< 0.05), and 35.20% for oxytocin-induced contractions (n=6; p<0.05). conclusion: these results raise the possibility that hdaci's may have tocolytic potential, in addition to their current clinical indications. the inhibitory effect observed may be linked to the ability of hdac inhibitors to induce the expression of genes involved in the maintenance of myometrial quiescence via epigenetic mechanisms but may potentially also involve non-epigenetic pathways. progestin suppresses thrombin-enhanced interleukin-6 expression in term decidual cells: implications for abruption-induced preterm delivery. edward kuczynski, lynn f buchwalder, frederick schatz, charles j lockwood. obstetrics/gynecology reprod. sciences, yale university school of medicine, new haven, ct, usa. background and objective: decidual hemorrhage (abruption) promotes binding of factor vii to decidual cell (dc)-expressed tissue factor to generate thrombin. thrombin in turn induces several biological effects leading to preterm delivery via activation of cell surface protease-activated receptors (pars). interleukin-6 (il-6) is a pleiotropic proinflammatory cytokine induced by pars. this study assessed the separate and interactive effects of thrombin and medroxyprogesterone acetate (mpa) on il-6 expression in term dcs. methods: term decidua from stripped fetal membranes were isolated and the dcs were purified on a percoll gradient, grown to confluence and passaged until leukocyte-free. confluent dcs were primed in 10 -8 m estradiol (e2) of e2 +10 -7 m mpa for 7 days, then incubated in a defined medium (dm) with corresponding steroids ± thrombin. after 24 hours, il-6 levels in conditioned dm were measured by elisa and western blotting. in parallel 6-hour incubations, il-6 mrna levels were assessed by quantitative rt-pcr and normalized to -actin mrna. results: secreted il-6 levels were similar in cultures maintained in e2 alone (0.43 ± 0.14) and e2 + mpa (0.33 ± 0.06 pg/ml/ug protein; mean ± sem; n = 9). the addition of thrombin (2.5 u/ml) enhanced secreted il-6 levels by 14.3 ± 2.4 fold (p<0.05) in incubations with e2 and by 8.2 ± 1.4-fold (p<0.05) in incubations with e2 + mpa. the inhibitory effect of mpa was statistically significant (p<0.5). in confluent dcs incubated with e2 + mpa, exogenous thrombin (0.05-2.5 u/ml) elicited a concentration-dependent increase in secreted il-6 levels. hirudin acted as a pure thrombin antagonist, exerting no agonist effects alone, but counteracting thrombin-enhanced il-6 secretion. western blotting confirmed the elisa results. quantitative rt-pcr confirmed that il-6 m-rna levels corresponded to protein changes. conclusion: thrombin enhances il-6 mrna and protein expression in term dcs and progestin blunts these effects. since thrombin-generating abruption is closely associated with preterm delivery, anti-inflammatory effects induced by progestin inhibition of dc-derived il-6 may contribute to the protection against ptd, and may explain the reported protective effects of administration of 17 -oh-progesterone in recent clinical trials. introduction: catechol-o-methyltransferase (comt) catalyzes the methylation of the phenolic hydroxyl groups in a variety of catechols. during estrogen metabolism, this enzyme converts the catechol estrogen, 2-hydroxyestrogen (2ohe2), to 2-ethoxyestrogen (2meohe2). the comt substrate, 2-ohe2, can exhibit an anti-estrogenic effect in multiple biologic assays while the 2methoxyestrogen (2-meoe2) can exhibit an estrogenic effect. the biologic activities of these estrogen metabolites (2ohe 2meoe) depend upon their concentrations and tissue type. since comt activity ultimately controls levels of these metabolites, it appears to be a key factor in regulating the cellular estrogenic milieu. we have recently reported that amnion layers of human fetal membranes from laboring women exhibited 3 folds higher comt mrna expression when compared to non-laboring women (wentz et. al. sgi 2007) . objective: to investigate the impact of comt inhibition on prostaglandin e2 (pge2) production by human amniotic membrane explants. study design: explants consisting of 2-cm circular sections of the amnion layer (obtained from term pregnant women who underwent elective repeat cesarean section) were prepared and placed in tissue culture explants media at 37ºc. after a 4-hour incubation, explants were treated with the selective comt inhibitor ro 41-0960, at 10 and 50 m concentrations. the incubation media was harvested after 6 and 24-hour intervals. the levels of prostaglandin e2 (pge2) in the media were measured by sensitive elisa and were normalized against total protein concentration. results: ro 41-0960 comt inhibitor induced major reductions in pge2 production in media collected from amnion explants of human fetal membranes. in the group treated with 10 m of ro 41-9640 for 24 hours there was 72%±8% reduction of pge2 after 24 hours compared to untreated control (p<0.001). in the amnion explants treated with 50 m of ro41-9640, there was 74%±9% after 6 hours and 84%±11% after 24 hours of treatment compared to untreated control (p<0.001). conclusions: this finding indicates that comt activity in the amnion layer of human fetal membranes affects pge 2 production. by facilitating a pro-estrogenic milieu in human fetal membranes in late gestation, increased comt activity may indirectly increases production of factors associated with labor such as pge 2 . hypothesis: an extensive remodeling of the human cervical connective tissue takes place throughout pregnancy with a decrease in the total concentration of collagen and proteoglycans due to an altered higher metabolic turnover. we hypothesize that the profound changes in proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. we also hypothesize that proteoglycan production in cervical fibroblasts from preterm partal women are simmilar to the production in fibroblasts from partal women. method: cervical biopsies were obtained from 5 non-pregnant women, 5 women during elective cesarean section, 6 woman after spontaneous parturition and 4 after a preterm vaginal delivery. by explant technique fibroblasts were cultured from the biopsies. produced proteoglycans were metabolically labeled with s 35 during 24 hours and then purified by ion-exchange chromatography and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. results: the total proteoglycan production decreases with approximately 50% in partal and preterm partal cell cultures. the reduction of proteoglycans in preterm partal and partal cell cultures is significant compared to non pregnant fibroblast cultures. the distribution of biglycan and perlecan are similar in partal and preterm partal cells. biglycan are significantly reduced by around 40% and perlecan are significantly increased by around 60% compared to non pregnant cultures. preterm partal cervical fibroblasts secreates significantly more heparan sulfate proteoglycans compared to non pregnant cultures. the changes in total proteoglycan production in the human pregnant cervix can be seen in corresponding cervical fibroblasts as well. both partal and preterm partal cell cultures differ in their proteoglycan production compared to their non pregnant counterpart, suggesting a role for proteoglycans in cervical ripening. the role of the oxytocin/oxytocin receptor system is not well defined in human amnion. previous studies in rabbit amnion have demonstrated an up-regulation of oxytocin receptors in the end of pregnancy and have shown that there is a large increase in the ability of ot to stimulate pge 2 production. we and others have previously shown a role for nf-b in otr regulation. in whole genome array analysis of human amnion we found that otr was the gene with the second highest increase associated with activation of nf-b (the highest being cox-2). the present work was directed towards further understanding of otr expression and function in human amnion at term. we have shown that pge 2 release by pre-labour primary human amnion cells is significantly increased after oxytocin treatment for 6 hrs (ot: 10-7 m; n=3; triplicate samples; 40 fold increase p<0.05). the expression of otr in labour (+) and labour (-) primary amnion cells was measured with real time rt-pcr. we found a significantly higher level of expression in the labour (+) cells (n=3; duplicate samples; p<0.001). western blot analysis confirmed the upregulation of otr in labouring amnion. treatment with il-1b resulted in a significant upregulation of otr which peaked with a 17 fold induction after 6 hours (p<0.001). il-1 caused a 16 fold increase in otr mrna levels in labour (-) cells, bringing the expression level up to that found in labour (+) cells. our findings provide further evidence for a role of otr in human amnion. expression of otr in human amnion is significantly increased after the onset of labour. term non laboured amnion can be stimulated with il-1 to increase otr expression to levels of laboured amnion. one of the functions of otr in amnion cells is stimulation of prostaglandin synthesis. expression of antioxidant defence proteins in human myometrium before and after the onset of labour. vasso terzidou, mandeep s kandola, shirin khanjani, jan j brosens, phillip r bennett. parturition reserach group, irdb, imperial college, london, united kingdom. background oxidative stress is a result of an imbalance between the production of reactive oxygen species (ros) and the antioxidant defence mechanisms present in biological systems. parturition and infection-induced preterm labour resemble inflammatory processes that are linked to the production of ros including super-oxide (o 2 -), hydrogen peroxide (h 2 o 2 ) and peroxynitrite. low concentrations of ros can act as second messengers in the regulation of several cellular functions. in an attempt to maintain redox homeostasis cells are equipped with machineries to both produce and scavenge ros. enzymatic scavengers include superoxide dismutase (sod), glutathione peroxidise and catalase. sod is the first enzymatic step in the defence system against oxidative stress. nuclear factor-kappa b (nf-b), a transcription factor family classically associated with inflammation, is activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. labour is associated with an increase in nf-b activity in human myometrium and in fetal membranes. nf-b is known to regulate a range of genes associated with the onset of labour. in a study using affymetrix whole genome arrays analysis nf-b overexpression was associated with increased expression of sod-2 (3.5 fold). to determine changes in ros scavenging potential upon onset of labour, lower segment myometrial biopsies were taken at term before and after the onset of labour (n=7; each group). cdna was extracted from the tissues and real time rt-pcr was performed for sod-2, serum/glucocorticoidinduced protein kinase-1 (sgk-1) and the dna repair enzyme gadd45a. we found that labour onset is associated with a two fold increase in the levels of expression of each. oxidative damage at the fetomaternal interphase has been extensively studied in the placenta as part of investigations for first trimester pregnancy losses, iugr and pre-eclampsia. the role of ros is less well defined in human decidua and the underlying myometrium. our results suggest a role for oxidative stress and redox homeostasis in the maintenance of myometrial quiescene during pregnancy and onset of labour. plasma anandamide levels increase during labour induction and appear to delay labour progression. vijianitha nallendran, anthony h taylor, patricia mw lam, stephen c bell, david j taylor, justin c konje. cancer studies and molecular medicine, university of leicester, leicester, united kingdom. background: the evidence for a role of the endocannabinoid, anandamide (aea) in labour is conflicting. we previously showed elevated aea plasma levels in labouring women 1 whilst another group showed that activation of functional receptors for anandamide actually inhibited uterotonin-induced contractions through an adenylate cyclase pathway 2 . the aim of this study was to explore further the relationship between labour and plasma aea levels. methods: plasma aea levels in 20 women undergoing induction of labour for various indications at term, were measured by a sensitive isotope dilution method using hplc-ms/ms. each volunteer had an assessment of her cervix prior to the start of the induction when the first sample for aea was collected. once active labour was established (cervix 3cm dilated; contractions every 3-4 minutes), a second sample was collected. results: seventeen (85%) of the subjects were multigravida. the inductions were for postdates(n=7); decreased fetal movements(n=3), fetal growth restriction(n=2), symphysis pubis dysfunction(n=2), diabetes mellitus(n=1), pre-eclampsia(n=1), fetal cardiac complication(n=1), spontaneous rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes(n=1). plasma aea levels increased significantly once labour was established (figure) and demonstrated in 19 (95%) of the 20 cases. the median (interquartile range) plasma aea level increased from 1.35nm (0.99-1.61) at induction to 2.24nm (1.49-2.82); *p=0.01; mann-whitney u-test) at active labour. there was a positive correlation between plasma anandamide levels taken before induction and the time taken for the women to enter into active labour (r 2 =0.41, p=0.06 pearson correlation). plasma aea levels were higher in labouring women compared to non-labouring women after the induction of labour confirming our previous observations and suggesting a direct role for this endocannabinoid in labour. further studies are required to elucidate this role. nuclear factor-kappa b (nf-b) is a transcription factor family classically associated with inflammation, activated in response to infection and proinflammatory cytokines, such as those prevalent during labour. as a cytokineinducible transcription factor it plays a key role in the expression of a variety of genes involved in inflammatory responses and cell survival. nf-b dna binding and transcriptional activity plays an important role in labour associated gene expression in myometrium. in this study we examined the range of genes regulated by nf-b in myometrium using transient transfections and wholegenome array analysis. myometrial cells were extracted from myometrial biopsies taken at the time of elective caesarean sections at term. transient transfections of primary myocytes were performed using expression vectors for nf-b p65.the amount of dna was constant and psg5 was used as a control construct. cdna was made from myocytes transfected with either nf-b or psg5. affymetrix genechip u133 microarray was performed (n=3, each group). we found that 33 genes were significantly differentially expressed between control and overexpressed nf-b samples. twenty eight of these genes were upregulated with nf-b and 5 were down regulated. several chemokines and cytokines were identified in the upregulated group. interleukin 8 demonstrated the highest, 18 fold, induction in the upregulated group, followed by tumor necrosis factor, a-induced protein (tnfaip3), chemokine ligand 2 (ccl2), chemokine ligand 5 (ccl5), pentaxin related gene (ptx3), interleukin 6 (il6) and superoxide dismutase (sod-2). nine genes were present in the nf-b group that were absent in the control group. these included chemokine ligand 20 (ccl20), chemokine ligand 11 (ccl11), chemokine ligand 2 (cxcl2) and il1 . ingenuity pathway analysis demonstrated that immune response, inflammatory, cell growth and proliferation and cell death were the main pathways involved. standardisation experiments have been performed, and the microarray results were confirmed with real time rt-pcr in several candidate genes. our results provide further support in the role of nf-b in human labour and suggest its direct link in upregulation of inflammatory genes, cytokines and chemokines, consistent with the imflammatory nature of the biochemistry of labour. inflammation is widely accepted to be a key feature of human labor. secretory leukocyte protease inhibitor (slpi), an innate immune molecule, has been shown to be an antimicrobial and anti-inflammatory protein. the aims of this study were to verify its expression and localization in human myometrium methods: specimens were obtained at time of cesarean delivery with or without labor. expression and localization of slpi was detected using immunohistochemistry. slpi expression pattern relative to nf-b p65 subunit was compared between not in labor and in labor subjects, between different tissue sections as well as in in vitro model systems including myometrial explants, uterine smooth muscle cells (usmc) and ishikawa endometrial adenocarcinoma cells. slpi was predominantly localized to the nuclei of myocytes. the observed nuclear immunoreactivity of myocytes was increased during the labor relative to not in labor, paralleled with p65 nuclear translocation. the nuclear pattern of slpi is specific to myometrium since slpi immunostaining was present exclusively in the cytoplasm of all other tissues examined, including amnion, chorion, decidua and endometrium. slpi staining was also positive in macrophages, indicated by co-localization of slpi with cd68 positive cells. treatment with il-1 or tnf-induced nuclear translocation of p65 in myometrium explants and usmc, but not in ishikawa cells. in human myometrium, slpi is predominantly localized in the nuclei of myocytes and in macrophages. the nuclear expression pattern of slpi is myometrium-specific and increased following the onset of labor and correlated with nf-b activation. further understanding of its physiological significance may suggest new strategies aimed at preventing preterm birth. application of a new proteomic technology on amniotic fluid in prom. sara consonni, 1 niccolo bosso, 2 marianna andreani, 1 agnese pizzardi, 1 fulvio magni, 2 anna locatelli. 1 1 obstetrics and gynaecology, university of milano-bicocca, monza, italy; 2 experimental medicine, university of milano-bicocca, monza, italy. objective: mass spectrometry (ms) is the obligatory tool for proteomics studies. biological samples must be purified before ms analysis due to the matrix complexity. a recent approach combines active surface prepurification with maldi-tof (matrix assisted laser desorption) analysis. clinprot technology provides the prepurification of the sample through the use of magnetic beads (mb) with activated surface. this technique can be carried out by robot in an automated way on a large number of samples. a unique example of the use of mb before maldi-tof analysis to determine proteomic profiles of amniotic fluid (af) is reported for rapid detection of fetal aneuploidies (wang 2005) . the objective of our study was to verify the applicability of clinprot prepurification before maldi-tof analysis on amniotic fluid collected noninvasively in premature rupture of membranes (prom). methods: we sampled af from vaginal posterior fornix of women with preterm prom (group 1, n=10) and term prom (group 2, n=10). samples were prepared with mb and analyzed with ms maldi-tof in order to generate proteomic profiles. results: it was possible to generate average proteomic profiles in the two study groups and the observed profiles were different. samples of af non invasively collected in prom can be analyzed by ms maldi-tof after preparation with mb. this technique allows to retain part of the eluted sample for characterization of protein peaks of interest. due to the less laborious characteristics of this method in comparison with techniques based on bidimensional electrophoresis its application can be useful in clinical proteomics. progestins accentuate the maternal but not fetal inflammatory response of women with intra-amniotic inflammation. sonya abdel-razeq, irina a buhimschi, michael cackovic, guoyang luo, antonette dulay, victor rosenberg, mert bahtiyar, errol norwitz, edmund funai, catalin buhimschi. ob./gyn. reprod.sci., yale university, new haven, ct, usa. introduction: data from animal research suggests that progestins have a marked pro-inflammatory capacity. recent studies support the administration of 17 -hydroxyprogesterone caproate in women at risk for preterm birth. we sought to determine the impact of progestins during gestation on the extent of maternal and fetal inflammatory responses in pregnant women with intraamniotic inflammation. methods: amniotic fluid, placenta and cord blood were obtained from 52 women who delivered preterm (median[range], ga: 27 [18-33] wks). an amniocentesis was done to rule out infection. women exposed to progestins (n=17) within one week prior to amniocentesis were matched to 35 controls (crl) by age, parity, history of preterm birth ga, membrane status and interval to delivery. proteomic profiling of amniotic fluid [mass restricted (mr) score] identified the presence or absence of intra-amniotic inflammation. an mr score of 3 or 4 confirmed intra-amniotic inflammation. amniotic fluid and umbilical cord interleukin-6 (il-6) levels were measured by elisa. histological chorioamnionitis was graded based on recognized criteria. results: overall, women and their fetuses exposed to progestin did not exhibit an increased inflammatory response (table) . however, sub-analysis restricted to women with mr 3 or 4 (n=18) showed that in the context of intraamniotic inflammation, progestins were associated with significantly elevated amniotic fluid il-6 levels compared to unexposed women (progestins (n=6): 77 vs. crl (n=12): 12 [0.3-94] ng/ml, p=0.027). these relationships were maintained after correction for steroid and antibiotic exposure. such significance was not found for amniotic fluid glucose, ldh, wbc count or cord blood il-6. conclusion: our results suggest that progestins may amplify the maternal, but not the fetal inflammatory response of women with intraamniotic inflammation. objective: recently progestins have been shown to reduce the incidence of recurrent preterm labor in women. progestins have long been known to inhibit preterm labor in some species including rats and are also known to delay term labor. nitric oxide (no) donors, including ng, inhibit uterine contractility and have been used as tocolytics. the aim of this study was to examine the inhibitory effects of r5020 on preterm labor in rats induced with a progesterone antagonist (onapristone, zk) with and without ng. materials and methods: charles river s-d timed pregnant rats (n=6/group) were treated with zk (3 mg/rat, s.c.) alone or vehicle (controls) on day 17 of gestation. other groups of rats were treated with zk in combination with various doses of r5020 (0.3, 1 or 2 mg/rat s.c) with and without ng (2 mg s.c. pellet) from days 18 to 19 of gestation. all rats were sacrificed on day 20 of gestation and the number of fetuses and implantation sites were counted to determine the preterm delivery rate. one way anova was used for statistical analysis. p<0.05 was considered significantly different. results: rats treated with zk alone delivered all their fetuses prematurely compared with controls (p<0.05) treated with vehicle only (ca. 3% fetuses delivered). ng treatment alone did not affect the delivery rate (p>0.05) compared to controls. similarly zk + ng did not reduce the preterm delivery rate compared to zk alone (p>0.05). however r5020 dose dependently reduced (p<0.05 at all doses) the number of fetuses delivered prematurely in response to zk and the premature delivery rate was further reduced when treatment included the combination of r5020 plus ng (p<0.05). conclusions: zk effectively induces premature delivery. premature delivery produced by zk can be effectively reduced with a r5020, a progestin known to bind with high affinity to nuclear progesterone receptors. ng by itself, at the dosage used, does not reduce the prematurity rate caused by zk. however, r5020 and ng act synergistically to reduce the preterm delivery rate. this study indicates that combinations of a progestin with an no donor may be an effective treatment for preterm labor and delivery. monkeys. pl grigsby, 1 jp rasanen, 1,2 dw sadowsky, 1 m bertolino, 3 m carbonatto, 3 e gillio tos, 3 s canali, 3 j lacy, 4 a chollet, 4 mj novy. 1 1 reprod sci, oregon primate res ctr, beaverton, or; 2 ob/gyn, oregon health sci univ, or, usa; 3 rbm, merck serono, italy; 4 merck serono, switzerland. objective: to investigate the pharmacokinetics (pk) and pharmacodynamics (pd) of as606521, a novel orally active non-peptide oxytocin (ot) antagonist in rhesus monkeys. study design: dose finding and pk/pd studies were done in chronically instrumented non-pregnant (n=3) and pregnant (120-150 dga, term 165d; n=5) monkeys. maternal and amniotic fluid compartments were serially sampled during dosing and washout. treatment phases included: ot infusion control, ot infusion + as606521, and ot rescue therapy. ot infusions (2-64 mu/kg/hr, iv) were given to determine the lowest dose required to produce stable, sub-maximal uterine contractions in each animal. as606521 was administered p.o. at 5, 10, 20mg/kg and the effects on inhibition of uterine activity (hca, mmhg.sec/hr) were compared. to verify antagonist reversibility, objectives: infection such as chorioamnionitis is thought to be the cause of premature rupture of the membrane and induce uterine contractions leading to preterm delivery. however, the mechanism for enhancement of uterine contractility is not well understood. we have reported that non-selective cation channels (nsccs) regulate pacemaker potentials to generate rhythmical contractions and should be targets of magnesium ions used for tocolysis. the purpose of this study is to investigate the changes of the expression of nsccs during normal pregnancy and the effect of inflammation in preterm. methods; atp receptors (p2x) and transient receptor potential canonical (trpc) channels were examined as uterine nsccs. the mrna was extracted from the rat myometrium of non-pregnant and pregnant rats at days 15, 18, 20 and 22. the expression of each subtype of p2x and trpc channels was measured by real time rt-pcr (abi7700) with taqman probes (abi). as an inflammatory model lipopolysaccharide (lps; 0.2 mg/kg) was injected into intraperitoneal cavity at day 18 and the tissue was sampled after six hours. results; p2x4 and p2x7 were determined to be dominant subtypes of p2x channel. the expression of p2x4 was increased by 70% at day 22, compared with day 15 and that of p2x7 was enhanced by 90%. on the other hands, trpc3 and trpc4 were detected dominantly. the expression of trpc3 was increased three times in the late stages of gestation. however, trpc4 was suppressed by 57%. in the lps treated rat myometrium cox-2 mrna expression was measured to be 52 fold higher than that of the control rat, showing inflammatory effects in the myometrium. in this model the expressions of p2x4, p2x7 and trpc3 were enhanced by 7.4, 18.6 and 24.7 times, but trpc4 was not changed. the mrna expression of p2x4, p2x7 and trpc3 channels in rat myometrium was increased in the late stages of pregnancy. these channels are suggested to be concerned with onset of labor. in the inflammatory model the expression of these channels was accelerated dramatically and these values were much higher than those in normal pregnancy. this finding supposed inflammation may enhance some types of nsccs to accelerate uterine contractility and induce preterm delivery. anandamide ( to determine the mechanism of rho protein activation during oxytocin and carbachol induced contraction, freshly prepared myometrial strips in krebs henseleit buffer were treated with oxytocin (10 nm) and carbachol (100 μm) under isometric and tension free conditions. control strips were exposed to buffer only. treated myometrial strips were solubilised and separated into membrane and cytosolic extracts and equal aliquots were immunoblotted with rhoa and rhob antibodies. rhoa translocated to the membrane after oxytocin and carbachol stimulation under both isometric and tension free conditions (p<0.05). there were no significant changes in rhob membrane to cytosol ratios relative to control. agonist induced contraction in human myometrium is associated with rhoa but not rhob membrane translocation. during pregnancy components of the intracellular camp signalling pathway show increased gene expression resulting in the maintenance of myometrial quiescence until term where a substantial decrease in expression of these genes is observed. protein kinase a regulatory subunit riia (rii ) is upregulated at both the mrna and protein levels in the human myometrium during pregnancy. this particular subunit is membrane-bound and by directing phosphorylation to myometrial cytoskeletal proteins may affect contractile machinery thus playing a role in maintaining uterine relaxation. acetylation of histones promotes a favourable chromatin environment for transcriptional activity of many genes. this process is largely inhibited by histone deacetylases (hdacs), whereby its activity leads to transcriptional repression. hdacs can be recruited to the promoter region of a gene by other transcription factors such as sp proteins. since the rii promoter contains three sp1-4 consensus binding sequences in its proximal part, we investigated whether this gene is a target for transcriptional regulation by hdacs. using dna precipitation assays we found that sp1, 3 and 4 as well as hdac1 and 2 form complexes with biotin-labelled fragments relating to sp1-4 elements in the promoter region of rii . additionally, treatment of myometrial primary cell cultures with hdac inhibitor trichostatin a (tsa), or with the methyltransferase inhibitor 5-azac resulted in increased mrna and proteins levels. further studies with full and truncated luciferase constructs of the promoter region of the rii gene in transiently transfected myometrial cells confirmed that all three sp1-4 elements are involved in the transcriptional regulation of the gene. this process involves hdacs, as 6h treatment with tsa significantly increased the luciferase signal. changes in the binding of sp proteins and hdacs to the promoter after tsa and azac treatment were investigated employing chromatin immunoprecipitation assays. alterations in the methylation status of the promoter after treatment were examined by bisulfite modification and dna sequencing. together, this study highlights the importance of chromatin modifications in the maintenance of uterine quiescence during pregnancy as well as identifying a potential mechanistic target for drugs that may reduce the incidence of preterm labour. objective: progesterone maintains pregnancy by promoting myometrial quiescence. typically progesterone effects are thought to be mediated through the classic genomic pathway. there is evidence, however, that progesterone also acts via a non-genomic pathway by interacting with specific membrane progesterone receptors (mprs) and in particular progesterone receptor membrane component -1 (pgrmc-1). the role of non-genomic progesterone actions in human pregnancy and parturition is not clearly understood. the goal of this study was to measure the extent of mpr expression in biopsy specimens of human myometrium obtained at cesarean delivery, and to determine whether expression changes with advancing gestation or the onset of labor. study design: lower uterine segment myometrial biopsies were obtained at the time of delivery from consenting women who were at term and not in labor (n=4), preterm and not in labor (n=7) and term and in labor (n=5). protein extracts were prepared and subjected to polyacrylamide gel electrophoresis and immunoblot analyses for pgrmc-1 and gapdh. abundance of pgrmc-1 protein relative to gapdh was determined by digital densitometry. we also performed immunohistochemistry (ihc) to determine the cellular localization of pgrmc-1 in the human pregnancy myometrium. results: pgrmc-1 protein was identified in each biopsy specimen. there was a 2-fold increase in pgrmc-1 protein in term compared with preterm biopsies (relative to gapdh p<0.04). the relative level of pgrmc-1 protein was not different between biopsy specimens from laboring and non-laboring women at term (compared to gapdh, p=0.8). pgrmc-1 immunoreactivity was localized to granular cytoplasmic staining. conclusion: this is the first description of the presence of pgrmc-1 protein in the human myometrium during pregnancy. its presence suggests that progesterone may influence contractility non-genomically via these receptors. the functional significance of the gestational age associated two fold increase in pgrmc-1 is unclear. changes in expression of this receptor during pregnancy may be important for the hormonal control of parturition and can be the focus of future studies. (ruddock et. al., abstract smfm, 2008) . the aim was to examine the mechanism of p4 inhibition. methods: uterine tissues from women (n=55) at term with cesarean section, were suspended in organ chambers and exposed to various agents or solvents. contractility was registered, stored, analyzed and compared before and after addition of agents or kcl. tissues were treated with p4 alone (10 -10 to 10 -3 m) or p4 bound to bovine serum albumin (bsa/p4, 10 -6 to 10 -3 m p4), a progestin with low affinity to mpr (r5020, 10 -7 -10 -3 m), or a non-sex steroid (cholesterol, 10 -5 to 10 -2 m). other tissues were pretreated with selective inhibitors of adenylate cyclase (sq 22536, 10 -5 m), guanylate cylase (odq, 10 -5 m), phosphodiesterase (rolipram, 10 -5 m), nitric oxide (no) synthases (l-name, 10 -4 m) or a nuclear p4 receptor antagonist (mifepristone, mif, 10 -5 m), followed by p4. data were analyzed by anova for statistical differences (p<0.05). results: p4 rapidly (<1 hour), effectively (100%) and dose-dependently inhibits spontaneous and kclinduced contractility (ed 50 of <10 -5 m incubation of strips with zd-6416, at concentrations up to 1 m for one hour prior to the experiment, led to no significant reduction in the total work done. however, incubation of strips with l-798106 with a concentration of 100nm for one hour prior to the experiment, led to a statistically significant reduction in the total work done after one and a half hours. furthermore, when increasing concentrations of pge 2 (10 -10 to 10 -6 ) were added to l-798106 (100nm) pre-treated strips, total work done per contraction was comparable to that of non-treated controls. similar effects were not observed in zd-6416 (100nm) pre-treated strips. since the reduction in contractility caused by l-798106 was greater than that caused by zd-6416, it is likely that the stimulating effect of pge 2 acts predominantly via ep3 receptors. taken together with our previous data showing an increase in ep3 at labour, this data shows that targetting an ep3 receptor may be a useful strategy in managaing pre-term labour. recently a truncated 46kda isoform of the er-has been described in human endothelial and testicular cells. we describe the presence of this 46kda erisoform in pregnant and immortalized non-pregnant human myometrial cells. methods: myometrial tissue obtained from non-laboring pregnant women undergoing cesarean section at term (n=4) was dissected prior to being finely minced. a portion of the tissue was placed in pbs and sonicated, while the remainder of the tissue was dissociated with collagenase prior to filtration and placement on culture plates. cells were then cultured in mem w/10% fetal bovine serum (fbs) until confluence. cultured cells were then dissociated with 0.05% typsin/edta, subsequently re-plated and grown to confluence. immunohistochemistry directed toward smooth muscle protein was used to verify myometrial phenotype.) protein was extracted and quantified. western blot was performed with mouse monoclonal anti-er-receptor antibody (santa cruz biotechnology) to the f-10 domain of the human er-receptor. an immortalized non-pregnant human myometrial cell line provided by ann word, htert, was cultured and isolated as described above. results: htert cells expressed both the truncated (46kda) and the full length (66kda) er-isoforms. fresh and cultured myometrial cells from non-labored term pregnant patients also expressed both isoforms of er-. subsequent subcultures of myometrial tissue continued to express the 46kda erisoform, yet the expression of the 66kda isoform was lost. a representative blot is below. conclusions: we demonstrate, for the first time, the presence of a 46kda erisoform in cultured and non-cultured pregnant and cultured nonpregnant human myometrial tissue. discovery of this isoform of er-in myometrial tissue could provide insight into the molecular mechanisms involved in parturition. the action of prostaglandin f 2 (pgf 2 ), a potent uterotonic stimulant that is associated with labour at term and preterm, is mediated by its receptor, ptgfr. myometrial ptgfr mrna levels fall during pregnancy and this likely plays a role in uterine quiescence. however, the mechanisms by which this occurs are poorly understood. we previously reported that pgf 2 downregulates fp mrna expression in cultured human myometrial ultr cells in a protein kinase c (pkc) dependent manner. in addition to the downregulation of mrna levels, receptor desensitization may also represent another mechanism of decreasing ptgfr activity because ligand binding to g protein coupled receptors often results in receptor internalization. we therefore hypothesized that pgf 2 treatment of ultr cells also results in pkc dependent ptgfr internalization. methods: near confluent cultured human myometrial ultr cells were treated +/-10 -7 m or 10 -6 m pgf 2 for 1, 3, 6 or 24 hr. cells were fixed with formaldehyde and visualized for localization of ptgfr by immunofluorescence. to examine the potential involvement of pkc in the process, ultr cells were treated +/-10 -6 m pgf 2 and +/-10 m myristoylated pkc inhibitor (20-28) and examined for ptgfr cellular localization as described above. results: pgf 2 treatment resulted in a dose dependent decrease in ptgfr membrane signal at 1, 3, 6 and 24 h. this decrease was dependent on pkc as cotreatment with the myristoylated pkc inhibitor (20-28) prevented the pgf 2 induced decrease in membrane ptgfr at 1 h treatment. there was no visible effect of the pkc inhibitor on ptgfr membrane signal on its own. conclusion: we conclude that pgf 2 decreases membrane levels of ptgfr protein in human myometrial ultr cells in a pkc dependent manner. these results suggest that pkc may be required for both the pgf 2 induced internalization and desensitization of ptgfr protein and downregulation of ptgfr mrna in human myometrial ultr cells. therefore pkc may play a crucial role in downregulating ptgfr expression and activity and maintaining uterine quiescence during pregnancy. rhoa is a small gtpase that acts as a molecular switch to control a variety of signalling pathways in smooth muscle, including contractility. it is thought that increases in rhoa-gtp levels facilitates phosphorylation of target proteins such as cpi-17, promoting contractility at pre-term and term labour in humans. however, in situations of acute or chronic hypoxia in the uterus, it is important that myometrial contractility and subsequent labour is not facilitated prematurely. given the importance of rhoa to a cell's response to hypoxia in other cell types, it was hypothesised that rhoa plays a central role in the mechanism controlling smooth muscle contraction in the uterus too. following acute hypoxia (0.5% o2) for one to six hours, rhoa mrna, total protein and activation (rhoa-gtp) levels were analysed, using semi-quantitative pcrs and western blot, and compared to normoxic non-pregnant human uterine smooth muscle control cells. next, we investigated whether reduced oxygen conditions affected oxytocin induced activation of rhoa, following a two hour treatment of 10 nm oxytocin. firstly, our results demonstrate that the rhoa itself is significantly activated under low oxygen conditions, resulting in phosphorylation of myosin phosphatase, myosin light chain and cofilin, three proteins known to be central in contraction and actin filament organisation. secondly, hypoxia significantly reduced the coupling of oxytocin to rhoa activation under the conditions examined. we observed a significantly reduced level of rhoa expression and activation which correlated with an increase in the level of another rhogtpase protein, rhoe. we propose that rhoa inactivation occurs through a rhoe-mediated mechanism, suggesting a balance in the activity of these two antagonistic rhogtpases in oxytocin-induced hypoxic human uterine smooth muscle cells. these results provide a possible explanation for the reduced coupling of oxytocin as a stimulant of myometrial contractions during slowly progressing labours. oxytocin-induced release of calcium ions (ca 2+ ) from sarcoplasmic reticulum (sr) and sensitisation of contractile proteins to ca 2+ have been suggested to mediate the oxytocin-induced potentiation of myometrial contractions. objective: we investigated the effects of oxytocin in the presence of nifedipine, a known inhibitor of the l-type calcium channel (ltcc). method: samples of myometrium were obtained from women undergoing term caesarean section with the approval of the local ethics committee. a standard organ bath system (ad instruments, uk) was employed to analyse contractile activity. stable spontaneous contractions were recorded for 40-60 minutes before addition of nifedipine. results: in agreement with our previous findings, application of oxytocin to spontaneously active strips produced a two-component effect: a transient tetanus-like contraction, followed by prolonged augmentation of phasic contractions. nifedipine (1um) rapidly abolishes spontaneous contractions, subsequent addition of 100nm oxytocin produced an initial, transient rise in force, approxiamately 20% compared to oxytocin alone, followed by high frequency oscillations in >50% of strips. calcium-free solutions were used to confirm that oscillations were due to ca 2+ entry. disabling the sr store using thapsigargin (1um) had no effect on oscillations, confirming the sr not to be involved. the t-type calcium channel blocker, mibefradil (1um ) showed no inhibition of oscillations. an ip 3 receptor and store-operated calcium channel inhibitor, 2-aminoethyldiphenylborate (2-apb) 50um also had no effect on oxytocin-induced oscillations. the store-operated calcium channel inhibitor skf-96365 (10um) showed partial inhibition of oscillations. conclusions: based on these results, we propose that the most likely mechanism of ca 2+ entry producing oxytocin-induced oscillations in the presence of nifedipine is the transient receptor channel-c (trpc) channel, known to be present in the human myometrium. further work needs to be completed to clarify this further. identification of stim and orai in human myometrium. a new paradigm in store-operated calcium signaling. evonne c chin-smith, 1 mark r johnson, 2 rachel m tribe. 1 1 division of reproduction and endocrinology, king's college london, london, united kingdom; 2 department of maternal fetal medicine, imperial college school of medicine, chelsea and wesminster hospital, london, united kingdom. background: recent reports have suggested that two novel proteins, stim and orai, are involved in the regulation of store-operated calcium entry. we have previously reported that members of the trpc family, putative basal and store operated calcium entry channels, are present in human myometrium and regulated by labour associated stimuli il-1beta and mechanical stretch. although stim and orai isoforms have been reported in other smooth muscle cell types, there are no published reports of stim and orai expression in human myometrium. the aim of this study was to identify mrna expression of stim1, stim2, orai1 and orai2 in human myometrium. methods: human myometrial biopsies were obtained from women undergoing elective caesarean section at term (prior to labour) with informed written consent and institutional ethics committee approval. whole myometrial tissue was either snap frozen and stored at -80 o c or used for cell culture. rna was extracted from whole tissue (n=5), primary cultured myometrial cells (n=4) and passaged (p2) myometrial cells (n=5) and stim1, stim2, orai1 and orai2 mrna expression was assesed by quantitative real-time pcr. results: all four genes were expressed in whole myometrial tissue and cells. stim1 and stim2 mrna expression in cultured myometrial smooth muscle cells (primary and passaged) was significantly reduced compared to myometrial tissue expression (p<0.05). however, there was no significant difference in either orai1 or orai2 expression in whole tissue versus cultured myometrial smooth muscle cells. conclusion: to our knowledge this is the first report of stim1/2 and orai1/2 mrna expression in human myometrium. these genes may contribute to the regulation of calcium signalling in human myometrium, but the functional significance of their expression remains to be determined. funded by the bbsrc. obstetrics and gynecology, national university of ireland, galway, galway, ireland. objective: the ability of uterine smooth muscle cells to stimulate collagen contraction has been well established as an in vitro model of myometrial contractility. devost and zingg (2007) reported that the contractility of human myometrial cell lines layered onto collagen matrices was increased by oxytocin while another group described the stimulation of human uterine smooth muscle cell contractility, cultured within collagen lattices, with endothelin-1 (dallot et al., 2003) . our study investigated the response of human primary uterine smooth muscle cells cultured within collagen lattices, to various compounds, including the non-specific depolarizing agent potassium chloride (kcl), the inflammatory cytokine tnf , the rock-1 inhibitor y-27632, oxytocin, and oxytocin plus its clinically used antagonist, atosiban. methods: human primary uterine smooth muscle cells (utsmc) (lonza) were maintained in dmem high glucose media. cells (150,000 per well) passage 3-8, were embedded in 1.5 mg/ml collagen, in 0.5 ml aliquots, in dmem-f12 media on 24 well plates (invitrogen) (dallot et al., 2003) . effects on contraction were studied by monitoring changes in gel area (alpha innotech imager, image j software (nih)). statistical significance was determined by the student t test. results: the utsmcs displayed basal contraction of the collagen gels while the non-contractile cell line hek293 cells, did not. kcl (20nm) stimulated an 11% increase in contractility (n=3, p=0.0262) while 10 nm tnf resulted in an 8.4% increase (n=3, p=0.0205), in comparison to unstimulated cells embedded in gel. the rock 1 inhibitor y-27632 (10 m) inhibited contractility, with a 17.5% decrease in collagen gel contractility (n=3, p=0.011). a 13% increase (n=4, p= 0.0146) in utsmc embedded collagen contractility was observed with 10 nm oxytocin, which was antagonized by atosiban (1 m) (n=4). conclusion: this study highlights the importance of the development and optimisation of a reproducible human in vitro myometrial contractility model, to evaluate the effect of various known labor-associated, and also unknown compounds. this should aid in our understanding of the many complex biochemical pathways involved in myometrial contractility at labor, and ultimately contribute to the prevention of preterm labor. changes in intra-mural myometrial blood flow during spontaneous labour using 3d power doppler angiography (pda). nw jones, 1,2 nj raine-fenning, 2 h mousa, 1 mj taggart, 3 k jayaprakasan, 2 gj bugg. 1 1 nottingham university hospitals nhs trust, united kingdom; 2 nottingham university, united kingdom; 3 university of newcastle upon tyne, united kingdom. methods 3d pda was used to measure the percentage (%) change in the vascularization index (vi), the flow index (fi) and the vascularization flow index (vfi) 1 in a volume of myometrium at the uterine fundus of 20 nulliparous women at term, in the first stage of uncomplicated spontaneous labour. 3d data sets were obtained during a single cycle of uterine relaxation (r1), contraction and subsequent relaxation (r2). measurements were made independantly by two authors (nwj and gjb) using vocal® (ge kretz) and the mean value was used for analysis. the results from each woman are presented as a % change of r1. data is presented as medians [interquartile range (iqr)] and analysed using non-parametric tests. the median volume (cm 3 ) of interest for r1 was 17.9cm 3 (12.6-24.4cm 3 ), for the contraction was 17. (fig.1) . the % change for all three indices between r2 and r1 was not significantly different. however, there was a significant difference between the contraction and r2 (p< 0.01). the mean intra-class correlation coefficient and 95% confidence interval (ci) of vi, fi and vfi for the authors (nwj and gjb) were 0.99 (0.98-0.99), 0.99 (0.98-0.99) and 0.98 (0.96-0.98), indicative of good inter-observer reliability. conclusion 3d pda is a useful and reliable tool in the assessment of changes in intramural myometrial blood flow, which was found to reduce significantly during a contraction but increase again during the following uterine relaxation. 1. raine-fenning nj, et al. the inter-observer reliability of 3d pda acquisition within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. 196 the role of pgf2 on myometrial contractility; studied with a selective fp antagonist. shankari arulkumaran, 1 andre chollet, 2 phillip r bennet. 1 1 imperial college parturition research group, institute of reproductive and developmental biology, hammersmith hospital campus, london, united kingdom; 2 merck serono, geneva, switzerland. objectives: human myometrial strips established in culture will usually begin contracting after one hour. we have previously shown that stretch upregulates prostaglandin synthesis and have therefore hypothesized that spontaneous contractions occur because of stretch-related prostaglandin synthesis. methods: experiments were performed using 5x2mm human pre-labour, lower uterine segment myometrial strips in a dmt myograph 800ms in oxygenated kreb's solution, with adi powerlab software. spontaneous contractions required stretch force and initial experiments determined that the maximum number of strips attaining spontaneous contractions was greatest at a force of 5-6g. a novel antagonist to pgf2 (fpa) was studied in this model. the fpa has a ki of 6nm for fp and is 10-100 fold selective for fp compared with other prostanoid receptors. results: addition of pge 2 and pgf2 after the commencement of spontaneous contractions caused a statistically significant increase in the total work done by the strips. the effect of pge 2 was being greater than that of pgf2 . pre-incubation of the baths with a novel and selective fp antagonist (fpa) with concentrations up to 1 m (10 -6 ) did not affect the total work done by spontaneous contractions compared to non-treated controls. however, increasing concentrations of the fpa (10 -8 to 10 -5 ) decreased the total work done by 5-fold on strips treated with pgf2 beforehand in comparison the pgf2 -treated strips alone. conclusion: these data suggest that stretch and synthesis of prostaglandins is essential for spontaneous contractility in human myometrial strips. since fpa is able to block pgf2 induced but not spontaneous contractions, it is likely that pgf2 does not play a role in spontaneous myometrial contractility in vitro, although it may do so in vivo. because other factors may combine to increase contractility, the combination of an fp antagonist with other inhibitors may therefore, be a more effective strategy in reducing pre-term deliveries. objectives: brain natriuretic peptide (bnp) is synthesized in fetal membranes and inhibits oxytocin-induced contraction of preterm human myometrium. we hypothesized that bnp may be a paracrine mediator of human myometrial quiescence. we showed bnp content is higher in membranes from preterm pregnancies absent labor and significantly decreased with idiopathic preterm labor. while bnp activates natriuretic peptides receptors a (npr-a), b (npr-b), and its clearance receptor (npr-c), we have shown bnp does not inhibit myometrial contraction via npr-a or npr-b. herein, we test in part the hypothesis that bnp inhibits myometrial contractions by activating npr-c by quantitating npr-c in human myometrium at different gestations and labor status. methods: myometrial samples were obtained at the time of cesarean section after informed consent from 4 groups of patients: preterm not in labor (pt-nl), preterm in labor (pt-l), term not in labor (t-nl) and term in labor (t-l). myometrial samples were obtained from women 30-34 weeks' gestation, and term between 38 and 41 weeks. mrna for npr-a, b and c were semiquantitated by realtime pcr (normalized by 18s mrna), and npr-c protein by western blot. results: natriuretic peptide receptors a, b and c mrnas were identified in all 4 groups. while there were no differences in mrna levels among groups, npr-c protein was increased in samples from term laboring women. conclusion: since bnp inhibits the contraction of human preterm but not term myometrium independent of npr-a and npr-b, we have previously speculated that bnp activates another "unknown" receptor. herein we find that myometrial npr-c protein but not mrna increases during human labor at term. the increase in npr-c at term could compete with the unknown quiescent receptor to functionally reduce the availability of bnp and thus permit or promote myometrial activation/contraction. (supported by a grant from chilean government fondecyt 1020675). objective: bnp is synthesized within human chorion and amnion and inhibits oxytocin-induced contraction of human myometrium. bnp activates guanylate cyclase (gc) natriuretic peptides receptors a (npr-a) and b (npr-b). bnp also stimulates the clearance receptor (npr-c) whose action is not mediated by gc. the intracellular pathway of bnp/npr-c inhibition is not known. we determined that bnp does not inhibit myometrial contraction via npr-a or -b. we hypothesized that bnp inhibits myometrium by npr-c activation. we test aspects of our hypothesis by determining whether bnp/npr-c pathway inhibits myometrial contractions via myometrial nos pathway. methods: bnp and canp (specific npr-c agonist) were added to primary human myometrial cell cultures and enos/inos activity/expression determined. cell cultures (n=6) were prepared from myometrial samples of nonlaboring, term pregnant women. after confluence (10d), the cells were incubated (6h) with 100nm bnp and/or canp. nos activity was measured ( l-citruline assay) and transcription/translation semi quantitated (realtime pcr and western blotting). results: bnp had no effect on either nos expression or activity. canp significantly reduced inos but not enos mrna level. canp did not alter the protein level of nos but significantly reduced nos activity. co-incubation with bnp and canp reduced both mrna levels (p< 0.05) and significantly decreased enos, but not inos, protein without change in overall nos activity. within the female pelvis. ultrasound obstet gynecol. 2004; 23:501-8. conclusion: the activation of npr-c by canp reduces nos activity and expression. the same effect was not observed by bnp. the difference may be explained because bnp (but not canp) activates all natriuretic peptide receptors, and they may have opposite actions on nos pathway. we conclude unlikely bnp inhibits human myometrial contraction by npr-c activation via the nos pathway. ( introduction: ultr is a retroviral immortalized human uterine smooth muscle cell line which we use as a model for uterine hypertrophy studies. however, this cell line has limited usage due to a reduced rate of cell division and eventually replicative senescence in culture. one of the mechanisms of human somatic cellular senescence is un-compensated shortening of telomeres, the specialized dna structures located at the ends of eukaryotic chromosomes. introduction of human telomere reverse transcriptase (htert) has been shown to induce telomerase activity and telomere elongation, and extend life-span of normal human cells. objective: to recover ultr cell division without altering the phenotypic characteristics of smooth muscle cells by introducing htert, therefore improving ultr cell line. methods: ultr cells were transfected with a modified htert expression vector at a relatively early stage (passage 28) in the presence of selective antibiotic. ultr cell growth rate, with (ultr-ht) or without transfection, was determined by plating cells in multiple plates at a fixed density and counting cell numbers after 7 days. single cell clones of stably transfected cells were further isolated by plating in 96 well plates. expression of htert, smooth muscle specific genes (smc-sgs), and target genes was identified by rt-pcr of total rna extracted from ultr and ultr-ht cells. results: rt-pcr demonstrated successful introduction of htert into ultr cells. the growth rate of ultr-ht cells was increased and cell morphology was improved (free of the typical aneupoid appearance). at passage 37, ultr-ht cells grew 3.1 fold (p<0.0001) and 26.9 fold (p<0.0001) faster than ultr cells at passages 28 and 34, respectively. currently 8 single cell clones have been selected. ultr-ht cells express a set of smc-sgs, including -actin, caldesmon, calponin, myosin heavy chain, sm22, and smoothelin, confirming that the smooth muscle phenotype is preserved. a panel of genes involved in angiotensin ii signalling, including angiotensin ii receptors (at1/2), nox family (nox1, 4, 5 and duox1), nox-associated genes (p22phox, p47phox, p67phox, and rac1/2), was also preserved. conclusion: this is the first report of rescuing uterine smooth muscle cell replicative senescence via activation of telomerase. we have established a human uterine smooth muscle cell line which will provide an improved in vitro model for studying human myometrium. at term, myometrium is characterized by spontaneous contractions which vary in the amplitude, frequency and duration depending on specie and hormonal status. repetitive depolarization of plasma membrane followed by transient elevation in [ca 2+ ] i is thought to underlie the contractions. however, the detailed pathways which regulate the spontaneous contractility remain unclear. objective: this study was designed to elucidate the effect of protein kinase c (pkc) on the spontaneous contractions and [ca 2+ ] i transients in the myometria from term pregnant mice and women. methods: the human samples were obtained from women undergoing cesarean section with the approval from the institutional review board committee while mice myometria were dissected from the 18 days pregnant mice sacrificed according to the animal study protocol. the isometric force and [ca 2+ ] i were measured simultaneously in fura-2 loaded myometrial strips using spectrofluorometer equipped with force transducer. results: the pkc activator phorbol 12,13-dibutyrate (pdbu) applied at 10 -7 m first stimulated the amplitude of spontaneous contractions in mice and human myometria followed by their inhibition. under the pdbu treatment the frequency of the contractions was first increased and then decreased in both species. at the same time, pdbu didn't initially increase the amplitude of [ca 2+ ] i transients but attenuated it over time. however, the frequency of the transients was first increased and later decreased upon the pdbu exposure. in addition, pkc activation with pdbu resulted in the elevation of the uterine basal tone without corresponding changes in the basal level of [ca 2+ ] i in human myometrium. in mice myometrium, on the contrary, pkc activation didn't result in an increase of the muscle tone and the basal level of [ca 2+ ] i . conclusions: we propose that pkc causes bi-phasic effect on uterine spontaneous contraction in mice and human first to potentiate the amplitudes and the frequencies and later decreases them. the amplitude of [ca 2+ ] i was not initially potentiated by pdbu suggestive of the dissociation of the contractile and [ca 2+ ] i response. the stimulatory effect of pdbu on the basal muscle tone in human myometrium and the absence of the effect in mouse suggest different mode of pkc action on uterine contraction in human and mice. introduction: mechanical stretch of uterine myocytes is detected through integrins on the cell surface which form part of the focal adhesion complex, this signals through mapk, ultimately leading to the up-regulation of various pro-labour genes including pghs-2 and il-8. on integrin activation fak is recruited to the focal adhesion complex and activated by autophosphorylation at tyr-397, creating a src binding site. this promotes further fak phosphorylation at tyr-576, 577 and 925, enhancing fak catalytic activity. however fak can also act as a scaffold protein and its exact role in the expression of pro-labour genes is uncertain. in this study we used inhibitors for the kinase activity of fak and mek 1/2 in order to test the affect of fak kinase activity on expression on pghs-2 mrna. methods: primary human myometrial cell cultures were grown from myometrial biopsies taken from women undergoing elective caesarean section. cells were plated onto 6-well flexible bottom plates coated in type i collagen. cells were subjected to 11% static stretch for up to 60 minutes. cells were also incubated with either the rho kinase inhibitor y27632 or the mek 1/2 inhibitor u0126 prior to being stretched. western blots were performed using antibodies to fak phospho-397, fak phospho-925 and erk 1/2 phospho-202/204, -actin was used as a loading control. rna was also extracted and levels of pghs-2 measured using q-pcr. results: stretch increased levels of phosphorylation at both tyr-397 and tyr-925 with the greatest increases occurring at 30 and 60 minutes. however the increase at tyr-397 appeared to be greater than at tyr-925. incubation with the rho kinase inhibitor reduced phosphorylation of fak-397, however this did not affect phosphorylation of erk 1/2 or stretch induced up regulation of pghs-2 mrna. in contrast incubation with the mek 1/2 inhibitor reduced erk 1/2 phosphorylation and expression of pghs-2 mrna, whilst also reducing fak phosphorylation (n=4; p=0.05). conclusions: fak has previously been shown to be important in activation of the stretch induced mapk cascade and pghs-2 expression. however these data suggest that while stretch causes fak phosphorylation fak kinase activity is not essential to pghs-2 expression. this suggests fak may be acting as a protein scaffold. . our aim was to examine the effects of both natural progesterone and 17 hp on spontaneous myometrial contractions. myometrial biopsies were taken with informed consent and ethics approval from non-labouring women at elective caesarean section 37 weeks gestation. strips of myometrium 15 mm long ,2 mm wide were cut and suspended under a resting tension of 20 mn in organ baths of krebs gassed with 95% o 2 / 5% co 2 within 12 hours of collection. progesterone or 17 hp were added in a cumulative manner at 20-minute intervals. changes in amplitude were recorded. results were compared using anova. following equilibration for 2 hours, myometrial strips contracted in a rhythmic manner (amplitude 91.6 ±16.1 mn, n=8 pairs). progesterone (10nm-30 m) produced a concentration dependent inhibitory effect on myometrial contractions (fig1), which was greater than that of vehicle (p<0.05). maximum inhibition measured 93.3 ± 2.0% and 67.2 ±14.2% for progesterone and vehicle, respectively. 17 hp exerted an inhibitory effect, this was not significantly different from the vehicle (p>0.05). progesterone exerts an inhibitory effect on myometrial contractility in vitro. this is apparent within minutes suggesting a nongenomic action. our data are in agreement with some reports in the literature but conflict with others[3]. this acute inhibition of myometrial contractility may contribute to the mechanism by which progesterone prevents preterm birth. in contrast, we were unable to demonstrate an inhibitory effect of 17 hp on contractility despite its demonstrated ability to reduce the incidence of preterm delivery [4] . our data suggest that natural progesterone may be a more effective tocolytic agent in the acute setting than 17 hp. obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: chemokines has been shown to play an important role in regulating uterine function. recent evidence demonstrates that monocyte chemotactic protein (mcp) 1 level in amniotic fluid increases during spontaneous labor. the aim of this study was to examine the expression of mcp 1 in human myometrium. methods: myometrial biopsies were taken from nonpregnant women undergoing hysterectomy and term pregnant women undergoing cesarean section followed written consent and local ethics committee approval. elective cesarean section was performed before the onset of labor while emegency section was done after the onset of labor. immunolocalization (n = 5 each) was performed on paraffin sections by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. reverse transcriptionpolymerase chain reaction (n = 10 each) using gene specific primer against mcp 1 and mcp 1 receptor was performed to identify mcp 1 messenger(m) rna in human myometrium . results: immunohistochemical findings demonstrated mcp 1 in human myometrial cells from nonpregnant, term pregnant women before and after the onset of labor. mcp 1 was labelled on plasma membrane and cytoplasm of myocytes from these three groups of women. similarly, mcp 1 and mcp 1 receptor mrna were found in nonpregnant and term pregnant women before and after the onset of labor. in this prospective study a group of 4075 fetuses was consecutively enrolled. one ultrasound examination was performed to each patient within 6 days from delivery. we considered fetal macrosomia a birthweight 4000g and big babies a birthweight 4500g. cut-off points for identifying the best value of fetal abdominal circumference for fetal macrosomia prediction were chosen by receiving operator characteristics' curve (roc) analysis. using the best cut-off indicated by roc analysis, specificity and sensitivity were calculated. mean gestational age at delivery was 38 +3 weeks (3 +3 sd). 1015 neonates weighted less than 2500 g, 2818 had a weight range between 2500 and 3999 g and 242 weighted more than 3999 g. the fetal ac measurement was the selected criterium to evaluate the risk of fetal macrosomia. to identify macrosomic fetuses the roc curve analysis identified a cut off of ac > 350 mm which allowed to select 867 fetuses. analysing this population we found 3183 true negative cases, 25 false negatives, 650 false positives and 217 true positives, with a sensitivity of 89.6% and a specificity of 83%. to detect big babies the roc curve analysis identified a cut off of ac > 361 mm (n.452 fetuses), reaching a sensitivity of 100% and a specificity of 90%, without false negative cases and with 413 false positives (39 true positives, 3623 true negatives). the 141 cases that weighted between 4000 and 4499 g, were included in the 413 cases considered false positives by this cut-off. conclusions. these results clearly indicated that ultrasounds alone can not be used to manage a pregnancy suspected for fetal macrosomia or big babies. in fact, to avoid shoulder dystocia and all other complications strictly related to macrosomic fetuses, clinicians should perform a large number of useless elective cesarean sections. . pathway analysis of differentially expressed genes (pa; z-score 2.0) showed up-regulation of axon guidance, folate biosynthesis, nitrogen metabolism and down-regulation of steroid biosynthesis, insulin signaling, oxidative phosphorylation, tgf-beta signaling, and ubiquinone biosynthesis pathways in cm vs cf. comparison of mnr vs c in f showed 320 genes up-and 354 down-regulated (n=3,3; p<0.05). pa showed up-regulation steroid biosynthesis, fatty acid metabolism, glycolysis/gluconeogenesis, phosphatidylinositol signaling, ketone body metabolism, and ubiquinone biosynthesis pathways and down-regulation of bile acid biosynthesis, cell adhesion molecules, dna polymerase, notch signaling and ti diabetes mellitus pathways in mnr vs c. comparison of mnr vs c in m showed 525 genes up-and 869 down-regulated (n=3,3; p<0.05). pa showed up-regulation of jak-stat signaling, autophagy, renin-angiotensin system (ras), and ubiquinone biosynthesis pathways and down-regulation of apoptosis, basal transcription, folate biosynthesis, nitrogen metabolism, protein export, and snare interaction pathways in mnr vs c. only the ubiquinone biosynthesis pathway up-regulation of mnr vs. c was common to both m and f. conclusions: ta and pa demonstrate sex-specific transcriptome expression in fetal kidneys at 0.9g. in addition, these results show sexspecificity in response to mnr. we have seen similar effects of mnr on gene expression for autophagy, apoptosis, ras, ubiquinone and cell adhesion pathways at 0.5g, suggesting these pathways may contribute to persistent affects of mnr. finally, we postulate that stress in utero may contribute to sex differences in risk of hypertension in adult life. leptin and neuropeptied y protein expression paradoxally increased in gestationall food restricted dams. louiza belkacemi, chun-hung chen, andrea jelks, michael g ross, mina desai. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: placental insufficiency is associated with marked increase in placental leptin production. this results in a rise in maternal leptin levels that serves as an early index of placental dysfunction. further increased placental leptin and suppressed neuropeptide y (npy) are associated with preeclampsia. leptin, an anorexigenic hormone and npy, an orexigenic peptide regulate food intake. importantly, leptin also serves as a placental and fetal growth factor. we have shown that maternal food restriction (mfr) results in intrauterine growth ... sf ()to:!)) df(,..)l) ...._ bf(jtolo) ... -m bf(,..lj) =-7(30.4) <(u.)) 14(48.1) )(6.7)"" 1'0~.1) !(11.7)' d.'cbom< 7(104) 2f(81 ))""" s(17l) background teenagers are more likely to deliver small-for-gestational age (sga) infants than adults, even after adjustment for socioeconomic factors 1,2 . previous studies of mostly black and hispanic subjects in the usa have suggested that maternal growth may contribute to reduced infant birthweight, due to preferential nutrient partitioning to the mother 3 . the impact of maternal growth on birthweight and nutrient partitioning in pregnant teenagers in the uk has not been examined. methods skeletal growth (change in knee-height from 1 st to 3 rd trimester), weight gain and skinfold thicknesses were measured in pregnant teenagers (n=369, 45% non-white) in london and manchester. key mediators of nutrient partitioning and metabolism: insulin-like growth factor(igf)-1, igf binding protein(bp)-1 and leptin, were measured in maternal plasma (28 weeks gestation). results maternal growth (defined as increase in knee-height >2mm/90days) was detected in 36% of pregnant teenagers. this growth was not associated with sga birth; in fact these mothers were more likely to deliver large-forgestational age (lga) infants (p<0.05). maternal weight gain and fat accrual at peripheral and central sites were greater in growers (p<0.01). these parameters correlated positively with maternal igf-1 and leptin but negatively with the igf inhibitor, igfbp-1 (p<0.001 for all). subjects delivering sga infants gained significantly less weight (p<0.01) and had lower igf-1 levels (p<0.01) than those delivering non-sga infants. conclusion maternal growth in teenage pregnancy was not associated with reduced birthweight. indeed the increased weight gain and fat accrual observed in growing teenagers may protect against sga birth and promote fetal growth. igf-1 and leptin promote fetal growth, primarily through effects on maternal metabolism and nutrient partitioning to the fetoplacental unit. these data suggest that higher maternal igf-1 and leptin in growing teenagers may provide an anabolic drive for both maternal and fetal growth. background. umbilical oxygen uptake (o 2 umb uptake) has been estimated in human pregnancies only in acute experiments at the time of caesarean section. the recently developed possibility to measure umbilical blood flow by ultrasound in utero, prompted us to study normal and iugr pregnancies in order to evaluate fetal oxygen uptake utilizing the fick principle, i.e. uptake equals umbilical blood flow times (a-v) differences. methods. thirty-six iugr pregnancies were studied at the time of elective caesarean section and compared to twenty-one controls (c) (gestational age: c=38.9±0.2 and iugr=32.0±0.5 wks). an ultrasound examination was performed within 4 hours from the caesarean section in all the recruited patients. umbilical vein absolute volume flow (qumb) was measured as the result between umbilical vein area and the time-averaged peak velocity * 0.5. blood samples from umbilical vein (uv) and artery (ua) were obtained after the delivery and blood gases and acid-base balance were evaluated. umbilical oxygen uptake was calculated as o 2 umb uptake = qumb*(uv-ua) o 2 content. results. as expected, average fetal and placental weights were significantly different in the studied groups (1287±83 and 254±32 g in iugr vs 3274±62 and 474±16 g in n) . iugr pregnancies showed a significant reduction in qumb (98.5±6.6 vs 234.8±12.1 ml/min; p<0.01) but no differences in the qumb/kg of fetal weight. iugr fetuses showed a significant reduction in o 2 sat, o 2 cont and po 2 in both uv and ua compared to n. (uv-ua)o 2 content (1.79±0.72 vs 3.3±0.24 mmol/l; p<0.001) and o 2 umb uptake/kg (0.12±0.01 vs 0.24±0.01 mmol/min/kg; p<0.01) were significantly reduced in iugr. conclusions. we here report an evaluation of fetal oxygen uptake that proved surprisingly similar to the values reported in chronically catheterized animals. however, iugr fetuses showed a significant reduction in both blood and oxygen supply: this latter was reduced more that 50% on a per kg basis. iugr fetuses therefore utilize less oxygen than normally grown fetuses. circulating levels of vitamin d and il-6 in pregnancies with iugr. calvin j hobel, 2 chander p arora, 1 adegoke adeniji, 1 priya arora, 1 susan e jackman, 1 olga miadel, 1 baldjyan lilit. 1 1 ob-gyn, cedars-sinai medical center, burns ans allen research institute, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: any condition resulting in under exposure to sunlight, including the use of sun block or poor nutrition may result in insufficiency (37.5-80 nmol/l) or even deficiency of vitamin d (<37.5 nmol/l).vitamin d regulates placental development and function. vitamin d deficiency has been linked to increased risk of serious chronic and inflammatory diseases. objective: to determine if the circulating levels of vitamin d and interleukin -6 (il-6) in maternal plasma correlates to pregnancies resulting in fetus with intrauterine growth restriction (iugr). hypothesis: the metabolism of vitamin d initiates the biochemical cascade of events leading to the expression of il-6 and the inflammatory response in iugr births. study design: in a behavior in pregnancy study, plasma samples at all three time points were analyzed in a cohort of women for 25(oh)d using elisa. the samples were also analyzed for il-6 at three stages of pregnancy: t1 (18-20 weeks), t2 (28-30 weeks) and t3 (34-36 weeks). iugr was defined as birth weight below the tenth percentile for gestational age. none of the iugr cases had spontaneous preterm birth. results: iugr was diagnosed in 18 of 528 women with available samples from a behavior in pregnancy study (bips) . out of these subjects, 22 were selected as matched case controls. circulating levels of vitamin d (25(oh)d) were significantly lower in iugr cases at each visit (p<.001). the levels indicated deficient (29.4±4.6nmol/l) vitamin d in iugr group at t1 but sufficient vitamin d levels (83.8±5.8 nmol/l) in controls. subsequent visits also showed lower levels in the iugr cases compared to the control group (t2: 19± 3.1 nmol/l vs 45±3.6nmol /l; t3: 22 ± 4.1nmol/l vs 50± 2.9nmol /l). at all three time intervals, significantly (p<.001) higher levels of il-6 were associated with the iugr cases (415pg/ml, 1329pg/ml and 2526 pg/ml respectively) as compared to the controls (60pg/ml, 330pg/ml and 1240pg/ml respectively). conclusions: vitamin d deficiency or even insufficiency may be an unrecognized cause of iugr. it is possible that a primary non-infectious inflammatory process is activated by vitamin d deficiency. combined assessment of vitamin d deficiency and il-6 expression during different stages of pregnancy may facilitate the resognition of the risk of developing iugr. response to global 30% maternal nutrient restriction (mnr). nathan drever, thomas j mcdonald, peter w nathanielsz, cun li. obstetrics and gynecology, university of texas health science center at san antonio, san antonio, tx, usa. background: igf-ii is a major growth factor in the developing pancreas and studies in the human fetus demonstrate that the peptide localizes to b cells of the islets (j endocrinology 165:2). rodent studies show decreased fetal pancreatic growth and igf-ii and ins abundance and increased apoptosis with mnr (j endocrinology 140:10). we previously demonstrated a fall in most components of the placental and fetal baboon liver igf systems with mnr. here we have evaluated fetal pancreatic igf-ii and ins changes in response to mnr. methods: pregnant baboons were fed ad lib (ctr, n=12) or 70% of wt adjusted ctr diet (mnr, n=11) from 0.16 gestation (g) and fetuses were recovered at c-section under general anesthesia at 0.5 (n=9; 5 ctr and 4 mnr) and 0.9g (n=14; 7 ctr and 7 mnr). igf-ii and ins expression were determined by immunohistochemistry (ihc) and quantified by image analysis for fraction (area immunostained/area of the field x 100%) and density. data are expressed as mean + sem; ctr data are expressed] first; comparison made with two tailed t-test. results: at 0.5g there was no difference in igf-ii or insulin fraction or density between groups. at 0.9g, igf-ii fraction (2.57 ± 0.42 vs 1.44±0.27,p<0.05) and density (1.02x10 7 ±1.90 x10 6 vs 5.19x10 6 ±9.50x10 5 , p<0.05) and insulin fraction (2.51 ± 0.25 vs 1.58 ± 0.05, p=0.05) were reduced. conclusion: moderate mnr decreases abundance of fetal pancreatic igf-ii and ins at 0.9g. in the fetal baboon and supports the extant evidence for impaired pancreatic development with mnr seen in rodents. maintenance of liver growth in the hypoxic growth restricted fetal sheep: a role for intrahepatic glut1? sheridan gentili, janna l morrison, i caroline mcmillen. sansom institute, unisa, adelaide, south australia, australia. objective: we have previously demonstrated that there is a differential tissue response to chronic placental and fetal growth restriction. growth of fetal tissues such as the brain and the adrenal are consistently spared in the face of chronic substrate restriction, whilst we have demonstrated that the growth of the fetal liver may be either maintained or reduced. it is unclear whether the growth response of the fetal liver to hypoxia and hypoglycemia are determined by intrahepatic metabolic adaptations. hypothesis: we hypothesize that there will be a differential profile of hepatic expression of glut1, 11 hsd1, the gluconeogenic and glycolytic enzymes pepck and g3pdh and the transcription factors pgc1 and ppar in animals in which liver growth is maintained or reduced. methods: carunclectomy was performed in 19 non-pregnant ewes to induce placental restriction (pr). vascular catheters were inserted in 19 pr and 9 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. mean gestation po 2 <17mmhg was defined as hypoxic (h; normoxia, n). post mortem was performed at 140-145d. hepatic mrna expression of glut1, 11 hsd1, pepck, g3pdh, pgc1 and ppar was determined using qrt-pcr. results: four experimental groups were defined by fetal po 2 and liver growth (c-n, pr-n, pr-h and pr-h-reduced liver growth). fetal weight correlated with mean gestational po 2 (r 2 =0.67, y=0.21x+0.5, p<0.01). liver weight was significantly lower in a cohort of pr-h fetuses (c, 24.1±1.6; pr-n 22.9±0.9; pr-h 22.0±1.6; pr-h-rlg 15.8±0.3g:kg; p<0.05). glut1 expression was highest in those pr-h fetuses in which liver growth was maintained, whilst the expression of 11 hsd1, pgc1, ppar and pepck was highest in the pr-h fetuses in which liver growth was reduced (p<0.05). conclusions: in the pr-h fetuses in which liver growth was reduced, the increase in 11 hsd1 expression may be associated with an increase in hepatic exposure to cortisol and an associated increase in pepck, pgc1 and ppar . interestingly the compensatory increase in hepatic glut1 expression did not occur in fetuses in which liver growth was reduced. predicting the trajectory of fetal growth. racine n edwards-silva, 1 jeffrey gornbein, 2 calvin j hobel. 3 1 obstetrics gynecology, los angeles, ca, usa; 2 biomathematics, david geffen school of medicine at university of california, los angeles, ca, usa; 3 obstetrics gynecology, david geffen school of medicine at university of california, los angeles, ca, usa. objective: to evaluate twelve potential predictors of fetal growth trajectory defined as the rate of fetal weight change over time. study design: a longitudinal prospective study of singleton fetal growth trajectory. the twelve potential predictors considered were: fetal gender, gestational age, parity, race, bmi, age, weight at 1 st clinical exam, cumulative weight gain at each exam, smoking, and alcohol use. estimated fetal weight was computed using the hadlock formula and 4 sonographic fetal biometric parameters at 18-20 weeks, 28-30 weeks, and 34-36 weeks. the rate of change in fetal weight was defined as 60 x (fetal wt at exam j -fetal wt at exam i )(j > i)/ (gestational age at exam j -gestational age at exam i ). bivariate statistical analysis included the non-parametric spearman rank correlation and wilcoxon rank sum test. all factors were assessed multivariately using multiple linear regression. results: there were 454 multi-ethnic women included in the study, after 112 were excluded. they underwent a total of 1,277 exams. fetal gender (p=0.0048), maternal weight at 1 st exam (p=0.0055), and cumulative maternal weight gain at exam 3 (p < 0.001) were significant predictors of fetal growth trajectory. in this model, male fetuses had an average rate of fetal weight change of 46.9 grams per 60 days higher than females. the rate of fetal weight change increased by an average of 8.8 grams per 60 days for each 10 lb increase in maternal weight at the 1 st exam. the fetal growth rate increased 2.2 grams per 60 days for each 1 lb of cumulative weight gain at the 3 rd exam. conclusions: in this study, maternal weight at the 1 st exam and cumulative maternal weight gain were the significant determinants of fetal growth trajectory. adequate initial maternal weight and cumulative gestational weight gains probably ensure sufficient nourishment for normal placental growth, uteroplacental blood flow, and fetal nutrient uptake. this supports the emphasis on periconceptual nutritional counseling for all pregnant women. the implication of this study is that similar to fetal programming of adult diseases, there is nutritional programming of fetal growth trajectory. 216 high risk patients. other predictors include the known risk factors of age <15 and >40, single, tobacco/alcohol use, and african american race. interestingly, hispanic and native american patients have a lower lbw rate compared to other groups. introduction: catecholamines released by the sympathetic nervous system and adrenal medulla act via b-ars to regulate glucose and insulin function in liver, pancreas, adipose and muscle tissue. b-ar knock out mice show increased fat mass and glucose intolerance (asenslo et al.,diabetes,2005) . mnr animal models have increased sympathetic activity. we, therefore, evaluated effects of mnr on baboon fetal liver b1-ar. methods: baboons were fed as ad lib controls (ctr) or 70% of wt adjusted ctr diet (mnr) from 0.16 gestation(g) with fetuses retrieved at c-section under general anesthesia at 0.5 or 0.9g. protein expression determined by immunohistochemistry for b1-ar in the central liver lobule was quantified by image anaysis and expressed as fraction = area immuno-stained/area of the field x 100%. all data are expressed as mean + sem with ctr data presented first. liver glycogen expression was determined by the periodic accid schiiff (pas) method. comparisions were made with student's t-test with alpha level set at 0.05. results: fetal body and liver wts were not changed by mnr at either age. pas stained liver glycogen at 0.5g = 197.5 ± 1.7 vs. 162.8 ± 11.7%, p< 0.05 . b1-ar fraction was lower following mnr at 0.5g and at 0.9g (p< 0.05; fig 1) conclusions: mnr decreased b1-ar over 50% at 0.5g and 20% at 0.9g. decreased fetal liver b1-ar in mnr alters glucose metabolisam and may result in reduced lipolysis predisposing to fatty liver. infection with noncytopathic bovine viral diarrhea virus (ncpbvdv) during early bovine pregnancy (<150d gestation) results in fetal immunotolerance, persistent infection (pi) and intrauterine growth restriction (iugr). in contrast, infection after the development of adaptive immune competence (>150d gestation or postnatal) results in a transient infection (ti). we have previously reported an iugr in pi fetuses presenting as decreased body weight and ponderal index. a growth defect can be the result of many factors, including placental insufficiency and nutrient restriction, however it was hypothesized that the iugr seen in bvdv pi fetuses may be an immunopathological effect caused by the persisting virus. our two part experimental design examined the relationship between bvdv and its pi host. in experiment (exp) 1, blood cell mrna was collected from pi steers (n=2; confirmed by virus isolation), or uninfected control steers (n=3) and used to identify differentially expressed genes using microarray (affymetrix) and qtrt-pcr approaches. in exp2, bvdv naïve pregnant heifers (n=6 per group) were not infected (control) or infected with ncpbvdv on d.75 or d.175 of pregnancy creating pi and ti fetuses, respectively. fetuses were collected by c-section on d.190; infection was confirmed by elisa and qtrt-pcr. histology of placental tissue revealed no placentitis or pathology, and glucose and lactate levels in fetal serum were normal. microarray analysis revealed 294 genes that were differentially regulated in pi vs. controls (p<0.05, >1.5 fold). qtrt-pcr of steer and fetal blood revealed a significant upregulation of activators and products of the antiviral type-i interferon (ifn-i) pathway. as ifn-i can act as a growthsuppressive cytokine, a long-term upregulation may contribute to the iugr seen in persistent bvdv infection and in other viral infections observed during pregnancy. nricg 2006-03907 from the csrees. patients with a short cervix are at an increased risk for spontaneous preterm delivery. therefore, it is possible that women with a short cervix during pregnancy are also at risk to deliver an sga neonate. this study was conducted to address this question. study design: patients >14 weeks of gestation were prospectively enrolled into an observational study (08/2002 to 06/2007). transvaginal sonographic examinations were performed every 2 weeks until delivery. the shortest cervical length between 14-32 weeks was used for analysis. sga was defined as less than the tenth percentile of birth weight. results: 303 asymptomatic patients were studied. 19.8% of patients delivered an sga neonate (60/303). the median cervical length was 21 mm (0 to 55mm); 184 patients had a cervical length <25mm. of these, 17% (32/184) delivered an sga neonate. similarly, 17% (17/98) patients with a cervical length <15mm had an sga neonate. no relationship was found between a short cervix and sga (short cervix was defined as either < 25mm and <15 mm). the frequency of sga was not significantly different between women with a short cervix and those with a long cervix [17% (32/184) vs. 24% (28/119); p=0.34]. newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced background folate is an essential micronutrient for cellular growth. recommendations on periconceptional folic acid use are mainly focussed on prevention of neural tube defects, despite growing evidence that folic acid use may have positive effects on birth weight. objective to examine associations between folic acid use, intrauterine fetal growth and birth weight. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods information on folic acid use was obtained by questionnaires and categorized into three groups: 1) preconception start of folic acid use; 2) start of folic acid use in first ten weeks of gestation; 3) no folic acid use at all. fetal growth measurements included head circumference, abdominal circumference and femur length measured in mid-and late pregnancy, i.e., gestational age 18-25 and >25 weeks, respectively, and birth weight. fetal weight in mid-and late pregnancy was estimated using the haddock method. results data from 6,719 pregnant women were available. overall, folic acid use was positively associated with fetal growth. preconceptional folic acid use resulted in an increased growth of 6 grams (95%ci 2.92-9.01, p<0.001) per week from late pregnancy to birth, compared to no folic acid use. similarly, start of folic acid use in the first ten weeks of gestation resulted in an increased growth of 5 grams (95%ci 2.19-7.88, p=0.001) per week from late pregnancy to birth. both preconceptional folic acid use and folic acid use started in the first ten weeks of gestation resulted in higher birth weights of 69 grams (95%ci 39-99) and 51 grams (95%ci 23-80), respectively, compared to no folic acid use. a tendency was found for an increased risk of birth weight less than 2500 grams when folic acid was not started preconceptionally (or 1.5, 95%ci 0.9-2.4). conclusion periconceptional folic acid use is significantly associated with increased fetal growth resulting in a higher birth weight. ductus venosus isovolumetric relaxation in severely premature growth-restricted fetuses. jason l picconi, katherine drennan, farhan hanif, michael kruger, giancarlo mari. obstetrics and gynecology, wayne state university/dmc, detroit, mi, usa. objective: ductus venosus (dv) doppler waveforms are characterized by two periods in which blood velocity decreases. the first represents the isovolumetric relaxation (ir) at the end of ventricular systole and the second represents atrial contraction at the end of ventricular diastole (a). ductus venosus reversed flow (dvrf) occurring at the time of the a-wave is considered a risk factor for intrauterine fetal demise (iufd). we have previously reported that absent or reversed a-wave flow can be present for weeks before iufd occurs or delivery is performed for non-reassuring fetal testing. the guiding hypothesis for this study is that decreased flow at the time of ir in combination with absent or reversed a-wave flow allows a more accurate prediction of fetal outcome than a-wave absent or reversed flow alone. material and methods: ductus venosus doppler was serially studied in 17 severely premature iugr fetuses (estimated fetal weight < 10 th percentile and umbilical artery pulsatility index > 95 th percentile) from diagnosis until demise or delivery. ductus venosus waveforms were assessed quantitatively for peak systolic velocity (psv), isovolumetric relaxation velocity (irv), and end diastolic velocity (edv). the psv/irv + edv were compared to fetal and neonatal outcome. a kruskal-wallis one way anova, mann whitney u post-hoc test, and a roc, were used for statistical analysis. a p < .05 was considered statistically significant. results: all fetuses were delivered at < 32 weeks. six cases resulted in iufd, five cases resulted in neonatal demise (nd), and six cases resulted in neonatal survival (ns) at the time of discharge from the hospital. the psv/irv+edv correlated better than a-wave reversal of flow with perinatal outcome. a psv/irv+edv score less than -1.8 resulted in iufd, whereas a score greater than -1.8 resulted in live birth. live births segregated based on estimated gestational age, where those fetuses at less than 28 weeks resulted in nd and those fetuses at or greater than 28 weeks resulted in ns. all results were statistically significant. conclusions: the isovolumetric relaxation velocity is a novel doppler parameter in the assessment of severely premature iugr fetuses. these data indicate that assessment of irv should be considered part of the evaluation of severely iugr fetuses. background: fetal growth restriction has been linked to an increased incidence of chronic hypertension, which may be the result of extracellular matrix changes (ecm) within the vascular tree. in previous studies, ecm changes were observed in the umbilical arteries of preterm growth restricted infants. objective: to determine if there are alterations in collagen subtypes within the umbilical cords from growth restricted fetal rat pups after a period of maternal nutrient restriction. methods: timed pregnant sprague-dawley rats were fed either a 50% food restricted diet (mfr; n=5) or were fed ad libidum (control; n=5) from d10 until d21 of gestation. litter size, fetal weights and placental weights were then noted and umbilical cords from 3 randomly selected pups in each litter were snap frozen. gene expression for collagens i, iii, xiv and decorin was evaluated by real-time rt-pcr with normalization to the gadph housekeeping gene. data were analyzed from fitting general linear regression models with estimation based on the quasi-likelihood estimation (generalized estimating equations) to account for clustering of responses within litters. data are shown as cycles to amplification (ct), which is inversely proportional to mrna levels. results: no difference in median litter size was detected. however, fetal and placental weights in the mfr group were significantly less than those from control dams. no significant differences in umbilical cord gene expression for collagens i, iii, xiv or decorin were detected between mfr and control pups. conclusions: maternal food restriction does not result in any detectable alterations in collagen or decorin expression within the intact umbilical cord, despite causing significant reductions in fetal growth. control (n=5) p-value litter size (median, range) 11 (8,15) 12 (9,15) 0.59 fetal weight (mean ± sd) g 3.29 ± 0.12 4.0 ± 0.1 <0.001 placental weight (mean ± sd) g 0.73 ± 0.05 0.90 ± 0.03 0.007 collagen i (mean ± sem) ct 17.9 ± 0.13 18. we have previously demonstrated that the restriction of placental and fetal growth results in fetal hypoxia and fetal brain sparing suggesting a redistribution of cardiac output. furthermore, placentally restricted (pr) hypoxic fetuses are more dependent on their sympathetic nervous system for the maintenance of blood pressure during late gestation. nerve growth factor (ngf) plays a significant role in sympathetic innervation. hypothesis: we hypothesize that the expression of ngf will be higher in the aorta and femoral artery of the pr hypoxic compared to the control fetus. method: carunclectomy was performed in 6 non-pregnant ewes to induce pr. vascular catheters were inserted in 6 pr and 4 control (c) fetuses at 103-117d and arterial blood samples were collected for blood gas analysis. all pr fetuses introduction elevated sflt-1 levels have been shown to be a feature of pre-eclampsia and is considered to play a significant role in the pathogenesis of the condition. the role of angiogenic factors in fetal growth restriction has not been as well established. this study evaluated the levels of circulating vascular endothelial growth factor (vegf) and its soluble receptor sflt-1, in normal pregnancies and isolated placental vascular disease without evidence of pre-eclampsia. method 42 maternal peripheral venous samples were collected antenatally from two groups of pregnant women between 25-40 weeks of gestation. group a: uncomplicated normal pregnancies (n = 20) and group b: pregnancies complicated by isolated placental vascular disease( n = 22) as defined by birth weight less than 10 th centile for gestation and umbilical artery doppler s:d ratio above the 95 th centile for gestation, with no evidence of maternal preeclampsia or pre-existing hypertension. plasma vegf and sflt-1 levels were measured using standard eliza techniques. comparison between groups were performed by using one way analysis of variance. the maternal plasma sflt-1 levels (figure1) in group a: normal pregnancies increased with gestation (p < 0.001). the sflt-1 levels in group b: isolated placental vascular disease were significantly higher throughout all gestations (p = 0.002) and did not show a significant variation with gestation ( p = 0.47). the plasma vegf levels were below the detectable levels of the assay in all samples except 3 normal pregnancies. the significantly increased maternal plasma sflt-1 levels in established isolated placental vascular disease without evidence of pre-eclampsia suggest a disease of placental origin. the dysregulation of angiogenic factors may be part of a repair and regeneration process in the placenta. objectives: production of 5 -reduced neurosteroids is critical for reducing fetal vulnerability to stressors in pregnancy and inhibition of 5 -reductases (5 r) increase acute hypoxia-induced apoptosis in the fetal brain (yawno et al 2007 neurosci 146:1726 . we have developed a model of placental insufficiency that results in fetal growth restriction (gr) in the guinea pig (palliser et al 2007 repro sci 14 #381). the aim of the present study was to determine the effects of suppression of 5 -reduced steroid synthesis on apoptosis in vulnerable regions of the fetal brain and on neurosteroid synthetic enzymes in pregnancies compromised by chronic placental insufficiency. methods: placental insufficiency was induced in guinea pig dams by surgical ablation of uterine artery branches at mid gestation (term 68d). sham operated or gr dams received finasteride (a 5 r inhibitor; 25mg/kg/day) during late gestation (55d until term). activated caspase-3, a marker of apoptotic cell death, was measured by immunohistochemistry and steroidogenic enzymes, 5 r1 and cytochrome p450 side chain cleavage (p450scc) were measured by real time pcr and western blotting in fetuses (65d) and neonates 24h after birth. results: placental insufficiency significantly reduced fetal body and organ weight by 30% whilst sparing brain weight. the number of activated caspase-3 positive cells was significantly increased in the fetal hippocampus of gr fetuses and further increased in the cortex of gr fetuses receiving finasteride. the neonatal brain also exhibited changes in caspase-3 activation following gr and finasteride treatment. the fetal adrenal and the placenta responded to the compromise with increased expression of p450scc mrna and 5 r1 protein, respectively. conclusion: the combination of placental insufficiency and suppressed neurosteroid system leads to markedly increased apoptotic cell death in the fetal brain which continues to affect the neonatal brain. the fetus responds to these conditions by increasing steroid synthetic enzyme expression in the placenta and adrenal glands suggestive of a possible neuroprotective feedback process. to study the effect of smoking by mothers on the fetal and neonatal brain using non-invasive magnetoencephalography technique (meg). materials and methods: using fetal magnetoencephalography, cortical auditory evoked responses (aer) were measured from 21 fetuses ranging from 27 to 39 weeks gestational age for a total of 69 recordings. 29 measurements were taken from 10 mothers with a history of smoking (sm) and 40 from 11 mothers with no smoking history (ns). after delivery, five sm and five ns newborns had meg aer measurements twice for a total of 20 recordings. aer was quantified by cross-correlation analysis and its significance was assessed by boot-strap technique. results: aers were detectable in 35 out of 40 fetal recordings in the ns group and 23 of 29 in the sm group. the neonatal response rate was 90% for each group. the latencies were divided into three components: c1 (100-300 ms), c2 (301-600 ms) and c3 (601-900 ms). in both fetuses and neonates as well, there was a statistically significant difference (p<0.05) between the two groups in the c2-component and the sm group showed faster aer compared to the lr group. conclusion: meg technique provides a non-invasive approach to study the effects of smoking on developing fetal and neonatal brain. the observed decrease in the latency of fetuses and neonates of the smoking mothers could indicate a hypersensitive cortical response to auditory tone. adenosine (ado) modulates metabolism in adult mammals through multiple mechanisms that involve ado a1 and a2a receptors. objective: this study was designed to test the hypothesis that ado a1 and a2a receptors participate in fetal metabolic homeostasis. methods: experiments were performed in chronically catheterized fetal sheep (>0.8 term). intravascular infusion for 1 h of dpcpx (a1 receptor antagonist) or zm241385 (zm, a2a receptor antagonist) was performed alone or in concert with ado administration. the highly selective ado receptor antagonists were also infused in fetuses in which hypoxia was induced for 1 h by having the ewe breathe a hypoxic gas mixture (fio2 = 0.10). data were analyzed by two-way repeated measures of anova. results: blockade of ado a1 receptors (n=8) increased significantly (p < 0.05) fetal concentrations of glucose [control (c): 15.6 ± 1.0 (se)]; experiment (e): 17.38 ± 1.1 mg/dl] and lactate (c: 16.5 ± 2.4; e: 22.5 ± 4 mg/dl) without significantly altering insulin levels and arterial blood gases or ph. antagonism of ado a2a receptors (n=6) did not affect plasma levels of glucose, lactate, or insulin. intravenous infusion of ado (n=11), which did not alter pao2 or paco2, increased concentrations of glucose (c: 18.0 ± 0.8; e: 22.6± 1.3 mg/dl) and lactate (c: 12.2 ± 1.0; e: 20.4 ± 1.6). zm (n= 7), but not dpcpx (n=9), abolished ado-induced rise in glucose and lactate concentrations. isocapnic hypoxia (pao2 13 torr), which increases (2-3 fold) fetal plasma ado levels to those similar to ado infusion, decreased arterial ph (c: 7.331 ± 0.016; e: 7.140 ± 0.032), and increased fetal levels of glucose (c: 16.2 ± 1.3; e: 33.7 ± 7.8 mg/dl) and lactate (c: 13.3 ± 1.4; e: 97.3 ± 7.7 mg/dl) without altering changing insulin concentrations. these effects of hypoxia were not altered by dpcpx or zm. conclusions: 1) a1 receptors modulate plasma levels of glucose and lactate in normoxic fetuses; 2) a2a receptors mediate the adoinduced rise in plasma glucose and lactate; and 3) a1 and a2a receptors are not significant modulators of hypoxia-induced changes in plasma levels of glucose and lactate. supported by usphs hd-18478. objective: to investigate the dynamic changes of the relationship between leptin, adiponectin and resistin in maternal and fetal circulation during pregnancy and in the early post-natal period. materials and methods: thirty pregnant women with uncomplicated singleton pregnancy delivered at term. maternal and fetal/neonatal venous blood samples were obtained at delivery and at 72 hours from birth. leptin, adiponectin and resistin were measured by specific elisa assays. neonatal anthropometric measurements, glucose metabolism and lipid profile, blood pressure information were obtained. statistical analysis was performed by anova followed by student t test or duncan's test whenever appropriate. correlations were calculated by using the pearson coefficient. results: adipokines concentration at birth and at 72h from birth was showed in table. multivariate regression analysis showed that fetal leptin levels were positively associated with female gender and adiponectin levels, but not with anthropometric characteristics. fetal leptin and adiponectin levels were not correlated with maternal concentration, whereas fetal and maternal resistin levels were. fetal and maternal resistin concentration was positively associated with gestational age and birth weight and fetal resistin levels correlated negatively with lipid profile. after birth leptin concentration in maternal and neonatal circulation decreased dramatically and the correlation between leptin and adiponectin levels was lost. a positive correlation between resistin and leptin concentration in neonatal circulation was found at 72h from birth. conclusions: in contrast to adult life a positive correlation between leptin and adiponectin is present in fetal life. placental secretion of leptin, but not of adiponectin and resistin, contributes significantly to maternal and fetal circulating levels. significant changes in the relationship among adipokines occurred immediately after birth and may affect growth and development in early post-natal period. adipokines concentration in maternal and fetal circulation ) antagonism has been shown to normalize placental perfusion and fetal growth in several rat models of fetal growth restriction. however, direct administration of et a antagonists to newborn rats within 3 hours of delivery has been consistently associated with neonatal demise due to failure of the ductus arteriosus to close, raising concerns about the safety of their use late in pregnancy. perinatal exposure to et a antagonists (maternal administration in late gestation) and its impact on rat pup survival and oxygen saturation has not been investigated. objective: to determine the impact of a maternally administered et a antagonist on oxygen saturation in newborn and 7-day-old rat pups. methods: timed pregnant sprague-dawley rats were treated with fr139317 (12 mg/kg/day; et a antagonist) or 4.2% nahco 3 vehicle, by subcutaneous osmotic pump connected to an intravenous catheter, from gestational day 14 (term=22 days) through parturition. all five pregnant rats in each group delivered spontaneously and nursed their pups through postpartum day 7. oxygen saturation of each rat pup was measured by pulse oximeter on postpartum days 1 and 7. results are presented as means ± se. newborn 7-day neonate vehicle 92.6 ± 0.9 94.4 ± 0.8 eta antagonist 94.7 ± 0.8 91.6 ± 1.1 there were no statistically significant differences between the treatment groups. maternal administration of an et a antagonist from gestational day 14 through parturition, at a dose sufficient to ameliorate fetal growth restriction, has no adverse impact on oxygen saturation in neonatal rat pups. helen l torrance, 1 jan b derks, 1 martijn a oudijk, 1 avnesh s thakor, 2 tereza cindrova-davies, 2 frank van bel, 1 gerard ha visser, 1 graham j burton, 2 dino a giussani. 2 1 perinatal center, university medical center utrecht, netherlands; 2 department of physiology, development neuroscience, university of cambridge, united kingdom. introduction: the management of perinatal asphyxia remains a major concerns in obstetrics. umbilical cord compressions (ucc) induce fetal asphyxia and ischaemia-reperfusion (i/r). i/r increases reactive oxygen species, for instance via activation of the xanthine oxidase (xo) pathway, which may promote oxidative stress in the fetal circulation. while treatment with allopurinol of asphyxic human neonates reduced free radicals and improved cardiovascular status, treatment started postnatally was deemed too late to prevent oxidative damage (benders et al. arch dis child 91:163, 2006) . consequently, in complicated pregnancy, recommendations to treat the fetus via the mother, rather than the neonate, with allopurinol are being entertained today. we investigated the effects of maternal allopurinol treatment on indices of oxidative stress in the fetal heart following repeated ucc in sheep. methods: at 0.8 of gestation,10 surgically instrumented sheep fetuses were submitted to i/r (5 x 10 min repeated ucc) under maternal allopurinol (n=5) or saline vehicle (n=5) infusion. fetal hearts were collected 48h after i/r and snap frozen for measurement of (anti)oxidant proteins by western blot. hearts from 5 uninstrumented fetal sheep at 0.8 gestation served as controls. statistical comparisons were made using one-way anova. results: i/r episodes led to increased expression of cox-2, enos, hsp90 and decreased expression of sod and gluthatione peroxidase (gpx) in the fetal heart, findings consistent with cardiac oxidative stress. maternal treatment with allopurinol ameliorated these effects (fig. 1 objectives: hypoxic-ischemic fetal brain injury (hie) is a major cause of neonatal death and morbidity. evidence suggests that the brain cell injury associated with chronic hpx (in contrast to an acute ischemic reperfusion injury) is a complex process reflecting a series of adaptive intracellular events that are duration and gestational age dependent. we applied advanced proteomic tools as a next step toward ascertaining a more complete understanding of the impact of hpx on the fetal brain proteome. methods: time-mated guinea pigs were housed in a chamber beginning on day 50 for 14d, breathing either room air (nmx), or 10.5% or 12% o 2 (12% o 2 hpx or 10.5% o 2 hpx). on day 65 (term), the fetal brains were removed and preserved for study. total protein was extracted, and the proteome first characterized by 2d gel electrophoresis. the density of the resulting protein spots were acquired and analyzed using the gs800 densitometer and pdquest software. identified spots of interest were trypsin digested and subject to maldi mass spectrometry using the 4700 proteomics analyzer mass spectrometer for peptide mapping or sequencing. the hpx-induced protein spots were identified based on a minimum of a 2x change from nmx. results: hpx had a clear effect on the fetal brain proteome. superoxide dismutase (sod), heat shock protein 70 (hsp70), actin (actg1), interleukin-1 (il-1), and glutamine synthetase (gs) were each up regulated, while cofilin-1, brain-type creatine kinase (bb-ck), and -1 gtp binding protein were each down regulated. all protein changes were proportional to the hpx (12% o 2 hpx vs nmx, 12% o 2 hpx vs 10.5% o 2 hpx, p<0.05). conclusions: this initial application of proteomic techniques confirms that fetal brain damage secondary to chronic hpx (in contrast to acute hpx) is a complex processes characterized by fetal adaptations mediated by multiple protein activations and inactivations. while sod, il-1, and gs have each been previously investigated, the potential roles of actg1, bb-ck, beta-1 gtp binding protein, and cofilin-1 in the fetal response to hpx and the associated brain damage are unclear and represent strong candidates for future investigation. acknowlegement: this study was supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). intermittent umbilical cord occlusion occurs in 5% of human pregnancies.given that elastogenesis within the vascular wall is in part mediated by hemodynamic conditions during development, the blood pressure response to acute hypoxic insults such as cord occlusion may alter arterial composition. elastin content of a central and peripheral artery and blood pressure responses in fetal sheep exposed to varying degrees of cord occlusion were determined. methods: over a 4 day period, near term fetal sheep received total umbilical cord occlusion (uco) lasting 1 min/ hour (mild group; n=5), 2 min/hour (moderate group; n=4), 3 min/hour (severe group; n = 6) or no occlusion (control group; n=7). fetal arterial blood samples were drawn 5 min prior to and at the end of cord occlusions. mean arterial pressure (map) was monitored continuously. the carotid and superior mesenteric arteries were excised and a colorimetric assay (biocolor) performed for determination of elastin content. results are presented as mean ± sem. results:umbilical cord occlusions produced decreases in fetal arterial oxygen pressure and oxygen saturation that were progressively more pronounced across mild, moderate and severe uco groups (p < .01). lactate concentration rose during occlusions in the moderate and severe groups, but not the mild group (p < .01).elastin content of the superior mesenteric artery did not differ between the 3 experimental groups and the control group. elastin content of the carotid artery and map response elastin content (μg/mg tissue) max ∆ in map duration of rise in map (min) control 5.7 ± 0.4 4.0 ± 1.7 -mild 7.0 ± 0.7 21.06 ± 2.5 ** 10.39 ± 0.6 moderate 7.4 ± 0.7 32.4 ± 1.6 ** † 14.3 ± 0.5 † severe 9.5 ± 1.0 * 34.5 ± 0.6 ** † † 17.4 ± 1.2 † † * p < .05; ** p < .01: experimental groups compared to control † p < .05; † † p < .01: compared to mild group the max in map was positively correlated with elastin content (r 2 = .56, p < .05). conclusions:the transient rise in blood pressure and preferential blood flow to the brain that occur in response to acute hypoxemia during severe intermittent umbilical cord occlusion induce an increase in elastin synthesis in the carotid artery.this may give rise to adaptive programming of postnatal central arterial compliance. electrocortical activity during repetitive umbilical cord occlusions (uco) with worsening acidemia in the ovine fetus near term. martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 obgyn, chri, univ of western ontario, london, canada; 2 obgyn, harbor-ucla med ctr, torrance, ca. objective: uterine contractions during labour can restrict umbilical blood flow compromising fetal oxygenation and leading to adverse neonatal outcome including newborn encephalopathy/subsequent cerebral palsy. while electronic fetal heart rate (fhr) monitoring is widely used for assessment during labour, abnormal fhr patterns as used clinically have a poor positive predictive value for concerning/significant acidosis at birth. there is limited study of fetal electrocortical activity (ecog) in animal models with induced hypoxia/ acidemia as might be seen during labour. thus, we aimed to induce repetitive ucos in fetal sheep leading to worsening acidemia to determine the predictive value of ecog activity for fetal compromise. methods: near-term fetal sheep (n=10) underwent chronic preparation with artery catheters, ecg/ecog electrodes and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) ucos with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood samples for blood gases and ph were taken at selected time points during the baseline, ucos, and recovery periods. arterial blood pressure (abp) and fhr were continuously monitored and spectral edge frequency (sef) was calculated from ecog. correlation of sef with fhr change was calculated for time intervals using sef maxima and fhr minima during uco series. data are presented as means±sem. results: repetitive ucos led to development of a marked acidosis (ph 7.36±0.03 to 6.91±0.12, p<0.001). 52±13 min prior to fetal ph drop <7.0, the sef of ecog began to increase abruptly during each fhr deceleration from 3±1 hz up to 23±2 hz (p<0.001) and was correlated to both fhr change and a decrease in fetal abp during each fhr deceleration at this time (p<0.001). conclusion: our findings suggest that in the animal model studied (1) fetal ecog activity is impaired with progressive acidemia accompanied by fhr decelerations and pathological abp decreases; (2) there is a consistent temporal relationship between the occurrence of ecog alterations and the subsequent critical drop of ph <7.0. these findings could contribute to improvement in the clinical ability to predict fetal compromise during labour using ecog/fhr monitoring. background: the ductus arteriosus (da) plays a pivotal role in fetal development and circulation. during gestation, patency of the fetal da is maintained by nitric oxide (no) and prostaglandins (pg). no and pgs are downstream effectors of estrogen (e2) and progesterone (p4). however, the roles of e2 and p4 in da regulation have not been studied. objective: we hypothesized that: e2 and p4 have opposite effects in the da; that rising e2 and falling p4 levels help trigger da closure via specific hormone receptors; and that no and/or pgs mediate da responses to e2 and p4. methods: expression of er , er , pra, prb, and the putative membrane receptors mpr-, -, -, pgrmc-1, -2, and serbp-1 was examined by rt-pcr and qpcr on days 15, 17, 19 (term), and p1. the effects of e2 and p4 (10 -9 -10 -4 m) on the d19 fetal da were examined in a cannulated microvessel myography system. e2 and p4 effects were also studied in the presence of receptor antagonists (ici 182780, ru486) and no and pg inhibitors (l-name, indomethacin). results: er and pr receptors were expressed at low levels; pgrmc-2 expression was stronger than other membrane prs, and increased with advancing gestation. under fetal o2 conditions, e2 induced rapid, concentrationdependent dilation at 10 -6 -10 -4 m (n=11); p4 induced progressive vasodilation at all doses (n=10). e2 and p4-induced dilation occurred within 60-180 seconds of exposure. while e2-dilated das did not respond to ici, ici-pretreated das failed to dilate to the same dose of e2 (n=10) suggesting antagonism of e2 effects. the vasodilatory effects of e2 were partially inhibited by pretreatment with l-name. p4-dilated das did not respond to ru486, whereas ru486pretreated das showed a small, significant response to p4 (n=10), suggesting partial antagonism or signaling via alternative, ru486-independent pathways. pre-treatment with indocin did not block the vasodilatory effects of p4. conclusions: contrary to our expectation, both e2 and p4 have dilating effects on the fetal da, via no, pgs and non-no, non-pg pathways. expression studies and the rapid response of ex vivo das are consistent with non-genomic actions via membrane receptors. hormone shifts in parturition may have longterm effects on da preparation for postnatal closure, but strategies to maintain fetal da patency or treat newborns with pda will require better understanding of this process. background. for the fetus, arterial blood gases are critical in the regulation of cerebral blood flow (cbf) and cerebral oxygenation. however, the relation of cbf, cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity to arterial o 2 tension (pao 2 ) are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that in the near-term fetus, acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with ecog state. methods. by use of a laser doppler flowmeter with a fluorescent tissue o 2 probe, and with fluorescent-labeled microspheres, and with ecog electrodes, in near-term fetal sheep (n = 8) we measured laser doppler cbf (ld-cbf), tpo 2 , ecog (root mean square (rms) voltage with high voltage low frequency, hvlf versus low voltage high frequency, lvhf) and spectral edge frequency-90% (sef 90 ) in response to 40 min moderate isocapnic hypoxia. results. ld-cbf, cerebral o 2 delivery, tpo 2 , and several other variables correlated highly with ecog state. in the normoxic control fetus, in association with a shift from hvlf to lvhf ecog activity, tpo 2 decreased briefly to 7±1 from a control value of 10±1 torr; however, as ld-cbf increased 18±5%, and sef 90 increased to14±2 from 7±1%, tpo 2 returned to near normal value. with acute hypoxia (pao 2 = 12±1 torr) when in the lvhf state ld-cbf increased only 23±14%, as opposed to a 52±10% increase when in hvlf ecog state. with this degree of hypoxia, tpo 2 decreased to 3±1 torr, sef 90 remained at 8±2%, and cerebral metabolic rate for o 2 (cmro 2 ) decreased 32±5% (p<0.05). conclusions. for the near-term fetus, normoxia with changes in ecog state was associated with brief periods of decrease in tpo 2 , which were restored quickly by increased ld-cbf. in contrast, acute hypoxia was associated with a significant depression of cortical tpo 2 , cmro 2 , and ecog state, with increased ld-cbf failing to restore cortical tpo 2 . thus, we reject the null hypothesis that in such fetuses, hypoxia demonstrates no compromise in cerebral oxygenation. (supported by usphs hd-03807). releasing hormone and arginine vasopressin in the ovine fetus. charles a ducsay, 1 kanchan m kaushal, 1 malgorzata mlynarczyk, 1 kimberly hyatt, 2 dean a myers. 2 1 ctr. for perinatal biol., loma linda univ., loma linda, ca; 2 ob/gyn, univ. oklahoma hlth. sci. ctr., oklahoma city, ok. background: long term hypoxia (lth) profoundly affects the hypothalamicpituitary-adrenal axis of the fetal sheep. we previously showed that lth causes augmented corticotrope function. the present study was designed to test the hypothesis that lth enhances sensitivity to the acth secretagogues; cortiocotrophic releasing hormone (crh) and arginine vasopressin (avp) resulting in increased anterior pituitary corticotrope secretion of acth. methods: pregnant ewes were maintained at high altitude (3,820 m) from day 40 to 130-131 of gestation (dg), when they were returned to the lab and a maternal tracheal catheter was implanted. maternal po 2 was maintained at a level comparable to that observed at altitude ( 60 mmhg) by nitrogen infusion. on 132 dg, lth (n = 4) and age-matched, normoxic control (n = 4) fetuses were implantated with vascular catheters. each fetus received a 15 min infusion of either saline vehicle, 100 ng/kg of ovine crh or 20 ng/kg of avp (estimated body weight/min) in a randomized order over 3 consecutive days (137-139dg). blood samples were collected at 0 min (baseline prior to infusion), 15, 30, 60 and 90 min following the start of the infusion and analyzed for acth, as well as the acth precursors pro-opiomelanocortin and the major processing intermediate 22 kda proacth. results: vehicle had no effect on any of the measured parameters. with crh infusion, acth (pg/ml) increased in both groups over the course of the study. however, peak concentrations (at 90 min) were significantly higher in the lth group compared to control (240±17 vs. 110±14, respectively; p<0.01). acth precursor secretion (pm) was greater in lth fetuses compared to controls during the experiment (p<0.01). in response to avp, peak acth concentrations were also higher in the lth fetuses compared to control (209±51 vs. 67±20 respectively; p<0.01), however peak levels were reached at between 15 and 30 min after start of infusion with levels in both groups returning to pre-infusion values. a similar pattern was observed with precursor levels (102.1±27.9 vs. 66.1±10.2, p<0.05, lth vs. control). conclusions: lth significantly increases pituitary sensitivity to both crh and avp. this enhanced sensitivity may be mechanism of our previously observed enhanced corticotrope function. (supported my nih grants hd33147 and hd31226). martin g frasch, 1 roy mansano, 2 michael g ross, 2 robert gagnon, 1 bryan s richardson. 1 1 ob/gyn, chri, university of western ontario, london, canada; 2 ob/gyn, harbor-ucla med. ctr., torrance, ca. objective: repetitive uco leading to worsening fetal acidosis with fetal heart rate (fhr) decelerations (fhr dec ) are accompanied by pathological decreases of fetal arterial blood pressure (bp). we hypothesized this bp change may be caused by the bjr, a vagally mediated reflex with bradycardia and hypotension to reduce cardiac workload, via stimulation of cardiac chemoreceptors during systemic acidemia. methods: ten near-term fetal sheep (125 ± 2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. after a control period, fetuses underwent a series of mild (1 min every 5 min), moderate (1 min every 3 min) and severe (1 min every 2 min) uco each lasting 1 h or until ph decreased to < 7.0. fetal arterial blood samples were taken at selected time points during the control and uco periods. bp and fhr were continuously monitored. individual fhr nadir during each fhr dec and accompanying bp change ( bp = [bp at the time of a fhr nadir] -[bp at baseline preceding a uco series]) were determined during all uco. data are presented as means±sem. results: control period ph (7.36 ± 0.03), fhr (163 ± 5 bpm) and bp (45 ± 4 mmhg) were within the physiological range. average depth of fhr dec for all uco was 93 ± 11 bpm and increased with higher lactate concentrations (r = 0.40, p < 0.05) and lower ph (r = 0.34, p = 0.08). bp during all uco demonstrated an initial hypertensive response to fhr dec , which decreased with lower ph (r = 0.49, p < 0.05). bp increased 11 ± 2 mmhg (p < 0.05) during mild uco (ph to 7.32 ± 0.03), 4 ± 3 mmhg during moderate uco (ph to 7.19 ± 0.04, p < 0.05) and 2 ± 4 mmhg during severe uco (ph to 6.91 ± 0.12, p < 0.05). conclusion: these results suggest that bjr, a short-acting, ph dependent depressor reflex, blunts the physiologic hypertensive response to cord occlusion insults leading to worsening acidemia as might be seen in the human fetus during labour with repetitive variable fhr dec . the failure to increase bp may prevent optimal blood flow distribution responses necessary for preservation of vital organs. role of nos and pde5 in placental dysfunction following fetal bypass. mitali basu, 1 r scott baker, 1 christopher t lam, 1 kenneth e clark, 2 pirooz eghtesady. 1 1 cardiothoracic surgery, cincinnati children's hospital, cincinnati, oh, usa; 2 ob/gyn, university of cincinnati, cincinnati, oh, usa. introduction rising placental vascular resistance following fetal cardiopulmonary bypass (bypass) remains the achilles' heel of fetal cardiac surgery. we have previously shown in real-time that nitric oxide (no) production rises during bypass and falls post-bypass, while cgmp levels rise throughout. using immunohistochemical and western analysis of placenta, we examined the involvement no pathway components in this placental vascular pathophysiology. ovine fetuses at 100-110 days gestation were placed on bypass for 30 minutes and followed post-bypass for 2 hours. placental samples were collected immediately prior to bypass and at 30 and 120 min post-bypass (n=7) and compared to a group of similarly instrumented controls, (n=6). placental enos, inos and pde5 protein expression was measured using standard methods and relative expression normalized to beta-actin. statistical analysis utilized students t-test, and anova for trend and group-wise analysis, (significance at p=0.05). pre-bypass protein expression did not differ between groups. pde5 protein levels and phosphorylated pde-5 expression were both elevated 30 min post bypass, and reduced 120 min post bypass compared to sham, (p=0.01). enos levels in the bypass group increased linearly from pre-bypass to 120 min postbypass (p<0.01), and were also elevated compared with shams (p<0.01), while shams had declined significantly by 120 min post bypass, (p<0.01). similarly, phosphorylated enos expression in the bypass group increased linearly from pre-bypass to 120 min post-bypass, (p=0.058), while shams trended towards decline by 120 min post-bypass. simultaneously, placental inos expression remained stable within groups, but was lower in the bypass group at 30 and 120 min post-bypass, (p<0.04). the preceding data correlated with observed immunohistological changes in the same placental cotyledons. fetal bypass leads to significant increases in placental protein levels of pde5, phosphorylated pde-5, enos and ostensibly phosphorylated enos. increased pde5 expression may be a response to increased no and the generated cgmp. this data suggests a compensatory upregulation of pde5 and enos that eventually fails, leading to increasing placental vascular resistance and subsequent lethal placental dysfunction. angiotensin receptors (rat-ii) in neuronal nuclei of the brainstem have been implicated in integration of baroreceptor responses. in near term fetal sheep, we have previously shown that 5 days of mild chronic hypoxemia increases heart rate (hr) and blood pressure (bp). the aim of the present study was to investigate the effects of chronic mild hypoxemia on the expression of rat-ii in the medulla oblongata and on baroreflex control of the circulation. methods: at 125 days of gestational age, pregnant sheep were submitted to 5 days of hypoxemia (75% of fetal arterial po 2 ; at day 5 fetus 14±1 vs 20±0.5; mother 67±5 vs 116±3 mmhg). hr and arterial bp were continuously recorded from mother and fetus in control (n=10) and hypoxemic (n=10) animals. baroreflex sensitivity (brs) as well as hr and diastolic bp variability were analyzed (nevrokard software). brainstem was collected and rat-ii expression in the nucleus tractus solitarius (nts) and dorsal motor nucleus of the vagus (dmnx) was measured by autoradiography using 125 i-sarthran labeling. data are shown as mean±sem and were analyzed by t-test. results: hypoxemia induced a greater total binding of 125 i-sarthran in nts and dmnx resulting from an increased expression of at1 receptors. in the time-domain analysis of brs hypoxemia induces lower baroreflex sensitivity in the fetus (brs in ms/mmhg; 8.5±0.6 vs. 11.5±1.2, p<0.05). in the frequency domain, hypoxemia changes the relative power of low frequencies (lf: 0.04-0.15 hz) and high frequencies (hf: 0.15-0.4 hz) of rri and dbp with an increased value of the lf/hf ratio (rri lf/hf 3.3±0.4 vs 2±0.5, p<0.05; dbp lf/hf 2.7±0.3 vs 1.8±0.2, p<0.05). also the alpha index for baroreflex sensitivity is decreased in hypoxemic animals (alpha lf 9.2±1 vs 15.3±2.6, p<0.05; alpha hf 8.5±1.4 vs 19.8±5.3, p<0.05). no differences were observed in maternal variables. conclusions: our results suggest a link between a prenatal insult, alterations in cns receptors and functional alterations of baroreflex responses. a higher sympathetic outflow, suggested by a greater lf/hf ratio, and impaired reflex gain, both possibly mediated by increases in nts rat1, may have potential long-term consequences for the development of hypertension. hd37885;hd047584;hl51952. objective : we evaluated velocity profiles, relative wall distension rate (rwdr), and wall shear rate (wsr) of fetal descending aorta (fda) in uncomplicated singleton gestations. study design: ninety seven uncomplicated singleton fetuses were studied throughout gestation using multigate spectral doppler analysis (msda) working with gasp software. this consists of a personal computer (pc) add-on board including a single high-speed digital signal processor. the analysis of echo-signals backscattered from 256 range cells located along the axis of the interrogating ultrasound (us) beam. post-processing was accomplished using gasp software. statistical analysis consisted of spearman correlation and chi-square test. results: velocity profiles, wall distension, wall shear rate were obtained from fetal descending aorta throughout gestation establishing gestational age specific norms. wdr[%] is highly correlated with gestational age in appropriate growth fetuses (2.5±1.6 , rs=0.51 p<0.01) with linear regression with standardized coefficient of 0.4 (p<0.01). in contrast, wsr (421±97) is unchanged during the first , second and third trimesters (p=0.8).conclusion: we speculate that the relative wdr changes observed during gestation, in normally grown fetuses, may be secondary to adaptive vascular and autonomic responses and the evolving composition of the vessel wall, particularly with respect to elastin. conversely, the mean wsr for the study group was independent and constant throughout the gestation. these findings suggest that there is an increase in the diameter of the fetal aorta, which provides adaptation to the progressive flow demands, while preserving other key hemodynamic parameters. in this study, we have established normative values of rwdr and wsr, which are new rheological parameters that may be useful in distinguishing normal and pathologic hemodynamic states. objective: placental dysfunction is a key barrier to successful fetal cardiopulmonary bypass (cpb) for repair of congenital heart defects in utero. endothelial cells regulate vascular tone during fetal cpb through interactions of vasodilation by nitric oxide (no) and endothelin-1 (et-1)-mediated vasoconstriction. the objective was to determine the time during fetal cpb when endothelial cell-mediated changes occur. methods: human umbilical vein endothelial cells (huvec) were cultured in media containing 10% serum collected from ovine fetuses (n=3) that underwent 30 min of cpb, then were maintained for 120 min. serum was collected before cpb, from pump prime before initiation of cpb, 30 min on cpb, or 30 and 120 min after fetal cpb. control cells were cultured in normal fetal serum. cells were harvested 24 and 48 hr after addition of fetal serum. no production was measured in real time with an electrochemical detection system (inno-t, harvard apparatus). et-1 was measured in the culture media by elisa. results: no production by huvec after 24 and 48 hr was stimulated above control levels by fetal serum collected during and up to 120 min after fetal cpb (p< .01). serum collected from fetuses that were surgically instrumented, but not yet subjected to cpb, decreased no levels below controls (p<.01). stimulation of et-1 after 24 and 48 hr of huvec culture peaked with serum collected at 30 min after fetal cpb (p<.01 compared with control), but was elevated above control levels at each collection time point (p<.05, table 1 ). conclusions: fetal cpb releases serum proteins that elevate endothelial cell no and et-1 production during and for at least 120 min after cpb. although the specific regulatory proteins remain to be identified, the no and et-1 pathways share circulating mediators and participate in a feedback loop to modulate vascular tone. to test the hypothesis that hypoxia-induced upregulation of no is linked to cardiac inos expression, a selective inos inhibitor, l-nil (l-n6-(1-iminoethyl)-lysine), was administered in vivo to fetal guinea pigs and no levels measured in fetal hearts. methods: pregnant guinea pigs were exposed to either normal room air (normoxia; 21%o 2 ) or 10.5%o 2 (hypoxia) in a hypoxic chamber for 14 days prior to term (term=65d). l-nil was administered to pregnant normoxic and hypoxic guinea pigs via their drinking water at a dose of 1-2mg/kg/d for 10 days. at 60d gestation, pregnant sows were anesthetized and near-term fetuses removed via hysterotomy. the fetal hearts were excised, weighed, and normalized to their respective fetal body weights. left cardiac ventricles were obtained and frozen in liquid n 2 and stored at -80 o c until ready for analysis. the effect of total no product (no 2 and no 3 -, nox) of left ventricles of fetuses exposed to normoxia (n=4), hypoxia (n=5) and hypoxia plus l-nil (n=4) was quantified by a commercial fluorometric no assay kit. results: intrauterine hypoxia significantly reduced fetal body weight by 18% and increased placenta/fetal body wt by 33% as expected for hypoxic stress. hypoxia induced a slight increase in heart/fetal body wt by 4%. fetal cardiac nox levels (pmoles/mg) were increased by hypoxia (68.4+11.2) by 1.8 fold compared to normoxic controls (37.3+12.1). l-nil significantly decreased (p<0.05) nox levels in hypoxic hearts by 58% (68.4+11.2 vs 29.0+9.3; hypoxic vs hypoxic+l-nil, respectively). conclusion: l-nil inhibits inos-derived no generation in the hypoxic fetal guinea pig heart. since previous study in our lab showed a significant increase in inos expression but a decrease in enos and no change in nnos expression, we hypothesize that hypoxia upregulates cardiac no generation via the inos pathway. further study is needed to identify the important role of cardiac inos in the adaptive response of fetal hearts to chronic intrauterine hypoxia. objective: fetal asphyxia-mediated metabolic acidosis results in a ph decrease and an increase of lactate and base deficit in blood (bd blood ) and extracellular fluid (bd ecf ). it is not clear whether bd blood and bd ecf are similarly altered with worsening fetal acidosis. in the present study we sought to study the dynamic relations of bd blood and bd ecf to lactate in the ovine fetus subjected to repetitive uco with worsening acidemia. methods: ten near-term fetal sheep (125±2dga) underwent chronic preparation with brachial artery catheters and placement of an inflatable umbilical cord occluder. following a baseline recording period, fetuses underwent a series of mild (1min every 5min), moderate (1min every 3min) and severe (1min every 2min) uco with each series lasting 1h or until fetal arterial ph decreased to <7.0. fetal arterial blood was sampled at baseline, immediately before and during the first mild, moderate and severe uco and at 20min intervals during the moderate and severe uco. bd gap (bd blood -bd ecf ) was studied in relation to lactate. presented as means±sem. results: lactate correlated to bd blood (r=0.92, p<0.001). bd ecf correlated strongly with bd blood (r=1, p<0.001). bd ecf increased by 0.55±0.03 mmol/l more than bd blood from baseline to ph nadir, with bd gap therefore decreasing with increasing lactic acidemia ( fig. 1) . lactate, bd blood , bd ecf and bd gap correlated to ph (r=0.86, r=0.95, r=0.95 and r=1, respectively, all p<0.001) and ph could therefore be predicted with any of the four acid-base parameters. conclusion: the increases of bd blood and bd ecf and the decrease of bd gap with increasing lactic acidemia suggest a relatively more rapid accumulation of [h+] in ecf during uco of increasing severity. this may be because the metabolic build-up of acidosis occurs primarily in the tissues and the endothelial permeability for [h+] is impeded during increasing acidosis with atp depletion thus decreasing [h+] movement from ecf to plasma. previous studies have shown that intraperitoneal transplantation of human umbilical cord blood (hucb)-derived mononuclear cells led to the specific 'homing' of these cells to a hypoxic-ischemic brain lesion in perinatal rats. motor deficits resulting from the lesion were alleviated upon transplantation. thus, the presence of hucb cells at the lesion site seems to be a major prerequisite for their potential beneficial effect. however, the mechanisms of cell 'homing' are still unclear. in this study, we focused on elucidating mechanisms underlying the specific migration of hucb-derived mononuclear cells to the brain lesion. one possibility to induce cell 'homing' are chemotactic signals present at the lesion site. the cxc chemokine stromal derived factor-1 (sdf-1), which was previously shown to be a potent chemoattractant for directed migration of other stem and progenitor cells, is a putative candidate in our lesion paradigm. therefore we investigated the spatial and temporal expression of sdf-1 in brain hemispheres with or without hypoxic-ischemic lesion. sdf-1 expression was substantially increased at the lesion site during the investigated period of fourteen days after the insult. furthermore, hla-positive hucb cells were mainly detected in sdf-1 expressing brain regions and we were able to show that these cells express the sdf-1 receptor cxcr4 on their surface. the functional implication of sdf-1 in directing hucb cell migration was determined by application of neutralizing sdf-1 antibodies in vivo, resulting in a reduced number of hucb-derived mononuclear cells at the lesion site. with these functional effects, together with the observed timing and location of its expression, the involvement of the chemokine sdf-1 in hucb cell 'homing' seems conceivable. novel pathways in inflammation-induced fetal brain injury. michal a elovitz, jinghua chai. obstetrics and gynecology; crrwh, university of pennsylvania, philadelphia, pa, usa. intro: while survival of extremely preterm infants continues to improve, the number of children with cognitive impairment and/or cerebral palsy is increasing. if preterm birth (ptb) cannot be prevented, then strategies to identify and treat fetal brain injury in the setting of a ptb must be investigated. these studies were performed to explore novel pathways involved in fetal brain injury in the setting of inflammation-induced ptb. methods: cd-1 mice on e15 receive intrauterine injection of lipopolysaccharide (lps) or saline. 6 hours after injection, fetal brains from the left upper horn were harvested. 2 fetal brains were removed from each dam with 6 dams per treatment group. 12 separate rna samples were prepared and used for microarray (ma) analysis. all protocols were conducted as described in the affymetrix genechip expression analysis technical manual in the ma core facility using the moe 430av2 chip. data analysis was performed using significance analysis for microarray (sam). the brain samples from the same dam were considered as dependent samples. pathway analysis and functional annotation clustering tools were used with david. validations studies using qpcr with fetal brain samples (n=10-14 per group) were performed. results: while there was significant differences in gene expression between lps and saline exposed fetal brains, variability existed even between pups from the same dam. 542 genes were significantly differentially expressed between lps and saline brains (p<0.01). with a p value of <0.0001, 40 genes were differentially expressed. pathway analysis revealed significant involvement of 1) the cadeherin, cadherin-like, cell fraction and calcium binding (enrichment score 4.03) and 2) ribosomal processing, rna metabolism, pyrimidine metabolism (enrichment score 2.2). specifically, genes involved in neurogenesis, synaptic function, and neuronal and glial metabolism were most differentially regulated. qpcr confirmed observed fold changes in 6/10 genes analyzed. inflammatory pathways were not differentially regulated. conclusions: current theories regarding fetal brain injury in ptb focus on activation of inflammatory processes as essential events. this data suggests that long-term neurological injury in a ptb may be secondary to altered neuronal function, metabolism and/or communication. disruptions in these pathways should be explored as key mechanisms to adverse neurological outcomes in preterm neonates and should be targets for future investigations. regulation of microglial activation in the developing murine brain. mariya hristova, virginia zbarsky, daniel cuthill, adam wallace, donald peebles, gennadij raivich. institute for women's health, university college london, london, united kingdom. introduction: the aim of this study was to assess the process of microglial differentiation in developing white matter, an area of the brain that is particularly vulnerable to damage pre-myelination (approx 32 weeks gestation). microglia form a distinctive non-neuronal component. although related to peripheral macrophages they undergo highly specific processes of regional maturation and differentiation inside the brain with a slimming of the cell body, development of very elaborate crenulated arborised branches and downregulation of most macrophage activation markers. this process is relatively rapid in most grey matter brain regions, but is retarded in and around the subcortical white matter (swm) giving rise to the phagocytic fountains of microglia (fom). methods:we examined the process of deactivation and morphological differentiation in the cortex and swm of mice 0-28 days after birth (p0-p28) using confocal microscopy for monoclonal antibodies against alpha and beta integrin subunits and the costimulatory factor b7.2, colocalised with standard microglial marker iba1. results: strikingly, only the fom macrophages, but not cortical microglia, strongly expressed typical activation markers alpha-5, alpha-6, alpha-m, alpha-x, beta-2 and b7.2. the data for alpha-x are shown as an example in the figure; cortical microglia are shown in the grey bars and swm in black. fom activation was maximal at p0, decreased linearly over p3 and p7 and disappeared at p10. this process followed the presence of ingested phagocytic material but correlated only moderately with ramification, demonstrated by non-ramified but inactive p0 cortical microglia and formation of stubby processes in p3 fom. conclusion: these data describe strong and selective biochemical activation of fom phagocytes in p0-p7 swm, roughly equivalent to early 3 rd trimester human foetal development. this presence of highly active phagocytes in the neighbourhood of vulnerable wm could play an important role in the genesis of axonal damage in the foetus and premature neonate. 1997, pp. 13-25) . mbp is expressed in mature oligodendrocytes (jakovcevski and zecevic, glia 2005) . although myelination in the fetal sheep brain, a model extensively used to evaluate fetal development, has been described using conventional staining techniques (barlow, j comp neurol 1967) the use of specific mbp staining allows a more precise determination of the onset of myelination (antonow-schlorke et al., reprod sci 14.1, 2007, 248a) . aim: to use mbp expression to determine the trajectory of development of different white matter tracts of fetal sheep brain. methods: the ontogenetic profile of mbp expression was estimated in 47 healthy fetuses at 0.27 (n=6), 0.40 (n=5), 0.53 (n=5), 0.63 (n=6), 0.75 (n=12), 0.87 (n=9) and 0.93 (n=4) of gestation (term 150 days). after brain fixation and embedding in wax, sections at the level of the optic chiasm were stained with a monoclonal anti-mbp antibody using the abc-technique. immunohistochemical distribution of mbp was morphometrically assessed in the dorsal internal capsule and cortical white matter tracts, i.e. the lateral centrum semiovale, superficial white matter and median corpus callosum using an image analysis system (scion image 6.21, nih, usa). results: mbp expression of various fetal white matter structures started at different time points; initially in the internal capsule at 0.53 of gestation followed by the cortical white matter structures at 0.63 of gestation. cortical myelination advanced from the cortical deep white matter via superficial white matter to the corpus callosum reflecting different rates of progression. conclusions: the onset of mbp expression estimated here explicitly antedates previous observations in sheep (barlow, 1969) the way the embryonic heart functions before cardiac morphogenesis is completed is still subject of many studies. to gain more insight into the early cardiac structure-function relationship, doppler blood flow velocity waveforms at four different locations in the embryonic chicken heart during cardiovascular development were assessed. we collected waveforms using high frequency ultrasound biomicroscopy with a 55-mhz transducer at hh stages 18, 21 and 23 which are comparable to humans at 5 to 8 weeks of gestation. waveforms were obtained at the inflow tract, the primitive left ventricle, the primitive right ventricle, and at the outflow tract in ten different embryos per stage. by exploring the time relation between the waveforms, cardiac cycle events were outlined. our results demonstrate that stage and location dependent, intracardiac blood flow velocity waveforms can be obtained in the chicken embryo which reflect stage dependent pumping mechanisms. the blood flow profiles assessed at the four locations in the embryonic heart demonstrated a developmental related increase in velocity. in the primitive ventricle the passive filling wave decreased, whereas the active filling wave increased resulting in a decreased p to a ratio in the course of time. high frequency derived cardiac blood flow velocity characteristics support previous findings that the embryonic heart functions like a suction pump at early development and transforms towards another pumping mechanism at later developmental stages. these findings are of importance for the interpretation of human first trimester cardiac velocimetry studies. figure 1a . changes in the perimeter of the thymus with gestational age by fetal gender are depicted in figure 1b . background. for the fetus, the roles of arterial blood gases are recognized to be critical in the regulation of cerebral blood flow (cbf) and cerebral metabolic rate for oxygen (cmro 2 ), cortical tissue po 2 (tpo 2 ), and electrocorticographic (ecog) activity. in addition, metabolites such as adenosine (ado) are important in this regard. nonetheless, the relation of adenosine and its metabolites to these indices of cerebral oxygenation are not well defined. in an effort to elucidate these interrelations, we tested the null hypothesis that acute hypoxic-associated cerebral oxygenation and related variables are not closely associated with adenosine. the most common cause of perinatal brain injury is chronic hypoxia/ischemia. the mechanisms are complex and poorly understood. applying proteomics tools, we identified a set of hypoxia-related proteins in the fetal guinea pig (gp) brain (companion abstract). one protein, cofilin-1, which regulates the rapid cycling of actin assembly and disassembly, was dramatically altered by chronic brain hpx. herein, we test the hypothesis that confilin-1 modifications during hpx contributes to brain damage via apoptosis and is an example of an adaptive response that becomes maladaptive. methods: time-mated gps were housed in a chamber from 50d to 65d (term) with either room air (nmx) or 10.5% or 12% o 2 . fetal brains were removed and sections prepared for double staining specific to bax and dephosporylated cofilin-1. total protein was isolated and equal amounts loaded on a sds gel, separated by electrophoresis, and transfered to pvdf membranes probed with primary antibodies to dephosphorylated and phosphorylated cofilin-1, bax and bcl-xl, and incubated with second antibody. protein signals were detected, quantified by densitometry, and normalized to -actin. total rna was isolated, reverse-transcribed, and quantified by real-time pcr using sybr green i labeling. expression was calculated by the ct method using 18s for control. melt analysis was performed to confirm specificity of the amplification, and efficiency determined by the slope of the standard curve. the mitogen activated protein kinase (mapk) signalling pathway is upregulated in perinatal hypoxic-ischaemic brain. the role of the extracellular signal-regulated kinase (erk) cascade, a pivotal component of mapk signalling, remains unclear with reported actions ranging from mitogenic and trophic effects to a neuronal death-promoting role. the aim of this study was to assess the role of neuronal erk activity using selective neuronal inhibition in a perinatal model of hypoxic-ischemic brain injury. methods: we explored the effects of selective neuronal inhibition of erk activation using transgenic mice expressing dominant-negative (dn) mek1, the upstream activator of erk1/2, which was under the control of the pan-neuronal talpha1 alpha-tubulin promoter. p-erk immunoreactivity was quantified following a hypoxic inschemic (hi) insult in p7 c576bl/6 mice, caused by unilateral occlusion of the carotid artery, followed by hypoxia with 8% oxygen for 60 mins at 36°c. the volume of surviving brain on the affected hemisphere was expressed as a % relative to the control side. results: expression of the mek1 dn was associated with a reduction in erk1/2 activation following hypoxia ischaemia, as assessed by p-erk immunoreativity. compared with the wild type, littermate controls, mek1dn animals exhibited significantly decreased volume of forebrain damage brain following unilateral, 60 min hi insult (*p<0.05, **p<0.01, anova). similar protective effects of mek1dn were observed in cerebral cortex, hippocampus, thalamus and striatum when compared to wild type littermate controls and correlated with a significant reduction in microglial activation in all brain areas. conclusion: overall, these results suggest that neuronal mek1 and its downstream signals have an important death-inducing role in this model of perinatal brain injury and could serve as potential targets for therapeutic intervention. oup, 2004) . however, whether melatonin protects placento-fetal development during hypoxic or undernourished pregnancy is unknown. we investigated in rats the effects on placental and fetal growth of maternal treatment with melatonin in hypoxic and undernourished pregnancy. methods: on pregnancy day 15, wistar rats were divided: control (21% o 2 ) + melatonin (5 g.ml -1 drinking water), hypoxia (10% o 2 ) + melatonin, and undernutrition (40% reduction in food intake) + melatonin (n=7 per group). on day 20, dams were anaesthetised, the pups, placentae and fetal brain were weighed. results: relative to controls (fetal weight: 3.73±0.05g; brain/body weight ratio: 46.2±1.2mg/g; n=76), both hypoxic (2.94±0.03g; 58.4±1.1mg/g, n=80) and undernourished pregnancy (3.05±0.04g; 53.0±1.1mg/g, n=80) promoted asymmetric iugr (p<0.05), without affecting placental weight. melatonin treatment had no effect on iugr, but it decreased placental weight in normoxic (0.43+0.01, n=95), hypoxic (0.44±0.01, n=98) and undernourished (0.41±0.01, n=81) pregnancies relative to untreated controls (0.48±0.01, n=76; p<0.05). when fetal body weight was expressed relative to placental weight, a measure of placental efficiency, melatonin prevented the fall in the ratio in undernourished, but not hypoxic pregnancies (fig.1 objective: midkine (mk) is a 13-kda heparin-binding growth factor with various functions including cell proliferation, migration, differentiation and angiogenesis. mk expression is strictly regulated in temporal sequence; it is highly expressed during midgestation. we recently studied mk expression in the midgestation human fetus, and showed that the highest mk expression were observed in the adrenal gland, brain, lung and kidney. in the present study, we investigated the profile of mk in human amniotic fluid (af) and amnion (am). methods: amniotic fluid and amniotic membranes were collected at diagnostic amniocentesis, preterm no labor, and term no labor. mk protein levels were analysed by western blot. expression of transcripts encoding mk and putative mk receptors were examined by rt-pcr and real-time quantitative rt-pcr (qpcr). results: 1) western blot analysis demonstrated abundant mk protein in the human am; mk levels were higher at midgestation than at term (3-fold: 16wks vs. 37wks). 2) tissue transglutaminase known to polymerize mk was abundant in af. 3) qpcr revealed that mk mrna was not expressed in am whereas it was highly expressed in the fetal adrenal, kidney and lung (positive controls). 4) among the receptors implicated in mk signaling, lowdensity lipoprotein receptor-related protein and syndecan-4 were expressed in am while protein-tyrosine phosphatase, anaplastic lymphoma kinase, and syndecan-3 were not. conclusions: abundant mk protein in the midgestation af is likely to be derived from the fetus. mk in af may play a role in feto-amniotic communications and/or development of fetal organs exposed to af. short term hfix expression. we hypothesized that long term hfix expression could be achieved in fetal sheep using an aav vector, without stimulating an immune response to the transgenic hfix protein. we injected aav8 hfix vector (1 -9 x 10 12 p/kg) into the peritoneal cavity of fetal sheep under ultrasound guidance in early (n = 3) or late (n = 4) gestation. fetal blood was retrieved by ultrasound guided sampling from the intra-hepatic umbilical vein. fetal and lamb blood was tested for hfix expression using elisa, antibody responses using functional assays, and for liver damage up to a year after birth. vector spread was detected in maternal and fetal tissues by quantitative pcr analysis. results: the highest level hfix was detected 18 days after late ip injection (44% and 28% normal human levels). early gestation ip injection gave 8.7% and 1.8% at 3 and 21 days after injection. hfix levels dropped rapidly correlating with the increase in size of the fetal liver and lamb. however, hfix was detectable at a low levels (0.7%) 1 year after birth in early and 4 months after birth in late gestation injected lambs. up to 1 year after birth, liver function tests and bile acid levels were normal, showing no evidence of liver pathology. no functional antibodies to the hfix protein or aav vector were detectable. high vector levels were detected in the fetal liver, and other peritoneal organs; no vector was present in fetal gonadal tissue. conclusion: hfix expression is detectable up to 1 year after delivery of aav vector to the fetal sheep using a clinically applicable method. this is the first study to show long term hfix expression after fetal gene therapy in a large animal. further work will include testing for immune tolerance to exogenous hfix protein in these animals. objective: placental estrogens play a pivotal role in the endocrine control of pregnancy and may be involved in the key changes occuring during parturition. it has been established that crh interacting with crh receptor 1 has a positive effect on estrogen production throughout pregnancy. urocortin 2 (ucn 2), a novel peptide of the crh family binding exclusively crh receptor 2, is expressed by human placenta and the aim of the present study was to evaluate the influence of ucn 2 on estrogen biosynthesis in cultured trophoblast cells. methods: cultured term placental cells were treated with various concentrations of ucn2 in the presence of the estrogens precursors dehydroepiandrosterone sulphate (dhea-s), androstenedione or testosterone. estradiol secretion was measured in the culture medium using a specific elisa, p450arom mrna and protein expression were evaluated by real time pcr and western blot analysis respectively. results: the addition of ucn2, in presence of dheas significantly increased e2 levels at 4, 8 and 12 hours treatment, while in presence of androstenedione and testosterone an increase in e2 secretion was detected only at 12, 24 and 36 hours (at different ucn2 doses). both p450arom mrna and protein were up-regulated in presence of each estrogen precursor and the addition of ucn2 caused a synergistic increase. anti-sauvagine 30 (a crh type 2 receptor antagonist) resulted in a significantly attenuated ucn2 effects on e2 secretion and on p450arom mrna and protein expression. conclusions: the present study supports a possible role of ucn2 on placental e2 biosynthesis: e2 secretion and p450arom transcript and protein expression were significantly increased after ucn2 treatment. in conclusion, the crf family may play a major role on placental steroidogenesis, stimulating dheas secretion in fetal adrenals by crh and controlling placental estrogen biosynthesis through ucn2. a possible influence on the mechanisms of parturition may be hypothesized. increased expression of kiss-1 gene in preterm placenta. letizia galleri, michela torricelli, chiara voltolini, fernando m reis, felice petraglia. department of pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. introduction placenta expresses a large number of peptides involved in delivery. neuropeptides, cytokines, are expressed and secreted by human placenta at the time of preterm delivery. human placenta is a major source of kiss-1, a 54-amino-acid peptide encoded by a putative metastasis suppressor gene. kiss-1 acts on its placental g protein-coupled receptors (kiss-1r); this peptide stimulates release of oxytocin in rats, the most potent known uterine stimulant, suggesting a possible role of kiss-1 in the mechanisms of labor. aim of study the aim of this study was to evaluate placental expression of kiss-1 at preterm labor. material and methods placental tissue and plasma samples (both maternal and fetal) were collected at term in the absence of labor (tnl), at term spontaneous vaginal delivery (tl), and at preterm labor (ptl). changes in placental mrna expression were determined by real-time quantitative rt-pcr analysis. kiss-1 protein levels were measured by specific immunoenzymatic assay (elisa). results placental kiss-1 mrna expression was significantly higher (p<0.0001) in ptl than in tnl and in tl. however, maternal and fetal plasma kiss-1 levels did not differ among tnl, tl, and ptl samples. conclusion the present study showed that placental kiss-1 mrna expression is increased in preterm delivery. further studies are needed to better understand the role of kiss-1 on cascade leading term and preterm labor. placental angiogenesis is essential for placental development, which occurs under a hypoxic environment ( 2-8 % o 2 ) during normal pregnancy. it has been reported that in transformed human dermal microvascular endothelial cells, hypoxia activates erk1/2, which stabilizes hypoxia-inducible transcription factor-1 (hif-1 ). however, signaling mechanisms governing placental angiogenesis under hypoxia is largely unknown. herein, we tested whether hypoxia affected fgf2-and vegf-stimulated cell proliferation partly via activating erk1/2 and stabilizing hif-1 protein levels in human placental artery endothelial (hpae) and transformed human placental microvascular endothelial (hpme) cells. methods: cells cultured under normoxia ( 20% o 2 ) or hypoxia (3% o 2 ) for 2 days were treated with fgf2 and vegf. after 3 days, the number of cells was determined. activation of erk1/2 and levels of hif-1 protein were determined by western analysis. results: under normoxia, fgf2 and vegf stimulated (p < 0.05) cell proliferation and induced ( 5 min, p < 0.05) erk1/2 phosphorylation in hpae and hpme cells. hypoxia promoted (p < 0.05) fgf2-and vegf-stimulated cell proliferation in hpae cells, whereas hypoxia blocked (p < 0.05) such actions in hpme cells. hypoxia enhanced fgf2-, but not vegf-induced erk1/2 phosphorylation in hpae cells. in contrast, hypoxia promoted (p < 0.05) vegf-, but not fgf2-induced erk1/2 phosphorylation in hpme cells. hypoxia increased (p < 0.05) hif-1 levels in the hpae cells: first detected at 0.5 hr and maintained up to 24 hr. hpme cells had high basal levels of hif-1 (3 folds; p < 0.05) compared with hpae cells. hypoxia did not alter hif-1 levels up to 4 hr and decreased (p < 0.05) hif-1 levels at 24 hr in hpme cells, conclusions: in hpae cells, hypoxia enhances fgf2-and vegf-stimulated cell proliferation possibly partially via promoting activation of different protein kinases. this stimulatory effect is associated with increased hif-1 protein levels. however, inhibition by hypoxia on fgf2-and vegf-stimulated hpme cell proliferation is associated with relatively high hif-1 levels in the first 4 hr and decreased hif-1 levels at 24 hr. thus, these data suggest that different signaling mechanisms are involved in hypoxia-modulated growth factor-induced placental angiogenesis in which an increase in hif-1 levels plays a critical role. objective: corticotrophin releasing factor (crf) is a well established neurohormone involved in the initiation of the stress response. we recently documented that rat placenta is analogous to human placenta in the expression of crf-mrna and protein, and that placental crf release may contribute to in utero meconium passage. time-of-day variation in crf expression is a highly recognized phenomenon at the brain cellular sites of crf synthesis. we sought to determine whether crf expression in rat placenta is subject to time-of-day variation. method: time-dated pregnant rats were obtained on day 12 of gestation (term=22 days), housed in under 12h-12h light-dark conditions (lights on 0600). lab chow and water were continuously available. on day 18, pregnant rats were quickly anesthetized by exposure to isoflurane, abdomen opened and fetuses and placenta exteriorized either at 0545-0600hr (zeitgeiber time, zt0) or 1245-0100hr (zt6). individual placentas were processed either for rna extraction or immunohistochemical investigation. the levels of crf-mrna were assessed by pcr using rat specific pcr primers. pcr bands were subsequently cloned and sequenced. bouin's solution fixed paraffin sections of placenta were subjected to crf immunohistochemistry with antibodies specific to rat species and intensity of immunostaining was analyzed using image pro-plus software and expressed in arbitrary units (au). results: one specific pcr band of 339 bp size was consistently identifiable in placenta harvested at zt6 but not at zt0. nucleotide sequence of the pcr band confirmed its identity as crf-mrna. intensity of crf-immunostaining was significantly greater at zt6 in giant trophoblast (gt) cells (gt-crf: zt0= 0.176 ± 0.122 au, zt6= 0.272 ± 0.014 au, p=0.007) but not in spongiotrophoblast cells (stc) (stc-crf: zt0 = 0.218 ± 0.025 au, zt6= 0.162 ± 0.011au, p=ns) or labyrinth cells (lbc) (lbc-crf: zt0= 0.148 ± 0.003, zt6 = 0.149 ± 0.015 au, p=ns) . conclusion: similar to adult brain, rat placenta expresses crf mrna and protein. time-of-day variation of crf expression originally seen in central nervous system neurons is also identifiable in giant trophoblasts cells at zt6. these findings suggest that stress-mediated placental crf release, and potentially fetal meconium passage, may be dependent upon time-of-day. objectve: transplacental water flow is essential for the provision and maintenance of fetal body water and amniotic fluid. water flow across membranes is regulated by aquaporin (aqp) water channels in many tissues. thus aqp1, expressed in the placenta, is a candidate to regulate maternal to fetal fluid exchange. maternal beta-mimetics have been hypothesized to augment fetal growth by increasing the availability of nutrients and perhaps water. as camp acts as a second messenger to increase expression of selected aqps in other tissues, we sought to determine if betamimetics acting through camp could modulate placental aqp1, and potentially influence placental water transfer. methods: trophoblastic cell cultures were established in first trimester-derived extravillous htr-8/svneo cells and term placenta-like trophoblast carcinoma cell line jeg-3. cultures were treated with sp-camp, a membrane-permeable and phosphodiesterase resistant camp, and forskolin, an adenylate cyclase stimulator, in doses of 0.5, 5 and 50 m for 2 hrs (aqp1 mrna expression) and 20 hrs (aqp1 protein expression). for time course experiments, cells were incubated with 5 μm of either sp-camp or forskolin for 2, 10 and 20 hrs at 37°c, 5% co 2 . after cell harvest, mrna and protein expression were assayed using real time pcr and western blotting. results: sp-camp and forskolin increased aqp1 mrna expression in both cell lines after 2 hrs (p<0.05) in a dose-dependent manner. protein expression paralleled the increase seen in the mrna. 5 m of sp-camp and forskolin stimulated aqp1 mrna expression after 2 hrs in htr-8/svneo cells and after 10 hrs in jeg-3 cells (p<0.05). 5 m of either sp-camp or forskolin stimulated aqp1 protein expression in both cell lines after 10 hrs (p<0.05) and the expression remained high at 20 hrs. conclusion: aqp1 gene expression in trophoblast cells is up-regulated by camp agonists. these results suggest that maternal beta-adrenergic agonists or antagonists may modulate maternal-fetal water flux via modulation of aqp water channels. rat placenta expresses corticotrophin releasing factor-binding protein and mrna. jayaraman lakshmanan, thomas r magee, bindu cherian, sharon k sugano, hanalise huff, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor-binding protein (crf-bp), a 37 kda secreted glycoprotein, was originally isolated from human plasma. it binds crf and urocortin i with an equal or greater affinity than does the crf receptor. crf-bp expression has been reported in target tissues such as brain and placenta. based on its ability to inhibit crf actions in vitro, it is speculated to function as a "gate keeper" for crf-initiated stress responses. we recently documented expression of crf and urocortin-1 in rat placenta. in the present study we sought to establish the expression of crf-bp in rat placenta by immunohistochemical and pcr-analyses. methods: placental tissues (n=20) collected from sprague-dawley pregnant rats on day 21 of gestation (term=22) were either fixed in 4% paraformaldehyde solution (ph 7.4) and paraffin embedded or processed for total rna extraction. paraffin sections of five micron thickness were subjected to immunohistochemical analysis with goat polyclonal antibodies to human crf-bp precursor (sc-1824 santa cruz biotechnology, ca) by avidinbiotin-peroxidase complex technique. the structure of cloned human crf-bp precursor exhibit significant amino acid sequence homology among all species studied. immunoreactivities on the sections were quantified using image pro 4.01 software and staining intensity (od/area) expressed as arbitrary units (au). all values are expressed as mean±sem. for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, clone into plasmid vector and dna sequenced. results: the crf-bp antibody elicited strong positive staining in decidua, giant trophoblasts, spongiotrophoblasts and labyrinth cells. the results of image analyses revealed (au): decidua: 0.191±0.010, giant trophoblast cells: 0.139±0.029, spongiotrophoblasts: 0.120±0.003, fetal membranes: 0.128±0.009. pcr analyses identified a single 434 bp band, consistent with crf-bp. conclusion: our study establishes for the first time that rat placenta is analogous to humans in that both express crf-bp mrna and protein. immunohistochemical findings reveal that crf-bp protein expression occurs at multiple sites within the placenta. expression of per2 clock protein in rat placenta: an internal timer for placental functions. jayaraman lakshmanan, reuben lakshmanan, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: corticotrophin releasing factor (crf) expression time-of-day variation occurs in giant trophoblast cells on the maternal side of the rat placenta. this temporal change in crf expression pattern is very similar to that of central nervous system neurons, suggesting that placental cells may use a similar mechanism to read time of the day. the self-sustaining rhythm generating capacity of the suprachiasmatic nuclei is believed to be derived from cell-autonomous, transcriptional feed-back loops dependent on a number of canonical clock genes. in the present study we sought to determine whether rat placenta expresses period gene 2 (per 2), one of the clock/cycle related genes. methods: time-dated pregnant sprague-dawley rats were received on day 13 of gestation and housed in a controlled environment (0600-1800h lights on) with free assess to food and water. placentas collected on days 20 and 22 of gestation (term=22days) between 0930 and 11.30 am were fixed in 4% paraformaldehyde and paraffin embedded. for each gestational ages a total of 12 placentas from 6 different pregnant rats were used. paraffin sections (3 sections per placenta) were subjected to immunohistochemical analysis with antibody to per2 (1:200, santa cruz biotechnology, ca) by abc technique and 3, 3'diaminobenzidine as a chromagen. sections were examined under microscope, imunostaining quantified by image pro 4.01 software and expressed in arbitrary units (au). data are presented as mean ± sem. statistical significance was analyzed by anova with a p value < 0.05 as significant. conclusion: the present results indicate that rat placental cells, devoid of any neural innervations, express per2 clock protein, similar to central nervous system neurons. based on the differences in the relative intensities between trophoblasts and labyrinth cells on day 22 of gestation, we conclude that fetalmaternal interactions in per2 regulation disappear at term (or at the time of birth.). our findings imply that per2 may function as internal physiological modulator in placenta. to determine the mechanism(s) underlying placental time-of-day crf variation, we examined the effect of maternal administration of betamethasone (a synthetic glucocorticoid) on nuclear gc receptor expression in placental cells. method: time-dated pregnant rats (n=6) on day 21 of gestation were given a single subcutaneous injection (at 5:45 am) of betamethasone (350μg/kg body weight) while control pregnant rats (n=6) received saline. dams were exposed to isoflurane anesthesia at 10:45 am, and placentas harvested, fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analysis with gc-receptor antibody (sc-8992, santa cruz biotechnology), with immunoreactive material identified by abc technique using 3, 3' diaminobenzidine as a chromagen. percentages of gc-nuclear receptor (gc-nr) positive cells and their intensities (od/area) were quantified using image pro 4.01 plus software. all values are expressed as mean ± sem. statistical analysis was by anova with p<0.05 considered significant. results: in placentas of saline exposed pregnant rats, isolated labyrinth (lb) cells (<1%) were positive to gc-nr. no positive staining for gc-nrs was seen either in giant trophoblasts (gt) or in spongiotrophoblasts (st). betamethasone administration was associated with a significant and marked increase in gc-nr staining in all three placental cell types (p<0.05). among the cells, gt (69±10%) demonstrated a greater percentage of cells expressing gc-nr than did st (34±5%) or (lb=38±5%) (p<0.05), though there was similar immunoreactive intensities (gt: 0.216±0.017, st: 0.238±0.031, lb: 1802±0.022 au; p=ns) conclusion: our findings indicate that all placental cells respond to gc with upregulation of gc-nr with an enhanced response among gt cells. these results suggest that endogenous or exogenous maternal stress-induced gc exposure may influence signaling responses within both maternal and fetal placental compartments. ovine placenta at very-preterm gestation expresses corticotrophin releasing factor (crf), crf-binding protein (crf-bp) and glucocorticoid receptors (gr). jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: in humans, the placenta is the major source of maternal and fetal plasma crf. based on its critical functions, crf is considered as a "placental clock" of parturition. we recently reported that ovine fetal as well as maternal plasma contain measurable amounts of crf at near-term but not at very preterm gestation. we interpret the absence of crf in plasma at very preterm gestation is either due to lack of placental crf expression and/or placental crf release. here, we examined the expression status of crf, crf-bp (a known specific binding protein for crf and a known regulator of crf functions in human placenta) and gr (a known positive regulator of crf expression in human placenta) in ovine placenta collected at very-preterm gestation. method: placenta harvested from time-dated pregnant ewes on 118±2 days gestation were fixed in bouins's solution and processed for paraffin embedding. paraffin sections were subjected to immunohistochemical analysis with polyclonal antibodies specific to ovine crf(1:300-1:500, phoenix pharmaceuticals, ca,) crf binding protein (1:100-1:200, sc 20630) and gr (1:100-1:200, sc: 8992, santacruz biotechnology, ca). immunoreactive material on the sections were identified as brown staining by abc technique using diaminobenzidine as chormagen. immunoreactive material was quantified by image analysis using image pro 4.01 plus software and the immuoreactive intensity (od/area) expressed as arbitrary units (au). results: strong positive staining for crf (0.363±0.033 au), crf-bp (0.505±0.003 au) and nuclear-gr (0.383±0.015au) were noticed in syncytiotrophoblast cells in all placental sections obtained from pregnant ewes at118 days gestation. control sections exhibited no positive staining. conclusion: our findings indicated that ovine placenta at very-preterm gestation expresses crf, crf-bp and gr. these results suggest that absence of measurable crf in maternal and fetal plasma at very-preterm gestation is not due to lack of placental crf expression but rather due to the absence of regulated crf secretory mechanisms. rat placenta expresses urocortin i protein and mrna. thomas r magee, michael g ross, john d richard, sharon k sugano, jayaraman lakshmanan. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: urocortin i (ucn-1), a 40 amino acid neuropeptide belongs to corticotrophin releasing factor (crf) stress hormone family. in humans, the placenta and several gestational tissues have been reported to express ucn-1 protein and ucn-1 mrna. a number of published studies indicate that ucn-1 is similar to crf in expression pattern and biological functions. we recently reported that rat placenta is a site of crf protein and crf mrna expression. in the present study we sought to determine whether rat placenta expresses ucn-1 protein and ucn-1 mrna. methods: placenta (n=18) collected from pregnant rats at 21 day gestation were either fixed in bouin's solution and processed for paraffin embedding or placed in rna later preservative and frozen for rna extraction. for immunohistochemical localization, five micron thickness paraffin sections were cut and immunostained with rabbit polyclonal antibodies to ucn-1 (1:500 to 1:750, sigma) by standard abc technique. control sections were incubated with omission of ucn-1 antibody. immunoreactive materials on the sections were identified using 3, 3' daminobenzidine as chromagen. immunostaining intensity (od/area) was quantified by image-pro plus software and expressed in arbitrary units (au). for pcr, cdna was synthesized and pcr amplified with a one step rt-pcr kit. fragments were gel purified, cloned into a plasmid vector and dna sequenced. all values are given as mean ± sem. results: ucn-1 polyclonal antibody elicited strong immunostaining in placental trophoblast cells with variable intensity. the pattern of immunostaining is as follows: giant trophoblast cells: 0.334±0.319 au, spongiotrophoblast cells: 0.318±0.013 au and labyrinth cells: 0.233±0.139 au. the relative intensity in labyrinth cells was significantly lower than the other two cell types (p< 0.01). pcr analyses revealed the presence of a single band of 379 bp, consistent with ucn-1 mrna. conclusion: similar to human placenta, rat placenta expresses ucn-1 protein and ucn-1 mrna, with most expression localized to the maternal side trophoblast cells. these results support our hypothesis that rat placenta can be used as a model to understand the role of this peptide in feto-maternal stress. the role of the nuclear hormone receptors fxr, pxr and car in placental bile acid homeostasis in normal and pathological pregnancy. victoria geenes, peter dixon, selina raguz, jenny chambers, kishore bhakoo, catherine williamson. imperial college, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder characterised by raised maternal serum bile acid levels and associated with adverse fetal outcome. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from an accumulation of bile acids in the fetal circulation. bile acids are the toxic end products of hepatic cholesterol metabolism and are synthesised from 12 weeks gestation. in common with other waste products, accumulation in the fetal compartment is prevented by excretion across the placenta into the maternal compartment. however, studies of maternal and cord serum from normal and oc pregnancies have suggested that bidirectional transfer of bile acids is possible. hepatic bile acid transport and metabolism is regulated by members of the nuclear hormone receptor family, namely fxr, pxr and car, but the mechanisms for regulating placental transfer are unknown. objectives: this study used placenta from normal and cholestatic pregnancies to investigate the expression of genes involved in hepatic bile acid homeostasis. methods: villous trophoblast samples from 6 oc and 7 normal pregnancies (np), and 3 human livers were collected and preserved in rnalater. explant cultures were prepared from a further 4 np placentas and cultured for 6 days at 8% oxygen. on day 5 they were treated with chenodeoxycholic acid, lithocholic acid or vehicle for 24 hours prior to fixing in rnalater. total rna was extracted using trizol, and reverse transcribed to cdna. quantitative real-time pcr was performed using sybr green. target gene mrna abundance was calculated from a standard curve and normalised to l19. results: the expression of fxr, pxr and car, the nuclear hormone receptors responsible for hepatic bile acid homeostasis and several fxr target genes (shp, mdr3 and bsep) was found to be very low in np, and unaffected by the presence of maternal cholestasis. furthermore, the expression of these genes could not be induced by bile acid treatment in an in vitro model. here we have shown that the nuclear hormone receptors (fxr, pxr and car) involved in hepatic bile acid homeostasis are expressed at very low levels in normal and pathological pregnancy and are thus likely to be of limited importance in placental bile acid transfer. a placental phenotype for obstetric cholestasis. victoria geenes, 1 jenny chambers, 1 jo wyatt-ashmead, 2 kishore bhakoo, 1 catherine williamson. 1 1 imperial college, london, united kingdom; 2 hammersmith hospital, london, united kingdom. background: obstetric cholestasis (oc) is a pregnancy specific liver disorder that affects 0.7% of pregnant women in the uk. biochemically, oc is characterised by liver dysfunction with elevated maternal serum bile acids (sba), and clinically by an increased risk of fetal complications including fetal distress, meconium staining of the amniotic fluid, preterm labour and sudden intrauterine death. the aetiology of oc is complex and not fully understood, but the fetal complications are likely to result from the toxic effects of bile acids, which can cause vasoconstriction in the placenta and fetal cardiac dysrrhythmias. furthermore, in a rodent model of oc, bile acids cause oxidative stress in the placenta and a reduction in litter size. these changes are absent in animals treated with ursodeoxycholic acid (udca), a drug used to treat oc. however human data are lacking. objectives: this study used whole placenta and explant cultures to investigate the effects of bile acids on human placental architecture and to establish whether udca protects the placenta against bile acid induced damage. methods: villous trophoblast samples were collected from 15 oc and 5 normal pregnancies (np) and fixed in formalin. explant cultures were prepared from a further 4 np placentas, and cultured for 6 days at 8% oxygen. on day 5 a subset were treated with udca overnight, and on day 6 the explants were treated with taurocholic acid, taurochenodeoxycholic acid or vehicle for 6 hours prior to fixing. 5 m sections were stained with haematoxyllin and eosin. slides were reviewed by a perinatal pathologist and syncytial knots (sk) counted. results: oc tissues showed marked alterations in morphology, including congestion of the terminal villi, and loosening of the stroma and fibrotic change of the membranes of the stem villi. there were significantly more sk the oc samples (mann-whitney u test p=0.024). furthermore, sk were increased in explants treated with bile acids, but not those treated with udca prior to the addition of bile acids. conclusions: here we describe several morphological abnormalities of the placenta associated with maternal cholestasis. these changes were confirmed using a placental explant model, which also showed that udca protects the placenta against bile acid induced damage. in summary, this study indicates that udca is likely to protect the fetus in oc. the placenta has complex metabolic and endocrine activities and is essential for the growth and survival of the fetus in utero. ultrasound is the most sensitive and less invasive method to evaluate placental size, morphology and function. the three-dimensional approach allows to calculate placental volume in the i and ii trimester of pregnancy. pregnancies from assisted reproduction technologies (art) show an increased rate of pathologies potentially related to placental insufficiency such as intrauterine growth restriction and preterm delivery. aim. to determine placental volume in art pregnancies in a cross sectional study in the first trimester of gestation. design. using three-dimensional ultrasound machine (ge 730) placental volume measurement from art pregnancies (n=26; 10-13 wks) were compared with data from normal controls (n=26) matched for gestational age. data were analysed with software stata 9. results. mean placental volume was 46.2 ml (sd±23.9) in art pregnancies and 58.4 ml (sd±22.9) in normal controls. mean difference resulted in 12.12192 ml (-1-25.19835) and was statistically different (p=0.047). multiple linear regression analysis showed a statistically significant interaction (p=0.04) between gestational age and case status, i.e. differences in placental volume increase significantly with advancing gestational age between cases and normal controls. mean gestational age at birth was not essentially different between the two subsets (39.1 weeks ± 1.6) however a statistically significantly lower mean fetal birthweight was found in art pregnancies: 2945 g (sd±537) vs 3226.(sd±294.4) (p=0.044). conclusions. placental volume is slightly decreased in art pregnancies. measurements of placenta volume by 3d ultrasound may play a role in identifying the degree of placental growth early in gestation in a risk population. in vitro effects of adenovirus-mediated gene transfer of insulin like growth factor-1(ad-igf-1) in trophoblast cells. mounira habli, 1 datis alae, 2 foong yen lim, 2 jignesh parvadia, 2 timothy crombleholme. 2 1 obstetrics and gynecology; 2 pediatric surgery, university of cincinnati/children's hospital, usa. objective:recently, ad-igf-1 corrected both fetal and placental weights in rat model of fetal growth restriction (fgr) . to elucidate the mechanism of action of ad-igf-1;we examined the effect of ad-igf-1 on trophoblast proliferation, differentiation and invasion. methods: rcho-1 trophoblast cell line derived from rat choricarcinoma was used. ad-igf-1 and galactosidase transgene (ad-lacz) were given at moi of 10:1 and 100:1 in all the experiments.transduction efficiency was assessed 24 hr after infection with ad-lacz by galactosidase enzyme activity. the invasiveness of rcho-1 was measured by using bdmatrigel invasion chamber kit. rcho-1 proliferation cell density was assessed by crystal violet assay. morphologic analysis of rcho-1 differentiation in response to treatment (differentiated cells are multinucleated giant cells) was assessed byimunocytochemistry staining using placental lactogen-1 antibodies(specific hormone produced by giant cells). results: 100% efficiency of gene transfer of ad-lacz to rcho-1 cells was observed at both moi's.there was no significant difference in proliferation between treated control group regardless of the dose at 12 and 24 hrs. ad-igf-1 induced morphologic differentiation of trophoblast cells compared to control (fig1) at 24 hrs.ad-igf-1 induced higher rate invasion in a dose dependent fashion as compared to control(ad-lacz) group(16% at 100moi vs 0.8% in control p<0.008)(fig2). conclusion: ad-igf-1 gene transfer induces differentiation and invasiveness of trophoblast cells. these may be the mechansim of correction of fgr in rat model of placental insufficiency. placental gene transfer may be an effective treatment strategy for placental insufficiency. types of human placenta. martina dieber-rotheneder, ursula hiden, gernot desoye, mila cervar-zivkovic. department of obstetrics and gynaecology, medical university graz, graz, austria. background and hypothesis: endothelin-1 is a polypeptide with a wide range of functions. in the placenta it acts as a potent regulator of vasotonus on endothelial and smooth muscle cells, whereas on the trophoblast it regulates cell proliferation, invasion and apoptosis. endothelin-1 action is differently signaled through two endothelin receptor (etr) subtypes, etr-a and etr-b. several alternative splice variants of etr were identified. here we hypothesize that the etr-a splicing varies with gestational age and is different in trophoblast vs endothelial cells of the human term placenta. methods: mrna of placental tissue from first trimester (pregnancy terminations, missed abortions) and term of gestation, first trimester and term trophoblasts, arterial and venous placental endothelial cells as well as cell lines representing first trimester trophoblast (ach3p) and term placental endothelial cells (hpec) were analyzed for full length etr-a and known as well as unknown splice variants by sqrt-pcr using primers spanning the exons 2-5. the predominant dna bands in agarose gel were excised and sequenced. results: all tissues and cells expressed full length etr-a. in all tissues an additional 3 4-deletion variant was identified which was not found in the isolated cells. trophoblasts expressed another yet unidentified splice variant. the endothelial cells expressed a 3-deletion variant and a novel splice variant, which was identified as partial 2 -partial 3 deletion. this novel splice variant was found regardless of the arterial or venous origin of the cells as well as in the cell line (sv40-transformed). in tissues und trophoblast no difference in splicing was found between first trimester and term. conclusion: gestational age does not alter splicing of endothelin receptor-a. splicing is different between trophoblasts and endothelial cells. the endothelial cells contain a novel splice variant. the differential splicing may allow maternal and fetal endothelin-1 to induce different effects on the two major cell types of the placenta. (supported by grant 11258, jubilee fund, austrian national bank, vienna). adrenomedullin production and secretion by human trophoblast cells are regulated by glucocorticoids and hypoxia. francesca ciardo, 1 katia pacioni, 1 emanuela marinoni, 1 giovanna corona, 1 massimo moscarini, 1 alfredo patella, 2 romolo di iorio. 1 1 department of gynecology, perinatology and child health, university "la sapienza", rome, italy; 2 department of obstetetrics and gynecology, university of ferrara, ferrara, italy. objectives: adrenomedullin (am) is produced by intrauterine tissues and is involved in the regulation of implantation, placental hemodynamics and endocrine function. circulating am is increased in pregnancy complications such as preeclampsia and intrauterine growth retardation. we investigated whether am output by human trophoblast cells is regulated by hypoxia and/or glucocorticoids. study design: trophoblast cells obtained by human placentas at term (n=7) were cultured in presence or absence of hypoxia (3% o 2 ) and treated with or without betamethasone at the dose of 10 -5 , 10 -6 , 10 -7 m. media and cells were collected at 48h and at 2, 8 and 24h from syncytiotrophoblast cultures. am was measured in cultured media by specific ria kit. protein expression in trophoblast cells was evaluated with immunohistochemistry and western blot. results: hypoxia stimulated am output and protein expression by cytotrophoblast and syncytiotrophoblast cells. betamethasone induced an increase in am production and secretion in a time-and dose-dependent manner (figure). effects of hypoxia were partially reversed by betamethasone in a dose and time-dependent fashion. conclusions: am production in trophoblast cells is up-regulated by hypoxia and glucocorticoids, independently. increased am levels in pregnancy complications characterized by placental insufficiency might derived by an increase in am placental secretion stimulated directly by hypoxia or indirectly by an increase in fetal cortisol levels. preeclampsia complicates 5-8% of pregnancies, and while associated with significant maternal and fetal morbidity and mortality, the mechanisms responsible for preeclampsia are not completely understood. recent advances suggest there are imbalances of pro-and antiangiogenic factors, present from the time of implantation affecting vascular responsiveness of the fetal placental unit and maternal vasculature. in this study, we investigate vasomotor responsiveness of placental arteries from normal and preeclamptic (pet) pregnancies using an in vitro muscle bath contractility assay. transverse rings of placental arteries were cut and equilibrated in the muscle bath containing a bicarbonate buffer aerated with 95% o 2 /5% co 2 at 37°c. viability was demonstrated by contraction to 110mm kcl prior to all experiments. cumulative logarithmic dose dependent responses to four different contractile agents: pgf2 , serotonin, carbochol, and norepinephrine were compared. placental arteries precontracted with pgf2 at half maximal concentration were relaxed with three vasorelaxants: sodium nitroprusside (snp), forskolin (fsk) and lactate (lct). agonist studies revealed contraction to both pgf2 and serotonin but not to carbochol or norepinephrine. there were no statistically significant differences between the responses of normal and pet arteries. both normal and pet arteries demonstrated heightened responsiveness to sodium nitroprusside compared with forskolin and lactate. this study reveals that the placental vessels are more sensitive to sodium nitroprusside, that mediates relaxation through nitric oxide -cyclic gmp signal transduction pathway, than forskolin that induces relaxation through the cyclic amp pathway. cancer complicates approximately one in 1,000 pregnancies. depending on the type of cancer and trimester, patients can terminate the pregnancy or choose treatment such as chemotherapy. since there is limited knowledge on the safety of chemotherapy on fetal tissues in utero, clinicians cannot efficiently analyze the risks of particular anticancer agents when deciding on a course of therapy. since carboplatin is among the most common anticancer agents used for treatment of cancer during pregnancy the primary objective of this study was to evaluate if carboplatin will readily cross to placental barrier and determine total potential platinum fetal exposure. this project utilizes an ex vivo placenta perfusion model to determine the concentration of carboplatin that crosses the human placental barrier. placentas are obtained within 15 minutes of spontaneous delivery and re-perfused. two carboplatin concentrations were selected of 1000 ng/ml and 5000 ng/ml to represent clinically relevant maternal plasma concentrations. antipyrine was used as the internal control. serial samples were collected every 10 minutes for total 60 minutes in open-open model then experiment repeated with closed circuit model. antipyrine concentrations were determined by hplc methods. platinum concentrations in media samples were determined with a validated atomic absorption assay. a cell culture-based approach will be used to determine whether cell cycle or apoptotic proteins are altered (p53, bax, bcl-2, aif, and pro-caspase-3) using western blotting as a detection method. a total of two placentas have been completed at the low carboplatin concentration and one at the high concentration. the mean carboplatin clearance was 0.67 +/-0.01 ml/min and 1.04 +/-0.15 ml/min at the low and high carboplatin concentrations, respectively. an estimated 50% increase in the transport fraction was observed in the high concentration experiments compared to the low concentrations model. fetal carboplatin exposure ranged from 60 to 300 ng/ml. the placental perfusion experiments conducted at carboplatin 1000 and 5000 ng/ml indicate that carboplatin crosses the placenta through simple diffusion. the toxicology assays are ongoing to determine potential effect on fetal tissues. preterm delivery represents one of the predominant causes of perinatal mortality and morbidity, and is one of the most unpredictable gestational disturbances. urocortin is a peptide expressed by trophoblast and gestational tissues (amnion and chorion), whose maternal levels correlate with gestational length, since they are increased at preterm delivery and decreased in post-term pregnancy, when compared to term pregnancy. the addiction of urocortin enhances contractility of human myometrial strips, suggesting a possible role on uterine contractility. high maternal urocortin levels in threatened preterm delivery correlates with the timing of delivery, suggesting a possible role in the predictivity pf preterm delivery. in the present study urocortin concentrations in amniotic fluid collected at mid gestation for amniocentesis were correlated with gestational age at delivery. a case-control study with amniotic fluid obtained from healthy 60 women undergoing amniocentesis for genetic indications was performed; urocortin concentrations were measured by a specific elisa. amniotic fluid urocortin concentrations resulted significantly lower (p= 0.0025) in women delivering preterm (n=20; ucn= 1.23 ± 0.07 pg/ml) (m ± se) than in those delivering at term (n= 40; ucn= 0.77 ± 0.11pg/ml). in conclusion, the present preliminary data showed that amniotic fluid urocortin concentrations at mid gestation may represent predictive marker of preterm delivery. human placentas throughout pregnancy. annunziata mastrogiacomo, 1 elisabetta federico, 1 francesca caprio, 1 maria teresa schettino, 1 gabriele coppola, 2 antonio de luca, 2 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 department of medicine and public health, section of anatomy, second university of naples, nales, italy. hypothesis apoptosis is intimately involved in placental homeostasis, growth and remodeling and the apoptotic rates increase progressively during normal pregnancy as part of normal placental development. moreover, apoptosis increases in pregnancies complicated by some pathologies such as preeclampsia, fetal growth restriction, diabetes. in the present study, we describe differences in the expression of pro-apoptotic protein bax, in first trimester voluntary termination of pregnancy, first trimester abortion (reserved abortion), caesarean birth, spontaneous birth, preeclampsia and diabetes. material and methods human placental samples were obtained with informed consent from patients undergoing surgery such as first trimester voluntary termination of pregnancy (n=15), first trimester abortion (miscarriage) (n=15), delivery section (n=15), spontaneous birth (n=15), preeclampsia (n=15) and diabetes (n=15). the gestation period ranged from 5 to 40 weeks. the specimens were immediately fixed in formalin for immunohistochemistry. we first observed a strong increase of bax expression in the cytotrophoblast, stroma, endothelial cells and decidua of placentas of the first trimester abortion compared to the low/moderate bax immunopositivity in all the placental compartments during the first trimester voluntary termination of pregnancy. secondly, we showed a more intense immunopositivity for bax in the third trimester spontaneous birth respect to the third trimester caesarean birth. thirdly, we observed an increase of bax expression in preeclamptic placentas compared to the normal full-term placentas. on the contrary, we observed a moderate bax expression in diabetic placentas only slightly decreased compared to the normal full-term placentas. our results seem to suggest that deregulation of apoptotic turnover may lead to placental dysfunction and pathologies. objectives: maternal endothelial activation in preeclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. the application of proteomic technologies such as mass spectrometry promises the reward of identifying and characterising these factors. using a placental explantconditioned culture media model system we have developed a proteomics workflow to obtain relative quantification of proteins released into placental explant culture media. methods: term villous explants were cultured in serum-free conditions and exposed to differing oxygen concentrations (6% 1%) to mimic physiological and non-physiological intervillous o 2 tensions. the d4 media was concentrated, immunodepleted to remove fibrinogen (using beckman igy-12 columns) and proteins labelled using an itraq (applied biosystems) kit following the manufacturer's protocol. labelled peptides were fractionated by strong cation exchange and then analysed using lc-maldi on a 4800 maldi-tof/tof (applied biosystems) mass spectrometer. data was analysed using protein pilot 2.0 (applied biosystems). results: when matched pooled (n=6) media samples were subjected to our proteomics workflow over 590 proteins were identified with a calculated false positive identification rate of <1%. of these proteins a total of 29 display a statistically significant difference in protein levels between the 1% 6% o 2 samples (p=<0.001). from a list of 40 proteins of interest we selected 7 proteins for validation using elisa/western blotting to measure their relative abundance in both pooled and individual explant media samples. interleukin 8 is an example of a low-abundance differentially expressed protein identified in our proteomic workflow and subsequently validated by elisa; il-8 (pg/ml) in 6% o 2 : 43.7, range 30.8 to 47.3; 1% o 2 : 103.0, range 57.6 to 141.0; values are median with interquartile range. *** p<0.0001, mann-whitney test (n=40) conclusions: we demonstrate a successful reduction to practice of a relative quantitative proteomics strategy applied to placental explant-conditioned culture media. having optimised and validated this proteomic workflow we will apply the same methods to compare proteins released by normal and preeclamptic placentas. a proteomics screen of human placental microvillous syncytiotrophoblast (stb) revealed the expression of dysferlin (dysf), a membrane repair protein associated with certain muscular dystrophies (vandré et al., 2007) . a second ferlin protein, myoferlin (myof), was also discovered which has not yet been characterized in the placenta. in human c2c12 myoblasts, dysf and myof are reciprocally expressed as a function of differentiation into multinucleate syncytial myotubes, with dysf predominating in differentiated cells. we hypothesized that dysf and myof would show a similar reciprocal expression pattern in human trophoblasts as a function of syncytialization. methods term placentas from uncomplicated pregnancies were obtained with informed consent (n=8) and either fixed for immunofluorescence (if) labeling or flash frozen for subsequent immunoblotting (ib). human trophoblastic (bewo, jar, and jeg-3) and other cell lines (mrc-5, hl-60, huvec) were cultured in the absence or presence of 20 μm forskolin or solvent control for 0-3 days. dysf and myof expression was examined by rt-pcr, ib, and if. results ib validated proteomics data showing myof expression in term placenta. by if, myof labeling was predominant in apical and basal stb plasma membranes. myof was expressed in trophoblastic cell lines (bewo, jar, and jeg-3), cultured endothelium (huvec), and a fibroblast cell line (mrc-5), but was not detected in a leukemic cell line (hl-60). dysf was constitutively expressed in jar, but minimally expressed in unstimulated bewo. following forskolin-induced syncytialization, we observed a time-dependent increase in dysf expression in bewo concomitant with increasing syncytin-1 levels. by if, dysf expression was restricted to bewo cells in syncytial structures. in contrast, myof expression was robust in mononuclear bewo cells and not down-regulated over the course of 3 days of differentiation. conclusions two ferlin-family genes are expressed in trophoblasts. fusion-competent bewo cells behave similarly to cultured myoblasts and ctbs with regard to dysf expression, which is restricted to syncytializing cells. myof, in contrast, is expressed constitutively in bewo and other models. while both proteins are likely to function in stb plasma membrane repair (e.g., following syncytial sprouting), the relative contributions of each to this process awaits clarification. features of chronic placental insufficiency are significantly more common in small for gestational age placentas from infants with intrauterine growth retardation. emily king, 1 violetta kolesnikova, 1 carri tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: there is a strong association between intrauterine growth restriction (iugr) and small for gestational age (sga) placentas (heinonen et al. placenta (2001), 22:399-404) . the cause is likely multifactorial, but we hypothesize that chronic uteroplacental insufficiency may play a role. our objective was to test whether sga placentas from human neonates with iugr show significantly more pathologic features of placental insufficiency compared to controls. design: we performed a retrospective review of 242 consecutive singleton placentas submitted to ohsu pathology (2005-06), excluding elective terminations and spontaneous abortions before 22 weeks gestation. clinical records were reviewed and pregnancy outcomes recorded, including: 1) neonatal sex; 2) weight (iugr calculated by routine methods), 3) trimmed weight of the placenta (sga calculated by routine methods), 4) gross placental infarctions, 5) gestational age at delivery, and 6) maternal features (e.g. race, gravida). controls were defined as cases within the series without sga or iugr (e.g. submitted for meconium, infection, etc). histologic sections were scored by two pathologists while blinded to clinical diagnoses as positive or negative for features of placental insufficiency, including accelerated villous maturation (avm), chorangiosis, and microscopic infarctions. significant associations were tested by 2 analysis and logistic regression for multiple variables. results: similar to prior reports, we observed a strong association between iugr and sga placentas ( 2 30.5; p <0.0001). this relationship was independent of maternal race, fetal sex, and parity, although it was more common in primigravidas. avm and placental infarctions were significantly more frequent in sga placentas with superimposed iugr. controls ( introduction: messenger rna expression peripheral blood cells (pbc) has been recently used as biomarkers of environmental exposures (ionizing radiation or tobacco), physiological conditions (stress) and diseases (hypertension, neurological disorders). pbc evaluation is a useful diagnostic tool in an era of individualized medicine. since there is an urgent need for non-invasive methods for determination of fetal (f) and placental (p) function, this study was designed to evaluate the genes differently and commonly expressed in p tissue and leukocytes in maternal (m) and f circulation.material and methods. p (n=5), f (n=3) and m blood (n=3) were obtained during cesarean section in pregnant baboons at term. total rna from a buffy coat pellet was isolated using a modified procedure of the qiagen rneasy mini kit (qiagen). anti-sense rna (arna) was synthesized and purified using the illumina rna amplification kit (ambion, usa). hybridization of arna to illumina sentrix human whole genome (wg-6)beadchips and subsequent washing, blocking and detection was performed using illumina's beadchip 6´2 protocol. samples were scanned on the illumina beadarrayer 500gx reader using illumina beadscan image data acquisition software (ver. 2.3.0.13). differential gene expression data analysis was performed using illumina beadstudio software (ver. 1.5.0.34). results. the detection level of gene transcripts using illumina methodology with control human rna was 6000-7000 at p<0.0001. 4329 gene transcripts were detected in f blood and 2957 in maternal blood. 1452 transcripts were uniquely expressed in fetal blood. 130 transcripts were found in m, but not in f leukocytes. the number of gene transcripts expressed in p tissue was 4825 and of these 2953 genes were not expressed in m leukocytes. conclusion. despite white blood cells trafficking through the p barrier there is a set of unique genes expressed only in p or in the m or f circulation. the application of these genes as the biomarkers of p barrier function still need to be evaluated. objective: to evaluate if a relationship exists between duration of placental exposure to meconium in vivo and histologic evidence of severity and extent of meconium uptake by macrophages. study design: from a cohort of 353/7069 (5%) consecutive singleton liveborn infants delivered at term with thick meconium-stained fluid, 193(55%) had placental histologic examination performed, and in 50/193 the timing of meconium appearance after membrane rupture was documented. placental histologic examination quantitatively evaluated the intensity of meconium uptake by resident macrophages based on the number of macrophages/field, and the extent of uptake based on histologic location, graded in a score 0 to 3. results: mean interval between meconium appearance and delivery was 136.7± 126.7 min (range 10-510). after exclusion of 6 cases in which severe placental inflammation interfered with analysis, meconium uptake by macrophages was documented in 40/44 cases at the level of amniochorionic membranes, in 34/44 cases at the placenta, and in 18/44 cases at the umbilical cord. there was no correlation between the interval meconium appearance-to-delivery in relation to presence of meconium in the membranes (p=0.53), in the placenta (p=0.78), in the cord (p=0.71), or score of severity of meconium uptake (p=0.76). the results did not change after correcting for gestational age, oligohydramnios, presence of placental acute inflammatory or vascular lesions. conclusion: there is no relationship between duration of placental exposure to meconium and the extent and intensity of its uptake by macrophages in cases with exposure up to 8.5 hours. transfer of bisphenol a across the human placenta. biju balakrishnan, 1,2 kimiora henare, 1,2 eric thorstensen, 1,2 murray d mitchell. 1,2 1 the liggins institute, university of auckland; 2 national research centre for growth and development, auckland, new zealand. introduction: there are growing concerns over the effects of developmental exposure to the xenoestrogen bisphenol a (bpa). animal studies have shown that bpa is transferred through the placenta and can cause deleterious effects to the fetus. the presence of bpa in feto-placental tissues in humans has also been reported. however, a detailed study of the time-course of bpa transfer across the human placenta has not been performed. the aim of this research was to study the transfer of bpa in ex-vivo perfused human placental tissues. methods: a dual recirculating single cotyledon perfusion was used to monitor the placental transfer of bpa. bpa (10ng/ml), antipyrine (ap) (40μg/ml), and fitc dextran (fitc-dx) (12.5μg/ml) were added to the maternal perfusate, and perfusion was continued for 4 hours. perfusate samples were collected from both reservoirs at timed intervals and analysed. bpa, ap, and fitc-dx were determined by hplc with fluorescent detection, hplc with uv detection, and spectrofluorometry respectively. the viability and metabolic activity of the placentae were assessed by measuring -hcg (elisa), glucose utilization, and lactate production (autoanalyzer). fetal pressure, ph, flow rates and fluid shifts were monitored continuously. results: the biochemical validation parameters (glucose consumption, lactate production and -hcg secretion) indicated that the placental tissue was metabolically active and viable throughout the 4-hour perfusion period. the physical parameters observed (fetal pressure <50 mmhg, ph range 7.2-7.6) were in concordance with other published works for placental perfusion. membrane integrity was confirmed by fluid shifts from either circuit of <5ml/hr, and by <2% materno-fetal transfer of fitc-dx. an observed ap transfer of 20-30% further validated our model. bpa first appeared in the fetal compartment within 30 minutes of perfusion and reached a peak of about 25-30% of maternal concentration within 3 hours of perfusion. this figure is likely to be an underestimate since it does not include conjugated bpas. conclusion: this first study of bpa transfer in ex-vivo perfused human placental tissue shows that our model can serve as a useful tool to study the transfer kinetics and metabolism of bpa in human term placentae. we found that bpa rapidly crosses the human placenta at environmentally-relevant doses, with potentially harmful effects on the human fetus. angiogenic growth factor secretion by uterine natural killer cells in co-culture with extravillous trophoblast. gendie e lash, 1 katsu naruse, 1,2 barbara a innes, 1 stephen c robson, 1 judith n bulmer. 1 1 institue of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom; 2 obstetrics and gynaecology, nara medical university, nara, japan. objectives. uterine natural killer (unk) and extravillous trophoblast (evt) cells have been proposed to play roles in remodeling of uterine spiral arteries through secretion of various angiogenic growth factors. we have previously demonstrated that unk cells are a significant source of angiogenic growth factors within the decidua, many of which alter with gestational age. however whether the secretion of these proteins is altered by interactions of unk cells with evt is unclear. hypothesis. unk cell angiogenic growth factor secretion is regulated by evt in early pregnant decidua. methods. placental and decidual samples were collected from women undergoing termination of pregnancy with written informed consent (8-10 and 12-14 weeks gestation, n=12 each group). 0.2×10 6 cd56 + unk cells were positively selected from decidua and co-cultured with evt (0.2×10 6) or cytotrophoblast (ctb; 0.2×10 6 ) purified from the same placenta for 24h in direct or indirect contact (n=6 each group). angiogenin, ang2, pdgf-bb, fgf-basic, timp1, icam1 and vegf-a were measured by fast quant ® angiogenesis multiplex assay system, and vegf-c, ang1 and plgf by elisa. results of unk co-culture with evt or ctb (negative control) at each gestational age were analyzed with wilcoxon rank test. in addition, the effect of direct and indirect co-culture of unk cells with evt at each gestational age was compared with mann whitney u test. results. at 8-10 weeks gestation unk secretion of ang2 (p=0.03), icam1 (p=0.05) and plgf (p=0.03) was increased in the presence of evt compared with ctb. no differences were observed at 12-14 weeks gestation. in addition, at 8-10 weeks gestation unk secretion of ang2 (p=0.004), vegf-c (p=0.007) and ang1 (p=0.004) was increased in direct co-culture with evt compared with indirect co-culture. at 12-14 weeks gestation unk secretion of timp-1 (p=0.03) was reduced in direct co-culture with evt compared with indirect co-culture. conclusions. unk cell secretion of several key angiogenic growth factors was altered by direct culture with evt. these data suggest that a membrane bound molecule (such as hla-g) mediates this modulation of unk cell activity. abnormalities in placentation and impaired placental circulation can lead to fetal growth restriction. 3-d ultrasound can be used to evaluate this through the quantification of the power doppler signal that may be expressed as a percentage of colour voxels within a user-defined volume (vi: vascularisation index). we aimed to test the hypothesis that increased fetoplacental blood flow correlates with an increased vi, using the in vitro dual perfusion model of the human placental lobule. three term lobules were dually perfused through both circulations with earles bicarbonate buffer (ebb), and supplemented on the fetal-side only with adult erythrocytes, prepared to a 5% haematocrit. following initial equilibration perfusion at normal flow values, fetal-side flow was varied between 1 and 10 ml/min, whilst maternal-side flow was held at 14 ml/min. images were obtained with a 'voluson i' ultrasound machine and a neonatal transducer (pulse repetition frequency = 0.6hz, wall motion filter = low 1, and gain = 0.0). three 3-d datasets were acquired at each flow rate from each placental lobule and these were measured in triplicate using vocal. a sphere was centred on a visibly perfused cotyledon along the chorionic-decidual axis, with a diameter corresponding to placental thickness. linear regression analysis was used to assess the relationship between the total fetal-side flow and mean vi. the mean vi showed a high degree of correlation with total fetal-side flow for each lobule ( figure 1 ) suggesting increased vascular perfusion and the inclusion of perfused vessels that cross the detection threshold with increased flow. this data provides qualifying information for translation to a clinical application, where early gestational fetoplacental blood flow will be assessed to predict the onset of fetal growth restriction. anthony n imudia, 1 brian a kilburn, 1 anelia petkova, 1 samuel s edwin, 2 roberto romero, 2 d randall armant. objective survival of first trimester cytotrophoblast cells depends on their upregulation of heparin-binding egf-like growth factor (hbegf), which is downregulated in placental tissues diagnosed with preeclampsia. we have examined the expression and cytoprotective activity of hbegf in term villous explants subjected to hypoxic stress in vitro. non-pathological placentas were collected by cesarean section at term (n=6). chorionic villous explants were prepared and cultured at either 20% or 2% o 2 and treated with the hbegf antagonist crm197 or recombinant hbegf. paraffin sections were assayed for trophoblast cell death by the tunel assay, proliferation by immunohistochemical labeling of nuclear ki67 and hbegf expression by semi-quantitative immunohistochemistry. data were compared using anova and the student-newman-keuls posthoc test. results crm197 (10 mg/ml) increased trophoblast cell death after culturing villous explants 8 h at 20% o 2 (p<0.05), but only slightly affected proliferative capacity. culture at 2% o 2 increased trophoblast cell death 100% above explants incubated at 20% o 2 (p<0.05). trophoblast cell proliferation decreased after 24 h in explants cultured at either 20% or 2% o 2 (p<0.05). exogenous hbegf (1 nm) prevented the elevation of cell death during hypoxia (p<0.05) and maintained nuclear ki67 expression at 20%, but not 2%, o 2 . contrary to first trimester trophoblast, hbegf was not upregulated by hypoxia in term trophoblast. the failure of term trophoblast to elevate hbegf expression in response to hypoxia could contribute to their decreased survival at low o 2 compared to early gestation. endogenous hbegf signaling appears to facilitate survival of term trophoblast during villous explants culture. exogenous hbegf supplementation prevented cell death due to hypoxia and maintained trophoblast proliferation rates under in vitro conditions. therefore, hbegf, which is downregulated in preeclampsia, could have significant impact on trophoblast survival during late gestation. supported by the intramural research program of nichd. conspicuous meconium-laden macrophages in the chorionic plate are an independent predictor of clinically significant fetal distress. violetta kolesnikova, 1 emily king, 1 carrie tillotson, 2 jean-marie guise, 3 terry morgan. 1 1 pathology; 2 center for biostatistics; 3 obstetrics and gynecology, ohsu, portland, or, usa. background: meconium is a common indication for placental examination, present in approximately 30% of placentas routinely submitted to pathologists (beebe et al. obstet gynecol 1996; 87:771-8) . prolonged meconium exposure leads to accumulation of meconium-laden macrophages in the chorionic plate (estimated to require at least 3 hours). our objective was to test whether prolonged meconium exposure is associated with clinically significant fetal distress. design: retrospective review of 250 consecutive singleton placentas submitted to ohsu pathology (2005-06) was performed, excluding abortions before 22 weeks gestation, and placentas without representative sections of the chorionic plate. clinical records were reviewed and pregnancy outcomes recorded, including: 1) evidence of fetal distress prompting c-section (non-reassuring fetal heart rate), 2) gestational age, 3) maternal diagnosis, 4) apgar scores, and 5) neonatal length of hospital stay. routine histologic sections were independently scored by two pathologists while blinded to clinical diagnoses and outcomes. cases were scored as positive or negative for: 1) diffusely conspicuous meconium-laden macrophages in the chorionic plate (at least 1/ hpf), 2) chorioamnionitis, and 3) features of placental insufficiency. significant associations were tested by the mann-whitney u test for paired comparisons, x 2 analysis, and logistic regression for multiple variables. results: conspicuous meconium staining of the chorionic plate was common (86/250, 34%) and was significantly more frequent in c-sections performed for fetal distress (19/35 cases, 54%) (x 2 7.1; p-value <0.01). logistic regression modeling showed that this association was independent of chorioamnionitis and features of placental insufficiency. there was no association between meconium and neonatal outcome, including no difference in apgar scores or length of hospital stay. conclusions: our data support the hypothesis that meconium-laden macrophages in the chorionic plate are associated with fetal distress prompting c-section. whether meconium is the cause or consequence of this distress is uncertain. however, given the acute time frame between clinical diagnosis and c-section, we suspect prolonged meconium exposure may be a significant cause of non-reassuring fetal heart rate changes. apoptotic index in normal and intrauterine growth-restricted rat placentas. elissa scotland, tri nguyen, s chiang, radmila runic. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: intra-uterine stress caused by maternal food-restriction may have adverse fetal and placental effects. we sought to assess the effect of maternal food restriction during pregnancy on placental apoptosis. methods: rat placentas were analyzed from control dams fed ad libitum and dams 50% food-restricted from day 10 of gestation. placentas were harvested at embryonic days 16 and 20 (n=3). placentas were fixed in 4% pfa and two methods of analysis were utilized to determine the proportion of apoptotic to non-apoptotic cells within maternal food restricted and control rat placentas: immunohistochemistry (ih) using fas-ligand and in situ tunel (terminal deoxynucleotidyl transferase biotin-dutp nick end labeling). tunel: after rehydration, slides were subjected to tdt enzyme. dab was used to visualize brown apoptotic nuclei. dna-se treated slide was used as a positive control. ih: primary rabbit polyclonal fas-ligand antibody was used at 1:100 dilution, after which secondary antibody linked to peroxidase and dab staining was performed. results: food-restricted placentas demonstrated 9.29% relative apoptotic index when compared to 2.95% in the control group. the iugr iod (integrated optical density) per unit area in the placental membrane was higher than in the control group. fasl demonstrates an iod of 0.112 + 0.025 in food restricted placentas as compared to 0.049 + 0.003 per μm2 surface area in controls. apoptosis was seen in the amnion as well as trophoblast (syncytialtrophoblasts and some cytotrophoblasts). conclusion: the increased apoptotic index in maternal food restricted placentas suggests that the accompanying iugr may be a result of both maternal/fetal nutrient restriction and increased fetal stress. this study found that maternal food restriction during pregnancy affects placental apoptosis. there is more apoptosis in the iugr placental bed at e16 than at e20, with the reverse being true for the placental membrane. more research is required to statistically validate the data. the suggested next step is to evaluate fas and fas-l and to address possible mechanisms of placental apoptosis. cord. jayaraman lakshmanan, 1 avish arora, 1 lilit baldjyan, 2 sharon k sugano, 1 olga miadel, 2 adegoke adeniji, 2 michael g ross, 1 calvin j hobel. 2 1 ob-gyn, harbor-ucla medical center, torrance, ca, usa; 2 ob-gyn, cedars-sinai medical center, los angeles, ca, usa. background: crf-bp is a 37kda protein, that specifically binds corticotrophin releasing factor (crf). the structure of cloned crf cdnas in all species examined predicts that the precursor is larger than the 37kda in size and contains one n-glycosylation site. marked reductions in plasma crf-bp levels seen in pregnant women prior to both preterm and term delivery led to the notion that crf-bp is a "gatekeeper" of crf responses. recently we identified crf-bp expression in term human umbilical cord (uc) by immunohistochemical analyses. objective: to characterized the expression of crf-bp by immunohistochemical and biochemical analyses in human uc. methods: freshly obtained human preterm (28 to <37 weeks, n=6) umbilical cord (6 pieces of 3mm thickness taken at 1-2 cm intervals close to placenta) were fixed in bouin's solution and paraffin embedded. also, pieces of ucs were dissected, arteries and vein separated, weighed and frozen at -80c. for immunohistochemical localization, uc sections were subjected to immunostaining with polyclonal antibodies to human crf-bp precursors. for western blot analyses, whole uc as well as isolated arteries and vein were homogenized in a buffer containing detergents and protease inhibitors. the homogenate supernatant proteins were subjected to western analyses by standard protocol. immunoreactive protein bands were identified by chemiluminescent reagent. results: both goat and rabbit crf-bp polyclonal antibodies elicited weak to moderate positive staining in uc epithelial layers, vascular musculature and barely endothelial cells. they identified a strong 50kda band in whole uc as well as in isolated artery and venous preparations. in addition minor immunoreactive protein bands of 37, 25, 20kda in size were noticed in all three preparations. conclusion: we conclude that preterm uc expresses crf-bp. we postulate that the 50kda major band is either glycosylated crf-bp precursor or glycosylated 37kda mature protein. the low molecular weight immunoreactive proteins likely represent proteolytically processed, glycosylated crf-bp precursor or a proetolytically processed mature 37da crf-bp. uc obtained at delivery could be useful as a tool to understand the critical functions of crf-bp in feto-placental unit. objective: adenosine, known to be released from inflammatory sites and tisse ischemia, has many important biologic roles. four specific adenosine receptors have been cloned to date, termed a1, a2a, a2b, and a3. recently our study has shown that increased a3 receptor in the trophoblast of preeclamptic pregnancy was noted and non-vascular and trophoblast-mediated a3 receptor may play an important role in the pathogenesis of preeclampsia. there are evidences of impaired trophoblast invasion related to matrix metalloproteinase (mmp) in preeclampsia and the relationship between adenosine receptor and mmp in other fields. the objective of this study is to evaluate the effect of mmp expression by adenosine a3 receptor in preeclamptic villous explants at different oxygen conditions. methods: placental villous explants from normal (n=10) and preeclamptic (n=10) pregnancies were cultured at high (20%) and low (3%) oxygen levels for 5 days. explants were analyzed for mmp-2/-9 and timp-1/-2 by rt-pcr and western blot. preeclamptic villous explants in hypoxic culture condition were treated with a3 receptor agonist, cl-ib-meca and a3 receptor antagonist, mre. mmp-2/ -9 expression was determined in a time-and dose-dependent manner by rt-pcr, western blot. also mmp-2/-9 activity was evaluated by zymogram assay. results: there were significantly increased a3 receptor intensity and reduced mmp-2/-9 and timp-1/-2 expression at low oxygen level in normal and preeclamptic villous explants. interestingly, in preeclamptic villous explants, after high oxygen culture mmp-2/-9 and timp-1/-2 expression were recovered to almost same level compared to those in normal villous explants. treatment of preeclamptic villous explants with cl-ib-meca in low oxygen level resulted in a time-and dose-dependent enhanced expression of mmp-2/-9. this cl-ib-meca-induced expression of mmp-2/-9 was inhibited by pretreatment with mre. conclusion: to our knowledge, this study is the first to evaluate modulation of mmp secretion by adenosine a3 receptor in preeclamptic villous explant. our results provide evidence for the existence of functional adenosine a3 receptors in the trophoblast and suggest that adenosine a3 receptor will be investigated as a therapeutic target in preeclampsia. murray d mitchell, 1 timothy a sato, 1 anderson wang, 1 jeffrey a keelan, 1 anna p ponnampalam, 1 michelle glass. 2 1 liggins institute; 2 department of pharmacology and clinical pharmacology, university of auckland, auckland, new zealand. introduction: it is well established that prostaglandins play critical roles in multiple aspects of pregnancy and that the fetal membranes are an important site of intrauterine prostaglandin production. endocannabinoids have been implicated in the maintenance of pregnancy and parturition in women and are a source of arachidonic acid which is a substrate for the production of prostaglandins. the aim of the present study was to determine the effects of endocannabinoids on the production of prostaglandins in extraplacental membranes -amnion, chorion and decidua. methods: explants of term amnion and choriodecidua were established and treated with endogenous endocannabinoids 2-arachidonoyl glycerol (2ag) and anandamide (aea) and with the synthetic cannabinoid cp55,940, to determine the ability of these substances to modulate prostaglandin e 2 (pge 2 ) production. pge 2 was measured by radioimmunoassay. the explants were also treated with cp55,940 in the presence of either sr141716a (a selective antagonist of the cannabinoid receptor cb1) or ns398 (a cox-2 inhibitor), to determine whether any observed stimulation of pge 2 production was mediated through cox-2 activity and/or the cb1 receptor. cox-1, cox-2, cpla2 and pgdh protein levels were measured by western blotting. results: all three cannabinoids caused a significant increase in pge 2 production in amnion but not in choriodecidua. however, separated fetal (chorion) explants responded to cannabinoid treatment in a similar manner to amnion, whereas maternal (decidual) explants did not. the enhanced pge 2 production caused by cp55,940 was abrogated by co-treatment with either sr141716a or ns398, illustrating that the cannabinoid action on prostaglandin production in fetal membranes is mediated by cb1 agonism and cox-2. preliminary data from western blotting show that cannabinoid treatment results in the up-regulation of cox-2 expression. however, there was no change in cox-1 expression and no evidence either for up-regulation of cpla2 or for down-regulation of pgdh expression. conclusion: this study demonstrates a potential role for endocannabinoids in the modulation of prostaglandin production in late human pregnancy, with potentially important implications for the timing and progression of term and preterm labour and membrane rupture. inactivation of vegf receptor-2, but not vegfr-3 or vegfr-1, during the peri-implantation period prevents normal pregnancy development in the rodent through disruption of uterine angiogenesis. nataki c douglas, 1 hongyan tang, 1 raul gomez, 1 bronislaw pytowski, 2 daniel j hicklin, 2 jan kitajewski, 1 mark v sauer, 1 ralf c zimmermann. 1 1 division of reproductive endocrinology and infertility, columbia university, new york, ny, usa; 2 imclone systems, inc., new york. objective: vegf is involved in the regulation of uterine angiogenesis and implantation in both rodents and non-human primates. vegfr-1, r-2, and r-3 are expressed in the uterine decidua and are involved in the regulation of vessel formation in many systems. to determine if these receptors have a functional role in the regulation of post-implantation angiogenesis and pregnancy development, we examined the effects of blocking vegfr-1, r-2, and r-3 function. design: prospective animal laboratory material and methods: to avoid effects of vegf receptor neutralization on ovarian function, we utilized a progesterone replaced, ovariectomized mouse model. vegf receptor blocking antibodies were administered on ed 3.75, prior to embryonic expression of these receptors. embryonic development was evaluated on ed 10.5, blood vessel density and apoptosis on ed 7.5, and cellular proliferation on ed 5.5 (n=5 per time point in each group). anova with bonferroni correction was used to compare sample means. results: see tables. conclusions: neutralization of vegfr-2 and vegfr-3, but not vegfr-1 resulted in a significant reduction in cellular proliferation and decidual angiogenesis. vegfr-3 mediates decidual angiogenesis, but is not required for normal pregnancy development. in contrast, an intact vegf/vegfr-2 pathway is required for the decidual angiogenesis that mediates early pregnancy development. effect of vegfr neutralization on ed 10.5 control anti r-1 anti r-2 anti r-3 no. of implantation sites 10.7 +/-1.2 11 +/-0.9 11.0 +/-1.1 14.0 +/-0. hoxa10 encodes a transcription factor required for endometrial receptivity and embryo implantation. our objective was to identify and to characterize those molecular markers regulated by hoxa10 in multiple cellular model-systems. using microarray technologies, we identified putative hoxa10 target genes involved in early implantation. liposome-mediated transfection delivering either empty vector or the same plasmid constitutively expressing hoxa10 was introduced into newly impregnated mice during laparotomy or layered onto cultured human endometrial stromal-cells (hescs). rna products from these in vivo and in vitro transfections were used to identify targets and to validate the microarray screen employing semi-quantitative real-time pcr (qrt-pcr). we identified 82 statistically-significant genes regulated by hoxa10 overexpression of which 57 genes were down-regulated greater than 2-fold when compared to controls. cellular ontogenies of differentially-expressed genes include: cell adhesion molecules, signal transduction factors as well as metabolic regulators. furthermore, we identified the 3-phosphoglycerate dehydrogenase gene, (pgdh) whose products are regulated by hoxa10 during implantation in both murine model systems in and cell culture. this genes codes for an enzyme critical to de novo l-serine biosynthesis via a phosphorylation-dependent pathway. microarray analysis demonstrated a 2fold expression decrease when hoxa10 is overexpressed. this diminution in pgdh expression was noted in the validation experiments using qrt-pcr and corroborated in hesc cells where the mrna levels decreased to 40% when compared to controls. the repression of pgdh during the implantation window may represent a conservation of activity as secretory-phase protein synthesis may be suppressed in order to promote cellular differentiation and resultant implantation. these regulatory relationships identified in mouse implantation likely function to enhance uterine receptivity and may have a role in human implantation. objective: hoxa10 is expressed in endometrium, where it is regulated by sex steroids and is necessary for endometrial receptivity and implantation. hoxa10 is also expressed in leukocytes. here we hypothesized that hoxa10 would be regulated by sex steroids in both cell types. we further hypothesized a correlation between expression of hoxa10 in peripheral blood cell (pbcs) and endometrium in both mice and in humans. methods: real-time pcr was used to determine differential expression of hoxa10 mrna in u937 cells, a monomyelocytic cell line, in response to increasing concentrations of estradiol. to determine if hoxa10 is expressed in leukocytes in vivo, peripheral leukocyte hoxa10 mrna expression was measured over sequential estrus cycles in mature cd1 nulliparous mice and correlated to vaginal smear-cytology. additionally, peripheral leukocytes were isolated either from pregnant or normally-cycling women to assess hoxa10 mrna expression. results: there was a direct, dose-responsive correlation between exposure to increasing estradiol and hoxa10 mrna expression levels in u937 cells. in a murine model, we demonstrated that hoxa10 mrna expression-levels varies throughout the estrus cycle with a marked increase in expression following vaginal plug detection. the nadir of hoxa10 expression is prior to proestrus and increased up to 2-fold during the receptive phase. this increased expression continues throughout gestation. the heightened expression in murine leukocytederived hoxa10 mrna also is demonstrated across species. our preliminary data suggests that the greatest fold-increase of expression occurs during the window of implantation of the secretory phase in normal, cycling women. this level was sustained in pregnancy. there appears to be a trend with the highest levels of expression associated with viable gestations. women with attenuated expression profiles had non-viable gestations. conclusions: hoxa10 expression is regulated by sex steroids in both leukocytes and endometrium. the temporal pattern of peripheral hoxa10 transcript expression demonstrated in mice and humans mimics the differential rna expression documented within the uterus. leukocyte hoxa10 expression during the reproductive cycle in mice and humans is a marker of endometrial receptivity. peripheral leukocyte expression of hoxa10 mrna may correlate with implantation success. carolien m boomsma, annemieke kavelaars, marinus jc eijkemans, gijs teklenburg, bart cjm fauser, cobi heijnen, nick s macklon. reproductive medicine and gynaecology and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands. the purpose of this study is to assess the association between the intra-uterine cytokine expression profile at the time of embryo transfer and successful embryo implantation following ivf/ icsi treatment. materials and methods 210 women undergoing ivf/ icsi underwent endometrial secretion aspiration prior to embryo transfer. 16 known soluble mediators of implantation were measured using a multiplex immunoassay, namely il1ß, il5, il6, il10, il12, il15, il17, il18, tnf , vegf, ifn , eotaxin, mcp-1, ip-10, dkk-1 and hbegf. mif was determined using an elisa. the total protein concentration was measured for normalization purposes. data were log transformed to obtain normal distribution. multivariable logistic regression analysis with a backward elimination procedure (p<0.1) was used, potential confounders (age, blood contamination, embryo quality) were included in a forward stepwise model. ten mediators of the 17 analysed were detectable in 90-100% of the samples. il15 was detectable in 76% of samples, dkk-1 68%, il-10 56%, il-17 54%, il-5 35% and hbegf was detectable in 23% of samples. ifn-was not detectable in any of the samples. multivariable logistic regression showed only logmif concentrations to have a significant correlation with achieving clinical pregnancy (p =0.036). higher mif concentrations were correlated with a higher chance of conceiving. endometrial secretion analysis represents a novel means of assessing the intra-uterine milieu encountered by the embryo and offers new perspectives in the study of endometrial receptivity. in this large prospective study assessing an array of cytokines, mif was found to be significantly correlated with pregnancy. mif, macrophage migration inhibitory factor, is a cytokine with numerous proinflammatory, immunomodulatory, angiogenic and tissue remodelling properties. mif induces the synthesis and secretion of matrix metalloproteinases by endometrial cells, which may contribute to embryo invasion. its expression is particularly increased during the secretory phase, suggesting a role in reproductive processes. analysis of aspirated endometrial secretions offers a direct clinical test of endometrial receptivity which can be applied during treatment cycles without disrupting implantation. endometrial receptivity and secretory differentiation require progesterone (p). it has been hypothesized that low p levels result in delayed endometrial differentiation and infertility due to reduced receptivity. objective: test the effects of low luteal p on histologic and molecular markers of differentiation and function. methods: normal cycling women (n=12) were treated with daily leuprolide (1.0 mg/d) beginning in the midluteal phase and continuing through the protocol. after menses, subjects received transdermal estradiol (e, 0.2 mg/d) for 20 days. after day 10 of e, subjects also received daily i.m. injections of p, randomized to 10 mg/d (sub-physiological) or 40 mg/d (physiological). endometrial biopsy was performed after 10 days of combined e and p treatment. additional untreated women had biopsies performed 10 days after spontaneous lh surge. endometrial histologic dating was performed by two individuals according to the criteria of noyes et al. mrna levels were assessed using real-time rt-pcr. results: mean(s.d.) of peak and trough p serum concentrations in the 40 mg/d p group were 18.2(5.1) and 9.4(4.8) ng/dl, respectively, while those in the 10 mg/d group were 7.0(3.0) and 3.4(1.0) ng/dl. there were no differences between treatment groups for histologic dating; the mean(s.d.) histologic date was 25.7(0.8), 24.4(0.9), and 24.8(1.3) for the 10 mg, 40 mg, and spontaneous cycle groups, respectively. there also were no differences among the three groups in mrna levels of ten functional markers (er , pr, 3 integrin subunit, osteopontin, cyr61, egr-1, fkb52, c-fos, and cd55), although variability of gene expression was greater in those who received 10 mg/d p than in those who received 40 mg/d p. there was also no correlation between serum progesterone level and gene expression or histologic date. conclusions: sub-physiological levels of progesterone, in the range seen in ovulatory women, do not induce detectable changes in expression of marker genes or histological dating, although low p levels were associated with greater variability of gene expression. these data suggest that abnormalities in endometrial histologic development and function likely result from intrinsic abnormalities rather than from low levels of p secretion. (supported by unc nova carta fund and nih u54 hd-35041). endometrial ip10 attracts trophectoderm through cxcr3 interaction. simcha yagel, 1 caryn greenfield, 1 hen sela, 1 jacob hanna, 2 irit manaster, 2 orna singer, 3 ronit haimov-kochman, 1 shira natanson-yaron, 1 diana prus, 1 benjamin reubinoff, 2 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical centers, jerusalem, israel; 2 lautenberg center for immunology, jerusalem, israel; 3 human embryonic stem cell research center, jerusalem, israel. introduction: implantation is initiated in part by attraction of the blastocyst to the endometrium lining the uterus. we hypothesized that this process is partly accomplished by chemokines expressed by the endometrium interacting with chemokine receptors on the blastocyst, suggesting that they play an important role in implantation. materials and methods: chemokine receptors were characterized in jeg cells, placental villi, primary trophoblast cell culture, trophectoderm cells derived from human es cells, blastocyst trophectoderm, and 1 st trimester placental tissue sections. expression of chemokines was tested in decidua, endometrium, and ishikawa and hec-1 cell lines. immunohistochemistry, intracellular staining, elisa, facs analysis, and rt-pcr were employed to characterize chemokine receptor and ligand expression. functional testing was performed using transwell migration assays and in a nude mouse model using a matrigel gel plug cell attraction assay followed by facs analysis. results: trophoblasts demonstrated expression of various chemokine receptors, most prominently cxcr1 and cxcr3. immunohistochemistry of trophoblast from placental villi plated on matrigel expressed cxcr3 and cxcr1 as well as hla-g. noteworthy is that trophectoderm cells derived from hes cells treated with bmp-4 and jeg cells, and blastocyst trophectoderm expressed principally cxcr3. elisa and immunohistochemistry showed that decidua and endometrium expressed chemokines ip10 and il8. migration assays demonstrated that ip10 significantly attracted various trophoblast and trophectoderm cells in vitro, and in the mouse model in vivo. taken together these results demonstrate the interaction between trophoblasts and endometrial cells is mediated by cxcr1 and cxcr3, and il8 and ip10. these interactions are important in the attraction of trophoblasts at the feto-maternal interface. however, ip10-cxcr3 is the most relevant to early implantation as only cxcr3 is expressed consistently by trophectoderm. the endometrial proteome: changes from proliferative to secretory phase. lois a salamonsen, jenny i-c chen, xian mak, peter j stanton, david m robertson, andrew n stephens. prince henry's institute of medical research, melbourne, vic, australia. global gene analyses have demonstrated major changes across the menstrual cycle, but which of these are reflected in the proteome is not known. this study aimed to globally assess proteins differentially expressed in the endometrium between the proliferative and. secretory phases. 2d page analysis with dige minimal dye labelling was conducted across the pi range 4-7 on endometrial tissue from either the mid-proliferative or midsecretory phases (n=4/group). profiles were assessed using samespots software. differentially expressed proteins were identified using maldi-tof ms and the interrelationship of proteins examined using ingenuity software. a total of 1010 spots were detected: 196 were differentially expressed (p < 0.05) with 17 spots having an overall false discovery rate <5% (q <0.05). hierarchical clustering analysis revealed that these 17 proteins lay within three main branches in the protein dendrogram. one cluster had proteins upregulated in the proliferative phase and two contained proteins up-regulated in the secretory phase. the unique protein profiles were also revealed using principle component analysis (pca): proteins clustered into two main groups, according to cycle phase. pca thus indicated similar unique protein signatures as suggested by hierarchical clustering. thirty one of the differentially expressed proteins were identified using maldi-tof ms. these proteins could be grouped into seven categories, which included structural (7), transport (4), regulatory (2), membrane (2), enzyme (2), motor (1) and others (2). proteins involved in matrix assembly and those needed for subsequent establishment of secretory endometrium were up-regulated in proliferative endometrium. proteins important for cellular organisation and communication as well as products responding to environmental stress and the immune system were highly up-regulated during the secretory phase. biological pathways were constructed based on the proteins identified. the top network for secretory endometrium clustered around tgf-b. others related to inhibition of cell death / cell viability and leukocyte extravasation. these studies provide a global approach to the cyclic changes of the endometrium and highlight the complex dynamics of protein expression in human endometrium. hormonal regulation of prokineticins in the human fallopian tube: potential regulators of embryo transport. aw horne, 1 hn jabbour, 2 p lourenco, 1 s wright, 2 s battersby, 2 arw williams, 1 hod critchley. 1 1 reproductive and developmental sciences, university of edinburgh, edinburgh, united kingdom; 2 mrc human reproductive sciences unit, queen's medical research institute, edinburgh, united kingdom. background: understanding the factors regulating embryo transport in the fallopian tube (ft) has important clinical implications. embryo retention in the tube due to ft dysfunction is thought to lead to tubal pregnancy, a considerable cause of morbidity and occasional mortality. transport of the embryo through the ft is, for the greater part, accomplished by smooth muscle contraction. a group of multi-functional proteins and their receptors, called prokineticins, have been shown to affect smooth muscle function in other tissues, such as the intestine. the expression pattern of prokineticins, and their receptors, was examined in normal human ft obtained throughout the menstrual cycle, and the effect of the sex steroids on prokineticin expression was examined in an in-vitro model of the ft. methods: ft biopsies (n=18) and sera (for measurement of oestradiol and progesterone for endocrine staging) were collected from women undergoing gynaecological procedures for benign conditions. using a combination of quantitative taqman rt-pcr and immunohistochemistry, the mrna and protein expression pattern of prokineticins, and their receptors, were examined in the ft throughout the menstrual cycle. tubal explant culture was established using surgical tissue from the biopsies and exposed to varying concentrations, and time courses, of oestrogen and progesterone. results: prokineticin 1 (pk1) and prokineticin 1 receptor (pkr1) mrna are up-regulated in the progesterone-dominant mid-secretory phase. pk1 and pkr1 protein are expressed in the epithelium, smooth muscle and around the blood vessels of the ft. stimulation of tubal explant cultures with a physiological concentration of progesterone showed an up-regulation of pk1 and pkr1. conclusions: prokineticins show temporal variation in expression in human ft and appear to be regulated by progesterone. their role in embryo transport needs to be investigated to further understanding of pregnancy complications, such as tubal pregnancy. translating mouse to human: a dynamic model of xenografted human endometrium. alex j polotsky, liyin zhu, nanette santoro, jeffrey w pollard. albert einstein college of medicine, bronx, ny, usa. in the mouse endometrium, the hormonal environment controls cellular proliferation and cell cycle activity. estradiol (e2) inhibits glycogen synthase kinase 3 beta (gsk-3 ), resulting in nuclear accumulation of cyclin d1 and progression of the cell cycle, as well as dna replication licensing. in utero administration of the gsk-3 inhibitor, licl, results in epithelial cell proliferation in the absence of e2 (zhu, pollard. pnas. 2007; 104:15847) . in this study, we derived a functional model of xenografted human endometrium to perform mechanistic studies of human endometrial proliferation. methods: human endometrial samples were obtained from volunteers aged 18-45. immuno-compromised mice were transplanted with disaggregated/ recombined human epithelial glands and stroma under the kidney capsule. after 6 weeks of out-growth, mice were ovariectomized, and replaced with e2 or licl. xenografts were harvested and processed for immunohistochemistry (ihc) and glandular labeling index (li). t test was used to compare group means. results: 40-50% of engraftments were successful, resulting in a vascularized endometrium with characteristic architecture (a, b). hoechst 33258 staining confirmed that xenografts were made up of human cells ©. e2 (e) induced significantly greater proliferation compared to control (d) as assessed by ihc for ki67, with li of 25.9±1.3 and 8.8±3.3, respectively, p =0.02). minichromosome maintenance-2, a protein involved in dna replication licensing, was more sensitive for e2-treated cells synthesizing dna than was ki67 staining (i). estrogen (g) and progesterone receptors (h) were expressed in xenotransplant tissue, the latter being up-regulated by e2. licl (f) induced proliferation similar to e2 and greater than control (li =18.4±6.4, p= 0.2 for e2 vs. licl). conclusion: xenografted human endometrium provides a dynamic model of endometrial proliferation that is well suited for translational studies. administration of licl in the absence of e2 induced glandular proliferation, supporting the notion that similar mechanisms are operative in human proliferation as in the mouse. gnrh analogs have been extensively used in assisted reproduction. although the main effects of gnrh analogs are via gnrh receptors on the pituitary gonadotrope, gnrh and gnrh receptors have been identified in many reproductive tissues including human endometrium, suggesting their potential action at endometrial level. in the present study, we examined the potential regulatory action of gnrha, la on sex steroid mediated gene expression in human endometrium. human endometrial surface epithelial (hes) cells and isolated endometrial stromal (esc) cells were used as in vitro models. all experiments lasted for 24h. the cells were treated with estradiol (e 2 , 10 nm, 24h), progesterone (p4, 10 nm, 24h), gnrha (la 1 μm, 24 h), e 2 (12h) followed by p4 (last 12h) and gnrha plus e 2 plus p4 where, gnrha added either first, second or last in order at 8h intervals. total rna was extracted, reversed-transcribed and subjected to real-time pcr simultaneously, measuring the expressions of il-1 , il-1 , il-2, il-4, il-5, il-8, il-10, il-12p35, il-12p40, il-15, ifn-and tnf . both hes and esc expressed majority of these cytokines with the exception of low to undetectable levels of il-2, il-4, il-5 and ifn-. treatment with e 2 and p4, either alone, or in combination significantly upregulated the expression of many of these cytokines at varying extend as compared to controls. however, gnrha either alone or in combination with e 2 and p4 significantly diminished e 2 , p4 or e 2 plus p4 induced mrna expression of cytokines. the suppressive effects of gnrha on some of the cytokines varied significantly by the order which gnrha was introduced into the culture medium. we conclude that gnrha by acting directly on the endometrial cells effectively suppresses the mrna expression of several key proinflammatory cytokines upregulated by ovarian steroids. our results imply that gnrha therapy during assisted reproduction may modify endometrial receptivity via downregulation of proinflammatory cytokines induced by estradiol and progesterone. supported by nih grant hd37432. introduction: endometrial angiogenesis is characterized by a rapid increase during the early proliferative phase that peaks midcycle, followed by a gradual decrease in the secretory phase, while menses involves generalized endometrial inflammation, necrosis, and vascular thrombosis. vascular endothelial growth factor (vegf), whose expression is regulated by sex steroids, is a mediator of endometrial angiogenesis. recently, myoferlin, a 230 kd transmembrane protein, was identified in endothelial cells where it mediates vegf-dependent endothelial cell proliferation, migration, and nitric oxide synthesis by affecting vegf receptor-2 function and stability. myoferlin also appears to play a role in vesicle trafficking and membrane repair. objective: to characterize myoferlin protein expression in endometrium. methods: western analysis of cultured human endometrial stromal cells (hesc) and human endometrial endothelial cells (heec) treated with physiologic concentrations of estradiol and progesterone was performed using a polyclonal rabbit antibody against myoferlin. subsequently, immunohistochemistry (ihc) was performed on human endometrium samples obtained from various stages of the menstrual cycle. results: myoferlin protein was expressed in hescs and heecs, but expression was not affected by sex steroids. ihc staining for myoferlin was specific, intense, and localized to the apical membrane of glandular epithelial cells and endothelial cells, with less intense staining in the stroma. h-score quantification showed that in endometrial endothelial cells, myoferlin protein expression was highest during the early proliferative and early secretory phases, while glandular and stromal myoferlin expression peaked during the late proliferative/early secretory phase. conclusion: myoferlin expression in human endometrium correlates with periods of greatest endometrial angiogenesis, and expression is not limited to endothelial cells, but also includes glandular and stromal cells. given the involvement of myoferlin in vegf signaling as well as membrane repair, understanding its role in human endometrium may further elucidate an understanding of endometrial development both under physiologic and pathologic conditions. the human embryo is the primary regulator of embryo-endometrial molecular cross talk during early implantation. gijs teklenburg, 1,2 cobi heijnen, 1 esther baart, 1 karima amarouchi, 1 carolien boomsma, 1 janet carver, 2 helen mardon, 2 annemieke kavelaars, 1 nick macklon. 1 1 reproductive medicine and gynaecology, and laboratory of psychoneuroimmunology, university medical center, utrecht, netherlands; 2 nuffield department of obstetrics and gynaecology, university of oxford, women's centre, john radcliffe hospital, oxford, united kingdom. introduction: uterine receptivity and implantation are controlled by locally acting trophic factors and cytokines. in humans, the regulation of the embryo-endometrial dialogue beyond the early blastocyst stage is poorly understood. we hypothesized that the interaction between a healthy conceptus and the receptive endometrium is associated with a distinct local regulation of cytokine production favouring implantation. methods: 97 human embryos, cryopreserved at day 4 after fertilization and donated for research, were thawed and cultured under standard conditions until day five. forty-two embryos from 18 donors developed to the blastocyst stage. following removal of the zona pellucida, they were placed in individual coculture on a confluent monolayer of endometrial stromal cells. on day 8, the developmental potential of each embryo was assessed as early arrested, late arrested or developing. culture supernatants were analysed for concentrations of il-1 , il-5, il-6, il-10, il-12, il-15, il-17, il-18, tnf-, mcp-1, ip-10, eotaxin and hb-egf using a multiplex immunoassay. day 8 supernatants from 29 culture systems in which no embryo had been placed were also analysed as controls. results: 11 out of 42 (26%) embryos continued to develop and were able to attach and invade into the stromal cell compartment of the co-culture environment. twelve late arrested embryos showed signs of degradation on day 8 and the totally disintegrated embryos were assigned to the early arrested group. supernatants from both early and late arrested embryo cultures contained significantly lower levels of a number of cytokines and growth factors in comparison to developing embryo cultures. moreover, the levels of these mediators in co-cultures were significantly lower than those in non-embryo control stromal cell culture supernatants. conclusion: these data suggest a pivotal role of the embryo in embryoendometrial cross talk. whether reduced mediator expression in co-cultures reflects a selective down regulation of stromal cell cytokine and growth factor production is now being investigated. epithelial cell dynamics during human implantation. hiroshi uchida, tetsuo maruyama, toru arase, masanori ono, takashi kajitani, maki kagami, hideyuku oda, sayaka nishikawa, yasunori yoshimura. department of obstetrics and gynecology, keio university school of medicine, shinjuku, tokyo, japan. epithelio-mesenchymal transition (emt) is thought to play a role in functional differentiation of endometrial epithelial cells during human implantation. molecular mechanism of epithelial sheet remodeling caused by embryo invasion remains elusive. to address this, we investigated cellular dynamics of n-cadherin and vimentin, the two representative major markers of emt, during implantation. in in vitro implantation assay using a human endometrial epithelial cell line, ishikawa, and a human choriocarcinoma cell line, jar (uchida et al., hum reprod 2007), we pre-treated human ishikawa cells with or without ovarian steroid hormones (17 -estradiol + progesterone; ep), fa-5 (n-cadherin blocking ab), or suberoylanilide hydroxamic acid (saha), one of histone deacetylase inhibitors, which has a potential to improve in vitro implantation (ibid). implantation or treatment with or without ep or saha enhanced the expression of n-cadherin and vimentin but down-regulated ecadherin. furthermore, treatment with ep or saha accelerated ishikawa cell motility and increased the number and spreading area of jar spheroids. in vitro implantation assay, the most prominent staining intensity of n-cadherin was observed just around the adhered spheroid from which its intensity decreased away. functional blockade of n-cadherin by fa-5 resulted in the complete suppression of ishikawa cell motility, the unique distribution of n-cadherin around jar spheroids, and the spreading area of jar spheroids, while it did not affect the number of the adhered spheroids. human implantation consists of the multiple steps, including apposition, adhesion and penetration. thus, these results collectively indicate that emt may take place after the apposition and that n-cadherin may be required for the remodeling and emt of the epithelial sheet during embryo invasion. n-cadherin may enhance the recruitment of spheroid-neighboring cells, suggesting its role in the covering-up of the invading embryo through acceleration of epithelial cell motility. endocannabinoid regulation in human endometrium. jessica g scotchie, marc a fritz, steven l young. obstetrics and gynecology, university of north carolina, chapel hill, nc, usa. background: research in mouse models demonstrates two cyclically regulated endocannabinoids produced in murine endometrium, anandamide and 2-arachidonoyl glycerol (2-ag); both play critical roles in murine embryonic implantation. no studies of endocannabinoids in human endometrium have been performed. objectives: determine menstrual cycle expression and localization of synthetic and degradative enzymes for anandamide and 2ag in human endometrium. methods: human endometrium was collected from volunteers across the menstrual cycle (n=43). quantitative rt-pcr was performed analyzing the expression of: n-acylphosphatidylethanolamine (nape) and fatty acid amide hydrolase (faah), the synthetic and degradative enzymes for anandamide, respectively; sn-1-diacylglycerol lipase-a (dagla), and b (daglb), the synthesis enzymes for 2ag; and monoacylglycerol lipase (magl) and cyclooxygenase-2 (cox2), the degradative enzymes for 2ag. the constitutive gene ppia was used for comparison. immunohistochemical localization of nape protein was performed using nape-pld polyclonal antibody (cayman chemical, #10005430). anova and student's t-test analysis performed on samples grouped by proliferative (pro), early, mid-, and late secretory (es, ms, ls) phases. results: mrna expression of all enzymes responsible for synthesis and degradation of anandamide and 2ag was detected throughout the cycle. no significant cyclic change in nape, faah, or daglb gene expression was seen. a decrease in dagla gene expression in the ms and ls phases compared to the pro and es phases (p=0.004) was seen. magl gene expression was higher in the secretory phase than the pro phase (p=0.004). cox2 gene expression was detected at low levels in the pro, es and ms phases, with marked increase in the ls phase (p<0.05 for all comparisons). protein localization of nape showed a cytoplasmic epithelial location, with increased staining on pro and es samples compared to ms and ls samples. immunolocalization of remaining proteins is ongoing. conclusion: this is the first report documenting the presence of endocannabinoid synthetic and degradative enzymes in human endometrium. genes controlling anandamide expression do not fluctuate significantly across the cycle. however, 2ag's degradative enzymes increase in the secretory phase, suggesting that lower 2ag levels may be advantageous for embryo implantation. our findings suggest that human endometrial endocannabinoid regulation differs from murine regulation. interleukin-1 beta (il-1 ) regulates il-6 signaling in decidua-implication in the pathophysiology of preeclampsia (pe). sj huang, 1 cf yen, 2 cp chen, 3 f schatz, 1 cj lockwood. 1 1 obstetrics, gynecology and reproductive sciences, yale university, new haven, ct, usa; 2 ob/gyn, chang gung memorial hospital, tao-yuan, taiwan; 3 ob/gyn, mackay memorial hospital, taipei, taiwan. objective: previously, we found much higher cytoplasmic immunoreactive il-6 levels in the preeclamptic decidual cells than in adjacent interstitial trophoblasts. such decidual cell-derived il-6 contributes to the systemic endothelial cell dysfunction that elicits the proteinuria and hypertension of the maternal syndrome. il-6 promotes the transition from innate to adaptive immunity. moreover, by skewing monocyte differentiation from a dendritic to a macrophage phenotype, decidual il-6 may promote the macrophage excess observed in the preeclamptic decidua. macrophages impair trophoblast decidual invasion to foster incomplete spiral artery remodeling that elicits placental ischemia and hypoxia. the current study: 1) localized il-6 mrna levels in preeclamptic versus normal decidual sections; 2) evaluated mechanisms regulating il-6 synthesis by targeting intracellular signaling pathways with specific inhibitors; 3) identified potential il-6 targets by immunolocalizing the il-6 receptor (il-6r) to specific cell types in placental bed biopsies. methods: in situ hybridization localized il-6 mrna in normal versus preeclamptic decidua. il-6r was immunolocalized in placental bed biopsies. leukocyte-free first trimester decidual cells were incubated with e2 and mpa ± il-1 (1 ng/ml) ± an inhibitor of p38 mapk (sb203580) or protein kinase c (calphostin c) or nf b (activation inhibitor iii) for 24 hrs. an elisa measured secreted il-6 levels. results: il-6 mrna was present primarily in decidual cells with increased il-6 mrna levels observed in pe. preferential expression of the il-6r was observed on decidual cells in placental bed biopsies. compared with basal il-6 levels (0.22 ± 0.14 pg/ml/ g cell protein) by decidual cells, il-1 enhanced il-6 output by decidual cells (364.55 ± 160.29 pg/ml/ g cell protein). only the p38 mapk inhibitor significantly reduced this output to 39.50 ± 8.09 pg/ml/ g cell protein (n=4, p<0.05). our results indicate that inflammatory cytokine enhances il-6 synthesis in decidual cells of the preeclamptic decidua by a mechanism involving p38 mapk. such il-6 is likely to act as an autocrine/paracrine effector via decidual cell-expressed il-6r to contribute to the macrophage excess observed in the preeclamptic decidua. heparanase is up-regulated by estrogen and during the secretory phase of the human endometrium. ronit haimov-kochman, 1 shira natanson-yaron, 1 caryn greenfield, 1 achinoam lev-sagie, 1 lichtenstein michal, 2 haya lorberboum-galsky, 2 israel vlodavsky, 3 simcha yagel, 1 arye hurwitz. 1 1 ob/ gyn, jerusalem, israel; 2 cellular biochemistry human genetics, hadassah hebrew university medical centers, jerusalem, israel; 3 cancer and vascular biology research center, technion school of medicine, haifa, israel. introduction: heparanase is an endoglycosidase that cleaves heparan sulfate (hs) proteoglycan of the extracellular matrix. the full-length proheparanase is activated by cleavage into an active isoenzyme, resulting in the release of hsbound cell-differentiation factors, such as hb-egf. the cycling endometrium involves remarkable steroid hormone-induced tissue remodeling. in vivo, increasing exposure to unopposed estrogen may lead to endometrial malignant transformation. aim: to investigate heparanase expression and regulation in the cycling endometrium. materials and methods: heparanase mrna levels were measured by quatitative rt-pcr in naturally menstruating women and in hec1a, estrogen receptor (er)-negative and ishikawa, er-positive endometrial carcinoma cell lines exposed to increasing doses of estradiol. heparanase isoenzymes were localized by immunohistochemistry using specific anitbodies in murine endometrium and human normal, hyperplastic and malignant endometrium. results: heparanase mrna level increased 100fold in secretory phase (d21) compared to proliferative phase (d10) endometrium. heparanase transcript levels increased 14fold during 8 hr culture in er positive adenocarcinoma cell line exposed to increasing doses of estradiol, but not in hec1a, er negative cell line. both heparanase isoforms were localized to murine glandular endometrium. human glandular endometrium at both proliferative and secretory phases was immunoreactive with the active isoform of heparanase. proheparanase was detected in basal membrane of endometrial glands and endometrial stroma during secretory phase. along with malignant transformation of the endometrium the presence of proheparanase increased dramatically from none in stroma of normal and hyperplastic endometrium to abundance in malignant tumors. conclusions: heparanase gene expression is higher during the window of implantation and up-regulated with estrogen in endometrial cells via er in vitro and vivo. heparanase is differentially localized in the secretory phase of the endometrium compared to the proliferative phase, suggesting a role for this molecule during the window of implantation in man. interleukin-18 (il-18) system mrna and protein expression in the human fallopian tube with ectopic implantation. hong-yuan huang, 1,2 tien-hung huang, 2 chin-jung li, 1 chyi-long lee, 1,2 hsin-shih wang, 1,2 yung-kuei soong. 1,2 1 obstetrics and gynecology, chang gung memorial hospital, kwei-shan, tao-yuan, taiwan; 2 obstetrics and gynecology, chang gung university and school of medicine, kwei-shan, tao-yuan, taiwan. objective: ectopic pregnancy, an abnormal implantation of a fertilized ovum outside the uterine cavity, has been increasing in number at a staggering pace of all pregnancies. il-18 system is one of the major cytokines involved in human endometrium during embryo implantation and might perform a defensive role against maternal immune response. very little information is available regarding the expression and synthesis of cytokines in the pathogenesis of fallopian tube with ectopic gestation. the purpose of this study is to investigate il-18 system expression in human fallopian tubes with ectopic pregnancy. methods: paired segments of human fallopian tubes with ectopic implantation site and side portion close to ectopic gestation (n=7) were collected from women undergoing laparoscopic salpingectomy after informed consent and irb approval. segments of fallopian tubes from women undergoing tubal ligation (n=4) were used as control groups. total extracted rna was reverse transcribed and amplified by pcr using specific primers for gapdh (94 bp), il-18 (144 bp), il-18bp (188 bp) and il-18r (307 bp). quantitative il-18 and il-18bp mrna expression in human fallopian tube was determined by real-time pcr. to determine the presence of il-18 system proteins, tissues were fixed and processed for immunohistochemical study. data analysis was done with anova and pearson's correlation. results: il-18 and il-18bp as well as il-18r mrna were all expressed in tubal ectopic implantation and normal tubes. according to real-time pcr with c t value quantification and 2 -ct method, a significantly higher il-18 expression in tubal ectopic implantation and lower ratio of il-18 antagonist to agonist in portion close to ectopic implantation is demonstrated in comparison to normal tubes (p<0.05). immunoreactive il-18 system at the protein levels was also present in human fallopian tubes with ectopic implantation and normal tubes. conclusions: these results suggest that fallopian tube il-18 system expression may play a crucial role during the process of early embryonic implantation. the expression and ratio of antagonist to agonist in fallopian tubes may indicate an earlier "dialogue " in human fallopian tubal gestation prior to uterine implantation. replacement. marcia c ferreira, ines kd cavallo, fernando m reis. gynecology, ufmg, belo horizonte, mg, brazil. activin a is a growth factor expressed in the endometrium, where it modulates tissue remodelling and enhances decidualization. the effects of activin a are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (fstl3). while the endometrial expression of activin a increases during the secretory phase of menstrual cycle, the effects of ovarian steroids on these proteins and their mrnas has not been assessed yet in postmenopausal women or in ovariectomized animals. we have evaluated the effects of estrogen alone or estrogen plus progestin on the endometrial expression of activin beta-a subunit, follistatin and fstl3 in ovariectomized rats. adult female wistar rats (n=21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n=7) or associated with depot medroxyprogesterone acetate (2.4 mg/kg body weight, i.m. injection, n=7), or oil vehicle (control group, n=7). one week after the hormone or placebo treatment, the animals were sacrificed and their uteri were removed and processed by immunohistochemistry and real-time pcr. data were normalized to the expression of ribosomal phosphoprotein p0 (rpp0) and analyzed with the delta-delta ct method, anova and newman-keuls test. activin beta-a subunit mrna levels increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, p<0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, p<0.05). this was accompanied by increase of beta-a subunit immunostaining in estradiol and estroprogestin-treated rats, which was noted only in the surface endometrial epithelium. follistatin mrna expression, conversely, showed a significant decrease in the groups treated with estrogen alone (0.5 fold compared to controls, p<0.05) and estrogen plus progestin (0.4 fold compared to controls, p<0.05), while follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprotestin-treated rats compared to controls. fstl3 expression was similar in the 3 groups. in conclusion, the expression of activin beta-a subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. endometrial nk cells are a unique inert nk subset until pregnancy. simcha yagel, 1 irit manaster, 2 jacob hanna, 2 ronit haimov-kochman, 1 miri godin, 2 yuval bdolach, 1 caryn greenfield, 1 shira natanson-yaron, 1 arye hurwitz, 1 debra s goldman-wohl, 1 ofer mandelboim. 2 1 obstetrics and gynecology, hadassah-hebrew university medical center, jerusalem, israel; 2 lautenberg center for tumor immunology, hadassah-hebrew university medical center, jerusalem, israel. introduction: we recently demonstrated that nk (natural killer) cells play a critical role in trophoblast migration and angiogenesis at the fetal maternal interface. nk cells populate the endometrium at the secretory phase of the menstrual cycle, the time of anticipated blastocyst implantation. peripheral blood (pb) nk cells and decidual nk (dnk) cells express a variety of activating receptors, including nkp44, nkp30 and nkp46, collectively known as natural cytotoxicity receptors (ncrs), and nkg2d which regulate nk cell killing and growth factor production. to compare endometrial nk cell (enk) activating receptor expression and function to pbnk and dnk cells and endometrial ligand expression, with a focus on their roles in blastocyst implantation. patients and methods: subjects were ivf patients undergoing natural menstrual cycles. endometrium samples were collected on treatment days 10 and 21. a lymphocyte profile of the endometrial cells and pb was performed. facs analysis was performed on isolated endometrial nk cells, pbnk cells and dnk cells for cd56, cd16, nkp44, nkp30, nkp46 and nkg2d. ncr ligand expression was characterized on adherent endometrial cells using ncr-ig fusion proteins and nkgd2-ig and nkg2d specific ligands as well as control ccmi-ig. redirected killing assays and cytokine secretion assays of ifn , vegf, plgf, and il-8 with and without il-15 were performed. results: endometrial lymphocytes of day 10 and 21 in these women are mostly cd56 bright cd16-nk cells, with a significant amount of t cells, similar to pbnk cells and in marked contrast to dnk cells. unlike pbnk and dnk cells, enk receptors do not express nkp30, nkp44. nkp46 and nkg2d are the only activating enk receptor expressed. like decidual cells, adherent stromal endometrial cells expressed the ligands for nkp30, nkp44 and nkg2d, suggesting that these nk cells have potential for activation. finally enk cells could not kill or secrete cytokines. conclusions: these findings of a unique activating receptor profile on endometrial nk cells, unlike that of dnk and pbnk, suggest that enk cells are a special local population of nk cells that change dramatically and are activated at the onset of pregnancy. variation in platelet activation throughout the menstrual cycle. fiona c denison, 1 amy o robb, 1 imogen b smith, 1 nicholas l mills, 2 hilary od critchley, 1 david e newby. 2 1 centre for reproductive biology; 2 centre for cardiovascular sciences, the university of edinburgh, united kingdom. background: platelet-monocyte aggregation (pma) is a sensitive and novel measure of platelet activation with important proinflammatory consequences including release of cytokines and chemokines. previous studies using less sensitive techniques suggest that platelet activation alters during the menstrual cycle in response to circulating concentrations of sex steroids. the effect of sex steroids on circulating (c) pmas during a single menstrual cycle is not known. objective: to determine whether cpmas, platelet surface (ps) p-selectin and plasma (p) p-selectin vary through the menstrual cycle in response to changes in circulating sex steroid concentrations. methods: 10 healthy, nulliparous, pre-menopausal, non-smoking women (mean age 31 years), with regular menses (27-29 days) were studied. subjects gave written informed consent and the study had ethical approval. serial venous blood samples were taken at menstrual, follicular, periovulatory and luteal phases of a single cycle (days 1-3, 6-9, 13-15 and 20-22). cpmas (monocytes positive for the platelet marker cd42a) were measured by flow-cytometry. psp-selectin expression was calculated on cd42a positive cells. isotype-matched controls were used. serum oestradiol (e) and progesterone (p), plasma and pp-selectin were measured by elisas. data were analysed by one-way anova with repeated measures and bonferroni's post-tests for multiple comparisons. results: luteal phase p was >30 nmol/l in all women. numbers of cpmas and expression of psp-selectin were both significantly higher during menstrual compared with periovulatory phase of the menstrual cycle (25.59±2.15 vs. 16.65±2.88%, p=0.01 and 3.95±0.77 vs 2.10±0.37%, p<0.05, respectively). there was no significant difference in pp-selectin concentration during the menstrual cycle (p=0.8). there was no correlation between levels of serum e or p and numbers of cpmas, expression of psp-selectin or pp-selectin concentration. conclusions: numbers of cpmas and expression of pspselectin are maximal at menstruation with neither numbers of cpmas nor expression of psp-selectin correlating with serum e or p levels. this study suggests that activated platelets may potentially contribute to the inflammatory response at menstruation by releasing inflammatory mediators. by angiogenic factors and peri-cellular proteases in decidual secretory endometrium (dse), decidua parietalis (dp), and basalis (db) of miscarriage patients and matched controls. comparison of these parameters between the two groups enabled hypothesizing about their correlation with the occurrence of miscarriages. methods: decidua was obtained during 1 st trimester termination of pregnancy (control group) and vacuum aspiration of missed abortions (case group). vascularization was studied by cd34-immunohistochemistry. the expression of vascular endothelial growth factor-a, placental growth factor, flt-1, kdr, angiopoietin-1, angiopoietin-2, tie-2 and the membrane-type matrix metalloproteinases mt1-, mt2-, mt3-and mt5-mmp were determined at mrna and antigen level and cd56-positive unk cells, cd68-positive macrophages, proliferation (ki67) and apoptosis (activated caspase-3) were evaluated by immunohistochemistry in consecutive serial sections. results: the decidual vascularization pattern showed differences between cases and controls: i.e. fewer vessels with larger circumference in cases, and this correlated with the differential expression of various angiogenic factors and proteases at mrna and antigen level. moreover, the endothelial protein expression of flt1, kdr, mt2-and mt5-mmp was increased at the implantation site of cases. ki67 and active caspase-3 showed similar levels in the two groups and also the immune cells, both unk cells and macrophages, showed no differences at the implantation site between both groups. conclusion: differences between cases and controls appeared not to be based on altered proliferation, apoptosis, and/or inflammation. the differences in vascularization pattern and in the expression of angiogenic factors and proteases between both study groups suggest a correlation between decidual vascularization and the occurrence of miscarriages. respond to adrenomedullin. yaun-lin dong, 1 hong y wen, janice endsley, 2 alison hogg, 2 hui-qun wang, 3 manubai nagamani, 1 chandra yallampalli. background: natural killer (nk) cells are the predominant lymphocytes present in human implantation site. decidual nk cells express perforin, an essential molecule required for lysis. formation of the placenta involves cooperation between maternal nk cells and fetal trophoblast cells that remodels the blood supply; however, the interaction between trophoblasts and decidual nk cells is largely unknown. adrenomedullin (adm) has been implicated in regulating early placental function and fetal growth. objective: to determine the role of multifunctional peptide adm in the decidual nk cells and fetal trophoblast cells interactions. methods: decidual and placental tissues were obtained from normal firsttrimester pregnancies terminated for social reasons. ethical approval to use these tissues was obtained from the irb of university of texas medical branch. cell preparations containing all decidual mononuclear cells were isolated by collagenase enzymatic disaggregation. cd56 decidual nk cells were purified by magnetic bead isolation. results: 1) immunohistochemical analysis showed that adm is expressed primarily in decidual cells and trophoblast cells at the human implantation site; 2) confocal imaging analysis demonstrated that decidual nk cells, which were identified by anti-cd56 staining, express adm receptor components crlr/ramp 2 /ramp 3 and their mrna expressions were futher confirmed by rt-pcr; 3) k562 target cell killing assay indicates that adm inhibits cytokine il-12/il-15-induced decidual nk cell cytotoxicity; and 4) immunofluorescent labeling and flow cytometric analysis revealed that adm suppresses perforin expression by decidual nk cells. conclusion: trophoblast-derived adm inhibits decidual nk cell cytotoxicity via suppressing perforin expression, thus, our results provide evidence for a new paradigm of embryonic-maternal communication involving a adm mediated interaction between decidual nk cells and fetal trophoblasts. leandro g oliveira, gendie e lash, judith n bulmer, barbara a innes, roger f searle, stephen c robson. institute of cellular medicine, newcastle university, newcastle upon tyne, tyne and wear, united kingdom. background: we have previously demonstrated that co-culture with extravillous trophoblast cells (evt) (expressing hla-g) alters cytokine secretion by uterine natural killer (unk) cells, particularly at 12-14 weeks gestation. we have also reported that unk cells can stimulate evt invasion, but only at 12-14 weeks gestation (not at 8-10 weeks gestation). in addition, unk cell cytokine profiles alter with increasing gestational age. other reports have suggested that evt or hla-g expressing cells may alter the expression of cytokines and angiogenic growth factors by unk cells. hypothesis: hla-g expressing cells alters unk secretion of cytokines. methods: cd56 + unk cells were isolated from early pregnancy decidua (8-10 and 12-14 weeks gestation, n=10 each group) using enzyme digestion and positive immunomagnetic bead separation. the human b lymphoblastoid 721.221 transfected with either hla-g1 (221g) or a mock cdna (221cdna) were obtained as a kind gift from mr r apps (university of cambridge, uk). isolated unk cells were cultured in the presence or absence of the two cell lines in either direct or indirect contact (n=5 each group and each gestational age) for 24 hours. cell supernatants were analysed for cytokines using a fastquant® th1/th2 multiplex protein assay (il-6, il-1 , il-10, il-2, il-5, tnf-, il-13, il-4, ifn-) or by standard elisa (tgf-1). the effect of direct co-culture of unk cells with 221g compared with co-culture with 221cdna at each gestational age was tested using mann whitney u test. the effect of co-culture of unk cells with 221g in both direct and indirect contact was also tested using mann whitney u test. results: there was no difference in the level of cytokines secreted by the 221g or 221cdna cells. cytokine secretion by unk cells was not altered after direct co-culture with either 221g or 221cdna cells at either gestational age. in addition, direct or indirect co-culture of 221g or 221cdna with unk did not alter cytokine secretion at either gestional age. conclusions: hla-g does not alter the secretion of cytokines by unk cells from either 8-10 or 12-14 weeks gestation. other evt or decidua derived factors (including hla-e) may be responsible for the alteration in secretion of cytokines by unks with increasing gestational age. introduction: natural killer (nk) lymphocytes are central to innate immunity and contribute to tissue homeostasis by eliminating altered cells. their nkg2d receptor pathway plays a fundamental role in target elimination through binding nkg2d ligands on the cell surface. reduction in the nkg2d ligand, ulbp2, expression is associated with immune resistance in neoplastic processes. we have previously shown that fibroblasts from adhesion tissue (at) are characterized by increased extracellular matrix molecules and inflammatory cytokines compared with normal peritoneal (np) fibroblasts. objective: to determine if there is a difference in nk lymphocyte-mediated elimination between np and at fibroblasts and to investigate potential role of nkg2d pathway in this process. material and methods: expression of nkg2d ligands; ulbp2, ulbp3, mica, and micb was evaluated by flow cytometry and western blot in primary cell cultures of fibroblasts from np and at, established from two patients. peripheral blood nk lymphocytes (cd56+cd3-) from three healthy volunteers were isolated using macs system with purity greater than 90% and kept in interleukin 15 overnight. fibroblast elimination with and without ulbp2 blocking was investigated following 3-hour co-incubation with allogeneic nk lymphocytes using our established flow cytometric cell mediated cytotoxicity assay. paired t test was used in statistical analysis. results: the flow cytometry studies showed that nkg2d ligands (ulbp2, ulbp3, mica and micb) were lower in at compared to np fibroblasts, reaching a statistical significance in ulbp2 expression (p = 0.039). western blot analysis also revealed a lower ulbp2 protein level in at than np fibroblasts. furthermore, nk lymphocyte-mediated elimination was 20% lower in at in comparison with np fibroblasts. blocking ulbp2 expression resulted in decreased nk lymphocyte-mediated np fibroblast elimination by 27%, supporting the role of nkg2d receptor pathway in the process. conclusions: our results demonstrate that nkg2d pathway is operational in at fibroblast resistance to immune elimination, and extends our prior observations of the potential role of immunological mechanisms in the pathogenesis of adhesion development. objective: galectin-1 is an anti-inflammatory lectin that has pleiotropic regulatory functions at the crossroad of innate and adaptive immunity. human galectin-1 is expressed in the placenta and immune privileged sites and it has been implicated in establishing immune tolerance. the aim of this study was to examine the evolution and placental expression of the lgals1 gene in primates. methods: seven primate nucleotide sequences were generated, aligned to 27 vertebrate orthologs from all classes and subjected to phylogenetic analysis. deduced amino acid sequences were analyzed for functionally important substitutions. placental galectin-1 expression was studied by immunohistochemistry and western blot. results: 1) the lgals1 gene had high sequence identity among all investigated species. 2) phylogenetic analysis revealed that intense purifying selection had been acting on the lgals1 gene in placental mammals (dn/ds=0.11); 3) residues responsible for sugar binding or molecule stabilizing were highly conserved in primates. 4) immunostaining showed a uniformly abundant and ubiquitous galectin-1 expression pattern in human, old and new world monkey and prosimian placentas, regardless the type of placentation. the lgals1 gene has conserved sequence and placental expression pattern in primates that may suggest its important function in maternal-fetal immune interactions. these results support the view that immune interactions at the maternal-fetal interface evolved in concert with invasive placentation and that these interactions have been maintained regardless of the degree of placental invasion in primates and other mammals. expression of interleukin-23 in human endometrium throughout the menstrual cycle and early pregnancy. yesim h uz, 1,2 william murk, 1 umit a kayisli, 1 aydin arici. 1 1 department of obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 department of histology and embryology, trakya university school of medicine, edirne, turkey. background: interleukin-23 (il-23) is a recently discovered heterodimeric cytokine, comprised by a novel p19 subunit and a p40 subunit shared by il-12. it has biological activities that are similar to but distinct from il-12, and is known to be involved in th1/th2 cell class switching and the regulation of cytokines such as ifn-gamma, il-12, tnf-alpha, and il-17. early pregnancy is associated with alterations in the maternal immune response, such as changes in cytokine expression, and leukocyte recruitment and subtype switching. we hypothesized that expression of il-23 in the human endometrium is menstrual cycle-and pregnancy-dependent. materials and methods: endometrial samples from women (n=44) undergoing surgery for benign gynecologic conditions, and decidual tissues from women (n=8) with clinically normal pregnancies terminated voluntarily in the first trimester, were obtained after receiving informed consent. endometrial samples were grouped according to menstrual phase. paraffin sections were stained with il-23p19 antibodies and evaluated semi-quantitatively with hscore. statistical analysis of the data was done using anova, with p<0.05 considered significant. results: il-23 immunoreactivity was predominantly located in the cytoplasm of both endometrial stromal (esc) and glandular (egc) cells. escs showed mild il-23 immunoreactivity without significant changes in intensity throughout the menstrual cycle. on the other hand, first trimester decidual cells showed significantly stronger il-23 staining compared to escs from non-pregnant endometrium (p<0.001). il-23 immunoreactivity in egcs was high in the late proliferative phase, as compared to other cycle phases and first trimester tissues (p<0.001). moreover, egcs from the early secretory phase (p<0.05) and first trimester tissues (p<0.01) showed higher il-23 immunoreactivity compared to the early proliferative and late secretory phases. conclusions: this is the first study describing il-23 expression in the human endometrium and decidua. these results suggest that il-23 has a cycledependent expression in endometrial cells and may be involved in regulating cytokine expression and immune cell modulation during the menstrual cycle and early pregnancy. gercel-taylor, douglas d taylor. obstetrics, gynecology, and women's health, university of louisville, louisville, ky, usa. objective: estrogen appears appear to be a critical regulator of the immune system. since hypoestrogenism is present in the postmenopausal woman, our objective was to determine whether t cell activation and function, defined as il-2 production and signaling molecule expressions at the transcriptional and translational levels, were affected by a low estrogen environment. design: prospective study in a university research laboratory. materials and methods: jurkat 6.1 t cells, initially grown in estrogen free media, were incubated in 4pm (representing postmenopausal levels) or 40pm (premenopausal levels) of estradiol (e 2 ) for 48 hours. cells were either resting or activated with a phorbol ester, 4 -phorbol 12 -myristate 13 -acetate (pma), and ionomycin. enzyme-linked immunosorbent spot assay (elispot) was performed to analyze production of il-2. expression of signaling protein components, cd3 and jak, were determined by western immunoblotting. real time-polymerase chain reaction was performed to quantify cd3 , jak2, and jak3 gene expression. a p value of <0.05 was considered significant. results: jurkat cells exposed to 4pm e 2 and activated exhibited significantly diminished numbers of il-2 producing colonies compared to t cells exposed to 40pm (55.7±1.2 vs. 75.3±0.7 colonies, p<0.0001). analysis of cellular cd3 and jak2 protein demonstrated that jurkat cells incubated in 4pm e 2 expressed a 1.48-fold decrease in cd3 and 1.64-fold decrease in jak compared with cells incubated in 40pm e 2 (p<0.001). these diminished protein levels appeared to be the consequence of suppressed transcription, as the mrna levels of cd3 , jak2 and jak3 were significantly decreased in jurkat cells incubated in low levels of estrogen (11.3, 213.3, and 13.3 fold, respectively, compared to 40pm). conclusions: jurkat cells exposed to low postmenopausal estrogen levels produce significantly less il-2 following activation, which was associated with a significant decrease in signaling proteins. the diminished levels of signaling proteins appear to result from decreased cd3 , jak2 and jak3 gene expression in the presence of low estrogen. these findings support the observation of decreased cellular immune response in postmenopausal women and may provide a basis for the increased risk of infections and cancer proliferation associated with aging. support: dept. of ob/gyn research seed fund. the expression pattern of 2 novel cytokines (il-24 and 29) in human fetal membranes. judith eckardt, 1,2 stephen j fortunato, 1 holger maul, 2 ramkumar menon. 1 1 the perinatal research center, nashville, tn, usa; 2 womens' hospital, university of heidelberg, heidelberg, baden-wuerttemberg, germany. objective: interleukin (il) 24 and 29 are novel cytokines produced by various immunological cells in response to microbial antigens. the functions of these cytokines in reproductive system is unknown. this study examines the expression pattern of il-24 and il-29 in human fetal membranes from preterm and term births and in in vitro in normal term membranes in response to bacterial endotoxin (lipopolysaccharide-lps). methods: fetal membranes collected (n=12) from cesareans at term (normal, not in labor) were placed in an organ explant system for 48 hours and were stimulated with lps for an additional 24 hrs. fetal membranes were also collected (n=10) either at preterm or term after vaginal deliveries. in a case -control study (preterm birth vs. normal term deliveries) amniotic fluids (af) (n=200) were collected to document the role of il-24 and il-29 in ptb. tissue expressions of il-24 and il-29 were studied by rt-pcr using specific primers. elisa documented culture media and af cytokine concentrations. statistical analysis was performed using non-parametric mann-whitney u test. results: both il-24 and il -29 expressions were seen in fetal membranes in culture (in vitro) regardless of stimulation. in vivo in membranes from preterm and term deliveries and membranes at term not in labor also documented the expression of these cytokines. culture media analysis documented higher concentration of il-24 after lps stimulation (lps-65.5 vs. 7.7 pg/ml; p=0.006) whereas no difference was noticed in il-29 concentration (lps-22.8 control-19.5 pg/ml; p=0.5) between the two groups. af analysis, regardless of the status, did not document detectable concentrations of either of the cytokines (lower limit 7.65 pg/ml for both). conclusion: this is the first study to document the expression of two novel cytokines in laboring and non laboring human fetal membranes and also in membranes from preterm deliveries. il-24 production was stimulated by lps whereas il-29 was not affected. these cytokines are not physiological components of af and their role in fetal membranes is unclear. higher il-24 concentration in response to lps but lack of its presence in term or preterm af is suggestive of an autocrine immune response during pregnancy in response to a microbial antigen. evidence for a selective migration of fetus specific cd4 + cd25 bright regulatory t cells from the peripheral blood to the decidua in human pregnancy. tamara tilburg, 1,2 dave l roelen, 2 barbara j van der mast, 1 godelieve m de groot, 1 carin kleijburg, 1 sicco a scherjon, 1 frans hj claas. 2 1 department of obstetrics, leiden university medical centre, leiden, netherlands; 2 department of immunohematologie and bloodbank, leiden university medical centre, leiden, netherlands. during pregnancy the maternal immune system has to tolerate the persistence of fetal alloantigens. many mechanisms contribute to the prevention of a destructive immune response mediated by maternal alloreactive lymphocytes directed against the allogeneic fetus. murine studies suggest that cd4 + cd25 + t cells provide mechanisms of specific immune tolerance to fetal alloantigens during pregnancy. previous studies by our group demonstrate that a significantly higher percentage of activated t cells and cd4 + cd25 bright t cells are present in decidual tissue in comparison with maternal peripheral blood in human pregnancy (1) . in this study we examined the phenotypic and functional properties of cd4 + cd25 bright t cells derived from maternal peripheral blood and decidual tissue. depletion of cd4 + cd25 bright t cells from maternal peripheral blood demonstrates regulation to a 3rd party umbilical cord blood cells comparable to non-pregnant controls, whereas the suppression capacity to umbilical cord blood cells of her own child is absent. furthermore, maternal peripheral blood shows a reduced percentage of cd4 + cd25 bright foxp3 + and cd4 + cd25 bright hla-dr + cells compared to peripheral blood of non-pregnant controls. in contrast, decidual lymphocyte isolates contain high percentages of cd4 + cd25 bright t cells with a regulatory phenotype that are able to down regulate fetus-specific and non-specific immune responses. these data suggest a preferential recruitment of fetus-specific regulatory t cells from maternal peripheral blood to the fetal-maternal interface where they may contribute to the local regulation of fetus specific responses. (1) tilburgs t, roelen dl, van der mast bj, van schip jj, kleijburg c, de groot-swings gm, kanhai hh, claas fh, scherjon sa. differential distribution of cd4 (+) cd25 (bright) and cd8 (+) cd28 (-) t-cells in decidua and maternal blood during human pregnancy. placenta. 2006 apr;27 suppl a:s47-53. 3 alicia del toro-arreola, 2 lourdes nunez-atahualpa, 4 juan velazquez-rodriguez, 5 laura gonzalez-lopez, 6 jorge i gamez-nava, 3 adrian daneri-navarro. there is evidence that the maternal immune system is influence by changes in the hormonal levels during the menstrual cycle (mc). so far, the information related to the levels of t, treg, nk cells and receptors of activation and inhibitors is scarce. aim: to analyze the populations of t, treg, nk cells and their receptors of peripheral blood of healthy women and their correlation with hormones during mc. material and methods: we studied to 10 women not using hormonal contraceptives in the day 5th of the follicular phase and 21st of the luteal phase of the mc. pbmc subsets and their receptors were determined by flow cytometry and hormone levels by chemiluminescence method. we found that the progesterone and prolactin were positive correlated (rho= 0.802, p<0.05 and rho= 0.757, p<0.05, respectively) with cd94/nkg2 in t cells and negative correlated (rho= -1.00, p<0.01 and rho= -0.857, p<0.05, respectively) with nk cells. meanwhile cortisol was positive correlated (rho= 0.857, p<0.05) with the receptor nkg2d expressed in nk cells. the results observed in this study in the luteal phase of mc on the expression of cd94/nkg2 inhibitor receptor and nkg2d activator receptor were related to a particular hormone (progesterone, prolactin and cortisol) might contribute to understanding the physiological role of the neuroendocrine axis on the expression of some receptors of the immune system in order to keep the homeostasis milieu of the mc. objective: sp-d, a key component of the innate immune system, is detected in amniotic fluid (af) and believed to originate in the fetal lung. however, sp-d is produced by other cells and therefore extra-pulmonary sources must be considered. the objective of this study was to determine the maternal and fetal plasma and af concentrations of sp-d to gain insight into the behavior of this natural antimicrobial peptide in pregnancy. moreover, we studied sp-d expression in maternal and fetal peripheral leukocytes. methods: maternal and fetal plasma and af samples were obtained from patients in the following groups: 1) term not in labor (tnl; n=20); 2) term in labor (til; n=31); 3) preterm labor without histologic chorioamnionitis (ptl; n=30) and 4) preterm labor with histologic chorioamnionitis (ptl-hc; n=27). sp-d concentration was measured by elisa. sp-d mrna expression in maternal and fetal leukocytes was evaluated by real-time qrt-pcr. flow cytometry and confocal microscopy were used to study the localization of sp-d in leukocytes. results: 1) the af sp-d concentration increased as a function of gestational age (mean, til: 52,055.7 ng/ml vs. ptl: 31,371.0 ng/ml; p<0.05); 2) in contrast, the maternal and fetal plasma sp-d concentrations decreased with advancing gestational age (mean, til: 375.8 ng/ml vs. ptl: 672.9 ng/ml; p<0.05, and til: 366.9 ng/ml vs. ptl: 1606.8 ng/ml; p<0.001, respectively); 3) the maternal plasma sp-d concentration was lower than that of fetal plasma (mean: 506.1 ng/ml vs. 1054.4 ng/ml; p<0.001); 4) however, sp-d mrna expression in maternal leukocytes was 4.8 fold higher than that of fetal leukocytes (p<0.001); 5) neutrophils (both maternal and fetal) expressed sp-d as demonstrated by flow cytometry and confocal microscopy. conclusion: 1) the concentrations of sp-d in the maternal and fetal circulation decreased with gestational age while the af concentration increased; 2) the expression of sp-d mrna is higher in maternal leukocytes than in fetal leukocytes; 3) we report for the first time that maternal and fetal neutrophils are a source of sp-d and propose that this molecule plays a role in host defense against infection and in the modulation of the maternal and fetal immune response. introduction intrauterine insemination (iui) is a fertility technique that allows for couples to have intercourse after the procedure is performed. it has been postulated that intercourse after iui may increase the pregnancy rate by either endometrial stimulation or because it may represent a second spermatic flow in the periovulatory period. in the present study we evaluate the effect of intercourse on the pregnancy rate of patients undergoing iui. material and methods: from 2004 to 2006 675 patients were enrolled in the study. every couple undergoing iui was instructed at the moment of insemination to decide whether to have or not intercourse on the same day of the procedure. all couples were abstinent three to seven days before iui. the information regarding intercourse was recorded the day after treatment. ovulatory, insulin resistant, cervical, male, tubal and endometrial factors as well as parity and time of infertility were compared between the two groups. all these factors were analyzed based on number of follicles that ovulated in each group. our principal outcome was to determine the pregnancy rate. intercourse was practiced by 65.8% of the couples. the global pregnancy rate was 15.4%. the pregnancy rate for the couples who had intercourse was 13.3% and 19.5% for those who did not have intercourse (p<0.05). even though age, parity, time of infertility and stimulation protocols were similarly distributed in both groups, the proportion of tubal and endometrial factors were higher among those who had intercourse (p<0.01). when subjects with tubal and endometrial factors were excluded, the pregnancy rate between both groups (n=463) was similar (16.1% vs 19.6% for positive and negative intercourse, respectively). the average number of ovulatory follicles was 2.12 + 1.1 for the group that had intercourse and 2.17 + 1.3 for those who did not. according to our results, intercourse after iui does not improve pregnancy rate after this procedure is performed. furthermore our study indicates that iui does not interfere with sexual intimacy since almost 66% of the couples decided to have intercourse on the same day of the procedure. . we sought to investigate the effects of gravidity on mmc and fmc in healthy, parous women. methods: mc was assayed in dna extracted from peripheral blood mononuclear cells (pbmc). hla-genotyping was first conducted and mc quantified employing a q-pcr assay targeting a non-shared maternal-or fetalspecific hla polymorphism. gravidity was dichotomized as a history of one pregnancy compared with two or more, and the prevalence of mc was analyzed using logistic regression. possible confounders were included as appropriate, including subject age and time since last pregnancy. adjustment for possible correlation between values was also made when there were repeated measures for the same subject. results: for the mmc analysis, there were 35 subjects with 73 observations. for the fmc analysis, there were 68 subjects with 145 observations. table 1 provides a summary of mmc and fmc by gravidity. mmc was significantly decreased with higher gravidity. fmc was not affected by gravidity. gravida 1 gravida 2 or more adjusted* or (95%ci) mmc 7/14 (50%) 5/48 (10%) 0.19 (0.04-0.79) fmc 13/30 (43%) 36/115 (31%) 0.49 (0.21-1.17) *adjusted for possible correlation between values within a subject, subject age, and time from last pregnancy, as appropriate. increasing gravidity is significantly associated with a decreased prevalence of mmc. despite additional sources of fmc, there does not appear to be an increase in fmc prevalence with increasing gravidity. the biology of mc is incompletely understood, and the nature of mmc and fmc are likely to be different given that acquisition of the former, but not the latter, occurs within a nascent immune system. these data raise interesting questions when considered as interactions of acquired grafts within a host, including whether emergence and persistence of one dominant source of mc may be most advantageous for an individual. anti-igd antibody treatment as a novel immunosuppressive agent for autoimmune diseases and its effects on th1/th17 gene expression. tommie g nguyen, 1 eileen d gallery, 1,2 jonathan m morris. elevated t-helper cell type-1 (th1) and type-17 (th17) cytokine expression have a role in autoimmune diseases, allograft rejection and pregnancy-related complications. thus, molecules that can shift the immunity away from th1/th17 responses toward a th2 response represent a novel therapeutic treatment for these conditions. we have previously demonstrated that pregnancy is associated with a suppression of t-bet in peripheral t cells. in this study, we examined a novel effect of anti-igd antibody on t-bet expression, th1/th17 gene expression in human peripheral blood mononuclear cells (pbmc) and its therapeutic effects in an animal model of collagen-induced arthritis. methods: human pbmc were isolated and then cultured in the presence of anti-igd antibody at various time points followed by stimulation with pma/ionomycin (p/i) for 5 hrs. gene expression was examined by rt-pcr, western blotting and elisa. for in vivo study, arthritis-prone dba/j1 mice were induced to undergo joint inflammation by intradermal injections with bovine type-ii collagen. these mice were then given 3 daily doses of 10 mg/kg of intravenous injection with anti-igd antibodies as preventive or therapeutic treatments (n = 10 per group). results: treatment with anti-igd antibodies significantly suppressed p/iinduced expression of t-bet (a master regulator of th1 immunity), tnf-(a classical pro-inflammatory th1 cytokine), and il-17 (a critical proinflammatory th17 cytokine) in human pbmc. this suppression is highly specific to these genes because anti-igd antibodies have no effects on the expression of ifn-g and il-2 (two classical th1 cytokines). in vivo experiment showed that anti-igd antibody treatment markedly reduced clinical severity of joint inflammation when comparing the clinical score of control mice group (7.8 ± 1.1, mean ± s.e.m), preventive group (5.4 ± 1.2) and therapeutic group (3.5 ± 1.3) . conclusions: our study has demonstrated that suppression of t-bet by anti-igd antibodies, similar to the changes seen in human pregnancy is a novel in vivo anti-inflammatory effect. given the essential role of t-bet, tnf-and il-17 in the pathogenesis of human autoimmune diseases, anti-igd antibodies may represent a novel immunosuppressive treatment that needs further studies and evaluation. objective: women with circulating anti-phospholipid antibodies (apl) are at risk for recurrent miscarriage, preeclampsia and preterm labor. apl antibodies directly target the placenta by binding to phospholipids or phospholipid-binding proteins expressed on the surface of viable trophoblasts. the objective of this study was to determine the effects of apl antibodies on first trimester trophoblast cells. methods: two mouse igg1 anti-human beta 2 -glycoprotein i monoclonal antibodies (mabs), designated id2 and iic5, were used in these studies. the first trimester trophoblast cell line, htr8, was incubated with either medium, a mouse igg1 control, id2 or iic5 (5-40 g/ml), in the presence or absence of unfractionated heparin (100ng/ml). trophoblast cell death and apoptosis was determined using a viability assay, hoechst staining and a caspase activity assay. cytokine production was evaluated by multiplex analysis. results: following a 48 hour incubation, significant trophoblast cell death was induced by iic5 (34.5 ± 2.8%) and id2 (46.4 ± 3.2%) at the high dose of 40 g/ml, when compared to the medium and mouse igg controls (p<0.001). hoechst staining showed that id2-and iic5-induced trophoblast cell death was a result of apoptosis. moreover, id2 and iic5 induced a significant increase in caspase-3, -8 and -9 activity (p<0.001). treatment of trophoblasts with heparin significantly inhibited the effects of iic5 and id2 on cell death by 46.5 ± 12.8% and 52.6 ± 8.6% %, respectively (p<0.005). following a 72 hour incubation at lower concentrations (20 g/ml), treatment of trophoblast cells with id2 or iic5 resulted in a significant upregulation of il-8, mcp-1, gro production (p<0.05), and a significant reduction in il-6 secretion (p<0.05). conclusion: this study demonstrates that at low levels apl antibodies can modulate trophoblast cytokine production, while at higher levels, the same antibodies induce trophoblast apoptosis in a caspase-dependent manner. these findings shed new light on the mechanisms by which apl antibodies may impact placental survival and function. antigenic targets for the diagnosis of premature ovarian failure. hc bohler, c gercel-taylor, lt ku, st nakajima, dd taylor. obstetrics, gynecology, women's health, university of louisville, louisville, ky, usa. objective: premature ovarian failure (pof) is a premature depletion of ovarian follicles before the age of 40, affecting approximately 1% of women <40 years. the involvement of autoimmune mechanisms in pof has been suggested and similar mechanisms have been postulated for other ovarian pathologies, including idiopathic infertility, polycystic ovary syndrome (pcos), or endometriosis. while the association of autoantibodies has been demonstrated for these ovarian pathologies, variation in specificity and frequency of false positivity have limited the diagnostic use of autoantibodies. the objective of this study was to develop an antigen array to differentiate antibody recognition patterns of pof from other infertility pathologies. design: prospective study in a university research laboratory. materials and methods: patients diagnosed with infertility were included in this pilot study: endometriosis (n=6), pcos (n=4) and pof (n=5). autoantibodies were assayed by dot immunoblotting using an antigen array derived from endometrial and ovarian cells. for the cellular antigen preparations, solubilized total proteins were separated by reverse phase-hplcquid chromatography and the individual proteins were blotted onto nitrocellulose membranes and reactivity visualized by peroxidase-labeled antihuman igg. results: patients with pof, endometriosis, and pcos all exhibited autoantibodies reactive with these cellular proteins. while some antigenic reactivities were shared by all infertility patients, the pattern of antigen recognition was distinct for patients with pof. patients with pof all recognized a common 6 antigenic proteins (row 2, antigens a,b,d-g). conclusions: alterations in autoreactivity are observed in patients with the diagnosis of infertility; however, distinct patterns of autoantibody recognition can be demonstrated for patients with different pathologies. while this study needs to be expanded to reliably establish the specificity, sensitivity and positive and negative predictive values, patients with pof clearly exhibit a shared recognition pattern that may be useful a diagnostic marker. cicek gercel-taylor. ob/gyn, university of louisville, louisville, ky, usa. objective: americans' consumption of nutraceuticals is one of the most rapidly expanding health markets, growing at a rate of 25% annually. multiple nutraceuticals containing phytoestrogens have been marketed as "immune boosters" despite suboptimal evidence-based medicine to support such statements. as immunomodulatory therapies should affect downstream cytokine expression, the relative effects of estradiol and genistein in regulating expression of cd3-and jak3 were tested. these markers were chosen since they are central to t cell signaling. cd3-is a critical transducer of tcr activation and regulates t cell proliferation and cytokine production. jak3 upregulation is a specific marker for hematopoietic cell stimulation. methods: to test the immunomodulatory effects of phytoestrogens and estrogen, jurkat 6.1 (t cell leukemia) cells were grown in estrogen-free, phenol red-free media for 72 hours. these cells were then exposed for 48 hours to 0 pm, 4 pm (postmenopausal), or 40 pm (premenopausual) of estradiol in the presence of increasing concentrations of genistein (0, 0.4, 1.5, 6.25, 25, and 100 m). cells were then solubilized and cellular protein quantitated. protein concentrations were standardized and western blots for each set of culturing conditions were run in triplicate. cd3-and jak3 expression were quantitated following visualization with chemiluminescence by digital pixel quantification. results: our findings show that in the absence of estradiol and at postmenopausal levels of estradiol, genistein induced a dose dependent increase in cd3-reaching a maximum of 20 fold. although cultivation of t cells in 40 pm of estradiol significantly increased the levels of cd3-and jak3 relative to hypoestrogenic conditions, the genistein mediated dose response was not observed. conclusions: these in vitro results indicate that genistein can at least partially reverse suppression of signaling molecules observed in postmenopausal estrogen environments. clinically, this suggests that phytoestrogens may have greater immunomodulatory properties for postmenopausal females than those that are premenopausal. maternal serum il-6 as a biomarker of acute immunologic rejection of pregnancy. joaquin santolaya-forgas, 1 juan deleon-luis, 2 isabel galan. 2 1 obstetrics and gynecology, brigham and women's hospital, boston, ma, usa; 2 amarillo women's health research institute, texas tech university health science center, amarillo, tx, usa. objective: markers of acute rejection of pregnancy are very scarce. in this study we aimed at determining if rapid changes in maternal serum concetration of a variety of biomarkers could be used for this purpose. we used an established baboon model for in utero stem cell therapy to introduce at 36-44 days from conception and via ultrasound-guided celocentesis, human hematopoietic stem cells with different proportions of natural killer t-cells (nk). maternal blood was collected before and 24 hours after celocentesis for quantification of hormones and il-6 using solid phase, enzyme labeled, chemiluminescent sequential immunometric assays. pearson correlation analysis was used for determination of significant changes from baseline (p<0.05). results: all 9 animals survived their pregnancies. seven animals receiving <5% concentration of nk delivered at term ( 180 days gestation) while 2 animal receiving more than 7% concentration of nk had dead fetuses on ultrasound evaluation 24 hours after celocentesis. table 1 depicts mean maternal serum concentration of the biomarkers investigated (all n.s.). figure 1 shows mean il-6 changes from baseline in continuing (n.s.) and rejected pregnancies (p<0.05). conclusions: we have described a model in which in utero graft vs host disease can be studied. these preliminary results suggest that of all the biomarkers investigated, il-6 might be the most sensitive for detection of an acute rejection of pregnancy. biomarkers of acute immunologic rejection of pregnancy biomarker unit pre-celocentesis (9) the activity of cytotoxic cd8+ t cells during pregnancy protects the mother and fetus from infection. however, pregnancy's effect on the proliferation and apoptosis of cd8+ t cells has not been clearly defined. objective: to determine if normal pregnancy changes the number of proliferating or apoptotic splenic cd8+t cells. methods: female c57bl/6 mice were used unmated (um) or underwent timed mating. one day prior to harvest, mice were i.p. injected with bromodeoxyuridine (brdu), which is incorporated into replicating dna. harvested spleens were homogenized, enumerated, and stained for cell surface expression of cd8 and t cell receptor beta chain (tcr ). apoptotic cells were detected by treatment with terminal transferase and fitc-dutp (tunel). the numbers of cd8+tcr + cells that were brdu+ or tunel+ were calculated from the percentage of positive cells obtained by flow cytometry and the absolute number of cells counted. for each experiment, the ratio of the number of positive cells in pregnant to um mice was compared by anova with dunnett's post-test. results: at day 5 of pregnancy (n=5), the number of brdu positive cd8+ t cells was two fold higher than that found in um (n=12, p<0.01). the number of proliferating cd8+ t cells continued to be non-significantly elevated at day 8 (1.6x, n=5), day 10 (1.4x, n=7), and day 12 of pregnancy (1.5x, n=6) compared to um. by day 15 of pregnancy (n=5) the number of proliferating cd8+ t cells returned to the um level, however by this time the total number of splenic cd8+ t cells was 1.5 fold higher than um (n=12 p<0.001). on gestational day 18, the number of proliferating cd8+ t cells declined further (0.3x, n=4), and the number of splenic cd8+t cells returned to the um level (n=12, p>0.05). compared to um mice, there was no significant difference in the number of cd8+ t cells undergoing apoptosis at any gestational day examined (0.7-0.9x, p>0.05). in normal murine pregnancy, the number of cd8+ t cells is increased in late gestation, and then returns to baseline at the end of pregnancy. this is due to an early increase then gradual decline in cd8+ t cell proliferation, accompanied by a steady rate of apoptosis. this argues that the maternal immune system undergoes dynamic homeostatic changes, and is not globally suppressed. supported by nihro1hd043-185 niht32ai055402. arturo cerbulo-vazquez, 1 cun li, 1,2 gene hubbard, 2 natalia e schlabritz-loutsevitch, 1,2 peter w nathanielsz. objectives: early thymocyte (t) maturation occurs in the cortex while later stages occur in the medulla. thymic epithelial cells (tec) synthesize gc and t express gr. tec may influence t cell maturation by regulating apoptosisinduced gc-gr interactions. igf-1 (also synthesized by tec) may support thymocyte prolifetarion. human fetuses of mothers in premature labor are exposed to gc. gc administered to pregnant baboons at 0.6, 0.65, and 0.7 gestation (g) alters fetal lymphocyte populations at 0.95 (g) (j repro immunol, 2006, 69:149) . we determined if fetal gc exposure alters thymic 1) structure; 2) gr and igf-i protein. methods: pregnant baboons received saline (control ctr; n=6) or betamethasone i.m. (175 μg/kg daily for two days at 0.6, 0.65 and 0.7g; gc group; n=6), c-sectioned at at 0.9g under general anesthesia and thymic gr and igf-i evaluated by immunohistochemistry. results: gr localized to medulla and a few cortical cells. igf-i localized to cortex with little medullary expression. medullary necrosis was greater in ctr than gc fetuses. t gr was located in cytoplasm. no gross differences were observed between ctr or gc fetuses for either igf-i or gr. conclusions: a) early thymocyte maturation may be supported by igf-1, b) later differentiation involves gr, and c) after exposure to gc doses equivalent to human therapy, no gross effects were detected on gr or igf, but d) natural thymic necrosis was inhibited. lindsay s christensen, peyman bizargity, elizabeth a bonney. ob/gyn, university of vermont, burlington, vt, usa. background: the exact mechanism by which bacterial products trigger preterm delivery and the immune cellular circuits involved remain unclear. our recent data in normal c57bl/6 (b6) or recombinase deficient c57bl/6 mice (rag-ko) indicates that t and b cells are not critical for lps-induced preterm delivery and stresses the importance of related innate mechanisms. rag-ko mice are more susceptible to lps, suggesting that t or b cells may control the innate response. macrophages are vital to innate immunity and produce proinflammatory cytokines that can activate prostaglandin synthesis and myometrial contraction. we questioned whether differences in susceptibility between the strains are due to differences in the uterine macrophage response to lps and thus examined macrophages at the maternal-fetal interface early after injection. objective: to compare uterine macrophages levels at 2 and 6 hours after lps injection in pregnant b6 and rag-ko mice. methods: b6 and rag-ko mice were mated and on gestation day 15, females were injected intraperitoneally with 3μg lps in 200 l saline (pbs) or 200 l pbs alone. euthanasia and uterine harvest occurred 2 or 6 hours after injection. frozen uterine sections were stained with the macrophage marker f4/80 or an isotype-matched control followed by an alexafluor 546-conjugated secondary and a nuclear stain (dapi). sections were visualized by fluorescence microscopy. for each mouse, f4/80+ dapi+ and total dapi + cells were counted in 3 areas of 1 representative section and the percentage of f4/80+ dapi+ cells was calculated. the mean percentage for at least 6 representative areas per experimental group was analyzed by anova. results: two hours post-injection, macrophages levels were similar in b6 and rag-ko mice injected with pbs (b6, n=3, 2.9±1.6; rag-ko, n=3, 2.7 ± 1.2 % + per area). lps injection increased macrophages (p<0.05) in both strains (b6, n=3, 4.4±2.9; rag-ko, n=3, 5.5 ± 1.8,); no difference was evident between strains. the percentage of f4/80+ cells was similar 6 hours post-injection (b6, n=3, 3.4± 1.9 v. rag-ko, n=3, 3.6± 2.8), and not elevated relative to the 2 hour time point. objective. decay-accelerating factor (cd55), is expressed in the plasma membranes and protects mammalian cells against the lytic action of serum complement. phosphoinositide 3-kinases (pi3ks) are involved in the regulation of cell functions by synthesizing a second messenger molecule ptdins (3,4,5) p3. akt, a serine-threonine kinase acts downstream of pi3k and regulates cell survival, growth and proliferation. the pi3k-akt activity is controlled by tumor suppressor gene pten. in this study we assessed whether the pi3k-akt activity affects the expression of cd55 in human endometrial and cervical cells. methods. endometrial and cervical cell lines which differ in the constitutive pi3k activity were used in this study. ishikawa and rl95-2 endometrial cell lines harbor pten mutation and have high levels of phosphorylated akt (p-akt). hec-1-a and kle endometrial cell lines and hela cells express wild-type pten and have minimal or no demonstrable levels of p-akt. the expression of cd55 was evaluated by rt-pcr, immunoblotting and flow cytometry. the pi3k activity was assessed by immunoblotting with anti-p-akt antibodies. the effect of inhibition of pi3k-akt pathway on cd55 expression was evaluated in cells treated with wortmannin (400 nm), ly294002 (25 mm), or with akt inhibitor sh5 (5 mm). results. immunoblotting densitometry and measurements of mean fluorescence intensities showed that the level of cd55 expression correlates with the status of pi3k-akt pathway. the cd55 expression was lowest in hec-1-a, ishikawa and rl95-2 cells which constitutively express p-akt. higher cd55 expression was found in hela cells and kle cells which express wild-type pten product and has no detectable phospho-akt. mean fluorescence intensities were 3.4-fold higher for kle cells and 12-fold higher for hela cells compared to hec-1-a cells. treatment of cells with akt inhibitor led to 1.1-1.4-fold increase in cd55 expression. the 1.1-5.7-fold increase following treatment with pi3k inhibitor wortmannin was found in ishikawa cells, rl95-2 and kle cells. conclusions. human endometrial cell lines with elevated pi3k-akt activity express lower level of cd55 compared to cell lines with intact pten gene function. these findings may indicate that structural alteration at the dna level and resultant overexpression of pi3k-akt pathway are involved in the downregulation of cd55. endometrial and cervical cells. pawel goluszko, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. objective. cell shape is determined by the cytoskeleton, which provides the mechanical support and is involved in cellular signaling. apoptotic cells undergo major morphological changes such as rounding and contraction, a process regulated by caspases, the cysteine proteases responsible for events controlling the cell disassembly. the motifs in certain cytokeratins make them substrates for caspase degradation. anti-apoptotic serine/threonine kinase akt provides a survival signal by phosphorylating downstream effector molecules including caspase-9. while studying the akt distribution in human endometrial cell line we found that akt shows filamentous pattern of staining resembling cytoskeleton organization. in this study we evaluated whether akt staining correlates with the microfilaments (mf), microtubules (mt) or intermediate filaments. endometrial ishikawa, rl95-2, hec-1-a, kle and cervical hela cell lines were used in the study. incubation with cytochalasin d, (1ug/ml) or nocodazole (1mg/ml) and labeling with bodipy fl phallacidin, anti -tubulin or anti-akt antibody were used to assess whether cytoskeleton disruptors affect akt distribution and mf and mt organization. for colocalization, cells were stained with anti-cytokeratin 18 mouse antibody followed by anti-mouse alexa 488 conjugate, and then stained with anti-akt rabbit antibody and anti-rabbit alexa 549 conjugate. the scans collected with laser scanning confocal microscope from channels filtered for alexa 488 and alexa 459 were combined digitally and evaluated with imaris colocalization analysis software. filamentous pattern of akt staining was most pronounced in ishikawa and less obvious in hec-1-a cells. treatment with cytochalasin d or nocodazole resulted in disruption of mf and mt but had no effect on cytokeratin organization and akt distribution. double staining with anti-cytokeratin-18 and anti-akt antibody showed overlapping staining for cytokeratin and akt. analysis of digitally acquired images showed highest correlation for colocalized channels in rl95-2 cells (0.87) followed by ishikawa (0.79) and hela cells (0.72). lowest correlation was found for kle (0.65) and hec-1-a cells (0.59) conclusions. this study indicates a strong colocalization pattern of serine/ threonine kinase akt with cytokeratins, and suggests a mechanism by which cytokeratins might be protected against cleavage by caspase-9 and caspase-3 in the early apoptotic stages. background: cd4+ cd25+ t regulatory cells (t-reg), express foxp3,suppress antigen-specific immune responses and are important for allograft tolerance. during pregnancy the mother tolerates an allograft expressing paternal antigens (the fetus), requiring substantial changes in immune regulation over a programmed period of time. the presence of t-reg cells (cd4+ cd25highfoxp3+) was assessed in the peripheral venous blood of 25 non-pregnant, 63 pregnant and seven postnatal healthy women by flow cytometry. human decidua was obtained by surgical termination of pregnancy in the first (n=26), second (n=16) and third (n=5) trimester of human pregnancy. paraffin sections were immunostained for foxp3 and cd25. foxp3 +cells were quantified in 5 x400 fields and results compared between first, second and third trimester samples and according to the presence of extravillous trophoblast. results: fluorometric studies of blood samples indicate an increase % of circulating cd4+ cd25highfoxp3 t-cells in pregnant (3.06% [range 1.21-4.8%]) vs. non-pregnant controls (2.58% [range 1.1 -5.03%]; p<0.05). a progression from 1st, 2nd and 3rd trimesters indicated the % of cd4+ cd25highfoxp3 t-cells was 3.01%, 2.88% and 3.14%, respectively. low numbers of foxp3+ cells were detected in all decidua samples and their distribution mirrored that of cd25+ cells. in 1st trimester samples, foxp3+ cells were often detected in lymphoid aggregates adjacent to endometrial glands. increased numbers of foxp3+ cells were detected in 1st (21.9±4.3) compared with 2nd trimester decidua (9.9±3.2; p<0.05) but there was no difference between 1st and 3rd trimester (12.0±3.1), nor between 2nd and 3 rd trimester decidua. in 1 st trimester decidua, numbers of foxp3+ cells were increased in areas without extravillous trophoblast. conclusion: normal human pregnancy is associated with an increase in the number of circulating cd4+ cd25highfoxp3 t-cells. the presence of foxp3+ cells in early gestation human decidua may be important in the initiation of materno-fetal tolerance at an autocrine level. (supported by mrc). aims: -defensins are small cationic peptides with antibiotic and antimicotic activity. hyaluronan and its degradation products have been described as endogenous ligands for tlr2 and tlr4, whose involvement in -defensin expression has been reported in different epithelial tissues and cell lines. we aim to investigate weather low molecular weight hyaluronic acid induces -defensin 2 release by keratinocytes, via tlr2 and tlr4. methods: the induction of -defensin 2 production following in vitro treatment of human keratinocytes with a low molecular weight hyaluronic acid solution was evaluated by pcr-analysis and elisa techniques. studies on the involvement of tlr2 and tlr4 in -defensin 2 production have been performed using specific blocking antibodies. results: pcr and elisa revealed an intense -defensin 2 production following hyaluronic acid treatment in human keratinocytes. the -defensin 2 production induced by hyaluronan was abolished following block of tlr2 and tlr4 by specific antibodies, demonstrating the involvement of these receptors. the same hyaluronic acid treatment did not induce activation of inflammatory genes, such as il-8, tnf-, il-1 and il-6. conclusion: our data show that hyaluronic acid is an efficient inducer ofdefensin 2 production in keratinocytes, via tlr2 and tlr4. this observation might be important to open new perspectives in the development of possible topical products containing hyaluronic acid, to improve the release ofdefensins by keratinocytes, ameliorating the self-defence of the skin in case of skin infections. therefore, one of the possible applications for this kind of topical products might be the treatment of infective vulvitis, one of the most distressing gynaecological diseases for adult women. pregnancy outcome? kiera von besser, 1 serena wu, 1 mary d stephenson. 1,2 1 obstetrics and gynecology, university of chicago, chicago, il, usa; 2 obstetrics and gynaecology, university of british columbia, vancouver, bc, canada. objective: to investigate whether gender of an ongoing pregnancy impacts the probability of a successful outcome, and, to ascertain whether the gender of prior live birth(s) impacts subsequent pregnancy outcome, among women with rm/aps. materials and method: cohort-control study. rm subjects were evaluated by mds between 1992 -2004 (stephenson, 1996 . couples who met aps criteria (wilson et al, 1999) , restricted to rm only, were followed prospectively. cohort data was compared to live birth data from the vital statistics agency of british columbia from 1999-2004. secondary sex ratios (ssrs) among successful pregnancy outcomes were calculated by dividing the number of male live births by female. sex ratios were calculated for all pregnancies 24 weeks, regardless if they ended in success or demise. pearsons 2 test with yates continuity correction was applied. results: 388 subjects were identified. 226 subjects had 319 prior live births of known gender (174 male/145 female), giving a ssr of 1.20. there were also 25 prior fetal demises 24 wks of known gender (13/12) giving a sex ratio for all prior pregnancies at 24 wks of 1.19. 252 subjects delivered 301 subsequent live births of known gender (158/143), giving a ssr of 1.08. there were also 6 subsequent fetal demises (3/3) giving a sex ratio for all subsequent pregnancies of 1.10. 112 subjects delivered both prior and subsequent live births. the ssr was 1.36 (83/61) among their prior and 1.24 (68/55) among their subsequent live births. including fetal demises, 119 subjects had ongoing prior and subsequent pregnancies. the sex ratio was 1.32 among their prior pregnancies and 1.10 among their subsequent. as the control, a ssr of 1.06 (124,982/118,093) was calculated from vital statistics data. when the prior and subsequent ssrs of the cohort were compared to each other, as well as to the control, there were no statistically significant differences. conclusions: our findings, from the largest study of its kind to date, suggest that, in patients with rm/aps, the gender of an ongoing pregnancy does not significantly affect the probability of a successful outcome, to any greater degree than it does in the general population. also, the gender of a prior ongoing pregnancy does not significantly impact the likelihood of developing rm/aps. oocyte maturation. jk friend, fb bezirci, e seli. ob gyn, yale u., new haven, ct, usa. introduction: oocyte maturation is associated with repression of transcription. during oocyte maturation, fertilization, and early embryo development until the onset of zygotic gene expression, proteins are synthesized from maternallyderived mrnas. the regulation of protein expression from these maternal mrnas is post-transcriptional, and occurs mainly via poly(a)-tail elongation. the embryonic poly(a)-binding protein (epab) is the predominant poly(a)binding protein before the activation of the zygotic genome, and plays a critical role in the activation of certain maternal mrnas, those bound by cpeb and probably pumilio. we are characterizing additional functions of epab during the process of oocyte maturation. methods and results: our model system is the xenopus laevis oocyte where oocyte maturation is induced by the addition of progesterone. our preliminary findings indicate that epab is phosphorylated, and that levels of phosphorylated epab increase upon progesterone-induced oocyte maturation. moreover, glycerol gradient centrifugation revealed that nonphosphorylated and phosphorylated epab are contained in distinct complexes that change mobility upon oocyte maturation. furthermore, oligo-dt selection for poly(a)-containing mrnas strongly suggests that these mrnas are bound exclusively by phosphorylated epab. using affinity purification, we have determined that nonphosphorylated epab exists primarily in a large protein complex prior to oocyte maturation that is later disassembled after the addition of progesterone. conclusions: based on these preliminary findings, we conclude that prior to oocyte maturation, the bulk of epab is nonphosphorylated and is found in a protein-rich complex separate from poly(a)-containing mrnas. upon oocyte maturation (when certain maternally-derived mrnas are activated for translation), the majority of epab becomes phosphorylated, and this phosphorylated form of epab is likely bound to translationally-active mrnas. we are currently investigating what kinase phosphorylates epab and whether this phosphorylation plays a role in translational up-regulation of epab-bound mrnas. introduction: reactive oxygen species (ros) play important roles in all aspects of cellular fate. nadph oxidase isoforms, a family of genetically preserved enzyme complexes, have been shown to be the main sources of ros in various cell types. however, the role of nadph oxidase isoforms in human myometrium proliferation and differentiation has not been defined. in the myometrium, different smooth muscle phenotypes maybe associated with specific physiologic functions. we have shown that angiotensin ii (angii) stimulates hypertrophy but not cell proliferation in ultr cells, an in vitro model of human myometrium. ultr cells at greater than 30 passages display a replicative senescent phenotype. by introducing human telomerase reverse transcriptase (htert) gene, we have obtained a stable cell line (ultr-ht) which has a significantly increased division rate and distinct cellular morphology than the original ultr cells. objective: to determine the relationship of expression of nadph oxidase to ultr cellular fate. methods: early and late passages (p26-34) of ultr and ultr-ht (p31-38) cells were grown on either plastic or collagen iv (cn4)-coated surfaces. ultr-ht cells were further stimulated with angii (0.1 um) for 24 hrs. expression of nadph oxidase core (nox1-5 and duox1/2) and associated subunits (p22phox, p47phox, noxo1, p67phox, noxa1, p40phox, and rac1/2), and angii receptors at1/2 was identified by rt-pcr from cellular total rna. fluorescent immunohistochemistry (ihc) was employed to determine protein expression and localization. results: the mrna level of house keeping gene -actin was unchanged by any cellular manipulation. the senescent phenotype of ultr cells was accompanied by an apparent down-regulation of nox1, p22phox, and noxa1 genes, and an up-regulation of at1/2. overexpression of htert did not reverse nox1, p22phox and noxa1 expression while cell division rate was increased. however, there was a down-regulation of nox4, at1/2 and rac2. plating ultr-ht cells on cn4 induced nox4/5 down-regulation and up-regulation of duox1/2, with no apparent change of at1/2. however, exposure to cn4 re-directed cellular response to angii such that only nox5 was induced by angii stimulation. conclusion: expression of nadph oxidase isoforms nox1, 4, 5, and duox1/2 are correlated with ultr cell differentiation and cell fate control. data also suggests that ang ii-induced myometrial hypertrophy involves nox5 mediated ros generation. fluids from reproductive women -the influence of aging. eriko y fujii, 1 masahiro nakayama. 2 1 women's health, national center for child health and development, tokyo, japan; 2 aska reproductive clinic, nara, japan. [introduction] receptor for advanced glycation end products (rage) is a multiligand type glycoprotein, and is characterized based on its ability to bind ages, adducts formed by non-enzymatic glycation and oxidation of protein and lipids. this process occurs during normal course of aging. ages/rage interaction regulates various physiological function, such as inflammation, angiogenesis through vegf inducement. a soluble form of rage (srage) works as a decoy in the body and inhibits intracellular signaling. [objectives] the balance of these factors may contribute to reproductive dysfunction by aging. we aimed to measure the ages, srage and vegf concentrations in plasma and follicular fluids from reproductive women, and examined the differences of those factors between young group and old group. [material and methods] patients' plasma and follicular fluid were collected with consent based on regulations of the ethical committee, and we measured ages (pentosidine, cml), srage and vegf in duplicate using commercially available elisa kits (fushimi co, r d and cyelex). concentrations were calculated from each standard curve, and compared between the young group under 34 years old, and the old group over 35 y.o. data were evaluated for the difference in two groups by student's t test , and the significance was determined by p<0.05. [results] 1) srage in plasma, 1517±642 pg/ml (mean±sd), n=40 in the young group was significantly higher than 1268±530 pg/ml, n=51 in the old group. there was no significant difference in plasma vegf. 2) vegf in follicular fluid was 45±22 pg /mg protein, n=37 in the young, and 56±27 pg /mg protein, n=63 in the old was increased significantly. 3) we could not see statistical difference of pentosidine nor cml concentrations between two groups in plasma and follicular fluid samples. [conclusions] it has been reported that higher concentration of vegf in follicular fluid may relate to worse pregnancy rate in art. there was a significant decrease of plasma srage in older group in our result, and because of this decrease of 'decoy', focal ages-rage-vegf signaling might be activated in older women. our results showed the possibility that ages/rage and vegf regulation may contribute to the reproductive dysfunction by aging. and leiomyosarcoma cells. qun pan, xiaoping luo, nasser chegini. ob/ gyn, university of florida, gainesville, fl, usa. as a part of a novel endogenous rna silencing machinery, a noncoding short rna strand referred to as "microrna" (mirna) has been identified to regulate the stability of the target gene expression through mrna degradation and repression. we have identified the expression of many of these mirnas in leiomyoma, myometrium, their isolated smooth muscle cells (lsmc and msmc), transformed lsmc (t-lsmc) and sklm (leiomyosarcoma cell line), including mir-21 which is predicted to target the expression of many genes, including tgf-b and tgf-b type ii receptor (tgf-brii). however, the biological significance of these mirnas in various cellular processes remains to be established. as such in the present study we examined the expression, regulation and function of mir-21 in lsmc, msmc, t-lsmc and sklm. we found that mir-21 is expressed and regulated by 17b-estridiol and medroxyprogesterone acetate (10 -8 m) in these cells (p<0.05). we further assessed the regulatory function of gain of and loss of function of mir-21 on the expression of tgf-brii. transfection of lsmc, msmc, t-lsmc and sklm with pre-mir-and anti-mir-21 oligonueclotides resulted in a significant increase and/or inhibition of mir-21 expression in these cells, respectively as determined by realtime pcr (p<0.05). over-expression of mir-21 resulted in a significant reduction, while transfection with antimir-21 increased the expression of tgfbrii mrna and protein in these cells as compared to controls (p<0.05). we concluded that mir-21 is expressed in leiomyoma and myometrial cells, its expression is regulated by the ovarian steroids and it functions by targeting the expression of tgfbrii and possibly other genes with key regulatory action on cell growth, angiogenesis, transcription regulation, ecm turnover and apoptosis that results in leiomyomas growth and regression. (supported by nih grant hd37432). objective: placenta and a number of gestational tissues are well recognized to express corticotrophin releasing factor (crf), urocortin 1 (ucn1), ucn2, ucn3 and crf -r1 and crf-r2 receptor subtypes together with crfbinding proteins locally. ucn2 and ucn3 are implicated in the reversal of stress responses initiated by crf. in the present investigation, we evaluated functions of crf and ucns by quantifying their contents in venous smooth muscle layers using image pro 4.01 in human umbilical cords collected at preterm and term gestation methods: umbilical cord specimens (3-4mm thickness, 6 pieces per umbilical cord) collected at preterm and term (n=6 each) at delivery were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochemical analyses with polyclonal antibodies to crf (1:300), ucn1 (1:250), ucn2 (1:250) and ucn3 (1:250) (peninsula laboratory, pa and sigma aldridge, ms) by standard abc technique. immunoreactive materials on the sections were identified using 3, 3'-diaminobenzidine as a chromagen. immunostaining intensities (od/area) on uc-sections were quantified by image pro 4.01 software and expressed as arbitrary units (au). all values were expressed as mean ± sem. differences between groups were evaluated by anova, followed by the post-hoc tukey test for multiple comparison. results: antibodies to crf and ucns elicited positive immunostaining of variable intensity in venous smooth muscle layers in uc-sections of preterm and term gestations. immunostaining intensity (au) of venous smooth muscle layers at preterm (pt) and term (t) are as follows: crf-pt= 0.0451±0.0043 vs crf-t= 0.1027± 0.0242 (p<0.5); ucn1-pt=0.1736 ± 0.0108 vs ucn1-t= 0.0959± 0.0116 (p<0.01); ucn2-pt= 0.0964± 0.0063 vs ucn2-t= 0.0724 ± 0.1632 (p=ns); ucn3-pt= 0.1214 ± 0.0034 vs 0.0732± 0.0050 (p<0.05). conclusion: crf content in venous smooth muscle layer markedly increased at term, while ucn1 and ucn3 contents significantly decreased and no significant change occurred in ucn2 content. based on the opposing changes in crf vs ucn1 and ucn3 immunostaining, we speculate that crf, but not ucn1 or ucn3, is the major key player of vasodilation and function locally at the level of venous smooth muscle cells at term. ucn1 and ucn3 may perform cytoprotective functions at preterm. physiol. 1947; 150: 511-519, am j physiol. 1952; 170:72-76) . these studies showed that when ad libitum (al) feeding was resumed in cr females, fertility was sustained well beyond the age at which al-fed females became infertile. however, much of what is known on the effects of cr on fertility derives from models in which cr was initiated at weaning. further, there is large variation in how the experiments were conducted. objective: herein we tested if adult-onset cr could delay age-related infertility in females. methods: cr (40%, nia protocol as described in j geront. 1999 54a:b492-b501) was initiated in c57bl/6 female mice at 4 months and continued until 15.5 months of age, at which time al feeding was resumed. matings were initiated at 10 months of age. for mating during cr, a male mouse was housed overnight in a cage with a female and removed the next morning, so that the female mice could be fed their dietary food ration. al-fed and cr females followed the same mating regimen. the number of offspring born and that survived to weaning (day 21) were recorded. results: fertility was lost in 3 of 11 al-fed females by 10 months of age and continued to decline through 15.5 months of age. age-matched females maintained on cr during the same period exhibited poor fertility, with a total of 9 pregnancies achieved out of 12 females. although cr females showed poor fertility while on cr, their fertility improved dramatically after the reinitiation of al feeding at 15.5 months of age. while only 1 out of 8, al-fed females achieved a pregnancy between 15.5-18.5 months of age, 6 out of 10 age-matched cr-then-al-fed females achieved a total of 10 pregnancies in this 3-month time. notably, 79% of the pups born to cr-then-al-fed females between 15.5-18.5 months of age survived and developed to weaning without complications. conclusions: adult-onset cr allows maintenance of female fertility into advanced maternal age after the reinitiation of al feeding. how long fertility can be maintained and the minimum time needed for the beneficial effects of cr to be realized remain to be investigated. nonetheless, these observations suggest that there may be ways to safely extend fertility in females at ages when reproductive function is suboptimal (nih r37-ag012279). bile acids in human ovarian follicular fluid. laura p smith, 1,2 kaila deiorio-haggar, 1 jason reindollar, 3 alan s penzias, 1,2 anny usheva-simidjiyska. 3 1 ob/gyn reproductive endocrinology infertility, bidmc, boston, ma, usa; 2 boston ivf, boston, ma, usa; 3 endocrinology, bidmc, boston, ma, usa. introduction: bile acids are known to serve important functions in the hepatobiliary and gastrointestinal systems. the presence of bile acids in the human ovary and relation with fertility potential have never been previously evaluated. methods: human follicular fluid (ff) from large follicles and small follicles was obtained at vaginal oocyte retrieval. human serum was obtained before and 36 hours after human chorionic gonadotropin (h-cg). follicular fluid and serum samples were analyzed for total bile acids by spectrophotometry. bile acid concentrations were correlated with age, number of retrieved and mature oocytes, number of fertilized oocytes, and pregnancy. results: bile acid concentrations were analyzed and compared to the normal human serum bile acid concentration which ranges from 0 to 15 micromoles/l. bile acids are present in follicular fluid with a mean concentration of 15.28 micromoles/l in large follicles and 14.22 micromoles/l in small follicles (p=0.108). pre and post h-cg serum bile acid concentrations differed significantly (13.88 micromoles/l vs. 7.85 micromoles/l, p=0.004). there was a trend toward higher bile acid concentration in large follicles of young patients < 35 years old compared to older patients 40 years old (16.22 ± 5.55 vs. 13.56 ± 2.26, p=0.051). there was also a trend toward higher pre h-cg serum bile acid concentrations in older patients (4.47 ± 1.92 vs. 2.90 ± 0.72, p=0.079). there was no correlation between serum and follicular fluid bile acid concentrations and number of oocytes retrieved or number of mature oocytes, but there did appear to be a positive correlation between pre h-cg serum bile acid concentration and number of fertilized oocytes (spearman's correlation coefficient 0.678, p=0.005). conclusions: this is the first demonstration of the presence of bile acids within human ovarian follicular fluid. there may be a relationship between bile acid concentration and fertility potential. the precise function of bile acids in human ovarian follicular fluid is under investigation. objective to investigate the impact of ovarian hyperstimulation treatment on the biomarkers of the homocysteine pathway in blood and follicular fluid, and their association with the follicle diameter as a measure of follicular maturation. methods in 201 women undergoing ivf/icsi treatment blood samples were collected on cycle day 2 and the day of hcg administration. during oocyte retrieval in each woman the diameter of two follicles was measured and the corresponding follicular fluids were collected. in blood and follicular fluid total homocysteine (thcy), folate, cobalamin and pyridoxal'5'phosphate (plp) concentrations were determined. women with a serum folate 22.5 nmol/l were classified as folic acid supplemented. results ovarian hyperstimulation significantly decreased thcy and cobalamin blood levels (both p 0.001). the blood and follicular fluid concentrations of thcy, folate, cobalamin and plp were significantly correlated (all p 0.001). in the total group, a two-fold increase of thcy in follicular fluid resulted in a 0.06 mm decrease of the follicular diameter (p 0.05). in non-supplemented women this decrease was 1.64 mm (p 0.01). in supplemented women a twofold increase of follicular fluid folate resulted in a 0.74 mm decrease of the follicular diameter (p 0.05). conclusions ovarian hyperstimulation reduces thcy blood levels independent of folic acid supplementation. however, high follicular fluid thcy and folic acid supplementation may have detrimental effects on the maturation of the follicle. 2), post-fixed for 40 rain in 2% w/v osmium tetroxide in the same buffer, quickly dehydrated in a series of ethanol solutions, and embedded in epon. thin sections were stained with uranyl acetate followed by lead citrate and were observed at electron microscope. results: ten blastocysts from each group were collected and analyzed. compared to the in vivo embryos, blastocysts generated in vitro exhibited significant differences. the surface of their trophectoderm (te) layer had a reduced number of microvilli, the number of the apoptotic cells in the inner cell mass (icm) was higher and the presence of non functional mitochondria was elevated. in this study we have, for the first time, compared the ultrastructure of the in vivo and in vitro conceived blastocysts. taken together, these results suggest that both, the higher rate of apoptosis and the morphological alterations in the mitochondrial structure in ivf embryos, are associated with stress during in vitro embryo culture. therefore, these parameters can be used, in the future, as markers for the assessment of the embryo wellbeing in the ivf settings. deteriorating oocyte quality is a critical hurdle in the management of infertility, especially one associated with advancing age.here, we explore a newly discovered role of nitric oxide (no) in the sustenance of oocyte quality. methods: sibling oocytes from superovulated mice were subjected to intracytoplasmic sperm injection (icsi) with cauda-epididymal spermatozoa following exposure to either the no donor, s-nitroso n-acetyl penicillamine (snap, 0.23 m/min); a soluble guanylyl cyclase inhibitor, 1h-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one (odq, 100 m) or an no synthase inhibitor, n w -nitro-l-arginine methyl ester (l-name, 1 mm). their sibling oocytes were subjected to icsi either before (young) or after culture for the corresponding period (old). outcomes of fertilization, cleavage and development to the morula and blastocyst stages were compared. some embryos from each subgroup were also subjected to tunel assay for apoptosis. results: oocytes deteriorated in their ability to undergo normal fertilization and development to morulae/blastocysts after aging in culture, as compared to their sibling cohorts subjected to icsi immediately after ovulation (p<0.05). this deterioration was prevented after oocyte exposure to snap. while, exposure to l-name or odq resulted in a significant compromise in fertilization and development to the morulae/blastocysts (p<0.05) with detection of apoptosis, which was also noted in embryos derived from aged oocytes but not in those from young or snap exposed oocytes. conclusions: no is essential to sustain oocyte fertilizability and developmental ability, and to prevent blastomere apoptosis. objective to investigate the effects of the levels of the biomarkers of the homocysteine pathway on ivf outcome. methods from 201 women undergoing an ivf or icsi procedures, two blood samples and two mono follicular fluid samples were collected for determination of folate, cobalamin, and total homocysteine (thcy). total protein was determined in follicular fluid to adjust the biomarker concentrations for follicular maturation. primary endpoint of the study was oocyte quality measured by fertilization and embryo quality (range 1-5; with 1 being best quality). secondary endpoint was the occurrence of pregnancy. results 67% of the included women used a folic acid supplement (serum folate 22.5 nmol/l). in non-supplemented women higher cobalamin levels in follicular fluid correlated with a better embryo quality (estimate -0.87; p 0.05) and higher thcy levels (median 7.1 mol/l, range 4.0-47.0) correlated with a worse embryo quality (estimate 1.01; p 0.05). in supplemented women higher follicular fluid thcy (median 6.4 mol/l, range 3.5-73.6) correlated with better embryo quality (estimate -0.58; p 0.05). the follicular fluid folate level of oocytes that did not fertilize was 1.1-fold higher than in the fluids of a fertilized oocyte (95% ci 1.00 -1.18; p 0.05). a two-fold increase of follicular folate corresponded with a 3.3 higher chance to achieve pregnancy (95% ci 1.09-9.71). conclusions cobalamin levels in follicular fluid are correlated with embryo quality. folic acid supplementation modifies the thcy and folate levels in follicular fluid and thereby affects oocyte quality. the level of folate in follicular fluid is important in the fertilization process. we recently reported that increasing relative oocyte immaturity is associated with worsening outcome, and that cycles with many immature oocytes are more common in younger women. (moore et al, asrm annual meeting, 2007) to further investigate this trend, we conducted a case/control analysis of patients with repeated cycles of high-level oocyte immaturity (hloi). methods: oocyte maturity data was collected on all icsi cycles starting in 2002. we defined a cycle with hloi as having >50% immature oocytes (>2 sd's above the median). a case was defined as a patient with hloi on more than one cycle. control subjects were age-matched and defined as having </= 35% (1sd above median) immature oocytes on all cycles. results: from 450 subjects, we identified 8 cases of recurrent hloi (comprising 19 icsi cycles) and 240 control subjects. at baseline, cases had more oocytes retrieved per cycle than controls (22.5 vs. 16.3, p<.0001). all other baseline characteristics were similar. all case subjects were younger than 37. outcomes are shown in table 1 . discussion: clinical pregnancy and live births were reduced in the case group, but more than expected based on % immaturity. during 19 cycles, there were no live births among the case group. patients with recurrent hloi represent a previously undescribed group of young patients with a very poor prognosis during icsi treatment. this study may suggest that the problem underlying the immaturity may relate to overproduction of oocytes during stimulation, a factor which may be modifiable. however, the additional trend (though not significant) that even the mature oocytes from the case patients tend to have poor outcomes suggests the possibility of a maturation defect and warrants further consideration. data on response to gonadotropins, fert rates, and implantation rates will be presented. amongst those, 23 were performed for an initial diagnosis of premature ovarian aging (poa), 6 for the diagnosis of premature ovarian failure, and 11 for repeated pregnancy loss. in women under age 42, b-fsh levels over 12 miu/ml and up to < 50 miu/ml denoted a diagnosis of poa, while levels of 50miu/ml were considered diagnostic for pof. all patients signed consent permitting review of medical records for clinical research purposes. results: day 3 fsh levels ranged from 5.6 to 30.0 miu/ml (mean 13.6 ± 7.2 miu/ml). amh levels ranged from < 0.1 to 2.7 ng/ml (mean 0.8 ± 0.8) and allele repeats ranged from 17 to 49, (mean for allele-1, 27.2 ± 4.3; allele-2, 32.0 ± 6.4). mixed model analysis of variance for all patients, adjusted for age, race, and allele-1 revealed a statistically significant correlation between b-fsh levels and number of repeats in allele-2 ( figure 1 ; p < 0.01). we did not observe a significant linear relationship between serum amh levels and cgg repeat counts, however cgg repeat counts above 32 were significantly associated with serum amh levels less than 1 ng/ml (chi square = 0.04). conclusions: triple repeats above 30, a level currently considered well within normal, are associated with evidence of premature decline in ovarian function. the evaluation of triple repeats in frm1 gene offers a first objective assessment of ovarian function and should be predictive of autologous reproductive life span in most women. objective: the levels of mis in the serum and in the ovary have been demonstrated to decrease in response to increasing doses of cisplatin. we hypothesize that ovarian stimulation also will reveal dose related decreases in ovarian and serum mis levels after cisplatin exposure, further demonstrating the follicular damage from chemotherapy. methods: adult female sprague-dawley rats were treated with saline, 4.5 mg/kg cisplatin or 6.0 mg/kg cisplatin as two weekly injections. five days following the last cisplatin injection, the rats were injected with pregnant mare serum gonadotropin (pmsg) and euthanized 54 hours following the pmsg injection. serum was collected and both ovaries were obtained for protein analysis. serum and ovarian levels of mis were determined using an elisa kit. western blot analysis, immunohistochemical analysis, and tunel analysis were also performed on the ovarian proteins. results: stimulated mis levels declined in a linear fashion in response to increasing cisplatin doses (p<0.001). ovarian protein levels measured by elisa and western blot both indicated that stimulated ovarian mis levels also decrease in a linear fashion (p=0.001 and p<0.001 respectively.) immunohistochemical analysis revealed that while the total number of follicles remains the same, both the total number of mis positive follicles, and the percentage of mis positive follicles declines in a linear fashion (p=0.001 for both). tunel analysis reveals that there is no change in the incidence of apoptosis in the stimulated ovaries from any treatment group. conclusions: the administration of pmsg after treatment with cisplatin causes a dose dependent decrease in the levels of mis in both the serum and in the ovary. serum mis levels following ovarian stimulation may be useful as a serum biomarker in this animal model to assess ovarian damage following the administration of cisplatin. correlations of micro-rnas 23a, 23b, 17-5pp, 211 and 542-3p with inflammatory and steroidogenic factors in human granulosa/cumulus cells. tannaz toloubeydokhti, orhan bukulmez, kenneth c drury, r stan williams, nasser chegini. obstetrics and gynecology, university of florida college of medicine, gaineville, fl, usa. as part of the novel endogenous rna silencing machinery, noncoding short rna strands, referred to as micrornas (mirnas), have been identified to regulate the stability of the target gene expression through degradation and repression. the expression of many of these mirnas has been identified in human tissues however, their biological significance in various cellular processes remains to be elucidated. in this cross-sectional study of human granulosa cells, we aimed to study the potential correlations between five selected mirnas, mir-23a, mir-23b, mir-542-3p, mir-211 and mir-17-5p, and mrna expressions of some of their predicted target genes namely, cyclooxygenase (cox)-2, il-1 , steroidogenic acute regulatory protein (star), aromatase and estrogen receptor (er) . granulosa/cumulus cells were obtained from 26 women (age range 23-42; mean age 34.8 ± 0.5) undergoing oocyte retrieval for assisted reproduction. total rna was extracted from these cells and reversed-transcribed and real-time pcr was performed to measure steady-state levels of mirnas and mrna transcripts. we observed that the expression of mir-23a, mir-542-3p, mir-211 and mir-17-5p displayed a significant and positive correlation with the expression of cox-2. moreover, mir-23b significantly correlated with star mrna expression, but not with other genes tested (p<0.05). with advancing age, the expression of mir-17-5p and cox-2 was significantly decreased, with cox-2 expression displaying a positive correlation with aromatase, star and er mrna expressions (p<0.05). none of the factors tested showed any significant correlations with peak estradiol levels and number of oocytes retrieved. receiver operating characteristic curve analysis revealed that female age cut-off value of 31.5 y best predicted whether mir-17-5p was below its mean arbitrary value. in conclusion, given the importance of mirnas' regulatory function in gene expression, our results suggest that mir-23b may play an important role in gene expression related to granulosa cell luteinization, while variation in mir-17-5p expression with female age may suggest its potential role as a predictor of oocyte quality and granulosa cell function. given the importance of mirnas in gene regulation, the role of mirnas in ovarian physiology and aging requires further investigation. support: nih grant 37432. introduction: a symbiotic relationship between ovarian granulosa cells (gc) and the enclosed developing oocyte is critical to reproductive efficiency. genetic modulations in gc's can lead to reproductive insufficiency, highlighting the critical role of gc's in reproductive competence. defects in the oocyte-gc repertoire in women with dor include lower e2 levels, luteal deficiency, poor fertilization rates, higher incidence of aneuploidy, lower pregnancy and higher miscarriage rates. utilizing global gene expression analyses in cumulus gc's, we attempt to enhance our understanding of biological mechanisms that may contribute to poor reproductive capacity in young women with dor. methods: 8 infertile women (<38 years old) undergoing ivf were prospectively enrolled into two groups based on ovarian reserve (normal reserve, fsh<10 dor, fsh >10miu/ml). at egg retrieval, cumulus gc's were isolated, rna extracted transcribed into cdna. microarray targets were generated cdna hybridized to affymetrix human genome u133 plus 2.0 genechips. microarray data were analyzed (array assist) and normalized (robust multichip analysis). a difference in gene expression of > 2 fold was considered biologically relevant. results: of the 8,846 genes identified to be differentially expressed in young women with dor compared to normal reserve, 215 genes demonstrated consistency of expression across five different normalization schema; 94 were down regulated and 121 up-regulated. expression of gremlin, a member of the dan family of genes known for its highly regulated expression pattern during folliculogenesis, was noted to be down-regulated 3-fold over two probe sets (-3.08) in women with dor versus normal reserve; this down-regulation was confirmed by real-time pcr (-5.33) . conclusions: this is the first demonstration linking differential expression of gremlin with etiology of infertility in women. gremlin is a downstream effector of oocyte-derived gdf-9 which facilitates cumulus cell expansion, a critical event in reproductive physiology. our finding of a significant downregulation of gremlin expression in cumulus gc's associated with dor may partly explain the physiology of poor reproductive performance. nih k24cd41978. nih 5k12rr17672. ferring pharmaceuticals. the objective of the present study was to investigate the effects of the gnrh antagonist ca on expression of mis and aromatase (cyp19) using luteinized human granulosa cells obtained during in-vitro fertilization cycles and an immortalized human granulosa cell line (hgl5). the granulosa cells were cultured +/-dibutyryl camp (1 mm) and incubated +/-the gnrh antagonist, ca, for 24-48 h. rna was isolated, reversed-transcribed and real-time pcr was performed to measure mrna transcripts for ovary-specific cypiia and mis. we observed that camp markedly induced aromatase mrna in both the primary and immortalized human granulosa cells. interestingly, camp treatment of these cells also caused an upregulation of mis mrna. objectives: bisphenol a (bpa), a known endocrine disruptor, is a chemical used as a plasticizer in the manufacture of polycarbonate plastics and epoxy resins and is present in multitude products, including the interior coating of food cans, milk containers, and baby formula bottles. bpa can leach into foods during heat processing and is known to exert a variety of endocrine-like effects on different cell types acting as an estrogen because it contains two hydroxyl groups in its diphenyl structure. in this study we focused on the effects of bpa on aromatase expression and estradiol production in the human granulosa kgn cell line. we also evaluated its effects on several transcription factors crucial in cyp19 expression. materials and methods: kgn cells were cultured in f-12dmem and were starved for 48 h before experiments. subsequently they were treated for 48 h with vehicle (control), fsh (100ng/ml), and/or bpa (20, 40, 60, 80, 100um) . messenger rna expression was quantified by real time pcr and estradiol secrection was measured in supernatants by elisa. results: fsh induced a 10-fold increase in aromatase expression. bpa induced a dose-dependent decrease in cyp19 production, with the greatest effect at 100um (p<0.001), resulting in 91+/-3 % (mean+/-sem) inhibition, compared to aromatase expression induced by fsh alone. bpa also reduced levels of estradiol secretion in a dose-dependent manner, with the greatest inhibition at 100um (p<0.01) resulting in 90+/-5% decrease. we also evaluated expression of transcription factors known to be involved in regulating the activity of the ovary-specific aromatase proximal pii promoter. interestingly factors known for induction of aromatase such us steroidogenic factor-1 and gata-4, mimic the pattern of cyp19 expression after bpa treatment, whereas, other receptors previously reported to act as aromatase inhibitor, such as ppar gamma were up-regulated by the addition of bpa. moreover, expression of creb remained virtually intact, suggesting that most likely mechanisms governing endocrine disruption by bpa are highly selective. conclusions: bpa inhibited fsh stimulated aromatase expression and downstream estradiol secretion. we propose that constant exposure to this chemical may result in endocrine disruption which may contribute to reduced fertility and early ovarian senescence. oocyte maturation occurs during folliculogenesis as a result of complex cell-tocell communications between the granulosa cells and the oocyte. maintaining the granulosa cells' spherical structure and network of gap junctions surrounding the oocyte is critical. this project tests the ability of a novel substrate-free threedimensional culture system to form viable granulosa cell spheroids. methods: after irb approval, freshly obtained follicular fluid from in vitro fertilization was obtained and granulosa cells were purified by percol gradient. nonadhesive agarose hydrogels, containing 822 cylindrical round bottom recesses 200 m in diameter, were cast from micro-molds designed using computer-assisted rapid prototyping. granulosa cells seeded at a density of 800,000 cells per gel were incubated for up to 10 days. cellular viability was assessed with live:dead assay. results: after three days in culture, granulosa cells formed spheroids of densely packed cells that were difficult to disperse with multiple pipettings. the cells remained viable for at least 10 days. conclusions: granulosa cells can be cultured in a novel substrate-free threedimensional culture system. the cells form tightly adherent spheroids that remain viable for extended culture. the cohesiveness of the cells suggests the formation of gap junctions. this is under investigation with immunohistochemistry and electron microscopy. these experiments suggest that a substrate-free threedimensional hydrogel culture system may be ideal to reassemble follicular structure important for future in vitro evidence testing and oocyte maturation. known to function as responders to pathogens, inflammation, and tissue injury. previous studies in our laboratory demonstrated that saa was produced in mouse granulosa and production was regulated by cytokines. ovulation has long been considered an inflammatory reaction and patients with chronic inflammatory conditions often experience infertility. the present study was undertaken to explore the role of saa in human ovarian function. methods: ovarian granulosa and luteal cells were obtained from surgically removed specimens and mural and cumulus granulosa-luteal cells were obtained from ivf aspirates. rna was extracted from fresh or cultured cells. some cells were treated in vitro with tnf or other cytokines for 24h. expression of saa was assessed by quantitative rt-pcr. in addition, serum levels of saa were determined using a commercial elisa in women undergoing ovulation induction (oi) and art. saa levels were measured at baseline, during oi, on the day of hcg administration and at the time of the pregnancy test. results: saa mrna was expressed in theca, granulosa, and granulosa-luteal cells. in granulosa-luteal cells both saa1 and saa2 mrnas were expressed at higher levels in cumulus compared to mural granulosa. expression of saa1 and saa2 in theca was increased following treatment with tnf in vitro. serum levels indicated that patients with ovulatory dysfunction had increased levels of saa at the time of hcg injection while patients without ovulatory dysfunction had lower saa levels as compared to the baseline level. in addition, patients undergoing oi who achieved pregnancy exhibited increased levels of saa at the time of the pregnancy test compared to baseline levels, whereas patients who did not become pregnant had lower post-cycle levels of saa. interestingly, saa levels did not change in art patients that became pregnant without undergoing oi (donor egg or frozen embryo transfer) conclusions: human ovarian cells express saa mrnas which can be altered in vitro. serum levels of saa may correlate with the responsiveness of the ovary to gonadotropins as evidenced by altered levels in women with ovulatory dysfunction, and by an increase in pregnant patients following ovarian stimulation. yeh, beom su kim, felicia hercules, jennifer peresie, armando arroyo. gynecology-obstetrics, university at buffalo, buffalo, ny, usa. objective: cisplatin is a common chemotherapeutic agent given to women for treatment for a wide variety of cancers. we hypothesize that one mechanism by which cisplatin may cause damage to ovarian structures is by decreasing the amount of anti-oxidant activity in the ovary. we examined super-oxide dismutase 2 (sod2), a critical anti-oxidant enzyme that has been shown to be affected by cisplatin in other tissues, in the ovaries of cisplatin treated animals. methods: adult female sprague dawley rats were injected with saline, cisplatin 4.5 mg/kg or cisplatin 6.0 mg/kg as 2 weekly doses. five days following the last injection, the rats were euthanized and both ovaries were excised. one ovary was processed for immunohistochemistry and the other was processed for protein analysis using western blot techniques for sod2. the anti-sod2 antibody was purchased from santa cruz. the immunohistochemical sections were scored using a semiquantitative h scoring method. results: immunohistochemistry analysis of the expression pattern of sod2 following cisplatin administration revealed that there was a significant linear decrease in a dose response pattern in the expression of sod2 in antral follicles and in corpora lutea (p<0.01 for both). no change was found in the h score of sod2 in other ovarian structures. western blot analysis of sod2 in the ovaries following increasing doses of cisplatin revealed no changes in the overall protein levels of sod2 in the ovary. conclusions: this is the first report that administration of cisplatin causes changes in the expression pattern of sod2 in antral follicles and in copora lutea. cisplatin decreases the amount of sod2 available in these structures, possibly leading to increased oxidative stress and free radical damage, thereby leading to ovarian damage found after cisplatin administration. is there evidence for aromatase activity in the stroma of postmenopausal ovaries? mf landay, rh fogle, rb allen, s patel, fz stanczyk, rj paulson. ob/gyn, usc, keck school of medicine, los angeles, ca, usa. background: following menopause, the ovaries continue to secrete androgens and estrogens. we have recently confirmed the production of androstenedione, testosterone and estradiol (e 2 ) up to ten years after menopause by measuring gradients from ovarian venous effluent and peripheral blood. anti-müllerian hormone (amh) and inhibin b have been shown to be markers of follicular activity. peripheral levels of these hormones have previously been found to be undetectable in menopause, suggesting the absence of follicular activity in the postmenopausal ovary. objective: to investigate if the postmenopausal ovary continues to demonstrate evidence of follicular activity as the source of steroid production. design: observational study materials and methods: eight subjects aged 53 ± 2.7 yr (range 48-56) were enrolled. postmenopausal status was confirmed by preoperative fsh levels of more than 40 u/l and/or amenorrhea greater than 12 months. serum was collected from the ovarian veins during total abdominal hysterectomy and bilateral oophorectomy. peripheral blood was also collected pre-operatively, intraoperatively and postoperatively. all samples were analyzed for amh and inhibin b using elisas with sensitivities of 0.05 ng/ml and 7 pg/ml, respectively. androgen and estrogen levels in these samples have previously been reported, and documented a gradient between ovarian venous effluent and peripheral serum in all cases. results: 1) six patients had no detectable follicular activity by amh and inhibin b levels. 2) one patient demonstrated detectable inhibin b levels with an 7-fold gradient between ovarian venous effluent (226 pg/ml) and peripheral blood (31 pg/ml), however no amh was detected. 3) in one patient, aged 52 and 120 months postmenopause, both amh and inhibin b were detected. peripheral inhibin b levels were high at 912 pg/ml. amh was detectable at levels of 0.95 ng/ml. conclusions: 1) in the majority of patients, continued e 2 and androgen production in the ovary occurs in the absence of follicular activity as detected by amh and inhibin b production. 2) some patients have evidence of follicular function up to ten years after menopause. 3) since e 2 is produced in post-menopausal ovaries in the absence of follicular activity, these data provide evidence for aromatase activity in the stroma of post-menopausal ovaries. to elucidate the process by which prostaglandin f2 (pgf2 ) mediates luteal regression, this study examined the role that the transcription factor yin yang 1 (yy1) and histone deacetylase 1 (hdac1) play in altering luteal cholesterol uptake by the scavenger receptor class b type i (sr-bi), intracellular cholesterol transport by steroidogenic acute regulatory protein (star), and cholesterol processing by p450 side chain cleavage enzyme (p450scc) expression. rat luteal cells (10 days post-ovulation) were treated with pgf2 (24 hr) and trichostatin a (tsa; 24 hr), a potent hdac inhibitor. protein expression was measured post-treatment by western blot and cholesterol was localized via filipin staining. star and sr-bi promoter activation was also assessed in hek 293t cells treated with yy1, myy1, a deletion mutant lacking an essential region required for transcriptional repression, and tsa. pgf2 caused a significant 2-fold decline in star (p<0.005), and smaller declines in sr-bi and p450scc which occurred concomitantly with an increase in yy1 (4-fold, p<0.001) and intracellular lipid staining (20% increase). in contrast, 100 nm tsa treatment caused a dose dependent increase in sr-bi, star, and p450scc protein levels, 3.5-fold (p<0.005), 1.4-fold (p<0.05), 4.1-fold (p<0.005), respectively, and a 2.5-fold decline (p<0.05) in intracellular lipid levels. tsa prevented the pgf2 -induced decline in sr-bi, star, and p450scc expression. promoter analysis demonstrated that wildtype yy1, but not myy1, repressed sr-bi and star activation while the addition of tsa overcame the repressive effects. this study shows the critical role that yy1 plays in pgf2 induced luteal regression by recruiting a histone deacetylase to block three essential processes in steroid production. in luteal cells yy1-mediated global repressive action prevents cholesterol metabolism by inhibiting cholesterol uptake, intracellular transport, and processing thus leading to functional and structural luteal demise. supported by nih hd35163 and nih hl78817. regulation of intermedin expression in cycling rat ovary. madhu chauhan, rebekah elkins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd) is a ct/cgrp family peptide involved in a variety of physiological functions, including vasodilatation and fetoplacental growth. this peptide is expressed in a variety of tissues such as stomach, placenta, uterus, pituitary and ovary suggesting its different functions including in reproduction. imd gene has an estrogen response element and we have shown that the plasma concentration of immuno-reactive imd is elevated in rats with pregnancy. however, expression of imd in the ovary and its regulation during estrous cycle is not known. we hypothesize that imd is expressed in the ovary and its expression is hormonally regulated throughout the estrous cycle in rat. objective: 1) to assess expression of imd mrna and its receptors components calcitonin receptor like receptor (crlr), and receptor activity modifying proteins, ramp1 and ramp3 mrna in rat ovary; on diestrus, proestrus and estrus stages of rat estrus cycle and ; 2) to demonstrate immunohistolocalization of imd, crlr, ramp1, ramp2 and ramp3 in rat ovary. methods: groups of 4 sprague-dawley non-pregnant and pregnant rats on day 18 of gestation were used in this study. non-pregnant rats were sacrificed on diestrus, proestrus and estrus stage. ovaries were collected and total rna was extracted using trizol method. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). immunohistolocalization of imd, ramp1, ramp2 and ramp3 proteins were assessed in tissue sections of ovaries from pregnant rats sacrificed on day 18. results: 1) imd mrna is regulated in rat estrus cycle and its expression is significantly downregulated in estrus stage compared to the diestrus and proestrus; 2)expression of imd receptor crlr is highest in the diestrus stage, followed by a decline in proestrus which further declined during estrus; 3) expression ramp1 mrna is higher in proestrus compared to diestrus and estrus (p<0.05) but there is no significant change in the ramp 3 expression during the estrus cycle and ; 4) imd, ramp1, ramp2 and ramp3 are immunolocalized in rat ovary in granulose cells of all follicles and granulosa cells of the corpus luteum. conclusion: imd mrna and protein are expressed in rat ovary suggesting a possible role for imd in ovarian function. defining and defying deterioration in oocyte quality with advancing chronological age: role of nitric oxide. pt goud, 1 ap goud, 1 mp diamond, 1 b gonik, 2 hm abu-soud. 1 1 div reprod endocrinology, dept ob/gyn, wayne state university, detroit, mi, usa; 2 div maternal and fetal medicine, sinai grace hospital, wayne state university, detroit, mi, usa. nitric oxide (no) is a ubiquitous free radical essential for oocyte maturation, function and sustenance of oocyte quality post-ovulation. the current study investigates the role of no insufficiency in the causation of oocyte quality deterioration with advancing chronological age. methods: in set 1, oocytes were retrieved from the following superovulated b6d2f1 mice: (a) young breeders (yb, n=109); (b) retired breeders (rb, n=40), and © old animals (oa, n=16), aged 6-12, 45-52, and 70-75 weeks respectively; and studied for zona pellucida dissolution time (zpdt), spindle ( -tubulin fluorescence immunocytochemistry, sigma) and chromosome morphology (dapi, vector), ooplasmic microtubule (mt) dynamics (omd) in response to taxol [1], and cortical granule (cg) status (rhodamine conjugated lectin, vector). in set 2 (n=56), oocytes from retired breeders were studied after exposure in vitro to an no-donor, s-nitroso acetyl penicillamine (snap in m-16, 0.23 m no/min , n=23, 3 h, 37°c, 5% co 2 ), while their sibling control oocytes were cultured for corresponding period under identical conditions without snap. [1]. zpdt, spindle and chromosome morphology, omd and cg status were assessed with a confocal microscope and compared between the subgroups using anova, chi square and/or fisher's exacts test and appropriate post-hoc tests. results: in set 1, a significantly fewer oocytes were retrieved per animal (mean) in rb (10) and oa (4) compared to yb (27.3, p<0.05). advancing age was also associated with a significant increase in zpdt, omd and cg loss in rb compared to yb (p<0.01). furthermore, significantly fewer oocytes from rb than yb had normal spindle and chromosome morphology (p<0.05). oocytes from oa had significant spindle and chromosome disarray, a near total cg loss and significantly harder zp (p<0.01). these oocytes also exhibited diminished omd in response to taxol although, metaphases were exquisitely sensitive to disruption with taxol. in set 2, exposure to snap in oocytes from rb resulted in a significantly lowerzpdt, omd and cg loss, and significantly higher incidence of normal spindles ( the gamma-aminobutyric acid (gaba), its biosnthetic enzyme gad and gaba-a receptors have been found in the oviduct and ovary. objective: this study examined the expression of gaba-a receptor subunit genes and gad and whether the gaba-a receptor could alter cytosolic ca2+ in gc. methods: for qrt-pcr, both human cumulus and mural gc were obtained at the time of oocyte retrieval for ivf and cultured separately. total rna was isolated separately from each gc type and from human brain ( positive control). the total rna from 16 patients was pooled for each gc type and all rna was treated with dnase i. two-step qrt -pcr was performed using gene-specific lux™ primers for all 18 gaba-a receptor subunits, gad65 and gad67 plus gapdh. single and specific qrt-pcr products were verified by melting curve analysis, gel electrophoresis, and dna sequencing. for ca2+ study, gc were cultured on coverslips. gc were loaded with fura-2-am and changes in ca2+ concentration of gc were studied using a dynamic digital ca2+ imaging system. gaba-a agonist, muscimol, was used to study any dose-dependent effects on gc. gaba-a antagonist, 10 m bicuculline, were perfused 1 min. prior to and during application of muscimol. the responses were represented as changes in the 340/380 nm fluorescence ratio over time. 50 m atp was used as positive control. results: the qrt-pcr results indicated that all gaba-a receptor subunits were expressed to various degrees in both types of gc, with the 5 expressed highest in both cell types. gaba-a receptor subunits showing the next highest expression in both cell types were 3, 3 and 2. gad67 isoenzyme was more abundantly expressed in cumulus and mural gc than gad65. ca2+ imaging showed that muscimol, had the ability to increase ca2++ in gc, about 19% gc (n=6) cells responded to muscimol. muscimol increased intracellular ca2+ in a dose-dependent manner. the muscimol responsive cells was reduced by bicuculline, from 19% to 1 % (n=6, p< 0.05). conclusion: qrt-pcr indicates that gaba-a receptor subunit gene and gad67 expression occurs in both cumulus and mural gc. the ability of bicuculline to inhibit the ca2+ response to muscimol suggests the activation of gaba-a receptor. the current study confirms the presence of functional gaba-a in gc for the first time, and suggests that gaba may exert trophic effects in the ovary via gaba-a receptor. the present study test the hypothesis that administration of l-nitro-l-arginine methyl ester (l-name), a nos inhibitor, prior to hypoxia prevents the hypoxiainduced activation of p38 mapk, erk and jnk in the cerebral cortex of the guinea pig fetus during gestation. to test this hypothesis 34 guinea pig fetuses at 35 and 60 days gestation were assigned to normoxic (nx, n=6), hypoxic (hx, n=6) and hypoxic pretreated with nos inhibitor (hx+l-name, 30mg/kg i.p., n=5) groups. hypoxia in the fetuses was induced by exposing the pregnant guinea pigs at both gestational ages to an fio 2 of 0.07 for 60 min. cerebral tissue hypoxia was documented biochemically by determining the tissue levels of atp and phosphocreatine (pcr). neuronal nuclei were isolated, purified and proteins separated by sds-page, and then probed with specific phosporylated erk, jnk and p38 antibodies. in the 35days gestation group: expression of p-p38 was 39.5±3.4 (nx), 79.1±2.8 (hx) 47.1±4.1 (hx+l-name). p-erk expression was 59.7±3.2 (nx), 109.6±6.3 (hx), 72.4±2.7 (hx+l-name). p-jnk expression was 68.3±7.2 (nx), 94.6±3.8 (hx), 55.8±9.0 (hx+l-name). in the 60 days gestation group: expression of p-p38 was 108.7±3.6 (nx), 231.1±8.3 (hx), 116.5±9.8 (hx+l-name). p-erk expression was 94.1±12.9 (nx), 226.5±9.1 (hx), 139.1±12.6 (hx+l-name). p-jnk expression was 83.2±9.5(nx), 218.2±6.9 (hx) 105.3±10.8 (hx+l-name). the data show that administration of l-name prior to hypoxia decreased the expression of phosporylated p38, erk and jnk at both gestation ages however expression of phosporylation was higher at term as compared to preterm. since a nos inhibitor prevented the hypoxia-induced phosphorylation of p38, erk and jnk in both gestational ages, we conclude that the hypoxia-induced activation of p38, erk and jnk in the cerebral cortical nuclei of preterm and term guinea pig fetus is no-mediated. we speculate that no-mediated modification of cysteine residue leading to inhibition of map kinase phosphatases results in increased activation of p38, erk and jnk in the guinea pig fetus. (nih-hd 20337, nih-hd 38079 and st. christophers foundation for children). the the endocannabinoid, anandamide (aea), is involved in the hormone-cytokine network that regulates implantation and early pregnancy maintenance with levels at the endometrial level considered a major checkpoint 1 . high levels (28nm) in culture are associated with embryo death 2 while plasma levels (>4nm) at 6 weeks in women undergoing ivf-et are associated with failed pregnancy 1 . what is uncertain is whether systemic aea levels after spontaneous conception in women presenting with threatened miscarriage are predictive of pregnancy outcome. our aim was therefore to measure plasma aea levels in women presenting with threatened miscarriage and to relate these to outcomes. methods: plasma aea levels were measured using a sensitive and previously validated hplc-ms method 3 at 6-12 weeks gestation in 45 women (nonsmokers, bmi <30kg/m 2 ) presenting with threatened miscarriage and in whom a viable pregnancy was confirmed by ultrasound scan. results: nine of the 45 women subsequently had a miscarriage. the plasma aea levels in those women who had live births was 2.85-fold lower than that in those who subsequently miscarried (1.21 ± 0.12nm versus 3.45 ± 0.31nm, mean ± sem; respectively; p<0.0001 unpaired student's t-test). the roc analysis revealed an area under the curve of 0.972 ± 0.025 with a sensitivity of 100% (95%ci of 66.3% to 100.0%) and a specificity of 94.4% (95% ci of 81.34% to 99.3%). using an aea level of 2.0nm as the optimal cut-off point, a single plasma aea measurement provided a sensitivity of 100% and a specificity of 94% with a negative predictive value of 100% and a positive predictive value of 78% for subsequent miscarriage. conclusion: these findings suggest a possible predictive role for plasma aea in women presenting with threatened miscarriage. the data also indicate that systemic aea levels may reflect local endometrial levels and therefore the local hormonal milieu in the early stages of normal pregnancies and in those complicated by threatened miscarriage. introduction: follicle stimulating hormone (fsh) mediates cyclic follicle growth and development and is widely used for controlled ovarian stimulation. the ovarian response of different women to fsh is variable, ranging from poor response to ovarian hyperstimulation and has been partly attributed to two common variants of the fsh receptor (fshr). we have previously identified four abnormal fshr splicing products (3 exon deletions and 1 intron insertion) in women with low and high response to fsh. two of the splice variants, deletion of exon 2 and deletion of exon 6, showed a correlation with low and high response to fsh, respectively. in the current study, we evaluated the functional competence of the mutant fshrs in vitro. methods: we established stable hek293 cell lines expressing wild-type (wt) and splice-variant (del) fshr under the control of the inducible tet on/off system. the cells were transfected with a camp-responsive luciferase reporter plasmid and stimulated with fsh. results: the subcellular distribution of all splicing variants was the same as the controls and the protein localized mainly on the cell surface. all four splicing variants showed markedly decreased camp activation compared to controls when stimulated with fsh. however, all variants were able to form functional heterodimers with the wt receptor when co-expressed. interestingly, the heterodimer containing the form of fshr lacking exon 2, found in patients with decreased response to fsh, resulted in lower intracellular camp compared with the wild-type homodimer. conclusion: our findings suggest that fshr variants can contribute to abnormal response to stimulation in certain women undergoing ivf treatment. the variants require the presence of wild-type receptor in order to initiate signaling in response to fsh. further analysis of this signaling cascade in granulosa cells is underway to estimate the final production of estrogen from these heterodimeric receptors. objective: to compare the proteome of human endometrium in the prereceptive versus the receptive phases of the menstrual cycle. m m: endometrial biopsies were collected 2 and 7 days after urinary lh surge in the same menstrual cycle from three fertile women (n=3). proteins were extracted using sample grinding kit (ge healthcare) and interfering substances removed using 2-d clean-up kit (ge healthcare). labelling was performed with cydye dige fluors (ge healthcare) and proteins were separated using difference gel electrophoresis (dige). for the isoelectric focusing, 24 cm ipg-strips in the nonlinear range of ph 3-11 were used. the second dimension was carried out using sds-page. differentially expressed proteins were detected by image analysis using decyder v6.5 and the statistical module eda (ge healthcare). the spots of interest were subject to protein identification based on in-gel digestion, maldi-tof/tof mass spectrometry and database searching. western blot analysis were performed in the same biopsies in order to validate some candidate proteins. results: table 1 displays the differentially protein abundance between days lh+2 and lh+7 (> 2-fold change). of these proteins, 10 were increased at lh+7 in comparison with lh+2, whereas only 2 proteins were decreased. stathmin was found more than 2-fold decrease in lh+7 compared to lh+2 in the three patients studied in the validation studies performed by western blot. conclusions: this study shows that the human endometrium has a differential protein repertoire during the window of implantation compared to the prereceptive phase. the role of these proteins in the molecular events directed to embryo implantation is under research. differential protein abundance in human endometrium ( the extracellular signal-regulated kinases 1 and 2 (erk1/2) are a mitogen-activated protein kinase (mapk) subfamily that act as key links in eukaryotic intracellular signaling transduction. activated by phosphorylation in response to specific stimuli, erk1/2 is known to play a role in the regulation of cellular proliferation and survival. the human myometrium is a tissue known to undergo cycle-dependent proliferative and apoptotic changes in response to sex steroids. we hypothesized that erk1/2 activity is involved in mediating menstrual cycle-dependent changes in the myometrium. materials and methods: immunostaining for phospho-erk and total-erk was performed on myometrial tissues obtained from normal women (n=20) at different phases of the menstrual cycle. staining intensities were evaluated by hscore. myometrial smooth muscle cells were isolated and cultured from normal women and treated with vehicle, estrogen (10-8m), and progesterone (10-8m) for 5 and 15 minutes, and then subjected to western blot analysis for p-and t-erk. statistical analysis was performed using one-way anova. results: tissue staining revealed that p-erk was mostly nuclear in all tissues, while t-erk was cytoplasmic and nuclear. p-erk staining was significantly stronger in the secretory phase and strongest at the early secretory phase, compared to other phases (p<0.05). t-erk staining intensity was moderatehigh without variation across the menstrual cycle. in cultured myometrial cells, progesterone significantly increased p-erk levels within 5 and 15 minutes (p<0.05) when compared to control. our results suggest that erk activity in the human myometrium is regulated in a menstrual cycle-dependent manner. the increased phosphorylation in the secretory phase suggests the involvement of progesterone in erk activation, as supported by our in vitro results. this increased erk activity may play a role in regulating myometrial proliferation. measures of insulin resistance (ir) including: fasting serum insulin, gir and homa. measures of plasma levels of endothelin-1 (et-1), soluble intercellular adhesion molecule-1 (sicam-1), soluble vascular cell adhesion molecule-1 (svcam-1) and high sensitivity c-reactive proteins (hscrp). results: women with pcos exhibited significantly higher levels of et-1 (p < 0.05), sicamp-1 (p < 0.05), svcam-1 (p < 0.001) and hscrp (p < 0.001) as compared with age-matched controls, respectively. positive correlations were evident between et-1 and fai (r = 0.41; p < 0.01) but et-1 negatively correlated with shbg (r = -0.36; p < 0.05). svcam-1 positively correlated with total t (r = 0.62; 0.001), hscrp correlated with: bmi (r = 0.73; p < 0.001), and homa (r = -0.39; p < 0.05), respectively. conclusions: women with pcos exhibited abnormal levels of endothelial and inflammatory markers, which appear to be inter-related to hyperandrogenaemia. (1), and a higher expression of fatty acid amide hydrolase (faah) in peripheral lymphocytes post-ovulation (2), suggest an involvement of the endocannabinoid system in menstrual cycle control. our aims were to investigate changes in plasma aea levels and in endometrial faah expression throughout the menstrual cycle. methods: plasma aea levels were measured using a hplc ms/ms method from 47 women, median age 34yrs (range 20-45) and bmi 22kg/m 2 (range 19-27), with regular menstrual cycles, with no medical problem and not on any medication for the preceding 6 months. the menstrual cycle was divided to early follicular d2-7(n=11), late follicular d8-11(n=7), ovulatory d12-15(n=10), early luteal d16-22(n-10) and late luteal phases d23-28(n=9). uterine biopsies were taken from hysterectomy specimens taken for benign conditions and subjected to immunohistochemistry for faah with polyclonal antibodies. results: aea levels were significantly higher around ovulation in comparison to the pre-ovulatory or post-ovulatory phases as shown in the figure. faah expression in the endometrial stroma was unchanged throughout the follicular phase but increased during the mid to late luteal phase reaching a maximum in the late luteal phase. a high aea level in the early follicular phase and during ovulation suggests a role for aea in ovulation. the lower levels of aea in the luteal phase, essential for successful implantation, may be regulated by increased faah expression at the uterine level. the modulation of plasma aea levels during the menstrual cycle strongly suggests that it is regulated by gonadal steroid hormones. (1 , 5, 10, 15, 20, 25, 50 and 120, as well as at pregnancy. binding activities of igfbp and their protein levels (igfbp1,2,3,4,5,6) were assessed by western ligand blot (wlb) and western blot (wb), respectively. the glyscosylation and phosphorylation status of igfbps were examined by deglycosylation treatment. our results showed that both glycosylated and unglycosylated igfbp2 elevate in fetal and newborn rat and graduately decline to a lower level at day 15 and kept lower constant level since then. interestingly, biding activity of glycosylated igfbp2 was not detected by wlb assay. the igfbp-2 cleaved products were observed after rat day age day 15, which associated with a decrease in full length of igfbp-2, suggesting endoproteolytic processing may involved in decreasing igfbp content. igfbp-3, with heterogeneous glycosylation, were appeared after age day 25 and disappeared during pregnancy, recurrence again postpartum. glycosylation of igfbp3 has no effect on its binding activity. unglycosylated and glycosylated igfbp were constant in life time. physiologically constant igfbp5 were detected by wb in protein, but not by wlb in binging activity. conclusion: highly elevated circulation igfbp2 suggest physiological role in new born and early postnatal development. its binding activity are well regulated by its posttranslation modification, such as glycosylation of igfbp2 in inactivating binding activity and possible involvement of active endoproteolytic processing in maintaining binding active igfbp-2 at low background: cigarette smoking affects hormone biosynthesis, storage, release, binding, transport and clearance, resulting in changes in circulating hormone levels. we used metabolomics to analyze the effects of cigarette smoking and second hand smoke (shs) on changes in hormone levels in women of childbearing age. methods: this is a three arm study; women aged 18-44years who are active smokers, exposed to shs (passive smokers), or non-exposed were recruited from the washington d.c. area. all women completed a detailed staffadministered questionnaire probing their medical history, occupational, lifestyle factors and diet. blood and urine samples were collected at the follicular phase. hormone profiles were determined using metabolomics for serum thyroxine and triiodothyronine and 17 steroid hormones, as well as for cotinine using isotopedilution tandem mass spectrometry (lc/ms/ms-api 5000). in addition, all samples were analyzed for serum lh, fsh, tsh and creatinine. results: the relationships of cigarette smoking, age, relative weight, and dietary intake to serum thyroxine, triiodothyronine, estradiol (e2), estrone (e1), progesterone (p), 17-hydroxyprogesterone (17ohp),dehydroepiandrosterone (dhea), dehydroepiandrosterone sulfate (dheas), androstenedione, cortisol, 11-deoxycortisol, corticosterone, testosterone, aldosterone and vitamin d3 were analyzed using lc/ms/ms. mean tsh in non-exposed, shs-exposed and smokers: 1.32, 0.86, and 1.02miu/ml respectively. similarly, mean t4 9.8, 12.23 (23% increase) (p=0.05), 9.41μg/dl (-7%) in active smokers; (active vs. exposed p=0.03). shs increased dhea levels (33% higher, p = 0.02), dheas (23% higher, p = 0.07), cortisol (21% lower, p = 0.01), aldosterone (63% lower, p = 0.02) and androstenedione (20% higher, p = 0.01). these data suggest that active smoking and shs can have a profound effect on serum t4, adrenal steroid and sex hormone concentrations in women of childbearing age. objective: to determine the prevalence and predictors of the metabolic syndrome (mbs) among in saudi women with polycystic ovary syndrome (pcos) in comparison to women without pcos and to assess the role of androgens and insulin resistance (ir) in mbs development. design: a prospective case control study. setting: tertiary referral university hospital. subjects: six hundreds saudi women living in the jeddah area were classified as follows: 300 with pcos and 300 age-matched women without pcos. interventions: blood samples were collected from all women with or without pcos between 8:00-11:00, after an overnight fast. main outcome measures: measures of abdominal obesity, blood pressure and that of serum levels of lh, fsh, tsh, ft 4 , 17-ohp, 4-a, dheas, total t, free t, shbg, insulin, hdl-c, triglycerides and plasma levels of glucose. measures ir including: fasting serum insulin, gir and homa. results: age-adjusted prevalence of mbs was higher in women with pcos (53.5%, 95% ci: 37.6-61.2%) as compared with women without pcos (14.7%, 95% ci: 10.2-18.6%) (p<0.000). in the same age group, the risk of mbs in women with pcos was greater than that for women without pcos (p<0.001). markers of ir were significantly abnormal in women with both pcos and mbs in comparison to those without mbs (p<0.001). the most common abnormal components of mbs in women with both pcos and mbs (after adjustment for age) were: decreased hdl-c (83.1 ± 10.5%); increased triglycerides (53.4 ± 7.7%); and increased bmi (38.2 ± 4.6%), respectively. the prevalence of mbs from lowest to highest tertile of free t level was 20.1, 33.7 and 54.2%, respectively; in women with both pcos and mbs. in women with pcos, 9% exhibited all 5 components of mbs; 14.1% had 4 components, and 40.5% had 3 components. conclusions: women with pcos exhibited significantly higher prevalence of mbs (3.6-fold) as compared with age-matched control without pcos. ir is a possible common pathogenetic factor for both mbs and the pcos. it is suggested that more intensive screening and/or therapy monitoring of mbs among women with pcos should be part of the therapeutic modalities of the condition. case of sisters with complete androgen insensitivity syndrome and discordant mullerian remnants. jennifer l nichols, jennifer j gell, eric j bieber. reproductive endocrinology and infertility, geisinger medical center, danville, pa, usa. complete androgen insensitivity is an x-linked recessive disorder resulting in the abnormal expression of the androgen receptor. affected individuals are most commonly phenotypically female but genotypically male. the prevalence of this disorder is 1 in 20,000 live male births. we present a case of complete androgen insensitivity in 2 members of the same family with differing residual mullerian tissue. sister a presented at age 17 for evaluation of primary amenorrhea. a karyotype revealed 46,xy. an mri of the pelvis showed a hypoplastic uterus but no ovaries. this patient underwent laparoscopic bilateral gonadectomy and hemihysterectomy. on examination under anesthesia, she was noted to have a normal vagina with no cervix noted. at laparoscopic evaluation, she was noted to have bilateral elongated gonads and what appeared to be a remnant of uterine tissue. pathology revealed portions of immature testicles and fragments of smooth muscle in what grossly appeared to be the uterine remnant. the patient's total testosterone following surgery was noted to be elevated at 2.91 ng/ml. other laboratory evaluation showed fsh 4.5 miu/ml, lh 7.04 miu/ml, free testosterone 3.9 pg/ml, and estradiol 24.6 pg/ml. approximately two years later sister b presented at age 17 for evaluation of primary amenorrhea. no uterus or ovaries were visualized on pelvic ultrasound. again a karyotype revealed 46,xy. laboratory evaluation demonstrated fsh 1.0 miu/ml, lh 13.28 miu/ml, estradiol 37.3 pg/ml, total testosterone 9.25 ng/ml, and free testosterone 11.1 pg/ml. she underwent a laparoscopic bilateral gonadectomy. no uterus, cervix or pelvic masses were identified on exam under anesthesia. at laparoscopy, both gonads were visualized and removed without difficulty but no uterus was visualized. pathology reported two testicular and epididymal-like structures. this case demonstrates the presentation and laparoscopic results of complete androgen insensitivity syndrome discovered in two siblings. although both girls are genotypically male, they differ in the presence of vestigial mullerian tissue. the case shows with complete androgen insensitivity, as an x-linked defect, one should consider apparent sisters of affected individuals, as well as offspring of unaffected individuals with a family member diagnosed. background: anandamide (n-arachidonoylethanolamine, aea) is an endocannabinoid that binds to and activates the cannabinoid receptors, cb1 and cb2 and may have important roles in the regulation of human reproduction. the traditional lipid extraction methods used for aea 1 are cumbersome, slow and of low efficiency. the aim of this study was to determine whether the use of a solid phase (spe) method of aea extraction from human plasma would offer any advantages over the traditional liquid phase (lpe) method. methods: pooled human plasma was obtained from the local blood transfusion unit and aliquots stored at -20°c prior to extraction. an internal standard of 2.5pmol of deuterated aea (aea-d 8 ) was added to each plasma sample and aea extracted from 5 aliquots on each occasion over three days using the lpe 1 and spe methods. spe was performed with waters oasis hlb 1cc/30mg cartridges on a waters vacuum manifold. cartridges were activated with methanol and water, the samples applied and washed with 40% methanol at 1ml/min. aea was eluted with 1ml acetonitrile and the eluents dried under a stream of nitrogen before reconstitution in 80 ml acetonitrile. aea levels were measured using uplc-ms/ms against authentic standards. results: these are shown in the table. conclusion: the spe method was 3-fold more efficient at extracting aea compared to the traditional lpe method. the intra-day and inter-day assay variability were similar for both techniques, although the spe method was quicker, cheaper and required less plasma to generate data similar to that from the traditional lpe method, suggesting that the spe method may be more efficient than the lpe method, and thus making it more suitable for routine analysis of multiple plasma aea samples. background: the precise role of the endocannabinoid, anandamide (narachidonoylethanolamine, aea) in reproduction has been hampered by difficulties in its accurate measurement. aea levels have previously been measured by tlc, gc-ms and hplc-ms but these are laborious. our aim was to improve the analysis of aea using uplc and tandem ms/ms with a standard isotope-dilution method 1,2 . methods: authentic non-labelled and deuterium-labelled aea (aea-d8) diluted in acetonitrile were maintained at 4°c before analysis and separation by uplc on a c 18 (2.1 x 50mm) column maintained at 40°c using a gradient of 80% a, 0.5min: 80% a, 1.5min: 0% a, 2.5min: 80% a, 3.5min: 80% a with a flow rate of 0.7ml/min. the mobile phases were a (2mm ammonium acetate, 0.1% formic acid) and b (acetonitrile, 0.1% formic acid). the analytes were quantified using multiple-reaction monitoring in positive ion mode with a quattro premier mass spectrometer. entry, collision and exit energies were -2, 17 and -17ev, respectively. calibration curves were performed in triplicate with 2.5pmol aea-d8 as the internal standard. transitions employed were 348.3>62.3 and 356.3>63.3 for aea and aea-d8, respectively. results: calibration curves (1.66 to 133fmol aea on column; n=15) were linear, producing a mean (±sd) gradient of y = 2.48±0.14x, crossing the y-axis very close to the origin (0.004±0.04 units). variability was limited, with an r 2 =0.999. measurements were precise, 133fmol aea produced a cv of only 3.7%, and the retention time was consistent at 1.67±0.0009min (n=20). the limit of quantification (signal/noise>10) was 0.22fmol on column and the limit of detection (lod) was 0.055fmol on column (signal/noise=3). accuracy for 3.33fmol, 6.65fmol and 133fmol aea was 97.5±9.5%, 98.5±6.1% and 104.5±3.2%, respectively. conclusions: the method described is an improvement over other lc-ms/ms methods 1,2 with a lower lod [0.055fmol v. 25fmol 1 or 43fmol 2 ] and shorter run time [4min v. 15min 1 or 5.4min 2 ]. thus, an improvement in terms of speed, increased sensitivity and better reproducibility will allow for a more accurate assessment of aea concentrations in a number of biological samples. objectives: the gata family of transcription factors consists of six zinc-finger proteins with a critical role in tissue-specific and developmentally-regulated gene expression. gata factors exert their effects alone and through interactions with cofactors, such as friend of gata (fog), as well as with nuclear hormone receptors, including steroidogenic factor-1 (sf-1), a well-described activator of gonadotropin gene expression. the objective of these studies was to define the role of gata family members in the gonadotrope. methods: 1) total rna was extracted from the gonadotrope cell line, l t2, and analyzed by standard rt-pcr analysis. 2) the cv-1 fibroblast cell line was transiently transfected by the calcium phosphate precipitation method with a rat -207/+5 lh promoterreporter vector as well as cmv-driven expression vectors for gata-2, gata-3, dngata-3, fog-2 and/or sf-1. results: gonadotrope l t2 cells were found to express transcripts encoding gata-2, gata-3, and gata-4 as well as fog-1 and fog-2. gata-2 and gata-3 stimulated lh gene promoter activity by approximately 5-fold (p<0.05) and synergistically increased the sf-1 response (40-fold versus 10-fold for sf-1 alone; p<0.05). the gata-mediated increase in lh gene expression was nearly eliminated with co-transfection of fog-2. similarly, co-transfection with a gata-3 dominant negative construct blunted wild-type gata-3 effects in a dose-dependent fashion. conclusions: these data demonstrate expression of both gata and the functionally related fog proteins in a well-characterized gonadotrope cell line. furthermore, they demonstrate a functional role for these factors in regulation of gonadotrope function, specifically expression of the lh gene. low-dose dexamethasone (dex) therapy early in pregnancy is used in fetuses with suspected risk of congenital adrenal hyperplasia. several adverse neurological events in prenatally treated children have been reported and the fetal hypothalamic-pituitary-adrenal (hpa) axis may be involved. aim: to investigate the immediate and long-term effects of early maternal dex administration on fetal growth and pituitary-adrenal activity in sheep. method: pregnant ewes carrying singleton fetuses (total n=119) were randomized to control (2ml saline/ewe) or dex treatment (0.14mg/kg ewe weight) consisting of four intramuscular injections at 12-hourly intervals over 48 hours on 40-41 days of gestation (dg). at 50, 100, 125, and 140dg fetal weights were recorded. i 125 -ria, qrt-pcr and in-situ hybridisation were used to measure fetal plasma cortisol and acth levels and to analyse adrenal and pituitary mrna expression. results: dex-exposed fetuses were lighter than control animals at 100dg*, but not at other times; in general fetal organ weights were similar between treatment groups. fetal plasma acth was unaffected by dex and did not differ between genders. similarly, pomc and pc-1 mrna in pars distalis were unaltered after dex. however, fetal plasma cortisol was reduced after dex in both male and females at 50dg*, was similar at 100 and 125dg, then elevated at 140dg*. plasma cortisol in female fetuses in control and after dex was significantly higher than in males. the increases in cortisol after dex at 140dg* were associated with increased fetal adrenal expression of p450c17 and 3bhsd mrna in females, reduced expression of mc2r in males, but no difference in star mrna. conclusion: we conclude that in sheep, early dex programs the developmental trajectory of the fetal hpa with increased activation directly of the adrenal, but not pars distalis function. in females this effect may be attributed to increased fetal adrenal steroidogenic activity. the effect of dex in increasing cortisol in males, albeit at a significantly lower level than in females, appears to be independent of the enzymes that we have measured.*p<0.05. synaptophysin and gonadotropin-releasing hormone (gnrh) are colocalized in rat hypothalamus. armando arroyo, 1 beom su kim, 1 amanda biehl, 1 blenna cl bett, 1,2 john yeh. 1 1 gynecology-obstetrics, university of buffalo, buffalo, ny, usa; 2 physiology and biophysics, university at buffalo, buffalo, ny, usa. the cellular and molecular mechanisms that control gonadotropin-releasing hormone (gnrh) release are not completely understood. gnrh is stored in synaptic vesicles and released by exocytosis at gnrh nerve terminals. there are currently nine families of synaptic vesicle proteins that are involved in neurotransmitter release by exocytosis. synaptophysin is one of the most common synaptic vesicle proteins present in synaptic vesicles in neurons. the hypothesis of this study is that synaptophysin is expressed in gnrh neurons. we obtained sections from the hypothalamus of female sprague dawley rats. double-label fluorescence immunohistochemistry was performed on free-floating sections. sections were incubated with a mixture of mouse monoclonal antibody against gnrh (1:200-chemicon international) and with a rabbit polyclonal antibody against synaptophysin (1:100-santa cruz biotechnology) for 16 h. after incubation the sections were washed and incubated with a mixture of alexa 488 conjugated goat antimouse and alexa 594 conjugated goat antirabbit (1:1000; molecular probes) for 2 h. slices were visualized with confocal microscopy (zeiss lsm-510). fifteen out of a total of fifteen gnrh cell bodies in the medial preoptic area and most gnrh neuron terminals in the median eminence showed intense immunostaining for synaptophysin. this is the first study to demonstrate that synaptophysin is expressed in rat gnrh neuron terminals. this suggests that synaptophysin is present in gnrh neuron vesicles. thus, gnrh release by exocytosis may be mediated by synaptic vesicle proteins. objective: our aim was to identify the effects of early gestation gc exposure on fetal and postnatal hpa axis development and function in postnatal life. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dex groups (0.14 mg/kg), consisting of four at 12 h intervals on days 40-41 of pregnancy. at 7 months postnatal age, catheters were implanted; a bolus injection of crh (0.5 mg/body weight) and avp (0.1 mg/body weight) were administered and arterial blood samples were taken at -30, -15, 0, 5, 10, 20, 30 60, 90, 120 and 180 min. levels of hepatic cbg mrna were determined by qpcr, expressed relative to 18s rrna. plasma cortisol and cbg levels were measured by radioimmunoassay. results: both total and free cortisol levels in the dex females (n=5) were lower than in dex males (n=7) from 30 to 90 min (p 0.05) and lower than in control females (n=6) at 30 minutes (p 0.04), also in the dex-m group were higher than in control males (n=5) at 60 min (p 0.04). plasma cbg levels and cbg mrna expression were not altered by dexamethasone exposure or sex. conclusions: these findings suggest that prenatal glucocorticoid exposure alters the development of the hpa axis differentially according to the sex of the exposed fetus. to determine maternal injections of synthetic glucocorticoids in early gestation can alter expression of hippocampal corticosteroid receptors at 7 months postnatal age. method: pregnant ewes carrying singleton fetuses were randomized to control (2 ml saline/ewe) or dexamethasone treatment (0.14 mg/kg) consisting of four injections at 12 h intervals on days 40-41. hippocampal was collected from the offspring at 7 months postnatal age. levels of mrna of gr and mr were determined using qpcr and levels relatived to 18s rrna. results: dexamethasone-treated male animals (n=7) had significantly higher levels of mr gene expression than both control males (n=5; p=0.028) and females (n=6; p=0.037). gr gene expression levels were higher in treated vs. control males (p=0.043), but in females levels in treated and control animals were similar. total body, brain and hippocampus weights were similar. conclusions: maternal dexamethasone administration in early pregnancy resulted in gender-dependent changes in hippocampal gene expression when measured in the offspring seven months after birth. objective: diminished ovarian reserve (dor) affects many younger women. we analyzed superovulation with intrauterine insemination (so) cycles in vitro fertilization (ivf) cycles of women with dor to determine if women <35 with dor differ from their older counterparts. method: irb approved retrospective review of so ivf cycles from 1/2005-6/2007 with follicle stimulating hormone (fsh) levels 10 based on age <35 or 35. results: 49 so cycles were performed in 18 women. no differences were noted in clinical pregnancy. women <35 were more likely to have inseminates with a lower mean tms/ins, median tms/ins was 71.0 among pregnancy cycles compared to 28 for unsuccessful cycles. for ivf, mean total gonadotropin dosage was significantly lower in women <35, mean number of follicles 15 mm peak e2 were significantly higher in women <35. so ivf measures are in tables 1 2, respectively. conclusions: similar clinical pregnancy outcomes were seen despite age. in ivf, women <35 required less gonadotropins and generated more follicles but with no significant difference in number of mature oocytes or clinical pregnancies. of note, pregnancy rates for so in women <35 with dor are substantially lower than expected in our clinical practice. as ivf yielded a substantially higher chance of pregnancy, consideration should be made to expedite progression to ivf. to assess the effects of intraovarian injection of ad-fshr on the reproductive system of fshr (-/-) mice. methods: about 50 l containing 3x10 9 pfu of ad-hfshr were injected directly into each ovary of treated group and same amount of ad-lacz were injected into the ovaries of control animals. vaginal smears were collected and body weight was measured daily. four weeks after the injection animals were sacrificed and all organs were weighted and evaluated by h e. fsh, e2 and p4 measured before and after treatment. results: ad-hfshrtreated mice showed obvious estrogenic changes in vaginal smear while in control animals vaginal smear remained at diestrus stage. significant increase in total body weight and estrogen dependent organs weight (uterus, ovary, vagina) was observed in treated animals compare to control group (p<0.02). no significant weight changes were observed in other organs. h e evaluation of the ovaries showed significant increases in both the total number of follicles and the collective diameter of the follicles in treated animals compare to controls. on average 18 follicles/ovary were observed in ad-hfshr-treated group of which 4 follicles were at the antral stage while only 2 follicles observed in ad-lacz control group, with zero follicles at antral stage. a 2.5 to 3 folds increase in e2 and about 50% decrease of fsh observed in treated animals compared to control mice. there was no significant change in serum progesterone level between treated and control groups. conclusion: intraovarian injection of an adenovirus expressing human fshr gene is able to restore folliculogenesis and resume estrogen hormone production in female forko mice. objective: the sentinel issue surrounding multiple gestations following ivf is the inability to precisely estimate the reproductive potential of individual embryos with the currently used embryo grading systems based on embryo cleavage rate and morphology. recently, metabolomic profiling of spent culture media using raman and near-infrared spectroscopy have been reported to predict reproductive potential of embryos. in this study we applied proton nuclear magnetic resonance (¹h nmr) spectroscopy to analyze metabolomic profile of embryo culture media and to identify components of the media that correlate with reproductive potential. methods: eighteen spent media samples from embryos that failed to implant, and 16 samples from embryos that resulted in pregnancy and delivery were individually collected after embryo transfer on day 3, and evaluated using ¹h nmr. the spectra obtained were analyzed using a selective genetic algorithm (ga) to determine regions predictive of pregnancy outcome as determined by logistic regression. to avoid random correlations, a leave-one out crossvalidation was used. sensitivity and specificity of predicting pregnancy (described as implantation and delivery) were calculated. results: using the ga, two areas in the ¹h nmr spectral region were identified as most discriminatory between the two groups. viability indices calculated by ¹h nmr using these regions were significantly higher in culture media of embryos with proven reproductive potential (0.66±0.13) compared to those that failed to implant (0.33±0.16) (p<0.001). compiled outcomes from the leave-one-out cross-validation of the logistic regression using the ¹h nmr measurements resulted in a sensitivity of 96% and a specificity of 77.8%. quantification by integration showed significantly decreased glutamate levels (p=0.002) and a trend toward an increase in pyruvate levels (p=0.1) in culture media of embryos that did not cause pregnancy. conclusion: metabolomic profile of spent embryo culture media using ¹h nmr correlates with the reproductive potential of embryos. the lower glutamate levels detected in culture media of embryos that failed to implant could potentially be due to the toxicity associated with increased embryonic glutamate uptake. additional studies using complementary approaches are needed to further delineate molecular components associated with reproductive potential. objective: beta-carotene and other carotenoids are known antioxidants previously identified in human follicular fluid (ff). in addition to their inherent antioxidant properties, carotenoids have been identified as precursors of the antioxidant retinol in bovine ff. high retinol levels in bovine ff are associated with non-atretic follicles. this would suggest a possible role for retinol and its carotenoid precursors in follicular heath and the general oxidative state of the follicle. we sought to measure carotenoids and retinol in the ff of women undergoing ivf and correlate these levels with normal fertilization as a marker of follicular/oocyte health. design: prospective cohort study materials and methods: ff from a single 18-20 mm follicle was obtained from 24 women (age 29-43) undergoing ivf and intracytoplasmic sperm injection (icsi). serum was also obtained at the time of oocyte retrieval. retinol, vitamin e ( , , and tocopherol) and carotenoids ( -carotene,cryptoxanthin, lycopene and lutein/zeaxanthin) were measured using hplc. we correlated ff carotenoid and retinol levels with oocyte fertilization status following icsi. results: as previously reported, retinol, vitamin e and carotenoids were all identified in the ff. each fat-soluble vitamin level was significantly lower in ff compared to serum (p<0.001 for all analytes) and the levels were strongly correlated (r2>0.45, p<0.003 for all analytes). mean levels of ff -carotene were significantly higher in those follicles that resulted in a fertilized oocyte (0.08 +/-0.04 g/ml vs. 0.01 +/-0.01 g/ml, p=0.015). other carotenoids, retinol, and vitamin e levels did not correlate with fertilization outcomes. conclusions: our finding of a strong association between ff -carotene concentration and subsequent normal fertilization of the oocytes suggests an important antioxidant role for -carotene in the health of the human ovarian follicle/oocyte. the lack of correlation with other carotenoids, retinol and vitamin e suggests that the antioxidant properties of -carotene act by preventing singlet oxygen and scavenging the peroxyl radical and may directly influence oocyte competence. mature oocytes obtained during egg retrieval have been shown to undergo spindle depolymerization as a result of cooling. for this reason, ivf programs strive to maintain constant temperature during follicle aspiration. we sought to elucidate if there was a difference in temperature of fluid aspirated into a hand held (hh) or heating block (hb) encased collection tube. the experiment was performed in an ambient temperature of 23°c. thermistors, pinhead sized sensors with an accuracy of 1%, were used to monitor temperature differences between control fluid (cf) (sterile water 37ºc) and aspirated fluid. data was recorded using an 8-channel data acquisition system from dataq instruments. one thermistor was placed into the cf, the other was placed into an empty collection tube set in the heating block or held in a gloved hand. a 33cm, 17 gauge, single lumen needle connected to 70 cm of tubing was used to aspirate into the empty collection tube. temperature was recorded 2x/second, each experiment was repeated 3 times. baseline empty collection tube temperature was significantly cooler in the hh vs the hb group (32.99±1.39°c vs 36.61±.16°c, p=.016). two seconds after aspiration, the lowest aspirate temperature was observed, (hh 30.34±.14°c, hb 30.48±.23°c, p>.05) no difference between groups. in both groups, temperature quickly increased as aspiration progressed (hh 34.57±.38°c, hb 34.86±.33°c, p>.05). the hb group took an average of 5.28 min to return to baseline (37°c), the hh group never returned to baseline. substantial cooling of aspirated fluid occurs during oocyte retrieval, with a mean temperature decrease of 6.59±.19°c corresponding to 30.41°c. considering this dramatic decrease, the difference between temperatures in the hh vs. the hb group is negligible. current aspiration systems poorly regulate temperature, thus the choice of aspirating into a test tube warmed by hand or by heating block is inconsequential. clinical management of twin gestations with recurrent preterm labor symptoms. lesley de la torre, 1 luis roca, 1 niki b istwan, 2 debbie j rhea, 2 gary j stanziano, 2 victor hugo gonzalez-quintero. 1 1 obstetrics and gynecology, university of miami medical center, miami, fl, usa; 2 clinical research, matria healthcare, marietta, ga, usa. objective to examine pregnancy outcomes in women with twin pregnancies receiving nifedipine tocolysis (nt) who experienced recurrent preterm labor symptoms (rptlsx). study design twin pregnancies enrolled for outpatient preterm labor (ptl) surveillance services prescribed nt following an initial episode of ptl were identified from a database (n=1421). eligible for inclusion were patients later hospitalized with acute rptlsx (n=862). included were those <35 weeks' gestational age (ga), with intact membranes, remaining undelivered for >48 hours after rptlsx . pregnancy outcomes of women resuming nt (rnt group, n=418) following hospitalization were compared to those having an alteration in treatment (alttx group, n=238) to continuous subcutaneous terbutaline. per normality assumptions, either independent student´s t or mann-whitney u test statistics were used for continuous variables; pearson´s chi-square for categoric. all p-values presented as two-sided, significant at <0.05. results overall, 862 (60.7%) of twin pregnancies prescribed nt experienced rptlsx; 206 (23.9%) were not eligible for continued tocolysis. pregnancy outcomes are presented in table. conclusion rptlsx are common. in twin pregnancies receiving nt, alteration of tocolytic treatment following rptlsx had a positive impact on pregnancy prolongation and neonatal outcomes. routine cervical dilatation during elective cesarean section -is it really necessary? arie koifman, avi harlev, eyal sheiner, fernanda press, arnon wiznitzer. obstetrics and gynecology, soroka university medical center, ben-gurion university of the negev, beer-sheva, israel. objective: the purpose of this study was to examine the necessity of routine cervical dilatation during elective cesarean section. material and methods a retrospective cohort study was performed, including all cases of elective cesarean sections performed at a tertiary medical center during 2005. stratified analysis, using the mantel-haenszel technique, was done to control for confounders. results out of a total of 666 elective cesarean deliveries (cd), 348 underwent routine cervical dilatation. the overall rate of febrile morbidity was 4.2%. no significant differences in postpartum febrile morbidity were noted between the groups (5.1 and 3.1%; p=0.071). in addition, hospitalization duration did not differ between the groups (4.1±1.4 and 4.1±2.0 days p=0.95 ). about 31 % of all elective operations were repeated cd. there was no difference in febrile morbidity between the groups even in that subgroup of the elective cd. likewise, there was no difference in anemia rate between the two groups (hemoglobin 9.50 ±0.73 mg and 9.54±0.65mg p= 0.91 ). controlling for a previous vaginal delivery, using the mantel-haenszel technique, no significant association was noted between cervical dilatation and fever (weighted or=2.2; 95% ci 0.8-6.4; p=0.161). nevertheless, in a subgroup of patients following a previous vaginal delivery, cervical dilatation was significantly associated with post-operative fever (or=5. the majority of women with an unfavorable cervix undergoing iol at term deliver vaginally, even with a prolonged first stage of labor. this is important information to discuss with women prior to iol when establishing labor expectations. providers should consider the ongoing success of labor induction when contemplating a diagnosis of "failed induction". objective: this study was performed to investigate and compare changes of the lipid peroxide levels and the protein carbonyls formation in the maternal venous plasma of preterm premature rupture of membrane (pprom) during antibiotics administration. materials and methods: thirty-six pregnant women with pprom between 25 and 32 weeks of gestation were chosen for this study. eighteen patients (group 1) were treated with amoxicillin and erythromycin for 7 day period while the other 18 patients (group 2) were treated with 3 rd generation cephalosporin and erythromycin for the same period. samples of maternal blood were obtained from the two groups at before the antibiotics administration, day 3, and day 7 after the antibiotics administration. lipid peroxide levels were measured by thiobarbituric acid reaction and protein carbonyl contents were determined by the 2,4-dinitrophenylhydrazine method. results: 1. the lipid peroxide levels and protein carbonyls formation in the maternal venous plasma of pprom was significantly higher than that of normal pregnancy (4.77±0.36 vs 7.11±0.41 nmol/mg protein, p<0.01), (3.55±0.22 vs 5.69±0.30 nmol/mg protein, p<0.01). 2. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the maternal venous plasma with pprom mixed and incubated by amoxicillin, cefodizime, and erythromycin (in vitro). 3. there were no significant differences in the lipid peroxide levels and protein carbonyls formation of the venous plasma of group 1 among before the antibiotics administration, day 3, and day 7 after the antibiotics administration. 4. the protein carbonyls formation in the venous plasma of group 2 was significantly decreased at day 3 and day 7 after the antibiotics administration than that of before the antibiotics administration (6.04 ±0.44, 5.53±0.37, 7.04±0.51 nmol/mg protein, p<0.01). conclusion: in the maternal venous plasma of pprom, the lipid peroxide levels and protein carbonyls formation were increased. the results suggest that reactive oxygen species formation by inflammatory reaction is suppressed by combined treatment of 3 rd generation cephalosporin and erythromycin. objective: the aim of the present prospective cohort study was to evaluate the correlation between blood flow pulsatility index in fetal middle cerebral artery (mca) and the onset of spontaneous labor. study design: doppler evaluation of fetal mca were performed between 24 and 41 weeks gestation in 664 consecutive pregnant women with a singleton pregnancy and known gestational age. the study population was divided according to the mca pi (< 0.74 or >/= 0.74 mom) and, using survival analysis and cox regression, the two subgroups obtained were compared. in these analyses, the event was delivery (following spontaneous labor) and the time variable was time elapsed since doppler exam. results: the median time elapsed between doppler evaluation and spontaneous labor was significantly shorter in the women with mca pi lower than 0.74 mom (5.5 days, interquartile range 2-10) in comparison with the women with mca pi higher or equal than 0.74 mom (22.5 days, interquartile 5-37.5 days (p<0.001, mann-whitney test). after correction for birth weight and umbilical artery pi, survival analysis and cox regression confirmed that mca pi was independently associated with the number of days elapsed from doppler to spontaneous labor and delivery (p< 0.001, exp(b) 2.77, ci 95% 1.95-3.90). conclusions: the present data show that, at term of pregnancy, fetal cerebral resistance reduction anticipates the onset of spontaneous labor. novel calcium sensitizers and human uterine contractility. mark p hehir, audrey t moynihan, terry j smith, john j morrison. department of obstetrics and gynaecology, national university of ireland, galway, ireland. objective: the factors regulating contractility of uterine smooth muscle are central to the occurrence of preterm labour and delivery. calcium sensitizers are a novel class of drugs with unique molecular actions. levosimendan, the best characterized of these compounds, is used in the treatment of acute and chronic heart failure and is a compound which exerts a number of effects on smooth muscle. it can exert an inotropic effect via sensitization of myofilaments to calcium and also exerts a relaxant effect by opening atp-dependent potassium channels. for these reasons we hypothesized that levosimendan may have an effect on myometrial contractility and investigated its action on both spontaneous and agonist induced contractions. method: biopsies of human myometrium were obtained at elective caesarean section (n=16). dissected myometrial strips suspended under isometric conditions, undergoing spontaneous and oxytocin-induced contractions, were exposed to cumulative additions of levosimendan in the concentration range of 1 nmol/l to 100 mmol/l. two sets of control experiments were performed simultaneously as follows: 1. strips exposed to either physiological salt solution (pss) only, for spontaneous contractions, or 0.5 nmol/l oxytocin; 2. strips exposed to pss/oxytocin and vehicle for levosimendan. results: levosimendan exerted an inhibitory effect on spontaneous and agonist induced contractions, compared to control strips. the mean maximal inhibition values were as follows: 45.34 ± 5.92% for spontaneous contractions (n=6; p< 0.05) and 41.88 ± 5.40% for oxytocin-induced contractions (n=6; p<0.05). no significant difference was found between control 1 and control 2 for both spontaneous and oxytocin induced contractions. conclusion: the calcium sensitizer levosimendan exerted a potent relaxant effect on uterine contractility in vitro. this action was seen in both spontaneous and agonist induced contractions. the results from this study raise the possibility of calcium sensitizers holding potential tocolytic properties in vivo and further studies are required to investigate the potential benefits of this novel class of drugs. to determine to what degree extensive patient movement affects uterine emg signals and toco signals. study design: 11 pregnant term labor patients were recorded using both uterine emg and toco simultaneously. baseline recordings were obtained when patients were still, and these were used as control records. test recordings for both devices were obtained from all patients by asking the patients to perform movements. area under the rectified-voltage amplitude curve of the uterine emg signals and area under the curve for the toco signals were found. mean %-increase was calculated for the uterine emg and toco devices (each device %-increase values were averaged over all 11 patients). mann-whitney rank-sum test was used to look for any statistical differences in %-increase in area for uterine emg vs. toco methods (p < 0.05 considered significant). results: there was a large increase in activity (artifact) on both devices' signals during patient movement (figure 1) . both devices' traces eventually returned to baseline after the patient movement stopped. toco movement artifact was significantly higher than emg movement artifact ( table 1) . conclusions: both uterine emg and toco signals experience artifact when patients move the uterine emg electrodes and toco pressure transducer, respectively. toco seems to be more adversely affected by such movements. uterine emg may be a preferred method for monitoring contractions of laboring patients in the clinic. supported by grant nih r01-hd037480. results: 68% of obstetricians completed the questionnaire. none would counsel patients against labour unless there were contraindications. the majority would recommend labour for all indications for previous caesarean section investigated although in all instances, personal preferences were lower (p<0.04); after a failed instrumental delivery, 74% obstetricians would recommend labour but only 44% would choose that option for themselves (p<0.01). overall, female obstetricians would contemplate and recommend labour more readily than male obstetricians. labour augmentation and induction were recommended to patients more frequently (66% and 57% respectively) than chosen for personal care (57% and 52%). reluctance for labour augmentation and induction was greatest among younger consultants. conclusion: consultants have responded to consumerism and aim to meet the requests of their patients. they more readily recommend labour than they would choose for personal care, and a majority would recommend labour induction when necessary. informed patient choice is paramount rather than attaining a target figure for women attempting to labour, and the views of those requesting delivery by caesarean section should be respected. (table) . cd increased in nullipara singleton, cephalic, term pregnancy in spontaneous labor, mostly due to dystocia; nullipara singleton, cephalic term pregnancy with induced labor, mostly due to non-reassuring fetal status and dystocia; singleton, cephalic term pregnancy with previous cd, mostly due to elective cd; singleton cephalic preterm. no changes in neonatal outcome were observed. conclusion: clinical audit was useful to keep under control cd rate in our institution in comparison with italian reality, but it was not sufficient to maintain stable cd rate suggesting the need of other, multifaceted strategies. elective delivery at 39 weeks' gestation is a common obstetric intervention. the purpose of this study is to estimate the maternal mortality rates (mmr) associated with this acog -approved intervention. there are no reliable us data describing mmr by mode of delivery. therefore, we base our estimates on british data indicating a procedure related mmr of 2.06/100,000, 5.8/100,000 and 18.2/100,000 for vaginal, elective cesarean section (c/s) and emergency-unplanned c/s deliveries, respectively. a decision tree model was constructed assuming that all eligible patients (approx. 2,500,000/annually in the u.s.) would be delivered at 39 weeks by either an elective c/s or an induction of labor. we further assumed that 50% of inductions would result in a vaginal delivery. our estimates show that the annual, delivery-related maternal mortality associated with an elective delivery of all patients at 39 weeks would be 145 and 253 for elective c/s and induction of labor, respectively. because vaginal delivery results in the lowest mmr, we performed a oneway sensitivity analysis to identify the impact of changing the success rate of induction on the estimated mmr. the results indicate that once the success rate exceeds 77%, this intervention would be associated with a lower mmr as compared to elective c/s. our estimates indicate that although the overall mmr associated with elective delivery at 39 weeks is relatively low (approximately 12.5/100,000 deliveries), it is certainly not negligible. in addition, the mmr is highly dependent on the likelihood of a successful vaginal delivery following induction of labor. when the success rate for induction of labor falls below 77%, an elective c/s appears to be the safer delivery method. background: the first obstetric visit is an opportunity to provide the pregnant patient information regarding substances that can cause potential harm to the pregnancy. little is known about how obstetric care providers handle these topics. objective: to examine patient-provider communication about substance use during the first prenatal visit. methods: we audiotaped first prenatal visits and qualitatively analyzed those tapes in which patients disclosed substance use. we invited patient participants to return for a semi structured interview during which they reviewed their audiotaped conversations and described their reactions to the providers' communication styles. results: 29 providers (21 residents, 5 midwives, 3 nurse practitioners) and 51 patients participated. providers asked about smoking, alcohol and drug use in all 51 (100%) visits. 25 patients reported being smokers, 4 reported alcohol use, and 11 reported drug use. provider responses to smoking disclosures included brief discussions of smoking effects on pregnancy, encouragement to quit/cut down, and referral to smoking cessation programs. provider responses to alcohol or drug disclosures included only general statements regarding effects on pregnancy (e.g., "we find that this is bad for babies.") and referral to ultrasound/genetics for reassurance. few alcohol or drug discussions assessed whether the patient had intentions or concerns regarding continued use during the pregnancy. few discussions addressed strategies for behavioral change. none included assessment for motivations, readiness, or barriers to change. in follow up interviews, patient participants said they expected to be asked about substance use but advised providers to ask non-judgmentally. those who used alcohol/drugs wanted more information on potential effects of these substances on the pregnancy/fetus and appreciated the reassurance from referrals to ultrasound/genetics. discussion: counseling for risky behaviors in the first obstetric visits contained only limited discussion of the effects of the risky behaviors and primarily focused on referral-which may be a proxy for avoiding a difficult and time consuming conversations. background. there are conflicting results regarding the association of maternal psychiatric disorders or psychological distress due to stressful life events with pre-term birth and low birth weight. aims. to investigate the association between maternal psychiatric disorders and/or stressful life events and intrauterine growth abnormality, low birth weight or preterm birth. method. three mutually exclusive and homogeneous groups of pregnant women (20 with actual psychiatric disorder, 20 with stressful life events, and 40 healthy comparisons) underwent serial fetal ultrasound examinations and uterine and umbilical artery doppler velocimetry at 20 (+1), 28 (+1) and 34 (+1) weeks of gestational age. subjects were recruited from all consecutive women attending two antenatal clinics. the presence of any maternal medical illness, drug treatments, fetal chromosomal and/or structural malformations, were exclusion criteria. all women recruited underwent a structured interview at 18-20 weeks for the psychiatric diagnosis (mini international neuropsychiatric interview -mini) (sheehan et al, 1998) ; moreover, the 17-item hamilton rating scale for depression and the hamilton rating scale for anxiety were included in the assessment. the person who obtained obstetrical clinical data was blind to the results of psychological evaluations. results. the three groups were comparable for: age, parity, socioeconomic status, smoking, alcohol consumption, body mass index. gestational age at birth was not different in the three groups. infants of women with psychiatric disorders had significantly lower birthweight (3084 + 414 g) and higher percentage of birth weight below the 10 th centile for gestational age (30%) than infants of healthy mothers (3408 + 453 g and 5%, respectively). there was also a trend towards lower mean birth weight (3242 + 400 g) and higher incidence of birth weight below the 10 th centile (10%) in the stressful life event group. there was no significant difference among groups in the percentage of abnormal uterine or umbilical doppler results. conclusions. maternal psychiatric disorders are associated with a lower birth-weight, but the effect is unlikely to be due to abnormal utero-placental or feto-placental vascularisation. objective: the purpose of this study was to evaluate antenatal ultrasound as a tool for the detection of intrauterine growth restriction (iugr) and small for gestational age (sga) infants among subjects with elevated human chorionic gonadotropin (hcg) levels on second trimester serum screening. although iugr has been linked to elevated hcg levels, the optimal screening regimen for antenatal sonographic surveillance has not been previously established. methods: a retrospective cohort study was performed at saddleback memorial medical center where serial ultrasounds from 26 weeks-delivery are generally recommended for patients with hcg levels > 2.0 mom. all pregnancies were dated by second trimester ultrasound ± last menstrual period. subjects with an hcg > 2.0 mom, who had at least one antenatal ultrasound evaluation for iugr, were identified from an electronic ultrasound database used for clinical report generation. ultrasound data were then linked to an obstetrical birth outcomes database for relevant demographic/delivery information using unique identifiers. iugr was defined as a sonographic estimated fetal weight (efw) <10%ile for the estimated gestational age (ega). sga was defined as an actual birthweight <10%ile for the ega at the time of delivery. results: from 1999-2007, there were 659 subjects with elevated hcg levels who underwent antenatal ultrasound surveillance for iugr and who had known delivery information. a total of 1708 ultrasound examinations were performed. the median number of examinations per subject was 3 with a range from 1-5 examinations per subject. the incidence of iugr and of sga were 5.0% (n=33/659) and 7.3% (n=48/659), respectively. no fetus with iugr demonstrated absent or reverse end diastolic umbilical artery doppler flow. antenatal ultrasound examinations only identified 31.3% (n=15/48) of sga infants. however, the sensitivity for the detection of sga was 100% when an efw cut-off of 75% was used. conclusions: although the majority of sga infants did not demonstrate growth restriction on antenatal ultrasound, a sonographic efw > 75%ile appears to be a safe cut-off to rule out fetuses at risk for sga. neonatal dry lung syndrome after pprom: reason to change intrauterine referral practice in the netherlands? ellen s hoogakker, 1 christiaan v hulzebos, 2 gerda g zeeman. 1 1 obstetrics and gynecology, university medical center groningen, groningen, netherlands; 2 pediatrics, division of neonatology, university medical center groningen, groningen, netherlands. background: neonatal dry lung syndrome (dls) is a distinct clinical entity following pprom, mimicking pulmonary hypoplasia but with dramatic respiratory improvement during the first 24-48 h . its pathogenesis implies complete collapse of small airways to a degree that capillary forces impede distension by ordinary ventilatory pressures. in the netherlands, women with pprom remain admitted at a level iii nicu perinatal center until they reach 32 wks. when they are referred back to their community hospital, unless they live in the neighborhood of the tertiary center. we question this referral pattern because we still see severe respiratory problems occur > 32 wks. we sought to determine the prevalence of such morbidity, in particular dls, in women with pprom who deliver > 32 wks. methods: retrospective descriptive study of neonatal outcome data of singleton pregnancies complicated by pprom between 24-32 wks, who deliver > 32 wks (latency > 24 h) , during the 4-yr period of 2002-2006 at a single academic center. data were extracted from medical records and electronic department databases. results:108 pprom pregnancies were identified. all but 2 received at least 1 full course of steroids. 27 (25 %) delivered > 32 wks. 3 newborns born at the community hospital needed emergency transportation to a level iii nicu for respiratory morbidity. neonatal outcome data (means ± sd) are listed in the table. there were no cases of late onset sepsis, nec or perinatal mortality. conclusions: respiratory morbidity still occurs after pprom with delivery > 32 wks. further investigation of pregnancy-related characteristics, such as the presence of anhydramnios and the latency period, with regards to dls is needed. modification of current referral practice depends upon complete data derived from all dutch level iii perinatal care centers (n =10). tertiary care center (n = 8) to determine if an association exists between macrosomia (birthweight > 4000g) and perinatal outcomes in women with and without gestational diabetes mellitus (gdm). study design: this is a retrospective cohort study of 36,241 singleton pregnancies. the study cohort was stratified by the diagnosis of gdm, with the presence or absence of macrosomia as the dependent variable. perinatal outcomes examined included neonatal hyperbilirubinemia, hypoglycemia, respiratory distress syndrome (rds), shoulder dystocia and erb's palsy. chisquare tests were performed as well as multivariable analyses controlling for confounders, using p<0.05 to indicate statistical significance. results: in women diagnosed with gdm, macrosomia is associated with a higher frequency of hypoglycemia, respiratory distress syndrome, shoulder dystocia and erb's palsy. though the prevalence of these outcomes is relatively decreased in patients without gdm, they are still more prevalent in macrosomic patients. macrosomia is associated with a higher prevalence of adverse perinatal outcomes in women with and without gdm. therefore, it is important to evaluate neonates with birthweights greater than 4000 grams for hypoglycemia and unrecognized erb's palsy. conclusion a significant proportion of our obstetrical patients are obese and many do not perceive themselves to be obese. while our finding of an inverse correlation between level of education and bmi may be confounded by socioeconomic status, our results suggest that in order to address the problems of obesity in our population an important first step will be improving the education of our reproductive age women regarding normal weight gain and nutrition in pregnancy. this may have a significant impact on improving pregnancy outcomes of today's obstetrical population. objective: the aim of the study was to evaluate the impact of a "flat" oral gct upon the outcome of pregnancy. the gct was considered flat when the difference between the basal value and the after load value was 10%. methods: we prospectively analyzed the outcomes of 410 pregnancies who delivered at our department. inclusion criteria were singleton pregnancy; bmi <30 kg/m 2, absence of major risk factors for diabetes and of pregestational diabetes. 50 g 1-hour oral gct was performed at 24 -28 weeks of gestation. women were subdivided into 3 groups according to the result of the gct: group 1= negative (load glucose >10% and <140 mg/dl) 327 women (79.8%); group 2=flat (load glucose 10% than basal and <140 mg/dl) 51 women (12.4%); group 3=positive gct/negative ogtt, 32 women (7.8%). data are mean ± sd. differences were calculated with the student t test for unpaired samples and 2 test. regression analysis were performed by the least squared method. p-values were considered significant at p<0.05. results: the characteristics and obstetric outcomes in the three groups are presented in the table 1 . in all patients there was a significant linear relationship between the load and basal glucose values (load value=66.8+0.6 basal value; r=0.2; p<0.001) and between birthweight and load values (birthweight=2978.3+2.8 load value; r=0.15; p<0.02). the relationships were significant also in group 1 and 2 separately but not in group 3. in this preliminary study we found no major outcome differences in women with flat gct compared to women with normal and gct positive/ ogtt negative results. it seems important, however, to futher investigate the meaning of a flat curve in a bigger population and/or by means of metabolic studies with the use of stable isotopes. results characteristic of the study population and obstetric outcomes are presented in table 1. the probability to be primiparous and to deliver babies <10° centile decreased significantly with bmi whereas the risk of cesarean section, of post partum hemorrhage at vaginal delivery and to deliver babies >90° centile increased significantly. also the risk to develop preeclampsia and gestational diabetes was increased, although not significantly, with bmi. compared to lean and normal, obese were more likely to be hypertensive and diabetic before pregnancy (p<0.001) and to start pregnancy without any medical and obstetrical risk but obesity (85.6% vs 90.5 and 90.2%; p<0.01). african women exhibited the highest bmi and the highest rate of obesity (table 2 ). conclusions we confirm that obese women have an increased risk of prepregnancy and pregnancy complications: less than 50% have an uncomplicated pregnancy. at delivery there is an increased risk of cesarean section and post partum haemorrhage. objective: overweight and obese women often give birth to larger babies, which is associated with traumatic birth injuries and an increased risk to develop obesity, diabetes and hypertension in childhood and later in life. the mechanisms underlying fetal overgrowth in obese pregnant women are largely unknown. the aim of this study was to establish a mouse model of obesity/high fat diet in pregnancy. we hypothesized that a moderately high fat diet prior to and during pregnancy would result in increased maternal adiposity, fetal overgrowth and a metabolic profile similar to that of obese newborn offspring with persistent pulmonary hypertension, despite enhanced pregnant women. method: c57bl/6j female mice were fed control (c, 11% of energy from fat) or isocaloric high fat (hf, 32% of energy from fat) diets ad libitum for 8 weeks prior to mating and during gestation. at gestational day 18.5 maternal blood samples were obtained to measure adiponectin, leptin and cytokine levels and maternal fat pads were isolated and weighed. in a sub set of animals measurements of transplacental transport of neutral amino acids and glucose were performed in vivo under ketamine anesthesia using 3 h-meaib, and 14 c-glucose. results: no significant differences were observed in maternal pre-pregnancy bodyweight, total caloric intake, weight of the dam at e18.5 or litter size between treatment groups. fetal weight was increased in the hf group by 45% (p<0.05). maternal adiponectin levels were significantly (p<0.01, n=18) decreased (hf 45 ± 8, c 69±15 g/ml) and leptin levels increased by 60% in animals fed a high fat diet, but this difference did not reach statistical significance (n=6). adiposity (fat pad weight) was increased by 7% (p<0.05, n=10 in the dams fed high fat diet, however no difference was observed in maternal il-6 levels and neither group had measurable levels of tnf-. maternal red blood cell lipid profiles were altered in high fat animals with an increase in stearic and linoleic acids but decreased oleic acid levels. preliminary data showed that placental uptake and transfer to the fetus of glucose and meaib were increased by at least 50% in dams fed high fat diet. conclusion: this murine high fat diet model has several features consistent with human obesity in pregnancy and the maternal metabolic environment is similar to that seen in the human. the increase in placental uptake and transfer of nutrients constitute a key mechanism underlying fetal overgrowth in overweight/obesity in pregnancy. objective: epidemiological studies have demonstrated a positive relationship between maternal overnutrition and the development of the metabolic syndrome in the offspring. we previously reported that lambs of well fed ewes have increased plasma glucose levels in early life, this may be a consequence of altered hepatic glucose production. increased 11 hsd1 expression is associated with an increase in intracellular cortisol, promotion of hepatic insulin resistance and a consequential increase in gluconeogenic activity. hypothesis: we hypothesised that maternal overnutrition in late gestation in the sheep results in increased hepatic expression of 11 hsd1 in the lamb before and after birth. methods: ewes were provided with either 100% (control,c) or 160% (well fed, wf) of maintenance energy requirements from 115d gestation until delivery. post-mortem was performed on either 139±141d gestation (c=6,wf=8) or and postnatal day 30 (c=12,wf=9). plasma glucose and leptin concentrations in the fetuses and lambs were determined. the relative hepatic mrna expression of 11 hsd1 and reference gene rpp0 were determined by qrt-pcr. results: relative liver weight was significantly higher in lambs of wf ewes compared to c at 30d (p<0.05). the expression of 11 hsd1 mrna was significantly higher in the postnatal compared to the fetal lamb (p<0.001) independent of maternal nutritional treatment. however, there was no effect of maternal overnutrition on the hepatic expression of 11 hsd1 mrna before or after birth. there was no effect of prenatal nutrition on fetal or postnatal plasma cortisol concentrations. the expression of 11 hsd1 in the liver of lambs of wf, but not c ewes, was inversely related to plasma glucose concentrations in the first 24hrs after birth (r 2 =-0.78, p=0.002). we have therefore demonstrated that exposure to prenatal overnutrition results in an inverse relationship between 11 hsd1 mrna expression in the liver at 30d and plasma glucose concentrations on the first day of life. this suggests that exposure to high glucose levels before and immediately after birth results in a reduced expression of hepatic 11 hsd1. we would expect a reduction, rather than promotion of intra-hepatic cortisol production, and therefore it is unlikely to explain the hyperglycemia present in lambs of wf ewes in early life. ninety-seven (38%) of mothers were classified as obese (bmi>30) based on their first pregnancy weight. glycemic control at 36 weeks was superior in the non-obese group (table 1) . there were no differences in glycemic control during the last week of pregnancy. obesity was significantly associated with increased maternal weight gain during pregnancy. mean birth weights, ponderal indices and rates of macrosomia were significantly higher in infants born to obese women when compared to non-obese (table 1) . primary cesarean deliveries rates were comparable. the rate of neonatal hypoglycemia, hyperbilirubinemia, phototherapy and neonatal icu admissions did not differ between obese and non-obese diabetic women (table 1) . although not statistically significant, there was a trend towards an increased rate of birth injuries in the obese group. a similar comparison between obese and non-obese women treated with medication (glyburide or insulin) demonstrated a higher mean birth weight (3675g+593 vs. 3434g+520, p=.005) and higher rate of macrosomia (11 vs. 9%, p=0.04). there were no differences in glycemic control, cesarean delivery rates and other neonatal outcomes between the obese and non-obese treated with medication. conclusion: obese women with type ii and gdm give birth to larger infants than their non-obese counterparts and have a higher incidence of fetal macrosomia. there was a trend towards increased rate of birth injuries in the obese group. despite these differences other maternal and neonatal outcomes were similar which may be a reflection of glycemic control. introduction: tlrs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. the placenta, and more specifically the trophoblast, may play an important role in the response to infection. previously, we described the expression of tlr-3 by human trophoblast and their ability to respond to polyinosinic-polycytidylic acid (polyi:c), a synthetic double strand rna which mimics viral rna. in the present study we evaluate the effect of polyi:c in mouse pregnancy and characterize the local and systemic response. material and methods: human first trimester trophoblast cell line, htr8, was treated with polyi:c. c57b/6 wild type and tlr-3 knock out mice were injected intraperitoneally with polyi:c at 16.5 gestation day. cytokine and chemokine level were determined in supernatant and lysates using the bio-rad multiplex assay and analysis was done using the bio-plex100 is. results: polyi:c induced cytokine (il6, il1 and il1 ) and chemokine (il8, rantes, gro , mcp1 and mip1 ) secretion and production by human cultured trophoblast in a time (24-48 h) and a dose (2-25 g/ml) dependent manner. injection of polyi:c to c57b/6 wild type mice induced preterm delivery within 24 h at a dose of 9 mg/kg body weight. no effect was observed in tlr-3 knock out mice. a robust systemic (spleen and serum) and local (placenta and amniotic fluid) inflammatory response was observed 2 and 4 h following polyi:c treatment. trophoblast cell cultures from tlr-3 ko mice confirmed that the response to polyi:c is tlr-3 dependent. conclusion: we demonstrate that viral infection may trigger an immune response leading to preterm labor. furthermore we show that the trophoblast is able to recognize and respond to viruses through the expression of tlr-3. our findings provide a novel mechanism of pathogenesis of preterm labor associated with tlr-3 mediated inflammatory response. introduction: adverse neurological outcome is a major cause of neonatal morbidity after a preterm birth (ptb). a growing body of evidence demonstrates the involvement of inflammatory pathways in ptb and implicates these pathways as a causative factor in fetal brain injury. however, activations of cytokine pathways in normal labor (whether iatrogenic preterm or at term) has been observed. what remains understudied is the effect of labor on the preterm fetal brain and whether an inflammatory stimulus is essential for fetal brain injury. methods: 2 mouse models were utilized: (1) a model of intrauterine inflammation (lps into uterine horn) (n=9); controls received intrauterine saline (n=9) and (2) autism is a neurodevelopmental disorder with a strong genetic component and several known environmental risk factors, such as infection. in addition, its onset of etiology is likely to occur during prenatal development. we propose that subjecting fetal sheep via amniocentesis to the bacterial endotoxin lipopolysaccharide (lps) injected to the amniotic fluid at gestational day (gd) 110 will result in morphological alterations in the offsprings' cerebellum resembling alterations found in the cerebellum of patients with autism. using high precision design-based stereology, we investigated mean total-and layer-specific volume and mean total granule and purkinje cell (pc) number in the cerebellum of 6 lps infected animals and 3 controls. the results of the present study showed preserved volumes of the total cerebellum as well as of the molecular layer, outer and inner granular cell layers and white matter. interestingly, compared to controls, the lps infected brains showed a statistically significant increase (+20.4 %) in the mean total number of granule cells, whereas the pcs did not show any difference between the groups. these seemingly paradoxical results might be explained by (1) the so-called time of origin of these neurons, i.e. the pcs develop prenatally whereas the granule cells develop postnatally or (2) the direct correlation between pcs and granule cell number in the cerebellum. these results might contribute, as an animal model, to our understanding of the biological basis for interindividual differences in morphological alterations found in the brains of patients with autism. the previous studies have shown that there is a higher incidence of spontaneous preterm birth and poorer neonatal outcome in pregnancies with a male fetus. in vitro studies also report a sex dependent pattern in different placental enzymatic systems. we have shown previously that lactobacillus rhamnosus gr-1 supernatant is able to antagonize the actions of lps on cytokines and ptgs2 in placental trophoblasts. we hypothesize that fetal sex will influence the production of cytokines and prostaglandin regulating enzymes in lps and lactobacilli treated placental trophoblast cells. methods: term placentae were collected from women undergoing elective caesarean section. placental trophoblasts were isolated using established primary culture protocols. cells were pretreated with lactobacilli supernatant and subsequently treated with lps. pgdh and ptgs2 expression levels were measured by western blot analysis and tnf-, and il-10 concentrations measured by elisa. results: lps stimulation caused a marked increase in production of tnf-(30.9 pg/ml to 1175.3 pg/ml, n=11, p<0.05), an effect that was greater in placentae of the male fetuses (1516.6 pg/ml, n=6) compared to female fetuses (562.5 pg/ml, n=5). lactobacilli supernatant abolished this response in both sexes. lps-activated trophoblasts from placentae of the male fetuses showed an increase in il-10 production (n=5, p<0.05) and ptgs2 expression (n=4, p<0.05). however, there was no response to lps in placentae of the female fetuses. lactobacilli supernatant up-regulated pgdh (n=8) by 63%, and this effect was greater in placentae of the female fetuses (n=4). conclusion: we conclude that human placentae from pregnancies carrying male fetuses are more responsive to lps by producing more pro-and anti-inflammatory cytokines, as well as ptgs2. conversely, placentae of the female fetuses upregulate pgdh with lactobacilli treatment. these findings may explain the underlying mechanism for the higher incidence of preterm birth and adverse pregnancy outcomes seen with male fetuses in the clinical setting. , 2007) . this study investigates whether iai is associated also with altered expression of neuropilin-1. methods: (i) immunohistochemistry (ihc) was performed on tissue sections of term decidua with or without clinical / histologic evidence of iai (n=3 for each). neuropilin-1 expression was scored by an investigator blinded to the identity of the samples. (ii) cultured term dscs were retrieved from elective cesarean (n=6), purified, and depleted of leukocytes. after treatment with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days, dscs were stimulated with il-1b (1-10 ng/ml), tnfa (1 ng/ ml), or thrombin (2.5 iu/ml) for 24h. since no elisa exists for neuropilin-1, protein expression was determined by immunocytochemistry (icc). (iii) total rna was extracted and the effect of il-1b on neuropilin-1 mrna expression measured by real-time rt-qpcr and corrected for b-actin mrna. results: neuropilin-1 expression in term decidua was increased in tissues with iai vs controls (p<0.05), and localized primarily to dscs. using icc, an increase in neuropilin-1 was noted after stimulation with il-1b and tnfa, but not thrombin. il-1b increased neuropilin-1 mrna expression in dscs by 6.5±1.6-fold (from 28.0±17.7 to 240.9±189.4 neuropilin-1 mrna/b-actin mrna; p=0.031). conclusions: iai is associated with increased expression of the vegf receptor, neuropilin-1, in term decidual tissues. il-1b and tnfa (but not thrombin) stimulated neuropilin-1 expression in term dscs, and this effect appears to be mediated at the level of gene transcription. since aberrant vegf function alters vascular permeability, these data provide a mechanism by which iai can promote 'decidual activation' and preterm labor. inflammation, university of glasgow, glasgow, united kingdom. objective: asthma is associated with inappropriate activation of airway smooth muscle, chemokine expression and accumulation of mast cells which drive smooth muscle reactivity. labour is similarly associated with smooth muscle activation and expression of cxcr1 and cxcr2 ligands. the role of mast cells in human parturition is unknown; however, mast cell products can stimulate myometrial contractions and preterm labour in animal models. we have quantified mast cells in association with human labour and determined whether they express cxcr1 and cxcr2. methods: lower segment myometrial and cervical biopsies were taken at term caesarean section from women not in labour (nil) (myometrium n=8; cervix n=9) and in labour (il) (myometrium n=8; cervix n=9). mast cells were localised in myometrial and cervical sections by icc with a primary antibody against c-kit. the number of cell transects in 10 randomly selected high-powered fields (400x) was quantified blindly by two observers for each specimen, with median density and interquartile range (iqr) calculated. backto-back icc was performed to determine whether c-kit co-localised with the chemokine receptors cxcr1 and cxcr2. results: mast cells were in close association with myometrial smooth muscle in non-labouring lower segment myometrium. labour was associated with a significant influx and increase in mast cells numbers (nil median 11.9, iqr 6.7 -19.8; il median 26.9, iqr 16.1 -40.2, p=0.025). in contrast no significant increase in mast cells was observed in cervical tissue in association with labour (nil median 19.0, iqr 4.6 -35.5; il median 7.9 iqr 3.5 -10.4, p=0.17). analysis of chemokine receptor expression demonstrated co-localisation of cxcr1 to c-kit positive cells present within the myometrium. conclusions: human labour at term is associated with an increase in mast cells within the myometrium, with close approximation to smooth muscle bundles. these mast cells express the chemokine receptor cxcr1, the ligands of which we have previously shown to be up-regulated in labouring myometrium. mast cells are not accumulated in cervix in association with labour suggesting a less critical role in cervical ripening. further analysis of the role of mast cells in modulating myometrial smooth muscle physiology is warranted. progestins and the glucocorticoid receptor in human myometrial and amnion-derived wish cells. alison j tyson-capper, stephen c robson. reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. background and objectives: progesterone (p) can reduce the risk of preterm birth in some high risk women. in this context there is accumulating evidence that this, at least in part, may be due to anti-inflammatory and immunoregulatory properties of p. target tissue responsiveness to p is considered to be determined by the progesterone receptors (pr) and nuclear co-factors that directly interact with pr. pr and glucocorticoid receptors (gr) share several structural and functional characteristics, including similarities in dna sequence recognition by binding to the same hormone response elements. pr and gr interact with similar chaperones in the absence of ligand and with a similar group of co-activators in the presence of hormones; both can display comparable anti-inflammatory activities under specific physiological conditions. in this study we aimed to investigate whether the anti-inflammatory properties of progestins may be mediated by pr and gr signalling. methods: primary cultures of non-pregnant and term pregnant human myometrial (passage 1-2) and wish cells were serum starved for 24hrs and treated with 17-hydroxyprogesterone (17-hp), progesterone (p), dexamethasone (dex) and immunofluorescent staining and immunoblotting analyses performed. in some experiments cells were pre-treated with ru486 (a pr/gr antagonist) or org 31710 (a pure pr antagonist). results: in the absence of hormone gr appeared to be predominantly cytoplasmic, whereas, upon treatment with 17-hp and p (1 and 10 m) and dex (10nm) gr was abundant within the nuclei of myometrial cells. immunoblotting analyses demonstrated that levels of gr progressively increased within the nuclear fractions of both pregnant myometrial and wish cells in response to increasing concentrations of p (100nm to 10 m), and decreased sequentially within cytoplasmic fractions. in the presence of org31710 gr protein levels remained constant within the cytoplasm. there also appeared to be a slight increase in gr expression, though not statistically significant (p>0.05) within both cells types in response to p. conclusion: in this study we show that gr is activated by 17-hp and p and translocates to the nuclei of human myometrial and amnion-derived cells. in addition, levels of gr increase in response to p. whether p and 17-hp act as agonists or antagonists for gr in the regulation of hormone response genes associated with the onset of term and preterm labour remains to be elucidated. objective: using a mouse model of inflammation-induced preterm birth (ptb), we have demonstrated dramatic cytokine elevations in the uterus and placenta with concomitant, though less dramatic, cytokine elevations in the fetal liver and brain, associated with neuronal injury. because precise mechanisms of fetal injury in ptb remain unclear, we sought to examine inflammatory cell trafficking, and target organ damage by histopathologic assessment of the placenta, fetus, and fetal brain. study design: 6 hours after intrauterine infusion of saline or lps into the right lower uterine horn of cd-1 mice, the left upper horn, with the gestational sacs(gs) in situ, was removed en bloc(n=6 per group) each with 2-4 gs with15 fetuses/treatment group. specimens were fixed, bisected and processed for histology and ihc. inflammatory and hematopoietic cells were quantified using pas, gata-1 (erythroid precursors), cd3, and bm8 (macrophage-mp) within the placenta, liver, extremity mesenchyme, brain and leptomeninges. the presence of hemorrhage, necrosis, and apoptosis (h2ax stain) was assessed. erythopoietin (epo) levels were measured in brain and liver by elisa. results: more neutrophils were present in maternal decidual vessels in lps compared to saline (p=0.02). in lps-exposed, fetal mp were increased in the placenta (p=0.007), fetal extremity mesenchyme (p=0.018), fetal liver (p=0.005) and leptomeninges (p=0.013) but not in the brain or spinal cord compared to saline. no necrosis, hemorrhage or increased apoptosis was noted in the fetal brains. 69% of lps-exposed fetuses and 0% of saline-exposed had liver hemorrhages (p< 0.001). increases in nucleated erythrocytes and erythroid precursors were found in fetal vasculature of the placenta in lps-exposed (p=0.004). epo levels were not elevated in either group. conclusion: intrauterine lps infusion induces acute inflammation predominantly in the maternal circulation of the placenta. in the fetus, there is widespread mp activation, liver hemorrhage and increased erythroid precursors seen in the fetal circulation of the placenta. although histologic evidence of cns damage was not evident, the increased mps present in the leptomeninges may play an important role in inflammatory-mediated cns damage. non-toll-like innate immune proteins: do they change during pregnancy? juan m gonzalez, hua xu, ella ofori, michal a elovitz. obgyn; crrwh, university of pennsylvania, philadelphia, pa, usa. introduction: trem-1 (trigger receptors expressed on myeloid cells) is an important regulator of innate immunity. the natural ligand for trem has not been identified. activation of trem-1 in the presence of toll-like receptors results in substantial amplification of the host inflammatory response (klesney-tait et al nature immunology 2006). since inflammatory pathways are implicated in adverse pregnancy outcomes, this novel mediator of inflammation may play a critical role in preterm birth (ptb). therefore, we sought to determine trem-1 expression in the uterus, cervix, and placenta across gestation and to determine if trem-1 levels are altered by intrauterine inflammation. methods: in cd-1 mice, trem-1 was investigated in non-pregnant (np) and throughout gestation e 15, e 18 (n=3 -6 per group). uterine, cervical, and placental tissues were harvested. using an established mouse model of inflammation-induced ptb, uterine tissue was collected 6 hours after intrauterine infusion of saline (n=3) or lipopolysaccharide (lps) (n=3). for a non-pregnant model, using cd-1 mice, lps (n=3) or saline (n=3) was injected into the uterine horn following same procedures as with pregnant mice. uteri were harvested 6 hrs later. quantikine ® mouse trem-1 immunoassay was utilized for these studies. statistical analysis was performed using oneway anova followed by pair-wise comparison if statistical significance was reached (p<0.05) results: trem levels are significantly different between np and pregnant uterine tissues (p=0.002). e15 and e18 trem expression is significantly increased 2.3 and 2.5-fold compared to np (p=0.004 and 0.001 respectively). trem-1 levels in the placenta and cervix were not significantly different between e15 and e18. trem levels increased about 4-fold in the uterus after intrauterine infusion of saline or lps compared to e15 controls. in nonpregnant, trem levels were significantly different (p=0.05) with a 17-fold increase in trem expression in uteri exposed to lps or saline compared to controls. conclusions: non-toll-like innate immune proteins are differentially regulated during pregnancy compared to the non-pregnant state. the role of trem-1 in inflammation-induced ptb requires further study. research is warranted to determine if uterine up-regulation of trem in gestation is associated with an increased likelihood of responding to pathogens or severe as a protective mechanism. reduced plasma levels of vitamin d in caucasian women at term are associated with increased rate of infection. chander p arora, 1 adegoke adeniji, 1 susan e jackman, 1 babak forooghi, 1 isaac mostadim, 1 phillip yadegari, 1 calvin j hobel. 2 1 og-gyn, cedars-siani medical center, los angeles, ca, usa; 2 university of california los angeles, los angeles, ca, usa. background: vitamin d plays an important role in human pregnancy by acting as a regulator of immunity at the fetal-maternal interface. inflammatory changes associated with pro-inflammatory cytokines were reduced by vitamin d while anti-inflammatory cytokines were increased in t lymphocytes. vitamin d status has been defined as deficiency (<37.5nmol/l), insufficiency (37.5 to 80 nmol/l) and sufficiency (>80nmol/l) . objective: to assess the involvement of vitamin d in the occurrence of maternal infection during pregnacy in women with term deliveries. hypothesis: vitamin d metabolism could affect the rate of infection during pregnancy. study design plasma levels of 25(oh)d were determined by elisa and the rate of infection was recorded in a behavior in pregnancy study. in this study, 626 ethnically diverse women were evaluated at 18-20 weeks (t1), 28-30 weeks (t2) and 34-36weeks (t3). maternal infections were documented at each stage as well as at baseline visit with history of infection in current pregnancy. of these subjects who delivered at term two groups (58 with no infection during pregnancy, 101 with infection or history of infection) were matched further for non-smoking status, non-diabetics, ethnicity and maternal age. plasma from these were assayed for 25(oh)d and analyzed for the rate of maternal infection using fisher´s exact test or chi-square test. results although the women delivered at term, the levels of 25(oh)d in caucasians were significantly lower in the subjects with infection than the ones without (p<.001). women with vitamin d insufficiecy in the first trimester were more likely to develop infection during pregnancy (46.2 nmol/l ± 6.8 at t1, 28.4 nmol/l ± 4.1 at t2 and 30.2 nmol/l ± 3.6 at t3; all p<.001) but not subjects with sufficient vitamin d at t1(87.3 nmol/l ± 5.4 at t1, 27.9 nmol/l ± 3.8 at t2 and 32.8 nmol/l ± 4.6 at t3; all p<.001). conclusion the results reveal a positive association between 25(oh) d concentrations and greater risk of infection. vitamin d deficiency or even insufficiency may, therefore be involved in the pathogenesis of maternal infection during pregnancy. it is probable that vitamin d deficiency or even insufficiency could modulate the maternal susceptibility to infection during pregnancy by a proinflammatory mechanism. in vitro and in vivo observational data suggest that infection leads to caspase activation and apoptosis in the placenta and membranes, however currently there are no data on the role of apoptosis in the pathogenesis of infection associated preterm delivery. here we used group b streptococcus (gbs) as a model pathogen and examined the role of caspase dependent and independent apoptosis in preterm delivery. methods: we injected (7.5x10 8 ) heat killed group b streptococcus organisms (hk-gbs) intraperitoneally (i.p.) in 14.5 day pregnant c57b/l6 mice. the mice were euthanized at 5 hr (n=4) and 14 hr (n=6), the placentas and membranes were removed and assessed for apoptosis by tunel staining. caspase 3 activation and expression were determined by immuno-histochemistry (ih) and western blotting. the effect of apoptosis on preterm delivery was assessed by i.p. treating the pregnant mice with pbs (n=3), dmso (n=4) or pancaspase inhibitor z-vad-fmk (n=6) prior to hk-gbs and observing the animal for delivery for 48 hrs. results: there was a time dependent, gbs-induced increase in apoptosis by tunel assay and caspase 3 activation in the placenta and membranes. in addition hk-gbs-induced the expression of caspase 3 and caspase independent m-calpain in the placenta. z-vad-fmk (10 mg/kg), at the maximum concentration that did not induce maternal illness, did not prevent hk-gbsinduced preterm delivery. conclusions: caspase dependent and independent mechanisms are activated in the placenta upon exposure to gbs. systemic adminstration of a pan-caspase inhibitor did not impact upon the occurance or timing of bacterially induced preterm delivery. further studies are needed to assess the role of apoptosis in the pathogenesis of infection associated preterm delivery. early and 25 (3.2%) had a late sptb. the mean + sd gestational age at blood draw was 12 + 2 weeks. the median level of bb was higher in women with early as compared with late sptb or term births (p= 0.047 for trend). women with bb in the top quartile were 4.7 times more likely to have an early sptb as compared with women who had lower levels of bb (95% ci 1.5 to 14, p = 0.003). there was no association between bb and late sptb (rr= 0.8, 95% ci = 0.3 to 2). the adjusted or of an elevated bb for early sptb was 4.3 (95% ci = 1.3 to 14, p= 0.02). when the analysis was restricted to the 38 women with sptb the rr of an elevated bb for early sptb was 3.1 (95% ci 1.3 to 7.5, p = 0.03). conclusions: a significant relationship was found between an elevated bb in early pregnancy and early sptb suggesting inflammatory events in early pregnancy, perhaps infection-related, are part of the pathogenic mechanisms. objective: genital tract infection and/or inflammation appears to contribute to the majority of ptds preceding 30 weeks of gestation. ptd in humans has been associated with colonization and/or infection with a variety of different organisms including gram positive and negative bacteria, mycoplasma, ureaplasma, trichomonads, malaria parasites and viruses. the innate immune response to these pathogens is produced by a family of pattern-recognition cell membrane receptors known as the toll-like receptors (tlrs). these studies sought to characterize the tlr isoforms expressed in the preterm mouse uterus, and their modulation during lipopolysaccharide (lps)-induced ptd. methods: using sterile surgical technique, day-15 pregnant cd-1 mice underwent intrauterine injection of 250 g lps. subsequently, the mice were euthanized at 0, 2, 6, 12, 18 and 24 hours after lps injection. uterine tissue was harvested and placed in rnalater; subsequently total rna was isolated using the trizol reagent and genomic dna was removed using turbo dnafree. cdna was made using iscript cdna synthesis kit. pcr primers were designed using published mouse tlr gene sequences. real-time quantitative rt-pcr was performed using the power sybr green master mix and an abi prism 7000 multicycler. to confirm tlr amplicon sizes, the rt-pcr products were visualized on a 2% agarose/tbe gel stained with gelred. results: these studies have confirmed mrna expression of all 12 of the reported mouse tlr isoforms. these tlr amplicons range in size from 83 to 200 bp as expected; amplicon sequences are pending. quantitative rt-pcr studies performed using uterine tissues from five mice at each time point demonstrated that at 6 hours after lps injection, tlr3 increased 3-fold and tlr6 increased 2-fold (both p<0.05). in contrast, the expression of tlr4 (the ligand for lps) remained stable during the 24 hours after lps; the expression of tlr1, 5, 7, 8, 9, and 13 also remained stable. tlr2 expression trended upward and tlr11 trended downward, although neither was statistically significant. conclusions: these studies have confirmed expression of all 12 tlrs within the preterm pregnant mouse uterus, along with characterization of their modulation during lps-induced ptd. these observations are important because they contribute to our understanding of the immunologic signaling events leading to ptd. (funded by nih hd044747). udp-glucose and its receptor p2y14 as a new innate immune system in the female reproductive tract. toru arase, tetsuo maruyama, hiroshi uchida, takashi kajitani, masanori ono, maki kagami, hironori asada, yasunori yoshimura. obstetrics and gynecology, keio university, shinjukuku, tokyo, japan. objective: innate immune system involving toll-like receptors has recently emerged in the female reproductive tract (frt). we hypothesize that there may exist new other mucosal immunity in frt. recently, it has been reported that p2y14, a g protein-coupled p2y receptor for udp-glucose (udp-g), is involved in the lung epithelial immune system. the aim of this study is to investigate whether udp-g and p2y14 have a potential as the defense immune system in frt, in particular endometrium. materials and methods: we obtained human endometrial tissues from consenting reproductive-aged patients. the spatiotemporal expression of p2y14 in human and mouse endometrial tissues was analyzed using rt-pcr and ihc. isolated human endometrial cells and a human endometrial epithelial cell line, ishikawa, were cultured, treated with udp-g, and subjected to rt-pcr analysis for il-8 mrna expression. we also measured the il-8 secretion using elisa. small interfering rna was used to knock down p2y14 expression. the chemotactic activity of udp-g on neutrophils was tested using transwell assay with ishikawa cells. lastly, mouse uterine tissues were incubated with udp-g and subjected to rt-pcr analysis for mrna expressions of kc and mip-2, the il-8 homologues in mice. results: p2y14 was exclusively expressed in the glandular and luminal epithelium both in human and mouse uteri. treatment with udp-g induced the secretion of il-8 in ishikawa and human endometrial glandular cells, but not stromal cells, in a dose-and a time-dependent manner. p2y14 knockdown abrogated udp-g-induced il-8 production. treatment with udp-g also significantly increased the chemotaxis of neutrophils, which was attenuated by co-addition of anti-human il-8 neutralizing antibody. in the mouse uterus stimulation of udp-g significantly up-regulated the expressions of kc and mip-2 mrna. conclusions: udp-g is an endogenous molecule and released into the extracellular environment in a lytic manner after cell damage. taken together, our results collectively substantiate a model in which udp-g released from endometrial cells damaged by infection stimulates il-8 production via p2y14 in endometrial glandular epithelium, which, in turn, recruits neutrophils thereby preventing the progression of infection. thus, udp-g and its receptor p2y14 may be involved in the trans-species mucosal immune system in frt. (jsgi 2007; 14, 2(suppl) , abst #31) but in utero effects on fetal lung remain to be established. we have examined the relationship between duration of iai and subsequent azi treatment on the severity of fetal lung histopathology. we hypothesized that early treatment would prevent the development of advanced lesions, while late treatment may reduce the severity of lung damage. study design. thirteen chronically instrumented rhesus monkeys received intraamniotic inoculation of u. parvum (serovar 1; 7-14 x 10 7 cfu) at 130 ± 1.2 days gest. age (dga, mean ± sem, term=167 d). six of these animals received maternal i.v. azi (12.5mg/kg q12h or q6h for 10 d) either alone (n=3) or in combination (n=3) with dexamethasone (dex; 4mg/kg/d i.v. for 4d) and indomethacin (indo; 100mg/d p.o for 5d). tissues were obtained at cesarean section for histopathologic assessment. leukocytic infiltration of aveolar spaces and septal walls, type ii pneumocyte hyperplasia and peribronchiolar lymphocytic aggregates were scored as absent (0), minimal (1), moderate (2) and severe (3). results. inoculation-to-delivery interval was 18-25d for combined treatment groups and was similar to long duration infection without treatment (15-21d). treatment effects were tabulated as mean scores and compared as follows: control (n=3), score 1; short duration infection (3-8d; n=4), score 8; long duration infection (15-21d; n=3), score 12; short duration infection + treatment (n=5), score 4; long duration infection + treatment (n=1), score 5. conclusions. histopathologic findings of fetal pneumonia progressively worsen with duration of u. parvum iai. early maternal azi treatment prevents development of advanced lung lesions, while later treatment may reduce the severity of fetal lung damage. our results suggest that in utero treatment of iai may lower the risk of neonatal bronchopulmonary dysplasia. support: nih hd06159, rr00163. brain associated with adverse neurodevelopmental outcome. lps triggers proinflammatory responses through toll-like receptor-4 (tlr4). mitogen activated protein kinases including c-jun-n-terminal kinase (jnk) have been reported to be implicated in tlr4 signalling pathways and play important role in both onset of labor and brain injury. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of specific inhibitor of jnk signaling, d-jnk inhibitory peptide (d-jnki) can (i) inhibit jnk activity in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr-4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. in vitro kinase assay demonstrated the activation of jnk in response to lps in the maternal uterus and fetal brain. furthermore, the brain specific jnk3 was found to be the major jnk isoform activated by lps in the fetal brain. co-administration of d-jnki with lps to pregnant mice delayed significantly (p<0.007) lps-induced preterm delivery and reduced pup mortality up to 15%. this was associated with inhibition of jnk activity in both maternal uterus and fetal brain. in addition, we have found that treatment with lps significantly up-regulated cox-2, cxcl1 (il-8 equivalent) and ccl2 in myometrium and this is significantly suppressed after co-administration of d-jnki. we conclude that specific inhibition of jnk signaling may have a potential of controlling preterm labor and preventing fetal brain damage as a result of infection/inflammation. the prostaglandin 15-deoxy-12,14 -prostaglandin j 2 delays lps-induced preterm delivery and reduces mortality in the mouse. intrauterine infection is a common trigger for preterm birth, and is also a risk factor for the development of neurodevelopmental abnormalities in the neonate. bacterial lipopolysaccharide (lps) binds to toll-like receptor-4 (tlr4) to activate pro-inflammatory signaling pathways. the transcription factor nuclear factor kappa b (nf-kb) is a key player in the orchestration of the inflammatory response and has a central role in parturition. we have previously shown that exposure to the anti-inflammatory cyclopentenone prostaglandin 15-deoxy-12,14 -prostaglandin j 2 (15d-pgj 2 ) inhibits il-1b-induced nf-kb activity and cox-2 expression in human myometrial and amnion epithelial cells in vitro. in the present study, we used a mouse model of intrauterine infection-associated preterm labor to determine whether the administration of 15d-pgj 2 can (i) inhibit nf-kb in vivo, (ii) delay lps-induced preterm delivery, and (iii) improve neonatal outcome in the presence of intrauterine inflammation. intrauterine administration of tlr4 specific lps to cd1 pregnant mice at 16 day of pregnancy caused preterm delivery after 18 to 24 h with 70% pup mortality. co-administration of 15d-pgj 2 with lps to pregnant mice delayed significantly (p<0.008) lps-induced preterm delivery and conferred protection from lps-induced fetal mortality up to 5%. we have looked at the expression profile of several labor associated genes in myometrium 6 hours after lps administration. (otr, connexin 23 and 46, cox-1, cox-2, cxcl1 (il-8 equivalent) and ccl2). we have found that treatment with lps significantly up-regulated cox-2, cxcl1 and ccl2 and this is significantly suppressed after with co-administration of 15d-pgj 2. western analysis for ser536-phosphorylated p65 and ikkb in-vitro kinase assay has demonstrated that lps induced activation of nf-kb at both 1 h and 6 h. co-administration of 15d-pgj 2 was associated with inhibition of nf-kb activation in both the maternal uterus and the fetal brain. conclusion 15d-pgj 2 may have potential as a therapeutic agent in the management of preterm labor and, by targeting the player nf-kb, may have the added advantage of preventing detrimental effects to the fetus that may result from infection/inflammation. synergistic macrophage response to co-activation of tlr-2 and tlr-3: mechanisms and implications for bacterial/viral co-infection. vladimir ilievski, 1 emmet hirsch. 1,2 1 department of ob/gyn, evanston northwestern healthcare, evanston, il; 2 department of ob/gyn, feinberg school of medicine, northwestern university, chicago, il. background: toll-like receptors (tlrs) recognize structural components of pathogens and initiate host defenses. tlr-2 responds to gram-positive organisms and peptidoglycan (pgn), a gram-positive cell wall constituent. tlr-3 is activated by viral infection in response to double-stranded rna. polyinosinic-cytidylic acid (poly(i:c)) is a tlr-3 ligand. we have shown that pgn and poly(i:c) have a synergistic effect on the expression of downstream genes for both tlr-2 and tlr-3. here we identify mechanisms underlying this synergy. methods: mouse peritoneal macrophages or a mouse macrophage cell line (raw 264.7) were treated in tissue culture with either pgn (1 g/ml), poly(i: c) (10 g/ml) or both pgn and poly(i:c) either simultaneously or sequentially for 5-10 hours. total rna was extracted and duplex rt-pcr was performed for inducible nitric oxide synthase (inos), interleukin 1 (il-1 ), tumor necrosis factor (tnf), the chemokine rantes and tlr-2, normalized to the housekeeping gene gapdh. results: compared to stimulation with either pgn or poly(i:c) alone, costimulation of raw 264.7 cells with both pgn and poly(i:c) resulted in synergistic expression of inos, il-1 , tnf and rantes (p<0.05 for all) at 5 and 10 hours . sequential stimulation with either pgn or poly(i:c) for 5h followed by incubation for an additional 5h with the alternate ligand also induced synergistic expression of the same rnas, albeit at lower levels than were elicited by simultaneous stimulation. in contrast, incubation with either pgn or poly(i:c) for 5h followed by medium for 5h induced minimal to no gene expression. both pgn and poly(i:c) induced tlr-2 mrna after 5h but not 10h. tlr-3 mrna was not detectable by rt-pcr. in primary peritoneal macrophages, similar synergy due to pgn and poly(i:c) was seen. conclusions: simultaneous or sequential exposure to pgn and poly(i:c) exerts a synergistic effect on the expression of inflammatory mediators in macrophages. interestingly, either one of these two tlr pathways can prime cells for activation of the other pathway. a possible mechanism for this effect may be induction of tlr-2 by either tlr-2 or tlr-3 activation. these observations have implications for bacterial/viral co-infection. this is a secondary analysis of a prospective cohort study. after irb approval, daily blood samples were obtained from pprom subjects and analyzed for il-6 by elisa. paired maternal serum il-6 levels from 34 subjects were divided into 2 groups: il-6 levels obtained 12-120 hours prior to completion of antibiotics and those obtained 12-120 hours after completion of antibiotics. the wilcoxon signed rank test was used to perform the data comparison on the analyze-it statistical software program. statistical significance was defined as p<0.05. of the 34 pprom subjects, the maternal age was 26.8 yrs; gestational age at admission was 26.7 weeks; latency was 13.1 days; gestational age at delivery was 30 weeks; and infant birth weight was 1409 grams. median maternal serum il-6 levels obtained off antibiotics were significantly higher when compared to those on antibiotics (4.9 vs. 2.7 pg/ml, p<0.02). the results of this investigation suggest that maternal serum il-6 levels rise after discontinuation of antibiotics. the optimal duration of antibiotics administration in the setting of pprom is unknown. this data suggests a role for continuation of antibiotics in women with pprom in order to prolong the latency period and potentially decrease neonatal morbidity. to identify clinical and pathologic factors associated with fetal inflammatory responses in the placenta from term parturients. methods: retrospective cohort study of consecutive term parturients with submitted placentas in 2005. placentas with histologic chorioamnionitis were divided into two cohorts: group 1-maternal inflammatory responses only, and group 2-maternal and fetal inflammatory responses. maternal and fetal inflammatory responses in the placenta were classified according to guidelines established by the amniotic fluid infection nosology committee of the perinatal section of the society of pediatric pathologists. selected demographic, intrapartum, newborn and placental characteristics were analyzed with t-tests and chi-square tests as appropriate. multiple logistic regression was used to identify independent predictors of fetal inflammatory responses in the placenta. results: of 351 consecutively submitted placentas, 210 had histologic chorioamnionitis: 155 with maternal inflammatory responses only (group 1) and 55 with both maternal and fetal inflammatory responses (group 2). fetal inflammatory responses observed in group 2 were associated with higher stages of maternal inflammatory responses (p<.001). 80% of fetal inflammatory responses were stage i (acute chorionic vasculitis or phlebitis).group 2 patients were more likely to have had amnioinfusion (29% v 13%, p=.006) and less likely induction of labor (22% v 39%, p=.02). group 2 was more likely to have had intrapartum fever (56% v 40%, p=.04) and maternal tachycardia (58% v 39%, p=.02). newborns from group 2 were more likely to have been observed for sepsis (69% v 37%, p <.001) and have an apgar score 6 at 5 minutes (7% v 1%, p=.02). a logistic regression model showed that stage ii or greater maternal inflammatory responses (or 6.3) and amnioinfusion (or 2.9) were independent predictors of fetal inflammatory responses. conclusion: higher stages of maternal inflammatory responses in the placenta and amnioinfusion were independent predictors of fetal inflammatory responses in term parturients with histologic chorioamnionitis. objective: previous studies have shown that sulfasalazaine (sasp) inhibits lipopolysaccharide (lps)-induced nf-b activation and decreases lpsstimulated interleukin-8 (il-8) production by cultured explants of placenta, amnion and choriodecidua with no effect on cell viability. bacteria-induced il-8 production in the cervix is a potential mechanism for premature cervical ripening that may lead to preterm birth. it is unclear, however, whether sasp can inhibit il-8 production by endocervical cells. therefore, we performed a series of in vitro studies to determine if sasp inhibits il-8 production by endocervical epithelial cells stimulated with bacterial pathogens associated with preterm birth. methods: human endocervical epithelial cells were incubated with 0-1.6 mm sasp overnight and then stimulated with 9 ccu/ml ureaplasma parvum, 10 8 cfu/ml escherichia coli, or 2 x 10 6 cfu/ml gardnerella vaginalis for another overnight incubation in 96-well cultures. conditioned medium was then harvested and production of il-8 was quantified by elisa. viability of the cells was ascertained at the end of the experiment with the mtt-assay. each treatment was applied in quadruplicate wells and experiments were repeated 3 times using different batches of cells for each pathogen. results were evaluated using the general linear models procedure of sas for a randomized block design. results: sasp had no detectible effect on il-8 production by endocervical cells not treated with bacteria. at the highest concentration tested (1.6 mm), sasp significantly inhibited il-8 production by cultures stimulated with e. coli (p<0.001), u. parvum (p<0.001), and g. vaginalis (p<0.001). viability of the cells, however, was significantly reduced by sasp at 0.8 and 1.6 mm in the both the presence and absence of bacteria for all experiments. conclusion: although high concentrations of sasp inhibit il-8 production by cultures of endocervical cells stimulated with pathogens associated with preterm birth, this effect may be due to toxicity of the antibiotic on the cells. the effect of 17 -hydroxyprogesterone caproate on lipopolysaccharide stimulated peripheral blood mononuclear cells from pregnant women. richard h lee, aimin li, frank z stanczyk, jinwen i lin, t murphy goodwin. obstetrics and gynecology, university of southern california, los angeles, ca, usa. introduction: 17 -hydroxyprogesterone caproate (17-ohpc) reduces the rate of recurrent preterm birth in high-risk women. however, there are lines of evidence to suggest that 17-ohpc alters inflammatory response in the setting of gram-negative infection. in a mouse model, 17-ohpc decreased the rate of preterm birth but was associated with significantly increased maternal morbidity when mice were exposed to lipopolysaccharide (lps). in non-pregnant women, 17-ohpc pre-treatment of whole blood exposed to lps significantly increased the production of tnf-. objective: to determine if 17-ohpc has an effect on the production of proinflammatory cytokines from peripheral blood mononuclear cells (pbmc) in pregnant women. methods: pbmc were isolated from fresh whole blood samples of 10 pregnant women between 16 and 24 weeks. pregnant women had no prior history of preterm birth. cells were treated with 17-ohpc (1 m) and escherichia coli lipopolysaccharide (lps, 1 g/ml) alone or in combination. after 6 and 24 hours of culture, supernatants were collected and tested for tnf-and il-6 levels by enzyme-linked immunosorbent assay (elisa). statistical analysis was performed using non-parametric tests. p < 0.05 was considered significant. results: pbmc exposed to lps significantly increased tnf-and il-6 secretion compared to untreated controls (p=0.008 and p=0.008). tnf-concentrations were not significantly different between lps and lps+17-ohpc treated cells at both 6 and 24 hours (p=0.86 and p=0.17). il-6 production was significantly increased after 6-hour treatment with lps+17-ohpc compared to lps alone (2,730 [2,150-5,090] background. recent clinical trials indicate that progestins reduce the incidence of preterm birth in some high risk pregnancies. it has been proposed that progesterone promotes uterine quiescence, in part, via its antiinflammatory properties with inhibition of pro-inflammatory gene expression. it is intriguing that progestins are clinically effective given the considerably increased background circulating levels of the hormone during pregnancy. we hypothesised that non-classical progesterone signalling pathways contribute towards mediation of the anti inflammatory effects of progestins. to the endotoxin lipopolysaccharide (lps) by activation of inflammatory pathways. myometrial cell cultures were treated with lps (1 g/ml)+/progestin (50nm). two progestin compounds were investigated. natural progesterone (p) is known to have a strong affinity with progesterone receptor (pr) analogues in contrast to 17-hydroxyprogesterone (17-hp) which, in the absence of esterification with caproate or acetate, has been reported to have no progestogenic activity at pr. the effect of p and 17-hp on the activation of two inflammatory genes known to be associated with labour (cycloxygenase 2 [cox-2], toll-like receptor 4 [tlr-4]) was evaluated. cox-2 and tlr-4 were detected at the protein and mrna levels using sds-page and rt-pcr. results. lps-induced expression of cox-2 and tlr-4 proteins were significantly inhibited by both p (p<0.01 and p<0.05, respectively) and 17-hp (p<0.01 and p<0.05, respectively). furthermore, preliminary results indicate that co-incubation with the anti-progesterone mifepristone, fail to abrogate the anti inflammatory effect associated with progestin treatment. conclusion. non-classical progesterone signalling pathways have a role in mediating the anti-inflammatory properties of progestins. further elucidation of the molecular actions of progestins may allow novel approaches to ameliorate the inflammatory response associated with preterm labour. detection and transcriptional expression of tlr-2, tlr-4 and tlr-9 at the maternal-fetal interface. jacqueline p tilak, karen zakharian, alexandra tungol, gabriel o schubiner, david m svinarich. patient care research, providence hospital, southfield, mi, usa. preterm labor and delivery remains a leading cause of neonatal morbidity and mortality and bacterial infection is considered to be the most common etiology. toll-like receptors (tlr's) and the associated components of the innate immune system may represent a first line of defense against these pathogens. tlr's belong to a family of pattern-recognition receptors that bind to highly conserved pathogen-associated molecular patterns (pamps) including lipopolysaccharide (lps), lipotechoic acid (lta) and cpg dna, and are a key component of the innate immune system. this study was undertaken to characterize the transcriptional expression and regulation of tlr-2, tlr-4 and tlr-9 within gynecologic and gestational tissues. human first trimester trophoblasts, endometrial mesoderm, ectocervix, choriocarcinoma and neonatal epithelium, were cultured in media alone or in the presence of either lps (1mg/ml) or lta (10 mg/ml) for 0, 2, 4, 6, 8 and 24 hours. total rna was isolated and semi-quantitative rt-pcr was performed using intron-spanning amplimers to tlr-2, tlr-4, tlr-9 and gapdh. following gel electrophoresis, the integrated optical densities were determined for the corresponding pcr products and normalized with respect to gapdh levels. inducible transcription of tlr-2 with lta was observed in choriocarcinoma cells (6-fold, 2h), and endometrial mesoderm cells (19-fold, 4h). tlr-4 induction with lps was observed in ectocervical cells (4-fold, 4h), choriocarcinoma cells (4-fold, 4h) and endometrial mesoderm cells (7-fold, 24h). tlr-9 induction with lps was observed in choriocarcinoma cells (32fold, 4h) and neonatal epithelial cells (11-fold, 4h). all cell lines showed at least constitutive levels of transcription for tlr-2, tlr-4 and tlr-9. these data demonstrate that tlr-2, tlr-4 and tlr-9 are transcriptionally expressed either constitutively or inducibly in both gynecologic (endometrial mesoderm, ectocervix) and gestational (chorion, trophoblast), tissues. translation of these receptors suggests that the innate immune system may function at the maternal-fetal interface to help protect the fetus against infection and prevent or diminish the likelihood of prematurity. objective: further to our development of a sheep model of intrauterine ureaplasma spp infection, we aimed to examine the capability of ureaplasma colonization in the amniotic fluid to infect the fetus and alter lung and brain development. methods: at 50 days of gestation (d, term=150 d) ewes bearing single fetuses were given a single ultrasound-guided intra-amniotic injection of (a) 2x10 7 colony forming units (cfu) of u parvum (serovar 3, n=7; serovar 6, n=8), (b) 2x10 4 cfu of u parvum (serovar 3, n=6; serovar 6, n=8) or (c) media control (n=6). at 125 d, fetuses were delivered by cesarean section. amniotic fluid and umbilical arterial blood samples were collected. fetal body weight was recorded, fetal cerebrospinal fluid (csf) collected and a descending pressurevolume curve constructed after inflation of the lungs to 40 cmh 2 o. fetal membranes were immersion fixed and the fetal brain was perfusion fixed with 4% paraformaldehyde. the fetal brain and membranes were blocked, paraffin embedded, stained and viewed under the light microscope. results: chronic intra-amniotic colonisation with u parvum serovar 3 or 6 (ureaplasmas) did not result in fetal abortion or death. amniotic fluid ureaplasma titers at delivery were not dose-dependent. chronic ureaplasma exposure did not affect fetal body or brain weights, or result in a fetal systemic inflammatory response. umbilical arterial blood gases at delivery were similar between ureaplasma-and media-exposed fetuses. chronic intra-amniotic exposure to ureaplasmas resulted in higher inflammatory cell scores in the fetal membranes compared to media controls (p<0.05). lung compliance was increased in ureaplasma-exposed fetuses compared to controls (p<0.05). there were no gross anatomical changes observed in the white or grey matter in the cerebral hemispheres of fetuses that had been exposed to ureaplasmas; even in animals (n=3) that had csf ureaplasma colonisation. conclusion: chronic ureaplasma colonisation enhances fetal lung compliance without gross anatomical changes in the fetal brain. background: an important source of vitamin d is its synthesis by skin from uv solar radiations. the skin pigment melanin absorbs uv photons thus reducing the vitamin d synthesis by more than 90% making african americans at high risk for vitamin d deficiency. low maternal vitamin d status during pregnancy has been linked to molecular pathways for adverse outcomes. objective: the nf b transcription factor regulates genes involved in inflammation and immune processes, and is proposed to play a major role in the successful outcome of pregnancy and labor. prior immunohistochemical (ihc) and biochemical studies have been conflicting regarding nf b activation in human intrauterine tissues. the aim of this study was to quantify nuclear localization of p65, the major tranactivating nf b subunit, in full-thickness fetal membranes (fm) and myometrium using ihc. methods: a retrospective analysis of paired fm, decidua, and myometrial samples was conducted using tissues collected from women in 4 cohorts: preterm no labor (pnl, n=20), preterm labor (ptl, n=20), spontaneous term labor (stl, n=19), and term no labor (tnl, n=22). subjects were delivered between gestational ages 26-36 weeks (preterm) and 37-44 weeks (term) by cesarean. paraffin sections were stained with polyclonal (rabbit) anti-human p65 and microscopically examined. myometrial samples were categorized in a blinded fashion to 3 groups of staining: no nuclear p65 (-), rare nuclear p65 (+), and >50% nuclear p65 (++). a p65 nuclear labeling index (nli; % nuclear p65/total cells) was calculated for each histological layer in full-thickness fm specimens using a blinded targeted randomization scheme consisting of 5 non-overlapping low-magnification fields. results: nuclear p65 labeling was rare in amnion and inconsistently present in chorion. in decidua, p65 nuclear labeling was observed in all cases; however, decidual nli did not vary significantly across cohorts [pnl-40% (24-55%); ptl-45% (39-57%); tnl-37% (25-43%); stl-41% (30-59%); all values are median (iqr)]. in myometrium, ++ p65 nuclear labeling was significantly associated with labor, but present only in a portion of cases (ptl-26%; stl-50%). despite a trend, decidual nli was not significantly correlated with myometrial nuclear p65 labeling: myometrial specimens classified as -, +, and ++ corresponded with decidual nli of 40% (32-55%) [median (iqr)], 37% (28-47%), and 56% (40-58%), respectively. conclusions: in a comprehensive ihc analysis, significant nuclear p65 immunolabeling was observed in myometrial cells following labor. nuclear p65 labeling in decidua was present in all cases, and did not vary significantly among the cohorts. the inflammatory cytokines interleukin-1 and tnf-increase g-csf expression in term decidual cells. objectives: chorioamnionitis (cam) elicits premature rupture of the membranes and pre-term delivery. previously, we found that the decidua from cam patients contains much higher neutrophil numbers than control decidua, but macrophage numbers are similar in both groups. granulocyte colony-stimulating factor (g-csf) enhances granulocyte colony formation chemoattraction and activation. the amniotic fluid during cam contains elevated tnf-and il-1 levels. to account for the marked influx of neutrophils infiltration in cam-complicated decidua, tnf-and il-effects were assessed on g-csf expression in term decidual cell (dc) monolayers. methods: confluent leukocyte-free term dcs from normal term deliveries were primed with 10 -8 m estradiol (e2) + 10 -7 m medroxyprogesterone acetate (mpa) for 7 days, then switched to serum-free defined medium (dm) with steroids ± tnf-or il-1 . after 24h, aliquots of conditioned dm supernatants, cell lysates and extracted rna from 6h parallel incubations were frozen. secreted g-csf was measured by elisa in conditioned dm and normalized to cell protein and mrna was assessed by quantitative real time rt-pcr and normalized to -actin mrna. results: in cultured first trimester dcs, basal g-csf levels in conditioned dm were 0.13± 0.06 pg/ml/ug cell protein [mean ± sem, n=8] and did not differ from e2+mpa basal levels. the addition of 1ng/ml of tnf-or il-1 significantly elevated g-csf output to 2.22 ± 0.97 and 454 ± 122 respectively (p<0.05), representing more than a 10-fold and 4,000-fold change; respectively. western blotting confirmed the elisa results. quantitative rt-pcr demonstrated corresponding changes in g-csf mrna levels as found for the elisa measurements. concentration-dependent effects for both tnfand il-1 from 0.01 to 10.0 ng/ml were observed. conclusions: when extrapolated to the placental milieu of cam, the marked increase in g-csf elicited in term dcs by tnf-and il-1 may provide a mechanism to account for the selective increase in decidual neutrophils versus macrophages. background. the immunological mechanisms of the female reproductive tract are unclear. toll-like receptors (tlrs), innate immune receptors that combat microbial infections and establish adaptive immunity, may play a role. infection in pregnancy has been associated with preterm birth and tlrs may modulate the host response to such infections. we hypothesised that the distribution of tlr2 and tlr4 in cervical epithelial cells may differ with pregnancy, and that oestrogen may contribute to the modulation of these receptors. aims and objectives. 3. to compare tlr2 and tlr4 protein expression, using immunocytochemistry, in the cervical epithelium of pre-menopausal non-pregnant women with pregnant women at term. methods. fresh non-pregnant (n=18) and pregnant (n=11) human cervical cells were obtained by smear and tlr2 and tlr4 expression investigated by immunocytochemistry. cervical epithelial cells from nonpregnant women obtained by smears were then coincubated with varying concentrations of estradiol, and tlr2 and tlr4 protein expression quantified by immunocytochemistry. results. using pixel-based image analysis software, we demonstrated a reduction in tlr2 expression in late pregnant compared with non-pregnant cervical epithelium (p=0.0001), whilst tlr4 did not appear to differ. there was an apparent up-regulation of tlr2 protein expression in response to 17beta-oestradiol (n=5) objective: to evaluate in vitro antimicrobial activity of cranberry-exposed urine against common urinary pathogens. subjects were pregnant women enrolled in a clinical trial evaluating the effect of daily cranberry juice cocktail or matching placebo on asymptomatic bacteriuria and other urinary tract infections. methods: 4-hour urine samples from 28 pregnant women who were randomized to cranberry or placebo in three treatment arms: a. cranberry two times daily with meals (c, c; n=10), b: cranberry in the am, then placebo at dinner (c, p; n=10), c: placebo two times daily with meals (p, p; n=8). we identified 15 non-pregnant, reproductive-aged controls, randomized them to the same treatment groups and collected 4-hour urine specimens from them. the ph of all urine specimens was adjusted to ph=7 and filtered. aliquots of each were independently inoculated with overnight culture of 10 2-3 cell/ml each of single strains of e. coli with fimbriae type i and type ii, k. pneumoniae, and c. albicans. after 24 hours of incubation for e. coli and k. pneumoniae, and 48 hours for c. albicans cfu/ml of each specimen were enumerated by subculture with quantitative plate counts in duplicate. results: there were no differences for any of the antimicrobial activities between pregnant and non-pregnant groups, or based on treatment allocation. we were able to perform antimicrobial assays on the urine of women exposed to cranberry juice or placebo, but unable to demonstrate differences based on treatment allocation or pregnancy. this may be due to beta-error. further studies are planned. supported by r21dk65827-01; clinical trials registration nct00506025. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• objective: to measure compliance, we sought to evaluate the use of a bioassay for cranberry in the urine of women enrolled in a clinical trial designed to determine the effect of daily dosing of cranberry juice cocktail or matching placebo on the incidence of asymptomatic bacteriuria (asb) and other urinary tract infections (uti) during pregnancy. methods: we collected 4-hour urine specimens from 34 pregnant women who were randomized to ingest cranberry or placebo in three treatment arms: a: cranberry two times daily with meals (n=11), b: cranberry in the am, then placebo at dinner (n=12), c. placebo two times daily with meals (n=11). we identified 14 non-pregnant, reproductive-aged controls, randomized them to the same treatment regimens (group a: n=6, group b: n=3, group c: n=5), and collected 4-hour urine specimens from them. bacterial anti-adhesion effects of the cranberry-exposed urine were evaluated utilizing a human red blood cell hemagglutination assay specific for uropathogenic p-fimbriated e. coli. activity was quantified as 0, 50, and 100%. results: when combining all subjects and dosing regimens, the sensitivity of the assay was 25%, range 17% in the pregnant group assigned single daily dose of cranberry to 100% in the non-pregnant group assigned to multiple daily doses. the specificity ranged from 63% to 100%. conclusions: these data indicate that the bioassay can be applied to the pregnant patient population, although the sensitivity of the assay is variable. higher daily dosing appears to confer greater sensitivity. further studies are needed to evaluate the utility of this bioassay for compliance. supported by r21dk65827-01. clinical trials registration nct00093938. objective: increasing evidence suggests that inflammation plays a crucial role in initiation of labour both at term and preterm. we have previously shown upregulation of pro-inflammatory cytokines in myometrium, cervix and membranes at term labour. we have also shown that myometrium and cervix is invaded by leukocytes at the time of labour, and these leukocytes express pro-inflammatory cytokines. in this study, we aimed to test the hypothesis that pro-inflammatory cytokines and toll-like receptor 2 and 4 (tlr2 and 4) mrna expression are up-regulated in circulating leukocytes during term and preterm labour. methods: peripheral blood samples were taken from 37 pregnant women: 28-36 weeks gestation (w) preterm not in labour (ptnl) n=10; 28-36 w preterm in labour (ptl), n=7; 37-42 w term not in labour (tnl) n=10; and 37-42 w term in labour (tl) n=10. leukocytes were isolated using dextran sedimentation. total rna was isolated and reverse transcribed using high capacity cdna archive kit, and mrna expression determined by real-time pcr (abi, taqman). student's t-test was used to compare outcomes between groups. results: messenger rna expression of il-8, icam-1, mcp-1, tlr2 and tlr4 was significantly increased in tl compared to gestation matched tnl. the expression levels of il-1b, il-8 and tlr-2 were significantly greater in ptl compared with gestation matched ptnl. (table i) . conclusions: we show up-regulation of pro-inflammatory cytokine production in circulating leukocytes in both term and preterm labour. thus, the pathophysiology of labour seems to involve the upregulation of proinflammatory cytokine production in peripheral blood leukocytes, followed by their invasion into the myometrium and cervix. this work further contributes to our understanding of the pathophysiology of parturition, and suggests that peripheral blood leukocytes may be potential targets for therapeutic agents aimed at modifying the time course of parturition. introduction. the mechanisms of innate immunity and tolerance in the female reproductive tract (frt) are ill-understood but involve the pattern recognition toll-like receptors (tlrs). we have previously demonstrated high expression levels of tlr2 gene and protein in fresh human cervical epithelium. aims. in order to gain insight into the immunological role of cervical epithelial cells (cecs), we sought to determine cec responses to the following tlr-2 agonists: macrophage-activating lipopeptide (malp-2), pam 3 csk 4 , and peptidoglycan. methods. cecs were isolated by smears and compared between 7 pregnant (3 rd trimester) and 7 nonpregnant women. following cell preparation, flow cytometry was performed using a direct staining procedure with mouse anti-human tlr2 primary antibody and its igg 1, isotype control, to determine tlr-2 protein expression. a further 7 nonpregnant smear samples were each prepared, and seeded at a concentration of 100,000 cervical epithelial cells/ml into 24-well cell plates. cells were incubated at 37°c in 5% co 2 overnight with the tlr2 agonists malp-2 and pam 3 csk 4 (at concentrations of 10 and 1000ng/ml), peptidoglycan(50ng/ml), il-1 (10ng/ml, positive control) and rpmi 1640 + 1-glutamine media only (vehicle). following centrifugation, all resulting supernatants were stored at -80°c until the concentration of il-8 was determined by elisa and an array of cytokines by bead assays. results. both pregnant and nonpregnant cecs expressed tlr2 (specific mean fluorescence 4.7 vs 3.3 respectively) but did not differ (p = 0.2). unstimulated cells incubated with buffer alone demonstrated high il-8 levels (6602 pg/ml), which did not differ following stimulation with malp-2 (6217 pg/ml), pam 3 csk 4 (6449 pg/ml) or peptidoglycan (4867 pg/ml). results of an array of pro-and anti-inflammatory cytokines following incubation of cells stimulated with tlr2 agonists are pending. conclusion. high basal il-8 levels suggest constitutive expression by cecs but the physiological relevance of this intriguing observation is unclear. that cec stimulation with tlr-2 agonists did not lead to further release of il-8 may epitomise the resistance of these cells to antigenic challenge by the vaginal commensal flora. cecs may play a pivotal role in modulating the immunological tolerance necessary to minimise inappropriate inflammation in the cervix. there are several epidemiological and clinical studies that omega-3 fatty acids and related fish oils can decrease the premature labor and birth. however, the scientific mechanisms are not well elucidated. this study was carried out to investigate the effects of omega-3 fatty acids on producing pge2 and il-6, in human umbilical vascular endothelial cell(huvec) media with artificial inflammation as an infection-induced premature labor tissue model. also, if there are some significant effects with omega-3 fatty acids, we want to investigate the possible mechanisms. materials and methods; human umbilical vascular vascular cell primary culture was done. in the adequate cell confluence in each cell dish, pretreatment with various concentrations of epa, dha and troglitazone (another ppar-ligand) and incubation were done for 24 hours in 37°c, co 2 incubator. after that, 10 g/ml conc. of lipopolysaccharide(lps) was treated to the each cell dishes and incubated for 8 hours. the cell media were collected, and pge 2 and il-6 concentration were checked by elisa. the each cell lysates were collected and western blot anaysis for cyclooxygenase(cox)-2 were done. on the other hand, nuclear factor kappa b (nf b) luciferase vector was transfected and then did the same pretreatment with epa, dha and troglitazone and lps treatment to each cell dishes. after that, nf b luciferase activity was checked with luminometer. statistical analysis was done by student t-test. results: epa, dha and troglitazone decreased the pge2 (p<0.05) and il-6(p<0.01) significantly in elisa. cox-2 expression revealed the significant reduction with pretreatment of epa, dha and troglitazone in higher concentration (50, 100 m) in the western blotting (p<0.01). nf b luciferase activity were also significantly decresed with pretreatment of epa, dha and troglitazone in higher concentration (p<0.01). conclusion; this study offers some scientific mechanisms, by which omega-3 fatty acids (epa, dha) and troglitazone may be one kind of the preventive medicine for infection-induced preterm labor and delivery. also, these effects may come from the common mechanism of epa, dha and troglitazone, ppar-ligands. the next study would be how the cox-2, nf b and pparare related. (2mg/ml). cytokine expression was measured in medium using the bio-plex suspension array system (bio-rad). mean expression was compared between the two groups using t test. results: 2-aminopurine significantly inhibited lps stimulated cytokine production by human myometrium. in contrast, there were no significant differences in expression after mpa treatment, although a trend towards inhibition of proinflammatory cytokine and an increase in il-10 production was noted. introduction: intrauterine infection is now recognised as a major antecedent of white matter injury (wmi) in the preterm infant brain, which can manifest later as cerebral palsy or as varying degrees of cognitive dysfunction. wmi in these infants is characterized by damage to immature oligodendrocytes, and has been linked both to microglial activation and to elevated levels of tnfa, il-1b and il-6. we have developed a fetal sheep model for diffuse and focal wmi, based on repeated administration of e. coli lipopolysaccharide (lps) as the stimulus for an inflammatory response. methods: surgery to implant fetal vascular catheters was performed on pregnant ewes carrying single fetuses at d89-90 of gestation. fetuses received repeated iv injections of lps (100ng/kg estimated fetal weight/day) between d95 and d99. plasma levels of pro-inflammatory cytokines were determined in fetal arterial blood samples taken between d94 and d104. at d105 ewes and fetuses were euthanized and fetal brain tissue samples collected for histological analysis. results: five days after final administration of lps to fetuses we observed a pattern of wmi similar to that seen clinically, ranging from focal to diffuse injury within the periventricular region, impairment of white matter (cnpase; marker for immature/mature oligodendrocytes) tracts, and thinning of the corpus callosum, characterised by oligodendrocyte disruption and microglial proliferation in the surrounding and sub-cortical white matter. we also found a progressive rise in fetal plasma tnf levels between days 97 and 103 (day two of treatment to third day following final dose of lps). background: plasma fatty acid (fa) levels are associated with altered host inflammatory responses, blood pressure regulation, and insulin resistance, characteristic features of pregnancy-induced hypertension (pih). most studies compare the n-3 and n-6 polyunsaturated fatty acids (pufas). in addition, recent data demonstrate that saturated and trans-fas promote inflammation. based on the immunomodulatory activity of various fas, we explored their effects on placenta inflammatory responses ex vivo. methods: placenta cells were isolated from fresh human (anonymous), term placentas and treated with/without lipopolysaccharide (lps) with various fas, including saturated fas, trans-fas, and n-3 and n-6 pufas (at physiological concentrations). after an overnight treatment, tnf-alpha (tnf) production was determined. data were analyzed by analysis of variance (anova) followed by the dunnett's test. results: oleic acid (18:1n-9), a cis-monosaturated fa common in the mediterranean diet, did not induce significant placenta tnf production (fig. a) . by contrast, elaidic acid (18:1n-9), a trans-monosaturated fa, induced tnf production by 300-fold compared to vehicle (fig. a) . likewise, palmitic acid (18:0), a saturated fa, induced placenta tnf production by 800-fold (fig. a) . to mimic inflammation, placenta cells were treated with lps ex vivo in the presence/absence of fas. docosahexanoic acid (22:6n-3, dha) reduced placenta tnf production by up to 90% following stimulation (fig. b) . similarly, treatment of placenta cells with linoleic acid (18:2n-6, la) or arachidonic acid (n20:4n-6, aa) suppressed tnf production by up to 87% and 98%, respectively (fig. b) . conclusions: both saturated fas and trans-fas, which positively correlate with hypertension, induce placental tnf production. the n-6 fa precursors to prostaglandins, aa and linoleic acid, reduce placental tnf production following stimulation. likewise, dha, a product of n-3 fa metabolism commonly consumed in fish oil and associated with improved blood pressure, suppresses tnf production by stimulated placenta cells. vegf and flk1 mediated glomerulogenesis in offspring exposed to maternal hypernatremia. roy z mansano, mina desai, ahmed abdel-hakeem, thomas r magee, tazmia q henry, cynthia nast, john s torday, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: growth restricted human and animal offspring, resulting from maternal nutrient restriction or uteroplacental insufficiency, consistently demonstrate reduced glomerular number and a predisposition to adult systemic hypertension and renal compromise. in contrast, maternal water restriction (wr) produces newborns with a unique programmed phenotype of increased glomerular number. glomerulogenesis is dependent, in part, on appropriately timed and quantified vascular development. as vegf and its receptor flk1 have crucial stimulatory effects on renal vasculogenesis, we hypothesized that maternal wr-induced morphologic renal changes are secondary to vegfmediated vasculogenic effects. methods: from day 10 to term gestation, pregnant rats received either ad libitum food and water (control, n=7), or wr to produce an increment of 6 meq/l in plasma sodium (n=7). following delivery, all dams received ad libitum food and water. at d1 after birth, offspring kidneys were extracted for mrna. vegf and its receptor, flk1, mrna levels were determined using real-time rt-pcr (presented as fold difference normalized to 18s). at d21 after birth, offspring glomerular number were determined in formalin fixed 5 m sections using histomorphometric analysis. values are means±se. results: wr offspring (day 21) had higher glomerular number than control (wr 25±1, control 22±1 per mm2, p<0.01). flk1 mrna expression was increased in wr offspring kidneys (wr 6.4±2.3, control 1.0±0.3, p<0.05) as compared to controls. in contrast, vegf mrna expression in wr offspring kidney was comparable to control (wr 0.9±0.1, control 1.0±0.2, p=0.8). conclusion:prenatally wr offspring demonstrate significantly increased glomerular number. as vegf recruits angioblasts to the developing glomerulus via its receptor flk1, increased receptors (flk1) with the same level of the ligand (vegf) suggest that enhanced vasculogenesis may represent a putative mechanism for increased nephrogenesis in wr offspring. modulation of newborn vasculogenesis via vegf and flk1 expression may represent a therapeutic option for growth restricted offspring with decreased glomerular number. objective: maternal food restriction (mfr) during gestation (embryonic day 10 to birth) reduces rat kidney glomerular number by 19% at 3 weeks of age. undernutrition during human gestation leads to similar impaired nephrogenesis and increased hypertension in adults. renal development may be delineated into stages including ureteric bud branching, mesenchymal to epithelial transformation and glomerulogenesis. previously, we detected dysregulation of genes controlling nephrogenesis at gestational ages, e16-e20, suggesting that mfr has significant effects on ureteric bud branching. in the present study we evaluated the effect of this dysregulation on early nephron development in e16 fetal kidney explants from dams with mfr. methods: e16 fetal kidneys were collected from 50% mfr pregnant rats and ad libitum control rats (n=3 per group and time point), incubated in dmem/f12k medium for 0, 1, 2, 3, and 4 days, and fixed with 4% paraformaldehyde. kidneys were stained with fluorescein-labeled dolichos biflorus agglutinin (dba), images captured and terminal ureteric buds quantitated. all values are presented as mean ± sem. differences were considered significant at p<0.05. results: mfr ex-vivo kidneys at day 0 demonstrated an initial moderate reduction in terminal branches versus control (c) samples (mfr: 78±9 vs c: 86±3 terminal branch ends per kidney). both mfr and control (c) kidneys demonstrated significant (p<0.05) increases in branching in explant culture to maximum values at 4 days (mfr: 147±16 vs c: 217±16). with increasing culture days, the percent reduction in mfr branching increased with terminal branch point decreases of 9% at day 0, 14% at day 1, 24% at day 2, 20% at day 3, and 32% at day 4 (p<0.05, mfr vs c at day 4). conclusion: kidney explant cultures from mfr treated fetuses display basal and culture-based decreased branching compared to controls. this decrease in terminal ureteric buds, in combination with our previous findings of dysregulation of branching-associated kidney transcription and growth factors, suggests mfr induces early (e16) dysregulation of branching as a major mechanism of the associated nephropenia. these results indicate that early programming events in kidney development induce nephropenia and renal disease in adults. intrauterine growth restriction (iugr) has important implications for the neonate not only at the time of birth, but also as an adult. in humans and animals with iugr, the elevated risk of developing hypertension is thought to involve an upregulation of the renin-angiotensin system (ras). however, the link between the intrauterine insult and enhanced ras is not known. a novel mechanism leading to hypertension has emerged recently involving two orphan g-protein coupled receptors (gcr91 and gcr99) and their endogenous ligands, succinate and -ketoglutarate. infusion of succinate into adult mice induces hypertension, an effect which was eliminated in gcr91 knockout mice or by pretreatment with ace inhibitors. interestingly, succinate levels have been shown to increase in the circulation under conditions of oxidative stress, one of the hypothesized mediators of developmental programming of hypertension. thus, we hypothesized that gcr91 and gcr99 are upregulated in a rat model of iugr and may contribute to in utero programming of hypertension. timedpregnant rats were fed either control (c, 20% casein) or low protein (lp, 8% casein) diet throughout gestation. kidneys were collected on embryonic day 19 (e19), post-natal day 30 (d30) or 130 (d130), n 3. real-time pcr was used to compare gcr91, gcr99 and renin expression. data were standardized to a housekeeping gene (s15) and are expressed as fold increase/ decrease compared to control. a student's t-test was used to determine significance between groups (p<0.05). offspring from lp dams were significantly smaller at birth and did not display catch-up growth. in kidneys from lp fetuses, both gcr91 and gcr99 were significantly elevated compared with controls (3.6 fold and 4.7 fold respectively; p=0.02), whereas renin was 0.78 fold lower. on post-natal d30, there was a trend towards increased expression of both gcr91 (1.7 fold; p=0.07) and renin (1.6 fold; p=0.09) in offspring from lp dams. at d130, there were no significant differences between groups. in conclusion, these preliminary data suggest that in iugr offspring from lp rats, the gcr91/ 99 pathway may be enhanced, indicating a novel mechanism for the programming of hypertension. objective: in addition to excess adipose tissue, obesity is accompanied by increased fat storage in organs such as the liver. peroxisome proliferatoractivated receptors (ppars) are ligand-activated transcription factors involved in the regulation of lipid metabolism, and lipid-associated inflammatory response. obesity represents a state of chronic low-level inflammation, with ppar and ppar implicated in this process. we have previously shown that nutrient restriction in pregnancy results in intrauterine growth restricted (iugr) newborns which develop adult obesity with elevated c-reactive protein (crp) levels. as crp is derived from the liver, we hypothesized that iugr-induced obesity inhibition of hepatic ppar and ppar is associated with an increased inflammatory response. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21 to produce iugr newborns. all pups were nursed by control dams and weaned at 3 weeks to ad libitum feed. at 1 day and 9 months of age, male offspring were analyzed for hepatic ppar , ppar and crp mrna (real time rt-pcr) and protein (western blot) expression. data was normalized to -actin and presented as fold change for protein levels. at 9 months, hepatic triglyceride content was determined enzymatically. results: at 1d of age, iugr pups showed significant downregulation of ppar (0.2-fold) and ppar (0.4-fold) expression, though crp expression was significantly upregulated (4-fold). findings persisted at 9 months of age, with continued downregulation of ppar and ppar (0.5-fold) and upregulation of crp expression (5-fold). furthermore, iugr adults had significantly increased hepatic triglyceride content (600±60 vs. 373±28 mg/g liver, p<0.05). conclusions: reduced expression of hepatic ppar and ppar in iugr offspring may contribute to elevated hepatic crp levels and triglyceride content. thus, developmental hepatic dysregulation leads to programmed obesity-induced inflammation in iugr offspring. offspring. mina desai, 1 ederlen casillas, 1 guang han, 1 darran n tosh, 1,2 michael g ross. 1 1 dept. of ob/gyn, labiomed at harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of physiology, univ. of adelaide, australia. objective: leptin and insulin mediate central anorexigenic signaling responses via different receptor molecules: leptin binds to the obrb receptor activating jak-stat3 and pi3k pathways, whereas insulin activates pi3k pathway by binding to its receptor (ir) and substrate (irs2). maternal food restriction in pregnancy results in iugr newborns that develop hyperphagia and adult obesity. the iugr newborns have significantly decreased plasma leptin levels with increased hypothalamic expression of basal obrb, stat3 and decreased expression of ir and irs2, suggesting altered anorexigenic pathways. we studied the response of hypothalamic leptin/insulin signal molecules to peripheral leptin in iugr newborns. methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to 21. 1 day old male offspring were given saline or leptin (1 g/g, i.p). hypothalamus was dissected at 15, 30 and 45 minutes and protein expression of total stat3, phosphorylated stat3 (p-stat3), inhibitor of leptin signal (socs3), ir, irs2, total akt and phosphorylated akt was determined by western blot (normalized to -actin). data is compared between leptin and saline treatments in iugr and controls. results: in response to peripheral leptin, iugr newborns showed marked dysfunction in stimulated hypothalamic leptin and insulin signaling responses. (1) jak-stat3: leptin-treated controls show progressively increased pstat3 (4-fold) with initial suppression of socs3 (0.5-fold) as compared to salinetreated controls. conversely in leptin-treated iugr, the pattern is reversed such that there is sustained decline in pstat3 expression (0.3-fold) with failure to downregulate socs3. (2) pi3k pathway: leptin-treated controls showed a significant reduction in irs2 (0.3-fold) and pakt (0.5-fold) as compared to saline-treated controls. however, leptin-treated iugr newborns exhibited a paradoxical increase expression of irs2 (3-fold) and pakt (2-fold). conclusion:the iugr offspring demonstrate persistent upregulation of leptin receptor, a reduced phosphorylated stat3 (p-stat3) response in conjunction with an enhanced socs-3 response. the persistent increase in insulin responses indicates a dysfunction in dynamic signaling, leading to altered anorexigenic response and development of programmed obesity. we have previously demonstrated that maternal food restriction (mfr) in rats induces a marked increase in the expression of vegf protein in the aorta and mesenteric arterioles, accompanied by an increase in tgf-and collagen in both vessel types in adult rat offspring (am j physiol, 2007) . the aim of this study was to determine if this in vivo finding could be reproduced in an in vitro preparation. methods: two types of preparations were used in this study. we isolated endothelial cells from 3 week old male control rat aortas. these cells were used after the third passage. staining with von willerbrand factor demonstrated that these cells were pure endothelial cells. the second type of preparation was aortic explants from 3 week old male control rats. endothelial cells and aortic explants were transfected with a vegf adenovirus (106-109 viral infective particles) or a -galactosidase-adenovirus as a control. after 24 hours of culture protein was isolated from cells and explants for western blot analysis using rat specific antibodies. culture media was assayed for vegf by elisa. results: transfection of vegf adenovirus induced a dose-dependant increase in the expression of vegf protein in primary endothelial cells and aortic explants. the transfection of vegf into the endothelial cells showed a bell shaped curve, and was accompanied by an increase in media levels of vegf protein. maximal secretion of vegf was found with 107 viral infective particles. vegf adenovirus transfection induced a dose-dependant increase in c-reactive protein (crp) (inflammatory marker), and tgf-protein in both aortic explants and primary endothelial cells. these results indicate that upregulation of vegf in blood vessels induces inflammation and tgf-expression which in turn can induce collagen synthesis. thus the increased collagen expression and reduced compliance previously reported by us in vessels of mfr offspring can be explained by the over-expression of vegf which we reported. therapeutic intervention aimed at prevention of the increase in vascular vegf expression in mfr offspring could potentially prevent programmed hypertension. maternal regulation of high fat nourishment during lactation period reduce a hypertension of male offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med.univ., fukushima, fukushima, japan. objective: exposure to undernutrition or high fat nourishment during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of maternal feeding regulation during lactation period on blood pressure of offspring is unclear. our objective was to investigate the effects of either high-fat diet (hfd) during gestation to lactation period or restrictive fed a hfd during lactation period on blood pressure in male rat offspring. we use 3 types pregnant wistar rats as fed with normal nutrition (group a), with a high fat diet (hfd) during gestation to lactation period (group b) and with hfd nutritionally restricted by feeding with 30% of the normal lactationmatched dietary intake from the day of delivery to the end of lactation period (group c). the male offspring was measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using oneway annova. results: body weight was significantly reduced in c offspring compared to a and b in male offspring at day 28 after delivery (p<0.01). at 12 weeks old, the body weight of c offspring was no difference to catch up compared to a and b offspring. systolic and diastolic blood pressures were significantly elevated at all 12, 24 and 60 weeks in offspring of b > c >a. (p<0.01, vs. a) conclusions: under high-fat nutrition during gestation to lactation period induced hypertension in male rat offspring. maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. background: uterine artery (uta) doppler velocimetry has been validated in populations of heterogeneous parity in the second trimester for prediction of obstetric outcomes requiring preterm delivery to include: fetal growth restriction, fetal demise, hypertensive disorders of pregnancy, abruption, and indicated preterm delivery. understanding that parity may affect uta doppler indices in subsequent pregnancies, we sought to validate these predictive values in the first trimester in a homogeneous population of multiparous women. study design: multiparous women undergoing first trimester screening of singleton pregnancies were enrolled and followed prospectively until delivery (n=91). these women were divided into controls, ri < 0.78 (n=74) and cases, ri 0.78 (n=17), based on prior studies. demographic, clinical, and sonographic data (including uta indices and assessment of notching) were obtained. statistical analysis included student's t test and chi square. results: cases were not significantly different from controls in terms of maternal age, ethnicity, bmi, or medical history. uta doppler indices were significantly different between the two cohorts in terms of the presence of unilateral or bilateral notching (48% vs, 55% p<0.001 and 22% vs. 68%, p<0.001, respectively). in contrast to that observed in patients of heterogenous parity previously, ri 0.78 was not associated with adverse pregnancy outcomes despite an average ri of 0.86, significantly above this threshold. conclusions: in this multiparous cohort ri was not predictive of adverse obstetrical outcome, in contrast to that observed in cohorts including nulliparous patient. parity may affect uta vasculature and obscure the ability of doppler velocimetry to predict adverse obstetric outcome in multiparous women. presence of uta doppler notching in the first trimester remained a robust predictor of adverse obstetric outcomes in multiparous patients. objective: epidemiological studies have shown that offspring exposed to preeclampsia during fetal development are more susceptible to airway disease later in life. we have shown previously that gender, but not sflt-1 over-expression during pregnancy determines higher reactivity in the offspring airways at 6 months of age. the objective of this study was to examine the effect of preeclampsia on the trachea from female and male offspring in our model of sflt-1 induced preeclampsia at 1 year of age and compare responses between the two age groups. methods: cd-1 mice at day 8 of gestation were injected via the tail vein with adenovirus carrying sflt1 (adsflt1, 10 9 pfu/100 l) or mfc (admfc, 10 9 pfu/100 l). mice were allowed to deliver. tracheas were isolated from female and male offspring at 6 months and 1 year of age, and rings were mounted in organ chambers for isometric tension recording. responses to potassium chloride (kcl, 60 mm), the mast cell degranulating agent compound 48/80 (48/80, 100 g/ml), and concentration-responses curve to acetylcholine (10 -9 -10 -5 m) were obtained. results: there was no significant difference in responses to acetylcholine, kcl, or compound 48/80 between 1 year old offspring born to the sflt1 and mfc groups. when comparing offspring within the same pregnancy exposure groups, responses to acetylcholine in adsflt1-treated group were significantly higher in 1 year old females than males. comparison between age groups by pregnancy exposure revealed that in the mfc group, 1 year old male offspring had higher responses to compound 48/80 and acetylcholine than 6 months old males. responses to kcl were significantly higher in 6 months old males than 1 year olds independent of maternal treatment during pregnancy. in females, the only difference between age groups was observed in the mfc group, where 6 months old offspring demonstrated significantly higher responses to acetylcholine compared to 1 year old offspring. conclusions: our findings did not show that airways of 1 year old offspring born to mice with a preeclampsia-like syndrome induced by sflt-1 over-expression have airway hyperreactivity. however, sex and age differences in airway responses dependent on maternal exposure during pregnancy were observed, and needs to be explored further to elucidate underlying mechanisms. objective: maternal food restriction (fr) results in iugr newborns that when normally nursed exhibit rapid catch-up growth and adult obesity. continued fr during nursing delays catch-up growth and prevents adult obesity. igf-1, which modulates growth and is secreted by the liver, may contribute to these morbidities. igf-1 is epigenetically regulated involving two promoters, alternative exon splicing and multiple transcription termination sites. we determined if hepatic igf-1 mrna levels correlate with obesity, and whether these changes are due to programmed epigenetic modification. methods: control pregnant rats received ad libitum food from gestation day 10 to 21 and lactation, whereas study dams were 50% fr. fr pups were nursed by either control (fr/adlib) or fr dams (fr/fr) and weaned to ad libitum feed. at 1 day and 9 months, male livers were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. results: at 9 months, obese fr/adlib males showed increased mrna levels of igf-1a, igf-1b, igf-1 exon 1, and igf-1 exon 2 as compared to controls (134±5, 165±6, 149±6, and 146±7 %). comparing fr/adlib 9 month to newborn offspring, h3/k4 was increased at igf1-promoter 1, promoter 2, exon 5, utr#3 and utr#4 (610±157, 731±167, 345±66, 1597±412, and 794±208 %), though there was no differences between control 9 month and newborns. in contrast, 9 month fr/fr males had comparable mrna levels to the controls except for igf-1b (% of control: 147±19). further, fr/fr 9 month h3/k4 was only different from newborns at utr#4 (% of newborn: 289±69). conclusion: iugr newborns with rapid catch-up growth and adult obesity have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. conversely, iugr with delayed catch-up growth and absence of adult obesity have levels similar to that of controls. thus, modulation of the rate of iugr newborn catch-up growth may protect against igf-1 epigenetic modifications. introduction: igf-ii is synthesized as a pro-hormone (proigf-ii; 156-amino acid peptide) which is then processed into its active forms: "big" igf-ii (1-87) and mature igf-ii (1-67). these active forms are essential for placental and fetal development and have also been shown to persist into postnatal life. since maternal smoking is known to adversely affect feto-placental growth and postnatal development, we postulated that these effects might be mediated through nicotine-induced alterations in igf-ii processing. methods: in the present study, nulliparous female wistar rats (200-250g) were given nicotine (1mg/kg/day) or saline for 14 days prior to mating, during pregnancy, and throughout lactation. at gestational day 15, 18 and 21, dams were euthanized and we collected serum (fetal and maternal), amniotic fluid and recorded fetal body weight. a subset of dams were allowed to deliver at term. following parturition, serum samples from the offspring were collected at birth (pnd1) and weaning (pnd21). body weight was recorded weekly from birth to weaning. pro, "big" and mature igf-ii levels were determined by western blot analysis. results: maternal nicotine exposure during pregnancy resulted in a significant reduction in fetal body weight by gestational day 21. however, there was no effect of nicotine on fetal serum or amniotic fluid igf-ii levels at any gestational age examined. in maternal serum, mature igf-ii in control animals decreased with advancing gestational age such that igf-ii levels were lowest at gestational day 21. nicotine administration prevented this decline, which resulted in significantly higher mature igf-ii levels in nicotine-exposed mothers at gestational day 21. in postnatal life, nicotine exposed offspring had significantly lower levels of "big" igf-ii expression at weaning (pnd21). conclusions: these data demonstrate that nicotine can alter the amount of the active forms of igf-ii in the mother and the newborn. dysregulation of maternal igf-ii occurs concomitantly with suboptimal fetal growth. results from this study suggest a mechanism by which maternal smoking causes impaired fetal growth and adverse postnatal health outcomes. objective: maternal food restriction in pregnancy results in iugr newborns which develop adult metabolic syndrome. programming of both increased appetite-mediated hyperphagia and enhanced adipogenesis contribute to the development of obesity. transcription factors, peroxisome-proliferatoractivated-receptor (ppar 2), ccaat/enhancer binding-protein (c/ebp ), and sterol regulatory element binding-protein (srebp1c) regulate adipogenesis and lipogenesis. although iugr offspring exhibit acute upregulation of the adipogenesis signaling cascade prior to the development of obesity, we determined if this increased adipogenic potential was an intrinsic cellular response, and thus maintained in cell culture. we further examined the responses to adipocyte stimulators (ppar activator-ligand rosiglitazone) and inhibitors (ppar repressor-ligand badge). methods: control dams received ad libitum food, whereas study dams were 50% food-restricted from pregnancy day 10 to term. adipocytes from 1 day old iugr and controls were isolated and cell proliferation rate was determined (mtt). primary adipocyte cell cultures were established and following 100% confluence, iugr and control adipocytes were treated to two doses (1 and 10 m) of either rosiglitazone or badge for 24h. mrna and protein was extracted for expression of ppar , c/ebp , srebp1c. data was normalized to -actin and compared to the respective untreated cells. results: iugr adipocytes had significantly increased protein expression of ppar (3.5-fold) and c/ebp (2-fold) as compared to control adipocytes, though srebp1c levels were unchanged. mrna levels showed similar changes in iugr newborns. importantly, iugr adipocytes exhibited increased cell proliferation (125% of control, p<0.05) and showed greater response to rosiglitazone (2.5-fold), though similar response to badge, as the control adipocytes conclusion: iugr primary adipocytes cell culture exhibit basal phenotypic characteristic of programmed upregulation of adipogenic transcription factors which promote adipose cell proliferation. the enhanced response to the adipogenic stimulant is further evidence of the predisposition to obesity. in contrast, the normal suppressive response to the inhibitor suggests that iugr adipocytes may respond to pharmacologic approaches to prevent obesity during this period. objective: maternal nutrient restriction results in intrauterine growth restricted (iugr) newborns which develop programmed obesity despite a normal post-weaning diet. the epidemic of obesity has been attributed in part to programmed "thrifty phenotype" and exposure to "western" diets. hepatic igf-1 is epigenetically regulated involving two promoters, alternative exon splicing, and multiple transcription termination sites. iugr offspring with normal post-weaning diet have increased postnatal hepatic igf-1 mrna levels, likely a result of igf-1 histone and chromatin structure modifications to h3k4 trimethylation. we hypothesized that iugr newborns that develop programmed obesity would demonstrate discernable hepatic igf-1 changes which are distinct from diet-induced obesity. we determined igf-1 hepatic mrna levels and epigenetic characteristics in programmed (iugr) and dietinduced (dio) offspring. methods:: control pregnant rats received ad libitum food whereas study dams were 50% maternal food restricted from day 10 to 21. all pups were nursed on ad libitum fed dams. controls were weaned to high-fat (fat, 16%) diet whereas iugr were weaned to normal ad libitum diet (fat, 9%) to produce diet-induced (dio) and programmed obese groups, respectively. at 9 months, male hepatic igf-1 were analyzed for igf-1 mrna variant levels (real time rt-pcr). chromatin immunoprecipitation (chip) was performed using the antibody for h3k4 dimethyl and h3k4 trimethyl, and associated levels of each igf-1 species were measured by pcr. result: relative to dio control males, iugr had increased mrna of igf-1a, exon 1 and exon 2 (336±4, 267±4, 143±5%). chip with h3k4 dimethyl showed increased igf-1 exon 6 (215±20%) and with h3k4 trimethyl, increased igf-1 promoter 1 and promoter 2 (282±26, 1183±23%) as compared to dio controls. conclusion: adult obese iugr males exposed to normal postweaning diet have increased hepatic igf-1a mrna and h3k4 dimethylation and trimethylation of igf-1 than dio controls. changes in igf-1 in adulthood from a prenatal insult thus suggest that igf-1 is programmed during the fetal period and may be associated with programmed adult obesity. rebekah elkins, 1 pandu gangula, 2 chandra yallampalli. 1 1 obstetrics gynecology, university of texas medical branch, galveston, tx, usa; 2 internal medicine, university of texas medical branch, galveston, tx, usa. objectives: we previously reported that the offspring of rats fed 6% protein during gestation develop hypertension at two to four months and that the hypertension is exacerbated in males. this study is to evaluate: 1) changes in estrogen receptor (er) angiotensin ii subtype receptor 1 (at 1 -r) and endothelial nitric oxide synthase (enos) in the mesenteric artery and aorta of offspring and assess if these changes, if any, are gender specific. methods: pregnant sprague dawley rats were fed either 20% protein (ctrl) or 6% protein (lpd) from day 1 of gestation. the offspring were evaluated for hypertension by means of systolic blood pressure measurements. at four months for the males and nine months for the females, mesenteric artery and aorta were collected in rnalater. expression of estrogen receptor a (er-a) and b (er-b), at 1 -r, and enos were analyzed by western immunoblotting and rt-pcr and expressed relative to b-actin or 18s. results: mesenteric artery shows no differences between ctrl and lpd female offspring in at 1 -r, enos, er-a or er-b. similarly mesenteric artery shows no diet exposure related changes in at 1 -r, er-a or er-b in male offspring. however, enos expression was lower in mesenteric artery of lpd male offspring. on the other hand, in the aorta both er-a and er-b levels are lower in lpd female offspring while there were no changes in at 1 -r or enos. no changes in at 1 -r, er-a or er-b were observed in male offspring aorta of ctrl and lpd rats. conclusion: the in utero exposure to lpd results in adult hypertension in both male and female offspring. some mechanisms for hypertension include the decrease in er-a and er-b but not at1-r or enos in females, and the decrease in enos but not at 1 -r or er in males indicating gender related differences. offspring. hidenori takahashi, toshiaki okawa, keiya fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. our objective was to investigate the effects of either severe undernutrition during late gestation or lactation period on blood pressure and the development of vascular function in male rat offspring. we use normal pregnant wistar rats (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b) and 30% restricted after delivery to the end of lactation period (group c). the offspring was measured blood pressure at 12 and 24 weeks by using indirect tail-cuff method. rings of thoracic aorta with intact endothelium from the male offspring of a and b at 8 weeks, were equilibrated at 2 g passive tension in organ chambers filled with krebs-henseleit solution continuously bubbled with 5%co2 in air (37°, ph 7.4) for isometric tension recording. concentration-response relationships to norepinephrine (ne) and angiotensinii(atii) were obtained in the absence or presence of n(omega)-nitro-l-arginine methyl ester (l-name) or a selective atii type-1 receptor blocker (valsartan). responses to cumulative concentrations of sodium nitroprusside (snp) and to 10-5m oxyhemoglobin (hb, nitric oxide scavenger) were also determined. contractions were expressed as a percent of the reference contraction induced by potassium chloride (60 mm). statically analysis was performed using one-way annova. results: body weight was significantly reduced in b offspring compared to a and c in male offspring at day 1 (p<0.01). at 12 weeks the body weight of offspring of b was no difference to catch up compared to a and c offspring. systolic and diastolic blood pressures were significantly elevated at both 12 and 24 weeks in offspring of b > c >a. ne concentration-dependently stimulated tension of aortic rings from in a and b offspring, which was not significantly (n=6). maximal contractions to ne were significantly stimulated by l-name in a (p<0.05), but not b offspring. valsartan significantly inhibited aortic contractions by ne in r (p<0.05), but not a offspring. there was no significant difference on responses of aortic rings by atii, snp and hb in a and b offspring. conclusions: severe under nutrition during not only late gestation but also lactation period induced hypertension in male rat offspring in adulthood. fetal origin of adult hypertension might be vascular endothelial dysfunction. fujimori, akira sato. obgyn, fukushima med. univ., fukushima, fukushima, japan. objective: exposure to undernutrition during fetal life has been proposed as an underlying cause of adult hypertension, but the effect of either high fat nourishment or undernutrition during lactation period on blood pressure is unclear. our objective was to investigate the most effective maternal nourishment and feeding period for offspring induced adulthood hypertension in using high-fat diet (hfd). study design: we use 5 types pregnant wistar rats as fed with normal nutrition (group a), nutritionally restricted by feeding with 30% of the normal gestation-matched dietary intake from day 17 of gestation to delivery (group b), 30% restricted after delivery to the end of lactation period (group c), with a high fat diet (hfd) during gestation to lactation period (group d) and with hfd nutritionally 30% restricted from the day of delivery to the end of lactation period (group e). the offspring was measured body weight (bw) and measured blood pressure at 12, 24 and 60 weeks by using indirect tail-cuff method. statically analysis was performed using one-way annova. results: bw was significantly reduced in b offspring compared to another (a, c, d, e) male offspring at day 1 (p<0.01). at day 28 after delivery, bw was significantly reduced in c, e offspring compared to a, d in male offspring (p<0.01). at 12 weeks old, bw of all type offspring was no difference. systolic and diastolic blood pressures were significantly elevated at 12 weeks in offspring of d> b > e > c >a. (p<0.01, vs. a). at 24 weeks, hypertensive offspring as b>d>e>c>a (p<0.01, vs. a). at 60 weeks, d> e > b > c >a (p<0.01, vs. a). conclusions: maternal high fat environment make a hypertensive offspring, but regulation of fat feeding during lactation period may reduce adulthood hypertension. in case with normal food, restrictive feeding during late gestation is more effective than lactation period for inducing hypertensive male offspring. regulation of maternal feeding not only during late gestation but also lactation period may control adulthood hypertension. the strongest epigenetical factor of maternal nutrition is high fat feeding during pregnancy to lactation period for f1 blood pressure, respectively. or residence at high altitude, impacts fetal growth and development. in a preliminary study, we observed a significant decrease in birth weight, subsequent compensatory postnatal growth, and an increase in relative right ventricular (rv) weight at postnatal (pn) day 30 in female offspring of rats exposed to hypoxia (14,000 ft; 11.6% po 2 ) from days 12 thru 15 of gestation (dga). thus, our objective was to further elucidate the impact of prenatal hypoxia on fetal growth and postnatal development. methods: pregnant dams (hx, n=15) were hypoxic from 12-15 dga with additional control dams either fed ad libitum (al, n=10), pair-fed with the hx dams throughout gestation (pg, n=12), or only pair-fed during the window of hypoxia (ph, n=12). female offspring from hx, pg, and ph dams were cross-fostered onto additional al dams (n=8/litter) by 48 h after birth. results: at birth, there was no difference in litter size; however, body weight (bw) of the hx, pg, and ph pups was lower (p<0.05) than that of al pups, and hx pups were lighter (p<0.05) than ph pups. weight of hx offspring remained lower (p<0.05) than al pups until the termination of the study at pn120, while the pg and ph pups reached weights comparable to the al offspring by pn90. relative to bw, heart weight and left ventricular/septal (lvs) weight was not different among groups; however, right ventricular weight (rv/bw) was greater (p<0.05) in the hx offspring at pn120 as was rv/lvs (p<0.05). cardiac function was evaluated by echocardiography at pn174. rv wall thickness was 47% greater (p<0.05) in hx pups as compared to al pups, confirming the significantly higher relative rv weight observed at necropsy. pep, pep/at, and pep/et were 36%, 14%, and 31% higher respectively in the hx offspring relative to the al offspring. lv end diastolic and end systolic diameters were smaller (p<0.05) in hx and ph offspring relative to the al group. myosin heavy chain (mhc) and mrna concentrations in the rv were evaluated by qrt-pcr, and the mhc / mrna ratio was greater (p<0.05) in the hx pups. conclusion: prenatal hypoxia from 12-15 dga impacted both fetal and postnatal growth, altered postnatal heart development and function, with the primary impact being on the rv. supported by nih hd48902. reproductive sciences; 2 pharmacology experimental therapeutics, university of maryland, baltimore, md, usa. background: exposure to nicotine (nic) is a significant risk to normal fetal development. fetal nic, which readily crosses the placenta, can be acquired from pregnant mothers by smoking or nicotine replacement therapy. the impact of nic on fetal organs may be mediated directly and/or via intrauterine hypoxia (hpx) via constriction of the uterine circulation. in adult hearts, both nic and hypoxia stimulate gene expression of matrix metalloproteinases (mmp), although the study of nic and hypoxia on gene expression in fetal organs remains incomplete. because mmps are involved in the regulation of extracellular matrix turnover and cardiac remodeling, we tested the hypothesis that prenatal nic and intrauterine hpx upregulate protein expression and activity of mmp2 in the fetal guinea pig heart. methods: pregnant guinea pigs were placed in either normoxia (nmx) or hpx (10.5%o2 in chamber) for 14d prior to term (65d). in two separate groups, nic was also added to the drinking water ( 18 mg/kg/d) for 10d at a dose that generates fetal nic levels (88ng/ml cotinine) equivalent to a moderate smoker. anesthetized near-term fetuses ( 63d) were excised and weighed. left ventricles of hearts were obtained and frozen at -80c for storage. mmp2 protein levels and enzymatic activity were measured by western analysis and gel zymography, respectively. results: nic alone (nmx+nic) decreased (p<0.05) fetal body wt by 12%, increased (p<0.05) the relative fetal brain wt (brain wt/fetal body wt ratio) by 14.2% and had no effect on relative placental or fetal heart wts. hpx alone decreased (p<0.05) fetal body wt by 20.3%, increased the relative fetal brain wt by 12% but, in contrast to nic alone, increased relative placental wt by 33.8%. both mmp2 protein levels (mmp2/a-actin density values) and activity (clear band density) were increased (p<0.05) by nic alone (by 1.6 and 1.8 fold, respectively) and hpx alone (by 1.5 and 1.3 fold, respectively). in addition, both protein and activity levels of hpx hearts were further increased by nic (by 1.6 and 1.3 fold) compared to hpx alone. conclusion: prenatal nic upregulates mmp2 expression in nmx fetal hearts and is potentiated by hpx. this suggests that under conditions of intrauterine stress cardiac remodeling by mmp activation may be an important mechanism by which nic and hpx affect fetal heart function. objective: in addition to peripheral hypoglycemic effects, insulin induces central anorexigenic responses via stimulation of the phosphoinositide-3 kinase (pi3k) pathway and cellular growth by mitogen activated protein kinase (mapk) pathway. the pi3k signaling cascade is activated by insulin binding to its receptor (ir), recruiting ir substrate 2 (irs-2), and phosphorylating pi3k. activated pi3k in turn causes phosphorylation of protein kinase b/akt which subsequently modulates hypothalamic anorexigenic responses. in contrast, the mapk (erk1/erk2) signaling pathway likely involves irs-1. further, insulin signaling is inhibited by the lipid phosphatase pten. we have previously shown that maternal food restriction (mfr) during pregnancy results in iugr newborns that develop hyperphagia, obesity and insulin resistance as adults. we sought to determine if altered hypothalamic basal insulin signaling expression of pi3k and mapk pathways contribute to reduced satiety responses and thus enhanced growth in iugr newborns methods: pregnant control dams received ad libitum (n=5) food, whereas study dams were 50% mfr (n=5) from pregnancy day 10 to 21 to produce iugr newborns. at day 1, hypothalamic region was dissected and analyzed for mrna levels (real time rt-pcr) of insulin signaling components via pi3k (ir, irs2, pi3k and akt) and mapk (irs1, erk1, erk2) pathways, and pten. data is presented as fold difference normalized to beta-2-microglobulin. results: at 1d of age, iugr pups exhibited downregulation of the entire pi3k pathway with significantly decreased (p<0.05) mrna levels of ir (0.6-fold), irs-2 (0.5-fold), pi3k (0.6-fold) and akt (0.6-fold). further, iugr pups showed similar decreased mrna expression of erk1 (0.6-fold) and erk2 (0.5-fold). however pten expression was similar to the controls. conclusion: reduced insulin-mediated pi3k signaling likely contributes to the suppressed anorexigenic responses and development of obesity in iugr offspring. reduction of central mapk signaling suggests a potential maldevelopment of additional neuronal pathways in iugr offspring. objectives: in the rat, uteroplacental insufficiency restricts fetal growth and impairs mammary development further compromising postnatal growth. both male and female growth-restricted offspring have a reduced nephron endowment but only males develop hypertension with glomerular hypertrophy, which can be reversed by improving the lactational environment. this study used cross-fostering to assess the influence of the prenatal and postnatal environments on renal development and nephrogenesis. methods: bilateral uterine vessel ligation (restricted, r) or sham surgery (control, c) was performed on day 18 of gestation in wky rats. control and restricted pups were cross-fostered onto c or r mothers on postnatal day 1 (pn1). post mortem was carried out on pn1 (c and r) and pn7 (c-on-c, c-on-r, r-on-c, r-on-r). results: body and kidney weights were decreased in r and r-on-r pups on pn1 and pn7 (p<0.05). there was some evidence of accelerated pup growth for r-on-c relative to r-on-r on pn7. male, but not female, relative bmp4 mrna expression on pn1 was higher in r than c (p<0.05) while gdnf, tgf 1 and at1 receptor were not different. on pn7, wnt4 (but not at1r, vegf-a) mrna expression (males only) was relatively higher in r-on-r (p<0.05) when compared to c-on-c (p<0.05). this and the histological analyses suggests an up-regulation of nephrogenic activity with more immature nephrons (males and females) in r-on-r (p<0.05) when compared to c-on-c, while r-on-c remained intermediate. intrauterine growth-restricted pups were born lighter and with smaller kidneys. this was partially rescued by improving lactational nutrition (r-on-c) at pn7. higher bmp4 mrna expression indicates impaired branching morphogenesis in pn1 r male, but not female kidneys, suggesting the timing and/or molecular mechanisms underlying the nephron deficit may be sex specific. at pn7 there was evidence of extended and increased nephrogenic activity in r-on-r, however, this was unable to restore the later nephron deficit. improved lactation for r-on-c, which prevented the adult nephron deficit and hypertension, increased and extended nephrogenesis to a lesser degree than r-on-r suggesting that the restoration of nephron endowment was likely to have occurred prior to pn7. amy m tetrault, sarah b lieber, marya shanabrough, tamas l horvath, hugh s taylor. obstetrics, gynecology and reproductive sciences, yale university school of medicine, new haven, ct, usa. objective: classically recognized for its role in energy balance, body weight and appetite, ghrelin has also been implicated in reproduction. ghrelin (-/-) mice are infertile while administration of ghrelin to wt mice results in decreased litter size and constrained embryonic growth. here we investigate the effect of maternal ghrelin deficiency on in utero developmental programming of the female reproductive tract. hox genes determine developmental identity of the paramesonephric duct. we determined that hoxa10 is regulated by ghrelin in vitro and that in utero ghrelin deficency alters f2 hoxa10 gene expression and reproductive success. methods: wild-type females mice parented by ghrelin +/-b6d2f1 (ghrellin deficient) mice were analyzed for litter size, oocyte, and corpus luteum number. rna was extracted from the uterus of mice exposed to ghrelin deficiency in utero. ishikawa cells were treated with ghrelin with/without receptor (ghsr) antagonist, or saline and rna extracted. in both hoxa10 expression was analysed by real time rt-pcr normalized to -actin and also determined by ihc. experiments were repeated in triplicate and mrna expression compared by student's t-test. results: wild-type female offspring of ghrelin deficient dams had smaller litter sizes than controls (n=5, 4.7±3.3 pups; n= 4, 8.7±2.3 pups, respectively; p<0.01). no differences were seen in oocyte or corpus luteum number suggesting a uterine defect. hoxa10 mrna and protein expression were decreased in the uterus of the f2 females. ghsr was expressed in uterine endometrium. treatment of ishikawa cells with 1nm to 100nm ghrelin resulted in a 30 to 80% increase (p<0.01) in hoxa10 expression. treatment with ghrelin and ghsr antagonist resulted in similar increases in hoxa10 expression indicating a non-receptor mediated mechanism. conclusion: ghrelin contributes to reproductive tract developmental programming; in utero ghrelin deficiency compromises reproduction in female offspring. the developmental effects of ghrelin were mediated by alteration in hox gene expression and not through the classic ghsr receptor. obesity and decreased ghrelin may lead to defects in developmental programming of the reproductive tract. these findings demonstrate the importance of nutrition, energy utilization and appropriate ghrelin levels on normal uterine development. we have previously studied the deleterious effects of lack of the endothelial nitric oxid synthase (nos3) in mouse dams and their offspring. our laboratory demonstrated that adaptive responses in subsequent pregnancies may offset the harmful effects of the genetic deficiency of nos3. in this study we aimed to determine hepatic and renal histopathologic damage in nos3 deficient pregnant mice comparing animals carrying their first versus their second pregnancy. study design: gravid nos3 +/+wt and nos3 -/-ko mice during their first (p0) or second (p1) pregnancy were sacrificed at day 18 of gestation. livers and kidneys were stained and analyzed for the presence and extent of histopathologic lesions. results: nos3 +/+wt dams displayed a low incidence of significant renal or hepatic lesions in either the first or the second pregnancy. in nos3 -/-ko mice the incidence of liver necrosis and inflammation during the first pregnancy was 44% and 49%, respectively. in nos3 -/-ko dams sacrificed during the second gestation the incidence rates for the same lesions were 6% and 19%, respectively (p<0.05). this correlation persisted when we analyzed the relative severity of hepatic lesions between p0 and p1 animals. although a similar trend was observed, the difference between p0 and p1 animals with regards to kidney lesions did not reach the level of statistical significance in our study. conclusions: a second pregnancy in this animal model of hypertension was associated with a significantly improved hepatic histopathology compared with the first pregnancy. this observation is consistent with our previous studies showing a decrease in systemic vascular resistance in p1 versus p0 nos3 -/-ko mice. the beneficial effects of a prior pregnancy may partially underlie the phenomenon of a decreased risk of preeclampsia in multiparous versus nulliparous women. further studies are required to delineate the counterregulatory mechanisms leading to improved cardiovascular function in subsequent pregnancies in these genetically modified animals. maternal hypomethylation is associated with congenital heart defects in down syndrome. lmjw van driel, 1,2 r de jonge, 3 wa helbing, 2 bd van zelst, 3 j lindemans, 3 eap steegers, 1 rpm steegers-theunissen. 1,2,4,5 1 obstetrics gynecology; 2 pediatrics; 3 clinical chemistry; 4 epidemiolog y biostatistics; 5 clinical genetics; 6 erasmus university mc, rotterdam, netherlands. background: maternal age and hyperhomocysteinemia are risk factors for having a child with down syndrome (ds) and congenital heart defects (chds), respectively. evidence is rising that ageing is associated with a state of hypomethylation. objectives: to investigate whether the risk of a child with ds and chd is associated with maternal hypomethylation. methods: we conducted a case-control triad study at 16 months after the index-pregnancy. case-children (n=24) were included if they had ds and chd. children (n=251) without a major congenital malformation served as controls. the concentrations of s-adenosyl methionine (sam), s-adenosyl homocysteine (sah), sam/sah ratio, and homocysteine in maternal blood were measured as biomarkers for methylation. the data were analyzed using the mann-whitney u test and a logistic regression model. results: maternal age was included in the model as potential confounder. the levels and the crude and adjusted or(95%ci) of the biomarkers are shown in table 1. an increase of the sam/sah ratio with 1 unit decreases the risk of a child with ds and chd with 30 percent. moreover, every increase of 1 mol/l of homocysteine 1.1 fold increases this risk. conclusions: maternal hypomethylation is significantly associated with an increased risk of having a child with ds and chd. since, the effects are confounded by maternal age, hypomethylation can be considered as feature of ageing. the developmental origins hypothesis postulates that during critical ontogenetic periods, transient environmental stimuli perturb developmental pathways and induce permanent changes in gene expression, metabolism, and chronic disease susceptibility. one likely mechanism is via early nutritional influences on epigenetic gene modification consisting of the presence of a methyl group on the carbon 5 of a cytosine residue. this modification is responsible for an important form of gene regulation in eukaryotes. in the present study, we have tested the hypothesis that maternal low-protein diet altered epigenetic regulation of specific gene of the offspring. c57bl/6 female mice were mated and on the day the plug was detected, these females were then randomly allocated to be fed isocaloric diets consisting 18% protein or 9% protein. at delivery, offspring were killed and the livers were removed immediately, frozen in liquid nitrogen and stored at -80c. genomic sequencing after bisulfite modification is used to study site-specific dna methylation. dna methylation status of oct-4 and sphk-1 gene upstream regions in the mouse liver was analyzed. hepatic oct-4 or sphk-1 promoter methylation was not significantly different between both groups. however, dna methylation pattern of the genomic dna is specific in low-protein diet group. aberrant oct-4 and sphk-1 gene expression may cause perturbations in cell differentiation. we suggest that the epigenetic mechanism consisting of dna methylation underlies the fetal programming theory. primary human cytomegalovirus (hcmv) infection during pregnancy can have devastating consequences for both the mother and fetus. hcmv infection has been implicated in the development of pre-eclampsia and intrauterine growth retardation (iugr), as well as congenital cmv syndrome in newborns exposed in utero. previously, we have shown that hcmv infection of placental cytotrophoblasts inhibits their normal invasion, proliferation, and migration. however, the mechanisms occurring during early establishment of placental infection are largely unknown. we assessed the impact of hcmv infection on cytotrophoblasts by performing immuno-based assays for various cytokines and cellular growth factors. we detected significant cytokine dysregulation at both 24 and 48 hours after in vitro hcmv infection of cytotrophoblast cells. soluble cytokines involved in recruitment of monocytes and macrophages (gro-a, mcp-3) were downregulated at both 24 and 48 hours after infection. sdf-1, which is chemotactic for lymphocytes during early inflammation, was also decreased. these results suggest that recruitment of cells involved in the anti-viral immune response is being interrupted early in the course of infection by hcmv. additionally, a large decrease in the amount of soluble hgf was seen. hgf normally induces migration of cytotrophoblasts along the invasive pathway, and downregulation of this factor could severely affect these processes. finally, we saw increased amounts of soluble icam-1, contrasted by decreased amounts of vcam-1, indicating dysregulation of adhesion molecules that are necessary for successful placental invasion to occur. all together, these data indicate significant alterations in cytokine profiles as early as 24 hours after hcmv infection, which could provide important clues to the pathogenesis of hcmv in placental invasion and inflammation. additional studies will further elucidate this dysregulation, and determine whether these effects are due to alterations in pre-existing cellular factors or if transcriptional alterations are involved. method: studies were performed in pregnant ewes (n=6 in each group) with twin fetuses at 118-120 days (very preterm) and 140-142 days (near-term). neither mother nor fetuses were instrumented prior to the time of blood collection. maternal blood was collected from the jugular vein before sedation. anesthesia was then induced with isofluorane, the abdomen was opened, fetuses exteriorized and blood collected from umbilical cords. blood samples were transferred to plastic tubes containing ethylene-diamino tetraacetic acid and reduced glutathione, plasma separated and stored at -80c. commercial radioimmunoassay kits for ovine-crf (phoenix pharmaceuticals, b90: 7.2 ± 1.5 pg/ml) and cortisol (diagnostic laboratory, b90: 1.2 ± 0.4 ng/ml) were used according to the manufacturer's instructions. results: in very-preterm gestations, maternal and fetal plasma crf levels were undetectable. maternal, but not fetal, plasma cortisol levels were measurable (33±4 ng/ml). in near-term gestations, both cortisol and crf were measurable in maternal (crf: 86±6 pg/ml; cortisol: 55±1 ng/m) and fetal plasma (crf: 730±68 pg/ml; cortisol: 5±2 ng/ml). plasma crf levels were higher in nearterm fetuses than in their maternal ewes (p <0.001). conclusion: ovine plasma crf levels are measurable in maternal and fetal plasma in near-term but not very-preterm gestations. the absence of crf in preterm plasma, perhaps due to reduced placental expression and/or placental crf release, may contribute to the rarity of in utero meconium passage in preterm gestations. interleukin objectives: interleukin-6 (il-6) is a pro-inflammatory cytokine produced in adipose cells. recent studies suggest il-6 may be a marker of maternal obesity and in utero fetal programming. our hypotheses were 1) il-6 correlates with maternal obesity and 2) il-6 mediates the effect of maternal obesity on infant birth weight. methods: the parity, inflammation, and diabetes (pid) study is a longitudinal study of adipokine levels in a diverse sample of pregnant women. we present a cross-sectional analysis of first trimester il-6 levels from 173 non-diabetic women who underwent a live birth. the independent variable was il-6 (pg/ml), measured with monoclonal antibody elisa assays. the dependent variable was infant birth weight (gms). maternal bmi categories were: normal/underweight (< 25 kg/m 2 ); overweight (25-30 kg/m 2 ); and obese (> 30 kg/m 2 ). data on demographic and clinical factors, nutrition and physical activity were collected at baseline. average il-6 levels were compared across bmi categories using anova. the association of il-6 levels with infant birth weight was estimated using multiple linear regression, adjusting for covariates. results: average il-6 levels were significantly higher in obese women (2.6± 1.6) compared to overweight (1.2± 0.4) and normal/underweight women (1.1± 0.7) [p< 0.001]. after adjustment, il-6 levels was positively correlated with pre-pregnancy bmi [regression coefficient (rc)0.12; 95% ci: (0.05, 0.2)]. as shown in the table below, elevated levels of il-6 were statistically significantly associated with a 0.5 gm higher infant birth weight after full adjustment. each 1 unit increase in pre-pregnancy bmi was associated with a 8 gm higher infant birth weight. table 1 . association of il-6 with infant birth weight characteristic regression coefficient 95% confidence interval interleukin-6 0.5 0.02, 0.7 pre-pregnancy bmi 8 0.7, 38 gestational weight gain -3 -15, 10 bmi = body mass index; coefficients adjusted for demographics, clinical factors, pre-pregnancy bmi, gestational weight gain, non-fasting first trimester glucose levels, gestational age, nutritional intake and physical activity conclusion: il-6 and pre-pregnancy bmi were associated with infant birth weight after adjustment for covariates. our findings suggest that the effect of maternal obesity on infant birth weight may be mediated through il-6 or an alternative independent pathway. we have demonstrated that 9 -tetrahydrocannabinol (thc), in physiologically relevant concentrations, inhibits the growth and tight transcriptional control of the bewo trophoblast cell line 1 . the mechanism involved the decreased expression of the transcriptional regulator histone deacetylase 3 (hdac3). in these experiments we sought to answer the question, 'does anandamide (aea) work in the same manner as thc?' methods: the first trimester human trophoblast cell, bewo were plated at 1 or 4 x 10 5 cells /well to 96-well and 6-well plates for growth and rna experiments, respectively. after growing to 70-80% confluence, cultures were treated with varying concentrations of aea up to a maximum of 30 m for 48hr. cell numbers were determined using the xtt apoptosis/proliferation assay. total cellular rna was prepared and the relative levels of hdac3 and gapdh determined by end-point rt-pcr. aea exhibited an inhibitory effect on the bewo cell cultures, but only at concentrations in excess of 3 m where confluency was significantly reduced from 75% at 3 m to 50-60% at 15 m and 30 m (*p<0.05; one-way anova with tukey's hsd test; n= 4). cultures treated with 15 m and 30 m aea did not exhibit increased cell death or failure to attach to the substratum, as evidenced by the lack of increase in the shedding of cells into the spent medium. bewo cells treated with aea showed a dose-dependent decrease in hdac3 mrna expression with a significant effect at 0.3 m (*p<0.05; oneway anova with tukey's hsd test; n=9). at this dose, the effect of aea had reached an effective maximum decrease in hdac3 mrna levels (48%) because hdac3 mrna levels were not decreased further by either 3 m aea (45%) or 30 m aea (49%). the alteration of hdac3 gene expression by aea in bewo cells with its associated decrease in cell number suggest that the trophoblast cell may be an important target for circulating endocannabinoids during the 1 st trimester of pregnancy. the data indicates that although exocannabinoids and endocannabinoids both inhibit bewo cell growth, they do so using different transcriptional mechanisms. further understanding of the mechanism(s) by which aea alters placental physiology may lead to new strategies for the prevention of pregnancy complications such as 1 st trimester miscarriages. (1) taylor background: anandamide (aea) exerts its effects by acting on two cannabinoid receptors, cb1 and cb2 with the main regulator for aea levels being the metabolising enzyme, fatty acid amide hydrolase (faah). aea, cb1, cb2 and faah constitute the endocannabinoid system and previous studies have shown that faah and cb1 are expressed in term human placenta(1) suggesting that the endocannabinoid system might be present earlier in gestation. this study aimed to document changes in faah, cb1 and cb2 expression in the placenta during the first trimester of pregnancy. methods: first trimester samples (7 to 12 weeks gestation) were fixed in 10% neutral-buffered formalin for 4 days before embedding into paraffin wax or frozen in liquid nitrogen for rna analysis. for immunohistochemistry, faah polyclonal antibodies were used at an optimal dilution of 1:2000 in pbs, cb1 at 1:4000 and cb2 at 1:500. transcripts were measured using q-pcr with gene-specific primers. results: immunoreactive faah, cb1 and cb2 were detected in all samples. faah immunoreactivity in the syncytiotrophoblast increased between the 7th and 10th gestational week and by week 11 faah was barely detectable within large parts of the placenta. simultaneously, faah immunoreactivity increased in the mesenchymal core of the developing villi. immunoreactive cb1 and cb2 localised to the syncytiotrophoblast, cytotrophoblast and mesenchymal core with cb1 immunoreactivity showing diminished intensity after week 10, although this did not reach significance at the transcript level. cb1 immunoreactivity was absent from fetal blood cells and infiltrating maternal plasma cells, whereas cb1 and cb2 immunoreactivity was detected in endothelial cells but not in the vascular smooth muscle cells of blood vessels. the intensity of cb2 immunoreactivity in the syncytiotrophoblast differed from that of cb1 and faah in that it remained constant throughout. conclusion: the data suggest that placental faah and cb2 levels do not alter significantly during the first trimester, but alter their cellular distribution from the syncytiotrophoblast to the mesenchymal core. the significant loss of cb1 expression from the syncytiotrophoblast after the 10th week of gestation, a point of critical alteration in the developing placenta, suggests that its retention may be detrimental to normal placental development. reference: park, b. et al., (2003) hankins, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: numerous angiogenic proteins synthesized in the placenta are thought to be involved in placental vascularization and development; however, the molecular mechanism modeling the angiogenic process in early pregnancy remains elusive. calatonin gene-related peptide (cgrp) is a multifunctional peptide expressed at the human implantation site; but its influence on in vitro angiogenesis by human micro vascular endothelial cells is not known. objective: the present study was designed to determine the influence of cgrp on angiogenesis by human dermal microvascular endothelial cell (hdmvec) in vitro. methods: hdmvecs (vec technologies) were cultured in mcdb-131 complete solution containing cgrp (10 -8 m), cgrp plus its antagonist cgrp 8-37 (10 -7 m), in 6 well plates with 2x10 5 cells per well. the existence of cgrp receptor components calcitonin receptor-like receptor (crlr) and receptor activity modifying protein 1 (ramp1) was determined using immunofluorescent staining. cell proliferation was examined using methylthiazoltetrazolium (mtt) assay. the pro-angiogenic bioactivity of cgrp was evaluated using cell migration and capillary like tube formation on the matrigel. results: 1) immunofluorescent staining showed that cgrp receptor components crlr and ramp1 are abundantly expressed by hdmvecs. replacement of the primary antibodies with preimmune serum resulted in a negative staining; 2) cgrp dose-dependently (10 -10 to 10 -7 m) stimulated hdmvec proliferation, and this effect was totally blocked by cgrp antagonist, cgrp 8-37; 3) quantitative analysis for cell migration revealed that cgrp increases hdmvec migration in a dose and time-dependant manner; and 4) cgrp promotes hdmvec capillary like tube formation, and the length of capillary tube induced by cgrp (10 -8 m) was significantly increased over that of the untreated controls. this increase was observed at 12 hours of treatment and further increase was noted at 24 hours of culture. conclusion: cgrp induces in vitro angiogenesis by promoting microvascular endothelial cell proliferation, migration and capillary like tube formation. therefore, trophoblast derived cgrp at the implantation site may play a role in placental angiogenesis and fetal growth. over-expression of socs-3 gene promotes il-10 production by jeg-3 trophoblast cells. qin dong, 1,2 ruping fan, 2 yang gu, 2 david f lewis, 2 yuping wang. 2 1 biochemistry, harbin medical university, harbin, heilongjiang, china; 2 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: suppressor of cytokine signaling-3 (socs-3) plays an important role in negative regulation of inflammatory response in biological cells. evidence has shown anti-inflammatory cytokine il-10 expression was significantly reduced in trophoblasts of preeclamptic (pe) placentas. we sought to determine if over-expression of socs-3 in placental trophoblasts could promote il-10 production. methods: full-length socs-3 open reading frame (socs-3 cdna) was generated by rt-pcr from total rna samples isolated from human leukocytes and cloned into a pzsgreen1-n1 vector, which encodes a green fluorescent protein zsgreen1. successful socs-3 cloning was confirmed by sequencing. for transfection, jeg-3 cells were placed into 6 well/cluster plates at a concentration of 1.2 x105/well. the tranfection was carried out with 1.0 g of socs-3/zsgreen1 plasmid (psocs-3/zsgreen1) for 6 hours when cell reached 45-50% confluence. siport lipid were used. jeg-3 cells transfected with zsgreen1 plasmid (pzsgreen1) only was used as control. after approximately 60 hours of transfection, cells were treated with il-6 at 1 and 10ng/ml. medium was then collected and measured for il-10 by elisa. il-10 production was calculated as the percentage of increase by psocs-3/zsgreen1 transfected cells compared to the cells transfected with pzsgreen1 only. result: il-10 production was increased by psocs-3/zsgreen1 transfected jeg-3 cells compared to the cells transfected with pzsgreen1 only when stimulated by il-6, control: 9.9% increase; 1ng/ml il-6: 28.5% increase and 10ng/ml il-6: 140% increase, p< 0.05, respectively. data are means from three independent experiments. conclusion: over-expression of socs-3 gene could promote il-10 production by placental trophoblast cells. reduced sil-6r release and increased ratio of sgp130/sil-6r production by placental tissues from women with preeclampsia. shuang zhao, 1 ruping fan, 1 jingxia sun, 2 yang gu, 1 david f lewis, 1 yuping wang. 1 1 obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa; 2 obstetrics and gynecology, first hospital, harbin medical university, harbin, heilongjiang, china. objective: placental tissue/trophoblasts release more inflammatory cytokines (il-6, il-8 and tnf ) in preeclampsia (pe) than in normal pregnancies. however, the reason for increased inflammatory cytokines released by pe placentas is not clear. soluble il-6 receptor (sil-6r) and membrane receptor il-6/gp130 complex play an important role in the negative regulation of cytokine signaling in suppressor of cytokine signaling (socs) pathway. in contrast, soluble gp130 (sgp130) is an antagonist for il-6/il-6r trans-signaling. this study was undertaken to determine sil-6r and sgp130 production by villous tissues from normal and pe placentas. methods: placentas delivered by 8 normal and 9 pe pregnant women were used in this study. placental explants were incubated with dmem for 48h. the culture medium was collected. placental villous tissue productions of sil-6r and sgp130 were measured by elisa. all samples were assayed in duplicate. data are expressed as mean ± se and analyzed by mann whitney test. a p level <0.05 was considered statistically different. results: placental tissues from pe produced significantly less sil-6r than tissues from normal pregnancies, 3.56±0.61 vs. 5.84±0.34 pg/mg of wet tissue, p<0.01. soluble gp130 production was relatively compatible between pe and normal placental tissues: 162.29±21.71 vs. 162.83± 5.56 pg/mg of wet tissue. the ratio of sgp130/sil-6r release was significantly higher in pe than in normal placentas, 49.84±6.93 vs. 28.62±0.02, p<0.01. conclusion: reduced sil-6r production and/or increased ratio of sgp130/sil-6r production by pe placentas suggest less cytokine inhibitory activity in pe placentas, which may contribute to the increased toxic cytokine production in placentas from pe. using chemiluminescence immunoassays. peter s uzelac, 1 jing dai, 2 frank z stanczyk, 2 daniel r mishell, jr. 2 1 obstetrics and gynecology, university of louisville, louisville, ky, usa; 2 obstetrics and gynecology, university of southern california, los angeles, ca, usa. objective: characterizing human chorionic gonadotropin (hcg) levels throughout normal pregnancy is critical to its use as a bio-marker for abnormal gestations. there is a paucity of data describing hcg trends during pregnancy and most of the relevant studies use older, less specific assays. our first objective was to characterize hcg levels throughout normal gestation using two different contemporary chemiluminescence immunoassays. our second objective was to compare hcg patterns in healthy gestations with pregnancies affected by diabetes, a common obstetrical complication. methods: a single blood sample was collected from 137 healthy pregnant women and 170 diabetic pregnancies. gestational age was confirmed by ultrasound. serum hcg levels were quantified by chemiluminescence immunoassays using the acs-180 and immulite systems. data was grouped in 4-week intervals until 16 weeks of gestation and 8-week intervals thereafter, with 21 to 27 samples in each interval for healthy women and 6 to 56 samples in each interval for diabetic women. paired t-test and wilcoxon rank sum test were used for statistical analysis. results: using the acs-180 system, mean hcg levels (miu/ml) for healthy pregnant women were 64,489 at 5-8 weeks, 100,421 at 9-12 weeks, 52,724 at 13-16 weeks, 16,741 at 17-24 weeks, 16,580 at 25-32 weeks, and 22,535 at 33-41 weeks. mean hcg levels obtained from the immulite system were similar. for diabetic pregnancies, mean hcg levels (miu/ml) were 45,165, 59,899, 45,448, 19,920, 20,372 and 19,525 , respectively. between 9-12 weeks of gestation, the hcg levels were significantly lower in diabetic pregnancies (p=0.02) compared to healthy controls. 3) compared to healthy pregnancies, diabetic gestations have significantly lower peak hcg levels. the cause of this difference is an area deserving further investigation. background: mouse and human placentae share several cellular and molecular features, including a haemochorial interface, allowing the murine labyrinth to be compared with the human fetal placenta. there is an autonomous embryonic ras from at least the time of implantation. ace converts angiotensin i to the active angiotensin ii (angii). angii's actions as a pro-inflammatory agent, in promoting cell migration, angiogenesis and cellular growth and apoptosis, strongly suggest a rôle in placentation. hypothesis: there would be counterregulation of the placental ras if the effect of ace were removed. this study investigated for the first time whether the knockout of somatic ace affected the expression and localisation of various components of the ras in the placentae of wild-type (wt/wt), heterozygous (wt/4) and ace knockout (4/4) mice. methods: immunohistochemistry (dako envision plus) was used to localise and semiquantify ang type 1 receptor (at1r), ang type 2 receptor (at2r), ace, and ace-2 in the three genotypes (n=3/group). placental sections were blinded to genotype; a score range of 0-4 was used. the 2 test was used (spss version 15.0) to analyse the difference in staining score by genotype. results: immunoreactivity of all antigens increased in the placental labyrinth of 4/4 mice compared to wt/wt and wt/4 mice (at1r p<0.05; at2r p<<0.001; ace p<<0.001; ace-2 p<<0.001). at2r and ace-2 displayed increased staining in the fetal vascular endothelium of 4/4 mice (at2r p<<0.001; ace-2 p=0.05), and in the cells lining the maternal central artery (at2r p<<0.001; ace-2 p<<0.001). ace-2 expression was very high in cytotrophoblast lining the maternal blood space in all genotypes. no gross structural differences were seen. comment: the antibody used did not differentiate between ace and the membrane-bound "testicular"-ace (tace). immunohistochemically-identified ace expression was upregulated in 4/4 placentae despite the loss of somatic ace, suggesting that t-ace can be expressed placentally. we believe this is the first demonstration of such expression. ace and t-ace are catalyticallysimilar in converting angi to angii. furthermore, angii acting via the at2rs is vasodilatory and ace-2 catalyses production of the vasodilatory ang (1-7); placental blood flow was presumably well-maintained and pregnancy outcome is normal in 4/4 mice. it is generally accepted that prostaglandin production plays a crucial role in both term and preterm parturition, and recently the administration of 17 alpha hydroxyprogesterone acetate has been shown useful in preventing preterm labor. what is not clear is the biochemical pathway of prostaglandin production during labor and what, if any, effect 17 alpha hydroxyprogesterone acetate has on this pathway. in order to address this question, we used immunohistochemical staining techniques to evaluate the effect of treating human placental amnion and chorion decidua with 17 alpha hydroxyprogesterone acetate. membranes from unlabored patients were obtained at cesarean section and immediately seperated into control and drug treated specimens. controls were from the same placenta and were subjected to all experimental procedures except for the addition of 17 alpha hydroxyprogesterone acetate to the culture media. specimens were compared at zero, six, and twenty four hour intervals. at the appropriate time, each specimen was formalin fixed and then paraffin blocked. tissue sections were then mounted on slides which were immunohistochemically stained using appropriate primary and secondary antibodies and standard techniques. the slides were then analyzed via light microscopy for changes in staining of three enzymes involved in prostaglandin production--cyclooxygenase 1 (cox-1), cyclooxygenase 2 (cox-2), and 15-hydroxy prostaglandin dehydrogenase (pgdh). compared to control, the slides treated with 17 alpha hydroxyprogesterone acetate had differing amounts of enzyme expression. cox-1 was relatively unchanged and pgdh was only slightly increased, but cox-2 was noticeably decreased in the treated slides. these results were time dependent. this data suggests that 17 alpha hydroxyprogesterone acetate decreases prostaglandin production in fetal membranes primarily by downregulation of the cyclooxygenase 2 enzyme. objectives: in the human placenta, proliferation, differentiation and fusion of cytotrophoblasts (ct) are essential events in the formation of the multinucleated syncytiotrophoblast, however the regulation of these processes is poorly understood. using an explant model of human first trimester placenta we have established that both igf-i and -ii enhance ct proliferation, differentiation and survival mediated via igf1r signalling. we have also shown that non-specific inhibition of protein tyrosine phosphatases inhibits igf-mediated signalling in trophoblast; therefore, we have now used sirna-mediated knockdown to investigate the role of the tyrosine phosphatase shp-2 in this pathway. methods: amaxa nucleofector technology was used to deliver shp-2 or scrambled sirna (100nm) to bewo cells or first trimester villous tissue fragments. knockdown was confirmed by q-pcr and western blotting. transfected cells and tissue were maintained in culture for 72 hours, then treated with igf-i or igf-ii (10nm) for a further 24 hours before immunohistochemical (ihc) analysis for cell proliferation (ki67, brdu) or apoptosis (m30). results: sirna-mediated knockdown of shp-2 in bewo cells (95% reduction on western blot) demonstrated that igf-induced proliferation was reduced from 67.3±3.5% to 41.9±2.4% (p<0.001, n=5). ihc analysis of tissue demonstrated that shp-2 is localised to ct. following knockdown (93% decrease by q-pcr), igf-i-and igf-ii-induced ct proliferation was decreased by 60.02±7.4% and 55.9±9.6 % respectively (p<0.001, n=4). furthermore, the ability of igf-i-and igf-ii to prevent ct apoptosis (m30 staining) was reduced by 48.3±10.1% and 39.1±7.9% respectively (p<0.05, n=4) after shp-2 knockdown. conclusions: igf stimulation of cytotrophoblast proliferation is mediated by shp-2. exogenous igf rescues cytotrophoblast from apoptosis, and this pathway is also shp-2-dependent. villous endothelial cells. emily j su, zhi-hong lin, ping yin, scott reierstad, joy innes, serdar e bulun. obstetrics and gynecology, northwestern university feinberg school of medicine, chicago, il, usa. background: within the human vascular system, estrogens have been shown to enhance vasodilatation in both normal and abnormal endothelium. estrogenic function occurs by activation of one or both of two estrogen receptors, estrogen receptor-alpha (esr1) and estrogen receptor-beta (esr2). these estrogen receptors are expressed in a wide variety of tissues. within the vasculature, estrogen receptors regulate the expression of multiple vasodilator and vasoconstrictor proteins. specifically, esr2 has been shown to be critical in maintaining normal vascular physiology in a murine model, where esr2 knock-out mice demonstrate significant systolic and diastolic hypertension. we hypothesize that within placental endothelium, estrogen plays an important role in maintaining normal vascular function that is critical for normal fetal growth and development. methods: term placentas from uncomplicated pregnancies were obtained, and the decidua was removed. an iv cannula was inserted into the umbilical vein, which was perfused with a collagenase/dispase solution. the perfusate was collected and subjected to further purification. these cells were cultured in complete medium, and after the initial passage of these cells, purity was confirmed via immunofluorescence and flow cytometric studies. estrogen receptor expression was determined in these cells via western blotting. additionally, these endothelial cells underwent treatment with varying doses of estradiol (10 -9 m to 10 -6 m), and quantitative real-time pcr was performed thereafter for mrna levels of various genes important in prostanoid production. results: western blotting demonstrated that esr2 is the only estrogen receptor expressed within villous placental endothelial cells. estradiol induced cyclooxygenase-2 (cox-2) mrna levels 3-to 6-fold, as quantified by real-time pcr (p<0.001). conversely, there was no effect of estradiol on cyclooxgenase-1 (cox-1). conclusion: these results suggest that estradiol and esr2 are important in mediating the balance of prostanoid production that is essential in maintaining placental vascular health. future studies will further delineate estrogenic effects on prostanoid production within placental endothelial cells in health and disease. supported by the smfm/aaogf scholarship award and the nih grant u54-hd40093. previously we established that proteins secreted by the decidua promote the differentiation of extravillous trophoblasts (evt) from a proliferative phenotype (characterized by cx40, her-1 and alpha5 integrin protein expression) to an invasive phenotype (characterized by her-2 and alpha1 protein expression). the ability of decidua-conditioned media (dcm) to induce trophoblast differentiation was inhibited in the presence of the her-1 receptor antagonist, ag1478. furthermore, dcm-induced jar cell migration was also attenuated in the presence of ag1478. thus, the purpose of this study is to define the role of her signaling in evt differentiation and invasion. methods: evt differentiation was assessed in placental villous explant outgrowths and jar cells using antibodies against markers of the proliferative and invasive phenotypes. trophoblast migration was assessed using jar cells in transwell migration assays. results: treatment of placental villous explants with egf, a her-1 ligand, resulted in the downregulation of her-1 and an upregulation of her-2 expression, as well as an induction of alpha1 integrin expression. pre-treatment of placental villous explants with ag1478 blocked this effect. in the jar cell line, egf treatment mimicked the differentiation-promoting effects of dcm by downregulating her-1 and upregulating her-2 expression, effects that were both blocked when jar cells were pre-incubated with ag1478. in contrast to dcm however, egf stimulation did not induce jar migration. stimulation of jar cells with hb-egf, a her-1/her-4 heterodimer ligand, induced jar migration in a dose-dependent manner. analysis of dcm using antibody arrays confirmed the presence of many members of the egf family including hb-egf. immunohistochemical assessments of placental villous explants verified the expression of her-4 in evt outgrowths and in jar cells; her-4 expression was not affected by stimulation with either egf or hb-egf. conclusions: her signaling is an important and necessary component of the invasive evt differentiation cascade. our data supports a role for her-4 signaling in the induction of the invasive evt phenotype. chandrasekhar thota, chandra yallampalli. obstetrics gynecology, university of texas medical branch, galveston, tx, usa. background: parathyroid hormone related peptide (pthrp) is expressed in trophoblast cells and may play a role in placental growth and function. studies conducted in pregnant rats using pthrp antagonist showed decreases in fetoplacental growth during mid gestation. objective: to assess the effects of pthrp silencing on the expression of growth factors in immortalized first trimester trophoblast cells (htr-8/svneo cells). methods: htr-8/svneo cells cultured at 37º c and 5%co2 in rpmi-1640 medium supplemented with 5% fbs were transiently transfected with 100nm of three different sirna sequences of pthrp, si1, auaccuaacucaggaaacuuu; s i 2 , g a g c u g u g u c u g a a c a u c a u u ; a n d s i 4 , caagauuuacggcgacgauuu. for control, a scrambled sirna sequence was used. at 90% confluency, cells from each well were split and transfected again in triplicates with respective sirna sequences. total rna was isolated using trizol reagent 24 h after transfection, and protein was extracted using lysis buffer 72 h after transfection. the isolated rna and protein were subjected to reverse transcription and polymerase chain reaction (rt-pcr) and western analysis, respectively, using primers and antibodies specific for pthrp, plgf, vegf and lif. the results are expressed relative to either 18s for changes in mrna expression or -actin for changes in protein expression. results: rt-pcr of total rna obtained from htr cells subjected to double transfection with sirnas for pthrp showed a significant decrease in pthrp expression. expression of growth factors plgf, vegf and lif showed decreases with all the three sirna sequences used compared to the scrambled sequence. western analysis of cell lysates obtained from htr cells subjected to transfection with sirnas for pthrp showed a significant decrease in protein expression of pthrp and vegf. however the protein expression for plgf, and lif decreased in cells transfected with only si4 sequence of pthrp. conclusions: our studies showed that transient transfection of htr cells with sirna for pthrp caused decreases in both mrna and protein expression of pthrp. our results further suggests that decrease in pthrp peptide in transfected cells resulted in a decrease in vegf, plgf and lif suggesting that pthrp may play role in regulating trophoblast cell functions. objective. maternal obesity poses an increased risk to the fetus during pregnancy, and has long term consequences for the progeny. we tested the hypothesis that maternal caloric excess effects growth-related gene expression changes in the murine placenta. methods. female c57bl/65 mice were fed a hypercaloric diet (20% fat, 38% sugar) or standard chow for six weeks prior to mating and throughout pregnancy. near-term (day 18 gestation) the dams (4 controls, 4 overfed) were sacrificed. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on regulated genes. results. maternal overfeeding was associated with a two-fold increase in body fat mass. 41 probe sets, corresponding to 37 genes showed differential expression (p < 0.01); twenty-seven of which were up-regulated, and ten down-regulated, as compared to the placenta of control fed dams. of note, several genes related to obesity, diabetes, dna methylation, and the tgf beta pathway were differentially expressed. conclusions. diet-induced obesity in mice was associated with altered placental gene expression, including genes involved in tgf beta signaling and dna methylation pathways. these findings may have important implications for placental growth and epigenetic regulation. (supported by usphs hd-03807, earnest eu framework 6, tommy's the baby charity, uk). objective. maternal dietary protein restriction has been shown to have deleterious effects on placental development, and has long-term consequences for the progeny. to comprehend more completely stress responses to maternal protein restriction, we measured gene expression changes in the mouse placenta. methods. pregnant fvb/nj mice were fed an isocaloric diet containing 50% less protein than normal chow (10% vs. 20% protein content) from embryonic day 10.5 (e10.5) to e17.5. following placental rna extraction, we used the affymetrix mouse 430a_2.0 array to measure gene expression changes. we performed pathway analysis on the regulated genes, and used both qrt-pcr and immunohistochemistry to verify the results. results. the weights of the e17.5 pups were decreased 15% (p<0.05). 244 probe sets, corresponding to 235 genes, were regulated by protein restriction (p<0.001); ninety-one being up-regulated and 153 down-regulated. of particular note, several genes related to the p53 pathway were up-regulated. along with p53 itself, positive regulators of p53 (zmiz1, jmy, hipk2) and genes activated by p53 (inpp5d, cebpa) were induced. for selected genes we confirmed these results using qrt-pcr and immunohistochemistry. conclusions. by microarray analysis, we have described the genetic response to maternal protein deprivation in the mouse placenta. we observed that pups were growth restricted, and genes related to the p53 pathway were regulated. we propose a model through which intrauterine growth restriction is triggered, in part, by activation of the p53 pathway. (supported by usphs hd-03807 and the sgi medical student grant to cpg). purpose: human placental villous tissue cultures have been underused in the study of placental drug disposition. thus we assessed the utility of this model by studying the effect of time in culture on the viability and integrity of the tissue and the, expression and function of proteins involved in the formation and efflux of 1-chloro-2,4-dinitrobenzene (cdnb) conjugate 2,4-dinitrophenyl-s-glutathione (dnp-sg) as a model system for phase ii metabolism and cellular efflux. methods: placental tissue samples were obtained within 30 minutes of cesarean deliveries following normal pregnancies in three patients. villous tissue was cultured in m199 medium to 48hr. at 2, 4, 6, 10, 24, and 48hr post culture, villous tissue was preincubated without or with atpase inhibitor sodium orthovanadate, exposed to 100 μm cdnb, rinsed and incubated in buffer at 37°c to determine formation and efflux of dnp-sg, which was assayed by hplc. changes in expression of gstp1-1, abc transporter isoforms b1, c2 and g2 (abcb1, abcc2, and abcg2, resp.) were assessed by immunoblotting. lactate dehydrogenase (ldh) release, methyl tetrazolium thiazolyl blue (mtt) incorporation, and total tissue glutathione content were monitored up to 48hr. villous tissue morphology was assessed by immunohistochemistry. results: villous tissue structure and protein expression of glutathione-stransferase isoform p1-1 (gstp1-1) and abcg2 remained unchanged over 48hr in culture. expression of abcb1 and abcc2, and total tissue glutathione decreased with culture time. ldh release was unchanged up to 24hr and increased at 48hr, while mtt incorporation remained constant to 10hr and decreased at 24 and 48hr suggesting a decline in tissue integrity and viability at 48hr. however, dnp-sg formation, dnp-sg buffer/tissue ratio, and the extent of inhibition of dnp-sg efflux by sodium orthovanadate remained unchanged through 48hr. sodium orthovanadate decreased the dnp-sg buffer/tissue ratio by 70.5 ±6.90% (p<0.05), consistent with inhibition of apical abc transporters. conclusions: these results support the use of this model to study the coordinated function of metabolizing enzyme gstp1-1 and apical abc transporters in the formation and efflux of the model substrate dnp-sg. the model may be useful to study metabolism and transport of other compounds. syncytiotrophoblast. shauna f williams, ewa fik-rymarkiewicz, stacy zamudio, nicholas p illsley. obstetrics, gynecology and women's health, umd-new jersey medical school, newark, nj, usa. introduction: multiple inputs influence placental protein synthesis. nutritional, endocrine and metabolic factors have been implicated but its regulation has not been investigated. one of the factors shown to be associated with the inhibition of protein synthesis is hypoxia. the goal of this study was to determine the effects of hypoxia on a marker of placental protein synthesis. eukaryotic initiation factor 2 (eif2 ) is a subunit of eif2 which is required for initiation of translation however when phosphorylated, eif2 is unable to participate in the assembly of the initiation complex. hypoxia has been shown previously to cause increased phosphorylation of eif2 . we hypothesized that hypoxia would increase the levels of phosphorylated eif2 in term syncytiotrophoblast, thus inhibiting protein synthesis. methods: primary syncytiotrophoblast cultured from term cytotrophoblast were incubated for 18 hr in atmospheres of 1, 3, 5 or 10% o 2 or in the presence of the hypoxia-mimetic, dimethyloxalylglycine (dmog, 0.02-2.0 mm) in 20% o 2 . cell extracts were analyzed by western blotting to determine the degree of eif2 phosphorylation. results: incubation in 1, 3, or 5% o 2 did not increase eif2 phosphorylation relative to the 10% o 2 control (n=4, separate placental preparations). incubation in dmog concentrations up to 0.5 mm did not affect eif2 phosphorylation however incubation in 2.0 mm dmog increased eif2 phosphorylation by 58 ± 15% (p < 0.05, n=3). conclusions: contrary to our expectations, inhibition of protein synthesis via the eif2 regulatory pathway was not apparent except when induced by the highest concentration of dmog, consistent with severe hypoxia. thus while eif2 phosphorylation does occur, we did not observe changes at dissolved oxygen levels of 1%. these data suggest that a reduction in syncytial protein synthesis via the eif2 pathway takes place only under severe hypoxic stress. (supported by nih hd46982). the increasing prevalence of overweight and obese women of childbearing age is a growing public health concern. the impact of maternal obesity on placental aa transport, which is essential for normal fetal development, remains poorly defined. there are three sub-types of the placental na-dependent system a transporter, snat1, 2 and 4 which mediate neutral aa transport. snat2 is ubiquitously expressed in mammalian tissues and is likely responsible for the majority of placental system a activity. objective: to examine the impact of maternal obesity and over-nutrition on the fetal: maternal (f:m) aa ratio and placental protein abundance for snat2. methods: nonpregnant ewes were randomly assigned to a control (c, 100% of nrc recommendations) or obesogenic (ob, 150% of nrc) diet from -60 to 75 days of gestation (dg). under isofluorane anesthesia, maternal and fetal blood samples were collected for aa analysis by hplc from five twin bearing ewes in each dietary group. after euthanasia, placental cotyledonary (cot) tissue was separated from caruncular tissue, frozen in liquid nitrogen and stored at -80 0 c for western blot analysis. results: fetuses from ob ewes were 26% heavier (p<0.05) than those from c ewes at 75 dg (234± 7 vs. 186±7g). blood concentrations of asn, thr, cit, arg, tau, tyr, trp, val, phe, leu and orn were higher (p<0.05), or tended to be higher (met and lys, p<0.10) in ob than c ewes. in contrast, f:m ratios, for asn, ser, gln, his, gly, thr, cit, arg, b-ala, tau, ala, tyr, trp, met, val, phe, leu, orn and lys were reduced (p<0.05) in ob compared to c ewes. snat2 content in cot tissue was reduced in ob when compared to c ewes (0.5±0.1 vs. 1.0±0.2 arbitrary units; p<0.05). conclusions: maternal obesity in pregnancy reduced expression of placental snat2 protein and efficiency of placental aa transport in ewes, providing a mechanism whereby fetuses may mitigate excessive delivery of aa under conditions of maternal obesity and over-nutrition. decreased aa transport to the fetus may play a role in altered cellular structure and function. nih inbre 1p20rr16474. early gestation utero-placental hemodynamics in an ovine model of fetal growth restriction. lucia dohnal, james s barry, henry l galan, randall b wilkening, russell v anthony. perinatal research center, university of colorado health sciences center, aurora, co. objective: fetal growth restricted (fgr) pregnancies, during late gestation, exhibit altered placental hemodynamics, and reduced capacity for o 2 and nutrient transfer. it was our objective to examine utero-placental hemodynamics and o 2 uptake during early gestation in an ovine model of fgr. methods: singleton-bearing ewes were instrumented with uterine artery flow probes, uterine venous and femoral artery catheters before being placed into a highambient temperature (fgr; n=9) or normothermic (con; n=6) environment at 40 days of gestation (dga). maternal arterial and venous blood, uterine artery flow, heart rate, arterial pressure and respiration rate was collected until 55 dga, at which time umbilical venous blood, fetal weight, placental weight and tissue were harvested. data reported here were analyzed by students t-test. results: maternal respiration rate (153.3±5.4 vs 91.6±9.2 breaths/min) and arterial po 2 (91.0±1.2 vs 85.8±1.0 mmhg) were increased (p 0.01), whereas maternal heart rate (74.3±1.83 vs 88.8±0.03 beats/min), blood pressure (83.7±1.3 vs 94.0±3.2 mmhg) and arterial pco 2 (30.2±1.1 vs 35.6±0.9 mmhg) were reduced (p 0.01) in fgr pregnancies. at 55 dga, fetal weight was not different (p 0.10), but placental (total placentome) weight (85.5±15.1 vs 146.7±27.0 g) was reduced (p 0.05) in fgr pregnancies. while uterine artery (pregnant horn) flow (115.5±13.4 vs 178.4±54.3 ml/min) tended (p=0.064) to be reduced in fgr pregnancies, relative uterine artery flow (4.6±0.5 vs 5.8±0.5 ml/min/g fetus; 157.6±25.8 vs 128.1±17.3 ml/min/100g placenta) was not different (p 0.10). uterine o 2 uptake (mmol/min), relative uterine o 2 uptake (ml/min/g fetus or ml/min/100g placenta) and uterine o 2 extraction (%) were not different (p 0.10) between fgr and con pregnancies. at 55 dga, umbilical vein po 2 (mmhg), o 2 content (mm) and o 2 capacity (mm) were also not different between fgr and con pregnancies. conclusions: reduction in absolute uterine artery flow (ml/min) did not impact utero-placental o 2 uptake or transfer to the umbilical vein, and may have resulted from reductions in maternal cardiac output. relative uterine artery flow was not reduced, suggesting that uterine blood flow and delivery of o 2 to the conceptus does not mediate the ongoing placental growth restriction initiated during early gestation. supported by nih hd43089. barton c staat, 1 anna maria marconi, 2 cinzia paolini, 2 alex cheung, 1 henry l galan, 1 frederick c battaglia. 1 1 obstetrics gynecology and pediatrics, univ of colorado at denver health sciences center, aurora, co, usa; 2 dept of obstetrics and gynecology, san paolo institute of biomedical sciences, university of milano, milano, italy. objective: to determine relative contributions of transplacental flux vs fetal production for myo-inositol and mannose in normal term pregnancies using stable isotopic methodolgy. background: myo-inositol and mannose are important in biologic functions. an external supply of mannose may be required for glycoprotein synthesis. low maternal myo-inositol is associated with spina bifida. mannose concentrations are known to be higher in the mother than the fetus. in contrast, myo-inositol concentrations are higher in the fetus than the mother. what remains unknown is whether fetal levels of these polyols are a result of direct maternal transport or from conversion of glucose. design: four term uncomplicated pregnancies undergoing an elective ceasaran section were infused with 13 c labeled isotopes of glucose, myo-inositol and mannose over 2 hours prior to delivery. maternal samples were obtained prior to infusate being administered, and at 1 hour (h), 1.5h and 2h. fetal concentrations were measured from umbilical artery and vein plasma. the concentrations of labeled and unlabeled glucose, mannose and myo-inositol were measured using high pressure anion exchange chromatogrpahy permitting detection of 10 polyols and sugars at concentrations in the μm range. the feto-maternal molar percent enrichment (mpe) ratio was calculated for each glucose, mannose, and myo-inositol as the ratio between fetal plasma enrichment and the maternal plasma enrichment at steady state. steady state was calculated as the mean of the three maternal samples taken during infusion. results: the feto-maternal mpe ratios of mannose (0.985 ± 0.05, p=0.03) and glucose (0.917± 0.06, p=0.002) were not significantly different from 1.0, consistent with transplacental supply. the feto-maternal ratio for myo-inositol (0.1 ± 0.05, p=0.02) indicates little transplacental flux (10% of fetal inositol derived from maternal plasma). conclusion: in normal term pregnancies, fetal mannose and glucose concentrations are dependent upon maternal transplacental supply. in contrast, fetal myo-inositol concentration is not dependent upon transplacental supply, but fetal demands are met by placental conversion, likely from glucose. christian wadsack, 1 manuela augsten, 1 christian guelly, 2 ursula hiden, 1 ingrid lang, 3 manfred moertl, 1 uwe lang, 1 gernot desoye. 1 1 clinic ob/gyn; 2 center of med res; 3 inst cell biol, histol embryol, med univ graz, austria. background placenta and fetus need lipids for growth and synthesis functions. part of the lipids is supplied from maternal sources by transplacental transfer. recently, we identified in human placenta the high density lipoprotein (hdl) receptor scavenger receptor class b type i (sr-bi). among other functions it mediates hdl-induced ser1177-phosphorylation of endothelial nitric oxide synthase (enos) resulting in enos activation. this mechanism allows hdl to contribute to regulation of vasotonus in arteries. we hypothesized that term placental endothelial cells (ec) express sr-bi at levels different between arteries and veins. methods sr-bi was localized by ihc and quantified by qrt-pcr in rna isolated from arterial and venous vessels. arterial (eca) and venous (ecv) placental ec were rigorously characterized. sr-bi levels were measured by qrt-pcr and immunoblotting. hdl binding and uptake was measured in eca and ecv with 125 i-labelled hdl. hdl from human donors was used to stimulate ser1177 enos phosphorylation. epigenetic regulation was studied by methylationspecific pcrs for 4 cpg-rich promoter regions of sr-bi. pdzk1 a key adaptor for sr-bi mediated enos activation was measured by sqrt-pcr. in situ analyses (ihc, qrt-pcr) showed more sr-bi in arteries than in the vein. the differential expression persisted in vitro in isolated eca and ecv even after 7 passages and culture under same conditions suggesting epigenetic mechanisms regulating sr-bi. however, no methylation was found in eca or ecv. sr-bi was functional since hdl cell association was 2-fold higher in eca than in ecv (89 ± 0.4 vs 49 ± 4.1 ng hdl/mg prot). hdl did not induce ser1177 enos phosporylation in eca or ecv, which was stimulated by ionomycin about 3-fold in both cell types. pdzk1 was undetectable in eca and ecv, whereas it was expressed in placental tissue. conclusion more sr-bi is expressed in ec from arteries than from veins in situ and in vitro. this is not the result of different methylation of sr-bi promoter and, hence, unlikely an epigenetic phenomenon. mechanism of differential expression and its functional consequences for vasotonus regulation is yet unknown. the lack of pdzk1 may account for the failure of hdl to activate enos. (grants 10053, 10896, 11165 oenb). cells. juan a arroyo, brad ziebell, mi-hye park, henry l galan. obstetrics and gynecology, university of colorado andgealth sciences center, aurora, co, usa. introduction: mtor is a protein that regulates cell growth in response to nutrients and growth factors. downstream effectors of the mtor pathway include the p70 and the 4ebp1proteins. activation by phosphorylation of these proteins increases protein synthesis. given that various signaling pathways are regulated by hypoxia in human trophoblast and that mtor is expressed in human trophoblast, our objective was to determine the effects of hypoxia in the activation of mtor, p70 and 4ebp1 in cultured human trophoblast. study designs: trophoblast cell were isolated from term uncomplicated placentas using a trypsin, dnase and disapase solution. cytokeratin immunocytochemistry confirmed trophoblast cells culture purity. trophoblast cells were treated with hypoxia (2% o 2 ) or normoxia (21% o 2 ) for 24 and 48 hours. western blot for p-mtor, mtor, p-p70, p70, p-4ebp1, and 4 ebp1 were done for each time studied. results: trophoblast cells demonstrated: 1) positive staining for cytokeratin, 2) non significant differences for mtor at either 24 (1.5-fold; p= 0.128971) or 48 hours (1.4-fold, p= 0.13181), 3) no differences in p70 protein at 24 (1.1fold; p= 0.128971) or 48 hours (1.3-fold, p= 0. 0.153458), 4) no differences for 4ebp1 at either 24 or 48 hour. conclusion: we conclude that the mtor pathway is not regulated under hypoxic conditions in cultured trophoblast, which suggests that hypoxia does not affect protein synthesis in cultured human trophoblast. however, this may not reflect what happens in vivo in iugr. (supported by nih grant r01 hl071990-01a1). increased expression of phospho-mtor, phospho-p70, phospho-akt and phospho-erk in an ovine model of fetal growth restriction. juan a arroyo, brad ziebell, henry l galan. obstetrics and gynecology, university of colorado and health sciences center, aurora, co, usa. objective: both phosphorylated (p) mtor and p70 are known to be involved in protein synthesis and are regulated by physiological conditions such as fetal growth restriction (fgr). in a hyperthermic (ht) ovine model of fgr we hypothesize that mtor, p70, 4ebp1, erk and akt will be phosphorylated (activated) in the placentae of 130 age (dga) animals. study design: 4 ewes were exposed to ht conditions for 80 days to induce iugr and 4 were placed in ambient conditions. at necropsy (130 dga), placentomes were separated into the maternal (caruncle) and fetal (cotyledon) components and frozen for western blot analysis with antibodies against (p) mtor, mtor, (p) p70, p70, (p) 4ebp1, 4ebp1, (p) erk, erk, (p)akt and akt. results: compared to control animals, fgr animals had smaller fetuses (2914±201g v. 1718±433g; p=0.03) and smaller placentae (349±21g v. 169±22g; p=0.03) at 130 dga. fgr cotyledon showed an increase in p-mtor (1.8-fold; p=0.01), p-p70 (1.8-fold; p<0.008), p-erk (1.4-fold; p<0.008) and p-akt (2.6-fold; p<0.02). in contrast, caruncle (maternal) did not show any changes for the mtor pathway. conclusion: in fgr ovine pregnancies, the fetal placental tissues (cotyledons) showed upregulation of the mtor pathway for protein synthesis via phosphorylation of the p70 but not 4ebp1 while this was not seen in the maternal (caruncle) tissues. in addition neither the cotyledon or caruncle tissues at mid-gestation (95 dga) showed changes in these endpoints, which is prior to the exponential fetal growth that starts at mid-gestation. (supported by nih grant r01 hl071990-01a1). hyperuricemia has long been recognized as a common clinical finding in preeclamptic (pe) women. to date, elevated uric acid concentrations in these women have been considered a marker of disease severity. however preeclamptic pregnancies with hyperuricemia, are associated with an increased frequency of preterm birth and fetal growth restriction. over the past decade several pathogenic roles for uric acid have become evident, raising the possibility of a role(s) for uric acid in the altered vascular and placental functions associated with pe. objective: examine the effects of syncytial uric acid uptake on system a amino acid transport across the human placenta using a primary placental villous explant model. methods: placental villous explants from placentae of healthy, term pregnancies were incubated for 2 hours with uric acid (8.3 mg/dl), corresponding to concentrations of uric acid observed in pe women. these experiments were conducted in the presence or absence of probenecid (10 m), a uric acid cellular uptake inhibitor. system a amino acid transport was subsequently assayed using a radiochemical assay in which na+-dependant uptake of radio-labeled system a substrate, [14c] methyl-amino-isobutyric acid, was measured over 20 minutes. data were analyzed using a paired student's t-test and presented as mean ± sem. results: uric acid attenuated system a amino acid placental transport by 33% (±0.06%, p<0.05). this inhibitory effect of uric acid on system a activity was prevented by probenecid. conclusions: uric acid reduces placental amino acid transport at concentrations observed in pe women. this inhibitory effect of uric acid is dependant upon syncytial uptake of uric acid, being inhibited by the uric acid transporter inhibitor probenecid. these results may be relevant to the increased frequency of fetal growth restriction observed in hyperuricemic pe. additionally the results of this study, indicating a detrimental effect of hyperuricemia on placental function, also suggest a role for uric acid in the pathophysiology of pe. hyperuricemia, a well-documented clinical finding in preeclamptic women, is associated with pre-term birth and intrauterine growth restriction. uric acid is higher in women destined to develop preeclampsia as early as 10 weeks of gestation at a time when cytotrophoblast are invading decidua and myometrium and remodeling uterine spiral arterioles. we propose that elevated concentrations of uric acid may have detrimental effects on placental development in part through inhibition of trophoblast invasion through the decidua. objective: examine the effects of increasing concentrations of uric acid on trophoblast invasion through a reconstituted extracellular matrix. methods: using the in-vitro matrigel invasion assay, the effects of increasing concentrations of dissolved uric acid (4.5 mg/dl, 6.4 mg/dl and 8.3 mg/dl) on the ability of immortalized first trimester extravillous trophoblast cells (htr8-svneo) to invade through a reconstituted extracellular membrane were assessed. the concentrations of uric acid used were comparable to those measured in healthy pregnant women and preeclamptic women with an increase in uric acid of two or four standard deviations above normal. cells that successfully invaded through the matrigel membrane within 48 hours were fixed with methanol, stained with hematoxylin and counted. data were analyzed using a one-way analysis of variance with fisher's post-hoc analysis. results: uric acid attenuated trophoblast invasion in a dose-dependent fashion (p<0.05), with decreases of 29% (± 5.6%), 58% (± 3.6%) and 71% (± 2.2%) respectively compared to untreated controls. conclusions: exogenous uric acid, at physiological and pathological concentrations, is capable of attenuating trophoblast invasion through a reconstituted extracellular membrane in a dose dependent fashion. these results suggest uric acid is a potential contributor to the pathophysiology of altered placental perfusion in preeclamptic pregnancies. previous studies have shown that transforming growth factor (tgf)-is a key inhibitory factors in the invasion of early trophoblast cells, suggesting therefore that overcoming tgf-beta signaling may be necessary for successful implantation. smad ubiquitin regulatory factor 2 (smurf2), a hect type e3 ubiquitin ligase, is a key regulator of tgf-signaling pathway, targeting tgf-receptors and various smads for proteasome-mediated degradation. in this context, smurf2 has been shown to play important roles in embryonic development, cell senescence and tumor formation. as a key regulator of tgfbeta signaling, we wished to determine whether smurf2 has a physiological role during embryo implantation, especially in trophoblast invasion. we have examined the spatio-temporal expression of smurf2 in human placental villi during pregnancy. we have also investigated the possible function of smurf2 in trophoblast cell migration and invasion in a model system involving a human extravillous trophoblast cell line, htr8/svneo. our results showed that expression of smurf2 in placental villi was the highest during the first trimester and the expression decreased in the 2nd trimester. expression of smurf2 was lowest in placental villi at parturition. overexpression of smurf2 in htr8/svneo cells reduced tgf beta type i receptor levels and attenuated the inhibitory effect of tgf-on cell migration and invasion. conversely, rnai-mediated down-regulation of smurf2 resulted in significant increase of tgf-type i receptor protein levels. in contrast, the levels of smad2, another potential target of smurf2, was unchanged. in conclusion, the present study suggests that smurf2 participates in trophoblast cell migration and invasion by down-regulating the expression of tgf-type i receptor. our previous data demonstrated the extensive expression of mmp-26 in various kinds of trophoblast cells in human placenta at the early pregnancy. however, the modulation of the enzyme in trophoblasts is largely unclear. in the present study, the effects of the two types of gonadotropin releasing hormone (gnrh) on mmp-26 expression were examined in an immortalized human cytotrophoblast cell line, b6tert-1 that has been established in this lab. real-time quantitative pcr and western blot analysis revealed that both types of gnrh (gnrh i and gnrh ii) could increase mmp-26 mrna and protein levels in b6tert-1 cells in time-dependent manners. in particular, regulatory effect of gnrh i on mmp-26 expression was concentration-independent, whereas that of gnrh ii was dose-dependent. moreover, both gnrh i and gnrh ii could evidently activate jnk kinase, and sp600125, an inhibitor of a jnk kinase, reversed the up-regulation of mmp-26 induced by either gnrh i or gnrh ii. on the other hand, it is not likely that erk1/2 pathway participates in the signaling of gnrh i or gnrh ii. collectively, our observations suggest that gnrh i and gnrh ii elicit their modulation effects in human trophoblastic cells through jnk pathway leading to up-regulation of mmp-26. during the first trimester of pregnancy, the oxygen tension of the developing trophoblast cells is less than 5%. however, the majority of studies on primary trophoblast cell development have been performed at 20% oxygen. primary third-trimester trophoblast cells are believed to be nonproliferative syncytiotrophoblast cells. we have previously demonstrated that low oxygen tension dramatically affects the differentiation pathway of these cells. we now hypothesize that cell culture in low oxygen tension will improve cell growth and restore proliferation. methods: primary trophoblast cells were purified from third-trimester placenta by enzymatic dispersion and cd-9 negative selection and cultured at 20%, 5% or 2.5% oxygen tension for up to 5 days. the number of cells in culture was assessed by cell counting and by measuring genomic dna. live:dead and mtt assays were used to determine viability. proliferation was assayed with brdu and immunohistochemistry for proliferating cell nuclear antigen. to assess cellular activity, radioactivity of protein precipitated from cells cultured in the presence of tritiated leucine was measured. results: there were no obvious morphologic changes in the cells cultured in different oxygen tensions. the amount of cell loss was directly proportional to oxygen tension: at 20% oxygen 60% of the cells remain in culture; at 2.5% oxygen tension 80% of the cells remained. the cells at 2.5% oxygen tension were proliferating and had a five-fold increased metabolic activity. conclusions: it was previously believed that third-trimester trophoblast cells are non-proliferative. we have demonstrated that low oxygen tension increases the survival of primary third-trimester trophoblast cells. this may reflect the change in the differentiation pathway of these cells. however, the cells also begin to proliferate and increase their metabolic activity. trophoblast cells in vivo form a three dimensional structure which promotes critical complex cell-to-cell interactions that cannot be studied with traditional monolayer cell culture. we developed a substrate-free three-dimensional trophoblast culture system capable of studying cellular interactions without a confounding artificial matrix. methods: nonadhesive agarose hydrogels containing 822 cylindrical recesses 200 m in diameter were cast from molds designed using computer-assisted prototyping. tcl trophoblast cells were seeded into the gels (800,000 cells per) for up to 10 days. viability and cellular stress were assessed and the threedimensional structures of the spheroids were analyzed. results: tcl trophoblast cells formed uniform spheroids within three days of seeding. the spheroids remained intact after being removed from the mold. when placed in traditional cell culture dishes the cells adhered to the plate within one hour and rapidly proliferated into a monolayer. repetitive reseeding allowed easy transition between monolayer and spheroid without affecting cellular morphology. serial sectioning on days 3, 7 and 10 revealed central vacuolization forming a trophoblast vesicle with an outer rim 12.3 m (+/-1 m) thick. this rim size remained constant for at least 20 days. live:dead assay demonstrated that the outer cells remained viable and staining against proliferating cell nuclear antigen demonstrated that the cells were proliferating. the inner cells undergo apoptosis as demonstrated by caspase-3 staining. there is an abundance of vegf staining in the cells remaining in the on the inside of the sphere suggesting a gradient of nutrient or oxidative stress. the formation of a vesicle has been confirmed with confocal imaging. em imaging revealed the structure of the rim. conclusions: trophoblast cells cultured in a novel substrate-free three dimensional system form trophoblast vesicles within 7 days of seeding. these vesicles remain viable after long-term culture and can be repeatedly reformed with repetitive seeding. this new cell culture technique allows us to better study placental cell-cell interactions with the potential of forming microtissues. the transcription factor glial cell missing-1 (gcm1) mediates cell cycle arrest and differentiation of human trophoblast progenitors into villous syncytiotrophoblast and invasive extravillous cytotrophoblast (evt). micro-array analysis of total rna extracted from cultured bewo cells, in which gcm1 mrna and protein were repressed using sirna, identified tissue inhibitor of metalloproteinase-4 (timp-4) in the highest (4-fold) upregulated group of genes. confirmatory rtpcr demonstrated a 7-fold mrna induction. in placental villi, gcm1 acts as a transcription factor promoting expression of the fusogenic protein syncytin1 that mediates syncytial fusion into the overlying syncytiotrophoblast. by contrast, syncytial fusion is uncommon in evt. rather these cells invade several millimeters into the distal myometrium where they transform spiral arterioles. to investigate the role of timp4 and gcm1 in the trophoblast we assessed its mrna by qrt-pcr and protein by western blot in cellular extracts from both bewo cells grown under standard cultivation conditions (synchronized by prior thymidine exposure) and floating cultured first trimester villous explants cultured in 8% oxygen with prior exposure to either gcm1 sirna or anti-sense oligo-nucleotides to gcm1. gcm1 inhibition in the bewo system was associated with a 50-70% increase in timp-4 protein expression and alteration of cell proliferation and differentiation in both models. we are presently utilizing the explant model of evt invasion (explant tips cultured on matrigel in 3% oxygen) to test the hypothesis that gcm1 mediates metallo-proteinase expression and evt invasion via timp-4. presently we conclude that gcm1-mediated evt differentiation involves more than an arrest of mitosis and may include promotion of invasion via repression of timp-4. funding: cihr. scott h purcell, 1 jeremy d cantlon, 1 virginia d winn, 2 russell v anthony. 1,2 1 colorado state university, fort collins, co; 2 university of colorado health sciences center, aurora, co. background: periattachment factor (pf) is a nuclear protein first described in the bovine conceptus. our research in sheep has shown pf mrna concentration peaks when the conceptus is undergoing elongation and initial apposition to the endometrium, and that pf is a nuclear protein localized to the trophoblast. in silico analysis identified a human homolog, hprr15. objective: the objective of this experiment was to determine if pf was expressed in the human placenta, and to develop short-hairpin (sh) rnas for hpf to begin investigating its function. materials and methods: immunohistochemistry was performed on paraffin embedded first and second trimester human placental samples. placental sections were immuno-stained using rabbit polyclonal antiovine pf or anti-human cytokeratin-7. cytotrophoblasts from first trimester pregnancies (n=5) were subjected to an in vitro invasion assay and rna was harvested following 0, 3, and 12 h. quantitative rt-pcr was performed on these samples with intron-spanning primers for hpf, and normalized on hs15 mrna concentrations. based on the human pf sequence, four putative shrna constructs were generated and cloned into a lentiviral expression vector. bewo human choriocarcinoma cells were treated with one of four shrna contructs or an empty vector for 72 h and then rna was harvested from cells for analysis by quantitative rt-pcr. results: periattachment factor was present in the nuclei of both first and second trimester cytotrophoblasts. hpf mrna concentration increased as invasion occurred from 0, 3, to 12 h in all samples; while hypoxia decreased expression at 18 h of invasion compared to 18 h under normoxic conditions. the four lentiviral vectors expressing shrna against hpf resulted in hpf mrna concentrations at 2, 18, 83 and 97% of hpf mrna concentration with the control vector. conclusion: the presence of pf in the human placenta and the increase in pf mrna during cytotrophoblast invasion may indicate this gene plays a role during implantation. we have developed shrnas against pf that result in greater than 98% mrna knockdown and will be using these to begin to elucidate the function of pf in the human placenta, specifically during the invasion process. recently we demonstrated that infusion of imd antagonist (imd 17-47 ) in rat caused distorted labyrinth indicative of a deficient vasculature in placenta. we hypothesize that imd has a role in migration of first trimester trophoblast cell (htr-8/svneo) via regulating human leukocyte antigen (hla-g) and stimulating mek1/2/ erk1/2 phosphorylation. objectives: 1) to asses the effect of imd on migrating capacity of htr-8sv/neo cells using scratch assay in presence or absence of mek and ras/raf inhibitor, u0126 and manumycin a, respectively ; 2) to assess the effect of imd peptide on phosphorylation of mek1/2 and erk1/2 in first trimester htr cells 3) to analyze the effects of imd on the expression of human leukocyte antigen, hla-g, a critical factor involved in the invasion and vascular remodeling of spiral uterine arteries and subsequent pregnancy in human. methods: htr-8sv/neo cells were used to assess the effect of imd (10 -8 m) on the expression of hla-g mrna and phosphorylation of erk 1/2and mek1/2 protein by reverse transcriptase polymeration chain reaction (rt-pcr) and western blot analysis respectively. scratch wound assay was used to determine the migration capacity of htr cells. total rna was isolated from cells using trizol reagent and processed for rt-pcr and results are expressed relative to 18s mrna. trichloroacetic acid was used for the extraction of total protein for western blot analyses. results: our data demonstrates that, 1) imd enhances the migrating capacity of htr cells (compared to the untreated cells) and these effects are inhibited by mek and ras/raf inhibitors, u0126 and manumycin a, respectively; 2) imd (10-8 m ) stimulates phosphorylation of erk1/2 and mek1/2 proteins in htr cells, 3) imd increases the expression of hla-g mrna in htr cells. conclusion: imd promotes migration of first trimester htr cells through mek/ erk signaling pathway and modulates the expression of immunoregulatory molecule, hla-g in these cells. chymotrypsin-like protease promotes the placenta tissue release of sflt-1. yang gu, shuang zhao, david f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: the placenta is a major source of soluble vegf receptor-1 (sflt-1) in the maternal circulation during pregnancy. increased placental release of sflt-1 is believed to play an important role in the pathophysiology and pathogenesis in pe. however, the mechanism of increased placental sflt-1 release in pe is unknown. we recently reported increased chymotrypsin-like protease (clp) activity and expression in placental trophoblasts from pe. in this study, we tested if proteolytic effects of chymotrypsin may play a role in promoting sflt-1 release by placental trophoblasts. methods: placentas delivered by normal pregnant women (n = 5) were used. we tested if chymotrypsin could promote sflt-1 release by placental tissue, in which villous explants were cultured with dmem containing chymotrypsin at 1.0, 2.5, and 5.0 g/ml for 6 hours. the culture medium was then collected for measuring sflt-1. we then determined the specificity of chymotrypsin induced sflt-1 release. villous tissues were cultured with or without chymotrypsin inhibitor (ci) in culture and then the medium was collected and measured for sflt-1. soluble flt-1 was measured by elisa. all samples were assayed in duplicate. data are presented as mean ± se and analyzed by anova. a p level < 0.5 is considered as statistically different. results: 1) sflt-1 concentrations in the medium were increased when chymotrypsin was present in culture and the increased sflt-1 release induced by chymotrypsin was in a concentration-dependent manner: control: 8.25±1.39; 1.0 g/ml: 10.51±1.89; 2.5 g/ml: 13.03±2.06; and 5.0 g/ml: 16.12±2.23 (p<0.01) pg/mg tissue/hour. 2) ci could attenuate sflt-1 release. this inhibitory effect was also revealed in a concentration-dependent manner: control: 5.67±0.92; ci 0.5 g/ml: 4.96±1.30; and ci 5.0 g/ml: 3.08±0.47 (p<0.05) pg/mg tissue/hour. conclusions: increased placental sflt-1 release stimulated by chymotrypsin and decreased placental sflt-1 release inhibited by chymotrypsin inhibitor suggest that the proteolytic effect of clp may play a role in sflt-1 generation. therefore, increased clp activity in placental trophoblasts may contribute to the increased placental sflt-1 production in pe. (supported nih grants hl65997 and hd36822). the change of autophagy-related proteins, lc3 and beclin-1, by tnfa stimulation in cultured primary trophoblasts. soo-young oh, kyung hee kim, suk-joo choi, jong-hwa kim, cheong-rae roh. department of obstetrics and gynecology, samsung medical center, sungkyunkwan university school of medicine, seoul, korea. objective: our previous work have demonstrated that the expression of lc3, but not beclin-1, was increased in placentas from pregnancies complicated by severe preeclampsia (sgi 2007abstract #209) . to understand the regulatory mechanism of these autophagy-related proteins in trophoblast cells, we investigated the changes in these proteins in response to cytokine or hypoxic stimulation in cultured primary trophoblast. material and methods: primary human cytotrophoblasts obtained from normal term placenta were cultured with stimulation of tnf-a or cocl 2 for a given time and the changes of beclin-1 and lc3 were assessed using immunoblot analysis. paired t test was used for statistic analysis. results: tnf-a stimulation induced a significant increase of the expression of lc3-ii in cultured primary trophoblasts while decreasing the expression of beclin-1 (p<0.05 for each). however, cocl 2 stimulation did not induce a significant change of both lc3-ii and beclin-1. conclusions: our data suggests that tnf-a stimulation in cultured primary trophoblasts is associated with increased autophagic activity. background: thyroid hormones play vital roles in the development of the fetal brain. mutations in mct8, recently recognised as a specific thyroid hormone transporter, define a novel syndrome of severe x-linked psychomotor retardation accompanied by elevated serum t3. we previously reported that mct8 expression in n-tera-2 (nt2) cells (a human embryonal cell line with characteristics of cns precursors), as well as mct8-null jeg-3 choriocarcinoma cells, resulted in markedly reduced cell proliferation. further, the s448x mct8 mutation, as reported in males affected by severe psychomotor impairment, resulted in a similar repression of proliferation to wild type, whereas the l471p mutant failed to influence cell turnover compared with control. methods: we now examine the effect of "knocking down" mct8 via sirna and evaluate the effects of cell proliferation (mtt and [ 3 h]-thymidine assays) and tri-iodothyronine (t 3 ) uptake. results: repression of endogenous mct8 expression in nt2 cells by 90 % caused a significant increase in proliferation compared to matched-dose nonspecific sirna treatment, independent of t 3 concentration (8.6 %, 7.2 % and 8.3 % induction at 0, 10 and 50 nm t3, n=3, p<0.02). we also sought to examine the role of mct8 in t3 uptake. in jeg-3 cells, wild type mct8 induced a 1.7-fold increase in the uptake of 125 i-labelled t 3 . by contrast, mutants s448x and l471p failed to significantly augment t 3 uptake, though r271h caused a mild but significant 1.17 fold induction in uptake, hence retaining approximately 25% of wt activity. in parallel experiments, co-transfection of mu-crystallin, a t 3 binding protein, resulted in a similar increase in t3 uptake compared with control (1.9-fold; n=6; p<0.001), implying that mct8 plays only a minor role in thyroid hormone efflux in jeg-3 cells. mutants s448x, l471p and r271h showed analogous responses to those in the absence of mu-crystallin. conclusion: these results further extend the evidence of a potential role for mct8 in the modulation of cell proliferation, independent of t 3 transport. to determine predictors of failure for labor induction in women with preeclampsia. we conducted a retrospective cohort study to examine cesarean delivery rates in all the preeclamptic women at a single institution undergoing labor induction between 1987-2001 with a singleton pregnancy >=24 weeks gestational age (ga). bivariate analyses informed the creation of multivariable logistic models to predict the risk of cesarean delivery using multiple predictors (maternal age, race/ethnicity, unfavorable cervix, gestational diabetes, diabetes, and gestational age). analyses were stratified by parity. our study population included 1,123 preeclamptic women undergoing labor induction. in the bivariate analyses, the risk of cesarean delivery ranged from as low as 10.5% (p=0.01) among multiparous women 24-28 weeks ga to as high as 70.8% (p<0.01) among nulliparous women with diabetes. a total of 1,019 women had adequate data to be included in the multivariable analyses. odds ratios of the predictors are presented in the objective: preeclampsia and cardiovascular disease share many risk factors, and women with preeclampsia are at increased risk of cardiovascular mortality later in life. we investigated whether risk factors associated with cardiovascular disease and preeclampsia remain elevated months postpartum. methods: we measured plasma sflt1, endoglin, plgf, cellular fibronectin (cfn), uric acid, homocysteine, and asymmetric dimethylarginine (adma) in 30 women with uncomplicated normotensive pregnancies compared to 20 women with preeclampsia in samples collected at pre-delivery and again several months postpartum (average 10.6±6.2 months). data are mean±sd or median (interquartile range). statistical analysis was by wilcoxin rank-sum or students unpaired t-tests with statistical significance accepted at p<0.05. results: the mean concentration of sflt1, endoglin, plgf, homocysteine, adma, cfn, and uric acid were all significantly different in samples collected pre-delivery in subjects with preeclampsia compared to controls (table). adma, cfn and uric acid remained significantly higher postpartum in subjects with previous preeclampsia compared to postpartum controls (table) . conclusions: biological markers associated with altered vascular function or cardiovascular risk are elevated in women with preeclampsia, and some remain significantly higher in postpartum preeclamptic women. these data suggest that vascular dysfunction persists in women with previous preeclampsia, and may contribute to the increased risk of future cardiovascular disease. funded in part by national institutes of health nih-5mo1-rr00056 and nih-2po1-hd30367. (ajp, 2007) . as leptin is a known potent angiogenic factor we hypothesized that leptin deficiency and/or resistance to leptin-induced vegf expression might be a mechanism for reduced angiogenesis in mfr offspring. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. mfr and control offspring were sacrificed on day 1 of life (p1). some tissues were used to determine the expression of leptin by western blot analysis. for culture experiments, thoracic aortas were dissected, cut into 1-2 mm explants and incubated with leptin (25-100ng/ml) in dmem (10% fbs). after 24 hours of culture, rna was extracted from the tissues and subjected to real time rt-pcr using specific rat primers for vegf, vegfr1 and r2, and ob-ra, stat3 and socs3 (18s mrna as control). culture media was analyzed for vegf protein by elisa. results: expression of leptin mrna and protein in p1 mfr aortas was significantly reduced. in culture, leptin significantly increased expression of vegf, vegfr1 and vegfr2 mrna in explants of aortas obtained from the control but not mfr tissues. as expected, control but not mfr aortic explants secreted significantly more vegf in vitro. to determine the mechanism for resistance to leptin-induced vegf in mfr offspring, we assessed expression of leptin receptor (ob-ra) in explants treated with leptin. leptin was found to induce the expression of ob-ra in aortas from both dietary groups. this upregulation of leptin receptor was accompanied by significant upregulation of stat3 and socs3 mrna in the control tissues. in contrast, in mfr explants only the 50ng/ml concentration of leptin induced an increase in stat3 mrna, and the magnitude of socs3 mrna increase by both concentrations of leptin was significantly less in the mfr explants. conclusion: these results indicate that reduced angiogenesis in mfr vessels is in part due to reduced leptin expression and ability of leptin to stimulate vegf expression. although in vitro leptin induced the expression of its receptor in both groups, it was only in the mfr group in which leptin up-regulated vegf and its receptors. our results suggest that this defect in leptin receptor function in mfr vessels is due, in part, to defects in jak/stat signaling. objective: women with a history of early-onset preeclampsia are at increased risk of developing major cardiovascular disease (cvd) related events, that have a detrimental effect on their long-term health and life expectancy. in this follow-up study, we measured established risk factors predictive of first cvd events after early-onset preeclampsia. study design: over a 10-year interval, 243 primiparous women with a history of early-onset preeclampsia (delivery <34 weeks gestation) were included and tested for major cardiovascular risk factors at least six months after delivery, in addition to a population-based control group of 374 healthy non-pregnant women. women with chronic hypertension were excluded. results: mean age was 30.5 years for cases compared to 28.3 years for controls (p<.001). after adjustment for age, we observed significantly increased mean values for weight (p=.002), body-mass index (p<.001), systolic blood pressure (p<.001), diastolic blood pressure (p<.001), total cholesterol (p=.006), ldl cholesterol (p<.001), triglycerides (p=.027), fasting blood glucose (p<.001), and lower hdl cholesterol (p<.001) in women with previous early-onset preeclampsia. no difference was found for height, smoking, diabetes, and ethnicity. estimated 10-year risk of first cvd events by framingham risk scores remained <10% for all women (low-risk). nonetheless, at mean (sd) 0.7 (1.0) years after early-onset preeclampsia, 15% of women met the criteria for metabolic syndrome, 89% of women exhibited >=1, 51% of women >=2 and 19% of women >=3 major cvd risk factors. conclusion: the majority of women with a history of early-onset preeclampsia exhibit at least one modifiable risk factor for future cvd. although most of these women are classified as low-risk according to the current aha guidelines, this is mainly due to their young age masking other, mostly modifiable, major risk factors. our data thus support life-style intervention programs aimed at primary prevention of cvd in women with a history of early-onset preeclampsia. it has recently been shown that antihypertensive drugs can stimulate cytokine release in normal and hypertensive pregnancy. there is evidence that these cytokines alter the secretion of inhibin a. inhibin a and activin a levels are increased in pre-eclampsia (pe), but it is not known if antihypertensive therapy can affect their secretion. patients and methods we recruited 129 women with hypertensive disorders in pregnancy (63 pe and 66 non-proteinuric hypertension [ht]) and 129 matched normotensive controls. inhibin a and activin a levels, before and 24-48 hours after initiating antihypertensives, were measured in serum and urine, using an elisa. the same markers were measured using validated assays in 84 placentas delivered at cesarean section at similar gestational age (29 pe, 24 ht and 31 controls). analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. marker levels before and after antihypertensive therapy were compared using paired t-test. we compared placental concentrations between the group which received antihypertensive therapy and the group which did not, using an independent t-test. data were analysed using spss®. in pe, both serum and urine levels of inibin a and activin a were increased at all gestations (p<0.0001). activin a (but not inhibin a) level was also increased at all gestations in ht (p<0.0001). after 28 weeks' gestation (but not before), antihypertensive treatment was associated with a significant fall in both inhibin a and activin a serum levels, and urinary inhibin a, in both pe and ht. the placental concentration of inhibin a, but not activin a, was significantly higher in women with pe compared with controls (p<0.0001). there was no significant difference in either marker between controls and women with ht. the fall in serum levels of inhibin a and activin a following antihypertensive treatment after 28 weeks' gestation may indicate that these drugs have an effect on the pathophysiology of pe other than their known antihypertensive action. pre-eclampsia (pe) is a placental disease of unknown etiology. anti-angiogenic factors, such as soluble fms-like tyrosine kinase 1 (sflt-1) and soluble endoglin (seng), and pro-angiogenic factors, such as vascular endothelial growth factor (vegf) and placental growth factor (plgf), are believed to play an important role in its pathophysiology. maternal plasma concentrations of these markers are altered in pe, even weeks before the clinical manifestations. the aim of this study was to compare the concentration of these markers in placental extracts of normotensive pregnant women, and women with pe and non-proteinuric hypertension (ht). patients and methods placental samples were collected at cesarean section from women with pe (n = 29), ht (n = 24) and normotensive pregnancies of similar gestational age (n = 32). these samples were stored at -80ºc. the frozen tissue samples were homogenised and these four markers measured by specific, validated enzymelinked immunosorbent assays. analysis the three study groups were compared using anova multiple comparisons with bonferroni post hoc testing. the data were normally distributed after logarithmic transformation. data were analysed using spss®. the concentrations of both sflt-1 and seng were significantly higher in the placentas of women with pe, but not ht, compared with controls (p=0.0002). there was no significant difference in plgf concentration between controls and women with pe or ht. placental vegf concentrations in both pe and ht were higher than in controls (p<0.0001); there was no significant difference between the levels in pe and ht (p=0.3). the fact that placental concentrations of sflt-1 and seng mirrored the known rise in serum levels in pe suggests that the placenta is the main source of these circulating factors. although sflt-1 was significantly raised in pe, plgf was not reduced. this suggests that the lower levels of free plgf found in the serum of women with pe are not the result of impaired placental production or secretion, but are due to increased binding by (the increased levels of) sflt-1 in the serum. objective. the purpose of this study was to evaluate whether systematic screening with uterine artery doppler (utad) and serum biochemical markers of oxidative stress, endothelial dysfunction and vasculogenesis performed during the first trimester predict efficiently pre-eclampsia (pet), specifically early-onset pet, in an unselected chilean population. methods. this nested case-control study involved 2831 asymptomatic pregnancies scanned at 11 +0 -13 +6 week of gestation. the subjects for biochemical testing were women who were delivered due to pet (n=43) and normotensive controls (n=129) that were enrolled during the first trimester scan. mean pulsatility index (pi) of the utad was calculated. blood samples were obtained and stored at -84 o c until biochemical analysis of oxidative stress, endothelial dysfunction and vasculogenesis were performed. normally distributed data were analysed by the unpaired t test, and non-normally distributed data by the mann-whitney rank sum test. chi-square tests were used for the comparison of categorical variables. a probability level of p<0.05 was considered significant. multiple logistic regressions were used to develop a combined predictive index. results. there was 13% and 22% significantly increased of the mean pi utad in women who later developed pet or early-onset pet compared to control pregnancies during the first trimester scan. although oxidative stress and endothelial dysfunction biochemical markers were not different between all pet pregnancies and control groups, plasma levels of sflt1 (1762.1 ± 397.5 vs. 1169.4 ± 71.1 pg/ml, p<0.05) and placenta growth factor (22.0 ± 3.5 vs. 49.8 ± 5.4 pg/ml, p<0.016) were significantly higher in women who subsequently developed early-onset pet compared to controls. multivariate logistic regression showed that a combination between abnormal utad and both biochemical markers of abnormal vasculogenesis were the best predictor test for early-onset pet, being its detection rate 56% with 10% false positive rate. conclusion. this study has shown early and selective changes in markers of impaired placentation and angiogenic state in women who later developed earlyonset pet, without alteration in oxidative stress and endothelial dysfunction. supported by fondecyt 1050482. currently it is unknown whether maternal inflammatory changes are specific to pregnancy or reflect an innate susceptibility to inflammation. c-reactive protein (crp) and interleukin-6 (il-6) are markers of the acute-phase inflammatory response and predictive of future cardiovascular events. we compared crp and il-6 levels after influenza vaccination, as an in vivo model for lowgrade inflammation, in non-pregnant women with a history of early-onset preeclampsia and controls with only uneventful pregnancies. methods: forty-four women with a history of early-onset preeclampsia (delivery <34 weeks' gestation) and twenty-nine controls with at least one uneventful pregnancy received an influenza vaccination. we then compared plasma levels of crp and il-6 at baseline, 1.3 days and 3.3 days after vaccination. results: median baseline crp and il-6 levels of women with a history of early-onset preeclampsia were comparable to controls (1.6 versus 0.8 mg/l; p=0.44 and 5.0 versus 3.4 pg/l; p=0.34, respectively). however, high crp and il-6 responses to vaccination were more common in cases (ors for response >75th, >80th, >85th, >90th and >95th percentile based on the distribution of control values of 2.3, 2.7, 3.1, 4.3 and for crp [p for trend 0.11] and of 0.9, 1.4, 1.9, 2.6 and 4.5 for il-6 [p for trend 0.043], respectively). the relationship between high il-6 responses and early-onset preeclampsia persisted after adjustment for body-mass index (p for trend 0.048). conclusion: women with a history of early-onset preeclampsia more frequently exhibit an innate pro-inflammatory phenotype not specific to pregnancy. background altered maternal inflammatory responses play a role in the development of preeclampsia and the hemolysis, elevated liver enzymes and low platelets (hellp) syndrome. we examined whether allelic variants of the innate immune receptors toll-like receptor 4 (tlr4) and nucleotide-binding oligomerization domain 2 (nod2), that impair the inflammatory response to endotoxin, are related to preeclampsia and hellp syndrome. we determined five common mutations in tlr4 (d299g and t399i) and nod2 (r702w, g908r and l1007fs) in 340 primiparous women with a history of early-onset preeclampsia, of whom 177 women developed hellp syndrome and in 113 women with a history of only uneventful pregnancies as controls. in addition, we assessed plasma levels of pro-inflammatory biomarkers c-rp, il-6, sicam-1, fibrinogen and von willebrand factor in a subset of 214 women included at least six months after delivery. after adjustment for maternal age and chronic hypertension, attenuating allelic variants of tlr4 were more common in women with a history of early-onset preeclampsia than in controls (or 2.9 [95% ci 1.2-6.7]). highest frequencies for tlr4 variants were observed in women who developed hellp syndrome (adjusted or 4.1 [95% ci 1.7-9.8]). in addition, high levels of il-6 and fibrinogen were associated with a history of early-onset preeclampsia. combined positivity for any of the tlr4 and nod2 allelic variants and high levels of il-6 was 6.9-fold more common in women with a history of early-onset preeclampsia (95% ci 2.1-23.2) compared to controls. we observed an association of common tlr4 and nod2 gene variants, and pro-inflammatory phenotype with a history of early-onset preeclampsia and hellp syndrome, that suggests involvement of the maternal innate immune system in severe hypertensive disorders of pregnancy. thus, we sought changes in pbef in the serum of patients with mild and severe pe, compared with matched controls. immunodistribution of pbef in fetal membranes and placentas from similar patients was also studied. methods: serum samples (68) were collected with clinical data including; gestational age, medications, ethnicity and recognized complications. patients in labor or infection were excluded. the standard bp and proteinuria criteria was utilized to classify cases for pe grouping; no pe (n=45), mild pe (n=8 bp; 140/90-160/110, proteinuria; trace to 1+ or 5-300mg/24hr urine) and severe pe (n=15 bp; >160/110, proteinuria; >2+ or >5g/24hr, or other associated symptoms). pbef concentration was determined by eia (phoenix pharmaceuticals) in accordance with the manufacturers instructions. fetal membranes and placentas of additional patients, no pe (n=4) and with pe (n=5) were fixed and embedded in paraffin. sections (7um) were immunostained with pbef antibody 1/250 (pheonix) and treated with abc reagent (vector labs) followed by dab (0.5% mg/ml), washed, counterstained, mounted and viewed under brightfield microscopy. results: the concentrations of pbef in serum were between 5-150 ng/ml and were significantly higher in those patients with mild pe (p=0.027) and further significantly elevated in those with severe pe (p=0.003) compared with the matched controls. pbef was detected by immunocytochemistry in the placental syncytiotrophoblast and in the amnion and choriodecidua of the fetal membranes. conclusions: pbef was elevated in the serum of patients according to the degree of pe severity and may be derived from the placenta and/or fetal membranes. (supported by nih #u54rr14607-08 to the pacific research center for early human development, university of hawaii). background: platelet-monocyte aggregation (pma) is a novel sensitive measure of platelet activation and indicates a proinflammatory state (cytokine release). less sensitive techniques demonstrate platelet activation during pregnancy and pre-eclampsia (pe) but platelet activation has not been assessed by pma.objective: longitudinal study of pma in normal pregnancy and pe. methods: 25 healthy, non-smoking primigravida with an uncomplicated pregnancy and 16 primigravida women with pe were studied. pe was defined by standard definitions. informed consent was obtained and the study had ethical approval. serial venous blood collected at 16, 24, 32,37 wks in controls, at time of diagnosis in pe cases and 6 wks post-natal (pn) in all. pma, platelet surface p-selectin (psp-sel) and monocyte cd40 expression (mcd40) were analysed by flow-cytometry and plasma (p) p-sel by elisa. results: groups were matched for mean age and bmi. in controls, pmas, psp-sel and mcd40 expression and pp-sel increased with gestation and decreased post-natally (table 1) . for pe analysis, data was divided into pre-term (sampled at mean 30 wks), and term (mean 38 wks). pp-sel was lower in pre-term pe than control (normal pregnancy 32 wks; p=0.03) there was no significant difference in other measures between pe and control ( objective: much effort has been put into the evaluation of novel markers to identify pregnant women at risk for the development of pre-eclampsia. soluble endoglin (seng) and soluble fms-like tyrosine kinase 1 (sflt1), two antiangiogenic agents, appear to be involved in the pathogenesis of pre-eclampsia. despite several studies describing higher midtrimester serum concentrations of these markers in women with subsequent pre-eclampsia, information on first trimester serum levels is scarce. the aim of this study was to assess seng and sflt1 as first trimester serum markers for the prediction of pre-eclampsia. methods: sera were obtained between 11+2 and 13+6 weeks of gestation from 46 women who later developed late-onset pre-eclampsia and from controls matched for gestational age, maternal age, maternal pre-pregnancy weight, and storage. using commercially available microplate enzyme immunoanalytical methods, seng and sflt1 were determined and the results analyzed using non-parametric statistical tests. results: the serum concentration of seng was found to be increased in women with subsequent pre-eclampsia when compared to controls (mean ± sd, 5.57 ± 1.18 ng/ml versus 5.02 ± 1.01 ng/ml, p = 0.009, unpaired mann-whitney test). similarly, the serum levels of sflt1 were higher in women later developing late-onset pre-eclampsia (1764 ± 757 ng/ml) when compared to controls (1537 ± 812 ng/ml, p = 0.036). sensitivities and specificities for predicting pre-eclampsia were 63% and 57% for seng and 64% and 56% for sflt-1, respectively. the combination of the two markers by multiplication yielded a sensitivity of 64% and a specificity of 55%. conclusion: seng and sflt1, showing increased first trimester serum levels in women with subsequent pre-eclampsia, might both fulfill the characteristics of first trimester markers to predict pre-eclampsia. the combination of the two, however, did not improve the sensitivity nor the specificity compared to their individual determinations. moderate sensitivities and specifities, however, limit the clinical use of these molecules as single markers. pregnancy is associated with increased monocyte/platelet aggregate formation in whole blood. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is associated with increased rates of platelet clearance, changes in platelet function and platelet activation. the goal of the current study was to examine basal levels of platelet activation through pregnancy beginning prior to conception, and to examine the association of platelet activation with the development of hypertensive complications during pregnancy. methods two indices of platelet activation, platelet cd63 expression (%cd63) and monocyte/platelet aggregate (%mp) formation, were measured in whole blood by flow cytometry using specific, fluorescentlylabeled monoclonal antibodies in 16 healthy, nonsmoking women during the follicular phase of their menstrual cycle (pp, cycle day 7.5+0.9). all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 wks) and late pregnancy (lp, 31-34 wks). five of these women were diagnosed with hypertensive complications (4 hypertension, 1 preeclampsia) at term although hypertension was not observed at any study time point. data are expressed as mean±sem. p<0.05 was accepted for significance. results subjects were 29.1+0.9 years old with a bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. a significant increase in the %mp formed over time of pregnancy was observed (p=0.015). there was little change in the %mp formed between pp and ep (pp, 6.0±3.8 % and ep, 10.4±3.8 %, p=0.384). however, the %mp increased significantly in lp (20.9±3.8 %) as compared to pp (p=0.005) and ep (p=0.041). this increase occurred independent of the development of hypertensive complications (p=0.657) and independent of pp platelet activation status. although statistically significant increases in cd63 expression were not observed, the change in cd63 expression over pregnancy correlated with the change in %mp over pregnancy (r=0.56, p=0.024) and cd63 expression correlated with %mp in lp (r=0.57, p=0.020). conclusion these combined observations suggest that pregnancy is associated with increases in levels of unstimulated platelet activation and that these increases occur in the presence or absence of subsequent hypertensive complications. furthermore, we observed a correlation between the changes in two distinct platelet activation events, %mp and cd63 expression, during pregnancy. supported by nih hl 71944. backgound sildenafil citrate (sc) has been proposed as a therapy to improve uterine perfusion in pregnancies complicated by iugr or preeclampsia. we sought to determine the effects of sc on uterine vascular resistance, uterine blood flow and cardiac output in young healthy nulliparous women. methods eleven young healthy nulligravid women were studied during the luteal phase (cycle day 22 + 5) of the menstrual cycle. women were randomized in a double-blind fashion to receive placebo (pl), or sc at a dose of 25 or 100 mg. uterine artery vascular resistance, uterine artery volumetric flow, brachial artery volumetric flow and cardiac output were measured at baseline and at 1 and 3 hours post dosing employing color doppler ultrasound. comparisons were made by anova between those randomized to pl (n=5) versus sc (n=6). p<0.05 was accepted for significance. data are expressed as mean + s.e.m. results there were no significant differences in subject age, cycle day, body mass index, uterine blood flow, brachial blood flow or cardiac output at baseline comparing the two groups. there was a tendency towards increased uterine blood flow in subjects randomized to receive sc (68% increase) compared to pl (no change), changes in uterine blood flow, brachial blood flow and cardiac output are outlined in the objective reduced maternal plasma volume in the third trimester has been associated with both fetal growth restriction and preeclampisa. we sought to determine the degree to which third trimester plasma volume is dependent on plasma volume prior to pregnancy. methods sixteen young (29.1 + 2.9 years) healthy nulligravid women had their plasma volume measured during the follicular phase (cycle day 7.5 + 3.6) of the menstrual cycle and subsequently conceived. subjects were predominantly caucasian (15/16) with a mean prepregnancy bmi of 23.5 + 3.5 kg/m 2 . plasma volume was re-estimated at 32-34 weeks gestation. all patients were placed on sodium and total calorie balanced diets for 3 days prior to each plasma volume determination. plasma volume was determined employing evans blue dilution with multiple post injection sampling time points. data are expressed as mean + s.d. results baseline prepregnant plasma volume was 2,868 + 446 ml or 123 + 18 ml/unit bmi. third trimester plasma volume was 4,306 + 663 ml representing a 51 % increase (p<0.001). the range of plasma volume expansion was 20-84% dependent upon prepregnant plasma volume. plasma volume in the third trimester of pregnancy was strongly correlated to prepregnant plasma volume r= 0.65 (p=0.006). plasma volume expansion was consistent across the range of prepregnancy plasma volume. conclusions pre-pregnancy plasma volume contributes approximately 42% of the variance in third trimester plasma volume. the observed increase is plasma volume is independent of prepregnancy volume resulting in a greater percentage increase for those starting at the lower end of the plasma volume range. as third trimester plasma volume is strongly associated with pregnancy outcome the correlation of prepregnancy plasma volume to third trimester plasma volume suggests that prepregnancy status contributes to these adverse reproductive events.this work was supported by nih hl 71944. background: hydatidiform mole is a rare disorder of pregnancy and may predispose the mother to severe morbidity. molar pregnancies are known to be associated with high risk for the development of early onset preeclampsia. in recent years, the expression of sflt-1 (soluble vegfr-1) was found to be increased in preeclampsia, and contributes to the pathogenesis of the maternal systemic disease. the objective of the present study was to examine the expression of sflt-1 in placentae from molar pregnancies. methods: placental samples from unique cases of twin pregnancies with complete molar pregnancy in one sac and developing fetus in the other sac were prospectively collected (n=2). the first set delivered at 23 weeks due to excessive bleeding. the second set delivered at 26 weeks due to severe iugr and elevated blood pressure. mrna level of sflt-1 was measured by quantitative real-time pcr using specific taqman primers and probe. protein expression of sflt-1 in placental tissue lysates were measured by western blot analysis using a polyclonal antibody against flt-1. immunohistochemistry of paraffin embedded samples was performed using specific antibody for sftl-1. results: mrna level of sflt-1 was increased by 2.3 fold in the molar placentae compared to matched controls. the placentae of the developing fetuses which were growth restricted exhibited 2.7 fold increase compared to controls. sflt-1 protein expression in the molar placentae was increased by 7.7 fold compared to controls, while the co-twin placentae exhibited a 1.4 fold increase compared to controls. immunohistochemistry revealed strong positive immunoreactivity for sflt-1 in the trophoblast layer of both molar pregnancies and iugr co-twin relative to controls. conclusion: our data suggest that sflt-1 expression is increased in placentae from molar pregnancies and thus may explain the increased risk for developing early onset preeclampsia. the expression of sflt-1 in the growth restricted twin placenta is also increased compared to controls and support our previous observation (supported by cihr and owh/igh). (n=145); 2) neonates of patients with pe (n=104); and 3) sga neonates (n=48). cord blood was collected immediately after delivery and pz plasma concentrations were measured by elisa. pz deficiency was defined as a cord plasma concentration <5 th percentile of the normal pregnancy group. non-parametric statistics were used for analysis. results: 1) cord plasma pz concentration differed significantly among the 3 study groups (kruskal wallis, p<0.001); 2) neonates of patients with pe and sga neonates had a significantly lower median cord plasma pz concentration than those delivered after normal pregnancy (pe: median 0.45 g/ml, range 0.05-1.68, p<0.001; sga: median 0.46 g/ml, range 0.06-3.56, p=0.011; normal pregnancy: median 0.6 g/ml, range 0.04-1.17); 3) there were no differences in the rate of pz deficiency among the groups; and 4) there was no relationship between placental histologic findings and median cord plasma pz concentrations between and among the sga and pe groups. conclusions: 1) at the time of delivery, the median cord plasma pz concentration was lower in sga neonates and those born to women with pe than in neonates born to normal pregnancies; 2) there was no difference in the rate of pz deficiency among the study groups, suggesting that the lower median pz cord blood concentrations in pe and sga groups may result from activation of the coagulation cascade rather than an inherited pz deficiency. objective: periconceptional multivitamin (mv) use may be related preeclampsia risk. we examined the relation between timing and frequency of periconceptional multivitamin use and the risk of preeclampsia. methods: women in the danish national birth cohort who delivered singleton liveborn infants (n=26,133) reported upon enrollment at 10.8 weeks (sd 3.6) the number of weeks of regular multivitamin use during a 12 week periconceptional period (lmp-4 to lmp+8). preeclampsia cases were identified using icd-10 codes (n=605, 2.3%). logistic regression was used to estimate the effect of frequency (number of weeks of use) and timing of use to lmp+2] and post-conception [lmp+3 to lmp+8]). results were stratified a priori by overweight status. results: overall, 18,551 women (71%) reported mv use in the periconceptional period. after adjustment for bmi, smoking, parity and chronic hypertension, infrequent mv use (<4 weeks of use) had no relation to risk (or 1.15; 95% ci 0.92,1.44) but regular use (>=8 weeks) was associated with modestly reduced risk (0.84 (0.67,1.06). similarly, when mv use was modeled as a continuous variable, each additional week of use was related to reduced risk for preeclampsia (or 0.83, 95% ci 0.67-1.02). this potential dose effect of periconceptional mv use appeared to be limited to normal weight women (bmi <25 kg/m², or 0.74; 95% ci 0.55-1.01), with no apparent effect among overweight women (bmi 25 kg/m², or 0.93; 95% ci 0.68-1.26). a total of 7,332 women reported regular mv use in both the preconception and post-conception periods, and 5,630 women reported regular use only in the post-conception period. among normal weight women, regular use in the preconception period had no effect on preeclampsia risk (or 1.29, 95% ci 0.89-1.85). in contrast, use in the post conception period was associated with reduced risk for preeclampsia (or 0.56, 95% ci 0.39-0.82). conclusions: regular periconceptional mv use was associated with a modestly reduced risk for preeclampsia among normal weight women. if causal, mv use immediately after conception appeared to be the critical exposure window. background: pre-eclampsia (pe), a disorder of pregnancy characterized by maternal inflammation, results in immune, cardiovascular and metabolic dysfunction. in non-pregnant persons, inflammatory disorders are treated with and prevented by pharmaceuticals and lifestyle methods such as physical activity (pa). while most pharmaceuticals are contraindicated for pregnant women, pa during pregnancy has been found safe, healthy and beneficial for both mother and baby. clinical evidence has found pa can beneficially affect pregnancy outcome, decrease excessive inflammation and decrease the risk of pe. epidemiological studies indicate that pa may be useful in preventing pe. unfortunately, previous studies have quantified pa based on recall of postpartum women and have not controlled for differences in women's interpretations of amount, type or intensity of pa. however, investigating pa utilizing a laboratory-based exercise intervention to control these variables inflicts difficulties translating the intervention into a community-based program that attracts and retains pregnant women in order to enhance public health. method: a retrospective study was performed to determine the rates of pe among the 9,160 women who gave birth at yale-new haven hospital (ynhh) during 2004 and 2005, and a 90 person subset of this group who performed prenatal pa in a community-based program that is evidence-based and standardized, thereby controlling for type and intensity of pa. additionally, the program is established in the community, has been offered to the public for 30 years and is internationally known. results: during 2004 during -2005 , the pe rate for the general population at ynhh was 7.8%. for the pa group, the rate was 2.2%. two women in the pa group were diagnosed with pe in the last month of pregnancy and delivered normal infants at term. no pe was observed in this group (pa group) during the second or early third trimesters nor was there any prematurity in this group. significance: these findings support the hypothesis that adequate physical activity provided in a standardized community-based group setting may provide a non-pharmacological approach for preventing pe. growth restriction. margreet plaisier, 1 esther streefland, 2 pieter koolwijk, 3 frans m helmerhorst, 1 jan jaap hm erwich. 2 1 department of gynecology and reproductive medicine, leiden university medical center, leiden, netherlands; 2 department of obstetrics and gynecology, university medical center groningen, groningen, netherlands; 3 department of physiology, vu univerity medical center, amsterdam, netherlands. objective: disturbances in decidual and placental vascular development may play a role in the pathogenesis of pregnancy complications, like pre-eclampsia (pe) or fetal growth restriction (fgr). whether the regulation of decidual vascular adaptation to implantation is altered in these illnesses, is not elucidated yet. the present study focused on the role of first-trimester angiogenic factors in the pathogenesis of pe and or fgr. methods: first-trimester decidua samples were obtained during routine chorionic villous sampling. the expression of vascular endothelial growth factor (vegf-a), placental growth factor (plgf), flt-1, kdr, angiopoietin-1 (ang-1), angiopoietin-2 (ang-2) and tie-2 mrna was determined by rt-pcr. the expression of the angiogenic factors was related to the pregnancy outcome, i.e. uncomplicated, pe or fgr. results: the first-trimester decidual tissues expressed all angiogenic factors. mrna levels of vegf-a, plgf, kdr, ang-1, ang-2 and tie-2 appeared increased in fgr cases compared to matched controls. in addition, plgf, ang-1 and tie-2 mrna appeared increased in pe cases compared to matched controls. the differential expression of angiogenic factors was more pronounced in cases with fgr than pe. the large inter-individual variation disallowed a significant outcome. conclusions: various angiogenic factors showed differential mrna expression in 1 st trimester decidua of patients developing pe or fgr in later pregnancy compared to their matched controls. the first-trimester decidual samples provided a unique opportunity to obtain information regarding the onset of pe and fgr. early 1 st trimester changes in angiogenic factor expression may well occur as a compensatory mechanism. in turn, this may set the stage for increased non-branching angiogenesis and altered decidual and placental vascular adaptation, which may be part of the pathogenesis of pe and/or fgr. complications. beth a bouchard, 1 adrienne schonberg, 2 gary j badger, 3 ira m bernstein. 2 1 biochemistry; 2 obstetrics and gynecology; 3 medical biostatistics, univ of vt, burlington, vt, usa. background preeclampsia is characterized by endothelial dysfunction. the goal of the current study was to prospectively measure plasma levels of the soluble endothelial cell adhesion molecules, sicam-1 and svcam-1, beginning prior to pregnancy and determine if subjects destined to develop hypertension complicating pregnancy had differences in the concentrations of these molecules. methods serum levels of sicam-1 and svcam-1 were measured in 16 healthy, nonsmoking women (cycle day 7.5 + 0.9, prepregnancy) by elisa. all women subsequently conceived singleton pregnancies and were re-examined in early (ep, 11-16 weeks) and late pregnancy (lp, 31-34 weeks). five of these women developed hypertensive complications (4 gestational hypertension, 1 pre-eclampsia) near term. all subjects were normotensive at all study time points. data are expressed as mean ± sem. p<0.05 was accepted as significant. results subjects were 29.1+0.9 years old with a mean bmi of 23.5+0.9 kg/m 2 at the time of prepregnancy studies. significant differences in sicam-1 levels as a function of pregnancy were observed (p=0.046) and are outlined in table 1 p=0.662 differences were dependent upon the stage of pregnancy in those women who were not diagnosed with hypertensive complications with a decrease in sicam-1 levels in ep (p=0.024) followed by an increase in sicam-1 levels in lp (p=0.044). in women with hypertension in pregnancy, these differences in sicam-1 levels were not evident (p=0.66). there were no differences in sicam-1 levels comparing women with or women without hypertensive complications prior to pregnancy (p=0.971). in contrast to sicam-1, we observed no significant differences in svcam-1 levels over pregnancy or between those with and without hypertension. conclusions these combined observations suggest that levels of the soluble adhesion molecule sicam-1 change significantly over time in normal pregnancies. subjects destined to develop hypertension did not demonstrate the early pregnancy reduction in sicam-1. supported by nih hl 71944. yuval bdolah, 1 uriel elchalal, 1 shira natanson-yaron, 1 hadas caspi, 1 tali bdolah-abram, 1 angelika bord, 1 caryn greenfield, 1 debra goldman-wohl, 1 ariel milwidsky, 1 franklin h epstein, 2 s ananth karumanchi, 2 simcha yagel, 1 drorith hochner-celnikier. 1 1 ob/ gyn, jerusalem, israel; 2 ob/gyn medicine, beth israel deaconess medical center, harvard medical school, boston, ma, usa. objectives: nulliparity is a risk factor for preeclampsia (pe) with a reported incidence of up to 4-5 times higher than multiparous pregnancies. soluble fms-like tyrosine kinase-1 (sflt1), a circulating anti-angiogenic molecule of placental origin plays a pivotal role in pe by antagonizing placental growth factor (plgf). increased sflt1 and sflt1/plgf have been shown to antedate clinical signs in pe. we therefore hypothesized, that the higher risk of pe in nulliparous pregnancies is associated with high sflt1 (or sflt1/plgf). methods: maternal serum samples from nulliparous (n=68) and multiparous (n=159) term singleton pregnancies without pe, at the time of admission to delivery room, were used. serum samples were analyzed for levels of sflt1 and plgf by elisa. statistical analysis was performed applying t-test and the kruskall-wallis test and using spss software. results: for nulliparous and multiparous pregnancies, the mean serum sflt1 levels were 13,385 ± 960 and 10,584 ± 722, (p=0.021), the mean serum plgf levels were 215 ± 15 and 249 ± 14 (p=0.117), and the mean ratios of sflt-1/plgf were 98 ± 13 and 65 ± 6 (p=0.025), accordingly. in a subgroup of 10 multigravidous nulliparous pregnancies, sflt1 levels were 14,063 ± 2295. correcting for maternal age did not alter the results. moreover, results did not differ between multiparous pregnancies with a 5-10 years interpregnancy interval compared with a 1-5 years interval. conclusions: in nulliparous pregnancies, circulating sflt1 levels and sflt1/ plgf ratios are significantly higher than in multiparous pregnancies. these findings suggest that the increased risk of pe in nulliparous pregnancies may involve anti-angiogenic imbalance. nulliparity may be more substantial than primigravity, as a risk factor for pe, suggesting that first semester abortions in primigravidas may not protect from pe in a subsequent term pregnancy. nevertheless, even 5-10 years intervals from the previous gestation do not increase the risk for pe. different normograms of angiogenesis should be used, when assessing the risk for pe in multiparous versus nulliparous pregnancies. objective: we determined whether maternal serum levels of angiogenic proteins namely soluble fms like tyrosine kinase (sflt-1), soluble endoglin (seng), and placental growth factor (plgf) -measured during the first trimester are associated with the subsequent development of placental abruption. methods: we performed a prospective, nested case-control study of women enrolled in the massachusetts general hospital obstetric maternal study (moms). first trimester serum samples from 34 placental abruption cases and 250 normal pregnancies were measured for angiogenic factors. cases and controls were matched by body mass index and age. placenta abruption was diagnosed by standard clinical findings and pathological examination of the placenta. women with confirmed preeclampsia or chronic hypertension were excluded. results: compared to controls, cases had more pregnancies, delivered infants at an earlier gestational age and with lower birth weight. first trimester levels of seng were significantly increased in cases compared to controls: 7.95 ± 0.48 ng/ml vs. 6.48± 0.15 ng/ml, p <0.05. there were no significant difference in serum levels of plgf, 40.31± 6.22 ng/ml versus 40.04± 1.79 ng/ml, p=ns, although sflt1 levels were lower in cases: 1.86 ± 1.7 ng/ml vs. 2.7±0.98 ng/ml, p=ns. in logistic regression analysis adjusted for age, race, smoking, number of pregnancies, gestational age at delivery, gestational age of blood sampling, and blood pressure at first prenatal, seng levels remained independently associated with subsequent risk (odds ratio 1.3, 95% ci 1.1-1.6) of placental abruption. examining this relationship by tertiles of seng, in the unadjusted model, women in the second (or 7.4, 95% ci 1.6-33.5) and third (or 8.8, 95% ci 1.9-39.1) tertiles were at increased risk of developing placental abruption compared with women in the lowest tertile. after adjusting for known risk factors of placental abruption, women in the second (or 13.1, 95% ci 1.3-129.09) and third (or 13.3, 95% ci 1.9 95.3) tertiles remained at increased risk for placental abruption. conclusion: increased first trimester maternal serum levels of seng are associated with increased risk of subsequent placental abruption. 4 ob/gyn, univ of vermont. shear stress is the most potent physiologic stimulus for elevating endothelial no production for flow-mediated vasodilatation. we measured in vivo shear stress during the proliferative and secretory phases and at 12 and 32 weeks of gestation hypothesizing that uterine blood flow (ubf) elevations in turn increase shear stress in early and late gestation. methods: during proliferative, secretory phases, and at 12 and 32-34 weeks of pregnancy ua blood velocity and internal radius were measured bilaterally using color doppler ultrasound. blood viscosity was measured at shear rates in excess of 60/sec. results: compared to the proliferative phase viscosity was decreased at 12 weeks and more so at 32 weeks gestation (p<0.05). 0.096 ± 0.002 0.096 ± 0.002 nsd 0.13 ± 0.004*** 0.17 ± 0.008*** ua velocity (cm/sec) 9.3 ± 0.57 15.3 ± 1.0*** 31.4 ± 3.0*** 55.7 ± 7.7*** ubf (ml/min) 15.0 ± 0.9 27 ± 2*** 101.8± 12.9*** 294.2 ± 40.3*** shear stress (dynes/cm 2 ) 20.7 ± 1.5 37.8 ± 2.62*** 37.1 ± 3.8*** 47.9 ± 6.8*** internal ua radius was not altered by the menstrual cycle, and was greater at 12 weeks (+35%) and 32 weeks (+77%). compared to proliferative, secretory phase showed significant rises in unilateral ubf and velocity that rose progressively during gestation. in contrast, shear stress increased in secretory (80%) and did not rise further in early pregnancy but by 32 weeks shear stresses was further elevated 131%. conclusions: equivalent rises in shear stress during the secretory phase and 12 weeks gestation demonstrate increases in radius and profound remodeling of uas that reflect the physiologic process of "normalization of shear". by late gestation, continued but modest rises in radius illustrate that further increases in shear stress occur almost solely due to rises in ubf via falls in down stream impedance. continued rises in shear stress into late gestation provide progressive stimuli for no production by ua endothelium. nih hl49210, hd38843, hl63101 background: hypertension during pregnancy is associated with altered uterine vascular reactivity and blood flow, although its effects on arterial myogenic behavior have not been explored. the purpose of this study was to evaluate the effects of hypertension and no inhibition on myogenic tone in pregnancy, as the ability of a vessel to constrict and dilate in response to pressure plays a key role in regulating blood flow to the uterus. methods: three groups of sprague dawley late pregnant (day 20) rats were used: control (n=5), hypertensive (0.5g/l l-name in the drinking water, n=9), and treated with l-name and hydralazine (also in the drinking water, 0.272 g/l, n=5) to prevent the blood pressure increase, yet maintain no inhibition. resistance-sized radial arteries (<150 m) were mounted in a pressure arteriograph and equilibrated at 60 mmhg (in pss containing l-nna and indomethacin) to induce a myogenic response. vessels were then subjected to pressure steps from 20 to 200 mmhg. tone (%) was calculated by comparing the vessel diameter at each pressure with the passive diameter at the same pressure (determined by incubation with 0.1mm papaverine and 10 m diltiazem). results: myogenic tone developed in controls (46±5% maximal), and was maintained over a broad range of transmural pressures . arteries from the l-name group did not develop tone at any pressure. co-treatment with hydralazine reinstated tone (33±4% maximal) over the same range of pressures as in the control group. the reduction in average placental weights in the l-name group (401 vs. 425mg, p<0.05) was restored by hydralazine (433mg, p<0.05 vs. l-name). average fetal weights were also reduced in the l-name group (1.96 vs. 2.22g, p<0.001), but only partially restored by hydralazine co-treatment (2.11g, p<0.05 vs. control and l-name groups). conclusions: surprisingly, radial uterine arteries from the l-name group did not develop tone over any range of pressures, despite the fact that matched arteries from late-pregnant controls developed significant myogenic tone. this abolishment of tone was reversed by hydralazine, which also had beneficial effects on fetal and placental growth. these results implicate hypertension rather than no inhibition as the key factor in the suppression of myogenicitiy and dysregulation of uterine blood flow. charles r rosenfeld, timothy roy. division of neonatal-perinatal medicine, ut southwestern medical center at dallas, dallas, tx, usa. background: upbf b rises 30-fold in ovine pregnancy; but the mechanisms responsible for the rise and maintenance are unclear. we (jsgi 2005) reported that uterine vascular smooth muscle bk ca k + channels contribute to uterine vasodilation and upbf b maintenance; but up-stream activators are unclear. uterine vascular prostacyclin synthesis increases in pregnancy, but cyclooxygenase inhibition does not alter upbf b (ajp 1992) . vascular nitric oxide synthase (nos) also increases; but acute inhibition with l-name decreases upbf b only 25% (jci 1996) . it is unclear if l-name doses were insufficient or if prolonged nos inhibition has a greater effect. objective: to determine if local nos inhibition with l-name dose-dependently decreases upbf b and if prolonged inhibition exerts a greater effect on upbf b . methods: 11 pregnant ewes were studied at 100-148d gestation age (ga). 4 had doseresponse studies with uterine artery l-name infusions to achieve 0.025-1.0 mg/ml over 30min. 7 had 72h arterial l-name infusions to achieve and maintain levels of 0.01 mg/ml at 107 (n=6), 122 (n=7) and 135d (n=7) ga, while measuring arterial pressure (map), heart rate (hr) and upbf b before, during (2,4,6,24,30,48,54 and 72h) and after (72, 96h) infusions. uterine arterial and venous cgmp were measured. data were analyzed by anova. results: acute nos inhibition decreased upbf b 0-20%, but was not doserelated. 72h arterial l-name infusions decreased upbf b by 2-6h at each ga (p 0.025, anova) and values returned to baseline by 96h postinfusion. sensitivity did not differ between ga (p=0.1, anova), upbf b falling 20-30% at each ga. contralateral upbf b was unaffected at all ga. map rose and hr fell during infusions at 107 and 122d ga, p 0.03; but were unaffected at 135d ga. venous-arterial cgmp concentration differences were seen at 107d and absent at 72h of l-name infusion, p=0.04. conclusions: uterine vascular nos increases in ovine pregnancy, but its inhibition decreases upbf b 30%, suggesting study doses were insufficient to fully inhibit vascular nos activity. alternatively, nos contributes to the maintenance of upbf b , but other mediators, not yet identified, are more important in activating bk ca and regulating upbf b . notably, l-name reached the systemic circulation, and although further diluted, map rose 5-10%, suggesting the systemic vasculature may be more sensitive to nos inhibition than upbf b . charles r rosenfeld, xiao-tie liu. division of neonatal-perinatal medicine, university of texas southwestern medical school, dallas, tx, usa. background: uteroplacental blood flow (upbf) rises 40-fold in ovine pregnancy, reflecting increases during pre-implantation, placentation and finally, in the last third of gestation. we reported that bk ca density in uterine vascular smooth muscle (uvsm) increases in ovine pregnancy (sgi 2007) and inhibition with tetraethylammonium ( 0.3 mm) dose-dependently decreases basal upbf 80% in the last third of pregnancy (jsgi 2005) . it is unclear how bk ca subunit expression changes and activity is regulated in pregnancy; since uterine vascular nitric oxide is increased, signaling via cgmp-dependent protein kinase g (pkg) is one potential pathway. objective: to determine simultaneous changes in uvsm bk ca density and regulatory 1-subunit expression and the cgmp signaling pathway for activation in ovine pregnancy. methods: endothelium-denuded segments of 2 nd generation uterine arteries obtained from nonpregnant (n=3), pregnant (n=14, 56-145d gestation; term 150d) and postpartum (n=8, 2-56d) sheep were used to measure expression of bk ca -and regulatory 1-subunits and signaling proteins in uvsm by immunoblot analysis and immunohistochemistry. results: uvsm bk ca density, reflected as a change in the 100 kda -subunit, rose 70% with placentation (p<0.05) and was unchanged thereafter. expression of the 39 kda regulatory 1-subunit paralleled the rise in bk ca density during placentation, increasing 50% (p<0.001), but increased another 2-fold and exponentially in the last third of pregnancy (p<0.001, r 2 =0.99, n=13). changes in subunit immunostaining in uvsm paralleled increases in protein. although uvsm soluble guanylyl cyclase was unchanged in pregnancy (p=0.8, r=0.08, n=14), pkg expression rose 2-fold (p=0.002, r=0.86, n=11) and gradually returned to nonpregnant levels by 30d postpartum (p=0.008, r=0.83, n=9). uvsm cgmp is being measured. conclusions: these are the 1 st data suggesting that increases in ovine upbf during placentation involve vascular growth and bk camediated vasodilation. the rise in upbf in the last third of pregnancy reflects bk ca -mediated vasodilation due to enhanced channel activation via increases in uvsm pkg, bk ca phosphorylation and changes in subunit stoichiometry due to an exponential rise in the regulatory 1-subunit. it is unclear what initiates and directs these changes in bk ca expression and sensitivity. relaxin (rlx) is a hormone traditionally associated with changes in the female reproductive tract during pregnacy. recent evidence suggests that rlx may play a pivotal role in regulating cardiovascular function during gestation. analogous to pregnancy, administration of recombinant human relaxin (rhrlx) to nonpregnant female rats reduces systemic vascular resistance, as well as increases global arterial compliance. additionally, we demonstrated that, concurrent with rlx´s influence on overall cardiovascular function, small renal arteries (sra) from rhrlx-treated mice and rats are characterized by reduced passive stiffness and increased arterial wall area whereas external iliac arteries (eia) are not. we hypothesized that rlx regulates arterial passive mechanical properties by altering the cellular and biochemical composition of the arterial wall. nonpregnant female mice were administered rhrlx for 5 days after which sra and eia were isolated. we measured arterial collagen, elastin, and total protein using the sircol collagen assay, the fastin elastin assay, and the pierce bca protein assay, respectively. additionally, we quantified arterial smooth muscle cell (smc) density using immunofluorescent techniques. sra isolated from rhrlx-treated mice were characterized by a significant reduction in collagen to total protein ratio (0.13±0.01 vs 0.19±0.01 μg collagen/μg protein; mean±sem; p<0.02) as well as a significant increase in smc density (6.1±0.1 vs 5.0±0.1 cells/1000 μm 2 ; p<0.001) compared to control mice with no significant change in elastin content (104±4 vs 97±14 μg elastin/mg dry weight). in contrast, there were no significant changes in collagen to total protein ratio (0.25±0.02 vs 0.28±0.04 μg collagen/μg protein), smc density (3.9±0.2 vs 4.0±0.2 cells/1000 μm 2 ) or elastin content (89±12 vs 83±5 μg elastin/mg dry weight) in eia from the rhrlx-treated mice compared to control mice. of note, comparable results were observed for rlx knock-out (rlx -/-) and wild-type mice with rlx -/mice exhibiting increased arterial collagen and decreased smc density. we conclude that the rlx-induced decrease in passive stiffness of sra that we previously reported is, at least in part, due to rlx-induced alterations in arterial wall cellular and biochemical composition. further, our findings suggest that these vessel wall remodeling effects of rlx are artery-type specific. relaxin is a peptide hormone that emanates from the corpus luteum of the ovary and circulates during pregnancy. this hormone plays an important role in renal vasodilation and hyperfiltration, two fundamental maternal adaptations in pregnancy. chronic administration of recombinant human relaxin (rhrlx) to both virgin rats and mice inhibits myogenic reactivity and increases compliance of small renal arteries, thus further mimicking pregnancy. we hypothesize that these arterial responses to rhrlx are mediated by the lgr7, and not the lgr8 receptor. both lgr7 and lgr8 receptor-deficient, and wild-type virgin mice were investigated. the mice were chronically infused with rhrlx or vehicle (veh) for 5 days. small renal arteries were isolated and mounted in a pressure arteriograph and myogenic reactivity was assessed (% change in diameter over baseline in response to a 20 mmhg step increase in intraluminal pressure). small renal arteries from rhrlx-infused lgr7 wild-type mice showed inhibited myogenic reactivity with a 6.1 ± 0.5% increase in diameter whereas the arteries from rhrlx-infused lgr7 knock-out mice exhibited robust myogenic reactivity with only a 1.2 ± 0.4% change in diameter (p=0.001). in contrast, myogenic reactivity of small renal arteries was inhibited in both the rhrlx-infused lgr8 knock-out and wild-type mice. the veh-treated lgr7 and lgr8 mice, regardless of genotype, exhibited robust myogenic reactivity. arterial compliance was also assessed for each genotype/treatment group. rhrlx infusion increased arterial compliance of small renal arteries from lgr7wild-type, but not from lgr7 knock-out mice (p=0.01). in contrast, the arteries from rhrlx-infused lgr8 wild-type and knock-out mice showed increased compliance relative to veh-infused animals. conclusion: relaxin-induced inhibition of myogenic reacitivity and increase in compliance of small renal arteries is mediated by the lgr7, and not the lgr8 receptor. smoking is associated with adverse pregnancy outcomes including fetal growth restriction. pathologic effects of smoking on maternal vasculature is a potential mechanism leading to fetal growth restriction. the objective of this study was to determine whether cigarette smoke exposure during pregnancy affects the functional properties of uterine and peripheral arteries using a gravid murine model. study design: pregnant and virgin c57bl/cj mice were exposed to whole body side stream smoke using an inhalational chamber for 4 hours/day. smoke exposure was increased from day 4 of gestation until late pregnancy (day 16-19) with mean total suspended particle levels of 63 mg/m3, representative of moderate to heavy smoking in humans. control animals were exposed to ambient room air. late pregnant and virgin mice were sacrificed and uterine, mesenteric, and renal arteries were isolated and studied in a pressure arteriograph system (n=3-6 in each group). plasma cotinine was measured by elisa. means were compared using t-test or analysis of variance. results: fetal weights were significantly reduced in mice exposed to smoke compared to control fetuses (0.88 ± 0.1g vs 1.0 ± 0.08g, p=0.02), while litter sizes were not different. cotinine levels in smoking mice were significantly elevated compared to control mice (51.9 ± 9.2 vs 4.2 ± 0.8ng/ml for virgin mice and 33.2 ± 21 vs 2.8 ± 0.4ng/ml for pregnant mice). there was no significant difference in phenylephrine responses between groups. endothelial mediated relaxation responses to methacholine were significantly impaired in both the uterine and mesenteric vasculature of pregnant mice exposed to cigarette smoke during gestation. no difference in endothelial-mediated relaxation was seen in isolated renal arteries in pregnant mice exposed to cigarette smoke, however relaxation was significantly reduced in renal arteries from smokeexposed virgin mice. conclusions: passive cigarette smoke exposure is associated with impaired vascular relaxation of uterine and mesenteric arteries in a gravid murine model. functional vascular perturbations during pregnancy, specifically reduced uterine blood flow and impaired peripheral vasodilation, may be a mechanism by which smoking results in fetal growth restriction. human chorionic gonadotropin (hcg) is essential during early human gestation for "rescue" of the corpus luteum. however, its potential contribution to the widespread maternal vasodilation of pregnancy that occurs at this stage of pregnancy remains uncertain. our objective was to use the renal circulation of conscious rats as an experimental model in which to test the vasodilatory potential of hcg. in addition, we investigated both myogenic reactivity and relaxation responses in small renal and mesenteric arteries isolated from rats, as well as in small human subcutaneous arteries using a pressure arteriograph. we chronically instrumented 10 rats for measurement of renal function. five were ovariectomized, and 5 sham-ovariectomized. after 7 days of surgical recovery, baseline glomerular filtration (gfr) and effective renal plasma flow (erpf) were measured on two separate days and the values averaged. then, an osmotic minipump containing hcg (25 iu/min) was implanted s.c. and renal function was again assessed 2 and 5 days thereafter. gfr and erpf significantly increased and calculated effective renal vascular resistance decreased from baseline in the intact (p<0.001 vs. baseline), but not ovariectomized (p=ns) rats on both days 2 and 5 of hcg administration. in the intact, but not ovariectomized rats, plasma osmolality declined and progesterone increased (both p<0.001 vs baseline). plasma hcg concentrations were 2,500 miu/ml and comparable in both groups of rats. incubation of small renal arteries from rats with recombinant human relaxin (rhrlx, 1-100 ng/ml), but not hcg (2,500-200,000 miu/ml) in vitro inhibited myogenic reactivity and relaxed phenylephrine (pe)-constricted arteries. in contrast, both rhrlx and hcg inhibited myogenic reactivity and relaxed pe-constricted small mesenteric arteries from rats and small human subcutaneously arteries. in conclusion, consistent with our earlier work showing a virtually exclusive role for relaxin in mediating the renal circulatory changes of pregnancy, hcg is likely to play little or no role. in contrast, hcg and relaxin are both likely to contribute to the vasodilation of other organ circulations during pregnancy. pierre-andre scott, 1,2 michele brochu, 2 jean st-louis. 1,2 1 research centre, chu ste-justine, montréal, qc, canada; 2 pharmacology, université de montréal, montréal, qc, canada. uterine vasculature undergoes major structural and functional changes during pregnancy. estrogens have been shown to induce increased uterine blood flow in this circulation. we have reported that estradiol (17 -e2) induced direct vasorelaxant response on smooth muscle of the uterine arteries that, for it major part, is not mediated through tissue nitric oxide. to investigate the cellular effectors mediating this vasorelaxant effect of 17 -e2, we set-up uterine arteries of non-pregnant rats in wire myograph systems for microvessels. 17 -e2 and 17 -e2 were equipotent in relaxing phe (1 μmol/l)-preconstricted uterine arteries, although the later produced significant smaller relaxations. genistein, a phytoestrogen presumed to inhibit phosphatases, also produced uterine artery relaxation with significant lower potency. to try interfere with the vasorelaxant effect of 17 -e2, tissues were preincubated with different substances. cycloheximide (protein biosysthesis inhibitor), ici 182,780 (estrogen receptor modulator), and kt5720 (pka inhibitor) did not significantly influenced the response to 17 -e2. rp-8-pcpt-cgmps (pkg inhibitor) slightly displaced the concentration-relaxation curve to 17 -e2 to the right. inhibitors of potassium channels, penitrem a (bkca) and glybenclamide (katp), showed opposite effects for 17 -e2 concentration-response curve; the former producing a right shift and the latter a small not significant left shift. these data indicated that the direct acute effect of 17 -e2 in uterine artery is the result of complex interactions within smooth muscle cells, involving potassium channels, and protein kinases and phosphatases. the renin-angiotensin-aldosterone system is paradoxically activated during pregnancy, since blood pressure decreased. earlier data showed that the high levels of aldosterone present during pregnancy may be involved in cardiovascular adaptation to pregnancy. in order to delineate this effect of aldosterone on vascular tone, potassium canrenoate, an antagonist of mineralocorticoid receptors (mr), was administered (20 mg/kg/day) to pregnant rats from the day 15 to 22 of gestation. rats were sacrificed at day 17, 19 and 22 of gestation together with untreated and day 14 pregnant rats. the thoracic aorta was quickly removed and set up in tissue baths as ring (2-3mm) preparation. as observed previously, canrenoate enhanced responsiveness to phe for all time of treatment tested, but only at day 17 (2 days treatment) for kcl responsiveness. aortic contractile responses to tea (tetraethylammonium) gradually decreased during pregnancy to almost disappear at the end of gestation. in the present results, canrenoate treatment made the response statistically different by day 17 of pregnancy. finally, the activity of na/k-atpase was measured by the relaxant effect of kcl added to physiological solution without potassium. the activity of the pump was decreased when approaching parturition compare to day 14 of pregnancy. canrenoate treatment abolished this effect. the present data show that vascular changes that occurred during pregnancy are markedly modified by treatment of rats with an antagonist of mineralocorticoid receptors, suggesting that aldosterone may be involved in vascular adaptation to pregnancy. background impaired endothelium-dependent vasodilatation has previously been demonstrated in small myometrial arteries from women with gestational diabetes. this impairment may play a role in mediating the complications observed in diabetic pregnancies. it is not known which mechanisms of endothelium-dependent vasodilatation are affected in myometrial arteries by gestational diabetes. aim to investigate mechanisms of endothelium-dependent vasodilatation in uterine arteries using a mouse model of pregnancy complicated by diabetes. methods diabetes was induced in female c57bl6/j mice (streptozotocin; 200 mg/kg) prior to mating. mice were culled at day 19 of pregnancy (term) and uterine arteries dissected, mounted on a wire myograph, normalised to 55mmhg and equilibrated (37°c; 5%co 2 /air). arteries were constricted with phenylephrine (10 m) and a concentration-response curve to the endotheliumdependent vasodilator acetylcholine (ach; 0.1nm-1 m) constructed in the presence and absence of a nitric oxide synthase inhibitor (l-nna; 10 m), a cyclooxygenase inhibitor (indomethacin; 10 m) or a combination of the two to determine the contribution of nitric oxide (no), prostacyclin and endothelium derived hyperpolarising factor (edhf) to vasodilatation. results sensitivity to ach was comparable between diabetic and vehicle treated mice (ec 50 31.4 ± 8.0nm vs 41.9 ± 15.9nm). the contribution of individual endothelium-dependent vasodilators was significantly altered in arteries from diabetic mice. at 1 m ach, edhf-mediated relaxation was significantly reduced (p=0.03, one-way anova) compared with controls (13.17 ± 4.87 vs 67.23 ± 13). in comparison, no-mediated relaxation was significantly increased (p=0.04, one-way anova) compared with controls (64.88 ± 10.78 vs 17.54 ± 5.77%). conclusions endothelium-dependent relaxation was not reduced in uterine arteries of diabetic mice compared with controls. however, there was a profound change in the contribution of endothelium-dependent vasodilators in arteries of diabetic mice. this may alter compensatory capacity as disease progresses. supported by mfn training grant (cihr). raf-1 serine/threonine protein kinase has been extensively studied as the upstream kinase linking ras activation to the mek/erk module. mek/erk has been shown to play a role in the modulation of vascular contraction. however, the role of raf kinase in vascular contraction and its possible involvement in alteration of maternal vascular function during pregnancy is not known. objectives: to determine (1) if raf kinase contributes to phenylephrine (pe)induced contractile response, (2) the role of raf kinase inhibitor (gw5074) in regulating vascular tone during pregnancy, and (3) mechanism by which gw5074 produces vasodilatation in rat mesenteric arteries. methods and results: conscious non pregnant (np) and pregnant (p) sprague dawley rats received increasing doses of pe in the absence or presence of gw5074. pe induced a dose-dependent increase in map in both np and p rats but responses were substantially depressed in pregnancy. gw5074 shifted the pe-induced dose response to the right in both np and p rats. gw5074 itself did not affect basal blood pressure. isometric tension studies in mesenteric arteries showed that gw5074 did not change the kcl-evoked contraction but significantly inhibited the contraction to pe in both np and p arteries. interestingly, at a given concentration, gw5074 produced greater inhibition of pe-induced contractile response in p than in np arteries. also, in p arteries the inhibitory effects of gw5074 were greater as the pregnancy progressed from day 18 to day 20. raf kinase expression and activity was significantly decreased in arteries of p compared to np rats. in mesenteric vascular smooth muscle cells (vsmcs), pe stimulated activation of raf kinase as indicated by phosphorylation of its immediate target, mek1/2 in a time-and dose-dependent manner. measurement of [ca 2+ ] i with fura-2 showed that gw5074-mediated inhibition of pe-induced contraction was not associated with decrease in [ca 2+ ] i . vsmcs treated with pe exhibited higher levels of the contractile proteins, p-mypt1 and p-mlc20 which was inhibited by gw5074. conclusion: to our knowledge, for the first time we show that raf kinase plays an important role in the regulation of vascular contractility. decreased expression and/or activity of raf kinase during pregnancy may explain in part, for decreased systemic vascular reactivity during late gestation. the mechanism(s) that contribute to reduced vascular sensitivity to phenyleperine (pe) during pregnancy is not well understood. pe, in addition to activating the classical contractile pathways, also stimulates growth factor pathways that results in activation of ras/mitogen-activated protein kinases. it is not clear whether these pathways play a role in the modulation of vascular contraction. hence, the present study was designed to determine 1) if ras is involved in mediating the pressor response to pe in non pregnant (np) and pregnant (p) rats, 2) if so, the mechanism by which ras protein contributes to pe-induced vascular contraction, and 3) any differential expression and/or activity of ras during pregnancy that might contribute to altered vascular response. methods: 1) mean arterial pressure (map) was measured in conscious np and p rats. 2) isometric tension was measured in mesenteric arteries of np and p rats. 3) expression of contractile proteins, p-mlc20 and p-mypt1 were studied in cultured vascular smooth muscle cells (vsmcs). 4) expression and activity of ras in mesenteric arteries of np and p rats were measured by western blot analysis. results: (1) intravenous administration of pe resulted in a dose-dependent increase in map but responses were substantially depressed in pregnancy. inhibiting ras activation with manumycin, decreased pe-induced increase in map in both np and p rats. manumycin by itself had no effects on basal map. (2) isometric contraction studies in myograph with mesenteric arteries showed that manumycin shifted pe-induced dose response curve to the right with a decrease in e max in np and p rats. higher concentration of manumycin was required to inhibit pe-induced contraction in np ( 10 μm) compared to p (1 -3 μm) rats. (3) pretreatment with manumycin inhibited pe-induced increase in the extent of phosphorylation of both mlc20 and mypt1 in mvsmcs. (4) compared to p rats, mesenteric arteries of np rats revealed increased basal and pe-induced expression and activity of ras. reduced expression of this contractile ras pathway might contribute to decreased vascular reactivity during pregnancy. conclusion: ras protein plays a role in regulation of vascular contraction. the decreased amount and activity of vascular ras may be a novel mechanism that explains the reduced vascular resistance during pregnancy. overweight affects the relaxin-induced response of mesenteric arteries. joris van drongelen, 1 arianne van koppen, 2 marc ea spaanderman, 1 paul abm smits, 2 frederik k lotgering. 1 1 obstetrics and gynecology, radboud university nijmegen medical centre, nijmegen, netherlands; 2 pharmacology and toxicology, radboud university nijmegen medical centre, nijmegen, netherlands. objective: overweight affects pregnancy-induced vascular adaptation and predisposes to gestational hypertensive disease. relaxin plays a key role in normal gestational vascular adaptation by increasing endotheliumdependent vasodilation and compliance, and reducing myogenic reactivity. we hypothesized that overweight blunts the vascular response to relaxin. the vascular responses to flow and pressure of mesenteric arteries after pre-treatment to human recombinant relaxin (rlx) or placebo (plac) were examined in overweight (ow) and normal weight (nw) rats in a pressure-perfusion myograph. overweight was established by a high-fat diet. the endothelium-dependent vasodilatation was measured in response to flow: e 50 (flow inducing 50% dilatation) and e max (maximal dilative effect). active contractile (myogenic reactivity) and passive dilative (compliance) vascular responses to pressure were determined. all vascular responses were calculated as the proportional change in diameter to 40% precontraction with u46619. results: in nw rats rlx decreased e 50 and increased e max to flow and attenuated myogenic reactivity without affecting vascular compliance. in ow plac-treated rats, compared to nw rats, an upregulated vasodilative state was present (decreased e 50 and increased e max , and lower myogenic reactivity). in ow rats rlx increased e 50 to flow and unaffected e max . rlx increased myogenic reactivity mildly in absence of changes in vascular compliance. values are presented as mean±sem; * p<0.05 between nw/ow, # p<0.05 within nw/ow. whereas rlx stimulates endothelium-dependent vasodilation to flow and myogenic reactivity in nw rats, ow overturns the flow-induced response and decreases myogenic reactivity to a lesser extent than present in nw rats. we speculate that these overweight-induced adverse effects of rlx prelude to vascular maladaptation and gestational hypertensive disease. compared to the luteal (lut) phase, uterine blood flow is increased in vivo during the follicular (fol) phase and more so during pregnancy (preg). both of these are physiologic states of high estrogen and shear stress. endothelial cells express enos and produce greater amounts of nitric oxide (no) in response to elevations of shear stress. phosphorylation of enos is a signaling marker of activation. we have recently validated lut, fol and preg uaec culture models for evaluating enos phosphorylation responses to shear stress and vascular mediators. we hypothesized that uaecs derived from fol and preg sheep will show greater enos phosphorylation than lut phase uaecs, and with more robust responses in the presence of estrogen (e 2 ). methods: uaecs were cultured until 80% confluence, and then subjected to 0 (static control), 3 or 15 dynes/cm 2 for 48hrs in the absence or presence of e 2 (10nm). western analysis was used to compare optical densities of ser635-penos (penos) normalized to total-enos (mean + sem). results: in lut uaec penos was equally increased two fold by 3 and 15 dynes/cm 2 (from 1.26 + 0.5 to 2.4 + 0.008 and 2.56 + 0.06, respectively). compared to lut uaecs, the static control fol uaecs appeared to have higher penos levels (2.2 + 1.5) and this was further increased 1.5-1.8 fold with 3 dynes/cm 2 (3.2 + 0.8) and 15 dynes/cm 2 (3.9 + 0.38). as seen with fol uaecs, preg uaec static penos (1.8 + 0.4) appeared higher than lut uaec, but unexpectedly, neither 3 nor 15 dynes/cm 2 significantly raised these levels of penos (1.76 + 0.05 and 1.6 + 0.04). regardless of shear stress level, e 2 replacement in the culture media only increased the penos levels in the nonpregnant, but not the pregnant derived uaecs. conclusions: increasing amounts of shear stress have a corresponding increase in the ratio of enos that is phosphorylated at serine635 in lut and fol uaecs. pregnant uaecs do not appear to increase the constitutive ratio of serine635 phosphorylation of enos at either 3 or 15 dynes/cm 2 . in contrast to our hypothesis, chronic treatment with e 2 for 48hrs did not augment the ratio of enos phosphorylation in pregnancy. nih hl49210, hd38843, hl87144. cells. honghai zhang, wu xiang liao, dong-bao chen. reproductive medicine, university of california san diego, la jolla, ca, usa. s-nitrosylation (sno) is a rapid, reversible, and nitric oxide (no) dependent post-translational protein modification critical for signal transduction. estrogen stimulates endothelial cell (ec) no production but yet to be determined is if this leads to increased formation of sno-proteins in ec. hypothesis: we hypothesize that estrogen stimulates the formation of sno via a receptor and endogenous no dependent pathway in huvec or ovine uterine artery ec. methods: cells on glass coverslips were treated with 1 mm of no donor gsno or dea nonoate, estradiol-17 (10 nm) in the presence of l-name (1 mm) for 30 min. the cells were fixed with methanol. in situ sno-proteins were detected by a rapid biotin derivatization method by blocking thiols by methylmethanethiosulfonate (mmts), followed by ascorbate reduction and labeling with texas red-labeled ethylmethanethiosulfonate (mtsea) and examined by fluorescence microscopy. cells ( 3x10 6 /group) were lysed in hen buffer, and then similarly prepared but labeled with biotin-mtsea. a portion of the samples were separated on sds-page and blotted with anti-biotin antibody for total sno-protein patterns. the biotin-labeled sno-proteins were captured by avidin, followed by immunoblotting with specific antibodies. results: basal sno-protein labeling was apparent in both ec types. exogenous no donated by gsno or dea nonoate significantly increased red fluorescence labeling of sno-proteins in the cytosol of both ec types. treatment with estradiol-17 also increased total sno-protein labeling in both ec types. pretreatment with l-name significantly reduced sno-protein labeling. sds-page and immunoblotting with anti-biotin antibody analyses identified multiple bands of sno-proteins. in avidin captured biotinylated samples, multiple proteins were indentified. conclusion: exogenous no donated by gsno or dea nonoate and endogenous no upon estrogen stimulation increased s-nitrosylated proteins in ec (supported by nih ro1 hl70562 and 74947). background and objective: the renin-angiotensin system (ras) functions both systemically and locally as a primary regulator of blood pressure and fluid volume and is therefore involved in the etiopathogenesis of essential hypertension as well as preeclampsia, a pregnancy-induced hypertensive disorder in pregnant women. while much progress has been made in the development of strategies to block the systemic ras and thus treat essential hypertension, relatively less advance has been achieved in the development of effective local ras inhibitors to treat preeclampsia, primarily due to the fact the conventional systemic ras inhibitors generate potential teratogenic risks to pregnant women during critical stages of their pregnancies. ginger has been widely and effectively used in pregnant women to treat pregnancyinduced nausea and vomiting with minimal adverse effects. the present study was designed to investigate whether ginger could down-regulate renin secretion by human decidual cells (the major source of renin in the tissue-based uteroplacental ras), as an initial step toward a long-term goal to develop potential safe and effective ras inhibitors for use in obstetric patients. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were treated with ginger juice at various doses in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. the ginger juice used was freshly prepared from the ginger roots purchased from a local grocery store and different batches of juice preparations were standardized based on their optical density values at a wavelength of 400 μm on a spectrophotometer. results: a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the control (i.e. 0%), ginger juice decreased the renin protein contents in the culture supernatants in a dose-dependent manner. no significant difference in the cell morphology was observed between the control and treatment groups. conclusion: ginger root juice down-regulates renin secretion in human decidua, showing a great potential of a novel ras inhibitor, particularly, in obstetric patients. noninvasive quantification of the autonomic nervous system throughout pregnancy. dietmar schlembach, 1 karoline pickel, 1 daniela ulrich, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 manfred g moertl. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. introduction: the analysis of heart rate variability (hrv), blood pressure variability (bpv), and baroreflex sensitivity (brs) has become a powerful tool for the assessment of autonomic control. although hrv analysis was initially developed for risk stratification in cardiology, the field of clinical application has broadened in recent years. the aim of our study was to investigate the adaptation of autonomic control during pregnancy based on analysis of heart rate variability and baroreceptor sensitivity. methods: 20 patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i made by cnsystems. all measurements were performed in supine position under standardized resting conditions. autonomic parameters were recorded at rest and within the "deep breathing method" as a "cardiovascular challenge test". results: throughout pregnancy a shift to higher sympathetic activity respectively a lower parasympathetic activity was observed. , and 16.15±5.54 [>30 wks]) (figure 1). during the deep breathing maneuver we could show an increase of the sympathetic / parasympathetic ratio (figure 2). the noninvasive determination of autonomic parameters throughout pregnancy is possible. these results can be used as basic parameters for classifying and assessing autonomic changes in pathological conditions in pregnancy such as hypertensive disorders. how far the characterization and challenge of these autonomic functions can be utilized for diagnosis and prediction of preeclampsia has to be shown in future studies. hemodynamic parameters measured noninvasively throughout pregnancy. daniela ulrich, 1 manfred g moertl, 1 karoline pickel, 1 philipp klaritsch, 1 isa alkan, 2 uwe lang, 1 dietmar schlembach. 1 1 obstetrics and gynecology, medical university of graz, graz, styria, austria; 2 cnsystems medizintechnik ag, graz, styria, austria. background: hemodynamic changes throughout pregnancy have been measured predominantly by invasive techniques. the discussion on the valence und the low acceptance of these invasive procedures by pregnant women demands a noninvasive method for evaluating and distinguishing cardiovascular adaptative mechanisms throughout normal and especially complicated pregnancy. method: 20 healthy patients with uncomplicated pregnancy were measured with the taskforce® monitor 2040i (cnsystems, austria) at different time points throughout pregnancy. cardiovascular parameters were recorded in supine and left lateral position under standardized conditions. results: throughout pregnancy an increase of global parameters such as heart rate (hr), blood pressure (bp), total peripheral resistance (tpr) and total peripheral resistance index (tpri) were observed. stroke volume (sv), stroke index (si), cardiac output (co), contractility index (ci), acceleration index (aci), and left ventricular ejection time (lvet) decreased throughout pregnancy (table). discussion: the noninvasive determination of cardiovascular parameters throughout pregnancy is possible and the results of this pilot study can serve as basic parameters for classifying and assessing cardiovascular changes in pathological conditions in pregnancy such as hypertensive disorders. assigning pregnant hypertensive women into hyper-and hypodynamic groups may aid in planning individual therapeutic strategies. objective: both gnrh i and gnrh ii are expressed in humans. gnrh i is produced by the hypothalamus under the regulation of gonadal steroids and stimulates pituitary gonadotropins. during pregnancy gnrh i is also produced by the placenta and affects hcg. gnrh ii, the ancient isoform, is produced by numerous human tissues including immune and reproductive tissues and circulates in blood in quantifiable levels. we have demonstrated that gnrh ii analogs directly affect fertilization and uterine function, and propose that it acts via immune regulation. during pregnancy it is known to regulate numerous hormone productions, yet the levels of gnrh ii has not been reported. in these studies we have determined the circulating levels of gnrh ii throughout pregnancy and in early pregnancy loss. study design: thirty-three women having normal pregnancies were followed prospectively. plasma samples were drawn at 8, 10, 12, 14, 16, 28, 36 weeks gestation and during labor. plasma was also collected from patients have spontaneous abortions (n=8). circulating gnrh ii and crh and gnrh i were measured by specific radioimmunoassays. results: circulating gnrh ii increased from non-pregnant levels (65+/-2 pg/ ml, mean+/-sem) to 83+/-2 pg/ml by 10-week lmp. gnrh ii concentrations continued to increase through 16 weeks gestation over the 10-week levels, although a significant increase was only attained by 28 weeks. gnrh ii continued to increase in late term, attaining levels of 99+/-1 pg/ml which was significantly higher than that of early gestation. during labor and delivery gnrh ii in maternal plasma was further increased to 106+/-2 pg/ml, i.e., 1.5 times that of 8-week gestation (73+/-4 pg/ml). circulating gnrh ii did not parallel gnrh i in early pregnancy but did in late gestation. gnrh ii did correlate with crh in early pregnancy but not in late gestation. patients having spontaneous abortion had increased circulating gnrh ii at 8-weeks lmp (85+/-3 pg/ml) as compared to normal pregnancies (73+/-4 pg/ml). conclusion: gnrh ii increased throughout pregnancy attaining highest concentrations during labor and delivery. patient with early pregnancy loss had increased gnrh ii expression. the function of gnrh ii or factors affecting this increased gnrh ii in normal or abnormal human pregnancy should be investigated. introduction: plasma levels of the endocannabinoid, anandamide (aea) decrease during the luteal phase of the menstrual cycle and early pregnancy and increase during parturition. 1 high plasma aea levels at 6 weeks in women undergoing ivf-et was associated with a failure to achieve an ongoing pregnancy 2 . what is uncertain is what happens to aea levels when the pregnancy has already failed. we aimed to quantify aea levels in women presenting in early pregnancy with a diagnosed non-viable pregnancy and to compare them to those of a viable pregnancy. methods: plasma aea was measured by a sensitive isotope dilution hplc-ms/ms method from 45 women in early pregnancy (5-12 weeks) of whom 25 had a viable pregnancy and 20 had a non-viable pregnancy. serum hcg and progesterone were measured in blood samples by standard elisa methods. results: the ages and bmi of both groups were similar (29 ± 4.9 versus 28 ± 4.7 years; mean ± sd; p=0.45; student's unpaired t-test and 24 ± 2.7 versus 25 ± 2.9 kg/m 2 ; p=0.91) respectively. plasma aea levels in women with non-viable pregnancies were significantly higher than those in women with viable pregnancies (1.68 ± 0.77 versus 1.15 ± 0.30nm; p=0.007). there was no correlation found between aea levels and hcg or progesterone levels p=0.272 and r=0.274; p=0.075, respectively) , despite progesterone being significantly lower in the non-viable group (40.0 ± 35.1 versus 61.0 ± 26.3ng/ml; p=0.027). conclusion: higher plasma aea levels were associated with early pregnancy failure, and this association appeared to be independent of serum hcg or progesterone concentrations. these data suggest that plasma aea levels are linked with pregnancy failure through a mechanism that does not involve hcg or progesterone production. the precise involvement of anandamide in early pregnancy needs to be investigated further. 6n-3) and arachidonic (aa, 20:4n-6) acids, the major brain long-chain polyunsaturated fatty acids (lc-pufa) are generated by elongation-desaturation of dietary essential fatty acids (efa). the maternal liver is principally responsible for efa elongation-desaturation. objetive. we determined effects of protein restriction in pregnancy on expression of 5d, 6d and elongases 2 and 5 (elov 2 and 5) in the maternal liver rat. methods. pregnant rats were fed control (20 % casein; c) or restricted (10 % casein; r) isocaloric diets. at day 19 gestation maternal blood and livers were collected. serum triglycerides, cholesterol and glucose were determinate with the synchron cx autoanalyser and leptin and insulin by ria. liver fat determination was performed by the soxhlet method. liver ara and dha were calculated by gas chromatography based upon retention times from methyl ester standards. liver 5d, 6d, elov 2 and 5 mrna were measured by rt-pcr and northern blot. data are m ± sem; analysis by t-test. results. liver weights did not differ in c and r. total liver fat (760 ± 39 vs 543 ± 22 mg) serum leptin (5 ± 0.1 vs 7 ± 0.7ng/ml) and insulin (0.2 ± 0.04 vs 0.9 ± 0.09 ng/ml) differed in c and r respectively (p<0.03). serum triglycerides, cholesterol, and glucose did not differ. desaturase and elongase mrna and % of maternal liver aa and dha were lower in r (fig 1) . conclusion. low liver fat content and desaturase and elongase mrna in r indicate impaired lc-pufa synthesis which may adversely impact fetal development, especially the brain. objective: to test the hypothesis that obstetrical dic results from an excessive leak of fetal material into the maternal circulation. a secondary objective was to assess maternal morbidity. methods: all cesarean hysterectomy cases for hemorrhage at our hospital from 1993 to 2002 were included. intravascular presence of fetal material was determined by two pathologists, blinded to each other and any clinical information. the percentage of those with any fetal material in the maternal circulation was calculated for each diagnosis for hemorrhage. for a given diagnosis, the percentage of intravascular fetal material in those with the given diagnosis was compared to those without that diagnosis using fisher's exact test. most patients had multiple hemorrhage diagnoses. a two sample t-test was used to evaluate the difference in mean blood loss between those with and without intravascular fetal material. results: primary outcome* 89% of patients received blood products and 40% received clotting agents. the average blood loss was 2650 cc. there were no maternal deaths and 4 injuries to adjacent organs, all to the bladder. conclusions: there was no association between the presence of intravascular fetal material and any specific hemorrhage diagnosis or amount of blood loss. although the power to detect a relationship was low, the excessive leakage of fetal material as a specific and exclusive mechanism of obstetrical dic, as postulated, seems unlikely. while 90% of the population was transfused, there were few intraoperative complications and no maternal deaths. hypertension, tel-aviv university, sheba medical center, ramat-gan, israel. objective: the joint national committee on high blood pressure (jnc7) determined blood pressure of 120-139/80-89 in adults to be prehypertension that requires health promoting life style modifications to prevent cardiovascular disease. similarly, we hypothesize that gestational prehypertension in pregnancy is associated with increased risk of pregnancy complications and its management would improve pregnancy outcome. methods: prospective and retrospective recruitment resulted in a study group consisting of 40 patients who were diagnosed in the first and early second trimester (<15 weeks) with blood pressure of 120-130/80, and a control group consisting of 87 patients with blood pressure < 120/80 at similar gestational age. comparisons between the two groups were accomplished for demographic characteristics and outcome measures using the chi-square test statistic and two-sample t-tests for categorical and continuous variables, respectively. results: all outcome measures analyzed resulted in poorer results being associated with the study group compared with control as follows: 10% versus 2% of the study and control group pregnancies, respectively, experienced preeclampsia (p=0.07); and 14% versus 0% of the study and control group, respectively, were associated with gestational hypertension (p=0.0005). 10% of the control group deliveries were associated with admission to the nicu, compared to only 3% in the control group (p=0.16); 17% versus 10% of the study and control group deliveries, respectively, were preterm (p=0.36); twenty-nine percent of the study group were cesarean deliveries, compared to 38% in the control group (p=0.05). on admission mean sbp and dbp was 132 and 76 mmhg, respectively, in the study group, compared to 122 and 70 mmhg in the control group (p=0.0003 for sbp and p=0.03 for dbp). conclusions: "gestational pre-hypertension" may be a real pregnancy condition that when is recognized, physician attention, patient close monitoring during prenatal care and delivery and early intervention in patients at risk, may all promote improved pregnancy outcome. larger scale study is in progress to evaluate and confirm these findings in the larger pregnant population. are contractions at 24-32 weeks gestation less painful than those at full term? kristy a ruis, karin blakemore, abimbola aina-mumuney, valerie jones. gynecology and obstetrics, johns hopkins hospital, baltimore, md, usa. objective to determine if gestational age plays a role in severity of pain associated with uterine contractions. study design self-reported pain from women in labor, on a scale of 1-10, is assessed and recorded in an obstetrical database by nurses at regular intervals at two hospitals within our medical system and was retrospectively reviewed from 2002-2007. pain at various dilatations (3-10cm) of women who were laboring and then delivered at 24-32 weeks' gestation was compared to women in labor at term. maternal demographics of age, race, education level, bmi, presence of support person, request for analgesia at any point in labor, and epidural placement were abstracted from maternal records. categorical data were analyzed using chi square or fisher exact test; continuous variables using student t-test. results 99 laboring patients between 24-32 weeks gestation and 409 at term were identified. term and preterm patients did not differ with respect to maternal demographics. pain reported by preterm patients was significantly less at 3-4 cm dilatation (4.8 vs. 6.3‚ p=0.01) and significantly greater at 9-10 cm (4.2 vs. 2.3‚ p=0.03). pain reported at 5-6 cm and 7-8 cm was not statistically different between the groups. of note, fewer of the preterm patients received an epidural despite the request for analgesia. conclusion preterm laboring patients between 24-32 weeks gestation may perceive less pain at 3-4 cm dilatation compared to term laboring patients; however, their pain perception at advanced dilatation was comparable to those at term despite the fact that they were less likely to have an epidural in place at the time of pain evaluation. in light of these findings, the widely accepted etiology of labor pain as the result of cervical dilatation may need to be re-examined. also, factoring in the patient's discomfort level into the evaluation for onset of early active labor may not be valid in the preterm patient. background and objective: asthma is a risk factor for adverse pregnancy outcome. this has been attributed to the effect of allergy in pregnancy. mast cell mediators can induce uterine contractility. the objective of the study was to test the hypothesis that pregnant women with asthma have different uterine electrical signals from those generated by normal pregnant women. materials and methods: uterine electromyography (emg) recordings from gestational age matched pregnant women at term were analyzed. the cases consisted of patients with asthma and the controls, those women without asthma. emg was recorded for approximately 30 minutes from surface electrodes placed upon the maternal abdomen, with the electrical signals filtered in the uterine-specific range of 0.34 to 1.00 hz to remove noise components. the recordings were analyzed in their entirety first by power spectrum analysis and then by lyapunov analysis. the power-spectrum-largest-peak frequency and the largest lyapunov exponent were calculated for each patient, and then the mean and standard deviation of these parameters was compared using student's t-test. results: patients with asthma had significant lower power spectrum frequency and higher lyapunov exponent than women without asthma. see 1) the power spectrum frequency and the lyapunov exponent used to analyze uterine emg signals were different in women with and without asthma; 2) the results suggest that uterine electrical activity is altered in women affected by asthma; 3) these findings may explain the observed increased frequency of preterm birth in women with asthma. further studies are required to dissect the mechanisms. fetal microchimeric cells trafficked into the maternal circulation persist in blood and tissues for years after pregnancy. increasing data suggest that microchimerism occurs after every pregnancy, but the biological role of fetal microchimerism or the cell types involved is unclear. while persistent fetal cells were initially implicated in autoimmune disease, animal studies suggest these fetal cells play a broader role in response to tissue injury. methods: appendix specimens were acquired from 9 women undergoing appendicectomy during pregnancy. detailed reproductive histories were obtained. fluorescence in situ hybridisation (fish) with two different probes allowed investigation of the presence of male presumed-fetal cells and nested pcr amplification of sry gene confirmed male dna in the appendix. immunostaining was used to determine the fetal cell phenotype. results: male cells were identified in appendix tissues from women with known male pregnancies (n=7) and also from a woman with no sons and a previous miscarriage of undetermined gender (n=1). no male cells were observed in the control (n=1), a woman with 3 daughters. male cells of presumed fetal origin were evenly distributed in the muscle, mucosa and submucosa layers of the appendix. morphology and co-localisation analysis suggested the identified male cells had differentiated primarily into muscle cells or lymphocytes. combined immunostaining and y-fish demonstrated male desmin+ muscle cells and cd3+ and cd19+ lymphocytes. finally, the presence of male dna in the appendix specimens was confirmed by nested pcr. male cells of presumed fetal origin were identified in the appendix of pregnant women. microchimerism frequency varied according to the reproductive history and the degree of inflammation. microchimerism rates were higher in the appendix from women with current male pregnancies than in those with previous male pregnancies and were higher in those with histological acute inflammation, when compared to milder cases. male microchimeric cells identified were of haematopoietic and mesenchymal origin. this study suggests that fetal cells are present in sites of tissue injury and may participate in tissue repair during pregnancy. collagen i 2 and collagen-binding integrins mrna expression in rat cervix during gestation. huiling ji, tanya l dailey, vit long, edward ks chien. obstetrics and gynecology, maternal fetal medicine, women and infants' hospital of rhode island, providence, ri, usa. objective cervical remodeling is associated with cell proliferation and extracellular matrix (ecm) remodeling. integrins are a family of multifunctional cell adhesion receptors which mediate ecm-cell interactions including binding collagen. of the 24 integrin (i) heterodimers, 1 1, 2 1, 10 1 and 11 1 are primary receptors for collagen. the predominant cervical collagen is collagen type 1. the purpose of this study is to investigate collagen and integrin expression in the pregnant rat cervix. the cervix was harvested from timed pregnant sprague-dawley rats. non pregnant (np)and timed pregnant day 12, 16, 18, 20, 21 and 22 animals were euthanized using a protocol approved by the iacuc. four animals were sacrificed on each day of gestation. quantitative rt-pcr was used to evaluate mrna expression and normalized to -actin. a standard curve was generated from a single sample. data was analyzed using anova and multiple comparisons testing. collagen i 2 mrna expression increased through mid-gestation but decreased to np levels on day 22. a similar pattern was seen with the integrin beta 1 subunit. the pattern of integrin alpha subunit expression was different for each subunit during pregnancy. the changes in collagen, integrin alpha 1, 10, 11 and integrin beta 1 were found to be statistically significant. (figure) . the pattern of collagen binding integrin alpha subunit expression during the rat gestation appears to vary independently of each other. the expression of the 1 subunit paralleled col 1 2 expression. collagen binding integrins may play independent roles in signaling and biomechanics during gestation. funding: women infants' hospital research fund; phs nih-ncrr p20 rr018728 p20 rr017695. background. the effects of chemotherapy on human fetal development are poorly studied, mainly due to the lack of adequate animal models in which placentation and embryological development resembles humans and pharmacokinetic studies, prenatal sonography as well as histological studies can be performed. aim of the study. to test the baboon as model for studying pharmacokinetics and fetal effects of chemotherapy administered during pregnancy. methods. experiments were performed in 11 pregnant baboons at a mean gestational age of 134 (+/-10) days. detailed ultrasound examination (biometry, doppler, screening) was performed 3 days before, at and 1 day after the drug administration. the administration of different schemes of chemotherapy and the fetal samplings occurred under endotracheal anaesthesia. maternal blood samplings, as well as percutaneous ultrasound guided fetal blood (2ml) and amniotic fluid (4ml) samplings were performed at least once immediately after drug administration. in case of fetal demise, the fetus underwent detailed macro-and microscopic examination. in the other animals mother and fetus were euthanized 24h after the experiments, with collection of blood, amniotic fluid and tissues for further analysis. results. all 11 mothers survived the experiments, one mother developed paralitic ileus. none of the fetuses died during the acute phase of the experiment but 3/11 (27%) fetuses died within 24h following the experiment. none of the animals showed significant clinical or histological signs of infection or anaemia. a total of 50 ultrasound examinations were performed in 11 fetuses, allowing the creation of sonographic growth charts in this model. at the time of drug administration the mean maternal weight was 16.8kg (range 14.0-20.2) and the estimated fetal weight was 405gr (range 317-484). sixteen out of 18 cordocenteses (89%) and all 18 amniocenteses (100%) were successful in obtaining the required samples for analysis. conclusion. the pregnant baboon can be used as a model for studies on pharmacokinetics and fetal effects of chemotherapy administered during gestation. prenatal ultrasound is comparable to the human situation, and amniocentesis and cordocentesis can be performed with a high success rate and short term survival. therapy to prevent preterm birth is limited". moreover, 17-hydroxyprogesterone (17ohp) caproate is a category d drug according to the fda (evidence of fetal harm). p is produced in the adrenal glands, gonads and brain. after 8 weeks gestation p is secreted from the placenta independently to the mother and the fetus. p is the precursor of aldosterone and after conversion to 17ohp, of cortisol and androstenedione. baboons have similar reproductive system and development to humans and are used to study mechanisms of labor and prevention of preterm labor. currently, there are no normative data on the concentration of p, 17ohp, cortisol ©, estradiol (e2) or inhibin (i) throughout their pregnancy. therefore, a doseresponse or the teratogenic effects of p or 17ohp caproate cannot be studied in a controlled manner in this animal model. the aim of this study was to generate reference values for these hormones during baboon gestation and early postpartum life. material and methods: 43 hormonal quantitative measurements were performed (immulite analyzer) results: the mean concentrations of these 5 hormones are depicted in figure 1 (maternal serum, from conception to term 180 days) and figure 2 (newborn serum) conclusion: this study provides reference values for 5 hormones quantified during the baboon gestation and early postpartum life. this may be useful for future research concerning the effects of p and 17ohp administration during pregnancy. (1)non-pregnant women eligible for ivf treatment and (2)pregnant women receiving prenatal care. the pre-9/11 samples were drawn in the year prior to 9/11/01; the post-9/11 samples were drawn within 6 months after. the pre-9/11 and post-9/11 normal pregnant samples were drawn at 20+2 weeks' gestation. the non-pregnant samples were from ivf candidates with serum e2 levels<75 newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced pg/ml and fsh serum levels<12.5 miu/ml. the samples were assayed for acth and cortisol by a commercially available immulite™ system. hsp70 and hsp90 were measured by commercially available elisa. these results were compared in the patient populations before and after 9/11. t-tests were used for analysis. statistical significance was set at p<0.05. results: in the pregnant subjects, there were no statistical differences in the mean levels of acth, cortisol, and hsp70 pre-and post-9/11 (table 1) . only serum hsp90 levels were significantly increased in the pregnant women post-9/11.* mean serum acth, cortisol, hsp70, and hsp90 levels were all significantly different in non-pregnant subjects pre-and post-9/11. conclusions: except for hsp90, serum markers for stress were unchanged in pregnant subjects after the stress of 9/11/01 in comparison to non-pregnant women whose serum hsp90, hsp70, cortisol, and acth levels were significantly different. the lack of change in serum markers of stress in pregnant women suggests that the hormonal milieu of pregnancy may buffer against acute environmental stressors. objective: in human pregnancy, maternal body composition provides an indicator of maternal nutritional status and metabolic capacity. previously, we reported that 11ß hsd-2 activity was significantly reduced in term placentas from thin women and women with a smaller mid-upper arm circumference before conception. this suggests that these fetuses may have been exposed to inappropriate levels of maternal cortisol, which is a mediator of gestation length. in this study, we hypothesize that the duration of gestation will be shorter in women who tend to be thin and have a lower mid-upper arm circumference prior to conception. methods: within the longitudinal, population-based southampton women's survey (sws), analyses were performed on 1301 women whose estimated date of conception was set using an algorithm that combined menstrual and early ultrasound scan data and who had a spontaneous onset of labour and delivered after 37 weeks of gestation. linear regression was used to examine the relationships between maternal body composition and the duration of gestation. results: within the 1301 women surveyed, lower maternal body mass index, mid-upper arm circumference and arm muscle area before conception were all associated with shorter duration of gestation at delivery (r=0.12, p=0.00001; r=0.10, p=0.0002; and r=0.09, p=0.0009, respectively). a lower subscapular skinfold thickness, sum of skinfolds and height were also associated with shorter duration of gestation (r=0.08, p=0.006; r=0.07, p=0.009 and r=0.06, p=0.03, respectively). mother's age, own birthweight and ratio of subscapular/triceps skinfold thickness were not related to the duration of gestation. conclusions: in this study, we found that thinner women with a lower midupper arm circumference and arm muscle area tended to have a shorter duration of gestation. our findings of shorter gestation length in thinner women are in keeping with our previous observation of lower 11ß hsd-2 activity in term placentas from thinner women. we conclude that metabolic capacity prior to conception could influence duration of gestation through mechanisms that include alteration in placental metabolism and fetal cortisol exposure. fecal incontinence (fi) is a debilitating condition affecting 2-10% of the us. our prior studies found that 29% of women report new onset fi after childbirth. the goal of our study was to examine the impact of fi on postpartum quality of life (qol). women reporting fi on a statewide survey who agreed to participate in a 2-year study of qol were included in the analysis. women were considered to have fi based upon the nih definition of fi. the quality of life survey was based upon the uebersax incontinence impact questionnaire and was administered every 6 months for 2 years. qol in women with fi was examined using chi square, and impact of severe fi (stool incontinence) was determined by multivariate logistic regression. results 2907 women with fi were surveyed and of those, 1247 (43%) returned at least 1 survey during the study period, completed all survey questions, and were included in the final analysis. among women with fi, 51% felt frustrated due to fi, 26% reported fi impacted their emotional health, 18.5% reported fi impacted child-caring abilities, and 16.7% reported a negative impact on social activities. qol was similar across survey periods. one out of three women with fi reported severe symptoms (incontinence of stool). women with severe symptoms were 4-7 times more likely to report negative impacts on qol compared to milder (e.g. flatus) fi after adjusting for age, parity and urinary incontinence. 33.9% felt their stool was stored elsewhere before bowel movements, 13.9% reported using digital defecation and more than half (52%) reported symptoms of urinary leakage. despite the substantial impact on postpartum quality of life, few women sought medical help with only 10% of women at 6 months, 13.5% at 1 year, and 16.7% at 2 years ever reporting their symptoms to a medical provider. postpartum women report that fecal incontinence has a substantial negative impact on their qol after delivery including their emotional health and ability to care for their newborn. despite this profound impact, few women will discuss fi with medical providers. these data suggests there may be a benefit for providers to inquire about fi at postpartum visits. objective: the objective of this study was to determine what characteristics contribute to racial/ethnic differences in breastfeeding rates. study design: a retrospective cohort study of all women who delivered a viable infant (n=26,781) was conducted. the primary outcome was breastfeeding upon discharge of the hospital. we first examined the association between race/ ethnicity and breastfeeding. next, we conducted stratified analyses examining a variety of predictors of breastfeeding within the racial/ethnic groups including maternal demographics, obstetric interventions, and perinatal complications. results: we found that both asians (or 0.53, 95% ci 0.47-0.61) and blacks (0.32, 95% ci 0.27-0.37) had statistically significant lower rates of breastfeeding, while latinas (or 1.18, 95% ci 0.99-1.41) showed a trend towards higher rates of breastfeeding. in latinas, we found that 3 rd and 4 th degree lacerations were significantly associated with lower rates of breastfeeding, but were not predictive of breastfeeding in the other racial/ethnic groups. epidural use was predictive of lower rates of breastfeeding in caucasians and blacks, but was not predictive in latinos and asians. some of the other predictors which differed between the racial/ethnic groups were obesity and induction of labor (table 1) . conclusion: race/ethnicity is significantly associated with breastfeeding, with blacks and asians having the lowest rates of breastfeeding at discharge. a variety of other factors are associated with breastfeeding and interestingly, their effect appears to differ between the racial/ethnic groups. future studies might elucidate the sociocultural and biomedical reasons that explain the differences. these results help focus our efforts during the peripartum period to advocate for mothers who have factors that might impede breast feeding. epidemiology, erasmus medical centre, rotterdam, netherlands; 4 pediatrics, erasmus medical centre, rotterdam, netherlands; 5 public health, erasmus medical centre, rotterdam, netherlands; 6 clinical genetics, erasmus medical centre, rotterdam, netherlands. background recommendations on folic acid use to prevent neural tube defects are launched in several countries. however, the adequate use of folic acid supplements during the periconception period seems to be low. objective to assess the prevalence of adequate folic acid use, defined as the preconception start of supplements, and to identify its determinants in a multi-ethnic population. design the study was embedded in the generation r study in rotterdam, the netherlands, a population-based prospective cohort study from early pregnancy onwards. methods from all women in the cohort who delivered between april 2002 and january 2006 information on folic acid use and potential determinants was obtained by questionnaires and physical examination. logistic regression models were used to identify determinants of periconception folic acid use. results. data from 6,940 pregnant women were available. of all women 37% adequately used folic acid supplements. the most important risk factors for inadequate use were unplanned pregnancy (or 9.5, p<0 .001), nonwestern ethnicity (or 3.5, ci 2.9-4.3, p <0.001) and a low educational level (or 2.5, ci 1.8-3.6, p<0.001). other risk factors were single marital status, smoking, multiparity (all p<0.001) and alcohol use (p<0.05). prior spontaneous abortion was associated with increased adequate folic acid use (p<0.001). conclusion adequate periconceptional folic acid use is low. improved preconception care and public health education programs are necessary to improve the uptake of folic acid. although methamphetamine (ma) use has been front and center of the united states drug control policy since 2005, little attention has been given to ma use in pregnancy, despite the fact that pregnant admissions to drug treatment for ma have been rising sharply. to determine trends in the prevalence of ma treatment admissions during pregnancy, we undertook a secondary analysis of the treatment episode data set (teds), an administrative data set that captures at least 70% of all known treatment admissions in the us. in particular, we investigated risk factors for ma use and how these characteristics have changed over time. demographic, geographic and substance use data were collected for the 219,335 pregnant admissions captured between 1994 and 2005. logistic regression models were constructed by year. confounding was assessed via backwards elimination with a change-in-estimate criteria of 0.1 considered substantial. trend results were reported as adjusted proportions and represented graphically. overall ma prevalence, reported as the primary drug of use upon admission, rose from 8.1% in 1994 to 24.5% in 2005. although white women had 3 times the odds of using ma in 1994 (adjusted or (95%ci) 3.2 [2.8, 3.8]), this had dropped to 1.9 [1.7, 2.1] by 2005, mostly due to an increase in latina ma use in pregnancy. pregnant women admitted for ma had fewer prior treatment admissions, were more likely to be unemployed, and less likely to use alcohol or marijuana. we found great geographic variability in use. ma was more common in the pacific region and least common in new england. there was little change in regional variation over the study time period. less is known about the perinatal effects of ma compared with other substances, yet it is the primary drug of choice for pregnant women admitted into treatment. as pregnant women occupy a unique place in drug treatment, analysis of national trend data is essential to guide both policy and research. background: epidemiologists have grouped the multiple disorders that lead to extremely preterm delivery in a variety of ways. we sought to identify characteristics that would support the combining or dividing of the disorders that lead to preterm delivery. methods: we enrolled 1,006 women who delivered a live born singleton infant between 23 and 27 completed weeks gestation at 14 tertiary centers in the united states. each delivery was classified according to the complication that prompted presentation: preterm labor, preterm premature rupture of newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced fetal membranes (pprom), preeclampsia, placental abruption, cervical incompetence, and fetal indication/intrauterine growth restriction (iugr). we compared these entities on the frequency of characteristics identified by standardized interview, chart review, histological examination of the placenta, and culture of placenta parenchyma. results: the percents of women who presented with each antenatal complication were: preterm labor (40), pprom (23), preeclampsia (18), placental abruption (11), cervical insufficiency (5), and fetal indication/iugr (3). after considering antecedents and correlates of the processes leading to preterm delivery, we observed two overarching epidemiologic patterns. the first pattern, characterized by recovery of organisms from the placenta and by histologic chorioamnionitis, tended to be associated with preterm labor, pprom, placental abruption, and cervical insufficiency. the second pattern, characterized by a paucity of organisms and inflammation and the presence of histologic features of dysfunctional placentation, tended to be associated with preeclampsia and fetal indications/iugr. conclusions: disorders leading to preterm delivery can be categorized broadly into two groups: those associated with intrauterine inflammation and those with aberrations of placentation. predictors of compliance with tuberculosis screening in pregnancy. nadav schwartz, 1 sarah wagner, 1 sean keeler, 1 may tam, 1 julian mierlak, 1,3 aaron caughey. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny; 2 obstetrics and gynecology, ucsf, san francisco, ca; 3 obstetrics and gynecology, gouverneur health care services, new york, ny. objective: poor compliance with tuberculosis screening and treatment is a major obstacle to the containment of this disease. we sought to identify predictors of ppd and cxr compliance during pregnancy. methods: a retrospective cohort study at a single institution which serves a largely immigrant population in nyc, from november 1, 2001 through june 30, 2006. data on maternal age, ethnicity, country of origin, level of education, ppd and cxr status were collected. results: of the 4,049 pregnancies, asian and hispanic race/ethnicities accounted for 50.4% and 44.0% of the population, respectively, with 11.2% being us-born. the mean age was 27.02 years and the overall ppd+ rate was 50.3%. there was 5% non-compliance with ppd testing, and 5% of ppd+ patients were non-compliant with their chest x-rays. asian women were more likely to be ppd-compliant than hispanic or caucasian women. ppd+ asian women were also more likely to be compliant with cxr. us-born women were significantly less likely to be compliant with their ppd or with their cxr. women >40 years old were less likely to be compliant with their cxr, while women with an elementary school education or less were more likely to be compliant. (see table for odds ratios and 95%ci) conclusions: age, education, immigrant status and other cultural and ethnic factors appear to play a role in compliance with tuberculosis screening. further elucidation of these effects may help clinicians target at-risk subpopulations and improve overall compliance, working towards better control of this disease. background: in the us, differential outcomes in cervical cancer among medically underserved women are linked to multiple barriers impacting loss to follow-up and failure or delay in diagnostic resolution. prior studies have found risk factors for acquisition of hpv and for inability to obtain a pap smear. no study has explored health literacy and physician communication as a key factor. methods: this research represents the formative phase of a randomized controlled trial of african american (aa) and hispanic women aimed to improve communication and abnormal pap smear follow-up in chicago. semi-structured interviews and focus groups were conducted face to face in spanish or english. each interview lasted 45 minutes, and each focus group lasted 60-90 minutes. we recruited 20 patients (10 hispanic, 10 aa) and 20 providers from a purposive sample representative of two large clinics that serve low-income women. results: all interviews were transcribed by two investigators. each interview was coded by two investigators separately and then a third investigator reviewed the transcripts and coding to achieve triangulation. codes for these themes were developed and the responses were tabulated using the coding scheme. atlas ti was utilized to analyze all qualitative data. from these data, we uncovered provider-patient challenges in cancer communication including: the providers' trade off between medical accuracy and/or literacy when communicating with their patients. for the patients, the word cancer was important to hear since they wanted the truth and needed to hear this word in order to encourage them to respond more quickly. however, many providers believed that the word cancer was too "scary" and to "extreme" of a word that may communicate too much exaggerated information. both the hispanic and aa patients did not seem to differ in their responses. conclusion: results exemplify not only the importance of health literacy and patient provider communication, but demonstrate the wealth of information gained from qualitative research-a method of data collection seldom utilized in ob/gyn investigations. funding: women's reproductive health research award: hd050121-02; r21 ca126450 (spring). population. kristine beckers, 1 isabelle guelinckx, 2 greet vansant, 2 roland devlieger. 1 1 obstetrics and gynaecology, university hospital gasthuisberg, leuven, belgium; 2 clinical nutrition, katholic university leuven, leuven, belgium. objective: to generate reference charts for weight gain during pregnancy for the different bmi-categories (underweight, normal weight, overweight, obesity), based on recent data in a homogeneous caucasian population. methods: in a retrospective study at the department of obstetrics of the leuven university hospital (belgium), weight gain and pre-pregnancy bmi were determined in 605 belgian pregnant women with accurately dateable, uncomplicated singleton pregnancies. centile curves for the different bmicategories were constructed using of the linear mixed model, one set of charts based on the absolute weight gain, another set based on the relative (expressed as percentage) weight gain. the effect of parity on weight gain was examined. results: overall mean weight gain was 14.8 ± 4.7 kg (32.63 ± 10.36 lbs). mean weight gain was 15.4 ± 4.1 kg (33.95 ± 9.04 lbs) in the underweight population, 15.1 ± 4.5 kg (33.29 ± 9.92 lbs) in the population with normal weight, 13.7 ± 5.3 kg (30.20 ± 11.68 lbs) in the overweight population and 12.0 ± 5.9 kg (26.46 ± 13.01 lbs) in the obese population. weight gain (pattern and amount) of the underweight and normal weight patients differed significantly of the overweight and obese patients. parity had a statistical, but no clinical significant influence on amount and evolution of weight gain. conclusion: by using strict inclusion criteria, bmi-category-specific reference charts were generated representing the optimal gestational weight gain, rather than the mean weight gain. this enables the weight charts to be used as a clinical tool during the counselling of pregnant women. further studies are required to assess the effectiveness of this clinical tool. female fshr(-/-) mice are sterile, because of a block in folliculogenesis at the primary follicle stage, show decrease in e2 and elevated fsh. this animal model is an appropriate model for studying hypergonadotropic ovarian dysgenesis and infertility, caused by c566t mutation in fshr gene. objective: to investigate the effects of bmt on serum hormonal levels, follicular maturation and fertility of fshr(-/-) mice. methods: fshr (-/-) mice at 6-10 weeks of age were randomized into treated versus control groups.bm from 1 syngenic female donor was injected into the tails of 2 recipients. control group received vehicle alone. vaginal smears were collected, body weight was measured daily. sample animals were sacrificed at 0, 1, 2, 3, and 4 weeks post bmt. all organs were weighted and examined by h e. fsh, e2, and p4 were measured before and after treatment. for donor cell tracking, dna was extracted from various organs. specific primer sets were designed for normal and mutant hfshr gene. pcr amplification was done and pcr products were analyzed in 1% agarose gel. results: total body weight significantly increased in treated animals(p< 0.02). significant increase in both the total number of follicles, and the collective diameter of the follicles in treated animals observed(p<0.03 and p< 0.002). six out of 8 treated animals showed estrogenic changes in daily vaginal smear. e2 level increased 2.5-7.5 times and fsh level dropped to 50% in treated animals. normal (donor allele) fshr gene was amplifiable in 6 out of 8 recipient mice, and was detected only in the ovaries and uterus but not in any other tested organs.control group did not show any changes in vaginal smear, hormonal level, and normal fshr allele. conclusion: intravenously injected syngeneic bone marrow cells were able to home to the ovaries of female fshr (-/-) mice and restore folliculogenesis and resume steroid hormone production. potential mechanisms for these observations will be discussed. conclusion: neural stem cells (nscs) give rise to progenitors, which expand by rapid proliferation until cell cycle arrest followed by differentiation along one of 3 cns cell lineages: neurons, astrocytes and oligodendrocytes. increased differentiation of neuronal cells with ins and even more with leptin suggests that iugr-associated reduction of these growth factors may contribute to impaired hypothalamic pathway development. objective: to develop a technology of modulating hucb in order to achieve neuronal cells for future potential replacement of damaged neuronal tissue. design: we developed a two-dimensional (2d) tissue culture technology for isolation and differentiation of collagen-adherent hucb neural progenitors (hucbnps). we further used the extracellular matrix protein collagen, organized in a three-dimensional (3d) gel, supplemented with neuronal conditioning medium and nerve growth factor (ngf), to facilitate ex-vivo long term neuronal differentiation of hucbnps. we developed a stable green fluorescence protein (gfp)-pc12 cell model, to be used as a positive control for monitoring neuronal outgrowth for proper evaluation of the hucbnps growth in the 3d scaffold, mimicking a neural tissue organization. results: our experimental data indicate that 3d collagen environment is neuronal biocompatible, supporting attachment, long-term survival, proliferation and differentiation of both hucbnps and gfp-pc12 cells. conclusions: improvement of the 3d technology with cultures of hucbnps that is in progress in our laboratory might be the first step in validating the concept for the feasibility of generating a neuronal 3d tissue construct for future potential treatment of neurodegenerative disorders. piane, justin tsai, gnanaratnam giritharan, emin maltepe, paolo rinaudo. reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: ivf embryos are often used to generate stem cells. however, it is unknown if stem cell lines originated from in vitro generated embryos are different from stem cell originated from in vivo embryos. trophoblastic cells, due to their external position within the embryo, are most susceptible to environmental factors encountered in vitro. furthermore, our previous data showed that trophoblast transport functions may be impaired after culture in vitro and that the number of trophoblastic cells is reduced in the embryo after ivf. in this study, we assess the differentiation characteristics of ts cell lines obtained from in vivo and in vitro fertilized embryos. methods: oocytes were isolated from superovulated c57bl/6j female mice and in vitro fertilized with sperm from male c57bl/6j mice. the resulting late-cavitating blastocysts were harvested. in vivo controls were obtained by flushing the blastocysts from the uteri of superovulated pregnant mice 4 days post hcg. ts cells from the two groups were allowed to develop and maintained in vitro in the presence of fgf4 and heparin, using a feeder layer of human placental fibroblasts. ts cells were then allowed to differentiate without fgf4 and heparin, fixed on day 8, stained with -tubulin and zo-1 antibodies and then observed under fluorescence microscopy. three ts cell lines per group were analyzed and their differentiative capacity was evaluated using morphological criteria. results: in vivo and ivf derived ts exhibit a similar differentiation pattern. in particular, the number and timing of trophoblast giant cells and spongiotrophoblasts derivation is similar in the two groups. there are no obvious abnormalities in the immunologic staining morphology of the different cell lines at different time points. conclusion: there are no apparent morphological alterations in ts cells lines derived from ivf embryos as compared to in vivo embryos. this finding in an animal model increases our confidence in the reliability of human stem cells derived from ivf. as a further investigation, markers of trophoblast giant cells, spongiotrophoblast, syncytiotrophoblast and chorionic trophoblast cells will be examined and compared in the two different groups by northern blot hybridization. genomewide high density snp-cgh reveals several new deletion copy number variants on the x chromosome in pof patients. erik ah knauff, 1 cisca wijmenga, 2 ruben van 't slot, 2 lude franke, 2 bart cjm fauser. 1 1 reproduction gynecology, university medical centre, utrecht, netherlands; 2 complex genetics group, university medical centre, utrecht, netherlands. introduction: around 1% of women have a post-menopausal hormonal profile before age 40, referred to as premature ovarian failure (pof). pof could act as a genetic model for accelerated follicle loss and may render useful information about the polygenetic background of major individual differences in menopausal age. macrodeletions on the x chromosome are associated with pof but karyotyping has a maximal resolution of 10mb. when using genotyping whole genome arrays it is now possible to detect submicroscopic deletions and duplications (copy number variants (cnv)) up to 50 kb effective resolution. we have screened for microdeletions on the x chromosome in a well-phenotyped cohort of pof patients. methods: our study included 108 caucasian, 46,xx patients with spontaneous secondary amenorrhea before age 40, fsh > 40 iu/l and absence of (low) 45x/46 xx mosaicism and fmr1 premutations. dna analysis was performed using illumina 370k cnv arrays containing 370,404 probes. 12,556 probes were located on the x chromosome ( 1 probe for every 12 kb), of which 1,348 probes were specifically designed to detect known cnvs. ten samples with call rates <99% were removed from the analysis and one sample gave inconsistent x probe intensities. we screened for deletions ( 60 kb) using an algorithm detecting at least five consecutive probes with intensities (logr > -0.20) below the mean probe intensity. we identified a total of 183 x chromosomal microdeletions, divided in 84 loci and varying between 60-200 kb in size. 46 of the 97 samples showed at least one deletion on the x chromosome. 27% of the identified deletions had already been recorded in the database of genomic variants. in the newly identified newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced loci, we found 26 coding regions containing 38 genes. eight of these are established or potential pof candidate genes, including five that are clustered on the terminal xq critical pof region. conclusions: we observed abundant variation in the cnv regions, a proof-of-principle for this specially designed cnv-chip. snp comparative genomic hybridization revealed possible new deletions specific to pof on the x chromosome. data on duplications, validation and whole genome cnv analysis, compared to a control cohort, will become available soon and will be presented at the sgi annual scientific meeting. unexplained intrauterine fetal death (iufd) is associated with long qt syndrome. irene cetin, 1 patrizio antonazzo, 1 sabrina cozzolino, 1 stefania calabrese, 1 lia crotti, 2 francesca ferrari, 3 roberto insolia, 2 fabio facchinetti, 3 peter j schwartz. 2 1 dept. of obst/gynecol, univ. of milano, milano, italy; 2 molec cardiol lab, policlinico san matteo, pavia, italy; 3 mother-infant dept., univ. of modena/reggio emilia, modena, italy. introduction: in developed countries, nearly 1 in every 200 pregnancies ends in late fetal loss. many iufds can be attributed to maternal disorders, fetal pathology, placental pathology and fewer to complications of labor and delivery. however, 25-50% of cases remain unexplained. we recently demonstrated that 10% of sudden infant death syndrome (sids) cases carry functionally relevant genetic variants in long qt syndrome (lqts) genes. aim of the study was to analyze whether lqts genes are associated with iufd. materials and methods: 60 patients with iufd were enrolled in two years, as part of an italian multicentre study. iufd was defined as fetal death at 22 weeks or more of gestation according to the definition of late fetal death of the who. 32 out of 60 cases were classified as "unexplained stillbirths" according to the wigglesworth and aberdeen classifications. at birth placental and/cord samples were collected and dna extracted. the main lqts genes kcnq1, kcnh2, scn5a, kcne1 and kcne2 were screened through dhplc and sequence analysis. any amino-acid substitution identified in the samples was checked for in a control population of 122 caucasian women with uneventful pregnancies. preliminary data on the first 17 cases (gestational age of death: 23-38 weeks) are reported. results: a total of 3 missense mutations were identified in 3 of 17 stillbirths (18%), two on scn5a and one on kcnh2. the two mutations on scn5a (v1951l; p2006a) were observed in two iufd occurred at term; they had been previously associated to sids and shown to increase the late sodium current. the mutation on kcnh2 is a novel genetic variant absent in 244 reference alleles, never described in any control populations; this mutations was present in a case of iufd diagnosed at 33 weeks of gestation. we are currently performing the electrophysiological cellular studies to define its functional effect. conclusions: these preliminary data indicate that a potentially significant number of currently unexplained iufd might be caused by ion channel diseases such as lqts. the potential prevention of sids or iufd recurrence and the identification of other affected family members could have important implications for the affected families. alternative splicing of epab is regulated by exonic splicing enhancers. e seli, a yaba, o guzeloglu-kayisli, md lalioti. ob gyn, yale u., new haven, ct, usa. introduction: alternative splicing is an important mechanism by which the genome gives rise to the observed diversity of proteins. embryonic poly(a) binding protein (epab), expressed exclusively in oocytes and early embryos, mediates translation of maternal mrnas. we identified an alternatively spliced form of epab lacking exon 10 (cex10del), and investigated its regulation as a model for alternative splicing in early development. specifically, we evaluated: imprinting (expression from maternal or paternal allele only); rna editing (post-transcriptional single nucleotide substitution); and exonic splicing enhancers (eses, exonic sites that bind splicing proteins). methods: a single nucleotide polymorphism (snp) detected in exon 9 (c1290a/g) served as a marker for the parental origin of the spliced form. snp genotyping was performed by pcr amplification of exon 9 followed by restriction enzyme digestion. to evaluate imprinting, we characterized heterozygous mice (a/g) that inherited the snp from either the mother or the father. to test for rna-editing and exonic enhancer contribution we tested mice homozygous for the exon 9 snp (a/a or g/g). efficiency of alternative splicing in different genetic backgrounds was tested using real-time pcr normalized to actin expression. results: in mice heterozygous (a/g) for the exon 9 snp, cex10del was only expressed from the (a) allele. however, this was independent of the parental origin of the allele, ruling out imprinting. in mice homozygous (g/g) for the exon 9 snp, the cex10del variant also contained (g). therefore, rna editing did not occur. further sequence analysis led to the identification of an additional snp in exon 10 (c1383g/c) that co-segregated with the exon 9 snp. presence of c1383g led to the formation of an ese that binds splicing regulatory protein srp40, leading to efficient exclusion of exon 10. real time pcr revealed a five-fold increase in the expression of the cex10del alternative splicing variant in animals carrying the enhancer (homozygous g/g) for the exon 10 snp compared to those that did not (homozygous c/c) at the same locus (p < 0.001). conclusions: in this study, we found that eses mediate the alternative splicing of oocyte-specific transcripts. our findings suggest that single nucleotide polymorphisms may alter the ratio between alternative splicing variants of oocyte-specific proteins. the role that these subtle differences play in determining individual reproductive outcome remains to be identified. epigenetics and chromosomal abnormalities in human oocytes. ilse van den berg, 1,2 joop se laven, 1 robert jan galjaard, 2 j hikke van doorninck. 1,2 1 obstetrics gynaecology; 2 clinical genetics, erasmus medical center, rotterdam, netherlands. humans have a low fertility rate compared to other mammalian species. moreover their fertility declines with increasing age. both phenomena are largely due to an increasing number of chromosomal abnormalities in human oocytes during life. identifying factors that cause aneuploidy in oocytes may offer possibilities to diminish these abnormalities in vitro or in vivo. chromosomal segregation errors can result from aberrant recombination but little is known about epigenetic factors that may cause aneuploidy. epigenetic modifications such as histone acetylation and subsequent de-acetylation in oocytes are necessary for a correct progress through meiosis. if disturbed it may lead to aberrant segregation of chromosomes or chromatids due to decreased kinetochore function and imperfect spindle figures resulting in aneuploidy. mice oocyte data have shown a correlation of abnormal histone de-acetylation and aneuploidy and a correlation between age, remaining histone acetylation and aneuploidy (akiyama et al., 2006) . our research focused on human oocytes and investigated whether a similar relationship between histone acetylation, age and aneuploidy is present. human oocytes were surplus from standard ivf/icsi treatments (ic+). human oocytes showed immunostaining for histone 4, lysine 12 acetylation (h4k12) at the germinal vesicle stage and complete deacetylation at the mii stage in 45% of the oocytes, while 55% keep high levels of h4k12 acetylation. treatment of germinal vesicle oocytes with a histon deacetylase inhibitor (tsa) during in vitro maturation until mii stage resulted in high levels of acetylation. remaining acetylation in tsa treated oocytes was correlated significantly with abnormal spindle figures (tubulin staining), a hallmark of developing aneuploidy. similarly, 80% of oocytes with naturally remaining h4k12 acetylation showed abnormal spindle figures. in contrast 80% of the normal de-acetylated oocytes showed normal spindles (p=0.002). this suggests that defective de-acetylation of h4k12 in human oocytes leads to abnormal spindle figures and subsequent aneuploidy. advanced maternal age is associated with a reduction in de-acetylation during in vitro maturation and an increase in spindle abnormalities. these results may stimulate the development of assays for histone modifications as biomarkers to follow oocyte quality in in vitro maturation studies or in optimizing general ivf treatments. objective: racial disparity in preterm birth (ptb) is unexplained and genetic risk factors are suspected as a major cause. this large scale candidate gene study examines differences association of single nucleotide polymorphisms [snps] in caucasians (c) and african americans (aa) to help elucidate racial disparity in preterm birth (ptb) methods: in this case (preterm birth <36 weeks) control study (term birth > 37 weeks) maternal and fetal dna from 370 (172 cases and 198 controls) c and 279 (82 ptb and 197 term) aa were collected. a high-throughput candidate gene association study was performed examining 1442 snps in 130 genes of selected from hypothesized ptb pathways. single locus association analyses were performed separately on maternal and fetal samples. results: snps in 23 genes associated with ptb (p<0.01) were common between races with both maternal and fetal dna analyses. however, snps in 24 genes in c and 32 in aa in both maternal and fetal dna differed in its association with ptb. in c maternal dna, the single strongest association between ptb and snp was in plasminogen activator tissue (plat) gene (c-4443t; rs879293) at both allelic (p = 2.00x10 -3 ) and genotypic (p=2.0x10 -6 ) level with an odds ratio (or) of 2.80 ]. the single strongest effect in c fetal dna was observed in a snp in the interleukin-10 receptor antagonist gene (a18792g; rs17121510) for both allele (p=0.01) and genotype (p=3.34x10 -4 ), or 1.92 ). in aa, the strongest associations were in interluekin-15 (c13929t-rs10833, allele p=2.91x10 -4 , genotype p=2.0x10 -3 ) in maternal dna with an or=0.54 [ci 0.37-0.78] and in fetal dna interleukin-2 receptor b (a14481g; rs84460, allele p=1.4x10 -4 , genotype p=6.3x10 -4 ) with an or 2.36 ]. conclusion: large scale high throughput analyses of snps in candidate genes of ptb pathway reveals significant differences between races at both maternal and fetal levels. we found that the strongest single locus associations differed in the two races in both maternal and fetal dna samples. these findings support the hypothesis that underlying genetic predispositions may differ between these populations, perhaps partly explaining racial disparity in preterm birth. candidate gene association study indicates differential etiologies in gdm and in t2dm. johann urschitz, tarik sultan, kenneth ward. obgyn, jabsom, university of hawaii, honolulu, hi, usa. objective: recently several genome-wide association studies have associated multiple single nucleotide polymorphisms (snps) in multiple genes with increased risk of type 2 diabetes. as risk factors are similar between t2dm and gestational diabetes (gdm), we investigated a possible association of those 18 snps with gdm. study design: blood was collected from caucasian (100 cases, 364 controls) women who met coustan-carpenter criteria for gdm and non diabetic controls. dna was extracted and a candidate gene association study was performed (taqman, abi). results: chi 2 contingency tests were used to analyze genotype and allele frequencies in controls and gdm affected pregnancies (see table below). none of the snps showed a significant association after bonferroni correction. conclusion: several polymorphisms which displayed highly significant associations with type 2 diabetes are not associated with gestational diabetes. these results suggest that gdm is not a simple unmasking of a t2dm predisposition by the metabolic demands of pregnancy; rather it appears that different biological mechanisms are responsible for the respective diseases. introduction: histone acetylation/deacetylation plays an important role in the regulation of gene expression. histone deacetylase inhibitors (hdaci), in general, maintain gene expression, although in some cases they cause repression of specific genes. in this context we have previously shown that the hdaci tsa suppresses activation of the pro-contractile gene cox-2 in human myometrial and amnion-derived cells [reproductive sciences, vol 14, no 1 (supplement, #9)]. we have also shown that expression profiles for hdacs (1-6, 8) differ significantly within upper and lower regions of the myometrium during pregnancy and in labour. objectives: since changes in both acetylation and phosphoryation status can influence the activity of individual hdacs we aimed to define whether there are any changes in the levels of acetylation and phosphoryation of myometrial hdac 1, 2 and 8 during pregnancy and labour. methods: protein homogenates prepared from non-pregnant, term and labouring myometrium were treated +/-shrimp alkaline phosphatase and subjected to sds-page and western immunoblotting (wb) using antibodies to hdac1, 2 and 8. the acetylation status of the hdacs was assessed by a co-immunoprecipitation assay using anti-hdac and anti-acetylated lysine antibodies. results: distinct patterns of acetylation were observed for the individual hdacs; hdac 1 and 2 appeared to be more acetylated in non-pregnant and labouring lower tissues when compared to pregnant myometrium. in contrast, hdac8 appeared to be slightly more acetylated in lower uterine samples from pregnant and labouring myometrium. in experiments to evaluate changes in phosphorylation status we observed that 1) myometrial hdac1 appeared to less phosphorylated in both upper and lower labouring samples when compared to non-pregnant and pregnant samples; 2) hdac2 appeared to be more phosphorylated in labouring samples than non-pregnant and pregnant myometrium; 3) no changes in phosphorylation levels were observed for hdac8 using this test system. conclusions: the difference in hdac acetylation and phosphorylation levels in the human myometrium may indicate differential regulation of the activity of the hdacs within the distinct myometrial regions, perhaps leading to the alteration of their epigenetic effect on genes related to myometrial quiescence and contraction and the subsequent onset of both term and preterm labour. objective: native hawaiians and pacific islanders experience a higher perinatal morbidity secondary to the increased incidence and earlier onset of heart failure. this increased incidence is likely multi-factorial and includes co-morbidities and genetic factors. mutations in the human adrenergic receptor gene may play a role in the determination of heart function. mutations in the b1-adrenergic receptor (adrb1) located on chromosome 10 have been identified as a cause progressive cardiomyocyte loss leading to a dilated cardiomyopathy. the minor allele frequencies for most of these polymorphisms have been determined for caucasian, japanese, african-american and chinese as part of the hapmap project. the frequencies have not been determined in the pacific islander groups. this study helped determine the hawaiian allele frequency and determine the allele frequency in the presence of cardiac or other vascular co-morbidities such as hypertension, preeclampsia, and gestational diabetes. study design: real time pcr technology (taqman genotyping assays, applied biosystems) was used to screen maternal dna (n= 1315) from the phenotyping sample core of the pacific center for early human development (prcehd) for the rs1801253 single nucleotide polymorphisms (snps). genotype frequencies were also determined in 100% complete ethnic controls as determined by a four grandparent descent. results: chi square was used to analyze allele and genotype frequencies differences between controls and affected pregnancies. the results are summarized in the following tables. conclusion: the rs1801253 snp was not significantly associated with hypertension, preeclampsia or gdm in our overall hawaiian population. the genotype frequency in the 100% pacific islander group was significantly different from the caucasians (p=0.04) and asians (p=0.008) in our overall hawaiian population. angiotensin-converting enzyme and -adducin polymorphisms in preeclamptic mothers and fetuses. c mando, 1 p antonazzo, 1 s tabano, 2 f colleoni, 1 a martinelli, 1 s calabrese, 1 c benedetto, 3 l marozio, 3 f facchinetti, 4 m miozzo, 2 i cetin. 1 1 inst. obstetrics and gynecology, fondaz. irccs policlinico mangiagalli regina elena, univ. milan, milan, italy; 2 dept. biology and genetics, univ. milan, italy; 3 dept.obstetrics and gynecology, univ. torino, italy; univ. modena and reggio emilia, modena, italy. background the genes encoding angiotensin-converting enzyme (ace) and -adducin (add1) share the potential of influencing blood pressure. previous studies demonstrated in humans the association of hypertension with the combined effect of both ace insertion/deletion (i/d) polymorphism, which leads to a different activity of the enzyme, and add1 g460w non-sense single nucleotide polymorphism (snp). ace i-i genotype has been associated with low serum ace activity. controversial studies concerning the association of ace polymorphism with preeclampsia (pe) were reported and the possible combined effect of both ace i/d and add1 g460w polymorphisms yet remains to be investigated. population association study we genotyped 2 polymorphisms (ace i/d and add1 g460w) in 497 women: 197 with pe (119/197 with severe pe) and 300 controls. moreover, we investigated a subset of their fetuses: 24 from severe pe, 17 from mild pe and 300 controls, in order to identify specific maternal and/or fetal genotypes conferring a higher risk to develop pe. we both evaluated the single and the combined effects of ace and add1 genotypes on mother-fetus couples and singularly on mothers and fetuses. ace i/d genotype was analyzed using a pcr method; an allelic discrimination approach was performed to detect the add1 snp. in mother-fetus genotype couples, neither ace nor add1 polymorphisms are associated with pe, nor are the combination of their genotypes separately in mothers and in fetuses. nevertheless in mothers with mild pe ace i-i genotype is significantly less frequent (8% vs 13%; p<0.05) and ace i-d is significantly more frequent (59% vs 42%; p<0.02) compared to controls. ace i allele frequency is not significantly different in mild pe compared to controls (37% vs 38%). in women with mild pe the i allele seems to move from i-i to i-d genotype. it leads to the increase in mild pe women of those genotypes with a higher ace activity conferring a higher susceptibility to develop pe. this association with mild pe and not with severe pe could be due to the contribution in the latter of many other genetic and environmental factors. introduction: maternal mrnas stored in the oocyte are critical for early development as transcription ceases upon oocyte maturation, and gene expression until zygotic genome activation (zga) is mediated by translation of maternal transcripts. cytoplasmic polyadenylation element binding protein (cpeb) plays a central role in this process by stabilizing maternal mrnas and regulating their timely activation. this function has been well characterized in xenpous, and a similar role in mammals is suspected as the cpeb knockout mouse displays infertility as a result of arrested oogenesis. in order to investigate if translational regulation of maternal transcripts by cpeb is maintained in mammals, we characterized the spatial and temporal expression of cpeb-1 in the mouse and human. methods: ten different somatic tissues, testes, and ovaries were tested by rt-pcr for the expression of cpeb mrna in mouse and human. cpeb mrna expression in mouse was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. in human, pi and mii oocytes, 8-cell embryos and blastocysts were evaluated. sequencing of the pcr products was performed to confirm specific amplification of cpeb. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results and discussion: highest level of cpeb mrna expression was detected in ovaries and testis of both mouse and human. in addition, differential expression of cpeb in somatic tissues was also observed. among these, prominent expression was present in brain, where a role for cpeb in facilitating gene expression through cytoplasmic polyadenylation has been proposed. these data would suggest that translational control of mrna by cpeb may be a mechanism utilized by multiple somatic tissues to regulate gene expression. in the mouse, cpeb was expressed in pi and mii oocytes and 1-cell and 2-cell embryos, and became undetectable in 4-cell or more advanced embryos. human cpeb was expressed in pi and mii oocytes, but not in 8-cell embryos or blastocysts. zga occurs at late 2-cell stage and 4-to-8-cell stage, in mouse and human, respectively. the tightly controlled temporal expression of cpeb prior to zga in both mouse and human is consistent with a conserved role for cpeb in the regulation of maternal mrna expression during early development in mammals. background embryonic stem cells (esc) express specific transcription factors that are indicative of their ability to maintain pluripotency. these factors are activated during preimplantation embryo development. the interplay between the transcription factors pou5f1 (oct3/4) and caudal-homeobox domain 2 (cdx2) is thought to regulate inner cell mass (icm) and trophectoderm (te) differentiation; however, the mechanism of cell fate determination in mammalian embryos is poorly understood. genetic ablation of oct3/4 in mice prevents icm development, while cdx2 knockout embryos fail to implant. esc deficient in nanog, a second key regulator of pluripotency, fail to maintain pluripotency and undergo differentiation. expression of nanog in primate embryos has not been investigated, therefore the localization of oct3/4, nanog and cdx2 was determined in rhesus macaque blastocysts. methods oocytes were collected from rhesus macaque females, fertilized and cultured in vitro to the blastocyst stage. embryos were collected 144 hours post insemination, then fixed prior to incubation with primary antibodies directed against oct3/4, nanog and cdx2. following incubation with cy3 conjugated secondary antibodies, nuclei were counterstained with dapi and embryos visualized using an olympus bx41 fluorescence microscope. results nanog protein was restricted to the icm of monkey blastocysts. unlike the mouse embryo, oct3/4 protein was detected in both the icm and te, based on two different antibodies. the expression of cdx2 was localized specifically to the te. conclusions the ubiquitous pattern of oct3/4 expression is consistent with observations in human, cow and pig embryos. significantly, lack of restricted oct3/4 protein, and icm localization of nanog in primate blastocysts, suggests that nanog more specifically determines cell fate in primate embryos. these results contrast markedly with current mechanistic hypotheses, although other factors may lie upstream of nanog. importantly, this difference may underlie observations that regulatory mechanisms in esc differ between mice and primates. further investigations will focus on determining the onset of marker expression, and the upstream regulators of nanog activation. supported by nih grants 1r01hd045966 1r21rr021881, and the intramural research program of nichd. in vitro-conceived mice tend to be smaller at birth and throughout their life. luisa delle piane, annemarie donjacour, francesca di sebastiano, gnanaratnam giritharan, paolo rinaudo. obstetrics, gynecology and reproductive sciences, university of california san francisco, san francisco, ca, usa. objective: epidemiological evidence indicates that ivf is associated with an increased incidence of low birth weight. this phenomenon has not been studied in a mouse model; in addition, it is unknown if the resulting offspring show different growth pattern later in life. we therefore created an ivf mouse model to follow growth patterns till adulthood. methods: we generated 3 experimental groups of b6 mice: one cohort of in vivo generated mice (in vivo group), one cohort of in vitro generated animals (ivf group) transferred to cd1 foster mothers and one cohort of animals fertilized in vivo and transferred to cd1 foster mothers (flushed blastocysts group, fb). the fb group was generated because our preliminary results showed that embryo transfer to b6 foster mothers was not successful. all pups were delivered at term, measured and weighed at birth and then weekly up to 18 weeks. parametric tests (anova with bonferroni correction) were used as appropriate. a mixed model was used to compare growth curves. results: average litter size was 6.3 (in vivo), 3.7 (fb) and 3.5 (ivf). birth weight of male mice both ivf (1.57 mg) and fb (1.73 mg) was larger than male in vivo mice (1.30 mg) (p<0.05). ivf mice tended to be smaller than fb mice in both sexes (p= 0.0533). the bmi (body mass index) was not different among all the groups. male fb growth curves were different from in vivo mice (p<0.0001) and more importantly from ivf growth curves (p=0.0516) (fig. 1) . conclusion: the method of conception and the maternal environment play a significant role in determining birth weight in this mouse model, emphasizing that the fb group is the best control for the effects of ivf in this model. these preliminary results confirmed for the first time an ivf effect on growth and interestingly the ivf males did not show a catch-up growth. the finding of smaller ivf mice when compared to fb is both noteworthy, because it confirms human data, and worrisome, because lower birth weight is associated with long term sequelae according to the barker hypothesis. the effect of betamethasone exposure in mid-gestation on renal sodium excretion in male sheep. lijun tang, 1 luke carrey, 1 nancy valego, 1 philip deibel, 1 james perrott, 1 jorge figueroa, 1 mark chappell, 2 james c rose. 1 1 obestetrics and gynecology, wake forest university, medical school, nc, usa; 2 hypertension center, wake forest university, medical school, nc, usa. objective: whereas prenatal exposure of ovine fetuses to clinically relevant doses of glucocorticoids during the time of peak nephrogenesis results in a reduction in nephron number in adulthood, there is little information about its effect on sodium excretion. in the present study, we evaluated the effect of exposure to betamethasone on renal sodium excretion in adult male sheep. methods: we studied nineteen conscious adult rams at 1.5 years of age which were exposed to either vehicle or betamethasone at 80-81 days gestation. we implanted vascular and bladder catheters and then allowed the animals a 5-7 days recovery period prior to study. inulin and para-aminohippuric acid (pah) clearances were performed for estimating glomerular filtration rate (gfr) and renal plasma flow (rpf) respectively. following determination under basal conditions, an acute hypertonic sodium load was administrated intravenously by a continuous infusion of nacl (0.0275 meq/kg/min at 0.55 ml/min) for 60 minutes. urine was continuously collected for determination of na + excretion. results: basal gfr was decreased in steroid exposed adults (2.267 ± 0.10 ml/min/kg) compared with the vehicle animals (1.935 ± 0.08 ml/min/kg) (p=0.028). rpf was similar in the vehicle and steroid exposed group (15.483 ± 0.40 ml/min/kg vs 13.561 ± 0.80 ml/min/kg). at basal conditions, na + excretion (u na v) was similar in vehicle and steroid exposed group (16.60 ± 5.37 μmol/h vs 14.37 ± 2.46 μmol/h). this similarity was also present after normalization by body weight (0.278 ± 0.09 μmol/h/kg vs 0.234 ± 0.04 μmol/h/kg). the vehicle group excreted 53.65 ± 9.71% of the dose of na during the experiment while the betamethasone exposed group excreted only 37.07 ± 4.42% (p<0.05). gfr and prpf did not change during the experiments. these results suggest that prenatal exposure to glucocorticoids affects renal function in adult male sheep which results in a decreased basal gfr and an attenuated ability to excrete on acute sodium load. the elevated blood pressure previously observed associated with this prenatal steroid treatment may be related to the alteration in renal function. the study is supported by nih grants hd 47584, hl 68728 and hd17644. characterisation of human fetal cardiac stem cells. marah alfakir, nicholas dawe, annette meeson, stephen c robson. school of surgical reproductive sciences, newcastle university, newcastle upon tyne, united kingdom. objectives: for many patients with severe cardiac disease treatment is limited to surgical intervention and/or heart transplantation. the heart has a limited regenerative capacity but it is insufficient to repair extensive damage. cellular therapies might provide an alternative treatment. we have identified stem cells (sp cells) in the adult mouse and human heart and have shown that the mouse cardiac sp cells can differentiate along a cardiac lineage. sp cells can be identified using facs combined with hoechst 33342 dye efflux. this ability to efflux hoechst dye is due to expression of abcg2 (an abc transporter). we have hypothesized that the fetal heart would contain more cardiac stem cells, relative to the adult, and the aim of this project was to determine the spatial and temporal expression of known stem cells markers in the embryonic/fetal heart. methods: 15 human fetal hearts were collected aged between 6-16 wk. we used rt-pcr, using primers for several stem cell (abcg2, cd34, cd45) and cardiac specific (mlc-2v, mhc) markers, and ihc on frozen and paraffin sections, using a panel of antibodies (abcg2, cd34, cd45, islet1). cdna was generated from the following regions of the heart at different developmental stages: right and left atrium, right and left ventricle, and outflow tract. results: abcg2 mrna was highly expressed in all regions of the fetal heart between 7-10 wk. expression was down regulated at 11-15 wk especially in the la and lv. a similar pattern of expression was observed for cd34. cd45 showed low/moderate expression in all heart regions examined; however, this expression was absent in the lv at 13-15 wk. mrna and protein analysis showed similar results. whilst protein expression of abcg2 was robust at 7 wk (approximately 3-6%), it declined with increasing gestational age. cd34 was also strongly expressed at 7 weeks of age. there was no co-localisation between abcg2 and islet1. conclusion: abcg2 and cd34 are both expressed between 7-10 wk of age. based on this analysis, further studies are underway to isolate and characterise both the abcg2 and cd34 expressing fetal cardiac cells which might be a potential source of cardiac stem/progenitor cells. xanthine oxidase plays a significant role in the fetal cardiovascular defence to hypoxia. ja hansell, a kane, e herrera, da giussani. physiology, cambridge university, united kingdom. prenatal hypoxia remains a major concern in obstetrics. the fetal defence to hypoxia includes redistribution of the cardiac output, away from peripheral and towards essential ciculations, such as those perfusing the brain (cohn et al . ajog 120:817,1974) . the physiology underlying this response is well characterised and involves chemoreflex and endocrine responses (giussani et al. fet mat med rev 6:17, 1994) . more recently, local factors such as nitric oxide (no) and reactive oxygen species (ros), and the interaction between them, have been shown to play a role. antioxidants, such as vitamin c, scavenge hypoxia-induced ros, maintain no high and thereby depress peripheral constriction (thakor et al., sgi 2006) . in this study, we address the source of hypoxia-induced ros production and investigated the effects on the in vivo fetal femoral constrictor response to hypoxia of maternal treatment with the xanthine oxidase inhibitor allopurinol in sheep. methods: under anaesthesia, 8 sheep at 0.8 gestation, were instrumented with maternal and fetal catheters and a fetal femoral transonic probe. five days later, all animals were subjected to 2 h normoxia, 0.5 h hypoxia (mat fio 2 to reduce fetal pao 2 to ca. 10 mmhg) and 1 h recovery, either following maternal i.v. treatment with vehicle or allopurinol in low (30 mg.kg -1 over 20 min) or high (150 mg.kg -1 over 30 min ) doses. treatments finished 100 min prior to hypoxia. the low allopurinol dose was adopted from human studies (benders et al. arch dis child 91:163, 2006) . the timing of the hypoxic challenge coincided with peak concentrations in fetal sheep plasma of oxypurinol (active metabolite). we evaluated the effect of bpa exposure on uterine gene expression in a nonhuman primate model. method: a total of nine vervet monkeys (cercopithecus aethiops sabaeus) were ovariectomized. three monkeys were treated with bpa via alzet pumps (50 microgram/kg/day), two with estradiol benzoate and three received combined treatment with bpa and estradiol. the remaining monkey was untreated and served as a control. following 28 days of treatment the monkeys were hysterectomized and immunohistochemistry performed to examine endometrial gene expression. we evaluated hoxa10, proliferating cell nuclear antigen (pcna), and caspase iii gene expression as markers of differentiation, proliferation, and apoptosis respectively. differential expression was evaluated by h-score. results: exposure to a combination of estradiol and bpa resulted in increased expression of hoxa10 and pcna compared to control (p<0.01). lower, but increased, levels of hoxa10 and pcna gene expression were seen in the specimens exposed solely to estradiol (p<0.05). those specimens exposed to bpa alone demonstrated a small induction in hoxa10 expression (p<0.05) with no change in caspase iii or pcna expression when compared to the control. conclusion: bpa induced the expression of hoxa10 in the uteri of a non-human primate. exposure in the presence of endogenous estrogen further augmented estrogenic stimulation confirming that bpa is a xenoestrogen. bpa's effect of developmental gene expression may alter uterine differentiation. bpa acts as an endocrine disruptor by altering estrogen regulation on a gene required for fertility. contractility and meconium passage. jayaraman lakshmanan, 1 john d richard, 1 guo l liu, 1 sharon k sugano, 1 ahmet karadag, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: endogenous glucocorticoids (gcs) increase with advancing fetal age suggesting that gcs function as a hormonal modulator of organ maturation. although fetal colonic motility and meconium passage primarily occur near term, the role of gc in colonic maturation is poorly understood. we sought to examine gc-nuclear receptor (gc-nr) expression in ovine fetal distal colon smooth muscle from very-preterm to term gestation to define the cascade of gc initiated biochemical changes as a prerequisite for maturation-associated meconium passage. methods: ovine fetal distal colonic segments removed at very-preterm (vpt: 118-120 d gestation), preterm (pt: 130-132 d), near term (nt: 140-142 d) and term (t: 146-147 d) (n=6 fetuses in each group) were fixed in bouin's solution and paraffin embedded. sections were subjected to immunohistochmical analysis with gc-receptor antibody (1:200, sc-8992, santacruz biotechnology, ca) using standard abc regimen. immunoreactive material identified by 3,3'diaminobenzidine as chromogen. the percentage of gc-nr staining in smooth muscle-enteric unit was analyzed by counting dark brown immunostained nuclei at 10 different fields at 400x magnification. all values are expressed as mean ± sem. results: the gc-receptor antibody immunostained nuclei both in smooth muscle and enteric units and the observed pattern (expressed as percentage of total nuclei) are as follows: nuclear gc expression varies with gestational age with maximal expression occurring near-term in smooth muscle and enteric neurons, in a synchronized manner. a near total disappearance of gc-nr expression occurs at term. these results suggest that peak gastrointestinal gc-nr mediated effects may occur prior to term with secondary signaling effects resulting in distal colonic motility maturation and meconium passage. the rna-binding protein hud co-localizes with choline acetyl mechanisms of in utero meconium passage. jayaraman lakshmanan, john d richard, guo l liu, sharon k sugano, octavio balbuena, michael g ross. dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa. objective: we have previously speculated that crf, acting through its receptor crf-r1, increases gastrointestinal (gi) motility and potentiates in utero meconium passage. patients with paraneoplastic syndrome with auto-antibodies to hud, a neuronal rna binding protein, develop severe gi dysmotility, indicating a role for hud in gi motility. hud binds mrna for actylcholinesterase implying a role in the cholinergic neurotransmitter system. based on these known functions, we hypothesized that hud may regulate post-transcriptional activity in fetal colonic cholinergic neurons expressing crf-r1. method: bouin's solution-fixed paraffin-embedded sections of distal colonic segments were prepared from very preterm (vpt: 118-120 days), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (146-147 days) ovine fetuses (n=6 at each age). sections were immunostained with anti-human neuronal protein huc/hud antibodies (1:800 to 1:1000) with abc reagents and examined at 400x. neurons positive for hud staining were quantified. double immunofluorescence and laser confocal analyses evaluated co-expression of hud in neurons expressing peripheral choline acetyl transferase (pchat) (a marker for peripheral cholinergic neurons) and crf-r1 receptor. results: hud immunostaining was seen in either entire cytoplasm (c) or cytoplasm and nuclear regions (c+n) in both submucosal and myenteric neurons. a greater percentage of submucosal (vpt: 55±3, pt: 53±3, nt: 54±3, and t: 57±3 %) than myenteric neurons (vpt: 35±3, pt: 42±3, nt: 38±4, t: 31±2 %) exhibited positive staining at all ages. the percentages of neurons with c+n staining in submucosal neurons were significantly lower at very-preterm gestation. confocal studies co-localized the hud staining with pchat and crf-r1 receptor immunoreactivity both in submucosal and myenteric neurons. conclusion: in the fetal enteric nervous system hud may function both as a rna-binding protein and as a nuclear cytoplasmic shuttling protein. colocalizaion studies suggest that both pchat-mrna and crf-r1 mrna species are target transcripts for hud in myenteric neurons. we speculate upregulation of hud contributes to in utero meconium passage. meconium passage during mild stressors. jayaraman lakshmanan, 1 guo l liu, 1 john d richard, 1 sharon k sugano, 1 raina khan, 1 octavio balbuena, 1 kimberly chap, 1 virender rehan, 2 michael g ross. 1 1 dept. of ob/gyn, harbor-ucla med. ctr., torrance, ca, usa; 2 dept. of pediatrics, harbor-ucla med. ctr., torrance, ca, usa. objective: mr exhibit a greater affinity to glucocorticoids (gc) than do glucocorticoid receptors (gr). thus low circulating gc levels may preferentially bind mr during mild-stress periods, while the high gc levels may bind gc-receptors. mr antagonism increases gc-mediated responses, suggesting that mrs have a regulatory impact on gc-mediated stress responses. we recently documented that fetal in utero meconium passage is a neurovisceral stress response. to test our hypothesis that mrs play a primary role in mediating suppressive gc effects, we examined the expression patterns of mr in ovine fetal distal colon. methods: bouin's solution fixed paraffin sections of distal colonic segments collected from ovine fetuses (n=6 for each gestational ages) at very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142 days) and term (t: 146-147days) were subjected to immunohistochemistry with polyclonal antibodies to mr. digital photos (10 field per colonic ring, 6 rings each gestational age) taken at 400x were used to count the number of intensely stained neurons, referred to as "mr-capped neurons". differences over time were determined with anova. results: mr antibody elicited a punctate staining pattern in all layers of ovine fetal distal colon. significant immunoreactive intensity was observed in submucosal and myenteric ganglia at all ages: submucosal ganglia: vpt=0.130 ±0.009, pt=0.154±0.002, nt=0.151±0.005, t=0.169±0.016; a subpopulation of enteric neurons exhibited dense staining ("mr-capped"). advancing gestation was associated with a significant decreased in the percentage of mrcapped myenteric (vpt = 49±7, pt = 27±5, nt = 19±3, t = 22±1%; p<0.05), though not submucosal ganglia neurons. results indicate a significant decrease in the percentage of mr capped myentric neurons with advancing gestation. in view of the potential inhibitory effect of mr on gc-mediated stress responses, these results suggest that the decrease in mr expression may contribute to near term and term in utero meconium passage. the ambiguity of myometrial progesterone receptor expression in pregnancy and labour. alison j tyson-capper, elizabeth a shiells, stephen c robson. surgical reproductive sciences, institute of cellular medicine, newcastle university, newcastle upon tyne, united kingdom. background and aims: in contrast to many other species, human parturition is not due to a reduction in circulating levels of progesterone (p) but appears to be related to changes in expression, ratios or signalling events of p receptors (pr-a and pr-b). the literature on pr expression in human myometrium in pregnancy and labour remains conflicting. we hypothesise this is due, at least in part, to differing specificities of the various pr antibodies employed. we carried out a comprehensive analysis of pr expression using ten 'pr-specific' antibodies that recognise different amino acid epitopes within the pr proteins. two phosphorylation-specific antibodies for pr were also included to define whether phosphorylation status of myometrial pr changes in pregnancy and in labour. methods: western immunoblotting and semi quantitative rt-pcr were undertaken using protein lysates and rna prepared from myometrial tissue from: -first (n=4) and second (n=5) trimesters, paired upper and lower segment myometrium from preterm (22-28 wks, n = 4), term not in labour (n = 10), term spontaneous labour (n = 8) and non pregnant (n = 10). results: using the same set of myometrial lysates for each antibody, we found that the specificity of individual pr antibodies, in particular those raised against internal and c-terminal epitopes of the pr proteins, varied considerably resulting in different patterns of expression. there also appeared to be temporal and spatial differences in levels of myometrial pr proteins (90-120kda/60-70kda) in pregnancy and in labour with use of the phosphorylation-specific pr antibodies, however, it remains to be resolved which pr isoforms these may be. in contrast, data using four antibodies all of which react with the amino terminal domain of pr consistently indicated that expression of pr, in particular pr-b, decreased significantly at term (p < 0.001) and in labour (p < 0.001) when compared to non-pregnant levels. a decrease in levels of pr-b protein was also observed when comparing preterm levels to non-pregnant levels. rt-pcr using primers specific to pr-b consistently indicated that levels of pr-b mrna decreased at term and in labour. conclusion: interpretation of gestation-related changes in myometrial pr expression and phosphorylation status must take into account the pr antibodies used. further studies are now underway to validate the specificity of the pr antibodies. objective to test the hypothesis that expression of fshr in mural gl cells from ivf follicles varies with the infertility diagnosis and correlates with the outcome of ovulation induction (oi). materials and methods 86 women undergoing ivf were classified as: 1. "no ovarian factor", (tubal or male factor and egg donors, nof;n= 37); 2. endometriosis (endo; n=17); 3. poor responders (pr; n=19); 4. polycystic ovary syndrome (pcos; n=13). ovulation induction was carried out using a long or microflare or antagonist protocol based on clinical parameters and gonadotrophin doses were selected based on ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual response. after ultrasound guided egg retrieval, mural gl cells were isolated from pooled follicular fluids from each patient using a percoll gradient and anti-cd45 immunobeads to eliminate wbcs, viability was assessed by trypan blue. fshr was measured by rt-pcr as relative expression compared to beta actin. statistical analysis was performed with the spss using pearson´s correlation, one way anova and student´s t-test. results fshr expression in nof (137±33.6) was statistically significantly higher than in pr (57±15.2) and lower than in pcos (290±77.2). both endo (85±30.2) and pr levels of fshr were statistically significantly lower than in pcos. analysis of all cycles showed that fshr expression correlates positively with the number of total (r=0,26; p< 0,05) and mii (r=0,35; p< 0,01) oocytes and negatively with the units of fsh (r=-0,31; p< 0,01) and lh (r=-0,24; p< 0,05) administered for oi. separate analysis of nof showed a positive correlation with the number of total and mii oocytes retrieved and with estradiol levels on day of hcg but not with the total dose of gonadotropins received during oi. fshr expression correlates negatively with day 3 fsh levels in all patients except in pcos (r=-0,25; p<0,05). conclusions these results suggest that expression of fshr in the hyperstimulated ovarian follicle is "average" in normoresponders, high in high responders (pcos) and low in poor responders (pr and endo). knowledge of the level of expression of fshr might be useful to individualize gonadotrophin doses and oi protocols thus improving pregnancy rates. comparison of intracellular atp levels between non-vitrified and surviving post-vitrification/thawed human oocytes. somjate manipalviratn, 1 zhi-bin tong, 1 eric widra, 2,3 alan h decherney. 1 1 reproductive biology and medicine branch, nichd/nih, bethesda, md, usa; 2 department of obstetrics and gynecology, georgetown university hospital, washington, dc, usa; 3 shady grove reproductive science center, washington, dc, usa. objective: to compare intracellular atp level in non-vitrified and surviving post-vitrification/thawed human oocytes using an atp bioanalysis assay. design: prospective match-controlled laboratory study material and methods: all oocytes were obtained from women undergoing controlled ovarian stimulation for ivf/icsi. oocytes were discarded due to nuclear immaturity at the time of planned icsi. all immature oocytes (gv and mi) were incubated overnight. oocytes from patients with 2 or more discarded eggs which matured to mii stage were used in this study. oocytes from each women were randomly divided into 2 groups. in group 1, the oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. in group 2, the oocytes were vitrified using 15% ethylene glycol, 15% dimethyl sulphoxide and 0.5 m sucrose as cryoprotectant. these oocytes were kept in liquid nitrogen for 5-7 days before thawing with a rapid thawing method. oocyte survival was determined by morphological assessment after 90 minutes of incubation then oocytes were placed in 50 l of ultrapure water and kept at -80 o c for further atp analysis. intracellular atp level was determined using luciferin-luciferase bioluminescent assay. result: ninety-one discarded human oocytes were obtained from 23 women. 47 oocytes were vitrified/thawed before measuring for atp content. the other 44 oocytes were measured for their atp content without undergoing vitrification/thawing process. oocyte survival rate after vitrification/thawing is 72.34% (34/47). mean oocyte atp levels in non-vitrified oocytes is significantly higher than surviving post-vitrification/thawed oocytes (table 1). coefficient of variation of luciferin-luciferase bioluminescent assay is less than 5%. conclusion: oocyte atp level in surviving post-vitrification/thawed human oocytes is significantly lower than non-vitrified oocytes. objective to study the expression of genes involved in cell proliferation and steroidogenesis in the human follicle (kl1, kl2, fshr, papp, p450) and its relationship with ivf outcome. materials and methods 89 patients with different infertility diagnosis underwent ovulation induction with either a long or microflare or antagonist protocol based on clinical parameters; the dose of gonadotrophin used for ovulation induction was selected based on the ovarian reserve (day 3 fsh and e2 and basal antral follicle count) and adjusted to the individual patient response. ultrasound guided egg retrieval was performed 36 hours after administration of 10.000 iu of hcg. mural granulosa-lutein cells (gl cells) were isolated from pooled follicular fluids (ff) from each patient using a percoll gradient and anti-cd45 immunbeads to eliminate wbcs, viability was assessed by trypan blue. the genes under study were measured by rt-pcr as relative expression compared to actin. statistical analysis was performed with the spss statistical software using pearson´s correlation and mann-whitney u. results kl2 expression correlates positively with kl1 levels (r=0,86; p< 0,01) and with genes implicated in granulosa cell function such as fshr (r=0,36; p< 0,01), papp (r=0,54; p< 0,01) and p450 (r=0,39; p< 0,01). the number of mii oocytes obtained is positively correlated with both fshr (r=0,35; p< 0,01) and papp (r=0,28; p< 0,05) expression, but not with the other genes. kl2 expression in women who became pregnant during the ivf cycle studied was higher (25,8± 10) than in non pregnant women (16,6 ± 3,1).(n=80, mann-whitney u p<0,05). conclusions patients who become pregnant have increased expression of kl2, which is associated with optimal granulosa cell proliferation and maturation. this is in accordance with the presence of lower apoptosis in granulosa cells in the same patients. syndrome during assisted reproduction treatment. k jayaprakasan, r jayaprakasan, h al-hasie, js clewes, bk campbell, ir johnson, nj raine-fenning. school of human development, university of nottingham, nottingham, united kingdom. objective: to test the hypothesis that an increased pre-treatment ovarian blood flow is associated with the development of ovarian hyperstimulation syndrome (ohss) and to evaluate ovarian vascularity as a predictor of ohss during invitro fertilization (ivf). methods: 150 subjects undergoing first cycle of ivf had 3d transvaginal ultrasound in the early follicular phase of the menstrual cycle preceding ivf. 50 of them developed ovarian hyper-response, defined as retrieval of 15 oocytes and ohss. 100 subjects had normal ovarian response with retrieval of 4 to 15 oocytes in the absence of ohss. antral follicle count (afc), ovarian volume (ov), and ovarian vascularity (vascularisation index, vi; flow index, fi and vascularisation flow index, vfi) were measured and an unpaired t-test was used to compare these parameters between the ohss and the control groups. multiple logistic regression analysis was used to assess the predictive value of these variables against age, bmi and basal fsh for the development of ohss. results: the ovarian vi (9.64 ±7.94 vs. 8.64 ± 7.31), fi (37.95 ± 5.5 vs. 37.95 ± 5.5) and vfi (3.77 ± 2.68 vs. 3.51 ± 3.1) were similar in both the groups. afc and ov were significantly higher (p<0.01) in the ohss group (28.90 ± 14.55 and 10.40 ± 3.77 cm 3 respectively) than in the control group (19.23 ± 9.91 and 8.94 ± 4.7 cm 3 respectively). afc was the only significant (p=0.001) predictor of ohss on multiple regression analysis (table 1) . conclusion: women developing ohss during ivf do not demonstrate an increased pre-treatment ovarian blood flow as measured by 3d ultrasound but do have a significantly higher afc, which is the only significant predictor of ohss. compared to other species, little is known about the steroid biosynthetic capacity and gene expression profiles of human cumulus cells. objective: to examine the ability of human cumulus cells in primary and long-term culture to synthesize steroids and respond to gonadotropin or camp-dependent stimulation. methods: human cumulus cells were isolated from cumulusoocyte complexes during assisted reproductive technology (art) and placed in primary culture or propagated to third passage. at subconfluence, cells were transferred into serum-free conditions in the presence of vehicle, forskolin, fsh, or hcg. at 48h, media was collected and progesterone (p4) and estradiol (e2) levels were determined by eia. aromatase activity was measured by the tritiated water assay. aromatase (cyp19) and cholesterol side-chain cleavage (cyp11a1) mrna abundance was determined by quantitative real-time pcr (qrt-pcr). transcriptional regulation of the cyp19 and cyp11a1 promoters was investigated by transient transfection of cumulus cells with promoter luciferase constructs. results: substantial amounts of p4 and e2 were synthesized by cumulus cells in culture, which were further elevated by forskolin, hcg, or fsh treatment. the presence of cyp19 and cyp11a1 mrna in cumulus cells was confirmed by qrt-pcr, and the relative mrna abundance of both transcripts was induced by forskolin, hcg, or fsh treatment. changes in cyp19 and cyp11a1 gene expression in response to camp and gonadotropin stimulation were associated with increased transcriptional regulation of both the cyp19 and cyp11a1 gene promoters. our studies demonstrate that human cumulus cells are sites of significant p4 and e2 biosynthesis and respond to camp-and gonadotropin-stimulation in vitro. the ability to examine human cumulus cells in primary and long-term culture provides a unique model system to investigate cumulus cell function, and the paracrine role of cumulus cells in oocyte development, maturation, and subsequent fertilization. background: there is increasing evidence suggesting that events occurring during fetal development may result in increased predisposition to adult conditions, such as diabetes. in vitro fertilization (ivf) is a new environmental stressor and the long-term effects of these manipulations are currently unknown. there is some evidence that lower number of insulin-secreting cells at birth signals predisposition to diabetes later in life. in this study, we compare areas of pancreatic insulin-secreting cells in newborn mice conceived in vivo versus in vitro. methods: oocytes were collected from super ovulated cf-1mice and fertilized in vitro with cauda epididymal sperm from b6d2f1/j mice. fertilized eggs were cultured in whitten medium under 5% co2 in humidified air at 37 °c for 96 h. blastocysts were transferred to the uteri of pseudo-pregnant recipients. control mice were allowed to conceive in vivo. the newborn animals were sacrificed within 24 hours of birth. pancreases were sectioned, immunostained with anti-insulin, and total areas and stained areas were compared using t-test with two-tailed distribution. p value of <0.05 was considered significant. results: there were total of 12 newborn animals: 5 females (2 ivf, 3 controls) and 7 males (3 ivf, 4 controls). percentage of total pancreatic area occupied by insulin-secreting cells was lower in female ivf (0.145 mm 2 , 0.69%) animals compared to female controls (0.079 mm 2 , 0.97%) and higher in male ivf (0.019 mm 2 , 0.40%) animals compared to controls (0.138 mm 2 , 0.25%). the average of males and females was slightly lower for ivf (0.082 mm 2 , 0.54%) than controls (0.109 mm 2 , 0.61%). none of these differences were statistically significant. there was a trend in newborn female mice towards lower amount of insulin-secreting cells in the ivf offspring, suggesting possible predisposition to diabetes later in life. this effect was not observed in newborn males. while our study failed to demonstrate consistent and significant differences in the areas of insulin-secreting cells between newborn mice conceived in vitro and in vivo, the number of animals in the study may have been too small to show significant results. larger studies are needed to further investigate this question. peter s uzelac, phyllis risch, kassi shelton, steven t nakajima. obstetrics and gynecology, university of louisville, louisville, ky, usa. objective: several fertility preservation methods currently available may not be viable options to certain patients due to their high cost and limited number of centers offering these services. for women undergoing bilateral oophorectomy, in vitro maturation (ivm) of oocytes retrieved from unstimulated whole ovary specimens may represent a more practical solution. here we describe out initial experience in offering ivm as a fertility-preserving measure to two patients undergoing total abdominal hysterectomy and bilateral salpingo-oophoectomy (tah-bso). case one was a 29 year old nulligravid with chronic pelvic inflammatory disease unresponsive to medical management. case two was a 39 year old nulligravid with stage ia endometrial carcinoma. surgery was planned for the mid-follicular phase in order to collect prophase i oocytes before the onset of late-follicular phase atresia. upon surgical removal, suction aspiration of all identifiable follicles was performed. ovaries were then serially sectioned and all tissue was examined for the presence of prophase i oocytes. results: total number of prophase i oocytes retrieved was 9 and 6, maturation rate after 24 hours was 33% (2/6) and 44% (4/9), fertilization rate after 24 hours was 100% (2/2) and 25% (1/4), maturation rate after 48 hours was 50% (2/4) and 40% (2/5) and fertilization rates after 48 hours 50% (1/2) and 50% (1/2), for case 1 and case 2 respectively. ivm of oocytes retrieved from unstimulated whole ovary specimens may be a simple and inexpensive approach to fertility preservation in women undergoing bilateral oophorectomy. by requiring minimal adjustments to in vitro fertilization protocols, this treatment could easily be implemented in centers which currently have no fertility preservation program. patient age at time of retrieval may be predictive of developmental potential. continued patient enrollment and follow-up studies on the developmental potential of embryos derived from this technique are necessary to fully evaluate its potential for a role in fertility preservation. the routine use of progesterone as luteal phase support in women with a diagnosis of polycystic ovary syndrome (pcos) undergoing ovulation induction cycles with oral agents has not been fully elucidated. we hypothesize that women with pcos utilizing either clomiphene citrate or letrozole, an aromatase inhibitor, should administer intravaginal progesterone in the luteal phase to increase pregnancy rates. design: retrospective chart review. materials and methods: cycle data from women with pcos undergoing ovulation induction with clomiphene citrate or letrozole at the university of cincinnati medical center from 2002 to 2007 were evaluated. diagnosis of pcos was based on rotterdam criteria and all other infertility diagnoses were excluded. clinical pregnancy rates (presence of fetal cardiac activity on ultrasound at 6-7 weeks gestation) in women who received intravaginal micronized progesterone (200 mg bid) following ovulation induction (with and without intrauterine insemination) were compared to those who did not receive progesterone. results: no significant differences were noted in demographic parameters, including patient age or bmi. a total of 182 cycles were evaluated in 72 women treated with clomiphene citrate. clinical pregnancies were documented in 19.5% (18/93) of cycles in the progesterone group compared to 9.9% (9/91) of the non-progesterone group (p < 0.08). forty-three cycles were evaluated in 24 patients treated with letrozole. clinical pregnancies were documented in 17.2% (5/29) of cycles in the progesterone group compared to none (0/14) in the non-progesterone group (p < 0.02). conclusions: patients with pcos who used letrozole for ovulation induction had superior clinical pregnancy rates when using intravaginal micronized progesterone compared to women who did not receive luteal phase support. there was a trend toward increased clinical pregnancy rates in pcos patients utilizing luteal support following clomiphene citrate for ovulation induction. therefore, in women with pcos undergoing ovulation induction with oral agents, luteal supplementation with progesterone should be strongly considered, especially in those using letrozole. were measured and an unpaired t-test was used to compare these parameters between poor and normal responders. multiple logistic regression analysis was used to assess the predictive value of these variables against age and basal fsh for poor ovarian response. results: the ovarian vi (7.5 ± 5.3 vs. 8.6 ± 7.9), fi (38.9 ± 6.9 vs. 37.9 ± 6.5) and vfi (3.2 ± 2.6 vs. 3.5 ± 3.4) were similar in both poor and normal responders. afc and ov were significantly lower (p<0.01) in poor responders (9 ± 3.3 and 6.3 ± 3.5 cm 3 respectively) than in normal responders (19.2 ± 9.9 and 8.9 ± 4.7 cm 3 respectively). afc was the best (p<0.001) predictor of poor ovarian response on multiple regression analysis ( objective: gay male couples increasingly seek parenthood through in vitro fertilization using an oocyte donor and a gestational carrier, but no studies describe this unique experience. the purpose of this study was to determine medical and psychosocial issues unique to gay men using art. design: qualitative analysis of semi-structured interviews with gay male couples seeking parenthood through art. materials and methods: sixteen gay males (eight couples) entering an art program were assessed through the use of a semi-structured interview. characteristics evaluated included age, relationship status, duration of their relationship, psychological health and stability, how the decision was made concerning who would donate the sperm, the decision to use an anonymous or known oocyte donor, and whether their gestational carrier was someone previously known to them or not. results: the average age of the men in this study was 40 years. all eight couples were in a committed relationship and had been together for an average of 7.3 years. five of the couples (69%) had been joined in a civil union which is legal in the state of connecticut. all subjects were psychologically stable and in good health. six couples (75%) were very clear about which partner would inseminate the oocytes. of those couples, two felt that the older partner should donate; two felt that the partner who cared more about a genetic connection to the child should donate; and two felt that the partner with "better genes" should donate. the remaining two couples chose to inseminate equal numbers of oocytes in order to transfer an embryo from each partner. all couples chose an anonymous oocyte donor, two couples chose relatives as their gestational carriers, while the others chose carriers recruited through an agency. conclusions: participants in this study were determined to become parents through assisted reproduction. they had given thoughtful consideration to the medical and psychosocial issues unique to this process including which partner would be the genetic parent, and why. clomiphene superovulation. rb allen, 1 az steiner, 2 rh fogle, 1 mj kalan, 1 rj paulson. 1 1 ob/gyn, usc keck school of medicine, la, ca, usa; 2 ob/gyn, unc, chapel hill, nc, usa. intro: micro-dose hcg has been demonstrated to increase ovulation and pregnancy rates following clomiphene administration in women resistant to clomiphene. since micro-dose hcg stimulates growth in follicles expressing the lh receptor, we hypothesized that its use following clomiphene in women with unexplained infertility would increase the number of follicles that ovulate and subsequently, pregnancy rates. m m: irb approval was obtained for this prospective pilot study of women with unexplained infertility. on day #3 a baseline ultrasound was performed and serum fsh and e2 levels were measured. subjects were given 100mg of clomiphene daily from days 3-7 and then returned for serial ultrasounds on day #8. when at least 2 follicles 12mm were present, 200 iu of hcg im daily was initiated. cycles were monitored by ultrasound every 2 days until a +lh surge occurred. all subjects had previously undergone a cycle using clomiphene alone for superovulation followed by iui and these cycles were used for comparison. results: 5 subjects, aged 33.2±2.5 yrs (mean±sem) and bmi 25.8±1.6 kg/m 2 with 7.6±3.2 yrs of infertility were enrolled. the mean fsh and e2 levels were 8.9±1.4 miu/ml and 37.4±5.9 pg/ml, respectively. there was an average of 6.2±0.2 days from starting clomiphene to the attainment of 2 follicles 12mm. 4 out of the 5 subjects had 2 follicles 12mm by day #8. a mean of 10.4±0.7 days of treatment were required until ovulation occurred. there were twice as many follicles in the micro-dose hcg cycles, although due to the small sample size this did not reach statistical significance (2.8±0.4 follicles, measuring 20.6±0.9 mm in the micro-dose hcg cycles, compared to 1.4±0.3 (p=0.05), measuring 18.6±2.4 mm (p=0.47) in the control cycles). there was no difference in the endometrial thickness at the time of ovulation between the micro-dose hcg cycles and the control cycles: 8.4±2.0 mm vs. 10.0±2.3 mm (p=0.30), respectively. all subjects had at least 2 ovulatory sized follicles in the study cycles. 40% of subjects had an iui performed, however there were no pregnancies resulting from this study. conclusions: 1) the addition of micro-dose hcg to clomiphene may allow more follicles to reach an ovulatory size, which should theoretically increase pregnancy rates 2) this pilot study demonstrates that adding micro-dose hcg may increase the effectiveness of clomiphene when given in a superovulatory protocol. jessica salas, donald maier, john nulsen, claudio benadiva, lawrence engmann. obstetrics gynecology, university of connecticut, farmington, ct, usa. objective: to evaluate the antepartum, intrapartum, and neonatal complications in patients undergoing ivf using a gnrh-antagonist protocol where a gnrhagonist was used to induce final oocyte maturation. materials and methods: a retrospective review of data from high responders undergoing their 1st or 2nd ivf cycle using a gnrh-antagonist protocol who were triggered with leuprolide acetate (study group) or hcg (control) and achieved at least a singleton pregnancy reaching the third trimester. patients younger than 40 years old were included. both groups received luteal phase support with intramuscular progesterone. the study group received estrogen patch supplementation. outcomes measured were antenatal and intrapartum complications, order of gestation, gestational age at delivery, birth weight, neonatal adverse outcomes, and congenital anomalies. pearson chi square, fisher's exact test, or independent t-test were used as appropriate. results: the baseline characteristics were different (table 1) . maternal antenatal and intrapartum complications were similar in both groups (23.4% vs 39.4%, p=0.23). there were more singletons in the study group (84.9% vs 63.8%, p<0.05). a subgroup analysis of gestational age at delivery, birth weight, neonatal complications and congenital anomalies in singletons showed no difference (table 2) . conclusions: this is the first study reporting the maternal and perinatal outcomes after gnrh-agonist trigger. differences in response to ovarian stimulation may dictate use of gnrh-agonist triggering for prevention of ohss, which may explain the differences in baseline characteristics. maternal and neonatal complications remain unaffected. table 1 lupron (n=33) objective: moderate to severe ovarian hyperstimulation syndrome (ohss) occurs in 1-5% of assisted technology cycles and carries the potential for severe complications. traditional management consists of hospitalization with intravenous fluids (ivf), bedrest, and close monitoring. early and aggressive paracentesis is an alternative method of treatment for ohss in an outpatient setting, but the economic consequences of the two treatment regimens have not been compared. design: cost-effectiveness analysis materials and methods: two scenarios, outpatient management with transvaginal paracentesis and conservative therapy with hospitalization, were compared. potential initial outcomes were analyzed for the conservative group to include hospitalization either in a ward hospital bed or the intensive care unit (icu) for an average of seven days. costs included ivf administration, ultrasound, and daily bloodwork. initial outcomes for the outpatient management group included no further therapy beyond the initial transvaginal paracentesis, bloodwork, ivfs, and ultrasound versus admission for an average of three days to a ward bed with similar management. the probability of the negative outcome for the conservative group was set at 5% (icu admission) and 10% for the outpatient group (regular hospitalization). results: the cost of conservative therapy including first tier complications ranged from a low of $8,399 to a high of $14,615. the cost of outpatient management with aggressive paracentesis and its first tier complications ranged from a low of $940 to a high of $2,060. this resulted in an estimated cost burden of $7,459 to $12,555 for conservative management with hospitalization. the main improvement factor was that patients in the outpatient management group had a much lower likelihood for prolonged hospitalization than the conservatively managed group. conclusions: aggressive outpatient treatment of moderate to severe ohss with early paracentesis appears to be a cost-effective strategy. induction. zeynep alpay, michael p diamond, michael l kruger, elizabeth e puscheck. obstetrics and gynecology, division of reproductive endocrinology and infertility, hutzel hospital, wayne state university, detroit, mi, usa. introduction: aromatase inhibitors (ai) are a new method of ovulation induction and is proposed to replace clomiphene citrate (cc) due to their reported advantages. their superior effect is thought to be due to up-regulation of the estrogen receptors and increase in the sensitivity of the endometrium, resulting in better proliferation despite low estrogen levels in the circulation. objective: to compare the effect of different ovulation induction methods, including ai, cc and gonadotropins (gt) on the endometrium in infertility patients. methods: we reviewed 321 ovulation induction cycles performed in our institution in the last one-year period retrospectively. there were 149 gt, 145 cc and 27 ai cycles. age, gravida, day 3 (d3et) and midcycle (mcet) endometrial thickness, endometrial pattern (ep) on day of hcg injection, endometrial growth during the induction cycle (d-et), which was calculated by the difference between mcet and d3et, and the pregnancy rates (pr) were compared. the ep was categorized as type a (homogenous and hyperechogenic), type b (intermediate isoechogenic pattern and a poorly defined central echogenic line), and type c (multilayered triple-line). chi-square, anova, t test and pearson correlation were used for statistical analysis. results: in all cycles, ep was closely related to the d-et (p < 0.001). this correlation appeared to be more pronounced when observed ep was compared with d-et (r = 0.311; p < 0.001) rather than with mcet (r = 0.168; p = 0.002). there was a reverse relationship between the age of the patients and ep (r = -0.215; p < 0.001). a negative correlation was also found between the gravida and ep (r = -0.28; p < 0.001). pregnancy rate was significantly correlated with ep (r = 0.193; p < 0.001). pregnancy rate was 2% in type a ep, 5.2% in b, 16% in c when all cycles were analyzed. aromatase inhibitors and gt treatments each resulted in 52% type c pattern in contrast to 33% with cc (p = 0.014 for ai vs. cc; p = 0.05 for gt vs. cc). the d-et was greater in gt cycles compared with ai or cc (p < 0.001). conclusion: these results show that ep has a strong correlation with pr in ovulation induction cycles. additionally, ai and gt treatments have similar effects on ep. both ep and the growth of the endometrium during the induction (d-et) are good prognostic variables for the successful pregnancy initiation. ovarian response in patients undergoing ovarian stimulation after myomectomy. hyacinth browne, 1,2 desiree mccarthy-keith, 1,2 barbara stegmann, 1,2 alicia armstrong. 1,2 1 reproductive biology and medicine branch, nichd, nih, bethesda, md, usa; 2 reproductive endocrinology and infertility, walter reed army medical center, washington, dc, usa. objective: the impact of myomectomy on ovarian function has not been wellstudied. other surgical treatments of fibroids, such as hysterectomy and uterine artery embolization, have shown an increase of fsh into the peri-menopausal range. the objective of this study is to examine ovarian response in infertile women undergoing ovarian stimulation after abdominal myomectomy. design: retrospective analysis. materials and methods: a retrospective analysis of all infertile women with known fibroids who had a failed art cycle, from january 2000 to 2007, followed by an abdominal myomectomy and a subsequent art cycle was performed. women served as their own controls. ovarian function pre and post-myomectomy was assessed by age, day 3 and 10 fsh levels, days of stimulation, total gonadotropins used, peak estradiol level, number of oocytes retrieved, embryos obtained, and high-grade embryos, and pregnancy outcome. quantitative results are presented as mean + sd. results: four women had a failed art cycle and underwent an abdominal myomectomy prior to a subsequent art cycle. the mean age was 35 and 36 pre-and post-myomectomy, respectively. all subjects had uterine factor infertility. two of these women also had tubal factor infertility, and one had endometriosis and male factor infertility. we found no difference in ovarian response pre and post-myomectomy. pre-myomectomy post-myomectomy age (years) 35 +3.6 36 +3.6 day 3 fsh (u/l) 6.9 + 1.7 6.1 + 0.96 day 10 fsh (u/l) 6.1 +1.9 6.2+ 2. objective: anti-mullerian hormone ( amh) is produced by developing primordial follicles. amh levels are stable through the menstrual cycle and therefore can be drawn randomly. the objective of this study was to assess the association of anti-mullerian hormone ( amh) and oocytes obtained at ivf retrieval. design: cross-sectional cohort study. materials and methods: the study cohort is comprised of thirty women in 30 cycles of in vitro fertiization (ivf). serum levels antimullerian hormone (amh) were drawn before starting an ovulation induction cycle for ivf. standard ovulation induction was performed with leuprolide flare and 450 miu/ml per day of follicle stimulating hormone. we performed linear regression of log convert4d oocyte number against the log converted amh level. serum amh was measured using an enzymatically amplified two-site immunoassay dsl-10-14400 active mis/amh elisa. statistical analysis was performed using spss version 15.0. continuous values are presented as mean std error. results: the log number of oocytes retrieved was directly related to the log value of amh (p = 0.036). conclusions: our data demonstrate an association between serum amh and oocytes produced in response to ovulation induction. using amh levels in addition to fsh levels in evaluating for ovarian reserve and pregnancy outcome may help improve predictions of reproductive performance. support: foundation for reproductive medicine. context: prediction of outcome after in vitro fertilization (ivf) can be difficult due multiple factors. human chorionic gonadotropin (hcg) levels correlate with pregnancy outcome, but data that can be used to easily counsel patients on their possible outcome is lacking. objective: to investigate the use of hcg levels along with other significant factors to predict the likelihood of an ivf pregnancy progressing to the point of detection of cardiac activity by ultrasound design: retrospective data analysis of 1377 ivf cycles performed from january 1997 to july 2007 resulting in 665 pregnancies. multiple logistic regression analysis modeling was performed to determine the factors most predictive of an ongoing early pregnancy and to assess possible confounding variables. setting: an academic fertility center. patients: patients undergoing in vitro fertilization using autologous fresh embryos. intervention: none main outcome measure: pregnancy continuation to the documentation of cardiac activity by ultrasound. results: maternal age, day 14 (post-oocyte aspiration) hcg level, and day 16 hcg levels were significant in predicting pregnancy outcome. day 14 and day 16 hcg levels were highly correlated and can be considered proxies for each other. the most accurate predictive model used only a single day 14 hcg level and maternal age. the type of fertilization method used, the cycle number for that patient, and the number of embryos transferred were not found to be significantly different. ongoing pregnancy rates were directly proportional to day 14 hcg level, and inversely proportional to maternal age. the incidence of multiple pregnancies also increased proportionally to the initial hcg level. 99% of ongoing pregnancies had hcg level > 23 miu/ml. conclusions: a single day 14 post-oocyte aspiration hcg level and maternal age are the most predictive of an ongoing ivf pregnancy. there was no difference in outcome between fertilization methods or number of embryos transferred. there is no benefit to obtaining serial hcg levels after the initial one. erk activation with u0126 (10 m)) reduced fsh stimulated cyclin d2 mrna expression by 50%. fsh has also been shown to stimulate mtor, a regulator of growth and proliferation of many cell types. inhibiting mtor activation with 10nm rapamycin for 15 min significantly reduced fsh-mediated cyclin d2 mrna expression. dht exposure for 24 hours also inhibited fsh stimulated mtor signaling, as shown by a 40% reduction in the phosphorylation of its down stream target p70s6kinase. furthermore, pretreatment with dht resulted in significantly reduced fsh-mediated tsc-2 phosphorylation, which is an upstream regulator of mtor pathway. further studies revealed that fsh mediated tsc-2 phosphorylation and mtor signaling is regulated by erk, but independent of akt. based on these results we conclude that elevated 5 reduced metabolites of androgens inhibit fsh-stimulated pka-erk pathway resulting in the inhibition of multiple mitogenic signaling pathways leading to defective follicle maturation culminating in anovulation. thus, rescuing the erk activation might serve as a potential therapeutic target to restore normal granulosa cell proliferation in hyperandrogenic states. (supported by nih grant hd-38424). searching pcos-genes: results of a genome screen for pcos in a dutch founder population. olivier valkenburg, 1 annemarie g mulders, 1 aida bertoli-avella, 2 ben a oostra, 2 joop se laven. 1 department of gynecology and obstetrics, erasmusmc, rotterdam, netherlands; 2 department of internal medicine, erasmusmc, rotterdam, netherlands. context: although the etiology of pcos is not yet fully understood, evidence has accumulated for a complex genetic background i.e. a combination of multiple genetic and environmental factors. to reduce genetic heterogeneity, this study was conducted in a genetically isolated population in the netherlands . objective: in order to identify genomic loci that are associated with pcos, a whole genome screen using highly polymorphic microsatellite markers was performed in a founder population. subjects and methods: 72 pcos patients (2003 rotterdam criteria) were identified in the founder population (rucphen, the netherlands) of ± 20.000 inhabitants. patients underwent a standardized screening procedure that included clinical, ultrasound and endocrine evaluation. all patients plus 141 unaffected first-degree family members were genotyped using 366 polymorphic markers on (microsatellites) from the abi prism ® linkage mapping set md-10 (average spacing 10 cm). association-analysis was performed with the transmission disequilibrium test (tdt, genehunter software). linkage analysis was performed in 11 clusters of 2 or more closely related patients (simwalk2). results: there was an average number of 10.1 alleles per polymorphic marker. the tdt identified two non-adjacent markers on chrome 6 (d6s262 and d6s308) that showed significant association with pcos (p 0,01). other markers that showed significant association were positioned on chromosomes 1 (d1s450), 3 (d3s1565), 4 (d4s406) , 7 (d7s530), 8 (d8s264) and seventeen (d17s784) (all p values 0.01). linkage analysis in 11 sub-pedigrees revealed no significant linkage for these, or other, loci with pcos. moreover the results of a prior report of linkage of a marker on chromosome 19 (d19s884) could not be confirmed. conclusions: the lack of consistent results suggests the absence of a consistent genetic background in this select group of pcos patients. this supports the hypothesis of a complex genetic background for pcos that allows relatively small contributions of multiple risk-genes to be involved in the pathogenesis of this syndrome. in this founder-population the genetic heterogeneity was not sufficiently reduced to find risk-loci in a genome wide screen using highly polymorphic markers with an average spacing of 10 cm. objectives: to objectively quantify uterine and endometrial blood flow in women with polycystic ovarian syndrome (pcos) and to examine if this was different in women with different phenotypic expressions of the disease. methods: transvaginal 2d and 3d ultrasound was performed in 36 women with pcos, as defined by the rotterdam criteria, and sub-group analysis conducted based on the subjects' body mass index (bmi), ovulation status, and hirsutism score. anova was used to compare the mean values between the groups. results: pcos women with clinical hyperandrogenaemia had significantly lower endometrial and subendometrial blood flow than their anovulatory normoandrogenic counterparts (table 1 ). there were no differences between lean and obese women or between anovulatory and ovulatory women with pcos. the pulsed wave doppler parameters were similar in all three phenotypic groups. conclusions: hirsute women with pcos have impaired endometrial perfusion compared to their normoandrogenic counterparts which is only evident with 3d ultrasound and not conventional pulsed wave doppler. nusayba a bagegni, 1 jill blaine, 1 anuja dokras. 2 1 obstetrics gynecology, university of iowa hospitals clinics, iowa city, ia, usa; 2 obstetrics gynecology, university of pennsylvania, philadelphia, pa, usa. background: there is conflicting evidence on the association between pcos and early and late obstetric complications. it is unclear if the reported risks are independent of bmi, preexisting hypertension and diabetes. we examined the risk of early and late obstetrical complications in a large group of women with pcos compared to controls. methods: we reviewed pregnancy records of women with pcos (rotterdam criteria, n=130) and controls (tubal infertility, n=130) after in vitro fertilization at university of iowa from 1994-2006. the wilcoxon rank sum test and fisher's exact test were used to evaluate differences between variables and logistic regression analysis was used to determine the independent risk of pcos. results: subject demographics and medical history are shown in table. the first trimester miscarriage rate was 18% in women with pcos and 15% in controls. after logistic regression analysis pcos was not associated with miscarriage (p=0.495). the prevalence of gestational dm (gdm) was similar in both groups 12% pcos vs 11% controls. pcos was not associated with gdm after adjusting for age and bmi (p=0.678). however, bmi was significantly associated with gdm after adjusting for age and pcos (p=0.012). risk of both pre-eclampsia and pih was 10% in pcos and 5% in controls, but not statistically significant after adjusting for age, bmi and twin gestation. preexisting htn showed a significant association with preeclampsia (p<0.01). there was no significant difference in preterm delivery, cesarean section, twin gestation, intrauterine fetal death and intrauterine growth restriction in the 2 groups. conclusion: despite adequate power, our study did not detect an increased risk of miscarriage in women with pcos. obesity was a significant contributor to late obstetric complications, namely gdm. these findings may warrant aggressive counseling of women with pcos on the potential benefits of weight loss prior to pregnancy. objective: to assess the prevalence of polycystic appearing ovaries (pcao) as defined by the rotterdam criteria; and to determine whether metabolic parameters differ between regularly cycling women with pcao and those with normal ovaries. studies have demonstrated a population frequency of pcao of 21-33%, with 7-24% among women with regular cycles. most were performed in a young reproductive age population; none examined the impact of age. all were performed prior to adoption of the rotterdam criteria. recent studies have shown an increased prevalence of metabolic syndrome among women diagnosed with polycystic ovarian syndrome (pcos). however studies focused on women in their 20s found no increase in bmi or fasting insulin with pcao but regular menses. participants: 222 women aged 25-45 with regular cycles (every 25-35 days) enrolled in the ova study, a population-based study of ovarian aging. each subject underwent a transvaginal ultrasound for assessment of ovarian volume and antral follicle count (afc). outcomes collected included waist measurements and hdl, ldl, total cholesterol, triglycerides, fasting glucose and insulin. pcao were determined by the rotterdam criteria (afc of 12 on one ovary or ovarian volume > 10cc). student's t-test and chi-square tests were used to assess differences between those with and without pcao for continuous and categorical variables, respectively. the prevalence of pcao decreased with increasing age (table 1) . of the 69 women with pcao, 62% met the afc criterion only, while 19% met the volume criterion only, and 19% met both. women with and without pcao did not differ in waist measurements, or in fasting lipids, insulin, or glucose. the rotterdam criteria, while less subjective than those described by adams in 1986, have led to an increased prevalence of pcao among women with regular menses, over 1/2 of women in their 20s. women with pcao do not differ from those with normal ovaries in metabolic parameters associated with pcos. consideration should be given to adopting an age-adjusted criterion for afc, or a combination of afc and ovarian volume, for diagnosing pcos. background: pcos, especially accompanied by obesity, has been reported to be associated with a characteristic dyslipidemia comprising elevated triglycerides (tgs) and depressed hdl, especially the hdl-2 fraction. this is an atherogenic profile; in fact, studies suggest low hdl-2 may correlate most strongly with cardiovascular disease risk. weight loss is a mainstay of treatment and improves all manifestations of the disease, but the optimal diet to recommend remains undetermined. preliminary studies show a high protein diet may improve total hdl levels and insulin responses. however, the effect of weight loss and dietary composition on hdl-2 levels in pcos has not been investigated. objective: to evaluate the fasting lipid profile in newly diagnosed obese pcos patients and to determine the effects of a high-protein diet with or without metformin on weight loss, hdl-2 and other lipoproteins, and menstrual cyclicity. methods: in this pilot retrospective observational study, the fasting lipid profile of 42 obese women newly diagnosed with pcos was determined. they were then placed on a high protein (80-100 g/day), low carbohydrate (30-50 g/day) diet with or without metformin (76 and 24%, respectively) and followed monthly for an average of 13 months (range 5-31). results: at diagnosis, 66% had low hdl and 89% had low hdl-2; only 29% had elevated tgs. on the diet, the patients demonstrated an average weight loss of 24.7 lbs (212.01 to 187.4, p<0.001) and decreased bmi of 4.2 kg/m2 (34.2 to 30.0, p<0.001). hdl levels increased significantly (18% increase from 46.6 to 55.0, p<0.001), especially the hdl-2 fraction (34% increase from 10.9 to 14.6, p<0.001). triglycerides decreased as well (135.0 to 107.2, p<0.027). ldl decreased but did not reach statistical significance. 29 resumed menstrual cycles, 11 were started on oral contraceptives, and 1 had a hysterectomy. 2 pregnancies occurred. no difference was seen with metformin use. conclusion: a majority demonstrated decreased hdl and hdl-2 at diagnosis. the high protein diet resulted in significant weight loss and improvement in hdl-2 levels, as well as improvements in total hdl, tgs and menstrual cyclicity. metformin produced no added benefit. prospective trials based on these data will help determine the optimal diet to reduce the significant short-and long-term morbidity in the large population of women with pcos. resistin is an adipokine that has been associated with obesity and insulin resistance in animal models. studies on the role of resistin on insulin resistance in humans have been controversial. recently resistin has been shown to exert atherosclerotic effects and elevated resistin levels have been observed in women with coronary heart disease (chd). women with polycystic ovary syndrome (pcos) are at high risk for chd. our present study investigates potential association of resistin and markers of inflammation, c-reactive protein (crp) and insulin resistance in women with pcos. methods: thirty two women with pcos participated in the study. all were hirsute and had irregular cycles. nineteen women with normal ovulatory cycles who matched the pcos patients in bmi served as controls. fasting glucose, insulin, resistin and crp levels were measured in all women. after a high carbohydrate diet for 3 days, a standard oral glucose tolerance test (ogtt) was performed. blood samples were collected for glucose and insulin before and 1, 2 and 3 hrs after 75g oral glucose. the area under the curve (auc) for insulin and glucose was calculated. women with overt diabetes were excluded from the study. results: fasting insulin levels (24.9 + 2.2 μu [±se] /ml) and insulin response to oral glucose (auc) (527.9 + 52.2μu/ml) were higher (p < 0.001) in women with pcos compared to controls. all were insulin resistant with homa-ir value > 3.9 mol x μu / l 2 . resistin levels in women with pcos (15.6 ±1.3 ng/ml) was significantly (p < 0.02) higher compared to control women (10.7 ± 1.4 ng/ml). crp levels in women with pcos (9954.2 ± 918 ng/ml) was also significantly (p < 0.001) higher than the controls (2396.2 ± 579 ng/ml). there was a significant positive correlation between resistin and crp levels (r = 0.362, p < 0.04). there was no correlation between resistin levels and fasting insulin levels or insulin auc. there was also no correlation between resistin levels and fasting glucose or glucose auc. conclusions: our results indicate that (1)women with pcos and insulin resistance have increased resistin levels, (2) there is a strong association between resistin and crp (3) elevated resistin and crp levels may predict women who are at increased risk for chd (3) ) it is unlikely that resistin plays a major role on insulin resistance in women with pcos. vuk p jovanovic, 1 enrico carmina, 2 prati vardhana, 1 michel ferin, 1 rogerio a lobo. 1 1 department of obstetrics and gynecology, columbia university, new york, ny, usa; 2 department of internal medicine, university of palermo, palermo, italy. current diagnostic criteria for pcos includes both ovulatory (ov) and anovulatory or "classic" (c) phenotypes. in an effort to further characterize differences and /or similarities between these 2 phenotypes, we studied 48 hyperandrogenic women with pcos (age 27.3±6.4, bmi 27.1±5.6) and 20 age matched controls (age 27.7±4.5, bmi 26.8±3.9). women with pcos were divided into weight matched (ov) n=14 and (c) n=34 groups. fat and weight distribution were assessed by dexa (total fat, r1 fat, trunk fat) as well as fasting levels of lh, e2, mis/amh, kisspeptin and testosterone, the adipocytokines (leptin, adiponectin, visfatin and retinol-binding-protein-4 rbp4) and serum glucose, insulin and crp. the hyperandrogenic pcos groups had characteristically altered hormone profiles compared to matched controls. although total fat mass was comparable, women with c-pcos had a significantly larger waist circumference (93.2 14.9 vs. 86 85 cm, p<0.05) trunk fat, r1 fat and %trunk and %r1 fat compared to ov-pcos (p<0.05). leptin, rbp4 and visfatin did not significantly differ among the pcos subgroups although adiponectin was lower in the c-pcos group (p<0.05). quicki was significantly lower in c-pcos (0.33±0.02 vs. 0.35±0.02, p<0.05) and insulin was higher (14.9±5.76 vs. 9.7±3.5, p<0.01). serum lh was also higher (11.3±7.8 vs. 6.5±1.7, p<0.05) but kisspeptin, testosterone and estradiol were similar. mis/amh (8.3±5.2 vs. 6.8±4.9 ng/ml, not significant) and crp (2.78±1.5 vs. 1.3±0.5, p<0.05) were higher in c-pcos. significant correlations (p<0.05) were noted among kisspeptin/rbp4 (r 0.426), mis/testosterone (r 0.451), insulin/ %trunk fat (r 0.616, p<0,01), total fat/leptin (r 0.772, p<0.01), %trunk fat/adiponectin (r -0.522). in conclusion, women with c-pcos when compared to similarly hyperandrogenic women with ov-pcos with similar bmi, have increased abdominal fat, and appear to have differences in serum lh, crp and increased insulin resistance. the latter, as well as subtle differences in the adipocytokines may explain these differences in anthropometric findings. problems of normal oogenesis and folliculogenesis but also disturbed oogenesis and folliculogenesis in polycystic ovaries are not fully understood. oocyte specific genes play an essential role in oogenesis and folliculogenesis. there are suggestions about possible role of some oocyte specific genes in etiopathophysiology of pcos. zona pellucida 4 gene (zp4) is recently identified gene, which belongs to zona pellucida genes such as zp1, zp2 and zp3. the role of zp4, contrary to above-mentioned other zp genes is not well described. the aim of this study was to analyze zp4 coding sequence and expression in patients with polycystic ovary syndrome. material included blood received from 29 patients (mean age 24,2 +/-3,23 years; mean bmi 31,4 +/-4,54 kg/m 2 ) with polycystic ovary syndrome. all patients with pcos were diagnosed with the use of eshre/asrm criteria from 2003. dna was isolated from blood cells( after separation of blood cells from serum) using a dna isolation kit (qiagen ). genomic dna was used for in vitro amplification by pcr with a specific set of primers complementary to the coding sequence of the zp4 gene. products from each pcr reaction were examined by sscp method. samples with changes detected by sscp in comparison to control probes were cloned into plasmid vector and then automatically sequenced from a total of 29 patient samples with pcos, we identified 5 nucleotide changes in the zp4 coding seguence : 4 silent nucleotide changes in exons 1,2,5 ,7, and 1 nucleotide change in the exon 5 ( position 114, t>g). the mutation in exon 5 ( t>g ) results in substitution of cystein for glycin of amino acid in position 223 of zp4 protein. in summary, our data demonstrate that zp4 nucleotide changes account for 15% of patients with pcos. relationship between serum mullerian inhibiting substance levels and insulin in women with polycystic ovary syndrome. rebecca a chilvers, shilla chakrabarthy, summer james, xin ma, manubai nagamani. ob/gyn, university of texas medical branch, galveston, tx, usa. müllerian-inhibiting substance (mis) is a member of the transforming growth factor-superfamily of growth factors. it is expressed exclusively in granulosa cells and is believed to play a role in the regulation of follicle selection and maturation. women with pcos have anovulation and most of them have hyperinsulinemia. the purpose of our study is to investigate possible association between insulin and mis in the dysregulation of folliculogenesis in pcos. methods: twenty one women with pcos who had anovulatory cycles and hyperandrogenism were recruited for the study. sixteen women with ovulatory cycles and matched the pcos patients in age and bmi served as controls. an oral glucose tolerance test (ogtt) was performed in all women. blood samples were obtained at fasting and 1, 2 and 3 hours after glucose ingestion for measurement of glucose and insulin. to investigate the effect of hyperinsulinemia on mis secretion, mis levels were measured in ten patients during the ogtt. fasting mis, testosterone, dheas, fsh, and lh levels were measured in all patients. results: fasting insulin levels (16.4 + 2.4μu/ml) vs 9.9 + 1μ u/ml [+ se]) (p < 0.03) and area under the curve (auc) of insulin (267.9 + 33.0 vs 134.0 + 12.2μu/ml) (p <0.001) were significantly increased in women with pcos compared to control women, while the glucose levels were normal indicating insulin resistance. mis levels were significantly (p < 0.001) increased in women with pcos (6.5 + 0.9 ng/ml) compared to controls (1.9 + 0.2 ng/ml). there was no correlation between age and mis levels in pcos patients while there was a highly significant negative correlation between the age and mis levels in the control women ( r = -0.822, p< 0.0001). there was significant negative correlation between mis and fasting insulin levels (r = -0.462, p < 0.03) and insulin auc during the ogtt (r = -0.485, p <0.02). there were no changes in mis levels during ogtt. conclusions: results of our study indicate that in women with pcos, (1) there is an increase in secretion of mis, (2) higher insulin levels are associated with lower mis levels, (3) acute increase in insulin levels has no effect on mis levels, (4) lower mis levels in women with severe hyperinsulinemia could be due to associated increased follicular atresia and a decrease in the ovarian reserve. further studies are needed to investigate the role of insulin on mis secretion. hiroyuki asakura, noriko tanaka, kyoko nishio. ohgimachi ladies' clinic, osaka, japan. objective: elevation of serum anti-müllerian hormone (amh) levels among polycystic ovary syndrome (pcos) patients has been reported. however, the regulatory factors of amh in pcos remain unknown. we examined correlations between amh values and various indices of insulin resistance in pcos women. methods: 40 infertile women compatible with rotterdam criteria for pcos were recruited under informed consent. the subjects underwent 75g oral glucose tolerance test (75ggtt, sampling at 0, 60, 120 minutes), and 2-step elisa for amh (sensitivity 0.7 pmol/l, inter intra-assay c.v. :12.3%, 14.2%, respectively). p<0.05 was considered as statistical significance. results: average amh level of the subjects (age:31.9+3.9 years, bmi: 21.1+3.0 kg/m 2 , mean+s.d.) was 50.4+31.1pmol/l, which was higher than normo-ovulatory women (16.7+10.5 pmol/l, n=16). amh levels had significant positive correlation with total ovarian volume by ultrasound (18.2+11.4 cc), but not with bmi, waist-hip ratio (0.85+0.06), and levels of serum lh (5.5+3.6 iu/l), total testosterone (67.3+32.8 ng/dl). although fasting glucose levels (83.1+4.9 mg/dl) were positively correlated with total ovarian volume (r=0.41), amh levels had no significant correlation with fasting insulin (4.3+2.3 iu/l), fasting glucose/insulin ration (22.8+8.4), homa-ir (0.90+0.51), and the sum of insulin levels (98.6+42.1 iu/l)during 75ggtt. conclusions: increased serum amh level of pcos and its positive correlation with total ovarian volume implies that determination of serum amh level would aid in confirming diagnosis of the ovulatory disorder. absence of relationship between amh and various clinical indices of insulin resistance suggests alternative regulatory factors of amh gene expression in the follicular compartment. reproductive tissues. amisra a nikrodhanond, keeley l mui, helen h kim. ob/gyn, university of chicago, chicago, il, usa. background: although primarily a neuroendocrine hormone, gonadotropinreleasing hormone (gnrh) is also produced in reproductive tissues, where it acts locally. the study of gnrh regulation in these tissues is limited by low levels of expression. objective: to elucidate mechanisms that regulate gnrh expression in male tissues, our objective was to identify regions of the gnrh gene that target expression in vivo. methods: transgenic mice were generated with 2 different fragments of the mouse gnrh gene promoter (-3446/+28 and -249/+28 bps), fused to the luciferase reporter gene. in these mice, luciferase activity (detected as light) reflects gnrh promoter activity. using bioluminescent imaging, gnrh promoter activity was assayed in live gnrh-luc mice and in their reproductive tissues ex vivo. to confirm imaging results, luciferase activity was also measured as relative light units (rlu) in tissue homogenates. for each dna construct, 5 male mice were examined. with whole-body imaging, bioluminescence was detected in the genital region of the gnrh-luc transgenic mice (fig.a) . in mice that incorporated the -3446luc transgene, examination of reproductive tissues ex vivo revealed bioluminescence in the prostate and penis, but not in the seminal vesicles, epididymis, or testes (fig.b) . in contrast, in the -249luc mice, bioluminescence was detected in the testes, but not in other tissues (fig.c) . examination of luciferase activity in tissue homogenates from -3446luc mice confirmed gnrh promoter activity in the prostate (3876 ± 624 rlu) and penis (576 ± 151 rlu) and absence in the testes (39 ± 11 rlu). in the -249luc mice, however, luciferase activity was detected only in the testes (1283 ± 97 rlu) and not in the prostate (65 ± 25 rlu) or penis (48 ± 10 rlu). conclusions: our studies demonstrate that gnrh is present in male reproductive tissues, but may be differentially regulated in the testes vs. prostate/penis. promoter elements, contained within the proximal -249 bp of the mouse gnrh gene, are sufficient to mediate testicular gnrh expression while -3446 bp of the gene promoter are necessary to direct expression to the prostate and penis. characterization of mouse ringo/speedy homologues. z walton, s uckac, o guzeloglu-kayisli, md lalioti, d sakkas, e seli. ob gyn, yale u., new haven, ct, usa. introduction: ringo/speedy (ringo/spy) is a recently discovered cyclindependent kinase (cdk) activator that functions similar to cyclins in controlling the cell cycle. ringo/spy plays a crucial role during meiotic maturation in xenopus by inducing meiotic g2/m progression and germinal vesicle breakdown (gvbd). more recently, padmanabhan and richter demonstrated that ringo/spy mrna is repressed in the xenopus oocyte cytoplasm by pumilio 2. upon meiotic reactivation, pumilio 2 loses its interactions with ringo/spy permitting its translation. ringo/spy is then expressed, leading to activation of cpeb by phosphorylation, which in turn elicits polyadenylation and translation activation of the mrna for a critical oocyte maturation factor, the mos kinase. in this study, we investigated the expression of ringo/spy in mouse. methods: ten different somatic tissues, testes, and ovaries were tested by reverse transcription-polymerase chain reaction (rt-pcr) for the expression of ringo/spy homologues and their alternative splicing variants. ringo/spy mrna expression was also tested in prophase i (pi) and metaphase ii (mii) oocytes, 1-cell, 2-cell, 4-cell, 8-cell embryos and blastocysts. amplification with actin primers provided a positive control and allowed semi-quantitative analysis. results: we analyzed the two previously identified homologues of ringo/spy (a and b). the two alternative splicing variants of ringo/spy a (a1 and a2) were separately studied for their expression profile. we also identified an additional alternative splicing variant of ringo/spy b and evaluated similarly. ringo/spy a (1 and 2) were expressed in testes, ovaries, and certain somatic tissues including brain, and spleen. ringo/spy b (both variants) were only expressed in testis. ringo/spy a or b were not present in mouse oocytes or early embryos. conclusions: our findings indicate that the previopusly described ringo/spy homologues a and b are not expressed in mouse oocytes or early embryos suggesting that either an alternative cdk-activator or a yet to be identified homologue of ringo/spy may be mediating meiotic g2/m progression in mouse. testis specific expression of ringo/spy b, and previously described role of ringo/spy in meiosis suggests a role for this protein in male gametogenesis in mouse. treated with glyburide. jennifer aguayo, gladys a ramos, alethea hanley, carri r warshak, thomas r moore. reproductive medicine, university of california, san diego, san diego, ca, usa. objective: to determine the risk factors that may predict inadequate response to first-line glyburide monotherapy in pregnant women with type 2 diabetes mellitus (dm) or gdm and to assess if non-responders are at increased risk of adverse pregnancy and neonatal outcomes. study design: this was a retrospective cohort of 146 women diagnosed with type ii or gdm initially treated with glyburide at a single institution from 2001-2004. maternal characteristics, adequate glycemic control defined as more than 66% of fasting glucose <95 mg/dl and one-hour post-prandials <135 mg/dl, and neonatal outcomes were assessed. non-responders were defined as failure to achieve adequate glycemic control on maximum daily doses of glyburide or intolerance due to side effects, necessitating switch to insulin therapy. statistical methods included bivariate analyses. results: of the 146 women initially treated with glyburide, 25 (17%) failed to achieve adequate glycemic control or did not tolerate glyburide. reasons for glyburide non-response were maximum dose (20 mg/d) reached (56%), maternal hypoglycemia (28%) and other side effects (16%). there were no statistically significant differences between non-responders and responders with respect to family history of diabetes (75% vs. 60%, p=0.25), prior history of gdm (24% vs. 32%, p=0.63) and macrosomia (>4000 g) (28% vs. 24%, p=0.80) or 1 hour glucose challenge test (204+51 vs. 180+36mg/dl, p=0.07). non-responders had a higher rate of obesity (bmi>30) (71% vs. 44%, p=0.02) and earlier gestational age at initiation of therapy (24+7.7 vs. 29+7.7 wks, p=0.01). there were no differences between the groups in the mean 36-week fasting (96+17 vs. 90+14 mg/dl, p=0.15) and post-prandial glucose values (133+26 vs. 127+19 mg/dl, p=0.87). no significant differences were observed in incidence of pre-eclampsia, primary cesarean delivery or birth weight. neonatal outcomes including ponderal index, neonatal hypoglycemia, nicu admission, and birth injuries also did not differ between the groups. conclusion: women who are obese and who require earlier initiation of glyburide therapy are at increased risk of non-response to glyburide monotherapy. however, despite non-response, these women can be managed with subsequent insulin therapy to achieve similar glycemic control and pregnancy and neonatal outcomes. a different diagnostic strategy using the 100gram, 3-hour glucose tolerance test for the diagnosis of gestational diabetes mellitus. yvonne w cheng, ingrid block-kurbisch, jennifer lydell, aaron b caughey. obstetrics, gynecology and reproductive sciences, university of california, san francisco, san francisco, ca, usa. objective: to examine whether a simplified strategy using the 100gm, 3-hour glucose tolerance test (gtt) may be useful for the diagnosis of gestational diabetes mellitus (gdm). methods: this is a retrospective cohort study of women with singleton pregnancy who received the 50-gm, 1-hour glucose challenging test (gct) for initial screening and the 100gm, 3-hour gtt as confirmatory tests for the diagnosis of gdm between 1988 and 2001. various combinations of the 100gm, 3-hour gtt results were examined and compared to the diagnostic criteria for gdm established by the carpenter and coustan criteria using the receiver-operator characteristic (roc) curves. perinatal outcomes of women who would have had a false-positive or false-negative test results were compared to those who did not have gdm using the carpenter and coustan criteria. potential confounding factors were controlled for using multivariable regression models. results: 2,535 women had 100gm, 3-hour gtt results available for analysis during the study period. using gdm diagnosed by the carpenter and coustan criteria as reference, various diagnostic strategies was compared using roc curves (figure 1) . summation of the 1-hour and 2-hour gtt results with a diagnostic threshold of 334mg/dl yielded the most optimal balance between sensitivity (82.1%) and specificity (93.2%). when compared to women without gdm, women who were diagnosed with gdm by c c criteria but not by summation of 1-hour and 2-hour gtts had higher odds of operative vaginal delivery (aor=1.81, 95% confidence interval [ci] 1.03-3.27) and neonatal birthweight >4000gm (aor=1.91, 95% ci 1.03-3.57). conclusion: using only the summation of only the 1-hour and 2-hour gtt results of the 100gm 3-hour gtt offers an alternative test strategy which may be more convenient and less costly for the diagnosis of gdm. however, women who would have gdm by the carpenter and coustan criteria but not by the summation method have higher odds of having an operative vaginal delivery and neonatal birthweight >4000gm. outcome of induction of labor indicated for term prom among women with or without a uterine scar. yifat ochshorn, avital skornick rapaport, adi reches, joseph b lessing, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. objective: we aimed at comparing the outcome of induced term deliveries presenting with prom with or without a previous uterine scar. methods: the computerized files of 973 women delivered following induction of labor due to term prom, were reviewed. perinatal outcome parameters such as the mode of delivery, indication for cesarean section, rate of low 5' apgar scores and nicu admissions were compared between women with and without a previous cesarean. results: during the study period 973 women delivered in our institution following prom and induction of labor , 49 of them had a previous uterine scar. parturients of both groups (with and without a scar) were similar with regard to age, gestational age at delivery and parity. cesarean section rate was higher for the previous scar group (28% vs 17%, p<0.05). the most common indication for cesarean was arrest of dilatation and/or descent among women with previous scar accounting for 43% and 27% (p<0.05) for women with and with no uterine scar, respectively. no differences were noted in neonatal outcome parameters such as rate of low 5' apgar scores and nicu admission rate between the two groups. conclusion: induction of labor due to prom culminates in higher cesarean rate in women with one previous scar compared with parturients with no scar. perinatal outcome is similar between the two groups. acid base balance and immediate perinatal outcome of vertex compared with breech presentation in elective cesarean section. avital skornick rapaport, yifat ochshorn, joseph b lessing, yuval yaron, michael kupferminc, ariel many. obstetrics gynecology, lis maternity hospital, tel aviv sourasky medical center, tel aviv, israel. backround: apgar scores, umbilical blood ph and bicarbonate are generally lower and pco 2 levels are higher in vaginally delivered breech neonates compared to cephalic deliveries. although cesarean delivery improved apgar scores there is a debate wether it improved the acid base status. this retroprospective study compared umbilical cord blood acid-base values and perinatal outcome of elective cesarean breech-delivery with those of elective cephalic cesarean delivery and to determine whether a different metabolic status and perinatal outcome should be expected in neonates in breech presentation. study design: the study group included singleton pregnancies delivered by elective cesarean section at term between january 2003 and march 2006. computerized files of singleton breech presentation elective cesarean sections were compared to those of singleton vertex neonates delivered by elective cesarean section. demographic data included: maternal age, pregnancy week at delivery and parity. perinatal outcome measures checked were: birth weight, apgar scores at 1'and 5', umbilical cord venous and arterial ph and base excess. results: during the period between january 2003 and march 2006 there were 1411 singleton elective cesarean sections, 404 of them were breech and 1007 vertex. the mean age, gravida and parity were significantly different between groups (33.5 vs.31.8, 2.95 vs. 2.19. and 1.26 vs.0.64 respectively, p<0.001). the birth weight was significantly different -3414.8 gram for the vertex and 3189.9 gram in the breech deliveries (p<0.001) there were no differences in either apgar scores or umbilical ph between the breech and vertex neonates delivered by cesarean section at term (38-40+6w). venous and arterial po 2 and pco 2 levels were significantly different, though the differences were very small and we doubt if these differences have any clinical importance. conclusion: although vaginal breech deliveries are associated with increased risk of asphyxia during delivery, elective cesarean breech deliveries are not at increased risk for lower ph levels or apgar scores. the clinical significance of bleeding during the second trimester of pregnancy. arie koifman, 1 amalia levi, 2 yaron zaulan, 1 avi harlev, 1 eyal sheiner. 1 1 obstetrics gynecology, soroka university medical center, ben gurion university of the negev, beer-sheva, israel; 2 epidemiology and health services evaluation, faculty of health sciences, ben gurion university of the negev, beer-sheva, israel. objective: this study aimed at investigating clinical importance and pregnancy outcome in women suffering from bleeding during the second half of their pregnancies. methods: a population based study including all deliveries which took place in the soroka university medical center between the years 1988-2005 were examined. comparison was performed between patients with and without second trimester bleeding pregnancies terminated before 22 weeks, multiple gestations and women lacking prenatal care were excluded . stratified analysis, using the mantel-haenszel technique, and a multiple logistic regression model were performed . results: during the study period, 175,093 singleton deliveries occurred in our institute. of these, 2010 (1.1%) were complicated with bleeding upon admission during the second half of pregnancy. the cases were attributed to placental abruption (63.5%; n=1276) and placenta previa (36.5%; n=734). independent risk factors associated with bleeding, were oligohydramnios, polyhydramnions, (odds ratio [or]= 1.6; 95% confidence interval [ci] 1.2-2.0; p=001 and 1.5;1.2-1.8;p<0.01 respectively ), suspected intra uterine growth restriction (iugr, 3.2; 2.6-4.0; p<.001), gestational age, previos abortions and maternal age. these patients subsequently were more likely to deliver by cesarean section (cs, 72.9% vs. 12.1%, or=19.5; 95%ci 17.6-19.5; p<0.001). perinatal mortality among patients admitted due to second half bleeding was significantly higher as compared to patients without bleeding (p<.001). conclusion: bleeding upon admission during the second half of pregnancy is an independent risk factor for perinatal mortality. careful surveillance, including fetal monitoring, is suggested in these cases in order to reduce the adverse perinatal outcome. crude and adjusted odds ratios for perinatal mortality among patients with vaginal bleeding. characteristics or 95% ci p crude or for perinatal mortality 7.9 6.6-9.4 <0.001 or adjusted for: <0.001 iugr 7.5 6.3-9.0 <0.001 oligohydramnios 7.6 6.3-9.1 <0.001 premature rupture of membranes 7.9 6.6-9.7 <0.001 the predictors included neonatal pi, bmi, or birthweight while the outcomes included hypoglycemia, shoulder dystocia, acidemia and hyperbilirubenemia. roc curve analyses were utilized to evaluate the relationship between sensitivity and specificity for each of the predictors and outcomes, with an area under the curve (auc) significantly greater than 0.5 indicating a screening test that is better than chance. results: neonatal pi, bmi and birthweight are poor predictors of nicu admission, acidemia and hypoglycemia with all auc values not statistically significantly different than chance alone. they are a reasonable predictor of shoulder dystocia, with birthweight functioning the best (p<0.001). in general, neonatal anthropometric measurements do not appear to be good predictors of short term neonatal outcomes. although they are a reasonable predictor of shoulder dystocia, they are not useful clinically for this outcome since they cannot be calculated until after birth. in future studies, new models of neonatal anthropometrics should be created to better predict adverse neonatal outcomes. neonatal anthropometric measurements and their relationship to perinatal outcomes expressed as auc and 95% confidence intervals history of at least one spontaneous preterm delivery were offered protocolbased prenatal care including first-or second trimester bacterial vaginosis screening, repeated ultrasound cervical length assessment, and supportive care by specialized nurses. women with a positive test for bacterial vaginosis were treated with oral metronidazol therapy, and women with a cervical length below 2.5 cm (before 25 weeks of gestation) underwent a vaginal cerclage. progesterone administration was not provided. data on obstetrical and medical history, pregnancy, delivery and neonatal outcome were prospectively collected and evaluated. results: in total, 104 pregnant women were prospectively followedup. eighty-seven women had experienced a preterm delivery in their last pregnancy, 52 of whom had delivered before 32 weeks. three women were diagnosed previously with a bicornual or septal uterus. twelve women received metronidazol therapy and 18 women underwent a vaginal cerclage. eighty-four women (80.8%) delivered at 37 weeks or beyond. one-hundred and three women (99.0%) delivered no earlier than 30 weeks, and only one woman delivered at 23 weeks. two women gave birth to a twin (at 30 and 35 weeks, respectively). two neonatal deaths due to pulmonary hypoplasia were recorded. conclusion: women at high risk of preterm delivery who were offered protocol-based prenatal care not including progesterone therapy had a better pregnancy outcome than would be expected from previous studies. our findings may question the reported beneficial effects of prenatal progesterone administration. premature rupture of membranes. tracy a manuck, alexandra g eller, m sean esplin, robert m silver. obstetrics gynecology, university of utah health sciences, salt lake city, ut, usa. objective: historically, outcomes following expectant management of midtrimester preterm premature rupture of membranes (pprom) have been uniformly poor. thus, many patients elected pregnancy termination. however, outcomes may be improved with recent advances in neonatal medicine. our purpose was to assess outcomes in expectantly managed early pprom in an era of improved maternal and neonatal care. study design: this is a retrospective cohort of patients from 2 tertiary healthcare systems from 2002-2007 experiencing pprom 24.0 weeks gestation. patients electing immediate termination of pregnancy, carrying fetus(es) with lethal anomalies, or delivering within 12 hours of pprom were excluded. survival without major morbidity was the primary outcome. data were analyzed using student t-test and chi-square as appropriate. results: a total of 92 women carrying 117 fetuses (73 singleton, 15 twin, 2 triplet, 2 quadruplet) met inclusion criteria. only the fetus in the "ruptured sac" was studied. pprom occurred at a mean of 20.7 (+/-2.6, range 13.6-24) weeks. the average latency period was 31.8 (+/-29.4, range 0.5-105) days, with a mean delivery gestational age of 25.2 (+/-4.3, range 15.4-34) weeks. this communication provides the first report of fundal uterine necrosis following placement of multiple uterine compression sutures. only two previous published cases report occurrence of uterine necrosis following application of a uterine compression suture --both identified as the b-lynch technique --and neither of these cases report necrosis confined to the fundus. in our case, uterine atony was refractory to pharmacologic therapy and manual compression of the uterus appeared to decrease the amount of blood loss. therefore, we applied a traditional b-lynch which effectively contracted the majority of the uterus, while the fundus remained atonic. a second horizontal, square suture was then placed between the cornua and carried over the fundus (see figure one: red reflects b-lynch suture; blue represents second fundal, square stitch). the patient subsequently experienced significant, persistent fevers despite antibiotic therapy. a repeat laparotomy was performed, at which time we found uterine necrosis confined to the uterine fundus (see figure 2 : photograph taken at the time of hysterectomy). the placenta showed no histologic evidence of chorioamniotis --hence, the patient's main risk factor for fundal necrosis was the compression sutures. physicians should be aware of the risk of uterine necrosis, especially after placement of multiple compression sutures and, more specifically, after placement of a compression suture confined to the uterine fundus. background: migraines are far more common in women than in men. the incidence of migraines increases in girls after puberty, reaching an incidence of 25% in women who experience migraine at least once a year around middle age. migraine has been postulated as one of the major risk factors for stroke during pregnancy and the puerperium.triptans are a class of serotonin receptor agonists used in the treatment of migraine headaches. triptans administered in combination with other drugs have been known to precipitate serotonin syndrome, a rare but potentially life-threatening condition clinically manifested by a triad consisting of mental-status changes, autonomic hyperactivity, and neuromuscular abnormalities. objective: to determine whether triptan monotherapy is associated with serotonin syndrome methods: using data mining techniques we analyzed the entire food and drug administration's (fda) adverse event reporting system (aers) database. results: after excluding reports of concomitant serotonergic medication or other potentially confounding medication, eleven reports remained of serotonin syndrome associated with triptan use without concomitant serotonergic medication. the mean age for these eleven cases was 39.9 years; nine cases occurred in females and two occurred in males. there were no apparent instances of overdose among these eleven cases. conclusions: serotonin syndrome is a rare but serious occurrence with the use of triptan monotherapy. such cases were seen with eletriptan, rizatriptan, sumatriptan, and zolmitriptan. because of the spontaneous nature of voluntary reporting to aers, the actual number of occurrences of serotonin syndrome in patients using triptans is probably higher and cannot be assessed from aers data. users of triptan in combination with an ssri or snri should be warned of this rare but serious adverse effect. during periods of drug initiation, dose escalation, or the addition of another serotonergic agent, patients should be particularly vigilant for symptoms of concern and seek urgent medical attention if any occur. the offending agents should be withdrawn and the patients closely monitored and treated with supportive measures as required. rising maternal mortality in the midst of modern technology. oormila p kovilam, 1 jane khoury, 2 padmini c sekar, 3 ralph c buncher. 3 1 obstetrics gynecology, university of cincinnati, cincinnati, oh, usa; 2 center for epidemiology and biostatistics, cincinnati children's hospital medical center, cincinnati, oh, usa; 3 environmental health, university of cincinnatic, cincinnati, oh, usa. introduction: maternal mortality rate (mmr) is an index of overall wellbeing of the community and safe motherhood should be given utmost priority in obstetric care. as per 1990, who estimates 1% of maternal mortality occurs in developed countries 1 . this rate has established without much decline in recent years. the mmr for ohio for 1987 to 1996 was reported by cdc to be 6.3 per 100,000 live births with 95% confidence interval 5.1 to 7.6. objective: our objective was to audit the trend in maternal mortality in the state of ohio for the last 20 years. methods: information from the death certificates completed by attending physicians, medical examiners, coroners, funeral directors filed with ohio state registration offices were analyzed. we only used women who were residents of the state of ohio at the time of death, and cause of death was coded as a "complication of pregnancy, childbirth and the puerperium", icd9 codes 630 to 677 and icd10 codes o0 to o9. five year maternal mortality pattern and long term trend was assessed. results: the number of maternal deaths recorded as due to pregnancy complications varied over the years from a high of 20 in 1981 to a low of 4 in 1996 and 1997. in general the five-year mortality rate was decreasing over time until the last four years 2000 to 2003, when we may be seeing an upward trend compared to 1995 to 1999 (p=0.07). the rates and associated 95% confidence intervals (ci) are shown in the table below. years mmr 95% ci 1980 ci -1984 ci 7.9 4.6-11.1 1985 ci -1989 6.0 3. 2-8.8 1990-1994 5.8 3.0-8.6 1995-1999 4.5 1.6-7.4 2000-2003 8.4 3.3-13.5 the rate of maternal mortality is on the rise after a period of downward trend. we should develop a multidisciplinary approach to analyze and target the root causes of maternal death. introduction. women with thrombophilia are at higher risk of vte during pregnancy due to the acquired hypercoagulable state. it is likely that not only maternal veins but also placental vessels are more prone to the development of thrombosis. our objective was to compare maternal and fetal placental circulation disorders in women with intra-uterine fetal death (iufd) and thrombophilia and women without thrombophilia. methods. in a dutch multi-centre study on iufd, during the period 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. factor v leiden and prothrombin g20210a were tested at induction of labour. panel classification of cause according to the tulip classification 1 was performed by assessors after individual investigation of structured patient information. we studied the cause of death group "maternal and fetal placental circulation disorders": placenta bed pathology with abruption or infarction as origin of mechanism and placental parenchyma pathology with fetal thrombotic vasculopathy and massive perivillous fibrin deposition as origin of mechanism. results. of the 689 women tested for factor v leiden, 46 (6.7%) were carriers. of the 261 deaths caused by "maternal and fetal placental circulation disorders" 18 (6.9%) mothers were carriers of factor v leiden. of the 428 deaths with another cause of death 28 (6.5%) mothers were carriers of factor v leiden (p=0.88). of the 691 women tested for prothrombin g20210a, 21 (3.0%) were carriers. of the 264 deaths caused by "maternal and fetal placental circulation disorders" 9 (3.5%) mothers were carriers of prothrombin g20210a mutation. of the 427 deaths with another cause of death 12 (2.8%) mothers were carrier of prothrombin g20210a mutation (p=0.65). in women with iufd and factor v leiden or prothrombin g20210a mutation "maternal and fetal placental circulation disorders" did not seem to cause iufd more often than in non-carriers. , university medical center groningen, groningen, netherlands; 2 pathology, university medical center groningen, groningen, netherlands; 3 trial coordinating center, dept of epidemiology, university medical center groningen, groningen, netherlands; 4 hematology, university medical center groningen, groningen, netherlands. introduction. growing evidence suggests that women with thrombophilic defects may be at higher risk of fetal loss. although some paternal components to the predisposition of preeclampsia have been demonstrated it is not known whether paternal components contribute to intra-uterine fetal death (iufd). our objective was to investigate the relation between paternal thrombophilic defects and iufd. methods. in a dutch multi-centre study on iufd, from 2002-2007 we studied 750 singleton deaths > 20 weeks of gestation for which the diagnosis of iufd was determined before labour. we tested male partners of women with iufd for antithrombin (at), protein c, protein s type i and iii, factor v leiden, prothrombin g20210a (factor ii) and factor viii: ag. standard tests were performed in one laboratory. normal ranges were determined in healthy male blood donors. we compared prevalence of thrombophilic defects to reference values from the literature. . of the 642 men tested for factor v leiden, 26 (4.0%) were carrier versus 618 (96.0%) non-carriers. prevalence of factor v leiden in the normal population is 5% (p=0.27). 642 men were tested for prothrombin g20210a, 6 (0.9%) were carriers and 636 (99.1%) non-carriers. all were heterozygous. prevalence of prothrombin g20210a mutation in the normal population is 3% (p=0.002). decreased levels of antithrombin, protein c, protein s type i and iii and increased levels of factor viii: ag were observed significantly more often in male partners of women with iufd compared to the normal population. conclusion. in our iufd group the prevalence of male factor v leiden carriers was comparable to the normal population, prevalence of prothrombin g20210a mutation was lower. the difference in other factors imply an as yet unexplained association of male thrombophilia with fetal death in their partner. objective fetal macrosomia, the most important complication of gdm, increases the risk of shoulder dystocia, erb's palsy and intrauterine hypoxia. we compared frequencies of fetal macrosomia and erb's palsy in two gdm cohorts with different severity of glycemic disturbance and in healthy controls. methods we studied 898 consecutive gdm women with singleton childbirth and 2 or 3 abnormal values in 2-h ogtt with 75 g of glucose. abnormal plasma glucose values of the ogtt were: fasting 5.1, 1-h 10.0 and 2-h 8.7 mmol/l. insulin treatment was started when 2 values were 5.5 preprandially or 7.8 mmol/l postprandially in the 24-h glucose profile done within 7 days of diagnosis. if the same woman had more than 1 childbirth during the study period, only the last pregnancy was included. the control group consisted of 798 women from a nearby town with singleton childbirth in the same hospital. women with diabetes were excluded from controls. macrosomia was defined as birth weight (bw) >2.0 sd above the mean of a standard population. erb's palsy was diagnosed by pediatric surgeons. results gdm women were older, more obese, had more childbirths and more often a previous child with bw >4000g than controls. insulin was started in 367 gdm women (41.9%). c/s rate was 18.7% in controls, 27.0% in diettreated and 42.0% in insulin-treated gdm women. macrosomia rate was 2.3% in controls, 4.6% in diet-treated and 18.4% in insulin-treated gdm women (p<0.001 compared with controls or diet-treated gdm women). the frequency of macrosomia did not differ between the diet-treated gdm and control women. erb's palsy occurred in 0.3% of controls, in 1.7% of diet-treated and in 1.6% of insulin-treated gdm women. the frequency of erb's palsy was significantly higher in both gdm groups than in controls (p=0.013). by regression analysis, previous child with bw >4000g (p<0.0001), previous c/s (p=0.0006), mother's age (p=0.012), bmi (p=0.015), insulin treatment (p=0.02) and fasting plasma glucose of the 24-h profile (p=0.027) were significant independent predictors of fetal macrosomia. conclusions the 24-h glucose profile done shortly after the diagnosis of gdm clearly distinguishes between low-risk (diet-treated) and high-risk (insulintreated) gdm pregnancies for fetal macrosomia. unfavourable fetal body composition of diet-treated gdm women is the likely explanation for the high rate of erb's palsy in this group. 0.264 1'-:·:tpartq,-, 1'-:·:tpartq,-, 1'-:·:tpartq,-, 25% p=0.001). first trimester uta doppler indices were similar in the two cohorts in terms of the resistance and pulsatility indices (ri 0.63 vs. 0.67, p=0.39; pi 1.29 vs. 1.44, p=0.42) . however, bilateral notching was much more common in the cohort of prior adverse outcomes (22% vs. 45% p=0.05) as well as in patients destined to have a subsequent poor outcome (p=0.001). conclusions: not surprisingly, prior poor obstetric outcome was strongly associated with recurrent adverse obstetric outcome. uta notching was robustly associated with prior poor obstetric history as well as a recurrent poor outcome. this clinical history had no discernable influence on ri or pi. reassurance may not be offered based on first trimester uta ri and pi. anti-retroviral therapy is associated with increased blood loss and uterine atony for patients undergoing primary cesarean section. carey eppes, alice cootauco, melissa russo, jessica bienstock. gynecology and obstetrics, johns hopkins university school of medicine, baltimore, md, usa. objective: anti-retroviral therapy has been associated with gastrointestinal smooth muscle dysfunction. it has also been hypothesized that myopathies can occur secondary to anti-retroviral therapy due to mitchondrial alterations. in our experience, we have noticed an increased incidence of uterine atony in our hiv patients on anti-retroviral therapy. we sought to examine the incidence of postpartum hemorrhage and uterine atony in patients currently on anti-retroviral therapy. study design: a retrospective case-controlled study was conducted on all hiv positive pregnant women on anti-retroviral therapy undergoing a primary low segment transverse cesarean section from 1996 through 2007. these patients were obtained from an irb approved database containing hiv positive patients within our institution. patients' medical records were abstracted for demographic data, use of uterotonics, preoperative and postoperative hematocrits, and incidence of blood transfusions. controls were matched for age, parity, gestational age and surgical indication. data was analyzed using the t-test, fischer's exact test and chi square. results: there were no differences in demographics, incidence of chorioamnionitis or magnesium sulfate use between groups. patients on anti-retroviral therapy had a statistically greater decrease in hematocrit and estimated blood loss compared to controls. they also had an increased need for uterotonics and blood transfusions. conclusion: anti-retroviral therapy may impact uterine smooth muscle contractility in pregnancy, as evident by the increased incidence of uterine atony and change in hematocrit. additional research is needed to elucidate the mechanism. clinicians should be aware of the potential for uterine atony and excessive blood loss in patients on anti-retroviral therapy. background: adolescent pregnancy is frequently associated with adverse outcomes, especially small-for-gestational age (sga) deliveries. some studies have implicated maternal nutritional status in these poor outcomes. methods: in a prospective longitudinal study, 500 ethnically-diverse, pregnant adolescents were studied from booking to parturition, with collection of anthropometric and nutritional variables. blood samples (28-32 weeks' gestation; n=305 subjects) were assayed for a spectrum of nutritional biomarkers. logistic regression was used to determine significant associations between studied variables and pregnancy outcomes. results: median age at recruitment was 17.8 years (iqr:17.1-18.4). outcome data was available for 478 subjects. 214 (44.8%) had uncomplicated pregnancies. median birthweight was 3,200g (iqr: 2,831-3,530g) and median birthweight centile was 37.8% (iqr: 14.3-64.2%). 84 (17.6%) infants were born sga and 43 (9.0%) were preterm. spontaneous vaginal deliveries occurred in 71.0% of cases. there were 14 (2.9%) cases of pre-eclampsia and 34 (7.1%) admissions to neonatal care. 31.9% of subjects reported smoking at booking. iron deficiency anaemia was prevalent in 49.4% of subjects by 28-32 weeks and was strongly associated with higher infant birthweight centile (p<0.0001). 48.4% had serum 25-hydroxy vitamin d concentrations <15ng/ml, although this was not associated with any outcomes. low folate status, as indicated by low red cell folate (p=0.001), low serum folate (p=0.032) and high serum homocysteine (p=0.027) concentrations, was associated with higher rates of sga birth. conclusion: adolescent pregnancy in inner-city populations is associated with a high risk of sga and preterm birth, increasing the likelihood of health problems in later life and perpetuating social disparities in health. the association between anaemia and higher birthweight is unexplained. impaired maternal folate status may contribute to impaired fetal growth. we suggest that sga birth in this population could be reduced by the use of antenatal supplements or the mandatory fortification of flour with folic acid. amount of cigarette use pre-pregnancy and during pregnancy were self-reported and subgrouped into nonsmokers, quitting during pregnancy, and continued smoking throughout pregnancy. categorical outcomes were compared using chi-square test. multivariable logistic regression analyses were used to control for potential confounders (continued smoking compared to quitting during pregnancy). results: women who quit smoking during pregnancy had higher rates of pregnancy-associated hypertension and cesarean delivery but lower rates of preterm delivery and neonatal birthweight <2500gm compared to non-smokers. compared to women who quit smoking during pregnancy, those who continue to smoke have lower odds of pregnancy-associated hypertension and cesarean delivery, but higher odds of preterm delivery, neonatal birthweight <2500gm, and apgar score <7 at 5 minutes (see table) conclusion: quitting cigarette smoking during pregnancy appears to reduce undesirable neonatal outcomes, though an increase in pregnancy associated hypertension. these findings should be emphasized to women who are smoking during pregnancy. to achieve effective tamponade the balloon was inflated initially up to 150-200ml with incremental increases of volume by 50 ml until bleeding stops. to measure intrauterine balloon pressures we used a standard pressure transducer (edwards lifesciences) connected to the balloon port and to a pressure monitor (ge dash 3000). the pressure transducer was mounted on the bedrail at uterine level, primed with sterile saline and then connected to the balloon port of the catheter. the system was zeroed to atmospheric pressure, the stopcock of the intrauterine balloon port was opened and the pressure was recorded (p1: the total pressure on the fluid). to verify the accuracy of measurements we used another balloon catheter (reference), inflated with the same amount of saline, connected to the pressure transducer and zeroed to atmospheric pressure. the reference balloon measured p2 (the intrinsic balloon pressure caused by distension at that volume). the system was then zeroed to the reference balloon. the pressure transducer was then reconnected with the intrauterine balloon and the actual intrauterine pressure was recorded (p3). we calculated the correlation coefficient between p3 and systolic, diastolic and mean blood pressure using a linear regression (statsdirect statistical software there is a growing body of evidence to suggest that peripartum assessment of fetal or neonatal lactate levels are as good as or better than standard blood gas analysis in the prediction of neonatal outcome. in this study we have evaluated the ability of umbilical cord blood gases and lactate levels in the prediction of neonatal hypoxic-ischaemic encephalopathy (hie). department of neonatal paediatrics, king edward memorial hospital, perth, western australia, australia; 3 women and infants research foundation, king edward memorial hospital, perth, western australia, australia. objective: current evidence suggests that umbilical cord ph at delivery provides the most sensitive reflection of birth asphyxia. paired umbilical artery/vein blood gases have been routinely collected at king edward memorial hospital over the last 5 years. the objective of this study was to determine: local reference ranges; accuracy of sampling; and the rates of metabolic acidosis. of the 19,426 births (2003) (2004) (2005) (2006) , accurate paired results were available on 64% of births. over the study period there was a progressive improvements in accuracy rates of paired sampling (p<0.05). the median (2.5 th , 97.5 th centile) values for cord arterial blood gases were: ph 7.27 (7.08,7.37); po2 16.3mmhg (4.6,31.0); pco2 55.1mmhg (39.0,79.0); base excess -3.0 (-10.8, 2.7); and lactate 3.7mmol/l (1.8,7.8). there was a progressive improvement in all blood gas measures over the 4 years of this study (all p<0.05). moreover, there were significant reductions in all measures of metabolic acidosis (see table) . the progressive improvement in the measures of metabolic acidosis remained significant after multivariate analysis including obstetric, fetal, and demographic factors associated with metabolic acidosis. the introduction of universal umbilical cord blood gas analysis to all births is associated with significant improvements in all markers of metabolic acidosis. together with other compelling evidence in the literature, these data support the routine use of cord arterial and venous gases at all births; however, improved accuracy rates on paired sampling requires an ongoing education program umbilical artery indicators of acidosis, percentage of total ph < 7.0 ph < 5th centile* objective: intrapartum pcn prophylaxis aims to prevent early-onset gbs sepsis by interrupting vertical transmission from colonized mothers to their newborns. however, despite its wide clinical use, systematic pharmacokinetic evidence in support of the current pcn dosage regimen is lacking. current cdc guidelines recommend intensified surveillance and testing of infants exposed to <4h of prophylaxis. our goal was to examine the relationship between maternal time of exposure to pcn and fetal serum pcn levels among maternal-fetal dyads exposed to short durations of pcn prophylaxis (<4h) compared to those exposed to longer durations. study design: ninety-eight laboring gbs positive women were administered 5 million units (mu) of intravenous pcn to be followed by 2.5 mu every 4 hours until delivery (cdc 2002) . subjects with renal disease, multiple gestation, and preterm delivery (<37wks) were excluded. umbilical cord blood samples were collected at delivery and pcn levels measured by high-performance liquid chromatography. intra and inter-assay coefficients of variation were <3%. results: the pcn concentrations (mean±sd) by duration of prophylaxis were: <1 h, 11.6±4.5 g/ml (n=10); 1-2h, 9.7±3.4 g/ml (n=15); 2-3h, 6.6±3.8 g/ ml (n=15);3-4h, 3.6±1.8 g/ml (n=17);4-8h, 6.9±3.7 g/ml (n=11);>8h 4.1±2.6 g/ml (n=24); and for those without a second dose after 4h, 2.3±0.9 g/ml (n=6). fetuses exposed to short duration (<4h) had higher levels of pcn than those exposed to >4 h (p=0.003). in multivariable linear regression analysis, fetal pcn levels were determined by total duration of exposure, time since last dose, dosage, and number of doses, but not maternal bmi. pcn levels in cord serum increased linearly until 1 hour; thereafter, they decreased rapidly, but all groups were significantly above the minimum inhibitory concentration (mic) for gbs (0.1 g/ml)(p<0.002). furthermore, every sample individually remained 10-179 fold above the mic. conclusion: in this study, even short durations of prophylaxis achieved levels above the mic, suggesting a benefit to prophylaxis even in precipitous labors. the data also suggests that the current cdc designation of infants exposed to <4h of pcn prophylaxis as particularly at risk for gbs sepsis may be inaccurate from a pharmacokinetic standpoint. objective: 3-oh aa is a metabolite of tryptophan with pro-oxidant and proapoptotic properties and has been shown to increase in umbilical cord blood in pregnancies with intra-uterine infection. since labour-related events may also activate inflammatory pathways, we sought to determine the placental release of 3-oh aa into the umbilical circulation in labouring vs non-labouring patients at term. methods: twenty-six patients were studied (term labour n=18, and term elective cesarean section n=8) with blood sampling from a clamped segment of umbilical cord after delivery of the fetus and from the cord at its insertion into the placenta after delivery of the placenta, with subsequent measurement of blood gases/ph and 3-oh aa (isocratic hplc using fluorometric detection with assay sensitivity at 1 pmol). results: 3-oh aa measurements from respective umbilical and placental cord vessels were all variably higher in the labouring group vs the elective cesarean group patients (table 1) . for labouring group patients, the 3-oh aa levels from the umbilical vein were significantly higher than those from the umbilical artery, indicating net release from the placenta into the fetal circulation. placental vein levels were also significantly higher than those from the umbilical vein, indicating continued placental release of 3-oh aa into the cord blood after delivery of the fetus. conclusion: labour at term is associated with changes in the placental metabolism of tryptophan resulting in the increased release of 3-oh aa into the fetal circulation with the potential for pro-oxidative and apoptotic effects in many tissues, including the brain. obstetrics, gynecology, and reproductive sciences, new brunswick, nj, usa; 2 department of obstetrics and gynecology, mineola, ny, usa. objective: ischemic placental disease (preeclampsia, small for gestational age, sga and placental abruption) is a major contributor to pregnancy-related morbidity. although the placenta is considered a fetal organ, it is accepted that ischemic placental disease (ipd) can present clinically with either fetal or maternal manifestations. we hypothesized that the pattern of diagnosis (maternal versus fetal) varies by gestational age and would provide insights in to origins of indicated and spontaneous preterm birth. methods: this was a retrospective cohort study utilizing the maternallylinked reproductive history data for missouri residents , restricted to singleton live births. women who experienced spontaneous onset of labor and subsequently delivered preterm were classified as spontaneous preterm birth. medically indicated preterm birth included women who delivered preterm through a labor induction or (prelabor) cesarean delivery. ipd was classified as maternal (preeclampsia only), fetal (sga only) or both (preeclampsia with sga or abruption, and all 3 conditions). results: among term births with ipd, 22.6% presented as maternal disease only, 69.5% as fetal disease, and the remainder (7.4%) as both. among spontaneous preterm births with ipd, a greater proportion were of fetal presentation ( bethesda, md, usa; 2 dept ob/gyn, wayne state univ, detroit, mi, usa; 3 dept pathology, wayne state univ, detroit, mi, usa; 4 dept ob/gyn, soroka univ medical center, israel. objective: hemoglobin (hg) and its catabolic products have been observed in cases of amniotic fluid (af) discoloration, which is a risk factor for intraamniotic infection/inflammation (iai). the study aimed to determine the association between af fetal hg concentration and gestational age, term and preterm labor and iai. study design: this cross-sectional study included: 1) mid-trimester (n=60); 2) term not in labor (tnl) (n=21); 3) term in labor (tin) (n=47); 4) preterm labor (ptl) who delivered at term (n=89); 5) ptl without iai (n=74); 6) ptl with iai (n=78); 7) preterm prelabor rupture of membranes (pprom) with (n=48) and without iai (n=48). af fetal hg concentrations were determined by elisa. results: 1) fetal hg was detected in 80.4% of all af and 31.7% of mid-trimester samples; 2) women at tnl had a higher median af fetal hg concentration than patients at mid-trimester (19.5 ng/ml, iqr 0-49 vs 0.0 ng/ml, iqr 0.0-19.9, p=0.008); 3) no differences were found in median af fetal hg concentration among patients with and without labor at term (til: 19.1 ng/ml, iqr 0-36.2; p=0.4); 4) median af fetal hg concentration was not significantly different among the 3 ptl subgroups [ptl with iai: 114.6 ng/ml, ptl who delivered preterm: 109.6 ng/ml, ptl without iai who delivered at term: 134.21 ng/ml, p=0.71(kruskal wallis) ]; 5) in pprom, there were no differences among patients with and without iai (148.4 ng/ml, respectively; p=0.074) ; 6) median af fetal hg concentrations were significantly higher in ptl or pprom, with or without iai than in pregnant women at term, with and without labor (p<0.001 for all comparisons). conclusions: 1) immunoreactive af fetal hg increases with gestational age; 2) among women with ptl or pprom, the median af fetal hg concentration is not associated with iai; 3) the median af fetal hg concentration is higher in pregnancies complicated with ptl or pprom than in term pregnancies. the impact of latency time to delivery after preterm premature rupture of membranes (pprom) on neonatal outcome. dan nayot, 1 deborah penava, 1 barbra de vrijer, 1 orlando da silva, 2 bryan s richardson. 1 1 obstetrics and gynaecology; 2 pediatrics, university of western ontario, london, on, canada. objective: there continues to be controversy as to the management of pprom with conservative management to advance gestational age (ga) versus aggressive management with early induction to avoid chorioamnionitis. we have therefore used the perinatal and neonatal databases of a large regional patient population to determine the association of pregnancy variables with latency time to delivery after pprom and the impact of latency duration on adverse neonatal outcomes. methods: the perinatal/neonatal database of st. joseph's health care, london, ontario was used to obtain demographic and neonatal outcome information for all patients with pprom >25 and <37 weeks gestation, singleton and no major anomalies, delivering between january 1, 1996 and december 31, 2005. patients were grouped according to ga at pprom stratified for latency time <72 hrs vs >72 hrs with incidences for those pregnancy related variables and neonatal outcomes available from the database then compared with the use of logistic regression analysis. results: there were 1535 patients who met the inclusion criteria of whom 10%, 19%, and 71% had pprom at 25-28 wks , at 29-32 wks, or at 33-36 wks, respectively, and with these pprom groupings showing a stepwise decrease in the percentage of patients with latency to delivery >72 hrs, at 67%, 42%, and 10%, respectively. pregnancy related variables and neonatal outcomes for these patient groupings are as shown in table 1 . conclusion: despite a 2 to 3 fold increase in the incidence of chorioamnionitis with latency to delivery >72 hrs, a policy of conservative management to advance ga after pprom will result in decreased severe infant morbidity until 32 weeks, and moderate infant morbidity until 36 weeks. hospital. outcomes studied were prolongation of latency period with intact membranes and prom using magnesium sulfate (mgso 4 ), nifedipine, terbutaline and 2 tocolytic agents. secondary outcomes examined were maternal complications. statistical analysis performed were t-test and 2 . comparisons were expressed as odds ratios (95% ci). results: in a 9-year period, 183 twin gestations were admitted in ptl and administered 1 tocolytic agent(s) with mean gestational age of 28.8 weeks. when 2 tocolytic agents were used, maternal complications were: 7 (9.5%) with intact membranes and 1 (2.8%) with prom had pulmonary edema (or=2.67, ci 0.58-12.29); 11 (14.1%) with intact membranes and 2 (5.6%) with prom had chorioamnionitis (or=1.3, ci 0.41-4.15); 2 (2.6%) with intact membranes and 2 (5.6%) with prom had postpartum hemorrhage (or=0.43, ci 0.05-3.62). conclusion: there is no difference in prolongation of latency period achieved in twin gestations in ptl with intact membranes or prom using 2 tocolytic agents. similarly, there is no difference between the 2 groups with latency period 48 hrs. there is an increased likelihood of pulmonary edema and chorioamnionitis with use of 2 tocolytic agents. however, results are not significant due to type ii error. mean latency period and latency ≥48 hrs using single or multiple tocolytic agent ( introduction: high sensitivity crp (hscrp) is a serum marker of inflammation and has proven clinical utlility in predicting cardiovascular disease (cvd). considering the hypothesized association between preeclampsia (pre) and inflammation and cvd, it is plausible that hscrp may have utility in predicting pre. prior to widespread utilization of this marker, the affect of labor on levels of crp needs to be clarified. we assessed the association between labor and hscrp levels in term deliveries and between elevated hscrp and adverse perinatal outcomes. a secondary analysis comparing hscrp in women with preeclampsia (pre) to those without was performed. methods: women presenting for term delivery or pre were prospectively identified as part of a case-control study. clinical data and serum were collected for all subjects. a standard immunoturbidimetric assay was used to measure hscrp levels. women presenting for induction of labor or planned cesarean delivery (non-labor) were compared to women presenting in labor. a secondary analysis comparing non labor women with and without pre was performed. serum was collected prior to labor induction in the non-labor group. nonparametric comparisons were made using wilcoxon rank sum tests. mvlr was used to evaluate dichotomous outcomes and control for confounders. results: 344 women were included (non-labor group (n=146), labor group (n=120), non-labor pre (n=78)). the median and mean hscrp levels were 6 and 9 and 15 and 21 in the non-labor and labor groups respectively (p=0.005). elevated levels of hscrp in these term deliveries were not associated with chorioamnionitis (p=0.3), maternal postpartum complications (endometritis, hemorrhage, transfusion) (p=0.67), mode of delivery (p=0.3), or admission to the nicu (0.96). levels of hscrp were significantly greater in the non-labor pre group compared to non labor without pre (mean 34.5 vs.20.1, median 27 vs.6, p<0.001). conclusion: use of hscrp as a biomarker may improve clinical prediction of obstetrical complications such as pre. levels of hscrp are affected by labor and this should be taken into account when studying the utility of this biomarker. further, crp levels are elevated in women with pre even after excluding patients in labor. further investigations to determine if crp elevation in term labor is associated with adverse outcomes may be warranted. the role of hscrp as a valid and discriminating biomarker in pre should be assessed. review of the electronic labor record facilitated collection of maternal demographic, intrapartum, and newborn data. data analyzed with t-tests, chi-square tests, and calculated odds ratios with 95% cis as appropriate. a forward stepwise logistic regression analysis was used to identify predictors of histologic chorioamnionitis. results: of 351 submitted placentas, 210 had histologic chorioamnionitis (cases) and 141 did not (controls). the groups were similar with respect to age, race, gbs status, and mode of delivery. gestational age, birthweight, duration of labor and ruptured membranes, and number of vaginal exams were greater in the cases (p .01). the cases were more likely to have had epidural anesthesia (or 2.9), internal monitoring (or 2.2), fever (or 4.8), maternal tachycardia (or 1.9) and fetal tachycardia (or 3.8) and less likely to have had induction of labor (or 0.49). newborns in the histologic chorioamnionitis group were more likely to have been observed for sepsis (or 3.8 severe sepsis defined as sepsis associated with acute respiratory distress syndrome (ards) or cardiovascular dysfunction(cvd) or with 2 or more other organ dysfunction.all patients were resuscitated with fluids and treated with broad spectrum antibiotics and supportive care as needed. outcome data were: etiology, management, maternal complications, duration of icu stay and perinatal survival. results patients were young (mean age=23.6± 7.6 years) with a mean gestational age at delivery 29.5±8.9 weeks. etiologies were pyelonephritis(n=8), septic abortion(n=4), endomyometritis (n=3), chorioamnionitis (n=3), ruptured appendix(n=3), pneumonia( n=1) and one unknown. eighteen (78%) were diagnosed during antepartum and 5 (22%) postpartum period.there were 2(9%) maternal deaths and high rate of major morbidities (table) . among the 18 antepartum patients,there were,4 abortions,4 iufd,2 neonatal death for a perinatal survival rate of only 39%. conclusion pregnancies complicated with severe sepsis/septic shock are associated with substantial maternal and perinatal morbidities. the low maternal mortality rate in our study as compared to previous reports is attributed to early diagnosis and aggressive management of maternal complications. fetal loss rate, however continues to be high when septic shock develops antepartum. and tnfa levels are elevated. we developed a dynamic computer (in silico) model of pregnancy (uterine myometrial environment -infection/inflammation and endocrine crosstalk). mathematical differential equations were used to describe the interactions between molecules. infection was represented as increased levels of activated, nuclear transcription factor nf-kb. in the model ru486 inhibited both the glucocorticoid receptor and progesterone receptors. simulations were run adding different concentrations of ru486 (1.5 um= dose used in patients, 0.5 um, 0.05um) at different time points during infection (before, at the time of or after nf-kb activation). ru486 degradation kinetics was also included. the effect of ru486 on nf-kb induced il-6 and tnfa levels was assessed. results: infection induced nf-kb activation led to increased il-6 and tnfa levels. there was a subsequent increase in cortisol that led to dampening of nf-kb activation, il-6 and tnfa levels. in the presence of ru486, il-6 and tnfa levels continued to rise. the effect of ru486 on nf-kb induced il-6 and tnfa was dose dependent and was more prominent in slow metabolizers who had ru486 in the system for a longer time. addition of ru486 after the onset of nf-kb activation led to increased il-6 and tnfa levels above those observed without ru486. the addition of misoprostol (prostaglandin e1 analogue), at the concentrations used together with ru486 for medical abortion, did not add to the effect of ru486 on nf-kb induced il-6 or tnf. conclusions: ru486 has dose dependent effects on infection induced immune activation and may contribute to the pathogenesis of c sordelii induced sepsis syndrome. the increased susceptibility of neonates to infection remains a major clinical problem. we previously found that after intraperitoneal (ip) listeria monocytogenes infection, neonatal mice have an ld 50 that is 5 orders of magnitude lower than adults. we also found that the inflammatory response of neonatal mouse macrophages, but not neutrophils, in the peritoneal fluid was deficient, and that this correlated with low levels of macrophage chemokines mcp-1 and rantes. given that the liver and spleen are important organs in listeria infection, we sought to characterize the innate immune response in these organs. a sublethal dose of listeria was injected intraperitoneally into balb/c 4-6 week old adult mice and 3-5 day old neonatal mice. liver and spleen was collected at 0, 24, 48 and 72 hours, sectioned serially for staining with hematoxylin-eosin and primary antibodies (rabbit anti-listeria monocytogenes igg; mhc class ii rat anti-mouse igg, a marker for activated macrophages; f4/80 rat antimouse igg, a marker for macrophages) and secondary antibodies (cy-3 goat anti-rabbit igg; af488 goat anti-rat igg) were applied. negative controls used only secondary antibody. real time pcr was used to compare the levels of the chemokines mcp-1 and rantes in adult and neonatal liver. after ip infection, both adults and neonates showed similar influx of neutrophils to the sites of infection within the liver and spleen. at 24 hours post-infection with listeria, adult liver and spleen showed increased staining for f4/80 and class ii, which increased further and became confluent surrounding microabscesses at 48 and 72 hours. in contrast, the listeria infected neonatal mouse showed some increase in f4/80 around microabscesses but no apparent increase in staining for mhc class ii. the neonates showed greater staining for listeria at each time point. mcp-1 and rantes levels were higher in infected neonatal liver compared to adults. in the neonatal mouse, the innate immune response in the liver and spleen was characterized by a deficiency of activated macrophages. this deficiency was not correlated with hepatic expression of 2 macrophage chemokines. is there a seasonal pattern in the incidence of post-cesarean endometritis? tamula m patterson, alan tn tita, william w andrews. obstetrics and gynecology, the university of alabama at birmingham, birmingham, al, usa. objective: several theories, including one suggesting a peak in july coincident with resident turnover, postulate seasonality in post-cesarean infections. we assessed whether there is seasonal variation in endometritis. a retrospective cohort study of post-cesarean endometritis at our university-based institution using our obstetric computerized database to compare annual variation in monthly incidence patterns from 1992 to 2006. prior to establishing an average aggregate seasonal pattern for all years, years were assessed separately for a recurrent pattern of peaks and nadirs in incidence. peak incidence (or nadir) was defined as any monthly incidence that differed from the mean incidence for the year by over 25%. results: a total of 22.4% (10,966) of 48,913 deliveries from 1992 to 2006 were by cesarean. annual cesarean rates increased by an absolute rate of over 10%; while, post-cesarean endometritis rates decreased from 23% to 2%. monthly incidence of post-cesarean endometritis did not reveal a consistent recurrent pattern of peaks (figure1) or nadirs. the month of july accounted for only 4 out of a total of 40 peaks for all years. the adjacent months of june and august accounted for only 3 each. the month with the highest number of peaks was april with only 7. these findings contraindicated the establishment of an average aggregate monthly pattern for all years. the incidence of post-cesarean endometritis did not follow a seasonal pattern. chi-square analyses were used to compare associations between race (black (bl) vs non-black (nbl)) and dichotomous characteristics. student´s t-test was used to compare continuous variables. results 1,030 patients were evaluated (439 cases and 591 controls). 85% and 15% of cases and 74% and 26% of the controls were bl and nbl respectively. the baseline prevalence of chtn in bl and nbl controls was 6.15% and 2.65% (p=0.09). when comparing bl and nbl cases, bl women had a higher mean systolic blood pressure and screening bmi. bl women also had a trend toward being discharged on post partum blood pressure medicine when compared to nbl women. there was no difference in chtn, diabetes, severity of disease, iugr, or delivery <34 wks between the two groups (table) . to determine the leading causes of death in a case series of stillborn infants examined in a large hospital autopsy service, and to describe the most common post-mortem observations. study design: one hundred and sixty one stillborn infants were examined. gross pathology observations were recorded at autopsy and during the placental exam, and tissue sections were collected and examined microscopically. immediate and underlying cause of death (cod) were recorded, along with contributory cod, concomitant/significant cod, and incidental findings. statistical analysis was conducted using the software spss v.11.5. results: the immediate anatomic cod could be determined in 33.5% of all infants examined. in over 50% of these cases, cod was attributable to placental or umbilical cord findings affecting the maternal-fetal blood supply. the most prevalent among these findings were placental lesions (maternal floor infarction, placental abruption, fetal thrombotic vasculopathy), umbilical cord lesions (entanglement, true knot, compression, excessive length/twisting), and infectious/inflammatory processes (chorioamnionitis, chronic villitis objective: advanced maternal age (ama) is associated with increased risk of intrauterine fetal demise (iufd). antenatal testing (at) is widely used in clinical practice to prevent iufd due to uteroplacental insufficiency and has been suggested to reduce the risk of iufd in ama women. we sought to assess the impact of at on obstetrical interventions and compliance with new practice recommendations. methods: retrospective cohort of ama women (40 and older at their due date) who delivered at or after 32 weeks. non-exposed women delivered from july 2003 to december 2004 (when at for ama was not routinely recommended); exposed women delivered from july 2005 to december 2006 (after at for ama was introduced at our institution). subjects were identified through the perinatal database; records were abstracted for demographics, medical history, and labor/delivery variables. outcomes included rates of at and induction of labor (iol) and mode of delivery. associations between at for ama and outcomes were tested using t test and chi square. assuming a baseline rate of iol of 20%, we had 80% power to detect an increase to 35% after the introduction of at. results: 276 women met the inclusion criteria: 147 delivered before the introduction of at (non-exposed=before at) and 129 delivered after the introduction of at (exposed=after at). baseline clinical characteristics were similar in both groups. as anticipated, at was more common in the after at group than in the before at group (92% vs 27%; p<0.0001). 86 women were not eligible for or declined trial of labor, thus not "at risk" for iol and not included in all analyses. ob intervention rates were increased after at compared to before at (table 1) . the corrected iufd rates were similar in both groups (8/1000 vs 7/1000). conclusion: at an academic center, compliance with new practice recommendations was excellent. introducing at testing for a new indication seemed to increase iol and cs rates. these findings should be considered when assessing the risks:benefits ratio of antenatal testing. . we stratified indications for cesarean into the following: failed induction (cervix <4 cm dilated), arrest of dilation, arrest of descent, failed operative delivery, fetal intolerance of labor (fil), and "other" reasons (e.g., pre-eclampsia, abruption, chorioamnionitis, malpresentation). both fil and "other" reasons were more likely to occur among the medically indicated group (rr 1.7, . physicians in solo practice had higher rates of elective inductions (p<0.0001), but there was no association between cesarean and practice type. conclusions: inductions accounted for 13.1% of cesareans. these results suggest an increased risk for cs for patients undergoing medically indicated inductions at our institution. there was no association between cesarean and type of practice, whether solo or group, suggesting institutional clinical policies may be more important than practice type in determining delivery outcome after induction. further research is needed to understand how age, race/ethnicity, or other unmeasured patient factors may impact these findings. given rising rates of both cesarean delivery and inductions, this information may be pertinent to women considering elective induction prior to 41 weeks. objective: fetal demise can lead to a consumptive coagulophathy ("fetal death syndrome") traditionally attributed to the release of "tissue thromboplastin", now known as "tissue factor" (tf). tf is the most potent activator of coagulation. despite the appeal and acceptance of this proposed pathophysiology, there is no evidence supporting this view. this study was undertaken to determine if fetal death prior to development of fetal death syndrome is associated with changes in maternal plasma concentration of cd40l (a marker of platelet activation), tf and its soluble inhibitor (tfpi). methods: a cross-sectional study included the following groups: 1) women with normal pregnancy (n=71) and 2) patients with fetal demise without disseminated intravascular coagulation (n=50). plasma concentrations of scd40l, tf and tfpi were measured by elisa. standard coagulation tests were performed. non-parametric statistics were used for analysis. results: 1) patients with fetal demise had a higher median maternal plasma scd40l concentration than women with normal pregnancy (median 1213.3 pg/ml, range 118-3818 vs. median 369.5 pg/ml, range 63.5-1848.7, p<0.001); 2) there was no significant difference between the groups in the median maternal plasma tf concentration and 3) in contrast, the median maternal plasma tfpi concentration was significantly lower in patients with fetal demise than in women with normal pregnancy (median 45.6 ng/ml, range 13.6-115.2 vs. median 66.7 ng/ml, range 37.4-86.5, p<0.001). conclusions: 1) a change in the plasma concentration of tf was not demonstrated; 2) a change in the ratio of tf/tf inhibitor pathway may predispose to thrombin generation and activation of the coagulation cascade; 3) however, maternal platelet activation is present in patients with a fetal demise without fetal death syndrome and 4) the role of tf in fetal death syndrome remains to be proven. lipoic acid inhibits matrix metalloproteinase 9 production, activity and prostaglandin e 2 secretion by cultured amnion epithelial and mesenchymal cells. r moore, j novak, d kumar, j moore. case western reserve university, cleveland, oh, usa. introduction: cytokines, free radicals, matrix metalloproteinases (mmp) and prostaglandins (pg) have been implicated in processes of fetal membrane rupture and labor. dietary anti-oxidant supplementation has been suggested as a possible therapy for high risk patients, however, clincal evidence supporting the efficacy of agents such as vitamin c or n-acetylcysteine remains controversial. in fact, we have previously shown that vitamin c increases matrix metalloproteinase (mmp) 9 activity in isolated fetal membrane fragments and fails to inhibit tumor necrosis factor (tnf) induced fetal membrane weakening in vitro. in this study, we examine the effect of the naturally occurring anti-oxidant, -lipoic acid, on tnf induced mmp9 activity/protein and pge 2 secretion in isolated amnion epithelial and mesenchymal cells. methods: amnion epithelial and mesenchymal cells were pre-treated with increasing doses of -lipoic acid (0-1mm/6h), then with increasing doses of tnf (0-50ng/ml/24h). medium and cells were analyzed by gelatin zymography/western blotting for mmp9/mmp2 activities/protein. pge 2 output was determined by immunoassay. results: tnf induced a dose dependent increase in mmp9 production, secretion and activity in amnion epithelial cells. tnf (50ng/ml) induced an 18 fold increase in cellular active mmp9 production and 3 fold increase in secreted mmp9 enzyme activity by amnion epithelial cells. these increases were reduced 62-100% following 6h pre-treatment with 0.5-1.0mm -lipoic acid. mmp2 protein/activity and pge 2 secretion by amnion epithelial cells were barely detectable and unaffected by tnf and/or -lipoic acid treatment. in striking contrast, mesenchymal cells exhibited little basal or tnf induced mmp9 protein/activity. mmp2 protein/activity in mesenchymal cells were unaffected by either tnf and/or -lipoic acid. however, tnf treated mesemchymal cells exhibited a dose dependent increase in pge 2 production (13 fold increase/50ng/ml tnf/24h) that was inhibited by 70%-100% following 0. objective: nearly fifty years after the discovery of microphthalmia-associated transcription factor (mitf), its gene was identified as a specialized transcription factor that dictates cell-specific differentiation. unique mitf isoforms are generated from alternative promoter usage. an isoform of mitf (mitf-cx) is down-regulated in cervical stromal cells of the ripened cervix. further, mitf-cx inhibits il-8 gene expression and thereby suppresses signaling of the final pathway in cervical ripening. since mitf binds to canonical eboxes (canntg) in promoter regions of target genes, we sought to determine if mitf regulated its own promoter through ebox motifs. methods: the 2 kb genomic dna sequence upstream of the mitf-cx transcription start site was cloned into pgl3 luciferase reporter vectors which were co-transfected with wild type or mutmitf-cx (impaired dna binding) into cervical stromal cells or hek293 cells. at 48 h, promoter activity was determined and normalized for transfection efficiency. results: gel-shift assays conducted with oligonucleotides corresponding to 11 eboxes in the mitf-cx promoter revealed two strong binding sites (eboxes 3 -983 to -977 and 9 -329 to -324 ). specific binding was established using oligonucleotides with or without mutated ebox, antibody supershift experiments, competition with cold probe, and absence of binding to mutmitf-cx. binding specificities were confirmed in nuclear extracts from cells that overexpressed mitf-cx, but not control or mutmitf-cx. reporter gene studies indicated that mitf-cx, but not mitf-m, increased mitf-cx promoter activity 4-to 5-fold. whereas mutations in ebox 3 or 9 resulted in significant decreases in mitf-stimulated promoter activity, mitfinduced increases in promoter activity were abolished by mutations in both eboxes. conclusions: collectively these experiments indicate that mitf-cx is a vital regulator of its own promoter activity and acts in a positive feedforward loop through two specific binding sites in its promoter. moreover, isoform-specific amino acids are important to mediate mitf-induced mitf-cx promoter activity. decreasing mitf protein or mutating its promoter would interrupt this loop resulting in rapid reduction of mitf synthesis. since mitf-cx suppresses il-8 gene expression in cervical stromal cells, we suggest that preservation of mitf-cx-induced mitf gene expression is an important mechanism to maintain the "brake" on cervical ripening and ensure cervical competency during pregnancy. the role of cd44 in cervical remodeling. denisse sanchez, brenda timmons, mala mahendroo. obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa. objective: prior to the onset of parturition, the uterine cervix undergoes a remodeling process from a closed, rigid structure, to one that is soft and dilatable. many changes occur, including increases in hyaluronan (ha), a glycosaminoglycan that facilitates loosening of the collagen matrix. in the postpartum period, the concentration of ha is reduced to that of the nonpregnant state. cd44, a transmembrane glycoprotein expressed in hematopoietic and epithelial cells, is a receptor for ha. cd44 expression by immune cells is important in extravasation of leukocytes into tissue. cd44 may also be required for ha catabolism through the action of hyaluronidases 1 and 2. in the cervix, cd44 is expressed in the endo-cervical epithelia as well as in immune cells localized in the stromal matrix. to study the importance of cd44 in cervical remodeling, mice with a null mutation for cd44 (cd44 -/-) were evaluated during pregnancy, parturition and postpartum. methods: changes in ha amount and size distribution were assessed using ha molecular weight gels and fluorophore assisted carbohydrate electrophoresis. to identify defects in cervical remodeling in the cd44 -/mice, differences in expression for genes regulated in the cervix were studied by quantitative real time pcr . to determine whether cd44 plays a role in the recruitment of immune cells during cervical ripening and postpartum repair, immunohistochemistry with antibodies against leukocytes was done. in the postpartum period, there is a higher ratio of high molecular weight ha relative to low molecular weight ha in the cd44 -/mice. this suggests that postpartum breakdown of ha in cd44 -/cervices is delayed. as compared to wt cervix, there was a significant increase in hyaluronidase 2 (hyal-2) mrna in the postpartum period. the distribution and relative numbers of immune cells in the cd44 -/cervix was similar to wt. conclusion: these studies provide evidence that cd44 may play a role in remodeling of the postpartum cervix back to the nonpregnant state. our current data suggests that the catabolism of ha after birth is delayed in the mutant mice and upregulation of hyal2 may compensate to allow ha removal. furthermore, the activity of hyal-2 may be dependent on cd44. little difference in the recruitment of immune cells between cd44 and wt animals suggest that cd44 expression is not required for this process. these experiments provide a greater understanding for the role of cd44 and ha in cervical remodeling. in background: during in vitro experiments we have shown that separation of amnion from choriodecidua occurs as an integral part of the process of fetal membrane (fm) rupture. although spontaneous amnion and choriodecidual separation is seen in fm after both svd and elective c/s deliveries, its etiology is uncertain. biochemical degradation at the amnion-choriodecidua interface may be a key contributing factor. our previous biomechanical studies have demonstrated that separated fm require less physical work to rupture than intact membranes. the purpose of this study was to determine whether fm separation was associated with clinical differences in the birth process. hypothesis: during term, normal labor, spontaneous separation of fm is associated with differences in the clinical parameters of labor and delivery. study design: fm from consecutive term deliveries were cut off the placental disk. separated areas of fm were cut from the intact areas. both were weighed and their weight ratios determined. maternal medical, pregnancy, and delivery data were collected and analyzed. results: 221 term fm had the following characteristics: maternal age 26±6.4 yr, gravida 3.1±2.2, gestation 39±1.1 wks, elec. cs 10.0%, duration of rom 403±417 min, duration of contractions 642±440 min, african american 45%. 40% of the fm had <10 % separation; 11% had more than 95 % separation. srom fm with >10% separation (vs. <10%) had significantly shorter duration of rom (p=0.03) and admission to birth (p=0.05) times. srom fm with >95% separation (vs. <10%) had even shorter rom (p=0.006), duration of contractions (p=0.002) and admission to birth (p=0.0003). the >95% group (vs. <10%) was further along, gestationally (p=0.05). srom fm (vs. arom) had shorter admission to birth (p=0.008), but longer rom to birth (p<0.0001) times. absence of epidural (p=0.001), srom mode of rupture (p=0.04), svd (vs. elec. c/s) (p=0.06), and the presence of meconium (p=0.002), were all associated with increased fm separation. conclusion: spontaneous separation of fetal membranes is nearly universal and is associated with increased gestation, spontaneous rupture of membranes, shorter duration of contractions, and svd. speculation: we speculate that programmed biochemical changes initiate fm separation which then facilitates rom and childbirth. whole genome array and si-rna investigation of the function of nfkb in human amnion epithelial cells. sheri e lim, 1 shirin khanjani, 1 yun s lee, 1 tg teoh, 1,2 philip r bennett. 1 1 institute of reproductive and developmental biology, imperial college, london, united kingdom; 2 maternal fetal medicine, st. mary's hospital, london, united kingdom. introduction: labour is associated with activation of nfkappab in the amnion. nfkappab increases prostaglandin synthesis through the upregulation of cyclooxygenase-2 (cox-2), which is essential to the labour process. cox-2 mrna expression increases with gestation in the amnion. primary amnion epithelial cells cultivated from tissue collected prior to the onset of labour display a spectrum of nfkappab activation, similar to the spectrum of cox-2 expression presumably relating to the nearness of labour. our aim was to investigate the full range of genes under nfkappab control in amnion epithelial cells by using whole genome arrays. methods: amnion from 20 women undergoing elective caesarean section was collected and primary cell cultures established. total rna and protein were extracted from each culture. nuclear localization of nfkappab is required for its activation. western analysis of nuclear p65 was therefore performed to identify the samples displaying the lowest and highest nfkappab activity. the corresponding rna samples displaying the three lowest and three highest nuclear p65 protein concentrations were used for whole genome analysis using affymetrix u133 arrays. results and conclusions: we identified 919 significantly regulated genes. the gene with the highest fold change was cox-2 (x44.4) followed by oxytocin receptor (x24.1), ch10orf (x19.6), integrina2 (x17.7), and connective tissue growth factor (x14.9). other significant genes included interleukin-8 (il-8) (x6.3). pathway analysis revealed the majority of other nfkappab associated genes were involved in cell signaling, turnover and proliferation. we used real-time pcr (rtq-pcr) to validate cox-2 and il-8 expression. to prove that cox-2 is directly regulated by nfkappab, primary amnion epithelial cells were then transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna pool. western blot analysis confirmed knockdown of nfkappab p65 associated with inhibition of cox-2 demonstrating that nfkappab is essential for cox-2 expression. objective: premature birth is a major public problem accounting for over 13,000 deaths and 30,000 surviving infants with life-long morbidity yearly. in order to develop a rational basis for treatment and prevention of premature fetal membrane (fm) failure, we first need to understand the sub-failure fm structural and mechanical behavior at near full term. methods: we utilized planar biaxial mechanical testing, which approximates the physiologic loading state, for mechanical evaluation of the fm, and a structural constitutive model approach was used to offer insight into the structure-strength of the fm by integrating information on tissue composition and structure. small angle light scattering (sals) was used to nondestructively quantify the collagen fiber architecture of both intact and separated fm layers. results: in the stress free state, the gross collagen fiber architecture of the fm and separated layers were not homogenously align but exhibited small regions of fiber alignment. the amnion layer displayed the greatest alignment. the model fit the equi-biaxial strain data well (r 2 = 0.99) and indicated that fm collagen fibers were rapidly recruited and straightened well below failure stress levels. collagen fibers were gradually recruited followed by a drastic increase in fiber recruitment. conclusion: this study provided the first data on the effective collagen fiber stiffness in the intact fm under physiologic biaxial loading, which was related to quantitative collagen fiber architectural measures. modeling results indicated that the collagen fibers became fully loaded and straighten well below physiological loading levels. failure did not occur during physiological loading, indicating that fibers do not begin to fail until all collagen fibers are fully straightened and bearing load. this result suggested modest structural reserve in the fm collagen architecture, and may be an important aspect of its failure properties. previously, we demonstrated that a physically "weak zone" exists overlying the cervix in the fm, evident of collagen remodeling and cellular apoptosis. we are currently extending the present study to include the "weak zone" tissues, allowing us to elucidate the micro-mechanical mechanisms that facilitate failure in this newly identified fm zone. supported by nih 48476. the extracellular matrix of the cervix undergoes extensive remodeling during parturition. hyaluronan (ha) is a major constituent of the extracellular matrix of the term pregnant cervix. the onset of labor is preceded by an increase in ha and after delivery, the concentration of cervical ha gradually decreases to that of the non-pregnant state. these dramatic changes suggest that ha plays an important role during parturition. hyaluronan synthase 2 (has2) is one of three known ha synthases and the most abundant isoform in the pregnant cervix. transcripts for has2 are regulated by two alternative promoters, one upstream of the first coding exon (proximal promoter) and another upstream on an untranslated exon 1 (distal promoter). the focus of the current study is to further our understanding of the transcriptional regulation of has2 during cervical ripening. methods: rna blotting was carried out using transcript specific probes corresponding to the distal and proximal promoter of the mouse has2 gene. the regulation of has2 was evaluated in a cervical epithelial cancer cell line (caski cells) which we have previously shown to express endogenous has2. regulation of has2 expression by epidermal growth factor was assessed in the caski cells by western blotting and quantitative real time pcr assessment of transcripts. results: has2 mrnas in the nonpregnant (np) and pregnant cervix are transcribed from the distal promoter upstream of exon 1. two transcripts of approximately 4.1kb and 2.8kb were detected that arise from use of 2 polyadenylation sequences. as compared to np, the expression of has2 is increased 4, 23, and 9 fold on gestation days 15, 18 and shortly postpartum respectively. has2 mrna expression is increased upon treatment of caski cells with epidermal growth factor (30ng/ml) and is suppressed in cells treated with ag1478 (20 m), an inhibitor of egf receptor phosphorylation. maximal stimulation was observed at 4 and 8 hours of treatment. conclusion: has2 is the major ha synthase expressed during cervical ripening and the majority of transcripts are driven by the distal promoter in the has2 gene. in vitro studies using caski cells suggest has2 is regulated in part by the egf signaling pathway resulting in a several fold increase in has2 expression. these results provide an understanding of has2 gene regulation at the time of cervical ripening which will ultimately enhance our understanding of the molecular mechanisms important to cervical ripening. chorion and decidua cells. chad a grotegut, bernard j canzoneri, liping feng, phil heine, amy p murtha. obstetrics and gynecology, duke university, durham, nc, usa. objective: preterm premature rupture of the fetal membranes accounts for approximately 30% of all preterm deliveries. cigarette smoking independently carries a fourfold increase risk for pprom. our laboratory has previously demonstrated that the chorion layer undergoes apoptosis in women with pprom. this study was conducted to determine if extract of cigarette smoke causes cell death in specific cells of the fetal membrane. fetal membranes were collected at the time of elective cesarean section from women without labor and at term. the chorion and decidua layers were separated and purified on a gradient spin column and then plated near confluence. cigarette smoke extract (cse) was collected in cell media and used to treat chorion and decidua cells in culture at concentrations ranging from 1% to 100% in 96-well plates. cell viability was determined at 24, 48 and 72 hours following treatment with a non-radioactive cell viability assay. data were analyzed using paired t test (analyse-it, leeds, uk). chorion and decidua cells underwent cell death when exposed to cse in a dose dependent fashion. increasing concentrations of cse resulted in increased cell death at 48 hours in both cell types (figure 1 ). at 48 hours, chorion exhibited greater percent cell death compared to decidua at concentrations of 20, 40 and 60% cse (p=0.003, 0.019, and 0.069, respectively). for any given concentration of cse, the degree of cell death increased with increasing length of exposure (36, 48 and 72 hours) for each cell type. chorion cells routinely exhibited greater percentage of cell death following treatment with cse at concentrations ranging from 20-60% compared to decidua cells. human chorion and decidua cells in primary cell culture exhibit a dose-response and time dependent cell death in the presence of cse. human chorion cells show greater sensitivity to cell death when compared to decidua cells. further studies are needed to determine the mechanisms through which these cell types undergo cell death and the implications for the differential sensitivity to cigarette smoke. yoon ha kim, 1 tae-bok song, 1 cheol hong kim, 1 jong woon kim, 1 moon kyoung cho, 1 sung yeul yang, 2 bong whan ahn. 2 1 obstetrics gynecology, chonnam national university medical school, gwangju, korea; 2 biochemistry, chonnam national university medical school, gwangju, korea. objective: to investigate the lipid peroxide levels and protein carbonyls levels in the amniotic fluid of pregnant women with preterm premature rupture of membranes (pprom). the lipid peroxide levels in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were newborn offspring with persistent pulmonary hypertension, despite enhanced newborn offspring with persistent pulmonary hypertension, despite enhanced measured by thiobarbituric acid reaction. the protein carbonyl contents in the amniotic fluid of normal pregnancy (n=20) and pregnant women with pprom (n=20) were determined by the 2,4-dinitrophenylhydrazine method. after amniotic fluid of them were mixed and incubated up to 5 hours with 0.2ml of 1mm moxalactam, cefodizime, amoxacillin, erythromycin, the lipid peroxide levels and protein carbonyl contents in them were measured. results: 1. the lipid peroxide levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (9.74±0.48 vs. 7.20±0.38 nmol/mg protein, p<0.01). 2. the protein carbonyl levels in the amniotic fluid of pregnant women with pprom was significantly higher than that of normal pregnancy (13.0±0.33 vs. 11.27±0.17 nmol/mg protein p<0.01). 3. the lipid peroxide levels and protein carbonyls formation by moxalactam in the amniotic fluid of pregnant women with pprom was significantly higher than basal level (12.08 ± 0.81 vs. 9.74±0.48 nmol/mg protein, 20.08 ± 0.66 vs. 13.0±0.33 nmol/mg protein, p<0.01). 4. the lipid peroxide levels and protein carbonyls formation by cefodizime in the amniotic fluid of pregnant women with pprom was significantly lower than basal level (5.04 ± 0.33 vs. 9.74±0.48 nmol/mg protein, 9.76 ± 0.35 vs. 13.0±0.33 nmol/mg protein, p<0.01). 5. there were no significant differences in the levels of lipid peroxide and protein carbonyls by amoxacillin and erythromycin in the amniotic fluid of pregnant women with pprom between antibiotics-induced and basal levels. background: chorioamnionitis (cam) is a major antecedent of preterm delivery (ptd) associated with elevated amniotic fluid tnf and il1 . we hypothesized that these cytokines enhance the term decidual cell (dc) expression of the matrix metalloproteinases (mmp) 2 and 9, which can then promote ptd by degrading the extracellular matrix of the decidua, fetal membranes, and cervix. methods: immunostaining for mmp-2, mmp-9, and vimentin (a dc marker) was performed on cam-complicated (n=5) and gestational age-matched control decidua (n=5), and staining intensities were evaluated by hscore. confluent, leukocyte-free term dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-2 and mmp-9 levels were measured by elisa (n=8), and quantitative rt-pcr assessed mmp-2 and mmp-9 mrna levels (n=4). results: tissue staining revealed that mmp-2 and mmp-9 levels in cam-complicated decidua (hscore mean±sem: 203±14 and 198±12, respectively) were significantly higher than in control decidua (136±6, and 114±6 respectively; p<0.05). in cultured term dcs incubated with e2, tnf and il1 significantly increased secreted levels of mmp-9 compared to e2 alone (pg/ml/ g protein: 162.3±110.1 and 20.4±6.5, respectively, vs. 0.5±0.2; p<0.05). in parallel incubations with e2+mpa, basal mmp-9 output was lowered by 50%, and tnf -and il1 -elicited mmp9 levels were blunted by 70% and 40%, respectively. rt-pcr confirmed that tnf and il1 increased mmp9 mrna levels (p<0.05), although mrna levels in e2+mpa incubations were not different from those of e2 alone. mmp2 levels in all treatments were similar. conclusions: mmp-2 and mmp-9 are elevated in cam decidua compared to controls. our in vitro results suggest that mmp-9 expression is enhanced by the high levels of il1 and tnf associated with cam, and that mpa may be able to blunt this effect. we have previously found a similar regulatory mechanism of mmp-1 and mmp-3 and their over-expression in cam-complicated tissues. synergy among these mmps may represent a potent pathogenic mechanism of cam that can be targeted through the therapeutic use of progestins in preventing cam-induced ptd. domerudee preechapornprasert, 1 patama promsonthi, 1 wasun chantratita, 2 mana rochanawutanon, 2 patcharee karnsombut, 2 chutatip srichunrusami, 2 stephen j lye, 3 boonsri chanrachakul. 1 1 obstetrics and gynecology, ramathibodi hospital, mahidol university, bangkok, thailand; 2 pathology, ramathibodi hospital, mahidol university, bangkok, thailand; 3 obstetrics and gynecology, samuel lunenfeld research institute, toronto, canada. objective: cervical ripening is an inflammatory process involving chemokines, cytokines and various mediators. recent study has shown that the level of monocyte chemotactic protein (mcp) 1, a chemokine, increases in amniotic fluid during spontaneous labor. the aim of this study was to examine the localization and expression of mcp 1 in human cervix before pregnancy, during pregnancy and after the onset of labor. methods: this study was approved by the local ethics committee and written informed consent was obtained from each participant. cervical biopsies were taken from 4 groups of women; nonpregnant women, first trimester pregnant women, term pregnant women with and without labor. tissue samples were fixed in 10% formal saline for paraffin section. immunohistochemistry (n = 5 each) was performed by avidin biotin complex (abc) technique using monoclonal antibody specific to human mcp 1. the mcp 1 messenger(m) rna was identified by reverse transcription-polymerase chain reaction using gene specific primer against mcp 1 and mcp 1 receptor (n = 10 each). results: immunohistochemistry demonstrated mcp 1 in cervical tissues from all four groups of women. mcp 1 was localized on plasma membrane and cytoplasm of both squamous epithelial and columnar cell lining of endocervical gland. mcp 1 and mcp 1 receptor mrna were identified in nonpregnant, first trimester and term with and without labor human cervix. conclusion: mcp 1 and mcp 1 receptor were located in cervical tissues of nonpregnant and pregnant women at different gestation both before and after the onset of labor.ongoing studies are investigating the role of this chemokine during pregnancy and labor. whether preterm cervical ripening is just an aberrant regulation in timing or whether divergent mechanisms and pathways are involved in preterm versus term cervical ripening remains to be elucidated. methods: cervical tissue was collected from 3 groups of cd-1 mice. group 1: mouse model of ptb that utilizes intrauterine infusion of lipopolysaccharide (lps). group 2: e15 dams representing preterm controls. group 3: e18.5-19 dams selected from a timed pregnant batch where half of the dams had delivered representing term cervical ripening. n=6 dams/treatment group. 18 separate rna samples were used for microarray analysis (ma). significance analysis for ma and partek software was used for biostatistical analysis. pathway analysis was performed using david. quantitative pcr was performed to confirm the most differentially regulated genes. results: using a cut-off of 64-fold change with p value of <0.0001, 368 genes in the cervix were differently regulated between the 3 groups. principal component analysis revealed three distinct groups (see graph). functional annotation clustering demonstrated the following pathways: 1) in preterm cervical ripening (e15 lps vs e15): immune and inflammation response, defense response 2) in term cervical ripening compared to preterm controls: negative regulation of cellular process and biological process, ecm, cell-cell communication. qprc confirmed the highly significant differences found in ma (see table) . conclusions: the molecular mechanisms and pathways governing preterm and term cervical ripening are distinctly different. elucidating these unique pathways can lead to improved therapeutics for prevention of ptb as well as for postdate pregnancies. cervical ripening at term involves activation of apoptotic enzymes. maria kb sennstrom, 1 valentina ciani, 2 gunvor e ekman. 1 1 obstetrics and gynecology, women and child health, karolinska institute, stockholm, sweden; 2 obstetric and gynecology, university of siena, siena, italy. aim: to investigate if the human cervical ripening at term involves programmed cell death. during the final cervical ripening the extracellular matrix dominated cervix undergoes an extensive remodelling of the tissue. inflammatory mediators such as the cytokines increase in ripening cervical tissue at term. programmed cell death, apoptosis, has been suggested as important in this process. apoptosis can be induced by inflammatory mediators such as cytokines. we also looked upon the distribution of inflammatory cells in cervical tissue. materials and methods: cervical biopsies from pregnant women at term and post partum women with fully ripened cervix were studied. biopsies from non-pregnant women served as controls. immunohistochemical analysis of the apoptotic enzyme caspase-3 and the inflammatory cell marker cd 45 was performed on paraffin embedded sections of cervical tissue. double staining was performed. mann-whitney u-test was used for statistical analysis. results: there was a significantly higher frequency of caspase-3 staining in the post partal sections from ripened cervical tissue compared to tissue from term pregnant (p=0.002) with unripe cervix and from non-pregnant patients (p=0.00067). the inflammatory cells staining for cd 45 increased in post partal and term pregnant sections compared to non-pregnant (p=0.016). there was a higher frequency of caspase-3 positive cells in the post partal tissue than of cd45 positive cells (p=0.00011). the localization of cd-45 positive staining was highest in the epithelia and basal lamina while caspase-3 staining was most pronounced in stromal tissue and around vessels. conclusion: our data show apoptotic activity in stromal tissue in fully ripened human cervix at term of pregnancy, suggesting that apoptotic mechanisms are involved in the extracellular matrix remodelling at term. the apoptotic activity is not co-localized with inflammatory cells suggesting non-infectous inflammation with apoptosis as important for cervical ripening at term. objective: cervical biomechanical responses are important for accommodating the increased stress induced by an enlarging uterus. a mechanical testing system was modified to evaluate the stress relaxation response in the pregnant cervix. methods: tissue harvested from the non pregnant and timed pregnant (days 12, 16, 20, 21, and 22) sprague-dawley rats underwent tensile testing using an instron 5800 material testing system. the testing regimen consisted of tissue extension to near maximal strain over 60 seconds followed by a period of constant strain for 60 minutes, then return to rest over 60 seconds. this cycle was repeated 2 additional times with a 60 minute rest period between cycles. strain and force measurements were recorded at 1 second intervals. 4-6 animals were used for each time point. in addition, stress and strain at the yield point was also determined for each gestational time point. results: the pregnant and non pregnant samples exhibit marked differences in response in both stress and strain. the timed pregnant tissue demonstrated progressively increased compliance and lower nominal stress compared to non pregnant tissue. peak nominal stress declined with each successive cycle. this was demonstrated in the peak nominal stress and strain values at the yield point. conclusion: the cervix becomes more distensible (compliant) but less resistant to force with increasing gestational age. ...,.. e. coli 1.2x 10 8 .:!: 8.5x 10 8 1.2 x 10 8 .:!: 3.0 x 10 8 group a (c, c) group b ( c, p) 2.3x 10 8 + 2.7x 10 8 9.4 x 1 0 7 + 4.8 x 10 8 .33 .81 .83 group c (p, p) 1.1 x 1o"::!>s x 1o" 1.1 x 10 8~ 1.ox:1o• k. ~neumoniae group a ( c, c) 5.3 x 10 7 + 3.4 x 10 7 9.2 x 10 7 + 6.7 x 10 7 .88 .87 .91 group b (c, p) 6.0 x 10 7 + 4.8 x 10 7 6.3 x 10 7 + 3.0 x 10 7 group c (p, pl 4.7 x 10 7~2 .3x 10 7 6.9x 10 7~4 .6x 10 7 c. al bicans group a (c, c) 1.1 x 10 '+1.8x 10 7 1.9 x 10 8 + 1.0 x 10 8 group b (c, p) 1.5x 1o•+ 1.0x 10 7 2.4 x 10 8 + 1.1 x 10 6 .75 . 87 .60 group c (p, p) 4.7x 10 7~2 .3x 10 7 2.1 x 1 0 7~5 .9 x 10• introduction: a growing body of evidence supports that inflammatory processes are implicated in spontaneous preterm birth (ptb). using an inflammatory and non-inflammatory mouse model of ptb, we sought to determine if activation of these inflammatory pathways are essential for ptb and/or cervical ripening to occur. methods: timed pregnant cd-1 mice were used in these two models of ptb: 1) a model of intrauterine inflammation where lipopolysaccharide (lps) is injected into the uterine horn (n=6); controls for this model received intrauterine saline (n=6) and 2) a non-infectious model of ptb using ru486 sq (150ugrams/dam) (n=5); controls for this model received no intervention (n=6) were used for these studies. for both models, 6 hours later uterine and cervical tissues were harvested. the tissues were processed for protein and rna studies. elisas were performed to assess il-6 and tnf-alpha in the uterine tissue. mrna expression of ifn , il-6, il-1 , il-10, tnf-alpha were assessed in cervical tissue from both models by quantitative pcr. results: in uterine tissues, both il-6 and tnf were significantly elevated in the lps-induced ptb when compared to control, saline, and non-infectiousinduced preterm birth (p<0.001) (figure 1). in cervical tissue, an increase in il-6, il-1beta and tnf mrnawas observed in both models, while ifn-gamma and il-10 were only increased in the lps model. (figure 2 ). conclusions: up-regulation of pro-inflammatory cytokines in the uterus do not appear to be essential for ptb. cytokine expression in the cervix is greater in an inflammatory model of ptb but is also present in a non-infectious model. these studies suggest that targeting a cytokine response in the cervix may hold the most promise in prevention of ptb. the initiation of labor at term and preterm is associated with an inflammatory response, with increased interleukins in amniotic fluid (af) and infiltration of the myometrium by neutrophils and macrophages (m ). whereas, in preterm labor, intra-amniotic infection may provide the stimulus for increased af interleukins and inflammatory cell migration, the stimulus for these events at term has remained uncertain. in studies using pregnant mice, we observed that the m that invade the maternal uterus near term arise from the fetus. furthermore, we obtained compelling evidence that surfactant protein-a (sp-a), a developmentally regulated c-type lectin secreted by the fetal lung into af near term, activates af m , which migrate to the uterus where they promote an inflammatory response culminating in labor. we propose that interactions of m surface receptors with sp-a, at term, or bacterial lipopolysaccharide at preterm, initiate changes in m phenotypic properties, resulting in the enhanced expression of genes that promote their migration to the uterus. the objectives of the present study were to analyze the numbers and phenotypic properties of mouse af m during late gestation and to identify their putative tissue source(s) of origin. to assess changes in the number of m in af during late gestation, af cells were isolated and stained for the m marker f4/80. the density of adherent f4/80 + cells greatly increased in equivalent volumes of af between 15.5 and 18.5 days postcoitum (dpc) (19.5 dpc = term). interestingly, the f4/80 + cells at 18.5 dpc were highly similar in morphology to those present in 18.5 dpc fetal lung, but distinctly different from those in fetal liver, suggesting their pulmonary origin. the af m were foam cell-like, suggesting the presence of lipid inclusions, a property shared by adult alveolar m . to further analyze gestational changes in the m population(s) in mouse af, we used flow cytometric analysis. in our initial studies, cells isolated from af were stained for f4/80 and for cd45, a pan-leukocyte marker. we observed that af from 15.5 and 18.5 dpc mice contained two sub-populations of cd45 + f4/80 + cells. studies are in progress to analyze these af m populations for expression of cell surface antigens indicative of their maturity, activation state and chemotactic properties in association with the developmental induction of sp-a synthesis and secretion by the fetal lung. april bleich, patrick keller, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. the role of prs, proinflammatory cytokines, cell adhesion molecules, cox-2, and toll-like receptors in mediating cervical ripening prior to labor is not clear. the objective of this study was to quantify pr isoforms and determine the relative expression of certain inflammation-related genes in cervical stroma from nonpregnant and pregnant women in early gestation (eg), term before and after cervical ripening, and during labor. methods: standard curves of pr-b, -a, pra+b and qpcr were used to quantify total and pr-b in cervical stroma from 9 nonpregnant (proliferative, n = 4; progestin treatment, n = 5) and 35 pregnant women undergoing hysterectomy (eg, n = 7; term before ripening, n = 10; after ripening, n = 7; in labor, n = 11). cervical status was determined by modified bishop scoring. results: total pr expression was maximal in nonpregnant women in the proliferative phase (47.6 ± 8.7 pg/ug cdna) and decreased 80% by progestins (8.6 ± 2.7 pg/ug cdna). this level was maintained in stroma from pregnant women before labor (6.6 ± 1.3, eg.; 9.9 ± 1.8 before ripening; 12.5 ± 2.9 after ripening pg/ug cdna). in contrast, total pr was decreased significantly in the dilated cervix (2.4 ± 0.6 pg/ug, p < 0.05). interestingly, whereas pr-b was 30 ± 2% that of total pr in the nonpregnant cervix, pr-b mrna levels were 52 ± 4% to 70 ± 6% in cervical tissues from all pregnant women and did not vary with labor status. using immunoblot analysis and pr-specific antibodies (pgr1294), pr-b immunoreactivity was 50% that of total pr in all samples from pregnant women, and both pr-a and -b were downregulated significantly in the dilated cervix (from 44 ± 9 to 19 ± 8 units/atub). decreased expression of pr in the dilated cervix was accompanied by significant increases in il-8, mcp-1, tlr-2, cd62l, cox-2, and s100a9 mrna (all p < 0.05, anova) but not cd11b or tlr-4. pgdh mrna was decreased significantly in the dilated cervix. with the exception of cox-2, expression of these genes was similar before labor regardless of cervical ripening. conclusions: both pr and pr-b are decreased proportionately in the dilated cervix, but not during cervical ripening. further, a number of inflammatory gene products are increased dramatically in the cervix during labor, but not before. taken together, the results suggest that cervical ripening is distinct from cervical dilation and involves upregulation of cox-2 but not il-8, mcp-1, or toll-like receptors. ifn-y il-6 il-1,6 il-10 tnf-01 "p value <0.05 lps/saline ru486/controls 10.3"" 0.63 68" 6.6" 15.7* 24" 12" 5.9* 1.8 3.1" 722 association between interleukin-6 (il-6) and ilto examine association of amniotic fluid interleukin 6 (il-6) concentration with il-6 and its receptor il6-r haplotypes in term and preterm caucasians (c) and african americans (aa) samples. methods: in this study case (preterm birth -ptb [<36 weeks]) and control (term [>37weeks]) amniotic fluid (af) il-6 concentrations were analyzed for association with haplotypes of the il-6 and il6r genes in aa and c separately. in il-6, eight, and in il-6r, 22 single nucleotide polymorphisms (snps) were examined. aa and c maternal and fetal genotypes were assessed (aa:35 maternal:cases-57 controls-34 fetal: cases-54 fetal controls-; c: maternal cases-88, controls-40, fetal:cases-86; controls=36). haplotype associations were performed by using a sliding window with outcome il-6 concentration. analyses were performed separately on maternal and fetal dna. results: the strongest haplotype associations were observed in il-6r rather than in il-6. in c fetal dna il-6r haplotypes defined by markers 16311-21909 bp from the transcription start site associated most strongly with af il-6 concentrations (global p=1.6x10 -3 ) and in aa maternal il-6r haplotype markers at 16311-21909-22214-26274 (global p=2.30x10 -3 associated with il-6 concentrations. in the c fetal cases the 16311-21909 haplotype with the highest concentration was a-g (log(cytokine) = 3.67 pg/ml). in aa maternal samples the highest concentration was observed for haplotype t-t-g-c at 16311-21909-22214-26274.this was seen in both cases and controls at (case log (cytokine) = 3.84; control log(cytokine) = 3.49 pg/ml). significant associations from haplotype analyses converged on three regions of the il-6r in both races. no strong differences were observed between the haplotypes of cases with and without microbial invasion of the amniotic cavity (miac), with the exception of aa fetal samples that showed two overlapping haplotypes in il-6 that associated in cases with miac but not in cases without miac (-661 and -636; -636 and -237)(both with p < 1x10 -3 ) conclusion: differences in the af il-6 concentration in ptb do not result from single snp effect on il-6 but are a result of complex relationships between il-6 and il-6r haplotypes. these associations exhibit racial disparity. the role of tnf-in parturition. helen alexander, 1 amanda tattersall, 1 mark tattersall, 1 suren sooranna, 1 peta grigsby, 2 leslie myatt, 2 mark johnson. 1 1 obstetrics gynaecology, imperial college, london, united kingdom; 2 obstetrics gynaecology, university of cincinnati college of medcine, cincinnati, oh, usa. introduction: tumour necrosis factor-alpha (tnf-) is thought to play a role in inflammation-induced preterm labour since the decidua produces tnfin response to bacterial products and amniotic fluid tnf-concentrations are increased in the presence of intra-amniotic infection. the aims of this study were to (i) investigate the expression of myometrial tnf-and its receptors in relation to the onset of preterm and term labour; (ii) to identify which intracellular pathways are activated by tnf-; and to investigate the effect of tnf-alone and in combination with il-1 or il-8 on gene expression in uterine myocytes. methods: biopsies of human myometrium were taken at caesarean section from women before and after the onset of preterm and term labour and analysed for tnf-and its receptor mrna expression. a further 6 samples were obtained before the onset of labour (n=6) from which myocytes were isolated and cultured in 6-well plates. when cells were 85-95% confluent either tnf-at a concentration of 1ng/ml was added to the cells for 7, 15, 30, 60 and 120min and the cells analysed by western blotting or tnf-at concentrations of 0, 0.1 and 1ng/ml was added either alone or in combination with similar concentrations of il-1 or il-8 to cells for 6 hours and mrna was extracted and converted to cdna to determine il-8 and gapdh gene expression by qpcr. results: there was no change in tnf-mrna expression in relation to the onset of labour, but the expression of tnfr1 and tnfr2 mrna levels were significantly increased with gestation and further increased with the onset of labour. incubation of uterine myocytes with tnf-(1ng/ml) activated all three mapk substypes: erk, jnk, and p38, activation peaked between 7-15 minutes. preliminary data suggest that tnf-induces il-8 mrna expression but exposure to il-1 or il-8 itself did not enhance this response. conclusions: although there is no significant increase in tnf-concentration from baseline during labour we have shown tnf receptor mrna levels do increase with labour at term. exposure of isolated uterine myocytes to tnfcauses activation of all mapk subtypes and an increase in il-8 mrna expression. this enhanced mapk-dependent il-8 expression at term may be mediated via increased myometrial sensitivity to tnf-through increased tnf receptor expression. remodeling. brenda c timmons, 1 anna-marie fairhurst, 2 mala s mahendroo. 1 1 obstetrics and gynecology, ut southwestern medical center, dallas, tx, usa; 2 immunology, ut southwestern medical center, dallas, tx, usa. objective: the molecular mechanisms involved in cervical ripening are not well understood. immunohistochemical studies from our lab report a recruitment of inflammatory cells to the cervical stroma one day before birth in the mouse using a neutrophil/monocyte marker (neutrophil 7/4). in this study, we sought to identify and quantitate inflammatory cells migrating into the mouse cervix and to determine if this recruitment was affected by changes in progesterone levels. peripheral blood was also evaluated to see if changes in the cervix was paralleled in blood. methods: flow cytometric analysis was performed using cervical cells and peripheral blood obtained before and during cervical ripening along with 2-4h postpartum. dispersion of cervical cells was optimized. these cells were stained with a panel of fluorescent conjugated antibodies directed against leukocyte antigens and analyzed on an lsrii flow cytometer. cells were also sorted and stained to visualize cell morphologies. to determine the effect of progesterone on the migration of leukocytes, gestation d15 mice were treated for 13h with a progesterone receptor (pr) antagonist prior to tissue collection. results: neutrophils do not appear to increase in the cervix until after birth. monocyte (mo) numbers do increase during cervical ripening (late day 18, d18.75) and remain high through postpartum (pp). macrophages (mø) are present prior to cervical ripening and steady state levels are maintained during labor and pp. pr antagonist treatment on d15 resulted in a premature increase in mo but not neutrophils or mø. in contrast to the cervix, mo and neutrophil numbers do not significantly increase in the peripheral blood until pp. results from lymphocyte studies suggest a low level of b and t cells in the cervix. in the peripheral blood, b cells remain consistent through parturition and the t cells decrease by d18.75 and continue to decrease pp. conclusion: tissue mo are increased in the cervix during ripening. this recruitment is dependant on loss of pr function. in contrast, neutrophils are increased in the pp cervix while mø numbers appear constant. timing of changes in mo and neutrophil numbers in the peripheral blood differed from that observed in the cervix suggesting quantitation of these cell types in blood is not reflective of what is occurring in the cervix during ripening, dilation and pp repair. novel interactions between nf-b and other labour-associated transcription factors identified by a tf-tf array. shirin khanjani, yun s lee, suren r sooranna, mark r johnson, phillip r bennett. irdb, imperial college, london, united kingdom. introduction: external stimuli lead to changes in cellular gene expression through activation of inducible transcription factors. nf-b is a ubiquitous transcription factor classically associated with inflammation, which is activated in response to infection and proinflammatory cytokines such as those prevalent during the labour. the tf-tf interaction array uses a novel technology for detecting interactions between transcription factors based on binding of tfs to their own consensus dna binding sequence. materials and methods: primary myometrial cells were grown until 90-95% confluent and stimulated with 1ng/ml il-1 prior to nuclear protein extraction. the nuclear extracts were incubated with the provided set of biotin-labeled, double-stranded oligonucleotide probes, which represent a known library of cis-elements. during the incubation step, these tf probes bind to their specific tfs in the nuclear extract. next, immunoprecipitation was performed using an antibody against nf-bp65, which pulled out nf-bp65 and any tfs bound to it, bound to corresponding cis-elements. normal igg was used in a parallel experiment to represent a negative control. free cis-elements and nonspecific binding proteins were washed away and the cis-elements were finally eluted and hybridized to the array membrane, which is spotted with different tf consensus sequences. results: table 1 shows the different tfs interacting with nf-bp65 based on the degree of binding stimulated by il-1 compared to no-il-1 control. conclusion: these data show that il-1 stimulation causes nf-b to bind to a wide variety of other treansciption factors. of particular interest in the area of parturition is binding to the other pro-inlammatory tfs such as c/ebp and ap-1, which are known to regulate labour-associated genes in synergy with nf-b. the lack of association between nf-b and pr without il-1 stimulation supports the concept that with the onset of labour inflammation leads to functional progesterone withdrawal, rather than pr acting to inhibit inflammation. table 1 high ap-1, c/ebp, cbf, creb , c-myb, e2f-1, ets, ets-1/pea3,fast-1, gas/isre, hse, mef-1, mef-2, myc-max, nf-1, nfatc, nf-e1, nf-e2, pax-5, objective: pre-partum cervical ripening involves remodeling of collagen structure and inflammatory immune cell activity (jsgi 10:323,2003; reprod biol endo 3:39,2005) . although parturition is associated with systemic or local progesterone withdrawal (ajog 196:289,2007) , effects of a decline in progesterone on cervical ripening is not known. the present study tested the hypothesis that progesterone withdrawal promotes collagen degradation, innervation, and immune cell trafficking in the cervix of nonpregnant mice. methods: adult virgin female c57bl6 mice received capsules (sc) with oil vehicle (v) or estradiol (e) and progesterone (p) to simulate concentrations in pregnancy (hum reprod 12:602,1997) . after 15 days, mice in the v and e+p groups were euthanized. the p capsule was removed from some mice on day 17 (e-p) and groups killed on days 18 and 19. cervix sections were stained for collagen, nerve fibers, macrophages, or neutrophils (n=6/group/day; 3 sections/ cervix; biol reprod 61: 879,1999; jsgi 12:578,2005) . stained macrophages and neutrophils were counted (image pro-plus 6, media cybernetics). results: e+p treatment for 15 days promoted hypertrophy of the cervix compared to v controls, i.e., collagen content and structure diminished, cell nuclei density declined, and nerve fibers increased. removal of p did not affect these endpoints. for immune cells, e+p for 15, 18, or 19 days decreased immune cell numbers. by contrast, p removal increased macrophages and neutrophils in the cervix on days 18 and 19 (p<0.05, e-p vs e+p groups, respectively). the census of resident immune cells in e-p groups at 24 and 48 h after p removal equaled that in the v group. conclusions: mimicking gonadal steroid concentrations in circulation during pregnancy promotes hypertrophy and suppresses immigration of immune cells in the cervix. in this non-pregnant murine model for parturition, progesterone withdrawal recruits immune cells, but fails to promote further remodeling or hyperplasia of nerve fibers in the cervix. the findings raise the possibility that ripening of the cervix requires not only recruitment, but also activation of immune cells. whether proinflammatory activities by specific immune cells affect nerve fiber hypertrophy or neural activity, as part of the mechanism for ripening of the cervix, remains to be determined. we have previously identified and characterized a truncated pr (pr-m), that localizes to the mitochondrion by multiple experimental techniques, including confocal imaging of a recombinant gfp fusion protein, western blot analysis after cellular fractionation of nuclear pr negative t47d-y breast cancer cells and western blot analysis of purified human heart mitochondrial proteins. initial studies with nuclear pr negative mcf-10a breast epithelial cells shown to express pr-m demonstrated an increase in mitochondrial membrane potential (mmp) with progesterone/progestin treatment. these studies led to the hypothesis that progesterone modulates cellular respiration via the mitochondrial receptor, pr-m. objectives: the present studies sought to further localize pr-m to the outer, inner or matrix portion of the mitochondrion and to correlate the increase in mmp with total cellular atp production. additionally, the potency of progesterone and synthetic progestins on the change in mmp was evaluated. methods: the location of pr-m was determined by western blot analysis after fractionation of human heart mitochondria with digitonin treatment and differential centrifugation. mmp was determined in mcf-10a breast epithelial cells and a673 rhabdomyosarcoma cells by the change in fluorescent emission of jc-1. total atp was determined by a bioluminescent assay. results: western blot analysis after mitochondrial fractionation showed pr-m localization exclusively in the outer membrane. a dose-dependent increase in mmp was seen in cell lines with 30-60 min treatment with progesterone and r5020 which were inhibited by a specific pr antagonist, rti-6413-049b, and not affected by the translational inhibitor, cycloheximide. similar changes in mmp were seen with the same concentration of progesterone, mpa and r5020. progesterone/progestin treatment for 120 min led to an increase in total cellular atp without a change in cell number. conclusions: progesterone/progestin treatment results in an increase in cellular respiration in cells expressing an outer mitochondrial membrane pr and known to lack nuclear pr expression. this may represent a mechanism whereby progesterone enhances cellular energy production to meet the demands of pregnancy. introduction pge 2 is a major product of the fetal membranes, decidua and myometrium and plays an important role in cervical ripening and myometrial contractions. there are four pge 2 receptors, ep-1 and ep-3 mediate contractions whilst ep-2 and ep-4 mediate quiescence. ep-4 contains multiple consensus sequences for transcription factors known to be of importance in labour, in particular nfkappab. we therefore performed experiments to determine the effect of activation and inhibition of nfkappab upon ep-4 expression. myocytes plated in 6 well plates were treated with il-1 (1ng/ml), to activate nfkappab. myometrial cells were also transiently transfected with nfkappab p65 sigenome smart pool and sicontrol non-targeting sirna to knock down nfkappab p65. rna was extracted for amplifying ep-4 using quantitative rt-pcr with amplification of l19 as a control to normalise data. il-1 caused an increase in expression of ep-4. knock down of nfkappab using si-rna resulted in a further increase in ep-4. this data suggests that although il-1 stimulates expression of ep-4 it does so through an nfkappab independent mechanism. the upregulation of ep-4 with sirna knock down of p65 suggests that activation of nfkappab would inhibit ep-4 expression consistent with the concept that activation of nfkappab at term causes the myometrium to adopt a more contractile phenotype. introduction: a role for the pro-inflammatory cytokine il-6 is suggested in preterm and term birth, independent of the presence of infection. we previously showed that mice with a null mutation in the il-6 gene (ko) delivered one day later than wild type (wt) mice due at least in part to altered timing of uterine expression of prostaglandin (pg) f 2 receptor, ptgfr, mrna. we also observed differences in mrna expression of other uterine activation proteins (uaps), pg h synthase (pghs)-2, oxytocin receptor (otr) and connexin-43 (cx-43), suggesting multiple physiological effects for il-6 in term delivery. objective: to examine the effect of il-6 deficiency on the peri-partal uterine mrna expression of the pge 2 receptors (ep) 2, 3 and 4; the post-partum changes of all uaps; and the relationship of these to serum progesterone (p 4 ) concentrations. methods: gestational length was observed in pregnant c57bl/6 wt (n=61) and ko (n=39) mice. uap mrna levels were measured by real time rt-pcr in wt and ko dams sacrificed from d15 through delivery and up to 24h postdelivery. serum p 4 was determined by ria. data were analyzed by one-way and two-way anova using the holm-sidak test to differentiate treatment effects at p<0.05. results: birth was delayed in the il-6 ko mice (20.7±0.2d vs. 19.7±0.1d), and this affected the timing of peri-partal changes for all uaps similarly. both ep2 and ep4 (relaxatory receptors) were elevated at d18 in ko dams, but returned to low levels before delivery and were elevated at delivery (ep2) or afterwards (ep4). ep3 levels did not change. otr increased several hours before delivery in all dams. cx-43 increased at delivery, then fell, while pghs-2 increased at delivery and remained elevated afterwards. ptgfr mrna increased 3-4-fold at delivery and a further 2-3-fold after delivery, suggesting loss of pgf 2 permitted enhanced ptgfr expression. p 4 serum concentrations fell pre-partum in both groups. conclusions: il-6 ko alters the expression pattern of several pregnancy and parturition-related genes and may delay the pre-partum p 4 fall, suggesting a potential ovarian effect. a uterine role for il-6 in regulating the timing of normal term parturition cannot be ruled out. objective: recent studies have highlighted the prevalence of vitamin d deficiency in pregnant women, particularly in those from ethnic groups with darker skin who require higher levels of uv light to make parental vitamin d. as the active form of vitamin d, 1,25-dihydroxyvitamin d 3 (1,25(oh) 2 d 3 ) is a potent immunomodulator, we postulated that vitamin d deficiency may lead to dysregulated placental immunity. both trophoblast and decidua express the enzyme 1 -hydroxylase (cyp27b1) which catalyzes synthesis of 1,25(oh) 2 d 3 from the inactive pro-hormone 25-hydroxyvitamin d 3 (25ohd 3 ). in view of the role of trophoblast as a barrier site protecting the fetus against infection, we investigated the impact of cyp27b1, 25ohd 3 and 1,25(oh) 2 d 3 on innate immune responses in trophoblastic cells. methods: 3a trophoblast cell line was used to assess the effect of vitamin d on innate immune responses. the cells were treated for 24 hrs with various concentrations of 1,25(oh) 2 d 3 (1-100 nm) and antimicrobial cathelicidin expression was assessed by real time pcr. to assess whether expression of cyp27b1 was affected by pathogenic stimuli, 3a cells were treated with ligands for toll-like receptor (tlr) 1-9, cyp27b1 expression (real time pcr) and 25ohd 3 utilization were assessed. results: 3a trophoblast cell line; which shows temperature-sensitive differentiation, revealed expression of cyp27b1 with higher levels of enzyme activity under conditions of syncytiotrophoblast development. cells treated for 24 hrs with 1,25(oh) 2 d 3 showed dose-dependent induction of the antimicrobial defensin cathelicidin expression (3.5-17 fold induction), whilst cells treated pro-hormone 25ohd 3 (100 nm) showed 2.5-fold induction of cathelicidin expression. activation of tlr3 (poly i:c) and tlr4 (lipopolysaccharide) enhanced the expression of cyp27b1 (2-and 2.5-fold) and increased the sensitivity to 25ohd 3 as a consequence. conclusion: these data show that autocrine synthesis of 1,25(oh) 2 d 3 from 25ohd 3 can stimulate trophoblast immune responses in a similar fashion to macrophages. as 25ohd 3 is the major circulating form of vitamin d, we hypothesize that trophoblast innate immunity may be significantly compromised under conditions of vitamin d deficiency. introduction: secretory leukocyte protease inhibitor (slpi) is a potent 12-kda protein inhibitor of neutrophil elastase and it is a mediator of mucosal immunity and an inhibitor of nfkb regulated inflammatory responses. however, its source, function and regulation within the uterus during pregnancy and at parturition are not well defined. it has previously been shown to be present in fetal membranes and cervical mucus and in amniotic fluid, where its levels is increased from second trimester to term and with a further increase at parturition. slpi has also been shown to be responsive to progesterone in human epithelial cells. our aim was to determine the effects of il-1 on slpi gene expression in human myometrium. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates and when cells were 70-80% confluent they were serum starved overnight and incubated with 1 m 6methyl-17 -hydroxy-progesterone acetate for 48h and with or without 1ng/ml il-1 for a further 6 hours. at the end of incubations rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of slpi, gapdh and beta-actin were measured by qpcr. results: 6 h incubation of uterine myocytes with 1ng/ml il-1 caused a marked increase in slpi by 201% (n=12; p<0.015). incubation of uterine myocytes with 1 m progesterone for 48h also increased slpi by 139% (n=12; p<0.05). incubation with il-1 for 6h in the presence of 48h with progesterone increased slpi: gapdh mrna ratio from 10.50 ± 3.37 to 33.33 ± 10.72 (mean ± sem; p<0.004). slpi expression was similar in the upper and lower segment myometrium in preterm patients. in term myometrium the slpi: beta-actin mrna ratio was increased by 50-and 200-fold in term labour versus term non labour samples in the lower and upper segment respectively (mean ± sem; p<0.05). these data show slpi is present in human myometrium and that it is increased by il-1 . progesterone also increases its expression. its expression is increased in term labour where its anti-bacterial, anti-fungal and anti-viral properties could allow it to act as an endogenous block to infections. objective: pre-b cell colony-enhancing factor (pbef) downstream mapk signaling and transcription factor activation. introduction: pbef is expressed in all layers of the human fetal membranes and the myometrium and is upregulated by labor, infection, nf-kb, ap-1 and stretching. pbef has the ability to protect a variety of cells from apoptosis, however it also appears to act as an insulin mimetic via the insulin receptor (fukuhara et al. science 2005: 307; 426-430) . its levels are elevated in obese patients, those with type 2 diabetes and in gestational diabetes. because pbef has poorly understood biological activities an investigation of mapk signaling and transcription factor activation induced by pbef has been undertaken in order to gain insights into its mechanisms of action. methods: primary aec were isolated from fetal membranes and treated with rhpbef (100ng/ml) for 1h, lysed and the resultant proteins labeled with cy3 or cy5 (amersham). there were used on a protein microarray containing 64 constituents of the mapk signaling pathways (sigma). isolated aec were transfected with luciferase constructs for nf-kb, ap-1, cre, hse and gre response elements using the exgen500 reagent. the cells were treated with rhpbef (0.1, 1.0, 10, 100 ng/ml) 4 hrs, lysed and 50ul of sample was analyzed for luciferase activity with dual-luciferase reporter assay system (promega). co-transfection with gfp and sv40 luciferase constructs to control for transfection efficiency and cell number (respectively) was also performed for each experiment. results: pbef significantly upregulated 20 mapk signaling components including 9 functioning as part of the erk pathways and 5 belonging to p38 signaling. it also activated nf-kb and camp response elements. however, it significantly down regulated 3 signaling molecules belonging to the jnk pathway that resulted in decreased c-jun phosphorylation. conclusions: pbef signaling in aec is consistent with its anti-apoptotic ability and its upregulation of the pro-inflammatory cytokines. although some mapk components responsive to pbef could be associated with insulin receptor signaling, some may not. therefore, it appears likely that pbef interacts with another potentially unique, but currently unidentified receptor. was the first effector to be characterised, but camp is now known to have other effectors, including camp receptor protein, cyclic nucleotide-gated channels and camp-guanine nucleotide exchange factor/exchange protein directly activated by camp (camp-gef/epac). two epac's have been identified and they consist of 4 functional domains: camp-binding domains, a dep domain, a ras exchange motif and a gef domain. our aim was to determine the presence of epac's in human myometrium and to study the effect of camp responses on prolabour genes such as il-8, pghs-2, otr and fp. methods: primary human uterine myocytes were isolated from non labouring myometrium and cultured in 6 well plates until 70-80% confluent. cells were serum starved overnight and incubated with 1μm methyl progesterone, 0.2mm sodium 8-bromo-camp, 0.1mm forskolin, 1μm kt5720, 75ng/ml brefeldin a and 0.1mm 8-pmeopt-2'o-me-camp either alone or in combination for 48h. rna was extracted and converted to cdna. paired upper and lower segment myometrial tissue was collected at caesarean section either before or after the onset of term or pre-term labour and frozen for extraction of rna (n=6 for ptnl, ptl, tnl and l). copy numbers of epac1, epac2, il-8, pghs-2, otr, fp, gapdh and -actin were measured by qpcr. results: epac1 and epac2 were present in the upper and lower segment of myometrium with epac1 levels being some 10-fold higher than those of epac2. there was no change with the onset of labour at or before term. treatment with il-1 for 6h significantly increased epac2 (n=12; p<0.012) but had no effect on epac1. when conditions mimicked pregnancy, (the presence of forskolin and progesterone), brefeldin a, an epac antagonist, increased basal pghs-2 and fp mrna expression (n=6; p<0.05), but had no effect on il-8 and tended to reduce otr. the il-1 -induced increase in pghs-2 and il-8, but not fp, was greater with the epac antagonist. conclusions: these data show epac's are present in human myometrium and that camp acts via epacs to reduce pghs-2 and fp expression during pregnancy. background: inflammatory events have been implicated in the process of labour. glucocorticoids mediate strong anti-inflammatory effects through binding to the glucocorticoid receptor (gr), which on activation translocates to the nucleus and either increases or decreases the expression of responsive genes thereby suppressing inflammation. objective: to characterise the expression profile for gr protein and mrna in human myometrium during fetal maturation and parturition. methods: western immunoblotting (wb) was employed to characterise gr protein expression in first (n=4) and second trimester myometrium (n=5), and in paired upper and lower segment pregnant (non-labouring, p, n=8) and labouring (l, n=8) myometrium; as compared to non-pregnant (np, n=8) control samples. immunofluorescence staining with confocal microscopy and rt-pcr were also undertaken. results: detection of gr protein by wb revealed two bands at 90-95 kda, representing the alternatively spliced isoforms, gr-and gr-. densitometric analysis showed that gr levels decreased significantly during pregnancy and remained at very low levels at term and in labour when compared with np samples (p<0.001); this decrease was seen for gr-and gr-. no significant temporal variations were observed in gr protein levels at term or during labour. gr protein was localised by immunofluorescence staining to the nuclei and cytoplasm of cells. less intense staining was apparent in p compared to np tissues; consistent with wb data. rt-pcr showed a consistent predominance of gr-to gr-mrna in all the tissues used. the observed decrease in protein expression was also mirrored at the mrna level: gr-mrna levels appeared to gradually decrease throughout gestation (p<0.05). differences between the upper and lower myometrial regions were only observed in the labouring samples, where gr-mrna levels were significantly decreased in the lower segment (p<0.05). nested pcr was additionally used to amplify gr-, but no consistent pattern of mrna expression was obtained. conclusions: these data are the first to characterise gr expression in human myometrium. spatial and temporal variations have been found with expression evolving through the three trimesters of pregnancy and in labour. further studies are now underway to evaluate whether gr contributes to the sequence of inflammatory events implicated in triggering term and preterm labour. these studies sought to determine the mechanism by which pas modulate the immune response. methods: 1) an in vitro co-culture model mixed with human cervical epithelial hela cells and pma-induced human macrophage u937 cells at epithelial/macrophage cell ratio of 5:1 was employed. 2) the co-culture was pre-treated with medroxyprogesterone acetate (mpa), progesterone (prog) and dexamethasone (dex) at concentrations of 1, 10, 100 and 1000 nm for 2 hrs followed by 2 day stimulation of 10 g/ml lipopolysaccharide (lps). these experiments were repeated using hela cells transfected with sirna for glucocorticoid receptor (gr). the production of cytokines il-1 , il-6 and il-8, il-10 and tnf were determined using elisas. 3) the co-culture was pre-treated with 100nm of each pas for 2 hours prior to lps stimulation at 10 g/ml for 0.5, 1.5, 6, 12, 24 and 48 hours. the phosphorylation of p38 mapk and gr and the expression of mapk phosphatase 1 (mkp1) were determined by western-blotting. results: il-1 , il-6 and il-8, il-10 and tnf were elevated by 3.8 fold, 1.7 fold, 1 fold, 4.9 fold and 4.6 fold in the co-culture model in response to lps(p<0.001 for all). pretreatment of mpa and dex, but not prog, inhibited the lps-induced cytokine production. the anti-inflammatory effect of mpa occurs in a dose-dependent manner. in the absence of gr, the inhibitory effect of mpa and dex on il-6, but not on il-1 , was lost. mpa and dex, but not prog, induced the phosphorylation of gr and expression of mkp1. p38 mapk was activated by lps for up to 24 hrs and pretreatment of mpa and dex attenuated this response. the ability of mpa and dex to suppress the inflammatory response in cervical tissues appears to be mediated through a gr-dependent pathway, specifically though p38 mapk. our studies suggest that prog is not a significant immunomodulator in these tissues. background: progesterone (p4) has been shown to play a critical role in maintaining pregnancy and preventing preterm birth. these effects are likely related to its immunomodulatory properties. we investigated whether camp plays a role in the immunosuppressive action of progesterone on fetal mononuclear cells. methods: umbilical cord blood mononuclear cells were isolated using density gradient centrifugation. to establish a p4 effect and optimize concentrations, cells were pretreated with p4 (10 -4 m-10 -6 m), dexamethasone (3um) or vehicle for 1 hour prior to overnight lps (10ng/ml) stimulation. supernatants were assayed for tnf-using elisa. ldh assays confirmed the absence of a cytotoxic effect. next, cells were pre-incubated with forskolin (adenylate cyclase activator) or db-camp (camp agonist) prior to lps to determine the effects of camp on tnf production. finally, cells were pretreated with rp-camp (camp antagonist) prior to p4 incubation and lps stimulation to determine whether the p4 effect was reversed by a camp antagonist. results: p4 significantly inhibited lps-induced tnf production in a dose dependent manner, with maximum suppression observed at 10 -4 m. both forskolin and db-camp suppressed lps-induced tnf production in a doserelated manner (fig 1) . finally, a dose-dependent partial reversal of tnf production was observed when cells were pretreated with rp-camp. (fig 2) conclusions: progesterone suppresses the inflammatory response in fetal mononuclear cells as measured by tnf expression. this effect is mimicked by adenylate cyclase activators and camp agonists and partially reversed by camp antagonists. this implicates the camp pathway in mediating the immunomodulatory actions of p4. objectives estrogen improves endothelial function after vascular injury via largely unknown mechanisms. endothelial progenitor cells (epcs) are known to be implicated in various vascular events requiring endothelialization. we hypothesized that estrogen and progesterone could influence the function and the change of epcs in menstrual cycle. material and methods peripheral blood mononuclear cells (pbmcs) were isolated from peripheral blood of healthy young women in each menstrual period by density gradient centrifugation with ficoll separating solution. pbmcs were seeded in endothelial basal medium with or without estrogen or progesterone. on the 4th day in culture, nonadherent cells were removed. on the 7th day in culture, adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. the number of ldl-and lectin-positive cells was measured as epcs using flowcytometry. the expression of estrogen and progesterone receptor mrna in epcs were measured by real time pcr in menstrual and luteal period. the number of epcs was significantly increased in the menstrual and luteal period compared with the follicular phase. estrogen and progesterone significantly increased the number of adherent epcs dose dependently in menstrual period, but not in luteal period. the expression of estrogen-alpha receptor in menstrual period was higher than luteal period. also, estrogen-beta receptor in luteal period was strongly expressed compared with menstrual period. these results suggest the expression of estrogen receptor and progesterone receptor play important roles to regulate epcs' proliferation during menstrual cycle. objective: elevated levels of fetal plasma avp are associated with the development of oligohydramnios, in part a result of avp-mediated reduction in fetal urine production. as avp urinary concentration effects are mediated via upregulation of renal tubular aqp1 water channels, we propose that avp modulates placental aqp channels, influencing bidirectional maternal-fetal water flow. we sought to study the effect of avp on trophoblast aqp1 gene expression using the first trimester-derived extravillous htr-8/svneo cells and the term placenta-like trophoblast carcinoma cells jeg-3. methods: cultures of both cell lines were treated with a physiological concentration of avp (0.1 nm) to determine aqp1 mrna and protein expression. negative controls consisted of cells incubated in medium supplemented with 1% fbs without avp. to determine whether avp regulation of aqp1 occurs by a camp signaling pathway, jeg-3 cells were preincubated with 100 μm 9-(tetrahydro-2'-furyl) adenine (sq22536), a cell-permeable camp inhibitor, before being treated with avp (0.1 nm). cells were incubated for 10 hrs for aqp1 mrna expression and for 20 hrs for protein extraction at 37°c. after harvest, real time pcr and western blotting analysis were used to detect the aqp1 mrna and protein expression levels, respectively. results: avp increased aqp1 mrna expression in both cell lines by 1.8 and 2.8 fold after 10 hrs. aqp1 protein expression paralleled the increase seen in the mrna (p<0.05). pretreatment of jeg-3 cells with sq22536 inhibitor completely blocked the stimulatory effect of avp. conclusion: aqp1 gene expression is up-regulated by avp in first trimester and term trophoblast cells, with a higher induction in the later. avp activation of aqp1 gene expression occurs via a camp mediated pathway, as the adenyl cyclase inhibitor blocked avp effects on aqp1 gene expression. these results suggest that increased fetal plasma avp may contribute to oligohydramnios by an increase in aqp-mediated fetal to maternal water flow. objective: changes in expression, ratio and activity of progesterone receptors within human myometrium have been proposed as potential contributory mechanisms for the functional progesterone withdrawal effect which precedes labour. progesterone receptor c (pr-c) is an n terminally truncated isoform of the full length progesterone receptor; pr-c has been shown to be abundant in fetal membranes, placenta and upper segment of laboring myometrium (1,2). the function and regulation of pr-c is at present unclear. in this study we aimed to investigate the regulation of pr-c in cultured human myometrial and amnion-derived wish cells. methods: myometrial primary cell cultures were prepared from non pregnant and term pregnant uteri. both myometrial and wish cell cultures were treated with natural progesterone (0, 100nm, 1mm, 10mm, 100mm) for 2, 6 and 24hrs. western immunoblotting was employed using nuclear and cytoplasmic extracts prepared from treated cells to observe the effect of progesterone on a) expression and b) subcellular localisation of pr-c within both cell types. immunofluorescent staining and confocal microscopy were also employed. experiments were also undertaken whereby nuclear and cytoplasmic extracts were subjected to shrimp alkaline phosphatase treatment to evaluate the phosphorylation status of pr-c in response to hormone. results: data indicates that a 60kda cytoplasmic protein, representing pr-c is abundant in myometrial and amnion cell cultures. we show that expression of pr-c increases in both cytoplasmic and nuclear fractions within both cell types in response to progesterone. evidence also suggests that pr-c is phosphorylated in a progesterone-independent manner. concurrent treatment with progesterone and the pr-antagonists ru486 and organon 31710 in amnion-derived cells still led to an increased abundance of pr-c but seemed to alter the phosphorylation status of pr-c. conclusion: pr-c expression and subcellular localisation within myometrial and wish cells alters in response to progesterone. speculation is generated about potential role of pr-c in reproductive tissues and its contribution to functional progesterone withdrawal. 1. taylor smooth muscle responses have been studied, data on regional blood flow (bf)distribution are scarce. we have studied the effect of a single dex course on relative bf distribution within the brain, skeletal muscle, heart, omental fat, placenta and adrenal in fetal sheep at 0.75 g, the equivalent of 30 weeks. methods: fetal sheep received amniotic, jugular, carotid and femoral artery and vein catheters at 104 days g (dg). experiments started at 110 dg in 8 dex (2 mg) and 8 saline treated controls (ctr), each receiving 4 injections 12 h apart. fetal blood pressure was recorded continuously (windaq pro+). microspheres (1.86x10 6 total; sterispheres, biopal) were injected at 11:00am into fetal jugular and femoral veins before maternal i.m. injection (t0) and 2 h after the 3rd injection (t3) in both groups. tissues were collected 12 hours after the 4th injection for assessment of bf (biopal). results: fetal bp increased above baseline after dex (6.8+1.5 mmhg; p=0.007) with no change in ctr (-0.1+0.4 mmhg). regional bf distribution is presented as a percent of the total counts per 100 g tissue (table 1) and after normalization within each animal to placentomal flow, shown previously to be unchanged by dex, in table 2 . no significant differences in flow were found. discussion: although dex altered bp, regional distribution of bf among key organs was unaffected. whether gc does not affect regional bf, or the rise in bp is the result of a non-specific, system-wide vasoconstriction, or the impact of gc on bf is similar in all organs, will be subject of further investigation that will include determination of regional vascular resistance and absolute flow. stimulator while crf-r2 is an inhibitor of gi motility at times of stress. we recently hypothesized that stress-induced in utero meconium passage in fetuses is analogous to stress-induced defecation in adult rats and likely mediated by crf pathway. in support, we demonstrated hypoxia-induced meconium passage in conjunction with marked increases in fetal plasma crf levels. as the mouse is a common experimental model, with known genome, we sought to determine the presence of crf-r1 and crf-r2 receptors in mouse fetuses. methods: time-dated cd-1 pregnant mouse were anaesthetized and fetuses (n=10) were collected at e17 (term = e19). whole gi tracts were dissected, fixed in bouin's solution, paraffin embedded and sectioned at five micron thickness. sections were immunostained with rabbit polyclonal antibodies to crf-r1 and crf-r2 by abc technique with vector abc reagents. immunoreactivity was identified as brown staining using 3',3 diaminobenzamidine as chromagen. slides were counter stained with mayer's hematoxylin and examined under the microscope. immunoreactive signals in the smooth muscle layers of gi tracts were quantified by image analysis using image pro 4.01 plus software. results: immunostaining for both crf-r1 and crf-r2 was seen in the smooth muscle layers of all lumens examined (7 lumens per section). the immunostaining intensity expressed as intensity over density (iod) in arbitrary units for crf-r1 (maximal iod: 0.369±0.024 au and minimal iod: 0.177±0.13au) and for crf-r2 (maximal iod: 0.318±0.015 au and minimal iod: 0.240±0.020 au) greatly varied between lumens. the mouse fetal gi tract expressed both crf-r1 and crf-r2 receptors, suggesting that the mouse is an appropriate model for fetal-stress induced neurovisceral motor responses. the non-uniform distribution of crf receptors in the fetal gi tract suggests the existence of regional differences in the expression patterns of crf-receptors of both types. we speculate that mouse devoid of crf and crf-r1 or r2 receptors will be useful to confirm the participation of crf pathway in stress-induced in utero meconium passage. antenatal exposure to glucocorticoids (gc) is associated with a reduction in nephron number and hypertension in adult life. one of the mechanisms for the development of hypertension is thought to be the long term effect of single nephron hyperfiltration. however, in most studies there is only a 20-30% reduction in nephron number with no change in gfr. thus, although a reduction in nephron number may be a contributing factor it is unlikely the only variable. the aim of the present study was to evaluate nephron mass, renal function and blood pressure in a cohort of adult sheep exposed antenatally to betamethasone. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hs apart at 80 days gestational age and allowed to deliver at term. at 1.5 yr of age in female (f) and male (m) offspring, glomerular filtration rate (gfr) was measured as inulin clearance and effective renal plasma flow (erpf) as pah clearance. blood pressure was measured though an indwelling catheter in the femoral artery over a 48-hour period. nephron number was measured by the acid maceration technique. data mean±sem were analyzed by anova and/or two sample t test. results: antenatal bm was associated with an elevation in arterial blood pressure and a reduction in nephron number in both f and m offspring. in contrast, a reduction in gfr and erpf was present only in males. plasma and urinary electrolytes as well as urinary protein excretion were not different from ctr. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on blood pressure regulation. interestingly, while the decrease in nephron number was of similar magnitude in m and f, evidence of alterations in renal function was present only in males. these suggest that the decrease in renal function observed in males is not solely a consequence of the decrease in nephron number. furthermore, it is possible that different mechanisms are responsible for the elevation in arterial blood pressure observed in m and f. hl 68728; hd p01 hd04784. clr l>t-x to evaluate the effects on blood pressure, urine output, sodium excretion and gfr of the intrarenal infusion of angiotensin 1-7 in the male sheep with or without prenatal exposure to b. we studied male sheep which had received either b (n=4) or vehicle (n=3) at 80-81 days gestation and were born at term. catheters were placed in the femoral artery, femoral vein, left renal artery and the bladder. the right kidney was removed. after 5 days recovery the sheep received an infusion of ang 1-7(1ng/kg/min) with or without its antagonist, [d-ala 7 ]-ang-(1-7)(d-ala, 10ng/kg/min), into the renal artery for 24 hours. blood pressure, gfr, urine output and sodium excretion were measured before and after the infusion. two-way analysis of variance (anova) was used to test mean values between the groups. with or without d-ala, map and urine output didn't change significantly during ang 1-7 infusion. however, the infusion of ang 1-7 resulted in a decrease (change from baseline) (p=0.029) in na + excretion of 0.41±0.15 meq/kg/24h in the vehicle animals and 0.12±0.05 meq/kg/24h in the b animals. there was no significant change in gfr during ang 1-7 infusion. gfr was lower in the b group during the combined ang 1-7 and d-ala infusion (91.5±3.5ml/min vs 117.0±15 in vehicle group, p=0.03). intrarenal infusion of ang 1-7 at 1ng/kg/min is followed by a significant decrease in sodium excretion but no significant change in urine output and gfr. the decrease tended to be greater in the vehicle animals and was not markedly attenuated by the antagonist d-ala. this suggests the effect of ang 1-7 may be mediated by a receptor other than the mas receptor for ang 1-7 and may be of lesser magnitude in animals exposed to b before birth. and norepinephrine (0.1-0.6 g/kg/min) and to an l-name (2 mg/kg) bolus were studied. vascular reactivity was analyzed by curve fitting of either the blood pressure or hr responses to obtain the maximal response. data are expressed as mean±sem of change from baseline. statistical significance was established using t test. results: bm-exposed animals displayed a higher blood pressure response to at-ii (atii max 142±4 vs 101±5 % of baseline, p<0.05, figure left panel). no differences in blood pressure or heart rate responses to norepinephrine infusion were detected in bm when compared to control animals (figure 1 right panel) . maximal response to l-name was also elevated in bm-exposed animals but did not reach statistical significance with the current sample size. conclusion: we have shown that antenatal gc treatment significantly increases blood pressure in sheep. here we show an enhanced in vivo response to at-ii. this response may contribute to the increased blood pressure observed. the greater response to l-name most likely represent an increased no production as a compensatory mechanism to the higher blood pressure observed. hl 68728. women at risk of delivering preterm are frequently treated with glucocorticoids to facilitate fetal lung maturation. animal studies have revealed that prenatal exposure to glucocorticoids may alter aspects of hypothalamic-pituitary adrenal functioning in later life. in this study we examined the effects of clinically relevant prenatal betamethasone treatment on the responsiveness of pituitary cells (in terms of adrenocorticotropin, acth, secretion) isolated from adult sheep. time-dated pregnant sheep were injected with betamethasone (0.17 mg/kg) or vehicle on days 80 and 81 of gestation. thereafter pregnancy was allowed to continue unimpeded and offspring were born. pituitaries were isolated from adult sheep aged between 6 and 9 months and cells were dispersed and plated at a density 2×10 5 cells per well in 48 well plates. after 48h, cells were stimulated with normal medium, 10nm arginine vasopressin (avp), 100nm avp, 1nm corticotropin releasing factor (crf) or 10nm crf for 2h. medium was collected and analyzed for acth using a commercially available kit. acth secretion (given as % increase from untreated cells) was similar between the control and betamethasone groups following 10nm avp (21.9 ± 4.3 vs. 20.4 ± 6.6) and 1nm crf (10.0 ± 2.8 vs. 10.7 ± 5.0). at higher stimulatory concentrations of both avp and crf, acth secretion remained similar in control cells (24.1 ± 4.9 and 11.8 ± 5.9 respectively), but was significantly decreased in betamethasone cells (9.9 ± 7.5 and 2.4 ± 4.3 respectively). these findings suggest that prenatal exposure to clinically relevant doses of betamethasone can alter pituitary responsiveness to both avp and crf in adulthood. this research was supported by nih grants hd47584 and hd11210. lcc is supported by an rjr-leon golberg fellowship in pharmacology and toxicology. objective: newborn meconium (mec) passage normally occurs within the first 24-48 h after birth. in contrast, more than half a million infants born annually in the us pass mec in utero. neither the physiological mechanism(s) nor causes for in utero mec passage are well understood, though both fetal maturation and stress are associated. we recently reported in utero mec passage and marked increases in plasma crf levels in term fetal rats exposed to maternal hypoxic stress. we hypothesize that stress-induced in utero mec passage is mediated by the crf pathway in a manner analogous to stress-induced defecation in adult rats. in the present investigation we examined placental crf content prior to and following maternal hypoxia, to determine the source of increased plasma crf. methods: time-dated pregnant rats (n=6) were exposed to a paradigm of graded, stepwise hypoxia on day 22 (term=22) (pediatr. res. 61: 176-179, 2007) . control pregnant rats were exposed to 21% oxygen for a similar duration as experimental animals. at the end of the study, placentas were harvested, fixed in 4% paraformaldehyde and paraffin embedded. sections (n=6 per placenta) were subjected to immunohistochemical analyses with rat/human crf antibody (peninsula laboratory) by abc technique. immunoreactivities on placental sections were quantified using the image pro 4.01 software and intensity expressed as arbitrary unit (au). all values are mean± sem. results: in control maternal rats exposed to normoxia, positive staining for crf was seen in decidua (0.206±0.015 au) and in all three major placental cells types (giant trophoblast cells: 0.143±0.019 au, spongiotrophoblast cells: 0.133±0.003 au and labyrinth cells: 0.083±0.008 au.) in contrast, in animals exposed to hypoxia, there was a total absence of crf staining in the placental cells, with only positive crf staining seen only in the decidua (0.197±0.006 au). conclusion: the total absence of crf staining in both the basal and labyrinth zones in placentas of pregnant rats subjected to hypoxic stress suggests that placental cells rapidly release crf into the maternal and fetal circulation in response to hypoxic-stress. our findings support our hypothesis that the peripheral crf pathway mediates in utero mec passage. offspring. angela g massmann, jie zhang, jorge p figueroa. department of obstetrics and gynecology, wake forest university school of medicine, winston-salem, nc, usa. objective: in rats and sheep, exposure to glucocorticoids (gc) in the perinatal period induces hypertension in adult life. recent evidence suggests that endothelin b (etb) receptor plays an important role in the regulation of sodium balance and blood pressure. renal medulla is an important site of expression and action of etb receptor. furthermore, in kidney it is the medulla (km) where the highest concentrations of immunoreactive et-1 and etb receptor are found. the aim of this study was to measure eta and etb expression in adult sheep kidney exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (v) 24 hours apart at 80 days of gestational age and allowed to deliver at term. at 1.5 yr of age, male and female offspring exposed to either vehicle or bm were euthanized and the kidneys harvested. kidney medulla (km) was obtained by sharp dissection. eta and etb protein and mrna expression were evaluated by western blot and rnaase protection assay. data are expressed as mean±sem and were analyzed by two way anova. results: a significant decrease in etb (f=5.166, p=0.030) but not eta protein was observed in km of bm exposed male and female adult sheep. at the mrna level, bm exposure also affected etb, but not, eta expression. however, bm treated animals had higher mrna levels than control sheep (f=7.05; p=0.01). conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects. the activation of etb receptor by et-1 inhibits sodium transport function in the collecting duct and the selective gene deletion of the etb in medulla results in hypertension.therefore, our finding of a significant reduction in etb protein suggests that there may be an impairment in the kidney's ability to regulate sodium reabsorption. the functional relevance of the alterations in etb protein expression as well as the discrepancies in mrna regulation need to be established. hl 68728; hd p01 hd04784. in the placenta, 11 -hydroxysteroid dehydrogenase type 2 (11 hsd2) regulates the transfer of cortisol. maternal glucocorticoid (gc) administration has been shown to affect the ovine fetal growth trajectory and regulate 11 hsd2 expression in mid and late gestation. however, little is know about the effects of gc administration in early gestation. we hypothesized that maternally administered low-dose dexamethasone (dex) in early gestation would affect 11 hsd2 expression, which will subsequently alter fetal birth weight. pregnant ewes were randomized and received 4 intramuscular injections consisting of either saline (2ml saline/ewe) or dex (0.14 mg/kg) given 12 hours apart starting on 40 days of gestation (dg). the animals were sacrificed at 50, 100, 125, and 140dg and fetal weights were recorded and placental tissue was collected. qrt-pcr was used to measure 11 hsd2 placental mrna expression. statistical analysis was done by anova with student's t-test posthoc. overall, there was no significant effect of dex treatment on placental 11 hsd2 mrna expression across gestation. however at 50dg, there was a significant increase in 11 hsd2 expression after dex (p=0.026). there was an increase in placental 11 hsd2 mrna progressively from 50dg (mean = 0.45) to 140dg (mean =1.48), independent of treatment. overall, a positive correlation between 11 hsd2 and fetal weight (r 2 =0.43) was apparent at 50dg and at 140dg (p=0.065) in both control (p=0.01) and dex (p=0.13). significant positive correlations between placental 11 hsd2 and fetal weight (r 2 =0.45) were found in females (p=0.01) and a similar trend was found in male fetuses (p=0.07). we conclude that in the sheep placenta, 11 hsd2 expression increases throughout gestation and may respond, at least transiently in early gestation, to elevations in maternal gc. the positive correlation between 11 hsd2 and fetal weight is consistent with the thesis that the fetal growth trajectory may be influenced by maternal cortisol levels and that placental 11 hsd2 is an important mediator of these effects. antenatal gc exposure induces hypertension in the young adult rat (neuroendocrinol; 1996; 64:412) and increases femoral vascular resistance in the lamb (jphysiol; 2003; 547:61) . unpublished own examinations in preparation of this study have shown age dependency of the vascular reactivity. vascular contractility of the middle cerebral artery (mca) decreased in response to k + and noradrenaline (na) between 3mo and 2,5y of age (p<0.01). vascular relaxation to acetylcholine (ach) but not to pge2 decreased during the same time (p<0.01). vascular contractility of the renal artery (ra) increased in response to k + (p<0.05). aims: to examine if antenatal gc alter vascular reactivity in the aged individual when cardiovascular diseases are predominant. we studied the ra because its vascular tone is involved in modulation of arterial pressure control (amjphysiol;2002;283:r441) and the mca because depressive disorders that are associated with dysregulation of the hpa axis (jcem;1997;82:234) increase stroke mortality for unknown reasons (amjpsychiatry; 2003; 160:1823) . to be clinically relevant, we administered dexamethasone at the dose used to enhance lung maturation in babies threaten premature labor. methods: pregnant dams received saline (n=6) or 170μg/kg dexamethasone (n=6) i.p. at day e19/20 equivalent to 2x12mg dexamethasone administered to a 70kg pregnant woman. at 2.5 years of age, vascular response of the ra and mca to endothelium-dependent and independent mediators was measured using wire myography. vessels were inspected histologically for intact endothelium. results: basal vasoconstrictory and dilatory responses to all mediators were less pronounced in the mca than in the ra probably reflecting autoregulatory properties of cerebral vessels (p<0.05). after prenatal dexamethasone exposure, contractility but not sensitivity to k + and na was enhanced in the mca and even more pronounced in the ra (p<0.05). relaxation to ach and pge2 was similarly diminished in both vessels after precontraction with na (p<0.05). conclusions: prenatal dexamethasone exposure at the dose used clinically increases renal and cerebral vascular tone in the aged rat by endotheliumdependent and independent mechanisms. this effect may be a potential mechanism of fetal programming of cardiovascular diseases in later life. betamethasone (bmz) therapy during pregnancy to improve fetal lung maturity may result in long term side effects, including hypertension, during adulthood. the kidney is an important organ which regulates systemic blood pressure via the local renin angiotensin system (ras). the major enzymes of the intrarenal ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the hypothesis of this study is that prenatal bmz exposure will result in alterations of the enzymes regulating the intrarenal ras. specifically, sheep that are treated with bmz in utero will have higher amounts of ace activity in kidney cortex compared to control animals. pregnant sheep of known mating date were either treated with vehicle (n=7) or with two doses of bmz (n=6), 0.17mg/kg, 24 hours apart at 80 days of gestation. renal cortex from the male offspring was harvested at 19-24 months of age. cortical membranes were then solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were separated by reversephase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results: bmz treatment resulted in a significant increase in ace activity compared to control animals (p=0.0073). ace2 and neprilysin levels were not significantly different between treatment groups. when expressed as a ratio of ace/ace2 activity, bmz treated animals exhibited a 2.5 fold greater proportion of ace activity versus control animals (p=0.04). this study demonstrates that bmz exposure during fetal life results in a significant increase in renal ace activity during adult life. this increase in enzyme activity would be expected to be associated with increased levels of ang ii in the kidney and na + retention, possibly underlying the development of hypertension. hd47584, hd17644. we recently hypothesized that stress-induced in utero meconium passage in term fetuses utilizes peripheral and/or central crf pathways in a manner analogous to stress-induced defecation in adult rats. as participation of central crf pathway requires the crf circuitry system in brain-gut axis, we exploited immunohistochemical techniques to address whether term fetal colon is wired by crf-nerve fibers. methods: fetal distal colon segments (n=6) were dissected from term ovine fetuses (146-147 d gestation). in addition, lumbosacral spinal cord with spinal roots and dorsal root ganglia (drg) (n=3) were dissected from ovine fetuses, and vagal nerve trunk with nodose ganglia (n=3) were dissected from mouse fetuses. paraffin sections fixed either in bouin's solution or zamboni's were subjected to immunohistochemistry with rabbit polyclonal antibodies specific to ovine-crf (ocrf). immunoreactivity on the sections was identified by standard abc technique, immunostaining quantified by image-pro plus software and expressed (intensity over area) as arbitrary units (au). results: ocrf antibody elicited strong positive staining on the serosal surface (port of entry for extrinsic nerve fibers) in distal colonic segments objective: we hypothesize that stress-induced in utero meconium passage is mediated by the crf pathway. ucn-i, similar to crf, is a potent contractility inhibitor of preterm ovine fetal distal colon, which has abundant crf-r2 receptors. both ucn-1 and crf thus may inhibit colonic motility and prevent preterm in utero meconium passage via crf-r2 receptors. however, ucn-i is reported to stimulate contractility of adult rat colonic smooth muscle strips more strongly than crf. we sought to study the pattern of ucn-i innervation in distal colon of ovine fetuses to delineate whether ucn-1 functions as a facilitator or inhibitor of colonic propulsive motility. methods: bouin's solution fixed, paraffin sections of very preterm (vpt: 118-120 days gestation), preterm (pt: 130-132 days), near term (nt: 140-142) and term (t: 146-147days) ovine fetal distal colon rings were subjected to immunohistochemistry with polyclonal antibodies to human ucn-1 (1:500 sigma) by abc system. intensity of ucn-1 immunostaining in smooth muscle layers and myenteric neurons were quantified by image-pro plus software and expressed (intensity/area) in arbitrary units (au). differences over time were assessed with anova. in all groups very preterm was significantly greater than term (p<0.05). conclusion: ucn-1 immunoreactivity in all three muscle layers, as well as myenteric and submucosal neurons, is highest at very preterm as compared to more mature gestations. these results suggest that the reduction of ucn-1 inhibitory function may facilitate stress-induced meconium passage at term. to evaluate the effect on blood pressure (bp), urinary output (uop), sodium excretion (nae) and gfr of the intrarenal infusion of ang ii in the male sheep after prenatal exposure to b. methods: we studied male sheep which had received either b (n=3) or vehicle (n=3) at 80-81 days gestation. catheters were placed in the femoral artery and vein, left renal artery and in the bladder. a right side nephrectomy was performed. after 5 days of recovery, the sheep were infused with ang ii (1 ng/kg/min) with or without candesartan (10 ng/kg/day) or pd123319 (pd 500 g loading dose and 10 ng/kg/min infusion) into the renal artery over 24 hours. bp, gfr, uop and nae were measured before and after the infusion. two-way analysis of variance was used to test mean values between the groups. results: bp and uop did not change with ang ii infusion with or without candesartan. the infusion of pd did not affect uop but did increase bp in the b sheep (p=0.03). ang ii decreased the nae in both groups(p=0.0007). candesartan increased nae among controls(controls 0.16 and b 0.56 meq/kg/h, p=0.048). pd infusion decreased nae among controls but this was not significant. baseline gfr was higher among vehicle compared to b animals (p=0.0172). during ang ii infusion there was a decrease in gfr among control compared to b sheep (-71.17 vs. -18.46 ml/min, p=0.0081). this difference was not seen during candesartan or pd infusion. conclusion: ang ii infusion led to a decrease in nae in both groups and gfr among controls but not b animals. infusion of ang ii with candesartan increased nae while pd decreased nae among controls but not b animals. this suggests that prenatal steroid exposure can alter at2 receptor mediated response in renal function by decreasing the effect of ang ii on renal sodium excretion. the differences in gfr suggest that this may be due to an effect on tubular sodium reabsorption rather than glomerular perfusion. antalarmin antagonism of acth-induced cortisol secretion by ovine adrenal cells probably not at acth receptor. nancy k valego, james c rose. center of research for ob/gyn, wake forest u. school of medicine, winston-salem, nc, usa. in addition to regulating the hpa axis, crh and its type 1 receptor (crhr-1) occur peripherally and in the female reproductive system. late in human pregnancy, a surge in placental crh probably stimulates adrenal secretion of cortisol and dheas requisite to parturition. we previously reported that, in dispersed fetal or adult ovine adrenal cortical cells, 24 hour incubation with the specific crh-r1 antagonist, antalarmin (ant), significantly reduced both cyclicamp and cortisol responses to acth, suggesting an interaction with acth-r (like crh-r1, a g-protein-coupled membrane receptor). however, forskolin (fsk; direct stimulant of adenylyl cyclase)-stimulated cortisol secretion was also attenuated by 24 hour co-incubation with ant. our objective was to clarify the effect of ant on binding of acth to its receptor after a short (2.5 hours) treatment. method: acth binding: the adrenals from adult sheep were obtained at necropsy and the cortex cells dispersed and plated @ 200000 cells/well. after 48 hours in culture, wells were rinsed 2x and refilled with serum-free dmem/ f12 containing vehicle (dmso) with or without ant. after 2.5 hours, wells were washed very gently and the binding assay (adapted from rainey et al; j biol chem,264:21474,1989) completed. triplicate vehicle or ant wells were treated with i 125 -tyrosine 23 human acth (1-39) with or without 10 -6 m acth (for non-specific binding). after 1 hour, wells were chilled, washed, and the cells lysed and counted on a gamma counter. fsk treatment: cells were treated as above except that fsk (10 -6 m) was added to the wells. after 2.5 hours, medium was removed and stored @ -80ºc for camp eia and cortisol ria. results (mean±se) of 2.5 hour incubation on acth binding and fskinduced secretion. vehicle antalarmin acth binding (net cpm) n=4 3024±415 2966±463 cortisol (ng/ml/2.5hr) n=7 144±20 126±19* (p=.012) camp (pmol/ml/2.5hr) n=12 9.1±2.0 4.8±1.1* (p=.003) * indicates significant difference from vehicle alone. conclusion: the crh-r1 antagonist, antalarmin, attenuates acthstimulated cortisol secretion but not by preventing acth receptor binding. however, its effect is early in the secretory process and is associated with a reduced camp response. objective glucocorticoids are often administered to pregnant women to prevent neonatal respiratory distress syndrome. prenatal steroid treatment increases blood pressure in adult sheep. exposure to excess corticosteroids before birth is hypothesized to be a key mechanism underlying the fetal origins of adult disease hypothesis and effects on the renin-angiotension system (ras) may modulate the steroid-induced increases in blood pressure. we therefore sought to determine if renin processing and secretion were altered in adult female sheep exposed antenatally to betamethasone ( ) and to compare them with data from males studied previously. methods pregnant sheep were randomized to receive 2 doses of 0.17 mg/kg of or vehicle, at 80 and 81 days of gestation; the offspring were studied at 6 and 18 months of age. in 17 female offspring, active renin concentration (arc) and total renin concentration in plasma were measured by ria of angiotensin i generated by incubation with excess substrate. prorenin concentration (prc) is the difference between total and active renin. nine or control exposed female animals born at term (142-148 days of gestation) were brought from the farm at 12-32 months of age, and had vascular and bladder catheters placed. five days after surgery, a sodium load of hypertonic nacl (0.0275 meq/kg/min at 0.55 ml/min) was given for 60 min. blood samples were obtained. data are expressed as mean sem and were analyzed by t test. the prc was significantly higher in the females than in the males (17.74± 1.15 vs 8.90± 1.46 p<0.0001) but there was no effect of prenatal steroid treatment. arc was similar in both genders. however, arc was a significantly greater percent of the total plasma renin concentration in the males (33.9± 12.7 vs 13.88± 3.06 p<0.0001) during the na infusion experiment, the exposed females had lower arc than did the control females (0.40± 0.08 vs 1.28± 0.20 p<0.05). conclusion the data suggest that prenatal exposure to didn't alter the processing and secretion of renin in adult female sheep. prenatal steroid treatment does not appear to alter the effect of gender on plasma renin levels in adult sheep.it seems unlikely that the elevated blood pressure seen in adult ewes after prenatal exposure is the result of increased secretion or processing of renin. supported by hd 47584 and hd17644. background: mrap is a recently discovered protein with alpha and beta isoforms, a common amino terminal region and divergent c-terminal sequences generated by alternative splicing. in human and mouse mrap plays an essential role in the generation of a functional, g-protein coupled acth receptor (melanocortin-2 receptor; mc2r) but has not been described for ruminants. we previously reported that lth fetal sheep exhibited elevated basal acth 1-39 yet decreased expression of key steroidogenic enzymes and the mc2r in the adrenal cortex while basal cortisol levels were not different from control. we hypothesized that mrap could play a key role in mediating the effect of lth as well as developmental regulation of fetal adrenal cortisol synthesis in fetal sheep. the goals of the present study were to determine the ontogenic pattern of mrap expression in the sheep fetus and to determine if lth alters mrap expression. methods: we searched the bovine genomic sequence database (www.ncbi.nlm. nih.gov/genome/guide/cow/) and found a sequence with >90% homology to the human mrap n-terminal 68 residues, with partial homology to the beta isoform in the carboxyl region ( 65%). using primers based on the bovine sequence, we confirmed the presence of mrap in the ovine fetal adrenal cortex. adrenal cortical tissue was collected from sheep fetuses from 105-120 (n=5) and near term (142-145; n=5) days of gestation (dg) as well as from lth (n=6) fetuses (exposed to high altitude hypoxia from 40 to 139-141 dg) and age-matched normoxic controls (n=6). cyclophilin was used as a housekeeping mrna. data are expressed in fg mrna/50 ng total rna. results: the expression of mrap, based on quantitative rt-pcr, was low between 105-120 dg (11.72 ± 4.2) and increased (p<0.05) approx. 5-fold near term (140-145 dg; 50.6 ± 7.3). levels of mrna for mrap were highly correlated to cyp17 mrna in individual samples. mrap expression in control (25.1 ± 2.5) and lth (26.5 ± 4.2) did not differ between groups. gender differences in hypertension are well described and there is growing evidence that the regulation of the renin angiotensin system (ras) is influenced by sex hormones. the major enzymes of the ras include angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin. the peptide products of these enzymes have opposing actions. angiotensin ii (ang ii), the product of ace, is a potent vasoconstrictor. on the other hand, the peptide products of ace2 and neprilysin, ang (1-7) and ang (1-4), exhibit vasodilatory properties. thus, modifications in the relative proportions of these enzymes and their peptide products can result in alterations of systemic blood pressure. the purpose of this study was to describe the gender differences in angiotensin converting enzyme (ace), angiotensin converting enzyme 2 (ace2), and neprilysin (nep) enzyme activities in adult sheep kidney cortex. renal cortex from male (n=5) and female (n=8) sheep were harvested at 15-31 months of age. the tissue membranes were solubilized and incubated at 37°c with either 125 i-ang i or 125 i-ang ii in the presence or absence of lisinopril to inhibit ace activity, mln4760 to inhibit ace2 activity, or sch39370 to inhibit neprilysin activity. the metabolic products were then separated by reverse-phase high performance liquid chromatography (rp-hplc). the rate of enzyme activity was then quantified by calculating the area under the curve for each product and converted to fmol of product per mg protein per minute of incubation. statistical analysis was performed using student's t-test. p<0.05 was considered significant. results ace activity was over 4 times greater (1282 ± 103.5 vs. 303.3 ± 118.4 fmol/mg/min, p<0.0001), ace2 activity was 2.5 times greater (1826 ± 312.5 vs. 721.2 ± 86.4 fmol/mg/min, p=0.02) and nep activity was nearly 2 times greater (761.3 ± 73.09 vs. 401.5 ± 39.81 fmol/mg/min, p=0.0081) in female kidney cortex. in adult sheep, key enzymes of the intrarenal ras have signficantly greater activity in female kidney cortex. these findings suggest that there are fundamental, gender specific, differences in the regulation of the enzymes of the intrarenal ras. the physiologic significance of these findings remain to be elucidated. hd47587, hd17644. in rats and sheep exposure to glucocorticoids (gc) in the perinatal period is associated with a reduction in nephron number and hypertension in adult life. furthermore, antenatal exposure to gc alters glucose tolerance in animals and in people. the aim of the present study was to determine 1) if insulin resistance is a contributing factor for the development of hypertension in adult sheep exposed antenatally to gc and 2) if diet-induced obesity has a more pronounced effect in sheep exposed antenatally to gc. methods: pregnant sheep were treated with two im doses of betamethasone (bm, 0.17 mg/kg) or vehicle (ctr) 24-hours apart at 80 days gestational age and allowed to deliver at term. at 9 mo of age, female sheep were randomly allocated to be fed at either 100% of recommended nutritional allowance or ad libitum for three months. sheep were chronically instrumented under general anesthesia to place intravascular catheters. insulin sensitivity was evaluated both by iv glucose tolerance test (ivgtt) and euglycemic clamp (hec)techniques. for the ivgtt a 0.25 g/kg glucose bolus was used and for the hec 4mu/kg human insulin was used. data mean±sem were analyzed by anova and/or two sample t test. results: ad lib fed sheep gain > 50% of the original weight. antenatal bm was associated with an elevation in basal and ivgtt plasma insulin values. as shown on the figure, diet-induced obesity significantly increased insulin plasma levels during ivgtt in bm-exposed adult female sheep (f=18.982;p<0.001). insulin sensitivity derived from hec was significantly decreased by obesity in bm-exposed adult female sheep. conclusion: our data show that prenatal exposure to a single course of gc at 0.55 gestation has long-term effects on glucose metabolism regulation. bm-exposed sheep exhibit alterations in glucose tolerance, hyperinsulinemia and insulin resistance. these abnormalities are exagertated by diet-induced obesity. hl 68728. syngergistic induction of 11 -hydroxysteroid deyhdrogenase type 1 expression by cortisol and interleukin-1 in human fetal lung fibroblasts. z yang, 1 p zhu, 1 cm guo, 1 l myatt, 2 k sun. 2 1 school of life sciences, fudan university, shanghai, china; 2 obstetrics and gynecology, university of cincinnati, cincinnati, oh, usa. objectives: glucocorticoids acting via glucocorticoid receptor (gr), serve as crucial hormones in fetal lung maturation. glucocorticoids and proinflammatory cytokines to induce 11b-hydroxysteroid dehydrogenase type 1 (11b-hsd1) which converts inactive cortisone to active cortisol, but their effect on 11b-hsd1 expression has not been addressed in human fetal lung. we examined the interactions and mechanism of cortisol and interleukin-1b (il-1b) effect on 11b-hsd1 in human fetal lung fibroblasts (hfl-1 cells). methods: the expression of 11b-hsd1 in hfl-1 was examined with immunocytochemistry and pcr. 11b-hsd1, prostaglandin h synthase-2 (pghs-2) and cytosolic phospholipase a2a (cpla2) mrna levels in cultured human fetal lung fibroblasts treated with cortisol and il-1b were measured with real time pcr. the roles of gr and c/ebps in the effect of cortisol and il-1b were studied using a gr antagonist (ru486) and transfection of plasmid carrying c/ebp-specific dominant-negative gene (cmv500-a/cebp). results: hfl-1 cells expressed a high level of 11b-hsd1. both cortisol (10 -8 -10 -6 m) and il-1b (0.1-10 ng/ml) induced 11b-hsd1 mrna expression in a concentration-dependent manner, an effect blocked by the mrna transcription inhibitor 5,6-dichlorobenzimidazole riboside (75 μm). ru486 (10 -6 m) blocked the induction of 11b-hsd1 by cortisol. induction of 11b-hsd1 mrna expression by cortisol (10 -6 m) was synergistically increased by co-treatment with il-1b (0.1-10 ng/ml) in a concentration-dependent manner. in contrast, the induction of cpla2 and pghs-2 expression by il-1b was inhibited by cortisol, suggesting a different mechanism of interaction. transfection of the cells with c/ebp-specific dominant-negative plasmid attenuated induction of 11b-hsd1 mrna expression by either cortisol or il-1b. these data suggest induction of 11b-hsd1 expression by cortisol is a gr dependent process involving c/ebps, which also mediate induction of 11b-hsd1 expression by il-1b. conclusions: cortisol and il-1b synergistically induce 11b-hsd1 expression in human fetal lung fibroblasts. this would lead to greater local cortisol production, perhaps providing either a self-attenuating mechanism for control of inflammation or a mechanism for enhancing fetal lung maturation when the fetus is exposed to cytokines e.g. with infection-induced preterm labor. do alterations in placental 11 -hydroxysteroid dehydrogenase (11 hsd) activities explain differences in fetal hypothalamic pituitary adrenal function following periconceptional undernutrition or twinning in sheep? kl connor, 1 pl van zijl, 2 cw rumball, 2,3 al jaquiery, 2,3 je harding, 2,3 mh oliver, 2,3 fh bloomfield, 2,3 jrg challis. medicine, university of toronto. periconceptional undernutrition (pcun) leads to activation of fetal hypothalamic pituitary adrenal (hpa) function, whereas twinning results in delayed fetal hpa activation. we hypothesized that these differences in fetal hpa activity were the result of altered patterns of expression of placental of 11 hsd isozymes and hence of the maternal glucocorticoid (gc) effect on the fetus. we developed a mass spectrometric assay for the measurement of 11 hsd-1 and -2 activities and validated this method against a widely used thin layer chromatography method. sheep were randomly assigned to ad libitum (n control) concentrates throughout gestation or were undernourished (un) from 60 days before until 30 days after mating to reduce maternal body weight by 15%, with ad libitum feeding thereafter. placentomes were collected on days 50, 85, 120, and 131 of gestation (term, 147d) and 11 hsd-1 and -2 activities were determined by measuring the rate of interconversion between cortisone to cortisol. with un, there was a trend towards lower 11 hsd-2 activity at d50 (p=0.1), a significant reduction at d85 (p<0.05), but no difference at d120 or d131. 11 hsd-1 activity was not different between n and un animals at any time. there was no effect of twinning on 11 hsd-1 or -2 at d50. however, with twins both 11 hsd-1 (p=0.01) and -2 (p<0.05) activities increased at d85. 11 hsd-1 activity was reduced in twins (p=0.06) at d120 but was higher than any singleton pregnancies at d131 (p=0.06). 11 hsd-2 was not different between singletons and twins at either d120 or d131. overall, 11 hsd-2 was lower in placentae of male compared to female fetuses in late gestation; 11 hsd-1 was higher in male than female fetuses. there was no interaction with un in either sex. we conclude that pcun and twinning result in alterations of placental 11 hsd activities in sheep. modifications in these enzymes during critical periods of fetal development may affect transplacental transfer or placental generation of gcs reaching the fetus potentially influencing the timing of activation of the fetal hpa axis, fetal maturation and later life development. methods: pregnant sprague-dawley rats had 50% mfr from day 10 of gestation until delivery. control animals had ad libitum food. offspring were sacrificed on day 1 of life (p1) and at 6 months (n= 6-8 per group). adrenals were dissected and snap frozen in liquid nitrogen for later extraction of rna. real time rt-pcr using specific rat primers was used to quantify mrna levels (18s as control). we evaluated the expression of 11-beta hydroxylase (cyp11b1), aldosterone synthase (cyp11b2), acth receptor (mcr2), p450 side chain cleavage enzyme (cyp11a1), star protein, 11 -hydroxysteroid dehydrogenase type 1 (hsd1) and type 2 (hsd2), glucocorticoid receptor (gr), and mineralocorticoid receptor (nr3c2). fold changes in mrna expression in controls and mfr offspring was compared by student's t-test. results: there was a marked downregulation in expression of cyp11b1 (p=.01), cyp11b2 (p=.04), hsd2 (p=.03), p450 (p=.05), acth receptor (p=.05), star (p=.01) and nr3c2 (p=.02) mrna in p1 mfr offspring, with no changes in hsd1 and gr. gender specific differences were found in the adult mfr offspring. in the male mfr offspring the expression of hsd1 and gr were significantly upregulated with a trend towards an increase in acth receptor (p=.07) whereas in the female mfr the expression of acth receptor (p=.02) was increased and nr3c2 (p=.03) and cyp11b2 were decreased (p=.04). in combined data for adult male and female offspring the expression of acth receptor was significantly (p=.02) increased. conclusion: these results indicate that mfr has a suppressive effect on steroidogenic enzymes of the newborn offspring regardless of gender. this may be an adaptive mechanism in the fetus/newborn to offset the high circulating maternal glucocorticoids in response to undernutrition. in adult male mfr offspring, increased hsd1 indicates an increase in gc synthesis whereas in the females there were no changes in gc synthesizing enzymes with a suppression of mc synthesizing pathway. the common finding of an increased acth receptor expression in both genders would suggest an increased sensitivity of adult mfr offspring adrenals to the effects of stress. objective: during gestation the fetal adrenal undergoes phases of active growth (40-90d), quiescence (100-120d) followed by reactivation (>135d) before birth. insulin like growth factors play an important role in stimulating adrenal growth throughout late gestation. interestingly the prepartum activation of the adrenal is delayed in the female compared with the male fetus, but the mechanisms underlying this delay are unknown. hypothesis: we hypothesize that there are gender specific differences in the gestational profile of adrenal igf2 mrna expression between male and female fetuses. methods: a total of 37 twin fetuses were used in this study. post mortem was performed at either 110-112d, 125-134d or 137-145d gestation. adrenal mrna expression of igf1, igf2, igf1r, igf2r and cyp17 was determined by qrt-pcr. results: fetal weight was not different between males and females, and increased (p<0.0001) with increasing gestational age. the relative adrenal weight was lower however after 125d when compared to the earlier gestation age group (p<0.0001). adrenal igf1 mrna expression was lower (p<0.0001) at 137-145d when compared to the earlier gestation age groups. interestingly, igf1r expression was highest at 125-134d (p<0.0001). there was an interaction between the effects of age and gender on adrenal igf2 and igf2r mrna expression such that the expression was higher in males compared to females at 125-134d (p<0.01), but was not different to either the earlier or later gestational ages. there was no effect of either gender or gestational age on adrenal cyp17 mrna expression. conclusions: it has been speculated that a delay in prepartum activation of the adrenal in female fetuses may be due to gender specific differences in the intra-adrenal bioavailability of igf2. in this study we have demonstrated there is an increase in both igf2 and igf2r mrna expression in males compared to female fetuses. this may be evidence that adrenal igf2 expression plays a role in the earlier activation of the adrenal gland in male fetuses. we have previously shown that in response to a secondary stressor, in vivo cortisol secretion is elevated in long term hypoxic (lth) ovine fetus despite lower acth receptor mrna expression, and no differences in plasma adrenocorticotropic hormone (acth) levels when compared to normoxic controls. the present study was designed to determine the potential mechanism(s) of this enhanced cortisol secretion. specifically we tested the hypothesis that post receptor signaling events including camp production and expression of steroidogenic acute regulatory protein (star) are enhanced following lth. methods: for the lth group, pregnant sheep were maintained at high altitude (3,820 m) from day 30 to near term. on days 138-141 (term = 146 days), fetal adrenal glands were collected from lth (n=6) and age-matched, normoxic signal transduction pathways that control embryogenesis, cell differentiation, cell proliferation and cell death. the roles of extracellular signal-regulated kinase1/2 (erk1/2) and p38 map kinase in the differentiation and invasion of human trophoblasts have been studied. however, the in vivo expression and activation of erk1/2 and p38 at the placental bed has not been elucidated. the study group consisted of placental bed biopsy tissues obtained from the pregnancies without preeclampsia (n=24) and with preeclampsia (n=8) between 31 and 40 weeks of gestation. we evaluated the expressions and phosphorylations of erk1/2 and p38 map kinase in the invasive trophoblasts in the placental bed tissues using immunohistochemistry. results: p38 and phospho-p38 map kinase were not detected in invasive trophoblasts in cases or controls. erk1/2 and phospho-erk1/2 were positive in invasive trophoblasts albeit with variable staining. phosphorylation of erk1/2 was significantly less frequent in invasive trophoblasts in placental bed biopsies from women with preeclampsia compared with normotensive controls. conclusion: these findings suggest that preeclampsia is associated with decreased activation of erk1/2 in invasive trophoblasts in vivo. objective: placentae from preeclamptic pregnancies are characterized by an excess of immature hyperproliferative trophoblast cells, however the molecular mechanisms regulating cell cycle progression in this pathology are unclear. our aim was to examine the expression of g1 phase cell cycle regulators in normal and preeclamptic placentae and to establish whether the hyperproliferative state of trophoblast cells in preeclampsia may result from a developmental delay. methods: human placental samples were collected from normal pregnancies throughout gestation (n=60) and from severe early onset preeclamptic (n=20), and age-matched control placentae (n=15). protein expression of cyclin e1, cyclin d1, cyclin d3 and cell cycle inhibitors, p15, p16, p21, and p27 was assessed by western blot analysis. spatial and temporal localization of cyclins e1, d1, d3, p21 and p27 was determined by fluorescence immunohistochemistry. expression of cyclin e1 and cyclin d1 mrna was evaluated by qpcr analysis. results: immunohistochemical analysis showed cyclin e1 and cyclin d3 to be localized to cytotrophoblast (ct) cells of the chorionic villi; additionally cyclin e1 was also expressed in the extravillous trophoblast cells of the anchoring columns. in contrast, cyclin d1 expression was predominantly restricted to the villous stroma. cell cycle inhibitor p27 was expressed in both ct and syncytiotrophoblast (st) cells whereas p21 was restricted to the st. during normal placentation, levels of both cyclin e1 and cyclin d3 were high in the first trimester and decreased with advancing gestation. the expression of cyclin d1, p27 and p16 showed an inverse correlation to cyclins e1 and d3 whereby their expression increased towards term. levels of p21 and p15 remained constant throughout pregnancy. preeclamptic placentae showed a significant increase in both cyclin e1 and p27 and a decrease cyclin d1 and p15 expression, as compared to age-matched control tissues. interestingly, preeclampsia was associated with an increased number of cyclin e1 positive progenitor ct cells in the floating villi and an increased expression of p27 in the endothelial cells lining the villous vessels. conclusion: preeclampsia displays an altered expression of g1 phase cell cycle regulatory molecules portraying an expression profile that closely resembles that of first trimester placentae. (supported by cihr, owh/igh). we have also demonstrated elevated, hif-1-mediated placental and circulating sflt-1 at high altitude. we sought to correlate circulating levels of plgf, free vegf and sflt-1 with maternal and fetal oxygen tensions. we hypothesized that circulating sflt-1 and free vegf would be increased at 3600 m, and that plgf, known to be up-regulated by higher oxygen tension, would be decreased. methods: we collected both serum and plasma samples, the latter treated with inhibitors of platelet activation. maternal and umbilical samples were obtained from 77 and 65 healthy mother-infant pairs living at 400 m and 3600 m respectively. plgf, free vegf and sflt-1 were measured in both serum and plasma using commercially available elisa kits (r d). data were log-transformed and analyzed by unpaired student's t test or anova as appropriate. results: plgf did not differ between altitudes. free vegf (7±2 pg/ml vs. 12±2 pg/ml, p<.05) and sflt-1 (25.7±2.4 vs. 31.4 ±2.2 ng/ml) were increased at 3600 m. however concentrations of all the angiogenic growth factors were reduced when platelet activation was prevented (-35±3% plgf; -85±5% free vegf; -24±2% sflt-1, p<.001). cord blood free vegf was 100-fold greater than in the mothers, but inhibition of peripheral cell activation abolished detectable levels in 95% of the babies. similar results were obtained for sflt-1. thus, while free and total maternal circulating vegf and sflt-1 are elevated at high altitude, these increases are due to activation of peripheral blood cells. moreover, free vegf does not exist in detectable amounts in human pregnancy. there was no relationship between variation in the angiogenic growth factors and maternal or fetal oxygen tensions. the objective of this study is to identify candidate genes responsible for the variance between severe preeclampsia (spe) and hellp syndrome (hs). placental biopsies from spe (n=6) and hs (n=2) were collected. diagnosis of spe was confirmed by blood pressure and protein criteria. hs was diagnosed in preeclamptic patients who developed characteristic laboratory abnormalities. placental tissues were embedded in oct for sectioning, h e staining and rna isolation (invitrogen, carlsbad). gene expression data was obtained by hybridizing fluorescently labeled reverse transcription products to spotted cdna microarrays. the microarrays were produced by the laboratory of microarray technology at the van andel institute (grand rapids, mi) using a custom microarrayer. microarrays were scanned using an agilent g2505b scanner. images were analyzed using genepix 5.0 (axon). limmagui was used to generate lists of discriminating genes for these data, while david provided functional annotations. there were 310 differentially expressed genes between spe and hs (p<.05). among these candidate genes, 233 were up-regulated and 77 were downregulated. the most up-regulated genes are 5(3)-deoxyribonucleotidase (dnt-2), superoxide dismutase 3, hydroxy-delta-5-steroid dehydrogenase, caspase 9, and general transcription factor iih. further analysis of functional groups revealed the most enriched gene categories are related to cellular energy (mitochondria), cell cycle regulation, and protein metabolism. the underlying role of the placenta in the development of hs is currently unknown. this study shows that there is a relatively mdest number of genes that are differentially expressed between hs versus spe placentals. thes data provide the molecular context for placental changes seen in this variant of preeclampsia and provides an opportunity to study the role of the effected genes in disease variance. (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and control group (29 patients) uncomplicated pregnancies. total rna was isolated and reversely transcribed to c-dna. the mrna level of tert was detected with a probe-specific real-time quantitative pcr assay using ß-actin as the reference gene. crossing point (cp) values were obtained during the pcr amplification and the relative expression level of htert equals to 2 (cp htert -cp ß-actin) . statistical analysis was performed using the student's t test. immunohistochemistry (ihc) staining was employed to localize htert protein on placenta tissue sections using abc method, incubation with rabbit anti-htert antibody followed by application of a goat anti-rabbit antibody with results evaluation using microscope. objective tgf s are involved in the regulation of trophoblast differentiation and invasion, and we have previously reported that tgf-3 is over expressed in pre-eclamptic placentae. tgf s signal via a receptor complex composed of type ii (t r-ii) and type i (t r-i) receptor. to date, seven type i receptors, designated as activin receptor-like kinase (alk1-7) have been identified. our aim was to investigate the expression pattern of alk1 and alk5 receptors, known to exert tgf signaling, in preeclamptic and iugr placentae. methods human placental tissue throughout gestation was used in order to determine the development profile of the receptors. in addition, placental tissue from preeclamptic and iugr pregnancies and from age matched controls was collected. all iugr pregnancies were characterized by absence of end diastolic velocity in the umbilical artery and had no evidence of preeclampsia. expression of alk1 and alk5 mrna was measured by real-time pcr analysis, and protein by western blot analysis using alk1 and alk5 antibodies. immunoblot analysis demonstrated a unique developmental profile whereby alk1 expression increased with advancing gestation. in preeclamptic placentae alk5 expression was significantly increased compared to preterm and term controls, whereas alk1 expression was significantly decreased. preeclamptic placentae also exhibited decreased phosphorylation of smad1, a tgf signaling molecule, which is activated by alk1 and increased phosphorylation of smad2, which is triggered by alk5. the expression of alk1 and alk5 in iugr placentae differed from that of preeclampsia as both alk1 and alk5 mrna levels were significantly increased in iugr compared to preterm and term controls. however, only alk5 expression was significantly increased in iugr placentae at the protein level, while no differences in alk1 protein levels were noted between iugr and controls. conclusions imbalance between alk1 and alk5 signaling pathways might play a role in the pathogenesis of preeclampsia and iugr. as tgf signaling via either alk1 or alk5 has been found to differentially regulate vasculogenesis, changes in these signaling pathways may contribute to the altered vasculogenesis found in these pregnancy-related disorders. (supported by cihr and owh/igh). (14), severe preeclampsia (10), hellp syndrome (4) and eclampsia (4) and 29 patients, the control group who had uncomplicated pregnancies. total protein was isolated from placental tissue. gadd45a and its downstream signal proteins--phospo-p38, phospho-mkk3/mkk6 were assessed by western blot. immunohistochemistry (ihc) staining was employed to localize the expression of gadd45a and sflt-1 proteins in placenta tissue sections using abc method. huvec cells were cultured to 80-90% confluence and were divided into 3 groups: control, stress induction (sorbitol, 0.3m, 4h), p38 inhibition (sb-203580, 1ug/ml, 5ug/ml and 10ug/ml, 1 hour) + sorbitol (0.3m, 4h). total protein was isolated from cells and the supernatant of huvec was collected. western blot was processed to detect the induction of gadd45a and phospho-p38. supernatant sflt-1 was measured with an eia kit and the results were read at 450nm wavelength. results: gadd45a protein was elevated in the preeclamptic placentas with its downstream proteins (mkk3 and p38) activation compared with control. overexpression of gadd45a and sflt-1 in preeclamptic placentas was observed with ihc staining. in huvec cells, gadd45a was induced by sorbitol, triggering the activation of the downstream p-38 pathway and the accumulation of sflt-1 in the supernatant. the up-regulation of sflt-1 secretion by inducing gadd45a was depleted when treated with p-38 inhibitor. conclusions: our study reveals that gadd45a and its down stream p-38 pathway were activated in preeclamptic placentas and this stress inducible signal pathway regulates the secretion of sflt-1, which is a key player in preeclampsia. it provides novel evidence that links placental stress to sflt-1 secretion via the gadd4. trophoblast adipose triglyceride lipase (atgl) expression is upregulated in preeclampsia. beth a plunkett, 1 jennifer a doll, 2 emily j su, 1 serdar e bulun, 1 mona cornwell, 2 susan e crawford. 2 1 obstetrics and gynecology, northwestern university, chicago, il, usa; 2 pathology, northwestern university, chicago, il, usa. objective: preeclampsia is characterized by placental endothelial cell dysfunction and elevated maternal triglyceridemia (tg). tg traverse the placenta in a process of uptake followed by lipolysis with subsequent release of fatty acids to the fetus. although three placental lipases (hormone sensitive lipase, endothelial lipase and lipoprotein lipase) have been identified, they do not account for all lipolytic activity. here, we introduce a new lipase, adipose triglyceride lipase (atgl), which is responsible for the hydrolysis of triglycerides to diglycerides in adipocytes and was recently identified as a receptor for endothelial cell modulator pigment epithelium-derived factor. the purpose of this study is to determine if expression of atgl, a potential modulator of both lipid metabolism and vasculature, is upregulated in preeclampsia. methods: immunohistochemical studies were performed on placental tissues from normal pregnancies (n=5) and those complicated by severe preeclampsia (n=5) with anti-atgl antibodies. the degree of positivity in the trophoblasts and endothelial cell was scored (1=none, 2=spotty, light, 3=consistent, dark). to determine if atgl is a product of placental endothelial cells (plec), microvascular cells were isolated from normal placental tissue. purity of the sample was confirmed using flowcytometry (>2/3 positivity for factor 8 antigen). atgl was detected immunohistochemically and via western blot using anti-atgl antibodies. results: mean atgl expression in preeclamptic trophoblast was significantly higher than normal placentas (3.0 + 0 standard deviation versus 2.3 + .39, p = 0.018). endothelial expression was not significantly different in preeclamptic (2.6 + .41) versus normal placentas (2.3 + .39, p>0.05). atgl stained intensely and demonstrated a beaded pattern in the endothelial cells, suggestive of a lipid droplet pattern. immunohistochemistry of plec and western blot analysis of cell lysates revealed strong immunopositivity for atgl, although at a smaller size than anticipated. conclusion: these findings demonstrate that a novel lipase, atgl, is produced by trophoblasts and is upregulated in severe preeclampsia. plec express high levels of atgl, suggesting that atgl could prove to be important in the vascular dysfunction and lipid abnormalities characteristic of preeclampsia. increased endothelial chymotrypsin-like protease (chymase) expression is responsible for endothelial activation in preeclampsia. yuping wang, yang gu, yanping zhang, david f lewis. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: endothelial (ec) activation is an important component of inflammatory phenotypic changes in preeclampsia (pe). our previous study showed enhanced chymotrypsin-like protease (clp)/chymase expression in the maternal vessel endothelium in women with pe. in this study, specific effect of placental-derived clp on ec activation was examined. methods: human uterine microvascular endothelial cells (utmvecs) were used. placental conditioned medium (cm) was prepared by culturing villous explants from normal and pe placentas. confluent utmvecs were treated with placental cm with or without depletion of chymotrypsin. ec adhesion molecule expressions for icam, vcam, p-selectin and e-selectin were determined by a colorimetric assay at od 450nm. depletion of chymotrypsin from cm was performed by immunoprecipitation. to further determine if activation of endogenous clp/chymase in ecs is responsible for up-regulation of p-selectin and e-selectin expression, chymase sirna was applied to ec culture before the cells were treated with normal or pe cm and then ec adhesion molecule expressions were examined. data was expressed as mean ± se and analyzed by anova. a p level < 0.05 was set for statistically different. results: 1) expressions of vcam, p-selectin and e-selectin, but not icam, were significantly increased in pe-cm treated utmvecs compared to those of normal-cm treated cells and untreated controls, p<0.01; 2) there was no difference for adhesion molecule expression in utmvecs between normal-cm treated with untreated controls; 3) utmvecs transfected with chymase sirna significantly reduced p-selectin and e-selectin expressions when exposed to pe-cm, p<0.05. conclusion: placental-derived clp/chymase is responsible for activating ecs and inducing ec adhesion molecule expression. activation of ec chymase may be directly related to the inflammatory phenotypic changes that occur in ecs in pe. (supported nih grants hd36822 and hl65997). corin is a transmembrane serine protease that is important in processing natriuretic peptides (nps) and maintaining normal blood pressure. genetically modified mice without corin function develop a syndrome during pregnancy similar to preeclampsia. corin is present in the pregnant uterus and a deficiency in the enzyme may lead to hypertension and preeclampsia. we tested the hypothesis that corin expression was increased in human myometrium from women with preeclampsia. methods: myometrium was obtained from 4 groups of women at the time of primary cesarean section: (i) preterm no labor with preeclampsia (n=3, ptspe, 28.9 weeks); (ii) preterm labor (n=3, ptl, 30.9 weeks); (iv) term no labor (n=3, tnl, 39.6 weeks); (v) term labor (n=3, tl, 39.7 weeks). microarray gene profiling was performed using affymetrix human genome u133 plus 2.0 arrays. women who were not in active labor at the time of delivery (tnl) served as the control group. conventional and real time pcr was performed to verify corin expression in the arrays was directionally accurate. corin protein expression was examined by western blot. results: compared to tnl control, corin levels decreased nearly 2-fold in myometrium from women in term labor (tl). there was a small increase in corin gene expression of preeclamptic women (ptspe) and little-to-no change in myometrium from women in preterm labor (ptl). these results were confirmed by pcr analysis. conclusion: blood volume surges during pregnancy, increasing the potential for hypertension and preeclampsia in the mother. the corin-np control system could contribute to this condition. however, there are no reports on corin message or protein levels in women with preeclampsia. our preliminary results suggest that preeclampsia is not a consequence of a corin deficiency (decreased corin preeclampsia). acknowlegement: supported by grants from the phs (r01 hl049041-12, cpw) and cdc grant (u7dp00187-03, cpw). the expression of gp130 at implantation site: implication for the pathogenesis of preeclampsia. objective: gp130 is a common shard signal transducing subunit of the il-6 cytokine family which is critical for implantation, and viewed as marker of endometrial blastocyst receptivity. preeclampsia (pe) is highly related to the restricted trophoblast invasion, which leads to impaired spiral artery remodeling. our hypothesis was that the poor placentation of pe is associated with the altered expression of gp130 at the implantation site. methods: human decidua from patients with pe and uncomplicated term deliveries (n = 9, respectively) were immunostained for gp130. the intensity and distribution of immunostaining on decidual cells and extravillous traphoblasts were evaluated with hscore. statistical analysis of the data was performed using student's t-test and kruskal-wallis one way anova on ranks followed by post hoc test. results: immunostaining of gp130was significantly higher in decidual cells of patients with pe compared with normal specimens, with hscore (median, [interquartile range]) value 180 [142.5-185.0] and 120 , respectively (p<0.05). in pe specimen, the hscore of decidual cells was also significantly higher than that of trophoblast (40 [30-50]; p<0.05). there were no difference in the comparison of hscore of trophoblasts between the pe and normal specimens, and in normal specimens between decidal cells and trophoblasts. conclusions: the increase of gp130 expression in the decidual cells of preeclamptic placenta may be implied to the pathogenesis of poor placentation in preeclampsia. we have previously demonstrated that decidual cells are the major source of renin at the human uteroplacental interface, but little is known regarding the human decidual renin expression in hypoxic conditions. therefore, the present study was undertaken to determine the effect of hypoxia on renin secretion by human decidual cells in vitro. methods: full-term normal human placentas were obtained within one hour of vaginal deliveries or cesarean sections. decidual cells were isolated from the decidua parietalis. after an initial culture for 2 3 days in a serum-containing medium, the decidual cells were exposed to normoxia or hypoxia (10% or 3% oxygen) in a serum-free medium for 24 hours. the culture supernatants were then harvested and subject to western blot analyses of renin protein contents. a dominant band of renin at approximately 47 kd was detected in all samples. when compared with the cells cultured in the normoxic condition, the cells cultured in both hypoxic conditions (i.e. 10% and 3% oxygen) had significantly lower renin protein contents in their culture supernatants. conclusion: our data for the first time show that hypoxia down-regulates renin secretion in human decidua, suggesting a link between the uteroplacental ras and oxygen tension during human gestation. the effect of nucleated fetal red blood cells derived from preeclamptic patients on endothelial progenitor cell proliferation. keiichi matsubara, emiko abe, yuko matsubara, shinji hyodo, masaharu ito. obstetrics and gynecology, ehime university school of medicine, toon, ehime, japan. objectives inadequate uteroplacental circulation results in placental ischemia and the development of preeclampsia (pe). endothelial progenitor cells (epcs) are thought to be a key player in the fetal angiogenesis. vascular endothelial growth factor (vegf), which is up regulated in pe, is involved in epcs proliferation. recently, it was reported that fetal nucleated red blood cells (nrbcs) have the capability to generate vegf. we hypothesized that nrbcs could influence epcs proliferation in the placenta and may be involved in the pathogenesis of pe. material and methods mononuclear cells (mncs) were isolated from the umbilical venous blood of normal pregnant women and preeclamptic patients by density gradient centrifugation. mncs were incubated with anti-cd71 antibody conjugated with microbeads. nrbcs were collected using a mini macs separator. nrbcs were incubated for 24 hours with or without angiotensin ii (ang ii) and erythropoietin (epo). vegf and placental growth factor (plgf) concentrations in the supernatant were measured using elisa. also, pbmcs without nrbcs were seeded in endothelial basal medium with or without nrbcs using a boyden chamber. these samples were incubated for 7 days with or without ang ii and epo. the adherent cells were incubated with di-ldl, fixed with paraformaldehyde, and stained with fluorescein isothiocyanate-labeled lectin. di-ldl and lectin positive cells was considered to represent epcs and the number was measured using flowcytometry. the number of nrbcs derived from umbilical venous blood was significantly increased in pe. both ang ii and epo significantly increased vegf concentration in the supernatant of nrbcs derived from normal pregnant women. however, ang ii and epo did not influence the nrbcs' vegf production in pe patients. plgf was not detectable in the supernatant. the number of epcs in the umbilical venous blood was significantly decreased in pe and the number was not changed by nrbcs. on the other hand, the number of epcs was significantly decreased in the culture with nrbcs. epo significantly increased the number of epcs in pe at the lower concentration of epo compared with normal pregnant women. conclusions it appears that fetal nrbcs may inhibit fetal epcs proliferation in pe. since epo reduced the inhibitory reaction of nrbcs without vegf production epo may affect epcs proliferation independently of nrbcs. relationship between the hypoxia-inducible factor-2 (hif-2 ) and the receptor svegf-r1/sflt-1: implication for phatophysiology of preeclampsia. julio e valdivia-silva, 1,3 juan c gonzalez-altamirano, 2 keisy lopezthe trophoblast invasion is critical for the establishment of the uteroplacental circulation. at early phases of this process local oxygen pressure in the placenta is lower, that pathologically in preeclampsia remain constant. because of this, is important to understand the response of placental cells against these stimuli. in the present work, we use primary cultures of trophoblast cells, fibroblasts of the villous, and human umbilical endothelial cells, isolated of preterm and term placentas (with and without preeclampsia), to explore the effect of the oxygen pressure in the expression and synthesis of vegf, svegfr-1/sflt-1, hif-1 and-2 . our results show that the low pressure of oxygen resulted in a significant increase of the mrna and the protein of the receptor svegf-r1 selectively in the cts. the vegf's expression and synthesis was raised in three cellular types, but the free protein (not bounded to svegf-r1) of the cts was diminished. on the other hand, the expression of the arnm of hif-1 or -2 in cells was comparable in all the types of placentas, nevertheless, the protein hif-2 was more increased in the cts of preeclamptic placentas. to evaluate the relation of hif-2 and the increase of the receptor svegfr-1, we used sirna-hif2 . in response to the inhibition, the expression of the receptor svegf-r1 diminished dramatically. the blockade of hif-2 did not alter vegf's expression. our data are the first that propose that the protein of the factor of transcription hif-2 is one of the molecules involved in the selective expression of the receptor svegf-r1 in trophoblast cells during hypoxia. figure: reduction of the expression to soluble receptor flt-1/svegfr-1 after transfection with sirna-hif2 in trophoblast cells. sirna= interference rna, lu=luciferase. luc-sirna was used as control. effects of estradiol on synthesis, secretion, and activation of von willebrand factor in endometrial endothelial cell. shumei zhao, chainarong choksuchat, michael s scholfield, todd d deutch, thomas d kimble, david f archer. obstetrics and gynecology, eastern virginia medical school, norfolk, va, usa. objectives: heavy menstrual bleeding (hmb) is a serious clinical condition affecting 30% of women. due to inefficient and ineffective medical interventions, women with hmb often elect endometrial ablation or hysterectomy to eliminate hmb symptoms. morbidity and loss of fertility linked to these surgical treatments support the search for more effective medical remedies. von willebrand factor (vwf), a principle initiator of blood clotting produced by endothelial cells. women with von willebrand disease (vwd), have a high incidence of hmb indicating poor clotting. these findings suggest vwf heavily impacts the amount of blood loss during menstruation. estrogen stops/reduces hmb and has been used to treat hmb in women with vwd, although the mechanism(s) is unknown. this proposal addresses the hypothesis that estradiol (e 2 ) increases the synthesis and activation of vwf, promoting clotting. materials and methods: immortalized human endometrial endothelia cells (heecs) were used for the studies. to determine if e 2 increases synthesis of vwf, we treated heecs with e 2 at 0.001μm, 0.01μm and 0.1μm for 24 hours. vwf protein and mrna levels were determined by western blotting and real time pcr, respectively. to establish if e 2 can convert vwf from an inactive to an active conformation, the release of activated vwf by cells into culture medium will be assessed by elisa. decreased adamts13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) activity results in increased amounts of active vwf. rrelease of activated adamts13 will be determined by fret. to ascertain if e2 regulated vwf secretion is by genomic pathway, heecs will be exposed to the estrogen receptor antagonist ici 182,780. elisa and fret will assess the release of active vwf and adamts13, respectively. results: western blotting demonstrated e 2 increases vwf mrna and protein levels in a dose-dependent manner in heecs.conclusion: the project will determine if e 2 acts at the heecs to increase synthesis, secretion and activation of vwf. preliminary results show e 2 increases intracellular vwf protein and mrna levels in heecs, supporting our hypothesis that e 2 increases synthesis of vwf in heecs in vitro. if e 2 increases activated vwf, it could reduce/stop hmb by increasing activated vwf, providing justification for a clinical trial of e 2 to treat hmb. over-expression of vegf-d does not induce lymphangiogenesis in the mouse endometrium. peter a rogers, 1 jacqui f donoghue, 1 marc g achen, 2 steven a stacker, 2 jane e girling. 1 1 obstetrics gynaecology, monash university, melbourne, victoria, australia; 2 ludwig institute for cancer research, melbourne, victoria, australia. background: the human endometrial functionalis has reduced lymphatics compared to the basalis and myometrium 1 . this study examines the distribution of lymphatics in mouse uterus and investigates if over-expression of the lymphangiogenic growth factor vascular endothelial growth factor-d (vegf-d) stimulates growth of new endometrial lymphatic vessels. methods: the distribution of uterine lymphatics was examined in c57bl/ 6jxcba mice collected during the oestrus cycle, early pregnancy and following oestrogen and progesterone treatment. human 293ebna cells with/without stable transfection of vegf-d were injected into the uterine horn of nod/scid mice. uteri were collected after 4 weeks. serial sections were immunostained with lyve-1 and/or vegfr3 (lymphatic endothelial cell markers), cd31 (blood endothelial cell marker), mab286 (human vegf-d), mab1278 (human mitochondria) and pcna (proliferative cell nuclear antigen). results: lymphatic vessel profiles were mostly found in the connective tissue between the longitudinal and circular muscle layers of the myometrium. they were rare in the endometrium and only observed in 24% of the sections. when present in endometrium, lymphatic vessel profiles were usually situated adjacent to the endometrial/myometrial border. 293ebna tumours formed inside and outside the uterine horn of both the control (n=4 of 6) and vegf-d group (n=3 of 6). localization of 293ebna cells within the mouse uterus was confirmed by anti-human mitochondrial expression. vegf-d immunostaining confirmed that transfected 293ebna cells expressed vegf-d in vivo. over-expression of vegf-d did not stimulate endometrial lymphangiogenesis, although there was an increase in vessel diameter of lymphatics in the myometrium adjacent to tumours. initial analysis shows no significant effect of vegf-d 293ebna cells on endometrial blood vascular density or endothelial cell proliferation. conclusions: minimal lymphatics are present in the mouse endometrium, as is the case for lymphatic vessels in the human endometrial functionalis. the lack of endometrial lymphangiogenesis in response to vegf-d suggests the presence of an inhibitory factor limiting lymphatic growth in this tissue. in early pregnancy, decidual-derived vegf mediates angiogenesis and is required for implantation and placentation. the role of decidual vegf in later pregnancy is poorly understood. decidual hemorrhage (placental abruption) generates excess thrombin and is a major risk factor for pprom and preterm birth. this study compares immunohistochemical (ihc) localization of vegf in decidual tissue sections from term pregnancies complicated by abruption and gestational age-matched controls, and investigates the effect of thrombin on vegf expression by cultured human term decidual stromal cells (dscs). study design: ihc was performed on serial sections of term placental tissues (no labor) with (n=5) and without (n=5) abruption. purified term dscs were passaged until >99% free of cd45+ cells by facs. confluent dscs were primed with 10 -8 m estradiol (e2), 10 -7 m medroxyprogesterone acetate (mpa), both, or vehicle for 7 days. after 24h incubation in defined medium with corresponding steroids ± thrombin (0.5-2.5 iu/ml), conditioned supernatants were analyzed for vegf by elisa. extracted total rna was used to assess vegf mrna levels by quantitative rt-pcr using established primers. results: vegf expression was localized by ihc primarily to dscs in placental tissue sections, and was increased in tissues from placental abruption vs controls. in term dscs, thrombin increased vegf secretion in a dosedependent fashion irrespective of the hormonal milieu (eg, 2.17-fold stimulation by 2.5 iu/ml thrombin from 5.05±1.23 to 12.62±3.01 pg/ml per mcg protein for e2+mpa; p=0.034). this effect was abrogated by the thrombin inactivator, hirudin. vegf mrna were similarly increased by thrombin with or without steroid hormones (eg, 2.9-fold for e2+mpa; p<0.05). conclusions: placental abruption is associated with increased vegf expression in term decidual tissues in vivo with thrombin enhancing vegf mrna and protein expression in term dscs in vitro. excess thrombinmediated vegf expression in term decidua aberrantly increases endothelial cell permeability to further generate thrombin by continuous exposure of tissue factor-expressing decidual cells to circulating factor vii. thrombin-enhanced matrix metalloproteinase expression in term dscs would degrade decidual and fetal membrane extracellular matrix to induce pprom and preterm birth. basal directional release of angiotensin ii by endothelial cells stimulated by chymotrypsin-like protease (clp)/chymase. yuping wang, david f lewis, yang gu. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: chymotrypsin-like protease (clp)/chymase is a serine protease which plays a major role in angiotensin ii (ang ii) generation in the human heart. our previous study showed a higher clp activity in the maternal plasma in women with pe than in normal pregnancies. we also found enhanced chymase expression in the maternal vessel endothelium in women with pe. in this study, we determined if clp could promote endothelial cell (ec) generation of ang ii. methods: we specifically examined basal directional release of ang ii by cultured ecs. ecs were grown on cell culture insert (6 well/plate, 8 micron pore size). when ecs reached confluence, chymotrypsin (chy) at concentrations of 0.25, 0.5, 1.0, 2.5, and 5.0 g/ml were added to the upper chamber of the cell insert. after 24 hours of culture, medium in the lower chamber was collected. medium concentrations of ang ii were measured by enzymelinked immunoassay (eia). all samples were measured in duplicate. data are expressed as mean ± se and analyzed by anova. a p level < 0.05 was considered statistically different. results: chymotrypsin produced a concentration-dependent increase in basal directional release of ang ii by cultured ecs, control: 1.016 ± 0.065 g/ml; chy 0.25: 1.284 ± 0.281 g/ml; chy 0.5: 1.204 ± 2.06 g/ml; chy 1.0: 2.726 ± 0.829 g/ml; chy 2.5: 4.499 ± 1.467 g/ml (p<0.01); chy 5.0: 5.159 ± 1.087 g/ml (p<0.01), respectively. data are means from 6 independent experiments. conclusion: apical exposure of ecs with chymotrypsin-like protease could promote basal directional release of ang ii. our result implicates that in pe, elevated clp levels in the maternal circulation are very likely to affect ec generation of ang ii. basal directional released ang ii may bind to its receptor on underlying vascular smooth muscle cells and contribute to the increased vasoconstriction in pe. (supported nih grants hd36822 and hl65997). vegf stimulates angiogenesis and vasodilation critical for dramatic rises in materno-feto interface blood flows directly linked to fetal growth/survival. extracellular signal-regulated kinase (erk2/1) pathway mediates partially vegf-induced angiogenic and vasodilatory responses in placental endothelial cells (ec). it is, however, unknown how this vegf-induced signaling is organized in placental ec. objectives: ovine fetoplacental artery ec (ofpaec) and its transformed counterpart, sv40-of to test whether: 1) vegf-activated erk2/1 signaling is compartmentalized in the caveolae and disruption of caveolae interferes vegf-induced erk2/1 activation and; 2) caveolin-1, the structure protein of caveolae, regulates vegf-stimulated erk2/1 phosphorylation. methods: ofpaec or sv40-of cells were cultured in mcdb-131/10% fbs/antibiotics. serum-starved subconfluent ( 80%) cells were treated with rhvegf (0 to 100 ng/ml) for various times. caveolae were disrupted by -cyclodextrin ( -cd, 10 mm, 60 min) or caveolin-1 scaffolding domain (cav-sd, 5 m, 2 hr). sv40-of cells were used for fractionation of caveolae membranes by discontinuous sucrose gradient (45%/35%/5%) ultracentrifugation. activation of erk2/1 signaling pathway were analyzed by western-blotting with specific antibodies. results: in total cell extracts, vegf stimulates erk2/1 phosphorylation in a time-and dose-dependent manner. erk2/1 phosphorylation maximized by vegf (10 ng/ml) at 5-10 min, which was abrogated by -cd or cav-sd. all the molecules for compromising the erk2/1 signaling module, plc 1, pkc , src, ras, raf-1, mek2/1 and erk2/1, were detectable in purified caveolae membranes positive for various markers including caveolin-1, enos, flotillin-1, and -adaptin. in caveolae, vegf dramatically increased phosphorylated erk2/1 without altering total erk2/1 in a time-dependent manner similar to that in total cell extracts, which also maximized at 5-10 min. pretreatment with -cd or cav-sd blocked vegf stimulation of erk2/1 phosphorylation in caveolae. conclusion: vegf activates the erk2/1 signaling pathway in caveolae and caveolae integrity is essential for vegf-activated erk2/1 signaling pathway. we conclude that caveolae/caveolin-1 serves as a platform for compartmentalizing the vegf-induced erk2/1 signaling pathway in placental ec (hl74947 and hl70562). hypoxia upregulates gcm1 in human placenta. david mccaig, fiona lyall. institute of medical genetics, university of glasgow, glasgow, united kingdom. introduction: studies in transgenic mice have shown that a variety of genes regulate the differentiation of trophoblast cells. these genes include gcm1. gcm1 is also expressed in the human placenta. placental gcm-1 protein has been reported to be reduced in pre-eclampsia, in view of the close link between hypoxia, hypoxia-reoxygneation, pre-eclampsia, placental development and the reported reduction in gcm1 we hypothesised that gcm1 expression would be affected by hypoxia. aim: the aim of this study was to determine the effects of hypoxia on gcm1 expression in the human placenta. two model systems were used; (1) free floating villous explants and (2) cultured primary cytotrophoblast and syncytiotrophoblast cells as described previously*. methods: explants or cell cultures were exposed to either hypoxia or hypoxia followed by re-oxygenation. western blot analysis was used to assess gcm1 protein levels. bands on the gels were quantified using scanning densitometry. statistical differences (n=6 experiments for both models used) were calulated by anova and turkey's post-hoc test. results: gcm1 protein was detectable at a low level in villous explants maintained for 7h in 20% o 2 . a striking increase in gcm1 protein was observed when villous explants were incubated for 1h in 0% o 2 (p<0.002). incubation of villous explants for 1h in 0% o 2 followed by re-oxygenation for 6h in 20% o 2 resulted in a marked decline in gcm1 protein (p<0.002). expression of gcm1 was also analysed in primary cytotrophoblast and syncytiotrophoblast cultured in 18% o 2 or reduced oxygen (2% o 2 ) conditions. gcm1 protein was not detected in any of the experimental conditions used. discussion: the present study has shown that acute hypoxia increases gcm-1 protein in villous explants. the experiments with purified trophoblast do not support a role for hypoxia increasing gcm-1 in these cells under the experimental conditions used. the present findings are in keeping with the complex effects of oxygen depending on the conditions used. the observed hypoxic effects on gcm1 warrant further investigation. the effect of acute alcohol exposure on histone3-lys9 modification in the mid-gestation embryonic lung. xiangyuan wang, 1 debra wolgemuth, 1,2,3 laxmi baxi. 1 1 ob/gyn; 2 genetics development; 3 human nutrition, columbia university medical center, new york, ny, usa. objective maternal alcohol abuse during pregnancy produces an array of birth defects comprising fetal alcohol syndrome. lung development depends on a balance between cell proliferation and apoptosis. we have previously shown that the acute alcohol exposure in the mid-gestation embryo can delay lung development and induce apoptosis. acute exposure to ethanol of selected tissues in mouse embryos has been reported by others to initiate apoptosis within 12 hours after exposure and result in histone modifications. specifically, histone acetylation and deacetylation are involved in transcriptional activation and repression, respectively, but can also involve apoptosis. in the present study, we have investigated the effect of alcohol on acetylation of histone3 at lysine9 (ach3lys9) in the mid-gestation embryonic lung. pregnant c57bl/6j mice at day 13.5 of gestation (e13.5) were injected intraperitoneally with 2 doses of 25% ethanol (3.75g/kg ), 4 hr apart (alcoholexposed: ae) or with ringers solution (controls: c). ae and five c fetuses were retrieved 12 and 24 hr later and the lungs were fixed and processed for morphological evaluation and staining with rabbit polyclonal anti-ach3ly9 antibody. the entire lung tissue field was evaluated for the levels of ach3lys9 staining and scored as (-) to (++++). three areas were selected randomly from each sample and the total number of cells and staining positive cells were counted in the bronchial epithelium and in the mesenchyme. twelve hr after alcohol exposure at e13.5, the morphology of ae embryonic lungs was normal. however, high levels of ach3lys9 were detected in 50% of the bronchial epithelial cells and 50% of the mesenchymal cells. the expression level in both lineages decreased 24 hr after alcohol exposure. in the controls, the expression of ach3lys9 was virtually undetectable in both the bronchial epithelium and the mesenchymal cells. our previous study showed that ae e13.5 lungs significantly increased apoptotic cell in both bronchial epithelium and mesenchyme 16 hours after alcohol treatment. we now observe that elevated expression of ach3lys9 in the embryonic lung preceded the observation of apoptosis, suggesting that alteration in the acetylation of h3 could be one of the molecular mechanisms involved in the induction of apoptosis following acute alcohol exposure. objective: our purpose was to evaluate the effect of vitamin c and e supplementation on lipid peroxide levels, total antioxidant ability, and antioxidant levels in the umbilical venous plasma. materials and methods: women at risk for preeclampsia (nullipara, previous preeclampsia, chronic hypertension) were recruited at 15 to 20 weeksgestation and randomly assigned to receive either 1000 mg of vitamin c and 400 iu of vitamin e (study group, n=20) or placebo (control group, n=20) daily until delivery. umbilical venous blood were collected after full term delivery. lipid peroxide levels, oxygen-radical absorbance capacity (orac) values, antioxidant levels were measured by each method (thiobarbituric acid reaction, cao's method, and high performance liquid chromatography). results: 1. the lipid peroxide levels in the umbilical venous plasma of study group were significantly lower than that of control group (2.22±0.06 vs. 2.56±0.12 nmol/mg protein, p<0.05). 2. the orac values in the umbilical venous plasma of study group were significantly higher than that of control group (15010.1±649.4 vs. 11804.6±463.7 u/ml, p<0.05). 3. the -tocopherol levels in the umbilical venous plasma of study group were significantly higher than that of control group (130.2±10.5 vs. 78.7±4.4 nmol/ml, p<0.01). 4. there were no significant differences in the ascorbic acid, uric acid, -carotene, retinol, and -tocopherol levels in the umbilical venous plasma between control and study group. conclusion: this suggested that maternal vitamin c and e supplementation may affect the oxidant-antioxidants balance of the utero-placenta unit and fetus. placental tnf-related apoptosis-inducing ligand (trail) in normal pregnancy and pre-eclampsia. xilian bai, jenny e myers, philip n baker, john d aplin, ian p crocker. maternal and fetal health research group, the university of manchester. introduction: enhanced placental trophoblast apoptosis is well known to occur in pre-eclampsia. however, the potential role of membrane associated or soluble trail, an apoptosis-inducing ligand, and its death receptor, dr5, is not known. we tested the hypotheses that the trail/trail-receptor system is compromised in pre-eclampsia and that soluble trail is a circulating factor which triggers the vascular complications of pre-eclampsia. method: this study was conducted on placental samples and plasma (edta) from women with uncomplicated pregnancies (n=16) and with pre-eclampsia (n=16) at 35-40 weeks gestation. protein expression levels and tissue localisation of trail and dr5 were defined in villous tissue by western blotting and immunohistochemistry. soluble trail (strail) was measured in maternal plasma from both groups using a commercial elisa (diaclone). results: placental villous trail and dr5 protein was unaltered in preeclampsia compared to normal pregnancy. whilst there was differential distribution of trail and dr5 within the component cells, this also was unaffected in pre-eclampsia. within the villi, trail was mainly confined to the cytoplasm and perinuclear regions of cytotrophoblast, syncytiotrophoblast, stromal cells and the fetal capillary endothelium. conversely, dr5 was restricted to trophoblast only, distributed evenly between cytoplasm, plasma membranes and nucleus. strail was present in plasma from non-pregnant women of childbearing age [391.6 (312.4-646.8) , median (interquartile range), pg/ml, n=6]. however, there was no significant increase either in pregnancy [372.3 (319.6-442. 3) pg/ml, n=12] or pre-eclampsia [301.8 (209.0-435.5 ) pg/ ml, n=13] (kruskal-wallis test, p>0.05). conclusions: these results suggest that placental villous trail is not adversely regulated in pre-eclampsia. the absence of dr5 in stromal and endothelial cells may increase resistance to apoptotic stimuli in cells of the villous core. the co-localisation of trail and dr5 in trophoblast suggests a role in autocrine regulation of cell turnover in this cell type. introduction: preeclampsia is associated with apoptosis of the syncytial layer of placenta and the release of particulate and soluble factors which are deleterious to maternal endothelial function. the goal of the current study was to determine whether laser capture microdissection (lcmd) and western blotting could be used to assess levels of syncytial fas ligand (fasl), a key protein in the apoptotic cascade. methods: frozen sections of term placenta delivered from uncomplicated pregnancies at term were used for study (n=3). following staining with mayer's hematoxylin (n=3), lcmd (leica instruments) of an intact terminal villus was carried out using a focused laser pulse directed to the area of interest using a microscope. in the first round of microdissection the placental villus core consisting of fibroblast, macrophages, fetal vessels, and connective tissue, were removed. in the second round of lcmd, the syncytial layer of that same villus was removed and collected in lysis buffer containing detergent and protease inhibitors, and the number of nuclei per sample was recorded. this procedure was repeated until syncytial tissue from 10-15 villi were collected. electrophoretic separation of lysate proteins was then carried out, and western blotting and immunodetection using an anti-fasl antibody was performed. results: we observed that fasl was detected in microdissected syncytial specimens at a molecular weight of approximately 37 kda ( figure) , consistent with our previous reports. it is of note, that western blotting of samples containing approximately 5000 (lane 1), 2000 (lane 2), 1000 (lane 3), and 500 (lane 4) nuclei revealed that fasl could be reliably detected in specimens containing as few as 1000 nuclei. conclusions: since fasl is a cytokine of low to moderate abundance in placenta, this suggests that lcmd coupled with western blotting will be a valuable methodology to elucidate pathways of syncytial apoptosis and pathophysiology in pregnancies complicated by preeclampsia. supported in part by nih grant hd33909. the placenta of preeclamptic pregnancies shows oxidative and nitrative stress. we have shown low protein abundance but paradoxically high activity of inducible nitric oxide synthase (inos) in the placenta with severe preeclampsia compared with normal pregnancies. protein nitration and phosphorylation are post-translational modifications possibly involved in nos activity. objectives: examine inos localization and tyrosine phosphorylation in the preeclamptic placenta and the effect of a peroxynitrite generator (sin-1) on inos expression in primary human placental microvascular endothelial cell (hpmec) cultures. methods: placental lysates from normal (n=3), mild (n=2) and severe (n=3) preeclamptic pregnancies were western blotted for inos and what are the roles played by peroxynitrite in preeclamptic women? yoshikatsu suzuki, 1 tamao yamamoto, 1 yoshimasa watanabe, 2 takeo itoh, 2 hidetaka izumi. 3 1 obstetrics and gynecology, nagoya city university, nagoya, japan; 2 pharmacology, nagoya city university, nagoya, japan; 3 obstetrics and gynecology, izumi women's hospital, fukuoka, japan. aim preeclampsia is characterized by hypertension plus proteinuria. it is hypothesized that the endothelial cell function might be activated by placental faculty in early pregnancy and the activation might cause the vascular disease in preeclampsia. peroxynitrite, which is produced by combination with superoxide (o 2 -) and nitric oxide (no), is strong oxidant as well as o 2 -.we investigated whether or not the localization of peroxynitrite in both placenta and resistance artery might play important roles of developing preeclampsia. omental arteries or placentas were obtained from severe preeclamptic and term-normotensive pregnant women at cesarean section. they were stained by ant-nitrotyrosine (nt, a marker of peroxynitrite) antibody. furthermore, the concentration of nt was measured in omental arteries. in formed consent was obtained from all patients in written. preeclampsia was diagnosed according to the criteria of japan society of obstetrics and gynecology. the localization of nt was seen in placentas obtained from sever preeclamptic women (7/10), although it was not seen from normotensive pregnant women (0/8). the localization was also seen in placentas from 5 of 7 severe preeclamptic women with intrauterine fetal restriction. in omental arteries from both groups, the localization of nt was seen to same degree, and the concentration of nt was similar (0.788±0.161 for sever preeclampsia and 0.984±0.209 for normotensive pregnant women). from these results, it was suggested that an increase in o 2 production, a decrease in no as well as peroxynitrite production in the placentas might cause the vascular dysfunction and subsequently damage uteroplacental blood circulation in preeclampsia. however, the role played by peroxynitrite in resistance artery might be different and more complicated. background: preeclampsia (pe) is associated with shallow cytotrophoblast (ct) invasion of the decidua, leading to impaired vascular transformation and poor uteroplacental perfusion. ct invasion requires the selective proteolysis of the peri-decidual cell (dc) extracellular matrix. we hypothesized that il1 and tnf , cytokines that have been linked to pe, may induce aberrant expression of the matrix metalloproteinases (mmp) 1 and 3 in dcs, thereby preferentially degrading the decidual ecm and interfering with sequential ct invasion. methods: immunostaining for mmp-1, mmp-3, and vimentin (a dc marker) was performed on decidua from normal (n=5) and preeclamptic (n=4) women, and staining intensities were evaluated by hscore. confluent, leukocyte-free first trimester dcs were primed with 10-8 m estradiol (e2) or e2 + 10-7 m medroxyprogesterone acetate (mpa), and then switched to a defined medium with e2 +/-mpa with or without 1 ng/ml of il1 or tnf . secreted mmp-1 and mmp-3 levels were measured by elisa (n=8) and confirmed by western blotting. quantitative rt-pcr assessed mmp-1 and mmp-3 mrna levels (n=3). results: tissue staining revealed that mmp-1 and mmp-3 levels in preeclamptic decidua (hscore mean±sem: 217±41 and 205±11, respectively) were significantly higher than in normal decidua (140±22 and 148±25, respectively; p<0.05). in cultured first trimester dcs incubated with e2, tnf increased secreted mmp-1 and mmp-3 levels by 13±2 and 8±2-fold, respectively, while il1 increased them by 17±3 and 28±13 fold, respectively (p<0.05). in parallel incubations with e2+mpa, basal mmp-1 and mmp-3 output were lowered by approximately 70% and 60%, respectively, while tnf -and il1elicited mmp-1 and mmp-3 levels were blunted by 40-60%. western blotting confirmed the elisa results, and mrna levels corresponded to changes in mmp-1 and mmp-3 secreted protein levels. conclusions: over-expression of mmp-1 and mmp-3 in decidual cells may promote pe by disrupting decidual ecm and impairing normal ct invasion. the high levels of il1 and tnf associated with pe may be contributing to this over-expression. our in vitro observations that mpa blunts tnf -and il1 -elicited mmp expression suggests that exogenous progestin may offer a novel therapeutic approach in preventing pe. to produce 2-methoxyestradiol (2-me), a compound with diverse biological activities including inhibition of hif-1 , a transcription factor that mediates cellular response to hypoxia. circulating levels of 2-me and placental comt activity are significantly reduced in preeclampsia, raising the possibility that reduced production of 2-me contributes to the pathophysiology of preeclampsia by altering placental response to hypoxia. genetic variation in the comt gene is linked to comt activity and has been associated with intrauterine fetal growth restriction. we determined if a snp in exon 4 of the comt gene (rs4818), which does not change amino acid sequence (136leu136), but reduces comt mrna translation, was associated with preeclampsia. we analyzed comt genotypes in paired dna samples extracted from maternal and cord blood from normal pregnancies and pregnancies complicated by preeclampsia by allele discrimination. the study population was predominantly (>98%) african-american. the frequency of the minor rs4818 "g" allele, which is associated with low comt activity, was similar in maternal cases and controls (cases: 19%; controls: 23%, p=0.5), but was significantly greater in fetal dna from 35 pregnancies complicated by preeclampsia compared to 55 control pregnancies (g allele frequency cases: 34%; control: 16%; p<0.001). likewise, fetal carriage of the rs4818 "g" allele conferred a significantly greater risk of preeclampsia (odds ratio: 1.74; 95% c.i.: 1.22, 2.48, p<0.0045). there was also a significant discordance between paired maternal and fetal rs4818 genotypes with significantly greater discordance for the "g" allele in fetuses hosted in preeclamptic pregnancies (odds ratio: 3.17; 95% c.i.: 1.19, 8.49). we conclude that genetic variation in the comt gene is associated with risk of preeclampsia, possibly through a mechanism involving reduced production of 2-me. supported by nih p60md002256. smoking is associated with elevated adma in preeclampsia. michael p frank, 1 robert w powers. 1,2 1 magee-womens research institute, pittsburgh, pa, usa; 2 obstetrics gynecology, university of pittsburgh, pittsburgh, pa, usa. objective: cigarette smoke exposure paradoxically reduces the risk of preeclampsia. asymmetric dimethylarginine (adma) is an endogenous competitive inhibitor of nitric oxide synthase (nos), an independent risk factor for cardiovascular mortality, adma is elevated in women who develop preeclampsia, and adma has been reported to be both higher and lower in smokers. the objective of this study was to investigate the concentration of adma in pregnant smokers and nonsmokers with and without preeclampsia. study design: case-control study of 120 women with uncomplicated pregnancy (controls), and 57 women with preeclampsia matched for gestational age at sample collection. adma was measured by hplc. cigarette smoke exposure was determined by questionnaire and confirmed by plasma cotinine. data are mean±sd. analysis was by two factor anova with fishers post-hoc testing, significance accepted at p<0.05 results: as previously reported, maternal plasma adma concentrations were higher in women with preeclampsia compared to controls (p<0.001). in addition, the concentration of adma was significantly higher in preeclampsia smokers compared to controls and preeclampsia nonsmokers (p<0.05). in contrast, there was no difference in adma concentration between control smokers and nonsmokers. conclusion: these data may suggest a differential effect of cigarette smoke exposure on circulating adma concentrations between women who do and do not develop preeclampsia. previous data has suggested that cigarette smoking is associated with lower adma in low risk elderly patients, and higher adma in high-risk subjects with diabetes. therefore, the data in preeclamptic and non-preeclamptic subjects may be consistent with these studies, however, the underlying biological explanation for this differential effect has yet to be determined. objective: eclampsia is similar to hypertensive encephalopathy in which an acute elevation in blood pressure causes autoregulatory breakthrough, hyperperfusion and edema formation. we previously reported that the pressure of breakthrough was similar between nonpregnant (np) and late-pregnant (lp) rats, but only lp animals developed edema. this study tested the hypothesis that lp animals have decreased in cerebrovascular resistance (cvr) and hyperperfusion in response to breakthrough vs. np. we further hypothesized that acute hypertension would cause greater blood-brain barrier (bbb) permeability in lp rats due to elevated hydrostatic pressure. methods: in vivo models of bbb permeability and cerebral blood flow (cbf) were used in np (n=16) and lp (d19-20; n=16) rats that were either normotensive or hypertensive (np-htn, lp-htn) by infusion of phenylephrine to raise mean arterial pressure. permeability was determined in anterior and posterior brain regions by calculating the flux of 70kd dextran into the brain tissue, measured by a fluorescent spectrophotometer after flushing the vasculature with saline. cbf and cvr were measured by infusion of 15 m fluorescent microspheres and determined based on the flow rate and fluorescence intensity of a reference sample for each animal. animals were ventilated to maintain blood gases within normal ranges (po 2 >100mmhg, pco 2 =35-45mmhg). results: although the pressure change was similar between np and lp ( 83 and 85 mm hg), lp animals responded to acute hypertension with hyperperfusion. cbf increased from 86 ± 10 to 242 ± 22 ml/100g/min in np (181%) and 94±9 to 318±31ml/100g/min in lp (238%; p<0.05 vs. np). hyperperfusion in lp animals was associate with decreased cvr vs. np (0.7±0.07 vs. 0.5±0.05 mm hg/(ml/100g/min); p<0.05). bbb permeability was significantly increased in lp animals at breakthrough vs. np in both anterior and posterior brain regions. the flux of dextran in anterior and posterior brain regions for np vs. lp animals was: 113±50 vs. 362±87 for anterior (p<0.05) and 167±58 vs. 545±140 for posterior (p<0.05). conclusions: these data demonstrate that pregnancy decreases cvr and causes hyperperfusion of the brain during acute hypertension. because increases in cvr is a protective function in the brain, impairment of these mechanisms during pregnancy may predispose the brain to edema when blood pressure is elevated, as in eclampsia. introduction: this study used the in vitro dually perfused human placental lobule to test the hypothesese that placental release of vegf and the fetoplacental vasodilatory response to exogenous vegf-165 are altered by tissue oxygenations that mimic healthy and preeclamptic pregancies. methods: lobules were dually perfused for six hours under one of two oxygenation conditions, representing 'normoxia' and 'hypoxia' (n = 6 each): delivering maternal side inflow perfusate at oxygen concentrations of 15.6 % and 8 %, respectively, distributed via 22 cannulae; and fetal side inflow perfusate oxygen concentration of 2-3 % in both systems. venous perfusates were sampled and assayed, appropriate to side of release, for erythropoietin (epo), macrophage inflammatory protein-1 alpha (mip-1 alpha) (reference oxygen sensitive hormones), free vegf, svegfr-1 and plgf. in separate perfusions, fetoplacental vasodilation in response to 550 pm vegf was investigated, following preconstriction of the fetoplacental vasculature to steady state fetal-side inflow hydrostatic pressure (fihp) with the thromboxane mimetic, u46619, (n = 6 each). results: maternal-side mip-1 alpha release was higher in the 'hypoxic' than the 'normoxic' system (680 ± 119 and 531 ± 93 pg/ml, respectively, at 6 hours, mean ± se; 2-way anova: p<0.01). maternal-side epo and fetal-side soluble free vegf and svegfr-1 release were not different between groups. fetal-side release of plgf was higher in the 'hypoxic' group than the 'normoxic' group (0.13 ± 0.10 and 0.03 ± 0.01 pm, respectively, at 6 hours, mean ± se; 2-way anova: p < 0.05). there was no difference in the vasodilatory response to vegf-165 in the fetoplacental vasculature between the groups (64.8 ± 5.2 and 63.5 ± 6.0 % change in fihp, mean ± se). discussion: differences in mip-1 alpha and plgf release provide evidence for metabolic separation of the adapted systems, caused by a changed oxygen environment. our failure to observe differences in epo, vegf and svegfr-1 release may be explained by longer lag-times for up regulation of their gene expression. vegf associated endothelial signalling appears to be unaffected by 'hypoxia' in the placenta over the time course studied here. background: there is a placental renin-angiotensin system (ras) from very early pregnancy. ang iv mediates various effects by binding to its specific receptor, the at4r, the active site of which is an insulin-regulated aminopeptidase (irap). there is at4r expression in both endothelial and smooth muscle cells. ang iv at low concentrations is vasodilatory, increasing blood flow via the at4rs; it also stimulates nf-kappa beta and modulates glut-4. to date at4r expression has not been investigated in the placenta. we propose that at4r plays a part in the placental vascular development necessary for successful pregnancy, and that reduced at4r expression may be associated with inadequate vascular adaptation contributing to pre-eclampsia (pe). aim: to identify and locate at4r expression in both np and pe placentae. methods: the study had hospital ethical approval; written informed consent was obtained from all women. placental samples were obtained from 4 np and 4 pe at delivery (gestational ages: 39 and 38 weeks respectively). samples were taken from 3 areas, near cord, middle and outer edge of the placenta. paraffin embedded sections were immunostained for irap reactivity using a rabbit polyclonal antibody (gift from professor david james, garvan institute, australia). immunoreactivity of trophoblast and uterine cell populations was assessed using a semi-quantitative grading system. grade 0 = no positive labelling, 1 = 1 -25%, 2 = 26 -49%, 3 = 50 -74% and 4 = 75 -100% of cells positively labelled. median (max, min) are shown. results: 1) at4r immunostaining was prominent in the syncytiotrophoblast and hofbauer cells of all placental villi examined, with no differences in expression between sampling sites. 2) at4r positivity was reduced in near cord pe samples (2.2 (2.4,1.2)) compared to np (3.3 (4.0,2.5) p=0.029). conclusion: we have shown for the first time, dense at4rs in syncytiotrophoblast and hofbauer cells in np placentae, and their down-regulation in pe. reduced ang iv/at4r binding may contribute to increased placental vasoconstriction resulting in increased ischemia/reperfusion. this in turn may stimulate xanthine oxidase, which is itself stimulated by angii, leading to increased superoxide production. further work is needed to clearly define the role of this newly identified component of the renin-angiotensin system in normal pregnancy and pe. pre-eclampsia is associated with lower percentages of regulatory t cells in maternal blood. jr prins, 1 hm boelens, 1 jj hm erwich, 1 j heimweg, 2 s van der heide, 2 ajm van oosterhout, 2 aej dubois, 3 jg aarnoudse. 1 1 obstetrics, university medical center groningen, groningen, netherlands; 2 laboratory of allergology and pulmonary disease, university medical center groningen; 3 pediatric allergology, beatrix children's clinic, university medical center groningen. pre-eclampsia is a serious disease of human pregnancy and immunological mechanisms play a role in its pathophysiology. normal pregnancy is associated with an increase in regulatory t (treg) cells and with a predominant th2 immune profile. treg cells are a subpopulation of cd4+ lymphocytes and are specifically characterized by the lineage specific transcription factor foxp3. treg cells seem to induce immunological changes that have a protective role in maintaining normal pregnancy. we hypothesised that percentages of treg cells are decreased in pregnancies complicated by pre-eclampsia. methods in total, 18 women with pregnancies complicated by pre-eclampsia and 26 healthy pregnant controls were enrolled. to obtain control umbilical cord blood as well, control group i consisted of eighteen healthy pregnant women at term. in addition, since 13 women with pre-eclampsia delivered preterm, control group ii (peripheral blood only) consisted of women during normal pregnancy with a gestational age matched for the preterm pre-eclamptic group. treg cells were measured from whole blood using four-color flow-cytometry. women with a pregnancy complicated by pre-eclampsia had a significantly lower percentage of cd4+ foxp3+ treg cells (4.5 vs 8.8%; p<0.05). in the pre-term group the pregnancies complicated by pre-eclampsia showed a significantly lower percentage of cd4 + foxp3 + cells in the peripheral blood as compared to the healthy pregnant controls. at term this percentage was also lower but not significantly so. between pre-term and term pregnancies both complicated by pre-eclampsia no significant difference was found in the percentage of cd4 + foxp3 + treg cells. no difference was found in umbilical cord blood (7.2 vs 7.9%). conclusions our data suggest that pre-eclampsia is associated with a diminished percentage of treg cells in peripheral blood. we conclude that a deficiency of regulatory t cells may play a role in the pathophysiology of pre-eclampsia. background: preeclampsia and eclampsia are significant causes of maternal and fetal death. however, the pathophysiology of these conditions is unclear. we and others have reported that inhibition of endogenous nitric oxide (no) synthesis produces symptoms similar to preeclampsia in pregnant rats. several studies demonstrate that fetoplacental weights are altered in pregnancies of spontaneous hypertensive (shr) rats. in addition, impaired synthesis of tetrahydrobiopterin (bh4), a major co-factor for endothelial nitric oxide synthase (enos) activity and enhanced expression of enos has been observed in the pathogenesis of hypertension. in the current study, we examined whether supplementation of bh4 and sepiapterin (sep, a precursor for bh4 biosynthesis in the salvage pathway) reduces increased blood pressure and improves fetoplacental weights in shr pregnant rats. methods: groups (4-6) of shr pregnant rats were either treated with bh4, sep (20 mg/k.g body weight/day/rat, oral tablets) or vehicle (normal diet) beginning from day 12 of pregnancy until day 20 of gestation. animals were sacrificed on day 20 of gestation and fetoplacental weights were recorded immediately. western blot analysis was performed to determine vascular enos expression (enos/gapdh). results: significant (p<0.05) elevations in blood pressure (bp, mmhg) were observed in shr (shr, 141±1.42 vs. wky, 106 ± 3.04 mmhg) compared to wistar-kyoto (wky) group. supplementation of either bh4 or sep (128±2.19), significantly (p<0.05) reduced elevated bp beginning from day 14 of pregnancy. fetal but not placental weights were significantly (p<0.05) reduced in shr (2.39±0.03 grams) compared to wky (3.10±0.04) rats. bh4 (2.51±0.04) treatment partially (p<0.05) increased fetal weights compared to the shr group. vascular enos expression is significantly (p<0.05) elevated in shr (1.0±0.07) compared to wky rats (0.56±0.15). further, treatment with bh4 (0.58±0.1) but not sep (0.92±0.1) significantly (p<0.05) reduced elevated enos protein expression in shr rats. conclusions: bh4 may be beneficial treatment of preeclampsia to reduce blood pressure and improve fetal perfusion to increase fetal weights. genetic risk factor for severe preeclampsia: significance of endothelial nitric oxide synthase gene t-786->c and missense glu298asp variants. toshihiro yoshimura, 1 michihiro yoshimura, 2 masafumi nakayama. 3 1 obstetrics and gynecology, kumamoto university school of medicine, kumamoto, japan; 2 cardiovascular medicine, jikei university school of medicine, tokyo, japan; 3 cardiovascular medicine, kumamoto university school of medicine, kumamoto, japan. introduction: we recently identified two endothelial nitric oxide synthase (enos) gene polymorphisms, a glu298asp missense variant in exon 7 and a t-786-->c variant in the 5'-franking region, which are associated with coronary spasm and myocardial infarction in japanese population. and we also identified a missense glu298asp variant is associated with severe preeclampsia and placental abruption. our objective was to analyze the association between the t-786-->c and severe preeclampsia. materials and methods: the study participants included 62 patients with histories of severe preeclampsia. this is a preliminary study, therefore, the comparisons were made with the 345 general normal population. results: the analyses revealed that the frequency of the missense glu298asp variant (n=18/62, 29%) was significantly higher than the general population (n=44/345, 13 %), as we previously published. however, the frequency of the t-786-->c variant (n=9/62, 14%) was not different from the general population (n=61/345, 18 %). interestingly, only one patient had both t-786-->c and missense glu298asp variants, and she developed placental abruption. conclusion: although our sample size is small, it is very unlikely that the t-786-->c variant is associated with severe preeclampsia. the t-786-->c variant may not be a genetic susceptibility factor to severe preeclampsia. the t-786-->c may have some reproductive significance in combination with missense glu298asp variant, however huge number of patients would be needed to analyze such rare (2.5%) combination of the variance. the association between the development of preeclampsia and methylenetetrahydrofolate reductase, angiotensinogen, vascular endothelial growth factor single nucleotide polymorphism genotype combinations. hyun soo park, 1 jong kwan jun, 2 chan-wook park, 2 joong shin park, 2 bo hyun yoon, 2 hee chul syn. 2 1 obstetrics and gynecology, dongguk university international hospital, goyang-si, gyeonggi-do, republic of korea; 2 obstetrics and gynecology, seoul national university college of medicine, seoul, republic of korea. objective this study was conducted to investigate if there exists any genotype combination of multiple single nucleotide polymorphism (snp)s which is frequently found in preeclampsia patients. study design one hundred sixty two preeclampsia patients and 199 normotensive pregnant women were included in this study between jan 1998 and jul 2004. diagnosis of preeclampsia and assignment of severity were made according to the criteria by national high blood pressure education working group and american college of obstetricians and gynecologists. the patients were reclassified as early (30 weeks or before) and late-onset (31 weeks or beyond) disease. genotypes were measured with pcr-rflp for methylenetetrahydrofolate reductase (mthfr) c677t, angiotensinogen (agt) m235t, vascular endothelial growth factor (vegf) c936t with the dna extracted from maternal blood. case-control study for each snp was done and the frequencies of genotype combination were compared. anova, t-test, chi-square test, fisher's exact test and logistic regression analysis were used for statistical analysis. a p value of <0.05 was considered statistically significant. results genotypes of mthfr polymorphism showed significant difference between late onset preeclampsia and control (cc+ct/tt, or: 1.82, p<0.05) but agt and vegf polymorphism did not show statistical difference between any case-control combination. only 20 out of possible 27 genotype combinations were found and there was no statistical difference in the frequencies of genotype combination between case and control group. conclusion mthfr polymorphism might be associated with the development of preeclampsia, but there was no combination of mthfr, agt and vegf polymorphisms which is associated with the development of preeclampsia. diffuse staining and vascular smooth muscle (vsm) staining. resistance-sized vessels (10-200 μm) were evaluated. results: for mpo, the intensity of staining (fig) and the % vessels with neutrophil, diffuse and vsm staining was significantly greater for obese than for overweight or normal weight patients: % diffuse staining (64.6 ± 4.2 vs. 40.4 ± 3.9 vs. 21.8 ± 4.7, p<0.001); % vsm staining (30.2 ± 6.1 vs. 20.0 ± 5.3 vs. 4.0 ± 2.6, p<0.01). for mmp8, obese and overweight patients had a greater (p<0.001) % vessels with neutrophil, diffuse and vsm staining than normal weight patients: % diffuse staining (63.0 ± 5.5 vs. 55.2 ± 4.3 vs. 26.0 ± 3.0); % vsm staining (36.8 ± 2.9 vs. 27.4 ± 3.1 vs. 2.8 ± 1.2). conclusions: neutrophils infiltrate the systemic vasculature of obese women and release mpo and mmp8. speculation: obesity may put women at risk for pe because their vasculature may already be dysfunctional due to neutrophil infiltration and release of mpo and mmp8. hl069851. collagen is an important protein that maintains the structural integrity of tissues. disruption of vascular smooth muscle collagen could result in vascular dysfunction in women with preeclampsia. recently, neutrophil infiltration of the systemic vasculature was demonstrated in preeclamptic women. neutrophils produce inflammatory mediators, such as reactive oxygen species (ros) and tnf-. we hypothesized that neutrophils, ros and tnf-would alter expression of collagen regulating genes. methods: primary cultures of human vascular smooth muscle cells (vsmc) were seeded into t-25 flasks (40,000 cells/flask) and grown for 3 days to 70% confluence. the cells were treated for 24 hours with medium control, ros (hx, 0.05 mm + xo 0.003 u/ml), tnf-(1 ng/ml); and neutrophils (60,000) activated with arachidonic acid, 50 μm, (1:16 ratio of neutrophils to vsmc). rna was extracted from cell homogenates and analyzed for gene expression with an rt2 profiler pcr array system for human extracellular matrix genes (superarray). to determine the fold-change of gene expression, the results were first normalized to a housekeeping gene and then ct was calculated across two rt-pcr arrays where group 1 was the control and group 2 was the experimental treatment. results: table 1 . conclusions: neutrophils, ros and tnf increased mmp1 expression. interestingly, genes involved in collagen synthesis (col1a1) or inhibition of mmp-1 activity (timp1) were either not affected or down-regulated. these data suggest that neutrophil infiltration in preeclamptic women could cause vascular dysfunction by creating an imbalance between collagen synthesis and collagen breakdown favoring breakdown. hl069851, fogarty 5d43tw007692, p60md002256. objective: hypoxia increases membrane attack complex (mac) binding to cultured human trophoblasts, and mac enhances apoptosis in trophoblasts exposed to low compared to normal fio 2 . trophoblast microparticles and cellular fragments released into the maternal circulation in vivo may contribute to the systemic pathophysiology of preeclampsia. we tested the hypothesis that hypoxia induced mac deposition on cultured human trophoblasts yields microparticles and fragments coated with mac. study design: primary cytotrophoblasts from term human placentas (n=4) were cultured 24 h in 1% and 20% oxygen in dmem with 10% human serum with active mac or heat inactivated serum (control). media were centrifuged to obtain pellets of microparticles and cell debris which were immunostained for mac or exposed 6-12 h to confluent, phorbol myristate acetate differentiated u937 macrophages. the percentage of macrophages that ingested trophoblast debris was quantified by counting the number of macrophages with immunofluorescence for trophoblast cytokeratin 7 filament staining, as assessed by confocal microscopy. results: cultures exposed to normal human serum, but not heat inactivated control serum, showed mac immunofluorescence on microparticles and fragments in medium, with the highest level of mac in cultures exposed to extreme hypoxia. the maximal percentage of macrophages that ingested the trophoblast debris coated with mac from cultures with 1% oxygen was 56.2%, not different from the 52.6% from cultures exposed a 1% fio 2 and control serum. conclusion: trophoblasts exposed to hypoxia and active complement release microparticles and cellular fragments coated with mac into the extracellular environment. mac coating does not influence phagocytic removal of the debris by macrophages suggesting that placental derived, membrane bound mac could circulate to yield systemic affects on maternal endothelium. supported by nih hd29190 and hd045675. is there a role for fatty acids in the pathogenesis of pre-eclampsia? nicola j robinson, 1 laura j minchell, 1 jenny e myers, 1 philip n baker, 1 carl a hubel, 2 ian p crocker. 1 1 maternal and fetal health research group, the university of manchester; 2 obstetrics, gynecology and reproductive sciences, university of pittsburgh. objectives: women with pre-eclampsia (pe) display altered lipid metabolism as characterized by elevated circulating triglycerides and non-esterified fatty acids (nefa) and these changes are evident before the disease is clinically apparent. we have tested the hypothesis that the increased circulating levels of nefa contribute to endothelial dysfunction in pe. methods: human umbilical vein endothelial cells were incubated for 24h with pooled plasma (2%) from normal or pe pregnancies, or with palmitic, oleic and linoleic acid in culture media at the concentrations and molar ratios to albumin identified in normal (100, 113, 37 m, ratio 0.9) and pe pregnancies (140, 172, 53 m, ratio 1.6) 1,2 . lipid droplet accumulation was determined using an oil red o absorbance assay. endothelial metabolism was measured using the mtt test and mitochondrial membrane potential determined by jc-1 assay as a marker of early apoptosis. results: plasma from pe pregnancies increased endothelial cell lipid droplet accumulation compared to normal plasma (p<0.01, wilcoxon signed ranks, n=9). this change was replicated following exposure to nefa at the combined concentrations found in pe compared to normal pregnant controls (p<0.05, n=7). plasma from women with pe caused a significant decrease in mitochondrial dehydrogenase activity (mtt test; p<0.01, n=8) and a reduction in jc-1 fluorescence (p<0.05, n=7), compared to normal plasma, suggestive of mitochondrial membrane depolarization and increased cellular apoptosis. again these effects were replicated using nefa in culture medium at the levels found in pe compared to normal pregnancies (mtt test: p<0.05, n=9; jc-1 assay: p<0.05, n=10). conclusions: in endothelial cell cultures, plasma from women with pe caused increased lipid droplet accumulation, decreased cellular metabolism and increased apoptosis. these changes to cellular function were mirrored using nefa in culture medium at the concentrations and molar ratios to albumin previously reported in pe. these findings provide evidence that the changes in endothelial cell function induced by plasma from women with pe may be due to the increased nefa circulating levels and that increased palmitic, oleic and linoleic acid, in combination, could play a role in pathogenesis of pe. 1 lorentzen (1995) bjog; 2 endresen (1992) ajog. background: skewing of the maternal endothelial phenotype in pre-eclampsia (pe) is attributed to the release of unknown factors from a hypoperfused placenta. we hypothesise that factors secreted from pe placental tissue will impair endothelial cell function. we have tested the effect of soluble factors by menopausal status, there was a significant increase in bmi in pre-but not in postmenopausal women. in postmenopausal women there was insufficient power to note a statistically significant change in bmi. results are summarized with the follow-up for each group represented by "n" in the graph below. premenopausal patients were divided into hysterectomy with oophorectomy (pbso) versus hysterectomy alone (ph). the ph group showed an increase in bmi that plateaus at time 3. in the pbso group the bmi continued to increase over time. subgroup analysis comparing ph to pbso demonstrates initial weight loss in pbso but a significant increase in bmi from baseline at time 3 compared to ph. conclusions: hysterectomy appears to be associated with an increase in bmi over time. subgroup analysis suggests that, in premenopausal women, oophorectomy is more strongly associated with continuing weight gain than hysterectomy alone. purpose: obesity is implicated as a key risk factor in chronic disease, but no studies have associated central obesity to the presence of chronic abdominal and/or pelvic pain. we set out to identify the prevalence of chronic abdominal/ pelvic pain in an underserved, primarily latina population by a cross-sectional study in the olive view-ucla outpatient gynecology clinic. we sought to identify an association between the presence and severity of abdominal/pelvic pain and central obesity. methods: nonpregnant women presenting to the gynecology clinic were prospectively evaluated and grouped according to the presence of abdominal/ pelvic pain ('none-mild' or 'moderate-severe' pain). body mass index (bmi) and abdominal circumference (ac) were measured. patients with 'moderate-severe' pain completed standardized questionnaires for pelvic pain and global health scores. results: 207/276 (75%) of patients has 'none-mild' pain, and 69 (25%) had 'moderate-severe' pain. pain prevalence was not significantly associated with bmi (mean: 'none-mild' 30.5+7.7 kg/m2; 'moderate-severe' 30.6+7.6 kg/m2, p=0.95), nor was pain severity (p=0.58). pain prevalence was significantly associated with ac (mean: 'none-mild' 37.1+7.7 inches; 'moderate-severe' 40.2+7.0, p=0.004). a borderline positive association exists between ac and pain severity (p=0.077). conclusions: we demonstrate an association between both the presence and severity of chronic abdominal/pelvic pain and central obesity, independent of bmi. ac appears to be a more relevant factor than other traditional measures of habitus in patients with this chronic malady. to improve preventative care in women's health management, further evaluation of the role of central obesity in the pathogenesis of chronic pain is necessary. aims: vulvitis is one of the most frequently diagnosed gynaecological infections. we aim to assess the efficacy against infective vulvitis of a new topical medical device containing low molecular weight hyaluronic acid (lmw-ha). the ability of this molecule to stimulate -defensin 2 release in keratinocytes has been recently shown. methods: we report preliminary data regarding 20 women suffering from infective vulvitis, as assessed by a gyneacologist: patients were randomly selected to receive sphg800 (10 patients), a cream containing low-molecular weigh hyaluronic acid, or vehicle (10 patients). patients were asked to apply the cream to the vulva twice-daily for 7 days. at the end of treatment, evaluation of efficacy, tolerability and acceptability of the cream was assessed by a specific questionnaire. results: preliminary results show that patients receiving sphg800 report a significative improvement of vulvitis symptoms, in terms of itch, redness of the skin and burning, in comparison to vehicle. sphg800 also showed a good tolerability, cosmetic acceptability and symptomatic relief perceived by patients. conclusion: sphg800 seems to be efficacious in ameliorating the symptoms of infective vulvitis. this activity may be probably related to the lmw-ha presents in this formulation: in fact, low molecular weight hyaluronic acid has been recently shown to induce -defensin 2 production by human keratinocytes. since this peptide exerts antimicrobial and antimicotic activity, the improvement of symptoms assessed in patients receiving sphg800 might be linked to a reduced infective charge, attributable to the activity of -defensin 2. background: allogeneic hematopoietic stem cell transplant (hsct) is a treatment used for many malignant and nonmalignant diseases of the bone marrow and immune system. hsct may be complicated by chronic graft-versus-hostdisease (cgvhd) in 60 to 70% from matched unrelated donors. genital cgvhd complicates about 25% of hsct and may uncommonly result in labial fusion. case: 22 year old woman with a history of ewing's sarcoma and acute myelogenous leukemia, had received chemotherapy and total body irradiation (tbi) followed by a matched unrelated donor hsct. menarche occurred at 13 years of age after normal pubertal development. she menstruated regularly until cancer diagnosis. premature ovarian failure resulted after chemotherapy and tbi and oral contraceptive pills were used for hormone replacement. after transplant, she developed chronic gvhd involving the skin, eyes, mouth and joints, and concomitantly complained of vulvar pruritus. she was presumed to have a yeast infection which was treated with fluconazole without a pelvic exam. she was evaluated by a gynecologist when she was unable to insert a tampon. pelvic exam revealed dense labia minora adhesions from the clitoris to urethral meatus and posteriorly leaving a 1 cm opening at the urethra. pelvic mri revealed a normal uterus and ovaries. after 2 weeks of topical estrogen cream, the adhesions remained dense and were lysed under general anesthesia. vaginal examination revealed pale, minimally rugated, normal mucosa. cervical cytology was normal. post-operatively she used daily topical estrogen and hydrocortisone creams. at 3 months after surgery, her urinary stream was stronger. on pelvic exam, the labial opening was 4 cm, but a small posterior forchette adhesion elicited severe pain. after using dilators coated with topical steroids and estrogen, she was able to insert a tampon. conclusion: genital gvhd should be considered in women with genital tract complaints after hsct. labial fusion secondary to chronic gvhd may be treated successfully with surgery and medical therapy. support: rbmb/nichd/nih. dana r ambler, mazen e abdallah, rahi victory, michael p diamond, elizabeth e puscheck, jay m berman. obstetrics and gynecology, wayne state university/detroit medical center, detroit, mi, usa. objective: to determine which factors are predictive of a ruptured ectopic pregnancy, and whether endometrial stripe thickness can be used as an alternative to such criteria in the diagnosis of an ectopic pregnancy. design: retrospectively collected ectopic pregnancy database. setting: detroit medical center, detroit, michigan. patients or participants: 413 women with a diagnosis of an ectopic pregnancy were studied, with 90 surgically confirmed tubal rupture cases. interventions: abstracted data included hcg(iu), gestational age (days), presenting symptoms of pain and/or bleeding, hemoglobin (hgb), hematocrit (hct), historical risk factors, ultrasound-determined ectopic size, endometrial stripe thickness (mm), amount of cul de sac fluid (cds), tubal rupture at time of surgery, and estimated blood loss (ebl)(ml). covariates included demographics. results were significant when p<0.05. results: chi square analysis revealed that there is a relationship between endometrial stripe thickness and hcg levels. logistic regression models demonstrated that endometrial stripe thickness was not predictive of ectopic rupture, (or 0.98, p=0.96) . however, logistic regression, both forced and forward stepwise analysis, demonstrated that hcg (or=6.8, p<0.001), a large volume of cul-de-sac fluid (or=4.2, p<0.001), and increasing pain (or=3.5, p=0.004) were associated with increased risks of rupture. gestational age, and ectopic related risk factors were also not predictive of rupture. conclusions: endometrial stripe thickness is not a useful predictor for the diagnosis of a ruptured ectopic pregnancy. serum hcg measurement, cds fluid volume and the presence of pain are much stronger diagnostic indicators of ectopic pregnancies. clinical profile of migraineurs in a university hospital gynecology department in japan. [objectives] the changes in hormonal milieu associated with menarche, pregnancy, menopause, and post-menopause are frequently accompanied by changes in the patterns and frequency of migraine. little is known on the relationship of women's issues of migraine though the balance between estrogen and progesterone is critical in the elimination of migraine. our aim was to investigate the relations among the prevalence of migraine, the reproductive stage, and gynecologic diseases. [materials and methods]197 female patients (average age: 44.6 years old) who consulted a physician and agreed to answer about the questionnaires during september, 2006 -june, 2007 . migraineurs were diagnosed with the migraine screener by the japanese headache medical treatment promotion committee in 2005. and the patients answered questionnaires that screened about menopausal disorder and abnormal menstruation at once. they were conducted a survey in the form of a questionnaire and sometimes were taken blood samples. [results] 52 in 197 patients had migraine (26.4% average age: 40.6 years old). prevalence were 33.3% in one's twenties, higher than another ages. most patients with migraine was complicated by menstruation disorders (premenstrual syndrome 67%, dysmenorrhea 52.6%), sterility (42.4%), and severe menopausal disorder (80%). when trh and the lh-rh test were examined for the sterility patient, migraineurs had higher prolactin basal level and lh level after lord but lower fsh level before and after lord than nonmigraineurs. on the other hand, the prevalence of migraine for postmenopausal women and women who had gynecology cancer treatment was low, and there was no relation between migraine and pregnancy history. [conclusions] this study provides migraine headache is influenced by reproductive stage and that women with migraine are frequently complicated by menstruation disorder. it is thought that migraine account for abnormality of hormone milieu including abnormality of the hypothalamus-pituitary system. objective: this study evaluated the efficacy of 3 doses of estradiol (e 2 ) gel 0.1% (divigel ® ), a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel, to reduce frequency and severity of vasomotor symptoms and signs of vulvar and vaginal atrophy (vva) associated with menopause. design: 488 postmenopausal women were evaluated in a 12-week study comparing placebo to e 2 gel 0.1% at doses of 1.0 g/day, 0.5 g/day, and 0.25 g/day with estimated nominal daily deliveries of 0.027 mg, 0.009 mg, and 0.003 mg of e 2 respectively. endpoints included mean change from baseline in daily frequency and severity of moderate to severe hot flashes (msvs). vaginal ph and % superficial cells were collected at baseline and end of study. results: e 2 gel 0.1% showed statistically significant improvements from placebo gel as early as week 2 (table) that were maintained throughout treatment. signs of vva (vaginal ph and % of superficial cells) showed statistically significant improvements from baseline with all 3 doses of e 2 gel 0.1% compared to placebo. conclusion: e 2 gel 0.1% significantly decreased the frequency and severity of msvs at all doses evaluated in this trial. e 2 gel 0.1% offers multiple dosing options to individualize patient therapy, including the lowest effective dose that was studied (0.25 mg e 2 , delivering 0.003 mg e 2 /day), to treat vasomotor symptoms associated with menopause. estradiol (e 2 ) gel 0.1% (divigel ® ) is a novel formulation of e 2 consisting of 1 mg e 2 per 1 g transdermal gel for the treatment of vasomotor symptoms associated with menopause. safety and tolerability of e 2 gel 0.1% were evaluated in a large placebo-controlled trial. design: 488 postmenopausal women participated in a 12-week study comparing placebo to 0.25 g/day, 0.5 g/day, and 1.0 g/day of e 2 gel 0.1%. circulating e 2 and estrone (e 1 ) concentrations were measured. safety analyses included the incidence of adverse events (aes) and clinical laboratory evaluations, including plasma levels of sex hormone binding globulin (shbg). application site tolerability was assessed using the draize scale. results: all doses of e 2 gel 0.1% produced physiologic e 2 :e 1 ratios similar to those seen in premenopausal women. e 2 :e 1 increased from a baseline mean of 0.17 to 0.49, 0.69, and 0.95 with, respectively, the 0.25, 0.5, and 1.0 g/day doses of e 2 gel 0.1%. the most frequently reported aes were breast tenderness and postmenopausal bleeding that appeared to be dose-related and would be expected with increased circulating estrogen concentrations. there were no remarkable changes in hematology, blood chemistry, urinalysis, lipid, coagulation, and carbohydrate values following treatment with e 2 gel 0.1%. shbg levels remained unchanged after 12 weeks of treatment at all doses. the vast majority of patients had no evidence of skin irritation throughout the treatment period. mean draise scale scores after 4, 8, and 12 weeks of treatment were 0.0 for all treatment groups except for a mean value of 0.1 ± 0.34 for the 0.5 g dose group after 4 weeks of treatment. conclusion: e 2 gel 0.1% is a safe and well-tolerated therapy for the treatment of menopausal symptoms. design: pharmacokinetic parameters, dosing, efficacy, and safety information for divigel ® , elestrin ™ , evamist ™ , estrogel ® , and estrasorb ® were obtained from current prescribing information (obtained from manufacturer websites) and the data were compared. results: together, these new transdermal therapies offer multiple dosing/ delivery options and contain a wide range of e 2 (0.25 mg to 4.6 mg), with the lowest systemic daily delivery of e 2 attained by the divigel ® 0.25 g dose. following 12 weeks of treatment, across the dosing options, each treatment significantly reduced both the frequency and severity of moderate to severe vasomotor symptoms (msvms) compared to placebo. change in frequency of msvms ranged from -6.0 (divigel ® 0.25 g) to -11.1 (estrasorb ® ); change in severity scores for msvms ranged from -0.3 (divigel ® 0.25 g) to -1.7 (divigel ® 1.0 g). all treatments are safe and well tolerated. breast tenderness was the only adverse event reported in 5% of subjects, occurring with all therapies. conclusions: current treatment guidelines recommend using the lowest effective dose of estrogen for the treatment of menopausal symptoms. this side-by-side comparison of the recently available e 2 non-patch transdermal options to the standard transdermal therapies is meant to assist physicians in individualizing treatment for their patients. introduction: cdb-2914 (cdb) is a relatively new progesterone receptor modulator being clinically evaluated for contraception and treatment of fibromas. its use in an intrauterine device/system (ius) has not been reported. in this study we prepared cdb-filled intrauterine devices (cdb-ius) and evaluated their effects on endometrial growth and bleeding patterns in rhesus macaques. methods: short (1.0-1.5 cm) lengths of silastic tubing (od 2.4 mm), either empty (n=3),or filled with silicone rubber matrix containing 50% of micronized cdb (n=5), were inserted into the uterine lumens of 8 ovariectomized rhesus macaques. animals were induced to cycle by sequential treatment with systemic estradiol and progesterone (p) as reported (brenner et al, ann ny acad, 2002) . after 3.5 cycles, at the end of the follicular phase, the uterus was removed and processed. results: when systemic p treatment was withdrawn at the end of each cycle, animals with empty ius menstruated normally, while animals with cdb-ius bled little or not at all. over the whole 3.5 cycles, animals bearing a blank ius bled for an average of 11.66 ± 0.88 days while cdb treated animals bled for an average of only 1 ± 0.45 days. at the end of treatment, animals exposed to blank ius had mean endometrial wet weights of 409 ± 112 mg while the cdb-treated endometria weighed only 188 ± 30 mg. the proliferation markers ki-67 and phospho-h3 were substantially lower in the cdb-ius treated than in the blank treated animals. histologically, the cdb exposed endometria were atrophied with evidence of glandular degeneration while the blank controls were proliferative and normal. summary: in cycling rhesus macaques, a cdb-ius prevented progestational development, blocked menstruation after p withdrawal, and suppressed endometrial proliferation. if these effects are confirmed in women, the cdb-ius could provide estrogen-free and bleed-free contraception, and could help control heavy menstrual bleeding. supported by rr00163, hd 43209 and the population council. introduction: irregular uterine bleeding is a major side effect and cause for discontinuation of ltpoc use. while endometria of ltpoc-exposed women display abnormally enlarged, fragile blood vessels (bv), decreased blood flow and evidence of oxidative stress, the mechanisms by which structural and vasomotor endometrial dysfunction occurs remains unknown, in part by the difficulty of manipulating hormone levels in women. the aims of this study were 1) to validate the guinea pig (gp) as a model to study uterine effects of ltpoc and 2) to investigate ltpoc-effects on endometrial histology and oxidative stress markers. methods: oophorectomized gps were implanted s.c. with time release pellets containing either placebo (crl,n=6); estradiol (e2,n=3); medroxyprogesterone acetate (mpa,n=6) or e2+mpa (n=3). after 21 days, uterine horns were weighed and frozen or paraffin embedded. angiogenesis was assessed by quantitative image analysis of vonwillebrand factor staining and included bv density and size. oxidative stress was detected by 8isoprostane and 8-oh-deoxyguanosine (8oxog). apoptosis was investigated by the tunel method. statistical analysis was by 1-way and 2-way anova. results: gp uteri were enlarged by both e2 (p<0.001) and mpa (p=0.025). effects of mpa on uterine weight differed significantly depending on e2 levels (p<0.001), where mpa opposed the e2 effect in combined treatments. angiogenesis parameters were similarly impacted upon. thus, mpa alone increased bv density (p=0.036) and bv average area (p=0.002). the presence of e2 significantly decreased these parameters (bv density mean± sem: crl: 9.4±1.0%, e2: 10.3±1.6%, mpa: 13.6±1.1%, e2+mpa: 6.0±0.7%, p=0.002). these changes were associated with highly elevated 8-isoprostane content in e2+mpa-treated uteri compared to all other groups (p<0.001). abnormalities in the e2+mpa group were consistent with chromatin redistribution, nuclear pyknosis, karyolysis and increased nuclear 8oxog staining and a marked increase in tunel labeling. conclusions: ltpoc exposure alters endometrial vascular and tissue morphology consistent with oxidative stress and apoptosis in a complex interplay with endogenous estrogens. the gp is an excellent model for the study of ltpoc effects on the uterus. physiologic and psychological symptoms associated with injectable and oral contraceptive use. abbey b berenson, susan odom, carmen r breitkopf, mahbubur rahman. ob/gyn, utmb, galveston, tx, usa. objective: to compare physiologic and psychological symptoms over 24 mo among users of depomedroxyprogesterone acetate (dmpa), an oral contraceptive with 20 micrograms ethinyl estradiol (oc), and non-hormonal (nh) contraception. methods: a total of 604 women reported the presence of 16 symptoms prior to initiating contraception (217 dmpa, 216 oc, 171 nh) and every 6 mo thereafter for 24 mo. longitudinal relationships between symptoms and contraceptives (reference: nh), as well as race/ethnicity (non-hispanic black, non-hispanic white, and hispanic) were assessed by gee-analysis after adjusting for age, visits and baseline status of symptoms. persistence, resolution, and new development of symptoms were noted by method in 6 mo increments for 24 mo and compared with that of nh controls. the gee analyses showed that oc was protective against mastalgia (or= 0.7), cramping (or=0.5), hair loss (or=0.6), acne (or=0.4), nervousness (or=0.5) and mood swings (or=0.7). when race was considered, oc was protective for all women against acne, for whites against mastalgia and cramping, and for non-whites against nervousness. for whites, it was a risk factor for bleeding between menses. oc use resulted in resolution of acne within 6 mo in nearly 50% of those with it at baseline, but no significant resolution after 6 mo. also mastalgia (29% at baseline) was less likely to persist at 18 and 24 mo. bleeding between menses was reported for the first time at 18 mo by 11% of oc users. dmpa users of all races had an increased risk of missed periods (or=95.1), bleeding between menses (or=3.6), bleeding >20 d (or=13.6) and loss of libido (or=2.2) relative to nh users. it reduced cramping (after 12 mo) and bloating (all 24 mo). racial differences were observed with dmpa protective against mastalgia and mood swings in whites, cramping in whites and hispanics, and bloating in non-whites. it was a risk factor for loss of energy in whites only. neither method affected depressive symptoms. conclusion: side effects of these two methods are mostly related to abnormal bleeding. very low dose pills can be protective against symptoms (mastalgia, cramping, hair loss, acne, nervousness and mood swings) commonly associated with pills while dmpa protects against cramping and bloating. knowledge about racial differences will allow physicians to individualize therapy. counseling should include that some symptoms can develop after 18 mo while resolution often occurs within 6 mo. methods: twenty-four women were enrolled in irb approved prospective, randomized, cross-over trial comparing 2 months of oc (ortho cyclen) vs. tc ortho evra). the daily oc administers 35 micrograms of ee; the weekly tc contains .75 milligrams of ee. each treatment was followed by 2 months of washout and 2 months of the alternative contraceptive. blood was drawn at baseline and final week of treatment for each arm of the study. ee was quantified by ria, with preceding organic solvent extraction and celite column partition chromatography. data were analyzed by t test with boferroni's correction, p < .05. results: after two months of treatment the mean (+/-sem) ee levels for tc = 81.9 pg/ml (+/-12.0) and oc = 94.1 pg/ml (+/-9.8). the ee level is not significant different for the two medication (p = 0.42). conclusions: there is no difference in ee levels with the use of these oral and transdermal contraceptives. this suggests the transdermal contraceptive, despite the lack of the hepatic first pass effect, has similar levels of ethinyl estradiol compared to oc. the continuous elevation in ee seen with the tc route of administration, versus the episodic increases seen with the oc route, raises concerns of constant exposure to ee. this may explain the increased risk of estrogen-induced thrombotic events with this tc route of administration. impact of paracervical block, in combination with general anesthesia, on post-abortion pain. gweneth b lazenby, tod aeby. obstetrics and gynecology, university of hawaii, honolulu, hi, usa. objective to evaluate the impact of paracervical block with a long-acting local anesthetic, in conjunction with general anesthesia, on post-operative pain. methods a power analysis determined 35 patients per arm were needed to demonstrate a significant difference of 2 in mean pain scores. seventy-two patients were allocated to one of two arms using urn randomization. all patients received standardized anesthesia; intravenous sedation for gestational age under 12 weeks and general anesthesia for over 12 weeks. thirty-nine patients were randomized to receive a paracervical block with 0.5% bupivacaine and thirtythree were randomized to no local anesthesia prior to surgical abortion. patients completed visual analog scales for pain and anxiety prior to the procedure, upon awaking, 30 and 60 minutes post-operatively, and prior to discharge. data were analyzed using an anova and students t-test the experimental and control groups were equivelent in age, ethnicity, gravidity, parity, prior abortions, prior vaginal deliveries, prior c-sections, gestational age, number of laminaria, pre-operative and intra-operative dilation, operative time, estimated blood loss, and reported complications. pain and anxiety were not significantly affected by placement of a paracervical block. these data do not support the hypothesized benefit of local anesthesia, prior to surgical abortion under general anesthesia, to reduce post-operative pain. we do not recommend the routine use of a paracervical block to decrease post-operative pain. age, parity, history of abortion and contraceptive choices affect the risk of repeated abortion. oskari heikinheimo, 1 mika gissler, 2 satu suhonen. 1 1 ob gyn, university of helsinki, helsinki, finland; 2 national research and development centre for welfare and health, helsinki, finland. objective the rate of repeat abortion varies from 30 to 38% in northern europe. however, risk factors for repeat abortion are poorly understood. we characterized risk factors (demographic, as well as those related to abortion and postabortal contraception) of repeat abortion. design a prospective cohort study of 1269 women undergoing medical abortion between august 2000 and december 2002. the subjects were followed by means of finnish registry on induced abortions until december 2005; the follow-up time (mean ± sd) was 49.2 ± 8.0 months. results altogether 179 (14.1%) of the subjects requested repeat abortion within the follow-up time. in univariate analysis previous abortion, parity, young age, smoking and failure to attend the follow-up visit were associated with increased risk of repeat abortion. immediate -in contrast to postponed -initiation of any contraceptive method was linked to lower risk of repeat abortion. in comparison to combined oral contraceptives, use of intrauterine contraception was most efficacious in reducing the risk of another pregnancy termination. in multivariate analysis the effects of young age, parity, previous abortion and type of contraception on the risk of another abortion persisted. conclusions increased focus on young, parous and those with the history of an abortion may be efficacious in decreasing repeat abortion. contraceptive choices made at the time of abortion have an important effect on the rate of reabortion. postabortal use of intrauterine contraception, specifically that of lng-ius, might decrease the rate repeat abortion. objective. to determine the rate of failure and to analyze factors associated with failure for the essure permanent birth control device at the detroit medical center (dmc). methods. a chart review was conducted on patients who underwent essure placement at the dmc from january 2003 through june 2007. patient demographics, past medical and surgical history, anesthesia type, procedure time, intraoperative complications, and procedure failures were noted. data were analyzed for statistical significance using spss. results. there were 316 essure procedures attempted at the dmc from january 2003 through june 2007. of the 316 attempted procedures, there were 25 failures (7.9%). 13 of the 25 failures were attributed to difficulty visualizing the ostia (52%). other causes of failure included expulsion of the device (2), tubal spasm (1), uterine perforation (1), and tubal ostia too large for the device (1). there were 3 cases of failed placement for undocumented reasons, one case requiring a laparoscopic tubal ligation secondary to postprocedure tubal patency, and 3 post-procedure pregnancies. age, race, body mass index, gavidity, parity, history of sexually transmitted infections, medical history, history of cesarean section, tobacco or illicit drug use, anesthesia type, and physician experience with the procedure were not significantly associated with placement failure or difficulty visualizing the ostia. a longer procedure time was significantly associated with failure (40.6 vs 26.6 min, p = 0.004), and history of ectopic pregnancy was significantly associated with difficulty visualizing the ostia (33.3% vs 4.7%, p = 0.003). conclusion. the failure rate for placement of the essure permanent birth control device at the dmc is 7.9% with a pregnancy rate of 0.95%. the majority of failures may be attributed to difficulty visualizing the ostia. a history of ectopic gestation was significantly associated with difficulty visualizing the ostia; thus, it may be reasonable to advise these women that success in essure placement may be reduced. introduction. the essure permanent birth control device is a relatively new form of minimally invasive sterilization for women. under hysteroscopic guidance, a dynamically expanding micro-insert is introduced into the proximal portion of the fallopian tube. the micro-insert induces local fibrosis and ultimately occlusion of the tubal lumen. a hysterosalpingogram (hsg) is performed three months after the procedure to confirm bilateral tubal occlusion. objective. to determine the follow-up rate for the post-essure hsg for a clinic population. methods. a retrospective chart review was conducted on university health center (uhc) patients who underwent placement of the essure permanent birth control device at the detroit medical center from january 2003 through june 2007. follow-up for the post-essure hsg as well as the result of the hsg were noted for each patient. results. placement of the essure permanent birth control device was attempted in 83 uhc patients of which 79 were successfully completed. of the 79 patients, ten underwent a post-essure hsg (12.7%). the hsg was performed three to six months after placement of the essure permanent birth control device. bilateral tubal occlusion was documented in all ten patients. conclusion. despite counseling patients prior to their procedure that a hsg is needed and providing an information sheet, the follow-up rate for the post-essure hsg for this clinic population is only 12.7%. for those in whom a hsg was performed three to six months after essure placement, bilateral tubal occlusion was confirmed in all. steps and or approaches to improve compliance with post-procedure confirmation of tubal occlusion should be employed to increase follow-up in the future. towards fibroids gene therapy: adenovirus mediated delivery of herpes simplex virus 1 thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the eker rat model. memy hassan, 1 dong zhang, 3 salama salama, 1 cheryl walker, 2 hala el-mazar, 1 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. aim: assessment of the efficacy of gene therapy of uterine leiomyoma in the immune-competent eker rat model using adenovirus mediated delivery of herpes simplex-1-thymidine kinase gene followed by ganciclivir treatment (ad-tk/gcv). method: female eker rats with mri-confirmed uterine fibroid lesions were randomized to a single treatment with direct intratumor injection of ad-tk/gcv, ad-lacz/gcv, or medium.the tumor volume was evaluated by serial mri scanning and confirmed with caliper measurement at time of euthanasia. sample rats were selected randomly and killed at the following time points; 10, 20 and 30 days post treatment. samples were collected from tumors, other body organs and blood to assess the safety and efficacy of the treatment. results: ad-tk/gcv treatment produced dramatic shrinkage of the total uterine fibroid volume by 75% ±16, 59% ±6 and 68%±28 of pretreatment volume at days 10, 20 and 30 respectively. the tumor size in negative control animals receiving ad-lacz/gcv continued to grow by +26%±10, +66%±23, +102% ±38 while receiving media continue to grow by + 20% ± 6 , +70% ± 8 and +110% ± 15 at same time points. ad-tk/gcv induced significant increase in caspase 3 activity, bax expression, decrease in bcl2 and parp proteins expression and increased tunnel apoptosis index. additionally ad-tk/gcv treatment decreased cyclin d1and pcna expressions. ad-tk/gcv did not produce any significant change in liver function tests or relative uterine horns weight to total body weight. the adenovirus transfection did not disseminated significantly to other distal organs except to liver and myometrium in limited number of animals. h e staining of non targeted organs did not revealed any sign of tissue damage. ad-transfection increased local cd4 and cd8 expressions as well as serum anti-ad antibodies. conclusion: ad-mediated delivery of hsv1tk gene by direct intra-tumor inoculation followed by sc treatment of gcv for ten days effectively shrinks uterine leiomyoma lesions in eker rats. this effect is mediated via induction of apoptosis and decreasing the proliferation. the treatment regimen is well tolerated. these studies provide essential preclinical data for the development of gene therapy as an alternative non-surgical treatment option for women with symptomatic uterine fibroids. inhibitors of catechol o-methyl transferase shrinks uterine fibroids in the eker rat model. memy hassan, 1 dong zhang, 3 hala el-mazar, 1 cheryl walker, 2 ayman al-hendy. 3 1 ob/gyn, utmb; 2 md anderson; 3 ob/gyn, meharry medical college, nashville, usa. background :the sex hormone dependent pattern of uterine leiomyomas and their high content of catechole -o-methy transferase (comt) raise the possibility for the development of novel treatment option using comt inhibitors.aim : to assess the potential therapeutic utility of a synthetic comt inhibitor (ro 41-0960) in the eker rat model of uterine leiomyoma. methods female eker rat were evaluated by mri to confirm the prescence uterine fibroid lesion, then randomized for sc treatment with ro 41-0960 150 mg /kg ,twice/ day for 28 days versus vehicle injection.fibroid tumor burden was evaluated by serial mri measurement and confirmed by direct caliper measurement at time of euthanasia. sample animals were euthanized at 2 and 4 weeks. at that time tumor tissue, blood and most of animal organs including long bones were collected and subjected for further evaluations. 24 hours urine samples were collected for evaluation of estrogen (e2) metabolites and bone resorption marker. results:animals treated with ro 41-0960 exhibited significantly lower uterine fibroid tumor burden (86% and 105%) of pretreatment volume at 2 and 4 weeks post treatment respectively. conversely, the tumor size in control animals continued to grow and reached 300 %, and 300 % of pretreatment size at the same time points. ro 41-0960 treatment resulted in an increase in urinary 2/16 hydroxy e2 metabolite ratio. in addition ro 41-0960 increased bax expression and decreased parp , pcna and cyclind1 experssions . all ro 41-0960 treated animals tolerated the treatment protocol with no signs of toxicity. h e staining of different body organs did not reveal any signs of tissue damage. furthermore, there was no significant change in both liver function tests (alt, ast, billirubin) and bone resorption marker , deoxypyridinoline croslinks, between treated and control rats. conclusion ro 41-0960, a synthetic selective comt inhibitor, caused immediate arrest of the growth of eker rat uterine leiomyoma. this effect might be in part due to modulation of various estrogen dependent genes regulating leiomyoma apoptosis (parp, bax) and proliferation (pcna, cyclin d1). this anti-estrogenic effect is due to the accumulation of the the antiestrogenic metabolite 2 hydroxy estrogen secondary to comt inhibition.comt inhibitors might present an alternative non-surgical option for the treatment of women with symptomatic uterine fibroids. background development of uterine leiomyomas (fibroids) is the most common pathological feature in the female reproductive tract. they negatively impact patients of virtually every gynecologist. despite such morbidity, leiomyoma development is poorly understood. we have recently demonstrated that leiomyomas have a genomic expression pattern that limits retinoic acid (ra) exposure. our group and others have demonstrated that tgf-beta regulation is altered as well. these two pathways likely play central roles in leiomyoma development. the central feature of uterine leiomyomas, the extracellular matrix (ecm), is regulated by both all-trans retinoic acid and tgf-3, focusing on versican as a critical ecm component. human uterine leiomyoma and patient-matched myometrium were obtained from surgical specimens under an irb-approved protocol. these tissues were immortalized and treated with either all-trans retinoic acid, tgf-3, or anti-tgf-3 antibody. rna was isolated for qrt-pcr. human immortalized leiomyoma cells demonstrated the same increased template expression of tgf-3 (2.96±0.71 fold), retinoic acid metabolizing protein (cyp26a1; 16.81±4.50), and versican variant v0 (7.75±2.2 fold) as was found in the progenitor tissue. when treated with all-trans ra, expression of versican variant v0 decreased to levels found in myometrial cells (0.96±0.04 fold). conversely, when treated with tgf-3, expression of versican variant v0 increased 2.98±0.22 fold. to confirm that tgf-3 was central to the overexpression of v0, we treated leiomyoma cells with anti-tgf-3 antibody, and found that baseline over-expression of v0 template was decreased to expression levels similar to untreated myometrial cells. finally, we elucidated a link between the ra and tgf-pathways by assessing the impact of ra treatment of tgf-3 expression, demonstrating that tgf-3 template decreased to levels comparable to myometrial cell expression (0.84±0.12 fold). the disrupted leiomyoma ecm, of which versican is a central component, defines the leiomyoma phenotype. in this study, the leiomyoma fibrotic phenotype regressed when treated with ra and increased when treated with tgf-3, providing the basis for novel therapeutic interventions directed at cell differentiation and ecm formation. objectives: uterine fibroids are the leading cause for hysterectomies in the us. the lack of an appropriate in vitro cell model for the initiation of fibroid growth has hindered advancement in understanding the cellular and molecular basis for the development and progression of uterine fibroids. fibrosis is the underlying mechanism of uterine fibroid formation and myofibroblasts cells are the principal fibrogenic cell type in the uterus. we sought to develop a myofibroblast in vitro cell model for analyzing the initiating molecular events of uterine fibrosis. methods: smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and cultured in the presence of 10% serum until 70% confluent. for the next 72 h cells were cultured in serum-free media followed by replacement with serum containing media. cells were fixed at 0, 10, 20, 30, 60 m later. cell fine structure and cytoskeletal organization were evaluated by transmission electron microscopy. smooth muscle specific alpha-actin ( -sma) and progesterone receptors (pr) were detected by western blot. results: we observed smc differentiation into myofibroblasts, marked by the presence of notched nuclei ( figure) and the increased expression of -sma 20 m after serum replacement. pr-a and pr-b were detectable at 10, 20 and 30 m. conclusions: the development of myofibroblasts is important in wound healing and fibrosis. we show for the first time that uterine myofibroblasts can be derived in culture from myometrial smcs. thus, these cells will be utilized as a model for developing "in vitro fibroids". this model will enable the study of myofibroblast activation, cytokine signaling, intracellular regulation of uterine fibrogenesis, production of extracellular matrix proteins and development of antifibrotic drugs. the presence of prs in our model enables us to evaluate pr mediated events in fibroid pathogenesis and treatment. this model will be more useful in determining the molecular biology of fibroid initiation than cell models derived from established fibroids that are already well past their initial stages of development. novel approach to genome-wide expression profiling analysis. liping feng, morgan walls, insuk sohn, millie behera, sin-ho jung, phyllis leppert. obgyn; biostatistics and bioinformatics, duke university medical center, durham, nc, usa. background: 10 microarray studies have examined the differential gene expression between uterine fibroid and normal myometrium. all previous studies considered the fibroid as a whole and analyzed only fold changes. we have developed a novel statistical approach to genome-wide expression analysis comparing two fibroid tissue sites to myometrium. methods: using affymetrix tm u133a genechip, we have compared the gene expression between c and e and matched adjacent m. data has been analyzed by considering the 3 specimens per subject and 5 subjects as individuals. we used a block one-way anova method to test if each gene was differentially expressed among the three sites. the p-value is calculated using a permutation method accounting for possible dependency among three lesions. the multiple testing issue was addressed by controlling the false discovery rate. expression values were calculated using the robust multichip average (rma) method. rma estimates are based upon a robust average of background corrected perfect/mismatch (pm) intensities. normalization was done using quantile normalization. expression values were then transformed by taking logarithm base 2. confirmatory rt-pcr was performed. results: we applied a hierarchical clustering analysis to all raw data sets and then displayed a dendrogram, where the height of each branch point indicates the similarity level at each generated cluster. identical gene expression among sites clusters together. due to a strong site effect, m tissues clustered separately from e and c combined. 45 genes were differentially expressed when we used a 0.1 q-value cut off. expression data revealed concordant changes in genes regulating cholesterol biosynthesis, gene transcription, estrogen and extracellular matrix formation when both e and c were examined. cyp19 was detected and we report for the first time that scc-112 (a cell cycle-regulated molecule) folliculin and l-selectin are differentially expressed suggesting that they may be involved in the regulation of cell growth and proliferation of uterine fibroids. conclusions: our novel robust analysis of gene expression provides new clues to the relevant pathways of fibroid development. this new statistical approach that can be used in clinical and/or translational studies to identify differentially expressed genes comparing treatment regimens, cells or tissues. objectives: thrombospondin-1 (tsp-1) is a large matricellular glycoprotein secreted by many cell types. matricellular proteins modulate interactions between cells and their environment, regulate cell adhesion and are expressed during tissue formative processes. they are especially important in fibrosis. tsp-1 plays an important role in angiogenesis and is an activator of tgf -3. in a previous study, we found that differential expression of tsp-1 in uterine fibroids may contribute to an altered healing process leading to fibrosis. this alteration in tissue response to injury initiates the development of abundant, nonaligned collagen fibrils and changes in other components of the ecm. methods: we measureed the pattern of mrna and protein expression of tsp-1 by rt-pcr and western blot in an in vitro serum-deprived differentiated myometrial cell (myofibroblast) model. specifically, smooth muscle cells (smcs) were enzymatically isolated from the myometrium of non-pregnant women and grown in primary culture to 70% confluence. then smc were serum deprived for 72 h and treated back with 10% serum for 10, 20, 30, and 60 m. cells were collected for rna and protein, and tsp-1 expression was evaluated. in addition, cells were stained using the combination of anti-cd45 and anti-smooth muscle -actin or combination of anti-cd42d and anti-smooth muscle -actin as well as the appropriate single and double negative controls. stained sections were analyzed using zeiss axio imager widefield fluorescence confocal microscopy. results: tsp-1 mrna and protein was present in cells in this serumstarvation model after the addition of serum and the expression level remained elevated for 60 m following the addition of serum. fluorescence staining analysis indicated that these cells were positive for human smooth muscle -actin, but negative for leukocyte antigen cd45 and platelet marker cd42d suggesting that the myofibroblasts cells themselves were the source of the tsp-1. conclusions: unlike skin wounds, where tsp-1 is derived from the blood macrophages, monocytes and platelets, differentiated myometrial cells appear to produce tsp-1. elucidating the roles of tsp-1 in myometrium physiology and pathobiology will increase our understanding of the etiology of uterine fibroids and may lead to improved therapies. further studies utilizing this cell model to determine the role of tsp-1 in the activation of tgf -3 are indicated. phospholipid s1p via gi, rac and rho pathways. yoel smicun, armando wu pimentel, jennifer gilman, david a fishman. obstetrics gynecology, new york university school of medicine, new york, ny, usa. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of ovarian cancer patients. we have demonstrated that low concentration s1p enhances while high dose s1p inhibits invasion of epithelial ovarian carcinoma (eoc) cells in a dose and attachment mode dependent manner. we sought to further dissect the pathways by which s1p affects invasion, using specific inhibitors. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi, p38-mapk, rac and rock, thereafter cells were detached and tested for invasion towards 40 m lpa chemoattractant in matrigel-coated chambers. conditioned media from pretreated cells and invading cells were quantified for upa activity using colorimetric assays. the significance of results was calculated by student's t-test. results: inhibition of gi mildly increased invasion of both control ( p=0.019) and 20 m s1p treated cells ( p=0.0002). inhibition of both p38-mapk and rac did not affect 20 m s1p treated cells, in contrast invasion of control cells was mildly increased (p=0.025, 0.034). inhibition of rock, a protein effector downstream of rho, highly elevated invasion of both control and 20 m s1p treated cells (8 fold, p=0.0035, 23 fold, p=0.00037). both upa and gelatinase activities were higher in conditioned media of invading cells than of attached cells. gelatinase activity was enhanced by both concentrations of s1p (p=0.002, 0.001). ptx fully inhibited gelatinase activity of control and 0.5 m s1p treated cells, and partially of the 20 m s1p treated cells (p<0.00007). 0.5 m s1p significantly increased upa activity of attached (p=0.011) but not of invading cells. this increase was sensitive to ptx and rac inhibitor. 20 m s1p inhibited upa in both attached and invading cells (p=0.0004), this inhibition was rock dependent. conclusions: these findings suggest a strong inhibition of invasion and upa by the rho pathway, of both control and 20 m s1p treated cells. this inhibition is induced partially by upstream gi protein. increased invasion by 0.5 m s1p is associated with elevation of gelatinase activity through gi, rac and rock pathways. this suggests that attached cells and invading cells affect upa activity through different pathways. objectives: sphingosine-1-phosphate (s1p) levels are elevated in serum and ascites of epithelial ovarian cancer (eoc) patients. we have demonstrated that invasion of attached eoc cells differentially react to s1p as compared to invading cells. we examined the impact of the inhibitors for gi and rac on attached and invading eoc. methods: dov13 eoc cells were pretreated for 4-hrs with vehicle, 0.5 m or 20 m s1p and with inhibitors for gi (pertussis toxin (ptx)), and rac (nsc23766, (rac-i)), and cells were detached and assayed for invasion towards 40 m lpa in matrigel-coated chambers. to distinguish the response of attached from invading cells, inhibition of cells pretreated with inhibitors was either continued or not in the invasion chambers. conditioned media (cm) of invading cells were quantified for upa and gelatinase activity by fluorometric and colorimetric assays. significance of results was calculated by student's t-test. results: the significant (p=0.004) increase of invasion by 0.5 m s1p was inhibited by both ptx and rac-i, either by pretreatment alone or by continuous treatment (p=0.0024-0.015). however, the invasion was higher in cells inhibited continuously than cells inhibited only in dishes. both inhibitors did not affect cells treated with 20 m s1p. control cells invasion was increased (2.3 fold, p=0.0019) by continuous rac-i treatment. 0.5 m s1p increased gelatinolysis in cm of invading cells. this and control cells activity was inhibited by ptx pretreatment. continuous treatment with ptx or rac-i elevated 3 and 2 fold gelatinase activity of control and 20 m s1p treated cells. in contrast upa activity was inhibited by both 0.5 m and 20 m s1p. activity of control cells was inhibited by both pretreatment and continuous treatment with both ptx and raci. upa activity of cells treated with 0.5 m s1p was increased only by pretreatment with ptx and raci. in contrast, in cells treated with 20 m s1p upa was inhibited only by continuous inhibition with ptx and raci. conclusions: invasion of s1p induced eoc cells correlated with the gelatinase and upa activities in their microenvironment. ptx and rac-i affect attached and invading cells in different manner, inhibition of invasion and gelatinolysis of attached and increased invasion of invading cells. this suggests a dual effect of the gi-rac pathway, inhibiting attached and stimulating invasion via gelatinolysis of invading cells. lysophosphatidic acid (lpa) levels are elevated in the ascites and plasma of early-and late-stage ovarian cancer patients, underscoring the unique role this bioactive lipid plays in the pathobiology of epithelial ovarian cancer (eoc). lpa binding to its cognate g-protein-coupled receptor can transactivate the receptor tyrosine kinase, egfr, which is often overexpressed in ovarian tumors. in the current study, we investigated the role that lpa activation of egfr plays in the processing of the metalloproteinase, mmp-2, and eoc dissemination. lpa stimulates tyrosine phosphorylation of egfr in ovarian cancer cells, and egfr kinase activity is required for optimal lpa induction of pro-mmp-2 activation. lpa-dependent pro-mmp-2 activation does not require the egfr ligands, amphiregulin, b-cellulin, egf, hb-egf, and tgf-a. we previously reported that when cells are cultured at high density, lpa represses rhoa activity to induce loss of stress fibers, and these changes in actin microfilament organization contribute to pro-mmp-2 activation. in the current study, inhibition of rho-kinase/rock with y-27632 reversed the repression of lpa-stimulated pro-mmp-2 processing observed with treatment of the egfr kinase inhibitor, ag1478. correspondingly, lpa induced the loss of stress fibers, while inhibition of egfr kinase restored stress fiber formation in lpa-treated cells. this suggests that lpa acts through egfr to modulate microfilament organization and pro-mmp-2 processing. finally, lpa-induced cellular haptotactic migration and invasion are abrogated when egfr kinase activity is blocked. taken together, these results suggest that egfr signaling plays a critical role in lpa regulation of metastatic pathways by mediating changes in the cytoskeleton which impact protease activity. objectives: membrane-derived vesicles are active modulators of tumor dissemination; they promote and contribute to extracellular matrix degradation and tumor cell invasion. we have isolated vesicles from ascites of ovarian cancer patients and from ovarian cancer cells in vitro. here we analyzed the functional consequences of exposure of cancer cells to vesicles derived from a different type of malignancy in order to evaluate their proinvasive properties. methods: ovarian cancer (dov13), breast cancer (mda mb 231) and mesothelioma (hp-1) cells were grown in media supplemented with 10% fbs. after serum deprivation, 10% vesicle-free fbs was added for 4 hr to induce vesicle release. vesicles were isolated from media by differential centrifugation and quantified with a bradford assay. fusion to cells was followed by fluorescence of the lipophilic tracer dii. each cell line was stimulated with 1 and 10 g/ml of each type of vesicles and tested in a matrigel invasion assay. changes in proliferation were evaluated in an mts assay. gelatin zymography was used to assess matrix metalloproteinase activity of vesicles. results: zymographic analysis showed that vesicles contained mmp-2 and 7. fusion experiments showed that all vesicles fused to all cell types. all vesicles induced the invasion of their respective cell types in a dose-dependent manner. when vesicles were used at 1 g/ml they induced significantly high levels of invasion in all cell types tested. at 10 g/ml they significantly inhibited invasion in different cell types. both concentrations of vesicles stimulated cell proliferation in all cell types tested. conclusions: membrane derived vesicles are potent mediators of invasion for mesothelioma, breast and ovarian cancer cells in vitro. this effect occurs in a dose-dependent manner when vesicles induce invasion of the same cell type from which they were isolated. when vesicles are used to induce a different cell type, low concentrations induce invasion while high concentrations inhibit invasion, this effect was independent of cellular proliferation. these results suggest that a novel mechanism may be at play where activation of recognition of self and non-self specific pathways may determine the invasive potential of the tumor cell upon fusion to microvesicles. the identification of signaling pathways responsible for this heterotypic signaling is a current effort. vegfr-2 as a potential target to inhibit lpa-induced epithelial ovarian cancer (eoc) invasion. sonia dutta, fengqiang wang, elaine barfield, david a fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objective: vegf and vegf receptors (vegfrs) play important roles in ovarian cancer metastasis. in this study, we examined the expression profiles of vegf and vegfrs (vegfr1, vegfr2, co-receptor nrp1 and nrp2) in established eoc cells lines (dov13, r182, ovca429, skov3) and an immortalized normal ovarian epithelium (iose-29). the effect of lysophosphatidic acid (lpa) on vegf and vegfrs expression and the effect of vegfr-2 silencing by rnai on lpa-induced invasion were also evaluated. methods: vegf and vegfrs expression in ovarian cancer cell lines and normal ovarian epithelium was quantified by real time pcr. cell invasion differentiate into either a pro-tumor or anti-tumor phenotype depending on the specific tumor microenvironment. previously, we described a subgroup of epithelial ovarian cancer (eoc) cells with a functional tlr-4-myd88-nf-b pathway (type i eoc cells). these cells have constitutive nf-b activity and constitutively secrete il-6, il-8, mcp-1, and gro . we hypothesize that type i eoc cells, but not type ii, can promote macrophage differentiation into a tumor-supporting immune cell. methods: eoc cell conditioned media (cm) was prepared by incubating eoc cells in log-phase growth in optimem for 48h. migration assay was done using an in vitro cell culture insert with 8 m-size pore and pkh26 red fluorescentlabeled thp-1. cytokine profile of thp-1 cells co-cultured with eoc cell cm was determined using xmap technology. modulation of response to apoptotic bodies was determined by "pre-educating" thp-1 cells with eoc cell cm for 24 h prior to exposure to apoptotic bodies (1:1 ratio with thp-1 cells). results: monocytes migrate toward eoc cells. however, migration is significantly higher towards type i eoc cells. type i, but not type ii, eoc cells alter monocytes' cytokine profile by inducing the secretion of high levels of progrowth and angiogenic cytokines il-6, il-8, mcp-1, and gro . furthermore, type i eoc cells modify monocytes response to apoptotic bodies by inducing a significant increase in the secretion of il-6, il-8, mip-1 , mip-1 , and gro (18-fold, 2.5-fold, 2-fold, 3-fold, and 11-fold respectively). conclusion: we demonstrate for the first time a differential interaction between two subtypes of eoc cells and monocytes. we showed that the microenvironment created by type i eoc cells is able to modify the function and differentiation of immune cells towards a tumor supporting phenotype. understanding the molecular mechanisms mediating this tumor-immune interaction will help to design appropriate immune therapies that will take into consideration the tumor microenvironment. objectives: the standard of treatment for patients with ovarian cancer (oc) is intravenous combination chemotherapy (ct) after primary cytoreductive surgery. although initial response is above 80%, most of these patients experience recurrence. the only approach for these patients is salvage ct which may prolong their lives for months. early detection of patients who are not responding to current therapy or are at risk of experiencing an early relapse of disease might improve response rates and survival if alternative therapy is possible. no single predictive marker has yet been proven sufficiently sensitive or specific to find a place in the daily clinic. in the present study we use a newly described biomarker test for the detection of oc (visintin et al 2007 clinical cancer research) and evaluated the ability of the test to monitor ct response methods: 55 patients with oc, figo stage i-iv, were included in this study. all patients recieved postsurgery first line combination ct (paclitaxel/carboplatin). samples were evaluated at 1) baseline (mean 35 days after surgery), prior to the first cycle of ct, and 2) after 3 cycles of ct. 20 μl serum samples were analyzed by multiplex assay (beadlyte® cancer biomarker panel kit) for six markers. changes during ct and differences in markers between patients with short time to progression (ttp) and patients with long ttp were determined. results: positive test for oc was observed in 96 % of the patients evaluated at baseline. all patients had residual disease after surgery. from patients with long ttp, 15/20 patients (specificity 75 %) had a negative test after 3 cycles. from the patients with short ttp, 13/14 had a positive test (sensitivity 93 %) p=0.000096 ( ²). the risk of experiencing a ttp shorter than 6 months when having a positive test after 3 cycles of ct was 72 %. conclusion: we describe for the first time the use of a panel of biomarkers for oc that might have an application for monitoring ct response and risk of relapse. the test detects the presence of residual disease following debulking surgery, and differentiates between long term and short term progression. a longitudinal study is performed to determine how early during ct the test can identify responder versus non responder. ksp inhibitor (arry-520) as an alternative for paclitaxel in myd88-positive epithelial ovarian cancer cells. ki h kim, 1 ayesha b alvero, 1 yanhua xie, 1 david trollinger, 2 gil mor. 1 1 obstetrics, gynecology, and reproductive sciences, yale university school of medicine, new haven, ct, usa; 2 array biopharma inc., boulder, co, usa. background: we previously described that epithelial ovarian cancer (eoc) cells ubiquitously express tlr-4, but not the adaptor protein myd88. when treated with paclitaxel, a known tlr-4 ligand, myd88-positive eoc cells exhibited nf-b activation, increased secretion of il-6, il-8, mcp-1, and gro , and increased p-erk levels . since majority of eoc patients are given paclitaxel in combination with a platinum drug, it is not only important to distinguish those patients that should not be given paclitaxel; it is also important to identify alternative chemotherapy agents that would benefit this sub-group of patients. the objective of this study is to determine if the ksp inhibitor, arry-520, can be an alternative for paclitaxel in myd88-positive ovarian cancer patients. methods: myd88-positive and myd88-negative eoc cell lines isolated from either ascites or tumor tissue were treated with increasing concentrations of arry-520 (3 to 3000 nm) or paclitaxel (0.2 to 20 m) for 24, 48, and 72 hours and cell viability was determined using the celltiter 96 aqueous one solution cell proliferation assay. cytokine profiling was performed from supernatants using xmap technology. nf-b activity was determined using a luciferase reporter system. p-erk levels were measured by western blot analysis. results: arry-520 and paclitaxel exhibited the same cytotoxic effect on myd88-negative and positive eoc cells. the ic 50 at 48h for myd88-negative eoc cells was 0.003 m and 0.2 um for arry-520 and paclitaxel, respectively. for myd88-positive eoc cells, the ic 50 at 48h was 3 m and 20 m for arry-520 and paclitaxel, respectively. however, unlike paclitaxel, arry-520 did not induce nf-b activation or enhance the secretion of il-6, il-8, mcp-1, and gro , and did not induce erk phosphorylation on myd88 positive cells. conclusions: administration of paclitaxel to patients with a myd88-positive tumor could have detrimental effects due to the paclitaxel-induced enhancement of cytokine production which promotes chemoresistance and tumor growth. arry-520 has similar anti-tumor activity in eoc cells as that of paclitaxel. however, unlike paclitaxel, it does not induce cytokine production in myd88positive eoc cells, and therefore, the ksp inhibitor arry-520 may represent an alternative to paclitaxel in this subgroup of eoc patients. introduction: nf-b activation has been associated with cell proliferation, angiogenesis, metastasis and suppression of apoptosis in ovarian cancer. in addition, nf-b activity induces the production of pro-inflammatory cytokines which may contribute to chemoresistance. inhibition of nf-b may represent a new approach to prevent tumor growth and reverse chemoresistance. in the present study we described the characterization of a novel nf-b inhibitor, eriocalyxin b (erib) that is able to re-establish the apoptotic cascade in chemoresistant ovarian cancer cells by suppressing pro-inflammatory cytokines and antiapoptotic proteins. materials and methods: eoc cell lines were isolated from malignant ovarian cancer ascites. caspase activity was determined by caspase-glotm assay. cytokine production and secretion were determined using multiplex assay. erib effect on cancer cells was evaluated in a time and dose dependent manner using celltiter 96 cell proliferation assay. protein expression was determined by western blot analysis. combination studies were done with paclitaxel and carboplatin in addition to erib treatment. nf-b activity was determined by monitoring the expression of a nf-b luciferase reporter. results: erib decreased cell viability of ovarian cancer cells with an ic50 of 0.5-1μm in 48 hours and was associated to increasing levels of caspases 8,9 and 3 activity. intracellular changes induced by erib included: 1) inhibition of nf-b activity; 2) decrease in cytokine production; 3) down regulation of anti-apoptotic proteins xiap and flip, and reversal of resistance to tnf-and fasl-mediated cell death; and 4) chemosensitization to carboplatin and paclitaxel. at present, evidence is accumulating regarding the existence of unique populations of specialized tumor-initiating, stem-like cells within various tumor types of distinct origins. these cancer stem cells (csc), with characteristics reminiscent of normal stem cells, are thought to be responsible for driving tumor growth. we propose that ovarian cancers arise from csc, and are using microarray-based technology to identify specific genes/cell surface markers associated with ovarian csc that will permit distinction of these rare cells from the remaining tumor bulk. to identify unique gene signatures associated with an ovarian tumor-initiating cell population, we have utilized an in vivo serial transplantation model. this model selects for primary human ovarian tumor cells with increased tumorigenic capacity, given that time to tumor formation decreases with successive serial transplant despite fewer cells injected. our initial studies used cells derived from a human ovarian clear cell carcinoma, serially transplanted for three passages in nod/scid mice. rna derived from these transplanted tumors was analyzed on human genome microarrays. from these analyses, several differentially expressed genes were identified. the differential expression noted for potential genes of interest is currently being validated by rt-pcr. of particular interest, expression of the transmembrane glycoprotein muc4 was found to increase both at the rna and protein level with successive transplant of this clear cell carcinoma. further studies are ongoing to determine the functional significance of muc4 and other identified differentially expressed genes in ovarian clear cell cancer. in addition, we are carrying out parallel microarray analyses in other ovarian tumor subtypes. background recent observations suggest a decreased incidence of neoplastic lesions in hiv infected individuals treated with highly active anti-retroviral therapy comprised of protease inhibitors, such as ritonavir. the polycomb group protein ezh2 is associated with aggressive human malignancies via transcriptional suppression of dna repair proteins. objective objective of the present study was to assess the antineoplastic impact of ritonavir on epithelial ovarian cancer (eoc) cell lines. methods eoc cell lines (mdah 2774 and skov-3) were treated with serial dilutions of ritonavir (1-10 mm) dissolved in dmso. normal diploid human fibroblasts served as controls. growth curves, apoptosis and cell cycle analysis were performed with cell counting kit-8, annexin v and flow cytometry. signal transduction was studied with western blotting. dna double strand breaks (dsb) were induced with 0.5 mm etoposide treatment for 16 hours. homologous recombination (hr) repair of dna damage was measured by assessment of rad51 foci formation in the nucleus of the cells as visualized by fluorescence microscopy according to previously published criteria. untreated eoc cells expressed higher levels of ezh2 and lower levels of rad51 and xrcc2 as compared to controls and in turn, had lower prevalence of rad51 repair foci formation in response to dsb. serial treatments of eoc cells with ritonavir resulted in a decrease in expression of ezh2 and an increase in expression of rad51 and xrcc2 when compared to untreated eoc cells. after induction of dsb, the rad51 repair foci formation was significantly more prevalent in eoc cells treated with ritonavir as compared to untreated eoc cells. in addition, ritonavir induced apoptosis in ovarian cancer cell lines by down-regulation of akt pathway and caused g1 cell cycle arrest mediated by down modulating levels of prb phosphorylation and depleting the cyclindependent kinase 4 and 6. ritonavir effectively induces apoptosis, cell cycle arrest and improves repair of dna damage by hr in ovarian cancer cell lines. as impaired hr is a key event in causation and progression of neoplastic lesions, ritonavir may have a role in chemoprophylaxis and treatment of human malignancies. a rare case of ovarian cystic lymphangioma. tomer singer, 1 tamer seckin, 1 noa feldman, 1 susan jormark, 2 michael divon. 1 1 department of obstetrics and gynecology, lenox hill hospital, n.y., ny, usa; 2 department of pathology, lenox hill hospital, n.y., ny, usa. cystic lymphangioma (cl) is a rare, benign malformation of the lymphatic system whose exact etiology remains uncertain. cl may arise in different sites: the spleen, the mediastinum, the axillary region, the retroperitoneum, and the mesentery. retroperitoneal cl is extremely rare and its true incidence is unknown. the majority of cases are symptomatic during childhood. clinical presentation of adult cl is variable and may be misleading. typically, this is a slow-growing tumor and it remains asymptomatic for a long period of time. it is most often found incidentally during abdominal or pelvic imaging studies, surgeries or autopsies. total surgical removal of the lesions with microscopically clear margins is the best approach when it is possible. we report, for the first time, a case of cystic lymphangioma arising from the ovary in a post-menopausal woman and present the feasibility and the advantages of laparoscopic surgery, allowing accurate diagnosis, optimal treatment and minimal risk for the patient. lgsc) is a chemoresistant ovarian neoplasm that has been molecularly linked to low malignant potential tumor (lmp), which often behaves in a non-invasive fashion. micropapillary features within lmp (lmp-mp) are associated with increased invasive behavior. the aim of this study was to determine the differential gene expression of lmp, lmp-mp, and lgsc to identify genes involved in malignant transformation and carcinogenesis. methods: laser capture microdissection was used to isolate epithelial cells from snap-frozen lmp (n=18), lmp-mp (n=9) and lgsc (n=11). rna was extracted, amplified, reverse transcribed to cdna and hybridized to affymetrix u133 plus 2 arrays. data was analyzed by significance analysis methods: medical records were reviewed for patients who underwent hysterectomy for all indications at wsu hospitals from 1/1/05 -4/30/06. pathology reports were reviewed to identify the incidence of adenomyosis. data obtained from medial records included: age, race, insurance, bmi, social history, medical history, and presenting symptoms. the correlation between adenomyosis and all the above factors was tested using the appropriate statistical methods. results: 850 patients were included. adenomyosis was confirmed by pathology in 375 patients, an incidence of 44.1%. incidence was not significantly different among races after controlling for parity. 46.3% in african americans, 35.3% in caucasians, and 40.9% in others. incidence was not statistically different between uninsured(28.0%), privately insured (47.7%), and medicaid (41.9%) patients. p=.065. incidence of adenomyosis was not different between smokers (38.9%) and nonsmokers (44.6%). p= .386. table i shows the correlation between adenomyosis and different risk factors. conclusion: adenomyosis was diagnosed in 44.1% of hysterectomy specimens. race, socioeconomic status or social habits didn't affect its incidence. diabetes and endometrial cancer were negative risk factors for adenomyosis, whereas htn, hypothyroid, breast cancer, fibroids, polyps and endometriosis didn't affect its incidence. menorrhagia, dysmenorrhea, dyspareunia, and chronic pelvic pain but not metrorrhagia had a positive correlation with adenomyosis. there is a protective adipocytokine profile. we hypothesize that this finding may precede some ill-defined threshold of fat mass and/or insulin resistance after which adiponectin decreases. the objective: to correlate reproductive hormone production with menstrual bleeding patterns among women in the menopause transition. methods: a sub-cohort of the swan study consisting of 848 women age 42-52 was studied. each woman collected daily first void urine samples for one complete menstrual cycle or 50 days (whichever came first) once a year for 3 years. urine was assayed for excreted levels of fsh, lh, estrogen metabolites and progesterone metabolites which were normalized for creatinine concentration. ovulation was detected by a validated algorithm. menstrual bleeding parameters were derived from daily calendars. correlations between bleeding characteristics, hormone concentrations, and other potential clinical predictors were analyzed using multivariate logistic regression models. results: the cohort was ethnically diverse with a median age of 47 and with 73% in the early perimenopause at the start of the study. 21% of all cycles were anovulatory. short cycle intervals (< 21 days) were associated with the early perimenopause (or 5.2, ci 2.1, 12.9) and with anovulation (or 20.3, ci 7.2, 56.8). long cycle intervals (36+ days) were associated with late perimenopause (or 8.7, ci 1.0, 79.1)and with anovulatory cycles (or 2.6, ci 1.9, 3.5). both short (1-3 days) and long (8+ days) duration of menstrual bleeding were significantly associated with anovulation (or 2.4 and 2.6, respectively). women with anovulatory cycles were less likely to report heavy menstrual bleeding than women with ovulatory cycles. menorrhagia was not associated with steroid hormone concentrations but was associated with obesity (or 4.7, ci 2.6, 8.5) and with the self-reported presence of fibroids (or 4.1, ci 1.8, 9.5). conclusions: among women in the menopause transition, abnormalities in timing of menstrual bleeding (cycle intervals or bleeding duration) have a hormonal basis and are frequently associated with anovulation. in contrast, abnormally heavy periods do not have a hormonal basis and are less likely following anovulatory cycles. heavy periods are associated with obesity and fibroids. to undetectable levels. the use is vaginal e is contraindicated, although these women have even higher rates of av. topical testosterone (tt) has successfully treated vulvar atrophy; testosterone receptors are also present in the vaginal epithelium. objective: assess the effect of tt on vaginal maturation index (mi) and relief of av symptoms. methods: 10 postmenopausal women on aromatase inhibitor with symptomatic atrophic vaginitis were enrolled in prospective, irb approved study. estradiol (e) and testosterone (t) levels, av questionnaires (score 0-3; none to most severe symptoms of dryness, irritation, and dyspareunia), gynecologic exam, and vaginal smears (for ph and vaginal maturation index, mi, by meisels criteria) were performed at baseline and after 1 month of daily treatment with 300 mcg of tt. data was assessed by t test and fischer's exact test; significant p < .05. results: e levels were undetectable at baseline and following treatment. t levels increased (mean +/-sem) from baseline (16 +/-0.8) to treatment ( 38 +/-10.9); the difference was not significant; p = .8, although one patient had an appreciable rise in serum t level. two av symptoms improved significantly with tt use; comparing baseline to treatment scores: vaginal dryness (2.2 vs. 0.75; p = .03) and dyspareunia (2.3 vs. 1; p = .02 saliva analysis is a convenient, non-invasive and rapid method for assessing estradiol (e2) levels. however, particularly in postmenopausal women, the low salivary e2 levels are often near or below the sensitivity of available assays, compromising both accuracy and precision. we present results using an extraction step prior to e2 assay, which concentrates the sample to increase sensitivity and removes potentially interfering substances. morning saliva samples were obtained from premenopausal (mid-luteal phase, n=4,651) and postmenopausal women (n=1,770) not taking hormones, and from postmenopausal women receiving oral conjugated equine estrogens (cenestin, n=119; premarin, n=439), oral micronized e2 (estrace, n=145; compounded e2, n=1618), transdermal e2 patches (climara, n=623; vivelle, n=1619); or topical e2 cream (compounded e2, n=107). e2 levels were determined by an automated enzyme immunoassay (eia) after solid phase extraction. the functional sensitivity of the assay was determined to be 0.8 pg/ml, compared with >2 pg/ml without extraction. 3.9 0.5 5.7 1.0 9.6 1 oral conjugated equine estrogens; 2 oral e2; 3 e2 patch; 4 topical cream salivary e2 levels corresponded with the hormone dosage, suggesting a reliable assessment of unbound e2 levels with each formulation, dosage and type of estrogen therapy. extraction prior to eia in an automated assay dramatically increased precision and accuracy at low concentrations. omitting the extraction step may have contributed to poor serum versus saliva correlations in other studies. this method may therefore allow reliable monitoring of estrogen therapy without the need for expensive and inconvenient blood tests. the role of fibulin-3 in pelvic organ prolapse. david d rahn, 1 jesus f acevedo, 1 patrick w keller, 1 lihua marmorstein, 2 r ann word. 1 1 ob-gyn, ut southwestern, dallas, tx, usa; 2 visual sciences, univ arizona, tuscon, az, usa. objectives: fibulin-5 (fib-5) is crucial for normal elastic fibers in the protein showed a statistically significant reduction in its expression (p= 0.01). lox, loxl1, 3 and 4 proteins were detected by immunohistochemistry in all three layers of vaginal skin biopsies: (1) stratified squamous epithelium; (2) the lamina propria and (3) the muscularis layer from both patients with pop and controls. significantly, in both groups we detected a numerous macrophages scattered throughout the vaginal thickness which displayed a very strong immunostaining to loxl1. patients with severe pop showed reduced expression of proteins regulating collagen and elastin biogenesis. our finding raises the possibility that failure of ecm homeostasis could underlie the etiology of pop in women. fibroblast proliferation is regulated by hoxa11: molecular implications for pelvic organ prolapse. kathleen a connell, marsha k guess, richard bercik, hugh s taylor. obstetrics, gynecology reproductive sciences, yale university school of medicine, new haven, ct, usa. objectives: the integrity of the extracellular matrix (ecm) is maintained by a delicate balance between collagen synthesis and degradation. previously, we have demonstrated that hoxa11 is essential for the development of the uterosacral ligament in mice and regulates the expression of collagen type iii and mmp2. we have also shown that hoxa11 is deficient in the uterosacral ligament of women with pelvic organ prolapse (pop) compared to women with normal support. the exact mechanisms by which hoxa11 regulates pelvic organ integrity and repair remains to be elucidated. the aim of this study was to determine the effect of hoxa11 expression on fibroblast proliferation, and its potential role in pop. methods: nih 3t3 cells, a murine fibroblast cell line, were seeded onto a six well plate (1x 10 5 cells/well) and transfected with either a vector carrying a hoxa11 cdna or empty vector alone as a control. immunohistochemistry using bromodeoxyuridine (brdu) was performed to evaluate cell proliferation. cell division in the uterosacral ligament (usl) was also compared in women with and without pop. usl specimens were obtained at the time of hysterectomy for benign disease. immunohistochemistry was performed on paraffin embedded sections of the usl to evaluate expression of two mitotic markers, cyclin b1 and phospho-histone 3. results: constitutive expression of hoxa11 in murine fibroblasts resulted in significantly higher proliferation. cells transfected with hoxa11 had a mean brdu incorporation of 40.8+ 8.8 cells/ 100 cells compared with 32.2 + 7.5 cells/ 100 cells in controls (p=0.03). in the usl obtained from women with and without pop, cell proliferation as determined by cyclin b1 and phospho-histone3 expression was not significantly different. cyclin b1 and phosphor-histone 3 were expressed in comparable numbers of cells in both groups. conclusion: hoxa11 is necessary for usl development and promotes proliferation of adult fibroblasts. hoxa11 may have a similar function in vivo in usl, and may regulate fibroblast proliferation during growth and the acute phase response following trauma when fibroblasts are activated to proliferate and remodel the ecm. it is likely that hoxa11 mediated proliferation of usl fibroblasts contributes to the tensile strength and resilience of these structures and thereby prevents pop. supported by nichd wrhr: 5k12hd047018-03. connective tissue composition, in term of collagen i, iii and proteoglycans content, in support and nonsupport structures of women with uterine prolapse. elisabetta trabucco, 1 gennaro acone, 1 sara d'avino, 1 marco torella, 1 gennaro trezza, 2 annamaria marenna, 1 nicola colacurci, 1 luigi cobellis. 1 1 department of obstetrics and gynecology, second university of naples, naples, italy; 2 loreto mare hospital, naples, italy. objective. connective tissue consists mainly of collagen and elastic fibers, glycoproteins and proteoglycans (pgs) and is considered an important factor of the support and nonsupport structures of the genitourinary region. it has been already demonstrated altered morphologic features in the pelvic support connective tissue in women with genital prolapse. however, analysis of nonsupport tissue may provide a more accurate reflection of body collagen. the objective of our study was compare the expression of collagen i, iii and four pgs (decorin, fibromodulin, lumican, biglycan), essential for synthesis and regular assembly and diameter of the collagen fibrils, in uterosacral ligaments and in a nonsupport tissue, the uterine cervix, between premenopausal women with uterine prolapse respect to age matched controls. we characterized uterosacral ligaments and uterine cervix of 25 premenopausal women with uterine prolapse and 16 controls. this immunohistochemical study was performed on paraffin-embedded sections. results. uterosacral ligaments of the prolapsed uteri are characterized by a higher expression of collagen iii, decorin, fibromodulin and byglican and a lower quantity of collagen i. no differences in the immunoreactivity of lumican between the two patients groups. the abnormalities of support connective tissue composition are not observed in the uterine cervix of patients with prolapse. conclusions. our results suggest an altered remodeling of connective tissue in the ligaments of premenopausal patients with prolapse, with a significant decrease in collagen i content and an increase in collagen iii and pgs expression . in the prolapse patients this abnormal collagen metabolism and organization, mainly related to the observed change in pgs expression, might affect significantly the tensile strength of the connective tissue and consequently the support that is provided by the suspensory apparatus to the uterus. the alterd remodeling of support tissues, not detectable in nonsupport tissues, may suggest a predominant role of local biomechanical stresses (childbirth, chronic straining) in the pathogenesis of prolapse respect to systemic connective tissue deficiency. objective: to achieve vaginal delivery with minimal maternal injury, vaginal smooth muscle (sm) contractility likely decreases. the objective of this study was to characterize changes in rat vaginal sm contractility in pregnancy that affords circumferential vaginal distension. methods: a tubular segment of the vagina of 8 virgin, 8 late pregnant , 8 immediate and 8 late post vaginal delivery rats were mounted in an organ bath between a force transducer and an adjustable support block. dose response curves to norepinephrine (ne) and potassium (k+) were used to assess contractility. relaxation capacity was determined in ne preconstricted vagina, using cumulative doses of sodium nitroprusside (snp). differences in active vs. passive length-tension curves were used to measure sm contractility relative to the vaginal wall forces. sm -actin levels were measured using quantitative confocal immunofluorescence. results: cumulative doses of ne induced a maximum constriction force of 33.1 ± 6 mn/g in virgin vagina while no measurable force was generated in response to ne in late pregnant or postpartum vaginas. virgin vagina relaxed with cumulative doses of snp; no measurable relaxation response was observed from pregnant or postpartum animals. in the presence of the high dose k+ (124mm), virgin vagina generated the greatest contractile force (43.92 ± 3 mn/g) vs. late pregnant vagina (22.6 ± 2.4 mn/g, p<0.001) or immediate post partum vagina (26.81 ± 4 mn/g, p=0.033). four week postpartum k+ induced forces were similar to virgin levels (43.89 ± 3 mn/g). sm force generation (difference in active vs. passive length tension curves at 3mm of displacement) was decreased in late pregnancy (15.3 ± 4 mn/g) compared with virgin (112 ± 29 mn/g, p < 0.023) and 4 week postpartum vagina (172 ± 34 mn/g, p < 0.004). the expression of sm -actin was lowest in late pregnancy (15 ± 1.5, p < 0.001) and immediate postpartum vagina (23 ± 1.5, p < 0.001) relative to virgin (33 ± 3) with a return to virgin levels 4 week postpartum (56 ± 2). conclusions: vaginal sm contractility diminishes in pregnancy. the altered contractility is mirrored by a decrease in sm -actin protein expression. near complete recovery to pre-pregnancy levels occurs by 4 weeks post partum. these functional and biochemical changes likely represent maternal adaptations designed to minimize trauma during passage of the fetus(es). background: diagnosis or exclusion of endometriosis usually requires laparoscopy and peritoneal biopsy. we have described a novel and consistent observation of small nerve fibers in the functional layer of eutopic endometrium in women with endometriosis (tokushige et al, 2006) . these nerve fibers are not present in women without endometriosis. this finding allows the possibility endometriosis in the nude-mouse and inhibited mmp-3 expression in human endometrial stroma. the present findings may lead to the development of novel treatments of endometriosis involving statins. supported by nih u54hd052668. k-ras actived immunocompetent retrograde menstruation model of endometriosis. ching-wen cheng, 1,2 di licence, 1,2 cristin g print, 1,3 d stephen charnock-jones. 1,2 1 pathology, university of cambridge, cambridge, united kingdom; 2 obstetric gynaecology, university of cambridge, cambridge; 3 department of molecular medicine pathology, university of auckland, auckland, new zealand. objective: to establish a new murine model of endometriosis that mimics the retrograde menstruation theory using immuno-competent mice. method: ovariectomised donor female k-ras v12+/-/cre +/+ /rosa26r-lacz +/+ transgenic mice were treated sequentially with steroid hormones. to induce decidualization and activation and k-ras transgene, 16 mg/kg b-nf dissolved in maize oil was injected into the uterine lumen. tissue degeneration mimicking menstruation was induced by hormone withdrawal. menstruating endometrial tissue was collected from donor mice 60 hours after last hormone treatment, re-suspended in matrigel, and implanted subcutaneously in female c57bl/6 recipients. immunohistochemical and morphometric methods were used to characterize the endometriosis-like lesions. microarray analysis (illumina, n=6 in each group), was used to study the molecular changes in "menstruating" uteri following k-ras activation. result: simple transplantation of decidualised endometrial tissue into immunocompetent animals does not lead to endometriotic lesion development but using tissue with the genetic modification described here overcomes this. viable endometriosis-like lesions are visible 28 days after implantation. these lesions are histologically similar to those seen in man with intact glands, functional blood vessels, leukocyte infiltration and collagen deposition. statistical analysis revealed that 220 transcripts were differentially regulated in the ras activated and control tissue. gene ontology (go) analysis indicated that transcripts associated with epithelial cell function and differentiation were over represented within this gene set (chloride transport and epidermis development) as were those associated with the acute inflammatory response and neutrophil chemotaxis. we developed a new model of endometriosis using immunocompetent mice to mimic retrograde menstruation induced endometriosis. this permits for the first time, the ready use of transgenic and knock-out tools to investigate the cellular and molecular mechanisms underlying endometriosis. since the animals have an intact immune system it also allows the testing of therapeutic agents that modify the inflammatory response. stra6 is expressed and induced by progesterone in the endometrium and is absent in endometriosis. mary ellen pavone, serdar e bulun, scott s reierstad, joy innes, magdy p milad, you-hong cheng. obstetrics and gynecology, northwestern univeristy, chicago, il, usa. objective: retinoic acid (ra) plays important roles in development, growth and differentiation by regulating the expression of target genes. in the mouse endometrium, ra deficiency leads to hyperkeratinization, while high concentrations of retinoids promote secretory characteristics. this leads many to believe that ra mediates important actions of progesterone in the endometrium and may account for progesterone resistance in endometriosis. the mechanism for regulation of ra production by progesterone is unknown. moreover, the conversion from retinol to retinoic acid is not different between normal endometrium and endometriotic tissue. we hypothesize that retinol intake into cells rather than conversion of retinol to ra is the critical step that determines ra activity in the endometrium and endometriosis. stra6, a widely expressed multitransmembrane domain protein and member of a group of "stimulated by retinoic acid" genes, has been identified as the retinol binding protein receptor. it is strongly expressed in adult mice in several tissues including the female genital tract. we hypothesize that ra activity in the endometrium may be regulated by stra6. here we investigate the differential expression of stra6 in the endometrium and endometriosis. design: we studied primary stromal cells isolated from the eutopic endometrium of disease free women and walls of ovarian endometriomas from women with endometriosis with respect to stra6 expression and regulation by progesterone. materials and methods primary culture of eutopic endometrial and endometriotic stromal cells were treated with 10 -7 m estradiol (e 2 ), 10 -7 m r5020, a progesterone agonist, or vehicle for 24 hours. real-time pcr was employed to quantify stra6 mrna levels. we treated cells for 48 hours and quantified protein levels by immunoblotting. results basal mrna of stra6 levels in endometrial cells (n=8), were >1,000 fold higher than those in vehicle-treated endometriotic cells (n=8, p<.001). in endometrial stromal cells (n=8), r5020 stimulated stra6 mrna levels by 1.7-3 fold. r5020 induced stra6 protein levels in endometrial stromal cells (n=3). conclusions stra6 is highly expressed and regulated by progesterone in endometrial stromal cells, whereas it is practically undetectable in endometriotic cells. stra6 may mediate important actions of progesterone in endometrium. upregulation of aromatase expression in endometrial cells disseminated into the peritoneal cavity (pc) may influence their survival and persistence via local estrogen synthesis. the inducing factors for the upregulation of aromatase in endometriosis are not well defined, but increased expression of sf-1 has been suggested to play an important role. given that the aromatase substrate androstenedione (a4) is the predominant steroid in peritoneal fluid, we aimed to determine whether a4 has a role in the regulation of aromatase expression in human endometrium. we found that culture of primary endometrial stromal cells and explants with a4 (5-10 nm) for 24 h significantly upregulated expression of aromatase mrna transcripts containing exon iia at their 5'-ends. moreover, in human endometrial surface epithelial cells (hes), dose-response studies with a4 (5-100 nm) revealed that 10 nm a4 maximally upregulated expression of both aromatase and sf-1. when tissue samples were evaluated from women with endometriosis and control endometrium, expression of aromatase mrna mirrored sf-1. treatment of hes cells (24 h) with tritiated a4 demonstrated its metabolism to estradiol (e 2 ), testosterone (t), dihydrotestosterone (dht) and androstanediol (a-diol). although equimolar concentrations of a4, t and e 2 upregulated aromatase and sf-1 mrna expression in hes cells, the nonaromatizable androgens, dht and a-diol, had no effect. a positive feedback role of estrogen in aromatase upregulation was suggested by the finding that the estrogen receptor antagonist, ici 182,780, markedly diminished aromatase and sf-1 mrna expression induced by a4. finally, chromatin immunoprecipitation revealed that a4 significantly enhanced recruitment of sf-1 to its response element (-136 bp) upstream of cyp19 exon iia. collectively, our findings strongly suggest that exposure of endometrial cells within the pc to c 19 -steroids may cause an acute upregulation of cyp19 gene expression through their aromatization to estrogens. thus, estrogen may play a critical positive feedback role in the pathogenesis of endometriosis. supported by nih r01-dk031206. the hpa axis and depression/anxiety scores in chronic pelvic pain patients. b stegmann, 1 b frank, 2 j gemmill, 1 g chrousos, 1 m ballweg, 3 p stratton. 1 1 rbmb, nichd/nih, bethesda, md; 2 som, wake forest university, winston-salem, nc; 3 endometriosis association, milwaukee, wi. stress, pain, anxiety and depression adversely affect the hypothalamic-pituitaryadrenal (hpa) axis. we examined the influence of depression and anxiety on the hpa axis response to corticotropin-releasing hormone (crh) testing in women with and without chronic pelvic pain (cpp). methods: healthy women, aged 18-50, with without cpp, with regular menses and off hormonal contraception were studied. none had pelvic infections, untreated depression, manic depression, fibromyalgia, or chronic fatigue syndrome. after ovine crh injection (1 mcg/kg), serial blood samples (-5, 0, +15, +30, +45 minutes) were obtained for acth and cortisol measurements. depression and anxiety were scored using the duke quality of life questionnaire. hpa response was abnormal if peak acth levels were > 55 pg/ml without a rise in cortisol levels. 4 subject groups were: no pain and normal acth response (npnr), pain and normal acth response (pnr), no pain with an abnormal acth response (npar) and pain with an abnormal acth response (par). student t-test was used for comparisons. results: 31 women (21 cpp, 10 controls) had a mean age of 33.5 ± 9.1 years (range: 21-47). 18 women responded normally (10 pnr, 8 npnr) and 13 women responded abnormally (11 par, 2 npar). peak and absolute rise in acth were significantly higher in abnormal (a) vs normal (n) responders; (peak: 85.2 a vs 33.5 pg/ml n, p<0.001; absolute: 51.2 a vs 27.5 pg/ml n, p=0.024). there was a significant difference within groups: peak acth: 88.5 par vs 37.1 pg/ml pnr, p=0.001; 67.5 npar vs 20.3 pg/ml npnr, p<0.001; absolute acth: 72.2 par vs 28.2 pg/ml pnr, p=0.001, 56.1 npar vs 20.4 pg/ml npnr, p<0.002). however, total cortisol level was not different between or within the 2 groups (23.2 a vs 20.0 mcg/dl n, p=0.08). mean depression score did not differ among cpp patients (34.7 par vs 35.2 pnr, p=0.95), but differed among controls (25 npar vs 4.3 npnr, p=0.01). anxiety scores did not differ between or within groups (29.5 npar vs 8.4 npnr p = 0.12; 40 par vs 39.7 pnr, p=0.66). conclusions: chronic pelvic pain is associated with an abnormal hpa response, regardless of anxiety or depression symptoms. abnormal hpa responses in control women, however, appear to be influenced by depression. the mechanism and clinical significance of these findings should be explored. support: rbmb/nichd/nih and endometriosis association. objective: the eutopic endometrium in women with endometriosis demonstrates altered endometrial receptivity and altered gene expression. it is unknow if the endometrium is defective giving rise to a predisposition toward endometriosis or alternativly if the endometriosis causes the altered endometrial receptivity. here we created experimental endometriosis in a mouse model through allotransplantation of the uterus to the peritoneal cavity in immunocompetent mice and examined the expression of several markers of endometrial receptivity in the eutopic endometrium. materials and methods: the uterus of 8-week-old cd1 female mice was transected at cervicovaginal junction and each horn divided and transplantated into the abdomidnal cavity of eight cd1 mice. seven controls received sham surgery only. after 14 weeks the uterus was removed, snap-frozen in trizol. total rna was extracted and cdna generated. quantitative real time rt-pcr using sybrgreen was performed and normalized to -actin. fold changes in normalized hoxa10, hoxa11, bteb1, emx2, igfbp1, integrin -3, total progesterone receptor (pr-ab) and progesterone receptor-b (pr-b) were assessed. all experiments were conducted in duplicate, repeated at least three times and compared using mann-whitney rank sum test. results: hoxa10, hoxa11, bteb1, emx2, and igfbp1 mrna expression showed 0.57-fold (p=0.046), 0.25-fold (p<0.001), 0.50-fold (p=0.043), 0.23fold (p=0.038), and 0.09-fold (p<0.001) decrease in the uterus of mice with experimental endometriosis compared with controls. pr-ab and pr-b mrna showed 2.7-fold (p=0.016) and 8.39-fold (p=0.006) increase in endometriosis group compared to controls, respectively, however the ratio of pr-b to pr-ab (b/ab) showed no significant change in both groups (20.98 vs. 7.53 , endometriosis vs. control, p>0.05). there was no significant change in integrin -3 mrna expression (1.09-fold, p>0.05). conclusion: we demonstrate significant changes in multiple markers of endometrial receptivity in eutopic endometrium after induction of endometriosis. these findings suggest that origionally normal endometrium can develop defects with the creation of endometriosis; an abnormal endometrium is not a prerequisite for the development of endometriosis or associated abnormalities. this data also suggest the existance of a signal conduction pathway from endometriosis that alteres endometrial gene expression. endometrial expression patterns of relaxin and relaxin receptor mrna suggest involvement of relaxin in endometriosis. sara s morelli, 1 felice petraglia, 2 gerson weiss, 1 stefano luisi, 2 pasquale florio, 2 jeff gardner, 1 andrea wojtczuk, 1 laura t goldsmith. 1 1 obstetrics, gynecology and women's health, new jersey medical school, newark, nj, usa; 2 pediatrics, obstetrics and reproductive medicine, university of siena, siena, italy. objective: in normal endometrium, relaxin is a potent inhibitor of matrix metalloproteinases, which have been implicated in the invasive process of endometriosis. we tested the hypothesis that relaxin plays a role in endometriosis by comparing expression of relaxin and its lgr7 receptor in normal human endometrium to levels in samples from patients with endometriosis. materials and methods: total rnas, extracted from ectopic (n=8) and eutopic (n=11) endometrium of patients with endometriosis and from endometrium of normal controls (n=12), were reverse transcribed into cdnas. real-time pcr was performed using primers previously shown to identify human lgr7 relaxin receptor mrna, h2 relaxin mrna, and beta-actin mrna, with sybr-green detection of double stranded dna products. the comparative c t method (2 -ct ) determined relative lgr7 and relaxin mrna expression (normalized to beta-actin mrna expression). relaxin mrna was detectable in normal endometrium from 9 of 12 (75%) control patients. in contrast, relaxin mrna was detectable in a lower proportion of samples [9 of 19 (47.4%)] from patients with endometriosis, among whom relaxin mrna was detectable in a lower proportion of ectopic samples [2 of 8 (25%)] than in eutopic samples [7 of 11 (63.6%)]. lgr7 relaxin receptor mrna was detectable in all samples, with lower expression in endometriosis samples than in endometrium from normal controls. relaxin receptor lgr7 mrna levels vary with cycle phase, with greater expression in the secretory phase (sp) than in proliferative phase (pp): in normal controls 5.9-fold higher levels in the sp than pp; and in endometriosis patients 3.6-fold higher levels in the sp than pp. in both phases, lgr7 mrna levels were lower in ectopic samples than in either eutopic samples (6.2-fold lower in pp and 19.0-fold lower in sp) or endometrium from normal controls (6.5-fold lower in pp and 15.6-fold lower in sp). eutopic endometrium had similar lgr7 mrna expression to controls throughout the cycle. decreased local expression of relaxin and relaxin receptor mrna in ectopic endometrium from patients with endometriosis throughout the menstrual cycle suggests that relaxin may be protective against endometriosis. ovarian reserve after laparoscopic cystectomy of endometriotic ovarian cysts. shinichi hayasaka, takashi murakami. obsterics and gynecology, tohoku university, sendai, japan. objective laparoscopic ovarian cystectomy is generally recommended for endometriotic ovarian cysts because it has been associated with a higher pregnancy rate and a lower recurrent rate. however, residual ovarian function after laparoscopic cystectomy of endometriotic ovarian cysts has been questioned. in this study, we retrospectively evaluated ovarian response to hyperstimulation in women selected for ivf and icsi cycles who previously underwent laparoscopic cystectomy of a monolateral endometriotic ovarian cyst. methods a retrospective review of the patients between january 2003 and september 2007 was performed. the operated ovary and contralateral intact ovary were compared in terms of number of oocytes retrieved, number of follicles with a mean diameter > 15mm at the time of hcg administration. the patients who had recurrent endometriotic ovarian cysts were excluded. in total, ten patients were identified. the mean(±sd)number of oocytes retrieved was 4.1±2.4 in the control ovary and 1.1±1.4 in the previously operated ovary(p=0.031). the mean(±sd)number of follicles with a mean diameter > 15mm was 5.6±2.5 in the control ovary and 1.8±1.1 in the previously operated ovary(p=0.0004). the age of patients and diameter of the operated ovary had little influence on the difference between the response of the control ovary and one of the previously operated ovary. laparoscopic cystectomy of endometriotic ovarian cysts is associated with a significant reduction in ovarian reserve. background pre-eclampsia (pe) is associated with systemic maternal endothelial dysfunction, which is thought to result from the presence of circulating factors released following placental damage. it is hypothesised that this occurs as a result of reduced oxygen (o 2 ) delivery. some features of placental pathology seen in pe, such as increased apoptosis, can be reproduced by culture of placental explants in 1% o 2 . metabolomics operates to study 'all' metabolites within a biological system. this strategy has previously identified differences in maternal plasma between normal pregnancies and those complicated by pe. in these experiments we used metabolomics to investigate differences in the metabolic profile of explants cultured in different o 2 tensions. hypothesis the metabolic profile of placental villous explants is altered by culture in different o 2 tensions. methods placental villous explants were cultured with either 10% serum (n=10) or in serum-free conditions (n=6) for 96h in 20%, 6% and 1% o 2 . after 96h, conditioned culture medium and tissue were collected and snap frozen. tissue was homogenised in 50% ice cold methanol prior to analysis. samples were analysed using gas chromatography-mass spectroscopy (gc-ms). samples cultured at 20%, 6% and 1% o 2 were compared to identify concentration differences in metabolites in conditioned cultured medium and tissue lysate. kruskal-wallis test was used for statistical analysis. due to the large number of metabolites identified, a p value of 0.001 was considered significant. results the mean intra-assay variability was 9.1%. there were no differences in the metabolic profile of conditioned culture medium from 6% and 20% o 2 . several metabolites differed in culture medium from 1% compared to 6% and 20% o 2 including: deoxyribose, glycerol and threionic acid. these changes were present in both serum and serum-free conditions. using these metabolites alone, culture in 1% o 2 could be completely discriminated from 6% and 20% o 2 . deoxyribose was also elevated in tissue lysate from 1% o 2 compared to 6% o 2 . conclusion this metabolomic strategy can identify differences in metabolic profile in placental tissue cultured in 1% o 2 . these novel compounds may provide further insight into pathophysiology of pe. objective a strategy of metabolic footprinting (the study of extracellular metabolites, which are related to intracellular metabolism) was used to detect a wide array of low molecular weight metabolites. metabolic profiles were employed to differentiate between placental explants cultured at different oxygen tensions. two separate studies were combined to validate initial observations. methods metabolic footprints of placental villous explants, obtained from uncomplicated pregnancies, (n=5) were cultured for 96h in 1%, 6% and 20% oxygen. conditioned culture medium was then collected and frozen, prior to analysis by uplc/ltq-orbitrap mass spectrometry in both negative and positive ion mode. samples cultured at 1%, 6% and 20% were compared to identify differences in the metabolic profiles. this procedure was repeated for 6 different explants and the results compared across the 2 studies in order to validate initial findings. approximately 350 unique peaks were detected in both sample sets in negative ion mode and 300 peaks in positive ion mode. a number of metabolites were identified as being significantly different using kruskal-wallis (pval<0.01) under different oxygen tensions. some differences were seen between the results obtained from both runs due to separate batch preparation of the medium but good reproducibility was obtained for many metabolites between batches. metabolites of biological interest included amongst others 2-deoxyribose, valine, tyrosine and aspartic acid. the largest differences were those seen between o 2 1% and o 2 6%. comprehensive metabolic profiles were detected and employed to identify differences in the metabolic footprints of explants cultured under differing oxygen conditions. a number of biologically interesting metabolites were characterized. objective: brain weight and dna content were reduced in leptin deficient (lep ob /lep ob ) mice postnatally. leptin is detected in the sera and its receptors are expressed in the brain of the mouse fetus. we examined the role of leptin in the proliferation and differentiation of neural lineage cells in the mouse fetus. methods: the number of total cells in the cerebral cortex was compared between (lep ob /lep ob ) and wild type fetuses from embryonic day (e) 14 to 18. the number of brdu positive cells was also compared on e14 and e16. brdu uptake, the ratio of viable colony number to plated cell number, the proportion of multipotent, neuronal or glial progenitor colonies, and the expression levels of hes mrna were compared between leptin-and non-treated neurosphere cells. moreover, we examined stat3 phosphorylation by leptin stimulation with elisa to investigate whether or not jak/stat3 pathway transduces the leptin signal in the prenatal period as in the adult. results: lep ob /lep ob fetuses had reduced total cell numbers in the ventricular zone (vz) on e16 and e18, and in the cortical plate on e18. the number of brdu-positive cells was reduced in vz of e14 and e16 lep ob /lep ob fetuses. brdu uptake and the number of viable colonies were increased by 2 days-leptin treatment in the neurosphere culture. the proportions of glial-restricted and multipotent precursor colonies were increased by leptin, whereas that of oligodendrocyte precursor colonies were decreased. hes1 mrna expression was enhanced in neurosphere cells by leptin. neither the amount of phosphorylated stat3 nor that of stat3 was increased in neurosphere cells by leptin stimulation. conclusion: our study suggests that leptin maintains the neural stem and glial-restricted precursor cells through upregulation of hes1 expression and increases the number of cerebrocortical cells in the mouse fetus, however, jak/stat3 pathway does not mediate the leptin signal in undifferentiated neural lineage cells. 311a vaginal wall, and >90% of fib-5 knockout (ko) mice develop pelvic organ prolapse by 20 weeks of age. in contrast, fib-1 and -2 deficiencies do not result in prolapse, and fib-4 kos die shortly after birth. herein, we evaluated the role of fib-3 in pelvic organ support. methods: two observers serially measured the degree of vaginal, perineal, and anal prolapse in 227 fib-3 ko (fib3 -/-) and in fib3 -/-mice severity of prolapse was significantly related to age, but not parity, and the average age at diagnosis was 37 wks. fib3 -/-animals with advanced prolapse had attenuated uterosacral ligaments and descent of the bladder and uterus caudal to the symphysis. pro-mmp2 (2-fold, p=0.03), active mmp2 (2-fold, p=0.06), and prommp9 (3-fold, p=0.05) were increased in vaginal tissues from mice with gross prolapse compared with age-, strain-, and cycle-matched controls. this increase in protease activity, however, was also accompanied by increased expression of fib-5 and tropoelastin (1.7-fold, p<0.001). regardless of prolapse maternal morbidtties tf23 ards-n(%) 12 (52) acute ren~m f11lure-n (%) 10(43) car~tovascular ~ysfunrtion-n ("/o) 15 ( 65) hepatc fa..lure-n (%) dt sst'dlm atl:d inlfavascul..-coagul tipalhy-n(%) 6 (26) durallon of !cu suy (days. mean+sd) 9 21:t 12 .8 hospital my (days.. me411+sd) 2. condon, j. c., hardy, d. b., kovaric, k., and mendelson, c. r. (2006) mol endocrinol 20(4), 764-775. objective: innervation of the uterine cervix is important for the process of parturition (physiol beh 63:929,1998; j histochem cytochem 52:1249 ,2004 jsgi 12:578,2005) . the hypogastric nerve is a major pathway that innervates the uterine cervix, yet its contribution to processes associated with cervical ripening and parturition is not known. the objective of this study was to determine the effect of hypogastric nerve transection on processes associated with cervical remodeling and parturition. methods: time-dated pregnant rats were sham-operated or the hypogastric nerve bilaterally transected just anterior to the inferior mesenteric ganglion on day 15 post-breeding. live pups were born spontaneously by all rats at term on days 22-23 post-breeding. on the day of birth, the cervix was excised, postfixed overnight, sectioned, and processed to evaluate collagen content and structure (nih image j). sections were also processed by immunohistochemistry to assess cell nuclei density, the census of resident macrophages, and area of tissue that contained nerve fibers. results: hypogastric neurectomy did not affect cell nuclei density, the number of macrophages, or density of nerve fibers in the cervix. the failure of hypogastric nerve transection to affect indices of cervical remodeling is consistent with the finding that duration of pregnancy and timing of birth were not different in sham-operated and hypogastrectomized rats. conclusions: nerve fibers in the hypogastric nerve that innervate the lower uterus and cervix, including sympathetic and neuropeptidergic projections, are thus not essential for birth. these novel findings provide support for the contention that innervation of the uterine cervix other than through the hypogastric nerve contributes to processes associated with cervical ripening and parturition. receptor system in prostaglandin synthesis in pregnancy. eugenie r lumbers, 1,2 della m yates, 2 carolyn m mitchell, 2 jonathan j hirst, 1,2 tamas zakar. 2,3 1 school of health sciences, university of newcastle, newcastle, nsw, australia; 2 mothers and babies research centre, hunter medical research institute, newcastle, nsw, australia; 3 obstetrics and gynaecology, john hunter hospital, school of medical practice and public health, university of newcastle, newcastle, nsw, australia. the fetal membranes and decidua contain prorenin, which requires proteolytic activation. ngyuen (j clin invest. 109:1417) described a renin/prorenin receptor that conformationally activates prorenin, so that it can form ang i from aogen. in addition, receptor-bound prorenin can stimulate intracellular pathways directly. prostaglandins (pgs) participate in the control of term and preterm labour, and renin can stimulate pg production by amnion and decidua when angiotensin's action is blocked mitchell placenta 17:299) . the prorenin receptor may mediate these effects. our aim was to find out if the prorenin receptor gene is expressed and colocalised with prorenin and prostaglandin h2 synthase-2 (pghs-2) in human placenta, amnion, chorion and decidua. we have also determined if there is any change in the expression of the prorenin and prorenin receptor genes with labor. placentae with attached membranes were collected after term birth either by elective caesarian section or by spontaneous labor, and prorenin, prorenin receptor and pghs-2 mrna levels were quantitated relative to beta-actin mrna by real-time rt-pcr in amnion, chorion, decidua and placenta. before labor, mean prorenin mrna levels were 13.8 times higher in decidua and 4.0 times higher in chorion and placenta than in amnion (p<0.001). there were no significant changes with labor. proenin receptor mrna was expressed in all tissues. proenin receptor mrna levels in prelabor amnion, chorion and placenta were similar, while levels in decidua were 50% of those in amnion (p=0.02). this low level of prorenin receptor mrna in decidua persisted after labor (p<0.001). pghs-2 mrna expression was highest in amnion; in decidua and placenta it was only 6 and 4% of amnion. similar differences were present after labor. we conclude that the decidua is the principal source of prorenin within the pregnant uterus, and all gestational tissues are targets for prorenin. decidual prorenin may affect pghs-2 expression in amnion through the prorenin receptor forming a maternal-fetal paracrine system that stimulates pg production at labor. the immuno-suppressive effects of progesterone on umbilical cord blood mononuclear cells and the effect of culture media estradiol content. nadav schwartz, 1 xiangying xue, 2 christine n metz. 2 1 obstetrics and gynecology, nyu school of medicine, new york, ny, usa; 2 feinstein institute for medical research, manhasset, ny, usa. objective: progesterone (p4) is known to supress the maternal immune response and similar effects have been seen with fetal cells. we sought to ascertain whether p4 action was modulated by the phenol red and/or estrogen content of the culture media. methods: umbilical cord blood mononuclear cells were isolated using a density gradient centrifugation. the cells were incubated with p4 (10 -4 m) 1 hour prior to overnight stimulation with lps. supernatants were assayed for tnf-using elisa. the following culture media were used: a)'red' media: rpmi+10% fbs, b)'yellow' media: phenol red-free rpmi+10% fbs, and c)'clear' media: phenol-free rpmi+10% dextran/charcoal treated fbs. finally, the 'clear' media experiments were repeated with estradiol add-back. results: p4 suppressed tnf production in all medias. tnf levels were suppressed to 39.4% (p<0.06) of controls using 'red' media and to 42.0% (p<0.03) in 'yellow' media. the degree of suppression was not as substantial using 'clear' media where tnf levels were 64.5% (p<0.02) of control. (fig 1) adding estradiol to the 'clear' media had little or no effect on the degree of tnf suppression. (fig 2) conclusions: progesterone exerts an immuno-suppressive effect on lpsstimulated fetal mononuclear cells which is more pronounced when using media containing untreated fetal bovine serum. the phenol-red/estradiol content of the culture media did not modulated the p4 effect. dextran/charcoal treatment of fbs appears to deplete the media of factors other than estradiol that are necessary for p4 to exert its full effects. culture conditions should be optimized when studying progesterone's effects on immune response. 743 ovine fetal regional blood flow following prenatal dexamethasone (dex) at 0.75 gestation (g). antonine d van heesewijk, 1 jan g nijhuis, 1 susan l jenkins, 2 peter w nathanielsz, 2 mark j nijland. 2 1 ob/gyn, academisch ziekenhuis maastricht, maastricht, netherlands; 2 ob/gyn, cpnr, uthscsa, san antonio, tx, usa. background: pregnant women at risk for premature labor receive antenatal glucocorticoids (gc) to reduce neonatal morbidity and mortality. while fetal blood pressure (bp), heart rate and vascular endothelial and control fetuses (n=6). dispersed adrenal cortical cells (2.5 x10 5 cells/tube; in duplicate) were challenged with 10 -8 m acth. samples were collected at time 0 (baseline), 5, 15, 30, and 60 min after acth treatment, and tissue and media samples were frozen for determination of cortisol, camp, and star. results: cortisol output (ng/2.5x10 5 cells) was higher in the lth group compared to the control, (p< 0.01) at 15min (3.3 ± 0.5 vs. 1.0 ± 0.1), 30min (7.7 ± 0.6 vs. 1.9 ± 0.1), and 60min (10.9 ± 0.7 vs. 2.5 ± 0.1). peak camp levels (fmol/2.5x10 5 cells) were observed at 30 min but did not differ between control (81.8 ± 10.4) and lth (77.9 ± 17.8) adrenal cells. western analysis demonstrated that star protein expression was higher in the lth adrenal cortex compared to control (p<0.05) at 0 min (1.02 ± 0.07 vs. 0.59 ± 0.05), and at 60 min (1.26 ± 0.04 vs. 0.65 ± 0.05), relative optical density units. conclusions: results from the present study taken together with those of previous in vivo studies suggest that the enhanced cortisol output in lth fetuses is the result of increased adrenal acth sensitivity. this enhanced sensitivity is not due to differences in camp generation. star, which is a key element involved in cholesterol transfer at the level of the mitochondrial membrane appears to play a key role in the enhanced cortisol response to acth in the lth group. (nih grants hd31226, hd33147 and llu-nih imsd 2r 25gm060501-05). expression. hiroaki itoh, 1 makoto kawamura, 2 shigeo yura, 2 haruta mogami, 2 tsuyoshhi fujii, 2 norimasa sagawa. 3 1 obstetrics and gynecology, national hospital organization osaka national hospital, osaka, japan; 2 gynecology and obstetrics, kyoto university graduate school of medicine, kyoto, japan; 3 obstetrics and gynecology, mie university school of medicine, tsu, japan. objective: epidemiological evidences suggested that undernutrition in utero develops risk factors for adult cardiovascular disorders, such as blood pressure increase. the present study was designed to prove the hypothesis that maternal iso-caloric high protein diet alleviates blood pressure increase in the adult offspring by affecting placental 11beta-hydroxysteroid dehydrogenase type 2 (11beta-hsd2) expression. methods: the 30 % calorie restriction was applied to pregnant mice by using either standard protein diet (spd; 20% casein protein; spd-un) or high protein diet (hpd;40% casein protein; spd-un). in some groups, these pregnant mice were sacrificed at 18.5 d.p.c.; then maternal and fetal plasma as well as placental tissues were corrected. while in other groups, systolic blood pressure was measured in the offspring at 16 wks. fetal and maternal corticosterone levels were measured by elisa. the placental 11beta-hsd2 gene expression was measured by quantitative taqman pcr. results: systolic blood pressure in the spd-un offspring at 16 weeks (106 mmhg) was higher than that in spd-nn offspring (99.3 mmhg). by contrast, systolic blood pressure in the hpd-un offspring (97.5 mmhg) was similar to that in spd-nn offspring. maternal calorie restriction with spd and hpd caused similar elevation of maternal plasma corticosterone concentrations. by contrast, fetal plasma corticosterone levels in hpd-un offspring (151 ng/ml) was lower than those in spd-un offspring (208 ng/ml), indicating the amelioration of fetal exposure to high corticosterone by maternal hpd. the placental 11beta-hsd2 gene expression in the hpd-un was 72% higher than that in spd-un, suggesting that maternal hpd augmented placental 11beta-hsd2 gene expression in the placenta and probably facilitated the inactivation of maternal cortocsterone in the process of placental transfer to fetal circulation. conclusion: the preset study suggests that maternal high protein ingestion may elevate placental 11beta-hsd2 gene expression and ameliorate blood pressure increase in the adult offspring via modification of fetal exposure to maternal corticosterone. elevated cortisol levels at birth exert critical maturational effects through action at glucocorticoid receptors, gr. in contrast, the low cortisol concentrations in the preterm fetus, before the time of increased fetal cortisol secretion, are near to the kd for cortisol at the higher affinity mineralocorticoid receptor, mr. we hypothesized that endogenous cortisol in the fetus exerts physiologic actions at mr in tissues without appreciable cortisol-inactivating enzyme, 11 hsd2, including the fetal lung and brain. we therefore tested the effect of infusion of a mr-antagonist, ru26752 (mra, 1.68 mg/h over 12h) into 120-130 day fetal sheep. we tested for effects on lung liquid production rate, lung liquid and plasma electrolytes, plasma acth and cortisol, and hematocrit at 10-12h after start of the infusion. although there was no significant difference in the liquid production rate in fetuses infused with ru26752, the ratio of na + to k + in lung liquid was significantly greater in mra-treated fetuses than in the control fetuses (34.7±1.6 vs 30.2±1.7). plasma acth was significantly greater in the mra-treated fetuses than in control fetuses at 12h (657±262 vs 171±20 pg/ml); in the mra-treated fetuses, acth concentrations at 12h were greater than in the same fetuses at 0h (124±16 pg/ml), wheras there was no increase in plasma acth in the control fetuses (0h: 100±19 pg/ml). similarly, plasma cortisol concentrations at 11 and 12h were greater in the mra infused fetuses than in control fetuses (12h: 18.2±5.5 vs 7.0±1.4 ng/ml), and cortisol increased significantly over time in the fetuses infused with mra, but not in control fetuses (0h, mra: 4.7±1.5, control: 2.9±0.5 ng/ml). infusion of mra did not alter fetal plasma electrolyte, fetal blood pressure or fetal heart rate. however, fetal packed cell volume was significantly increased at 9-12h of infusion of mra (12h: 37±1 vs 32±1 %) and fetal pco2 was significantly greater at 12h of infusion of mra as compared to control fetuses (61.0±2.0 vs 53.5±0.8). these results suggest that endogenous cortisol concentrations in the preterm fetus exert negative feedback control of acth via action at mr in the fetal brain, and play a role in regulation of fetal lung liquid composition and in fetal fluid balance. preparturient increases in fetal acth secretion are cyclooxygenase-2 (cox-2) dependent. charles e wood. dept. physiology and functional genomics, university of florida college of medicine, gainesville, fl, usa. maturation of the fetal hypothalamus-pituitary-adrenal axis is critical for the timely somatic development of the fetus and readiness for birth. in sheep, increased preparturient activity of the fetal hpa axis appears to be responsible for triggering parturition itself. this study was designed to test the hypothesis that prostaglandin generation, mediated by cox-2 in the fetal brain, is critically important for stimulation of the preparturient increase in fetal acth secretion. singleton fetal sheep were chronically catheterized with vascular, amniotic, and lateral cerebral ventricular catheters. nimesulide, a selective cox-2 inhibitor, was infused (1 mg/day, n=9), and vehicle (50% dimethylsulfoxide, n=6) was infused. arterial blood samples were drawn from fetuses at 48 hour intervals for measurement of plasma hormone concentrations and arterial blood gases. in the vehicle-treated fetuses, fetal plasma acth, pomc, and cortisol concentrations increased exponentially (p<0.001 by anova) before spontaneous parturition at 146±0.3 days. in the nimesulide-treated fetuses, fetal plasma acth and pomc were constant and did not increase prior to parturition (p=ns by anova). in contrast, fetal plasma cortisol increased independent of acth (p<0.001 by anova) and the fetuses were born spontaneously on 148±0.3 days gestation. the slight delay in spontaneous parturition in the nimesulide-treated fetuses was statistically significant (p<0.01 by mantel-cox test). intracerebroventricular nimesulide infusion did not decrease fetal plasma concentrations of prostaglandin e2, demonstrating that the action of the nimesulide was restricted to the fetal brain. fetal blood gases were normal in all of the fetuses, and there were no differences in blood gases between groups. we conclude that the spontaneous increase in fetal acth and pomc prior to parturition is cox-2 dependent. however, we also conclude that the increase in fetal plasma cortisol concentration can occur independent of increases in fetal acth, suggesting that the increase in cortisol can result from increases in adrenal sensitivity to acth. the results of this study demonstrate that the timing of parturition in the sheep is not dependent upon increased acth secretion, and the results suggest that parturition is regulated primarily by changes in adrenal sensitivity. expression of extracellular signal-regulated kinase1/2 and p38 mitogen-antiphosphotyrosine. immunolocalization of inos was performed in the same tissues. hpmec were used to determine effect of sin-1 (1mm) on inos protein expression (30 min, 4 and 8 h). results: inos protein was detected in microvascular endothelium, smooth muscle and syncytiotrophoblast of the placenta. inducible nos levels in placentas from severe preeclampsia were significantly decreased compared to normal (p<0.05) but were not different from mild preeclampsia. aligning western blots of anti-phosphotyrosine and inos revealed a phosphorylated band corresponding to the molecular weight of inos. the proportion of tyrosine phosphorylated inos was reduced by 35% in severe preeclampsia compared with normotensive. preliminary data with ip for phosphotyrosine and crossblotting with inos confirmed this finding. sin-1 treatment decreased inos protein abundance at 4 and 8 h in hpmec. conclusions: our results demonstrate decreased tyrosine phosphorylation of inos in preeclamptic placenta. phosphorylation of tyrosine in inos has been reported to negatively regulate its activity. we therefore postulate that decreased phosphorylation of inos may be responsible for the increased catalytic activity of the enzyme that we have previously observed. the decreased levels of inos observed on sin-1 treatment may be due to nitration, an effect analogous to preeclampsia, where the presence of peroxynitrite has been well established. it remains to be seen if protein nitration has an effect on enzyme stability. objective: 8-isoprostane (8-iso), a prostaglandin-like compound formed in vivo, is a reliable measure of oxidative stress which occurs in response to many different stimuli, including infection. periodontal disease is a chronic oral infection associated with fetal growth restriction and preeclampsia. our objective was to determine if maternal periodontal disease is associated with oxidative stress as measured by 8-iso. methods: a secondary analysis was conducted using prospective data from the oral conditions and pregnancy study. a cohort of healthy women enrolled <26 weeks underwent oral health examination and serum sampling. maternal periodontal disease was categorized as healthy, mild, or moderate-severe by clinical criteria. maternal serum was analyzed for 8-iso by ultra-sensitive elisa. elevated 8-iso was defined by a value 75 th %tile. maternal factors associated with elevated 8-iso were determined using chi-square or t-tests as appropriate. a logistic regression model was created to determine adjusted odds ratios (95 th %ci) for elevated 8-iso. results: 791 women had complete data for analysis. the median 8-iso level (iqr) was 1,806 (16 -81,870 pg/dl). using bivariate analysis, moderate-severe periodontal disease, non-white race, use of wic/food stamps, unmarried status, obesity, and lack of insurance were associated with elevated 8-iso. the logistic regression model for elevated 8-iso is shown below. adjusted or (95 th ci)* mild periodontal disease 1. maternal periodontal disease and utilization of public assistance are associated with oxidative stress in pregnancy. the relationship between periodontal disease, social hardship, oxidative stress, and adverse pregnancy outcome remains to be determined. antioxidant therapy could represent novel therapy for the prevention of adverse pregnancy outcome. severe transient immunological thrombocytopenia in a preeclampsia patient whose bone marrow production of platelets had been restricted. toshihiro yoshimura. obstetrics and gynecology, ntt-west kyushu general hospital, kumamoto-city, kumamoto, japan. introduction: idiopathic myelofibrosis is a chronic myeloproliferative disorder in which clonal haemopoetic stem cell proliferation is accompanied by reactive fibrosis. case report: a 23-year-old primiparous woman was referred to our hospital for prenatal care after 12 weeks of gestation. her medical history was significant for idiopathic myelofibrosis, and congenital agenesis of the spleen. the laboratory workup showed the patient's white blood cell count to be 5,500/ l; hemoglobin, 8.0 gm/dl; and platelet count, 38,000/ l. her bleeding time was normal (3 min). until the 34th week, the patient's pregnancy was uneventful. during the 35th week, however, the patient's platelet count declined to 16,000/ l, when proteinuria became evident. her bleeding time was prolonged to 13 min. the coagulation system was normal. by the 37th week, the patient's blood pressure was elevated to 150/100 mmhg, and her urinary protein excretion exceeded 2 g/day. our initial diagnosis was thrombocytopenia due to bone marrow suppression. the patient had no history of platelet transfusion, but we expected it would increase the platelet count, particularly since in the absence of destruction by the spleen, the lifespan of the platelets would be prolonged. this was not the case, however. elective induction of labor was carried out after 38 weeks. at the same time, the patient was transfused with 10 units of platelets, but her platelet count remained unchanged (20,000/ l). it was then that we realized that immunological thrombocytopenia may be involved, and massive doses (400 mg/kg) of gamma globulin were given intravenously for one day. thereafter, platelets (15 units) were again transfused, this time raising the platelet count to 110,000/ l. cesarean section was promptly carried out under general anesthesia. we later found that the patient's serum platelet associated immunoglobulin (paigg) level was positive, though antiplatelet and antinuclear antibodies were negative. the postoperative course was uneventful. the maternal platelet count declined to the pretransfusion level within 2 days after delivery, but gradually increased to the prepregnancy level by 2 weeks. comment: this idiopathic myelofibrosis patient in whom bone marrow production of platelets had been severely restricted demonstrates that immunological destruction may play a major role in the development of thrombocytopenia in preeclampsia. early l-arginine therapy improves notably the fetal growth in preeclamptic women. a randomized controlled trial. keisy lopez-molina, 1 juan c gonzalez-altamirano, 2 julio e valdivia-silva. 1,3 1 oncoinmunología y biología vascular, facultad de medicina, universidad nacional san agustín, arequipa, peru; 2 cardiología y cirugía de tórax, hospital nacional case essalud, arequipa, peru; 3 inmunología, instituto de investigaciones biomédicas, méxico, distrito federal, mexico. objective: to assess the benefit of early administration to l-arginine, the precursor to nitric oxide, on fetal growth. methods: one hundred women with preeclampsia were randomized to receive either l-arginine or placebo until day 1 postpartum. 96 live singleton infants were born after preeclamptic pregnancies and compared those with other 50 control infants. birth size was expressed as the ratio between observed and expected birth weights, and infants smaller than two standard deviations from expected birth weights were classified as small for gestational age (sga). all the women had neither previous precedents of the preeclampsia nor other factors that they cause sga. results: no significant differences existed between the groups with preeclampsia before randomization. preeclampsia was associated with a 8% (95% confidence interval [ci] 6%, 9%) reduction in birth weight. 4 women with hellp syndrome had to leave the study. the risk of sga was three times higher (relative risk [rr] = 3.4; 95% ci 2.8, 7.0) in infants born after preeclampsia without l-arginine therapy than in control pregnancies, and two times higher (relative risk [rr] = 1.8; 95% ci 0.7, 2.5). in infants born after preeclampsia with l-arginine therapy. conclusion: fetal growth improve markedly with l-arginine therapy in women with preeclampsia. introduction: although the pathogenesis of severe preeclampsia (pe) and intrauterine growth restriction (iugr) is poorly defined, inadequate remodeling of uterine spiral arties may promote reperfusion injury leading to focal infarction and reduced nutrient flow between mother and fetus. our goal was to determine whether an intravillous inflammatory cytokine cascade was associated with pe and iugr. methods: immunohistochemistry (ihc) examined il-1 and il-1 expression in 3 preterm placentas with severe pe+iugr, and 3 idiopathic preterm controls (ptc) with no evidence of clinical or histological chorioamnionitis. cultures of fibroblasts (fibs) and syncytiotrophoblasts (scts) from term placentas (n=5) were treated with il-1 and cytokine levels in conditioned media were determined using elisa or multiplex array. results were analyzed by student's t test or anova. results: the gestational age at delivery was not significantly different in pe+iugr and ptc groups (28.3±2.2 vs 27.6±2.8 wks). ihc of pe+iugr samples revealed intense villus core staining of il-1 adjacent to infarcts. villi distal to infarcts stained with a lower intensity. in contrast, staining was virtually undetectable in ptc. the pattern of il-1 staining was nearly identical in pe+iugr and ptc groups, was localized to the syncytium, and did not differ with respect to distance from infarct. to identify cellular targets of il-1 action, primary cultures of fibs and scts were incubated for 48 h in serum-free medium ± 1 ng/ml il-1 . by elisa we observed that il-1 treatment of fibs increased il-8 levels from 4±1 to 2161±346 pg/ g protein (p<0.001). similarly, multiplex array revealed that levels of il-4, il-6, and il-11 were induced 24-, 269-, and 12-fold, respectively in fibs. the presence of 200 ng/ml il-1 receptor antagonist reduced the il-1 -mediated increase in il-8 levels 95±3% (p<0.001), indicating that this response was mediated through il-1 receptor. in marked contrast, il-1 treatment did not significantly affect levels of il-4, il-6, il-8 or il-11 in scts, indicating that this response was cell type-specific. conclusions: these results indicate that increased expression of il-1 in the placental villus core in pe and iugr may promote an inflammatory cascade in fibs at this site leading to focal infarction and reduced flow of nutrients to the fetus. introduction: in human pregnancy, decreased responsiveness to angiotensin ii (angii) starts in week 10, promoting an expanded vascular bed. at the same time levels of the vasodilatory hemopexin increases 1 . during pre-eclampsia the vascular bed is contracted and the responsiveness upon angii persists. this may be due to the increased levels of extra cellular atp, a natural inhibitor of hemopexin 1 . in the present study we tested whether extra cellular atp is toxic in the pregnant condition. methods: pregnant rats were infused with either atp (750 g/kg bw; n=9) or saline (n=5) on day 14 of pregnancy (permanent jugular vein cannula/1 hr infusion). non-pregnant rats (n=8) were infused with atp identically. one day before and 1, 3, 6 days after infusion, 24 hr urine and blood samples (wbc count and hemopexin activity) were collected. seven days after the infusion, rats were sacrificed and kidney tissue processed for immunohistology. results: urinary albumin excretion was increased in pregnant atp infused rats only (fig 1) . wbc were also increased only in pregnant rats infused with atp (days 15 and 17 vs pre-infusion value). in pregnant atp infused rats intraglomerular influx of monocytes was increased, which correlated with urinary albumin excretion on the same day (r 2 = 0.66). staining of glomeruli for the angii receptor (at-1r) showed decreased at-1r expression in control pregnant rats as compared to non-pregnant rats, while at-1r expression in atp infused pregnant rats was increased as compared to control pregnant rats. hemopexin activity was increased on days 17 and 21 in control pregnant rats as compared to all other groups. discussion: these data support the notion that atp is toxic exclusively in the pregnant condition. it may be suggested that atp induced an inflammatory response, although the exact mechanism by which atp induced its effects needs further investigation. background: hepatocyte growth factor (hgf) is a multifunctional cytokine that is known to promote division, motility, invasion and morphogenesis of a wide range of cell types and to inhibit apoptosis. the effects of hgf are mediated through its interaction with the tyrosine kinase receptor c-met. in pregnancy hgf/met system is involved in the physiologic growth and development of the feto-placental unit. hgf/met effects are counteracted by transforming growth factor-1 (tgf-1), that is known to promote progressive fibrosis in human tissues. moreover tgf-1 plays a major role in trophoblast growth and differentiation. tgf-1 inhibits hgf expression as well as hgf inhibits tgf-1 expression. an understanding of the mechanisms regulating placental balance of these growth factors may provide insights into the processes that occur in complications of pregnancy, such as preeclampsia (pe) and fetal growth restriction (fgr). objective: to verify the hypothesis that hgf, c-met and tgf-1 are differently expressed in tissues of normal and pe placentas. methods: we studied 13 placentas from pregnancies complicated by pe (7 with normal fetal growth and 6 with fgr) and 5 placentas from normal pregnancies. from each placenta random samples were excised and rna was extracted; quantitative real-time rt-pcr analysis was used to investigate mrna expression of hgf, c-met and tgf-1 in pe (with or without fgr) and in normal placentas. results: gestational age, neonatal weight and placental weight were, as expected, lower in pe groups. the mrna expressions of the three molecules are higher in pe than in normal placentas, but the difference is statistically significant only for c-met. the higher values are mainly due to the increase in the pe group without fgr for c-met and tgf-1 and the increase in the pe-fgr group for hgf. conclusion: increased levels of expression of hgf/met system in pathological placentas could be explained as an attempt to repair placental damages; nevertheless placental regeneration could be inhibited by the increase of tgf-1, that promote fibrosis. placental expression of hgf, c-met and tgf-1 is increased in all pe placentas, but particularly in placentas of pe with normal fetal growth, where placental tissue is probably less compromised. obesity is a risk factor for preeclampsia (pe), but the reason for this risk is unknown. previously, we found that neutrophils infiltrated into the vasculature of pe women released myeloperoxidase (mpo) and matrix metalloproteinase 8 (mmp8), products that can cause oxidative stress and vascular dysfunction. if neutrophils infiltrate the vasculature of obese women and release mpo and/or mmp8, this may help explain why they are at risk of developing pe. hypothesis: systemic vascular tissue of obese women will have a significant presence of mpo and mmp8 as a result of neutrophil infiltration. methods: subcutaneous fat, which is highly vascularized, was obtained at abdominal surgery from 5 normal weight, 5 overweight and 5 obese women. formalin fixed, paraffin embedded 8 μm sections of fat biopsies were stained using immunohistochemistry with specific antibodies for mpo and mmp8. data were evaluated for intensity of vessel staining by visual score (0-4), density of staining using image analysis software, and % vessels with neutrophil staining, liberated from serum-free placental villous explant cultures from normal and pe pregnancies on human endothelial cells, and identified candidate mediators of their differential effects on metabolism and behaviour. methods: term placental villous tissue from normal (n=9) or pe (n=10) pregnancies were explanted for 4 days at 6% oxygen. conditioned 24h medium (day 3-4) was applied to human umbilical vein endothelial cells (huvecs). an angiogenesis assay was conducted in which tubule length and number were measured by morphometric imaging following seeding on 80% matrigel. the effect of explant conditioned medium on endothelial cell metabolism was determined by mtt and bioluminescent atp assay. the release of vasoactive metabolites (nitrite, endothelin-1, prostacyclin) from huvecs exposed to this medium was also measured. finally, a luminex bead array was used to screen the explant media for a panel of 15 chemokines/cytokines. results: in the angiogenesis assay, tubule length (p<0.05, mann-whitney u test) and number (p<0.05) were significantly decreased in pe compared to normal pregnancy medium. there was no change in mtt reduction, but endothelial cellular atp levels were also significantly reduced following exposure to pe explant medium (p<0.05). both normal and pe-derived medium stimulated huvecs to produce vasoactive metabolites. the following cytokines were detectable in the explant media: interleukin (il)-6, il-8, gro-, monocyte chemotactic protein-1 and macrophage inflammatory protein-1 (mip-1 ). higher levels of both il-8 and gro-were present in the normal medium compared to pe (both p<0.0001). mip-1 was present in pe conditioned media but undetectable in media generated from normal placental explants. conclusions: these results suggest that pre-eclampsia stimulates the release of soluble factors from the placenta which have adverse effects on endothelial cell angiogenic potential and metabolism. altered levels of several cytokines were detected in the explant medium, and the effects of these on endothelial cell function are currently being addressed. yang gu, davis f lewis, yuping wang. obstetrics and gynecology, lsuhsc-shreveport, shreveport, la, usa. objective: placentas from women with preeclampsia (pe) release more chymotrypsin-like protease (clp). the purpose of this study was to determine if placenta-derived clp was responsible for altering endothelial (ec) barrier function in pe. approaches: endothelial junctional protein complex ( -catenin/ve-cadherin/ p120) expression and junctional protein ve-cadherin distribution were examined in ecs treated with pe placental conditioned medium. methods: 1) confluent ecs were treated with pe placental conditioned medium (cm). the association of ec junctional protein complex ve-cadherin/catenin/p120 was examined by a combined immuno-precipitation (ip) and immuno-blotting (ib) assay, in which total cellular protein was immunoprecipitated with monoclonal antibody against -catenin and then immunoblotted with antibodies against ve-cadherin or p-120. 2) confluent ecs grown on cover slips were exposed to pe placental cm with or without depletion of chymotrypsin. ec junction protein ve-cadherin distribution was examined by fluorescent microscopy. results: 1) ve-cadherin and p120 are expressed in control ecs but not in ecs exposed to pe cm, which indicate that the junctional protein complex vecadherin/ -catenin/ p120 is lost in ecs exposed to pe cm. 2) ecs exposed to pe placental cm showed a discontinuous distribution and reduced expression for ve-cadherin at cell contact areas. the zipper like structure was lost and cleft was formed at cell-cell contacts. these observations indicate that the homotypic cell-cell adhesion and junction protein intracellular partner complex are disrupted. these disruptive phenomena in cells treated with pe conditioned medium were not present in control cells and in cells treated with cm after depletion of chymotrypsin. conclusion: clp released by the placenta could be a candidate agent that is responsible for disrupting ec integrity and inducing endothelial permeability in pe. (supported by nih grants hl65997 and hd36822). introduction: chronic pelvic pain (cpp) syndromes such as endometriosis, irritable bowel syndrome, and interstitial cystitis are associated with visceral hyperalgesia, and often coexist in the same patient. one possible explanation for this phenomenon is viscero-visceral cross-sensitization in which increased nociceptive input from an inflamed pelvic organ sensitizes neurons that receive convergent input from an unaffected organ to the same dorsal root ganglion (drg). nociception induces upregulation of perk and substance p. the purpose of this study was to determine, in a rodent model, whether uterine inflammation increased the number of perk-and sp-positive neurons in sensory ganglia innervating both uterus and colon. methods: colonic and uterine drgs were retrogradely labeled with fluorescent tracer dyes micro-injected into the colon and uterus. ganglia were harvested, cryoprotected and cut for fluorescent microscopy. results:approximately 7% neurons were colon-specific and 11% were uterus-specific. among these uterus-or colon-specific neurons, up to 5% of labeled drg neurons in the l1-s3 levels innervated both visceral organs. uterine inflammation increased the number of perk and sp-immunoreactive neurons in drg neurons innervating colon, uterus, and those innervating both organs. furthermore, this effect was specific as non-retrograde labeled drg neurons did not manifest a significant increase in perk or sp immunoreactive cells. conclusions: localized uterine inflammation leads to increased expression of sp and perk in uterine afferents as well as dichotomizing afferents innervating both uterus and colon, suggesting that viscero-visceral convergence is present at the level of drg primary afferent cell bodies. this visceral sensory integration may underlie the co-morbidity of female pelvic pain disorders and may provide basic information regarding the etiology of cpp syndromes. purpose: success rates of medical and surgical modalities for the treatment of severe fecal incontinence due to obstetric trauma are modest. we tested whether the intrasphincteric injection of autologous myoblasts is clinically safe and feasible for postobstetric fecal incontinence. methods: using ultrasound guidance a suspension of autologous myoblasts was injected in the anal sphincter muscle complex in three women with severe postobstetric fecal incontinence. main outcome measures were safety, feasibility and the wexner grading score. secondary outcome measures were incontinence episodes, rockwood fecal incontinenece quality of life scale, external anal sphincter morphology and anal pressure values. results: all procurement procedures and injections were performed without complications; there were no clinically or laboratory signs of infection or inflammation. three months post myoblast injection, wexner continence grading scores and rockwood incontinence scales were markedly improved and incontinence episodes reduced in all three patients. no change could be observed in sonographic anal sphincter morphology. mean anal squeeze pressure improved. conclusion: autologous myoblast injection for fecal incontinence is clinically feasible and safe; further studies will evaluate the efficacy of this approach. objective: to evaluate whether hysterectomy is associated with a change in postoperative bmi. methods: a retrospective cohort study was conducted of 548 hysterectomies performed for benign indications from june 2001 to june 2006. institutional review board approval was obtained. the data were collected by a medical student blinded to the hypothesis. basic demographic data were recorded. body mass index (bmi) was determined at 4 time points: time 0 (immediately postoperatively), time 1 (2 weeks to 5 months postoperatively), time 2 (5 months to 1 year postoperatively) and time 3 (1 to 2.5 years postoperatively). one-way anova was performed using ncss statistical software. a bonferroni multiple comparison test (p<0.05) was used to compare median changes in bmi from baseline. the starting bmi served as the control group. results: there was a statistically significant increase in bmi at time 3 compared to baseline in all women (31.9 to 33.6). when sorted was measured by matrigel invasion assay. small interference rna targeting vegfr-2 were predesigned by ambion inc. and cells were transfected using ambion siport neofx according to the optimized protocol. results: of the four receptors (vegfr-1, vegfr-2, nrp-1 and nrp-2), vegfr-2 was the predominant receptor that expressed in the more invasive cell lines (dov13, skov3 and r182). lpa, at 10-20 m, significantly induced vegfr-2 expression in dov13 and skov3 cells (p<0.05), without significantly affecting vegfr-1 expression. lpa (1-20 m) also significantly induced the expression of vegf 121 and vegf 165 (p<0.05) in dov13 and skov3 cells. by small interference rna (sirna) transfection, we demonstrated that inhibition of vegfr-2 expression could significantly decrease dov13 cells' invasiveness (p<0.001) and moderately decrease skov3 cells' invasiveness. moreover, silencing of vegfr-2 by sirna significantly suppressed lpa-induced dov13 and skov3 invasion. conclusion: these results suggest that knocking down vegfr-2 expression by rnai may be an effective strategy to inhibit lpa-induced ovarian cancer metastasis. cancer. fengqiang wang, david fishman. obstetrics and gynecology, new york university school of medicine, new york, ny, usa. objectives: expression of matrix metalloproteinases, such as mmp-1 and mmp-7, has implicated in epithelial ovarian cancer (eoc) invasion and metastasis. osteopontin (opn) was also expressed in various human cancers and associated with tumor progression, invasion and metastasis. in the present study, we examined the correlation of mmp-1, mmp-7 and osteopontin expression with tumor stage in 41 ovarian tumor tissues and 7 normal ovaries using a commercial tissue scan array. methods: complimentary dna (cdna) from normal ovaries (n = 7) and ovarian tumors (n = 41) of different stages (stage i, n = 16; stage ii, n = 3; stage iii, n = 19; stage iv, n = 3) was amplified by real time pcr using gene specific primer pairs for mmp-1, mmp-7, and osteopontin. gapdh was also amplified as a reference control. the expression level of mmp-1, mmp-7 and osteopontin was calculated as relative expression normalized to that of gapdh in each tissue sample, and the expression in sample that has the lowest target gene expression was arbitrarily set as 1. the average expression of mmp-7 in ovarian tumor tissues (14630 ± 30606) is 49 fold of that in normal ovaries (301 ± 641, p = 0.0047). using an arbitrarily set standard, 1 of 7 normal ovaries (14.3%) has elevated mmp-7 expression; in ovarian tumor tissues, the percentage is 82.9% (34 of 41), significantly higher than in normal tissues (p = 0.0008). in early stage tumors (stage i/ii, n = 19), 17 of them have elevated mmp-7 expression (89.4%, p = 0.0008 vs. normal ovaries), whereh the average expression of mmp-7 is 63 fold of that in normal ovaries (p = 0.069) and 1.8 fold of that in late stage tumors (stage iii/iv, n = 22). the mmp-1 expression in ovarian tumors (n = 41, 154 ± 632) is 28 fold of that in normal ovaries (n = 7), however, the difference is not significant (p > 0.05) due to the extremely high expression of mmp-1 in two tumor tissues. osteopontin expression in ovarian tumor tissues is 11.2 fold of that in normal ovarian tissues (11 ± 12.51, n=7 vs. 123.6 ± 160.0, n = 41, p<0.0001). in early stage (i/ii) ovarian tumors, osteopontin expression is 8.1 fold of that in normal ovaries, while in late stage (iii/iv) ovarian tumors, its expression is 13.9 fold of that in normal ovaries (p<0.05), suggesting an increase trend associated with disease stages. conclusions: our results suggest that mmp-1, mmp-7 and osteopontin alone or combined may have clinical value for ovarian cancer detection. objectives: -tubulin acetylation has been proposed to control the dynamic nature of microtubule stability. acetylated -tubulin plays a role in regulating microtubule functions in mitosis and cell migration. here, we sought to identify the relationship between post-translational -tubulin acetylation and the expression of epithelial cell adhesion molecules (ep-cam) after exposure to microtubule interacting agents in ovarian cancer cells. methods: epithelial ovarian cancer cell line, hey was treated with microtubule stabilizing agents (taxol, epothilone b and discodermolide), microtubule-destabilizing agent (vinblastine), hdac6 inhibitor trichostatin a (tsa), anti-metabolite fluorouracil (5fu), or alkalyting agents (cisplatin and carboplatin). cells were separately treated with either ic 50 or 10-fold ic 50 of each agent, and incubated at 37°c for 1 h, 24 h and 48 h. acetylation oftubulin and pan--tubulin were evaluated by western blot analysis followed by protein quantification. cell surface ep-cam expression was examined by flow cytometry. results: increased acetylation of -tubulin was seen with taxol, epothilone b, discodermolide, vinblastine and tsa. -tubulin acetylation was time and dose dependent. the highest level of -tubulin acetylation (2.5-fold) was observed with vinblastine at 10-fold ic 50 after 48 h. exposure to microtubule interacting agents and tsa resulted in increased cell surface expression of ep-cam in a time and dose dependent manner. the highest level of cell surface ep-cam expression (8.5 fold) was observed with 10-fold ic 50 of vinblastine at 48 h. the increase in acetylated -tubulin and ep-cam expression was clearly detectable after 1 h treatment. this data reveals a similar dose and time dependent increases between the acetylation of -tubulin and the increase of ep-cam expression. conclusions: these data demonstrate the promotion of -tubulin acetylation and cell surface ep-cam expression by a microtubule destabilizing agent and by microtubule stabilizing agents. interestingly, vinblastine induces the highest -tubulin acetylation and ep-cam expression. acetylation of alpha-tubulin may be associated with redistribution of cell surface antigens in ovarian cancer cells. objective: epothilone b (epob) is similar to taxol in its ability to enhance tubulin polymerization and to stabilize microtubules. epob is currently being evaluated as an antitumor agent and is in phase iii clinical trials. the tumor suppressor gene, p53 plays an important role in the induction of apoptosis by a variety of anticancer drugs. p38 mitogen-activated protein kinase is activated by a wide array of stress stimuli including chemotherapeutic agents and promotes apoptosis. since both p38 and p53 activation induces apoptosis, we hoped to evaluate the relationship between p38, p-p53 and parp cleavage, an early indicator of apoptosis, in ovarian cancer cells after treatment with epob. methods: hey cells were treated with epob (5 to 200nm) for 16h. parp cleavage product p85 as well as p-p38, p53, phospho-p53 (ser-15), p21 and survivin were determined by western blot analysis. a wild type ovarian cancer cell line hey, was treated with or without 5 m sb203580, a specific inhibitor of p-p38, followed by treatment with epob (20 or 50 nm) for 16h. lysates were prepared and western blot analysis was performed with polyclonal phospho-p38 and anti-phospho-p53 antibodies. results: epob induces p53 activation and apoptosis demonstrated by increased parp cleavage product. time course studies indicated that phosphorylation of p53 precedes phosphorylation of p53 in hey cells. the expression of p53 targeted gene, p21 (survivin) were differentially expressed depending on the dose of epob and the duration of drug exposure. pretreatment with specific inhibitor of p38 markedly inhibited the p53 phosphorylation at serine 15. conclusions: 50 nm epob (a concentration that triggered mitotic arrest) causes a decrease in p21 expression and an increase in survivin expression. epob induces parp cleavage. p38 inhibitor sb203580 inhibits epo-b -induced p53 phosphorylation. these results suggest that phosphorylation of p38 may lead to p53 activation and these signaling events may be related to epo-b induced cell death in ovarian cells. conclusions: nf-b has been shown to control the expression and function of anti-apoptotic proteins and pro-inflammatory cytokines. in the present study we demonstrated that specific inhibition of nf-b by erib reversed the anti-apoptotic state of chemoresistant ovarian cancer cells, therefore may provide a new potential venue for the treatment of ovarian cancer patients. expression in cervical cancer. madhu chauhan, chandra yallampalli. gynecology, university of texas medical branch, galveston, tx, usa. background: intermedin (imd)/adrenomedullin2 is a ct/cgrp family peptide. imd is expressed in a variety of tissues such as pituitary, stomach, placenta, uterus, and ovary .we have shown that imd plays a role in a variety of physiological functions, including vasodilatation and fetoplacental growth regulation. further we have data indicating an angiogenic activity of imd in first trimester trophoblast cells. we hypothesize that imd is expressed in cervix and may have a role in the pathology of cervical cancer objective: 1) to analyze expression of imd mrna in human cervix, rat cervix from non-pregnant and pregnant rats; 2) to assess the differences in the expression of imd in normal human cervix and cervical carcinoma tissues and 3) to analyze the effect of imd on the expression of tnf-alpha and il-1beta in human epithelial cervical carcinoma cells (hela). methods: groups of 4 sprague-dawley, non-pregnant and pregnant rats on day 18 of gestation were used in this study. cervical tissues were collected from the non-pregnant and pregnant rats, women undergoing hysterectomy and from women diagnosed with cervical carcinoma. total rna was extracted using trizol method. hela cells were grown to 80% confluency in rpmi medium supplemented with 10% fbs. the cells were starved in 1%fbs for 4 hrs followed by treatment with imd (10 -8 m) in presence or absence of imd antagonist, imda (10 -5 m). cells were further incubated for 24hrs and total rna was extracted using trizol reagent. rna was treated with dnase1 before performing the reverse transcriptase polymerase chain reaction (rt-pcr). the rt-pcr data was normalized to that of 18s. results: 1) imd mrna is expressed in rat and human cervix and in hela cells. 2) expression of imd is elevated in pregnant rat cervix as compared to the non-pregnant. 3) imd has no effect on tnf-alpha expression in hela cells treated with imd but caused a decline in the expression of il-1beta mrna and, 4) expression of imd mrna is significantly elevated (p< 0.05) in cervical carcinoma as compared to the normal cervix. conclusion: imd is expressed in both rat and human cervix and is elevated in pregnant rat suggesting that it may have a role in cervical function during pregnancy in rat. in addition we demonstrate that imd is involved in the pathology of cervical carcinoma and thus may have a clinical significance in the pathology of cervical cancer. objective: there is minimal information about the impact of treatment delays or dose reductions on chemotherapeutic treatment responses and overall outcomes in ovarian cancer patients. the primary objective of this study was to quantify the impact of treatment delays and dose reductions in an in vivo xenograft ovarian cancer model and to evaluate if the growth factors could improve overall survival. methods: heya-8 and skov3-ip xenograft mouse models to determine the effect of treatment delays or dose reductions on tumor response. results: the results indicated that full dose of paclitaxel (20 mg/kg) and carboplatin (50 mg/kg) delivered on timeevery 21 daysachieved better tumor response in both aggressive (heya-8) and metastatic (skov3-ip1) human ovarian tumors compared to the non-treatment and vehicle groups. in heya-8 mice, paclitaxel/carboplatin alone and paclitaxel/carboplatin followed by growth factor support agents including pegfilgrastim (1000 μg/kg) and darbepoetin (10μg/kg) improved overall survival rate. growth factors also improved the tolerability in heya-8 model. for skov3-ip1 mice, the treatment delays resulted in a significant reduce in overall survival time compared to full dose, on time treatment (p< 0.01). for the dose reduction group, there was a significant different survival comparing to full dose, on time treatment (p< 0.05). conclusion: treatment delays had a negative impact on tumor response and overall survival compare with treatment controls. in addition, use of growth factor agents also improved treatment response and tolerability of chemotherapy and ultimately overall survival. -415) . median age at recurrence was 73 (33-104), and median interval to recurrence 13.5 months (1-415). 81 (56%) patients died of recurrent disease and 10 (7%) of other causes. site of recurrence and was local in 92, groin in 30 and distant in 22. 58 of the local recurrences were managed with wide local excision (wle) 12 radical surgery (6 radical excisions with skin flaps, 4 posterior exenterations, 1 anterior exenteration, 1 total exenteration), 10 wle and radiotherapy (rt)/chemort, 6 chemo/chemort/rt, and 6 palliation. 12 groin recurrences were managed surgically (5 adjuvant rt), 3 rt alone, 1 chemotherapy alone, and 1 palliation. 2 patients with distant metastases were managed surgically, 10 with rt/chemo/chemort, and 9 palliation. overall median survival after recurrence was 17 months. median survival (months) by site of recurrence was: local 56, groin 12, distant 4, groin or distant 6. there was a significant difference in survival in local vs groin node recurrence (p<0.0001), local vs groin and distant (p<0.0001), and groin node vs distant (p 0.0075). 3 and 5 years after recurrence survival rates were: local 56% and 46%, groin 13% and 13% distant 0,0. conclusion patients with vscc remain at risk of recurrent disease therefore long term follow up these patients is essential. patients with local recurrence often have a good prognosis. outcomes for groin and distant recurrences are poor, but a proportion of those with groin recurrences can be salvaged. therefore early identification of local and groin recurrences is essential for improving outcomes in recurrent vscc particularly in cases with more conservative management of primary disease. introduction: endocrine disrupting chemicals (edcs) are environmental agents possessing hormone-like activity. exposure to edcs during differentiation can interfere with hormonal signaling, resulting in predisposition to some cancers. it has been suggested that some of these effects may be epigenetic, mediated by changes in dna and histone methylation. we hypothesized that 2 edcs, diethylstilbestrol (des), and bisphenol-a (bpa), may act by altering the expression of dna and histone methyltransferases (dnmts and hmts). methods: ishikawa (endometrial) and mcf7 (breast) cells were cultured in steroid-free, phenol-free media with bpa (25 m), des (5 x 10-8 m) or vehicle for 48 hours. pregnant cd-1 mice were treated with intraperitoneal injections of des (10 mg/kg) or vehicle on days 9-16 of gestation. 2 weeks after birth, offspring were sacrificed and tissue obtained. mrna was extracted, cdna was generated and quantitative real time rt-pcr was performed and normalized to -actin. all experiments were conducted in triplicate, repeated three times and compared using anova. results: 3 dnmts (dnmt1, 3a, and 3b) and 2 hmts (mll and ezh2) were screened after in vitro exposure to edcs (table 1) . ezh2 mrna expression was significantly increased in ishikawa cells after treatment with des or bpa. a similar response in ezh2 expression was seen in mcf7 cells treated with des or bpa. due to the consistent induction of ezh2 in all cells with either treatment, we examined the effect of des exposure on ezh2 expression in vivo. in adult mice exposed to des in utero, ezh2 expression was persistently increased (3.0±0.66 fold, p<0.05). conclusions: breast and uterine cell lines show increased expression of ezh2 in response to bpa or des exposure. this differential expression persists in the adult offspring of mice exposed to des in utero. ezh2 has been identified as a risk for neoplastic progression in the breast and for increased proliferation in uterine cancers. des induced ezh2 expression may be a mechanism for the increased incidence of breast and reproductive tract cancers seen in des exposed women. defects in cellular programs that control apoptosis lead to an imbalance of cell proliferation and cell death in ovarian cancer. recent evidence suggests that the use of some anti-inflammatory drugs decreases risk of ovarian cancer. natural curcumin-based anti-inflammatory therapies were shown to be beneficial in preclinical models of ovarian cancer (lin et al., 2007) . in this study, we examined the effects of plant derived curcumin on cell proliferation, apoptosis, and vegf expression in cultivated ovarian cancer cells. ovarian cancer igrov1 cells were cultured under standard conditions to study the effects of curcumin on cell kinetics and on vegf expression. cell proliferation was measured by srb and mtt assays. apoptosis was determined by measuring cytoplasmic histone-associated-dna-fragments (cell death detection elisa, roche, germany). vegf gene promoter-reporter activation and real-time quantitative reverse transcription polymerase chain reaction were used. conditioned media concentrations of vegf were measured with a commercially available enzyme-linked immunosorbent assay (quantikine, r d systems, minneapolis, mn). we observed that curcumin dose-dependently suppressed cell growth in igrov1 cancer cells (ic 50 =10um). treated cells showed a 2-3 fold increase in dna fragmentation compared to controls. curcumin also resulted in a significant decrease of vegfmrna expression and vegf protein secretion into the conditioned media in a dose-dependent manner. in this study, we have demonstrated that curcumin induces apoptosis, suppresses growth, and inhibits vegf gene and protein expression in an ovarian cancer cell line. experiments are underway to identify specific mechanism of curcumin action. curcumin may act as a chemosensitizing drug by potentiating the antitumor effects of standard treatments including taxols and platins in ovarian cancer. supported by the caldwell family foundation and the vesa w. and william j. hardman, jr. charitable foundation inc. daniel r christie, 1 faheem m shaikh, 2 john a lucas iii, 1 susan l bellis. 2 1 obsterics and gynecology, university of alabama at birmingham, birmingham, al, usa; 2 physiology and biophysics, university of alabama at birmingham, birmingham, al, usa. introduction: previous work has shown that an upregulation of the enzyme st6gal i, which is responsible for the 2,6 sialylation of 1 integrins, confers a more tumorigenic/metastatic phenotype to colon carcinoma cell lines. it is not known, however, if this is unique to colon cancer, or if it is more broadly applicable to other forms of cancers. quantitatively increased expression of st6gal i has been reported in ovarian cancers versus controls, but no biochemical or functional assays have been described to date. objective: because 1 integrins are involved in cell adhesion and migration, and because metastasis of epithelial ovarian cancers is largely an intraperitoneal dissemination, we hypothesized that upregulation in the expression of st6gal i in an ovarian cancer cell line would enhance binding with the extracellular matrix, increase invasiveness, and alter migration. methods: in the present study we forced a stable transduction of st6gal i into the ov4 ovarian tumor cell line, which we found to be lacking the st6gal i enzyme, establishing a parental (par), an empty lentiviral vector (ev), and an st6gal i expressing (st6) cell line. a collagen i cell adhesion assay was performed and quantified by staining adherent cells and measuring absorbance. a haptotactic collagen i cell migration assay was performed by seeding cells in boyden chambers lined with collagen i concentration gradient, and quantified with cell staining and absorbance measurement. cell invasion through a reconstituted basement membrane (matrigel) was quantified as previously described for the cell migration assay. results: we were able to demonstrate successful creation of the ov4-st6gal i cell line by western blot analysis. functional assays demonstrated increased adhesion to collagen i (p < 0.05), increased haptotactic collagen i cell migration (p < 0.01), and increased invasiveness (p < 0.05) in the st6 cell line as compared to par and ev when analyzed by one-way anova. conclusion: this initial study into st6gal i in ovarian cancer may have future therapeutic implications, and, in addition, lend greater insight into the intraperitoneal dissemination of disease.of microarrays (sam), arrayassist and binary tree structured vector quantization. differentially expressed genes were integrated with a database of known predicted protein-protein interactions (ophid) and key genes are being validated with real-time pcr and immunohistochemistry. results: unsupervised hierarchical clustering revealed collective clustering of all tumors, irrespective of their pathological classification. sam analysis has highlighted 47 probe sets as differentially expressed between lmp and lmp-mp, 134 probes between lmp and lgsc, and 180 probes between lmp and lmp-mp+lgsc. no differential gene expression was detected between lmp-mp and lgsc. ophid analysis demonstrated gene members of the egfr and mapk1/3 pathways to be differentially regulated between the non-invasive and the invasive tumors. to date, we have successfully validated 2 members of the mapk1/3 pathway-poly adp ribose polymerase1 (ppol) and traf family associated nf kappa b activator (tank)-using real-time pcr. conclusion: our data demonstrate that although the 3 tumors have related genetic profiles, lmp-mp and lgsc are similar to each other and different from lmp, in keeping with their clinical behavior. members of the mapk1/3 and egfr pathways appear to play a key role in low grade serous cancer. identification of novel genes associated with malignant transformation, may lead to development of more effective targeted therapy for lgsc. background: approximately 50% of pap smears with the ambiguous diagnosis of atypical squamous cells, cannot exclude high grade (asc-h), are negative for dysplasia in follow up colposcopic examination and biopsy. although hpv testing provides excellent negative predictive value (npv) (95%), the prevalence of high risk hpv infection is high in young women and the positive predictive value (ppv) in asc-h pap smears is no better than cytologic diagnosis alone (50%). recent studies have shown that immunostaining for p16 ink4a , or proex tm c, supports a diagnosis of dysplasia in surgical biopsies. our objective was to determine whether staining for p16 or proexc provides sufficient predictive value to reliably distinguish high grade dysplasia in asc-h pap smears. design: we retrospectively collected samples from liquid based pap smears diagnosed as asc-h at either ohsu or portland oregon kaiser permanente (2005-07). known hsil and negative pap cases were included as immunostaining controls. asc-h cases with followup cervical biopsies (n=90) were included for subsequent immunostaining according to manufacture's instructions. samples were also sequestered for hpv testing (pending). immunostained slides were scored as positive or negative by two independent cytopathologists (as and tm) while blinded to pap diagnoses and surgical biopsy outcomes. results: we observed excellent agreement between pathologists' scores (pairwise kappa statistic 0.85). the correlation between p16 and proexc scores was moderate (kappa 0.40). chi-square analysis comparing staining to biopsy outcome revealed a significant association between proex c positivity and cervical dysplasia (*** p<0.001 jing lin, zhenmin lei, ch v rao. ob/gyn women health, university of louisville health sciences center, louisville, ky, usa. introduction: matrix metalloproteinases (mmps) are a family of highly homologous zinc-dependent endopeptidases, which degrade all kinds of extracellular matrix proteins. the degradation process is required for tissue growth and remodelling. these enzymes are probably involved in uterine growth and devolopment and its differentiated functions. ovarian steroid hormones, estradiol (e 2 ) and progesterone (p 4 ), are known to regulate some of the uterine mmps. since it is now known that lh/hcg can directly regulate uterine growth and devolopment and its functions, we questioned whether these gonadotropins could also regulate mmps in the uterus. methods: sixty day old wild type (wt) and lh receptor knockout (lhrko) mice and 21-day e 2 and p 4 treated 30-day old animals were used. in addition, primary cultures of uterine epithelial cells from wt animals were treated for 24 hrs either with no hormone or with a single or a combination of 100 pg/ml of e 2 or p 4 , 100 ng/ml of hcg. then mmp-2, -7 and -10 mrna levels were quantified by the semiquantitative rt-pcr. results: while the uterine mmp-2 mrna levels were unaffected, mmp-7 mrna levels significantly decreased and mmp-10 mrna levels significantly increased in lhrko animals as compared with wt siblings. e 2 and p 4 treatment reversed mmp-7 and mmp-10 changes in lhrko animals. treatment of wt type primary endometrial epithelial cell cultures with hcg had no effect on mmp-10 mrna levels, but it did increase mmp-7 mrna levels. this increase was synergistic with both e 2 or p 4 . conclusion: while lh/hcg do not regulate uterine mmp-2 and mmp-10, they seem to co-regulate mmp-7 with ovarian steroid hormones. cancer. radhika gogoi, christine ardalan, dorota popiolek, leslie gold, john curtin, stephanie v blank, bhavana pothuri. new york university, new york, ny, usa. objective: megesterol, a synthetic progestin with strong androgenic properties, is used in the medical management of patients (pts) with atypical endometrial hyperplasia (aeh) or endometrial carcinoma (emca). our hypothesis was that androgen receptor (ar) expression in the endometrium of aeh and emca pts would correlate with degree of response to treatment. methods: pre-and post-treatment endometrial biopsy specimens were obtained from 8 pts treated with megesterol for aeh or emca. ar expression was investigated by immunohistochemical (ihc) analysis with appropriate positive and negative controls. ihc staining was scored for intensity (1-4) and percentage of positive cells (1-4) in the glandular and stromal compartments by a pathologist, blinded to clinical response data. a composite score utilizing both intensity and percentage of positive cells was calculated. we evaluated pre-and post-treatment ar expression in responders and nonresponders as well as ar expression in emca, aeh and normal endometrium. the mann whitney u and the wilxocon signed rank tests were utilized for statistical analysis. results: eight pts' pre-and post-treatment samples were obtained; 4 with emca and 4 with aeh. three pts had no response, 1, a partial response and 4, complete responses. ar expression in emca samples when compared to the normal endometrium was significantly lower in both the glands (mean 2.8 vs. 8; p<0.01) and the stroma (mean 2.3 vs. 5; p<0.05). although there was no statistically significant difference in glandular ar expression in the pretreatment biopsies of responders compared to nonresponders (mean 4.6 vs. 2.7; p=0.14), there was a significantly higher level of glandular ar expression in the post treatment biopsies of responders compared to non-responders (mean 7.4 vs. 3; p<0.05). furthermore, we noted a trend towards higher levels of glandular ar expression in the post-treatment versus the pre-treatment biopsies in the responders (mean 7.4 vs. 4.6; p=0.07). we noted a significant decrease in ar expression in emca compared to the normal endometrium. increased ar expression after treatment in responders suggests an important role of the ar in pts treated conservatively with progestational therapy, and needs further prospective validation as a means to predict treatment response in these pts. objectives: study the correlation between adenomyosis and some potential non-surgical risk factors. objective: o 6 -methylguanine-dna methyltransferase (mgmt) acts to repair dna damaged by alkylation of guanine residues. mgmt promoter methylation and gene silencing is seen in a variety of cancers and pre-cancerous changes. the loss of mgmt activity is associated with increased sensitivity to alkylating agents and is a favorable prognostic indicator in gliomas. we sought to determine if mgmt promoter methylation plays a role in endometrial cancer. methods: one hundred and twenty primary endometrial cancers were analyzed for mgmt promoter methylation by combined bisulfite restriction analysis (cobra). the cohort included 77 endometrioid endometrial cancers, 43 endometrial tumors of adverse histologic type, and 6 endometrial cancer cell lines. twenty one endometrioid and mixed endometrioid ovarian cancers were also analyzed. a subset of the primary tumors was analyzed for mgmt expression by immunohistochemistry. results: no mgmt promoter methylation was seen in the 120 endometrial cancers evaluated or the 6 endometrial cancer cell lines. one of the 21 ovarian cancers showed methylation. immunohistochemistry for mgmt expression is ongoing. conclusion: mgmt promoter methylation is an infrequent event in endometrial cancer. mgmt expression in tumor cells and repair of alkylguanine residues could explain in part the limited response of endometrial tumors to alkylating chemotherapy. objective: uterine sarcomas (1% of gynecological malignancies) originate from uterine mesenchyma. patients with high-risk disease (i.e. high grade) or at advanced stages have poor 5-years overall survival (< 10-30%). pelvic radiation and/or chemotherapy have not demonstrated to improve survival. identification of new therapies for this malignancy is a major goal. few in vitro models have been established to test therapeutic agents for uterine sarcoma. here we sought to establish a new human uterine sarcoma cell line and to test the effects of chemotherapeutic drugs: tnf-related apoptosis inducing ligand (trail) used alone or in combination. methods: tissue sample was obtained from a woman with uterine sarcoma undergoing hysterectomy. sarcoma cell lines were established using a published protocol for endometrial cancer. phenotypic characterization was made through the different passages (1 to 13) by western blot. levels of estrogen (er), progesterone (pr) and trail receptors were also studied (rt-pcr, w-b). cells were incubated with chemotherapy agents (cisplatin, paclitaxel and doxorubicin and trail) and cytotoxicity (mts assays) and apoptosis (flow cytometry, parp cleavage by w-b, and dna laddering) measured. results: human uterine sarcoma cell line was established from a highgrade uterine sarcoma. through the different passages the cell line remains expressing cytokeratin, vimentin, tissue factor, caveolin-1 and -actin. this cell line expresses low er and pr levels. trail receptors (r1 and r2) were also detectable (rt-pcr, w-b). cisplatin, paclitaxel and doxorubicin (5um for 24 h) produced low cell cytotoxicity (< 20%). trail (200 ng/ml for 18 h) induced about 30% cell cytotoxicity. apoptosis was confirmed by parp cleavage. doxorubicin significantly enhances trail mediated cytotoxicity (up to 80%), this was demonstrated by a significant increase in the sub g0/g1 region in the dna histogram. conclusions: we establish a human uterine sarcoma cell lines using protocols for endometrial cancer. more importantly, we demonstrated that doxorubicin enhances trail effect on this uterine sarcomas cell line. thus, this combination might be considered as a treatment for high-risk uterine sarcomas. (financial support fondecyt 1050744). defining the tumor initiating capacity of cd133 + human endometrial cancer cells. anne m friel, 1 petra a sergent, 1 christine l cummings, 2 rosemary foster, 3 current data suggest rare subpopulations of cells with tumor initiating capabilities are a common feature of solid tumors. several investigators have recently identified cd133, a cell surface marker expressed on primitive cells of neural, hematopoietic, endothelial and epithelial lineages, as a potential tumor initiating cell (tic) marker in solid tumors of the brain, colon and pancreas. in our efforts to investigate such a population in human endometrial cancers (enca), we developed an in vivo model that is based on serial transplants of tics from endometrial tumor explants. serial transplant experiments were initiated in nod/scid mice that were injected with primary human enca cells. a subset of cells derived from the generated tumor was subsequently transplanted into new recipients. tumors formed following serial transplantation retained the original histological phenotype of the primary enca, and the number of cells required to initiate tumor formation was reduced at each successive transplant stage suggesting an increase in the ratio of tics to non-tics. we exploited this model in our initial efforts to investigate the tumor initiating potential of cd133-expressing enca cells. to evaluate the tumorigenic potential of cd133 + cells, the tumor initiation capacity of xenograft derived cd133 + and cd133cells were compared following subcutaneous injection of each population into nod/scid mice. only the cd133 + fraction was tumorigenic consistent with the hypothesis that tumors are generated and maintained by a subpopulation of cells with phenotypically distinct profiles. we are further investigating the functional significance and characteristics of this fraction in vitro and in vivo. objectives. central issues in tumor biology are the understanding of factors that control tumor cell proliferation and the identification of extracellular matrix cues controlling the signaling transducing repertoire that make cancer cells proliferate and invade the host tissues. among these factors, small leucine-rich proteoglycans (srlps):decorin, fibromodulin, lumican, biglycan, are emerging as powerful modulators of angiogenesis and cell growth, by affecting several key elements including matrix assembly, growth factor binding, and receptor tyrosine kinase activity. recently has been demonstrated that srlps can act as a pan-erbb ligand and, in doing so, down-regulate the activity of one of the most potent oncogenic proteins, erbb2, whose overexpression is linked to poor prognosis and increased cancer mortality in breast, ovary, and prostate. since srlps have not been previously investigated in the endometrial cancer biology, we investigated their role in the benign and malignant endometrial neoplasia. method. the sampling has been obtained in women (n=60; mean age 50± 2.7) with endometrial hyperplasia (n=20), polyps(n=20), and cancer(n=20), during therapeutic and diagnostic procedures (hysterectomy, colpohysterectomy, hysteroscopy). physiological endometrial samples (n=20) were obtained from menopausal women (n=20; mean age 51± 2.5), during procedures for others gynaecologic indications. immunohistochemestry was the biology technique used for the detection of srlps.results. in the physiological endometrial samples immunoreactivity for decorin, fibromodulin and biglycan was intense (+++), while it was weak in polyps and hyperplasia (+) and absent (-) in cancer. no significant difference in the staining of lumican between the physiological and pathological samples. conclusions. these results could provide a mechanism by which naturally occurring proteins normally synthesized by fibroblasts and smooth muscle cells, the two key components of the tumor stroma, may play a protective role in the genesis and progression of endometrial neoplasia counteracting the growth of neoplastic cells and suppressing tumor cell-mediated angiogenesis. although further studies are necessary to understand mechanisms whereby srlps might influence endometrial cell growth and survival, these molecules may represent potential target for pharmacological cancer therapy. myostatin is a member of the tgf-beta superfamily of protein and a wellknown inhibitor of skeletal muscle proliferation. the muscular component of the uterus is the myometrium, a tissue that regulates its mass in response to different physiologic conditions under the influence of steroids. we determined the expression of myostatin mrna in immortalized pregnant human myometrial (phm1) cells and we verified its biological activity. functional assays showed myostatin induced phosphorylation of smad-2 and reduction of proliferation of phm1 cells in a time and dose-dependent manner. to investigate the physiological relevance of our in vitro findings, the expression of myostatin in rat uterus was examined at various phases of the estrous cycle. for the first time we report that myostatin is expressed in rat uterus and that levels of myostatin mrna change during distinct phases of the estrus cycle. uterine levels of myostatin peaked during late estrous and were the lowest at pro-estrous. to further examine the role of steroids in myostatin regulation, we examined the effects of gonadal steroid administration in ovariectomized (ovx) rats. ovaryectomy increased myostatin expression compared to normal cycling controls. estrogen treatment strong decreased myostatin levels while progesterone produced less robust effects on myostatin expression. these findings taken together suggest an important role for myostatin in the regulation of myometrial functionality. her2neu over-expression and pi3kinase/akt pathway activation in paget's disease of the vulva. amy stenson, 1 bita behjatnia, 1 jaime shamonki, 1 jianyu rao, 1 amer karam, 1 jonathan berek, 2 oliver dorigo. 1 1 ob/gyn and pathology, ucla, los angeles, ca, usa; 2 ob/gyn, stanford university, palo alto, ca, usa. background: paget's disease of the vulva is rare with high recurrence rates. treatment of recurrent disease is challenging due to its extent and location. non-surgical approaches have limited clinical efficacy, obviating the need for novel therapies. in contrast to paget's of the breast with well-described overexpression of her2neu, molecular features of vulvar paget's are poorly characterized. our objective was to study therapeutic targets in vulvar paget's, including the her2neu protein and the phosphorylated oncoprotein akt (pakt). in addition, detailed clinical characteristics were correlated with molecular expression. methods: specimens with vulvar paget's disease were retrieved from ucla's department of pathology. protein expression was evaluated by immunohistochemistry on slides from paraffin embedded tissue using the hercep test (dako) for her2neu expression and a standard protocol to assess expression of activated pakt. slides were scored by two independent pathologists based on a nominal scale of 0 (negative) to 3 (strongly positive). clinical data was retrieved via chart review. results: between 1995 and 2007, 25 patients with vulvar paget's were identified. median age was 65 yrs (36-83 yrs). a family history of cancer was found in 14/25 (54%), 7/25 (28%) were smokers and 13/18 (72%) had a history of hormone use. intraepithelial lesions accounted for the majority (15/25, 60%), while 6/25 (24%) demonstrated invasion and 4/25 (16%) were associated with underlying gi malignancy. 16/25 (64%) had at least one recurrence, with median time to recurrence 24 months. so far, 13 specimens were stained for her2neu and pakt. overexpression of her2neu was found in 10/13 (77%.) positive staining for pakt was evident in 10/13 (77%.) statistical analysis suggested a correlation between her2neu and pakt expression. conclusions: our study demonstrates that overexpression of her2neu and activation of the pi3kinase/pakt pathway are important features of vulvar paget's disease. to the best of our knowledge, this is the largest series evaluating these molecular pathways in vulvar paget's. our data suggest that her2neu and pakt may be important molecular targets for novel therapies using for example the monoclonal antibody trastuzumab directed against her2neu, or a pi3kinase pathway inhibitor like rapamycin. introduction: uterine leiomyomas are the most frequent tumor of the female reproductive tract and are the primary indication for hysterectomy in women worldwide. these tumors occur in up to 70% of adult women, and their prevalence is especially high in africa-american women. currently there is no effective and safe medical treatment for uterine fibroids and surgery is the main stay. epigallocatechin gallate (egcg) constitutes the main solid extract of green tea and is believed to contributes most of the antioxidant and antiinflammation capacity of green tea. egcg has been shown to have beneficial effects on prostate cancer and breast cancer by inducing apoptosis and inhibiting proliferation of cancer cells. in this study, we investigated the ability of egcg to inhibit proliferation of human leiomyoma cells (hlm) in vitro. methods: human immortalized leiomyoma cells were cultured at 37ºc in a humidified atmosphere of 5% co 2 -95% air in smbm medium supplied with 5%fbs, 0.1% insulin, 0.2% hfgf-b, 0.1% ga-1000 and 0.1% hegf(lonza). the cells were plated at density of 2×10 5 cells/well in 6-well plates and grown under the same conditions above. the monolayer cultures at approximately 70% confluence were treated with various concentrations (0, 0.1, 1.0, 10, 50, 100 and 200μm) of egcg (sigma) for 7 days. a fluorometric assay using hoechst 33258 dye (sigma) for dna quantitation was conducted at the following designed time points, day 1, 3, 5, and 7 post egcg treatment. the cells were lysed and dna content was determined using hoechst dye solution (1μg/ml). fluorescence was measured after excitation at 355nm and emission at 458nm. results: egcg exhibited marked anti-proliferation effect on the growth of hlm cells. compared with untreated control, the inhibitory effect of egcg on hlm cells was observed at 10 μm and peaked at 100 μm concentration. the difference was statistically significant (p = 0.002). evaluation of the mechanism of action of egcg is cuurently under investigation in the lab.conclusion: egcg significantly inhibited the proliferation of human leiomyoma cells in a dose-depended pattern. egcg may present a potential effective and safe medicinal treatment for uterine fibroids. objective: choriocarcinoma is an aggressive form of germ cell tumor that exhibits rapid growth with early metastases. choriocarcinomas autonomously secrete hcg which acts as an autocrine/paracrine growth factor in these cancers. we hypothesize that a novel hcg antagonist (hcg-ant) can limit tumor expansion by blocking hcg-induced expression of pro-invasive genes. we investigated if hcg-ant could alter rna expression of matrix metalloproteinases (mmp-1 and mmp-7), which facilitate basement membrane degradation and hence invasion, and metastin (kisspeptin), a strong suppressant of metastasis, in the choriocarcinoma cell line jeg-3. design: in vitro experiments using the jeg-3 cell line. materials and methods: after plating jeg-3 cells in a 24 well tray overnight in rpmi media containing 10% fbs, cells were washed twice with pbs and then cultured in 500 ul of serum-free rpmi media containing one of four treatments: 1) saline; 2) hcg (50 iu); 3) hcg (50 iu) plus hcg-ant (100 iu); or 4) hcg-ant (100 iu). rna was extracted from each well using trizol and reverse transcribed using sensiscript (qiagen). the relative expression of mmp-1, mmp-7, and metastin mrna was quantified using sybr-green based real time pcr. the expression of the housekeeping gene hprt was used to normalize expression data. results: treatment of jeg-3 cells with hcg-ant vs. hcg alone reduced expression of mmp-1 (51.95 ±15.51 vs. 85.99 ±10.56) and . hcg-ant reduced mmp-1 and mmp-7 expression by 40% and 43%, respectively (p<0.05). treatment with hcg-ant vs. hcg increased metastin expression (5.44 ±2.29 vs. 4.63 ±1.32). metastin expression was increased by 17% following hcg-ant treatment. conclusion: treatment of the jeg-3 choriocarcinoma cell line with hcg-ant reversed the increased expression of mmp-1 and mmp-7 following treatment with hcg. metastin expression was increased by hcg-ant. this data suggests that hcg antagonism is capable of altering gene expression thereby inhibiting invasion in a choriocarcinoma cell line. the role of hcg-ant as an adjuvant therapy in hcg sensitive tumors offers promise. retinoids, but not progesterone, directly induce differentiation and apoptosis of endometrial cancer cells. you-hong cheng, serdar e bulun. ob/gyn, northwestern university feinberg school of medicine, chicago, il, usa. objectives: the opposing actions of estrogen and progesterone during the menstrual cycle regulate endometrial proliferation, differentiation and secretion. the unopposed action of estrogen contributes to the development of type i endometrial cancer. however, the mechanisms for progesterone protection of estrogen-induced carcinogenesis in endometrium remain unclear. methods and results: in the current study, we demonstrated that retinoids (9-cis retinoic acid (ra) or all-trans ra (atra)) significantly inhibited basal and hormone-stimulated ishikawa cell proliferation by over 60% using mtt assay. the same experiment indicated that estrogen had no significant effect, whereas progesterone slightly induced, on cell proliferation. cell cycle analysis indicated that atra significantly increased the g1/g0 cell population by 20% and decreased s phase cells by 20%, suggesting that ra induces cell cycle arrest at the s phase. knock-down of rar alone or both rar and rxr significantly increased proliferating cell nuclear antigen (pcna) levels in epithelial ishikawa cells, suggesting that ra signaling via rar/rxr activation is critical for normal endometrial growth and differentiation. to determine whether retinoids are naturally secreted by the endometrial stromal cells, we cultured primary stromal cells and analyzed the culture media using hplc. we found that retinol is the predominant retinoid form secreted by stromal cells. the average concentration of retinol in the cultured media of eutopic endometrial stromal cells was approximately 4 to 6 ng/ml/10 5 cells (n=5). although there was less than 0.25 ng/ml/10 5 cells of all-trans retinal or atra in the cultured media, we did find a small peak for all-trans retinal and atra in the media using hplc analysis. furthermore, progesterone significantly increased secreted retinol levels from eutopic endometrial stromal cells, but decreased retinol levels secreted from endometriotic stromal cells. retinol is a precursor for ra that is converted to retinal and then to ra. conclusions: we demonstrated that progesterone signaling via pr induces endometrial stromal cells to secrete paracrine retionids that in turn control the phenotype of adjacent epithelial cells. conversely, this interaction is disrupted in diseased endometrial tissues, such as endometrial cancer and endometriosis. the effects of hormonal contraceptives on antimullerian hormone by obesity status. anne z steiner, 1 frank z stanczyk, 2 stan patel, 2 alison edelman. 3 1 ob/gyn, university of north carolina, chapel hill, nc, usa; 2 ob/gyn, usc keck school of medicine, los angeles, ca, usa; 3 ob/gyn, oregon health and sciences university, portland, or, usa. background: antimullerian hormone (amh) is emerging as a predictor of reproductive potential. serum levels of fsh, a commonly used measure of ovarian reserve, are suppressed with the use of oral contraceptives (ocs) thereby limiting its use. the impact of ocs and on serum amh levels in normal and obese women is unknown. objective: to examine the impact of ocs on serum amh levels by obesity status in reproductive-age women. materials and methods: ovulatory women, ages 18-35 years, of normal (< 25 kg/m 2 ; n = 10) and obese (> 30 kg/m 2 ; n = 9) body mass index (bmi) received a low dose oc (20 mcg ethinyl estradiol/100 mcg levonorgestrel) for two cycles. serum was obtained at three time points: after 21 days of active pills (cycle 1, day 21), at the end of the 7-day hormone-free interval (cycle 1, day 28), and during the first week of active pills in cycle 2 (cycle 2, day 5,6, or 7). amh levels were quantified by elisa; fsh and lh levels were determined by chemiluminescent immunoassay. amh at the three time points was compared using repeated measures anova. models were used to assess the relationship between amh and cycle day by obesity status. amh and gonadotropin levels were compared using spearman's correlation. results: amh levels did not differ by oc cycle day (p anova = 0.90) or by active versus placebo pill use (2.79 ng/ml ± 1.3 vs. 2.85 ng/ml ± 1.4, p=0.93) among normal bmi women. among obese women, amh levels differed by oc cycle day (p anova = 0.04), but not by use of active or placebo pill (1.63 ng/ml ± 1.2 vs.1.71 ng/ml ± 1.2, p = 0.88). across the cycle, cv(standard deviation/mean) averaged 20.2%± 3.1 in the obese and 8.8%±1.3 in the normal bmi women ( p=0.003). modeling to determine differences in amh throughout the cycles based on obesity status showed a significant interaction (p = 0.02) and lower amh levels in the obese group (p<0.001). among both bmi groups, serum amh and fsh levels did not correlate during active pills or after 7 days of placebo pills. conclusions: in young, normal bmi women serum amh levels do not appear to fluctuate during oc use. among obese women, amh levels are lower and fluctuate significantly. these fluctuations do not appear to mirror changes in gonadotropins and may complicate clinical interpretation of amh. background: menopausal hormone therapy (ht) is a confusing topic for many clinicians and patients. objective: to assess comprehension of basic clinical trial features among prospective participants for the keeps trial, designed to study the effects of ht initiated within 3 years of menopause on chd markers. methods: screening materials were provided giving an overview of study purpose, duration, medications, likelihood of receiving drug vs. placebo, ht related risks and side effects. at a subsequent interview, a 10-item questionnaire assessed the participant's level of comprehension. a score of 80% was adequate to complete the informed consent process. those scoring <80 % were re-counseled and retested. demographic variables determining the likelihood of an initial score >80% were evaluated by univariate and multivariable analyses. results: 32% (24/76) scored >80% on initial testing. all women scored >80% after re-counseling and retesting. likelihood of an initial response >80% correct was unrelated to age or time since menopause. ability to correctly respond was influenced by highest educational level attained. none of 8 women whose furthest educational level was high school scored >80% on initial testing, significantly less than those with a college education (p=0.04). a higher proportion of college graduates (13/32) scored >80% compared to those attaining further education (11/35) (p=0.43). comprehension was greatest for study purpose and duration (64/76 and 70/76 correct responses respectively) and least for questions related to results of the whi hormone trial breast cancer and chd. that e alone was not associated with an increased risk of chd (41%) or breast cancer (50%) was poorly understood. conclusion: comprehension of the risks and benefits of ht by potential research subjects is low despite provision of reading materials prior to the informed consent process. (supported by the montefiore medical center/albert einstein college of medicine site for the kronos longevity research institute and k24-hd41978 to ns). variation in menopausal symptoms within a sample of hispanic women: swan, the study of women's health across the nation. robin r green, 1 alex j polotsky, 1 aileen p mcginn, 1 carol a derby, 1 rachel p wildman, 1 lhasa ray, 1 kavitha t ram, 1 gerson weiss, 2 nanette f santoro. 1 1 albert einstein coll of med, bronx, ny; 2 univ of med and dent of new jersey, newark, nj. background: menopausal symptoms are experienced by over 75% of women. purpose: to describe symptom frequency in a sample of midlife hispanic women from different countries of origin. methods: the study of women's health across the nation (swan) recruited 277 women at baseline who self-identified as hispanic. their baseline responses to validated questionnaires regarding common menopausal symptomatology were examined. symptoms were reported over the previous two weeks and scored on a frequency scale ranging from 1 (not at all) to 5 (every day). for all analyses, symptoms were dichotomized into "absent" vs. "present" variables. responses were correlated with acculturation (4-item scale: marin, hisp j behav sci 2:183, 1987) and analyzed by sub-ethnicities: central/south american (c/s am), puerto rican (pr), dominican (dr), and cuban (cu). associations between symptoms and sub-ethnicity were tested by chi-square. logistic regression was used to test for associations with acculturation and being us-born. there was significant variation in several menopausal symptoms. while puerto-rican women had the highest likelihood of reporting trouble sleeping (or=2.2, 95%ci: 1.1-4.3) and headaches (or=2.7, 95%ci:1.4-5.3), dominican women were most likely to report vaginal dryness (or=2.8, 95%ci: 1.1-7.1) acculturation and being us-born did not explain the variation between subethnicities in any of the models tested conclusion: there appear to be significant differences among hispanic women with respect to menopausal symptomatology. these differences were not readily explained by measures of acculturation. (supported by the study of women's health across the nation (swan) has grant support national institutes of health, dhhs, through the national institute on aging, from the national institute of nursing research and the nih office of research on women's health (grants nr004061; ag012505, ag012535, ag012531, ag012539, ag012546, ag012553, ag012554, ag012495 and cd41978). purpose: to compare the relative effects of conjugated equine estrogen, raloxifene, and tamoxifen therapies on cognition among women aged 65 years or older. participants and methods: annual modified mini mental state (3ms) examinations were used to assess global cognitive function among the 7,212 women enrolled in the two randomized placebo-controlled clinical trials of the women s health initiative memory study (whims) and 300 women enrolled in co-star, the cognitive substudy of the nsabp's study of tamoxifen and raloxifen (star) trial who had baseline 3ms testing. associations between baseline cognitive risk factors common to both trials and baseline 3ms scores were assessed and interactions used to examine whether risk factor relationships differed between cohorts. factors for which relationships were similar were used as covariates in analyses comparing ontrial 3ms scores. factors for which relationships did not appear to be similar were used to stratify analyses. results: compared to placebo, each of the active therapies was associated with a small mean relative deficit in 3ms scores of 0.5 units or less, which was fairly consistent between women with and without prior hysterectomy. overall, relative deficits appeared to be most marked for tamoxifen (unadjusted p=0.003) but were also evident for raloxifene (p=0.06) and cee (p=0.02). conclusions: while unmeasured differences between trials may have confounded our analysis, these findings suggest that both tamoxifen and raloxifene may adversely affect cognitive function in older women. weight gain and increased abdominal fat have been found in women after menopause and is associated with higher levels of leptin, and decreased levels of the cardioprotective adipocytokine adiponectin. at the same time, bmd characteristically decreases. in an effort to determine the evolution and correlates of these changes, we studied 25 postmenopausal women (pm) within 4 years of menopause (age 58.4±1 yrs) of normal weight (bmi 26.3±1) and compared them to 20 weight matched (bmi 27.4±1) premenopausal (pre) controls (age 26.4±1yrs.) all subjects had bmd and body composition studies by dexa and measurements of leptin, adiponectin, visfatin and retinol-binding protein 4 (rbp4.) while total fat was similar in the 2 groups, pm had more trunk and abdominal fat (10516±800; 818±67g) compared to pre (8626±493g p<0.05; 577±33g p<0.01) pm also had greater %trunk fat and %central abdominal fat compared to pre, p<0.01.serum leptin (31.8±0.5 vs 28±5 pg/ml) and visfatin ( 10.4±1 vs. 9.4±0.5 ng/ml) were similar but adiponectin (17±1 vs. 13.3±1 g/ml) and rbp4 (28.7±2 vs. 22 .3±1 ng/ml) were higher, p<0.05 in pm. while in pre, abdominal fat correlated negatively with adiponectin (p<0.01) in pm only leptin correlated with various parameters of fat mass, p<0.02, and adiponectin did not correlate but correlated positively with age (r 0.47, p<0.05.) as expected, pm had reduced bmd at the lumbar spine and hip (0.84±0.025 vs. 0.99±0.025 g/cm2; 0.72±0.025 vs. 0.87±0.025 g/cm2, p<0.01) but there was a correlation between total and trunk fat in pm and lumbar bmd (r 0.69, 0.66, p<0.01) but not with hip bmd; or any correlations in pre. there was a correlation between leptin and lumbar bmd in pm (r 0.47, p<0.02) but not in pre. in summary, in normal weight early pm, abdominal fat is increased, but only adiponectin and rbp4 are altered with an increase in the former correlating with age. lumbar bmd correlated with fat mass in pm and is partially explained by increases in leptin. it is suggested that in spite of increasing abdominal fat in normal weight early pm, (which correlates with a higher lumbar bmd) david d rahn, jesus f acevedo, r ann word. ob-gyn, ut southwestern, dallas, tx, usa. objectives: matrix metalloprotease (mmp) activity is increased in the postpartum vagina of wild type (wt) animals, and this activity is accompanied by a burst in elastic fiber synthesis. the mechanisms that precipitate these changes are unclear. the goals of this study were to determine how vaginal distention (such as in parturition) affects elastic fiber homeostasis in the vaginal wall and the potential significance of these changes in the pathogenesis of pelvic organ prolapse. methods: nonpregnant (18) and 20 pregnant (d14) wt mice underwent either vaginal distention with a 750μl balloon x 40 min (to simulate parturition) or sham procedure. tissues were obtained at 24 and 48 h for immunoblot analysis, zymography, and histology. distention was also performed in 30 young fbln5 -/-, fbln5 +/-, and wts, and prolapse was quantified for 8 weeks. results: distention resulted in marked increases in mmp activity in nonpregnant animals (prommp9, 5.6-fold; active mmp9, 3.5-fold; prommp2, 2.4-fold; active mmp2, 3.7-fold; all p < 0.05 compared with sham) which was accompanied by fragmented and disrupted elastic fibers in the vaginal wall. abundant amounts of tropoelastin and fibulin-5 in the nonpregnant vagina were not increased further by distention. in pregnant animals (which normally have suppressed vaginal wall fibulin-5 and tropoelastin), however, distention resulted in 3-fold upregulation of both proteins (p<0.05). distention also induced increased mmps in pregnant animals (mmp-9, 1.7-fold; prommp-2, 1.7-fold; active mmp-2, 2.3-fold; all p < 0.05). thirteen young fbln5 -/-(4-6 wks prior to age of prolapse development), 11 het siblings, and 6 age-matched wts underwent serial exams after ballooning. distention induced rapid increases in perineal bulge and vaginal diameter (within 3 d) in fbln5 -/mice which never recovered. conclusions: in pregnant mice, vaginal distention results in increased protease activity in the vaginal wall but also increased synthesis of proteins important for elastic fiber assembly. distention may thereby contribute to the burst of elastic fiber synthesis in the postpartum vagina. the finding that distention results in accelerated pelvic organ prolapse in fbln5 -/animals, but not wt, indicates that distention results in loss of pelvic organ support if elastic fiber synthesis is compromised. further, the data suggest that elastic fiber synthesis is crucial for recovery of the vaginal wall from parturition/distention-induced increases in vaginal protease activity. the molecular etiology of pelvic organ prolapse (pop) is complex and not yet well understood. defects in the connective tissue, such as a decrease in extracellular matrix (ecm) protein content may predispose women to pop. our objective was to study the expression and the enzymatic activity of matrix metalloproteinases (mmps) and their tissue inhibitor (timps) in vaginal tissue of patients with advanced pop and controls. after informed consent, pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q), and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer. western immunoblot analysis was performed (patients: n=7, controls: n=6) to study the protein expression of mmp-1, -12, timp-1, -2, -4. gelatin zymography was used to quantify the activity of mmp-2 and -9. immunohistochemical analysis for timp-2 and -4 was performed on pfa-fixed vaginal biopsy tissue (n=3) in each group. both patient and control vaginal biopsy samples expressed latent and active forms of mmp-12, and mmp-1. the protein expression of the 45 kda active form of mmp-12 was significantly increased in patients with pop compared to controls (p=0.03). zymography detected the enzymatic activity of the proform and active form of both mmp-2 and mmp-9. we found a significant increase in gelatinolytic activity of both 68 kda pro-form and 62 kda active forms of mmp-2 (p=0.01 and p<0.001 respectively) as well as 87 kda active form of mmp-9 (p<0.001) in patients with pop compared to controls. timp-1 protein expression in vaginal tissue showed a significant reduction in pop patients compared to controls (p=0.002). timp-2 and -4 immunostaining were mainly localized in the smooth muscle cells at the muscularis layer of the vaginal biopsies. in vaginal tissue of patients with pop, we have shown a decrease in timp protein expression paralleled by an increase in mmp protein expression and activity. these findings reflect an active ecm remodelling that may compromise the structural integrity of the pelvic floor leading to pop. aberrant elastin and collagen synthesis may play a role in the pathogenesis of pelvic organ prolapse (pop). the lysyl oxidase (lox) family of enzymes direct cross linking of collagen and elastin polymers, however to-date no information is available on the expression and localization of these proteins in human vaginal tissue. our objectives were to study the expression and in situ localization of lox, loxl1, loxl3 and loxl4 proteins in the vaginal tissue of patients with advanced pop and asymptomatic controls. pre-menopausal caucasian patients affected by pop ( grade 3 by pop-q) and control patients (no pop) matched for age and bmi, undergoing vaginal and abdominal hysterectomy respectively were recruited. full thickness anterior vaginal epithelial tissue was obtained from the surgical cuff of patients and controls in the proliferative phase of the menstrual cycle. total protein was extracted using ripa lyses buffer and western immunoblot analysis was performed (patients: n=7, controls: n=6). pfa-fixed vaginal biopsy tissues (n=3 for each group) were used for immunohistochemical study. vaginal biopsy samples from both patient and control groups expressed all four members of lox family proteins: lox (47 kda pro-form and 35 kda active form), loxl1 (47 kda pro-form and 35 kda active form), loxl3 (83 kda) and loxl4 (84 kda). the expression of all lox family proteins was reduced in patients with pop compared to controls; however only the pro-form of lox of making the diagnosis of endometriosis from an endometrial biopsy. objective: to evaluate the diagnostic value of examining endometrial biopsy specimens for small nerve fibers in women with pelvic pain or infertility in a double-blinded prospective comparison with laparoscopy. methodology: we undertook to compare the detection of endometrial nerve fibers with laparoscopy and peritoneal biopsy for the diagnosis of endometriosis in 38 women (aged 34.4 years; range 22-45 years) who presented with chronic pelvic pain or infertility. small nerve fibers were detected in the functional layer of endometrium using immuno-histochemical staining with the highly specific pan-neuronal marker pgp9.5, using a carefully validated technique and blinded assessment of nerve fiber density. laparoscopic assessment of the presence of endometriosis and peritoneal biopsies was undertaken by three experienced gynecologic endoscopic surgeons. data from these assessments were recorded independently of endometrial findings and maintained under blinded coding until the codes were broken. results: small nerve fibers were detected in all 23 of the women in whom endometriosis was surgically diagnosed and in none of the 15 women in whom endometriosis was excluded at laparoscopy, giving the specificity and sensitivity of 100%. the density of nerve fibers in the endometriosis cases was 3.4 per mm²± 4.3 (mean ± sd). conclusions: endometrial biopsy provided a reliability of detection or exclusion of endometriosis which was equal to that of diagnostic laparoscopy carried out by experienced gynecologic laparoscopists. background: erk1/2 are mapk intracellular signaling proteins involved in cell survival and differentiation. endometriosis requires angiogenesis for ectopic implant growth. we hypothesized that the endometriotic peritoneal environment, known to be high in estrogen (e2), vegf, and cytokines such as il-8 and mcp-1, stimulates angiogenesis in human endometrial endothelial cells (heec) through erk signaling. methods: serial sections from normal (n=24) and ectopic (n=25) endometrium were immunostained for total-(t-) and phospho-(p-) erk1/2 and cd34 (an endothelial progenitor cell marker); results were quantified by computer densitometry and grouped by menstrual phase. cultured normal heec were treated with control, e2 (10-8m), il-8, mcp-1, and vegf (all 2 ng/ml) for 5 15 min, and western blotted for p-/t-erk (n=3). heec were treated with peritoneal fluid (pf; diluted 1:3 in basal media) from normal (n=4) and endometriotic (n=4) women, with or without pd98059 erk1/2 inhibitor (20 m) for 48 h, and cell viability was analyzed by mtt assay. statistical analysis was done with one-way anova. results: tissue staining revealed that ectopic cd34+ endothelial progenitor cells undergoing angiogenesis (vessel sprouting and/or angiogenic cell cord formation) exhibited the strongest levels of p-erk. heec of ectopic foci showed moderate-high p-erk staining, while surrounding mesothelial capillaries were weak for p-erk. in normal endometrium, p-erk was cycledependent, with low levels in the late secretory phase vs. other phases (p<0.05). p-erk of ectopic heec showed no cycle dependence, with moderate-high levels in all phases. t-erk in all tissues was high and invariable. in cultured heec, treatment with pf from endometriotic women significantly increased heec viability after 48 h compared to normal pf (p<0.05). this effect was abrogated by erk1/2 inhibitor. among factors known to be high in endometriotic pf, vegf increased p-erk in cultured heec in 5 and 15 min (p<0.05), while e2, il-8, and mcp-1 had no effect. conclusions: high p-erk found specifically in sprouting endothelial progenitor cells and focal ectopic capillaries suggests that erk1/2 is involved in endometriotic angiogenesis. the peritoneal microenvironment of endometriosis may be persistently stimulating erk-mediated endometrial angiogenesis through vegf. further investigation into erk1/2 inhibitors may offer a novel treatment of endometriosis. the events leading to the development of post operative adhesions are unknown, though recent observations emphasize the crucial role played by the superoxide ion generated by hypoxia. exposure of normal peritoneal fibroblasts to hypoxia caused the development of the adhesion phenotype, which is characterized by increased extracellular matrix molecules and inflammatory cytokines as well as high protein nitration and low nitric oxide. superoxide dismutases (sod) are a family of metalloenzymes that eliminates superoxide by converting it to hydrogen peroxide, protecting mammalian organisms against superoxide. objective: to determine whether sod is differentially expressed between normal peritoneal and adhesion fibroblasts and whether its expression is modulated by hypoxia. methods: fibroblasts from normal peritoneal and adhesion tissues were cultured under normal (20% o 2 ) and hypoxia (2% o 2 ) conditions for 24 and 48 hours. real time rt/pcr was utilized to measure the absolute mrna levels for sod in both cell lines before and after hypoxia exposure. results: normal peritoneal fibroblasts exhibited significantly higher basal mrna levels for sod (49.7 pg/ grna, p<0.05) as compared to adhesion fibroblasts (38.1 pg/ grna, p<0.05). short exposure to hypoxia resulted in a significant increase in sod mrna levels to 63.3 and 52 pg/ grna in normal and adhesion fibroblasts respectively, p<0.05. in contrast, long exposure to hypoxia resulted in a significant decrease in sod mrna levels to 41.5 and 25.7 pg/ grna in normal peritoneal and adhesion fibroblasts respectively, p<0.05. conclusion: sod mrna levels are lower in adhesion as compared to normal fibroblasts, both basally and following short and longer exposure to hypoxia. reduced sod expression creates an milieu with greater free radical levels, which leads to the development of the adhesion phenotype. enhancement of sod levels and/or function are likely to be of benefit for reduction of postoperative adhesion development. objectives: development of endometriosis requires ectopic attachment and proliferation of endometrial tissue. the invasive process required to establish endometriosis may involve matrix metalloproteinases (mmps), including mmp-3. recently, we have demonstrated that statins inhibit proliferation of endometrial stroma. this study evaluated the effects of simvastatin on a nude mouse model of endometriosis and on modulation of mmp-3. methods: proliferative phase human endometrial biopsies were established as organ cultures or utilized for stromal cell isolation. to establish endometriosis in the nude mouse, endometrial tissues were maintained in 1 nm estradiol (e) for 24 hrs and subsequently injected intraperitoneally into ovariectomized nude mice. mice were treated with e (8 μg, silastic capsule implants) and simvastatin (5-25 mg/kg/day) by gavage for 10 days beginning 1 day after tissue injection. control mice received e+vehicle. subsequently, animals were sacrificed and endometrial implants were evaluated. studies of endometrial stroma involved plating the cells in the presence of e (1 nm) or e+medroxyprogesterone acetate (mpa; 50 pm) with and without simvastatin (1-10 μm). some cultures additionally received interleukin-1 (il-1 , 200 ng/ml). conditioned media was subjected to western analysis for expression of mmp-3. results: in vivo studies demonstrated a dose-dependent inhibitory effect of simvastatin on number and volume of endometrial implants in mice. at the highest dose of simvastatin (25 mg/kg/day), the number of endometrial implants was decreased by 83% and the volume by 98% in comparison to the control group. isolated stromal cells expressed abundant levels of mmp-3 following treatment with e, but minimal levels in cultures additionally receiving mpa or simvastatin. although il-1 induced a dramatic increase in mmp-3 secretion from cells pretreated with e alone, treatment with either mpa or simvastatin prevented this induction. cultures receiving e+mpa+simvastatin were the most resistant to mmp-3 induction by il-1 . objective: to increase awareness of potential presentations of adenomyosis and the differential of a uterine mass. material and method: in a tertiary medical center, a patient was being evaluated for a uterine cystic mass and cyclic dysmenorrhea. the patient is a 26 year old nulliparous woman who has been complaining of cyclic dysmenorrhea for several years. the pain starts with the onset of menses and lasts around 2 weeks. the patient did not improve on contraceptives. the patient had prior laparoscopy and imaging studies which misdiagnosed the mass as either a leiomyoma or an adnexal hemorrhagic cyst. the patient underwent exploratory laparotomy and resection of the mass. results: on ultrasound, the mass appeared as an echodense cyst within the uterus. intraoperatively, a 3 cm thick-walled well circumscribed uterine chocolate cyst was identified. the resection of the cyst was performed in similarly to an ovarian cystectomy. tissue examination confirmed the diagnosis of cystic adenomyosis. following surgical intervention, the patient reported significant improvement of symptoms. conclusion: this case highlights an unusual presentation and treatment of adenomyosis and cyclic dysmenorrhea. cystic adenomyosis should be on the differential of uterine cystic mass, particularly in young women with cyclic dysmenorrhea and menorrhagia. the earlier misdiagnosis probably resulted from the unfamiliarity of physicians with this condition. similar clinical presentations may occur with congenital uterine anomalies (with an obstructed uterine segment) and cystic degeneration of a leiomyoma. the incidence and pathogenesis of cystic adenomyosis are unknown. oral contraceptives may be helpful as a first line therapy. however, resection of the adenomyotic cyst appears to be more effective, particularly in women seeking fertility. objective: it is hypothesized that abnormalities within the eutopic endometrium of females with endometriosis can cause the ectopic growth of the endometrial tissue at extrauterine sites. previous studies have shown that gene expression in eutopic endometrium of women with endometriosis is markedly different from disease-free females. inflammatory cytokines and receptor-dependent kinase pathways are widely recognized as therapeutic targets for immune disorders, which is believed to be the underlying pathogenesis of endometriosis. in this study we asked whether responses of primary eutopic endometrial cell cultures are dysregulated between females with or without endometriosis. methods: a total of 9 biopsies of endometriotic eutopic endometrium (eee) and disease-free endometrium (dfe) were obtained from proliferative phase females. the primary cell cultures established from these biopsies were treated with tnfa to induce expression of inflammatory mediators. parallel cultures were also treated with kinase inhibitors of p38, mek, pi3k and ikk. after a period of 24 or 48 hours, concentrations of il-6, mcp-1, and gm-csf in cell culture supernatants were analyzed by elisa. results: in eee, basal concentrations of mcp-1 and gm-csf were 2-3 times higher, while il-6 was 10 times higher compared to dfe. as expected, tnfa treatment stimulated higher levels of cytokine secretion in dfe mimicking disease state, however, the same treatment had almost no effect on eee. kinase inhibitors were very effective in lowering the cytokine levels of dfe, however, their effect on eee were less dramatic. conclusion: eee expresses higher levels of inflammatory cytokines under basal conditions, which is in concert with the current literature. our results validate that high il-6 levels in endometrium are diagnostic for females with disease. the increase of gm-csf, il-6 and mcp-1 following tnfa treatment was expected in dfe however; tnfa's effect was blunted in eee, which implies that eee are already highly activated. the effect of kinase inhibitors on cytokine production from eee was unaltered relative to dfe, which implies that tnfa stimulated kinase pathways are modified even in eee. endometrial cancer is the fourth leading cause of cancer in women, and hyperplasia and adenocarcinoma are more commonly seen in women with polycystic ovary syndrome (pcos). mig-6, a negative inhibitor of egf signaling, is expressed in normal secretory phase endometrium and associated with hyperplasia in mig-6 knockout mice. objective: we examined and compared endometrium from normal and pcos women for mig-6 expression and characterized its regulation using in vivo and in vitro models. methods: immunohistochemistry (ihc) and real-time pcr were performed in endometrium from normal (n=20) women and pcos (n=15) women. regulation of mig-6 was studied in ishikawa and ecc-1 endometrial cell lines, treated with sex steroids or egf. endometrial xenografts from normal and pcos women were implanted in ovxd rag2-(c) immunocompromised mice, treated with e 2 or e 2 + p pellets to mimic a natural cycle. results: mig-6 protein expression was low in the functionalis of the proliferative phase and high in the secretory phase; this pattern was reversed in the basalis layer. pcos secretory phase endometrium had significantly less mig-6 protein and mrna than normal endometrium (p < 0.01). xenografts using pcos samples had paradoxically elevated expression of mig-6 compared to normal, and appeared unresponsive to steroid treatments. mig-6 expression in endometrial cell lines was regulated by egf but not by ovarian steroids, e 2 or e 2 + p. conclusions: mig-6 expression is low in proliferating endometrium and regulated by egf. risk of hyperplasia or cancer in pcos women can be explained by altered expression of mig-6. reduced expression mig-6 provides insight into endometrial function and may lead to better treatment options for disorders of endometrial proliferation including endometriosis, adenomyosis, endometrial hyperplasia and cancer. supported in part by nih-u54-hd35041(sly), u54hd055764 (lcg) and u54hd77495 (fjd). could androgens influence human luteal cells function? anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. in pcos patients reproductive dysfunctions are frequently observed even if ovulation occurs. an impaired luteal function could partially explain this subfertility, since luteal steroidogenesis deficiency and premature luteal degeneration have been described in pcos patients. based on the frequent observation of high plasmatic levels of androgens in pcos, we investigated whether hyperandrogenism could negatively affect luteal function.to this aim, we tested the in vitro effects of androgens on progesterone (p) and on vascular endothelial growth factor (vegf) production by human luteal cells. indeed, vegf is essential for normal luteal development and function being an important regulator of angiogenesis and vascular permeability. since prostaglandins (pgs) play a central role in modulating luteal function, the influence of androgens on pge2 and pgf2a secretion was also investigated. in order to investigate whether testosterone and androstenedione act directly or after local aromatisation, we tested the in vitro effects of estriol, estrone and 17--estradiol on p, vegf, pge 2 and pgf 2 secretion by human luteal cells. moreover, we tested the effects of testosterone and androstenedione in presence of exemestane -an aromatase inactivator. highly purified human luteal cells were cultured for 24 h with medium alone (control) or in presence of increasing concentrations of testosterone or dihydrotestosterone or androstenedione (from 10 -10 to 10 -6 m) or in presence of increasing concentrations of estriol, estrone and 17--estradiol (from 0.1 to 10 ng/ml). moreover, testosterone and androstenedione 10 -7 m were tested in presence of exemestane (from 4 to 400 nm). in the culture medium vegf, p, pge 2 and pgf 2 secretion was assayed by commercially available elisa kits. in order to evaluate the influence of androgens and estrogens on vegf mrna expression on luteal cells real-time rt-pcr has been performed. our results demonstrated that testosterone, androstenedione and dihydrotestosterone were all able to significantly reduce vegf secretion in human luteal cells, while no effect was seen on vegf mrna expression. androgens were also able to significantly decrease p and pgf 2 secretion, while an increase was observed on pge 2 production. moreover our preliminary results demonstrated that in human isolated luteal cells estriol, estrone and -estradiol at all tested doses are able to mimic androgens effects on p and pge 2 production. on the contrary estrogens were able to increased vegf release. estrogens seemed to have no effect on pgf 2  released. data regarding exemestane inhibition of testosterone and androstenedione are still in progress. increased levels of androgens were able to decrease luteal vegf, p and pgf 2 release and might be involved in pcos-luteal phase defect. nevertheless, the observed effects could probably be attributed -at least in part -to estrogens locally produced via the aromatase enzyme, rather than be directly mediated by testosterone and androstenedione. the in vitro effect of proghrelin-derived peptides on purified human luteal cells. anna tropea, 1 mariateresa orlando, 1 maria francesca gangale, 1 federica romani, 1 isamaria loiudice, 1 federica tiberi, 2 stefania catino, 3 antonio lanzone, 3 rosanna apa. 1 1 cattedrà di fisiopatologia della riproduzione, università cattolica del s. cuore (ucsc), roma, italy; 2 istituto scientifico internazionale (isi) "paolo vi", ucsc, roma, italy; 3 istituto di ricerca "associazione oasi maria ss onlus", troina (en), italy. ghrelin, well known mediator of neuroendocrine effects, has been demonstrated to have a role as signal for energy insufficiency. several evidences suggested that ghrelin might also operate as regulator of reproductive function. indeed, we recently demonstrated that both p and vegf released were significantly decreased by ghrelin in isolated human luteal cells. moreover ghrelin was also able to reduce prostaglandin (pg) e 2 and increase pgf 2 luteal cells release. in the present work we investigated whether two ghrelin-related peptides derived by the same ghrelin precursor (unacylated ghrelin and obestatin) might affect human isolated luteal cells function. conventionally regarded as an inert form of ghrelin, unacylated ghrelin has been recently proven as biologically active in specific cellular contexts. obestatin has been suggested to directly control porcine ovarian cell functions. in these assumptions, we investigated whether unacylated ghrelin and obestatin could directly affect luteal progesterone (p) release. moreover, since vascular endothelial growth factor (vegf) is known to play a critical role in luteal development and function, the influence of unacylated ghrelin and obestatin on luteal vegf, pgf 2 and pge 2 production was also analysed. highly purified human luteal cells were incubated for 24h with medium alone (control) or with hcg (100 ng/ml) or with cocl 2 (10 μm) or in presence of increasing concentrations of unacylated ghrelin (1-100 nm) and obestatin (1-500 nm). in the culture medium vegf, pgf 2 , pge 2 and p secretion was assayed by commercially available elisa kits. moreover real-time rt-pcr has been performed in order to evaluate whether unacylated ghrelin and obestatin could affect vegf mrna in human luteal cells. our preliminary results demonstrated that either unacylated ghrelin or obestatin are able to negatively affect luteal steroidogenesis. moreover, both peptides seemed to increase the release of two luteotropic factors -vegf and pge 2and to reduce pgf 2 release -a luteolytic prostanoid -from isolated human luteal cells. finally data regarding the expression of vegf mrna are still in progress. our results suggest that unacylated ghrelin and obestatin -two ghrelin-related peptides -could play a role in regulating luteal function affecting both luteal steroidogenesis and luteotropic/luteolytic imbalance. the mechanisms responsible for labor progression have yet to be fully elucidated. in a previous study, over-expression of small conductance calcium-activated k + channel isoform 3 (sk3) in transgenic mice compromised parturition, suggesting a role for sk3 channels in this process. based on these findings, we hypothesized that sk3 channel expression is reduced late in pregnancy to enable the uterus to produce the forceful contractions required for parturition. we investigated the effects of sk3 channel expression on gestation and parturition by comparing transgenic mice over-expressing mice sk3 (sk3 t/t ) mice to wild-type (wt). in wt mice, sk3 transcript and protein are significantly reduced during pregnancy. the force produced by uterine strips from sk3 t/t mice on the day of delivery was significantly less than wt or sk3 knockout control (sk3 dox ), and the contractile force reached wt levels by application of the sk3 channel inhibitor, apamin. moreover, lipopolysaccharide and ru486, which induce pre-term labor in wt mice, failed to result in completion of delivery in sk3 t/t mice. thus, stimuli that initiate parturition under normal circumstances are insufficient to coordinate the uterine contractions needed for the completion of delivery when sk3 channel activity is in excess. the mechanism(s) down-regulating this channel in the uterus during pregnancy is unknown. the sk3 gene promoter region contains two specificity protein (sp) binding sites; 1) sp1, is a transcription factor known to enhance the transcription of genes in response to estrogen, and 2) sp3, which competes for the same binding motif as sp1, reduces gene expression. sp1 protein expression in mice uteri dramatically decreases in late pregnancy, while sp3 protein level remains consistent. our data indicate that sk3 channels must be downregulated for the gravid uterus to generate labor contractions sufficient for delivery in both term and preterm mice. the changes in sk3 channel expression may be transcriptionally regulated by sp1 and sp3. key: cord-348855-lnltoj1n authors: iannaccone, giulia; scacciavillani, roberto; del buono, marco giuseppe; camilli, massimiliano; ronco, claudio; lavie, carl j.; abbate, antonio; crea, filippo; massetti, massimo; aspromonte, nadia title: weathering the cytokine storm in covid-19: therapeutic implications date: 2020-06-29 journal: cardiorenal med doi: 10.1159/000509483 sha: doc_id: 348855 cord_uid: lnltoj1n background: severe acute respiratory syndrome coronavirus 2 (sars-cov-2) recently emerged in wuhan, hubei-china, as responsible for the coronavirus disease 2019 (covid-19) and then spread rapidly worldwide. while most individuals remain asymptomatic or develop only mild symptoms, approximately 5% develop severe forms of covid-19 characterized by acute respiratory distress syndrome (ards) and multiple-organ failure (mof) that usually require intensive-care support and often yield a poor prognosis. summary: the pathophysiology of covid-19 is far from being completely understood, and the lack of effective treatments leads to a sense of urgency to develop new therapeutic strategies based on pathophysiological assumptions. the exaggerated cytokine release in response to viral infection, a condition known as cytokine release syndrome (crs) or cytokine storm, is emerging as the mechanism leading to ards and mof in covid-19, thus endorsing the hypothesis that properly timed anti-inflammatory therapeutic strategies could improve patients' clinical outcomes and prognosis. key messages: the objective of this article is to explore and comment on the potential role of the promising immunomodulatory therapies using pharmacological and nonpharmacological approaches to overcome the dysregulated proinflammatory response in covid-19. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) recently emerged in wuhan, hubei-china, as responsible for the coronavirus disease 2019 (covid-19) and then spread rapidly worldwide. it was declared a pandemic by the world health organization (who) on march 11, 2020 [1, 2] . while most individuals remain asymptomatic or develop only mild symptoms, up to 15-20% require hospitalization and less than 5% develop a critical illness characterized by acute respiratory distress syndrome (ards) and multiple-organ failure (mof) that usually need intensive-care support and often yield a poor prognosis [3] . in most cases, patients presenting to the emergency room have not undergone prehospital ambulatory testing or, as a consequence, any ongoing treatment intended to reduce the severity of disease [4, 5] . the pathophysiology of covid-19 is far from being completely understood, and the lack of effective treatments has led to a sense of urgency to develop new therapeutic strategies based on pathophysiological assumptions. the sars-cov2 spike protein initiates cellular infection by binding angiotensin-converting enzyme (ace)-2 on human cells [6] . cellular infection and viral replication cause activation of the inflammasome in the host cell, leading to the release of proinflammatory cytokines and cell death by pyroptosis with ensuing release of a damage-associated molecular pattern, further amplifying the inflammatory response [7] [8] [9] . the exaggerated cytokine release in response to viral infection, a condition known as cytokine release syndrome (crs) (fig. 1 ) or cytokine storm, is emerging as one of the mechanisms leading to ards and mof in covid-19 [7] . in line with this, recent studies have shown that patients with covid19 have high levels of inflammatory cytokines, such as interleukin (il)-1β, il-2, il-6 il-7, il-8, il-9, il-10, il-18, tumor necrosis factor (tnf)-α, granulocyte colony-stimulating factor (g-csf), granulocyte-macrophage colony-stimulating factor, fibroblast growth factor, macrophage inflammatory protein 1, compared to healthy individuals [10] ; circulating levels of il-6, il-10, and tnf-α also correlated with illness severity as they were significantly higher in intensive care unit (icu) patients compared to mild/moderate cases. in particular, il-6 may suppress normal t-cell activation [11] , and tnf-α can promote t-cell apoptosis via interacting with its receptor tnf receptor 1 [12] , and their upregulation may in part contribute to lymphocytopenia, a feature often encountered in covid-19, with a more pronounced decline in severe cases [13] . as such, a recent study found that, in icu patients due to covid-19, tnf-α and il-6 concentrations negatively correlated with total t-cell, cd4+, and cd8+ counts [14] . furthermore, ace-2 consumption by viral entry interrupts angiotensin ii (angii) metabolism, resulting in an initial increase in local angii concentrations that may enhance proinflammatory cytokine release and foster diffuse microvascular dysfunction and a prothrombotic milieu [15, 16] . antiviral treatment may play a role in the management of covid-19 but, especially in more severe forms, immunomodulatory treatments blunting cytokine release may turn out to be beneficial if appropriately timed. the objective of this article is to explore and comment on the potential role of the promising immunomodulatory therapies using pharmacological and nonpharmacological approaches to overcome the dysregulated proinflammatory response in covid-19 (fig. 1) . despite being extensively used empirically in the treatment of severe forms of ards, the role of systemic corticosteroids (cs) remains controversial [17] . cs are the cornerstone of treatments for cytokine storms and macrophage activation syndrome in autoimmune/autoinflammatory diseases [18] ; in the covid-19 scenario they may be useful in the more severe forms of crs to curb the systemic inflammatory response and prevent the occurrence of ards, if appropriately timed [10, 19] , 20]. on the other hand, the early use of cs seems to be outweighed by their adverse effects, such as impaired virus clearance and an increased rate of secondary bacterial or fungal infections [21] . in fact, a recent systematic review and metaanalysis in adult patients with influenza pneumonia demonstrated that cs increase not only mortality but also the rate of secondary infections and prolong the icu length of stay [22] . for these reasons, the who does not recommend routine administration of cs in covid-19 patients outside of clinical trials; their adjunctive use may be indicated on an individual basis or unless indicated for other conditions, such as chronic obstructive pulmonary disease, asthma, and septic shock [23] . fig. 1 . cytokine storm consequent to sars-cov2 infection is emerging as the main mechanism leading to ards and mof in covid-19. identification of patients with a hyperinflammatory response through cytokine profiling could direct the choice of a specific anticytokine drug or even combined/sequential regimens; in selected cases, implementation of broad-spectrum anti-inflammatory therapies, such as ivig and blood purification, could be considered. aak1, adaptor-associated protein kinase 1; ccr5, c-c chemokine receptor type 5; t cd, t-cell cluster of differentiation; fgf, fibroblast growth factor; gm-csf, granulocyte-macrophage colony-stimulating factor; g-csf, granulocyte colony-stimulating factor; mip1, macrophage inflammatory protein 1. the antimalaria drugs chloroquine (cq) and hydroxychloroquine (hcq) have been proven to interfere with the viral entry process through alteration of ph-dependent endosome-mediated viral endocytosis and could inhibit sars-cov entry through change of the glycosylation of the ace2 receptor and spike protein. hcq is less toxic than cq and can be used for long periods of time [24] . moreover, both cq and hcq have been proven to have immunomodulatory activities by interfering with toll-like receptor signaling pathways, reducing cytokine production, and inhibiting mhc class ii expression, antigen presentation, and immune activation through the reduction of cd154 expression in t cells [25] [26] [27] . the ability of cq/hcq to inhibit certain coronaviruses, such as sars-cov-1, has led to inclusion of the use of cq/hcq for the treatment of covid-19. high-quality studies are, however, urgently needed to provide information on its effectiveness, address the optimal dose and duration of treatment, and explore its side effects and long-term outcomes. since the earliest phases of the pandemic, cq and hcq have been empirically used alone or in combination with macrolides for the treatment of covid-19 with the intent of reducing hospitalizations and deaths. although several multicenter randomized controlled trials are underway, a recently published large observational multinational registry showed a decreased in-hospital survival and an increased frequency of ventricular arrhythmias in patients with covid-19 requiring hospitalization treated with cq/hcq alone or in combination with a macrolide. however, these data do not apply to the use of cq/hcq in the ambulatory and outpatient settings and, due to the observational study design, the possibility of unmeasured confounding biases cannot be excluded. on the basis of this study, at present the use of cq and hcq is not recommended in hospitalized patients other than in the context of randomized trials. il-1β, il-6, and tnf-α levels are upregulated in patients with covid-19, especially in severe forms, and may play a central role in determining diffuse tissue damage associated with crs [28] , thus leading to the assumption that drugs inhibiting these molecules could be beneficial in reducing this exaggerated inflammatory response. anakinra, an il-1 receptor antagonist that blocks the biologic activity of il-1, is a promising agent in this setting since it has already been shown to be effective in severe sepsis complicated by crs macrophage activation syndrome [29, 30] . recently monteagudo et al. [31] reported 5 cases of a cytokine storm (2 with an identified viral trigger) in which continuous intravenous anakinra infusion resulted in prompt improvement in laboratory and clinical parameters. there are limited data with specific il-1 blockers in patients with covid-19. cavalli et al. [32] reported on 29 patients with covid-19 and ards managed with noninvasive ventilation outside of the icu treated with high-dose anakinra; the treatment was shown to be safe and it was associated with clinical improvement in 72% of the patients [32] . in a recently published cohort study by huet et al. [33] , including 52 patients in the anakinra group who were prospectively enrolled and 44 patients in the control group who were retrospectively selected, subcutaneous anakinra (100 mg twice a day for 72 h and then 100 mg daily for 7 days) reduced both the need for invasive mechanical ventilation in the icu and mortality among patients with severe forms of covid-19, without serious side effects [33] . obviously, despite the promising results of these studies, controlled trials are required in order to obtain confirmation of efficacy. swedish orphan biovitrum, the manufacturer of anakinra, announced upcoming trials of anakinra in covid-19 in a recent press release [34] . a small retrospective analysis of 10 patients treated with canakinumab, an il-1β antibody (novartis), 10 patients with severe covid-19 pneumonia and hyperinflammation showed rapid improvement of the inflammatory response and resolution of the hypoxemia [35] , supporting the central role of il-1β and the inflammasome in covid-19. a phase 3 clinical trial with canakinumab in severe covid-19 disease is ongoing (nct04362813). anti-il-6 agents are a class of drugs with anti-inflammatory properties that act by targeting either the il6 receptor or il-6 itself. studies have shown that tocilizumab, an il-6 receptor antibody, could reverse crs in the setting of chimeric antigen receptor t-cell therapy for cancer [36] . because of the greatly elevated serum levels of il-6 in patients with covid-19 [3] , its off-label used in the early stage of the diseases suggests a favorable impact on the course of covid-19 [37] . moreover, in 2 small retrospective studies of covid-19 patients in china suffering from severe lung injury it was observed that the use of this agent could provide significant clinical improvement without important side effects [38, 39] . a press release from a network of hospitals in france announced that the corimuno-toci (nct04331808) trial of tocilizumab versus placebo in patients with severe covid-19 pneumonia not requiring mechanical ventilation met the primary endpoint of reduction in the proportion of deaths or the need for mechanical ventilation at 14 days [40] . another ongoing clinical trial (nct04327388) aims to assess the efficacy of sarilumab, another il-6 receptor inhibitor, in adult patients hospitalized with severe or critical covid-19. a press release from regeneron announced the results of the phase 2 study and the decision to proceed with enrollment in phase 3 only in patients defined as "critical," i.e., those requiring high-flow oxygen therapy or mechanical ventilation. the press release showed encouraging data for sarilumab (400 mg) versus placebo in the reduction of death or the need for ventilator on day 29, with intermediate results for the 200-mg arm, hence the decision to continue with only the 400-mg arm in phase 3 [41] . furthermore, an ongoing clinical trial (nct04380961) is evaluating the response to sirukumab, an antibody targeting il-6, administered as a single intravenous dose, compared to placebo in severe or critical covid-19 on top of standard of care. a clinical trial (nct04330638) is also testing whether combined il-1 and il-6 blockade using anakinra, tocilizumab, and siltuximab (another antibody that binds to il-6), alone or in different combinations, may produce additional clinical benefits (in terms of death, days hospitalized, icu days, etc.) compared to standard-of-care or single anticytokine therapy. tocilizumab is now already included in many practice guidelines for covid-19 management, especially for the treatment of critically ill patients with severe refractory hypoxemia in a later stage after the high-viral-load initial phase all over the world, while we wait for more definite data from multiple ongoing clinical trials [42] . the viral spike protein of sars-cov2 seems to induce a tnf-α-converting enzyme (tace)-dependent alteration of ace-2, which allows virus penetration into host cells, and the more tnf-α levels are increased the more this pathway is facilitated [43] . tnf antagonism in high cytokine states is a relatively unexplored field, with a case report suggesting a potential benefit in macrophage activation syndrome caused by herpes virus simplex infection [44] . a clinical trial evaluating the effects of the tnf-α blocker adalimumab in covid-19 infection is currently ongoing (chictr2000030089). the janus kinase (jak) family, and in particular the adaptor-associated protein kinase 1 (aak1), plays a role in viral particles endocytosis; aak1 inhibitors have been suggested as possible candidates for the treatment of covid-19. barcitinib, a jak inhibitor with a high affinity for aak1-binding, has been the first to be considered due to its relative safety [45] . however, there is the risk that jak inhibitors could affect the activity of a variety of inflammatory cytokines, including inf-α, a potent mediator of the antiviral response. at present, there are 2 clinical trials (chictr2000030170 and chictr2000029580) the results of which will hopefully provide further insights into jak and aak1 inhibition effects in covid-19 patients. c-c chemokine receptor type 5 (ccr5) is expressed on the surface of white blood cells, especially t-cd4+ cells, and mediates macrophage migration into areas of inflammation, favoring the release of inflammatory cytokines and amplification of the immune response [46] . a phase 2 trial is currently evaluating leronlimab, a humanized monoclonal antibody inhibiting ccr5 which is already under study in hiv infection [46] , to determine whether blockage of this pathway is able to reduce mortality at 28 days in severe forms of covid-19 (nct04347239). mesenchymal stem cells (msc) possess potent immunomodulatory activities [47] . several in vivo studies in animal models and ex vivo human lung models have proven that msc can prevent lung injury, reduce inflammation, regulate immune responses, and foster alveolar fluid clearance [47] . moreover, msc secrete antimicrobial and painkiller molecules [47] . the in vivo safety of in vivo local and intravenous administration of msc has been demonstrated in multiple human clinical trials, including studies of ards [47] . on the basis of these positive results, msc use has been proposed to treat lung injury due to covid-19, and a recent pilot trial by leng et al. [48] including 7 patients showed that intravenous transplantation of ace-2 msc is safe and effective for the treatment of sars-cov2-related pneumonia, especially in patients in critically severe condition. at present, there are several ongoing clinical trials testing the application of mesenchymal stromal stem cells (sc) (nct04361942 and nct04345601), adipose-tissue-derived sc (nct04366323), human dental pulp sc (nct04336254), and bone marrow-derived sc in covid-19 patients, which hopefully will provide further insight into the potential beneficial/harmful roles of msc. intravenous immunoglobulin (ivig) has already been proven to be beneficial in patients with autoimmune and chronic inflammatory diseases [49, 50] . a new perspective is the use of ivig-containing polyclonal immunoglobulin g (igg) isolated and pooled from healthy donors to provide short-and medium-term therapies to covid-19 patients [51] . ivig may exert many anti-inflammatory and immunomodulatory effects which could accelerate virus clearance and prevent entry into target cells. in a small chinese case series from jin yin-tan hospital (wuhan, china) a high dose of ivig administered within the first few days of clinical deterioration proved to be a valuable option to block covid-19 cascade progression, thus obtaining significant improvements in terms of clinical outcomes and recovery rates; the timing, however, was found to be crucial, as no beneficial effects were observed if lung injury and systemic damage had already occurred [52] . despite it being promising, ivig use in covid-19 requires evaluation in dedicated prospective and randomized clinical trials. convalescent plasma therapy (cpt) is based on the transfusion of plasma collected from patients who have recovered from sars-cov2 infection, and developed antibodies, to susceptible individuals in order to confer them immediate immunity. plasma from healthy donors provides neutralizing antibodies, limiting viral amplification and immunomodulatory effects via the infusion of anti-inflammatory cytokines and antibodies that block complement, inflammatory cytokines, and autoantibodies [53] . cpt has already been proven to be beneficial both as postexposure prophylaxis and/or treatment for different infectious diseases, including in the context of sars-1 and mers outbreaks [54] . recently, in a case series of 5 critically ill patients with covid-19-related ards, convalescent plasma transfusion with a sars-cov-2-specific antibody (igg) [52] led to improvement in patients' clinical status. moreover, a systematic review by rajendran et al. [55] including 5 studies on the use of cpt in covid-19 individuals showed that this therapy may reduce mortality in critically ill patients, increase neutralizing antibody titers, allow the disappearance of sars-cov-2 rna in a large number patients, and lead to significant symptom relief. of note, the cpt benefit is greater when it is used in a timely manner in the early viremic phase as its prevalent action is through direct neutralization of the virus, whereas the use of ivig administration may be useful even in a more tardive phase as its principal mechanism is to counteract the deleterious effects of the dysregulated immune response. on the basis of these encouraging results, there are several ongoing clinical trials testing the efficacy of cpt in covid-19 patients, and their results may provide further insights into the proper timing and modality of use of this promising therapy. a nonpharmacological option for covid-19 to counteract the dysregulated proinflammatory response could be represented by blood purification techniques [56, 57] . nevertheless, the application of these techniques in covid-19 patients is still not widespread and further investigation is needed to confirm their beneficial effects. therapeutic plasma exchange is an extracorporeal treatment performed by filtrating a volume of plasma equivalent to the estimated plasma volume that selectively removes circulating pathogenetic substances, such as auto-reactive antibodies, immune complexes, paraproteins, lipoproteins, and inflammatory mediators, like cytokines. therapeutic plasma exchange was proven to be effective as rescue therapy to mitigate the cytokine storm in a small case series of patients suffering from ph1n1 influenza a-associated respiratory failure and hemodynamic shock, reducing the need for vasopressors and improving the pao 2 /fio 2 ratio [58] . hemadsorption is performed using dedicated blood adsorber devices, with a highly porous biocompatible polymer able to bind hydrophobic compounds with a molecular weight between 10 and 55 kda, a range where most cytokines reside. in europe cytosorb © cartridges (cytosorbents corporation, usa) are among the most diffused extracorporeal therapies with the aim of obtaining cytokine blood purification, and it has been proven to be beneficial in controlling the dysregulated inflammatory response in patients who have undergone cardiac surgery or are critically ill [59] . in a case series of septic patients requiring renal replacement therapy, an adjunctive hemoadsorption strategy resulted in rapid hemodynamic stabilization and a sensible decrease in blood lactate, with a reduction of the mortality rate compared to the mortality rate predicted by acute physiology and chronic health evaluation ii (apache ii score), especially when started within 24 h of a sepsis diagnosis [60] . this approach provides a nonspecific cytokine removal, thus leading to the assumption that the possible underlying mechanism explaining its efficacy lies in the fact that cytokines that are more concentrated and, therefore, more likely to be harmful are eliminated to a greater extent [59] . moreover, according to the "cytogenetic model," clearance of circulating cytokines could allow redirection of the host immune response to the source or sites of inflammation [61] . nevertheless, at present, cytokine hemoadsorption therapy is not a standardized strategy and there is still a lack of control trials proving its real effectiveness in clinical outcome improvement and short-and long-term survival in humans. further research is needed to clarify the mechanisms of action, indications, and clinical benefits of this therapy [19, 62] . however, in the absence of resolutive options for covid-19 treatment, the use of hemoadsorption therapies in critical cases has recently been proposed, as it could help to contain the inflammatory response responsible for crs in these patients. another adjunctive potential of extracorporeal therapy includes plasmapheresis using highaffinity matrixes of lectin for coronavirus trapping, thus leading to a viremia reduction [63] . in patients who fail other treatments, extracorporeal membrane oxygenation (ecmo) remains the last resource [64] . an ongoing trial (nct04324528) is currently testing whether veno-venous (vv) ecmo in combination with a cytokine adsorption (cytosorb adsorber) device, versus vv-ecmo alone may blunt inflammation parameters and improve patient survival in those with severe covid-19 infection. recently, a small case series of covid-19 patients treated with blood purification therapies showed a reduction of inflammatory markers, and 2 out of 3 patients survived with no early complications [65] . nevertheless, the high cost and relatively low availability represent a significant limitation for the use of this option for a pandemic viral disease, highlighting the need to accurately select the target patients. in the covid-19 pandemic, the timing and patient selection will 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extracorporeal therapies in intensive care: si vis pacem para bellum hemoadsorption by cytosorb in septic patients: a case series clinical review: blood purification for sepsis extracorporeal blood purification therapies for sepsis lectin affinity plasmapheresis for middle east respiratory syndrome-coronavirus and marburg virus glycoprotein elimination extracorporeal membrane oxygenation for critically ill patients with coronavirus-associated disease 2019: an updated perspective of the european experience potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill covid-19 key: cord-022940-atbjwpo5 authors: nan title: poster sessions date: 2016-09-07 journal: febs j doi: 10.1111/febs.13808 sha: doc_id: 22940 cord_uid: atbjwpo5 nan ** each poster has been given a unique number beginning with the letter p; the next part relates to the session in which the poster will be presented. moreover, klf5 is also acting on cellular processes such as cell migration, apoptosis, inflammation, angiogenesis and differentiation. previous studies showed a novel role for klf5 as a regulator of proliferation and differentiation in skeletal muscle stem cells. detecting klf5 at the protein level harbored technical obstacles. commercially available antibodies exhibited low affinity, low specificity and failed to recognize post-translationally modified forms that are directly relevant to the function. thus, these obstacles prevent further functional protein studies such as western blots, protein co-immunoprecipitation and chromatin immunoprecipitation (chip) assays. therefore, we used crispr/cas system to establish a stable cell line which carry v5 epitope tag into the n-terminal of klf5 gene. insertion into the target side of klf5 gene via crispr-cas9 system provided an opportunity to overwhelm the above mentioned obstacles. v5 epitope tag would not interfere with the function of the klf5 and also enable us to recognize endogenous klf5 via anti-v5 antibody in the mouse myoblast cell lines (c2c12). we confirmed the targeted insertion into the exon 1 of the klf5 gene both at the dna and protein levels. the conformational dynamics of structural domains plays an important role in functioning of many proteins. the reca proteins from e. coli are known to be the central catalyst of homologous recombination and repair in bacteria. it forms a helical filament on ssdna capable to bind homologous dsdna and catalysis of the exchange of the complementary strand. significant mobility if its c-terminal domain has been observed experimentally by cryo-electron microscopy. however its potential significance for reca protein activities still remains unclear. in this work we investigated this question by construction of a mutant reca protein with artificial disulfide bridge between central and c-terminal domains. the wild type protein has no disulfide bonds, and therefore its native mobility can be restored in vitro, by addition of b-mercaptoethanol. our data suggest that the s-s bridge decreases both the rate of atp hydrolysis in vitro and the e. coli resistance to uv in vivo. thus, our experimental results indicate that the flexibility of the c-terminal domain significantly affects recombination activity of reca protein in vivo and in vitro. hydroxiurea (hu) is an inhibitor of ribonucleotide reductasethe enzyme that catalyzes the process of free dntps synthesis in living cells. treating cells with hu causes diminishment of the nucleotide pool. as a result, single-stranded dna regions are generated, which leads to s-phase checkpoint activation. the progression of replication forks is blocked and the completion of dna replication is prevented. this results in s-phase cell arrest. nevertheless, our results demonstrate that after prolonged hu treatment, the saccharomyces cerevisiae cells seam to escape the arrest and continue the progression of their cell cycle. we show that when cells re-enter the cell cycle, mrc1, but not ctf4 is detached from chromatin. our data also shows that meanwhile, rad53 checkpoint activity is diminished in order to allow s-phase checkpoint escape and completion of the cell cycle. moreover, cells not only continue the cell cycle, but steadily surmount in the presence of hu. all this data indicates that cells have made the decision to compromise s-phase checkpoint and to adapt to the novel environmental conditions in order to survive. as both mrc1 and ctf4 are known to be responsible for polymerase and helicase harmonization during replicative arrest, our data indicates that mrc1 has a more specific role in the process of adaptation. our data demonstrates that mrc1 is a leading protein to regulate the stability of s-phase checkpoint arrested replication forks. zinc finger domain is the most common dna binding domain in metazoa. almost 100 drosophila proteins with c2h2 zinc fingers also have zinc finger associated domain (zad). several proteins with zad (zw5, pita and zipic) were found to interact with cp190 and act as insulator proteins. for some of the zad-containing proteins (for example, weekle and grauzone) it was shown that their zad domains can form dimers with each other. the ability of these proteins to dimerize appears to be especially important in the light of the model suggesting that dna-binding insulator proteins can support genome looping and organization of chromatin structure via interaction with each other. in this work we aimed to understand the role that zadmediated protein-protein interactions play in maintenance of dna loops, focusing on proteins: zw5, pita, zipic and cg6808. first, we performed co-precipitation and yeast two hybrid assays to confirm dimerization of isolated zads in vitro. we observed that only zads from the same protein can specifically interact with each other (homodimerization) and they are unable to interact with zads from different proteins (heterodimerization). we confirmed homo-but not heterodimerization of zads in vivo with coimmunoprecipitation experiments in s2 cells. furthermore, we found that zad proteins can support longdistance interactions in transgenic constructs in flies. using model system with cg6808 protein, we demonstrated that zad is essential for these interactions. proteins without zad cannot maintain loop formation. finally, analysis of chip-seq experiments for zw5, pita, zipic and cg6808 revealed that binding sites of zad proteins often overlap with regions of inter-chromosomal contacts known from hi-c experiments. we conclude that zad-containing proteins can support longdistance genomic interactions and dimerization of zads is necessary for these interactions. this study was supported by the russian science foundation (project №14-24-00166). over the years a large body of clinical knowledge has accumulated on pharmacological effects of drugs on thyroid function. antipsychotics are administered over long periods in humans; therefore their possible adverse side effects should be taken into consideration. the aim of this study is to evaluate the effects of haloperidol and clozapine on plasma t3 and t4 concentrations in adult male wistar rats. fifty rats aged between 14 and 15 weeks (270 ae 30 g) were divided into five groups (n = 10 in each group), and drugs were administered each day intraperitoneally (ip) for 28 days. the first group was a sham group. the other four groups were considered as low and high treatment doses of the drugs. after a one-week habituation period, animals was administered haloperidol (0.05 mg/kg, n = 10 and 2 mg/kg, n = 10) and clozapine (0.5 mg/kg, n = 10 and 20 mg/kg, n = 10). the rats were anesthetized with ether, and bloods were collected by direct cardiac puncture 24 hours after the last injection. the t3 and t4 plasma concentration levels were analyzed with chemiluminescent immunoassay. statistical analysis was performed with ibm spss v20.0. kruskal-wallis and bonferroni tests were used. t4 plasma concentration levels significantly differ between sham (median=8.25 mg/kg) and haloperidol (2 mg/kg) (median=7. 00 mg/kg), haloperidol (0.05 mg/kg) (me-dian=7.70 mg/kg) and clozapine (20 mg/kg) (median=8.32 mg/ kg), haloperidol (2 mg/kg) (median= 7.00 mg/kg) and clozapine (20 mg/kg) (median= 8.32 mg/kg) groups (p < 0.05). however, no significant differences between the groups regarding to t3 plasma levels were observed. in conclusion, haloperidol and clozapine increased the t4 plasma concentrations, but didn't have any significant effect on t3 plasma concentrations. p-02.09.1-003 isolation of lipase producing strains of bacillus obtained from olive wastewater and screening for substrate specificity in this work, wastewater samples of an olive factory from yusufeli (artvin, turkey) were collected carefully. after a centrifugation period of samples, supernatants were applied to a 0.45 lm filter and incubated on lb agar medium for 24 hours. based on differencies of colony morphologies, 13 isolates were selected and purified for identification. 16s lipase activity assay was carried out by rhodamine b. all of the 13 strains exhibited lipase activity. for determining the substrat specificity of isolates, 5 different substrates were used; 4nitrophenyl-butyrate, 4-nitrophenyl-caprylate, 4-nitrophenyl-laurate, 4-nitrophenyl-myristate, 4-nitrophenyl-palmitate. results were measured spectrophotometrically at 405 nm. all of the strains hydrolyzed 4-nitrophenyl-butirat, while there was no activity with 4-nitrophenyl-palmitate. bacillus sp. l3 was the most efficient strain that hydrolyzed all of the substrates. the gene encoding for lipase of bacillus sp. l3 will be cloned and expressed for more analyses and industrial applications. p-02.09.1-004 some quantitative aspects of hair follicle layers differentiation e. vsevolodov 1,2 , v. golichenkov 3 , a. mussayeva 1,2 , i. latypov 2 1 llc "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 lomonosov's moscow state university, moscow, russia in the course of stable hair growth the differentiation of hair bulb cambium cells to several layers with dissimilar cytochemistry and morphology takes place. this means the activation of different genes in the cells of different layers. depending upon the hair diameter some layers may be absent (medulla in the thin hairs). the hair diameters of the carpet sheep breeds vary widely even within the same square mm of the skin. we compared the different layers thicknesses proportions for the follicles with varying hair diameters. the follicle layers were measured on 155 microphotos of transverse histological sections of the follicles made under the standard magnification. all follicles belonged to the same skin biopsy. the measurements were made at the levels just below the fissure separating the hair and inner root sheath appeared. the empirical regressions of the layers thicknesses and of ratios of different layers against hair diameters were counted. the computer model was made on the basis of these regressions which allowed to obtain the absolute parameters of the layers as well as ratios of these parameters for every chosen hair diameter. using this model we found an essential trend in changing the proportions in relative layers dimensions as we choose the follicles with more and more thick hair. when we change the follicles with 30 mcm hair diameter for those with the hair diameter 100 mcm the ratio of hair medulla diameter to hair diameter increases from 0.07 to 0.76. the ratio of hair diameter to the diameter of inner root sheath increased from 0.70 to 0.84. it means that the thicker is the hair the higher proportion of cells produced by cambium are spent to build innermost layers (medulla layer within the hair or hair within the complexinner root sheath + hair). these data may throw some light on positional information mechanism of layers differentiation. lignin is a heterogeneous polymer that constitutes 30% of woody plant cell walls. microorganisms that degrade lignin are fungi, actinomycetes and to a lesser extent, bacteria. in case of industrial applications, the use of fungi is not feasible due to the structural hindrance caused by fungal filaments, requirement of particular culture conditions such as humidity, aeration which are not compatible with industrial processing environments. bacteria are worthy of being studied for their ligninolytic potential due to their immense environmental adaptability. environmental concerns and increasingly stringent emissions standards have led the pulp and paper industry to devise ways to decrease the level of chlorinated lignin residues in its effluents through both production process changes and improved treatment technologies. bleaching with the enzymes is the most promising because the enzymes may be very efficient, and can be used under industrial conditions. the main objective of this study was to investigate the adequency of klebsiella pnuemonia gst (glutathione-s-transferase) pretreatment for bleaching of calabrian pine kraft pulp. for this purpose the following conditions were investigated: enzyme loadings from 3 to 10 u/g pulp basis and the consistecy of the pulp was between 3 and 10%. enzyme at the desired concentration was added to the pulp and the mixture was incubated at 40°c for 2 hours. after the enzymatic pretreatment to determine the optimum conditions the kappa number of all reactions were analyzed according to tappi standarts. as a result of this study we determine the optimum conditons as 5% pulp consistecy, 8u/g enzyme for pulp treatment. after the enzymatic treatment carried out under optimum conditions we are planning to submit a short bleaching sequence and analyze for physical properties such as viscosity and brightness. owing to this bleacing sequence we are going to able to compare the enzymatic and chemical treatments of pulp in bleaching prosess. p-02.09.1-006 biochemical characterization of lipase from bacillus subtilis strain a10 from olive waste water f. ay sal, m. kac ßagan, s. c ß anakc ßi, a. o. beld€ uz karadeniz technical university, trabzon, turkey lipases (triacylglycerol acyl hydrolases, ec 3.1.1.3) are regarded as mild and environment-friendly biocatalysts for triacylglycerols hydrolysis. in addition to this hydrolytic reaction, they also catalyze reverse reactions of esterification, transesterification, and interesterification in non-aqueous environments. substrate, stereo-, regio-and enantio-specificities, and chiral selectivity are certain unique attributes of lipases that make them industrially attractive. these properties are often exploited in the manufacturing of detergent formulations, synthesis of fine chemicals, useful esters and peptides, food processing, paper manufacturing, degreasing of leather as well as in bioremediation. in this study, lipase from bacillus subtilis strain a10 is partially purified and characterized. bacillus subtilis strain a10 is isolated from olive factory from soke (aydin, turkey) and identified with 16s rrna analysis. lipase activity is screened on petri supplemented with rhodamine b. bacteria was grown in lb medium supplemented with 1% olive oil (vol/vol) for 48 hour at 37°c. after incubation, cells were harvested by centrifugation at 11,000 rpm for 5 minutes, resuspended in 50 mm tris-hcl (ph 8.0) buffer, followed by sonication with sartorius labsonic m to release intracellular proteins. q-sepharose is used as ionexchange column chromatography for lipase purification. effects of temperature on activity and stability were determined spectrophotometrically using p-nitrophenyl laurate as the substrate. effects of ph on activity and stability were also determined. the effects of various metal ions and other reagents on the hydrolytic activity were assayed at 37°c. the enzyme was active and stable in the broad ph range of 5.0-10.0 and temperature range of 24-50°c. bacillus subtilis strain a10 have high lipolytic activity. after cloning this enzyme to an expression vector and detailed characterization, this may suggests its usefulness in industrial applications. p-02.09.1-007 investigation of pin1 as a nuclear factor one binding partner s. saritas, a. e. yilmaz, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey the nuclear factor one (nfi) proteins are important regulators of gene expression in the developing embryo and in adult stem cell niches. this transcription factor family has four members: nfia, nfib, nfic, and nfix. nfi proteins bind a consensus sequence on gene regulatory regions as homo or heterodimers. each member of nfi family has a highly conserved n-terminal dna binding and dimerization domain and a diverse proline rich c-terminal transcriptional activation/repression domain. as knockouts of nfi genes display distinct developmental phenotypes, we hypothesized that specificity of nfi protein function may arise from their interactions with binding partners. a yeast-two hybrid screen identified protein interacting with never in mitosis a1 (pin1) as a potential nfib interactor. pin1 is a ubiquitously expressed protein that specifically recognizes and binds to a phospho-serine or a phospho-threonine followed by a proline (ps/pt-p motif), and catalyzes isomerization of peptidyl-prolyl bonds. interestingly, both n-terminal and c-terminal domains of four nfi isoforms contain several conserved putative ps/pt-p motifs and some of these are reportedly phosphorylated. we looked for nfi pin1 interactions in vitro by gst-pulldown and co-immunoprecipitation assays. while gst-pin1 fusion protein interacts with all of four nfi isoforms, it binds nfib most strongly, nfia and nfic moderately, nfix most weakly. moreover, deletion of the cterminal domain leads to loss of nfi affinity for pin1 implicating this domain in nfi-pin1 interactions. co-immunoprecipitation assays where we co-expressed various epitope tagged nfi and pin1 proteins in hek 293t cells showed that pin1 precipitates nfib, as well as other nfi isoforms and nfib can, in turn, precipitate pin1. we are currently carrying out site-directed mutagenesis on nfib to identify the specific residues that pin1 recognizes. we will further explore if this interaction regulates nfi function during embryonic development. that pre-adapt migrating fish to the life in seawater. among others, smoltification induces intense growth of fish that enter the ocean at a size where risk of predation is significantly reduced. skeletal muscle growth depends on a tightly controlled balance between protein synthesis and degradation. protein synthesis driven by hormone regulation is well studied in smoltified atlantic salmon; while less is known on protein degradation occurring via a number of pathways including cytosolic ubiquitin-proteasome system and calcium dependent calpains. the aim of this study was to compare calpain and proteasome enzymatic activities in the skeletal muscles of s. salar parr, pre-smolts and smolts. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from indera river (kola peninsula, russia). our results demonstrated the significant differences in studied protease activity levels between parr and smolts. calpain and proteasome activities in s. salar smolt muscles showed a significant drop compared with that of parr. the negative correlation between proteases activity levels in the muscle tissue and overall fish growth rate was shown. so, our data indicated life stage specificity in skeletal muscle protein degradation capacity in migrating fish. we suppose that intense muscle growth in s. salar pre-smolts is supported by various mechanisms including accelerated muscle protein accretion through the reduction of protease activities. obtained results enhance our knowledge in the mechanisms of atlantic salmon smoltification. the work was supported by the russian scientific foundation, project no. 14-24-00102. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] the sociodemographic characteristics of the pregnant women who double and triple prenatal screening test h. d€ ulger, s. yabanci€ un meram medical faculty, n.e.university, konya, turkey double and triple prenatal screening tests which are applicable during first and second trimesters of pregnancy predict existent abnormalities at early stage. the aim of this study is to investigate the relationship between positive results of double and triple tests, further confirmatory tests during prenatal phase, postnatal status of babies and maternal age. in this study, double and triple test results of pregnant women who were admitted to meram faculty of medicine during 2009-2013 period were scanned from archive and test results indicating risk were detected. from these results, those which were above cut-off values for down syndrome, trisomy 18, open spina bifida were determined. a questionnaire was carried out with voluntary participants by reaching to these individuals. positive-negative result ratio of all double and triple test results and sociodemographic features such as age, occupation, presence of consanguineous marriage were investigated. all data from archive and answers from survey questions were assessed statistically. participants of the study were 18 to 46 years old and their average age was 29.89 ae 6.56. 219 ofthem (69.30%) were under 35 years of age whereas 97 of them (30.70%) were above 35 years of age. number of pregnancies were scaling between 1 to 13 with an average of 2.56 ae 1.56. 207 of 316 mothers (65.50%) were not undergone amniocentesis, whereas 6 babies with chromosomal abnormalities were detected among 109 mothers who were undergone amniocentesis. in conclusion, there may be regional, sociological and such that reasons for those who were not undergone amniocentesis despite positive double and triple test results. 6 (5.50%) chromosomal abnormalities were detected among pregnancies with increased risk assessment with positive double and triple results. p-02.09. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the effects of oil on the growth and development of amphibians l. sutuyeva, t. shalakhmetova, a. ondassynova al-farabi kazakh national university, almaty, kazakhstan currently, the pollution of ecosystems by oil and oil products is increasing everywhere. the oil gets into water and ground during oil production, transportation and accidents. as a result, terrestrial animals and hydrobionts are exposed to oil contamination. thus, populations of animals decline. it can be assumed that the most sensitive to the effects of pollutants are animals in early stages of development. amphibians have established themselves as the most convenient bioindicator species. since lake frog (rana ridibunda) and green toad (bufo viridis) are the bioindicator species in kazakhstan, the study of the effects of oil on their larvae was carried out. we used water-soluble fraction of the oil from zhanazhol field (aktobe region) in our test. the larvae of control group were kept in pure water, and larvae of test groupsin aquariums with 0.05, 0.5 and 1% concentrations of the oil fractions. the concentrations were chosen in accordance with the level of pollution of kazakhstan's water bodies with oil. mortality of larvae, morphometric parameters and morphogenesis were studied. it was found that high mortality of larvae is the most visible reaction when exposed to oil. this indicator rose noticeably depending on the doses (0.05, 0.5% and 1%) in both species with percentages 16%, 51% and 78% in r. ridibunda and 22%, 61% and 83% in b. viridis, respectively, while in the control group it was about 10%. furthermore, delayed larval development was detected. thus, the larvae from the control and 0.05% oil group reached gosner stage (gs) 36, tadpoles from 0.5% and 1% groups were at gs-32 and gs-29, respectively, by the 30th day of life. moreover, behavioral abnormalities (sluggish movements) and decreased sensitivity to mechanical stress (touch) were observed under the influence of high concentrations of oil fractions. thus, oil in low concentrations alters the growth and development of tadpoles of anurans, and causes their increased mortality in high concentrations. p-02.09.1-011 effect of catechin loaded plga nanoparticles on glioma cell line histone h1t is a linker histone which binds to dna and contribute in chromatin condensation as well as regulation of specific genes through spermatogenesis. replacement of this histone h1 subtype and hyperacetylation of histone h4 tail, facilitate the replacement of histones with sperm chromatin condensing proteins of tnps and prms. ethical approval and informed patient consent was gained from 12 infertile men referred to royan institute. testicular biopsies were collected from patients through assisted reproductive techniques (art) procedure. based on pathological results samples were classified into the following three subgroups: obstructive azoospermia (as positive control), complete maturation arrest and sertoli cell only syndrome (negative control). chromatin of tissues evaluated for presence/absence of histone h1t protein in regulatory regions of tnps and prms genes using chip-real time pcr. results showed lower incorporation of h1t protein on regulatory regions of tnps and prms genes in two spermatogenic failure group versus positive control. in this study, it can be concluded that the decreased levels of h1t histone variant in testis tissues and failure in chromatin condensation have significant association with male infertility. p-02.09.1-013 serum dickkopf-1 levels in obese children and adolescents that obesity is detrimental to bone health despite potential positive effects of mechanical loading conferred by increased body mass on bones. the wnt/b-catenin pathway is essential for normal osteogenesis. serum dickkopf-1 (dkk-1) is one of the most important inhibitors of the wnt//b-catenin pathway. the aim of this study was to investigate the serum dkk-1 levels in obese and non-obese children and adolescents. materials and methods: the study included 30 obese children and adolescents (14 males and 16 females) aged from 7 to 17 years and 30 healthy normal-weight controls (13 males and 17 females) aged from 6 to 17 years. serum dkk-1 levels were measured by elisa method using commercially available kit. results: body mass index of the obese children was significantly higher than that of non-obese children (p = 0.000). however, there was no significant difference between dkk-1 levels of the groups. (these results are preliminary and the study is continuing). discussion and conclusion: our result showed that serum dkk-1 levels were not changed in obese children and adolescents. p-02.09.1-014 transcriptional regulation of cdo by nuclear factor one proteins b. kutay, c. lektemur, v. g€ uler, a. kumbasar department of molecular biology and genetics, istanbul technical university, istanbul, turkey nuclear factor one (nfi) transcription factors play important roles in regulation of central nervous system development. three of the four members of nfi family, nfia, nfib, and nfix are expressed in neural progenitors, as well as neurons and glia in the embryo. inactivation of these genes in mice show that they function in development of neocortex and hippocampus in the forebrain, cerebellum, spinal cord and precerebellar nuclei of the hindbrain, regulating neurogenesis, gliogenesis, as well as neuronal migration, axonal outgrowth and guidance. all three neural specific nfis are expressed in precerebellar neuroprogenitors, however, only deletion of nfib leads to a delay in development of precerebellar neurons. investigation of misregulated genes in nfib à/à precerebellar neuroprogenitors identified cell adhesion associated, oncogene regulated (cdo) as a potential downstream target of nfib. interestingly, this gene has been reported to be upregulated in nfia à/à hippocampus as well. cdo, a cell surface glycoprotein of the ig superfamily, has been found to regulate neurogenesis in vivo, is highly expressed in the developing brain and can induce neural differentiation by promoting heterodimerization of basic helix loop helix transcription factors with e proteins. bioinformatic analysis of the 5 kb human cdo promoter region identified five nfi binding sites: one cluster in the first 1 kb region, another in the 3.5 kb upstream region. electrophoretic mobility shift and supershift assays showed that nfib binds to all five sites. furthermore, nfib, along with the other neural nfis, inhibits the proximal cdo promoter driven luciferase activity by up to 85% in hek293t cells. preliminary data indicate that nfis bind to sites in both clusters in human neural stem cells (hnscs) suggesting that these sites are functional in vivo. we are currently investigating this possibility through nfi overexpression and silencing experiments that will examine regulation of cdo in hnscs. differentiation. the aim of this study is to investigate bdnf and drd2/ankk1 gene variants in eos development. in this study, 111 eos patients and 138 healthy controls were used. genomic dna extraction was performed from peripheral blood leukocytes. drd2/ankk1 taq1a (rs1800497) and bdnf val66met (rs6265) polymorphisms were determined by real-time polymerase chain reaction (rt-pcr). positive and negative syndrome scale (panss) was used to determine eos severity. for drd2/ankk1 rs1800497 polymorphism, there was a significant difference in the genotype frequencies between patients and controls for the co-dominant model (p = 0.05, or = 1.723; 95% ci: 0.996-2.98). however, no significant relationship was observed in the genotype frequencies of bdnf val66met polymorphism between eos patients and controls (p = 0.489). these results indicate that, drd2/ankk1 rs1800497 genotypes may affect eos development. however, bdnf val66met polymorphism may not be associated with eos. lack of association of bdnf val66met polymorphism may be due to limited number of patients. our findings need to be confirmed by further studies. various dyes used in the textile industry are discharged in large quantities to the receiving environment in the manufacturing process. this is the beginning of a process that is difficult to compensate for environmental and human health. therefore, contaminated areas should be cleaned. in addition, technologies with high polluting potential should be integrated with biological approach and thereby the impurities consisting of dyes should be reduced. in this experiment; burdirect black meta konz (c.i. direct black 38) was intended to decolorization using laccase. firstly, enzymatic decolorization of the dye was determined using spectrophotometry. the wavelengths of maximum absorption of burdirect black meta konz (c.i. direct black 38) was determined between 200 and 800 nm. then, optimization studies have been done. for optimization studies; dye concentration, laccase activity, ph, buffer concentration, temperature, mediator effect, mediator concentration and time parameters were determined. lastly, in optimal conditions, atr-ftir and gc-ms analyzes of ensuring decolorization of dye were analyzed. decolorization of burdirect black meta konz (c.i. direct black 38) was performed successfully and the absence of any metobolite in the decolorization medium has been provided by atr-ftir and gc-ms analyzes. assessing in terms of application, it can be easily applied by provided the reaction conditions in textile factories. laccase is a tool of decolorization of dyes in environmental friendly process. thus for the development of spermatids into mature sperm able to fertilize the oocyte.one of the causes of male infertility is in fact impaired sperm fertilization capacity due to sperm chromatin abnormalities and aberrant protamine replacement.recent research has focused on protamine biology,including protamine gene and protein structure,mechanisms of protamine expression regulation and involvement of the protamines in male fertility.various studies reported abnormal expressions of protamine (prm) genes in sperm of infertile men.the aim of the study is to investigate the gene expression of prm1, prm2 and their relationship with defective spermatogenesis. materials and methods: this study has been performed on 50 infertile and 3 fertile turkish men.total rna was extracted from the sperm pellet using trizol reagent.after rna extraction and cdna synthesis,real-time quantitative polymerase chain reaction (rt-qpcr) was used to determine the expression of prm1 and prm2. results: distinct levels of spermatozoal prm1 and prm2 mrna were found in infertile patients compared to fertile control groups.we found that the mrna levels of prm1 was reduced in 23 (%47), and the mrna levels of prm2 was reduced in 33 (%64) out of 50 infertile patients.in the current study,we found statistical significant association between the prm1 expression and infertility (p < 0.05).although prm2 gene expression was decreased in most of infertile patients compared to fertile control groups,the differences between the groups were statistically insignificant (p > 0.05). discussion: the results of the study suggested that, the protamine expressions which were associated with spermatogenesis may be important in infertility treatment. further studies are required in a large series of different populations to clarify the role both prm1 and prm2 themselves and their mrna expression on male fertility. the study was conducted to characterize the processes of muscle growth in atlantic salmon (salmo salar l.) of different ages inhabited rivers indera and varzuga (kola peninsula, russia) in summer and autumn. the expression levels of genes myosin heavy chain myhc, myostatin (mstn), and myogenic regulatory factors myf5, myogenin) in white muscle were studied in salmon parr of age groups 0+, 1+, 2+ in june and october. the changes in expression levels of mrfs, myhc and mstn indicating the extent of hyperplasia, hypertrophy, and restriction of muscle growth at different ages of parr were revealed. the pattern of age-related changes differed between seasons. especially, the expression of genes myod, myogenin and myhc peaked in yearling parr (1+) in summer, that indicated the high rate of hyperplastic and hypertrophic muscle growth in yearlings (1+). at the same time, the mstn expression level, the negative regulator of muscle growth, was highest in parr at age 1+. possibly, it is the necessary regulation mechanism to attenuate hyperplasia and hypertrophy and control muscle growth. in autumn, the expression level of myhc and myogenin were higher in salmon of age 0+ and 1+ then in 2+, indicating the higher intensity of hypertrophy in parr at both first ages in comparison to 2+. there was no differences in expression level of myod, myf5 and mstn between age groups in autumn. moreover, the expression levels of genes studied were lower in autumn than in summer. thus, it indicated the decrease of muscle protein synthesis and muscle growth rate in autumn. these findings expand knowledge on age-and season-related features of muscle development in young atlantic salmon in their natural habitat. the study was supported by the grant of the russian science foundation no. 14-24-00102. p-02.09.1-020 lmp2 and lmp7 gene polymorphisms in the southeastern anatolia population of turkey d. mihc ßioglu 1 , f. ozbas gerceker 2 1 sanko university, gaziantep, turkey, 2 gaziantep € universitesi, gaziantep, turkey introduction: the low molecular weight polypeptide 2(lmp2) and low molecular weight polypeptide 7(lmp7) genes are located in the class ii region of the major histocompatability complex (mhc) locus on chromosome 6. these genes encode peptides forming the large components of the proteosome complex which degrades short-lived cytoplasmic proteins. due to the significant role of lmp products antigen presentation, these genes can be accepted as strong candidates of susceptibility factors for different diseases. population genetic studies can also contribute to understanding of the possible role of lmp gene polymorphisms. the aim of this study was to determine the allele and genotype frequencies of the lmp2 and lmp7 gene polymorphisms in southeastern anatolia population and to compare these with the frequencies in other populations previously reported. material and methods: a total of 110 healthy and unrelated individuals participated in this study. polymorphism analyses were done by polymerase chain reaction (pcr)-restriction fragment length polymorphism (rflp) method and allele/ genotype frequencies of lmp2 and lmp7 genes were determined. results: a deviation from the hardy-weinberg equilibrium (v2 = 17.97,p < 0.05) was found for the genotype distribution of lmp2 gene polymorphism, while the lmp7 genotypes found to be distributed (v2 = 0.43,p > 0.05). discussion: available allele frequency data for different populations were used to calculate genetic distances and to construct a neighbor-joining tree. among the included populations, nahuas (mexico) population was found to have the lowest genetic distance from the southeastern anatolia-turkey population. conclusion: it can be concluded that, more studies using different types of genetic markers are needed to clarify the filogenetic relationships of southeastern anatolia population with other populations and also the number of population studies on lmp2 and lmp7 genes should be increased to understand their effects as a genetic marker. p-02.09.1-021 investigation of in vitro antioxidant activity of quercetin loaded plga nanoparticles pharmacological effects. but its usage is restricted because of low aqueous solubility, poor bioavailability, poor permeability and instability in physiological medium. these problems can be overcome with encapsulation of quercetin into nanocarriers such as biodegradable plga based nanoparticles. polymeric nanoparticles which have 1-1000 nm particle size and providing controlled released of biological active agent are prepared by using biodegradable and biocompatible polymers. in this study, encapsulation of quercetin molecules into plga nanoparticles was carried with using the single emulsion (w/o) solvent evaporation method. size measurements of the obtained nanoparticles were performed by zetasizer and their size were found 882. 1; 189.25; 436 .9 nm respectively. the morphological features were examined by sem images. antioxidant activities of q5, q7 ve q10 nanoformulations have been investigated by dpph and no (nitric oxide) methods. it is thought that the nanoparticular formulations that is developed in this study can be useful model for the other antioxidant molecules and will provide a significant contribution to the food and pharmaceutical industry. "this research has been supported by yıldız technical university scientific research projects coordination department. project number: 2014-07-04-gep03". p-02.09. the effect of environmental enrichment on spatial memory and certain nmdars, and 5ht2a expressions in rat pups introduction: the aim of the study was to investigate the effect of environmental enrichment exposed during whole childhood on spatial learning and memory and certain nmdars, and 5ht2a in the hippocampi of pups. materials and methods: four-weeks old, male, weaning rats were randomised into 2 groups as enviromental enrichment (ee, n = 12) and standard cage control (scc,n = 12) groups. eeg housed in an enriched environment and sccg were kept in standard cages for 8 weeks. following the experiment the rats were trained and tested in the morris water maze (mwm) , open field test (oft) and forced swim test (fst) in order to assess the neurobehavioural effects of ee. nr2a, nr2b, 5ht2a protein levels were analyzed by western blotting from hippocampi of rats. results: the positive effect of ee was seen at the learning phase in the mwm as 'latency to locate the hidden platform' between groups thoughout the 4 training days showed that eeg located the hidden platform significantly earlier than sccg on days 2, 3 (p = 0.006, p < 0.0001). also eeg significantly spent lower time in the outer zone of the maze on days 2, 3 which was the sign of low anxiety level (p = 0.011, p = 0.049). the parameters of oft which indicated increased locomotion, exploration and low anxiety were significantly higher in eeg (p < 0.05), in fst comparison of groups showed no difference (p > 0.05). the levels of nr2b and 5 ht2a were significantly increased as compared to sccg as well (p < 0.0001, p = 0.003). discussion & conclusion: these findings showed that exposure to ee throughout the whole childhood causes several neurobehavioural effects like increased exploration and low anxiety. these effects may lead to improvement in speed of learning. increase in the nr2b and 5ht2a concentrations which are the receptors that are related to learning and memory in the hippocampi accompanied these changes which may be basis of the neurobehavioural improvements or may provide contribution to positive neurobehavioural effects. p-02.09.1-023 effects of monosodium glutamate exposure during prepubertal term on several biochemical parameters in rats h. i. b€ uy€ ukbayram, d. kumbul doguc ß, i. ilhan, a. y. ismail s€ uleyman demirel university, isparta, turkey monosodium glutamate, which is commonly used in processed foods as flavor enhancer, is considered 'generally recognised as safe' by fda; however many studies have revealed the negative effects of msg.we aimed to evaluate the effects of msg in childhood on several serum parameters. sixty-six rats, (4 weeks old) were divided into 3 groups as control (cg, n = 22; 11 + 11, male+female) , experiment 1 (msg-low dose, e1g, n = 22; 11 + 11, male+female) and experiment 2 (msg-high dose, e2g, n = 22; 11 + 11) groups. msg was administered at 25 mg/kg/d to e1g, 2.5 g/kg/d to e2g for 6 weeks by oral gavage. the rats were sacrified and blood samples were collected from aorta. the blood samples were centrifuged, the serum samples were separated and glucose, alt, total protein, albumin, creatinine, cholesterole and triglyceride levels were analysed by beckmann au 5800 autoanalyser. level of total protein was significantly increased in e1g and e2g groups when compared to cg (p < 0.05). level of alb€ umine was also increased in both egs but significant difference was seen in e2g as compared to cg. creatinine levels were significantly increased in egs when compared to cg (p < 0.05). although the glucose levels in both egs were increased, the increase in e2g was statistically significant (p < 0.05). the alt levels of in egs were also increased but the significant increase was seen in e2g (p < 0.05). the effect of msg seem to be dose dependent and especially effect on carbonhydrate metabolism. increasing doses caused increase in glucose level, and tendency to glucose intolerance. increasing doses of msg also caused increase in creatinine and urea. another apparent effect of msg was detected on alt activity. in conclusion the negative effect of msg on glucose level, liver and kidney functions depends on daily dose intake. consumption of msg seem to be inevitable it has to be restrained in children otherwise early metabolic problems may be future problems for these children. (700 mg/kg) + tartrazine (750 mg/kg) + brilliant blue fcf (600 mg/kg) + ponceau 4r (70 mg/kg) + azorubine (400 mg/kg) + indigotine (500 mg/kg) + erythrosine (10 mg/kg). artificial food color mixture were administered to g 2 and g 3 and drinking water was applied simultaneously to g 1 by oral gavage per day for 3 weeks. after application all rats were sacrificed, the total oxidant (tos)/antioxidant (tas) capacity were analyzed in rats' brain, liver, kidney homogenate and serum with rel tos-tas diagnostics assay kit.the statistical analysis was carried out by using kruskal wallis test. tas and tos levels in liver homogenate were not found significantly different between all groups (p > 0.05). in serum and kidney and brain homogenate, tas levels were not significantly different between all groups. tos levels in g 3 were higher than g 1 and g 2 in serum and kidney and brain homogenate (p < 0.05). exposure to synthetic food colors may increase oxidative stres in vitale organs such as brain, kidney in female rats. these alterations differ according to organ and dose. parallel with increasing trends on healthy eating habits, consumption of prebiotics and probiotic microorganisms have been popular due to their benefits on human health. functional dairy foods such as probiotic yoghurt and cheese are the most common foods including probiotic microorganisms. due to some considerations such as standardization and quality in bulk production, starter cultures are used in industrialised fermentative food production to start fermentation. starter culture basically refers the microorganisms which induce and maintain fermentation of the fermentative foods and starter cultures including probiotic microorganisms are called as probiotic starter cultures. in this study, probiotic cheese starter cultures as a microbial community were investigated using computational systems biology tools. a metabolic network model of probiotic cheese starter culture was reconstructed using microbial community network modeling approach. literature-based genome-scale metabolic models of commonly used lactic acid bacteria were used for the microbial community metabolic model. the microbial community metabolic model simulated metabolic interactions of the microorganisms in the probiotic starter culture. metabolic flux values computed by the metabolic network model also predicted the metabolic pattern of the glycolysis (conversion of lactose), lipolysis (conversion of fat) and especially amino acid catabolism which are associated cheese flavor metabolism. simulations obtained by metabolic network-based analysis of cheese starter cultures can also be used for other fields like genetic engineering, upstream processing of the functional cheese production. p-03.01.1-002 er quality control protein network in cf to modulate f508del-cftr rescued phase ii xenobiotic metabolizing enzymes convert parent compounds into more hydrophilic metabolite by catalyzing conjugation reactions including glutathione and amino acid conjugation, glucuronidation, sulfation and acetylation. this study was aimed to describe the best cell line model for studying phase ii xenobiotic metabolizing nqo1 and gst-pi enzymes. for this purpose, mrna and protein expression of nqo1 and gst-pi enzymes were analyzed in ht29 and sw620 (colon); hepg2 and huh7 (liver); pnt1a and pc3 (prostate) cell lines by qrt-pcr and western blotting techniques, respectively. protein expression analysis revealed that nqo1 protein was expressed in all cell lines and relative protein expression is highest in the hepg2 (100%) and pnt1a (97%) while huh7 (31.9%) showed relatively low expression of nqo1. in addition, nqo1 mrna expression was relatively high in ht29 (1.68 fold) and pnt1a (1.92 fold) when compared with liver cell line hepg2 (1.00 fold). gst-pi protein expression was found very high in huh7 (100%) while there was no expression in hepg2. gst-pi mrna expression was relatively higher in pnt1a (3.56 fold) and ht29 (2.47 fold) when compared with huh7 (1.00 fold). according to these results, choosing the best cell line as model depends on the purpose of the research. for studying metabolism of a chemical by nqo1 and gst-pi or effect of a chemical on translational regulation of these enzymes, it is better to consider protein expression of the cell lines for choosing best model. however, if the aim is to study effect of a chemical on transcriptional regulation of these enzymes, it is better to choose a cell line that expressing highest mrna of gene of interest. in conclusion, considering both mrna and protein expression levels together, the best model cell lines for studying phase ii xenobiotic metabolizing nqo1 and gst-pi are ht29 and huh7, respectively. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] the studies on the pancreatic cells' surface glycoconjugates profiles in rats fed with high fat with lectin labelling methods by flouresans microscopy y. mater, s. beyhan ozdas gebze technical university, kocaeli, turkey in this study, the backbone of the cellular adhesion-recognition mechanism, located in the cell membrane. the study material selected pancreas tissue, has a privileged structures. the pancreas is one of the main organs to aid in digestion. the pancreas functions as an exocrine gland and role in digestion. in addition, the pancreas also functions as an endocrine gland, secreting several hormones into the blood that control the blood levels of glucose and other nutrients. due to the pancreas have been selected for this unique feature. thus, different types of cells in the same sample will be able to study the structure of the surface glycoconjugates. generally researches about determination of carbohydrates in the cell, glycoproteins or/and glycolipids are cut with enzymes. next step, the oligosaccharide mixture obtained, than establishing the complete structure of oligosaccharides and polysaccharides requires determination of branching positions, the sequence in each branch, the configuration of each monosaccharide unit, and the positions of the glycosidic links. this is a more complex problem than protein and nucleic acid analysis. these processes are indispensable for the understanding of the chemical structure of the sugar. whereas in cells using labeled lectins specific sugars, it is possible to accurately determine. in this study, was used triticum vulgaris (wga) labeled with fluorescein (fitc). thus, the cells located on the cell surface and neu5ac (sialic acid) for wga sugar residues were investigated. according to preliminary results of this study, wga labeled with fitc is specifically binding of these sugars. when this study is completed, the differences of sugar on the surface of different type of cells in the pancreas can be distinguished in micrographs. thus, in the cells of the pancreas, the sugar units involved in adhesion-recognition can be possible to determine specifically. large scale gene networks could be topologically analyzed in order to obtain possible global system-level structure cancer gene co-expression networks can have lower connectivity as compared to normal samples. using colorectal tissue gene expression datasets, we observed that tumor specific networks are less connected than normal networks. functional enrichment analysis suggested that cell cycle genes and methylation-associated cell adhesion genes can specifically play a role in the connectivity loss of carcinoma samples. literature confirmation provided a gene network including significant genes playing roles in the intersections between cell cycle, cell adhesion, and cell skeleton dynamics. this network can provide novel insight to our understanding of the molecular mechanisms of colorectal cancer. p-03.01.1-012 tf-mirna circuits specific to epithelial cancers y. oztemur, a. aydos, b. gur dedeoglu ankara university biotechnology institute, ankara, turkey cancer is the most common cause of death in the world but there are still a lot of uncertainties about the exact mechanism taking roles in regulation of it. cancers can be classified according to cell type; in which they start. carcinomas are the most prevalent types of cancer and start in epithelial tissues. they are also named as epithelial cancers (ecs) and make up about 85 out of every 100 cancers. over the past few years, many studies are concentrated on mirnas, which have emerged as important regulators of gene expression like transcription factors (tfs). tfs are regulators at transcriptional level while mirnas are post-transcriptional regulatory key-elements. otherwise the transcription of mrnas and mirnas are known to be regulated by tfs and tfs are the targets of mirnas. therefore, it is crucial to characterize the relation of tfs, mirnas and their targets by building circuits in diseases such as in ecs. for this study, mirna and mrna expression studies including epithelial tumors and normal samples searched in geo and array express microarray databases. 4 mrna studies and 4 mirna study, which were designed for 4 different ecs (breast, lung, ovary and colorectal) were selected to be analyzed. differentially expressed (de) mrnas and mirnas between epithelial tumors and normal samples were extracted (p ≤ 0.05, 2 fold change). among de genes, transcription factors and mirnas were identified and listed for epithelial tumor vs. normal comparison. circuit analysis resulted with remarkable circuit, which was common for all the types of ecs that includes klf4 transcription factor and hsa-mir-145. in the literature hsa-mir-145 and klf4 are known as important regulators in different types of cancer, which indicated that the motifs involving tfs and mirnas might be useful for understanding the regulation of ecs. as a conclusion finding out new and common circuits may aid us in predicting new or alternative diagnostic and/or prognostic biomarkers for ecs. mesenchymal stem cells (mscs) are multipotent stromal cells that can differentiate into a variety of cell types which are used in cell therapy. although they are the most attractive cell type for cell therapy studies, primary mscs lose their differentiation potential with increasing time in culture and passage so they are of limited use. due to this disadvantage, msc lines are more suitable for in vitro researches owing to their immortality. in this study we compared primary bone marrow-derived msc (bm-msc) with bone marrow derived msc line (rcb2153) in terms of cell characteristics and gene expression profiles to determine the functional differences among mscs types. firstly, mscs were identified by using cd29, cd70, cd90 and cd105 as positive markers and cd34 as a negative marker. gene expression profilling was investigated using affymetrix hg-u133-plus2 arrays. the significant go biological process terms and kegg pathway enrichment analyses of the identified degs were performed using david (p < 0.001, fold change≥2). the analysis showed similar pathway clustering in both cell types. the resulting quantitave transcriptome of 754 genes were identified that differentially expressed in msc line versus primer mscs (538 upregulated and 216 down-regulated). functional classification of changed genes was mainly clustered in cell cycle, cell death and mismatch repair. kegg pathway analysis revealed that the genes were significantly enriched in pathways including "cell cycle, dna replication and focal adhesion" pathways. in conclusion, our results indicate that msc lines can be used instead of primary mscs. these quatitative results provide an important basis to adapt cell lines to more closely resemble physiological conditions as oppossed to animal experimentation. this could help to minimize the use of animals in research. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] association between loss of 18q21, gain of 20q13.33 and progression in sporadic colorectal cancer n. belder 1 , b. savas 2 , m. a. kuzu 2 , i. pak 3 , h. s€ umer c ß elebi 1 , a. ensari 2 , h. € ozdag 1 1 biotechnology institute, ankara university, ankara, turkey, 2 school of medicine, ankara university, ankara, turkey, 3 ankara oncology training and research hospital, ankara, turkey colorectal cancer (crc) is one of the most diagnosed cancer and the third leading cause of cancer deaths throughout the world. identifying of copy number variation (cnv) profiles between early and late stage cancers can be useful to understand the progression and aggressiveness of cancer. the main goal of this study was to construct a comprehensive insight of association between cnv and sporadic crc stages in order to identify novel candidate targets which may contribute to tumor progression. affymetrix 6.0 genechip snp arrays were used for characterization of cnvs in tumor and matched normal formalin-fixed, paraffin-embedded (ffpe) tissues from 5 stage i, 17 stage ii and 25 stage iii samples. paired cnv analyses were performed using partek genomic suite 6.6 and genomic segmentation algorithm was performed using a minimum of 10 markers per segment, a signal-to-noise ratio of 0.3 and the cut-off value for the gain and loss was set of 2 ae 0.3. the adjusted p-value ≤0.05 were considered to be significant. whole genome cnv analysis revealed that amplification of 20q13.33 with 4 genes was found the most frequent (76.5%) in stage ii tumors. the most frequent (36%) amplifications were 13q12.2 and 7p22.2 in stage iii tumors. while deletion of chromosome 18q21.2 in stage iii with a frequency of 36% was found the most frequent loss, deletion of 18q21.1 was seen the most frequent (64.7%) in stage ii tumors. two tumor suppressor genes smad2 and smad4 which are found in these deletion regions were common genes between stage ii and stage iii. our results showed that gain of 20q13.33 might have a significant role in the progression of cancer. loss of 18q21 comprising two tumor suppressor genes is also another important finding. 18q21 loss can be a significant prognostic value in colorectal cancer even though validation of target genes requires additional study and larger sample size. this work was supported by tubi-tak project no:109s477. p-03.01.1-016 meta-analysis based mirna signature discriminates cervical cancer from normal samples a. yucel polat, y. oztemur, a. aydos, b . gur dedeoglu biotechnology institute, ankara university, ankara, turkey gynaecological cancers are common problems in female health. squamous cell carcinoma (scc) is a type of these malignancies. this tumor type is derived from pre-cancerous lesions, which is called cervical intraepithelial neoplasia (cin). cin is classified as cin1, cin2 and cin3 according to their dysplasia grade in the cervical tissues. mirnas are small non-coding rnas that were shown to have important roles in the development and progression of various cancers. the aim of this study is identifying mir-nas, which are playing a part in progression of cervical lesions by a ranking based meta-analysis approach. two mrna and three mirna expression studies, which include normal, cin1, cin3 and scc samples were selected from arrayexpress and gene expression omnibus (geo) databases. three mirna studies were combined with anova dependent ranking based meta-analysis program which was developed in our laboratory to find out a mirna signature that can discriminate cin1, cin3 and scc samples from normal samples. the top five mirnas with the highest ranks in meta-list were selected for further analysis. predicted targets of these mirnas were identified by mirdb target prediction tool. additionally two mrna datasets were selected for mirna-target validation studies. common genes, which were obtained from meta-mirna targets and differentially expressed genes between normal and cin1, cin3 and scc groups from two independent studies, were identified and they were subjected to pathway enrichment analysis. pathway enrichment analysis that was performed with 338 common genes showed that these targets were significantly enriched (p < 0.05) in especially cell proliferation, cell survival and cell cycle pathways, which are the key players of cancer development and progression. the meta-analysis results together with validation analysis of their targets may point out the potential roles of mirnas as biomarkers for the diagnosis and the treatment of cervical cancer. p-03.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] the hypoglycaemic and regenerative activity of thymbra spicata in alloxanized-diabetic rats thymbra spicata (labiatae), a carvacrol and thymol containing plant, is one of the medicinal herbs used by diabetic individuals to reduce blood glucose in turkey. we investigated the hypoglycaemic and anti-lipemic effects of the aqueous extract prepared from dried leaves and flowers of this plant in alloxanized-diabetic rat model. rats were divided as: diabetic control (group 1), dia-betic+glibenclamide (group 2), diabetic+plant extract (group 3), untreated control (group 4) and control+plant extract (group 5) groups (n = 6 for each group). serum glucose, lipid levels and body weight changes were mesasured and pancreas and liver histology of the rats were examined. each rat in all groups were administered the plant extract (30 mg), and the reference drug glibenclamide (2 mg/kg) by gastric gavage every day for 8 weeks. in group 1, blood glucose, serum alt, ast, triglyceride, cholesterol and ldl cholesterol levels increased while body weights decreased. in group 2, serum glucose, alt, ast, triglyseride and hba1c levels decreased compared to group 1 while cholesterol and ldl levels were high. in group 3, serum alt, ast, trigliserit, cholesterol, ldl levels decreased significantly but serum glucose and hba1c were higher compared to group 4. body weights increased except group 1 and hdl levels were not altered. histologically degenerative changes observed on pancreas of group 1 were decreased in groups 2 and 3. there was no difference on liver histology of the groups. in conclusion, thymbra spicata showed a protective and regenerative effect on diabetic pancreas. the hypo-lipidemic effect of the plant extract was also more effective than glibenclamide possibly due to the flavonoids, saponins and triterpenoids contents in the extract. its hypoglycaemic and protective activity should be tested for different doses and extract preparations and for longer periods. our study suggests that thymbra spicata is an excellent candidate for future studies on diabetes mellitus. with three different transcriptome data sets from the public gene expression omnibus database: time dependent data of dphop mutant, dargr mutant and wild type strain. the dynamic data spanned both primary and secondary phases of the metabolism. statistical results of transcriptome data were used for reporter metabolite analysis and reporter pathway analysis, which identify the metabolites (or pathways) with a significant coordinated transcriptional change in response to gene deletion perturbation in phosphate and nitrogen metabolisms. further, the production of actinorhodin, a pharmaceutically important compound, was modeled in the two deletion strains by calculating the metabolic fluxes subject to transcriptional level constraints on enzyme-coding genes. the metabolic switch from primary to secondary metabolism was highlighted in terms of the activity of pathways and fluxes as a result of the computational analyses in this work, leading to a better understanding of the role of phosphate and nitrogen metabolisms in increasing production levels. introduction: as a member of legume family licorice (glycyrrhiza glabra l.) has been widely used by human kind for many years as food constituent. especially by folks in rural sites licorice consumed widely. beside food constituent licorice has been used for medical purposes as well. licorice found effective with scientific datas on peptic skin infections, ulcers, inflammation, eczema, alzheimer disease, liver disease, and cancers. it also has been used as natural sweetener and food additive for preparing candies, chewing gum and beverage since ancient times. like all other medicine it has not been free of adverse event or toxicological effects. material and methods: alcoholic extracts of plant obtained by maceration process. for in vitro examination of anti-oxidant profile of licorice dpph free radical scavenging, abts cation radical scavenging and cupric ion reducing antioxidant capacity assay applied. application of extract made by oral route to rats for a week. anti-oxidant profile has been evaluated by myeloperoxidase (mpo), arylesterase (ares), total oxidative stress (tos) and total antioxidant status (tas) of serum levels. determination of toxicological effects alt, ast, ldh and alp values studied. histological investigation applied on liver and kidney tissues. results and discussion: results compared with control and standarts. antioxidant potential of licorice has been observed by in-vitro assays. serum mpo and ares values also compared with in-vitro results and correlation between them has evaluated. toxicological investigations made after evaluation of ast, alt, ldh and alp values. conclusion: in vitro assays has showed that licorice has potential anti-oxidant effect. investigation revealed that a mild toxic effect of licorice by biochemical tests. toxicological profile compared with control group and alt, ast values found slightly decreased and a mild elevation has been seen in ldh and alp values. for further and detailed investigation is needed. p-03.01.1-020 on the applications of a metabolic network model of mesenchymal stem cells h. fouladiha, s. a. marashi, m. a. shokrgozar, m. farokhi mesenchymal stem cells (mscs) have several applications in tissue engineering and regenerative medicine. mscs can be very useful in stem cell therapy, because they can be isolated bone marrow or adipose of an adult. these cells have also been used as gene or protein carriers. therefore, maintaining them in a desire metabolic state has been the subject of several studies. here, we have used a genome scale metabolic network model of bone marrow derived mscs for exploring the metabolism of these cells. then, we try to validate the computational results by experimenal tests. we analyzed metabolic fluxes in order to increase stem cell proliferation using the metabolic model. consequently, the experimental results were in consistency with computational results. in the present work, the applicability of the metabolic model was successfully approved. therefore, this metabolic model can be useful in biomedical researches of stem cells. p-03.01.1-021 qtl analysis for body weight and fatness in bxd recombinant inbred mouse strains a. dogan 1 , c. neuschl 2 , r. alberts 3 , g. a. brockmann 2 1 school of medicine, istanbul kemerburgaz university, istanbul, turkey, 2 department for crop and animal sciences, humboldt-universit€ at zu berlin, berlin, germany, 3 helmholtz-zentrum f€ ur infektionsforschung, braunschweig, germany genetic variation in body weight and composition is under the influence of many genes and have different genetic architectures. in the present study, the genetic factors contributing to body weight and fatness were examined under energy rich feeding conditions. growth traits, lean and fat weight, fat mass gain were analyzed to map qtls in a set of bxd ri strains. genome-wide analyses were revealed several genomic loci that control body weight and associated bodily changes in a sex and age-specific manner. the genetic data provided evidence for significant qtls on chromosome (chr) 4, 5, 14, and 16. most likely candidate genes within or near the regions with the highest significance levels were identified. the genes 1700048f04rik, gbe1, a830060n17, and four genes cenpc1, stap1, uba6, gnrhr for example, are suggested as most likely positional candidates accounting for the qtl effects on chr 5 for fat mass, on chr 16 for fat mass gain and on chr 16 for lean weight, and chr 16 for body weight, respectively. our results showed that body composition and fatness are highly complex that many genetic factors regulating and suggested candidate genes, which may help for studies of human fatness. related to serotonergic and gaba systems in response to hormonal changes. the nutrients involved in neurotransmitter synthesis may be the cause of relationship between diet and premenstrual syndrome. therefore, the aim of this study was to investigate the effect of various nutrients and premenstrual syndrome. this study was conducted to 29 healthy women aged 20-37 years. participants were asked to fill in premenstrual assessment form. dietary intakes (three days in each phases) were recorded during premenstrual, menstrual and postmenstrual phases. energy, protein, amino acids, iron, calcium, and magnesium intakes were estimated. statistical analyses were performed using the spss software. friedman tests were conducted and differences were considered to be statistically significant for p-values lower than 0.05. 60.9% of the participants reported premenstrual symptoms and premenstrual symptoms related nutrient intake were increased in these women. it was determined that energy (p = 0.03) and protein (p = 0.001) intakes were higher in the premenstrual phase. during premenstrual phase; tyrosine (p = 0.002), isoleucine (p = 0.001), leucine (p = 0.001), lysine (p = 0.008), methionine (p = 0.008), cysteine (p = 0.008), tryptophan (p = 0.000), and glutamic acid (p = 0.005) intakes were higher than other phases. likewise, iron intake was higher on premenstrual phase (p = 0.054). on the other hand, intake of other potential premenstrual syndrome related nutrients like fat, cholesterol, calcium, magnesium, and vitamin b6 were not significantly different within the menstrual phases. amino acids including tyrosine, tryptophan, glutamine, and vitamin b6 are involved in neurotransmitter synthesis and might be related to premenstrual symptoms. consequently, elevated intakes of dietary protein and some amino acids during premenstrual phase may be related to premenstrual syndrome symptoms. until now far uv cd spectra of only two potexviruses were published. the papaya mosaic virus (papmv) spectrum, measured by leclerc and co-authors contained no obvious anomalies and was similar to the spectrum of isolated papmv coat protein (cp). but measured 30 years earlier by homer and goodmanfar uv cd spectrum of potato virus x (pvx) itself had anomalous character and differed strongly from the spectrum of isolated pvx cp. in the present work we measured far uv cd spectra for two more members of potexvirusgenus: alternanthera mosaic virus (altmv) and potato aucuba mosaic virus (pamv) and their free cps. the altmv virion and altmv cp spectra were similar to each other and to the spectra of papmv and its cp. the pamv spectrum resembled the pvx spectrum in anomalously low ellipticity of the negative band at 208 nm, but in contrast to pvx, did not have additional peak at 228 nm. homologous modeling showed that cp of the three viruses is very similar in the core structure, and the observed difference may be explained by differences in disordered parts of proteins. possible reasons of potexvirus structural variability are discussed and it is suggested that the intravirus potexvirus cps may assume different conformations in different virions of the same preparation or even along the length of one virus particle. this work was supported by the russian science foundation (grant 14-24-00007). the antimicrobial potential of different phenolics was tested on pectobacterium in search of possible mode of action. in this respect, biofilm formation, exoenzyme activity, gene expression and virulence on its natural host (potato, cabbage, calla lily) were performed. also computational approach to show interaction between phenolic compounds and target protein was carried out using docking tools. the virulence determinants of pectobacterium were significantly impaired, at compound concentrations that did not affect bacterial cell growth. these observations suggested a mechanism which specifically interferes with bacterial virulence. since, these virulence determinants in pectobacterium are controlled by quorum sensing (qs), we focused on the effect of phenolics on the qs system in pectobacteria. the study revealed an inhibiting effect of the tested compounds on the expression level of central qs system and controlled genes, using qrt-pcr. also, there was a prominent reduction in the level of qs signal molecules n-acyl-homoserine lactone (ahl) accumulation. in addition infection capability was also practically blocked, which was completely recovered by application of exogenous-ahl. these results were supported by a potential interaction of plant phenolics with qs targets, as shown by molecular docking tool. collectively, results suggest the potential interference of phenolic compounds with qs central components (expi/expr proteins). moreover, it holds potential for future development of control measures against pectobacterium, and possibly other pathogens with similar mode of virulence. saccharomyces cerevisiae has been a key experimental organism for the study of infectious diseases, including double-stranded rna (dsrna) viruses. the l-a dsrna virus family of s. cerevisiae is widely distributed in nature. several versions of l-a virus are described and new ones continue to be discovered. some s. cerevisiae strains along with l-a dsrna possess smaller dsrnas, called m satellites. these dsrnas encode a sole secretable protein, known as k1, k2, k28 and k-lus toxin. l-a genome encodes the gag major structural protein and gag-pol fusion protein, formed by ribosomal frameshifting. gag-pol has transcriptase and replicase activities are necessary for maintenance of both l-a and m satellite dsrnas. so far, it's not known whether certain l-a virus has evolved to maintain a distinct type of satellite dsrna or this phenomenon lacks inherent specificity. we developed universal strategy to obtain full length l-a and m dsrna genomes from s. cerevisiae. complete viral dsrna genomes can now be cloned, as evidenced by l-a-28 dsrna, analyzed and sequenced directly from any yeast strain by means of enzymatic manipulations on total or fractioned rna content. we have identified previously undescribed l-a variant from different yeast strains specifically associated with certain type of m satellites. moreover, we identified for the first time full 5 0 -utr and 3 0 -utr sequences of m2 satellite. highly conserved sequence regions along with variable fragments were discovered at protein level, revealing clear trend to form clusters among different l-a gag-pol proteins. the obtained data suggest that each l-a virus variant can specifically maintain a distinct type of satellite dsrna. p-04.01.1-004 physic-chemical characterization of plga adjuvants for immunization per os t. chudina, d. kolybo palladin institute of biochemisry of the national academy of sciences of ukraine (nasu), kyiv, ukraine antibodies against diphtheria toxin play the most important role in the immunity against corynebacterium diphtheriae. all current diphtheria vaccines have parenteral route of administration. undoubtedly, oral administration of antigens would be the most patient-friendly way of immunization. however, the efficacy of free antigens oral administration is limited by their degradation in the gastrointestinal tract and poor absorption by m-cells. biodegradable and biocompatible polymers, like poly (d,l lactide-co-glycolide) (plga), are widely used for the design of mucosal immunizing agents. importantly, that the way of particle preparation plays an important role in plga biodegradation and antigen release. the aim of this work was to characterize the main physic-chemical properties of two types of plga particles: with immobilized antigen (plga 1) and with encapsulated antigen (plga 2) . we have prepared two types of plga particles containing egfp-sbb proteins (non-toxic recombinant fragment b of dt fused with egfp). the antigen loading efficiency of particles was determined based on the ratio of protein concentration in solution before and after loading and shown better results for plga 2 particles (plga 1 -72.05%, plga 2 -90.02%). the flow cytometry results demonstrated that 99% of plga1 particles conjugated with egfp-sbb, and only 92.2% of plga 2 particles conjugated with protein.the particle sizes had the slight difference by the results of two different techniques (ntanumber based, the software tracks individual particles; dls -scattering intensity weighted), however demonstrate similar patterns. dls data showed that the mean plga 1 particles size was 203.3 nm and plga 2 -211.6 nm. nta data also showed that mean plga 1 particles size a little smaller than plga 2 (183.8 nm and 192.8 nm respectively) . demonstrated differences in the properties of synthesized particles may have an influence on the immunogenicity of the used for oral immunization antigen. p-04.01.1-005 a suitable system for studying the functionality of a plasmodial protein in mammalian cell lines cho-mt58, a mutant cell line was proved to be an appropriate tool for investigating intracellular function of cct. in this cell line, the endogenous cct activity decreases dramatically at 40°c, blocking membrane synthesis and ultimately leading to apoptosis. we have studied the rescuing potential of pfcct in cho-mt58 cells with the isogenic cho-k1 cells as a control. cells after transient transfection were incubated at 40°c and then analysed by facs using the fluorescence of egfp fused to pfcct. the proportion of cells undergoing apoptosis was determined by propidium-iodide staining. we have demonstrated for the first time that heterologously expressed pfcct is able to complement endogenous cct activity in mammalian cells. thus, a suitable system has been established for functional investigation of structural elements of pfcct. in order to reveal the role of different protein sequences in enzymatic function, we redesigned the structural gene of pfcct obtaining a modular system where different domains are easy to be removed or exchanged. here we designed a series of different truncation and deletion constructs to reveal the role of plasmodium specific sequences. in parallel, heterologous expression experiments of different constructs in the mutant cho-mt58 and the wild type control cell lines are performed to validate the reported model system. p-04.01.1-006 host-pathogen interactions: is there a relationship between tlr 4 polymorphisms and tuberculosis in a group of turkish patients? introduction: tuberculosis (tb) is a global health problem and according to world health organization (who) each year more than 2 million individuals die from tb and each year20,000 cases of tb are notified in turkey. malatya is the third largest city in east anatolian region of turkey and tb incidence rate is higher (28.5/100,000) comparing to the general population of the country. for this reason it is important to determine the factors that lead to tb in this population. disease agent can stay in the latent phase for long periods of time after infecting the individuals. while some infected individuals show the symptoms some others never do and even 90% of these never develop clinical disease. various mechanisms take place during the host response to infectious agents. toll-like receptor (tlr) genes are shown to be candidate genes in these responses. materials and methods: in this study 49 tb patients and 50 healthy controls were included. tlr 4 genotyping for rs4986790, rs4986791 was performed by using a commercial taqman snp genotyping assay kit. data were summarized by count and percent. hardy-weinberg equilibrium was tested by chi-square distribution with 1 df. differences between groups due to allelic and genotypic distributions were analyzed by pearson's exact or fisher's exact tests. in all comparisons significance level was considered to be 0.05. results: the single nucleotide polymorphisms (snps) which were subject of this study haven't been screened in turkish population earlier. no significant association was found between tb and the snps we screened in our group of patients. discussion and conclusion: unlike other populations results we couldn't find a significant association between the disease and the genotypes of our patients. the study should be performed in bigger populations in order to confirm the results. p-04.01.1-007 lytic action of bacteriophages as a tool for the obtaining of images p. boltovets 1 , r. radutny 2 , t. shevchenko 3 1 institute of semiconductor physics nas of ukraine, kyiv, ukraine, 2 scientific and technical center of advanced technologies nas of ukraine, kyiv, ukraine, 3 taras shevchenko national univercity of kyiv, kyiv, ukraine obtaining of images by different types of bacteria now became a very special branch of skill at the interface between science and art. however the authors did not found any scientific article, where bacterial lawn was used as the background and the image was formed by the lytic action of the virus (bacteriophage). whereas the mentioned approach could be used not only with artistic aims but for the practical use. the aim of this work was to demonstrate a possibility to obtain the image on the bacterial lawn by the lytic action of the bacteriophage. the bacterial lawn was obtained by the standard metod using the 1.5% agar with the nutrient medium and the 0.7% agar containing escherichia coli culture. stencils with the preparation of the bacteriophage t4 were applied. samples were incubated during the twenty-four hours at +37°c. after that stencils were removed and the samples were stained by coomassie blue r-250 or fuchsine (with further fixation by the 7% acetic acid). several approaches to obtain the image by the lytic action of the virus were applied. first of all stencils made from printing paper and filter paper were compared. it was demonstrated, that the use of filter paper stensil allows to obtain more accurate and controllable images, than the use of the printing paper stensil. in the next series of the experiment the possibility of the reversed stencil use (where the image is formed not by the lytic zone but by the zone of bacterial growth) was demonstrated. also the possibility of the partial staining of the obtained image was explored. it gives an opportunity to obtain polychrome images using available colorants. summarizing the above it should be noted, that it was the first time when the graphical image was obtained by the lytic action of the virus on bacteria. this approach could be used not only for the artistic aims but as well for the practical use, for example, for the restriction of the action of microorganisms in out-of-theway places. burgdorferi the identification and characterization of possible antigens is essential for the improvement of current laboratory diagnostics for lyme disease and vaccine development. in this study, several recombinant b. burgdorferi outer surface proteins have been obtained and their antigenic properties have been evaluated in an effort to characterize novel immunodominant antigens. because b. afzelii and b. garinii are the most prevalent species in latvian ticks, proteins with conserved domains were included in this study. a panel of serum samples of lyme disease patients with early and disseminate disease stage was used. the controls were matched by age and sex to the patients and represented the same geographic area. the results show that proteins of several b. burgdorferi gene families have properties with respect to their candidacy as a subunit assay for a novel lyme disease immunodiagnostic. especially, the difference in their size in a range on the western blot assay may provide good discrimination between protein bands. however, they have potential for diagnosis if used in combination with other antigens but not as a "stand alone" test. in conclusions, this study showed the existing challenges in serological testing of early lyme disease. the conservation of the sequence of antigen between species of b. burgdorferi complex is essential for the most successful serodiagnostic marker candidate. the presence of homologous proteins in treponema species could lead to the cross reactivity in syphilis patients, and should be carefully evaluated. antimicrobial resistance is one of the greatest challenges in modern medicine. there is a pressing need for better understanding of the specific mechanisms that contribute to resistance to optimize existing therapies. in 2013 in georgia extended-spectrum beta-lactamase (esbl)-producing e. coli strain was isolated from the post-surgical sample obtained from gallbladder of the patients with chronic calculous cholecystitis which belongs to the sequence type 23 (st23) complexes with ctx-m 55 gene. is this strain characterized by other differences on a proteome level? are antibiotics against which the strain is resistant inducing the changes in bacterial proteome? the present work was aimed (i) to study the differences on a proteome level (i) between e. coli 92-1917/13-g and attc e. coli-reference strain and (ii) to compare the proteomes of 1917 strain at two conditions: with and without antibiotics. 1917 strain was grown in the presence of three antibiotics: rocephin (ceftriaxone), fortum (ceftazydym) and claforan (cefotaxime sodium) together. proteomic expression was analyzed using two-dimensional gel electrophoresis and mass spectrometry. significant differences were found for several proteins, including putative abc trnsporter arginine protein 2, cystine-binding periplasmic protein, fkbp-type peptidyl-prolyl cis-trans isomerase, outer membrane protein a, d-galactose binding periplasmic protein and some others. the importance of these differences for anti-microbial resistance will be discussed. p-04.01.1-010 molecular characterization of resistance and virulence features in staphylococcus aureus clinical strains isolated from cutanaeus lesions in patients with drug adverse reactions i. lupu 1 , i. gheorghe 2, 3 , m. popa 2, 3 , a. ion 3 , m. mihai 1 , v. lazar 2, 3 , m. c. chifiriuc 2, 3 1 carol davila" university of medicine and pharmacy, bucharest, romania, 2 research institute of the university of bucharest-icub, bucharest, romania, 3 faculty of biology, university of bucharest, bucharest, romania patients treated with epidermal growth factor inhibitors often experience cutaneous adverse reactions. however, the infectious complications of these toxic effects and the contribution of specific pathogens, such as the community emergent methicillin resistant staphylococcus aureus strains. the present study was aimed to identify the types of sccmec and virulence genes profile in clinical s. aureus isolated from cutaneous lesions of different severity degrees in patients with dermatologic toxic effects. this study was conducted on a total of 42 s. aureus clinical strains isolated in 2016 from acneiform reactions pustulae and periungual lesions in patients with drug cutaneous adverse reactions. multiplex pcr was performed on genomic dna from isolates in order to identify the sccmeccentral elements and the virulence genes: bbp (bone bound sialoprotein), ebps (elastinbinding protein), fnbb, fnba (fibronectin-binding proteins), fib, clfa, clfb (clumping factors a and b), cna (collagen-binding protein), luk-pv (panton-valentine leucocidin), hlg (haemolysin), tst (toxic shock toxin). the mrsa phenotype was genetically confirmed by the presence of meci gene in case of 19.04%, meca in 14.28%, sscmec type ivd element in 11.90%, ccrb2 in 7.17% and sccmec types i, iii, iv in 4.76% of the studied s. aureus strains. regarding the virulence genes encountered in s. aureus strains, the most frequent was clfa (90.47% of the isolates), followed by clfb (88.09%), fib (35.71%), hlg (16.66%) and bbp (14.28%). these results confirm the high prevalence of mec i and sscmec type iv elements, usually encountered in communityacquired mrsa strains, in cutaneous isolates from patients with dermatologic toxic effects. more data on the virulence and genetic background of these local strains are needed to appropriately assess the risk of such infections and avoid the inappropriate administration of beta-lactams. p-04.01.1-011 analysis of toxicogenic properties of staphylococcus aureus strains isolated from cows with subclinical form of mastitis in the central area of russian federation. pore-forming toxins and enterotoxins), which are present in s. aureus strains isolated from clinically healthy cows. staphylococcus strains were isolated from cow's milk. disk diffusion method was used to determine the sensitivity to antibiotics. pcr analysis was used for detection of meca, mecc genes and genes of toxins. investigated strains were resistant to oxacillin (14%), vancomycin (8%) . it was found that all strains, which contain meca and mecc genes, showed resistant to more than 5 antibiotics. it was determined that among the investigated strains 13% contained meca, 13% -mecc, 9% contained both meca and mecc. some strains contained genes of panton-valentine leukocidin (pvl) or alpha-hemolysin and several strains contained both types of genes. enterotoxin a (sea) gene was detected in 26.7% of cases, sed -5%, seg -10%, sei -35%. genes of staphylococcal toxins b, c, e, h were not found. the presence of phenol soluble modulin biosynthesis genes was determined: genes of alpha peptide synthesis were found in 93% of strains, beta peptide toxin genes in 73%, delta toxin gene in 100%. it was determined, that clinically healthy animals are carriers of s. aureus strains that cause mastitis. high level of antibiotic resistance was found in strains containing meca and mecc genes. the major part of the strains carried genes of phenol soluble modulin biosynthesis. the role of phenol soluble modulins as well as of pvl and alpha-hemolyzin in the development of mastitis is not completely clear. we conclude that pore-forming toxins have dominant role in the latent form of mastitis. p-04.01.1-012 impact of lactoferrin on the hydrophobicity and adherence to the inert substratum of staphylococcus aureus strains isolated from patients with cutaneous drug reaction skin healing is a complex biological process that requires the involvement of different cell types and humoral effectors. one of the main factors are aggravating and delaying the healing process is represented by the supra-infection with pathogenic or opportunistic microorganisms that grow in specialized consortia embedded in a self-produced extracellular polymeric matrix, called biofilms, which are extremely resistant to any antimicrobials and host immune response. lactoferrin (lf) is an ironbinding glycoprotein which promotes skin healing by enhancing the initial inflammatory phase, but also by inducing an overabundant immune response. the aim of this study was to investigate the influence of lf, one of the main components of innate, humoral anti-infectious immunity on some microbial features, involved in the first steps of the infectious process, such as hydrophobicity and adherence of staphylococcus aureus strains isolated from maculo-pustular lesions in patients with adverse reactions to epidermal growth factor inhibitors. for hydrophobicity measurement the bacterial suspensions were grown in the presence or absence of lf, and then, the "microbial adherence to hydrocarbons test" (math) was performed. the capacity to develop biofilms on inert substrata and the influence of lf on this feature was spectrophotometrically quantified using an adapted microtiter method, after crystal violet staining. our results showed that lf decreased the hydrophobicity and limited the biofilm development of all 42 s. aureus tested strains, in a dose and time dependent manner. the decreasing effect on the microbial hydrophobicity was accompanied by a lowering effect on the adhesion of microbial strains to the inert substratum. in conclusion these observations indicate that lf exhibits a wound pro-healing effect, by limiting the microbial colonization and biofilm formation and thus, the occurrence of infectious complications of skin lesion. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] host-specificity determinants of bacteriophage vb_ecom_fv3 considered vehicles of s.aureus intoxication in humans throughout the world. the objective of the present study was to assess the presence of enterotoxigenic and methicillin-resistant s. aureus in water buffalo milk and dairy products. a total of 100 water buffalo milk and 100 dairy products (50 water buffalo cream and 50 water buffalo cheese) were collected from different dairy farms, smallholders and local bazaars in samsun, turkey. all samples were analyzed using the standard procedure en iso 6888-1 and isolates were confirmed for the presence of the 16s rrna and nuc gene by polymerase chain reaction. s. aureus was identified in 30 of 100 water buffalo milk (30%), 9 of 50 water buffalo cream (18%), and 17 of 50 water buffalo cheese (34%). a total of 99 isolates were confirmed as s. aureus by pcr. genotypic methicillin resistance was evaluated using pcr for the meca gene. out of 99 isolates, 9 (9%) were found to be methicillin resistant (meca gene positive) by pcr. the enterotoxigenic s. aureus was identified in 12 out of 99 (12%) isolates by the mpcr technique. five isolates produced staphylococcal enterotoxins sea (5/12; 41.6%), two isolates produced sec (2/12; 16 .6%), one isolate produced (1/12; 8 .3%) sed, one isolate produced (1/12; 8.3%) see and three isolates produced sec+sed (3/12; 25%) . none of samples were positive for seb. in conclusion, the presence of enterotoxigenic and methicillin-resistant s. aureus in milk and dairy products is of significant for public health concern and also these enterotoxin genes sea and sed are predominant toxins that can cause staphylococcus intoxication in humans. this study was funded by ondokuz mayıs university, samsun, turkey, scientific research project programs (project no: pyo. vet -1904.12 .004) and this article was part of a phd thesis. p-04.01.1-015 identification and biochemical characterization of an immune modulating protein from helicobacter pylori b. kaplan t€ urk€ oz faculty of engineering, department of food engineering, ege university, izmir, turkey helicobacter pylori is able to achieve persistent infection with minimal immune response. the first line of defence during h.pylori infection is through gastric epithelial cells which present toll like receptors (tlr). a family of bacterial proteins which share homology with the toll/il-1 receptor (tir) domain were identified. the structure of btpa from brucella showed that bacterial tir proteins (btp) mimick human tir domain proteins and act on myd88 signaling pathways to suppress tlr signaling. h.pylori might also produce a similar protein. a putative h. pylori tir protein was found based on sequence homology and the corresponding gene; hp1437; was cloned in fusion with an n terminal cleavable 6his-tag. the recombinant protein, 6his-1437 was purified using nickel affinity chromatography. 1437 was subjected to limited proteolysis and the bands were analyzed by peptide mass fingerprinting (pmf). oligomerization of 1437 was investigated by in vitro pull-down and size-exclusion chromatography. 1437, a 239 amino acid protein, has a predicted c terminal tir domain similar to other btps and sequence alignments verified the presence of tir domain signature regions. recombinant 6his-1437 was produced with a yield of 10 mg/l culture. a structurally stable 25 kda fragment was obtained from limited proteolysis which contained the tir domain as verified by pmf. in vitro pull down assays showed 1437 interacts with itself forming dimers as shown by size-exclusion chromatography. tir domain proteins function by interacting with themselves and other tir domains. our results showed that 1437 also form dimers, supporting that it is a btp. current research is focused on solving the structure of 1437 and investigating its interaction with myd88. 1437 might play a direct role in reduced immune response against h.pylori by binding to myd88 analogous to other btps. further characterization of 1437 will provide the first solid evidence of presence of a tir domain protein in h.pylori. p-04.01. [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] lipopolysaccharides with different lipid a acylation status from vibrio cholerae and campylobacter jejuni contribute differently to il6 production by bone marrow-derived macrophages k. korneev 1,2 , e. sviriaeva 1, 2 lipid a is a biologically active part of lipopolysaccharide (lps) from gram-negative bacteria that is responsible for the activation of the innate immunity through interaction with toll-like receptor 4 (tlr4) and subsequent production of proinflammatory cytokines. bacteria frequently transform their lipid a so that its recognition by tlr4 is not sufficient for induction of effective antibacterial immune response. we compared biological activity of various lps from pathogenic bacteria vibrio cholerae and campylobacter jejuni. we purified r-form lps for each strain by hydrophobic chromatography. the biological activity of lps preparations was evaluated by their ability to activate production of proinflammatory cytokine il6 by bone marrow-derived macrophages from c57bl/6 mice, using tlr4-deficient macrophages to control for specificity of tlr4 signaling. lps from e. coli and inactive lps from f. tularensis were used as positive and negative controls. lps from v. cholerae demonstrated biological activity similar to that of lps from e. coli, consistent with the presence of highly acylated lipid a in both strains. however, the former was a slightly weaker activator than the latter, because lipid a from v. cholerae had on average shorter acyl chains. lipid a from c. jejuni had on average longer acyl groups than in e. coli, while degree of acylation was lower, and as a result its lipid a displayed significantly lower biological activity. our study demonstrates importance of functional groups of lipid a in the ability of lps to activate production of il6 by macrophages. in line with our previous reports, we confirmed a direct correlation between biological activity of various lps species with their lipid a acylation status: the biological activity increases with increase in the length and in the number of the acyl chains. excess proinflammatory cytokine production through tlr4 activation can cause sepsis, while inefficient activation may result in the failure to clear bacteria. clostridium perfringens phospholipase c (cpplc) is the most toxic extracellular enzyme produced by this bacterium and it is an essential virulence factor in the pathogenesis of gas gangrene. cpplc may lead to cell lysis at concentrations that causes extensive degradation of plasma membrane phospholipids. however, at sublytic concentrations it induces cytotoxicity without causing evident membrane damage. the results of this work demonstrated that the cytotoxic effect of cpplc requires its internalization and the activation of the mek-erk pathway. cpplc internalizartion occurs through a dynamin-dependent mechanism and in a time progressive process: first, cpplc colocalizes with caveolin both at the plasma membrane and in vesicles, and later it colocalizes with early and late endosomes and lysosomes. the results also showed that cpplc requires endocytosis in order to activate mek-erk, because treatment with the dynamin inhibitor, dynasore, prevents cpplc endocytosis, erk 1/2 activation and cytotoxcity. cholesterol sequestration as well as inhibition of actin polymerization also prevents cpplc internalization and cytotoxocity, involving endocytosis in the signaling events required for cpplc cytotoxic effect. once internalized, cpplc induces reactive oxygen species production through the activation of pkc, mek/erk and nfjb dependent pathways. inhibition of either of these signaling pathways prevents cpplc's cytotoxic effect. in addition, it was demonstrated that nfjb inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of cpplc in mice. these data provide new insights about the mode of action of this bacterial phospholipase c, previously considered to act only locally on cell membrane. understanding the role of these signaling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridial myonecrosis. p-04.01.1-020 apoptosis induced by clostridium perfringens phospholipase c is mediated by reactive oxygen species m. flores-d ıaz 1 , l. monturiol-gross 1 , m. j. pineda padilla 1 , c. araya-castillo 1 , a. alape-gir on 1 bacterial phospholipases are lipolytic esterases surface associated or secreted by a wide variety of bacterial pathogens. clostridium perfringens, the most broadly distributed pathogen in nature, secretes a prototype phospholipase c (plc), also called a-toxin, which plays a key role in the pathogenesis of gas gangrene. this toxin causes death to cultured cells and extensive myonecrosis when injected intramuscularly in experimental animals. the results of the present study showed that c. perfringens plc (3-5 ng/ml) induces morphological and biochemical changes characteristic of apoptosis in cultured cells, as determined by scanning electron microscopy. nuclei condensation and fragmentation were observed by fluorescence microscopy and a typical ladder fragmentation pattern of genomic dna was detected by dna in agarose gels. cell death was prevented by the caspases inhibitors z-devd-fmk and z-vad-fmk. c. perfringens plc induces oxidative stress in cultured cells as determined by fluorescence microscopy and flow cytometry using the membrane permeable probe dcfda. different antioxidants including the gluthation precursor nac, several iron chelators and the free radical scavengers tiron and edaravone prevent cell death induced by c. perfringens plc in cultured cells or in mice challenged intramuscularly with 1.2 lg of that toxin. thus, this work provides compelling evidence that superoxide, hydrogen peroxide, and the hydroxyl radical are involved in the cytotoxic and myotoxic effects of c. perfringens plc. furthermore, the data demonstrated that edaravone, a clinically used hydroxyl radical trap, reduced the myonecrosis and the mortality caused by c. perfringens in a murine model of gas gangrene, induced by intramuscular bacterial injection of 10 8 bacteria. this knowledge provides new insights for the development of novel therapies to reduce tissue damage during clostridial myonecrosis. lectins are ubiquitous proteins able to recognize mono-and oligosaccharides with high specificity and low affinity. lectins do not have any catalytic activity, unlike enzymes, and they are not products of the immune system in contrast to antibodies. lectins play a crucial role in cell interactions on molecular level showing their importance in various physiological and pathophysiological processes as well as both mutualistic and parasitic interactions between microorganism and hosts. photorhabdus luminescens is a gram-negative bacterium from the family enterobacteriaceae. the bacteria have a complex life cycle that involves mutualistic and pathogenic interaction with two different invertebrate hosts. it is highly pathogenic towards insect larvae. in addition, p. luminescens lives in the intestine of infective juveniles of nematode heterorhabditis bacteriophora, together forming an effective entomopathogenic complex. we have identified several soluble lectins produced by p.luminescens. in this study, we focus on 4 proteins from p. luminescens, which show a high sequence homology with each other. a wide range of methods was used for structural and functional studies of photorhabdus lectins, e.g. surface plasmon resonance, isothermal titration calorimetry, analytical ultracentrifugation and x-ray crystallography. all lectins from p.luminescens recognize l-fucose and d-mannose. despite being closely related, they differ in fine binding specificities. to determine their biological function, knock-out mutants of p. luminescens are being prepared to study its interaction with axenic nematodes and insect larvae. breast cancer is the major disease of women in developed countries occuring predominantly after the age of 65. triple negative breast cancer (tnbc) is a typical subtype of epithelial breast cancer which lacks estrogen receptor (er), progesterone receptor (pr) and human epidermal growth factor receptor 2 (her2) all together. although various researches have been focused on characterizing tnbc and enlightening different molecular markers with the aim of improving the overall outcome, currently the sole affective therapy action for tnbc is chemotherapy. thus chemoresistance is the main clinical challange and accounts for 90% of failures in terms of treating the disease. multidrug resistance (mdr) is defined as simultaneous resistance towards the drugs which do or do not demonstrate structural resemblance and have different effects on their molecular targets. p-glycoprotein (p-gp) is a membrane protein coded by abcb1 (mdr-1) gene. p-gp is an atp-dependent pump which pumps a wide range of drugs out of the cells including chemotherapeutic agents such as doxorubicin (dox) and pactilaxel. in the present study, tnbc cell line mda-mb-231 was treated with increasing doses of dox, cell viability was examined with srb assay and development of mdr was investigated through mdr assay and rt-pcr. results demonstrated that cell viabiliy decreased significantly with the treatment of higher doses. mdr was shown to be increased when cells were treated with 50, 200 and 800 nm of the drug respectively along with 15 lm of p-gp inhibitor verapamil. rt-pcr results were obtained to be consistent with mdr assay results and indicated increased mdr-1 gene expression with the treatment of dox. especially after 400 nm of dox treatment, mdr-1 was overexpressed to be 59 fold when compared to control. in conclusion, it was demonstrated that mda-mb-231 cells have shown to display elevated resistance to higher doses of dox. p-05.01.1-005 targeting dna damage response pathway in cancer cells under heat stress and the mechanical effect of ultrasound y. furusawa 1 , t. kondo 2 1 toyama prefectural university, imizu-shi, japan, 2 university of toyama, toyama, japan ultrasound (us) has been widely utilized for diagnosis and therapy in many medical fields. the biophysical modes of us are divided into three classes, thermal, cavitation and non-thermal non-cavitation effects. in clinical use for cancer therapy, the thermal effect was utilized for hyperthermia therapy with focusing us on cancer to rise the temperature from 41°c to 44°c, or further which could induce thermal ablation of cancers. cavitation leads to a variety of mechanical stress such as shear stress, shock wave, high pressure, and chemical stress such as free radical formation, both of which have been inferred to act simultaneously on all biological materials. it has been indicated that us induces cell killing, cell lysis, loss of viability, and loss of clonogenicity. recently, we found that heat stress as well as us without thermal effect induce not only dna single-strand breaks but also dna double-strand breaks, a most cytotoxic region of dna, in chromatin dna detected by both gammah2ax staining and neutral comet assay. in response to the stresses which induce dna damage, the dna damage sensor protein kinase, ataxia telangiectasia mutated (atm), atm and rad3 related (atr), and dna-dependent protein kinase (dna-pk) become activated form to initiate signal transduction pathways activating cell-cycle checkpoints, dna repair, and apoptosis. the molecules consisting of dna damage response pathway were expected as therapeutic targets because defects in the response to dna damage agents can be lethal. this work was designed to explore the possible therapeutic targets of the molecules in dna damage response pathways for future us-aided therapy. finally, several kinases (e.g., checkpoint kinase) on dna damage response pathway seems to be the targets for hyperthermia and us therapy. (ural branch) , ekaterinburg, russia, 2 shemyakin and ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia based on the recently synthesized (s)-(2-aminopurin-6-yl) amino acids (gly, ala, val, phe, pro), we obtained a series of novel modified nucleosides using the transglycosylation reaction. for the first time, it has been demonstrated that the corresponding nucleobases are good substrates for the genetically engineered recombinant e. coli purine nucleoside phosphorylase (conversion to nucleosides reached 90-98%). nucleosides, such as ribosides, 2-deoxyribosides, and arabinosides were obtained in high yields (70-88%). it has been found that yield in the transglycosylation reaction does not depend on the structure of the amino acid fragment. the nucleosides synthesized are considered as potential inhibitors of intracellular adenosine deaminase (ad), the increasing activity of which is observed in hepatitis, cirrhosis, hemochromatosis, obstructive jaundice, prostate and bladder cancer, hemolytic anemia, rheumatic and typhoid fever, gout, and cooley's anemia. cytotoxicity of the synthesized nucleosides was tested in the jurkat (model of human t-lymphoblastic leukemia) and el-4 (model of mice t-lymphoblastic leukemia) cell lines. the compounds studied did not exhibit cytotoxic activity compared to the activity of the known antitumor agent nelarabin. the work was financially supported by the russian science foundation (grant 14-13-01077). p-05.01.1-007 dna binding, dna cleavage, antimicrobial activities, antimutagenic and anticancer studies of a schiff base and its complexes n. yildirim 1 , n. demir 2 , m. yildiz 3 1 health services vocational school, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 department of biology, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 department of chemistry, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. the rational design and synthesis of new schiff bases and their metal complexes have been drawing great interest because of their diverse biological and pharmaceutical activities. so, exploring and designing novel molecules that have biological activities and capable of interacting with nucleic acids has a great significance for disease defence and to discover new dna-targeted anticancer drugs for chemotherapy. in this study, we report the synthesis and characterization of a novel schiff base and its ni(ii) and cu(ii) complexes. the minimal inhibitory concentration (mic) of the compounds was screened in vitro against bacteria and yeast cultures using broth micro dilution test. dna binding and dna cleavage activity of the compounds were investigated by uv-vis spectroscopy and agarose gel electrophoresis. antimutagenic activity of compounds were tested in the absence of microsomal enzymes (s9-). also, cytotoxicity of the compounds against hepg2 cell lines was assayed by the mtt (3-(4,5-dimethylthyazolyl-2)-2,5-diphenyltetrazolium bromide) method. consequently, uv-vis spectroscopy studies indicated that the compounds interact with calf thymus dna (ct-dna) via intercalative binding mode. dna cleavage activity studies showed that the cu(ii) complex can effectively cleave pbr322 plasmid dna. compounds inhibited the base pair mutation with high inhibition rate in the absence of s9. also, schiff base complex had cytotoxic activity towards hepg2 cell line, that it was found to be more potent than the control cisplatin. p-05.01.1-008 single particle electron tomography of rnap elongation complex, stalled at position +24 genome in vivo is constantly exposed to the damaging effects of the environment. single-strand breaks (ssbs) are the most frequently occurring dna lesions. accumulation of unrepaired ssbs can interfere with the cells metabolism and increase genomic instability. in vivo, ssbs are repaired in specific pathway, but, in eukaryotic nuclei, dna is organized in chromatin that could affect the accessibility of lesions to sensor proteins. breaks in a template strand induce arrest of rna polymerase ii (polii) in vitro and in vivo and can be revealed in a transcription-dependent manner. our recent biochemical studies identified two key intermediates formed during transcription through a nucleosome by rnap that are nearly homogeneous, active and stable by biochemical criteria (complexes stalled after entering 24 or 42 bp into the nucleosome; ec+24 or ec+41, respectively). hear we produced two complexes, both stalled in the +24 position, one without break in the dna, and the other with introduced ssb at position +12 of a non-template dna strand. complexes were purified using affinity chromatography and applied to a carbon-coated, glow-discharged em grid. tomographic studies were performed at ae70°in a jeol microscope at 200 kv accelerated voltage. images were recorded using a gatan ccd camera. image analysis was performed using the imod software. the resulting structure of the ec+24 complex with no break in dna consist of two domains, connected by a single dna string. the complex with a break introduced into the dna has a more compact appearance and its two domains were connected by two dna strings, thus forming an intranucleosomal dna loop. our data suggest that ssbs in a non-template strand can induce the formation of stable non-productive transcription intermediate. the inhibitory effect of ssbs onto transcription may suggest a possible mechanism for their recognition in vivo with a transcription-dependent pathway. this work has been supported by the rsf grant #14-24-00031. colorectal cancer (crc) is one of the leading causes of cancerrelated deaths in the developed countries. according to 2014 who report new incidence rate of crc in turkey is 6.5% among other cancer types. owing to difficulty of the low allele frequency variations detection, genetic association profiles of crc have not been entirely identified. low allele frequency variations mlh1 à93g>a (rs1800734) promotor substitution, mlh1 415g>c (rs28930073) exonic substitution, mthfr c677t (rs1801133) and apc 1307 t>a (rs1801155) were investigated in this study. these 4 snps "rs1800734, rs28930073, rs1801133, rs180115" are located on 1p36.3, 5q21, 3p22 respectively. colonoscopic investigations were performed on both cancer and control group. the 4 snps were genotyped using kompetitive allele specific pcr technology in 1014 cases and 805 healthy controls. statistical analysis was carried out with cochran-armitage chi-square test. in this study these 3 of the 4 snps in mlh1, mthfr genes were examined for the first time in turkish sporadic crc cases. statistical analysis showed no significant association within our turkish sporadic crc population. percentage of mlh1 à93aa genotype in group aged ≥ 65 was found to be 5.7% in cancer versus 2% in control group. moreover apc 1307a, mlh1 415c alleles were detected only 1 and 3 allele respectively. previously, apc 1307a allele was determined in 53.3% of a turkish cohort. however in the present study apc 1307a allele was detected on 1 allele only. studies showed mlh1 à93 promoter variation as a risk factor for microsatellite instabile crc but for the current study this data is not available. in spite of literature mthfr c677t and mlh1 415g>c snps were not found to be associated with sporadic crc in turkish population. this research demonstrates that importance of population based studies in multifactorial disease. p-05.01.1-010 excision of damaged bases from transcription intermediates by fpg/nei superfamily dna glycosylases k. makasheva, d. zharkov sb ras institute of chemical biology and fundamental medicine, novosibirsk, russia oxidative lesions are abundant due to constant presence of reactive oxygen species in living cells. repair of oxidative base lesions is initiated by dna glycosylases. for example, bacterial fpg and nei dna glycosylases excise oxidized purines and pyrimidines, respectively, from dna. their human homologs, neil1 and neil2, have been reported to show preference towards oxidized lesions in dna bubbles. from these observations, it had been hypothesized that neil proteins may be involved in the repair of lesions in dna bubbles generated during transcription. however, it is not presently clear how neils would behave on bubbles more closely resembling transcription intermediates (e. g., containing the rna strand), and bacterial homologs fpg and nei had never been investigated with bubble substrates. we have studied excision of either 8-oxoguanine (8-oxog) or 5,6-dihydrouracil (dhu) by e. coli fpg and nei and human neil1 and neil2 from single-strand oligonucleotides, perfect duplexes, bubbles with different number of unpaired bases (6 to 30), d-loops with dna or rna and from complexes with rna polymerase. fpg, neil1 and neil2 efficiently excised dhu located inside a bubble. fpg and neil1 was generally more active than neil2 in excision of 8-oxog from ssdna and bubbles. nei, on the other hand, was active only on dhu located in dsdna (either perfect duplex or dna/dna d-loop). fpg and neil1 also have shown activity in d-loops with rna. the presence of an additional unpaired 5 0 -tail of the third strand of d-loops didn't affect the glycosylases activity. the activity of fpg was observed in pre-assembled transcriptional complexes with e. coli rna polymerase and depended on the position of the lesion in the transcription bubble, possibly reflecting local accessibility of the lesion within the elongation complex. this work was supported by rsf (14-24-00093). nucleotide excision repair (ner) is a multistep process that eliminates a wide range of lesions in dna, including uv photoproducts and base modifications by many carcinogenic and chemotherapeutic agents. one of the advanced approaches to ner process investigation is based on reproducing the repair reaction by mixing protein extracts from mammalian cells with model linear dnas, bearing lesions. long linear dnas (137 bp) containing efficiently recognized and processed by ner system lesions (fluoro-azidobenzoyl photoactive lesion fab-dc, nonnucleoside lesions nflu and nant) in both strands have been synthesized. we have demonstrated that dnas containing closely positioned lesions in the both strands represent difficult-to-repair (fab-dc/nflu(+4), fab-dc/nflu(à3)) or unrepairable (nflu/nflu (+4), nflu/nflu(à3), nant/nflu(+4), nant/nflu(à3)) structures. besides, it has been shown that model dnas bearing 2 bulky lesions in opposite positions (fab-dc/nflu(0), nflu/nflu(0)) represent unrepairable structure as well. the model substrates with increasing distance between lesions in the duplex demonstrated the full recovery of substrate properties in ner process (fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(à21), nflu/nflu (+8), and nant/nflu(+8)), whereas the level of specific excision from nflu/nflu(à10), nflu/nflu(à21) and nant/nflu(à10), nant/nflu(à21) was approximately 50% of the nflu/dg or nant/dg dna respectively. it has been shown that modified dna-duplex (54 bp) with fab-dc has decreased structurally dependent affinity for xpc-hr23b compared to duplexes containing lesions in both strands being analyzed (fab-dc/dg, fab-dc/nflu(+4), fab-dc/nflu (à3), fab-dc/nflu(+8), fab-dc/nflu(à10), fab-dc/nflu(-21)) and increased compared to umdna. the data provide an argument that the ner system of higher eukaryotes recognizes and eliminates injured dna fragments on a multi-criteria basis. it is well known that dna plays crucial role in the biological system because of including all the genetic information for cellular function. therefore, the interaction of molecules with dna has gained interest in the medicinal chemistry to explore new anticancer agent. photodynamic therapy which is alternative cancer treatment method depends on free radicals and singlet oxygen to destroy tumor tissue via necrosis and apoptosis. phthalocyanines (pcs) are used for photodynamic therapy because of their absorption of high wavelength light ability and they have high triplet quantum state yields and long lifetimes in triplet states. also they do not have any toxic effect without light. in this study the novel synthesized 4[2-(2morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanines were used. the potential properties of phthalocyanine compounds for photodynamic therapy were purposed to reveal by the preliminary work. for this aim, the mode of dna binding, photocleavage and topoisomerase i inhibition of these compounds were investigated. 4[2-(2-morpholin-4-ylethoxy) ethoxy]phthalonitrile substitued zinc(ii), manganese(ii) and copper(ii) phthalocyanine compounds have been synthesized. the interaction of novel pcs compounds with calf thymus (ct) dna was investigated by using uv-vis spectroscopy, thermal denaturation studies and viscosity measurements. additionally, dna photocleavage and topoisomerase i inhibition studies were performed to pbr322 dna by using agarose gel electrophoresis. the interaction studies indicated that pcs compounds powerfully bound via an intercalation mechanism with ct-dna. these compounds showed efficiently dna photocleavage under irradiation at 650 nm. the all of pcs inhibited topoisomerase i in a dose-dependent manner. all the experimental studies showed that pc compounds might be used agents for photodynamic therapy. p-05.01.1-014 target search by base excision repair dna glycosylases e. dyatlova, g. mechetin, d. zharkov institute of chemical biology and fundamental medicine, novosibirsk, russia the problem of rapid target search in dna is faced by transcription factors, restriction endonucleases, dna repair enzymes and other sequence-or structure-specific dna-binding proteins. theoretically, the fastest target search in dna can be achieved by combining one-dimensional diffusion along the dna contour (processive search) and three-dimensional diffusion (distributive search). the balance between these search modes depends on many factors affecting dna-protein interactions, such as the presence of mono-and divalent cations, competing proteins, crowding effect, etc. presently, the mechanisms of target search are understood only for a handful of enzymes. we have recently developed an assay to study target search by dna repair enzymes, based on cleavage of oligonucleotide substrate containing two targets. thus, the distance between the targets can be precisely controlled, and any modification can be introduced into dna. subsequently, the probability of correlated cleavage (p cc ) is estimated, reflecting the efficiency of enzyme transfer between the specific sites. in this work, we have investigated five repair enzymes: e. coli endonuclease viii (nei), its human homologs neil1 and neil2, and uracil-dna-glycosylases (ung) from e. coli and vaccinia virus. as expected, p cc of all enzymes depended on the ionic strength of the solution and the presence of mg 2+ . ung from vaccinia virus was the most sensitive to these factors, raising questions about its proficiency as a suggested processivity factor of viral dna polymerase. nei, neil1 and neil2 showed a peak of p cc at low but non-zero ionic strength indicating that nonpolar interactions contribute to binding of these proteins to nonspecific dna. this conclusion was also supported by analyzing amino acid conservation in the catalytic core of nei. introduction of bulky fluorescent group between two specific sites greatly reduced the ability of glycosylases to slide along dna. this work was supported by rsf (14-24-00093). p-05.01.1-015 does causes 1800 mhz magnetic field application kras and p53 mutations in colon?: occurences histopatologically and microbiologically changes in colon determination of kirsten rat sarcoma (kras) and p53 gene mutations in colon. materials and methods: in this study, three groups were prepared as control,sham and electromagnetic field (emf) group.1800 mhz radiofrequency (rf) radiation was produced by using an electromagnetic energy generator.the emf group rats were exposed to electromagnetic field for 12 weeks as 45 minutes per day.at the end of experiments, rats were sacrificed under ethyl ether anesthesia and the rat colons were dissected.fecal speciments were collected.fecal dna (for detection of fusobacterium and bacteroides) and colonic dna (for detection of kras and p53 mutations) were isolated.rt-pcr tchnique was used for detection of bacterias and mutations. results: no any differences was observed histopathologically between control and sham groups.erosions and partial losses were observed at mucosal epitelium in the emf group.the corrupted gland structure, the mucosal edema and the inflammatory cell infiltration were observed.the amout of collagen was increased and fibrosis was detected in emf group.goblet cell number decreased statistically significant when compared to control and sham groups (p < 0.05).the amount of fusobacterium increased significantly in emf group compared to controls.the difference was not detected between groups in the amount of bacteroides.all the samples analysed for kras and tp53 mutations in the colon tissue were found to be wild type.no significant difference was observed between the control group and the emf applied group. discussion and conclusion: in conclusions,for 12 weeks 45 minute/day exposure to 1800 mhz emf caused histopatological damage in rat colon.the amount of fusobacterium is increased.emf exposure did not caused to kras and p53 mutations in colon tissue. p-05.01.1-016 synthesis, antimicrobial activity, genotoxicity, dna binding and dna cleavage studies of new glycine methyl ester derivative schiff base there has been an increasing focus on the binding study of small molecules to dna during the last decades, since dna is an important genetic substance in organisms. therefore, the current growing interest in small molecules that are capable of binding and cleaving dna is related to their utility in the design and development of synthetic restriction enzymes, new drugs, dna agents, and also to their ability to probe the structure of dna itself. in recent years, schiff bases have found increased application in pharmaceutical research, organic synthesis, and bio-processes. schiff bases are considered as favored and the most widely used ligands, due to their metal complexes having variety of applications as antibacterial and anticancer agents. in this study, we report the synthesis and characterization of a novel glycine methyl ester derivative schiff base. the minimal inhibitory concentration (mic) of the compound was screened in vitro against bacteria and yeast cultures using broth micro dilution tests. antimutagenic activity of compound was tested in the absence of metabolic activation. also, dna binding and dna cleavage were investigated of compound by uv-vis spectroscopy and agarose gel electrophoresis respectively. consequently, this compound differs significantly in its activity against tested microorganisms. this difference may be attributed to the fact that the cell wall in gram-positive bacteria is a single layer, whereas the gram-negative bacteria cell wall is a multilayered structure, and the yeast cell wall is quite complex. the compound inhibited the base pair mutation in the absence of s9 with high inhibition rate. uv-vis spectroscopy studies of the interactions between the compound and calf thymus dna (ct-dna) showed that the compound interacts with dna via intercalative binding. to date a large number of the sequences in the human genome (g4 motifs) with the potential to form a spatial structure, gquadruplexes is known. g4 motifs were found in the promoter regions of most of the known oncogenes. recent experimental studies have shown that genome instability directly related to the non-canonical dna structures, including g-quadruplexes. in this work we study the distribution of somatic snvs within the g4 motifs in tumor samples with the aim to identify involvement of the motifs in the process of mutagenesis in pancreatic cancer. using the access kindly provided by the international icgc consortium to the database, we analyzed 68 samples of pancreatic ductal adenocarcinoma and 27 samples of pancreatic endocrine neoplasms. we considered only the promoter regions as the richest with g-quadruplex motifs. we found that quadruplex sequences have the ability to focus somatic snvs. this could be explained by the errors of polymerase during replication through secondary dna structures. furthermore, the snvs occur much more often in loops of g4 motifs than in g blocks, without changing the motive. in addition, t>g(a>c) and t>c(a>g) substitutions occur significantly more likely in loops which in turn stabilize the g-quadruplex structure. the cancer-related mutations tend to increasing the length of g blocks. the conservation of g4 motifs may indicate an important functional significance of g-quadruplex structures in human genome. supported by project no. 16-14-10396 of the russian science foundation. background: multiple myeloma (mm) is a rare, leading to bone destruction and marrow failure, largely incurable malignant disease of plasma cells. anemia (mostly normocytic normochromic) is seen in most patients. mean platelet volume (mpv) is a laboratory marker of platelet function and activity, the most accurate measure of platelet size. the aim of this study was to investigate the mean platelet volume (mpv) values in this disease. materials and methods: whole blood samples were collected from 60 healthy controls and 105 patients with mm. the mean age for controls and patients were 54.5 ae 8.5 and 57.4 ae 8.1 years, respectively. mpv levels were calculated with cancer is a chronic disease in the world which is the second leading cause of death, after cardiovascular diseases. benzimidazoles have been known to act as antiproliferative or anticancer agents in chemotherapeutic drug research area. in this regard we aimed to investigate the cytotoxic and apoptotic properties of novel benzimidazole derivatives bearing pyridyl/pyrimidinyl piperazine moiety against a549 lung adenocarcinoma cells. a549 lung adenocarcinoma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by mtt assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic 50 values of the compounds were determined for a549 cell line. compounds 4, 7 and 12 which were including 4-chlorophenyl, 4-nitrophenyl on pyridine ring; 4-fluorophenyl on pyrimidine moiety, had significant cytotoxic activity with ic 50 values lower than 55.33 ae 23.40 lg/ml. compound 12 showed the highest cytotoxic activity with a ic 50 value of 16.67 ae 2.24 lg/ml, whereas cisplatin ic 50 values were 14.0 ae 2.0 lg/ml lg/ml against a549 cells. cytotoxic activity of compound 4 and 7 with a ic 50 value were 55.3 ae 23.4 and 21.7 ae 8.4 lg/ml, respectively. also, compound 12 showed the highest population of early apoptotic cells (39.4%) of the tested compounds which was 5.88-fold higher than for cisplatin. compound 7 produced a comparable population of apoptotic cells with a percentage of 9.5%, respectively according to cisplatin's percentage of 6.2%. it was determined that synthesized compounds 4, 7 and 12 had considerable anticancer activity against a549 cell lines compared to cisplatin. compound 12 including 4-florophenyl on pyrimidine ring was the most cytotoxic compound against the a549 cell line. our study results demonstrated that compound 7, 12 also induced apopototic pathway on a549 cells. p-05.01. in vitro/in vivo antimitotic activity and structure-activity relationships of new glaziovianin a isoflavone series glaziovianin a (gva), isolated from the leaves of the astelia glazioviana, demonstrated cytotoxicity, disrupting microtubule structure and dynamics of hl-60 cells. the aim of the present work was to devise a concise synthetic route toward gva and its derivatives in order to expand structure-activity relationship studies and to investigate their anti-mitotic effect. a concise six-step protocol for the synthesis of gva and its alkoxyphenyl derivatives 9 starting with readily available plant metabolites from dill and parsley seeds was developed. the sea urchin embryo tests confirmed that gva directly affects tubulin/ microtubule dynamics and structure. the b-ring substitution pattern of gva derivatives exhibited strong effects on activity. according to the assay results, the anti-mitotic activity decreased in the following order: gva > myristicin ≥ 3,4,5-trimethoxyphenyl = 4-methoxyphenyl > dillapiol > 3-methoxyphenyl> 3,4dimethoxyphenyl > 2,3,4,5-tetramethoxyphenyl derivatives. a methylenedioxy moiety was essential for the activity of compounds substituted with four b-ring alkoxy groups. the mts assay of the limited panel of cancer cell lines shows that gva displayed the highest inhibitory activity, with ic50 values ranging from 0.27 (a375 cells) to 2.2 lm (mda-mb-231 cells). compounds, containing 3,4,5-trimethoxy and apiol-derived b-rings, respectively, were less active. other isoflavones did not affect cancer cell growth up to 10 lm. anti-proliferative effects of isoflavones 9 observed in both the sea urchin embryo model and human cancer cell lines correlated well. importantly, none of the synthesized isoflavones 9 demonstrated cytotoxicity in human pbmcs, up to 10 lm. in summary, gva and its analogues were synthesized via a scalable six-step reaction sequence. the gva and its analogues containing 3,4,5-trimethoxy and apiol-derived b-rings were found to be promising anti-mitotic microtubule destabilizing agents with low toxicity against human pbmcs. bag-1 is a multifunctional protein which has interactions with a number of cellular proteins; nuclear hormone receptors, bcl-2, hsp70/hsc70 family, growth hormone receptors, raf-1, ubiquitin machinery and dna to regulate cell survival. for this reason, bag-1 is a critical molecular player in the regulation of cell survival signaling and apoptosis mechanism. elevated expression levels of bag-1 are associated with progression of cancer. in the treatment of breast cancer, silencing tools as a promising combined therapy strategies in the presence of classical chemotherapeutics gain importance to investigate interaction networks of cell death and survival signaling pathways. therefore, we aim to understand potential role of bag-1 silencing in the treatment of breast cancer cells with apoptotic agents; cisplatin or paclitaxel. our results showed that, silencing of bag-1 enhanced cisplatin or paclitaxel-induced apoptosis in mcf-7 cells by down-regulating antiapoptotic and upregulating proapoptotic bcl-2 family proteins, changes on cell cycle, upregulation on subg1 phase, activating caspases and cleavage of parp. in addition, knockdown of antiapoptotic bag-1 has a suppressive role in pi3k and akt signaling pathway in mcf-7 breast cancer cells through inhibition of akt phosphorylation and downregulation on pi3k. investigation targets of akt pathway showed that mtor cell survival pathway also affected through bag-1 silencing. bag-1 silencing inhibited mtor signaling via downregulating both rictor and raptor proteins which are the members of rapamycininsensitive mtorc2 and rapamycin-sensitive mtorc1 complexes, respectively. knockdown strategies of bag-1 is important to enlighten the network interactions of bag-1 and clarify its interaction partners in the cells. therefore utilization of bag-1 targeted strategies might further increase therapeutic efficiency of drugs through inhibiting cell survival machinery in the treatment of metastatic breast cancer. p-05.01.1-024 biological activity evaluation of new 3,5,6trisubstituted triazine derivatives bearing different heterocyclic rings against lung cancer cell lines l. yurttas, g. akalin c ß iftc ßi, h. e. temel, b. demir anadolu university, eskisehir, turkey cancer is one of the major death causing disease worldwide. among the various cell types occurs on different organs, lung cancer is one leading cause of cancer death accounting for approximately 26% of all female and 28% of all male cancer deaths in 2013. the resistance development, cytotoxicity and inadequacy are the main encountered problems by the treatment with existing chemotherapeutic agents. therefore, there is continuous need to discover new active and non-toxic molecules. 1-[4-(5,6-bis(4-substituted phenyl)-1,2,4-triazin-3-yl)piperazin-1-yl]-2-[benzimidazole/benzoxazole/benzothiazole-2-yl)thio]ethanone (1-9) derivatives were synthesized with a four-step synthetic procedure using toluil, anisil and 4-chlorobenzil as starting materials. the anticancer activity of the compounds was evaluated using the methods mtt (3-(4,5-dimethylthiazol-2-yl )-2,5-diphenyltetrazolium bromide), brdu (bromodeoxyuridine) assays and flow cytometric analysis against lung cancer cell lines. the lipoxygenase enzyme inhibition activity of the compounds were also investigated using the method described by baylac and racine. compounds was found to have (inhibition concentration) ic 50 values between 135-500 lg/ml. the early and late apoptotic cell percentage was determined as 6.6 for compound 8 by flow cytometric analysis. the lox inhibition activity was found 48.35 ae 3.08 for compound 1. compound 8 bearing 8-chlorobenzil and benzoxazole moieties was found as the most active compound when we evaluate anticancer potential of all compounds. the lox enzyme inhibition was indicated for the compound 1 including methyl substituent on phenyl rings. the dna synthesis inhibition of the compounds has been still studied at the concentrations ic 50 /2, ic 50 and ic 50 x2. p-05.01.1-025 single amino acid substitutions and deletions modulate the drp-lyase activity of human dna polymerase iota n. miropolskaya, i. petushkov, a. kulbachinskiy, a. makarova institute of molecular genetics, moscow, russia dna polymerase iota (pol ι) is a y-family dna polymerase that possesses an unusual combination of properties. due to the special organization of the active site pol ι has a very low accuracy of dna synthesis but possesses an ability to bypass a variety of dna lesions. in addition to the dna polymerization activity, human pol ι also possesses an intrinsic 5 0 -deoxyribose phosphate (drp)-lyase activity. removal of the drp group is a pivotal step in base excision repair (ber) in vivo. although pol b plays a key role in the drp group cleavage and dna synthesis during ber, pol ι was shown to complement the in vitro single-nucleotide ber deficiency of pol b null cell extracts and was suggested to be involved in ber under oxidative stress. the drp-lyase active site in pol ι is still not known. to address the mechanism of the drp-lyase activity of pol ι we obtained a series of pol ι mutant variants including point mutations of conserved lysine residues and deletions in different locations. we purified human pol ι variants from yeast saccharomyces cerevisiae and tested the effect of mutations on the cleavage of an internal 5 0 -drp group in oligonucleotide dna substrates in the presence or absence for me 2+ ions. the experiments revealed several point amino acids substitutions that significantly affected the drp-lyase activity of pol ι, thus suggesting a possible location of the drp-lyase active site. furthermore, we showed that deletions in the n-terminus of pol ι and metal ions modulate its drp-lyase activity, which may play an important role in the regulation of pol ι activities in vivo. this work was supported by russian foundation for basic research grants 15-04-08398-a and 15-34-70002-mol-a-mos and by the russian academy of sciences presidium program in molecular and cellular biology. rosmarinus officinalis, commonly known as rosemary, is an aromatic plant belongs to lamiaceae family. from past to now, rosemary have been used as a traditional medicine to cure for various illnesses such as diabetes, rheumatism and cancer. recent studies have shown that rosemary is effective for various cancer types. in this study we aimed to investigate the effect of rosemary in glioblastoma cells (gbm) by comparison with etoposide and the effect of rosemary by concurrent application with the etoposide. gbm cells (u87 mg) were seeded into the 24 well plates and cultured with dmem supplemented with 10% fetal bovine serum. rosmarinus officinalis tea was prepared just as traditional usage and filter sterilized. at the second day of the culture rosemary in 1/75 (v/v) dilution ratio was given to first group, 40 lm etoposide was given to second group, 1/75 (v/v) diluted rosemary and 40 lm etoposide together were given to third group. after one day incubation cell viability was measured by neutral red assay. it was observed that rosemary reduced the viability of gbm cells by nearly %38, etoposide reduced the viability by nearly % 49 and rosemary with the etoposide reduced the viability by nearly %50. the results showed that rosemary was able to reduce the viability of gbm cells but hadn't got an increasing or inhibiting potential over the etoposide's cytotoxic effect. from our previous studies we know that rosemary increases the proliferation of mouse embryonic fibroblasts. it is considered that rosemary might have a protection potential from dna damages and when rosemary is used with etoposide during the cancer treatment, it might reduce the side effects on healthy cells. in conclusion rosemary promises hope for developing new cancer treatment strategies and reducing the side effects of chemotherapeutics. for further studies it is aimed to examine the effects of rosemary with other chemotherapeutics and if rosemary has got a protection potential from the genotoxic stress. morpholine moiety has been found to be an excellent pharmacophore in medicinal chemistry and a number of molecules possessing morpholine skeleton are the clinically approved drugs. in this present study, we aimed to investigate the possible underlying apoptotic mechanism for the cytotoxicity of new morpholine dithiocarbamate derivatives bearing 1-(2-aryl-2-oxoethyl)-2-substituted benzimidazole moiety on c6 glioma. c6 glioma cell lines were used in the studies. the cytotoxic activities of the tested compounds were determined by cell proliferation analysis using standard (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (mtt) assay. detection of apoptosis was performed using annexin v-fitc apoptosis detection kit bd, pharmingen according to the manufacturer's instruction. all measurements were performed on a facs-calibur cytometer. the ic50 values of the compounds were determined for c6 cell line. compounds 1, 2, 8, 9, and 10 , which were including hydrogen, 4-methyl, 3-methoxy, 3-chloro and 3-floro substituents on phenyl acetyl moiety, had significant cytotoxic activity with ic 50 values lower than 55 lg/ml. compound 10 showed the highest cytotoxic activity with a ic 50 value of 28 lg/ml, whereas cisplatin ic 50 values were 15 lg/ml against c6 cells. cytotoxic activity of compound 1, 2, 8, 9 and 10 with a ic 50 value were 42, 55, 50 and 30 lg/ml, respectively. compound 2, 9 and 10 showed the highest population of early apoptotic cells as 11.5, 10.1, and 10.3% respectively compared to cisplatin (6.2%). also, compounds caused dna synthesis inhibition depend on their ic50 values by brdu assay. conclusions: it was concluded that synthesized compounds had considerable anticancer activity against c6 cell lines. however, compound 2, 9 and 10 including 4-methyl, 3-chloro and 3-floro substituents were the most active compounds against the c6 cell line. also our study results showed that compound 2, 9, 10 induced apoptosis in c6 glioma cells. rutin is a glycosided flavonoid and known to have antioxidant and anti-inflammatory properties.trail induces the apoptosis of tumor cells and has no significant toxic effect on normal cells. although trail is a promising anticancer agent, trail resistance is a major barrier to effective cancer therapy. this study was conducted to examine the utility of the combined use of rutin and trail in prostate cancer cells. pc-3 and du145 prostate cancer cells were treated with rutin (1-1000um) and/or trail (20 ng/ml), cell viability and migration were examined. cell viability was determined by trypan blue exclusion and mtt assay. cell migration was determined by wound healing assay. furthermore, lactate dehydrogenases (ldh) levels of medium were determined as biochemical markers of cell viability. pc-3 and du-145 prostate cancer cells were treated with rutin for 24 and 48 hours incubation and ic 50 doses for 48 hours incubation were determined 250um and 1000um respectively. treatment with rutin, pc-3 cells is more sensitive than du145 cells. rutin and rutin plus trail inhibit prostate cancer cell growth in a dose-dependent manner. treatment with trail has no effect at inhibiting growth of pc-3 and du145 prostate cancer cells. the combination of rutin and trail elicit a synergistic antitumor effect on pc-3 and du145 prostate cancer cells. there is a significant increased in rutin and rutin+trail treatments group of ldh activities with respect to control and trail group. conclusion: present data show that rutin efficiently enhanced trail effects in prostate cancer cells. combined treatment with rutin and trail is more effective than the individual treatments of trail at inhibiting growth of prostate cancer cells. p-05.01.1-030 determination of antigenotoxic, proliferative and cytotoxic properties of ellagic acid since ancient time, people use plant for traditional treatment. plants or fruits are produced different type of secondary metabolites. particularly phenolic phytochemicals from plants play an important role in the prevention and treatment of radical damage by inactivating the reactive oxygen compounds due to their antioxidant properties. however, the structure and the activities of many herbal products are not fully elucidated yet and there are several studies about the toxicity of herbal antioxidants and their possible risks to human health. ellagic acid, phenolic compounds, is an important substance. ellagic acid is a naturally occurring plant phenol found in numerous fruits, including blackberries, raspberries, strawberries, cranberries, walnuts, pecans, pomegranates and wolfberries. different researchers give some information about the biological activities of ellagic acid. in this study, we aimed to determine the cytotoxic, proliferative and antigenotoxic effects of ellagic acid, which is phenolic compounds found in natural products. cytotoxic effects of ellagic acid on huvec is investigated by lactate dehydrogenase (ldh) and cell proliferation (wst-1) methods; and antigenotoxic effects against ccl 4 on human lymphocytes is investigated by single cell gel electrophoresis (comet) methods. the rusults showed that high concentration (100 and 200 lm) of ellagic acid has cytotoxic and mutagenic effects, but showed antiproliferative effects. on the contrary, low concentrations (4, 8, 12.5 lm) of ellagic acid has anticytotoxic and antimutagenic effects. as a conclusion, low concentrations of ellagic acid might be use treatment of some disease. but high concentrations of ellagic acid constitute a risk factors for people. keywords: cytotoxicity, antiproliferation, wst-1, ldh, rtca-sp the constitutive nuclear factor kappa b (nf-kb) activation is widely found in diverse types of hematologic malignancies such as acute myeloid leukemia (aml) and chronic myeloid leukemia (cml) as well as solid tumors. inhibition of nf-kb signaling via proteasome inhibitors such as bortezomib can induce apoptosis in myeloid leukemia cell lines. however it is not clear whether the cytotoxic effects of bortezomib on myeloid leukemia cell lines is due to direct inhibition of nf-kb or another pathway, such as dna damage. in this study, cml cell line k562 and aml cell line hl-60 were treated with bortezomib (bor) , etoposide (eto) and camptothecin (cpt) alone or in dual combination with these drugs, following by measuring the effects on cell viability, apoptosis and signal pathways. the effect on cell viability was determined using the mtt assay. the data were used in combination index and isobologram analysis. the expression levels of apoptototic genes (bcl2, bax and caspase 3), the related dna damage genes (atm and atr) and the involved genes in nf-kb signaling (rela and p50) were determined by real time rt-pcr. we showed that combinations of bor with topoisomerase inhibitors (cpt and eto) exhibited synergistic cytotoxic effect in k562 cell line but not in hl-60 cell line. the combination treatment increased apoptosis and dna damage response. dnadamage-sensing kinases were detected in k562 and hl-60 cells following treatment with bor as similar as topoisomerase inhibitors. bor increased the mrna levels of atm and atr dramatically, which indicated active dna damage in the myeloid cell lines. furthermore, bor induced apoptotic cell death by decreasing bcl2 and increasing bax and caspase 3 levels. these effects of bor were observed to correlated with increasing the p65 expression levels. this study on the mechanism of action of bor indicates that this compound affects several pathways involved in the control of cell cycle progression, apoptosis and dna damage. p-05.01.1-032 analysis of molecular cytogenetic alterations in gastric and colon carcinoma by array-based comparative genomic hybridization (array cgh) introduction: genomic dna regions are frequently lost or gained during tumor progression. we aimed to evaluate tumor samples of patients with gastric cancer and colorectal carcinoma to show these genetic alterations by array-based comparative genomic hybridization (array cgh) method. materials and methods: dna isolation was performed from the tumor samples obtained from sixteen patients with primary gastric adenocarcinoma and twelve patients with colon adenocarcinoma. then, agarose gel electrophoresis was performed in those dna samples. following electrophoresis of dna, array cgh procedure was performed to four patients with gastric adenocarcinoma and three patients with colon adenocarcinoma who had dna breaks with 100-200 kb. results: after array-cgh study, many common genetic changes in gastric and colon cancer genome were determined. in gastric cancer dna samples, common losses were detected in chromosome 1p34. 1, 1p13.3, 1q22, 3q29, 5p13.2, 6q24.1, 7q11.23, 7q22.1, 8q 12.1, 9p12, 11q13.1, 12 q24.31, 14q32.31, 16q22.1, 17q21.2, 19q13.3, and 20q11 .23, and also common gains were detected in chromosome 3p26.1, 6q27, 8q12.1, 15q11.2 and xq25. in colon cancer dna samples, common losses were detected in chromosome 1p34. 1, 3q29, 7p22.2, 7p11.21, 9q34.11, 12q24.31, 17q21.2, 17q25.1, 19p13.3, 19p13.33, 20q11.21, and 20q11.23 , and also common gains were detected.in chromosome 14q32. 33, xp22.33, xp22.11, xp13.3, xp11.1 and xq25 . both in gastric and colon cancer dna samples, common losses were detected in chromosome 12q24. 31, 17q21.2, and 19p13.3 , and common gains were detected in xq25. discussion and conclusion: we think that these common changes, generally in dna loss areas harboring tumor suppressor genes and dna gain areas harboring oncogenes, may important in gastrointestinal tumorigenesis. the dna of every cell is under a constant attack by various mutagenic factors which damage the dna and can cause cell cycle arrest and even cell death. accumulation of dna damage is the basis for cancer development and one of the reasons for aging of the organisms. in order to preserve the integrity of its dna cells have evolved an impressive array of dna repair pathways, which are precisely coordinated with the progression of the cell cycle. one of the first events at the site of dna damage is poly(adp-ribose) polymerase 1 (parp1) recruitment which is a sensor for single strand breaks in dna. parp1 catalyzes the synthesis of poly(adp-ribose) or par which is needed for the recruitment of many other dna repair proteins by means of par-binding domains. we used high speed confocal spinning-disk microscopy of living cells to obtain precise kinetics of recruitment of par-dependent proteins to the sites of laser induced dna damage. our results show that the investigated par-dependent proteins are recruited to dna damage sites in the matter of seconds, they reach peak intensities for 20 to 30 seconds after damage infliction and start dissociating. the recruitment of the proteins is entirely dependent on par because addition of parp inhibitor abbrogated their recruitment. the use of spinning-disk microscopy of living cells allowed us to obtain the kinetics of recruitment of the studied proteins to the sites of dna damage. the results are consistent with the fact that parp1 and par-dependent proteins are quickly recruited to damage sites and generation of par is essential for other dna repair protein recruitment. the precise kinetic curves may serve as a basis for investigating how they will change or if they will change at all when cells are put in different conditions or treated with various chemical substances affecting dna metabolism and repair. introduction: chronic myeloid leukemia (cml) is a myeloproliferative disease associated with reciprocal translocation between chromosomes 9 and 22. bcr-abl fusion gene which exhibits constitutively active tyrosine kinase activity has a main role in cml. the tyrosine kinase inhibitor imatinib is used as a first line treatment in cml patients, but imatinib resistance leads to failure in therapy. the application of imatinib in combination with other anticancer agents may be a strategy to increase the antileukemic effect of imatinib. in this study, we have investigated the antiproliferative effect two novel agents: a benzamide derivative xt5 and a benzoxazole derivative xt2b in combination with imatinib. these molecules were investigated in imatinib-sensitive (k562s) and imatinib-resistant (k562r) cml cell lines. materials and methods: antiproliferative and apoptotic effects were assessed by mtt assays and flow-cytometry, respectively. we also evaluated the effects of these compounds on the expression of apoptosis-related genes bax, bcl-2, bad, bim, bcl-xl and mcl1 by real-time quantitative pcr. results: treatment of k562 cells with xt5 increased the expression levels of the pro-apoptotic genes bax, bad and bim in both sensitive and resistant cells. however, xt2b was not found to have similar effects on k562r and k562s cells. combined application of xt5 increased cell death in the mtt assay. mtt assay demonstrated that ic50 for xt5 treated cells in k562r with imatinib (ic50 = 3.5) is lower than k562r without imatinib (ic50 = 8.5). discussion and conclusion: our results showed that combining xt5 with imatinib has more antiproliferative and apoptotic effect on a cml cell line. as a result combination of xt5 with imatinib can be an alternative approach to overcome imatinib resistance. introduction: the mmr(mismatche repair) system recognizes base-base mismatches and insertion or deletion loops in doublestranded dna, and it degrades the error-containing region of the newly synthesized strand, allowing the polymerase to correctly resynthesize the second strand according to the template sequence. the human mmr system includes the mlh1 and msh2. alteration in expression or a defect in mlh1 or msh2 can cause resistance to anti-cancer drugs used in chemotherapy. the attempt of the mmr system to detect drug induced dna damage, triggers the activation of apoptosis, a mechanism which may enhance the cytotoxicity of chemotherapy. loss of the mmr system would make the neoplastic cell less able to initiate apoptosis. inability to initiate apoptosis could be a mechanism of resistance to drugs. chronic myeloid leukemia (cml) is a clonal disease originating from aberrations in hematopoietic stem cell. imatinib, a tyrosine kinase inhibitor has significantly improved clinical outcome for cml patients. however, patients develop resistance when the disease progresses to the blast phase (bp) and there are several mechanisms involved in imatinib resistance. in this study we investigated the role of mmr system in imatinib resistance. materials and methods: k562s (sensitive) and k562r (resistance) were grown in rpmi-1640. k562r cells were maintained in rpmi-1640 medium supplemented with 5 lm imatinib rna isolation, cdna synthesis, rt-pcr was performed respectively. results: the results demonstrated that expression of mlh1 in k562r cells is dramatically lower than equal amount of imatinib treated k562s cells, whereas msh2 expression level did not change in both cell lines. conclusion: it can be suggested that alteration and down-regulation of mlh1 genes leads to imatinib resistance. p-05.01.1-037 characterization of interaction between rad51 inhibitor dids and human serum albumin d. velic, s. henry, c. charlier, m. popova, p. weigel, j. masson, i. nabiev, f. fleury cnrs/university of nantes, nantes, france 4 0 -diisothiocyanostilbene-2,2 0 -disulfonic acid (dids) has been largely used during the last 30 years for its inhibitory effect on anion transporters and channels. more recently, ishida and colleagues have described a possible mechanism by which dids inhibits rad51-mediated homologous pairing and strand exchange, key processes in dna repair by homologous recombination. thus, dids could act as a potential revertant of radioand chemo-resistance in cancer cells, which is the major cause of failure during therapeutic protocols. new drugs targeting rad51 protein have since been developed with potential use for medical applications. in this context, we attempted to determine the behaviour of dids towards blood and plasma proteins such as serum albumins. firstly, we analysed the effects of several environmental factors such as solvent polarity, which may affect the stability of the molecule. secondly, we analysed the spectroscopic properties of dids in the presence of human or bovine serum albumin proteins. uv-visible absorption, circular dichroism, fluorescence spectroscopy and isothermal calorimetry were used. here we show for the first time that dids can interact with both serum albumins. we have also determined the characteristics of these interactions. the comparison of several dids derivatives led us to identify the essential chemical moiety of this compound involved in the interaction. moreover, by using site competition approaches we show that the main binding site for this molecule is in subdomain ib of the protein. these findings show that the binding of dids to serum albumin proteins may change the equilibrium between the free and bound dids forms, thereby affecting its bioavailability and efficiency against the rad51 recombinase protein. p-05.01.1-038 mechanism of tap73 beta-mdm2 autoregulation p73 is a transcription factor which is the member of a p53 family. it regulates many cellular processes, such as apoptosis, cell cycle, and senescence. in contrast to p53, p73 is rarely mutated in tumors and elevated p73 expression is observed in many types of cancers including hepatocellular carcinoma, neuroblastoma, and lung. defining regulatory mechanisms which control p73 protein abundance and activity will be crucial for the development of new therapeutic strategies for cancers. mdm2 is known as the key player in regulation stability and activity of p53. in addition, p53 induces mdm2 transcriptional activity, and caspase-2, 3 activations which cleave mdm2 n-terminal at asp 367. cleaved form of mdm2 binds p53 and promotes its stabilization. mdm2 suggested as a candidate to modulate p73 activity and stability too. however, an interaction between p73 and mdm2 has not defined well. in this study, we aimed to analyze the role of mdm2 in p73 stability. to define this relationship, firstly, we overexpressed the tap73beta isoform using trex system in hep3b. tap73 beta and mdm2 protein levels were determined by western blot. to examine whether mdm2 mediate tap73 beta protein degradation by the proteasomes, cells were treated with proteasome inhibitor, mg132 for 4 hours prior to analysis. previous studies showed that p53-induced caspase-2 and caspase-3 activation cleaves mdm2. considering this, we firstly examined caspase-3 activation by western blot in hep3b tap73 beta cells. then we analyzed expression of cleaved mdm2 and tap73 beta levels following caspase inhibitor, z-vad-fmk treatment. as a conclusion, tap73 beta-induced full-length mdm-2 expression. furthermore, tap73 beta enhanced cleavage of mdm2 via increased caspase-3 activation. in addition, inhibition of caspase-3 activation caused a decrease in cleaved-mdm2 levels in parallel with tap73 beta expression repression. our results suggested positive regulation between mdm2-tap73 beta. hepatocellular carcinoma (hcc) is one of the most common type of liver cancer and third leading cause of cancer related deaths in worldwide. discovery of new targets is important in survival of hcc patients. p73 is a transcription factor which is the member of p53 family. it has two promoters; while p1 promoter expresses apoptotic ta isoforms, p2 promoter expresses anti-apoptotic dn isoforms. in addition, alternative splicing in c terminal creates many isoforms of ta and dn p73. it has been shown that both tap73 and dnp73 isoforms are expressed in hcc patient tissue and cell lines. the ratio between tap73 and dnp73 affects the apoptotic response, drug response and prognosis. accordingly, identification of the role of p73 and its targets are important in discovery of new treatment strategies in hcc. to understand the role of p73 isoforms in hcc, firstly we performed mtt assays following dna-damaging drugs and multikinase inhibitor, sorafenib treatment to categorize hcc cell lines as resistant or sensitive. after that, we analyzed the expression levels of tap73 isoforms via western blot in all hcc cell lines. then we overexpressed the tap73beta isoform using trex system in hep3b and snu449 cells. these two clones were analyzed for dna damaging drug response by mtt, cell cycle and apoptosis by flow cytometry, and tumor formation by in vitro and in vivo experiments. in scope of our study; 1. only tap73 alpha isoform is expressed in a few hcc cell lines. 2. there is no correlation between basal expression of p73 isoforms and drug responses in hcc cell lines. 3. there is no change in expression of p73 isoforms after treatment of drugs. 4. we showed that the ectopic expression of tap73beta in hep3b arrested the cell cycle in g1/ s and decreased the colony formation. therefore, the capacity of tumor formation of the cells dramatically decreased in scid mice. as a result, we revealed that tap73 beta play role in tumor formation, cell cycle arrest, dna damage responses in hcc. p-05.01.1-040 biochemical characterization of exonuclease iii-family ap endonuclease point mutants reveals role of conserved amino acid residues in the nir-specific enzymes a. mursalimov, z. koshenov, t. yeleussizov, m. redrejo-rodriguez, a. ishchenko, b. t. matkarimov, m. saparbaev national laboratory astana, astana, kazakhstan oxidative dna damage caused by reactive oxygen species is believed to be a major type of endogenous cellular damage. oxidatively damaged dna bases are substrates for two overlapping repair pathways: dna glycosylase-initiated base excision (ber) and apurinic/apyrimidinic (ap) endonuclease-initiated nucleotide incision repair (nir). in the ber pathway, an ap endonuclease cleaves dna at ap sites and 3 0 -blocking moieties generated by dna glycosylases, whereas in the nir pathway, the same ap endonuclease incises dna 5 0 to a number of oxidized bases. majority of characterized ap endonucleases possess classic ber activities and about half of them are able to catalyze nir activity. at present, the molecular basis of dna substrate specificities of various ap endonucleases remains unclear. here, we examined amino-acid sequence requirement of the nir activity of human major ap endonuclease 1 (ape1). amino acid sequence alignment of various ap endonucleases including e coli exonuclease iii (xth), human ape1 and archaeal mth212 revealed conserved amino acid residues in the nir-specific ap endonucleases ape1, mth212 and exoa that are absent in xth. based on these data, we constructed four ape1 point mutants y128h, n174q, g231s and t268d and examined their dna substrate specificities. results obtained from biochemical characterization of ape1 mutants are discussed in the light of the evolutionary conserved dna repair functions of ap endonucleases and whether these functions can be mutationally separated from. since its discovery some 40 years ago, cisplatin has evolved for its efficacy in one of the most used drugs in treatment of various cancer types. huge effort was invested in understanding the action of cisplatin and development of more potent drugs. they target mainly neighboring purine bases of nuclear dna forming covalent intra-or inter-strand cross-links that affect inhibition of replication and transcription, cell cycle arrest, and attempted repair of the damaged nucleotides. if such damage cannot be removed the cell dies. we have studied the details of the binding site of the short oligonucleotide modified by a platinum compound using complementary solution techniques used in modern structural biology, including raman spectroscopy with dft calculations aided interpretation of the obtained vibrational spectra. moreover, the calculated structure of the dna duplex was verified using saxs (small angle x-ray scattering) curve. in our contribution, we will present an nmr structure of a dna cross-linked with a cisplatin derivative containing a cyclohexane ring. at this atomic level resolution, structural features probably influencing cytostatic effects are described and compared with previously published structures. common structural features of previously determined structures are: a significant roll (25-60°) of the guanine bases involved in the cross-link, bending and unwinding of the double helix at the site of cross-link and orientation towards the major groove. also, the platinum-guanine plane angle varies between 19 and 54°. although the experimental structures were often used as the starting models for molecular dynamics (md) simulations, results of these md still leave many questions unresolved. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. p-05.01.1-043 ercc2/xpd polymorphisms and colorectal cancer risk: a case control study in a north eastern iranian population j. mehrzad islamic azad university, neyshabur, iran excision repair cross-complimentary group 2 (ercc2) is one of the important dna repair genes.ercc2 codon 751 and 312 polymorphisms has been shown to modulate cancer risk. we therefore assessed the relationship between the ercc2 polymorphisms and the susceptibility to colorectal cancer in a case-control study. there were 105 lung cancer cases and matched healthy controls in this study. information concerning demographic and risk factors was obtained, each person donated 2 ml blood for biomarker testing. ercc2 genotypes were determined by t-arms-pcr method. all of the statistical analyses were performed with spss (v 20.0). there was significant difference between the frequencies of ercc2 polymorphism in cancer cases and controls (p < 0.05). the frequencies of ercc2 751 gln allele were 6.2% in controls and 13.8% in cancer cases. the individuals with lys/gln+gln/gln combined genotype were at an increased risk for lung cancer as compared with those carrying the lys/lys genotype (adjusted or=2.80, 95%=ci 1.21à6.48). the above findings indicate that the genetic polymorphism in the ercc2 codon 751 is associated with the risk of colorectal cancer in an iranian population (neyshabur citizenship). peptide pore blockers are potent tools to study structure and function of potassium voltage-gated channels (kv). kcsa-kv1.x chimeras, in which a ligand-binding site of eukaryotic kv-channel is inserted into bacterial kcsa channel, mimic properly the pore domain of kv-channels. a fluorescence-based approach to study the binding of peptide blockers with kcsa-kv1.1-kcsa-kv1.3 chimeras was developed by us. this approach rested on high-level expression of kcsa-kv1.x chimeras in e.coli inner membrane, binding of fluorescently-labeled toxin at the surface of the spheroplast and analysis of competitive binding of studied ligands by laser scanning confocal microscopy (lscm). here we report on a new analytical system for search and study of kv1.6-channel blockers that combines bl21 (de3) cells expressing kcsa-kv1.6 and rhodamine-labelled agitoxin 2 (rh-agtx2) as a fluorescent probe. by tuning cultivation conditions, the high-level of membrane expression of kcsa-kv1.6 was achieved. it was found that lowering both the growth temperature and the concentration of inducer resulted in significant increase in membrane-embedded kcsa-kv1.6. for system validation, wellknown kv1 channel blockers were studied by the method of competitive binding, and equilibrium dissociation constants were estimated for agtx2, osk1, and kaliotoxin. a new system was applied to study molecular determinants of peptide-kv1.6 channel binding using a number of agtx2 mutants constructed by us, whose affinities to kcsakv1.6 were measured. a new bioengineering fluorescent system is a robust and sensitive assay for assessing the binding activity of kv1.6 channel blockers. it can be used to study interaction interfaces of toxinchannel complexes, to search for novel peptide blockers and to develop new potent and selective kv1.6-blockers for scientific and medical purposes. the work was supported by the grant 14-14-00239 from russian science foundation. asparagus racemosus root extracts (ar) have been exhibited to show a wide range of pharmacological benefits. in this study, liposomes of ar were developed and assessed their physicochemical properties and anti-inflammatory activity in monocytic leukemia cell line (thp-1). liposomes containing ratios of ar to lipid and phosphatidylcholine to cholesterol ratio were synthesized by thin-film hydration (tf), reverse-phase evaporation (rev), and polyol dilution (pd). the in vitro anti-inflammatory activity was assessed in terms of inhibition of tumor necrosis factor alpha (tnf-a) in lipopolysaccharide activated thp-1 by elisa. the size of ar liposomes prepared by tf were larger, whereas those prepared by rev and pd were smaller. ar to lipid ratio was shown to have no influence on particle size, whereas zeta potential enhanced with increasing ar to lipid ratio. ar liposomes with lipid ratio of 1:5 achieved the highest value of entrapment efficiency and were at the highest with polyol dilution method. ar was found to have no toxic effects on thp-1 cells. the anti-inflammatory activities of ar and ar liposomes in terms of tnf-a in thp-1 cells were was exhibited to possess the highest values of around 52% at ar concentration of 1 lg/ml and % tnf-a inhibition tended to decline with the increasing amount of ar. this result may be attributed to the increased amount of liposomal particles being uptaken into the cells as a result of the increasing ar concentrations. it can be suggested that ar liposomes could be an alternative choice of topical/transdermal drug delivery for anti-inflammatory activity. p-mis-002 inhibition of ire1 signaling enzyme increases the expression of tumor suppressor genes and modifies their hypoxic regulation in u87 glioma cells d. tsymbal, o. minchenko palladin institute of biochemistry of the national academy of sciences of ukraine (nasu), kyiv, ukraine gliomas constitute one of the most aggressive groups of malignant neoplasms with poor survival prognosis and scarce therapeutic options. plentiful studies have proven the connection between endoplasmic reticulum stress and malignant growth. we have studied the effect of inhibition of ire1 (inositol requiring enzyme 1), which is a central mediator of endoplasmic reticulum stress and controls cell proliferation and tumor growth, on hypoxic regulation of the expression of different proliferation related genes in u87 glioma cells. it was shown that inhibition of ire1 leads to up-regulation of the expression of krt18, cd24, mest, cenpu, myl9, ing1, ing2, mybl1, and mybl2 genes at the mrna level in u87 glioma cells, with more profound changes for mest, mybl1, and cd24 genes. hypoxia leads to up-regulation of the expression of cd24, ing1, and ing2 genes and to down-regulationof krt18 gene in glioma cells. at the same time, inhibition of ire1 modifies the effect of hypoxia on the expression of all studied genes: suppresses effect of hypoxia on ing1 gene, eliminates hypoxic regulation of krt18, cd24, and ing2 genes in glioma cells. the present study demonstrates that inhibition of ire1 enhances the expression of all studied genes and modifies the hypoxic regulation of these gene expressions in gene specific manner and thus possibly contributes to slower glioma cell proliferation, but several aspects of this regulation remain to be further clarified. amplification and clonig of dna polymerase 1 (pol1) of thermus scotoductus k1 isolated from an armenian goethermal spring a. saghatelyan, h. panosyan, a. trchounian, n. birkeland yerevan state university, yerevan, armenia the most important enzyme ''mined'' from thermophilic microorganisms is dna polymerase, which widely used in molecular biological studies. although dna polymerase produced by thermus aquaticus (taq polymerase) was launched into the market long back, isolation of more processive, reliable and stable dna polymerases from other species is a demand. the purpose of this work was to amplify and clone the pol1 gene of t. scotoductus strain k1 recently isolated from an armenian geothermal spring. the draft genome sequence of strain k1 was deposited under accession number ljjr00000000.1. genomic dna was isolated using genelute bacterial genomic dna kit. primers for the pol1 gene were designed manually. the gene was amplified using pfu polymerase, and amplicons (~2.5 kb) were ligated into the pet-21b(+) vector (novagen) and transformed into chemically competent top10 escherichia coli. inserts were sequenced with t7prom and t7term primers, which showed that the gene sequence was correct and in the right reading frame and could be expressed in mesophilic e.coli. dna polymerases patented form different species of thermus are mostly comparable, suggesting that only limited natural variations in taq-like dna polymerase may be discovered. the pol1 gene from k1 shares 99% and 83% similarity with pol1 of t. scotoductus sa-01 (2.0 kb) and t. aquaticus, respectively. although the difference is not huge at sequence level, possible functional differences (e.g. stability, proofreading activity, resistance to different pcr inhibitors etc.) may occur. therefore, it is important to express and purify dna polymerase from strain k1 for further investigations. peptide ligands of the immunoglobulin g fc region identified by screening phage libraries and site-directed mutagenesis n. kruljec, p. molek, t. bratkovic young researcher, ljubljana, slovenia affinity chromatography based on immunoglobulin (ig)-binding proteins, such as staphylococcal protein a and streptococcal protein g, typically represents the initial step in therapeutic antibody purification process. however, this approach suffers from high cost, poor ligand stability and the requirement for relatively harsh elution conditions that can negatively impact activity and immunogenicity of antibodies. compared to protein ligands, peptides represent an interesting alternative due to higher stability and less expensive production. furthermore, the expected lower affinity for immunoglobulins should allow for elution under milder conditions. the aim of our research was to identify short peptide ligands for the fc region of human iggs. we have screened three commercially available phage display libraries of random cyclic and linear peptides for binding to human fc region in solution using an optimized biopanning approach. five non-homologous linear peptides were shown to specifically interact with the fc portion of immunoglobulins as verified by a set of phage elisa assays. individual phage-displayed peptides were able to recognize specific subclasses of igg. the highest-affinity peptide (12l-19fc), which competed for fc binding with protein a, was subjected to mutagenesis studies. we displayed on phage several variants of 12l-19fc with individual amino acid residues exchanged for alanine as well fragments of the parent peptide of different lengths and evaluated binding to fc with phage elisa to identify the minimal binding motif. binding characteristics of the minimized peptide were further analyzed using spr biosensor. the details will be disclosed at the meeting. diverse effects of ganoderma lucidum in combination with tamoxifen citrate and doxorubicin in mcf-7 breast cancer cells ganoderma lucidum, an edible medicinal fungus, has been known with its anti-metastatic, anti-carcinogenic bioactivities and widely used in asian countries in complementary and alternative medicine. however, there is no information regarding its combined usage with tamoxifen and doxorubicin in breast cancer treatment. we investigated the interactions between ganoderma lucidum and tamoxifen or doxorubicin in mcf-7 human estrogen receptor positive breast cancer cell line. anti-proliferative properties of six extracts were assessed by wst-8 method. the most effective extract in inhibition of mcf-7 cell viability was then evaluated in terms of its anti-metastatic activity by boyden chamber assay. apoptosis and cell cycle assays were performed by flow cytometry. ganoderma lucidum ether extract (g.ether) was the most effective extract on inhibition of cell viability among others with ic (50) values of 100 lg/ml and 12.82 lg/ml at 48 h. and 72 h. respectively. we found that g.ether is capable of inducing apoptosis and changing cell cycle dynamics. however, incubation with g.ether did not affect mcf-7 cell motility significantly. we then assessed the interactions between g.ether and tamoxifen or doxorubicin in mcf-7 cells. the interactions between g.ether and cancer therapeutics were examined by combination index analysis and macsynergy ii software. interestingly, g.ether increased the anti-proliferative effect of tamoxifen although exhibited strong antagonism with doxorubicin in mcf-7 cell line. testing the best matrix/analyte combination for maldi tof mass spectrometric detection of steroid hormones, amino acids, vitamins and carbohydrates in spite of numerous advantages, there are serious drawbacks of the application of matrix assisted laser desorption/ionization time-of-flight mass spectrometry (maldi tof ms) for smallmolecule analyses (below 500 da) and quantification. the main problem is the background interference from commonly used maldi matrix materials. the aim of this work is to evaluate maldi tof mass spectra of physiologically relevant small molecules: steroid hormones, vitamins, amino acids and carbohydrates, acquired with several organic, traditional matrices. small volume, 0.5 ll, of each sample solution (testosterone, progesterone, estradiol, l-cysteine, l-alanine, dl-methionine, glutathione, d-(+)-glucose, d-(+)-maltose, vitamin a, vitamin e) was mixed on the sample plate with the same volume of organic matrix solutions (dhb, thap, chca, 9-aa). for each molecule/matrix pair, we determined quantitative and qualitative parameters of ms analysis. to calculate within day and day-today variation we used excel tools (anova tests). in addition, homogeneity of the sample/matrix distribution on the target was also calculated and expressed as the coefficient of variation of a series of measurements. our results show selectivity of the detection of individual molecules related with the matrix applied. the statistical analysis of certain molecule/matrix pairs gave within and day-to-day variations less than 15%. additionally, homogeneity of the sample/ matrix mixture distribution on the target plate was with some matrices, also less than 15%. some of the used matrices have a great potential for the analysis of small molecules with good analytical parameters, with low variations and high homogeneity of samples on the maldi target plate. these results hold potential for quantification of metabolically-significant small molecules and are very promising for future applications of maldi tof ms analyses. stress causes different expression of mitochondrial biogenesis markers in rat steroid-producing cells of adrenal gland and testes i. starovlah, s. radovic, t. kostic, s. andric faculty of science univeristy of novi sad, novi sad, serbia functional mitochondria of steroid producing cells of adrenal cortex and leydig cells of testes are essential for steroid hormones biosynthesis and regulation. the aim of this study was to determine transcriptional profile of mitochondrial biogenesis markers in adrenal cortex and leydig cells by applying in vivo and in vitro studies. immobilization stress (imo), was performed for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) consecutive days. in in vitro studies, primary cultures of purified leydig cells from undisturbed rats were stimulated with stress hormone adrenaline, propranolol (nonselective b-adrs-blocker) and prazosin (the selective a1-adrs antagonist). rq-pcr results showed that the transcription of the main regulator of mitochondrial biogenesis, ppargc1a and ppargc1b, significantly decreased in adrenal cortex of 10ximo rats. oppositely, the significant increase of the same transcript was registered in leydig cells from the same rats. in parallel, transcription of ucp1, the mediator of regulated proton leak, decreased in adrenal cortex, but increased in leydig cells of the same group of rats. incubation of leydig cells with adrenaline, increased transcription of the main markers of mitochondrial biogenesis (ppargc1a, ppargc1b, nrf1 and nrf2a). nonselective b-adrsblocker attenuated this effect. the selective a1-adrs antagonist did not change adrenaline-induced stimulation of ppargc1a, ppargc1b, nrf1 and nrf2a transcription in leydig cells, indicating that the most of the effects are probably mediated by b-adrenergic receptors, not by a1-adrs of leydig cells. in summary, the results suggest that reduction of transcription of mitochondrial biogenesis markers could be a possible mechanism that protects body from excessive glucocorticoid production from adrenal glands in stress conditions, while at the same time stimulation of mitochondrial biogenesis markers transcription in leydig cells could serve as mechanism to preserve testosterone production. p-mis-008 generation of new mitochondria is possible protection mechanism of basal steroidogenesis in leydig cells s. radovic, i. gak, t. kostic, s. andric faculty of science, university of novi sad, novi sad, serbia mitochondria are the most important component of stress response in all cells and for steroid-hormones-producing cells they are the starting point for steroid biosynthesis. here we investigated the parameters of mitochondrial biogenesis in these cells from rats exposed to the psychophysical stress by immobilization (imo). imo stress was applied for 2 hours daily for one (1ximo), two (2ximo) or ten (10ximo) days.hormone levels were measured employing eia, elisa kit or ria. mitochondrial membrane potential (δwm) was measured by tmre fluorescence, mitochondrial mass was detected by quantitative analysis of mitotracker-green fluorescence as well as relative intensity of fluorescence, since number of mitochondria and mitochondrial architecture were defined using transmission electron microscopy. relative gene expression and proteins analyses were performed by rq-pcr and western blot. there was positive correlation between δw m of leydig cells and androgens production of leydig cells. both of them were reduced in all stressed rats but partially recovered in 10ximo group. the mitochondrial mass in leydig cells from 10ximo group was increased. transmission electron microscopy analyses showed that acute and two times repeated stress altered architecture of mitochondrial cristae, while 10ximo increased number of mitochondria and recovered mitochondrial architecture. there was significant increase in the expression of the all markers of mitochondrial biogenesis in leydig cells from 10ximo rats compared with other groups. accordingly, stress-triggered mitochondrial biogenesis represents an adaptive mechanism and does not only correlate with but also is an essential for testosterone production, being both events depend on the same regulators. supporting the evidence that stress, a constant factor in life of humans, induces mitochondrial biogenesis in leydig cells, our results indicate this mechanism probably protects the basal steroid production in stress conditions. targeting survival pathways in leukemic cells through synergism of metformin and thymoquinone u. glamoclija, m. suljagic international university of sarajevo, sarajevo, bosnia and herzegovina generation of resistance to current treatment options is common problem in the therapy of many hematological malignancies. combined therapies utilizing compounds with low toxicity that act synergistically, are proposed to overcome this problem. metformin and thymoquinone (tq) are two molecules which have proven safety profile and represent potential candidates for treatment of hematological malignancies. there are more than 150 clinical trials, at different stages, exploring metformin anticancer activity. metformin activates amp activated protein kinase (ampk) leading to inhibition of the mammalian target of rapamycin (mtor) and induction of apoptosis in different cancers. however, human leukemic cells with increased basal protein kinase b (akt) phosphorylation were shown to be resistant to metformin-induced apoptosis. it was found that activity of metformin can be enhanced by combination with akt and/or nuclear factor 'kappa-lightchain-enhancer' of activated b-cells (nf-jb) inhibitors. tq is phytochemical compound that has shown inhibitory capacity on both of these targets. wst-1 assay was used to evaluate the effects of metformin and tq in dhl4 (b cell lymphoma) and k562 (chronic myelogenous leukemia) cell lines. compusyn software was used in order to calculate the combination index (ci). the ci value indicates whether two drugs have synergistic (ci<1), additive (ci=1) or antagonistic effects (ci>1). we have shown that separately, metformin and tq, exhibit dose dependent inhibition of dhl4 and k562 cells. in combinatorial study with fixed constant ratio and simultaneous drug exposure, in dhl4 and k562 cell lines, ci values were 0.68 and 0.66, respectively. to our knowledge, this is the first report showing synergistic effects of metformin and tq in lymphoma and chronic myelogenous leukemia derived cell lines. these promising data are currently being investigated in order to obtain the insight into their molecular mechanisms. for the last decade many methods of calculating and analysing the physical characteristics of dna has been developed. these methods allow to estimate distributions of free energy, propensity to bend, stress-induced duplex destabilization (sidd), electrostatic potential (ep) etc. and most of them have been used for prediction of genomic regulatory site positions. the main idea of such approach is that proteins recognize genome regulatory sites by these physical and chemical properties, so the physical characteristics are used to predict the location of regulatory sites. most of the characteristics mentioned above describe properties of dna at equilibrium or steady state, but we propose to use characteristics of internal dna dynamics. in this work we used the coarse-grained model of dna, developed recently, to simulate dynamics of the dna open states. with this model we were able to calculate trajectories of the open states moving along the molecule and their dynamical characteristics, such as: open state activation energy, size, half decay time and sound velocity in dna. we use distribution of four dynamical characteristics around transcription start site of experimentally found e.coli promoters taken from regulon db to organise them in stable clusters. clusterization was made with ward method and consensus clustering technique was applied to clusterization results for analysis of its consistency. the same procedure was applied to equilibrium dna characteristics for comparison. distribution of go functions among clusters was also analysed. stable promoter clusters obtained with different physical properties share some similarity. it was not surprise that clusters obtained with dynamical characteristics of dna more similar to sidd clusters then to ep clusters. the data highlights the possible role of dna dynamical properties in transcription initiation and its applicability to promoter identification together with other physical and textual properties of dna. chromium complex with 5-hydroxyflavone acts on metabolic pathways the development of novel therapeutic strategies for obesity treatment are urgently required as obesity is currently the main leading cause in type ii diabetes and insulin resistance. among natural compounds, flavonoids have recently gained interest due to their positive role in maintaining blood glucose levels and insulin secretion. their association with trace elements, wellknown for their capacity in increasing the efficiency of insulin, might potentiate flavonoids biological effects. in this context, the aim of our study was to investigate the in vitro changes in energetic metabolism related genes expression profile in the presence of a chromium complex with 5-hydroxyflavone. dna microarray technology was used for a large scale screening of differentially expressed genes in human adipose stem cells (hascs) after 3 weeks of adipogenic induction in the presence of the chromium complex with 5-hydroxyflavone. moreover, perilipin expression was assessed by flowcytometry. the chromium complex with primuletin negatively regulates the expression of key genes involved in adipogenesis and also modulates the expression of the genes associated with triglyceride synthesis and subsequent fat storage in mature adipocytes. consequently, the chromium complex with 5-hydroxyflavone can be further employed in studies on animal models to investigate the possible improvement of metabolic disorders. deinococcus radiodurans is a highly radioresistant and stress-resistant bacterium. despite extensive studies, the mechanisms of transcription regulation that contribute to the stress-resistance are still poorly understood. d. radiodurans encodes multipe stress-related proteins including three members of the gre-family of transcription factors: grea, gfh1 and gfh2. while grea is a universal bacterial factor that stimulates rna cleavage by rna polymerase (rnap), the functions of lineage-specific gfh proteins remain unknown. we cloned, expressed and purified d. radiodurans rnap and gfh factors and their mutant variants and analyzed their properties using various in vitro transcription approaches. we tested gfh effects on rnap activity in promoter, elongation and termination complexes assembled on natural and synthetic dna templates under different conditions. we found that the gfh factors strongly enhance site-specific pausing and intrinsic transcription termination by d. radiodurans rnap but do not act on active transcription complexes and do not compete with the grea factor. uniquely, the pause-stimulatory activity of gfh is greatly enhanced by manganese ions, which are accumulated in d. radiodurans cells under stress conditions, and is modulated by the secondary rna structure. we revealed functionally important regions in the gfh factors and the rnap active site involved in transcriptional pausing. we propose that gfh factors inhibit rna extension in paused complexes through binding within the secondary rnap channel, coordinating metal ions in the rnap active site and stabilizing an inactive enzyme conformation. this may serve as a sensitive mechanism to regulate transcription under stress conditions and coordinate it with dna repair and replication. our data suggest that gre and gfh proteins target different structural states of the transcription elongation complex and reveal functional diversity of the factors that bind within the secondary channel of rnap. from planktonic to biofilm state of growth, flagella formation is turned off, and the production of fimbriae and extracellular polysaccharides is activated. bola protein is widespread in nature and has been associated with several cellular processes. using high-troughput techniques we showed that bola protein is a new bacterial transcription factor, which regulates the switch between motile and sessile lifestyle. it negatively modulates flagellar biosynthesis and swimming capacity in escherichia coli. moreover, bola overexpression favors biofilm development, involving fimbriae-like adhesins and curli production. our recent results show that bola action in these pathways is related with cdi-gmp a relevant intracellular signaling molecule involved in biofilm formation. we demonstrate that bola contributes to a fine-tuned expression of different diguanylate cyclases and phosphodiesterases and c-di-gmp has a negative influence in the bola mrna transcription. herein we propose that bola is a key player in motile/adhesive transcriptional switch, contributing to a fine-tuned regulation of these important pathways. background: deep venous thrombosis (dvt) is an important health problem worldwide. its pathophysiology is multicausal and involves environmental, genetic and acquired factors. factor v leiden (fvl), prothrombin g20210a (pt g20210a), and methylenetetrahydrofolate reductase (mthfr) gene mutations are to predispose to venous thrombosis. the aim of this study was to compare the frequency of fvl, pt g20210a and mthfr polymorphisms between patients with dvt and healthy controls. methods: this study was conducted at the bozok university hospital. total 220 participants were included in this study, 126 patients with dvt and 94 healthy blood donors. in order to identify fvl, pt g20210a, mthfr c677t and mthfr a1298c, the polymerase chain reaction (pcr) method was utilized combined with the amplification refractory mutation system. results: in 126 patients fvl was present in 54 (42.8%) patients while in controls fvl was present in only 18 (19.1%). frequency of fvl was significantly higher in cases as compared to controls (p < 0.05). pt g20210a mutation was present in 13 patients (10.3%) and in 5 healthy participants (5.3%). mthfr c677t and mthfr a1298c polymorphisms were almost equally distributed among patients and healthy participants. however, the concomitant presence of fvl and double heterozygous polymorphisms of mthfr c677t/a1298c was found in 11 patients (8.7%) and in 2 healthy controls (2.1%), showing significant association with deep venous thrombosis. conclusion: in this study, the frequencies of fvl and pt g20210a polymorphisms were found significantly higher in patients with dvt than those in healthy participants. thus, fvl and pt g20210a polymorphisms have a contributory role on the development of dvt in contrast, mthfr c677t and mthfr a1298c genotypes were not associated with a predisposition to development of dvt. but, a combination of double heterozygous polymorphisms of mthfr c677t/a1298c with fvl may be associated with increased risk of dvt. p-mis-016 self-assembling micellar clusters comprising drugs, nanoparticles and fluorescent compounds for bilogical applications when designing drug carriers, the drug-carrier ratio is an important consideration, because the use of wrong drug-carriers relation can result in toxicity as a consequence of poor metabolism and elimination of the carriers. solubility problem of various substances also plays an important role in many aspects of fundamental science and practical field. specifically, it is an important parameter as well as bioavailability, which determines the required concentration of drug in the body needed to achieve a pharmacological response. among the variety of solubilization methods micellar solubilization is widely used as an alternative to the dissolution of poorly soluble drugs. here, we show a specific approach based on sequential selfassembly of nonionoc detergent micelles (t 9 100, tx114) followed by enacpsulation of various nanoparticles (noble metals, magnet etc.), drugs, fluorescent compounds leads to the formation of stable micellar nano-amd microcomplexes. we propose 3 ways of micellar clusterisation. in the first one micelles are modified by semi-hydrophobic chelator followed by addition of metal ion to make cross-linking. the second way is similar to the first one and suggests application of the metal complex with incresed denticity instead of naked metal ion, and the third one involves micelles clusterisation by semi-hydrophobyc metal complex directly. therefore, one can stabilize micellar network by means of 'interactions on interface': semi-hydrophobyc metal complexes are embedded inside micelle due to hydrophobyc interactions. hydrophobic fluorescent compounds-loaded micellar complexes demonstrates better optical response in aqueous media without crystallization. such obtaining clusters are also very flexible and can be modified by nanoparticles to obtain various nanocomposites, such as fluoromagnetic clusters. this work was supported by russian foundation of basic research grants no. 15-43-03214 r_center_a (2015 no. 15-43-03214 r_center_a ( -2016 no. 15-43-03214 r_center_a ( ) and 15-33-20002 mol_a_ved (2015 no. 15-43-03214 r_center_a ( -2016 . lamellipodia and membrane blebs utilize different signalling pathways to induce directional movement of walker carcinosarcoma wc 256 cells in a physiological electric field clear if those reactions are mediated by similar mechanisms. to establish that, we performed proteomic analysis and subsequent investigation of the role of differential signalling pathways in electrotaxis of cells representing various strategies of movement. cells were exposed to ef in galvanotaxis apparatus and their reaction was recorded. in some experiments cells were pre-incubated with erk1/2 or btk-1 inhibitors. the phosphorylation of erk1/2 and btk-1 was determined by western blot analysis. proteomic analysis was performed by ultimate 3000rs lc nanosystem coupled with a q-exactive mass spectrometer. both blebbing (bc) and lamellipodial (lc) cells show cathodal migration in a physiological ef (1 v/cm). comparative analysis of bc and lc cells proteomes revealed about 150 differential proteins. functional analysis in ingenuity analysis pathway allowed to determine the statistically significant signalling pathways in which these proteins are engaged. among the most distinctively regulated pathways are tec kinase and erk/ mapk signalling activated in lc but not bc. it was found that btk-1 is required for directional movement of lc but not for bc cells. moreover, ef induced stronger and faster btk-1 phosphorylation in lc than bc cells. in contrast erk 1/2 activity was not necessary for electrotaxis of lc cells and ef did not induce erk 1/2 phosphorylation. our results reveal that both lamellipodia and membrane blebs can efficiently drive electrotactic migration of wc256 cells but it is mediated by different signalling pathways. this work was supported by a grant from the national science centre 2012/07/b/nz3/02909, poland. newborn screening for congenital hypothyroidism in turkey: a regional evaluation € o. demirelce 1 , n. y. saral 1 , f. b. aksungar 1,2 , a. coskun 1,2 , m. serteser 1,2 , i. unsal 1,2 1 acibadem labmed, istanbul, turkey, 2 acibadem university, istanbul, turkey congenital hypothroidism (ch) is the most common congenital endocrine disorder and the most important cause of preventable mental retardation. it is important to begin the treatment within 2 weeks before the development of brain damage. tsh based newborn screening programs are shown to be useful for implementing early treatment of ch. in this study, regional results of ch screening program in turkey between 2006 and 2015 were assessed retrospectively. we have evaluated the results of marmara, central anatolia, aegean and mediterranean regions in which our laboratories are located. screening was based on tsh determination in dried blood spot specimens. tsh limits determined to be 10 lu/ml for cut off point and 25lu/ml for clinical decision point. tsh was measured using enzyme immune assay (eia). blood spot tsh data for 65857 newborns during this time period were evaluated. permanent or transient ch was determined according to the results of thyroid function tests. confirmed ch cases were based on local endocrinologists' report and initiation of thyroxine treatment. the frequency of neonatal tsh levels were found to be under the cut off level of 10lu/ml in 63513 (96.44%), between 10 and 25lu/ml in 2287 (3.47%) and above the level of 25lu/ml in 57 (0.09%) babies, respectively. recall rate was 3.5%. ch cases of neonatal tsh levels greater than 25lu/ml were 26. the incidence of ch of this group was 1:2532. there were no significant differences in the number of congenital hypothyroidism between males and females (p > 0.05). the preliminary results of our study indicate that the incidence of ch in our region is higher than the worldwide reports as has been proved by preceding studies. iodine deficiency, dyshormonogenesis, highly consanguineous population, may contribute to the high incidence of ch in turkey. newborn screening of ch must be developed for detecting true cases and tsh cut off point must be reviewed for decreasing redundant recall rate. in silico analysis of the first complete genome sequence of lactobacillus acidipiscis species k. papadimitriou 1 , m. kazou 1 , v. alexandraki 1 , b. pot 2 , e. tsakalidou 1 1 agricultural university of athens, athens, greece, 2 institut pasteur de lille, lille, france introduction: lactic acid bacteria (lab) constitute a significant group of microorganisms for the food industry, as they play a key role in food fermentation and consequently in human health. lactobacillus acidipiscis aca-dc 1533 is a gram-positive, motile, rod-shaped lab isolated from traditional greek kopanisti cheese. here we present the in silico analysis of the first complete genome sequence of l. acidipiscis in order to explore the biology of the species. materials and methods: sequencing of l. acidipiscis genome was performed using the hiseq 2000 and pacbio rsii sequencing platform technologies and the genome assembly was validated against an nhei optical map of the l. acidipiscis genome. protein-coding sequences were predicted by glimmer, rrna genes by rnammer and trna genes by the trnascan-se server. potential genomic islands were detected using the island-viewer software tool, prophage regions by phast and the subsystem-based annotation by rast server. finally, the circular representation of l. acidipiscis genome keyed to the cog groups was constructed by cgview server. results: the sequencing analysis resulted in one continuous genomic scaffold of 2,678,726 bp with a g+c content of 39.75%. the genome contains 2,525 protein-coding genes on the chromosome covering up to 82.09% of the genome sequence, 63 trna and 18 rrna. according to the subsystem-based annotation, 1,562 protein-coding genes were assigned to 310 metabolic subsystems. the most abundant of the subsystems are related to carbohydrates (n = 287, 18.37% of total protein-coding genes) and protein metabolism (n = 173, 11.08% of total protein-coding genes). furthermore, three prophage regions were detected; one intact (43.5kb), one incomplete (14.7 kb) and one questionable (29.3 kb). discussion and conclusion: the whole genome analysis of l. acidipiscis aca-dc 1533 provided interesting information about a not well-studied species. investigation of serum irisin levels of patients with metformin taking new onset type 2 diabetes mellitus increases glucose tolerance and energy expenditure and improves carbohydrate homeostasis. metformin is a biguanidine class antidiabetic drug which inhibits liver gluconeogenesis and decreases insulin resistance and is frequently recommended in treatment of new onset type 2 diabetes mellitus (t2dm). irisin has a role in the regulation of energy metabolism pathways and its level in blood of persons with t2dm has been reported to decrease. regarding this relationship, it was aimed to reveal the effect of metformin on serum irisin levels. 20 patients with impaired oral glucose tolerance test were included to this investigation. they were recommended to take metformin and to change their life style, such as exercise and diet. their blood were taken at the beginning and after 1 month. also, a healthy control group (n = 20) was formed from persons with similar age and sexual distribution as the patient group. irisin levels of their sera were measured by enzyme-linked immunosorbent assay (elisa) method. statistical evaluation of the measurements showed no significant difference (p = 0.780) between the irisin levels of the patients at the beginning and after 1 month treatment. a similar result was found between the control and the treated groups (p = 0.170), while a significant difference (p = 0.002) was observed between the control and untreated patients groups. the results obtained from this study do not show a clear and significant change in the blood irisin levels of the patients with new onset t2dm taking metformin together with life style change. a longer period of treatment and a higher number of patients may be needed for more reliable results. thermodynamics of dna ligands binding at specific sites of telomeric g-quadruplex dna g-quadruplexes are a perspective target for anticancer therapy. for example stabilization of the telomeric g-quadruplex dna formed by single-stranded ends of the chromosomes leads to inhibition of telomerase, which is active in 90% of cancer cells. similarly, small molecules targeted to a specific g-quadruplex would inhibit various cellular processes. stoichiometry and affinity of interaction of these compounds to dna is determined by specific structural motifs within a g-quadruplex. rational design of novel chemical compounds requires an in depth knowledge of interactions between known ligands and g-quadruplex structures. experimental methods that are used for determination of thermodynamic binding parameters, such as isothermal titration calorimetry, differential scanning calorimetry, ultraviolet absorption and circular dichroism spectroscopy provide a collective characteristic for all of the ligand molecules bound to dna, while the information on ligand affinity to individual dna binding sites is lost. we propose a complimentary method for detailed analysis of thermodynamic parameters of ligand binding based on the introduction of fluorescent probes in the structure of g-quadruplex. monitoring fluorescence quenching of the fluorescent labels allows to derive binding constants of the dna-ligand interaction at a specific binding site. temperature dependence of the fluorescence quenching determines the thermodynamics of the dnaligand complex formation. since only a proximal ligand is able to quench the fluorescence, this method allows characterization of the ligand binding to a particular site the g-quadruplex structure. the study was supported by project no. 16-14-10396 of the russian science foundation. the correlation between biochemical and dynamic surface tension parameters of calves blood serum during the animal ontogenesis, as well as by various pathologies or poor diet, the imbalance of protein, mineral, lipid components is observed (the changes in all parameters of biological liquids are accompanied of these metabolism peculiarities). the dynamic surface tension (dst) of serum essentially depends on these factors and (in combination with the biochemical parameters) can provide the valuable information for evaluation of the physiological and biochemical status of the organism (can be used as an express test for animal diagnostics in future). the aim of the work was to study dst and biochemical parameters of calve serum, as well as their correlations, as the main indicators of the animals. ) of calve serum were in the range of the normal values for healthy animals and can be considered as reference data for animal science and practice. the obtained results enable us to establish correlations between the dst and biochemical parameters of calves serum. this work was supported by the russian scientific foundation (grant 14-16-00046). the middle strong correlations of dst values of calves serum with the level of total protein, albumin, billirubin, some enzymes and cholesterol, whereas only weak correlations with the other biochemical parameters (urea, calcium, magnesium, phosphorus, etc.) were found. in the veterinary science and practice such correlations are important for the estimation of the organism physiologicaland biochemical status, for general inspections of cattle before vaccination (immunization) or slaughter, for "quick separation" of healthy and ill animals in the case of infection, etc. role of protein kinase c in the regulation of astrocytic glutamine transporter sn1 in ammonia-exposed mouse cortical astrocytes (bisi; 1 lm). total pkc activity was analyzed by a direct pkc assay and phosphoserine detection by western blot (wb) analysis. protein level of sn1 and sn2, second astrocytic gln transporter belonging to system n, in a membrane fraction was also analyzed. the total uptake and system n-mediated (l-ala and l-leu-inhibitable) gln uptake was tested. treatment of astrocytes with ammonia resulted in a decrease of pkc activity, whereas pma treatment increased pkc activity in ammonia-independent way. bisi treatment reversed fully, and ammonia partially, the pma-induced pkc activity. pma treatment resulted in only a slight decrease in sn1 protein level in both control and ammonia-treated astrocytes, while a decrease of total and system n-mediated gln uptake were noted in control astrocytes, an effect not exacerbated by ammonia. in turn, cotreatment with pma and bisi reversed the decrease of total gln uptake and showed tendency towards increase in system nmediated gln transport. the results suggest that: a) ammonia changes the dominating direction of system n transport from release to uptake, which may be related to decreased phosphorylation or to alterations in relative phosphorylation by different pkc isoforms. this inference remains to be verified in further studies; b) changes in system n transporter function induced by ammonia appear to involve mechanisms other than changes in transporter expression. evidence for human ghrelin ghs-r1a and orexin ox1 heteroreceptor complex formation in a heterologous system ghrelin and orexin are two peptides implicated in the regulation of energy balance and modulation of food-related motivation at the level of the midbrain dopamine reward system. their function in the hypothalamic arcuate nucleus and the ventral tegmental area (vta) has already been described, but the modulation at the level of receptors remains unclear. the action of these peptides is mediated by g-protein-coupled receptors (gpcrs): ghrelin 1a and 1b (ghs-r1 a , ghs-r1 b ) for ghrelin, and orexin 1 and 2 (ox 1 , ox 2 ) for orexin. traditional approaches to know the mechanism of neurotransmission of dopaminergic neurons in the mesolimbic system have focused on targeting neuronal receptors as single entities. from the discovery that gpcrs for neuromodulators may form heteroreceptor complexes, our hypothesis is that ghrelin and orexin receptors may interact and form novel functional units that may specifically participate in the central regulation of food intake and energy balance. as a proof of concept we have investigated the potential of human ghs-r1 a and ox 1 receptors to form heterocomplexes. formation of ghs-r1 a -ox 1 receptor heteromers in transfected hek293t cells was detected by bioluminescence resonance energy transfer (bret) and proximity ligation (pla) assays. furthermore, a negative crosstalk was identified in cells co-expressing both receptors by assessing mitogen-activated protein kinase (mapk) and adenylyl cyclase (camp) pathways, and by a label-free dynamic mass redistribution assay. experiments in sources endogenously expressing ghs-r1 a and ox 1 receptors are needed to know the functional relevance of the heteromer. from the negative crosstalk here identified, it is tempting to speculate that ghs-r1 a -ox 1 receptor heteromers are important players in mediating the response to the combination of different orexigenic signals. lysosomal storage diseases which are related to deficiency of specific lysosomal hydrolases resulted to clinical aspects due to accumulation of substrates in different tissues. since dried blood spot (dbs) is non-invasive, low-cost, easy transportable, acceptable enzyme stability compared to leucocyte and/or fibroblast culture, it's recommended as a first screening test. however the false positive rate with dbs sample is higher compared to other samples. we aimed to investigate any possible effect of leucocyte number on enzyme activity in dried blood samples in a retrospective study. we re-evaluated the lysosomal enzyme activity results in regard to leucocyte number among data within last 1 year. enzyme activities had measured by using fluorometric and lc msms method. we determined the correlations between the lysosomal enzyme activities of alpha glycosidase, glycocerebrosidase, alpha galactosidase, sphingomyelinase, galactocerebrosidase and alpha-l-iduronidase in healthy population (n = 220). while glycocerebrosidase and galactocerebrosidase positively correlated with the number of neutrophils, alpha galactosidase, sphingomyelinase and alpha-l-iduronidase positively correlated with the number of lymphocytes. alpha glycosidase activity showed a correlation both lymphocytes and neutrophils. the patients having the glycocerebrosidase enzyme activity which was lower than 0.6 nmol/ml/hour (which is accepted as the cut off value to recall the patients) existed significantly lower number of leukocyte, lymphocyte and neutrophil compared to those of patients having higher enzyme activity than 0.6. our data indicated that the enzyme activity in dried blood samples including low leucocyte number might be found lower than reference intervals resulting in false positive diagnosis. therefore we suggest that the laboratory scientists should evaluate the number of leucocyte levels while they were interpreting data. using dna-markers for estimation of genetical variability of two kazakh sheep breeds a. mussayeva 1,2 , b. bekmanov 1,2 , a. amirgalieva 2 , k. dosybaev 1,2 , z. orazymbetova 1,2 , r. zhapbasov 2 , a. zhomartov 2 , n. zumadillaev 3 , n. zumadillaev 3 1 llp "kazcytogen", almaty, kazakhstan, 2 "institute of general genetics and cytology" sc mes, almaty, kazakhstan, 3 branch "scientific research institute of sheep" llp "kazakh research institute of animal husbandry and feed", almaty, kazakhstan to compare the frequencies of different microsatellite loci in sheep breeds subpopulations genomic structure of edilbay and kazakh archaromerinos was investigated. different methods for homogeneity testing of two breeds were elaborated. inter simple sequence repeats (issr) pcr analysis of the breeds studied displayed species and breed specific fragments with different frequencies (population frequency more than 0.4) there were found rarely met fragments (frequency lower than 0.4). the combinations of these fragments present the specific issr-spectra which arrange genofond profiles of breeds. using panels of microsatellits (recommended by isag) 2 breeds (5 populations) were characterized. informative value and resolving capacity of the sum of 32 str-loci were estimated. wide polymorphism of alleles length was demonstrated both when the breeds were compared and within the breeds. 10 informative markers were chosen for both two breeds, 7 markers being used for both breeds, while other 3 markers were informative for one of the breed only. when the animals of one breed were compared unique alleles which were met only within one of populations were of much interest. for example the allele 174 of bm1824 was met in birlik population of edilbay breed as often as in 59% of animals while in two other populations there were no this allele. in kumtekey population one can meet 70% animals having particular locus (dyms1), while in the other population (cf ablay) this locus was not met at all. basing on genetical distances obtained using fragment analysis phylogenetic relationships between populations were estimated. so for example edilbay population of cf ajar has the larger distance from two other populations (birlik and bayserke-agro) than each of them from one another. two subpopulations of kazakh arkharomerinos breed (cf kumtekei and cf ablay) also have the genetical difference. how preeclampsia affects oxidant status and antiinflammatory potential of breast milk? preeclampsia is a pregnancy syndrome associated with hypertension, proteinuria and edema, leading to maternal morbidity/mortality and preterm delivery. in this study we aimed to investigate if the breast milk of preeclamptic mothers is effected in oxidative status and anti-inflammatory activity in comparison to the breast milk of mothers with healthy pregnancies. for the aim of the study, hyaluronidase and myeloperoxidase activities (mpo), total oxidant status (tos), total antioxidant status (tas), oxidative stress index (osi) and tbars levels were measured in breast milks of 20 preeclamptic mothers and 20 mothers with healthy pregnancies as control group. when the control group and preeclamptic group were compared, hyaluronidase activity, tas, tos and osi levels showed statistically significant differences in the preeclamptic group. hyaluronidase activity was significantly higher in the preeclamptic mothers' breast milk (734 vs 594 u/ml, p = 0.046). while tos levels were significantly higher in the preeclampsia group (7.48 vs 4.51 lmol/l, p = 0.001), the tas levels were significantly higher in the control breast milks (0.557 vs 0.813 mmol/l, p = 0.011). as expected osi levels (tos/tas ratio) were significantly higher in the preeclampsia group. even though the mean levels were higher in preeclamptic group, the difference in mpo activities and tbars levels did not show statistic significance. oxidant status parameters also suggest that preeclampsia effects in both ways by increasing oxidant status and also decreasing antioxidant capacity shifting the balance to the increased oxidant stress side. as the results showed that the preeclampsia group had higher hyaluronidase activity, this can be interpreted as preeclamptic mothers' milk have higher inflammatory potential as this enzyme enhances inflammation by catalyzing the depolymerization of certain acidic glycosaminoglcans. p-mis-030 investigation of relationship between postprandial lipemia and erythrocyte membrane cholesterol level postprandial lipemia is a metabolic condition related to an increase in plasma triglycerides. remnant-like lipoprotein particles are predominant in postprandial phase and they play an important role in development of atherosclerosis. cholesterol is a prominent component of erythrocyte membranes and regulates the membrane functions such as viscosity and permeability. free cholesterol derived from erythrocytes is thought to participate in the atherosclerotic plaque formation. in the current study, it was aimed to investigate the relationship between postprandial lipemia and erythrocyte membrane cholesterol level in healthy subjects. study group included 87 subjects (39 female and 48 male with age range of 18-55 years). then these individuals were divided into three groups according to the values of area under curve (auc) calculated by using triglyceride levels at the fasting state and at 2nd, 4th and 6th hours after the high fat diet (ottt). lipid and erythrocyte membrane cholesterol (emc) values were compared between groups with low and high ottt response. while tc, tg, ldl-c and emc were significantly higher, hdl-c was significantly lower in high ottt response group than low ottt response group. it was not observed any statistically significant difference when compared emc values between women and men study groups. on the other part, it was seen positive correlation between emc and auc (r = 0.33, p = 0.002), tg (r = 0.26, p = 0.016), tc (r = 0.30, p = 0.005), ldl-c (r = 0.29, p = 0.007) in the total study group. it was concluded that, postprandial lipemia may show atherosclerotic tendency not only with atherogenic lipid profile but also with increasing emc. p-mis-031 eu-openscreen: the european infrastructure for chemical biology b. stechmann, p. gribbon eu-openscreen, fmp leibniz institute for molecular pharmacology, berlin, germany small molecules that can be applied as chemical 'tool' compounds (or 'probes') have become indispensable in basic research for the elucidation of fundamental biological mechanisms. they act directly with the protein-of-interest and often allow for the interrogation of biological processes that cannot be properly studied with traditional genetic or rna interference approaches. eu-openscreen (www.eu-openscreen.eu) is the largest emerging academic chemical biology research infrastructure initiative in europe and will provide access for molecular and cell biologists to screening infrastructure, well-characterized highquality chemical libraries, and facilities for medicinal chemistry services for compound optimization. molecular biologists who have a robust and suitable biological assay and are interested in collaboratively developing chemical tool compounds to validate their targets-of-interest are welcome to work with eu-openscreen. selected assays are screened against a collection of more than 100,000 compounds, incl. confirmatory and counter screening, ic/ec50 determination, sar (structure-activity relationships) and qc of confirmed hit compounds. eu-openscreen will start operations in 2017, but it can already look back on a growing number of transnational activities: joint screening projects, exchange of local compound libraries, development of new design principles for its compound collection; exchange of experimental data through its pilot database etc. steps towards an arthrobacter nicotinovorans based biotechnology for production of 6hidroxy-nicotine as the archetypal agonist of nachr, nicotine stands up as a powerful scaffold for developing new alzheimer disease therapeutic agents in form of nicotine derivatives. in this context, arthrobacter nicotinovorans pao1 and its wide range of nicotinederivatives produced when grown on nicotine have a huge biotechnological potential. indeed, the metabolic intermediate 6-hydroxy-nicotine (6hnic) produced by a. nicotinovorans pao1 was shown to bind to the nachrs, and by modulating their function, to sustain spatial memory formation in a rat model of ad. the current work presents the first attempts to produce and isolate 6hnic by using a genetically engineered a. nicotinovorans strain. the growth and the 6hnic accumulation were compared for two strains: 1. a. nicotinovorans pao1 wild type strain and 2. a genetically engineered a. nicotinovorans pao1 strain (part2ndh) containing the nicotine-dehydrogenase (ndh) genes cloned in the nicotine inducible part2 vector. the growth curves were followed spectrophotometrically. the consumption of nicotine and accumulation of 6hnic were monitored by hplc using a mn nucleodur 100-3 c18ec column and 0.1 m sulfuric acid at a flow rate of 1 ml/minute. the growth curve of the part2ndh strain shows that the bacteria grow slower when compared with the wt. as a result, in the wt strain, the nicotine is quickly depleted from the medium and only low amounts of 6hnic are observed. although the sds-page analysis of the total protein extracts from the part2ndh strain did not show clear signs of ndh overexpression, the enzyme is produced and is active, allowing a 5 fold accumulation of 6hnic in the growth medium. the first attempts to purify ndh from the part2ndh strain using imac were nevertheless unsuccessful. in conclusion, using the part2ndh strain for 6hnic production is feasible. further improvements of the growth condition and strain are envisioned (i.e. knocking the ndh downstream genes; adding inhibitors for the downstream enzymes). studies on the impact of butyrylcholinesterase (bche) on the symptoms and progression of cognitive impairments like alzheimer's disease (ad) or other neurodegenerative disruptions speak in favour of selective bche inhibitors as a new approach in future ad pharmacotherapy. some derivatives of quinine and quinidine, present in the cinchona species bark, have already been identified as selective bche inhibitors with respect to acetylcholinesterase (ache); therefore, further investigation of these compounds might result in promising leads for enhanced anti-ad drugs. we synthesised ten quaternary derivatives of cinchonines and their corresponding pseudo-enantiomeric cinchonidines. quaternization of quinuclidine moiety was carried out with groups diverse in size: methyl and differently meta and para substituted benzyl groups. all of the compounds were prepared in good yields, characterized by standard analytical spectroscopy methods, and were tested for their bche and ache inhibition potency. the inhibition potency of the compounds was defined by the dissociation constants of the enzymeàinhibitor complex (ki). all of the tested compounds reversibly inhibited both human bche and ache. the compounds inhibited bche with ki constants in the range of 0.04-30 lm, and ache in the range 2.5-70 lm. five cinchonidines displayed a 95-510 times higher inhibition selectivity to bche over ache, and four of them were potent bche inhibitors with ki constants up to 100 nm. bche affinity toward the studied compounds depended on the size of the substituent on the nitrogen of the quinuclidinium part of the molecule and on the resonance stabilization of the substituent at the quaternized nitrogen. based on the presented results, cinchonidine cd-(pbr) can be pointed out as a potent and selective bche inhibitor that could be considered for further research in alzheimer disease pharmacotherapy. exposure to nmda (100 lm) for 72 h increased the expression of kir4.1 mrna and decreased that aqp4-and gs mrna. the expression of kir4.1 was decreased by 72 h exposure to glu (2 mm) and tnfa (50 ng/ml). at 8 h incubation, nmda induced a decrease of kir4.1 expression in the presence but not in the absence of calcium in the medium. nmda did not alter the expression of nmda receptor subunits. tnfa increased the expression of the nr1 subunit, and decreased that of nr2b mrna. glu decreased the expression of 3 out of 7 subunits. the study demonstrates, to our knowledge for the first time, that prolonged exposure of astrocytes to nmda alters the expression of mrna coding for critical astrocytic proteins. the dependence of the decrease of kir4.1 mrna expression on extracellular calcium suggests the ionotropic nature of nmda receptor stimulation. the effects of nmda receptor stimulation occurred by a mechanism bypassing changes in subunit composition of the nmda receptor. experiments are under way to establish whether the tnfa-induced changes in the expression of nmda receptor subunits contribute to modulation of nmda receptor stimulation by inflammation. the importance of education in reducing preanalytical errors the preanalytical phase includes the request of test, the preparation of patient, the obtaining of sample from the patient, the transport of the sample to the laboratory, and the pretreatment of sample. the preparation of patient and the obtaining of sample are considered as the most common error sources. in order to reduce preanalytical errors, we aimed to provide training for phlebotomists and to also determine their knowledge level about the preanalytical phase before and after these training. it was given the training related with preanalytic phases to 130 pediatric nurses and 350 adult nurses, other phlebotomists in march. the surveys which are made before and after the training were consisted of 20 questions that are related with demographyic features and preanalytic phases. in order to determine the effects of training to the preanalytic phase, the preanalytic error rates before (in february) and after (in april) traning was calculated with the formula of: (the number of rejected samples/the number of total samples)x100. the average age of participants was 35 ae 9 years. it was not found significant difference between their correct answers rate before the training and the education degree of the participants. the correct answer rate before the training was 59% and after the training it was 92%, which showed an increase of 55%. the preanalytic error rates which were 0.61% in february were decreased to 0.40% in april. in our study, the positive results were obtained through the training aimed to reduce the preanalytical errors. by providing regular training to the phlebotomists and also providing pretraining to the beginners, the updating of their information about preanalytic phase can be achieved. in this way, the loss of labor and economic related to preanalytical errors can be avoided and the accurate results can be obtained in short time. curcumin, the active compound of turmeric (curcuma longa) has antiinflammatory, antioxidative and antitumour effects. unfortunately, curcumin has a poor absorption and low stability. both can be solved by encapsulation of curcumin using a proper technique like electrospray. it was reported that piperin, the active compound of black pepper, enhances the intestinal absorption of curcumin and thus its bioavailability. due to these facts it was aimed in this study to nanoencapsulate turmeric extract in order to enhance its absorption and stability. for that purpose, it was encapsulated with the maize protein zein, chitosane and black pepper extract by varying the voltage and flow rate of electrospray and the concentration of the compounds. the nanocapsules were characterised by measuring their particle size and with help of sem photographs. the particle size of the final nanocapsule formulation was 550 nm and had a sufficient stability over a period of 6 months, visually determined. by encapsulating turmeric extract into double layer nanocapsules with help of black pepper extract, zein and chitosane, the turmeric extract could be protected from degradation, which was observed for the pure turmeric extract in form of clearing its yellow colour. analysis of the human genome reveals that potential g-quadruplex sequences are enriched in promoters of the oncogenes. growing body of evidence suggests that g-quadruplexes (g4) may play putative roles in various biological processes, such as the regulation of gene expression. consequently, targeting the oncogenic g-quadruplexes using small molecules is an alternative strategy for the potential treatment of cancers. porphyrin derivatives are promising class of drug in this respect, being nucleic acids binders and generators of reactive oxygen species under visual light irradiation. interaction between porphyrin derivatives and g4 dna from oncogene promoter region has been studied in vitro. we applied chemical probing, circular dichroism spectroscopy and uv melting techniques in order to map the oxidized bases, monitor structural rearrangements and evaluate stability of the resulting dna structures. specifically, we observed that g4 dna is considerably more susceptible to lightinduced modification than duplex dna; 5 0 -terminal tetrads of the g4 dna are preferably oxidized; structural changes induced by oxidation result in decrease of the thermodynamic stability of the g4 dna. irreversibility of these effects on dna make porphyrin derivatives perspective lead compounds for rational design of ligands targeting human oncogenes. the study was financially supported by project no. 16-14-10396 from the russian science foundation. resistin levels in denervated obese rats n. saglam 1 , t. ahmedi rendi 1 , c. kahraman 2 , a. alver 1 1 department of medical biochemistry, faculty of medicine, karadeniz technical university, trabzon, turkey, 2 school of health, d€ uzce university, d€ uzce, turkey the sympathetic nervous system is an important factor affecting the metabolic and secretory function of the white adipose tissue. resistin is mainly expressed by mononuclear cells, also it is expressed by adipocytes, pancreatic cells, and muscle. resistin induces insulin resistance and glucose intolerance in mice. resistin plasma levels depend on fat depots size and sex. resistin levels decrease in short-term fasting in mice, then it increase refeeding. also, it increase as a response to fed with the high fat diet. in our study we aimed to determination of the effect of high-fat diet and denervation on serum resistin levels in rats. in this study 4 experimental groups were formed each consisted of 8 rats. during 10 weeks, first two groups are fed with high-fat diet and other two groups are fed with standart diet which they purchased from research diets company. at the beginning of the feeding periods, retroperitoneal fat tissiues of animals assigned to the first and the third groups were denervated. second and fourth groups were not denervated. at the end of the 10 week feeding periods, blood collected from rats and blood resistin concentration was determinated by elisa. in denervated and fed with high fat diet groups serum resistin levels higher than control groups (p < 0.05). according to our literature research, there are no studies demonstrating the relationship between resistin and the sympathetic nervous system. also, denervation may lead to increase in serum resistin levels. the amount of resistin is possibly reduced by b -adrenergic activation. in conclusion, it was concluded that there is differences on serum resistin levels depending on diet in bilateral denervation of retroperitoneal fat tissues of rats. stress activated protein kinases regulates the ribosomal frameshift rate in est3 gene, encoding subunit of telomerase s. t€ urkel, s. sarica uludag university, bursa, turkey est3 gene (ever shorter telomere) of s. cerevisiae encodes one of the essential subunits of telomerase enzyme. expression of est3 gene is regulated at the translation level by +1 programmed ribosomal frameshift (prf). it is known that the physiological stresses affect telomere length. in this study, we have investigated the effects of stress activated protein kinases snf1p (ampk) and gcn2p (eif2 kinase) on the prf rate in est3 gene. prf rate of est3 gene was quantified in plasmid based expression system. expression vectors were transformed in to the wild type and mutant yeast strains that deleted for snf1, or gcn2 genes. yeast cells were grown in normal conditions or subjected to acid stress, osmotic stress, or glucose limitations to activate protein kinases gcn2p, and snf1p, respectively. prf rate of est3 gene was measured as 13% in the normal growth conditions in the wild type cells. but, the prf rate of the wild type strain grown in glucose limited conditions decreased more than 10-fold, giving less than 1% prf rate. contrary to glucose limitation, osmotic or acid stress activated frameshift rate by 2-fold in the wild type cells and prf rate increased to 25%. when the prf rate was analyzed in gcn2 and snf1 mutants, frame shift rate of est3 was 4-5% in normal growth conditions. when these mutants were subjected to acidic or osmotic stress, prf rate activated slightly. we have also shown that gcn1p and gcn20p, positive regulator of gcn2p, is also essential for the regulation of prf in est3 in response to stress conditions. it is clear that the basal level expression of est3 is highly dependent on the gcn2p kinase complex. gcn2p is also associates with ribosomes, indicating that gcn2p might have a significant function in connecting the stress signals to biosynthesis of the full length est3 peptide. this regulation might also link the biosynthesis of functional telomerase and telomere replications to cell physiology through protein kinases such as snf1p and gcn2p. inflammation might have a role in erosive esophagitis but not in non-erosive reflux disease the relationship between inflammatory activation mechanisms and acid-peptic injured esophageal tissue is not clear. we evaluated whether there are differences between inflammation and tight junctional proteins such as e-cadherine among subtypes of gastroesophageal reflux disease. the aim of this study was to investigate any possible role of inflammation in pathologic mechanism of reflux disease by determining the inflammatory markers in injured esophageal tissue as well as serum of patients. three groups (erosive-ee, n = 18; nonerosive-nerd, n = 12; healthy controls-hc, n = 13) were evaluated with upper gastrointestinal endoscopy. the esophageal biopsies and blood samples were collected. serum e-cadherine levels, nfkb, chitotirosidase (chit), myeloperoxidase (mpo) activities in serum and homogenized tissues were determined. nkfb levels in tissue was significantly higher in subjects with ee (4.9 ae 2.53 ng/mg.prt) versus hc (2.95 ae 1.51 ng/mg.prt, p = 0.018). mpo tissue activities in ee group were significantly lower (0.07 ae 0.06 u/mg.prt) than hc (0.23 ae 0.22 u/mg.prt, p = 0.025) while mpo serum levels were higher in ee (1.15 ae 1.63 ul) versus hc (0.56 ae 0.69 ul, p = 0.045). tissue chit levels were three fold increased in ee versus hc (p = 0.071). none of these measurements showed any differences in nerd group. nfkb and mpo levels had a negative correlation (r=à0.408, p = 0.005) in tissue. nfkb and ecad levels had a positive correlation in serum (r = 0.642, p < 0.0001). inflammatory process might play a pivotal role in injured mechanism only in erosive esophagitis but not in nerd. noninflammatory mechanisms might be responsible such as hypersensitivity in patients with non-erosive reflux disease. d-dimer (a fibrin degradation product) test is used to aid in the diagnosis of intravascular coagulation. the aim of this study is to investigate the correlation between d-dimer levels and other inflammatory markers including procalcitonin. anonymized data on d-dimer, fibrinogen, hscrp, wbc, neutrophil% (neut%) and procalcitonin levels from 50,107 patients (mean age ae sd, 53.37 ae 20.6) were used for the correlation (excel analyze-it v4.60.4) and linear regression (pasw statistics 18 v18.0) analysis between the measured parameters. there was a significant (p < 0.05) age-dependent increase in d-dimer levels between different age groups. patients with the highest d-dimer levels were also found to have an increased frequency of hscrp levels. d-dimer levels showed a significant correlation with hscrp, wbc and neut%. a model describing the positive association between these parameters were built. the resulting equation is as follows: d-dimer = (hscrp*0.054) + (0.011*age) + (0.006*wbc) + (0.04*neut%)à0.929. correlation analysis between procalcitonin and d-dimer levels gave pearson's correlation coefficient of 0.159. our results suggest that the age-dependent variations should be taken into account while interpreting d-dimer test results. in addition, neut% ratio was found to be the most important parameter for estimating d-dimer levels. our equation can be used when the d-dimer test is not available or for control purposes only. in the field of cancer research great hope lies in finding more powerful and selective way for the direct elimination of cancer cells. this task can be solved by means of nanobiotechnology. recent progress in this field has arisen interest in a carbon nanostructurefullerene c 60 . fullerene exhibits not only unique physico-chemical properties and biological activity but also a significant potential to serve as a nanocarrier for selective drug delivery into cancer cells. the aim of this study is to analyze a unique tool for cancer therapy. the main idea is realized by the non-covalent conjugation of c 60 with the well-known anticancer drug -doxorubicin (dox). two types of conjugate with different c 60 -dox ratio (1:1 and 2:1) were studied. conjugates absorbance and fluorescence, size distribution as well as a mass data were recorded utilizing optical and analytical equipment (microplate reader, zetasizer, lc-ms/ms and maldi-tof). in vitro studies were performed including evaluation of c 60 -dox conjugate effects on human leukemic cells (jurkat, ccrf-cem, thp1 ad molt-16) viability. conjugates accumulation and distribution within cancer cells was monitored using fluorescent microscopy accompanied with fluorescence-activated cell sorting. it was evidently proven that both c 60 -dox conjugates were stable and could be used as reliable candidates for biological application. cellular accumulation and distribution studies showed that conjugation of dox with fullerene promoted its entry into leukemic cells. accumulation of dox in the form of conjugates within cancer cells was intensified compared to the free drug. the results show that conjugated dox is more cytotoxic and the value of its ic50 are lower compared with the free dox. obtained results confirm nanocarrier function of fullerene c 60 and the perspective of its application for optimization of doxorubicin efficiency against leukemic cells. comperative investigation of protective effects of tea and tea-related wastes on reducing potaential of h2o2-induced erythrocytes tea processing waste (tpw) formed during the tea production process in tea factories is up to 50,000 tones/year in turkey. tpw is one of the abundant available phenolic biomass among plantal wastes. in this study, black and green teas and their wastes were used. the aim of the study is to determinate the phenolic content and the radical scavenging activities of the samples, and to measure their effects on hydrogen peroxide-induced erythrocyte damage due to analyzing the reducing potential of erythrocyte involving glutathione reductase (gr), glutathione peroxidase (gpx) activities and reduced glutathione (gsh) content. total polyphenol content of samples was determined as mg catechine per dry mass by using folin-ciocalteau reactive and dpph radical scavenging activity was estimated by cuendet method as equivalent catechine standard. in erythrocyte, gsh level was measured by method of sedlak and lindsay while gr and gpx activities were assayed by the methods of bergmeyer and beutler, respectively. the highest phenolic content was observed in green tea and its wastes (p < 0.01) whereas black fiber waste had the lowest phenolic content. therefore, the highest radical scavenging activity and gsh level were detected in green tea and its wastes (p < 0.01). erythrocyte with the extracts of the teas and their wastes had the similar enzyme activities for both gpx and gr. in sum, the teas and wastes have antioxidant activity but, green tea and its leaf waste hade higher antioxidant activity than other samples. the tea wastes might be evaluated as many of protective health products, particularly in cosmetic fields thus, these by-products no application for any area is expected to become an economical value. fluorouracil (5-fu) is a chemotherapeutic drug classified as an "antimetabolite". it works through irreversible inhibition of thymidylate synthase. chemical derivatization of 5-fu with carbohydrtates is being investigated widely in order to enhance its bioavailability, therapeutic efficiency and to reduce its toxicity. however, water solubility of the newly derived compounds is usually very low. so, in order to obtain a pharmaceutically relevant formulation they need to be formulated appropriately. in this study, we prepared micellar delivery system for the new tetra-o-acetylglycose derivative of 5-fu synthesized via "click reaction", namely f1-[{1 0 -(2″,3″,4″,6″-tetra-o-acetyl-b-dglycopyronosyl)-1 0 h-1 0 ,2 0 ,3 0 -triazole-4 0 -yl}methyl]5-fluorouracil. since the water solubility of this compound is very limited, we tested its solubility in several pharmaceutically relevant solvents by visual estimation after stiring increasing amount of the compound in 1 ml of solvent for 48 h. to estimate the carcinogenic potential of this compound, salmonella/microsome mutagenicity assay (ames test) was performed in four histidine-requiring strains of s. typhimurium, tester strains ta98, ta 1537 (for the detection of frameshift mutations) ta100 and ta 1535 (for detection of base pair substitutions) according to the oecd guideline 471. the drug was solubilized (500 lg/ml) with no precipitation in lutrol ò -f68/ethanol/water (2.25:2.25:5.5 , wt/wt) micelles (7.3 ae 1.0 nm). the results of ames test were negative so the compound neither produced frame shift mutations nor base pair mutations in s. typhimurium strains. the results imply that the new compound can be dissolved in aqueous micellar delivery system in order to be used for further studies, and that it was not mutagenic in the tested s. typhimurium strains. in conclusion, the formulation of the newly synthesized compound is not carcinogenic, and can be evaluated for anticancer activity in vitro and in vivo. integral metabolism parameters of dairy goats during reproductive cycle periods d. solovyeva, e. zarudnaya, s. zaitsev moscow savmb, moscow, russia study of the goat metabolism at different periods of the reproductive cycle allows to correct feeding ration, to increase the age of the productive use of animals and to receive high-quality products. the aim of the work was to determine the metabolic parameters of blood serum of goats, expressed in terms of biochemical parameters and interfacial tensiometry and study their relationship to metabolic processes in the body goats depending on the age and the period of the reproductive cycle. the 90 healthy goats were divided into 5 groups. the dynamic surface tension (dst) parameters were obtained from dependences of a surface tension (r) vs. time (t): at t?0 (r 0 ), at t = 0.02 s (r 1 ), t = 1 s (r 2 ) and t?∞ (r 3 ). this work was supported by the russian scientific foundation (14-16-00046). all animals had 4-5% fat content. the contents of total protein (5.4%), albumin (8.4%) and urea (8.8%) are higher for the lactating animals as compared to the normal goat values. the levels of total cholesterol (18.0%) and creatinine (6.2%) are higher for the lactating animals. in lactating animals have the highest level of, which along with high phosphorus level talks about the intensification of energy processes during lactation. the correlations were found between the biochemical and dst parameters of the goat blood: lipids or cholesterol levels with r 0 (r = à0.40), r 1 (r = à0.72), r 2 (r = à0.89); total protein or albumin levels with r 1 (r = à0.42), r 2 (r = à0.56), r 3 (r = à0.90); aminotransferase activity with r 2 (r = à0.47), r 3 (r = à0.63). the correlations were found between the total protein and albumin levels with k 0 (r = 0.54), k 1 (r = 0.44); glucose levels and r 1 (r = 0.47), r 2 (r = 0.43). thus, the dst and biochemical parameters of goats have strong correlation relationships that are important for biomedical and veterinary applications. the relation of the severity of atherosclerotic disease with oxidative stress in patients with stable coronary artery disease h. sezen harran university, sanliurfa, turkey introduction: because, to the best of our knowledge, the relationship of total oxidant status (tos) and total antioxidant status (tas) with the severity of stable coronary artery disease (cad) has not been investigated in the literature so far, the present study was conducted to address this issue. materials and methods: this study consisted of 349 consecutive patients and controls who underwent coronary angiography. for each patient, the total gensiniscore (gs) was calculated andthose with a gs of >20 were classified as the high gs group (hgg), and those with a gs less than 20 were defined as the low gs group (lgg). the total oxidant status (tos) and total antioxidant status (tas) levels were measured using the erelmethod. the osi, which is an indicator of the oxidative balance, was calculated as the percentage ratio of tos to tas. results: the tas was lower in the hgg than lgg. the tos and osi were higher in the hgg than lgg. the correlation analysis showed that gs was negatively associated with the tas and positively with the tos and the osi. the multivariate analysis showed that age, tos, and hdl-c were independent variables for a high gs. the cut-off level of 10.9 lmol h 2 o 2 equiv./ l for serum tos levels predicted high gs with a sensitivity of 70% and a specificity of 56%. discussion: information on the severity of atherosclerosis is requiredtopredicttheprognosis of an individualpatientandtodetermine the proper treatment modality. the gs system has beenproventodemonstratethe severity of atherosclerotic disease. inthepresentstudy, thepatientswith a high gs had increasedlevels of oxidants. inaddition, tos was an independentindicator of theseverity of atherosclerosis. the optimal cut-offvaluefor tos topredict high-gens score was 10.9 (sensitivity 70% and specificity 56%). conclusions: the results suggest that the severity of atherosclerosis in stable cad is associated with increased oxidative status. evaluation of roemerine as a multidrug resistance pump inhibitor f. g. avci 1 , c. velioglu 1 , e. recber 1 , c. unsal 2 , g. gulsoy 2 , b. sariyar akbulut 1 1 marmara university, istanbul, turkey, 2 istanbul university, istanbul, turkey efflux by multidrug resistance (mdr) pumps is a common defense mechanism used against antimicrobials. by pumping the drugs out, these pumps significantly reduce the efficacy of drugs. one approach to overcome this limitation is offered by the combinatorial therapies where drugs are co-administered with together with pump inhibitors. by simply preventing the efflux of the drug, the presence of inhibitors enhance drug efficacy. (-)-roemerine is an aporphine type alkaloid with significant antibacterial (against bacillus cereus, escherichia coli) and antifungal (against candida strains) activities. interestingly, (-)-roemerine was also found to enhance the cytotoxic response mediated by vinblastine in multidrug-resistant kb-v1 cells. in the same study, this finding was linked to its possible interaction with p-glycoprotein, a eukaryotic mdr pump. taking this finding as the starting point, the current study investigates the potential of roemerine as an inhibitor of the p-glycoprotein homologue pump, bmra, in bacillus subtilis 168. the antimicrobial agent berberine was used as the model agent since its efficacy is reduced by efflux through mdr pumps. to this end, bacillus subtilis 168 cells were subjected to 100 lg/ml berberine, a value well below the mic. this concentration only slightly retarded growth for 2 hours but then cells resumed their regular growth. upon addition of 25 lg/ml (-)-roemerine to the bacillus subtilis 168 cells treated with berberine, growth pattern changed, indicating possible interaction with bmra. further investigation for the change in the expression of bmra was achieved with real time pcr analysis. glucose oxidase is an enzyme that catalyzes the oxidation of glucose to d-glucono-1,5-lactone and hydrogen peroxide. we hypothesized that enzyme would cause a double negative effect on cancer cells, by reducing the presence of glucose in cancer microenvironment and producing reactive oxygen species. to increase enzyme stability and enhance cellular uptake we encapsulated the enzyme with a thin acrylamide layer. the purpose of this work was to optimize the synthesis of these glucose oxidase nanoparticles and investigate their effect on cancer cells. nanoparticles containing single glucose oxidase were synthesized in two steps; first by introducing the vinyl groups onto the surface of enzyme by acyloylation followed by polymerization step with acrylamide monomers. encapsulated enzymes are approximately 150 nm in size and retain most of their activity. after the optimization of nanoparticles, the anticancer potency of these nanoparticles was in vitro tested in mcf-7 breast cancer cell line. according to results, both nanoparticles and free enzyme are capable of inhibiting viability of cancer cells in a similar manner at very low concentrations. currently we are investigating mechanisms involved in this viability inhibition. initial results demonstrated that glucose supplement does not rescue cells from death induced by the activity of glucose oxidase, suggesting an oxidative stress related cause of inhibition. further studies are required to elucidate the exact mechanism. until now there is no determined advantage of glucose oxidase encapsulation against proteolysis. however, encapsulation may induce the accumulation of enzyme in cancer microenvironment. furthermore results suggest that glucose oxidase has a high effect on the viability of mcf-7 breast cancer cells indicating that this enzyme may have a potential use in cancer treatment. studies on the interaction of human phospholipid scramblase 1 with c-terminal domain of topoisomerase iia u. sivagnanam, s. n. gummadi applied and industrial microbiology lab, bhupat and jyoti mehta school of biosciences, indian institute of technology madras, chennai, india human phospholipid scramblase 1 (hplscr1) is a multifunctional protein that plays key roles in several cellular processes including apoptosis, tumorigenesis, anti-viral defense, cell signalling and several protein-protein interactions. it has been shown that hplscr1 interacts with the c-terminal domain of topoisomerase iia (topo iia) and enhances its decatenation activity in vitro. the interacting region in topo iia was identified but till date, no reports exist on the binding region in hplscr1. this study aims to identify the region of hplscr1 that interacts with topo iia. to identify the topo iia interacting sites in hplscr1, nterminal deletion constructs of hplscr1 viz δ25-hplscr1, δ50-hplscr1, δ75-hplscr1, δ100-hplscr1 and δ160-hplscr1 were generated by pcr, cloned, overexpressed and purified to homogeneity using ni 2+ -nta purification. the cterminal domain (ctd) of topo iia was cloned in pgex6p-1 and was expressed as a gst fusion protein. gst pull down assays will be performed with the deletion constructs of hplscr1 and the gst-ctd-topo iia. the binding region in hplscr1 will be confirmed by peptide competition assays. our initial results show that the decatenation activity of topo iia was enhanced when the topo ii was pretreated with hplscr1. δ100-hplscr1 did not show any enhancement of the decatenation activity compared to full length hplscr1. hence, the binding region could be in the 1-100 region of hplscr1. further deletions were done in the 1-100 region of hplscr1 as described earlier. gst-pull down assays and decatenation assays will be performed for the deletion constructs to narrow down the region of hplscr1 that binds to topo iia. we conclude that hplscr1 interacts with and enhances the activity of topo iia and the 1-100 region of hplscr1 is critical for enhancement of decatenation activity. further work is under progress to identify the exact topo iia binding region of hplscr1 and the physiological relevance of this interaction in the cell. a. ugur kurtoglu 1 , v. aslan 2 , e. kurtoglu 2 1 department of biochemistry, antalya education and research hospital, antalya, turkey, 2 department of hematology, antalya education and research hospital, antalya, turkey beta-thalassemia is a common autosomal recessive disorder resulting from over 200 different mutations of the beta-globin genes. our aim was to creat a mutation map of beta thalassemia in province of antalya, turkey. in this study, mutation analysis of a total 150 of beta-thalassemia patients followed up at the thalassemia center of the antalya education and research hospital, antalya, turkey, were included. according to our results, the ivs 1.110 is the most frequent mutation type in our province same as other geographical regions of turkey. the most frequent mutations in heterozygous or homozygous patients are ivs 1.110, ivs 1.6, ivs 2.1 and ivs 1.1. our results indicate the importance of micromaping and epidemiology studies of thalassemia, which will assist in establishing the national prevention and control program in turkey. keywords: beta-thalassemia, beta-globin gene, mutation p-mis-052 investigation of the in vitro effects of some antibiotics on the purified beta-glucosidases from the rat liver n. t€ urkmen 1 , h. kara 2 1 karadeniz technical university medical biochemistry department, trabzon, turkey, 2 balikesir university veterinary faculty biochemistry department, balikesir, turkey beta-glucosidases catalyzes the hydrolysis of the glycosidic bonds to terminal non-reducing residues in b-d-glucosides and oligosaccharides.b-glucosidases are widely distributed in the living world.b-glucosidases which in mammals, primarily found in the liver and kidneys;lysosomal b-glucosidase (gba1),non-lysosomal b-glucosidase (gba2), cytosolic b-glucosidase (gba3),intestinal lactase-phloriz the hydrolase (lph). liver tissues of wistar-albino rats were homogenized with homogenizer in the extraction buffer and crude extract was obtained after centrifugation.ammoniumsulfate precipitation range designated crude extract was purified by sepharose 4b-ltyrosine-1-naphthylamine hydrophobic gel.commercially availabled antibiotics were prepared with substrate buffer.it was investigated inhbition effects of cefuroximesodium, ampicillin-sulbactam, amoxicillin trihydrate/potassium clavulanate, cefazolin sodium, gentamicin sulfate and ceftriaxone disodium antibiotics onto gba2.inhibition types and k i values of related antibiotics were determined with p-npg substrate.lineweaver-burk plot was used for that purpose. rat liver gba2 was purified at 30.2-fold with 43.4% yield.gba2 was illustrated 58 and 110 kda at sds-page.ic 50 value of ampicillin/sulbactam antibiotic for gba2 was found 62.97 mg/ml with competitive type inhibition and other antibiotics didn't inhibit. purfication methods are being used in the literature for the purified b-glucosidase from different sources.purified gba2 was illustrated 58 and 110 kda at sds-page.about molecular weight of bglucosidases is presented different information in the literat€ ure. this has been reported because of acid beta glucosidases are abnormal migration at the acrylamide or agarose gels.it was investigated inhbition effects of various antibiotics onto purified gba2.ampicillin/sulbactam antibiotic inhibited to purfied gba2 at the competitive type.similiar antibiotics studies have been made in the literature for different enzymes. effect of glutamine on insulin resistance and endoplasmic reticulum stress g. aydogdu 1 , p. b. sermikli 1 , a. abbasi taghidizaj 2 , e. yilmaz 2 1 ankara university, institute of science, ankara, turkey, 2 ankara university, biotechnology institute, ankara, turkey obesity and diseases are one of the most important public health problems of the world.excess fat storage in adipocytes leads to the release of increased amounts of non-esterified fatty acids, glycerol, hormones, cytokines, which are factors involved in the development of insulin resistance that cause type 2 diabetes. one of the major differences between obese and lean individuals is the amino acid concentration in the circulation. although there are many studies about the amino acid metabolism associated with insulin resistance in obese individuals, the effect of glutamine metabolism in insulin resistance mechanisms are not well understood yet. glutamine can be used as fuel and its levels in tissues and circulation can regulate cell responsiveness to insulin and cellular metabolism. therefore, glutamine is a potentially important factor that might help us better understand insulin resistance and type 2 diabetes. to determine whether glutamine effect on insulin resistance and endoplasmic reticulum stress, 3t3-l1 cell is treated with different concentration of glutamine and analyzed by western blot for er stress markers. our results indicated that glutamine reduced endoplasmic reticulum stress and related with that attenuated insulin resistance. in case of transport of amino acids, insulin resistance, how it is affected when we have the information about the important tips on energy requirements and metabolism reach insulin resistance and type-2 diabetes treatment is likely to reveal a possible new targets. how does different lead levels affect tsh, ft3, ft4, vitamin b12 and folate? e. ozkan ankara occupational disease hospital, ankara, turkey exposure to heavy metals is increasing with the industrialization of society. one of the most intense exposure to heavy metals is pb on this issue. this study was aimed to determine the relationship between different blood pb levels and serum thyroid hormones (th), vit b12, folate. the cases were 20-65 years old, male individuals who admitted to our hospital between april 2012-march 2016 for periodic inspections because of occupational exposure to pb. the parameters of the cases were retrospectively retrieved. according to their pb levels, exposed workers (n: 9400) were divided into four subgroups; group (g) 1: 0-9.99 lg/dl, g 2: 10-19.99, g 3: 20-39.99, g 4: ≥40 . from these, the number of cases whose th levels were measured (n:3894) given respectively; g 1:3062, g 2:178, g 3:334, g 4:275 cases. also the number of cases whose vit b12 and folate levels were measured (n:2807) given respectively; g 1:2139, g 2:143, g 3:276,g 4:249 cases. levels of pb were determined by icp-ms. th, vit b12, folate were determined by cmia. between the groups formed according to pb levels, there was no significant difference in terms of average t3, tsh and vitamin b12 (p > 0.05). on the other hand there was statistically significant difference between t4 and group 1,3,4 (p < 0.05) but there was no difference between group 2 (p > 0.05). the average folate belongs to the first group was about 10% higher than the other 3 groups, and found that the difference was statistically significant (p < 0.05). there are many publications which have various results between pb levels and t3,t4, tsh. but this study is important to compare the effect of different levels of pb. up to day there was no publication about the relation between different pb levels and vit b12, folate. it was seen that there was no significant clinical relation between different pb levels and thyroid parameters, vit b12. but the low levels of folate in the high pb levels groups shows us that we need further studies about this relationship. fluorescent study of in meso crystallization of membrane proteins with the introduction of membrane protein in meso crystallization 30 years ago by landau and rosenbusch, a new era of membrane protein structural research has emerged (1). since that time this method became associated with a number of major breakthroughs in the field (2) including exceptional successes in structural studies of microbial rhodopsins and g-protein coupled receptors (3) . here we used fluorescence microscopy to study in meso crystallization process of bacteriorhodopsin. several observations provide new insights into the in meso crystallization process. the crystallization starts with formation of microcrystals, followed by growth of a dominating crystal at the expense of smaller ones and formation of a depletion zone around it. these observations demonstrate an ostwald ripening mechanism of the in meso crystal growth. the depletion zone formed around the growing crystal is consistent with the previously proposed analogy relating in meso crystallization with the crystallization in a microgravity convection-free environment. this work is supported by rsf 14-14-00995. (1) landau, e. m.; rosenbusch, j. p. proc. natl. acad. sci. u. s. a. 1996, 93, 14532à14535. (2) caffrey, m. acta crystallogr., sect. f: struct. biol. commun. 2015, 71, 3-18. (3) katritch v., cherezov v., stevens r.c. (2013) . annu rev pharmacol toxicol 53, 531-556. p-mis-058 stamp1 is critical for both ar and mtor signaling in prostate cancer cells x. sheng, y. jin, f. saatcioglu university of oslo, oslo, norway androgen receptor (ar) signaling plays a central role in the initiation and progression of prostate cancer (pca), including when the disease progresses to castration-resistant pca (crpc). the second central signaling pathway in pca, similar to various other cancers, is the pi3k-akt-mtor signaling. importantly, these two oncogenic pathways cross-regulate each other in pca cells by reciprocal feedback, thereby maintaining tumor cell survival even when one is suppressed. we have previously identified that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk mapk signaling. human pca cell lines lncap and vcap were used in the study. colony formation, soft-agar growth, prostatosphere formation assays were performed. for in vivo xenograft experiment, the cells were implanted subcutaneously into the flanks of nude mice. here, we show that stamp1 knockdown caused defects in colony formation, anchorage-independent growth and prostatosphere formation in lncap and vcap cells both in vitro, as well as tumor formation and growth in vivo. this may be due to the impaired ar and mtor signaling in these cells upon stamp1 knockdown. interestingly, in the crpc cell line 22rv1, where-stamp1 knockdown did not affect mtor signaling, there was a remarkable repression of tumor take rate and growth. these results clearly indicate that stamp1 is essential for both ar and mtor signaling, and is crucial for pca growth in vitro and in vivo. however, the detailed molecular mechanism requires further investigation. taken together, these data unveil a critical role for stamp1 in coordinating the ar and mtor signaling pathways in pca cells, solidifying the basis for its pro-survival effects in pca, including in advanced disease. quantification of 2d thin layer chromatograms using 2d gel analysis software and gel documentation system o. kaynar, m. ileriturk, d. kaynar, s. kurt ataturk university, erzurum, turkey introduction: thin layer chromatography (tlc) is an important chromatographic technique that is widely used as a cost-effective method for rapid-sensitive analysis of compounds in plants, animals, and humans. however, one dimentional (1d) tlc is not sufficient for the separation of complex compounds. therefore, two-dimentional (2d) tlc was developed. the quantitative evaluation of plates are performed with tlc scanners or documentation systems. however, these systems specific for 1d plates, and cannot be adapted to quantitative evaluations of 2d plates. in this study, the applicability of the gel documentation systems and 2d analysis software for the analysis of 2d tlc plates were examined. material and method: 2d tlc of lipids: 1st dimension: methyl acetate/n-propanol/chloroform/methanol/0.25% kcl (25/ 25/28/10/7 v/v); 2nd dimension: chloroform/methanol/acetic acid/ water (90/40/12/2 v/v); detection: charring. 2d tlc of aminoacids: 1st dimension: 1.5% (v/v) formic acid; 2nd dimension: toluene/glacial acetic acid (10:1 v/v); detection: uv. phospholipid and aminoacid standards, each include 6 different classes were developed by 2d tlc. plates visualized with biorad geldoc xr, and band volumes on plates were calculated with biorad pdquest 2d gel analysis software. for the method validationa) 5 plates containing same 6 lipid classes were developed in the same day, and results were used for the calculation of intra-assay cv;cv% = average of each sample standard deviation/mean of sample 9 100 b) 6 plates containing same 6 lipid classes were developed in 6 different days, and results were used for the calculation of interassay cv; cv% = standard deviation of each sample average/mean of the plates 9 100 results: volume of each phospholipid and aminoacid had less than 10% intra and inter-assay cv. conclusion: gel documentation system with 2d gel analysis software can be used for the quantitative analysis of the 2d tl plates both at uv and visible light. the role of na + k + atpase activity in the vasodilatory effect of n-acetylcysteine introduction: spasm occurred at the stage of and after the preparation of arterial grafts used in coronary artery bypass surgery (cabg) is effective on morbidity and mortality in the first 24 hours of postoperative patients. n-acetyl cysteine (nac) that vasodilatory effect is known,may be considered as a suppressor agent for vasospasm developing during cabg. however, for the prevention of complications that may arise during or after cabg mechanism of these vasodilatory effects should be described. this study was aimed to investigate the role of atpase enzyme on the vasodilatory effect of nac. materials and methods: in this study, 28 adult male wistar albino rats were used. rats were separated into four groups as control rats (g1), 2 mm nac (g2), 5 mm nac (g3) and 10 mm nac (g4). a portion of the thoracic aorta isolated from rats was used for the relaxation response recording, and the other portion was used for measurement of nakatpase activity. isolated smooth muscle rings are suspended in the 20 ml organ bath containing krebs solution for relaxation responses. in all groups, level of smooth muscle contraction were allowed to reach a plateau by adding 60 mm kcl to the organ bath. then, in the first 10 minutes of application relaxation responses which created by adding nac to the medium were recorded and the maximum relaxation responses were measured. nak atpase activity was determined using the mazzanti method. groups means were compared by one-way analysis of variance (anova). the threshold for statistical significance was set at .05. results: the contraction force decreased in all nac dose groups compared to control group and this reduction was statistically significant (p < 0.05). similarly, nak atpase activity is also decreased in a dose dependent manner (p < 0.05). the findings obtained in this study suggest that vasodilator effect of nac formed in thoracic aortic smooth muscle was associated with the activity of the enzyme na k atpase. in the presented study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic activity. a bacillus uk69, which was isolated from the soil samples taken from farmland on kahramanmaras sutcu imam university campus, showed high keratinolytic activity when cultured on feather meal medium. the enzyme activity was studied in the ph range of 5.0-12.0. the optimum temperature for keratinase activity was investigated by varying the incubation temperature between 20°c and 80°c. optimum keratinolytic activity was observed at 60°c and ph 10.5. the enzyme was stable at 60°c. the activity was investigated in the presence of some chemicals, including sds, tween 80, dmso, triton x-100, edta, nacl, zncl2, cacl2, glucose. the keratinolytic activity was inhibited by all chemicals tested to some degree. the molecular weight of keratinase was determined by polyacrylamide gel (10%) using standard molecular weight marker and estimated about 37 kda by sds-page. the keratinase isolated from bacillus uk69 could be used in biotechnological processes i.e. feather degradation, wastewater treatment and in industrial processes, such as detergent, food and leather industries. introduction: hemoglobinopathies, including thalassemia, abnormal hemoglobins, constitutes a major group of inherited disorders of hemoglobin synthesis. the reduced or absent of the beta (b) or alpha (a) globin chains of the adult human hemoglobin molecule results beta or alpha thalassemias, leading to imbalanced a-globin/non a-globin chains. the aim of this study was to give a quik desicion with a/b-globin mrna ratio for sequencing of a or b gene, when the anemia is not detectable. materials and methods: mrna and cdna extraction of 25 bthalassemia and 15 a-(including two of 3.7 kb del./hbs) thalassemia subjects and normal controls were accomplished using the high pure rna isolation kit and transcriptor first strand cdna synthesis kit, respectively, following the manufacturer's instructions. we used cdna as a template in the real-time pcr amplification using primers specific for a, b globin genes. amplification was performed in a lightcycler ò 480 instrument. the a/b-globin mrna ratio of each sample was calculated based on the 2 àddct method. results: a/b-globin mrna ratios calculated in a-thalassemia subjects relative to normal control as a result of numbers of defective a-globin genes. the a/b-globin mrna ratio was found higher in b-thalassemia subjects. coinheritance of a-thalassemia in hb s subjects concluded a stabile a/b-globin mrna ratio as per a-thalassemia or b-thalassemia subjects. discussion and conclusion: instability in a/b-globin chains is a significant factor of thalassemia disease severity and can be used before deciding type of gene sequencing when the anemia is not detectable. this study indicates that imbalance in globin gene expression could be demonstrated by measuring a/b-globin mrna ratio, which was conveniently and accurately determined by qrt-pcr and give an information about globin gene function which gene should be correct to investigate an individual for globin gene mutation. p-mis-067 self-assembling peptides mimic supramolecular biochirality r. garifullin 1,2 , m. o. guler 1 1 bilkent university unam, institute of materials science and nanotechnology, ankara, turkey, 2 kazan federal university, institute of fundamental medicine and biology, kazan, russia supramolecular chirality is rooted in asymmetric spatial arrangement of structural elements (e.g. molecules or units with higher hierarchy). self-assembled systems giving rise to this kind of chirality are of great importance because they closely resemble natural biological systems and potentially can lead to new advanced functional materials. in the process of self-assembly, both molecularly chiral and achiral structural units can organize into chiral nanostructures. chiral arrangement of chromophore molecules in space is known to result in emergence of chiroptical properties of a chromophore. organization of pigment-protein complexes into macrodomains in green plants gives rise to biochirality emanating from long-range chiral order of complexes. owing to this order, macrodomains start to absorb circularly polarized light intensively and thus exhibit huge circular dichroism (cd) signal. in our study, a simple approach which was aimed at mimicking the biochirality phenomenon makes use of self-assembling peptide amphiphiles and their interactions with pyrene chromophore. designed peptide amphiphiles are capable of self-assembly into nanofibers with chiral interior, which in principle gives an opportunity to achieve long-range chiral order. two modes of interactioncovalent and noncovalentwere utilized in order to induce supramolecular chirality. covalent interaction mode included direct covalent attachment of pyrene to peptide sequence. upon self-assembly of peptide amphiphile into nanofibers intense circular dichroism phenomenon was observed. noncovalent interaction mode envisioned encapsulation of pyrene molecules in the hydrophobic core of nanofibers of another peptide amphiphile. co-assembly of peptide amphiphile and pyrene molecules led to chiral order and intense cd signal. in addition, it was possible to control the sign of cd signals by using either of peptide isomers, l or d. p-mis-068 pon1 activity in hdl subgroups of obese, overweight and normal weight subjects objective: the aims of this study were isolation of hdl-c subgroups by using precipitation method, determination of pon-1 activity in both total and hdl3 subgroups, and evaluation of performance characteristics of pon-1 activity measurement method in newly diagnosed obese, overweight and normal subjects. material and methods: the study population consists of newly diagnosed 71 obese, 40 overweight and 30 normal subjects. fasting morning blood samples were taken from all study groups. hdl3 subgroup was obtained by heparin-mn-dextran sulphate precipitation method and cholesterol was measured with direct (homegenous) hdl-c method. hdl2-c concentrations were calculated with the subtraction of hdl3-c from total hdl-c. hdl3-c and total pon-1 activity were determined by using eckerson method. non-hdl3 pon-1 activitiy was calculated with subtraction of hdl3 pon-1 activity from total pon-1 activity. results: total hdl-c, hdl2-c and hdl3-c concentrations and the activity of total pon-1 and hdl3 pon-1 were found lower in obesity according to overweight and normal subjects (p < 0.001). negative correlations were found between body mass index and hdl3-c, total pon-1 and hdl3 pon-1 (r = à0.244, p < 0.005; r = à0.247, p < 0.005; r = à0.199, p < 0.05, respectively). conclusion: our findings indicated that hdl-c metabolism and lipoprotein associated antioxidant defense mechanisms were adversely affected with obesity. in conclusion we think that precipitation method using for separating hdl3 subgroup, is simple and cost effective for routine applications in clinical laboratories. besides hdl3-c measurements, pon1 activity, measurement of total and hdl3-c subgroup might be helpful to evaluate the atherosclerotic process in obese subjects. keywords: obesity, body mass index, paraoxonase, hdl subgroup, cholesterol p-mis-069 hepatitis e virus antibody prevelance among persons who work with animals in north cyprus introductions: hepatitis e infection is a major cause of viral hepatitis in many developing countries. the objectives of the present study was to determine the seroprevelance of hev infection in peoples who work with animals in northern cyprus. materials and methods: prevelance of hev infection were determined in study 4 group population: persons without occupational exposure to animals; persons who work with animals; veterinarian and butcher. a total of 400 blood samples were collected. all serum samples were tested elisa using a commercially available kit according to the manufacturer's instructions. ti-test were used for istatistical analyses. p > 0.05 was accepted as significant value. results: in a study of 400 blood donors (334 male, 66 female), the overall prevelance of anti-hev igg antibodies were 3.0%. the blood samples were collected 5 different areas. the prevelance of anti-hev igm antibodies was 0.25% and he was 44 years and acting a butcher during 20 years. the prevelance of anti-hev igg of women were approximately two fold higher than men. no significant difference in anti-hev prevelance was observed between the age of the blood donors. according to the anti-hev igg prevelance, the without occupation expose to animal animal were 1%, the animal husbandry were 7% and the veterians and the butcher were 2% were found. discussion: the prevelance of anti-hev in the north cyprus (3%) was found low such as the prevelance of the turkey (5%). the prevelance of anti-hev igg in animal husbandry were higher that the other groups because of they may be more spend of time and contact with animals. the prevelance of igm results suggested that the possibility of outbreaks may be low in north cyprus. conclusion: this study was the first seroprevelance analysis of north cyprus according to the population number.the further studies could be included the seroprevelance of anti-hev from the animals. most errors in the clinical laboratory occur in the preanalytical phase the aim of this study was to investigate the causes and rates of rejected samples, regarding to certain test groups in our laboratory. this study was designed on the rejected samples between january 2015 and january 2016. clinical chemistry, coagulation, hormone, cardiac markers, total urine evaluation and other (ethanol level, hba1c, hb electrophoresis, neonatal bilirubin, drug level, blood gas, fecal occult blood) test groups were included. the total number of specimen and rejected samples was obtained from the hospital information system retrospectively. types of inappropriateness were evaluated as follows: erroneous coding, clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen. it was determined that 873343 blood samples were sent to our laboratory in one-year period. 0.37% of them were rejected because of preanalytical errors. erroneous coding was found as the most common rejection cause (33%). rejection rates of clotted specimen, hemolysis, insufficient volume, incorrect patient, incorrect tube and inappropriate specimen were found to be 11%, 15%, 19%, 2%, 4% and 16% respectively. in our study, erroneous coding was the most common cause of preanalytical errors. education of medical secretaries is relevant and important as can be seen in the decrease of sample errors and the resulting quality improvement. glycosylated hemoglobin test (hba1c) is important for screening, diagnosing, and monitoring diabetes and prediabetes. however, hba1c levels may dependent on patient ethnicity suggesting that the diagnostic cut-offs should be evaluated for specific populations. therefore, our aim in this study was to evaluate the efficiency of hba1c for predicting diabetes in comparison to oral glucose tolerance test (ogtt) results for turkish population. the study included anonymous lab results (acibadem labmed laboratories in turkey) of 6270 patients (3913 female, 2351 male) aging 40.4 ae 11.6 years (15-86) who had an initial diagnosis of diabetes. glucose and insulin levels during ogtt were measured after the initial administration of 75 g sugar (0hour), 1-hour and 2-hour. these parameters were statistically analyzed in comparison to simultaneous hba1c results. glucose measurements at 1 hour had better distinction power (p < 0.05) between these individual groups than initial and 2-hour glucose measurements. the average hba1c (%) levels for healthy, pre-diabetic and diabetic individuals were 5.4 ae 0.4, 5.7 ae 0.4 and 6.2 ae 0.7, respectively. roc curve analysis showed 23.4% sensitivity and 98.2% specificity for the clinically accepted hba1c cut-off value of 6.5%. hba1c cut-off value of 5.9% had a higher sensitivity of 67.8% and comparable specificity of 85.7%. the highest discrimination power between healthy, pre-diabetic and diabetic individuals was observed at glucose concentration at 1-hour after sugar administration in ogtt test as opposed 2-hours generally used for diagnosis. low sensitivity was observed for the clinically adapted 6.5% cut-off value of hba1c. the cut-off value of 5.9% for hba1c was found to be more sensitive with comparable specificity than the 6.5% cut-off values for diabetes screening in our population. our results suggest that 5.9% for hba1c should be considered for diabetes cutoff value for turkish population. induction of the glutathione-dependent detoxification capacity is involved in the hepatoprotective effect of silymarin against acetaminophen-induced hepatotoxicity y. kim, d. kwon, c. ahn seoul national university, seoul, south korea recent findings in this laboratory showed that silymarin was capable of promoting hepatic glutathione (gsh) synthesis via a modification of the transsulfuration reactions in the liver. to investigate its pharmacological significance, we examined the hepatoprotective effect of silymarin against liver injury induced by acetaminophen (apap). adult male mice were treated with silymarin (200 mg/kg, po) every 12 hours for a total of 3 doses prior to an apap challenge (500 mg/kg, ip). the apap-induced liver injury was assessed by histopathological examination and measurement of changes in plasma enzyme activities, lipid peroxidation and formation of nitrotyrosine protein adducts in the liver. plasma levels of apap and its major metabolites were monitored for 24 hours to estimate the metabolic transformation of apap. also protein and activity of the major cyp subtypes involved in the metabolic activation of apap into a toxic metabolite were determined in liver of the mice treated with silymarin only. silymarin pretreatment attenuated the apap-induced liver injury significantly when determined 24 hours later. plasma concentrations of apap, apap-glucuronide or apap-sulfate in plasma were not changed, but thiol conjugates of apap, such as apap-glutathione, apap-cysteine and apap-n-acetylcysteine, were elevated significantly in the mice pretreated with silymarin. however, silymarin treatment did not affect protein expression of cyp2e1, cyp1a2, or cyp3a11 in the liver. also hepatic microsomal enzyme activities measured using p-nitrophenol, ethoxyresorufin and erythromycin as substrates, were not increased by silymarin, indicating that the elevation of apap-thiol conjugates should be attributed to an augmentation of the gsh conjugation capacity. it is suggested that silymarin may protect the liver against an electrophilic substance-induced toxicity by increasing gsh availability which would enhance the detoxifying capacity of liver cells. prostate cancer (pca) is the second leading cause of death among men in western countries. we have previously found that the six transmembrane protein of prostate 1 (stamp1) promotes pca cell proliferation as well as inhibits apoptosis through, at least in part, regulating the erk/mapk signaling. we also found that stamp1 is highly mobile in pca cells and shuttles between the plasma membrane and the golgi, often found in vesiculotubular structures in the cytosol. using advanced imaging techniques, we have now characterized the trafficking of stamp1 from the plasma membrane to early endosomes in lncap cells, by analysing its dynamic targeting to the three main endocytosis pathways: clathrin-mediated endocytosis, caveolae/lipid rafts, and the arf6-dependent pathway. we found that stamp1 fused to cyan fluorescent protein (cfp-stamp1) is present at the plasma membrane where it accumulates in punctate structures. live cell confocal imaging showed that these puncta were dynamic over time indicating that stamp1 may be constitutively delivered to the plasma membrane and removed from it by endocytosis. co-expression of cfp-stamp1 with various fluorescent protein markers revealed that cfp-stamp1 puncta corresponded to lipid rafts that were labelled with caveolin-1-rfp or antibodies against flotillin. live cell imaging showed that cfp-stamp1 and caveolin-1-rfp disappeared at the same time from the same region of the plasma membrane suggesting that lipid rafts are likely to be responsible for stamp1 internalization. notably, stamp1 was absent from other endocytosis structures such as clathrin-coated pits/vesicles. further work is needed to determine whether stamp1 internalization is required for its function, such as its link to erk signaling, and whether interference with lipid rafts influences stamp1 effects on pca cell proliferation and survival. antithrombin-iii, mpv and plasma total homocysteine levels in behcet's disease introduction: behcet's disease is a multi-systemic and chronic inflammatory vasculitis of unknown etiology characterized by recurrent oral and genital ulcers, uveitis, arthritis, arterial aneurysms, venous thrombosis and skin lesions. platelet indices such as mean platelet volume (mpv) is a standart indicator of platelet function in disease pathophysiology. antithrombin, a glycoprotein synthesized in the liver, is the major plasma inhibitor of thrombin thus modulating blood coagulation. antithrombi-iii (at-iii) is a enzyme even moderate deficiency significantly increases the risk of thrombosis. homocysteine (hcy), that is formed during the metabolism of methionine. several clinical studies have clarified that elevated blood hcy levels are related to atherosclerotic disease. in our study, we investigated ovocystatin is one of the best characterized members of cystatin superfamily of protease inhibitors, and it has been frequently used for pathophysiological studies as the model protein, representative for this superfamily. its application has been supported by high structural similarity to human cystatin c as well as several common biological activities. as regard to biological activity, cystatins, including ovocystatin, are best characterized as inhibitors of cysteine proteases of papain family (c1), such as cathepsins b, h, l and s. these inhibitors participate in intra-and extracellular control of proteolytic events, both in physiological and pathological states. in the recent decade also new activities of cystatins, not assigned to inhibition of papain-like cysteine cathepsins, were found. these activities are associated with an alternative active center for legumain-type proteases in the molecule. here we report a chemical modification of ovocystatin that disables the anti-papain activity of the inhibitor but does not affect its anti-legumain activity. the chemical knockout has been obtained by reaction with 2-hydroxy-5-nitrobenzyl bromide (hnbb) that covalently modifies the trp104 residue in the molecule. the reaction has been monitored by uv-vis and fluorescence spectroscopy. the anti-papain activity of the inhibitor has been measured colorimetrically against bana as a substrate. the anti-legumain activity was assessed fluorometrically using z-ala-ala-asn-amc. the reacted inhibitor exhibited an additional, characteristic for hnbb, band at 410 nm in uv-vis scan. accordingly, an ablation of trp fluorescence was also observed. the molecule fully retained the anti-legumain activity, while only residual antipapain activity (10%) was observed. the modified ovocystatin can be a useful molecular tool for studying the physiological and pathological processes specifically associated with legumain activity. 3 departments of medicine (hematology/oncology) and biochemistry and molecular biology, university of louisville, james graham brown cancer center, louisville, ky, united states 6-phosphofructo-2-kinase/fructose-2,6-bisphospatase (pfkfb) family of enzymes are responsible for the conversion of fructose-6-phosphate (f6p) to fructose-2,6-bisphosphate (f2,6bp) and vice versa, and f2,6bp is an allosteric activator of phosphofructokinase-1 (pfk1), a rate-limiting enzyme of glycolysis. among the four identified pfkfb isozymes (pfkfb1-4), pfkfb2 is the least studied isozyme in human cancers. there exists two different splice variants of pfkfb2, variant-1 and variant-2, coding two different isoforms, isoform a and b, respectively. in this study, we first analyzed the effect of k-ras(g12d)induced oncogenic transformation on pfkfb2 expression in pancreatic duct cells. we found that oncogenic k-ras induction in immortalized pancreatic duct cells (ipde) was associated with decreases in total pfkfb2 mrna and protein expressions (mrna; ipde: 1 ae 0.15; ipde+kras: 0.78 ae 0.24 and protein; ipde: 1 ipde+kras: 0.55). we then, checked individual expressions of splice variants and observed that while pfkfb2 splice variant-1 (p2-v1) expression was reduced by k-ras induction (ipde:100; ipde+kras:81.50), pfkfb2 splice variant-2 (p2-v2) expression was increased (ipde:100; ipde+kras:125.70). then, we checked effects of p2-v1 and p2-v2 on glycolytic phenotype of ipde and ipde+kras cells. over-expression of pfkfb2 variants increased f2,6bp concentration (p2-v1: 1.96; p2-v2: 1.72 fold; compared to empty vec), glucose uptake (p2-v1: 16%; p2-v2: 30%) and glycolysis (p2-v1: 20%; p2-v2: 30%) in ipde+k-ras cells. we next analyzed the subcellular localizations of pfkfb2 isoforms and observed that both pfkfb2 isozymes localize to the nucleus, with more prominent nuclear localization of p2-v1 compared to p2-v2. also, nuclear localization ratio of p2-v2 increases after oncogenic transformation with mutant k-ras. taken together, these results suggest that pfkfb2 may have a role in the glycolytic phenotype of pancreatic cancers characterized with hyperactive k-ras signaling. effects of p38 map kinase inhibitors on mda-mb-231 cell line introduction: p38 mapk phosphorylates serine and/or threonine residues of the target proteins. the activation of p38 mapk leads to cell growth, differentiation, survival or apoptosis. in this study, we tested the effect p38 mapk sb203580 and sb202190 on mda-mb-231 cells to further elucidate the controversial role of p38 mapk on cell proliferation or cell migration. materials and methods: mda-mb-231 cancer line was cultured in rpmi-1640 supplemented with 10% fbs. the cytotoxic and cell migration effects of sb203580 and sb202190 inhibitors were tested by mtt assay and wound assay, respectively. the effects of both inhibitors on proliferation and adhesion of md-mb-231 cells were determined by icelligence system. results: it was found that sb202190 p38 map kinase inhibitor was more effective than sb203580. however, no significant effects of low doses of 1 lm and 5 lm of both inhibitors were seen on cell proliferation as compared to the dmso-treated control cells for up to 96 hours as determined by icelligence system. on the other hand, both sb203580 and sb202190 significantly prevented cell proliferation at a concentration of 50 lm. both sb203580 and sb202190 significantly reduced cell migration in a time-dependent manner at a concentration of 50 lm. then, we tested whether each p38 mapk inhibitors have any effect on cell adhesion during a treatment period of 3 hours using icelligence system. only 50 lm concentration of sb202190 reduced cell adhesion for about 1.5 hour (p < 0.001). conclusion: p38 mapk inhibitors sb203580 and sb202190 differentially affect cell proliferation, survival and migration. acknowledgements: this study is financially supported by dumlupınar university, scientific research project no 2015-85. mutagenicity of a series efficacious benzoxazine derivativesa new approach to evaluate ames test data e. foto 1 , f. zilifdar 1 , s. yilmaz 2 , t. sarac ßbasi 1 , i. yalc ßin 2 , n. diril 1 1 hacettepe university, ankara, turkey, 2 ankara university, ankara, turkey testing safety of drug candidates is as crucial as evaluating their efficacy in early drug development. we previously synthesized a series of 1,4-benzoxazine-3-one derivatives showing significant antimicrobial, in vitro anticancer, topoisomerase i inhibitory activities and studied their several mechanisms of action. in this present study, we have evaluated mutagenic activities of these compounds and their potential metabolites. moreover, we aimed to develop a new statistical algorithm available for structureactivity relationship analysis to identify the regions responsible for the activity. to evaluate mutagenicity of the compounds, ames salmonella/microsome test was used. salmonella typhimurium ta98 and ta100 strains were used to detect for frameshift and basepair substitution mutagens, respectively. additionally, mutagenicity of potential metabolites of them were evaluated by adding metabolic activation system (s9) which was prepared from a pool of male sprague dawley rats. results were evaluated with student's-t test. following regression model estimation analysis, we detected minimum mutagenic doses of all tested compounds for generating a 3d-common features pharmacophore model with hiphop method. according to the results, only bs12, bs13, bs16 and bs17 exhibited strong mutagenic effects on both strains in the presence and absence of s9. additionally bs10, bs7, bs1 and bs15 (in the absence of the s9), bs18, bs4 and bs7 (in the presence of the s9) showed weak mutagenic effects on ta98. hiphop analysis results revealed that mutagenicity was increased in the presence of aromatic desactivating groups which might form hydrogen bonds at the position of r3 and hydrophobic groups at the position of r2 of the benzene ring in the structure of benzoxazine. the new statistical approach developed in this study can be useful for assessing the ames test data available for structure activity relationship analyses. background: recently more than thirty different diseases can screen simultaneously with expanded newborn screening (nbs) programs by tandem ms.expanded nbs with tandem ms is performed routinely at akdeniz university hospital central laboratory since 2008.the aim of this study was to evaluate our nbs results with some second-tier and confirmatory tests. materials and methods: nbs results (n = 1863) were evaluated in dried blood samples which sent to our laboratory for the study between august 2014 and august 2015. electrospray ionisation (esi)triple quadrupole mass spectrometer (shimadzu lc-ms/ms 8030,japan) was used for nbs analysis,acylcarnitine and amino acid profile were screened with mrm (multiple reaction monitoring) spectrum within 2 minutes.second-tier tests were performed as urine organic acid analysis by gas chromatography-mass spectrometry (gc-ms),plasma and urine quantitative amino acid analysis by high pressure liquid chromatography (hplc).pathological nbs results were assessed in three separate groups as amino acid metabolism disorders, fatty acid oxidation defects and organic acidemias. results: metabolic diseases were found in 69 (3.70%) patients by the second-tier tests performed.there were detected amino acid metabolism disorders in 13,organic acidemia in 42,fatty acid oxidation defects in 14 patients. conclusions: the reason of high positive results in our laboratory could explain that our study includes both screening and monitoring of previously diagnosed metabolic patients.nbs is performed in only a few centers in turkey although there were the national screening programs included nbs in many foreign countries.more expanded nbs programmes in our country is required to start treatment of patients before irreversible damage is not occured. although many reports indicate the involvement of calpain in several human pathologies, it is not yet clarified how the protease can recognize the substrates to digest and how can escape to its natural inhibitor calpastatin. answers to these questions have been obtained by identifying specific intracellular localizations of calpain and its substrates and analyzing the interactions of the protease with calpastatin. these studies were carried out using human sknbe neuroblastoma cells. protein-protein interactions and intracellular localization of calpain and the related proteins were determined by immunoprecipitation and isolation of membrane microdomains. we have observed that small amounts of calpain-1 are localized in lipid rafts microdomains together with n-methyl-d-aspartate receptor (nmdar) containing nr1/nr2b subunits. immunoprecipitation experiments have demonstrated that nmdar containing nr1/nr2b subunits, calpain-1, hsp90 and neuronal nitric oxide synthase (nnos) but not calpastatin and calpain-2 are present in specific protein complexes. thus, in this localization calpain activity is regulated by hsp90 that reduces the affinity for ca 2+ of the protease. cell stimulation with nmdar agonists induces calpain activation that specifically cleaves the subunits nr2b of the receptor promoting changes in lipid rafts organization and internalization of nmdar without affecting cell viability. moreover, in these conditions, also nnos is digested and converted in the active form by calpain-1. our data suggest a physiological role of calpain-1 at specific cell sites. the protease inserted in lipid rafts microdomains is in strict contact with its targets and escapes to calpastatin which is not inserted in these structures. following an increase in ca 2+ influx, the activated protease regulated by hsp90, promotes the removal of nmdar from the plasma membranes, decreasing ca 2+ entrance through this receptor-channel and protecting cells from ca 2+ overloading. tissue transglutaminase 2 (tg2) is a multifunctional protein complex that can act as a crosslinking enzyme, gtpase/atpase, protein kinase and protein disulfide isomerase. at the cell surface, tg2 was shown to be involved in adhesion, migration, invasion, growth, epithelial mesenchymal transition and hence implied in the metastatic development of many different tumor types. renal cell cancer (rcc) is one of the most common type of cancer in adult males that generally grows as a single tumor within a kidney. our previous findings indicate that the increased expression of tg2 in rcc results in tumor metastasis with a significant decrease in disease-and cancer-specific survival outcome. herewith, the role of tg2 in cell migration of rcc was investigated in this study by transducing the model rcc mouse cell line renca with a series of tg2 mutant constructs. renca cells were transduced by lentiviral particles encoding wttg2, transaminase-defective tg2-c277s form with low gtpbinding affinity, gtp-binding deficient form tg2-r580a, and transaminase-inactive tg2-w241a. in order to investigate the role of tg2 transamidating and gtpase activity in cell migration, scattering assay was used where 7 colonies for each mutant clone was followed for a time interval of 24 hours. our results showed that non-transduced control and tg2-c277s mutant renca cells demonstrated a similar migration pattern with a 20% of scatter activity. on the other hand, 52% colonies formed by renca cells overexpressing wttg2 and tg2-w241a mutant scattered away from each other. a small insignificant increase in scattering was seen in 28% of the total number of 10 colonies for renca cells overexpressing tg-r580a construct. data from this study supports that gtp-binding activity of tg2 is the drive force in migration driven scattering of renca cells, suggesting that inhibitors targeting the gtp-binding activity of tg2 may serve as a new therapeutic approach in the treatment of rcc. background: in this study, we aimed to investigate the relationship between level of vitamin d with subclinical hypothyroidism and subclinical hyperthyroidism. material and metod: study groups planned as three groups such as euthyroid (n = 11966), subclinical hypothyroid (n = 1040), subclinical hyperthyroid (n = 795). serum tsh, free t4 (ft4) and free t3 (ft3) levels were determined by chemiluminescence immunoassay and serum 25-hydroxy (oh) vitamin d 25 (oh) d level were determined by liquid chromatography-tandem mass spectrometry. euthyroidism was defined as a normal level of tsh (range, 0.4 to 4.2 miu/l), ft4 (range, 0.8 to 2.3 ng/dl) and ft3 (range, 2.3 to 4.2 ng/dl). subclinical hypothyroidism is defined as an elevated serum tsh level associated with normal total or free t4 and t3 levels. subclinical hyperthyroidism is defined as low serum tsh levels associated with normal free t4 and free t3 levels. results: subclinical hyperthyroid group had significantly higher 25 (oh) vitamin d levels compared to the euthyroid and subclinical hypothyroid groups (p < 0.05). 25 (oh) vitamin d levels in subclinical hypothyroid group was not statistically significant when compared with the euthyroid group. food processing wastes provide carbon sources in high amounts for fermentative microorganisms to produce energy. converting carbon-rich biomass into bioethanol through fermentation by microorganisms both provides energy requirement for humankind and also decrease pollutant gases like co 2 , no x and so x (ghorbani et al., 2011) . fermentation processes for bio-ethanol production could be achieved by saccharomyces cerevisiae, zymomonas mobilis, and escherichia coli. bacterial hemoglobin (vitreoscilla hemoglobin, vhb) is the first and best characterized prokaryotic hemoglobin molecule. the function of vhb is supporting the cellular respiration through binding to oxygen at microaerobic environment, transferring it to the terminal respiration oxidases (geckil et al., 2004) and thus improving growth and productivity of the microorganisms. in this study, e.coli strains fbr5, ts3 and ts4 were used as ethanologenic microorganisms. expression of vhb in ts3 is lower than in ts4 strain. for the efficient ethanol production effect of different inoculum sizes, sugar species and sugar concentrations in the growth medium were investigated. vhb expression increased effectively the viability of ts3 strain by up to 1.8x10 9 cfu per ml of fructose (3%, w/v) supplemented lb medium starting with small inoculum for fermentation. this indicates that vgb expression should be at the certain level to maintain sufficient the cell growth for ethanol production. geckil h, gencer s (2004) . production of l-asparaginase in enterobacter aerogenes expressing vitreoscilla hemoglobin for efficient oxygen uptake. applied microbiology and biotechnology 63: 691-697. ghorbani, f., younesi, h., sari, a. e., najafpour, g. (2011) . cane molasses fermentation for continuous ethanol production in an immobilized cells reactor by saccharomyces cerevisiae. ethanol production from dairy industry by product using bacterial hemoglobin t. sar, g. seker, a. g. erman, m. yesilcimen akbas gebze technical university, depertment of molecular biology and genetics, kocaeli, turkey bioethanol production from biomass has a great potential to reduce greenhouse gases emissions. ethanol has several applications in industries (chemical, medical, pharmaceutical, food etc.) in the form of raw material, solvent and fuel. one of the most abundant liquid wastes is cheese whey generated from dairy industries. whey powder is concentrated form of whey and contains lactose and also protein, lipid, minerals and vitamins. vitreoscilla hemoglobin (vhb) is the first bacterial hemoglobin. the main function of this molecule is to improve oxygen transfer to cellular oxidases and thus supporting cellular growth and productivity at low oxygen levels (kallio et al. 1994) . in this work, e. coli strains fbr5, ts3 (low level vhb expressing) and ts4 (high level vhb expressing) were used as ethanol producing microorganisms. fermentation medium containing whey powder supplemented with lb material was inoculated with these strains and incubated for 48 hours at 37°c and 180 rpm in a 1000 ml erlenmayer flask. the ethanol production was improved over 300% by using lower vhb expressing strain. the ethanol levels (v/v, %) were determined as 1.08, 4.99 and 1.51 for fbr5, ts3 and ts4 strains respectively. it is shown that the certain levels of vhb could be useful tool to increase the growth and productivity of ethanol from dairy industry wastes. kallio p.t., kim d.j., tsai p.s. and bailey j.e. (1994) . bioethanol is usually produced from cellulose, hemicellulose and lignin. the lignocellulosic wastes should be hydrolysed into fermentable sugars by using enzymes or dilute acids before microbial fermentation. acidic hydrolysis methodology is cheaper than enzymatic hydrolysis but it can cause production of some inhibitors like aliphatic acids, which affect the growth of microorganisms. vitreoscilla hemoglobin (vhb) is the first described prokaryotic hemoglobin. the recombinant strains carrying vgb gene (e. coli, p. aureginosa) which encodes vhb showed increased bacterial growth, productivity of metabolites compared to untransformant counterparts under low oxygen concentrations [nasr et al., 2001; geckil et al., 2004] . in this study, ethanologic e. coli strains fbr5, its derivative strains ts3 (vgb+) and ts4 (vgb+) were used. ts4 was constructed in such that it could express more vhb than ts3. bioethanol production by these strains in presence of lignocellulosic hydrolysates derived inhibitors was investigated. different acetic acid concentrations (2.5-10 mm) were used as inhibitors from lignocellulose hydrolysate. 10.0 mm acetic acid was used as an inhibitor. the growth of vhb expressing ts3 and ts4 strains was inhibited about 31% after 48 hours fermentation time. strain fbr5 was inhibited as high as 76% by using the same inhibitor including growth medium. it was shown that the expression of vhb could improve growth and productivity in presence of lignocellulosic inhibitors. differentiation of preadipocyte, also called adipogenesis, leads to the phenotype of mature adipocyte. however, excessive adipogenesis is closely linked to the development of obesity. thus, any drug or chemical that can inhibit adipogenesis may have preventive and/or therapeutic potential against obesity and related diseases. azd1208, an inhibitor of the family of pim kinases, is known for anti-cancer activity. here we investigated the effect of azd1208 on adipogenesis in 3t3-l1 preadipocytes. notably, azd1208 treatment led to a concentration-dependent inhibition of both lipid accumulation and triglyceride (tg) synthesis during the differentiation of 3t3-l1 preadipocytes into adipocytes with no cytotoxicity. on mechanistic levels, azd1208 strongly reduced not only the expression levels of ccaat/enhancer-binding protein-a (c/ebp-a), peroxisome proliferator-activated receptor-c (ppar-c), fatty acid synthase (fas), and perilipin a but also the phosphorylation levels of signal transducer and activator of transcription-3 (stat-3) during adipocyte differentiation. furthermore, azd1208 largely decreased leptin, but not adiponectin, mrna expressions during adipocyte differentiation. collectively, these results demonstrate that azd1208 inhibits adipogenesis in 3t3-l1 preadipocytes and the inhibition is largely attributable to the reduced expression and/or phosphorylation levels of c/ebp-a, ppar-c, fas, perilipin a, and stat-3. effect of intrauterin exposure to artificial food colourings on dna damage in rats in many research genotoxic potential of food additives has been investigated. however there are few findings about the effect of artificial food colourings (afc) on dna. in this experimental study, we aimed to analyze whether in utero exposed artificial food colourings would have effect on dna and cause damage.thirteen female rats were included to the study which were equally divided into two groups as control (cg, n = 15) and food colouring (fcg, n = 15) groups. a mixture of nine food colours were given daily to fcg by oral gavage from preconception to birth. no adverse effect level (noael) of artificial food colourings for each colouring was administered to fcg. three months after the birth, 6 offspring from each group were selected randomly as control (cg) and experiment (eg) groups. then they were sacrified under anesthesia. for performing the alkaline comet comet assay leukocytes were seperated from whole blood samples. the alkaline comet assay was performed. the extent of dna damage was assessed from the length of dna migration derived by subtracting the diameter of the nucleus from the total length of the image and graded into 5 categories and these grades were converted into arbitrary unit (au). differences between the means of data were compared by independent samples t test. the results were given as the meanaesd, p values of less than 0.05 were considered as statistically significant. although the extent of dna damage was higher in eg, the comparison of experiment (13.50 ae 1.76) and control (11.66 ae 1.36) groups showed no statistical difference (p = 0.072). relationship between glucocorticoid receptor gene polymorphisms and recurrent depression l. aydogan, i. benli, z. c. ozmen, i. butun gaziosmanpasa university medical faculty, department of biochemistry, tokat, turkey objective: sensitivity to glucocorticoids varies between individuals and these differences have been implicated in the etiology of psychiatric diseases such as depression. recent studies have found relationship between common glucocorticoid receptor (gr) gene (nr3c1) polymorphisms and unipolar or bipolar depression. the nr3c1 gene is a candidate gene affecting depressive disorder risk and management. the aim of the present study was to evaluate the relative distribution of specific polymorphisms of nr3c1 (bcl1 and rs33388) in recurrent depressive disorder (rdd) patients. methods: our study included 100 volunteers with recurrent depressive disorder and 100 healthy individuals without any mental illness. depression was assessed by hamilton and madrs depression scale. nr3c1 gene polymorphisms were detected by real-time pcr, with hybridization probe method. allele and genotype frequencies at two loci (bcli and rs33388) were investigated in rdd patients and controls. results: genotype distribution among rdd patients and the control group for bcl-1 (g/c) were as follows: cc 3% and 5%, gc 27% and 34%, gg 70% and 61%, respectively. there was not a significant difference when the frequency of the allele (p = 0.204) and genotype frequency (p = 0.382) were compared between the patients and the control. genotype distribution in the rs33388region (a/t) of the patients and controls were tt 29% and 25%, ta 49% and 54%, aa 22% and 21%, respectively. allele frequency (p = 0.841) and the genotype frequencies (p = 0.754) were not significantly different among the groups. conclusion: numerous nr3c1 gene polymorphisms were previously reported in association with modification of depressive disorders. the results of our study showed no association between gr genotype and recurrent depressive disorder. nr3c1 polymorphism does not play a role in the development of recurrent depressive disorder. thymoquinone (tq) has been shown to supress the proliferation of various tumor cells, while it is minimally toxic to normal cells. the aim of this study is to investigate the potential therapeutic effects of tq on cell proliferation, apoptosis, invasion, migration, colony formation and wound-healing in sh-sy5y human neuroblastoma cell line. sh-sy5y cell line treated with 5-400 lm tq by solving medium for 24, 48 and 72 h considering a time-and dose-dependent manner. the cytotoxic effect of tq was determined by mtt method. total rna was isolated by trizol reagent. cdna synthesis was performed by using commercial kit. mdm2, p53, p21, akt, pten, cdk4, cyclin d1, caspase-3, -8, -9, -10, bcl-2, bax, parp, bcl-xl, bid, dr4, dr5, puma, noxa, mmp-2, -9, timp-1, -2 and gapdh gene expression profiles were analysed by real-time pcr method. effects of tq in sh-sy5y cells on invasion, colony formation and cell migration were detected by matrigel-chamber, colony formation assay and woundhealing assay, respectively. statistical analysis were performed with rt 2 profiles array data analysis by using student's t test. ic 50 value of tq in sh-sy5y cells was detected as 15 lm at 48 th hours. by rt-pcr results, it was determined that tq caused a decrease in the expression of mdm2, akt, cdk4, cyclin d1, bcl-2 and mmp-2. it is also observed that tq caused a significant increase in the expression of p53, pten, caspase-3, -10, bid, dr4, puma, noxa and timp-1. it was also found that tq in sh-sy5y cells suppressed invasion, migration and colony formation by using matrigel invasion chamber, wound healing and colony formation assay, respectively. in conclusion, we demonstrate that tq significantly effect cell cycle, apoptosis, invasion, migration and colony formation of sh-sy5y cells. tq may be a potential candidate as chemotherapeutic agent for the treatment of neuroblastoma. more studies have to be performed to profile the mechanisms and genome wide effects of tq to prove its therapeutic potential. dna aptamers can achieve a very high affinity to the target due to the potential of developing broad target-binding interface. however, classic strategy selection of aptamer binders is a challenging task requiring multiple rounds of panning and post-selection optimization. we have developed fast and convenient technique for the selection of dna aptamers based on the offrate selection and tandem affinity purification (tap). we constructed and produced in e.coli recombinant chimeric protein, comprising two affinity tags (his6 and gst) separated from each other and from the target protein (anthrax protective antigen domain iv, padiv) by sumo protease recognition polypeptide and synthetic cleavage site for the anthrax lethal factor (lf). the protein bound to aptamer library is first captured by imac resin, cleaved by sumo protease, captured by gst resin and eluted by lf following the lines of the tap method. the gst-captured aptamer-target complexes were subjected to the off-rate selection using soluble padiv as the competitor. multiple selection rounds are cumbersome and can result in carryover. high abundance of moderate affinity aptamers in the resulting pools obtained by classic selection approaches suggests that the procedure to counter-select them at the beginning of panning is needed. reduction of the contact duration between the aptamer library and the target was crucial for efficient selection of high-affinity binders. on the other hand, tap prevents contamination, and bundled with the off-rate selection, allows for clean isolation of high-affinity binders with affinity in the low nanomolar range. the developed technique is applicable for efficient selection of high affinity dna binders to soluble recombinant proteins and their fragments. dna aptamers obtained will be further used for the development of diagnostic and therapeutic tools for the detection and treatment of anthrax. the work was supported by russian science foundation research grant no. 14-15-00630. the role of macab efflux pump in protection of serratia marcescens against antibiotics and oxidative stress the emergence of bacterial multi-drug resistance is a growing problem of public health worldwide. bacterial drug efflux pumps are membrane protein complexes that function to expulse drugs from the cell. they play a crucial role in the rising rates of antibiotic therapy failures. the homolog of macrolide-specific pump macab was identified in opportunistic pathogen serratia marcescens and was used in this study to characterize its role in protection against antimicrobials and other processes beyond the active efflux of antibiotics. here we used method of serial dilutions to determine minimum inhibitory concentration (mic) for s. marcescens sm6 wild type (wt) and its isogenic δmacab mutant strains. we also used h 2 o 2 survival assay to evaluate the ability of wt and the mutant strain to withstand an oxidative stress. finally, we used b-galactosidase assay to evaluate macab promotor activation in the reporter strain and followed macab expression by western blotting analysis using macab-6xhis strain. we show that in contrary to its e. coli homolog, macab pump in s. marcescens is not involved in the protection against macrolides but instead it is required for protection against aminoglycosides. we further show that similar to its salmonella typhimurium homolog, s. marcescens macab is essential for protection of bacteria against h 2 o 2 . transcriptional analyses demonstrate that while low level of macab promotor activity can be detected after 2 hours of growth in lb-broth there is at least 5-fold increase in expression in response to the presence of h 2 o 2 . on the protein level macab can be detected starting from 3 hours of growth in lb-broth and it reaches maximum expression on 16 hour of growth. our data suggest that macab pump in s. marcescens is involved in protection of bacteria against aminoglycoside antibiotics and is crucial for protection against reactive oxygen species. we are currently working on identification of macab substrate with anti-h 2 o 2 properties. antiproliferative and apoptotic effects of noscapine on mcf-7 and mda-mb-231 human breast cancer cell lines approximately 10-17% of breast cancers are negative for estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. these are most aggressive tumor and a clinical problem because of lack of targeted therapies. noscapine is an alkaloid from opium. noscapine is a microtubule-interfering agent. it causes mitotic arrest, induces apoptosis. in this study, we aimed to investigate the effects of noscapine in mcf-7 and mda-mb-231 human breast cancer cell lines. the cytotoxic effects of docetaxel, tamoxifen, and noscapine on the mcf-7 and mda-mb-231 cell lines were analyzed by roche xcelligence system. the cells were cultured in 10% fetal bovine serum containing dulbecco's modified eagle medium at 37°c in a humidified atmosphere containing 5% co 2 . 24 h after seeding, the cells were treated with 4 different doses of docetaxel (12.5 to 100 nm), tamoxifen (12.5 to 100 lm), and noscapine (12.5 to 100 lm). cultured cells were harvested, fixed with 10% formalin, and centrifuged. pellet was blocked, fixed, and embedded in paraffin. paraffin-embedded cells blocks were sectioned at 4 lm thickness and stained with h&e, ki-67, bcl-2, cyclin-d1, and bax. sides were assessed under a light microscope. quantification of the analyzed proteins were evaluated by the percentage of positive cells. all drugs showed cytotoxic effects on both cell lines. all drugs inhibited the proliferation of breast cancer cells, but effects were dependent on time and dose. all drugs were especially more effective on mcf-7 cells. immunohistochemical examinations revealed that tamoxifen was more effective on mcf-7 cells, hovewer docetaxel and noscapine were more effective on mda-mb-231 cells. tamoxifen has more apoptotic and antiproliferative effects on mcf-7 cells. docetaxel and noscapine showed more apoptotic and antiproliferative effects on mda-mb-231 cells. noscapine may be an effective anticancer agent due to antiproliferative and apoptotic effects on breast cancer cells. negative selection of dna aptamers to reduce non-specific binding in solid-phase-based selection procedures carryover by binders specific to the components of the selection system can be a serious issue in hampering the aptamer selection campaign. solution-or "mass"-based techniques still cannot substitute classic phase-separation strategies. one approach to prevent selection of "passenger" phase-specific (plastic, beads) or blocking agent specific aptamer species is their depletion from the initial library pool. our aim was to develop the universal technique for removal of such aptamers exemplified by bsa-and casein-specific binders, while preserving the initial library complexity. the dna aptamer library was subjected to three rounds of depletion using magnetic beads with covalently attached casein and bsa. to ensure high depletion efficiency, beads were pelleted in a 15-ml centrifuge tube by a neodymium magnet through a 10-cm cushion of 20% sucrose, thus preventing weakly bound aptamers from re-populating the library. high complexity of the input library helped to avoid pcr amplification after depletion rounds preventing the library bias introduced by dna amplification. the depletion effciency was confirmed by real-time pcr. resulting oligonucleotide sub-library was analyzed for binding to the targets using solid-phase real-time pcr assay. we have shown that three rounds of panning under the conditions employed provided full depletion of the initial dna pool from nucleic acid structures capable of binding to protein competitors and hampering the process of aptamer selection. we compared selection efficiency of aptamers specific to type a botulinum neurotoxin light chain in depleted vs undepleted library. the yield of the target-specific aptamers was 10-fold higher in the library subjected to the depletion procedure. removal of undesired binders from aptamer libraries appears an important step of solid-phase selex procedure. it can become a useful approach in optimizing solid-phase selex. the work was supported by russian science foundation research grant no. 14-15-00630. epithelial mesenchymal transition (emt) is a critical trans-differentiation program driving cancer metastasis. patients showing signs of emt or presence of distant metastasis have poor prognosis. another well-known feature of decreased cancer-associated survival is the lack of anti-cancer immune responses. thus we hypothesized that the emt and anti-tumor response should be linked via altered secretion of soluble factors by metastatic cells. all cell lines were grown in dmem. emt status of crc cell lines were assessed by investigating canonical markers of emt. cytokine/chemokine expression of crc cells was performed using r&d systems antibody arrays and validated using ccl5 sandwich elisa and rt-pcr. the mechanism of action of zeb1/2 on ccl5 promoter has been studied by luciferace assay and chip. ccl5 coding region was cloned into pcdna3.1 and stably transfected into dld-1 cells. ccl5 deficient ct26 cells were generated using lentivirual shrna transduction. cells overexpressing or knock/down ccl5 were injected orthotopically into mice. t lymphocyte (til) infiltration in respect to ccl5 and sip1 expression was studied using ihc or flow cytometry. emt status catagorised 13 crc cell lines into epithelial, intermediate epithelial, intermediate mesechymal and mesenchymal. cytokine/chemokine antibody arrays showed a significant increase in ccl5 in induced dld-sip1 cells. elisa, multiplex assays and rt-pcr confirmed a significant increase of secreted ccl5 in the induced dld-sip1 cells as well as mesenchymal crc cells as compared to epithelial ones (p = 0.027). promoter studies showed that zeb1/2 bind to ccl5 promoter and and activate ccl5 gene expression. no metastasis was observed for dld-1 cells overexpressing ccl5 but significant alterations of tumour associated lymphocytes were identified in syngeneic orthotopic crc models. our data shows that ccl5 is up-regulated by emt inducing transcription factor sip1, and mesenchymal (metastatic) crc cells secrete significantly more ccl5 compared to epithelial (non-metastatic) ones. ccl5 did not induce emt per se but abundant secretion of ccl5 by metastatic crc cells was a crucial regulator of immune infiltrate in crc. inhibiting ccl5 in metastatic crc may have a therapeutic potential. barley (hordeum vulgare l.) belongs to the grass family, poaceae (gramineae). it is the fourth most important cereal crop after wheat, maize and rice and is among the top ten crop plants in the world. talbina was used to be recommended for the sick and for one who is grieving over a dead person. talbina is made by adding one or two tablespoon of barley flour (must be 100 percent wholegrain barley flour) to one-and-a-half cups of water and placed on low heat for 10-15 minutes (optional: add milk or yoghurt and sweeten with honey). the main objectives of this investigation were determine the a-tocopherol contents and antimutagenicity activity of talbina (hordeum vulgare l.). our results showed that the total tocopherol content was in the range of 0.25 to 1.03 lmol/g fw. talbina extract was shown to have greater antimutagenic activity observed in the 2500 lg/plate concentration s. typhimurium ta98. at all the doses antimutagenic response was significant at (p < 0.01) against both the strains with a percent mutagenicity decrease from 40 to 25 for ta98 followed by ta100 with percent antimutagenicity from 30 to 11. the results of the study concluded that talbina is a better antimutagenic agent than vitamin e and combination of vitamins did not produce any synergistic activity. the compounds containing thiadiazoles have diverse applications as antifungals, anticancer agents, antibacterial, antiinflammatory drugs, antidepressants and carbonic anhydrase inhibitors according to literature. in this study some novel thiadiazole compounds [(1,4,10,13)-tetrathia[4.4] (2,5)-1,3,4-thiadiazolophane; (4,16)dioxo-1,7,13,19)-tetrathia[7.7](2,5)-1,3,4-thiadiazolophane; (4,7,19,22)-tetraoxo(1, 10, 16, 25 )-tetrathia[10.10](2,5)-1,3,4-thiadiazolophane and (4,7,10,22,25,28)-hexaoxo(1, 13, 19 ,31)-tetrathia [13.13] (2,5)-1,3,4-thiadiazolophane] were used to evaluate the cytotoxicity on healthy human lymphocytes and the antibacterial activities. cytotoxicity tests were perfomed using mts assay and the trypan blue test. cells were incubated with the compounds for 72 hours. at the end of the each 24 hour, cell vitality was assessed by measuring the absorbance (490 nm) of each well using a microplate reader for mts assay. in addition, viability percents of the cells were determined after trypan blue test. as a result, the compounds showed cytotoxicity in a dose dependent manner. for the concentrations of 1:1000 of 0.5 mg/ml, the cytotoxic effect was eliminated. also, antioxidant capacity was determined using 2,2-diphenyl-1-picrylhydrazyl (dpph) reagent. moreover, the antibacterial activities of the compounds were analyzed using a microdilution test against e. coli and s.aureus. compounds having various concentrations showed different antibacterial effects against these two bacteria. arabidopsis thaliana ecotypes vary in their ability to utilize organic p substrates insufficient quantity of inorganic phosphorus in soil is an evergrowing problem that affects many fields of agriculture. unlike inorganic phosphates, organic phosphorus compounds are very common in many soil types, but plants are often unable to efficiently utilize them. to better characterize the extent of natural variation in the ability of the model plant arabidopsis thaliana to grow on organic phosphorus compounds, we grew 19 arabidopsis ecotypes on several organic and inorganic sources of phosphorus. plants were grown in liquid or solid media containing naphosphate, phytate and atp as the sole supply of phosphorus or in absence of any phosphorus source. after several weeks of growth, plants were assayed for changes in their morphological and physiological characteristics. phytate was shown to be the least preferred source of phosphorus compared to inorganic phosphate and atp. the rate of biomass accumulation in all ecotypes decreased in the following order from inorganic phosphate to atp to phytate. lateral root formation was markedly reduced in the absence of any phosphorus source or in the presence of phytate. we also showed that phosphomonoesterase activity in intact roots increased when plants were grown on atp and phytate. overall phosphorus content in leaves and roots was similar when plants were grown on atp or inorganic phosphate, but it was markedly reduced on phytate. substantial differences between ecotypes were also observed in root length, p content in ash and phosphomonoesterase activity in intact roots. our analysis of the ability of arabidopsis ecotypes to grow on several different phosphorus sources provides a unique opportunity to investigate the degree of natural variation in this plant's ability to adapt to different nutritional environments. analysis of many important morphological and physiological changes observed in these plants can further extend our understanding of the full range of plant responses to phosphorus availability. laboratory tests are important in terms of confirmation of diagnosis given by clinics and implementation of appropriate treatment protocols for patients. laboratory tests used by the clinics have been increased in parallel with time.there are many reasons for increased use of the test such as increase of elderly population, increase in standard of care, lack of information and shortening of turn around time. unnecessary laboratory testing also constitute one of the reasons for increased use of laboratory tests. in our study we aimed to investigate the unnecessary laboratory testing for fpsa test. fpsa tests which are ordered with total psa tests that values of less than 4 ng/ml or greater than 10 ng/ml were accepted as inappropriate initial testing. 9759 fpsa tests were evaluated as unnecessary laboratory testing. the clinic which ordered the maximum unnecessary laboratory testing with 3315 was urology within all the clinics. although to the restrictions about the ordering of total psa and fpsa tests there were no decrease in the number of unnecessary laboratory testing. unnecessary usage of laboratory testing may cause increase of false positive results, increase in the use of invasive testing, unnecessary drug consumption and increase of healtcare costs. some precautions may be effective in reducing unnecessary tests such as to inform clinicians about the cost of laboratory tests, to increase the clinician education programs and to develop usage of disease specific diagnostic algorithms about test ordering. local clinical validation of blood collection tubes although the tubes with gel and clot activator are widely used due to the advantages, there are ongoing discussions about the effects of the blood collection tube on clinical outcomes in the analysis of biochemical parameters. therefore, we aimed to prove the local clinical validation of the new produced blood collection tubes with low-volume. the blood samples of 40 patients who referred to the hospital phlebotomy unit were collected using holder into the 3 different tubes. first tube was 5 ml glass tube and with no additive, second was 5 ml tube with gel separator, third was 1 ml tube with gel separator. serum was separated and immediadiately analysed for 25 biochemical parameters. the difference between the analyte amounts in the different tubes was evaluated using paired t-test. the clinical significance was evaluated using significant change limit. bias (%) between the other tubes with the reference tube was also evaluated according to the ''allowable total error". when we compared the other test tubes to a glass tube which was assumed reference tube, total protein, albumin, amylase, calcium, triglyceride, cholesterol, hdl-cholesterol, total and direct bilirubin, iron, gamma glutamyl transferase, magnesium, phosphorus results were statistically significant. but the results of all the analytes were within the significant changes limit and the allowable total error was not significant. while a biochemical parameters have analysed, it may be absorbed into the gel and this may caused from factors such as the chemical structure of the gel, analyte itself, the residence time in the gel, storage temperature and volume of the sample e.g. as well as the leaking of gel material to the sample was reported to be another factor for affecting the analysis. despite these factors, we observed that neither gel-clot activator tube with low nor high volume affect the clinical results. the research of the frequency of interference in thyroid function tests interference is defined as the effect of substance in the sample which changes the correct value of laboratory results. the frequency of interference in immune techniques is varied. the frequency of interference depends on population of the study, technique for detecting the reaction and researcher's method. unexpected or inconsistent results with clinical findings should suggest the possibility of interference. in this study it is aimed to investigate the frequency of interference in thyroid function tests (tsh, ft3, ft4) which are the most common requested laboratory tests. thyroid function tests of 47915 patients are analyzed in ankara numune education and research hospital in october 2014-may 2015. five samples which had the incompatible results with clinical findings are re-evaluated just because of the suspicion of interference. the detection of interference included; repetition of test via different immune techniques, serial dilution, polyethylene glycol (peg) precipitation and incubation with heterophilic blocking tubes (hbt). the results of two different immune techniques and before/ after incubation with hbt showed no significant difference. linear curves had observed in serial dilution. after peg precipitation; below 40% of recovery had obtained in one sample, therefore it is interpreted as macro-tsh. the frequency of interference in thyroid function tests for 8-month study period was 0.01%. no information is found about the best test for defining the cross reaction. it is also aforethought that interference should not be excluded by using any single procedure. p-mis-105 development of polyclonal and monoclonal antibodies against fatty acid binding protein 4 (fabp4/ap2) a. abbasi taghidizaj, g. aydogdu, b. p. sermikli, e. yilmaz ankara university, ankara, turkey recombinant proteins and antibodies can be use for therapeutic or diagnostic purposes which produced in many different host organisms. the technique for the production of immortal cell making single antibody, fusing target antibody-forming b lymphocyte precursor with a suitable myeloma cells. the fused hybrid cells (called hybridomas), as a cancer cell will reproduce rapidly and will produce large amounts of the desired antibodies. fatty acid binding protein (fabp4) is a well characterized intracellular lipid transport protein and plays a key role in the intracellular fatty acid transport and adipose tissue metabolism. fabp4 as a adipokine that regulates glucose homeostasis and has various features for metabolic syndrome associated with obesity. in this study, production of monoclonal antibodies against immunogenic fabp4 protein made by recombinant dna technology. recombinant his-fabp4 was expressed in e.coli and purified. balb/c mice used for immunization and serum anti-fabp4 antibodies determined by enzyme-linked immunosorbent assay (elisa). hybridoma cells created by fusion of splenocytes and myeloma partner cells. after selection of antibody producing cell clones, injecting hybridomas into the peritoneal cavity in balb/c mice ascites fluids was obtained. we have selected fifteen hybridoma clones that produced antibodies specific for fabp4, as shown by western blotting and immunocytochemistry. as a result we produced mabs that will be useful for the scientific community working on fatty acid binding proteins and lipid metabolism. in near future, therapeutic approach for this antibody maybe a possibility in metabolic syndrome. thioridazine, an anti-psychotic drug, inhibits migration, invasion and epithelial mesenchymal transition in breast cancer cell lines thioridazine (thz), an antipsychotic drug, exhibits anti-angiogenic effects on breast cancer cell lines. however the mechanistic insight in exerting antiangiogenic effect is not clearly understood. the objective was to investigate the role of thz in epithelialmesenchymal transition (emt) by using cell migration assay, scratch assay, western blot (wb) and immunocytochemistry. thz treatment reduced cell viability on mda-mb-231, mcf-7 and cd44 + /cd24-cells and ic50 values of thz were found to be 9 lm, 16.4 lm and 18 lm respectively, at 24 hours. invasion potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells were determined as 29%, 24%, 16.5% when compared to relevant treatment controls. migration potency of mcf-7, cd44 + /cd24-and mda-mb-231 cells was determined as 28.5%, 63.2%, 61% respectively. among the three cell lines mda-mb-231 cells display enhanced invasive and migration ability when compared to other cell lines. western blotting results demonstrate that thz significantly increases e-cadherin, cytokeratin-18, b-catenin, while inhibiting n-cadherin, vimentin, fibronectin. immunocytochemistry studies revealed decrease in e cadherin and a concomitant increase in vimentin level for all three cell lines upon treatment with thz. moreover thz significantly inhibited the cell migration, invasion and emt in mda-mb-231, mcf-7 and cd44 + /cd24 cell lines by suppressing mesenchymal markers. in conclusion, these data suggest that thz might be a novel anti-proliferative and anti-metastatic agent for treatment of breast cancer. effect of seasonal temperature and humidity on urine density in children environmental heat and humidity are important factors affecting hydration status in childhood. hereby, we aimed to investigate the effects of seasonal climate changes on urine density of children living in mediterranean climate, cyprus. 1700 healthy 0-18 year children's (850 girls, 850 boys) age, sex and urine density results were collected retrospectively for three consecutive years. the correlation of urine density with each seasonal and 12 months' average temperature and humidity has been analysed. the urine density results had a positive correlation with temperature (r = 0.083, p = 0.001) and a negative correlation with humidity (r= à0.072, p = 0.003). mean urine density in spring was higher than that of autumn (p = 0.02) and winter (p = 0.00). mean value of summer was higher than autumn (p = 0.03) and winter (p = 0.00). 0-24 months age group had lower urine density. evaluation of urine density based on gender and puberty revealed no statistically significant difference. seasonal mediterranean climate changes have an impact on urine density in children which may affect hydration status especially in infants < 2 yrs of age. during high temperature seasons ensuring adequate water intake is essential in this age group in mediterranean climate. p-mis-108 implementation related to the use of antibiotics and data sources by community pharmacists in north cyprus as the resistance to antibiotics is gaining importance in today's world;the solution to this problem is possible through a common consciousness of the doctor who prescribes antibiotics,the pharmacist who sells and the patient who consumes antibiotics. irrational use of drugs is an economic and medical problem in many developed and developing countries around the world.the aim of this study is to determine the sales ratio of non-prescription antibiotics in pharmacies which is the biggest category of the antibiotic group sold as well as the indications that lead to its' prescription. eighty-four pharmacies out of 168 pharmacies located in north cyprus were involved in the study with 50%stratified systematic sampling, questionnaires were filled and a consent form was signed by the participating pharmacists. the pharmacists involved in the study stated that non-prescribed antibiotics were demanded from the pharmacists and all except two (97.6%),responded positively to this demand. it has also been identified in the study that 41.5% of the daily sale of antibiotics in the first half of the year 2014 was non-prescribed. the most purchased antibiotics either with or without prescription was found to be the penicillin and its derivatives with 76.2% and upper respiratory tract with 86.9%. when the level of selfawareness of the pharmacists was examined, the rate is found in north cyprus to be (41.5%),compared with the studies conducted in greece,italy,malta and spain 47% and egypt 50.4%that designated the non prescribed antibiotics purchased from the public pharmacies. the rate of sale of non-prescribed antibiotics in north cyprus has been found to be at a higher level compared to the rates in many developed and developing countries. furthermore, the upper respiratory tract infections are amongst the most common viral causes which lead to a high consumption of both prescribed and non-prescribed antibiotics. this study was supported by turkish viral hepatitis prevention society. acrylamide has cytotoxic, antiproliferative and apoptotic effects on human lung adeno carcinoma cell line a549 acrylamide (aa), a widespread substance in many fields, forms in foods during high temperature processing such as baking, roasting, frying. aa is a potent neurotoxic, genotoxic and clastogenic agent being a strong electrophile and forming adduct with biological molecules or potent nucleophiles. up to now, several studies confirmed the toxicity of acrylamide to several organs. on the other hand, aa is reported to have inhibition effects both on proliferation and differentiation of different cancer cells in a time and dose-dependent manner. in addition, natural and synthetic acrylamide derivatives are also used as potent anti-cancer agents. moreover, inhibition concentration (ic50) values of aa against these cancer cells have not been investigated in detail yet. thus, the goal of this study is to investigate the cytotoxicity of aa on a549 cells including with ultrastructural and morphological effects. ic50 value of aa on a549 cells for 24 h was detected with mtt (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h-tetrazolium bromide) colorimetric assay. we evaluated morphological changes under confocal microscopy and ultrastructural changes under transmission electron microscopy (tem). our results demonstrate that aa inhibits the proliferation of a549 cells in dose-dependent manner and ic50 on a549 cells was found to be 4.6 mm for 24 hours. confocal microscopy evaluations showed that aa caused nuclear condensations, fragmentations, cytoskeleton lacerations and membrane blebbing. tem results revealed membrane blebbing, chromatin condensations and cell shrinkage. although aa is a probable carcinogen substance, it drastically inhibited cell viability in dose-dependent manner. from microscopic assessments, aa is suggested to induce apoptosis in a549 cells. in conclusion, the present study confirms the high potential of aa for cytotoxic, antiproliferative and apoptotic activity on a549 cells. however, appropriate aa dose is critical to prevent its possible adverse effects. effect of hemolysis and lipemia on some immunochemical tests in beckman coulter unicell dxi 800 immunoassay analyzer c. yilmaz, s. yildiz, m. senes, v. fidanci, d. y€ ucel ankara training and research hospital, ankara, turkey the aim of the study was to investigate the effects of in vitro hemolysis and lipemia on 25 immunoassays studied by the beckman coulter unicell dxi 800 immunoassay analyzer. we prepared a serum pool without hemolysis, lipemia and icterus. baseline serum pool concentrations of 25 tests were measured by the beckman coulter unicell dxi 800. d _ ifferent serum pools, six for hemolysis and five for lipemia, were spiked with increasing concentrations of hemoglobin (0.6, 1.2, 2.4, 4.5, 6.6 and 8.6 g/l hemoglobin) and intralipid (0.625, 1.25, 2.5, 5 and 10 g/l intralipid). the hemolysate was prepared by osmotic shock method. intralipid (20%, baxter, deerfield, il) was used to mimic the effect of lipemia. the hemolysis (h), lipemia (l) and icterus (i) indices were measured on beckman coulter au 5800. after spiking the pools, the 25 tests were measured again in duplicate on beckman-coulter dxi 800 analyzer. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. we observed a positive interference due to hemolysis for folat, vitamin b12, testosterone and by lipemia for cortisol. there was a negative interference of hemolysis for ca 19.9, ca 125, ca 15.3, insulin, pth and e2, and of lipemia for progesterone, ca 19.9, vitamin b12 and pth. we found clinically significant effect (>total analytical error) of hemolysis on folate and insulin, and lipemia on cortisol. investigation of the effect of two different p38mapk inhibitors in rats subjected to isoproterenol-induced acute myocardial injury: an experimental study objective: acute myocardial infarction is a serious acute condition. in the current study, we aimed to investigate the possible effect of two different mitogen-activated protein kinase (p38mapk) inhibitors in rats subjected to isoproterenol (iso)induced myocardial injury. materials and methods: a total of 32 male wistar-albino rats were equally and randomly seperated into four groups as follows: control, iso, iso plus sb203580 andiso plus tak-715. treatment agents were orally administered and myocardial injury was induced by subcutaneous injection of iso. serum cardiac troponin-i (ctni), ischemia modified albumin (ima), heart fatty acid binding protein (hfabp) levels and paraoxonase-1 (pon-1) activity, tissue tos (total oxidant status), tas (total antioxidant status), tt (total thiol), tumor necrosis factor-a (tnf-a) levels, superoxide dismutase (sod) and glutathione peroxidase (gsh-px) activity levels were measured. tissue mrna levels of nf-jb, p38 mapk and nuclear factor erythroid 2-related factor 2 (nrf2) were analyzed. heart tissues were also immunohistochemically and histopathologically evaluated. results: both compounds have led to a decrement in myocardial damage, apoptosis, ctni, ima, hfabp, tos, and tnf-a levels, nf-jb, p38 mapk, phosphorylated c-jun n-terminal protein kinase (pjnk 1/2) expressions. on the other hand, the applied treatment increased sod, gsh-px, tas and tt levels, as well as phosphorylated extracellular signal-regulated kinase (perk 1/2) and nrf2 expressions. conclusion: data established from the current study suggest that administered agents have protective effect against cardiac injury induced by iso, which was more prominent in rats received sb203580 treatment. p38mapk inhibitors may constitute a useful choice as cardioprotective agents due to their antiinflammatory, antioxidant and anti-apoptotic effects. keywords: _ isoproterenol, myocardial infarction, myocardial ischemia, p38 mitogen-activated protein kinases, sb203580, tak-715. silicosis composes the vast majority of occupational lung diseases. silicosis, caused by inhalation of crystalline silica, is a chronic lung disease characterized by parenchymal nodules and pulmonary fibrosis. the susceptibility of patients with silicosis to infection is thought to be due to toxic effects of silica on pulmonary macrophages. ada activity is considered as a nonspecific marker of t cell activation and cellular immunity. this study aimed to compare the serum ada activity in silicosis patients with spirometric values. in this study there were 35 males in each groups which contained patients with silicosis (group 1), individuals having similar symptoms with silicosis from same occupational area (group 2) and healthy subjects (group 3). routine hematological and biochemical parameters were also measured. the serum ada activity and spirometric values (fev1, fev1%, fev1/fvc, fev1/ fvc%, fef25-75 and fef25-75%) were compared. the average age of group1, 2 and 3 are 38.5 ae 10.6, 39.5 ae 2 and 51 ae 10.5 years, respectively. there was a significant difference between group 1 and 3 in terms of the ada level (p < 0.05). there was a negative correlation between ada activity and fev1, fev1%, fev1/fvc, fev1/fvc%, fef25-75 values. elevated serum ada activity has been shown in many diseases with induced cellular immunity. despite initially toxic effects were lead to a little immunological reaction in patients with silicosis, continuation of this immunological response is important in some chronic manifestations of silicosis. the release of chemotactic factors and inflammatory mediators cause the migration of polymorphonuclear leukocytes, t lymphocytes and macrophages. in this study, the ada activity was significantly higher in patients with silicosis than others. increased immunity in patients with silicosis is being considered, increasing ada activity might be help of earlier recognition of these patients and to take better quality of life. atlantic salmon (salmo salar l.) is an important model system in evolutionary and conservation biology that provides fundamental knowledge into population persistence, adaptive response and the effects of anthropogenic change. the role of behavioral and body size variation in environmental adaptation of atlantic salmon is well known, by contrast, the underlying biochemical mechanisms are largely unknown. intracellular proteases, such as cathepsins b and d in lysosomes and calpains and proteasome in cytosol, due to their metabolic and regulatory role may contribute to phenotyping speciation of salmon young. we examined the activity of intracellular proteolytic enzymes in skeletal muscles of atlantic salmon parr from two local habitats of the varzuga river (the main channel and small tributaries) differing in hydrological and feeding parameters. calpain and proteasome activities were determined by casein or suc-llvy-amc hydrolysis in the skeletal muscles of s. salar from varzuga river (kola peninsula, russia). it is known that salmon parr originated from a common hatch became phenotypically divergent during the settle in the biotopes. reliable difference in studied enzyme activities in the salmon parr from two local habitats was found; furthermore, calpain and cathepsin b proteolytic activities were found to negatively correlate with parr body size. muscle proteolytic activity data support an idea on protease contribution to environmentally-driven adaptation and speciation process in fish. the work was supported by the russian scientific foundation, project no. 14-24-00102. the phylogenetic analyses of anthriscus (apiacea) species from turkey based on non-coding "trn" regions of chloroplast genome p. yilmaz sancar 1 , m. tekin 2 , s. civelek 1 1 firat university, elazig, turkey, 2 cumhuriyet university, sivas, turkey anthriscus pers. (apiaceae) species belongs to apiaceae family and is represented by 16 genus on the world and by 8 genus in turkey. anhriscus species are used extensively for treatment various disease such as asthma, alzheimer and show anti-tumoral, anti-microbial, antioxidant features. for determining exact species which treat disease it is necessary sorting species correctly with molecular markers to support morphological features. anthriscus species were defined by examining insufficient quantity of samples in turkey flora. besides, no detailed study was found in our country after flora study. for this reason a revision study was made with the aim of solving some systematical problems in 2013 by tekin. the result of the study provided important contribution to the systematic of the species in turkey. however a molecular study was also required for building the obtained results on a more solid ground. in this study, the aim to reveal systematic and phylogenetic relationship among species of anthriscus in turkey, by using trnl-f region in chloroplast genome. dna was isolated by ctab method and amplified in pcr by using e-f primaries. the obtained data was evaluated by mega 7.0 program and phylogenetic tree was prepared by using maximum likelihood method. according to the phylogenetic tree that we prepared by using the sequence line up of trnl-f section, it was observed that a. cerefolium, a. caucalis and a. tenerrima species completed their speciation and an isolation with other species in terms of speciation was provided. it was also observed that a. kotchi, a. sylvestris subs. sylvestris, a. sylvestris subs. nemarosa and a. lamprocarpa'nın provided hybridization among themselves but they did not complete their speciation. it was determined that a.lamprocarpa var. chelikhii which is one of the two different varieties of a. lamprocarpa is actually a new sub-species. this fact was supported by molecular data obtained from the study we made after morphologic data. introduction: excessive production of androstenedione can becaused by defects of adrenal steroid biosynthesis, tumors of ovarian and adrenal origin, polycystic ovarian syndrome, increased peripheral sensitivity to androgens, and increased peripheral production of androgens. most epidemiologic studies use enzyme-linked immunosorbentassay (elisa) to measure sex steroid hormones because they have acceptable turnaround times and arerelatively inexpensive. mass spectrometry-based methods are currently the most specific quantitative analytical methods for steroid determination. mass spectrometry methods are independent of matrix effects or cross-reactivity. in this study, a new liquid chromatography-tandem mass spectrometry (lc-ms/ms) method was developed. materials and methods: for serum androstenedione measurement, 50 ll of internal standard (d5-11 deoxycortizol) in methanol was added to 250 ll standart or serum and centrifuged at 4.500 rpm for 10 minutes to remove the precipitated proteins. supernatant was transferred to clean tubes and this procedure was performed twice. the supernatant was collected and dried under a nitrogen gas flow at 60 • c and dissolved in mobile phase. 60 ll was injected in to the ultra performance liquid chromatography analytical column for chromatography. elisa study was conducted with drg (lot. no. 50k074) brand kit. results: method comporison between lc-ms/ms and elisa was found slope value 18,412, intercept value à22.87 and r² value 0.1033. the regressione quation was elisa= à2.861782 + 4.905103 lc-ms/ms. discussion and conclusion: method comparison study presented higher results in elisa compared to lc-ms/ms. in our opinion, this might due to the interference in elisa systems. our lc-ms/ms method allows rapid, sensitive and specific determination of androgens in plasma and serum.the specificity of liquid chromatography-tandem mass spectrometry (lc-ms/ ms) offers advantages over immunoassays. heparins play an important role in cell growth, differentiation, migration and invasion. however, the molecular mechanisms of heparin mediated cellular behaviors are not well defined. to determine the effect of heparin on gene expression, we performed a cdna microarray in a hepatocellular carcinoma cell line and found that heparin regulates transcription of genes involved in glucose metabolism. in this study, we showed a new role of heparin in the regulation of thioredoxin interacting protein, which is a major regulator of glucose metabolism, in hepatocellular carcinoma cell lines. we determined the importance of a unique carbohydrate response element located on its promoter for the heparin-induced activation of thioredoxin-interacting protein and the modulatory role of heparin on nuclear accumulation of carbohydrate response element associated proteins. we showed the importance of heparin mediated histone modifications and downregulation of enhancer of zeste 2 polycomb repressive complex 2 expression for heparin mediated overexpression of thioredoxininteracting protein. when we tested biological significance of these data; we observed that cells overexpressing thioredoxininteracting protein are less adhesive and proliferative, however they have a higher migration and invasion ability. interestingly, heparin treatment increased thioredoxin-interacting protein expression in liver of diabetic rats. in conclusion, our results show that heparin activates thioredoxin-interacting protein expression in liver and hepatocellular carcinoma cells and provide the first evidences of regulatory roles of heparin on carbohydrate response element associated factors. this study will contribute future understanding of the effect of heparin on glucose metabolism and glucose independent overexpression of thioredoxin-interacting protein during hepatocarcinogenesis. prolidase activity in chronic obstructive pulmonary disease and asthma t. g€ uc ßl€ u 1 , h. s€ urer 1 , g. bilgin 2 , d. y€ ucel 1 1 ankara training and research hospital, medical biochemistry department, ankara, turkey, 2 ankara training and research hospital, chest diseases department, ankara, turkey chronic obstructive pulmonary disease (copd) is a consequence of an underlying chronic inflammatory disorder of the airways that is usually progressive and causes dysregulation in the metabolism of collagen. and asthma is a disease where there is an accumulation of collagen in the reticular basal membrane of the airway leading to chronic inflammation. prolidase has an important role in the recycling of proline for collagen synthesis and cell growth. we measured and compared prolidase activity in healthy individuals with copd and asthma patients to find out that whether its activity might reflect disturbances of collagen metabolism in the patients. 60 patients with copd, 60 patients with asthma and 20 healthy control subjects with similar age range and sex were included in our study. the patient and control groups do not have any other chronic disease. serum prolidase activity was measured in the patient and control groups. ferritin and alpha-1 antitrypsin concentrations were also compared. there was no significant difference between serum prolidaz activities of asthma and copd patients. serum prolidase activities of both copd and asthma patients were significantly lower than those of the control subjects (p < 0.05). there was no significant difference for ferritine and alpha-1 antityripsin levels between the groups. the prolidase activity is significantly lower in asthma and copd patients comparing with control subjects. the collagen metabolism may be undergone to a change in these patients. hence, there may be an effect on the accumulation of collagen in the reticular basal membrane. the results suggest that collagen turnover are altered by the development of copd and asthma in human lungs, and prolidase activity may reflect disturbances of collagen metabolism in these pulmonary diseases. monitoring serum prolidase activity may be useful in evaluating fibrotic processes and in the chronic inflammatory lung diseases in human. acyclovir molecule in the active site of e. coli purine nucleoside phosphorylase (on the basis of x-ray study) i. kuranova 1,2 , v. timofeev 1,2 , n. zhukhlistova 1 , y. abramchik 3 , t. muravieva 3 , r. esipov 3 1 shubnikov institute of crystallography of fsrc "crystallography and photonics" ras, moscow, russia, 2 national research centre "kurchatov institute", moscow, russia, 3 shemyakin-ovchinnikov institute of bioorganic chemistry, russian academy of sciences, moscow, russia e. coli purine nucleoside phosphorylase (pnp), which catalyzes the reversible phosphorolysis of purine ribonucleosides, belongs to the family i of hexameric pnps. due to key role in the purine sulvage pathway pnps are attractive targets for drug design against some pathogens. they also used widely in biotechnology for the synthesis of nucleoside analogues as well as for the activation of the prodrugs in anti-cancer gene therapies. the acyclovir (acv), acyclic derivative of guanosine, is antiviral drug for the treatment of some human viral infections. the crystalline complex of e. coli pnp with acyclovir was prepared by co-crystallization using counter diffusion in capillary through the gel layer. the set of x-ray data at 100 k from single crystal grown in space (sp. group p6 1 22) was collected on the spring-8 synchrotron-radiation facility (japan) and the structure was solved at 2.32 a resolution, using the molecular replacement method (pdb id 5i3c). acv molecule was located in the nucleoside binding pocket of the enzyme in two conformations. the phosphate binding site was occupied by so4 ion. the hydrogen bonds network and hydrophobic interactions stabilising acv molecule in the active site as well as the conformational changes upon ligand binding were described. the comparison of e. coli pnp/acyclovir complex and the similar complexes of bacillus subtilis pnp (pdb id 4da7) and human pnp (pdb id 1pwy) allowed to establish the peculiarities of acv binding of in the e. coli enzyme. gonadotropins are glycoprotein hormones that regulate normal growth, sexual development, and reproductive function. these are large, up to 40 kda proteins, which are synthesized and secreted by the gonadotropic cells of the anterior pituitary gland. these hormones may vary in the level of glycosylation depending on the tissue and the metabolism cycles. follicle-stimulating hormone (fsh) and upon binding to fsh receptor, a g-protein coupled receptor (gpcr), regulates the development, growth, pubertal maturation, and reproductive processes of the body. human chorionic gonadotropin (hcg) and luteinizing hormone (lh) act via a shared gpcr (lh receptor) and regulate mechanisms essential for ovulation, early pregnancy and placental function in females as well as spermatogenesis and testosterone production in males. activation of gpcrs by these hormones can be measured by monitoring formation of cellular cyclic adenosine monophosphate (camp). the level on camp was measured using a f€ orster resonance energy transfer (fret)-based biosensor tepacvv (h74) kindly provided by dr, kees jalink. the biosensor was expressed using the developed bacmam gene delivery system (recombinant baculoviruses carrying the transgene under a strong mammalian promoter). kgn cells expressing the fsh receptor and cos7 cells expressing the lh receptor served as study objects. monitoring of specific gpcr activation in living cells, allows detection of only the biologically active agonists, which has real impact in quantification of large hormones. differences in levels of hormone glycosylation may affect their biological function. investigation of this phenomena is planned for near future. detection of biological activity of gonadotropins is of importance for pharmaceutical industry, where today the concentration of recombinant proteins is mostly estimated using immunological assays only. development of a colorimetric aptasensor for the detection of peanut allergen protein ara h 1 in food samples b. bora ege university, izmir, turkey food allergy, especially peanut allergy is a life-threatening problem, and severe reactions against these foods can be observed. since unintnded consumption of non-labeled foods is the most dangerous risk, any residual allergen protein should be tested and labeled by the manufacturers. an aptamer based colorimetric test is a powerful alternative to commercially available rt-pcr and elisa test kits. the main objective of this study is to develop an aptamer based colorimetric test fort he detection of major peanut allergen protein ara h 1. ara h 1 aptamer was used to recognize any residual peanut major allergen protein ara h 1 in food samples. recombinant ara h 1 protein was produced and puirifed to be used as a target. ara h 1 aptamer was used in combination with a blocking sequence, to prevent non-specific binding event, a biotinylated complementary strand to the blocking sequence, and finally strp-hrp interaction in order to facilitate colorimetric reaction. optimal blocking sequence length was optimized and introduced to the 3 0 site of aptamer sequence to construct an aptamer-hairpin structure. liberation of the blocking sequence allows biotinylated complementary strand to bind to the blocking sequence and consequently str-hrp conjugate to achieve color development that is proportional to the target concentration. since, the aptasensor will be used for the detection of ara h 1 in food samples, total protein extraction from chocolate samples was also optimized. in order to lower the detection limit of aptasensor, aptamer coupled magnetic bead based pre-enrichment assay was aslo optimized for the total protein extraction. as a result, a sensitive, fast and reliable aptamer based colorimetric assay was developed for the detection of peanut allergen protein from food samples. moreover, the assay has the advantages like ease of application and low cost which makes the assay a promising and a powerful alternative to commercially available rt-pcr and elisa tests. the association between lipid parameters and waist circumference in female university students in turkey s. ozen, a. cort sanko university, department of nutrition and dietetics, gaziantep, turkey a high waist circumference is associated with an increased risk for type 2 diabetes, dyslipidemia, hypertension, and cvd in patients with a bmi in a range between 25 and 34.9 kg/m 2 . monitoring changes in waist circumference may be helpful, in addition to measuring bmi, since it can provide an estimate of increased abdominal fat even in the absence of a change in bmi. objective of the study was to find an association between plasma lipid profile and anthropometric parameters (waist circumference percentage of body fat and body mass index (bmi)) in abdominal obesity in turkish university students. lipid profile and anthropometric parameters of obesity were studied in a sample of 30 women. students with high bmi (>24) had higher values of low-density lipoprotein (ldl), triglycerides (tg) and cholesterol (c) than students with low bmi (<24) but these differences were not significant. high-density lipoprotein (hdl) levels were non-significantly higher in low bmi (<24) student group. waist circumference, percentage of body fat was higher in high bmi (>24) group than low bmi (<24) group. waist circumference, percentage of body fat was positively correlated with bmi in both samples (bmi (>24) and bmi (<24)). students were grouped depend on their waist circumference. healty individuals who had lower than 80 cm waist circumference had decreased tg levels compared to cardiovascular risk group who had higher waist circumference than 80 cm. this study shows an association between waist circumference, percentage of body fat, body mass index and lipid parameters in young female university students. with regard to the relationship, the screening females for central obesity to prevention of cardiovascular disease are recommended. a new biotechnological product from propolis with low allergen: anti-inflammatory effect propolis is extensively used in food industry due to its special medical properies (antioxidant, antimicrobial, antiseptic, antibacterial, anti-inflammatory and antimutagenic effects). even these positive properties it may cause some allergic reactions in consumers with allergic predispositons. previously, we demonstrated that biotransformation of propolis by some special strains of lactobacillus plantarum (10, 8014, aatc strains) might decrease the allergenic molecules in propolis. in this study, we aimed to investigate the effect of biotransformation of popolis on it's antiinflammatory activities. before biotransformation, propolis samples were treated with different solutions (10% ethanol and polyethylene glycol -peg 40%) and different method (ultrasonic treatment 300 w/25 o c/ 30 minutes) in order to facilitate solvation of solid samples which are very dense and not suitable for fermentation. fermantations were performed at 30 o c/48 hours under constant agitation conditions. the anti-inflammatory activity was determined in-vitro conditions using hyaluronidase's analysis and the xanthine oxidase activity. the highest inhibition (%) of radicals produced by xanthine oxidase was determined in solid samples treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (88.43%), followed by liquid samples treated by ultrasonic method prior to transformation (86.21%). concernig the results of hyaluronidase activity (%) inhibitions, the best value were determined in the solid sample treated by peg prior to biotransformation and using of l.plantarum 8014 strain during fermentation (93.93%). results indicated that the anti-inflammatory activities of analysed samples are quite high and depending of used extraction methods prior the biotransformation and used specific strain of l.plantarum could be optimized in terms of other required parameters. faceanti-mullerian hormone is not predictive for poor neonatal outcome aim: anti-mullerian hormone (amh) is a growth factor specific to ovaries. it is commonly used to predict ovarian reserve and outcomes of fertility treatments. recently, low levels of amh have been shown to be related to hypertensive diseases of the pregnancy and the risk of preterm labor. the aim of this study was to investigate the diagnostic performance of amh levels of mothers to predict poor neonatal outcome in term pregnancies and the relationship between amh and birthweights of the newborns. materials and methods: 187 patients, having delivery beyond 37 weeks, and who did not have any other medical problems were included in the study. the patients had normal 50 g. oral glucose tolerance test results. they were divided as 3 groups, based on their newborns' birthweight as "2500 g. and 4000 g.". level of amh was determined by elisa method. results: there was not any relation with the amh of the mothers and the poor neonatal outcome of the newborns, in all 3 groups. also no siginificant difference was observed in amh levels of the patients having delivery in early term and late term periods. when the patients of the same group were evaluated; amh levels were irrelevant to age, gravidy, delivery week, body mass index, the weight gain during pregnancy, and poor neonatal outcome. conclusion: amh is not a predictive factor for poor neonatal outcome and it is not a determinant of the weight of the newborn. objectives: the aim of the study was to investigate the effects of differing amounts of hemolysis on serum high sensitvity troponin i (hs-tni), ck-mb mass and myoglobin measurements. materials and methods: we prepared serum pools having troponin i, ck-mb and myoglobulin concentrations at low (5.33 ng/l,1.5 ng/ml and 34.3 ng/ml respectively), normal (39.25 ng/l, 3 ng/ml, 197.9 ng/l respectively) and high (7345 ng/l, 22 ng/ml, 574 g/ml respectively) values. the osmotic shock method was utilized to prepare a hemolysate. hemolysate was added into serum pools increasing concentrations of hemoglobin (0.65, 1.3, 2.5, 5, 7.26 and 9 .42 g/l hemoglobin). troponin i, ck-mb (mass) and myoglobin concentrations were measured in duplicate by beckman coulter access 2 analyzer. the hemolysis indices were measured on beckman coulter au 5800. a change of 10% from baseline results was taken as evidence of interference and the interfered tests were also evaluated according to total analytical error based on analytical imprecision and intraindividual biological variation. results: we found a positive interference due to hemolysis for ck-mb (mass) at low concentrations (1.5 ng/ml), and a negative interference for myoglobin at low concentrations (34.3 ng/ml) and high concentrations (574 ng/ml). conclusions: ck-mb increase and myoglobin decrease in hemolyzed samples with hemoglobin ≥9.42 g/l, but the bias might not be clinically significant (< total analytical error) in samples. a retrospective study to determine a reliable marker for selective screening of pompe disease lysosomal storage diseases (lsd) are rare inherited metabolic disorders caused as consequence of a deficiency in a specific enzyme required for lysosomal function. pompe disease is one of these disorders with deficiency of a-1,4 glycosidase enzyme with an incidence of 1:4,500-1:33,000. as enzyme replacement therapies are available nowadays, early diagnosis is crucial and selective screening is a rational method to reach pompe patients among people who administer to healthcare with lsd suspected symptoms. this study aims to examine the relationship between basic biochemistry parameters and a-1,4-glycosidase activities retrospectively, in order to find a key parameter for selective screening of pompe disease. for this reason a-1,4glycosidase, creatine kinase (ck), creatine kinase-mb (ck-mb) activities calcium, phosphate levels of those who had been suspected to be lsd patients and administered to our laboratory for analysis are examined retrospectively. out of 134 patients's examined,14 of them were diagnosed with pompe disease depending on clinical findings & low a-1,4glycosidase activity. enzyme activities of pompe patients were 0.337 nmol/ml/hour as lsd suspected patients'activities had a mean of 2.78 nmol/ml/hour (p = 0.00).comparison of ck activity was compared results showed significant difference between pompe patients and lsd suspected patients. even though ck activity levels of the lsd suspected patients were much higher (400vs41-171u/l) than reference interval, the levels of the pompe disease patients' were still more than twice of the lsd suspected group (956vs400u/l, p = 0.02). ck-mb, ca, p levels didn't show a significant difference. a strong (-) correlation (p = 0.005 r=à0.240) was observed between a-1,4-glycosidase and ck activities (n:134). selective screening is a rational way to diagnose rare diseases. this study's results show that ck activity can be used as a key parameter to determine patients for selective screening of pompe disease within lsd suspected population. the functional effect of stem cells on the reproductive organs infertitility is considered as a major health problem of recent century. importance of stem cell is increasing so it is searched new features and supposed to be involved in the infertitility treatment where oxidative stress and apoptosis play importany role. we aimed to investigate the beneficial effect of the stem cells related to free radicals and cell death on testis and ovary. biopcy model of wound healing was created in the rat testis and ovary with ppd syringe where stem cells were delivered by injection. rats were divided into four groups including controls, sham, wound healing and wound healing with stem cell. after the creation of the wound, bone marrow-derived mesenchymal stem cells from the tibia of the mature rats and medium were administered to ovaries and testes. following the applications, ovary and testis samples were investigated for oxidative stress and apoptosis by immunohistochemistry. in comparison with the medium and stem cell applications without a medium support, it was meaningfully determined that healing effect in testicles and ovaries were spotted specifically on the seven day. tissues were analysed for these staining by h-score and h-score results were determined using one-way anova test statistically. our results show the positive effects which clinic applications can bring by displaying the great contribution of the stem cell application in the treatment of testicle and ovary damage. these findings suggest that transplantation of the mesenchymal stem cells may help to promote better enviroment for the reproductive organs by the effect on oxidative stress and apoptosis. the further studies of these results in the molecular level can lead the way to solve the problem of infertility, to increase the percentage of success in the ivf and icsi techniques and more importantly to perform a differentiation from a somatic cell to a germ cell. the antimicrobial activity of 2 (5h)-furanone derivative on staphylococcus aureus nosocomial infections caused by methicillin-resistant staphylococcus aureus strains are known to be a reason of many infectious diseases like osteomyelitis, endocarditis, sepsis etc. being organized in biofilms these bacteria become extremely resistant to antimicrobials and host immune system leading to difficulties in treatments. here we report the effect of 2 (5h)-furanone derivative possessing sulfonyl group and l-menthol moiety (f105) on biofilms formed by s. aureus atcc29213 and mrsa cells. while exhibiting relatively high minimal inhibiting concentration -mic (16 mg/l), clear synergy with a number of antibiotics was found in the checkerboard assay. thus, in the presence of 2 mg/l of f105 the mic of kanamycin was decreased 4-fold, and the mics of both erythromycin and ampicillin were lowered 2-fold. at the concentration of 80 mg/l f105 also completely inhibited the biofilm formation by s. aureus; the cell growth was suppressed by two orders of magnitude as judged by differential fluorescent staining with syto9 and propidium iodide. the addition of f105 to preformed 24 h-old biofilms increased the fraction of red-stained (dead) cells of both s. aureus atcc29213 and mrsa strains uniformly throughout the whole profile of the biofilm. the quantitative analysis of clsm microphotographs revealed that f105 at concentration of 80 mg/l led to death of up to 98% of biofilm-embedded cells. this fact suggests that f105 efficiently penetrates into the biofilm matrix and kills the cells without visible damage of biofilm structure. in summary, furanone f105 seems to be a promising compound for drugs design to treat biofilm-embedded s. aureus. this work is supported by the russian science foundation, project №15-14-00046 and the german academic exchange service (№91531398). pneumonia is an inflammatory lung disease which can be associated with inadequacy of host defense system and the proliferation of various pathogenic microorganisms into the lower respiratory tract. community acquired pneumonia (cap) is one of the leading causes of death in elderly. the incidence of pneumonia in people aged 65 and over is 3-5 times more than young adults. creactive protein (crp) is an acute-phase protein of hepatic origin that increases following interleukin-6 secretion by t cells and macrophages. procalcitonin (pct) is a peptide precursor of the hormone calcitonin, the latter being involved with calcium homeostasis. it is composed of 116 amino acids and is produced by parafollicular cells (c cells) of the thyroid and by the neuroendocrine cells of the lung and the intestine. the level of pct rises in a response to a proinflammatory stimulus, especially of bacterial origin. the aim of this study was to compare crp and pct levels in young and elderly patients with pneumonia. recently diagnosed 43 young and 46 elderly patients with pneumonia and their respective aged matched controls (n = 41, n = 42) were enrolled this study. crp and pct levels were by immunoturbidometric and by elisa methods respectively. crp and pct levels for young control and patients and elderly control and patients respectively are 2.32 ae 2.25 mg/l, 0.26 ae 0.13 ng/ml, 20.71 ae 34.61 mg/l, 0.64 ae 1.09 ng/ml, 2.32 ae 2.14 mg/l, 0.27 ae 0.07 ng/ml and 31.41 ae 31.0 mg/l, 0.49 ae 0.83 ng/ml. young patients with pneumonia have significantly higher crp and pct levels than their controls (p < 0.001 and p < 0.028). elderly patients with pneumonia have significantly higher crp levels than their controls (p < 0.001). crp and pct are important markers in the diagnosis of pneumonia. effect of serum albumin concentration on total and ionized calcium z. adiyaman, c. yilmaz, s. a. peker, d. y€ ucel ankara training and research hospital, ankara, turkey objective: the aim of the study is to investigate in vitro effect of albumin concentration on total and ionized calcium concentrations. materials and methods: a serum pool with low albumin (3.10 g/dl) and normal calcium (9.79 mg/dl) concentrations was prepared from leftover sera. from this serum pool, two parts, each of 10 ml were aliquoted. purified albumin, 0.3 g, was added to one of these pools and albumin concentration was determined as 6.1 g/dl. the low and high albumin pools were mixed at different ratios and pools with 3.89, 4.78, and 5.37 g/dl albumin concentrations. total calcium and albumin concentrations of these 5 pools were measured at a beckman-coulter au5800 analyzer and ionized calcium was measured at a radiometer abl 800 blood gas analyzer in triplicate. total and ionized calcium concentrations were evaluated as compared to those of the original pool with an albumin concentration of 3.10 g/dl. results: total calcium concentrations are increased with the increasing albumin concentrations: 1.2%, 1.58%, 2.85%, and 3.59%, respectively. whereas, ionized calcium concentrations were decreased with increasing albumin: 3.0%, 6.1%, 7.4%, and 8.6%, respectively. conclusions: when total allowable error limits based on biological variation were considered, total calcium concentrations are significantly increased at >5 g/dl albumin concentrations. ionized calcium is significantly affected by 0.8 g/dl and over albumin concentrations. a regression equation based on albumin concentration may be useful for corrected ionized calcium concentrations. relationship between lipoprotein (a) and hba1c in patients with type ii diabetes , is a complex lipoprotein consisting of ldl and apolipoprotein(a). lp(a) is a risk factor for coronary artery disease and stroke. the relationship between lp(a) and diabetes mellitus is not clear. in this study, the relationship between lp(a) and glycemic parameters such as hba1c and fasting glucose concentration was investigated. lp(a), hba1c, fasting glucose, triglyceride, total cholesterol, ldl-and hdl-cholesterol concentrations were screened retrospectively from july 2013 to july 2016. there were 1831 patients with these test results at the same time. the patients were grouped according to hba1c values: group i < 5.7% (n = 468), group ii 5.7-6.4% (n = 739), and group iii >6.5% (n = 624). the relationship between these parameters were statistically within each group and all groups. there was not a statistically significant difference between the lp(a) concentrations of group i and group ii. lp(a) concentrations of group i and ii were significantly higher than those of group iii.. _ in total, lp (a) was negatively correlated with hba1c (r = 0.09; p < 0.01), but there was not a significant correlation with fasting blood glucose. _ in groups, there was a significant and negative correlation between lp(a) and fasting glucose in only group i. the negative correlation between lp(a) and glycemic parameters is interesting in patients with diabetes. despite lp(a) is an independent risk factor for cardiovascular diseases, on the contrary to expectations, lp(a) concentrations are decreased in diabetes. effect of blood collection through intravenous lines on hemolysis erroneous results are one of the most important causes of medical errors and may lead to unnecessary investigations or inappropriate interventions. total testing process consists of preanalytical, analytical and postanalytical phases. hemolyzed specimens that one of the most common source of preanalytical errors are frequently observed in laboratory practice and associated with incorrect laboratory results. blood collection through intravenous lines frequently results in hemolysis especially at eds and icus. in this study, we aimed to compare the effect of blood drawing by using bd luer-lock adapters and injector on the hemolysis rates at the ed. 60 patients who has been admitted to the ed were included in this study. all samples were drawn from newly inserted iv lines. the first blood sample was drawn with injector and the second one was drawn with luer-lock adapters to vacuum tubes. after the centrifugation routine chemistry tests and hemolysis indices were analysed on a beckman coulter au680 analyzer for each serum tube. the statistical significance of differences between two tubes was calculated with paired samples t test and statistical significance was accepted as p < 0.05. there were statistically significant differences between the two groups of tubes for the following parameters: ldh, ck, ast, k + , total bilirubin, protein, albumin, alp, calcium and hemolysis index (p < 0.05). the use of luer-lock adapters instead of injector could reduce the hemolysis rate. because of it reduces false results and unnecessary investigations, this approach will be more appropriate and cost-effective in ed. hemolysis and test rejection: are we following a reliable process? introduction: in laboratories, some blood samples are rejected due to hemolysis. we usually cancel only some of the tests that are affected by hemolysis. however, the frequency of the test cancellation may be relative. each test is affected in different degrees of hemolysis; some of them are not even affected at all. in this study, we aim to investigate unnecessary cancellations and explain the relationship between hemolysis and test results according to their kit inserts. materials and methods: we measured hemoglobin levels of 50 hemolyzed serum using drabkin method (abbott). interference studies are conducted using clsi protocol nccls ep7-p is written in kit inserts. target values (100%) and their change due to different degree of hemolysis have been defined. results: hb concentration ranges of hemolyzed sera were found from 44 to 849 mg/dl. according to kit inserts, aspartate aminotransferase (ast) test results deviate 5.7% from the target when the degrees of hb are 62 mg/dl. when the degree of hemoglobin is 125 mg/dl, the test strays about 11.6%. potassium levels increase (108%) at 125 mg/dl hb while this increase reaches to 17.9% at 250 mg/dl hb. sodium, calcium, ck, crea, total bil, lipase are not significantly affected even at 2000 mg/dl. in lactate dehydrogenase (ldh) tests, test reporting is not allowed at any hemolysis level. alt increases 11%, at the 1000 mg/dl hb. ast and potassium results were excluded from patients' reports even though those samples had low hb. some of them were reported despite of excess hemolysis. some tests are even blocked without ever being studied. discussion: prior to the approval of the lab specialist, technicians decide whether to cancel the tests affected by the hemolysis according to the visible hemolysis based on their personal knowledge. conclusion: we should use the hemolysis index, in which standards would be defined via guidelines. this way, all technicians and specialists could know which results are false. the dna-binding hu-proteins are present in all bacteria and belong to the family of nucleoid-associated proteins. these proteins can be considered precursors to eukaryotic histones. gene knockout of hu-proteins partially inhibits the growth of bacteria, their ability to resist various stressing factors and in some cases leads to their death. since the spatial structure of hu-proteins is highly conserved it is possible to create inhibitors that will affect them in a broad spectrum of pathogenic bacteria. in the present work the preparation of the recombinant hu protein from mycoplasma gallisepticum, crystallization of this protein, and x-ray diffraction study of this protein has been reported. the crystallization conditions for studying protein were found by the hanging-drop vapor-diffusion method. found conditions have been adapted to the counther-diffusion method in the capillary. the x-ray diffaction dataset from grown crystals have been collected using synchrotron radiation. 3d-structure of the hu protein from mycoplasma gallisepticum have been determined with 3a resolution. structural features of the investigated protein are described. this work is supported by russian scientific fund (15-14-00063). a novel sensitive disposable indium tin oxide (ito)-based electrochemical immunosensor was developed for simple, rapid and sensitive biomonitoring of sox2. sox2 is a cancer biomarker and used for detecting of small cell lung cancer, lung adenocarcinoma, squamous cell carcinoma, skin cancer, prostate cancer, and breast cancer. in this study, indium thin oxide (ito) thin film was used as working electrode. carboxyethylsilanetriol was also used for electrode modifying so as to obtain self-assembled monolayers. the formed self-assembled monolayers were activated with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (edc)/n-hydroxysuccinimide (nhs) chemistry. edc was used as a heterobifunctional crosslinker. nhs was used in conjunction with the crosslinker edc. anti-sox2 antibody was used as a biorecognition element and it was covalently immobilized onto the ito electrode modified with carboxyethylsilanetriol. immobiliztion steps were characterized by cyclic voltammetry (cv), electrochemical impedance spectroscopy (eis), and scanning electron microscopy (sem). the optimal immobilization conditions for the best sensitivity of the new immunosensor were investigated. under optimal conditions, this immunosensor demonstrated a wide linear range (0.0025-2 pg/ml) with a detection limit as low as 0.02 ng/ml sox2. furthermore, the developed sox2 immunosensor had good storage stability, repeatability and reproducibility. in this work, we successfully fabricated disposable ito thin film based electrodes for sensing the interaction between sox2 antigen and anti-sox2 antibody by electrochemical impedance spectroscopy and cyclic voltammetry. and our developed immunosensor has an acceptable performances for the detection of sox2 antigen, exhibits low detection limit, has selective and reproducible results in immunoreaction analysis. we are thankful for the support from t € ub _ itak (the scientific and technological research council of turkey, project number: 113 z 678). applying multiple linear regression model to determine the relationship between anti mullerian hormone with age, luteinizing hormone, follicle stimulating hormone and estradiol: a data mining study introduction: anti mullerian hormone (amh) has a widely used in our life because it is a good indicator of reproductive age to estimate the time of menopause. the purpose of this retrospective data mining study is the estimate of ovarian reserve by using amh and determines relationship between other indicators which are luteinizing hormone (lh), follicle stimulating hormone (fsh), estradiol and age. materials and methods: 25.294 women members were included this retrospective data mining study who were applying to acıbadem labmed laboratory. multiple regression analysis of age related changes of amh (18-45) and lh, fsh and estradiol were investigated. beckman gen ii elisa kit was used for amh and the technique of electrochemiluminescence and roche elecsys cobas analyzer were used for the measure of other hormones. results: amh shows meaningful correlation between lh, fsh, estradiol and age but also seen there is no correlation between progesterone. after the multiple linear regression analysiz amh= 11.018-(0.220 9 age)à(0.066 9 fsh) + (0.044 9 lh)à(0.004 9 estradiol) is detected and the model's r 2 = 0.627 is also detected. conclusion: nowadays there are lots of methodology were developed the estimate the function of ovary and biological age of ovarian. age, fsh, lh and estradiol show ovarian reserve by indirectly. this study shows the mathematical relationship between amh and the other indicators and results are thought to lead to future developments. antioxidant and anticancer effect of artemisia absinthium extract on colon and endometrium adenocarcinoma cells plants have always been among the common sources of medicines that have many phytochemicals with various bioactivities, including antioxidant and anticancer activities artemisia absinthium (ar) has been used as an antipyretic, antiseptic, anthelmintic, tonic, diuretic, and for the treatment of stomachaches in turkish folk medicine. this study aimed to investigate antioxidant, cytotoxic, genotoxic and apoptotic effect of methanol extracts of ar activities on the human colon (dld-1) and endometrium (ecc-1) adenocarcinoma cell line. total phenolic, flavonoid content, and antioxidant activities were determined using suitable methods (abts, cuprac i.e). cytotoxic effects of ar on cells were determined by mtt and neutral red uptake assays. genotoxicity was evaluated by comet assay and, apoptosis induction were detected by apoptosis elisa and acridine orange staining methods at the half maximal inhibitory concentrations (ic50) levels. it was determined that extract have shown antioxidant activity in all tests and that they could be considered as a source of natural antioxidants. cytotoxic effects were concentration-time dependent. specifically, apoptotic and genotoxic effect increased at 100 and 200 lg/ml concentrations by 48 hours. we found that ar extract had antiproliferative, genotoxic and apoptotic effects on the human cancer cell lines dld-1 and ecc-1. however, further studies at molecular level are required to support our findings and to elucidate chemopreventive and chemotherapeutic effects of ar on colon and endometrium cancers. keywords: artemisia absinthium, antioxidant, anticancer, apoptosis, genotoxicity introduction: colorectal cancer is considered as a major gastrointestinal. this cancer is the second cancer related cause of death after lung cancer in worldwide. we designed a vaccine chimeric including cea and ca19-9 against colorectal cancer (ce-ca). materials and methods: the construct were analyzed by bioinformatics softwares. in this study, the ce-ca gene was optimized using the codon bias of e.coli and synthesized by biomatik company. then construct (ce-ca) was cloned into an expression vector and recombinant constructs transferred to e.coli bl21de bacterium and desired recombinant protein was expressed. recombinant protein was purified using ni-nta affinity chromatography. the content of secondary structures was obtained by circular dichroism (cd) spectrum. then recombinant protein was confirmed using western blot analysis and indirect elisa method. results: sds-page analysis showed that the recombinant protein was highly expressed and purified. western blot analysis confirmed recombinant protein. also cd spectrum confirmed predicted structures by bioinformatics tools. the elisa results showed significantly high affinity toward recombinant ce-ca protein. discussion: based on many studies, cea as potential immunogenic candidate could be considered in vaccine studies. also ca19-9 is a cell-surface antigen that has significant increase of expression in colorectal cancer, thus as marker of colorectal cancer. based in available data, these two antigens, in combination can provide specificity for production of colorectal cancer vaccine. conclusion: these findings suggest that ce-ca as potential immunogenic candidate which could be considered in future vaccine studies and detection of colorectal cancer. flow cytometric cell cycle and apoptosis analyses of some wild animal species a. tas, e. koban bostanlar tubitak, marmara research center (mrc), genetic engineering and biotechnology institute (gebi), animal genetic and reproductive biology laboratory, kocaeli, turkey cell biobanking; more specifically cryopreservation of biological diversity, is promising as a tool to preserve wild animals as well as domestic ones via nuclear transfer. in this study, we investigated the viability and cell cycle characteristics of 5 wild animal species (fallow deer, red deer, wild sheep, wolf, wild goat). auricular tissue samples were maintained in pbs+2%psa. tissues were seeded on 35 mm petri dishes containing dmem/high glucose supplemented with 20% (v/v) fcs and incubated 5%co2 in air at 95% relative humidity and at 37°c. after seeding, the medium was unchanged for 7 days and then it was changed in every 2 days for 25 days at maximum. once the cells were obtained; flow cytometric cell cycle and apoptosis analyses were done. in terms of apoptosis, all the groups showed high viability rates (over 95%) in culture when compared with the negative control (38%). the cell cycle comparisons were made between serum-starved cells and roscovitine treated cells, both for which untreated cells were used as control, which revealed different results for different species. there was no difference found between serum-starved cells and roscovitine treated cells for red deer and wolf. the serum-starved cells resulted in higher g0/g1 phase for fallow deer and wild goat. on the contrary, roscovitine treated cells resulted in higher g0/g1 phase for wild sheep. as a result; the cells obtained from wild animals had high viability and g0/g1 phase rates. therefore, they may serve as a donor cell source for nuclear transfer studies.(grant: tubitak kamag, turkey, 109g016). the interaction of different types of antibiotics with endothelial cells in the presence of nanoparticles the interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. the aim of this study was to investigate the interaction of nanoparticles (fe 3 o 4 ) or nanoparticles fused with different antibiotics with cell membranes in order to reveal changes in the membrane organization. endothelial cells were used to determine the effect of different antibiotics (gentamicin, kanamycin, amikacin, penicillin, polymyxin, neomycin, cefotaxime, bacitracin, moxicillin, erythromycin, streptomycin and vancomycin) on the membrane organization. for recording the anisotropy of cell suspensions treated with antibiotics or nanoparticles fused with antibiotics we used 1-4-trimethyl-6-phenyl 1, 3, 5 hexatrien p-toluenesulfonate (tma-dph). we decided to use nanoparticles fused with antibiotics because they contain small amounts of antibiotics which makes them less toxic than simple antibiotics,which is very important in patients with genetic diseases such as cystic fibrosis, that should be treated with antibiotics for a long time. our results showed that at temperatures between 31 and 35°c simple nanoparticles decreased the membrane fluidity. at physiological temperatures (37-39°c) nanoparticles fused with antibiotics (gentamicin, vancomicin, cefotaxim, bacitracin, amoxicillin) increase more the membrane rigidity compare with simple antibiotics or nanoparticles.erythromycin, polymyxin and penicillin increase the membrane rigidity at 37°c, and at 39°c the same effect was obtained in the presence of nanoparticles fused with these antibiotics,suggesting that the nanoparticles are dependent to temperature for penetrating the membrane. in conclusion the membrane fluidity does not depend on antibiotics types, the modification are present in many antibiotics irrespective of class type.the presence of nanoparticles fused with antibiotics is very important for long term treatment. objectives: hypertension is an important cardiovascular risk factor for the development of atrial fibrillation (af). increased atrial electromechanical coupling time interval measured by tissue doppler is accepted as an important factor for prediction of af development in hypertensive patients. monoamine oxidases (maos), are enzymes which catalyze the oxidation of monoamines. 8-isoprostane is considered as an indicator of oxidative stress. mao activity and 8-isoprostane levels were measured in some diseases. however, there are no information on 8-isoprostane levels and mao activity in newly diagnosed patients with stage 1 hypertension has not been observed in a study of literature. aim: this is the first study, we aimed to evaluate the levels of mao and 8-isoprostane in newly diagnosed patients with stage 1 hypertension. the study included 60 newly diagnosed stage 1 hypertensive patients with no other systemic disease. 30 patients were selected as randomized (17 women, 13 men; range of age 46-74 years) and 30 healthy individuals as control (15 women, 15 men; range of age 44-72 years). all the underwent tissue doppler echocardiographic examination. blood samples were taken from patients and controls and, the levels of mao and 8-isoprostane in serum samples were measured by elisa. results: baseline blood pressures, electrocardiographic and echocardiographic findings, and atrial electromechanical coupling were similar in both groups (p > 0.05). compared to the control group, the activity of mao and 8-isoprostane levels were found significantly higher in patients (p < 0.05). conclusion: increased 8-isoprostane level indicate that there is oxidative stress in newly diagnosed patients with stage 1 hypertension. also, increased mao activity may be biochemical biomarkers for the diagnosis of hypertension. keywords: hypertension, monoamine oxidase, 8-isoprostane p-mis-147 determining the indirect reference intervals for complete blood count parameters in bursa, turkey reference intervals (ris) for laboratory test results are defined as the most commonly used diagnostic tool in medicine. therefore, careful determination of ris by the laboratory for use is a very important task. although c28-a3 guideline recommends the direct ris (dris) calculated from healthy subjects, ris can be calculated from laboratory data which are called as indirect ris (iris). the study was carried out at the central laboratory for clinical chemistry, teaching and research (uludag university, bursa, turkey) . the results of the laboratory analyses from 142,591 males, 215,658 females, stored for approximately one year, were used for statistical analysis. data for hospitalized patients and for ambulatory patients from the intensive care unit were eliminated. furthermore, we used evidence based criteria to enrich the health-related values. a modified bhattacharya procedure was used to estimate the iris from hospital patient data. the nested anova was used to evaluate variations among genders and ages. cell dyn analyzer (abbott diagnostics, il, us) was used for the measurements of complete blood count. the obtained iris were also compared the dris determined in our previous ri study and the ris suggested by the manufacturer. we found that the ris of rbc, hb and hct required strong gender partition and calculated the ris of rbc, hb and hct separately. the observed iris for wbc, sub-fractions of wbc and plt in both genders are in good accordance with the dris reported in previous study. age-related changes were noted for rbc, hb, and hct. the calculated iris for rbc, mcv and rdw are different from the ris suggested by the manufacturer. we believe that, using this relatively easy technique, every laboratory can produce its own iris, divided, where possible, according to sex and age and according to local conditions. these ranges can be complementary to dris obtained for reference individuals according to the ifcc recommendations. the principal sigma 70 subunit, involved in transcription of most house-keeping genes in escherichia coli, was also shown to induce rnap pausing during transcription elongation, by interacting with promoter-like motifs in the transcribed dna. such pauses were proposed to play important roles in the regulation of phage and cellular genes. e. coli contains six alternative s subunits but little is known about their ability to induce transcriptional pausing. we expressed and purified alternative s subunits of the sigma 70 family and tested their effects on transcription elongation in vitro on natural and synthetic dna templates containing consensus promoter motifs. the structure of the paused complexes was analyzed by dna footprinting methods. in vivo analysis of transcription was performed using reporter genes placed under the control of corresponding promoters. we demonstrated that the stationary phase sigma 38 subunit induced efficient rnap pausing on both synthetic and natural dna templates containing promoter-like motifs in initially transcribed regions. in contrast, the sigma 32 and sigma 28 subunits did not affect rna elongation. we showed that the sigma 38 -induced pausing depends on sigma contacts with both nontemplate dna strand and rnap core. the pausing results in formation of backtracked transcription elongation complexes which can be reactivated by gre factors that stimulate rna cleavage by rnap. our results for the first time reveal transcriptional pausing induced by an alternative s subunit. analysis of sigma 38 -dependent promoters shows that a substantial fraction of them contains potential pause-inducing motifs suggesting that such pausing may be a widespread phenomenon. we propose that sigma 38 -dependent pauses may play important roles in genetic regulation and modulate the binding of transcription repressors or activators to promoter regions. the crosstalk between streptococcus pneumoniae rnase r, ribosomes and translation c. b arria, s. domingues, c. arraiano instituto de tecnologia qu ımica e biol ogica, lisbon, portugal ribonucleases (rnases) are enzymes that ensure maturation, degradation and quality control of rna thus, contributing to the maintenance of the optimal amount of each transcript in the cells. escherichia coli rnb family of enzymes is present in all domains of life and includes rnase r, rnase ii and the eukaryotic rrp44/dis3, dis3l1 and dis3l2 proteins. in streptococcus pneumoniae only rnase r was identified. rnase r, encoded by the rnr gene, hydrolyzes rnas starting from the 3 0 end. rnase r level is increased in several stress conditions such as heat shock, stationary phase or cold shock, conditions in which most of the proteins translation is blocked. moreover, rnase r is the only exoribonuclease able to degrade highly structured rnas without the help of a helicase which is critical at low temperatures. here, we investigated the role of this enzyme by comparing the wild type strain with an rnr mutant strain. for that purpose we performed northern blots analysis of transcripts involved in translation. also, we investigated rnase r connection to the ribosome and polysome fractions using sucrose gradient polysome separation and western blots. in this study, we highlight the importance of s. pneumoniae rnase r in translation. we show that this enzyme interacts with ribosomes mostly with the 30s subunit at 37°c. moreover, in the absence of this enzyme we have observed a decrease in the amount of the 70s ribosomal subunit, concomitantly with the increase of 30s and 50s subunits. rnase r seems also to modulate the amount of the elongation factors ef-tu and ef-g transcripts. nevertheless, preliminary results further suggest other roles of rnase r in translation. modified nucleotides are present in many rna species in all domains of life. the biosynthetic pathways of such nucleotides are well studied. however, much less is known about the degradation of rnas and the salvage of modified nucleotides, their respective nucleosides or heterocyclic bases. using an e. coli uracil auxotrophic strain, we screened the metagenomic libraries for genes, which would allow the conversion of 2-thiouracil to uracil and thereby lead to the growth on a defined synthetic medium. we show that a novel gene encoding previously uncharacterized domain of unknown function (duf) is responsible for such phenotype. we have purified this recombinant protein and demonstrated that it contains a fe-s cluster. the substitution of cysteines, which have been predicted to bind such clusters, with alanines abolished the growth phenotype. we conclude that this domain is required for conversion of 2-thiouracil into uracil in vivo. this work is supported by the research council of lithuania (lmt, mip-103/2015 modified nucleotides are present in almost all classes of rna. they have great chemical diversity and are critical for rna folding, stability, interaction with cellular proteins and thereby for various cellular processes such as translation, stress response, and signaling pathways. biosynthesis of pyrimidine nucleotides and their modified derivatives in rna is well studied. nonetheless, not much is known about the cellular degradation of these compounds and the enzymes catalyzing such processes. using an e. coli uracil auxotrophic strain, we screened metagenomic libraries for genes encoding isocytosine deaminases. three novel genes were obtained, one of which encodes a protein similar to 8oxoguanine deaminases. the other two encode proteins resembling hydroxydechloroatrazine ethylaminohydrolases. we confirmed that these proteins are functional in vivo, allowing growth of e.coli on minimal medium with isocytosine. we also demonstrated that such purified recombinant enzymes catalyze the conversion of isocytosine, but not cytosine, into uracil in vitro. natural products display special attributes in the treatment and prevention of various human diseases, including cancer. a significant number of organic compounds from plants exhibit anticancer properties as attested by in vitro and in vivo studies. emerging evidence supporting the antineoplastic activity of natural compounds has rendered them promising agents in the fight against cancer. in this study, skin from limnio grape, a red greek grape variety that is indigenous to the greek island of lemnos, was extracted using mixtures of methanol, water and acetone; the apoptosis-inducing properties of these extracts were studied in the human ovarian malignant adenocarcinoma cell lines tov-21g and tov-112d. for this purpose, tov-21g and tov-112d cells were treated with limnio grape skin extracts at a range of concentrations, at 37°c, for 24, 48 and 72 hours. untreated cells incubated for the same time intervals served as controls. cell viability was determined by measuring metabolic activity (colorimetric mtt assay) and observing cell membrane integrity (cell staining with trypan blue). after the determination of the optimal concentration of the extract, total rna was extracted from treated and untreated (control) tov-21g and tov-112d cells. after determination of rna concentration and subsequent first-strand cdna synthesis, mrna expression analysis of apoptosis-related genes was performed with rt-pcr using gene-specific primers. an increasing percentage of non-viable cells was observed by increasing cell exposure time and extract concentration. distinct modulations of the expression of apoptosis-related genes at the mrna level were also observed, mainly concerning bcl2, bclx, bax, bak1 and bcl2l12, along apoptosis induction. in conclusion, the cytotoxic properties of limnio grape skin extracts against ovarian malignant adenocarcinoma cells merit further investigation. the intrinsic apoptotic pathway seems to be the major mechanism of action induced by these plant extracts. almost all eukaryotic mrnas are polyadenylated by a complex machinery that recognizes the poly (a) signal, cleaves the mrna and adds the poly (a) tail. 70% of human genes harbor multiple poly (a) signals. alternative polyadenylation (apa) generates transcript isoforms with different 3 0 utr (untranslated region) lengths due to the use of proximal or distal poly (a) signals. hence, tightly regulated apa has been observed in normal physiological settings as well as in diseases. considering that 3 0 utr shortening cases have been linked to increased protein levels, we hypothesized deregulated apa to be one of the potential cancer related mechanisms. we investigated the 3 0 utr alterations in er(+) breast cancer patients and cell models compared to normal breast tissue, using gene expression data and a probe-based quantification tool, apadetect. based on means of proximal to distal probe sets, slr (short-long ratio) were calculated as an indication apa. significance analysis of microarrays (sam) determined significant genes. the gse numbers of the datasets are gse2034, gse7390 and gse9761. we analyzed two datasets of er(+) breast cancer patient samples (n = 209, n = 135) compared to normal breast tissue (n = 82) using apadetect and sam. a total of 184 3 0 utr shortening and 253 3 0 utr lengthening events were detected in breast cancer samples compared to normal breast tissue. ontology analysis suggested almost all the 3 0 utr shortening genes were proliferation related and were indeed reported to be upregulated in breast cancer. to further investigate the connection between apa and era status, we used data from a cell line model; wild type or era transfected mda-mb-231 cells that are otherwise of triple negative nature. our results suggested that most of the genes are 3 0 utr shortened or lengthened via direct binding of era to dna. our results suggest involvement of apa mechanisms in era action mechanisms. possible link between era regulated transcription and apa remains to be elucidated. contamination of nucleic acids (na) as a result of na extraction protocols may result an inaccurate measurement of dna copy number. agarose gel electrophoresis and spectrophotometric methods are commonly used to check dna purity. however, the resolution of these methods may not be good enough for special applications such as determination of dna copy number and separation of base pairs (bp) that are close in their bp number. in this study, we have developed a new method for separating na's ranging between 75-20000 bp also detecting the impurities in dna solution in 1%, 5% and 10% ratios to the dna of interest. the developed method was validated using the in-house dna fragments of 100, 150 and 200 bp. the dna mixture analyzed using analytical hitachi elite lachrom hplc using the guard and analytical columns tskgel dna-npr, 2.5 lm, 4.6 mm id 9 0.5 cm and tskgel dna-npr, 2.5 lm, 4.6 mm id 9 7.5 cm, respectively. the validation of the analysis was performed by running each sample five times on three different days. the linearity of the detector response was established by plotting a graph to quantity versus area of 200 bp dna. the lod and loq were then measured by calculating the minimum level at which analyte can be readily detected and quantified. the ratios calculated with hplc were compared to the ratios calculated by quant-it kit. recovery values were calculated for each measurement and the uncertainty were calculated for each ratio. the method was found linear for 200 bp in the range of 0.4 ng to 800 ng dna with the regression coefficient of r 2 =0. 9992. lod and loq for the 200 bp dna was found to be 0.39 ng and 1.56 ng, respectively. the recovery values for the 1%, 5% and 10% impurity ratios were found 101.74. 97.41 and 99.47, respectively. the purity of the synthetic dna was determined by hplc and related uncertainty was calculated. the developed method is a simple alternative to electrophoresis and spectrophotometric methods with higher resolution and separation range. physical and chemical factors can disturb the conformation of proteins maturing within the cellular secretory pathway. in response to unfolded proteins the cell activates several stress signaling and adaptive response mechanisms. the aim of our study was to investigate small non-coding rnas as the potential regulators of cellular response to unfolded proteins (upr). for this, we conduct the next generation sequencing of small rna and transcriptome analysis of mrna from jurkat cells exposed to dithiothreitol (dtt), which reduces protein disulfide bounds. analysis of mirnas reveals the differential expression of 104 mirnas. we observe a decrease in the normalized amount of reads aligned to mirna loci in stressed cells. affymetrix analysis with subsequent gsea reveals downregulation of reactome mirna biogenesis pathway (fdr = 0.096). the length distribution of small rnas revealed 32 nt-peak corresponding to trna-derived fragments, amount of which was increased by 2.6-fold under dtt treatment. the trna isotypes that gave rise to almost 76% and 86% of all 32nt rna fragments in stressed and control cells, respectively, include glycine, glutamic acid, aspartic acid and valine. the vast majority of 32nt fragments produced from these trnas are precisely phased 5 0 halves with the characteristic cleavage patterns generated by rnase a angiogenin (ang). observed upregulation of tirna in stressed cells is accompanied with upregulation of ang mrna and down-regulation of angiogenin inhibitor 1 (rnh1). we speculate that translational repression, associated with observed tirna, is an additional mechanism of reducing global protein synthesis in response to dtt-induced stress. collectively, our findings reveal the increase in tirna, the differential regulation of mirna expression together with the global mirna downregulation as the most prominent small rnome reprogramming events and possible fine-tuned levels of post-transcriptional regulation upon dtt-induced cellular stress response. global gene expression changes after spinal cord injury j. k. hyun 1,2,3 , j. kim 1,2 , j. y. hong 1 1 dankook university, cheonan, south korea, 2 institute of tissue regeneration engineering (itren), cheonan, south korea, 3 the neuronal regeneration is hardly achieved spontaneously after spinal cord injury (sci), and the restoration of somatic and autonomic functions after sci is also challenging in the clinical field. the pathophysiology of sci is extremely complex and many in vitro and in vivo studies continued to report opposite results each other in spite of the same treatments, therefore a fundamental analysis such as an extensive assay of global gene expression is required to find a way for spinal cord regeneration. in this study, we aimed to detect the changes of global gene expression after spinal cord contusion in rats according to the time sequence. the spinal cord tissues at contusion site were sequenced after spinal cord contusion in rats using rna-sequencing technology. for time sequence analysis, five time points was determined; 1 hour, 1 day, 1 week, 1 month and 3 months after spinal cord contusion, and sham operated rats at each time point were used as controls. quantitative rt-pcr analysis was also performed to validate expression changes of candidate genes in each category. we found that the pattern of changes in gene expression at acute and subacute stages was quite different from that at chronic stage, especially genes associated with with neurotrophin signaling and apoptosis pathways. most of gene expression levels of inflammatory cell markers were increased and peak during acute stage (1 hour to 1 week) and maintained until chronic stage. some of regeneration-associated genes (rags) including brain derived neurotrophic factor, glial cell derived neurotrophic factor and ciliary neurotrophic factor were increased at 1 hour or 1 day after sci. we concluded that the information of gene expression level according to the time sequence after sci might be useful to determine treatment strategies for spinal cord regeneration especially in chronic stage. p-01.02.2-011 3 0 utr length isoform generation profile in a differentiation model alternative polyadenylation (apa) is the regulated selection of a specific poly(a) signal among other proximal and/or distal signals on the 3 0 utrs (untranslated region) for the endolytic cleavage and addition of a poly(a) tail to form the mature mrna. consequently, position of the poly(a) site determines the length of the 3 0 utr which is known to harbor microrna and rna binding protein sites. such apa isoforms have already been linked to altered protein levels and even functions. therefore we hypothesized apa to be one of the mechanisms to generate isoform diversity in proliferating and differentiated cells to better understand the molecular basis of cancer. we used a combinatorial in silico and in vitro approach to analyze a well known enterocyte differentiation model; caco-2 cells. initially we analyzed gene expression datasets for the proliferative and differentiated caco-2 cells using a probe based apa detection tool. to better understand the significance and to validate these results, we used proliferating and differentiated (day10) caco-2 cells and tested sample apa events by rt-qpcr. 3 0 utr isoforms were identified by using 3 0 race pcr. we identified 43 genes (32% of all apa events) to undergo 3 0 utr shortening in differentiated cells compared to proliferating cells. on the contrary 91 genes (68% of all apa events) went through 3 0 utr lengthening events. several genes have been validated to follow the pattern that was seen in apa detection tool so far. to begin understanding the mechanism behind these observations, we are investigating potential inducers of apa during the complex events of differentiation. our next aim will be to further validate and investigate the consequence of such isoform generation events both in the context of differentiation in colon cancer cells. recognition of phosphorylated threonine-4 of rna polymerase ii c-terminal domain by 3 0end processing apparatus o. jasnovidova, m. krejcikova, k. kubicek, r. stefl central european institute of technology, masaryk university, brno, czech republic rna polymerase ii has evolved an array of heptad repeats with the consensus sequence y1-s2-p3-t4-s5-p6-s7 at the c-terminal domain (ctd) of its largest subunit, rpb1. phosphorylation of serines (s2, s5, and s7) and tyrosine-1 orchestrate the binding of rna processing and transcription factors in the site of transcription. several recent studies showed that also threonine-4 site can be phosphorylated which has a number of functional consequences. to reveal the structural basis for the recognition of threonine-4 phosphorylated ctd, we set out to investigate several proteins factors that were implicated with a high levels of threonine-4 ctd phosphomarks using integrative structural biology. one of them, a factor involved in the 3 0 -end processing and transcription termination, showed a high affinity to the phosphothreonine ctd. using nuclear magnetic resonance spectroscopy (nmr), we determined its structure bound to the ctd phosphorylated at threonine-4 that reveals a direct read-out of the phosphothreonine. altogether, our data provides the first insights into the recognition of this poorly understood ctd mark that plays important role in the ctd code of rna polymerase ii. the results of this research have been acquired within ceitec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. introduction: the treatment of brain tumor glioblastoma (gbm) is still one of the greatest challenge. anti-inflammatory drug indomethacin (ind) mainly acting through the inhibition of cyclooxygenase (cox) has also anti-cancer activity including brain tumors. the aim was to investigate how ind effects an immortality enzyme telomerases' activity. materials and methods: monolayer and spheroid cultures of t98g human gbm cell line were used to evaluate the effects of ind (100 lm) on cell proliferation, viability, apoptosis, cell cycle, camp levels, the levels of apoptotic and anti-apoptotic proteins, morphology (sem) and ultrastructure (tem) for 72 hours. results were analyzed using the student's t-test. results: ind decreased cell proliferation (p72 < 0.01), cell viability (p72 < 0.000001), cell rate at s phase (p72 < 0.000001) and g2 + m phase (p72 < 0.001), camp levels (p72 < 0.01), the levels of pdgfr-a (p72 < 0.001), mrp-1 (p72 < 0.05), nf-jb (p72 < 0.01) and cox-2 (p72 < 0.01) in comparison to control group. ind mildly increased apoptosis (p72 < 0.001) and caspase-3 levels (p72 < 0.01). interestingly, ind increased htert levels (142%, p24 < 0.001; 154%, p72 < 0.0001). sem evaluation showed that ind led to decreased and shortened microvilli, the lost of cell interactions and the conversion of many cell shapes from spindle to oval. many cell remnants in the intercellular area, intact cell membranes, many dense lipid droplets and few autophagic vacuols in the cytoplasm were observed under tem. discussion and conclusions: the effect of ind on telomerase activity can only be found in 8 publications at pubmed research that they only showed its' inhibitory effect in colon, gastric, head and neck cancers. in contrast to previous studies, it was shown for the first time that ind increased telomerase activity in gbm cells and this increase was independent from cox-2 and other tested factors. p-02.02.2-002 interaction between fibrinogen and insulin-like growth factor binding protein-1 under physiologic conditions and influence of diabetes mellitus type 2 on this interaction n. gligorijevic, o. nedic institute for the application of nuclear energy, university of belgrade, belgrade, serbia fibrinogen is plasma glycoprotein and principle participant in blood coagulation. it interacts with many proteins, including insulin-like growth factor binding proteins (igfbps). one of them, igfbp-1, is controlled by insulin. metabolic changes due to diabetes mellitus (dm) affect igfbp-1. besides glucose regulation, igfbp-1 stimulates wound healing. we have investigated complexes formed between fibrinogen and igfbp-1, their change in dm type 2 (dm2) patients and involvement in fibrin clot. samples from adult healthy persons and dm2 patients were studied: plasma, isolated fibrinogen and fibrin. the amount of igfbp-1/fibrinogen complexes was determined using immunoblotting. immunoprecipitation and lectin affinity chromatography were used to confirm interaction between fibrinogen and igfbp-1. in vitro incubation of fibrinogen with excess glucose or methylgyoxal (mgo) was employed to demonstrate influence of glyco-oxidation on complexes. results have shown that igfbp-1/fibrinogen complexes can be differentiated from igfbp-1 oligomers and igfbp-1/alpha-2macroglobulin complexes. the amount of igfbp-1/fibrinogen complexes was lower in patients with dm2. complexes participated in fibrin clot formation, the amount being significantly lower in patients' samples. the quantity of igfbp-1 monomer in fibrin clot was greater in patients' samples. in vitro experiments revealed that complexes undergo glyco-oxidative modifications leading to their reduced formation, cross-linking and increased acidity (faster electrophoretic movement). isolated fibrinogen from patients with dm2 was additionally able to bind exogenous igfbp-1. since igfbp-1 stimulates wound healing, directly and by delivering igfs, igfbp-1/fibrinogen complexes may be seen as igfbp-1 storage instrument, ready to participate in fibrin formation and to assist in damage repair. reduction of complexes due to glyco-oxidative stress in patients with dm may be part of the mechanism responsible for impaired coagulation process. human interferon gamma (hifnc) is a proinflammatory cytokine involved in the regulation of nearly all phases of immune and inflammatory responses. its abnormal expression is associated with the aetiology of many inflammatory and autoimmune diseases. recently we have been exploring the idea to counteract the over-expression of the endogenous hifnc by competitive inhibition with inactive hifnc mutants. they are designed to have preserved affinity to the hifnc receptor, but to be deprived in their capability to trigger the intracellular signal transduction. to this end a library of mutants was created and two potential hifnc antagonists were selected for further investigations: a single point mutant k88q (q substitution for k in position 88) and a double mutant with additional substitution in the n-terminus. both mutants and the wild type hifnc were expressed in e. coli employing the established by us methodology for large scale production of aggregation-prone proteins in soluble native form. the purified mutants were screened for interferon activity (antiproliferative assay), binding affinity (isothermal titration calorimetry) and ability to compete with the wild type for the hifnc receptor (competition assay on wish cells). the selected mutants demonstrated 100 (single mutant) and 1000 (double mutant) times lower antiproliferative activity than the wild type. measuring the binding thermodynamic parameters, we proved that the receptor binding affinity of both mutants was preserved, which is an indication for their potential to compete with the wild type hifnc for its receptor. finally, the biological assay performed on wish cells showed a distinct dose-dependent competition between the wild type hifnc and the mutants. based on the results presented in this study we conclude that the two hifnc mutants are potential candidates for autoimmune therapy based on selective suppression of the endogenous hifnc activity. mesencephalic astrocyte-derived neurotrophic factor (manf) is an er (endoplasmic reticulum) stress-inducible protein and widely expressed in mammalian tissues. it has been identified as a secretory protein that protects cells against er stress-induced damage. er-stress is one of the main mechanisms that play a role in ischemia/reperfusion (i/r)-induced renal injury. recent studies demonstrated that manf can protect cardiac myocytes and cortical neurons against i/r-induced injury. moreover, it has been suggested that it has a restorative effect in ischemic injury. nevertheless, the function of manf in i/r-induced renal injury is still not known. in the present study, we investigated the function of manf by manipulation its expression level in ischemic acute renal failure model established in proximal tubular kidney cells (hk-2 cells). for this purpose, the cells were transfected with either manf sirna or manf encoding plasmids for silencing or over-expression of manf, respectively. then, the cells were exposed to hypoxia-reperfusion (h/r) induction for indicated times. evaluations of cell viability were determined with wst-1 reagent. the changes in protein levels of h/r-induced stress markers were analyzed byimmunoblotting. the results showed that the overexpression of manf has provided a significant resistance to h/r-induced cell death, whereas silencing of manf has rendered the cells more susceptible to death. it was also determined that the pretreatment of cells with manf conditioned medium caused a decrease in cell death. additionally, oxidative/nitrosative stress (os/ns) and er stress levels were decreased with over-expression of manf and increased by silencing of manf in hk-2 cells. taken together, our study suggests that manf may have a protective role against h/r-induced renal cell injury, possibly through the reducing effects on os/ns and er stress. p-02.02.2-005 his-flag tag as a fusion partner in insect expression systemgain or loss? e. krachmarova 1 , m. tileva 1 , k. maskos 2 , i. ivanov 1 , g. nacheva 1 1 institute of molecular biology "roumen tsanev", sofia, bulgaria, 2 proteros biostructures, martinsried, germany human interferon gamma (hifnc) is a glycoprotein playing major role in the regulation of innate and adaptive immunity. glycosylation is not essential for hifnc activity but is important for its stability, half-life and protease resistance in blood. the commonly used hifnc in therapy and research is produced in e. coli and therefore is not glycosylated. bearing in mind the above mentioned shortcomings of the non-glycosylated hifnc we expressed it in mammalian cells and transgenic mice, however very low yields were achieved. to obtain glycosylated hifnc, here we employed a secretory expression of n-terminal his-flag fusion protein in baculovirus-infected insect high five ò cells. this small hydrophilic tag is designed to not affect the proper folding of the target protein and to facilitate the detection and purification procedures. in parallel the same fusion was expressed in e. coli cells. the fusion proteins were purified to high degree of purity by affinity and size-exclusion chromatography. bioassay carried out on wish cells showed that the antiproliferative activity of both fusion proteins was 500 times lower than that of the native hifnc. this result shows that, in contrast to the generally hold view, the n-terminal his-flag tag interferes with the biological activity of hifnc despite of the protein glycosylation. in order to restore the biological activity we attempted to remove the his-flag tag enzymatically. surprisingly, we found that the fusion protein obtained from insect cells was resistant to enterokinase, independently of the enzyme source and experimental conditions, whereas the protein isolated from e. coli was susceptible and the tag-free protein showed fully restored biological activity. we are prone to explain the enterokinase resistance of the fusion protein from insect cells with either the specific conformation of the glycosylated protein or with the interaction of the carbohydrate residues with the enzymatic activity of the enterokinase. p-02.02.2-006 development of fluorescence assay for highthroughput screening system based on flow cytometry for directed evolution of cellobiose dehydrogenase cellobiose dehydrogenase (cdh) is an enzyme produced by phanerochaete chrysosporium and it has been already successfully cloned in other organisms. one of the most important roles of cdh is removing products of cellulose degradation. cdh is very important for biofuel and biosensor industry. for improvements of enzyme properties we have used directed evolution. the most important step is to develop screening system that reflects properties of interest. screening in microtiter plates (mtp) is expensive, time-consuming and has low throughput with a small number of variants detected (10 3 -10 4 in months). the aim of this work was the development of screening system for mutant libraries of cdh expressed on surface of yeast cells based on fluorescent enzymatic assay and flow cytometry. the screening method should be capable of screening cellobiose dehydrogenase variants mutated for higher activity and higher thermostability by error prone pcr. the fluorescent assay was beta-galactosidase (ec 3.2.1.23) also known as lactase is the enzyme that typically catalyzes hydrolysis of beta-1,4-d-galactosidic linkages in beta-d-galactosides, including disaccharide lactose, with glucose and galactose as end reaction products. this enzyme is able to catalyze synthesis of oligosaccharides, in particular galactooligosaccharides via galactosyl transfer reaction. arthrobacter sulfonivorans beta-galactosidase of unique for prokaryotes extracellular localization may find application in food industry for manufacturing lactose-free dairy products and in pharmacology as bioactive principle of medicines prescribed for patients suffering from lactase deficiency. the study was aimed at cloning of the gene encoding a. sulfonivorans beta-galactosidase, purification and characterization of the enzyme. a novel extracellular beta-galactosidase from a. sulfonivorans was recovered with an overall 207-fold purification, a 7.7% yield and specific activity 16 300 uámg à1 protein. the subunit molecular mass of the enzyme determined by sds-page analysis equalled 125 kda. it was found that the enzyme displays pi 5.35, prefers ortho-nitrophenyl-beta-galactoside as substrate (km 27 mm) and shows maximum activity at 40°c and at ph 7.5-9.5. the beta-galactosidase gene was isolated from the genomic dna library of a. sulfonivorans, sequenced, cloned and deposited in the genbank database under accession number km277894.1. it was established that the gene carries an open reading frame consisting of 3132 bp (1043 amino acids) and encodes beta-galactosidase referred to glycosyl hydrolase family 2 (cazy database). p-02.02.2-009 different splice-forms of tdrd7 protein mutated in cataract's and glaucoma's interacts with s6k1/2 o. skorokhod, v. filonenko department of cellular signalling, institute of molecular biology and genetics nas of ukraine, kyiv, ukraine ribosomal s6 kinases (s6k) are important players in cellular pi3k/mtor signalling network, deregulation of which has been associated with methabolic disorders, inflammation and cancer. previously we had identified a novel binding partner of s6k1 -tdrd7 (trap). tdrd7 is a scaffold protein detected in complexes involved in the regulation of cytoskeleton dynamics, mrna transport, protein translation, non-coding pirnas processing, transposons silensing. it was reported recently that mutations in human tdrd7 result in cataract and glaucoma formation, defined by elevated intraocular pressure (iop) and optic nerve damage. the aim of our study was to confirm s6k-tdrd7 interaplay and study its role in cells. bioinformatical analysis of tdrd7 sequence revealed the presence of potential phosphorylation sites of s6k2. using in vitro kinase assay, we have demonstrated that recombinant s6k2 phosphorylate 3 from 5 fragments of tdrd7. formation of s6k2-tdrd7 complexes in vivo was further confirmed by coimmunoprecipitation using anti-s6k2 and anti-tdrd7 antibodies generated previously in rat brain lysates. this interaction was further confirmed by confocal microscopy, oleksandr had shown that tdrd7 co-localize with s6k2 in hepg2 cells, predominantly in perinuclear region, enreached for one of the tdrd7 isoforms identified previously. moreover, we have detected that c-terminal synthetic peptides of s6k2 with methylated arg interfere with tdrd7 from hepg2 lysates. the physiological characteristics of s6k2-tdrd7 interaction and the role of this complex formation in neuropathology's development need further investigation. many biological function of placenta are performed not just a set of individual proteins, but also different oligomeric structures and complexes. herewith, activities of complexes may considerably differ from activities of individual proteins. therefore, identification and characterization of placental multi-protein complexes is an important step to understanding the placenta function. the aim of the present work was to investigate a composition and biological functions of the very stable high molecular mass multi-protein complexes (spc) from placenta of healthy mother. we isolated spcs (~1000 kda) from the soluble fraction of three human placentas. light scattering measurements and gel filtration showed that the spc is stable in presence salts, acetonitrile and triton x-100 in high concentrations, but efficiently dissociates in the presence of 8 m urea and 50 mm edta. such a stable complex is unlikely to be a random associate of different proteins. it was shown the spc includes a number of proteins with molecular weights of 2 to 180 kda. several protein components of the spc were identified, including serum albumin, transferrin, iggs, annexin a5 and other proteins. serum albumin, transferrin and protein with molecular weight 14,1 kda are the main proteins of the complex. it was shown high the spcs from three placentas possesses dnsase and catalase activities. an addition, investigation of cytotoxic effect on human cancerous cell lines has shown that the spcs reveal high cytotoxicity. antibody-cytochrome b5 fusion protein, characterization and applications for antibody development process antibodies have recently become an essential tool being a part of immunodiagnostics, therapeutics and as a valuable instrument in life science research. an enormous number of options utilizing a various tags were used to create a universal antigen-binding domain, which can be easily detectable, highly soluble and might be produced in high yields with low costs, but no multipurpose solution exists yet. we addressed the question whether a single tag could be found for enhancing solubility of recombinant fab antibody fragment and providing its detection and accurate quantification by rather simple method. a new application for hemeproteincytochrome b 5 as the antibodies fusion partner were proposed. we have constructed of recombinant fab antibody fragment cytochrome b 5 fusion protein. we have shown that cytochrome b 5 enhance expression of fab antibodies fragments in bacterial system, and could be a versatile tool for recombinant proteins folding, redox (oxidation) state studies and for their precise concentration determination in the turbid solutions. fusion fab-b5 protein has a stable red color and characteristic absorbance spectrum with the maximum absorbance at 413 nm in oxidized environment. cytochrome b 5 change its spectrum maximum depending on environmental redox potential and its folded state, so one can track these events in real time spectrophotometrically. binding activities of fab-b 5 fusion protein and hybridoma secreted immunoglobulin were measured by biolayer interferometry and elisa. no significant difference between them was revealed. due to this feature we can distinguish the chimeric protein of interest in complex mixtures and control the process of recombinant proteins expression and purification in real-time. besides, cytochrome b 5 fusion tags multiples recombinant antibody yield (from 2 to 3 times) and doesn't affect antigen-binding properties. the bb125-135 site of fibrin molecule is the site of fibrin protofibrils lateral association l. urvant palladin institute of biochemistry nas of ukraine, kyiv, ukraine previously we showed that fibrin-specific monoclonal antibody i-3c (monab i-3c) inhibited the fibrin protofibrils lateral association. we suggested that the epitope of monab i-3c in bb118-134 of coiled-coil region of fibrin molecule coincides with the site involved in fibrin protofibrils lateral association. the aim of this study was to localize the site of protofibrils lateral association in fibrin molecule using the synthetic peptides bb121-138, bb125-135 and both their scrambled version, and bb109-126 peptide. monab i-3c was isolated from hybridoma culture medium by affinity chromatography on fibrin-sepharose. turbidity analysis was used to study the effect of synthetic peptides on fibrin polymerization. the interaction between peptides and monab i-3c was investigated by spr method using plasmon-6 device. we investigated the effect of synthetic peptides which corresponded to amino acide sequences of fibrin molecule bb109-126, bb121-138, bb125-135, and the scrambled versions of bb121-138 and bb125-135 peptides on a binding to monab i-3c and on the fibrin polymerization process. in spr analysis was showed that bb121-138 and bb125-135 peptides, but not their scrambled version, binds to monab i-3c, immobilized to a chip. turbidity data showed that only bb121-138 and bb125-135 peptides caused the 2-fold decrease of the rate of the lateral association of protofibryls at the concentration 2.2 9 10 à4 m and 2.7 9 10 à4 m, respectively. both of them decreased the final clot turbidity. our data let us to suggest that the bb125-135 site is the site that involved in protofibryls lateral association. it has been recently shown that irisin immunoreactivity was altered in gastrointestinal cancers. as known hematological malignancies was one of the most common malignancies through world, but no study was present how irisin was changed in this type of cancers. therefore, purpose of this was to investigate how immunoreactivity to irisin was altered in hematological malignancies (blood cancers). we used an antibody from phoenix to demonstrate how a 12 kda band after deglycosylation of irisin altered in hematological malignancies. here we first time showed that irisin tissue immunoreactivity from acute lymphoblastic leukemia (all) and acute myelogenous leukemia (aml) patients was increased when compared with unaffected biological tissue parts. from the immune-histochemical (ihc) investigations it is concluded that hematological tissue and blood cells may be another source of irisin and increased with cancer, thus this finding might help to enlighten pathophysiology of hematological malignancies. the value of urine neutrophil gelatinaseassociated lipocalin (ngal) in acute heart failure n. serdarevic clinical centre, sarajevo, bosnia and herzegovina introduction: renal dysfunction is very common in heart failure (hf) and neutrophil gelatinase-associated lipocalin (ngal) is used as an early marker of acute renal tubular injury. recent studies have been reporting that ngal is inhibitor of inactivation of matrix metalloproteinases (mmp-9) which results in enhanced proteolytic activity with prolonged effects on collagen degradation. due to its relation to extracellular matrix degradation in myocardium and infammation, we hypothesized possible increased ngal expression in hf besides it renal dysfunction etiology. patients and methods: in study were included 30 patients hospitalised with signs and symtoms of ahf. urine samples for ngal analysis were collected at admission and analysed by the chemiluminescent microparticle immunoassay (cmia) for the quantitative determination of neutrophil gelatinase-associated lipocalin in human urine (abbott, architect analyzer). refferent range for urine ngal is 0-135 ng/ml. on admission blood samples for bnp (brain natriuretic peptide) analysis were drawn and tested by architect bnp chemiluminescent microparticle immunoassay (cmia), abbott laboratory. results: the mean age of the patients (male= 30, female= 30) was 68.86 years (sd 10.92 years). among them 18 (30%) patients was diagnosed as a hf-pef (hf with preserved ejection fraction) while 42 (70%) as a hf-ref (hf with reduced ejection fraction). mean bnp values was 1616.71 pg/ml (sd 1511.15 pg/ml) and mean lvef was 33.66% (sd 14.19%). mean urine ngal was 60.91 ng/ml (sd 78.72 ng/ml). we found significantly positive, but weak correlation among ngal and bnp only by pearson correlation test (r = 0.139, p = 0.464, wilcoxon signed rank test z = à4.782 p < 0.5). conclusion: bnp levels are elevated in hf with reduced and preserved ejection fraction. urine ngal is not elevated in acute heart failure, but it is slightly positively correlated with serum bnp values. converging evidence implicates the intermediate and medial mesopallium (imm) of the domestic chick forebrain in memory for a visual imprinting stimulus. a number of learning-related changes have been found in plasma membrane and mitochondrial proteins of imm. for broader analysis of these changes we employed two-dimensional gel electrophoresis/mass spectrometry approach and identified differentially expressed proteins in membrane-mitochondrial fraction of the imm across chicks with different estimated levels of imprinting 24 h after training. we further inquired whether the amounts of those proteins in the imm and a control region (posterior pole of the nidopallium, ppn) are correlated with memory for the imprinting stimulus. learning-related increase in the amounts of the following proteins was demonstrated in the left imm, but not in the right imm or left and right ppn: (i) membrane cognin;(ii) a protein resembling the p32 subunit of splicing factor sf2;(iii) voltage dependent anionic channel-1;(iv) dynamin-1; (v) heterogeneous nuclear ribonucleoprotein a2/b1. obtained results indicate that the molecular processes involved in learning and memory of imprinting cover a wide range of cellular activities, including stabilization of protein structures, increased mrna trafficking, synaptic vesicle recycling and specific changes in the mitochondrial proteome. the aim of this work is to study the substrate and inhibitory properties of uridine derivatives in the reactions catalyzed by e.coli up in order to shed some light on the substrate's conformation in the productive complex with the enzyme. we studied the e.coli up-catalyzed phosphorolysis of uridine and its derivatives modified in the heterocyclic base and the sugar moiety. the kinetic constants (km, ki, kcat ) of the phosphorolysis reaction of near 30 uridine derivatives were determined. the combined kinetic (nnna, 35, 2016) and structural data (acta crystallogr., d70, 3310, 2014) provide clear evidence that up binds uridine in the most energetically unfavorable conformation, which, to the best of our knowledge, has no precedents in the enzymes of nucleic acid metabolism. this is possible due to multiple interactions between the substrate and the protein environment (active site residues) mainly through hydrogen bonds. these results are important for understanding the mechanism of action of this class of enzymes. an analysis of the conformations of nucleosides in solution and rotational barriers suggests that the energy difference between the ground state of uridine and uridine complexed with up may be high as 63-69 kj/mol. the binding in a high-energy conformation results in the weakening of the glycosidic bond. the observed conformation of uridine complexed with sulfate (mimetic of phosphate) may be very similar to its conformation in the transient state. until now, foxp1 (forkhead box p1) has been identified as a tumor suppressor in several correlation studies in breast cancer. although, foxp1 is defined as a transcriptional repressor that interacts with other transcription factors in various mechanistic studies, there is no study that explains its repressor functions in breast cancer biology. here we demonstrate the repressor function of foxp1 on nfat (nuclear factor of activated t cells) and the migratory effect of this repression in mda mb 231 breast cancer cells. we performed co-immunoprecipitation experiments for the investigation of protein-protein interaction between two transcription factors. protein-protein interaction on dna was investigated with emsa and transcriptional effects of foxp1 on nfat, lusiferase reporter assay was performed. wound healing assay was used to analyse the effects of overexpression of foxp1 on tumor cell migration. our results showed that foxp1 has protein-protein interaction with nfat on dna and enhances breast cancer cell migration by repressing nfat transcriptional activity and foxp1 shows oncogenic function by regulating breast cancer cell motility. introduction: phosphodiesterase 5 (pde5) is one of phosphodiesterase lead to hydrolyzing cgmp.the cgmp signaling pathway has an important role in proliferation of cells. previous studies showed pde5 was increased in cell lines cancers thus pde5 inhibitors can used as efficacious therapeutic option for treatment of cancers. the current study was to investigation the effect of hydroalcoholic achillea.wilhelmsii extract (hawe) on the pde5 gene expression and cgmp signaling in the mcf-7 er + and mda-mb-468 er à . methods and materials: the ed50 of the hawe on both cell lines were examined by using mtt viability test then the expression of pde5 and cgmp concentration were measured in timedependent manner (in the ed50) by real-time rt-pcr and colorimetric assay respectively. results: treatment with the hawe showed, 25 lg/ml is ed50 for both cell lines and the hawe lead to reduction in pde5 mrna expression and evaluation of intracellular cgmp showed an increase pattern in the time-dependent manner. conclusion: our results showed that the hawe has anti-proliferative property in the mcf-7 and mda-mb-468, cell lines of breast cancer through the cgmp pathway, these data suggested that the hawe can be potential source for the isolation of effective anti-proliferative molecules. keywords: achillea.wilhelmsii, breast cancer, anti-proliferative, phosphodiesterase, cgmp signaling pathway. outer membrane protein g (ompg) is a stable monomeric porin having 14-stranded beta barrel form from e.coli. its exact function is not fully understood; however, it allows the passage of molecules up to 900 da in neutral ph but the pore is closed by going through a conformational change under ph 5.5. as being monomeric and having ph-dependent gating characters, it is suitable for biosensor and targeted drug delivery applications. an attempt on ompg is to create a larger pore while its stability is undisturbed. ompg-16s is obtained by adding 38 amino acids to the primary chain in order to have a 16-stranded beta barrel porin. ompg-16sl is formed by further adding 6 amino acids to loop l6 and by replacing 7 lysines with arginines. ompg-16s and ompg-16sl mutants are investigated by fourier transform infrared spectroscopy (ftir) and compared with ompg-wild type (wt) in terms of ph-dependent conformational changes and thermal stability. each mutant is prepared in na-phosphate buffer pd 5.5/7.5 and infrared spectra are recorded. further, temperature profiling are recorded for the range between 22 to 106°c. results show that both mutants are responsive to ph changes. while turning the ph from acidic to neutral, beta sheet signals shift to lower wavenumbers showing difference in secondary structure, implying the existence of closed and open states. on the other hand, mutant proteins show structural differences compared with the wt protein. porins are known for their remarkable thermal stability. the mutans retain this character by having transition temperature of $ 75°c, although this is less than the wt transition at $ 85°c. in conclusion, two mutants show signs of open and closed states as ompg-wt and even if the mutants are less stable than ompg-wt. this study shows that the attempted alterations in ompg structure are successful in terms of ph-response but it needs improvement in terms of stability when necessary. nad is a key factor in the regulation of mitochondrial metabolism. besides its vital role as redox carrier, nad serves as substrate for protein adp-ribosylation and deacetylation, modifications which modulate enzyme activities in mitochondria. these functions depend on how nad levels are maintained in this organelle. in human cells, mitochondrial nad is segregated from the cytosolic pool and can be synthesized from nmn, which is probably imported into the matrix. here, we tested whether the nudix pyrophosphatase nudt13 participates in the regulation of the mitochondrial nad pool. this enzyme has a predicted nadh pyrophosphatase zinc ribbon domain and a mitochondrial targeting sequence at its n-terminus. however, it has not yet been functionally characterized. we overexpressed nudt13 endowed with a c-terminal flag-epitope in human cells. to evaluate changes in the mitochondrial nad concentration, we used a reporter system which includes the overexpression of the catalytic domain of poly(adpribose) polymerase 1 (parp1) within the organelles (mitoparp). thereby mitochondrial nad is converted into protein-bound poly(adp-ribose) (par). the extent of par formation correlates with the mitochondrial nad availability and is detected by western blotting. our results established that nudt13 is indeed a mitochondrial protein, as it was localized exclusively to these organelles. moreover, when nudt13 was overexpressed along with the mitoparp detector system, a dramatic decrease of par was observed. the obtained results indicate that nudt13 is enzymatically active upon overexpression in the mitochondrial matrix and that it might cleave nad, thereby modulating its organellar level. however, at this point we cannot exclude the possibility of direct par cleavage by nudt13. further characterization of nudt13 will define its substrate specificity and clarify its role in mitochondrial metabolism. the incidence of increase in colorectal cancer (crc) worldwide has become a major health problem. early diagnosis and treatment of crcs are of importance for improving survival. in the present study, it was aimed to investigate chemopreventive effect of rosmarinic acid and evaluate the angiogenesis process in azoxymethane (aom)-induced crc model. male sprague-dawley rats were randomly divided into a control group, aom-induced rat colorectal cancer group (15 mg/kg body weight aom; ip, weekly for four weeks), and rosmarinic acid (5 mg/kg body weight; oral, daily for four weeks)-treated group. in addition to the standart diet of the all groups 15.8% peanut oil was added throughout the experiment. the all rats were sacrificed at the end of 30 weeks. biochemical examinations were performed in rat plasma. histopathological adenocarcinoma rates were observed in 87.5% of aom group. the incidence of adenocarcinoma was showed a reduction in the treatment group. significant increases in plasma tos and mcp-1 levels were found in the aom group compared to controls. these increases were reduced in the treatment groups but no significant. a significant increase was detected in tas levels in the treatment group when compared to the aom group. significant decreases in plasma adiponectin levels were found in the aom and the treatment groups compared to controls. in conclusion, treatment with rosmarinic acid reduced the occurrence of inflammation and was helped to maintain the oxidant-antioxidant balance in the model of aom-induced rat colon cancer. mitochondrial genome, while being strongly reduced in course of evolution, still codes for several proteins. the vast majority of them are components of the respiratory chain complexes. to produce these proteins, the system of mitochondrial translation is presented in the organelles, which is in common close to that in bacteria. translation initiation in bacterial cells is orchestrated by three protein factors called if1, if2 and if3. the orthologs of the two latter proteins are commonly found in mitochondria. however, mitochondrial if3 could not been identified in several groups of organisms, including s.cerevisiae, for a long time. recently we have shown that baker's yeast protein aim23p possesses a function of mtif3. however, the mitochondrial translation has not been stopped in the yeast strain without aim23p which is surprising taking into account the fact that if3 is obligatory for the translation in bacterial systems. instead of blocking of mitochondrial protein synthesis in absence of aim23p, we observed the translational imbalance: the synthesis rate of the complex v subunits was increased while the synthesis rate of the complex iv subunits was repressed. thus, in addition to its general role in translation initiation, aim23p might specifically affect the biosynthesis of individual mitochondrial-encoded protein species. our genetic experiments have revealed that, indeed, aim23p is almost indispensable for cox1p synthesis, and that it affects the translation of cox1 mrna through its 5 0 -utr, like classical mitochondrial translational activators. this is in accordance with our measurements of complex iv activity which is several times less in yeast lacking aim23 gene than in the wild-type. taken together, our results point on the multiple role of aim23p in mitochondrial translation: in addition to its function as mitochondrial if3, it specifically regulates the amount of complex iv subunits and its activity. p-02.02.2-024 the circulating betatrophin and irisin levels in polycystic ovary syndrome patients with and without insulin resistance introduction: polycystic ovary syndrome (pcos) is the most common endocrine/metabolic disease in women around the world, characterized by oligo-or anovulation, polycystic ovary, and/or hyper-androgenism. insulin resistance (ir) and obesity are common findings in patients with pcos. irisin is a recently identified myokine secreted from skeletal muscle in response to physical activity. irisin has been postulated to induce the differentiation of white fat tissue into brown fat tissue. betatrophin is a currently discovered new hormone proposed to stimulate b-cell proliferation. in this study we investigated the levels of irisin and betatrophin in pcos patients. materials and methods: our study group was consisted of 40 patients with pcos and 20 healthy volunteers. patients group was divided into two subgroups according to presence of ir. (pcos+ir and pcos-ir). the oral glucose tolerance test (ogtt) and the homeostatic model assessment (homa-ir) were performed to assess glucose tolerance and insulin sensitivity. irisin and betatrophin levels were measured by elisa method. results: circulating irisin was significantly higher in the pco-s+ir subgroup than the control group (p < 0.022). circulating betatrophin was significantly lower in both patients subgroups than the control group (p < 0.008). there was no negative or positive correlation between irisin and betatrophin levels. discussion: these data suggest that irisin and betatrophin may act a role together in the ir mechanism in pcos patients. butyrylcholinesterase (bche) synthesized in liver has long been associated with hyperlipidemia, type 2 diabetes and obesity. there are also reports on bche knockout mice becoming obese. the exact involvement of how bche interacts with lipids is still not clear. previously we displayed a correlation between leptin, waist circumference, fat mass and bche levels. recently, we have also shown that bche overexpression in hepg2 cells is regulated by alpha linoleic acid. as the next approach on the analysis of lipid metabolism and bche interaction, we considered the capability of bche to hydrolyze lipids. human serum bche was purified by subsequent deae-tris-acryl m and procainamide chromatography. the purified bche was utilized in a modified acid lipase assay with the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). as the second alternative substrate trioleic acid was utilized. the triolein hydrolysis was measured by the nefa kit. verification that bche hydrolysis of these lipid substrates was not due to another esterase was done by iso-ompa inhibition studies. also, lectin binding studies with bche and rca were carried out to rule out non-specific esterase activity. using purified human serum bche and hepatic lipase as control enzyme we found that bche is able to hydrolyze the acid lipase substrate 4-methylumbelliferyl palmitate (4-mu-palmitate). we found that bche hydrolyses this molecule at ph 8 rather better than at ph 7.4. at ph values, purified human bche has a km value that was 10 times bigger than that of human pancreatic lipase. with the bigger molecule the triolein, the difference between the km values of bche and pancreatic lipase was smaller. bche seems to hydrolyze triolein with an efficacy comparable to approximately 25% that of human pancreatic lipase. our results display that another function of bche may be its lipid hydrolyzing activity. p-02.02.2-026 determination of regional reference ranges for erythropoietin with laboratory data mining serum erythropoietin (epo) levels are the main regulator factor of erythrocyte production and increase in response to hypoxia. our region is a location dominated by hypoxic conditions due to the high attitude. in this study we aimed to investigate the mean serum epo levels in the living conditions of our region. two hundred and eighty epo results from our laboratory data whose hemoglobin levels were normal were evaluated in the study. mean serum epo levels were analyzed via chemiluminescence method in beckman coulter dxi 800 auto analyzer. the epo levels of samples was 12.26 ae 9.32 mul/ml (ranged between 3.00 and 48.57) mul/ml. when we performed ae 1 sd for the studies population we determined normal serum epo levels were as 2.94-21.58 mul/ml. the upper limit determined by our results was 17% higher than that of determined by the manufacturer as 18.5 mul/ml and the lower limit determined by our results was 17% higher than that of determined by the manufacturer as 2.59 mul/ml. normal serum epo levels were considerable for our region and the upper and lower limits were higher than those of determined by the manufacturer. more detailed studies considering the physical properties of participants including a higher number participants are necessary. subclinical hypothyroidism is the precursor to hypothyroidism because it has a tendency to transform into hypothyroidism. subclinical hypothyroidism is considered one of the risk factors causing metabolic syndrome. metabolic syndrome can be characterized by plasma level of apelin released from adipocytes. in the present study, we aimed to measure serum apelin level of patients with subclinical hypothyroidism and compare them with serum apelin level from healthy individuals. our study group included 31 patients diagnosed with subclinical hypothyroidism and 23 healthy volunteers. serum samples were obtained from each participant for the measurement of apelin. these were then stored at à20•c until the time of analysis. serum apelin concentrations were determined using an enzymelinked immunosorbent assay. the mean serum apelin levels of subclinical hypothyroidism and control groups were 79 ng/l, control group 60 ng/l respectively. there was no statistically significant difference in terms of the mean apelin levels between the groups (p > 0.05). apelin levels didn't show significant correlation with bmi (p > 0.05). in the present study, no significant difference of serum apelin level was observed between patients with subclinical hypothyroidism and healthy control subjects. however, the apelin levels were higher in the patients with subclinical hypothyroidism than in the control group. the possible relationship between thyroid hormones and apelins is critical to understanding the etiopathogenesis of metabolic disorders. the mitochondrial erv1/mia40 import system does not impact cytosolic fe-s cluster protein maturation and iron regulation erv1 is a sulfhydryl oxidase that partners with the import receptor mia40 to import small cysteine-rich proteins into the mitochondrial intermembrane space. it has also been suggested that erv1 has an additional role in maturation of cytosolic fe-s cluster proteins and regulation of iron homeostasis in s. cerevisiae. however, these studies were performed on one particular erv1 mutant strain (erv1-1) that we discovered has additional defects in glutathione (gsh) metabolism. since gsh is required for iron regulation and cytosolic fe-s cluster assembly, this complicates our understanding of erv1 0 s role in these processes. we discovered that the erv1-1 strain originally tested for fe-s cluster defects was the only strain to exhibit defects in the cytosolic fe-s enzymes. mitochondrial and cytosolic fe-s protein activities in the other erv1 and mia40 mutants tested were similar to the wt control. in addition, while all the erv1 and mia40 mutants tested exhibit temperature-dependent defects in mia40 oxidation, only the erv1-1 strain has significantly reduced gsh levels and more oxidized gsh: gssg redox state. we determined that the cause of gsh depletion in the erv1-1 strain is an additional mutation in the gene encoding the glutathione biosynthesis enzyme (gsh1) that compromises gsh1 protein folding and/or stability. to address whether gsh deficiency in the erv1-1 mutant is the underlying cause for the cytosolic fe-s cluster defects and iron misregulation for this strain, we measured fe-s protein activity, iron-regulated gene expression, and iron accumulation in erv1 and mia40 mutant strains. only the erv1-1 strain exhibited iron misregulation and accumulation of mitochondrial iron, while exogenous gsh rescued these defects. these results demonstrate that the defects in cytosolic fe-s enzymes and iron homeostasis in erv1-1 are due to gsh depletion and neither erv1 nor mia40 play significant roles in cytosolic fe-s cluster assembly and iron homeostasis. human c-peptide is a 31 amino acid polypeptide, which is secreted into blood from b-cells in the pancreas where pro-insulin undergoes a post translational modification and cleaved into insulin and c-peptide. human c-peptide concentrations in blood plasma and urine reflect the level of insulin resistance associated b-cell function and can point out insulin secretory failure. the reference intervals in blood plasma and urine are 0.5-10.0 ng/ml and 40-150 ng/ml respectively. c-peptide measurement in urine and plasma provides a guide for therapy in diabetes. this study describes a method for the development and validation of picaa (peptide impurity corrected amino acid analysis) method for the determination of the purity of the human c-peptide which could be used as a reference material to measure cpeptide concentrations in plasma. two different methods were performed for the picaa; aaa-id-ms/ms for quantification of constituent amino acids following hydrolysis of the material and rp-hplc-esi-tof ms for determination of the peptide related impurities. the result of the aaa id ms/ms method was corrected for the amino acids originating from the impurities. id ms/ms-aaa was performed with zivak ò hplc and zivak ò tandem gold triple quadrupole ms equipped with a phenomenex ez:faast 4l aaa column (250 9 2 mm i.d). the mobile phase was composed of, a: 20 mm ammonium formate (af) in water, b: 10 mm af in acetonitrile (acn). the intact peptide analysis was performed by a hitachi lachrome elite hplc and bruker microtof-q mass spectrometer equipped with a capcell pak mg-ii c18 column (150 9 2 mm i.d., 5 mm particle size). the purity of the synthetic c-peptide was determined by picaa analysis and related uncertainty was calculated. traceability to si was established using the amino acid standards of which the purity was determined by tub _ itak ume using qnmr analysis. picaa is a simpler alternative to the full mass balance approach which requires large quantities of the peptide material. p-02.02.2-034 heat shock proteins: complementary therapies in brain tumors with viscum album e. onay-ucar, s. n. biltekin 1 istanbul university, faculty of science, department of molecular biology and genetics, vezneciler, istanbul, turkey cancer is one of the lethal diseases in the world. different cancer types possess overexpressed hsps levels. viscum album extracts with their anticancer and antioxidant properties are being used in cancer therapies. biochemical composition of this plant is known to vary its features depending on the host trees and time of harvest. in our previous study, it has been found that v.album inhibited hsp27 expression and induced caspase-dependent apoptosis in c6 rat gliomas. the aim of the current study is to find out whether different v.album extracts have different effects on hsps expression level and apoptosis in c6 glioma cell line or not. in this study, three different extracts of v.album were compared for their potential inhibition effects on hsps. the cytotoxic effects of extracts have been determined via mtt test. different experiment groups were set up subjected to heat shock and/or incubated without any heat shock application. overexpression of hsps was induced by heat shock at 42°c for 1 h in c6 cells. expression levels of hsps were determined by western blot analysis. the apoptosis inducing effect was also evaluated via caspase-3 activation in c6 glioma cells. pretreatment of the cells with non-toxic dose (10 lg/ml) of v.album extracts prior to heat shock, reduced significantly the expression levels of hsps. similarly, pretreatment with the extracts prior to heat shock increased apoptosis via caspase-3 activation in c6 glioma cells. these results will be utilized in the determination of the relation between extract composition and stress protein expressions. these results suggest that different extracts of v.album are able to down regulate expression of hsps, and induce apoptosis. this warrants further exploration as a potential resource of bioactive compounds that can be used in cancer therapy. future studies targeting hsps for the development of chemosensitizers may help improve the treatment of cancer in combinational therapy. biological drugs (biologics) are the fastest-growing category of therapeutics among those approved by the agencies for drugs regulation. most biologics are proteins designed for parenteral use. however, proteins are characterized by poor pharmacokinetic and safety profiles. peg-coating (poly-ethylene glycol coating) of biologics provides several benefits, including an increased half-life related to reduced renal clearance, an increased stability to degradation, and a reduced immunogenic/antigenic response. preservation of the three-dimensional structure and activity of the pegylated form is a strict requirement for human use. the recombinant proteins used for this studies (as-sod, superoxide dismutase; mmp12, matrix metalloproteases 12; ansii, l-asparaginase ii) were cloned and then over-expressed in escherichia coli. pegylation reactions were performed using commercial reagents. all the protein samples were purified and analyzed by solution and solid-state nmr (fields from 700 mhz to 950 mhz). we developed new protocols to prepare samples of pegylated proteins, demonstrating that solid-state nmr spectra of exceptionally good quality can be obtained for pegylated proteins in the sedimented state (obtained by either ultracentrifugation or rehydration of freeze-dried samples); surprisingly, sedimentation of pegylated proteins to this end has never been attempted. the spectral quality is comparable toor better thanthat of the corresponding crystalline samples. the excellent quality of the solid-state nmr spectra would make it possible to perform extensive resonance assignment and even a conventional full structure determination of biologics. the proposed method is based on the comparison of a standard twodimensional solid-state nmr spectrum of the sedimented pegylated protein with that of the crystalline state of the native proteinfor which the x-ray structure is available. all eukaryotic creatures hereditarily have natural defense mechanisms and are protected from the infections with this defense mechanism. antimicrobial peptides (amp) contain 10-50 amino acid content, are positively charged with amphipathic feature. the antimicrobial activities of amps are thought to be depended on the microbiocidal effects by binding to the surface of microorganisms and creating pores in their membranes. defensins are both effector and mediator small antimicrobial peptides of the immune system. these peptides in cationic and amphipathic structure have broad spectrum antibacterial, antifungal and antiviral features. defensins regulate the innate and acquired immune systems by suppressing proinflammatory responses during infection. mammals have three structural subfamilies of defensins. these show differences according to the trisulfide arrays in their structure and are classified as a,b, h defensins. human beings have tissue-specific six functional a defensins. human hnp-1 and hnp-4 encoded by defa1, defa3 and defa4 genes are firstly expressed in neutrophils. human hdp5 and hdp6 encoded by defa5 and defa6 are firstly expressed in paneth cells in the intestines and play important role in the defense and homeostasis. human beings have many pseudogenes such as defap and deftp in addition to these functional genes. according to literature data, defensins play an important role in defense against microbial placements on mucosal surfaces. in addition, the antimicrobial spectra of defensins include gram negative and gram positive bacteria, fungi and viruses. in addition to their antimicrobial efficiency, they can accelerate the wound healing due to their mitogenic effects on epithelium cells and fibroblasts. bile salt hydrolase (bsh) enzyme catalyzes the hydrolysis of glycine and/or taurine-conjugated bile salts into amino acid residues and the free bile acids that reduce cholesterol. however, some intestinal bacteria have an excessive deconjugation of tauro-conjugated bile salts and production of secondary bile acid having potential harmful side effects to the host. the catalytic mechanism and substrate preference of such bsh enzyme is not clear. in this study, bsh gene from lactobacillus plantarum gd2 strain was cloned, expressed, characterized in escherichia coli blr(de3) strain, and then val-58 and phe-129 amino acids, supposed to be responsible for substrate preference, were substituted for met-58 and ile-129 amino acids respectively by site directed mutagenesis. the hydrolysis activities and stability of the mutant recombinant bsh (mrbsh) enzymes were examined along with six different bile acids by ninhydrin assay and sds-page respectively. ninhydrin test results indicated that wild-type recombinant bsh (wrbsh) hydrolyzed six major human bile salts with an apparent preference towards glycine-conjugated to tauro-conjugated bile salts. however, the activities of mrbsh/phe129ile enzyme are 29%, 23%, 13%, 14%, 0% and 0% of the activity of wrbsh against to glycocholic acid (gca), glycodeoxycholic acid (gdca), glycochenodeoxycholic acid (gcdca), taurocholic acid (tca), taurodeoxycholic acid (tdca) and taurochenodexycholic acid (tcdca) respectively. the activities of val58met mrbsh enzyme are 79%, 93%, 74%, 43%, 44% and 28% of wrbsh against to gca, gdca, gcdca, tca, tdca and tcdca respectively. our findings support the suggestion that bsh enzymes recognize their substrates predominantly at the amino acid moieties and not at the cholate moieties. however, further pcr-based site-directed mutagenesis and structure-driven computational and theoretical approaches are required for the precise determination of their substrate specificities and the selection of probiotic bacteria. we deposited bacteriorhodopsin in purple membranes under applied electrical field onto ito (indium tin oxide) support. purple membranes film, highly oriented in one direction, was placed between two ito electrodes. we studied dependence of electrical properties of these films on light illumination. we argue that this setup can be used for functional studies of microbial rhodopsins. in opposite to already published results where this system was used as a photocondensor for studying functional properties of bacteriorhodopsin, we studied electric properties of such systems and we found strong light dependence of resistivity of bacteriorhodopsin in purple membranes films. optogenetics is already used in study of neuronal cells cooperation in vitro and in vivo by means of microbial rhodopsinsion pumps and channels incorporated in membranes of neurons changing their electrical potential while receiving a light quantum by laser or led source. best perspectives optogenetics will give after successful transfer to medical applications, such as the treatment of blindness, treatment of disorders like parkinson's disease etc. but to achieve these we need a broad set of tools, optogenetics tools, highly specialized to solve specific problems of neurophysiology. to the creation of such tools our work is dedicated. new optogenetic tools can be made by mutations in existing ones altering their properties (mainly spectral characteristics, selectivity and conductivity) or some promising mutations in conserved residues can be found in existing organisms. a halophilic archaeon halosimplex carlsbadens is a host of protein of our interest. according to the theoretical data based on the alignment with br and the 3d structure model of this novel protein, we suppose this protein functions like the light-driven h+ pump: all the key residues are the same or at worst have the similar properties, except one in the position 96leucine instead of the aspartic acid. a gram-positive bacteria deinococcus-thermus phylum syntheses rhodopsin with substitution of this aspartic acid to alanine. sphingomonas paucimobilis has rhodopsin where aspardic acid in position 96 is changed to serine residue. and one yet uncharacterised guillardia theta rhodopsin even has the same as br motif (d85, t89, and d96) but according to alignment is closer to chr2 even the last one motif is e85, t89, n96. it is expected that all of them will show us new properties. though the further experimental data are essential. the work is supported by rsf 16-15-00242. evaluation of some thymus proteins in patients with crimean congo hemorrhagic fever i. b€ ut€ un 1 , s. sahin 1 , f. duygu 2 1 university of gaziosmanpasa, department of biochemistry, tokat, turkey, 2 oncology education and reasearch center, ankara, turkey crimean congo hemorrhagic fever (cchf) is a tick-borne viral zoonotic disease. it has a high fatal rate (%5-30). tokat is one of the cities having the most reported cchf cases, in turkey. clinical presentation of the disease varies widely among patients. thymic peptides are small molecules synthesized by thymic epithelial cells. they play role in the immune response, as well as anti-inflammatory process. fourty patients referring to the hospital with tick-contact history and/or presenting clinical manifestations consistent withcchf and with positive pcr results for cchf virus in blood samples were included to the study. the wbc and platelet values at application and before the patients were discharged were recorded. the healthy control group consisted of age and gender matched healthy volunteer adults free of any chronic disease. thymosin alpha1 (ta1), thymuline and thymosin beta4 (tb4) were studied by the elisa method in this study. biochemical parameters were also analysed. ast and alt values were significantly higher (p < 0.01) and plt and wbc levels were significantly lower in the cchf group (p < 0.05). levels of tf, ta1 and tb4 were found to be significantly higher in cchf (p < 0.01). there was no mortality during the study period. duration of hospitalization was 6.73 ae 2.74 days. levels of tb4 were significantly correlated with duration of hospitalization (r= à0.351, p = 0.036). alt levels were significantly correlated with tf levels (r = 0.413, p = 0.010). 17 patients received ffp and apheresis for the supportive treatment, while 5 patients received only ffp and 2 patients got only apheresis. 16 patients did not get any of these blood products. there was not a statistically significant differences in thymus peptides among these treatment groups (p > 0.05). we report 40 survived cchf patients with elevated thymic peptides. pathogenesis of cchf has many points to be highlighted. thymic peptides may play role in the clinical situation of the patients with the disease. the effect of methocarbamol on the peroxiadse activity of human erythrocyte hemoglobin hemoglobin is released to blood circulation, after red blood cells lysis. it is carried in circulation by binding to haptoglobin. in normal persons, no free hemoglobin is observed in the blood, because most of hemoglobin is in the form of haptoglobin complex. in some diseases that are accompanied by hemolysis, the amount of released hemoglobin is higher than its complementary haptoglobin. as a result, free hemoglobin appears in the blood, which is a toxic compound for these patients. free hemoglobin has been showed to have peroxidase activity and considered a pseudoenzyme. in this research, the effect of methocarbamol on the peroxidase activity of human hemoglobin was studied. our results showed that the drug inhibited the pseudoenzyme by un-competitive inhibition. both k m and v max decreased by increasing the drug concentration. k i and ic 50 values were determined as 6 and 10 mm, respectively. molecular docking results showed that methocarbamol did not attach to heme group directly. a hydrogen bond connected nh 2 of carbamate group of methocarbamol to the carboxyl group of asp126 side chain. two other hydrogen bonds could be also observed between hydroxyl group of the drug and ser102 and ser133 residues of the pseudoenzyme. p-02.02.2-042 dca reduces viability and down regulates mapk protein activations in human malignant mesothelioma cells and pericardium. microarray analyze results performed in mm patients revealed that one of the most prominent changes is upregulation of many genes involved in glycolysis and the krebs cycle. dichloroacetate (dca) is an inhibitor of pyruvate dehydrogenase kinase (pdk) that enhances the oxidative activity of cells by activating pyruvate dehydrogenase (pdh) in mitochondria. dca has shown as a promising anti-neoplastic agent that re-sensitizes cancer cells to apoptosis. the aim of this study is to elucidate the coupling between pdk inhibition and mm cell proliferation and cell cycle. human malignant mesothelioma (spc212) cell line was used as a model for dca treatments. cell viability was measured by mts assay; mapk protein activations and expressions were assessed by western blotting; cell cycle profile was analyzed by flow cytometry. statistical analysis was performed by utilizing one-way anova test. results showed that dca reduced viability of spc212 cells in a concentration and time dependent manner. protein analysis indicated that mapk pathway was down regulated at concentrations greater than 50 mm. moreover, primary cell cycle analysis has indicated arrestment at g2/m phase in 24 hours. our findings corroborate with recent reports where dca treatments resulted in reduction of viability and g0/g1 and g2/ m arrest in other cell lines. abnormalities in mapk signalling play a critical role in the progression of cancer. here, we showed for the first time that dca decreased mapk activation in 24 h. our results suggest that dca is an anti-prolifertive agents for mm cells in vitro. however, it requres extra analysis with other mesothelial cells. future study will focus on investigating relation between mapk and mitochondrial apoptosis. adrenomedullin (adm) is a vasodilator peptide consisting of 52 amino acids. adm is synthesized in many tissues. and is a biologically active peptide that has various effects including vasodilatation, the regulation of vascular endothelial function and adjusting adipogenesis. hypoxia inducible factor 1 alpha (hif 1a) is a subunit of a heterodimeric transcription factor hypoxia inducible factor 1. it is the master transcriptional regulator of cellular and developmental response to hypoxia. the dysregulation and overexpression of hif1a by either hypoxia or genetic alternations have been heavily implicated in cancer biology as well as a number of other pathophysiologies. in our research, the adm and hif 1-a levels in heart, kidney and lung tissues of rats were investigated in control, hypoxia, control+adm and hypoxia+adm groups. rats in hypoxia groups were provided hypoxic environment containing of 10-12% oxygen and 88-90% nitrogen for 1 week. rats in adm groups were injected intraperitoneally in a dose of 1.25 nmol/kg for four days before the collection of the tissues. the control group was oxygenated with normal air. the control and treatment groups were formed from 7-8 animals and adm, hif 1-a levels were measured in taken tissues with immunoassay method. the aim of this study was to investigate the reaction of the organism when exposed to hypoxic conditions and the effect of adm over hif 1-a level. adm levels and hif 1-a in heart tissue were found decreased in hypoxia group, and adm levels increased in hipoxia+adm group. hif 1-a levels decreased in hypoxi+adm group. adm levels in liver tissue were found decreased in hipoxia and control+adm groups than control group. hif 1-a levels were higher in control+adm group. adm has a role in angiogenic process, and our experiment showed that adm reacts earlier than hif 1-a, and affects its synthesis. organism increases its vascularization as a reaction to hypoxic condition, and adm treatment may provide a rapid adjustment. p-02.02.2-044 covalent conjugation and characterization of immunogenic protein of toxoplasma gondii and polyacryclic acid as vaccine candidate r. c ß akir koc ß yildiz technical university, department of bioengineering, istanbul, turkey toxoplasmosis is a major medical and veterinary disease caused by toxoplasma gondii which infect approximately half of the world's population. this infectious disease especially gains importance in pregnant women and immunodeficient individuals. also t. gondii infection has economic importance. however, there are only one attenuated-live t. gondii vaccine for veterinary uses and no vaccine against t. gondii is available for humans. therefore development of an effective vaccine would be extremely valuable for preventing disease in human and veterinary medicine. subunit vaccines are very attractive vaccine candidates but there is low antigenicity problem when they are used alone. polymers themselves don't stimulate immune response while they used with antigenic structure of various infective agents enhance immune response because when proteins are covalently conjugated with hydrophilic polymers, (1) their circulatory-lives and stability (in different ph and temperature values) enhance (2) binding to proteases and clearance by the reticuloendothelial-system decreases. in this study, immunogenic protein of t.gondii and polyacrylic acid with immune stimulant properties was covalently conjugated and conjugation was demonstrated by size-exclusion chromatography (sec) and fluorescence spectroscopy. it is significant to detect time of death in case of a sudden death for medical and legal concerns. there is no known method that can be used for post mortem time detection. based on this deficiency pmi detection in narrow time frame is a big problem. in this study, we aimed to investigate and determine timedependent expressional changes of apoptotic markers by western blot technique. 14 postmortem skeletal muscle were analyzed 12hour periods in first 36-hour after death. 2nd and 3rd 12-hour periods were statistically significant (p < 0.005). keywords: post mortem interval, time of death, apoptosis. hyaluronidases are excessively found in nature and involved in numerous biological functions. hyaluronidases primarily degrade hyaluronic acid (ha) and have significant role in fertilization during acrosomal reactions. therefore, the measurement of hyaluronidase enzyme activity may provide valuable information about acrosomal function and the fertilizing ability of the sperm. the aim of this study was to investigate the semen hyaluronidase enzyme activity changes among four different sheep breeds (akkaraman, suffolk, merino, and kıvırcık). in this research, ten ram testis tissues from each sheep breed, a total of 40, were cut and collected on ice. ovine testicular hyaluronidase of four different sheep breeds was purified from a crude ammonium sulfate-precipitated fraction of an extract of ram testis. the semen hyaluronidase enzyme activity differences between the sheep breeds were examined by spectrophotometrically monitoring the appearance of ha at 232 nm. analysis of variance test was used to examine the possible mean differences among the four different sheep breeds. the observed mean differences in enzyme units for kıvırcık, suffolk, akkaraman, and merino were as follows 891.60, 817.60, 729.40, and 681.60, respectively. the observed mean differences in absorbance values for kıvırcık, suffolk, akkaraman, and merino were as follows 0.4455, 0.4088, 0.3648, and 0.3408, respectively. the results showed that the observed mean differences in enzyme units and absorbance values among the four different sheep breeds were not statistically significant. despite that, in average kıvırcık had higher values for the activity of each sample and yet it had the smallest values for standard deviation. therefore, in order to achieve higher enzyme activity and more homogenous samples kıvırcık breed should be preferred. what is extra to learn from protein drying measurements? hydrations of soluble proteins are crucial for their functionality. therefore elucidating the details of protein hydration is still of interest in the proteins' action mechanisms. this is the motivation of the present study. in order to study protein hydration, changing concentrations of the well-studied serum albumin protein was measured with the spectroscopic techniques like uv-vis and ft-ir spectroscopy. spectral data is analysed and calculations were performed on the data to extract the relevant changes in the protein. experimental parameters' variation in association with the spectral changes implies the involvement of protein structure and hydrogen bonding in the drying process. the protein's reactions may not be merely a feature of the protein structure in the common sense but it could be related directly to the protein hydration states as well. this is understandable since it is already known that enzymatic proteins lose their functionality when they are dried while this drying may or may not involve dramatic structural changes. on the other hand, here it is claimed that the role of water in gaining the functionality that was lost in the dried state is not just about enhanced diffusion processes and the dynamicity but could be related to the functionality of water in the energy transfer processes as well. investigating the cellular effects of the aldoketo reductases akr1b1 and akr1b10 in hct-116 colon cancer cells b. taskoparan, e. g. seza, m. s. ceyhan, s. banerjee middle east technical university, ankara, turkey aldo-keto reductases (akr) are nad(p)h dependent oxidoreductases are best characterized as glucose reducing agents, and have been implicated in diabetic pathophysiology. increased expression of akr has been associated with tumors of lung, breast, prostate, cervix, ovarian and colon. two members of the akr superfamily that have been associated with different cancer types are akr1b1; aldo-keto reductase family 1, member b1, and akr1b10; aldo-keto reductase family 1, member b10. both are 36-kda cytosolic reductases that are similar in both amino acid sequence identity (71%) and tertiary structure with the (a/b) 8 barrel topology. while hct-116, a colorectal cancer cell line, cells expresses akr1b1 robustly, there is no expression of akr1b10. in this study, we have stably knocked down akr1b1 through shrna technology and overexpressed akr1b10 in hct-116 cells. comparisons were made with a known akr inhibitor sorbinil. with the knock down of akr1b1, we have observed reduced cellular proliferation, enhanced apoptosis, delay in cell cycle progression, reduced expression of mitogenic proteins and a decrease in activation of the inflammatory transcription factor nuclear factor kappa b (nf-kappab). interestingly, although akr1b10 overexpression did not affect cell proliferation, apoptosis or cell cycle, some effect was observed with nf-kb signaling. our data indicate that, although closely related, akr1b1 and akr1b10 have very different contributions towards signaling pathways in colorectal cancer. comparison of different nisin quantification methods and optimization of nisin production by lactococcus lactis z. girgin ersoy, g. demir, m. f. cesur, s. tunca gedik gebze technical university, kocaeli, turkey nisin, which is produced by certain strains of lactococcus lactis, is the only bacteriocin approved by world health organization (who) as a food additive. it prevents the growth of foodborne bacteria which cause food spoilage. nisin research and applications necessitates developing an accurate and reproducible method for its quantification. the agar diffusion bioassay is the most widely used method for quantifying nisin, although it has limitations especially diffusion-related difficulties of the active substance. in the present study, "agar diffusion bioassay", "enumeration of colony forming units", "colorimetric assay" and "flow cytometry" methods were compared with each other to determine antibacterial activity of nisin on micrococcus luteus. moreover, this study also covers the results about the effect of different cultural conditions to optimize nisin production by l. lactis. galactose, lactose and their combination in m17 medium (ph 7) boosted nisin production at 25°c, as the addition of 1.5 lg/ml hemin into the fermentation broth. to our knowledge, this is the first study showing the usage of "flow cytometry" method to determine nisin activity of fermentation broth filtrates. p-02.02.2-051 coronaviral nucleocapsid protein is an antiviral target for drug development institute of genomics and bioinformatics, national chung hsing university, taichung, taiwan between 2003 and 2004, the severe acute respiratory syndrome (sars)-cov caused a worldwide epidemic and had a significant economic impact in the countries affected by the outbreak. recently, the middle east respiratory syndrome human coronavirus (mers-cov) was found in patients with severe acute respiratory tract infections in the middle east and south korea. as is true for all coronavirus infections, there are no efficacious therapies currently available against coronaviral diseases, making the development of anti-coronavirus compounds a priority. the cov genome consists of positive-sense, single-stranded rna approximately 30 kb, and it contains several genes encoding several structural and non-structural proteins that are required for progeny virion production with a conserved order. the n proteins exist in the center of the viral particle and represent a helical structure complex. nucleocapsid protein is most abundant structural protein of covs, binds the viral rna genome to form the virion core, leading to the formation of a ribonucleoprotein (rnp) complex or to a long helical nucleocapsid structure, that is important for maintaining the rna in an ordered conformation for replication and transcription. the cov n protein is also involved in the regulation of cellular processes, such as gene transcription, interferon inhibition, actin reorganization, host cell cycle progression, and apoptosis. two strategies to inhibit oligomeric n protein function have been reported. the first strategy is to discover antiviral agents that target the rna-binding site. the second one is to impair normal n protein function by interfering with monomer-oligomer equilibrium. our recent studies suggest that n proteins in infections caused by coronaviruses will be useful antiviral drug targets because they serve many critical functions during the viral life cycle. post-translational modification of vascular endothelial growth factor (vegf) in colon cancer cells s. tunc ßer, e. solel, s. banerjee middle east technical university, ankara, turkey vascular endothelial growth factor a (vegf-a), commonly referred as vegf, is a potent secreted mitogen crucial for tumor initiation and progression. the gene for vegf is translated into a number of splice isoforms that lead to 121, 165, 189 and 206 amino acid proteins, with different receptor-binding and matrixbinding properties. in the present study, we discuss the functional significance of post-translational modification/processing of vegf 165 isoform in hct-116 colon cancer cells. we also focus on the role of calcium in the post-translational modification of vegf 165 . we show that vegf 165 undergoes n-linked glycosylation in hct-116 cells. perturbation of cellular calcium may affect vegf driven malignant phenotypes. p-02.02.2-053 novel methods for modulating the activity of bcl2 family proteins in apoptosis p. rowell, j. miles, a. wilson, t. edwards apoptosis, also known as programmed cell death, is an essential cellular process, but is implicated in several human diseases, including diabetes and cancer, when it is up-or down-regulated respectively. bcl-2 family proteins are major players in the control of intrinsic, or mitochondrial apoptosis; they respond to intracellular stress signals, function through protein-protein interactions and converge on the mitochondrial outer membrane to cause membrane permeabilisation, release of cytotoxic molecules, and initiation of an apoptotic cascade that leads to cellular demise. our work aims to identify molecules able to bind and modulate the activity of several key players in the bcl-2 family, including the pro-survival members bcl-2, bcl xl and mcl1, and the death promoting family member bax. adhirons, novel non-antibody peptide display scaffolds developed at the university of leeds, have been used to construct a phage display library containing over 10 10 clones, and form a key part of the strategy to identify such molecular modulators. adhirons able to selectively bind individual bcl-2 family members have been identified, in vitro assays carried out to test for modulatory activity, and xray crystallography used to elucidate details of how they interact with their target proteins. more recently, studies have been carried out to identify adhirons able to target multiple bcl-2 family members, with the aim of selectively inhibiting defined groups of proteins in cells. this work provides opportunities to differentiate the activities carried out by different bcl-2 family proteins in apoptosis, enabling us to better understand how their dysregulation contributes to human disease. biophysical and evolutionary study of the structural flexibility of adp-dependent sugar kinases from mesophilic and psychrophilic archaea r. zamora 1 , v. castro-fern andez 1 , c. a. ramirez-sarmiento 1 , e. a. komives 2 , v. guixe 1 1 facultad de ciencias, universidad de chile, santiago, chile, 2 department of chemistry and biochemistry, university of california, san diego, united states of america the capability of extremophiles microorganisms to live at low temperatures is mainly attributed to the high structural flexibility of its enzymes. several sequence and structure features have been associated to a high structural flexibility that enables metabolic processes to occur at low temperatures. during evolution, the general mechanism adopted by these enzymes has been to reduce the free energy of the transition state rather than the michaelis constant, k m . increased structural flexibility and decreased affinity for its substrates in psychrophilic enzymes is compensated by an increase in the catalytic rate, k cat . few psychrophilic enzymes have been reported to performance the optimization of their catalytic efficiency (k cat /k m ) by decreasing k m values. we use the adp-dependent kinase sugar family of archaea as a model, to identify particular structure and sequence features of a psychrophilic enzyme that would make this enzyme more flexible than their thermostable homologues. we characterize the bifunctional psychrophilic enzyme phosphofructokinase/glucokinase from methanococcoides burtonii (mbpfk-gk) and the bifunctional mesophilic enzyme phosphofructokinase/glucokinase from methanococcus maripaludis (mmpfk-gk) by spectroscopic, biophysical and computational techniques. the comparison showed that the absence of two ion pairs is primarily responsible for the increased structural flexibility accounted in the psychrophilic model. this increase in structural flexibility is reflected in the exponential increase in the k m values with temperature. additionally, we reconstruct the sequences of all ancestral enzymes between the current enzymes and their last common ancestor, which was used to trace the occurrence of these electrostatic interactions during evolution in the adp-dependent sugar kinase family. our results suggest that electrostatic interactions are a dominant feature in the transition from psychrophilic to thermophilic environments. fondecyt 1150460. elucidating the domain swapping mechanism of the forkhead domain of human foxp1 fox transcription factors control gene transcription during key processes, such as embryogenesis and immunity, and feature a conserved dna-binding domain known as forkhead. while most forkhead domains are monomeric, solved structures of members from the p subfamily (foxp) show that they can oligomerize by domain swapping (ds), a mechanism where protein segments are exchanged between subunits leading to an intertwined dimer. the biological relevance of ds in foxp has been stressed by disease-causing mutations that impair this process. however, for many proteins ds takes days to occur and requires global protein unfolding. thus, the current mechanism impedes a conciliation of the occurrence of ds of foxp1 in a biological context. here, we elucidate the biological feasibility of this process by biophysically characterizing the ds mechanism of the forkhead domain of foxp1 using size exclusion chromatography (sec), circular dichroism, and hydrogen-deuterium exchange mass spectrometry (hdxms). our results show that ds of foxp1 occurs at micromolar protein concentrations, within hours and is energetically favored. remarkably, dimeric foxp1 follows a threestate n 2 « 2i « 2u folding mechanism, where dimer dissociation into a monomeric intermediate (i) precedes protein unfolding, in contrast to other ds proteins. using sec and hdxms, we show that the i state of foxp1 largely resembles the native state, but has a larger hydrodynamic radius and a higher deuterium uptake in regions that maintain the compact monomer, suggesting that the i state is an 'open' conformation en route of ds. finally, we compared the local flexibility of the dimer and monomer of foxp1, showing that only the hinge region connecting ds segments exhibits different deuteration rates. our results show that ds in foxp1 follows an unusual threestate folding mechanism that proceeds through transient structural changes rather than needing protein unfolding as in most ds proteins. (fondecyt 1130510, 11140601). p-02.02.2-057 the sustained release of growth factor proteins following their implantation in tissue engineering j. jang bone tissue engineering has become a promising approach for bone regeneration. however, insufficient vascularization during bone regeneration, particularly with large bone defects, results in poor and unsustainable bone formation due to central necrosis. therefore, vascularization following implantation in vivo is essential to the successful formation of new bone tissue. we evaluated the release profile of vegf from bgs using a novel fluorescence-based retention assay, which revealed that vegf loaded on bgs can be released in a sustained manner without an initial burst (near zero-order cumulative release) with a controlled release rate of 13.6% per week for up to 7 weeks. in contrast, an elisa-based release assay showed vegf to have an early burst-release profile for the first week. however, the biological activity of vegf released from the bgs was preserved over the 7-week release period, which is consistent with the sustainedrelease profile observed in the fluorescence-based retention assay. we developed a novel fluorescence-based retention assay to evaluate the release of vegf from bgs. this fluorescence-based retention assay, which detects the vegf that remains on bgs, reveals that vegf loaded on bgs can be released in a sustained manner, with a minimal initial burst, for up to 7 weeks. these results indicate that the sustained biological activity of the vegf released from bgs over the full 7-week period promotes bone regeneration, and suggest its potential use for bone tissue engineering. irisin is a recently discovered myokine which regulates energy metabolism and is associated overweight. we aimed to evaluate serum irisin levels in the patients with morbid obesity. a total of sixty patients with morbidly obese and thirty healthy control subjects were included in this study. all participants were measured body weight and height, the lipid profile, and plasma glucose, hba1c, insulin and irisin levels. irisin levels were measured by elisa method. serum irisin levels were significantly lower in morbidly obese patients than healthy controls (p < 0.05). there was no statistically significant difference in terms of age or gender. serum irisin was negatively correlated with bmi, insulin levels, and homa-ir (p = 0.006, p = 0.046, p = 0.048, respectively). our study revealed lower irisin levels in morbidly obese patients with respect to control subjects. the lower irisin levels observed in morbidly obese patients might suggest a loss of browning of subcutaneous adipose tissues. p-02.02.2-059 pka inhibition restores adenosine uptake in renal tubular epithelial cells under high d-glucose conditions w. garrido, j. catal an, s. alarc on, r. san mart ın universidad austral de chile, valdivia, chile introduction: diabetic nephropathy (dn) is the leading a cause of end-stage renal failure whose pathogenesis must to be elucidated. the progression of dn has been associated with elevated levels of adenosine. extracellular adenosine availability is regulated by the activity of the equilibrative nucleoside transporters (ents). due to the ents have putative sites of phosphorylation our objective was to evaluate the role of pka and ck2 kinases on ents activity. methods: adenosine uptake (10 lm adenosine, 60s, 22°c) was assayed in hk2 cells preincubated with 5 mm or 25 mm d-glucose for 24 h and exposed to 10 lm 8-br camp, 10 lmh89 or 100lm tbb for 1 h. plasma membrane and intracellular proteins were fractionated by the biotinylation method and ent1 and ent2 contents were quantified by western blot. results: high d-glucose in hk2 cells inhibited the uptake of adenosine. this effect was reversed using a pka inhibitor (h89) through an increased ent2 uptake activity. we noticed this pka inhibitor did not regulate the plasma membrane localization of ent1 or ent2 under normal d-glucose (5 mm) or high d-glucose conditions (25 mm). also, we saw that tbb (ck2 inhibitor) decreased the activity of ent1 and ent2 under normal glucose conditions, decreasing the localization of ent1 at cell surface, while the membrane localization of ent2 decreases under the effect of tbb and high d-glucose conditions. conclusions: pka inhibition reversed the effect of high d-glucose, increasing the uptake of adenosine mediated by ent2. this could be a new target for the restoration of adenosine levels in dn. relation between serum lipo (a), plasma fibrinogen, red cell distribution width and mean platelet volume in healthy adult men the aim of this study was to investigate the relationship between the serum lipo (a) and plasma fibrinogen, red cell distribution width (rdw) and mean platelet volume (mpv) in healthy adult men. for this purpose, 37 healthy adult men have normal physical examination and laboratory findings and not use any drug were included to the study. serum lipo (a) levels and hematologic parameters (fibrinogen, rdw-sd and mpv) were measured by autoanalyzer and commercial kits. the mean of the age of the persons was 27.2; body mass index was 24.2; serum lipo (a) level was 0.21 and plasma fibrinogen level was 1.61. there was significant positive correlation between the serum lipo (a) and plasma fibrinogen levels (r = 0.246; p = 0.025), significant positive correlation between the serum lipo (a) and rdw-sd (r = 0.267; p = 0.004) and significant negative correlation between lipo (a) and mpv (r= à0.205; p = 0.027). the plasma fibrinogen and the serum lipo (a) levels have been known as the risk factors for cad (coronary artery disease) increase together in healthy adult men. similar findings have been reported in cad patients. it has reported that elevated rdw is associated with intracoronary thrombotic burden and may be associated with the severity and instability of acute myocardial infarction. in addition, mpv is predictor of severe atherosclerosis and may be used for the prediction and identification of cardiac risks in cad patients. our findings show that elevated rdw and decreased mpv may predict to increased risk of cad in the future, in healthy adult men. follicular fluid is rich in peptides, which significantly influence the growing oocyte. due to existence of a link between kisspeptin (metastin) cells and gonadal steroids kisspeptin might manipulate the gonadotropin axis and folliculogenesis. in this context, the study was planned to investigate for the first time that the follicular fluid (ff) and serum concentration of kisspeptin in high and poor responders undergoing in vitro fertilization (ivf)/intracytoplasmic sperm injection (icsi). biological samples were collected from twenty infertile women with polycystic ovary syndrome (pcos) and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone (gnrh) antagonist protocol for ivf/icsi treatment. kisspeptin concentrations were measured in serum and follicular fluid by using elisa, whereas fsh and lh levels were detected by routine laboratory methods. it was found that kisspeptin levels were significantly lower in serum and follicular fluid of infertile women with pcos. kisspeptin levels were correlated with fsh and lh level in infertile women with pcos. it can be concluded that low level of kisspeptin might inhibit gnrh release that might cause to the inhibition of fsh and lh release and might disrupt folliculogenesis. decline in serum and ff levels of kisspeptin might be possible cause of anovulation and subfertility in pcos subjects. cryptococcus albidus d24 is a newly identified yeast isolates from petroleum area in _ izmir as a lipase producer. the molecular weight of the enzyme is 36.31 kda as found. optimum temperature was 40°c and half-life times were 180, 27, 9 and 0.59 min at 30, 40, 50 and 60°c, respectively. optimum ph value was 8.0. however, it shows significant ph stability at ph values 4.0 and lower. the existence of acetone in the solution as a solvent enhanced lipase activity. cryptococcus albidus d24 lipase was able to catalyze the esterification reaction between fructose and palmitic acid to produce fructose palmitate using acetone as the solvent. due to its stability in organic solvents, we propose that in order to increase the yield of fructose palmitate, we could immobilize d24 lipase. therefore, the effect of immobilization on kinetic parameters of d24 lipase was investigated. different immobilization materials and methods were used to find efficient support materials for d24 lipase immobilization. additionally, fructose palmitate production processes will be optimized with immobilized lipase. introduction: the diagnosis of osteoarthritis (oa) is based on clinical symptoms and radiographic findings. it is known that the pathologic changes at the molecular level in the joint cartilage tissue start before symptoms appear in oa. c1q tumor necrosis factor-related protein 1 (ctrp-1), c1q tumor necrosis factor-related protein 3 (ctrp-3) and kallistatin are related to many different cellular processes including bone and cartilage tissue metabolism. the aim of this study was to investigate any probable association between the serum ctrp-1, ctrp-3 and kallistatin levels and diagnosis and radiologic staging of knee oa patients. materials and methods: this study included 60 patients with knee oa and 30 healthy individuals for control purposes. the patient group was divided into four stages radiologically. ctrp-1, ctrp-3 and kallistatin levels were measured in serum samples of patient and control groups with elisa method, and the differences between the groups were analyzed with statistical methods. results: the levels of serum ctrp-1 in the patient group were significantly higher than in the control group (p = 0.001), serum ctrp-3 and kallistatin levels were not statistically different (in order of p = 0.251, p = 0.160). in the patient group, there was not a significant difference between serum ctrp-1, ctrp-3 and kallistatin levels and radiologic stages (respectively p = 0.811, p = 0.715, p = 0.202). there was a significant positive correlation between the radiologic stage and patient's age, body mass index, western ontario and mcmaster universities arthritis index and visual analogue scale values (respectively p = 0.001, r = 0.510; p = 0.010, r = 0.331; p = 0.001, r = 0.683; p = 0.001, r = 775). discussion and conclusion: serum ctrp-1 levels were detected significantly increased in patients with knee oa, but there was no significant difference in ctrp-3 and kallistatin levels. there was not a significant association between the radiologic stage and levels of ctrp-1, ctrp-3 and kallistatin. enzymes in microorganisms, especially termophilic bacteria are more attractive in biotechnology and molecular biology due to the higher catalytic activity. turkey is rich in geothermal resources and it is important to determine unknown microbial content of geothermal sources. in this study, water and sludge samples were taken from ayder, kizilcahamam and gonen hotsprings. firstly, ph, temperature, salt concentration, gram reaction, mobility, endospore formation, oxidase and catalase tests were carried out as conventional characterization. molecular characterizations of isolates were achieved by fames, rep-pcr and 16s rrna sequencing. finally, test isolates were evaluated according to enzyme production capability by petri dish. as result of conventional tests, isolates were determined as gram positive, mobile-rod shaped, aerobic, oxidase, catalase and endospore positive. the growth range for ph and temperature of the isolates were determined as 5-9 and 50-65°c. in consequence of the salt test, the test isolates were grown at 2-10% nacl. 19 of thermophilic isolates were selected by rep-pcr and according to 16s rrna sequencing analysis test isolates were belonging to bacillus, geobacillus, anoxybacillus and brevibacillus genus at a range of 98-99%. enzyme tests showed that, some of the isolates were able to produce protease (f17, f31, f76, f6, f99 and f19), amylase (f41, f76, f42, f98 and f10), cellulase (f17, f41, f31, f6, f99 and f19), xylanase (f17, f31, f76, f99 and f19) and lipase (f17, f31, f6, f99 and f19). it can be concluded that, geothermal regions are rich in bacillus and related genera. fame analysis was particularly insufficient for diagnosis of thermophilic microorganisms, but rep-pcr was successful in separation of organisms at species and even subspecies levels. most of our bacterial isolates have industrially important enzyme production capacities. it is a pioneer result to use bacteria for industrial applications which need higher temperatures. p-02.02.2-065 warburg effect was investigated by studying various metabolic molecules and assays in mammalian cell lines z. i. kanbagli, b. kiratli, h. cimen yeditepe university, istanbul, turkey majority of the different cancer cells switch their metabolism from oxidative phosphorylation pathway to glycolytic pathway; in order to meet excessive energy requirement, which is also called warburg effect. acetylation is one of the most crucial post-translational modifications playing key roles in metabolic reprograming. in this study, the relationship between acetylation dependent changes in energy metabolism and apoptotic pathways were investigated in pnt1a, du145, hela, hep3b, hek293t, shsy5y. immunoblotting experiments were applied by using antibodies against acetylated-lysine to examine the changes in overall acetylome. candidate proteins displaying elevated acetylation was identified with mass spectrometry based-proteomic analysis. glucose transporter 4 (glut4) was used to detect insulin-stimulated glucose transport, total oxphos rodent antibody cocktail to identify variations in complexes which are responsible for most of the atp production. caspases (casp-3, -9) to unreveal different activation levels of apoptotic pathway among the cell lines. mitochondrial membrane potential was measured by using rho-damine123 by employing confocal microscopy. the expression level of respiratory chain complex iv subunit mtco1 and casp-3 was higher in hek293t compared to other cell lines. casp-9 was upregulated in cancer cell lines, mostly in hep3b. glut-4 levels were downregulated in cancer cell lines in contrast to healthy cell lines. findings imply that these proteins might have significant roles leading to variable metabolic and apoptotic activity of each cell line during energy production. due to the results, mtco1 might be important in adaptation of different cell lines to regulate the overall respiratory chain complex activity. reduction in glut4 level demonstrates insulin desensitization in cancer cell lines, which might lead to metabolic defects in these cells. besides, since p53 has a repressive effect on glut4, it also can lead us to study about p53 levels. the effect of inhibition of pi3k/akt/mtor signaling pathway on receptor tyrosine kinase expression in breast cancer cells g. tunali, a. l. dogan department of basic oncology, hacettepe university cancer institute, ankara, turkey the increased expression and activation of receptor tyrosine kinases (rtk) (egfr, her2, her3) play important roles in breast cancer pathogenesis. her2/her3 interaction is the most potent heterodimer and it causes oncogenic pi3k/akt activation. inhibitors of pi3k/akt pathway (akt inhibitor and pi3k/mtor dual inhibitors) lead to increase in rtk levels and activities while blocking signaling pathway. in this study, the time dependent effect of dual pi3k/akt inhibitor pi-103 on receptor tyrosine kinase expressions' in breast cancer cells was investigated. two breast cancer cell lines, mda-mb-468 cells (which has egfr overexpression and pten deficiency) and skbr-3 cells (which has her2 overexpression) were evaluated for the effect of dual inhibitor. these cells were treated with dual pi3k/akt inhibitor pi-103 for different time periods (1-24 h) . egfr, her2 her3 total rtks expression and pi3k/akt pathway inhibition (p-akt and p-p70s6k expression) were evaluated by western blot. in mda-mb-468 cells, there were significant decrease in p-akt and p-p70s6k proteins' expression during the first 3 h. this inhibition was followed by reactivation of the signaling pathway after 6 h. in skbr-3 cells, p-akt and p-p70s6k proteins' expression were significantly decreased during the first 6 h. the pi3k/ akt signaling pathway in these cells were reactivated after 12 h. basal expression of egfr and her3 in mda-mb-468 cells and basal expression of her2 and her3 in skbr-3 cells were found to be very high. transient inhibition of akt and mtor protein kinase activation in tumor cells followed by reactivation of signaling pathway did not result in a time-dependent difference on egfr, her2 and her3 expression levels. these results suggest that dual pi3k/mtor inhibiton by pi-103 may trigger receptor tyrosine kinase reactivation due to the signal distruption without affecting total protein expression level. site-specific bioorthogonal reactions are one of the significant tools for discovering different aspects of biological systems in live cells. the reactions should be highly stable and rapid in physiological conditions. various chemical tools can be used in bioorthogonal reactions to monitor biological systems, therapeutics, microscopy and diagnostic applications in live cells. synthetic covalent chemistry in the study of biological systems has been used to label biomolecules selectively in their native environment. for example, small synthetic fluorophores can be added to biomolecules without disturbing other molecular biological pathways. aldehydes or ketone-based functional handles can be attached onto protein at specific sites via chemoenzymatic reactions. labeling of carboxy terminus of a-tubulin has been successfully studied in our previous studies by replacement of wild type tyrosine with unnatural amino acid 3-formyltyrosine in the presence of tubulin tyrosine ligase enzyme (ttl)-as its role can suggest whether certain cancer cells might grow more aggressively than others. in this work, we highlight the synthesis and spectroscopic properties of azacoumarin chemical probes to study tubulin-tubulin tyrosine ligase (ttl) system in live cells. significant increase in fluorescence quantum yield or a red shift on absorption and emission maxima is observed when the conjugated product is formed. bioorthogonal fluorescent labeling is such a favorable reaction to perform rapid kinetics, localization and high site-specificity in cell environment. newly synthesized azacoumarin fluorophores should therefore not only be useful for studying ttlbased biological systems, but also would enable broad range of high-yielding and fast diagnostics for future biolabeling applications in biochemistry, cell biology and beyond. binase is an extracellular ribonuclease from bacillus pumilus which shows antiviral and antitumor activities in cell cultures. however, the action of binase on intracellular functions and processes has not yet been identified. here, for the first time we report the whole transcriptome analysis of binase-treated human lung adenocarcinoma epithelial a549 cells. a plasmid-based reverse genetics system and colorimetric cell viability assay were used to identify the binase internalization and binase cytotoxicity towards a549 cell line, respectively. for the whole cell transcriptome analysis a549 cells were treated with 100 lg/ml of binase for 24 h followed by mrna extraction and library preparation. sequencing was performed on solid 5500xl wildfire next-generation sequencer. we found that binase internalized into a549 cells after 2 h of incubation. the binase at 100 lg/ml was absolutely non-cytotoxic towards a549 cell line and was active in the cell culture medium during 48 h incubation. the analysis of rna-seq data showed that among 13 thousands of protein coding transcripts 791 transcripts were up and down regulated by binase, among them 79 transcripts were induced and repressed. binase repressed the production of s100a16 and tnxb which act cancer biomarkers, scn8a and drd4 which play a crucial role in cancer metastasis and responsible for pediatric tumors, respectively. the induction of transmembrane protein transcript abcb11 by binase can help binase to internalize into the cell as abc transporters are often account for transporting drugs across the cellular membrane. binase induced the production of nlrp3, rasgrp1and alpk2 transcripts which can activate apoptosis, cytokine or t cell activation in cancer cells. thus, binase exerts different effects in cancer cells. the rnaseq data obtained will help to understand the mechanism of binase anticancer action. .72) is a specific group of phosphatases capable of hydrolyzing myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate) with the formation of less phosphorylated inositol derivatives (from mono-to pentaphosphate). three major types of phytases are recognized on the basis of the first phosphate group hydrolyzed by the enzymes: 3-phytase, 4/6-phytase, and 5-phytase. due to the stereospecific way of phosphate release from the phytate molecule by the action of phytases, these enzymes by themselves and their composition may serve as a potential alternative for production of myo-inositol phosphate isomers with therapeutic properties. chemical synthesis of these compounds is inefficient and costly. pantoea sp. strain 3.5.1 showing high phytase activity was isolated from the forest soil sample of the republic of tatarstan, russia. in this study the main objective was the cloning and expression of pantoea sp. 3.5.1 phytase gene in e. coli. first, we amplified the phytase gene (agpp) from the genomic dna of the bacteria using specific primers phmh_dir and phmh_rev. size of phytase gene corresponded to 1729 base pairs. during the optimization of amplification conditions it was found that the optimum temperature for primer annealing was 66°c. this temperature increases the specificity and efficiency of annealing. then the pcr-product of agpp gene was cloned into the pbad myc/ his vector first. on the next step we carried out subcloning of the agpp into a pet28a expression vector. multicopy plasmid pet28a/agpp contained the sequence of the phytase gene of pantoea sp. 3.5.1 under t7 promoter. the corresponding recombinant protein was expressed in e. coli as a fusion with a 6 his-tag and was detected by western blotting. recombinant phytase was purified via affine chromotography on the ni-nta column and displayed high phosphomonoesterase and phytase activities. bag-1 (bcl-2 associated athanogene) is a multifunctional protein that interacts with diverse array of cellular targets and modulates a wide range of cellular processes, including proliferation, cell survival, transcription, apoptosis, metastasis and motility. in human cells bag-1 exists as three major isoforms (bag-1s, bag-1m and bag-1l) derived by alternative translation initiation from a single mrna, which allows interactions with various molecular targets such as hsp70/hsc70 molecular chaperones, components of the ubiquitylation/proteasome machinery, bcl-2, raf-1 kinase, nuclear hormone receptors and dna. our work aims to investigate how altered bag-1 expression levels affect cell survival in mda-mb-231 (er, pr and her2/neu negative) breast cancer cell lines. we first cloned bag-1l gene to a cloning vector, later we transfected mda-mb-231 cells for overexpression of bag-1. we also used bag-1 sirna to silence bag-1 gene. western blot analysis was applied to demonstrate relative expression levels of bag-1, its interacting partners and certain proteins which are important for apoptosis pathway. we performed xtt cell viability assay for bag-1 overexpressed cells to checkbag-1's impact on cell survival, and observed enhanced survival rates on cells compared to that of the untreated cells with bag-1 overexpression. in addition, our study revealed that once bag-1 forms a complex with c-raf/b-raf/hsp70/akt/bcl-2, modulation of cell survival was observed. we believe that once the exact localization and involved molecular mechanisms of bag-1 and its isoforms are found, the role of each bag-1 isoform in cell survival can be understood better. this can further provide routes to study tumor development. the aim of this study is testing the recombinant glp1 encapsulation into a biocompatible material. we also tested if it can be a therapeutic candidate drug for type 2 diabetes. the incretin hormones, which are also named as endogenic peptide hormones have become a more attractive therapy for type 2 diabetes because of different physiological effects. in circulation, glp1 is cleaved by ddp4 in a very short time. if glp1 can be protected from cleaving, the effective time of glp1 would be increased and by this way it can replace the therapy of insulin. chemical synthesis methods of peptides are limited because of low efficiency and high cost. the production of peptides by recombinant e. coli is an alternative way because of effective production, simplicity and low cost. however, the major disadvantage derived from the recombinant e. coli is the frequent formation of inclusion body. for that reason, extra methods are needed for obtaining soluble recombinant peptides. glutathione s-transferase (gst) tag is commonly used as affinity and solubility tag to improve the solubility of recombinant peptides. in this study, we cloned and heterologously produced glp1 using the gst fusion system in e. coli. affinity purification of recombinant protein was achieved by using glutathione immobilized columns. characterization of the gst-tagged glp1 was performed by sds-page. the purity of fusion protein was found to be 65%, as confirmed by glp1 elisa kit. then, the fusion protein was encapsulated in a chitosan coated polygalacturonic acid. the different ph stability and in vitro release tests also in different phs was studied. morganella morganii is an opportunistic pathogen capable of causing a wide range of clinical infections. it is known that microbial metalloproteases play an important role in the development of bacterial infections. thus, investigation of m. morganii metalloproteases has a particular interest. bacteria were grown in lb medium at 37°c. as a bioinformatics tool blast was used. for molecular biological experiments, thermo scientific kits and sibenzyme enzymes were used. pbad/myc-his plasmid was used as an expression vector. bacterial transformation was carried out by heat shock method. bacterial cells were disrupted by sonication. gene expression products were analyzed by western blotting. to analyze the actinolytic activity of bacterial extracts sds-page electrophoresis was used. the putative metalloprotease gene (an cp004345.1) has been found in the genome of annotated strain of m. morganii kt. its amino acid sequence has partial homology (37%) with actin specific metalloproteases grimelysin from s. grimesii and protealysin from s. proteamaculans. using specific primers the gene with 99% homology was identified in the genome of clinical isolate of m. morganii 4. rt-pcr analysis showed that this gene had the maximum expression at 48 h of growth. in addition, the cellular extract of m. morganii 4 had small actinolytic activity. cloning of the gene into e. coli dh5a cells led to the synthesis of the 35 kda protein. it is known that the highest expression of serratia proteases is observed at 48 h of growth, and the molecular weight of the mature proteins is 32 kda. it was shown that metalloprotease gene of m. morganii 4 expressed at the same time of growth. moreover, the recombinant e. coli cells synthesized protein with the similar weight (35 kda) which perhaps is a mature form of the metalloprotease from m. morganii. as a result, in the genome of m. morganii 4 the metalloprotease with similar properties to grimelysin and protealysin proteases was identified. the preliminary characterization of p-ii like protein glnk from lactobacillus brevis z. iskhakova 1 , d. zhuravleva 1 , a. laykov 1 , k. forchhammer 2 , a. kayumov 1 1 kazan federal university, kazan, russia, 2 eberhard-karls university tuebingen, tuebingen, germany the p-ii proteins in bacteria, archaea and plants regulate the activity of a variety of proteins in response to specific metabolic signals which affect their structural state and interaction ability. among various bacteria belonging to lactobacillus only some species have genes encoding pii protein in the genome. here we report the preliminary characterization of pii-like protein lbrglnk from lactobacillus brevis. while the amino acid sequence alignment revealed only 50-70% homology of lbrglnk with other well studied pii proteins, lbrglnk also has the atp binding motive gdgk. trimeric structure of lbrglnk was confirmed in vitro by size exclusion chromatography, suggesting possible similarities of lbrglnk properties with pii proteins. the isothermal titration calorimetry revealed a preferential binding of adp (kd = 50 lm) over atp (kd = 357 lm) suggesting that they compete for binding to lbrglnk. neither 2-oxoglutaric acid nor other nucleotides were interacting with lbrglnk in itc measurements. the mutation gly91ala in the atp binding motive completely abolished the interaction with both adp and atp. the pull down of lbrglnk with l. brevis cell extract allowed identification of chaperonin grol, transketolase and glnr-like transcriptional regulator from merr family as most probable partner proteins for interactions with lbrglnk. this work was supported by the russian foundation for basic research (project no. 15-04-02583a background: hemophilia is a bleeding disorder due to the deficiency in coagulation factors viii (hemophilia a) or ix (hemophilia b). hemophilia patients are essentially treated with intravenous replacement of the missing or dysfunctional factors fviii or fix by recombinant proteins. these therapies often induce the generation of acquired antibodies, and thus, novel approaches are needed. most recent hemophilia strategies target the tissue factor pathway inhibitor (tfpi), which is the major inhibitor of the coagulation cascade, particularly of the extrinsic tenase complex. anti-tfpi agents have been empirically developed such as aptamers, peptides, monoclonal antibodies. we have followed a structure-based strategy, to design a mutated fxa that would show more affinity for tfpi, and thus trap this inhibitor. tfpi exists as two isoforms are folded as multi-kunitz domains related by linkers. the second kunitz type domain of tfpi (tfpi-k2) is known to bind the catalytic site of fxa. methods: the molecular complex of tfpi-k2-fxa was modeled and submitted to molecular dynamics (md), allowing the identification of low-spots interaction. modified fxa with theoretically stronger interaction with tfpi-k2 were predicted using md. the mutants and wild type proteins were expressed in hek cells, and their processing status was checked. they were tested by western blotting, by chromogenic activity using a specific substrate of fxa, by thrombin generation assay in fviii depleted plasma. finally, their binding to a tfpi-k2 peptide array was compared. results: the mutants showed better efficiency to restore thrombin generation in plasmas from hemophiliacs and displayed stronger binding to tfpi-k2 than the wild type fxa. conclusions: the proof of concept of the synergistic approaches of md and mutagenesis was obtained and an efficient tfpi trap was designed. the mutated fxa is a candidate for a new hemophilia therapy. organophosphorus acid anhydride (op) nerve agents are potent inhibitors which disrupt the mechanisms of neural transmission. organophosphorus acid anhydrolase (opaa; e.c.3.1.8.2) is a class of enzyme that potentially acts on phosphorus anhydride bonds, reported intracellularly in diverse organisms, albeit notably the enzyme belongs to alteromonas species are more extensively studied. whereas mass-transfer problem is a major issue in native whole cell biocatalysis, new anchor system derived from the n-terminal domain of ice-nucleation protein from pseudomonas syringe inav (inav-n) was used for the first time to display opaa onto the cell surface. tracing of the recombinant protein and its activity assay showed a successful presentation of opaa and its significant ability for biodegradation of organophosphorus compounds. further studies on bacterial fractions confirmed that opaa is remarkably located on the outer membrane. the specific activity of recombinant bacteria to degrade diisopropylfluorophosphate (dfp) was measured at 260 u/mg of cell wet weight, which almost all was observed in the outer membrane fraction. recombinant cells could also degrade chlorpyrifos (cp) compound in 195.6 u/mg activity. it can be concluded that inav-n anchor is efficient for targeting opaa on the cell surface and can effectively eliminate the masstransfer problem in native whole cell bioconversion system. proper spatial and temporal organization of proteins involved in cell signal transduction is crucial for the specific and efficient information transfer. scaffold proteins coordinate the action of signaling molecules by their physical binding and organization in multiprotein complex assemblies. multiple protein binding is often mediated through intrinsically disordered regions of the scaffold, where the interaction epitopes are defined by linear peptide motifs. using a hub scaffolding protein axin as a paradigm, we have employed peptide microarray technique to identify the binding epitopes for axin interaction partners at high resolution. this enabled us to design axin-derived peptides corresponding to the respective binding epitopes that compete for the interaction in vitro. by transfection of chemically stabilized competitive peptides directly into the cells, we have shown the effect of specific interaction blocking on axin-mediated signaling in vivo. our data demonstrate a proof of concept for a rational design of inhibitors of protein-protein interactions that allow specific intervention with single function of the targeted protein (i.e. recruitment to the axin complex). contrary to the inhibitors that completely disrupt the protein function (e.g. inhibition of a kinase catalytic site), this approach provides a tool for investigating specific action within the axin complex, while the other cellular functions of the targeted protein remain preserved. the results of this research have been acquired within cei-tec 2020 (lq1601) project with financial contribution made by the ministry of education, youths and sports of the czech republic within special support paid from the national programme for sustainability ii funds. de novo design of an artificial biocatalyst using immunoglobulin template became rather routine procedure due to the achievements of molecular biology and crystallography. recently the 'reactibody' approach was developed based on the chemical selection of catalytic repertoires from immunoglobulin library followed by expression of these biocatalysts in eukaryotic system. in this study we structurally characterize the a5 antibody, its kappa and lambda variants, in order to understand the difference on the active site between a5 and a17 which although there are two antibodies sharing very high homology and sequence identity their active residues are located in a different region of the antibody. the structures of the a17 antibody kappa and lambda variants have been already determined, there was no structural information though about the a5 antibody. the structural analysis revealed dramatically different angle in position of nucleophilic residue tyr33 and area of solvent accessible surface. the structural difference of active center reflects on kinetics of the a5 organophosphate modification. both variants of antibody bind with organophosphate throw induce-fit mechanism, but rate of the step of induce fitting is different (k obs are 35 s à1 and 16 s à1 for a5kappa and a5lambda respectively). this observation may hint at novel means of regulation of velocity and specificity of artificial biocatalysts. this study was supported by grant #rfmefi61614x0009. translation elongation factor 1ba (eef1ba) is a component of a heavy form of translation elongation factor 1 (eef1h). it functions as a catalyst of gdp/gtp exchange in translation elongation factor 1a (eef1a) restoring its active conformation appropriate for aminoacylated trna binding. eef1ba forms a tight complex with translation elongation factor 1bc (eef1bc) via the n-terminal domain, while its c-terminal domain executes the catalytic activity. eef1bc has been shown to enhance the attributed to the c-terminal domain catalytic activity of eef1ba. this suggests that the eef1ba n-terminal domain may influence the guanine nucleotide exchange process. to test this hypothesis we prepared a set of n-terminal truncated forms of human eef1ba and checked their activity in the guanine nucleotide exchange assay on both isoforms of eef1a, eef1a1 and eef1a2. we showed that recombinant eef1ba is a non-globular monomeric protein in solution with an elongated shape by analytical ultracentrifugation approach. the truncation of the dispensable for the catalytic activity n-terminal domain of eef1ba resulted in significant acceleration of the rate of guanine nucleotide exchange in eef1a2 comparing to full-length eef1ba. similar effect on the catalytic activity of eef1ba was observed after its interaction with eef1bc. in contrast, the effect of full-length eef1ba and its truncated forms on the rate of guanine nucleotide exchange in eef1a1 was similar but relatively modest compared to eef1a2. this can be explained by higher rate of spontaneous gdp dissociation from eef1a1 comparing to eef1a2 and lower affinity of eef1a1 to eef1ba. thus, we propose that the n-terminal domain of eef1ba via flexible linker region may interfere with eef1a binding to the cterminal catalytic domain that results in a decrease of the overall rate of guanine nucleotide exchange reaction. the formation of a tight complex between eef1bc and eef1ba n-terminal domains abolishes this inhibitory effect. p-02.02.2-079 assessment of quantitative proteomics results in large-scale data-independent with fragmentation spectra reproducibility measure reduces variation and allows to use lowintensity signals organisms with reduced genomes that lack the vast majority of transcriptional or translational regulation systems tend to adapt to changing environment with a variety of subtle changes in protein abundances. as soon as relative changes for most proteins fall below 50%, the power of traditional label-free proteomic analysis rapidly becomes insufficient for robust profiling of hundreds of samples. intoduction of frament-by-fragment and sample-by-sample signal quality assesment in mrm and dia experiments helps to increase accuracy of methods and at the same time reintroduce cases which could have been excluded during bulk quality assessment due to lower signal-to-noise ratio for several fragments. 240 samples of mycoplasma gallisepticum were acquired in data-independent manner on sciex tripletof 5600+ mass-spectrometer (swath acquisition) during the year. the samples were produced from mycoplasma gallisepticum culture cultivated at different temperatures. the signals for each fragment were extracted with vendor-supplied software with the theoretical fragment ions for each peptides instead of spectral library. the results were used for relative protein quantitation in two manners the first conventional method included direct use of peptides with top 3 most intense signals. the second included selection of peptides and ions for quantification for each pair of samples based on the reproducibility of fragmentation patterns after computing the areas of chromatographic peaks for each ion. spectral angle was used as a distance measure for fragmentation patterns for clustering. further, a base set of detected ions was selected for each peptide and a subset for comparison of each pair of runs. the first method resulted in quantitation of 354 proteins across all samples with variation across lc-ms replicates was 21% on average, and the second approach led to quantitation of 515 proteins in total, 390 of them across 75% of samples, all with the variation about 11% on average. p-02.02.2-080 interaction of plasminogen fragments k 1-3 and k 5 with fibrin fragment dd t. yatsenko, v. rybachuk, l. kapustianenko, s. kharchenko, o. yusova, t. grinenko palladin institute of biochemistry of nas of ukraine, kyiv, ukraine introduction: plasminogen interaction with specific binding sites in c-terminal d-domains of fibrin molecule initiates the activation process of proenzyme and subsequent fibrin clot lysis. the sites are exposed under fibrin polymerization. plasminogen kringle domains ensure the proenzyme interactions with fibrin clot. in this study, we investigated the binding of human plasminogen kringle fragments k 1-3 and k 5 with human fibrin fragment dd and their effect on glu-plasminogen interaction with dd. results: kringle-containing fragments k 1-3 and k 5 reduce plasminogen activation by tissue-type activator on fibrin fragment dd. the level of glu-plasminogen binding to dd is decreased by 50-60% in the presence of k 1-3 and k 5. fragments k 1-3 and k 5 have high affinity to fibrin fragment dd (dissociation constant is 0.02 lm for k 1-3 and 0.054 lm for k 5). analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with c-c-chains of fragment dd. k 1-3 interacts with complex of fragment dd-immobilized k 5 as well as k 5 with complex of fragment dd-immobilized k 1-3. the plasminogen fragments do not displace each other from binding sites located in fibrin fragment dd, but can compete for the interaction. analysis of k 1-3 and k 5 binding to fibrin fragment dd with reduced disulfide bonds showed the interaction of both plasminogen fragments with aand c-chains of fragment dd. conclusions: widely known specific plasminogen-binding site located in aa148-160 region of fibrin molecule is not a single binding sequence of fibrin peripheral domains or plasminogenbinding site is not linear and contains amino acid residues from other polypeptide chains of fibrin d-domains. fibrin fragment dd contains different binding sites for plasminogen kringle fragments k 1-3 and k 5, which can be located close to each other. possible plasminogen kringle-binding sites are located in aand c-chains. p-02.02.2-081 implementation of budded baculovirus particles for characterization of ligand binding to g protein-coupled receptors a. allikalt, a. rinken university of tartu, tartu, estonia g protein-coupled receptors (gpcrs) constitute the largest class of membrane receptors involved in regulation of signal transduction into the cell in response to various extracellular stimuli. for that reason, gpcrs have become important targets for variety of drugs. as these receptors are present in native tissues at very low concentrations, efficient recombinant expression systems are needed to produce sufficient amounts of protein. we have shown that budded baculovirus particles, which display gpcrs on their surfaces can be used as a source of receptors for the investigation of ligand-receptor interactions. this expression system can be used for radioligand binding assay as well as for fluorescence anisotropy-based assay (fa). we have validated the system with budded baculovirus particles produced in spodoptera frugiperda (sf9) cells expressing human dopamine d 1 receptors using [ 3 h]sch-23390 and bodipy-fl-skf-83566 as reporter ligands for corresponding assays. this system has many advantages, for example good signal to noise ratio, homogeneity of the receptor, high expression levels and long-term stability of the receptor preparation. fa method allowed real-time monitoring of reporter ligand binding in the absence and presence of different dopaminergic ligands, giving information about their kinetic properties. association, as well as dissociation of the bodipy-fl-skf-83566 itself were rapid with an apparent half-life of t 1/2 = 38.5 ae 0.3 s for association (2 nm) and t 1/2 = 73.4 ae 3.8 s for dissociation. we determined the pharmacological profiles of different dopaminergic ligands in displacement binding assays with membranes of sf9 cells or budded baculovirus particles. the data were in good agreement for both membrane preparations tested in radioligand binding as well as in fa assay. obtained results indicate that budded baculovirus particles can be proposed as a source of gpcrs for performing fluorescence anisotropy as well as radioligand binding assays. gastrointestinal (gi) cancer includes a variety of cancer types affecting the structures and functions of the gi system, encompassing the gi tract and the accessory organs of digestion, from the esophagus, stomach, biliary system and pancreas to the intestine, rectum and anus. despite the significant advances however, much remains to be learned in the spectrum of gi cancer. several investigators have shown that both gas6 and its receptors, axl, sky, and mer are expressed in various types of cancers. however, the expression level of gas6 in gi cancer remains unclear. the aim of the study was to determine and compare plasma gas6 levels in gi cancer patients. 15 female and 27 male patients were included in the study (n = 42): 21 colorectal, 8 gastric, 4 pancreatic, 3 liver, 2 ampullary, 2 gall bladder and 2 esophageal. from all gi cancer patients, 2 ml venous blood was collected in citrate tubes before surgery. blood samples were centrifuged at 3000 g for 10 min, and plasma samples were carefully removed and stored in à80°c prior to use. the level of plasma gas6 was measured using a commercial developmental elisa kit (r&d systems, minneapolis, mn) which is validated by our laboratory. plasma gas6 levels in cancer patients were determined as follows: 1.84 ae 1.1 ng/ml in colorectal; 1.16 ae 1.1 ng/ml in gastric; 1.63 ae 0.61 ng/ml in pancreatic; 3.91 ae 0.89 ng/ml in liver; 2.15 ae 0.26 ng/ml in ampullary; 1.34 ae 0.46 ng/ml in gall bladder and 2.32 ae 1.7 ng/ml in esophageal cancer. preliminary findings indicate that there is a relation between gi cancers and plasma gas6 levels. taken together, these results suggest that gas6 may be a candidate biomarker for diagnostic use in gi cancer. inhibition of gas6 would be an attractive therapeutic target for slow down the progression of gi cancer. monday 5 september 12:30-14:30 computational biology p-03.03.2-001 computational approaches as an assay for blactam hydrolysis in class a b-lactamases c. tooke university of bristol, bristol, united kingdom b-lactam hydrolysing enzymes, in particular carbapenem-hydrolysing enzymes, are an increasing clinical threat. herein we show that molecular dynamics (md) and combined quantum mechanics/molecular mechanics (qm/mm) approaches are a predictive tool of carbepenemase activity in class a b-lactamases. b-lactam drugs are the most prescribed class of antibiotics worldwide, especially in the treatment of gram-negative pathogens such as klebsiella pneumoniae and escherichia coli. these organisms produce b-lactamases, enzymes which hydrolyse the b-lactam ring, a key resistance mechanism. class a b-lactamases have the ability to hydrolyse carbapenems, termed 'last resort' antibiotics. in particular, the kpc (klebsiella pneumoniae carbapenemase) family are the most clinically important, and recently identified natural kpc variants show increased hydrolytic activity against ceftazidime, a third generation cephalosporin. here we use computational simulations of b-lactam hydrolysis by b-lactamases. in particular, molecular dynamics (md) combined with qm/mm approaches have been used to model the deacylation of the carbapenem meropenem across 8 class a b-lactamases. this method has been extended to model cephalosporin hydrolysis across class a b-lactamases, including kpc variants. these approaches calculated the free energy barriers and correctly distinguished carbapenemases from carbapenem-inhibited enzymes. preliminary results suggest this protocol is also a predictive tool for ceftazidime hydrolysis. further, md simulations of 5 kpc variants (single and double amino acid changes) were analysed to identify structural changes in the active site, highlighting that variants differ in the size of the active site opening, corresponding with experimentally derived kcat values. these computational assays provide a predictive tool of b-lactam hydrolysis and has potential to provide insights into important mechanistic differences both across class a b-lactamases and within the same families. p-03.03.2-002 computational design of a novel polyglutamic dendrimer-based platform as an anticancer therapeutic approach poly (glutamic acid) (pg)-dendrimer as potential nanocarriers for cancer therapies, to specifically deliver tumor associated antigens (taa)mannosamine and melanato target cells and to modulate cancer antigen intracellular trafficking within the cytoplasm to promote an efficient and selective antitumor immunotherapeutic effect. the theoretical structures were obtained using x-plor software. the molecular dynamics simulation of pg-g4-dendrimer and taas was performed using desmond. the electronic properties of the structures were determined by semi-empirical methods using mopac. docking studies of taa to pg-g4-dendrimer to mannose receptor (mr1) were performed using hex 8.0.0 software. taa lumo atoms were conjugated to homo atoms of pg-g4 dendrimer using maestro software. results showed that pg-g4-dendrimer displays 64 carboxylic end groups available for covalent interaction with taas. the homo molecular orbitals of the dendrimer was located on the a-carbon of the carboxylic acid groups from backbone chain and it preferentially interacts with lumo molecular orbitals of amine group from taas. no differences in the gap energy of homo/lumo of all pg-g4-conjugates. taas bind preferentially to a-carbon of cooh of backbone chain instead of cooh from side chain. docking results showed that majority of taa conjugated pg-g4-dendrimer binds to the core of the mr1 receptor. increasing of the number of mannosamine conjugated to pg-g4-dendrimer more close and stable is the conjugated to the receptor. this system shows promising results as a novel functionalized pg-dendrimers for cancer therapy. theileria parva is one of the the economically important protozoan of the theileria genus belong to apicomplexa phylum which include plasmodium spp. and toxoplasma gondii, causative agents of malaria and toxoplasmosis respectively. this parasite is the disease agent of tick-borne east coast fever (ecf) ranks first among the tick-borne diseases of cattle in sub-saharan africa. the disease caused by the parasite affects a large proportion of domestic and wild animals and leads serious economic losses in the world. major problems in dealing with this illness are the high cost of drugs, development of resistance, and absence of effective vaccines. thus, it is important to develop an efficient and affordable antitheilerial agent. for this aim, 1-deoxy-d-xylulose-5 phosphate reductoisomerase (dxr) which subjected to identify novel drug aganist malaria and toxoplasmosis, of theileria parva was selected as potential target for improving novel inhibitors aganist ecf. a computational molecular modeling approach was conducted to determine the 3d structure of tpdxr by phyre2. energy minimisation and root mean square deviation (rmsd) was performed by 3drefine and superpose servers. to ensure the quality of modelling, stereochemistry, energy profile and residue environment of modelled structure were checked by different servers and possible ligand binding pockets were identified by metapocket 2.0 server a reliable 3d model for dxr from t. parva was modeled by using 3au9 as a template. the ca rmsd and the backbone rmsd deviations for the model and the template crystal structure were found to be 0.85 and 0.86 a, respectively. the ramachandran plot for the predicted model by rampage reveals that model shows an acceptable stereochemistry. top three considered possible binding pockets have been identified. these results have important implications for future screens aimed at finding new and safe molecular entities active against tpdxr through docking studies. p-03.03.2-004 molecular binding profile of protoberberine alkoloids on amyloid precursor proteincleaving enzyme 1 (bace1) as a drug candidate for alzheimer's diseases g. yalcin 1 , i. yildiz 2 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmaceutical chemistry, faculty of pharmacy, ankara university, ankara, turkey alzheimer's disease (ad) is the most prevalent neurodegenerative disorder that leads to dementia and nowadays over 46 million people live with dementia worldwide. because of the prevalence and economic burden of the disease, drug development studies have picked up speed and scientists especially focused on natural products. ad is basically characterized with tau hyperphosphorylation and accumulation of amyloid b (ab) proteins. ab proteins are generated from sequential cleavages of amyloid precursor protein (app) by b and c secretases, and b-site app cleaving enzyme 1 (bace1) is a b secretase essential for ab production. the alkaloids represent a very extensive group of secondary metabolites, with diverse structures, distribution in nature and important pharmacological activities. protoberberine alkaloids, which belongs to isoquinoline alkaloid class, are widely arranged in many species of the berberidaceae, annonaceae, fumariaceae, papaveraceae, and other plant families. recent searches showed that some of the protoberberine alkaloids such as berberine, palmatine, jatrorrhizine, columbamine, magnoflorine prevents the progress of neurodegenerative disorder. however, the mechanisms of them are not absolutely clear. therefore, we have aimed to elucidate the binding and affect mechanism of these alkaloids on the bace1 open and closed forms in here. for this purpose, molecular docking studies were applied for these natural products to the both forms of bace1 by using autodock vina and it was subjected to explicit solvent simulations by amber molecular dynamic package. our preliminary studies indicate that gly34, thr72, gln73, phe108, tyr198, lys224, thr232, arg235, thr329 residues of binding pocket have affiliations with all of the mentioned alkaloids and the binding of them generates alterations on closed form of bace1. the complexity of animal milk needs to apply numerous approaches and methods for its investigations. an understanding of the processes occurring in the milk can be used, for example, for quality control of the products. fat and protein are main components of milk which have a significant influence at its colloid properties, such as dynamic surface tension (dst). the application of regression-correlation analysis to milk data enables to develop a reliable quantitative model. the aim of our investigation was to perform the regression analysis to establish the relationship between above-mentioned parameters. for this purpose, we used a statistical software packages r version 3.1.2. dst was determined by bpa -1p tensiometer. milk fat (f) and protein (p) contents were measured by analyzer bentley 150. this work was supported by the russian scientific foundation (grant 14-16-00046). obtained formulas characterized the degree of influence of fat and protein contents of a milk sample for each of the dst parameters (r 0 , r 1 , r 2 , r 3 , k 0 , k 1 ): r 0 = 61.1538 + 0.8908 * p à 1.2953 * f r 1 = 63.3297 + 0.5658 * p à 1.4132 * f r 2 = 55.4274 + 0.4585 * p à 1.0143 * f r 3 = 45.6265 + 0.34634 * p + 0.05197 * f k 0 = 6.0986 + 0.1543 * p + 0.1351 * f k 1 = 10.7832 à 0.1470 * p à 1.0302 * f these formulas show that the maximum total effect of fat and protein contents influences at r 0 and r 1 . a significant coefficient (> 1) before the fat is observed in the formula, which describes the value of the tilt of final part of the tensiogram (k 1 ). the resulting regression equations have fundamental importance. with their help it is possible to calculate the dst parameters without their experimental determination, positioning fat and protein contents data. obtained dst parameters promote more complete characterization of the properties of the milk that may be used for dairy products. p-03.03.2-006 molecular studies of scorpion toxin and its mutants interactions with voltage-gated potassium channels the voltage-gated potassium kv1.3 channel is mostly expressed in neurons and immune cells. its blockage has a high therapeutic potential, for example, specific inhibitor shk toxin is undergoing clinical trials on psoriasis. goal of the current study was an interface analysis in complexes of hybrid channel kcsa-kv1.3 with peptide blockers agitoxin and its mutant forms. 3d structure was generated by homology modeling method using complex of mutated kcsa channel with charybdotoxin (pdb-code 2a9h) as a template and equilibrated by molecular dynamic simulation in gromacs software. analysis of hydrophobic and stacking interactions, hydrogen and ionic bonds of the toxin and potassium channels was performed for representative frames with optimal toxin orientations using program platinum and apbs software package. we performed contacts energy characteristics estimation to predict key toxin residues for binding process and possible mutation sites for changing selectivity against kv1.x channels. the results of investigation are in good agreement with the experimental values of binding constants, obtained by competitive binding assays. results of the conducted investigation may find an application in fundamental science and drug design. the research was supported by the russian science foundation grant no. 14-14-00239. simulations were performed using the supercomputing center of lomonosov moscow state university. p-03.03.2-007 homology modeling and molecular docking study of the paraoxonase-1 and its polymorphic variants q/r 192 and m/l 55 for non-statin lipid lowering drugs paraxonase-1 (pon1) enzyme is an hdl associated ester hydrolase exhibiting paraoxonase, arylesterase and lactonase activity, and reduces the formation of atherosclerosis blocking the ldl oxidation and reducing levels of oxidized lipids. in this study, molecular docking approach and molecular dynamics simulation were applied for finding the affinity of non-statin lipid-lowering drugs to pon1 and its polymorphic structures pon1 q/r 192 and m/l 55. fibrates (bezafibrate, ciprofibrate, clofibrate, fenofibrate, gemfibrozil), phytosterols (beta-sitosterol, brassicasterol, campesterol, stigmasterol) and other lipid lowering drugs (ezetimibe, niacin, orlistat, probucol, and sibutramine) was obtained from pubchem database. x-ray crystallographic structure of human pon1 and its polymorphic variants pon1 q/r 192 and m/l 55 was generated via 'modeller', homology modelling software, from human-rabbit hybrid x-ray crystal structure of pon1 (pdb code: 3sre). 10 ns molecular dynamic simulation analysis was performed using gromacs 4.5.5. affinity of lipid lowering drugs to pon1 and its polymorphic variants was predicted by molecular docking approach using autodock 4.2 suite. unlike other lipid lowering drugs that they have negative δg values for affinity, probucol, orlistat and betasterol was calculated by positive δg values (18.7, 12.3 and 1.4 kcal/mol). these values suggest that they may have no affinity to pon1 q/r 192 polymorphic structure. in all drug groups, brassicasterol and stigmasterol to pon1-m/l 55 and sibutramine to pon1 q/r 192 were calculated as the highest affinity. in generally, phytosterols predicted by high affinity to pon1 and m/l 55 polymorphic structures in comparison to other lipid lowering drugs. our study demonstrated that phytosterols predicted as high affinity compounds on pon1 structures may reduce the activity of antioxidant pon1 enzyme. this study need to be supported by in vitro and in vivo detailed studies. prolactin and its cognate receptor, prolactin receptor (prlr), are involved in over 300 distinct functions in mammalians. the mammalian prlr gene consists of 10-13 exons and several 5 0 and 3 0 regulatory sequences. in this study, gaps and annotation errors in the rat prlr gene were corrected by comparing the genomes of mammals and rodents and new putative exons were identified. the rat prlr gene sequences from two different sources (rnor_6.0, nc_005101.4 and rn_celera, ac_000070.1) were used and primary analysis showed that both sequences contain several gaps (varying from 0.35 to 4 kbp), corresponding to about 5.6% (10-11 kbp) of the gene. using the rat known prlr mrna exon sequences, it was found that the rnor_6.0 prlr gene has two exon-10 (one is about 2 kbp long and the other immediately after this). comparisons of mammalian and rodent prlr gene structures showed that the 2 kbp stretch is an assembly artifact. by comparing both gene sequences (and also other available rodent prlr genes), the gaps in the rat prlr gene were reduced from 5.6% to 3.8% (from 11 kbp to 7 kbp). functional annotation of the gene revealed that r. norvegicus prlr gene could have two more additional exons, exon-12 and -13, similar to mus musculus prlr gene. in mammals, prlr mrnas contain non-protein coding exons in the 5 0 utr (exon-1 and -2). in rats, there are 5 exon-1 variants, resulting from alternative promotor usages. studies on the rat and mouse prlr genes revealed that both rodents share 4 common non-protein coding exon-1 variants. in conclusion, it is found that the rnor_6.0 version of the prlr gene has the highest number of unidentified base pairs (corresponding to 5.6% of the gene) and the second exon-10 is the assembly artifact. the rat prlr genes in both databases have several gaps and our corrected version is the best available and characterized form of the rat prlr gene. in silico affinities of some statins to paraoxonase-1 enzyme the structure of the statins (atorvastatin, fluvastatin, lovastatin, mevastatin, pitavastatin, pravastatin, rosuvastatin and simvastatin) was obtained from pub chem database, and x-ray crystal structure of pon1 (pdb id:3sre) from protein data bank. modeller software was used for homology modeling of pon1 and its polymorphic variants that's called as pon1 q/ r 192 and m/l 55. amino acid sequence of human serum pon1 (uniprot: p27169) was used as the modeller template. all molecular dynamics simulations were carried out with gro-macs 4.5.5 software. molecular docking calculations on each of the polymorphic structure of the pon1 was performed with auto dock4.2. suite. for each substrate, y71 residue showed open conformation in pon1 and m/l 55 polymorphic structures while q/r 192 polymorphic structure showed closed conformation. in comparison between structures of pon1 variants, in most cases statins had lower affinity to q/r 192 polymorphic structure than to the other variant. in this study, among statins, atorvastatin showed lowest but simvastatin highest affinities to pon1. by considering that the high affinity drugs can have reducing effect of pon1 activities, it may be more appropriate to use the low affinity statins in hyperlipidemia treatment. however, these findings need to be supported with in vivo and in vitro studies. p-03.03.2-010 self-assembly of lipidoids for sirna uptake and release mechanisms studied by molecular dynamics simulations o. acar 1 , d. alpay 2 , a. r. atilgan 1 , c. atilgan 1 1 sabanci university, istanbul, turkey, 2 northwestern university, evanston, united states small interference rna (sirna) has the ability to bind a specific mrna which provides silencing of selected genes. nanocarriers made out of self-assembled lipidoids encapsulate sirna and deliver them into target cells effectively. in this study, a library of lipidoid structures is constructed and studied by molecular dynamics (md) simulations in different solvents, including sodium acetate, to ferret out their self-assembling mechanisms. the effect of the protonation state of the head group of lipidoids on the final shape of the self-assembled carrier is also studied. we further examine the role of the size of hydrocarbon tails in the packing. we study the final topology and the geometry of the self-assembled lipidoids both in the presence and in the absence of sirna. we find that stable clusters form with as few as 20 chains. for lipidoids having neutral head groups, clusters are in the form of dense bundles, while those with charged head groups form spherical capsids which are depleted of the salt on the inside and having a salt rich phase on the outside. in the self-assembled structure, lipidoids are arranged so as to expose the nitrogen and oxygen atoms to the solvent. while partial capsids with these properties also form at lower lipidoid numbers, 200 chains are necessary to form a fully closed capsid. in the presence of the sirna, the capsid assembles around the nucleotide. the free energy to remove the sirna from the assembly is calculated via repeated steered md calculations utilizing jarzynski's equality relating it to the irreversible work along and ensemble of trajectories. we therefore determine an optimal tail length for the most stable nanostructure, paving the way for designing nanocarriers with high efficacy. milk is one of the most valuable products for humans and attracts a lot of interest of researchers in various fields such as biochemistry, biology, food science and technology. the methods of milk study are quite varied. we chose the combination of the ultrasonic and dynamic surface tension (dst) measurements with the possible correlations among the obtained parameters. the aim of this work is to study the correlation between the parameters of milk, such as a content of fat, protein, lactose, minerals, dry milk solids and dst parameters. for this purpose we used milk analyzer 'klever-1m' and tensiometer 'bpa-1p'. three groups of animals were formed from clinically healthy holstein cows at the age of 4-5 years according to the fat content in the milk sample. group i -5 cows (milk fat content 4.01 ae 0.41%), group ii -6 cows (milk fat content 3.32 ae 0.14%), group iii -11 cows (milk fat content 2.73 ae 0.20%). this work was supported by the russian scientific foundation (grant 14-16-00046). the biochemical parameters of the milk samples of all three groups are in the range of the 'normal' values for healthy holstein cows: protein content varies from 3.0% to 4.2%, lactose and mineral content varies from 4.6% to 0.7%, respectively. the dst parameters (r1, r2 and k0) for the group i have strong positive correlations with the fat content in the studied milk samples. at the same time for the groups ii and iii the fat content in the milk indicates only medium positive and weak positive correlations with the r1, r2 and k0. obtained absolute values of the dst parameters of the milk samples showed some differences between all three groups. thus, the dst parameters are changing in direct proportion to fat content in the milk sample that can be explain by the primarily role of the milk lipids in the formation of the water/fat surfaces (such as fat globules, lipid-protein particles, etc.). p-03.03.2-012 exploration of allosteric paths in caspase molecules using energy dissipation e. n. bingol, o. sercinoglu, p. ozbek sarica marmara university, istanbul, turkey caspases are highly regulated aspartate-specific cysteine proteases that have major roles in programmed cell death; apoptosis. effector caspases are at the terminal step of the pathway, hence they are considered as death switches. with the discovery of the presence of allosteric sites, these molecules attracted the attention of the pharmaceutical studies and became drug targets. as a result of the binding of small molecules to the dimeric interface, active site loops are shifted to an unfavorable position. this is associated with a network between distal allosteric sites and the active site loop. an energy dissipation model was applied in order to analyze this matter in further detail. perturbation of specific residues enable us to determine a possible signaling network in proteins using external energy as an input, while focusing on the dispersion of this energy between residues throughout the structure. molecular dynamics simulations were performed with and without energy perturbation using namd software with charmm27 force field. energy perturbation was applied by increasing the velocity of a chosen residue at the desired time step of the initial md simulation. energy change of each residue was calculated upon the application of perturbation. as a result, residue response times, corresponding to the time of the response of a residue after the perturbation of another chosen residue, are obtained. combining reponse time data with a residue interaction network, it is possible to construct a final network that shows the communication started by perturbation within the molecule. it is shown that perturbation of allosteric sites result in the disruption of the catalytic sites given in literature. our findings support this and also gives a little detail of the possible communication between distal allosteric site and the active site loops. this finding enables the usage of this methodology for similar structures where the exact allosteric mechanism is yet not known. p-03.03.2-013 effect of complex mammalian membrane models with multiple membrane components on ras protein nanoclustering a. farcas 1,2 , l. buimaga-iarinca 2 , c. floare 2 , l. janosi 2 1 faculty of physics, babes-bolyai university, cluj-napoca, romania, 2 national institute for research and development of isotopic and molecular technologies, cluj-napoca, romania ras proteins are essential for the cellular signal transduction that regulates cell proliferation and differentiation and act as binary switches between gdp and gtp forms. a wide range of human tumors are associated with defective ras protein signaling. the production of permanently activated ras proteins is correlated with mutations in ras genes. experiments and computer simulations have shown that membrane-bound ras proteins form nonoverlapping dynamic nanosized subdomains (nanoclusters) in activation state-/isoform-dependent manner. we performed coarse-grained molecular dynamics simulations to investigate the effect of complex mammalian membrane models on formation and evolution of ras nanoclusters. a fundamental part of the plasma membrane is the phospholipids bilayer, which contains phosphatidyl-choline (pc), phosphatidylethanolamine (pe), phosphatidyl-serine (ps), sphingomyelin (sm) and cholesterol (chol). the nature of lipid-lipid and protein-lipid interactions was studied in binary (pc:chol) and quinary mixtures (pc:pe:ps:sm:chol). because the polar lipids are not uniformly distributed between the two leaflets of the membrane, the construction of the plasma membrane with five-component lipid mixtures took into account the asymmetry between the outer and inner mono-layers. the phospholipids chain saturation (combined with the presence of cholesterol) constitute the dominant factor in phase separation and was, therefore, modeled in different lipid tail combinations for various headgroups. using microsecond timescale simulations of membraneembedded ras proteins, we have shown that the nanoclusters are spontaneously forming dynamic structures whose behavioral characteristics is modulated not only by the ras isoform, but also by the complexity of the membrane model. furthermore, we showed that variations in the plasma membrane lipid composition have important implications in the localization of ras protein nanoclusters. optogenetics comprises biological methods to achieve gain or loss of function of well-defined events in specific cells of living tissue by means of targetable control tools that respond to light and deliver the effector function. microbial rhodopsins (mrs) have been established as powerful light-sensitive tools for optogenetics. acting as ion pumps or channels, mrs are used to induce cell (de)polarization to control neuronal activity in a wide range of living organisms. mrs are membrane proteins found in a large clade of organisms, including eukaryotes, bacteria, and archaea. they share a common architecture of 7 transmembrane a-helices and a covalently linked retinal, which is employed to absorb photons for energy conversion or the initiation of cellular signaling. major efforts are put into screening of natural and generating of synthetic mrs with desirable properties for optogenetics, e.g. ion selectivity. however, experimental study of mrs is difficult and resource consuming owing to, among other factors, low expression levels and protein stability. thus, there is a need in developing of computational tools for identification of mrs with desirable properties. we used non-redundant atomic structures of mrs taken from protein data bank to develop a set of numerical descriptors that reflects functional properties of mrs. then, we calculated the descriptors for non-redundant sequences of mrs with known function taken from the uniprot database, resulting in the feature matrix. we applied the support vector machine and the 5fold cross-validation procedure, using the feature matrix as the training set. as a result, we obtained the classifier that discriminates mrs in terms of the ion selectivity, e.g. na + , h + , or cl à pumps, with high precision. finally, we used the derived classifier on a test set of proteins and identified mrs for the further experiment in vivo. rational design of peptides with required stability and functional activity properties becomes a real instrument for the new generation drug development. the reca bacterial protein (and human rad51 homolog) is considered to be the central catalyst of homologous recombination, a mechanism essential for the accurate repair of double-strand dna breaks. dna repair via homologous recombination requires reca nucleoprotein filaments assembly. using seqopt (http://mml.spbstu.ru/seqopt/), a novel method for a-helix sequence optimization we present the successful design of peptide sequences capable to maintain a very stable a-helix structure and to inhibit reca activity. novel a-helical 18 amino acids peptide is constructed based on reca-dna complex structure. we observed in vitro inhibition of reca atp hydrolysis, dna strand exchange reaction and reca filament formation. also, we observed lower e. coli resistance to uv and sos-response suppression in vivo. computational identification of promiscous enzyme activity for the morita-baylis-hillman reaction k. ozturk, s. sayin, n. celebi olcum yeditepe university, istanbul, turkey enzyme promiscuity attracts considerable attention in terms of enzyme evolution, protein engineering and biocatalysis. especially, development of highly efficient novel biocatalysts starting from promiscuous enzymes that have the catalytic machinery to perform desired chemistry is an intense area of research in recent years. in this work, we computationally explored the catalytic promiscuity of natural enzymes for the synthesis of morita-baylis-hillman (mbh) adducts, which display antitumoral activity against human cervical cancer cells, by mining structural protein databases using quantum mechanically optimized theoretical active site models (theozymes). catalytic interactions in the active sites of selected hit proteins with potential mbh activity were evaluated in solvated dynamic environment using molecular dynamics simulations. computational screening of the protein data bank for the quantum mechanically determined optimal arrangement of catalytic functional groups for the target mbh reaction successfully identified an enzyme with experimentally determined promiscuous mbh activity. ras proteins mediate a wide variety of signal transduction pathways that regulate cell growth, proliferation and differentiation. these proteins are small gtpases that act as binary switches between gdp-bound 'off' and gtp-bound 'on' states. oncogenic point mutations of ras are associated with~15% of all cancers and up to 90% in specific tumors and many developmental disorders. both experimental and in silico results showed that the membrane-bound ras proteins form non-overlapping dynamic nanosized subdomains called nanoclusters in an activation state-/ isoform-dependent manner. we performed coarse-grained molecular dynamics simulations in order to investigate the formation and evolution of ras nanoclusters in mammalian model membranes. ras proteins were inserted into the cytoplasmic side of the plasma membrane model (di-c16:0-phosphatidyl-choline: di-18:2-phosphatidyl-choline: cholesterol 5:3:2) where they formed highly dynamic nanoclusters, both in size and in composition. furhermore, we found that the presence of ras protein nanoclusters has a significant impact on the model membrane behavior. properties such as phase behavior, diffusion coefficient, surface tension and lipid tails order parameter are also influenced by the temperature variation of the model membrane. we have investigated dynamics in three different crystal forms of ubiquitin, as well as ubiquitin in solution, with particular emphasis on (i) conformational exchange between b turn type i and ii in the region 51-54 and (ii) rocking dynamics where protein molecules as a whole undergo subtle reorientational motion within the confines of the crystal lattice. experimentally, both motional processes have been probed using relaxation dispersion techniques, including recently developed near-rotary-resonance dipolar relaxation dispersion experiments. thereby it has been determined that rocking motion in one of the crystal forms (pdb id 3n30) occurs on the time scale of tens of microseconds, whereas the conformational exchange has characteristic time constant of ca. 100 ls. using molecular dynamics simulations, we have shown that the similarity of motional time scales is not accidental: bi↔bii exchange and rocking motion appear to be coupled. we have investigated the mechanisms of this coupling and predicted a number of point mutations that are expected to abrogate (or enhance) rocking. the crystals of ubiquitin containing these mutations have been modeled in silico. we have also investigated the interactions (in particular, crystal contacts) that control the balance between bi and bii conformations in different crystal forms. finally, we have used md simulations as a basis for chemical shift calculations and illustrated how relaxation dispersion effects can emerge as a function of bi↔bii exchange in conjunction with the rocking motion. the md simulation study was supported by rsf grant 15-14-20038. serine/threonine kinases are attractive targets in targeted cancer therapy due to their overexpression in several forms of cancer. flavonoids are highly bioactive plant secondary metabolites that are important in human health due to their antioxidant property. quercetin, a natural flavonoid derivative, has been shown to regulate several signal transduction pathways and is in phase i clinical trial as an anticancer drug. this study explored the inhibitory potential of quercetin and its derivatives using in silico methods like molecular docking and molecular dynamics simulations. quercetin and its derivatives were observed to bind to several serine/threonine kinase family proteins with binding energy significantly better than other known inhibitors and commercially available drugs. this study thus sheds light on the atomic level interactions that define the polypharmacological nature of quercetin and its ability to interfere with a number of cancer pathways. introduction: noninvasive prenatal diagnosis (nipd) of the fetal rhd status by rhd genotyping of the maternal plasma was initially applied in alloimmunized pregnant women. fetal rhesus d status detection for management of rhd incompatibility using circulating cell-free fetal dna from maternal plasma or serum is now accepted by many obstetricians in europe as reliable and useful. the aim of the study was to detect fetal rhd specific antibodies in maternal plasma using a nanopolimer based electrochemical biosensor. materials and methods: a three-electrode system, consisting of a gold electrode, an ag/agcl reference electrode and a pt counter electrode, was accommodated in a 10-ml electrochemical cell. anilin and jelatin were used for immobilization of rhd antibody. the polimerization was occured at 319 nm uv light. antibodies of rhd antigen were detected using differential pulse method at between 0.4 and 0.6 v potentials by observing the differentiations in the current values. results: the rhd status of the fetus was predicted in 20 rhdnegative pregnant women (8-36th week of pregnancy). rhd antibody were detected in maternal bood using biosensor in 15 of the fetuses. the results were confirmed with real-time pcr. the fetuses found rhd (+) for exon 5 and 7 of rhd gene by multiplex real-time pcr. discussion and conclusion: biosensors based studies might be useful, because they allow to monitor the molecular interactions in real-time providing qualitative and quantitative information, through kinetics, affinity and concentration analyses. we found that more advantages in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, specific, economic, practical and less time-consuming. fetal rhd detection at low concentrations and in the early week of pregnancy is possible with this method. p-03.03.2-022 investigation of phylogeography of cricotopus sylvestris (diptera: chironomidae) using mitochondrial and nuclear molecular markers the family chironomidae is one of the most widely distributed insect families of diptera, and this family is distributed in all continents and all habitats from the tropics to the arctic in lakes, streams and puddles. in this study, we aimed to determine the dispersal of c. sylvestris using molecular phylogenetic markers not only in turkey but in the world and to reveal from where this species may have entered to turkey in the past. c. sylvestris larvae were collected from 8 lakes across turkey. after total genomic dna extraction from body of larvae, fragments of two mitochondrial genes, cytochrome c oxidase subunit i (coi) and cytochrome b (cytb), and one nuclear gene, carbomyl phosphate synthase domain (cps) of cad, were amplified and sequenced. in addition, several sequences of these three genes of c. sylvestris from different countries of different continents such as south corea, japan, canada, denmark, and sweden were obtained from genbank. all sequences were aligned using mega 6 and bioedit version 7.0.9.0 and were used for phylogenetic analyses. neighbour-joining (nj) tree was created in mega 6 and paup 4.0b10 with 1000 bootstrap replicates. maximum likelihood (ml) analysis was performed in raxmlgui 1.0 using gtrgamma model with 1000 bootstrap replicates. beast v1.8.0 was used for bayesian analysis. our phylogenetic analyses indicated that the japanese, south corean and american c. sylvestris were different from european and turkish members. turkish members of c. sylvestris were closely related to european ones according to our bayesian, nj and ml analyses. in turkish members, c. sylvestris collected from hazar and c ß ıldır lake was more ancient than those from marmara, sapanca, c ß ıldır, aygır, beys ßehir, e girdir and sıhke lakes. in conclusion, our results clearly suggest that several transoceanic dispersal events among the continents may have occurred and that the entrance of turkish c. sylvestris to turkey may have been from southeast and northeast of the country. metagenomics is providing great help to explore world of unculturable microorganisms in the natural samples to enhance our knowledge about microbial diversity. here, we have performed metagenomic analysis of fresh water lake bacterial community using 454 pyrosequencing techniques. we have observed a wide array of bacteria from phylum proteobacteria and family enterobacteriaceae as well as very few viruses from podoviridae, siphoviridae and unclassified phages. we have conducted a metagenomics analysis with the primary focus on the examination of the community of bacteria in a fresh water lake ecosystem. roche gs flx software gave us total 156 253 reads (with an average read length of 795.274 bp). there were 15 226 contigs having > 100 bp sequence length whereas 10 481 contigs with > 500 bp sequence length. for further analysis we have taken contigs with > 500 bp only. further, we have analyzed the microbial community composition using blastn/blastx against nt/nr databases with e-value cutoff of 10 à5 . ≥70% of total contigs were mapped to the reference with ≥60% contig match coverage. the community analysis revealed that domain bacteria is predominantly present (99.8%) in the water sample, followed by eukaryota (0.02%), viruses (0.08%) and other sequences (0.02%). most abundant phyla was proteobacteria (99.8%) and the most dominant family was enterobacteriaceae (89%) followed by xanthomonadaceae (10%), vibrionaceae (0.4%), pasteurellaceae (0.1%), shewanellaceae (0.07%). we performed functional analysis of all 5974 contigs using rapid annotation using subsystems technology (rast) 4 which detected 15 319 coding sequences and 197 rnas in 619 subsystems. among the classified cds from rast showed major cds hits for enzymes involved in the subsystems amino acids and derivatives and the carbohydrate metabolism. the great diversity of microorganisms present in the lake may reflect the human activity in the area. maldi-tof mass spectrometry is a ubiquitous and widespread tool for protein identification. once the protein sequence is unavailable, unambiguous identification cannot be performed, and predictability is limited by the presence of sequenced homologous proteins. we present a statistical approach to predict a number of structural, localization and functional properties of unknown proteins by direct analysis of mass distribution shapes of their post-cleavage fragments obtained from maldi-tof mass spectrometry data. secondary structure of proteins is best predicted by their specific cleavage at the inertial hydropathy group amino acid residues (filmv), with thermolysin (afilmv) being the closest commercially available reagent, leading to distinguishing between proteins with presumably ahelixes or b-sheets with 90% accuracy. cellular localization of proteins is best predicted by their specific cleavage at the external hydropathy group amino acid residues (dehknqr), exemplified by gluc(phosphate)+lysc(dek) cleavage. protein location in the cell membrane and its localization character (monotopic/ transmembrane, single-pass/multi-pass transmembrane) are predictable with~75% accuracy by this single cleavage, with optimal combination of 3-4 cleavages improving the accuracy tõ 80%. functional prediction of proteins is the best among membrane-associated proteins with characteristic structural conformations. we attribute the differences in the mass distribution shapes to the characteristic clustering of amino acids residues with respective hydropathy properties that are involved in the formation of 3d structural conformations of proteins. the suggested approach allows for a non-parametric statistical prediction of uncharacterized proteins from their maldi-tof mass spectrometry data without knowledge or reconstruction of their primary sequence. potential applications include proteomic studies of organisms with unavailable genomic sequences and highly variable proteins analysis. the cancer genome atlas (tcga) represents a comprehensive database of genomic, transcriptomic and epigenetic alterations across more than 20 tumor types. earlier we developed cross-hub tool aimed at multi-way analysis of tcga data in the context of gene expression regulation. in the present work, the software was updated with new features that are described below. crosshub is a python-based application. one of the features of crosshub is the combining tcga rna-seq co-expression analysis to encode chip-seq data in order to reveal most possible transcription factor (tf) targets and coupling mirna-mrna co-expression to several algorithms of mirna target prediction in order to enhance its efficacy. the key feature of the updated crosshub version is the analysis of the associations between expression ratio of tf to its targets and tf mutation status. this allows identification of tfs that are functionally (in)activated with driver mutations in a particular cancer type. the second novel feature of crosshub is the analysis of associations between 'tf-to-targets' expression ratio and tumor characteristics (tnm classification, pathological stage), patient follow-up, etc. in turn, this analysis may result in the identification of 'tf-targets' functional relations that are important for disease progression, tumor invasion, response to chemotherapy. thus, crosshub was supplemented with new features that can be useful for comprehensive tcga data analysis. the updated version of crosshub is freely available at http://sourceforge.net/ projects/crosshub/. this work was financially supported by the russian foundation for basic research (grants 15-04-08731, 16-16-00114 and 15-34-70055) and ras presidium program 'molecular and cellular biology'. p-03.03.2-026 mutations leading to increased rnase production and streptomycin resistance in bacillus pumilus bacillus pumilus strain 3-19 which was derived from soil-isolated b. pumilus 7p using chemical mutagenesis is characterized by resistance to streptomycin (str, up to 500 lg/ml) and ability to produce extracellular enzymes in quantities almost 10-fold higher than the parent strain. these features make the 3-19 strain suitable for industrial production of rnase (binase) which is known for its antitumour and antiviral properties and can be used as an rna-degrading tool in molecular biology. the whole genomes of both mutant and wild-type b. pumilus strains were sequenced recently. to reveal the exact genetic features responsible for rnase overproduction and str resistance we have fulfilled comparative genome analysis of b. pumilus 7p and 3-19 strains. facilities of rast server, edgar platform and additional bioinformatics tools (plasmid finder, prophinder, bl2seq) were used. it is found that both b. pumilus genomes under study contain an intact prophage, while only wild-type strain bears a 6 kb cryptic plasmid. none of the systems is inactivated in mutant strain according to the results of metabolic reconstruction. 3.4% of total cdss differ in 3-19 strain in comparison to 7p one, 36% of them are hypothetical. the altered genes are involved in membrane transport, cell wall composition, chemotaxis, spore formation, carbohydrate metabolism, dna metabolism, translation and transcription regulation. mutation (k56n) leading to str resistance is identified in 30s ribosomal protein s12p. regulatory and coding regions of binase gene have no modifications. candidate genes which can account for binase overproduction are selected. mutation k56n is classical in str resistance and leads to enhancement of decoding accuracy while decreasing elongation speed. rnase overproduction is brought about by non-specialized mechanism since other hydrolases are also overproduced in mutant strain. genes encoding extracellular serine protease, sporulation initiation phosphotransferase f, gnat-family acetyltransferase and cell wall modifying enzymes are reported previously to increase production of degradative enzymes. the action of encoded by them proteins lead to increase of stability and release of secreted proteins to environment and to derepression of their transcription from negative regulators bacillus pumilus strain 7p has been identified on its ability to produce ribonuclease and different extracellular proteases. in order to increase inherent biosynthesis of proteases the 7p strain was screened on culture medium supplemented by streptomycin. derivative b. pumilus strain 3-19 gains the resistance to streptomycin and also shows the increased ribonuclease activity. we used genomes of both these strains to explore streptomycin susceptibility and increased activity of hydrolytic enzymes. whole-genome shotgun sequencing was performed using a combination of pyrosequencing and ion semiconductor sequencing, which provided 23x (7p) and 30x (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) overall genome coverage. assembled genome sequences of 7p and 3-19 strains included 9 and 8 scaffolds > 500 bp with a calculated genome size of 3 577 758 bp and 3 572 739 bp, respectively. the gc content was 42%. both draft genomes have been deposited at genbank (jojx00000000.2 for 7p and jhud00000000.2 for [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] . detailed comparative genomic analyses of strains have been performed. we calculated average nucleotide identity (ani) values between the genomes of our strains and 9 completed b. pumilus genomes deposited in ncbi database. two b. pumilus strains (sh-b9 and safr-032) revealed the max. ani value (95.17% and 94.65%, respectively). b. pumilus sh-b9 strain has been used as a reference for snp calling in strains 7p and 3-19. 3828 snps for the 7p strain and 862 for the 3-19 strain were classified as nonsynonymous variants. 20 radical nucleotide substitutions from the 3-19 genome were not found in 7p genome. among them, the mutation in the 56 codon of rpsl gene (coding 30s ribosomal protein s12) is probably associated with resistance to streptomycin. also, two mutations in rpob and nusa genes (coding rna polymerase and transcription termination factor rho, respectively) may be related to increased enzymes activity. both our strains contain 143 protease-coding genes. twelve of them are encoding extracellular proteases. here we propose an algorithm that can predict an antibodies mutant forms with desired specificity. this algorithm allows to determine the position and type of amino acid residue for mutagenesis. approach is based on a hybrid method of quantum and molecular mechanics (qm/mm) that allows to understand the reaction mechanism and the role of active center amino acids. catalytic antibody a17, that is able to hydrolyze pesticide paraoxon, was selected as a model. however, the hydrolysis efficiency of paraoxon by a17 antibody is only 9 m à1 min à1 , that is insufficient for using this antibody as antidote. the main fundamental goal of our study is to determine the necessary conditions for improving the binding reaction of paraoxon by catalytic antibody a17. the hybrid qm/mm method allows to study the reaction mechanism of interaction of a17 with paraoxon. it was shown that the reaction proceeds via the classical s n 2 mechanism. the key step of the reaction is the proton transfer from the catalytic residue tyr-37 to paraoxon. qm/mm approach determines position for mutagenesis -leu-47 in light chain. for one of the mutant in this position -leu47argwere predicted (i) increased probability of formation of a hydrogen bond between the catalytic moiety and paraoxon compared to the wild type antibody and (ii) smaller value of the diffusion coefficient, which reflects the best positioning of paraoxon in the active center. steady-state kinetic analysis shows that leu47arg exhibits a 70-fold increase in k 2 /k d compared to a17 (90 m à1 min à1 vs. 1.35 m à1 min à1 ). double mutant leu47arg/ser35ala also has improved constants of interaction with paraoxon in comparison with the wild type antibody, however, a single mutant leu47arg still binds paraoxon three times better, that may be due to the fact that the serine in 35 position increase the nucleophilicity of tyr37. thus, our results are in line with our computed predictions. this work was supported by rfmefi60414x0069. due to high prices of meat and meat products, low quality raw materials like offal tissues are commonly used in turkey. in the retrospect of the studies for evaluating and detection of unwanted tissues in the sample is basic histological examination. the light microscopy techniques are very strong method if a researcher qualification is enough. a new researcher-independent method must be developed. therefore, different tissues and organs constitute of unique mrna and protein. our method is based on this event, so the antigenic sites of the tissues can be detected by selected antibodies. the first set of the antibodies are for detecting muscle and adipose, consist on muscle actin and adipose triglyceride lipase. this set is used for calibration on standard meat sample. the second set of the antibodies are detecting of offal tissues, consist on trrap and casein. anti-casein antibody is selected because the mammary gland usage in grinded-meat is very common. immuno-staining started with hier (heat mediated epitope retrieval), then classical ihc method applied to slides with dab-chromogen. after all process completed the slides were photographed by las (leica application suite) on microscope. the capture settings were remained same on both sets. image capture size is 1392x1040 pixels and field of view (fov) is 449 9 335 lm. all the image files were converted to binary for threshold operation. the threshold values of first set and second set were calculated and their ratios were compared. the formula is based on the distribution (dst) of pixel intensity (int) over threshold (thrs) values on all fov (axis: the results are good enough to detect the unwanted micro-structures on 80% raw meat and 20% offal tissue. calculations proofed with imagejò. future application of this method and opencv-based software algorithm is to port the source code to a single board computer (sbc) with a digital microscope connected. monday 5 september 12:30-14:30 mechanisms of pro-inflammatory diseases p-04.02.2-001 the effects of raas inhibition in rate limiting step by aliskiren on testicular torsion injury in rats testis torsion is a urological emergency condition that results in necrosis of the testis if the condition is not treated. unfortunately treatment of testis torsion is not fully understood, therefore clinical and experimental studies are performed continuously. reninangiotensin-aldosterone system (raas) contributed to pathophysiology of several diseases. aliskiren (als) inhibits the renin on the first step of this system. our aim is to investigate the protective effect of aliskiren on unilateral testis damage caused by experimental testis torsion and detorsion. the forty-eight rats were separated into eight groups of six animals: sham, sham+als 200 mg/kg (oral) group, torsion group (tor), torsion/detorsion group (tor/det), tor+als 100 mg/kg (oral) group, tor+als 200 mg/kg (oral) group, tor/det+als 100 mg/kg (oral) group, tor/det+als 200 mg/kg (oral) group. in the tor and tor/det groups, the left testes were rotated 720°clockwise together with the spermatic cord and tunica vaginalis in the scrotal space. the left testes of the rats were subjected to torsion and detorsion during 2 h. after experimental procedures, testicular tissues were examined by histopathologic and molecular analyses. the il-1b and inos mrna expressions were increased in tor and tor/det groups when compared with sham group. both doses of aliskiren administration decreased these expressions in tor/det groups. the stereological results revealed that aliskiren administration promote the numerical density of mature spermatids in tor and tor/det groups. the numerical densities of tor/det+als100 and tor/det+als200 groups were similar and these two groups have significant difference when compared to the tor and tor/det groups. the administration of als may be useful for preventing ischemic damage on unilateral testes injury in rats. this study supported by a tubitak 3001 project, coded 114s048. 3 ) has recently been recognized as a potent immunomodulator which acts through regulation of gene expression involved in immunity response thus affecting various inflammatory and autoimmune diseases. the study was aimed at investigating hepatoprotective role of d 3 in vdr-mediated regulation of pro-inflammatory factors in diabetic liver. materials and methods: type 1 diabetes was induced in male c57bl/j6 mice by i.p. injection of high-dose stz (150 mg/kg b.w.). after 2 weeks of stz-induced diabetes animals were treated with/without d 3 (15 iu/mouse per os) for 8 weeks. blood serum 25ohd 3 was assessed by elisa. rel-a, vpf, inos and vdr expression was measured by qrt-pcr and/or western-blot. results and discussion: diabetes caused two-fold reduction of serum 25ohd 3 level, indicative of d 3 deficiency. significant alterations in d 3 -endocrine system were found as is evident from reduced expression of cyp27a1, cyp2r1, vdbp and vdr at transcriptional and translational levels. these changes were accompanied by a marked increase in mrna and protein levels of inflammation markers rel-a, vpf and inos in hepatic tissue of diabetic mice. diabetes also led to structural lesions in liver tissue. complete restoration of 25ohd 3 content and partial normalization of liver tissue structure were achieved after d 3 treatment. d 3 administration partially normalized expression of cytochromes involved in d 3 metabolism and hepatic pro-inflammatory factors. d 3 treatment prevented overexpression of rel-a and phosphorylated p65/rel-a translocation to hepatocellular nuclei that is most likely mediated through 1,25(oh) 2 d 3 and vdr. conclusion: study confirmed that diabetes-induced liver abnormalities are associated with chronic inflammation that can be linked to impaired d 3 metabolism and deficiency. our findings demonstrate protective vdr-mediated effect of vitamin d 3 against diabetes-induced liver injury. p-04.02.2-003 lavandula stoechas extract increased glucose uptake and protein levels of key signaling molecules in insulin resistant c2c12 muscle cells s. savranoglu 1 , h. ipek 2 , s. arslan 3 , h. delig€ oz 4 , a. r. t€ ufekc ßi 5 , i. demirtas 5 , t. boyunegmez t€ umer 6 1 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 2 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey, 3 deparment of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 4 department of chemical engineering, faculty of engineering, pamukkale university, denizli, turkey, 5 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, turkey, 6 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: the aim of this is to identify remedial effects of lavandula stoechas, anatolian traditional medicine, against metabolic disorders developed on the ground of insulin resistance. ethyl acetate extract (eae) of l. stoechas was investigated in c2c12 myotubes which were made insulin resistant by free fatty acid (ffa) treatment, for its effects on glucose uptake and as well as on the activation of akt-1 (by pakt/ akt ratio) molecule which plays a central role in insulin signaling through serine (473) phosphorylation. in addition, the protein level of lipoprotein lipase (lpl) enzyme was also evaluated. material and methods: c2c12 cells were made insulin resistant by palmitic acid (ffa) and effects of eae on p-akt (ser473)/akt ratio and lpl level were determined by sds-page/western blot. the effect of eae on glucose uptake in insulin resistant cells were determined by the 2-deoxyglucose uptake assay. results: eae at 25 and 50 lg/ml significantly increased the glucose uptake 120 and 182% compared to insulin resistant control cells. metformin at 2 mm increased this parameter up to 132%. eae increased pakt ser473/akt level 43-37% and lpl expression 50-92% for 25 and 50 lg/ml, in insulin resistant myotubes, respectively (p < 0.05). discussion: eae of l. stoechas improved impaired insulin sensitivity through both enhancing glucose uptake and activation of akt1 molecule through ser473 phosphorylation. in addition, it also considerably increased lpl level which has very important function in lipid metabolism. conclusion: overall, results demonstrated that l. stoechas contain phytochemicals which can be effective for the prevention and also treatment of insulin resistance and associated conditions. our research group is on the way for the identification of these 'bioactive' molecules with bioassay guided fractionation studies. tubitak (projectid: 112t442) supports this study. achievement of complete pain control is very difficult task, which requires a search for new molecular targets during the analgesic substances development. considering the importance of glial cells and their signaling molecules, development of new gliotropic therapeutic methods is a promising direction in pain treatment. polyunsaturated fatty acids, including docosahexaenoic acid demonstrating anti-inflammatory and antioxidant activity are of considerable interest. docosahexaenoic acid (dha, 22:6 n à 3) analgesic activity was studied using a chronic constriction injury (cci) rat model. animals were subcutaneously injected with dha emulsion at a dose of 4.5 mg/kg (125 mm/kg) daily during 2 weeks after surgery. collection of material for subsequent immunohistochemistry investigation was performed on day 28. we clearly demonstrated that the activation of neurokinin neurotransmission and nnos synthesis are coincided with the astroglial activation in the spinal cord dorsal horn (scdh) superficial lamina during neuropathic pain development. however, the detailed mechanisms of interaction between substance p (sp)-, no-ergic systems and astrocytes in the spinal cord remain to be elucidated. systemic administration of dha to cci animals reduced neurogenic pain intensity and duration, leading to an earlier stabilization of paw weight distribution and preventing the development of degenerative changes in denervated limb. this drug treatment reduced the level of the sp-and no-ergic neurotransmission and decreased astrocytosis in the scdh superficial lamina. thus, the ability of dha to affect nociception is a promising and safe alternative to current pharmaceutical therapeutics. immunohistochemistry studies carried out with the russian science foundation financial support (agreement no. 14-50-00034), obtaining dha and all manipulations with animals of the material was funded by rfbr according to the research project no. 16-34-00023 mol_a. p-04.02.2-005 circulating endothelial-derived apoptotic microparticles and aopps are related to highsensitive troponin t in patients with chronic hepatitis c infection the aim of this study was to evaluate non-standard risk factors for cardiovascular events, such as endothelial dysfunction assessed by endothelial-derived microparticles (emps) (cd144 +/ cd31 + ), advanced oxidation protein products (aopps), and low-grade inflammation, that are potentially associated with elevated levels of high-sensitivity troponin t (hs-tnt) and n-terminal pro-brain natriuretic peptide (nt-probnp) in patients with chronic hepatitis c (chc). methods and results: eighty-six chc patients and 60 healthy control subjects were enrolled in the study. circulating levels of hs-tnt, nt-probnp, aopps-albumin (the ratio of aopps to albumin content), emps (cd144 +/ cd31 + ), hs-crp, and tnf-a were assessed. compared with chc patients, the chc patients with diabetes mellitus (dm) had higher levels of emps (cd144 +/ cd31 + ) and aopps-alb, which were associated with elevated hs-tnt levels (≥13.3 pg/ml). nt-probnp positively correlated with tnf-a level in all chc patients and this correlation was stronger in diabetic patients. in multivariate logistic regression analysis, the independent factors associated with the presence of elevated hs-tnt levels were the presence of dm (p < 0.001) as well as high levels of aopps-alb, apoptotic emps (cd144 + /cd31 + /an-v + ), and nt-probnp (p = 0.04, p = 0.03, p = 0.04 respectively). conclusion: the prevalence of elevated hs-tnt were increased significantly in the diabetic patients with chronic hepatitis c. hs-tnt was related to non-standard risk factors for cardiovascular events, and circulating endothelial-derived apoptotic microparticles (cd144 + /cd31 + /an-v + ) level was an independent predictor for elevated hs-tnt levels, potentially indicating some abnormalities in the myocardium. apnea; and healthy individuals; and assessing the connection between the pain and the dimension of the sleep disorder. material and methods: 60 patients who were diagnosed with obstructive sleep apnea and 40 healthy individuals who were similar in terms of age and gender were included in this study. the patients, who were diagnosed with obstructive sleep apnea with the examination and sleep tests, were assessed according to the 1990 american college of rheumatology (acr) criteria in terms of fms. serum d vitamin level was measured by using the ultra performance liquid chromatography method. findings: when the fibromyalgia syndrome and obstructive sleep apnea and pure obstructive sleep apnea patient groups are compared with the control group, the vitamin d level was found to be low at a significant level (p = 0.038, p = 0.001, respectively). no significant difference was found between the vitamin d levels in fibromyalgia syndrome, obstructive sleep apnea and pure obstructive sleep apnea patient groups. a negative correlation was found between the number of the sensitive points and vitamin d levels in fibromyalgia syndrome patients (p = 0.013). results: it has been concluded that the obstructive sleep apnea and fibromyalgia syndrome patients have low vitamin d levels, and this situation must be considered in treatment modalities. on the other hand, the results obtained in the study make us consider that vitamin d metabolism is not influential in the pathogenesis of the fibromyalgia syndrome and obstructive sleep apnea togetherness. p-04.02.2-007 decreased chitotriosidase activity and levels in familial mediterranean fever discussion: familial mediterranean fever is an inflammatory disease. several cytokines and inflammatory mediators are playing role on pathogenesis of the disease. although ıt has been demonstrated that the increased concentrations of cht in patients with fmf. we found lower cht activity and concentrations in patients with fmf. conclusion: serum cht enzyme activity and concentrations may not be considered as a biomarker in fmf patients taking colchicine. new studies are needed to evaluate the changes of the enzyme activity, concentration and the role of cht in patients with colchicines negative patients. chronic hyperglycemic state leads to an increase in subclinical systemic inflammatory response in diabetes mellitus type 2 (dmt2) patients. inflammation-based scores, neutrophil to lymphocyte ratio (nlr), platelet to lymphocyte ratio (plr) and red blood cell distribution width to platelet ratio (rpr) are biomarkers able to quantify systemic inflammation. the aim of the study was to investigate association of the inflammation-based scores with short-and long-term glycemic control markers, and whether they could be used as indicators of glucoregulation in dmt2 patients. the cross-sectional study included 92 dmt2 patients, treated at the primary health care centre zenica from december 2015 to april 2016, distributed into groups according to glycated hemoglobin (hba1c) values: a (n = 59, hba1c ≤7.0%) and b (n = 33, hba1c > 7.0%). complete blood cell count, fasting blood glucose (fbg) and hba1c measurements were determined at the primary health care centre zenica and at the department of laboratory diagnostics, cantonal hospital zenica by standard laboratory methods. all statistical tests were performed using spss 19.0. p values fasting blood glucose and hba1c were significantly higher in the group b compared to the group a (p < 0.0005). there was no significant difference of nlr, plr and rpr between the groups (p = 0.50; p = 0.220; p = 0.525, respectively). significant correlation of inflammation-based scores with fbg and hba1c was found only between plr and hba1c in the group a of dmt2 patients (r = 0.328, p = 0.011). inflammation-based scores could gather meaningful clinical information, either diagnostic or prognostic, on a variety of hyperglycemic, inflammatory, cardiovascular and thrombotic disorders. since there was no statistically significant difference of nlr, plr and rpr between dmt2 patients with good and poor glycemic control, we conclude that these scores could not be used as indicators of glucoregulation in dmt2 patients. chronic inflamation plays a central role in the development and progression of diabetes and in the pathogenesis of its comlications. the neutrophil-lymphocyte ratio (nlr) and platelet-lymphocyte ratio (plr) are indicators of subclinical inflamation. mean platelet volume (mpv) is one of the platelet function indices that reflects the platelet production rate and stimulation. we investigated the association of nlr, plr and mpv with prediabetes and type 2 diabetes mellitus (t2dm) and determine whether or not these are reliable markers for diagnosis. we evalueted 76 people's results who were carried out oral glucose tolerance test (ogtt). acording to 2-h values of plasma glucose in the ogtt; 1. group (normal glucose tolerance: ngt): under 140 mg/dl (n = 42), 2. group (prediabetic: impared glucose tolerance (igt)): ranging from 140 mg/dl to 199 mg/dl (n = 25), 3. group (firstly diagnosed diabetic by ogtt): above 200 mg/dl (n = 9). 4. group is clear diabetic without complication (taking treatment) group (n = 34). we compered nlr, plr, mpv and some biochemical markers between four groups. there are significantly differences between all groups in nlr (p = 0.004) and plr (p = 0.021) values. nlr values are significantly higher in prediabetic (1. it is recognized that a chronic low-grade inflammation and an activation of the immune system are involved in the pathogenesis of insulin resistance and type 2 diabetes mellitus (t2d). this study aimed to analyze the long-term impact of altered metabolism in female t2d patients at the level of mediators of inflammatory response. this study included 65 femalet2d patients and 107 control subjects, which were recruited at the clinical center university of sarajevo and the general hospital tesanj. in this study the effects of glycemic control on markers of the inflammatory response crp, fibrinogen, leukocytes, sedimentation, and cytokine il-6, were analyzed. all subjects included in this study were free of evidence infections, surgery, thyroid disease, polycystic ovarian syndrome, active liver and kidney damage. all biochemical analyses were performed by employing standard ifcc protocols. results from this study demonstrated significant increase of fibrinogen (p = 0.0001), crp (p = 0.001), il-6 (p = 0.013), leukocytes (p = 0.0001) and sedimentation rate (p = 0.008) in female t2d population compared to control subjects. interestingly, a significant correlation was shown between crp and hba1c (p = 0.035), il-6 and glucose (0.032), il-6 and bmi (0.007). in our study, female t2d compared to the healthy population had significantly higher levels of fibrinogen, leukocytes, il6, crp and sedimentation. other studies conducted in female population associated elevated levels of il-6 and crp with t2d independent of other risk factors for diabetes. crp being most robust predictor of diabetes. studies have shown that crp is an important predictor of t2d for the female but not the male population. thus, our data suggest that inflammation play an important role in the pathogenesis in female diabetic population. a more detailed study on a far larger number of subjects should point out fact if they can effectively be used as biomarkers in the primary prevention of t2d in this population. objectives: bone and mineral metabolism disorders hold an important place among the complications after renal transplantation. the purpose of this study was to demonstrate the relationship between vitamin d, calcium, phosphorus metabolism with graft function and to measure 1,25(oh) 2 d 3 levels with lc-ms/ ms in renal transplant recipients. design and methods: this study included 30 renal transplant recipients (10 female, 20 male; mean age: 40.30 ae 12.86) from living related donors were transplanted. blood samples were collected immediately before and after transplantation at month 6. serum creatinine, bun, calcium, phosphorus, alkaline phosphatase, glucose, albumin, pth, 25(oh)d and 1,25(oh) 2 d 3 levels were measured. gfr values were estimated by ckd-epi. plasma 1,25(oh) 2 d 3 levels were determined in a lcms-8040 triple quadrupole tandem mass spectrometer (shimadzu corporation, japan) by mrm. spss 20.0 software was used for statistical analysis. results: although plasma 1,25(oh) 2 d 3 levels significantly increased (p = 0.0001), we did not find any significant differences for serum 25(oh)d levels after transplantation. when posttransplant levels of serum phosphorus, pth, creatinin, bun and alp levels were found to be significantly decreased (p = 0.0001, p = 0.011 for alp), we observed significantly higher calcium and gfr values (p = 0.0001). vitamin d insufficiency was present 13.3%, deficiency 36.7%, severe deficiency 50% before transplantation, insufficiency was also seen 26.7%, deficiency 50%, severe deficiency 23.3% after transplantation at month 6. conclusions: in our study, all patients were found to vitamin d deficiency and insufficiency. determination of vitamin d deficiency and consequently treatment with vitamin d supplements could lead to better graft surveys. free fatty acids (ffa) represent important link between obesity, insulin resistance, type 2 diabetes (t2d), and dyslipidemia. increased adiposity, as approximated by body mass index (bmi), correlates well with increased serum levels of leptin-adipocyte derived hormone implicated in the regulation of adipose mass and alterations in insulin action and secretion. the main objective of the present study was to investigate the potential association of serum ffas with leptin levels in healthy and newly diagnosed type 2 diabetic subjects. this study involved 13 newly diagnosed type 2 diabetics and 13 healthy subjects. all participants in the study were free of evidence of hepatitis, viral infection or active liver and kidney injury. for biochemical analyses of glucose, glycosylated hemoglobin (hba1c), and lipid profile, standard ifcc protocols were used. analysis of free fatty acids (ffas) was done by gas chromatography, while serum leptin levels were determined by the elisa kit. in addition to the expected differences in glucose, hba1c, and bmi, our results also showed significant differences in leptin, myristoleic, palmitic, linolenic, arachidic, and arachidonic acids between t2d and control subjects. in healthy subjects, a significant correlation was demonstrated between glucose and lauric, arachidic, arachidonic acid levels, body weight, and bmi. newly diagnosed diabetics showed significant association between glucose and lauric, myristoleic and linolenic acid levels; with leptin being associated with myristic and palmitoleic acid levels. interestingly, in all participants, significant association was found between glucose and hba1c, glucose and leptin, myristoleic, arachidic, and bmi as well as between leptin, arachidic acid, and bmi. thus, our data point out association of different types of ffas with leptin levels in newly diagnosed type 2 diabetics. however, further studies should be done in larger number of patients to confirm our results. rheumatoid arthritis (ra) and ankylosing spondylitis (as) are chronic inflammatory diseases with distinct clinical manifestations in many ways. the aim of this study is to evaluate the serum levels of molecules which may be used as markers for angiogenesis and vascular leakage in the processes of two clinically different pictures, ra and as. 30 ra patients, 30 as patients and 30 healthy volunteers with mean age of 30-50 were included in the study. serum levels of vegf, angiopoetin-2 and tie-2 were measured by enzymelinked immuno-sorbentassay (elisa) using a commercially available kit. serum nitricoxide levels were evaluated by the griessreaction. serum vegf, ang-2 and no levels were significantly higher in the as group ml, p < 0.001; p < 0.001; p < 0.05). no differences were found between as and ra for tie-2 (p > 0.05). vegf, ang-2, tie-2 and no levels were positively correlated in both as and ra patients (p < 0.001), but no correlation was detected between clinically activation index das-28, basda _ i scores and laboratory measurements such as sedimentation, crp and anti-ccp (p > 0.05). when the diagnostic performance of the parameters were evaluated with the roc analysisonly the performance of the ang-2 in as patients was sufficient (auc (95% cl): 0.718, p < 0.05). elevation of angiogenic factors in the serums of as and ra patients supports the role of angiogenesis in the etiopathogenesis of these diseases. however, lack of relationship between disease activity leads to not to use these factors as a marker for clinical follow-up. only ang-2 measurements may be useful for the differantial diagnosis. the evaluation of ischemia modified albumin as an early biomarker of acute myocardial infarction introduction: acute myocardial infarction (ami), remains a leading cause of morbidity and mortality worldwide. early diagnosis of ami is very important because early treatment may reduce the extent of injury to the myocardium. currently, biomarkers of myocardial necrosis such as myoglobin, ck-mb and troponins are highly sensitive and exhibit good specificity. however, these biomarkers increase after tissue injury, approximately 4-6 h after the cardiac event and detect only the consequences of prolonged ischemia. recently, ischemia modified albumin (ima) has been assessed and found to be very useful for the diagnosis of myocardial ischemia and it is considered as a serum biomarker. the aim of the present study was to evaluate the serum level of ima and determine the relation between patients with ami and control group, in order to verify its potential as a novel marker for early detection of mi. materials and methods: the study was performed with 25 patients and 37 healthy controls. blood samples from all subjects were collected by venipuncture in plain tubes, and immediately centrifuged at 4000 g for 10 min at 4°c. the serum samples were stored at à20°c until analysis. the serum levels of ima were determined using the cusabio biotech human ischemia modified albumin, elisa kit according to manufacturer's instructions. the results are given as international units/milliliter (iu/ml). results: our findings revealed that ima showed no significant difference between the groups. conclusion: our results suggest that ima assay is not a sensitive marker for early detection of ischemic hearth disease and cannot be used alone for the diagnosis of ami. prospective studies are needed to identify ima's potential as a biomarker for ami. p-04.02.2-015 neutrophil-to-lymphocyte ratio and platelet-tolymphocyte ratio in polycystic ovary syndrome polycystic ovary syndrome is a complex and multifactorial disease with metabolic dysfunction and the etiopathogenesis is not well established. emerging data suggest that adiposity and chronic low-grade inflammation are involved in the development of the metabolic dysfunction. neutrophil-to-lymphocyte ratio (nlr) and platelet-to-lymphocyte ratio (plr) have recently been investigated as two new inflammatory markers used in the assessment of systemic inflammation in many diseases. the purpose of the study was to investigate their relation with pcos patients. the study population consisted of 44 patients with polycystic ovary syndrome and 23 healthy women controls. nlr and plr obtained by dividing absolute neutrophil to absolute lymphocyte count and absolute platelet count to absolute lymphocyte count, respectively. the neutrophil count (4.41 ae 1.6 vs. 3.77 ae 1.23, p < 0.05) and platelet count (320.34 ae 55.11 vs. 283.96 ae 52.17, p < 0.05) were higher in patients with pcos compared to the control group. lymphocyte count was 2.08 ae 0.43 in pcos patient and 2.30 ae 0.57 in control group. the nlr and plr of pcos patients were significantly higher compared to those of the controls (2.18 ae 0.92, 1.69 ae 0.57 p < 0.05, 158.31 ae 32.85, 128.59 ae 31.94 p < 0.05, respectively). in this study we found nlr and plr were significantly increased in patients with pcos compared to healty control. nlr and plr were two useful inflammatory markers for assessment of patients with pcos. imbalance in neurotransmission in conjunction with neuroinflammation contribute to neurological dysfunction observed during acute liver failure (alf). own observations indicate that alf in a mouse model is associated with altered expression and/or intracellular distribution of synaptic proteins. since neutralization of tgf-b1 appears to improve the neurological score in alf mice, we examined the possibility that increased levels of tgf-b1, caused by liver damage, may affect the expression of selected synaptic proteins. expression and/or cytoplasmic vs. membrane distribution of a number of functionally critical synaptic proteins in cerebral cortex and blood tgf-b1 were measured in c57bl6 mice with alf induced by single i.p. injection of aom (100 mg/kg of b.w.) and after neutralization of tgf-b1 induced by single i.p. injection of ab-tgf-b1 (1 mg/kg) 2 h before aom injection. in alf mice, blood tgf-b1 was increased, and the expression of presynaptic proteins: synaptophysin and synaptotagmin was increased in the cytosolic (s2) fraction by~45% and 30%, respectively, but was slightly depressed in the membrane (p2) fraction by~20% and~15%. aom induced an increase of postsynaptic proteins: psd-95 and nnos by~40% in p2 fraction. tgf-b1 neutralization resulted in a reduction in the expression of presynaptic proteins by~30% in s2 fraction and~20% in p2 fraction, in control animals and normalized their amount in the cytosolic fraction after aom injection, but was ineffective with regard to psd-95 and nnos. the results indicate that in alf mouse, neutralization of cytokine tgf-b1 normalizes synaptophysin and synaptotagmin expression in the synaptoplasm, without affecting their synaptic membrane content. effect of tgf-b1neutralization appear to be confined to the presynapse. 25% of acute pancreatitis (ap) patients develop severe acute pancreatitis (sap), which is is resulted in multiple organ dysfunction syndrome. an extensive inflammatory response occurs due to inflammatory mediators synthesized and secreted during sap. since preventing the inflammation in sap is important in the prognosis of the disease, new drug candidates having strong antiinflammatory effects will provide a new concept for therapeutic strategies against acute pancreatitis. non-steroidal anti-inflammatory drugs (nsaids) show their effects by inhibiting cyclooxygenases (cox-1 and cox-2) and they play an important role in the pathogenesis of acute pancreatitis. since conventional nsaids inhibit both cox-1 and cox-2, they have serious side effects on gastrointestinal system. therefore, new highly selective cox-2 inhibitors having fewer side effects are needed. in the present study, selective cox-2 inhibitory activities and cytotoxic effects of new series of 2-benzoxazolinone and thiazolo [3,2-b ]-1,2,4-triazole derivatives previously synthesized as specific cox-2 inhibitors with no side effects on gastrointestinal system were investigated. permeability of the compounds was tested by pampa using caco-2 cells. compounds were found highly selective, non-toxic and permeable. ap was induced in rats via retrograde injection of stc into the pancreatic duct system. rats were pre-treated with saline or celecoxib or the new compounds before stc injection and sacrificed 24 h later. the severity of ap was evaluated using biochemical and histopathological analyses. edema, inflammation, hemorrhage and acinar cell necrosis were detected in the pancreatic tissue of sap group. sap was remarkably increased serum lactate dehydrogenase, ast, alt, lipase and amylase activities and serum tnf-a, il-1b, il-2, il-6 and il-8 levels. tissue myeloperoxidse activity was also increased. pretreatment with the novel compounds reserved all these biochemical and histopathological parameters. alopecia areata (aa) is an inflammatory disease which is affects hair follicles, and sometimes nails. it is suggested that cytokinemediated immunity plays an important role in etiopathogenesis of aa. this study was planned to evaluate the serum ykl-40 and tgf-b1 levels of patients with aa. 40 patients with aa and 40 healthy volunteers were recruited into the study. fasting venous blood samples were collected from the participants and serum was obtained by centrifugation. serum ykl-40 and tgf-b1 levels were measured by enzyme linked immunosorbent assay (elisa). serum tgf-b1 levels in the patient group were significantly lower compared to the control group whereas serum ykl-40 levels were significantly higher in patient group. tgf-b1 levels of men and women with aa were found to be significantly lower than that of controls. while serum ykl-40 level of male control group is higher than the male patients, there were no significant differences between women groups. the increased serum ykl-40 levels in aa patients suggests that ykl-40 plays a crucial role in the pathogenesis of aa. arterial immune mediated inflammation participates centrally in all stages of the development of atherosclerosis, from the initial lesion to the end-stage thrombotic complications. although emerging evidence supports augmented cardiovascular morbidity and mortality in cutaneous psoriasis (psc) and psoriatic arthritis (psa) as compared to the general population its underlying mechanism is poorly understood. here we analyzed the inflammatory burden in recent onset of psa patients without traditional cardiovascular risk factors (cvrf) in a transversal study measuring carotid intima media thickness (cimt) (measured with ecodoppler), and proatherogenic inflammatory molecular markers like c-reactive protein (crp), interleukin 6 (il-6), and soluble intercellular adhesion molecule-1 (sicam-1) in comparison with control patients. cimt values are similar in psa (0, 59 ae 0.045*) and psc (0, 62 ae 0.10) patients. however, both of them were significant increase compared with control (0.436 ae 0, 05). regarding inflammatory markers il-6 serum levels in patients with aps was higher than pcs (18 ae 2.9) and healthy controls (16, 3 ae 2.5) but the difference did not achieve statistical significance (*p > 0.05). on other hand mean of sicam-1, value from patients with recent onset of psa is significant higher than controls. psc remain without significant changes compared to control (*p > 0.05). in addition mean value from patients with recent onset of psa is significantly higher than in controls (*p < 0.05) and psc group. overall, preliminary findings suggest for the first time that patients with early psa, without evident traditional cvrf have significant increased values of cimt, sicam-1 crp against the general population control group. this data strongly supports that early cv molecular markers are increased after the first symptoms and signs of this disease even in the absence of traditional cardiovascular risk factors. furthermore, this give new windows for a proper treatment. p-04.02.2-020 protective effect of trail against proinflammatory cytokines on pancreatic beta cells correlated with decrease in dr5 and increase in dcr1 expressions universitesi, antalya, turkey introduction: proinflammatory cytokines are known to have destructive effects on beta cells, which contribute to type 1 diabetes (t1d) development. the combinatory effects of three of these cytokines in particular, namely tnf-alpha (tnf-a), ifngamma (ifn-c), and il-1beta (il-1b), are claimed to render beta cells prone to t cell-mediated destruction. the recently identified anti-inflammatory feature of tnf-related apoptosis-inducing ligand (trail), its possible protective role in this process. in this study, the effects of applications of trail with tnf-a, ifn-ᵞ, and il-1b on beta cell viability and correlation of these effects with trail receptor expression patterns were investigated. methods: glucose-responsive insulin-secreting nit-1 mouse beta cell lines were treated with tnf-a, ifn-ᵞ, il-1b, and soluble trail (strail) individually and in various combinations. cell viabilities were determined at 24 and 48 h by mtt assay. trail ligand and receptor expression profiles on nit-1 cells, and alterations in receptor expression levels following cytokine applications were determined by western blotting analysis. results: trail treatment did not have any cytotoxic effects on nit-1 beta cells at 48 h, while increasing cell viability following il-1/ifn/trail and il-1/tnf/trail combined applications. substantial levels of death receptor 5 (dr5) expression were detected on nit-1 cells before applications, yet it displayed decreased levels at 48 h of trail treatment. lower levels of decoy receptor 1 (dcr1) expression detected prior to treatments increased significantly in contrast. discussion: the fact that trail co-treatment with tnf-a, ifn-ᵞ and il-1b increased cell viability in nit-1 beta cell lines along with reduction in dr5 death receptor expression and an increase in the decoy receptor dcr1 expression, points out to a possible protective effect of trail in insulitis, and strengthens its potential as a putative therapeutic molecule in prevention of beta cell loss. behc ßet's syndrome (bs) is a multisystemic inflammatory disorder with a strong and complex genetic background. being a prevalent disorder both in turkey and also in the ancient trade road 'silk road' countries, bs is an important cause of impairment and disability owing to its chronic and relapsing nature. besides, bs is reported to be an important cause of mortality among the young male patients. while the epidemiology of bs is substantially well documented, currently, the etiology, the molecular mechanisms underlying its pathogenesis, and the classification of the disorder remain to be elucidated. our aim was to disclose the disease mechanisms at molecular level in turkish bs patients by obtaining, comparing, and analysing the transcriptome data of bs patients with age and gender matched healthy controls. for this purpose, by using the affymetrix hg u133 plus 2.0 microarrays, peripheral blood cell mrna profiles of 30 bs patients (b) and 15 matched healthy controls (c) were obtained. following bioinformatics, gene ontology, and pathway analysis, validation experiments of the identified prominent mrnas were performed by qrt-pcr methodology. the comparison of b vs. c yielded differentially expressed gene numbers of 30 and 625 for the chosen fold changes of 2.0 and 1.5 respectively (p ≤ 0.05 for both). during gene ontology and pathway analysis, immune system process, immune system diseases, systemic lupus erythematosus, arthritis, and intestinal immune network for iga production categories/pathways were significantly enriched. clustering analysis revealed a molecular signature which accurately distinguished b and c samples, while the qrt-pcr analysis successfully validated the chosen mrna transcripts. this study documented differential expression of a large number of immune system and immune disease related genes in bs patients. the uncovering of the molecular disease mechanisms of bs will point to novel candidate molecules to be targeted for the treatment of the disorder. obesity is a public health problem in developed countries and worldwide with increasing prevalence through a relationship primarily with atherosclerotic cardiovascular diseases as well as several metabolic disturbances such as increased insulin resistance and diabetes. although several studies identified obesity as an independent risk factor for atherosclerotic cardiovascular diseases, the mechanism underlying the increased cardiovascular risk in obese patients has not been clearly delineated. adma, no, endothelin-1 and homocysteine are an indicator of endothel disfunction that plays an important role in the pathophysiology of atherosclerosis. in our study, obese children and the control group were compared in terms of adma, no, endothelin-1 and homocysteine, we also investigated whether there is a correlation between these parameters. 58 obese and 30 healthy children, participated in the study. when the obese group was compared to the healthy controls, the adma level of the obese group were significantly higher than those of the control group but there was no statistically significant difference in no, endothelin-1 and homocysteine. increased adma level might trigger the pathogenesis of atherosclerosis starting from the childhood years onward. that is why controlling obesity in this age group with diet and other treatment modalities will prevent the mortality and morbidities that will be seen in adult years. inh deficiency leads to the formation of bradykinin causing to dilation of blood vessels. furthermore, the study conducted by shagdarsuren, on the damage done by c1-esterase, demonsrates that the complement system and triglyceride levels are affected. we investigated lipid oxidation and fetuin a levels in patients with c1 _ inh deficiency. materials and methods: 44 people with c1 _ inh and 44 people without any illnesses were taken into the study. fetuin a was studied using an el _ isa kit from raybio (usa). ferrous ion oxidation-xylenol orange test was used to find looh serum levels. sh (free thiol groups) test was studied with regards to ellmans method modified by hu. _ ibm spss 20.0 was used for statistical results. results: in assessments made between the healthy and the illness groups, there was significant differences in the levels of fetuin (p = 0.035), looh (p = 0.000) and sh (p = 0.047). when pearson correlation analysis was performed, we detected a significant positive correlation between fetuin a and looh levels (r: +0.326) discussion and conclusion: in these patients, lipids is secreted from the adipose tissue. in response, anti-atherosclerotic fetuin a levels were risen. patients also possessed increased lipid peroxidation, this increase shows positive correlation with fetuin a levels. in conclusion, we identified that sh with antioxidant properties have increased levels. aim: high fructose corn syrups are found in soft drinks, juice beverages, breakfast cereals, most of the processed foods. it has been shown that high dose of fructose intake may lead to a reduction in the number of hepatocytes, deterioration of liver function, increasing reactive oxygen species and liver steatosis. the aim of this study was to explore whether caffeic acid phenethyl ester (cape) has any potential protective effect on high fructose diet-induced fatty liver model. materials and method: totally fifty rats were divided into five groups. control group, %10 fructose administered group, cape group, %10 fructose + cape administered group and ethanol group. after 6 weeks, liver oxidant and antioxidant status, and blood tnf alpha, il-6, and il-8, tissue nfkb levels were quantified. protein levels were investigated against, nfkb and p-nfkb antibodies and normalized and analyzed against b-actin antibody by western blotting. results: serum tnf-alpha, il-6, il-8 levels were found to be increased in fructose group compared with the control group (p < 0.05). in liver tissue of 10% fructose administered group, mda, protein carbonyls and no levels were higher than control group. however sod activity did not show any difference among the groups. in the fructose administered group, caspase 3 showed liver apoptosis and was considered as positive. acquired data revealed that nfkb protein level was decreased in the presence of cape while increment in nfkb protein level was observed in the fructose administered group compared with control group. in case of pnfkb antibody, increment observed in fructose only and both cape and fructose administered groups, respectively. in cape only administered group, there was a decrement in the level of pnfkb protein. conclusion: depending on further analysis, experimental findings are expected to implicate the role of cape as a protective agent on high fructose diet-induced fatty liver model in relation of inos, nfkb and p-nfkb pathways. the investigating association of hepcidin levels with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction a. g. agg€ ul 1 , n. uzun 1 , e. c ß inar tanriverdi 2 , h. € un 1 1 agri ibrahim cecen university, agri, turkey, 2 nenehatun maternity hospital, erzurum, turkey this study was designed to investigate hepcidin levels and their associations with iron homeostasis and inflammation variables in pregnant women with intrauterine growth restriction (iugr). a total of 88 pregnant women were included in this study. pregnant volunteers were divided into two groups (30 healthy pregnant women and 58 pregnant women with iugr). serum hepcidin, total free iron, ferritin, transferrin, transferrin receptor and interleukin-6 (il-6) levels were measured by elisa. also, hemoglobin (hb) and c-reactive protein (crp) levels were determined in serum samples from the healthy pregnant women and the pregnant women with iugr. there were significant differences in hepcidin, ferritin, transferrin receptor, crp and il-6 levels between healthy pregnant women and pregnant women with iugr. hepcidin, ferritin, crp and il-6 levels in pregnant women with iugr were significantly higher than healthy pregnant women (p). the mediators of systemic inflammation in lipopolysaccharide-induced neonatal sepsis rat model sepsis is an excessive inflammatory response that causes shock, multi-organ failure and high mortality. foreign bacterias and lipopolysaccharides lead to stimulation of endothelial cells to produce biologically active mediators such as proinflammatory cytokines and chemokines, cell adhesion molecules, and growth factors. then these mediators could be act on targets, which were involved in the initiation of systemic inflammation in neonatal sepsis. our aim was to indicate a protective role of thalidomide and etanercept, which have anti-tnf-a activity on systemic inflammatory response in lipopolysaccharide (lps)-induced neonatal sepsis rat samples. thirty 7-day-old wistar rats were randomly divided into five groups: a control group that received normal saline, a sepsis group that received lps, thalidomide, etanercept and both thalidomide and etanercept treatment group that were administered with therapeutic agents 6 hrs after lps injection. the rats were sacrificed at 24 hrs after lps or normal saline injection (n = 6). hepatic tissue tnf-a, il-6, icam-1 and pdgf levels were determined by enzyme-linked immuno sorbent assay (elisa) method in all groups. in sepsis group, tissue tnf-a, il-6, icam-1 and pdgf levels were statistically significantly higher than in controls (p < 0.001). at same time, pretreatment with both thalidomide and etanercept were found statistically dramatically decreases the levels of tnf-a, il-6, and pdgf when compared to sepsis group (p < 0.001). there were no significant differences in the icam-1 levels between the all treatment groups and the sepsis group. higher liver tissue tnf-a, il-6, icam-1 and pdgf levels are associated with severe bacterial infection. these proinflammatory cytokines and angiogenic factors may be important in the endothelial dysregulation seen in sepsis. therapeutic agents used in the present study can be help to avoid devastating effects of neonatal sepsis. n-stearoylethanolamine (nse)is saturated minor compound of natural origin that represents the large family of signaling lipids n-acylethanolamines, which belong to endocannabinoid system. considering the crosstalk between obesity-induced inflammatory response and its key role in synaptic dysfunction and neurodegeneration, our current study aimed to investigate the biological effect of nse on brain tissue under high fat diet-induced insulin resistance. previously we found that nse administration to insulin resistant rats caused normalization of liver and pancreas lipid composition followed by the improvement of glucose tolerance and insulin sensitivity (decline in serum insulin level and homa-ir value). moreover, this effect of nse correlated with inhibition of nf-kb translocation into the nucleus of peritoneal macrophages and decreased pool of serum tnfa level in obesity-induced insulin resistant rats. further experiments showed that fat overload triggered significant reduction in the level of main phospholipids (phosphatidylethanolamine, phosphatidylcholine and sphingomyelin), while there were no changes in cholesterol content. nse at a dose of 50 mg/kg during 2 weeks of administration to insulin resistant rats showed a tendency to restore the phospholipid level that was accompanied by increased neural cell survival (91%) compared to rats without treatment (84%). neuroinflammation accompanied by intensive reactive oxygen species (ros) production impairs neurotransmission in a wide range of neurodegenerative pathologies. flow cytometry is used for quantitative analysis of global dna methylation, but fluorescence microscopy is mostly preferred to qualitatively reveal intranuclear localisation of dna methylation and its copattern with other markers. both methods use a similar immunostaining protocol. in this study, we aimed to compare these methods concerning the detection of the global amount of dna methylation. for this, mouse embryonic fibroblasts were cultured either with or without phenol red and then stained for dna methylation or positive controls (histone, betaactin, phosphoakt) by specific antibodies, or nonspecific control antibodies. some cells were incubated with trypan blue before or after the addition of antibodies. fluorescence intensities were measured by the green fluorescence channel (530/30 nm). autofluorescence spectrum of cells was analysed, and fluorescence channel used for dna methylation detection was changed to red (650 nm lp). a poor discrimination between signal and noise was detected due to cellular autofluorescence interfering with specific detection of dna methylation by flow cytometry but not by microscopy. it was also the case for the other markers examined. conventional advances to reduce autofluorescence such using phenol red free culture media or trypan blue quenching were not effective, but using the red channel regarding autofluorescence spectra allows detecting specific staining of dna methylation by flow cytometry. but, green channel did work well for microscopy analysis. findings show that flow cytometry detection of dna methylation requires much attention to quench cellular autofluorescence compared to detection by fluorescence microscopy. one reason could be that flow cytometry detects all cellular content, but manual image-based analysis can exclude cytosolic components. these results suggest the usability of flow cytometry and microscopy as complimentary methods for dna methylation detection, but optimisation to reduce autofluorescence is crucial for flow cytometry. objectives: lung cancers are divided in two main groups as small cell lung cancer (sclc) and non-small cell lung cancer (nsclc) . docetaxel (dtx) and cisplatine are chemotherapeutic that has an anti-tumor activity against various solid tumors. the growing resistance against dtx and cisplatine (cis) still continues to be the biggest obstacle for the treatment success of nsclc patients. deguelin (deg.) is a natural plant derivative and has an encouraging activity against a lot of human cancers. the comparison of the treatment activity of the separate and combined usage of deg., which is a potential chemotherapeutic agent, and dtx, cis which are used in standard treatment, is aimed in this study. material-method: the ic50 doses of dtx, cis and deg. on the a549 and h1299 nsclccell lines were determined via the cell vitality tests in our study. the active concentrations determined were applied to nsclc cell lines as deg., dtx, cis and their combinations. the impacts of the medicine are studied by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, colony formation, migration and angiogenesis analyzes on the treated cells and measuring the oxidative stress index (osi). statistical analyse program, rstudio (v.0.98.501) and the r-script language were used to examine the differences between the agents. the states in which the pvalue was lower than 0.05 were accepted as statistically meaningful. results: we found that deg. has pro-apoptotic, anti-migratory and cytotoxic potential on lung cancer cells. deg. amplified cis and dtx-related anti-cancer efficacy (increased apoptotic cell content and cytotoxicity, reduced migration). also, deguelin pretreatment sensitized the cells dtx-treatment (reduced ic50 values). these effects were remarkable in p53-mutant cells. conclusion: deguelin, solely, has anti-cancer potential on nsclccells. both deguelin pre-treatment and combinantion with standart chemotherapeutics result in enhanced anticancer efficacy. the 83% of the lung cancers are non-small cell lung cancers (nsclc). despite docetaxel (dtx) and cisplatine (cddp) are agents used in the standard treatments of these patients and the recent improvements in the treatments, the response and remission rates observed on the patients are relatively nominal. selenium (se) is an essential diet component and is introduced to have a preventive impact on different levels of cancer. the aim of our study is to investigate the impacts of selenium addition on anticancer feature and tumor prevention before or/and during nsclc standard treatment. the ic50 doses of dtx, cddp and selenium on the a549 and h1299 (p53 mutant) nsclc cell lines were determined via the cell vitality tests in our study. the active concentrations determined and the stipulated available concentrations were applied to cell lines as dtx, cddp, se combinations. the impacts were compared by applying flow cytometric analyzes (apoptosis, cell cycle), glutathione and reducted glutathione, western blot analyzes on the treated cells and measuring the oxidative stress index (osi) and thioredoksin reductase activity. selenium pre-treatments reduced dtx-related ic50 concentrations at lower doses in both nsclc cells. however, cddprelated ic50 concentrations reduced dose-dependent manner. selenium supplementation also altered cell-cycle charactheristics at several concentrations and combination regimens. the remarkably higher osi values were observed after dtx treatment and osi levels were found to be lower in selenium pre-treated nsclc cells. selenium sensitizes nsclc cells to dtx treatment at lower concentrations. however, this effect is obtained dose-dependent fashion for cddp regimen. breast cancer is the most common female malignancy worldwide. human epidermal growth factor receptor 2 (her2) is overexpressed in 30% of breast cancers in association with aggressive phenotypes. the prognosis of metastatic breast cancer remains poor in spite of advances in therapy. as such, her2 has long been studied as a potential target for anticancer drugs. the modulation of intracellular signaling pathways leads to altered cell metabolism that triggers tumorigenesis and adapts cells to cancer cell metabolism. this characteristic hallmark of cancer metabolism is known as warburg effect meaning energy production via enhanced glycolysis. despite of several studies in breast cancer metabolism, little detail exists on the link between her2 overexpression and warburg effect. we have committed examining the nature of aerobic glycolysis in her2 overexpression. in breast cancer cell line mcf7, her2 overexpression (mcf-her2) results in mitochondrial dysfunction with low mitochondrial membrane potential (δᴪm) and ros accumulation. intracellular iron levels are also higher in mcf7-her2 cells than vector control (mcf7-vec). additionally, mcf7-her2 cells show enhanced levels of atp and lactate in association with increase in glucose levels. we have found that complex i activity increases in mcf7-her2 and decreases in knockdown of her2 in hcc1954 cells that is her2 positive breast tumor cell line. based on these results, we conclude that there is a link between her2 overexpression and metabolic indicators of warburg effect. expression and methylation analysis revealed 11 microrna genes deregulated by methylation and new potential target genes of mir-375 and mir-127-5p in breast cancer micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to assess the contribution of methylation to expression level alterations of 20 mirna genes and to search for novel potential targets of these mirnas. to analyze alterations in expression we used qpcr technique with 2 references (rnu6, rnu48) and 30 paired (tumor/normal) breast cancer (bc) samples. for methylation analysis a methylation specific pcr and the same set of bc samples were used. significant downregulation was shown for mir-125b-5p, -129-5p, -132-3p, -193a-5p, -34b-3p, -212-3p, -127-5p, and -17-5p (p ≤ 0.05, fisher's exact test) in bc. we observed 10 mirna genes to be hypermethylated and mir-191hypomethylated. hypermethylation for 3 of these mirna genes was shown for the first time: mir-132, -137, and -1258 (41-34% of bc cases). a significant correlation between methylation and expression alterations was revealed for 5 mirnas with downregulation: mir-125b-5p, -129-5p, -132-3p, -193a-5p, and -34b-3p (spearman's correlation coefficient (rs) was in the range à0.71 to à0.81, p ≤ 0.01), and for 3 mirnas with both scene (down-and upregulation) as well: mir-148a-3p, -203a, and -375 (rs = à0.72 to à0.93, p ≤ 0.01). comparative analysis of the data on expression alterations of 20 mirna genes and 6 protein-coding genes, which were predicted as targets by mirwalk 2.0, revealed the negative correlation between expression levels for some potential mirna-mrna interaction pairs. for example, for pairs mir-375/rhoa, mir-375/rassf1(a), mir-127-5p/dapk1 (rs = à038 to à0.46, p ≤ 0.05). thus, both mirnas and methylation affect regulatory networks in bc. novel potential mirna-mrna interaction pairs could be useful in the development of bc therapy approach. this work was financially supported by grant 14-15-00654 from the russian science foundation. the authors thank the n.n. blokhin cancer research center for tissue samples. clear cell renal cell cancer (ccrcc) with metastases has pour prognosis: 5-year survival is about 9%. micrornas (mirnas) and methylation of mirna genes play a great role in epigenetic deregulation in malignant tumors. the aim of our study was to find out mirnas which methylation contributed to ccrcc progression, including metastasis, and to reveal potential target genes of these mirnas. to analyze methylation status, we used a methylation specific pcr as a method and a representative set of 70 paired (tumor/ normal) ccrcc samples. we also used 19 post-mortal renal tissues from individuals without cancer history as additional control. for expression analysis we used qpcr method and 45 paired ccrcc samples. we observed 9 mirna genes (mir-124a-1/-2/-3, -9-1, -9-3, -34b/c, -129-2, -193a, -107) to be hypermethylated, (p ≤ 0.05, fisher's exact test), 3 mirna genes to be hypomethylated and mir-203a with both scene (hyper-and hypomethylation was detected). methylation of 7 mirna genes (mir-124a-2/-3, -34b/c, -129-2, -107, -148a, -203a) correlated with advanced stage and/or tumor size and/or dedifferentiation. hypermethylation of mir-129-2, mir-203a, and mir-107 significantly correlated with metastasis presence (p < 0.05, fisher's exact test). besides, preliminary data revealed the positive correlation between hypermethylation of mir-129-2 and up-regulation of 3p protein-coding genes: rarb(2), rhoa, nkiras1, and chl1, which were predicted as targets by mirwalk 2.0 (spearman's correlation coefficients (r s ) was in the range 0.35-0.53, p ≤ 0.05). in conclusion, novel supposed interactions of mir-129-2 with target genes could be useful as missing chains in signaling pathways. tests for hypermethylation of mir-129-2, mir-203a, and mir-107 could be suggested as markers of metastasis and pour prognosis of ccrcc. because of difficulty in diagnosis and treatment hc is a clinical problem: early symptoms of hc are often non-specific and surgical resection is the only curative treatment for hc. it is well known that epigenetic alterations are linked to cancer development. the purpose of this study was to determine potential mechanisms of epigenetic regulation of genes related to energy metabolism in hc. we have performed bioinformatics analysis of the cancer genome atlas (tcga) project rna-seq data with crosshub software and found a number of genes involved in glycolysis and differentially expressed in cholangiocarcinoma. qpcr analysis revealed significantly decreased expression of pgm1 and eno3 genes in a majority of hc samples which were known as up-regulated in other human cancers according to the literature date. on the basis of tcga methylation dataset (450k illumina microarrays) we supposed that cpg methylation of pgm1 and eno3 promoters may play a role in their inactivation. using bisulfite sequencing study we identified several regions within the gene promoters (pgm1:~750 bp and 330 bp upstream tss; eno3:~750 bp downstream tss) that are frequently methylated in hc samples (up to 60%, 12/20) with down-regulated pgm1 and eno3 expression. thus, we demonstrated frequent and significant pgm1 and eno3 down-regulation associated with hypermethylation of the specific regions within the gene promoters in hc. the pattern of pgm1 and eno3 gene promoter methylation suggests a possibility of ones to be used for the hc diagnosis and development new strategies for therapy. this work was financially supported by grant mк-8047.2016 .4 from the president of the russian federation. the work was performed using the equipment of eimb ras 'genome' center. introduction: the development of stomach cancer is a multifactorial and complex process and includes multiple epigenetic, genetic alterations and dietary/non-dietary factors. iodine as an antioxidant may play a protective role against gastric cancer. the aim of this study was to investigate the changes in iodine level in rat with stomach cancer induced by n-methyl-n 0 -nitro-n-nitrosoguanidine (mnng). materials and methods: a total of 62 sprague dawley rats were randomly divided into six groups. 55 rats were administered with mnng (200 lg/ml) by oral gavage on days 0, 14 and 21 to initiate stomach cancer. during the experiment, 28 rats died and those surviving were sacrificed in the 3rd, 5th, 7th, 10th and 12th months of the experimental period (group i, ii, iii, iv, v, respectively). the control group (group vi) contains 7 rat which are given only food and water for 12 months. the stomach tissue was examined histopathologically. and also, iodine levels in stomach tissue was determined using the foss method. results: a decrease in iodine level was determined in stomach cancer tissue of rats in group i-v compared with normal healthy stomach tissue in group vi. when the control (group vi) iodine level was taken as % baseline, the % iodine levels of all groups were determined as follows 71.78, 52.79, 40.76, 25.33 and 11.96 for groups i-v, respectively. the pathological diagnosis of gastric cancer was adenocarcinoma. discussion and conclusion: the iodine levels of group i were higher than those of group ii (p < 0.01) and of groups iii, iv and v (p < 0.001) and also were lower than in the control group (p < 0.001). iodine deficiency as one of the risk factors of stomach cancer strongly supports the necessity for the application of effective iodine prophylaxis in the areas with iodine deficiency. iodine supplementation might be useful in stomach cancer therapy and therefore, further research is warranted. this study was supported by ataturk university (project number: 2005/147). effect of water extract of turkish propolis on mitochondrial membrane potential in human laryngeal epidermoid carcinoma cell lines propolis is the generic name for the resinous substance collected by honeybees from the buds of various plant sources and it is used by bees to seal holes in their honeycombs, smooth out the internal walls, and protect the entrance of bee hive against intruders. the aim of this study is to investigate what kind of changes the turkish propolis cause on mitochondrial membrane potential (mmp) of human laryngeal epidermoid cell lines (hep-2), by considering its anticancer features. water extract of turkish propolis (wep, 1-3 mg/ml) and ethanolic extract of turkish propolis (eep, 0.075-3 mg/ml) were prepared and incubated with hep-2 cell lines (24, 48, and 72 h). mmp was investigated with a flourometric method by using dioc 6 (3,3 0 -dihexyloxacarbocyanine iodide). the most significant mmp decrease was seen on 72rd hour. both wep and eep extracts at all concentrations decrease mmp according to that of control. the recent studies have shown that propolis extracts have induced apoptotic cell death by decreasing mitochondrial membrane potential in various cancer cells. it was concluded that both wep and eep decreased mitochondrial membrane potentials on hep-2 cell series according to control (0 concentration) depending concentration and time. there are numerous transcription factors involved in the regulation of the inducible gene expression. thus, transcription of proinflammatory genes, steroid hormone receptors, etc. is controlled by the group of factors triggering gene expression which includes nf-kb. another group of factors is involved in the formation of the structure of the chromatin of the inducible genes regulatory regions, providing competence for gene expression. it is expected that this group of factors includes the proteins of nf1 (nuclear factor 1) family. there are few data suggesting that the nf1 factors maintain potentially active state of the chromatin of the hormone-dependent gene promoter regions. these findings initiated studies of the correlation between presence of the nf1 transcription factors on the chromatin of a gene regulatory region and the functional state of the gene in vivo. as a model we chose the rat tryptophan dioxygenase (tdo) gene which is expressed tissue-specifically in the liver under control of glucocorticoid hormones. three constitutive dnase i-hypersensitive regions are identified in the regulatory region of this gene. to conduct the study we used rat liver and kidney. the basic methods were electrophoretic mobility shift assay (emsa), immunoblotting assay and chromatin immunoprecipitation combined with real-time pcr (chip-qpcr). using emsa we found that the proteins of nf1 family interact with the constitutive dnase i-hypersensitive regions in vitro. immunoblotting assay of the protein fraction from rat liver used in emsa experiments showed the presence of the nf1-b1 isoform. chip-qpcr revealed statistically significant differences in the level of the factor nf1 enrichment of the tdo gene regulatory region between the rat liver and kidneys at p < 0.05. these data suggest the involvement of the nf1 proteins in the formation of the chromatin structure of the rat tdo gene promoter region. reciprocal (9;22) translocation and bcr-abl fusion protein that is responsible for developing leukemia are observed in more than 95% of chronic myeloid leukemia (cml) cases. epigallocathecin-3-gallate (egcg) is a green-tea flavonoid and egcg is proposed as a natural anti-cancer agent. histone modifications which contain histone deacetylases (hdac) and histone acetyltransferases (hat) are parts of epigenetic regulations. hdacs play important roles in different human malignancies including leukemia via activation of abnormal signaling pathways. hdac inhibitors have become remarkable therapeutic molecules for malignancies. the aim of this study is to determine the expression changes of leukemia-related hdacs with the treatment of egcg in k-562 cells. the cytotoxic effect of egcg on k-562 cells was determined in time and dose dependent manner by wst-1 analysis. total rna was isolated from k-562 cells. reverse transcription procedure was performed for cdna synthesis and gene expressions were detected by rt-qpcr. the expression level of hdac4, hdac6, hdac11 gene that supports cell proliferation was down-regulated 2.29, 2.56, 3.81 folds in k-562 cells treated with ic50 dose of egcg, according to control, respectively. our current findings suggest that is a polyphenol egcg may be a hopeful agent in treatment of cml by hdac inhibitory effect. chronic lymphocytic leukemia (cll) is a disorder of morphologically mature but immunologically less mature lymphocytes and is manifested by progressive accumulation of these cells in the blood, bone marrow, and lymphatic tissues. carbonic anhydrase (ca) is a metalloenzyme which is widely distributed in the living world, and it is essential for the regulation of acid-base balance. anti-ca antibodies have been reported in many disorders, such as systemic lupus erythematosus, sj€ ogren's syndrome, rheumatoid arthritis, endometriosis, idiopathic chronic pancreatitis, type 1 diabetes and graves' disease. the goal of this study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in cll. 38 patients with cll and 39 healthy controls were included in the study and ca i and ii autoantibody levels were investigated by elisa. the ca i autoantibody levels of cll group were significantly higher than the healthy group (p = 0.006) while there was no statistical difference between serum ca ii autoantibody levels of the groups (p = 0.301). we found a significant positive correlation between hemoglobin and hemotocrit levels in patients with cll (r = 0.931, p = 0.0001). cut-off value of 0.072 absu for anti-ca i was associated with 92% sensitivity and 41% specificity and a cut-off value of 0.047 absu for anti-ca ii was associated with 48% sensitivity and 85% specificity for predicting cll. the ca i autoantibody levels in patients with cll were found higher compared to control group and the results suggest that ca i autoantibody may be involved in the pathogenesis of cll. genetic and epigenetic aberrations can lead to the activation of oncogenes and inactivation of tumor-suppressor genes (tsgs) followed by the development of malignant tumors. in the present work we evaluated the frequency of alterations of cpg island methylation and dna copy number in 47 paired (tumor/normal) breast cancer (bc) samples using comparative dna hybridization on noti-microarrays and original niman software. the microarrays contained 180 noti-clones associated with 188 chromosome 3 genes. expression alterations were assessed with the use of qpcr technique, ddct method and original atg software. in total, 35 noti-sites with high (15-38% of cases) hypermethylation/deletion (hm/d) frequency were revealed in bc. among genes associated with these sites, there are both known tsgs and tsg-candidates (aldh1l1, vhl, ctdspl, etc.) as well as genes, which involvement in breast oncogenesis was shown for the first time (lrrn1, foxp1, prickle2, etc.). noti-microarray data were verified selectively using bisulfite sequencing for vhl, nkiras1, itga9, lrrc3b, and ctdspl genes. several genes with high hm/d frequency (aldh1l1, ephb1, itga9, and ropn1) were tested for expression alterations using qpcr. frequent (57-90% of cases) and significant (> 2-fold) down-regulation was shown for all of them in bc. the most significant expression loss was observed for aldh1l1 geneon the average 50-fold mrna level decrease in 90% of samples. the involvement of the majority of genes with high hm/d frequency in breast oncogenesis was shown for the first time. these genes are novel tsg-candidates in bc. functional hypermethylation associated with expression loss was shown for aldh1l1, ephb1, itga9, and ropn1 genes thereby strengthening the speculation on tumor suppressor abilities of these genes. methylation and expression analyses of genes, that were revealed by noti-microarrays, were financially supported by grant 14-15-00654 from the russian science foundation. functional hypermethylation of a number of chromosome 3 genes was revealed in colon cancer using noti-microarrays cancer is a disease of genome caused by genetic and epigenetic aberrations. noti-microarrays, that were developed by prof. e.r. zabarovsky, is a unique tool that allows us to simultaneously detect hypermethylation of cpg islands and dna deletionstwo major reasons of inactivation of tumor suppressor genes (tsgs). in the present work, the frequency of chromosome 3 genetic and epigenetic alterations in colon cancer (cc) was evaluated. noti-microarrays, that contained 180 noti-clones associated with 188 chromosome 3 genes, were used for comparative (tumor/normal) hybridization of dna from 24 paired cc samples. data analysis was performed using original niman software. expression alterations were evaluated using qpcr technique and original atg software. in total, 24 noti-sites with 20% and above hypermethylation/ deletion (hm/d) frequency were revealed in cc. among genes associated with these sites, there are several known tsgs and tsg-candidates (for example, vhl, ctdspl, and itga9), but for the majority of genes, involvement in colon oncogenesis was shown for the first time (for example, lrrn1, nbeal2, and ube2e2). the highest hm/d frequency was observed for ankrd28, nkiras1/rpl15, itga9, cmtm6, and gor-asp1/ttc21a genes -38-42%. expression alterations were evaluated for 3 genes with high hm/d frequency (plcl2, prickle2, and ppp2r3a) and significant mrna level decrease (> 2-fold) associated with hypermethylation was shown for all of them in the majority of samples. a number of novel potential tsg-candidates was revealed in cc. functional hypermethylation associated with expression decrease was shown for plcl2, prickle2, and ppp2r3a genes thereby enhancing the suggestion on tumor suppressor function of these genes. this work was financially supported by in many countries, radon is the second leading cause of lung cancer, which accounts from 3% to 14% of cases. it is obvious that the population of all the developed and industrial countries in the world spend most f their time, almost 80%. therefore it is necessary to explore the obtained radiation dose, because of the presence of radon in a room due to the radon emanation from the soil and exhalation from a variety of building materials. the developed countries solve this problem of radon pollution as well as create a special monitoring services. the paper presents some data of 8 genes molecular-genetic analysis from patients with lung cancer who live in almaty located in a foothill area of tectonic faults. the object of research were blood samples obtained from patients diagnosed with lung cancer who are receiving a treatment at the almaty oncology center and living in the city of almaty, where the level of radon activity exceeds the norm approved by the international commission on radiation safety. as a control group relatively people living in the plains, characterized by a lower radon emanation have been considered. to determine mutations in the genes polymerase chain reaction with a subsequent analysis of restriction fragment length polymorphism has been conducted. the pcr products were subjected to hydrolysis by bstni restriction endonucleases haeiii, ras i. disturbances in the genes under consideration to variour types of cancer development. the analysis showed that examinees do not have mutations in the kras gene codons 12-13, which corresponds to a control group consisting of 90 people living in the city of balkhash. on the whole, molecular genetic studies have shown that 44 examined patients do not have mutations in the kras gene. one mutation was been found in the egfr gene. aim: polyps are abnormal growths of tissue that can be found in gastro intestinal system. they are most often found in the colon and rectum. most polyps are noncancerous (benign) however, because of abnormal cell growth, they can eventually become cancerous. the aim of this study is to determine the concentrations of trace element contents in colon and rectum polyp tissues and whether there is any relationship between polyp tissue element levels and the disease. material and method: the present study was conducted on total of 82 individuals including 57 patients and 25 healthy subjects. while receiving normal intestinal tissue from healthy control group; from the patient group both normal tissue and polyp samples were taken during colonoscopy procedure. the concentrations of the elements (al, cr, mn, fe, co, ni, cu, zn, as, se, ag, cd, hg and pb) were determined with induced coupled plasma-mass spectrometer. results: the mean concentrations of cr, mn, ni, se and ag in colorectal polyp tissues of patients were significantly higher than in colorectal tissues of control subjects (p is less than 0.05). on the other hand the mean concentration of cd and pb in colorectal polyp tissues of patients were significantly lower than in control colorectal tissues of control subjects (p is less than 0.05). there was no any significant difference between the groups in terms of concentrations of al, fe, co, zn, as and hg (p is more than 0.05). conclusion: the differences found in some elements between polyps and a control tissues may provide an indication about the role of trace elements in the early stage (polyps) in the colon carcinogenic process and encourages further studies to confirm the involvement of such elements in neoplastic processes. the use of herbal medicines is steadily growing, with approximately 40% of the population use herbs to treat various illnesses in the western world. vitex agnus-castus has been used since ancient times as a remedy. the aim of this study was to investigate the in vitro anticancer activities of vitexagnus-castus oil. for this purpose, the cytotoxicity of vitexagnus-castus oil in sh-sy5y cells was investigated by crystal violet staining. ec50 was found to be 0.5%(w/w) vitexagnus-castus oil for this cell line. this dose was applied to the cell for 24 h, and the cells were harvested for further studies. vitex agnus-castus oil treatment increased bax and p38 mrna levels. on the other hand, bcl-2, bcl2 l1, erk-2, jnk, caspase 3 and 8mrna expression levels were reduced significantly withvitexagnus-castus oil treatment while p53 and pten remained unchanged. these results indicate that another effector caspase such as caspase 6 or 7 may be involved apoptosis process which remains to be elucidated. moreover, mapk pathways, p38 and erk, may be involved in vitexagnus-castus oil induced apoptosis in sh-sy5y cells. these initial observations suggest that this agent might not be useful in treating cancers. further detailed studies should be carried out to elucidate the exact mechanism of vitexagnus-castus oil in neuroblastoma cell lines. melanoma is a skin cancer with a melanocyte origin that can occur in any part of the body that contain melanocytes. while melanoma is less common than other skin cancers, it causes the majority of deaths related to skin cancer. several gene expression databases have shown that interferon regulatory factor 4 (irf4) is upregulated in melanomas, and genome wide association studies linked variation at irf4 locus with skin cancers. irf4 was first identified to have roles in lymphocyte development and function. studies have identified a 'non-oncogene addiction' of malignant cells to irf4 in various hematopoietic cancer types. the aim of this study is to investigate the role of irf4 in melanoma cell lines. lentiviral vectors were used to reduce irf4 levels in melanoma cell lines. a gfp competition assay was performed to study the competitive fitness of melanoma cells with irf4 knockdown (gfp positive cells) over melanoma cells with normal irf4 levels (gfp negative cells). cell cycle profiles were investigated in melanoma cells with irf4 knockdown by propidium iodide staining. migration potential was assessed as well by wound healing assay. our preliminary data showed a decreased competitive fitness for cells with decreased irf4 levels. cell cycle profiling showed increased g0/g1 and decreased g2/m levels in irf4 knockdown cells compared to controls. wound healing assay results showed no difference between controls for cells with reduced irf4 levels. taken together, these results indicate that irf4 knockdown affects the melanoma cell lines' survival and cell cycle profile, suggesting a non-oncogene addiction of melanomas to irf4. these observations are largely similar to previous observations in hematopoietic cancers. unravelling the role of irf4 in melanoma will increase our knowledge about melanoma development and progression and thereby may lead to targeted therapy in melanoma treatment. humans are exposed to various chemicals having beneficial or toxic effects at a time in their daily lives. 7,12-dimethylbenz[a] anthracene (dmba) is a carcinogenic compound produced during the incomplete combustion of carbon-containing compounds. endosulfan is an organochlorine pesticide used against insects on food. morin is an antioxidant, antiinflammatory and chemoprotective flavonoid. this study is aimed to determine the effect of morin in the presence of dmba and endosulfan. for this purpose, 56 adult wistar male rats weighing 170-255 g were randomly selected and divided into eight groups. 25 mg/kg body weight (b.wt.) morin and 5.0 mg/kg b.wt. endosulfan were given to morin and endosulfan treated groups three times in a week. the rats in dmba treated groups were gavaged with 30.0 mg/kg b.wt. dmba three times during the administration period (54 days). cytochrome p4501a (cyp1a) associated 7-ethoxyresorufin o-deethylase (erod) and glutathione s-transferase (gst) activities were measured in rat liver cytosols and microsomes. in addition, liver tissues were evaluated by histopathological analysis. erod activities of control, morin, endosulfan, dmba, morin+endosulfan, morin+dmba, dmba+endosulfan and morin+dmba+endosulfan groups were 71 ae 7, 112 ae 6, 114 ae 8, 126 ae 6, 121 ae 9, 156 ae 12, 151 ae 15 and 185 ae 13 pmol/min/mg protein, respectively. all treatments increased erod activities. gst activities of these groups were 365 ae 14, 430 ae 27, 365 ae 24, 651 ae 57, 460 ae 10, 577 ae 25, 585 ae 23 and 649 ae 33 nmol/min/mg protein, respectively. histopathological studies showed that endosulfan and dmba induced inflammation in the liver tissues and morin reduced their effects. in conclusion, morin treatment increased the metabolism of dmba and endosulfan by inducing cyp1a activity. gst activities of morin+dmba+endosulfan group were not significantly different from those of dmba group. histopathological studies indicated that morin administration reduced the toxic effect of endosulfan and dmba in the liver cells. hepatocellular carcinoma (hcc) is the sixth most common cancer and third most frequent cause of cancer-related death worldwide. molecular mechanisms of hepatocarcinogenesis is still unclear. the impairment of epigenetic mechanisms is implicated in the development of multiple cancers, including hcc. transforming growth factor-beta has been shown to play both tumorsuppressive and tumor promoting roles. transforming growth factor-beta signaling pathway involves activation of smad2 and smad3 by the type i receptor and formation of smad2/3/4 heteromeric complexes that enter the nucleus to regulate transcription. 3-deazaneplanocin a is an inhibitor of the histone methyltransferase ezh2. we aimed to reveal the effect of 3-deazaneplanocin a on transforming growth factor-beta /smad pathway in hepg2 cell line. hepg2, a human liver cancer cell line cultured in dulbecco's minimal essential medium supplemented with 10% fbs. the cells were seeded the day before 3-deazaneplanocin a administration and then the cells were treated with 5 lm 3-deazaneplanocin a for 3 days. expression levels of genes were analyzed by roche lightcyclerò 480. gapdh was used as housekeeping gene. apoptosis assay was performed by the muse annexin v and dead cell assay kit. the unpaired t-test was used to compare variables and p < 0.05 was accepted as statistically significant. 3-deazaneplanocin treatment was significantly reduced transforming growth factor-beta, smads 2-7 in hepg2 cells (p < 0.05). we also found that 3-deazaneplanocin induces apoptosis in treated cell line (p < 0.05). as a result, 3-deazaneplanocin a may take place in treatment of hepatocellular cancer by its inhibitory effect on transforming growth factor-beta /smad pathway and inducing apoptosis in liver cancer cells. brefeldin a (bfa) is a lactone antibiotic first isolated from the fungus eupenicillium brefeldianium. bfa inhibits the transport of secreted proteins from endoplasmic reticulum (er) to golgi apparatus, leading to disruption of golgi function, accumulation of unfolded and not fully incompletely processed proteins in er. bfa also inhibits cell proliferation, phosphorylation and migration of cancer cells. therefore in this study, we investigated the effects of bfa on breast cancer cell proliferation of various phenotypes. in we observed that bfa inhibited the proliferation of all three phenotypes of breast cancer cells, but the effects of bfa were seen at different times and doses. according to time and dose, bfa was observed more effective to mcf-7 compared to other cell lines. physiological, pathological and physical factors. moreover, nlr may represent the two opposing inflammatory and immune pathways that exist together in cancer patients. we aimed to investigate nlr in breast cancer in our population. methods: using data retrieved from the medical records, 66 women diagnosed primary breast cancer met our study inclusion criteria as they had a complete blood count with leukocyte differential performed before any anti-cancer therapy. and 44 women with benign mammary neoplasm/disease, followed up in the outpatient clinics of mammary disease and confirmed with sonographical/histopathological examination, made up our controls. exclusion criteria included laboratory evidence of white blood cells count (wbc) > 10.5 9 10 9 /l. differential leukocyte counts were obtained by bc 6800 (mindray medical international ltd., china), we examined wbc, neutrophil, lymphocyte, platelet counts, and hematocrite, nlr, mean platelet volume values. results: although there is lack of evaluation of tumor-associated neutrophils and lymphocytes, higher nlr median values and lower lymphocyte mean counts (lymphopenia) were shown in women with breast cancer (p < 0.0001). there was a weak negative correlation in breast cancer between nlr values and platelet counts (r s = à0.274; p = 0.026). holmboe] is distributed throughout southern mediterranean europe from spain to the eastern mediterranean on anatolian peninsula of turkey. present study was designed to investigate the in vitro anti-cancer activities of turkish black pine essential oil. the essential oil was extracted by steam-hydrodistillation and its chemical composition analyzed by gc-ms. the major components of the essential oil were a-pinene, b-pinene and trans-b-caryophyllene, respectively. the crystal violet staining method was used to investigate the cytotoxicity of essential oil in sh-sy5y cells. ec50 was found to be 0.75% (w/w) essential oil for sh-sy5y cells. neuroblastoma cells were incubated at 37°c for 24 h. after 24 h, cells were harvested for further studies. bax and p38 mrna levels were significantly elevated in essential oiltreated cells. on the other hand, bcl-2, bcl2 l1, casp-3, casp-8, erk-2 and jnk expression were significantly downregulated. unlike these proteins, p53 and pten mrnas were not changed. in this study, apoptosis was enhanced by turkish black pine essential oil treatment which was activated by the involvement of another effector caspase subfamily, like casp-6 and casp-7. additionally, erk and p38 mapks may be associated with upregulation of the level of bax. based on these results, we suggest that p. nigra subsp. pallasiana essential oil might not be well-suited in cancer treatment. however, further detailed research is necessary to establish the exact role of p. nigra subsp. pallasiana essential oil in sh-sy5y cells. p-05.02.2-026 the protective effect of newly derivatized compound naringenin-oxime and relative to naringenin against cisplatin-induced nephrotoxicity and genotoxicity in rat background: the aim of this study was to evaluate the possible protective effect potentials of newly derivatized compound naringenin-oxime (ng-ox) relative to efficacy of free naringenin (ng) on cisplatin (cis) induced nephrotoxicity and genotoxiticity in rat. methods: totally, fifty six male wistar albino rats were equally divided into eight groups as follows: control; cis treatment (7 mg/kg b.w., i.p.), ng and ng-ox (20 mg/kg b.w., i.p daily for 10 days) alone treatment; cis + ng (20 or 40 mg/kg b.w., i.p daily for 10 days) and cis+ng-ox (20 or 40 mg/kg b.w., i.p daily for 10 days) combination treatment. at the end of the study total antioxidant capacity (tac) levels, total oxidant status (tos), lipid peroxidation (lpo), total thiol, catalase (cat) were studied in homogenate kidney. peripheral lymphocyte cell dna damage was investigated with comet assay results: the results suggest that cis induces oxidative stress resulting in increased tos and lpo reduction thiol, tac and cat in kidney and increased peripheral lymphocyte cell dna damage. the treatment with naringenin and naringenin oxime alone or with cis treatment showed a protective effect against the toxic influence of cp on peroxidation of the membrane lipids and an altering of the total thiol status in the kidney of rats. from our results we conclude that naringenin and naringenin oxime functions as a potent antioxidant and suggest that it can control cp-induced nephrotoxicity and genototoxicity and ng-ox was found more protective than that of ng on cisplatin induced toxicity in rats. keywords: naringenin, naringenin-oxime, antinephrotoxic, antigenotoxic, comet assay. introduction: oxidative damage is considered to play a pivotal role in ageing, several degenerative diseases, and carcinogenesis. lung cancer is the most common type of cancer, resulting in over 1.3 million deaths each year worldwide. accurate and reliable determination of superoxide radicals has been widely investigated using spectrophotometric, electrochemical, amperometric, polarimetric, piezoelectric technologies. among these methods, electrochemical detection is a most promising approach to achieve accurate, separate and rapid superoxide radicals monitoring with using biosensor system. materials and methods: we used a new technic for detecting superoxide radicals in samples. superoxide dismutase (sod) enzyme immobilized on the surface of gold electrode with the help of gelatin, bovin serum albumin (bsa) and glutaraldehyde (ga) crosslinker. for the biosensor preparing benzoquinone selected as a mediator in working buffer and measurements were carried out at à0.7 v. result: for the optimization studies, effect of the bsa, gelatin, glutaraldehyde, ph, buffer concentration on biosensor response. characterization of the biosensor commitment to the work process and answer reproducibility were evaluated. the analytical characteristic of the biosensor were evaluated by measuring the steady state current response to superoxide radical concentrations. the electrochemical response of the enzyme electrode was linearity gradually leveled of at higher concentration. we found that crosslinking of the sod (e.c.1.15.1.1) with glutaraldehyde could be achieved over a wide range of relative mole ratios in 50 mm phosphate buffer at ph 7.5, glutaraldehyde concentration of %2.5. discuss and conclusion: in this study, a new technique for developed sod biosensors has been developed, which features effective combination of sod/gelatin/bsa/ga modified electrode, trapping of sod and glutaraldehyde cross-linking. this technique is reliable and cost effective. the effect of astaxanthin on apoptosis and cell arrest in u87 brain cancer cell line f. s€ og€ utl€ u, b. € ozmen yelken, c ß . kayabasi, a. asik, s. gonca, r. gasimli, s. yilmaz s€ usl€ uer, c ß . biray avci, c. g€ und€ uz department of medical biology, izmir, turkey a brain tumor is a collection, or mass, of abnormal cells in your brain. brain tumors can be cancerous (malignant) or non-cancerous (benign). the brain is one of the least accessible organ that active pharmacological compounds cannot be delivered. the two physiological barriers control and block the entry and exit of endogenous, exogenous compounds. one of these is the bloodbrain barrier and the other is the blood-cerebrospinal fluid barrier. this structures maintain protection of the brain. when there is a cancer case, it can lead to problem. astaxanthin with potent antioxidant properties can cross blood-brain barrier. in our study, we aimed to evaluate the effects of astaxanthin on apoptosis, cell cycle and also migration in brain cancer cell line. in present study, xcelligence real-time cell analyzer was used so as to determine cytotoxic effect of astaxanthin in u87 cell line. changes of apoptosis and cell cycle in u87 cell line exposured to ic 50 dose of astaxanthin (19.5 nm-80 lm) are detected with annexin v-egfp apoptosis detection kit and cycle test plus dna reagent kit with facs, respectively. the result of apoptosis and cell cycle test was analysed in flow cytometry. the group to which active substance was not treated was used as controlled. the wound healing assay performed in order to measure migration ability of u87 cell line to which astaxanthin was treated or not. ic 50 dose of astaxanthin was calculated as 9.20 lm at 48 h by xcelligence rtca sp based on time and dose. astaxanthin decreased the migration ability at rate of 25% in u87 cells treated by ic 50 dose of astaxanthin. astaxanthin had no apoptotic effect on viability in u87 cell line and astaxanthin caused an increase of g2/m phase arrest (1.15 fold) and s phase arrest (1.41 fold). astaxanthin has cytotoxic effects in brain cancer. it determined that astaxantin decreases cell cycle potential at g2/m even a little. the effect of anticancer of astaxanthin should be researched further. interferon regulatory factor 4 (irf4) is a critical transcription factor in development and survival of different cell types including immune cells and melanocytes. furthermore, it has been demonstrated that irf4 expression levels are elevated in several lymphoid cancers, and irf4 is one of the key transcription factors for the survival of these cancers. several genome-wide association studies identified irf4-linked genetic variants to increased melanoma incidence. in addition results from our lab and elsewhere have shown high levels of irf4 expression in melanoma cell lines. furthermore our preliminary results suggest melanoma cells are sensitive to irf4 expression levels. however, there are no published studies about irf4 target genes in melanoma cells. in this study, we are investigating the genome-wide target genes of irf4 in melanoma cell lines via high-throughput sequencing of immunoprecipitated chromatin (chip-seq). we have identified possible irf4 binding regions in loci with known key roles in development of melanocytes from neural-crest cells. one such key factor is mitf, which is the master regulator in melanocyte development and also plays critical roles in melanoma. integrating chip-seq and rna-seq data suggests irf4 as a transcriptional regulator of genes related to progression of melanoma. objectives: aim of this study was to evaluate prognostic importance of selected laboratory parameters (c-reactive protein (crp), gama glutamiltransferaz, ferritin (fer), potassium, chloride, calcium, phosphorus, magnesium, total protein, aspartat aminotransferaz, alanin aminotransferaz (alt), ifn-c, il-6, tnf-a) in non-small cell lung cancer (nsclc). material and methods: 20 patients with nsclc who were treated with chemoradiotherapy (crt) prospectively evaluated. all patients were newly diagnosed tumour. heparinized blood samples were taken from the patients before and after the completion of crt. fer analyzed by chemiluminescence method on beckman coulter dxi 800; ifn-c, il-6, tnf-a were analyzed with elisa kits (boster biological technology) and other biochemical parameters analyzed on abbott architect c16000. post-crt and pre-crt levels compared with survival. results: the lr cox regression analysis revealed that pre-crt ferritin was significantly associated with survival of patients with nsclc (hazard ratio (hr) = 1.007, p = 0.023, 95%ci; 1.001-1.013). it was also demonstrated by lr cox regression analysis, high levels of pre-crt crp was associated with worse outcome of patients (hr = 1.017, 95%ci;1.001-1.032, p = 0.035). after crt, mean alt level was determined as 16.71. there was survival difference in nsclc patients with high post-crt alt (hr = 0.912, 95%ci; 0.841-0.990, p = 0.027). conclusions: there exists a clinically relevant relationship between pre-crt fer concentration and the prognosis of survival in patients with nsclc. elevated fer is the result of inflammation rather than body iron overload. ferritin showed negative correlation with survival so it could be a useful biomarker to indicate bad prognosis of the patients with nsclc. additionally, crp which is easy to detect and feasible for the use in the routine clinical practice should be considered in the prognosis of nsclc patients. keywords: ferritin, nonsmall cell lung cancer, survival, c-reactive protein. epigenetic therapy tries to reverse the aberrations followed to the disruption of the balance of the epigenetic signaling ways through the use of natural and synthetic compounds, active on specific targets, such as dna methyltransferases (dnmts). we previously synthesized some benzoxazole and benzamide derivatives which might have anticancer activities on account of their heterocyclic structure. our studies showed that not only these compounds caused selective cytotoxicity towards cancer cells (hela) with little or no toxicity on normal cells (l929) but also were not genotoxic. in this study, we aimed to test whether these compounds changed global demethylation profile of normal and cancer cells. we used methylation specific comet assay (msc assay) to determine global methylation levels of cells. cells were treated with the tested compounds at ic 50 concentrations for 48 h. slides were prepared as did in alkaline comet assay, then they were incubated with methylation specific restriction enzymes (mspi, hpaii) before electrophoresis. differences in global methylation levels between nontreated control cells and cells treated with compounds were compared by using tail moment data. 5-aza-c, a demethylating agent, was used as reference drug. msc assay results revealed that none of the tested 9 compounds caused hypermethylation on both cell lines. however, global methylation levels decreased statistically (p < 0.05) through both cells treated with c-2 and c-8. only c-3 decreased methylation level on l929 but not on hela. consequently, c-2 and c-8 caused demethylation on hela cells similarly with 5-aza-c at low concentrations. for the reason that dna methylation is regulated mainly dnmt enzymes in the cell, c-2 and c-8 might cause global demethylation in the cell by inhibiting dnmt activity. further studies will be done to support this prediction. overall, macrophages and some subtypes of lymphoid cells are found in tumour stroma. these cells secrete a variety of growth factors, proinflammatory cytokines and chemokines, esp. tnf-a, il-1b and il-6, causing the formation of inflammatory microenvironment around tumour cells. tnf-a and il-1b signaling increases activity of nf-kb pathway. at the same time, il-6, triggers jak-stat signaling pathway, which effector is stat3. nf-kb and stat3 activity facilitates hyperexpression of mir-nas mir-155, mir-181 and mir-21 as well as down-regulates expression of mirnas mir-15/16, mir-199 and let-7. this investigation aims to identify in what way these shifts in mirnaome can lead to epigenome reorganization supporting the cell transformation. mirna targets within gene transcripts were predicted in silico using targetscan software. transcripts of hdac2/4/8/9 and sirt1/5 genes encoding histone deacetylases carry targets for at least one of up-regulated mirnas mir-155, mir-181 or mir-21. also, these mirnas can silence ezh1, mll, mll3, nsd1, setd6/7/8, smyd1, suv39h2 genes encoding histone methyltransferases. mirna mir-21 suppresses gene encoding de novo dna methyltransferase dnmt3b. at the same time, down-regulation of mirna mir-15/16 can allow hyperexpression of gene encoding acetyltransferase elp3. these shifts impair dna and histone methylation, cause the increase of overall level of chromatin acetylation and expression and, therefore, create epigenetic background for reactivation of silent transposons, oncogenes as well as other genes important for cell transformation. immune system can paradoxically facilitate the tumour growth instead of healing. cancer-related inflammation leads to the mir-naome and epigenome shifts contributing to the tumour promotion and progression. lysine acetylation is one of the key mechanisms to regulate chromatin structure and transcriptional activation. acetyl-lysine modifications are recognized by bromodomains, which are small interaction modules found on diverse proteins including histones. among these acetyl-lysine reader proteins is the family of the bet (bromodomain and extra-terminal) proteins which contain tandem bromodomains (bd1 and bd2). the recent discovery of potent and specific inhibitors for the bet family proteins has stimulated intensive research activity in diverse therapeutic areas, especially in oncology, where bet proteins regulate the expression of key oncogenes and anti-apoptootic proteins. several bet inhibitors are currently in clinical trials and reported to exhibit promising clinical activities. however, pleiotropic nature of bet proteins regulating tissue-specific transcription has raised safety concerns and suggested that attempts should be made for domain-specific targeting. here, we report the recent progress in the development of bet inhibitors in korea research institute of chemical technology (krict). we have identified the bet inhibitors with a novel scaffold different from the previously reported diazepine and azepine scaffolds and specific for first bromodomains (bd1s). a medicinal chemistry effort is currently made to optimize the pharmacokinetic properties of these lead compounds for further drug development. the experimental data from the biochemical and cell-based assays for these bd1-selective bet inhibitors will be presented. family of small c-terminal domain serine phosphatases (scp), which includes ctdspl, ctdsp1, and ctdsp2, plays a regulatory role in a number of vital processes. in particular, it is shown that ctdspl is capable to activate the retinoblastoma protein (rb) which is well-known tumor suppressor and one of the key cell cycle regulators. although the question on whether ctdsp1 and ctdsp2 dephosphorylate rb is open, high similarity of sequences and three-dimensional structures of phosphatases may indicate the similar function of these enzymes. in the current study expression of scp genes was evaluated by quantitative pcr in 28 non-small cell lung cancer (nsclc) samples. using original crosshub software, that combines an analysis of high-throughput sequencing data of the cancer genome atlas project (tcga) and databases of mrna-mirna interactions (targetscan, mirtarbase, etc), the involvement of mir-183-96-182 microrna cluster in co-regulation of ctdspl/1/2 genes in nsclc was predicted. the significant (5-fold on the average) and simultaneous decrease of mrna levels of ctdspl/1/2 genes was revealed in the majority of nsclc samples (82%, 23/28). such unidirectional expression change and strong positive correlation between phosphatase expression levels (r s = 0.53-0.62, p ≤ 0.01) allowed us to suggest a common mechanism of their inactivation. we evaluated the expression of predicted co-regulators of scp gene expression, mir-183-96-182 family, in examined nsclc samples. as a result, the simultaneously increased levels of all three mir-nas in most nsclc samples (82%, 23/28) and negative correlation with phosphatase gene expression was shown. the results suggest the ability of investigated phosphatases to exhibit tumor-suppressive activity and the involvement of mir-183-96-182 micrornas in the regulation of rb protein activity via inactivation of ctdspl/1/2 in nsclc. cancer is one of the leading causes of death in all around the world. cancer is defined as a disease involving abnormal cell growth with the potential to invade or spread to other parts of the body. tumor markers are substances that are produced either directly by the tumor or as an effect of the tumor on healthy tissue. tumor markers can be used for screening, determining prognosis and monitoring effectiveness of therapy and disease recurrence. the aim of this study is to investigate the frequency of tumor markers orders and the appropriateness of these requests. laboratory information systems data for 2015 were reviewed. for 2015, a total of 35874 patients and 55539 tumor marker requests were included. carbohydrate antigen 19-9, cancer antigen 125, cancer antigen 15-3, prostate specific antigen, alphafetoprotein and carcinoembryonic antigen were measured by chemiluminescence method. according to the data from the year of 2015, both positive tumor markertest resultsratio and the positive patient ratio were 14%. in the patients group with increased marker levels, 48% of the patients had no history of cancer. in the patients group with tumor marker levels in referenceranges, 40% patients with diagnosed cancer history in remission. the ratios of positive tumor markers were 13% forca 19-9, 19% for ca 125, 7.9% for psa, 25%for ca 15-3, 8.4%for afp, and 9.56% for cea. in conclusion; unnecessary test requests increase laboratory work load and health expenses. laboratory and clinical staff collaboration is crucial to increase the appropriate use of tumor markers. dna methylation is an epigenetic modification that is involved in both normal biological and disease states. hypermethylation of promoter regions of tumor suppressor genes have a role in tumor development. therefore, the measurement of promoter methylation of genes can be used for diagnosis and prognosis purposes of cancer. to detect dna methylation alterations in a sample (biopsy, blood, saliva, etc.), sensitive detection systems and optimization of the methods are needed. as a part of a collaboration project between national metrology institute of korea (kriss) and national metrology institute of turkiye (tubitak ume). dna methylation status of apc and gstp1 genes were studied. dna methylation measurements were performed using stepone real-time pcr system and results were analyzed using hrm (high resolution melting) software. the parameters effecting the quantification of dna methylation were found as primers, annealing temperature, pcr cycle number, fluorescence dye and the commercial dna methylation standards used for quantification of dna methylation. since, the accurate measurement of dna methylation is very critical in early diagnosis of cancer and choosing the right therapy, optimization of the method is required. cancer is a disease that includes heterogenic and complex molecular changes. anti-carcinogenic effects of resveratrol, a natural polyphenol, have been proved in a variety of cancer cells. considering the effects of resveratrol, the influence of the signal transduction pathways in the presence or absence of p53 of colon cancer cells is gaining importance. our aim was to investigate the effects of resveratrol in the presence or absence of p53 on cell viability, apoptotic cell death ratio and fold changes of proliferative or anti-proliferative gene expressions, which may have important effects on colon cancer, in hct116 colon carcinoma cells. ic50 doses of resveratrol were determined by wst-1 assay. the apoptotic cell death ratios in treatments of resveratrol were determined by annexin-v-fitc/pi assay for flow cytomety . the changes of ccnd1, fra1, ppard, egfr, birc5, pcna, mcl1, stat3, fos, jun, p27, atf4, trail, puma, gadd45a, rb1, faslg, tnf, socs3 , stat1 gene expressions were evaluated by real time pcr. all data were statistically analyzed by student's t test. our research has revealed that resveratrol (60 lm) causes decrease in cell viability and increase in apoptotic cell death in hct116 p53(+/+) and hct116 p53(à/à) cells significantly (p < 0.05). the fold changes of the gene expressions have shown that resveratrol has significant (p < 0.05) and different effects on the expressions of the genes related with the existence of p53 in hct116 cell lines. therefore we proposed that resveratrol might show proliferative or apoptotic effects related with p53 mutation of colon cancer cells and we predicted that unconscious consumption of resveratrol in colon cancer patient might cause adverse effects. introduction: colorectal cancer (crc) is the third most common cancer worldwide. alterations in methylation profiles of tumor suppressor genes (tsgs) have been recognized as a key mechanism in colorectal cancers. in the current study, we investigated the hypermethylation status of 25 tsgs in colorectal cancer tissues. materials and methods: formalin-fixed paraffin-embedded (ffpe) tissue samples obtained from patients with crc. methylation specific-multiplex ligation dependent probe amplification (ms-mlpa) technique was used to assess the methylation status of 25 tsgs. the findings were evaluated in terms of age, mortality, survival, positive lymph node status, lymphovascular invasion, and perineural invasion. results: hypermethylation-detected patients and hypermethylation-undetected patients were called as group 1 and group 2, respectively. hypermethylation was detected in atm, cdkn2a, and gata5 genes. mortality rate was (12.5%) in group 1 and group 2 (p > 0.05). mean 5-years survival rate in group 1 was 45 ae 3 months and mean 5-years survival in group 2 was 44 ae 3 months (p > 0.05). positive median lymph node count was 10 ae 2 for group 1 and 5 ae 1 for group 2 and the difference was statistically significant (p < 0.05). frequencies of perineural invasion and lymphovascular invasion rate in two groups were 25% (p > 0.05). discussion and conclusion: our findings suggest that tsg hypermethylation found in crc patients may increase the lymph node metastasis. further investigations with larger sample size are required to support our results. boron (b) is known to be important for cell replication and development, but the underlying mechanism remains obscure. recently b has also become important in some specific anticancer processes. some recent reports advise using of some boron compounds for the treatment of specific forms of cancer. for instance, boron-based drugs (bortezomib) are now being developed for use as therapeutic agents with anticancer activities and several other boron-based compounds are in various phases of clinical trials. it has been shown that bortezomib disrupts the regulation of cell cycle and induces apoptosis in both hematologic and solid tumor malignancies except for colon carcinoma. colorectal cancer (crc) is the third most common cancer in men and the second in women, accounting for 10% of all tumour types worldwide. cytotoxic effects of boron compounds on crc cells and changing of its effects related with p53 mutation, which is mutated 50% of cancer cases, have not take part in literature yet. for this purpose; the aim of the study was designed to investigate the effects of borax pentahydrate and disodium pentaborate decahydrate compounds on cell viability, apoptotic cell death ratio and parp protein expressions in p53 (+/+) and p53 (à/à) hct116 colon carcinoma cells lines. the effects of the boron compounds on cell viability were assessed by xtt assay and apoptotic effects and parp protein expression of the compounds were evaluated by flow cytometry and western blot analysis respectively. our results showed that borax pentahydrate (6 mm) and disodium pentaborate decahydrate (12 mm) significantly causes nearly 50% reduction of cell viability at 48 h (p < 0.05). apoptotic cell death ratios and parp expressions revealed that both of the compounds might have a potential for a candidate of anticancer agent. epithelial-mesenchymal transition (emt) is a significant event for metastasis, and could be mediated by several pathways such as pi3k/akt, map kinases and many epigenetic regulators. satb2 is an epigenetic regulator involved in emt and osteoblastic differentiation. since preliminary results indicate that there is a crosstalk between p38 and akt pathways in nsclc cells, we aimed to determine whether this crosstalk has a regulatory effects on emt and satb2 expression in nsclc cells. we used a549 and h1650 cells as a model to evaluate the effects of the crosstalk between p38 and akt on emt of nsclc cells. therefore, cell culture, inhibition of p38 activation via sb203580, transient expression assay for (ca-akt), western blot analysis, sirna transfection for satb2, wound healing and invasion assay were performed in this study. firstly, the expression statues of e-cadherin, satb2, p-p38, p38, p-akt and akt was examined in a549 and h1650 cells by western blot analysis. we observed that e-cadherin and satb2 are downregulated in a549 cells (highly active p38, lowly active akt) compared to h1650 cells (lowly active p38, highly active akt), suggesting that e-cadherin and satb2 are associated with the crosstalk between p38 and akt pathways. our results demonstrated that p38 inhibition in a549 cells leads to decreased pten expression and subsequently increased akt activation. then, we found that p38 inhibition upregulated satb2 expression, and reversed emt in a549 cells. furthermore, alone satb2 knockdown is sufficient to induce emt, and prevented the effects of p38 inhibition on emt. all these results strongly indicate that the crosstalk between p38 and akt pathways might determine satb2 expression and epithelial characters of nsclc cells, and satb2 is a critical epigenetic regulator for emt in nsclc cells. therefore, it is also need to explore how p38 and akt signalling pathways could regulate satb2 expression. this work was supported by tubitak (114s007, 215z283). introduction: lung cancer is a disease characterized by uncontrolled cell growth in the lung tissues. the most common causes of lung cancer are tobacco smoke, radon gas, asbestos, air pollution, and genetic factors. nitric oxide (no) has potential mutagenic and carcinogenic activity and may play important roles in lung cancer. endothelial no, synthesized from l-arginine by endothelial no synthase (enos), inhibits apoptosis and promotes angiogenesis and tumor cell proliferation. the aim of the present study was to examine the possible relationship between enos gene intron 4 vntr and exon 7-g894t (glu298asp) the stressful ecosystems exert strong adaptive pressure and proteins that facilitate these adaptation processes are candidate drug targets. nucleotides are the core of biochemical pathway required for cancer cell growth and replication and genetic changes will lead in oscillation in their pools. although it is questionable whether the warburg effect actually causes cancer, impairing dglucose uptake and metabolism induces oxidative metabolism. lproline (lproline) homeostasis is critical in a constellation of human diseases, in parametabolic linkage between cancer, epigenetics (phang et al. 2013 ) and bioenergetics (pallotta 2013) where degradation and biosynthesis are robustly affected by oncogenes or suppressor genes that can modulate intermediates involved in epigenetic regulation. lproline-fueled mitochondrial metabolism involves the oxidative conversion to l-glutamate by a flavin dependent lproline dehydrogenase/oxidase and a nad +dependent l-d 1 -pyrroline-5-carboxylate dehydrogenase. in saccharomyces cerevisiae an important test tube, put1p and put2p respectively help cells to respond to changes in the nutritional microenvironment by initiating lproline breakdown after mitochondrial uptake (pallotta 2014) . in this preclinical study, low molecular weight compounds were tested for inhibiting lproline mitochondrial transport and put1p/put2p catalytic activities. thus, in seeking for natural bioactive compounds targeting lproline pathway and its substrate channeling (becker's group 2016), we report data using in silico screening and in vitro researches in saccharomyces cerevisiae with genetic background atcc18790 but different phenotypic landscape induced by nutritional stress/ ph changes. cells vitality, dψ measurements, nad(p) + /nad(p) h pool and flavine turnover were determined in spectrofluorimeter microplater reader and via hplc (pallotta et al. 1998 (pallotta et al. , 1999 (pallotta et al. , 2004 pallotta 2011; di martino pallotta 2011) thus in supporting of future cancer therapies with decreasing side effects. evaluation of lymphocyte to monocyte ratio (lmr) in patients with colorectal cancer introduction: inflammation may play an important role in cancer progression and a high neutrophil to lymphocyte ratio (nlr) has been reported to be a poor prognostic indicator in several malignancies. the aim of this retrospective study was to evaluate the prognostic value of nlr, lymphocyte to monocyte ratio (lmr) and platelet to lymphocyte ratio (plr) in patients with colorectal cancer (crc). : 107 patients who were diagnosed with colorectal cancer between january 2010 and january 2016; were evaluated retrospectively. the cutoff value was determined using receiver operating characteristics curve analysis. survival analysis was performed using the kaplan-meier method and log-rank test. the cox proportional hazard model was used to identify the influence of factors related to survival. (tnm stage, tumor differentiation, age, tumour size and lmr) results: receiver operating characteristic curves showed that lmr was superior to plr and nlr as a predictive factor in patients with colorectal cancer. the cutoff value for lmr was 1.82. cancer-specific survival was not significantly different between the high-and low-lmr groups (p = 0.786). age was identified as independent prognostic factor in colorectal cancer (hazard ratio: 3.50; 95% confidence interval: 3.14-351.82; p = 0.004). discussion and conclusion: our preliminary study showed that the lmr was not an independent prognostic factor in crc patients, but additional large sample sized prospective studies will be needed to confirm these findings. the aim of this study is to investigate the effects of luteolin treatment on enzymatic activity of arginase, and ornithine and polyamine levels (putrescine, spermidine spermine) in serum and cancer tissues of ehrlich ascites breast cancer model. balb/c female mice were divided randomly into following groups: healthy control, healthy treatment, cancer control, treatment 1 and treatment 2. 0.2 ml ehrlich ascites tumor cells was inoculated subcutaneously to medial part of left hind leg. healthy treatment and treatment 1 groups, and the treatment group 2 were given 5 mg/kg and 10 mg/kg dose of luteolin, intraperitoneally, for a 12 days period, respectively. luteolin has a hydroxylated flavonoid structure and shows potent antioxidant, anti-inflammatory, and anticarcinogenic properties. luteolin not only leads to cell death in various tumors by suppressing cell survival pathways and stimulating apoptotic pathways, but also sensitize them to cytotoxic therapy. supporting various previous studies, tumor implantation to healthy mice resulted in statistically significant elevation of serum arginase and polyamine levels (p < 0.05) indicating the tumor cells as the main source of this production. furthermore, luteolin treatment abolished this increase in serum arginase and polyamine levels (p < 0.05). tissue measurements of arginase and polyamine levels indicated that luteolin treatment resulted with an increase in these parameters of tumor tissue while the serum levels of them showed a significant decrease. our results revealed that increased tissue arginase and polyamine levels might be related with estrogenic agonistic effect of luteolin on utilized tumor model in this experiment; and decreased serum levels of these parameters while there is a significant increase of them in tissue levels might be a result of a suppression of polyamine efflux from the tumor tissue by inhibitory effect of luteolin on plasma membrane polyamine transporters. hepatocellular carcinoma (hcc) is the third most common cause of cancer-related deaths. around 30-40% of hcc patients are diagnosed at an early stage of the disease. hepatic resection, liver transplantation are common strategies in hcc treatment. even if, most of the patients present advanced-stage tumors and have a restricted survival rate. for the reason, resistance against existing tumor stress conditions have been demonstrated in hcc. hypoxia, hyperglycemia are general stress sources in hcc and result in aggressive cell phenotype, resistance to apoptosis and therapeutic drugs. thioredoxin interacting protein (txnip) regulates cellular responses under stress conditions. over-expression of txnip results activation of oxidative stress and apoptosis. in cancer models txnip is considered as a tumor suppressor gene. however, its role in the development, progression of hcc and mechanisms behind it warrant further investigation. in this study expression levels of txnip were examined in 12 hcc cell lines by rt-pcr and western blotting. txnip expression was significantly high in poorly-differentiated snu-182, snu-387 and snu-423 than the well-differentiated hcc cell lines such as huh-7, hepg2 and plc/prf/5. besides, expression of txnip was examined in 16 non-hcc and 79 hcc tissue samples by immunohistochemical staining. txnip positivity was observed in 40% of well and 80% of poor differantiated hcc tissues. however, no txnip positivity was observed in non-hcc tissues. to investigate whether txnip might be involved in biological responses such as cell proliferation, motility and invasion, we used overexpression and silencing strategies. overexpression of txnip minimally inhibited adhesion and proliferation, whereas boyden-chamber motility and invasion assay showed that invasiveness of cells were increased. our findings suggest that txnip expression is increased in hcc and txnip over-expression is important for invasive phenotype during hepatocarcinogenesis. cardiovascular diseases are the leading cause of morbidity and mortality in the western world. it was shown that ischemic tolerance of the heart can be enhanced not only by ischemic or pharmacological conditioning (pre-and postconditioning), but also by adaptation to chronic hypoxia. different studies have indicated that these cardioprotective phenomena may at least partly share the same signaling pathways. the jak/stat signaling pathway has been demonstrated to participate in the development of cardioprotection by conditiong apparently through the inhibition of gsk-3b. the aim of our present study was to determine whether this pathway also takes part in cardioprotection induced by adaptation to chronic hypoxia. we investigated the effect of inhibitor of jak2 kinase (ag-490) on myocardial infarct size and the jak2/stat3 signalling pathway and other effector molecules that may participate in cardioprotection conferred by adaptation to hypoxia. adult male rats were adapted to intermittent normobaric hypoxia (10% o 2 , 3 weeks, 8 h/day) and part of them recieved ag-490 (3 mg/kg) 15 min before ischemia. control rats were kept under normoxia. infarct size was assessed in isolated perfused hearts. relative expression of the key components of the jak2/stat3 signalling system and other proteins was detected using western blotting. preliminary data indicate that administration of the jak2 inhibitor ag-490 caused a significant increase in infarct size in hypoxic rats. western blot analysis revealed changes in phosphorylation of jak2, stat3 and some other proteins involved in cardioprotection (akt, erk1/2, gsk3b). these results suggest that the jak/stat signaling pathway could participate in the development of a cardioprotective phenotype in rats exposed to chronic hypoxia. however, further research will be needed to clarify in more detail the role of this signalling pathway in the cardioprotective mechanism. p-06.01.2-002 detrimental effect of hypertension on myocardium was reversed by liver x receptor agonist gw3965 hypertension is a cardiovascular disease that causes functional and structural changes in the heart. nuclear liver x receptors (lxrs) are involved in the control of cholesterol and lipid metabolism. however, effect of lxr activation on the hypertensive heart is not well characterized. in this study, the effects of lxr agonist gw3965 on hypertension-induced damage of myocardium were investigated. hypertension was induced by deoxycorticosterone acetate (doca) injection (20 mg/kg, twice a week) following the unilateral nephrectomy in male 8-week-old wistar albino rats for 6 weeks. blood pressure was measured by using tail-cuff method. gw3965 (10 mg/kg/day, i.p.) was administered last 1 week. expression of various markers (grp78, perk, p-perk, ikb-a, nf-kb p65, tnf-a, bax, bcl-2, mmp-2) in the ventricular tissue were examined by western blotting. inflammation and fibrosis were evaluated in histopathological examination. gw3965 treatment reduced systolic blood pressure of hypertensive animals. expressions of endoplasmic reticulum stress markers grp78 and p-perk were increased by hypertension and gw3965 treatment reversed them. hypertension-induced increase in nuclear nf-kb p65 expression and decrease in ikb-a expression were reversed by gw3965 treatment. while bcl-2 expression was lower, bax level was higher than control in the hypertensive animals. in hypertensive group, fibrosis marker mmp-2 expression was augmented and gw3965 treatment reversed this elevation. hypertension-induced increase in interstitial and perivascular collagen deposition and inflammatory cell infiltration in left ventricle were prevented by gw3965 treatment. these results suggest that lxr activation by gw3965 restored the hypertension-induced structural changes of heart in the doca-salt hypertension. methylphenidate (mph) is a psychostimulant prescribed for the treatment of attention deficit hyperactivity disorder (adhd), one of the most common neurobehavioral disorders of childhood and adolescence. in fact, despite the widespread use of mph the full comprehension of its cellular/molecular mechanisms is still elusive, including its effect on blood-brain barrier (bbb). this barrier is a key structure in the central nervous system since it protects the brain and its dysfunction has been described as a critical event in several brain diseases. thus, the aim of the present study was to clarify the effects of mph on the bbb function in both physiological and adhd conditions. for that, we used a rat model of adhd, the spontaneously hypertensive (shr) rats, and wistar kyoto (wky) animals as inbred comparator strain. also, to mimic a clinical dosing schedule for adhd treatment, rats were administered for monday-friday with vehicle or mph (1.5 mg/kg/day or 5 mg/ kg/day, per os) from p28-p55. chronic mph treatment (5 mg/kg/day) promoted cortical bbb permeability in both wky and shr animals; however, more prominent in wky rats. this effect can be explained by the downregulation of claudin-5 and collagen-iv, tight junction and basal lamina protein, respectively. noteworthy, wky animals also showed an increase in the expression of caveolin-1 and in both vascular cell and intercelular adhesion molecules. these bbb alterations led to subsequent infiltration of peripheral immune cells, including cd169 + macrophages. furthermore, hippocampal bbb disruption was only observed in wky rats with 5 mg/kg of mph. here, mph decreased collagen iv expression and upregulated caveolin-1, with no alterations in claudin-5. overall, our results show that chronic exposure to mph can led to brain vascular alterations particularly under physiological conditions. this highlights the importance of an appropriate mph dose regimen for adhd, and also that mph misuse can have a negative effect. regulators of g proteins signaling (rgs) serve several cellular functions varying from tolerance, dependence, neuroprotection, transcription and tumorgenesis. despite their initial role as gtpase activating proteins, evidence suggests that rgs proteins are localized in the nucleus, interact with transcription factors thus regulating transcriptional responses. it was shown that rgs4 directly interacts with and interferes in opioid receptor (or) signaling. rgs4 is mostly expressed in brain and is implicated with brain structural alterations; however, the molecular mechanisms of how rgs4 could be involved in cellular differential functions remains unclear. based on these observations we examined whether rgs4 can regulate transcriptional responses mediated by the stat5b transcription factor. isolated neural stem cells from rgs4 à/à mice were immunostained for the mitosis marker ph3 and the mrna levels of antiapoptotic genes were determined. proliferation assays were performed with brdu staining in neuro2a cells stably expressing rgs4. the functional assays of stat5b transcriptional activation were performed in hek293 expressing either the erythropoietin receptor (epo-r) or the delta opioid receptor (d-or). the present data demonstrate that rgs4 blocks stat5b phosphorylation and transcriptional activation by interfering in stat5b heterodimerization upon epo-r or d-or activation triggered by cytokines or opioids administration. lack of functional rgs4 results in increased mrna levels of stat5b target genes such as the members of the bcl anti-apoptotic family bcl-2, bcl-6 and bcl-xl. this upregulation of stat5b inducible gene transcription results in an increased proliferation rate of neural stem cells. this study demonstrates for the first time a non-canonical function of rgs4 in stat5b mediated transcriptional responses and a novel selective role of rgs4 in transcription. role of the pre-molten globule structure in amyloid fibril formation a. eshaghi department of biology, faculty of science, islamic azad university, mashhad branch, mashhad, iran the major factor that caused extensive research on the protein fibrillation is their crucial roles in important diseases known as the amyloidosis diseases. neurodegenerative diseases, including alzheimer's, parkinson's, diabetes and huntington are the most important types of this disease. understanding the mechanisms of fibril formation and ways of treatment can be useful in reducing this type of disorder. in this project, the fibrillation of carbonic anhydrase protein was investigated as a model. carbonic anhydrase creates two stable intermediated known as pre-molten and molten globule, in different ph solution. this protein at ph between ph 3-4 molten globule structures was formed while the pre-molten form took place under ph 3. in our tests at ph 3.5 when the protein in molten globule structures only the amorphous aggregates were formed. instead, at ph 2.4 in pre-molten globule structure amyloid fibrils formed in the protein. there some reports, indicated the protein from pre-molten globule structure go toward amyloid assembly. even intrinsically unstructured proteins such as alpha-synuclein first took a structure similar to pre-molten globule and then made amyloid fibrils. it seems pre-molten globule structure have the major role in promoting to amyloid fibrils. perhaps drugs that prevent the formation of premolten globule structure have an important role in inhibiting amyloid fibrils. identification of compounds preventing the biochemical changes that underlie the epileptogenesis process and understanding the mechanism of their action is of great importance. we have previously shown that myo-inositol (mi) daily treatment for 28 days prevents certain biochemical changes that are triggered by kainic acid (ka)-induced status epilepticus (se), [1, 2] . however in these studies we have not detected any effects of mi on the first day after se. in the presented study we broadened our research and focused on ka induced other early molecular and morphological changes and influence of mi treatment on these changes. the increase in the amount of voltage-dependent anionic channel-1 (vdac-1), mitochondrial-plasma membrane cofilin and caspase-3 activity was observed in the hippocampus of ka treated rats. administration of mi 4 h later after ka treatment abolishes these changes, whereas under the same time schedule diazepam treatment has no significant influence. the number of neuronal cells in ca1 and ca3 subfields of hippocampus is decreased after ka induced se and mi post-treatment significantly lessens this reduction. no significant changes are observed in the neocortex. obtained results indicate that mi post-treatment after ka induced se could successfully target the biochemical processes involved in apoptosis, reduces cell loss and can be successfully used in the future for translational research. references 1. r. 2010. neuroscience letters, vol. 468, no. 3, pp. 277-281. 2. r. solomonia, et al; 2013. cell. mol. neurobiol. vol. 33, no extracellular deposits of amyloid-b peptide (ab) in brain parenchyma via proteolytic processing of amyloid precursor protein (app) are one of the typical characteristics of alzheimer's disease (ad). these aggregates mainly occur as a result of an increase in ab production or a decrease in its degradation. it was found that the neurotoxicity of ab aggregates is accelerated by acetylcholinesterase (ache). besides, ab-ache complex has a prominent neurodegenerative effect in brain. thus, cholinesterase inhibition and preventing ab production are current treatment strategies for ad. recent studies have shown that methylene blue (metb), a cholinesterase inhibitor with phenothiazine structure, inhibits the formation of amyloid plaques and neurofibrillary tangles. azure b, the major metabolite of metb, has been shown to inhibit ache and bche with ic50 values of 0.486 lm and 1.99 lm, respectively. in the present study, we tested whether azure b, may effectively lower the levels of ab 40/42 . we treated chinese hamster ovary cells, which stably express human wild type app and presenilin-1 (ps70) with 0-15 mm azure b or vehicle for 24 h. to determine the effect of azure b treatment on ab 40/42 levels, we used separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. azure b treated ps70 cells were also assessed by propidium iodide in flow cytometry for cellular toxicity. we observed a significant decrease in both extracellular ab 40 and ab 42 levels with a dose range treatment of azure b in ps70 cells. ab 40 levels were reduced by 89.2% in 10 lm and 94.1% in 15 lm azure b-treated cells when compared to control. additionally, ab 42 levels were decreased by 83% in 10 lm and 93.5% in 15 lm azure b-treated cells when compared to control. overall, these preliminary results suggest that azure b may have beneficial effects for the treatment of ad. the effect of green silver nanoparticles (agnps) on the amyloid formation in alphalactalbumin and chaperone action of alphacasein a. ghahghaei, m. dehvari, j. valizadeh formation and deposition of protein fibrillar aggregates in the tissues is associated with several neurodegenerative diseases such as alzheimer's and parkinson's disorders. molecular chaperones are a family of proteins that are believed to have ability for inhibiting protein aggregation. in the present study the effect of different concentrations of green synthesis silver nanoparticles (agnps) from pulicaria undulate l. on the amyloid formation of a-lactalbumin (a-la) and chaperone action of a-casein have been investigated. the effects of the agnps were determined using light scattering absorption, tht binding assay, intrinsic fluorescence assay, ans binding assay, cd spectroscopy and sds-page. light scattering and tht assay results showed that agnps have the ability in preventing aggregation of a-la in a concentration-dependent manner. consistent with these results, sds-page results represented that by increasing the concentration of agnps the adsorption and interaction between agnps and protein have increased. light scattering and tht assay results, also, revealed that the amyloid fibrilation decreased in the presence of both agnps and a s -casein compared to presence of a s -casein alone. fluorescence results, however, show that agnps have no effect on the chaperone ability of a-casein and in fact they perform their protection of protein aggregation action independently. consistent with the above experiments, cd spectroscopy also revealed that agnps have decreased structural changes in reduced a-la in absence and presence of a-casein, both the tertiary and the secondary structure of the proteins. our finding represented that agnps have preventing effects on protein aggregation and have no effect on the chaperone ability of a s -casein. in the main, results of this study show that biosynthesized agnps mediated by >pulicaria undulate l. maybe could be affective as a therapeutic agent for inhibiting aggregation in treatment of amyloidosis disorders. pink1 is a mitochondrial kinase with multiple cellular functions. while loss-of-function mutations of pink1 gene lead to early onset parkinson's disease, its over-expresion is associated with cancer development. parkinson is a multifactorial neurogenerative disease, with a complex aethiology including various cellular stressors. it is now known that genotoxic stress also triggers the release of soluble factors able to induce changes in neighboring cells enhancing the initial lesions, process known as bystander phenomena. althrough the mechanisms are still unclear, recent studies point towards a role for mitochondria in this process. our work investigates pink1 role in intracellular and intercellular stress response, comparatively in various models: fibroblasts (mefs) and neuroblastoma (sh-sy5y) used as a tumoral model or differentiated to a neuronal phenotype. pink1 role in this process was analyzed using genetically engineered pink1 deficient cells exposed to a genotoxic agent, bleomycin. the modified cell lines showed a reduced level of basal atp production. pink1 proved to be involved in cellular vulnerability to stress. despite differences in cellular sensibility between our models, genotoxic treatment of pink1 deficient cells induced consistently higher lesions compared to corresponding wild type variant. pink1 deficient cells showed altered intercellular signaling of stress, impairing bystander phenomena induction, by suppression of signal formation in treated cells, but also by altering the capacity to respond to the signals in neighboring cells. our hypothesis is that pink1 contributes to the management of cellular stress being involved in bystander transmission of detrimental effects through intercellular communication. this is determined mainly by its role in maintaining mitochondrial homeostasy and atp levels, pink1 deficient cells lacking the amount of energy required for rapid dna repair and stress signaling transmission. p-09.02.2-008 intranasal administration of synthetic fragments from receptor for advanced glycation end products prevents memory loss in olfactory bulbectomized mice the receptor for advanced glycation end products (rage) is a member of the immunoglobulin protein superfamily. activation of rage causes brain inflammation, oxidative stress and secretion of beta-amyloid that has been recognized as an essential phase in the development of alzheimer's disease. it is known that the receptor soluble isoform (srage) which lacks the transmembrane and cytosolic domains binds to ligands and prevents negative effects of the receptor activation in in vivo and in vitro experiments. we proposed that potential ligand-binding peptide fragments from srage would demonstrate the same biological activity. we have selected and synthesized 10 peptide fragments from unstructured surface-exposed regions of rage. synthetic peptides were intranasally administrated into olfactory bulbectomized (obe) mice with neurodegeneration of alzheimer's type. we have found that only administration of rage fragment (60-76) effectively prevents the obe murine memory from impairment, leads to decrease of beta-amyloid level and blocks the development of neuronal pathology in the brain of experimental mice. six overlapping fragments of rage (60-76) peptide were synthesized in order to find a site, responding for the therapeutic effect. tests in obe mice carried out with these fragments showed that only the n-terminal part of the molecule is responsible for preventing obe mice memory from impairment. all fragments which do not include n-terminal 60-61 dipeptide have been fully inactive in these experiments. we have proposed that active peptides can interact with beta-amyloid or s100b protein preventing these ligands from binding with rage. this interaction can inhibit the development of neurodegeneration. the aim of this study was to examine effects of social isolation, enriched environment and exercise on learning in rats. the study included 36 female 25 day old wistar rats. the rats were randomly divided into four different groups; control, exercise, social isolation and the enriched environment groups. the social isolation group and the enriched environment group were housed under their specific conditions and the exercise group and the control group were housed in standard conditions during 6 weeks. the rats in the exercise group swam for 6 weeks. after 6 weeks, the rats were evaluated in the morris water maze. brain and blood samples were taken and the hippocampus tissue was dissected. bdnf and ngf levels were measured in these samples. in conlusion, while enriched environment was a positive effect on spatial learning, social isolation was a negative effect on spatial learning and increase thigmotactic behaviors. according to the analysis results ngf and bdnf levels in the hippocampus and plasma did not change with environmental conditions and exercise. time of exposure to social isolation, procedures of the enriched environment, time of exposure to the environment, type and duration of exercise and gender may affect the results. alzheimer's disease (ad) was characterized by dementia that typically begins with subtle recognition failure and poor memory. it slowly becomes more severe and, eventually, incapacitating. the cholinergic system seemed particularly susceptible to synapse loss, especially in cortical regions associated with memory and executive function (1) . recent studies showed that the main cause of the loss of cognitive functions in ad patients was a continuous decline of the cholinergic neurotransmission in cortical and other regions of the human brain (2) . acetylcholinesterase (ache) and butyrylcholinesterase (bche) are hydrolytic enzymes that act on acetylcholine (ach) to terminate its actions in the synaptic cleft by cleaving the neurotransmitter to choline and acetate. both enzymes are present in the brain and detected in neurofibrillary tangles and neuritic plaques. it was suggested that ache predominates in the healthy brain, with bche considered to play aminor role in regulating brain ach levels. however, bche activity progressively increases in patients with ad, while ache activity remains unchanged or declines. both enzymes therefore represent legitimate therapeutic targets for ameliorating the cholinergic deficit considered to be responsible for the declines in cognitive, behavioral, and global functioning characteristics of ad (3). we initiated a study to screen their acetylcholinesterase (ache, ec 3.1.1.7) inhibitory activities, which are the key enzymes taking place in pathogenesis of ad. newly synthesized chiral benzimidazole derivatives with thioure structure showed ic 50 values in the range of 11.55-36.00 nm for ache. this study was financed by turkish research council-tubi-tak (kbag 115z837). p-09.02.2-012 f3-a13 h, novel fingolimod derivative, activates camp-dependent signalling pathway in sk-n-sh cell line g. celik turgut 1 , a. sen 1 , d. doyduk 2 , y. yildirir 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 department of chemistry, faculty of sciences, gazi university, ankara, turkey fty720, a sphingosine 1-phosphate (s1p) receptor modulator, is the first oral disease-modifying therapy to be approved for the treatment of relapsing-remitting multiple sclerosis. in this study, we have synthesized and characterized novel derivative of fty720, namely f3-a13 h, and have determined its underlying camp regulation in sk-n-sh cell lines. for this purpose, we first determined the regulation of the camp response element (cre) activity and camp concentration by f3-a13 h along with fty720 using pgl4.29 luciferase reporter assay and camp immunoassay, respectively. then, we have determined their effect on camp/pka-related gene expression profiles using custom arrays along with fty720 treatment at non-toxic doses. it was found that f3-a13 h significantly activate cre and increase camp concentration in the sk-n-sh cell line, indicating that it activates camp pathway through cell surface receptors as fty720 does. furthermore, f3-a13 h modulates the expression of the pathway related genes that are important in camp signaling pathway. in summary, our data demonstrate that the novel fty720 derivative act as a modulator of camp ultimately by influencing the gene expression via the camp and downstream transcription factor cre pathway. in conclusion, f3-a13 h might contribute future therapies for multiple sclerosis. alzheimer disease (ad) results in memory impairment and accompanied by neuroinflammation, cholinergic deficit and amyloid-beta (ab 1-42 ) accumulation in brain. we found that bacterial lipopolysaccharide (lps) injections or mice immunizations with extracellular a7 nicotinic acetylcholine receptor (a7 1-208 nachr) domain resulted in astrogliosis, decrease of a7 nachr density, accumulation of ab 1-42 in brain and episodic memory impairment. the aim was to reveal main event triggering ad-like symptoms development. c57bl/6 mice were injected with lps, immunized with recombinant a7 1-208 or a7 1-208 endoglycosidase treated to remove carbohydrates. two immunizations with 3 week interval were performed. control mice obtained complete freund's adjuvant injections. mice were tested for memory performance, blood sera were examined for presence and fine specificity of a7 1-208 -specific antibodies and brain preparations were studied for a7 nachr, ab 1-42 and il-6 levels. the original a7 1-208 ('glyc') was more immunogenic than 'deglyc', and their epitopes were recognized with different efficiency. in contrast to lps and 'glyc' a7 1-208 immunization with 'deglyc' a7 1-208 did not stimulate il-6 elevation in brain and had no proinflammatory effect. immunizations with 'glyc' or 'deglyc' a7 resulted in similar a7 nachrs decrease and ab 1-42 accumulation in brain and significant episodic memory decline comparable to those after lps injections. a7 nachr interacts directly with amyloid-beta precursor protein and facilitates its proper processing and metabolism. our data indicate that decrease of a7 nachr density caused by a7 1-208 -specific antibody is critical for ab 1-42 accumulation and episodic memory impairment while pro-inflammatory capacity of a7 1-208 -specific antibody plays secondary role in ad-like symptoms development. in vitro antioxidant and antiacetylcholinesterase activity of achillea millefolium alzheimer diseases (ad) is a neurodegenerative condition without a current effective treatment. increase in reactive oxygen species and lipid peroxidation or decrease in total antioxidant capacity causes oxidative stress-induced tissue damage. it has been suggested that decrease in oxidative stress and inhibition of acetylcholinesterase (ache) activity play a major role in the prevention and slowing of cognitive symptoms of ad. recently, studies have been directed for the discovery of medicinal plants and natural substances that are known to have natural antioxidants. achillea millefolium (a. millefolium) is a traditional herbal medicine that contains natural compounds with antioxidant activity and has been used as a carminative, diuretic, menstrual regulator and wound healer, however the mechanism of its actions are unclear. the aim of our study was to investigate the effects of a. millefolium extracts on free radical production, acetylcholinesterase (ache) activity and lipid peroxidation in vitro. methanol (me) and ethanol (ee) extracts of a. millefolium were prepared to determine (a) in vitro antioxidant capacity (by using 2,2-diphenyl-1-picrylhydrazyl assay, radical scavenging activity, phosphomolibdenum-reducing antioxidant power, ferricreducing antioxidant power, and total phenolic-total flavonoid contents), (b) effects on ache kinetics (by using a colorimetric assay) and (c) effects on sodium nitroprusside-induced lipid peroxidation in mice brain homogenates. me showed a higher antioxidant activity compared with ee in the biochemical assays tested. similarly, me demonstrated significant inhibition of ache activity that was potent than ee. both extracts dose-dependently decreased malondialdehyde content in mice brain homogenates suggesting a strong inhibition of lipid peroxidation. these results showed that a. millefolium has a high antioxidant capacity and antiache activity, indicating a potential use as an adjuvant therapy in ad. b-cells are known to play a key role in multiple sclerosis (ms) progression and autoimmune response. cxcr5 is the main b-cell chemokine receptor that under normal conditions directs their migration to specific areas of secondary lymphoid organs. in ms, areas of demyelinating lesions have been reported to attract bcells due to overexpression of cxcl13, the cxcr5 ligand. we aimed to determine whether snp rs630923 located in the promoter of cxcr5 gene and associated with high risk of multiple sclerosis could have a direct effect on of cxcr5 promoter activity. mef2c binding to dna was assessed using pull-down assay. b-cell stimulation was performed using lps, pma and ionomycin. activities of variants of cxcr5 promoters containing different rs630923 alleles were estimated using luciferase reporter assay. we determined that minor rs630923 allele creates functional mef2c-binding site within one of the regions required for the basal activity of the cxcr5 promoter. cxcr5 promoter containing minor rs630923 variant that is statistically associated with low risk of ms showed significantly decreased activity in stimulated human b-lymphoblastoid cell lines. mef2c has been reported to play an essential role in b-cell survival and b-cell responses. we determined mef2c as the main regulator of rs630923-dependent modulation of cxcr5 promoter activity in b-lymphoblastoid cell lines. this link may be directly related to pathogenic b-cell activities in multiple sclerosis. introduction: parkinson's disease (pd) is the second most common neurodegenerative progressive brain disease with increasing prevalence in aging population. the etiopathogenesis involves many cellular procesess, but is not fully elucidated yet. treatment of pd is based on levodopa and dopamine agonists, but mao-b inhibitors, comt inhibitors, amantadine or anticholinergics may be used as initial monotherapy or as adjuvant therapy. treatment related adverse drug reactions (adrs) are frequent, but cannot be predicted and/or prevented. non-motor adrs, such as nausea, somnolence, hallucinations and hypotension are frequent in dopamine agonist therapy, while dyskinesias along with motor fluctuations are the most common late adrs with levodopa. the aim of our study is to combine clinical data with genetic and epigenetic biomarkers in the algorithm for personalized approach to pd management. materials and methods: we are planning a clinical study to assess the combined impact of selected clinical, genetic and epigenetic factors on the progression of pd, adrs and treatment response. our study will have a retrospective and prospective arm. we will collect peripheral blood samples of pd patients and clinical data. single nucleotide polymorphisms (snps) in the genes involved in dopamine, neurotransmitter and drug metabolism and transport, receptors and signalling pathways will be genotyped. snps within inflammatory, neurodevelopmental, antioxidative defense, synaptic transmission and immune response pathways will also be analysed. in the prospective arm we will isolate the exosomes and check their mirna content at the time of diagnosis and after the treatment initiation. the combined effects of clinical, genetic and epigenetic factors will be analyzed using lasso penalized regression analysis. conclusions: we hope to identify genetic and/or epigenetic biomarkers that may predict progression of pd, adrs and treatment response and may support personalized tratment of pd. most evidence indicates that g protein-coupled receptors form heteromers between them and with other receptors. by allosteric mechanisms, them acquire a multiplicity of unique pharmacological and functional properties. recently, we discovered that dopamine d1 receptors (d1r) and histamine h3 receptors (h3r) form heteromers through which h3r ligands can inhibit d1r function. d1rs also physically interact and modulate ionotropic glutamate nmda receptors (nmdar). in the present work, we investigated if nmdar, d1r and h3r form a heterotrimeric complex in brain. the heteromer expression was studied in slices from both rat and mouse brain cortex by co-immunoprecipitation (co-ip) and proximity ligation assays (pla). the ability of d1r and h3r to interact with nmdar in transfected hek cells was analyzed by bioluminescence resonance energy transfer (bret) with bimolecular fluorescence complementation (bifc) experiments. heteromer properties were studied by analyzing erk1/2 phosphorylation and cell death in cortical slices. endogenous d1r-h3r heteromers were detected in rat and mouse cortical slices, where h3r ligands decreased d1r signaling (erk1/2 pathway) and were also able to block the cell death induced by overstimulation of either d1r or nmdar. by bret experiments in transfected hek cells, we demonstrated that both d1r and h3r form heteromers with nmdar subunit 1a in the presence of subunit 2b. d1r-h3r-nmdar heteromers were detected by bret with bifc. endogenous d1r-h3r-nmdar heteromers were observed in rat and mouse cortex by pla. many systems, including the glutamatergic and dopaminergic, are involved in neurodegeneration. our innovative finding is that d1r, h3r and nmdar form heteromers that may be a point of intervention for cognitive disorders in neurodegeneration. d1r-h3r-nmdar heteromers are expressed in brain cortex and a complex interaction exists between protomers in the heteromer, where h3r ligands act as a 'molecular brake' for d1r and nmdar signaling. studies conducted on obesity and hfd (high fat diet) revealed hypothalamus have crucial roles on development of metabolic diseases. after chronic over nutrition or high fat diet, as a neurodegenerative condition, premise inflammation, neural stress and development of functional impairments are observed. these studies generally focused on changes in neurons, but it's effects on brain vessels are still unknown. in this study, as a neuronal damage infrastructure, changes in hypothalamic vascularity investigated. experiment initiated with 5 weeks old total 40 male wistar rats. in order to acquire obese phenotype, the rats were fed either cafeteria diet as hfd, or normal/chow (standard diet, sd) for 9 months. intravenous glucose tolerance tests performed before sacrification. animals were exposed for 10 s to co2 and then decapitation was performed with guillotine. isolated brains were directly immersed into liquid nitrogen and stored at à80°c. the hypothalamic sections were acquired with the cryostat instrument at different. immunofluorescence was performed on serial sections through the hypothalamus using the antibody smi-71 and cd31. changes in tight junction (tj) proteins (occluding and zone occludin-1) are evaluated via western blot (wb) analysis. the hfd-treated consumed significantly more food than did control animals, when examining average food consumption per day and rats that received the hfd diet weighted significantly more at the end of 9 month diet treatment. there were no differences acquired for glucose tolerance tests. however, after hfd treatment, wb analysis have shown that tj proteins decreased even if hypothalamic micro vessel number increased and smi-71 staining have shown that increased. our primary results have shown that hfd diet can affect hypothalamic vascularity and such changes might initiate neurodegenerations and functional impairments as observed in neuroretinal degeneration in relation to vasculopathy in diabetic patients. defects of mitochondrial trafficking are common problem in many neurodegenerative diseases. its dysregulation can contribute to changes in bioenergetics profile of the cell and can lead to cell death. in our study we investigate distribution of mitochondria and their transport in primary fibroblasts derived from patients with sporadic form of alzheimer's (ad) and parkinson's (pd) diseases. our data revealed that in the most cases the velocity of mitochondrial movement is lower in ad and pd cells in comparison to the control. the most intense differences between ad, pd patients and control group are observed in the case of movement of large mitochondria. owing to the fact that mitochondrial trafficking depends on mitochondrial state, we investigated the 'age' of mitochondria. we observed a diminished mitochondrial turnover in ad and pd fibroblast. evaluation of the mitochondrial distribution within the cell in all 3 groups (ad, pd and control) showed that in the perinuclear area are accumulated 'old' and 'worn out' mitochondria, probably dedicated to remove from the cell. because mitochondrial biogenesis, shape and size depends on fusion/fission proteins we assessed their level within the cell. to summarise, our results revealed alterations in mitochondrial trafficking in fibroblasts derived from patients with alzheimer's and parkinson's diseases in comparison to the healthy control cells. carbonic anhydrases (cas, ec 4.2.1.1) is a zinc metalloenzyme that catalyzes the reversible reactions of co 2 and water. carbonic anhydrases (cas, ec 4.2.1.1) form a family of metalloenzymes that play an important function in various physiological and pathological processes. therefore, many researchers work in this field in order to design and synthesize new drugs. carbonic anhydrase activators are important as much as inhibitors. caas have polar groups to make hydrogen bond in the main body and the activation property of enzyme increaase in this way. caas are have polar groups to make hydrogen bond in the main body and the activation property increaase in this way. furthermore, recent studies suggest that ca activation may provide a novel therapy for alzheimer's disease. in this study ca activators are determined. human carbonic anhydrase isozymes ca i and ca ii are isolated from human blood erythrocyte. hca-i and hca-ii isoenzymes were purified using sepharose-4b-l tyrosine-sulfanilamide affinity colum. finally, hca-i and hca-ii isoenzymes were eluted with appropriate elution buffers. enzyme purity was checked by sds-page. the enzyme activity system contained 0.05 m tris-so 4 ph 7.4, r-nitro phenol in 1 ml total volume. effects of some macrocyclic thiacrown ethers derivates were investigated. enzyme activities were measured at constant substrate and different activator concentrations to find ac 50 value. these compounds are thought to be useful for treating alzheimer's disease. introduction: gender differences in stress models are not studied in detail. we compared different stress conditions on brain bdnf levels, in social isolation (sit) and predator scent tests (pst) in rats. bdnf levels in cortex, hippocampus and amygdala were compared, effects of chronic fluoxetine (flu) treatment were evaluated. methods: 128 rats were used. for sit, animals were kept individually for 1 month and for pst, rats were exposed to dirty cat litter for 10 min at the first day of 1 month stress. flu was given (5 mg/kg, ip) through stress. controls, stress and treated groups were evaluated in elevated plus maze (epm), anxiety scores were calculated. brain bdnf levels were determined in cortex, hippocampus and amygdala by western blot. p < 0.05 were considered significant. results: sit and pst induced anxiety in both male and female rats, females having greater anxiety scores than males (p < 0.05). flu restored anxiety scores in both sexes (p < 0.01) in two settings. male and female rats exhibited reduced cortical bdnf levels in sit (p < 0.001). pst reduced cortical bdnf in females, but increased in males. hippocampal bdnf expression was lowered in sit (p < 0.01) and pst (p < 0.001) in both sexes. female rats had 40% lower bdnf expression than males in amygdala in sit. flu did not restore cortical bdnf in females in both tests, but reduced incresed bdnf levels in males (p < 0.001). flu did not restore reduced brain bdnf in males in hippocampus and amygdala, but restored in hippocampus, in females. discussion: our findings indicate that sex differences must be considered in studies related to mood disorders of animal models, and suggest that bdnf expression in different brain regions are altered differentially in a gender-dependent manner in rats. antianxiety effect of flu is not mediated through increasing bdnf activity in cortex in both genders. increased bdnf in hippocampus and amygdala may reflect its antidepressant effect in female rats, but not in males. perineuronal nets (pnns) are special forms of neural extracellular matrix found around neuron bodies and neurites. hyaluronan and proteoglycan link protein1 (hapln1) is one of the major elements of pnns. hapln1 interacts with tenascins and aggrecan which are other essential pnns components. in most of neurodegenerative disorders caused by neuritogenesis defects, disrupted pnns structure and decreased expression of hapln1 were observed. however, the role of hapln1 in neural differentiation is unknown. the aim of this study is to determine mrna and protein levels of hapln1 during differentiation using pc12 cell line as a neural differentation model, derived from rat pheochromocytoma. after pc12 cells were stimulated to differentiate into neurons by nerve growth factor on days 3, 5 and 7; cells were collected, qrt-pcr and western blot were performed. also, in order to find out whether there is a physical interaction between hapln1 and proteins related to neuritogenesis defects, spinal muscular atrophy (sma) was used as a neurodegenerative disease model. therefore, a detailed hapln1 and survival motor protein 1 (smn1) network analysis were performed in-silico. as a result, we analyzed 3 fold increase in hapln1 mrna level compared to undifferentiated state. on the other hand, a decrease in protein level was detected. this decrease in cellular hapln1 level suggests that, hapln1 is required for formation of pnns structure, thus secreted to extracellular environment at early stage of differentiation. in addition, according to in-silico analysis, an indirect path between hapln1 and smn1 through fibulin2 (fbln2) was detected. fbln2 was also found to be an interaction partner between different matrix molecules such as aggrecan and hapln1 which form a macromolecular meshwork. the results of this study will pave the way for investigating the role of hapln1 and fbln2 in neurodegenerative disease models. also it will help us to understand the mechanism of neuritogenesis defects. determination of properties of bone marrow and tissue-derived mesenchymal stromal/stem cell population in neurofibromatosis type 1 patients neurofibromas, complex tumors deriving from schwann cells and containing fibroblasts, vascular structures and mast cells, are part of the clinical picture in nf1. the risk of malignancy is increased in nf1, wound healing is delayed and keloid formation is frequent. because multiple tissues are involved in malignant and non-malignant manifestations of nf1, we considered the mesenchymal stem/stromal cells (msc) carrying the nf1 mutation might play a role in the microenvironment. mscs affect the biological behaviour of other cells: they alter their proliferation, apoptosis and migration through various secreted growth factors, cytokines, chemokines, or by direct contact. we examined the msc of nf1 patients. methods: the adipogenic and osteogenic differentiation potential of mscs from nf1 and healthy subjects was examined in vitro and by rt-pcr. msc's migration potential was measured in the scracth assay. mscs' interaction with schwann cells and their effect on tumorigenesis was examined in co-culture by apoptosis markers on flow cytometry. results: nf1-mscs' adipogenic and osteogenic differentiation potential was lower than healthy controls as assessed by staining aizerin red s and oil red o and rt-pcr for osteopontin and collagen1. mscs cultured from dermal neurofibroma showed faster closing of the scratch compared to the same patient's normal and caf e au lait skin. on the other hand, mscs obtained from plexiform neurofibroma healed late, while mscs derived from the same patient's caf e au lait skin showed the fastest healing. hepatic encephalopathy with ammonium ions accumulation is accompanied by some disorder in the brain due to toxic material concentration being usually detoxified in the liver. one of the reasons for hyperammoniemia could be some imbalance in brain glutamine metabolisation induced by the key enzymes glutamine transferases (gts), which catalyze the reaction of glutamine transamination resulting in neurotoxic product of a-ketoglutaramate (akgm). akgm is hydrolyzed to a-ketoglutarate and ammonia by x-amidases. in the study, the dynamics of the enzymes activity in the tissues and biological liquids of experimental animals with hepatic dysfunction induced by thioacetamide (taa) was under investigation. white laboratory rats of wistar line (female, weight of 140 g) chronically intoxicated with hepatotrophic toxine of taa. every 2 weeks, some biological samples were collected to assess gt-k and x-amidase activities. x-amidase activity was the highest in the kidney tissue in the control and decreased by 70% in the experimental group. in the experimental hepatic x-amidase activity decreased by 240% compared to those in the control. the average x-amidase activity in the blood serum (0.015 nmols/ mg/min) and in the brain (0.005 nmols/mg/min) differed faintly. maximal gt-k activities were revealed in the kidneys; in the controls, it was about 250% higher than those in the experimental animals. the difference between average enzyme activities in the liver of the control and experimental animals reached 350%. the average gt-k activities in the blood serum and brain of the control and experimental animals were rather similar. the decrease in x-amidase and gt-k activities obtained in the study during hepatic pathology development could testify to imbalance of glutamine metabolism, possibly aimed at declining the level of akgm neurotoxicant under the hepatic dysfunction. acknowledgments: supported by the russian federation ministry of science and education (grant no rfme-fi60414x0116). wilson disease is an autosomal recessive disorder of copper metabolism characterized as neurodegeneration and liver abnormalities. it is caused by defects in the atp7b gene. atp7b is responsible for the sequestration of cu into secretory vesicles, and this function is exhibited by the orthologous ccc2p in the yeast. we aimed to characterize clinically-relevant novel mutations of p.t788i, p.v1036i and p.r1038g-fsx8 in yeast lacking the ccc2 gene. the patients with these mutations have copper storage abnormalities in different parts of their bodies; p.t788i mutation mainly affects the liver and the nervous system, p.v1036i mutation affects the nervous system, and p.r1038g-fsx83 mutation causes damages to the liver. to better understand the effects of these mutations on normal functions of atp7b, we cloned human atp7b gene onto a yeast expression vector and created the same mutations by site directed mutagenesis. then, wild type and mutated forms of atp7b genes were transformed into yeast cells lacking the homologous ccc2 gene for functional comparison. first, we analyzed the expression of atp7b and its variants in yeast cells by a real time pcr approach and western blot to make sure that transformed cells express the plasmids. expression of human wild type atp7b gene in ccc2d mutant yeast restored the growth deficiency and copper transport activity, however, expression of the mutant forms did not restore the copper transport functions and only partially supported the cell growth. our data support that p.t788i, p.v1036i and p.r1038g-fsx83 mutations cause functional deficiency in atp7b functions and suggest that these residues are important for normal atp7b function. in recent years, attempts were made to develop miniaturized potentiometric biosensors which is particularly important to reduce the amount of enzyme and reagents needed. the miniaturization of a biosensor is possible by using an all solid-state polymer membrane ion selective electrode which is cheap, easy to prepare and allow micro-sized construction. the use of all solidstate polymer membrane ion selective electrode as the basic sensing element also has the advantage of providing biosensors that are easy to fabricate, exhibit rapid response and have long life-times. they are also mechanically stable and allow flow-through configuration. genetical and chemical modifications for the alteration of enzyme molecule characteristics are gained considerable importance. enzymes can be modified chemically by using water-soluble polymers or some chemicals. conjugates of natural and synthetic macromolecules with enzymes provides wide usage in medicine and in many fields of biotechnology. in this study, enzyme-polymer conjugates with different molar ratios were synthesized using urease enzyme. in this study micro sized potentiometric urea sensitive biosensor has been developed in which urease-polymer conjugates were immobilized on polymeric membrane ammonium ion selective electrodes whether pvc or derivatized-pvc via glutaraldehyde cross linking reaction. biosensor is not include inner reference electrode and inner reference solution. potentiometric performance of biosensor will be examined with a computer-controlled measurement system designated. the most important features of the obtained micro sized urea biosensor by using enzyme-polymer conjugations were being highly sensitive, having long life-time, easily built at a low cost, and having short response time when compared with conventional potentiometric urea biosensor. also, these biosensors were easily built at micro-construction. this study was supported by grant from the tub _ itak research fund (project number: 114z138). creative drama technique as a new tool to increase enthusiasm and to achieve learning objective for medical students e. y. sozmen 1,2 , e. erem 3 1 faculty of medicine, ege university, izmir, turkey, 2 center for continuing education, ege university, izmir, turkey, 3 recently drama in education techniques have been implemented successfully in education program of primary and secondary high school and positive effects of these techniques on learning ability and attitude of students have been shown. the aim of this study was to organize an education program based on drama in education techniques in a special module of ege university medical faculty and to test any effect of this technique on achievement of learning objectives and student's perspectives on drama. the special module program was on the oxidative stress and antioxidants. the program covered the drama in education sessions (improvisation, role play, game) linked with learning objectives (understanding of free radical generation and free radical reactions in body, evaluation of the effect of free radical reactions in diseases as well as increase the ability usage of scientific information), laboratory work (antioxidant activity determination) and searching a special scientific topic on literature. 12 students (in 3rd year of faculty) who had taken theoretical lecture on this subject a year ago, participated in this special module. the opinions of the students on the program were obtained through a questionnaire form and the increase in knowledge was evaluated via pre/posttest. the mean of pretest point was 2.7/10, that increased to 7.4/10 in the posttest evaluation. 50% of students pointed that they enjoyed participating in drama activities in the pre-questionnaire, this rate was 100% in the final questionnaire. they all remarked that implementation of drama in education was beneficial for their communication skill, helping them to learn more about science and increased their enthusiasm to learn and discuss the scientific information. although the preparation process might take more time and need to hard work for teachers, we concluded that the drama methods as a new tool to increase of participant's interest might be proposed for students in higher education. laboratory-based performance assessment in medical education: an opportunity for connection between scientists and medical students h. tuncel cerrahpasa medical faculty, istanbul university, istanbul, turkey number of medical students who interested in basic medical sciences is declining and medical sciences literacy is falling, it is crucial to develop ways for students and scientists to connect. students need to know that science is an intensely human endeavor, and scientists need mechanisms to bring that truth to the community at large. based on continued interest and experience on the part of faculty, and on student feedback, the development of a more effective and stimulating interactive learning tool was undertaken. an in-depth knowledge of laboratory medicine principles is vital to all practicing physicians. great variation exists in the ways that medical students learn the principles of laboratory medicine. there are a number of programs for electronic media that emphasize laboratory-related skills. some of these are appropriate for medical students in the clinical years. programs that teach skills in common laboratory procedures, such as interpretation of peripheral blood smears and microscopic examination of urine sediment, have been shown to improve student performance. to ensure that important principles are addressed, medical schools should establish goals and objectives specifically related to laboratory medicine and experiment with optimal teaching and assessment methods. we also hope that this study will inspire dialogue among primary care and specialist physicians as to the proper degree of education in this area. ideally, it will encourage scientific studies that address evidence-based possibilities for improving critical laboratory medicine educational outcomes, that is, the training of physicians who optimally use laboratory diagnostics and therapeutics. engaging medical students in scholarly scientific activities and producing clinically competent and research-oriented medical workforces are essential demands, particularly in developing countries. an experimental special study module for medical undergraduate students: learning western blot analysis and detection of b-actin protein expression in tissue and cell culture samples learning, introduce basic principles of laboratory research and to present the results.b-actin is one of six actin isoforms which is mainly expressed in all eukaryotic cells. western blotting is a widely used laboratory technique to determine specific proteins and to evaluate protein expression in tissues and cells. in our study, different concentrations of rat spleen homogenates (25, 50, 75 lg/well) and 50 lg protein/well of human lung and ovary cancer cell lysates were used. the proteins were seperated by 10% sds-page, transferred to pvdf membrane, incubated with specific b-actin antibody and then with hrp-conjugated antibody. protein bands were detected with ecl and densitometric analysis of proteins was quantified by imagej software. differences in protein band intensities were compared using one way anova.a value of p < 0.05 was considered statistically significant. we detected b-actin expression in rat spleen homogenates, human lung and ovary cancer cell lysates, as a 43 kd protein. the protein band intensities were in correlation with protein concentrations. the highest concentration resulted in the highest signal intensity in rat spleen homogenates.b-actin level was higher in ovary cancer cells than in lung cancer cells, although both proteins were loaded equally. the feedback showed that students were very satisfied with this laboratory ssm. they developed their independent study skills, planned a research, worked in a laboratory, learned and performed a technique, western blotting. the hands-on experience is very important for medical undergraduate students for their future scientific career. three-dimensional structure of truncated human kv10. voltage-gated potassium channel kv10.2 belongs to the ether-ago-go family. it has been proved that its mutants are involved in development of neurological diseases and some types of tumor. directed drug design needs knowledge of details of the threedimensional channel structure. the members of the kv10-12 subfamilies are characterized by extremely long n-and c-terminal intracellular tails, which possess a number of structural domains. the n-terminal pas domain in kv10 plays an important role in activation, and is thought to alter the rate of deactivation, possibly by binding at or near the s4-s5 linker at the inner mouth of the pore. here we present an improved 3d structure of the truncated human kv10.2 channel, obtained by single particle em. this channel lacks its cytoplasmic pas domain but it still forms tetrameric particles. earlier we showed that the full length kv10.2 channel is build according to the 'hanging gondola' type, and its cytoplasmic and transmembrane parts are connected by linkers. the cytoplasmic part includes the interconnecting pas and cnbd domains. deletion of the pas domain leads to the conformational change in the cytoplasmic part of the channel. for interpretation of the 3d structures we used homology modeling and molecular dynamics simulation. there are several templates available to the moment including eag domain-cnbhd complex of the mouse eag1 channel, full-length shaker potassium channel kv1.2, c-linker/cnbhd of zelk channels and others. but there are still no templates for many fragments that led to necessity of partial de novo modeling. analysis of molecular trajectory allowed estimating dynamical characteristics of channel, supposing interdomain interactions. results of the conducted investigation have a great interest at both the academic and the industrial levels. the objective of this task program is to enable students to gain scientific attitude and skills for them to be able to deal with the problems they'll confront in future research careers. it's been emphasized in various studies that medical students are keen on conducting scientific research, and it's been stated that they need to be supported in this respect, as they lack the adequate fund of knowledge. this study aims to share information throughout a 5-year performance of the research training program (rtp) conducted at ege university, faculty of medicine. rtp is an educational program applied in addition/parallel to the bachelor's degrees for establishing scientific thought, attitude, proceeding and knowledge of the willing medical students, and enabling them to adopt study skills. the dynamic program produced by the rtp committee in 2011 has been developing each and every year via feedback received from the students. an operating principles, a manual for advisor and an introductory booklet have been laid out. applications are being accepted at the end of first academic year, making announcement to the freshmen in advance. students are being admitted to the program, taking the assessment criterias into consideration. second and third year medical students attend the didactic part of the program during the terms devoted to special study modules. thereafter, they go through the project management phase, and accomplish their projects under supervision of a faculty member until their graduation. 12 first graduates of the program, accomplishing their projects, received their certificates at the graduation ceremony of 2015 graduation. currently, there are 61 students in total from all classes who perpetuate their studies within the program. an inventive training pattern of ege university faculty of medicine, rtp experience is being maintained as a dynamic process and successfully keeps the students advised of conducting scientific researches, cultivating scientific awareness. objectives: objectives selection and verification of blood collection tubes is an important preanalytical issue in clinical laboratories. in this study comparison with the reference glass tube of ayset plastic tubes containing separator gel and assessment for routine clinical chemistry laboratory testing in samples were aimed. materials and methods: thirty-four volunteers were included in the study. samples were taken into two different tubes by two experienced technologists according to the clsi protocol [tube1: z (becton dickinson and company, franklin lakes, nj, usa); tube2: ayset (lot10069, turkey)]. glucose, urea, creatinine, ast, alt, total-cholesterol, triglycerides and high density lipoprotein-cholesterol were analyzed subsequently (olympus au2700) and randomised samples stored at 2-8°c for 24 and 48 h. 0th hour sample was analyzed immediately after collection and accepted as the reference for the comparison of the other samples. a paired t-test and wilcoxon signed rank sum test were used to test the significance of differences between the reference tube and test tubes. results: the difference between the results of reference tube and test tubes for glukose, urea, creatinine, ast, alt, total cholesterol, tg and hdl-cholesterol at 0, 24 and 48 h were statistically no significant (p = 0.09, p = 0.07, p = 0.20, p = 0.16, p = 0.13, p = 0.09, p = 0.05, p = 0.54, p = 0.58, p = 0.46, p = 0.19, p = 0.13, p = 0.66, p = 0.72, p = 0.34, p = 0.11, p = 0.16, p = 0.06, p = 0.08, p = 0.39, p = 0.23, p = 0.63, p = 0.58, p = 0.54, respectively). conclusions: no significant difference was observed between ayset tubes' results and the reference tube's results. e. akin c ß akir d€ uzen laboratuvarlar grubu, istanbul, turkey insulin resistance underlies the development of obesity which is a global health problem. obesity is a main concern of scientists because it's associated with type 2 diabetes, hypertension and some cancers. recently, inflammation centered mechanisms are deeply investigated as well as the effects of anti-inflammatory diets which are highly rich in vitamins, minerals, fibers and healthy oils. these diets are proposed to inhibit or supress the secretion of the inflammatory mediators and also improve the intestinal microflora. the aim in this study is to highlight the increasing trend of publications in regard to insulin resistance and inflammation based obesity along with the effects of antiinflammatory diets used for its treatment in the last decade. we performed a pubmed search with key words of 'obesity and insulin resistance and inflammation' (01/01/2005-15/05/ 2016) (search #1). besides, we performed another search with key words of '(anti-inflammatory diet or dietary supplement) and (obesity or insulin resistance)' (search #2) to highlight the value of anti-inflammatory diets and dietary supplements in combating inflammation based obesity and insulin resistance. search #1 revealed 6763 articles; of these 341 were clinical trials, 18 were observational studies. human studies were 4 552 while animal studies were 2 580. overall, there were 2 229 review articles and 5 meta-analysis in the field. search #2 revealed 4 255 articles of which 796 were clinical trials, 958 were review articles, 46 were meta-analysis. human studies were 2 610 and other animal studies were 1 931. the relationship of metabolic diseases with a low grade inflammatory state has opened a new area of research to understand the consequent causes of inflammation in the human body. the increasing scientific data in the field indicates that antiinflammatory diets may serve as powerful tools to solve the inflammation and the consequent insulin resistance and obesity. medical and biological illustrations for life sciences education: is 'a picture worth a thousand words' in visualizing medicine and science? medical and biological illustrations (mbi) convey complex ideas with just an image and they are powerful intersections of science and art. the clarification of complex pathways via illustrations can be effective means in education as they help the student to visualize the biomolecular world and understand the mechanisms. our aim is to illustrate how a mbi is developed over the example of mechanism of insulin action, via the phosphoinositide (pi) 3 kinase-protein kinase b (akt) pathway. organising one's thoughts and clarifying relationships and then using the optimal complexity level to illustrate the pathway most clearly are the basics of mbi. thus, we made a thorough investigation of insulin mechanism on glucose uptake in skeletal muscle and adipose tissue; a biochemical process that includes insulin receptor (ir), ir substrate, pi3 kinase, pi-dependent kinase 1 and akt. then, we found the 3d structures of molecules via protein databanks and accordingly created drawings and diagrams of each component in both molecular and macrolevels by adobe photoshopò software. graphics tablets and a compatible pc were also used in the production phase. the use of computer hardware/software enables unlimited detail in images and provides the flexibility that classical drawing techniques can not. thus, the final diagram clarifies the underlying molecular mechanisms of a biochemical pathway along with the physiologic actions. recent improvements of computer technology have resulted in the creation, and reproduction of high-quality lower cost medical art. mbi's can be used in education to explain concepts/pathways to students to enhance learning. similarly, mbi's are great tools to show mechanism/procedures to enhance patients' understanding of their medical condition. considering their unquestionable contribution to education, research and patient care, the creation of mbi's should be promoted as a graduate level course and the discipline should be represented at academic level. biochemistry is a compelling field with broad applications in many disciplines like medicine, dentistry, pharmacy and bioengineering. biochemical research increasingly combines ideas from genetics, molecular biology, etc. and collaborates with many disciplines. our objective is to conduct a scientometric analysis of the last decade's postgraduate theses in the field of biochemistry (ptb) in regard to number, collaborations, subject area distribution, etc. to discover the characteristics and trends in turkey. we searched the turkish higher education council's theses database (2006) (2007) (2008) (2009) (2010) (2011) (2012) (2013) (2014) (2015) which includes master of science (msc.), doctorate (ph.d.) and specialization (s) theses in all disciplines. an electronic search with the keyword of 'biochemistry' (in the thesis subject area) was conducted, thus it brought all theses either in the biochemistry discipline or theses in other disciplines but have a biochemistry component. we performed data cleaning and further quantitative analyses in excel. we also executed word count analysis on the titles of theses to derive the main subject areas in ptb. of the total of 6374 ptb (2215 s, 2781 msc, 1339 phd theses) 37.6% was in natural sciences while 62.4% was in health sciences. the theses output-growth measured by the compund annual growth rate was 82% over the 10-years. the top 3 clinical disciplines in collaboration with biochemistry were pediatrics, surgery and cardiology, and the top 3 science disciplines were biotechnology, bioengineering and biology. oxidants-stress and antioxidants (1008), endocrine-metabolism (655) and enzymology (608) were the top research areas in ptb, followed by genetics (305) and cancer (252). scientometrics is a powerful tool to understand the direction of science and research. our ptb analysis indicated that prominent areas like stem cell, biosensors, geriatrics are somewhat lagging in turkish biochemistry research while postgraduate education and research in total is growing fast with sound collaborations. the 1st turkish in vitro diagnostic symposium evaluation objective: in vitro diagnostic (ivd) medical laboratory tests and the equipment are closely related the public health, patient safety and the safety of all who utilize these tests. it depends on auditing of the process from the production to the consumption of these materials, that they do not pose a risk to individuals and society. upon these basic requirements; 'turkish in vitro diagnostic symposium: medical laboratory tests' was held in february 2016, organized by the cooperation of turkish biochemical society branch of izmir, and dokuz eylul university health sciences institute. it was intended to shed light on some questions such as, what is the place and importance of ivd in turkey? what are the responsibilities of educational institutions?, what is the role of ministry of health? to put across the conditions of preparing a substructure that may provide achieving success in producing ivd medical devices and in this sector, in our country. material-method: 39 invited speakers attended the symposium, along with the participation of both as lecturers and attendees; ministry of health, turkish standards institution; representatives of manufacturer enterprises; representatives of enterprises manufacturing in turkey; scientists conducting considerable researches on health technology; students and representatives from some of the non-governmental organizations. in addition to the presentations, gathering up the written opinions of the participants, a report was prepared. results: the symposium that lasted for 3 days was realized with 120 participants in total,55 of which from universities;38 representatives of their companies;17 from chamber-institute-public establishments and 10 of which from public hospitals. 94 of the participants were from izmir,26 of them were coming from out of izmir. conclusion: at the end of the symposium,40% of the participants gave feedback. among the feedback selected; 86.2% of the participants evaluated the symposium overall, as successful. 75.6% of them found the symposium successful with regard to its scientific content. their feedback were 55.2% positive in terms of the symposium's contribution to the situation assessment on ivd in turkey, and 44.8% of them stated they would consider participating in the second of the ivd symposium if it is to be organized. perceptions of molecular life science master's students on their scientific and academic competencies and prospective plans for professional development the master's education (me) in molecular life sciences (mls) is aimed at strengthening the knowledge and skills base of the young scientist, preparing him for the competitive academia/industry positions. the rapidly developing pace of science and research forces the master's student (ms) to play a central role in monitoring and guiding his scientific education and professional development (pd). thus, the aim of our study was to examine the perceptions of ms of mls, regarding their scientific and academic competencies. with this data, we planned to analyse if this awareness channels ms to take action and/or matches with their prospective plans. we developed a 15 item online survey with 3 sections (demographic data, current data-contributions of me, competencies and prospective data-action for pd, future plans) and distributed it via e-mail to various postgraduate institutes. at the end of the 10-day period, 138 ms students (in the thesis phase) answered the questionnaire (female: 66.7%, male: 33.3%). the most highly rated activities that contributed to their scientific knowledge and skills gained in the me were laboratory work (73%), visits with their mentors (70%) and theoretical lessons (65%). ms expressed low levels of sufficiency both in theoretical scientific knowledge and laboratory skills (only 43% and 33% sufficiency, respectively). communication skills (80%) and team work (78%) were rated as the highest professional competencies followed by literature search and research planning (both 73%). it was striking that ms perceived themselves as quite insufficient in scientific writing (50%), data analysis (60%) and project writing (61%) while proficiency in english (55%) was the first area they wanted to take action. despite their perceptions of insufficiencies in many areas, a majority (69%) wanted to continue to phd education. these and similar surveys may lead to an increase in selfawareness in ms and the data may contribute to the revision of me. the report of the 1st turkey in vitro diagnostic symposium results the following aspects and suggestions took place in the results report prepared after the symposium: about the national ivd-tc r&d, production, forming quality assurance and innovation strategy and policy the cooperation of the university and the industry is not sufficient most of the industrialists cannot take enough advantage of the support provided by the institutions like tubitak, the ministry of science, technology and industry and the ministry of development the statistics on the ivd-tc in turkey should be carried out as soon as possible national standards should be determined in parallel with the international standards the vat rate of the exported raw materials that would be used in ivd production should be decreased. about the education and training, the job titles and positions the related graduate programs, which would focus on all steps of the whole life cycle related to the ivd-tcs one by one, are not widespread there is no 'postdoc' application in turkey. 'postdoc' staff is needed for insufficient component human resources the lecturers should not be restricted to one discipline only graduate programs on laboratory medicine are needed to be established and spread, in order to train component labor specified on the ivd-tc about research and development there are almost no researches related to product development. this should be associated with the education and training institutions about the research centers currently, there is a real infrastructure on health technology in turkey, but there are difficulties in its instituonalism the insufficient cooperation of the university and the industry does not allow the inventions to turn into products the cooperation supports of r&d, being restricted to tech-nopark and r&d centers, are open fields for improvement p-edu-014 phd training in medical education: career profiles and satisfaction levels of graduates from dokuz eylul university graduate school of health sciences this survey was carried out within the scope of special study modules that is entitled 'phd training in medical sciences' by a group of medical students in deu. the purpose of the survey to investigate the members of health sciences that have successfully completed their phd training in terms of the levels of satisfaction and the status of their career. from this scope we generated two hypothesis: we expected that graduate phd graduates are mostly involved in academy and find their satisfaction levels at moderate level as to phd education. the study was designed as cross-sectional. we reached 166 phd graduates who had graduated from deu graduate school of health sciences between 1991 and 2002 from 12 different departments via e-mail. the survey was included 27 questions, which were prepared in the light of the existing literature. among the 166 phd graduates, 55 (30%) participated in the study. through this survey, perception of phd students on supervisors' scientific and educational abilities, opinions on phd training, productivity of phd training, number of articles published, their position and related satisfaction levels after graduation were investigated. according to the results, more than half of the graduates (%52.7) are well satisficed from the education they had taken. beside this, interestingly we found that %94.5 of graduates prefers staying in the academic positions and %64.8 of them sustains their communication with their supervisors. in conclusion, most of phd graduates were contented with phd training and their career profiles. as a result of this survey, we produced a novel and precise contribution to the literature. in a further study, this survey may extend to other parts of turkey and compile the results in order to get more accurate and inclusive data. phd is an international degree that is reached by conducting an original research after finishing bachelor or master's degree. doctoral degree (phd) can open the door of academic career; on the other hand, a person with a doctoral degree is equipped to carry out important work in research, industry, or public sector. today, gradually increasing number of phd students have brought some problems in phd training. the purpose of this study is to investigate and review activities that have been done by the following international organizations: orpheus: (organization for phd education in biomedicine and health sciences) eua-cde: european university association-council on doctoral education. febs education committee: federation of european biochemical societies these three organizations have done workshops on phd training to pay attention to the following points: *a phd student must take some courses and trainings outside her/his institute, should not be limited to the institute. *the phd training programme should include transferable skills courses. *clinicians, if involved in phd training, should be given free time from their clinic duties. *with regards to potential problems with the supervisor, the institute should provide the student an advisory system. *students should be encouraged to participate in the management of doctoral programmes in the institute. *the students should be given be opportunity to select their own supervisor (thus their thesis area). the phd training has gained quality thanks to these organizations. it is advised that graduate school of health sciences should follow the recommendations and report from these organizations. itsn1 and itsn2 are genes encoded adaptor proteins with multiple isoforms participating in clathrin-mediated endocytosis (cme), mapk signaling and reorganization of actin cytoskeleton. changes in itsns expression can lead to different neurodegenerative disorders and cancers. to date little is known about regulation of itsn genes on posttranscriptional level. the aim of our work was to predict and experimentally confirm target sites for micrornas that could potentially regulate itsns expression. using 8 web servers we analyzed 3 0 utrs of short and long isoforms of human itsn mrnas and found conservative target sites for mir-34, mir-19, mir-129, mir-103/107, mir-194, mir-181 and mir-30 in 3 0 utr of itsn1-s, predicted by 5 servers, mir-203 predicted by 5 servers for itsn1-l, and mir-153, mir-148/152, mir-27, mir-144 and mir-128 predicted by 5-6 servers for itsn2-l. to elucidate potential impact on cme, mapk signaling and actin cytoskeleton regulation by these mirnas we performed enrichment analysis by diana-mirpath server and found that mir-34, mir-19, mir-103/107, mir-181, mir-30 and mir-148 were highly enriched for all analyzed pathways. using regrna 2.0 and scan for motifs services we predicted 12 types of different regulatory elements in 3 0 utr of itsn1 and itsn2: k-box and brd-box, musashi binding element for rbps musashi1 and musashi2, gu-rich element (gre) and au-rich elements (are) that regulate mrna decay. to confirm itsn1-s regulation by micrornas we cloned 3 0 utr of itsn1-s into luciferase reporter vector and transfect hek293 cells by this construction and mir-181a, mir-30a and mir-19a. for mir-181 transfected cells, we found 25-40% decrease of expression of itsn1-s 3 0 utr-bearing construction. for other mirs we did not obtain strong reproducible effect in luciferase assay. these data may confirm mir-181 target site in 3 0 utr of itsn1-s mrna but needed additional research. objective: stroke is an acute neurological disorder that is mostly caused by ischemia in central neural system. 30% of stroked patients lose their life in 1 year, and remaining 1/3 of the living patients continue to their lives as dependent to others. nihss and mrs are two scales which are used in prognosis studies because they can show stroke intensity and after stroke functional recovery. microrna's which have effects on transcription and posttranscription gene regulations are small rna molecules. their roles have been investigated on pathophysiology and treatment of diseases. in this study, it was aimed to detect changes in blood serum levels of mir-146a, -155, -210, 181b, -31, 126, -92a and let-7f of ischemic stroke patients and to investigate role in predicting prognosis methods: 21 patients diagnosed by acute ischemic stroke admitted to neurology service of g€ oztepe hospital and 16 healthy individual were included in the study. after stroke patients' blood samples were taken periodically in 1st day, 1st week, and 3rd month, and at the same time nihss and mrs scores were determined. set 8 mirna blood serum levels were measured by rt pcr results: when compared to the control group, we found that after stroke 1st day peripheric blood levels of mir-146a,-31,-92a and let-7f were significantly low; when 1st week and 1st day serum records were compared there was a significant increase in mir-126 level; and when 1st week and 3rd month records were compared we noted that there was a significant increase in mir-146a,-126 and let-7f levels. from prognosis point of view; after ischemic stroke measurements showed that mir-181b in the 1st day, mir-146a and mir-210 in the 1st week showed positive significant correlation with 3rd month mrs scores (p = 0.002, p = 0.05, p = 0.002, respectively) conclusion: according to the outcomes of this study, after stroke 1st day mir-181b, 1st week mir-146a and 1st week mir-210 levels can be stipulated to use in predicting patients' 3rd month prognosis p-01.03.3-004 inhibitory rna aptamer against lambda ci repressor showed the transcriptional activator activity s. ohuchi, b. suess tu darmstadt, darmstadt, germany because of the variety of functionalities on gene regulation and the easiness of molecular engineering, functional rnas are promising parts for the construction of genetic circuits. artificial affinity rna, or rna aptamer, is one of such genetic parts. in the previous study, an inhibitory rna aptamer against a repressor protein, tetr, was developed as a transcriptional activator [1] . the expression of this aptamer abolishes the repressor activity of tetr, resulting in the elevated gene expression under the control of tetr. because of simplicity of the mechanism, similar transcriptional activators can be generated by using rna aptamers against other repressor proteins. here, we examined the generation of an activator based on an rna aptamer against one of the most frequently applied repressor proteins, lambda phage ci. in vitro selection (selex) was performed targeting a recombinant ci protein employing an rna pool containing 40-nucleotides of a random region. after 6 rounds of selex, the pool rna showed the affinity, as well as the inhibitory activity, against ci in vitro. then, rna aptamers with the transcriptional activator activity were screened from the enriched pool in vivo employing a reporter plasmid on which the expression of a reporter gene, lacz, is repressed by ci. when the variants of the rna pool were transformed to e. coli cells harboring the reporter plasmid, about half of the transformants showed the elevated reporter expression. interestingly, all of these desired rna clones shared the same sequence. quantitative analysis indicated that 35-fold induction of the reporter expression was achieved upon the aptamer expression. our results suggested that diversity of artificial transcriptional activators can be extended by employing rna aptamers against repressor proteins to broaden parts for the construction of genetic circuits. [1] hunsicker, et al. (2009) an rna aptamer that induces transcription. chem. biol., 16: 173-180. p-01.03.3-006 microrna expression signatures between non-atherosclerotic plaque and atherosclerotic plaque in cad with humans, and parallels whole blood rnas and represent a new important class of gene regulators. the present study was designed (i) to investigate the mirna expression profile in human atherosclerotic plaques from peripheral arteries aorta as compared to non-atherosclerotic left internal mamary artery (lima); (ii) to examine the expression levels of mirna in whole with correlation mirnas of aorta tissue. material and methods: thirty-one patients with cad were enrolled in study. lima and aorta tissue samples were obtained during coronary artery bypass surgery. whole blood samples were collected before cabg surgery. each patient with cad was obtained from whole blood, aorta and limas tissues. these tissue samples were immediately soaked in rnalater solution and homogenized using a magnalyser. the rna was extracted using the trizol reagent and the mirneasyò mini-kit. the expression profiles of 738 mirnas were evaluated using highthroughput qrt-pcr. results: we found that mir-497-3p was expressed only in aorta. mir-431-5p and 433 were expressed both aorta and whole blood. 6 mirnas were significantly up-regulated in aorta when compared to lima tissue (fc > 2). 59 mirnas were significantly down-regulated in aorta compared to lima. conclusion: in conclusion, our study suggests that mir-497-3p, mir-431-5p and 433 might be a potential for cardiovascular disease development. also mir-431-5p and 433 might serve as novel non-invasive biomarkers for cad p-01.03.3-007 mir193b regulates cell proliferation and colony formation in pancreatic ductal adenocarcinoma n. gurbuz, e. isildar due to the strong metastatic potential, pancreatic cancer has the highest mortality rate of all major cancers. therefore, the investigators are in urgent need of developing the new alternative therapeutic approaches for the prevention of pancreatic cancer. mirnas, small noncoding rnas, regulates as an inducer or inhibitor in expression of key mediators related molecular mechanisms in cancer promotion. to investigate the effect of mir193b on pancreatic ductal adenocarcinoma cells, we performed the cell viability and clonogenic assays by mts and crystal violet dye, respectively, in panc-1 and miapaca-2 cells transfected with mir193b mimic. our data revealed that mir193b led to decrease the cell viability depending on enhanced mir193b doses, which are 25, 50, 100 and 150 nm, as the ratio of % 23, 60, 82 and 87, respectively, in miapaca-2 cells and as the ratio of % 40, 58, 77 and 89, respectively, in panc-1 cells compared with control condition of each cell. this inhibition mediated by mir193b was also obtained in colony formation both of pancreatic cancer cells. when the induced effect of mir193b on the death of pancreatic cancer cells was compared with gemcitabine, which is currently used as a clinical drug for pancreatic cancer patients, we determined that mir193b was more effective than gemcitabine. based on our findings, it is clearly shown that mir193b serves as a tumor suppressor in pancreatic ductal adenocarcinoma cells. we strongly believed that mir193b gene therapy might be more effective and targeted approach than classical gemcitabine therapy for pancreatic cancer patients. *this work was supported by tubitak (the scientific and technological research council of turkey) grant 114s501. breast cancer is the most common cause of cancer death in women. trastuzumab is a therapeutic agent frequently used against her2+ breast cancers, which has role in approximately 20% of invasive breast cancers. with the discovery of their activity in cancerogenesis, micrornas (mirnas) have become potential candidates to mediate therapeutic actions by targeting genes that are effective in drug response. recent studies have showed that mirnas are induced by targeted therapies. in this study, we aim to find out mirna-mediated mechanism, which is driven by common trastuzumab responsive micro-rnas in her2+ breast cancer. for this purpose, the common trastuzumab responsive mirnas were determined in treated bt474 and skbr3 cells by microarray profiling. two datasets were intersected to find out common mirnas for both cell lines. the overlapping predicted targets of common mirnas were provided by two different mirna-target prediction databases and then a mirna-gene network was built in cytoscape by using networkanalyzer plugin. the most shared target genes were chosen to be analyzed in the ebi-embl gene expression atlas for their expression patterns in breast cancer. 25 common mirnas were found to have overlapped targets in two target prediction algorithms that were used to build the mirna-gene regulatory network. 14 overlapped targets were determined as the most shared genes in the mirna-gene network. expression pattern of each shared gene in the gene expression atlas showed that 12 out of 14 the most shared target genes were strongly dysregulated in several breast cancer types. our results suggest that mirnas might show a common response to the targeted therapies and network analysis can be profitable to have a better explanation of the regulatory mechanisms between drug responsive mirnas and their target genes. revealing the mirna-potential target interactions might provide novel key players that mediate the mechanisms of action in drug treatment. chronic myeloid leukemia (cml) is a clonal disease of primitive pluripotent stem cells that identified with a specific t(9;22) reciprocal translocation that encoding bcr-abl oncoprotein. resveratrol (res) is a natural phytoalexin found in grapes and induces apoptosis, erythroid differentiation and autophagy in leukemic cells. micrornas are small, single strand, non-coding rna molecules that regulate post-transcriptional gene expression via disrupting the stabilization of target transcripts or inhibiting protein translation. in our study we aimed to determine cytotoxic effect of res in k562 human cml cell line and to evaluate the expressions of mirnas that are associated with genetics of leukemia after treatment with res; to investigate target genes of mirnas which show significant expression alterations and molecular mechanisms of res treatment. k562 cells were treated with 100 lm (ic50 dose) res during 72 h and cytotoxicity was evaluated by using wst-1 assay. the rt-qpcr is used for mirna and gene expression analysis. results showed that; res up-regulated tumor suppressor mir-214-3p level 2.87 fold and significantly downregulated hdac1 gene expression (p = 0.003) and upregulated p62/ sqsmt1 gene expression (p = 0.001), according to the control cells.p62/sqstm1 interacts with lc3 and plays role as a critical player in the autophagic degradation of the bcr-abl fusion protein. our findings showed that resveratrol acts as a hdac inhibitor targeting hdac1 and p62/sqsmt1 gene expression level. treatment with hdac inhibitors results apoptosis, cellcycle arrest, cell differentiation, anti-angiogenesis and autophagy. downregulation of hdac1 provides post-translational modification for expression of tumor supressor genes and leads to cell cycle arrest and increases apoptosis. these results could be linked to hdac1 dependent induction of gene associated with autophagy like p62/sqsmt1 and resveratrol could be a therapeutic candidate as a hdac inhibitor for cml treatment. the mirna used in this study are hsa-mir-145, hsa-let-7a, hsa-mir-29b and hsa-mir-155. thereafter, we bought pre-mirnas and their mirnas commercially. we apply them to the a549 cell line in different combination and different concentrations. these mirnas applied solely onto cells or in combination as; four of them, let7 + 145, let7 + 29b, let7 + 155, 145 + 29b, 145 + 155, 145 + let7 + 29b, 155 + let7 + 29b. the cell viability was detected by wst-1 kit in a 96 well plate elisa reader. cells were seeded as 10 000 per well, mirnas incubated with cells for 24 h in an appropriate atmosphere. according to our results some combinations and mirnas didn't alter viability, however 145 + 29b and 145 + 155 combination increased the cell viability dramatically. on the other hand let7 + 29b and 155 only applications decreased the cell viability. the other applications' viability results are not different from the control cells significantly. in this study, we used a549 cell line also called non-small lung cancer (nsclc) cell line and possibly effective mirnas on lung cancer. it is important to exhibit the mirna combinations should be effective on cancer cells' viability. the prospect combinations were determined which is crucial to develop new strategies for cancer treatment. competing endogenous rnas (cernas) act as molecular sponges for the same pool of micrornas through their mirna response elements (mre), titrate mirna levels and thereby regulate gene expression post-transcriptionally. smad interacting protein 1 (sip1), a member of the zeb family is a regulator of epithelial-to-mesenchymal transition (emt) program, which is active during embryogenesis and tumor invasion and metastasis. hence, we investigated the regulation of sip1 by cernas in hepatocellular carcinoma (hcc) cells. among hundreds of sip1 cernas listed at competive endogenous mrna database (cerdb), 14 transcripts (pten, zeb1, ptch1, creb5, acvr2b, enah, robo2, erbb4, e2f3, foxo1, rictor, klf3, ets1, cdk6) sharing at least 9 common mre sites with sip1 were selected and their expression in 9 hcc cell lines were determined by qrt-pcr. ets1 was found to be highly correlated with sip1 in hcc. furthermore, repressing sip1 expression by shrna in hcc cells resulted in decreased expression of ets1, pten and zeb1. our results suggest a possible bidirectional and post-transcriptional regulation of sip1 and its cer-nas in hcc. a meta-analysis for the identification of common microrna signatures in colorectal cancer n. sahar 1,2 , n. belder, h. ozdag 1 biotechnology institute, ankara university, ankara, turkey, 2 comsats institute of information technology, islamabad, pakistan colorectal carcinogenesis (crc) has quite frequent incidence and mortality rates worldwide, despite being studied for decades now. new biomarkers are needed to be identified in addition to the existing ones, due to heterogeneous nature of this disease. the regulatory molecular machinery of a cell, including micrornas (mirnas), contributes to this heterogeneity upon aberrant expression. herein, for a mechanistic understanding of differential gene expression in crc tissue we analyzed mirna expression profiles of 78 crc tumors against 62 normal colorectal mucosa samples, using raw data from e-mtab-752 and e-geod-35834 (affymetrix microarrays), and gse35982 and e-mtab-813 (agilent microarrays) datasets obtained from gene expression omnibus and arrayexpress. raw samples were normalized (different platforms separately) using quartile normalization in brb-array-tools. differential expression of mirnas was identified using cut-off values of p ≤ 0.05, fold change ≥ 1.5 and stringent false discovery rates. mirtarbase and mirwalk2.0 databases were explored to identify validated targets. we found thirty (including mir-21 and mir-183) and thirteen (mir-1, mir-139, mir-375, etc.) mirnas commonly upregulated and downregulated respectively, in both affymetrix and agilent microarray results. predicted targets of these mirnas frequently belong to pathways related to cancer like b-catenin, phosphoinositol-3kinase, and transforming growth factor-b, to name few. moreover, the target genes were significantly enriched in clusters related to cell cycle, cell differentiation and regulation of apoptosis. these promising results will further be compared with differentially expressed gene profiles from a cohort of turkish crc patients. hence the integrated study will refine the panel of diagnostic and prognostic crc markers. hsa-mir-x modulates motility and invasion in triple breast cancer cell line s. noyan 1 , h. g€ urdal 2 , b. g€ ur dedeoglu 1 1 biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacology, ankara university, ankara, turkey breast cancer is a heterogeneous disease and expression levels of certain receptors have demonstrated subtypes which characterize clinically distinct breast tumors. a triple-negative phenotype lacks expression of er, her2 and pr and is known as basallike carcinoma. micrornas are a class of small non-coding rnas that participate in the gene expression in many biological processes. e-cadherin is an important mediator of adhesion in epithelial tissues, and loss of e-cadherin can play a critical role in tumor invasive behavior. another key player of cell integrity pip (3, 4, 5) triphosphate is generated at the leading edge of the cell and leads to cell polarization. pip3 is generated by hydrolysis of pip (4,5) bisphosphate, which is synthesized by pip5k1. any dysregulation in these molecules may support the invasive behavior of the cells. the aim of this study is to find out the role of mirna precursor (hsa-mir-x) in invasion and motility in triple negative breast cancer cells. in this study a triple-negative breast cancer cell line bt-20 was transfected with hsa-mir-x or scrambled control sirna. to check its role in motility and invasion, wound healing and invasion assays were performed respectively. cell invasion was monitored over a period of 24 h by xcelligence real-time cell analyzer using a double-plate and measuring impedance-based signals. additionally emt markers were analyzed by qrt-pcr to explain the molecular mechanisms beneath motility and invasion. we observed that cell motility and cell invasion diminished after transfection of bt-20 cells with mimic for hsa-mir-x. furthermore, qrt-pcr experiments indicated that transfection of hsa-mir-x decreased the expression level of pip5k1c while increasing the e-cadherin expression level. wound healing and invasion assays together with qrt-pcr results support the role of hsa-mir-x in cell motility and invasion. this process might be explained via e-cadherin mediated met or gsk-3-beta related inhibition of invasion. expression level of five micrornas as diagnostic markers in glioblastoma situated in the main brain lobes, but can also be found in other brain regions. while microrna (mirna) as non-coding rnas, play a crucial function in the post-transcriptional regulation of gene expression by mrna degradation or translational repression. in the present study, we aimed to investigate the contribution of gene expression of the five mirnas and to unravel their role in brain tumor cell lines, the mirnas to the risk of gbm tumor. the five gbm cell lines (crl-2365, crl-2366, crl-2948, crl-1690 and htb-15) were evaluated with non-malignant (normal) brain cell line (hcn-2) . determinations of expression level of five mirnas (mir-21, mir-101, mir-138, mir-196, and mir-222) were evaluated by monitoring quantitative rt-pcr (qrt-pcr) technique. the expression levels of four mirnas (mir-101, mir-138, mir-196, and mir-222) were significantly decreased while the expression level of mir-21 was increased in gbm cell lines according to hcn-2 cell line. consequently, these five mirnas can potentially be used as biomarkers for gbm tumor; further studies are mandatory to a better understand and confirm our preliminary findings. background: noncoding rna are known to be crucial molecules with diverse regulatory roles in neural oncology and neurodegenerative disease. the recent study suggested that lncrna anril play role in the development of neuroblastoma and alzheimer disease via binding disease-specific micrornas. material and methods: we used lncrnadisease, hmdd v2.0, mir2disease to predict lncrna-and mir-associated disease in our study. in addition, we utilize tardetscan to search lncrna-mirna interaction. results: disruptions of lncrna anril expression (also named as cdkn2b-as, locus cdkn2a/b (ink4/arf), chromosome 9p21) have been associated with the development of neuroblastoma and alzheimer disease. here, we predicted interactions between noncoding transcripts encoded by locus cdkn2a/b and their molecular partners -microrna. anril can act as decoy while containing sequences that mimic mirna target sites to titer these mirs away from their primary targets thereby act as molecular sponge. using targetscan 7.0, we predicted target sites for hsa-mir-15-p/16-p/195-p/424-p/497-p/6838-p and hsa-mir-125-5p/4319 in anril 3 0 utr. then, we used hmdd v2.0 and mir2disease databases to define if any of these mirs participate in alzheimer disease and neuroblastoma. according to both databases, mir-125 is implicated to alzheimer disease and mir-15 to neuroblastoma. as soon as anril participate in the development of both abovementioned disorders and can have microrna sponge activity, it could potentially positively regulate mir-125 and mir-15 targets by competing with them for micro-rna binding sites thus restoring the expression of target genes. in our further research we plan to experimentally validate predicted microrna sites in anril 3 0 utr. conclusions: we predicted sites for mir-125 and mir-15 in 3 0 utr of anril lncrna that could uncover its possible sponge activity in the development of neuroblastoma and alzheimer disease. aim: matrine excracted from saphora flavescens root and demonsrated that indicates pro-apoptotic and anti-proliferative effect in many types of cancer. acute lymphoblastic leukemia (all) is an acute form of leukemia, or cancer of the white blood cells which characterized by the overproduction and accumulation of lymphoblasts. mirnas play important roles in deregulated cell death mechanisms. we aimed to investigate the effects of critical mirnas during the development of matrine resistance on all cell line ccrf-cem. material-method: ccrf-cem cells were treated with different (0.5-3 mg/ml) concentrations for 72 h and cell viability measurements were carried out with xcelligence device to determine the cytotoxic effects of matrine. mirnas were extracted from treated and untreated ccrf-cem cells using the mirna isolation kit. cdna was synthesised using allin-one first strand cdna synthesis kit. expressions of 44 selected mirnas were analysed with miprofiletm custom mirna qpcr array using the applied biosystem 7500 fast real-time pcr system. results: according to the cytotoxicity assay, it was determined that 'treatment with increasing concentrations of matrine, decreased the viability of the ccrf-cem cell line. expression levels of 44 different mirnas were studied for indicated passages in two replicates. our results showed that hsa-mir-376b-3p (-37,099 fold, p = 0.008), hsamir-106-3p (-16,6795 fold, p = 0.028), hsa-mir-20a-3p (-15,926 fold p = 0.0148), hsa-mir-519a-3p (-11,7398 fold, p = 0.00534), hsa-mir-204-5p (-10,9536 fold, p = 0.0012), hsamir-30b-5p (-9,0631 fold, p = 0.0221), hsa-mir-15b-5p (-8,8971 fold, p = 0.0339), hsamir-106b-5p (-8,8561 fold, p = 0.021), hsa-mir-885-3p (-8,6139 fold, p = 0.00006), hsamir-30a-5p (-8,594 fold, p = 0.009) expression were decreased during the development of matrine resistance. conclusion: these data suggested that especially hsa-mir-376b-3p plays a critical role in the matrine response. ericd (e2f1-regulated inhibitor of cell death) is a newly found lncrna. it is located at 8q24.3. it has two exons and its transcript size is 1745 bp. ericd is regulated by e2f (transcription factor 2) and modulates the cellular response to dna damage. arid3a is a family member of the at rich interaction domain (arid) dna-binding proteins that participate in diverse biological processes like development, cell cycle control, chromatin remodeling and epigenetic regulation. both, ericd and arid3a have just opposite roles in apoptosis in case of dna damage indicating a probability of reciprocal interaction between each other. till now, there is no work related to the interaction between lncrna and arid3a in cancers. herein we try to find a probable interactive role between these in cancers. in this study, 12 different cancer cell lines, 1 osteoblast cell line and 19 different types of normal human tissues rnas were selected for expression analysis of ericd and arid3a. after rna isolation, cdna was converted from their rnas. expression profile analysis of ericd and arid3a in different cancer cell lines and normal tissues was done using imagej program for semiquantitative and 2 (-ddct) method for quantitative rt-pcr. among used cancer cell lines, ericd was highly expressed and arid3a had lower expression in u-2os (osteosarcoma), a-172 (glioblastoma) and a549 (lung cancer). at the same time, ericd expression was lower and arid3a had high expression in hfob 1.19 (osteoblast cell line) and normal tissues like brain and lung . both ericd and arid3a are cell cycle regulated and are commonly regulated by e2f. they have just opposite roles in apoptosis during dna damage. these two genes have a probability of reciprocal interaction between each other in cancer. our results indicate that both ericd and arid3a might have opposite roles in lung cancer, glioblastoma and osteosarcoma. ericd and arid3a might act as cancer promoting and tumor suppressor genes respectively in these cancers. the importance of mirna expressions in infertilty implantation process is controlled with endometrium, factors secreted by the embryos and in accordance with these factors embryo and/or endometrium via receptors on. more than 700 human microrna (mirnas) that are small noncoding rnas were shown to play an important role in intracelluler cycle regulation in both normal and pathological conditions. in this study we aim to identify mirnas and controlling molecules expressions in different time period of endometrium in fertile and infertile cases. the endometrial samples were taken from fertile and infertile patients in proliferation and early secretion periods. the samples are fixed and stained either with hematoxylen-eosin for morphological analysis or with immunohistochemistry for distributions of anti-dicer, anti-drosha, anti-eif2a and anti-eif2c. mir-17-5p, mir-23a, mir-23b, mir-542-3p, mir-21, mir-199a*, mir-705, mir-20a, mir-26a, mir-125b, mir-200a/b/c were analyzed with qrt-pcr. while dicer immunoreactivity was detected weakly only proliferation phase of fertile group, this immunoreactivity were detected strongly in both proliferation and early secretory phases of infertile group. drosha immunoreactivitiy was also weakly detected in the proliferation phase of fertile group, it was moderately detected in both proliferation and early secretory phases of infertile group. eif2a immunoreactivitiy was similar in each groups but there were a few differences between fertile and infertile group. eif2c immunoreactivitiy was negative in all groups. mir-21, mir-199a* and mir-23a were highly expressed in proliferation phase of fertile group, mir-23a and mir-125b were highly expressed in early secretion phase of infertile group. in conclusion, dicer and drosha immunoreactivities and different expression of mirna's were detected in all groups. implantation problems may be reason for different mirna expression which controlling with dicer and drosha in the infertile endometrium in both proliferation and early secretory phases. wheat is an important agricultural crop with an over 615.8 million metric tons harvesting capacity annually. drought and salinity are environmental stress factors that affect yield and quality of wheat, dramatically. there are different defense mechanisms against these stress conditions in plants. altering gene expression profiles by micrornas at post-transcriptional level is one of the most conserved mechanisms among plants. micrornas are an extensive class of noncoding rnas, approximately 22 nucleotide length which regulates the expression of genes by binding to the 3 0 -untranslated regions of specific mrnas. micrornas implicated under salt and drought stress have widely been reported in numerous plant species and wheat genomes in the last years; however, studies on einkorn wheat (triticum monococcum spp. monococcum) are not yet available. the goal of this study is identification of conserved micrornas from einkorn wheat using next generation sequencing technology and bioinformatic analysis. in this study, small rna molecules were extracted from pooled plant samples grown under normal, drought and salinity conditions. sequencing analysis revealed 75 164 unique small rna sequences obtained from 15 139 448 raw reads. after bioinformatic analysis based on comparative genomics approaches, we identified 168 putative mature microrna sequences belonging to 142 distinct microrna families. since chromosomal sequence data is not available for triticum monococcum spp. monococcum, we used available sequences from triticum urartu, a close relative, as template to extract precursor microrna sequences. 111 of precursor sequences showing 100% homology to triticum urartu genome were analyzed for secondary structure prediction using mfold software. data provided in this study is critical to investigate transcriptional regulation of genes involved in stress metabolism in einkorn wheat. the role of vim-as1, a natural antisense transcript, in cancer coding or non-coding transcript. in this regard, vim-as1 is nats located antisense to vimentin gene. in the present study, we aimed to determine tissue and cell line distribution of vim-as1. materials and method: for the tissue expressions analysis, human total rna master panel ii (containing 20 different human tissue samples) was used. total number 14 cancer cell lines and 5 normal cell lines included in the study. for the expression analysis rt-pcr and qpcr methods were used. results: as a result, expression levels of vim-as1 were found to be tissue specific. particularly, while vim-as1 was highly expressed in lung and thyroid gland tissues, its expression was not observed brain, stomach and adrenal gland tissues. also, vim-as1 was also found to be differentially expressed in cancer cell lines. vim-as1 expression was found to be lost in cal29, pc3, and hct116 and highly diminished a549 cancer cells. also, it is found to be highly expressed in bcpap, panc1 and beas2b cells. discussion: results of the current study suggest that vim-as1 may have significant role in the regulation of vim gene in thyroid gland tissues, as it is highly expressed in both thyroid gland tissues and bcpap thyroid cancer cells. moreover, vim-as1 and vim axis can be involved in the formation of lung tumors because vim-as1 was highly expressed in normal lung tissues and beas2b cells and expressed very low levels in a549 lung cancer cells. lung cancer is the leading cause of cancer related deaths in the world and approximately 90% patients with lung cancer ultimately die from metastatic disease. metastasis is the most dangerous step of cancer. in our recently published work showed that akt/nf-kb pathway is continuously active and induces cellular invasion and pten suppresses cellular invasion via inhibition of akt/nf-kb pathway. in this study we aimed to show nf-kb mediated induction of mirna expression can responsible for inducing nsclc invasion. we used chromatin immunoprecipitation (chip) assay kit for detection of tnf-a induced nf-kb mediated mirnas. therefore, h1299 and pc14 cells treated by tnf-a (30 ng/ml) for chip assay. chromatin regions, reading with chip-seq, were analyzed using bioinformatics tools. we also performed additional bioinformatics search to find nf-kb related mirnas which potentially take a role in nsclc invasion. we investigated the effects of mirna which determined at the bioinformatics analysis results on invasion using invasion chamber method. we found 16 mirnas which potentially induced by nf-kb and related with nsclc invasion. our invasion results indicate that mir-548a-3p, mir-548as-3p, mir-8078, mir-1915, mir-6814-3p, mir-548q mimics can induce cellular invasion on h1299, mir-548v, mir-548 h-5p, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 mimics can induce cellular invasion on pc14. we also verified our results by qrt-pcr, because we want to sure that mirnas which can induce invasion, can also transcriptionally regulated by nf-kb or not. we found that mir-548q, mir-548a-3p, mir-584as-3p, mir-1915, mir-8078 in h1299, mir-138-5p, mir-548a-3p, mir-548as-3p, mir-8078 in pc14 can induce cellular invasion by nf-kb. as a conclusion, our investigation indicate that nf-kb can induce nsclc invasion via mir-548a-3p, mir-548as-3p and mir-8078. (this study is supported by tub _ itak grand number 112s636). to understand of the molecular mechanisms of cellular invasion is very important for fight against cancer mechanisms and first step of invasion is emt. cells change phenotypical and genotypical in emt progress. in this study, we focussed on the explore molecular mechanism of tnf-alpha induced cellular invasion of nsclc. we use western blot, qrt pcr and mirna transfect methods for this purpose. we find that tnf-alfa treatment reduce the expression of pten and induce e cadherin expression in a549 cells. when we inhibit nf-kb activity by p65 targeted sirna tnf-alpha can not reduce pten expression means that tnf-alpha inhibits pten expression through on nf-kb. because tnf/nf-kb pathway change the transcriptional level of mir-548as and pten 3 0 untranslated region has recognition site for mir-548as. therefore; we transfected a549 cells by mir-548as. mir-548as transfection leads to inhibition of pten expression. our results indicate that tnf-alpha mediated activation of nf-kb can inhibit pten expression on induction of emt through on mir-548as. (this study is supported by tubitak grand nummber 112s636). introduction: the corpus luteum (cl) is an ephemeral tissue whose regulated secretion of progesterone is essential for maintenance and/or timely termination of pregnancy in rodents. our previous finding that cl of pregnant rats does not possess fas/ fasl system suggests that this tissue may undergo autophagic, but not apoptotic, cell death during regression. we here investigated the presence of autophagic system in cl and its any implications in rodent pregnancy and parturition. materials and methods: lc3 (-i and -ii) expression in cl was estimated by western blot analysis in comparison with progesterone secretion and luteal mass throughout pregnancy. lc3 was also tested by immunocytochemistry. functional implication of autophagy in this tissue was examined by local treatment with bafilomycin a1 (autophagy and v-atpase inhibitor, 6.23 pg/ 0.1 ml/ovary) on day 19 of pregnancy. reproductive, biochemical, and morphological outcomes were evaluated. results: while the expression of cytosol-associated lc3-i was not significantly altered throughout pregnancy, that of autophagosome-associated lc3-ii increased significantly from day 15, showed a peak on day 21, and decreased on day 23 (day of normal parturition). lc3-ii / i ratio had positive correlations with steroidogenic activity and cell size. immunoreactive lc3 was found to be present in the cytosol of steroidogenic cells and showed marked aggregation in cells on day 21. in vivo treatment with bafilomycin a1 resulted in unaltered delivery, but in significant reductions in steroidogenic cell size and neutrophil infiltration compared to vehicle-treated control groups. discussion and conclusion: the ratio of lc3-ii / i in cl was markedly up-regulated in the late phase of pregnancy. manifestation of this autophagy parameter was temporally matched with further structural and functional activation of cl. autophagy may contribute to activation, but not regression, of rodent cl and thus their female reproduction. apoptotic and necrotic effects of low dose bisphenol a in shsy5y neuroblastoma cells b. ayazg€ ok, t. t€ uyl€ u k€ uc ߀ ukkilinc ß faculty of pharmacy, hacettepe university, ankara, turkey bisphenol a (bpa) is a commonly used chemical in industry to make plastics. 'low-dose' term has been expressed for the first time in studies with bpa in 2001. the value of low dose bpa was received as <1lm. in this study, matrix metalloproteinases (mmps) together with their tissue inhibitors (timps), involved in tissue remodeling after i-131 therapy, have been examined in 51 patients (8m/46f) with ptc and 38 (3m/38f) with ptc+ht. peripheral blood samples were collected just before and, subsequently, at 4 days after i-131 administration (3.7 gbq). ptc+ht patients had positive titers of anti-thyroglobulin autoantibodies (tgab). the serum levels of tgab, mmp-2, mmp-9, timp-1 and timp-2 were measured by elisa. there were no significant changes in serum concentrations of mmp-2, timp-2 and mmp-2/timp-2 ratio after i-131 in the two groups. in ptc patients, i-131 administration resulted in an increase with 26% in timp-1 level (p = 0.005) and a reduction with 44% in mmp-9/timp-1 ratio (p = 0.003). in ptc+ht patients it has been observed an increase with 18% in tgab level (p = 0.001), 5% in mmp-9/timp-1 ratio (p = 0.003) and unchanged timp-1 serum concentration. tgab titers were positively correlated with mmp-9/timp-1 ratio (r = 0.51, p < 0.001). our data suggest that radioiodine therapy for ptc patients, but not for ptc+ht, modulates the balance of mmp-9/timp-1 for anti-invasion and anti-migration by augmenting timp-1. in criminal cases, the determination of the time of death of the bodies step in the analysis of events is making a big contribution. today, forensic and molecular methods utilized time of death rather than provide clear information offers potential death time interval. principally, 'time of death' is a term that should be avoided. in forensic science, the postmortem 'interval' is the term to be considered. nowadays, there is no precise molecular method that can be used alone in the determination of pmi. aim of this study is to analyze the usability of ecm components, adamts1,5 and 9 and mmp1,2 and 9 to determine pmi. for this purpose, with iliopsoas muscle tissues provided by ethical board of the ankara institute of forensic medicine, 21 cases have been included in this study, divided into 3 groups according to their pmis: '0-12 h', '12-18 h' and '18-27 h'. from these tissues, western blotting technique is used to analyse the expression of adamts1,5 and 9 and mmp1,2 and 9. it is determined that when pmi increases; adamts-1 and 9 amounts decrease. on the other hand adamts-5 amounts are examined to increased related to the interval., especially on the '12-18 h' dataset. additionally, considering metalloprotease levels, mmp-2 and 9 amounts decrease as pmi increases. unlike mmp-2 and 9; mmp-1 levels increase proportional to the interval, especially on the '18-27 h' dataset. these results are the first data on pmi determination from iliopsoas muscle tissue. in this study, it is suggested that adamts-5 and mmp-1, proteases responsible for ecm degradation, can be used to determine pmi as markers. here we present the first observations of postmortem variation of mmp and adamts activities in skeletal muscle. in recently, popular mmps and adamts s pathways of the relationship between time-dependent changes to investigate the post mortem time interval determination to shed light on the future. the role of functional polymorphisms of matrix metalloproteinases 2 and 9 in spontaneous abortion samples capillaries, which are responsible for maintenance of implantation and placental nutrition, have major effects on mechanisms underlying abortion. matrix metalloproteinases (mmps) function in various cellular pathways. they play a role in patholohical conditions, metastasis; as well as normal physiological functions like tissue and bone regeneration, physiologic function of uterus, ovulation, embryogenesis and embryo implantation. mmp2 and mmp9 (gelatinases) have key roles at organisation of extracellular matrix and affect endometrial implantation. previous studies have shown that mmp2 -1306c>t and -735c>t polymorphisms cause loss of gene function, and mmp9 -1562c>t polymorphism causes a decrease in gene expression, and also gene expression levels are lower in abortion specimens, compared to control specimens. the goal of this study was to investigate the potential effects of functional mmp2 -735c>t and -1306c>t polymorphisms, and mmp9 -1562c>t polymorphism on etiology of abortion. restriction fragment length polymorphism (rflp) method was used to analyze the polymorphisms those evaluated in the study. study group consisted of samples collected from 80 spontaneous abortion specimens, and control group consisted of peripheral blood blood samples collected from 100 healthy subjects. the risk of abortion was 2.2-fold higher in patients with heterozygous -1306c>t polymorphism (p = 0.043). combined genotype analysis showed that the risk of abortion was 3.7-fold higher for patients with normal -735c>t polymorphism, and heterozygous -1306c>t polymorphism (p = 0.021). functional polymorphisms of mmp2 and mmp9 may have a role in etiology of abortion. p-02.07.5-011 changes in the specific extracellular matrix proteins and expression of adamts proteinases and effects of hypoxia in the adriamycin-induced renal fibrosis model renal fibrosis is a common cause of renal dysfunction with chronic kidney diseases. this process is characterized by excessive production of extracellular matrix (ecm) or inhibition of ecm degradation. adamts proteinases, which are widely presented in mammals, have very critical roles in ecm remodeling. we aimed to study the role of adamts proteinases in the pathogenesis of renal fibrosis and the effets of hypoxia by studying adamts expressions in rat kidney after adriamycin (adr) administration. adr was administered intravenously in consequtive two doses (2.5 and 4 mg/kg) to the rats. in addition to control and adr groups, another rats were assigned into three groups as sham, 15 min and 45 min ischemia-reperfusion. after 2 months following the first dose, the expression of adamtss were determined in the renal tissues using western blot analysis. additionally, renal nephropathy and fibrosis were assessed pathological and immuno-histochemical staining methods. in the adr group rats developed renal dysfuntion and tissue damage in favor of renal fibrosis pathologically. it is observed that occurred remarkable changes in the expression of adamtss. it is showed that hypoxia and hypoxia time enhanced tubulointerstitial fibrosis in the rat kidney tissues. expression differences were absorved also in the hypoxia groups, and especially the expression of adamts-1 and -15 genes were showed an increase in various rates. the restricted and different expression pattern of adamts protein profiles in the adr-induced renal fibrosis suggest that adamts family members are related with development and progression of fibrosis. moreover, our findings raise the possibility that the hypoxia promotes renal fibrogenesis. the monitoring of adamts proteinases might contribute to the early diagnosis of renal fibrosis and chronic kidney disease. adams (a disintegrin and metalloproteas) are transmembrane proteins that contain a pro-domain, a metalloprotease, a disintegrin, a cysteine-rich, an epidermal-growth factor like and a transmembrane domain, as well as a c-terminal cytoplasmic tail. they are involved in cell adhesion and they have protease activities. previous studies showed that some adam proteins act in a highly diverse set of biological processes, including fertilization, neurogenesis, myogenesis, embryonic tgf-a release and the inflammatory response. although there are more researches about adam proteins, still the function of all adam proteins remain unclear. we aimed to investigate the potential link of infertilty with adam9, -10, and -17. in this study twenty four patients were included. the patients were classified as normozoospermia (ns; n = 8), oligozoospermia (os; n = 8), azoospermia (as; n = 8) groups. adam9, -10 and -17 protein levels in infertility indviduals were analysed by western blot. adam9 protein level was 1.7 fold lower in the os and as groups than in the ns group. adam10 protein level was 1.36 fold higher in the as group than in the ns group. adam17 protein level was 2 fold lower in the as group accourding to ns group. we observed no change between protein level of adam10 and adam17 of os and ns groups . in conclusion, adam proteins may have a potential role in male infertility. our study is a preliminary and first study on this issue. keywords: adam, infertility. the role of tissue metalloproteinase inhibitors thymus chemokine and thrombospondin-1 on prognosis of crimean-congo hemorrhagic fever s. bakir, m. bakir, s. ersan, a. engin cumhuriyet university, sivas, turkey crimean-congo hemorrhagic fever (cchf) is a disease which is caused by an arbovirus carried by ticks and characterized by the sudden onset of high fever, severe headache, dizziness, back and abdominal pain. cchf pathogenesis is still not resolved today to fully open. therefore, in this study, to determine the level of tck-1, timp-1 and tsp serum samples obtained from cchf patients and the control group is intended to be examined for the pathogenesis and prognosis of the disease. the study sample was created 45 patients with diagnosis of cchf. 45 healthy volunteers were chosen control group matched for gender and similar to in terms of age. tsp, tpc-1 and timp-1 levels of patients and a control group were analyzed using the human elisa kits. serum timp-1 tck-1 and tsp levels in cchf patients were measured significantly higher than the in the control group. cchf pathogenesis of today still have not provided fully open. therefore, it reveals the importance of this work. in our study, presence of high timp-1, tck-1 and tsp levels indicate important direction for pathogenesis and prognosis of cchf disease. p-02.07.5-015 activation of calpain 1 and protein kinase ca promotes a constitutive expression and release of matrix metalloproteinase 9 in peripheral blood mononuclear cells from cystic fibrosis patients matrix metalloproteinase 9 (mmp9) is physiologically involved in remodeling the extracellular matrix components but its abnormal release has been observed in several human pathologies, including cystic fibrosis. we have studied if the alteration in intracellular ca 2+ homeostasis, observed in peripheral blood mononuclear cells (pbmcs), isolated from cystic fibrosis (cf) patients homozygous for deletion of phenylalanine 508 in gene of cystic fibrosis transmembrane conductance regulator (f508del-cftr), could be involved in the abnormal presence of mmp9 in the extracellular fluids of cf patients. pbmcs were isolated from 23 healthy donors and 26 cf patients homozygous for f508del-cftr. mmp9 levels were evaluated following 2 h of cell incubation. our investigations show that all cf pbmcs analyzed constitutively express and release high levels of mmp9; conversely, in pbmcs from healthy donors, expression and secretion of mmp9 are undetectable but both events can be evoked, after 12 h of cell culture, by a possible paracrine stimulation. we have demonstrated that in cf and 12 h-cultured control pbmcs mmp9 secretion is prevented by chelation of intracellular ca 2+ and mediated by the concomitant activation of calpain and protein kinase ca (pkca) and also that mmp9 expression is mediated by the sequential activation of pkc and extracellular signal-regulated protein kinases 1 and 2 (erk1/2). moreover, the rescue of active f508del-cftr reduces the extent of mmp9 secretion, correlating the channel defect to the [ca 2+ ] i dysregulation of cf pbmcs. our results indicate that the high level of intracellular ca 2+ concentration in cf pbmcs, promoting the aberrant activation of both calpain and pkca, induces a constitutive release of mmp9. these data characterize new alterations in mononuclear leukocytes of cf patients that may be of primary importance in the progression of the disease and indicate that pbmcs may contribute to the accumulation of mmp9 in fluids of cf patients. p-02.07.5-016 aebp1/aclp is upregulated in differentiation, injury repair and fibrotic degeneration of skeletal muscle c. € ozdemir 1,2 , u. akpulat 1 , i. onbasilar 3 , c ß . kocaefe 1 1 department of medical biology, faculty of medicine, hacettepe university, ankara, turkey, 2 department of stem cell, institute of health, hacettepe university, ankara, turkey, 3 laboratory animal breeding and research unit, faculty of medicine, hacettepe university, ankara, turkey aebp1/aclp is an ambiguous gene with several attributed functions and cellular events, adipogenic differentiation, cell adhesion, pattern development and fibrosis are the well-understood. aebp1 is the short isoform that acts as a transcriptional repressor by targeting the ap2 promoter and aclp, which is the long isoform that harbors a leader sequence that directs the peptide to the extracellular compartment. the latter is known to be associated with development of the connective tissue, injury repair and fibrosis in certain pathological conditions. aebp1/aclp displays a moderate expression in skeletal muscle where the role is not known. we have investigated the spatial and temporal expression of aebp1/aclp in defined models of skeletal muscle differentiation, injury repair and fibrosis. aebp1/aclp expression is present in steady state dividing myoblasts. this basal expression is upregulated 4 folds upon the induction of differentiation in c2c12 cells. considering that differentiation and post-natal injury repair share several common aspects, we also investigated the expression of aebp1/aclp in acute injury-repair model. in the course of cardiotoxin induced injury, aebp1/aclp expression peaks up to 5 folds in the 6th day of regeneration. this time point concomitantly corresponds to tissue remodelling. since aebp1/aclp is also known to be associated with fibrotic events in chronic pathological conditions, we also have investigated its expression in tenotomy induced skeletal muscle degeneration which mimics endomysial and perimysial fibrosis. aebp1/aclp expression is upregulated up to 10 folds in early time-point samples. these results depict a novel role for aebp1/aclp in extracellular remodeling of the skeletal muscle during injury repair as well as fibrotic degeneration. the source of this expression might come from fibroadipogenic precursers which reside in endomisial area of muscle. our current efforts are focused on presenting of this endomysial expression. the mprbp gene from b. pumilus 3-19 encoding a novel secreted metalloproteinase was identified. based on the primary structure the enzyme has been classified as metzincin metalloproteinase that combines the features of two families: astacin and adamalysin. representatives of the adamalysin family previously have not been described for bacilli. the aim of the work was to elucidate the mechanisms of the gene expression regulation of a new bacillus pumilus 3-19 extracellular metalloendopeptidase. promoter region analysis revealed the presence of potential binding sites for transcription factors spo0a (sporulation) and degu (biodegradation). study of mprbp expression in strain defective in regulatory proteins degs and degu shows that the productivity of metalloproteinase biosynthesis decline in average 60% compared with the strain with a complete degs-degu system. we also studied mprbp expression in strains with a mutation in the gene degu, leading to stabilization of degu~p protein. it is known, that this mutation leads to a multiple increase in the gene expression level, positively regulated by degs-degu system. our data shows a 10-fold increase in metalloproteinase productivity in the recombinant strain. thus, deg-system participates in control of the proteinase synthesis but not only in the regulation of mprbp gene expression. the mprbp expression in the strain deficient in regulatory protein spo0a remained at the level with expression in the strain with the complete spo0a. a similar pattern we observed in the study of mprbp gene expression in strains defective in other spore-specific regulatory proteins (spo0b, spo0f, spo0k, spo0j, sigf, sigh, sigk). these data indicate that mprbp gene expression is free of spo-regulatory proteins. on this basis, we concluded that the expression of metalloproteinase gene is not correlated with the sporulation. p-02.07.5-019 paricalcitol attenuate activity and expression of matrix metalloproteinases in a rat model of renal ischemia-reperfusion injury matrix metalloproteinases (mmps) are endopeptidases involved in the degradation of extracellular matrix. they have been postulated to have a role in the pathogenesis of ischemia-reperfusion injury (iri). in the present study, we investigated the effect of paricalcitol, a synthetic vitamin d analog, on mmps in renal iri. 21 wistar albino rats were divided into three groups: sham operated, ischemia-reperfusion, and paricalcitol-pretreated. iri model was induced by bilateral clamping of renal arteries for 45 min followed by 24 h of reperfusion. the analysis of serum creatinine levels and activities/expressions of mmp-2 and -9 were performed after 24 h of iri. the effects of paricalcitol on activities and expressions of mmp-2 and mmp-9 levels were investigated by gelatin zymography and immunohistochemistry, respectively. the pathological examinations were performed to score tubular damage by light microscopy. creatinine levels increased significantly in the iri group. rats in the paricalcitolpretreated group showed significant decrease in expressions and activities of mmp-2 and mmp-9 during iri. moreover, pathological examinations displayed significantly lower score of tubular damage in paricalcitol-pretreated group. in conclusion, paricalcitol attenuated iri by downregulating the expressions and activities of mmp-2 and -9. p-02.07.5-020 the changes of matrix metalloproteinase 2, 9 activity and hyaluronic acid level in rat's heart and serum under cadmium influence o. fomenko 1 , o. shaulska 1 , y. kot 2 , g. ushakova 3 , a. shevtsova 1 1 dnipropetrovsk medical academy, dnipropetrovsk, ukraine, 2 kharkiv national university, kharkiv, ukraine, 3 dnipropetrovsk national university, dnipropetrovsk, ukraine the changes in the molecular mechanisms of the extracellular matrix degradation under toxic factors are not well known. the main goal of work was the investigation of the mmp2 and mmp9 activity and hyaluronic acid level in the heart and blood serum under cadmium influence at different doses. the 18 wister rats divided to 3 groups were used for the experiment. cdcl 2 x2.5h 2 o in doses 0.1 lg/kg and 1 lg/kg was given to rats intragastrically in drinking water during 36 days. the rats were decapitated under isoflurane anesthesia according to ethical rules; the heart was quickly removed. the relative activity [in arbitrary units (au)] of pro-and active forms of mmp9 and mmp2, total protein (tp) and hyaluronic acid levels were calculated. it was shown that low doses of exogenous cadmium (0.1 lg/ kg) lead to reduced activity of pro-and active forms of mmp9 in myocardium (7.3 ae 0.6 au/mgtp and 7.1 ae 0.6 au/mgtp compare to the 9.67 ae 0.4 au/mgtp and 9.7 ae 0.5 au/mgtp in the control rats accordingly) and in serum (0.95 ae 0.2 au/mgtp and 0.35 ae 0.05 au/mgtp compare to the 1.54 ae 0.05 au/mgtp and 1.49 ae 0.05 au/mgtp in the control rats accordingly), but pro-mmp2 activity in heart was increased (14.5 ae 1.6 au/mgtp compare to the 9.8 ae 0.6 au/mgtp in the control rats); level of ha was decreased in both tissues (0.69 ae 0.16 lg/ml and 3.63 ae 0.3 lg/ml compare to the 1.0 ae 0.13 lg/ml and 3.91 ae 0.3 lg/ml in the control rats accordingly). high doses of cadmium (1 lg/kg) caused a reliable increase of both gelatinase activity in the myocardium: mmp2 increased from 9.65 ae 0.4 au/mgtp to 14.1 ae 0.8 au/mgtp, prommp9to 12.6 ae 1.5 au/mgtp, mmp9to 15.4 ae 1.6 au/mgtp. ha level was increased in serum (4.28 ae 0.1 lg/ml) and decreased in heart (0.49 ae 0.09 lg/ml). the results indicate the dose-dependent and tissue-specific effect of cadmium on mmp-depended protein degradation and level of hyaluronic acid. a disintegrin-like and metalloproteinase domain with thrompospondin-1 repeats (adamts) are a large family of proteoglycanase that show proteolytic activity towards proteoglycans like aggrecan, brevican, neurocan, and versican. interleukin-33 (il-33) is an il-1 cytokine family member that uniquely plays a role as a cytokine and nuclear factor. it is released by necrotic epithelial cells and activated innate immune cells as an alarming danger signal. adamts and il-33 implicated in brain cancer pathogenesis. we aimed to seek the amount of adamts15 in u118 proteolytic enzymes are able to speed up wound healing by removal of the necrotic tissues and fibrin. several investigations have reported that proteases damage also the microbial biofilms formed by opportunistic bacteria including staphylococci on surfaces of chronic and acute dermal wounds. therefore, proteases are seemingly perspective enzymes for biofilm eradication by hydrolysis of both matrix proteins and adhesins, proteins providing cells attachment onto solid surface and other bacteria, as well as by the cleavage of signalling peptides of intercellular communication of gram-positive bacteria. here we report that ficin, a plant protease, efficiently degrades the structural components of biofilm matrix formed by s. aureus and s. epidermidis at concentrations of 10 lg/ml while trypsin and chimotrypsin are used as 1-2 mg/ml solution. the spatial structure of the biofilm was analyzed by atomic force microscopy. after ficin treatment, the biofilm structure became porous, with reduced viscosity. the congo red staining of the treated biofilms confirmed the hydrolysis of the protein component of the matrix. moreover, the biofilm treatment with ficin increased the antimicrobial efficiency of ciprofloxacin against biofilm-embedded cells of s. aureus and s. epidermidis. while 24 h antibiotic treatment did not lead to the increase of dead cells of neither s. aureus nor s. epidermidis embedded into the biofilm matrix, in the presence of ficin the fraction of viable cells decreased significantly. accordingly, soluble ficin appears beneficial for outer wound treatment biofilm eradication and reduces the reinfection risk. the wound-healing activity of ficin requires further investigations. this work was supported by the russian science foundation (project no 15-14-00046). resveratrol (resv) is an antioxidant that belongs to the group of plant compounds, called polyphenols. resv is defined as an antimicrobial substance that is produced by several plants (red grape skin, peanuts and berries) to protect them from rough environments like excessive ultraviolet light, infections and climate changes. as an antioxidant, this polyphenol protects the body against cardio-vascular and cancer diseases. besides, resv has anti-inflammatory, neuro-protective, anti-diabetic and other pharmacological effects. although the positive pleiotropic effects of this polyphenol are well documented, there is a huge need to understand its influence on the biophysical properties of lipid bilayer. in the present work, the interaction of resv with membranes composed of palmitoyl-docosahexaenoyl phosphatidylcholine (pdpc) or palmitoyl-oleoyl phosphatidylcholine (popc), sphingomyelin (sm) and cholesterol (ch) was investigated by means of fluorescence spectroscopy. generalized polarization of the fluorescent probe laurdan (gp) as a function of temperature was used to probe the changes in the fluidity of lipid bilayer induced by different resv concentration. the obtained results showed decreased lipid ordering from 50 to 100 lmol resv and opposite effect from 200 to 500 lmol in pdpc/sm/ch mixtures as compared to the control without resv. the interaction of resv with popc/sm/ch mixtures caused only an increase in the lipid ordering as a function of resv amount. popc and pdpc have the same head group but different fatty acid chains at sn-2. since resv changes the physicochemical properties of lipid bilayer by different ways one might suggest that the interaction of polyphenol with the membrane depends on the level of fatty acid unsaturation. this specific effect of resv on lipid organization could be related to its unique properties to prevent the cell from oxidative stress. neurodegeneration is the umbrella term for the deseases including progressive loss of structure or function up to death of neurons. beta-amyliod peptide is proteolytic fragment of the amyloid protein. the spontaneously formation of selective, voltage-dependent, ion-permeable channels in the lipid bilayers was reported as one of amyliod peptide toxicity mechanisms. the aim of our study was the investigation of the influence of the flavonoids (phloretin, phlorizin, quercetin and genistein) on the membrane activity of amyliod peptides. virtually solvent-free bilayer lipid membranes were prepared from mixtures of phospholipids in 0.1 m kcl (ph 7.4) using monolayer-opposition technique. using spectrofluorimetry we estimated prepared from phospholipids by extrusion the liposomal membrane permeability for calcein. we found that the addition of phloretin into membrane bathing solution led to an signicant increase in the channel forming activity of fragments 25-35 of amyloid peptide, fragment 25-35 of [gly35]-amyloid peptide and 106-126 of human prion protein. addition of other flavonoids did not cause any changes in the steady-state amyloid-induced current. it was found that the effect was caused by electrostatic interaction with the peptide. we found that time course of amyloid induced leakage calcein from liposome's is characterized by two components: the fast one is related to sorption of b-amyloid peptide on the membrane and the slow one is related to the oligomerization of the peptides on the surface of the lipid bilayer. addition of the phloretin simultaneously with b-amyloid peptide to the suspension of liposomes caused significant reduction in time parameters characterizing fast and slow components. from this results we can proposed that phloretin compensates the positive charge of the b-amyloid peptides and leads to the changes in their oligomerization status. the study was supported in part by rfs (14-14-00565) and sp-69.2015.4. ferritin nanocarriers: a focus on a metal-based drug encapsulated inside the protein cavity s. ciambellotti 1 , c. bernacchioni 2 , f. scaletti 3 , p. turano 1 1 cerm (magnetic resonance centre), florence, italy, 2 department of biochemical sciences, university of florence, florence, italy, 3 chemistry department 'ugo schiff', florence, italy ferritin is one of the main player involved in the iron metabolism. the biological role of ferritin is the storage of iron atoms inside the cavity preventing the uncontrolled accumulation of toxic species inside cells. ferritins are polymers constituted by 24 subunits that self-assemble giving rise to an almost spherical nanocage. in vertebrates, ferritins are formed by two distinct subunits, the heavy chain (h), with oxidoreductase activity, and the light chain (l) without catalytic activity. ferritins are promising nanocarriers for the delivery of contrast agents for diagnosis and of drugs for therapeutic purpose. their endogenous origin and the possibility to stabilize and protect the enclosed cargo inside the cavity, make ferritin a biocompatible vehicle. moreover, there are specific receptors on cells that recognize and incorporate ferritin by endocytosis, prospecting a kind of targeted-delivery. following the increasing interest in nanotechnology, we studied the interaction between different isoforms of ferritin with an antimetastatic drug, called nami-a, which is the first ruthenium derived anti-cancer drug to have entered clinical evaluation. this molecule is a metal-based prodrug that can release the metal ion ligands. here, we describe nami-a hydrolysis in the presence of recombinant homopolymers of ferritin followed spectrophotometrically. thanks to characteristic time dependent changes of spectral profile in the uv-visible region, we could detect differences in the hydrolysis process. the formation of a ru-adduct with hferritin was established by uv-visible and circular dichroism spectroscopies, as well as by kinetics measurements that showed inhibition of the ferroxidase activity of h-ferritin. crystallization trials are in progress to identify the binding site of ru by solving the x-ray structure of the complex. finally, we planned to test the cytotoxicity of ferritins pre-incubated with nami-a, using different cancer cell lines. a. cort 1 , t. ozben 2 , a. sansone 3 , s. barata-vallejo 4 , c. chatgilialoglu 5 , c. ferreri 3 1 sanko university, gaziantep, turkey, 2 akdeniz university, antalya, turkey, 3 cnr, bologna, italy, 4 universidad de buenos aires, buenos aires, argentina, 5 national center for scientific research 'demokritos', athens, greece liposomes as biomimetic models of cell membranes were used for examining some novel aspects of drug-metal induced reactivity with unsaturated lipids under oxidative conditions. the chemical basis of cis to trans transformation was ascertained by liposome experiments, using bleomycin-iron complexes in the presence of thiol as a reducing agent that by incubation at 37°c gave rise to the thiyl radical-catalysed double bond isomerisation of membrane phospholipids. the effect of oxygen and reagent concentrations on the reaction outcome was studied. as a chemical biology model for antitumoral strategies, liposomes highlight the role of cell membranes, which are not spectators but important targets of the drug effect, with synergic roles for chemotherapeutic effects. indeed, fatty acid recruitment and membrane formation are attracting a lot of interest in cancer, and in this context the loss of the natural cis geometry and the oxidationinduced lipid remodelling are worthy of deeper studies in antitumoral strategies. furthermore, the interaction between drugs and lipids can be suggestive of novel aspects of chemical reactivity for liposome carriers when circulating in vivo. gpr17 is a g-protein coupled receptor (gpcr), expressed in cells of brain, heart and kidney. it is related to the leukotriene and purinergic subfamilies of the rhodopsin-like class a gpcrs. gpr17 plays controversial role in the brain and spinal cord cells recovery after injuries. it is assumed that gpr17 is one of the cell death regulators immediately following an injury but at later stages it also takes part in tissue regeneration. there are also data implying some role of gpr17 in glucose homeostasis. drugs targeting gpr17 may help with treatments of multiple sclerosis and ischemia. the damage of rat's brain in artificially induced ischemia disease decreased after gpr17 inhibition. in addition, gpr17 takes part in myelin sheath formation, the lack of which is known to be the reason of multiple sclerosis. to better understand functional role of gpr17 and enable design of more efficient ligands we initiated structural studies of this receptor. to improve receptor stability and facilitate crystallization we created a series of chimeric constructs using different fusion partnerssmall soluble proteins inserted into the native amino acid sequence. preliminary experiments were carried out to evaluate the influence of different ligands on the receptor stability and cell surface expression in insect sf9 cells. this work was supported by russian federal target sterols are significant for the structural and dynamical features of cell membranes. among them, cholesterol is the major sterol in mammalian cell membranes whereas stigmasterol is the predominant sterol in plant membranes. stigmasterol varies structurally from cholesterol in having both an ethyl group at carbon 24 and an additional trans double bond between carbons 22 and 23. dimyristoylphosphatidylcholine (dmpc) is a widely studied synthetic lipid, which has a neutral (zwitterionic) pc headgroup and two symmetrical 14-carbon fatty acyl chains. the studies on the interactions of cholesterol and stigmasterol with dmpc membranes at molecular level are very limited. in the present study, a calorimetric comparison of the effects of the animal sterol cholesterol and the plant sterol stigmasterol on zwitterionic dimyristoylphosphatidylcholine (dmpc) multilamellar vesicles (mlvs) was investigated for the first time by using differential scanning calorimetry (dsc). our dsc studies indicate that with the inclusion of increasing cholesterol and stigmasterol concentrations from 5 to 40 mol% into pure dmpc mlvs, the pretransition disappears, the main phase transition shifts to lower temperatures and then disappears at cholesterol and stigmasterol concentration above 25 mol%. the main phase transition enthalpy (dh) is progressively reduced whereas full width at half maximum (dt ½ ) gradually increases with increasing cholesterol and stigmasterol concentrations. moreover, the main phase transition peak consists of overlapping sharp and broad components, indicating the hydrocarbon chain melting of sterol-poor and sterol-rich dmpc domains, respectively. in conclusion, this study shows clearly that both cholesterol and stigmasterol interact effectively with dmpc membranes and cause changes in their structural and functional properties. p-03.03.3-007 trh receptor mobility in the plasma membrane is affected by its interaction with its cognate signaling molecules and by cholesterol depletion r. moravcova, b. melkes, j. novotny department of physiology, faculty of science, charles university in prague, prague, czech republic g protein-coupled receptors (gpcrs) play a fundamental role in transferring information from extracellular environment to the cell interior. some gpcrs are supposed to form signaling complexes with their cognate g proteins and possibly other accessory proteins, which may facilitate the activation of g proteins and thus accelerate signal transmission. here we investigated the role of some components of thyrotropin-releasing hormone (trh) receptor signaling in hek293 cells stably expressing yfp-tagged trh receptor using fluorescence recovery after photobleaching (frap). we observed significant changes in values of the diffusion coefficients if expression of b 2 -arrestin or gb 2 subunit were suppressed by sirna. results of our frap experiments indicated significant difference between control and trh-treated cells as the diffusion coefficient markedly decreased after agonist stimulation. on the other hand, the same decline of the diffusion coefficient value was found after silencing with sirna and subsequent treatment with trh for most of the screened proteins. treatment of cells with 10 à5 m trh led to fast internalization of trh receptor, which was revealed by real-time confocal microscopy. it is known that cholesterol is an essential component of the cell membranes and it exerts modulatory effects on the functioning of various membrane proteins. disruption of plasma membrane integrity by cholesterol depletion using b-cyclodextrin significantly reduced the apparent diffusion coefficient values. interestingly, addition of trh to cells treated with b-cyclodextrin did not further reduced trh receptor mobility. it can be concluded that stimulation with agonist and/or sirna silencing of some components of the trh receptor signaling cascade significantly affects the mobility of trh receptor in the plasma membrane. p-03.03.3-008 l-opioid receptor mobility in the plasma membrane is diversely affected by biased ligands b. melkes, l. hejnova, j. novotny opioid receptors belong to the large family of g protein-coupled receptors (gpcrs), which are currently considered among the most important targets for pharmacological manipulations. during the past few years, a great deal of attention has been devoted to biased agonism. this phenomenon reflects the ability of different ligands to selectively affect the functioning of some gpcrs so they can display marked differences in their efficacies for g protein-or b-arrestin-mediated signaling. here we decided to investigate the effect of different agonists of the l-opioid receptor (l-or) on the lateral mobility of this receptor in the plasma membrane of hek293 cells which were stably transfected with l-or tagged with yellow fluorescent protein (l-or-yfp). it has been found previously that damgo stimulates g-protein-dependent signaling, endomorphine 2 stimulates arrestin-dependent signaling and morphine does not show any significant bias towards these two signaling pathways. in our experiments, we used the fluorescence recovery after photobleaching (frap) method to estimate the diffusion coefficients of l-or-yfp in the resting state and after addition of the above mentioned specific agonists. we observed that addition of damgo increased the value of the diffusion coefficient and addition of endomorphin 2 decreased the value of diffusion coefficient of l-or-yfp. addition of morphine or the l-or antagonist naloxone did not change the value of the diffusion coefficient compared to the resting state. these results indicate that different biased agonists of l-or may differently affect the mobility of this receptor in the plasma membrane. these findings provide new insights into the dynamics of l-or in the plasma membrane and contribute to better understanding of the mechanism of biased agonism at gpcrs, which is of central importance for receptor homeostasis and fine regulation of receptor activity. color tuning and adding potassium selectivity to a light-driven sodium pump k. kovalev 1,2 , v. polovinkin 1,2,3 , v. shevchenko 1,2 , v. gordeliy 1,2,3 1 moscow institute of physics and technology, dolgoprudniy, russia, 2 research centre j€ ulich, j€ ulich, germany, 3 institut de biologie structurale, universit e grenoble alpes, cnrs, and cea, grenoble, france recently a light-driven sodium pump has been discovered, characterized and tested as an inhibitory optogenetic tool. sodium pumping rhodopsin from dokdonia eikasta kr2 has an absorption maximum at 523 nm at ph 7.5 and to create more redshifted variants we analyzed available structures of the kr2 (pdb codes: 4xtl, 4xtn) and did the rational mutagenesis of residues in the retinal proximity region (i.e. m149, g153 and s254). the mutants of kr2 under investigation were: m149a, g153v, m149a/g153v, s254a, s254f, s254g, s254l, s254m, s254n, s254t, s254v, s254y. the protein mutants were expressed in escherichia coli c41 strain, expression was induced by the addition of 1 mm isopropyl b-d-1-thiogalactopyranoside. the cells were then washed trice with unbuffered 100 mm nacl or kcl solution. finally, the ph changes in cell suspensions (od600 = 8.0) were monitored. ph changes upon the addition of 30 lm of protonophore carbonylcyanide m-chlorophenylhydrazone were also measured. the following mutants completely abolished the protein function and were not used for further characterization: s254f, s254l, s254m, s254n, s254v. the remaining mutants have shown sodium pumping activity and s254a mutant has gained an additional potassium pumping activity. all functionally active mutants were purified using ni-affinity chromatography and the absorption spectra were collected for them at ph 7.5 (50 mm na/na-pi, 100 mm nacl). m149a mutant absorption maximum is blue-shifted to 519 nm. g153v and m149a/g153vblue-shift to 470 nm. s254a, a potassium pumping variant,red-shift to 545 nm. s254g, s254yred-shift to 545 nm. s254tno change in absorption maximum position. based on structural analysis of kr2 we discovered another potassium pumping variant and provided the variants with absorption maximum blue-shift up to 53 nm and red-shift up to 22 nm. human endothelin receptors belong to the g-protein coupled receptors (gpcrs) superfamily. this class is pharmacologically important, with more than 40% drugs targeting it. the human endothelin system, which includes endothelin receptors types a and b (etb and eta), plays a highly important role in the blood pressure regulation. endothelium cells produce peptides, known as endothelins 1-4, which activate endothelin receptors and launch cascades of reactions that lead to vasoconstriction or vasodilatation depending on the receptor subtype and the tissue. additionally, endothelin receptors take part in such processes as transmission of nerve impulses, development of neural crest, and regulation of acid-base-salt balance in kidneys. in order to stabilize etb receptor for crystallization trials we introduced a compact soluble protein, apocytochrome b564ril (bril), is the third extracellular loop of the receptor. bril is known to be an effective crystallization driver for gpcrs. the engineered protein was expressed using baculovirus system in sf9 insect cells, purified and subject to variety of pre-crystallization assays. localization of the overexpressed protein in insect cells was visualized via the confocal microscopy. thermal stability of the protein in the presence and absence of ligands was measured by the thermal shift assay. finally, the mobility of the receptor in lipidic cubic phase (lcp) at many different conditions was probed by the lcp-frap (fluorescence recovery after photobleaching) assay. these tests showed that the obtained protein is thermally stable, functionally active and diffuses well in lcp at certain conditions, making it a suitable candidate for proceeding to in meso crystallization trials. this work was supported by the russian science foundation (project no. 16-14-10273). mitochondrial oxidative phosphorylation is the key metabolic pathway for the production of atp. mitochondrial respiratory chain (rc) defects are some of the most commonly diagnosed inborn errors of metabolism with a diverse spectrum of clinical phenotypes. the aim of the study is to evaluate the rc enzyme activities and histopathological findings in muscle biopsies of patients with suspected mitochondrial disease. muscle biopsy samples were collected from 48 pediatric patients. the samples were homogenized in seth buffer using a glass/glass homogenizer. the activities of citrate synthase (cs) and rc enzymes (complex i, ii-iii, and iv) were measured in tissue homogenates by kinetic spectrophotometric assays by schimadzu uv spectrophotometer (uv-1800). non-collagen protein was determined by the modified lowry assay. activities of complex (c) i, ii-iii and iv (cox) were normalized by cs. histopathological investigations included h&e, modified gomori trichrome, periodic acid schiff, oil-red-o, nadh, sdh, cox and atpase stains. deficiency of rc complexes was detected in 39 biopsies (81%). c iv deficiency was most common (60%), followed by c i (40%) and c ii-iii (27%). multiple complex deficiency was present in 40% and isolated deficiency was present in 42% of the biopsies (20% c i, 20% c ii-iii, 60% c iv). cs activity was elevated in 44% of the biopsies. decreased c i/cs, c ii-iii/cs and c iv/cs ratio was observed in 44%, 42% and 52%, respectively. comparing with histological data, biochemical analysis revealed additional findings in 50% of biopsies. complex iv deficiency, either isolated or accompanied by other complex deficiencies, is most common in our cohort. rc analysis may reveal additional findings to histopathological results and careful clinical investigation with correlation of clinical, biochemical and histopathological data is mandatory for the challenging diagnosis of mitochondrial disorders in childhood. investigation of adipocytokines, activity of glut and na + /k + -atpase in rats fed glucose, fructose, starch-based sugars objective: all over the world, shows a significant increase in obesity and diabetes. intake of foods that contain fructose, glucose and starch-based sugar is a potential risk for metabolic syndrome. obesity and diabetes are important effects of high fructose corn syrup (hfcs). we aimed to research, activity of na + /k + -atpase in addition to glucose transporter (glut) 2, resistin, adiponectin and other biochemical markers in rats fed glucose, fructose and starch-based sugars. materials and methods: study was performed on rats and 3 groups were included in the study. rats were fed with chows that were given either normal diet for control group (70% carbohydrate, 20% protein and 10% fat), high fructose (70% carbohydrate (87% fructose and 13% starch), 20% protein and 10% fat), or high sucrose (70% carbohydrate (87% sucrose and 13% starch), 20% protein and 10% fat). rats were fed with chows for 8 weeks. in this process, the weight of the rats were followed. at the end of the experiment, blood is taken in all groups. level of hba1c, glucose, resistin and adiponectin were studied. glut2 and na + /k + -atpase activity were studied in the liver tissue. results: a significant increase in adiponectin levels were determined in rats fed both hfcs and sucrose (p < 0.001). a significant decrease in level of na + /k + -atpase activity were determined in rats fed both hfcs and sucrose (p < 0.001). there was no significant differance level of hba1c, glucose, resistin and glut2 in rats fed sucrose or hfcs (p > 0.05). conclusions: fructose-rich diet has an effect on changes in the atpase activity and is a major risk factor for obesity. keywords: adiponectin, fructose, glucose, high-fructose corn syrup, na + /k + -atpase, resistin. p-03.03.3-014 nucleic acid-biomembrane lipid selfassemblies: from primordial soup to novel genome organization model and cellular nonviral nanotherapies f. k. demirsoy 1 , n. eruygur 2 , e. s€ uleymanoglu 3 1 biotechnology central laboratory, biotechnology institute, ankara university, ankara, turkey, 2 department of pharmacognosy, faculty of pharmacy, cumhuriyet university, sivas, turkey, 3 department of pharmaceutical chemistry, faculty of pharmacy, gazi university, ankara, turkey turkey nucleic acid-cell membrane complexes has attracted research interest as models in gene regulation, cell cycle and division, as biosensors designs, as well as in molecular evolution. zwitterionic phospholipids, complexed with polyribonucleotides by divalent metal cations (mg2+) are considered as genosome vehicles. their formations are studied by spectroscopic, thermodynamic, interfacial and microscopic approaches to build thermodynamic and kinetic models of their structural transitions. dna forms a mg2+-driven ternary complexes with neutral liposomes both at the airwater interfaces and at vesicle surfaces. the described self-assemblies form relevant models for nuclear pore complexes and their further implications in gene expression and functions. such membrane contacts could be considered also in prokaryotic nucleoids important in bacterial segregation, whereas in eukaryotes these complexes can be regarded as focal points for transcription-translationtranslocation processes. the effects of ozone/oxygen mixture on citrate synthase and mitochondrial complex activities of striated muscle tissue of healthy mice we investigated the effects of ozone/oxygen mixture and oxygen on citrate synthase (cs) and succinate dehydrogenase (sdh) activities of striated muscle tissue of healthy mice. thirty-six mice were included the study. firstly muscle samples were taken from all mice's left thigh muscle in under anesthesia (group 0). secondly mice were randomly divided in four groups as: group 1: oxygen was given once at 3 days for 7 days, group 2: ozone/oxygen was given once at 3 days for 7 days, group 3: oxygen was given once at 3 days for 30 days, group 4: ozone/oxygen was given once at 3 days for 30 days. ozone/oxygen mixture and oxygen were given at a dose of 1 mg/ kg groups (1) (2) (3) (4) . after treatment animals were sacrificed, and muscle samples were taken and stored in à80°c for until the analyses. muscle tissues were homogenized in 0.05 m tris-hci and 0.15 m kci. cs and sdh activities were measured with spectrophotometer. cs and sdh activities were expressed as lmol/min/g tissue. cs and sdh activity results were given as mean ae sd. cs activity has been found in group 0 (37.8 ae 11.7), group 1 (46.9 ae 10.8), group 2 (34.7 ae 6.8), group 3 (32.9 ae 7.5) and group 4 (33.9 ae 8.5). sdh activity has been found in group 0 (2.50 ae 1.03), group 1 (2.37 ae 0.77), group 2 (2.52 ae 1.45), group 3 (2.24 ae 1.06) and group 4 (2.59 ae 1.25). there was no statistically significant difference among all groups in terms of cs (p > 0.130) and sdh activities (p > 0.997). there was no difference between all groups in terms of inflammation, muscle fiber size, regeneration or necrosis. vascular structures, connective tissues, lipid and glycogen content of fibers were normal. cytochrome oxidase activity was also normal. ratio of ragged blue fibers of all groups were less than 0.3%, so they were scored as 1. there was no difference among groups for ragged red fiber content. we have not found a significant effect of ozone/oxygen mixture and oxygen on cs and sdh activities of striated muscle tissue of healthy mice. lipidic cubic phases (lcps) consist of bicontinuous lipid bilayers and water channels. lpcs are widely used for membrane proteins crystallization and further determination of their spatial structures by means of x-ray crystallography. this approach was successfully used for studying g-protein coupled receptors structures. usually crystallization initiates by adding the precipitants (buffers with high salt concentrations). here we propose to use photo-switchable analogs of 1-monoolein to change lattice parameter of lpc. we synthetized a number of novel diazo-analogs of 1-monoolein. their structures were confirmed by nmr-spectroscopy and mass-spectrometry. being incorporated in phospholipid vesicles or detergent micelles they subjected to trans-to cis-isomerization under the light exposure at 365 nm. also we characterized the lcp's structures and properties by small-angle x-ray scattering on the rigaku instrument. one of the synthetized compounds, 3-(4-{-[4-(octyloxy) phenyl] diazenyl} phenoxy) propane-1,2-diol (1% mol), was incorporated into the 1-monoolein cubic phase. according to small-angle x-ray scattering data the structure of the monoolein cubic-pn3 m phase with lattice parameter 106.3 angstrem was not affected by insertion of this photo-switchable monoolein's analog. after the light exposure at 365 nm we observed trans-cis-isomerization. in the same time the cubic-pn3 m phase was not changed but the lattice parameter reduced to 98.8 angstrem. this effect on monoolein lpc is similar to the addition of a precipitant to initiate protein crystallization process. the spontaneous return to the initial lattice parameter was completed after 3 days in dark. thus, we demonstrated the possible controlling of the monoolein cubic phase lattice parameters by adding the photo-switchable diazo-derivatives of monoglyceride analogs, which can be used for crystallization of membrane proteins. evaluation of certain protein and phosphoprotein expression levels by using western blot technique in head and neck squamous cell carcinoma a. kalayci yigin 1 , t. cora 2 1 cerrahpasa faculty of medicine, istanbul university, istanbul, turkey, 2 faculty of medicine, selcuk university, konya, turkey introduction: head and neck squamous cell carcinoma (hnscc) is a primer tumor type in head and neck cancers, characterized by aggressiveness, early recurrence and metastasis. while alcohol and smoking play an important role at pathogenesis of disease, deregulation of some signaling pathways, genes and protein levels related to these pathways are considered as important at contribution of development of hnscc. materials and methods: in this study, protein and phosphoprotein expression levels of the frequently phosphorylated sites (egfr, pegfr, igf-ir, pigf-ir, pdgfrb, ppgdfrb, pten, ppten, akt and pakt) were investigated by using a western blot to confirm the expression of mrna in the context of protein levels at normal-tumor tissues of hnscc and sccl-mt1 that is a hnscc tumor cell line and hek-293 that is a normal cell line. results: as a result of western blot analysis egfr, pdgfrb and igf-1r were detected as highly overexpressed cell surface receptors in tumor tissues of hnscc. discussion and conclusion: the findings of this study revealed the overexpression of other cell surface receptors as well as egfr in hnscc. in conclution, potential pathways were identified by determining the cell surface receptors overexpressed in hnscc, these data support each other and may be important in pathogenesis of hnscc. introduction: the investigation of final products of lipid peroxidation is considered as the main mechanism involved in development of pathogenesis, diagnostics and prognosis of various parasitic illnesses. materials and methods: the concentration of lp-ap in the blood was determined in the study group considered of 129 women (65%), and 69 men (35%). results: before antiparasitic treatment, women infected with g. intestinalis showed a statistically significant 1.5 times increase of gpx activity levels; and 1.7 times ada level increase compared to the control group. after the treatment, the cat activity showed a sharp increase, whereas the ada activity decreased by 1.4 times, compared to the average level before the treatment. the results of the blood samples of the infected men with giardiasis, show the statistically significant increase in the level of all the studied parameters of lipid peroxidation, except the total primary production (tpp). the exception was the mda level, remaining significantly increased, in contrast to the control group and to the condition after antiparasitic treatment. in infected men, the level of cat activity was more than 5.8 times higher than that noted in control group. after treatment the levels of ada activity and gpx returned to the values of the control group, while the level of cat activity remained elevated. conclusion: an accumulation of primary and secondary metabolites in the course of giardiasis seems to confirm its involvement in the induction of oxidative-antioxidative homeostasis. antiparasitic treatment in giardiasis leads to normalization of the ap parameters studied in women and men, except the mda content in the blood of men. therefore, additional antioxidant treatment is advised for the antiparasitic therapy of men. in vitro effects of ethanol on rat brain synaptosome and dose-dependent antioxidative role of boric acid ethanol is a psychoactive drug that is very large and used frequently today. it has suppressive effects brain's comminication pathway. depending on its acute or chronic use and the dose, ethanol increase membrane fluidity and it causes oxidative stress. this study deals with the in vitro effects of ethanol toxicity (200 mm) and potential protective effect of different doses of boric acid (ba) (5, 10 and 25 mm) on rat brain synaptosomes. with this aim, five male spraque dawley rats are killed by decapitation under anesthesia. after the frontal cortexes of the rats are taken out, each of them is divided into four pieces. these pieces were used as a sample in five groups (control, ethanol, ethanol+5 mm ba, ethanol+10 mm ba, ethanol+25 mm ba) which include six samples. the synaptosomal fractions are prepared by the homogenization of the frontal cortex pieces and centrifugation for each samples. as markers of ethanol-induced oxidative stress in the synaptosome of the rats, malondialdehyde (mda), nitric oxide (no) and catalase (cat) levels were measured. mda levels in the ethahol group were a quantity increased compared with those in the control group but it unchanged significantly as statistically (p < 0.05). however the mda level in the ethanol+ boric acid (25 mm) group was shown to be significantly decreased compared with that in the ethanol group (p < 0.05). the cat activity of the ethanol group was significantly higher than that in the control group and cat activity of the ba (5 mm,25 mm) groups were close compared with control groups (p < 0.01). no levels in ethanol groups were decreased but unchanged compared with control groups as statistically. neverthless, no levels in ethanol+ boric acid (25 mm) groups were increased (p < 0.05). these results demonstrate that ethanol (200 mm) is capable of triggering damage to rat brain synaptosome and ba could be influential in antioxidant mechanisms against oxidative stress resulting from ethanol exposure. acute myeloid leukemia (aml) is the most common form of acute leukemia in adults and its incidence increases with age. carbonic anhydrases (cas) are zinc-containing enzymes. these enzymes catalyze a very simple physiological reaction, the inter conversion between carbon dioxide and the bicarbonate ion, and are thus involved in crucial physiological processes connected with respiration and transport of co 2 /bicarbonate between metabolizing tissues and lungs, ph and co 2 homeostasis, electrolyte secretion in a variety of tissues/organs, and biosynthetic reactions and many other physiologic or pathologic processes including reproductive tract. investigation of autoantibodies in aml patients have been popular research area in recent years. the aim of the current study was to investigate carbonic anhydrase i and ii (ca i and ii) autoantibodies in the serum of subjects with aml based on the information and considerations of autoimmune relation of acute myeloid leukemia. anti-ca i and ii antibody levels were investigated by enzyme-linked immunosorbent assay method (elisa) in serum samples of thirty patients with aml and thirty healthy peers. anti-ca i and ii antibody titers of aml group were significantly higher compared with the control group (p = 0.000), (p = 0.034), respectively. we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in patients with aml (r = 0.613, p = 0.000). we found significant positive correlation between anti-ca i antibody and anti-ca ii antibody titers in women and the men (r = 0.851, p = 0.000), (r = 0.503, p = 0.080), respectively. at an anti-ca i cut-off point of 0.123 absu, sensitivity was 80% and specificity 100%. at an anti-ca ii cut-off point of 0.097 absu, sensitivity was 60% and specificity 77%. the ca i and ca ii autoantibody levels in patients with aml were found higher compared to control group and the results suggest that ca i and ca ii autoantibodies may be involved in the pathogenesis of aml. aim: behc ßet's disease (bd) is a systemic autoimmune disease. recurrent oral and genital mucosal ulcers, uveitis, and skin lesions are characteristic findings for bd. platelet-lymphocyte ratio reflects a novel marker for romatological diseases. the aim of this study was to investigate the platelet/lymphocyte ratio in behc ßet's disease. methods: whole blood samples were collected from 50 healthy control and 21 patients with behc ßet's disease. the mean age for controls and patients were 40 ae 1.0 and 37 ae 13, respectively (p = 0.639). patients with chronic disease and inflammatory disorders were excluded. thrombocyte and lymphocyte counts were analyzed with abbott cell dyne heamotolgy analyzer. statistical analysis was performed with ibm spss v20. results: platelet counts were higher but not statistically significant in patients with behc ßet's disease compared to control group (289 ae 100 vs. 257 ae 57) (p = 0.088). lymphocyte counts were lower in patients with behc ßet's disease compared to control group (2.56 ae 0.58 vs. 2.69 ae 0.85) (p = 0.513). platelet/lymphocyte ratio was higher but not statistically elevated in patients with behc ßet's disease compared to control group (118 ae 41 vs. 106 ae 49) (p = 0.344) conclusions: platelet/lymphocyte ratio (nlr) and mean platelet volume (mpv) as inflammatory markers recently became popular because of their simplicity, cost effectivity and their advantages to predict clinical prognosis of specific diseases. according to this study's results, platelet/lymphocyte ratio must be analyzed in vast scale patient populations to identify the disease. objectives: systemic lupus erythematosus (sle) is a chronic relapsing autoimmune disease characterized by production of autoantibodies against a series of nuclear antigens and by chronic inflammation. in recent years, neutrophil-lymphocyte ratio (nlr) was determined to be a good indicator of inflammatory status. nlr can be easily calculated from a whole blood count. introduction: neuroblastoma, an embryonal malignancy, is the most common extracranial solid tumor of childhood. untreated neuroblastoma tumors and cell lines are reported to have reduced hla class i expression, rendering them potentially susceptible to natural killer cell killing due to lack of engagement of hla class i-specific inhibitory killer cell immunoglobulin-like receptors (kirs). the aim of this study was to investigate whether kir genes could influence the risk of neuroblastoma and prognosis of the patients. materials and methods: study group consisted of 50 neuroblastoma patients (15 male, 21 female, median age: 36 months) followed at the pediatric oncology clinic of c ß ukurova university medical faculty. control group consisted of 100 healthy children. 14 patients had stage 1, 2, 3 or 4s disease, 36 patients had stage 4 disease. 16 different kir genes were analysed by sequence specific oligonucleotide probe (ssop) analyses. statistical analysis were done using fisher's exact test. results: compared to the control group, neuroblastoma patients had lower expression of activating kir gene, kir2ds3 (p = 0.005), and higher expression of inhibitory kir gene 2dl3 (p = 0.038). additionally kir2ds3 genes were more common in patients with early stages (stage 1, 2, 3 or 4s) (p = 0.023) and kir2dl3 genes were more common in patients with stage 4 (p = 0.044). furthermore, there were no statistically significant differences between the rate of aa and bx genotypes and their centromeric/ telomeric regions of patients and controls. discussion: kir2dl3 gene can have a triggering effect in neuroblastoma pathogenesis; whereas kir2ds3 can have a protective role. investigating nk cell infiltration and kir receptors in neuroblastoma tissue samples will give more insight to the pathogenesis p-04.03.3-008 neuroprotective and immunomodulatory effects of urtica urens s. arslan 1 , g. terzioglu 1 , b. kabalay 1 , a. r. tufekci 2 , a. sen 1 , i. demirtas 2 1 department of biology, faculty of arts and sciences, pamukkale university, denizli, turkey, 2 deparment of chemistry, faculty of arts and sciences, c ß ankiri karatekin university, c ß ankiri, turkey urtica urens (small stinging nettle) has been used for medical purposes in turkey as an alternative therapy. it has been used in the treatment of inflammation that is early, non-specific immune reaction to tissue damage or pathogen invasion, plays an important role in the initiation of neurodegenerative disorders such as multiple sclerosis. however, there are limited studies that investigate anti-inflammatory activity of urtica. therefore, aim of this study is to find out theanti-inflammatory effect of chloroform extract in caco-2 cell line. for this purpose, firstly, chloroform extract of urtica leaves was prepared. chemical composition of extract was determined by lc-ms. the effect of chloroform extract on selected pro-inflammatory and inflammatory proteins such as tumor necrosis factor-a (tnfa), nuclear factor kappa b (nf-jb), c-x-c motif chemokine 10 (cxcl10), and 11 (cxcl11) were determined. whole genome transcriptome analysis was performed by using human ht-12 v4 beadchip. extract treatment caused 35% and 40% increases in protein and mrna levels of nf-jb, respectively. on the other hand, tnf-a protein and mrna levels decreased significantly (24% and 12%, respectively). similarly, cxcl10 and cxcl11 mrna levels decreased 45% and 38%. transcriptome analysis showed that 233 probes were significantly changed (p < 0.05). pathway analysis revealed that the extract altered a group of genes involved in immune response, calcium ion homeostasis and transport, potassium channel complex, g-protein coupled receptor protein signalling pathway, etc. it is well established that calcium is very critical for brain cell death and formation of many brain disease including multiple sclerosis. these observations suggests that urtica maybe used in neurodegenerative diseases. in order to further test this hypothesis experimental autoimmune encephalomyelitis experimentsand activity guided fractionations have been still continuing. this work is supported by tubitak 111t515. p-04.03.3-009 linear low molecular weight a-1,6-glucan from bifidobacterium bifidum bim b-733d is implicated in pathogenesis of celiac disease the bifidobacteria are recognized as human commensals and widely used as probiotics. earlier, we have found (kiseleva et al., benef. microbes, 2013, 4(4) : 375-391) that bifidobacterium bifidum bim b-733d contains low molecular mass (5-7 kda) a-1,6glucans (g anti-tpo and g anti-tg ) that interact selectively with human autoantibodies to thyroid peroxidase (anti-tpo) and thyroglobulin (anti-tg), recognized markers of autoimmune thyroid disease (atd). the aim of the study was isolation and identification of b. bifidum bim b-733d biopolymers (bps) interacting selectively with autoantibodies to tissue transglutaminase (anti-ttg) and antibodies to gliadins (anti-gl), recognized markers of celiac disease (cd). we used affinity chromatography with anti-gl, size exclusion chromatography, 1 h and 13 c nmr spectroscopy, elisa with immobilized bps, tissue transglutaminase (ttg) and gliadins (gl) as positive controls. the bp isolated by affinity chromatography with anti-gl (as more available marker of cd) and size exclusion chromatography was identified by two-dimensional nmr spectroscopy as 5-7 kda linear a-1,6-glucan identical to g anti-tpo and g anti-tg . the functional activity of the bp named g anti-gl , viz., ability to interact selectively with anti-ttg and/or anti-gl was proven by elisa with (i) serum samples of cd patients containing either both anti-ttg and anti-gl without anti-tpo and anti-tg or anti-gl without anti-ttg, anti-tpo and anti-tg vs. serum samples of healthy donors without four types of antibodies and (ii) pure anti-gl vs. pure total igg (without anti-ttg, anti-gl, anti-tpo, anti-tg). since (i) serum samples of cd patients do not contain anti-ttg without anti-gl and (ii) pure anti-gl isolated by affinity chromatography with gliadins (gl) cross reacts with tissue transglutaminase (ttg), additional studies with pure anti-ttg are necessary to find out which of the two antibodies, anti-ttg and anti-gl, bind g anti-gl . in conclusion, we proved that b. bifidum bim b-733d cells contain linear low molecular mass a-1,6-glucan, g anti-gl , that interacts selectively with anti-ttg and/or anti-gl. since g anti-gl is identical to earlier found g anti-tpo and g anti-tg , we hypothesize that the a-1,6-glucan is implicated in pathogenesis of both autoimmune diseases, cd and atd. influences of elevated serum ferritin levels on insulin resistance and non-insulin-dependent diabetes mellitus (niddm) have predicted either because of increased body iron stores or influenced by several inflammatory diseases. low serum 25 hydroxyvitamin d is known to perturb cellular function in many tissues, including the endocrine pancreas, which are involved in obesity and niddm. we planned to investigate the association between 25hydroxyvitamin d with hematologic parameteres and iron status in obesity vs. diabetic patients. study groups consist of control, non-diabetic obese, obese-diabetic and lean-diabetic groups. serum triglycerides, total cholesterol, ldl-c, hdl-c, fasting glucose, hba1c, uric acid, creatinine, ggt,25-hydroxyvitamin d, insulin, crp, esr, total blood count and iron status. apart from the three parameters, there were no significant difference (p > 0.05) between groups. serum ferritin and mchc levels were significantly higher in lean-diabetic patients (p < 0.05). on the other hand, rdw are determined to be significantly lower (p < 0.001) in the non-diabetic obese group. no difference was detected in 25-hydroxyvitamin d levels between the control and the study groups (p > 0.05). non-diabetic obese patients had significantly (p < 0.05) higher levels of tg and lower levels of hdl compared to obese-diabetics. insulin levels were higher in nondiabetic obese and obese-diabetics than lean-diabetics (p < 0.05). this study provides evidence that lean diabetic patients show higher ferritin and mchc levels than obese patients. the increase in serum ferritin and mchc levels is related with altered iron metabolism at cellular level. at late mitosis, the mother cell divides, leaving two daughter cells connected by a thin intercellular bridge (icb). during abscission of the icb, the ingression of the cleavage furrow is formed, and the central spindle microtubules are compacted into the structure known as midbody (mb). the mb is situated within the icb, with the abscission usually occurring at one side of the mb. as a result, only one daughter cell inherits the post-mitotic mb. these mbs can then either accumulate in the cytoplasm or be degraded. recent studies have identified mbs as novel signaling platforms regulating stem cell fate and proliferation. indeed, stem cells as well as cancer cells were shown to accumulate post-mitotic mbs, resulting in reprogramming of the cell fate and conversion to highly-proliferative, stem cell-like phenotypes. it has been proposed that regulated macroautophagy may be playing a key role in mediating pots-mitotic mb degradation. therefore, the experimental approach involved studying the dynamics and function of a protein known as fyco1, which associates with postmitotic mbs and may regulate their degradation. in this study we identified fyco1 as a protein, which associates with post-mitotic mbs and may regulate their degradation. interestingly, fyco1 is also known to be present on autophagosomes, and overexpression of fyco1 can induce the formation of enlarged lc3-containing autophagocytic structures. here we demonstrate that fyco1 knock-down leads to defects in autophagic mb degradation, and that fyco1 functions by targeting endocytic membranes to the autophagic phagophore during early stages of mb degradation. additionally, we showed that fyco1 depletion leads to increased proliferation and cell growth in soft agar. based on all these data, we hypothesize that fyco1 mediates selective mbs degradation via endosome-dependent extension of the phagophore around the post-mitotic mbs, and that mbs may be the regulators of cancer proliferation and progression. p-05.03.3-002 proliferative effect of hypericine on human skin fibroblast cells and identification of the mechanism of action in molecular level to drawbacks associated with efficiency and viral genome integration. in order to improve reprogramming efficiency and compensate for viral transduction, new chemicals have been explored through ipsc research. the aim of this study was to investigate the proliferative effect of hypericine on human skin fibroblast cells (sf) in-vitro, and to identify the mechanism of action in molecular level. the proliferation was measured using the clonogenic and dimethylthiazol diphenyltetrazolium bromide (mtt) assays. real-time quantitative polymerase chain reaction (qrt-pcr) was performed to detect the mrna levels of cyclins (d1 and b1) and cell cycle controller genes (p53 and p21). sf cells were treated with different doses (1 nm-100 lm) of hypericine for 24 h and 48 h. a significant cell proliferation was observed in moderate concentrations (0.1-15 lm; %110-%134), but at high concentrations (25-50 lm) cytotoxic effects emerged in sf cells (ic50 = 23.62 m, r 2 = 0.915). qrt-pcr results revealed that the most proliferative dose of hypericine (15 lm) stimulates cyclin d1. the anti-proliferative activity of hypericine was accompanied by inhibition of cyclin b1 mrna, whereas it induced expression of p53 and p21 genes, and thus apoptosis was observed by dna laddering at the same dose (50 lm). overall results suggested that hypericine can compensate for viral transduction and improve reprogramming efficiency of ipscs by enforcing them in g1 phase. hence we report that hypericine can be a good candidate component for cocktails produced to trigger ipsc proliferation. glioblastoma (gbm) is the deadliest brain tumor. the mean survival time of gbm patients is approximately 12 months, increasing to 14 months after treatment with temozolomide, which is the gold standard chemotherapy. the resistance of gbm to chemotherapy seems to be associated with the blood-brain barrier (bbb) that limits the delivery of chemotherapeutics, and the presence of a population of cells that expresses stem cell-like properties, which are known to be chemo-and radioresistant,5 the glioblastoma stem cells (gscs). the difficulties imposed by these two factors could be reduced by the use of a targeted drugdelivery liposome-based strategy that allows bbb passage and reduces the side effects of chemotherapeutics. the present study evaluated the ability of the f3 peptide-targeted ph-sensitive lipid-based nanoparticle containing doxorubicin (dxr) to target gscs and non-gscs. we evaluated the expression of cell-surface nucleolin by flow cytometry, as well as of stem cell-like markers, in two gbm cell lines. we also determined the ability of gbm cell lines to specifically uptake the f3 peptide-targeted ph-sensitive lipid-based nanoparticles, by flow cytometry, and correlated it with the expression of stem cell-like markers. moreover, to ascertain the impact of intracellular delivery of chemotherapeutic drugs into gbm cell lines, cytotoxicity was further assessed by the mtt assay. our results showed that the f3 peptide-targeted ph-sensitive lipid-based nanoparticles successfully targeted glioblastoma cells and particularly gscs. in addition, the results also provided evidence of the nucleolin overexpression-dependency of this strategy, emphasizing the need to adapt the therapeutic strategy to the individual patient. this study showed that f3-targeted ph-sensitive liposomes may constitute an appropriate strategy to overcome the chemoresistance associated with glioblastoma cells. p-05.03.3-004 leukemic cell plasticiy as a resistance mechanism towards tyrosine kinase inhibitors chronic myelogenous leukemia (cml) is a hematopoietic stem cell disease characterized by the t(9;22)(q34;q11) translocation, which encodes the chimeric tyrosine kinase onco-protein, bcr-abl. the tyrosine kinase inhibitor (tki) imatinib is the first-line treatment for patients with cml. unfortunately drug resistance is one of the main problems observed. while secondary resistance is associated with bcr-abl kinase domain mutations, oncogene amplification and mechanisms interfering with intra-cellular drug concentrations; primary resistance mechanisms haven't been elucidated. we generated high dose imatinib-resistant k562 subclones (k562-ir) by clonal selection to study primary resistance mechanisms in vitro. drug resistance was shown by caspase 3 and annexin v/pi assays. we also showed cellular uptake and function of imatinib with western blot technics. k562-ir cells are not only resistant to imatinib but also to 2nd, 3rd generation tyrosine kinase inhibitors. we demonstrated that k562-ir cells have a highly adherent character, proliferate slowly and are resistant to drug-induced senescence. microarray analysis revealed that k562-ir cells differentially express tissue/organ developement and differentiation genes at high levels. we showed that k562-ir cells forms intact tumor spheroids in 3d cell culture conditions which is a marker of tumor initiating potential. cell surface maker analyses and protein analyses of k562-ir cell population, points towards an epithelial-mesenchymal plastic cell capable of adopting different morphologies. we hypotizied that imatinib and other tyrosine kinase inhibitors may cause the gain of phenotypic plasticity potential in leukemic cells, by interfering with signalling pathways; which in itself may lead to therapy resistance. hypoxia has multiple effects on cancer cells, which are critically involved in tumor progression. hypoxia leads to changes in tumor cell metabolism and can promote cancer cell survival, invasion and metastasis by its critically important role on maintenance of cancer stem cell (csc) phenotype. in this research, human cd133 + cscs isolated from human osteosarcoma cell line saos-2 using macs magnetic separation technique were characterized, and their stemness properties under hypoxic (1% o 2 ) and normoxic (21% o 2 ) conditions were compared in two and three dimensional culture conditions. two different 3d culture techniques (nanofibrous bacterial cellulose scaffolds and scaffold free microtissues) were used to evaluate effects of hypoxia on csc behavior, and the results were compared with the cell behavior in classical 2d culture systems. the morphologies of cells were examined by scanning electron microscopy (sem); rt-pcr and immunocytochemistry staining were used to examine the cancer stem cell phenotype maintenance under hypoxic and normoxic conditions. it is shown that hypoxia supports the expression of stemness markers such as oct3/4, nanog and sox2 compared to normoxic conditions in 3d cultures. although similar effects of hypoxia were observed in 2d cultured cscs, the expression levels of stem cell phenotypeindicative markers were significantly lower on 2d compared to 3d culture systems. this study is seen as an introduction to develop a more relevant 3d hypoxic cancer stem cell based tumor model to study csc behavior and tumor genesis in vitro for testing of novel cancer stem cell therapeutics and to understand signal transduction in cancer stem cells. prostate cancer (pca) is the second most frequent cause of cancer-specific mortality in the world. cancer stem cells (cscs) are a subpopulation of cells that involved in drug resistance, metastasis and recurrence of cancers. the efficacy of natural flavanone apigenin on cell survival, apoptosis and migration of cscs were evaluated. cd44 + cscs were isolated from human pca pc3 cells using a magnetic-activated cell sorting system. pc3 and cscs were treated with different concentrations of apigenin, docetaxel and combinations of the two agents for 48 h. apigenin dose dependently inhibited cscs and pc3 cell viability, and this was accompanied with a significant increase of the cell cycle inhibitors p21 and p27 (kip1). the flavonoid significantly induced apoptosis via an extrinsic caspase-dependent pathway by upregulating the mrna expressions of caspases-8, -3 and tnfa, but failed to regulate the intrinsic pathway as determined by the bax, cytochrome c and apaf-1 in cscs. in contrast to cscs, apigenin induced intrinsic apoptosis pathway as evidenced by the induction of bax, cytochrome c and caspase-3 while caspase-8, tnf-a and bcl-2 levels remained unchanged in pc3 cells. the ability of apigenin to inhibit the proliferation of cscs through apoptosis was confirmed by tali image-based cytometer. the flavanone strongly suppressed the migration rate of cscs compared to untreated cells. significant downregulation of mmp-2 and -9 exhibits the ability of apigenin treatment to suppress invasion. the expressions of pi3k/akt and nf-kb p105/ p50 were significantly decreased after 48 h apigenin treatment. taken together, these data demonstrated that flavonoid apigenin is an invaluable chemopreventive compound that inhibits proliferation, invasion and the stemness properties of cscs. this study was funded by the scientific and technological research council of turkey (tubitak, grant no. 115s356). (pi3k), are frequently found in patients with severe early-onset segmental overgrowth. whilst differences in timing and location of the founder mutation are likely to explain part of the observed disease heterogeneity, it is less clear whether and how quantitative differences in the strength and timing of pi3k activity contribute to phenotypic variability. our aim is to characterise pik3ca mutant-specific signalling as well as to explore the effects of varying the strength and/or temporal pattern of pi3k activation on downstream output specificity in the cell. we are currently employing crispr/cas9mediated gene editing in human induced pluripotent stem cells to generate isogenic disease models of three such activating pik3ca mutations. these cells will be used for signalome profiling by reverse-phase protein arrays (rppa) to compare and contrast mutant-dependent alterations to candidate signalling networks. in parallel, ongoing efforts focus on developing an endogenously expressed optogenetic p110a, allowing precise spatiotemporal control over pi3k signaling to unravel the extent to which pi3kdependent phenotypes are determined by strength of activation and/or dynamic encoding. ultimately, the outcome of this research will yield novel insight into fundamental aspects of pi3k signalling and potentially aid the development of targeted therapies for human diseases of pi3k hyperactivation. e. gov, n. kaya, k. y. arga cancer stem cells (csc) have been proposed to be the cancer initiating cells. because of their highly tumorigenic and drug resistant properties, cscs offer significant potential for developing novel anticancer drugs and therapeutic strategies. in the present study we analysed eight gene expression datasets for breast, ovarian, lung cancer and glioblastoma by comparing gene expression levels between stem cells and tumor cells and integrating them with genome scale biological networks. consequently, mutual molecular signatures (i.e: differential expressed genes, transcription factor, mirna) and biological characteristics were determined via integrative analyses, which might be feasible to uncover the mutual biological mechanism insights behind the cscs. it was identified twenty mutual differential expressed genes in four cancer types; jun and klf6 as transcription factors, egfr and cdk14 as receptors come into prominence as mutual signatures. molecules and pathways that were related to mapk, wnt, p53 signaling and pathways in cancer were the common indicators in csc types. our results provided similarities in gene expression profiles of various cscs and gave clues about the seed of tumorigenesis. this study proposed signatures and pathways that could be considered as effective therapeutic approaches in further experimental and clinical applications to eliminate subpopulation of csc. colorectal cancer (crc) is one of the leading causes of mortality worldwide. metastasis is associated with the presence of circulating tumor cells (ctcs) in the peripheral blood of cancer patients. ctc cut-off values have been shown to predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. in our study, ctcs were detected in blood samples of colorectal cancer patients, using with our modified convenient method for the strategies of ctc enrichment and detection. 7.5 ml peripheral blood samples were firstly collected and peripheral blood mononuclear cells (pbmcs) were isolated from the fresh blood samples by ficoll gradient separation. next, the leukocytes in pbmcs were removed by magnetic microbeads conjugated with cd45 for a negative selection. finally, the retained cells were labeled with anti-epithelial cell adhesion molecule (anti-epcam), cytokeratins (ck8, ck19) and the leukocyte-specific marker as anti-cd45. all samples were analyzed by bd facs aria iii flow cytometry. in total, 10 patients and 7 healthy people were included in this study. the results showed that ctcs were not detected in the blood samples of healhty volunteers, but 3-13 ctcs were detected with ck14, 15, 16, 19-based gating strategy in the blood samples of colorectal cancer patients. it is accepted that the cut off value is 3 ctcs for colorectal cancer and ctc is negative if it is below this value or ctc is considered as a positive, if it is equal to or above this value, which might be an indication for poor prognosis. thus ctc's detection may serve a representative surrogate tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. cells were grown in culture flasks in a humidified incubator at 37°c with 5% co 2 and were used at the proliferation and confluent stages. cultured cells were exposed to the pemf and prfe. the proliferations of the cells are measured by mtt assay for the effect of emf on the cancer cells. on the other hand the wound healing was investigated by closure of the wound by the cell proliferation with cell morphology using inverted microscope images. the proliferation decreased significantly by the effect of pemf on the semi confluent mcf-7 and mda-mb-231 cells. this effect was observed more prominent on mcf-7. considering prfe therapy this effect is much more pronounced especially for mda-mb-231 comparing with pemf. the phase contrast observations of these results were consistent with mtt analyses. similarly, this effect was seen less for pemf but the proliferation was more suppressed with prfe on the wound models. it was considered that the emf applications could be effective in cancer cells, but this effect has not been studied how it occurs in invasive cancers. in our cell culture study, the appropriate emf applications were found to be effective though the inhibition of proliferation of cancer cells even in invasive cancer but with lower effect. this means that emf applications may support the existing treatment methods of cancer patients and even people who suffer from invasive cancer. metastasis is the one of the most known causes of death in patients diagnosed with cancer. circulating tumor cells (ctcs) are shed from primary tumors and circulating in the bloodstream, and thought to play a key role in metastasis. a hypothesis that ctcs may contribute to metastasis was first introduced in the mid 19th century by thomas ashworth, an austrilian pathologist. in today's research, identification and molecular characterization of ctcs are thought to be a novel target for treatment of cancer and a key factor to understand the metastatic process. existing methods of ctc capture based on the cell search system, flow cytometers, laser scanning cytometers instruments, fiber-optic array scanning technology (fast), isolation by size of epithelial tumor cells (iset), and definition fluorescence scanning microscopy. ctcs are increasingly considered as a 'liquid biopsy' and when liquid biopsy is compared to tumor tissue biopsy, liquid biopsy for ctcs detection can be carried out routinely in patients due to accessibility and ease of blood collection. also, primary tumor sampling may not reflect the actual metastatic conditions, ctcs are thought to be a novel tumor biomarker for real-time monitoring of disease status and tailoring personalized therapy. with futher works, ctcs may be used as liquid biopsies and it might provide better understanding metastatic process, new approaches in cancer diagnostics and treatment. mesenchymal stem cells (mscs) are distributed all over the organism as a source of tissue formation and regeneration. glucose is vital for the proliferation and differentiation of mscs. glucose uptake is mediated by specific glucose transporters of two families, the na-coupled glucose transporters (sglt) and glucose transporter facilitators (glut). the presence and function of glut proteins in human placental amnion derived mscs (hamscs) is unknown. we aimed to investigate the presence of glut1, glut3, glut4 proteins and genes in hamscs isolated from term placentas. mscs were isolated from human term placenta amniotic membrane, the characterization of cells were provided by flow cytometry. mscs were used to assess their chondrogenic, osteogenic and adipogenic differentiation potential. the expression of glut1, glut3 and glut4 proteins was detected in hamscs by immunofluorescence. glut1, glut3, glut4 protein and gene expression in these cells were investigated by western blot and real-time pcr, respectively. flow cytometry analysis results of isolated cells showed that they were positive for cd44, cd90, cd73, cd105 (mesenchymal stem cell markers) and hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr were negative. the presence of glut1, glut3, glut4 proteins and genes were identified in hamscs. in this study, for the first time in literature, glut1, glut3 and glut4 gene and protein presence was determined in hamscs. therefore, gluts could mediate glucose transport in human amniotic membrane mscs. proliferation and differentiation of mscs in vitro are still not optimized. further studies are required to clarify the complex mechanisms regulating the relationship between glucose and mesenchymal stem cells. disclosure of this relationship may provide a better understanding of glucose-related pathologies such as diabetes. tumors have hierarchically organized heterogeneous cell populations and a small subpopulation of cells, termed cancer stem cells (cscs), is responsible for tumor initiation, maintenance as well as drug resistance. therefore, killing the cscs along with the other cancer cells is gaining an importance. in the present study, it was aimed to evaluate the cytotoxic and apoptotic activity of a novel platin (pt) (ii) complex [pt(hepy) 2 cl 2 ] on mammospheres obtained from mcf-7 human breast cancer line. elevated expression of stemness markers were determined by western blotting. cytotoxicity was assessed using the atp viability assay. effect of the pt (ii) complex on the formation and development of mammospheres was analyzed with sphere formation (sfa) assay. apoptosis was determined via cytofluorimetric analysis (caspase 3/7 activity, annexin-v-fitc and bcl-2 activity) as well as gene expression analysis. cytotoxicity was confirmed with the atp viability assay after the treatment with zvad-fmk (an apoptosis inhibitor) and necrostatin (a necroptosis inhibitor). in addition, alterations in mitochondrial membrane potential were evaluated by jc-1 staining. mammospheres exhibited increased oct-4 and sox2 (stemness markers) expressions compared to parental mcf-7 cells. cytotoxicity by pt (ii) complex was evident in a dose-dependent fashion (1.56-100 lm) . pt (ii) complex significantly prevented mammosphere formation and disrupted mammosphere structure in a dose-dependent manner. pt (ii)-induced apoptosis was determined based on the presence of caspase 3/7 activity, annexin-v-fitc positivity and bcl-2 inactivation. apoptosis was also confirmed with increased tnfrsf10a and hrk gene expressions. in addition to apoptosis, necroptosis was also present as evidenced with increased mlkl expression. mitochondrial membrane was depolarized. in conclusion, the pt (ii) complex seems to be a powerful apoptosis-inducing compound on cancer stem cells, thereby warrants further in vivo experiments. cancer is a disease which arises from destruction of growth and proliferation mechanisms in cells and is the second leading cause of death worldwide [1] . in the development of primary cancers, the head and neck cancer is accounting for approximately 550.000 new cases annually around the world [2] . laryngeal cancer is a type of head and neck cancer in which malignant cells arise from the mucosal tissues of the larynx [3] . cancer might spread from primary tumor by getting into the lymph and blood vessel system and forms secondary tumor. greater than 90% of deaths in cancer patients are attributed to metastasis [4] . circulating tumor cells (ctc's) provide an opportunity to understand the metastatic process of cancer patients. identification and molecular characterization of ctc's in the peripheral blood of cancer patients is a promising research area in the field of biomarker development and novel treatment targeting in today's cancer research [5] . the detection of ctc methods include cell search system, flow cytometry, high-definition fluorescence scanning microscopy, fiber-optic array scanning technology, isolation by size of epithelial tumor cells, and laser scanning cytometers [6] . in our study, 7.5 ml of peripheral blood samples were collected from larynx cancer patients and healthy volunteers and the samples were analyzed by bd facs aria iii flow cytometry via biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection [7] . according to the results of our study; ctcs were detected in larynx cancer patients by our newly modified method whereas there was no ctc's detection in the samples of controls. thus, this study may provide us monitoring of the treatment process of larynx cancer and this method might be used as diagnostic, prognostic, and predictive biomarkers in cancer therapy as a liquid biopsy. prostate cancer is the second most common cancer and the fifth leading cause of death from cancer in men 1 . circulating tumor cells (ctcs) present in the peripheral circulation of cancer patients with different solid malignancies including prostate cancer and have a potential as a liquid biopsy to monitor disease progression and response to therapies at cell and molecular level 2 . one of the general methods in ctc detection is flow cytometry 3 . radical prostatectomy is the most frequently applied procedure in the surgical management of localized prostate cancer. in this surgical operation, the surgeon removes the entire prostate gland with the seminal vesicles. a radical prostatectomy procedure can be done using the da vinci robotic system (intuitive surgical, sunnyvale, ca, usa) 4 . robotic surgery has been suggested to have fewer complications, lower risk of infections and shorter recovery period following robotic radical prostatectomy 5,6 . in this study, our aim was to detect ctcs before and after robotic radical prostatectomy in clinical localized prostate cancer patients. the ctc detection study was performed with our modified method in which 7.5 ml of peripheral blood samples were collected from each prostate cancer patient and healthy individual; the samples, using with biomarkers epcam, ck19, ck8 for positive selection and cd45 for negative selection, were analyzed by bd facs aria iii flow cytometry 7 . according to our results, we detected ctcs in the peripheral blood samples of prostate cancer patients before robotic radical prostatectomy. however, following this surgical procedure no ctc or decreased number of ctss was detected. our study might contribute to understand disease progression after robotic radical prostatectomy in clinically localized prostate cancer patients that warrants further research. keywords: circulating tumor cells, prostate cancer, flow cytometry, robotic radical prostatectomy. p-05.03.3-017 determination of effect cytotoxic, apoptotic, caspace-3 activity and mrna expression levels of apoptototic related genes of vulpinic acid on breast cancer cell lines n. kilic ß, s. aras, d. cansaran-duman ankara university, ankara, turkey breast cancer is the most common cancer types in women. several drugs used to treat breast cancer patients are developing resistance to the treatment for this reason success rate falls. therefore the discovery of alternative therapeutic agent and molecular detection of anticarcinogen effect because of treatment for cancer patients may be a source of hope for the contributions. in this study, different concentrations (1.562, 3.125, 6.25, 12.5, 25, 50 , 100 lm) vulpinic acid (va) lichen seconder metabolite was determined to cytotoxic, apoptotic effect and caspase-3 activity in breast cancer cells (mda-mb-231, mcf7, bt-474, sk-br3) and normal cell (mcf12a). in addition to the quantitative real-time pcr (qrt-pcr) using apoptose specific primers (tp-53, bcl-2, bax, birc-5, gapdh, caspase-3, caspase-7, caspase-8, caspase-9) and sybr green dye were performed to determine expression patterns of transcript level in cancer cell lines, using gapdh as a reference gene. the antiproliferative characterization of va effects identification of the gene set at molecular level and we determination role of va on apoptotic pathway. according to our study, va is demonstrated significantly (p < 0.05) effect cytotoxic, apoptotic, caspase-3 activity. beside this, dose dependent expression patterns decreased apoptose spesific genes (except of bcl-2) mrna levels from six to eleven fold change more than untreated va cell lines. va will be used as candidate molecule for effective treatment on breast cancer in the future. glycosylation largely determines the variety and functions of proteins. paucimannose, a mannosidic n-glycoepitope has long been thought to be specific for plants and invertebrates. recently, it has also been detected in mammalsin physiological conditions (stem cells) and in pathophysiological conditions (inflammation and cancer). in glioblastoma cells, paucimannose also seems to play a role in cell proliferation. glioblastoma is the most frequent brain tumor in adults with poor prognosis due to a lack of suitable treatments. we hypothesize that paucimannose could be a promising new biomarker as it is otherwise rarely found in mammals. therefore, paucimannose levels were investigated in different glioblastoma cell lines differing in their proliferation rate and tumorigenicity. the highest paucimannose levels were detected in low proliferating, nontumorigenic cells. furthermore, we found that modulation of paucimannose function by application of a specific antibody regulated cell proliferation and the capability of cells to form colonies in soft agar. these data support a functional role of paucimannosidic epitopes in tumorigenic processes. glioblastoma multiforme (gbm) is the most lethal type of malignant brain tumors. recently, gbm stem cells (gscs) have been studied in great deal and accepted that they have a legitimate role in tumor formation, development, chemo-resistance and recurrence. in this study, it is aimed to investigate new therapeutic targets within apoptosis related molecules to select and eliminate cd133+ gscs effectively. ten primary gbm cells were isolated from gbm tissue samples and they were cultured among with the 4 gbm cell lines (u87, u138, u118 and t98). cd133+ and cd133à cells were seperated by macs method via anti-cd133 (ac133) antibody from cultured cells and cell lines. rna isolation from cd133+ and (à) cells, cdna synthesis was performed. finally, by performing pcr array, mrna expression levels of 88 genes were detected. proper results were collected and analysed statistically. according to the results of pcr array; it has been found that cd133+ cells express approximately 223 fold tnfrsf21 and 20 fold tnfsf7 when they are compared with control cells. tnfsf7 binds to cd27 that is expressed on the surface of tcells. cd27 does not have a death domain, instead it has a cytoplasmic tail which binds to trafs. trafs act as adaptor molecules that are related with jnk and nf-jb signalling pathways. tnfrsf21 (dr6) is a death receptor which are known for transmitting the pro-apoptotic signals from outside to the inside of the cell. it negatively regulates t-cell activation and the release of few cytokines. as a conclusion, tnfsf7 and tnfrsf21 both are found on immune system cells, mostly on t-cells, which may mean that gbm stem cells act as a immune system cells to avoid the elimination by the immune system. to conclude, acting as an immune system cell and promoting survival via tnfsf7 and tnfrsf21, these molecules may be essential markers to target cd133+ gbm stem cells. the effect of docetaxel on p53, sin3a and mdm2 gene expression in mcf-7 breast cell line docetaxel is a cytotoxin effective in treating breast cancer. it stabilizes microtubules and causes catastrophic cell cycle arrest in g2/m. it also initiate signaling through cell death pathways that result in programmed cell death. in this study, it was aimed to investigate apoptotic and cytotoxic effects of docetaxel has on the mcf-7 breast cancer cells line. in this study, mcf-7 breast cell line was applied different doses docetaxel (10 nm, 100 nm, 1 lm, 10 lm, 100 lm) as 24 h and 48 h. mtt analysis was performed to the mcf-7 breast cancer line in control group and groups of docetaxel. afterwards, evaluation of apoptosis by tunel and levels of p53, sin3a and mdm2 gene expression by real-time pcr were determined in an order. it was observed cell variable was significant lower in docetaxel groups compared to control group (p < 0.05) in 24 h as mtt analysis. the lowest cell viabilty was determinated in group applied 100 lm docetaxel. while the lowest positive cell density was determinated in control group, it was observed apoptotic cell density gradually increased with increasing docetaxel concentration in groups treated docetaxel (p < 0.05). the highest p53, si3a and mdm2 expressions were apperared in 100 nm docetaxel group compared to control group. human alpha-fetoprotein (afp) and afp receptor binding domain (afprbd) are able to bind and internalize effectively by wide range of human tumor cells and tissues. as other vector molecules afprbd has insufficient quantity of chemical groups which can be conjugated with drugs or diagnostic agents. conjugation of vector molecules with macromolecular polymer carriers like dendrimers aims to solve this problem. our study describes influence of afprbd-dendrimer-doxorubicin conjugate surface charge on intracellular trafficking routes and toxicity. the amineterminated (g2) and acetyl-terminated (g2 12 ) 2nd generation pamam dendrimers carrying doxorubicin (dox) were used to synthesize conjugates with afprbd. unmodified by afprbd g2 and g2 12 dendrimer derivates labeled with dox were absorbed by the cells at 37°c with different efficiency. g2 12 -dox derivate characterized much slower internalization rate than nonacetylated g2-dox. only g2 12 -dox shown partial colocalization with lysosomal marker lamp2 after 4 h of incubation. internalization of afprbd-g2-dox and afprbd-g2 12 -dox did not show significant difference. at the same time, both conjugates contained afprbd wyкy almost fully associated with lamp2 already after 30 min of incubation. cytotoxicity results revealed that ic50 levels of g2 12 -dox and afprbd-g2 12 -dox coincided and demonstrated a bit higher activity against sensitive to dox skov3 and resistant skvlb cells than afprbd-g2-dox conjugate after 72 h of incubation. at the same time, after 1 h of incubation afprbd-g2-dox and afprbd-g2 12 -dox were much more than g2 12 -dox and g2-dox. we may conclude that there is significant difference in ways of dendrimers internalization by tumor cells depending on nature of surface chemical groups. on the other hand, chemical modification of dendrimer conjugated with does not afprbd influence dramatically on the protein trafficking and resulting cytotoxic effect. russian scientific foundation supported this study (no. 15-15-10013 1.40) , a key enzyme in glycolysis, catalyzes conversion of phosphoenolpyruvate (pep) into pyruvate with regeneration of adenosine triphosphate (atp). the key regulator of the metabolic alterations found in tumor cells is the glycolytic isoenzyme pyruvate kinase type m2 that is generally expressed in all proliferating cells and overexpressed in all tumor cells investigated to date. during carcinogenesis a shift in the pyruvate kinase isoenzyme equipment always takes place, such that the tissue-specific isoenzymes disappear, and m2-pk is expressed. breast carcinoma, the third most common cancer worldwide, accounts for the highest morbidity and mortality. breast cancer tissue analysis confirmed the upregulation of m2-pk in breast cancer, and high m2-pk levels were associated with poor prognosis of breast cancer patients. materials and methods: poly hema (mac) nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. pyruvate immobilization was actualysed with cross linking reagent glutaraldehyde. biosensor was developed by preparing pottasiumferrociyanide, selected as a mediator. results: cyclic voltammograms have been carried out at between~0.4 and 0.6 v potentials vs. ag/agci. m2-pk activty was detected by using differential pulse method at between 0.3 and à0.25 v potentials by observing the differentiations in the current values. in the optimization studies, some parameters such as optimum ph, temperature, concentration of glutaraldehyde and p-hema-mac, were investigated. discussion and conclusion: the method developed for the measurement of the tumor m2-pk activity by using biosensor. we found that more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. piruvat kinase tumor m2-pk activity determination at low concentrations is possible with this method. p-05.03.3-023 tie2/tek: a potential biomarker for targeting glioblastoma stem cells role in angiogenesis, endothelial cell survival, proliferation, migration and adhesion. therefore, tie2/tek could be a potential target for therapeutic strategies directed against glioblastoma stem cells and their microenvironment. in this study, we investigated the gene expression levels of tie2/tek in both cd133+ gscs and cd133à gbm cells. gbm primary cells were freshly isolated from glioblastoma tissue samples and cultured in dmem supplemented with 10% fetal calf serum and 1% penicillin-streptomycin at 37°c in 5% co 2 -humidified incubator. we isolated cd133+ and cd133à cells from 10 gbm primary cells using macs system. following rna isolation from healthy brain tissues, cd133à and cd133+ cells, cdna synthesis was performed. finally, according to microarray protocol, cell surface marker panel array was applied. expression levels were analyzed using the delta delta ct method. statistical analysis was performed using spss software for windows version 13.0. tie2/tek gene expression was determined as 50.07 fold higher in cd133+ gscs than normal brain tissue (p < 0.05). morever it was determined 7.52 fold higher compared to normal brain tissue in cd133à (p < 0.05). according to our results tie2/tek expression was higher in gscs, indicating that tie2/ tek may be a potential marker for targeting cancer stem cells in gbm. this research has been supported by the scientific and technological research council of turkey (no:114s189). adenosine inhibited the breast cancer stemlike cell population through erk1/2 pathway s. m. jafari, m. aghaie cancer stem cells (cscs) are immortal tumor-initiating cells that can self-renew and drive tumorigenesis in various cancers, including breast cancer and others solid cancers. in a study indicated that extracellular atp reduces tumor sphere growth and cancer stem cell population. but at present, there are no reports available in literature on the effect of adenosine on breast cancer stem cells. in this study we evaluated the effect of adenosine inhibition and its mechanism of action in breast cancer stem cells isolated from breast cancer cell lines. our result showed that adenosine significant reduces breast cancer stem cell population. reduction of erk1/2 protein levels was also observed after treatment cancer cells with adenosine. in conclusion, our results indicate that adenosine decreases the breast cancer stem-like cell population through erk1/2 pathway. taxanes are commonly used for the treatment of many cancers as chemotherapeutic drugs that resistance to these agents has become a major clinical obstacle. taxane based chemotherapy drugs such as paclitaxel, docetaxel and cabazitaxel bind microtubules and inhibit to microtubule polymerization appear to stimulate programmed cell death. taxane-resistance to cancer has not been clearly in progression and development of drug resistance. multiple mechanisms are involved in the drug efflux proteins as multidrug resistance protein, differences in amino acid sequences among the b-tubulin isotypes. we investigated taxane resistance with different doses of paclitaxel, docetaxel and cabazitaxel in prostate cancer stem cells. we compared the expression level of apoptotic proteins, and its functional role in resistance mechanisms in cd44 + /cd133 + prostate cancer cell lines. taxane drugs were categorized as concentration-dependent or time-dependent. cabazitaxel caused a time-dependent and dose-dependent reduction in cell viability in all tested cell lines. resistance activity was consistently higher in docetaxel in prostate cancer cells compared with the other drugs. there are many different response of clonogenic formation cd44 + /cd133 + cells with resistance to docetaxel, paclitaxel and cabazitaxel in prostate cancer stem cells. the innate of prostate cancer resistances are important characterization steps and critically limits treatment outcomes therefore novel drugs must be focus on antiresistance and molecular based combinations. mesenchymal stem cells (mscs) are self-renewing cells with ability to differentiate into organized, functional network of cells. mscs isolated from various tissues including adipose tissues, bone marrow, umbilical cord, placenta and pancreas have different differentiation and proliferation potential. good knowledge of the metabolism and proliferation mechanisms of stem cells is required for stem cell therapies. glucose is an important molecule in the culture of stem cells. glucose concentration affects the differentiation and proliferation potential of stem cells. the aim of the study was to investigate the proliferation status by identifying the proliferating cell nuclear antigen (pcna) expression under normoglycemic and hyperglycemic conditions in mscs. mscs were isolated from human term placenta amniotic membrane. characterization of the isolated cells was performed using flow cytometry. chondrogenic, osteogenic and adipogenic differentiation potential of these cells were investigated. characterized cells were cultured in normoglycemic and hyperglycemic conditions for 24 and 48 h and the expression of pcna protein expression in these cells were investigated by western blot. flow cytometry analysis showed that isolated cells were positive with mesenchymal stem cell markers cd44, cd90, cd73, cd105 and negative with hematopoietic markers cd34, cd11b, cd19, cd44 and hla-dr. western blot result of pcna protein expression statistically significantly increased in human amniotic membrane mscs under hyperglycemic conditions for 24 and 48 h culture. the glucose content of stem cell medium is important because glucose is an effective molecule of the proliferation of stem cells. proliferation of mscs in vitro are still not optimized. when the relationship between glucose and stem cells be understood, it will provide a better understanding for the glucose-related pathologies such as diabetes during pregnancy. prostate cancer (pca) is the second most common type of cancer among men in the world. it is revealed that some gene, protein and metabolite sets control the pca, however the whole metabolomics changes are not completely understood yet. pca is common among older men, and this is an important health problem in developed countries. sarcosine is the n-methyl derivative of the glycine amino acid. glycine n-methyl transferase produces sarcosine from glycine. besides, it is metabolized to glycine by sarcosine dehydrogenase. in 2009, high level of sarcosine in urine was associated with pca by sreekumar et al. they identified sarcosine as a pca biomarker that was significantly increased in urine during prostate cancer progression to metastasis. following this study, several studies have been published indicating sarcosine as a pca biomarker. in our study, a preliminary biosensor system was fabricated for determination of sarcosine in urine by using sarcosine oxidase. sarcosine oxidase was immobilized on au electrode surface using gelatin as an immobilization matrix. glutaraldehyde was used as a cross-linking agent to avoid the loss of the enzymegelatin mixture. optimization and characterization studies were carried out. sarcosine concentrations were detected carefully with the developed biosensor system. the fabricated preliminary biosensor is a promising system that can allow lower detection limits after surface modifications. activation of the epithelial-mesenchymal transition (emt) program in tumor cells is associated with invasiveness and stemness. recent studies implicate emt-inducing molecules in reprogramming energy metabolism. the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3 (pfkfb3) regulates glycolysis by producing fructose 2,6-bisphosphate (f2,6bp). given that pfkfb3 is induced by several established emt-inducers in tumor cells, e.g. hif-1a and ras, we hypothesized that pfkfb3 may be involved in regulation of the emt in tumor cells. silencing of pfkfb3 in pancreatic adenocarcinoma cell lines panc1 and s2vp10 was achieved using specific sirna molecules. mrna and protein expressions of the cdh1 gene (encoding e-cadherin, an established epithelial marker), as well as zeb1 and snai1 genes, by real-time quantitative (q)-pcr and western blot, respectively. immunfluorescence analysis was performed to visualize e-cadherin protein expression on plasma membrane. in order to test the effect of pfkfb3 on the invasive ability of the cells, a matrigel invasion assay was performed. ectopic expression of zeb1 was achived by transfecting cells with a plasmid carrying zeb1 cdna. cells that were depleted of pfkfb3 exhibited markedly increased cdh1 mrna and e-cadherin protein expressions and reduced snai1 and zeb1 mrna expressions. immunfluorescence analysis confirmed the upregulation of the e-cadherin protein on plasma membrane. silencing of pfkfb3 caused approximately 40% reduction in matrigel invasion, compared to non-targeting sirna. inhibition of the matrigel invasion caused by pfkfb3 depletion does not appear to be associated with reduced zeb1 expression, as ectopic expression of zeb1 did not reverse the effect of pfkfb3 silencing on invasion. taken together, these data suggest that pfkfb3 may be required for the maintenance of the mesenchymal phenotype and associated traits in pancreatic adenocarcinoma cell lines. introduction: leukemias are neoplasms that arise from hematopoietic cells initially proliferate in the bone marrow, and then disseminated in the peripheral blood, spleen, lymph nodes and eventually to other tissues. lymphomas occur primarily in the lymph nodes, but can be extended in peripheral blood and bone marrow infiltrate. aim: to determine the values of haematological parameters the control and test groups. to determine the prevalence of types of chronic leukemia in relation to the experimental group. compare haematological parameters in relation to the type of chronic leukemia. materials and methods: a prospective-retrospective study included subjects who were made laboratory hematology in oj clinical chemistry and biochemistry ukcs. blood tests conducted on the hematology analyzer siemens advia 2120 hematology system and abbott cell dyn 3700 and microscopic analysis of the peripheral blood smear. results and discussion: according to the age of respondents test group was established mild form of anemia, a red blood cell count is totaled 3.69 ae 1.13x10 12 , which is signifycantly lower compared to the control group. the average number of leukocytes was significantly higher in subjects studied groups and amounted to 136.91x10 9 , with a maximum value of 580 x10 9 . in the peripheral blood of patients with chronic leucosis has established a significantly higher number of cells compared to the control group (p = 0.001), while the number of monocytes was a significantly smaller. in the group of patients with chronic leukosis largest number had chronic lymphocytic leukemia (70%), and chronic myeloid leukemia had 30% of respondents. conclusion: subjects with cll were statistically older than patients with cml, and as regards the gender structure, men have dominated in cll and cml in women. white bloodline was found that the number of leukocytes in both forms of chronic leukemia high above the reference value. p-05.03.3-032 effect of enzymatic and non-enzymatic isolation methods of endometrial stem cells on their cell proliferative potential and mesenchymal stem cell characteristics human endometrial stem cells (hescs) are responsible for the monthly renewal of the basal layer of the human endometrium by facilitating stromal and vascular regeneration. in this study, hescs were isolated with three different isolation methods including non-enzymatic and enzymatic digestion using trypsin and collagenase type 1. the effect of these three isolation methods on the acquisition of mesenchymal stem cells (msc) and on hesc proliferative potential was evaluated through flow cytometric analysis of cd surface markers and wst-1 tetrazolium salt assay. our findings indicate that hescs isolated with these three methods have statistically similar cell proliferation rate at 24 h time point. however, at 48 h time point, hescs isolated with the non-enzymatic and collagenase type 1 method displayed a higher expansion in cell number when compared to the hescs isolated with trypsin. the late passage of hescs isolated with non-enzymatic and trypsin methods showed the highest proliferation rate in comparison to the hescs obtained via collagenase type 1 isolation method at 24 h, 48 h and 72 h. the three isolation methods for the early passages of hescs had a resemblance in their msc profile with no significant difference. on the other hand, late passage hescs isolated using trypsin non-enzymatic method showed a higher cd31and lower cd44 profile. moreover, late passage of hescs isolated with non-enzymatic method displayed a significant reduction in their cell surface cd90, cd73, and cd105 surface expression levels. only hescs isolated with collagenase type 1 did not present a significant shift in their mesenchymal cd marker profile from early to late passages, taken together results from this study suggest that the longterm maintenance of mesenchymal markers can only be achieved in cell isolation with collagenase type 1, while non-enzymatic method is more suitable to obtain higher msc cell yield for immediate use. hepatocellular carcinoma (hcc) abundantly arises on the viral and/or chemical-induced cirrhosis in liver. cirrhosis is defined as one of the premalignant stage hcc in which microenvironmental changes occurred such as uncontrolled production of collagen type i and activation of hepatocyte growth factor (hgf)/c-met signaling. it has been shown that epcam+/cd133+ subpopulation of cells isolated from hcc tissue can initiate tumor at very low concentration in xenograft model and behaves as hepatic cancer stem cells. however, the molecular mechanisms supporting hepatic stem cell activation are not well understood and knowledge about the role of hgf/c-met pathway in this process is not clear. in this study, we aimed to define effect of collagen type i and hgf induction on the cell behaviours of epcam+/ cd133. epcam+/cd133+ cells were sorted by magnetic separation from huh-7 cells. then proliferation and invasion of cells were analyzed under the hgf induction as well as branching morphogenesis in vitro. after hgf stimulation, phosphorylation level of c-met increased in epcam+/cd133+ subpopulation. moreover, presence of collagen type i enhanced significantly effect of hgf stimulation in the invasion of epcam+/cd133+ cells. we also have showed that hgf stimulation increased branching tubulogenesis capacity of epcam+/cd133+ subpopulation while it did not effect proliferation of cells. these effects of hgf reverted by c-met inhibitor, su11274, in vitro. all these findings showed that hgf and collagen type i regulates aggressive phenotype as microenvironmental changes via induction of invasiveness of epcam+/cd133+ subpopulation of huh-7. in conclusion, we showed that hgf/c-met signaling causes to get more metastatic phenotype based on invasion and tubulogenesis in epcam+/cd133+ hepatic cancer stem cells in hcc and it might be possible to use c-met inhibitors to target hepatic cancer stem cells during hepatocarcinogenesis. endometriosis is defined by the migration of endometrial mesenchymal stem cells (emscs) into the peritoneal cavity or other site of body rather than uterus in a retrograde fashion. its previously known intracellular crosslinking enzyme called tissue transglutaminase (tg2) was shown to play important roles in the extracellular matrix (ecm) modelling, fibrosis, cell adhesion and migration. we have hypothesized that tg2 might be expressed in emscs and take part in the formation of endometriosis. the difference in the proliferation capacity of emsc isolated from endometrial tissue with/without endometriosis was determined using wst-1 assay and tg2 activity and expression levels were analysed by btc assay and rt-pcr. the biosynthesis and activity for mmp-2 and -9 were investigated with zymography and rt-pcr, respectively. although tg2 activity was found to be 50% less in emscs isolated from endometriotic tissue, these cells showed 9 times higher tg2 protein expression than those isolated from the control tissue without endometriosis. emscs from endometriotic tissue have 2.3 times higher tg2 and 10.3 fold higher itgb1 mrna levels when compared to the cells of healthy group. similar results were observed in sdc-4 gene expression with a 2.5fold increase. endometriotic emscs demonstrated an average of 11.5-fold increase in the mmp-2 activity while a onefold increase was evident in mmp-9 activity when compared to the healthy emscs. emscs from patient group possessed a higher proliferative ability in comparison to that of healthy subjects within 96 h. the fact that emscs from the control tissue showed lower tg2 protein levels with a high enzyme activity suggested that tg2 might be important in the development of endometriosis not only by destabilizing ecm but also enhancing the cell migration. in this context, the upregulation of tg2 along with itgb1 and sdc4 was evident in emscs of endometriosis which was possibly associated with the increase in the activity of mmp-2 and -9. recent studies have indicated that pluripotent stem cells and some stromal stem cells such as mesenchymal stem cells (msc) are metabolically different from their differentiated counterparts. in this study, the cellular mechanisms controlling metabolic changes in stem cells was investigated using wharton jelly mesenchymal/stromal stem cells (wj-mssc). wj-msscs were isolated by the explant method and cultured in dmem-f12 with 10% fbs. endothelial differentiation was induced by the addition of vegf, egf, insulin and hydrocortisone for 6 days. neuronal differentiation was achieved by using commercial neuronal differentiation medium (millipore) for 3 days. in parallel experiments, cellular metabolic activity such as lactate production was measured. the msc characterization was performed by flow cytometry using antibodies against cd90, cd105, cd73 and cd44 (bd human msc analysis kit). the differentiation process was followed by measuring the expression of cd31, cd34 for endothelial and gfap, neu and tyrozine hydroxylase proteins for neuronal cells by immunofluorescence. for gene expression, nanog, cd34 and gapdh genes were analyzed by rt-pcr. differentiation stimuli to endothelial or neuronal cells resulted in a significant decrease in msc marker proteins. expression of stem cell markers other than cd73 were decreased to 2-20%. differentiation induced the expression of cd31, cd34 for endothelial and gfap and neu proteins for neuronal cells. in vitro lactate production was decreased following differentiation in both lineages. neuronal differentiation increased glucose consumption by~48% and the extracellular calcium concentration of these cells was significantly lower than synchronous undifferentiated cells. glycolytic activity is decreased during in vitro differentiation of wj-msscs. metabolic reprogramming and glucose uptake of cells may be an early indicator of the differentiation process in wj-msscs, supporting the view on their metabolic plasticity. store-operated ca 2+ entry (soce) activated by depletion of intracellular ca +2 stores has been shown to control intracellular ca 2+ homeostasis in many physiological and pathological events. stromal interactive protein, stim1, as endoplasmic reticulum (er) ca +2 sensor and orai protein as pore-forming subunit of soc channels play crucial roles in the activation of soce channels. stim1 and orai were reported to have pathophysiological roles especially in hepatocellular carcinoma (hcc). anticancer chemotherapy frequently falls back because of these tumor-initiating subpopulations, tentatively called 'cancer stem cells'. the purpose of this study was to investigate the roles of stim1 and orai on soce in differentiation of huh-7 hccs expressing epcam and cd133 surface adhesion molecules (epcam + cd133 + ). epcam + cd133 + subpopulations in huh-7 cells were separated via flow cytometry and transfected with stim1 and orai-1 over-expressing (oe) plasmids. expression levels were confirmed by rt-pcr. changes in intracellular ca 2+ concentration were monitored via dual wavelength spectrofluorimeter in fura 2-loaded cells. in epcam + cd133 + cells, er ca 2+ release increased without any change in soce compared to that of epcam à cd133 à cells. similar results were observed in stim1-oe epcam + cd133 + cells. on the other hand, increase in orai1 has no effect on either parameter. cancer is globally one of the most death causes. recently, huge improvements occurred in the cancer diagnosis and treatment due to advanced technology, however recurrence occurs almost 30-40% of patients and their survival times decreases. in this study, we aimed to investigate of relationship between the cancer stem cells which are strongly associated with chemotherapy and radiotherapy resistance and recurrence with the non-classical mhc i antigens which have immunosuppressive properties. for this purpose, we immunohistochemically evaluated the expression patterns of cd133, cd44, nanog, oct3/4, hla-g and hla-e in the advanced stage colorectal, gastric and breast cancer and also non malign biopsy samples. we detected that the cancer stem cell markers cd133, cd44, nanog and oct3/4 significantly increased in the advanced stage cancer tissues. however, the immunosuppressive hla-g and hla-e expressions increased only in the colorectal and gastric tumor tissue. in addition to the presence of cancer stem cell like cells in the tumor tissues, increased expressions of hla-g and hla-e may indicate an immune evasive adaptation of tumor cells. according to our findings, the hla-g and hla-e may be potential therapy targets to elimination of cancer stem cells of colorectal and gastric cancers. however, more detailed studies are needed to support our findings and also to determinate of clinical values of these markers. endocannabinoids increase sdf-1 release from human mesenchymal stem cells s. k€ ose 1 , f. aerts kaya 1 , d. uc ßkan c ß etinkaya 1 , p. korkusuz 2 1 stem cell research and application center, hacettepe university, ankara, turkey, 2 department of histology and embryology, hacettepe university, ankara, turkey lipid-structured endocannabinoids are endogenous morphine ligands and present widespread receptor-mediated effects at physiological and pathological levels on the nervous system as well as many other systems. these effects are partially realized through mechanisms affecting cell growth, differentiation, apoptosis and migration at the molecular level. the hematopoietic progenitor cells (hpcs) and mesenchymal stem cells (mscs) form a distinct niche in bone marrow where they interact with each other in harmony. the stromal cell-derived factor 1 (sdf-1/cxcl12) is a chemotactic factor in bone marrow and is released from mscs and their receptor cxcr4 is found in hpcs. with these rationale in mind, we asked if hpcs and msc interaction mediates sdf-1 release via endocannabinoidal system. bone marrow mscs obtained from healthy donors and passage 3 mscs were induced with 200 ng/ml lipopolysaccaride (lps) for 4 h. antagonists for cb1 (am281) and cb2 (am630) receptors were added to cultures for 4 days. after incubation with antagonists msc culture supernatants collected and processed with human sdf-1 beta in elisa medium. analyses demonstrated direct decreasing effect of endocannabinoid receptor antagonists on sdf-1 beta release from bone marrow mscs. in conclusion, endocannabinoidal system regulates sdf-1 release on mscs and directly act on hpcs mobilization in bone marrow microenvironment (niche). this may have a clinical implication on therapeutic mobilization strategies for hscs in hematology clinical applications. implantation is invasion of the embryo into the endometrium and occurs in three stages apposition, adhesion and invasion, via the complex cellular and molecular mechanisms. during these stages, both of maternal endometrium and embryo should be appropriate for the implantation which is the beginning of pregnancy. receptivity of uterine consists in the existence of growth factors such as tgfbeta-1, igfr1, vegf. it is indicated that damages of factors relesead from endometrium and blastocyst prevent implantation. recently, stem cells can be obtained from many sources to use for therapeutic purposes and mesenchymal stem cells derived from bone marrow are the most studied. in our study, it was aimed to investigate molecules play a role in blastocyst implantation after bone marrow derived mesenchymal stem cell application into the rat endometriyum. female rats were divided into three groups which were saline (sf, n:7), media (m, n:7), stem cell in media (m+bmsc, n:7). after vaginal smear technique, female rats in estrous cylcle were injected into the uterine and periton 200 ll saline, 200 ll culture media and 1 9 10 6 cell/200 ll culture media. the pregnant female rats on the 7 day were sacrified and uterine samples removed and were stained with heamatoxylin-eosin histochemically and anti-tgfbeta-1, anti-igfr1, anti-vegf and anti-pcna immunohistochemically and obseved under light microscope. h-score results were determined using one-way anova test statistically. it was found that intraperitoneal administration of stem cells with media, was increased tgfbeta-1, igfr1, vegf and pcna parameters when compared with the intrauterine administration of stem cells. in this study, it was revealed that distribution of molecules play role in implantation were changed due to stem cell application. it is supposed that stem cell treatments can be cured the molecules caused infertility. many unconventional biochemical factors remain to be investigated for their potential effects on stem cells. among others, endogenous gasotransmitter h 2 s, generated from l-cysteine and organosulfur-compounds (oscs) metabolisms, plays very important roles in the central nervous, respiratory and cardiovascular system. slow-releasing h 2 s donors are viewed as powerful tools for basic studies and innovative pharmaco-therapeutic agents for cardiovascular and neurodegenerative diseases. exogenous h 2 s administration is able to promptly scavenge ros, activate myocardial k atp channels and increase pro-cell survival signaling, very likely activating erk and phosphatidylinositol 3-kinase (pi3k)/akt pathways. the effects of h 2 s-releasing agents on the growth of stem cells are not yet widely investigated. therefore, stem cell therapy combined with h 2 s may have great clinical relevance in cell-based therapy for regenerative medicine. the effects of slow-releasing h 2 s agents on the in vitro cell growth and differentiation of human lin à sca1 + cardiac progenitor cells (hcpc) were here studied. in particular, the effects of h 2 s-releasing agents, such as na 2 s, gyy4137 and water-soluble gsh-garlic conjugates (gsgaws), on the cell viability and differentiation of hcpc were here investigated by colorimetric assay, immune-fluorescence microscopy and western-blotting analysis. the treatment with slow-releasing h 2 s donors increased the cell proliferation in a concentration dependent manner respect to the control. moreover, the treatment with gsgaws led to an up-regulation of the expression of proteins involved in the cell adhesion and differentiation processes. these preliminary results highlight on the effects of this gasotransmitter on the stem/progenitor cells and on the possibility to develop functional 3d-systems for cardiac tissue repair, that take into account the relevant biological role of h 2 s in the cardiovascular system. p-06.02.5-002 investigation of the protective effect of boric acid and omega-3 fatty acid in model of acute myocardial infarction changes in myocardial rats ischemic heart disease being the most common cause of the mortality and morbidity in worldwide commonly results from the occlusion or narrowing of the coronary arteries by atheromatous plaque and thus is named as coronary artery disease. male sprague dawley rats were used in the present study. rats were divided into 5 groups with 10 rats in each: control, mi, mi+boric acid, mi+omega-3 and mi+boric acid+omega-3 groups. control rats were treated with 2 ml/day saline, boric acid-treated rats received 100 mg/kg/day boric acid and omega-3-treated rats received 800 mg/kg/day for 28 days by oral gavage. for the experimental mi model, 200 mg/kg izoproterenol-hcl (iso) was administered subcutaneously two times with a 24-h interval in the last 2 days of the boric acid and/or omega-3 treatments. twelve hours after the second dose of iso, general anesthesia was induced. under general anesthesia and spontaneous respiration, ecg recordings were obtained by using a computerized data recording and analysis system (mp100, biopar) and d-ii recordings were used in the analysis. compared to the control group, serum ck-mb, bnp and tnf-a levels were higher in mi group (p < 0.001, p < 0.001 and p < 0.01 respectively). in the heart tissue homogenate, biochemically measured calpain activation and mda were increased (p < 0.01 and p < 0.001, respectively) and pon1 levels were decreased (p < 0.05). according to the ecg recordings, st wave and heart rate were found to be decreased (p < 0.001 and p < 0.001, respectively). on the other hand, all above mentioned parameters were found to be improved in rats treated with boric acid and/or omega-3 after induction of mi. moreover, histological analysis including light microscopy and tem revealed a significant histological improvement in rats treated with boric acid and/or omega-3 after induction of mi. results of the present study suggest that omega-3 and/or boric acid treatment significantly decreases the cellular damage in mi. this is study is aimed at measuring the level of serum heart-type fatty acid binding protein (h-fabp) in patients presenting with diabetic ketoacidosis (dka) and diabetic ketosis (dk) and to determine its role in identifying early period cardiac ischemia by comparing this level with the level of a control group at a comparable age this study was planned to be a prospective study and it included 35 patients diagnosed with dka, 20 patients diagnosed with dk and 20 voluntary pediatric and adolescent healthy control subjects. the h-fabp, creatine kinase-mb (ck-mb) and troponin-i levels were studied in patients with dka and dk as well as in the control group at the time of presentation. for dka patients, their h-fabp values were measured once again after acidosis correction and compared with the values they had at the time of presentation there were no differences among groups in terms of sex, age, height and weight. no statistically significant differences were found among groups with respect to troponin-i values (0.06 ae 0.08, 0.07 ae 0.04 0.04 ae 0.04; p = 0.457). no statistically significant differences were found among groups with respect to ck-mb values (1.48 ae 0.91, 1.55 ae 0.9, 2.09 ae 1.37; p = 0.229). the h-fabp values of dka patients at the time of presentation were found to be statistically significantly higher than those of dk patients and control group (1.17 ae 0.79; 0.79 ae 0.5 0.69 ae 0.36; p = 0.006). the h-fabp value of the dka group at the time of presentation was found to be statistically significantly higher than the value at hour 36 after acidosis correction (1.17 ae 0.79; 0.55 ae 0.28; p = 0.0001) the fact that h-fabp levels were found to be high in pediatric patients diagnosed with dka at the time of presentation suggested that myocardial ischemia had been triggered. in diabetic patients, every ketoacidosis attack may lead to cardiac ischemia, thereby accelerating progress to necrosis. in conclusion, we would like to propose h-fabp as a potential marker for indicating myocardial ischemia. p-06.02.5-004 genome-wide analysis of hypoxic stress response in human cardiomyocytes stress in human cardiomyocytes on a genome-wide scale remains poorly understood. this study aimed to identify the gene expression patterns of adaptive response of the human cardiomyocytes (hcm) to hypoxic stress. in vitro experimental models of hypoxia mimicking in-vivo coronary ischemia, are useful tools to identify molecular pathways involved in myocardial ischemia. in the current study, we cultured ac16-hcms in dmem/f12 with 10%fbs. to simulate hypoxia model, cardiomyocytes were plated in hypoxia chamber (1%o 2 , 5%co 2 , 94%n 2 ) for 3, 6, 12, 24 h and the control group was incubated in normal conditions (5%co 2 , 95%o 2 ). cell viability was determined using mttassay. annexin-v assay was used to monitor apoptosis. gene expression profiling was analysed with affymetrix-hg-u133-plus-2 arrays. following bioinformatic and statistical analyses differentially expressed genes (deg) were classified according to gene ontology using david and kegg pathway analysis tools. according to mtt, annexin-v and hif gene expression results, hypoxia time was determined as 3 h. we identified 649 genes (279 down-regulated and 370 up-regulated) (p < 0.001, fold change ≥1.5) were differentially expressed in hypoxic-ac16 vs. ac16. degs were mainly clustered in cell proliferation, regulation of cell death, cell adhesion and response to stress. furthermore, transcriptome analyses revealed that 'metabolic, cytokinecytokine receptor interaction, hif-1 signaling, tgf-beta, cell cycle and apoptosis' pathways were involved in the hypoxic stress response of human cardiomyocytes. this study provides molecular information regarding gene expression reprogramming of human myocardial hypoxia. the pathways identified in this study may pave the road for translational medicine. this study was supported by tub _ itak project number 111s189. autologous ips cells after reprogrammed into endothelial progenitor cells (epcs) may offer several advantages in the treatment of cardiovascular disorders because of their cardiogenic and vasculogenic differentiation potential. to reach that purpose, we differentiated and characterized mouse ips cells into flk1 + , a well-recognized epc marker. further maturation of epc was characterized by the expression of cd31 and cd133 markers. purified ips cells were differentiated into flk1 + cells with the use of differentiation medium on type iv collagen-coated dishes in the absence of lif. we then analyzed flk1 gene expression and protein levels with qrt-pcr, western blot and immunocytochemical methods on days 2.5 to 7.5. flk1 + cells isolated with macs system and then recultured these cells in differentiation medium with vegf to induce epc cells. following induction, cd31 and cd133 gene expression and protein levels were analyzed with genomic and proteomic methods. after isolating these cells and aggregate overnight, we cultured cells in three-dimensional condition in collagen type i and used differentiated medium including vegf and egf. we found that flk1 expressing cell number reached to a peak level (24%) on day 5.5 followed by a progressive decline subsequently. in the second step, cd31 and cd133 positive cells were generated and enriched during day 4 of induction. we showed optimal time for harvesting flk1 + cells is day 5.5 of initial differentiation. following isolation of flk1 + progenitor cells they were further matured into functional epcs by vegf within 4 days of induction. additionally for evaluation of angiogenic potantial differentiated cells, we monitored epcs behavior along vascular formation in 3d culture. our work demonstrates that epcs could be successfully derived from ips cells and these cells have vascular formation and angiogenic potential in 3d culture. epc drived ips cells play important role in the treatment of cardiovascular disease. p-06.02.5-006 electrophysiological, biochemical and genotoxic effects of luna experience on heart tissue in rat model pesticides are widely used for the control of agricultural, industrial and domestic pests. however, the uncontrolled use of pesticides has diverse effects on ecological system and public health. fungicides are one of the pesticide type used to kill fungi or fungal spores. in this study, the effect of different doses of luna experience, a fungicide, on the cardiac electrophysiology and genotoxicity in rats were investigated. among five groups (5 mg, 10 mg, 20 mg, control and positive control for comet assay) treatment groups received by gavage doses of luna experience for 30 days. electrical activity of heart were recorded using electrophysiological recording techniques. tissue activities of paraoxanase (pon) and arylesterase (are) and level of malondialdehyde (mda) were measured using biochemical methods. comet assay was performed on heart tissue. we calculated genetic damage index (gdi) and damaged cell percent (dcp) from comet assay. it was observed that there is a significant decrease in heart rate in all treated groups as compared with control group (p < 0.05). amplitude of p wave and qrs complex did not change (p > 0.05). in all treated groups, statistically significant differences were found for values of pon, are, mda, gdi and dcp when compared to control group (p < 0.05). according to our results, exposure to different doses luna experience have a probable hazard potential for the cardiac system. the macrocyclic cage complexes iron (ii) clathrochelates are of the interest due to their bioactivity; they are able to inhibit t-7 rna polymerase, possess toxicity to leukemia cells hl-60 and suppress amyloid fibril formation. their binding to serum albumins was reported; the extreme binding affinity to albumins is observed for the compounds bearing carboxy groups. upon this interaction, clathrochelates quench protein intrinsic fluorescence and gain optical activity inducing circular dichroism (cd) signal in 350-600 nm region. here we examine the effect of spatial arrangement (isomery of substituents) of clathrochelates on their binding to globular proteins. we study 6 bis-substituted clathrochelates bearing two same or different isomers (ortho-/meta-/para-) of carboxyphenylsulfid groups. their interaction with bovine (bsa) and human serum albumins, b-lactoglobulin and lysozyme are explored by cd and protein fluorescence quenching method. the binding of compounds to albumins evoked the cd bands of the same shape, but their intensities vary up to 45 times depending on substituents isomery. in the presence of b-lactoglobulin, the intensities, shape, and positions of the induced cdbands differ for the compounds with different isomer groups. the cd bands induced by the lysozyme in the case of di-para substituted clathrochelate are shifted relatively to the bands of other isomeric compounds. the pronounced quenching of protein fluorescence by clathrochelates was observed only in the case of bsa, its intensity depends on the geometry of substituents (9-17 times). the different spatial arrangement (isomery) of carboxyphenylsulfid substituents in clathrochelates causes the distinctions in both their cd-signal induced by interaction with proteins and their effect on the protein fluorescence. the geometry of ribbed substituents is important for their binding to biomolecules (particularly proteins) and is suggested to determine the structure of the formed guest-host complex. 3d bioprinting is a new technology that revolutionized the field of tissue engineering and regenerative medicine, allowing reconstruction of living tissue and organs preferably using the patient's own cells. using a 3d printer we can design biological structures by controlling exact deposition of cells, growth factors and extracellular molecules in a spatially-controlled manner. the aim of this study was to evaluate the differentiation of human amniotic fluid stem cells (afsc) into endothelial progenitor cells using a bioinkò hydrogel photopolymerized in a 3d network resembling vascular tissue. characterization of afsc was performed by flow cytometry, followed by sorting of the cd177 + stem cell subpopulation. cd117 + stem cells were stained with cell tracker red cmtpx and then mixed with bioinkò hydrogel. printing was done using a 150 lm diameter needle, under 1 bar pressure, and 150 mm/min speed. the network models with define distance apart were printed and analyzed by fluorescent microscopy. mtt test was used to evaluate the viability of the cd117 + stem cells. our results showed that afsc remained viable as shown by mtt assay. the fluorescent microscopy images confirm the viability biochemical test showing that the cd117 + cells viability is maintained after 7 days of cultured in bioinkò hydrogel. furthermore, histological section of hydrogel showed that cells have a relatively uniform distribution forming network interactions between cells. flow cytometry assay showed that cd117+ cells expressed endothelial markers such as cd31, cd105, cd133, cd144 and vegfr2. in conclusion 3d printers are useful tools for creating three-dimensional scaffolds that mimics the cell microenvironment where different types of cells could proliferate, differentiate and crosslink with each other forming tissue-like structures. this study aims to reveal the biocompatibility, biodistribution and immunomodulatory impact on the production of inflammatory citokines of magnetite (fe3o4) nanoparticles functionalized with natural compounds with proved antimicrobial and immunomodulatory effects. co-precipitation synthesized fe3o4 were functionalized with plant-derived compounds: eucalyptol, carvone, limonene and b-pinene. characterization was done by ir, sem and hr tem, while in vitro biocompatibility was tested using endothelial human cells (fluorescence microscopy and proliferation assay). in vivo biodistribution was tested in a balbc mouse model at 2 and 7 days post-intraperitoneal injection, followed by experimental organ removal. tissue sections obtained from vital organs were stained with hematoxylin-eosin. production of inflammatory cytokines was assessed by elisa. results demonstrated that, at concentrations of 500 lg/ml, all prepared nanosystems have a good biocompatibility in vitro and in vivo, allowing the development of cultured cells and also not affecting any visible behavior and organ morphology of the mice. microscopy evaluation of the organs sections revealed that nanoparticles are not present in vital organs such as brain, heart, kidney and liver, but aggregates were visible in the lungs and spleen. at 7 days post-injection no visible aggregated were found in the lungs, few dark-brown nanoparticles clusters being visible in the red pulp of spleen. elisa results revealed that fe3o4 functionalized with carvone and limonene significantly stimulated the production of il-2, il-10 and il-6, while reducing the production of tnfa. other nanosystems din not impact significantly on the cytokine production. functional fe3o4 nanoparticles are efficient drug delivery shuttles, able to stabilize pharmacological compounds, such as plant-derived bioactives, and their biocompatibility, specific biodistribution and limited immunomodulatory effects recommend their use in pharmacological formulations. p-07.01.3-004 new approach for cell imaging with fluorescent carbon nanoparticles m. dekaliuk, k. pyrshev, o. demchenko palladin institute of biochemistry, kiev, ukraine in the nanotechnology field, much interest was focused on the new carbon nanomaterials for cell imaging. recently discovered inorganic carbon nanoparticles ('c-dots') due to their excellent fluorescence characteristics and biocompatibility have ample opportunities for their use in imaging and functional transformations in living cells. their distinctive features, such as high brightness, small sizes, high biocompatibility, small negative charge on the surface and very easy methods of their preparation present a good alternative to other nanoscale materials. the focus of our research was to determine the possibility of using c-dots as the easily available probes for apoptotic cells detection. the carbon nanoparticles were prepared from alanine, citric acid, urea, etc by hydrothermal treatment at 180 c. the studies were performed with adherent epithelial vero and hela cell lines (atcc). with these tools we demonstrate that both native and apoptotic cells can be easily visualized. the cdots uptake occurs probably by endocytosis, which allows for much larger their number to accumulate in apoptotic cells. using the different methods of sample preparation, they show the ability for labeling various structural compartments of the cell. for living cells there are the intracellular vesicles and lysosomes. in contrast, in fixed cells the nucleus is labeled preferentially. the fact that apoptotic cells accumulate strongly increased amount of cdots can be efficiently used in flow cytometry for characterizing the cell populations regarding the relative amount of apoptotic cells in different experimental conditions. the application of such cheap and easily accessible nanoparticles provides more opportunities to simplify the popular methods of cell labeling and detection. previously, our studies showed the possibility of using these nanoscale fluorophores for super resolution method sofi. a new electrochemical microbial biosensor for the fast detecting of dopamine and epinephrine based on candida tropicalis immobilized in a carbon paste electrode (cpe) modified with single wall carbon nanotube (swcnt) was described in this paper. the immobilized cells were used as a source of polyphenol oxidase (ppo) to develop voltammetric epinephrine and dopamine biosensor. voltammetric determination of phenolic compounds like epinephrine and dopamine is a simple technique available. direct oxidation of phenols can be used, but the oxidation potentials of this compounds are similar and they can not be detected distinctively. another possibility is the use of biosensors based on the polyphenol oxidase (tyrosinase) enzyme that oxidises the phenolic compounds into their corresponding quinones. by this way phenolic compounds that epinephrine and dopamine that used in this study were detected at the different potential. the effect of varying the amounts of swcnt and microorganism on the response to epinephrine was investigated to find the optimum composition of the sensor. the effects of ph and temperature were also examined. increases in biosensor responses were linearly related to dopamine concentrations between 0.1 and 1.0 mm and epinephrine concentrations between 0.01 and 1.0 mm. limits of detection of the biosensor for dopamine and epinephrine were calculated to be 0.021 and 0.0029 mm, respectively. finally, proposed systems were applied to epinephrine and dopamine analysis in pharmaceutical drugs. objective: it has started a long time ago to search for a material that can replace blood. this material does not require special storage conditions, independently of the recipient's blood group and can be applied to all individual. milk, casein derivatives, starch, saline and ringer were used for this aim in the past. the determination of toxic effect of natural hemoglobin (hb) on human, researchers have focused on development modified blood. in this work, the development of an artificial biomaterial alternative of blood for using as preoperative and operative aims was aimed. material and methods: in our study, ultrapure hb molecules are immobilized on triethanolamine coated magnetic nanoparticles using various techniques. prepared nanoparticles were characterized by ft-ir, ctem, xrd and cyclic voltammetry (cv). the cytotoxic effects of artificial blood were tried on mtt cell proliferation. results: the characteristic peaks of hemoglobin were obtained from ft-ir spectra differently from support. particles size is concluded by using debye-scherrer equation as > 80 nm from xrd spectra. sem and ctem images supported xrd result. cv results showed that hb molecule has à0.418 v cathodic potential against ag / agcl standard electrode. significant differences were not observed in the mtt results (p < 0.01). conclusion: the nanoparticles were obtained in accordance with the intended desired method. it is determined that the hemoglobin molecules give the same potential with natural blood even after 3 weeks of immobilization and carrying oxygen as natural blood. there are statistical differences between results of mtt tests due to used concentration. but, it is considered that decantation advantage of the artificial blood minimized cytotoxic effects. proteoglycans are among the most abundant molecules of the inter-cellular structure and they are present in extracellular matrices of connective tissues. these glycosylated proteins contain one or more (gag) chains that are covalently attached to the core protein and their hydrodynamic function is mainly due to the physicochemical characteristics of this gag component which provides hydration and swelling pressure to the tissue. gag levels excreted via urine are used as a marker to monitor different diseases (chronic renal disease, renal fibrosis, glomerular filtration abnormalities, bladder stones, breast and lung cancers, hypertension and diabetes, etc.) besides the well known mucopolysaccharidoses. however, their detection by using chromatographic methods is hard, because of the high polarity of negative charges and different functional groups such as acetyl sulfates that generate microheterogenity. in this study, we developed molecularly imprinted chromatographic hplc columns for specific heparan sulfate (hsa), chondroitin sulfate (cs) and dermatan sulfate (ds) detection in urine. positively charged acrylamide monomers were first polymerized by precipitation polymerization, to produce polymers which will show specific recognition for gag's via electrostatic interactions and hydrogen bond formation. these gag selective polymers were then filled in the steel hplc columns and columns eluents were chemically degraded. degradation products of gag's were examined offline column coupled with tandem mass spectrometry. the results showed that our imprinted columns separated gag's specifically and sensitively. thus, urine gag's can be specifically determined by using a gag specific molecularly imprinted column. in this study internal standart weren't used because the matrix effect was lower than 5% for each urine samples. %cv of ds, cs and hsa was calculated as; supported lipid bilayers (slb) were started to be used for cell culture studies to focus on cell adhesion, cell signaling etc. testing the stability of slbs is essential to utilize them as cell culture platforms. in this study, the stability of phosphatidylcholine (pc) lipid bilayers on glass was investigated under milli-q water, phosphate buffer saline (pbs) and dulbecco's modified eagle medium culture (dme) medium supplemented with/without serum. the stability was also checked by enriching slb with different lipids. pc-liposomes were prepared by hydrating the dried thin lipid film with pbs and then by extruding the suspension through a polycarbonate membrane. a negatively charged phospholipid, phosphatidylserine (ps, 25%); a positively charged phospholipid, dotap (50%) and cholesterol (25%) were also used for liposome preparation. liposomes were fluorescently labelled and series of slb imaging were taken for a week. in all experiments in milli-q water and pbs, the stability was conserved for 7 days. pc bilayers in medium supplemented with serum showed hole formations on the second day and their number and size increased rapidly in time. when the bilayers were prepared in medium without serum, disruption was lowered but not completely removed as a result of other factors in medium. cholesterol providing an increased rigidity to the membrane caused higher stability. positively charged bilayer structures also showed increased stability. this can be explained by decreased mobility of bilayer as a result of electrostatic interaction between positively charged molecules and negatively charged glass surfaces. decreased mobility decreases the interactions within the medium. lastly, negatively charged bilayers did not show high stability. strong repulsive forces between the negatively charged surface and bilayer probably prevented the integrity of the bilayer and increased the deformation. in recent years the use of biopolymers has gained priority in tissue engineering and biotechnology, both as dressing material and for enhancing treatment efficiency. there is a demand for new biopolymers designed with protease inhibitors and antimicrobials. ll-37 is an important antimicrobial peptide in human skin and exhibits a broad spectrum of antimicrobial activity against bacteria, fungi, and viral pathogens. using lignin which is an abundant carbohydrate polymer in nature and a polyacrylic acid, we prepared a polymer film by plastifying caprolactone and polyacyrlic acid. films were actified to immobilize ll-37. the structure was elucidated in terms of its functional groups by fourier transform infrared spectroscopy (ftir), and the morphology of the film was characterized by scanning electron microscopy (sem) before and after the immobilization process. the amount of ll-37 immobilized was determined by elisa method. 99.9% of ll-37 peptide was successfully immobilized onto the films. antimicrobial activity was determined in the film samples by quantitative antimicrobial activity method. according to the results, ll-37 immobilized film samples were effective on test organisms; gram-positive staphylococcus aureus and gram-negative escherichia coli. in bio-compatibility assays, the ability to support tissue cell integration was detected by using 3t3 mouse fibroblasts. samples were examined under transverse microscope, non-immobilized sample showed a huge cellular death, whereas ll-37 immobilized film had identical cellular growth with the control group. this dual functional film with enhanced antibacterial properties and increased tissue cell compatibility may be used to design new materials for various types of biological applications. p-07.01. [3] [4] [5] [6] [7] [8] [9] [10] in vitro modulation of the cross-talk between macrophages and osteoblasts by titania nanotube-modified ti surfaces p. neacsu, a. mazare, p. schmuki, a. cimpean bone remodeling is a dynamic process that maintains a fine balance between bone formation and resorption, and is highly influenced by the inflammatory state of the local microenvironment. therefore, a proper modulation in the cellular interactions and cytokine expression is a promising approach to achieve enhanced bone healing. as the biomaterials surface has a major impact on cellular behavior, the goal of the current study was to investigate the influence of tio 2 nanotube-modified ti surfaces (ti/tio 2 ) on the cross-talk between raw 264.7 macrophages (mf) and mc3t3-e1 preosteoblasts (ob) in mono-and co-culture systems in comparison with flat ti (cpti). raw 264.7 and mc3t3-e1 cells were seeded on the test surfaces and grown in standard culture conditions for various periods of time. for co-culture studies, the cells were cultivated using a transwell system. inflammatory mediators released by raw 264.7 cells were measured using elisa technique, while the ob capacity to produce calcified bone matrix was evaluated by alizarin red staining. in co-cultures, lps-stimulated tnf-a, il-6 and mcp-1 release was significantly increased at 24 h, while after 7 days only il-6 exhibited higher amounts when compared with mf cultures alone. moreover, the secretion of these mediators by cells exposed to ti/tio 2 was diminished, especially in lps evoked conditions. also, alizarin red staining demonstrated the presence of calcium deposits when ob were co-cultured with mf for 24 h and 7 days, whereas the presence of the mf for 4 weeks significantly inhibited mineralization. on ti/tio 2 surface elevated calcified matrix was observed, as compared with cpti. this study reveals that the overall effect of inflammation suppression induced by ti/tio 2 may contribute to the enhanced mineralization. also, chronic inflammation may inhibit or delay the regeneration process. therefore, an adequate modulation of mf and ob interactions is vital for the biomaterials success in stimulating bone regeneration. p-07.01.3-011 synthesis and characterization of the branched magnetic polymer for drug delivery systems t. tarhan 1,2 , b. tural 2 , s. tural 2 1 mardin artuklu university, mardin, turkey, 2 dicle university, diyarbakir, turkey magnetic nanoparticles (mnp) have gained a lot of attention in biomedical and industrial applications due to their biocompatibility, easy of surface modification and magnetic properties. magnetic nanoparticles can be utilized in versatile ways, very similar to those of nanoparticles in general. however, the magnetic properties of these particles add a new dimension where they can be manipulated upon application of an external magnetic field. this property opens up new applications where drugs that are attached to a magnetic particle to be targeted in the body using a magnetic field. often, targeting is achieved by attaching a molecule that recognizes another molecule that is specific to the desired target area. in recent years, the development of the systems in which drug is delivered magnetically to the target is drawing considerable attention since it is a current issue. it is possible to eliminate the most of the problems caused by high doses of chemotherapy by using the magnetic drug delivery systems. therefore, it is important to design delivery systems with high drug loading capacity. it is necessary to increase the number of reactive groups on the surface of nanoparticles in order to increase drug loading capacity. in this study, we synthesized a novel magnetic surface for drug delivery systems. magnetic dextran-nta (md-nta) was synthesized by using magnetic o-carboxymethyl dextran (ocmd) and nana-bis (carboxymethyl) -l-lysine hydrate (nta) in order to increase the number of reactive carboxyl groups on the surface of biocompatible and biodegradable magnetic dextran. magnetic material (md-nta) which was prepared and characterized by the analysis of transmission electron microscopy (tem), scanning electron microscope (sem), vibrating sample magnetometer (vsm), fourier transform infrared spectroscopy (ftir) and x-ray photoelectron spectroscopy (xps). there are three subtypes of the tgf-b protein that has been reported to be involved in tissue repair process; scar tissue formation has been reported on tissues that has been affected by tgf-b1 and 2 due to high collagen synthesis. on the other hand the other isoform tgf-b3, suppresses the dense collagen production caused by tgf-b1 and prevents the scar formation. to be able to use these growth factors local or iv route, new drug transport systems are needed to protect the bioactivity during the treatment and controlled release. for this purpose poly(lactic-co-glycolic) acid polymer which is widely used in controlled release systems was chosen as the matrix material. aim of the project was to design, formulate, prepare and optimize tgf-b3 loaded plga nanoparticular and/or plga polymeric film drug delivery systems and to test their effect on cell proliferation. tgf-b3 loaded nanoparticles was prepared with emulsion-solvent evaporation method; whereas polymeric film systems was prepared with film castingsolvent evaporation method. following the preparation tgf-b3 loaded drug delivery systems was characterized. quantification and in vitro release of the growth factor tgf-b3 was studied with elisa. hepg2 cell line was used on mtt cell proliferation assay for both tgf-b3 loaded nanoparticles and films on a time course study. nanoparticles and films were prepared and loading efficiency of the nanoparticles were found to be 42.42%. particle size, zeta index and polydispersity index for this formulation were determined as 204.9 ae 10.3 nm, 0.0381 mw and 0.380, respectively. thickness of the prepared films were 286 ae 20.16 nm. additionaly prepared nanoparticles and films were found non-toxic. tgf-b3 nanoparticles and films which were prepared in this study are planned to be used as an effective treatment strategy for wound healing after injury. this project was supported by grand 112s541 from the scientific and technological research council of turkey (tubitak). polyvinylpyrrolidone (pvp) is a biodegradable material and natural polymeric biomaterial in such studies. ganoderma lucidum is a natural material containing triterpenes, polysaccharides, adenosine, polypeptides, and amino acids. these constituents have been shown to exhibit anti-cancer properties, enhance and regulate immunity, resist oxidation and ageing, and promote metabolism and cell proliferation. composites of polyvinylpyrrolidone (pvp) have been prepared by solution intercalation method using ganoderma lucidum at different loading amounts. the characterization of pvp/ ganoderma lucidum composites was made by x-ray diffraction (xrd) and scanning electron microscopy (sem); the interactions between ganoderma lucidum and pvp was determined by ftir-atr; the thermal stability was determined by simultaneous tg/dta. hemocompatibility of the prepared composite samples were investigated by a 96-well plate spectrophotometer. in addition, contact angles and antimicrobial activity of biomaterials were also determined. ftir-atr confirms interactions formed between ganoderma lucidum and pvp. xrd and sem results give evidence that ganoderma lucidum was well dispersed and homogenously in the pvp matrix. thermogravimetric analysis indicated that introduction of clay to the polymer network resulted in an increase in thermal stability. the results of in vitro hemocompatibility test were showed that pvp/ ganoderma lucidum composites are used as biomaterial. the development of synthetic materials, textured polymers and metals and their increasing use in medicine make research of biomaterials' hemocompatibility very relevant. composite material is a multi-phase system consisted of matrix material and reinforcing material. matrix material is a continuous phase and reinforcing material is a dispersed phase. the main two bioactive components of ganoderma lucidum can be broadly grouped into triterpenes and polysaccharides. despite triterpenes and polysaccharides being widely known as the major active ingredients at anti-cancer effect. this study describes the synthesis and characterisation of biocomposites of different molecular weight of peg (polyethylene glycol) as matrix with ganoderma lucidum as a filling material at different loading (%1, %2.5, %5 wt). the composites have been prepared by solution intercalation method using ground and sieved ganoderma lucidum at 25 micron scale. the characterization of composites was made by x-ray diffraction (xrd), scanning electron microscopy (sem) and fourier transform infrared attenuated total reflectance (ftir-atr) also in this study the hemocompatibility and antibactarial properties of composite investigated. when xrd and ftir-atr results discussed, all of the composites using the different loading amunt of ganoderma lucidum (%1, %2.5 and %5 wt) were shown a homogen distribution in the matrix (peg). and an interaction have occured between matrix and filling material. the sem photos have confirmed these results. peg and composites have been detected as hemocompatible. these results showed that they can be used as biomaterials. p-07.01.3-016 evaluation of the genotoxic potential of some nanocomposites by comet assay b. yilmaz 1 , s. dogan 1 , s. celikler kasimogullari 2 1 department of molecular biology and genetics, balikesir university, balikesir, turkey, 2 department of biology, uludag university, bursa, turkey due to its similar nature to the bone, nanohydroxyapatite is a biocompatible particle and poly(methyl methacrylate) (pmma) is a polymer that has been used in dentistry and orthopedic applications for years. in this study, genotoxic potential of pmma/nanohydroxyapatite nanocomposite films composed of polymers having different molecular weights and nanohydroxyapatite fillers in different concentrations (1, 2.5 and 5%) were investigated by comet assay which is a kind of gel electrophoresis that can be used to measure dna damage in individual cells. if the dna is damaged we expect broken ends to migrate apart from the head. at the end of the assay performed after incubation with lymphocytes of healthy humans, we measured the dna damage index (ddi) and percentage of damaged cells (pdc). in addition, to prove the morphological properties of the nanocomposites scanning electron microscope was used and an interaction between the matrix and nanoparticles with a homogeneous dispersion was observed. protein adsorption on stimuli-responsive mixed pdmaema/peo polymer brushes a. bratek-skicki 1,2 , c. dupont-gillain 1 1 universit e catholique de louvain, louvain-la-neuve, belgium, 2 j. haber institute of catalysis and surface chemistry, polish academy of sciences, krakow, poland smart polymer brushes are made of macromolecules that are sensitive to stimuli from the external environment, including ph, ionic strength, temperature, etc. when stimuli-responsive polymer brushes are introduced onto material surfaces, their properties can be adjusted by tuning the environmental stimuli. these brushes can find promising applications across many areas of research, including surface science, nanotechnology, and biotechnology. in our work, the adsorption of human serum albumin (hsa, molecular weight of 66.5 kda, isoelectric point ip at ph 4.7) and lysozyme (lys, molecular weight of 14.3 kda, ip~11) was studied on polymer brushes composed of poly(ethylene oxide) (peo) and poly (2-(dimethylamine) ethyl methacrylate) (pdmaema). peo is a protein-repellent polymer and pdmaema is a polyelectrolyte bearing a variable density of positive charges depending on ph. a gold substrate was modified by these thiolated polymers according to the 'grafting to' method. the obtained polymer brushes were characterized by xray photoelectron spectroscopy, static contact angle measurements and atomic force microscopy. polymer brush formation and protein adsorption were monitored by quartz crystal microbalance. surface characterization of the mixed brushes revealed the presence of both polymers at the surface. conformational changes of pdmaema/peo brushes were experimentally evidenced, and the results indicated that the brushes collapse at ph 9.00 (pdmaema is neutral in such conditions) and were swollen at ph 3.5 (pdmaema is positively charged). protein adsorption was performed at different ph values (3.5-9 .0) and salt concentrations (0.001-0.15m). it was shown that pdmaema has a high affinity to hsa at ph above its isoelectric point. however, the adsorption of positively charged lysozyme in a wide range of ph was not observed. these results indicate that pdmaema/peo brushes are promising candidates for selective adsorption from a mixture of proteins. clay-polymer nanocomposites (cpn) developed in recent years as a new type of inorganic-organic hybrid materials that were conceived for medical uses such as tissue engineering or drug delivery [1] , [2] . the understanding of the structure and physico-chemical properties of cpn is a first step in the investigation of biomaterials, but their potential in this respect is determined by their interaction with living tissue components. in this study, pure kaolinite was intercalated with dimethyl sulfoxide (dmso) and then intercalated kaolinite was modified pyridine, 2-amino pyridine and 2,6-diamino pyridine to expand the interlayer basal spacing. modified kaolinite samples as filler and poly(vinyl chloride) (pvc) polymer as matrix were used in the nanocomposite synthesis. nanocomposites of pvc have been prepared by solvent blending method using thf as a solvent. the material characterizations were carried out by xrd, afm, ftir-atr, dta/tg and dsc. the xrd results reveal the formation of intercalation/exfoliation of modified kaolinite in the pvc matrix. ftir and afm results confirm the presence of nanomaterial in kaolinite/pvc nanocomposites. tga data show that the modified kaolinit/pvc nanocomposites have significant enhanced thermal stability. the glass transition temperature (tg) of pvc nanocomposites is higher than that of pure pvc. in addition, the antimicrobial activity of clay-polymer composites were also determined. introduction: polyhydroxyalkanoates (phas) are biocompatible and biodegradable materials obtained from microorganisms. they are produced in the cytoplasm of several bacteria as energy reserve. the physical properties of poly(3-hydroxybutyrate) (phb), which is from the group of phas, make it a competitive source to petrochemical plastics. phb has potential in order to be used in a variety of application fields such as packaging industry, printing materials, agriculture and food industry. furthermore, phb meets expectations for tissue engineering applications, since it is biocompatible, biodegradable, non-toxic and has good mechanical properties. although its many advantages, blending approach could be needed in order to fulfill all expectations of a material. due to its flexibility, polycaprolactone (pcl) is a promising candidate to be blended with phb. the aim of this study is to construct a scaffold by using phb produced by extreme alkaliphilic b. marmarensis gmbe 72 t (dsm 21297) and commercial pcl as components and investigate its properties. materials and methods: electrospinning method was used in order to construct scaffolds from blend polymer solution containing phb from b. marmarensis and commercial pcl. results: nanofiber structures were observed on scanning electron microscope (sem) images and fourier transform infrared resonance (ftir) analyses have shown characteristic peaks for both phb and pcl. discussion and conclusion: phb could be blended with other polymers in order to enrich its properties. in addition, nanofiber structure of electrospun phb-pcl blend makes it a rewarding material as scaffold for several tissue engineering applications. q fever is a zoonotic disease that is encountered widely around the world, the most common acute form of q fever shows the following symptoms; a sudden fever, shivering, lassitude, headache, anorexia. because this disease does not show specific symptoms its diagnosis is possible with laboratory tests. current diagnostic kits lack effectiveness; this is why the main goal of our studies is to come up with a new diagnostic kit that does not have disadvantages that current diagnostic kits show. with this goal, nine mile i strain (rsa 493), s serologic virulent phase i, were obtained from slovak science academy, virology institute for rickettsia reference and research from who co-operation centre. these cells were purified and lipopolysaccharide (lps) isolation from coxiella burnetii was performed. the polymeric carrier, poly (n-vinyl-2-pyrrolidone-co-acrylic acid) [p(vp-co-aa)] was synthesized and characterized. physical complexes of obtained lps and p(vp-co-aa) with varying ratios. ternary complexes of lps-cu 2+ -p(vp-co-aa) were also synthesized with copper metal mediation. structure and interaction of lipopolysaccharide-p(vp-co-aa) complexes were investigated with zeta-sizer device using zeta potential analysis and ftir spectrophotometry according to the ratios of components, reaction environment conditions and chemical structure of the polymer. the best complex ratio according to analysis results will be used in the future studies for obtaining monoclonal antibodies which will be an important step for obtaining more effective and stable diagnostic kits that can be used for q fever. this 2513 in this study; new types of water soluble polymer-biomolecule conjugates were synthesized using covalent bonding techniques between polymers and co-polymers (varying monomers of polyacrylic acid and acrylic acid) with peptides. different compositions of polymers, varying ratios of biomolecule/polymer and different molecular weight of polymer has yielded new types of bioconjugates. conjugation mechanism, composition and structure were investigated with various physicochemical methods (uv, ftir, hplc, gpc, etc.). the peptide used in this study was the antigenic peptide epitope of sheep-goat pox disease (eakssiakhfslwksyadadiknsenk). whether this peptide series was bound to polymers or whether it was bound to polymer-protein carrier; peptide-specific immunogens that were capable of producing antibodies were synthesised. it is thought that using polymer-peptide bioconjugates that contain just peptide will yield a higher peptide-specific immunogenicity compared to traditional adjuvants. in vitro and in vivo investigations of bioconjugates effectivity is planned to be done in the future studies. p-07.01.3-026 bioinert fluorinated ethylene-propylene copolymer modified for keratinocyte adhesion surface properties are crucial when adhesion of a cell to a polymeric material is required for a biomedical application. one of the methods for polymer surface tailoring is argon plasma treatment. this simple and reproducible method alters the surface properties such as roughness and wettability without affecting the bulk properties of the material. for the modification of the bioinert fluorinated ethylene-propylene (fep), related to teflonò, we employed argon plasma treatment with the powers of 3 and 8 w for 20-240 s. the human keratinocytes of the hacat cell line served as a model cell line for biocompatibility testing. we studied adhesion, proliferation and morphology of the cells on modified fep matrices as well as controls (pristine fep and standard polystyrene tissue culture dish) by means of fluorescence microscopy. further morphological details were acquired by scanning electron microscopy. furthermore, fluorescence microscopy with immunochemical labelling was used to determine the size and distribution of focal adhesions in cells grown on the modified matrices. the overall effect of the matrices on metabolic activity of cultured hacat cells was also evaluated using the wst-1 reagent. the ar plasma treatment of fep matrices improved significantly cell adhesion and proliferation and promoted spreading of the hacat cells compared to the pristine fep, on which cells were not able to spread properly. also, increased metabolic activity rates for cells cultured on modified matrices were found in comparison to the pristine fep. altogether, we found that ar plasma treatment improved the surface properties of fep to such extent, that it allows cultivation of adherent cells on its surface. we therefore propose that ar plasma treatment is a useful method for fep surface modification. p-07.01.3-027 graphene oxide enriched biomaterials display potential for tissue engineering applications tissue engineering (te) requires more efficient systems that favor local tissue regeneration with minimum cytotoxicity. materials based on natural compounds ensure biocompatibility and have better effects for regeneration. graphene oxide (go) has been shown to enhance cell adhesion and to improve the rate of cell differentiation. in this context, the aim of this study was to evaluate if the addition of different concentrations (0.25-1 wt.%) of graphene oxide (go) improves the properties of cellulose acetate (ca) materials for biocompatibility and cell differentiation, in the prospective of using these films for te applications. go-containing ca films were tested for cytocompatibility by quantitative and fluorescence microscopy assays, and compared to the ca control. cell cytoskeleton architecture in contact with biomaterials was revealed by confocal microscopy. furthermore, bioconstructs were exposed to in vitro osteogenic and adipogenic induction and monitored for 21 days. histological stainings were performed to validate differentiation. osteogenic and adipogenic markers gene expressions were assessed via qpcr. ca/go 1 wt.% revealed best biocompatibility among all tested scaffolds. adhesion proved to be dependent on the percentage of go in material's composition. cells cultivated on ca/ go 1 wt.% expressed adipogenic and osteogenic markers earlier than cells cultivated on materials with lower go content. differentiation markers displayed an increasing profile of gene expression from 7 to 21 days post induction, with higher levels registered for materials with high go content as compared to films with low go content and to the control. go added to ca materials positively influenced cell survival, proliferation and cell differentiation. ca/go films represent potential candidates for te applications. the design of appropriate scaffolds remains one of the most important challenges for te. current idea is that the cell-scaffold interaction could drive cell differentiation and be linked to gene expression and protein organization. therefore, their quality is essential and should favour cell attachment, growth, migration, in situ vascularization and release of biochemical and physical factors able to address the cell fate. moreover, for an ideal scaffolding material an adequate and interconnected porosity is relevant for facilitating cell spreading and colonization of the inner layers. a combination of optimal mechanical and biochemical properties were here utilized to design a 3d composite hydrogel scaffold (3d-chs) in order to favour cell-scaffold interactions and promote a functional differentiation of human lin à sca1 + cardiac progenitor cells (hcpc). the biocompatible peg-diacrylate (pegda) was used to prepare hybrid protein-pegda hydrogels with embedded albumin-microspheres (ms) as protein component. ms were able to modulate the mechanical and biological behavior of the scaffold acting as air-reservoir, porogen agents and potential carriers of biomolecules. an increase of the hcpc viability in the ms-concentration dependent manner was observed. moreover, the microarchitecture of the 3d scaffold also plays a key role in the stability and functionality of cellularcomposite systems. therefore, pegda-honeycomb structures were fabricated using microstereolithography process and the hcpc viability and adhesion to the microstructures were assessed. 3d-chss were synthesized embedding honeycombstructures into ms-pegda hydrogel and the effects on cell proliferation, cell-cell interactions and cellular alignment were investigated. these results could be of relevant interest for expanding the knowledge on cell-scaffold interaction processes and to promote the development and the application of 3d-chs for tissue regeneration using the emerging bioprinting technologies p-07.01.3-029 gene expressions of mesenchymal stem cells after osteogenic induction on ceraform bone substitute a. kilic s€ uloglu, e. karacaoglu, h. akel, c ß . karaaslan hacettepe university, ankara, turkey ceraformò, is a synthetic calcium phosphate ceramic with the chemical composition of hydroxyapatite 65% and tricalcium phosphate 35%, with 60-85% pore volume and 100-400 lm pore diameter. in this study adipose tissue derived mesenchymal stem cells were differentiated into osteoblast cells and loaded on cer-aformò. in order to improve cell adherence, ceraformò was covered with fibronectin. the cells were cultivated for 28-day period by osteogenic induction medium. days 1, 7, 14, 21 and 28 were selected as specific intervals for incubations. total rna was isolated and cdna was synthesized. differences in the expression of runt-related transcription factor 2 (runx2), bone morphogenetic protein-2 (bmp-2), and osteocalcin (ocn) were determined by qpcr. the peptidylprolyl isomerase a (ppia) gene was used as an internal control. according to the qpcr results, ocn gene expression was observed on the day 14th, continued to increase in day 28. bmp-2 gene expression was increased in 14, 21, 28 day compared to 7 day. on the other hand, runx2 gene expression was increased only on days 21 and 28. these findings pointed out that the osteogenic induction was successfully activated on fn coated bone material. therefore, these results can be used in bone injury treatment and related disorders. p-07.01.3-030 on the in vitro cytotoxicity of graphene oxide nanomaterials v. grumezescu 1 , i. negut 1 , f. sima 1 , e. axente 1 , l. e. sima 2 1 national institute for lasers, plasma and radiation physics, magurele, romania, 2 institute of biochemistry, romanian academy, bucharest, romania during the last decade, graphene and its derivates have proven unique physicochemical properties, several applications being continuously developed. among them, electronic, catalytic, mechanical, optical, and magnetic properties have attracted huge interests. however, the merging of graphene and graphene oxide (go) with biotechnology is still in its infancy, many challenges remaining unexplored. potential applications are related to biosensors, drug delivery or gene therapy and cells imaging. in order to use gos as drug release matrices for cancer cells targeting, it is necessary to ensure that these molecules do not affect normal cells within tissues. it was shown that the cytotoxicity of graphene nanomaterials is highly dependent on surface functionalization. studies suggest that pristine and reduced go with fewer surface functional groups tend to be more toxic than go. in striking contrast, it has been reported that functionalized graphenes, can significantly reduce the cytotoxicity even at relatively high concentrations. in this study, we report on the comparison between go and protein functionalized go when submitted to in vitro cytotoxicity tests. bovine serum albumin (bsa) was used for the noncovalent go surface conjugation. three case-studies were investigated: aqueous nano-colloids consisting of serial dilutions of both go and go-bsa conjugates, dropcasted thin films and laser-assembled thin films on glass substrates. safe concentration windows were identified by live/dead staining and mts assays for different human melanoma cell lines, while melanocytes and human dermal fibroblasts were used as normality controls. the predominant melanoma subtype is represented by cells bearing braf (v600e) activating mutation. with a view to target this specific melanoma subpopulation, we embedded braf inhibitors into go laser-deposited scaffolds and tested their anti-tumoral effect. our results evidence the high potential of these nanomaterials for biomedical applications. osteoporosis is a skeletal disorder associated with low bone mass and increase in bone fragility due to increased osteoclastic activity. binding of rank ligand to its receptor on osteoclast precursor cells results in the osteoclast differentiation. sirna is a dsrna, used to inhibit the translation of the target gene. the aim of the study is to develop an injectable sirna-delivery system targeted to the bone for osteoporosis treatment. pei (polyethyleneimine) and rank complex was loaded into poly(lactic acid-co-glycolic acid) (plga) nanocapsules which are bound to hydroxyapatite (hap)-specific elastin-like protein (elp) for targeting to bone tissue. plga nanocapsules were prepared by w/o/w double emulsion technique. affinity of elp to hap was determined by ftir. elp was coated on the nanocapsules by using the transition temperature of elp. elp on plga nanocapsules were crosslinked by genipin and binding of elp on plga nanocapsules were studied by xps and tem. the optimum ratio of n (pei) to p (sirna) in the complexes to be loaded into plga nanocapsules were studied by etbr staining and zeta potential measurements with varying n/p ratios and finally pei-dnaoligo encapsulation efficiency of the capsules was determined by picogreen reagent. xps results of elp treated plga (elp-plga) nanoparticles indicated the presence of nitrogen atom (11.7%) in the sample which appeared as a fuzzy halo in tem micrographs. n/p ratios up to 5 show negatively charged particles. when n/p was 5, the zeta potential of complex was neutralized which also resulted in larger particles compared to the others. zeta potential moved to positive values when n/p was higher than 5. the migration of polyplexes with different n/p ratios (1-10) was analyzed by gel electrophoresis. dnaoligo complexes show the same patterns of complexation wih that of sirna and thus were used in the encapsulation efficiency studies instead of sirna. the encapsulation efficiency of pei-dnaoligo in plga nanocapsules was 29%. tuesday 6 september 12:30-14:30 aging p-09.03.3-001 novel benzenesulfonamides exhibit low toxicity on zebrafish embryonic development and selectively inhibit carbonic anhydrase ix with nanomolar affinity in xenopus oocytes introduction: the toxic effects of two recently discovered inhibitors (vd12-09 and vd11-4-2) that selectively and with extraordinary strong, picomolar affinity bind to human carbonic anhydrase (ca) ix, an anticancer target, were investigated on zebrafish embryonic development and in xenopus laevis oocytes. zebrafish has emerged as a promising animal model to evaluate the toxicity of the drug candidates. xenopus oocytes do not natively possess any ca activity and thus became a convenient in vivo model system to study the ph effects and the selectivity of synthetic ca inhibitors. materials and methods: morphological changes in zebrafish treated with the compounds were studied by light-field microscopy and histological analysis. ca activity in xenopus oocytes was monitored by measuring ph in the cytosol and at the outer membrane surface and confirmed by mass spectrometry of lysed oocytes. the toxicity studies showed lc50 values to be 13 lm for vd12-09, 120 lm for vd11-4-2 and 9 lm for ethoxzolamide (eza), a non-selective ca inhibitor commonly used in clinic. the zebrafish exposed to lc50 doses of vd12-09 and vd11-4-2 showed fewer phenotypic abnormalities and less morphological changes compared to the zebrafish treated with the corresponding dose of eza. vd11-4-2 exhibited 10-25 nm ic50 for both intracellularly and extracellularly expressed ca ix in xenopus oocytes while exhibiting strong selectivity over ca ii, ca iv and ca xii. discussion: interestingly, the compounds exhibited 10-fold lower toxicity, induced fewer side effects in zebrafish than eza and the amount of vd11-4-2 needed to cause complete inhibition of ca ix enzymatic activity in xenopus oocytes was 30-fold lower than eza. conclusions: vd compounds did not lead to deleterious effects on the zebrafish embryonic development and reached the ic50 of 10 nm for ca ix in xenopus oocytes. the compounds could be further developed as anticancer drugs. cacybp/sip is present in various cells and tissues, both normal and pathological. in normal tissues, e.g. stomach or colon, cacybp/sip is weakly or barely detected whereas in gastric or colon cancer this protein is expressed at a high level. there are also data indicating that the level of cacybp/sip expression correlates with tumor metastatic potency and multidrug resistance. taking into consideration data that suggest association of cacybp/sip with many vital cellular processes, in this work we decided to investigate the possible mechanism involved in regulation of cacybp/sip gene expression, mainly by transcription factors and, on the other hand, the influence of cacybp/sip on the expression of other genes. we have shown that nfat (nuclear factor of activated t cells) influences the cacybp/sip gene expression and that overexpression of cacybp/sip has an effect on the level of ap-1 and on the activity of nfat and ap-1 transcription factors. by analyzing the cacybp/sip gene promoter sequence we also found potential binding sites for transcription factors from the stat family, which are involved in interferon signaling. microarray data indicate that indeed overexpression of cacybp/sip affects levels of the stat proteins as well as of some interferons and interleukins. based on functional analysis we have found many genes the products of which are involved in immune response. to analyze in more detail the influence of an altered level of cacybp/sip on interferon signaling pathways as well as on factors involved in expression of interleukins, including nfkappab, we plan to apply methods such as luciferase assay, real-time pcr or immunocytochemistry. one of the pathological hallmarks of alzheimer's disease (ad) is the neuritic plaques occurred as a result of the extracellular accumulation of aß peptides formed from amyloid precursor protein (app) via the ß-amyloidogenic pathway. aß42 is more prone to aggregation to form plaques and more toxic to neurons than aß40. in addition to change in app metabolism, the decline in levels of neurotransmitter acetylcholine and cholinergic dysfunction are also observed in ad. thus, current strategies for ad treatment focus on compounds with inhibitory effect on cholinesterases as well as preventive effect on aß aggregation. in our earlier studies, toluidine blue o (tbo), a phenothiazine dye, was shown to be a highly effective inhibitor of cholinesterases with k i values in nm range. we also found that intracellular app and aß42 levels are reduced in human neuroblastoma cells after treatment with tbo. additionally, an earlier study revealed that tbo has a selective inhibitory effect on tau aggregation, the other pathological characteristic of ad. the aim of this study was to investigate whether tbo may effectively lower the level of extracellular aß40/42 in an ad-like cellular model. chinese hamster ovary cells that express human wild type app and presenilin 1, namely ps70, were treated with a dose range of tbo (0-15 lm) or vehicle control for 24 h. after treatment, aß40/42 levels in cell culture media were assayed by separate sandwich-based elisas and normalized to total protein levels, determined by bca protein assay. besides, biocompatibility of tbo was evaluated in the ps70 cells using cell viability assay for flow cytometry. strikingly, all dose ranges of tbo inhibited both aß40 and aß42 secreted into the cell culture media. significant reduction for both aß species was evident at 5 lm (p < 0.05), 10 lm (p < 0.001), and 15 lm (p < 0.001) of tbo vs. vehicle control. in conclusion, these results support the idea that tbo may be used as a therapeutic in ad. monitoring the changes of key molecules participating in the osmo-regulatory response of nucleus pulposus intervertebral disc cells during stress-induced senescence e. mavrogonatou, d. kletsas ncsr 'demokritos', athens, greece introduction: intervertebral disc cells are faced with a harsh extracellular milieu characterized by hyperosmotic conditions, nutrient and oxygen deficiency because of the absence of vascularization and oxidative stress due to the accumulation of their metabolism's by-products. we have previously shown that high osmolality is anti-proliferative for disc cells through the activation of the g1 and g2 cell cycle checkpoints by p53 and p38 mapk, respectively. in addition, we have shown the participation of nine solute transporters, with the a1 subunit of na + /k + -atpase being central in this response. here we assessed the changes in the expression of these key osmo-regulatory molecules during in vitro stress-induced senescence. materials and methods: changes in cell cycle progression were assessed using flow cytometry; overall transcriptional alterations were assessed by whole-genome arrays; differences in expression at the mrna and protein level were revealed by quantitative rt-pcr and western blotting, respectively; knocking-down of selected proteins was performed by sirna. results: high osmolality led to the differential expression of > 200 genes, including nine genes encoding transporters. p38 mapk and p53 were demonstrated to differently participate in the regulation of the aforementioned transporters, while knocking-down of three selected transporters had a distinct outcome on the overall cellular response towards hyperosmotic stress. these molecules were found to show differences in their expression in senescent cells. discussion: given that the presence of senescent cells has been demonstrated in the intervertebral disc in vivo and could most probably attributed to the prevailing stressful conditions, here we showed differences in the expression profile of known key molecules for osmo-adaptation during senescence. conclusion: understanding disc cells' physiology is of outmost importance when designing cell-based therapies for disc degenerative disorders. p-09.03.3-005 smad specific e3 ubiquitin protein ligase 2 (smurf2) and its potential effects on inhibitory transmission in aging adams 1, 2, 4, 5 1 interdisciplinary program in neuroscience, bilkent university, ankara, turkey, 2 national nanotechnology research center (unam), bilkent university, ankara, turkey, 3 department of molecular biology and genetics, bilkent university, ankara, turkey, 4 molecular biology and genetics zebrafish facility, bilkent university, ankara, turkey, 5 department of psychology, bilkent university, ankara, turkey smad specific e3 ubiquitin protein ligase 2 (smurf2) is part of the tgf-b signaling pathway associated with cellular proliferation, differentiation, genomic stability and senescence. moreover, smurf2, via its downstream partners, may regulate inhibitory synaptic transmission. our research group previously found that the smurf2 transcript is significantly higher in old zebrafish brains. thus, smurf2 may alter inhibitory synaptic transmission in aged animals. the focus of this study was to examine age-related changes in smurf2 protein levels and related key inhibitory synaptic proteins; gephyrin (gep), a scaffolding protein for gaba receptors, and gaba a , an ionotropic gaba receptor subtype. additionally, the levels of those proteins were studied in a mutant zebrafish line, which lacks acetylcholinesterase (ache) and is suggested to be a delayed aging model. whole brain tissues were isolated from young, middle-aged and old male and female zebrafish brains (ab/wildtype strain), as well as from old male and female ache mutant zebrafish (ache sb55/+ ). animals were maintained and raised in standard conditions. the extracted brain tissue was homogenized in ripa buffer and subjected to western blot analysis to determine differences in the relative protein expression levels. our preliminary data indicated that smurf2 and gep levels remain stable in the aging brain (p = 0.301, p = 0.335), and in the ache mutants gep levels are increased compared to the wildtype controls (p = 0.001). further analysis of the relationships between smurf2 and gaba a levels and brain aging is ongoing. we predicted that alterations in smurf2 levels would parallel changes in key synaptic inhibitory proteins during the aging process, which was the case for the gep levels. while smurf2 may regulate inhibitory synaptic transmission, the exact roles of those synaptic proteins in the context of normal and delayed brain aging are not known well-understood and the subject of continuing study. in recent years, express the hypothesis that aged individuals are vulnerable to infectious and other inflammatory agents and they become more prone to develop majority of severe age pathologies, including cardiovascular and oncology diseases, neurodegenerative diseases, type 2 diabetes mellitus and inflammatory diseases, etc. one of the central components of immune response is the family of toll like receptors (tlr). there are several opinions that single nucleotide polymorphisms (snp) leading to a loss of function of the respective tlrs can be associated with age and increase the risk of age related diseases, especially cardiovascular diseases (cvd). however, many available studies focusing on tlr snps and cvd are with conflicting results. the aim of this study was to assess the potential interaction between genetic variants of tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we evaluated 148 patients with ihd and 144 healthy subjects aged 45 years and over (ethnical kazakhs and russians living in republic of kazakhstan). polymorphic loci of the genes tlr2 rs5743708 and tlr4 rs4986790 were genotyped by pcr with subsequent restriction analysis. our results indicated that the genotype and allele frequencies of tlr2 (arg753gln) and tlr4 (asp299gly) were not significantly different between the 2 groups (p ≥ 0.05). statistical analysis didn't elicit any association between studied gene polymorphisms and predisposition to ihd in individuals over 45 years old (p ≥ 0.05). for these polymorphisms, age, fasting blood sugar and serum lipid levels were not also significantly different among different genotypes in the ihd and control groups. in conclusion, the data shows that there is no interaction between tlr2 and tlr4 and ischemic heart disease (ihd) in kazakhstan population over 45 years old. we plan to include other types of polymorphisms in tlr 2 and tlr 4 genes and increase the volume of patient cohort in our future studies. p-09.03.3-007 evaluation of prognosis with total oxidant/ antioxidant status and some oxidative stress parameters in patients with acute ischemic stroke stroke is the third most common cause of death after coronary heart disease and cancer. strokes are classified into two groups according to their pathology: ischemic stroke and hemorrhagic stroke. ischemic strokes make up 87% and hemorrhagic strokes 13% of all strokes. during ischemic stroke, oxidative stress has been shown to play a major role in the occurrence and progression, formed oxidants also affect cell membranes and genetic material such asdna, rna, and various enzymatic events, and they lead to cell damage. some studies have shown oxidant-antioxidant status but have not shown the relationship with prognosis. this study has investigated the relationship between prognosis and total oxidant/antioxidant status and biochemical parameters in patients with acute ischemic stroke 58 patients, with acute ischemic stroke and 37 healthy controls we reenrolled in the study. blood samples were taken within 1st and 7th days, and after 3rd months in the patient group for analysing serum total oxidant status (tos), antioxidant status (tas), catalase, arylesterase, and thiol. prognosis was evaluated with national institutes of health stroke scale (nihss) and-modifiedrankinscale (mrs) scores. there was no significantly difference between groups by means of serum tas, tos and catalase levels. but arylesterase (p: 0.07) and thiol (p: 0.031) levels were significantly higher in first 24 h blood samplingthancontrolgroup. statistically significant negative correlation was observed between the 3rd month values of tos and nihss score (r = 0.410, p = 0.037). but there was no correlation between mrs scores and serum tas, tos, catalase, thiol and arylesterase. similarly, our findings suggested some serum oxidant levels were increased in acute ischemic stroke patients and total oxidant status might be used in evaluation of prognosis but larger studies are needed. p-09.03.3-008 amylin and preptin regulate glucose homeostasis in infertile women with polycystic ovary syndrome and poor responders undergoing ivf/icsi disrupted glucose homeostasis leads not only metabolic disturbance such as polycystic ovary syndrome (pcos), but also influences oocyte growing. this study was designed to evaluate follicular fluid (ff) and serum levels of glucoregulatory hormones, amylin and preptin, in infertile women with pcos and poor responders undergoing ivf/icsi. human follicular and serum were obtained from 20 infertile women with pcos and 20 poor responder participants undergoing controlled ovarian stimulation (cos) with gonadotropin-releasing hormone antagonist protocol for ivf/icsi treatment. ff and serum amylin and preptin levels were measured by elisa. it was found that ff and serum amylin and preptin were lower in infertile women with pcos when compared with poor responder participants. ff amylin and preptin concentrations were lower than that of the serum amylin and preptin concentrations. decreased follicular fluid amylin and preptin levels suggest that amylin and preptin may have a physiological role in follicular maturation via controlling local glucose homeostasis. despite high serum levels of amylin and preptin in pcos their low concentration within the follicle may be main culprit of defective folliculogenesis seen in pcos subjects. similar to insulin resistance in pcos subjects existence of amylin and preptin resistance support the critical role of both peptides in follicular maturation in pcos. keywords: follicular fluid; amylin; preptin; polycystic ovary syndrome; infertility. the transcription initiation on p3 promoter of xp10 bacteriophage in presence of p7 protein, a modulator of rna-polymerase activity a. shadrin g.k. skryabin institute of biochemistry and physiology of microorganisms, ras pushchino, russia many bacteriophages are able to manage the transcription system of their bacterial host for their own needs. for example, bacteriophage xp10, in the early stages of infection of xanthomonas oryzae inhibits transcriptional activity of bacterial rna-polymerase on majority of promoters via p7 protein, except of bacteriophage p3 promoter responsible for expression of the bacteriophage 'middle' class genes. the focus of this work is to study the mechanism of action of p7 protein in the transcription initiation and identification of the role of the individual elements of p3 promoter of xp10 bacteriophage, enabling x. oryzae rna-polymerase escapes inhibition by p7 protein. we have designed a set of promoter probes representing the combination of sequences of p7-resistant p3 promoter and p7sensitive t5n25 promoter. using fret-based assay it was shown that the truncated probes corresponding to promoter dna downstream à26 position, relative to the transcription initiation start site, did not lead to dissociation of the sigma-factor. longer probes, containing à35 promoter element, induce dissociation sigma-factor. the in vitro transcription experiments show that the deletion of region 4, a sigma-factor domain responsible for interaction with à35 promoter element during the transcription initiation, is not critical for inhibition of rna-polymerase by p7 protein. promoter probe with up-element of p3 promoter had affinity to x. oryzae rna-polymerase a several times higher than a probe containing the consensus up-element for e. coli rna-polymerase . summing up the results, it seems like the transcription initiation on p3 promoter of bacteriophage xp10 can escape inhibition by p7 protein through a high affinity interaction between the up-element and c-terminal domains of the alpha subunit of rna-polymerase x. oryzae. p-09.03.3-010 distribution of soluble form of glial fibrillar acidic protein in the different areas of gerbils brain during development and aging y. kovalchuk, g. ushakova oles honchar dniepropetrovsk national university, dniepropetrovsk, ukraine astrocytes are the most abundant cell type within the cns and play an important role in cns homeostasis and function. glial fibrillary acidic protein (gfap) forms the main astrocytic intermediate filament (if). the overall level gfap in different parts of the brain uneven and depends on the number of astrocyte cells. gfap is very sensitive to any kind of neurodegenerative diseases and aging. during aging, a glial reaction is observed in the human brain, as well as in rat and mouse brains. the aim of our study was to investigate the quantitative astrocytes-specific protein gfap in different areas of the gerbils brain at the first stages of postnatal development and aging. for the study 30 gerbils brains were used and divided into 5 groups (n = 6): 1: newborn animals (1 day), 2-4: 30, 90 and 180 days respectively, 5: animals aged 2 years. the animals were decapitated under mild anesthesia (thiopental), with isolated brain three divisions: the cerebellum, thalamus and hippocampus, which are then used to produce cytosolic protein fractions. the level of gfap in the obtained fractions were determined according to the method of competitive elisa. newborn gerbils found no significant content of soluble form of glial fibrillar acidic protein in all investigated parts of the brain, and a sharp increase of amount within 30 days (in cerebellumamounted to 1.12 ae 0.03 lg/100 mg tissue; to 90-180 days increased to 1.67 ae 0.11 lg/100 mg tissue, and began to grow again in older individuals aged 2 years). unlike the cerebellum, the level of sgfap in hippocampus and thalamus reached the maximum at 30 days p.d. (1.5-1.7 lg/ 100 mg tissue), and unchanged for 180 days. these results revealed that the most intensive development of astrocytes in the cerebellum to 90 p.d. of gerbils, and in the thalamus and hippocampus are formed within the first month of life. the plastid-nucleus located protein whiry1 acts as an upstream regulator of leaf senescence binding to the promoter of senescence associated genes (sags) like senescence marker gene hvs40. in order to investigate the impact of whirly1 on drought stress-induced senescence, transgenic barley plants with a knockdown of whirly1 (hvwhy1kd) were grown under untreated and drought stress conditions. the leaf senescence evolution was monitored by physiological parameters and gene expression studies of senescence and drought stress related genes. to reveal the epigenetic indexing at hvs40 at onset of drought-induced senescence in wild type (wt) and hvwhy1kd lines, stress-responsive loading with histone modifications at 6 gene regions of hvs40 (2 regions in the promoter, one region around translation start site and 3 regions located in the gene body) was analysed by chip and quantified by rtq-pcr. in barley, drought treatment caused acceleration of leaf senescence in wildtype (wt) plants, whereas why1kd lines showed a staygreen phenotype. expression of senescence-associated and drought stress responsive genes expression was delayed in hvwhy1kd indicating that whirly1 protein acts as an upstream regulator of drought stress-induced senescence. the chip results showed that drought treatment is causing in wt a significant increase in the levels of h3k9ac all over the analyzed gene regions, correlating with a massive induction of hvs40 expression, while drought stress caused no substantial increase of h3k9ac in why1kd plants. the results suggest that drought induced expression of hvs40 is under epigenetic control, and furthermore that why1 is involved in this epigenetic control level. oncolytic viral therapy is based on the capabilities of selective lysis of tumor cells and is a prospective trend in cancer disease treatment. in vitro experiments showed that plant rhabdoviruses does not have any direct cytotoxic effect upon sarcoma 37 cells, causes induction of apoptosis in these cells and does not pose any threat to somatic cells of warm-blooded animals, which makes it possible to use this virus for therapy of malignant neoplasms. buckwheat burn virus (bbv), the prototypic member of the family rhabdoviridae, contains surface glycoprotein and which is lectin-active. its carbohydrate branch can aid adhesion of lymphocytes to tumor cells. the present study has addressed the effect of bbv on cancer cell viability. all studies were carried out after 1 week of inoculated with erlich cancernome (2 9 10 6 cells/animal, i. p.) in 2 months male balb/c mice treated at once with or without plant extract with bbv (15 mg/kg, i. p.). by fluorescent microscopy and using two due staining by acredine orange and propidium iodide it was found that in the 3rd day of administration of bbv lead to increasing of necrotic and apoptotic cells on 45% and 4% respectively versus to untreated group. at the same time the viability of investigated cells was impaired too and according to flow cytometry analysis using propidium iodide the amount of dead cells was elevated by fivefold (17.7% versus 3.5% in untreated group). also as was shown in previously reports bbv decreased activity of macrophages in the early stages after injection and it may have a positive effect when using this drug in tumor therapy. when using this drug appears to slow down the possibility of a sharp activation of macrophages, and as a consequence of the development of cytotoxic effect will be prolonged. key words: rhabdoviruses, buckwheat burn virus, cancer, cell viability. plants are considered as one of most promising sources for new antimicrobials, based on the evidence of their use in folk medicine to treat various infectious diseases since ancient times. despite relatively small area size, armenia has large diversity of flora with many endemic species. the main goal of this study was the screening of various parts of 28 herbs (widely being used in armenian folk medicine) for their antimicrobial activities in order to select most prospective plants for further comprehensive studies. plant crude extracts were obtained with maceration technique using five solvents: water, methanol, chloroform, acetone and hexane. agar well diffusion assay was used to evaluate antimicrobial properties of plant crude extracts at 500 lg/ml concentration against escherichia coli vkpm-m17, pseudomonas aeruginosa grp3, bacillus subtilis wt-a1, salmonella typhimurium mdc 1754 and staphylococcus aureus mdc 5233, candida albicans wt-174 and candida guilliermondii hp-17. statistical analysis was done using graphpad prism 5.03. crude extracts of all tested plant materials expressed antimicrobial activity against at least one test strain. most of the tested extracts inhibited growth of both gram-negative and gram-positive bacteria. in contrast, only some plant materials exhibited inhibitory activity against yeast strains. according to obtained data sanguisorba officinalis, rumex confertus, hypericum alpestre, lilium armenum and agrimonia eupatoria possessed the highest and broadest antimicrobial activity. moreover, the results showed that acetone was the most effective solvent for solubilizing antimicrobial compounds from plant materials followed by methanol, chloroform, hexane and water. the results demonstrated high antimicrobial activity of medicinal plants used in armenian traditional medicine. five plant species were selected for further comprehensive studies. besides, acetone was proposed as efficient solvent in antimicrobial screening protocols. p-02.08.5-004 effects of aluminum stress on photosystem-i apoprotein a2 gene (psab) transcription level in lichen xanthoria parietina (l.) th. fr. unal € ozakc ßa ege university, izmir, turkey in this study the effects of shortrerm aluminium (al) toxicity on the lichen xanthoria parietina (l.) th.fr. were investigated at physiological and transcriptional level. lichen thalli were treated with alcl 2 in different doses (0.25, 0.5, 1 and 5 mm). lipid peroxidation and chlorophyll integrity were determined by spectrophotometer. expression level of psab gene was also investigated. chlorophyll a content was significantly (p ˂ 0.05) decreased after 48 hours treatment with 1 mm and 5 mm of al, while chlorophyll b content was increased significantly due to treatment with increased concentration of aluminum. also treatment with 0.25 and 0.5 mm al for 24 hours increased the gene expression level of psab by 35.6% and 21.3% respectively. our results indicated that aluminum treatment has decreased the chlorophyll biosynthesis and increased the lipid peroxidation depending on time and concentration. this study also demonstrates that the psi can be readily photo-inhibited by aluminum stress. in conclusion, 5 mm al exposure for 48 hours could damage the electron transport in photosystem i. p-02.08.5-005 nigella sativa reduces paracetamol-induced nephrotoxicity and oxidative stress in rats: biochemical evaluation background: nigella sativa l. (ranunculaceae) (ns) is traditionally used to treat many conditions such as inflammation. this study evaluates the effects of ns seeds ethanol extract in paracetamol-induced acute nephrotoxicity in rats. material method: forty-eight female wistar albino rats were divided into eight groups: i = sham; ii = sham + 1000 mg/kg ns; iii = sham + 140 mg/kg (n-acetyl cysteine) nac; iv = 2 g/ kg paracetamol; v = 2 g/kg paracetamol + 140 mg/kg nac; vi, vii and viii = 2 g/kg paracetamol + 250, 500 and 1000 mg/kg ns, respectively. paracetamol administration (oral) was carried out 1 h after ns and nac administrations (oral), and all animals were sacrificed 24 h later. result: urea and creatinine levels were determined in serum, while glutathione, malondialdehyde levels and superoxide dismutase activity were determined in the kidney tissues. there were significant increases in the serum levels of urea and creatinine in the paracetamol-administered group. serum levels of urea and creatinine were decreased in all groups administered ns with paracetamol. ns administration dramatically restored sod, gsh, and mda levels in the kidneys. conclusion: the results suggest ns has a significant nephroprotective activity on paracetamol-induced nephrotoxicity. it may be suggested that the antiinflammatory and antioxidant effects of ns ethanolic extract originated from different compounds of its black seeds. p-02.08.5-006 the study of problems of preservation of the birches e. shadenova 1,2 , e. zhumabekov 2 , m. sembekov 2 , m. burchaeva 2 1 institute of general genetic and cytology, almaty, 2 laboratory of genetics and reproduction of forest culture, institute of general genetics and cytology, almaty, kazakhstan nature of deciduous trees have a whole range of various medicinal properties. instead of synthetic hormone substitutes, you can use medicinal infusions and decoctions of natural phytohormones are widely used in both folk and professional medicine. one of these plants is birch, its young leaves and buds. however, they also must be used with caution because overdose of these compounds is very dangerous, not only can you not get the desired effect, but also face the opposite of his action. in our research to mass replication of plants (different types of birches (betula ajanensis, yarmolenko, jacguemontii, maximowiczii, ulmifolia, middendorffii, kelleriana, tianschanica)) we use nutritional medium excluding the application of phyto promoters in order to prevent mutation. the object of research serve as the old, the sick, being on the verge of extinction, mature trees as explant meristema. since from the moment of calling experience and most cultivation occurs at nutritional medium without hormones. as a result of molecular analysis we get without virus, genetically identical plants. molecular certification of different types of birches of interest, both in terms of organizing, and in terms of selection and genetic improvement of valuable forms, identification of lines selected from natural populations and clones obtained in vitro. relationship between clones and installed parent form by comparing profiles amplific pcr products using issr-marking. according to the results of carried out works really recovered clones obtained from one source tree, indicating the potential for certification of clones studied forms of birches pcr. a study performed in the framework of the state grant project "conservation of breeding valuable species of birches". p-02.08.5-007 fractionated triterpenoid glycosides from sea cucumber inhibit invasion and metastasis in human cancer cells sea cucumbers are slow-motioned invertebrates. holothuria polii delle chiaje, 1824 is widely distributed sea cucumber in _ izmir coastline (turkey). it secretes saponins i.e. triterpenoid glycosides (ttg) as secondary metabolites. the aim of this study is to evaluate anti-invasive and anti-migrative effects of fractionated ttgs obtained from h. polii on ht-29, t84 and upci-scc-131 cancer cell lines. the semi-purified ttgs was extracted from h. polii collected from coast of _ izmir-dikili. the four different fractions (fraction a-d) were collected by using hplc (high-performance liquid chromatography) and characterizated with maldi-ms/ ms. the fractions obtained from h. polii extract include holothurin a (1243.50 m/z) and 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). anti-invasive and anti-migrative effects of the fractions on the cancer cell lines were detected with xcelligence rtca dp system. the results showed that fraction a-d inhibited migration and invasion of human cancer cell lines at 6th and 12th hours compared to control group. this study shows that holothurin a, 24dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer could be evaluated as promising anti-cancer agents for human cancers. acknowledgement: the authors acknowledged the scientific and technological research council of turkey (t € ub _ itak) for financial support (114z211). p-02.08.5-008 alternative splicing regulation of sr proteins in response to environmental stress in chinese cabbage serine/arginine-rich protein (sr protein) family, which acts as rna-binding protein, plays a major role in post-transcriptional regulation of pre-mrna, such as alternative splicing (as). these proteins cause pleiotropic effect by regulating as of pre-mrna in a tissue and developmental stage-specific and stress-responsive manner in arabidopsis. here, we identified 31 genes encoding sr proteins in chinese cabbage (brassica rapa chiifu-401) from brassica database and analyzed their phylogenetic relationship. b. rapa has 31 types of sr protein that are classified into common (sr, rsz and sc) and plant specific (scl, rs2z, rs and sr-like) subfamily similar with arabidopsis. interestingly, the as pattern of most sr genes changed at the late stage (14 and 21 days after germination). to verify the correlation between sr genes and environmental stress, we screened the as pattern of sr genes to various abiotic stress using rt-pcr and a microarray analysis. in particular, the expression level and the as pattern of bra015576 and bra018581 were affected significantly by heat stress. these results suggest that the as regulation by sr protein correlates with adaptation mechanism to the environmental stress in chinese cabbage. p-02.08.5-010 characterization of recombinant prolyl oligopeptidase from myxococcus xanthus and potential use in gluten hydrolysis e. k. kocaazorbaz, f. zihnioglu faculty of science, biochemistry department, ege university, izmir, turkey a recombinant prolyl oligopeptidase from myxococcus xanthus was purified with a specific activity of 224 u mg(-1) by using nickel-metal-chelate affinity chromatography and gel permeation chromatography. the recombinant enzyme had a monomeric molecular weight of 70 kda. its isoelectric point, determined by two dimension polyacryl-amide gel electrophoresis, was close to 6.3. the optimum ph and temperature was estimated as 7.5 and 37°c, respectively. the purified enzyme was stable from ph 6.0-8.5 and able to thermal stability up to 37°c. the k(m) and v(max) values were 0.2 mm and 3.42 micromol/ min per mg. the enzyme exhibited hydrolytic activity for suc-gly-ala-pna, suc-gly-pro-pna, z-gly-pro-pna, igf-1, substance p, whereas no activity for h-gly-pro-pna, h-val-ala-pna, h-arg-pro-pna, h-ala-pro-pna, glu-ala-pna, pro-pna, leu-pna. its proteolytic activity was inhibited by activesite inhibitors of serine protease, z-pro-prolinal pmsf, and metal ions, cd 2+ and hg 2+. the potential use of the enzyme was tested by the hydrolysis of the wheat gluten. the resulting gluten hydrolysate were characterized by means of their antioxidant, antibacterial, trypsin inhibition and prolyl oligopeptidase inhibition activities. keywords: serine protease, prolyl oligopeptidase, bioactive peptides, 2,4,6-trinitrobenzene sulfonic acid. proteomic analysis is probably the best approach to analyze seed germination. however, it is difficult to analyze complex samples and there are many obstacles that must be faced in order to achieve a reasonable proteome coverage. for example, the barley (hordeum vulgare) genome was fully sequenced in 2012, but the uniprot database contains less than 1000 reviewed sequences, which is approximately 16-fold less than for arabidopsis thaliana. here, to improve the barley proteome coverage, we employed several fractionation methods including polyethylene glycol precipitation, strong cation exchange chromatography, off-gel separation, sds-page and acetonitrile elution gradient. proteomic analyses were performed using an lc-ms-based analyses and an uhr q-tof mass spectrometer. the candidate peptides were targeted via selected reaction monitoring (srm) and triplestage quadrupole (tsq) mass spectrometer. in total, 4092 proteins were identified, which represents a three-to four-fold increase compared with the standard shotgun analysis of the same sample. out of these, 2240 were only accessible by one of the techniques and, besides, the detection limits were not similar. we hypothesized that an srm-based targeted analysis will allow detection and quantitation of most of these proteins, even without the application of proteome fractionation. we can conclude that all peptides from the library with ms/ms spectra of the total intensity above 10,000 are easily detectable in the total protein extracts. p-02.08.5-012 transcriptome sequencing based identification of alternative oxidase genes in white waterlily, nymphaea alba alternative oxidases (aoxs) are the terminal oxidases in the respiratory electron transport chain of plants. they reduce molecular oxygen to water with low proton translocation across the inner mitochondrial membrane. in plants, aoxs increase local tissue temperature to release volatile compounds thereby attracting pollinator insects and regulation of mitochondrial retrograde signaling pathway. regulation of retrograde signaling pathway is currently under investigation to improve cultivation studies in many plants. water lilies are aquatic ornamental and economically valuable plants classified under nymphaea family. nymphaea alba, white water-lily, has a special focus since its applications in landscaping of parks and gardens, farming as vegetable and medical applications. however, cultivation of n. alba is a challenging process. we hypothesized that by controlling alternative oxidases, success rate can be increased for n. alba cultivation. to identify alternative oxidase encoding genes in n. alba, we performed transcriptome analysis. by using transcriptome analysis data, aox gene sequences, subcellular localization of aox proteins and structural modelling of aox proteins were predicted. in 272934 transcripts, database search with trinotate tool revealed 77 transcripts with aox domains characterized in known alternative oxidases. blast analysis of these 77 sequences with known aox proteins revealed three distinct aox genes (nalba-aox1, nalba-aox2 and nalba-aox4). after subcellular localization analysis of three identified aox proteins by using targetp server tool, nalba-aox1, nalba-aox2 are predicted as mitochondrial while nalba-aox4 is localized in chloroplasts. template based structural modelling results showed that all identifed proteins are statistically similar to known structure models of corresponding aoxs. most environmental contaminants have toxic and mutagenic effects on living organisms as a result of the activation of free radical formation and inhibition of reparation activity. it is becoming relevant to search for protectors of natural origin from the effects of xenobiotics. many biologically active substances (bas) of inartificial origin are found to be antioxidants and can increase the body's resistance to the toxic and mutagenic effects of a wide range of pollutants. the aim of the study was to investigate the antioxidant and antimutagenic properties of bas from medicinal plants limonium gmelinii (plumbaginaceae) and inula britannica (compositae). the antioxidant potential of plant extracts was determined by the activity of superoxide dismutase (sod), catalase, and the content of malonic dialdehyde. mutagenic and anti-mutagenic properties of the extracts were determined in the test by counting chromosomal aberrations in root meristem of barley seeds. barley seeds were treated with an aqueous solution of unsymmetrical dimethyl hydrazine (udmh), which is highly toxic i class hazardous material, well known pro-oxidant. the results showed that udmh enhanced the process of lipid peroxidation and decreased the mitotic activity. treatment of barley seeds with extracts from i. britannica and l. gmelinii and their germination in the presence of stress factors stimulated antioxidant defenses in the primary roots of barley seeds. increase of the activity of sod and catalase, and reduction of peroxidation level of lipids were observed. cytogenetic study showed no mutagenic activity in plant extracts. when effects of plant extracts and udmh were combined there was a significant reduction in the frequency of structural mutations, induced by the toxicant. conclusion about the presence of the antioxidant and antimutagenic activity in the studied plant extracts is made. the work done within the framework of the mes project (no. gr 0115rk00378). p-02.08.5-014 comparative analysis of cytokinin dehydrogenase inhibition and trans-zeatin treatment in arabidopsis seedlings j. nov ak 1 , v. koukalov a 1 , z. medvedov a 1 , c. martin 1 , j. hradilov a 1 , l. sp ıchal 2 , b. brzobohat y 1 1 mendel university in brno, brno, 2 palack y university in olomouc and centre of the region han a, olomouc, czech republic cytokinins are plant hormones regulating many processes during plant life ranging from germination to senescence. manipulation of cytokinin levels and their impact on plant vitality, production and ability to defend against stresses is in great interest of agriculture. in this work we focused on comparison of inhibitor of the cytokinin degradation incyde (2-chloro-6-(3-methoxyphenyl)aminopurine) and exogenous application of trans-zeatin on arabidopsis thaliana seedlings. transcripts of genes regulating cytokinin metabolism were analysed by rt-qpcr analysis. classical cytokinin root essay revealed that incyde effect is comparable to that of trans-zeatin in a similar concentration-dependent manner. besides a negative effect on the primary root length, both substances induce flavonoid accumulation and an increase in the root hairs formation. histochemical staining of transgenic plants expressing glucuronidase (gus) under cytokinin-responsive promoter of arr5 gene revealed increased gus activity in cotyledons following incyde treatment suggesting diverse localization of cytokinin modulation upon trans-zeatin and incyde treatment, respectively. possible molecular differences originating in different cytokinin population and distribution following trans-zeatin or incyde treatments were monitored on the level of gene expression and via an lc-ms proteome analysis in roots and shoots of 14-day-old plantlets. rt-qpcr analysis revealed an alteration in cytokinin metabolism that could explain observed differences on the proteome level between incyde and trans-zeatin treated seedlings. pharmacologically inhibited cytokinin degradation could be very efficient tool for modulation of cytokinin levels. interestingly, the application of incyde and trans-zeatin shows a contrasting spatial and temporal pattern on molecular levels. incyde represents potent growth regulator with interesting properties useful for agriculture. p-02.08.5-015 the expression yield of prokaryotic alphaamylase is significantly magnified by molecular cloning techniques randomly hydrolyzing glycosidic bond alpha-amylase has been traditionally employed in bread and similar industries. in that regard, increasing the overall expression level of the enzyme is a crucial concern in biotechnology. to reach the goal, appropriate alpha-amylase producing species and expression vector were carefully selected. therefore, genome of bacillus subtilis was extracted and amplified by polymerase chain reaction (pcr) using specifically designed primers. subsequently, the extracted gene was inserted in expression vector pht43 and transferred to e. coli as intermediate host followed by bacillus subtilis host replacement. the recombinant vector was expressed in bacillus subtilis and the expression was evaluated by agarose gel electrophoresis. relative purification of the recombinant enzyme was performed by 50 kda filtration to remove impurities. to identify the biochemical characteristics, starch was used as specific substrate to measure enzyme activity and the enzyme was exposed to various ph and temperatures. the extra-cellular expression of alpha-amylase enzyme was successfully elevated by 5 folds in comparison to the native enzyme. the optimum temperature and ph for the enzyme was carefully determined as 70°c and 6, respectively. the enzyme was stable at 50°c, but thermal stability was dramatically decreased at higher temperatures up to 70°c. kinetic parameters were also measured; vmax was 1.998 u/ml min and km was 3.998 mg/ml. it is concluded that the elevated expression extent of recombinant alpha-amylase together with appropriate qualifications could make the clone a good choice for various industrial applications. flax seedlings of cultivars tmp1919, lira and lines g-1071/ 4_o, g-1071/4_k were treated for 4 and 24 hours with 500 lm alcl 3 solution or distilled water (control). twelve small rna libraries were constructed and sequenced using illumina gaiix. to identify known mirnas, obtained sequences were aligned with mirnas from mirbase (http://www.mirbase.org/). fold change value was calculated to identify up-and down-regulated mirnas under al stress. in total, about 40 million raw reads were obtained and 109 conserved mirnas from 26 families were identified. significant expression alterations in flax plants under al treatment were shown for mir319 and mir390. expression level of mir319 was varied in similar way in resistant and susceptible to al genotypes: mir319 was up-regulated after 4 hours of alcl 3 exposure and down-regulated after 24 hours. mir390 expression was increased after 4 hours of alcl 3 exposure and decreased after 24 hours in susceptible to al flax genotypes (lira, g-1071/4_o), while in resistant genotypes (tmp1919, g-1071/4_k) mir390 level was decreased after both 4 and 24 hours of al treatment. in other plant species, mir319 and mir390 were identified as al-responsive. mir319 targets mrna of tcp (teosinte branched/cycloidea/pcf) transcription factors, which control plant growth. mir390 targets mrna of tas3 protein, which regulates lateral root growth via degradation of arfs (auxin response factors). in flax, the involvement of mir319 and mir390 in response to al stress was shown for the first time. moreover, we revealed diverse expression alterations of mir390 in susceptible and resistant to al genotypes. this work was financially supported by grant 16-16-00114 from the russian science foundation. p-02.08.5-017 association genetics of phenylalanine ammonia lyase (pal) and cinnamyl alcohol dehydrogenase (cad) enzymes involved in lignin biosynthesis of european black poplar (populus nigra) b. taskiran, z. kaya middle east technical university, ankara, turkey populus nigra l. are considered as one of the most economically significant forest trees with respect to production of wood, biomass, and other wood-based products. while wood quality and biomass are directly associated with high cellulose content, lignin emerges as an undesirable polymer for both pulp and biofuel manufacturing industries. the aim of the study is by choosing the superior and eliminating the inferior clones to make a contribution to woody feedstock development and to improve wood quality of populus nigra. to estimate association genetics of pal and cad enzymes which have important functions in lignin biosynthesis, the important germplasms of populus nigra has been sampled from 3 year old poplar trees (285 clones x 2 replicates x 2 ramets) which were grown in behic ßbey plantation clone bank in ankara. additionally, five commercially registered clones and six foreign clones were included to the study to make comparison. the average mean values of cellulose, lignin and glucose content were calculated as 21.8 ae 16.29 lg/ml, 23 ae 4.64 lg/ml, and 35 ae 9.71 lg/ml, respectively. even though for pal and cad enzymes, data gathering process have been still resuming, particular clones have been separated from all in terms of pal and cad activities as expected. key words: populus, poplar, lignin, pal, cad, genetic variation, feedstock p-02.08.5-018 proteomic analysis of the molecular mechanisms of the response of plant seeds to pre-sowing treatment by stressors seed treatment with non-ionizing low-level radiation (nr), such as cold plasma (cp) or electromagnetic field (ef), is a modern eco-agricultural technology for stimulation of plant germination and performance. the molecular determinants of seed response to these treatments are not established and no genomic studies of plant seed response to nr have been reported. we studied the effects of pre-sowing seed treatment, using vacuum (7 min), radio-frequency ef (5-15 min) and cp (2-7 min), on germination and growth of non-oilseed helianthus annuus. to gain an insight into the molecular mechanisms underlying effect of nr on sunflower seed germination and dormancy, we estimated changes induced in the balance of plant hormones and differential protein expression. the results of the germination tests and estimation of seedling morphology showed that response develops in time and is stronger when sowing is performed in 7 days in comparison to 3 days after seed treatment. the 2d dige analysis revealed 38 differentially expressed proteoforms in kernels of seeds treated with cp or ef. proteins involved in biological processes of seed maturation, response to stress, response to abscisic acid stimulus, processes of organonitrogen compound metabolism and glucose catabolism were identified. while expression patterns for majority of the proteins were highly specific to cp and ef treated seed kernels, accumulation of several proteoforms of seed storage proteins (ssp), including vincilin-like, miraculin-like protein and albumin-8 were common for both experimental groups. this suggested that response to nr treatment could be at least partially associated to function of ssps in response to oxidative stress that protects proteins required for seed germination and seedling formation. variation of abundance of distinct proteoforms of helianthinin, vicilin-like and 11s globulin-like ssps suggested that post-translational modifications are involved in regulation of the function of ssps. p-02.08.5-019 suppression of lipopolysaccharide-induced inflammatory responses in raw 264.7 macrophages by tuber extract of cyclamen l. turkey is a prominent centre of plant diversity, being the meeting point of three main floristic zones. geophytes which have underground storage organs such as, tubers, bulbs and rhizomes. cyclamen l. is a tuberous geophyte traditionally used by some people for treating whooping cough, headaches or sinusitis, and confirmed to have antioxidant, analgesic and anti-inflammatory properties by several reports. a prolonged inflammatory response is often associated with chronic diseases such as cancer, arthritis and autoimmune disorders. recently, plant based products are used as an alternative and complementary treatment of these diseases. in this respect, the present study was aimed to determine the effects of three cyclamen tuber extracts on lps-induced inflammatory responses of murine raw 264.7 macrophages. firstly, c. cilicium (endemic), c. pseudibericum (endemic) and c. graecum subsp. anatolicum were collected from different localities of turkey. the tubers of plants were air-dried and grounded to fine powder and then extracted with ethanol. cell viability assay was performed to evaluate the nontoxic concentration in cell line by mtt assay. several measurements were performed including tnf-a, no and il-8 concentration assay by elisa after treatment compared to non treated cells to determine the anti-inflammatory activity. also, tnf-a and inos mrna levels were evaluated by quantitative rt-pcr. the cytotoxic activity which is considered safe on raw.264.7 cell were found as 0.5-5 lg/ml. studied cyclamen taxa inhibited tnf-a and il-8 release on lps stimulated-raw.264.7 in a concentration-dependent manner. among the three cyclamen tuber extracts evaluated, the highest nitrite-associated no inhibitory activity was obtained from c. pseudibericum compared to other two cyclamen l. taxa. collectively, these results suggest that cyclamen tuber extracts possess anti-inflammatory properties. p-02.08.5-020 in vitro hypoglycemic activity of ziziphus jujuba recent reports have indicated that continuous treatment with nutritional jujuba (ziziphus jujuba miller) fruit extracts in diabetic rats improved glucose utilization and produced a significant decrease in the blood glucose. in the present study, hypoglycemic activity of z. jujuba was investigated using various in vitro techniques. the hypoglycemic effect of z. jujuba in phosphate buffered saline which grown in balıkesir was studied by measuring glucose adsorption, glucose diffusion and glucose uptake by yeast cells. the glucose content in the solution measured by spectrophotometrically with commercially kits. the adsorption capacity of the z. jujuba was found to be directly proportional to the molar concentration of glucose. the glucose binding capacity of extract increased in higher glucose concentratrations. there was significant differences were observed between the adsorption capacities of z. jujuba and control samples (p < 0.05). the rate of glucose diffusion was directly proportional to the time. diffusion rate was significantly lower in the solution containing z. jujuba compared to control (p < 0.05). the extract demonstrated significant inhibitory effects on movement of glucose into external solution across dialysis membrane compared to control. the rate of glucose transport across cell membrane in yeast cells was observed to be inversely proportional to the molar glucose concentration. z. jujuba inhibited glucose transport across the yeast cells. the results showed that z. jujuba reduced glucose levels at least by three mechanisms. first by increasing glucose adsorption capacity during postprandial hyperglycemia; second by retarding glucose diffusion rate and third, at the cellular level by inhibiting glucose transport across the cell membrane. all of these decreased the absorption of glucose in the intestinal cells and the concentration of postprandial serum glucose. p-02.08.5-021 cucurbitacin b increased the anticancer effect of imatinib mesylate through inhibiton of matrix metalloproteinase-2 expression in colorectal cancer cells f. bakar ankara university, ankara, turkey several natural products have been investigated for their anticancer effects. among these, cucurbitacin b (cub) has been reported as its inhibitory effects on cancer cell proliferation. matrix metalloproteinases (mmps) belong to endopeptidase family and they are received as potential biomarkers for several types of cancer. the aim of this study is to investigate the effect of cub in combination with imatinib mesylate (im) on mmp-2 mrna expression of human sw480 colorectal carcinoma cells. the cytotoxicity analysis was performed via mtt assay. muse cytofluorimetric analysis system was performed to evaluate apoptotic cell population. the mmp-2 mrna expression was determined by quantitative real-time pcr. this study was supported by scientific and technological research council of turkey grant, sbag-114s871. data obtained from the cell culture experiments were expressed as mean ae sd and one-way anova test was applied for multiple comparisons. cub alone significantly inhibited cell growth at 10 lm and higher concentrations. the most potent effect was observed in cub-im combination treatment with 3.51 lm ic 50 value. in cub-im treated group, the apoptotic effect was higher than cub and im treated groups. cub-im induced apoptosis significantly at 10 lm concentration when compared to control and 1 lm (p < 0.05). cub alone showed inhibitory effects on mmp-2 mrna expression at 1 lm and higher doses significantly (p < 0.05). the results showed that the combination treatment of cub with imatinib synergistically inhibited human sw480 cell growth and induced apoptosis by increasing the anti-histone antibodybound nucleosom levels and annexin v binding. although cub could inhibit mmp-2 expression alone at higher treatment doses, it enhanced the inhibitory effect of im on mmp-2 synergistically in a dose dependent manner. in conclusion, this study suggests that cub combined with imatinib mesylate may enhance the effects of chemotherapy in patients with colorectal cancer. plants are most important parts of natural resources that alternatively referred to synthetic drugs for reasons such as being less side effects and lower costs. ziziphus jujuba miller (z. jujuba), a plant used in traditional medicine, is one of the most important ziziphus species belonging to rhamnacea family. the fruit and seeds of this plant are used different purposes such as antiinflammatory, antioxidant, immune-stimulant and wound healer. in this study, we investigated the antibacterial effects of z. jujuba. the aims of this study were to screen the antibacterial activity of z. jujuba. the extract was obtained from z. jujuba fruits pulverized with the aid of ball mill using 50% aqueous-ethanol solution. extracts were screened for antimicrobial activity against six different standard strains of bacteria by determining minimum inhibitory concentration (mic) according to clsi criteria. serial dilutions are made between 64 mg/ml and 0.031 mg/ml concentration range. the lowest concentration of wells that no visible growth has been accepted as mic value. materials in the mic and lower concentrated wells were transferred to 5% sheep blood agar petri dishes for calculation of minimal bactericidal concentration (mbc). the lowest concentration that no colony formation has been accepted as mbc value. jujuba showed the most potent effect on strain of s. aureus atcc 29213 is gram-positive cocci (mic: 2 mg/ml). the mic values of other gram-positive bacteria s. aureus atcc 43300, e. faecalis atcc 51219, l. monocytogenes f 1483 and m. smegmatis cmm 2067 were detected as 8, 16, 8 and 8 mg/ml respectively. mic values of gram-negative bacilli were detected as >64 mg/ml. consequently, z. jujuba was found to be effective on grampositive cocci bacteria (s. aureus atcc 29213, s. aureus atcc 43300 and e. faecalis atcc 51219). the strongest effect was observed on s. aureus atcc 29213 strain. in contrast, extract showed less effect on gram-negative bacilli. p-02.08.5-023 selective cytotoxic effect of morus rubra extract in human lung cancer cells through enhancing apoptosis and cell cycle arrest cancer is a disease that develops as a result of unlimited proliferation of abnormal cells that occurs due to loss of control over the mechanisms of normal growth and differentiation of cells. morus rubra, known as "red mulberry" belongs to family of moraceae. for many years, the fruits of morus species have been used to treat many diseases in traditional medicine. biological effects of morus species is predominantly attributed to its content of polyphenolic compounds. many studies have evaluated the cytotoxic effects of different morus species, but there is no study about cytotoxic effect of m. rubra. in this study, we aimed to evaluate the cytotoxic effect of m. rubra extract in human lung cancer cells (a549) with regard to apoptosis, cell cycle and mitochondrial membrane potential. cytotoxic effect of m. rubra extract on human lung cancer cells was determined using mtt assay. then, mechanisms of cytotoxic activity of m. rubra extract on a549 cells were examined in terms of cell cycle, apoptosis and mitochondrial membrane potential using flow cytometric methods. m. rubra extract exhibited selective toxicity against a549 cells compared to normal foreskin fibroblast cells. we determined that m. rubra extract increased cell cycle arrest at g 1 phase, the level of apoptotic cells and decreased mitochondrial membrane potential in a549 cells. our results showed that m. rubra extract has pro-apoptotic and antiproliferative effect in a549 cells. further studies are also needed to fully mechanisms underlying this effect of m. rubra extract. p-02.08.5-024 dipeptidyl peptidase iv inhibitory activity of arctium tomentosum l. a. zeytunluoglu 1 , f. zihnioglu 2 1 denizli vocational school of technical sciences, pamukkale university, denizli, 2 department of biochemistry, faculty of science, aegean university, izmir, turkey type 2 diabetes mellitus (t2dm) is rapidly growing metabolic syndrome of multiple aetiologies causing hyperglycaemia with insulin resistance at cellular level. a novel approach in the treatment of t2dm is based on preventing of rapid inactivation of the incretin hormone glucagon-like peptide-1 (glp-1) and glucose-dependent insulinotropic polypeptide (gip) by dipeptidyl peptidase-iv enzyme. in this study; dipeptidyl peptidase iv (dpp-iv; ec 3.4.14.5) inhibitory activity of the aqueous and methanolic extracts of arctium tomentosum leaves were successfully tested in vitro conditions. our study revealed that both aqueous and methanolic extracts obtained from test material had a significant dppiv enzyme inhibitory activity in changing ratio. the ic 50 values were also determined by nonlinear regression curve fit using graph pad prism 5.0 with appropriately diluted of lyophilized arctium tomentosum. diprotein-a (ile-pro-ile) was used as reference inhibitor. a. tomentosum aqueous extracts showed ic 50 4.6 lg/ml while the standard (positive control) diprotin a displayed the ic 50 value of 10.1 lg/ml. this study demonstrates that a. tomentosum aqueous extracts could be a good lead for further development as a new antidiabetic agent. p-02.08.5-026 dna recognition determinants of arabidopsis thaliana b3 transcription factors g. sasnauskas, k. kauneckaite, k. lapenas, v. siksnys institute of biotechnology, vilnius university, vilnius, lithuania transcription, one of the most important cellular processes, is regulated by transcription factors (tf), proteins that often directly interact with gene promoter sequences. tf binding to dna is mediated by various dna binding domains. the b3 tfs constitute a large, plant-specific protein family (approx. 10% of all tf proteins in the flowering plants), which is characterized by the presence of one or several small (approx. 110 amino acids) b3 dna binding domains. currently the b3 tfs are divided into four groups (lec2-abi3/val, arf, rav and rem). the preferred recognition sites were identified for representatives of all groups except the rem family. currently, only a single structure of a dna-bound b3 domain (arf1, arf family) is available, thus the mechanism of site-specific dna recognition by the lec2-abi3/val and rav b3 domains remains poorly understood. based on the arf1-dna structure (pdb 4ldx) we have built homology models of dna-bound b3 domains from a. thaliana abi3 (lec2-abi3/val family) and nga1 (rav family) transcription factors, mutated putative dna-interacting amino acid residues and characterized the dna binding ability of the purified mutants using electrophoretic mobility shift assay and a set of radiolabelled dna substrates carrying various variants of the optimal recognition site. we confirm the importance of several positively charged amino acid residues, which are conserved between the abi3/ nga1 b3 domains and structurally related dna-binding domains of bacterial restriction endonucleases ecorii, bfii and ngoavii; furthermore, we identify residues in the 'n-arm' and 'c-arm' loops that may be involved in specific interactions with the dna bases. our results therefore help us refine the homology models of the dna-bound b3 domains and in the future may help us predict the dna binding properties of currently uncharacterized b3 domains. p-02.08.5-027 immunohistochemical analysis of inhibitory effects of origanum hypericifolium oil on dipeptidyl peptidase iv in streptozotocininduced diabetic rats p. ili 1 , a. zeytunluoglu 2 1 denizli health services vocational high school, pamukkale university, denizli, 2 denizli vocational school of technical sciences, pamukkale university, denizli, turkey diabetes mellitus (dm) is a serious metabolic disorder with micro-and macro-vascular complications that result in a significant morbidity and mortality. glp-1 and gip have significant role in pancreatic beta cells and prevention of inactivation of them by dipeptidyl peptidase iv (dpp iv) inhibition is a novel approach to treatment of dm. origanum hypericifolium (lamiaceae) is an endemic turkish plant and its essential oil is mainly composed of monoterpenes including carvacrol and thymol. streptozotocin (stz) is used to induce diabetes in rats. the aim of this study is to investigate the inhibitory effects of o. hypericifolium essential oil on the dpp iv in stz-induced diabetic rats. the animals (female spraque-dawley rats) were assigned to four groups (group 1: control, group 2: stzinduced diabetic, group 3: o. hypericifolium injected, group 4: stz-induced diabetic and o. hypericifolium injected). dm was experimentally induced in groups 2 and 4 by a single intraperitoneal injection of stz at a dose of 45 mg/kg body weight. in groups 3 and 4, rats were intraperitoneally injected with o. hypericifolium oil at a daily dose of 1 ml/kg body weight for 42 consecutive days. at the end of the experimental period, all animals were sacrificed by cervical dislocation under ether anesthesia and liver and kidney tissues of each animal were rapidly excised. tissues were fixed in sainte-marie fixative. after routine histological processes, samples were embedded in paraffin, immunohistochemical staining for dpp iv was performed on sections and then they were photographed. the immunohistochemical reaction intensity differences were observed between the groups. in conclusion, the immunohistochemical distribution of dpp iv in the tissues that the test oil was applied in the diabetic rats may be important for the investigation of the inhibitory effects of oil on the enzyme. moreover, our findings suggest that o. hypericifolium oil may be used for prevention of diabetic diseases. introduction: all eukaryotic cells need microtubules for purposes of nuclear and cell division, organization of intracellular structure, and intracellular transportation, as well as ciliary and flagellar motility. microtubules are made of polymerized a/btubulin subunits. mec17 is important for microtubules, because it encodes the enzyme that adds acetyl groups to lysine 40 (k40) of tubulin. k40 is largely conserved in a-tubulins of many eukaryotes, and acetylation is thought to stabilize microtubule structure. in algae, the effect of acetylation by mec17 on flagellar motility and phototaxis has not been tested previously. materials and methods: in this study, mec17 mutant chlamydomonas reinhardtii cells were compared to wild-type cells to see the effect on flagellar motility and phototaxis. we tested phototaxis, eyespot size and quantity under the microscopy. in addition to this, we fixed cells and examined them by immunofluorescence microscopy using antibodies to tubulin, acetylated tubulin, and photoreceptor. results: we observed that some mec17 mutant cells contain more than one eyespot. we detected no acetylated-tubulin (ac-tub) by immunofluorescence. the cells still phototax and have normal motility discussion and conclusion: interestingly, mec17 cells still have the ability to phototax and they have normal flagellar motility, even though they contain occasional additional eyespots and no ac-tub. chlorella vulgaris as a model system for screening of plant growth modulators p. volynchuk 1 , e. marusich 1 , r. chuprov-netochin 1 , j. neskorodov 2 , y. mishutkina 2 , s. leonov 1 1 life sciences center, moscow institute of physics and technology, dolgoprudny, 2 center "bioengineering", russian academy of sciences, moscow, russia the discovery of new plant growth modulators became extremely important task as an alternative approach to overcome plants resistance to herbicides and pesticides, which leads to harmful action on plants and land rising, environmental and ecological problems. small molecules provide agricultural biotechnology with valuable tools, which help to circumvent the need for genetic engineering and offer unique benefits to modulate plant growth and development. we developed a system to explore molecular modes of action of plant growth modulators using chlorella vulgaris model. our model allows applying high content screening approach in 96well plate format for fast and robust effect assessment of large number of tested modulators. chlorella v. was grown in climate chamber under optimized constant temperature (20°c) and light conditions (16:8 hours/light:dark). modulating effect of tested compounds was estimated by spectrophotometric measurement of microalgae density at the beginning of the experiment (start point-0.1od) and 48 hours later. to validate our system we used known cytokinins and auxins (10 mm) as positive controls of growth stimulation. we showed that in presence of each compound the density of chlorella v. was increased in 7-11 times range, compared with only 2 times increase in control group. eight new chemicals (10 lm), which demonstrated modulation effect on nicotianatabacum l. pollen and arabidopsis thaliana models, were tested on developed chlorella v. model. positive controls showed no stimulating effect at this concentration, while tested compounds were confirmed as hits and increased the density up to 400%. we demonstrated that developed model system, based on chlorella v., is an effective system for primary screening of plant growth modulators. the main advantages of this system are short time of assay, simplicity of performance, possibility of automation and low cost. selected hits can be recommended as perspective candidates for future test on crop field. sunflower is under a big threat of downy mildew which is a fungal disease caused by plasmopara halstedii. the disease can cause up to an 80% yield loss in sunflower production. downy mildew resistance genes (r) denoted as pl has been discovered to date in sunflower. in recent years, single nucleotide polymorphism (snp) markers have become widely used in plant breeding programs. in this study snp markers have been currently developing for pl 6 , pl 8 , pl 13 , pl arg genes by competitive allele specific pcr (kasp) assay which enables bi-allelic scoring of snps and insertions/deletions (indels) at specific loci. in total 66 sequence tagged site (sts) sequences from ncbi were aligned for pl 6 , pl 8 , pl 13 , pl arg genes to identify conserved regions for each gene. based on the conserved regions, specific pcr primers were designed in order to make sequencing of these genes in five crosses and their f 2 progenies. sequence data will be used to design an allele specific primer maches the target snp and amplifies the target region with the common reverse primer provided by kasp genotyping assay. snp markers linked to pl genes which are being developed in this study, have the potential to be used in marker assisted selection (mas) for sunflower breeding programs. p-02.08.5-031 investigation of the antidiabetic effects of hibiscus sabdariffa, teucrium polium and myrtus communis in hepg2 cells line s. altundag 1,2 1 pamukkale university, denizli, 2 istanbul medeniyet university, istanbul, turkey some antidiabetic plants currently are used in alternative treatment of type ii diabetes. hibiscus sabdariffa, myrtus communis and teucirum polium plants are also known for their antidiabetic properties. hibiscus sabdariffa, myrtus communis and teucrium polium; depending on the impact on hepg -2 cells to investigate the possible mechanisms of type ii diabetes with researches on glucolysis and glucogenesis pathways gene expressions (pk m , glut-2,pepck). plants were obtained in dried state from reliable herbalists in denizli and mersin. plants treated with the extractor device. plants obtained aqueous extract was subjected to lyophilization process. human cancer cells have been used throughout the study. the cytotoxicity of the cells was measured by elisa plate reader . total rna was isolated using trızol ò solution was carried out according to the instructions of the manufacturer's (thermo scientific) recommended procedures were performed, but we have to optimize our own laboratory conditions. pk m , glut-2,pepck genes were synthesized by b _ ioneer. during our study the activation of certain genes (pk m , glut-2,pepck) were examined by real time pcr. in our study hibiscus sabdariffa, mrytus communis and teucrium polium plant to extract applied hepg -2 cell line in the gluconeogenesis and glycolysis pathways in involved in some important genes (pepck, pk m , glut-2) analyzed the expression levels . teucrum polium plant extract is applied in hepg -2 cells the glycolysis pathway genes (pk m glut-2) an increased expression also genes of gluconeogenesis pathway (pepck) were not decreased. however hibiscus sabdariffa and the expression of genes involved in glycolysis and gluconeogenesis pathway mrytus communis plants were observed to have a full effect as diabetic or hypoglycemic . in this context, it is considered that the plant teucrum polium on the line hepg -2 cells showed antidiabetic effect. p-02.08.5-032 purification of b-glucosidase from malatya apricot (prunus armeniaca l.) seeds and some of its biochemical properties h. kara 1 , s. sinan 2 , z. ekmekci 2 , y. turan 2 1 university of balikesir faculty of veterinary, balikesir, 2 university of balikesir faculty of arts and sciences, balikesir, turkey introduction: b-glucosidases are one of the key enzymes in carbohydrate metabolism and located in glycosyl hydrolases (ec 3.2.1) family. plant b-glucosidases have biotechnological significance as they are effective on glycosidic bonds of flavor and aroma precursors in plants. b-glucosidases that located in fruit seeds are important because they affect the amygdalin. aim of this study is purification and partially characterization of b-glucosidase from malatya apricot seeds. materials and methods: apricot seeds were homogenized with extraction buffer to prepare of crude extract. the enzyme protein was precipitated with 60% ammonium sulfate then purified by hydrophobic interaction chromatography using sepharose 4b-ltyrosine-1-naptylamine gel. para-nitrophenyl b-d-glucopyranoside (p-npglc) was used as substrate to determine biochemical properties of the enzyme. the optimum ph was determined using buffers between ph 2-12 and thermal optima was determined using 25-85°c temperature range. inhibitory effects was determined with 1 mm substances. results: the enzyme was 11.1-fold purified with yield of 14%. purified b-glucosidase from apricot seed was visualized about 60 kda molecular weight on sds-page. the kinetic parameters were determined against p-npglc substrate as km and vmax values of 2.5 mm and 58.1 eu, respectively. the optimum ph and temperature were determined 5.0 and 55°c respectively. effects of cacl 2 , kcl, nacl, mgcl 2 , k 2 so 4 , na 2 so 4 , cuso 4 , fecl 3 , pb(ii) acetate, agno 3 , zncl 2 and glucose on purified enzyme activity were investigated. kcl, na 2 so 4, pb(ii) acetat and cuso 4 reduced the enzyme activity. discussion and conclusion: in this study, b-glucosidase was purified from malatya apricot seed and some of its biochemical properties were determined. because this enzyme has pharmaceutical importance hydrolyzing amygdalin. the results showed that immobilized almond b-glucosidase was used to break amygdalin and release -cn compound that effective to shrink cancer mass. p-02.08.5-033 a new affinity gel for purifing polyphenol oxidase enzyme a. erg€ un 1,2 , o. arslan 2 1 balikesir university, science and technology application and research center, balikesir, 2 department of medical chemistry, faculty of science, balikesir university, balikesir, turkey polyphenol oxidase (ppo) enzyme, sometimes called as phenol oxidase, catecholase, phenolase, catechol oxidase or tyrosinase, is considered to be an o-dipenol. ppo (ec 1.14.18.1), a multifunctional copper containing metalloenzyme, is widely distributed in nature. ppo exists in many kinds of plants and fungi, such as banana, mushroom, butter lettuce, napoleon grape, potato, coffee, marula fruit, artichoke heads, longan fruit, tobacco, wheat flour. in this study, a novel affinity chromatography gel was synthesized for purifing ppo enzyme. the affinity chromatography gel was synthesized by coupling aniline as a specer arm to cnbr activeted sepharose-4b. then, p-amino benzoic acid was coupled to aniline as a ligand. ppo was purified from musa sapientum var. cavendishii (banana) by using sepharose-4b-aniline p-amino benzoic acid affinity chromatography gel. % 4.49 yield and 33.4 fold purification were achieved. sds-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent mw of 35 kda. the v max and k m of the purified enzyme were determined 42,628 u/ml min and 9.27 mm, respectively. p-02.08.5-036 phenolic content and antioxidant capacity of gamma irradiated olive leaves m. e. diken 1 , b. kocat€ urk 2 , s. dogan 1 , h. tuner 1 1 balikesir university, balikesir, 2 kavram vocational school, istanbul, turkey in this study, dried in diffirent ways (such as microwave, infrared, convection heaters and under normal atmospheric conditions) olive leaves has been used as experimental material. radiation has been applied to dried olive leaves in three diffirent dosages in room temparature. the amount of radiation to be implemented to the samples have 3, 5, 10 kgy/min. in this study, how gamma rays (radiation) effects phenolic components, total phenolic content and antioxidant capacity of dried olive leaves has been determined. the phenolic components, total phenolic content and antioxidant capacity were analysed with hplc, folin ciocalteu and dpph radical scavenging method, respectivelty. gamma rays is well known as a decontamination method for many foodstuffs and plant materials, being an environment friendly and effective technology to resolve technical problems in trade and commercialization. for this reason, nowadays it is utilized as an alternative method of sterilization. gamma rays are of ionizing radiation. when ionizing radiation interacts with matter, generally it causes a break in the molecular bonds and/or breaks the bonds between the molecules. these intermediates have unpaired electron and called free radical. gamma radiation or the radiation-induced free radicals would break or cause damage to the dna molecules of living organisms. gamma irradiation is predict to change phenolic content and antioxidant capacity in living tissues. phenolic compounds are secondary plant metabolites naturally present in fruits and vegetables. in recent years there has been a growing interest in food phenolics because of their potential health benefits mainly due to their antioxidant and free radical scavenging activity. despite the controversy about potential risks posed by genetically modified plants on human health, environment and microorganisms, cultivation area of these crops increases day by day. this increment has revealed concerns especially related to hgt. hgt studies indicated that antibiotic resistance genes in gm plants have a potential to transfer to soil microorganisms. in this study, hgt of widely used genetic elements such as regulatory sequences, from transgenic plants to bacteria was investigated. three soybean feed and four seed examples from turkish feed manufacturers' association were used for genetic analysis based on foreign gene determination. gmo analysis were conducted by camv 35s promoter-specific pcr in genomic dnas. in gm positive samples, genomic dnas sheared into appropriate fragment size by ultrasonication for the purpose of bacterial transformation. presence of 35s promotor region in fragmented dnas was proved by pcr. escherichia coli dh5a strain was transformed by fragmented dna samples according to cacl 2 method. 35s promotor sequence screened by using pcr in bacterial genomic dnas. as a result of gm screening, all feed and three of the seed samples were found to be transgenic. ultrasonication conditions were optimized for shearing dna's to 450-500 bp for bacterial transformation. fragmented dnas confirmed for carrying intact 35s promotor sequence. none of bacterial genomic dnas were found to be 35s-positive. according to the transformation results, absence of 35s promotor sequence in all tested bacterial genomic dnas, proved the dna samples belonging to gm plants can not transfer into compotent e. coli dh5 under laboratory conditions. for verification of this finding, transformation studies will continue with acinetobacter baylyi bd413 strain which is naturally competent soil bacterium for natural transformation. we believe that all of our findings will contribute to constitute transformation system which can be used as model in hgt studies. p-02.08.5-038 preliminary studies on differential methylation in a and d sub-genomes of upland cotton (gossypium hirsutum l.) four species of cotton (gossypium l.) provide raw materials for the textile industry. among the four species, two have diploid genome and another two have tetraploid genome. tetraploid genome consists of a and d sub-genomes. a sub-genome belongs to asian cotton while d-sub genome belongs to american cotton. previous studies revealed that d sub-genome of gossypium species contributes to the superior yield and quality of tetraploid gossypium l. species (atdt). dna cytosine methylation of four regions of dna sequences located on a and d sub-genomes of gossypium hirsutum l. texas marker 1 (tm-1) was investigated using bisulfite sequencing technique. among the regions studied two could not be located on subgenomes due to sequence identity match between a and d subgenomes. on the other hand two dna regions could be located on a and d sub-genomes using the blast searches. some of the dna sequences located on different sub-genomes showed polymorphic nucleotides including c/t and g/a polymorphisms. in silico analysis indicated that some alleles located on different sub-genomes of cotton have c/t and g/a polymorphisms. c/t polymorphisms between the sub-genomes could be thought as unmethylated cytosine using the bisulfite sequencing technique. this indicated that an extra attention needs to be paid in dna total cytosine methylation studies in polyploid species such as cotton using bisulfite sequencing, methylation sensitive amplification polymorphism (msap), whole-genome bisulfite sequencing (wgbs). otherwise, t in c/t polymorphism between the subgenomes could be thought as unmethylated cytosine. based on the two genomic regions we could conclude that a sub-genome may be more methylated than d sub-genome. differential methylation of genes located on different sub-genomes may provide another mechanism responsible for differential gene expression of genes located on different sub-genomes. p-02.08.5-039 cleaved minisatellite locus (cml) markers for fingerprinting of cotton cultivars grown in turkey e. u. gocer, m. karaca akdeniz university, antalya, turkey after their discovery by alec jeffreys in 1984, minisatellites have been used in genetic studies of many organisms. minisatellites, also called variable number tandem repeats (vntrs), are composed of arrays of longer repeats mostly dispersed throughout heterochromatin (centromeres and telomeres). direct amplification of minisatellite regions of dna using a single core primer is a powerful method to amplify minisatellites (damd-pcr). although the damd-pcr technique has been applied to many plant species, the level of polymorphisms in cotton (gossypium l.) is very low due to very narrow turkish cotton genetic base. the objective of this study was to improve the level of polymorphisms by cleaving minisatellite loci by restriction enzyme digestion. genomic dna samples of twenty-one turkish cultivars, pima 3-79, tm-1 and ps-7 were extracted. twenty-one minisatellite primers were screened using the damd-pcr technique. monomorphic amplicons were digested using several restriction enzymes. three to five micro liters of amplified products were digested with various restriction enzymes. digested products of minisatellite loci were separated in 3% high resolution agarose gels. comparison studies of digested and undigested markers revealed that cleaved minisatellite markers showed polymorphisms in those cotton lines that could not be differentiated by microsatellite and minisatellite markers. this approach was called cleaved minisatellite locus markers (cml). the amplification reactions of minisatellites used touch-down (td) cycling conditions. the use of td offered a simple and rapid means of optimizing polymerase chain reaction (pcr), increased specificity, sensitivity, and efficiency without the need for lengthy optimizations of minisatellite primers. the cml markers were obtained at a 55°c annealing temperature, which is a temperature higher than those used in random amplified polymorphic dna (rapd) inter-simple sequence repeat (issr) markers. p-02.08.5-040 association between cytosine methylation and tissue specific expression of microsatellites a. g. ince, m. karaca akdeniz university, antalya, turkey heritable covalent modification of dna, rna or protein without altering their primary sequences is defined epigenetics. because all biological events are influenced by epigenetics, it is one of the most important fields in science. dna methylation is one of the most important epigenetic mechanisms. dna cytosine methylation is a process by which methyl groups are added to cytosine bases of dna. microsatellites, also knows simple sequence repeats are dna sequences consisting of 1-6 nucleotide repeats. there is a large body of information regarding the relationship between microsatellite instability and abnormal gene expression, and between dna methylation and altered gene expression. however, there is limited information on cytosine methylation of microsatellites. in the present study, we investigated whether there is any association between cytosine methylation and tissue specific expression of microsatellites. genomic dna samples of various tissues and developmental stages of pepper line demre sivrisi (capsicum annuum l.) and cotton line tm-1 (gossypium hirsutum l.) were extracted. cdna samples were synthesized using mrna expressed in pepper tissues. cytosine methylation levels were investigated using bisulfite sequencing methods. screening studies of microsatellites revealed that some genes containing microsatellites were differentially expressed in tissues and developmental stages of pepper. microsatellite containing genes that expressed differently among tissues had also showed different methylation levels in cg, chg and chh (where h refers to a, c or t) contexts. methylation level differences between microsatellites were also observed. as far as our knowledge, it is the first report on differential expression of genes containing methylated microsatellites. p-02.08.5-041 pcr-lg: an alternative way to assign the chromosome location of genes/markers in cotton a. aydin, m. karaca akdeniz university, antalya, turkey assignment of genes and dna markers on chromosomes is very important in life sciences, especially for plant breeding and medicine. there are several methods for the assignment of a gene or dna sequence to a specific location on a chromosome. for example, the most widely used technique is the assignment of fluorescently-labeled gene or dna sequences (markers) on chromosomes using the fluorescently-labeled gene (for instance, fish technique). another example is the construction of a genetic map (linkage map) which orders the targeted genes along the dna strand based on recombination frequency. sequencing is the most precise technique in which coding (gene-containing) and noncoding dna region of genes could be located on a chromosome molecule. aneuploid lines could also be used to locate genes in a specific chromosome, but maintenance of these lines is difficult. here we report the use of chromosome substitution lines to indirectly locate genes/markers on chromosomes. we used chromosome substitution lines (csls) that carry a chromosome pair or chromosome arms from gossypium barbadense l. while the rest of chromosomes belong to g. hirsutum l. a total of 10 chls, a homozygous cotton line tm-1 and a double haploid line pima 3-79 were used as plant materials. twenty microsatellite primer pairs were utilized in touch-down polymerase chain reactions. we developed a method, called polymerase chain reaction to locate gene (pcr-lg), to assign genes/markers on chromosome or chromosome arm. with the use of pcr-lg approach any polymorphic genes/markers between tm-1 and pima 3-79 (g. hirsutum and g. barbadense) could be assigned to a chromosome or chromosome arm. results indicated that if 26 csls were used any polymorphic markers (genes) with known primer pairs could be assigned to cotton chromosomes. although we used cotton chromosome substitution lines to validate the proposed technique, it could be applicable all the species that have chromosome substitution lines. p-02.08.5-042 pecularities of genome variability of antarctic hairgrass deschampsia antarctica desv. from the maritime antarctic deschampsia antarctica desv. (poaceae) is the only grass species native to the antarctic region, adapted to harsh environmental conditions. however, reasons for its unique success remain unexplored. stressful environmental factors can influence a plant genome and cause changes in the chromosome number and morphology and increase genetic variation. therefore, the purpose of our research was to explore alterations in the d. antarctica genome both at the chromosomal and molecular levels and to investigate species genome stability using in vitro tissue cultures. plants used for the study were grown in vitro from seeds collected in the argentine islands region during 2008-2014. chromosome number was determined in plant root apical meristems and specimens prepared from tissue cultures. rrna genes localization were determined using the fish technique. moleculargenetic analysis was performed using pcr with polymorphic issr-primers. new forms of chromosome polymorphism, associated with aneuploidy (2n = 13-27), polyploidy (2n = 13-39) and the occurrence of additional b-chromosomes (2n = 26 + 1-3b), were observed. fish analysis also confirmed that genotypes with a different chromosome numbers varied in the number of 5s rdna and 25s rdna sites. assessment of genetic variation demonstrated a low level of diversity: differences between the plants with different chromosome numbers do not exceed the level of within-population variation. cytology analysis of d. antarctica cultured tissues revealed aneuploidy (up to 60.6 %) with predominance cells with diploid and near-diploid chromosome number irrespective of plant's initial karyotype (diploid, mixoploid or polyploid). we assume that discovered intraspecies chromosomal polymorphism is a manifestation of quick genome reaction to harsh antarctic conditions. whereas the results of molecular-genetic analysis and study of cell cultures of this species suggests on the relative stability of d. antarctica genome. p-02.08.5-043 angiotensin converting enzyme inhibitory activity of morchella esculenta (l.) pers a. zeytunluoglu 1 , i. arslan 2 1 biomedical equipment technology, pamukkale university, denizli, turkey, denizli, 2 biomedical engineering, pamukkale university, denizli, denizli, turkey hypertension is a multi-aetiological, chronic pathophysiology that leads to multi-organ dysfunctions like cardiovascular diseases, strokes, and renal complications. natural extracts play an important role in traditional medicines for the treatment of hypertension and are also an essential resource for new drug discovery. mushrooms are use as therapeutics in alternative and complementary medicine as functional food because of contain a large number of biologically active components that offer health benefits and protection against many degenerative diseases. morchella esculenta is one of the most highly priced edible mushrooms worldwide. it contains a wide range of active constituents which include tocopherols, carotenoids, organic acids, polysaccarides and phenolic compounds which exhibit a wide range of medicinal and pharmacological properties including anti-microbial, anti-inflammatory, immunustimulatory, antitumor and antioxidant. in this study; the in vitro angiotensin converting enzyme-i (ace-i) inhibitory activity of m. esculenta peptides were generated by alcalase hydrolysis were studied. the 5 kda < peptides < 10 kda in the ultrafiltration fractions displayed highest ace inhibition (85.9 ae 5.09% at 140 lg/ml). the results indicate that m. esculenta derived peptides may have potential as functional food ingredients in the prevention and management of hypertension. p-02.08.5-046 modulations of antioxidant enzymes, gsts and catalase, by salvia absconditiflora in hepg2 cell line d. irtem kartal 1 , a. altay 2 1 yuzuncuyil university, van, 2 erzincan € university, erzincan, turkey oxidative stress is considered to play a important role in the pathogenesis of aging and several degenerative diseases, such as cancer. in order to cope with an excess of free radicals produced upon oxidative stress, humans have developed several mechanisms for maintain redox homeostasis. these protective mechanisms either scavenge or detoxify ros, block their production, and include enzymatic and nonenzymatic antioxidant defenses. in enzymatic defenses include glutathione s-transferases and catalase enzymes. many epidemiological studies have revealed that there is a strong correlation between consumption of polyphenol-rich foods and the prevention of certain diseases like cancer, cardiovascular diseases and aging. phenolic compounds are abundant in all plants. so, they form an integral part of the human diet. salvia species, commonly known as sage, have been used since ancient times for more than 60 different ailments ranging from aches to epilepsy. there are around 900 species of salvia, 95 of which are represented in turkey including salvia absconditiflora. in this study, s. absconditiflora collected from metu campus (ankara, turkey) is extracted with methanol and water. effects of the water and methanol extracts on the mrna expressions of antioxidant enzymes gstm1 and catalase in hepg2 cells were investigated by q-rt-pcr technique. it was also monitored the effects of the extracts on the enzyme activities of gsts and catalase by spectrophotometrically. water and methanol extracts decreased gsts mrna expression in hepg2 cells for 48 hours and 72 hours incubation and methanol extract decrease catalase mrna expression for only 72 hours incubation. on the other hand, extracts highly increased the gsts and catalase activities in both hour incubation. overall, these results indicate that s. absconditiflora and/or its components have regulatory activities on antioxidant enzymes and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-047 transcriptomics and proteomics approach to drought stress mechanism in wheat b. cevher keskin tubitak marmara research center genetic engineering & biotech. institute, kocaeli, turkey birsen cevher keskin, yasemin yıldızhan, oktay kulen, bayram y€ uksel, selma onarıcı, _ ismail t€ urkan, as ßkım hediye sekmen, u gur sezerman, bu gra € ozer identification of novel stress-responsive genes and their role in drought response is an important area for the improvement of the crops. drought-related genes were investigated in leaves and roots of three wheat genotypes after different drought stress treatments by rna sequencing (rna seq) technology and de novo assembly was performed before comparative transcriptome analysis. analyzing 311 gigabases of 100 bp paired end illumina reads from a hexaploid wheat poly(a) rna library, we identified common and new differentially expressed transcripts. selected differentially expressed genes were confirmed by qrt-pcr. we also performed root proteome analysis with nano electrospray ionization source coupled to a high-performance liquid chromatography system (nanouplcàesiàqtofàms) to identify drought-related proteins. totally 191 proteins were differentially expressed in root tissues of tolerant and non-tolerant wheat genotypes. responses of antioxidative defense system to drought stress were comparatively studied in the same wheat cultivars. similarities between protein and rna levels help increase our confidence in novel biomarkers, differences may also reveal other post-transcriptional regulatory junctures. all these analyses will allow us to get a better idea about the possible role of these genes in the drought-response mechanism. the drought-related genes that are functionally characterized could be introduced into agronomically important wheat cultivars. this work offers a resource for accelerating drought-related gene discovery and improving this important crop. p-02.08.5-048 isolation and characterization of a hexose converter from olive s. altunok 1 , e. d€ undar 2 1 department of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey introduction: hexose sugars are key components of glycolysis and photosynthesis. the genes regulating their conversion into one another, is therefore, of great importance for the control of carbon metabolism. in this study, we report isolation and characterization of a cdna associated with conversion of hexoses in olive. the cdna putatively named aldolase based on bioinformatic and experimental analyses. material-methods: characterization based on nucleotide and amino acids were conducted using bioinformatic tools such as nucleotide and protein blast, bioedit, primer3, finchtv, clc genomic workbench, expasy, targetp, sosui and web promoter scan. comparison of the genomic and cdna sequence of the gene and detailed bioinformatic analyses including cellular location, hydropathy analysis, amino acid-nucleotide composition and predicted 3d structure were also conducted using the bioinformatics tools mentioned above. temporal expression pattern of the putative aldolase were conducted using real-time pcr experiments. sds and western blot analyses were completed while biochemical analyses are ongoing. oleocanthal is an important secondary metabolite that has been reported to be useful against important human diseases including cancer. the aim of this study was to identify and characterize the key biochemical and genetic components of oleocanthal biosynthesis. to determine the biochemical components of the pathway, multiple olive cultivars along with their spatial and temporal points were determined. the expression levels of multiple candidate genes were also aimed via real-time pcr. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes with that of other organisms) were conducted on ncbi web page. phylogenetic tree construction, amino acid composition analysis, nucleotide composition analysis, hydropathy analysis and translations through expasy were conducted. primer3 was used to design forward and reverse primers to amplify the target genes from different olive tissues at different times. analysis of the first candiate gene with bioedit program revealed that a+t ratio was more than g+c according to the nucleotide composition analysis. according to amino acid composition analysis isoleucine, lysine and leucine were more than other amino acids while kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. abundance of hydrophobic amino acids (leucine and isolecine) along with an abundant hydrophilic amino acid (lysine) suggest the existance of hydrophobic pockets in the protein which may mean a membrane bound protein or a sitoplasmic protein with a strong hydrophobic core. the molecular weight of the protein was 56 kda with a pi of 9.21. the protein was found to have a signal peptide. according to the sosui gramn , intracellular localization was found to be in the inner membrane. analysis of other candidate genes contiunes. acknowledgements: this study was supported by tubitak with grant number 110o108. key words: olive, olea europaea l., secologanin, polymorphism, allele diversity p-02.08.5-050 antioxidant potentials of propolis and its bioactive components, and their effects on cyp2e1 gene expression in ht-29 adenocarcinoma cell line a. altay 1 , d. irtem kartal 2 1 erzincan € university, erzincan, 2 y€ uz€ unc€ u yil university, van, turkey propolis is a resinous mixture that is collected by honeybees from plants, and is combined with beeswax and secretions from the bee's salivary glands plus some pollen. it is a rich mixture of polyphenols, flavonoid aglycones, phenolic acids and their esters. it has been used in traditional medicine for thousands of years because of these ingredients. propolis, just like honey, has been the subject of many studies due to its antimicrobial, antifungal, antiviral and hepatoprotective activities. the cytochrome p450 enzymes are involved in phase i xenobiotic and drug metabolism. cyp2e1 is present in high levels in human tumors demonstrated by qrt-pcr and immunohistochemical studies. in this study, propolis collected from datc ßa (mugla, turkey) is extracted with 70% ethanol. phenolic contents of the propolis were measured by lc-ms/ms. cytotoxic effects of the propolis on ht-29 human colon adenocarcinoma cell lines were examined via xtt colorimetric cell proliferation assay. effects of propolis extract and its main bioactive component caffeic acid on the expression of phase i detoxification enzyme cyp2e1 in ht-29 cells were investigated bu using q-rt-pcr technique. ic 50 values for dpph radicals scavenging activities of the extract was calculated as 0.042 mg/ml. tpc and tfc were determined as 168.6 mg gae/g extract and 141.8 mg qe/g extract, respectively. caffeic acid content of the extract was measured as 10.6 lg/g extract. ic 50 values for xtt assay in 48 hours incubation with the extract and caffeic acid were evaluated as 1.16 mg/ ml and 0.149 mg/ml, respectively. propolis extract and its main phenolic content caffeic acid significantly dicreased cyp2e1 mrna expression with 2.4 and 5.3 fold, respectively in ht-29 human colorectal adenocarcinoma cells for 48 hours incubation. overall, these results indicate that propolis and/or its components have regulatory activities on cyp2e1 expression and they may have a potential as a therapeutic agent in the treatment of cancer. p-02.08.5-051 effects of histone h3 lysine 9 inhibition on gene expression profile in tobacco (nicotiana tabacum l.) differentiation is the characteristic of multicellular organism. cellular dedifferentiation underlies topical issues in biology such as reprogramming in stem cell research, regeneration and nuclear cloning, and has common features in plants and animals. the state of dedifferentiation is evidenced by changes in cell morphology, genome organization and the pattern of gene expression as well as by the capability of plant tissues to differentiate into multiple types of cells depending on the type of stimulus applied. chromatin reorganization appears to be a fundamental theme in cellular dedifferentiation and reentry into the cell cycle both in plants and animals. the chemical modifications of histone proteins which are structural units of the nucleosome, generate 'codes' for the recruitment of proteins or protein complexes that affect chromatin structure and gene expression according to 'histone code' hypothesis. methylation of histone h3 at lysine 9 residue by specific methyltransferases suv39h1 in humans and kyp/suvh4 in arabidopsis generates a'code' for the recruitment of hp1 proteins. in this study, to enhance the dedifferentiation efficiency, chaetocin which inhibits suv39h1 has been used at the germination stage of tobacco seeds in in vitro conditions. our results showed that chaetocin induced callus formation from leaf discs of tobacco in the early stage of the inhibitor application. chaetocin can enhance reprogramming of plant cells in seed development treatment as callus induction. it is known that the formation of callus which is the non-differentiated cell community in plants, is a consequence of the changes in the gene expression profile. it has been found that epigenetic modifications play a crucial role in some of these changes. the definition of the genes related to callus formation by the inhibitation of an epigenetic modification -h3k9 methylation-in tobacco might be used to bear on various dedifferentiation-driven cellular processes. induction of tumor cell death by the use of some phytochemicals that consumed through diet, and derived from medicinal plants opens up new horizons for cancer treatment researches. rheum ribes species, which is studied in this research, is one of the commonly used herbs in pharmacological researches. the high content of phenolic compounds in r. ribes extracts were known to be responsible for the high antioxidant and antibacterial activities. our aim in this study is to assess cytotoxic and apoptotic changes by way of implementing methanol extract of the rheum ribes (root) to the mcf-7 breast cancer cell line. cytotoxic effect of rheum ribes extract was evaluated by using the xtt (2,3-bis(2-metoksi-4-nitro-5-sulfofenil)-2h-tetrazolyum) test. in order to determine the dose of ic50, plant extracts were applied as time and dose dependent in the range of 10-500ug. in 72nd hour, the ic50 value is determined as 400ug. to examine the apoptotic effects of the extract, total rnas were isolated from dose group and the control cells firstly, then cdnas were synthesized. expression profile of the target genes(caspase-3, caspase-7, caspase-8, caspase-9, bax, bcl-2, fas) are determined by qpcr. according to the results, when the control group compared with the cells, it was determined that, while 17.33 and 3.05-fold respectively increase in the gene expressions of caspase-3 and caspase-7 of dose group cells, 15.45-fold decrease in the gene expressions of bcl-2. no significant difference was observed in the other genes examined. based on the obtained data, we believe that methanol extract of the rheum ribes induces apoptosis by activating intrinsic pathway. as a result, this plant species can be a new and effective therapeutic candidate for the medical world in search of alternative anti-cancer approaches, and could shed light on the work to be done in this area. p-02.08.5-053 the effect of ferulic acid and 5-fluorouracil combination on apoptosis in pc-3 human prostate cancer cells prostate cancer is quite often seen in industrialized countries and has the second most common death rate due to cancer after lung cancer in men. 5-fluorouracil (5-fu) is a pyrimidine analog and cell cycle-targeting drug by inhibiting dna synthesis. it has been widely used for treatment of several cancers such as gastric, colorectal, and breast cancers. phenolic compounds found in foods are potential antioxidants of harmful oxidative processes related to cancer and also important due to induction of different mechanism such as apoptosis. ferulic acid (fa; 4-hydroxy-3-methoxycinnamic acid), a phenolic compound, is abundant in fruits and vegetables. the purpose of this study was to investigate combination effect of fa and 5-fu on apoptosis in pc-3 human prostate cancer cell line. the effects of 5-fu, fa, and combination of both of them on cell viability were determined by xtt method. total rna isolation was conducted using trizol reagent. expressions of important genes in apoptosis including casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs were investigated in four groups by qpcr. the ic 50 doses of fa and 5-fu were found to be 300 lm and 60 lm for 48 hours in pc-3 cells, respectively. in order to determine combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 40 lm 5-fu). according to qpcr results, a significant increase in the expressions of bax and cycs genes and a decrease in the expression of bcl2 were observed in the fa group, compared with the control group cells. after the treatment with 5-fu, the expression of casp8 was significantly increased compared with the control group. furthermore, combination of fa and 5-fu significantly increased expression of casp7, casp8, fas and cycs genes. in conclusion, comparing with the single treatments, it was observed that combination of fa and 5-fu affected expression of different genes in apoptosis in pc-3 cell line. p-02.08.5-054 combination of ferulic acid and gemcitabine affects expression of some apoptotic genes in pc-3 human prostate cancer cells prostate cancer, the second causing of cancer-related death in men, is an important cancer type due to the late age of onset, slow the progression, high incidence. gemcitabine is an effective agent in several cancer types including non-small cell lung cancer, pancreatic, bladder and breast cancer. it is known that gemcitabine is used single agent or as a part of combination treatment in prostate cancer therapy. nowadays, new treatment methods have been tested for cancer therapy including natural compounds with low toxicity. ferulic acid (fa) one of these agents, an abundant phenolic compound found in various fruit and vegetables. in this study, objective was to investigate combination effect of ferulic acid and gemcitabine on apoptosis in pc-3 human prostate cancer cell line. cell viability was determined by using xtt method after the treatment with gemcitabine, fa and combination of both of them. total rna was isolated with trizol reagent. expressions of casp3, casp7, casp8, casp9, bcl2, bax, fas and cycs genes are important in apoptosis, were evaluated in four groups by qpcr. the ic 50 doses of fa and gemcitabine were found to be 300 lm and 50 lm for 48 hours in pc-3 cells, respectively. for determination of combination effect, pc-3 cells were treated with <ic 50 doses (200 lm fa and 35 lm gemcitabine). when compared with the control group, qpcr results illustrated, a significant increase in the expressions of bax and cycs genes whereas a decrease in the expression of bcl2 gene in the fa treatment group. after the treatment with gemcitabine, the expression of casp3 and fas genes were significantly increased compared with the control group. furthermore, combination of fa and gemcitabine significantly increased expression of casp3, casp7, casp8, fas and cycs genes. in conclusion, it was observed that combination of fa and gemcitabine affected different genes in apoptosis compared with the single treatments in pc-3 human prostate cancer cell line. pistacia lentiscus linn. is widely distributed in mediterranean europe, morocco and turkey. the resin part of its known as mastic gum and plant called as mastic tree. it has been referred to over centuries as having medicinal properties to treat a variety of diseases. the aim of the present study are to evaluate antioxidant, cytotoxic and antimicrobial activities of heptane, chloroform and methanol extracts from p. lentiscus leaves and mastic gum. the antioxidant activities of chloroform and methanol extracts were assayed with various methods, including tpc, dpph free radical and abts radical cation scavenging activity. also, cytotoxicity of extracts was evaluated and screened against human mcf-7, hela, a549, and ht-29 cancer cell lines and nih-3t3 cells. the viability of cells in 100 lg/ml concentration of extracts was evaluated using mtt assay. antimicrobial activity of extracts were investigated against s aureus, s epidermidis, e coli, p aeruginosa, p vulgaris, k pneumoniae, c albicans, c glabrata, c guilliermondii, c tropicalis, c parapsilosis test organims by the agar well diffusion and broth microdilution methods . according to our results, the methanol extract of p. lentiscus leaves showed the highest dpph free radical scavenging activity (ic50 = 0.16 ae 0.02 mg/ml) and abts radical cation scavenging activity (1.33 ae 0.06 mg/ml). this study also exhibited that methanol extract of p. lentiscus leaves had the highest tpc (126.7 ae 5.7 mg gallic acid equivalents/g extract). the hexane extract of mastic gum showed antifungal activity against all of the tested candida species (6-16 mm / <0.25-8 mg/ml). the methanol extracts of p. lentiscus leaves showed the highest antimicrobial activity against all of the tested bacteria except k pneumoniae strain (8-14 mm/>256 mg/ml). on the other hand, p. lentiscus showed no cytotoxicity against cancer and normal cells. the results suggested that p. lentiscus may be natural source of antioxidant and antimicrobial activities. p-02.08.5-056 antibacterial activity of and chemical composition of alcoholic extract of marjoram against some human pathogens m. ahanjan, m. rahbar mazandaran university of medical sciences, sari, iran herbs enjoy a unique value and importance in sustaining healthy communities in terms of disease prevention (1) . in this regard, marjoram is a plant of the mint family which has antibacterial properties on microorganisms (2) . the current study aims to investigate the anti-microbial activity of the alcohol extracts (i.e., methanol or ethanol) of marjoram plants on the bacteria of staphylococcus (atcc: 25923), aureus e. coli (atcc: 25922), and salmonella enterica (atcc: 13076) and p. aeroginosa through utilizing disk diffusion method. also, the minimum inhibitory concentration and the minimum bactericidal concentrationwere measured through tube. the measurement of minimum inhibitory concentration and minimum bactericidal concentration of ethanol and methanol extracts on e.coli were equal with 100 and 120 milligrams per milliliter, subsequently. moreover, the measurement of the minimum inhibitory concentration and of the minimum inhibitory concentration of marjoram ethanol extraction on staphylococcus aurous was reported to be 90 milligrams and 100 milligrams per milliliter, subsequently. in addition, the amount of ethanol and methanol extracts on salmonella enteric and p. aeroginosa was equal with 80 and 90 milligrams per milliliter, subsequently. the results showed that marjoram alcoholic extract enjoy antibacterial properties. also, among the alcoholic extracts, the ethanol extract has demonstrated to be the most effective extract on salmonella enterica and e. coli and p. aeroginosa. p-02.08.5-057 molecular analysis of serine/arginine rich sc35 splicing factor from olive b. bas 1 , e. d€ undar 2 1 depertmant of biology, university of balikesir, balikesir, 2 department of molecular biology and genetics, college of arts and sciences, balikesir university, balikesir, turkey olive (olea europaea l.) is an evergreen fruit tree adapted to mediterranean climate and rich in tannins, essential oils and organic acids. serine/arginine rich splicing factors are essential in seqeunce specific splicing of pre-mrnas. in this study we report molecular characterization of an serine/arginine rich sc35 splicing factor (oesarsc35sf) that was isolated from a cdna library constructed from olive pedicels. nucleotide blast and protein blast (for comparison of the similarity of the candidate genes from other organisms) were conducted on ncbi web page. amino acid composition analysis, nucleotide composition analysis, hydropathy analysis, open reading frame determiantion, through, molecular weight and the isoelectric points calculations were conducted using online expasy software. primer3 was used to design forward and reverse primers to amplify the target gene from different olive tissues at different times. analysis with bioedit program revealed that a+t ratio was more than that of g+c. oesarsc35sf was a protein consisting of 267 amino acids. as expected, amino acid composition analysis revealed that serines and arginines were more than other amino acids. kyte&doolittle hyddropathy analysis revealed that the protein was hydrophilic. the molecular weight of the protein was 30 kda with an isoelectric point (pi) of 11.5. the protein was found to have a signal peptide. according to the predotar analysis results, intracellular localization was found to be in the mitochondrial. the combined results suggest oesarsc35sf might function as splicing factor as its homologs from the other plants. confirming this hypothesis with futher experimental characterization including biochemical function analysis continues. acknowledgements: this study was supported by tub _ itak with grant number 110o108. keywords: olea europaea l., oesarsc35sf, alternative splcing, pre-mrna splicing, bioedit, pedicel specific cdnas. p-02.08.5-058 application of three-phase partitioning for the purification of peroxidase from kiwano (cucumis metuliferus) z. denli 1 , g. arabaci 2 1 kto karatay university faculty of medicine, konya, 2 faculty of arts and sciences, sakarya university, sakarya, turkey peroxidases are enzymes able to catalyze reduction of h 2 o 2 and oxidize various substrates. the kiwano is an oval shaped fruit which has an orange skin with lots of tiny horns. in this study, peroxidase isolated from kiwano is purified with three-phase partitioning (tpp). kiwano fruit was homogenized and obtained crude enzyme extract. the extract was saturated with 50% (w/v) ammonium sulfate ((nh 4 ) 2 so 4 ) and added t-butanol with the ratio of 1:1.5 (v/v). the lower and interfacial layer was collected. the influence of percent saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85, 95%) and tbutanol ratios (1:0.5, 1:1, 1:1.5, 1:2) to the partitioning behaviour of peroxidase were analyzed. after dialyzed, the interfacial and lower phases were measured for peroxidase activity and protein content. the protein pattern of the peroxidases was evaluated by using gel electrophoresis. peroxidase activity recovery and the purification fold of interfacial and lower phases were 117.4, 1.7% and 0.84, 0.03. therefore, other experiments were continued with interfacial phase. at constant t-butanol with the ratio of 1:1.5, the enzyme activity recovery and purification fold of interfacial phase for saturations of (nh 4 ) 2 so 4 (50, 65, 75, 85%) were 40.9, 43.2, 39.2, 135% and 0.37, 0.31, 0.4, 0.91 . the interfacial phase was not dissolved in 95% (nh 4 ) 2 so 4 . at constant 85% (nh 4 ) 2 so 4 , the enzyme activity recovery and purification fold of interfacial phase for t-butanol ratio (1:0.5, 1:1, 1:1.5, 1:2) were 61.6, 48. 1, 127.6, 126.1% and 1.79, 0.6, 1.84, 1.41 . finally, at optimum conditions (% 85 (nh 4 ) 2 so 4 , t-butanol 1:1.5) after dialyzed interfacial phase, the enzyme activity recovery and the purification fold were 138.8% and 4.46. the results of gel electrophoresis showed that the molecular weight of enzyme was between 30-60 kda. the applications of tpp gave the maximum recovery of 138.8% and 4.46-fold purification. as a result, for purfication of peroxidases, tpp is a rapid, simple and economical technique. accumulating the most robust genes and proteins in elite genotypes without any harmful effect on the potential plant yield is an urgent need to enhance productivity under various stressors. among the stressors, drought is a major challenge for agricultural productivity. brachypodium distachyon, with its close relationship to agriculturally and economically important crops, is an important model plant species. although ongoing transcriptomic analyses in brachypodium distachyon available, proteomic analyses are required to obtain an integrated picture of drought response. in the current study, a comprehensive proteome analysis was conducted on brachypodium leaves under increasing levels of drought stress. to screen gradual changes upon drought stress, brachypodium leaves subjected to drought treatment for 4, 8 and 12 days were collected for each treatment day. the cellular responses were investigated through a proteomic approach involving two dimensional difference gel electrophoresis and subsequent combined tandem mass spectrometry. for the validation of transcriptional expression, the genes encoding selected proteins were examined through quantitative real-time pcr. spot detection on cye-dyed gels revealed a total of 497 distinct spots in brachypodium protein repertoire. a total of 13 differentially expressed proteins (deps), with at least 2-fold changes in abundance, were identified by mass spectrometry and classified according to their functions. the biological functions of deps included roles in photosynthesis, protein folding, antioxidant mechanism and metabolic processes, highlighting the significant degree of overlapping between metabolic alterations induced by drought stress. identified proteins in this study and understanding the molecular mechanisms of drought response and defense mechanisms in plants will contribute to the researches on development of drought tolerant crop species. p-02.08.5-060 immunohistochemical and electron microscopy investigation of tmv-based chimeric virus particles carrying conserved influenza antigen in nicotiana benthamiana recently we obtained and partially characterized set of viral vectors based on tobacco mosaic virus (tmv) genome displaying conserved influenza a m2e epitope from matrix m2 protein. purified chimeric virus particles (cvp) conferred protection of mice against lethal homologous and heterologous influenza virus challenge. we revealed significant difference in symptoms of infections caused by tmv-m2e recombinant viruses containing cysteine (cys) to serine (ser) or alanine (ala) substitutions in the human consensus m2e sequence. accumulation level of m2e-ala recombinant coat protein was significantly higher than m2e-cys/ ser (ratio 5:1). tmv-m2e-ser infection, in contrast to ala mutant, suspended growth and development of nicotiana benthamiana. non-inoculated leaves (14 d.p.i.) were fixed with ethanol and histological sections were incubated with mouse serum to m2e and secondary antibodies conjugated with either hrpo or fitc. cvps of all three mutants were detected in epidermal and stomata cells as well as in sieve elements and minor veins. electron microscopy analysis of mesophyll cells revealed typical rigid helical particles. cys/ser mutants mostly accumulated within ground cytoplasm as aggregates of discrete tubules in parallel arrangement, which were not delimited by lipid membranes. we discovered huge amount of cvps in the cytoplasm and lesser amount diffused in the central vacuole. essential part of ala particles was located in the cytoplasm, but mentioned aggregates were not found and only insignificant number of virions was revealed in vacuole. unlike wild-type tmv, none of the mutants was revealed in chloroplasts. diameters of cvps were as follows: sersingle particles in cytoplasm 13 ae 1. cyanidin is a natural anthocyanidin found in a variety of fruits (grapes, blackberry, blueberry, cherry and cranberry etc.) and vegetables (red cabbage, red onion). this polyphenolic compound is a flavonoid with significant antioxidant activity. cyanidin and its glycosides have vasoprotective effects and can interfere with inflammation, carcinogenesis, obesity, and diabetes. one important role of the macrophages is the release of pro-inflammatory mediators, such as nitric oxide, various cytokines, in response to activation signals, including chemical mediators, cytokines, and bacterial lipopolysaccharide (lps). in this study, we investigated the role of cyanidin chloride in inflammation. anti-inflammatory effects of cyanidin chloride were examined in lps-stimulated murine raw 264.7 macrophages. we observed the level of various inflammation markers such as nitric oxide (no), inducible no synthase (inos), cyclooxygenase-2 (cox-2), tumor necrosis factor-a (tnf-a) and interleukin-8 (il-8) under lps treatment with or without cyanidin chloride. cyanidin chloride inhibited not only no production but also the expression of cox-2 and inos, without any cytotoxicity. cyanidin chloride also attenuated pro-inflammatory cytokines and other inflammation-related markers such as il-8 in a dosedependent manner. in conclusion, cyanidin chloride may be beneficial for the prevention and treatment of anti-inflammatory diseases. p-02.08.5-062 the investigation of centranthus longiflorus plant extacts effects on cell proliferation and apoptosis activity in the cell lines of mcf-7 breast cancer h. askin ataturk university, erzurum, turkey introduction: in the u.s., breast cancer is the second most common cancer in women after skin cancer. current treatment of cancer can be done by surgery, chemotherapy, and radiation therapy. in addition, there is widespread use of complementary and alternative medicine in developed countries. plants and plant extracts play a critical role in the research into new anticancerogenic agents. centranthus longiflorus (cl) is used in alternative medicine for sedative and antispasmodic purposes. a plant of turkish origin, centranthus longiflorus used as traditional turkish medicine have remained uninvestigated for familial hypercholesterolemia, diabetes, coronary artery disease and cancer for their in vitro biological activity despite their use for sleep disorders. in this study, growth-inhibiting and pro-apoptotic effects of hexane, ethyl acetate and ethanol extracts of cl in mcf-7 breast cancer cell line were investigated. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from cl was analyzed using a gc-ms system. dose-and time-dependent cytotoxic and apoptotic effects of cl were evaluated by mtt cell proliferation kit and cell death detection elisa kit, respectively. manufecturer's protocol was followed for analyses. then, apoptotic genes; caspase3, bax and p53 and antiapoptotic genes; bcl-2 and pi3 expression levels were determined by rt pcr. results: according to our results, cytotoxic effect on mcf-7 cell was only observed in 20 and 50 lg/ml doses of cl. however, any of the application doses showed an apoptotic effect on mcf-7 cells. they exhibited a necrotic effect rather than the apoptotic effect. although alterations in expression levels of these genes were determined, this alterations was statistically insignificant. discussion and conclusion: consequently, we can say that cl have a cytotoxic effect on mcf-7 breast cancer cell lines. p-02.08.5-063 reduction of the chloroplast genome and the loss of photosynthetic pathways in the mycoheterotrophic plant monotropa hypopitys, as revealed by genome and transcriptome sequencing e. gruzdev, a. mardanov, a. beletsky, v. kadnikov, e. kochieva, n. ravin, k. skryabin institute of bioengineering, research center of biotechnology of the russian academy of sciences, moscow, russia genomes of parasitic plants represent interesting model systems to study effects of relaxed selective pressure on photosynthetic function. previous genomic studies of nonphotosynthetic plants revealed reduction of their chloroplast genomes, but the corresponding changes in their nuclear genomes are less known. here we present the data on the transcriptome and the chloroplast genome of the non-photosynthetic mycoheterotrophic plant monotropa hypopitys. the chloroplast genomes were sequenced for two specimens of m. hypopitys, collected in different regions of russia. the cpdnas are 34,800 bp (mon-2kalr) and 35,336 bp (mon-1volr) long and rearranged with respect to each other. both genomes contains genes encoding ribosomal proteins, infa, matk, and 4 ribosomal rna genes. 17 and 19 trna genes were predicted in two cpdna. genes encoding nadh dehydrogenase, plastid rna polymerase, all genes related to photosynthetic apparatus, clpp, ycf1, ycf2, accd, and some genes for ribosomal proteins are missing or became pseudogenes. the reduction of gene content is associated with extensive gene order rearrangement and the lack of inverted repeats. overall, the size and gene content of m. hypopitys cpdna indicates that it is close to the end of plastid genome degradation process. in order to get insights into the changes in the nuclear genome associated with the transition to nonphotosynthetic lifestyle, we sequenced and assembled the transcriptome of m. hypopitys. as expected for holoparasites, we did not found transcripts for the nuclear genes encoding the components of photosynthetic machinery, including photosystem i and ii, cytochrome b6f complex, and ribulose bisphosphate carboxylase. contrary to the holoparasitic plant phelipanche aegyptiaca, almost all genes of chlorophyll biosynthesis pathway from protoporphyrin ix were not found in the m. hypopitys transcriptome. this work was supported by the rsf grant 14-24-00175 and rfbr grant 14-14-00749 (mon-1volr cpdna sequencing). introduction: beta-sitosterol is a substance found in plants. chemists call it a plant sterol ester. it is found in fruits, vegetables, nuts, and seeds. it is used to make medicine. beta-sitosterol is used for heart disease and high cholesterol. the federal food and drug administration (fda) allows manufacturers to claim that foods containing plant sterol esters such as beta-sitosterol are for reducing the risk of coronary heart disease (chd). centranthus longiflorus (cl) is used in alternative medicine for sleep disorders. a plant of turkish origin, cl used as folk medicine have remained uninvestigated for familial hypercholesterolemia, coronary artery disease and preventing colon cancer for their in vitro biological activity despite their use for sleep disorders. we investigated of the chemical constituents from dried aerial parts of centranthus longiflorus. material and method: aerial parts of cl were collected in erzurum province. hexane, ethyl acetate and ethanol extraction were done by soxhlet extractor. the plant extracts obtained from the aerial parts of cl was analyzed using a perkin-elmer gc-ms system. results: ten compounds were obtained and identified as butanoic acid, hexadecanoic acid (palmitic acid), 7-methyl-z-tetradecen-1-ol acetate, octadecanoic acid (stearic acid), diisobutyl phthalate, 9-octadecenamide, octacosane, nonacosane, alfa amyrin and beta sitosterol. the latter two were obtained in all extraction (hexane, ethyl acetate and ethanol). discussion and conclusion: all of these compounds are isolated from centranthus longiflorus for the first time. these findings may shed light on the design of new drugs, the cholesterollowering effect. p-02.08.5-065 role of lutein for the high light-induced inhibition of photosystem ii related reactions in thylakoid membranes of arabidopsis thaliana, wt and lut2 k. dobrev, d. stanoeva, m. velichkova, a. v. popova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthetic reactions taking place in thylakoid membranes of higher plants are extremely sensitive towards different environmental stress conditions such as high and low temperature, high light intensity, uv radiation etc. carotenoids are intrinsic component of photosynthetic pigment-protein complexes and are involved in performing multiple important functions. their role of accessory pigments in absorbing sun light, participation in photoprotection via dissipation of excess absorbed light, deactivating of stress-induced reactive oxygen species and structural role are well documented and recognized. the role of lack of lutein in high light-induced alterations in structural organization and functional activity of the main pigment-protein complexes was evaluated using isolated thylakoid membranes of arabidopsis thaliana, wt and mutant lut2, deficient in lutein, subjected to photoinhibitory treatment for different periods of time. alterations in photochemical activity of photosystem i and photosystem ii were determined by a clark-type electrode in the presence of exogenous electron donors and acceptors. activity of oxygenevolving complex and of the grana and stroma situated photosystem ii reaction centers was evaluated by determination of flash oxygen yields and initial oxygen burst under constant light without donors and acceptors. low-temperature (77k) fluorescence was applied for unraveling of light-induced alterations in energy transfer and interaction between the main pigment-protein complexes. maximal quantum efficiency of psii was registered by pulse amplitude modulated fluorescence method. results obtained are discussed in respect to the importance of lutein for the organization and sensitivity of photosynthetic apparatus towards high light intensity treatment. modern agriculture relies heavily on phosphate rock fertilizer to improve phosphorus availability in many soils, but this approach is not sustainable long-term. phytate (myo-inositol hexakisphosphate) is an organic phosphorus compound often present in many soils. however, phytate can not be utilized by most plants, and its accumulation in soil leads to substantial ecological problems. phytases are enzymes that hydrolyze phytate and release inorganic phosphate. many microorganisms such as bacteria and fungi synthesize highly diverse phytases which are suitable for plant biotechnology. generation of transgenic plants expressing phytases of bacterial origin has been proposed as one option to improve plant phosphorus nutrition. in this study, we generated and characterized transgenic arabidopsis thaliana plants expressing a modified phytase gene paphyc from pantoea sp. under strong camv35s promoter. three individual transgenic a. thaliana lines expressing the bacterial phytase gene, as well as negative control plants harboring the camv35s promoter alone were identified. expression of phytase in plants was verified at both transcription and translation levels. phytase-expressing plants grown on media with phytate as the sole source of phosphorus demonstrated better than wild-type growth rates, shoot dry mass, shoot phosphorus content, as well as higher phytase activity in cell-wall extracts. overall, we show that plants expressing bacterial phytase are capable of better growth on phytate as the only source of phosphorus in laboratory conditions. further research investigating the applicability of using bacterial phytase expression to improve plant growth in soil is necessary to evaluate the different routes of solving the phosphorus deficiency problem in agriculture. p-02.08.5-067 the role of elevated temperature in photoinhibition and recovery of photosystem ii in thylakoid membranes from arabidopsis thaliana a. faik, m. gerganova, m. velitchkova institute of biophysics and biomedical engineering, bulgarian academy of sciences, sofia, bulgaria photosynthesis of higher plants is the principle process to transform light energy into biochemical usable energy. in nature, plants are exposed to the environment where light, temperature, uv-b radiation varied and very often their extreme values that are unfavorable for effective performance of photosynthetic reactions. plant are developed various strategies to cope with stress including radical scavenging enzyme system, accumulation of protective compounds, etc. pigment-protein complexes of photosystem i and photosystem ii and their light harvesting antenna, situated within thylakoid membranes, are involved in the primary reactions of photosynthesis -absorption of light, charge separation and electron transport. photosynthetic process is sensitive towards higher than optimal temperatures, the photosystem ii and oxygen evolving complexes being extremely sensitive to elevation of temperature. in present work pam fluorescence was applied to evaluate the effect of long term action of elevated temperature (38/30°c) on the quantum yield of photosystem ii, non-photochemical quenching and rdf, the latter quantifying the photosynthetic process. in addition, the activity of oxygen evolving complex was determined polarographically in the presence of exogenous electron acceptor 1,4 -benzoquinone. sds-page electrophoresis and western blot were applied to determine the damage of d1 -reaction center protein of photosystem ii. alterations of mutual organization within photosystem ii complex and its antenna and of energy interaction between them were followed by analysis of 77k steady state chlorophyll fluorescence spectra. the simultaneous application of high temperature and high light intensity resulted in a well pronounced reduction of non-photochemical quenching that restore to the initial values after recovery for 5 days at optimal conditions. d1 was also restored while quantum efficiency of photosystem ii did not recuperate to initial values. p-02.08.5-068 reorganization of the main pigment-protein complexes in thylakoid membranes from tomato (solanum lycopersicum) during long term exposure to elevated temperature the changes of earth climate resulted in unfavorable environment for plants. depending on the duration of influence of stress factors, the response of plants includes short and long term acclimation. the population, structure and organization of pigmentprotein complexes within thylakoid membranes are dynamic and flexible, thus providing for the acclimation of the photosynthetic apparatus to the changed environment. the main pigment-protein complexes, involved in energy transduction, are photosystem i, photosystem ii and light harvesting complexes. they are separated in grana and stroma regions of thylakoid membranes but it is well established that they can rearrange as a result of alterations of light intensity, temperature increase and decrease in order to balance the perception and utilization of excitation energy. in present work the effect of long term action of elevated temperature on organization and stoichiometry of main pigmentprotein complexes in the thylakoid membranes from tomato plants (solanum lycopersicum cv. m82) was investigated. three weeks old tomato grown at optimal conditions (22/20°c day/ night temperature and light intensity 250 lmol/m 2 /s) plants were exposed for 2 and 6 days to elevated temperature at 38/30°c. by means of blue-native electrophoresis the effect elevated temperature on the populations of psii (dimmer and monomers) and lhcii (monomers and trimers) was estimated and compared with the same parameters for control plants. the ability of plants to recover from this treatment was checked after 5 days under optimal conditions. the changes of content of chlorophylls and carotenoids were determined at every stage of treatment. based on the results obtained it can be concluded that one of the mechanisms for regulating the energy balance and maintenance of efficient photosynthetic process involves a change in the organization and stoichiometry of the photosystem ii and oligomer state of light harvesting complex ii. aim of the study, was to evaluate the anti-tumor effects of silymarin, curcumin and propolis on leptin-induced mcf-7 cells. mcf-7 cells were incubated various concentrations leptin (physiological, obesity and pharmacologically doses; respectively 10, 100 and 1000 ng/ml) . then different doses of silymarin (10, 20, 40, 80 lm), curcumin (10, 20, 40, 60 lm) and propolis (0.063, 0.125, 0.25, 0.5 mg/ml) were added. after 24, 48, 72 and 96 hours incubation periods 3 different area images were taken digital camera. then using dye release reagent we determined the intensity of apoptosis via colorimetric determination by elisa reader. absorbance was directly proportional the number of apoptotic cells (biocolor cell-apo percentageapoptosisassay). also, we examined the effect of these natural products on proliferation rate of leptin-induced mcf-7 cells for 1, 2, 3 and 4 hours (biovision cell proliferation kit) all experiments were carried out 3 different days, at least 3 times. all of three compounds were stimulate the apoptosis at all time points and all different doses of leptin. the differences was statistically significant at the level of p < 0.05 between 24 and 48 hours. it was found that there were not seen much cells at 72 and 96 hours time points. we thought that most of the cells were gone necrosis instead of apoptosis. the best effective doses on apoptosis of propolis was 0.063 mg/ml, silymarin and curcumin were 20 lm. also, we evaluated the effects of on proliferation rate the mcf-7 cells, we found that only propolis was effective of inhibiting proliferation at all doses of leptin induced mcf-7 cells in 1 hour. we hope this study will be a guide for the further studies in anti-cancer agent development field and show that the natural origin substances cause cancer cells apoptosis and provide targeted treatment for cancer therapy. p-02.08.5-070 investigation of some lichen-derived substances' cosmetic potential for skin protection against ultraviolet b due to the depletion of the stratospheric ozone layer and chronic exposure, the occurrence of various skin diseases have been increased in recent decades. thence, people and cosmetic companies have progressively given more importance natural sunscreen products for the protection from harmful sun rays, especially ultraviolet b rays. we, therefore, isolated some lichen-derived substances; 3-hydroxyphysodic acid and protolichesterinic acid from hypogymnia tubulosa and cetraria aculeate, respectively. chemical characterization and identification of the isolated lichen substances were accomplished by using ftir, 1 h-nmr and melting point analyses. the theoretical uv-vis spectra and 3d conformations of the isolated compounds were determined by using the gaussian 03 software with hf theory at the b3lyp/3-21g level. the dark toxicities and ultraviolet b protection capacities of the substances were lighted up as previously described [1, 2] on hacat human keratinocyte cell line by using mtt cell viability and ldh cellular membrane degradation assays. the obtained results from the assays showed that protolichesterinic acid has a more dark toxic activity on keratinocyte cells than 3hydroxyphysodic acid, and the toxic activities were found sufficient as much as 80% at the highest doses of the substances; 400 lm. however, it was observed that the cytotoxicity of the substances were reduced at the rate of approximately 15% by the irradiation. consequently, we think that the substances block the ultraviolet b rays but their cytotoxic feature is an important limitation to their usage in cosmetic industry. references [1] varol, m., t€ urk, a., candan, m., tay, t., koparal, a. t. (2016) photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes, phytotheraphy research 30:9-15. [2] varol, m., tay, t., candan, m., t€ urk, a., koparal, a. t. (2015) a great effort and financial supports have been consumed to explore and design novel sun protection factors due to the unhindered increase of malignant and non-malignant skin diseases caused by the chronic exposure and depletion of the stratospheric ozone layer. the testing of naturally produced compounds seems to be the best and inexpensive way to search for potentially photoprotective substances. on the other hand, as photo-resistant species, lichens are still poorly exploited. norstictic acid was, therefore, isolated from the acetone extract of pleurosticta acetabulum. ftir, 1 h-nmr and melting point analyses were performed to identify the chemical features of norstictic acid. gaussian 03 software with hf theory at the b3lyp/3-21g level was also performed to determine the theoretical uv-vis spectrum and 3d conformation of the isolated compound. the dark cytotoxicity and ultraviolet b-protection capacity of norstictic acid were comparatively tested as previously described [1, 2] by using mtt cell viability and ldh cellular membrane degradation assays. as a result of the experiments, it is observed that norstictic acid has a dark-cytotoxicity as less as 25% at the highest dose of the substance; 400 lm. however, ultraviolet b-induced damage on human keratinocytes was blocked by the lower concentrations of norstictic acid as 25, 50 and 100-lm, and 20% of cells were protected according to the control experiments of irradiated cells. consequently, we think that norstictic acid might be employed as a sun protection factor at the low concentrations, and further studies should be performed. years, researchers have focused on the lichen acids because of their biological activities. it is also suggested that lichens can be used as anticancer agents. vulpinic acid, an important lichen seconder metabolite, has antimicrobial activity and strong antimutagenic, anticancer and antioxidant capacity. nanotechnology has the potential to offer solutions to current obstacles in cancer therapies, because of its unique size (1-100 nm) and large surfaceto-volume ratios. so, in this study we aimed to determine the cytotoxic and proliferative effects of vulpinic acid and magnetic nanoparticles loaded with vulpinic acid (fe 3 there are four species of wild rice known around the world. zizania aquatica l., zizania palustris l. and zizania texana hitche are found in north america, whereas zizania latifolia (griseb) turcz) is native to east asia. cwr mainly grows in the areas along the yangzi river and the huai river in china without any cultivation and domestication. cwr was an ancient grain that has been used in chinese herbal medicine to treat a variety of ailments associated with nutrition, including gastrointestinal disorders and diabetes. our previous studies have demonstrated that consuming chinese wild rice can significantly improve blood lipid profiles and ameliorate high-fat/cholesterol diet-induced insulin resistance. however, compared to the well studied common dietary white rice, active composition and the associated proteomic information of chinese wild rice have yet to be investigated. in this study, we compared and analysed the different proteins between chinese wild rice and white rice by proteomics method. our study provides insights and experimental evidence for further exploration of this ancient medical food in disease prevention and therapy. the homology between cwr and n22 is 64%, but significant differences also exist between the two. we gained new insight by analyzing the biological function of the high reliability (credibility score 67 or higher, p < 0.05) peptide mass fingerprint of cwr2-de electrophoresis revealed differences in protein composition between cwr and n22. information obtained from the pmf indicates that glutelin precursor, caffeoyl coenzyme a (coa) omethyltransferase and putative bithoraxoid-like protein can provide gene evidences for its biological function. p-02.08.5-075 mir396 and growth-regulating factors interaction during maize leaf growth under low-temperature stress g. aktug, f. aydinoglu gebze technical university, kocaeli, turkey microrna (mirna) genes are a class of non-coding small rnas about 21 nucleotide-long which are revealed as regulators of plant growth and stress responses. the mirna mir396 targets and regulates growth-regulating factors (grfs) which are plant specific transcription factors family and this regulation machinery is conserved among plant species. plant growth is a result of cell division and expansion which took place as spatial gradient zones throughout maize leaf which are meristem, elongation and mature zones. cells proliferate in meristem, migrate to elongation zone and finally reach to mature zone to get its final size. it has been shown that mir396 affects cell division by regulating grfs and changes leaf size which are determined by cell number and cell size of leaf. this study aims to investigate the role of mir396 and grfs interaction during maize leaf growth under low-temperature stress. maize seedlings were grown under low-night temperature for stress treatment to generate growth retardation and control conditions as well to make comparative analysis. length of the third and fourth leaves of seedlings was measured every day and leaf elongation rate was calculated to observe stress effects on the leaves. growth zones of fourth leaves were harvested during steady-state growth phase for determining expression level of mir396s and their target by q-rt-pcr. we mined 8 mir396 genes sharing sequence similarity and 8 grf targets. the expression analyses of mir396s and grf5 are proceeding for different growth zones. in conclusion, this is the first study investigating the regulation network between mir396s and grf5 in different developmental stages of maize leaf under low-temperature stress. oeigpd cdna was isolated from a cdna library we constructed from olive pedicels. homology searches for nucleotide, amino acids and alternative open reading frames were conducted utilizing blastn, blastp, and blastx, respectively. nucleotide sequences of homologous genes from other plants were aligned using bioedit and the number of snps were detected. the alignment was then used to generate a phylogenetic tree using mega7 program. another alignment with amino acid sequences of the homologues proteins was also generated to construct a phylogenetic tree displaying oeigpd's position among other plants. various aspects of oeigpd including amino acid composition, hydropathy analysis, isoelectric point (pi) and three dimentional structure of the protein were determined using online software at expasy. multiple primer pairs to amplify the full length open reading frame of the gene, to clone the gene into the expressing vector plate51, and to detect expression through real-time pcr were designed using primer3. amino acid composition analysis revealed that oeigpd contained serine, arginine and isoleucine predominantly while hydropathy analysis suggested it was an hydrophilic protein. isoelectric point (pi) of the protein was calculated as 4.97. the molecular weight of the protein was calculated as 11 kda. analyses continue to determine the polymorphism of oeigpd among olive cultivars, and biochemical function of the gene in olive. p-02.08.5-077 cytotoxic effect of fractionated triterpenoid glycosides from holothuria polii in human cancer cells sea cucumbers are the members of class holothuroidea and they have more than 1100 described living species all around the world. sea cucumbers secrete special secondary metabolites from their body walls and they are called triterpene glycosides (tggs). in this study, cytotoxic activity of fractionated ttgs from h. polii on different cancer cell lines were carried out. h. polii delle chiaje, 1824 samples were collected from dikili-_ izmir. the semipurified extracts were fractioned by using hplc. four different fractions (fraction a-d) were obtained. in order to characterize the fractions, maldi-tof/ms was used. the cytotoxic activity of the fraction a-d were tested on ht-29, t84 and upci-scc-131 cell lines by using xcelligence rtca sp system. the cells were treated with three different concentrations of the fractions for 48 hours. the cell index data were compared with the control group. ic50 values of the fractions for three cell lines were calculated. according to the results, the fractions have holothurin a (1243.50 m/z), 24-dehydroechinoside a, scabraside a or fuscocinerosides b/c isomer (1227.50 m/z). the fraction d was the most effective on all cell lines with ic50 value of 10.46 ae 0.18 mg/l, 11.24 ae 0.57 mg/l and 11.13 ae 0.29 mg/l for ht-29, upci-scc-131 and t84, respectively. in conclusion, sea cucumber ttgs are promising agents for colon adenocarcinoma, oral squamous cell carcinoma and colorectal carcinoma (metastatic) treatment. p-02.08.5-078 effect of horse-chestnut (aesculus hippocastanum) seed extract on matrix metalloproteinases during diabetic wound healing impaired wound-healing in diabetics is a major public health problem. the expression and activation of matrix metalloproteinases (mmps) are also impaired in diabetic wounds according to previous studies. their main function is to degrade the various components of the extracellular matrix. also, they participate physiological processes such as inflammation, angiogenesis, tissue remodeling. horse-chestnut seeds (hc) are rich in saponins and flavonoids. it has been shown that hc has antiinflammatory, antioedema, vessel protective, and free radical scavenging properties. the aim of this study is to determine with molecular signs on cutaneous wound healing effects of the ethanol (50%) extract of hc (hce) seed in rats by excision wound model. this study was conducted on diabetic wistar albino rats, which were injected by a single dose (50 mg/kg i.p.) streptozotocin. diabetic treatment rats were applied topically 15% (w/w) ointment with hce and control rats were applied topically simple ointment, once a day during the experimental period. the gene expression levels of mmp-1, mmp-9 by qpcr and levels of nitric oxide (no), hydroxyproline and malondialdehyde in wound tissue investigated at the end of 3rd, 7th, and 14th days. wound closure was also measured. the hydroxyproline and no levels were significantly increased in the hce treated group versus control after the 3rd and 7th days. the malondialdehyde levels were significantly lower in the treatment group. mmp1 gene (associated with collagen processing and reepithelialization) expression levels in hce treated rats were increased in the 7th day while it was reduced in 14th day. mmp 9 gene (associated with inflammation and gelatinase) expression levels in hce treated rats were decreased in 7th, and 14th days compared to the control. these findings indicate that hce accelerated the cutaneous wound healing process in diabetic rats via mmp1 and mmp9 regulation. p-02.08.5-079 isolation and molecular molecular characterization of vps39/vam6 from olive b. celikkaya, e. dundar molecular biology and genetic at balikesir university, balikesir, turkey vps39/vam6 promotes aggregating and fusing of endosomes and lysosomes. it is a component of a protein complex that is found in vacuole membranes. this gene has been studied from various organisms including humans, drosophila, arabidopsis and rice. no studies on olive vps39/vam6, however, have been reported. the aim of this study is to report information of olive vps39/vam6 including expression pattern and biochemical characterization. vps39/vam6 was isolated from a cdna library we constructed from fruited olive leaves in july. to determine the putative name of the cdna, blast analyses were conducted for nucleotide, open reading frame and amino acid sequence comparisons. bioedit program was used to determine the nucleotide and amino acid composition along with its molecular weight and isoelectric point (pi). hydropathy analysis was conducted using kyte and doolittle program. phylogenetic analysis was done using mega7. cellular localization of the product was predicted using sosui gramn. the three dimentional structure of the protein was calculated using i-tasser and compared to previously known structures using cn3d. the blast and bioedit analyses revealed vps39/vam6 had 836 base pairs coding 120 amino acids with a molecular weight of 13.38 kda, and pi of 6.39. the at/gc ratio was very high (1.5) comparing to its homologs from other plants suggesting to expect significant differences of this gene's function from the others. amino acid composition analysis revealed high rates of serine, leusine and isoleucine indicating a hydrophobic property of the protein. the hydrophobic feature was confirmed by kyte and doolittle analysis while the cellular location was revealed to be extracellular. the hydrophobic nature despite extracellular location suggests it is a membrane associated protein which was confirmed by transmembrane domain analysis. as expected no signal peptide was detected. the 3d structure of the protein was similar to its previously reported homologs. p-02.08.5-080 isolation and characterization of the ribosomal l1 protein from olive s. cinarli, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey despite as a ribosomal protein, l1 is known as the inhibitor of the cellular aging gene and it has been reported to have roles in apoptosis. the ribosomal l1 protein is larger than the lsu of ribosome and contains 2 domains as 2-layered alpha/beta domain and 3-layered alpha/beta domain. in ribosomes, it functions in translocation and orientation of trnas. although the ribosomal l1 (rl1) gene has been studied in many plants, reports on olive rl1 (oerl1) are very rare. this study presents molecular characterization of rl1 gene from olive. oerl1 was isolated from a cdna library we constructed from unfruited olive leaves in july. homology analyses were conducted using blast programs. nucleotide and amino acid compositions, molecular weight, isoelectric point (pi) and at/gc ratio were determined using bioedit and expasy programs. cellular location of the l1 protein was determined using sosui-gramn program. signal peptide detection, transmembrane domain detection, three dimensional (3d) structure analysis, and phylogenetic analysis were conducted using signalp 4.1, thhmm, i-tasser/cn3d and mega7, respectively. oel1 was found to have an open reading frame of 909 base pairs coding 220 amino acids that constitutes a molecular weight of 24.9 kda and a high pi of 9.87. lysine, leucine and valine had higher rates. the hydrophilic nature suggested by kyte and doolittle analysis despite high rates of leucine and valine suggests an amphipathic nature of the protein that can bind to both hydrophilic and hydrophobic proteins and / or function in both media. a 1.59 at/gc rate is significant comparing to that of its homologs from other plants. sitoplasmic location predicted by sosui-grann is in agreement with the hypothesis suggesting an amphyphatic nature for oerl1. likewise, no signal peptide was detected and it was predicted to have at least one transmembrane domain. further characterization of oerl1 with respect to expression pattern and biochemical function continues. p-02.08.5-081 isolation and characterization of an olive splicing factor 3b subunit 1 m. nurcin, e. dundar department of molecular biology and genetics, balikesir university, balikesir, turkey splicing factor 3b subunit 1 (sf3b1) functions in the regulation of translation and gene expression. sf3b1 forms u2 small nuclear ribonucleoprotein complex (u2 snrnp). splicing factors 3a and 3b binds pre-mrna at the 5 0 site of the intron branching point. this binding joins u2 snrnp to pre-mrna. although sf3b1 from various plants have been widely studied, no studies on olive sf3b1 (oesf3b1) have been reported. this study reports information on various aspects of oesf3b1. oesf3b1 was obtained from a cdna library we constructed from fruited olive leaves in december. it was putatively identified as a splicing factor using blastn, blastp and blastx. to determine wheter oesfb1 was a sitoplasmic protein, sosui gramn was used. tmhmm was used to detect any transmembrane domains while signal peptide analysis was conducted by signalp. i-tasser and cn3d were used to generate the calculated 3d structure and to compare it with experimentally generated models, respectively. nucleotide and amino acid compositions along with the calculated molecular weight and isoelectric point (pi) were analyzed using bioedit and online expasy software. the phylogenetic trees revealed genetic relationship of olive among other plants based on oesfb1. the orf contained 1950 nucleotides coding 254 amino acids that produce a 28.2 kda peptide with a pi of 5.94. alanine, valine and leucine were found at high ratios suggesting a hydrophobicity which was also predicted by kyte and doolittle analysis. the at rich property of oesf3b1 is not unusual comparing to most plant genes. cellular localization of the gene was suggested to be in mitochondria with no signal peptide indicating oesf3b1 could be synthesizing in mitochondria. the predicted 3d structure of oesf3b1 was similar to experimentally produced structures while some hydrophobic pockets were predicted. further characterization of the gene with respect to temporal and spatial expression pattern and biochemical function continues. p-02.08.5-082 kafirin profile of turkish originated sorghum populations r. temizg€ ul, s. yilmaz, m. kaplan, t. akar department of biology, faculty of science, erciyes university, kayseri, turkey sorghum bicolor l. is the fifth important crop in the world with its high photosynthetic activity and resistance to unfavourable conditions as high temperature, drought, salt, and ph changes. sorghum has attracted great interest due to its intensive usage both as human and animal nutrition, and contribution to resistance against many diseases. some proteins of sorghum exerts reducing effect on nutrient digestion through making connetions with other proteins and/or carbohydrates. kafirin proteins have the highest proportion in grain with a range of 77-82%. they are grouped into a (23-25 kda), b (18 kda), c (28 kda) and d (13 kda) subunits depending on molecular weight, solubility and structure. in the current study, kafirin proteins from turkey originated 282 sorghum populations were acquired through sequential extraction; first, non-prolamines were removed through application of 5% naci concentration, and second, kafirins were obtained using tertiary butanol (60%) and reducing agents. sds-page was conducted for seperating and visualising the subunits of kafirins. the a, b, c, and d subunits of populations were respectively estimated as 97, 85, 95, and 60%. of the total proteins, 74% was identified as a, 12% b, 12% c, 1% d, and %1 non-prolamines. non-prolamin group of proteins were visualised as 18 different bands ranging from 15.6 to 230 kda. c and b group of proteins were only viewed when treated with reducing agents as 2-me and dtt suggesting that they are connected with complex cross-links. however, a group of proteins visualized without using these agents due to not having intra molecular disulphide bridges and inter molecular cross-links. non prolamins, except for 47.8, 45.7, 21.7, 20.3 and 15.6 kda, were able visualised in the presence of reducing agents. transcriptomic analysis of the genes encoding analysed proteins needs to be elucidated for better understanding of the genetic diversity and biochemical characteristics of sorghum. p-02.08.5-083 untargeted metabolomic profiling of romanian and uk tomatoes varieties by high performance liquid chromatography coupled with mass spectrometry c. socaciu 1,2 1 university of agricultural sciences and veterinary medicine, cluj-napoca, 2 center for applied biotechnology ccd-biodiatech at proplanta ltd, cluj-napoca, romania tomato flesh is a rich source of many phytochemicals of high nutritional value, including a large variety of carotenoid derivatives with health promoting properties. metabolomics became the most adequate technology for an accurate chemotaxonomic classification and discriminations between different varieties, based on untargeted profiling or targeted, quantitative analysis. different varieties of tomatoes (b-carotene-rich, lycopene-rich, ketocarotenoid-rich) cultivated in romania and uk were comparatively studied using enriched fractions obtained by a preliminary fractionation of the whole pulp homogenate. two methods were applied for carotenoid extraction: a mixture of hexane/ethanol (1) and chloroform/methanol (2) . the dried extracts were dissolved in ethyl acetate and analyzed by uv-vis spectrometry and hplc-esi(+)qtof-ms (bruker gmbh). the base-peak chromatograms were processed by specific biostatistics software (data analysis and profile analysis) and the molecular identification were determined by comparison with the data base lipidomics gateway (www.lipidmaps.org). the content of carotenoids were significantly higher using extraction (2) , ranging from 4.5 to 7.6 mg/100 g. the major carotenoid derivatives, were represented by lycopene, hidroxy-lycopene, all-trans or cis-beta-carotene, echinenone, all-trans retinyl palmitate, but also sterols, phospholipids, di/tri glycerides and ceramides. the romanian varieties were more rich in polar carotenoids and lipids, in general, while the uk tomatoes proved to be enriched in non-polar derivatives, especially esterified carotenoids, keto-carotenoids and glycerides. new molecules were identified, as good discriminatory markers of each tomato variety. acknowledgements. this work was supported by a grant of the romanian national authority for scientific research and innovation, cccdi -uefiscdi, project nr. 16/2015, pncdi3. glycogen is a multi-branched polysaccharide that serves as the main form of glucose storage in the body, where the main reserves are in the liver and muscle. it has been observed that glycogen metabolism is altered in many tumor types, and that glycogen content is inversely correlated with proliferation rate. in addition, it has recently been described that when glycogen accumulation is forced in glioblastoma u87 cells in hypoxia, senescence is induced and tumor growth is inhibited in vivo. our laboratory has various animal models with different parts of the glycogen metabolism pathway affected. most notably, we have two animal models lacking glycogen: muscle glycogen synthase (gys1 ko) and liver glycogen synthase (gys2 ko) knockout animals. we isolated mouse embryonic fibroblasts (mefs) from gys1 ko to perform replicative senescence assays. in addition, we induced hepatocellular carcinomas in gys2 ko animals via n-nitrosodiethylamine (den) injections in order to track tumorigenesis in animals lacking hepatic glycogen. lastly, we performed partial hepatectomies (phx), which involves the resection of two thirds of the liver, on gys2 kos to evaluate the effect of the lack of glycogen on hepatocyte proliferation. interestingly, we have observed that glycogen levels are increased in human and mouse fibroblasts under replicative senescence, and that mefs depleted of glycogen bypass senescence and immortalize faster than wts. we have also demonstrated that senescence pathways are down regulated in mefs lacking glycogen. furthermore, gys2 kos treated with den show higher tumor burden and mortality than controls. we also evaluated the effect of glycogen on hepatocyte proliferation after phx. gys2 ko mice present faster proliferation and liver regeneration rates, when compared to wt counterparts. collectively, our preliminary data suggest that glycogen metabolism plays a crucial role in the regulation of cell cycle in both physiological and pathological states. it is established that pineal is involved in circadian regulation of testosterone secretion from leydig cells. however, the precise routes of this regulatory involvement are still unknown. as cgmp has been also regarded as modulator of steroidogenesis we sought to study the effects of pineal removal on the circadian pattern of cgmp variations and expression of the genes that encode elements of no-cgmp signaling pathway in adult rat leydig cells. the analysis was performed on testicular leydig cells obtained from pinealectomised and shame pinealectomized rats, in six time point during 24 hours. the pinealectomy was confirmed by serum melatonin eia measurement. the androgen levels were measured by ria; cgmp by eia and gene expression was quantified by rq-pcr. all results were analyzed by cosinor method. data revealed circadian transcriptional pattern of nos2, nos3 (genes encoded no producers) and pde5a (gene for cgmp remover) in leydig cells from adult rats. pinealectomy significantly increased expression of nos2 which lost rhythm and increased and delayed amplitude of nos3 expression. further, pinealectomy initiated cyclic transcription of gucy1b3 and noncyclic transcription of gucy1a3 (genes encoded cgmp producers) and increased mesor and amplitude of pde5 transcription. the transcription of prkg1, the main effector in this signaling pathway was not affected with pineal abolition. additionally, pinealectomy did not influence the circadian transcription profile of coxi2 or other investigated genes (coxi1, nrf1, nrf2a, pgc1a) related to mitochondrial function and biogenesis. finally pinealectomy reversed phase of circadian cgmp oscillation in leydig cells, increased amplitude and slightly advanced peak of serum testosterone oscillation. results suggested pineal influence on circadian rhythm of no-cgmp signaling in leydig cells. further studies based on these data are needed to better understand the relationship between pineal and circadian rhythm of testosterone production. influenza is a contagious respiratory infection caused by a variety of influenza viruses. neuraminidase inhibitors is a new class of antiviral drugs that inhibit influenza viruses. the most popular antiviral agents is oseltamivir, having a commercial name of tamiflu, within anti-influenza antivirals. as well as tamiflu is a member of neuraminidase inhibitor group drug. therefore, this study was performed to determine the effect of tamiflu on cultured human peripheral blood lymphocytes. material and methods: for examining the presence of the indirect mutagenic effect of oseltamivir in iver s9 fraction mix was used. cells were treated with 0.5, 1 and 2 lg/ml oseltamivir, the tamiflu capsule ingradient, for 24 or 48 hours in the absence or presence of an exogenous metabolic activation system. the test chemical did not demonstrate any genotoxic effect dose-dependently but it showed a weak cytotoxicity on cells in this study. on the other hand, some concentrations of tamiflu induced sce and also decreased significantly the proliferation index (p ˂ 0.05) in the absence of s9 mix. result: tamiflu did not induce significant increases of ca or micronucleated cells in vitro in cultured peripheral blood lymphocytes under the treatment conditions used but week sce induction was observed. on the other hand, the weak cytotoxic effects observed disappeared in the cultures treated in presence of the s9 mix. discuss and conclusion: tamiflu weakly induced sce at the highest concentration with/without added s9 mix in cultured human peripheral lympocytes. it could be assumed to be a sce inducer. sces can be increased by several agents that attack dna. tamiflu decreased the proliferation index and nuclear division index at some concentrations thus interferring it as being weakly cytotoxic, though this effect disappeared in the presence of s9 mix applications. this finding is important for showing the inefficiency of tamiflu metabolites on the cell cycle. introduction: chronic renal failure as a result of the progression of diabetic nephropathy is the main cause of mortality in patients with type 1 diabetes. chronic hemodialysis is a life-saving therapy for patients with strong renal disorders. the main goal of hemodialysis is toxins removal from the patient. the monitoring of hemodialysis is the best way for biomedical evaluation of correctness and efficiency of this clinical treatment. according to the published data, the markers of development of diabetes complicated with renal failure are increased levels of glucose, urea and creatinine in the patient blood. today colorimetric and spectrometric methods are most commonly used for determination of the above metabolites in biological samples. however, these methods are complex in application, have low selectivity, and require pretreatment of samples. materials and methods: we propose for levels of glucose, urea and creatinine detection the potentiometric multibiosensor based on ph-sensitive field-effect transistors and immobilized enzymes developed in our laboratory. results: we developed a potentiometric multibiosensor and studied its main analytical characteristics. linear dynamic ranges of determination of substrates were following: 0.08 -1 mm of glucose, 0.1-10 mm of urea, and 0.02 -2 mm of creatinine. it was shown that the potentiometric multibiosensor had good reproducibility, and its bioselective elements were working independently from each other, because test of substrates cross-selectivity was negative. discussion and conclusion: very sensitive, fast and selective multibiosensor for simultaneous measurement of three metabolites in a single cycle based on ph-sensitive field-effect transistors and immobilized enzymes is developed. the developed potentiometric multibiosensor was verified by quantitative analysis of glucose, urea and creatinine in blood serum of patients with diabetic nephropathy. p-03.04.4-002 ph-dependent interaction of asymmetrically charged peptides with a protein nanopore over the past two decades, the ability to use natural or artificial nanopores to probe at uni-molecular level the structural and kinetic features of various bio-molecules (peptides, dna, rna) was successfully achieved. the operating principles of the nanopore-based single-molecule technique are simple: the single macromolecule capture, entry and subsequent translocations through a free-standing, voltage-biased nanopore, depend upon the physico-chemical and topological features of the analyte. the concentration, identity volume and charge of the analyte are then deduced from the analysis of the stochastic current blockade events caused by the trafficked analyte across the nanopore. herein, we used the a-hemolysin (a-hl) nanopore and set up an experimental model providing efficient control of a-hl-peptide interactions, in the presence of a ph gradient across the nanopore. for this, we engineered a 36 amino acids long peptide containing a neutral asparagines-containing sequence, flanked by oppositely charged aminoacid patches at the n-(glutamic acids) and c-termini (arginines), whose length was set as to span a single a-hl protein. when the ph of the solution in contact to the a-hl's b-barrel opening is changed from neutral to acidic values, the electrostatic interactions between the protein's mouth and either the n-or c-terminus end of the peptide occurs, and this influences strongly the dynamics of a peptide translocating the nanopore. we further proved that during the same experiment, peptide entry into the nanopore can be set to occur with either n-or c-terminus end head on, by simply changing the sign of the transmembrane potential across the nanopore. nanopores are emerging as a powerful and broadly applicable tool in biophysics, which allows one to study the features of charged macromolecules under confinement. a few noteworthy examples are: determining the electrophoretic mobility, effective charge and diffusion coefficients of charged molecules; exploring the folding and unfolding of peptides and proteins; analyzing biopolymers trafficking, protein transport, dna translocation, rna and dna sensing and sequencing. herein, we employ single molecule analysis techniques using a wild-type ahemolysin (a-hl) protein nanopore to study the capture and translocation behavior of a short cationic peptide (20 amino acids in length) at an extremely low ph value. our experiments revealed that an effective absorbing field is created by the electroosmotic flow, against the electrophoretic force, which enables the peptide capture inside the nanopore. furthermore, our findings show that the trajectory of a single peptide can be experimentally visualized and the main steps determined: the peptide capture, reversible translocation across the pore's vestibule and lumen regions, and the peptide release from the nanopore. also, the kinetic analysis of the main steps observed allowed us to describe the free energy profile of the peptide interactions with the protein nanopore. the presented work provides evidence for the ability of controlling the dynamics of a single-peptide, its capture and passage inside a a-hl nanopore, that underlie the processes naturally occurring in cells, thus proving a powerful approach for probing single molecule biophysics phenomena, in general. changes in the physical conditions of the cancer microenvironment driven by elevated tissue growth and angiogenesis, may introduce exposure of laminar fluid flow, which effect the key factors of cancer, such as progression, immune-escaping and metastasis. conventional experimental models fail to mimic the physical cues on tumor microenvironment. microfluidic culture techniques allow precise control of fluids, simultaneous manipulation and analysis of cultured cancer cells. here, we present a platform that can be used for the investigation of the role of flow mediated mechanical stimuli on cancer cells. microfluidic cell culture platform was fabricated using polymethyl methacrylate and double-sided adhesive films with 25 9 41 mm dimensions. ovary adenocarcinoma cells (efo-27 and onco-dg-1) were used for the optimization of the platform. to understand the fluid and gas distribution patterns, specific modeling was performed. dynamic microfluidic cell culture and static conventional cell culture conditions were compared for the differences of cancer cell phenotype, such as proliferation, viability, epithelial-mesenchymal transition. we confirmed that, the proliferation and viability of cancer cells are increasing under dynamic fluid flow. the proliferation rate of ovary adenocarcinoma cells was correlated with the increase of fluid flow rate. immunocytochemical analysis showed that fluid flow causes decrease in e-cadherin expression, and increase in n-cadherin and vimentin expressions, which indicate mesenchymal phenotype of cancer cells. our results showed that, cancer cells present different characteristics due to fluid flow of tumor microenvironment. to understand the role of physical dynamics by using microfluidic culture techniques, is a key to elucidate the mechanisms underlying disease progression, and may lead to new diagnostics and therapeutic approaches. (this study was funded by turkish scientific and technical research council (tubitak-214s334). high-sensitive detection of low-affinity antibodies by immuno-pcr with supramolecular olygonucleotide-streptavidin complex detection of low affinity antibodies in blood sera and cell surface outwashes is important both in the study of molecules that bind to cellular receptors (circulating tumor cell masking antibody, for example) and medicine (diagnosis of allergy). low affinity igm and ige antibodies can not sometimes be determined by conventional methods. we using supramolecular oligonucleotide-streptavidin complex formed from single-stranded synthetic oligonucleotide (60 n) contains biotin on 5'-and 3'-ends, and sterptavidin in molar ratio 1:1. this complex represents a structure with equivalent electrophoretic mobility of 600 bp dna and preferred "valency" of streptavidin is 2. this universal immuno-pcr approach make it possible to increase a signal by using several oligonucleotides per one antibody. after the method optimization we achieved 100-1000 times highter sensitivity than elisa. to reduce the matrix effect we used 100-500 fold dilutions of sera samples. this approach achieved a significant advantage, because it allows working with small-volume samples (need only 2 mkl of serum sample). antibodies to the disaccharide galb1-3glcnac (le c ) are typical of the natural antibodies. the igm anti-le c antibodies are found in almost healthy people without the epitope specificity variation. we have shown that the concentration of igm anti-le c antibodies was higher (p ≤ 0.0005) for health donor sera (n = 45; 31.3 ae 4.3 pg/ml) compared with sera from patients with breast cancer (n = 38; 17.0 ae 4.1 pg/ml). sensitivity of igm anti-le c antibodies detection was 100 pg/sample (50 mkl) ie 6.7 9 10 7 molecules. thus for the immuno-pcr detection of antibodies the 10 3 -10 4 tumor cells are sufficient. such amount of cells seems to be a realistic one for detection of antibodies masking circulating tumor cells. this study was supported by a grant from russian science foundation (#16-14-00136) and by russian federation president scholarships donated to d. yu. riazantsev (# sp4413.2015.4 bacterial pathogen detection and identification is of crucial importance for disease diagnosis, bacterial contamination surveys and water quality assessment. we propose herein a novel method for bacterial detection based on the interaction of single gram-negative bacterial cells (i.e.: escherichia coli and pseudomonas aeruginosa) with an ahemolysin (a-hl) protein nanopore embedded in a reconstituted lipid bilayer, at neutral ph. as a consequence of an applied voltage, the negatively charged bacteria suspended in saline buffer solution are electrophoretically driven towards the pore opening, inducing reversible blockages in the ionic current through a-hl. experiments were also performed in the presence of an antimicrobial peptide, cma3, as well as in acidic environment. statistical analysis of the frequency and duration of blockage events allowed us to discriminate between the two types of bacteria. the frequency of interactions was higher for escherichia coli with respect to pseudomonas aeruginosa. adsorption of cma3 peptides on the membrane of bacteria increased the frequency of interactions with the pore, contrary to the expected effect induced by lowering the net surface charge of the cells. in experiments performed at ph = 4, the frequency of blockage events was found to be two orders of magnitude higher, with longer interaction life-times. the net negative charge (7 uncompensated aspartate residues) localized at the entrance of the pore contributes an additional electrostatic repulsion interaction between negatively charged bacterial cells and a-hl. thus, adsorption of cationic peptides at the interface will reduce this repulsive interaction. the same effect was recorded at ph = 4, when the aspartate residues are partially protonated, confirming our understanding of the previously observed results. this method could be further developed and integrated with other techniques, making nanopore-based systems a fast and reliable bacterial detection and identification tool. this study was performed to analyze the effects of tunicamycin (tm) and taurohyodeoxycholic acid (tudca) on thle-3 cells. cells were treated with tm to induce endoplasmic reticulum (er) stress and tudca was administered as an er stress inhibitor. cytotoxicity was evaluated at different times of exposure by incubating cells with increasing concentrations of either tudca, tm or both. thle3 cells were cultured in fibronectin, bovine collagen i and bovine serum albumin coated plates. cell lines were grown in begm media supplemented with epidermal growth factor, phosphoethanolamine, fetal bovine serum, 100 u of penicillinstreptomycin and maintained in a humidified incubator at 37°c and a 5% co 2 atmosphere. cell viability was measured using the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay kit. cells were grown to confluence in 96well plates and incubated with 1 ll/ml dmso, 1-10 lg/ml tm, 0.1-5 mm tudca, or 10 lg/ml tm + 0.1-1 mm tudca for 18-48 hours. control cells were prepared in plates containing only medium. at the end of the incubation period, mtt was added to each well and incubation was carried out for 4 hours at 37°c. formazan production was expressed as a percentage of the values obtained from control cells. at all hours of incubation neither dmso or 1 mm tudca was cytotoxic. at 24 and 48 hours incubations 5 mm tudca and 10 lg/ml tm + 1 mm tudca were significantly cytotoxic compared to control, dmso and 1 mm tudca groups. treatment of cells with 0.5 mm tudca 8 hours before administrating 10 ug/ml tm significantly decreased the cytotoxic effect of tm. we conclude that tudca may show cytotoxic effects at 1 mm concentration when treated with tm. therefore 0.5 mm of tudca, administered 8 hours before tm treatment should be applied to protect against er stress. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; 214s223). recent studies reveals that history of preeclampsia is an independent risk factor for cardiac events and stroke. lipoprotein-associated phospholipase a2 (lp-pla2) is a vascular inflammatory marker associated with cardiovascular diseases (cvd). we hypothesize that vascular inflammation (lp-pla2 mass, activity, index) related genetic variations (pla2g7) increase the risk for developing future cardiovascular disease in women with pe. a group of 150 preeclamptic patients and 50 normal pregnant women were recruited from university of istanbul, cerrahpasa medical school, department of gynecology and obstetrics included into the study. the control group was matched for maternal and gestational age at time point of sampling. preeclamptic patients were starified into two groups; early-onset and late-onset according to the 32 gestational weeks. enzyme-linked immunosorbent assay procedure was used to determine the serum lp-pla2 mass level. lp-pla2 activity were determined by kinetic method. plag7 snp genotyping performed by using the sequenom massarray iplex. the rs1805017 tt genotype had a higher lp-pla2 index (p = 0.015) for early onset preeclampsia, cc genotype had a higher lp-pla2 mass and lp-pla2 index for late onset preeclampsia. no difference were found for control. the rs1809381475 gg genotype had higher lp-pla2 mass and index for late onset preeclampsia (p = 0.008, p = 0.012 respectively). stepwise logistic regression analysis performed to identify cardiovascular disease related variables that independently and significantly contributed to the presence of alleles of rs1805017 and rs1809381475 snps in early, late onset preeclampsia and control group. only lp-pla2 mass was independently and significantly associated with both snps in early onset preeclampsia. the association between lp-pla2 mass, index and rs1809381475, rs1805017 snps might be useful genetic markers to adress future cvd risk in patients with preeclampsia. introduction: b-thalassemia is one of the most monogenic autosomal recessive disorder characterized by defective production of the b-chain of hemoglobin. definition of the b-globin genotype is necessary for genetic counselling in the carriers, and for predicting prognosis and management options in the patients with thalassemia. dna-based diagnosis of b-thalassemias routinely relies on polymerase chain reaction (pcr) and gel electrophoresis. the aim of this study is to develop a new procedure, a dna-based piezoelectric biosensor, for the detection of b-thalassemia ivsi-110 mutation, the most common b-thalassemia mutation in turkey. materials and methods: b-globin gene of genomic dna isolated from whole blood, was amplified by pcr. bioactive layer was constituted by binding 2-hidroxymetacrilate metacriloamidoscystein (hema-mac) nanoploymers on the gold electrode's surface. single oligonucleotide probes specific for ivsi-110 mutation of b-thalassemia were attached to the nanopolymer via reactive cross-linker glutaraldehyde. the measurements were executed by piezoelectric resonance frequency which is caused by binding of pcr products in media with single oligonucleotide probe on the electrode surface. the results were confirmed by the conventional molecular method as arms. results: the piezoelectric resonance frequencies obtained by hybridization of the pcr products on bioactive layer were found 216 ae 12, 273 ae 6, and 321 ae 9 hz for the samples of normal b-globin, heterozygote, and homozygote of ivsi-110 mutation, respectively. discuss and conclusion: the developed biosensor serves as a specific result to ivsi-110 mutation. it could accurately discriminate between normal and ivsi-110 mutation samples. because of low costs, fast results, specificity and high detection/information effectiveness as compared with conventional methods, we can be offered this techique as an alternative to conventional molecular methods. the increasing use of nano-sized materials in the last several years has compelled the scientific community to investigate the potential hazards of these unique and useful materials. one of the most widely used nanoparticles is titanium dioxide. the objective of the research is to investigate the alterations in molecular and cellular responses in culture of primary lymphocytes to tio2 nps. human lymphocytes isolated from heparinized blood of healthy individuals were exposed to tio2 nanoparticles. viability, ros generation, the changes in the expression of genes encoding proinflammatory mediators tnf-a, il-1b and il-8 and dna damage were assessed. human lymphocytes were incubated with nanoparticles of different concentrations and viability was determined in 24 and 48 hours after treatment, respectively. cell viability was decreased by a treatment with nanoparticles in both a time-and concentration-dependent manners. the ability of tio2 to induce ros formation in lymphocytes was evaluated using dcf fluorescence as a reporter of oxidant production. the fluorescence intensity of oxidized dcf was increased in cells treated with nps. this means that ros generation occurred in response to the treatment with tio2. to investigate the expression level of mrna related to the inflammation responses in human lymphocytes real-time pcr was performed. the expression of il-1b, il-8 and tnf-a genes were increased by the exposure to nanoparticles of 10, 20 and 40 lg/ml for 24-48 hours. tio2 nanoparticles were shown to induce the dose-dependent fragmentation of dna strands. much evidence of hazardous health effects of nps has been reported. in this study, viability was reduced under the exposure to tio2. oxidative stress was elevated by the treatment with tio2 nps. oxidative stress can also trigger inflammation signals. induced by exposure to nanoparticles they may cause the translocation to the nucleus of transcription factors, which regulate proinflammatory genes, such as tnf-a, il-1b, il-8. background: endothelial cells (ec) represent one of the primary targets of the major pro-inflammatory cytokinetumor necrosis factor (tnf). development of the new approaches for the treatment of acute and chronic inflammatory conditions, including the strategies aimed to tnf neutralization, requires the usage of the adequate cellular models closely resembling the properties of the endothelium. the endothelium-derived ea.hy926 cell line expresses several inflammation and neoangiogenesis markers in response to activation factors however their expression can differ from the patterns demonstrated by primary ec. the aim of the current study was to compare the expression of the known endothelial cellular markers including receptor of vascular endothelial growth factor-2 (vegfr2) and a v b 3 -integrin on 2d and 3d cultures (spheroids) of ea.hy926. methods: the ea.hy926 cell line was used with permission from dr. edgell. the cells were cultivated in the presence of tnf (25 ng/ml) or vegf a (25 ng/ml) for 5 hours. mrna was isolated using rneasy kit from qiagen and reverse-transcribed with revertaid kit (fermentas). rt-pcr was performed with specific primers. expression of vegfr2 and a v b 3 -integrin was visualized by confocal microscopy using specific monoclonal antibodies and previously developed fluorescent hybrid proteins. results: the expression of a v b 3 -integrin and vegfr-2 increased on the 3d culture compared to 2d according to confocal microscopy and rt-pcr. the aforecited methods revealed elevated expression of a v b 3 -integrin in the 3d culture of the ea.hy926 cell line activated with tnf. also increased expression of vegfr2 in the 3d culture activated with vegf a. then by confocal microscopy, we analyzed our fluorescent hybrid proteins that bind a v b 3 -integrin and vegfr2 on the surface of 2d and 3d cultures as well as antibodies with fluorescent label. conclusions: 3d cultures of the ea.hy926 cell line represent a promising model for the inflammation studies. tumor necrosis factor (tnf) is a trimeric cytokine associated with the inflammatory response to tissue injury and found to possess a key role in rheumatoid arthritis pathogenesis. spd 304 is a highly toxic recently discovered tnf inhibitor that promotes trimer dissociation and lead to the inactivation of the protein. according to the traditional anti-tnf treatment of ra, we aim at extracellular inhibition of this pro inflammatory cytokine as an effective therapy. the project plan comprises design, synthesis and validation of candidate inhibitors (measurement of dissociation constant and aqueous solubility). because of the elevated percentage of insoluble compounds a solubility enhancement protocol has been developed. the experimental procedure was the following:: a. drug design. identification of novel drug compounds are based on two approaches: i) structure based drug design using the 3d structure of tnf and ii) design of more potent and less toxic spd304 analogues. b. drug synthesis. a series of spd304 analogues were in house synthesized while novel candidates discovered by in silico approaches were commercially available. the purity of the majority of the compounds exceeded 90%. c. solubility measurement and enhancement. samples were incubated under specific conditions that can enhance aqueous solubility and solubility measurement with a direct uv method pursued. d. measurement of the dissociation constant. a fluorescence binding assay was used in order to evaluate the inhibitory activity of the compounds. from our results it can be concluded that dmso, peg3350 and b-cyclodextrin can be used for solubility enhancement without interfering with fluorescence assay. however peg3350 -in contrast to dmso-is not suitable for isothermic titration calorimetry measurements. dissolution procedure also plays a crucial role in the levels of solubility reached. finally, it has been shown that some of the studied spd304 derivatives have better dissociation constants than spd304. the effect of exercises on serum bmp-6 levels of knee osteoarthritis cytokines. more complicated approaches are expected to focus on molecular proteins as bone morphogenetic proteins (bmps) of the transforming growth factor (tgf)-beta superfamily. bmps associated with many cellular functions, such as proliferation, differentiation, and apoptosis. bmp-6 is significantly important for the endochondral bone formation. inflamation can induced serum bmp levels in oa patients. the aim of this study is to evaluate the clinical findings of oa patients after the isokinetic exercise together with the serum levels of bmp-6 to sustain the molecular approaches for treatments. a total of 36 patients were included in this study. the groups are formed as follows: group 1, oa patients before the exercise; group 2, oa patients after the exercise; group 3, oa patients before the isokinetic exercise; group 4, oa patients after the isokinetic exercise clinical and biochemical findings were evaluated before and after 3 weeks of the exercise programme. self reported severity of pain was measured using the 100 mm visual analog scale (vas), womac scores were calculated and isokinetic knee muscle strength testing was measured using cybex dynamometer that a standardized protocol previously described was applied in a subject-specific range of motion. serum bmp-6 levels of all patients were studied by elisa method. results represented a better vas and womac scores for all exercise groups after treatment. the serum bmp-6 levels were significantly decreased in group 2 compared to group 1 (222.94 ae 44.66; 246.49 ae 38 .15 respectively, p < 0.01) and in group 4 compared to group 3 (246.74 ae 32.26; 256.23 ae 33.11 respectively, p < 0.05). there is not any statistically differences between group 2 and group 4 (p > 0.05). as a conclusion, the decreased serum levels of bmp-6 may be suggested as a biochemical marker for oa patients during exercise programmes. p-04.04.4-006 tnf-a blokade efficiently reduced severe intestinal damage in necrotizing enterocolitis c. tayman, s. aydemir, i. yakut, u. serkant, a. c ß iftc ßi g€ olbasi devlet hospital, ankara, turkey objectives: to ascertain the beneficial effects of infliximab an inhibitor of tumor necrosis factor alpha (tnf-a) on the development of nec in an experimental nec rat model. material and methods: thirty newborn sprague-dawley rats were randomly divided into three groups as nec, nec+ infliximab, and control. nec was induced by enteral formula feeding, exposure to hypoxia-hyperoxia and cold stress. pups in the nec+ infliximab group were administered infliximab at a dose of 10 mg/kg daily by intraperitoneal route from the first day until the end of the study. all pups were sacrificed on the 5th day. proximal colon and ileum were excised for histopathologic, immunohistochemical (tunel and caspase-3), and biochemical evaluation, including, total antioxidant status (tas), total oxidant status (tos), malonaldehyde (mda), and myeloperoxdase (mpo) and tnf-a activities. results: we observed better clinical sickness scores, weight gain, and survival rate in the nec+ infliximab group compared to the nec group (p < .05). histopathological and apoptosis examination (tunel and immunohistochemical evaluation for caspase-3) revealed lower damage in the nec+ infliximab group compared to the damage in the nec group (p < .01). tissue mda, mpo, tnf-a levels, and tos were significantly decreased in the nec+infliximab group, whereas tas was significantly increased in the nec + infliximab group (p < .01). conclusion: tnf-a blockade with infliximab efficiently reduced the intestinal injury and preserve the intestinal tissues from severe intestinal damage by its complex mechanisms on nec. therefore, it may be an alternative option for the treatment of nec.keywords: tnf-a; infliximab; necrotizing enterocolitis; newborn; protection; rat; treatment p-04.04.4-007 short-term diabetes causes cardiovascular inflammation: anti-inflammatory effect of resveratrol introduction: diabetes is a metabolic dysfunction and has been associated with various disorders including inflammation, cardiomyopathy and coronary artery disease. inflammation is a protective mechanism elicited by the host in response to infection, injury, and tissue damage. the aim of this study was to investigate the effect of intraperitoneally resveratrol administration on cardiac and vascular function in diabetic rats. materials and methods: diabetes was induced in sprague-dawley rats by using injection of streptozotocin (55 mg/kg, i.p.). rats were divided into group i: control, ii: control/20 mg/kg resveratrol; iii: diabetic/vehicle; and iv: diabetic/20 mg/kg resveratrol. histopathological examinations with masson's trichrome and verhoeff-van gieson staining were carried out to reveal cardiac and vascular tissue damage and inflammation. in addition to plasma glucose and cardiac & vascular mda levels were measured by standard enzymatic kits while tnf-a, il-1b, il-18 (mbl) were analyzed by elisa kit. results: final body weight decreased in all groups compared to control. in the diabetic rats, plasma glucose and vascular mda levels were enhanced while cardiac mda was unchanged compared to control. vascular tnf-a, il-1b and mbl and cardiac mbl were increased in the diabetic groups compared to control. discussion and conclusion: it has been found that resveratrol has greatly normalized altered parameters. taken together, resveratrol partly improved cardiac and vascular inflammation induced by diabetes. this may be due to the healing activity of resveratrol on pro-inflammatory markers. p-04.04.4-008 cytokine network is critical in growth hormone-induced resistance mechanism against curcumin which modulates jak/stat/ socs pathway in mda-mb-231 and mcf-7 breast cancer cells m. c ß elik, a. c ß oker g€ urkan, e. damla arisan, p. obakan yerlikaya, n. palavan unsal t.c. istanbul k€ ult€ ur university, istanbul, turkey curcumin (diferuloylmethane), a polyphenolic compound that triggers apoptotic cell death in various cancer cells such as prostate, colon, melanoma and breast cancer. a pituitary-derived hormone, growth hormone (gh) play role in elongation and differentiation of ductal epithelia into the breast terminal and buds. in this study, our aim is to determine the role of inflammation in curcumin induced apoptotic cell death via acting on jak/stat/socs pathway in wt and gh+ mda-mb-231 and mcf-7 breast cancer cell lines. according to mtt cell viability assay curcumin triggers cell viability loss in time and dose dependent manner in mda-mb-231 wt and mda-mb-231 gh+ breast cancer cell lines, respectively. selected concentrations of curcumin as 20 lm (for mcf-7) and 25 lm (for mda-mb-231) decreased cell proliferation and induced apoptosis through causing jak2 dephoshorylation, stat1, 3, 5 dimerization and acting on socs proteins expression in each cell lines. in addition, activated jak/stat/socs pathway, via forced gh expression has been suppressed following curcumin treatment for 24 hours. 20 lm curcumin-induced apoptotic cell death via dephosphorylating jak2 at tyr1007/1008 residues and decreased phospho-stat1, 3 level in both breast cancer cell lines. although curcumin dephosphorylated stat5 in both mda-mb-231 and mcf-7 wt cells, no significant effect has been observed in mda-mb-213 gh+ and mcf-7 gh+ cell lines. in consequence, although forced gh expression induced cell proliferation in mcf-7 and mda-mb-231 breast cancer cells, curcumin overcame gh-mediated resistance mechanism via acting on jak/ stat/socs signaling, which is related to pparg-induced inflammation. acknowledgment breast cancer is one of the highest cancer type among women worldwide. various enviromental and genetic factors such as age, gender, family history, metabolic diseases and gene mutations are involved in the breast cancer pathogenesis. growth hormone (gh), a pituitary derived hormone, has essential role on postnatal growth and development. it is also established that signalling route of gh and its receptor (ghr) activity is increased in different cancer types. curcumin, a nutraceutical deriatives from rhizomes of turmeric (curcuma longa), has potential therapeutic activity against cancer cells, including breast cancer. curcumin inhibits proliferation of cancer cells such as prostate, colon, melanoma, cervical and breast cancer via induction of apoptosis and inflammation. stat5, a major downstream target of gh/ghr signalling, is related to survival, proliferation and differentiation. in this study, our aim was to investigate curcumin-induced apoptotic cell death in gh overexpressed mda-mb-453 breast cancer cells via jak-stat/socs signalling and inflammatory response profile. according to mtt cell viability assay, curcumin decreased cell viability in time and dose dependent manner in wt and gh+ mda-mb-453 breast cancer cell lines. we found that 20 lm curcumin-decreased in apoptotic cell death through inactivity at jak2 which led to dimerization of stat1, stat3, stat5. concomitantly, curcumin affected stat regulating socs proteins in mda-mb-453 breast cancer cell line. in addition, we demonstrated that 20 lm curcumin induced pparg expression and altered inflammatory cytokine signalling cascade. consequently, although gh overexpression led to agressive profile in mda-mb-453 breast cancer cells, curcumin overcame this resistance. inflammation is involved in many systemic disturbances, including osteoarthicular or skin diseases, coordinating the signaling network that contributes to tissue injuries. the aim of our study is to reveal pro-inflammatory messengers at the cutaneous barrier (keratinocytes, fibroblasts, endothelial cells), simulating the dermal impact of active principles, especially polyphenols and flavones from vegetal sources: salvia officinalis, asculum hippocastanum and calendula officinalis. we focused on il6 and il8 cytokines as main mediators of inflammation progression, correlated in keratinocytes with il1a as skin irritation indicator and vegf as pro-angiogenic factor, as well as in endothelial cells with icam-1 and vcam-1 adhesion molecules expression. in order to in vitro mimic the inflammatory conditions, we used targeted stimuli for each type of cells: for fibroblasts and endothelial cells -tnfa, a systemic stimulus, single or combined with pma that activates protein kinase c and up regulates nadph oxidase, which lead to superoxide anion production; for keratinocytescontrolled uv-a and uv-b radiation, simulating the solar damages or potential uv interactions with active principles in light exposed skin. the main analysis technique was flow cytometry: beads bases assay for soluble factors and fluorescent antibodies staining. our results prove the different involvement of polyphenols and flavones in the anti-inflammatory mechanisms, depending of the vegetal source: active principles from salvia officinalis induce a strong inhibition of il6 and il8 in tnfa stimulated keratinocytes, fibroblasts and endothelial cells, reduce the icam-1 over-expression but have no effects on irradiated keratinocytes; biocomplexes from asculum hippocastanum inhibit only il8 release in stimulated fibroblasts, but protect keratinocytes from uv-a and uv-b radiation; compounds from calendula officinalis are active on il8 signaling in fibroblasts and counteracts only uv-b inflammation. ischemia and/or reperfusion injury is one of the most common causes of acute renal failure. ischemia-reperfusion associated with thrombolytic therapy, organ transplantation, coronary angioplasty, aortic cross-clamping, or cardiopulmonary bypass results in local and systemic inflammation. within the endothelium, ischemia produces expression of proinflammatory gene products (e.g. cytokines) and bioactive agents (e.g., endothelin), while preventing other "protective" gene products (e.g., thrombomodulin) and bioactive agents (e.g. nitric oxide). therefore, ischemia induces a proinflammatory state that increases tissue vulnerability to further injury on reperfusion. this experimental study was designed to investigate the protective effect of salvia l. extracts on kidneys from i/r injury. salvia lamiaceae have been used for treatment of some illnesses in turkish folk medicine. forty spraque dawley rats were divided into 5 groups (n = 8). right nephrectomy was performed to all groups. group i: control group; group ii: i/r group; group iii: i/r + 50 mg/kg salvia l.group; group iv: i/r + 100 mg/kg salvia l. group; group v: i/r + 50 mg/kg rosmarinic acid. group. salvia l. and rosmarinic acid for 7 days was given single dose as a gavage. 60 minutes ischemia, 60 minutes reperfusion were applied to groups except control. intracardiac blood samples were taken, high sensitive crp (hscrp), tumor necrosis factor-a (tnf-a), interleukin (il)-6 and interleukin ib (il-1b) levels were detected. serum hscrp levels were also determined in our clinical laboratory using routine standard methods. serum tnf-a, il-6 and il-ib levels were evaluated using an enzyme-linked immunosorbent assay technique. mean values were evaluated by statistical analysis. serum hscrp, tnf-a, il-6, and il-1b concentrations were significantly increased after renal i/r as compared to the control group. our treatment group 50 mg/kg salvia l. and 50 mg/kg rosmarinic acid especially 100 mg/kg of salvia l. were found to show a protective effect against renal structure and function. we concluded that salvia l. extracts could be beneficial in the treatment of renal ischemic injury. but 100 mg/kg salvia l. extract were more effective than 50 mg/kg salvia l. extract and used as synthetic 50 mg/kg rosmarinic acid. acne vulgaris is a common chronic inflammatory skin disease of unknown etiology. excess levels of secretory phospholipase a2 (spla2) contributes to inflammatory diseases and studies indicate that lipoprotein lipase (lpl) has differential effects on several inflammatory pathways. the aim of the present study was to assess serum activity of spla2, lpl and evaluate changes in circulating protein levels of angiopoietin-like protein 3 (angptl3), angptl4, cyclooxygenase (cox) and prostaglandin e2 (pge2). serum from 21 control subjects and 31 acne vulgaris patients with moderate and severe disease was evaluated for levels of spla2, cox, pge2, lpl, angptl3 and angptl4. disease activity was determined according to the national health service (nhs) lambeth and southwark clinical commissioning group guidelines for the management of acne. lipid profile, routine biochemical and hormone parameters were assayed by standard kit methods using autoanalyzers (beckman coulter au5800 clinical chemistry and unicel dxi 800 immunoassay systems). serum levels of spla2 and lpl were significantly increased in acne vulgaris patients compared to age and gender matched controls. no significant differences were found for cox, pge2, angptl3 and angptl4 levels between acne vulgaris patients and controls. the results of this study reveal the presence of a proinflammatory state in acne vulgaris as shown by significantly increased serum spla2 activity. increased lpl activity in serum of acne vulgaris can be protective in patients through its anti-dyslipidemic actions. to our best knowledge, this is the first study investigating spla2, lpl, angptl3 and angptl4 levels in acne vulgaris. future studies are aimed to understand the regulation of spla2 and lpl expression in acne vulgaris patients. acknowledgement: this study was supported by a grant from the scientific and technological research council of turkey (tubitak; #115s940). p-04.04.4-013 8-ohdg and hogg1 levels are as an oxidative dna damage markers in acne vulgaris treated with isotretinoin h. ecevit, m. izmirli, b. gogebakan, e. rifaioglu, d. sonmez, b. bulbul sen, t. sen, h. m. okuyan mustafa kemal university, hatay, turkey acne vulgaris is a skin disease that characterized by comedones, papules, pustules, nodules and cysts at face, back and body skin. isotretinoin is one of the treatment agents in acne vulgaris. about 4 weeks after drug treatment, the amount of sebum which is produced by sebaceous gland reduces keratinization disorder and the number of propionibacterium acnes normalizes. however, isotretinoin is known that has a wide range of side effects. in recent studies, isotretinoin treatment has been shown to increase the oxidative stress. 8-hydroxy-2 0 -deoxyguanosine (8-ohdg), an important indicator of oxidative dna damage, hydroxyl ion is bound at the 8th carbon of guanine. this structure is repaired through a base excision repair mechanism and the human 8-oxoguanine dna glycosylase 1 (hogg1) plays a key role in this processes. in this study we aimed to evaluate the dna damage and it's repair in acne vulgaris before and after 6 months of isotretinoin treatment by measuring 8-ohdg and hogg1 levels. the current study includes 43 acne vulgaris patients who are diagnosed in mustafa kemal university, department of dermatology. 8-ohdg and hogg1 levels were measured by enzymelinked immunosorbent assay (elisa) method for before and after 6 months of isotretinoin treatment. the commercial elisa kits (cloud-clone corp; usa and cell biolabs; usa) were used for the assessment of hogg1 and 8-ohdg, respectively.both 8-ohdg (p as a conclusion, isotretinoin increases dna damage and high serum 8-ohdg and hogg1 levels as a result of isotretinoin treatment may effect on the amount of reactive oxygen species. the pineal gland is a circumventricular organ which serves as a major neuroendocrine gland in the brain. its primary function is the production of melatonin which is controlled by signals from the suprachiasmatic nucleus. melatonin codes the length of the night and it is well recognized for its anti-inflammatory effects. lipopolysaccharide (lps) is the essential component in the outer surface membrane of gram-negative bacteria and act as a strong stimulator of natural and innate immunity in all eukaryotic species. furthermore, lps reduces melatonin synthesis and induces the expression of the serine protease inhibitor 3 (spi-3) in the stat3-mediated manner in pinealocytes. however, the precise function of stat3 in the cell signaling in the pineal gland is not yet known. here we investigated the effect of inhibition of stat3 on lps-induced changes in melatonin levels, expression of arylalkylamine n-acetyltransferase (aa-nat) and spi-3 in the pineal gland. experiments were performed in vitro using organotypic and primary cultures prepared from the rat pineal glands. levels of melatonin and spi-3 were determined from tissue homogenate by enzyme-linked immunosorbent assay (elisa). the pinealocytes were used to carry out sirna stat3 transfection. the successful transfection and subsequent decline in stat3 expression levels were proved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (sds-page). the changes in synthesis of aa-nat and spi-3 were studied by rt-pcr. in conclusion, lipopolysaccharide can affect the immunomodulators secreted by the pineal gland. the clarification of the effect of inhibition of stat3 on those immunomodulators is important from the clinical point of view because inhibitors of stat3 are nowadays used as tumour suppressors. silica nanoparticles have a great potential for a variety of industrial, diagnostic and therapeutic applications. in this study, we have evaluated the in vitro effects of amorphous silica nanoparticles (7 nm) using human lung mrc-5 fibroblast as model. cells were exposed to 62.5 lg/ml silica nanoparticles for 24, 48 and 72 hours. the cytotoxic and inflammatory response, and matrix metalloproteinase expression were examined. the pro-inflammatory cytokine il-1b, il-6, il-8, tumor necrosis factor (tnf-a), matrix metalloproteinases (mmp-2, mmp-9, mmp-1) and tissue inhibitor of metalloproteinase-1 (timp-1) were analyzed by western blot method. cytotoxicity was evaluated by lactate dehydrogenase (ldh) released into the culture medium by damaged cells. the level of ldh activity was increased after exposure to silica nanoparticles, in a time-dependent manner compared to control. the protein expression of il-1, il-6, il-8 and tnf-a as well as of mmp-1 and timp-1, was up-regulated whereas those of mmp-2, mmp-9 was down-regulated after 48 and 72 hours respectively. in conclusion, our data indicate that amorphous silica nanoparticles generate a cytotoxic and inflammatory response, as well as an imbalance in extracellular matrix due to the differential regulation of mmps and tissue inhibitor of metalloproteinase-1 in mrc-cells after 48 and 72 hours. p-04.04.4-017 association of fto gene variant (rs8050136) with markers of t2dm and obesity in population from bosnia and herzegovina and kosovo fto (fat mass and obesity-associated gene), recently discovered in a genome-wide association study for type 2 diabetes (t2d) encodes a 2-oxoglutarate-dependent nucleic acid demethylase and is mainly expressed in the hypothalamus. this gene may play important role in the management of energy homeostasis, nucleic acid demethylation, and regulation of body fat masse by lipolysis. the aim of this study was to analyze the association of this single nucleotide polymorphisms (snps) with clinical and biochemical parameters of obesity, t2d, prediabetes and at the level of healthy population from bosnia and herzegovina (bh). the study included 638 patients with t2d and prediabetes and 360 healthy controls both sexes, aged from 40 up to 65 years. patients were recruited at the clinical centre university of sarajevo, university hospital of clinical centre in banja luka, general hospital in te sanj and health centre in prizren. genotyping of analyzed polymorphism was performed by rt-pcr method in cooperation with the department of clinical chemistry, faculty of pharmacy, university of ljubljana (ljubljana, slovenia) and university hospital of charles university (hradec kralove, czech republic). our results did not show significant differences in genotype frequencies of the analyzed polymorphisms between patients with t2d, pre-diabetes and healthy population also, results of logistic regression analyses did not show significant association of risk a allele of fto gene polymorphism -rs8050136 with increased risk of t2d (or = 1.084, 95% ci 0.758-1.551, p = 0.659). a allele was significantly associated with higher values of hba1c, insulin, homa ir index, diastolic blood pressure and higher levels of inflammatory markers (fibrinogen and leukocytes). interestingly, a tendency of association of a allele with higher values of obesity markers (bmi, waist and hip circumference) was noted. further studies are needed on a larger population in order to confirm these results. the water extract of capparis ovata (cowe) has been shown to be used as an alternative medicine for the treatment of multiple sclerosis (ms). cowe was further fractionated and studied for additional anti-neuroinflammatory effects in sh-sy5y cells. for this purpose, the dichloromethane sub-fraction of the cowe extract was tested for its anti-inflammatory effects on selected anti-inflammatory genes believed to be important in ms pathophysiology using sh-sy5y cells. cell viability was assessed using lactate dehydrogenase (ldh) activity in the media conditioned by the crystal violet cell staining. in these cells, levels of the tumor necrosis factor-a (tnfa), nuclear factor kappa-lightchain-enhancer of activated b cells (nf-jb1), glial fibrillary acidic protein (gfap), c-x-c motif chemokine 9 and 10 (cxcl9, cxcl10), matrix metalloproteinase 9 (mmp9), chemokine (c-c) motif 5 (ccl5) and tyrosine-protein phosphatase non-receptor type 11 (ptpn11) were determined by quantitative reverse transcriptase-pcr assay (qrt-pcr). we have found out that the dichloromethane sub-fraction of cowe effectively inhibited the expression of all of the genes given above in sh-sy5y cells. thus, phytochemicals present in the dichloromethane sub-fraction of the cowe extract could be beneficial in preventing/treating neurodegenerative diseases in which neuroinflammation is part of the pathophysiology. studies are underway to identify the individual compound(s) in this subextract of the cowe extract contributing to these effects. this work is supported by tubitak 112s187 and pamukkale university paubap 2014fbe051. p-04.04.4-019 apigenin and luteoline were identified as active anti-inflammatory constitutents of lavandula stoeachas by bioassay guided fractionation h. ipek 1 , s. savranoglu 2 , a. r. t€ ufekc ßi 3 , f. g€ ul 3 , i. demirtas 3 , t. boyunegmez t€ umer 4 1 graduate program of bioengineering, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 2 graduate program of biology, institute of natural and applied sciences, c ß anakkale onsekiz mart university, c ß anakkale, 3 department of chemistry, faculty of sciences, c ß ankiri karatekin university, c ß ankiri, 4 department of molecular biology and genetics, faculty of arts and sciences, c ß anakkale onsekiz mart university, c ß anakkale, turkey introduction: lavandula stoechas, in the genus of lavender, has distinct therapeutic uses among anatolian people. rather than worldwide use of its essential oil in aromatherapy, specifically the aqeous portion as decoction has been traditionally used in anatolia against the components of metabolic syndrome, all of which share a state of chronic inflammation as an underlying cause. the anti-inflammatory constiutents of l. stoechas were isolated using a bioassay guided fractionation in lipopolysaccharide (lps) inflammed raw 264.7 macrophages. materials and methods: an aqeous extract was partitioned into ethyl acetate (eae) and n-butanol fractions. the eae, determined as bioactive extract was seperated into 12 subfractions by column chromatography. e6 was identified as active subfraction subjected to sephadex column to get pure compounds which were then applied to nmr, ir, and uv analyses for structure determination. in raw 264.7 cells, the effects of extracts/fractions/subfractions/compounds on lps induced no production was determined by using griess method. the potential inhibitory effects of each compound on lps induced inos expression were determined by qpcr and western blot. results: p-coumaric acid, apigenin and luteoline were found in the e6, and the first two compounds appeared to be primarily responsible for the anti-inflammatory activity. apigenin and luteoline at 50 lm decreased no production 66 and 80%-ic 50: 56 and 26 lm-by inhibiting inos gene expression 84 and 88% as well as protein expression 94 and 99%, respectively (p < 0.05). conclusion: this is the first time that luteoline and apigenin have been found in eae of l. stoechas, and the anti-inflammatory properties of the eae can be attributed, at least in part, to the presence of these two compounds. we are on the way to gain further insight for the action mechanism of these two active principles as anti-inflammatory agent. tubitak (project id:112t442) support this work. the role of tip60 in the inflammation process immun response generates the first line of host defense during inflammation and plays an important role inducing pro-inflammatory response by generating early response against pathogens. il-6 (interleukin 6) is one of the pro-inflamatory cytokines and its expression increases during the infection to activate the jak/ stat pathway. jak/stat pathway is regulated by hamp (hepcidin antimicrobial peptide). our previous study, we reported that hamp gene expression was decreased in liver-specific tip60 conditional knockout mice, so we thought that tip60 may have a direct or indirect role on inflammation mechanism. tip60 (tat interacting protein, 60 kda) is a member of the myst enzyme family of histone acetyltransferases (hats) and plays an important role in multiple function including cellular signaling, dna repair, cell cycle and apoptosis. in this study, the quantitative gene and protein expression of il-6 were investigated by using taqman real time pcr, western blot and immunohistochemistry analysis in control group, lpsinduced inflammation group and liver-specific tip60 conditional knockout group mouse liver. according to our preliminary results, the gene and protein expression of il-6 was increased in lps-induced inflammation group (p < 0.001, p < 0.001) and liver-specific tip60 conditional knockout group mouse liver (p < 0.05, p < 0.01). our initial data suggest that tip60 may be essential for the inflammation process. this work was funded by grants from the scientific and technological research council of turkey (tubi-tak) (grant number: 114z277). although intracellular reactive oxygen species (ros) level is necessary to maintain cellular homeostasis, elevated intracellular ros level with the impact of unfavorable environmental conditions leads to oxidative stress that may cause damage to dna, proteins and lipids. in case of inflammation, organism seeks to provide cellular homeostatis by increasing ros levels via antioxidant molecules and enzymes. therefore, it was thought that there can be a direct or indirect relation between inflammation and oxidative stress. in this study, inflammation was performed by intraperitoneal injection of lipopolysaccharide (lps). the gene expression and activity of antioxidant enzyme including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glutathione s-transferase (gst), glutathione reductase (gr) and glucose 6-phosphate dehydrogenase (g6pd). additionally, any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level are accepted as an indication for the accumulation of ros, the relative levels of them were also studied. to show our inflammation model was performed in mouse kidney with lps treatment or not, the expression of interleukin (il6), which is accepted as a inflammation marker, was investigated by real time pcr. the expression of il6 was significantly increased in lps treated group. while the level of mda and h 2 o 2 was elevated in lps treated group, gssg was decreased. no changes was seen for gsh level. the correlation was observed between enzymatic and molecular levels. while the gen expression and the enzyme activity of sod, cat, gst, gr, and g6pd were decreased, gpx was increased with inflammation. in conclusion, increasing ros level was observed in the inflammation process and, the antioxidant system was affected at the molecular and protein level. this work was funded by grants from the scientific and technological research council of turkey (tubitak) (grant number: 114z277). the aim of our study is to evaluate effect of vitamin d levels on hemogram parameters including neutrophil %, lymphocyte %, neutrophil % / lymphocyte % ratio (nlr) and mean platelet volume (mpv) in behcet's patients. fifty eight patients with diagnosis of behcet that applied to selcuk university faculty of medicine department of dermatology are recruited to the study. clinical and laboratory characteristics of the patients were obtained from hospital automation. t test was used to examine the differences between the parameters. p < 0.05 was taken to be statistically significant. there was a statistically significant difference between vitamin d values and age (p = 0.036) whereas difference was not significant between vitamin d and neutrophil %, lymphocyte %, nlr, mpv values. according to the literature, there are a lot of studies that show the relationship between vitamin d and hemogram parameters. however, contrary to the previous studies, we were unable to find any significant relationship between vitamin d and these hemogram parameters. these results serve the idea that the effects of vitamin d on the hematopoietic system should be further investigated experimentally and clinically. crimean-congo hemorrhagic fever is a tick-borne disease caused by the arbovirus and characterized by a sudden onset of high fever, severe headache, dizziness, back and abdominal pains. the exact pathogenesis of cchf has not been clarified yet. the aim of this study, clinical cases of cchf in cu, se and zn is to examine the relationship between the concentration of trace elements. the study sample consisted of 30 patients which have been diagnosed with cchf. matched for gender, 30 healthy volunteers were similar to the control group according to age. the patients and control groups, serum cu, zn and se levels were analyzed using atomic absorption spectrophotometer. cchf patients in the group, cu zn and se serum levels were significantly lower compared with the control group. in our study, the cofactor of the antioxidant enzyme cu, zn and se elements were lower. this shows us in cchf disease, a decrease in antioxidant enzyme activity, and suggest that they contribute to the immune system's degradation. p-04.04.4-024 inhibitors of mdm2 ubiquitin ligase as prospective modulators of autoimmunity e. bulatov, a. valiullina, r. sayarova, a. rizvanov kazan federal university, kazan, russia ubiquitin-proteasome system is seen as a pool of promising protein targets for therapeutic impact in many human diseases. mdm2 is an e3 ubiquitin ligase widely studied due to its wellknown role in cancerit negatively regulates p53 oncosuppressor that mediates apoptosis in tumour cells. inhibitors of p53/ mdm2 interaction have long been known as potential anticancer therapeutics. however, recent advances in the field suggest that both mdm2 and p53 might be playing a substantial role in autoimmune processes. we used a small molecule p53/mdm2 inhibitor nutlin-3a to test the effect of p53 activation on peripheral blood mononuclear cells (pbmcs) from both healthy volunteers and patients diagnosed with multiple sclerosis. in our study we employed a variety of molecular biology methods, such as immunoblotting, real-time pcr, mts cell proliferation assay, fluorescence flow cytometry and confocal microscopy. we demonstrated that disruption of p53/mdm2 interaction by nutlin-3a alters the p53 levels and also affects the lymphocyte subpopulations within pbmcs. our findings suggest that p53/mdm2 interaction inhibitors can potentially be used as prospective modulators of immune response in autoimmune diseases such as multiple sclerosis, systemic lupus erythematosus and other. the study was funded by rfbr research grant 16-34-60213 mol_a_dk. can ykl-40 be an inflammatory biomarker in vitamin d deficiency? object: the tumor necrosis factor (tnf) was found to be cytotoxic to tumor cells and to induce tumor regression in mice. except for one member, all receptors to the tnf superfamily bind tnf-related ligands and act mostly on inflammatory system. there are currently 19 tnf superfamily ligands. tnf superfamily ligands share several common features. tnf ligands are generally type ii transmembrane proteins whose extracellular domains are be divided by enzyme to create cytokines. the tnf superfamily currently consists of 29 receptors. tnf family receptors are type i or type iii transmembrane proteins that contain multiple extracellular domains. in this study, we investigated to presence, differences and effects of tnf superfamily and receptors genes in human and mice by using bioinformatics techniques. methods: the nucleotide and amino acid sequence of each protein in human and mice was determined using t-blast-n for homologous sequences. homologous sequences of human tnf family genes found an automated procedure by using psi-blast. the secondary structure of and three-dimensional of the protein were analyzed by psipred and ffas server. netcglyc 1.0 and netphos 2.0 program were used for post-translocation modifications. the web apoptosis database was also used for the lists of domains, proteins containing these domains and their associated homologs. results and conclusion: humans tnf ligands have 17 genes encoding proteins that contain a conserved carboxy-terminal domain. this family of proteins is highly conserved between humans and mice. humans contain 29 genes encoding tnffamily receptors. sequence data from the ncbi databases demonstrated the presence of 24 mouse tnf-family receptors with orthologs in humans and one additional receptor found only in mice. the differences and similarities in the tnfs genes in humans and mice will provide information for understanding the utility and limitations of the mouse models of disease and comparing of immunology outcomes. the development of left ventricular remodeling after acute myocardial infarction is a predictor of shock. the genetic influence on cardiac remodeling, and shock in the early period after acute myocardial infarction are unclear. the aim of the present study was to investigate the relationship between angiotensin converting enzyme (ace) gene polymorphism and modified shock index (msi) in the early period in patients with acute anterior myocardial infarction. overall 140 patients with a first acute ami were included in this study. dna was isolated from peripheral leukocytes. the id status was determined by pcr. based on the polymorphisms of the ace gene, they were classified into 2 groups: deletion/deletion (dd) genotype (group 1, n = 57), insertion/deletion (id), insertion/insertion (ii) genotypes (group 2, n = 83). blood pressure and pulse measurements were performed in all patients within 10 minutes admitted to coronary care unit. msi was defined as heart rate (hr) divided by mean arterial pressure (map). echocardiographic examinations were performed in accordance with the recommendations of the american echocardiography committee. one-way analysis of variance (anova) and chi-square analyses were used to compare differences among subjects with different genotypes. the study was approved by the local ethics committee, and each patient gave a written consent. there were no significant differences among clinical parameters of patients. msi was significantly higher in patients who have ace dd genotype than in patients who have ace id / ii genotypes (1.13 ae 0.52 and, 0.85 ae 0.37, p < 0.05). presentation time hypotension or developing hypotension during admission was reported to be an important predictor of intensive care unit admission besides other vital sign measurements. our results suggested that, ace gene i/d polymorphisms d allele may affect modified shock index in patients with a first acute anterior mi. glucocorticoids (gcs) are widely used in medicine, despite their side effects, e.g. osteoporosis. however, precise molecular mechanisms of gc action, especially on bone marrow (bm) cells, remain controversial. given the osteoprotective role of vitamin d 3 , the aim of our study was to examine prednisolone-induced changes in the rank (receptor activator of nuclear factor kappa-b)/rankl (rank ligand)/opg (osteoprotegerin) pathway of rat bm depending on the state of vitamin d endocrine system. female wistar rats received prednisolone (5 mg/kg b.w.) with and without 100 iu of d 3 (for 30 days). the levels of rank, rankl, opg, 1a-hydroxylase (cyp27b1) in bm were determined by western blotting. vitamin d 3 receptor (vdr) and rankl mrnas were measured by quantitative rt-pcr. 25ohd 3 content in the serum was assayed by elisa. rankand vdr-positive bm cells were quantified using flow cytometry and visualized by confocal microscopy. prednisolone induced a marked increase in rankl and rank levels, while opg level was shown to decrease. this reflects disturbances in cytokine-mediated regulation of bm progenitor cell function. data from flow cytometry indicated a significant growth in the number of rank-positive cells (hematopoietic osteoclast precursors) compared to control. these changes were accompanied by a decrease in the levels of vdr and cyp27b1, which is responsible for 1,25(oh) 2 d 3 synthesis, in bm and 25ohd 3 content in serum. co-localization of vdr and rank in mono-and multinuclear bm cells was observed, indicating a close relation between vitamin d 3 and rank/ rankl/opg pathway. vitamin d 3 co-administration prevented prednisolone-induced changes in bm cells through restoration of vitamin d 3 bioavailability and vdr signaling that resulted in a reduction of the osteoclast progenitor pool in bm. thus, prednisolone-induced imbalance in rank/rankl/ opg system components is associated with impairments of vitamin d endocrine system in bm and can be ameliorated by vitamin d 3 treatment. p-08.01.4-002 heat shock pathway in response to different stress factors the heat shock response is an emergency pathway of the cell, which mediates repair and protection from cellular stress and therefore guarantees the survival of the cell. this stress can range from heat or hypoxia to chemicals and heavy metals. it is highly conserved in all eukaryotic cells and plays an important role during atypical conditions. due to its high complexity, the pathway is not yet completely understood. most important, after activation of the pathway, is the refolding of proteins or, in case of severe misfolding, the depletion of proteins to maintain proteostasis. heat shock factor 1 encoded by the hsf1 gene is known as the main switch point in heat shock regulation. after activation it trimerizes and binds to heat shock elements in target gene promoters. one of these promoters is the hspa1a promoter (hsp72 promoter). the promoter was analyzed by dismantling it to its functional parts. especially three elements, the heat shock elements, were in the focus of this work. in first place parts of the promoter were multimerized and combined with different reporters, like luciferase, by cloning. also mutations in the natural promoter were designed by cloning. the focus now is on the heat shock elements, where hsf1 can bind as a trimer. the idea is that these different elements have various effects on different stressors like heat, chemicals (geldanamycine as hsp90 inhibitor, mg132 as proteasome inhibitor) or heavy metals (cadmium, arsenic, zinc) . this was tested on cells transiently transfected with those promoter variants. for promising variants stable cell lines were created. in these stable cell lines further experiments on mrna level can be conducted. in the last months experiments with the crispr/cas9 system were started. furthermore, experiments on transcriptional (qpcr) and translational (dual-luciferase assay) levels were done as well. in the end we hope to get a clear picture on the regulation of the hspa1a promoter by different stress factors. invasive cancer cells form membrane protrusions, invadopodia, that facilitate cell invasion and metastasis. key players invadopodia include the adaptor proteins tks4 and tks5, the actin regulators cortactin, wip and n-wasp, the kinase src and others. in spite that in the last two decades significant advances in our knowledge of the structure and development of invadopodia have been made, detailed mechanisms they are functioning is not yet available. we have identified a series of new tks4 binding partners including adaptor proteins itsn1, itsn2, crk and grb2, kinase src, amph1, bin1, plcg1 and also another member of the tks family -tks5. it may indicate the possible role of tks4 in transport and sorting of cell vesicles. current data are supported by interaction with the proteins of amph1 and bin1, as their main functions are membrane trafficking and remodeling. adaptor proteins crk, grb2 and itsns are important for the actin cytoskeleton rearrangements, endocytosis and signal transduction. moreover, we have identified and characterized new tks4 isoform -tks4-beta. we suggested that an active state of tks4 is regulated via intramolecular interactions between its proline-rich motifs and own sh3-domains. we have shown the interaction between itsns and other prominent component of invadopodia wip. data from immunofluorescent analysis revealed co-localization of itsn1 and wip at the sites of invadopodia formation and in clathrin-coated pits. we have also demonstrated that the key protein itsn1 and wip and n-wasp can form a complex in cells. together, these findings provide insights into the molecular mechanisms of invadopodia formation and identify itsns as scaffold proteins involved in this process. we have shown the interaction between itsns and other verprolin family members cr16 and wire which play an important role in the reorganization of the actin cytoskeleton. we have demonstrated that cr16 and wire interact with sh3domains of itsns in complex with actin. p-08.01.4-004 correlation between proteomic and phenazine profile of pseudomonas sp. phenazines are widely known compounds with huge variativity of biological activities which are produced by pseudomonas sp. and some other bacteria species. the results of our work shows the correlation between the changes of proteomic profile of pseudomonas aeruginosa caused by a mutagenesis and the secondary metabolism of antibiotics (phenazine) profile. different strains of pseudomonas aeruginosa were obtained using mutagenesis, after that bacterial cells were destroyed by ultrasound. protein-containing fractions were isolated using methanol-chloroform method as well as phenazines compounds were extracted from culture media using liquid phase extraction. obtained proteome was analysed by shotgun-proteomics technique. as the result of the liquid phase extraction phenazine compounds were mainly extracted to the organic phase. this phase was evaporated and re-dissolved in 100% methanol. after sample preparation obtained solutions were analyzed by hplc-agilent 1290 with quadrupole tof mass-detector. results of the analysis were compared with the library of known phenazine compounds mass-spectras generated by cfm-id online resource. obtained phenazine profiles were compared with each other and correlation with the changes in proteome was analyzed. received results promote better understanding of mechanisms of phenazine production. this data opens possibilities for targeted changes in the methabolic pathway in order to obtain phenazine compound with required biological activity. insulators are genomic elements which block enhancer-promoter interaction and prevent spreading of heterochromatin. cp190 protein is an integral component of most known drosophila insulators, it interacts directly with ctcf and pita dna-binding insulator proteins using dimeric btb-domain, but function of cp190 within insulators still remains to be elucidated. recently we described an interaction between cp190 btb-domain and cterminal domain of ctcf insulator dna-binding protein, subsequent deletion analysis allowed us to isolate 50aa fragment within ctcf c-terminal domain sufficient for interaction with cp190 btb, but deletions of flanking regions also lead to the loss of interaction with cp190 in vivo. at the same time crosslinking experiments suggest that a dimer of btb interacts with one molecule of ctcf, presuming that it could recognize two peptide fragments within ctcf c-terminal domain. we solved crystal structure of btb-domain from cp190 insulator protein at 1. 3 a resolution. overall structure is similar to other btb-domains. cp190 btb-domain has peptide-binding groove similar to that previously found in bcl6 btb domain. inspection of btb-domain surface revealed several possible binding sites for polypeptide fragments from ctcf protein. based on these observations a set of point mutations within peptide-binding groove of btb-domain has been designed and we tested ctcf-interaction abilities of these mutants using gst pull-down assay and yeast two-hybrid assay. the most significant impact was found with alanine-substitutions of hydrophobic residues whereas substitutions of hydrophilic amino acids were less effective. therefore our results support that cp190 btb-domain recognizes ctcf protein using peptide-binding groove. this study was supported by the russian science foundation (project №14-24-00166). p-08.01.4-006 comparative study of the fatty acid composition of lipids in the raw meat samples obtained from hybrid sheep one of the most important tasks in the animal biology and husbandry is to clarify the role of animal genetic diversity in providing nutrients to the diversity of animal products. the objective of our work was to study the chemical compositions of raw meat samples obtained from domestic (group i -purebred romanov sheep) and hybrid sheep (group ii -f 3 hybrids of romanov sheep with 12.5% of argali blood). the significant changes in fatty acid composition of the lipid fraction from the fat and muscle tissue of the hybrid sheep as compared to the control were found. the content of saturated fatty acids (sfas) in the fat samples of the hybrid animals was by 12.16% lower (55.12 ae 2.30%, p < 0.01), but polyunsaturated (pufas) or monounsaturated fatty acids (mufas) contents were by 0.64% and 9.43% higher (10.10 ae 0.43 and 28.78 ae 1.77 (p < 0.01), respectively) as compared to purebred romanov sheep. the most pronounced changes were found for palmitic acid (decreased from 27.70% to 14.24%) and for oleic, linoleic, arachidonic acids (increased from 20.37%, 5.58%, 0.35% to 29.98%, 6.10%, 0.70%, respectively). the last two acids together with the linolenic acids belong to the so-called essential acids and very important for the animal metabolism. a similar trend was observed on the composition of the lipid fraction of muscle tissue. sfas, pufas and mufas content in muscle tissue of hybrid sheep was 53.38 ae 0.34, 9.39 ae 1.00 and 34.16 ae 0.39%, that was 11.17% lower (p < 0.001), and 4.13% and 4.67% higher (p < 0.01) compared to purebred romanov sheep. these results emphasized the difference of the pufas/sfas ratios in fat and muscle tissues, respectively) and characterized the biological value of the lipid fraction of fat and muscle tissue. the obtained data gave evidence of the positive changes in the fatty acid compositions of the lipid fractions for the hybrid animals as compared to the purebred sheep. supported by the russian scientific foundation, no. 14-36-00039. foodborne illnesses resulting from the consumption of agricultural commodities contaminated with enteric pathogens are an increasing problem around the world. while various possibilities of produce contamination with pathogens exist, the global warming combined with a widespread use of animal manure in agriculture will likely contribute to an increased number of such outbreaks. thus, phages isolated from different agroecosystems may prove to be useful in detection/biocontrol of enterobacteria in produce. during the investigation of the impact of global warming on the diversity and co-evolutionary dynamics between microorganisms and viruses in lithuanian agroecosystems, a novel enterobacteria phage vb_ecos_nbd2 (nbd2) was isolated from agricultural soil using e. coli novablue for phage propagation. nbd2 genomic dna was isolated from cscl-purified phage particles, and was subjected to illumina dna sequencing. nbd2 is a virulent siphovirus that has a low-temperature plating profile (fails to form plaques at a temperature >30°c). the genome of nbd2 is $ 52 kb long, and has a total of 87 probable protein-encoding genes as well as 1 gene for trna ser . the genome analysis revealed that 20 nbd2 orfs encode unique proteins that have no reliable identity to database entries. among the orfs that encode proteins with matches to those in other sequenced genomes, 64 are similar to proteins from phages that infect different members of enterobacteriaceae, while 3 nbd2 orfs are most similar to those from bacteria. based on the similarity to biologically defined proteins, 32 nbd2 orfs were given a putative functional annotation, including 15 genes coding for morphogenesis-related proteins, as well as 14 associated with dna replication, recombination, and repair. phylogenetic analysis revealed that enterobacteria phage nbd2 is distantly related to phages belonging to the subfamily tunavirinae. this research was funded by a grant (no. sit-7/ 2015) from the research council of lithuania. p-08.01.4-009 the antibiotic novobiocin affects the composition of the escherichia coli proteome n. e. arenas 1 , j. williamson 2 , v. schw€ ammle 2 , s. douthwaite 2 1 universidad de cundinamarca, cundinamarca, colombia, 2 university of southern denmark, odense, denmark novobiocin (nov) is an aminocoumarin which competitively inhibits the atp binding site in the gyrase-b subunit of prokaryotic topoisomerase ii. nov remains a therapeutic choice for treating infections with bacterial pathogens that are resistant to more commonly used drugs. the aim of this study is assess the proteomic response of e. coli strain upon nov treatment. minimum inhibitory concentrations of nov were measured by standard assays. three different e. coli strains (as19, as19-rlma::aph and b) were grown aerobically in nutrient rich lb media at 37°c during one hour. the whole cell proteome (five biological replicates in each sample,) was assessed by lc-ms by using tmt labelling protocol. raw files were imported to proteome discoverer (thermo fisher scientific) and searched together with mascot against the uniprot e. coli reference proteome. mics for nov were determined to be >1000-fold higher the wild-type b-strain of e. coli than for the hypersusceptible as19 strains (1 lg/ml). whole genome comparison of the b and as19 strains were characterized by an increase in proteasome components (6 proteins), chaperones (3), error-prone dna polymerase components (4), ribosomal hibernation factors (3), heat shock response (2), electron transport coupled proton transport (8), pentose phosphate pathway (9), flagellar assembly (4), oxidative phosphorylation (10) and tca cycle (8). whereas ribosomal proteins (45), aminoacyl-trna synthetases (7), rnases (6), abc transporters (17), mismatch repair (3) and sec secretion pathway (4) were significantly down-regulated upon nov treatment. the three e. coli strains respond similarly upon nov treatment and their proteomes showed upregulation of heat shock response with changes in the components of translation and transcription, the proteasome and atp biosynthesis. the changes observed can be used to define the processes that are required for antibiotic tolerance and survival of e. coli against aminocoumarin antibiotics. postnatal growth is under control of pituitary derived hormone, growth hormone (gh) that triggers bone, fat tissue growth and development via acting on protein, carbohydrate and fat metabolism. gh functions on postnatal development by jak2/stat5 signaling following gh:gh receptor (ghr) dimerization. isolated growth hormone deficiency (ighd) is a medical condition of insufficient production of growth hormone (gh) that is caused by mutations on gh-n gene in different ethnic origin children. various mutations within gh has been determined in different populations so far, and glutamic acid to glycine (e33g), asparagine to aspartic acid (n47d), threonine to alanin (t-24a) missense mutations, alanine to serine (a13s) substitution, tryptophan to stop codon (w-7x), gaaa insertion in intron 1 of gh-n gene and both intron 1 (+83c) and deletion of 166. amino acid of gh protein phenylalanine (f166del) mutations were detected in turkish ighd children. the potential role of these mutations on cell growth, proliferation, emt via acting on gh signaling pathway has not been observed yet. all these mutations were performed on wild type gh-n gene inserted pc3.1 vector by site-direct mutagenesis and stable cell line of each gh gene mutations were generated by neomycin selection. although w-7x, e33g, f166del, a13s and n47d mutations suppresses gh signaling via acting on either jak2 dephosphorylation or stat5 downregulation, t-24a, gaaa insertion and deletion of +83c mutations have no significant effect on gh signaling. in addition, each mutation lead different growth suppression effect and colony formation potential and intracellular polyamine levels and odc expression profiles were essential role in emt potential of hek293 cell lines. as a result, w-7x, e33g, f166del, a13s and n47d mutations prevented gh signaling and cell growth and differentiation via polyamine metabolism. pulmonary embolism (pe) is a common cardiovascular emergency and affects a large number of patients. acute pe-induced oxidative stress can lead to the accumulation of specific nitroproteins that may play a role in disease progression. the impact of nitration of a single tyrosine residue often has broad implications on the activity of biologically critical proteins, which has become increasingly related to pathological conditions. in this study, we used a proteomic approach to analyze nitrated serum proteins in patients diagnosed with acute pe and healthy controls. nitrotyrosine (no2tyr)-containing proteins were immunoprecipitated from serum with a no2tyr affinity sorbent. precipitated proteins were separated by sds-page and visualized by coomassie blue staining and western blotting with mouse monoclonal anti-no2tyr antibody. among the numerous immunoreactive bands observed in disease patients, the 138 kda protein band was in-gel digested and analyzed by maldi-tof mass spectrometry (ms). mass fingerprint data sets obtained from the peptide fragment ions matched human collagen alpha-1 (iii) chain (co3a1_hu-man) with mascot algorithm analysis giving a score of 65 (p < 0.05). collagen alpha-1(iii) chain is a fibrillar collagen that is found in extensible connective tissues such as skin, lung, and the vascular system. altered metabolism of collagen and its excessive deposition in the matrix of the connective tissue is a hallmark of chronic interstitial lung diseases. collagen can be measured in serum and bronchoalveolar lavage fluid from patients with numerous chronic interstitial lung diseases. given these considerations, future studies are aimed understand the relevance of no2tyr modifications in co3a1 relating to changes in protein structure and function. recent studies have shown that the genes involved in dislipidemia represent potential loci to be associated with diabetes as a disease. recent genome wide association (gwa) studies have associated rs1260326 in gckr gene and rs4846914 in galnt2 gene with parameters of t2d and diabetic dyslipidemia. in this study, the association of these single nucleotide polymorphisms (snps) with t2d and dyslipidemia was tested in the population from bosnia and herzegovina (bh). our study involved 352 patients with t2d and 156 healthy subjects. biochemical and anthropometric parameters were measured in all participants. after dna extraction, sequenom iplex platform was used for the analysis of galnt2 polymorphism (rs4846914), while polymorphism in gckr (rs1260326) gene was analyzed by using real time pcr. our results demonstrated significant association of gckr rs 1260326 variant with waist circumference (p = 0.003) and fasting glucose levels (p = 0.003) in the control group. no such association was demonstrated for rs4846914 galnt2 gene. in the group of diabetic patients, significant association of gckr rs1260326 variant with levels of bilirubin (p = 0.004) and rs4846914 galnt2 variant with hba1c (p = 0.013) and triglyceride levels (p = 0.043) was also demonstrated. our results suggest an association of variations of gckr and galnt2 genes with specific markers of t2d and dyslipidemia. further studies would be needed in order to confirm these genetic effects in other ethnic groups as well. osteoporosis is the most common metabolic bone disorder affecting the normal bone turnover with low bone mineral density (bmd) and risk of fragility fractures. polymorphisms at the sp1 binding site of the collagen type 1 a1 (col1a1) gene is associated with low bmd. we examined the distribution of col1a1 gene polymorphism in 50 young osteoporotic women and in control group in turkish population. patients had low bmd with t score ≤2.5 sd and controls was 25 healthy women (35-57 years). mean age (51.28 ae 5.8) and (46.56 ae 6.15) respectively. the bmd, as g/cm 2 , was measured in the hip and the lumbar spine (l2-l4) with (dexa). dna was isolated from blood. col1a1 gene was analysed with genomica clinical array system. the x 2 test was used to compare allele and genotype frequences between patients and controls. mean of t score in patients was à3.06 ae 0.42. mean bmd (as g/cm 2 ) was 0.708 ae 0.073, and (1.009 ae 0.823) genotype distribution were18(36%) ss, 31 (%62)ss, 1(%2)ss for patients, and 12(48)ss, 9(36)ss, 4(16)ss for control . patients had 33(%66)s allele, 17(34%) s allele, controls had 67(67%)s allele, 33 (33%)s allele. when genotypes and bmd were compared in patients, there was no significant correlation between osteoporosis and genotypes. the allelic distribution was not significant between patients and controls p > 0.05. genotypic distribution in patients were significantly different. patients had a higher frequency of the ss(%62) than controls (ss %36) p < 0.05. this study shows that high prevalences of the ss genotype at the col1a1 locus, in osteoporosis . _ it is possible that the presence of the s allele causes variation col1a1 and col1a2 mrna's producing abnormal collagene protein. since collagen protein is major protein of bone, it is to be expected that a defect in this protein will produce bone fragility. col1a1 gene should be detected early to initiate preventative therapy for bone health. the biological activity of nigella sativa seeds is mainly attributed to its essential oil component which is pre-dominantly (30-48%) thymoquinone (tq). therapeutic effect of tq was exhibited in many diseases including inflammation, cancer, sepsis, atherosclerosis and diabetes. tq has been reported to exhibit antiproliferative effects on cell lines derived from breast, colon, ovary, larynx, lung, myeloblastic leukemia, and osteosarcoma and inhibited hormone refractory prostate cancer. tq induces apoptosis in tumor cells by suppressing nf-jb, akt activation, and extracellular signal-regulated kinase signaling pathways and also inhibits tumor angiogenesis. the aim of this study was to evaluate the anti tumor effects of tq on hepatoma cells. these antitumor assays include cell viability assay, clonogenic assay, scratch assay and molecular expression studies of death related genes. cells were treated with different concentration of tq in hep3b for cell proliferation by mtt and clonogenic assay. in addition, the metastatic character of tq was investigated by scratch assay in hep3b at 3-6 and 24 hours. the effect of tq was also evaluated at mrna level by real-time-pcr. tq was treated on the hep3b cells in three different concentration, namely 75-50 and 37.5 lm. tq showed the cell cytotoxicity in concentration and time dependent manner. the scratch assay revealed no healing in the scratched area due to the decreased cell viability. maximum permissible dose was 50 lm. proapoptotic genes, bax and bad, and autophagy genes, beclin-1 and lc3, were upregulated in hep3b cells after 24 hours treatment in contrast, antiapoptotic gene, bcl-2, expression level was decreased for hep3b cells after 24 hours. p-08.01.4-019 association of irs1 genetic variation with type 2 diabetes and insulin resistance in patients from bosnia and herzegovina insulin receptor substrate-1 (irs1) encodes the irs1 protein, a substrate for the insulin receptor tyrosine kinase and has a critical role in insulin-stimulated signaling pathways. previous studies showed that irs1 single nucleotide polymorphisms (snps) were associated with type 2 diabetes mellitus (t2d). this is the first study performed in a population from bosnia and herzegovina (bh) in which we examined the association of rs7578326 (g>a), rs2943641 (t>c) and rs4675095 (a>t) with t2d risk and related traits. our study involved 437 t2d patients and 252 healthy subjects. biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was done by spss 23. our results demonstrated a significant difference in frequency of rs4675095 (p < 0.001) and rs7578326 (p = 0.021) snps between t2d patients and control subjects. interestingly, here we showed a significant association of irs1 rs4675095 risk t allele with increased insulin levels (p < 0.001) and homa-ir (p < 0.001) in t2d patients. similarly, rs7578326 variant was also associated with the same markers of insulin resistance in diabetic patients, i.e. insulin levels (p = 0.029) and homa-ir (p = 0.025). no such association was demonstrated for rs2943641. however, this irs1 variant was associated with changes in lipoprotein levels, where risk c allele increased vldl (p = 0.006) and decreased hdl levels. our results suggest that irs1 variants are associated with t2d susceptibility in bh population, thus confirming similar findings in other population cohorts. furthermore, the associations of these variants with markers of insulin resistance and dyslipidemic metabolic changes point to their role as potential t2d biomarkers. the adra2a gene encodes alpha-2a adrenergic receptor which mediates adrenergic suppression of insulin. a genetic variant in adra2a was recently associated with defective b-cell function. the objective of this study was to analyze association of two adra2a polymorphisms (rs553668 a>g and rs10885122 g>t) with type 2 diabetes (t2d) and its related traits. in this study we have included 437 t2d patients and 252 healthy subjects from bosnia and herzegovina (bh). biochemical parameters, including but not limited to insulin, homa-ir, hba1c, glucose, and lipoprotein levels, were measured in all participants. genotyping analysis was performed by mass array sequenom iplex platform in cooperation with lund university diabetes centre, malmo, sweden. statistical analysis was performed by ibm spss statistics 23 software. our data showed that frequencies of both, rs10885122 and rs553668, variants were not significantly different between t2d and control subjects. however, rs553668 risk a allele appear to increase insulin levels (p = 0.0002) and homa-ir index (p = 0.00001). furthermore, this variant also seems to affect vldl levels (p = 0.004) and waist circumference (p = 0.02) in diabetic patients. the genotype analysis of rs10885122 variant demonstrated that risk g allele decreased hdl (p = 0.0004) and increased ldl levels (p = 0.014), as well as affected the waist circumference (p = 0.02) in diabetic patients. interestingly, haplotype analysis demonstrated the association of rs553668a / rs10885122g with higher homa-ir index. here we demonstrated that although both, rs10885122 and rs553668, adra2a polymorphisms were not associated with t2d risk in our cohort, they were associated with markers of dyslipidemic perturbations and insulin resistance in diabetic patients. further studies in larger cohorts are needed in order to explore these possible interactions and confirm our findings. smad-interacting protein 1 (sip1), also known as zeb2 is a member of zeb family transcription factors and was shown to regulate epithelial-to-mesenchymal transition, cell cycle, cellular senescence and cancer stemness. bipartite zing finger motifs at amino and carboxyl termini of the sip1 mediate its binding to ebox sequences in the genome. however, there are only limited data about sip1 target genes. by using a home-made anti-sip1 monoclonal antibody, clone 6e5, we conducted chip-seq in three hepatocellular carcinoma cell lines, namely snu398, plc/prf/5 and sk-hep-1 and found receptor tyrosine kinase-like orphan receptor 1 (ror1) as one of the targets. sip1 dnabinding consensus motif cacctg was found at +1 kb, +30 kb and +50 kb from ror1 transcription start site. chip experiments validated sip1 binding to all consensus motifs in the ror1 gene region. interestingly, the strongest enrichment was at +50 kb suggesting that long-range interactions play an important role in the regulation of ror1 by sip1. sip1 knockdown by shrna in high-sip1 expressing snu398 cells resulted in the repression of ror1 expression. ror1 is expressed in embryogenesis and fetal life, and is absent within most of adult normal tissues. however, overexpression of ror1 was observed in many human cancers, from hematological malignancies to solid epithelial tumors. ror1-positive cancer cells have enhanced proliferation, invasion and metastasis capacities, show resistance to apoptotic stimuli and display cancer stem cell characteristics. therefore, sip1 and ror1 act in similar pathophysiological processes. our finding that ror1 is regulated by sip1 at least in hepatocellular carcinoma cells adds another level of complexity to the molecular mechanisms of proliferation, invasion and stemness of cancer cells. hepatocellular carcinoma (hcc) is the most prevalent primary liver cancer and is one of the leading causes of cancer related deaths. smad interacting protein 1 (sip1), a member of the zeb family of emt inducers, is involved in cellular proliferation, senescence, invasion and metastasis in human tumors. however, genes regulated by sip1 in hcc are yet to be identified. we conducted a chip-seq study in high-sip1 expressing hcc cell line snu398 by using a home-made anti-sip1 antibody, clone 6e5. among 509 annotated genes, we selected six1 for further studies because of its increased expression in multiple cancers and its association with poor prognosis. sip1 dna-binding motif cacctg was found at -3 kb from transcription start site of six1 gene. chip qpcr experiment validated sip1 binding to this region with 19,7 fold enrichment. compared to healthy liver, six1 transcripts were upregulated in 8 of 9 hcc cell lines included in this study. knockdown of sip1 by shrna in snu398 cells caused upregulation of six1. immunohistochemistry studies in hcc tissue arrays showed increased expression of six1 in tumors and inverse association with sip1 expression in a tumor grade dependent manner. therefore, our results strongly suggest an inverse correlation of sip1 and six1 in hcc bone mineral density (bmd) and bone turnover are under genetic control and variations in the vitamin d receptor (vdr) are related to bmd. bmd is known to be affected by 25 -hydroxy vitamin d (25 (oh)d) and intact parathyroid hormon (ipth) levels. we aimed to determine correlation blood levels of vitamin d (vitd), ipth, and vdr gene effect in healthy turkish women. the subjects were 25 healthy women in age 35-57 years. the bmd was measured as a t score in the lumbar spine (l2-l4) with dexa. all subjects had normal t score between (-1.0 to 1.4) sd. vitd was measured by lc-20-at shimadzu. ipth was measured by chemiluminescence method, dna was isolated from blood. the fok i (vdrf-foki) and bsmi (vdrb-bsmi) polymorphisms of vdr gene was analysed with genomica clinical array system. the mean vitd level was (21.06 ae 14.54) lg/l, mean plasma ipth level was (57.96 ae 30.49) pg/ml. pearson correlation test showed no relation of vit d with bmd. there was moderately negative correlation between ipth and bmd (r = à0.3062). genotype distribution and allele frequency of subjects were as follows: 21(84%) ff), 1(4%) ff, 3 (12%) ff genotype in vdrf -fok1 gene, 7 (28%) bb, 14 (56%) bb, 4 (16%) bb in vdrb-bsmi gene. allele frequencies were f: 86%, f:14%; b:56%, b:44%. when fok1 and bsmi were combined, 52%(ff-bb) and 20% (ff-bb) were found as the most frequent genotypes. bsmi frequency was in hardy weinberg equilibrium (p > 0.5). but foki was not (p = 0). it was found that vit d, ipth levels and bmd were in normal levels in all carriers of ff genotype and in combined (ff-bb) type carrying healthy women (52%). the association vdr genotype and bmd may be different in various ethnic and geographical groups. therefore it is worthwhile to assess vdr polymorphism among turkish population. these type of distribution studies of vdr in healthy and in osteoporotic women may enlighten to earlier diagnosis and treatment planning. p-08.01.4-026 determination of hb a1c values in beta thalassemia f. g€ uzelg€ ul 1 , g. s. seydel 2 , a. e. yalin 3 , e. s€ onmez 1 , k. aksoy 1 1 c ß ukurova university, adana, 2 nigde university, nigde, 3 mersin university, mersin, turkey introduction: hemoglobinopathies are most commonly seen hereditary blood diseases worldwide. our aim was to compare the hba1c values measured on cation-exchange high performance liquid chromatography (hplc) in beta thalassemia cases. materials and methods: we collected 3 ml of whole blood k 3 edta containing tubes from forty-nine cases. arms, rflp and dna sequence analysis methodologies were carried out for determination of beta thalassemia mutations. hb a1c values were measured using the agilent 1100 hplc system. results: forty-nine diabetic and non-diabetic patients were diagnosed with beta thalassemias: twenty-one ivs1-110/ivs1-110, one ivs1-1/ivs1-1, one ivs1-5/ivs1-5, two ivs1-6/ivs1-6, two ivs2-1/ivs2-1, one fsc5/fsc5, one fsc44/fsc44, two -30/-30, two cd8/cd8, one cd36-37/cd36-37, one cd8-9/cd8-9, two cd82/cd83-g/ cd82/cd83-g, two ivs1-110/ ivs1-6, one ivs1-110/ ivs2-1, one ivs1-110/fsc44, one ivs1-110/cd39, one ivs1-110/cd8, one ivs1-110/cd8-9, one ivs1-110/ivs1-5, one ivs1-6/ ivs2-1, one ivs1-6/ ivs1-25, one ivs2-745/cd8 and one fsc5/cd37. cases were classified as diabetic (6), prediabetic (11) and non-diabetic (32) introduction: members of aurora kinase family aurora a, b and c are conservative kinases of cell cycle which are encoded by genes aura, aurb and aurc respectively. overexpression of aura and aurb was found in human cancers, especially in prostate cancer. moreover, there is the evidence that aurb interacts with one of the major oncogenic kinases -braf. little is known about implication of aurc in cancer, but it was demonstrated, that it can overlap aurb function and shares its location. we studied expression of genes of these kinases in urine of prostate cancer patients aiming to evaluate their involvement in this disease and their potential as tumor markers. materials and methods: 22 urine samples from patients with prostate cancer were gathered after prostate massage before surgical invasion. we used urine samples from 6 healthy men as control. we obtained cells from each urine sample by centrifugation and isolated rna using standard approach with phenol and guanidine thiocyanate. cdna was synthesized and taken to qpcr reactions. data was statistically analysed. results: expression of all studied genes was detected in urine of patients with prostate cancer and of healthy men. expression of aurb and aurc in cancer samples each was higher than expression of aura. the cumulative expression aurb and aurc was higher than expression of aura in 13 samples from 22. we observed positive correlation between expression of aurc and braf (rs = 0.548, p = 0.01). discussion and conclusion: previous investigation showed, that for normal prostate tissue 90% of aurora family expression was presented by aura. we suppose that presence of aurb and aurc cumulative overexpression means presence of cell cycle deviations in prostate tissue of these patients and might be further studied as prognostic marker. in this study we first showed the correlation between aurc and other carcinogenic kinase braf expression, which opens the perspective for investigation of role of aurc in carcinogenesis. bacillus marmarensis sp. nov. is an extreme obligate alkaliphile isolated from mushroom compost near marmara region of turkey. it can survive at extreme ph values up to 12.5. based on its genome sequence, metabolic pathways for 7 proteases, 6 amylases, 2 cellulases, 1 lipase, n-butanol and a biodegradable plastic poly-b-hydroxybutyrate were annotated. in addition to being a potential extracellular hydrolase producer, its ability to survive in the high ph range of 8.0 to12.5 makes it an attractive microorganism for different industrial applications. in the current study, the adaptation strategy of b. marmarensis sp. nov. to alkaline conditions was investigated using proteomic tools. the organism was grown at two different ph values, 10.0 and ph 12.0. for extraction of whole cell proteins, cells were disrupted with mp bio fast prep device. protein extracts were treated with protease inhibitors and a nuclease mix. salts were removed using a cleanup kit. obtained proteins were separated based of their isoelectric points in the first dimension and then based on their molecular weights in the second dimension. proteins maps of cells grown at these two extreme ph values showed significant differences in protein expression for alkaline adaptation. p-08.01.4-029 biochemical and proteomic analyses of normal human astrocytes and glioblastoma exposed to dichloroacetate treatment f. c. atilgan, h. cimen yeditepe university, istanbul, turkey glioblastoma (gbm) is an aggressive malignant tumor composed of astrocytes in brain tissue. gbm cells utilize glycolysis rather than oxidative phosphorylation to support rapid growth rate which is called warburg effect. dichloroacetate (dca) is an antiglycolytic agent that inhibits pyruvate dehydrogenase kinase (pdk) activity and induces apoptosis via normalizing the mitochondrial activity. this study aimed to demonstrate the metabolic alterations between the normal human astrocytes (nha) and gbm cell lines which are exposed to dca, and to identify the differentially expressed proteins by ms-based proteomic analyses. nha cell line, u87mg and u373 as gbm human cell lines were examined through analyzing the alterations in the glycolysis metabolism upon dca treatment by measuring the variations in the pyruvate levels, lactate dehydrogenase a, pdk3. mts was performed to investigate the effect of dca treatment on cell viability. immunoblotting of pgc1-a, oxphos complexes, and mitotracker green staining was employed to reveal the mitochondrial differences between normal and the cancer cells, and upon dca treatment of these cells. proteomic analyses were utilized for the identification of candidate proteins depending on the acetylation status. in this study, compared to nha, the pyruvate and ldha levels were elevated and pdk3 levels in u87mg were reduced by 14%. due to mts results, ≤10 mm dca treatment showed significant decrease in gbm cells compared to nha cells. immunoblotting and mitotracker green staining results showed increase in mitochondrial mass. elevation in the pyruvate and ldha levels and reduction in pdk3 level in u87mg and u373 cells indicates glycolysis dependent metabolic switch in energy metabolism. proteomic analyses demonstrate that most of the differentially expressed proteins comprised of metabolic enzymes. this study provides novel information about metabolic alterations existing between nha and gbm, which can inspire further studies for therapeutic applications. kidney stone is a complex disease resulting from environmental as well as hereditary factors and principally composes of approximately 75% calcium oxalate (caox) crystals, which are formed through a multi-step process. vitamin d receptor (vdr) gene encodes the nuclear hormone receptor for vitamin d3; downstream targets of this gene are chiefly contributed in mineral metabolism though the receptor regulates a variety of other metabolic pathways. calcium sensing receptor casr plays an important role in sustaining mineral ion homeostasis. the aim of this study is to profile the expression level of vdr and calcium sensing receptor (casr) genes and to unravel their role in rat kidney stone induced by ethylene glycol, in order to explain the underlying molecular mechanisms. total rna were extracted from paired sample before and after ethylene glycol treated of 50 rats. the mrna expression level of vdr and casr gene were measured employing quantitative rt-pcr (qrt-pcr). the mrna expression levels of both genes were significantly down-regulated according to before treated. in conclusion, our data suggest reduced mrna expression in vdr and casr genes might be a risk factor for kidney stone formation. further studies are necessary to verify these findings in different ethnic groups. p-08.01.4-031 apj receptor a445c gene polymorphism in turkish patients with coronary artery disease against different models of expected frequencies/counts to understand the evolutionary dynamics of saars in proteins. we obtained from ensembl the assemblies of genomes/proteomes of human and nonhuman primates (chimpanzee, gorilla, and rhesus monkey), rodents (mouse and rat), and birds (chicken and zebrafinch). the expected probabilities for the occurrence of saars based on their nucleotide frequencies in coding regions and amino acid frequencies in individual protein sequences or across the whole proteome were compared with the observed repeat occurrences. we found that with all three methods and in all eight species the correlation between observed and expected repeat counts decreased above a saar length threshold. the percentage of saar proteins for each amino acid also exhibited variability among species when both the repeat length and counts were taken into account. however, clustering based on saar characteristics generally reflected the known phylogenetic relationships between species. our comprehensive bioinformatics analyses reveal that saars show amino acid-specific occurrence patterns with respect to species as well as saar length. tissue proteins play important roles in biological metabolic processes. the qualitative and quantitative analysis of tissue proteins facilitates the understanding of molecular mechanisms that differentiate between physiologic and pathologic states. health and research institutions routinely prepare formalin-fixed paraffinembedded (ffpe) tissue blocks for histopathology. proteomics on ffpe tissue still requires standardization of tissue solubilization processes to overcome variability in protein extraction results. our aim is to compare the proteomic studies of fresh frozen and ffpe rat renal tissues. fresh frozen and ffpe preparations from renal tissues were included in this study. an adult rat was sacrificed and the dissected kidneys were divided two equal section. one immediately frozen in phosphate buffer, and the other tissue specimen not thicker than 5 mm to allow rapid penetration of the fixative put in 10% buffered formalin for 48 hours. the fresh frozen tissue was dissolved and homogenised in the cold phosphate buffer solution containing protease inhibitors. paraffin blocks were performed from formalin fixed tissue specimens. we have extracted the protein from the ffpe tissues using our previously verified method. we have utilized electrophoresis three times to compare protein yield, number, intracellular and intercellular of homogenised samples obtained from ffpe and fresh frozen kidney samples. the number of proteins identified from fresh frozen kidney tissue has generally been shown to be increased compared with ffpe tissue. decrease of the qualitative results in electrophoretic bands was found similar in all replicative studies. ffpe tissues undergo extensive cross linking between protein/ dna/rna molecules during formalin fixation, which creates inter-molecular crosslinks. on the other hand, ffpe tissues represent a valuable resource to carry out retrospective studies aimed to biomarker discovery in kidney cancer as well as other kidney diseases. background: development of atrial fibrillation (af) during the course of chronic primary mitral regurgitation (mr) is common and represents complex molecular mechanisms. however, the gene expression profile of human atrial fibrillation (af) in the setting of chronic primary mr remains uncharacterized. in the current study, we aimed to compare the gene expression profiles of patients with severe degenerative mr in sinus rhythm (sr) and af. methods: left and right atrial tissue samples were obtained from patients with chronic primary severe mr in permanent af (n = 30) and sinus rhythm (n = 30). we performed a novel micro-dissection technique for thin sections of atrial tissue samples and immediately fresh froze intra-operatively. transcriptomes of left and right atrial appendages of degenerative mitral regurgitation patients with sr versus af were compared by microarray analysis on affymetrix hgu-133 plus 2 platform. bioinformatics, data mining and pathway analyses were conducted on partek gs and webgestalt. genome-wide gene expression profiles were compared between af and sr groups among 54.675 transcripts representing 38.500 well-characterized human genes. differentially regulated genes were evaluated according to fold change (fc ≥ 1.5) with a p-value ≤0.05. results: most remarkable pathways altered in af atrial tissues compared to sr group, were extracellular matrix-receptor interaction; mapk, adipocytokine, and calcium signaling; apoptosis and cardiac muscle contraction pathways. conclusions: this is the first human study of comparative transcriptomics in left and right atrial tissues of patients with af versus sr associated with severe degenerative mr. the main findings of this multidisciplinary translational research provide novel candidate targets for the treatment and prevention of af. in order to acquire iron under iron-limiting growth conditions, bacteria employ specific mechanisms such as production and secretion of siderophores. siderophores are low molecular metalchelating compounds that contribute not only to iron scavenging, but also participate in other important processes including oxidative stress response and cell signaling. serratia marcescens, gramnegative bacterium, could be found in various environments, including wastewater, plant rhizosphere and hospital setting where s. marcescens can cause serious life-threatening infections. in this study, we performed a detailed characterization of the siderophores of the clinically important pigment-free s. marcescens strain sr41-8000 and environmental pigment-producing s. marcescens strain sm6. bioinformatic analysis of these genomes by antismash software revealed the presence of several clusters involved in non-ribosomal peptides synthesis (nrps). we found four nrps clusters in genome of s. marcescens sm6. cluster 1 has a low level of identity to enterobactin gene cluster typical for bacteria producing catechol-like siderophores. second cluster has only 4% of identity to xantholipin biosynthetic gene cluster. clusters 3 and 4 of nrps genes of s. marcescens sm6 did not show any homology to known nrps clusters. in contrast, the genome of s. marcescens sr41-8000 contains only one genetic cluster of nrps genes. this cluster does not have similarity to any of the known bacterial nrps genes. thus, genetic analysis of two isolates of s. marcescens allowed us to identify nrps genetic clusters and showed that the repertoire of these genes is different between strains. we hypothesized that the strain isolated from environment has competitive advantage over clinical isolate due to genetic diversity of siderophores. on the other side, clinical isolate has specific genetic cluster of siderophores which may promote s. marcescens growth and adaptation to the extreme niches present in medical facilities. p-08.01.4-037 the first glance on the genome's structure and activity in hibernator edible dormouse hibernation is a unique adaptive way of survival in extreme environmental conditions where mammals decrease their metabolic rate and demonstrate physical inactivity for prolonged periods of time (up to 6-8 months). remarkably, some hibernating animals have a long average lifespan and the ability to avoid muscle atrophy caused by disuse or immobilization. to identify main molecular pathways behind the protective musculoskeletal adaptation and genome structure in hibernator edible dormouse (glis glis), whole-genome analysis of mrna expression in muscles (m. soleus and m. edl) and lumbar spinal cord samples was conducted. three groups of the dormice: 1) active animals 2) hibernated animals and 3) animals immobilized for 2 weeks in laboratory, were examined. rna libraries have been sequenced using hiseq 2500 illumina platform. coupled with genome dna sequencing provided x10 coverage of the estimated genome, we have assembled de novo transcriptome of the dormice. differentially expressed genes in response to immobilization and hibernation were determined. transcriptional program of these phenotypes was similar. pathways enriched by differentially expressed genes were identified. gene expression of the key muscle proteins and muscle atrophy markers was analyzed. muscle-specific e3-ubiquitin ligases murf1 and mafbx revealed no changes in mrna expression. our study represents the first attempt to elucidate changes in transcription profiles of skeletal muscles and spinal cord during hibernation and hypokinesia in edible dormice. in corroboration to the gene expression data, they demonstrated minimal morphological evidence for muscle disuse atrophy during physical inactivity. edible dormice, thus, can be considered as a novel model organisms in investigation of the genetic mechanisms of hibernation and prevention of muscle atrophy. the work is performed according to the russian government program of competitive growth of kfu and supported by rfbr jsps_a no. 14-04-92116. in response to diverse environmental cues bacteria form complex structured communities called biofilms. the metabolic pathways activated by these cues are remarkably different depending on the species studied. however, they all lead to the formation of an extracellular matrix that holds the cells together. non-pathogenic gram-positive spore-forming soil b. subtilis strain is recognized as a model system for the study of biofilms. to discover the pathways regulating biofilm formation in b. subtilis, we studied the natural isolate of b. subtilis strain 168, and constructed the recombinant strains with knocked out genes of following regulatory proteins: abrb (global transcriptional regulator), degu (two-component response regulator of signal transduction system degs-degu), ccpa (regulator of carbon catabolism) and spooa (regulator of sporulation). in the minimal medium broth b. subtilis 168 wild-type strain forms biofilm with its maximum on 48th hour of culture growth. ph-optimum for biofilms formation by the wild-type strain is in the range of 7.4-8.0. the temperature optimum is in the range from 22°c to 45°c. this corresponds to the natural conditions of the b. subtilis habitat in rhizosphere. the level of biofilm formation by regulatory mutant strains with deleted abrb, degu, ccpa, spooa genes is on average 40% lower than by the wild-type strain. this indicates that global regulatory system controlls biofilm formation process. statistically significant differences in the levels of biofilm formation between regulatory mutants haven't been identified. ph and temperature optima of mutant strains are the same as for the wildtype strain -7,4-8 and 22°c -45°c respectively. the crataegus genus which is a member of rosaceae family, has approximately 200 species worldwide and 24 species in turkey. all plant species in this genus have the common name "hawthorn". crataegus microphylla (c. microphylla) c. koch which is characterised by having erect sepals in fruit and smaller leaves in comparison with the other species, is one of the wild edible fruits in turkey. crataegus species have been used as food and also in folk medicine for the treatment of various diseases. for this purpose, the potential biological properties of crataegus microphylla were aimed to reveal by the preliminary work. in this study, prevention of oxidative dna damage using supercoiled pbr322 plasmid dna, acetylcholinesterase, tyrosinase, a-glucosidase inhibition and antioxidant effects: 2,2diphenyl-1-picrylhydrazyl radical scavenging effect, phosphomolibdenum-reducing antioxidant power, ferric-reducing antioxidant power with total phenolic and total flavonoid contents of the c. microphylla leaves, stem barks and fruits that extracted with ethanol, methanol and water were investigated. the experiments of oxidative dna damage studies and antioxidant activities of c. microphylla extracts showed that methanol and ethanol extracts possessed a strong ability to prevent dna damage and significantly antioxidant activities. methanol extracts of stem barks from c. microphylla exhibited the highest acetylcholinesterase and tyrosinase activities (48.86 ae 4.06% and 85.57 ae 2.01%, respectively), at 200 lg/ml. in addition, ethanol extract of leaves from c. microphylla inhibited the a-glucosidase activity significantly when compared to acarbose. this study explained significant antioxidant, enzyme inhibitory, hypoglycemic, and neuroprotective activities of methanolic or ethanolic extracts prepared with stem bark and leaf from c. microphylla and also strong ability to prevent dna damage that corresponded to antioxidant potential of methanol extracts of leaf and stem bark. the yarrowia lipolytica species (yl) is nonconventional yeast widely used for recombinant protein expression due to its system of post-translation protein modification, which is the most similar to that of higher eukaryotes. yl appears the promising producer of recombinant proteins with much more complicated molecules compared to those of prokaryotic producers. however, an important feature for a producer strain of recombinant proteins is the genes, the expression of which undertakes under controlled conditions, and consequently, search of new effective inducible promoters in the yl genome is of great interest. proteome analysis of the yl cells grown at different ph values (4.0, 5.5, 9.0) showed that under alkaline conditions the amount of mitochondrial porine vdac (voltage dependent anion channel), one of the most abundant protein of the mitochondrial outer membrane, increased significantly. vdac is supposed to let reactive oxygen species out of mitochondria protecting the cell against oxidative stress. therefore, the por1 gene expression, encoding vdac should increase in the stress conditions. the promoter of the por1 gene was used to construct some new expression systems based on yl w29. a new genetic construct bearing a reporter bgalactosidase gene under control of the por1 promoter. b-galactosidase activity was assayed in the cells grown in various ph conditions and exposed to exogenous oxidants such as hydrogen peroxide, menadione, and methyl viologen. it was shown, that in h 2 o 2 and methyl viologen treated cells b-galactosidase activity increased 1.5-2.0 -fold reaching its maximum in the cells, grown at ph of 9.0. thus, we demonstrated high inducibility of the por1 promoter, which is essential for effective action of the expression system based on it and potency of application for transformed lines of producers. acknowledgments: supported by the russian foundation for basic research (grant no 16-34-00634 mol_a). aspergillus nidulans is able to detoxify and catabolize the toxic proline analogue, lazetidine-2-carboxylic acid in nature, azc serves as a plant protectant against infections and consumption. we have obtained evidence that azc is not only non-toxic for the model ascomycete aspergillus nidulans, but it can be utilized as a poor nitrogen source. in order to elucidate the molecular mechanism underlying azc detoxification, we have constructed and studied a. nidulans strains deleted in the cognate genes involved in azc detoxification in pseudomonas and saccharomyces cerevisiae. these genes, found by in silico analysis, encode a putative hydrolase, acha, and an azc acetyltransferase, ngn2, respectively. gene deletion was accomplished through double crossover. a cassette containing the~1500 bp 5' and 3' flanking sequences of each gene, with the afpyrg gene as a selection marker, was contructed. crossing the achad and the ngn2d strains isolated the achad ngn2d double mutant strain. rt-pcr was used for gene expression analysis in the wild type strain, area-loss of function and crea-derepressed mutant strains. our results clearly show that azc can be used as a poor nitrogen source by a. nidulans. this utilization requires a) acha, a putative azc hydrolase, and b) a fully active gaba catabolic pathway, as lack of either amdr or gata abolishes azc utilization. most importantly, the double mutant, achad ngn2d, shows azc toxicity, suggesting that ngn2 is a true orthologue of mpr1, able to detoxify azc, a phenotype that can be observed only in the absence of acha. as a final point, ngn2 was shown to be induced by the presence of azc and and to be under nitro the spatial genome organization plays a great role in the maintenance of the nuclear architecture and regulation of all processes occurring in the nucleus. this system is controlled by a set of special proteins having an architectural function. however, the mechanisms of their action remain unknown. among these proteins are, in particular, zad-domain-containing proteins. zinc finger-associated domain (zad) is a ubiquitous motif of c2h2 zinc finger proteins of drosophila. genes that encode zad proteins are specific for and expanded in the genomes of insects. only a few zad-encoding genes have known functions, and the role of zad is being discussed. up to date there was only one known crystal structure of zad-domain from drosophila transcription factor grauzone (grauzad). here, we present for the first time the crystal structure of the zad-domain of serendipity-d transcriptional activator of the egg-polarity gene bicoid. zad-domain was cloned, overexpressed in e. coli, purified and the structure was solved at 3.5 a by mad technique. detailed analysis of the structure proved that the protein exists in dimeric form and revealed unique spatial organization of the protein, different from those for grauzad. this work is supported in part by russian ministry of education and science grant (14.616.21.0066). mycoplasma gallisepticum is a convenient model object for studying the regulation of transcription because it has a reduced genome, lack of cell wall and many metabolic pathways and also easy to culture and non-pathogenic to humans. due to the nature of the genomic organization and the loss of many of the known regulators, the effect of disrupting the function of some proteins may be a useful tool for studying the regulation of transcription. the gene expression study was performed on agilent onecolor microarray with custom design and random-t7 polymerase primer for cdna synthesis. microarray represents 3366 probes for 678 orf including genes and ncrna. in this work, we have investigated the effect of changes in the level of gene expression of m. gallisepticum for two different types of conditions: a genetic knock-out mutants and the cell response to treatment with sub-lethal concentrations of antibiotics. we characterized transcription of m. gallisepticum when the cell responses to dysfunction of proteins with metabolic potential, possible regulators of expression, in violation of permeability of membrane by cccp, inhibition of ribosomal synthesis by tetracycline, dna gyrase by novobiocin and atp synthase by oligomycin. the data obtained allow to characterize the transcriptional response under different conditions and to identify groups of genes that change expression together. major transcriptional changes were observed in the response of cells under cccp treatment due to uncoupling of the proton gradient and further reducing the membrane potential, as well as under novobiocin treatment due to changing the topology of dna. global problem of oil pollution forces scientists to search for a new safe remediation technologies constantly. careful attention is paid to bacteria, some of which possess additional biotechnologically valuable properties, such as utilization of hydrocarbons and production of biofurfactants. in this regard, we carried out proteogenomic characterization of tsukamurella tyrosinosolvens strain ps2, which was isolated from chemical sludge and capable for alkane degradation and biosurfactant production. whole genome of the strain was sequenced on the miseq (illumina) platform, assembled and annotated. proteome on mineral medium with glucose, sucrose and hexadecane as a sole carbon and energy source was studied. shotgun proteomics approach was performed on hybrid chromatography-mass spectrometry machine (maxis impact). alkane oxidation genes (alkane-1-monooxygenase, rubredoxin and rubredoxin-reductase) under genome sequence, as well as two pathways of trehalose synthesis and genes for mycolic acids production were found. emulsification activity of cell-free culture liquid was about four times higher on hexadecane in comparison with sugars. proteomic profile was different at various culture conditions. all glycolysis genes, beginning with glucose-6-phosphate isomerase to pyruvate kinase, were found on the media with sugar. the medium with hexadecane helped to reveal enzymes involved in the beta-oxidation of fatty acids, for example 2,4-dienoyl-coa reductase, 3-ketoacyl-coa thiolase and enzymes of the initial mycolic acid synthesis pathways. thus we have established that the strain t. tyrosinosolvens ps2 utilizes sugar by glycolysis. also, the bacterium is capable for alkane oxidation followed by beta-oxidation of fatty acids. based on the proteogenomic data, we assume that the bacterium is able to synthesize trehalose lipids, namely, trehalose mycolates. obtained results could be useful to create conditions for increased biosurfactants production. gestational diabetes mellitus (gdm) is a glucose intolerance firstly diagnosed during pregnancy. in this study, we aimed to investigate the association between serum adiponectin, resistin levels and insulin resistance in gestational diabetic patients. a total of 80 patients; 40 healthy pregnant women (control group) and 40 pregnant women diagnosed with gdm (gdm group) were included in this study. serum adiponectin, resistin, glucose, insulin, hba1c levels and lipid parameters were measured. insulin resistance index homa-ir values were calculated. in this study, serum glucose, insulin, hba1c levels and homa-ir were significantly higher in gdm group compared to the control group (p = 0.038, p = 0.011, p = 0.001, p = 0.008, respectively). serum adiponectin levels were significantly lower (p < 0.001); whereas serum resistin levels were significantly higher (p = 0.004) in gdm group than in the control group. it can be concluded that resistin contributes to the formation of insulin resistance, adiponectin plays an important role in the regulation of this resistance and they also have effects on gdm pathophysiology. hematological cancers including acute myeloblastic leukemia (aml) and acute lymphoblastic leukemia (all) in terms of incidence and mortality, are the second most important cancer type in turkey. numerous studies show that cancer patients respond differently to treatment thus supporting the idea of personalized therapy need for individuals. renin angiotensin system (ras) have key roles in aml and all progression and it has been shown by many studies suggests that these system's genes might be good biomarkers for aml and all personalized therapy. we aimed to identify ras gene based homogeneous subgroups of acute leukemia and determine the most effective chemotherapoetic agent for each subgroup. after validation and verification of the results, more effective drugs can be recommended for the use in clinics for chemotherapy of aml and all. results of our preliminary studies showed that we are able to identify subgroups of aml and all as well as correlating each existing subgroup with fda approved drugs. considering the long and highly cost process of developing new drugs for cancer treatment makes the present study all the more valuable. in addition, there is a serious need for change in aml and all therapy since there is no highly effective chemotherapy protocol available for their treatment. welcome trust sanger (wts) and cancer cell line encyclopedia (ccle) databases will be used to determine subgroups of aml and all based on ras genes or whole genome expression using standard deviation and hierarchical clustering analysis. the most effective drugs for each subgroup will be identified using pearson's r correlation analysis with drug sensitivity data (ic50, ic50, amax, aare, etc.) available in same databases. further validation tests will be performed by in vitro validation using aml and all cell lines: drug sensitivity profiles will be determined and gene expression will be shown by q-rt-pcr. p-08.02.5-003 functional polymorphisms of ephx2 in a turkish population h. pinarbasi, i. sari cumhuriyet university, sivas, turkey soluble epoxide hydrolase (seh; ec 3.3.3.2) is encoded by ephx2 and catalyses the degradation of endogenous fatty acid epoxides generated by cyp450 epoxygenases. these fatty acid epoxides such as epoxyeicosatrienoic acids (eets) have been shown to posses vasodilator, anti-inflammatory, anti-platelet, anti-hypertensive, anti-apoptotic, anti-thrombotic and natriuretic effects. it has been reported that eet levels are associated with hypertension, stroke and cardiovascular diseases. individual differences in the ephx2 gene that affect the seh activity may alter the circulating levels of eets. k55r and r287q polymorphisms have been known to cause increased and decreased seh activity, respectively. therefore we aimed to determine the genotype frequencies of these two polymorphisms in a turkish population. k55r and r287q polymorphisms were determined by the real time pcr using double-dye hydrolysis probes or pcr-rflp method. the observed genotype frequencies for k55r polymorphism were 80.8% wild type (aa) and 19.2% polymorphic genotype (ag+gg) and for r287q polymorphism 81.4% wild type and 18.6% polymorphic genotype (ga+aa). the genotype distributions for both polymorphisms were in hardy-weinberg equilibrium. pregnancy is one of manifestations for thrombophilia factors, which in its turn leads to various complications of its course. one of the markers of hereditary thrombophilia is mutations in the folate cycle mtr, mtrr and mthfr genes. insufficient intake of folate during pregnancy disrupts the functioning of the genome, leading to miscarriage, violation of embryogenesis and various fetal malformations. however, results of studies on the role of hereditary thrombophilia in the occurrence of complications during pregnancy are rather contradictory. aim of this study was to determine the frequency of alleles and polymorphic variants of folate cycle genes mtr a2756g, mtrr a66g and mthfr c677t in women of kazakh ethnic group with pregnancy complications. we used real-time pcr. blood samples for dna isolation were obtained from 129 pregnant women. the main group consisted of women (n = 90) which had a history of two or more pregnancy complications in the form of pre-eclampsia, eclampsia, missed abortion, miscarriage, and etc. control group consisted of women (n = 39) with two or more normal pregnancy outcomes, and had no complications during pregnancy in history. average age of women in experimental group was 32.0 ae 0.50 years compared with control of the age 33.6 ae 0.33. the analysis of the frequency distribution of alleles of genes in experimental group of women with complications of pregnancy revealed no significant differences relative to the control group. analysis of the distribution of polymorphic variants of folate cycle genes showed significant difference between the study and control groups in the occurrence frequency of heterozygotes for the mutant allele g in the gene mtrr a66g (or = 2.89, ci 95% = 1.25-6.71; v 2 = 6.376, p < 0, 05). no significant differences in alleles between homozygous wild-type and homozygous mutant alleles were observed. this work was funded by the mes kazakhstan (gr 0115rk00287 project number). p-08.02.5-005 a study on the association between rs6918698 polymorphism in connective tissue growth factor gene and pseudoexfoliation syndrome pseudoexfoliation syndrome (pes) is a disorder of the extracellular matrix characterized by the production and progressive accumulation of an abnormal fibrillary material in many ocular tissues. pes prevalence is 11.3% above the age 40 in turkey. since pes is characterized by excessive synthesis of elastic microfibrillar components throughout the body, growth factors can have important roles in the pathophysiology of pes. human connective tissue growth factor (ctgf) is a protein expressed in a variety of tissues, including the anterior chamber of the eye. ctgf coding gene has several genetic polymorphisms. rs6918698 g/c single nucleotide polymorphism (snp) is found at position à945, in promoter region. the presence of a c allele for rs6918698 is critical for transcriptional suppression of the ctgf gene which would reduce ctgf production. aim of this study was to investigate if there is any association between pes and rs6918698 polymorphism of the ctgf gene. study population consisted of 60 patients with pes and 60 controls. blood samples were collected by g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. genomic dnas were isolated from whole blood samples using manual dna isolation. the frequency of ctgf rs6918698 polymorphic allele g was 0.442 in patients, and 0.450 in controls (0.967, p = 1.000). distribution of genotypes was gg: 31.7%, gc: 48.3% and cc: 20.0% among patients, while gg: 35%, gc: 40.0% and cc: 25.0% (or = 1.162, p = 0.689) in controls. statistical analysis showed that there is no significant relationship between ctgf rs6918698 snp and pes. these are the preliminary findings of a research project which is the first study analyzing the relationship between ctgf rs6918698 snp and pes. this work did not point out a role for ctgf rs6918698 in the risk for pes. a significant relationship might be found when the study population is enlarged. p-08.02.5-006 evaluation of rs11136000 single nucleotide polymorphism of clusterin gene in pseuodoexfoliation syndrome risk pseuodoexfoliation syndrome (pes), an age-related systemic disorder, is characterized by production and accumulation of abnormal fibrillar extracellular material in anterior structures of the eye. clusterin (clu) is a multifunctional glycoprotein produced and secreted by almost all cell types and is found in all body fluids and in accumulated pes material. under cellular stress conditions, clu provides inhibition of stress-induced precipitation and aggregation of misfolded proteins. clu expression level in pes patients is unexpectedly low and this could be due to single nucleotide polymorphisms (snp) on the gene coding for clu. rs11136000 c/t polymorphism has been found to be associated with alzheimer's disease and pathophysiology of alzheimer and pes are similar. this study aimed to determine whether rs11136000 snp of clu gene have a role in the development of pes. study population consisted of 60 patients with pes and 60 controls. blood samples were obtained from g€ ulhane military medical academy, department of ophthalmology, ankara, turkey. genomic dnas were isolated from whole blood of subjects using manual dna isolation. genotypes were assigned by pcr followed by restriction fragment length polymorphism analysis. t allele frequency of pes patients was 0.425 and that of controls was 0.442 (0.934, p = 0.794). the distribution of genotypes was cc: 30.0%, tc: 55.0% and tt: 15.0% among patients while cc: 28.3%, tc: 55.0% and tt: 16.7% (0.922, p = 0.841) in controls. there was no statistically significant difference between pes patients and controls in terms of tt genotype and t allele frequency. these are the preliminary findings of a research project which is the first study analyzing the relationship between clu rs11136000 snp and pes in turkish population. this work did not point out a relation for polymorphic genotype in the risk for pes. however, a relationship between clu rs11136000 polymorphism and pes can be found when we enlarge the study population. the tumor suppressor tp53 is the most frequently mutated gene in head neck squamous cell carcinoma cancer and represents a known transcription factor and tumor suppressor gene that regulates different microrna and target genes. the aim of our work is to construct the transcriptional and post-transcriptional network regulated by tp53 and to evaluate the difference at mrna and protein expression levels of the tp53 target genes in hpv negative head and neck squamous cell carcinoma (hnscc) patients with distinct tp53 mutation states and to elucidate the molecular mechanism that underlie the poor prognosis of tp53 mutation. to show the tp53 mutation landscape and its prognostic relevance for survival, we used cbioportal for cancer genomic analysis. we downloaded mutational profiles of 243 hpv negative hnscc patients. employing different databases we constructed the tp53 regulatory network. and then, to evaluate the effect on mrna, protein and microrna regulated by tp53 we used the mrna and protein expression profiles of patients from tcga. our results show that hotspot, truncating and missense mutations have statistical significance in the univariate analysis. the tp53 regulatory network show the involvement of important target involved in the progression of hnscc and the deregulation of protein expression of an important key epigenetic modifier ezh2 was significantly associated with tp53 mutational state. ezh2 is a member of the polycomb group protein enhancer zeste homolog 2 which is known to be directly repressed by tp53 and indirectly by the activation of mir-200a and mir-200b. we found a significant up-regulation of ezh2 that depend from tp53 mutation. it is important to understand the difference in mrna and protein expression of tp53 regulatory network that could depend from its mutational state. this finding suggest that ezh2 might be a potential therapeutic target for hnscc. p-08.02.5-008 next generation sequencing based approach for monitoring of minimal residual disease in acute lymphoblastic leukemia minimal residual disease (mrd) monitoring is widely used to evaluate efficiency of chemotherapy and to choose a strategy for further treatment in acute lymphoblastic leukemia (all). the most commonly used approaches for mrd detection are based on flow cytometry and qpcr. these methods have several important limitations including insufficient sensitivity, complicated experimental setup and false positivity. the newly developed next generation sequencing (ngs) approaches could overcome the existing limitations in mrd monitoring. here we describe a new mrd monitoring approach based on targeted deep sequencing of malignant rearrangements. first, we identified bcr/tcr rearrangements specific for the leukemic clones in initial bone marrow samples of 10 all patients. for this, we used sanger sequencing of the products of 19 multiplex pcr, performed with bcr/tcr specific primers combined according to the optimal frequency distribution of v/j-genes in healthy donors. second, we analyzed concentration of malignant clone rearrangements, identified at the first step, in dna samples obtained from bone marrow after 36 days of treatment. for this purpose, we performed ngs of 10 libraries for each identified leukemic rearrangement. four libraries were amplicons of bcr or tcr gene rearrangements generated using characteristic v and j segment specific primer combination. six additional libraries were amplicons of the same primer combination from the same dna sample which contained initial leukemic dna spike-in (in concentrations corresponding to 1 per 100, 1000 and 10,000 cells) for a calibration curve generation. using this approach, we analyzed 7 all clone specific rearrangements for three patients and calculated concentration of the leukemic clones by using the calibration curve. for one patient we didn't find any leukemic cells and for two patients we found 1 leukemic cell per 100,000 analyzed cells. znf365 is considered as a transcriptional target for p53 and plays an important role in the homologous recombination, mitosis, centrosome dynamics. as was shown by gwas some snps in znf365 have strong association with the risk of breast cancer (bc). however, it was unclear whether the same snps are associated with risk of bc in kazakhstan. therefore two polymorphisms (rs10995190, rs10761659) of znf365 were investigated in kazakh population in this study. the present case-control study was carried out with participation of 444 kazakh females with bc and 390 cancer-free donors. additionally, subtypes of bc, stratified by estrogen receptor (er+/à), progesterone receptor (pr+/à) and human epidermal growth factor receptor 2 (her2+/à) status were estimated. pearson p-value, odds ratio, 95% confidence interval tests were applied to data analysis. significant differences were found in allele frequency and genotype distribution at rs10995190 locus in znf365 between the patients and control groups (p = 0.013 for allele; p = 0.007 for genotype). moreover, significant association with bc was revealed for rs10995190 after dividing patients according to er+/ à, pr+/à and her2+/à status of the tumor. the g allele was associated with er+ (p = 0.01, or = 1.69, 95%ci:1.11-2.56), pr+ (p = 0.01, or = 1.78, 95%ci:1.13-2.79) and gg genotype with her2-bc carriers (p = 0.02, or = 1.66, 95%ci:1.04-2.63). also, g allele can be considered as a risk factors in er+/ pr+/her2-luminal type of tumor (p = 0.028, or = 1.79, 95% ci:1.06-3.05). our findings correlate with the data of several gwas where the association of the rs10995190 polymorphism with higher mammographic density and the risk of breast cancer have been shown. the obtained results allow us to consider g allele and gg genotype of rs10995190 as a marker of bc risk with predictive value, restricted to er, pr and her2 status of the tumor in the kazakh population. breast cancer (bc) is the most common cancer among women in most of countries. alternative variants of low-penetrance genes such as fgfr2 (rs2981582), tox3 (rs3803662), map3k1 (rs889312), lsp1 (rs381798) are shown to have high frequency in north america, south-east asia, australia, europe populations and a multiplicative effect on the development of bc. in this study was investigated assosiasion between alleles/genotypes combinations of these genes with increase/decrease of bc risk. the case-control study included 625 bc patients and 672 healthy women from kazakh and russian populations. genotyping was performed by pcr-rflp methods. combined effect of allele and genotype variations in four different genes on bc risk was assessed by apsampler algorithm. the fisher exact test, odds ratio (or) with 95% confidence intervals (95% ci) were applied to data analysis. according to obtained results combinations of allele c of tox3 rs3803662 and a of map3k1 rs889312 (p = 0.006, or = 2.2), also allele c of tox3 rs3803662 and c of lsp1 rs381798 (p = 0.02, or = 1.47) associated with increased bc risk in the russian population. consequently, combinations with c allele of tox3 rs3803662 contribute significantly to bc risk with p-value = 0.04, or = 1.86. on the contrary, tt genotype of tox3 rs3803662 with p = 0.04, or = 0.53 and its combination with allele t of lsp1 rs381798 with p = 0.03, or = 0.47 determine a bc risk reduction in russian population. in addition, a risk combination of allele c of lsp1 rs381798 and a of map3k1 rs889312 was found in kazakh population (p = 0.02, or = 1.35). studies have shown that a genetic predisposition to bc can be determined by the cumulative effect of individual alleles and genotypes and possible epistatic interactions of studied genes. obtained combinations of alleles and genotypes can be considered as complex genetic markers of bc and may be used as predictive. cancer that is caused by excessive proliferation of cells and reduced apoptosis is a pathological condition. currently, studies that are comitted with breast cancer are great important early detection and diagnosis of breast cancer. after the discovery of cisplatin as chemoterapy drug, new transition metal based complexes have been developed for treatment of cancer. in this study, anti-cancer activity of azo-azomethide ligand and its mononuclear metal complexes is studied on human cancer cell lines (mcf-7) and mouse fibroblast (l929) cell lines. cells were studied four different concentrations (12,5; 50; 75; 100 lm). xtt (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2h-tetrazolium-5-carboxanilide) protocol was applied after 24 and 48 hours. in our study 10% fetal bovine serum (fbs), 1% l-glutamine, 100 iu/ml penicillin and 10 mg/ml streptomycin in dmem (low glucose) were used. cancer cell lines in dmem medium is produced in 95% humidity and 5% co 2 incubator at 37°c. anti-cancer activity of synthesized complexes were determined on mcf-7 and l929. in the biological activity studies, synthesized compounds showed higher anticancer activity than positive control (5-fu). finally, our new synthesized complexes can be suggested that potent ajan for anti-tumuour for breast cancer drugs. large interindividual differences in response to chemotherapy present an important issue in cancer treatment. malignant mesothelioma (mm) is an aggressive tumor with poor prognosis, treated mostly with gemcitabine/cisplatin (gem/cis) or pemetrexed/cisplatin (pmx/cis) chemotherapy. as both clinical characteristics and genetic variability may affect treatment outcome, our aim was to construct and validate clinical-pharmacogenetic prediction models of treatment outcome in mm for both chemotherapy regimens and to develop an algorithm for genotype-based treatment recommendations. clinical-pharmacogenetic models were built on 71 gem/cistreated and 57 pmx/cis-treated mm patients. pharmacogenetic scores were assigned by rounding the regression coefficients. gem/cis model was validated on 66 independent mm patients. model predicting outcome of gem/cis chemotherapy included crp level, histological type, performance status, rrm1 rs1042927, ercc2 rs13181, ercc1 rs3212986, and xrcc1 rs25487. values ranged between 0 and 3.4; cutoff value of 0.75 had sensitivity of 0.62 and specificity of 0.81. patients with higher score had shorter progression-free and overall survival (p < 0.001). in the validation group, positive predictive value was 0.74 and negative predictive value was 0.56. model predicting outcome of pmx/cis chemotherapy included crp level, mthfd1 rs2236225, and abcc2 rs2273697 with scores ranging between 0 and 3.9. cutoff value of 2.7 had sensitivity of 0.75 and specificity of 0.61. patients with higher score had lower probability of good response and shorter progression-free survival (p < 0.001). clinical-pharmacogenetic models could enable stratification of mm patients based on their probability of response to gem/cis or pmx/cis and improve treatment outcome. this approach could be used for translation of pharmacogenetic testing to clinical practice as it would facilitate the selection of the best treatment option for each patient. p-08.02.5-014 evaluation of anti-diabetic potential of circiliol and circilineol using caco2 cell line diabetes mellitus is a metabolic disorder, and many people are suffering from this disease in the worldwide. oral hypoglycemic agents such as sulfonylureas and biguanides are currently used for the treatment. however, studies searching for a more effective anti-diabetic agents are being carried out continıously. based on that we aim to investigate the potential anti-diabetic effects of circilineol and circiliol isolated from teucrium alyssifolium extract, using in vitro cell culture models. for this purpose, the anti-diabetic actions were investigated by applying model caco2 (colorectal adenocarcinoma) cell line. we determined the level of ag (alpha-glucosidase), sglt1 (sodium-glucose transporter-1) and glut1-5 and glucose transport. neither ag activity nor sglt1 activity was increased with either circiliol or circilineol treatment in caco2 cells compared to positive control. similarly, neither the activity nor the expression level of glut1*-5 was increased in caco2 cell line with either circiliol or circilineol treatment relative to control. in conclusion, these results strongly suggest that circiliol and circilineol do not possess any anti-diabetic potentials.supported by tubitak 114z640 and paubap 2014fbe029 the purpose of this study was to characterize and assess the impact of a novel magnetite (fe3o4) nanosystem functionalized with the natural origin compound eugenol (e) on the pseudomonas aeruginosa virulence, biofilm formation and qs signaling in order to advance research aimed to find alternative and personalized therapeutic approaches for severe infections produced by this opportunistic pathogen. fe3o4 nanoparticles were obtained by a co-precipitation method and functionalized with analytical purity e. functionalized nanoparticles (fe3o4@e) were characterized by ir, sem, tga and hr tem. one laboratory and 9 p. aeruginosa clinical isolates were utilized in the study. growth and biofilm formation were assessed by an adapted microdilution method followed by absorbance reads and viable count analysis in dynamics (6, 12, 24 and 48 hours of treatment). soluble virulence factors production was assessed by enzyme activity evaluation of bacteria grown on specific media. the expression of qs core genes was analyzed by qrt-pcr and a luminescence assay. results demonstrated that the average size of the obtained nanosystem ranges 5-9 nm, particles are relatively homogenous and have a low tendency to form aggregates. subinhibitory concentrations of fe3o4@e limited biofilms formation in a time and strain dependent manner, and significantly inhibited the production of toxin pore forming enzymes (haemolysins and lipases) in most strains. the expression of lasi and lasr genes was three fold downregulated, while the expression of pqsr was upregulated in planktonic cultures suggesting that pqs signaling may be involved in virulence modulation after nanoparticle stimulation. the modulation of bacterial virulence and molecular signaling by functional nanoparticles utilized in subinhibitory amounts offer valuable perspectives to develop personalized antimicrobial approaches based on molecular communication control that clearly modulate pathogenicity and progression of the infectious process. p-08.02.5-016 specimen processing and handling for plasma ammonia measurement b. sarac ßligil 1,2 , s. abus ßo glu 2 , f. aky€ urek 2 , b. € ozt€ urk 2 1 selc ßuk medical school, konya, 2 biochemistry department, selcuk university faculty of medicine, konya, turkey objectives: ammonia requires special processing and handling conditions due to its' unstability. in this study, our aim to investigate a preanalytical factor (delayed analyze) affecting plasma ammonia measurement. design and methods: blood samples were obtained from 20 healthy volunteers. for determining different handling and storage conditions, the following protocols were applied: first protocol (a) transportation on ice and separation (centrifugation at 0°c) within 15 minutes of collection and analyze immediately. second protocol (b) transportation on ice and separation (centrifugation at 4°c) within 30 minutes and analyse refrigerated 2-8°c 2 hours (a1, b1) and 4 hours (a2, b2). all plasma ammonia levels was analyzed enzimatic glutamate dehiydrogenase methods by abbott architect c 8000 clinical chemistry analyzer. results: there were statistically alterations in all protocols compared to first protocol. prolonged centrifugation time for plasma ammonia lead to have higher results (38.6 versus 29.75 lg/dl, p < 0.001). in all protocols including a1, a2, b1, b2 also cause an elevation in plasma ammonia results (35.7, 37.4, 39.5 and 41.2; p = 0.032, p < 0.001, p < 0.001, p < 0.001, respectively). conclusions: ammonia concentration in the blood sample increases over time due to high concentrations in cells as erythrocyte or platelets (three fold). blood samples collected for ammonia determination should be stored on ice, and measured immediately. the wnt/b-catenin signaling pathway has been considered to be a factor in the development and progression of colorectal cancer. many studies have demonstrated that the presence of mutations or polymorphisms in ctnnb1(gene encoding b-catenin; mostly mutations in exon 3) can lead to aberrant activation of wnt/bcatenin signaling at the onset of various types of malignancies, including colorectal cancer. the aim of our study was to assess ctnnb1alteration in the patients with colorectal cancer and compared their tumor and normal tissues. a total of 30 paraffin-embedded colorectal tumor specimens were obtained from department of pathology in cerrahpasa medical faculty. also a total 30 paraffin-embedded normal tissue was used from same cases as a control group. ten-micrometer-thick tissue sections were placed on a glass slide and stained with he. then the tissue sections were dehydrated in graded ethanol solutions and dried without a cover glass. dna was extracted from the tissues with 100 ll of extraction buffer at 55°c over night. the tubes were boiled for 10 min to inactivate the proteinase k. ctnnb1 exon 3 was amplified by pcr. sscp is used to observe any difference between the 2 groups. genomic dna was isolated as described above. 10 ll of each pcr products mixed with 10 ll denaturating buffer and were denatured by heating at 95°c for 10 minutes in, and then were rapidly cooled on ice. the denatured pcr samples are run on 12% acrylamide/ bis gel in 0.59 tbe buffer for 3.5 hours at 200 v at room temperature with water cooling system. the gel was stained with silver staining method. migration and adhesion involve continuous modulation of cell motility, beta-catenin play major roles. beta-catenin gene alterations frequency range between 0 and 16% in colorectal cancer according to the different published studies. in our study no significant differences were found in the ctnnb1 exon 3 between the tumor group and normal groups. p-08.02.5-018 theranostic approach in agressive recurrent meningiomafirst experience in turkey m. o. demirkol 1 , b. uc ßar 2 1 koc ß university, istanbul, 2 american hospital, istanbul, turkey meningiomas arise from the meningothelial cells of the arachnoid membranes of the leptomeninx, which are attached to the inner layer of the dura mater. meningiomas can be classified into three grades (i-iii): grade i meningiomas which are benign, exhibit slow growth; grade ii (atypical) and grade iii (anaplastic) meningiomas, which have a much more aggressive clinical behaviour. meningiomas express non-steroid hormones, including somatostatin. in the brain, somatostatin-a cyclic tetradecapeptide neuropeptide-is believed to act as a neurotransmitter and neuromodulator. somatostatin performs its physiological functions by binding to specific receptors (sstri-sstrv). sstr2 exhibit high affinity for octreotate (tate). tate is a polar, watersoluble peptide that does not penetrate the intact bbb (brain-blood barrier). pet and scintigraphic imagings can only demonstrate somatostatin receptor positive intracranial lesions if the bbb is disrupted. in this aim, dotatate (dota-dphe1, tyr3-octreotate, tate) has been labelled with the positron emitter 68 ga and the beta and gama emitter 177 lu. in this case, we conducted a study to evaluate peptide receptor radionuclide therapy (prrt) planning based on pet/ct imaging of meningioma in the department of nuclear medicine and molecular imaging at the amerikan hospital. [ 68 ga] dota 0 -tyr 3 -oc-pet/ct has been established as the imaging modality of choice for the diagnosis and management of patient with skull-base malign meningioma (rapid progress -to 41 9 48 9 52 mm. from 31 9 37 9 35 mm. in 20 d.-after fifth operation). due to its high sstr2 selectivity, [ 177 lu]-tate showed significantly lower uptake/dose delivered to normal tissues, gatate-pet represents the imaging strategy of choice for an accurate assessment of sstr2 expression levels. although some studies have not shown a clear advantage over pet/ct, there is some evidence that it will have an advantage in selected body sites such as the head and neck, liver, and the pelvis. p-08.02.5-019 cardiovascular diseases can be treated by using 'tetr-odd-vp16' and 'hre' hypoxia inducible systems a. celik 1 , t. kaya 2 , s. cigdem 1 , m. g€ und€ uz 1 1 department of medical genetic, turgut ozal university, ankara, 2 faculty of medicine, turgut ozal university, ankara, turkey ischemia is an insufficient supply of blood to a tissue or organ, usually due to a blocked artery by a blood clot. up to now, the number one cause of death worldwide is caused by ischemia and related conditions such as heart attack or stroke. hif-1a is a transcription activator that functions as a master regulator of oxygen homeostasis. hif-1a protein levels increase under hypoxic conditions as a result of decreased o2-dependent prolyl-hydroxylation, ubiqutination and degradation. we aimed to break up clots in blood vessels and to prevent damage caused by ischemia by using hypoxia inducible systems. we added oxygen dependent degredation domain (odd) of hif1a between and in front of tetr dna binding domain and vp16 transactivation domain, so that tetr-odd-vp16 or odd-tetr-vp16 could activate transcription of tissue plasminogen activator (tpa) controlled by tetracycline response element (tre), in a hif1a independent manner. in addition, we also designed tissue plasminogen activator (tpa) under control of hypoxia response element (hre) of hif1a target genes. western blotting and immunofluorescence assay results showed the expression and nuclear localization of tetr-odd-vp16 and odd-tetr-vp16 constructs under hypoxic conditions, but not normoxic. in addition, using fluorometric reporter systems and tpa enzymatic assay we proved functionality of these constructs under hypoxic conditions. final apporoach to our project is predicting kinetic enzymatic activity of tpa during break up blood clots by using matlab. the results of the present investigation showed, the developed hypoxia responsible systems that can be engineered into endothelial cells to prevent ischemia related cardiovascular diseases. p-08.02.5-020 osteogenic potential assessment of some original scaffolds with magnetic properties new advances in bone tissue engineering demand the development of materials that can not only replace bone, but also regenerate the damaged tissue based on external or even internal stimulus. magnetic materials inside bone scaffolds are known to be a promoting factor for bone healing especially when the therapy is accompanied by application of external magnetic stimulation. based on a recent report, the presence of iron oxide in hydroxyapatite can improve the radio opacity and osteoblast proliferation. in this view, this study focuses on the development of silk fibroin-magnetite biocomposites for potential uses in bone tissue bioenegineering. such novel composites possess good mechanical properties, biocompatibility and biomineralization potential by in vitro tests and could become smart arhiectures, able to stimulate bone regeneration. a new culture model was developed by exposing a 3d cell/ scaffold bioconstruct to a continuous magnetic field during 3 weeks of osteogenic induction. in this view, mc3t3-e1 murine osteoblastes progenitor cells were seeded inside the novel silk fibroin-magnetite biocomposites and subjected to osteogenesys in a magnetic field during 21 days. osteogenic specific markers were evaluated every week in the presence and absence of the field. our results showed that the osteogenic marker's expression started earlier when mc3t3-e1 cells were exposed to the magnetic field. consequently, in our experimental model, the magnetic field had a benefic effect on the osteogenic differentiation process as mc3t3-e1 cells differentiated more efficiently in its presence. these results suggest that the bone healing process could be improved in the presence of a magnetic field. nevertheless, further in vivo studies on animal model should be employed for validation. p-08.02.5-021 impact of physical activity performed on different times of day on cardiac and skeletal muscle damage in trained and untrained male subjects s. algul, m. kara, o. ozcelik y€ uz€ unc€ u yil university, van, turkey introduction: physical activity elevates creatine kinase (ck) and creatine kinase myocardial band (ck-mb) levels which have been considered to be an indirect marker of skeletal and cardiac muscles damage. purpose: impact of physical activity performed on different times of day on serum levels of ck and ck-mb were investigated in trained and untrained male subjects. materials and methods: trained (n = 10, 18.3 ae 0.1 yr, 61.4 ae 2.3 kg) and untrained (n = 10, 18.6 ae 0.1 yr, 63.2 ae 2.4 kg) subjects performed three soccer matches (60 min) in field (30 m versus 50 m) in morning (m), afternoon (a) and at night (n) on separate days. the study protocol was approved by the local ethics committee. venous blood samples were taken at onset and at end of match. serum ck and ck-mb levels are measured using autoanalyser. data are expressed as mean ae s.e., compared by wilcoxon-signed rank and mann-whitney u-tests. p < 0.05 was accepted as statistically significant. results: ck and ck-mb levels increased in three matches in both groups (p < 0.05). importantly, there were significant increases in ck-mb levels in a and n exercises compared to m exercise (p < 0.05) in trained (10.6 ae 1.6 u/l versus 14.2 ae 1.9 u/l, 35% (m) 9.7 ae 1.3 u/l versus 18 ae 2.6 u/l, 66% (a) 9.5 ae 1.1 u/l versus 16.3 ae 2.0 u/l, 103% (n) and also untrained groups (8.6 ae 0.8 u/l versus 11.5 ae 0.8 u/l, 42% (m), 7.3 ae 0.5 u/l versus 11.9 ae 0.9 u/l, 85% (a) 7.9 ae 0.9 u/l versus 13.8 ae 0.9 u/l, 76% (n). discussion: increased metabolic stress or muscle damage during physical exercise elevate serum ck and ck-mb levels. however, higher percentage of increase in ck-mb levels in a and n exercise may reflects additional increases in cardiac muscle stress despite the similar skeletal muscle stress as indicated by ck levels. conclusion: considering the observation of higher percentage increase in ck-mb levels in untrained and also trained subjects, the caution should be taken while performing an exercise in a and n time especially in subjects who has cardiac weakness. p-08.02.5-022 regional assessment of hematological and discrimination indices of complete blood count for beta-thalassemia screening beta-thalassemia is one of the most common genetic abnormality causing health problems worldwide. blood count and film of beta thalassemia trait and iron deficiency anemia have similar features. therefore, several simple screening indices have been developed for differentiating between these diseases. it was to asaimed to assess the hematological parameters and discrimination indices in patients with betathalassemia trait who admitted to our hospital. the parameters of complete blood count (cbc) in 141 subjects (51 males and 90 females) diagnosed by mutational analysis (pcr, gene amplification, dna sequencing) between 2013 and 2015, were retrospectively screened and the thalassemia status of patients was assessed in terms of discrimination indices (eng-land&fraser (ef), green&king (gk), mentzer (m), ricerca (r), shine&lal (s-l), srivastava (s), ehsani and sirdah). the percentages of being above the cut off value were detected by ef 29.72%, gk 30.4%, m 26.35%, r 6.75%, s-l 8.78%, s 35.13%, ehsani 25.67% and sirdah 29.72%. the percentages of falsely negatives for the indices of ricerca and shine&lal were lower than others. morever, when the first three common mutations of our study were considered, 5 out of 23, 7 out of 21 and 4 out of 21 patients were up to the cut off values in terms of e&f, g&k, m, s, ehsani and sirdah indices for ivs-i-110 (g>a), ivsii-1(g>a) and heterozygous codon 8 deletion (-aa), respectively. the molecular diagnosis and prenatal detection for families at risk is important because of the difficulties of treatment in this disease. however, the use of discrimination indices may be valuable for distinguishing of thalassemia trait from iron defiency anemia when the equipment of molecular diagnosis are limited. in our study, ricerca and shine&lal had lower falsely negative results than others. nevertheless, further studies to detect diagnostic perfomance of discriminant indices should be conducted. p-08.02.5-023 novel therapeutic agents in the development of effective drug combinations to treat glioma s. avdieiev 1 , l. gera 2 , r. hodges 2 1 institute of molecular biology and genetics, kyiv, ukraine, 2 university of colorado, aurora, co, united states glial tumors are driven by multiple molecular aberrations that cannot be controlled by a single targeted agent. so, it is possible to expect that the combined multitarget anti-cancer therapy aimed simultaneously at different elements of tumor formation mechanisms will be more effective and will promote the extension of patients' life. to find out which drug combinations will enable the development of therapeutic regimens with improved effectiveness and decreased toxicity, the cytotoxic effects of several bradykinin antagonists (ba) were analyzed for different glioblastoma (gb) cell lines. among all the ba under investigation, bkm-570 appeared to be the most effective, with ic50 values of 4 lm and 3.3 lm in rat glioma c6 and human glioblastoma u251 cell lines, respectively. bkm-570 suppressed erk1/2 and akt1 phosphorylation in u251 cells. temozolomide (tmz), the firstline anti-gliomic drug used in clinics, has only a temporary positive effect and severe side effects in gb patients. we showed that the combination of bkm-570 and tmz led to significant potentiation of tmz cytotoxicity at sub-therapeutic concentrations. recombinant proteins with cytotoxic properties are promising agents for complex therapeutic applications. we revealed that the glioma-associated protein chi3l2 inhibited the viability of u251 cells more effectively than tmz. furthermore, the combination of chi3l2 and bkm-570 resulted in an additive cytotoxic effect. chi3l2-mediated decrease of cell viability was associated with a g1/s transition arrest. chi3l2 provoked the dramatic reduction of prb phosphorylation and a significant decrease of cyclin d1 expression, as well as a substantial increase in p53 level. in addition to the accumulation of p53, we observed the upregulation of cdk inhibitor p21. therefore, g1/s arrest in chi3l2-treated cells could be realized via activation of prb, downregulation of cyclin d, and activation of p53. harmful hereditary mutations in brca1 and brca2 are one of the most important risk factors of breast cancer. the aim of this study is to determine the mutations which are associated with breast cancer in people which is diagnosed breast cancer and/or have breast cancer-diagnosed family members by sanger sequencing and thus provide predictive and prognostic utility. our ongoing study is present the genetic variations in brca1 and brca2 genes in breast cancer-diagnosed 12 patients, that one of them is male, and 1 person yet healthy whose brca2 was sequenced by sanger sequencing. the data were analyzed by using seqscape software 2.6 and detected variations were compared with literature. in brca1, we determined 16 different benign genetic variations and 1 variation with unknown significance and 1 variation which has not in literature. in brca2 gene of 12 patient and 1 healthy person,14 benign variations,2 variations with unknown significance,7 variations which has not in literature and 1 mutation were determined. this mutation is c.3189-3192delgtca and is located in occr. c.9257-74a>c variation in brca2 gene, was determined in only male patient.c.32t>a variation in exon2 of brca2, was observed in only the youngest patient who has no family member with breast cancer and healthy person. while this variation takes place in literature as variation with 'uncertain clinical significance', an in silico program mutation taster speculated as 'disease causing' for this variation. also, almost all of variations with 'unknown significance' literature knowledge were determined in only one and different cases. this situation increases the possibility of being pathogenic of this variations. the our findings until now can contribute to variations with uncertain clinical significance in the literature. also the variations that have not in the literature but we suggest the possible relation with breast cancer as an estimate may be added to literature by expanding the study. p-08.02.5-025 inhibition of the recombinant human butyrylcholinesterase with paraoxon and coumarin analog of soman organophosphorus compounds (op) represent a class of extremely toxic chemicals that are used as warfare agents. uncontrolled utilization of op is highly dangerous due to their high potential to be efficient poisons in terrorist attacks. current therapy of op poisoning include intravenous administration of atropine and acetylcholine reactivators however, it does not completely eliminate brain damage effects. alternative experimental therapy against op poisoning is utilization of bioscavengers that irreversibly react with op and rapidly inactivate them. recombinant human butyrylcholinesterase (rhbche) is one of the most promising candidates as bioscavenger due to its pharmacokinetic characteristics and broad spectrum of op neutralizing activity. here we investigated in vitro inhibition of rhbche with two model oppesticide paraoxon (pox) and coumarin analog of soman (gd c ). both op lead to rapid and irreversible inactivation of rhbche that was monitored using ellman assay and fluorescence measurements. bimolecular inhibition rate constants dramatically differ between pox and gd c that could be explained by steric hindrance in soman analog. the next steps forward creation of catalytic bioscavengers based on rhbche should be done based on mechanisms of op-rhbche interactions. this work was performed in frame of grant rfme-fi60414x0069. non-hodgkin lymphomas (nhls) represent a heterogeneous group of malignancies that arise from the lymphoid system. at the present time exist a lot of drugs for the nhls therapy, but mostly all of them are unsafe and there is no consensus regarding the best treatment protocol. to increase the efficacy and safety of therapeutic b-lymphocyte depletion in lymphomas and leukemia's it would be preferable to induce the death of pathological b cells without affecting normal b cells to prevent side effects. similar to other types of cancer, nhls arise by a multistep accumulation of genetic aberrations that induce a selective growth advantage of the malignant clone. all b-cells of organism have a unique cell surface markerantigen b-cell receptor (bcr). we generate novel approach for personalized non-hodgkin lymphomas therapy based on peptide specific to malignant cells surface receptor. for this purpose we designed new lentiviral peptide library screening technique based on fluorescent reporter cells system. herein 7aa peptide library was used for screening of nhl's malignant receptor agonist. patients' lymph nodes biopsy samples mrna was used as a source of malignant bcr nucleotide sequence. variable domain of the lymphoma bcr was used for chimeric receptor generation, where bcr vh/vl part responsible for agonist recognition and bottom part of receptor was retranslate signal to the reporter gene. in this embodiment of the method, very large numbers of candidate 7aa peptides expressing lentivirus and eukaryotic reporter cells are packaged together in a format where each is capable of replication, thereby forging a direct link between genotype and phenotype. after four rounds of screening we discover peptides specifically interacted with malignant bcr's. selected peptide ligands were fused with chimeric antigen receptor for expression on t-cells. modified tcells selectively eliminate nhl's malignant cells ex vivo. this work was supported by grant rfmefi60714x0061. introduction: pesticides are used to prevent damage of unwanted insects, rodents, plant, moss and other pests. excessive use of pesticides may cause adverse effects in animals and humans. chlorpyrifosethylene (cpe) is an organophosphate pesticide, used in many agricultural products such as figs, cherries, olives worldwide; caused acute poisoning and chronically oxidative stress. rosadamascena mill (rosaceae) is a rose, used for production of rosewater (rw) and rose oil worldwide. rosaceae products are consumed in food and cosmetic industries. materials and methods: in this study, we investigated that cpe and rw effects on kidney tissues of rats. this study was included 32 adult male rats, divided into 4 groups. each group included 8 rats. i.group: control (regular feed), ii.group: cpe (0.3 mg/kg/day), iii.group: rw (100 mg/kg/day) and iv.group: cpe (0.3 mg/kg/day) + rw (100 mg/kg/day). following 15 days, kidneys were taken after sacrificed. analyzes were performed that malondialdehyde (mda), nitric oxide (no) as oxidant; superoxide dismutase (sod), glutathione reductase (gr) as antioxidant parameters. results: as compared with control, mda and no levels in cpe were a significant increase was determined (p conclusion: cpe is shown that significant increase on oxidant parameters, but significant decrease on antioxidant parameters. rw occurs opposite situation. similarly results of cpe, rw + cpe increased oxidant parameters, but decreased antioxidant parameters. these changes are lower than only cpe. these results showed that positive effects both rw and rw + cpe increasing on antioxidant parameters, also decreasing on oxidant parameters. we provide the comparative analysis of the reduced glutathione (gsh), reactive oxygen species (ros), a-tocopherol levels, an intracellular labile zn 2+ pool and esterase activity of red blood cells of patients with diagnosed components of the metabolic syndrome (ms)arterial hypertension and diabetes mellitus type ii (ah + dm + ). as the comparison groups were selected patients with one diagnosed component of msarterial hypertension (ah + dm à ) or without any diagnosed component of ms (ah -dm -). patients of all investigated groups were at the hospital treatment with a diagnosis coronary heart disease (chd) ii degree. human blood was obtained from normal donors and patients with chd ii stage. cytosolic esterase activity was assessed using calcein-am test. ros level was evaluated using cm-h 2 dcf-da. gsh level was estimated using lowry method. an intracellular labile zn 2+ pool was assessed using fluozin-3-am. investigations were performed on the specord m40, hplc system lc-20 prominence (shimadzu) and facscantoii (bd). a significant decrease of the intracellular level of labile zn 2+ in erythrocytes of patients with ah + dm + compare with ah + dm à and ah à dm à was shown. this fact confirms our assumption concerning the important role of zinc homeostasis in the etiopathogenesis of diabetes mellitus type ii. a direct relationship between the intracellular zn 2+ level modification and erythrocytes esterase activity of patients with chd ii degree was observed. moreover, in ah + dm + group of patients this relation was more marked. the unidirectional alteration in the erythrocytes redox state (gsh and a-tocopherol levels reduction, ros formation activation) was revealed at the whole of investigated chd patients groups (ah + dm + , ah + dm à , ah à dm à ). however, the pathological erythrocytes response on in vitro action of the different antioxidants (n-acetylcysteine, ascorbic acid, a-tocopherol, quercetin) had a diverse character that can be a significant test under antioxidant therapy prescription. it is known that long-term social isolation and the disorder of natural circadian rhythm is considered an important stress factors, which cause a variety of metabolic and mental disorders. it should be noted that the impact of the stresses takes up a larger area, according to, review of the action of their mechanism is one of the topical issues of modern science. it is estimated that as a result of stress the metabolic processes change in the organizm. because of this, we've studied the functionality of the antioxidant system in laboratory rat heart muscle cells and blood under psycho-emotional stress. it was found that quantitative changes of nitric oxide (no) was initiated the process of lpo, which caused oxidative stress in the cells and decreased antioxidant enzymes activity, such as catalase,sod, gpx and gr. the results suggested that psycho-emotional stress was accompanied by oxidative stress, causing a reduction in the intensity of energy metabolism in cardiac muscle cells, which was further strengthened by the fact that the activity of the enzymes involved in atp synthesis in mitochondria was reduced. also, we've studied exogenous creatine positive and negative affects on energy metabolism and blood lipid spectrum. based on this, we proposed that psychological stress is one of the factors contributing to the development of various cardiac diseases. the importance of free radical lipid transformations, which differ from the peroxidation processes, was pointed out for the first time in our laboratory studies. we have found that ros can induce free radical destruction processes of sphingolipids with c-c-bond cleavage [lipids, 2011, 46:271-276; lipid insights, 8, 1-9] . in case of acylated sphingolipids, they can undergo decomposition with c-c-bond cleavage upon direct uv irradiation [photochem. photobiol., 2012, 88:899-903] . it was of interest to establish the possibility of photosensitized decomposition reactions of not acylated sphingolipids, which do not absorb an ultraviolet. in this work we studied photosensitized reactions of sphingosines containing a free amino group, and low molecular compounds, which simulate their structure, such as aminoalcohols (serinol). as photosensitizers, the salts of transition metals, hydrogen peroxide, and acetone were used. oxygen was removed by bubbling with argon to reduce the probability of side reactions during photolysis of sphingolipids, such as oxidation processes (including oxygen reactions with alkyl radicals). we have shown that the action of uv-radiation on aminoalcohols and sphingosines in aqueous solutions in the presence of photosensitizers induces their destruction with c-c bond rupture. the main carbonyl product of sphingosines free radical destruction was an unsaturated aldehyde -2-hexadecanal. it was found, that 2-hexadecenal possesses a wide spectrum of biological activity: it promotes reorganization of the cell cytoskeleton and modifies the redox state of the cells [febs journal 282 (suppl. 1), abstracts: mem. biol. s5, lipid signaling & dynamics, p. 235.] . the results of this study can expand the frontier of research regarding free radical lipid damage, which could contribute to a better understanding of the origins of diseases associated with the activation of free radical processes in living organisms. structural basis for the 14-3-3 proteindependent inhibition of apoptosis signalregulating kinase 1 protein kinase ask1 (apoptosis signal-regulating kinase 1) is a member of the mitogen-activated protein kinase kinase kinase (map3k) family that plays a crucial role in immune and stress responses. the activity of ask1 is regulated through homo-oligomerization and interaction with other proteins including the 14-3-3 protein which binds to the phosphorylated motif located at the c-terminus of the kinase domain of ask1 and suppresses its catalytic activity through unknown mechanism. under stress conditions, ask1 is dephosphorylated at ser966 and the 14-3-3 protein dissociates. this dissociation is then one of the factors that lead to the activation of ask1. we performed low-resolution structural analysis of the kinase domain of ask1 (ask1-cd) bound to 14-3-3 using chemical cross-linking, analytical ultracentrifugation and small angle x-ray scattering. the low-resolution structural analysis shows that ask1-cd binds to the 14-3-3 protein in two to two stoichiometry through a small binding interface involving surface of 14-3-3 outside its central channel and several regions from the c-lobe of ask1-cd. the complex is dynamic and conformationally heterogeneous. phosphorus nmr and time-resolved fluorescence measurements, together with low-resolution structural analysis, indicate that binding of ask1-cd to 14-3-3 modulates conformation of ask1's activation segment. these results suggest that the 14-3-3 binding suppresses the catalytic activity of ask1 through direct structural modulation of its activation segment. our study provides new insight into the interaction between the kinase domain of ask1 and 14-3-3 and offers a plausible structural explanation for the 14-3-3 protein-dependent inhibition of ask1 kinase activity. introduction: thymoquinone (2-methyl-5-isopropyl-1,4-benzoquinone, tq) exerts a great antitumor activity against different types of cancer cells. a growing body of evidence indicates that reactive oxygen species (ros) generation followed by modulation of the akt and mapk pathways is a general mechanisms underlying the tq antitumor action. however, the data of tq effects on the mapk pathway are conflicting. to date, the activation or inhibition of the mapk protein family seems to depend on the cell type and on the tq concentration used. in order to elucidate the antitumor potential of tq against gliomas and the underlying molecular mechanism, tq influence on c6 rat glioma cells functioning was studied. results: it has been shown that the cultivation of c6 cells with tq in concentrations of 10 -100 lm during 24 hours strongly inhibits cell proliferation and induces cell death with id 50 of 60 lm. at the same time, tq induces ros generation and intracellular gsh depletion in a dose-dependent manner, that is followed by the mitochondrial potential decrease. interestingly, ros production has only cytoplasmic, but not mitochondrial origin in cells challenged with tq at the concentrations up to 100 lm. two-electron reduction of tq by dt-diaphorase attenuates tq anticancer efficiency whereby inhibition of dt-diaphorase by dicumarol increases tq-induced c6 cell death by 25 %. we analyzed mapk pathways involvement in c6 cells growth inhibition at tq treatment. it has been shown that inhibition of the erk pathway by pd98059 and jnk pathway by sp600125 does not influence on tq-induced effects. on the contrary, the specific phosphoinositide-3-kinase (pi3k) inhibitor (ly294002) abrogates tq-induced growth arrest. conclusion: antitumor effects of thymoquinone on c6 glioma cells is a result of ros generation and intracellular gsh depletion, that is followed by mitochondria disfunction, and growth arrest via pi3k pathway. assessment of oxidative stress and antioxidant defense activity parameters in patients with hiv-infection is of great importance, especially for hiv-positive women of reproductive age planning to have children. data of 53 women of reproductive age with hiv-infection analyzed: patients with hiv-monoinfection (n = 26) and patients with co-infection (hiv and hepatitis b and / or c) (n = 27). as a control we used the data of healthy women (n = 28). serum and hemolysate used as material for the study. lpo-aod products were determined by spectrophotometric and fluorometric methods. average value of initial lpo products -diene conjugates was significantly increased in the group with hiv-co-infected compared to control (1.57 times; p = 0.001) and group with hivmonoinfection (1.4-fold; p = 0.027). the level of secondary products -ketodienes and conjugated trienes increased in patients of both groups compared to control (2.16 times (p = 0.0002) and 2.43-fold (p < 0.0001), respectively). at the same time isolated double bonds and tba-active products content showed no significant changes (p > 0, 05). total antioxidant activity parameter decreased 1.28 fold (p = 0.0273) in the group with hiv-monoinfection compared to control. decrease in activity of the primary antioxidant enzyme -superoxide dismutase (p = 0.0077, compared to the control and p = 0.0035, compared with the group with hiv-monoinfection) and the level of a-tocopherol (1.22-fold to control and 1.25 fold to hiv-monoinfection) was detected in the group with hiv-coinfection. 1.29-fold higher content of retinol in hiv-coinfection group (compared to the control) revealed. in women with hiv-coinfection the oxidative stress was significantly higher than in women with hiv-monoinfection. suggested to include antioxidant supplements in the complex pathogenetic therapy in women with hiv-coinfection (hiv and hepatitis b and / or c), which will contribute to women's ability to bear children. p-09.04.4-008 acute different doses of malathion induce cholinesterase inhibition, glucogenic enzymes and histopathological change in rat liver malathion, which is an organophosphorus compound, is a widely used pesticide all over the world. despite its benefits, malathion has many toxic effects on many tissues including liver. we designed to evaluate the acute different doses of malathion on cholinesterase (che) inhibition, gluconeogenic enzymes and histopathological change of rat liver. for this purpose 4 groups were formed. rats in group 1 served as control group animals which were only given corn oil. group 2, group 3 and group 4 were administered 100, 200, 400 mg/kg of malathion, respectively, dissolved in corn oil by oral gavage. the rats were sacrificed after 24 hours following administration and the livers of rats were removed. the liver che, alanine aminotransferase (alt), aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) were studied using autoanalyzer. histopathological investigation was performed using microscope. the liver che activities of group 2, group 3 and group 4 were inhibition percentage of 53%, 43%, and 51% respectively, comparison of group 1's che activity. the liver alt, ast and ldh increased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). we also observed that there was vacuolar and hydropic degeneration in liver of group 4. according to our result, acute administrations of malathion result in hepatotoxic effects with increasing doses. background and aims: an imbalance between free radicals and antioxidants is closely linked to the onset of an acute myocardial infarction (ami). the aim of this study was to investigate the antioxidant status and the lipid peroxidation in patients admitted to hospital immediately after ami. methods: the study population comprised 19 patients with ami and 14 healthy subjects. patients that had an acute myocardial infarction (ami) less than 72 hours since onset were selected for this study. antioxidant status was assessed by lactonase activity of paraoxonase (pon1 dhc), trolox equivalent antioxidant capacity (teac) and plasma uric acid level. malondialdehyde was used as marker of lipid peroxidation. results: compare with the control group, the levels of mda and pon1 dhc were significantly higher in group with ami (p < 0.005, respectively p < 0.001). elevated levels of mda (p < 0.005) were found in patients with ami compare with the control subjects. ami patients had also statistically significant reduced levels of teac (p < 0.005) comparative with healthy subjects. no statistically significance was found for plasma uric acid level in subjects from our study. conclusion: a high lipid peroxidation correlated with a decreased teac activity suggest an exacerbated oxidative stress in subjects admitted to hospital immediately after an ami. p-09.04.4-010 dealing with copper toxicity: new insights into copper detoxification in yeast copper (cu), an essential metal, is a double-edged sword, as its essential nature is counterbalanced by the toxic effect that it can exert on cells when not properly controlled. as such, organisms have evolved defence mechanisms against cu toxicity, and in the yeast saccharomyces cerevisiae, the transcription factor ace1 orchestrates several of those mechanisms, by activating cu-detoxification genes. in s. cerevisiae iron (fe) uptake is partially dependent on cu, as the membrane multicopper-oxidase fet3, which is part of the high-affinity iron uptake system, requires cu as a cofactor. aft1, the low iron-sensing transcription factor in yeast, is known to regulate the expression of fet3 gene. however, we found that a strain lacking fet3 is more sensitive to cu surplus conditions than a strain carrying the aft1 gene disrupted. this finding suggests that under such conditions another regulator came into play and controls fet3 gene expression. we next evaluated whether ace1 could be the alternative regulator of fet3 under cu excess. to test this hypothesis we first constructed the double mutant aft1ace1 and assayed its fitness under cu surplus. as expected, the double mutant is much more sensitive to cu than the single aft1 or ace1 mutants. we next monitored the expression of fet3 gene in cells lacking ace1, using yeast-one hybrid and qrt-pcr approaches. our data clearly indicates that fet3 expression is dependent on ace1 when cu is overly abundant. altogether our data unveil a novel mechanism of cu detoxification relying on the activation of fet3 by ace1 in an aft1independent way. experiments to understand the consequences of this regulation in terms of cu detoxification are currently being undertaken. in joint degeneracy, reactive oxygen species manifest their toxicity both through intrinsic reactivity and the inflammatory process activation, leading to cartilage dysfunctions, injuries of matrix proteins and cytokines stimulation. the study is focused on the identification of oxidative balance modulation (enzymes and oxygen free radicals) by a bioactive extract obtained from small sea fish. the cellular support was represented by the chondrocyte line chon-001 derived from human long bones (atcc ò crl-2846 tm ), that assure the reproducibility of a standardised biological system. the oxidative stress was induced through stimulation with il1b, a cytokine-factor that promotes the protein catabolism and also with tnfa, the initiator of pro-inflammatory activation, combined with pma, the activator of protein-kinase c, triggering of oxygen reactive species generating cascades. the antioxidant effect was compared with known antioxidants: vitamin c, x3 fatty acid, n-acetyl -cysteine. the acellular antioxidant/antiradical screening was done using two complementary techniques for total antioxidant status evaluation and completed by measuring cellular catalase (cat) and superoxidedismutase (sod) activity, correlated with intracellular hydrogen-peroxide and superoxide anion monitoring through flow cytometry. the antioxidant properties of the bioactive extract proved in acellular systems are confirmed at cellular level by the involvement of the product in the enzymatic cascade cat -sod, potentiating the catalytic action of the enzymes, and by the decline of both intracellular reactive oxygen species (the hydrogen peroxide decrease with 50%, the superoxide anion is reduced with 31% compared with the stimulated control). the demonstrated antioxidant synergy assures a complete cellular protection induced by the small sea fish extract in human condrocytes. introduction: alzheimer's disease (ad) is a progressive neurodegenerative disorder characterized by memory loss, cognitive impairment. oxidative stress is a contributory factor in ad pathogenesis. glutathione (gsh) is the main antioxidant cellular defence. the ratio of gsh/gssg shows the redox status of gsh, and plays important role in maintaining intracellular redox homeostasis. the current study was carried out to determine oxidative dna damage and ratio of gsh/gssg which plays an important role in protection of target molecules from oxidation in the patients with ad. methods: the study subjects were consisted of patients with ad (n = 67) and age matched control group (n = 42) who were treated and followed in the cerrahpasa medical faculty hospital, department of neurology and department of internal medicine/ geriatrics. dna strand breaks and h 2 o 2 -induced dna damage were determined in lymphocyte dna with comet assay. the gsh and gssg levels in the erythrocyte lysates were measured by using a commercial spectrophotometric kit. the ratio of gsh/gssg were calculated. statistical analysis was performed with spss 22 software package. results: dna strand breaks and h 2 o 2 -induced dna damage were found to be higher (p = 0.000 for all), the ratio of gsh/gssg was found to be lower (p = 0.024) in the ad group than control group. there was no significant difference between male and female for dna strand breaks and h 2 o 2 -induced dna damage in the ad group, but ratio of gsh/gssg were higher in male when compared with female (p = 0.045). no significant difference was found between the men of ad group and men of the control group for gsh/gssg ratio whereas women of the ad have a lower gsh/gssg ration than those in the women of the control group (p = 0.009). conclusion: increased systemic oxidative dna damage and dna susceptibility to oxidation may be resulted from diminished gsh/gssg ratio in ad patients. this finding shows the importance of antioxidant support in ad management. p-09.04.4-013 validation of a liquid chromatography-tandem mass spectrometry method for the measurement of lipid peroxidation product 8iso-prostaglandin f2a in urine m. kant 1 , m. akıs ß 1 , h. _ is ßlekel 1,2 1 department of medical biochemistry, school of medicine, dokuz eylul university, izmir, 2 department of molecular medicine, school of medicine, dokuz eylul university, izmir turkey 8-iso-prostaglandin f 2a (8-iso-pgf 2a ) is a reliable indicator of lipid peroxidation resulting from oxidative lipid damage and is postulated as a gold standard biomarker for the evaluation of oxidative stress. the aim of this study was to validate non-invasive and highly accurate stable isotope dilution-multiple reaction monitoring liquid chromatography-tandem mass spectrometry (sid-mrm lc-ms/ms) method for identification and quantification of 8-iso-pgf 2a . twenty four hour urine samples were collected from healthy volunteers at medical school of dokuz eylul university for analytical performance studies. lc-ms/ms analyses were performed on conventional hplc coupled to a triple quadrupole ion trap mass spectrometer equipped with a turboionspray tm source. analyst software version 1.5 were used for data analyses. mrm transitions used were m/z? 250/164 for 8-iso-pgf 2a and m/z? 255/169 for 8-iso-pgf 2a-d 4 . analytical performance of the method was evaluated by linearity, selectivity, sensitivity, precision and accuracy studies using pure standards and samples extracted from urine of healthy volunteers. the linear calibration range for 8-iso-pgf 2a was determined as 20 ng/ml by using standards ranging from 0.25-50 ng/ml. analytical sensitivity of the method was determined by lod with s/n of 3 and loq with s/n of 10. lod and loq for 8iso-pgf 2a were 2.4 9 10 à2 and 38 9 10 à2 ng/ml, respectively. the assay stability and reproducibility were assessed by the precision and accuracy of intra-and interday measurements (n = 10). the intra-and interday cvs for 8-iso-pgf 2a were 4.5% and 1.2%, respectively. sid-mrm lc-ms/ms method for absolute quantification of 8-iso-pgf 2a was optimized and validated in our laboratory and therefore this highly accurate method can successfully be applied to clinical patient samples. p-09.04.4-015 synergistic anticancer effects of sulforaphane and cisplatin through the induction of apoptosis and autophagy following oxidative stress in malignant mesothelioma cells malignant mesothelioma is characterized by poor responsiveness to current chemotherapeutic drugs, usually owing to high resistance to apoptosis. here, we investigated chemosensitizing effects of phytochemical resveratrol, in combination with cisplatin, on malignant meothelioma cells. cell viability was evaluated using mtt assay. cell apoptosis was detected with dapi staining, caspase 3/7 activity assay and flow cytometry. cell cycle distributions, ros levels and mitochondrial membrane potential were determined using flow cytometry. the expression of cell cycle-, apoptosis-, and autophage-related proteins was measured with western blotting. the combination treatment of cisplatin and resveratrol (cis/res) synergistically induced apoptosis, as evidenced by typical cell morphological changes, the appearance of a sub-g 0 /g 1 peak, an increase in the annexin v(+) cells and the cleavage of caspase-3 and parp. cis/res increased ros production and depolarization of mitochondrial membrane potential with an increase in the bax/bcl-2 ratio. these changes were attenuated by pre-treatment with n-acetylcysteine, suggesting that cis/res induced apoptosis through oxidative mitochondrial damage. compared with msto-211h cells, cis/res was less efficient in killing h-2452 cells. h-2452 cells exhibited an enhanced autophagy to cis/res, as observed by an increase in viable cells exhibiting intense lysotracker red staining and up-regulation of beclin-1 and lc3a. inhibition of autophagy by bafilomycin a1 rendered cells more sensitive to cis/res-induced cytotoxicity and this was associated with induction of apoptosis. these data indicate that the increased resistance of h-2452 cells to cis/res is closely related to the activation of self-defensive autophagy, and provide the rationale for targeting the autophagy regulation in the treatment of malignant meothelioma. stress oxidative induced by chemotherapy with cyclophosphamide (cp) causes vulnerability in sperm and decline of fertility. this study was aimed to evaluate the role of ethyl pyruvate (ep) in the amelioration of fertility and growth of primitive embryo in animals that received cp. 24 adult male mice (6-8 weeks) were randomly divided into 3 groups: control group received normal saline (0. 2 ml/day, ip), cp group received cp (15 mg/kg/week,¬ ip), the cp+ep group received ep (40 mg/kg/day, ip) along with cp, were treated for 35 days. 8 mice from each group were arranged for evaluation of sperm quality and in vitro fertilization (ivf) too. afterward, the separated oocytes from 72 ovulation stimulated mice were conducted to evaluation of ivf and embryo development. the results revealed that cp caused notable decrease in percentage of fertilization in cp group, but administration of ep along with cp caused an increase in the percentage of fertilization in comparison to cp group. the percentage of the two cell zygotes in cp+ep group, and the percentage of blastocysts in control and cp+ep groups were higher than that in cp group (p < 0.05). results showed that the total number of arrested embryos in control and cp+ep groups was decreased in comparison to cp group (p < 0.05). the percentage of arrested embryos type i, ii, and iii in cp+ep group was decreased in comparison to cp group, but that decrease was significant only in types i and ii (p < 0.05). the average motility, viability, nucleus maturity and sperm morphology were decreased significantly in cp group in comparison with control and ep groups, whereas ep caused significant increase of these parameters (p < 0.05). also, the percentage of dna damage was increased significantly in cp group in comparison with control and ep groups (p < 0.05). the results of this study indicated that the ethyl pyruvate was able to suppress free radicals and enhance the ivf and quality of sperm in cp treated animals. malathion, which is a member of organophosphate chemical family, is used to control pests and is a widely used pesticide all over the world. however it is also known to be highly toxic on many tissues including pancreas. to test this we set 4 groups to administer a single dose of malathion dissolved in corn oil via oral gavage at the doses of 100 mg/kg (group 2), 200 mg/kg (group 3) and 400 mg/kg (group 4). only plain corn oil was given to control group (group 1). the rats were sacrificed 24 hours after administration of the chemical and the pancreases of rats were removed. in an attempt to evaluate the dose dependent response, we measured amylase and lipase activities, insulin, malondialdehyde (mda), total oxidant status (tos) levels in rat pancreases. all of the parameters were measured spectrophotometrically. we found that pancreas insulin levels significantly increased in group 4 compare to group 1, besides the insulin levels of group 3 and group 4 were significantly higher than group 2 (p < 0.013). pancreas amylase and lipase activities significantly decreased in group 3 and group 4 compare to group 1 and group 2 (p < 0.013). however, there was no significant change in pancreas mda and tos levels (p > 0.05). according to correlation analysis, when pancreas amylase levels declined, lipase levels were decreased simultaneously and there was a strong positive correlation between them (p < 0.01). in addition, when the comparison was evaluated as a binary, while pancreas amylase and lipase levels diminished, pancreas insulin levels increased and a strong negative correlation between them was found (p < 0.01). according to our result, acute administrations of malathion leads to alterations of insulin, amylase, and lipase levels with a dose dependent manner, while it does not to change oxidant status. the aim of this study is to evaluate the potential toxic effects of mancozeb on the stress biomarkers such as catalase (cat) activity, malondialdehyde (mda) level and protein levels in the brain tissue of zebrafish (danio rerio). mancozeb, is a synthetic fungicide contaminating aquatic environments as a potential toxic pollutants, was investigated in the present study for acute toxicity. zebrafish groups (m-low and m-high) were exposed to different doses of mancozeb (5 mg l-1 and 7.5 mg l-1) for 120 hours except the control group. catalase (cat) activity, malondialdehyde (mda) level and total protein levels were determined by spectrophotometer. the results showed that cat activity and mda levels were decreased in all experiment groups. protein levels were increased in experiment groups when compared to the control group. in conclusion, the changes in the cat activity and mda levels were time and as well as mancozeb dose-dependent. furthermore, the biochemical parameters of mancozeb exposure on zebrafish, showed that mancozeb has significant effect on the zebrafish and/or aquatic organisims. paracetamol (para), which is antipyretic and analgesic, is widely used around the world. paracetamol can be recommended for moderate or mild pains especially in pregnancy as an analgesic. it is known that, paracetamol can cause hepatotoxicity or nephrotoxicity. we were aimed that in this study to show potential hepatoprotective and nephroprotective effect of betaine against long term paracetamol using at therapeutic doses. it has been prepared 3 groups, control, para and para+-betain groups. paracetamol and betaine were administered by gavage to pregnant rats, from first day to the last day of pregnancy (aprox. 21 day). 2 ml physiological saline (%0.9 nacl solutions), 30 mg/kg/day paracetamol, 30 mg/kg/day paracetamol and 800 mg/kg/day betain was given by orally to control, para and para+betain groups respectively. the day of the birth, newborn rats anesthetized by ether and after decapitated. 8 newborn rat's liver and kidney tissues used for biochemical analysis [malondialdehyde (mda), reduced glutathione (gsh), nitric oxide (no) and paraoxonase-arylesterase (pon-are)] and 5 rat's liver and kidney tissues used for histological studies. we showed that, mda and no levels was significantly increased, while pon activities decreased. on the other hand gsh levels and are activities was decreased but these decline wasn't significant in the liver and kidney para group. these biochemical results showed hepatotoxicity and nephrotoxicity in neonates which can be formed in long term maternal paracetamol using at therapeutic doses. also our histological findings was support these biochemical results. we used betaine against potential hepatotoxic and nephrotoxic effect of long term maternal paracetamol using at therapeutic doses for neonates. betaine has antioxidant properties and also used as a methyl donor for transsulfuration reactions in the cell. biochemical and histological examinations showed that betaine protected the tissue injury relatively. p-09.04.4-020 lipid rafts are involved in modulation of ca 2+ responses induced by glutoxim and molixan in macrophages pharmacological analogues of oxidized glutathione (gssg) disulfide-containing drugs glutoxim ò (gssg disodium salt with dmetal nanoaddition, «pharma-vam», st. petersburg) and molixan ò (complex of glutoxim with inosine nucleoside) have found clinical application as a wide range immunomodulators and hemostimulators. however, the cellular and molecular mechanisms of these drugs action are still unclear. previously we showed for the first time that glutoxim and molixan cause biphasic intracellular ca 2+ concentration ([ca 2+ ] i ) increase due to ca 2+ mobilization from thapsigargin-sensitive ca 2+ stores and subsequent store-dependent ca 2+ entry in rat peritoneal macrophages. it is known that key proteins involved in ca 2+ signaling are localized in discrete plasma membrane lipid rafts domains. lipid rafts are cholesterol and sphingolipids enriched microdomains that function as unique signal transduction platforms. thus, the aim of the present work was to elucidate the possible involvement of lipid rafts in glutoxim and molixan effects on [ca 2+ ] i in macrophages. [ca 2+ ] i measurements were performed with fura-2am microfluorimetry. to examine the involvement of lipid rafts in the effect of gssg-based drugs on [ca 2+ ] i we used methyl-bcyclodextrin (mbcd), widely used to remove cholesterol from membranes, thus disrupting the lipid raft domains. we have shown for the first time that macrophage preincubation with 10 mm mbcd for 1 hours before molixan addition causes significant inhibition of both ca 2+ mobilization (on average, by 77.6 ae 9.2%) and subsequent ca 2+ entry (on average, by 82.3 ae 10.5%), induced by molixan. similar results were obtained in experiments with glutoxim. thus, we have demonstrated for the first time that mbcd significantly decreases both phases of glutoxim-or molixan-induced ca 2+ responses in macrophages. the results suggest that intact rafts are required to initiate complex signaling cascade activated by glutoxim or molixan and leading to [ca 2+ ] i increase in macrophages. plant-derived natural substances (phytochemicals) with potent pro-apoptotic activity towards cancer cells in vitro are considered as promising nutraceuticals in anticancer therapy. nevertheless, due to their relatively low bioavailability, administration of high doses of nutraceuticals that are not achievable in vivo seems to exert potentially negligible physiological effects in clinical trials. thus, there is a need for revealing novel cytophysiological effects of low doses of phytochemicals towards cancer cells. in the present study, we have considered thirty one nutraceuticals and selected four phytochemicals acting at low micromolar range (5 to 20 lm) against phenotypically different mcf-7, mda-mb-231 and sk-br-3 breast cancer cells, namely diosmin, sulforaphane, ursolic acid and betulinic acid. nutraceuticals inhibited cell proliferation and caused changes in the cell cycle that was accompanied by elevated levels of p53, p21, p16 and/or p27. apoptosis (annexin v staining, multicaspase and mitopotential assays) was observed exclusively when nutraceuticals were used at the concentration of 20 lm, whereas at the concentration of 5 lm, stress-induced premature senescence was noticed (sa-b-gal activity). nutraceuticals diminished the levels of glut-1 and selected glycolytic enzymes. nutraceuticals promoted oxidative and nitrosative stress as judged by increased production of total reactive oxygen species, total and mitochondrial superoxide, nitric oxide and protein carbonylation. nutraceuticals also stimulated dna single and double strand breaks that was accompanied by atm phosphorylation and to a lesser extent by histone h2ax phosphorylation and 53bp1 foci formation. taken together, several responses to nutraceutical treatment were observed in breast cancer cells that may reflect the heterogeneous nature of cancer cell populations. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. nucleolus is thought to be a stress sensor and oxidative and ribotoxic stimuli may cause the inhibition of rrna synthesis by the inactivation of transcription factor tif-ia/rrn3 that is accompanied by the relocation of nucleolar proteins and p53-based cell cycle arrest and/or apoptosis. as nutraceutical-mediated modulation of nucleolar activity may be considered an attractive anticancer strategy, in the present study, we have investigated the effects of three selected nutraceuticals, namely sulforaphane, ursolic acid and betulinic acid on nucleolus state in three breast cancer cell lines (mcf-7, mda-mb-231 and sk-br-3). we found that low dose nutraceutical treatment resulted in p21-mediated inhibition of cell proliferation, a decrease in rrna synthesis, shifts in lamin a/c and b1 pools, changes in the nucleolar protein levels and their carbonylation, and changes in nucleolus size and number. breast cancer cells differed in erk activity that resulted in different patterns of erk activation/inhibition, phosphorylation status of s6 and autophagy induction upon nutraceutical stimulation. nutraceuticals also affected dna methylation parameters, namely the levels of dnmt1, dnmt3a and dnmt3b that resulted in both global dna hypo-and hypermethylation. taken together, after nutraceutical treatment, nucleolus-centered cellular response was revealed in breast cancer cells of different phenotypic characteristic that may be considered a potential target of anticancer therapy. this study was supported by grant from the national science center, 2013/11/d/nz7/00939. p-09.04.4-023 rate of apoptosis in human macrophages infected with leishmania tropica promastigotes infection of the cells with parasites or exposing cells to heat stress induces a cellular stress. in the present study human macrophages are infected with leishmania tropica promastigotes and exposed to heat stress. the measurement of cytoplasmic histone-associated dna-fragments was carried out using elisa technique. visualization of apoptotic cells was performed by the terminal deoxynucleotidyl transferase dutp nick end labeling staining method (tunel). degree of oxidative stress on cell is evaluated by measuring nitric oxide (no), malondialdehyde (mda), reducte glutathion (gsh) levels and superoxide dismutase (sod) activities. results of the elisa technique showed that infection of macrophages with promastigotes induced apoptosis rate significantly (p < 0.001), heat stress however decreased the rate of apoptosis in infected macrophages remarkably (p < 0.001). high levels of apoptosis rate in infected macrophages and drastic decdrease in apoptosis in heat subjected macrophages infected with promastigotes are confirmed by visualisation of apoptotic cells using tunel method. levels of glutathion (gsh) in infected macrophages decreased significantly (p < 0.05), while malondialdehyde (mda) levels increased notably (p < 0.05). however, no statistical significant alterations were detected in the nitric oxide (no) values and superoxide dismutase (sod) activities. results of the present study indicates that infection of human macrophages with leishmania tropica induces a cellular stress response, characterized by decreased values of gsh and increased levels of mda. increased rate of apoptosis in infected macrophages may be due to the increased cellular stress caused by leishmania tropica amastigotes. decreased rate of apoptosis measured in heat exposed macrophages infected with promastigotes indicates an extention in life span of macrophages. measurements of the parameters characterizing the redox and inflammatory processes in blood are essential for diagnosis and prognosis of type 2 diabetes mellitus (t2dm), but also for monitoring the effectiveness of medical treatments. along with other biochemical effects, hormonal imbalance leads to modified transport function of erythrocytes due to changes in enzyme systems involved in upholding of cellular homeostasis through a rapid degradation of altered proteins, being the second line of defense against the free radicals, which degrade and eliminate the damaged molecules. some of these enzymes are hemoglobin peroxidase (pa) and esterase (ea). the aim of this research study was to identify new parameters with a potential role of biochemical markers in t2dm like hemoglobin peroxidase and esterase activity from erythrocyte. our data showed that pathological processes involved in t2dm imply an increased enzymatic activity of pa (73.85%), which correlates with increased levels of ea (47.9%) and glycated hemoglobin (hba1c) (13.51%), compared with control group. the variables hba1c, pa and ea are correlated: the identified pearson correlation coefficients r = 0.637 and r = 0.573 respectively, have an associated probability of p < 0.001 and p < 0.006 a value that indicates a strong positive correlation between the dependent variables pa and ea and independent variable hba1c. based on these results we concluded that together with glycosylated hemoglobin assay, all the studied parameters can be successfully used as extra test for diabetes associated with oxidative stress and disorders in erythrocyte functions or blood rheology. the radioprotective effects of propolis and caffeic acid phenethyl ester on radiationinduced oxidative/nitrosative stress in brain tissue s. taysi 1 , e. demir 2 , k. cinar 3 1 gaziantep university, school of medicine, gaziantep, 2 haran university, sanliurfa, 3 department of neurosurgery, medical school, gaziantep, turkey head and neck cancer patients treated with radiotherapy suffer severe side effects during and following their treatment. efforts to decrease toxicity of irradiation to normal tissue, organs and cells have led to searching for cytoprotective agent. investigations for effective and non-toxic compounds with radioprotective capability led to increasing interest in antioxidant such as propolis and caffeic acid phenethyl ester (cape). the aim of this study was to evaluate the antioxidant and radioprotective effects of propolis and cape on radiation-induced oxidative/nitrosative stress in the brain tissue. fourty sprague-dawley rats were randomly divided into five groups. group 1 (irradiation (ir) + propolis) received total cranium irradiation and propolis was given orally through an orogastric tube daily. group 2 (ir+cape) received total cranium irradiation plus cape, was dissolved in dimethyl sulfoxide (dmso) just before giving to the rats, intraperitoneally (ip) every day. group 3 (ir) received 5 gy of gamma irradiation as a single dose to total cranium plus 1 ml saline daily. group 4 received daily plain dmso. group 5 received daily plain saline. at the end of the 10 day time period, xanthine oxidase (xo), nitric oxide synthase (nos) activities, nitric oxide (no•) and peroxynitrite (onoo -) levels were significantly higher in ir group compared to all other groups. in conclusion, the results suggest the radioprotective ability of propolis and cape involving prevention of radiation-induced oxidative/ nitrosative damage. p-09.04.4-027 role of leptin and adiponectin in obesityassociated oxidative stress e. becer 1 , a. c ß irakoglu 2 1 near east university, nicosia, cyprus, 2 istanbul university, istanbul, turkey objective: increased oxidative stress is one of the major characteristics of obesity and obesity-related complications. adipokines also induce the production of reactive oxygen species and generating oxidative stress in physiological and pathological conditions of obesity. the aim of this study was to determine the association of leptin and adiponectin levels with body mass index, lipid parameters and oxidative stress biomarkers in obesity. methods: the study included 150 obese and 120 non-obese subjects. plasma leptin and adiponectin levels (ng/ml) were measured using commercially available enzyme-linked immunosorbent assay kits. serum lipid, superoxide dismutase, malondialdehyde and antropometric parameters were measured. results: obese and non-obese subjects did not differ in age, while plasma glucose, total cholesterol, triglycerides, ldl cholesterol and leptin levels were significantly higher and mean hdl cholesterol and adiponectin levels were significantly lower in obese than non-obese subjects. the plasma leptin (p < 0.001) and adiponectin (p = 0.003) levels were significantly correlated with bmi in both obese and non-obese subjects. in obese subjects, leptin levels were significantly correlated with superoxide dismutase (p < 0.01) and malondialdehyde (p < 0.01). strikingly, adiponectin was significantly correlated with superoxide dismutase (p = 0.02) and malondialdehyde (p = 0.0012) levels in obese group. conclusion: our results suggest that leptin and adiponectin levels are associated with defective antioxidant status and increased lipid peroxidation which may have implications in the development of obesity related health problems. clinical trials of biologically active plant substances show a significant preventive effect in cancer, cardiovascular diseases and peptic ulcer disease in the form of both nutritional supplements and therapeutic intervention. anthocyanins contained in dark berries show great antioxidant potential, with the most important including a reduction in oxidative degradation of lipids or tyrosine oxidation by peroxynitrite. our previous studies of the antioxidant properties of extracts from vaccinium corymbosum, aronia melanocarpa and sambucus nigra, however, indicated that their activities largely depend on the method of extraction. while quantitative determination of anthocyanins pointed to a disproportionately larger content of anthocyanins in isolates from lyophylized berries, their scavenging activities against hydroxyl radicals was surprisingly the lowest. inflammatory processes, vascular damage, atherosclerosis and others are caused by oxidativenitrosative stress, so we tested their efficiency to scavenge no degradation products. we found that only purified extracts of lyophilized berries showed the most significant effects against no degradation products, with efficacy of around 50%. an extract from aronia showed greater than 60% efficiency, and a net ethanol extract from all the berries showed a 30% effect. cleaned ethanol extracts showed the lowest effects, while aronia initiated production at a concentration of 25 mg/l. conversely, all acetone extracts consistently initiated no degradation products. these findings are in complete contrast to their determined action against reactive oxygen species. in summary, it follows that a particularly adjusted lyophilized extract of the berries could be responsible for the increased biological activity of no and the observed biological and pharmacological activities of anthocyanins in circulatory disorders. the study was supported by grant vega 1/0782/15 and 1/0018/16. p-09.04.4-032 attenuation of dysfunction, oxidative stress and apoptosis by resveratrol in benzo(a)pyrene exposed ins1-e 832/13 pancreatic beta cell line s. c ß elik, b. baysal faculty of medicine, afyon kocatepe university, afyonkarahisar, turkey diabetes is one of the most important problems in the world. this disease is a very important health problem due to affects many different organs and systems. it has been well established that, environmental pollutants had deleterious effects on glucose metabolism, and caused insulin resistance and type 2 diabetes. with this investigation, it was aimed to investigate the effects of benzo(a)pyrene on pancreatic beta cells and treatment affects of resveratrol. in this study, rat ins-1e beta cell line was used. after reaching the appropriate number of cells culture operations by cell culture, benzo(a)pyrene (20 lm, 24 hours) application have been made after resveratrol (80 lm, 48 hours) application. after incubations oxidative stress, insulin secretion (in cell and in medium), cell proliferation and apoptosis were analysed in cells by biochemical and molecular techniques. b(a)p application resulted in no increase and resveratrol also increased this level of no. resveratrol increased the tas levels decrased by b(a)p, and tos levels were also increased by resveratrol interestingly. osi levels determined with tas and tos levels, has no significant change between groups. gsh levels were decreased by b(a)p while resveratrol increased its' level to control level. mrna expression levels of beta cell functions related genes ins-1, ins-2 and sirt-1 were increased by resveratrol treatment. insulin levels in cell and in medium were increased after resveratrol treatment. mrna expression level of foxo-1 gene was increased while pdx-1 was decreased by resveratrol treatmeant. b(a)p suppressed the mrna expression of p53 gene, but resveratrol increased. the effects of b(a)p on pancreatic beta cells and the protective effects of resveratrol on this cells were investigated in vitro with this research firstly. the results obtained from this research showed that oxidative changes, functional impairment and carcinogenetic effects of b(a) p in pancreatic beta cells could be blocked by resveratrol. the protective effect of vitamin e (alphatocopherol) on ischemia-reperfusion injury in rat liver ischemia-reperfusion (i/r) process is usually used during transplantation and resection of the liver but liver dysfunction and cellular death due to lack of oxygen in tissues, changes in the balance of oxidant/antioxidant in favor of oxidants, and inflammatory response are inevitable during this process. in the present study, it was aimed to investigate whether vitamin e(alpha-tocopherol), an antioxidant agent, has a protective effect against liver ischemia reperfusion injury in rats by using morphometric methods. for this purpose, 21 adult sprague-dawley male rats were divided into 3 groups as; control, ischemia / reperfusion (i/r), and vitamin e+ischemia/reperfusion (evit +i/r). in experimental groups, the total hepatic ischemia was applied for 45 minutes followed by a 24 hour of reperfusion. in the treatment group, 7 days before ischemia 40 mg / kg dose of vitamin e was administered intraperitoneally once a day. after the termination of the reperfusion, the rats were perfused by cardiac way and liver tissues were dissected. following volume and weight calculations, the livers were subjected to the standard histological preparation methods and embedded in paraffin. serial sections at 5 lm thickness were obtained from these blocks, stained with hematoxylineosin, and analyzed with morphometric methods. in light microscopic examinations of the i/r group, irregularity in lobules, dilatation in central veins and sinusoids, extensive areas of necrosis and pycnotic nuclei were seen in hepatocytes. the volume density of sinusoids to liver parenchyma was estimated as 16% in the control group, whereas it was 36% in the i/r group. this value was decreased to 32% in the evit + i/ r group. however, no significant difference was found among the groups in the lobule area calculated by the point counting method. these results show that intraperitoneal vitamin e administration for 7 days prior to ischemia partially inhibits damage caused by ischemia-reperfusion injury in the liver. the leaves, fruit and bark of annona muricata, a member of the annonaceae family, are commonly used in the traditional medicine of tropical and subtropical regions. in recent years, many studies have shown their anti-cancer, anti-convulsant, antiarthritic, anti-parasitic, anti-malarial, and anti-diabetic activities. it should be noted that these characteristics have been described using different types of extracts from different parts of the plant. our studies have focused on the systematic characterization of activities most easily accessible from an aqueous extract of leaves, with hitherto documented antihypertensive and hepatoprotective effects. we found that the extract shows almost 54% efficiency against hydroxyl radicals. with increasing concentrations, the effectiveness weakened, reaching a second peak (40%) at a concentration of 50 mg/l. the scavenging activity against no degradation products maintained a continuously increasing trend with a maximum at a concentration of 100 mg/l. surprisingly, the extract initiated peroxynitrite production in a similar trend, except at 50 mg/l, where it scavenged peroxynitrites with relatively high efficiency, up to 34%. these findings are consistent with the elevated levels of reduced glutathione detected after incubation of liver mitochondria with extract to a maximum concentration of 50 mg/l, with subsequent sharp decline. the activity of glutathione s-transferase was decreased, although not significantly. this indicates a reduction of metabolic processes of compounds, allowing their action over a longer period of time. in a live system, even antihypertensive effects can be observed. however, a significant outflow of gsh to create the gsh adducts of active substances, and particularly s-nitrosoglutathione from increased production of peroxynitrites, can cause liver toxicity. the study was supported by grant vega 1/0782/15 and 1/0018/ 16. the role of polyamine metabolism in curcumin induced apoptosis via reactive oxygene species (ros) generation in growth hormone (gh) overexpressed t47d breast cancer cells r. genc ß, a. coker gurkan, e. d. arisan, p. obakan yerlikaya, n. palavan unsal, n. palavan unsal t.c istanbul k€ ult€ ur € universitesi, istanbul, turkey autocrine growth hormone (gh) signaling triggers cell proliferation, growth, metastasis and drug resistance in cancer cells. polyamines (pas) play an essential role in cell cycle, proliferation, growth and carcinogenesis of various cancer types such as prostate, colon and breast cancer. odc (ornithine decarboxylase) is the key enzyme in the pa biosynthetic pathway that is under control of antizyme (az) and antizyme inhibitor (azi) activity. pa catabolic enzymes polyamine oxidase (pao) and spermine/spermidine acetyle transferase (ssat) by-products triggers reactive oxygene species (ros) generation and apoptotic cell death. curcumin decreased cell viability in dose and time dependent manner in t47d wt and gh+ cells. although forced gh expression induced cell proliferation, 20 lm curcumin inhibited cell invasion. curcumin (20 lm) induced apoptosis by acting on intrinsic and extrinsic pathway in both cell lines. moreover, curcumin supressed odc, azi expression in wt and gh+ t47d cancer cells. although curcumin decreased az expression in t47d wt cancer cell, increased az expression was determined in t47d gh cancer cell. pao and ssat expressions were upregulated in t47d gh+ cells. concomitantly, putrescine levels were increased in t47d gh+ cancer cell compared to wt cells and curcumin depleted spermidine and spermine levels in wt and gh + t47d cells. curcumin induced-apoptotic cell death via ros generation and co-treatment of n-acetyl cysteine (nac) overcame curcumin effect. conclusion, forced gh expression triggers cell proliferation and growth via acting on polyamine metabolism and curcumin-triggered ros generation was prevented by nac treatment in t47d wt and gh+ breast cancer cells. acknowledgment: this study was supported by tubitak 1001 research project (project no: 113z791). the radio-protective effects of propolis and nigella sativa oil on oxidative/nitrosative stress in liver tissue of rats exposed to total head irradiation s. taysi our purpose is to investigate propolis and nigella sativa oil (nso) for their antioxidant effects on the liver tissue of rats exposed to ionizing radiation. a total of 32 sprague-dawley rats were divided into five groups to test the radioprotective effectiveness of of propolis and nso administered by orogastric tube. appropriate control group was also studied. biochemical parameters in liver tissue of rats were determined by spectrophotometer. xanthine oxidase (xo), nitric oxide synthase (nos), superoxide dismutase (sod) activities, nitric oxide (no•) and malondialdehyde (mda) levels were higher in ir group while glutathione-s-transferase (gst), glutathione peroxidase (gsh-px) level in the ir group were lower in this group when compared to the other groups. gst activity in ir plus propolis group was statistically higher than in all other groups. propolis and nso clearly protect liver tissue from radiationinduced oxidative stress. the effects of royal jelly on the antioxidant parameters in the breast tissues of the rats with breast carcinoma treated with paclitaxel or not effects of royal jelly on the breast tissue antioxidant parameters in rats with breast carcinoma treated with paclitaxel or not. 8-12 weeks old female sprague-dawley rats (n = 37) included in current study were divided into 5 groups: control group (n = 8) with healthy rats; breast cancer group (n = 8); breast cancer group (n = 7) treated with 15 mg/kg paclitaxel injection (once a week for 3 weeks); breast cancer group (n = 7) treated with 100 mg/kg royal jelly (by oral gavage for 30 days); and finally breast cancer group (n = 7) treated 100 mg/kg royal jelly in addition to 15 mg/kg paclitaxel injection. rats with breast carcinoma was obtained at 150th days after a single dose injection of 50 mg/kg n-methyl-n-nitrosourea (mnu). all cancer groups were followed by 30 days with treatment of paclitaxel and/or royal jelly. the antioxidant parameters in rat breast tissues, superoxide dismutase (sod) and catalase (cat) activities were determined by spectrophotometric colorimetric methods and glutathione (gsh) by high performance liquid chromatography (hplc). all the antioxidant parameters decreased in breast cancer group without any treatment (p < 0.05). but, statistically non significant increases were observed by paclitaxel and royal jelly treatment (p > 0.05). this study indicated that royal jelly supplementation can not be sufficient to increase the antioxidant parameters in breast cancer. we are going to continue to identify the effects of royal jelly on breast cancer detail. the effects of n-acetylcysteine on microsomal and serum paraoxonase 1 activities at high fat diet induced obese rats obesity is a chronic disease that develops from the interaction between genotype and environmental factors and increase in the accumulation of body fat. it is related with glucose and lipid metabolism disorders, cardiovascular diseases and increased oxidative stress. paraoxanase 1 (pon1) is an enzyme which plays a protective role in oxidative stress, inflammation and liver diseases. it has been suggested that pon1 has protective effects against high fat diet induced obesity and obesity related disorders. n-asetylcysteine (nac) is a potent antioxidant due to its ability to stimulate glutathione synthesis. the aim of this study was to evaluate the microsomal and serum pon1 enzyme activities (paraoxonase, arylesterase and lactonase) at high fat diet induced obese rats in the presence of nac. this study consisted of control, obese and nac-supplemented obese (2 g/l nac) groups. eighteen sprague-dawley rats were randomized into three groups (n = 6). control rats were fed by standart food and obese and nac groups were fed by high fat diet. the microsomal and serum paraoxonase, arylesterase and lactonase activities were determined by colorimetric methods. serum paraoxonase, arylesterase and lactonase activities decreased in obese and nac groups when compared to control groups. on the other hand microsomal paraoxonase, arylesterase and lactonase activities increased in nac group when compared to control and obese groups. however there was no statistically significant difference between the groups. it has been concluded that the microsomal and serum paraoxonase 1 enzyme activities did not change at high fat diet induced obese rats in the presence of n-asetylcysteine. reactive oxygen species, playing an active role in the early and late course of acute pancreatitis, lead to dysfunction of cell membrane and releasing of lysosomal enzymes, and thereby to the injury in pancreatic cells. gallic acid, found in vegetables such as green tea, is an active component which has antioxidant, antiinflammatory, antiviral, anticancer activities. the aim of this study was to investigate the effects of gallic acid in experimental acute necrotizing pancreatitis (anp) model in rats. eighteen male sprague-dawley rats were divided into three groups (6 rats in each group). group 1: sham + saline; group 2: anp induced by intraductal glycodeoxycholic acid and intravenous cerulein; and group 3: anp + gallic acid (100 mg/kg/day, intraperitoneal). at the end of 18th hours, pancreas histopathology was examined. the levels of serum amylase as a diagnostic marker of pancreatitis, interleukin-6 (il-6), total antioxidant status (tas), nitrite + nitrate, total thiols as antioxidant marker and thiobarbituric acid reactive substances (tbars) to measure malondialdehyde (lipid peroxidation product) were determined by spectrophotometric methods. serum amylase, il-6, plasma tbars levels were significantly higher but total thiols levels were lower than sham group in anp group without treatment (p < 0.05). however; tas and nitrite + nitrate levels did not show any significant difference (p > 0.05). on the other hand, while serum amylase, il-6 and tbars levels were lower, total thiols levels higher in gallic acid treatment group than in the untreated anp group, but statistically insignificant (p > 0.05). in conclusion, gallic acid treatment is beneficial but not sufficient to treat the acute necrotizing pancreatitis in rats. p-09.04.4-043 evaluation of oxidant/antioxidant system parameters, il-6 and il-10 levels in amniotic fluid of pregnancies with down sydrome b. _ imge erg€ uder 1 , s. bahsi 1 , t. bahsi 2 , v. topc ßu 2 , a. bakir 2 1 ankara universty faculty of medicinel, ankara, 2 zekai tahir burak research and training, hospital genetic center, ankara, turkey introduction and aim: down sydrome (ds) can be diagnosed at high-risk of down syndrome pregnancies by invasive prenatal testing. in this study we aimed to demonstrate antioxidant/oxidant system markers, il6 and il10 levels in amniotic fluid samples of pregnancies affected by ds. materials and methods: for this purpose we collected amniotic fluid samples from 18 pregnancies affected by down sydrome and 36 normal healthy pregnancies who applied to zekai tahir burak research and training hospital genetic center and were proceeded with amniocenthesis. in the amniotic fluid samples; malondialdehyde (mda), superoxide dismutase (sod), glutathion peroksidase (gsh-px) xhantine oksidase (xo), catalase (cat), adenozine deaminase (ada), nitric oxide (no), nitric oxide senthase (nos) enzymatic activities were evaluated by spectrophotometric methods, il6 and il10 levels are evaluated by elisa. for statistical analysis student's t-test and spearman corralation analusis are used. results: it was found that sod levels are significantly elevated in study group compared to control group (p < 0.05). besides this, in study group, cat and il-6 levels are found singnificantly lower than control group (p < 0.05). we couldn't find any significant difference between two groups in terms of mda, gsh-px, xo, no, nos, ada ve il-10 levels (p > 0.05). discussion and conclusion: there is an important decrease in inflammation compared to normal pregnancie in the amniotic fluid of pregnancies having ds. based on these results, sod enzyme may be used as a marker for prenatal diagnosis of ds. for this purpose these experiments should be tried on larger sample groups. the aim of our work was to compare prooxidant and antioxidant properties of linalool, which is the oxygenated monoterpene compound reported to be a major volatile component of the essential oil of several aromatic species, in hep g2 cells. cytotoxicity of linalool was assayed by celltiter-blue ò cell viability assay. malondialdehyde levels result in membrane damage in hep g2 cells were assayed by using fluorometric method. hep g2 cells were incubated with linalool at 24, 48 and 72 hours. the viability of hep g2 cells decreased in a manner dependent upon concentration and incubation time. the ic 50 values were calculated 81.5 lg/ml (24 hours), 72.7 lg/ml (48 hours) and 64.7 lg/ml (72 hours). but, cell viability of hep g2 cells increased when the cells preincubated with linalool at lower concentrations (˂ic 50 ) against h 2 o 2 cytotoxicity. membrane-damaging effects of linalool were increased with accelerating concentrations. on the other hand, membrane damaging effect of h 2 o 2 was decreased when the cells preincubated with linalool before h 2 o 2 incubation. oxygenated monoterpene linalool had both prooxidant and antioxidant properties showing membrane damaging and protective effects on hep g2 cells depend on concentration. postprandial lipemia is primarily characterized by increasing triglyceride levels after the lipid rich meal. postprandial lipemia may cause oxidative stress by increasing free radical production and increasing oxidative stress could be responsible for the development of many diseases. plasma oxidant-antioxidant status was evaluated in healthy individuals with postprandial hypertriglyceridemia generated by performing oral triglyceride tolerence test (ottt). the study group included 86 subjects (38 female and 48 male). ferric reducing ability of plasma (frap), total thiol and thiobarbituric acid reactive substances (tbars) levels were determined by colorimetric methods at fasting and 2nd, 4th and 6th hours following ottt. the levels of frap and thiol were significantly higher in males than females (p = 0.0001 and 0.042, respectively). thiol levels decreased significantly in both gender at postprandial 2nd, 4th and 6th hours as compared with fasting condition (p = 0.0001). while tbars levels increased at postprandial 2nd hour, that was only significant for male individuals (p = 0.0001). it has been concluded that postprandial lipemia may change oxidant-antioxidant balance in favor of oxidants and gender is an important criteria while assessing the oxidant-antioxidant status in postprandial lipemia p-09.04.4-046 ischemia modified albumin and c-reactive protein levels in prediabetes prediabetes is a state of abnormal glucose homeostasis characterized by the presence of impaired fasting glucose, impaired glucose tolerance, or both. the aim of this study was assess serum ischemia modified albumin (ima) in prediabetes and determine its correlation with other risk factors for chronic complications such as inflammation and hyperglycemia. glucose, insulin, total cholesterol, hdl cholesterol, triglycerides, albumin, c-reactive protein (crp) and ima were measured in 30 patients with prediabetes and 30 controls. prediabetes patients had higher levels of ima and crp in comparison with control subjects but there was no significant difference between groups for ima. significant positive correlation was observed between crp and fasting glucose, insulin. there was no significant correlation between ima levels and the parameters tested. we have shown higher level of crp in prediabetes. these results support the hypothesis that chronic inflammation may be involved in development of hyperglycaemia. p-09.04.4-048 the effects of s _ io 2 nanoparticles of rat uterine smooth muscle specially used in textile field sio 2 nanoparticles on uterus smooth muscle was aimed to be researched. in this study 64 wistar albino female rats were used. rats were separated in 4 groups as control, dose 1 (250 lg/ml), dose 2 (500 lg/ml) and dose 3 (1000 lg/ml). nanoparticle's size was chosen as 20 nm. preparations of four groups were evaluated for biochemical and histological examinations. all isolated uterine smooth muscle stripts except the controls were treated with sio 2 for two hours. in biochemical examinations in order to evaluate oxidative stress level of malondialdehit (mda), activity of superoxide dysmutase (sod) and glutathione peroxidase (gsh-px) were measured. in histological examinations via electron microscope ultrastructure of uterus was examined as well as apoptotic cells detected with immunofluorescent labeling method. intergroups differences were defined by statistical analysis. while mda level increased depending on the dosage, sod level was decreased depending on the dosage. gsh-px rate was decreased for each dosage with respect to control. however, no significant difference is detected between groups. in electron microscopic examination no changes were observed in uterus ultrastructure with compare to control. however, in immunofluorescent labeling it was detected that apoptosis increased in dosage groups with compare to control group. as a result, it was thought that application of sio 2 nanoparticles, in 20 nm size and in 250, 500 and 1000 lg/ml dosages caused of oxidative stress and apoptosis. this results suggested that sio 2 has toxic effects on uterine smooth muscle. uterine myomas are the most common benign pelvic tumors arising from myometrium. they are rarely associated with mortality but responsible for significant morbidity and have adverse effects on quality of life especially in reproductive age women. reactive oxygen species and superoxide dismutases, as well as sex steroids play important roles in the reproductive physiology processes. in addition, oxidative stress and impaired antioxidant defense system have been linked to pathophysiology of various diseases including malignant gynecological disorders. clinical investigations indicate that women with myoma may have increased risk of developing malignant tumors particularly sarcomas. the present study aimed to investigate the possible role of oxidant and antioxidant status in myomas. blood and urine samples of 21 myoma patients were collected. activities of erythrocyte antioxidant enzymes [copper-zinc superoxide dismutase (cuzn-sod), catalase (cat), glutathione peroxidase (gpx1)] and levels of lipid peroxidation biomarkers [plasma malondialdehyde (mda) and urine 8-epi-prostaglandin f 2a (8-epi-pgf2a)] were determined. the results were compared with those of 21 controls. the groups were matched in terms of age, body mass index, smoking habit, coexisting chronic diseases, menopausal status and sex steroid hormone levels. all antioxidant enzyme activities were higher (37% for cuzn-sod, p = 0.003; 52% for cat, p0.05) and the levels of lipid peroxidation biomarkers were lower (%59 for mda, p = 0.011 and 43% for 8-epi-pgf2a, p > 0.05) in myoma patients compared to controls. correlation analyses showed a significant negative correlation between erythrocyte cuznsod activity and plasma mda levels (r = -0.431, p = 0.005). the decreased lipid peroxidation may be the consequence of elevated antioxidant enzyme activities and the data suggests a protective role of activated antioxidants especially cuznsod and cat in patients with myoma. p-09.04.4-050 investigation of ischemia-modified albumin levels in patient with acute limb ischemia introduction: acute limb ischemia commonly occurs as a result of embolus caused by cardiac origin and which may end up with limb loss or even death if left untreated. thrombosis are usually seen where the arteries give branches and tendency to atherosclerosis is more serious at these sites. involvement of several arteries in either embolus or in situ thrombosis limits the adequacy of collateral circulation. restriction of blood flow due to arterial stenosis or occlusion often leads patients to complain of muscle pain on walking. any further reduction in blood flow causes ischemic pain at rest, which affects the foot. early recognition may prevent limb loss or death. ischemia can alter the capacity of the amino terminus of the albumin to bind free metal atoms such as cobalt, copper and nickel. this new, chemically changed albumin is called ischemia modified albumin (ima). ima is a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism, mesenteric ischemia and stroke. therefore, in this study it was aimed to investigate the ima level in acute limb ischemia. materials and methods: in this study, 24 patients with acute limb ischemia (li group; mean age 62 years) and 34 healthy individuals (control group; mean age 42 years) were included. ima levels were detected in control and li group by elisa (organo teknika, avusturya) using ima el _ isa kit. results: ima values were compared with nonparametric methods mann whitney u test, and significantly decreased ima level was statistically significantly different between li group and control group (p < 0.001). conclusion: there is a significant increase in serum ima in limb ischemia, so that alterations also might be clinically useful in the diagnosis of limb ischemia, but should be supported with further studies. object: polycystic ovary syndrome (pcos) is a multifaceted disorder with a pathogenetic pathway that is not fully understood yet. apart from hormonal derangements, insulin signaling defects and adipose tissue dysfunction, oxidative stress, defined as an imbalance derived from excessive formation of oxidants in the presence of limited antioxidants defenses, has been actively implicated in the etiology of the syndrome. the aim of this study was to determine of serum myeloperoxidase activity (mpo), paraoxonase 1 activity (pon1) and arylesterase activity (are) in patients with pcos. material and methods: the study was carried out on 150 women consisted of 103 patients with pcos and 47 healthy ones as control. serum pon1 activities were measured spectrophotometrically using diethyl-p-nitrophenylphosphate as substrate. phenylacetate was used as substrate for are measurement, and are activity was determined by measuring absorbance of the resulting phenol at 270 nm. molar absorptivity coefficients were used in the calculation of pon and are activities as 1 nmol phenol/ml serum/min. result: the mpo and are activities were significantly lower in the patient groups when compared with the control group (72.09 ae 60.66 -119.74 ae 90.81 u/ml p < 0.001, 47.43 ae 12.21 -83.93 ae 26.38 u/ml p < 0.001, recpectively). the pon1 activities are higher in the patient group (145.55 ae 98.23 u/ml) compared to the control group (153.33 ae 101.90 u/ml) are found, but are not statistically significant. conclusion: lower serum mpo and are activities might contribute to the increased susceptibility for the development of diseases risk in women with pcos. because free oxygen radicals are thought to contribute to the complication of many chronic diseases, the pcos may be related to oxidative stress. subclinical hypothyroidism, defined as an elevated serum thyroid stimulating hormone level associated with serum thyroid hormone concentrations within the reference range. free radicalmediated oxidative stress has been implicated in the pathogenesis of thyroid disorders. the ischemia-modified albumin (ima) has been proposed as a marker of protein oxidative damage, which has been found to reflect hypoxic stress. this study aimed to investigate the influence of subclinical hypothyroidism on serum ima levels. thirty-one subclinical hypothyroidism patients and 27 control subjects were enrolled in the study. albumin, ima were measured and ima/albumin ratio was calculated. to determine the ima levels the measurement method based on albumin cobalt binding assay was used. serum ima levels of patients with subclinical hypothyroidism were 0.58 ae 0.08 absu, ima levels of control subjects were 0.49 ae 0.08 absu. ima levels were significantly higher in patients with subclinical hypothyroidism patients than in control subjects (p < 0.0001). when ima values were normalized for albumin concentrations, the ima/albumin ratio was also significantly elevated in patient group compared to control group (p < 0.01). ima levels are increased in patients with thyroid dysfunction. elevated levels of ima can be a clinically useful marker of protein oxidative damage in subclinical hypothyroidism. p-09.04.4-054 the effects on endothelial dysfunction of quercetin in streptozocin-induced diabetic rats excessively produced in pathologic conditions. ultimately, imbalance between oxidants and antioxidants results with oxidative stress (os). in this study, we investigated some os parameters in standard (20% protein, 70% (7% sucrose) carbohydrate, 10% lipid) and sucrose (20% protein, 70% (35% sucrose) carbohydrate, 10% lipid) diet fed bdnf heterozygous mice liver tissues. the male c57bl6 strain wild type (+/+) and bdnf heterozygous (+/à) mice (5 weeks) were obtained. the animals were fed ad libitum by special standard and sucrose diets. twenty four mice were divided into four groups and each group consist six mice. all mice were fed for 16 weeks. first group involved in c57bl6 wild type mice and fed by standard diet. second group contained c57bl6 bdnf heterozygous mice and fed by standard diet. third group consisted c57bl6 wild type mice and fed by sucrose diet. fourth group involved in c57bl6 heterozygous mice and fed by sucrose diet. in first group, mda levels, sod and cat activities were higher than other groups. in second group, cat activities were lower than other groups. but, we could not find any statistically significant differences between all groups about mda, sod, cat levels in bdnf heterozygous mice liver tissues. in conclusion, standard and sucrose diet feeding may not affect mda, sod and cat levels in bdnf heterozygous mice liver tissues. brain-derived neurotrophic factor (bdnf) is member of neurotrophin family which plays critical roles in the development, differention, survival, maintenance of the central and peripheral nervous systems. bdnf also contributes to food intake and body weight control. bdnf heterozygous mice display increased body weight and mild hyperphagia. expression of bdnf is not limited to the brain, it also express some peripheral tissues like adipose tissue, liver, kidney, skeletal muscle, heart. even though roles of bdnf are well known relatively in central nervous systems, effects of this protein is not clear in peripheral tissues. as mentioned before, it is expressed in organs involved in energy, lipid and glucose homeostasis, including the liver, adipose and muscle tissues, but its role there remains unclear. in this study, we aimed to investigate role of bdnf on liver oxidative stress parameters in heterozygous mice model fed fat diet induced obese mice. in this study, we used c57bl/6 mice inbred strain wild type and bdnf heterozygous (+/à) mice. animals were divided to two groups: wild type (n = 5) and bdnf heterozygote mice (n = 5). the animals were fed ad libitum by high-fat diet during 4 month. weight gain was recorded every 15th days. in liver tissues were measured malondialdehyde (mda), superoxide dismutase (sod) and catalase (cat) by spectrophotometric methods. liver mda levels decreased in obese bdnf heterozygous mice compared to obese wild type group and statistically significant difference between groups. bdnf heterozygous mice cat activities were higher than the other group and this difference was statistically significant. there was no statistically significant difference between the groups in terms of sod activities. it has been concluded that the mda levels and sod enzymes activities changed at high-fat diet induced obese bdnf heterozygous mice compared to wild type mice liver tissues. p-09.04.4-060 disturbances of microelements profile in serum of overweight/obese adult females with acute and persistent pro-inflammatory chlamydia pneumoniae infection p-09.04.4-061 determination of reactive oxygen species induced dna damage using modified cupric reducing antioxidant capacity (cuprac) colorimetric method s. uzunboy, s. demirci c ß ekic ß, r. apak department of chemistry, istanbul university, faculty of engineering, istanbul, turkey reactive oxygen species (ros) term is a common name of a group of species. hydroxyl radical and singlet oxygen can be taken into account as ros samples. ros may be formed as a result of endogenous or exogenous reasons. although ros have some beneficial functions, they should be balanced by antioxidants (aox). excessive amounts of ros can attack biological macromolecules including dna. dna damage is usually related with mutagenic and carcinogenic changes. that's why determination of dna damage is so important and there are a great many studies in literature comprising different techniques. one of the most common of them is the 'comet assay'. but application of the method and interpretation of the results is not easy. investigation of certain dna damage products is also very common. these methods usually need expensive instrumentation such as using tandem mass spectrometry. on the other hand, depicting total dna damage on a certain product may cause misinterpretations. in the presented study, dna was decomposed by hydroxyl radicals produced by fenton method. in the study while dna is not cuprac reactive the oxidation products can react with the cuprac reagent. the effect of aox was also investigated. for this purpose, selected aox compounds were added to the reaction medium. because of their radical scavenging effect, the cuprac absorbance decreased in the presence of aox. in the presence and absence of aox, absorbance differences were calculated. the calibration graphs between final concentration and absorbance differences were drawn for each aox. gallic acid was determined as the most effective one among the tested aox samples. for statistical comparison with the presented study, tbars was used as reference method. direct use of dna as a probe material to determine oxidative damage may be an advantage to understand dna hazard in biological systems. the proposed method can be applied in all laboratories having a spectrometer as a cost-effective and simple procedure. p-09.04.4-062 effects of alpha-1 antagonists on oxidative system of rat heart tissue benign prostate hyperplasia is a progressive process occurring in the stromal and epithelial components of the prostate. alpha-1 receptor blocking agents are used for relaxation of the smooth muscles in the prostatic stroma. our aim was to investigate the effects of alpha-1 antagonists on oxidative system of rat heart tissue. 33 male wistar albino rats were divided into 5 groups randomly. 1) tamsulosin (1 mg/kd/day), 2) terazosin (5 mg/kg/day), 3) doksazosin (25 mg/kg/day), 4) alfuzosin (10 mg/kg/day), 5) control. all drugs were administered every other day single doze via oral. rats were sacrificed after 30 days. heart tissue was taken for biochemical analysis. malondialdehyde (mda), nitric oxide (no), protein carbonyl (pc) levels and superoxide dismutase (sod), glutathione peroxidase (gsh-px) enzyme activities were determined in supernatant samples. there was not an significant difference between terazosin, doxazosin, alfuzosin, tamsulosin groups in means of sod, mda and gsh-px levels. no levels were significantly different between tamsulosin group and the control group (p = 0.004). in addition, tamsulosin group and terazosin group were also significantly different (p = 0.032). according to these results we can say that tamsulosin group had higher no levels than control and terazosin group. tamsulosin's enhancer effect on no levels leads to relaxation of the heart muscle and vascular relaxation, and so fewer side effects than other alpha antagonists. the effect of rat liver tissue radical metabolism and the protective role of hippophae rhamnoides l. on cold and immobilization stress model cold and immobilization stress is a widely used model for study the changes that occur on oxidant-antioxidant balance. hippophae rhamnoides l. (seabuckthorn; sbt) a unique and valuable plant has recently gained worldwide attention, mainly for its medicinal and nutritional potential. this study was aimed to investigate the protective role of sbt which is a natural herbal product with high antioxidant content on oxidative and nitrosative stress induced by cold and immobilization stress in rats. 32 wistar albino rats were divided into 4 groups randomly. control (i.p. physiological saline), sbt (i.p. 200 mg/kg/48 hours sbt), stress (i.p. physiological saline; 6 hours cold + immobilization stress) and sbt + stress (i.p. 200 mg/kg/48 hours sbt; 6 hours cold + immobilization stress) groups were formed. 3nitrotyrosine levels were determined by elisa whereas total antioxidant capacity, total thiol, total glutathione, nitrite-nitrate levels and superoxide dismutase and glutathione peroxidase activities were measured by colorimetric methods. sbt + stress group nitrite-nitrat (p = 0.0001), total glutathione (p = 0.0001) levels and glutathione peroxidase activities (p = 0.0001) were found to be significantly higher whereas superoxide dismutase activity was found to be lower (p = 0.007) when compared to stress group. there was no significant differences between stress group total thiol and total antioxidant capacity levels compared with stress + sbt group. stress + sbt group oxidative and nitrosative stress marker 3-nitrotyrosine level was found to be significantly higher when compared with control group (p = 0.002) whereas there was no significant differences between stress and stress + sbt groups. all this data show that sbt has antioxidant properties on cold and immobilization induced oxidative and nitrosative stress in rat liver tissue. obesity is a major health problem with growing incidence and accompanying complications. its relation with diminished cognitive functions was reported. this study aims to evaluate the effects of obesity induced oxidative stress and metabolic alterations on the cognitive functions of children and adults. 33 children and adolescents with obesity (age: 8-18); and 33 age and gender matched healthy subjects were enrolled. all subjects completed the battery tests of cnsvs via computer. the scores were compared by using commercial software (ibm spss statistics 19). biochemical parameters, malondialdehyde (mda) and protein carbonyl (pc) levels were estimated. mda and pc levels were significantly higher in subjects with obesity (0.78 ae 0.16 lmol/l;198.30 ae 84.45 nmol/ml) than the controls (0.5 ae 0.10 lmol/l; 125.35 ae 43.52 nmol/ml) (<0.001). there was statistically significant difference between study and control groups on all cognitive performance domains. significant correlation was detected between mda, pc levels and the cognition indexes. children with obesity should be evaluated for the cognitive functions, together with the metabolic follow-up. obesity induced oxidative stress may be the reason of the diminished cognition in children as well as the changes in the lipid profile and inflammation, but we need larger study groups to lighten these complex process. p-09.04.4-067 relative contribution of nitric oxide synthase (nos) isoforms to oxidative/nitrosative stress in the cerebral cortex of rat with acute liver failure (alf) acute liver failure (alf) is associated with deregulation of nmda/cgmp/no signaling and oxidative/nitrosative stress in the brain. however, the relative roles of the different nos isoforms and the mechanisms underlying alterations in their activities during alf are not fully clear. here we investigated gene and protein expression of nos isoforms, nos activity, enos uncoupling and total no production in cerebral cortex of rats with thioacetamide (taa)-induced alf. sprague dawley rats (250-280 g) received three i.p. injections of taa (300 mg/kg) at 24 hours intervals. the brain cortex expression nos isoforms (enos/inos/nnos) was measured by real-time pcr and western blot, nos activity was tested by monitoring the conversion of radiolabeled arginine to citrulline. reactive oxygen species (ros) were quantified in the presence of nos substrate l-arginine, using the carboxy-h 2 dcfda probe. no was measured with the griess procedure. the enos expression was decreased, whereas the enos dimmer/monomer ratio and nnos/inos expression were elevated in taa treated rats. while the total nos activity was decreased, the inos activity was elevated and no concentration tended to increase. ros production was elevated by taa. unspecific nos inhibitors l-name and l-nna attenuated ros production in both control and taa rats, but with higher efficiency in the latter case. ca 2+ chelation had almost the same effect as pharmacological nos inhibition suggesting that ca 2+ -independent inos activity is not the main source of ros. incubation with high dose of tetrahydrobiopterin (bh4) with which is critical for enos dimerization and subsequent no production also reduced ros production indicating the enos uncoupling phenomenon in taa cortex. the study points to enos downregulation due to lowered protein expression and uncoupling as a novel mechanism contributing to enhanced superoxide o 2 anion formation, and confirms the role of inos/nnos in enhancing no synthesis in alf-affected brain. introduction: diabetes mellitus (dm) is an endocrine disorder of world which is characterized by altered blood glucose levels and related complications including hepatic injury. myrtus communis l. (mc) is widely used by diabetic patients in the folk medicine of turkey as well as they are used worlwide. it is known that of leaves, oil and fruit of myrtus communis l. (mc) have therapeutic effects on diabetes mellitus (dm). this study was aimed to analyze the possible antidiabetic and hepatoprotective effects of mc berries in streptozotocin (stz) induced diabetic rats. materials and methods: a total of thirty rats composed of six groups as each included five rats were used. 40 mg/kg stz was injected once to animals to induce dm. after stz injection, rats were exposed to three different ethanol extracts of mc berries (250, 500 and 1000 mg/kg) by oral gavage during 14 days. alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels were determined in serum and glutathione (gsh), malondialdehyde (mda) levels and superoxide dismutase (sod) activity were determined in liver tissue. results: mc administration provided significant reducement in the altered serum glucose, ast and alt levels in all diabetic groups. mc extract showed significant antioxidant activity by altering sod activity and gsh level and reducing mda levels in diabetic rats compared to controls (p < 0.05). serum ast and alt levels were reduced by mc administration in all diabetic groups. mc administration provided significance increment in sod activity and gsh level, and significant reduction in mda levels compared to controls (p < 0.05). the maximum hypoglycemic and antioxidant effects were observed at 1000 mg/kg dose of mc. background: human serum paraoxonase 1 (pon1) is a calcium dependent esterase that hydrolyzes organophosphates and also arylesters such as phenyl acetate. pon1 prevents ldl oxidation by hydrolyzing lipid peroxides. pon1 is inhibited by various chelating agents, heavy metal ions, and sulfhydryl reagents. in our study we investigated the effect of calcium on ldl oxidation of purified pon1 q192r isoenzymes. methods: pon1 q192r isoenzymes were partially purified from human serum. both allozymic forms were treated by preincubation with 1 mm edta for 15 minutes. ldl oxidation was induced by copper ions. formation of thiobarbituric acid-reacting substances (tbars) was used as a measure of lipid peroxidation. homocysteine thiolactonase (htlase activity) and arylesterase activities were measured spectrophotometrically by using homocysteine thiolactone and phenylacetate as the substrates. results: addition of 1 mm edta to partially purified hdl-pon1 q192r isoenzymes inhibited 100% of htlase and arylesterase activities. inactivation of pon1 for arylesterase/htlase activity by the addition of edta did not reduce the abilities of both allozymic forms in protecting ldl from oxidation. conclusion: ca +2 -dependent inhibition of pon1 q192r arylesterase/htlase by using the metal chelator edta, did not alter pon1's ability to inhibit ldl oxidation. pon1's ability to protect ldl from oxidation may not require calcium. p-09.04.4-070 evaluation of cholinesterase inhibitory effect, anti-radical and anti-lipid peroxidation activities of mentha pulegium i. hamad 1, 2 1 college of applied medical sciences, aljouf university, aljouf, saudi arabia, 2 faculty of medicine, bahri university, khartoum, sudan introduction: many studies indicated that intake of dietary and medicinal plants is effective in preventing or suppressing many diseases, therefore, there is a growing interest in plant'sbioactive compounds. mentha pulegium, is widely used in gulf countries in herbal teas or as additives in commercial spice mixtures for many foods to offer aroma and flavor. the aim of this study is to investigate the in vitro radical activity, the total phenol and flavonoid content, anti-lipid peroxidation and the cholinesterase inhibitory effects of mentha pulegium methanol extract. methods: the acetylcholinesterase and butyrylcholinesterase inhibitory potentials of extracts, were evaluated by colorimetric assay. the in vitro antioxidant activity was measured by dpph assay, the total phenols content was measured by folin-ciocalteau assay, the flavonoids content by the alcl 3 colorimetric method, and the protective effect of menthe mentha pulegium extracts against lipid peroxidation was evaluated using a liposome oxidation system. results: the methanol extract showed a scavenging activity nearly equivalent to vitamin c which is attributed to its high phenolic and flavonoid contents. the extract possessed protective effect against lipid peroxidation in a dose dependent manner. the methanol extract shows very little anticholinesterase activity as compared to the standard compound, physostigmine. conclusion: results presented here indicate that mentha pulegiumpossess strong antioxidant activity and protective effects and they can therefore be used as a natural additive in food, cosmetic and pharmaceutical industries. type 2 diabetes mellitus is a long term metabolic disorder that is characterized by hyperglycemia and insulin resistance. because of the hyperglycemia and free radicals, diabetes can cause cellular instability. micronuclei is a sensitive indicator of genetic damage and a marker of dna damage. micronuclei is also a morphological marker of chromosomal instability. in this study, we aimed to evaluate the frequencies of micronuclei in papanicolaou stained buccal cells of type 2 diabetic patients. a total of 30 type 2 diabetic patients and 30 healthy individuals were involved into our study. buccal smear samples that belong to these individuals were stained by using papanicolaou method for cytologic examination and the stained slides were evaluated by light microscopy (olympus bx-51). cells with micronuclei in each papanicolaou stained buccal smear sample were counted under light microscopy. the frequency of micronucleated epithelial cells were seen as significantly higher in type 2 diabetic patients than the control group (p < 0.05). one of the boron compounds is sodium perborate tetrahydrate (nabo 3 .4h 2 o), which the most widely used solid peroxygen compounds. it is used in safety bleach formulations, detergents and tooth powders. as known these products are commonly used in daily life. however, the actions on blood antioxidant defenses of sodium tetraborate against reactive oxygen species are not identified yet. it is reported that oxidative stress caused by ros damages. in this study, we searched enzyme activities of superoxide dismutase (sod), catalase (cat), glutathione-s-transferase (gst), glutathione reductase (gr), glutathione peroxidase (gsh-px) and glucose-6-phosphate dehydrogenase (g6pd) also the effect sodium perborate tetra hydrate on activities of these enzymes from human erythrocyte under in vitro conditions. according to our findings sodium perborate tetrahydrate caused significant (p < 0.05) increasing in the cat activity from red blood cell. the other antioxidant enzyme activities (sod, gst, gr, gsh-px and g6pd) did not show any changing by influence of sodium perborate tetrahydrate. metabolism of obese individuals could be exposed to risk of chronic low-grade pro-inflammatory effect and oxidative stress. some inflammatory and oxidative markers have been studied recently. plasma total antioxidant status (tas) and total oxidant status (tos) parameters can be non-invasive markers of diseases such as fatty liver disease, laparoscopic procedures (pneumoperitoneum), accompanying inflammatory condition like urinary tract infection, diabetic neuropathy, chronic hepatitis. the study groups have been comprised of two groups with normal to over-weight children. tas and tos levels were detected and the oxidative stress index (osi) was computed as a marker of the grade of oxidative stress. the over-weight group displayed higher levels of fasting glucose, insulin resistance, the body mass index. also, we know that insulin resistance leads to increased lipolysis and free fatty acid output. higher tos as well as crp is related to the group, also lower tas than other group is shown. crp levels in plasma were positively correlated with insulin and glucose levels. in addition, there was a significant relationship between osi and insulin resistance in the over-weight group. tas and tos are together more accurate sings of oxidative and antioxidative status of people. as well as a raise over weight-related subclinical inflammation and a fall antioxidant capacity is significant even in children. this condition may eventually develop the risk of long-term vascular damage. the effects of hydrogen peroxide pretreatment on antioxidant enzyme activities in calli tissues of two eggplant genotypes under salinity o. yasarkan 1 , e. aky€ uz 1 , g. baysal furtana 2 , s. s. ellialtioglu 3 , r. tipirdamaz 4 1 nezahat g€ okyigit botanic garden, istanbul, 2 department of biology, faculty of sciences, gazi university, ankara, 3 department of horticulture, faculty of agriculture, ankara university, ankara, 4 department of biology, faculty of sciences, hacettepe university, ankara, turkey the effects of hydrogen peroxide (h 2 o 2 ) pre-treatment on catalase (cat) and superoxide dismutase (sod) were investigated and lipid peroxidation measured as malondialdehyde (mda) content of the calli from salt-sensitive (artvin) and salt-tolerant (mardin) eggplant genotypes under salinity stress. the seeds from each genotypes were germinated on ms medium for 4 weeks and hypocotyl tissues from these plantlets were used as explants for calli induction on ms medium including 1 mg/l 2,4-d and 0.1 mg/l kinetin. as for the pre-treatment, the subcultured calli tissues were transplanted on the mediums containing 50 and 100 lm h 2 o 2 for 48 hours and then transplanted on the mediums including 150 mm nacl for 24 hours. antioxidant enzyme analysis and mda measurement was carried out for the control, nacl-only, h 2 o 2 -only and h 2 o 2 pre-treated tissues. pre-treatment with h 2 o 2 decreased the deleterious effects of salt stress on mda contents. in comparison with salt stressed groups, h 2 o 2 pre-treatment with or without nacl reduced mda content especially in artvin. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. comparing two genotypes, a decrease was observed on sod activity in artvin genotype and an increase in mardin genotype by comparison of salt stressed groups. higher increase on sod activity was observed in 100 lm h 2 o 2 + nacl groups on each genotypes. the result showed pre-treatment of 100 lm h 2 o 2 induced acclimation of the plants to salinity. in addition, 100 lm h 2 o 2 pre-treatment, as a stress signal, could trigger the activation of antioxidant enzymes in calli and in this way alleviated the oxidative damage in calli growth under salinity. the investigation of effects of ghrelin and cannabinoid cb1 receptor agonist and antagonist on oxidant and antioxidant mechanisms on brain tissues of penicillininduced epileptic rats the aim of this study is to investigate the individual effects of ghrelin and cannabinoid type1 (cb1) receptor agonist acea, the antagonist am-251 and the interaction of these two different systems on oxidant and antioxidant systems in the brain, cerebellum and brain stem tissues of penicillin-induced epileptic rats. in this study 56 male wistar albino rats were used weighing 20-250 g. each group was consisted of 8 rats. study groups: 1: control, 2:penicilin(500 iu), 3:penicillin(500 iu) + ghrelin(1 lg), 4:penicillin(500 iu) + am-251(0.25 lg), 5:penicilin (500 iu) + acea(7.5 lg), 6:penicillin(500 iu) + am-251(0.25 lg) + ghrelin (1 lg), 7:penicillin(500 iu) + acea(7.5 lg) + ghrelin(1 lg). than the levels of mda, gpx and sod are measured in plasma and tissue samples of these rats. penicillin was found to be induced lipid peroxidation in the brain, cerebellum and brain stem tissues in our study. ghrelin and acea, which both have anticonvulsant effects, were shown to be effective in reversing the oxidative damage caused by penicillin and proconvulsant am251 was found to further increase the oxidative stress caused by penicillin in these tissues. ghrelin also was found to suppress the oxidative stress caused by am251 in the cerebellum tissue but it did not contribute to antioxidant effects produced by acea. since the role of oxidative stress in epilepsy has been established, it may be suggested that ghrelin and acea may have anticonvulsant effects via their antioxidant features. the discovery of inhibitors for enzymes that metabolize endogenous ghrelin and cannabinoids through new studies may contribute to the improvement of seizure resistance in epilepsy. accelerated atherosclerosis in patients with ankylosing spondylitis (as) give rise to increased cardiovascular morbidity and mortality. endothelial dysfunction could be the initial process in the development of atherosclerosis. human endothelial cell-specific molecule-1 (endocan) is a novel human endothelial cell-specific molecule. therefore, we assessed serum endocan levels and carotid intimamedia thickness (cimt) as a surrogate marker of atherosclerosis in patients with as. a total of 57 patients with a diagnosis of as according to newyork ctriteria and 50 control subjects were included in our study. serum endocan, interleukin-6 (il-6), tumor necrozis factor-a (tnf-a), c reactive protein (crp) and cimt were measured in all participants. serum endocan, il-6, tnf-a levels were measured with elisa. the other parameters were done by routine biochemical methods. as patients exhibited increased serum endocan levels and cimt compared to matched controls (p < 0.05). whereas, serum il-6, tnf-a were similar between grous. in patient with as, there were no significant differences between active and inactive patients by means of il-6, tnf-a, endocan and cimt. in as group, cimt correlated with disease duration and age (r = 0.597, p = 0.000; r = 0.721, p = 0.000). we could not find any significant correlation between serum endocan levels and parameters studied. our study shows increased cimt in as patients without traditional risk factors such as increased bmi, lipid profile compared to controls. although we found increased circulating endocan levels in patients with as, the other factors could affect increased atherosclerosis in this population because of lack of correlation between endocan and cimt. probable biomarkers could be related to increased cimt in patients with as should be investigated in larger study groups. keywords: ankylosing spondylitis, atherosclerosis, carotid intima media thickness, endocan p-09.04.4-079 investigation of pentose phosphate pathway and oxidative stress in erthrocyte infected babesia ovis a. bildik, t. karagenc ß, p. a. ulutas, n. aysul, h. aksit adnan menderes university, aydin, turkey introduction: babesia infections occur in cattle, sheep, goat, horse, dog, cat pig and rodents. in this study, the effects of babesia ovis living and present in the erythrocytes to glucose metabolism was researched. at the same time, biochemical parameters were also associated with parasitemia. materials and methods: babesia ovis (israel) cell culture was provided from dr. abel martin gonz alez oliva (portugal). culture passaged 48 or 72 hours according to parasitemia state (8-10%). biochemical analyses were performed in erythrocyte culture in which parasitemia between 1% and 16%. cell counts and hemoglobin concentration of erythrocytes culture suspension were measured at cell counter instrument and than it was washed 3 times with physiological saline, erythrocyte suspensions were stored at-80oc analysis. gssg (oxidize glutathione), gsh (reducte glutathione), nadph, glukoz 6 p dehydrogenaz, gshpx (glutathione peroxidase), gshrx (glutathione reductase) were determined by commercial kits. all experiments were done in duplicate, the results were calculated by the number of erythrocytes. results: parasitemia was positively correlated with gsh, nadph and gshrx (p < 0.05). a correlation between other biochemical parameters was not observed. discussion: the pentose phosphate pathway in erythrocytes has an important role such as to provide pentose sugar required for the synthesis of nucleic acid, to reduce glutathione, to produce nadph and to protect from methemoglobin accumulation. in studie sthat naturally infected erythrocytes with babesia parasites, it was seen to be caused to oxidative stress, however gsh results in these investigation were obtained differently . conclusion: according to the results of this study that performed in vitro, it can be suggest that their glutathione metabolism and pentose phosphate pathway of parasites may active.key words: babesiosis, gsh, gssg, nadph, g6pdh, gshpx, gshrx p-09.04.4-080 in vitro protective effect of betaine on peroxidative injury caused by ethanol and aspirin exposure on rat brain synaptosomes i. sogut 1 , g. kanbak 2 1 istanbul bilim university vocational school of health services, istanbul, 2 eskisehir osmangazi university medical school department of biochemistry, eskisehir, turkey aspirin intake of specific daily doses are advised by doctors to postmenapausal women and men above 40 years of age to prevent heart attack and even cancer in recent times. in this study, the aim is to investigate the in vitro cytototoxic effects of different doses of ethanol (50 mm, 100 mm ve 200 mm) alone and together with 100 lg/ml aspirin, and possible protective role of 1 mm betaine on rat brain synaptosomes. 15 male sprague dawley rat forebrains were divided into equal pieces and pooled to form 10 study groups. synaptosomal fractions extracted from pooled rat brains were incubated with different doses of ethanol, aspirin and betaine, and malondialdehyde (mda) levels, an important indicator of cellular damage, were measured. a significant increase (p < 0.05) was observed in mda level of 200 mm ethanol group compared to control group. different doses of ethanol (50 mm, 100 mm ve 200 mm) + aspirin exposure significantly increased (p < 0.001) mda levels compared to controls, whereas betaine administration significantly decreased (p < 0.001) lipid peroxidation caused by ethanol + aspirin treatment. we conclude that ethanol and ethanol + aspirin administration increases lipid peroxidation in rat brain synaptosomes while betaine helps prevent this peroxidative membrane injury.keywords: aspirin, betaine, ethanol, malondialdehyde (mda) p-09.04.4-081 analyses of mitochondrial biogenesis in hepatocellular carcinoma treated with berberine f. aygenli, h. c ß imen yeditepe proteomics and mass spectrometry group (yediprot), genetics and bioengineering, yeditepe university, istanbul, turkey objective: berberine (bbr) has been demonstrated to have anticancer activities against various cancer types, particularly hepatoma. in this project, we aimed to reveal the effect of bbr treatment on mitochondrial biogenesis through sirtuins and hif-1a in hepatocellular carcinoma cell line, hep3b under hypoxia. method: hep3b cells were subjected to normoxia (21% o 2 ) and hypoxia (1% o 2 ) in the presence or absence of bbr treatment. the amount of bbr was optimized via cell viability (mts) assay under normoxia. then, immunoblotting experiments were performed to identify the effect of bbr on hif-1a, pgc-1a, and sirtuins involved in mitochondrial stress. the variation in the oxphos complexes and the level of reactive oxygen species (ros) were also measured to investigate the effect of bbr on mitochondrial energy stress state. results: here, we present that cell viability was significantly decreased at 25 lm. bbr treatment has shown significant reduction in hif-1a and sirt6 which responsible for up-regulation of glycolysis. also, succinate dehydrogenase (cii) and cytochrome c oxidoreductase (ciii) of the oxphos complexes were downregulated without any change in nadh dehydrogenase (ci) or atp synthase (cv). bbr significantly abolished to oxidative stress under hypoxia, which was demonstrated as a reduction in the level of reactive oxygen species by decreasing on sirt3 expression. bbr induces the overexpression of sirt1 and its deacetylated-pgc-1a, which might be an indicator of being a potent protective agent against hypoxia by normalizing mitochondrial function and inducing mitophagy in impaired mitochondria caused by deficiency of glycolysis and oxphos. conclusion: detailed information about the communication between hif-1a and sirtuins and their relation to mitochondrial energy production was provided with the alteration of their activity by bbr treatment. it is highly expected that bbr and its derivatives might become important during the development of supplemental therapies. introduction: reactive oxygen species are involved in a variety of biological phenomena, such as carcinogenesis, inflammation and aging. among the targets of ros, dna appears most important in tumor biology since it is firmly established that cancer is a genetic disease. ros induce several kinds of dna damage, including strand breakage and dna-protein cross-linkage. fruit of zizyphus jujuba, a traditional chinese herb widely consumed in asian countries, has been reported to possess several vital biological activities. this study intends to evaluate their antioxidant activity on glioblastoma cells. materials methods: cell survival was quantified by colorimetric mtt assay. human gliblastoma cells were pretreated with 100 lm h 2 o 2 after 30 minutes 100 lm ziziphus jujuba essential oil was added to the cells for three hours. then, the cell homogenates were taken and glutathione, total oxidant and total antioxidant capacity and nitric oxide levels were estimated using spectrophotometric methods. results: ziziphus jujuba treated cells could prevent intracellular glutathione from being depleted following an exposure of h 2 o 2 . also our data suggest that ziziphus jujuba is effective in preventing h 2 o 2 induced oxidative stress and nitric oxide levels. discussion: some research showed that h 2 o 2 was over produced in the pathological process of acute and chronic neuronal toxicity, the toxicity effect of b-amyloid on the cultured neuron and neuronal cell line was mediated by h 2 o 2 . the traditional medicine recommend several medicinal plants for providing relief from various inflammatory diseases. many research has been reported that the essential oil from seeds of helping to prevent the oxidative stress and neuronal diseases in brain. introduction: toxicity by oxygen radicals has been recommended as a major cause of cancer, heart disease, and aging. oxygen radicals and other oxidants appear to be toxic in large part because they start the chain reaction of lipid peroxidation. most of the analytical techniques for peroxide determination are generally time consuming and not very suitable for routine or on line analysis. we aimed to design a new biosensor for rapid determining of oxidant agent hydrogen peroxide. materials and methods: all reagents were of analytical grade unless stated otherwise, and were purchased from sigma aldrich. firstly, the 2-hidroxymetacrilate metacriloamidoscystein nanoploymers were immobilized by binding covalently with sulphur atoms on the gold electrod's surface. free nh 2 groups of catalase enzyme make schiff bases between nanopolymer's carbonyl groups, then immobilization was actualysed with cross linking reagent glutaraldehyde. we developed a biosensor system preparing ferrociyanide, selected as a mediator, in the buffer solution. results: polyhemamac nanopolymer and catalase complex were immobilized by glutaraldehite to construct a hydrogen peroxide biosensor. the responses of the biosensor are therefore proportional to the oxidation peaks of the complex at +0.019 v potential. the cyclic voltammograms obtained from the experiments showed that, pottasiumferrociyanide mediator complex positively affected the biosensor responses for hydrogen peroxide determination. discussion and conclusion: as a result of this study, the method developed by the catalase enzyme electrode was found to be more advantageous in comparison to other methods reported in the literature so far; it was determined that the method is sensitive, economic, practical and less time-consuming. since biosensor technology provides economical, practical, specific and sensitive results for the determination of hydrogen peroxide, it was improved very efficiently. p-09.04.4-085 impact of amoxicillin, gentamicin and cefazolin sodium antibiotics on antioxidant gene expression and enzymatic activities in mouse liver p. g€ uller 1 , h. budak 2 , m. sisecioglu 2 , m. c ß iftci 3 1 department of chemistry, faculty of science, atat€ urk university, erzurum, 2 department of molecular biology and genetics, faculty of science, atat€ urk university, erzurum, 3 department of chemistry, faculty of arts and sciences, bing€ ol university, bing€ ol, turkey reactive oxygen species (ros) are highly reactive molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. living organisms have the antioxidant defence systems to block harmful effects of ros. the imbalance between oxidants and antioxidants is termed oxidative stress. the antioxidant defence mechanisms are divided into two groups as enzymatic and nonenzymatic defences. enzymatic defence mechanisms consist of enzymes like superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), glucose-6-phosphate dehydrogenase (g6pd) and glutathione s-transferase (gst). the present study was designed to determine the effects of gentamicin, amoxicillin and cefazolin sodium antibiotics on the hepatic antioxidant system and to determine any possible correlation between enzymatic and molecular levels. for this reason, effects of these antibiotics on the transcription of the antioxidant system has been investigated by real time pcr, and then the enzyme activity of these enzymes have been measured in whole liver homogenate obtained from control group and the drug administered groups mice. our results demonstrate that administering antibiotics led to crucial inhibition of all antioxidant enzyme activity. while significant transcriptional activation for sod and cat was seen in the gentamicin treated group, the transcription of gst and g6pd was decreased. however transcriptional activation was seen for sod and cat in amoxicillin administered group, the transcription of gst was decreased as compared with the control group. in the cefazolin sodium treated group, while cat and gst transcription were elevated significantly, the expression of sod and g6pd were decreased. in conclusion, gentamicin, amoxicillin and cefazolin sodium affect the hepatic antioxidant system at the molecular and protein level. this work was supported by scientific research project of ataturk university of turkey (grant no: 2013/296). p-09.04.4-086 protective effects of curcumin supplementation on oxidant/antioxidant system changes created by organic phosphorus pesticide poisoning organic phosphorus pesticides (opp), widely used in agriculture or as insecticides in home, cause adverse health effects. chlorpyrifos is one of the most commonly used opp. we aimed to investigate the possible protective effects of curcumin (cur) supplementation, the principal curcuminoid of turmeric, on poisoning symptoms and oxidant/antioxidant system changes caused by chlorpyrifos. adult sprague-dawley rats were used. cur (30, 100 and 300 mg/kg) were administered orally for 5 days. on the sixth day, chlorpyrifos (279 mg/kg, s.c.) was administered. twenty four hours after chlorpyrifos administration, body weights, locomotor activities and body temperatures of rats were measured. following the measurements, rats were decapitated and the blood, brain and liver tissue samples were taken and prepared for the biochemical and histopathological measurements. chlorpyrifos administration increased the malondialdehyde (mda) levels but decreased catalase (cat), superoxide dismutase (sod), glutathione reductase (grx) concentrations and reduced/oxidized glutathione (gsh/gssg) ratio in the blood samples, brain and liver tissues compared with the control group (p < 0.05-0.001). the concentration of advanced oxidation protein products (aopp) were increased only in the brain tissue after chlorpyrifos administration (p < 0.001). cur administration reduced all of these changes (p < 0.05-0.001). similarly, cur at the doses of 300 mg/kg reduced the decreases in body weight, body temperature and locomotor activity with chlorpyrifos (p < 0.001). additionally, the histopathological damage scores induced by chlorpyrifos (p < 0.05-0.01) were decreased by the administration of cur (p < 0.05-0.01). our findings suggest that cur supplementation can ameliorate the poisoning effects of chlorpyrifos via supporting the antioxidant mechanisms and cur could be used for protective purposes against oxidative stress and tissue damage caused by chlorpyrifos. the effect of ogtt applied for screening in pregnancy on adenosine deaminase and xanthine oxidase activity in normal and prediabetic pregnant women z. c. ozmen, k. deveci, i. benli department of biochemistry, gaziosmanpasa university medical faculty, tokat, turkey objective: it was reported that the activities of adenosine deaminase (ada) were different in normal pregnant women and pregnant women with gestational diabetes mellitus (gdm). it was also stated that the activity of xanthine oxidase (xo) was increased in pregnant women with gdm. the objective of this study was to evaluate if glucose have effects on oxidative stress in prediabetic women by affecting ada and ox after 50 g ogtt which was applied in pregnant women for screening. methods: the serum specimens of 39 pregnant women who applied to the outpatient clinic of the obstetrics and gynecology department and had 50 g ogtt, were used in this study. ada and xo activities were analyzed in the serum specimens taken from the normal (n = 20) and prediabetic pregnant women (n = 19) in the 0th and 60th minutes of ogtt. ada and xo activities were measured with the spectrophotometric method and the u/l enzyme activity was calculated. findings: there was no significant difference between the 0th and 60th minutes regarding the ada activities in the normal and prediabetic pregnant woman groups. however, we observed a significant difference between 0th and 60th minutes regarding the xo enzyme activity in normal pregnant women (p = 0.001). in normal pregnant women, the median xo enzyme activity in the 0th minute was 0.29 (0.11-1.33) u/l and it was 0.8 (0.25-2.26) u/l in the 60th minute. nevertheless, there was no correlation between the xo activity and glucose level. as to the prediabetic pregnant women, there was no significant difference between the xo enzyme activities in 0th and 60th minutes. the results of our study showed that the xo activity increased as a response to ogtt in the normal pregnant women compared with the prediabetic pregnant women. this finding made us think that the oxidative stress caused by ogtt did not affect the xo response in prediabetic pregnant women and that there would be some adaptive mechanisms against the chronic exposure to high level glucose. rainbow trout (oncorhynchus mykiss) aquaculture continuously increases in turkey. the objective of the present study is to increase the productivity in fish farming of rainbow trout just via intervention in physical cases without the effects of any chemicals and investigate whether this conditions cause oxidative stress. in this experiment eight tanks were used and 44 rainbow trout larvae were placed in each tank and these tanks were illuminated with light in different wavelengths; natural sunlight, and incandescent long-wave (red light), medium-wave (green light) and shortwave (blue light) led lights. the experiment took 64 days. biochemical changes in rainbow trout exposed to light in different wavelengths (red, green, blue) were analysed via the variations in gr, gst, g6pd, gpx, sod and cat enzyme activities, which are significant for enzymatic antioxidant defence system and in ache activity, which plays an important role on central nervous system. maximum activity change in liver tissue was observed for gst and g6pd enzymes in fish grown under green light and for sod enzyme in fish grown under blue light. in gill tissue, sod and g6pd activities were affected the most, and in brain tissue, these were gst and sod activities. it was observed that the average weight of the fish increased 1.2 times under red and blue lights and 1.3 times under green light. the highest weight increase was observed under green light, however, antioxidant enzyme activities increased in the liver and gills and decreased in the brain tissue under this light condition. in conclusion, it was observed that productivity was 1.2 times under red light when compared with control group and it was determined that the fish grown under red light can tolerate oxidative stress more than other wavelength. p-09.04.4-089 effect of nigella sativa on biliary obstructioninduced oxidative stress and apoptosis in rats human safety concerns, since that these agents may not only cause acute toxicity via inhibition of acetylcholinesterase but they can also induce delayed toxicity in the nervous system. a key interest to the current work is the potential correlation between gene expression and cytoskeletal protein changes in differentiating neural cells exposed to sub-lethal neurite outgrowth inhibitory concentrations of specific ops, which was addressed by analysing the underlying changes in the levels of cytoskeletal gene expression and protein levels. to assess the molecular effects of op exposure, phenyl saligenin phosphate (psp), chlorpyrifos (cpf) and its metabolite chlorpyrifos oxon (cpfo) were applied at the point of induction of differentiation of rat c6 glioma and mouse n2a neuroblastoma cells and incubated for 24 hours. at sub-lethal concentrations (1, 3, 10 lm) all three ops used in this study were able to inhibit the development of neurites with no significant effect on cell viability, as determined by neurite outgrowth and mtt reduction assays. to understand the possible effects of ops on cytoskeletal gene expression, primers for genes encoding glial fibrillary acidic protein (gfap), biii tubulin, growth associated protein 43 (gap43) and neurofilament heavy chain (nefh) were optimized for qpcr analysis and the levels of the corresponding proteins were detected by western blot analysis. exposure to ops caused in most cases a reduction in the levels of cytoskeletal proteins, and the results from qrt-pcr analysis also indicated reductions in the gene expression of gfap in c6 cells, and of nefh and biii tubulin in n2a cells, in a dose dependent manner. thus, the observed changes in protein levels are at least partly due to altered gene expression. curcumin is extracted from a perennial herbaceous plant known as curcuma longa. in recent years, considerable interest has been focused on curcumin due to its use to treat a wide variety of disorders without any side effects. earlier studies have shown that curcumin has anti-apoptotic, anti-inflammatory, antiproliferative, antiangiogenic, anticancer and antiplatelet activities. the goal of the present study was to investigate the effects of curcumin on peroxy radical-induced oxidative changes in human platelets. 15 healthy volunteers were enrolled in the study. none of the study participants were on anticoagulation therapy. citrated venous blood samples were centrifuged at 200 g for 10 minute to obtain platelet-rich plasma (prp). the platelet pellet was washed and suspended with tris-nacl buffer. then, platelets were incubated with h 2 o 2 absence and presence of curcumin (50-500 lg/ ml) for 1 hours at 37°c. to determine the preventive effects of curcumin on the oxidative stress and apoptosis induced by peroxy radicals in human platelets were determined by measuring levels of of lipid peroxidation, total antioxidant capacity, caspase 3, 8 and 9 activities, and mitochondrial membrane potential. additionally, we also studied the effects curcumin on platelet aggregation induced by adp. pre-treatment of platelets with curcumin caused a marked reduction in oxidative stress, activation and apoptotic markers in a dose-dependent manner. on the other hand, pre-treatment of platelets with increasing doses of curcumin resulted in inhibition of platelet aggregation induced by adp. in the light of our findings, we suggest that curcumin may have a therapeutic potential to prevent platelet activation related disorders. people have been using mushrooms in the treatment of diseases as well as food, for centuries. most of the edible and inedible mushroom species were used in important medical studies and their effects were begun to be used in the treatment of diseases. this study focuses on the hepatoprotective effects of tricholoma anatolicum, which is endemic specie in turkey, against oxidative stress based on hydrogen peroxide (h 2 o 2 ) on hepg2 liver cancer cell line. t. anatolicum used in this study was extracted with the help of ultrasonication and fraction methods. then the cytostatic effects of extracts on hepg2 cells were explored and their hepatoprotective effects were determined. moreover, various concentrations of aqueous extract (ehta) of t. anatolicum were determined by hepg2 cells's 24-48-72 hours effect analysis on their cellular morphology, xtt and real-time cell analysis in of xcelligence device. ehta extract's cell pathway (apoptosis and necrosis) effects on hepg2 cells were determined with flow cytometry method with the help of annexin v-apc and 7aad fluorescent dye. finally, the phenolic compounds found in ehta extracts were determined with the help of hplc methods. according to xtt cytotoxicity analysis, the ehta extract values were determined as follows: 24 hours ic 50 > 2000 lg/ml, 72 hours ic 50 . furthermore, according to the real-time cell analysis made with xcelligence, ehta extracts were found to be; 24 hours ic 50 = 241.539 lg/ml, 48 hours ic 50 = 418.135 lg/ml, 72 hours ic 50 = 285.694 lg/ml. increasing concentrations of ehta extracts were determined to direct hepg2 cells to apoptosis. moreover, considering the hplc analysis -according to the reference point of 100 mg in 100 g sample-within ehta extracts, catechins and vanillic acid peaked. the final results revealed that t. anatolicum's effect on hepg2 was cytostatic at low doses, and cytotoxic at high doses. p-09.04.4-093 relationship between serum ceruloplasmin levels and coronary blood flow background: there is growing evidence that oxidative stres plays an important role in the development of the slow coronary flow (scf) phenomenon. ceruloplasmin (cp) is a copper containing metalloenyzme which has antioxidant functionthrough its ferroxidese 1 activity and is associated with cardiovascular diseases. we aimed to investigate the relationship between scf and serum cp level. methods: patients who underwent elective coronary angiography and had no significant epicardial coronary disease were included in the study. patients who had thrombolysis in myocardial infarction frame counts (tfcs) above the normal cutoffs were considered to have scf and those within normal limits were considered to have normal coronary flow (ncf). a total of 90 patients (55 subject as scf and 35 subjects as ncf) were analyzed. 5 ml blood samples were taken from the groups to study ceruloplasmin activity. serum ceruloplasmin levels were determined spectrophotometrically. results: the serum cp levels were statistically lower in scf group than in the ncf group (414 ae 79 versus 469 ae 99 ng/ml, p = 0.009). also there was a significant correlation between serum cp levels and tfcs (r = à0.378, p = 0.013). conclusion: the findings of this study suggests that patients with scf had lower serum cp levels correlated with tfcs. we concluded that reduced serum cp levels might represent a biochemical marker of scf. introduction: sleeve gastrectomy (sg) has been used for the surgical treatment of morbid obesity, as a first step or definitive treatment. alterations of thyroid hormones in gastrointestinal surgery were previously studied. the aim of the present study was to determine the effects of triiodothyronine (t3) supplementation on oxidative stress parameters in anastomotic tissue level. materials and methods: twenty-four male wistar albino rats were divided into control (n 12), and experimental (n 12) groups. rats were underwent a sg, with a hand-sewn suture. experimental group rats received a single dose of t3 (400 mg/100 g) in postoperative day. rats were sacrificed on postoperative day 7. serum thyroid stimulating hormone (tsh), free t3 (ft3), and free thyroxine (ft4) were analysed using elisa. each tissue was homogenized in ice-cold pbs (ph: 7.4) and centrifuged at 2000 rpm for 20 minutes (4°c) to avoid contamination with cellular debris. the supernatants were used to measure total oxidant status (tos), total antioxidant status (tas), nitric oxide (no) and malondialdehyde (mda) levels. all tissue parameters were analysed by spectrophotometric methods. oxidative stress index (osi) values were calculated. results: rats given t3 hormone had not decline in ft3 levels compared with the control groups. a significant decrease in ft4 levels was found in t3 given rats on postoperative day 7. whereas tissue tos levels did not alter by thyroid hormone treatment, tas levels significantly decreased. osi values were not statistically different in tissues. tissue no levels were also similar in both groups. mda levels increased in t3 given rats compared with the control group. discussion and conclusion: this study showed that anastomosis after sleeve gastrectomy is associated with decreased ft4 level. although tos levels and osi values were similar in both groups, t3 supplementation induced lipid peroxidation by increasing tissue mda levels that might deplete tissue antioxidant level. reactive oxygen species (ros) are reactive chemical molecules, which are produced by living organisms as a natural byproduct of the normal metabolism and environmental factors. although intracellular ros level is essential molecules for the signal transduction pathways, elevated intracellular level of ros leads to oxidative stress that causes damage to dna, proteins and lipids. therefore, excessive ros levels have to be eliminated by antioxidant defence systems. tip60 (tat interacting protein, 60 kda) is a histone acetyltransferases (hats) that catalyses multiple functions in metabolism such as dna repair, apoptosis, etc. we thought that if tip60 has a role in signal transduction and apoptosis, it might have direct or indirect relationship with the antioxidant system. the present study was designed to determine the impact of tip60 gene on the hepatic antioxidant enzymes including superoxide dismutase (sod), catalase (cat), glutathione peroxidase (gpx), and glutathione reductase (gr) both gene and protein level. for this reason, quantitative gene expression analysis on the antioxidant system has been investigated by real time pcr, quantitative protein expression has been investigated by western blot analysis, and then the activity of these enzymes have been measured in whole liver homogenate collected from control and liver-specific tip60 conditional knockout mice. additionally, since any change of reduced glutathione (gsh), oxidized glutathione (gssg), malondialdehid (mda), and hydrogen peroxide (h 2 o 2 ) level in the cell might be an indication for the accumulation of ros, the relative levels of them were also studied. our data showed that the absence of tip60 affects the antioxidan system both gene and protein level. in conclusion, our initial data suggest that tip60 may be essential for the (ros) homeostasis and redox regulation. curcumin is a major chemical component produced from the rhizome of the plant curcuma longa. lt has been demonstrated that curcumin has an antioxidant, anti-inflammatory, and antiproliferative effects and, protects tissues against ischemia/reperfusion (i/ r) injury. i/r has detrimental effects on transplanted organs including uteri. the major consequence of l/r injury is oxidative stress leading to the generation of ros. uterine transplantation (ut) has been gaining popularity around the worid in the past few years. the aim of our study was to examine the antiapoptotic effects of curcumin on uterine l/r injury. the rats were randomized into three groups of seven rats each, group i consisted of rats that did not receive any treatment, group ll exposed to 0.5 hour of lschemia and 1 hour of reperfusion, group iii of rats that received intraperitonealy curcumin (200 mg/kg) 0.5 hour before the induction of i/r. then, the rat uterine tissue levels of mda, tac, and activities of caspase 3, 8 and 9 were measured. furthermore, the apoptotic index was determined immunohistochemically by the tunel method using light microscopy. biochemical analysis results showed that curcumin decreased the mda and caspase-3 and 9 ieveis, and increased the uterine tissue levels of tac but, caspase 8 activity did not changed by curcumin suggesting that curcumin induces apoptosis via intrinsic apoptotic pathway. on the other hand, an high apoptotic index was observed in i/r group (27.37 ae 11.56%) and decreased after treatment with curcumin (8.69 ae 7.49%). in conclusion, we demonstrated the protective effect of curcumin on apoptosis immediately after reperfusion induction in uteri and we can say that curcumin could improve ir injury and decrease apoptotic index. we propose that curcumin may be a novel approach for improvement of uteri i/r injury. glutathione and the related enzymes belong to the defence system of the tissues against chemical and oxidative stress. these enzymes especially glutathione s-transferase are often overexpressed in tumor cells and are regarded as a contributor to their drug resistance and are thought to play an important role in cancer progression. the purpose of this study is to evaluate the protective effects of chlorophylline as an antioxidant molecule which has inhibitory effects on gst p1-1 on chemically-induced breast cancer model. in our previous work, we had observed that this molecule led to proliferation in breast cancer cells. in this study, n-methyl-n-nitrosourea (mnu) used for inducing carcinogenesis in eighteen, 21-day-old female sprague-dawley rats. chlorophylline and mnu solutions were injected intraperitoneally when the rats were 21, 28, 35 and 42 days old. their weight and tumor diameters were measured throughout the 5 months study period. at the end of the study, all animals were sacrificed and determined both glutathione levels and related enzymes activities (gluathione s transferase, glutathione reductase and glutathione peroxidase) in tumor and tissues such as liver, kidney, heart and spleen were studied and analyzed. as a result, in breast cancer model, glutathione and related enzyme activities were protected by chlorophylline treatment whereas mnu made them decreased compared to the control group. in conclusion, chlorophylline with antioxidant features decreased the toxic effect of mnu by regeneration of glutathione and enhancement of its related enzyme activities. the use of antioxidant molecules, because of proliferative effects and defence-oriented behaviours, should be discussed in cancer therapy. p-09.04.4-100 effect of overexpression of bacillus catalase on lactococcus lactis nisin production z. girgin ersoy, s. tunca gedik gebze technical university, kocaeli, turkey nisin, has been used commercially (e234) in food preservation for approximately 40 years. it's the only bacteriocin which is approved by world health organization as a food additive. the fundamental problem that limits nisin usage in food preservation is low product yield by producer strains. because of high commercial potential of nisin, studies about increasing the production efficiency of nisin is kept in the forefront in recent years. since nisin biosynthesis and bacterial growth are occuring in parallel to each other, conditions that promote growth are also expected to encourage nisin production. it is known that, when supplied with exogenous heme, lactococcus lactis cells can respire under aerobic conditions and produce higher energy which in turn cause higher biomass. however, aerobic conditions also cause oxidative stress since catalase enzyme, which detoxify hydrogen peroxide, is absent in l. lactis. in this study, to complete the missing component of the defence mechanism of l. lactis, catalase (kate) gene of aerobic bacterium bacillus subtilis was overexpressed in facultative anaerobe l. lactis cells. for this, kate gene of b. subtilis was amplified by polymerase chain reaction (pcr). plasmid constructions were established in e. coli by using an e. coli-l. lactis shuttle vector and then the recombinant plasmid was transferred to l. lactis cells by electroporation. the presence of catalase activity in the recombinant strain grown on the solid medium was first detected by dropping hydrogen peroxide directly on the cells, then with enzyme assays. fermentation studies are going on to determine nisin production of the recombinant strain. to the best of our knowledge, this study presents the first preliminary results that shows the effect of overexpression of catalase gene on nisin production. cancer is among the leading causes of morbidity and mortality worldwide. chemotherapy is one of the major cancer treatment strategies. remarkably, natural products have garnered increased attention in the chemotherapy drug discovery field because they are biologically friendly and have high therapeutic effects. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of humic acid. the present study investigated anticancer effects of ha in several human cancer cell lines. ha was purchased from sigma-aldrich. in this study, we used several human cancer cell lines: the human breast cancer cell line, mcf-7, colon cancer cell line, ht-29, lung adenocarcinoma cell line, a549, and servical cancer cell line, hela. the cells were maintained in dmem medium supplemented with 10% heatinactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ ml, 10 ug/ml, 5 ug/ml) were prepared using a stock solution of ha. the cell proliferation and migration was measured. on the other hand, the apoptotic mechanisms induced by ha in cancer cells were investigated using "apoptosis antibody array kit". the effects of ha on cancer cell lines were evaluated over 72 hours. according to our results, ha induced a decrease in ht-29, a549 and hela cell numbers in a dose-dependent and time-dependent manner. contrary to this, ha induced proliferation of mcf-7 cells in dose dependent manner. ha inhibited cell migration in a dose dependent manner except mcf-7 cell line. it was also determined apoptotic pathways in cancer cells induced by ha. it was concluded that ha has an inhibitory effect on certain some cancers. since the effect of ha on tumor progression is unknown, further studies are needed to clarify the rol of ha on cancer activity. p-09.04.4-102 chronic immobilization stress in rats: fluoxetine and amisulpride protects against chronic immobilization stress-induced biochemical alterations in the present study, the effects of amisulpride and fluoxetine on serum total sialic acid (tsa) and lipid bound sialic acid (lsa) levels was investigated in the rats exposed to chronic immobilization stress. the study was administered using 40 male wistar albino rats weighing 200-250 g. rats were divided into five groups (n = 8/ group). group i comprised the control group, group ii was exposed with saline + immobilization stress (30 minutes daily immobilization stress for 15 days and 0.5 ml saline was administered perorally 30 minutes before immobilization), group iii was exposed amisulpride (10 mg/kg/day) + immobilization stress, group iv was exposed fluoxetine (10 mg/kg/day) + immobilization stress and v. group was exposed amisulpride (10 mg/kg/ day) + fluoxetine (10 mg/kg/day) + immobilization stress. statistical analysis showed that the saline + stress, amisulpride + stress and amisulpride + fluoxetine + stress groups was significantly higher than the control group with regards to tsa levels (p < 0.05). whereas, the fluoxetine group was significantly lower than the group regarding tsa levels (p < 0.05). on the other hand, saline group was significantly higher than the control group with regards to lsa level (p < 0.05). whereas, no significant differences in lsa levels were observed in the amisulpride, fluoxetine and amisulpride + fluoxetine groups, as compared to the control group (p > 0.05). the present study demonstrated beneficial effect of fluoxetine and amisulpride on the concentration levels of lsa and tsa in stress. p-09.04.4-104 protective effect of borax and boric acid on total sialic acid and lipid-bound sialic acid levels against 3-methylcholanthrene and benzo(a)pyrene induced oxidative stress in rats s. ekin 1 , g. oto, f. g€ ok, y. karakus, d. yildiz 1 y€ uz€ unc€ u yil university, van, turkey the present study was performed to investigate total sialic acid (tsa) and lipid bound sialic acid (lsa) levels as possible in vivo chemoprotective effect of borax (bx) and boric acid (ba) again-st3-methylcholanthrene (3-mc) and benzo(a)pyrene (b(a)p) induced oxidative stress in rats. the rats were divided into nine groups of six rats each. group i: control, untreated animals were given % 0.9 nacl, group ii: the b(a)p were administered 25 mg/kg via ip. four times. group iii: the 3-mc-treated animals were administered 25 mg/kg via ip. four times, group iv: ba was given 300 mg/l/day with water. group v: bx was given 300 mg/l/day with water. group vi: b(a)p 25 mg/kg via ip four times + ba 300 mg/l/day dosage with water. group vii: 3-mc 25 mg/kg via ip four times + ba 300 mg/l/day with water. group viii: b(a)p 25 mg/kg via ip four times + bx 300 mg/l/ day dosage with water. group ix: 3-mc 25 mg/kg via ip four times + bx 300 mg/l/day with water. the experimental period was continued for 150 days. statistical analysis showed that the 3-mc + ba group was significantly higher than the control group with regards to tsa and lsa levels p < 0.001, p < 0.001,p p-09.04.4-105 effects of aluminum exposure on trace elements in rat tissues b. ozturk kurt, s. ozdemir department of biophysics, cerrahpasa medical faculty, istanbul university, istanbul, turkey aluminum (al) is the most abundant metal and the third most abundant element in the earth's crust. people are constantly exposed to al which is found in most rocks, soils, waters, air and foods, due to a result of an increase in industrialization and improving technology practices. the study was designed to examine the possible effects of aluminum exposure in different durations on trace elements in rat tissues. twenty-four healthy male wistar rats weighed 180-200 g were randomly divided into three groups: control group (gc) received only drinking water, short-term group (gs) and long-term group (gl). the study groups were orally exposed to 40 mg/kg body weight alcl 3 in drinking water for 8 and 16 weeks, respectively. at the end of the treatment period, rats were sacrificed and the kidney, liver, brain and cerebellum tissues were removed to analyse the levels of al, ar, b, ni, si, cr, cu, fe, mg, mn, se, cu and zn by icp-oes. the statistically significant increase were determined in cerebellum al, cu, as, b and cr levels in gl according to the gc. while as levels were statistically increased, ni levels were decreased in gl in the kidney and liver. while cu, mg and cr levels were higher, se and b levels were lower in the gs than gc in the brain. there were no significant difference in si and mn levels. as a result of our study, it may be concluded that al accumulation may lead to changes in tissue trace element levels. tacal, o., 266 tacal, ö., 321 take, g., 412 takic, m.m., 417 taldykbayev, z., 416 talim, b., 292 tamashevski, a., 382 tamer, f., 144 tamer, l., 245, 279, 395 taneva, s., 209 taniyan, g., 275, 306 tanrisev, m., 288 tanriverdi, e.c., 245 tanriverdi, k., 279 tarhan, m., 161 tarhan, t., 315 tartar, s., 387 tas, a., 204, 293 taskin, a., 406 taskin, t., 339 taskiran, e., 271 taskiran, b., 328 taskoparan, b., 221 taspinar, r., 301, 302 tastan, ö., 271 tatli, ö., 340 tauraite, d., 206 tay, t., 344 tayman, c., 354 taysi, s., 389, 392 teker, h.t., 270 tekes, s., 361 tekin neijman, s., 410 tekin, m.h., 188, 297 tekin, g., 191, 256 tekin, m., 196 tekin, n., 155 tekin, g., 256 telci, d., 187, 307 telefoncu, a., 151 temel, h.e., 158, 159, 160 temelie, m., 267 temizgül, r., 347 temlyakova, e., 168 teplova, v., 372 terashima, r., 284 tercan avci, s., 197 terekhov, s., 381 tereshenko, o., 304 terzi gulel, g., 149 terzi, e., 300, 302 terzi, e., 301 terzioglu, g., 296 terzioglu, o., 272 testoni, g., 348 tetik vardarli, a., 282 tevdoradze, t., 148 tevzadze, l., 148 tezcan, ö., 308 tezcanli kaymaz, b., 282 thielens, n., 228 thomaidou, d., 265 thomas, a., 228 thornton, j.d., 216 ticea, a.c., 168 tikhonova, a., 287 tileva, m., 209, 210 timofeev, v., 198, 202 timofeev, v., 202 timofeeva, e., 383 tipirdamaz, r., 401 tiryaki, m., 300, 302 tok, m., 140 tokay, e., 365 toker, a., 360, 402 tokgun, o., 284 toksoy öner, e., 138 tokyol, ç., 390 toman, r., 317 tomasi, a., 410 tombul, n., 333, 337 tooke, c., 231 topaloglu, h., 292 topbas, m., 200 topcu, b., 247 topcu, c., 292 topcu, g., 144, 358, 377 topçu, v., 393 topcu, c., 417 topçuoglu, c., 193, 416 topel, h., 197, 264 toprak, b., 223 toprak, m.s., 351 torac, e., 384 tosner, z., 383 tosun, m., 308, 354 toy, h., 143 toymentseva, a., 239 tozkoparan, b., 246 trabulus, d.c., 256 trantirek, l., 229 trantirkova, s., 229 trchounian, a., 165, 324 trizna, e., 200, 289 troshagina, d., 371 tro t, m., 269 truncaite, l., 149, 363 tsakalidou, e., 170 tsarkov, d., 233 tsarkova, m., 233, 235 tsigara, e.g., 206 tsigara, m.g., 206 tsverava, l., 266 tsvetkova, e., 253 tsyba, l., 362 tsyganov, d., 158 tsymbal, d., 165 tüfekçi, a.r., 241, 358 tufekci, a.r., 296 tufenkci, h., 139 tuglu, m.i., 300, 309 tuglu, i., 200 tuli, a., 142, 182, 237, 403 tuli, a., 352 tulubas, f., 247 tunali, g., 226 tunca gedik, s., 143, 408 tunca gedik, s., 221 tunçdemir, m., 396 tuncel, h., 378 tuncel, h., 273 tunçer, s., 222 tuncer, e., 161, 261 tuncer, z.s., 395 tuncer, b., 133 tuncer, e., 273 tuncez akyurek, f., 185 tunçez akyürek, f., 359 tuner, h., 333 tüney kizilkaya, i., 325 tural, b., 315 tural, s., 315 turan, i., 330 turan, m., 161, 261 turan, v., 173 turan, y., 333 turan, c., 411 turano, p., 217, 290 türel, s., 329, 341 turhan, t., 193, 242 turhan, y., 316, 317 turk, s., 373 turkan, a., 219 türkcan kayhan, c., 135 turkekul, k., 299 türkel, s., 176 turkeri, o.n., 245 turkeri, n., 141 turkkan, b., 257 turkmen, s., 401 türkmen, n., 180 turkon, h., 223 tutar, y., 161 tüten, a., 351 tutkun, e., 196, 275 tüylü küçükkilinç, t., 285 tuz, m.u., 321 twardovska, m., 335 tyapkina, o., 371 tzartos, s., 268 photoprotective activity of vulpinic and gyrophoric acids towards ultraviolet b-induced damage in human keratinocytes evaluation of the sunscreen lichen substances usnic acid and atranorin pleiotropic anticancer activity of selected nutraceuticals against mcf-7 bucharest, 3 national institute for marine research and development "grigore-antipa uk in some adult and elderly populations the acute and/or persistent infection with the common intracellular respiratory pathogen chlamydia pneumoniae (chl) may be associated with increased risk of developing obesity or cardiovascular disorders. thus, 19 microelements modifying oxidative stress status were determined by icp-ms/ms in the hno 3 diluted serum samples collected from chl-positive adult females (n = 39) living in urbanized area in poland. chl infection was confirmed by igg+ antibody elisa and real-time pcr assay. all females were classified under their body-mass index values to the normal-weight (nw), over-weight (ow) and obese group (ob) although there are many drugs currently used in the treatment of peptic ulcer, such a drug providing radical treatment without side effects is not available. since oxidative stress is involved in peptic ulceration, this study was designed to investigate antiulcerogenic and antioxidant effects of hippophae rhamnoides l. ether extract on indomethacine-induced stomach ulcer in rats. materials and methods: thirty-five sprague dawley male rats (weights ranging 180-220 g) were randomly divided into 7 groups, as each composed of 5 rats. after hippophae rhamnoides l. leaf ether extracts of 100 mg/kg, 250 mg/kg and 500 mg/kg doses and 20 mg/kg doses of famotidin orally administered, 25 mg/kg doses of indomethacine were orally applied to rats in order to make ulcer. on the sixth hour of indomethacin administration all rats were sacrificed using thiopental (50 mg/kg). the stomachs were removed, and ulcer areas were evaluated macroscopically. superoxide dismutase activity (sod), glutathione (gsh) and malondialdehyde (mda) levels in stomach tissues of rats were determined by elisa method with respective kits conclusions: we can conclude that the ether extract of hippophae rhamnoides l. leaves reduces free radical formation and has antiulcerogenic effects on stomach tissue control group; 2 hours torsion/4 hours detorsion group (t/d); all other groups were saturated for four days egcg, cape and egcg+cape (10lml/kg). sections were taken from bouine's-fixed and paraffin-embedded testicular tissue blocks and stained with h&e. immunohistochemistry was applied for the detection of pi3k, akt and mtorc. intensities were evaluated as mild (1), moderate (2) or strong (3). serum 8ohdg, plasma mda levels were analyzed using elisa method. results were analyzed by anova statistical test. testis samples in control group exhibited normal histological morphology. disorganization and separation of seminiferous tubule cells and accompanying interstitial edema and vessel dilation were while mda level decreased significantly in cape+egcg group, 8ohdg level showed significant increase in cape group. in conclusion, cape and egcg exerted protective effects on tt. effects may be achieved through pi3k/ akt/mtorc pathway involved in cell proliferation, angiogenesis, apoptosis. prophylactic use of egcg prior to tt surgery improved testicular morphology, therefore could prevent destructive effects of tt lmol/l), c18 (0.58 ae 0.278 lmol/l)), respectively. conclusions: this study is important for konya region, mitochondrial fatty acid b-oxidation disorders studies subject areas. this study is the first study to assess acylcarnitine levels of patients living in our region. we believe that our results will be useful for future studies. key words: acylcarnitine, mass spectrometry, dried blood spot p-09.04.4-137 binding of fas and cu(ii) ions to hsa changes its cys34 thiol group antioxidant capacity and carbonylation pattern with methylglyoxal binding of cu(ii) ion (0.1 mol/mol hsa) led to increase of k' value if fish oil extract was present, but for other fas k' value decreased. the content of free hsacys34-sh decreased for 10% after cu(ii) ion binding, and during 24 hours incubation at 37°c, it was further decreased for 10% (stearic acid, mixfas) and 20% (myristic, fish oil extract, oleic acid). carbonylation of fa-hsa-cu(ii) complexes with mg (20 mol/ mol hsa), lead to decrease in cys34-sh content depending on fa present: 30%-35% for myristic and stearic acid, 40% for oleic acid and mixfas and 40% for fish oil extract. carbonylation of fa-hsa-cu complexes could contribute to further enhancement of oxidative and carbonyl stress in diabetes as well as other diseases 151 pirto ek p-05.03.3-007 anti-proliferative and inducing apoptosis of the hydro alcholic achelia. wilhelmsii extract on human breast adenocarcinoma cell lines mcf-7 and mda-mb-468 background: vitamin d deficiency is associated with several conditions and/or diseases like inflammation, atherosclerosis, cardiovascular disease and mortality. several studies showed that lower vitamin d levels were associated with high serum levels of inflammatory biomarkers. ykl-40 is a glycoprotein, secreted by macrophages, neutrophils and different cell types. it is also associated with inflammation and pathological tissue remodeling. in this study, we aimed to evaluate relationship between the vitamin d deficiency and ykl-40 levels. methods: our study group includes 45 subjects with vitamin d deficiency (group 1) and 40 age and sex-matched healthy subjects with normal serum levels of vitamin d (group 2). plasma 25 (oh) vitamin d levels were measured with liquid chromatography-tandem mass spectrometry (lc-ms/ms) method. plasma ykl-40 analysis was performed by elisa. serum hs-crp levels were measured with nephelometric method. results: plasma vitamin d levels below 20 ng/ml were accepted as vitamin d deficiency. although we could not find any significant differences by means of serum hs-crp levels between groups (p > 0.05), plasma ykl-40 levels were significantly higher in group 1 than group2 (p < 0.05). conclusions: in literature, vitamin d deficiency is associated with inflammation. in our study, we found similar hs-crp levels between groups and higher ykl-40 levels in group 1. vitamin d deficiency may be related to increased ykl-40 levels in terms of causing chronic inflammation.keywords: vitamin d deficiency, ykl-40, inflammation. evaluation and comparison of tnf-family ligands and receptors genes in mice and humans by bioinformatics techniques stance called plaque builds up inside the coronary arteries. apelin is a novel endogenous peptide with inotropic and vasodilatory properties and is the ligand for the angiotensin receptor-like 1 (apj) receptor. we aimed to determine genotype and allele frequencies of apj receptor a445c gene polymorphism in turkish patients with cad and healthy controls by rflp-pcr. this study was performed on 159 unrelated cad patients and 62 healthy controls. we obtained aa, ac and cc genotype frequencies in cad patients as 41.5%, 49.1% and 9.4%, respectively. in the control group, frequencies of genotypes were found as 35.5% for aa, 48.4% for ac and 16.1% for cc. we did not observe difference in apj receptor a445c polymorphism between cad patients and healthy controls (v 2 = 2.178; df = 2; p = 0.336). the a allele was encountered in 66% (210) of the cad and 59.7% (74) of the controls. the c allele was seen in 34% (108) of the cad and 40.3% (50) of the controls. allele frequencies were not significantly different between groups (v 2 = 1.57; df = 1; p = 0.225). the frequencies of apj receptor a445c genotype were not significantly different between control and patients. individuals with cc genotypes had significantly higher weight, systolic and diastolic blood pressure levels and systolic blood pressure than other genotypes, p ≤ 0.05. in addition, hdl-c level was found decreased, but this reduction was not statistically significant. contrarily, the low levels of weight, sbp, dbp and tc were statistically significant in the subjects with aa genotype in cad. in conclusion, cc genotype carriers may have more risk than other genotypes in the development of hypertension in cad. we suggest that this polymorphism may not be a marker of cad, but it may cause useful in function of the apelin/apj system and may be a genetic predisposing factor for diagnostic processes and can be helpfull in finding new treatment strategies. p-08.01.4-032 comparative genomics/proteomics analyses of single amino acid repeat containing proteins across different vertebrate taxa a. g. keskus, o. konu department of molecular biology and genetics, bilkent university, ankara, turkeyconsecutive runs of single amino acids lead to overrepresentation of certain physicochemical properties in protein sequences. researchers also demonstrated a link between single amino acid repeat (saar) containing proteins and neurodegenerative diseases as well as biological functions. moreover, saar frequencies were shown to vary across species based on selected orthologous proteomes and/or proteins. hence, analysis of whole proteomes across multiple vertebrate taxa may provide additional species-and sequence-specific trends for saars. in addition, there is a need for testing the observed saar occurrencesthe aim of this study is to evaluate the effect of quercetin (q) on liver injury secondary to cerulein induced-acute pancreatitis (ap). for this reason, rats were randomly divided into four groups (8 rats for each group) control group received physiological saline, four time and dimethyl sulfoxide, two time, at 1 hours intervals, intraperitoneally (i.p.). cerulein group received cerulein (50 lg/ kg-rat weight, in physiological saline), four times, and dimethyl sulfoxide (1%), two times, at 1 hour intervals, i.p. quercetin pretreatment (q+cer) group received quercetin (100 mg/kg-rat weight, in dimethyl sulfoxide) one time, one hour before cerulein treatment and physiological saline, one time, six hour after cerulein treatment. quercetin post-treatment (cer+q) group received dimethyl sulfoxide, one time, one hour before cerulein treatment and quercetin, one time, six hour after cerulein treatment. cerulein treatment increased significantly vascular congestion in hepatic cells. quercetin treatment also decreased significantly vascular congestion. the liver mda and carbonyl levels in cerulein group were significantly higher than the control group (p < 0.01, p < 0.001, respectively). the mda and carbonyl levels in q+cer group decreased significantly compared to the cer group (p < 0.001). the mda, carbonyl, mpo levels in cer+q group were significantly lower than the cer group (p < 0.001). the gssg/gsh ratio of q+cer and cer+q groups were significantly lower than the cer group (p < 0.05, p < 0.001, respectively). the sod activity in cer group was significantly lower than the control group, but the sod activity in q+cer and cer+q groups was significantly higher than the cer group (p < 0.05).this study shows that quercetin treatment was reduced the severity of liver injury secondary to cerulein induced-ap as reflected by changes in the parameters of hepatic oxidant and antioxidant. p-09.04.4-029 identification of water extract of propolis components by using different columns in gas chromatography-mass spectrometry propolis is a natural material obtained by honey bees from various plants. protective effect of propolis against damages of free radicals is due to different compounds within propolis. the aim of this study is to identify qualitatively and quantitatively the chemical composition of water extract of propolis (wep) provided by erzurum region using rtx-1 and rtx-5 ms column of gas chromatography-mass spectrometry (gc-ms) and to compare with two columns.in this study, wep of 25 mg/ml was prepared, cleared by membrane filter of 0.45 lm and freezed at à80°c. then, it was lyophilized up to dry form and derivatized to apply for gaseous form. 7 mg of dry extract was reacted with 350 ll pyridin + 700 ll bis-trimethylsilyl trifluoroacetamide (bstfa) mixture including 1% trimethylchlorosilane (tmcs) and incubated for 30 minutes at 100°c. all analyses were performed with shimadzu gcms-qp2010 ultra by using a flame ionisation detector (fid). rtx-1 and rtx-5 ms capillary columns and helium for carrier gas at a flow time of 1 ml/minute were used. injection was applied on split mode at 250°c. derivatized propolis sample was injected as 2 ll, initial column temperature was adjusted as 60°c, then increased to 240°c with increments of 3°c. total analysis time was determined as 62 minutes. relative percent amount of separated compounds was calculated from total ion chromatogram with computerised integrator. all components were defined by using nist and wiley libraries.peaks obtained from rtx-1 column were much more than those of rtx-5 ms. on the other hand, analyses performing with both two columns have similar carbohydrate, aromatic acid and other acid contents.consequently chemical constituents of wep were determined qualitatively and quantitatively with gc-ms. it was concluded that rtx-1 column among both columns differentiating for polarities may produce more compounds in the propolis analysis. introduction: aquatic environment can be mostly contaminated by mixtures of metals. biochemical parameters have gain importance to characterize the effects of metals on aquatic organisms. glutathione s-transferase (gst) and its substrate glutathione (gsh) are important parameters of antioxidant defense system of fish metabolism due to their vital role in xenobiotic conjugation. objective: the goal of the current study to evaluate the changes in gst and gsh levels in response to cd, zn and cd+zn effects after 14 and 28 exposure days. materials and methods: fish were obtained from cukurova university fish culturing pools (turkey). fish were exposed to 1.0 lg/ml of cd (cdcl 2 .h 2 o) and zn (zncl 2 ), and their mixture, for 0, 14 and 28 days. at the end of the exposure period, liver tissues were dissected and homogenized in a phosphate buffer (ph 7.4). homogenates were centrifuged at 10,000 g (30 min, +4°c). supernatants were stored at à80°c until the analysis. one-way anova was used to compare data (mean ae se) followed by duncan's test (p < 0.05). results: gst and gsh changes were recorded as decreases after all metal exposures at day 14. although 28 day exposure was found as less effective, combined effects caused significant decreases in gst and gsh levels. also longer exposure durations were appeared to be more effective in that situation. conclusions: significant decreases in gst and gsh levels could be occurred due to increased reactive oxygen species caused by metals particularly their combined effects. metal type, their single and combined effects and also exposure duration should be also taken into account when considering the antioxidant system response. gst and gsh might be considered as sensitive biomarker in toxicity assessment of metal mixtures.financial support: this study was supported by a grant from c ß ukurova university (turkey).background/aims: the activation of lectin-like oxidized low density lipoprotein receptor-1 (lox-1) on endothelial cells leads to intracellular oxidative stress and inflammation and a feed-forward cycle of injury in diabetes, since both oxidized low density lipoprotein (oxldls) and glucose increase lox-1 expression. quercetin (qr) is part of a subclass of flavonoids called flavonols. polyphenolic compounds affect the development of atherosclerosis not only through antioxidant properties but also by modulation of serum lipids, thereby influencing the immune and inflammatory processes associated with the development of atherogenic diseases. we investigated the effects of dietary qr on endothelial dysfunction mediated by oxidized low density lipoprotein (oxldl)/lectin-like oxidized low density lipoprotein receptor-1 (lox-1) in animal model of type 2 diabetes mellitus. methods: we compared 4 groups of male adult wistar albino rats: a control group, an untreated diabetic group, diabetic groups treated with qr, and qr group. diabetes was induced by a single injection of stz (50 mg/kg). animals were kept in standard condition. in 30 day after inducing diabetic, serum was collected for biochemical parameters. glucose, lipid profiles, microalbuminuria, oxldl and lox-1 levels were determined. results: serum triglyceride, ldl, vldl levels in diabetic control group (without treatment) was significantly higher than control group (normoglycemic untreated group). supplementation with quercetin decreased serum total cholesterol and increased hdl-cholesterol compared with the control group. serum oxldl and lox-1 levels in diabetic control group (without treatment) were significantly higher than control group (normoglycemic untreated group). conclusions: consumption of quercetin reduced oxldl and lox-1 levels. thus, quercetin could be effective in improving hyperglycemia, dyslipidemia, and endothelial damage in type 2 diabetes. p-09.04.4-055 investigation of some oxidative stress parameters in bdnf heterozygous mice liver tissue a. bodur 1 , i. ince 1 , i. abidin 2 , a. alver 1 objectives: the aim of this study was to evaluate the possible protective effects of nigella sativa (ns) against cholestatic oxidative stress and liver damage in the common bile duct ligated rats. methods: a total of 24 male wistar albino rats were divided into three groups:sham control, bile duct ligation (bdl) and bdl+received ns; each group contain 8 animals. the rats in ns treated groups were given ns (0.2 ml/kg) once a day orally for 4 weeks starting 3 days prior to bdl operation. results: the changes demonstrating the bile duct proliferation and fibrosis in expanded portal tracts include the extension of proliferated bile ducts into lobules, mononuclear cells, and neutrophil infiltration into the widened portal areas were observed in bdl group. treatment of bdl with ns attenuated alterations in liver histology. the alpha smooth muscle actin (a-sma), transforming growth factor beta (tgf-b1) and the activity of tunel in the bdl were observed to be reduced with the ns treatment. bdl significantly increased the tissue hydroxyproline (hp) content, malondialdehyde (mda) levels, and decreased the antioxidant enzyme (superoxide dismutase (sod) and glutathione peroxidase (gpx)) activities. ns treatment significantly decreased the elevated tissue hp content and mda levels and prevented the inhibition of sod and gpx enzymes in the tissues. conclusion: the data indicate that ns attenuates bdl-induced cholestatic liver injury, bile duct proliferation, fibrosis, apoptosis and oxidative stress. the hepatoprotective effect of ns is associated with antioxidative potential. organophosphorous compounds (ops) are used widely as pesticides for agricultural purposes, as oil additives or as flame retardants. however, due to their widespread use, there are major introduction: in this study, the antiulcerogenic effect of a water extract (cawe) obtained from a spices sample, cinnamomum aromaticum, was investigated using indomethacin-induced ulcer models in rats. materials and methods: experimental groups consisted of six rats. antiulcerogenic activities of 50, 100, 200 and 400 mg/kg body wt. doses of the cawe were determined by comparing the negative (treated only with indomethacin) and positive (famotidine) control groups. results and discussion: although all doses of the cawe showed significant antiulcerogenic activity as compared to negative control groups, the highest activity was observed with 400 mg/kg body wt. doses (45%). the cawe showed similarly antioxidant activity when compared with trolox and ascorbic acids used as positive antioxidants. in addition, the activities of catalase (cat) and myeloperoxidase (mpo) enzymes were determined in the stomach tissues of rats and compared with those of the negative and positive control groups to expose the effects of these enzymes on antiulcerogenic activity. the enzymatic activities of cat and mpo and lipid peroxidation (lpo) level in indomethacin-administrated tissues were increased significantly by indomethacin in comparison to control groups. these enzymes and lpo level were decreased, however, by the cawe. in contrast to lpo level, cat and mpo activities, glutathione (gsh) level was decreased by indomethacin and increased by all doses of cawe and famotidine. the present results indicate that the cawe has a protective effect in indomethacin-induced ulcers, which can be attributed to its antioxidant potential. introduction: thiol groups (-sh) are important anti-oxidants and essential molecules protecting organism against the harmful effects of oxidative stress. the aim of our study is to evalute thiol-disulphide homeostasis with a novel and automated method in patients with prostate cancer (pc) before and after radical prostatectomy (rp). material and methods: 18 patients with prostate cancer and 17 healty control subjects were enrolled into the study. plasma samples were collected from patients before rp and 6 months after the rp operation. thiol-disulphide homeostasis was determined with a recently developed novel method. prostate specific antigen, albumin, total thiol, native thiol, disulphide and total antioxidant status (tas) were evaulated and compared between the groups. results: native thiol levels were 419.8 ae 54.87 lmol/l in the control group, 350.7 ae 46.35 lmol/l in the patients before rp, and 364.3 ae 46.88 lmol/l in patients after rp. native thiol, total thiol and tas levels were significantly higher in the control group than the patients before rp (p values < 0.001). native thiol, total thiol and tas levels were higher 6 months after rp compared to before rp in patients, but these changes were not significant statictically (p values 0.3, 0.3 and 0.09 respectively). discussion and conclusion: our study demonstrated that antioxidant defense mechanism was weakened as indicated by the decreased thiol levels in the patients with pc. increased oxidative stress in prostate cancer patients may cause metabolic disturbance and have a role in the pathogenesis of prostate cancer. p-09.04.4-108 is there any relation between 50 g oral glucose challenge test and serum total oxidant-antioxidant status in pregnant woman? the purpose of this study was to test the hypothesis that any degree of antepartum screening for gestational diabetes mellitus with oral 50 g glucose challenge test (gct) should be associated with oxidant-antioxidant status.in this prospectif study, oral glucose challenge test was applied to 25 pregnant women aged 25-40 years and at 24-28 weeks of gestation. plasma glucose concentrations were measured initial, 1 hour and in addition to test 2 hours after ingestion of 50 g glucose. at the same time serum insulin, cortisol, total antioxidant status (tas), total oxidant status (tos) levels were measured and the oxidative stress index (osi) was calculated.ten pregnant women (forty percent) had a positive glucose challenge test (gct). a positive moderate relation with initial and 1 hour serum total antioxidant status (tas) levels (r = 0.68) and the oxidative stress index (osi) (r = 0.67) was found. there was a positive weak correlation with initial and 2 hours total oxidant status (tos) levels (r = 0.22) but statistically significance difference was not found (p > 0.05).in this study after ingestion of 50 g glucose serum total antioxidant status (tas), serum oxidant status levels (tos) and serum oxidative stress index (osi) levels were higher than the initial levels.the results of this study suggest that antepartum screening for gestational diabetes mellitus with 50 g oral glucose challenge test (gct) weakly associated with oxidant-antioxidant status and to confirm this results the longer follow-up studies with more participants are necessary.wheat (triticum ssp.), cultivated for centuries in the middle-east, central asia, europe, and north-africa, is one leading staple crops around the world, and its marginally grown ancestor einkorn (triticum monococcum ssp. monococcum), possesses rich gene resources for wheat improvement and have bioactive compounds reducing and preventing chronic diseases such as diabetes, cancer, alzheimer, and cardio vascular diseases, beside their nutritional properties. however, as more attention has been given to wheat cultivars with strong gluten, protein content, starch composition, and resistance to biotic and abiotic stresses in bread wheat and yellow-colored pasta product in durum wheat health compounds such as fibers, phytochemicals, and bioactives have been underestimated so far. the aim of this study was, then, to examine the total phenolics and flavonoids, quantify their phenolic acids, a-tocopherol by high performance liquid chromatography (hplc), and their 2,2-diphenyl-1-picrylhydrazyl (dpph) scavenging activity of bread (triticum aestivum l.), durum (triticum turgidum ssp. durum desf.) wheat cultivars and einkorn (triticum monococcum ssp. monococcum) wheat populations collected from different provinces (bolu and kastamonu) of turkey. ferulic acid (148.67-764.04-lg/g), p-coumaric (5.06-54.09-lg/g), and total phenolic content (ranged 2.06-8.11-lmol gae/g) of einkorn populations were significantly higher than bread and durum wheat cultivars. results suggested the possibility of production of einkorn wheat populations, and hopefully cultivars rich in particular health beneficial component(s) may provide benefit to the consumers. in addition, higher phenolic content of einkorn may offer novel wheat genetic resources for the improvement of new wheat cultivars and the development of wheat-based functional foods. oxidative damage due to ischemia and acute kidney injury (aki) after coronary artery bypass graft (cabg) surgery are the leading complication during this process. in the kidney, ischemia/ reperfusion injury contributes to aki that is a clinical syndrome with rapid kidney dysfunction and high mortality rates. some animal and clinical studies have demonstrated an increase in serum and urinary neutrophil gelatinase-associated lipocalin (ngal) expression after renal ischemic injury.in this study, our aim was to investigate the relationship betwwen ngal and oxidative stress parameters due to ischemia caused by total perfusion time (tpt) in patients who have undergone on-pump cabg. materials and methods: the study was conducted in 30 patients who received on-pump cabg at university of istanbul, cerrahpasa medical faculty, department of cardiovascular surgery. blood samples were collected prior to surgery and after 2 hours following the termination of cardiac pulmonary bypass (cpb) . following centrifugation, serum samples were separeted and stored at -80 c until analysis. serum ngal, ima (ischemia modified alb€ umin), pco (protein carbonyl), nt (nitrotyrosine), lpd (lipid peroxide) levels were determined by elisa procedure. results and discussion: serum ngal, pco, nt levels in after 2 hours following cpb were significantly higher than the before surgery (p < 0.001, p < 0.001, p serum ngal levels in after 2 hours following cpb was found to have positive correlation with ima, pco, nt, and lpo levels (r = 0.59, p < 0.01; r = 0.77, p < 0.001; r = 0.68, p < 0.001; r = 0.77, p < 0.001 respectively). ngal levels were positively correlated with total perfusion time after cbp (r = 0.37, p < 0.05).the results of our study show that, increased ngal levels 2 hours after cpb were positively correlated with oxidative stress parameters and total perfusion time. investigation of the effects of thymoquinone against indomethacine induced gastric damage in rats introduction: incidence and high cost of acute stomach mucosa damages make this issue a very interesting issue for study. for this reason, it is aimed to investigate the effects of different dosage of thymoquinone (tq) against indomethacine induced gastric damage in rats. material and method: in our study, six groups of 36 wistrar male rats were used. groups are named as healthy, ind control, famotidine (40 mg/kg) and three different doses of tq (0.5, 1 and 2 mg/kg). while any treatment or drug administration will done on healthy group, model was generated to other groups by giving fam or tq with tap water via oral gavage. 5 min later, 25 mg/kg ind administrated to each rat. animals were sacrified about 6 hour later and stomach samples of each groups were collected for macroscopic study and gsh levels measurements. results: lower doses of tq is more effective and all tq groups exhibit reduced ulcer region with respect to the ind control group. gsh level of ind control group is lower than healthy group. the gsh level of tq, especially in lower doses, and fam groups statistically exhibit an increase in gsh level. conclusion: it is observed that ind induced gastric damage cause ulcer and increase in free radical. it is determined that lower doses of tq (0.5, 1 and 2 mg/kg) is also exhibit a protective effect on ind induced model. it is tought that quinone in tq structure have a strong redox feature and this feature clean up the free radicals caused by ind, it reduces the oxidative stress and protect the stomach from ulcer. anzer honey is the most famous honey in turkey with many endemic species flowers. the anzer plateau is located rize province of eastern black sea region. in this study, antioxidant and anti-hyaluronidase and anti-urease activities were investigated of the plateau bee pollen. the antioxidant capacity was determined by total phenolic content (tpc), total flavonoids contents (tfc), ferric reducing antioxidant power (frap) and 2,2diphenyl-1-picrylhydrazyl (dpph) radical scavenging activity. atherosclerosis is the leading cause of mortality worldwide, and as a chronic inflammatory disease, caused by a complex interplay between inflammatory and oxidative events.quercetin, a plant derived flavonoid and a well-known antioxidant, has shown great promises with regards to its protective effects against oxidative stress.however due to its physicochemical properties, the optimum pharmacokinetic behavior is a challenging issue.herein, we aimed to fabricate quercetin loaded solid lipid nanoparticle (quer-sln) to improve the bioavailability and therapeutics efficiency. furthermore the in-vitro capacity of quer-sln for ameliorating tnf-a induced oxidative stress in human endothelial vein cell (huvec) was evaluated. quer-slns were prepared by simple hot homogenizing method and characterized by means of drug loading (dl), encapsulation efficiency (ee), cytotoxicity, size, zeta potential and morphology. antioxidant activity of plain quercetin and quer-slns were then investigated using intracellular reactive oxygen species (ros) detection method (dcfh-da assay) by facs flowcytometryin conclusion, the results here showed superior control of oxidative stress by quercetin nanosystem as compared to plain quercetin. precirol based slns as a biocompatible/biodegradable lipid, may provide a novel drug delivery system for quercetin with improved beneficiary impact in atherosclerosis. objective: zinc is known as an antioxidant essential trace element. we aimed to evaluate the dose-dependent effects of zinc on the oxidant-antioxidant system in liver, kidney and brain tissues of rats and the histological alterations in the absence of oxidative stress (os). material and methods: thirty-nine female weighing about 150 gr wistar albino rats were divided into four experimental groups as ad libitum (al) diet (control), al diet + 3 mg/kg zn sulfate (low dose; group 1), al diet + 12 mg/kg zn sulfate (middle dose; group 2) and al diet + 25 mg/kg zn sulfate (high dose; group 3). zn sulfate solutions were administered 0.2 ml/day orally for 13 days and in day 14 rats were sacrificed and tissues were excised for detecting malondialdehyde (mda), advanced oxidation protein products (aopp), superoxide dismutase (sod), glutathione peroxidase (gsh-px), glutathione reductase (gr), and glutathione-s-transferase (gst) activities. histological evaluation was also performed to confirm the effects of zinc. results: in liver tissues aopp levels decreased in all groups receiving zinc as compared to the control group. liver mda levels were increased in group 1 and 3; sod and gsh-px levels were both increased while gst levels were decreased in all groups compared to control. gr levels were increased only in group 2. in kidney; aopp level was decreased only in group 1 and sod level was only decreased in group 2 as compared to control while gr levels were increased in all doses of zinc. in brain; aopp, gsh-px and gr levels were decreased in all groups receiving zinc as compared to control group. sod activity in brain tissues was increased by the administration of middle dose of zinc (group 2). gst level was decreased in only group 1 conclusions: the biochemical and histological findings of this study suggest that zinc has various effects on liver, kidney and brain tissues in the absence of os.key words: zinc, liver, kidney, brain introduction: this study aimed to investigate the effect of diabetes mellitus (dm) on oxidative stress and antioxidant capacity in humour aqueous (ha) and venous serum using total antioxidant capacity (tac) and total oxidative stress (tos) levels in serum and ha in cataract patients. materials and methods: in this study patients were divided into two groups. group 1 was composed of patients with type 2 dm and cataract and group 2 was composed of patients with cataracts who are not accompanied by dm and cataract patients who are not accompanied by systemic diseases. each group consisted of 20 patients, totally 40 patients were included in the study. the ha which was collected from the eyes at the beginning of the cataract surgery and venous blood serum collected from the same patients were analyzed. in both groups, ha and serum tac and tos levels were measured with elisa. results: : serum tac levels in the dm group were significantly lower than in the control group (p < 0.05). tos serum levels in dm group was statistically higher than the control group (p < 0.05). differences between tac and tos levels were not statistically significant when compared the two groups' ha results (p > 0.05). group 1, divided into two subgroups according to their hba1c levels, there was no statistically significant difference between the subgroups when hba1c levels were compared with the relationship between serum and ha's tac and tos levels (p > 0.05). there was not an association between the gender, age and the levels of tac-tos in both groups (p > 0.05). discussion and conclusion: presences of dm is the only risk factor for increase of oxidative stress and decrease of anti-oxidant capacity in patients without a systemic complication of dm and diabetic retinopathy. in our study, diabetic patients without retinopathy showed similar ha tos and tac levels to healthy individuals, this finding indicates that blood-aqueous barrier is protected in these patients. the effect of ferulic acid against testicular ischemia/reperfusion injury in rats u. sac ßik 1 , g. erbil 1 , z. c ß avdar 2 , c. ural 2 1 department of histology and embriyology, school of medicine, dokuz eyl€ ul university, izmir, 2 department of molecular medicine, health science of institute, dokuz eyl€ ul university, izmir, turkeytestis torsion is one of the urologic emergencies occurring frequently in neonatal and adolescent period. testis is sensitive to ischemia/reperfusion (i/r) injury and, therefore, ischemia and consecutive reperfusion cause an enhanced formation of reactive oxygen species (ros) that result in testicular cell damage and apoptosis. ferulic acid, known as an antioxsidant, is a phenolic acid found in seeds and leaves of the plants. we aimed to investigate potential protective effect of ferulic acid against testis i/r injury.thirty five wistar rats were randomly divided into 5 groups; control, ethyl alcohol, ischemia, i/r, i/r-ferulic acid groups. animals were exposed to 2 hours of ischemia followed by 2 hours of reperfusion. ferulic acid was administered (100 mg/ kg) before reperfusion intravenously. testicular cell damage was examined by h-e staining and pas. tunnel, active caspase-3, inos and mpo were evaluated by immunostaining. malondialdehyde (mda), glutathione (gsh) levels, glutathione peroxidase (gpx) and superoxide dismutase (sod) activities were assessed by biochemical methods.histological evaluation showed that ferulic acid pretreatment reduced significantly testicular cell damage and decreased tun-nel, caspase 3 positive cells; inos and also mpo expression. in addition, ferulic acid administration decreased significantly the mda levels increased by i/r. morever, ferulic acid increased significantly the sod activity levels, which was decreased by i/r. there were no statistically significant differences in the levels of gsh and gpx activity in all groups.the present results suggest that ferulic acid is a potentially beneficial agent in protecting testicular i/r. background: in heart failure (hf), angiotensin antagonists (aa), beta-blockers (bb), spironolactone, diuretics and acetylsalicylic acid are often used. top 3 pharmaceutical groups reduce mortality. on the increased oxidative stress (os) in patients with hf, it is known to have beneficial effects of certain groups of drugs. however, the net effect of these drugs in os is unknown. the aim of this study was to investigate the effects of drugs used in hf on os. materials and methods: 133 patients were included in the study. all of the patients had systolic heart failure and all of them were under treatment. drugs used by the patients were recorded. the levels of total antioxidant status (tos), total oxidant status (tas), the enzymatic activity of ceruloplasmin, paraoxonase-1 and arylestherase were measured according to erel's method. serum total thiol levels were measured with sh modified hu method and the lipid hydroperoxide levels were measured with the ferrous ion oxidation xylenol orange assay. the percentage tos / tas was determined as osi. results: in patients treated with acetylsalicylic acid (asa), spironolactone, beta blocker and furosemide, there were increased tos, decreased tas and osi (p < 0.05). in patients treated with angiotensin blockers, increased tas and looh, and decreased sh were found (p < 0.05). in patients treated with nitrates and ccb, tos and osi were found decreased. correlation analysis showed that increased tas correlated with the use of angiotensin blockers, asa, furosemide and beta blocker positively; and with the tos and osi level correlated with the use of spironolactone, asa spironolactone and furosemide (p < 0.05). conclusion: current medical agents that are being used in hf are effective in reducing os in hf patients. one of the effective mechanisms to reduce the mortality of some of these drugs may decrease os.key words: heart failure, drug use, oxidative stress p-09.04.4-119 across adjacent ring formed titanium phthalocyanine-mediated photodynamic therapy alters and degrades filamentous actin cytoskeleton and internal membranes photodynamic therapy (pdt) is widely accepted as a promising and minimally invasive treatment strategy due to its applicability on a wide range of cancer diseases. this clinically approved treatment method relies on the dramatic production of singlet oxygen and reactive oxygen species (ros) in target tissue to evoke apoptotic cell death [1] . we, therefore, focused on the intracellular ros accumulation, internal membrane degradation, filamentous actin cytoskeleton alteration and nucleus morphology changes induced by pdt-mediated across adjacent ring formed titanium phthalocyanine which was previously synthesized bis(ethane-1,1p-phenol-2,2-p-phenoxy) phthalocyaninatotitanium (iv).characterization of the synthesized metallophthalocyanine was accomplished by using uv-vis, ir, 1 h-nmr and maldi-tofmass spectroscopies. the dark and pdt-mediated activities of bare and phosphonolipids (max. 5%) charged titanium phthalocyanine (0.625, 1.25, 2.5, 5 and 10 lm) were determined on a549 human lung carcinoma and hacat human keratinocyte cell lines by using intracellular ros assay, dioc(6), tritc-phalloidin and dapi staining protocols. waltmann pdt 1200 l was used as the non-toxic light source at 100 j/cm 2 fluence and 150 mw/ cm 2 fluence rate. the experiments showed that pdt-mediated titanium phthalocyanine leads to significant and concentration dependent reactive oxygen species accumulation. moreover, internal membrane degradation, apoptotic bodies on nucleus and filamentous actin cytoskeleton alteration were observed. consequently, the activity mechanism of pdt-mediated titanium phthalocyanine seems to be in a tight relationship with ros accumulation-mediated internal membrane degradation, filamentous actin cytoskeleton alteration and apoptotic pathways activation. introduction: retinal vein occlusion (rvo) is a common retinal vascular disorder that can affect visual acuity and cause blindness in elder population. sulphur containing aminoacids such as cysteine (cys), cysteinylglycine, glutathione, homocysteine and c-glutamylcysteine are reported to be associated with the pathogenesis of rvo. thiols are organosulfur compounds that are formed of a carbon-bonded sulfhydryl group. sulphur containing aminoacids slightly contribute the composition of plasma thiol pool. thiols can undergo oxidation reaction via oxidants and form disulphide bonds our purpose is to research the relationship between a novel oxidative stress marker serum dynamic thioldisulphide homeostasis and retinal vein occlusion. materials and methods: 22 rvo patients and 22 controls were included in the study. native thiol, total thiol, disulphide levels are measured in the serum samples of rvo and control group by using an automated method described by erel et al. also disulphide/native thiol and disulphide/total thiol ratios were calculated. results: there were no significant difference between the rvo and control group in native thiol, total thiol, disulphide disulphide/native thiol and disulphide/total thiol ratios.(p > 0.05 for all) conclusion: our study is the first report evaluating the dynamic thiol-disulphide homeostasis in rvo patients by a newly developed method by erel et al. further large sample sized studies investigating the levels of sulphur containing aminoacids may additionally be planned to verify this study. purpose: the purpose of this study was to evaluate markers of systemic oxidative stress and antioxidant capacity in subjects with severity of osas. methods: a total of 106 osa patients were included in the study (18 controls, 14 with mild, 14 with moderate, and 60 with severe osa). patients were grouped according to apnea-hypopnea index (ahi) as mild, moderate and severe osa. patients with ahi<5 served as control group. known risk factors for oxidative stress, such as age, sex, obesity, smoking, hypelipidemia, and hypertension, were investigated as possible confounding factors. plasma arylesterase, total oxidative stress (tos), total antioxidant capacity (tac), total thiol, catalase (cat) levels were measured for all patients. results: the mean age was 52.49 ae 12.9 years and 40.6% (43/ 106) of the study population was female. plasma arylesterase, tos, tac, total thiol, and cat plasma values were not different between mild, moderate, severe osa groups and controls (p > 0.05). catalase levels were significantly lower in women patients with severe osa compared to healthy women controls (p < 0.05). there was a negative correlation between ahi and serum total thiol levels (r = à0.289, p < 0.05) in severe osa groups. conclusionthe present prospective study provides evidence that osa might be associated with decreased antioxidant burden possibly via catalase way. results: the sera 8-ohdg, mda and il-6 were significantly higher in diabetic group than control group (p < 0.05). although there was a notable positive correlation between mda and 8-ohdg, there was no a relationship between 8-ohdg or mda with il-6. discussion: in agreement with previous studies our data illustrated that high levels of oxidative stress is associated with increased production of oxidized lipids and nucleobases in diabetic patients compared to control group. also enhanced proinflammatory cytokine, il-6, induced inflammmation in these patients.conclusion: oxidative stress and inflammation play pivotal roles in the development of diabetes and can cause major complications in dm. so we suggest that early detection of these measurable indicatores can help to diagnosis the severity or presence of some complication in diabet. the effect of quercetin on erythrocyte glucose-6-phosphate dehydrogenase enzyme activity in ethanol treated rats in this study, we aimed to evaluate the effects of ethanol on erythrocyte (g-6-p-d) enzyme activity and the effects of quercetin on erythrocyte g-6-p-d activity in the recovery of the effects of ethanol.rats were randomly divided into four groups. the control group (n = 6) received physiological saline. the quercetin group (n = 7) received quercetin (120 mg/kg/ day) via i.g. route the alcohol group (n = 7) received ethanol (80% v/v, 1 ml/day) via i.g. route. the alcohol + quercetin group (n = 5) received 1 ml of ethanol (80% v/v) 2 hours after quercetin treatment (120 mg/ kg/day). experimental procedures were peformed for 30 days. erythrocyte g-6-p-d activity was found to be higher in the quercetin group than those in the alcohol group (p < 0.001). in the alcohol group, the erythrocyte g-6-p-d activity was found to be significantly decreased than those in the control group (p < 0.001). statistically significant differences were observed in erythrocyte g-6-p-d activity between the alcohol group and the alcohol + quercetin group (p < 0.001).as a conclusion, our results demonstrate that ethanol decreased erythrocyte g6pd activity and quercetin was found to be beneficial in the prevention of toxic effect raised by ethanol.key words: erythrocyte, ethanol, g6pd, oxidative stress, quercetin p-09.04.4-126 effects of the sulphasalazine to the cerebral hypoxia reperfusion injury in rat background: cerebral ischemia/ reperfusion (i/r) injury is still a difficult process to treat and rehabilitate today. this study was designed to investigate beneficial effects of sulfasalazine in cerebral i/r injury in rat. methods: except control group (n = 5), 20 wistar albino rats were divided into four groups for acute and chronic stage investigation of i/r injury, and temporary aneurysm clips were attempted to both internal carotid arteries for duration of 30 minutes. four hours later, except control, sham-a, sham-c groups, 40 mg/kg once a day sulfasalazine was administered to animals, orally. animals were sacrified and then necrotic neuronal cells of hippocampal ca1, ca2, and ca3 region, and cortical necrotic neurons, perivascular edema, pyknotic neuronal cells, irregularities of intercellular organization (iio) were counted and scaled histopathologically. tissue il-1b, il-6, malonyldialdehyde (mda), myeloperoxidation (mpo), no, and tnf-a levels were measured by using elisa, too. results: sulfasalazine could reduce perivascular edema, iio, cortical and hippocampal neuronal cell death in both stages. it could decreased mda in acute stage, but not reduce il-1b, il-6, mpo, no, and tnfa levels. it could increased il-1b levels in chronic stage but not affect to il-6, mpo, mda, no, tnf-a levels.conclusion: sulfasalazine could improve histopathological architecture of hypoxic tissue in both stages of i/r injury. it could inhibit lipid peroxidation cascades in acute stage but not affect to tissue mpo, no, il-6, and tnf-a levels in any stage in rat. these results suggested that therapeutic mechanisms of sulfasalazine should be investigated by using more specific laboratory methods in future studies.key words: antiinflamatory, cerebral hypoxia reperfusion injury, sulfasalazine, stroke. camel and horse milk xanthine oxidase (xo) was found to catalyze the reduction of nitrate and nitrite to nitric oxide (no) under aerobic condition. to date, mammalian nitrate reductase (nar) and nitrite reductase (nir) have not been identified. no, a gas, is found to control a seemingly, limitless range of functions in animals.one assay was used to determine nar and nir activities of milk xo: (1) nitrite formation from nitrate by nar, and (2) nitrite utilization by nir. nitrite concentrations were determined by using sulfanylamide and n-(1-naphtyl)-ethylenediamine, which form red color measured at 485 nm.these activities of the milk xo require nadh as a physiological electron donor. high xo, nar and nir activities are detected only after heat treatment (80°c, 5 min) of the fresh milk in the presence of molybdate. in both camel and horse milk nar activity of xo was almost two times higher than its nir activity. it is well known that xo can be reversibly converted from the dehydrogenase form to the oxidase through the oxidation of sulfhydryl groups. cysteine and, to a lesser extent, glutathione increased nir activity of milk xo but not its nar activity. the mechanism of this increase of nir activity remains unclear and is currently under study. substitution of tungsten for molybdenum under above conditions gave no detectable nar and nir activity of milk xo. the molybdenum site-directed inhibitor, tungsten inhibited in a dose-dependent manner. therefore, nitrate and nitrite are clear to interact with mo center of xo.camel and horse milk are traditional drinks in central asia and kazakhstan. therefore, it is very important that xo provide a mechanism for generation of no in camel and horse milk where nitric oxide synthase, no producing enzyme, does not exist. p-09.04.4-128 the influence of phytomedicine on metabolic processes of white rats undergone to ionized radiation the study of peroxide lipids oxidation (plo) process is used as one of stability parameters of organism's changes and as a key mechanism for understanding of adaptation reactions and of pathogenesis of different diseases. it's determined by high biological activity of products which are formed in the plo reactions, in this relation lipids with high contents of fat acids play important role. to investigate the influence of phytomedicine eminium regelii on the metabolic processes (peroxide lipids oxidation) of white rats' organism in conditions of ionized radiation.the animals were exposed to ionizing radiation (gamma-radiation 60 co) on the radiotherapeutic equipment teragam in a dose of 6 gy and received phytomedicine eminium regelii in a dose of 2.5 mg/kg orally within 14 days following the ionizing radiation exposure. gamma-rays caused the increase of lipid peroxidation (lpo) primary (dc) and secondary products' (mda) concentrations in spleen, liver, thymus and adrenal glands.treatment by phytomedicine resulted in contents of dc decreased in 3 times in spleen, in 7 times in thymus, in 5 times in adrenal glands, in liver in 4 times, in lymph nodes of small intestine it in 3 times. mda decreased in liver up to 6 and 3 times, in spleen in 3.2 times, in thymus in 9 times, in liver in 6 times, in adrenal glands in 12 time, no changes in lymph nodes of small intestine.the effect of phytomedicine treatment of organisms exposed to sublethal dozes of gamma-radiation results in the lpo primary and secondary products concentrations decrease in spleen, liver, thymus and adrenal glands. p-09.04.4-131 evaluation of serum levels of ischemia modified albumin (ima) in bipolar disorder patients k. € unal 1 , c. topc ßuoglu 2 , m. cingi 3 1 clinic of biochemistry, ankara polatli duatepe public hospital, ankara, 2 clinic of biochemistry, ankara numune training and research hospital, ankara, 3 clinic of psychiatry, ankara numune training and research hospital, ankara, turkey introduction: bipolar disorder is one of the most debilitating psychiatric disorders characterized by disruptive episodes of mania/hypomania and depression. considering the complex role of biological and environmental factors in the etiology of affective disorders; recent studies have focused on oxidative stress, which may damage nerve cell components and take part in pathophysiology. aim of our study is to contribute these data about oxidative stress in bipolar disorder, by detecting ischemia modified albumin (ima) levels of bipolar disorder patients in remission and also by comparing these results with healthy controls. methods: study population consisted of 35 patients meeting the diagnostic and statistical manual of mental disorders, fifth edition (dsm-5) criteria for bipolar disorder i. 36 healthy subjects were included as control group (hc). serum ischemia modified albumin (ima) levels of all participants were determined. results: statistical analysis on serum ischemia modified albumin (ima) levels did not show any significant difference between bipolar disorder patients in remission and healthy controls.conclusion: studies on oxidative stress in bipolar disorder have reached controversial results up till now. in this study, no statistically significant difference was detected between oxidative parameters of bipolar disorder patients in remission and healthy controls. in order to evaluate oxidative stress in bipolar disorder comprehensively, further studies are needed. keywords: bipolar disorder, ischemia modified albumin (ima), oxidative stress p-09.04.4-133 xanthine oxidase and adenosine deaminase activity in patients with familial mediterranean fever (fmf) objective: fmf is an autosomal recessive dissease which is characterized by recurrent fever and inflammation of serous membranes. in this study we measured serum adenosine deaminase (ada) and xanthine oxidase (xo) levels in fmf cases. method: serum ada levels were measured with a sensitive colorimetric method described by giusti and xo levels were analysed by the method of worthington in 30 fmf patients and 30 healthy controls. results: there was a significant difference in xo and ada levels between controls and cases. ada and xo levels were higher in patents with fmf. humic acid (ha) is a natural product which is forming during decomposition of organic matter in humus. in recent years, there are some resarches on the medical use of ha. the present study was undertaken in order to evaluate the anticancer properties of the ha using a prostate cancer and osteosarcome cell lines pc-3, sjsa1 as an in vitro model system.ha was purchased from sigma-aldrich. the cells were maintained in dmem medium supplemented with 10% heat-inactivated fbs and 1% penicillin/streptomycin. cells were grown in petri dishes in a humidified atmosphere containing at 37•c. five different concentrations (100 ug/ml, 50 ug/ml, 25 ug/ml, 10 ug/ ml, 5 ug/ml) were prepared using a stock solution of ha. we measured cell proliferation and migration to understand of progression effects of ha in pc-3 and sjsa1 cell lines in vitro.according to our results, ha treatment caused cytotoxicity and induced cell death in vitro in pc-3 cells with an ic50 value of 67.9 lg/ml. contrary to this, ha induced proliferation of sjsa1 cells in dose dependent manner. ha demonstrated the highest proliferatif activity against sjsa1 cells with an ic50 value of 100 > lg/ml. on the other hand, cell migration was reduced in pc-3 cell line and interestingly, migration was accelerated in sjsa1 cell line.our study may provide new insights into the regulatory effect of ha in cancer, but further studies are needed to clarify the role of ha in cancer pathogenesis. the febs journal 283 (suppl. 1) (2016)